Peptide Science - Present and Future

PEPTIDE

SCIENCE – PRESENT AND FUTURE

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PEPTIDE SCIENCE – PRESENT AND FUTURE Proceedings of the 1st International Peptide Symposium

Edited by

YASUTSUGU SHIMONISHI Osaka University, Japan

KLUWER ACADEMIC PUBLISHERS N E W Y O R K , B O S T O N , D O R D R E C H T, LONDON , MOSCOW

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0-306-46864-6

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0-792-35271-8

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Contents

xxxv

Preface First International Peptide Symposium

xxxvii

Acknowledgements

xxxix

Abbreviations

xli

Plenary lectures 1.

2.

From big molecules to smaller ones J.A. Wells, B.C. Cunningham, A. Braisted, S. Atwell, W. DeLano, M. Ultsch, M.A. Starovasnik and A.M. de Vos

1

Directions for solving the remaining problems in the synthesis of large peptides S. Sakakibara

6

General lectures 3. 4.

Peptide science: past, present and future D. Brandenburg

15

Quo vadis? Where is peptide chemistry headed? T. Shiba

21

Session A: Peptide mimetics and de novo design 5.

6.

7.

8.

Designing peptidomimetic antagonists for peptide receptors: topochemical differences of agonists and antagonists V.J. Hruby, S. Liao, G. Han, G. Bonner, M. Shenderovich, J. Slaninova, F. Porreca, H.I. Yamamura and M.E. Hadley

26

Self-assembling homopolymeric peptide tapes in aqueous solution A. Aggeli, M. Bell, A. Strong, S. Radford and N. Boden

30

Peptides containing nonnatural amino acids with functional side groups M. Sisido

34

Active carbonates of N-protected amino alcohols for synthesis of carbamate pseudopeptides J.B. Halstrøm

39

v

vi 9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Contents Convenient one-pot synthesis of cystine-containing peptides from protected peptidyl resins and its application to the synthesis of bifunctional anti-HIV compounds H. Tamamura, T. Ishihara, H. Oyake, A. Otaka and N. Fujii

41

Stereoselective synthesis of two type (E)-alkene dipeptide isosteres from a single substrate of β -aziridinyl- α,β -enoate H. Tamamura, M. Yamashita, H. Muramatsu, T. Ibuka, A. Otaka and N. Fujii

44

Design and synthesis of conformationally constrained dipeptide containing the α -fluoroglycine residue Y. Takeuchi, K. Kirihara, M. Kamezaki and N. Shibata

47

Screening of peptidomimetic tyrosine kinase inhibitors for inducing programmed cell death G. Kéri, L. Ôrfi, F. Hollósy, T. Vántus, J. Érchegyi, M. Idei, A. Horváth, I. Teplán, A. Gazit, I. Peták, Z. Szegedi and B. Szende

49

Molecular assembly of helix peptides vertically oriented on gold surface Y. Miura, S. Kimura, Y. Imanishi and J. Umemura

52

Gramicidin vesicles – self-assembly in water composed of gramicidin A as hydrophobic core of membrane D.-H. Kim, S. Kimura and Y. Imanishi

54

Design, synthesis, and characterization of a dimeric analog of small globular protein (SGP) in solution and its biological and channel-forming activities T. Kiyota, E. Matsumoto, S. Yamashita, S. Lee and G. Sugihara

57

Molecular design of cyclic peptides containing a non-natural amino acid: novel synthetic approach to artificial ion channels K. Donowaki, H. Ishida, Y. Inoue, Z. Qi, M. Sokabe, M. Azumano and S. Futaki

59

Structure based design and characterization of exocyclic peptidomimetics that inhibit TNFα binding to its receptor W. Takasaki, Y. Kajino, K. Kajino, M.I. Greene and R. Murali

61

A three-helix bundle metallopeptide destabilized at an acidic condition K. Suzuki and T. Tanaka

64

Contents 19.

20.

21.

22. 23. 24. 25.

26.

27.

28.

29.

vii

The alamethicin-leucine zipper hybrid peptide: modulation of the assembling of transmembrane peptides through the extramembrane peptide segments S. Futaki, M. Fukuda and M. Niwa

67

BCvir: backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in non-dividing cells. A. Friedler, N. Zakai, O. Karni, Y.C. Broder, L. Baraz, M. Kotler, A. Loyter and C. Gilon

70

Self-assembling homopolymeric peptide tapes in moderately polar organic solvents A. Aggeli, M. Bell, R. Owens, A. Smith and N. Boden

73

Substrate specificities of artificial flavo-enzymes K.-Y. Tomizaki, Y. Kakimoto, H. Akisada and N. Nishino

76

Catalytic activities of an artificial protein with designed loops K.-Y. Tomizaki, D. Oka, Y. Kakimoto and N. Nishino

78

Catalytic activities of an artificial heme enzyme K. Kaneko, K.-Y. Tomizaki and N. Nishino

80

A novel dioxopiperazine suitable as a β -turn constraint J.S. Davies, M. Stelmach-Diddams, R. Fromentin, A. Howells and R. Cotton

82

Amphiphilic β -structure formed by oligopeptides composed of alternating hydrophilic and hydrophobic residues S. Ono, K. Yamauchi, Y. Sugise, T. Tsutsumi, T. Okada, T. Fujii and T. Yoshimura

85

Design of cyclic antimicrobial peptides with low hemolytic activity L.H. Kondejewski, M. Jelokhani-Niaraki, E.J. Prenner, R.N.A.H. Lewis, R.N. McElhaney, R.E.W. Hancock and R.S. Hodges

88

Synthesis and characterization of linear and cyclic oligopeptides of 5-alkyl-substituted 3-aminobenzoic acids K. Endo, H. Takahashi, T. Kurusu, K. Kaji and A. Sida

92

Theoretical analysis of βαβαβ -type packing motifs formed by alternate intramolecular arrangements of three α -helices and three β -strands M. Oka and T. Hayashi

95

viii

Contents

30.

Theoretical analysis of β -type packing motifs formed by intramolecular arrangements of β -strands M. Oka and T. Hayashi

31.

32.

33.

34. 35.

36.

37.

38.

39.

98

Theoretical analysis of αββα -type packing motifs formed by intramolecular arrangements of two α-helices and two β strands M. Oka and T. Hayashi

101

Self-catalytic conversion of peptides from α -helix to α structural amyloid Y. Takahashi, A. Ueno and H. Mihara

104

Design, synthesis and characterization of heme-conjugated two- α -helix peptides S. Sakamoto, A. Ueno and H. Mihara

107

Self-assembly of heme-conjugated two-α -helix peptides I. Obataya, S. Sakamoto, A. Ueno and H. Mihara

110

Construction of porphyrin-binding peptides mimicking antiheme monoclonal antibody M. Takahashi, A. Ueno, T. Uda and H. Mihara

113

Construction of cyclodextrin–peptide conjugates and their molecular sensing properties S. Matsumura, S. Sakamoto, A. Ueno and H. Mihara

115

Interaction of bundled Ser-rich peptides with phospholipid bilayer K. Yoshida, N. Ohmori, T. Niidome, T. Hatakeyama and H. Aoyagi

117

5

Topographically designed analogues of c[D-Pen² , Pen , Nle 6 ]deltorphin I with stereoisomers of β -MePhe or β -iPrPhe in position 3 A. Misicka, J. Lin, K. Hosohata, H.I. Yamamura, P. Davis, F. Porreca and V.J. Hruby

119

Engineering novel mini-proteins by the transfer of active sites to small disulfide stabilized natural scaffolds C. Vita, E. Drakopoulou, J. Vizzavona, P. Garnier and A. Ménez

121

Session B: Peptide combinatorial libraries 40.

Preparation of heterocyclic positional scanning combinatorial libraries from peptides R.A. Houghten, S.E. Blondelle, C. Dooley, J.-P. Meyer, J.M. Ostresh and A. Nefzi

123

Contents 41.

42. 43.

44.

45.

46.

47.

ix

Design and synthesis of a lactam bridge stabilized coiled-coil template for library display M.E. Houston, R.T. Irvin and R.S. Hodges

128

Diversity of synthetic peptides Y. Konishi, J.J. Slon-Usakiewicz and G. Pietrzynski

132

Manual construction of combinatorial resin bound tripeptide library (one peptide on one bead) and screening to find antioxidative peptides T. Yasuhara and K. Nokihara

134

Selection of peptides that bind to α -lactalbumin from phage display library T. Nakamura, K. Hagiwara, K. Nitta and K. Takase

136

Identification of a novel analgesic peptide including an unnatural amino acid from peptide libraries synthesized with a multipeptide synthesizer I. Maeda, N. Kanemoto, K. Ogino, M. Kuwahara, Y. Goto, M. Adachi, T. Taki, M. Ohtani, Y. Muneoka, T. Kawakami and S. Aimoto

139

Artificial proteins screened from combinatorial chemical libraries with strong binding to double-stranded oligodeoxyribonucleotides: towards repressors to long terminal repeat controlled viral transcription. S.R.K. Hoffmann and B. Gutte

141

Synthesis, conformational analysis and application of tropanebased Fmoc-amino acid derivatives: novel antagonists of fibrinogen receptor, GPIIb/IIIa P.E. Thompson, D.L. Steer, K.A. Higgins, M.-I. Aguilar and M.T.W. Hearn

144

Session C: Novel methodologies in structural analyses 48.

49.

50.

Accurate peptide sequencing by mass spectrometry T. Takao, W. Mo, J. Fernandez-de-Cossio, J. Gonzalez, V. Besada, G. Padron and Y. Shimonishi

146

Characterization of sulfhydryl compounds attached to the cysteinyl residue of proteins or peptides by on line postcolumn derivatization K. Nokihara and T. Yasuhara

152

Regioselective hydrolytic cleavage of N-terminal myristoylpeptide K. Sugimoto, K. Okimura, N. Sakura and T. Hashimoto

154

x 51.

52.

53.

54.

55. 56.

Contents Selective removal of pyroglutamic acid residue from biologically active pyroglutamyl-peptides by aqueous methanesulfonic acid J. Kobayashi, K. Ohki, N. Sakura and T. Hashimoto

157

Hydrolytic removal of acetyl-amino acid from acetyl-peptide in methanesulfonic acid K. Ohki, N. Sakura and T. Hashimoto

160

Determination of the binding-site structure of the atrial natriuretic peptide receptor by stepwise-affinity alkylation K.S. Misono

163

Structure of glycoside of Bombyx mori prothoracicotropic hormone S. Nagata, J. Kobayashi, H. Kataoka and A. Suzuki

166

Photophysics of sterically constrained phenylalanines L. Lankiewicz, K. Stachowiak, A. Rzeska and W. Wiczk

168

Addressing combinatorial and peptide analysis by using overpressured layer chromatography in comparison with highperformance liquid chromatography and capillary electrophoresis G. Dibó, E. Mincsovics and Z. Majer

171

Session D: Peptides and proteins in signal transduction: Interaction with membrane receptors and intracellular role 57.

58.

59.

60.

61.

AVP(4-8)-induced branching signaling pathway was mediated by a putative GPCR in rat brain Q-W. Yan, L-Y. Qiao and Y-C. Du

174

The mechanisms of exocytosis induced by neurokinin peptides in mast cells H. Mukai, M. Kikuchi, Y. Suzuki and E. Munekata

179

Effects of dynorphins on free cytoplasmic calcium concentration and insulin release in ob/ob mouse pancreatic β cells S.V. Zaitsev, I.B. Efanova, A.M. Efanov and P-O. Berggren

183

The effect of magnesium ions on the uterotonic activity of oxytocin analogs J. Slaninová, L. Maletínská, M. ertová and Z. Procházka

185

Human thymopoietin beta-fragments: synthesis and antibody studies P.J.A. Weber, C.P. Eckard, S. Gonser, H. Otto, G. Folkers and A.G. Beck-Sickinger

188

Contents x i 62.

63.

64. 65.

66.

67.

68.

69.

70.

71.

72.

Cholecystokinin receptor parameters responsible for specific cellular signalling D. Gagne, N. Bernad, L. di Malta, R. Poosti, J-C. Galleyrand, S. Poirot, D. Fourmy and J. Martinez

190

Sweetness-suppressing activities of gurmarin analogues missing one disulfide bond M. Ota, Y. Shimizu, K. Tonosaki and Y. Ariyoshi

193

Modeling of the somatostatin receptor (hsstr3)-ligand complex J. Igarashi, X. Jiang and M. Goodman

195

Structural requirements of hyperalgesic nociceptin in receptor binding and activation T. Nose, R. Nakashima, R. Hatano, T. Sujaku, Y. Yamada, A. Nagahisa and Y. Shimohigashi

198

Activation of phosphatidylinositol 3-kinase via epidermal growth factor receptor in isolated rat hepatocytes T. Fujioka and M. Ui

200

Agonist-antagonist structure-activity relationships of thrombin receptor tethered ligand peptide T. Fujita, T. Nose, M. Nakajima, Y. Inoue, N. Nakamura, T. Inoue, T. Costa and Y. Shimohigashi

202

The first extracellular loop of the rat glucagon receptor contains determinants of glucagon binding C.G. Unson, A.M. Cypess, C-R. Wu, C.P. Cheung, B. Yoo and R.B. Merrifield

205

A novel type of rat brain δ opioid receptors differenciated by cyclopropylphenylalanine-containing enkephalin analog Y. Chuman, T. Yasunaga, T. Costa and Y. Shimohigashi

207

Synthesis of dimeric novobiocins as chemical inducers of dimerization for DNA gyrase-fused proteins M. Miyazaki, J. Kim, K. Oshikiri, K. Saito and S. Yokoyama

210

Computer biochemistry and molecular physiology of the natural peptide ligands: different functional groups A.A. Zamyatnin and O.L. Voronina

212

Bacterial peptide pheromone is imported where it directly binds to an intracellular receptor J. Nakayama, T. Horii and A. Suzuki

215

xii Contents 73.

74.

75.

76.

Grb2 SH2 Domain targeted inhibitors functioning in cellular signaling F-D.T. Lung, K. Rosenberg, L. Sastry, S. Wang, X-W. Wu, C.R. King and P.P. Roller

218

Interaction between heat-stable enterotoxin and its receptor (guanylyl cyclase C): influence of mutation at N-linked glycosylation sites on its ligand binding M. Hasegawa, Y. Matsumoto, Y. Hidaka and Y. Shimonishi

221

Receptor studies with novel insulin analogues and photoaffinity labelling techniques D. Brandenburg, M. Fabry, Y. Fischer, H-G. Gattner, J. Grötzinger, M. Hagelstein, C. Havenith, G. Kurapkat, S. Rütten, M. Siedentop and A. Wollmer

223

Radioligands for the receptor of mammalian melaninconcentrating hormone (MCH) E. Hintermann, R. Drozdz, H. Tanner, U. Zumsteg and A.N. Eberle

226

Session E: Structure, dynamics & folding of peptides and proteins 77.

78. 79.

80.

81.

82.

Using peptides to study the folding of a predominantly β -sheet protein K.S. Rotondi and L.M. Gierasch

228

α-Helical propensity of Ala-based peptides in organic solvents L-P. Liu and C.M. Deber

232

Effects on folding properties of endothelin/apamin by incorporation of selenocysteine into their cysteine framework S. Pegoraro, S. Fiori, S. Rudolph-Böhner, T.X. Watanabe and L. Moroder

235

Peptide conformational behaviour studied by high and low temperature reversed phase HPLC. M.T.W. Hearn, R. Boysen, Y. Wang and S. Muraledaram

240

Three-dimensional structure and receptor-recognition sites of bombyxin-II, an insulin-like brain-secretory peptide of the silkmoth K. Nagata, H. Hatanaka, D. Kohda, H. Ishizaki, K. Momomura, K. Tamori, T. Kadowaki, M. Tanaka, H. Kataoka, H. Nagasawa, A. Isogai, A. Suzuki and F. Inagaki

245

Cα -Methyl, Cα -phenylglycine peptides M. Doi, T. Ishida, E. Mossel, F. Formaggio, M. Crisma, C. Toniolo, Q.B. Broxterman and J. Kamphuis

249

Contents 83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

xiii

Graphite-mimetic peptides C. Toniolo, F. Formaggio, M. Crisma, T. Yoshikawa and T. Wakamiya

251

The E. Fischer’s octadecapeptide revisited after 90 years C. Toniolo, F. Formaggio, M. Crisma, S. Pegoraro and P. Rovero

253

Conformational analysis peptide fragments corresponding to domains of potassium ion-channel proteins P.I. Haris

255

Conformation and sweet taste of L-Asp- D -Xaa–OMe dipeptides Y.J. Kim and Y.K. Kang

257

Conformations of four cyclic tetrapeptides containing two Pro residues M. Tamaki, M. Yabu, A. Kaneko, S. Akabori and I. Muramatsu

260

Solution structure of the antimicrobial peptide ranalexin and a study of its interaction with dodecylphosphocholine micelles E. Vignal, A. Chavanieu, P. Roch, L. Chiche, G. Grassy, B. Calas and A. Aumelas

263

Formation of native disulphide bonds in endothelin-1: evidence for the involvement of a highly specific salt-bridge between the pro-sequence of endothelin-1 and mature sequence A. Aumelas, S. Kubo, N. Chino, L. Chiche, E. Forest, C. Roumestand and Y. Kobayashi

266

Structure–biological activity relationships of cecropin A (1–8)– magainin 2(1–12) hybrid and its analogues S.Y. Shin, J.H. Kang, D.G. Lee, S.Y. Kim, M.K. Lee and K.-S. Hahm

269

Structure-activity relationship of ω-agatoxin-TK, a peptide blocker of P/Q-type calcium channels T. Watanabe, M. Kuwada, Y. Nishizawa, K.Y. Kumagaye, K. Nakajima and N. Asakawa

271

Peptide scanning in structural-functional mapping of hemeproteins E.F. Kolesanova, J.G. Kiselar, S.A. Moshkovskii, S.A. Kozin, G. Hui Bon Hao and A.I. Archakov

274

xiv

Contents

93.

Structure analysis of the second transmembrane segment of the rat bradykinin receptor in solution and in micelles by CD and fluorescence spectroscopies E. Oliveira, A. Miranda, D. Andreu, S. Schreier, A.C.M. Paiva, C.R. Nakaie and M. Tominaga

277

Conformational analysis of antimicrobial peptide tachystatin A isolated from horseshoe crab hemocytes by two dimensional ¹H NMR spectroscopy N. Fujitani, S. Kawabata, K. Kawano, K. Hikichi and K. Nitta

280

Solution structure of α -conotoxin MI determined by ¹H-NMR spectroscopy and molecular dynamics simulation with the explicit solvent water H. Gouda, K. Yamazaki, J. Hasegawa, Y. Kobayashi, Y. Nishiuchi, S. Sakakibara and S. Hirono

282

Study of depsipeptide Boc–(Leu–Leu–Ala)2 –(Leu–Leu–Lac) 3 – OEt forming 310 -helix T. Ohyama, A. Hiroki, K. Kobayashi, K. Wakamatsu and R. Katakai

284

¹H Nuclear magnetic resonance analysis of [Ser6 , Ser 20 ]human-epidermal growth factor as a model for the intermediate of the disulfide folding pathway H. Murakami, N. Kobayashi, T. Shigematsu, K. Nihei and E. Munekata

286

PKa shift by NH ... S hydrogen bond in the hair-pin turn structure of Cys-containing oligopeptides N. Ueyama, S. Moriyama, T. Ueno and A. Nakamura

288

Effect of environment on the stability of helical Aib-model peptides H. Kodama, T. Hara, Y. Higashimoto, H. Yamaguchi, M. Jelokhani-Niaraki, S. Osada and M. Kondo

291

Structural analysis of mastoparan by solid-state nuclear magnetic resonance K. Fukushima, S-M. Kim, T. Fujiwara, T. Kohno, K. Wakamatsu, S-W. Kang and H. Akutsu

294

Four-helix bundle formation only through special pathway via micelle state H. Morii, H. Uedaira, M. Ishimura, S. Kidokoro, T. Kokubu and S. Ohashi

297

94.

95.

96.

97.

98.

99.

100.

101.

Contents x v 102.

103.

104.

105.

106.

107.

108.

109.

110.

Synthesis of peptides containing 9-aminofluorene-9-carboxylic acid by the modified Ugi reaction T. Yamada, K. Makihira, S. Suzuki, K. Yoneda, R. Yanagihara and T. Miyazawa

300

Effects of mutations on tetramer formation of tumor suppressor protein p53 Y. Higashimoto, M.S. Lewis, W.M. Poindexter Kennedy, A.M. Gronenborn, G.M. Clore, E. Appella and K. Sakaguchi

303

Disulfide-coupled folding of the circulating form of uroguanylin Y. Hidaka, M. Ohno, B. Hemmasi, O. Hill, W.-G. Forssmann and Y. Shimonishi

306

Solution structure of the extracellular domain of type-I receptor for bone morphogenetic protein-2/4 as studied by nuclear magnetic resonance spectroscopy T. Yamazaki, T. Hatta, Y. Nishiuchi, E. Katoh, T. Natsume, N. Ueno, Y. Kyogoku and Y. Kobayashi

309

Comparison of three-dimensional solution structures of dendrotoxin I, a potassium channel blocker, and calucicludine, a calcium channel blocker, determined by nuclear magnetic resonance spectroscopy E. Katoh, H. Nishio, T. Inui, Y. Nishiuchi, T. Kimura, S. Sakakibara and T. Yamazaki

311

Sequence, conformation, and function of peptides, retropeptides and sense and antisense gene products W.L. Duax, V. Pletnev, J. Bruenn and D. Ghosh

313

Ion conductance in a gramicidin channel: the right-handed double-stranded double-helical dimer B.M. Burkhart, N. Li, D.A. Langs and W.L. Duax

316

Protein folding intermediates and Alzheimer’s disease J.P. Lee, S.-S. Zhang, C. Clish, N. Casey, D. Hassell, E. Stimson, W. Esler, J. Maggio, Y. Lu, A.M. Felix and J.W. Peng

319

Topological isomers of human uroguanylin: function and conversion T. Yoshida, R. Tanabe, S. Kubo, N. Chino, T. Kimura, S. Sakakibara, M. Nakazato, K. Kangawa and Y. Kobayashi

322

xvi

Contents

111.

Topological isomers of human uroguanylin: structure determination by nuclear magnetic resonance R. Tanabe, T. Yoshida, S. Kubo, N. Chino, T. Kimura, S. Sakakibara, M. Nakazato, K. Kangawa and Y. Kobayashi

324

Homology modelling of the glycoprotein hormones: examination of the receptor recognition characteristics P. Gomme, J. Whisstock, P. Thompson, A. Lesk and M. Hearn

326

112.

Session F: Structural basis of peptide-protein & peptide-DNA interactions 113.

114. 115.

Prion protein structure and pathology of transmissible spongiform encephalopathies (TSE) K. Wüthrich, M. Billeter, R. Riek, G. Wider, S. Hornemann and R. Glockshuber

330

Peptide nucleic acid (PNA) P.E. Nielsen

335

Receptor based design, synthesis and biology of novel somatostatin analogs M. Goodman, X. Jiang, J. Igarashi, H. Li, T.-A. Tran and R.-H. Mattern

342

116. NMR structure of mastoparan-X bound to G protein α subunit H. Kusunoki, T. Kohno, K. Sato, T. Tanaka and K. Wakamatsu

346

117. Peptides and molecular recognition: head-to-tail self-assembly, formation of amphipathic surfaces and recognition of anionic superhelices E. Giralt, I. Dalcol, C. Ferrer, P. Gorostiza, T. Haack, A.D. Hamilton, D. Ludevid, E. Nicolás, M.W. Peczuh, X. Salvatella, J. Sánchez-Quesada and F. Sanz

351

118. Non-immobilised ligand interaction assay (NILIA) – the CD approach G. Siligardi and R. Hussain

357

119. Characterization and evolutionary aspect of a Trimeresurus flavoviridis serum inhibitor, PLI-I, against its venom basic phospholipase A2 isozymes I. Nobuhisa, S. Inamasu, M. Nakai, A. Tatsui, T. Mimori, T. Ogawa, Y. Shimohigashi, Y. Fukumaki, S. Hattori, H. Kihara and M. Ohno

360

Contents 120.

121.

122.

123.

xvii

A 24 mer peptide with activities of discrimination and recombination of nucleic acids N. Sugimoto and S. Nakano

362

Phosphorylation of synthetic peptides containing proline homologs by cdc2 kinase or cdk5 S. Ando, T. Ikuhara, T. Kamata, Y. Sasaki, S. Hisanaga, T. Kishimoto, H. Ito and M. Inagaki

364

Synthesis of oligomerization domain of p53 and its analogs and their association properties in buffer solution M. Miyabe, S. Lee, G. Sugihara, H. Sakamoto, K. Meno, Y. Aso and K. Sakaguchi

367

Stabilization of tyrosine-O-sulfate residues through the formation of a conjugate acid-base pair T. Yagami, K. Kitagawa, C. Aida, H. Fujiwara and S. Futaki

370

124. Circular dichroism and fluorescence study of the interaction of synthetic J protein from bacteriophage φ K with DNA S. Ono, T. Okada, S. Aimoto, T. Fujii, C. Shimasaki and T. Yoshimura

373

125. Unusual collagen assembly induced by acidic biopolymers in vitro H. Kitamura, C. Iwamoto, T. Hayami, S. Han, Y. Nodasaka, N. Sakairi, S. Tokura and N. Nishi

376

126. The role of nucleoplasmin – molecular chaperone protein – in the fertilization process N. Nishi, K. Hozumi, K. Iwata, A. Iihara, H. Kawahara and N. Sakairi

379

127. Conformation of chick DNA topoisomerase I as determined by limited proteolysis L.M. Assairi

381

128. The two adenosine triphosphate and nucleic acid binding sites of the calf thymus single-strand DNA-dependent adenosine triphosphatase L.M. Assairi

383

129. Induced helical structure in highly charged peptides A. Lombardi, L. Zaccaro, G. Ansanelli, C. Pedone and V. Pavone

385

Session G: New biologically active peptides 130. Nociceptin, a novel neuropeptide with multiple functions J.-C. Meunier

388

xviii Contents 131. An anti-HIV peptide T22 is confirmed to be a selective CXCR4/fusin inhibitor by comparative inhibition study of a chemokine SDF-1, synthesized by regioselective disulfide bond formation H. Tamamura, F. Matsumoto, K. Sakano, A. Otaka, T. Murakami, H. Nakashima, M. Waki, A. Matsumoto, N. Yamamoto and N. Fujii

398

132. Isolation of the nucleoplasmin-like molecular chaperone protein from salmon egg N. Sakai, K. Hozumi, K. Iwata, N. Sakairi and N. Nishi

403

133. Study of neurotoxic peptides from Chinese scorpion Buthus martensi Karsch M.-H. Ling, Y.-M. Xiong, Z.-D. Lan, H. Fei, D.-C. Wang and C.-W. Chi

407

134. Impervious peptides as growth hormone secretagogues R. Deghenghi, F. Boutignon, G. Muccioli, E. Ghigo and V. Locatelli

411

135. Effect of carnosine and its natural derivatives on apoptosis of neurons induced by excitotoxic compounds A. Boldyrev, D. Lawrence and D. Carpenter

413

136. Morphological differences action of the antibacterial CB-1, CB-2 and CB-3 on H.M. Chen, S.C. Chan,

416

observed by the different modes of peptides, cecropin B and its analogs bacterium and cancer cell W.L. Yau, D. Smith and W. Wang

137. Isolation and characterization of bioactive peptides from the sea cucumber, Stichopus japonicus M. Ohtani, E. Iwakoshi, Y. Muneoka, H. Minakata and K. Nomoto

419

138. Purification of peptide fractions with anticomplementary activity from red ginseng (Panax ginseng, C.A. Meyer) J.S. Ahn, T.H. Kwon, H. Park and K.H. Kim

421

5,12

7

, Lys ]-polyphemusin 139. HIV-cell fusion inhibitor, T22 ([Tyr II), and its related compounds H. Tamamura, M. Waki, A. Otaka, T. Murakami, H. Nakashima, A. Matsumoto, N. Yamamoto and N. Fujii

424

7

140. Efficient analogs of an anti-HIV peptide, T22 ([Tyr 5 , 1 2 , Lys ]polyphemusin II), having low cytotoxicity H. Tamamura, M. Waki, A. Otaka, T. Murakami, H. Nakashima, A. Matsumoto, N. Yamamoto and N. Fujii

427

Contents

xix

141. Cyclic RGD peptides with a novel mimetic G. Hölzemann, S.L. Goodman, C. Rölz and H. Kessler

430

1 4 2. Isolation and cDNA cloning of oryctin, a novel cysteine-rich antibacterial peptide, from the coconut rhinoceros beetle, Oryctes rhinoceros J. Ishibashi, H. Saido-Sakanaka, H. Nakazawa, M. Yamamoto and M. Yamakawa

432

143. Eumenine mastoparans. A new class of mast cell degranulating peptides in the eumenine wasp venom K. Konno, H. Takayama, M. Hisada, Y. Itagaki, H. Naoki, N. Kawai, A. Miwa, T. Yasuhara and Y. Nakata

434

144 . Direct evidence of the generation of lactoferricin from ingested lactoferrin H. Kuwata, T.T. Yip, M. Tomita and T.W. Hutchens

436

145. Human immunodeficiency virus type 1 Vif-derived peptides inhibit the viral protease and arrest virus production C. Gilon, A. Friedler, L. Baraz, I. Blumenzweig, O. Nussinuv, M. Steinitz and M. Kotler

439

146 . A novel cardioactive peptide of Aplysia contains a D-amino acid residue F. Morishita, Y. Nakanishi, S. Kaku, Y. Furukawa, S. Ohta, T. Hirata, M. Ohtani, Y. Fujisawa, Y. Muneoka and O. Matsushima

442

147 . Purification and primary structure of four novel neuropeptides in Aplysia kurodai K. Nakamaru, F. Morishita, Y. Furukawa, S. Ohta, T. Hirata, M. Ohtani, T. Takahashi, Y. Fujisawa, Y. Muneoka and O. Matsushima

444

148. Structure and function of fragments of antibiotic bactenecin 5 H. Aoyagi, M. Tsuiki, K. Yoshida, T. Niidome and T. Hatakeyama

446

149. Production of adrenomedullin in macrophages A. Kubo, N. Minamino, Y. Isumi, T. Katafuchi, K. Kangawa and H. Matsuo

448

150. Adrenomedullin production in fibroblast cell lines Y. Isumi, N. Minamino, T. Katafuchi, A. Kubo, K. Kangawa and H. Matsuo

450

151. Chemical and biological characterization of neuropeptides in the sinus glands of the kuruma prawn, Penaeus japonicus H. Nagasawa, W.-J. Yang, K. Aida and H. Sonobe

453

xx

Contents

152.

Preparation of a poly(ethylene glycol) hybrid containing two active fragments of laminin M. Maeda, K. Kawasaki, Y. Kaneda, Y. Mu, Y. Tsutsumi, S. Nakagawa and T. Mayumi

455

Modification of an anti-fungal peptide (ESF-1) for enhanced inhibitory activity and resistance to proteolysis J. Hastings, V. Vadyvaloo, G. Dykes, K. Tamura and S. Aimoto

457

153.

154. Structure determination and cDNA cloning of paralytic peptide in Bombyx mori S.-D. Ha, A. Suzuki, S. Nagata, M. Tanaka and H. Kataoka

460

155. Isolation and characterisation of a bacteriocin-like peptide produced by Brevibacterium linens ATCC 9172, an orange cheese coryneform bacterium C. Oliveira, A. Correia and M.T. Barros

462

156.

Synthesis, conformational analysis, and biological activity of opioid peptide analogs containing side chain fluorinated amino acids D. Winkler, N. Sewald, M. Gussmann, M. Thormann, H.-J. Hofmann, N.N. Chung, P.W. Schiller and K. Burger

465

1,3

157. Structural requirement tryptophan of tridecapeptide mating pheromone of Saccharomyces cerevisiae N.J. Hong, Y.A. Park and J.W. Lee

467

158. Synthesis and in vitro effects of short analogues of β - a m y l o i d peptide on resting transmembrane potential M. Zarándi, B. Penke, G. Laskay, E. Klement, I. Ocsovszki, K. Jost, J. Varga, K. Nagy, G.K. Tóth and K. Gulya

470

159. Macrotorials: synthesis and characterization of model compounds related to PTH (1–34) A.F. Spatola, W. Ma, A. Harvey, S. Chandrasekhar and J.A. Dodge

472

Session H: Progress in peptide and protein synthesis 160. Developments in peptide synthesis and peptidomimetic chemistry: HIV protease analogs and substrate-based inhibitors Y. Kiso

475

161. Thia zip reaction for the facile synthesis of large cyclic peptides J.P. Tam, Y.-A. Lu and Q. Yu

480

Contents

162. Synthesis of disulfide-bridged heterotrimeric collagen peptides. Conformational properties and digestion by matrix metalloproteinases J. Ottl, D. Gabriel, H.Y.C.D. Müller, H.-J. Musiol, W. Bode and L. Moroder 163. Combined solid-phase and solution approach to protein synthesis Y. Nishiuchi, J. Bódi, B.-P. Xiang, S. Isaka, T. Inui, H. Nishio, T. Kimura and S. Sakakibara 164. A simple linker for the attachment of aldehydes to the solid phase. Application to solid phase synthesis by the Multipin method. N.J. Ede and A.M. Bray 165. Stereoselective synthesis of dehydroaminobutyric acid P.M.T. Ferreira, H.L.S. Maia and L.S. Monteiro 166. Efficient preparation of dehydroalanine derivatives P.M.T. Ferreira, H.L.S. Maia and L.S. Monteiro 167. Totally stereocontrolled synthetic routes for (E)-alkene dipeptide isosteres N. Fujii, T. Ibuka, N. Mimura, H. Tamamura, A. Otaka, H. Ohno, H. Aoyama, K. Okano and M. Hirohashi 168. Synthesis of protected 2,3-epoxy amines utilizing aza-Payne rearrangement of activated 2-aziridinemethanols under basic conditions A. Otaka, Y. Umeda, H. Tamamura, T. Ibuka and N. Fujii 169. Convenient preparation of phosphopeptide fragments using High TFMSA system and its application to synthesis of long chain phosphopeptides K. Miyoshi, A. Otaka, M. Kaneko, H. Tamamura and N. Fujii 170. An efficient synthesis of tryptophan-containing cystine peptide using the silyl chloride–sulfoxide system Y. Fujiwara, T. Kimura, K. Akaji and Y. Kiso 171. Synthesis of an HIV-1 protease dimer analog covalently linked by a disulfide bridge M. Kishida, H. Matsumoto, N. Nishizawa, T. Kimura, Y. Fujiwara, K. Akaji and Y. Kiso 172. Enzymatic peptide synthesis using activated esters as acyl donors in organic media T. Miyazawa, E. Ensatsu, K. Tanaka, R. Yanagihara and T. Yamada

xxi

485

490

495 498 500

502

504

507

510

512

514

xxii

Contents

173. Facile synthesis of the acid-labile peptide amide linker containing the 10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5-yl group M. Noda

516

174. Synthetic approach to microsclerodermins S. Sasaki, Y. Hamada and T. Shioiri

519

175. Synthetic approach to caledonin, a natural bolaphile K. Lee and T. Shioiri

521

176. Synthetic approach to antillatoxin, an exceptionally ichthyotoxic cyclic lipodepsipeptide H. Fujiwara, F. Yokokawa and T. Shioiri

523

177. Desulfation vs. deprotection: direct solid-phase synthesis of Tyr(SO 3 H)-containing peptides using the SN1-type deprotection procedure K. Kitagawa, T. Yagami, C. Aida, H. Fujiwara, S. Futaki, M. Kogire, J. Ida and K. Inoue

525

178. Enzymatic syntheses of oligopeptides in organic solvents Y.-H. Ye, G.-W. Xing, G.-L. Tian, G. Chen and C.-X. Li

527

179. Studies of racemization in peptide synthesis for a phosphoryloxy-benzotriazin-one coupling reagent (DEPBT) H. Li, X. Jiang, Y.-H. Ye, C.-X. Fan and M. Goodman

530

180. Effect of polyethylene glycol on the synthesis of oligopeptides by proteases in an organic medium Y. Hirano, Y. Nagao and T. Terai

534

181. Synthesis of a cyclic tetrapeptide related to trapoxin B N. Nishino, K.-Y. Tomizaki, M. Komori, K. Ima-izumi, Y. Komatsu and T. Mimoto

536

182. Highly efficient solid phase cyclization of peptides by methylenedithioether or disulfide bond formation M. Ueki, T. Ikeo, K. Nakamura and T. Maruyama

539

183. 4-(2-(1-Hydroxyethyl)-1,3-dithiacyclohex-2-yl)-phenoxyacetic acid (HETPA): photolabile linker for Fmoc solid phase synthesis M. Ueki, S. Arimoto and R. Sasaki

542

184. Kinetic study of the pseudolysin-catalyzed synthesis of Z-Ala– Phe–NH 2 S. Rival, J. Saulnier and J. Wallach

545

Contents

xxiii

185. Solid phase peptide synthesis using 2-(4-nitrophenyl)sulfonylethoxycarbonyl (NSC) amino acids for Luteinizing hormone-releasing hormone (LHRH) S.H. Lee, J.I. Park, W.U. Chweh, Y.D. Kim and H.J. Kim

547

186. Novel synthesis through repeated S-cyanocysteine mediated segment condensations O. Ito, Y. Ishihama, Y. Oda, T. Takenawa, K. Yoshinari and M. Iwakura

550

187. Total synthesis of human pleiotrophin, a 136-residue heparinbinding neurotrophic factor having five disulfide bonds T. Inui, M. Nakao, H. Nishio, Y. Nishiuchi, S. Kojima, T. Muramatu, T. Kimura and S. Sakakibara

552

188. Engineering of snake venom cardiotoxin by chemical synthesis approach C.-Y. Wu, S.-T. Chen and K.-T. Wang

555

189. Specificity of Cardosins A and B in peptide synthesis A.C. Sarmento, M. Barros, L. Silvestre and E. Pires

558

190. A facile preparation of 2,2-difluoro-1,3,2-oxazaborolidin5-ones(I) and their application to side-chain protection of Asp, Glu, Ser and Thr J. Wang and W. Li

560

191. ESR-resin solvation approach for the investigation of the highly peptide-loaded synthesis protocol E.M. Cilli, G.N. Jubilut, S. Schreier and C.R. Nakaie

563

192. Synthesis of α-amino phosphonic acids by catalytic asymmetric hydrogenation M. Kitamura, M. Yoshimura, M. Tsukamoto and R. Noyori

566

193. Process impurity identification in MSI-78 J.L. Chang, J. Bai, J. Jiang, R. Pilgrim, W.-S. Chang, T.O. Kollie, R. Rasmussen, E. Tews, R.B. Miller and J.C. Tolle

569

194. Identification of unknown intermediate during methanolysis of Z-Lys(Boc)–OSu R.B. Miller, J. Jiang, Y. Liu and J.C. Tolle

571

195. Synthesis of phosphorylated polypeptide by a thioester method S. Aimoto, K. Hasegawa, X. Li, M. Seike and T. Kawakami 196. Synthesis of reaper protein by a thioester method using silver chloride as an activator T. Kawakami and S. Aimoto

574

576

xxiv

Contents

197. Preparation of a peptide thioester by a 9-fluorenylmethoxycarbonyl solid phase method X. Li, T. Kawakami and S. Aimoto

579

198. Chemical synthesis of barnase which possesses phosphoserine residue in place of the catalytic glutamic acid at position 73 H. Hojo, K. Yamauchi, M. Kinoshita, M. Go, T. Kawakami and S. Aimoto

581

199. Disulfide oxidation in water: investigation of CLEAR supports for on-resin cyclization K. Darlak, M. Darlak and A.F. Spatola

584

Session I: Glyco-, lipo-, phospho-peptides: Synthesis and biological activity 200. Non-natural peptide modifications using sugars and lipophilic side chains H. Kessler, F. Burkhart, M. Koppitz and E. Lohof

587

201. Diastereoselective synthesis of glycosyl-α -aminoacids and glycopeptide derivatives S. Bouifraden, M. El Hadrami J.-P. Lavergne, J. Martinez and P. Viallefont

595

202. Influence of glycation on cis/trans isomerization and tautomerization in novel morphiceptin-related Amadori compounds I. igrovi J. Kidri and Horvat

600

203. Chemoenzymatic synthesis of glycophosphopeptides J.E. Sander, T. Pohl and H. Waldmann 204. Design and synthesis of novel Src homology-2 domain inhibitors R.J. Broadbridge, R.P. Sharma and M. Akhtar

603

605

205. Chemo-enzymatic syntheses of eel calcitonin analogs having natural N-linked oligosaccharides T. Inazu, M. Mizuno, I. Muramoto, K. Haneda, T. Kawakami, M. Seike, S. Aimoto, K. Yamamoto and H. Kumagai

607

206. Chemo-enzymatic synthesis of bioactive peptide derivatives containing N-linked oligosaccharides K. Haneda, T. Inazu, M. Mizuno, R. Iguchi, K. Yamamoto, K. Fujimori, Y. Shimada and H. Kumagai

610

207. Optimization of N,N'-dialkyldiamide-type phosphate protecting group for phosphopeptide synthesis M. Ueki, M. Goto and J. Okumura

613

Contents

208. Solid phase synthesis of O-glycopeptide – Preparation of O-(N -acetylgalactosaminyl)-threonine derivative and its application to peptide synthesis T. Teshima, T. Urabe, T. Yamamoto, K.Y. Kumagaye, K. Nakajima and T. Shiba

xxv

615

Session J: Nonpeptide antagonists of peptide receptors 209. Design of novel, nonpeptide oxytocin receptor antagonists R.M. Freidinger, M.G. Bock, B.E. Evans, D.J. Pettibone and P.D. Williams

618

210. Development of potent non-peptide GPIIb/IIIa antagonists with tri-substituted β -amino acid derivatives as a new conformational restriction unit Y. Hayashi, J. Katada, T. Harada, K. Iijima, A. Tachiki, Y. Takiguchi, M. Muramatsu, H. Miyazaki, T. Asari, T. Okazaki, Y. Sato, E. Yasuda, M. Yano, I. Ojima and I. Uno

623

Session K: Protease inhibitors: Biology and inhibition 211. Design and synthesis of potent and selective inhibitors of the osteoclast-specific cysteine protease, cathepsin K D.F. Veber, R.W. Marquis, Y. Ru, D.S. Yamashita, H.-J. Oh, S.K. Thompson, S.M. Halbert, T.J. Carr, J.G. Gleason, T.A. Tomaszek, Jr., M.A. Levy, M. Bossard, D.G. Tew, I.E. James, J. Briand, S.A. Carr, D. Zymbryki, L. LeeRykaczewski, R.L. DesJarlais, M.S. McQueney, K.J. D’Alessio, B. Zhao, C.A. Janson, S.S. Abdel-Meguid and W.W. Smith

628

212. Difference of structural requisite in design of inhibitor of cathepsin B and papain: X-ray crystal structures of complexes T. Ishida, A, Yamamoto, K. Tomoo, K. Matsumoto, M. Murata and K. Kitamura

631

213. Synthesis of peptides with aziridine-2,3-dicarboxylic acid as an α-amino acid equivalent building block T. Schirmeister

634

214. Isolation and characterization of prolyl endopeptidaseinhibiting peptides from bovine brain S. Maruyama and T. Ohmori

637

xxvi

Contents

215. Structure based design and synthesis of novel orally active thrombin inhibitors M. Haramura, T. Shiraishi, T. Haneishi, K. Kuromaru, M. Ohta, J.-M. Kim, W.H. Nam, K.Y. Jung, G.-H. Jeon and T. Kozono

639

216. Design of bivalent plasmin inhibitors Y. Tsuda, Y. Okada, K. Wanaka, M. Tada, S. Okamoto, A. Hijikata-Okunomiya and Y. Konishi

642

217. Suicide-inhibition of human immunodeficiency virus type-1 protease by the gag precursor processing product (p2 gag peptide) S. Misumi, M. Tomonaga, A. Kudo, K. Furuishi and S. Shoji

644

218. Design of plasma kallikrein selective inhibitor and its application to affinity chromatography Y. Okada, Y. Tsuda, K. Wanaka, M. Tada, Y. Nakaya, U. Okamoto, S. Okamoto, N. Horie and A. HijikataOkunomiya

646

219. Structural essentials for novel inhibition of serine protease chymotrypsin Y. Shimohigashi, Y. Yamauchi, K. Ikesue, I. Maeda, M. Kawahara and T. Nose

649

220. KNI-764, A novel dipeptide-based HIV protease inhibitor containing allophenylnorstatine T. Mimoto, R. Kato, H. Takaku, S. Misawa, T. Fukazawa, S. Nojima, K. Terashima, H. Sato, M. Shintani, Y. Kiso and H. Hayashi

652

221. Self-association of cystatin E. Jankowska, B. Banecki, W. Wiczk and Z. Grzonka

654

222. The substrate specificity of microbial carboxyl proteinases B.M. Dunn and K. Oda

657

223. Allophenylnorstatine containing HIV-1 protease inhibitors: design, synthesis and structure–activity relationships for selected P2 and P2' ligands A.A. Bekhit, H. Matsumoto, H.M.M. Abdel-Rahman, T. Mimoto, S. Nojima, H. Takaku, T. Kimura, K. Akaji and Y. Kiso

660

Contents

224. Allophenylnorstatine containing HIV-1 protease inhibitors: design, synthesis and structure–activity relationships for selected P2 ligands H.M.M. Abdel-Rahman, H. Matsumoto, A.A. Bekhit, T. Mimoto, S. Nojima, H. Takaku, T. Kimura, K. Akaji and Y. Kiso

xxvii

662

Session L: Peptide pharmacology potential therapeutic application 225. Opioid peptide analogs with novel activity profiles as potential therapeutic agents for use in analgesia P.W. Schiller, G. Weltrowska, R. Schmidt, T.M.-D. Nguyen, I. Berezowska, Y. Chen, C. Lemieux, N.N. Chung, B.C. Wilkes and K.A. Carpenter

665

226. Peptide radiopharmaceuticals: studies of somatostatin receptors and ligand for tumor targeting S. Froidevaux, A. Heppeler, M. Behe, E. Jermann, A. Otte, J. Müller-Brand, H.R. Mäcke, P. Hildebrand, C. Beglinger and A.N. Eberle

670

227. Fatty acid acylated insulins display protracted action due to binding to serum albumin I. Jonassen, S. Havelund, P. Kurtzhals, J. Halstrom, U. Ribel, E. Hasselager, U.D. Larsen, J.L. Whittingham and J. Markussen

674

228. Melanotropin fragments containing difluoromethylornithine: synthesis and cytotoxicity H. Süli-Vargha, J. Bódi, H. Medzihradszky-Schweiger, L. Nagy, R. Morandini and G.E. Ghanem

678

229. Synthesis of RANTES-related peptides and their effect on human immunodeficiency virus-1 infection Y. Nishiyama, T. Murakami, K. Kurita and N. Yamamoto

681

230. The influence of structural motifs of amphipathic peptides on the permeabilization of lipid bilayers and the antibacterial and hemolytic activity M. Dathe, D. MacDonald, W.L. Maloy, M. Beyermann, E. Krause and M. Bienert

684

231. Proline at position 14 of alamethicin is essential for the hemolytic activity, catecholamine secretion from chromaffin cells and enhanced metabolic activity in endothelial cells M. Dathe, C. Kaduk, E. Tachikawa, H. Wenschuh and M. Melzig and M. Bienert

687

xxviii

Contents

232. Enkephalin metabolism in rat brain after bestatin administration N.A. Belyaev, E.F. Kolesanova, V.Y. Baronets, N.N. Terebilina and L.F. Panchenko

689

233. Synthetic antibacterial peptides derived from insect defensin isolated from a beetle, Allomyrina dichotoma H. Saido-Sakanaka, J. Ishibashi, A. Miyanoshita and M. Yamakawa

691

234. Do peptide antioxidants increase neuron viability by direct antioxidant effect? A. Boldyrev, P. Johnson, Y. Wei, C. Peters and M. Huentelman

693

235. Synthesis and characterization of ω -conotoxin TxVII T. Sasaki, M. Fainzilber and K. Sato

695

236. Design and synthesis of antifungal lactoferricin derivatives H. Matsumoto, K. Hashimoto, Z. Takatsu, S. Shimamura and M. Tomita

697

237. Incorporation of the nuclear localization signal (NLS) peptide–albumin conjugate to macrophages using pH-sensitive liposomes S. Futaki, R. Tachibana, M. Shono, M. Azumano, M. Niwa, H. Kiwada and H. Harashima

699

238. Dermorphin-dynorphin hybrid analogs: Opioid receptor selection and enzymatic stability A. Ambo and Y. Sasaki

701

239. Selective cytotoxicity of dermaseptins J. Ghosh, I. Kustanovich, Y. Shai and A. Mor

704

240. Synergism of antimicrobial peptides, magainin 2 and PGLa, in liposomes Y. Mitani, K. Tanaka, K. Akada, K. Miyajima, N. Fujii, M. Zasloff and K. Matsuzaki

707

241. Cell specific gene transfer using galactose-modified peptide T. Niidome, M. Urakawa, N. Ohmori, T. Hatakeyama, A. Wada, T. Hirayama and H. Aoyagi

709

242. Synthesis and biological activities of hPTH(1–34) analogues: modification of the middle part and C-terminal alkylamides J. Habashita, S. Nakagawa, T. Hamana, M. Kawase, S. Taketomi and T. Fukuda

711

Contents

xxix

243. Absolute importance of arginine residue at position 7 of dog neuromedin U-8 for contractile activity N. Sakura, K. Kurosawa and T. Hashimoto

714

244. Cyclic peptides having inhibitory site of α-amylase inhibitor tendamistat T. Hirano, S. Ono, H. Morita, T. Yoshimura, H. Yasutake, T. Kato and C. Shimasaki

716

245. Doxorubicin liposomal preparations coated with Laminin related peptides D. Polo, I. Haro, D. Cunillera and F. Reig

719

246. Importance of hydrophobic region in cationic peptides on gene transfer into cells and enhance effect of lysosomedisruptive peptide N. Ohmori, T. Niidome, T. Kiyota, S. Lee, G. Sugihara, A. Wada, T. Hirayama, T. Hatakeyama and H. Aoyagi

722

247. Synthesis and pharmacological properties of oxytocin antagonists containing conformationally constrained amino acids G.K. Tóth, K. Bakos, I. Zupkó, F. Fülöp, I, Pávó and G. Falkay

724

248. Identification of the structural elements responsible for high biological activity of dimeric opioid peptide biphalin A. Misicka, A.W. Lipkowski, D. Stropova, H.I. Yamamura, P. Davis, F. Porreca and V.J. Hruby

726

249. Orally active memory-enhancing peptides obtained by designing bioactive peptides derived from food proteins M. Yoshikawa, S. Sonoda, T. Matsukawa, Y. Jinsmaa, Y. Takenaka, M. Takahashi, S. Fukudome, H. Fukunaga, H. Kaneto and M. Takahashi

728

250. Dimers of bradykinin and substance P antagonists as potential anti-cancer drugs J.M. Stewart, L. Gera and D.C. Chan

731

Session M: Emerging functions and peptides 251. Ovine Leydig cell insulin-like peptide: a sheep relaxin? J.D. Wade, N.F. Dawson, P.J. Roche, L. Otvos, Jr., R.J. Summers, Y.-Y. Tan and G.W. Tregear

733

252. Self-assembling peptides in biology, materials science and engineering S. Zhang, M. Altman, R. Chan, P. Lee and R. Ma

737

xxx

Contents

253. Hybridization properties of nucleic acid analogs bearing peptide backbone M. Fujii, J. Hidaka and T. Ohtsu

745

254. Copper( II) complexes of the modified ACTH active center analogue hexapeptides – investigation by means of CD spectroscopy I. Vosekalna, B. Gyurcsik and E. Larsen

748

255. The structure–activity relationships of the sweet protein brazzein – roles of acidic amino acids M. Izawa and Y. Ariyoshi 256. Asymmetric synthesis of β -amino acids: dynamic kinetic resolution utilizing 2-oxoimidazolidine-4-carboxylate as a chiral auxiliary K.. Nunami, A. Kubo, H. Kubota and M. Takahashi

750

752

257. The function and insertion of a D-serine residue in ω-agatoxinTK M. Kuwada, T. Watanabe, T. Teramoto, Y. Shikata, Y. Nishizawa, K.Y. Kumagaye and K. Nakajima

755

258. Physiological role of the delta sleep-inducing peptide (DSIP) V.M. Kovalzon

757

259. Comparison of secondary structure and antibacterial activity of bactericidal peptides derived from bovine and Korean native goat lactoferrin K. Shimazaki, M.S. Nam, T. Tazume, H. Kumura, K. Mikawa, K.-K. Lee and D.-Y. Yu

759

260. Synthesis of enantiomerically pure non proteinogenic α -amino acids L. Lecointe, J.-M. Receveur, V. Rolland, M.-L. Roumestant, P. Viallefont and J. Martinez

761

261. Improvements of activity and stability against proteases of biologically active peptides by coupling of a cyclic peptide derived from an endothelin antagonist RES-701-1 K. Shibata, T. Suzawa, S. Soga, T. Mizukami, K. Yamada, N. Hanai and M. Yamasaki

764

262. Structure-activity relationship of cyclic depsipeptides, AM-toxin analogs M. Miyashita, T. Nakamori, T. Murai, H. Miyagawa, M. Akamatsu and T. Ueno

767

Contents

xxxi

263. Effects of chemical modification of Ca 2+ -dependent lectin CEL-III on its hemolytic and carbohydrate-binding activities H. Kuwahara, T. Funada, T. Niidome, T. Hatakeyama and H. Aoyagi

770

264. Coacervation of elastomeric polypeptides: microscopic observations and light scattering measurements K. Kaibara, T. Watanabe, K. Miyakawa, M. Kondo and K. Okamoto

772

265.

Preparation and properties of novel gramicidin S analogs possessing a polymethylene bridge between ornithine side chains K. Yamada, K. Ando, Y. Takahashi, H. Yamamura, S. Araki and M. Kawai

266. Effect of nitroprusside on amyloid β -peptide induced inhibition of MTT reduction in pheochromocytoma PC12h and C1300 neuroblastoma cells T. Takenouchi and E. Munekata 267.

Practical approach to enzymatic resolution of Fmoc- and Bocprotected amino acid racemates E. Marczak, A.W. Lipkowski, K. Czerwinski and J. Kazimierczak

268. Rapid translocation of amphipathic a-helical and β -sheetforming peptides through plasma membranes of endothelial cells J. Oehlke, A. Scheller, K. Janek, B. Wiesner, E. Krause, M. Beyermann and M. Bienert

775

777

779

782

Session N: Peptides in immunology/peptide vaccines 269.

270.

271.

Peptide analogues as vaccines and immunomodulators M.H.V. Van Regenmortel, N. Benkirane, H. Dumortier, G. Guichard, C. Mezière, S. Muller and J. P. Briand

784

Combinatorial peptide libraries and molecular recognition in T-cell mediated immune response B. Fleckenstein, K-H. Wiesmüller, M. Kalbus, R. Martin and G. Jung

788

On the puzzle of the acute phase reactants C-reactive protein and serum amyloid A E.J. Yavin, L. Preciado-Patt, H. Berger and M. Fridkin

792

xxxii 272.

273.

274.

Contents Design of peptidic and non-peptidic CD4 inhibitors as novel immunotherapeutic agents S. Li, T. Satoh, T.M. Friedman, J. Gao, A.E. Edling, R. Townsend, U. Koch, S. Choksi, X. Han, S. Shan, J.M. Aramini, M.W. German, R. Korngold and Z. Huang

797

Molecular design of T cell-costimulatory peptides for vaccine development T. Fukumoto, N. Torigoe, S. Kawabata, Y. Ito and K. Sugimura

801

The identification of dominant epitopes of h-FKBP-12 recognized by monoclonal antibodies, 3F4 and 2C1, against h-FK-506 binding protein 12(h-FKBP-12) M. Iwai, N. Shinkura, Y. Yamaoka, K. Nakamura, K. Tamura and M. Kobayashi

803

275. Contribution of peptide backbone atoms to binding of an antigenic peptide to class I major histocompatibility complex molecule N.G. Saito and Y. Paterson

805

276. B cell and T cell epitopes of Der f 2, a house dust mite major allergen of the Dermatophagoides farinae Y. Hantani, N. Uesato, G. Itoh, T. Ando, R. Homma, S. Ikeda, Y. Ino and M. Kurumi

808

277.

Conformational and binding studies of the peptide isolated from phage display library by anti-mouse CTLA-4 monoclonal antibody Y. Ito, Y. Oomura, K. Ezaki, S. Kojima, T. Fukumoto, N. Torigoe, S. Hashiguchi and K. Sugimura

278. Formation of DNA-anti-DNA antibody immune complex on collagen matrix M. Sasabe, H. Kitamura, K. Shindo, N. Sakairi and N. Nishi 279.

280.

810

813

Natural IgM antibodies against human endogenous retroviruses HERV-H demonstrated by means of synthetic peptides R. Pipkorn, A. Lawoko and J. Blomberg

816

Evaluation of region-specific antibodies for rat and human Chromogranin A with use of synthetic peptide N. Yanaihara, Y. Nishikawa, K. Iguchi, T. Mochizuki, M. Hoshino, S. Nagasawa, L. Jun, Y. Futai and C. Yanaihara

818

Contents 281.

282.

283.

284. 285.

Index

xxxiii

Design of synthetic antigen containing non-immunogenic T cell epitope core and simple non-native flanking regions F. Hudecz, K.A. Wilkinson, M.H. Vordermeier, J. Kajtεr, S. Jurcevic, R. Wilkinson and J. Ivanyi

820

Immune response to newly exposed regions of Band 3 protein of the red blood cell membrane during malaria infections U.A.K. Kara, L.H. Kwan and R.M. Kini

823

Peptide-induced antibodies as a tool to link genomes and proteomes T. Fröhlich and G.J. Arnold

825

Porphyrin-peptide conjugates with stabilized helices and sheets G.R. Geier, III, T. Baas and T. Sasaki

828

Molecular recognition of cyclic HIV protease inhibitors: highly orally-bioavailable DMP 851 P.Y.S. Lam, J.D. Rodgers, R. Li, C.-H. Chang, Y. Ru, P.K. Jadhav, C.G. Clark, J.A. Markwalder, S.P. Seitz, S.S. Ko, L.T. Bacheler, G.N. Lam, M.R. Wright, S. Erickson-Viitanen, P.S. Anderson and G.V. Trainor

831 833

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Preface In the late 1980s, Peptide Societies were established in Europe, the United States, and Japan, and more recently, in the Asian and the Pacific Rim regions including Australia, China, and Korea. At the time of the establishment of the American, European and Japanese Peptide Societies, the International Liaison Organizing Committee representing these Peptide Societies, along with the Australian Peptide Society, began discussions for holding international conferences which would supercede or be held in lieu of the numerous individual meetings, held by the peptide societies of each individual country or region. The representative of the Chinese Peptide Society participated in these discussion in the International Liaison Organizing Committee at the meeting of the American Peptide Symposium in Nashville, in June 1997. After lengthy discussions over several years, we agreed to organize and host the International Peptide Symposium in Japan. The First International Peptide Symposium (IPS’97) was held on November 30–December 5, 1997, in Kyoto, and was co-sponsored by four Peptide Societies. The attendance at this Symposium was 550 participants, including representatives from 32 different countries. We were very pleased with this outcome and anticipate an even larger attendance for forthcoming Symposia in future years. The revolution and advances in science and technology during the past two decades has caused traditional peptide chemistry to expand to peptide science, spreading from physical science to biology, pharmacology, and medicine. Furthermore, improvements in techniques for chemical and bio-syntheses, purification, analysis, etc., have removed the boundaries of size between small molecules and large molecules. Thus careful consideration should be given to these matters in the organization of peptide symposia and attempts should be made to meet the needs of participants in all respects. The scientific sessions, comprising 54 oral and 262 poster presentations, were programmed by the international program committee, the members of which communicated with me enthusiastically and very quickly via fax or e-mail. I am very appreciative of all the members of the program committee for their insights in all aspects of peptide science and their concentrated efforts to prepare the program. Drs James Well and Shumpei Sakakibara presented lectures highlighted in this Symposium, entitled “From Big Molecules to Smaller Ones” and “Directions for Solving the Remaining Problems in the Synthesis of Large Peptides”, both of which focused on the present status of peptide science. Two lectures, given by Drs Dietrich Brandenburg and Tetsuo Shiba, led us to reminisce about the century of peptide chemistry and the vast volume of peptide information on structures, activities, synthesis etc., which will be useful for the future development of peptide science. Other oral sessions, which comprised 19 invited and 25 selected lectures, dealt with the broad discipline of peptide science, including xxxv

xxxvi structure, peptide folding, synthesis, mimicry, de novo design, biological activity, and pharmacology. A considerable amount of fundamental research at universities involves the development of new instruments which are useful in the pharmaceutical and chemical industries. As a result, university–industry cooperation is increasing across the frontiers of countries and regions. IPS’97 represented a good opportunity to provide a forum, not only for the future of peptide research in basic research institutions and in industries but for international cooperation in peptide societies and the generation of new era in peptide communities. I very much appreciate the members, not only of the International Liaison Organizing Committee for planning the international organization of the Symposium but also of the Program Committee of the Symposium, for the time and effort spent in preparing the scientific program. The success of the Symposium was largely due to the close participation of all the members of these committees in the preparation of the Symposium. I am also greatly indebted to and wish to thank the members of the domestic organizing and executive committees for the preparation of the Symposium. The success of the Symposium was the result of the strong support of the local organizing as well as the executive committee. I am deeply indebted to the hard work of the members, who spent much time correcting, typing, and editing the materials required for the Symposium, including this Proceedings book. Finally, I sincerely express our appreciation to the Japanese pharmaceutical industries organization and several non-profit foundations for their financial support of the Symposium, who are listed on the following pages. Without their strong support this symposium would not have been possible. Yasutsugu Shimonishi

First International Peptide Symposium Kyoto Kaikan Hall & Kyoto International Exhibition Hall, Kyoto, Japan November 30–December 5, 1997

CHAIRMAN – Yasutsugu Shimonishi, Osaka University

PROGRAM COMMITTEE THE AMERICAN PEPTIDE SOCIETY Murray GOODMAN Martin KARPLUS Catherine D. STRADER Daniel VEBER

University of California, San Diego Harvard University Schering-Plough Research Institute Smith Kline Beecham Pharmaceuticals

THE AUSTRALIAN PEPTIDE SOCIETY Milton T. W. HEARN

Monash University

THE EUROPEAN PEPTIDE SOCIETY Alex N. EBERLE Günther JUNG Jean MARTINEZ Morten MELDAL

University Hospital Basel Eberhard Karls Universität Tübingen CNRS, Montpellier Carlsberg Laboratory

THE JAPANESE PEPTIDE SOCIETY Yoshiaki KISO Yuji KOBAYASHI Eisuke MUNEKATA Motonori OHNO Yasutsugu SHIMONISHI

Kyoto Pharmaceutical University Osaka University Tsukuba University Kyushu University Osaka University

INTERNATIONAL LIAISON ORGANIZING COMMITTEE Dietrich BRANDENBURG John DAVIES Yu-cang DU Jean E. RIVIER Peter W. SCHILLER Yasutsugu SHIMONISHI Takayuki SHIOIRI Geoffrey W. TREGEAR

Deutsches Wollforschungsinstitut, TA University College Swansea Shanghai Institute of Biochemistry The Salk Institute Clinical Research Institute of Montreal Osaka University Nagoya-City University University of Melbourne

xxxvii

xxxviii

DOMESTIC ORGANIZING COMMITTEE Masahiko FUJINO Terutoshi KIMURA Yoshiaki KISO Eisuke MUNEKATA Terumi NAKAJIMA Yoshio OKADA Motonori OHNO Takayuki SHIOIRI Yasutsugu SHIMONISHI Akinori SUZUKI Masaaki UEKI Noboru YANAIHARA

Takeda Chemical Industries, Ltd. Peptide Institute Inc. Kyoto Pharmaceutical University Tsukuba University Suntory Institute for Bioorganic Research Kobe-Gakuin University Kyushu University Nagoya-City University Osaka University The University of Tokyo Science University of Tokyo Yanaihara Institute Inc.

LOCAL ORGANISING AND EXECUTIVE COMMITTEE Saburo AIMOTO Ken-ichi AKAJI Tsunehiko FUKUDA Hiroshi KATAOKA Terutoshi KIMURA Hisakazu MIHARA Eisuke MUNEKATA Ki-ichiro NAKAJIMA Norio NISHI Yoshio OKADA Kazuki SAITO Kazuki SATO Yasuyuki SHIMOHIGASHI Yasutsugu SHIMONISHI Takayuki SHIOIRI Toshifumi TAKAO Tateaki WAKAMIYA

Osaka University Kyoto Pharmaceutical University Takeda Chemical Industry, Ltd. The University of Tokyo Peptide Institute Inc. Tokyo Institute of Technology Tsukuba University Peptide Institute Inc. Hokkaido University Kobe-Gakuin University Physical and Chemical Research Institute (RIKEN) Mitsubishi Chemical Industry, Ltd. Kyushu University Osaka University Nagoya-City University Osaka University Kinki University

Acknowledgements The Organizing Committee of the First International Peptide Symposium is grateful for the administrate and generous financial supports of the following organizations:

SPONSORS American Peptide Society Australian Peptide Society European Peptide Society Japanese Peptide Society

COOPERATION The Chemical Society of Japan The Pharmaceutical Society of Japan Japan Society for Bioscience, Biotechnology and Agrochemistry Protein Research Foundation Japan National Tourist Organization

DONORS Ciba-Geigy Foundation (Japan) for the Promotion of Science The Commemorative Association for the Japan World Exposition (1970) Osaka Pharmaceutical Manufacturers Association Suntory Institute for Bioorganic Research Uehara Memorial Foundation

CONTRIBUTORS FOR EXHIBITION Advanced Chem Tech Japan Bachem AG Biologica Co. Calbiochem-Novabiochem Japan Ltd. Chiron Technologies/Kurabo Industories CS Bio Co. CTC Laboratory Systems Corporation Genzyme Phamaceuticals Ito Ham Foods Inc. Jeol Ltd. Kokusan Chemical Works Ltd. Micromass UK Ltd. Nihon Perseptive Ltd. Nomura Chemical Co., Ltd. Peptide Institute, Inc./Protein Research Foundation Perkin-Elmer Japan Co., Ltd. PolyPeptide Laboratories, Inc.

xxxix

Chiyoda-ku, Tokyo, Japan Bubendorf, Switzerland Nagoya-shi, Aichi, Japan Minato-ku, Tokyo, Japan Neyagawa-shi, Osaka, Japan Billingham, Cleveland, UK Setagaya-ku, Tokyo, Japan Haverhill, Suffolk, UK Kitasouma-gun, Ibaragi, Japan Akishima-shi, Tokyo, Japan Chuoh-ku, Tokyo, Japan Wythenshawe, Manchester, UK Minato-ku, Tokyo, Japan Seto-shi, Aichi, Japan Minoh-shi, Osaka, Japan Urayasu-shi, Chiba, Japan Torrance, CA, U.S.A.

xxxx RSP Amino Acid Analogues, Inc. Shimadzu Corporation Thermo Quest K.K. Wako Pure Chemical Industries, Ltd. Watanabe Chemical Ind., Ltd. Wiley Japan YMC Co., Ltd.

Boston, MA, U.S.A Nakagyoh-ku, Kyoto-shi, Japan Suita-shi, Osaka, Japan Chuoh-ku, Osaka-shi, Japan Naka-ku, Hiroshima-shi, Japan Shinjuku-ku, Tokyo, Japan Nakagyoh-ku, Kyoto-shi, Japan

TRAVEL AWARDS American Peptide Society Travel Grant Supplement Deber, Charles M. Dibó, Gábor Grzonka, Zbignieu S. Houston, Michael Huang, Ziwei Jiang, Xiaohui Kéri, György Kondejewski, L.H. Konishi, Yasuo Lankiewics, Leszek Li, Haitao Misono, Kunio S. Pegoraro, Stefano Sharma, Ram P. Siligardi, Giuliano Spatola, Arno F. Stewart, John M. Unson, Cecilia C.

(Canada) (Hungary) (Poland) (Canada) (U.S.A) (U.S.A) (Hungary) (Canada) (Canada) (Poland) (U.S.A) (U.S.A) (Germany) (U.K.) (U.K.) (U.S.A) (U.S.A) (U.S.A)

Japanese Peptide society Travel Grant Supplement Aggeli, Amalia Ferreira, Paula T. Haris, Parvez I. Hastings, John W. Hudecz, Ferenc Kovalzon, Vladimir M. Ling, Minhua Makhoul, Cameel H. Misicka-Kesik, Aleksandra Monteiro, Luis S. Vosekalna, Ilze Winkler, Dirk Zamyatnin, Alexandez A. igrovi Ivanka

(U.K.) (Portugal) (U.K.) (South Africa) (Hungary) (Russia) (China) (Israel) (Poland) (Portugal) (Latvia) (Germany) (Russia) (Croatia)

Abbreviations Abbreviations used in the proceedings volume are defined below: βA ∆ AA ∇AA Aβ Abh Abu Ac ACE Acm Ac n c AcOH ACTH Ad AD Adoc ADP Afc AFM AgOTf Aib AIDS Aloc AM AMMTD

ANP ANS Apa APC Apns App APP Asa Atc ATP AVP AZT Bchla

β -amyloid peptide α β-dehydro-α -amino acid cyclopropylamino acid amyloid β -protein alkaloid epibatidine amino butyric acid acetyl angiotensin-converting enzyme acetamidomethyl 1-aminocycloalkane-1-carboxylic acid acetic acid corticotropin adamantyl Alzheimer’s disease adamanty loxycarbonyl adenosine diphosphate 9-aminofluorene-9-carboxylic acid atomic force microscopy silver trifluoromethanesulfonate α -aminoisobutyric acid acquired immune deficiency syndrome allyloxycarbonyl adrenomedullin (2S,3R,4S,5S,6S,11E)-3-amino-6methyl-12-(4-methoxyphenyl)-2,4,5-trihydroxydodec-11enoic acid atrial natriuretic peptide 1-anilino-naphthalene-8sulfonate 6-amino-penicillanic acid; apamin antigen presenting cell allophenylnorstatine amyloid precursor protein amyloid precursor protein azidosalicylic acid 2-aminotetralin-2-carboxylic acid adenosine triphosphate arginine-8-vasopressin 3'-azido-3'-deoxythymidine zidovudine bacteriochlorophyll a

BDNGF BHA BHP BHT BINAP BK BMP BN Boc, BOC BOI

Bom BOP

Bpa BPP BrZ BSA BSE Bz Bzl Cac CaM CaMKII CAMP, cAMP CAPS Car Cbz CCK CD Cdk CDR CDx CE CF CFA

xxxxi

brain-derived neurotropic growth factor benzhydrylamine t-butyl hydroperoxide butylhydroxytoluene 2,2'-bis(diphenylphosphino)-1,1'binaphthyl bradykinin bone morphogenetic protein bombesin tert-butyloxycarbonyl 2-(benzotriazol-1-yl)-oxy-1,3dimethylimidazolidinium hexafluorophosphate benzyloxymethyl benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate benzoylphenylalanine bradykinin potentiator 4-bromobenzyloxycarbonyl bovine serum albumin bovine spongiform encephalopathy benzoyl benzyl calcicludine calmodulin CaM-dependent protein kinase II cyclic adenosyl monophosphate 3-cyclohexylaminopropanesulfonic acid 1,2,3,4-tetrahydronorharman-1carboxylic acid carbobenzoxy; benzyloxycarbonyl cholecystokinin circular dichroism cyclin dependent kinase complementarity determining region cyclodextrin capillary electrophoresis carboxyfluorescein; cystic fibrosis complete Freunds adjuvant

xxxxii CFTR CG CgA cGMP CGRP Cha CHAPS

CHC CHEMS cHep cHex CHH CHL CHO Chol CHP cHx CID CIP Cit CJD CLEAR ClZ CNS COSY CPB CPD, CP cPen CP-MAS CRABP CRF CRP CsA CSPPS CTL CTX CZE Dab Dbt DBU DCC DCF

cystic fibrosis transmembrane conductance regulator chorionic gonadotropin chromogranin A cyclic guanosine monophosphate calcitonin gene related peptide cyclohexylalanine 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate central hydrophobic cluster cholesterylhemisuccinate cycloheptyl cyclohexyl hyperglycemic hormone chloroform Chinese hamster ovary cholesterol cumene hydroperoxide acid cyclohexyl collision-induced dissociation 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate citrulline Creutzfeld-Jakob disease cross-linked ethoxylate acrylate resin 2-chlorobenzyloxycarbonyl; 4chlorobenzyloxycarbonyl central nervous system correlated NMR spectroscopy m-chloroperbenzoic acid carboxypeptidase cyclopentyl cross polarization/magic angle sample spinning cellular retinoic acid binding protein corticotropin releasing factor C-reactive protein cyclosporin A convergent solid phase peptide synthesis strategy cytotoxic T-lymphocytes cardiotoxin capillary zone electrophoresis diaminobutyric acid dibromotyrosine 1,8-diazabicyclo-[5.4.0]-undec7-ene dicyclohexyl carbodiimide dichloro-dihydro-fluorescein

DCHA DCM DEAE DEPBT DEPS DFAB DFMO DIC DIEA, DIPEA DIPCDI DMA DMAP DMF DMPC DMS DMSO Dmt Dns Doc DOPE DOTA DPC Dph DPPA DPPC DQF DSIP DTT DTX Dyn EACA EADI EAE EC ECD ECEPP ECM ED 5 0 EDC

EDT EDTA EGF EGFR

dicyclohexylamine dichloromethane diethylaminoethanol 3-(diethoxyphosphoryloxy)-1,2,3benzotrazin-4(3 H )-one diethyl phosphocyanidate 4,4-difluoro-2-aminobutyric acid DL- α -difluoromethylornithine N,N' -diisopropyl carbodiimide diisopropylethylamine diisopropyl carbodiimide dimethylacetamide 4,4-dimethyl-4-aminopyridine dimethylformamide dimyristoyl phosphatidylcholine dimethyl sulfide dimethyl sulfoxide 2',6'-dimethyl-tyrosine dansyl 2,4-dimethylpent-3-yloxycarbonyl dioleoylphosphatidylethanolamine 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid dodecyl phosphocholine α,α-diphenylglycine diphenyl phosphorazidate dipalmitoyl phosphatidylcholine double quantum focused delta sleep inducing peptide dithiothreitol dendrotoxin dynorphin ε -aminocaproic acid (E)-alkene dipeptide isoster experimantal autoimmune encephalomyelitis endothelial cell extracellular domain empirical conformational energy program for peptides exracellular matrix median effective dose N-ethyl-N' -(3-(dimethylamino)propyl)carbodiimide hydrochloride ethane dithiol ethylenediamine tetraacetic acid epidermial growth factor epidermial growth factor receptor

xxxxiii ELISA EM EMCS EPG ES ESIMS ESMS ESR ET FABMS FD FITC FKBP FMDV FMOC, Fmoc FPLC

enzyme-linked immunosorbent assay electron microscopy N-(6-maleimidocaproyloxy)succinimide egg phosphatidylglycerol electron spray electrospray ionization mass spectrometry electrospray mass spectrometry electron spin resonance endothelin fast atom bombardment mass spectrometry field desorption fluorescein isothiocyanate FK-506 binding protein foot-and-mouth virus 9-fluorenylmethoxycarbonyl

fast protein liquid chromatography follitropin FSH FTase farnesyltransferase fourier transform infra red FTIR γ -aminobutyryl GAB γ -aminobutyric acid GABA (R)-3-hydroxy-4-aminobutyric GABOB acid gas chromatography GC guanylyl cyclase activating GCAP peptide GCase, GC guanylate cyclase GF growth factor GFP green fluorescent protein GH growth hormone GHRH, growth hormone releasing GRF hormone GHRP growth hormone releasing peptide GHS GH secretagogue Glc glycosyl GLP glucagon-like peptide GnRH gonadotropin releasing hormone GPCR G-protein coupled receptor GPH glycoprotein hormone GPI guinea pig ileum GRP gastrin releasing peptide GS gramicidin GSH reduced glutathione GSSG oxidized glutathione GST gluthatione S-tranferase

GTP GVHD Gyr h HATU HBTU

HDI HDL HEPC Hepes HETPA

HF HFIP hGH HIC HIF HIV HIVPR HLA HLE Hmb HMB, Hmb HMBC spectra HMC HMFS HMPA HMPB HOBt Hoc HOOBt Hph HPLC HPS HRP

guanosine triphosphate graft-versus-host disease gyrase human 1-hydroxy-7-azabenzotriazole uronium salt derivative O-benzotriazolyl- N,N,N',N'tetramethyluronium hexafluorophosphate hydroxyethylene dipeptide isoster high density lipoprotein hydrogenated egg phosphatidyl choline N-[2-hydroxyethyl]-piperazineN'-2-ethanesulfonic acid 4-(2-(1-hydroxyethyl)-1,3-dithiacyclohex-2-yl) phenoxyacetic acid hydrogen fluoride hexafluoroisopropanol human growth hormone hydrophobic interaction chromatography human gastric intrinsic factor human immunodeficiency virus human immunodeficiency virus protease human leukocyte antigen human leukocyte elastase N -(2-hydroxy-4-methoxybenzyl) hydroxymethylbenzoic acid heteronuclear multiple bond correlation spectra hydromethylcarbonyl N -[9-(hydroxymethyl)-2fluorenyl]succinamic acid hexamethyl phosphortriamide (256) 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid N-hydroxybenzotriazole cyclohexyloxycarbonyl 3,4-dihydro-3-hydroxy-4-oxo1,2,3-benzotriaine homo-phenylalanine high performance liquid chromatography N-hexadecyl- N,N-dimethyl-3ammonium-1-propylsulphate horse raddish peroxidase

xxxxiv HSA Hse Hsp hsst Hyp IC IEF IFN Ig IGF IGFBP IgG IL IP IR IRS Kd KLH Km LAP LC LC-MS LDH LFcin LH LHRH LPO LPS LSIMS MAb, Mab, mAb MALDI MALDI-MS

MALDITOF MAP, MAp MAPK MBHA MBP MCDP

MCH MD Me

human serum albumin homoserine heat shock protein human somatostatin hydroxyproline inhibitory concentration isoelectric focusing interferon immunoglobulin insulin-like growth factor IGF binding protein immunoglobulin G interleukin inositol phosphate infrared; insulin receptor insulin receptor substrate dissociation constant keyhole limpet hemocyanin Michaelis constant lamina-associated polypeptide liquid chromatography liquid chromatography-mass spectrometry lactate dehydrogenase lactoferricin luteinizing hormone; lutropin see GnRH linoleic acid peroxide lipopolysaccharide liquid secondary-ion mass spectrometry monoclonal antibody matrix-assisted laser desorption ionization matrix-assisted laser desorption/ionization mass spectroscopy matrix-assisted laser desorption/ionization time-offlight mass membrane-anchored protein MAP kinase methylbenzhydrylamine myelin basic protein 10-N-methylcarbonyl-3,7dimethylamino-10 H-phenothiazine melanin-concentrating hormone molecular dynamics methyl

MeOH MHC MIC MIF MIH MIP MK Mmt MOIH MP Mpa Mpt MS MSA MsCl MSH Mts MTT MVD MW Myr NADPH

NAH NB NBS NCA NCF NHSS Nip NK Nle NLS NM NMDA NMM NMP NMR NMU NO NOE NOESY

methanol major histocompatibility complex minimal inhibitory concentration mullerian inhibitory factor molt-inhibiting hormone macrophage inflammatory protein midkine 2'-methyl-tyrosine; methoxytrityl mandibular organ-inhibiting hormone mastoparan mercaptopropionyl dimethylphosphinothioyl mass spectrometry methanesulfonic acid methanesulfonyl chloride melanocyte stimulating hormone; melanotropin mesitylene-2-sulfonyl 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide mouse vas deferens molecular weight myristoyl nicotinamide adenine dinucleotide phosphate (reduced) 1,4-dihydronicotinamide novobiocin p-nitrobenzenesulfonyl; N-bromosuccinimide N-carboxyanhydride nerve growth factor N-hydroxysulfosuccimide sodium salt (±)-nipecotyl neurokinin norleucine nuclear localization sequence neuromedin N-methyl-D-aspartate N-methylmorpholine N-methylpyrrolidinone nuclear magnetic resonance neuromedin U nitrogen oxide nuclear Overhauser effect nuclear Overhauser enhanced spectroscopy

xxxxv Nphe NPY Npys Nsc Ntc 2α OA1 OAT ODS OEP OMe ONp OPLC Opp Osu OT O t Bu PAC, Pac PAGE PAL PAM Pbf PBS PC PCA PCR PDB PDGF PEG Pen PEP Pfp PG Phg PK PKC PLA 2 PLC PMB Pmc pNA PNA PrP PS PSD PTH PTN

N-benzylglycine neuropeptide Y S-3-nitro-2-pyridinesulphenyl N(α)-2-(4-nitrophenyl)sulfonylethoxycarbonyl nortropane-2α-carboxylic acid allyl ester azabenzotriazole ester octadecyl silyl octaethyl porphyrinate methyl ester p-nitrophenyl ester overpressured layer chromatography oligopeptide permease o -succinimide ester oxytocin tert-butyl ester phenacyl polyacrylamide gel electrophoresis photoaffinity labeling phenylacetamidomethyl 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl phosphate-buffered saline phosphatidylcholine passive cutaneous anaphylaxis polymerase chain reaction protein data bank platelet derived growth factor polyethylene glycol pentyl prolyl endopeptidase pentafluorophenyl phosphatidylglycerol phenylglycine protein kinase protein kinase C phospholipase A 2 phospholipase C polymixin B 2,2,5,7,8,-pentamethylchroman-6sulfonyl p-nitroaniline peptide nucleic acid prion protein polystyrene post-pource decay phenylthiohydantoin; parathyroid hormone pleiotrophin

PTTH PTX PY, pY PyBOP

RACE RIA RMS, rms RMSD, rmsd RNase ROE ROESY

ROS RP RP-HPLC, RPC Rpn

RT SAA Sar SAR SCL SCLC SCP SDF SDS SEAC SEC Sec SELDI SEM SH SH2 SLE SMPS SNBP SP SPPS SPR SRIF

prothoracicotropic hormone pertussis toxin phosphotyrosine benzotriazolyl- N-oxypyrrolidinium phosphonium hexafluorophosphate rapid amplification of cDNA ends radioimmunoassay root mean square root mean square deviation ribonuclease rotating frame nuclear Overhauser effect rotating frame nuclear Overhauser enhanced spectroscopy reactive oxygen species reversed phase reversed phase high performance liquid chromatography the ratio of positive peak intensity over negative peak intensity (absolute values) reverse transcriptase serum amyloid A sarcosyl; sarcosine structure-activity relationship synthetic combinatorial libraries small cell lung carcinoma small cardioactive peptide stromal cell-derived factor sodium dodecylsulfate surface-enhanced affinity chromatography size exclusion chromatography seleno-cysteine surface-enhanced laser desorption/ionization scanning electron microscope sulfhydryl Src homology-2 systemic lupus erythematoius simultaneous multiple peptide synthesis sperm nuclear binding protein substance P solid phase peptide synthesis surface plasmon resonance somatostain

xxxxvi ssDNA ST t-AMCHA

TASP TBS TBTU

t-Bu, But TCPP

TCR TEM TFA TFE TFMSA TGF THF ThT TIC, Tic TLC Tm TMPO TMS TMSBr TMSOTf

single stranded DNA heat stable enterotoxin trans-4aminomethylcyclohexanecarboxylic acid template-assembled synthetic protein tert-butyldimethylsilyl O-(benzotriazol-1-yl)N,N,N',N'tetramethyluronium tetrafluoroborate tertiary butyl meso-tetrakis(4carboxyphenyl)porphiyrin T-Cell receptor transmission electron microscopy trifluoroacetic acid trifluoroethanol trifluoromethanesulfonic acid transforming growth factor tetrahydrofuran thioflavin T tetrahydroisoquinoline carboxylic acid thin layer chromatography melting temperature thymopoietin trimethylsilyl bromotrimethylsilane trimethylsilyl trifluoromethanesulfonate

Tmt TNF TOCSY Tos TPA

TRCOSY TRH, TRF tRNA TRNOE Trt TSE TSH T-tBos UV UV-VIS VIH Vmax VP-1 VSMC WSCD WSCI

W T Xan Z

Nα ,2',6'-trimethyl-tyrosine tumor necrosis factor total correlation spectroscopy p-toluenesulfonyl 12-O-tetradecanoylphorbol-13acetate; tissue plasminogen activator transferred rotational correlated NMR spectroscopy thyrotropin releasing hormone transfer ribonucleic acid transferred nuclear Overhauser effect trityl transmissible spongiform encephalopathy thyroid stimulating hormone; thyrotropin tri-tert-butoxysilyl ultraviolet ultra violet-visible vitellogenesio-inhibiting hormone maximum velocity vasopressin vascular smooth muscle cell water-soluble carbodiimide water-soluble carbodiimide; 1-ethyl-3-(3-dimethylamino propyl)wild type xanthenyl carbobenzoxy; benzyloxycarbonyl

1 From big molecules to smaller ones J.A. WELLS 1 , 2 , B.C. CUNNINGHAM ¹ , A. BRAISTED¹ ,S. ATWELL 2 , 3 , W. DELANO1 , 2 , M. ULTSCH ³ , M.A. STAROVASNIK³ and A.M. DE VOS³ ¹Sunesis Pharmaceuticals, 3696 Haven Avenue, Suite C, Redwood City, CA 94063, USA ²Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA ³Department of Protein Engineering, Genentech, Inc., 1 DNA Way, S. San Francisco, CA 94080 USA

Introduction Much of cellular biology is regulated by protein-protein interactions. From the binding of extracellular hormones to specific receptors through signal transduction pathways, protein molecules are largely responsible for relaying extracellular information that effects intracellular changes in gene transcription and metabolism. Not only are protein-protein interactions fundamental to understanding how cells signal, but they are some of the most important targets in the pharmaceutical industry. Indeed, biotechnology is largely founded on providing protein hormones as injectable therapeutics. The intermolecular forces that govern binding reactions between proteins are poorly understood. Non-covalent binding energies are typically 100 to 1000-fold less strong than covalent bond energies, and there are not good algorithms for predicting binding free energies even when based on high resolution structures of bound complexes. Thus empirical methods, usually based on mutational analysis have been extensively employed to probe the energetics of protein-protein complexes. A better understanding of protein-protein interactions should facilitate rational design of small molecular weight mimics that may serve as orally active alternatives to injectable biopharmaceuticals.

Results and discussion A ‘hot spot’ dominates binding between hGH and its receptor Human growth hormone (hGH) in complex with its receptor represents one of the most thoroughly studied protein-protein complexes (for review see [1]). One hormone binds two molecules of its receptor, forming a 1: 2 complex. This receptor dimerization reaction is critical to activate the hGH receptor. Mutational and structural analysis show that formation of the complex occurs sequentially: Site 1 on hGH binds a first receptor and this assembles a complete binding site for binding of a second receptor to Site 2 on hGH. Antagonists of 1 Y. Shimonishi (ed): Peptide Science – Present and Future, 1–5 © 1999 Kluwer Academic Publishers, Printed in Great Britain

2 hGH have been produced by blocking binding at Site 2 while enhancing binding at Site 1 [2]. These analogs act to block receptor dimerization by wild-type hGH, and may be useful for the treatment of acromegly, a condition of excess hGH. To better understand the energetics of binding between hGH and its receptor at Site 1, we analyzed all the contact residues by alanine-scanning mutagenesis [3,4]. There are some thirty contact side-chains on each side of this interface. Alanine-scanning revealed that only a small set of residues (six to eight) focused near the center of the contact interface, dominated the affinity between these molecules. This patch, we call the ‘hot spot’, constituted only about one-quarter of the interface surface and was surrounded by residues that were far less important for binding. In fact, some of the residues that were mutated to alanine in the contact periphery even enhanced binding affinity. Most of the energetically important interactions were hydrophobic in character and made close van der Waals interactions across the interface. These studies raise the possibility that small molecules designed to mimic the hot spot may bind at these interfaces and block the protein-protein interaction. Structural plasticity at a remodeled interface The hGH receptor is structurally homologous to a large family of receptors, known as the cytokine receptor super family [5]. This family of receptors is notable for the diversity of immune, endocrine and neurological effects it modulates. The ligands that activate these receptors are one of the most lucrative groups of hormones in biotechnology. It is believed that all these receptors evolved from a common ancestor. They are thought to be activated by receptor oligomerization and bind to structurally similar regions of the hormone and receptor. Yet within the family the putative ‘hot spots’ of binding are not well conserved. For example, the most critical residues in hGH receptor are two tryptophans (Trp104 and Trp169) yet these are not often found in the other receptors. This may not be so surprising because as complexes co-evolve disruptive mutations on one side of the interface may be rescued by mutations in its binding partner. We wished to test how complementary mutations in hGH may respond to mutations in the hot spot of the hGH receptor. To do this we mutated Trp104 to alanine, a mutation which reduces affinity for wild-type hGH by > 2500-fold. We then randomly mutated five residues on hGH that normally contact Trp104 on the receptor. The pool of some 205 different hGH variants were displayed on filamentous phage particles as previously described [6], and sorted for binding to the mutated receptor [7]. After 6 rounds of binding selection we isolated a pentamutant of hGH that bound with high affinity (K d = 14 nM) to the mutant receptor and not to the wild-type receptor. The structure of the complex between the mutant receptor and hormone was solved at 2.1 Å resolution to determine how the structure of the complex

3 accommodated the remodeled interface [7]. Two of the selected residues in hGH (K172Y and D171T) positioned their side chains to fill the cavity created by the W104A mutation in the receptor. In addition, the D171T mutation broke a salt-bridge interaction with R43 on the receptor. Remarkably, this apparently deleterious change at the interface was accommodated by movement of Asp126 on the receptor to form an intramolecular salt-bridge with Arg43. This in turn removed a hydrogen bond between Asp126 and the main-chain amide of Trp104. The apparently unfavorable change allowed rotation of the main-chain next to position 104 to place the neighboring side-chain of Ile103 into the gap left by the W104A mutation. Thus, the hole created by the W104A mutation was filled by mutated side chains from the hormone as well as local structural reorganization on the receptor set up by a salt-bridge switch. These surprising structural changes are propagated throughout the interface. In some cases non-mutated residues shifted position by 2–3 Å relative to their position in the wild-type complex. Such large changes were carried even to the periphery of the contact epitope some 10 to 15 Å distant from the site of the mutations. In fact, of the 16 different hydrogen bond and salt-bridge pair across the interface, 7 switch to find new partners. Thus, these large complementary mutations were accommodated by a great deal of movement or ‘plasticity‘. Such plasticity at these interfaces may be a general phenomena. It is a property that makes rational design more challenging, but appears to allow interfaces more latitude to adapt to each other as they co-evolve. Minimization of protein A Given that a small set of residues are critical for binding, we wondered if a protein binding domain could be reduced in size and maintain structure and affinity. To evaluate this we investigated a simple protein-protein complex whose structure is known [8], that between IgG and a domain of Protein A, a three helix binding domain from Staphylococcus aureus. This domain binds with high affinity (K d ~10nM) to the Fc portion of IgG, using only contacts from the first two helices. However, attempts to delete helix-3 result in loss of structure and reduction in affinity to levels that are not detectable (Kd >> 1 mM). To restore structure and binding affinity to the first two helices we randomly mutated them, displayed the library of mutated proteins on filamentous phage and selected for binding to the Fc [9]. Mutations were directed to three surfaces on the two helix molecule: the exoface, the intraface and the interface. The exoface was a set of four hydrophobic residues on helix-1 and helix-2 that normally packs against helix-3. When these were mutated we isolated a double mutant with a Kd of 3 µM for the Fc. The intraface was a set of five residues that pack between helix-1 and helix-2. When these were mutated in the context of the exoface selectant we isolated a triple mutant which when combined with the exoface selectant bound with a Kd of 400 nM to the Fc. The interface was composed of the set of nineteen side-chains or Protein A that contacted the

4 Fc. These were mutated in quartets and when the best selectants were combined with the exoface and intraface selectant this produced a variant, we call mini-Z, with fifteen mutations that bound with a Kd of 50 nM to the Fc. This structure of mini-Z was less stable than the wild-type Protein A domain, but with the introduction of a single disulfide bond at the base of the two helices it could be brought to near wild-type stability [10]. This modification further improved the Kd to 10 nM. Moreover, the NMR structure of mini-Z showed it to fold virtually the same as the first two helices in the original three helix domain of Protein A. Recently we have solved the X-ray structure of the disulfide cross-linked mini-Z in complex with the Fc portion of an antibody [11], and have shown that the fold and fundamental contacts are preserved in the interaction. Thus, using protein structure to target mutations and phage display to select them we have reduced the size of the original Protein A domain by nearly a factor of two while accurately preserving the original binding interactions and stability in a two-helix module. Naive peptides to peotein A Several groups have shown that small peptides can be isolated for binding to proteins (for review see [12]). In one of the most striking examples of this, peptides ranging from 10-20 residues in length have been isolated that bind and dimerize the receptors for erythropoietin and thrombopoietin [13,14]. These were isolated by sorting first in a high copy display format on gene 8 and then a low copy format on gene 3. By taking advantage of avidity in the high copy format it was possible to isolate weak binding peptides which could be subsequently affinity matured in a low copy format. We wondered if a similar approach could be taken for isolating a peptide that binds to the Fc portion of the antibody and if so where would it bind [15]. A series of single disulfide containing libraries of random peptides was displayed on gene 8 having the format Y–C–X–C–Z, where X ranged from 4 to 10 and Y and Z carried the balance of the random residues to give a total of 20 including the two cysteines. From these we selected weak binding peptides ( K d ~ 10 µ M). The consensus peptide was transferred to gene 3 and further mutated to mature. By combining the best mutants we ultimately produced a peptide with a K d of 10 nM for the Fc. This peptide competes with the mini-Z domain for binding Fc yet shares no sequence homology. Thus, it was possible to isolate from completely random peptide libraries a peptide having no structural relationship to a natural binding peptide yet recognizes the same (or overlapping) portion of Fc.

Conclusions Protein-protein interfaces are considerably larger than small molecule-protein interfaces. Nonetheless, mutational studies show only a small portion of the

5 interface between hGH and its receptor is crucial for tight binding. This is likely a consequence of the fact that only a small number of contacts are necessary to generate high affinity. The interface is capable of adapting to mutational changes by reorganizing contact side chains. This plasticity affords the protein interface many options to adapt to mutational changes in its binding partner as they co-evolve. It is possible to reduce protein binding domains by suitable preservation of the binding determinants and restructuring to stabilize the smaller scaffold. It is also possible to isolate peptides that bind to the same hot spot but have completely different structural motifs. Thus, there are many solutions to binding, but interestingly many seem to be directed to similar regions of the protein surface. The use of molecular diversity methods coupled with structural insights is a powerful approach to redesigning binding peptides, and perhaps more importantly for discovering the rules that govern these interactions. Protein-protein targets have been generally intractable for identifying small molecule inhibitors by traditional pharmaceutical screening. Given the fact that small peptides can be isolated that block these interactions we may be on the verge of generating smaller molecular mimics for large protein signaling molecules.

References 1. Wells, J.A. and De Vos, AM. Annu. Rev. Biochem. 65 (1996) 609–634. 2. Fuh, G. Cunningham, B.C., Fukunaga, R., Nagata, S., Goeddel, D.V. and Wells, J.A. Science, 256 (1992) 1677–1680. 3. Cunningham, B.C. and Wells, J.A. J. Mol. Biol. 234 (1993) 554–563. 4. Clackson, T. and Wells, J.A. Science 267 (1995) 383–386. 5. Bazan, J.F. Curr. Biol. 3 (1993) 603–606. 6 . Atwell, S., Ultsch, M., De Vos, A.M. and Wells, J.A. Science 278 (1997) 1124–1127. 7. Lowman, H.B., Bass, S., Simpson, N., Wells, J.A. Biochemistry 30 (1991) 10832–10838; Lowman, H.B. and Wells, J.A. J. Mol. Biol. 234 (1993) 564–578. 8 . Deisenhofer, J. Biochemistry 20 (1981) 2361–2370. 9 . Braisted, A. and Wells, J.A. Proc. Natl. Acad. Sci. USA 93 (1996) 5688–5692. 10. Starovasnik, M., Braisted, A. and Wells J.A. Proc. Natl. Acad. Sci. USA 94 (1997)–10085. 11. Ultsch M., De Vos, A.M., Braisted, A. and Wells, J.A. unpublished results 12. Cunningham, B.C. and Wells, J.A. Current Opin. Struct. Biol. 7 (1997) 457–462; Clackson, T. and Wells, J.A. Trends in Biotechnol. 12 (1994) 173–184. 1 3 . Wrighton, N.C., Farrell, F.X., Chang, R., Kashyap, AK., Barbone, F.P., Mulcahy, L.S., Johnson, D.L., Barrett, R.W., Jolliffe, L.K., Dower W.J. Science 273 (1996) 458–463. Livnah, O., Stura, EA., Johnson, D.L., Middleton, S.A., Mulcahy, L.S., Wrighton, NC., Dower, W.J., Jolliffe, L.K., Wilson, LA. Science 273 (1996) 264–271. 14. Yanofsky, S.D., Baldwin, D.N., Butler, J.H., Holden, F.R., Jacobs, J.W., Balasubramanian, P., Chinn, J.P., Cwirla, S.E., Peters-Bhatt, E., Whithorn, E.A. et al., Proc. Natl. Acad. Sci. USA 93 (1996) 7381–7386. 15. Cwirla S.E., Balasubramanian, P., Duffin, D.J., Wagstrom, CR., Gates, CM., Singer, SC., Davis, A.M., Tansik, R.L., Mattheakis, L.C., Boytos, C.M., Schatz, P.J., Baccanari, D.P., Wrighton, N.C., Barrett, R.W.and Dower, W.J. Science. 276 (1997) 1696–1699. 16. DeLano, W. and Wells, J.A., Unpublished results.

2 Directions for solving the remaining problems in the synthesis of large peptides S. SAKAKIBARA Peptide Institute, Inc., Protein Research Foundation, Minoh-shi, Osaka 562, Japan

Introduction Peptide synthesis can be classified in two categories: one is the industrial synthesis of specific peptides in large quantities and the other is the synthesis of a variety of peptides for research purposes in small quantities. The points important for large-scale synthesis are: (1) how to reduce the total cost and (2) how to control the reproducibility of the synthesis. The points important for research-scale synthesis are: (1) the use of standardized procedures, (2) isolation of final products in highly homogeneous form, and (3) achievement of the entire synthesis within a short period. In recent years, over 90% of research peptides have been synthesized by a combination of solid-phase synthesis and HPLC. This works well for the synthesis of small peptides, but problems arise in the synthesis of large peptides. In this lecture, I would like to discuss such problems related to the synthesis of research peptides synthesized by the solidphase method, particularly when the size of the peptide is more than 100 residues.

Solid-phase synthesis Strategies for the synthesis of peptides by the solid-phase method have developed in two different directions: one is the original Boc-strategy isolating the final products from the solid support by HF, and the other is the Fmocstrategy isolating the products by TFA. The biggest drawback of the Bocstrategy is in the use of HF, which requires the use specially designed equipment for safe handling. However, every step of Boc-chemistry has been well established in terms of suppressing the formation of side products. In the Fmocstrategy, the final deprotection process with TFA is easier and safer than that with HF, but problems remain with respect to the removal of Fmoc groups. One problem is the trouble caused by the repeated treatment of peptides with a secondary base such as piperidine. Under the typical conditions of treating peptides with 20% piperidine in N-methylpyrrolidone (NMP), Asp(Ot Bu) residues tend to cyclize to a succinimide form, which is further epimerized and/or converted to the α or β -piperidide. The tendency toward succinimide formation is most serious when the carboxyl site of the Asp residue is occupied by Gly, 6 Y. Shimonishi (ed): Peptide Science – Present and Future, 6–14 © 1999 Kluwer Academic Publishers, Printed in Great Britain

7 but the same trouble occurs even when the Gly is replaced by Asn or Gln. The extent of this side reaction can be reduced to about one-tenth by treating the peptide with a mild base such as 10% morpholine in DMF, but the problem cannot be completely eliminated. One way to solve this problem is to incorporate a protecting group at the peptide bond participating in the formation of the succinimide structure, which is the so-called ‘backbone protection method.’ This idea was originally proposed by Sheppard et al. in 1994 for preventing the association of peptide bonds on the resin, for which the 2-hydroxy-4-methylbenzyl (Hmb) group was incorporated into the α -amino group of the following amino acid residue. This protecting group can be removed during the final treatment of the peptide with TFA, but the reactivity of such an imino group is so low that the time required for the coupling reaction with other Fmoc-amino acids is much longer than the case with normal amino group except the case of N-Hmb-Gly. A common problem in the solid-phase synthesis using either Boc- or Fmocstrategy is the formation of various truncated molecules during the elongation reactions. Thus, all the products obtained by the solid-phase method must be purified by strictly applying the most efficient procedures, and structures of the purified products must be confirmed using the most sensitive analytical techniques. At present, the most useful and efficient purification procedure is HPLC. In other words, we cannot synthesize homogeneous peptides by the solid-phase method without the help of HPLC.

High performance liquid chromatography Figure 1 shows the HPLC profile of a crude HF-product of a 67-residue peptide synthesized by the Boc-strategy in my laboratory. We were able to isolate an analytically pure peptide from this product after repeated purification by HPLC. Figure 2 shows the HPLC profile of a crude product of a 55-residue peptide after HF-reaction. We extended much effort toward isolating a homogeneous peptide from the HF-reaction product, but were not successful. The major reason for the formation of such miscellaneous side products is considered to be the association of growing peptide-chains on the resin. Recently, Kent proposed a new way to prevent this by leaving the newly formed amino

Figure 1.

Figure 2.

8 groups as trifluoroacetate after each cleavage reaction of Boc-groups by TFA, because the reagent is strong enough to break any inter- or intra-chain hydrogen bond. In the next step, a mixture of an activated Boc-amino acid and DIEA is added to the peptide resin, the TFA salts are neutralized with DIEA, and the next peptide bond is formed simultaneously [1]. He called this methodology the ‘in-situ neutralization method’ and recommended it for optimizing the yield of the final products, particularly when a large peptide is synthesized, but if the target peptide is larger than 100 residues, isolation of a homogeneous product may not be guaranteed in every case. In any case, application of HPLC is essential for synthesizing peptides by the solid-phase method. I would now like to review the history of HPLC from when it was introduced to peptide synthesis. After the introduction of automated amino acid analysis by Moore and Stein in 1958, the technique of high pressure liquid chromatography became popular. In late April 1974, an interesting roundtable discussion on the use of high pressure liquid chromatography was held at the 13th European Peptide Symposium. In the discussion, Ernst Bayer showed the separation profile of an octapeptide, synthesized by the solid-phase method, on a silica-gel column after labeling with dansyl groups, and then, Murray Goodman showed the possibility of separating dipeptide diastereomers on a Corasil II column. The first attempt at separating natural peptides by high pressure column chromatography was reported in 1974 by Greibrokk et al. for isolating prolactininhibiting factor from the extract of porcine hypothalami using several different types of columns [2]. Then, in 1975, Tsuji and Robertson reported the separation of a polypeptide antibiotic, bacitracin, in 10 different components on a µ Bondapack column [3]. In 1976, at the 14th European Peptide Symposium, Burgus and Rivier reported that HPLC could be an extremely powerful tool for detecting the closely related structural analogs in various synthetic peptides synthesized by the solid-phase method [4]. They also emphasized that HPLC would be useful not only for analytical purposes but also for preparative purposes. In the same year, Hancock independently published a similar report in the FEBS Letters [5]. Since then, many similar reports began to appear. Examining these documents shows that several researchers had already become aware of the power of HPLC for separating closely related peptides in 1974 or 75, but the usefulness of HPLC was formally reported by Burgus and Rivier for the first time in April 1976 at the Peptide Symposium. Although HPLC is very useful for separating closely related compounds, there are limits as shown in Figure 2. If we want to synthesize peptides much larger than 60 to 100 residues in a homogeneous form, we must apply some other ways to reduce the risk of the formation of closely related side products and also to enhance the performance of HPLC. Ligation of free or partially protected peptides In 1974, Corradin & Harbury demonstrated the re-synthesis of horse heart cytochrome c by binding two CNBr-fragments of native cytochrome c in an

9 aqueous medium. Then, a research group in Padua succeeded in reproducing the same reactions with fragments synthesized by the solid-phase method [6]. In 1981, Blake introduced an unique principle for coupling a peptide bond. One peptide containing a thioacid at the C-terminus was found to react with an amino group of another peptide in the presence of silver ions, and he applied the principle to the synthesis of bovine apocytochrome c [7]. Later, Aimoto improved the procedure and synthesized a 90-residue DNA-binding protein from four synthetic segments [8]. In 1994, Kent proposed the interesting idea for coupling the thioester of a free peptide with another free peptide containing Cys at the N-terminus. Applying the principle, he showed the possibility of synthesizing phospholipase A 2 (1–124) from the fragment (1–58) thioester with the fragment (59–124) in an aqueous medium [9]. A similar idea was also proposed by Tam in 1994, in which the carboxyl terminus of a component was modified to a glycolaldehyde ester, which was found to couple with a cysteinyl–peptide to form a product containing a thiazolidine ring, a Pro-mimic, in the middle [10]. These procedures should be important for synthesizing medium-size natural or unnatural proteins in aqueous media. Elongation of fully protected peptide segments on solid support Another way to synthesize large peptides is to assemble the complete sequence from fully protected smaller segments on a solid support instead of synthesizing the whole peptide by step-by-step elongation with individual amino acid derivatives. To realize this, the full sequence of the target protein is divided into several small segments, each of which is synthesized in a form with all the sidechain functional groups protected by permanent protecting groups, the terminal amino group protected by a temporary protecting group, and the terminal carboxyl group remaining free. If this can be done, the whole molecule may be assembled by the segment condensation reaction on an insoluble support. This procedure may be more advantageous than the ordinary solid-phase synthesis in terms of reducing the possibility of forming similar size side-products. The usefulness of this idea was demonstrated in 1968 in my laboratory for the synthesis of a collagen model and successfully obtained the product as single crystals after simple purification [11]. Later, several groups extensively studied this method [12]. In 1980, Kaizer introduced a 4-nitrobenzophenone oxime resin as a useful candidate for the synthesis of such segments; he synthesized 17 fully protected segments of RNase T1 applying the Boc-strategy and attempted to assemble them into the whole sequence. However, his product did not show particular enzymatic activity after HF deprotection reaction. Theoretically, the principle of segment condensations on a resin is attractive, but practically, in the case of Boc-strategy, there has been no successful example yet for the synthesis of large peptides. This may be due to the poor solubility of these segments in ordinary solvents suitable for elongation reactions.

10 With the Fmoc-strategy, a successful example was reported in 1991 for the synthesis of the 109-residue prothymosin α , for which several fully protected segments having only one carboxylic acid at each C-terminus were synthesized by using a 2-chlorotrityl resin followed by treatment with a mixture of acetic acid/trifluoroethanol/dichloromethane (1:1:8) [13]. Those segments in fivefold excess were coupled one by one on a resin using DCC/HOBt in DMSO to obtain the final protected protein. In 1996, Quibell et al. also reported the synthesis of HIV-1 tat protein (1–72) applying a similar principle, in which they applied the backbone protection method in order to prevent not only the association of growing chains on a resin but also the succinimide formation at Asp(Ot Bu) residues [14]. Segment condensation reactions in solution Two different strategies can be considered for the synthesis of peptides in this category. One is the Boc/Bzl-strategy and the other is the Fmoc/ t Bu-strategy as has been discussed in the previous section. Riniker et al. synthesized Fmoc/ t Bu peptide segments by the solid-phase method in 1993 using the newly developed 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyric acid (HMPB) resin and successfully applied the strategy to the assembly of human calcitonin (1–33), neuropeptide Y, and 70K S6 kinase (230–249) in solution [15]. In 1997, Kohmura and Ariyoshi also synthesized the B-chain (1–72) of a sweet protein Mabinlin II in solution by using the 2-chlorotrityl resin [16]. In the Boc/Bzl-strategy, the fully protected segments were not so useful as building blocks for the synthesis of large proteins on an insoluble support, but we had independently established the technique for coupling such fully protected segments in solution [17]. In the early stage of our study, little attention was paid to the solubility of the intermediate segments in solvent systems suitable for elongating these segments. However, during repetition of the experiments, we realized that almost all fully protected peptide segments were relatively soluble in ordinary solvents, except a small number of segments, particularly in the case of large peptides, which were hardly soluble in DMF or NMP. Later, we were able to overcome the insolubility problem by finding new powerful solvent systems, such as a mixture of chloroform (CHL) and trifluoroethanol (TFE) (3:1 v/v). All fully protected segments could be coupled smoothly without danger of epimerization in such a mixture of CHL/TFE when a water-soluble carbodiimide (WSCI), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was used as a coupling reagent in the presence of HOOBt [17]. Under these conditions, we succeeded in synthesizing a variety of biologically active peptides as well as a 121-residue protein, human midkine (MK) in solution [17, 18]. Synthesis of a 136-residue protein, human pleiotrophin (PTN), was also successful; the details of the synthesis will be reported at this Symposium in the poster session.

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In order to confirm the scope and limitations of the procedure, we decided to synthesize a 238-residue protein, green fluorescent protein (GFP), as our next target. For the synthesis of such a large protein, we decided to synthesize the starting individual protected segment by solid-phase method to save time because we could then check the purity of the segments, and if necessary, conduct further purification, before using them to assemble the whole molecule. Among methodologies reported for synthesizing the segments, we decided to adopt the method introduced by Giralt et al. in 1996 [19]. On examining their procedures in detail, we found that BrZ-group for protecting Tyr residues was too unstable under the conditions used to remove the protected segments from the resin. Therefore, before applying the procedure, we decided to develop a new protecting group for the Tyr residues, and finally found that the 3-pentyl (Pen) group was stable enough for removal of the segments from the resin and was itself removable under ordinary HF-deprotection conditions. Details of these findings will be reported by Nishiuchi in a later session of this Symposium. In order to accelerate the coupling reaction of every Boc-amino acid on the resin, we decided to use the HBTU/HOBt method in a presence of DIEA. Under these conditions, the amide groups of Asn and Gln are known to be dehydrated to nitrile; so, we decided to incorporate the xanthenyl (Xan) group to temporarily protect the amide group. In order to synthesize a large number of segments within a short period, we decided to use a semi-automatic synthesizer ACT model 90; we were able to synthesize two 10-residue segments a week. GFP is a 238-residue linear protein, which is isolated from the margin of the umbrella of a jellyfish, Aequorea victoria. The structure was deduced from the cDNA in 1994 [20], and reported to contain a chromophore with a dehydroimidazolone ring structure formed by the post-translational cyclization of the tripeptide unit of –Ser 65–Tyr 66 –Gly67 –[21]. From this, it was assumed that the protein is an autocatalytic enzyme. The molecular structure of recombinant GFP was determined by X-ray analysis to hold an 11-stranded β-barrel with a coaxial helix: the chromophore is formed from the central helix [22]. The primary structure is as shown on the next page. The synthesis was designed for assembly from 26 segments using the following side chain protecting groups: ClZ for Lys, cHex for Glu and Asp, Pen for Tyr, Xan for Asn and Gln, Tos for Arg, Bom for His, Acm for Cys, Bzl for Thr and Ser, and Hoc for Trp. Among them, segments 26 and 18 were synthesized in solution. We assembled the whole molecule following the scheme as shown on the next page, Some of the intermediates were only soluble in CHL/phenol (3:1) rather than in CHL/TFE; in the solvent containing phenol, the coupling reaction proceeded smoothly as in the case of CHL/TFE. After completion of the synthesis of four big segments, the terminal phenacyl (Pac) groups were removed using treatment with zinc dust in 20% acetic acid in HFIP, and the homogeneity of each big segment was confirmed by HPLC after deprotection in HF. Next, these big segments were coupled one by one to the fully protected GFP molecule as shown in the scheme. Although we could not confirm the result of the whole

12

segment-condensation reactions before this Symposium, we expected to obtain the final fully protected GFP molecule as in the case of MK or PTN synthesis smoothly since all large segments were highly soluble in CHL/TFE. Conclusion During the assembly of the GFP molecule, we were able to confirm the usefulness of the solid-phase method for reducing the time to synthesize pro-

13 tected segments having a size of 10 to 12 residues. However, here I wish to clearly emphasize that all the segment-condensation reactions must be carried out in solution; that is, every step of the segment-condensation reactions must be carefully monitored by either TLC or HPLC and progression to the next step should only be done after confirming the homogeneity of the product. If it is not done, highly homogeneous products cannot be obtained even by the solution procedures. By applying this principle, it should be possible to synthesize 100- or 200-residue peptides without any difficulty by the Boc/Bzl-strategy or probably even by the Fmoc/t Bu-strategy. The most important factor for realizing such a segment-condensation reaction in solution is the availability of powerful solvent systems such as mixtures of CHL/TFE, CHL/phenol, or CHL/HFIP, and a combination of useful coupling reagents, WSCD and HOOBt. I am optimistic about the total synthesis of GFP as we have not yet encountered any critical problem which would overturn our original expectations. By applying these methods, I am certain in general that we are able to synthesize an ordinary 100-residue protein within several months, and a 200-residue protein within a year. This approach should be the only possible way to synthesize such a large peptide in a homogeneous form by the chemical procedures. References 1. Schnölzer, M., Alewood, P., Jones, A., Alewood, D. and Kent, S.B.H., Int. J. Pept. Protein Res., 40 (1992) 180–193. 2. Greibrokk, T., Currie, B.L., Johansson, K.N.-G., Hansen, J.J. and Folkers, K., Biochem. Biophys. Res. Commun., 59 (1974) 704–709 3. Tsuji, K. and Robertson, J.H., J, Chromatogr., 112 (1975) 663–672. 4. Burgus, R. and Rivier, J., In Loffet, A. (Ed.) Peptides 1976 (Proceedings of the 14th European Peptide Symposium), Editions Univ. Bruxelles, Belgium, 1976, pp. 85–94. 5. Hancock, W.S., Bishop, C.A. and Hearn, M.T.W., FEBS Letters, 72 (1976) 139–142. 6. Di Bello, C., Vita, C. and Gozzini, L., Biochem. Biophys. Res. Commun., 183 (1992) 258–264. 7. Blake, J., Int. J. Pept. Protein Res., 17 (1981) 273–274; Blake, J. ibid., 27 (1986) 191–200. 8. Hojo, H. and Aimoto, S., Bull. Chem. Soc. Jpn., 64 (1992) 3055–3063; Kawakami, T., Kogure, S. and Aimoto, S., ibid., 69 (1996) 3331–3338. 9. Dawson, P.E., Muir, T.W., Clark-Lewis, I. and Kent, S.B.H. Science, 266 (1994) 776–779: Hackeng, T.M., Mounier, CM., Bon, C., Dawson, P.E. Griffin J.H. and Kent, S.B.H., Proc. Natl. Acad. Sci. USA, 94 (1997) 7845–7850. 10. Liu, C.-F. and Tam, J.P., Proc. Natl. Acad. Sci. USA, 91(1994) 6584–6588. 11. Sakakibara, S., Kishida, Y., Kikuchi, Y., Sakai, R. and Kakiuchi, K., Bull. Chem. Soc. Jpn., (1968) 1273; Sakakibara, S., Kishida, Y., Okuyama, K., Tanaka, N., Ashida, T. and Kakudo, M., J. Mol. Biol., 65 (1972) 371–373. 12. Lloyd-Williams, P., Albericio, F. and Giralt, E., Tetrahedron, 49 ( 1993) 11065–11133. 13. Barlos, K., Gatos, D., Kapolos, S., Papaphotiu, G., Schäfer, W. and Wenquing, Y., Tetrahedron Lett., 30 (1989) 3947–3950; Barlos, K., Gatos, D. and Schäfer, W., Angew. Chem. Int. Ed. Engl., 30 (1991) 590–593. 14. Quibell, M., Packman, L.C. and Johnson, T., J. Chem. Soc., Perkin Trans. 1, (1996) 1227–1234. 15. Riniker, B., Flörsheimer, A., Fretz, H., Sieber, P. and Kamber, B., Tetrahedron, 49 (1993) 9307–9320. 16. Kohmura, M. and Ariyoshi, Y., In Kitada, C. (Ed.) Peptide Chemistry 1996 (Proceedings of the 35th Japanese Peptide Symposium), Protein Res. Found., Osaka, 1997, pp. 101–104. 17. Sakakibara. S., Biopolymers 37 (1995) 17–28.

14 18. Inui, T., Bódi, J., Kubo, S., Nishio, H., Kimura, T., Kojima, S., Maruta, H., Muramatsu, T. and Sakakibara, S., J. Peptide Sci., 2 (1996) 28–39. 19. Rabanal. F., Giralt, E. and Albericio, F., Tetrahedron, 51(1995) 1449–1458. 20. Inouye, S. and Tsuji, F.I., FEBS Letters, 351(1994) 211–214. 21. Cody, C.W., Prasher, D.C., Westler, W.M., Prendergast, F.G. and Ward, W., Biochemistry, 32 (1993) 1212–1218. 22. Brejc, K., Sixma, T.K., Kitts, P.A., Kain, S.R., Tsien, R.Y., Ormö, M. and Remington, S.J., Proc. Natl. Acad. Sci. USA, 94 (1997) 2306–2311.

3 Peptide science: past, present and future D. BRANDENBURG Deutsches Wollforschungsinstitut an der RWTH Aachen, Veltmanplatz 8, D-52062 Aachen, Germany

Introduction Searching Medline for ‘peptides’ gives more than 20,000 entries for the last year – a remarkable growth since Emil Fischer and Franz Hofmeister proposed the CO–NH bond as structural characteristic of proteins in 1902, and since the name ‘Peptid’ was coined. Branching out of organic chemistry as a special field a century ago, peptide chemistry today is in the center of a science which lives through symbiosis with biology, biochemistry, physics and medicine. In this short account some features are outlined in a very general and, necessarily, fragmentary way.

Where are they found? By and by, a wealth of peptides was found in Nature with a vast spectrum of variation with respect to molecular size, structure and functions. From microorganisms, plants, animal and human sources peptides were isolated, ranging from antibiotics, alkaloids, and toxins to hormones and neuropeptides. In vertebrates, more than 100 peptides are active in the central and peripheral nervous system, in immunological processes, the cardio-vascular system and gastro-intestinal tract – reason enough for pronounced medical-pharmacological interest. Progress in isolation and structure determination, both hampered by often extremely low concentrations and amounts, critically depended on the development of highly sensitive techniques, like gas phase sequencing and mass spectrometry as major tools. In recent years, deduction of primary structure from the DNA sequence has been fast and convenient. However, analysis of peptides is still necessary in view of non-ribosomal pathways and posttranslational modifications.

How are they made? It took almost 50 years of rather slow, but systematic work in a few laboratories, until a new era began with the structure elucidation of oxytocin by Du Vigneaud, and of insulin by Sanger. New coupling procedures, protecting groups and deblocking methods were explored, and tactics and strategies for synthesis of 15 Y. Shimonishi (ed): Peptide Science – Present and Future, 15–20  1999 Kluwer Academic Publishers, Printed in Great Britain

16 oligo peptides in solution developed. Fighting the Damokles’ sword of racemization, overcoming other side reactions, extending the length of peptides were exciting, often frustrating, but always laborious experiences. A great analytical progress was succession of chromatography on paper by silica gel TLC. In the next two decades, the ‘golden age of peptide chemistry’ the art of making peptides came to full bloom. The spectrum of methods was widened, peptides grew longer, SH protection and disulfide formation were carefully investigated. Numerous analogues underwent biological testing, the first peptide drugs went to market. Bruce Merrifield’s ingenious concept led to an enormous further uprise in peptide chemistry – and opened the door for genetical engineering via chemical nucleotide synthesis. Solid phase synthesis was met with enthusiasm, but also scepticism, until the the advent of RP-HPLC made the final break-through possible. The many variants of carriers, anchoring groups, cleavage procedures ... finally led to the two main strategies of today: Boc/Bzl and Fmoc/t-butyl procedures. Attempts to render coupling yields as quantitative as possible have reached 99.5% per step. Synthesizers matured from manual shakers to electromechanical devices and further to fully automated instruments for batch or flow-through performance. The chemical methods were supplemented by biochemical approaches. Enzymatic peptide synthesis proved its value in a number of cases; it is indispensible for semisyntheses of insulin analogues. Recent advances are engineered enzymes (e.g. subtiligase) and zymogens as catalysts. The advent of recombinant DNA technology in the early eithties marked another area in peptide science. By now, already many polypeptides/proteins have been produced in large scale. Combination with chemical processing steps may be advantageous. In the last years, two technologies evolved out of solid phase synthesis, aiming at fast generation of large numbers of compounds for biological screening, and speedily gained grounds: Multiple, or multiple simultaneous PS, to give series of individual peptides in parallel syntheses. Combinatorial chemistry turned the principle of classical chemistry upside-down. Various strategies lead to generation of libraries containing thousands or even millions of peptides. These approaches found fast application in organic chemistry.

Proteins – peptides – mimetics Today, peptides are in the center of a continuum of compounds, boundaries are arbitrary. Solid phase and solution methods are available for fast and efficient coupling, deprotection, workup, purification, yet may need special adaption or modification to meet the individual character of a given peptide. Lipo-, phospho-, and glycopeptides as well as peptide nucleic acids (PNAs) widen the spectrum. Varying size and side chains, a huge number of defined

17 analogues has been synthesized for biological, conformational and many other studies. Moving to the larger species, it is remarkable to which extent the dogma ‘proteins cannot be chemically synthesized’ has been disproved. Insulin, ribonuclease, HIV protease, its all-D -form, barnase, human midkine (121 amino acids) are a few examples. In such total and also in semisyntheses, condensation of segments – obtained via solution or solid phase synthesis, or fragmentation of native proteins – is the method of choice. Successful ligations have been facilitated through auxiliary bonding and performed via non-peptide bonds. De novo Protein design is now a tempting field. Various strategies are being tested for subunit assembly, as covalent linear chain coupling or oligo-functional templates. On the other side, peptide mimetics are a very rapidly growing group of compounds. Such imitations (mimetics) of bioactive peptides range from peptide isoster to structures no longer recognizable as peptide. In order to achieve a stable/stabilized three-dimensional structure, computer-aided design, peptide and organic chemistry meet.

Ligands – structures – targets – effects Receptors, enzymes, and also DNA-sequences are the now known major targets of peptides. For many years, it was a problem to define the molecular sites upon which peptides act. Information on structure-function relationships was mainly derived from measurement of biological effects in laboratory animals, isolated organs, and later in isolated and cultured cells. The detection of receptors in the early seventies was a turning point. Highly sensitive assays, mainly based on radioactive labelled ligands, were developed, and correlations between receptor binding (usually with cells or membrane preparations) and bioactivity became possible. Extensive studies with large numbers of synthetic analogues of peptide hormones and neuropeptides gave a wealth of information leading, inter alia, to the concept that agonists interact with an activated conformation of the receptor, while antagonists firmly bind to the ground state of receptor. Since all molecular interactions occur between thee-dimensional structures, knowledge about static and dynamic spatial structures of ligands and receptors is of prime importance to understand ligand recognition, docking, and subsequent processes. This has been an area of extensive research. A large body of information on structure and conformation of peptides has been accumulated by X-ray crystallography and NMR spectroscopy. Computer techniques led to the construction of vivid molecular models. Receptors have been the rate-limiting factors, since native receptors were available in minute amounts only and thus allowed investigations not beyond the nanomolar level. Meanwhile, molecular biology is providing genetically

18 engineered native and mutated receptors, and primary structures continue to become known – yet the scale at which studies can be carried out is usually small. Only rarely has it been possible to discern the very details of interactions, as with GH and its receptor, where X-ray analysis of the complex revealed a 2 : 1 stoichiometry. I n most cases, indirect methods have to be used. Photoaffinity labelling is particularly valuable and could define contact sites (vasopressin, insulin). Molecular modelling helps to visualize such interactions. The situation is easier with enzymes and their substrates. From known X-ray structures the micro-environment in the substrate binding pocket is obvious. Based on the transistion state, the rational design of inhibitory analogues has advanced to high levels.

Towards application The overwhelming field of potential or already effective application has been, and certainly will be in the future, medicine. The principle of molecular pharmacology/molecular medicine, to reduce the very complex overall picture of a disease to the molecular level, directs drug design to ligands for these targets. The pronounced discrepancy between high biological importance of peptides, but low suitability for direct application is a tragedy, but also a challenge. Design of agonistic or antagonistic mimetics starting from the natural lead compounds aims, for instance, at stabilization against degradation, longer and enhanced action, and selectivity at the receptor. D-amino acids and cyclic structures have been effective elements. One decisive goal is the development of orally active peptide-derived drugs. Rational drug design is increasingly aided by computer simulations and molecular modelling. On the other hand: Molecules with native structures do have eminent therapeutic importance. The large-scale production of proteins via genetical engineering allows to verify the concept of therapy with endogeneous drugs (insulin, human growth hormone, tissue plasminogen activator, interferons). A successful field of great therapeutic value is the development of enzyme inhibitors, with reference to Captopril as the classical ACE inhibitor. Just one important further example are potential inhibitors for HIV protease. Finally, peptides play an important role in immunology. Epitope mapping, first with individual series of peptides, is now efficiently done with with libraries. In studies on the mechanism of immune response, natural peptide libraries, generated during protein processing with MHC molecules, have been analyzed through Pool sequencing. Peptide vaccines, for instance against foot and mouth disease, malaria or AIDS, are a very active area, and anti-peptide antibodies have become rather universal research tools.

19 Peptide activities Peptide science is performed in a wide field ranging from fundamental research at universities and other institutions to research, development and production in large pharmaceutical companies. In between, many activities came to life over the years, providing instrumentation and materials, custom syntheses and other services, thus contributing greatly to the advancement of science in the laboratories of their customers. The growth of peptide science is well reflected by the increasing size of the Peptide Symposia, the rising necessity to organize smaller spezialized meetings, and the birth of new journals. The five Peptide Societies (America, Australia, China, Europe, Japan) fulfill an important task in the international scientific exchange: The organization of symposia, the edition of journals on peptide science, supplemented by their highly informative newsletters, and finally provision of contacts via internet.

Conclusions – outlook In the last years, the picture of peptide science has changed. It is no longer the α-peptide bond alone, which dominates the scene, nor is it the diligent synthesis of an individual peptide and its subsequent bioassay. Diversity has become a catch-phrase. Mimetics and combinatorial libraries take their part, robot syntheses and high-througput screening search for new lead compounds. Peptide chemistry is, to a certain extent, returning to organic chemistry. The development in general, and this symposium in particular, show that peptide science is vividly alive. The demand of synthetic peptides for solving complex biological questions is steadily rising. It will be met in a supplementary way by peptide synthesis and genetical engineering. The advances which have been achieved allow progressing refinement of investigations, making use of the armament now at our hands – synthesis, analysis, isolation, structure elucidation, conformation analysis and molecular modeling – in an interdisciplinary collaboration between chemistry, biology, biochemistry, biophysics, pharmacology and medicine. While application-oriented research in the pharmaceutical industry has largely left the peptides, the future in fundamental research seems nevertheless promising. After all, the major aim of science is to aquire knowledge and to understand mechanisms of nature. There are many questions to be answered through peptides. When they are, then application is a logical next step.

Acknowledgment The author expresses his most cordial thanks to Hans-Dieter Jakubke, Leipzig; whose personal advice and book (see ref) were of invaluable help. Sincere

20 thanks are also due to Yasutsugu Shimonishi, Tetsuo Shiba, Günther Jung, Joachim Gante and Jochen Knolle.

References Reference is not made to individual sections, but rather to selected sources of general interest. Davies, J.S. (Ed.), Amino acids, peptides and proteins vol. 27, Royal Society of Chemistry Specialist Periodical Reports 1996, and preceeding volumes Jakubke, H.-D., Peptide, Chemie und Biologie. Spektrum Akademischer Verlag, 1966 Heidelberg Lloyd-Williams, P., Albericio, F. and Giralt, E., Chemical approaches to the synthesis of peptides and proteins. CRC Press, 1997 Jung, G. (Ed.), Peptide and nonpeptide combinatorial libraries. A handbook. VCH, Weinheim, 1996 Wieland, T. and Bodanszky, M. The world of peptides. Springer, Berlin 1991 Geisow, M.J. (Ed.), Perspectives on protein engineering ’96. BIODIGM 1996 http://www.biodigm.com Peptide synthesis database: http://chemlibrary.bri.nrc.ca/ Peptide information http://prfsun2.prf.or.jp/e-pi.htm, http://www.prf.or.jp/ Structural biology in Europe http://www.biodigm.com/strube.htm American Peptide Society: http://www.chem.umn.edu/orgs/ampepsoc/apshome.html European Peptide Society: http://www.swan.ac.uk/chemistry/eps/index.html

4 Quo vadis? Where is peptide chemistry headed? T. SHIBA Protein Research Foundation, 4-1-2, Ina, Minoh, Osaka, 562-8686, Japan

Introduction It is almost impossible to predict the future fate of peptide science. It is just as formidable a task to try to review all fields of this rapidly developing area. Nevertheless, I will try to map out a possible future for peptide science from a retrospective review of its history as well as literature data related to peptide science collected in our Foundation. I undertake this challenge with the hope that it will serve to encourage young researchers in this field.

Characteristic feature of peptide molecule First, let us re-examine the background of peptide chemistry. Natural peptides are composed of twenty kinds of amino acids whose arrangements are controlled by DNA triplets constituted of four kinds of nucleotides. We are very much indebted to the fact that there are only four code-composing nucleotides. If this were not the case, there would have been an unimaginable number of possible amino acids and their manipulation would have been impossible. Fortunately, only triplets of four DNA molecules were used as building block of life on earth. It is almost a miracle that this allows for the existence of all living organisms and also make possible their study from the standpoint of peptide research. However, recently peptide scientists are now venturing beyond this natural limitation by introducing of non-proteinic amino acids into peptides to create useful new molecules. The diversity of natural peptides is also guaranteed by the sequential combination of twenty amino acids. The possible number of decapeptides is 1013 and that of eicosapeptides could be 1026 . This means there remains much for peptide scientists to study in the future. Here we must not forget the importance of the definite amino acid sequence in a natural peptide. For instance, in the case of sickle cell anemia disease, the replacement of just one amino acid, Glu 6 , with Va1 6 out of 146 amino acid residues in the β -chain of hemoglobin is lethal. We are reminded that the raison d’être of peptide chemists lies here. The significance of the essential length of bioactive peptides in relation to receptors cannot be emphasized enough. As seen in the structure of TRF, which is composed of only three common amino acids, i.e., Glu, His and Pro, it is the striking and marvelous character of peptides that such a simple tripeptide 21 Y. Shimonishi (ed.): Peptide Science – Present and Future, 21–25 © 1999 Kluwer Academic Publishers, Printed in Great Britain

22 exhibits remarkable hormonal activity even at the extremely low picogram level, while each constituent amino acid alone shows no significant biological activity. This seems to suggest that the essential scale of a bioactive molecule is at least around 15 Å. Here we should note that among the many organic compounds, only peptide molecules fit this scale and yet offer a wide range of flexible conformations by taking numerous three- dimensional structures with φ and ψ angles, as well as side chain variations. Thanks to these intrinsic and marvelous properties of peptide molecules, the study of peptide science should remain essential as long as living organisms continue to exist on earth. Overview of peptide science The Institute of Protein Research Foundation (PRF) has been performing exhaustive surveys of extensive collection of research information in peptide and protein sciences since 1979. At present, more than 24,000 articles are accumulated from 120 current journals each year. This data collection can yield a wealth of statistical information concerning recent trends in peptide science. Figure 1 summarizes the total number of articles reported every year in the field of peptide science. It shows how active research activity in this field has been during the past two decades. Chemical syntheses of peptides Examination of the PRF data collection from another angle shows that the total number of peptide syntheses, particularly chemical synthese, is rapidly increasing recently; it is more than two times that in ten years ago (Figure 2). This tendency may be supported by the popular use of solid phase synthesis.

Figure 1. Number of papers in peptide science Biology Physiology, Pharmacology.

Review

Biophysics

Chemistry

Molecular

23

Figure 2. Peptide syntheses,

Chemical

Enzymatic

Others

Total chemical syntheses of enzymes are outstanding monuments in peptide chemistry. Historically, the first synthesis was performed for the ribonuclease molecule in 1969 (R.F. Hirshman). Solid phase synthesis was used for the same purpose (R.B. Merrifield, 1971), and crystalline ribonuclease A was finally obtained also by chemical synthesis (H.Yajima, 1980). Recently, in membrane biology, chemical synthesis is becoming an indispensable tool for providing activators or blockers in studies of ion channel mechanism. Alamethicin (C.E. Meyer, 1967), conotoxins (Y. Nishiuchi, 1982), endothelin (T. Masaki, 1988) and calciseptine (H. Schweitz, 1990) are representative ion channel peptides which have been successfully synthesized.

Noteworthy targets of peptide chemistry As rather special topics in peptide chemistry, we should note the work in invertebrate biology. It has led to the studies of endogenous antibiotics such as magainin from frog skin (M. Zasloff, 1987), cecropin from Cecropia moth (H.G. Boman, 1981), and lepidopteran from silkworm (T. Teshima, 1983). Cyclic peptides also belong to a historically important group of biologically active peptides. Cyclosporin A is now becoming an indispensable immunosuppressor in organ transplantation (A. Ruegger, 1976). A new antifungal peptide, aureobasidin A (K. Inami, 1996), is expected to be a promising drug for opportunistic infectious diseases such as candidasis or aspergillosis. Modified peptides such as glycopeptides, lipopeptides, and phosphopeptides are arousing strong interest due to their particularly important biologically activities. Muramyl peptide as one example of glycopeptides was found to possess a structure responsible for immunoadjuvant activity of the cell wall of microorganisms (S. Kusumoto, 1975).

24 Here I would like to propose a new term, ‘peptidoconjugate’, as a comprehensive name for these three modified peptides to emphasize the significance of the the peptide moiety. The term is analogous to the popular name of ‘glycoconjugate’ in carbohydrate chemistry. Further data collection should pay attention to this particular field, because of surge of report articles describing recent studies on such peptidoconjugates. Another important research direction is that of posttranslational modification peptides. Examples of this category include lanthibiotics such as nisin (E. Gross, 1871), ancovenin (T. Wakamiya, 1985), epidermin (G. Jung, 1991), which contain the lanthionine loop in the peptide chain. Nisin is now in practical use as food preservative in Europe and synthesized chemically (K. Fukase, 1992). Other post-ribosomal peptides worthy of note include 4-hydroxyprolinebradykinin (H. Kato, 1989), 6-hydroxydopa peptides (M. James, 1990), Damino acid-containing animal peptides (V. Erspamer, 1990, H. Minamikata, 1996), and hypusine (T. Shiba, 1971, H. Schümann, 1988). These peptides have not only been proven to play unique and important roles in view of their biological activities, but also are attracting much intensive attention with respect to their biosynthetic ways.

Other topics of peptide chemistry In view of the fascinating development of peptide chemistry, there are many more topics which should be mentioned here. However, owing to space limitations, I can only touch briefly on a few subjects. As mentioned earlier, the introduction of non-natural amino acids into peptide molecules is opening exciting new fields. The number of studies along this line has exploded since 1991 and tremendous development has been recently spurred by partnerships with combinatorial chemistry. Gene engineering based on site-directed mutagenesis also can develop further with the aid of combinatorial chemistry. What we should note here is that the basic methodologies of combinatorial chemistry together with the principle of solid phase synthesis as well as the presently available peptide libraries originated from the diligent and steady efforts of researchers in peptide chemistry. Therefore, the peptide chemist deserves to be honored as a pioneer of those newly developing fields. Another important area is that of peptidomimetics where the peptide bonds in natural peptide are replaced with other linkages in order to enable attack to enzyme inhibitors. The concept was initiated in as early as 1946 as a transition state analogue (L. Pauling). However, it has not been proven experimentally until R. Wolfenden studies on carboxy peptidase (1973). Captopril exploited by M. Ondetti was the most remarkable fruit in this line of studies, which developed into a typical drug for hypertension treatment today. Among rather recent contributions in this field, I should mention particularly the

25 studies on inhibitors of aspartic protease (D.H. Rich, R. Marshall, 1977), and HIV protease (J. Erickson, 1990, Y. Kiso, 1996). Scope for contribution of peptide chemistry to biomedical science Next, let us take a more targeted view of the future scope of peptide chemistry by concentrating on biomedical science. We are already familiar with the brilliant triumphs in this field, one of which was the development of an excellent peptide drug for prostatic cancer by Takeda Chemical Industries. Many other difficult diseases await decisive development in peptide chemistry. We need an epitope inhibitor for AIDS, a vaccine for multisclerosis as autoimmune disease, a vaccine for hepatitis B virus, an inactivator of oncogenes in cancer, a prevention drug of retinopathy in diabetes, a prevention drug of osteoporosis, an inhibitor of serine protease in Alzheimer’s disease, a vaccine against Plasmodium falciparum of malaria, an inhibitor of hemagglutinin on influenza virus, a non-addictive drug as an analgesic, and the list continues. Further challenging fields in peptide science Although peptide science already occupies a very important position in many fields of life science, much more important developments are now strongly anticipated in areas such as the following: antibody enzymes, conformational analysis, protein motifs, structure–activity relationships, peptide-membrane interactions, receptor action mechanisms, computer modeling, functional foods, and peptide engineering. Our data collection certainly indicates the growing importance of peptide science in those fields from the rapidly increasing numbers of studies over recent years. Particularly remarkable is research on peptide engineering and protein motifs. Conclusion In conclusion, from literature data as well as my personal observations, I am confident that peptide science, which has made rapid progress is still in its young growing stage and has a promising future in which it can make unique and important contributions to the welfare of mankind in future. We peptide scientists can look forward to an exciting future as we prepare to welcome the new millennium. Acknowledgements Thanks are due to M. Isoyama and Y. Nishiuchi of Protein Research Foundation for their kind help in the preparation and presentation of this lecture.

5 Designing peptidomimetic antagonists for peptide receptors: topochemical differences of agonists and antagonists V.J. HRUBY 1, S. LIAO 1, G. HAN 1, G. BONNER1 , M. SHENDEROVICH1, J. SLANINOVA 2, F. PORRECA 3, H.I. YAMAMURA 3 and M.E. HADLEY4 1

Department of Chemistry, University of Arizona, Tucson, AZ 85721 USA Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, 16610 Prague, Czech Republic 3 Department of Pharmacology, University of Arizona, Tucson, AZ 85721 USA 4 Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724 USA 2

Introduction The design of antagonists for peptide receptors based on the three dimensional structure of the native peptide ligands has been very difficult for several reasons: 1) agonists and antagonists have different structure requirements, that is, different structure-activity relationships; 2) agonists and antagonists have different dynamic structure requirements (for example, agonist-receptor interactions require structural changes for transduction, antagonist-receptor interactions do not); 3) partial agonist/antagonist activity is difficult to interpret for antagonist design; and 4) multiple binding modes (different structure-activity relationships) are possible for antagonists (how can this be factored into design?). Nonetheless, there are successful approaches that have been taken (Table l), but nearly always, success depends on finding a lead compound that demonstrates antagonist activity in a bioassay [1]. The appearance of partial agonist/partial antagonist activity and inverse agonism, not to mention differences in efficacy that often appear, makes it difficult indeed to determine what features of structure and dynamics are relevant to antagonist activity in any particular peptide ligand. We have explored the use of topographical side chain constraints of side chain groups (χ1 , χ 2 torsional angles, etc.) of critical pharmacophore residues in peptide and peptidomimetic backbone templates as a general method for obtaining potent antagonists for peptide receptors, and report here some of our recent results. Table 1 I. II. III. IV. V.

Successful approaches to finding peptide and peptidomimetic antagonists.

Classical peptide structure–activity studies – need a lead Amide bond replacements and peptoids Conformational constraints Topographical constraints Random screening – non-peptides often are not peptidomimetics

26 Y. Shimonishi (ed): Peptide Science – Present and Future, 26–29 © 1999 Kluwer Academic Publishers, Printed in Great Britain

27 Results and discussion Antagonists for opioid receptors Previously we demonstrated that somatostatin could be converted to µ opioid antagonists such as CTAP (DPhe-c[Cys-Tyr-DTrp-Arg-Thr-Pen]-Thr-NH 2 and TCTAP (DTic-c[Cys-Tyr-DTrp-Arg-Thr-Pen]-Thr-NH 2 by specific conformational constraint [3,4] coupled with topographical constraint [5] using D -Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylate). We have shown that by replacing the DPhe 1 or DTic 1 residue in CTAP and TCTAP, respectively, with the D -amino acid residues of β -methylphenylalanine, [(2R,3S ) and (2R,3R) respectively], potent µ opioid receptor antagonists are obtained [6], and in both cases the peptidomimetic analogues show δ agonist activity in the mouse vas deferens (MVD). Most interestingly both analogues show analgesic activity in the mouse tail flick assay, perhaps due primarily to their δ agonist activities (Table 2), and although [(2 S,3R) β -MePhe 1 ]CTAP has no MVD activity, it too has some analgesic activity. Antagonists for oxytocin (OT) receptors Previously we demonstrated that conformational constraint by bicyclization of the potent oxytocin agonist [ β -Mpa 1 ]OT, or the weak partial antagonist [dPen 1 ]OT, lead to potent oxytocin antagonists ([7,8], Table 3). Recently we have determined the bioactive conformation of the bicyclic oxytocin antagonists [9], and have demonstrated that these antagonists have different conformations and topographies than oxytocin agonists. A key pharmacophore residue for both agonist and antagonist biological activity is the Tyr 2 residue. To further explore the topographical requirements for potent antagonist activities at the oxytocin uterine receptor, we have introduced the 4 isomers of the highly topographically constrained tyrosine analogue β -methyl-2',6'-dimethyltyrosine (10, TMT) as their p-methoxy derivatives (MeOTMT) into the bicyclic oxytocin analogues (Table 3). Very interestingly it was found that the two bicyclic Table 2

Binding and Biological Activities of CTAP Analogues Biological activities Binding (nM) a

Bioassay (nM) b

Compound

µ

δ

MVD

GPI

Antinociception c

CTAP [(2R ,3S)β-MePhe 1 ] CTAP [(2R,3R)β−MePhe 1 ] CTAP [(2S,3R) β− MePhe 1 ]CTAP

2.1 20 21 5,400

5,300 1,200 1,300 13,000

0 Agonist Agonist 0

Antagonist Antagonist Antagonist 0

0 + ++ +

a b c

Rat brain membranes. µ vs [ 3 H]DAMGO; δ vs [ 3 H][p-ClPhe 4 ] DPDPE MVD = mouse vas deferens (δ); GPI = guinea pig ileum (µ) In vivo tail flick assay: + 10–30%; + + 30–40%

28 Table 3

Antagonist Potencies of Bicyclic Oxytocin Antagonist Analogue.

Analogue a

Potencies (pA 2 ) Uterotonic (no Mg +2)

[dPen 1,c(Glu 4 ,Lys 8 )]OT [dPen 1 ,(2S,3S)-MeOTMT 2,c(Glu 4 ,Lys 8 )]OT [dPen 1,(2R,3R)-MeOTMT 2,c(Glu 4 ,Lys 8 )]OT [dPen 1 ,(2R,3S)-MeOTMT 2,c(Glu 4 ,Lys 8 )]OT

8.2 8.3 7.8 ~5.5

a

MeOTMT = β-methyl-2',6-dimethyl-4-methoxytyrosine

oxytocin analogues containing the (2S,3S)- and (2 R,3R)-MeOTMT residues were highly potent oxytocin receptor antagonists (Table 3), whereas the (2R,3S)-containing analogue was a weak antagonist. Modeling studies and energy calculations suggested that a gauche(–) χ1 angle for the (2S,3S)-amino acid residue, and a gauche (+) conformation for the (2R,3R)-amino acid residue, are the lowest energy corformations for the binding pharmacophore for the antagonists. A trans χ 1 conformation, (the lowest energy χ1 conformation found in both the 2S,3R- and 2 R,3S-containing analogues), gives essentially inactive analogues. This suggests that antagonists of peptide receptors have very critical topographical requirements. Antagonists for melanotropin receptors Recently several new receptors for melanotropin peptides have been cloned. We have investigated methods for obtaining highly selective and potent melanotropin analogues for the melanocortin receptors. In one approach we took a super potent conformationally constrained analogue of a-melanotropin, namely Ac–Nle-c[Asp–His– DPhe–Arg–Trp–Lys]–NH 2 [11], and by using topographically constrained amino acids such as D -2'-naphthylalanine (Nal(2')) at the DPhe 7 -position obtained analogues that were potent antagonists at some melanocortin receptors, and equally potent agonists at other melanocortin receptors [12]. The spatial specificity of this conversion of a melanocortin agonist into an antagonist is shown by the following: 1) the DNal(2')7, but not the DNal(1') 7, analogue is an antagonist; 2) the D-pIPhe 7 - containing analogue, but not the 7 D-pClPhe - or D -pBrPhe 7- containing analogues, is an antagonist. Nevertheless, the DNal(1')7, D -pClPhe 7 , and D-pBrPhe 7 analogues are weak agonists at the frog skin receptor compared to the DPhe7 analogue. Molecular modeling of α-melanotropin (α-MSH) has suggested that the bioactive conformation of αMSH has a β -turn in the vicinity of the critical tetrapeptide pharmacophore sequence–His 6–Phe 7–Arg 8–Trp 9 –. Based on these concepts we have succeeded in converting a somatostatin analogue into melanocortin receptor antagonists. Examples of antagonists include H–DPhe–c[Cys–His–DTic–Arg– Trp–Cys]–Thr–NH 2 and H–DPhe–c[Cys–His–DPhe(p-I)–Arg–Trp–Cys]– Thr–NH 2 . Again in these cases it is remarkable that relatively small changes in topographical properties of a single amino acid residue (DPhe 7 ) can convert peptide receptor agonists into antagonists.

29 Conclusions Based on the results from these studies it is clear that the development of de novo approaches to peptidomimetic antagonist design presents very substantial challenges. Those features of structure and dynamic behavior that are critical to receptor binding and transduction must be carefully distinguished from those that are necessary only for specific binding/molecular recognition in both the ligand and the receptor. Thus, not only will true peptidomimetic antagonists require appropriate template structures that can mimic constrained peptide backbone conformations, but careful consideration of topographical constraint of side chain groups in the ligand also will be essential. We are now translating these findings into the design and synthesis of non-peptide peptidomimetic antagonist analogues.

Acknowledgements Support from the U.S. Public Health Service and the National Institute of Drug Abuse is gratefully acknowledged.

References 1 . Hruby, V.J., In Joosee, J., Buijs, R.M. and Tilders, F.J.H. (Eds.), Progress in Brain Research, Vol. 92, Chapter 18, Elsevier Sci. Publ., Amsterdam, 1992, pp. 215–224. 2 . Hruby, V.J., Drug Discovery Today 2 (1997) 165. 3 . Pelton, J.T., Gulya, K., Hruby, V.J., Duckles, S.P. and Yamamura, H.I., Proc. Natl. Acad. Sci. USA 82 (1985) 236. 4 . Kazmierski, W.M., Yamamura, H.I. and Hruby, V.J., J. Am. Chem. Soc. 113 (1991) 2275. 5 . Kazmierski, W.M. and Hruby, V.J., Tetrahedron 44 (1988) 697. 6 . Bonner, G.G., Davis, P., Stropova, D., Ferguson, R., Yamamura, H.I., Porreca, F. and Hruby, V.J., Peptides 18 (1997) 93. 7 . Hill, P.S., Smith, D.D., Slaninova, J. and Hruby, V.J., J. Am. Chem. Soc. 112 (1990) 3100. 8 . Smith, D.D., Slaninova, J. and Hruby, V.J., J. Med. Chem. 35 (1992) 1558. 9 . Shenderovich, M.D., Kövér, K.E., Wilke, S., Collins, N. and Hruby, V.J., J. Am. Chem. Soc. 119 (1997) 5833. 10. Qian, X., Russell, K.C., Boteju, L.W. and Hruby, V.J., Tetrahedron 51(1995) 1033. 11. Al-Obeidi, F.A., Hadley, M.E., Pettitt, B.M. and Hruby, V.J., J. Am. Chem. Soc. 111(1989) 3413. 12. Hruby, V.J., Lu, D., Sharma, S.D., Castrucci,A. de L., Kesterson, R.A., Al-Obeidi, F.A., Hadley, M.E. and Cone. R.D., J. Med. Chem. 38 (1995) 3454.

6 Self-assembling homopolymeric peptide tapes in aqueous solution A. AGGELI, M. BELL, A. STRONG, S. RADFORD and N. BODEN Centre for Self-Organising Molecular Systems, The University of Leeds, Leeds, LS2 9JT, UK

Introduction Peptide self-assembly can be exploited to engineer a wide variety of novel biopolymers. Here we present the design and characterisation of de novo oligopeptides which self-assemble into polymeric β-sheet tapes in water. Of special interest, the self-assembly of the peptides, and, consequently, the stability of the polymers, can be controlled by external triggers such as pH.

Results and discussion A set of design criteria have been formulated for the production of de novo, self-assembling, β -sheet, tape-forming oligopeptides [1,2]. These are: (i) highly co-operative intermolecular hydrogen bonds, (ii) cross-strand attractive forces, (hydrophobic, electrostatic, hydrogen bonding) between side-chains, (iii) tapetape repulsive forces to prevent aggregation, (iv) lateral recognition between adjacent β -strands to constrain their self-assembly to one-dimension, and (v) strong adhesion of solvent to the surface of the tapes to control solubility. These criteria have been exploited to produce de novo oligopeptides which self-assemble in water into polymeric, β -sheet tapes microns in length. At peptide concentrations above 5 mg/ml, the polymeric tapes, become entangled to produce a continuous, three-dimensional network, which transforms the initially fluid solution into a homogeneous self-supporting gel [1, 2]. The primary structure of these peptides can be chosen so that the gelation of their solutions can be controlled by chemical triggers. For example, an 11-mer peptide DN1-2E (Figure 1) has been designed so that its self-assembly is responsive to pH. At pH values less than 4, i.e. pH values lower than the pKa of the glutamate side-chains, the peptide molecules self-assemble into stable β -sheet structures and form gels. In contrast, at pH values higher than the pKa of the glutamate side-chains, the electrostatic repulsive forces between negatively charged glutamate side-chains, disrupt the polymeric β -sheet structure, and convert the gel into a clear fluid. The existence of polymeric β -sheets in the gel at pH 2 is confirmed by the FTIR spectrum (Figure 2), which shows an absorption maximum at 1625 cm –1, 30 Y. Shimonishi (ed): Peptide Science – Present and Future, 30–33 © 1999 Kluwer Academic Publishers, Printed in Great Britain

31 pH 2 : stable β -sheet tape ⇒ gel

pH 7 : unstable β -sheet structure → random coil ⇒ fluid

Figure 1. DN1-2E β-sheet dimers showing electrostatic charges on the side-chains present at low/neutral pH.

Figure 2.

FT-IR spectra of DN1-2E in its gel(pH 2) and fluid(pH 7) states.

32

Figure 3. CD spectra of DN1-2E. 110 µ M peptide solution in 10 µΜ phosphoric acid pH 2 (———), 260 µM peptide solution in 10 µ M phosphate buffer pH 9 (---).

characteristic of a β -sheet. The broad peak centred on 1665 cm –1 observed in fluid solution at pH 7 is indicative of a disordered random coil peptide. CD studies in dilute solution (Figure 3) reveal a characteristic β -sheet signal at pH 2, with a minimum ellipticity at approximately 220 nm whilst at pH 9, a spectrum indicative of random coil peptide is obtained. This confirms the information obtained by FTIR. It is interesting to compare the properties of these self-assembling peptide gels with the more classical biopolymer gels such as gelatin and agarose. The elastic (G') and dissipative (G") moduli are very similar. The stress response to strain for a peptide gel remains linear up to 230% strain, compared to conventional biopolymer gels which typically break at strains of 50%. The gels also show high thermal and chemical stability, and are both biodegradable and biocompatible. Furthermore, they are found to be stable in the presence of a variety of solutes, including biological proteins. This combination of properties of the polymeric peptide tapes, coupled with the ability to engineer functionality into the polymer by peptide design, make these materials attractive for the development of a wide range of applications. For example, switching between gel and fluid states may be used for drug delivery, where the drug molecule encapsulated in the polymeric gel network is released in response to a switch in pH.

Acknowledgements We acknowledge the financial support of EPSRC and of the Royal Society of Great Britain.

33 References 1. Aggeli, A., Bell, M., Boden, N., Keen, J.N., Knowles, P.F., McLeish, T.C.B., Pitkeathly, M. and Radford, S.E., Nature, 386 (1997) 259. 2. Aggeli, A., Bell, M., Boden, N., Keen, J.N., McLeish, T.C.B., Nyrkova, I., Radford, S.E. and Semenov, A., J. Mater. Chem., 7(7) (1997) 1135.

7 Peptides containing nonnatural amino acids with functional side groups M. SISIDO Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700, Japan

Introduction Nonnatural amino acids carrying a variety of functional side groups are finding increasing roles in the peptide chemistry as well as in the protein chemistry [1]. Because of the limited conformational freedom of the peptide skeletal, the side groups of nonnatural amino acids take constrained spatial arrangement and this provides unique opportunity for the functional groups to interact cooperatively. This will be exemplified in this article with the peptides containing electron donors, acceptors, and photosensitizers that are arranged along α-helical peptides in designed orders, distances, and orientations [2]. Also, introduction of nonnatural amino acids is a promising approach to extend the diversity of the peptide combinatorial library. Amino acids carrying long alkyl chains, polar aprotic side groups, hydrogen donors, and hydrogen acceptors may extend the modes of interactions against a variety of artificial ligands Amino acids carrying metal ligands may build artificial reactive centers in the peptides. Nonnatural amino acids have been linked to tRNA and the tRNA-aa*’s have been used for site-specific incorporation of nonnatural amino acids to proteins through in vitro biosynthesis. A variety of amino acids have been shown to be incorporated into proteins [3]. Examples of nonnatural amino acids that carry functional side groups are shown in Figure 1. There are fluorescent probes, electron donors, electron acceptors, and a photo-switch. Also there are a triplet sensitizer, a stable radical, and metal ligands. Amino acids carrying nucleic bases, sugars, a crown ether, long alkyl chains are also reported. Combining these amino acids together with naturally-occurring ones, we can prepare a large diversity of peptide samples using, in many cases, automated synthesizers. In this article, peptides carrying designed sequences of electron donors, electron acceptors, and photosensitizer are described. These peptides may exemplify the efficacy of the present approach. 34 Y. Shimonishi (ed): Peptide Science – Present and Future, 34–38 © 1999 Kluwer Academic Publishers, Printed in Great Britain

35

Figure 1.

Examples for optically active nonnatural amino acids carrying functional side groups.

Results and discussion Photoinduced electron transfer in peptides containing 1-pyrenylalanine1-Naphthylalanine-p-Nitrophenylalanine sequences Helical polypeptide is a stable molecular framework along which a variety of chromophores are arranged in designed order and designed interchromophore distances and orientations. In this study, attempts were made to built an effective path for electron transfer from an excited pyrenyl group to a p nitrophenyl group. The peptides synthesized for this purpose contain L 1-pyrenylalanine as a photosensitizer, L-1-naphthylalanine as a possible superexchange mediator, and L- p -nitrophenylalanine as an acceptor that are linked to helical poly(γ -benzyl L -glutamate) chain. The polypeptide was synthesized as follows: A heptapeptide Boc–pyrAla– napAla–ntrPhe–(Glu(OBzl)] 4 –OBzl was synthesized through liquid-phase method and the Boc group was deprotected. The latter was used as an initiator of the polymerization of Glu(OBzl)NCA in DMF. The polypeptide was fractionated through Sephadex LH60 gel/DMF and high molecular weight frac-

36

tions were taken up. The polypeptide was found to take right-handed α-helical conformation from CD spectroscopy. In the polypeptide I, the three chromophoric amino acids are directly linked and the triad sequence is linked to a Glu(OBzl) 4 unit that assures that the chromophoric sequence is located inside the helical region. The orientation of the side groups were predicted from molecular mechanics calculation assuming right-handed α-helical main chain. The result is shown in Figure 2. In the polypeptide II, the three chromophores are linked through two alanine units. Since the polypeptide has been found to take α-helical conformation from CD spectroscopy, the three chromophores are expected to take face-toface orientation (Figure 3), in contrast to the case of the polypeptide I in which these three takes side-by-side orientation (Figure 2). Quenching of pyrenyl fluorescence was observed for the two polypeptides as compared with the peptide containing single isolated pyrenyl group (P).

Figure 2.

Computer-predicted conformation of the polypeptide I.

Figure 3.

Computer-predicted conformation of the polypeptide II.

37

Figure 4. Fluorescence decay curves of the polypeptide containing single pyrenylalanine unit (P, top), the polypeptide II (middle), and the polypeptide III (bottom) measured in trimethyl phosphate.

Fluorescence decay curves also indicated accelerated quenching of pyrenyl fluorescence du to electron transfer to the nitrophenyl group. The decay curves for the two polypeptides and the peptide carrying isolated pyrenyl group are compared in Figure 4. The rate constant of electron transfer is calculated from the decay curve to be 5.3 × 107 s – 1 for I and 6.4 × 10 6 s – 1 for II. The slower rate constant for the polypeptide II is interpreted mainly in terms of shorter edge-to-edge distance between pyrenyl and nitrophenyl group in the polypeptide I (9.3-9.4 Å from Figure 2) than that in the polypeptide II (11.2–11.4 Å from Figure 3). The different rate constants for the two polypeptides indicate that the side-chain orientations are rather rigidly kept during the lifetime of pyrenyl fluorescence (200 ns) as shown in Figures 2 and 3. The decay curves of the two polypeptides are almost linear at least during the initial stage. This also indicates small fluctuations during the lifetime of pyrenyl fluorescence. The semirigid conformations of chromophore-linked α-helical polypeptides have been confirmed also in the energy-transfer experiments [4]. In the two polypeptides, a mediation effect by the intervening naphthyl group was expected. However, the rates of electron transfer in the polypeptide I and II were about the same as the corresponding peptides that lack the naphthyl group. This may indicate that the electron transfer proceeds mainly through bonds and the naphthyl group that is located in the space between the pyrenyl and nitrophenyl group does not affect the electron transfer.

38 References 1. 2. 3. 4.

Sisido, M., Prog. Polym. Sci., 17 (1992) 669. Sisido, M., Adv. Photochem., 22 (1997) 197. Hohsaka, T., Ashizuka, Y., Murakami, H. and Sisido, M., J. Am. Chem. Soc., 118 (1996) 9778. Kuragaki, M. and Sisido, M., J. Phys. Chem., 100 (1996) 16019.

8 Active carbonates of N-protected amino alcohols for synthesis of carbamate pseudopeptides J.B. HALSTRØM Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark

Introduction Pseudopeptide synthesis [1] is generally laborintensive, and does not usually lend itself to automation like solid phase peptide synthesis. However, elongation of the peptide bond by CH2 O to form the carbamate structure CH2 O C O N H , which confers flexibility while retaining H-bonding properties, makes possible the use of standard Boc- or Fmoc chemistry on a conventional peptide synthesizer. The reagents for such syntheses, Boc- or Fmoc-amino alkyl N -succinimidyl carbonates, are stable, crystalline compounds, which are easily accessible by treatment of Boc- or Fmoc-amino alcohols with di(N-succinimidyl) carbonate (DSC) in the presence of a suitable tertiary base (Figure 1). Fmoc-amino alkyl N -succinimidyl carbonates were previously reported (F. Albericio, personal communication), but were prepared in situ, and used without isolation.

Results and discussion The use of 2 equivalents of DSC in dry THF and 0.5–1 equivalent of polystyrene-linked DMAP as the tertiary base was found to facilitate the synthesis of the active carbonate at 20°C. It was subsequently purified by flash chromatography on silica gel 60, and crystallized from 2-propanol or mixtures of 2-propanol and petroleum ether. So far, all active carbonates were obtained crystalline (Table 1). The use of the above products in the synthesis of carbamate pseudopeptides was assessed by acylation of di- and tripeptides in DMF at 20°C. An excess of active carbonate of 5% was used and the yields were practically quantitative. The products were homogeneous by TLC. The tri- and tetrapseudopeptides formed were analyzed by plasma desorption or electrospray mass spectrometry in the positive mode, and the masses were found to agree with theory (Table 2). Peaks corresponding to M + Na and loss of Boc and/or tBu furnished additional evidence.

Figure 1.

Synthesis of active carbonate.

39 Y. Shimonishi (ed): Peptide Science – Present and Future, 39–40 © 1999 Kluwer Academic Publishers, Printed in Great Britain

40 Table 1

Physical data of active carbonates.

OSu Carbonate of

M.P.(°C)

[α] 20 D*

Yield(%)

Boc-Gly-ol Boc- D -Phe-ol Boc- L -Leu-ol Boc- L -Pro-ol Fmoc- L -Val-ol Fmoc- L -Tyr(tBu)-ol

122–123 104–106 102–103 130–132 139–141 53–59

– + 12.5° – 20.0° – 38.9° – 6.8° – 20.6°

60 73 78 87 77 55

*c = 1 in acetone Table 2

Mass spectrometrical analysis of synthesized pseudopeptides.

Pseudopeptide

M(theory)

M(found)

Boc-Glyψ [CH 2 OCONH]lle-Val-OH Boc-D-Phe ψ[CH 2 OCONH]Val-Leu-Ser-OH Boc-Pro ψ[CH 2 OCONH]Ala-Glu-OH Boc-Leuψ[CH 2 OCONH]Leu-Gly-Gly-OH Fmoc-Val ψ[CH 2 OCONH]Tyr-Leu-OH Boc-Leuψ[CH 2OCONH]Cly-Phe-OH Fmoc-Tyr(tBu)ψ[CH 2 OCONH]Leu-Gly-OH

417.5 594.7 445.5 488.6 645.8 465.6 659.8

417.4 594.4 444.9 487.5 645.5 465.0 659.2

The synthesis of 7 carbamate pseudopeptides (Table 2) illustrates the utility of the described crystalline Boc or Fmoc-amino alkyl N -succinimidyl carbonates. These reagents could be useful tools, e.g. in probing bioactive conformations by developing carbamate pseudopeptide libraries using standard Fmoc chemistry on a standard peptide synthesizer. Other obvious uses are preparative syntheses of selected Boc or Fmoc oligocarbamates for subsequent assembly into larger structures by conventional fragment condensation procedures.

Acknowledgements Per F. Nielsen is thanked for recording and interpreting the mass spectra.

Reference 1. Spatola, A.F., In Weinstein,B. (Ed.), Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Marcel Dekker Inc., New York, 1983, pp. 267–357.

9 Convenient one-pot synthesis of cystine-containing peptides from protected peptidyl resins and its application to the synthesis of bifunctional anti-HIV compounds H. TAMAMURA, T. ISHIHARA, H. OYAKE, A. OTAKA and N. FUJII Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Introduction To date, a variety of cystine-containing peptides have been isolated from natural sources and their peptide analogs have been synthesized in order to study structure-activity relationships. Thus we wished to develop a useful synthetic methodology for the rapid preparation of cystine-containing peptides. Recently several disulfide bond-forming reactions using sulfoxide-mediated oxidations have been developed [1–3]. For example, cystine formation by a 1 h treatment of S-Acm cysteine with trimethylsilyl chloride (TMSCl)-dimethylsulfoxide (DMSO)-TFA has been demonstrated. However, prior to oxidation, S-Acm cysteine peptides must be cleaved from the corresponding peptidyl resin and all protecting groups removed except for the Acm on the Cys(Acm). Simultaneous cleavage/deprotection with disulfide bond formation in a onepot manner would be desirable in order to facilitate the synthesis of cystinecontaining peptides. In the present study, cystine-containing peptides were found to be obtained in a one-pot manner by treatment of protected peptidyl resins with TMSCl-DMSO-TFA [4].

Results and discussion In order to evaluate the TMSCl-DMSO-TFA system in a one-pot synthesis of cystine-containing peptides, we initially confirmed the complete removal of the side chain protecting groups on several Fmoc-amino acid derivatives following treatment with TMSCl-DMSO/TFA. In addition the behavior of amino acids on two peptide linkers, p -Alkoxybenzyl alcohol linker (Alko linker) [5] and 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy linker (Rink amide linker) [6] was similarly examined. Next, two model peptidyl resins [T131(OH) and T131(NH 2 ) [7], These peptides are tachyplesin analogs having anti-HIV activity. The sequence of T131 (OH) is shown in Figure 1 (R = H).] were constructed using Fmoc solid-phase techniques. Alko linker and Rink amide linker were used for the synthesis of T131 (OH) and T131 (NH 2), respectively, 41 Y. Shimonishi (ed): Peptide Science – Present and Future, 41–43 © 1999 Kluwer Academic Publishers, Printed in Great Britain

42

Figure 1. The synthesis of a bifunctional anti-HIV compound, AZT + a tachyplesin analog using the TMSC1-DMSO-TFA.

with the following side chain protecting groups (Pmc for Arg, Acm for Cys, BOc for Lys, But for Tyr). One-pot treatment with TMSC1-TFA-DMSO resulted in the fully deprotected cystine-containing peptides. In detail, the given protected T131 (OH)-resin (50 mg, 9.6 µ mol) was treated with TMSC1 (0.105 cm³, 10 equiv.) in TFA (13 cm³) in the presence of anisole (0.15 cm³) at r.t. After 1 h, DMSO (1.9 cm³, 300 equiv.) was added to the reaction mixture at 4°C and the reaction was continued for 1 h. After removal of the resin by filtration, icecold dry diethyl ether (30 cm³) was added to the filtrate. The resulting powder was collected by centrifugation and then washed three times with ice-cold dry diethyl ether (20 cm³ × 3). Each crude product exhibited a sharp main peak on analytical HPLC (data not shown). HPLC purification afforded the corresponding disulfide forms of T131 (OH) and T131(NH2 ) in satisfactory yields (T131(OH): 33%, T131(NH 2 ): 56% from the corresponding protected peptidyl resin). The yields were comparable to those obtained in parallel syntheses of both peptides by a stepwise strategy of deprotection/cleavage and disulfide bond formation using 1 M TMSBr-thioanisole/TFA and TMSC1-DMSO/TFA or iodine oxidations, respectively. The present procedure was successfully applicated to the synthesis of bifunctional anti-HIV compounds, which were composed of tachyplesin analogs (an HIV second receptor CXCR4 antagonist) linked with AZT (a reverse transcriptase inhibitor) by a succinyl linker (unstable in aqueous neutral or basic media) (Figure 1) [8]. In conclusion, the present procedure provides a convenient methodology for the rapid preparation of cystine-containing peptides, including relatively complicated compounds such as bifunctional anti-HIV compounds. References 1. (a) Fujii, N., et al., In Jung, G. and Bayer, E. (Eds) Peptides 1988, Walter de Gruyter; Berlin, 1989, pp. 58–60; (b) Otaka, A., et al., Tetrahedron Lett., 32 (1991) 1223; (c) Tam, J.P., et al, J. Am. Chem. Soc., 113 (1991) 6657.

43 2. (a) Tamamura, H., et. al., Tetrahedron Lett., 34 (1993) 4931; (b) Tamamura, H., et al., Int. J. Peptide Protein Res., 45 ( 1995) 312. 3. (a) Akaji, K., et al., J. Am. Chem. Soc., 114 ( 1992) 4137; (b) Koide, et al., Synlett, ( 1991) 345. 4. Tamamura, H., et al., J. Chem. Soc., Perkin Trans. 1, (1996) 1911. 5. Wang, S.S., J. Am. Chem. Soc., 95 (1973) 1328. 6. Rink, H., Tetrahedron Lett., 28 (1987) 3787. 7. Tamamura, H., et al., Bioorg. Med. Chem., 6 (1998) 231. 8 . Tamamura, H., et al., J. Chem. Soc., Perkin Trans. 1, (1998) 495.

10 Stereoselective synthesis of two type (E)-alkene dipeptide isosteres from a single substrate of β -aziridinyl- α, β-enoate H. TAMAMURA, M. YAMASHITA, H. MURAMATSU, T. IBUKA, A. OTAKA and N. FUJII Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Introduction Among various dipeptide isosteres, the potential of (E)-alkene isosteres as backbone replacement of amide bonds in peptides has been well documented in the past few years [1]. One of the most important factors in progress in such a pharmacodynamics of peptide-lead drugs would be strongly rested on the development of a new efficient synthetic route. The γ ,δ-epiminoα,β -unsaturated esters, which are intermediates for the synthesis of isosteres, can be easily prepared as a mixture of the cis-( E)-, cis-(Z)-, trans- (E)- and trans(Z)-isomers (1–4) from the corresponding chiral amino aldehydes. Recently we reported that Pd(0)-catalyzed equilibrated reactions of various stereoisomers of γ ,δ -epimino- α,β− enoates (1–4) could afford predominantly the cis-( E)-isomer 1 [2]. In this study, the regio- and stereo-selective ring-opening reactions of N-2,4,6-trimethylbenzenesulfonyl(Mts)-protected aziridines bearing an α,β -unsaturated ester by TFA and methanesulfonic acid (MSA) have been found. MSA treatment of the cis-(E)-enoate 1 gave the mesylate5, which could be converted into the L , D-type (E)-alkene isostere 6 via organocopper-mediated anti-S N 2' reaction [3]. In sharp contrast, treatment of the cis- (E)-isomer 1 with the organocopper reagent exclusively gave the L , L -type (E)-alkene isostere 7 according to the reported method [4]. Results and discussion Treatment of aziridine 8 with MSA (10 equiv.) in CHCl3 at r.t. for 20 min gave exclusively the mesylate 9. Subsequent reaction of the mesylate 9 with 4 molar equiv. of BnCu(CN)MgC1 • BF3 in THF at – 78°C for 30 min afforded the protected L , D -type (2S, 5S) dipeptide isostere, Mts-L -Val- ψ [( E)–CH=CH]- D Phe-OMe, 10 in 94% yield based on 8 (diastereoselection > 99 : 1). In contrast, an anti-S N 2' reaction of the cis - (E)-enoate 8 with BnCu(CN)MgCl • 2LiCl yielded the L ,L -type (2R, 5 S) isostere, Mts- L -Val- ψ [(E)–CH=CH]– L -Phe–OMe, 11 (scheme 2). An important aspect of the ring-opening reactions is the inversion of configuration at the C-γ carbon via an SN 2 mechanism. Thus, cis-(E) enoates 44 Y. Shimonishi (ed): Peptide Science – Present and Future, 44–46 © 1999 Kluwer Academic Publishers, Printed in Great Britain

45

Scheme 1

afford syn-(E)-mesylates, which are converted into L , D-type isosteres with organocopper reagents. On the other hand, cis-(E)-enoates themselves give L ,L -type isosteres with organocopper reagents. In a comparable experiment, the trans(E)-enoate 12 was treated with MSA to obtain the anti - (E)-mesylate 13, which was converted by the organocopper reagent into the L , L-type isostere 11. I n contrast, the organocopper-mediated reaction of the trans- (E)-enoate 1 2 afforded the L ,D-type isostere 10. As a result, two type isosteres were stereoselectively synthesized from either cis- or trans- (E)-enoates. Likewise, other aziridinyl enoates gave the corresponding L , D-type isosteres and the L ,L- type isosteres with the MSA/organocopper and the organocopper treatment, respectively (data not shown). The (E) geometry of the double bond and the absolute configuration of the α -alkylated carbon centers in isosteres were well characterized by ¹H NMR and CD analyses, respectively [3]. Herein, regio- and stereoselective ring-opening reactions of N-Mts-protected aziridines bearing an α , β -unsaturated ester by TFA or MSA have been found [5]. The present ring-opening reactions provide useful approaches for the stereoselective synthesis of both L,L -type (or D ,D -type) and L ,D -type (or D ,L type) (E)-alkene dipeptide isosteres from either γ,δ-cis- or trans- γ,δ -epimino(E)- α,β -unsaturated esters. Taken together, complete stereocontrol in the

Scheme 2. Reagents: a, MeSO 3H; b, BnCu(CN)MgCl • BF3 ; c, BnCu(CN)MgCl • 2LiCl.

46 synthesis of L ,L -type, L ,D -type, isosteres can be established.

D , D -type

and

D , L -type

(E)-alkene dipeptide

References 1. Kaltenbronn, J.S., et. al., J. Med. Chem., 33 (1990) 838; Tourwe, D., et. al., Int. J. Peptide Protein Res., 39 (1992) 131; for a review of syntheses of (E)-alkene dipeptide isosteres up to May 1992, see: Ibuka, T., J. Synth. Org. Chem. Jpn., 50 (1992) 953. 2. Ibuka, T., et al., J. Org. Chem., 62 (1997) 2982. 3. Ibuka, T., et. al., J. Org. Chem., 56 (1991) 4370. 4. Fujii, N., et. al., J. Chem. Soc., Perkin Trans. 1 ( 1995) 1359. 5. Tamamura, H., et. al., Chem. Commun. (1997) 2327.

11 Design and synthesis of conformationally constrained dipeptide containing the α-fluoroglycine residue Y. TAKEUCHI,* K. KIRIHARA, M. KAMEZAKI, and N. SHIBATA Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194, Japan

Introduction Since the discovery of β -fluoro- D-alanine in 1970, a compound that has potent antibacterial activity comparable to that of tetracycline and chloramphenicol, considerable attention has been given to the design and synthesis of other fluorinecontaining amino acids. Various classes of fluorinated analogs of amino acids have been synthesized to date, including, for example, α -fluoromethylated amino acids, β -fluorinated amino acids, trifluoromethylated amino acids, ω -fluorinated amino acids, and those amino acids having fluorine(s) on an aromatic ring. Although the fluorine substitution at the α-position of α-amino acids would seem necessarily to profoundly influence their chemical and biological activity, no such α -fluoro- αamino acid has been reported in the literature. This undoubtedly reflects the chemical liability of the α -haloamine moiety. In 1991, the first synthesis of fully protected α-fluoroglycines was achieved in our laboratory using the Gabriel reaction of imidodicarboxylates with ethyl bromofluroacetate as a key step. Very recently, we have reported the first synthesis of α -fluoroglycine-containing dipeptides in fully protected form. As part of our research program directed toward the synthesis of α-fluoro amino acid derivatives, we have been interested in conformationally restricted dipeptides 1 which contain α -fluoroglycine as a constituent.

The use of such peptidomimetics is a powerful tool for analyzing the active site of enzyme and the secondary structure of peptides. As potential α -fluoroglycinecontaining peptidomimetics we focused on the cyclic compounds 1 in which the N and N’ atoms of the dipeptides are linked by a carbonyl group. This design is based on strategies to stabilize the carbon-fluorine bond by virtue of the electronattracting effect of the imide unit. We describe herein the general synthesis of 1 by the Gabriel reaction of hydantoins with ethyl bromofluoroacetate. We also 47 Y. Shimonishi (ed): Peptide Science – Present and Future, 47–48 © 1998 Kluwer Academic Publishers, Printed in Great Britain

48 present the synthesis of tripeptides that contain 1 in order to demonstrate the application of our strategy to the preparation of oligopeptides 2.

Results and discussion The α -fluoroglycine-containing dipeptidomimetics 1 were synthesized as described in the scheme (vide infra). First, the Gabriel-type reaction of the readily available diphenylhydantoin was performed by using NaH in DMF, followed by the addition of ethyl bromofluoroacetate. Although the desired 1a was the predominant product, obtained in moderate yield, this procedure resulted in the formation of a mixture of 1a and dialkylated product 4 accompanied by the starting material 3. In an attempt to overcome this problem of regioselectivity in the alkylation reaction, the conditions were optimized using several bases and additives in several solvents at different temperatures. The best result was obtained in the reaction of 3 with NaH, NBu 4 Br and ethyl bromofluoroacetate in THF at room temperature to give la in 91% yield. Other hydantoins which have different substituents at the 5 position were treated under the same conditions to give 1b,c in moderate to good yields.

We next demonstrated that our pseudo-peptides 1 can be incorporated into the oligopeptide 2 by normal peptide coupling techniques. Deprotection at the C-terminus of 1b was achieved with TFA/CH2 Cl 2 . Coupling of the carboxylic acid obtained with glycine ethyl ester in the presence of DCC/HOBT furnished the tripeptide 5 in 76% yield. Finally, N-terminal chain elongation was achieved by coupling 1b with N -Boc amino acids using the mixed anhydride method to give the tripeptides 6 in good yields.

12 Screening of peptidomimetic tyrosine kinase inhibitors for inducing programmed cell death G. KÉRI, L. ÔRFI¹, F. HOLLÓSY, T. VÁNTUS, J. ÉRCHEGYI, M. IDEI, A. HORVÁTH, I. TEPLÁN, A. GAZIT³, I. PETÁK², Z. SZEGEDI² and B. SZENDE² Peptide Biochem Res Unit., Dept of Medical Chemistry, ¹Institute of Pharmaceutical Chemistry, and ² 1st Institute of Pathology Semmelweis University of Medicine, Budapest, P.O.Box 260, Hungary-1444, ³ Dept. of Biol. Chemistry, The Hebrew University of Jerusalem, Israel-91904

Introduction Recently strong efforts have been directed toward the development of small molecular tyrosine kinase inhibitors as potential antitumor drugs. In our earlier work and also in the literature it has been demonstrated that tyrosine kinase inhibitors can induce apoptosis and we also found that some tyrosine kinase inhibitors induce a different kind of programmed cell death (PCD), which was named Clarke III type PCD [1]. In the present work we have screened a novel series of our peptidomimetic tyrosine kinase inhibitors for the induction of apoptosis or this Clarke III type PCD. Results and discussion We have synthesized a big series of peptidomimetic tyrosine kinase inhibitors including a series of quinazolines, quinoxalines and oxanyl hydrazide derivatives as part of our systematically built molecular library. The compounds were characterized by HPLC, NMR and MS as it has been described previously [2]. The tyrosine kinase inhibitory activity of the compounds were tested on receptor tyrosine kinases as it was published earlier [1, 3]. A series of Tyrphostins with various chemical structures were also selected to be tested in our assays since the Clarke III type PCD was first induced by a Tyrphostin. For control purpose we also synthesized some of the best known tyrosine kinase inhibitors like the PD153035 of Fry et al. and the best pyrrolopyrimidine compound [4]. The stained histological preparations were investigated for showing morphological signs of apoptosis or Clarke III type PCD, the number of such cells were counted and the relevant PCD index was calculated. Immunohistochemical reaction using Apop-Tag kit as well as FACS analysis was performed with selected compounds in repeated experiments under the same circumstances. 49 Y. Shimonishi (ed): Peptide Science – Present and Future, 49–51 © 1999 Kluwer Academic Publishers, Printed in Great Britain

50

Figure 1. The effect of various TK inhibitors (150 µM) on changes in DNA content (sub-G1 fraction) in HT29 human colon tumor cells, measured by FACS analysis after washing out broken DNA with at pH 7.8 phosphate-citrate buffer.

Our compounds were groupped around 6 lead structures, in focused sublibraries. All of these lead structures had strong tyrosine kinase inhibitory activity. We found that several of our potent tyrosin kinase inhibitors which formed part of an activity island showed a strong apoptotic effect causing 40–90% apoptotic cell death in the 150 µM dose range, however we had some compounds in both the quinazoline and the oxanyl hydrazide derivatives which had smaller tyrosine kinase inhibitory activity but still had significant apoptosis inducing effect. We also had some compounds with significant tyrosine kinase inhibitory activity but no apoptosis inducing effect. However every tyrosine kinase inhibitor which had an IC 50 below 10 µM had a strong apoptosis inducing effect (Figure 1). There was no correlation between the tyrosine kinase inhibitory activity and the Clarke III. type PCD inducing activity of the compounds. The strong apoptosis inducing activity of several tyrosine kinase inhibitors suggest their possible use as potent antitumor agents.

References 1 . Szende, B., Kéri, Gy., Szegedi, Zs., Benedeczky, I., Csikós, A., Ôrfi, L. and Gazit, A., Cell Biology International, 19 (1995) 903.

51 2. Ôrfi, L., Kökösi, J., Szász, Gy., Kövesdi, I., Mák, M., Teplán, I. and Kéri, Gy., Bioorganic & Medicinal Chemistry, 4 (1996) 547. 3. Strawn, L.M., McMahon, G., App, H., Schreck, R., Kuchler, W.R., Longhi, M.P., Hui, T.H. Tang, C., Levitzki, A., Gazit, A., Chen, I., Kéri, Gy., Ôrfi, L., Risau, W., Flamme, I., Ullrich, A., Hirth, K.P. and Shawver, L.K., Cancer Research, 56 (1996) 3540. 4. Fry, D.W., Kraker, A.J., McMichael, A., Ambroso, L.A., Nelson, J.M., Leopold, W.R., Connors, R.W. and Bridges, A.J., Science, 265 (1994) 1093.

13 Molecular assembly of helix peptides vertically oriented on gold surface Y. MIURA¹, S. KIMURA¹, Y. IMANISHI² and J. UMEMURA³ ¹Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Yoshida Honmachi, Kyoto 606-8501, Japan ²Department of Material Science, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan ³Institute for Chemical Research, Kyoto University, Gokanosho, Uji Kyoto 611-0011, Japan

Introduction α -Helix is a major structural unit of proteins, and frequently associates with other helices to form a helix-bundle structure due to self-assembling property of helices [1]. With using the assembling property of helix peptide, we have tried to construct various artificial peptide molecular assemblies. In the present study, we report on preparation of helix-peptide thin membrane on gold substrate with the helix orientation being controlled.

Results and discussion Hydrophobic helical peptides having functional groups were synthesized, and the peptide thin layer was formed on gold surface. In order to stabilize α -helical conformation the peptides were constituted by Ala and Aib residues. The counter part for the peptides was immobilized on the gold surface by formation of self-assembled monolayer (SAM) of the functional group-terminated alkyldisulfide or alkylthiol. The SAM was, then, incubated an alcohol solution of the peptides to form peptide thin layer on gold with help of specific interaction. The conformation and orientation of the peptides held on the surface were investigated by FTIR reflection-absorption spectroscopy (FTIR-RAS), in which an absorption band with the transition moment oriented vertically to the surface is enhanced. The molecular orientation of the helix peptide was estimated by the observed ratio of the amide I and II absorbances, D obs = A obs I/A obs II, in RAS spectra. Hydrophobic helical peptides having functional groups, HA16OH, HA16B, and BA16Cr formed the peptide layer of nearly vertical orientation to the substrate. Especially, HA16OH stood up vertically on the gold substrate. On the other hand, reference peptide having no functional group, BA16B, did not form oriented layer. Therefore specific interaction between the terminal function group of the peptide and the counterpart immobilized on the gold substrate played an important role in the formation of the peptide layer. To the best of our knowledge helix-thin layers with vertically 52 Y. Shimonishi (ed): Peptide Science – Present and Future, 52–53 © 1999 Kluwer Academic Publishers, Printed in Great Britain

53

Figure 1.

Molecular structure of peptides, alkyldisulfide and alkanetiol.

Figure 2. (a) FTIR-RAS spectrum of HA160H layer formed on SAM of MUA. (b) Schematic illustration of the peptide thin layer system.

orientation against surface have not been previously described. The use of specific interaction for anchoring self-assembled monolayer to substrate will also be effective to preparation of other self-assembling systems of various compounds. Furthermore, fabrication of self-assembled monolayer such as patterning on gold substrate may be possible by immobilization of one component in a prescribed pattern and molecular recognition at complexation of the other component in the membrane preparation. Reference 1. Otoda, K., Kimura, S., Imanishi, Y., J. Chem. Soc. Perkin Trans. 1,23 (1993) 3011.

14 Gramicidin vesicles – self-assembly in water composed of gramicidin A as hydrophobic core of membrane D.-H. KIM¹ , S. KIMURA¹ , and Y. IMANISHI² ¹Department of Material Chemistry, Kyoto University, Kyoto, Japan 606-8501 ²Department of Material Science, Nara Institute of Science and Technology, Ikoma, Japan 630-0101

Introduction Peptides, known as tailorable molecules by choosing appropriate amino acid residues for the sequence or by modifying their terminal or side chains with functional groups, are considered to be promising building blocks for supramolecular assemblies [1]. Peptide molecular assemblies in water will be prepared by amphiphilic peptides which are designed to balance the hydrophilicity and hydrophobicity in the molecule. The morphology of peptide molecular assemblies should be determined by this delicate balance. One of major properties of peptide molecular assemblies is an ordered structure of the constituent peptide molecules. This point presents a striking contrast with other molecular assemblies constituted by flexible alkyl chains or synthetic polymers with random-coil conformation. In this study, gramicidin A was chosen because of its hydrophobicity and rigid helical structure [2]. Gramicidin A was conjugated with poly(ethyleneglycol) (PEO) to make the peptide an amphiphilic compound. Gramicidin A/PEO conjugate was investigated on molecular assembly formation in water. Especially, we are interested in preparation of vesicular assembly by peptides, which are named ‘peptosome’ where peptides form thin membrane to be organized into liposomal structure (Figure 1) [1].

Results and discussion Three kinds of gramicidin A/PEO conjugates were prepared with varying molecular weight of PEO. The conjugates with PEO of lower molecular weights ( 150 or 300) were not dissolved in water. However, the conjugate with PEO of molecular weight of 600 was dispersed in water. The dispersion was analyzed by dynamic light scattering to show formation of peptide molecular assembly with diameter in the range of 80–90 nm. The morphology of the aggregates was spherically observed by transmission electron microscopy (TEM). Furthermore, the aggregates were suggested to take vesicular structure by TEM observation. The vesicular structure of the aggregates was also supported by 54 Y. Shimonishi (ed): Peptide Science – Present and Future, 54–56 © 1999 Kluwer Academic Publishers, Printed in Great Britain

55

Figure 1.

Schematic illustration of peptosome.

Figure 2.

Schematic illustration of gramicidin A/PEO aggregate.

encapsulation experiment of fluorescein-labelled dextran in aggregates. In the aggregates, the gramicidin A moiety took antiparallel double-helical conformation by CD spectroscopy [3].

Conclusion Gramicidin A/PEO conjugate was dispersed in water to form spherical molecular assembly (Figure 2). The peptide molecular assembly possessed inner water

56 phase to indicate formation of peptide vesicles. Gramicidin A/PEO conjugate organized molecular thin membrane with taking antiparallel double helix. This is a first example to demonstrate clearly the formation of vesicular peptide assembly, peptosome.

References 1. Imanishi, Y., Fujita, K., Miura, Y. and Kimura, S. Supramol. Sci., 3 (1996) 13. 2. Mou, J., Czajkowsky, D.M. and Shao, Z. Biochemistry, 35 (1996) 3222-3226. 3. J.A. Killian, Biochim . Biophys. Acta, 1113 (1992) 391–425.

15 Design, synthesis, and characterization of a dimeric analog of small globular protein (SGP) in solution and its biological and channel-forming activities T. KIYOTA¹, E. MATSUMOTO¹, S. YAMASHITA², S. LEE¹ and G. SUGIHARA¹ ¹ Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka 814–800, Japan, ² Departmnent of Forestry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

Introduction To investigate insertion mechanism of membrane proteins into membranes, we have designed and synthesized a small globular protein named SGP (1) . It consists of 69 amino acid residues which have four helices; a Trp-containing short hydrophobic helix in the middle surrounded by three Tyr-containing long basic amphiphilic helices. It has not only stable globular structure in buffer solution, but also strong membrane binding ability as well as ion channelforming activity. However, its channel-forming mechanism is not understood, yet. It is assumed that the peptide will form channels by aggregating into membrane. Thus, in the present study, we will report here that design and synthesis of a dimeric analog of SGP (d-SGP) dimerized through the Cys residues of Ac–Cys–Gly–Gly-sequence added to N-termini of SGPs (Figure 1).

Results and discussion CD study of SGP and d-SGP showed that in buffer solution, both SGP and d-SGP adopted α-helix structure where helical content of d-SGP (45%) was slightly lower than that of SGP (50%), while in the presence of egg-PC liposomes helical contents of both peptides slightly increased (65% for d-SGP and 60% for SGP). SGP Ac-A-A-A-A-A-A-W-A-A-A-A-G-P-N-G-L-Y-L-K-K-K-L-L-K-K-L-L-G-N-P-G-L-K-L-Y-K-K-L-L-K-K-L-L-L-K-L-G-N-P-G-L-L-K-L-Y-K-K-L-L-K-K-L-L-K-L-L d-SGP Ac-C-G-G-(SGP) | Ac-C-G-G-(SGP) Figure 1.

Primary structure of SGP and d-SGP.

57 Y. Shimonishi (ed): Peptide Science – Present and Future, 57–58 © 1999 Kluwer Academic Publishers, Printed in Great Britain

58

Figure 2. Circular dichroism and Trp fluorescence behavior of SGP and d-SGP in difference Gu · HCl concentrations. [ SGP] = 10 µM, [d-SGP] = 5 µM Ex. wavelength; 290 nm

To understand the stability of these peptides, CD and Trp-fluorescence spectra were measured as a function of guanidine hydrochloride (Gu · HCl) concentration (Figure 2). On CD, helical contents of both peptides gradually decreased with increasing in Gu · HCl concentration. The denaturation midpoints of SGP and d-SGP were estimated 4.8 and 5.6 M Gu · HCl, respectively. These indicate that the stability of α-helix structure of d-SGP was slightly higher than that of SGP. The denaturation curves of both peptides obtained from Trp-fluorescence were almost same, indicating that stability of tertiary structure of both peptides were not so different. The interaction of peptides with liposome was studied by monitoring Trpfluorescence of both peptides with increasing concentration of neutral (egg PC) liposomes and by quenching Trp-fluorescence with that containing 1,2,bis(9,10-dibromostearoyl) phosphatidylcholine. The results show that both peptides insert their central hydrophobic core into lipid bilayers. However, the significant difference between SGP and d-SGP was not observed, indicating that both peptides interacted with lipid bilayers in a similar manner. Finally, channel formation ability was measured using planner lipid bilayer method. SGP has possessed stable cation-selective ion channel into the membrane of PS/PE (1: 2). In the present study, d-SGP also has channel-forming ability in the same lipid bilayers, but the occurrence of channel formation is considerably less than that of SGP, suggesting that the dimerization of SGP works unfavorably for channel formation. Reference 1. Lee, S., Kiyota, T., Kunitake, T., Matsumoto, E., Yamashita, S., Anzai, K and Sugihara, G., Biochemistry, 36 (1997) 3782.

16 Molecular design of cyclic peptides containing a nonnatural amino acid: novel synthetic approach to artificial ion channels K. DONOWAKI¹, H. ISHIDA², Y. INOUE1,2, Z. QI³, M. SOKABE³, M. AZUMANO 4 and S. FUTAKI 4 ¹ Department of Molecular Chemistry, Osaka University, Suita 565, Japan ² Inoue Photochirogenesis Project, ERATO, JST, Kamishinden, Toyonaka 565, Japan ³ Department of Physiology, Nagoya University School of Medicine, Japan 4 Insititute for Medicinal Resources, Tokushima University, Tokushima, Japan

Introduction Interested in the structure-function relationship of natural and synthetic peptides, we have designed, synthesized, and evaluated a series of functional peptides containing rigid non-natural amino acid moiety(ies) such as 3-aminobenzoic acid [1]. The resulting functional peptides are restricted conformationally and, if cyclic, are expected to form a central cavity. Indeed, we have recently shown that the cyclic peptides (1–4) with long acyl chains are smoothly incorporated into a bilayer membrane and function as single ion channels [2], The same synthetic strategy, though applicable to the preparation of cyclic peptides consist of different amino acid moieties with similar side chains, cannot be extended to the syntheses of more sophisticated cyclic peptides carrying, for example, peptide side chains. We wish now to propose an advanced, more versatile methodology for preparing a wide variety of functional cyclic peptides. Using this synthetic strategy, we actually synthesized two cyclic peptides (5 and 6 ) carrying cholate groups or long peptide chains, which are expected to exhibit some novel ionchannel properties upon incorporation into a membrane.

59 Y. Shimonishi (ed): Peptide Science – Present and Future, 59–60 © I999 Kluwer Academic Publishers, Printed in Great Britain

60

Figure 1.

Synthetic schemes of the artificial ion channels, for 1–4 (A) and 5–6 (B).

Results and discussion In spite of the different ring sizes and chain lengths, the cyclic peptides 1–4 unexpectedly showed virtually the same properties as single ion channels. We therefore decided to replace the acyl side chains with some other functional groups. The cyclic peptides 1–4 are synthesized by stepwise coupling of the dipeptide components having a long acyl chain (Figure 1A). This procedure is advantageous as far as the cyclic peptides having the same acyl chains are concerned. However, this strategy is not suitable for the preparation of cyclic peptides with long peptide chains such as 6 , because the amino acid bearing a long peptide side chain cannot compatible with the peptide synthesis over supported resin. To overcome this synthetic problem, we designed an improved synthetic methodology as summarized in Figure 1B. In the procedure, the cyclic peptide framework, N'-terminals of which are protected with Fmoc groups, is constructed first, and then the peptide, or other, side chains are introduced to the free N'-terminals which are regenerated by eliminating the protecting group. First we checked the methodology by synthesizing a cyclic peptide bearing cholic acid in the side chain. The introduction of cholic acid was successful to afford the cyclic peptide (5) in 11% yield. The cyclic peptide with long peptide chains (6) was also synthesized by the same procedure in 5% yield. The ionchannel properties of these novel peptides are under investigation. Acknowledgment This work was funded in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture (HI, MS), by a grant from the Japan Society for the Promotion of Sciences (MS), and by the Naito Memorial Science Foundation (HI). References 1. Ishida, H., Suga, M., Donowaki, K., Ohkubo, K., J, Org. Chem., 60 (1995) 5374, Ishida, H., Donowaki, K., Suga, M., Shimose, K., Ohkubo, K., Tetrahedron Lett., 36 (1995) 8987. 2. Ishida, H., Donowaki, K., Inoue, Y., Qi, Z., Sokabe, M., Chem. Lett., (1997) 953.

17 Structure based design and characterization of exocyclic peptidomimetics that inhibit TNFα binding to its receptor W. TAKASAKI¹, Y. KAJINO, K. KAJINO, M.I. GREENE, R. MURALI Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA ¹ Present address: Analytical and Metabolic Research Laboratories, Sankyo Co. Ltd., Shinagawa-ku, Tokyo 140, Japan

Introduction Design of biologically active agents from critical sequences and structures of the authentic macromolecules have been used to discover active antagonistic small molecules that disable the function of several different macromolecules. The peptidomimetics offer one way to overcome some of the limitations of macromolecules. In this paper, we investigated the design of antagonistic peptidomimetics against tumor necrosis factor-α (TNF α) [1]. The design is based on atomic features deduced from the crystal structures of TNFα [2] and the TNF β /TNF-receptor (I) complex [3].

Results and discussion The critical sequences were cyclized and constrained with cysteine disulfide bridges in order to preserve the predicted configuration of the adjacent turn reversals, and aromatic residues were added to the termini of the cyclic constructs to improve binding efficacy. These aromatically modified exocyclic molecules (Table 1) resembled the authentic three-dimensional structures of critical binding sites of the TNF-receptor (I). The inhibition of 125 I-labeled TNF α binding to TNF-receptor was used to evaluate the mimetics (Figure 1). Peptidomimetics derived from the binding site-5 and -8 of TNF-receptor, WP5 and 8 series, exhibited some inhibitory activities. Binding site-9 was utilized as a template after replacement of the charged residue, Glu, with Gln (WP9Q). The WP9Q showed a 2-fold higher activity than the best WP5 binding site derived mimetic. Structural analysis was next undertaken and we superimposed TNFα to TNF β complexed with the TNF-receptor(I). Modification of other residues of WP9Q led to further improvement of inhibitory activities. The best antagonist, WP9QY, showed 5 µM inhibition of IC 50 in the binding assay, and biologically inhibited TNFα mediated apoptosis (Figure 2). 61 Y: Shimonishi (ed): Peptide Science – Present and Future, 61–63 © 1999 Kluwer Academic Publishers, Printed in Great Britain

62 Table 1. Sequences of critical binding sites on TNF-receptor( I) to TNFa and exocyclic peptidomimetics derived from the receptor

Figure 1. Inhibition of 125 I-TNFα and the receptor interaction by exocyclic peptidomimetics in competitive radioreceptor assay.

Figure 2.

Inhibition of TNF α -induced cytolysis of L929 cells by the antagonistic peptides.

WP9QY is the smallest anti-TNF peptidomimetic developed to date that might be used as a lead compound for the next generation of non-peptidic

63 inhibitors. While these results support a new general approach to novel drug discovery, the study also illustrates how exocyclic peptidomimetics are useful to understand atomic aspects of ligand/receptor interactions.

References 1. Takasaki, W., Kajino, Y., Kajino, K., Murali, R. and Greene, M.I., Nature Biotechnology 15 (1997) 1266. 2. Eck, M.J. and Sprang, S.R., J. Biol. Chem. 264 (1989) 17595. 3. Banner, D.W., D’Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.J., Broger, C., Loetscher, H. and Lesslauer, W., Cell 73 (1993) 431.

18 A three-helix bundle metallopeptide destabilized at an acidic condition K. SUZUKI and T. TANAKA Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan

Introduction There is a considerable interest in the design of topologically predetermined macromolecular scaffolds. Such systems possess substantial utilities in the design of peptide-based therapeutic agents. A lot of studies have been aimed at the construction of supramolecular structures and the addition of certain functions to the constructs. One of such functions is a pH dependent conformational change. Some designed peptides which form α-helix bundle or α -helical coiled coil change their conformations in a pH dependent manner [1, 2]. These peptides form the structures only at acidic conditions but not at neutral or basic conditions. However, peptides have been rarely reported to exhibit a drastic conformational change in the opposite manner. In this study, we designed a metallopeptide which forms a three-helix bundle at a neutral pH but which is drastically destabilized at an acidic pH. Results and discussion As a model peptide forming a three helix bundle, we designed and synthesized IZ-wild (Table 1) on the basis of the previous report of GCN4-pII [3]. The circular dichroism (CD) spectrum of IZ-wild was characteristic of an α -helical coiled coil structure in aqueous solution. Apparent molecular mass of IZ-wild determined by sedimentation equilibrium was 10,699, indicating a formation of the trimer. For the determination of helix orientation, we synthesized bipyridyl-modified IZ-wild. The (1:3) molar ratio of Fe2+ to the peptide was determined in the metal/peptide complex, which had the same elution profile of gel filtration with the trimerized IZ-wild. These results suggest that the helix orientation of the peptide is parallel. Next we designed and synthesized a metallopeptide, IZ-3adH (Table 1), in which two His residues existed at the a and d positions of the heptad repeat Table 1. Sequences of synthetic peptides arranged in heptads Peptide IZ-wild IZ-3adH

YGG YGG

defgabc

defgabc

defgabc

defgabc

IEKKIEA IEKKIEA

IEKKIEA IEKKIEA

IEKKIEA HEKKHEA

IEKKIEA IEKKIEA

64 Y. Shimonishi (ed): Peptide Science – Present and Future, 64–66 © 1999 Kluwer Academic Publishers, Printed in Great Britain

65 (these are positions i and i + 4) as metal binding sites (Figure 1). IZ-3adH had a random structure in aqueous solution and 5% trifluoroethanol (TFE). However, the peptide formed α -helix in the presence of Co 2+ in 5% TFE. From Co 2 + titration of IZ-3adH, three peptide molecules bound one Co2 + ion (pH 7). In contrast to the helix formation at neutral pH, CD spectra of the peptide showed a random structure in 5% TFE buffer (pH 5) in spite of the presence of Co 2 + (Figure 2). This result indicates that the protonated His residues at the acidic pH were dissociated from the metal, causing the destabilization of the α -helix. From CD measurements described above, it was found that IZ-3adH formed an α -helical Co(IZ-3adH)3 complex at a neutral pH in 5% TFE, and that the complex is drastically destabilized at an acidic pH. However, it was not identified whether the metallopeptide formed a bundle structure. To confirm the helix bundling of the metallopeptide, we coupled acridinyl group to the N-terminus of IZ-3adH. The acridinyl-modified IZ-3adH had a typical fluorescence spectrum of unassociated acridine in 5% TFE (pH 7). In the presence of Co 2 + , however, fluorescence of the metallopeptide was quenched, indicating

Figure 1.

Schematic representation of the structure of three-helix bundle metallopeptide.

Figure 2. and 5.

CD spectra of IZ-3adH (20

µM) in the presence of Co 2+ (6.7 µM) in 5% TFE at pH 7

66 the bundle formation of the metallopeptide. A pH-change from seven to five restored the fluorescence property because of the destabilization of the α-helix bundle. These results indicate that IZ-3adH formed a three helix bundle by the metal binding to His residues at neutral pH and that the structure was destabilized at acidic pH because of the dissociation of the metal from the protonated His residues.

References 1. Graddis, T.J., Myszka, D.G. and Chaiken, I.M. Biochemistry 32 (1993) 12664–12671 2. Kohn, W.D., Monera, O.D., Kay, C.M. and Hodges, R.S. J. Biol. Chem. 270 (1995) 25495–25506 3. Harbury, P.B., Zhang, T., Kim, P.S. and Alber, T. Science 262 (1993) 1401–1407

19 The alamethicin–leucine zipper hybrid peptide: modulation of the assembling of transmembrane peptides through the extramembrane peptide segments S. FUTAKI, 1,2 M. FUKUDA,¹ M. NIWA¹ ¹ Institute for Medicinal Resources, The University of Tokushima, Tokushima 770-8505, Japan ² Institute for Chemical Research, Kyoto University, Kyoto 611-0011, Japan

Introduction Many natural membrane proteins are composed of multiple subunits which assemble through non-covalent interaction to exert the protein function. To understand the mechanisms of the control of assembling is important not only for the understanding of ways of quarterly structural formation of the membrane proteins but also for the design for the artificial functional membrane proteins. Alamethicin is known as a typical ion-channel peptide which channel is formed by a random assembling of the transmembrane peptide to show multiple open states. The leucine zipper sequence is responsible for the stable dimer formation of GCN4, a transcriptional protein, in water. Using the alamethicin-GCN4 leucine zipper hybrid peptide (Alm–LeuZ), we have demonstrated here that modulation of the assembling of transmembrane peptides could be possible through the possible recognition between extramembrane peptide segments.

Results and discussion The hybrid peptide was designed as shown in Figure 1. The alamethicin sequence was placed at the N -terminus that is connected to the GCN4 leucine zipper sequence at the C-terminus via a (Gly) 4 linker. The peptide was prepared by Fmoc-solid-phase peptide synthesis followed by the acid deprotection and

Figure 1.

Design of the alamethicin–leucine zipper hybrid peptide (Alm–LeuZ).

67 Y. Shimonishi (ed): Peptide Science – Present and Future, 67–69 © 1999 Kluwer Academic Publishers, Printed in Great Britain

68 HPLC-purification. The fidelity of the product was ascertained by time of flight mass spectrometry (TOFMS). CD spectrum of Alm–LeuZ with liposomes (0.88 mM) prepared from egg yolk phosphatidylcholine in the presence of 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4) was suggestive of an helical structure ([ θ ] 222 : – 2.5 x 10 4 deg · cm²/dmol; peptide concentration: 7 µ M). The planer lipid membrane experiment (lipid: diphytanoylphosphatidylcholine; solution: 1M KCl) demonstrated that the hybrid peptide (50 nM) showed predominantly a single open state at + 100 mV whereas alamethicin amide (Ac-UPUAUAQUVUGLUPVUUEQF-amide, 10 nM), which lacks the leucine zipper sequence in its molecule, formed a channel with multilevel openings under the similar condition (Figure 2). This fact was suggestive that the number of the assembling alamethicin was modulated through the possible intermolecular interaction among the leucine zipper sequences. The observed conductance of Alm–LeuZ coincided with that deduced for the alamethicin tetramer [1]. Also, the conductance was comparable with that of a template-assembled alamethicin tetramer [2]. The way of assembling control was both voltage and concentration dependent. At the peptide concentration of 10 nM, the open state was estimated almost unique up to + 140 mV, whereas at 50 nM, up to + 120 mV. At the higher voltages, the conductance corresponding to the tetramer became less dominant probably owing to the voltage-dependent conformational change of alamethicin molecule, and larger conductance levels became dominant. Though further study should be necessary, this system would be

Figure 2. Schematic representation of the modulation of the assembly by the leucine zipper sequence (a); Channel current of alamethicin amide (b) and Alm–LeuZ (c).

69 promising not only to understand the role of extramembrane peptide segments of natural membrane proteins in its folding and functional mechanisms but also to create artificial functional membrane proteins.

References 1. Sansom, M.S. Prog. Biophys. Molec. Biol. 55 (1991) 139. 2. Matsubara, A., Asami, K., Akagi, A. and Nishino, N. Chem. Commun. (1996) 2069.

20 BCvir: backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in non-dividing cells. A. FRIEDLER1, N. ZAKAI 2, O. KARNI 2, Y. C. BRODER 2 , L. BARAZ 3, M. KOTLER 3 , A. LOYTER 2 and C. GILON 1 2

1

Department of Organic Chemistry, Institute of Chemistry, and Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel 3 Department of Molecular Genetics, The Hebrew University Hadassah School of Medicine, Jerusalem 91120, Israel

Introduction We have recently developed a general approach for the design and synthesis of small backbone cyclic (BC) peptides which mimic the structure and/or the function of active regions in proteins. This approach is based on the concepts of backbone cyclization [1] and cycloscan [2] and was designated ‘The BC proteinomimetic’ approach [3,4]. Backbone cyclization has been previously shown to convert peptides into selective and metabolically-stable peptidomimetics with enhanced biological activity as compared to the linear parent peptide. Cycloscan is a selection method based on conformationally constraint BC peptide libraries that allows rapid detection of the most active BC peptide derived from a given sequence. In this study we examined the feasibility of the BC proteinomimetics approach for the design and synthesis of BC peptides which mimic nuclear localization signal (NLS) regions in proteins. The nuclear import of HIV-1, which is mediated by the viral matrix (MA) protein, is crucial for the productive infection of non-dividing cells.

Results and discussion A library of BC peptides was designed based on the NMR structure of HIV-1 MA [5,6] (Figure 1a). The sequence of the NLS region in this protein is -KKKYK- and it is located within the outer strand of a β -sheet. The Tyr residue was replaced by a BC building unit. The library was synthesized by the SMPS method [7]. Screening of the library was performed by estimating its ability to competitively inhibit nuclear translocation of fluorescently labeled NLS-BSA in permeabilized cells [8]. 70 Y. Shimonishi (ed): Peptide Science – Present and Future, 70–72 © 1999 Kluwer Academic Publishers, Printed in Great Britain

71

Figure 1. General structure of the backbone cyclic NLS-mimetic peptides: (a) The first library: n = 0,2,3,4,6, m = 2,3,4,5, in peptide 19 n = 6, m = 4. (b) The second library and BCvir: peptide 19.1: AA = Ser, AA2 = Gly, 19.2: AA = Leu, AA2 = Gly, 19.3: AA = Met, AA2 = Gly, 19.4: AA = no amino acid, AA2 = Gly, BCvir: AA = Leu, AA2 = Val.

In the second library, the ring size and ring position were maintained as in the best peptide from the first library (peptide 19 Figure 1). The IC 50 values of the purified peptides from this library in comparison to that of the corresponding linear parent NLS peptides were determined in an ELISA-based assay [9] and are shown in Table 1. Peptide 19.2 was found to be the most potent inhibitor, with an IC50 value of 3 µ M. In order to further stabilize its β -structure, the Gly residue was replaced by a Val which is the best β-sheet former. The structure of the resulting new peptide, designated BCvir (peptide 22), is shown in Figure 1b. As anticipated, the purified BCvir was found to be highly active and its IC50 value reached 35 nM. BCvir (100 µ g/ml) also reduced HIV-1 production by 75% in infected non-dividing cultured human T-cells (Hut 78 cells). Since the MA NLS is essential for HIV-1 replication only in non-dividing cells, the effect of BCvir was studied in aphidicolin cell-cycle arrested cells. BCvir did not affect virus production in aphidicolin-untreated dividing cells. The metabolic stability of BCvir to proteolytic cleavage was also studied. The t1/2 value of BCvir digestion by trypsin is 7 h compared to that of the linear HIV-MA NLS parent peptide which is only about 1.3h. BCvir was able to inhibit nuclear import in in vitro assay systems as well as HIV – 1 replication in infected cultured cells. In addition, BCvir is resistant to proteolysis. These properties render BCvir and similar peptides potential candidates for the development of a novel class of anti-viral drugs based on blocking nuclear import of viral genomes.

Table 1.

IC 5 0 values for selected peptides.

Peptide name

IC5 0 (µM)

Peptide sequence/structure

HIV-MA NLS SV40 large T-antigen NLS 19.2 19.1 19.4 22 (BCvir)

12 0.001 3 >1000 150 0.035

Gly–Lys–Lys–Lys–Tyr–Lys–Leu–Lys–His–Cys–NH 2 Pro–Lys–Lys–Lys–Arg–Lys–Val–Cys–NH 2 See Figure 1b See Figure 1b See Figure 1b See Figure 1b

72 References: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Gilon, C., Halle, D., Chorev, M., Selinger, Z. and Byk, G., Biopolymers 31 (1991) 745–750. Gilon, C. (1997) submitted. Kasher, R., Bitan, G., Halloun, C. and Gilon, C., Lett in Pep Res 5 (1997) 101–103. Kasher, R., Barda Y. and Gilon, C., manuscript in preparation. Massiah, M.A., Starich, M.R., Paschall, C., Summers, M.F., Christensen, A.M. and Sundquist, W.I., J. Mol. Biol. 244 (1994) 198–223. Matthews, S., Barlow, P., Boyd, J., Barton, G., Russell, R., Mills, H., Cunningham, M., Meyers N., Burns, N., Clark, N., Kingsman, S., Kingsman, A. and Campbell, I., Nature 370 (1994) 666–668. Houghten, R., Proc. Natl. Acad. Sci. USA 82 (1985) 5131–5135. Broder, Y.C., Stanhill, A., Zakai, N., Friedler, A., Gilon C. and Loyter A., FEBS lett. 412 (1997) 535–539. Melchior, F., Paschal, B., Evance, J. and Gerace, L., J. Cell. Biol. 123(6)(1993), 1649–1659. Friedler, A., Zakai, N., Karni, O., Broder, Y.C., Baraz, L., Kotler, M., Loyter, A. and Gilon, C., Biochemistry 37 (1988) 5616–5622.

21 Self-assembling homopolymeric peptide tapes in moderately polar organic solvents A. AGGELI, M. BELL, R. OWENS, A. SMITH and N. BODEN Centre for Self-Organising Molecular Systems, The University of Leeds, Leeds, LS2 9JT, UK

Introduction The aim of our research is to exploit biological-like peptide self-assembly to engineer novel, functional, supramolecular structures [1, 2]. Here we show how a 24-residue peptide can be designed to self-assemble into homopolymeric tapes, microns in length, in moderately polar solvents.

Results and discussion K 2 4 ( N H 2 -KLEALYVLGFFGFFTLGIMLSYIR-COOH), modelled on the single transmembrane domain of IsK protein, was synthesised using standard automated solid phase synthesis protocols [3]. In very dilute solutions in methanol (below 5 µM), the peptide exists in a predominantly monomeric random coil state, as revealed by Circular Dichroism (CD) spectroscopy. Above a critical peptide concentration, ca 5 µM, the peptide molecules self-assemble into β -sheets, as observed by a characteristic minimum ellipticity at 216 nm in the CD spectra [2]. Clear, thermostable, self-supporting gels are obtained for peptide concentations in excess of 0.2% v/v (mM concentation). The polymeric structures giving rise to the gel network can be directly visualised by atomic force microscopy (AFM) and transmission electron microscopy (TEM). A typical AFM image of a K24 β -sheet polymer from a methanolic peptide gel is shown in Figure 1. The polymer is seen to be several micrometers in length, and to twist around itself to produce a distinctive loop. The thickness of the polymer (z axis, perpendicular to the plane of the image) was measured to be 0.5–1 nm, corresponding to the thickness of a single β sheet. The width of the polymer, ca 8 nm, was found to be in agreement with the expected length of a 24-residue peptide in a fully extended β -strand conformation. These observations show that the peptide β -strands self-assemble in register, with their long axes perpendicular to the long axis of the tapes (cross β structure). Specific peptide–peptide interactions are believed to drive the alignment of the peptide molecules during self-assembly. This is necessary in order to produce one-dimensional self-assembling elongated polymers, as opposed to 73 Y. Shimonishi (ed): Peptide Science – Present and Future, 73–75 © 1999 Kluwer Academic Publishers, Printed in Great Britain

74

Figure 1.

AFM image of a self-assembled polymeric β-sheet tape of K24 in methanol.

infinitely extending two-dimensional β -sheets. Polarised FTIR studies with partially oriented K24 polymers are consistent with the above proposed model. Tapes formed by self-assembly of the predominantly hydrophobic peptide K24 are soluble in moderately polar organic solvents such as methanol or ethanol/water mixtures, but not in water. In contrast, tapes formed by more polar peptides are soluble in highly polar solvents, including water [see abstract O-02 and reference 1]. Solubility is governed by the wetting properties of the tape surfaces: good solvents are essential to obtain stable dispersions of tapes.

Acknowledgements We acknowledge the financial support of EPSRC and of The Royal Society of Great Britain.

References 1. Aggeli, A., Bell, M., Boden, N., Keen, J.N., Knowles, P.F., McLeish, T.C.B., Pitkeathly, M. and Radford, S.E., Nature, 386, (1997) 259.

75 2. Aggeli, A., Bell, M., Boden, N., Keen, J.N., McLeish, T.C.B., Nyrkova, I., Radford, S.E. and Semenov, A., J. Mater. Chem., 7(7), (1997) 1135. 3. Aggeli, A., Boden, N., Cheng, Y.L., Findlay, J.B.C., Knowles, P.F., Kovatchev, P., Turnbull, P.J.H., Horvath, L. and Marsh, D., Biochemistry, 35, (1996) 16213.

22

Substrate specificities of artificial flavo-enzymes K.-Y. TOMIZAKI¹, Y. KAKIMOTO¹, H. AKISADA² and N. NISHINO³ ¹Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan ² Department of Environmental Chemistry, Kyushu Kyoritsu University, Kitakyushu 807-8585, Japan ³ Institute for Fundamental Research of Organic Chemistry, Kyushu University, Fukuoka 812-8581, Japan

Introduction Previously, we have synthesized a 53-peptide containing Cys(Fla) residues at the hydrophobic side of the four α -helix bundle structure (Figure 1). The 53-peptide exhibited the catalytic activity toward the oxidation of 1-benzyl-1,4-dihydronicotinamide (Bzl-NAH) in the presence of sodium dodecylsulfate (SDS) [1]. Instead of the addition of a surfactant, we modified the substrate with various alkyl chains to evaluate the specificity. In addition, in order to compare the positioning of Cys(Fla) on the amphiphilic α -helix, we synthesized the related 53-peptides with different sequences for Cys (Figure 1A).

Figure 1. (A) Amino acid sequences of four 53-flavo-peptides. (B) Top view of four α-helix bundle structure. (C) Structure of Cys( Fla). (D) Reaction scheme of flavin activity.

76 Y. Shimonishi (ed): Peptide Science – Present and Future, 76–77 © 1999 Kluwer Academic Publishers, Printed in Great Britain

77 Table 1. Kinetic parameters for the oxidation of 1-alkyl-NAHs by 53-fiavo-peptides. The decrease in absorbance of 1-alkyl-NAHs at 360 nm was measured in air-saturated 0.1 M Tris HCl buffer, pH 7.5 containing 12% methanol at 25C as reported method [2]. [1-alkyl-NAH] = 20–250 µ M and [53-flavo-peptide] = 0.84 µΜ. C8-NAH

C4-NAH

C12-NAH

k cat Peptides sec – 1

KM µΜ

k cat / K M M–1 sec – 1

k cat sec –1

KM µΜ

k cat / K M –1 M –1 sec

kcat sec –1

KM µΜ

kcat /KM M – 1 sec –1

53Fla-1 53Fla-2 53Fla-3 53(Fla) 2

250 200 220 210

770 850 810 2500

0.22 0.18 0.16 0.53

90 110 90 90

2500 1600 1700 6000

0.24 0.23 0.20 0.83

30 30 30 30

8300 7200 6500 25000

0.19 0.17 0.18 0.53

Results and Discussion The kinetic parameters for the oxidation of 1-alkyl-NAHs by 53-flavo-peptides are given in Table 1. With longer alkyl chain of substrates, kc a t /K M values of four 53-flavo-peptides were greater. These results suggest that the accommodation of 1-alkyl-NAHs depends on their hydrophobicity. 53Fla-1, -2, and -3, which contain single flavin moiety at different positions, showed similar activities. 53(Fla) 2 could catalyze the oxidation about 3 times more efficiently than 53Fla-1, -2, and -3. This fact implies the cooperative effect of two flavin moieties in the oxidation of 1-alkyl-NAHs.

References 1. Mihara, H., Tomizaki, K., Nishino, N. and Fujimoto, T., Chem. Lett., (1993) 1533. 2. Levine, H.L. and Kaiser, E.T., J. Am. Chem. Soc., 100 (1978) 7670.

23 Catalytic activities of an artificial protein with designed loops K-Y. TOMIZAKI¹, D. OKA¹, Y. KAKIMOTO¹ and N. NISHINO² ¹Department of Applied chemistry, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan ²Institute for Fundamental Research of Organic Chemistry, Kyuhu University, Fukuoka 812-8581, Japan

Introduction The bundle structure of four amphiphilic α -helix segments of polypeptide is one of the conformational models of natural proteins. In order to evolve the artificial proteins by de novo design, the catalytic function must be incorporated in futher rational design for enzymatic activity. We designed two loops between the amphiphilic α-helix segment to introduce His and Glu residues: as shown in Figure 1. The amphiphilic α -helix segments are designed according to our previous report as ELLKALAELLK [1]. In the loop segments between the first and second α -helices and the third and forth α -helices, respectively, the same amino acid sequence GGHGEGG was incorporated. The loops are expected to be arranged antipallally to organize the side chains of His and Glu in tetrahedral 2+ positioning in the presence of Zn (Figure 1B).

Results and discussion The 63-peptide (HEHE) showed 63% α -helicity in 0.1 M phosphate buffer, pH 7.5. The α -helicity increased slightly in >60% methanol solution, probably the second structure being more stabilized at the loop segments. The addition

Figure 1. Structure of 63-peptide (HEHE): (A) Amino acid sequence, (B) Illustration of four α-helix bundle structure with two designed loops, and (C) Possible tetrahedral coordination to zinc ion.

78 Y. Shimonishi (ed): Peptide Science – Present and Future, 78–79

© 1999 Kluwer Academic Publishers, Printed in Great Britain

79 Table 1. Kinetic parameters on the hydrolysis of decnoyl–Glu–ONp and Boc–Glu–ONp by 63-peptide (HEHE). The increase in absorbance of p-nitrophenolate anion at 400 nm was measured in 0.1 M phosphate buffer, pH 7.5, at 25°C.

Substrate

kc a t (s -1 × 10 3)

kM ( µ M)

1.6

360

4.5

3.7

220

17.4

kc a t / k M (M -1 s -1 )

of Zn 2+ induced the decrease in ellipticity at 208 nm; 208/222 ratio, 1.23 to 1.03. This fact suggests that the four α -helices are packed more tightly in bundle structure by fastening the interaction between two loops: as illustrated in Figure 1B. The 63-peptide (HEHE) was subjected to the hydrolysis of p -nitrophenyl esters. The hydrolysis of decanoyl–Glu–ONp was obviously enhanced with the addition of 63-peptide (HEHE); the k cat / K M value was determined as 17.4 M –1 s – 1 . Boc–Glu–ONp was less susceptible to the 63-peptide (HEHE) probably due to the lack of hydrophobic acyl group; the k cat / KM value, –1 4.5 M s –1. Broo et al. reported the second-order-rate constant at pH 6.5 as about 0.1 M – 1 s –1 for the acyl-transfer reaction from mono-p-nitrophenyl fumarate to the ornithine residue in the model peptide [2].

References 1. Mihara, H., Tanaka, Y., Fujimoto, T. and Nishino, N., J. Chem. Soc. Parkin Trans 2, (1995) 1915. 2. Broo, K., Brive, L., Lundh, A.-C., Ahlberg, P. and Baltzer, L., J. Am. Chem. Soc., (1996) 8l72.

24 Catalytic activities of an artificial heme enzyme K. KANEKO¹, K-Y. TOMIZAKI¹ and N. NISHINO² ¹ Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan ² Institute f or Fundamental Research of Organic Chemistry, Kyushu University, Fukuoka 812-8581, Japan

Introduction Heme proteins, which have an iron porphyrin at the active site, such as peroxidase and cytochrome P-450 catalyze various oxidation reactions efficiently. Hemes have been successfully introduced to polypeptides non-covalently [1] or covalently [2] so far. We also attempted to functionalize the de novo designed polypeptide, by introducing iron porphyrins. We utilized cysteine residues to combine synthetic porphyrin rings covalently via thioether linkages. In the present study, we demonstrate the peroxidase-like activity with an amphiphilic βαβα - structure with two iron porphyrins as an artificial heme enzyme.

Results and discussion A 49-peptide containing two iron porphyrins anchored on Cys residues (Figure 1) was synthesized. The 49-peptide (βαβα (Por(Fe)) 2) formed βαβα structure with porphyrin rings face to face in the hydrophobic core. The peroxidase activity of βαβα (Por(Fe)) 2 was examined toward the oxidation of 10-N-methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (MCDP) as a substrate in the presence of N - α-myristoyl-His-NH 2 . Various oxidants such a s H2 O 2, t -butyl hydroperoxide (BHP), cumene hydroperoxide (CHP), m-chloroperbenzoic acid (CPB), and linoleic acid peroxide (LPO) were employed and compared with each other in oxidation efficiency.

Figure 1. Design of βαβα(Por(Fe)) 2 . (a) Sequence of 49-peptide. (b) βαβα -Strand segment. (c)α Helix segment. (d) Por(Fe) bound to cysteine. (e) Illustration of βαβα(Por(Fe))2 .

80 Y: Shimonishi (ed): Peptide Science – Present and Future, 80–81 © 1999 Kluwer Academic Publishers, Printed in Great Britain

81 Table 1. Kinetic parameters for the oxidation of MCDP by βαβα (Por(Fe)) 2. [βαβα (Por(Fe)) 2 ] = 1 mM, [myristoyl–His–NH 2 ] = 20 µΜ, [MCDP] = 100 mM, 20 mM Tris • HCl buffer, pH 7.2 Oxidant

KM / µ M

k

H 2O2 BHP CHP LPO CPB

12500 1000 400 83 56

1.3 x 10 – 1 9.2 x 10 – 2 2.8 x 10 – 1 4.2 1.3

cat /sec

–1

k cat /KM /M – 1sec –1 1.0 9.2 7.0 5.1 2.3

x x x x x

10 ¹ 10 ¹ 10² 10 4 10 4

Figure 2. Dependence of the initial rate of oxidation of MCDP on methanol content. –
– : i n : in the absence of myristoyl–His–NH 2. the presence of 20 mM myristoyl–His–NH 2,

Table 1 shows kinetic parameters, K M, k cat , and Kcat /KM for various oxidants. H 2 O 2 did not give high activity. In contrast, hydrophobic oxidants, in particular LPO, were much more effective. Therefore, it is assumed that they are preferentially accommodated into the hydrophobic space of βαβα (Por(Fe))2 . The influences of methanol and guanidine hydrochloride on the oxidation of MCDP with CPB were examined in relation to the conformational changes in the reaction media. The β α β α (Por(Fe)) 2 exhibited the highest peroxidase activity in 80% methanol solution (6 times higher than that in H 2 O; Figure 2). Furthermore, the addition of guanidine hydrochloride enhanced the activity significantly. These results suggest that a pair of porphyrin rings are so tightly packed in the hydrophobic core of the peptide that the substrate and oxidant are interfered to have access to the heme.

References 1. (a) Robertson, D.E., Farid, R.S., Moser, CC., Urbauer, J.L., Mulholland, S.E., Pidikiti, R., Lear, J.D., Wand, A.J., DeGrado, W.F. and Dutton, P.L., Nature, 368 (1994) 425. (b) Choma, C.T., Lear, J.D., Nelson, MJ., Dutton, P.L., Robertson, D.E. and DeGrado, W.F., J. Am. Chem. Soc., 116 (1994) 856. 2. Nastri, F., Lombardi, A., Morelli G., Maglio, O., D’Auria, G., Pedone, C. and Pavone, V., Chem. Eur. J., 3 (1997) 340.

25 A novel dioxopiperazine suitable as a β-turn constraint J.S. DAVIES¹, M. STELMACH-DIDDAMS¹, R. FROMENTIN¹, A. HOWELLS¹ and R. COTTON² ¹ Department of Chemistry, University of Wales, Swansea, SA2 8PP, UK ² Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, SK10 4TG UK

Introduction In peptidomimetics, a number of rigid building blocks have been devised, as β -turn mimetics [1] and there are a number of ongoing studies on the use of templates [2] to induce β -sheet structures. In attempting to design a stabiliser which fits tightly at the β -turn without steric hindrance, and also preserves the ‘polar’ backbone, we speculated by molecular modelling, that creating a dioxopiperazine, as in Figure 1 should be considered. In essence it inserts a reverse cis-amide bond on to the peptide backbone.

Results and discussion Dioxopiperazines are not known for difficulties in their formation, as they are often undesirable by-products, but the example (1) proved difficult. Retrosynthetic analysis revealed that the starting materials had to be an orthogonally protected α ,α -diamino acid, coupled to a β -keto system. The former was synthesised as ZNHCH(NHBoc)COOH [3] from ZNHCH(CONH2 )CO 2 E t using an [I,I-bis(trifluoroacetoxy)iodo] benzene-induced Hofmann rearrangement in the presence of t-butanol. Coupling to diethylaminomalonate produced the dipeptide precursor. Yet having reached compound (3) in the Scheme, conventional cyclisation did not occur, unless the carboxyl group was activated (as an ONSu ester). An nmr study on (1) showed some stereoselection had

Figure 1.

A β turn/ β -sheet stabiliser unit.

82 Y. Shimonishi (ed): Peptide Science –Present and Future, 82–84 © 1999 Kluwer Academic Publishers, Printed in Great Britain

83 Table 1.

Summary of energy calculations computed for model dipeptides (4)

Energy E = ( H DKP –H X–OH )–( H dipeptide ) where H = Heat of formation A= B= C= A=

Option Chirality E kcal/mol

CHMe 2 , X = Me CHMe 2 , X= benzotriazole NHBoc, X= Me NHBoc, X = benzotriazole D

C

B

A LD

LL

LD

LL

LL

DL

LL

DL

– 0.980

0.270

–8.890

–7.500

3.780

4.170

–7.110

–6.570

occurred during cyclisation, as only the cis form was identified. A previous study [4] has suggested that an enhancement in the cis / trans ratio could be influenced by involvement of the side-chain.

(i) H 2 /Pd/C

(ii) When R¹ = R² = alkyl – no reaction; when R¹ = Et, R² = ONSu – 45% yield

The reluctance of dipeptides (3) to cyclise intrigued us, and initiated empirical energy calculations [5] on cyclisations of different combinations of the dipeptide (4). Only when the energy is negative is the reaction thermodynamically possible. From the table it can be seen that an active ester is very important (compare options A/C with B/D). The effect of BocNH- is to reduce the nucleophilicity of neighbouring atoms. The calculations are not subtle enough to explain chiral preferences. The results have been corroborated by a synthetic study of peptides A–D which showed that the benzotriazole ester D was required to yield dioxopiperazine (4) at room temperature.

Acknowledgements We are grateful to the EPSRC for studentships (MS-D and AH), and for a CASE Award from Zeneca Pharmaceuticals.

References 1. Friedinger, R.M., Perlow, D.S. and Veber, D.F., J. Org. Chem. 47 (1982) 104; Nagai, U. and Sato, K., Tetrahedron Lett. 26 (1985) 647; Hinds, M.G., Richards, N.G. and Robinson, J.A., J.Chem.Soc.Chem.Commun. (1988) 1447. 2. Nowick, J.S., Smith, E.M. and Pairish, M., Chem. Soc. Reviews. (1996) 401.

84 3 . Bock, M.G., Dipardo, R.M. and Friedinger, R.M., J. Org. Chem., 51(1986) 3718. 4 . Naraoka, H. and Harada, K., J. Chem. Soc., Perkin I (1986) 1557. 5 . MOPAC 93 JJP (Stewart and Fujitsu Ltd., Tokyo), obtained from QCPE, Dept. of Chemistry, Indiana Univ. Bloomington, Indiana 47,405 USA.

26 Amphiphilic β-structure formed by oligopeptides composed of alternating hydrophilic and hydrophobic residues S. ONO, K. YAMAUCHI, Y. SUGISE, T. TSUTSUMI, T. OKADA, T. FUJII and T. YOSHIMURA Department of Chemical and Biochemical Engineering, Faculty of Engineering, Toyama University, Toyama 930, Japan

Introduction β-Structure is commonly observed in natural proteins. However, the general comprehension of this secondary structure lags behind that of α -helical structure. This is due to the lack of a well-defined peptide model system for the investigations of β -structure, as described in the literatures [1]. We have designed and synthesized acidic model peptides 1 and 2 composed of alternating hydrophilic and hydrophobic amino acid residues (Figure 1) and investigated their pH-dependent behavior in aqueous solution using circular dichroism (CD) and fluorescence methods [2, 3]. As a result of the previous works, it was found that the model peptide 1 forms β -structural conformation depending on pH but not peptide 2. This finding suggests that the Trp residue introduced into peptide 2 affects unfavorably the formation of pH-dependent β-structure. Thus, to examine the effect of hydrophobic amino acid residues on the formation of pH-dependent β-structure, we synthesized model peptides 3–6 (Figure 1) containing Ala, Leu, Ile, and Phe residues other than peptides 1 and 2, and their β -structure forming abilities in aqueous solution and in phospholipid bilayer were evaluated using CD and fluorescence methods.

Results and discussion Peptides 1–6 were synthesized by the solution method using Boc-chemistry. The final products were purified by reverse-phase HPLC and established by Ac–Ser–Val–Glu–Val–Ser–Val–Glu–Val–NHCH3 Ac–Ser–Val–Glu–Val–Ser–Trp–Glu–Val–NHCH3 Ac–Ser–Val–Glu–Val–Ser–Ala–Glu–Val–NHCH3 Ac–Ser–Val–Glu–Val–Ser–Leu–Glu–Val–NHCH3 Ac–Ser–Val–Glu–Val–Ser–Ile–Glu–Val–NHCH3 Ac–Ser–Val–Glu–Val–Ser–Phe–Glu–Val–NHCH3 Figure 1.

Anionic amphiphilic β -structural model peptides.

85 Y. Shimonishi (ed): Peptide Science – Present and Future, 85–87 © 1999 Kluwer Academic Publishers, Printed in Great Britain

1 2 3 4 5 6

86 matrix assisted laser desorption ionization time of flight mass spectrometry. The peptide concentrations were determined by a quantitative amino acid analysis. CD spectra for peptides 1–6 were measured at peptide concentrations of 25, 50 and 100 µ M in 5 mM Hepes (pH 4.5–7.4). The strongest β -structure forming ability was observed for peptide 5 containing a Ile residue (Figure 2A). The β -structural content increased both with lowering pH and increasing peptide concentration. Peptides 1 and 4 containing Val and Leu residues, respectively, also took β -structural conformation depending on pH and peptide concentration (data not shown). However, peptides 2 and 3 containing Trp and Ala residues, respectively, took random structure under the all conditions tested (data not shown). Interestingly, peptide 6 containing a Phe residue showed CD spectra with a minimum (208 nm) and a shoulder (around 223 nm) at pH 4.5, suggesting a formation of α -helical conformation (data not shown). Comparing pH-dependencies for these peptides on their β -structural contents, it was concluded that the β -structure forming abilities of peptides 5, 1, 4, and 2 and 3 decrease in that order. CD spectra for these peptides in the presence of cationic phospholipid liposomes (egg-york phosphatidylcholine-sphingosine (3:1)) were also measured at various pH values (4.5–7.4) and at various peptide concentrations (25–100 µ M). Peptide 3 took a β -structural conformation in the presence of the phospholipid liposomes with lowering pH (Figure 2B), although it took a random structure in solution even at low pH. Similar induction of β-structural conformation by the addition of the cationic liposomes was observed for the all model peptides (data not shown). More detailed analysis to examine their β -strucure forming abilities in phospholipid bilayer should be required.

Figure 2. (A) pH-Dependent CD changes at 218 nm for peptide 5 in 5 mM Hepes at various peptide concentrations; (B) CD spectra for peptide 3 in the presence of the cationic liposomes. The concentrations of peptide and lipid were 100 and 900 µΜ, respectively.

87 References 1. For example: Choo, D.W., Schneider, J.P., Graciani, N.R and Kelly, J.W., Macromolecules, 29 (1996) 355; Schneider, J.P. and Kelly. J.W., Chem. Rev., 95 (1995) 2169; Tuchscherer, G. and Mutter, M., J. Peptide Science, 1(1995) 3. 2. Ono, S., Tazaki, K., Yaka, K., Ohta, M. and Kato, T., Peptides: Chemistry and Biology (Proceedings of the 12th American Peptide Symposium), ESCOM, Leiden, 1992, pp. 292–294 3. Yamamoto, Y., Ono, S., Sakai, Y., Yoshimura, T., Shimasaki, C. and Tsukurimichi, E., Peptide Chemistry 1994, Protein Research Foundation, Osaka, 1995, pp. 473–476.

27 Design of cyclic antimicrobial peptides with low hemolytic activity L.H. KONDEJEWSKI¹ M. JELOKHANI-NIARAKI¹, E.J. PRENNER¹, R.N.A.H. LEWIS¹, R.N. McELHANEY¹, R.E.W. HANCOCK² and R.S. HODGES¹ ¹ The Protein Engineering Network of Centres of Excellence and Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada ² The Canadian Bacterial Diseases Network, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada

Introduction We have utilized the cyclic decapeptide gramicidin S (GS) as the basis for our design of novel antimicrobial peptides. Results from our previous study [1] suggested that the lack of β -sheet structure and/or amphipathicity in cyclic GS analogs appeared to be responsible for conferring a high microbial specificity (a high therapeutic index) onto the peptides. In the present study we have utilized a highly amphipathic β -sheet peptide, GS14, as the basis of our design of peptides possessing high antimicrobial activity coupled with low hemolytic activity (a high therapeutic index). We have carried out systematic enantiomeric substitutions within the framework of GS14 to first define the optimum structural framework leading to desirable biological properties. Second, working within this structural framework, we have optimized the biological properties of the peptides by modulation of the effective hydrophobicity.

Results and discussion GS14, cyclo(Val–Lys–Leu–Lys–Val–dTyr–Pro–Leu–Lys–Val–Lys–Leu–dTyr– Pro), and its fourteen diastereomers were synthesized and purified as reported previously [1]. We have confirmed by both CD and NMR spectroscopy that GS14 exists in a β -sheet conformation silmilar to that of GS (data not shown). The alternating hydrophobic-basic residue pattern within the β-sheet framework of GS14 confers a high degree of amphipathicity to the molecule, with a hydrophobic face consisting of six hydrophobic residues (Val and Leu) and a basic face consisting of four Lys residues. Unlike GS14, all diastereomers exhibited CD and NMR spectra more typical of disordered structures, indicating that the β -sheet conformation was disrupted by the incorporation of a single enantiomeric substitution within the framework of GS14 (data not shown). 88 Y. Shimonishi (ed): Peptide Science – Present and Future, 88–91 © 1999 Kluwer Academic Publishers, Printed in Great Britain

89 GS14 and the diastereomers were resolved by reversed-phase HPLC, with all diastereomers eluting at shorter retention times compared to the parent molecule (data not shown). The diastereomers have the same sequence and therefore also the same intrinsic hydrophobicity as GS14. Any differences in retention time are therefore due to differences in their effective hydrophobicity, or in other words, the ability of the peptide to form a hydrophobic prefered binding domain for interaction with the hydrophobic surface of the HPLC matrix. Retention time on reversed-phase HPLC of the diastereomers is therefore a measure of the ability of the peptide to achieve an amphipathic nature in the presence of a hydrophobic surface. Binding studies utilizing bacterial-derived lipopolysaccharide (LPS) showed there was a direct correlation between LPS binding affinity and retention time on reversed-phase HPLC, with those diastereomers possessing the greatest retention times also exhibiting the greatest LPS affinity (data not shown). The biological activities of GS14 and the diastereomers were assessed as reported [1,2], and a clear relationship between retention time on reversed-phase HPLC and hemolytic activity was found. Those diastereomers which had the lowest retention times also exhibited the least hemolytic activity, indicating that high amphipathicity contributes to high hemolytic activity (data not shown). GS14 was found to exhibit essentially no antimicrobial activity against gram negative and gram positive microorganisms as well as yeast. This coupled with the very high hemolytic activity of GS14 resulted in this peptide possessing an extremely low antimicrobial specificity (therapeutic index). The diastereomers were found to exhibit a wide range of activities, with some possessing activities similar to GS14, to complete opposite activity profiles. As with hemolytic activity, there was a clear relationship between antimicrobial activity and retention time on reversed-phase HPLC, indicating that the least amphipathic peptides possess the most desirable antimicrobial activities. The high antimicrobial activities coupled with low hemolytic activities in those diastereomers with the lowest amphipathicity gave a number of analogs very favourable therapeutic indicies. The best analog, GS14K4, showed a 7000-fold increase in therapeutic index compared to GS14. It appears that the high LPS binding affinity of those GS14 diastereomers with high amphipathicity may be responsible for low antimicrobial activity. The high affinity to components of the outer membrane may decrease the ability of these peptides to penetrate to, and accumulate at their site of action on the inner membrane. A similar mechanism may be responsible for low antimicrobial activity against Gram positive microorganisms, where highly amphipathic molecules such as GS14 bind the outer surface (peptidoglycan layer) of Gram positive microorganisms with such high affinity which prevents their diffusion to their site of action at the inner membrane. The reasons for the decreased hemolytic activity of certain diastereomers on the other hand are not clear at this time. Peptide-induced formation of nonlamellar phases may be an important mechanism by which GS-like peptides cause membrane

90 lysis [3]. Our findings with GS14 diastereomers indicate that peptide-induced formation of nonlamellar phases correlates well with amphipathicity, with those diastereomers possessing a greater effective hydrophobicity also being more effective at inducing nonlamellar phases in zwitterionic lipids (data not shown). However, it is not clear whether this trend is attributable to variations in the peptides’ intrinsic capacity to induce the formation of nonlamellar phases or to variations in their capacity to partition into lipid membranes. The GS14 diastereomer possessing the highest therapeutic index was found to be GS14K4, an analog containing a D-Lys substitution in position 4 of GS14. This peptide was found to possess the optimum amphipathicity due to structural perturbation. Under the assumption that this optimum structure is retained (see below), the hydrophobic residues of GS14K4 were systematically replaced to create analogs which were either less hydrophobic or more hydrophobic than GS14K4 (Table 1). These substitutions were designed to optimize the intrinsic hydrophobicity of GS14K4. As well as exhibiting similar CD spectra, a linear correlation (R = 1.0) was found between predicted [4] and observed retention times on reversed-phase HPLC, indicating that all hydrophobicity analogs possessed essentially similar disrupted β -sheet conformations as GS14K4 (data not shown). As shown in Table 1, the analogs displayed a wide range of hydrophobicities. There was a linear correlation (R = 0.99) between the affinity for bacterial LPS and the amphipathicity of these analogs, with those possessing greater effective hydrophobicity also binding LPS with higher affinity (data not shown). The amphipathicity of the analogs also plays a role in hemolytic activity, with hemolytic activity dropping off sharply in all analogs having a lower retention time than GS14K4 (Table 1). Generally, antimicrobial activity against Gram negative microorganisms and yeast did not increase further with increasing hydrophobicity over that of GS14K4, although small increases in activity were seen with the majority of Gram positive microorganisms tested. The antimicrobial activty and specificity (therapeutic index) of GS14K4 hydrophobicity analogs against representative Gram negative and Gram positive microorganisms are shown in Figure 1. It is apparent that antimicrobial activity is greatly reduced below a certain critical amphipathicity value. Due to the reduced hemolytic activity, Table 1.

Linear sequences and properties of cyclic GS14K4 hydrophobicity analogs

Peptide

Sequence a

Y2/F2 V3/L3 V3/L3 Y2/F2 GS14K4 V3/A3 L3/A3 V3L3/A6

LKLKLFPLKLKLFP – – – LKLKLYPLKLKLYP – – – VKLKVFPLKVKLFP – – – VKLKVYPLKVKLYP – – – AKLKAYPLKAKLYP – – – VKAKVYPAKVKAYP – – – AKAKAYPAKAKAYP –

a

– –

Underlined residues represent D-amino acids.

Retention time (min.)

Hemolytic activity (µg/ml)

46.2 42.5 42.3 38.9 32.2 28.6 21.7

13 25 40 200 >800 >800 >800

91

Figure 1. Antimicrobial and hemolytic activities of GS14K4 hydrophobicity analogs. Antimicrobial activity (filled circles) and therapeutic index (open circles) are shown for A) Salmonella typhinurium C610 and B) Enterococcus faecalis ATCC 29212. Shaded boxes represent a therapeutic window within which lie peptides possessing optimum therapeutic indices for these microorganisms.

the therapeutic index of peptide V3/A3 is greater than that of GS14K4 for the Gram negative microorganism (Figure 1A) and marks an improvement in biological properties. For Gram positive microorganisms, GS14K4 represents the peptide with the greatest specificity due to the decreased antimicrobial activities of analogs which are less hydrophobic (Figure 1B). In both cases, however, a therapeutic window can be defined where both antimicrobial and hemolytic activities are optimized for a particular microorganism. Through smaller incremental changes in hydrophobicity it is likely that activity and specificity can be further optimized.

References 1. Kondejewski, L.H., Farmer, S.W., Wishart, D.S., Kay, C.M., Hancock, R.E.W, and Hodges, R.S., J. Biol. Chem., 271(1996) 25261. 2. Kondejewski, L.H., Farmer, S.W., Wishart, D.S., Hancock, R.E.W. and Hodges, R.S. Int. J. Peptide Prot. Res. 47 (1996) 460. 3. Prenner, E.J., Lewis, R.N.A.H., Neuman,K.C., Gruner, S.M., Kondejewski, L.H., Hodges, R.S. and McElhaney, R.N., Biochem. 36 (1997) 7906. 4. Guo, D., Mant, C.T., Taneja, A.K., Parker, J.M.R. and Hodges, R.S. J. Chromatogr., 359 (1986) 499.

28 Synthesis and characterization of linear and cyclic oligopeptides of 5-alkyl-substituted 3-aminobenzoic acids K. ENDO, H. TAKAHASHI, T. KURUSU, K. KAJI and A. SIDA Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan

Introduction Cyclic oligopeptides 1 have been designed and synthesized [1] from unnatural benzoic amino acids 2 [2]. A substituent X could be one of such functional groups as those of natural amino acid residues. The other substituent R is to modify the hydro or lipophilicity of 1 and also to restrict its flexibility. The peptide 1 may be reasonably rigid conformationally, and thus appropriate to quantitatively assess the roles of individual amino acid residues in the enzyme and coenzyme reactions.

Results and Discussion 5-Alkyl-3-aminobenzoic acids (2, X = CH2 CH2 OCH3 ) were prepared from 4-alkyldihydrocinnamic acids (5). Condensation of cuminaldehyde (3) and malonic acid, followed by hydrogenation yielded, on one hand, 4-isopropyldihydrocinnamic acid (5, R = i -Pr). Catalytic hydrogenation and Friedel-Crafts acylation of ethyl cinnamate (4), on the other hand, afforded

Figure 1.

Cyclic oligopeptides from unnatural benzoic amino acids.

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93

Figure 2.

Synthetic pathways of unnatural benzoic amino acids.

ethyl 4-decynoyldihydrocinnamate, which was further reduced and hydrolyzed to furnish 4-decyldihydrocinnamic acid (5, R = n -Dec). Both dihydrocinnamic acids (5, R = i-Pr and n -Dec) were converted to 7-substituted indanones and finally to 5-alkyl substituted-3-amino-2-methoxyethylbenzoic acids (2a), v i a 3-nitrolactones (6). The amino acids 2a are soluble in many polar organic solvents and solubilities of the decyl derivative in non-polar organic solvent such as n -hexane is greatly improved (0.5 mg/ml) compared with that of the isopropyl analog (98%). Each peptide was identified by MALDI-TOFMS and amino acid analysis. Circular dichroism (CD) spectra at the amide region showed that the peptides, His 6 -6L and -8L, took an α -helix structure in Tris HCl buffer (pH 7.4) with or without the heme. UV-VIS spectra of the heme were converted from the high-spin to the low-spin, which corresponded to the 6-coordinate form upon the addition of peptides (Figure 2A). These results indicated that both the peptides were able to bind the heme in the buffer. Furthermore, at the Soret band of heme in the presence of peptides, the strong induced CD spectra were observed with a positive peak at the Soret band and a negative peak at around 390 nm (Figure 2B). These suggested that the heme was highly oriented in the self-assembled peptides. Next, we examined a self-association state of the peptides by size exclusion chromatography. In the absence of heme, both peptides were assembled in an oligomeric state in the aqueous solution [ca. 6 mers, apparent molecular weight (MWa) = 30kDa]. When the peptide solutions containing heme were chromatographed, the heme remained to bind the peptides, suggesting that the heme tightly bound the peptides. Furthermore, the addition of heme induced a higher self-association state composed of more than 15 peptide molecules and heme (MWa > 70 kDa). Dynamic light scattering measurements also indicated that the diameters of the self-assembled peptide particles with heme were in the range of 150–180 and 80–100 nm for His6 -6L and -8L, respectively. The heme seemed to play a role as an assembling linkage between the peptide

Figure 2.

UV-VIS (A) and CD (B) spectra of heme bound to His6 - 8 L .

112 molecules through ligations. In conclusion, self-assembly of the amphiphilic α-helix peptides was accomplished by the heme binding.

References 1. McDermott, G., Prince, S.M., Freer, A.A., Hawthornthwaite-Lawless, A.M., Papiz, M.Z., Cogdell, R.J. and Isaacs, N.W., Nature, 374 (1995) 517. 2. Gibney, B.R., Rabanal, F., Skalicky, J.J., Wand, A.J. and Dutton, P.L., J. Am. Chem. Soc., 119 (1997) 2323. 3. Sakamoto, S., Sakurai, S., Ueno, A. and Mihara, H., J. Chem. Soc., Chem. Commun. (1997) 1221. 4. Rau., H.K. and Haehnel, W., Ber. Bunsenges. Phys. Chem., 100 (1996) 2052. 5. Mihara, H., Tomizaki, K., Fujimoto, T., Sakamoto, S., Aoyagi, H. and Nishino, N., Chem. Lett. (1996) 187. 6 . Mihara, H., Haruta, Y., Sakamoto, S., Nishino, N. and Aoyagi, H., Chem. Lett. (1996) 1. 7. D’Auria, G., Maglio, O., Nastri, F., Lombardi, A., Mazzeo, M., Morelli, G., Paolillo, L., Pedone, C. and Pavone, V., Chem. Eur. J., 3 (1997) 350.

35 Construction of porphyrin-binding peptides mimicking anti-heme monoclonal antibody M. TAKAHASHI1, A. UENO1, T. UDA2 and H. MIHARA1 ¹ Department of Bioengineering, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226 Japan ² School of Biosciences, Hiroshima Prefectural University, Nanatuka-cho, Shoubara, Hiroshima 727 Japan

Introduction An antigen binding site of antibody (complementarity determining region; CDR) might have some structural or sequential factors to bind antigen. It has been reported that synthetic peptides derived from CDRs sequences have binding properties similar to the intact antibody [1, 2]. On the basis of CDR sequence and structure of monoclonal antibody against heme (Fe III-protoporphyrin IX), we attempted to construct new type of porphyrin-binding peptides. The interaction between porphyrins and synthetic peptides was studied by absorption, fluorescence and circular dichroism (CD) spectroscopies.

Results and discussion It has been revealed that the heavy chain CDR2 (CDRH-2) of anti-heme monoclonal antibody, 2H5, performs the most important role in the recognition of heme [3]. Furthermore, it is known that there are only small repertories of main-chain conformations of CDRs (canonical structures [4]). According to the sequential and structural data in PDB, we predicted CDRH-2 structure of 2H5 as a β -strand-loop- β -strand (β -hairpin; Figure 1). On the basis of the above information, we synthesized peptides based on CDRH-2 of 2H5. These

Figure 1.

Predicted structure of CDRH-2 of 2H5 and sequences of synthetic peptides.

113 Y. Shimonishi (ed): Peptide Science – Present and Future, 113–114 © 1999 Kluwer Academic Publishers, Printed in Great Britain

114 Table 1. Binding constants (M – 1) of peptides with TCPP calculated from absorbance change of TCPP at 415 nm Py20C4 3.8 × 10

Py20C6 6

1.4 ×

10 6

Py20C8 9.4 × 10

Py20L 4

2.3 × 10

Py12C4 5

6.8 ×

105

Py12CL 4.8 × 10 5

peptides were modified with a pyrene (Py) moiety to detect the porphyrin binding by the spectroscopic measurements. An intramolecular disulfide bond was introduced to restrict the conformation. Shorter peptides, Py12C4 and Py12CL were also synthesized to examine necessity of the number of amino acids (Figure 1). We used m e s o-tetrakis(4-carboxyphenyl)porphyrin (TCPP) as ‘antigen’ to detect the interaction with peptides effectively. When these peptides were added to TCPP solution, remarkable hypochromicity and red shift of the Soret band of TCPP at 415 nm were detected. This change of spectra, however, was not detected by the addition of 1-pyreneacetic acid. This means that these peptides interact with TCPP. The binding constants of each peptide with TCPP were calculated from the decrease of absorbance at 415 nm (Table 1). As shown in Table 1, Py20C4 bound TCPP most effectively. Moreover, Py12C4 was not inferior to Py20C4 so much, suggesting that residues of C-terminus are not necessary to bind the porphyrin. Fluorescence intensity of pyrene in the peptides was remarkably decreased by the addition of TCPP. The binding constants calculated from variations of fluorescent intensity of pyrene at 378 nm were almost comparable to those from the absorption data (Table 1). We also measured CD spectra to obtain the structural information of these peptides. CD spectra of Py20C4, Py20C6, and Py20C8 seemed to indicate random structures. In contrast, the spectrum of Py12C4 suggested that the peptide formed a β -turn structure. Furthermore, CD spectrum at the Soret band of TCPP in the presence of Py20C4 showed the positive Cotton effects. This effect was not clearly detected with Py20C6 or Py20C8, but weaker one was detected with Py12C4. These effects might be related to the results from absorption and fluorescence measurements that Py20C4 has the most strong binding affinity to TCPP.

References 1. Saragovi, H.U., Fitzpatrick, D., Raktabutr, A., Nakanishi, H., Kahn, M. and Greene, M.I., Science 253 (1991) 792. 2. Smythe, M.L. and von Itzstein, M., J. Am. Chem. Soc. 116 (1994) 2725. 3. Uda, T., Okawa, Y., Umenobu, T., Hifumi, E. and Ogino, K., Chem. Lett. 11 (1993) 1923. 4. Chothia, C., Lesk, A.M., Gherardi, E., Tomlinson, I.M., Walter, G., Marks, J.D., Llewelyn, M.B. and Winter, G., J. Mol. Biol. 227 (1992) 799.

36 Construction of cyclodextrin-peptide conjugates and their molecular sensing properties S. MATSUMURA, S. SAKAMOTO, A. UENO and H. MIHARA Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226, Japan

Introduction Recent advances in de novo peptide design have provided the approaches in construction of various structural and functional motifs [1]. Nevertheless, it still remains difficulty to construct a hydrophobic pocket to catch small molecules in defined structures of peptides. To introduce a binding pocket on peptides, a cyclodextrin (CDx) would be a potent compound. Cyclodextrins are well-known host compounds and have been developed as fluorescence or absorption change indicators for various guests by the modification with appropriate probes [2, 3]. In the present study, we designed and synthesized a variety of α -helical peptides bearing a β -CDx as a binding site and a dansyl unit (Dns) as a fluorophore (Figure 1). The conformational and molecular sensing properties of the peptides with β -CDx and Dns in various positions were investigated.

Results and discussion The 17-peptide, originally designed by Marqusee and Baldwin [4], has three Glu/Lys pairs to form salt bridges in an α -helical form. On the peptide another pair of Glu and Lys residues were deployed at the 4th and 8th (EK3, EK3R) or 8th and 15th (EK6, EK6R) positions substituted for Ala to combine β - C D and Dns selectively on the side chains of these residues. The peptides were synthesized by the Fmoc solid-phase method, purified with reversed-phase HPLC, and identified by MALDI-TOFMS. Circular dichroism studies revealed that all designed peptides took an α -helical structure in a neutral buffer. EK3R and EK6R showed a higher helicity (90%) compared with EK3, EK6, and reference EK (50–60%). The

Figure 1. Designed structure of peptides conjugated with β-cyclodextrin and dansyl group.

115 Y. Shimonishi (ed): Peptide Science – Present and Future, 115–116 © 1999 Kluwer Academic Publishers, Printed in Great Britain

116

Figure 2. (A) Fluorescence emission spectra of peptides with various concentrations of 1-AdOH. [Peptide] = 10 µ M, in 20 mM Tris HCl buffer (pH 7.4), 25°C. (a) EK3R. (b) EK6. (B) Effect of αhelical structure on the sensing ability. [Peptide] = 8 µM, [1-AdOH] = 80 µ M, 25°C.

addition of 1-adamantanol (1-AdOH) as an exogenous guest, which has a high affinity for β -CDx, decreased the helicity in the cases of EK3R and EK6R. The fluorescence spectra of Dns in the peptides confirmed the intramolecular inclusion complexation of β -CDx and Dns (Figure 2A). EK3R and EK6R emitted more intensively ( λ em = 520 nm) than EK3 and EK6 did (λem = 526 nm, 530 nm, respectively). These results indicate that the complexation between βCDx and Dns is restricted on the peptides depending on their positions, resulting in the higher α-helical structure of EK3R and EK6R. The fluorescence intensity was decreased remarkably upon the addition of exogenous guests (Figure 2A). Thus, the sensitivity factors of peptides for various guests were examined using the decrease of fluorescence intensity in the presence of guest molecules. Although all the peptides had similar binding behaviors for each guest, each peptide showed a characteristic sensitivity. EK6 and EK6R showed the 4- to 20-fold higher binding constants for various guests compared to EK3 and EK3R, indicating that the sensitivity was dependent on the flexibility of the complex between β -CDx and Dns. Additionally, guanidine hydrochloride denaturation studies revealed that the sensing ability of peptides was decreased along with the decay of peptide secondary structure (Figure 2B). These results suggest that the binding and sensing abilities for the exogenous guests are controlled by the orientation of β-CDx and Dns which is regulated by peptide design.

References 1. Betz, S.F., Bryson, J.W. and DeGrado, W.F., Curr. Opin. Struct. Biol., 5 (1995) 457. 2. Ikeda, H., Nakamura, M., Ise, N., Oguma, N., Nakamura, A., Ikeda, T., Toda, F. and Ueno, A., J. Am. Chem. Soc., 118 (1996) 10980. 3. Aoyagi, T., Nakamura, A., Ikeda, H., Ikeda, T., Mihara, H. and Ueno, A., Anal. Chem., 69 (1997) 659. 4. Marqusee, S. and Baldwin, R.L., Proc. Natl. Acad. Sci. USA, 84 (1987) 8898.

37 Interaction of bundled Ser-rich peptides with phospholipid bilayer K. YOSHIDA, N. OHMORI, T. NIIDOME, T. HATAKEYAMA and H. AOYAGI Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852-8521, Japan

Introduction Design of bundled peptides has become of interest in recent year, because such peptides are useful not only as models of natural proteins but also as functional materials. We previously reported that an amphiphilic α -helical 24 mer peptide 4 6 showed strong activity toward phospholipid membrane [1]. Bundled peptides, 4α-4 6 composed of 4 fragments of the 46 chains and 4α-4 6 S in which 24 Ser residues are present instead of the Leu and Ala residues in 4α-4 6 , had much stronger membrane perturbation activity than 46 [2]. In the present study, we synthesized more hydrophilic bundled peptides containing 36 Ser residues, [Trp²]- and [Trp 12 ]-4α-4 6 S9, and examined their properties.

Results and discussion The structures of the synthetic peptides are shown in Figure 1. Tryptophan was introduced to monitor the peptide-lipid interaction. The CD measurement indicated that the peptides had α -helicity in a buffer solution (pH 7.4) and in the presence of DPPC vesicles. The helical content decreased with increasing the content of the Ser residue in the peptides. In the presence of DPPC/DPPG (3/1) vesicles, correct determination could not be done because of the turbidity owing to vesicle aggregation induced by the peptides. The membrane perturbation activity of the peptides was evaluated by the carboxyfluorescein (CF)

Figure 1.

The structures of [Trp²]- and [Trp12 ]-4 α-4 6 S 9 .

117 Y. Shimonishi (ed): Peptide Science – Present and Future, 117–118 © 1999 Kluwer Academic Publishers, Printed in Great Britain

118 leakage measurement. In the presence of DPPC and DPPC/DPPG (3/1) vesicles at 25°C, 4α-4 6 generally showed stronger activity than the Ser-containing peptides, indicating that hydrophobic interaction of the peptides with lipid is important for membrane perturbation. Since these peptides showed a moderate perturbation activity at low peptide concentration in the presence of DPPC/DPPG(3/1) vesicles, the electrostatic interaction of the cationic peptides with anionic DPPG may also contribute to the membrane perturbation. The leakage petterns at 50°C were much different from those at 25°C. The CF leakage reached to 100% at low peptide concentration. It is supposed that the hydrophobic region of the bundled peptides easily interacts with the less ordered methylene chains of lipids to penetrate into the membrane and disturb it. The membrane fusion activities of the peptides were determined and the results similar to those found in the CF leakage experiment were obtained. The binding study of the peptides with 1-anilino-8-naphthalenesulfonate (ANS) showed that the ANS fluorescence intensities increased with increase in the peptide concentrations. This means that the peptides formed hydrophobic pocket inside the structure formed by bundling. The binding constants were 3.7 × 1 0 4 and 4.2 × 10 4 M -1 for [Trp²]-and [Trp 12 ]-4 α-4 6 S9, respectively. The fluorescence properties of the Trp residues in the peptides were furthermore examined. No noticeable difference between the maximum wavelengths of the both peptides in the presence and absence of DPPC vesicles was observed, indicating that none of the Trp² and Trp12 residues were embedded in membrane. It is likely that [Trp²]- and [Trp12 ]-4α-4 6 S9 might lie on the surface of membrane, while 4α-4 6 S is thought to be perpendicularly embedded in membrane [2].

References 1. Agawa, Y., Lee, S., Ono, S., Aoyagi, H., Ohno, M., Taniguchi, T., Anzai, K. and Kirino, Y., J. Biol. Chem., 266 (1991) 20218. 2. Sakamoto, S., Mihara, H., Matsuo, E., Niidome, T., Anzai, K., Kirino, Y. and Aoyagi, H., Bull. Chem. Soc. Jpn., 68 (1995) 2931.

38 Topographically designed analogues of c [ D -Pen², Pen5 , Nle6 ] deltorphin I with stereoisomers of β -MePhe or β -iPrPhe in position 3 A. MISICKA 1,3,4 , J. LIN¹, K. HOSOHATA², H.I. YAMAMURA², P. DAVIS², F. PORRECA² and V.J. HRUBY¹ ¹ Departments of Chemistry and ²Pharmacology, University of Arizona, Tucson, AZ 85721, USA ³ Department of Chemistry, Warsaw University, 02-093 Warsaw, Poland 4 Medical Research Centre, Polish Academy of Science, 02-106 Warsaw, Poland

Introduction In an effort to determine the effect of the side chain conformational restriction on opioid receptor selectivity, cyclic disulfide-containing, δ opioid receptor selective deltorphin I analogue, c[D -Pen², Pen5 , Nle6 ] deltorphin I [1], was futher modified by all four stereo-isomers of the unnatural amino acids, respectively β -methylphenylalanine or β -isopropylphenylalanine in position 3. Theoretical study of the conformational space available for these constrained amino acids show that incorporation of β -MePhe into peptide chain has an effect on side chain rotamer population, where incorporation of β -iPr–Phe restricts or bias χ¹ and χ ², and may affect backbone φ, ψ tortion angles [2].

Results and discussion The asymmetric synthesis of all stereoisomers of β -MePhe and β -iPrPhe used in this work, were previously described [3, 4]. All of the analogues of cyclic analogue of deltorphin I were synthesized on MBHA resin, purified and characterized. The potency and selectivity of the synthesized analogues were evaluated by receptor binding asaays in the rat brain using [³H]CTOP (µ. ligand) and [³H][pClPhe4]DPDPE (δ ligand) (Table 1). The substitution of a β -MePhe or β -iPrPhe for Phe³ in cyclic analogues of deltorphin I have variable effects on the synthesuzed analogues depending mainly on the stereoisomer introduced. For β -MePhe³ isomers, both the 2S (L configuration) containing analogues show slightly better potency compare to the lead peptide, and the second one (2S,3R) is the most selective compound from the studied series. Both isomers with the 2R configuration are 10- to 20-fold less potent to the parent compound. The results are different for the β -iPrPhe³ analogues. Only one of them with 2S,3R configuration retains the high potency, but its selectivity is much reduced 119 Y. Shimonishi (ed): Peptide Science – Present and Future, 119–120 © 1999 Kluwer Academic Publishers, Printed in Great Britain

120 Table 1. Binding affinities of cyclic disulfide 2–5 deltorphin I analogues with diastereoisomers of β-MePhe or β-iPrPhe in position 3.

compared to the lead compound. Surprisingly the other analogue with 2S configuration is 10-fold less potent. Both of the synthesized β-iPrPhe³ analogues show much less selectivity compare to the respective β-MePhe³ analogues. The results demonstrate that topographical modifications of the side chain conformation of the critical structural moieties can significantly modulate both potency and receptor selectivity for ligands that have multiple sites of biological activity.

Acknowledgements This work has been supported by NIDA grant (VJH) and Chem. Dept., Warsaw University (AM).

References 1. Misicka, A., Lipkowski, A.W., Horvath, R., Davis, P., Yamamura, H.I., Porreca, F. and Hruby, V.J.,J. Med. Chem., 37 (1994) 141. 2. Hruby, V.J., Li, G., Haskell-Luevano, C. and Shenderovich, M., Biopolymers, 43 (1997) 219. 3. Dharanipragada, R., VanHulle, K., Bannister, A., Bear, S., Kennedy. L. and Hruby, V.J., Tetrahedron, 48 (1992) 4733. 4. Liao, S. and Hruby, V.J., Tetrahedron Letters, 37 ( 1996) 1563.

39 Engineering novel mini-proteins by the transfer of active sites to small disulfide stabilized natural scaffolds C. VITA, E. DRAKOPOULOU, J. VIZZAVONA, P. GARNIER and A. MÉNEZ CEA, Départment d’Ingénierie et d’Etudes des Protéines, C.E. Saclay, 91190 Gif-sur-Yvette, France

Introduction We have recently proposed a strategy to engineer novel active mini-proteins by the transfer of active sites to the small disulfide-stabilized scaffold of scorpion toxins [1, 2]. The chosen scaffold represents a structural motif evolutionary conserved in all scorpion toxins, insect defensins and is formed by an antiparallel β -sheet joined to an α -helix by three disulfide bonds [3]. According to the proposed strategy, we engineered a metal-binding protein [1] and a curaremimetic protein [4], which were obtained by transfer, within the β-sheet of the scorpion charybdotoxin, of the essential residues of the metal binding site of carbonic anhydrase and of the central core of the curaremimetic site of a snake neurotoxin, respectively. The two chimeric mini-proteins (37 and 31 AA, respectively) expressed the expected functionality even if the affinity for the target ligand (metals in the first case, acetylcholine receptor, in the second) was lower than that of the parent protein. Structure resolution by ¹H NMR [1, 5] revealed that the chimerae still preserved the original α / β fold of the scorpion toxin and that the transferred site maintained a native-like conformation. Here we report the engineering of a CD4 mimetic obtained by transferring an exposed β -hairpin (CDR2) of the human CD4, the principal receptor for HIV-1 infection [6], to the β -sheet of a truncated version (27 AA) of the scorpion scyllatoxin [1]. Results and discussion The new CD4 chimera, CD4M (Figure 1A), was obtained by solid phase synthesis, by using Fmoc-AA and HBTU coupling and purified by reversephase HPLC. The purified chimera is correctly folded, as assessed by CD and ¹H NMR analysis, is able to inhibit the CD4-gp120 interaction with an IC 50 of 2.5.10 –5 M (Figure 1B) and to protect peripheral blood lymphocytes from infection by HIV-1. The transferred site of the mini-CD4 was probed by the alanine scanning approach: this analysis clearly indicates a different role of each of the transferred residues and points to a Phe residue, present at the 121 Y. Shimonishi (ed): Peptide Science – Present and Future, 121–122 © 1999 Kluwer Academic Publishers, Printed in Great Britain

122

Figure 1. A. Sequence in one letter code of the scorpion scyllatoxin (ScTx), the CD4 mimetic (CD4M) and the CDR2-like region of human CD4. B. Inhibition of gp120 binding to recombinantCD4, coated on microtiter plate, by CD4M (
), soluble CD4 (∆) and the linear CD4M peptide () . The mouse anti-gp120 mAb NEA (NEN) was used for gp120 detection.

apex of the β -hairpin, as a ‘hot spot’ of the chimera active surface, in agreement with the literature data on mutagenesis of recombinant CD4 [6]. This and previous examples demonstrate that the transfer of functional sites to stable small presentation scaffolds represents a means to reproduce the ‘hot spot’ of the active surface of natural proteins. Such small, stable and functional miniproteins may be of great interest as tools to study the role of biologically relevant functional sites and as leads in the development of new drugs: designing new drugs on the basis of these smaller molecules will be easier that starting from the much larger natural proteins.

Acknowledgments We thank the Fondation pour la Recherche Medicale, Paris, France for a fellowship to E.D.

References 1. 2. 3. 4.

Vita, C., Roumestand, C., Toma, F. and Ménez, A., Proc. Natl. Acad. Sci. USA, 92 (1995) 6404. Vita, C., Curr. Opin. Biotechnol., 8 (1997) 429. Bontems, F., Roumestand, C., Gilquin, B., Ménez, A. and Toma, F., Science, 254 (1991) 1521. Drakopoulou, E., Zinn-Justin, S., Guenneugues, M., Gilquin, B., Ménez, A. and Vita, C., J. Biol. Chem., 271(1996) 11979. 5. Zinn-Justin, S., Guenneugues, M., Drakopoulou, E., Gilquin, B., Vita, C. and Ménez, A., Biochemistry, 35 (1996) 8535. 6. Sweet, R.W., Truneh, A. and Hendrickson, W.A., Curr. Opin. Struct. Biol., 2 (1991) 622.

40 Preparation of heterocyclic positional scanning combinatorial libraries from peptides R.A. HOUGHTEN, S.E. BLONDELLE, C. DOOLEY, J.-P. MEYER, J.M. OSTRESH and A. NEFZI Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA

Introduction The rapid parallel synthesis of large numbers of individual compounds or mixtures of compounds is now a widely used means to prepare combinatorial libraries of all types, including peptidomimetics, oligonucleotides, oligosaccharides and small organic compounds [1–3]. The design and development of strategies for the synthesis of individual and mixture-based combinatorial libraries of substituted heterocyclic compounds has been an area of intense research over the last 5 years. This family of compounds offer a high degree of structural diversity and have proven to be broadly and economically useful as therapeutic agents. One area of our research focuses on the synthesis and design of heterocyclic compounds using amino acids and peptidomimetics as starting materials [4]. Case studies illustrating the synthesis and identification of individual, highly active heterocyclic compounds are presented.

Results and discussion Through the application of the ‘libraries from libraries’ approach [4], modified dipeptides have been successfully used for the generation of a variety of heterocyclic compounds, such as hydantoin, thiohydantoin, cyclic urea, and cyclic thiourea combinatorial libraries [5,6]. Dipeptide synthetic combinatorial libraries (SCLs) having two positions of diversity have been selected as starting material for the solid phase synthesis of hydantoins and thiohydantoins. The reaction of the terminal amino group of resin-bound dipeptide with triphosgene or thiophosgene led to the intermediate isocyanate or thioisocyanate, which further reacted intramolecularly with the peptide amide bond to form the five-membered ring hydantoin or thiohydantoin in good yields (Figure 1). The successful solid phase synthesis of numerous individual compounds prepared as controls in the final library led us to devise a library containing approximately 5,832 hydantoins and thiohydantoins. The library comprised 72 mixtures (R¹ position), each containing 81 individual compounds (R² position). 123 Y. Shimonishi (ed): Peptide Science – Present and Future, 123–127 © 1999 Kluwer Academic Publishers, Printed in Great Britain

124

Figure 1.

Solid phase synthesis of hydantoins and thiohydantoins from resin-bound dipeptide.

In Figure 2 we show the LC-MS of the hydantoin A obtained from phenylalanine and valine (expected mass: 289), which is representative of the purities obtained for the compounds incorporated into the library. The hydantoin library was examined in a sigma competition binding assay using [ 3 H] pentazocine. The screening led to the identification of individual compounds showing excellent binding affinity (IC 50 values in the 100 nM range). Starting from a selectively monoalkylated resin-bound acylated dipeptide, the complete reduction of the amide carbonyls with diborane in THF yielded a triamine SCL having two available secondary amine functionalities. The treatment of this triamine SCL with carbonyldiimidazole or thiocarbonyldiimidazole affords the corresponding cyclic urea and cyclic thiourea in good yield and high purity (Figure 3). Following the initial synthesis of individual control compounds and using 37 amino acids for the first site of diversity, 40 amino acids for the second site, and 80 carboxylic acids for the third site, four separate positional scanning SCLs [7] were generated from the starting N-alkylated dipeptide SCLs having either a methyl or benzyl group on the C-terminal amide [each containing 118,400 (37 × 40 × 80) compounds]. These libraries were assayed for their ability to inhibit Candida albicans growth, which is a common opportunistic fungi responsible for infections, and it is the fungal infection most frequently associated with HIV-positive patients. Following the screening, four sets of individual compounds were prepared. Each set corresponded to all possible combinations of the building blocks defining the most active mixtures at each position of the given library. A total of eight N-benzyl amino cyclic thioureas, twelve N -methyl aminocyclic thioureas and twelve N-benzyl amino cyclic ureas were synthesized and assayed in a similar manner. Greater activities were found for the N-benzylated compounds relative to the N-methylated compounds (MIC values of the most active compounds varied from 8 to 64 mg/ml, and 64 to 125 mg/ml, respectively). Representative active compounds are the benzylated cyclic ureas, derived from L -asparagine, L -norleucine and cyclohexylacetic acid, and derived from D -cyclohexylalanine, L -norleucine and p-dimethylaminobenzoic acid (Figure 4).

125

Figure 2.

LC-MS spectra of hydantoin A.

Similar to the synthesis of cyclic ureas and thioureas, the treatment of a resin-bound N-acyl dipeptide with diborane affords a triamine having three available secondary amine functionalities. Following the cyclization with thiocarbonyldiimidazole, intermediate cyclic thioureas were formed initially, as described above (Figure 3). In contrast to previous synthetic routes, the presence of third secondary amines allowed the reaction to proceed through the formation of a highly active intermediate, which cyclized to afford a protonated bicyclic guanidine in good yield and high purity. Using 49 amino acids for the first site of diversity, 51 amino acids for the second site, and 41 carboxylic acids for the third, a library of 102,459

126

Figure 3.

Solid-phase synthesis of cyclic ureas and cyclic thioureas.

Figure 4.

Structures of two representative antifungal N-benzyl cyclic ureas.

(49 × 51 × 41) compounds has been synthesized in the positional scanning format. The screening of this library in a radio receptor assay selective for the kappa opiate receptor led to the identification of individual compounds showing excellent binding affinity (IC 5 0 value < 100 nM). Finally, the use of mixtures of compounds in the positional scanning SCL format versus the use of individual compound arrays clearly saves costs and time in the identification of highly active and specific individual heterocyclic compounds as shown above. A wide range of other heterocyclic pharmacophores and their corresponding SCLs are being prepared from resin-bound peptide SCLs. Acknowledgements The authors thank Eileen Weiler for editorial assistance. This work was funded by NSF grant CHE-9520142 and Trega Biosciences, Inc., San Diego, Calif. References 1. Houghten, R.A., Pinilla, C., Blondelle, S.E., Appel, J.R., Dooley, C.T., Cuervo, J.H., Nature, 354 (1991) 84.

127 2. Pinilla, C., Appel, J., Blondelle, S., Dooley, C., Dörner, B, Eichler, J., Ostresh, J. and Houghten, R.A., Biopolymers (Peptide Science) 37 (1995) 221. 3. Nefzi, A., Ostresh, J.M. and Houghten, R.A., Chem Rev, 97 (1997) 449. 4. Ostresh, J.M., Husar, GM., Blondelle, SE., Dörner, B., Weber, P.A. and Houghten, R.A., Proc. Nat. Acad. Sci. USA, 91 (1994) 11138. 5. Nefzi, A., Ostresh, J.M., Meyer, J.-P. and Houghten, R.A., Tetrahedron Lett., 38 ( 1997) 931. 6. Nefzi, A., Ostresh, J.M. and Houghten, R.A. Tetrahedron Lett., 38 (1997) 4943. 7. Pinilla, C., Appel, J.R., Blanc, P. and Houghten, R.A., Bio Techniques, 13 (1992) 901.

41 Design and synthesis of a lactam bridge stabilized coiled-coil template for library display M.E. HOUSTON1,2 R.T. IRVIN 1,3 and R.S. HODGES1,2 ¹Protein Engineering Network of Centres of Excellence, 713 HMRC, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada ² Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada ³Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada

Introduction Peptide libraries made up of millions of randomized peptide sequences have proven useful for the identification of novel ligands that maybe of interest for the development of medicinally active compounds [1, 2]. However, simple linear peptides libraries possess considerable conformational flexibility and can populate multiple random-like conformations. An alternative to linear peptide libraries is to incorporate randomized positions in stable secondary structural elements. To this end we envisaged utilizing the well characterized secondary structural element, the two-stranded α -helical coiled-coil as a template for peptide library development. The coiled-coil structure consists of two amphipathic helices wrapped around each other with a left-handed super twist. The coiled-coil is characterized by a heptad repeat denoted (abcdefg )n where positions a and d are typically occupied by hydrophobic amino acids (Figure 1).

Figure 1. Helical Wheel diagram of the coiled-coil template. Library Positions are denoted by the letter X and lactam bridges are indicated by lines linking E6 and K10 and E22 and K27 respectively. The italicized letter a to g represent the positions of the heptad repeat.

128 Y. Shimonishi (ed): Peptide Science – Present and Future, 128–131 © 1999 Kluwer Academic Publishers, Printed in Great Britain

129 These residues are aligned such that they create a hydrophobic interface which is responsible for the stability of this structure. This model has the advantage that it contains only one type of secondary structure, the α-helix. As shown by the wide variety of coiled-coil sequences existing in nature, the coiled-coil motif is quite tolerant to sequence variation. Moreover, the conformation can be readily determined by CD. In addition, the coiled-coil presents two exposeable surface to the aqueous environment per molecule.

Results and discussion The library template is illustrated in Figure 1. Five positions for library display were incorporated in the middle of the sequence where helical content would remain uniform. The sites are found at solvent exposed sites (positions b, c and f of the heptad repeat) of the coiled-coil template and away from the dimer interface. The dimer interface consists of Ile at position a and Leu at position d which have been shown previously to provide maximum stability to synthetic coiled-coils [3]. In addition, two Glu to Lys i, i + 4 lactam bridges where engineered at both termini to further stabilize the template. These bridges have been shown previously to enhance dimerization of synthetic coiled-coil sequences by destabilizing the unfolded form of the peptide [4]. To prevent dissociation of the two strands of the template a Cys–Gly–Gly linker was attached to the N -terminus such that an interchain disulfide bridge could be formed. As a proof of concept, we synthesized a library in which 18 of the 20 proteinaceous amino acids (excluding Pro and Cys) were incorporated at each of the 5 library positions. This gives rise to a library consisting of 18 5 or 1.89 million different sequences. The library was synthesized in two fragments using Fmoc chemistry. The C-terminal fragment (residues 11–27) housed the library positions and the C-terminal lactam. Incorporated into the N -terminal fragment was the Cys–Gly–Gly linker and N-terminal lactam bridge. The C -terminal segment

Figure 2. Fragment condensation of coiled-coil template. C-terminal fragment (residues 11–27) contains the C -terminal lactam bridge and library positions (denoted by X ). The N -terminal protected fragment (residues 1–10) contains the N-terminal lactam bridge and Cys–Gly–Gly linker.

130 was prepared on Rink amide MBHA RESIN and couplings were performed using HBTU/HOBt/NMM in NMP as described previously [4]. The side chain functionalities of the Glu and Lys residues of the lactam bridges were protected as OA1 and Aloc derivatives respectively. These allyl groups were efficiently removed using Pd(PPh3 )4 in CHCl3 /HOAc/NMM (37 : 2 : 1) under an atmosphere of nitrogen [5]. Lactam bridge formation was accomplished using a five-fold excess HBTU/HOBt and 7.5 excess of NMM in NMP. At library sites, the resin was divided into 18 equal portions and the individual Fmoc amino acids were coupled to the resin in separate reaction vessels. The extent of coupling was monitored using ninhydrin and where required multiple couplings were used. As the number of sequences within the library increased, multiple deprotection schemes were utilized to ensure complete removal of the Fmoc group. These schemes included 20% piperidine in NMP, 2% DBU, 2% piperidine in NMP and 20% piperidine in CH 2 Cl 2 /NMP/DMF (1: 1: 1) containing 1% Triton X. The N -terminal fragment was synthesized on SASRIN resin. Coupling of C -terminal FmocLys(Aloc) was accomplished using the 2,6-dichlorobenzoyl chloride method of Sieber [6]. Peptide chain elongation and lactam bridge formation were performed as described for the C -terminal fragment. Upon completion of the synthesis the protected peptide was cleaved from the resin using 1% TFA in DCM. The fragment was purified by reversed-phase HPLC using acetonitrile/water containing 0.05% TFA as the solvent system. The protected fragment (1.2 eq.) was coupled to Glu 11 of the resin bound C -terminal fragment using 2.4 equivalents of HBTU/HOBt in NMP over a 24 h period. The fully assembled protected peptide was then cleaved from the Rink amide resin using reagent K (TFA–phenol–water–thiophenol–ethanedithiol, 82.5 : 5 : 5 : 5 : 2.5). The complete library was prepared by air oxidization of the C -terminal Cys in 100 mM NH4 HCO 3 . The CD spectrum of the library under benign conditions (50 mM KH2 PO4 , 50 mM KCl, pH 7.0) of the library is shown in Figure 3. The CD spectrum is characterized by minimum at 221 and 208 and a maximum below 195 nm demonstrating the library is adopting a helical fold. The [θ] 222 value of –18,500 indicates that ensemble of sequences is 60% Helical. In summary, we have developed a coiled-coil template capable of maintaining itself in a helical conformation regardless of the amino acids (excluding Pro and Cys) occupying the 5 positions. This library may prove useful in the design of novel ligands and protein-mimetics.

Acknowledgments This research was funded by the Protein Engineering Network of Centres of Excellence of Canada

131

Figure 3 CD spectum of coiled-coil library.

References 1 . Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. and Cuervo, J. H., Nature 354 (1991) 84. 2 . Houghten, R. A., Appel, J. R., Blondelle, S. E., Cuervo, J. H., Dooley, C. T. and Pinilla, C., BioTechniques 13 (1992) 412. 3 . Zhu, B.-Y., Zhou, N. E., Kay, C. M. and Hodges, R. S., Protein Sci 2 (1993) 383. 4 . Houston, Jr., M. E., Wallace, A., Bianchi, E., Pessi, A. and Hodges, R. S., J. Mol. Biol. 262 (1996) 270. 5 . Kates, S. A., Baniels, S. B. and Albericio, F. Anal. Biochem. 212 (1993) 303. 6 . Sieber, P. Tetrahedron Lett. 28 (1987) 6147.

42 Diversity of synthetic peptides Y. KONISHI¹, J.J. SLON-USAKIEWICZ² and G. PIETRZYNSKI³ ¹ Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec, Canada, H4P 2R2 ² Advanced Bioconcept Ltd., 1440 St. Catherine W., Montreal, Quebec, Canada, H3G 1R8 ³ Supratek Pharma Inc., 275 St-Jacques, Montreal, Quebec, Canada, H2Y 1M9

Introduction The chemical diversity is a critical factor in many areas of medicinal chemistry including drug design. In most cases, the twenty coded amino acids are adequate to generate fairly diversified peptides and proteins. However, some specific biological functions are mediated through modifications of the coded amino acids, for example phosphorylation and glycosylation. Incorporation of unusual amino acids and other structural variation into synthetic peptides may improve drug-receptor interactions, pharmacokinetics, resistance to enzymatic degradation, solubility, mobility, tissue specificity, drug delivery, excretion and etc. [1]. In order to stimulate the use of uncoded amino acids in synthetic peptides and to promote their diversity, we made a database for peptide synthesis, which lists the commercially available coded and uncoded amino acids, and released it through Internet (http: //chemLibrary.BRI.NRC.CA) in 1996 [2].

Results and discussion The database for peptide synthesis currently lists 877 α -amino acids and 287 non- α-amino acids as well as 2124 carboxylic acids as N -terminal residue and 77 resins with amino acid attached from 42 suppliers. In addition, 254 building blocks are listed for combinatory library. Using these residues, ~2 million dipeptides or over 1 billion tripeptides are possibly generated, although the diversity of these residues and peptides has never been studied. The chemical and structural diversity of the residues are visualized by the 2D and the 3D structure displays, respectively. Since many unusual amino acids are homologous to the coded amino acids, the diversity is studied as modifications of the coded amino acids. The database contains 69 unusual amino acids as the analogues of the nonpolar aliphatic amino acids (Ala, Val, Ile, and Leu). They are then classified into 10 groups based on the side chain structure and/or the backbone modifications (Table 1). The analogues of other coded amino acids will be classified in similar ways. These unusual amino acids increase not only the diversity of the chemical and the three-dimensional structures, but also the diversity of the physical and chemical properties and of biological functions, 132 Y. Shimonishi (ed): Peptide Science – Present and Future, 132–133 © 1999 Kluwer Academic Publishers, Printed in Great Britain

133 Table 1. Classification of the Ala, Val, Ile, and Leu analogues listed in the database. Class

Description

Class I-1 Class I-2

Ala,Val, Leu, Ile and their D-isomers analogues with linear side chain

Class I-3

analogues with branched side chain

Class I-4

analogues with alkenyl or alkynyl side chain

Class I-5

analogues with non-aromatic cyclic side chain

Class I-6

analogues with backbone N-alkylation

Class I-7

analogues with Cα,α-dialkyl side chain

Class I-8 analogues with Cα, α-dialkyl cyclic side chain Class I-9 analogues with Cα, β -alkenyl side chain Class I-10 related amino acids

Number of amino acids listed in the database; example 8 amino acids 8 amino acids; α-aminobutyric acid, norvaline 8 amino acids; tert-butylglycine, homoleucine 5 amino acids; allyl-glycine, 4,5-dehydro-leucine 10 amino acids; cyclopentylglycine, β-cyclopropylalanine 12 amino acids; N-methylalanine, N-ethyl-leucine 7 amino acids; diethylglycine, isovaline, 2-aminobutyric acid 5 amino acids; cycloleucine 4 amino acids; 2,3-dehydrovaline 6 amino acids; sarcosine, N-ethylβ -alanine, N -cyclohexyl-glycine

which are critical for designing and incorporating them into synthetic peptides. For example, N-acyl- ∆ Ala has a unique activity to scavenge free radicals and is useful to reduce radiation toxicity and to inhibit radiation carcinogenesis [3]; and a minor structural modification of methionine to ethionine, i.e., an addition of a methyl group at the end of the side chain, changes the amino acid to a highly toxic compound. The literatures, which describe (1) the physical and chemical properties of the unusual amino acids, (2) the unusual amino acids in natural products, and (3) the use of the unusual amino acids in medicinal chemistry, are incorporated in the peptide synthesis database as a relational database. We believe that the classification of the unusual amino acids and their literature information will assist the proper incorporation of the unusual amino acids into the synthetic peptides and the peptidomimetics with their increased diversity.

References 1. Roberts, D.C. and Vellacio, F., In Gross, E. and Meienhofer, J. (Eds) The Peptides Vol. 5, Academic Press Inc., New York, 1983, pp. 341. 2. Pietrzynski, G., Slon-Usakiewicz, J.J. and Konishi, Y., In Peptides 1996 (Proceedings of the Twenty-Fourth European Peptide Symposium), in press 3. Buc-Calderon, P., Defresne, M.P., Barvais, C., Roberfroid, M. Carcinogenesis 10 (1989) 1641.

43 Manual construction of combinatorial resin bound tripeptide library (one peptide on one bead) and screening to find antioxidative peptides T. YASUHARA¹ and K. NOKIHARA² ¹ Junior College of Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagayaku, Tokyo 156, Japan ² Shimadzu Scientific Research Inc., Kanda-Nishikicho 1-3, Chiyodaku, Tokyo, 101, Japan

Introduction Several peptides have been shown to have antioxidative activities against peroxidation of lipids and fatty acids. As the formation of peroxides or radicals from polyunsaturated fatty acids can in some cases be fatal and may be responsible for aging, antioxidants are indispensable as food additives. Organic compounds, such as butylhydroxytoluene (BHT), are widely used in relatively high concentrations. Recently antioxidative peptides have been isolated from digests of soybean protein [1], from which a pentapeptide was selected as a lead compound and resulted in the discovery of a tripeptide, PHH, that was found to be highly antioxidative [2]. In the present study, we have systematically constructed and screened all possible tripeptide libraries, as tripeptides seemed to be the minimum requirement for practical antioxidative action.

Results and discussion Tripeptide libraries were constructed on the basis of a ‘one peptide on one bead’ approach, in which the C-terminus of the peptide was a randomized mixture of 19 amino acids (all natural amino acids except Cys) and was attached to 130 µm TentaGel-S beads. Manual synthesis using the Fmoc-strategy with PyBOP was carried out using a PCR-micro tube array. Needle or microcapillary connected a pump which was used to aspirate solvents and reactants. After incorporation of 19 different amino acid derivatives at the C-terminus, respectively, the resin was pooled, Fmoc-removed and divided in to 19 portions followed by incorporation of the individual 19 second amino acids, deprotected, and then each dipeptide resin was further divided into 19 portions with a total of 361 reaction tubes. After incorporation of the third amino acid, the resulting resin was deprotected, cleaved with a TFA-cocktail, washed and dried to give resin-bound tripeptides, A 1 –A 2 –X ( X = 19 natural amino acids except Cys), ca 150 nmol/tube. 134 Y. Shimonishi (ed): Peptide Science – Present and Future, 134–135 © 1999 Kluwer Academic Publishers, Printed in Great Britain

135 In the screening process antioxidative activities has been tested by the autoxidation of linoleic acid with ferric thiocyanate and detecting the generated linoleic acid hydroperoxide by HPLC [2]. As these methods required a long time to screen a large number of libraries, a rapid and higher through-put screening method with β -carotene and linoleic acid has been developed [3], which is the modified method presented by Tsushida et al. [4], to evaluate the activities of the resulting libraries. Resin-bound peptides were placed in a 96-well microtiter plates and incubated as described in [3]. The plates were sonificated for 2 min and the OD at 492 nm measured. Without sonification the difference between the peptides was small. Our previous results indicated that peptides showed antioxidative action at much lower concentrations than BHT and a plateau was found at a particular concentration, in contrast to BHT which increased in a concentration dependent manner. The exact peptide amount is not important when measured beyond the plateau concentration. Through the first screening it was shown that the favored amino acid residues are: Arg, Leu and Pro at the N -terminus and His, Trp at the second position. Consequently the parallel synthesis of 108 individual peptides, [L, P, R]– [H, W]–[18 different amino acids], were performed using a Module, SRM96 consisting of 96 micro reaction tubes with glass filters and glass titerplates within 12 h by the PyBOP/HOBt coupling protocol followed by cleavage to give 108 different free peptides. The resulting peptides were tested and their relative antioxidative properties were compared. Consequently, tripeptides containing the Trp residue at the second position showed higher activity and the tripeptides LHW, LWW, PHY, PWW, RHW, RHY, and RWW were particularly active. In addition to Tyr and His residues, which we have carefully investigated [2, 3], the Trp residue gave significant activity, although this residue is of relatively low abundance in proteins. The present results can be used to find proteins which contain the sequences and are expected to have antioxidative properties after partial digestion. The actual mechanism of antioxidative action in vivo seems to depend on combinatoral mechanisms such as metal ion chelating, active oxygen quenching and/or hydroxyl-radical scavenging actions. The detail was described in [5].

References 1. Chen, H-M., Muramoto, K. and Yamauchi, F., J. Agric. Food Chem, 43 (1995) 574. 2. Chen, H-M., Muramoto, K., Yamauchi, F. and Nokihara, K., J. Agric. Food Chem., 44 (1996) 2619. 3. Nokihara, K., Yasuhara, T., Muramoto, K., Ando, E. and Wray, V., In Kitada, C. (Ed.), Peptide Chemistry 1996, Protein Research Foundation, Osaka, 1997, p. 245. 4. Tsushida, T., Suzuki, M. and Kurogi, M., Nippon Shokuhin Kogyo Gakkaishi, 41 (1994) 611. 5. Chen, H-M., Muramoto, K., Yamauchi, F., Fujimoto, K., and Nokihara, K., J. Agric. Food Chem., 46 (1998) 49.

44 Selection of peptides that bind to α-lactalbumin from phage display library T. NAKAMURA1,2 K. HAGIWARA¹, K. NITTA², and K. TAKASE¹ ¹ Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba 305, Japan ² Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan

Introduction α-Lactalbumin (α-LA) is a milk protein involved in the regulation of lactose biosynthesis in the mammary gland [1]. It binds to galactosyltransferase and enhances the enzyme’s affinity for glucose so that the enzyme is converted to lactose synthase. α -LA is also known as a Ca 2 + -binding protein and its 2+ C a -depleted form assumes a molten globule structure observed as an intermediate of protein folding [2]. To use as probes to locate the binding site of galactosyltransferase and to study the mechanism of protein folding, we have obtained peptides that bind to α -LA by use of phage display system [3].

Results and discussion We used a phagemid library of 7-residue random peptides (pSKAN system, MoBiTec). Phages were selected by panning as described below. The wells of a microtiter plate were coated with 5% bovine α -LA overnight and blocked for 1 hour with blocking solution (2% gelatin and 107 p.f.u. helper phage M13K07 in 10 mM phosphate buffer (pH 7.0)) at room temperature. After washing for three times with 10 mM phosphate buffer, the wells were filled 11 with phage library (10 c.f.u.) diluted 1 : 1 with blocking solution and incubated for 2 hours at room temperature. The plate was washed ten times with 10 mM phosphate buffer. The bound phages were eluted with 100 µl of 100 mM glycineHCl (pH 2.2) and neutralized with 6 µ l of 2 M Tris. Eluted phages were used to infect E. coli W K 6 λ mutS log-phase cells. Phagemid particles were rescued from infected E. coli cells with helper phage M13K07, purified and concentrated by precipitation with polyethylene glycol. The phage preparation was resuspended in 10 mM phosphate buffer to 109 c.f.u./ml and used for a second round of selection. After two rounds of selection, phagemids were rescued from single ampicillin-resistant colonies of infected E. coli cells. The specificity of isolated clones was checked by enzyme-linked immunosorbant assay (ELISA) using a plate coated with various proteins at 1 mg/ml ( α -LA, bovine serum albumin (BSA), myoglobin, hen egg-white lysozyme). 36 136 Y. Shimonishi (ed): Peptide Science – Present and Future, 136–138 © 1999 Kluwer Academic Publishers, Printed in Great Britain

137 Phage clones were analyzed, and 5 of them were found to be more specific to α-LA than to BSA or myoglobin. However, these phases also reacted with lysozyme (Figure 1). The results are interesting in view of the fact that α-LA and lysozyme have similar three-dimensional structures despite their differences in the biological function and molecular charge [1]. It is possible that the peptides we selected recognize the structural domains common to the two proteins. We also screened the library against Ca 2 + -depleted α-LA. The method of screening is the same as native α -LA. 36 Phage clones were analyzed by ELISA, and 10 of them were found to be more specific to Ca2+ -depleted α -LA than to native α -LA or other proteins (Figure 2). The low affinity for lysozyme is noteworthy. Thus, these peptides recognize structural features not possessed by the native α -LA and lysozyme. The amino acid sequences of the selected peptides as determined by DNA sequencing will be presented elsewhere. Further study is under way to select phages with higher specificity. We also plan to study the effects of the α -LAbinding phages on the lactose synthase reaction and to seek for a chaperonelike function in these peptides.

Figure 1. The binding specificity of phages. Binding was determined by ELISA to a variety of proteins. 1 ~ 5 = phage clones selected for binding α -LA, 6 = helper phage M13K07. 7 = library. The ordinate represents ELISA readings.

Figure 2.

Specificity of phage clones selected for binding Ca2+ -depleted α-LA (1 ~ 10).

138 References 1. McKenzie, H.A. and White, F.H., Jr., Adv. Protein Chem. 41(1991) 173. 2. Okazaki, A., Ikura, T., Nikaido, K. and Kuwajima, K., Nature Struct. Biol. 1(1994) 439. 3. Smith, G.P. and Scott, J.K., Methods Enzymol. 217 (1993) 228.

45 Identification of a novel analgesic peptide including an unnatural amino acid from peptide libraries synthesized with a multipeptide synthesizer I. MAEDA 1,2 , N. KANEMOTO², K. OGINO², M. KUWAHARA², Y. GOTO², M. ADACHI², T. TAKI², M. OHTANI³, Y. MUNEOKA³, T. KAWAKAMI¹, and S. AIMOTO¹ ¹ Institute for Protein Research, Osaka University, Suita 565, Japan ² Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd, Tokushima 771–01, Japan ³ Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739, Japan

Introduction Combinatorial peptide libraries are powerful resources for developing pharmacologically active compounds such as, for example, potent agonists and antagonists for opioid receptors [1,2]. In a previous study, tetra- or pentapeptide libraries which contain Trp at the C-terminal position were found to show inhibitory activity on the rectum of a quail. Two types of peptides, YGHW–NH2 and TFVAW–NH 2 , showed strong activity in the assay [3]. These peptides also showed relaxant activity on the guinea pig ileum (GPI). In this study, penta–, tetra–, tri–, and di-peptide libraries which contain a Trp amide (or carboxyl) group at C terminus were synthesized with a multipeptide synthesizer in order to obtain more effective peptides for the GPI.

Results and discussion All peptide libraries were synthesized on multipeptide synthesizer ACT 396 (Advanced ChemTec Inc.) by standard Fmoc chemistry. At first, the libraries consist of 44 peptide mixtures, which are represented by the formulas, OXXXW–NH 2 (or –COOH), XXXW–NH 2 (or –COOH), XXW–NH 2 ( o r –COOH), and XW-NH 2 (or –COOH). O symbolizes the individual 19 natural L-amino acids except Cys, and X represents an equimolar mixture of the 19 amino acids. In total, the library was expected to contain 275,804 peptides (2 × 19 4 + 2 × 19³ + 2 × 19² + 2 × 19) in peptide combinations. When screening was performed by using GPI, four libraries, NXXXW–NH 2 , YXXXW–NH2 , XXXW–NH 2 , and XXW–NH 2, exhibited potent relaxant activity. On the contrary, the corresponding peptide libraries with a carboxyl group at the C terminus showed only weak activities in the assay. S e c o n d , 7 6 l i b r a r i e s ( 1 9 × 4 ) o f N O X X W – N H 2 , YOXXW–NH 2 , OXXW–NH 2 and OXW–NH 2 were synthesized and assayed by GPI. As a 139 Y. Shimonishi (ed): Peptide Science – Present and Future, 139–140 © 1999 Kluwer Academic Publishers, Printed in Great Britain

140 result, three types of libraries which have Asn residue at the N terminus, NQXXW–NH 2 , NXXW–NH 2, and NXW–NH 2 showed strong activity. NXW–NH 2 was taken as the first priority for further investigation, and the products were separated by reverse phase HPLC. Active fractions were subjected to amino acid sequence analysis and FAB Mass spectrometry analysis. The structure of the most potent tripeptide was found to be Asn(Trt)–Gln–Trp–NH 2 , whose trityl group was not removed. Since the activity of the peptide on GPI was inhibited by naloxone, analgesic activity was evaluated by the tail-pinch method. Intracisternal injection of Asn(Trt)–Gln–Trp–NH 2 into male mice resulted in dose-dependent analgesic activity. This peptide (10 µ g) produced complete analgesia and more than 90% of this maximal effect remained for up to 90 min after injection. In contrast, the analgesic effect by injection of 25 times amount of Leu-enkephalin persisted for only 15 min. Asn(Trt)–Gln–Trp–NH2 showed greater analgesic effects than equimolecular amounts of morphine. The activity of this peptide is comparable to strong opioid agonists in its potency and the duration of its effect. Opioid ( µ , δ and κ ) receptor binding assays were performed using guinea pig brain membranes as described previously [4]. Asn(Trt)–Gln–Trp–NH2 exhibited its highest selectivity for the µ -opioid receptor with a K i value of 54 nM, and had low affinities for the δ-and κ -receptor ( δ/ µ = 22 and κ /µ = 59). Several peptides which contain other amino acids at the C-terminal position or the position next to it were synthesized and examined for analgesic activity. No peptides showed greater activity than Asn(Trt)–Gln–Trp–NH2 . In addition, substitution of N-terminal Asn(Trt) for other amino acids with a trityl group, such as Gln(Trt), His(Trt) or Cys(Trt), resulted in only weak activity. When the structure of Asn(Trt)–Gln–Trp–NH 2 is compared with enkephalin, the trityl group may substitute for the Tyr phenol group of enkephalin. However, the substitution of this group with diphenylmethyl, benzyl or p-hydroxybenzyl groups caused a large decline in analgesic activity. It appears that all the structural groups on this tripeptide (including the trityl group) are required for activity.

References 1. Dooley, C.T., Chung, N.N., Wilkes, B.C., Schiller, P.W., Bidlack, J.M., Pasternak, R.A. and Houghten, R.A., Science, 266 (1994) 2019. 2. Dooley, C.T., Kaplan, R.A., Chung, N.N., Schiller, P.W., Bidlack, J.M. and Houghten, R.A., Pept. Res. 8 (1995) 124. 3. Ohtani, M., Muneoka, Y., Kanemoto, N., Ogino, K., Masui, Y. and Aimoto, S, Peptide Chemistry 1996 (1997) 205. 4. Maguire, P., Tsay, N., Kamal, J., Comenta-Morini, C., Upton, C. and Loew, G., Eur. J. Pharmacol., 213 (1992) 219.

46 Artificial proteins screened from combinatorial chemical libraries with strong binding to doublestranded oligodeoxyribonucleotides: towards repressors to long terminal repeat controlled viral transcription S.R.K. HOFFMANN and B. GUTTE Institute of Biochemistry, University of Zürich, Winterthurerstrasse 190 CH-8057 Zürich, Switzerland

Introduction The design of DNA-binding proteins that specifically recognize distinct sites on double-stranded DNA and control transcription is still a challenging problem [1a], but is of great interest because of potential therapeutic applications such as inhibition of cellular genes or entire viral genomes. In particular, human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) enhancerbinding proteins may suppress the transcription of the HIV provirus in infected cells on account of the known cis-acting regulatory function on transcription and replication of the viral genome. Due to the close sequence similarity between the recognition sites of bacteriophage 434 operator (ACAAG/ACAAT) and the enhancer sequences (ACAAG/ACTTT) of the HIV-1 LTR, the phage 434 repressor-operator system was used as a natural model for the design of a repressor for HIV-1 LTR controlled viral transcription. The interaction between the synthetic DNA-binding domain (residues 1–69) of phage 434 repressor and a synthetic double-stranded target DNA was very weak. However, when the recognition helix (residues 28–36) of the 434 repressor was extended at the Nand C -terminus by a tandem repeat of the positively charged segment 37–44, the resulting 42-residue polypeptide R42 showed strong HIV-1 enhancer binding [1b, c].

Results and discussion Based on this natural model, we show here how rational design strategies can be successfully combined with rational screening of combinatorial chemical libraries for the lead finding and optimization of linear and dendrimeric protein structures that act as artificial DNA-binding repressors (see Figure 1). The polypeptides were screened both with spatially adressable high-density compound arrays on continuous membranes and isotope mass encoded libraries prepared by the ‘one bead – one sequence’ methodology [2b, c]. 141 Y. Shimonishi (ed): Peptide Science – Present and Future, 141–143 © 1999 Kluwer Academic Publishers, Printed in Great Britain

142

143 For the efficient preparation of high-density compound arrays we have developed an automated pen directed computer plotter device. The method is adapted to the SPOT methodology [2a].To increase the performance, we have used here capillar ink pens for the delivery of compound solutions to porous or non-porous membranes. The automation of this procedure was done by a conventional 400 mm × 300 mm flat-screen computer plotter. For example, up to four 129 mm × 87 mm membranes or twelve 75 mm × 50 mm membranes can be simultaneously manipulated and up to 120 × 90 = 10,800 spots on a 129 mm × 87 mm membrane and 42 × 30 = 1260 spots on a 75mm × 50mm membrane can be established. The synthesis of the combinatorial chemical libraries prepared by the ‘One bead – One sequence’ methodology was started by derivatisation of the Novasyn TG  Amino resin with Fmoc-β Ala, followed by the coupling of BocSer-OH and the esterification of its side chain with Fmoc-Pro. After incorporation of Npys-L -Lys(Fmoc) or Dde-L -Lys(Fmoc), the side chain of lysine was deprotected and Fmoc-Phe was coupled, which made this anchor addressable for the attachment of an encoding TAG. The polypeptides were then assembled using the Boc/Bzl protection scheme as outlined in Figure 1). The coupling of an individual amino acid derivative within the ‘SPLIT and MIX’ procedure was followed by the removal of Fmoc from the TAG and coupling of the corresponding Fmoc protected amino acid or an encoding isotope mixture [2c]. In the screening process individual beads are isolated and the composition of the active polypeptide is determined by the release of the low molecular weight TAG and its analysis by mass spectroscopy or sequence analysis. Both types of libraries are competitively screened against a γ- [ 3 2 P ] - l a b e l e d 70 bp synthetic HIV-1 enhancer target DNA and non-specific calf thymus or herring sperm DNA. In preliminary competitive assays, strong binding of several proteins in high saline buffer (100 mM K-Pi (pH 7.5); 37.5 mM NaCl; 15 mM KCl; 4 mM MgCl2 ; 1.6% Ficoll; 0.08% Noni-det P40 ) even in the presence of a 6000- to 60,000-fold excess of competitor DNA (12 h to 24 h at 25°C to 37°C) is observed. The strongest binding mutants of R42 are given (Figure 1).

References 1. a. Pabo, C.O. and Sauer, R.T., Annu. Rev. Biochem., 61(1992) 1053; b. Hehlgans, T. et al., FEBS Lett., 315 (1993) 51; c. Städler, K. et al., Int. J. Peptide Protein Res., 46 (1995) 333. 2. a. Frank, R., Tetrahedron, 24 (1991) 1053; b. Furka, A., Xth Int. Symp. Med. Chem. (1988); Furka, A., Int. J. Peptide Protein Res. 37 (1991) 487; c. Lam, K.S., Nature, 354 (1991) 82; d. Geysen, H.M. et al., Chemistry & Biology, 3 (1996) 679.

47 Synthesis, conformational analysis and application of tropane-based Fmoc-amino acid derivatives: novel antagonists of fibrinogen receptor, GPIIb/IIIa P.E. THOMPSON, D.L. STEER, K.A. HIGGINS, M.-I. AGUILAR and M.T.W. HEARN Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton 3168, Victoria, Australia

Introduction As the gap is bridged between peptide and non-peptide drugs, molecular scaffolds are gaining prominence as a means to restrict and mimic peptide conformation. In this respect, non-peptide natural products, particularly those of medicinal value are providing a significant resource in the design of novel peptidomimetics. [1] The tropane alkaloids including cocaine, anatoxin-a and epibatidine, offer templates well suited to peptidomimetic application. The semi-rigid structure, diverse side-chain functionality and stereochemistry, established synthetic chemistry and applicability to solid phase synthesis are all desirable properties of such templates, and we have recently reported the synthesis of a range of tropane amino acids [2,3]. In order to demonstrate the utility of tropane amino acids we have incorporated them as glycine replacements into the ‘native’ GPIIb/IIIa inhibitor Arg– Gly–Asp–Ser (RGDS) [4] to establish if activity could be retained and equally as importantly to establish if useful structure function data could be gathered to expedite the generation of pharmacophores. Results and discussion Three tropane ligands have been synthesized and evaluated for inhibition of platelet aggregation. Racemic nortropane-2α -carboxylic acid (Ntc2α ), the opti-

Figure 1.

Amino acid structures and abbreviations.

144 Y. Shimonishi (ed): Peptide Science – Present and Future, 144–145 © 1999 Kluwer Academic Publishers, Printed in Great Britain

145 Table 1.

Inhibiton of thrombin-induced platelet aggregation by tropane derivatives.

1 2 3 4 5

Sequence

IC 50 (µM)

Arg–Gly–Asp–Ser Arg-(±)-Ntc 2α-Asp–Ser Arg-(+)-Ntc 2α-Asp–Ser Arg-(±)-Abh–Asp–Ser Arg-(±)-Nip–Asp–Ser

50 23 34 402 73

cally pure enantiomer derived from cocaine, (+)-Ntc2α , [2] and a ring contracted derivative derived from an intermediate in the synthesis of the alkaloid epibatidine (Abh) [3] – in addition ligands containing (±)-nipecotyl (Nip) residues were examined for comparison with recently reported work of Klein et al. [5]. Compounds 1–5 were assayed for their ability to inhibit the aggregation of a single washed platelet preparation by thrombin. Results are summarized in Table 1, and show the enhanced activity of Ntc containing ligands relative to the native sequence, RGDS, and that an optically pure isomer (+)-Ntc 2α a l s o retains activity. In contrast, the nipecotyl analogue shows marginally reduced activity, while the Abh ligand shows only poor inhibitory activity. The slightly reduced activity of the nipecotamide is in contrast to the highly potent nipecotyl isomers incorporating 4-guanidinocinnamic acid as an arginine mimic, [5] which are 10- to 40-fold more potent than the glycine containing analogue. The corresponding tropanes are currently under investigation. Generation of a tropane amino acid ‘construction kit’ analogous to the sugar amino acid kit described by Kessler et al. will provide an extensive survey of peptide conformation-activity relationships. [6] The tropane nucleus with its capacity for substitution, configurational diversity and relative synthetic accessibility offers significant potential in the generation of structurally defined pseudopeptide libraries. Acknowledgement This work was supported by the Australian Research Council. References 1. Barrow, C.J. and Thompson, P.E., In Abell, A. (Ed.), Advances In Amino Acid Mimetics and Peptidomimetics, Volume 1, JAI Press, Greenwich. 1997, pp. 251–293. 2. Thompson, P.E. and Hearn, M.T.W., Tetrahedron Lett., 38 (1997) 2907. 3. Thompson, P.E., Steer, D.L., Higgins, K.A., Aguilar, M.-I. and Hearn, M.T.W., In Kaumaya, P.T.P., Tam, J.P. (Eds.), Peptides: Chemistry, Structure and Biology (Proceedings of the 15th American Peptide Symposium), ESCOM/Kluwer, in press. 4. Ojima, I., Chakravarty, S. and Dong, Q., Bioorg. Med. Chem., 3 (1995) 337. 5. Klein, S.I., Czejak, M., Molino, B.F. and Chu, V., Bioorg. Med. Chem. Lett., 7 (1997) 1773. 6. Graf von Roedern, E., Lohof, E., Hessler, G., Hoffmann, M. and Kessler, H., J. Am. Chem. Soc., 118 (1996) 10156.

48 Accurate peptide sequencing by mass spectrometry T. TAKAO¹ , W. MO¹, J. FERNANDEZ-DE-COSSIO², J. GONZALEZ², V. BESADA², G. PADRON² and Y. SHIMONISHI¹ ¹Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan ²Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, Cuba

Introduction Mass spectrometry for the measurement of product ions produced upon collision-induced-dissociation (CID) or post-source decay (PSD) is a well accepted method for the characterization of biomolecules, especially for peptide sequencing and the analysis of post-translational modifications of proteins. However, the assignment of the product ions is not always definite, rendering the resulting data ambiguous. Moreover, the manual interpretation of the spectra requires considerable skill, without which, misassignments of ions can occur. To circumvent these issues, we [l–3] and others [4,5] earlier reported on the 18 O labeling of peptides for rapid and accurate sequence analysis of peptides and a software program designed to aid in peptide sequencing [6–9], which is based on low- and high-energy CID mass spectra. In this study, we describe an extension of this method to PSD experiments, using a high-resolution matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometer (TOFMS), along with a new version of the software, ‘SeqMS ’, which has been modified for the automated interpretation of PSD spectra.

Results and discussion Figure 1 shows a MALDI mass spectrum obtained for a lysylendopeptidase digest of horse myoglobin, which was carried out in a buffer containing 40 atom % H 2 18 O [1]. The spectra were acquired on a Voyager Elite XL timeof-flight mass spectrometer equipped with a delayed-extraction system with a 6.5 meter flight path in the reflectron mode. All the signals, which were isotopically resolved with an average resolution of 5000, indicate the partial incorporation of 18 O atoms into the peptides (see inset of Figure 1). Figure 2(a) shows the PSD spectrum from the signal at m /z 952.4 observed for peptide L13. The ions produced upon metastable decomposition were separated from undesired ones by rapid switching of an electrical deflector (timed ion selector). The product ions, which originated from the C terminus, partially retain the 18 O isotope, while those from the N terminus do not. Thus, the C -terminal ions, which have a distinctive appearance relative to isotopic distribution as the 146 Y. Shimonishi (ed): Peptide Science – Present and Future, 146–151 © 1999 Kluwer Academic Publishers, Printed in Great Britain

147

Figure 1. MULDI-TOFMS of a lysylendopeptidase digest of horse myoglobin (1 picomole) obtained in the reflectron mode. The signal labels denote the peptide fragments shown in the amino acid sequence. The signal marked by an asterisk is derived from the matrix. Relatively small peptides were not observed in this measurement because of the matrix suppression effect, which is often observed for complex mixtures.

result of 18 O-labeling, can be clearly differentiated from those derived from the N terminus (left inset of Figure 2(a)). The isotopic resolution of singly charged precursor ions and their product ions obtained for peptides up to ca. 2800 Da has been achieved using the delayed extraction method [10] equipped in a MALDI-TOFMS instrument, which permits the rapid identification and assignment of the 18 O-labeled and non-labeled ion species in the PSD spectra. The minimum amount of sample to provide sufficient precursor ions for PSD analysis is at the picomole level, although the issue of whether or not complete sequence information can be obtained is dependent on the molecular size and the nature of the sample. The spectrum was directly subjected to an improved version of SeqMS, a WINDOWS95-compatible computer program designed to aid in peptide sequencing by MS/MS [8]. The product ions observed in PSD analysis of peptides are quite similar to those obtained by low-energy CID mass spectra, e.g. the backbone fragmented ions and their rearranged ions, such as a–17,

148

149

150 b – 17, b – 18 and b n – 1 + H 2 O, which are significantly dependent on the structure of a peptide in the gas phase. The software can account for all these ions, N- or C -terminal blocking groups, gaps between signals, and 18 O-labeling of the C -terminal ions so as to efficiently interpret the PSD spectra. As a result, it was able to output the candidate sequences (Figure 2(b)) for peptide L13 (Pro 88–Leu–Ala–Gln–Ser–His–Ala–Thr–Lys 96 ), based on the peaks detected upon the threshold with the software. The product ions, calculated from the most likely candidate, were assigned to the intense signals observed (Figure 2(a)), suggesting the validity of the assignments. In addition, the gaps observed in the spectrum between the MH+ and y "7 ions and below 211.5 could be calculated to be Leu/Ile and Pro as the most likely amino acid combination (a relatively small gap can give rise to combination(s) of amino acids which coincide with this gap). The software has proved to be more efficient for obtaining a real sequence among the candidates with a high degree of probability when tested using the 18 O-labeled peptides observed in Figure 1. The present study demonstrates that accurate peptide sequencing, using a combination of stable-isotope labeling and high-resolution PSD analysis in conjunction with a modern MALDI-TOF instrument, can be achieved. The 18 identification of O-enriched ion species based on isotopic ion distributions allows for the facile and unambiguous assignment of the C-terminal ions, and furthermore, results in the rapid and reliable sequencing of peptides when the spectra are subjected to the automated spectral interpretation with the developed software, which is especially useful for the sequencing of unknown peptides. With the exponential growth of amino acid sequences derived from nucleotides or proteins, the present PSD analysis of peptides, aided by the software, provides the potential for rapid protein identification through data base searching and for the investigation of novel proteins.

Acknowledgement This work was supported, in part, by the Mitsubishi Foundation (to Y. S.) and SUNTORY INSTITUTE FOR BIOORGANIC RESEARCH (to T. T.).

References 1. Takao, T., Hori, H., Okamoto, K., Harada, A., Kamachi, M. and Shimonishi, Y., Rapid Commun. Mass Spectrom., 5 ( 1991) 312. 2. Takao, T., Gonzalez, J., Yoshidome, K., Sato, K., Asada, T., Kammei, Y. and Shimonishi, Y., Anal. Chem., 65 (1993) 2394. 3. Mo, W., Takao, T. and Shimonishi, Y., Rapid Commun. Mass Spectrom. 11 (1997) 1829. 4. Scoble, H.A. and Martin, S.A., Methods Enzymol., 193 ( 1990) 519. 5. Shevchenko, A., Chernushevich, I., Ens, W., Standing, K.G., Thomson, B., Wilm M. and Mann, M., Rapid Commun. Mass Spectrom., 11 (1997) 1015. 6. Eng, J.K., McCormack, A.L. and Yates, J.R. III, J. Am. Soc. Mass Spectrom., 5 (1994) 976. 7. Fernandez-de-Cossio, J., Gonzalez, J. and Besada, V., Comp. Applied Biosci., 11(1995) 427.

151 8. Takao, T., Fernandez-de-Cossio, J., Gonzalez, J., Shimonishi, Y., Padron, G. and Besada, V., Proceedings of the 45th ASMS Conference on Mass Spectrometry &. Allied Topics, (1997) pp. 1225 and 1349. 9. Taylor, J.A. and Johnson, RS., Rapid Commun. Mass Spectrom., 11(1997) 1067. 10. Vestal, M.L., Juhasz, P. and Martin, S.A., Rapid Commun. Mass Spectrom., 9 (1995) 1044. 11. Roepstorff, P. and Fohlman, J., Biomed. Mass Spectrom., 11 (l984) 601. 12. Johnson, R.S., Martin, S.A. and Biemann, K., Anal. Chem., 59 (1987) 2621.

49 Characterization of sulfhydryl compounds attached to the cysteinyl residue of proteins or peptides by on-line post-column derivatization K. NOKIHARA¹ and T. YASUHARA² ¹ Shimadzu Scientific Research Inc., Kanda-Nishikicho 1-3, Chiyodaku, Tokyo 101-8448, Japan ² Junior College of Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagayaku, Tokyo 156-0054, Japan

Introduction Cysteine residues in proteins and peptides play important roles in stabilizing the conformation of molecules through disulfide linkages and they also occur as reactive sulphydryl groups that contribute to oxidation/reduction processes in vivo. Small molecules possessing sulfhydryl group bind to cysteinyl residue(s) of proteins or peptides and are considered to play a roll in the folding process via disulfide interchange. To elucidate these biological pathways as well as to characterize the micro-heterogeneity of these cysteinyl residues in proteins, we have developed a rapid, facile, sensitive and reproducible quantitative analytical system for sulfhydryl compounds using an on-line post-column derivatization with Ellman-type reagents.

Results and discussion The present system consisting of a HPLC pump operated in an isocratic elution, an injector, an ODS-column followed by a coil-reactor, which were placed in an oven, and a UV-detector. Ellman-reagent containing reactant was continuously injected into the coil. Tri-n -butylamine solution was used as an ion-pair reagent. We have succeeded the determination of SH-compounds, such as Cys–Gly, Cys, homoCys, GSH, γ Glu–Cys, HSC 2 H 4 OH, SHCH 2 C O O H , H S C2 H 4 COOH, N-AC–Cys, and DTT, quantitatively within 25 min in the range 10 pmol ~ 1 nmol, especially glutathione (GSH) and its components could be analyzed within 10 min. Our method has significant advantages compared to those recently reported using capillary electrophoresis [1]. Derivatization is not necessary prior to the analysis and the sensitivity is not dependent on the particular compound. SH-compounds can be released from proteins by reduction with DTT and it is possible to perform preparative separations, in which sample can be obtained without derivatization, although any labelling reagent such as fluorescent reagent can be used. 152 Y. Shimonishi (ed): Peptide Science – Present and Future, 152–153 © 1999 Kluwer Academic Publishers, Printed in Great Britain

153 The present method has been applied to serum albumins. Recently, Ikegaya et al. have determined completely the 17 disulfide forms of human serum albumin (HSA) [2]. As HSA has 35 cysteinyl residues, one cysteinyl residue exists as a free sulfhydryl moiety (position 34) and this gives rise to the microheterogeneity. Here we report for the first time the quantitative characterization of the micro-heterogeneity of serum albumin. Different preparation methods are employed for different purposes. Consequently, the commercial preparations of BSA contain 55–70% SH-free molecules and the major substituted material is Cys (10–22%), Cys–Gly (6–10%) and the rest is considered to be dimer. On the other hand HSA is 30–37% Cys, 5–9% Cys–Gly, 60–70% intact sulfhydryl and almost no dimer. In both albumins there was neither GSH nor γ Glu–Cys. Additionally, freshly prepared HSA samples from healthy volunteers (separated by ethanol precipitation of plasma and characterized by the present method) contained Cys (0.46 nmol/mL serum) as the major substituent, together with Cys–Gly (0.24 nmol/mL) and very small amounts of GSH (0.02 nmol/mL). Hence, with regard to the sulfhydryl moiety, significant microheterogeneity has been demonstrated. Cysteine was found to be the major molecule attached to the sulphydryl group of the cysteinyl residue of serum albumin. Although glutathione could not be detected, peptidyl elements of the glutathione molecule were found. Detail of the analysis was published [3]. The present method can be used to reveal the microheterogeneity of proteins and is useful for quality control of both natural and recombinant proteins. Elucidation of the relationship between diseases and SH-compounds is also very interesting, hence the present method will make significant clinical contributions.

References 1. Russell, J. and Rabenstein, D.L., Anal. Biochem., 242 (1996) 136. 2. Ikegaya, K., Hirose, M., Ohmura, T. and Nokihara, K., Anal. Chem. 69 (1997) 1986. 3. Yasuhara, T. and Nokihara, K., Anal. Chem. 70 (1998) 3505.

50 Regioselective hydrolytic cleavage of N-terminal myristoyl-peptide K. SUGIMOTO, K. OKIMURA, N. SAKURA and T. HASHIMOTO Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181, Japan

Introduction The myristoyl (Myr) moiety was found in an amide linkage with the N-terminal Gly residue of a protein. We reported previously the regioselective cleavage of Myr–Gly–Ala–Phe–OH in HCl and H 2 SO 4 [1]. For further investigation of the regioselective cleavage of Myr–Gly–peptide, we examined the susceptibility and stability of the internal peptide bonds of Myr–Gly–X–Phe–OH (X = Ala, His, Gly) to several concentrations of methanesulfonic acid (MSA), and analyzed the hydrolysates by RP-HPLC. This paper describes the regioselective cleavage of Gly–X linkage of Myr–Gly–X –Phe–OH in aqueous MSA.

Results and discussion To analyze acid hydrolysates, a solution of a peptide at a concentration of 4 × 10 – 4 mol/l in 10 ~ 50% MSA in water:dioxane (1:l), or 70 ~ 90% MSA in water was maintained at 25 or 60°C for an appropriate period. After neutralization with 8 N NaOH, the solution was examined by RP-HPLC [column, YMC-Pack C 4 (4.6 × 150 mm); elution, 0 ~ 52% MeCN in 0.1% TFA; flow rate, 1 ml/mm; detection, 210 nm] to determine the amount of the starting material that remained. The remaining neutralized solution was lyophilized and then dissolved in the same amount of 1 N HCl, and the amounts of water-soluble hydrolysates were determined by RP-HPLC [column, Puresil™ C 18 (4.6 × 250 mm); elution, 6.4 or 8% MeCN in 0.1% TFA; flow rate, 1 ml/mm; detection, 210 nm]. To identify the hydrolysates, each peak on HPLC was collected, analyzed for amino acid composition, identified and confirmed by coelution with an authentic sample on HPLC. The peak areas of the starting material and the hydrolysates were compared with those of standard samples. We examined the susceptibility of the Myr–Gly, Gly–Ala and Ala–Phe bonds of Myr–Gly–Ala–Phe–OH to several concentrations of 10 ~ 90% MSA at 60°C for 3 h (Figure 1A). Hydrolysis resulted in the formation of one major and two minor hydrolysates. The hydrolysate contained H–Ala–Phe–OH as the major product, and H–Gly–Ala–Phe–OH and H–Phe–OH as the minor products. These results indicated that the higher the concentration of MSA 154 Y. Shimonishi (ed): Peptide Science – Present and Future, 154–156 © 1999 Kluwer Academic Publishers, Printed in Great Britain

155

Figure 1. Relationship between the concentration of aqueous methanesulfonic acid (MSA) and those of Myr–Gly–X –Phe–OH [X = Ala (A), His (B), Gly (C)] and its hydrolysates after incubation at 60°C for 3 h. ( ) Myr–Gly–X –Phe–OH, ( ) H–Gly–X –Phe–OH, ( ) H –X–Phe–OH,( ) H–Phe–OH.

during hydrolysis, the lower the yield of H–Gly–Ala–Phe–OH and H–Phe–OH. The yields of H–Ala–Phe–OH were highest after hydrolysis in 50 ~ 70% MSA. As the concentration of MSA increased over 70%, the rate of the hydrolysis reaction gradually decreased. The optimal concentration of MSA to selectively and rapidly cleave the Gly–Ala linkage seemed to 50%. These results were similar to the selective removal of pGlu–OH from pGlu-peptide [2] and of AC-amino acid residue from Ac-peptide [3] in aqueous MSA. The acid hydrolysis of Myr–Gly–His–Phe–OH (Figure 1B) and Myr–Gly–Gly–Phe–OH (Figure 1C) in aqueous MSA gave similar results. Thus, we examined the acid hydrolysis of Myr–Gly–X–Phe–OH (X = Ala, His, Gly) in 50% MSA at 60°C for 6 h. The acid hydrolysis (Figure 2) of the three analogs indicated that the m a j o r h y d r o l y t i c c l e a v a g e o c c u r r e d a t t h e G l y –X bond of Myr–Gly–X–Phe–OH, and Myr–Gly and X-Phe bonds were resistant to acid hydrolysis. T h i s s t u d y i n d i c a t e d t h a t t h e G l y - p e p t i d e l i n k a g e o f Myr–Gly–peptide is highly labile to 50% MSA compared with other peptide bonds, and is selectively hydrolyzed to give predominantly the cleavage product of the Gly-peptide linkage. These results should contribute to the development of a simple means of deblocking Myr–Gly–peptide prior to Edman degradation. Qualitative and quantitative analyses of not only Myr–Gly–OH but other fatty acid–Gly–OH in the MSA hydrolysate are currently under way in our laboratory.

Figure 2. Time courses of Myr–Gly–X –Phe–OH [X = Ala (A), His (B), Gly (C)] and its hydrolysates after incubation in 50% methanesulfonic acid (MSA) at 60°C for 6 h. ( ), Myr–Gly–X – Phe–OH; ( ), H–Gly–X –Phe–OH; ( ), H–X –Phe–OH; ( ), H–Phe–OH.

156 References 1. Okimura, K., Sugimoto, K., Sakura, N., Hashimoto, T., Peptide Chemistry 1996, Kitada, C. (ed.), Protein Research Foundation, Osaka, 1997, p. 89. 2 . Ohki, K., Sakura, N., Hashimoto, T., Chem. Pharm. Bull., 45, 194 (1997). 3. Ohki, K., Sakura, N., Hashimoto, T., Peptide Science – Present and Future, 164 (1988), ed. by Y. Shimonishi, Kluwer Academic Publishers.

51 Selective removal of pyroglutamic acid residue from biologically active pyroglutamyl-peptides by aqueous methanesulfonic acid J. KOBAYASHI, K. OHKI, N. SAKURA and T. HASHIMOTO Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181, Japan

Introduction A number of native proteins and peptides have been shown to possess a pGlu residue at the N-terminal, which is thought to afford protection against aminopeptidase digestion. Our recent studies [1–4] indicated that pGlu-peptide is highly sensitive to acid under mild conditions, generating not only the ringopened product of the pyrrolidone moiety of the pGlu residue, but also the cleavage product of the pGlu-peptide linkage, and pGlu-OH is selectively cleaved from pGlu-peptide in aqueous methanesulfonic acid (MSA). In this study, the selective removal of pGlu residue was examined from biologically active pGlu-peptides by aqueous MSA at 25°C.

Results and discussion The methods of acid hydrolysis used in this study were essentially the same as those described for highly selective cleavage of pGlu-peptide linkage in 70 and 90% MSA [1]. We examined the susceptibility of pGlu-peptide bond of dog neuromedin U-8 (d-NMU-8), LH-RH, a bradykinin potentiator (BPP-5) and physalaemin at a concentration of 10 – 3 mol/l in 70 or 90% MSA at 25°C for appropriate times. The acid hydrolysis of d-NMU-8 [pGlu-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH 2 ] (Figure 1B) in 90% MSA produced a major product of des-pGlu-peptide with deamidation of α- and β -carboxamides of the C-terminus and H–Glu-peptide as a minor product. However, 70% MSA hydrolysis (Figure 1A) gave predominantly the deamidated products. The des-pGlu-peptide predominated over the H–Glu-peptide at a molar ratio of about 11:1 in 70% MSA at 25°C for 12 h and about 14:1 in 90% MSA at 25°C for 4 days. Neither hydrolysate contained the cleavage products of the internal peptide bonds. The hydrolysis of a bradykinin potentiator, BPP-5 [pGlu-Lys-Trp-Ala-ProOH], in 70 and 90% MSA (Figure 1C) resulted in the superior formation of des-pGlu-peptide and small amounts of the ring-opened product. The molar 157 Y. Shimonishi (ed): Peptide Science – Present and Future, 157–159 © 1999 Kluwer Academic Publishers, Printed in Great Britain

158

Figure 1. Time courses of dog neuromedin U-8 (d-NMU-8) (A, B) and bradykinin potentiator, BPP-5 (C), and their hydrolysates during incubation in aqueous methanesulfonic acid (MSA) at 25°C. A, B; ( ) d-NMU-8, ( ) d-NMU-8 (2–8), ( ) d-NMU-8-OH, ( ) [Glu¹]-d-NMU-8. C; ( ) BPP-5, ( ) BPP-5 (2–5), ( ) [Glu¹]-BPP-5.

ratios of des-pGlu-peptide to H-Glu-peptide were about 62:1 in 70% MSA for 48 h and about 54:1 in 90% MSA for 3 days. The hydrolysis (Figure 2A) of LH-RH [pGlu-His-Trp-Ser-Tyr-Gly-Leu-ArgPro-Gly-NH 2 ] in 70% MSA gave the cleavage products of Trp3 -Ser4 linkage as the major products. However, 90% MSA hydrolysis (Figure 2B) predominantly produced des-pGlu-peptide with deamidated products of the C-terminal and decreased the cleavage products of Trp3 -Ser4 linkage. The hydrolysis of physalaemin (Figure 2C), which possesses one of the most acid-sensitive Asp-Pro peptide bonds, produced a complicated mixture of hydrolysates in 70 and 90% MSA, including des-pGlu-peptide as the main product, H-Glu-peptide, deamidated peptides and cleavage products of the Asp 3 -Pro 4 bond. Nevertheless, the hydrolytic cleavage of the pGlu-Ala bond was faster than that of the Asp3 -Pro4 bond. The present results indicated that pGlu-peptide bond in 70 or 90% MSA is the most susceptible among various peptides bonds and amide bonds at C-terminals and in the side chain of Asn residues. These results also suggested that this hydrolysis method may be applicable to selective removal of blocking groups of pGlu residues prior to Edman degradation.

Figure 2. Time courses of LH-RH (A, B) and physalaemin (C), and their hydrolysates during incubation in aqueous methanesulfonic acid (MSA) at 25°C. A, B; ( ) LH-RH, ( ) LH-RH (2–10), ( )LH-RH (4–10), ( ) [Glu¹]-LH-RH, ( ) LH-RH (1–3)-OH. C; ( ) physalaemin, ( ) physalaemin (2–11), ( ) physalaemin (4–11), ( ) [Glu 1 ]-physalaemin.

159 References 1. 2. 3. 4.

Ohki, K., Sakura, N., Hashimoto, T., Chem. Pharm. Bull., 45 (1997) 194. Hashimoto, T., Ohki, K., Sakura, N., ibid., 43 (1995) 2068. Hashimoto, T., Saito, S., Ohki, K., Sakura, N., ibid., 44 (1996) 877. Hashimoto, T., Ohki, K., Sakura, N., ibid., 44 (1996) 2033.

52 Hydrolytic removal of acetyl-amino acid from acetylpeptide in methanesulfonic acid K. OHKI, N. SAKURA and T. HASHIMOTO faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181, Japan

Introduction Acylation of the N-terminus of a protein with an acetyl (Ac) group is a common modification. Proteins and peptides with such blocking groups cannot be sequenced by Edman degradation. In the present study, we investigated the stability and susceptibility of the peptide bonds of Ac-X-Ala-Phe-OH (X = Gly, Ala, Pro, Tyr) towards aqueous methanesulfonic acid (MSA). This paper describes selective hydrolytic removal of Ac-amino acid from Ac-peptide in 70% MSA.

Results and discussion The hydrolysis methods used in this study were essentially the same as those described for highly selective cleavage of pGlu-peptide linkage in 70% MSA [1,2]. We examined the susceptibility of the Ac-Ala bond, and Ala-Ala and Ala-Phe peptide bonds, of Ac-Ala-Ala-Phe-OH (10 – 3 mol/l) to several concentrations of 10 ~ 90% MSA at 60°C for 90 min (Figure 1A). Hydrolysis resulted in the formation of two major and two minor hydrolysates. These peaks and the starting material peak were isolated by HPLC, and assigned on the basis of amino acid analysis and direct comparison with authentic samples on

Figure 1. Relationship between the concentration of aqueous methanesulfonic acid (MSA) and those of Ac-X-Ala-Phe-OH [X = Ala (A), Tyr (B), Gly (C)] and its hydrolysates after incubation at 60°C for 1.5 h.( ) Ac-X-Ala-Phe-OH, ( ) Ac-X-OH, ( ) H-Ala-Phe-OH, ( ) H-X-Ala-PheOH, (∆ ) H-Phe-OH.

160 Y. Shimonishi (ed): Peptide Science – Present and Future, 160–162 © 1999 Kluwer Academic Publishers, Printed in Great Britain

161 RP-HPLC [column, YMC-ODS-5-AM (4.6 × 150 mm); elution, 0 ~ 44% MeCN in 0.1% TFA; flow rate, 1 ml/min; detection, 210 nm]. The hydrolysate contained Ac-Ala-OH and H-Ala-Phe-OH as the major products, and H-AlaAla-Phe-OH and H-Phe-OH as the minor products. These results indicated that the higher the concentration of MSA during hydrolysis, the lower the yield of H-Ala-Ala-Phe-OH and H-Phe-OH. The yields of Ac-Ala-OH and H-Ala-Phe-OH were highest after hydrolysis in 60 ~ 70% MSA. As the concentration of MSA increased over 70%, the rate of the hydrolysis reaction gradually decreased. The optimal concentration of MSA to selectively and rapidly cleave the Ala-Ala linkage seemed to 70%. There are similarities between the selective removal of pGlu-OH from pGlu-peptide [1] and of Ac-amino acid residue from Ac-peptide in aqueous MSA. Hydrolysis of Ac-Tyr-Ala-Phe-OH (Figure 1B) and Ac-Gly-Ala-Phe-OH (Figure 1C) in aqueous MSA gave similar results. However, Ac-Pro-Ala-Phe-OH resisted hydrolysis to produce H-Phe-OH as the main product, and 73% of the starting material remained intact after 6 h incubation. Thus, we examined the acid hydrolysis of Ac-X-Ala-Phe-OH (X = Ala, Tyr, Gly) in 70% MSA at 60°C for 6 h and at 25°C for 3 days. Hydrolysis of AcAla-Ala-Phe-OH indicated that the major products, Ac-Ala-OH and H-AlaPhe-OH, predominated over H-Phe-OH at a molar ratio of about 5:1 at 60°C for 6h and about 11:1 at 25°C for 3 days. These results indicated that the XAla linkage was highly labile to 70% MSA hydrolysis at 60 and 25°C, whereas the Ac-Ala and the Ala-Phe bonds were resistant. The rate of hydrolysis was affected by the reaction temperature, and the cleavage reaction of the Ala-Phe bond was decreased at 25°C. These results also showed that acid hydrolysis at low temperature was superior to that at 60°C for highly selective cleavage of the X-Ala bond. This study indicated that the Ac-amino acid-peptide linkage is highly susceptible to 70% MSA compared with other peptide bonds, and is selectively hydrolyzed to give predominantly Ac-amino acid and deblocked peptide. These

Figure 2. Time courses of Ac-X-Ala-Phe-OH [X = Ala (A), Tyr (B), Gly (C)] and its hydrolysates during incubation in 70% methanesulfonic acid (MSA) at 60°C for 6 h. ( ) Ac-X-Ala-Phe-OH, ( )Ac-X-OH, ( ) H-Ala-Phe-OH, ( ) H-X-Ala-Phe-OH, (∆ ) H-Phe-OH.

162 results could be helpful in the development of a simple means of deblocking Ac-peptides prior to Edman degradation.

References 1. Ohki, K., Sakura, N., Hashimoto, T., Chem. Pharm. Bull., 45 (1997) 194. 2. Hashimoto, T., Ohki, K., Sakura, N., ibid., 44 (1996) 2033.

53 Determination of the binding-site structure of the atrial natriuretic peptide receptor by stepwise-affinity alkylation K.S. MISONO Department of Molecular Cardiology, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, U.S.A.

Introduction Atrial natriuretic peptide (ANP) is an antihypertensive peptide hormone secreted from the heart in response to high blood pressure. ANP dilates blood vessels, increases salt-excretion, and suppresses aldosterone and renin secretion, thus lowering blood pressure. These activities of ANP are mediated by specific cell-surface receptors coupled to guanylate cyclase (GCase). The ANP receptor is a type-I transmembrane protein containing an extracellular ANP-binding domain, a transmembrane sequence, and an intracellular ATP-binding regulatory-domain and a GCase domain. Binding of ANP to the extracellular domain activates the intracellular GCase domain by an as yet unknown mechanism. To elucidate how ANP interacts with the receptor and activates the GCase domain, it is essential to determine the structure of the ANP binding-site. To probe the binding-site structure, we have developed a new affinity-labeling method – stepwise affinity alkylation – that allows covalent and specific labeling of peptide-binding sites at high yields [1]. To facilitate the structural characterization, we have also produced by recombinant technique the extracellular ligand-binding domain of the ANP receptor (NPRA-ECD) in a water-soluble form. The recombinant NPRA-ECD has been purified by affinity chromatography and characterized. Using this material and the stepwise affinity alkylation procedure, we have identified segments of the receptor sequence that are at the ANP binding-site.

Results and discussion NPRA-ECD was expressed in COS cells after deleting the transmembrane sequence and the intracellular domains. NPRA-ECD, secreted into the culture medium by the transfected cells, was purified by ANP-affinity chromatography. The affinity-purified fraction gave a single band in SDS-PAGE as detected by Coomassie-Blue staining and by radioautography after photoaffinity labeling with azidobenzoyl- 125 I-ANF(4–28). NPRA-ECD bound ANP with the affinity (K d at 5 nM) comparable with that of the native ANP receptor. MALDI-TOF 163 Y. Shimonishi (ed): Peptide Science – Present and Future, 163–165 © 1999 Kluwer Academic Publishers, Printed in Great Britain

164 mass spectrometry gave a molecular mass of NPRA-ECD at 57,590, while the calculated mass of the expressed polypeptide is 48,380. The larger observed mass presumably reflects glycosylation. Titration of NPRA-ECD by varying concentrations of ANP followed by HPLC-gel filtration indicated a binding stoichiometry of unity. To probe the ANP receptor structure around the binding-site, we synthesized ANP peptide analogs that contained a reactive moiety, iodoacetyl-group (IAc-), at selected positions (positions 4, 18, and 29): [ 14 C] iodoacetyl-N-hydroxysuccinimide ester was reacted with the peptides, ANP(4–28), N 4a -acetyl[Lys 18 ]ANP(4–28), and N 4a -acetyl-[Lys 29 ]ANP(4–29), to obtain the (R4 α, shown below); N 4a-acetylreagents, N 4a -[ 14 C]IAc-ANP(4–28) N 18e -[ 14 C]IAc-[Lys 18 ]ANP(4–28) ( K18 ε); and N 4a -acetyl-N 29e -[ 14C]IAc[Lys 29 ] ANP(4–29) ( K29 ε); respectively. The stepwise affinity alkylation reaction was carried out as follows: 1) NPRAECD was incubated with a reagent at 0°C for 1 hr to allow binding; 2) the unbound reagent was removed by rapid gel filtration; and 3) the reagent: NPRA-ECD complex was incubated at 23°C overnight to effect covalent crosslinking. After the reaction, noncovalently associated affinity reagent was removed by gel filtration in 6M urea. The affinity-labeled NPRA-ECD was reduced and carboxymethylated, then digested with trypsin. Reverse-phase HPLC of the digest gave a single 14 C-radioactive peak, which was purified by additional HPLC steps. The purified peptide was then subjected to Edman degradation to obtain a binding-site sequence of the ANP receptor. The sequences obtained by these analyses were identified in the total sequence of the receptor deduced from the cDNA sequence. The binding-site sequences were localized in relatively hydrophilic regions in the extracellular domain. These experiments have identified the segments of the receptor polypeptide that constitute the ANP-binding site structure. The results have also indicated the topological orientation of the bound ANP relative to the elucidated bindingsite sequences. This approach may also be useful for studying the binding-site structure of other peptide hormone receptors.

Figure I. Schematic presentation of the structures of the stepwise affinity reagents for the ANP receptor. The arrows indicate positions of the reactive moiety, iodoacetyl-group.

165 Acknowledgments Able technical assistance by Xiaolun Zhang is gratefully acknowledged. This study is supported by grants RO1HL54329 from the NIH and GIA#95012310 from the American Heart Association National Center.

Reference 1. He, X., Nishio, K. and Misono, K.S., Bioconjugate Chemistry, 6 (1995) 541.

54 Structure of glycoside of Bombyx mori prothoracicotropic hormone S. NAGATA¹ , J. KOBAYASHI², H. KATAOKA¹ and A. SUZUKI¹ ¹ Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ² Faculty of Engineering Mie University. Tsu-City, Mie 514, Japan

Introduction Prothoracicotropic hormone (PTTH) is a brain neuropeptide that activates prothoracic glands to produce ecdysone, thereby playing a central role in the endocrine control of postembryonic development in insects. PTTH was first isolated from heads of the adult silkworm. Amino acid sequence was determined by peptide analysis and molecular cloning of cDNA [1,2]. PTTH of Bombyx mori is a homodimeric peptide which is held together by a disulfide bond [3]. Each chain consists of 109 residues and contains a carbohydrate chain bound to an asparagine at position 41. We here report the structure determination of carbohydrate chain of PTTH by using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) of glycopeptide before and after glycosidase digestions and HPLC analysis of pyridylaminated sugar chain.

Results and discussion The recombinant PTTH was expressed in E. coli and baculovirus systems to obtain non-glycosylated or glycosylated PTTH, respectively. The recombinant PTTH expressed in a baculovirus system (B-rPTTH) showed slightly higher molecular weight than that of the recombinant PTTH expressed in E. coli system (E-rPTTH) by MALDI-TOF-MS. When the lysylendopeptidase fragments of both recombinant PTTHs were compared by using reversed phase HPLC, only the fragments containing 41st asparagine had different retention times. In addition, MALDI-TOF-MS analysis of these fragment revealed that molecular weight of the fragment from B-rPTTH was larger than that from E-rPTTH by about 890 mass units, suggesting that the B-rPTTH was glycosylated at the 41st asparagine. To determine the structure of carbohydrate moiety, the glycopeptide obtained by lysylendopeptidase digestion of B-rPTTH was treated with several kinds of endo- and exo-glycosidases, and then the change of molecular weight of the glycopeptide was measured by MALDI-TOF-MS. From these analyses, 166 Y: Shimonishi (ed): Peptide Science – Present and Future, 166–167 © 1999 Kluwer Academic Publishers, Printed in Great Britain

167

Figure 1. The structure of carbohydrate moiety of PTTH.

the sugar chain of PTTH was determined as Man α–xMan β 1–4GlcNAc β 1–4 (Fuc- x)GlcNAc-(x indicates an undetermined linkage). This structure was also supported by the substrate specificities of glycosidases. In order to determine the two linkages between fucose and N-acetylglucosamine, and between terminal α -mannosyl residue and β -mannose, the sugar chain of PTTH was liberated by PNGase F, and labeled with 2-amino pyridine in reducing condition. Subsequently, the pyridylaminated sugar chain was subjected to HPLC analyses using ODS and TSK-amide columns. The elution time of pyridylaminated sugar chain after α -fucosidase and α -mannosidase treatments indicated that fucosyl and mannosyl residue were linked by the α 1–6 linkage. Finally, the structure of the glycosidic side chain on the B-rPTTH was determined as shown in Figure 1. Also, the structure of the sugar chain of natural PTTH purified from adult heads of Bombyx mori was determined by using the same methods. The main sugar chain on the natural hormone was identical to that of the B-rPTTH, but there existed the minor component whose fucose was lost. The biological activity of B-rPTTH in brainless Bombyx pupae was indistinguishable from that of the natural peptide, but E-rPTTH had about half activity of the natural peptide, indicating that the glycosidic side chain in PTTH stabilizes the hormone molecule in the hemolymph to reinforce the hormonal activity.

References 1. Kataoka, H., Nagasawa, H., Isogai, A., Tamura, S., Mizoguchi, A., Fujiwara, Y., Suzuki, C., Tshizaki, H. and Suzuki. A. Agric. Biol. Chem. 51(1987) 1067. 2. Kawakami, A., Kataoka, H., Oka, T., Mizoguchi, A., Kimura-Kawakami, M., Adachi, T., Iwami, T., Nagasawa, H., Suzuki, A. and Ishizaki. H. Science 247 (1990) 1333. 3. Ishibashi, J., Kataoka, H., Isogai, A., Saegusa, S., Yagi, Y., Mizoguchi, A., Ishizaki, H. and Suzuki. A. Biochemistry 33 (1994) 5912.

55 Photophysics of sterically constrained phenylalanines L. ANKIEWICZ, K. STACHOWIAK, A. RZESKA and W. WICZK Faculty of Chemistry, University of Gda k, Sobieskiego 18, Gda

k 80-952, Poland

Introduction In last years, there is a great interest in syntheses of sterically constrained amino acids and peptides in order to stabilize a conformation of peptides of interest, to improve receptor selectivity, and decrease enzymatic degradation. One of the most convenient amino acid to achieve conformation restrictions by various modifications is phenylalanine. And one of the most interesting conformational restriction of aromatic amino acids is based on the Pictet– Spengler reaction [1]. In order to use phenylalanine analogues in confomational studies of peptides by means of fluorescence it is necessary to know their photophysical behavior. In this communication we present photophysical properties of phenylalanine and its conformationally constrained analogues: tetrahydroisoquinoline3-carboxylic acid (Tic), (5-methyl) tetrahydroisoquinoline-3-carboxylic acid ((5-Me)Tic), (2'-methyl) phenylalanine ((2'-Me)Phe) and (2',6'-dimethyl) phenylalnine ((2',6'-Me2 ) Phe.

Results and discussion All studied analogues of phenylalanine (Tic, (5-Me)Tic, (2'-Me)Phe and (2',6'M e 2 )Phe) were prepared according to the procedures described in literature [2]. The chemical homogeneity of the synthesized compounds was assessed by ¹H NMR spectra, analytical reversed-phase HPLC and mass spectrometry (FD or FAB). Photophysical parameters were obtained as it is described in our previous similar studies of 7-hydroxy-tetrahydroisoquinoline-3-carboxylic acid and its derivatives [3]. The new phenylalanine analogues have shifted longwave absorption spectra and higher molar coefficients, what is advantageous for fluorescence studies. Incorporation of an one substituent into aromatic ring of phenylalanine analogue (e.g. (2'-Me)Phe and Tic) increases quantum yield (φ ) and decay time (τ ) (Table 1). On the other hand a presence of two substituents in the ring ((5-Me)Tic, (2',6'-Me2 )Phe) decreases quantum yield and decay time. Photophysical properties of unsubstituted Tic (a long decay time τ = 20 ns, quantum yield φ = 0.12 and batochromic shift of the spectrum) are making this 168 Y. Shimonishi (ed): Peptide Science – Present and Future, 168–170 © 1999 Kluwer Academic Publishers, Printed in Great Britain

169

Preparation of phenylalanine analogues.

Figure 1.

Table 1.

Photophysical parameters of phenylalanine and its analogues.

Compound

Quantum yield (φ)

Lifetime (τ) [ns]

k f • 10 – 6 [s –1 ] (k f = φ /τ)

kn r • 10– 8 [s –1 ] (k n r = ( 1 – φ) /τ)

Phe (2'-Me)Phe (2',6'-Me2 )Phe Tic (5-Me)Tic

0.020 0.046 0.012 0.120 0.048

6.48 6.17 2.57 20.04 12.94

3.09 7.45 4.67 5.99 3.71

1.51 1.55 3.84 0.44 0.74

particular amino acid suitable donor for energy transfer measurements. For comparison, very popular donor in such studies – tyrosine – has shorter decay time τ = 3.25 ns and comparable to Tic quantum yield φ = 0.14. Moreover, decay time and quantum yield for Tyr incorporated into a peptide chain strongly depend on microsurrounding – decay time becomes multiexponential and quantum yield decreases, what is not observed in the case of Tic. Fluorescence decay observed for Ac-Tic and Tic-NHMe is still monoexponential but decay time is slightly shorter. The conformational constrain in the case of Tic and (5-Me)Tic causes decrease of a rotational freedom of the chromophore’s linkers, what leads to more reliable estimation of interchromophoric distances.

170 Acknowledgements Supported by Polish Committee for Scientific Research (KBN).

References 1. Pictet, A. and Spengler T., Chem. Ber., 44 (1911) 2030. 2. Majer, P., Slaninova, J. and Lebl, M., Int. J. Peptide Protein Res., 43 (1994) 62. 3. Wiczk, W., Stachowiak, K., Skurski, P., ankiewicz, L., Michniewicz, A. and Rój, A., J. Am. Chem. Soc., 118 (l996) 8300.

56 Addressing combinatorial and peptide analysis by using overpressured layer chromatography in comparison with high-performance liquid chromatography and capillary electrophoresis G. DIBÓ¹, E. MINCSOVICS² and Z. MAJER¹ ¹ Department of Chemistry, Eötvös University, PO Box 32, Budapest 112, Hungary, H-1518 ² OPLC–NIT Engineering Co., Ltd., Andor u. 60., Budapest, Hungary, H-1119

Introduction The generation of libraries of short oligopeptides, peptidomimetics, peptoids or other small organic molecules in combination with high-throughput screening has become the method of choice for the production of new pharmacological leads for chemical optimization. Molecular diversity can be achieved in any desired form by using either the portioning–mixing [1] or parallel synthetic methods carried out manually in test tubes or on-the-spot, or automatically with the aid of sophisticated robotics [2]. However, characterization and identification of library composition have been lagging behind the synthetic and screening methodologies. This paper demonstrates the application of overpressured layer chromatography (OPLC) [3], a robust, high-throughput analytical technique for the rapid and efficient characterization of a simple mixture of model peptides.

Results and discussion Synthesis The peptides were synthesized by using polypropylene syringes fitted with a polyethylene filter disc as a reaction vessel. The syntheses were performed by standard Boc/Bz/Z chemistry on MBHA resin (substitution: 1.1 mmol/g). The acylation steps were carried out in dichloromethane using 3 equiv. HBTU, and 6 equiv. DIEA. The coupling reactions were monitored by Kaiser-test and repeated to completion when necessary. The peptides were removed from the resin by HF containing 10% p -cresol as scavenger. Overpressured layer chromatography OPLC was performed on Merck Silica gel 60 (20 × 20 cm) using the Personal OPLC Instrument (OPLC–NIT Engineering Co., Ltd., Budapest, Hungary). 171 Y. Shimonishi (ed): Peptide Science – Present and Future, 171–173 © 1999 Kluwer Academic Publishers, Printed in Great Britain

172

Figure 1. Overpressured Layer Chromatographic separation of synthetic model peptides. (From left to right) 1: Ac–Pro–Val–Val–Ser–Gly–NH 2 ; 2 : Ac–Pro–Val–Val–Ala–Gly–NH 2 ; 3 : Ac–Pro– Glu–Val–Ala–Gly–NH 2 ; 4: Ac–Pro–Glu–Val–Ser–Gly–NH 2 ; 5: Ac–Pro–Ser–Glu–Glu–Gly–NH 2 .

Several solvent systems were tested during optimization of the separation. Finally, a mixture of buthanol/pyridine/acetic acid/water (12:4:1:4, v/v/v/v) resulted in the best resolution. Other chromatographic parameters were also optimized: flow rate was set to 500 µl/min; eluent volume for the initial rapid development was 200 µ1; and total eluent volume was 5.2 ml. Terminal eluent pressure remained under 21 bar. After thorough drying, the chromatoplates were developed with chlorine–tolidine reagent. All components of the test mixture were completely resolved in a single run within 10 minutes. HPLC and CZE The same test mixture was submitted to HPLC separation using various gradients composed of acetonitrile and aqueous trifluoroacetic acid, and to

173 capillary zone electrophoresis at different pH (between 2–9) in phosphate, borate and citrate buffers. In these systems the five-component test mixture was unresolved or only separated into 2 or 3 overlapping peaks. These experiments show that OPLC can be orthogonally used with HPLC or CZE for the separation of highly related peptides.

Acknowledgements One of the authors (G.D.) was supported with a travel grant by the Soros Foundation.

References 1. Furka, Á., Sebestyén, F., Asgedom, M. and Dibó, G., Int. J. Peptide Prot. Res. 37 (1991) 487. 2. Cabilly, S., Combinatorial Peptide Library Protocols, Humana Press, 1997. 3. Mincsovics, E., Ferenczi-Fodor, K. and Tyihák, E., In: Sherma, J. and Fried, B., (Eds) Handbook of Thin-layer Chromatography, 2nd. Ed., Marcel Dekker, New York, 1996, pp. 171–203.

57 AVP(4-8)-induced branching signaling pathway was mediated by a putative GPCR in rat brain Q.-W. YAN, L.-Y. QIAO and Y.-C. DU Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China

Introduction In the past decade, the central effects of neurohypophyseal peptides (VP, OT and their metabolites) and the pharmaceutical properties of cloned receptors (V1a, V1b, V2 and OT-R) have been well documented [1]. However, the memory-enhancing mechanism of these neuropeptides in mammalian brain is still unknown. It has been found that among the AVP metabolites, the fragment AVP(4-8) did not possess the classical endocrine effects but showed extreme effectiveness on behavioral responses as its potencies in CNS were hundreds fold higher than those of AVP itself [2,3]. Moreover, the VP/OT receptors in the brain did not display affinity for this small molecule [2] and the AVP(4-8) binding sites in the limbic system could not be competed by AVP [4,5]. Thus it was suspected that AVP and its metabolite AVP(4-8) have separate special receptors in the brain. In fact, in a series of comparative studies, we have found that the biochemical parameters of AVP(4-8) usually differed from that of AVP, especially in the following aspects such as the consequent accumulation of the second messenger IP 3 [6], the stimulation of c-fos gene expression [7], the enhancement of neurotrophin gene expression [8] and the induction of long-term potentiation in rat hippocampal slices [9] etc. To clarify the characteristics of AVP(4-8) receptor, ZDC(C)PR, a synthetic antagonist, and pertussis toxin (PTX), a selective blocker of GTP-binding protein, were employed to investigate the signaling pathway of AVP(4-8). In the present work, experimental data suggested that AVP(4-8) receptor may couple with a G0 protein and its signaling pathway from IP3 to LTP and neurotrophin gene expression was respectively conducted by Ca 2+/CaM-dependent protein kinase II (CaMKII) and protein kinase C (PKC).

Results and discussion MAPK and CaMKII activations We have reported that both MAP kinase (p44 MAPK) activity and CaMKII autophosphorylation in rat brain were significantly enhanced by AVP(4-8) treatment and reached the maximum in 1–2 h after administration [10]. 174 Y. Shimonishi (ed): Peptide Science – Present and Future, 174–178 © 1999 Kluwer Academic Publishers, Printed in Great Britain

175 Furthermore, similar effects could also be observed in rat hippocampal slices. The exposure of rat hippocampal slices to AVP(4-8) rapidly increased MAPK activity and CaMKII autophosphorylation within 5–10 min, as shown by measuring the phosphorylation of the specific substrate MBP and in the presence and absence of an exogenous substrate respectively (Figure 1). ZDC(C)PR (0.5 µM) is an antagonist with Asp2 replacement of AVP(4-8). While it per se has no effect on MAPK activity and CaMKII autophosphorylation levels, nevertheless did markedly block 50 nM AVP(4-8)-enhanced protein kinases activation when present in the culture medium of hippocampal slices. On the other hand, 50-fold of ZDC(C)PR (5 µM) had no effect on the AVP( 0.1 µM)-induced MAPK activation. To see if a subfamily of AVP(4-8) receptor was involved, the hippocampal slices were pre-incubated with PTX for 30 min before the exposure to AVP(4-8). Results presented in Figure 2 revealed that the AVP(4-8)-stimulations of MAPK and CaMKII were both inhibited by PTX. However, our experimental data also indicated that AVP-induced MAPK activation was not sensitive to PTX. PKC and branching pathway To examine the role of protein kinase C (PKC) in the signaling pathway induced by AVP(4-8), PKC inhibitor (PMB) and activator (TPA) were employed. Treatment of hippocampal slices with TPA for 5 min elicited an increase of MAPK activity. Nevertheless, stimulating effect of by AVP(4-8) or AVP, together with TPA was not statistically different from that by each agent alone. The treatment with PMB for 5 min before exposure to the peptides completely abolished MAPK activation by AVP( 4-8).

Figure 1.

AVP(4-8) rapidly activated both MAPK and CaMKII in rat hippocampal slices.

176

Figure 2.

PTX and ZDC(C)PR blocked AVP(4-8)-stimulated MAPK and CaMKII activities.

The extent of CaMKII autophosphorylation induced by AVP(4-8) was attenuated by the simultaneous activation of PKC with TPA, but not affected by PMB treatment. Nevertheless, the extent of CaMKII activation was attenuated by TPA. KN-62 is a relatively selective inhibitor of CaMKII. The experiments were performed by pre-incubating hippocampal slices with KN-62 followed by the addition of AVP(4-8) for further incubation. Densitometry scanning of the autophosphorylation showed that the enhancement of total density of α and β subunits of CaMKII by AVP(4-8) stimulation was completely blocked by KN-62. The simultaneous measurement of MAPK activity indicated that the inhibition of CaMKII activity by KN-62 did not influence the activation of MAPK by AVP(4-8). Therefore, the signaling pathway from AVP(4-8) receptor to MAPK and to CaMKII were thought to be of two branches. PKC is between the receptor to MAPK but not that to CaMKII and there may be a negative cross-talk between PKC and CaMKII (see Figure 3.) CaM and its expression Enzymatic determination demonstrated that the free calmodulin concentration in the hippocampus readily increased by 3.4 fold at h-1 after treatment with exogenous AVP(4-8) and then gradually up to its maximum (5.2 fold) at h-3 in comparison with the control group. By using cDNA probe and dot hybridization analyses we found that CaM gene expression in the same tissue showed no significant change at h-1 after treatment but was markedly stimulated to a maximum (150%) at h-3. It is suggested that the increase of CaM may occur in two steps, i.e., a rapid delivery of CaM pool (GAP43) in the first stage and the expression of CaM gene in the later on. Based on the curves shown in Figure 4 we suggested that the former may initiate CaMKII activation and the later may be necessary for sustaining long-term potentiation.

177

Figure 3. Links in AVP(4-8)-induced signaling pathway in rat hippocampus.

Figure 4.

CaM gene expression replenished CaM pool and LTP.

GTP-binding protein and its coupled receptor By using 35 S-labelled GTP γ S we found that AVP(4-8) dose-dependently stimulated [ 35 S]GTP γ S binding on hippocampal synaptic membranes and this stimulation was blocked by PTX, a selective inhibitor of the G-protein-coupled receptor (GPCR), and by the antagonist ZDC(C)PR as shown in Figure 5. Since PTX and ZDC(C)PR can respectively block AVP(4-8) signaling pathway

178

Figure 5.

AVP(4-8)-stimulated GTP binding to G protein was blocked by ZDC(C)PR.

at the receptor and the GP level, it is suggestive that the AVP(4-8) receptor may belong to a GPCR family. Furthermore our results showed that in contrast to AVP(4-8), AVP-stimulation of MAPK activity could not be blocked by PTX. In view of the mentioned facts, i.e., AVP(4-8) binding can not be competed by AVP and VP/OT receptors in the brain do not display affinity for AVP(4-8), we believe that in rat hippocampus, there is a GPCR which is structurally distinct from known AVP receptors and it initiates memory-enhancing process via a branching signaling pathway as shown in Figure 3.

References 1. Burbach, J.P.H., Aden, R.A.H., Lolait, S.J., van Leeuwen, F.W., Mezey, E., Palkovits, M. and Barberis, C., Cell. Mol. Neurobiol., 15 (1995) 573. 2. De Wied, D., Diamant, M. and Fodor, M., Front. Neuroendocrinol., 14 (1993) 251. 3. Lin, C., Liu, R.Y. and Du, Y.C., Peptides, 11(1990) 633. 4. Du, Y.C., Guo, N.N. and Chen, Z.F., Acta Physiol. Sin., 46 (1994) 433. 5. Du, Y.C., Wu, J.H., Jiang, X.M. and Gu, Y.J., Peptides, 15 (1994) 1273. 6. Gu, B.X. and Du, Y.C., Chin, J. Biochem, Biophys., 24 (1992) 287. 7. Gu, B.X. and Du, Y.C., Acta Biochem. Biophys. Sin., 23 (1991) 537. 8. Zhou, A.W., Guo, J., Wang, H.Y., Gu, B.X. and Du, Y.C., Peptides, 16 (1995) 581. 9. Rong, X.W., Chen, X.F. and Du, Y.C., Neuroreport, 4 (1993) 1135. 10. Qiao, L.Y. and Du, Y.C., Acta Pharmacol. Sin., 18 (1997) 380.

58 The mechanisms of exocytosis induced by neurokinin peptides in mast cells H. MUKAI, M. KIKUCHI, Y. SUZUKI and E. MUNEKATA Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Introduction Neurokinin peptides consisting of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) are tachykinin family peptides isolated from mammals that have a wide variety of physiological functions such as the depolarization of primary sensory neurons, the contraction of smooth muscle, the exocrine secretion from pancreas and hypotension [1]. Most of biological functions of neurokinin peptides are mediated by three pharmacologically distinct cell membrane receptors classified as NK1 , NK 2 and NK 3 neurokinin receptors [1]. It has been already cloned functional cDNAs for three neurokinin receptors. Neurokinin peptides have a common Phe–X–Gly–Leu–Met–NH 2 structure (X is Phe or Val) at the C-terminus, and this structure is required for the activation of known neurokinin receptors. SP is also known to stimulate the secretion of histamine from peritoneal mast cells. As regard the mechanisms of exocytosis in mast cells induced by SP, it is already demonstrated that exocytosis stimulated by SP is not due to the activation of three known neurokinin receptors by the structure-activity studies using SP and its derivative peptides on the stimulation of exocytosis, that positively charged amino acid residues are important for the stimulation of exocytosis and a common Phe–X–Gly–Leu– Met–NH 2 structure at C-terminus is not required. In addition, it was suggested the direct activation of G i type of G protein in mast cells by SP as well as mastoparan (MP), that also stimulates exocytosis in mast cells [2,3]. It is already demonstrated that SP and MP induce the production of IP 3 and the activation of voltage-independent Ca 2+ channels indicating the importance of cytosolic Ca 2 + on the exocytotic pathway. However, for the sake of the difficulty in measuring the concentration of intracellular free Ca2 + ([Ca 2+ ] i ) n mast cells, it has not been clear the involvement of cytosolic Ca 2+ on the exocytotic pathway. In this study, we found the conditions that we could measure [Ca 2+ ] i change in rat peritoneal mast cells, and measured [Ca 2 + ] i increase induced by SP and MP to investigate the relationship between [Ca 2 + ] i increase and exocytosis in rat peritoneal mast cells. The secretion of β -hexosaminidase from mast cells was measured as the exocytotic secretion, because this enzyme was found in the same secretory vesicles that histamine was contained. Increase of [ C a 2+ ] i was measured using fura-2, a sensitive luminescent Ca2+ chelator. 179 Y. Shimonishi (ed): Peptide Science – Present and Future, 179–182 © 1999 Kluwer Academic Publishers, Printed in Great Britain

180 Results and discussion At first, we examined the effects of neurokinin peptides on the stimulation of β -hexosaminidase release. SP and MP stimulated β -hexosaminidase release in concentration-dependent manner, but NKA and NKB did not (data not shown). It was examined that the effect of SP and MP on the [ C a 2 +] i increase to investigate how [Ca 2+ ] i increase involved in the secretory pathway. As shown 2+ in Figure 1, 30 µM of SP as well as MP induced [Ca ] i increase even in the 2+ absence of extracellular free Ca , but the maximal increases were reduced to 2+ 37% and 20% of the levels seen in the presence of extracellular C a , respectively. In case of the stimulation of SP, only transient increase in [Ca 2 + ]i immediately after the stimulation was observed. However, the stimulation of MP induced sustained increase in [Ca 2+ ]i followed by the transient increase in the presence of extracellular Ca 2 + whereas this sustained increase did not 2+ 2+ occur in the absence of extracellular Ca . These results indicate that [Ca ] i increase induced by SP and MP was due to the combined mobilization of internal Ca 2 + and the entry of extracellular Ca2 + . Next, we measured the kinetics of the secretion induced by SP and MP in 2+ the presence or absence of extracellular free Ca . As a result, faster kinetics of secretion stimulated by SP and MP was observed in the presence of extracellular Ca 2 + than in the absence. T/2’s of secretion were about 4 and 10 sec after the stimulation of 30 µM SP, and 3 and 6 sec after the stimulation of 30 µM

Figure 1.

Effect of the presence of extracellular Ca 2 + on [Ca 2+ ] i increase induced by SP and MP.

181 MP in the presence or absence of extracellular C a 2 + , respectively. In addition, exocytotic events were done within 30 sec. These results demonstrated that the kinetics of exocytosis induced by SP and MP coincides with transient increase in [Ca 2+ ] i immediately after the stimulation suggesting that transient increase in [Ca 2 + ] i but not sustained one is responsible to the exocytotic secretion pathway. To confirm the involvement of [Ca 2+ ] i increase in the secretory pathway further, it was examined the inhibitory effects of pertussis toxin (PTX), which ADP-ribosylates Gi type of G proteins and renders them insensitive to regulation, on the stimulation of β -hexosaminidase release and the increase of [Ca 2+ ] i induced by SP and MP. Stimulation of β -hexosaminidase release by 30 µM SP and 20 µM MP was almost completely inhibited by pretreatment of mast cells with PTX as shown in Table 1. The increase in [Ca 2 + ] i induced by SP was blocked almost completely by pretreatment with PTX (Figure 2). In case of the stimulation by MP, only transient increase in [Ca2+ ] i was inhibited by the treatment with PTX, but sustained increase was not (Figure 2). These finding indicated that transient increase in [ C a 2+ ] i , but not sustained one involves in the exocytotic secretion pathway. These findings also suggest the presence of at least two different types of Ca 2 + channels: one is pertussis toxin sensitive channels stimulated by SP and MP that involve in transient [Ca 2 + ] i increase and exocytotic secretion and the other is pertussis toxin insensitive channels that involve in sustained [Ca 2 + ] i increase but not the secretion. 2+ In this study, it was measured [Ca ] i increase and exocytotic secretion induced by neurokinin peptides as well as MP in rat peritoneal mast cells to investigate the mechanisms of exocytosis, especially to elucidate the involvement of cytosolic Ca 2 + in exocytosis. SP and MP stimulated [ C a 2+ ] i increase and the secretion in concentration-dependent manner, but NKA and NKB did not. It was indicated that SP- and MP-induced transient increase in [ Ca 2 + ] i immediately after the stimulation was due to the combined mobilization of internal 2+ C a 2+ and the entry of extracellular Ca . MP, but not SP, caused not only 2+ transient [Ca ] i increase but also sustained one that was due to the influx of extracellular Ca 2 +. It was demonstrated that the kinetics of exocytosis induced by SP and MP coincides with transient increase in [ C a2 +] i , and these stimula2+ tion’s were prevented by PTX. However, sustained increase in [Ca ] i induced Table 1. Effect of preincubation of mast cells with pertussis toxin on β -haxosaminidase release induced by SP and MP. Peptide

Pertussis toxin

β -hexosaminidase release (%/total)

SP (30 µM)

– + – +

51.3 5.5 42.2 8.4

MP (20 µM)

Rat peritoneal mast cells were preincubated with 100 ng/ml of pertussis toxin before the stimulation by peptides.

182

Figure 2.

Effect of preincubation of mast cells with PTX on [Ca2 + ] i increase by SP and MP.

by MP was not inhibited by PTX. These results suggest that only transient increase in [Ca 2+ ] i induced by SP and MP involves in the mechanisms of exocytosis in mast cells. These findings also suggested the presence of at least two different types of Ca 2+ channels; one is PTX-sensitive channels stimulated by SP and MP that involve in transient increase in [ C a 2 + ] i and exocytotic secretion and the other is PTX-insensitive channels that involve in sustained increase in [Ca 2+ ]i but not the secretion.

References 1. Munekata, E., Comp. Biochem. Physiol., 98C (1991) 171. 2. Mukai, H. and Higashijima, T., Peptide Chemistry, 1992 (1993) 330. 3. Mukai, H., Higashijima, T., Suzuki, Y. and Munekata, E., In Kaumaya, P.T.P. and Hodges, R.S. (Eds) Peptides:Chemistry, Structure and Biology (Proceedings of the 14th American Peptide Symposium), Mayflower Scientific, 1996. pp 403–405.

59 Effects of dynorphins on free cytoplasmic calcium concentration and insulin release in ob/ob mouse pancreatic β -cells S.V. ZAITSEV 1,2 , I.B. EFANOVA 1,2 , A.M. EFANOV 1,2 and

P.-O. BERGGREN ¹

¹ The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, S-171 76 Stockholm, Sweden ² Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119899 Moscow, Russia

Introduction Opioid peptides of the dynorphin family have been found to be localized in the pancreatic islets of Langerhans, preferentially in glucagon and somatostatin secreting cells, adjacent to insulin producing β-cells [1]. Moreover, dynorphins have been shown to be tonic modulators of insulin secretion from the islets [1]. However, information about the mechanisms whereby these peptides affect insulin secretion is limited. The aim of the present study was to investigate the role of cytoplasmic free calcium concentration ([Ca2+ ] i ) in the effects of dynorphins on insulin secretion. Results and discussion Effects of dynorphin A (Dyn A), dynorphin B (Dyn B), dynorphin A 1–13 (Dyn A1–13 ), on [Ca 2+ ] i in pancreatic β -cells from ob/ob mouse have been studied. Measurements of [Ca 2+ ] i have been performed using the fluorescent Ca 2+ indicator fura 2AM, according to the procedure previously described [2]. At basal glucose concentration (3.3 mM) neither Dyn A nor Dyn A 1–13 , in the concentration range 10 –9 –10 – 6 M, had any significant effect on [Ca 2+ ] i (data not shown). Increase in glucose concentration in the medium to 6 mM gave rise to pronounced changes in [Ca 2 + ] i at 10 – 6 M concentrations of Dyn A, Dyn A 1–13 and Dyn B (Figure 1). The opioid peptides affected both the oscillatory pattern of [Ca 2+ ] i and increased the average [Ca 2+ ] i level. No significant changes in [Ca 2+ ] i have been seen in the presence of 10 – 9 –10 – 8 M of Dyn A under these conditions (data not shown). To check if opioid receptors were involved in the effects of dynorphins on [Ca 2 + ] i, experiments with the opioid antagonist naloxone were performed (Figure 1D). Naloxone (10 – 5 M ) inhibited the stimulatory activity of Dyn A 1–13 on [Ca 2+ ] i, suggesting the involvement of opioid receptors in the effects of dynorphins on [Ca 2+ ] i . Effects of Dyn A 1–13 on insulin secretion from ob/ob mouse pancreatic islets have been studied in the same medium as measurements of [Ca2+ ] i [ 2 ] , using 183 Y. Shimonishi (ed): Peptide Science– Present and Future, 183–184 © 1999 KIuwer Academic Publishers, Printed in Great Britain

184

Figure 1. Effects of 10 – 6 M DynA (A), 10 –6 M Dyn B (B), 10 – 6 M Dyn A1–13 (C), on [Ca 2 + ] i in ob/ob mice pancreatic β-cells in the presence of 6 mM glucose. D. Inhibition of the stimulatory effect of Dyn A 1–13 on [Ca 2+ ]i by 10 –5 M naloxone. Basal glucose level is 3.3 mM. Table 1. Effect of dynorphin A1–13 (DynA 1–13 ) on insulin secretion in the presence of 6 mM glucose in ob/ob mouse pancreatic islets. Test reagents

Insulin secretion (µU/islet/30 min)

p relative to 6 mM glucose

6 mM glucose + 10 – 5 M naloxone + 10 – 9 Dyn A1–13 + 10 –9 Dyn A 1–13 + 10 –5 M naloxone + 10 – 6 Dyn A 1–13 + 10 – 6 Dyn A 1–13 + 10 – 5 M naloxone

18 ± 4 17 ± 3 48 ± 10 52 ± 11 49 ± 7 51 ± 6

Ns p< p< p< p
10000 > 10000

620 ± 50 2000 ± 1100 120 ± 56 4000 ± 1700 3300 ± 670

> 10000 > 10000 5600 > 10000 > 10000

>10000 >10000 >10000 >10000 >10000

alanine ((p-F)Phe) was placed either at Phe-1 or Phe-4, a different effect became prominent (Table 1). Phe-1/(p-F)Phe substitution decreased the binding affinity (20–40%). In contrast, Phe-4/( p-F)Phe substitution slightly increased the affinity. Similar results were also obtained in the mouse tail-flick test. Nociceptin consists of a couple of basic amino acid pairs, namely two Arg–Lys sequences at positions 8–9 and 12–13. These pairs seemed to be processing sites for peptidases. Since nociceptins-(1–7) and -(1–11) were inactive, these basic pairs are not the sites for processing to produce active fragments (Table 1). When Arg–Lys was replaced by Ala–Ala, resulting derivatives of [Ala 8,9 ]- and [Ala 12,13 ]nociceptins were found to be almost completely inactive in the receptor binding assay. In the previous study, the analysis of nociceptin analogs with stepwise deletion of amino acid residues from C -terminus indicated that Asn-16 and Lys-13 were important [4]. Indeed, [Ala 16 ]nociceptin decreased its activity (13%). Recently, Butour et al. reported that C -terminal fragment of nociceptin, namely nociceptins-(6–17) and -(12–17) exhibits considerably high binding activity [5]. We synthesized and assayed both peptides, since the results suggest the insignificance of nociceptin N -terminal for receptor activities. However, no receptor binding and no receptor activation were observed for both peptides in this study. This clearly indicates that the N -terminal portion of nociceptin is crucially important for receptor responses. References 1. Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butoru, J.-L., Guillemot, J.-C., Ferrara, P., Monsarrat, B., Mazarguil, H., Vassart, G., Parmentier, M. and Costentin, J., Nature, 377 (1995) 532. 2. Reinschie, R.K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R.A., Bunzow, J.R., Grandy, D.K., Langen, H., Monsma, F.J. Jr. and Civelli, O., Science, 270 (1995) 792. 3. Shimohigashi, Y., Hatano, R., Fujita, T., Nakashima, R., Nose, T., Sujaku, T., Saigo, A., Shinjo, K. and Nagahisa, A., J. Biol. Chem., 271 (1996) 23642. 4. Hatano, R., Nose, T., Nakashima, R., Sujaku, T., Saigo, A., Nagahisa, A. and Shimohigashi, Y. In C. Kitada (Ed) Peptide Chemistry 1996 (Proceedings of the 34th Symposium on Peptide Chemistry), Protein Research Foundation, Osaka, 1997, pp. 201. 5. Butour, J.-L., Moisand, C., Mazarguil, H., Mollereau, C., Meunier, J.-C., Eur. J. Pharmacol., 321 (1997) 97.

66 Activation of phosphatidylinositol 3-kinase via epidermal growth factor receptor in isolated rat hepatocytes T. FUJIOKA and M. UI Ui Laboratory, The Institute of Physical and Chemical Research, Wako 351-0198, Japan

Introduction The epidermal growth factor receptor (EGFR) transduces mitogenic and other biological signals of EGF. The EGFR is the prototype of the type I subfamily, which includes three additional members: ErbB-2/Neu, ErbB3, and ErbB4. Phosphatidylinositol (PI) 3-kinase is a lipid and serine kinase that contains two SH2 domains and is specifically inhibited by a fungal metabolite, wortmannin [1]. In EGF signaling, PI 3-kinase does not appear to bind directly to the EGFR although EGF activates the kinase in a number of types of cells and cell lines. It was demonstrated that ErbB3 [2], p 1 2 0 cbl , a proto-oncogene product [3], and Gab1, the Grb2-binding protein [4], serve as the adapters between EGFR and PI 3-kinase. However, that was not the case with isolated rat hepatocytes. Thus, we investigated another signaling pathway for EGFstimulated PI 3-kinase activation in hepatocytes.

Results and discussion Hepatocytes were isolated from adult male rats by the collagenase perfusion method. Activation of PI 3-kinase of hepatocytes in response to receptor stimulation was determined by the following two methods. First, 32P-labeled cells were stimulated and the incorporation of 3 2 P into phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ), a product of the enzyme, was measured after separation of extracted cellular phospholipids on TLC. Second, the anti-phosphotyrosine (PY) immunoprecipitates prepared from the lysates of stimulated cells were assayed for PI 3-kinase activity with [γ -32 P ] ATP and PI as substrates. Stimulation of hepatocytes with EGF resulted in activation of PI 3-kinase, as detected by either of the two methods, in time- and concentration-dependent and wortmannin-susceptible manners. No PI 3-kinase activity was detected in tyrosine-phosphorylated EGFR, ErbB2, or ErbB3 immunoprecipitated with the individual antibodies as well as in the ErbB3, p120 cbl or Gab1 fraction precipitated with the respective antibodies. Hence, these proteins are unlikely to be involved in PI 3-kinase activation. The antibody raised aginst p85, the 200 Y: Shimonishi (ed): Peptide Science – Present and Future, 200–201 © 1999 Kluwer Academic Publishers, Printed in Great Britain

201 regulatory subunit of PI 3-kinase, immunoprecipitated several tyrosine-phosphorylated proteins including a protein with 190-kDa. It proved to be insulin receptor substrate-1 (IRS-1), which was tyrosine-phosphorylated in response to EGF stimulation of hepatocytes in a time- and concentration-dependent manner. The anti-IRS-1 immunoprecipitate was associated with p85 and exhibited PI 3-kinase activity that increased following EGF stimulation of cells. Thus, we show for the first time that EGF gives rise to tyrosine phosphorylation of IRS-1, which must mediate EGF-induced activation of PI 3-kinase, in hepatocytes. Involvement of insulin receptors in these EGF effects is unlikely because of undetectable tyrosine phosphorylation of insulin receptor β -subunits under the present conditions, although further studies will be required for a decisive role of IRS-1 in EGFR signaling to be proposed. In fact, roles of IRS-1 in cellular signaling arising from receptors of growth factors or cytokines other than insulin have been reported [5]. Since other proteins, such as IRS-2 [6], IRS-3 [7], IRS-4 [8], DOS [9], and Gab1 [5], that are structurally or functionally related to IRS-1 have recently been identified, differential roles of these members of ‘IRS family’ in cellular signaling will be the subjects of further extensive investigations.

References 1. Ui, M., Okada, T., Hazeki, K. and Hazeki, O., Trend. Pharmacol. Sci., 20 (1995) 303. 2. Soltoff, S.P., Carraway III, K.L., Prigent, S.A., Gullick, W.G. and Cantley, L.C., Mol. Cell. Biol., 14 (1994) 3550. 3. Soltoff, S.P. and Cantley, L.C., J. Biol. Chem., 271(1996) 563. 4. Holgado-Madruga, M., Emlet, D.R., Moscatello, D.K., Godwin, A.K. and Wong, A.J., Nature, 379 (1996) 560. 5. Yenush, L. and White, M.F., BioEssays, 19 (1997) 491. 6. Sun, X.L., Wang, L.M., Xhang, Y., Yenush, L., Myers, M.G. Jr., Glasheen, E., Lane, W.S., Pierce, J.H. and Whitae, M.F., Nature, 377 (1995) 173. 7. Lavan, B.E., Lane, W.S. and Lienhard, G.E., J. Biol. Chem., 272 (1997) 11439. 8. Lavan, B.E., Fantin, V.R., Chang, E.T., Lane, W.S., Keller, S.R. and Lienhard, G.E., J. Biol. Chem., 272 (1997) 21403. 9. Raabe, T., Riesgo-Escovar, J., Liu, X., Bausenwein, B.S., Deak, P., Maroy, P,, and Hafen, E., Cell, 85 (1996) 911.

67 Agonist-antagonist structure-activity relationships of thrombin receptor tethered ligand peptide T. FUJITA¹, T. NOSE¹, M. NAKAJIMA², Y. INOUE², N. NAKAMURA², T. INOUE³, T. COSTA4 and Y. SHIMOHIGASHI¹ ¹ Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan ² Research Division, The Green Cross Corp., Hirakata 573-1153, Japan ³ Institute of Genetic information, Kyushu University, Fukuoka 812-8582, Japan 4 Laboratory di Farmacologia, Istituto Superiore di Sanità, 00161 Roma 299, Italy

Introduction Serine proteinase thrombin plays the central role in blood coagulation, and has a specific receptor on the platelet surface. The receptor is activated when thrombin cleaves the receptor N-terminal extension to expose a tethered ligand. Synthetic heptapeptide SFLLRNP corresponding to this tethered ligand is feasible to activate the receptor without thrombin [1]. Systematic studies on structure-activity relationships between the ligand and the receptor have clarified several structural essentials of SFLLRNP for receptor binding and activation [2–4]. An antagonist of receptor can be a useful tool to understand the molecular mechanism of receptor activation, and for thrombin receptor, it would be a good remedy for thrombosis in therapeutics. To design a receptor antagonist, it is important to assess the role of each amino acid residue in ligand peptide in interaction with the receptor molecule. In the present study, in order to obtain an effective antagonist of thrombin receptor, we have designed and synthesized several SFLLRNP analogs loading various structural elements, that could be expected to establish new interaction with the receptor.

Results and discussion Since Ser-1 and Phe-2 of the ligand peptide are the most important residues for receptor activation, we first searched their specific binding sites in a transmembrane model of thrombin receptor. It was suspected that the sites for these amino acids exist at the junction between the second extracellular loop and the upper side of the fifth transmembrane domain where there is a cluster of aromatic residues. Since a cysteine residue adjacent to this putative binding site was found, we intended to capture this receptor cysteine. We thus synthesized several analogs of SFLLRNP containing a thiol group or its S-3-nitro2-pyridinesulphenyl (Npys) derivative at position 1. trans -Cinnamoyl group was also placed at the same position to evaluate the hydrophobic phenylvinyl 202 Y. Shimonishi (ed): Peptide Science – Present and Future, 202–204 © 1999 Kluwer Academic Publishers, Printed in Great Britain

203 Table 1.

Agonist and antagonist activities of ligand peptide analogs in platelet aggregation.

Peptides 1 2 3 4 5 6 7 8 9 10 11 12

SFLLRNP–NH 2 L -Cys/FLLRNP–NH 2 D -Cys/FLLRNP–NH 2 D -Cys(Npys)/FLLRNP–NH 2

(trans-Cinnamoyl)/( p-F)Phe/RLR–NH 2 [5] β Mp/( p-F)Phe/R/Cha/RY–NH 2 (Npys) β Mp/(p-F)Phe/R/Cha/RY–NH 2 (trans-Cinnamoyl)/( p-F)Phe/R/Cha/RY–NH 2 Thioglycollyl/( p -F)Phe/R/Cha/RY–NH 2 β Mp/(p-F)Phe/Cha/Y–NH 2 β Mp/( p -F)Phe/RY–NH 2 (Npys) β Mp/( p -F)Phe/RY–NH 2

Agonist Activity EC 5 0 ( µM)

Antagonist Activity a ) IC 5 0 (µM)

3.8 ± 1.3 inactive inactive inactive 64 ± 3.6 69 ± 8.9 inactive inactive 24 ± 14 inactive inactive inactive

– n.d. n.d. n.d. 2.6 ± 0.44 11 ± 12 34 ± 1.2 3.6 ± 1.2 16.6 ± 1.8 inactive inactive inactive

a )

Inhibition activity was examined against 6 µM SFLLRNP. β M p : β -mercaptopropanoyl n.d. : not determined

in receptor interaction. Furthermore, Phe-2 was substituted with para -fluorophenylalanine ((p-F)Phe), expecting to increase the receptor affinity due to the π – π interaction. Synthetic peptides were assayed in the test for human platelet aggregation, and the antagonist activity was evaluated by their ability to suppress the platelet aggregation induced by agonist SFLLRNP (Table 1). The peptides which exhibited agonist activity were analogs 5, 6 , and 9 . They were not so strong as SFLLRNP. All other peptides showed no activity in platelet aggregation. Then, all analogs were examined for their inhibitory activity against SFLLRNP (6 µM) in the platelet aggregation. Analogs 5 – 9 were found to inhibit this platelet aggregation. Analog 5 was reported previously as an effective antagonist [5]. This was confirmed in the present study (IC 50 = 2.6 µM), although we found that it is a partial agonist to induce aggregation. In this study, analog 8 was found to be an antagonist almost equipotent to analog 5, and devoid of appreciable agonist activity. The residual difference between peptides 5 and 8 is amino acids Leu and Cha at position 4. Thus, Cha-4 might be a determinant in antagonist activity. Analogs 5, 7 , and 8 have hydrophobic groups at their N-terminal, and therefore, a hydrophobic interaction between ligand and the receptor may be also a structural determinant for the antagonist activity. All analogs which showed inhibitory activity did not suppress platelet aggregation induced by adenosine 5'-diphosphate (ADP), indicating that they are antagonists specific for thrombin receptor. References 1. Vu, T.-K. H., Hung, D.T., Wheaton, V.I. and Coughlin, S.R., Cell, 64 (1991) 1057. 2 . Nose, T., Shimohigashi, Y., Ohno, M., Costa, T., Shimizu, N. and Ogino, Y., Biochem. Biophys. Res. Commun., 193 (1993) 694.

204 3. Shimohigashi, Y., Nose, T., Okazaki, M., Satoh, Y., Ohno, M., Costa, T., Shimizu, N. and Ogino, Y., Biochem. Biophys. Res. Commun., 203 (1994) 366. 4. Nose, T., Shimohigashi, Y., Okazaki, M., Satoh, Y., Costa, T., Shimizu, N., Ogino, Y. and Ohno, M., Bull. Chem. Soc. Jpn., 68 (1995) 2695. 5. Bernatowicz, M.S., Klimas, C.E., Hartl, K.S., Peluso, M., Allegretto, N.J. and Seiler, S.M., J. Med. Chem., 39 (1996) 4879.

68 The first extracellular loop of the rat glucagon receptor contains determinants of glucagon binding C.G. UNSON, A.M. CYPESS, C.-R. WU, C.P. CHEUNG, B. YOO and R.B. MERRIFIELD Rockefeller University, New York, New York 10021, U.S.A.

Introduction The first step in glucagon action is binding with high affinity to a specific glycoprotein receptor on the surface of the hepatocytes. The glucagon receptor belongs to a unique group of receptors in the superfamily of putative sevenhelical, transmembrane, G protein-coupled receptor proteins [1, 2]. Members of this subtype, which include receptors for related peptide hormones, glucagonlike peptide-1 (GLP-1) and secretin, are fairly homologous to each other, especially in their membrane-spanning helices. This suggests that specificity in the recognition mechanism lies not only in subtle variations in sequences of the ligands but also in structural subtleties within the receptors themselves. Extensive structure–activity studies of glucagon have identified specific residues in the peptide ligand that are responsible for either receptor binding or transduction. The complementary recognition site on the receptor which is the ‘switch’ that turns on the ensuing cascade of enzymatic events that constitute the transduction step, has yet to be deduced. Encouraged by our progress in the study of the peptide ligand glucagon using chemical synthetic methods, we have expanded our investigation to include the glucagon receptor protein, which will furnish additional information and complement ligand structure– function analysis.

Results and discussion Our previous work has implicated residues 126–137 from the membrane proximal region and 206–219 from the first extracellular loop of the receptor to be involved in glucagon binding [3]. Characterization of mutant receptor chimeras in which these regions had been replaced with analogous GLP-1 and secretin receptor sequences, confirmed that these sequences were part of the putative ligand binding pocket [4]. Interestingly, mutation of the 206–219 segment in the first extracellular loop region in either secretin/glucagon or GLP-1/glucagon receptor chimeras, had a more profound effect on both binding and activity than the replacement of the 126–137 sequence in the membrane proximal part of the N-terminal tail. This indicated that the first extracellular 205 Y. Shimonishi (ed): Peptide Science – Present and Future, 205–206 © 1999 Kluwer Academic Publishers, Printed in Great Britain

206 loop contained specific determinants of ligand binding. Close inspection of the loop sequence reveals a cluster of charged residues that may interact directly with glucagon. Given these initial observations, more rigorous structure– function studies by systematic mutation of specific amino acid residues within the first extracellular loop of the glucagon receptor were warranted. To continue our investigation, we altered candidate residues in the first loop that are potentially critical for forming the glucagon binding pocket. Residues 202, 206, 209, 210, and 219 of the glucagon receptor were each replaced by the analogous amino acid from the GLP-1 receptor. Mutant receptor genes were prepared by restriction fragment replacement ‘cassette mutagenesis’ using a synthetic rat glucagon receptor gene, and expressed following transient transfection in COS-1 cells [5]. Endoglycosidase H susceptibility analysis on immunoblots of membranes from transfected cells, showed that the mutant receptors were normally expressed on the plasma membrane surface [6]. The mutant receptors were assessed for the ability to bind ligand and to activate adenylyl cyclase relative to the native receptor. The IC 50 for competitive binding (31.6 nM) and the EC 50 for glucagon-stimulated adenylyl cyclase activity (3.2 nM) of the wild type receptor were consistent with reported values. Receptor mutant K206A displayed a threefold decrease in binding affinity while maintaining the ability to activate adenylyl cylcase. K206D bound glucagon just as well as the native receptor, but showed a threefold decrease in potency. D210H had properties similar to wild-type, while D210A lost eightfold affinity for glucagon. However both position 210 mutants activated cyclase as efficiently as native receptor. In contrast, mutants D219Y and R202D exhibited a significant attenuation of both the binding and activation function of the receptor. The triple replacement mutant [D209Q, D210 H, D219Y] exhibited a tenfold loss in receptor function. The data suggest that Arg202 and Asp 219 in the first extracellular loop of the receptor may be sites of interaction with glucagon. Arg 202 is located close to the membrane interface at the top of transmembrane helix II and may be a site of interaction for one of the negatively charged aspartic acid residues of glucagon. References 1 . Jelinek, L.J., Lok, S., Rosenberg, G.B., Smith, R.A., Grant, F.J., Biggs, S., Bensch, P.A., Kuijper, J.L., Sheppard, P.O., Sprecher, C.A., O’Hara, P.J., Foster, D., Walker, K.M., Chen, L.H.J., McKernan, P.A. and Kindsvogel, W. Science, 259 (1993) 1614. 2. MacNeil, D.J., Occi, J.L., Hey, P.J., Strader, C.D. and Graziano, M.P., Biochem. Biophys. Res. Comm., 198 (1994) 328. 3. Unson, C.G., Cypess, A.M., Wu, C.-R., Goldsmith, P., Merrifield, R.B. and Sakmar, T.P., Proc. Natl. Acad. Sci. U.S.A., 93 (1995) 310. 4. Unson, C.G., Cypess, A.M., Wu, C.R., Cheung, C.P. and Merrifield, R.B., In Tam, J.P. and Kumaya, P.T.P. (Eds) Peptides: Chemistry, Structure, and Biology (Proceedings of the 15th American Peptide Symposium), ESCOM Kluwer, 1997, in press. 5. Carruthers, C.J.L., Unson, C.G., Kim, H.N. and Sakmar, T.P., J. Biol. Chem., 269 (1994) 2932. 6. Unson, C.G., Cypess, A.M., Kim, H.N., Goldsmith, P.K., Carruthers, C.J.L., Merrifield, R.B. and Sakmar, T.P., J. Biol. Chem. 270 (1995) 27720.

69 A novel type of rat brain δ opioid receptors differenciated by cyclopropylphenylalanine-containing enkephalin analog Y. CHUMAN¹, T. YASUNAGA², T. COSTA³ and Y. SHIMOHIGASHI¹ ¹ Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan ² Manufacturing Process Development Division, Otsuka Pharmaceutical Co., Ltd., Saga Factory, Kanzaki, Saga 842-0103, Japan ³ Laboratorio di Farmacologia, Instituto Superiore di Sanità, 00161 Roma, Italy

Introduction The stabilization of bioactive conformation of peptides reinforces the ligandreceptor interaction, resulting in enhancement of biological activity. The incorporation of cyclopropylamino acids ( ∇AA) into a peptide chain is expected to constrain effectively its conformation due to the small φ and ϕ angles. In the opioid peptide-receptor system, the presence of δ and µ opioid receptors in brain was demonstrated by examining the binding affinities of radio-labeled ligands specific for each receptor subtype. When we assayed enkephalin analog containing E - (2R,3S)-cyclopropylphenylalanine ( ∇Phe), this analog exhibited a high affinity for δ receptors and a very weak affinity for µ receptors in rat brain [1]. However, it was completely inactive not only in guinea pig ileum (µ -prototype) but also in mouse vas deferens (MVD, δ -prototype) with no antagonist activity. Extremely weak affinity to MVD receptors was also evidenced by the binding assay [2]. Although these data suggested that E -(2R,3S)-∇ Phe-containing enkephalin distinguishes the δ receptors in the central and peripheral nervous systems, it is not clear yet whether or not a novel type of δ receptors exist either in the central or peripheral tissue. Cyclopropylphenylalanine possesses four stereoisomers in the E -(2R,3S)-, E -(2S,3R)-, Z -(2R,3R)-, and Z -(2S,3S)-configurations (Figure 1). In the present study, using highly receptor specific radio-labeled ligands, we have evaluated all of ∇ Phe-containing enkephalin analogs for their ability to bind to δ and µ opioid receptors in rat brain.

Results and discussion Four different stereoisomers of [ D -Ala 2,∇ Phe 4,Leu 5 ] enkephalins were assessed for opioid receptors in rat brain using various type of radio-ligands specific for µ or δ opioid receptors. For µ receptors, DAGO was most potent, and the 207 Y. Shimonishi (ed): Peptide Science – Present and Future, 207–209 © 1999 Kluwer Academic Publishers, Printed in Great Britain

208

Figure 1. Structure of cyclopropylphenylalanine-containing ehkephalin analogs.

isomers of Z-(2R,3R), Z -(2S,3S), E-(2R,3S), and E -(2S,3R ) followed in this o r d e r . I t s h o u l d b e n o t e d t h a t t h e Z -isomers are much more potent (60–1000-fold) than E-isomers. For δ receptors, several binding characteristics distinct from those in the µ assays became prominent. First, the Z -(2R,3R)isomer was found to be very potent and more active than the standard δ ligands. Table 1 shows the IC 50 values of all four enkephalin isomers. The most striking feature in binding to δ receptors is much increased affinity observed for the E-(2R , 3S)-isomer. The E-(2R,3S)-isomer was as active as the Z -(2S,3S)isomer (IC50 = ca. 10 nM) in the assays using [³H]DADLE, [³H]DSLET, and [³H]DEL (Table 1). It was also found that the binding curves of this E -(2R,3S)-isomer are very gently-sloping. For the high affinity binding site, the E -(2R,3S)-isomer binds very strongly with the IC 50 value of approximately 0.28 nM, whereas it binds weakly (IC 50 of approximately 20 nM) for the low affinity binding site. Scatchard analysis gave indeed a biphasic line, and the dissociation constants were estimated to be about 6.7 × 1 0 –12 M and 1.4 × 10 –10 M for the high (about 19% population) and low (about 81%) affinity binding sites, respectively. These results clearly indicated that two distinct δ receptors exist in rat brain and the E-(2 R , 3S)-isomer can discriminate them. DSLET was not able to differentiate these two subtypes, binding to both receptors equally well. Table 1. Binding affinities of four different stereoisomers of [ D -Ala², E-(2R ,3 S )-∇ P h e 4,Leu 5 ]enkephalins for µ and δ opioid receptors in rat brain. IC 50 (nM) Isomers Z-(2R,3R) Z -( 2S,3S) E-(2R,3S) E-(2S,3R)

[³H]DAGO (µ) 6.53 41.7 3290 6770

[³H]NAL (µ) 8.40 45.3 2410 3770

[³H]DADLE ( δ) 0.76 10.5 13.0 1290

[³H]DSLET (δ)

[³H]DEL (δ)

0.79 11.4 8.4 2560

1.12 5.22 12.7 1640

209 A confirmed finding in the present study is that the E-(2R,3S)-isomer binds to a novel class of δ receptors and discriminates this receptor from the ordinary δ receptor. This new type of δ receptor suspected to be a receptor which suppresses the thermal analgesia mediated through µ receptor [3].

References 1. Shimohigashi, Y., Costa, T., Pfeiffer, A., Herz, A., Kimura, H. and Stammer, C.H., FEBS Letters 222 (1987) 71. 2. Vaughn, L.K., Wire, W.S., Davis, P., Shimohigashi, Y., Toth, G., Knapp, R.J., Hruby, V.J., Burks, T.F. and Yamamura, H.I., Eur. J. Pharmacol. 177 (1990) 99. 3. Shimohigashi, Y., Takano, Y., Kamiya, H., Costa, T., Herz, A. and Stammer, C.H., FEBS Lett. 233 (1988) 289.

70 Synthesis of dimeric novobiocins as chemical inducers of dimerization for DNA gyrase-fused proteins M. MIYAZAKI, J. KIM, K. OSHIKIRI, K. SAITO and S. YOKOYAMA Yokoyama CytoLogic Project, ERATO, Japan Science and Technology Corporation, c/o RIKEN, Hirosawa, Wako-shi, Saitama 351-01, Japan

Introduction In the signal transduction of cells, dimerization of the biological receptors has been investigated as a general mechanism to control various intracellular signals. Synthetic molecules that bridge two target receptors have been developed as useful tools to examine the role of dimerization in the signal transduction [1]. Novobiocin (NB) is one of the most potent inhibitors against bacterial DNA gyrase. NB is known to bind the B subunit of gyrase (GyrB). The interaction between NB and GyrB seems to be very useful to develop a novel ligandinduced dimerization system, if it is possible to prepare divalent ligands of NB that dimerize GyrB-fused proteins. In the present study, to develop such a chemical dimerizer system for GyrBfused proteins, we designed and synthesized divalent analogs of NB (NB²). Their abilities as the dimer inducer were examined.

Results and discussion In the preparation of divalent NB analogs, it is important to determine which part is suitable to be cross-linked to each other [2]. Since early SAR study and crystal structure of GyrB-NB complex indicated necessity of novobiose and coumarin moieties for the binding [3, 4], terminal 3'-isopentenyl-4'hydroxybenzoic acid moiety was modified. The olefin was converted to a carboxyl group by oxidative cleavage using NaIO4 /KMnO 4 first [5], and then the carboxyl groups of two NB molecules were connected with oligo-glycine chain linked with ethylenediamine (Figure 1). The peptide bonds were formed by the WSCI-HOBt method. The final compounds, NB²s, were further purified by HPLC, and the structures were confirmed by NMR and MALDI-TOF MS. As a target receptor for the NB² system, we designed a molecular mutant of ErbB2 receptor which possesses GyrB instead of the extracellular domain II. This GyrB–ErbB2 chimera was expressed in HEK293 cells, and the dimer induced by NB²s was visualized by chemical cross-linking of the induced dimer using BS3 and by subsequent Western-blotting with an anti-ErbB2 antibody. 210 Y. Shimonishi (ed): Peptide Science – Present and Future, 210–211 © 1999 Kluwer Academic Publishers, Printed in Great Britain

211

Figure 1.

Structure of Divalent Novobiocin Analog (NB²).

NB²s showed better dimerization than the natural GyrB inhibitor Coumermycin [6]. Furthermore, NB²’s ability to form dimers depend on their spanner lengths of glycine linker. In conclusion, we suggest that the NB²–GyrB dimerization system developed in this study is useful to investigate the role of dimerization of the signaling proteins. Further studies are in progress in our laboratory.

Acknowledgements We thank Dr Michael A. Farrar for the GyrB construct, Dr Toshiyuki Kan for helpful discussion in synthetic chemistry, and Ms Yohko Kusano for the measurement of MALDI-TOF MS.

References 1. Crabtree, G.R. and Schreiber, S.L., Trends Biochem. Sci. 21(1996) 418. 2. Miyazaki, M., Kodama, H., Fujita, I., Hamasaki, Y., Miyazaki, S. and Kondo, M., J. Biochem. 117 (1995) 489. 3. Reusser, F. and Dolak, L.A., J. Antibiotics 39 (1986) 272. 4. Lewis, R.J., Singh, O.M.P., Smith, C.V., Skarzynski, T., Maxwell, A., Wonacott, A.J. and Wigley, D.B., EMBO J. 15 (1996) 1412. 5. Lemieux, R.U. and von Rudloff, E., Can. J. Chem. 33(1955) 1710. 6. Gormley, N.A., Orphanides, G., Meyer, A., Cullis, P.M. and Maxwell, A., Biochemistry 35 (1996) 5083.

71 Computer biochemistry and molecular physiology of the natural peptide ligands: different functional groups A.A. ZAMYATNIN and O.L. VORONINA A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, 33 Leninsky prosp., Moscow 117071, Russia

Introduction The similarity in the functional spectra of many endogenous regulatory oligopeptides [1] allows the assumption that these molecules possess some common physicochemical properties. These properties provide the basis for the interaction of an oligopeptide with a particular set of receptor structures, and this interaction is the initial step of the functional response. Of great interest is therefore to reveal common chemical features of these molecules, in particular their size and specific radicals, protruding out of the peptide backbone. The principles of computer biochemistry were used [2].

Results and discussion The object of investigation was the information of the data bank EROPMoscow (release 69) [3] on 2059 natural oligopeptides ranging in the size from 2 to 50 amino acid residues. The structural classification was performed by comparing step-by-step the linear sequences of amino acid residues and terminal radicals of all possible pairs of peptide molecules [4]. The homologous structures were grouped and these groups (families) were used for further analysis. 950 different structures and 322 (32 families) typical neuropeptides, 339 (31) releasing-factors, 145 (58) toxins including blockers of ionic channels, and 144 (42) antimicrobial peptides were extracted from the EROP-Moscow. The computer analysis of the common chemical features of differnt functional groups consisted in determining the average numbers of amino acid side radicals and of specific radical groups. They were compared with the corresponding data on 359 structurally different proteins [5]. Analysis has shown, that the content of individual amino acid residues in these functional groups of peptides was distinct from that of proteins. Some of them occur more frequently: Phe, Tyr, Pro, Arg, and Trp (neuropeptides); Trp and His (releasing-factors, Leu or Ile in monoamine-releasing hormones); Pro and Trp (toxins); Pro, Arg, and Lys (blockers of ionic channels); Pro, Arg, and Lys (antimicrobial peptides, Leu or Ile in one subpopulation of this functional group). Comprehensive analysis of individual peptides showed that in most cases, they contained at 212 Y. Shimonishi (ed): Peptide Science – Present and Future, 212–214 © 1999 Kluwer Academic Publishers, Printed in Great Britain

213 least one of these residues. Moreover, they are interchangeble in some families (e.g., Trp and His in releasing-factors). The positively charged radicals occur practically in all the molecules due to the presence of Arg/Lys residues and the N-terminal radical. The content of negatively charged Asp and Glu is substantially lower, and many C-termini are amidated (e.g., two thirds of releasing-factors). The results indicated that each member of all the functional groups of peptides contains a positively charged radical and at least one of the cyclic residues Trp, His, Tyr, Phe, Pro and/or highly hydrophobic radicals Leu or Ile. Hence, a common property of the natural peptide ligands is the occurrence of certain positively charged groups and cyclic/hydrophobic radicals. In this connection, it should be noted that most of classical transmitters are closely related chemical analogues of amino acids tryptophan, histidine, tyrosine, and phenylalanine respectively. They all possess both positively charged and cyclic radicals. Moreover, there are many examples that these molecules perform the functions of hormones. Therefore, it can be assumed that the side radicals of these amino acid residues participate in the binding of the peptide ligand with appropriate receptors. And the binding of both hormones and neurotransmitters proceeds by the same physicochemical laws. Owing to the high intramolecular motility of peptide, the majority of their chemical radicals are accessible for external interactions. It seems likely that positively charged and cyclic/hydrophobic radicals directly participate in interactions with some potential receptors of target cells. It can be assumed that the peptide ligand and the receptor are first non-specifically brought together as a result of the electrostatic interaction of a positively charged peptide radical with the negatively charged site of the receptor. After the partners come close together, a specific recognition occurs, which is due to fine fitting of the structural elements participating in this process. This fitting is accomplished by different types of weak interactions of peptide molecules with the receptors: formation of intermolecular hydrogen bonds, stacking interactions of cyclic radicals [6], or hydrophobic interactions. The specificity of peptide action, i.e. the extent of activity, is based on the unique mutual arrangement of radicals in the linear and spatial structures. The elucidation of the common chemical features is only the first step in the study of the structure-function relationships in endogenous peptides. At the next stage it would be necessary to carry out a special study of the spatial configuration of these molecules, which could give insight into the common features of the mutual arrangement of functionally important radicals in peptides with similar functional properties. Acknowledgements The authors thank Yu.V. Belokrylov, D.M. Vachadze, and O.V. Krut’ for the participation in designing specialized computer programs. This work was sup-

214 ported by the Russian Foundation for Basic Research (Grants No. 94-04-12411 and 95-04-12101).

References 1. 2. 3. 4. 5. 6.

Zamyatnin, A.A., J. Biochem. Organization, 1(1992) 2641–2644. Zamyatnin, A.A., Uspekhi Biol. Khimii, 35 (1996) 87–112 (in Russian). Zamyatnin, A.A., Prot. Seq. Data Anal., 4 (1991) 49–52. Zamyatnin, A.A., Prot. Seq. Data Anal., 4 (1991) 53–56. Nakashima, H., Nishikawa, K. and Ooi, T., J. Biochem., 99 (1986) 153–162. Zamyatnin, A.A., Ann. Rev. Biophys. Bioeng., 13 (1984) 145–165.

72 Bacterial peptide pheromone is imported where it directly binds to an intracellular receptor J. NAKAYAMA, T. HORII and A, SUZUKI Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Introduction Enterococcus faecalis, a gram-positive bacterium frequently involved in opportunistic infections and antibiotic resistances, produces a family of oligopeptide signaling molecules as bacterial sex pheromones. Conjugative transfer of a bacteriocin plasmid pPD1 is activated by a peptide pheromone cPD1 ( NH 2– Phe–Leu–Val–Met–Phe–Leu–Ser–Gly–OH) secreted from pPD1-free recipient bacteria (1) and is blocked by an oligopeptide inhibitor iPD1 (NH 2 –Ala–Leu– Ile–Leu–Thr–Leu–Val–Ser–OH) secreted from pPD1-carrying donor bacteria (2). In this study, we investigated how pPD1-carrying bacterial cell receives these peptide signals using a tritiated pheromone, [³H]cPD1.

Results and discussion A tritiated-cPD1, [4,5-³H–Leu2,6 ]cPD1 (denoted by [³H]cPD1), was obtained by catalytic reduction of a precursor peptide, [4,5-dehydro-Leu 2,6 ] cPD1, in the presence of tritium gas. [³H]cPD1 showed clumping-inducing activity comparable to that of the non-radioactive peptide and had sufficiently high specific radioactivity ( 11.7 TBq/mmol) for the binding experiments in this study. Donor cells rapidly incorporated [³H]cPD1. The cell-free extract but not the membrane fraction of the donor cells exhibited significant [³H] cPD1-binding activity, suggesting internalization of cPD1 and existence of intracellular binding molecule. The cell-free extract of a strain carrying the traA-bearing multicopy plasmid also exhibited high [ ³H] cPD1-binding activity. These results show that TraA is the binding protein for cPDI. Scatchard plot of [³H]cPD1-binding to GST-TraA, a fusion protein of glutathione-Stransferase and TraA, exhibited a dissociation constant (Kd) of 0.13 ± 0.01 nM against [³H]cPD1 (Figure 1A). The kd value is close to the minimum concentration required for pheromone induction (1), suggesting that TraA is a direct sensor for cPD1. In the presence of 0.3 nM of the inhibitor iPD1, the Kd value increased 0.61 ± 0.04 nM whereas Bmax was not significantly changed. This result shows that iPD1 acts an antagonist for TraA. None of the pheromones and inhibitors relating to other plasmid systems inhibited the 215 Y. Shimonishi (ed): Peptide Science – Present and Future, 215–217 © 1999 Kluwer Academic Publishers, Printed in Great Britain

216

Figure 1 (A) Scatchard analysis of [³H]cPD1-binding to GST-TraA. Closed circles, in the absence of iPD1; Open circles, in the presence of 0.3 nM iPD1. (B) Uptake of [³H]cPD1 into E. coli W3110(pGEX- traA) (closed circles) and W3110oppB: emr(pGEX-traA) (open circles). The cells were induced to express GST-TraA by adding 1 mM of IPTG to the culture at OD 660 = 0.5 and further incubation for 1 hr at 37°C. 0.3 nM of [³H]cPD1 was incubated with the cell and the radioactivity of the cells was measured.

[³H]cPD1-binding to GST-TraA. This imlies that the binding function of TraA is completely specific to the related pheromone and inhibitor, cPD1 and iPD1. A genetic study has demonstrated that an oligopeptide permease (Opp), that is a universal peptide transporter in bacteria, was involved in the uptake of pheromone (3). However, [³H]cPD1 was taken up into Opp-deficient Escherichia coli expressed with GST-TraA as done in wild-type E. coli expressed with GST-TraA (Figure 1B). This result suggests that cPD1 could permeate through the bacterial cell membrane in a manner independent of Opp.

217 Considering that the bacterial pheromones are hydrophobic, the pheromones may be permeable through the phospholipid bilayer and Opp may contribute to sense pheromone at a low concentration in nature. In our previous study, a gene disruption of traA resulted in a constitutive induction of the mating system, suggesting that TraA functions as a negative regulator for the cPD1 signal (4). Moreover, we have recently found that TraA was bound to a DNA fragment of pPD1 (J. Nakayama, unpublished). Taken together, it was speculated that TraA transduces cPD1 signal into a gene expression which triggers the mating system.

References 1. Suzuki, A., Mori, M., Sakagami, Y., Isogai, A., Fujino, M., Kitada, C., Craig, R. A, and Clewell, D. B., Science., 226 (1984) 849. 2. Mori, M., Tanaka, H., Sakagami, Y., Isogai, A., Fujino, M., Kitada, C., Clewell, D. B., and Suzuki, A., J. Bacteriol., 169 (1987) 1747. 3. Leonard, B. A. B., Podbielski, A., Hedberg, P. J. and Dunny, G. M., Proc. Natl. Acad. Sci. USA., 93 (1996) 260. 4. Nakayama, J. and Suzuki, A., Biosci. Biotech, Biochem., 61(1997) 1796.

73 Grb2 SH2 domain targeted inhibitors functioning in cellular signaling F-D.T. LUNG¹, K. ROSENBERG², L. SASTRY², S. WANG², X.-W. WU², CR. KING², P.P. ROLLER³* ¹ Department of Nutrition, China Medical College, Taichung, Taiwan, R.O.C. ² Lombardi Cancer Center, Georgetown University Medical Center, Washington D.C. 20007, U.S.A. ³ Laboratory of Medicinal Chemistry, Division of Basic Science, National Cancer Institute, National Institutes of Health, 37/5C-02, Bethesda, MD 20892, U.S.A.

Introduction The Grb2 protein plays an important role in growth factor(GF) initiated signaling in cells [1]. Specific tyrosines on GF-receptors became phosphorylated on mitogenic activation. The SH2 domain of Grb2 attaches to such pY-containing regions, thereby functioning in the assembly of multi-protein complexes required for activation of the cell’s machinery. Inhibition of Grb2-SH2 domain binding to pY-containing regions of cognate proteins has been demonstrated previously with the use of pY or pY-mimic containing peptides and peptidomimetics [2]. Using Phage library methodologies, we have recently discovered a novel non-phosphorylated disulfide-cyclized peptide, termed G1 (peptide 1 ), with good binding affinity to Grb2-SH2 [3]. This peptide has a new structure motif, in that it is non-phosphorylated, it has a unique sequence, the cyclic structure is required, and it binds with selectivity to Grb2-SH2 in comparison to Src-SH2. In more recent developments we synthesized a redox stable cyclic peptide analog 2, by replacing the disulfide linkage in 1 with a thioether moiety [3,4]. This redox stable analog showed equipotent affinity to the phage library generated peptide 1, but in addition, it was effective in inhibiting the interaction of the cellular Grb2 protein with the oncogenic GF-receptor ErbB-2, in cell homogenates. Whereas the X-ray structure based structural informtion is available to explain the mode of binding small Tyr-phosphorylated non-cyclic peptides to Grb2-SH2 [5], such data is not sufficient to explain the nature of binding by G1 and G1TE. For example, our alanine scan studies with peptide 1 would indicate that all amino acids, except Leu3 and Gly8 are essential for good activity [3]. Such is not the case for the earlier studied pTyr peptides. In the current study we synthesized pTyr analogs of our lead cyclic peptides 1 and 2 for structure/activity studies, and for comparison to other literature based pY-containing open chain peptides 8–11. 218 Y. Shimonishi (ed): Peptide Science – Present and Future, 218–220 © 1999 Kluwer Academic Publishers, Printed in Great Britain

219 Table 1. Comparison of Grb2-SH2 binding affinities of various phosphorylated and non-phosphorylated peptides.

Compound

Structure

IC 50 (micromolar) (a)

1. G1 phage lead peptide

10-25

2. G1TE, thioether analog

10-25

3. G1-pY4

0. 10

4. G1TE-pY3

0.13

5. G1TE-pY9

>100

6. SHC-Y(317)

>1000

7. SHC-pY (317)

1.0

8. BCR-ABL(177)

0.15 (b)

9. EGFR (1068)

1.0 (b)

10. library based

1.6 (c)

11. library based

0.2 (c)

(a) Competitive binding assays with SHC phosphopeptide 7, above, using surface plasmon resonance (SPR), unless noted otherwise; (b) Ref [5], using Elisa binding assays; (c) Ref [7], using SPR.

Results and discussion Cycic peptides 1–5 were synthesized using Fmoc based SPPS chemistry using PAL resin, with Trt protection on cysteines. Fmoc–Tyr(PO(OBzl)OH)–OH was incorporated for the synthesis of pTyr-containing peptides. Disulfide linkage in peptides 1 and 3 was formed by Fe(CN) 6 – 3 oxidation in solution at pH 8.0. Peptides 2, 4 , and 5 were synthesized by first preparing N-terminally chloroacetylated derivative of the open chain 10-mer precursors having a cysteine located in the C-terminal position, followed by intramolecular displacement of the chloride by the cysteine thiol [6]. Sequence comparison of peptide 1 to pY-containing peptides yields Y(pY)–X–N as the only consensus sequence. This suggests that Y4 in the G 1 peptide also contributes significantly to the binding affinity. Ala mutation studies confirm this observation. Significantly, none of the non-phosphorylated open chain peptide analogs of 7–11 showed detectable binding affinities [5, 7], in contrast to our phage library based cyclic peptides. Even with the lack of available structual information (NMR studies are in progress), current data suggests that the side chains of pratically all amino acids in 1 either contribute

220 to stabilize the favorable conformation, or participate significantly by directly interacting with the binding pocket of the Grb2-SH2 domain. Phosphorylation of Y9 in peptide 2 abolished the binding affinity. On the other hand, phosphorylation of Y4 in 1 , and Y3 in 2 improved the binding affinities by 50–100 fold, but the resulting peptides may not necessarily bind in the same manner in the binding pocket of the protein. Initial modeling studies, using free energy based molecular dynamics simulations, suggest an unusual arm-chair like bent conformation for peptide 1 . The conformationally constrained phosphorylated and non-phosphorylated peptides described here may serve as promising leads toward the development of inhibitory agents of deletirious cellular signaling functioning in cell proliferative diseases.

References 1. Schlessinger, J., Curr. Opin. Genet. Dev., 4 (1994) 23. 2. Burke, Jr., T.R., Smyth, M.S., Otaka, A., Nomizu, M., Roller, P.P., Wolf, G., Case, R. and Shoelson, SE., Biochemistry, 33 (1994) 6490. 3. Oligino, L., Lung, F.-D.T., Sastry, L., Bigelow, J., Cao, T., Curran, M., Burke, Jr., T.R., Wang, S., Krag, D., Roller, P.P. and King, C.R., J. Biol. Chem. 272 (1997) 29046. 4. Lung, F.-D.T., Sastry, L., Wang, S., Lou, B.-S., Barchi, J.J., King, C.R. and Roller, P.P., In Peptides: Chemistry and Biology; Tam, J.P. and Kaumaya, P.T.P. (Eds.), Kluwer Acad. Publishers, Dordrecht, The Netherlands, 1997, In Press. 5. Rahuel, J., Gay, B., Erdmann, D., Strauss, A., Garcia-Echeverria, C., Furet, P., Caravatti, G., Fretz, H., Schoepfer, J. and Grutter, M.G., Nature Struct. Biol., 3 (1996) 586. 6. Lindner, W. and Robey, F.A., Int. J. Peptide Protein Res., 30 (1987) 794. 7. Gram, H., Schmitz, R., Zuber, J.F. and Baumann, G., Eur. J. Biochem., 246 (1997) 633.

74 Interaction between heat-stable enterotoxin and its receptor (guanylyl cyclase C): influence of mutation at N-linked glycosylation sites on its ligand binding M. HASEGAWA, Y. MATSUMOTO, Y. HIDAKA and Y. SHIMONISHI Division of Protein Organic Chemistry, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan

Introduction Guanylyl cyclase (GC) C, identified from cDNA libraries of several mammalian small intestines, is a membrane-associated receptor for a heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli and an endogenous peptide, guanylin [1, 2]. The extracellular domain of guanylyl cyclase C, which includes the ligand-binding site, activates the cytoplasmic domain, catalyzing the conversion of GTP to cGMP as an intracellular signal. GC-C exhibits glyco-modifications and contains seven or eight potential N -glycosylation sites in the extracellular domain. To investigate the role of the N-glycosylation, each asparagine residue at the potential sites of porcine GC-C was converted to alanine by site-directed mutagenesis.

Results and discussion In a previous study, we established the expression system of the extracellular domain of porcine GC-C in insect cells using a baculovirus vector [3]. The recombinant extracellular domain was obtained as a soluble secretary protein and retained both ligand-binding ability and glyco-modifications. Asparagine residues at the eight potential N -glycosylation sites, positions 9, 20, 56, 172, 261, 284, 334 and 379, of the soluble extracellular domain (sECD) were converted to alanine residues by site-directed mutagenesis using standard PCR techniques. The transfer vector pVL1392 carrying the cDNA and the linearized baculovirus DNA (Baculo-Gold, Pharmingen) were cotransfected to Sf21 insect cells, resulting in high-titer recombinant viruses. To confirm the expression of the sECD mutants from Sf21 cells, the target proteins in the culture supernatants were radiolabeled by photoaffinity labeling using p -benzoylbenzoyl-[ 125 ITyr 4 ]STp(4–17) and analyzed by SDS-PAGE, following by autoradiography (Figure 1A). All mutants showed increasing rates of mobility compared with wild-type sECD on SDS-PAGE, corresponding to the absence of an oligosaccaride chain at each glycosylation site. The equilibrium binding experiments between [ 125 I-Tyr 4 ]STp(4–17) and the mutants revealed that the dissociation 221 Y. Shimonishi (ed): Peptide Science – Present and Future, 221–222 © 1999 Kluwer Academic Publishers, Printed in Great Britain

222

Figure 1. (A) The N -glycosylation mutants of sECD) in Sf21 cells culture supernatants. The sECD mutants were labeled by BB-[125 I-Tyr 4 ]STp(4–17). (B) Maximum ligand-binding capacities of the mutants.

constants (K D ) of the mutants were nearly identical to that of wild-type sECD (K D = 0.4–1.2 × 10 – 9 M). On the contrary, the maximum ligand-binding capacities (B max) decreased significantly for the case of N172A, N261A and N379AsECD (5–10%, compared with that of wild-type sECD), even though their expression level were similar to that of wild-type sECD. The B max of the other mutants except for N20A-sECD also decreased to 40–60% of that of wild-type sECD (Figure 1B). In addition, the N -glycosylation mutants of full-length GC-C expressed on 293T cell membranes, exhibited results which were comparable with the sECD mutants expressed in Sf21 cells. These results suggest that the N -glycans at Asn172, 261 and 379 play an important role in determining the activity of GC-C. Numerous functions for N-linked glycans have been implicated, including (i) promotion of proper folding, (ii) maintenance of protein conformation and stability, (iii) protection against denaturation and proteolysis, and (iv) modulation of biological activities. In the case of GC-C, it is not likely that glycomodification directly related to ligand binding, since previous studies suggest that the interaction between GC-C and the ligands is mainly the result of hydorophobic interactions [4, 5]. The N-glycans may function to support the stability of the conformation of the extracellular domain. N -glycosylaton at positions 172, 261 and 379, all of which may be located near the ligand-binding site, is critical and may serve to stabilize the tertiary structure. References 1. Schulz, S., Green, C.K., Yuen, P.S.T. and Garbers, D.L., Cell 63, ( 1991) 941. 2. Wada, A., Hirayama, T., Kitao, S., Fujisawa, J., Hidaka, Y. and Shimonishi, Y., Microbiol. Immunol. 38, (1994) 535. 3. Hasegawa, M., Kawano, Y., Matsumoto, K., Hidaka, Y., Fujii, J., Taniguchi, N. and Shimonishi, Y., In Kitada, C. (Ed.) Peptide Chemistry 1996 (Proceedings of the 34th Japanese Peptide Symposium), Protein Research Foundation, Osaka, 1997, p. 169. 4. Yamasaki, S., Sato, T., Hidaka, Y., Ozaki, H., Ito, H., Hirayama, T., Takeda, Y., Sugimura, T., Tai, T. and Shimonishi, Y., Bull. Chem. Soc. Jpn. 63 (1990) 2063. 5. Hasegawa, M., Kawano, Y., Matsumoto, K., Hidaka, Y., Sato, T. and Shimonishi, Y., Lett. Peptide Sci. 4 (1997) 1.

75 Receptor studies with novel insulin analogues and photoaffinity labelling techniques D. BRANDENBURG¹, M. FABRY¹, Y. FISCHER², H.-G. GATTNER¹, J. GRÖTZINGER³, M. HAGELSTEIN¹, C. HAVENITH¹, G. KURAPKAT³, S. RÜTTEN¹, M. SIEDENTOP¹ and A. WOLLMER³ ¹ Deutsches Wollforschungsinstitut an der RWTH Aachen, Veltmanplatz 8, D-52062 Aachen, Germany ² Institut für Physiologie, Klinikum der RWTH Aachen, D-52057 Aachen, Germany ³ Institut für Biochemie, Klinikum der RWTH Aachen, D-52057 Aachen, Germany

Introduction The insulin superfamily comprises a series of molecules with similar structure, but widely varying biological functions, ranging from the parent hormone to insulin-like growth factors (IGF) I and II and further to bombyxin. Understanding the interaction of these ligands and their receptors is of great fundamental as well as practical interest. The receptors for insulin and IGF-I show a high degree of homology, but different regions have been postulated for ligand binding. Our work aims at determining structure–function relationships, especially the structural requirements for receptor binding, and the definition of contact sites in the ligand–receptor binding regions.

Results and discussion Novel insulin analogues Continuing previous studies on the effect of chain length and side-chain variations in the C-terminus of the B-chain on activity and association [1,2], we have now replaced B26-tyrosine by D-alanine in des-(B27-B30)-insulinB26-amide (DTI). The shortened analogue D-Ala-DTI (1 ) was prepared by trypsin-catalyzed semisynthesis from des-(B23-B30)-insulin and the synthetic Gly-Phe-Phe- D Ala-NH 2 in a yield of 37% and 97% homogeneity (RP-HPLC). The first and so far only antagonistic insulin analogue is a covalent insulin dimer [3]. To test whether the marked discrepancy between receptor binding and biopotency can be increased, we introduced binding-enhancing Arg residues into insulin dimers. B29,B29'-suberoyl-(Arg A0 -insulin) 2 (3) was synthesized by crosslinking Arg A0 insulin with suberic acid bis-hydroxysuccinimide ester after protecting both N-termini with Msc groups. Deblocking with 20% piperidine and semipreparative RP-HPLC gave 3 in a yield of 20% and 99% homogeneity. Similarly, 223 Y. Shimonishi (ed): Peptide Science – Present and Future, 233–225 © 1999 Kluwer Academic Publishers, Printed in Great Britain

224 Table 1. Relative binding to receptors in cultured IM-9 lymphocytes, and stimulation of 2-deoxyD-[ 3 H ]-glucose uptake in cultured 3T3-L1 adipocytes (insulin = 100).

% Binding % Activity

1

2*

3

4**

5

842 967

51 3

90 6

63 32

71 132

* B29,B29'-suberoyl insulin dimer ** B1,B1'-suberoyl insulin dimer

B1,B1'-suberoyl-(Arg A0 -insulin) 2 (5) was obtained from A0,B29-(Msc) 2 -Arg A0insulin by crosslinking between the B-chain N -termini. Biological properties are compiled in the Table. D -Ala in position B26 gives rise to a dramatic increase in receptor binding and in-vitro activity. However, the NMR solution structure does not provide structural clues for a straightforward explanation of this superpotency. The addition of Arg to 2 increased receptor binding but, unexpectedly, also activity. It is striking that 3 can elicit maximal insulin effect in cardiomyocytes but, like 2, is unable to do so in 3T3-L1 cells (53%). We conclude that the monomeric and dimeric analogues are interesting lead compounds for refined receptor/activity studies directed towards signal generation and transduction in various cell systems.

Receptor studies applying photoaffinity labelling Recombinant human IGF-I was acylated with Asa(4-azidosalicyloyl)-OSu. The 3 monosubstitued derivatives were subfractionated by RP-HPLC to give pure N α-, N ε B28 -, and mixed N ε B65 /Nε B 6 8 -Asa-IGF-I. The sites were identified by MS after reduction, S-carboxymethylation, and tryptic fragmentation. Pure N αA1 and N ε B29 -Asa-insulins were prepared similarly. The radioiodinated probes could be specifically crosslinked to overexpressed receptors for insulin, IGF-I and 2 chimeric insulin/IGF-I receptors in 4 NIH3T3 cell lines. Tryptic digestion reveals characteristic labelling and fragmentation patterns which are currently analyzed further to determine the ligand–receptor contact sites [4]. Surprisingly, photoreactive insulin proved to be an excellent probe for IGF-I receptors with yields much better than expected on the basis of its known low cross-reactivity.

Acknowledgements Supported by Deutsche Forschungsgemeinschaft (DFG).

225 References 1. Spoden, M., Gattner, H.-G., Zahn, H. and Brandenburg, D., Int. J. Peptide Protein Res., 6 (1995) 221. 2. Leyer, S., Gattner, H.-G., Leithäuser, M., Brandenburg, D., Wollmer, A. and Höcker, H., Int. J. Peptide Protein Res., 46 (1995) 397. 3. Weiland, M., Brandenburg, C., Brandenburg, D. and Joost, H.G., Proc. Natl. Acad. Sci. USA, 87 (1990) 1154. 4. Fabry, M. and Brandenburg, D., In Meier, T. and Fahrenholz, F. (Eds) BioMethods 7, A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis, Birkhäuser, Basel, 1966, pp 65–81.

76 Radioligands for the receptor of mammalian melaninconcentrating hormone (MCH) E. HINTERMANN, R. DROZDZ, H. TANNER, U. ZUMSTEG and A.N. EBERLE Laboratory of Endocrinology, Department of Research (ZLF) University Hospital and University Children’s Hospital, CH-4031 Basel, Switzerland

Introduction Mammalian melanin-concentrating hormone (MCH), a peptide originally isolated from teleost fish posterior pituitaries, has recently been shown to be a hypothalamic neuropeptide whose neurons project into many different areas of the brain [1]. MCH influences several brain functions participating in the control of goal-oriented behaviors such as emotional responses, food and water intake, and in general arousal. In particular the effect of MCH on food intake receives increasing interest and several research groups undertake efforts to clone the MCH receptor.

Results and discussion We have recently succeeded in establishing the first MCH receptor binding assay using monoiodinated [Phe 13 , Tyr 19 ]-MCH [2], and we have biochemically identified MCH receptors on mouse and human melanoma cells by photocrosslinking with [D-Bpa 13 , Tyr 19 ]-MCH The binding studies revealed that high-affinity MCH receptors (KD approx. 0.1 nmol/l) exist in small numbers (< 1000 receptors/cell) on mouse B16, G4F and G4F-7 melanoma cells, on several human melanoma cell lines and on rat PC12 phaeochromocytoma cells [3]. The crosslinking experiments using the p-benzoyl-phenylalanine (Bpa) group as photolabel produced labeled bands of approx. 45 kDa in all these cell lines, indicating that the MCH receptor is part of the G protein-coupled receptor family. We have also shown that the MCH receptor is biochemically different from the melanocortin-1 (MC1) MSH receptor expressed on mouse B16 melanoma cells and that the MC receptor subtypes do not bind MCH radioligand in a specific way. Because of the limited stability and the extreme stickiness of the MCH radioligands used so far, we have now prepared several alternative MCH radioligand analogs by solid-phase synthesis and tested them in a radioreceptor assay. In these analogs, methionines and/or cysteines have been replaced or the configuration of specific residues has been changed yielding in part more potent tracer molecules. We also performed a structure-activity 226 Y. Shimonishi (ed): Peptide Science – Present and Future, 226-227 © 1999 Kluwer Academic Publishers, Printed in Great Britain

227 Table 1.

Structure and receptor-binding activity of MCH analogs for radioiodination. a

Peptide

Ki (nmol/l) ± SD

Relative activity

[Phe 13 ,Tyr 19 ]-MCH [Met(O) 4,8 ,Phe 13 ,Tyr 19 ]-MCH [Nle 4,8 ,Phe 13 ,Tyr 19 ]-MCH [Ser 4,8 ,D -Phe 13 ,Tyr 19 ]-MCH [D-Phe 13 ,Tyr 19 ]-MCH

16.7 ± 99.6 ± 6.5 ± 34.8 ± 2.4 ±

100 17 257 48 696

a b

3.9 15.9 0.9 10.4 1.0

b

K i determined by competition binding assay; n = 3. [Phe 13 ,Tyr 19 ]-MCH is defined as 100%.

study looking into the contribution of the different parts of the mammalian MCH molecule for receptor binding. Table 1 summarizes some of the structure-activity data using the competition binding assay with B16-G4F-7 cells. Replacement of Met4 and Met 8 in the [Phe 13 ,Tyr 19 ]-MCH structure by the corresponding S-oxides led to an approx. 6-fold reduction of the Ki : 16.7 nmol/l for [Phe 13 ,Tyr 19 ]-MCH as compared analog. to approx. 100 nmol/l for the [Met(O) 4,8 , Phe 13 ,Tyr 19 ]-MCH Replacement of the methionines by homoserines gave a Ki of 35 nmol/l for the resulting [Hse 4,8 , Phe 13 ,Tyr 19 ]-MCH, and replacement of the methionines by norleucines yielding [Nle 4,8 ,Phe 13 ,Tyr 19 ]-MCH further reduced the K i to 6.5 nmol/l. The latter peptide appeared to be very lipophilic according to its HPLC-elution profile and hence was not used as precursor for radioiodination. The observation with salmon MCH that oxidation or replacement of the methionines only marginally altered the biological activity [4] could not be confirmed for mammalian MCH containing Met(O) 4,8 , Hse 4,8 or Ser 4,8 with regard to their suitability as radiotracers. A better choice of structural change was to alter the configuration of the Phe 13 residue to its D-enantiomer which increased the potency of the parent [Phe 13 ,Tyr 19 ]-MCH analog by 7-fold with hardly any change in lipophilicity. Thus at present, [D -Phe 13 ,Tyr 19 ]-MCH is the most potent MCH radioligand.

Acknowledgement This work was supported by the Swiss National Science Foundation.

References 1. Baker, B.I., Trends Endocrinol. Metab., 5 (1994) 120. 2. Drozdz, R. and Eberle, A.N., J. Peptide Sci., 1(1995) 58. 3. Drozdz, R., Siegrist, W., Baker, B.I., Chluba-de Tapia, J. and Eberle, A.N., FEBS Lett., 359 (1995) 199. 4. Baker, B.I., Eberle, A.N., Baumann, J.B., Siegrist, W. and Girard, J., Peptides, 6 ( 1985) 1125.

77 Using peptides to study the folding of a predominantly β -sheet protein K.S. ROTONDI and L.M. GIERASCH Department of Chemistry, University of Massachusetts, Amherst, MA 01003, U.S.A.

Introduction While the primary sequence of a protein is sufficient to guide it to its folded state under native conditions from an ensemble of unfolded conformations in the presence of denaturant, we do not yet know the relative importance of local sequence-directed conformational preferences and global information such as the pattern of hydrophobic and hydrophilic residues. We have described the folding pathway of cellular retinoic acid binding protein I (CRABPI), a predominantly β -sheet protein with an internal cavity that binds retinoic acid, based on results from a variety of methods (stopped flow fluorescence and circular dichroism, quenched flow hydrogen exchange) [1,2]: An early kinetic phase (< 10 msec) appears to correspond to hydrophobic collapse and is accompanied by development of substantial native-like CD signal. The topology of the protein including presence of the central ligand binding cavity develops in a 100 msec kinetic phase [2]. Intriguingly, stable hydrogen bonding in the β sheets is present only after a longer kinetic phase (1 sec), which is also accompanied by the development of native tertiary interactions. A minor population of the molecules folds more slowly (15 to 20 sec) due to slow cis-trans isomerization around X -Pro bonds. We are now examining the importance of local sequence in this folding pathway in two ways: 1) by studies of peptide fragments; and 2) by studies of mutated versions of CRABPI. Thus far, we have found that the small helix–turn–helix of CRABPI has a strong locally driven folding tendency including a sequence motif called a Schellman motif [3]. In the present study, we have focused on the roles that local sequences in turns may play in the folding of CRABPI. The topology of a β -sheet protein requires that non-local segments of chain make specific contacts, yet local sequence must play a role early in folding to favor these interactions. The turns connecting strands are likely to facilitate the formation of the adjacent strand-strand contacts. In CRABPI, like many of its family members, tight turns connect most of the β strands. We have examined characteristics of the tight turns in CRABPI that may point out key local sequences for folding. If a turn is involved in guiding the chain towards the native topology, then one would expect it to be stabilized largely by interactions within its own sequence, even in the context of the native protein 228 Y. Shimonishi (ed): Peptide Science – Present and Future, 228–231 © 1999 Kluwer Academic Publishers, Printed in Great Britain

229 structure. Furthermore, those sequences critical for the folding of a protein should be conserved throughout a family of proteins in much the same way that functional elements are conserved. By these two criteria, the turn between strands 4 and 5 of CRABPI emerges as likely to play an important role in early folding steps. To assess the importance of this local sequence, we have studied an 11-residue peptide that encompasses this turn and find that it shows remarkable bias towards a native conformation.

Results and discussion There are seven tight turns in CRABPI (Figure 1). For purposes of this study, we define a turn region as the residues in the corner positions of the turn and two residues on either side (Table 1). Comparison of the number of intra-turn, interatomic contacts (distance between atoms less than or equal to the sum of the van der Waals radii) suggests that turn IV is largely stabilized by local sequence interactions. Turn II also appears to be a possible autonomous folding

Figure 1. Ribbon diagram of the crystal structure of CRABPI [4] showing the numbering of turns. The darkened area of the structure corresponds to turn IV and its flanking residues as studied as a peptide fragment. Table 1.

Summary of the turns in CRABPI.

Turn

Turn type

Sequence

Intraturn contacts †

Sequence conservation

I II III IV V VII VIII

βII' βI' βII Irr βI Irr Irr

QDGDQF STTVRT FKVGEGF ETVDGRK TWENENKI LANDEL GADDVV

5 5 3 7 4 4 5

17.2 7.8 19 18.6 7.5 8.7 10.5

†

Intraturn contacts are the number of residues which contact, not the total number of contacts.

230 sequence, as it has few contacts with extra-turn segments of the chain in the native fold. We determined the sequence conservation of the seven tight turns among the members of the lipid-binding protein family to which CRABPI belongs; there are thirty-four full sequences known in this family. The sequences were aligned using the programs Pile-up and Pretty (available in the Wisconsin GCG package). By increasing the stringency requirements for consensus, we were able to determine those sequences which were most conserved (Table 1). We found that turn IV was one of the most conserved motifs in this family. Based on these observations, we synthesized a fragment of CRABPI encompassing turn IV. We made the 11-residue peptide, EEETVDGRKSR, corresponding to CRABPI residues 72–82 with the conservative mutation of Cys 81 to Ser to avoid disulfide formation. The sequence selected for study includes a large number of conserved electrostatic contacts, including the intra-turn salt bridge between Asp 77 and Arg 79, and in the region adjacent to the turn, an acidic sequence comprised of Glu 72, Glu 73, and Glu 74 is juxtaposed to the basic and hydrogen-bond donating groups Lys 80, Cys 81, and Arg 82. This peptide behaves as a monomer, based on the lack of concentration dependence of its circular dichroism at 208, 218, and 222 nm over a 10 to 500 µM range and its proton 1D NMR signals (both chemical shift and line width) over a 0.1 to 1 mM range. Two-dimensional NOE spectroscopy on this peptide (12 C, 0.83 mM, pH 5.3, H 2 O/D 2 O 9 : 1 v/v) reveals a striking number of NOES for an 11 residue fragment, the intensities of which correspond well with the crystal structure distances in this segment in CRABPI (data not shown). In general, the NOE intensities are higher in the central residues of the peptide. The unusually structured nature of this peptide is supported by the observations of many NN NOES (observed for all residue pairs except 80–81 and 81-82). Two i–i + 2 NOES in the region of the turn also suggest considerable order in this portion of the fragment. Both are between Asp 77 and Arg 79, the pair of residues which participate in a salt bridge in the crystal structure. NOES were seen to build up on a 50 to 200 ms time scale, with no evidence for spin diffusion in mixing times as long as 400 ms. The temperature coefficients of the amide protons in the peptide (measured from TOCSY data obtained under the same conditions as above) also indicate that the peptide has a strong tendency to adopt a native-like conformation. Specifically, the low temperature coefficient of the Arg 79 amide (2.7 ppb/deg), relative to the temperature coefficients of the other amides (5.1 to 7.8 ppb/deg) is consistent with its involvement in an intramolecular hydrogen bond, as in the native protein. Many peptides are induced to favor a helical conformation by titration with trifuoroethanol (TFE); in fact, the resistance to this shift in conformational equilibrium is an indication of a strong bias against a helical conformation. The CD spectrum of the fragment of CRABPI studied (50 µM, pH 5.3) is

231 markedly insensitive to the addition of TFE up to 40% (v/v). In aqueous solution, the peptide CD has a minimum at 200, characteristic of a largely random coil ensemble, with weak negative intensity in the 210 to 235 nm region indicating some structure, albeit irregular. Titrating the peptide to 40% TFE caused a slight decrease in the negative maximum around 200 nm, but had no influence on the region from 208 to 230 nm, where the signature of helical conformation would be expected. The behavior of the isolated peptide is intriguing, given that CRABP I itself; upon titration with TFE, has the CD spectrum of a classical helix with clear minima at 208 and 222 nm. In conclusion, we have found that the turn IV region of CRABPI constitutes a local sequence with a high propensity to adopt a hairpin structure as it does in the context of the native protein. This sequence has a large number of intraturn contacts when it is in the folded protein, and constitutes a highly conserved region of the protein. As such it may play a critical role in the early folding stages of CRABPI. We are testing the role of this turn by mutagenizing CRABPI to remove turn IV and to replace it with a glycine linker. Interestingly, turn IV occupies a topologically significant position in the CRABPI molecule; it is approximately halfway through the sequence. By folding early it would have the greatest impact on the conformational entropy of the system. Turn IV is also the turn which joins the two orthogonal β -sheets together (Figure 1), and thus would be critical to formation of the overall chain topology.

Acknowledgements This research was supported by a grant from the NIH (GM27616). KSR was supported in part by a NIH Chemistry-Biology Interface pre-doctoral training grant (GM08515).

References 1. 2. 3. 4.

Clark, P.L., Liu, Z.P., Weston, B.F. and Gierasch, L.M., Folding and Design, 3 (1998) 401. Clark, P.L., Liu, Z.P., Rizo, J. and Gierasch, L.M., Nature Str. Biol., 4, (1997) 883, Sukumar, M. and Gierasch, L.M., Folding and Design, 2, ( 1997) 211. Kleywegt, G.J., Bergfors, T., Senn, H., Le Motte, P., Gsell, B., Shudo, K. and Jones, T.A., Structure, 2, (1994) 1241.

78

α-Helical propensity of Ala-based peptides in organic solvents L.-P. LIU and C.M. DEBER Division of Biochemistry Research, Hospital for Sick Children, Toronto M5G 1X8; and Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada

Introduction The α-helix is one of the main building blocks for protein structures. Previous studies indicate that individual amino acids have distinct helical preferences that influence the stabilities of protein secondary structures and folding in aqueous environments. However, in membrane environments, which provides a very different solvation matrix from water, the rules governing protein folding will tend to differ from those directing folding in the aqueous milieu. Yet, only modest progress has been made towards understanding the conformational preferences for the individual amino acids in membrane environments, largely because of the scarcity of high resolution structural data on membrane proteins. In the present work, a series of Ala-based model peptides are employed to address some aspects of this long-standing ‘puzzle’. The organic solvent 90% isopropanol, with dielectric constant between pure water and the hydrocarbon interior of biological membranes, was used to mimic the nonpolar environment of membranes. Results and discussion De novo designed 25-residue peptides with typical sequence Lys–Lys–Ala–Ala– Ala–X –Ala–Ala–Ala–Ala–Ala– X –Ala–Ala–Trp–Ala–Ala– X –Ala–Ala–Ala– Lys–Lys–Lys–Lys-amide were synthesized by continuous-flow Fmoc solid phase chemistry [1], where X residues were substituted with each of the 20 commonly-occuring amino acids. Circular dichroism (CD) was used to assess the secondary structures of peptides. Temperature-dependent CD spectra (Figure 1) of peptides (exemplified by the X = Val peptide) in 90% isopropanol all pass through an isodichroic point at 203 nm, suggesting that the peptide residues are likely in a two-state, helix-coil transition state in this environment [2]. Accordingly, when a two-state model is applied, the ∆ G values of helix formation for these Ala-based peptides can be calculated from the corresponding [ θ] values at 222 nm using the equation:

232 Y. Shimonishi (ed): Peptide Science – Present and Future, 232–234 © 1999 Kluwer Academic Publishers, Printed in Great Britain

233

Figure 1. Temperature dependence of CD spectra for the X = Val peptide in 90% isopropanol. Peptide concentration = 30 µM. (a) Curves obtained at temperatures, from bottom to top, of 5,15, 25, 35, 45, and 60°C, respectively. (b) CD spectrum comparison of the peptide sample upon cooling from 60°C to 5°C vs. that of the original measurement obtained at 5°C.

where [ θ ] helix = – 35,000 (deg cm² /dmole) [3], and [ θ ] c o i l = 0 (deg cm²/dmole) based on the random structure observed for the Pro peptide in aqueous buffer. Table 1 lists the ∆ G values, viz., the free energy of helix-formation for each of the twenty peptides. Table 1. Free energy of helix formation for KKAAAXAAAAAXAAWAAXAAAKKKK-amide peptides in 90% isopropanol (T = 25°C). X residues

– [ θ] × 10 –4 (deg cm²/dmole) 222nm

– ∆ G (kcal/mol)

L M F I V C A W T Y E D S R G Q N H K P

3.11 3.08 3.05 3.04 3.02 3.01 2.99 2.79 2.78 2.76 2.60 2.15 2.09 2.04 2.00 1.98 1.90 1.83 1.82 1.03

1.23 1.17 1.13 1.11 1.08 1.07 1.04 0.81 0.80 0.78 0.63 0.28 0.24 0.20 0.18 0.16 0.11 0.06 0.05 –0.50

234 Since the only difference among these peptides is the identity of the guest ‘X’ residues, it is reasonable to assume the difference in helix-formation observed in peptides is attributable to the conformational preference of the X-residue. Thus, the rank order of the helix-forming tendencies for individual amino acids in a nonpolar environment (90% isopropanol) is L > M > F > I > V > C > A > W > T > Y > E > D > S > R > G > Q > N > H > K > P, which is significantly different from those obtained in aqueous media [4]. For example, although β -branched amino acids (I, V, T) are typically α -breakers in aqueous environments, they display a considerable tendency to form α -helices in nonpolar media. Similarly, aromatic residues (F, W, Y) are poor helix formers in globular proteins due, in part, to loss of side chain entropy, whereas in membrane proteins, favorable hydrophobic interactions between the aromatic side chain and both the backbone and the non-polar solvent matrix likely outweigh this entropy loss, and thereby increase their helix-forming tendencies. The present studies highlight the environment-dependent intrinsic conformational preferences of the various amino acids, and represent a stride toward the development of sequence-dependent protein structure prediction in membrane environments.

Acknowledgements This work was supported, in part, by a grant to C.M.D. from the Natural Sciences and Engineering Research Council of Canada (N.S.E.R.C.). L.-P.L. held a Research Training Committee fellowship award from the H.S.C. Research Institute.

References 1. 2. 3. 4.

Liu, L.-P. and Deber, C.M., Biochemistry, 26( 1997)5476. Park, S.H., Shalongo, W. and Stellwagen, E., Biochemistry, 32 (1993) 7048. Chen, Y.-H., Yang, J.T. and Chau, K.H., Biochemistry, 16 (1974) 3350. Chakrabartty, A. and Baldwin, R.L., Adv. Prot. Chem., 46 (1995) 141.

79 Effects on folding properties of endothelin/apamin by incorporation of selenocysteine into their cysteine framework S. PEGORARO¹, S. FIORI¹ , S. RUDOLPH-BÖHNER¹, T.X. WATANABE² and L. MORODER¹ ¹ Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany ² Peptide Institute, 4-1-2 Ina, Osaka, Japan

Introduction In a previous study we have analyzed the effect on the redox potential of the active-site fragment 10–17 of glutaredoxin by replacing one or both Cys residues with seleno-cysteine (Sec). The redox potential of the bis-Sec-peptide (–381 mV) at neutral pH was found to be significantly lower than that of the mixed Sec,Cys-peptide (–326 mV), of DDT (–323 mV), glutathione (–205 mV) and the parent bis-Cys-peptide (–180 mV) [1]. These results clearly revealed that formation of mixed Se–S bridges in multiple-cysteine peptides is highly disfavored relative to the formation of the diselenide. Thus Cys→ Sec mutants could open an interesting new approach for the design of productive intermediates in oxidative folding pathways of synthetic peptides and proteins. To analyze this promising aspect of such chalcogen analogs, we selected a family of multiple-cystine peptides that has already been object of extensive refolding studies, i.e. endothelin-1 (ET-1)/apamin (APA) [2]. Both peptides are structurally characterized by the cystine-stabilized α-helix motif (CSH) [3] that is assumed to be responsible for the correct oxidative refolding of ET-1 into the ribbon (parallel disulfide bridges) and of APA into the globule isomer (crossed disulfide bridges).

Results and discussion Because of the existing small differences in electronegativities, covalence radii, bond lengths and angles of selenium and sulphur, our first aim was to establish the effect of S →Se replacements on the overall structure and thus on the bioactivities of the peptides. By the use of optimized procedures for the synthesis of Sec-peptides in terms of protection, facile β -elimination, deprotection and diselenide bridging [Sec³,Sec 11,Nle 7 ]-ET-1 was synthesized by regioselective Sec-Sec and Cys-Cys pairing procedures exploiting the stability of the diselenide group toward the equivalent amounts of tris(2-carboxyethyl)phosphine required for reductive cleavage of the Cys(StBu) derivatives [4]. A comparison 235 Y. Shimonishi (ed): Peptide Science –Present and Future, 235–239 © 1999 Kluwer Academic Publishers, Printed in Great Britain

236 of the CD spectra of ET-1 and [Sec³,Sec 11 ,Nle7 ]-ET-1 was strongly suggesting identical conformations, a fact that has been fully confirmed by 1 H-NMR structural analysis in aqueous acetic acid and DG calculations of the ET-1 analog as previously reported for ET-1 [5]. The preferred solution structure of the analog reveals the presence of the identical CSH motif with a possibly more constrained loop connecting the α-helix with the N -terminal portion of the molecule as shown in Figure 1. Similarly, the wt-endothelin and its bis-Secanalog exhibited the identical contractile activities in vitro (Figure 1) and pressor effects in vivo (data not shown). 7 Reduction of [Sec³,Sec 11 ,Nle ]-ET-1 with large excesses of DTT in TRIS buffer (pH 8.1) followed by oxidative refolding was found to regenerate quantitatively the correctly folded form as well assessed by the HPLC elution profile. Conversely, wt-ET-1 is known to refold into the correct ribbon and non-natural globule isomer at a ratio of 71/29. However, its N -terminal extension by the Lys-Arg propeptide portion leads exclusively to the native isomer due to a chaperon-like assistance of this dipeptide sequence. It has recently been demon–1 strated that this effect derives mainly from a salt-bridge between Arg a n d 8 Asp [6] that apparently drives the N -terminus to associate in antiparallel orientation to the α -helix with concomitant formation of a hydrophobic core. An equivalent effect is obtained in the ET-1 analog by the favorable redox potential of the pair of Sec residues.

Figure 1. Comparison of the solution structures of ET-1 [5] (A) and [Sec 3 ,Sec11,Nle 7 ]-ET-1 (B) in aqueous acetic acid and of the contractile activities of ET-1 and its bis-Sec-analog.

237 Unlike endothelin, apamin refolds under oxidative conditions quantitatively into the natural globule isomer. Extensive studies on this bis-Cys-peptide have shown that correct refolding occurs independently of the C-terminal tail in a very cooperative manner with a K ox of 0.033 M for Cys³/Cys 15 and 17 M for Cys¹/Cys 11. Thereby already in the Cys³/Cys 15 disulfide-bridged intermediate a native-like conformation is stabilized, whereas the per-Ser-analog as mimicry of the fully reduced apamin form exhibits only a nascent-type helix in the Glu 7 Arg 14 region [7]. To further study the folding mechanism of this model cysteinepeptide we have synthesized by regioselective procedures the [Sec³,Sec11 ]- and [Sec¹,Sec³]-apamin analogs as the non-natural ribbon (parallel disulfides) and beads (consecutive disulfides) isomer, respectively [4]. Upon reduction of wt-apamin and of the two bis-Sec-analogs with NaBH4 oxidative refolding at pH 7.0 was found to reproduce in almost quantitative manner all three starting isomers as shown by comparative HPLC and by the CD spectra reported in Figure 2. The high tendency of apamin to fold into the natural globule structure is largely compensated by the very high K ox value of the diselenide group which independently of sequence-encoded conformational preferences dictates formation of the related isomers. As expected, the CD spectrum of the ribbonisomer of apamin is very similar to that of ET-1, but surprisingly even the beads-isomer is apparently folded into α -helical conformation. In Figure 3 the solution structure of apamin [8] is compared with the preferred 3D folds of the [Sec³,Sec11 ]- and [Sec¹,Sec³]-apamin analogs determined by NMR in aqueous acetic acid and DG calculations. As suggested by the CD spectrum, the ribbon isomer B was found to exhibit an overall fold similar to that of ET-1 with the N -terminus aligned in antiparallel orientation to the α -helix, whereas in the natural globule isomer A it crosses the helix.

Figure 2. CD spectra of wt-apamin (A) and the [Sec³,Sec 11 ]- (B) and [Sec¹,Sec³]-apamin (C) and subsequent refolding by air oxygen upon reduction with NaBH 4 analogs ( Ο—Ο ) in 10 mM phosphate buffer (pH 7.0).

238

Figure 3. Solution structure of apamin [8] (globule isomer A), the [Sec³,Sec11 ]- ( trans-Pro 6ribbon isomer B) and [Sec¹,Sec³]-analog (beads isomer C) as determined by ¹H-NMR conformational analysis in aqueous acetic acid and DG calculations.

Most surprising was the overall fold of the beads isomer C where the β -turn type-structured N-terminus is again approaching with its diselenide-bridge in close contact the disulfide-bridged Ala 9 –Gln 17 portion which itself is folded into a distorted α -helix. Disulfide-bridged Cys residues in positions i and i + 4 are not compatible with the classic α-helix, but the C-terminal sequence portion of apamin apparently prefers ordered conformational states. Therefore, a highly distorted helix results from the disulfide bridging whereby the strain built-up on the disulfide bond could lead to an ‘activated’ disulfide which, if formed statistically in the refolding process, would immediately react with one of the N-terminal Cys residues. The observation that in solution the cis-Pro 6 isomer is more populated in the ribbon than in the globule structure might indicate that the trans → cis -Pro 6 isomerization is responsible for the highly dominant formation of the trans -Pro 6 -globule isomer in oxidative refolding of apamin most probably via the productive Cys³,Cys 15 -intermediate. To conclude, the Cys→ Sec replacements in endothelin and apamin clearly confirmed the absence of significant structural effects of these atomic mutations and thus the isomorphous character of the resulting analogs, but most importantly the ability of dictating the selective diselenide bridge in presence of additional cysteine residues. Correspondingly, the selenocysteine approach allows indeed to design productive and non-productive folding intermediates of multiple-cysteine peptides and proteins as tools for studying the oxidative folding mechanism.

Acknowledgements This study was supported by the DFG grant MO 377/7-2.

239 References 1. Besse, D., Siedler, F., Diercks, T., Kessler, H. and Moroder, L., Angew. Chem. Int. Ed., 36 (1997) 883. 2. Besse, D., Budisa, N., Karnbrock, W., Minks, C., Musiol, H.-J., Pegoraro, S., Siedler, F., Weyher, E. and Moroder, L., Biol. Chem., 378 (1997) 211. 3. Kobayashi, Y., Takashima, H., Tamaoki, H., Kyogoku, Y., Lampert, P., Kuroda, H., Chino, N., Watanabe, T.X., Kimura, T., Sakakibara, S. and Moroder, L., Biopolymers, 31(1991) 1213. 4. Pegoraro, S., Besse, D., Fiori, S., Rudolph-Böhner, S., Watanabe, T.X., Kimura, T. and Moroder, L., In Epton, R. (Ed.) Innovation & Perspectives in Solid Phase Synthesis and Combinatorial Libraries – 1998, Mayflower Scientific, Kongswinford, in press. 5. Tamaoki, H., Kobayashi, Y., Nishimura, S., Ohkudo, T., Kyogoku, Y., Nakajima, K., Kumagaye, S., Kimura, T. and Sakakibara, S., Protein Engineering, 4 (1991) 509. 6. Kubo, S., Chino, N., Nakajima, K., Aumelas, A., Chiche, L., Segawa, S., Tamaoki, H., Kobayashi, Y., Kimura, T. and Sakakibara, S., Lett. Peptide Sci., 4 (1997) 185. 7. Xu, X. and Nelson, J.W., Biochemistry, 33 (1994) 5253 and references therein. 8. Pease, J.H.B. and Wemmer, D.E., Biochemistry, 27 (1988) 8491.

80 Peptide conformational behaviour studied by high and low temperature reversed phase HPLC M.T.W. HEARN*, R. BOYSEN, Y. WANG and S. MURALEDARAM Centre for Bioprocess Technology Department of Biochemistry and Molecular Biology Monash University Clayton, Victoria, Australia 3168

Introduction The use of reversed phase high performance liquid chromatography (RP-HPLC) has become over the past two decades the separation tool p a r excellence for the isolation and purification of synthetic as well as naturally occurring peptides at both the laboratory as well as the process scale [1, 2]. In addition, application of RP-HPLC procedures at the analytical level, frequently now in conjunction with orthogonal methods such as capillary zone electrophoresis (CZE), electrospray mass spectrometry (ES-MS) or matrix assisted laser desorption time-of-flight mass spectrometry (MALDITOEFL-MS), represents the bulwark for characterisation of racemisation, deletion, truncation, incomplete deprotection, or chemical modification events that may occur with peptides produced by solid-phase synthetic protocols. The potential of RP-HPLC, however, goes well beyond such applications. Thus, in common with all high performance chromatographic methods, RP-HPLC also has other biophysical attributes. The well known characteristics of very high separation power and resolution can thus be exploited in a number of different ways, including the ability to derive amino acid descriptor coefficients which relate to the intrinsic hydrophobicity of the primary structure; to ascertain the partition/solubility properties in terms of the hydrophilicity/ hydrophobicity balance of the peptide under different solvent conditions; to generate linear free energy correlations which can be used in structure-function and structure retention correlations; and increasingly to assess the propensity of different peptides to form preferred secondary structures in hydrophobic environments [3–5]. Recent work from this laboratory has provided a conceptual as well as a practical framework to utilise this biophysical potential of RP-HPLC procedures [5, 6]. As part of these investigations the changes in the retention behaviour of all L- α -peptides and their retro-inverso-isomers over a broad range of temperature conditions have been determined. The present report provides some preliminary results and observations with such topologically and compositionally similar peptides, but which manifest reversal of their amino acid sequences. 240 Y. Shimonishi (ed): Peptide Science – Present and Future, 240–244 © 1999 Kluwer Academic Publishers, Printed in Great Britain

241 Results and discussions It is now well recognised that the both thermodynamic, as well as extrathermodynamic, relationships inter-link the dependencies of the retention behaviour of peptides and the chosen experimental conditions in RP-HPLC [1, 10]. In particular, under conditions of linear elution, isocratic chromatographic separation, the capacity factor, , where t R and t O are the retention time and column void time under a defined set of conditions) can be related to the change in Gibbs free energy, , for the association of the peptide with the solvated hydrocarbonaceous ligands immobilised onto a chromatographic sorbent by the dependency: (1) and thus k’ is related to the change in enthalpy and entropy through the Gibbs–Helmholtz relationships, i.e. through Eqns (2) and (3) as follows: (2) (3) and are the enthalpic and entropic changes associated where with the chromatographic migration process, respectively, R is the universal gas constant, T is the absolute temperature and Φ is the phase ratio. As we have shown elsewhere [10–12], under conditions whereby or and hence the change in the heat capacity, , of the system are independent of temperature, ie under conditions of isothermic separation, then the plots of the logarithm of the capacity factor (ln k') versus the reciprocal of the temperature (1/T) should be linear and classical van’t Hoff plot dependencies observed. However, when and show dependencies on temperature (the homothermic and heterothermic cases) then non-linear dependencies of ln k' versus 1/T are anticipated. With the homothermic and heterothermic dependencies, the relationship between ln k' and 1/T has been shown [10] to take the form of Eqns (4) and (5). Thus, when and are both dependent on T , but temperature-invariant heat capacity conditions occur, then the dependency of ln k'i on temperature can be approximated by the logarithmic expression (4) where the symbols T H and T S refer to the temperatures at which respectively. On the other hand, when temperature-dependent and and and conditions prevail, then the dependency of ln k'i on T can be more appropriately approximated by the polynomial expres-

242 sions as represented by Eqns (5) and (6), i.e. (5) (6) When these temperature-dependent homothermic and the heterothermic scenarios occur, the change in enthalpy can be thus represented by (7) whilst the change in heat capacity can be represented (with m = 2Rc, n = 6Rd, etc.) as (8) Under conditions of isothermic separation, the participation of secondary chemical or physical equilibria (such as ionisation changes or conformational transitions) represents negligible or only small contributions to the overall free energy changes, and the phenomenon of enthalpy–entropy compensation follows classical relationships. In contrast, when homothermic or heterothermic processes occur, then non-linear dependencies of ln k' versus 1/T are anticipated and non-classical enthalpy–entropy compensation effects will occur. Such behaviour often reflects the participation of conformational transitions associated with significant changes in the solvation of the peptide as well as changes in the order of the peptide–non-polar ligand complex. The use of peptide isomers containing L-α→ D - α-substitutions provides one avenue to gain key biophysical insight into the molecular origin of these thermodynamic and extra-therodynamic dependencies. In particular, the systematic use of peptidic isomers of the same composition but which bear chirality and/or topological relationships such as diastereomers with L-α - → D- α-substitutions (enantio-pairs); retromers with the same all L- α-amino acids but with the sequence reversed (retromeric pairs); and the retro-enantiomers with the same all L - α -amino acid residues substituted by the corresponding D - α-amino acid and with the amino acid sequence reversed (retroenantiomeric pairs) permits information on the underlying free energy relationships to be quantitatively determined and hence the propensity of particular amino acid sequences to adopt preferred secondary structures in the presence of the solvated hydrophobic ligands assessed. Through the expediency of employing the dependency of ln k' on 1/T as an experimental probe, studied at high and low temperatures, the relevant free energy terms can be quantitatively evaluated and insight into the origin of the dominant molecular contributions involved in the induction or destabilisation of secondary structure of the peptide in the presence of lipophilic environments determined. In Figs. 1(A)–1(B) are shown the plots of ln k' versus 1/T for the 20-mer peptide pair, DDALYDDKNWDRAPQRCYYQ (1) and retro-inverso isomer QYYCRQPARDWNKDDYLADD (2), analysed on a Zorbax 300 SB-C8 sor-

243

Figure 1. (A) Isocratic elution of the all L -α-peptide, 1. (B) Isocratic elution of the retro-inverso peptide, 2.

bent over the temperature range of 5–65°C using isocratic conditions encompassing the mole fraction, ψ , range of the organic solvent modifier (acetonitrile) from 0.17 < ψ < 0.21, whilst in Table 1 are shown the derived values for the and parameters. As evident from detailed corresponding regression analysis of these data, the van t Hoff plots for the peptide pair, 1 and 2, show slightly non-linear relationships of the retention (ln k’) and 1/T a s Table 1. Changes in enthalpy, , and entropy, for the peptide isomer pair, 1 and 2 , based on a isothermic retention process as determined from the corresponding van’t Hoff plot (i.e. log k' versus, 1/T). all-L -α -peptide, 1

ψ ACN 0.17 0.18 0.19 0.20 0.21

– – – – –

0.30 0.27 0.25 0.24 0.22

1.15 1.11 1.09 1.12 1.12

retro-inverso peptide, 2

– 0.27 – 0.24 – 0.22 – 0.22 –0.21

1.07 1.01 1.01 1.06 1.07

244 depicted in Figs. 1 (A)–1(B), indicating dependencies of ∆ H 0assoc,i and ∆ S 0assoc,i on T. The coefficients of the polynomial functions (a, b, c, etc., Eqn. (6)) and their regression coefficients (r = 0.987–0.999) confirmed that this 20-mer peptide pair at different solvent percentages satisfied a second order dependency in terms of the relationship between ln k' and 1/T. This finding is typical of peptides that are relatively highly stabilised in terms of their secondary structural features in the presence of n -alkyl ligands. Experimental data derived from circular dichroism measurements as well as from 2D-NMR studies in solvent environments analogous to the RP-HPLC conditions have confirmed [4,7] that both peptide 1 and 2 exist in α -helical conformations under such conditions, with the preferred sequence region of highest helical propensity corresponding to the residues extending through the D 6 to A13 region of peptide 1, and the residues extending through the region from A8 to Y 16 in the corresponding retro-inverso-isomer, 2. Overall, these results confirm that retro-enatiomeric pairs of peptides can have very similar interactive properties with lipophilic ligands. This conclusion is consistent with the ability of these peptides to assume similar topological features in solution or at lipid-water interfaces. Finally, these observations provide additional validation that the use of RP-HPLC under isocratic conditions, selected according to the molecular properties of the test peptides, provides a very useful adjunct to other physicochemical procedures to elucidate the thermodynamic and extra-thermodynamic effects associated with peptideligand interactions.

Acknowledgements These investigations were supported by the Australian Research Council.

References 1. Hearn, M.T.W., in Protein purification, principles, high resolution methods, and applications, (eds. J.C. Janson and L. Ryden), VCH Publ., New York, 1998, 239–282. 2. Aguilar, M.I. and Hearn, M.T.W., Meth. Enzymol., 1996, 270,1–25. 3. Wilce, M.C., Aguilar, M.I. and Hearn, M.T.W., Anal Chem., 1995, 34,1210–1219. 4. Higgins, K.A., Bicknell, W., Keah, H.H. and Hearn, M.T.W. J. Pept. Res., 1998, 50,421–435. 5. Zhao, L.X. and Hearn, M.T.W., 1998, submitted. 6. Boysen, R., Wang, Y. and Hearn. M.T.W., 1998, submitted. 7. Hearn, M.T.W. in Theory and Practice of Biochromatography (ed. M.A. Vijayalakshmi), Harwood Academic Publishers, Switzerland, 1998, in press.

81 Three-dimensional structure and receptor-recognition sites of bombyxin-II, an insulin-like brain-secretory peptide of the silkmoth K. NAGATA1 , 2 , H. HATANAKA1 , D. KOHDA1 , H. ISHIZAKI3 , K. MOMOMURA4 , K. TAMORI4 , T. KADOWAKI 4 , M. TANAKA2 , H. KATAOKA2 , H. NAGASAWA 2 , A. ISOGAI 2 , A. SUZUKI2 and F. INAGAKI1 1 Department of Molecular Physiology, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan 2 Department of Agricultural Chemistry, The University of Tokyo, Tokyo, Japan 3 Department of Biology, Nagoya University, Nagoya, Japan 4 The Third Department of Internal Medicine, The University of Tokyo, Tokyo, Japan

Introduction Bombyxin is a brain-secretory peptide of the silkmoth Bombyx mori, which shows 40% sequence identity to insulin (Figure 1) [1]. When injected into a brain-removed dormant pupa of a relative silkmoth Samia cynthia ricini, bombyxin promotes its adult development by stimulating ecdysteroid synthesis in the prothoracic glands [2,3]. Despite the sequence similarity, no cross-activity

Figure 1. Primary and tertiary structure comparison of bombyxin-II with human insulin and human relaxin 2.

245 Y. Shimonishi (ed): Peptide Science – Present and Future, 245–248 © 1999 Kluwer Academic Publishers, Printed in Great Britain

246 was observed between bombyxin and insulin [3]. To elucidate the structural basis of receptor specificity between them and to investigate the molecular evolution of insulin-superfamily peptides, we determined the three-dimensional structure of bombyxin-II, a representative molecular species of bombyxin, by NMR [4], and identified its functional sites by structure-activity relationship studies.

Results and discussion Bombyxin-II was synthesized by the solid-phase peptide synthesis and regioselective disulfide formation [5]. Two-dimensional ¹H NMR spectra (DQFCOSY, TOCSY, NOESY and PE-COSY) of 2–4 mM bombyxin-II dissolved in 30%/70% (v/v) acetic acid/water were measured on a JEOL α600 spectrometer at 28°C. Using these spectra, sequential assignments were made. The threedimensional structure was calculated by the simulated annealing protocol with X-PLOR ver. 2.1 using the 559 NMR-derived restraints (535 distances and 24 dihedral angles) [4]. The A-chain of bombyxin-II consists of two antiparallel helices (IleA2 to LeuA8 and ValA13 to TyrA19), which are connected by a loop (ArgA9 to SerA12) (Figure 1). The B-chain contains a less well-defined N-terminal region (pGluB( – 2) to HisB4), an extended arm (ThrB5 to GlyB8), a central helix (ArgB9 to AlaB22), and a C-terminal coil (GlyB23 to AspB25). The structure including the three helices is stabilized by the three disulfide bonds (CysA6–CysA11, CysA7–CysB7 and CysA20–CysB19) and a hydrophobic core (IleA2, LeuA16, TyrA19, TyrB6, LeuB11, LeuB15 and LeuB18). The overall main-chain fold of bombyxin-II in solution (Brookhaven PDB, 1BON) is similar to those of insulin in solution ( 1HIU), insulin in the crystalline T-state (4INS) and relaxin in crystal (6RLX) (Figure 1); the root-mean-square deviations are 1.31 Å, 1.28 Å and 1.45 Å, respectively, for the main-chain atoms (N, C α , C') within the common helical regions. The common structural features include (1) an A-chain with two helices joined by an extended loop, (2) a B-chain with an extended N -terminus followed by a central helix, (3) three disulfide bonds and (4) a hydrophobic cluster including the residues at A2, A6, A11, A16, A19, A20, B6, B11, B15, B18 and B19. Despite the overall structural similarity, the conformation of the B-chain C-terminal part of bombyxin-II is quite different from that of insulin (Figure 2). Bombyxin-II, like relaxin, adopts a helix and a coil, instead of a sharp turn and an extended β -strand as do insulin and IGFs. Bombyxin-II and human insulin are not cross-active to each other. In order to localize the functional specificity determinants, we synthesized hybrid molecules of bombyxin-II and human insulin and assayed the bombyxin-like activity (the prothoracicotropic acticity to the saturniid moth Samia cynthia ricini) and the insulin-like activity (the affinity to the human insulin receptor) [6]. Bonsulin, consisting of the bombyxin-II A-chain and the human insulin B-chain,

247 showed only the insulin-like activity, while imbyxin, consisting of the human insulin A-chain and the bombyxin-II B-chain, showed only the bombyxin-like activity. Therefore, the B-chain, but not the A-chain, determines either function. To localize further the important region for the bombyxin-like activity, we then synthesized chimeric molecules consisting of the bombyxin-II A-chain and a chimeric B-chain, and bioassayed the bombyxin-like activity [7]. Bonsy(6–18)lin, whose B-chain consists of the central part (B6 to B18) of the bombyxin-II B-chain and the N- and C -terminal parts (B1 to B5 and B20 to B30) of the human insulin B-chain, was shown to be a full agonist, while bonsy(7–18)lin and bonsy(6–17)lin were less active than bonsy(6–18)lin. Thus, the central part (B6 to B18) of the bombyxin-II B-chain is important for the bombyxin activity. In contrast, neither the N -terminal part (pGluB( – 2) to ThrB5) nor the C -terminal part (TrpB20 to AspB25) does not contribute to the bombyxin-like activity. To evaluate the importance of each side chain in the B-chain central part (B6 to B18), Ala-scanning mutagenesis was done. CysB7 and GlyB8 were not replaced because they were essential for maintaining the native conformation. The native Ala at B12 and B16 were replaced by the insulin-type amino acids, Val and Tyr, respectively. The bioassays showed that the side chains of TyrB6, LeuB11, AlaB12, ThrB14, LeuB15, AlaB16, AspB17 and LeuB18 contributed to the bombyxin-like activity, while those of ArgB9, HisB10 and ArgB13 did not. Other mutageneses by us showed that the N- and C-termini of the A-chain (GlyA1, ValA3 and CysA20–CysB19) were also important. The important residues for the bombyxin-like activity are mapped on the three-dimensional structure (Figure 2). They form two patches on the surface of bombyxin-II molecule. The patch I is a hydrophobic surface including the important residues, LeuB11, AlaB12, LeuB15, AlaB16, GlyA1, ValA3 and CysA20–CysB19, and putative important residues, IleA2 and TyrA19. The

Figure 2. Putative receptor-recognition sites of bombyxin-II. Right and left ovals represent the patches I and II, respectively.

248 patch II includes the important residues, TyrB6, ThrB14, AspB17 and LeuB18, and a putative important residue, ValA13. It is interesting that the location of the functional sites of bombyxin-II is similar to the putative receptor-recognition sites of human insulin [8] except for the C -terminal parts of the A- and B-chains; the residues at A21, B24 and B25 are also important for the activity of insulin, but not for that of bombyxin. They commonly involve the residues at A1 (Gly/Gly), A2 (Ile/Ile), A3 (Val/Val), A19 (Tyr/Tyr), A20 (Cys/Cys), B11 (Leu/Leu), B12 (Ala/Val), B15 (Leu/Leu), B16 (Ala/Tyr), and B19 (Cys/Cys) in the patch I and at A13 (Val/Leu), B6 (Tyr/Leu) and B17 (Asp/Leu) in the patch II. Therefore, we concluded that the functional patches I and II of bombyxin-II represent its receptor-recognition sites. Despite the similar location, their putative receptor-recognition sites show dissimilar features due to the different amino-acid residues in the central and C-terminal parts of the B-chain (B6, B12, B16, B17 and B24, B25) and the different conformations of the B-chain C -terminal part. Thus, we concluded that these structural differences in the central and C -terminal parts of the B-chain should make these molecules functionally different.

References 1. Nagasawa, H., Kataoka, H., Isogai, A., Tamura, S., Suzuki, A., Mizoguchi, A., Fujiwara, Y., Suzuki, A., Takahashi, S.Y. and Ishizaki, H., Proc. Natl. Acad. Sci. USA 83 (1986) 5840. 2. Ishizaki, H. and Ichikawa, M., Biol. Bull. 133 (1967) 355. 3. Nagasawa, H., Kataoka, H., Isogai, A., Tamura, S., Suzuki, A., Ishizaki, H., Mizoguchi, A., Fujiwara, Y. and Suzuki, A., Science 226 (1984) 1344. 4. Nagata, K., Hatanaka, H., Kohda, D., Kataoka, H., Nagasawa, H., Isogai, A., Ishizaki, H., Suzuki, A. and Inagaki, F., J. Mol. Biol. 253 (1995) 749. 5. Maruyama, K., Nagata, K., Tanaka, M., Nagasawa, H., Isogai, A., Ishizaki, H. and Suzuki, A., J. Protein Chem. 11(1992) 1. 6. Nagata, K., Momomura, K., Tamori, K., Kadowaki, T., Maruyama, K., Tanaka, M., Kojima, K., Nagasawa, H., Kataoka, H., Isogai, A. and Suzuki, A., In Yanaihara., N. (ed.) Peptide Chemistry 1992, ESCOM, Leiden, p. 416. 7. Nagata, K., Hatanaka, H., Kohda, D. Kataoka, H., Nagasawa, H., Isogai, A., Ishizaki, H., Suzuki, A. and Inagaki, F., J. Mol. Biol. 253 (1995) 759. 8. Schäffer, L., Eur. J. Biochem. 221 (1995) 1127.

82 α C -Methyl, Cα-phenylglycine peptides

M. DOI1 , T. ISHIDA1 , E. MOSSEL2 , F. FORMAGGIO2 , M. CRISMA2 , C. TONIOLO2 , Q.B. BROXTERMAN3 and J. KAMPHUIS4 Osaka University of Pharmaceutical Sciences, Osaka 569-11, Japan Biopolymer Research Center, CNR, Department of Organic Chemistry, University of Padova, 35131 Padova, Italy 3 DSM Research, Organic Chemistry and Biotechnology Section, P.O. Box 18, 6160 MD Geleen, The Netherlands 4 DSM Fine Chemicals, 6401 JH Heerlen, The Netherlands 1 2

Introduction Recent years have seen impressive advances in the design of peptides with predetermined secondary structures and physical properties. These studies have laid the groundwork for tayloring peptides capable of recognizing other bioorganic molecules in a sequence-dependent manner. To this aim the conformationally constrained Cα -methylated α-amino acids are among the most widely used structural units. In this connection we have already shown that peptides based on ( αMe)Phe (Cα -methyl phenylalanine) and ( αMe)Hph (Cα -methyl homo-phenylalanine) preferentially adopt β -turn and 310 -helical structures, and that turn and helix handedness is critically biased by the position of side-chain branching.

Results and discussion In order to complete the picture of the role played by the phenyl ring position in the C α-methylated α-aminoacid side chain on peptide preferred conformation, we have synthesized and examined by FT-IR absorption, ¹H NMR, CD and X-ray diffraction techniques a number of peptides, including homo-peptides, based on Cα -methyl, Cα -phenylglycine [(αMe)Phg]. Our conformational study in solution strongly supports the view that there are at least two significantly populated conformations present in the equilibrium mixtures (a fully-extended and a folded/helical conformation). The relative extent of the latter increases as peptide main-chain length is increased. In this Cβ-branched residue the relationship between α -carbon chirality and turn/helix handedness typical of protein amino acids seems to be slightly prevailing. We have also found that the conformational properties exhibited by ( αMe)Phgrich peptides in solution are well reproduced in the crystal state. An X-ray diffraction analysis shows that the ( αMe)Phg residue in Ac-L-( α Me)Phg–NHBzl (Ac, acetyl; NHBzl, benzylamino), Bz- L - (αMe)Phg–NHMe (Bz, benzoyl; NHMe, methylamino), and Z-L -(α Me)Phg-(D-Ala) 2 –OMe (Z, benzyloxycarbo249 Y. Shimonishi (ed): Peptide Science – Present and Future, 240–250 © 1999 Kluwer Academic Publishers, Printed in Great Britain

250

Figure 1. X-Ray diffraction structures of the tripeptides Z-L-(αMe)Phg-( L-Ala) 2 –OMe 1 and Z-L (αMe)Phg-(D-Ala) 2 –OMe 2 . The (αMe)Phg residue adopts the fully extended conformation in tripeptide 2, while it is folded in a type-I β-bend conformation in tripeptide 1. The intramolecular H-bonds are indicated by dashed lines. Numbering of the heteroatoms is also reported.

nyl; OMe, methoxy) (Figure 1) is fully extended, while a type-I β -turn is formed by Z- L-( α Me)Phg-( L -Ala) 2 –OMe (Figure 1) and a regular 310 -helix by pBrBz(Aib) 2 -L-( α Me)Phg-(Aib)2 –OtBu (pBrBz, para-bromobenzoyl; Aib, α -aminoisobutyric acid; OtBu; tert-butoxy) and its Nα -acetylated analog. Interestingly, in the former pentapeptide the helix is right-handed, while it is left-handed in the latter. The results already published and those described here allow us to conclude that the three C α -methylated, aromatic α-amino acids [(α Me)Phg, ( αMe)Phe, and ( αMe)Hph] are β -bend and helix-inducers, much stronger than their unmethylated counterparts. A comparison between the two Cα -methylated, β branched α-amino acids ( α Me)Val and ( αMe)Phg seems to indicate that the aliphatic residue has a more pronounced tendency to fold into bend/helical structures and to adopt the normal relationship between α -carbon chirality and bend/helix handedness.

83 Graphite-mimetic peptides C. TONIOLO1 , F. FORMAGGIO1 , M. CRISMA1 , T. YOSHIKAWA2 and T. WAKAMIYA2 1 Biopolymer Research Center, CNR, Department of Organic Chemistry, University of Padova, 35131 Padova, Italy 2 Department of Chemistry, Kinki University, Osaka 577, Japan

Introduction α,β -Dehydro- α -amino acid (∆AA) residues are frequently found in naturally occurring peptides of microbial and fungal metabolite origin and they are also constituents of polycyclic peptide antibiotics [1,2]. The presence of ∆ AA residues in peptides confers increased resistance to enzymatic degradation. Most of the conformational studies have been limited to peptides containing either ∆ Z Phe or ∆ Z Leu residues [2,3]. These two γ -branched ∆ AAs proved to be very effective β -turn/3 10 (α )-helical formers. On the other hand, only few conformational studies have been devoted to the conformation of ∆ Ala compounds (and they have been restricted to amino acid derivatives and dipeptides) [4, 5].

Results and discussion We synthesized the first model homo-peptide series from a Cα,β -dehydro- α aminoacid, pBrBz–( ∆ Ala) n –OMe (where pBrBz is para-bromobenzoyl, ∆ Ala is Cα,β -dehydroalanine, OMe is methoxy, and n = 1–6) by solution methods. The most relevant synthetic steps involve the preparation of the p BrBz-[A 2 pr(Z)] n – OMe (where A 2 pr is α,β -diaminopropionic acid, Z is benzyloxycarbonyl, and n = 1–6) oligomeric series, followed by removal of the Z N β -protecting group using 25% HBr in acetic acid, β -deamination via permethylation of the β amino group with a large excess of CH 3 I, and treatment of the resulting tetraalkylammonium group with a base (KHCO 3 ). The overall reaction yields from the [A 2 pr(Z)] n series to the (∆ Ala) n series range from 80% for the n = 1 oligomer, to 50% for the n = 2–4 oligomers and 20–25% for the n = 5, 6 oligomers. The conformational preferences of the terminally-blocked ∆Ala homo-oligomers were extensively analyzed by FT-IR absorption, ¹H NMR, and X-ray diffraction (using the latter technique we have solved the 3D structures of the n = 1–4 homo-oligomers). Our study showed that a multiple, consecutive, fullyextended conformation (2.0 5 -helix) largely predominates in CDCl 3 , solution. In this structure each of the NH groups is intramolecularly H-bonded to the 251 Y. Shimonishi (ed): Peptide Science – Present and Future, 251–252 © 1999 Kluwer Academic Publishers, Printed in Great Britain

252

Figure 1. The packing mode of the pBrBz–(∆Ala) 3 –OMe molecules in the crystal. The molecules pack as isolated entities. In particular, they do not form any intermolecular H-bond.

carbonyl group of the same residue. On the other hand, intermolecular H-bonds do not seem to play any significant role, even at high peptide concentrations. In analogy with the solution results the four homo-oligomers examined in the crystal state adopt single (n = 1) or multiple (n = 2–4) fully-extended conformations (Figure 1). Additional interesting properties are: (i) These peptide molecules are completely flat, including the amino acid side chains, and form graphitelike layers. The distance between two adjacent (∆Ala) n planes is ~ 3.30 Å, to be compared to the analogous distance of 3.35 Å in graphite. (ii) This n e w peptide structure is stabilized by two types of intramolecular H-bonds, Ni – (typical of the 2.0 5 -helix) and (characteristic of 2.05-helical ∆ Ala peptides and forming a six-membered ring). (iii) The average distance is 3.77 Å, the largest known separation of this type for any peptide structure established experimentally so far. In conclusion, we unambiguosly identified a new, completely flat peptide structure. For these graphite-mimetic peptides we foresee a bright future in biochemistry and material science as well.

References 1. Stammer, C.H., In Weinstein, B. (Ed.) Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Dekker, New York, 1982, pp. 33–74. 2. Jain, R. and Chauan, V.S., Biopolymers (Pept. Sci.), 40 (1996) 105. 3. Singh, T.P., Narula, P. and Patel, H.C., Acta Crystallogr., B46 (1990) 539. 4. Palmer, D.F., Pattaroni, C., Nunami, K., Chadha, R.K., Goodman, M., Wakamiya, T., Fukase, K., Horimoto, S., Kitazawa, M., Fujita, H., Kubo, A. and Shiba, T., J. Am. Chem. Soc., 114 (1992) 5634. 5. Pietrzynski, G., Rzeszotarska, B., Ciszak, E., Lisowski, M., Kubica, Z. and Boussard, G., Int. J. Pept. Protein Res., 48 (1996) 347.

84 The E. Fischer’s octadecapeptide revisited after 90 years C. TONIOLO1 , F. FORMAGGIO 1 , M. CRISMA1, S. PEGORARO 2 and P. ROVERO3 1 Biopolymer Research Center, CNR, Department of Organic Chemistry, University of Padova, 35131 Padova, Italy 2 Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany 3 Istituto di Mutagenesi e Differenziamento, CNR, 56124 Pisa, Italy

Introduction In 1907 Emil Fischer published in Ber. Deutsch. Chem. Gesell. [1] the synthesis and characterization of his longest (18-residue) peptide, H-L-Leu-(Gly)3 -L-Leu(Gly) 3 -L-Leu-(Gly) 8 -Gly-OH (mw = 1213.3), which represents a milestone in the history of peptide chemistry. Although advantaged by the introduction in the literature of sophisticated protecting groups and carboxyl activating methods, it took more than 50 years to peptide chemists to synthesize a peptide longer than the Fischer’s octadecapeptide. Results and discussion After 90 years we have resynthesized the Fischer’s octadecapeptide using the SPPS method on a PEG-PS resin containing an acid-labile 4-hydroxymethyl-3-methoxyphenoxyacetyl handle and using the Fmoc/OtBu chemistry. Fmoc deprotection was achieved by means of a DBU/piperidine mixture in DMF. Peptide bonds were formed in DMF using a 4-fold excess of the FmocAA-OH/TBTU/HOBt mixture, and double Fmoc-L-Leu-OH coupling. The peptide was removed from the resin by treatment with a TFA/EDT/H 2 O mixture, and purified by preparative HPLC. Characterization of the final product was achieved by analytical HPLC (Figure 1), amino acid analysis, and mass spectrometry. Rather surprisingly, despite the sequence would mainly consist of hydrophobic residues and long Gly stretches, neither incomplete coupling nor deprotection steps did represent any serious problem during the SPPS of the octadecapeptide. An attempt to assess the preferred conformation of the octadecapeptide in the solid state was made by means of the FT-IR absorption technique. The spectrum, with bands at 3305, 1660, and 1541 cm –1 , does reflect the occurrence of weakly H-bonded species, but this structure is clearly different from either the poly(Gly) n I conformation ( β -sheet structure) or the poly(Gly) n II conformation (3 1 -helix). 253 Y. Shimonishi (ed): Peptide Science – Present and Future, 253–254 © 1999 Kluwer Academic Publishers, Printed in Great Britain

254

Figure 1. Analytical HPLC chromatogram of the Fischer’s octadecapeptide prepared by the SPPS method. Column: Vydac C18 (0.46 x 15 cm); flow: 1 ml/min; UV detection at 210 nm; eluants: A = 0.1% TFA in H2O, B = 0.1% TFA in CH3CN; gradient: from 10% to 30% B over 20 min.

The far-UV CD spectrum of the octadecapeptide in aqueous solution at room temperature is characterized by a generally very weak dichroism with Cotton effects at 233 nm (negative), 212 nm (positive), and 193 nm (negative). On the basis of this pattern, we are inclined to exclude the formation of either the α -helical or the β -sheet conformation to any substantial amount under these conditions. Rather, the CD spectrum is reminiscent of an at least partially extended conformation. Cooling the aqueous solution to 3°C does not induce any significant change in the CD spectrum.

Reference 1. Fischer, E., Ber. Deutsch. Chem. Gesell., 40 (1907) 1754.

85 Conformational analysis of peptide fragments corresponding to domains of potassium ion-channel proteins P.I. HARIS Department of Biological Sciences, De Montfort University, Hawthorn Building, The Gateway, Leicester, LE1 9BH, United Kingdom

Introduction Voltage-gated potassium channels are large integral membrane proteins which conduct ions across biomembranes and are responsible for electrical excitability [1, 2]. They are thought to be constructed from four similar domains with each of the domains consisting of six membrane spanning segments, S1 to S6. The structural models of voltage-gated potassium channels are based solely on electrophysiological and mutagenesis experiments. From these experiments it is suggested that the H5 region is responsible for conductance and ion selectivity properties of the channel [for a review see Ref. 2]. More recent mutagenesis studies have indicated that the segment connecting the S4 and S5 putative transmembrane segments (S4–S5 loop) also constitute part of the ion-channel pore [3]. The S4 transmembrane domain is suggested to have a role in sensing the transmembrane electric potential [2]. Very little information is available regarding the conformation of ion-channel proteins. This is primarily due to the absence of sufficient quantities of such proteins for biophysical studies. Synthetic peptides provide an alternative approach for structural investigation of the structure of ion channel proteins. Previously we have used this approach to determine the structure of synthetic peptides corresponding to the pore (H5) and voltage-sensor (S4) domains of the Shaker channel [4, 5]. Here we report our studies on the structure of a peptide corresponding to the sequence that contains the S4 segment and part of the S4–S5 loop region (this peptide will be referred to as S4–S45) of this channel protein.

Results and discussion The sequence of the 34 residue S4–S45 peptide is as follows: Leu–Ala–Ile–Leu–Arg–Val–Ile–Arg–Leu–Val–Arg–Val–Phe–Arg–Ile–Phe– Lys–Leu–Ser–Arg–His–Ser–Lys–Gly–Leu–Gln–Ile–Leu–Gly–Arg–Thr–Leu– Lys–Ala 255 Y. Shimonishi (ed): Peptide Science – Present and Future, 255–256 © 1999 Kluwer Academic Publishers, Printed in Great Britain

256 The structure of the peptides was determined in an aqueous environment, organic solvent as well as in phospholipid membranes. The peptide is readily soluble in aqueous solution. The CD spectra recorded for the sample in H2 O shows a minimum near 195 nm. This indicates that the peptide is in a predominantly random coil conformation in this media. In contrast, the spectrum of the peptide in 100% trifluoroethanol shows a minimum near 208 nm and a shoulder near 220 nm which indicates the presence of α -helical structure. The peptide was also found to adopt an α -helical structure in phospholipid membranes. There was no evidence for the presence of any β -sheet structure for the peptide in solution and in membrane systems. The results of our study show that the conformation of the S4–S45 peptide is highly dependent on its surrounding media. It adopts a random coil conformation in aqueous solution which changes to a predominantly α -helical conformation in trifluoroethanol and in phospholipid membranes. These results are similar to what we have obtained previously for the S4 peptide [5]. The peptide investigated in this study contains the S4 domain as well as part of the S4–S5 loop that has been suggested to constitute part of the ion-channel pore [3]. In the absence of high-resolution structural data various models have been proposed for the structure of voltage-gated potassium channels. For example, it has been suggested that the ion-selective pore is composed of β -sheet structure [6–8]. Previously we have shown that a peptide corresponding to the H5 ionselective pore domain is predominantly α -helical in structure [4]. Here we have shown that another segment of the Shaker potassium channel, that is thought to be part of the ion-channel pore, adopts a predominantly α -helical structure. A detailed paper describing these studies, along with the NMR results, will be published elsewhere.

References 1. Noda, M., Shimizu, S., Tanabe, T., Takai, T., Kayano, T., Ikeda, I., Takahashi, H., Nakayama, H., Kanaoka, Y., Minamino, N., Kangawa, K., Matsuo, H., Raftery, M.A., Hirose, T., Inayama, S., Hayashida, H., Miyato, T. and Numa, S., Nature, 312 (1984) 121. 2. Pongs, O., Physiol. Rev., 72 (1992) S69. 3. Slesinger, P., Jan, Y.N. and Jan, L.Y., Neuron, 11(1993) 739. 4. Haris, P.I., Ramesh, B., Sansom, S.P., Kerr, I.D., Srai, K.S. and Chapman, D., Prot. Eng., 7 (1994) 255. 5. Haris, P.I., Ramesh, B., Brazier, S. and Chapman, D., FEBS Lett., 349 (1994) 371. 6. Yellen, G., Jurman, M.E., Abramson, T. and MacKinnon, R., Science, 251 (1991) 939. 7. Yool, A.J. and Schwarz, T.L., Nature, 349 (1991) 700. 8. Durrell, S.R. and Guy, H.R., Biophysical Journal, 62 (1992) 238.

86 Conformation and sweet taste of L-Asp- D -Xaa–OMe dipeptides Y.J. KIM¹ and Y.K. KANG² ¹Department of Chemistry, Seonam University Namwon, Chonbuk 590-711, Korea ²Department of Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea

Introduction The structure-taste relationships of sweet molecules have been studied by many groups and several models have been suggested to explain the structure-taste relationships of L -aspartyl dipeptide derivatives [1, 2]. In the previous work, we studied the relationship between the structure and taste of L -aspartyl dipeptide methyl esters (L - + HAsp– - L -Xaa–OMe) with the same chirality but different tastes in order to figure out the conformational preferences to elicit tastes [3]. Recently, Mattern et al. reported the relationship between topochemical arrays and the taste in aspartyl-based dipeptide taste ligands [4]. In the present study, conformational free energy calculations have been carried out on the L - + HAsp –- D -Xaa–OMe to describe low free energy conformations of the dipeptides in solution and to deduce conformational preferences to elicit the tastes.

Results and discussion The calculations were carried out on the L - + HAsp –- D -Xaa–OMe, where Xaa includes three type of residues, i.e., sweet (Abu, Ala, Ile, Ser, Thr, and Val), bitter (Leu, Phe, and Trp), and tasteless (Met and Tyr) residues. The tastes of dipeptides were taken from Refs. 1 and 5. The conformational free energy calculations were carried out with the ECEPP/3 [6]. The hydration shell model [7] was used to calculate the hydration free energy of each conformation of the dipeptide in the hydrated state. All the torsion angles of the peptide backbone, side chains, and end groups were allowed to move during minimization. The conformational entropy change was calculated using a harmonic method [8, 9]. The relative entropic contribution to the relative free energy was computed at T= 298 K. The starting conformations of L - + HAsp –- D -Xaa–OMe were obtained by combining the low energy conformations of L - + Asp– -NHMe and Ac-D -Xaa–OMe as described in our previous work [3]. The preferred conformations for sweet, bitter, and tasteless dipeptides are presented in Figure 1, in which each dipeptide is superposed on the reference 257 Y. Shimonishi (ed): Peptide Science – Present and Future, 257–259 © 1999 Kluwer Academic Publishers, Printed in Great Britain

258

Figure 1. The most preferred conformations for L - + HAsp – -D-Xaa–OMe: (a) sweet Abu, Ala, Ile, Ser, Thr, and Val dipeptides, (b) bitter Leu, Phe, and Trp dipeptides, and (c) tasteless Met and Tyr dipeptides. The reference conformation g– Fg – of aspartame is represented by dotted lines in (a)–(c). All hydrogen atoms are not shown for clarity.

conformation g – Fg – (dotted line) of the aspartame ( L - + HAsp – -L -Xaa–OMe) of our previous work [3]. The computed conformational preferences for sweet, bitter, and tasteless dipeptides in the hydrated state indicate to us that the conformation about the N–Cα bond of the Xaa residue, i.e., the orientation of the hydrophobic moiety with respect to the AH/B functionalities in the aspartyl moiety, seems to be crucial to elicit the tastes. Our results on the conformationtaste relationship for sweet L -+ HAsp – -D -Xaa–OMe dipeptides are consistent with the L-shape model proposed by Goodman’s group and similar to those o f L - + HAsp– -L -Xaa–OMe dipeptides studied by us previously. This work was supported by the Basic Science Research Institute Program, the Ministry of Education of Korea (BSRI-96-3448).

References 1. Walters, D.E., Orthoefer, F.T. and Dubois, G.E. (Eds) Sweeteners: Discovery, Molecular Design, and Chemoreception (ACS Symposium Series 450), American Chemical Society, Washington, DC,1991. 2. Yamazaki, T., Benedetti, E., Kent, D. and Goodman, M., Angew. Chem. Int. Ed. Engl., 33 (1994) 1437. 3. Kim, Y.J., Han, S.J., Kim, S.C. and Kang, Y.K., Biopolymers, 34 (1994) 1037. 4. Mattern, R.-H., Amino, Y., Benedetti, E. and Goodman, M., J. Peptide Res., 50 (1997) 286.

259 5. Miyashita, Y., Takahashi, Y., Takayama, C., Sumi, K., Nakatsuka, K., Ohkubo, T., Abe, H. and Sasaki, S., J. Med. Chem., 29 (1986) 906. 6. Némethy, G., Gibson, K.D., Palmer, K.A., Yoon, C.N., Paterlini, G., Zagari, A., Rumsy, S. and Scheraga, H.A., J. Phys. Chem., 96 (1992) 6472. 7. Kang, Y.K., Gibson, K.D., Némethy, G. and Scheraga, H.A., J. Phys. Chem., 92 (1988) 4739. 8. Go, N. and Scheraga, H.A., Macromolecules, 9 (1976) 535. 9. Kang, Y.K. and Jhon, M.S., Macromolecules, 17 (1984) 138.

87 Conformations of four cyclic tetrapeptides containing two Pro residues M. TAMAKI¹, M. YABU¹, A. KANEKO¹, S. AKABORI¹ and I. MURAMATSU ¹ Department of Chemistry, Toho University, Miyama, Funabashi, Chiba 274, Japan. ² Tokyo Bunka Junior College, Hon-cho, Nakano-ku, Tokyo 164, Japan

Introduction A Pro residue in naturally occurring bioactive peptides and proteins plays an important structural role in folding them. [1] In order to investigate the role of Pro residue in peptides and proteins, four cyclic tetrapeptides, cyclo(- L -ProNH–(CH 2 )n– CO–) 2 (n = 2, 3, 4, and 5) were synthesized [2], and the conformations in CHCl 3 and CH 2 Cl 2 were characterized by means of NMR, CD and IR spectra with the aid of conformational calculations.

Results and discussion CD spectra of these cyclic tetrapeptides in CD 2 Cl 2 were shown in Figure 1. These CD spectra in CH 2 Cl 2 were classified into two groups in relation to the number of methylene groups (n = 2–5) of amino acid residues (AA) used as a connector between two Pro residues. CD spectra of cyclic tetrapeptides having even-numbered methylene groups (n = 2 and 4) showed the one negative trough near 230 nm. The CD patterns are characteristic for having γ -turn structure. [2] On the other hand, cyclic tetrapeptides having odd-numbered methylene groups (n = 3 and 5) possessed the one negative trough near 210 nm and the one shoulder near 225 nm. In the NMR spectra of cyclic tetrapeptides having even-numbered methylene groups (n = 2 and 4) in CDCl 3 and CD 2 Cl 2 , the Pro α H resonance in CDCl 3 and CD 2 Cl 2 shifted to the low field (4.69 and 4.70, and 4.65 and 4.54 ppm, β respectively) and appeared as a double doublet due to the H 2 .The Pro β C in CDCl 3 and CD 2 Cl 2 fairly shifted upfield from a usual position for a trans Pro β C, and ∆ δ β−γ in CDCl 3 and CD 2 C l 2 were 0.97 and 0.50, and 1.44 and 1.51 ppm, respectively. In addition, the spatial NOE’S in these solvents were observed between AA α CH A and Pro δCH, and AA δNH and Pro αCH. These data indicated that cyclic tetrapeptides having even-numbered methylene groups (n = 2 and 4) in CDCl3 and CD 2 Cl 2 adopt two γ -turn structures consisting of all trans peptide bond with two trans Pro αC–C=O bonds [3]. In the NMR spectra of cyclic tetrapeptides having odd-numbered methylene groups (n = 3 260 Y. Shimonishi (ed): Peptide Science – Present and Future, 260–262 © 1999 Kluwer Academic Publishers, Printed in Great Britain

261

Figure 1.

CD spectra of cyclo(-L -Pro-NH–(CH 2 )n-CO-) 2

(n = 2, 3, 4, and 5) in CH 2 C l 2 .

and 5) in CDCl3 and CD 2 C l 2 , the Pro β C and γ C chemical shifts and Pro γ β C– C chemical shift difference ( ∆δ β – γ ) indicated the presence of two cis AA-Pro bonds in these solvents [3]. In addition, the spatial NOE’s in CDCl3 a n d CD 2 Cl 2 were observed between AA α CH A and Pro α CH, and AA γ NH and Pro δ CH. These data of NMR indicated that cyclic tetrapeptides having oddnumbered methylene groups (n = 3 and 5) have a C2 symmetric conformation consisting of the cis-trans-cis-trans peptide bond (with two cis AA-Pro bonds), α and two cis Pro C–C=O bonds in CDCl 3 and CD 2 Cl 2 . [3] The temperature coefficients of the chemical shifts (∆ δ / ∆ T ) of the NH resonances of AA preceding Pro residue, and IR spectra of the amide N–H stretch region in CDCl 3 and CD 2 Cl 2 of these cyclic peptides indicated that the amide protons of these cyclic peptides in CDCl 3 and CD 2 Cl 2 are shielded from the solvent and involved in a rigid intramolecular hydrogen bond, and suggested that the presence of the intramolecular hydrogen bonds in CDCl 3 and CD 2 Cl 2 of these cyclic peptides contributes to the stabilization of each predominant conformer. The molecular mechanics calculations and semi empirical calculation were performed on a UNIX workstation system: Silicon Graphics INDIGO 2. In the molecular mechanics calculations, each structure was optimized by Sybyl/X force fields using Spartan software. PM3 method was used for the semiempirical calculations. [4] The lowest energy conformation of these cyclic peptides are similar to the conformations proposed by the CD, NMR and IR measurements. Thus, cyclo(- L -Pro-NH–(CH 2 )n–CO–) 2 (n = 2, 3, 4, and 5) in CHCl 3 and CH 2 Cl2 showed two characteristic conformations containing cis or trans X-Pro

262 peptide bond in relation to the number of methylene groups (n = 2–5) of amino acid residues used as a connector between two Pro residues.

References 1. Smith, J.A., Pease, L.G., Reverse Turns in Peptides and Proteins In CRC Critical Reviews in Biochemistry; Fasman, G.D., Ed.; CRC Press, Inc.: Boca Raton, FL, 1980, Vol. 8, p 315. 2. Tamaki, M., Komiya, S., Yabu, M., Watanabe, E., Akabori, S. and Muramatsu, I., Peptide Chemistry 1996, (1997) pp. 393–396. 3. Deber, CM., Madison, V. and Blout, E.R., Acc. Chem. Res., 9 (1976) 106. 4. Clark, M., Cramer III, R.D. and Opdensch, N. van., J. Computational Chem., 10 (1989) 982.

88 Solution structure of the antimicrobial peptide ranalexin and a study of its interaction with dodecylphosphocholine micelles E. VIGNAL¹, A. CHAVANIEU² , P. ROCH³, L. CHICHE¹, G. GRASSY¹, B. CALAS², A. AUMELAS¹ ¹ CBS, UMR 9955, U414 INSERM, Faculté de Pharmacie, 34060 Montpellier, France ² CRBM, CNRS ERS 155, route de Mende 34033 Montpellier, France ³ DRIM, UMR 219, Place Eugeve Bataillon 34095 Montpellier, France

Introduction Ranalexin, a 20-residue peptide isolated from the skin of the bullfrog Rana catesbeiana, displays antimicrobial activity [1]. This peptide contains two cysteine residues in positions 14 and 20 linked by a disulphide bridge and was found to share structural similarities with polymyxin A. The solution structure of ranalexin was determined in water, in 30% TFE and in the presence of micelles of perdeuterated dodecyl-phosphocholine (DPC) by using CD, ¹H-NMR and molecular modelling techniques.

Results and discussion The reduced (RnxSH) and the oxidized (RnxSS) forms of ranalexin were synthesized by the solid phase method. RnxSS appeared as active as polymyxin B on gram-positive cocci, but eight-times less active than polymyxin B against gram-negative bacilli (Figure 1). On the other hand, RnxSH was two times less active than RnxSS against both bacteria, implying that the disulphide bond is not critical for the antimicrobial activity of ranalexin. The CD and NMR spectra clearly showed that RnxSH and RnxSS are mainly unstructured in water but display an alpha-helical structure spanning S. eperdimidis

RanaSH RanaSS Polymyxin B

E. coli

MIC

MBC

MIC

MBC

5 2.5 2.5

20 10 10

10 5 0.6

10 5 0.6

Figure 1. Sequence of ranalexin and antimicrobial activities of RnxSH, RnxSS and polymyxin B against Staphylococcus epidermidis (gram-positive) and Escherichia coli (gram-negative). (MIC) minimal inhibitory concentration and MBC (minimal bactericidal concentration) are given in µg/ml.

263 Y. Shimonishi (ed): Peptide Science – Present and Future, 263–265 © 1999 Kluwer Academic Publishers, Printed in Great Britain

264

Figure 2. Structure of RnxSS as determined in the presence of DPC micelles [(DPC)/(RnxSS) molar ratio = 28, 300 K, pH 3.7]. The backbone and the disulphide bond are displayed as thick and dashed lines, respectively.

residues 8–15 and 8–17, respectively, in 30% TFE, whereas a 3–10 helix sketch was identified in the 2–7 segment. Interestingly, RnxSS was found to interact with micelles of zwitterionic lipids assumed to simulate the membrane-water interface [2] and to adopt a helical structure from K7 to T 17 , which includes proline in position 10 and the 14–17 sequence of the macrocycle. In the presence of DPC the structure of RnxSS shows an uneven distribution of hydrophobic and hydrophilic side chains (Figure 2). A cluster of apolar side chains brings about a large hydrophobic surface mainly located on the same side of the helix and near the N -terminus, whereas two out of the three positively charged side chains belong to the macrocycle. Moreover, slow exchanging amide protons of residues V 9 , M 12 , I 13 and V 16 suggest that the hydrophobic face of the helix lies on the micelle surface thus burying these amide protons (Figure 2). Furthermore, during titration by DPC the large chemical shift variations experienced by the F¹ residue indicate that this residue also participates in the interaction with the micelle. This observation is strongly supported by the 64-fold decrease in antimicrobial activity measured for the analogue displaying F¹ deletion [1]. Indeed, due to the strong hydrophobicity of this aromatic residue [3] and its probable penetration in the micelle deduced by NMR spectra, an anchoring role in the membrane can be hypothesized for F¹. Taken together, our findings suggest that the disulphide bond does not strongly affect either the conformation or the antimicrobial activity of ranalexin. These results contribute to a better understanding of the interaction of ranalexin with the cell membrane and afford new insights into its mechanism of action at the structural level. Ranalexin is another peptide supporting the hypothesis of the requirement of hydrophobic–hydrophilic cluster proximity to induce antimicrobial activity [4, 5].

References 1. Clark, P.D., Durell, S., Maloy, W.L. and Zasloff, M., J. Biol. Chem. 269 (1994) 10849–10855. 2. Lauterwein, J., Bösch, C., Brown, L.R. and Wüthrich, K., Biochim. Biophys. Acta 556 (1979) 244–264.

265 3 . Wimley, W.C. and White, S.H., Nature Struct. Biol. 3 (1996) 842–848. 4 . Kini, R.M. and Evans, H.J., Int. J. Peptide Protein Res. 34 (1989) 277–286. 5 . White, S.H., Wimley, W.C. and Selsted, M.E., Current Opinion in Structural Biology 5 (1995) 521–527.

89 Formation of native disulphide bonds in endothelin-1: evidence for the involvement of a highly specific salt-bridge between the pro-sequence of endothelin-1 and mature sequence A. AUMELAS¹, S. KUBO², N. CHINO², L. CHICHE¹, E. FOREST³, C. ROUMESTAND¹ and Y. KOBAYASHI 4 ¹ CBS, UMR 9955, U414 INSERM, Faculté de Pharmacie, 34060 Montpellier, France ² Peptide Institute, Inc., 4-1-2 Ina, Minoh-Shi, Osaka 562, Japan ³ Institut de Biologie Structurale, 41, avenue des Martyrs, 38027 Grenoble, France 4 Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565, Japan

Introduction Endothelin (ET-1) gives at least 25% of the non-native disulphide arrangement. In contrast, addition of the Lys – 2 –Arg – 1 dipeptide, present in the pro-sequence, to the N-terminus of ET-1, to form a 23-residue peptide (KR-ET-1) has been shown to greatly improve formation of native disulphide bridges (96%) [1,2]. While the carboxylic acid state structure of KR-ET-1 is very similar to that of ET-1, the carboxylate state structure is more constrained and stabilized by the 8 Arg –1 –Asp salt-bridge unambiguously identified from the study of the [D 8 N ] , 10 [E Q] and [D 18 N] analogues of KR-ET-1 by CD, ¹H-NMR and mass spectrometry as a function of pH. Assuming that the formation of disulphide bonds occurs in a thermodynamically controlled step [3], we have hypothesized that the Arg –1 –Asp 8 salt bridge could be responsible for the increase in yield of the native isomer of KR-ET-1. To demonstrate this possibility, the formation of disulphide bonds for these carboxamide analogues was measured. In addition, the three-dimensional structure of the minor non-native isomer of KR-ET-1 was determined and its salt bridge potentialities evaluated. Results and discussion The fact that KR-ET-1 and the [E10 Q] and [D 18 N] analogues, which all display the Arg –1 –Asp 8 salt bridge, gave high yields of native disulphide bonds ( ≥ 90%), and that the [D 8 N] analogue, which does not display such a salt bridge, gave a native to non-native ratio similar to that of ET-1 (68%) strongly supports the direct involvement of the Arg –1 –Asp 8 salt bridge in the improved formation of the native disulphide bonds. In contrast to what was observed for the native isomer, the mass, CD and ¹H-NMR spectra of the non-native isomer were insensitive to carboxyl group 266 Y. Shimonishi (ed): Peptide Science – Present and Future, 266–268 © 1999 Kluwer Academic Publishers, Printed in Great Britain

267

Figure 1. Structures of the native (left) and the non-native (right) KR-ET-1 isomers. The two isomers differ by their disulphide pattern and therefore by the orientation of the N-terminal KRCSC sequence resulting in the presence or the absence of the Arg – 1 –Asp 8 salt bridge. The figure was prepared using MOLSCRIPT (Kraulis, 1991).

ionization, suggesting that there is no stable salt bridge involving carboxylate groups with Lys –2 or Arg –1 side chains in its structure. The structures of the two isomers (Figure 1) clearly show the conservation of the helical portion although for the non-native isomer, at pH 4.0, a lower helical content was observed. However, the two structures markedly differ about the orientation of the KRCSC backbone. In the native isomer, this backbone lays roughly anti-parallel to the axis of the helix, whereas in the nonnative isomer it is approximately perpendicular to the axis of the helix. Simulated annealing calculations on the latter isomer showed that distances between the Arg –1 guanidinium group and the Asp 8 or Glu 10 carboxylate groups in several conformations were compatible with salt bridge formation. However, in these conformations salt bridges are probably not stable enough to be experimentally observed probably due to the lack of efficient cooperativity between electrostatic and hydrophobic interactions. We conclude that the folding improvement in KR-ET-1, versus ET-1, is a direct consequence of the additional stability afforded by the salt bridge between two conserved residues, one belonging to the pro-sequence (Arg –1 ) and the other to the ET-1 sequence (Asp 8 ). Moreover, the involvement of the pro-sequence in the formation of native disulphide bonds strongly suggests that in the maturation pathway of ET-1, cleavage of the Arg 52 –Cys 53 amide bond occurs after formation of native disulphide bonds.

268 References 1 . Aumelas, A., Chiche, L., Kubo, S., Chino, N., Tamaoki, H. and Kobayashi, Y., Biochemistry 34 (1995) 4546–4561. 2 . Kubo, S., Chino, N., Nakajima, K., Aumelas, A., Chiche, L., Segawa, S., Tamaoki, T., Kobayashi, Y., Kimura, T. and Sakakibara, S., Lett. Pept. Sci. 4 (1997) 185–192. 3 . Gilbert, H.F. in Mechanisms of Protein Folding (1994) Pain R.H., Ed., pp 104–136, IRL Press, Oxford.

90 Structure–biological activity relationships of cecropin A (1–8)–magainin 2 (1–12) hybrid and its analogues S.Y. SHIN, J.H. KANG, D.G. LEE, S.Y. KIM, M.K. LEE and K.-S. HAHM Peptide Engineering Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST, P.O. Box 115, Yusong, Taejon 305-600, Korea

Introduction In order to design the peptides having potent antibiotic activity without hemolytic effect, cecropin A(1–8)-magainin 2(1–12) [CA-MA] and its analogues were synthesized and their antibiotic activities were evaluated. Secondary structures of the peptides in various condition were estimated by CD spectra.

Results and discussion The peptides take random structures in phosphate buffer, while showing a high α -helicity in 50% TFE or 30 mM SDS. CA-MA showed no hemolytic activity at 100 mg/ml, whereas CA-ME containing CA(1–8) and melittin(1–12) [1] showed a high hemolytic activity. A decrease in the positive charge at the two positions 18 and 19 of CA-MA by substituting with Thr (A1) induced a significant reduction in antitumor activity, but did not have a significant effect on antibacterial and hemolytic activity. The increase in hydrophobicity of Table 1.

Antibacterial and antitumor activities of the peptides in this study. MIC (µg/ml)

Peptides

Sequences

CA-MA CA-ME A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13

E. coli

B. subtilis

IC 5 0 (µg/ml) SCLC

12.5 25.0 25.0 25.0 6.25 1.56–3.125 100 3.125–6.25 100 25.0 50.0 12.5 12.5 6.25 25.0

6.25 6.25 3.125 3.125 3.125 0.78 25.0 6.25 50.0 6.25 12.5 6.25 6.25 3.125 6.25

79 90 116 107 24 12 86 24 84 26 34 24 12 26 86

269 Y. Shimonishi (ed): Peptide Science – Present and Future, 269–270 © 1999 Kluwer Academic Publishers, Printed in Great Britain

270 Table 2. Hemolytic activity and mean hydrophobicity of the peptides and % α-helicity of the peptides in various conditions deduced from CD spectra. % α -helicity

% Hemolysis

Peptides

Buffer

50% TFE

30 mM SDS

25 µg/µl

CA-MA CA-ME A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13

0.7 4.4 6.0 5.1 6.6 4.5 7.2 3.4 5.0 2.6 2.9 3.0 1.2 2.7 26.6

55.9 57.4 70.0 52.5 35.6 76.0 84.2 81.1 70.7 90.7 54.6 53.8 75.8 69.6 65.3

16.9 34.3 26.2 30.7 76.0 32.3 62.1 98.8 64.7 84.9 31.8 29.6 33.2 63.0 73.6

0 2.2 0 0 0 0 0 91.0 17.0 6.1 0 1.8 3.5 8.7 91.7

100 µg/µl 0 51.0 0 0 33.4 11.8 13.3 98.5 94.3 81.9 3.8 54.7 73.0 70.8 100

Mean hybrophobicity –0.154 –0.011 –0.062 – 0.086 – 0.072 –0.114 – 0.084 – 0.033 –0.047 –0.047 –0.149 –0.047 –0.100 –0.114 – 0.033

position 16 by substitution with Leu (A4) of Ser in CA-MA resulted in a remarkable increase in antibacterial activity as well as antitumor activity without a significant increase in hemolysis. In contrast, Phe replacement (A5) at this position had little influence on hemolytic activity, while causing a 4-fold reduction in antibacterial activity. These results suggested that the moderate hydrophobicity at position 16 of CA-MA plays a critical role in antibiotic activities. The α-helicity of the peptides is thus closely related to their hemolytic activity rather than antibacterial and antitumor activity. There is no correlation between the activity of the peptides against tumor cells and red blood cells. Also, there is no correlation between activity of the peptides against tumor cells and bacterial cells.

Reference 1. Andreu, D., Ubach, J., Boman, A., Wahlin, B., Wade, D., Merrifield, R.B. and Boman, H.G., FEBS Lett. 296 (1992) 190.

91 Structure-activity relationship of ω-agatoxin-TK, a peptide blocker of P/Q-type calcium channels T. WATANABE¹, M. KUWADA¹, Y. NISHIZAWA¹, K.Y. KUMAGAYE², K. NAKAJIMA² and N. ASAKAWA¹ ¹Eisai Tsukuba Research Laboratories, Tsukuba 300-26, Japan ²Peptide Institute Inc., Protein Research Foundation, Osaka 562, Japan

Introduction ω-Agatoxin-TK (ω-Aga-TK), a 48-amino-acid peptide isolated from the venom of the American funnel web spider (Agelenopsis aperta), is a selective and potent inhibitor of both the P-type and long-lasting Q-type (P/Q-type) calcium channels in mammalian brain neurons. This peptide has a unique structural profile including a high-density disulfide core structure formed by four disulfide bonds and an unusual D -form amino acid, D -Ser, at position 46 (Figure 1) [1]. A stereoisomer of this peptide, ω -[L-Ser 46 ]Aga-TK (ω -Aga-TK- L ), has also been found in the venom, which has the same amino acid sequence and disulfide pairings as that of the D -Ser toxin (ω -Aga-TK- D ) except for L -Ser at position 46 [2]. ω -Aga-TK- L has 80- to 90-fold less potency towards the calcium channels compared with ω -Aga-TK- D. Previously, we reported the conformational analyses of the two toxins using size-exclusion chromatography, circular dichroism (CD), and fluorescence spectroscopy [3]. The comparative analyses of the D -form and L -form toxins suggested that the D -Ser toxin may form a particularly compact folding involving β -sheet structures and the L -Ser toxin may have a relatively unfolded or extended structure at physiological pH and ionic strength. In order to further elucidate the structure-function of ω -AgaTK- D , we have examined the biological activity of the two toxins and a C-terminus truncated fragment by a patch-clamp technique, and analyzed the conformations of the peptides by the combination of CD measurements and hydrophobic interaction chromatography (HIC).

Figure 1 Primary structure of ω- agatoxin-TK- D

.

271 Y. Shimonishi (ed): Peptide Science – Present and Future, 271–273 © 1999 Kluwer Academic Publishers, Printed in Great Britain

272

Figure 2. Hydrophobic interaction chromatography of ω-Aga-TK- D and ω -Aga-TK- L. The chromatography was carried out on a TSK gel ether-5PW (0.75 × 7.5 cm) and with a linear gradient of ammonium sulfate in a phosphate buffer, pH 7.2. The flow rate was 1.0 ml/min.

Results and discussion During a hydrophobic interaction chromatography, ω -Aga-TK- D was eluted more slowly than ω -Aga-TK- L under neutral conditions as illustrated in Figure 2. This study clearly demonstrated that the molecular surface of ω -AgaTK-D has a relatively more hydrophobic nature compared with ω -Aga-TK- L under the neutral conditions, and the conformational difference is caused by the configuration of Ser 46 in the molecule. In order to study the role of the C-terminal region including Ser 46 i n t h e ω-Aga-TK- D , we have prepared a C-terminal truncated peptide of the D -form toxin by carboxypeptidase digestion, namely ω -Aga-TK- D -(1–47). Biological activity of ω -Aga-TK- D -(1–47) that lacks only a C-terminal Ala residue, was found to be much less potent than that of ω -Aga-TK- D , which was similar to ω -Aga-TK- L . Additionally, a conformational study using CD and HIC also indicated that ω -Aga-TK- D -(1–47) has a conformation distinct from ω -AgaTK- D , but has almost the same as that of ω -Aga-TK- L . These results revealed that this fragment had similar biological potency and conformational properties to ω -Aga-TK- L and the hydrophobicity of the C-terminal region may define the conformation and biological activity of ω -Aga-TK. Additional experiments to assess the biological importance of the carboxylterminal tail seem worthwhile. Studies are in progress to further characterize the carboxyl-terminal conformation of ω -Aga-TK.

References 1. Kuwada, M., Teramoto, T., Kumagaye, K.Y., Nakajima, K., Watanabe, T., Kawai, T., Kawakami, Y., Niidome, T., Sawada, K., Nishizawa, Y. and Katayama, K., Mol. Pharmacol., 46 (1994) 587.

273 2. Watanabe, T., Teramoto, T., Kuwada, M., Shikata, Y., Niidome, T., Kawakami, Y., Sawada, K., Nishizawa, Y. and Katayama, K., In Ohno, M. (Eds), Protein Research Foundation, Osaka, 1995, p.253. 3. Watanabe, T., Kuwada, M., Kumagaye, K.Y., Nakajima, K., Nishizawa, Y. and Asakawa, N., D. in Marshak, R. (Ed.), Techniques in Protein Chemistry VIII, Academic Press, 1997, p. 543.

92 Peptide scanning in structural -functional mapping of hemeproteins E.F. KOLESANOVA¹, J.G. KISELAR¹, S.A. MOSHKOVSKII¹, S.A. KOZIN², G.HUI BON HAO³ and A.I. ARCHAKOV¹ ¹Institute of Biomedical Chemistry, 10, Pogodinskaya ul., 119832, Moscow, Russia ²Brandeis Univiresity, 4125 South st., Waltham, MA, 02254-9110, USA ³INSERM-U310, Institut de biologie physico-chimique, 1,3 rue P. & M. Curie, 75005 Paris, France

Introduction Peptide scanning (PEPSCAN) is widely used for linear antigenic mapping of proteins, because it reveals almost all possible linear B-epitopes in a protein [1]. To our mind, linear antigenic determinants may have some common structural characteristics, thus allowing antigenic mapping data to be applied in structural studies of proteins. Besides that, an antigen-antibody interaction is only one example of protein-protein interactions, and PEPSCAN approach may be used for studies of other kinds of protein-protein interactions as well, contributing to structural-functional mapping of proteins. Results and discussion 511, 486, 409, and 90 partially overlapping hexapeptides, covering the whole sequences of cytochromes P450 (CYP) 1A2, 2B4, 101, and cytosolic domain of cytochrome b5, correspondingly, were synthesized on polyethylene pins [2–4]. Binding of the peptides with IgGs from antisera against corresponding proteins (including antisera against apo-CYP 2B4) was determined by ELISA. Search for common peptide fragments in cytochrome P450 sequences was performed using Cytochrome P450 DataBase [5]. Analysis of water acessibility of CYP101 and cytochrome b5 atoms was made with ONIX software [6]. Linear antigenic sites revealed by PEPSCAN occupy 22–66% of the mapped proteins sequences. CYP 1A2 had the lowest density of linear B-epitopes (22–24%) among the studied proteins, while B-epitopes of cytochrome b5 (cytosolic domain) composed 66% of its sequence. Use of antisera against CYP 1A2 and 2B4 from different host animals (mice and chickens) showed the existence of the so-called antigenie ‘core’ (antigenic sites revealed by both types of antisera) and the sites with somewhat occasional antigenicity (revealed with only one type of antisera). The former sites may be used for producing antipeptide antibodies with high probability of success, while the whole maps including both type of antigenic sites may be used for structural analysis of proteins. 274 Y. Shimonishi (ed): Peptide Science – Present and Future, 274–276 © 1999 Kluwer Academic Publishers, Printed in Great Britain

275 Antigenic sites of CYP 1A2 and 2B4 were classified into several groups depending upon their existence in molecules of other cytochromes P450. Monospecific linear B-epitopes are found only in the molecule of the protein under study, while group-specific B-epitopes are common for the corresponding subfamilies (CYP 1A and 2B) and even families (CYP 1 and 2). No epitopes common for different families were found. Analysis of B-epitopes location in CYP 101 and cytosolic domain of b5 with respect to the spatial structure of these proteins showed that almost all found linear antigenic sites are located at the boundaries of secondary structure elements and contain at least 50% water-accessible amino acid residues. This observation points out to the usefulness of the antigenic mapping data in the structural studies of proteins, e.g., in refining molecular modelling results. Comparison of antigenic mapping results of the holo- and apo-forms of CYP 2B4 showed only 43% identity in the structures of linear B-epitopes of both forms. Apo-CYP 2B4 had much less density of antigenic sites (23% of the sequence) than the holo-form (49%). Several B-epitopes characteristic for each of the two forms were revealed, pointing out to the possibility of producing specific antipeptide antibodies. These antibodies may be used for separate detection of the corresponding forms of the enzyme. Special modification of the PEPSCAN procedure was made in order to enable the mapping of CYP-interacting sites in cytochrome b5 and NADPHcytochrome P450 reductase (flavoprotein, FP). This modification consisted in adding the incubation of pin-linked overlapping hexapeptides covering regions 34–58 of cytochrome b5 and 201–225 of FP with CYP 2B4 (in the presence of 0.025% Emulgen 913 for the monomerization of this membrane protein) or 101. Several hexapeptides of cytochrome b5 bound CYP 2B4 and 101specifically, while no CYP-binding hexapeptide fragments were found in FP region 201–225. The revealed CYP-binding peptides of cytochrome b5 may be used further to model protein–protein interactions in monooxygenase multienzyme system. Thus, the presented results show the undoubtful usefullness and efficiency of different modifications of PEPSCAN approach (antigenic mapping, modelling of protein-protein interaction sites) in structural and functional studies of proteins.

References 1. Worthington, J. and Morgan, K., In Wisdom, G.B. (Ed.) Peptide Antigens. A Practical Approach. Oxford Univ. Press, Oxford, New York, Tokyo, 1994, pp. 181–217. 2. Kolesanova, E.F., Kozin, S.A., Lemeshko, A.O. and Archakov, A.I., Biochem. Mol. Biol. Int. 32 (1994) 465. 3. Kolesanova, E.F., Kiselar, J.G., Jung, C., Kozin, S.A., Hui Bon Hoa, G. and Archakov, A.I., Biochimie 78 (1996) 752. 4. Kolesanova, E.F., Kozin, S.A., Rumyantsev, A.B., Jung, C., Hui Bon Hoa, G. and Archakov, A.I., Arch. Biochem. Biophys. 341(1997) 229.

276 5. Archakov, A.I. and Bachmanova, G.I., Cytochrome P450 and Active Oxygen. Taylor & Francis, London, New York, Philadelphia, 1990, Supplement. 6. Ivanov, A.I., Rumjantsev, A.B., Skvortsov, V.S. and Archakov, A.I., J. Chem. Inf. Comput. Sci. 36 (1996) 660.

93 Structure analysis of the second transmembrane segment of the rat bradykinin receptor in solution and in micelles by CD and fluorescence spectroscopies E. OLIVEIRA¹, A. MIRANDA¹, D. ANDREU², S. SCHREIER³, A.C.M. PAIVA¹, C.R. NAKAIE¹ and M. TOMINAGA¹ ¹ Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua 03 de Maio, 100, São Paulo, SP, 04044-020, Brazil ² Department of Organic Chemistry, Universitat de Barcelona,Barcelona, Spain ³ Instituto de Química, Universidade de São Paulo, São Paulo, Brazil

Introduction In a recent paper [1], we investigated alternative strategies to minimize difficulties in synthesizing and purifying a long and hydrophobic transmembrane peptide sequence (Ac-CTVAEIYLGNLAGADLILASGLPFWAITANNFDNH2 , denoted TM2-34) corresponding to the (64–97) region of the second transmembrane segment of the rat BKB2 receptor [2]. Improved yield of synthesis was obtained by changing the solvent system during the coupling step, accordingly to our conjugated resin solvation-polarity of the medium strategy [3]. We also performed a preliminary conformational analysis of this 34-mer peptide and its minor fragment (74–94), denoted TM2-24, by circular dichroism (CD) in aqueous solution containing different amounts of hexafluoroisopropanol (HFIP). In this solvent, a well-known α-helical structure inducer, we have found a α-helical content lower than expected for this type of transmembrane fragment. Hence, to better investigate structural properties of these two peptides, taken as model of transmembrane fragment, CD and fluorescence spectroscopic methods were applied in aqueous solution, varying the pH and the proportion of trifluoroethanol (TFE), and an other secondary structure inducing solvent. This structural investigation was also carried out in presence of dodecylsulp h a t e - ( S D S ) a n d N -hexadecyl-N,N,dimethyl-3-ammonium-1 propylsulphate(HPS)-type micelles. Owing to the low solubility of TM2-34 analog, most of experiments in aqueous solution were performed with the TM2-24 transmembrane fragment.

Results and dicussion The CD spectra of TM2-24 (Figure 1) in aqueous media displayed remarkable pH dependence. In pH 3.5, the shape of the spectrum suggests that the peptide 277 Y. Shimonishi (ed): Peptide Science – Present and Future, 277–279 © 1999 Kluwer Academic Publishers, Printed in Great Britain

278

Figure 1. CD spectra of TM2-24 (24 µM) in different pH.

adopts a substantial proportion of β -structure. When the pH is increased from 3.5 to 7, the intensity of the signal at 215–220 nm decreases whereas in alkaline solution (pH 9.0), a random-coil structure is observed in higher extension. The steady state fluorescence spectra of this peptide showed an increase of the maximum emission intensity and the shift of maximum wavelength of emission as the pH increases (from pH 3.5 to 7.0). This result may indicate higher exposure of the Trp residue in pH 7.0 than in pH 3.5, probably as a result of conformational changes of the peptide. Secondary structures of TM2-24 and TM2-34 were also evaluated by titration with TFE. As expected, both transmembrane sequences assumed an increasingly α-helical structure as the concentration of TFE increases, reaching maximum values in neat TFE (about 70% of α -helical content in these two peptides). These results are in close agreement with the secondary structure predictions of these peptides performed by a system of neural networks by B. Rost, EMBL, Hildelberg, FRG [4]. Otherwise, we did not observe similar behavior of the TM2-24 peptide when in HFIP. In this case, the measured maximum helipcity was only about 30%, observed in aqueous 25% HFIP solution. The presence of SDS- or HPS-micelles in pH 7.0 changed the fluorescence spectra of TM2-24, reflecting the interaction of peptide with the hydrophobic micelle structure. In both types of micelles, a significant increase of the maximum emission intensity and blue shifted spectra were observed. Regarding CD studies, a clear conformation changes of the peptide was observed and α -helical contents of 44% and 55% was measured in SDS- and HPS-micelles, respectively. Thus, these findings confirmed that the secondary structure of peptides may be strongly affected by the nature of the solvating system and the α helipcity of transmembrane segment is enhanced when in presence of membrane mimetic micelle environment or in TFE.

279 References 1. Oliveira, E., Miranda, A., Albericio, F., Andreu, D., Paiva, A.C.M., Nakaie, C.R. and Tominaga, M., J. Peptide Res. 49 (1997) 300. 2. Cilli, E.M., Oliveira, E., Marchetto, R. and Nakaie, C.R., J. Org. Chem. 61(1996) 8992. 3. McEarchen, A E., Shelton, E.R., Bhakta, S., Obernolte, R., Bach, C., Zuppan, P., Fujisaki, J., Aldrich, R.W. and Jarnagin, K., Proc. Natl. Acad. Sci., USA 88 (1991) 7724. 4. Rost, B. and Sander, C., Proteins 19 (1994) 55.

94 Conformational analysis of antimicrobial peptide tachystatin A isolated from horseshoe crab hemocytes by two dimensional ¹H NMR spectroscopy N. FUJITANI¹, S. KAWABATA², K. KAWANO¹, K. HIKICHI¹ and K. NITTA¹ ¹ Department of Biological Science, Graduate School of Science, Hokkaido University, Sapporo 060, Japan ² Dpartment of Biology, Faculty of Science, Kyushu University, Fukuoka 812-81, Japan

Introduction Granular hemocytes in the horseshoe crab (Tachypleus tridentatus) hemolymph contain large and small granules. Large ones contain the coagulation factors and their regulators, and small ones contain the antimicrobial substances. Once lipopolysaccharide (LPS), an element of Gram-negative bacteria cell wall, contacts these hemocytes, body fluid coagulates rapidly. Tachystatin A is in the small granules and inhibits the growth of Gram-negative and positive bacteria, and fungi, and is expected to play an important role in the mechanism of the defense system of the horseshoe crab. Two isopeptides of tachystatin A were found and both consist of 44 amino acid residues containing three disulfide bonds of which position was determined only for Cys11–Cys29. One of them contained tyrosine at the COOH-terminal end, another contained phenylalanine at the corresponding site. [1] Tachystatin A shows weak sequence homology to ω-agatoxin-IVA of funnel web spider (Agelenopsis aperta), a potent blocker of voltage-dependent calcium channels. [2]

Results and discussion The solution structure of tachystatin A was determined by 2D-NMR spectroscopy. 2D-NMR spectra were acquired for tachystatin A at 293 K with a sample concentration of 2 mM. We used standard ¹H NMR methods, COSY, TOCSY and NOESY experiments, in D 2 O and H 2 O solutions. In the fingerprint region of COSY spectrum, of which peaks reveal the correlation between α protons and amide protons, we could observe 41 peaks expected for this molecule, and the chemical shift dispersion throughout the spectrum was sufficient to permit its complete assignment. 280 Y. Shimonishi (ed): Peptide Science – Present and Future, 280–281 © 1999 Kluwer Academic Publishers, Printed in Great Britain

281

Figure 1. Left: Three dimensional structure of tachystatin A in aqueous solution. Tachystatin A has a short antiparallel β-sheet and the topology of the β-sheet is (+2x, –1), corresponding with that of ω-agatoxin-IVA.; Right: The β-sheet structure in tachystatin A. NOE connections are shown with arrows and encircled protons represent slowly exchanging rate.

From the pattern of NOEs and of slowly exchanging amide protons, we expected a β -sheet structure consisting of 9–12, 28–31 and 39–42 residues. By calculating the low energy structures with the method of distance geometry and simulated annealing with restraints of NOES and dihedral angles obtained from spin-spin coupling constants between α protons and amide protons (³ J α N H ), we obtained a short anti-parallel β -sheet structure composed of 9–12, 29–32 and 38–41 residues (Figure 1). The topology of this β -sheet (+2x, –1) corresponds with that of ω -agatoxin-IVA. In other regions, there is no secondary structure similar to ω -agatoxin-IVA. From the calculated structure, we can assume the remaining disulfide bonds exist between Cys4–Cys24 and Cys23–Cys41.

References 1. Kawabata, S., et al., Peptide Chemistry, Protein Research Foundation, Osaka, 1997, p. 97–100 2. Kim J.I., Konishi, S., Iwai, H., Kohno, T., Gouda, H., Shimada, I., Sato, K. and Arata, Y., Journal of Molecular Biology. 250 (1995) 659–671

95 Solution structure of α -conotoxin MI determined by ¹H-NMR spectroscopy and molecular dynamics simulation with the explicit solvent water H. GOUDA¹, K. YAMAZAKI², J. HASEGAWA², Y. KOBAYASHI³, 4 Y. NISHIUCHI , S. SAKAKIBARA4 and S. HIRONO¹ ¹ School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku, Tokyo 108, Japan ² Developmental Research Institute, Daiichi Pharmaceutical Co., Ltd., Kitakasai, Edogawa-ku, Tokyo 134, Japan ³ Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565, Japan 4 Peptide Institute, Inc., Protein Research Foundation, Ina, Minoh, Osaka 562, Japan

Introduction Two-dimensional NMR experiments and simulated annealing calculations had provided the overall topology of α -conotoxin MI, a potent antagonist of the nicotinic acetylcholine receptor, but the resulting structures did not show an obvious structural feature. Therefore, the refinement procedure has been performed using molecular dynamics (MD) simulation with the explicit solvent water.

Results and discussion An MD simulation was performed by AMBER version 4.1 with its all-atom force field [1, 2]. One of structures obtained by simulated annealing calculations, which has the lowest energy value evaluated by the X-PLOR potential function, was solvated by 2517 TIP3P waters in a rectangular box whose dimensions were 48.2 × 45.0 × 40.1 Å. The simulation was run under SHAKE constraints, a 1 fs time step, and constant pressure. A cut-off radius of 9 Å for nonbonded interactions was applied. The interactions up to 14 Å were only calculated at every 10 steps. After two 3 ps of MD at 100 K and 200 K, 194 ps (total 200 ps) of MD was performed at 300 K. Potential functions for the NMR-derived constraints were added in order to make the trajectory satisfy these experimental data more closely. After about 100 ps, this simulation converged to a structure with average root-mean-square (RMS) deviations of 1.40 ± 0.07 Å from the initial structure and 0.48 ± 0.001 Å from the average structure over 100 to 200 ps for the backbone atoms excluding the N-terminal two residues. Figure 1 provides the average structure of α -conotoxin MI over 100 to 200 ps. It indicates the presence o f a 3 10 helix and a type I β -turn for residues Pro6 –Cys 8 and Gly9 –Tyr 12 , 282 Y. Shimonishi (ed): Peptide Science – Present and Future, 282–283 © 1999 Kluwer Academic Publishers, Printed in Great Britain

283

Figure 1. Stereopair of the mean structure over 100 to 200 ps of α -conotoxin MI. All heavy atoms are shown.

respectively. Recently, the high resolution X-ray structure of α -conotoxin GI, which has biological activity similar to that of MI, has been also reported [3]. The RMS deviation between the two structures was 1.14 Å for the backbone atoms. In addition, the X-ray structure of α -conotoxin GI shows the presence of a 3 1 0 helix for residues Pro 5 –Cys 7 and a type I β -turn or a distorted 310 helix for residues Gly 8 –Tyr 11 , as well as the case of MI. These results obviously indicate that the α -conotoxins MI and GI exhibit a common structural topology. It might be essential for the binding of two toxins to receptor sites.

Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

References 1. Pearlman, D.A., Case, D.A., Caldwell, J.W., Ross, W.S., Cheatham III, T.E., Ferguson, D.M., Seibel, G.L., Singh, U.C., Weiner, P.K., Kollman, P.A., AMBER 4.1, University of California, San Francisco, 1995. 2. Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz Jr, K.M., Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W., Kollman, P.A., J. Am. Chem. Soc. 117 (1995) 5179. 3. Guddat, L.W., Martin, J.A., Shan, L., Edmundson, A.B., Gray, W.R., Biochemistry 35 (1996) 11329.

96 Study of depsipeptide Boc–(Leu–Leu–Ala)2 –(Leu–Leu– Lac) 3 –OEt forming 31 0 -helix T. OHYAMA¹, A. HIROKI¹, K. KOBAYASHI¹, K. WAKAMATSU² and R. KATAKAI¹ ¹Department of Chemistry, ²Department of Biological and Chemical Engineering, Gunma University, 1-5-1, Tenjin-cho, Kiryu-shi, 376, Japan

Introduction 3 10 -Helices are much less common than α -helices, although they are by no means rare. A minority of the 310 -helices occur as an N- or C -terminal extension to an α -helix in globular proteins [1]. The reason and significance of its occurence at these locations are still not obviously. Short α -aminoisobutyric acid-containing peptides have been studied as models of peptide forming 3 10 helix [2]. We found that a depsipeptide Boc–(Leu–Leu–Ala) 2 –(Leu–Leu–Lac) 3 – OEt assumes 310 -helix. We suppose that the lack of hydrogen bondings at ester bonds plays an important role in forming 310 -helix for the depsipeptide. In this study, we have synthesized a depsipeptide Boc–(Leu–Leu–Ala)3 –(Leu–Leu– Lac) 2 –OEt in which the Lac 9 residue is substituted by Ala residue, for studying the influence of the ester bond on forming 310 -helix, and compared with the conformation of the depsipeptides by CD and 2D-NMR.

Figure 1 CD spectra for Boc–(Leu–Leu–Ala) 3–(Leu–Leu–Lac) 2 –OEt (a) and Boc–(Leu–Leu–Ala) 2 – (Leu–Leu–Lac) 3 –OEt (b) in 1-butanol.

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Figure 2. Summary of the ROE connectivity observed for Boc–(Leu–Leu–Ala)2 –(Leu–Leu–Lac) 3 – OEt (A) and Boc–(Leu–Leu–Ala) 3 –(Leu–Leu–Lac) 2 –OEt (B) in CDCl3 .

Results and discussion Depsipeptides were synthesized by the conventional solution phase method by using a segment condensation strategy. We used CD for conformational studies of depsipeptides in 1-butanol. The CD spectrum of Boc–(Leu–Leu–Ala)2 –(Leu– Leu–Lac)3 –OEt has a positive maximum at 192 nm with mean residue ellipticity [ Θ] = 14,500 deg cm2 dmol–1 and a negative maximum at 205 nm with [ Θ ] = –16,600 and an indistinct shoulder near 220 nm with [Θ] = – 4,000, is consistent with the theoretically predicted CD spectrum for the 3 10 -helices [3]. The CD spectrum of Boc–(Leu–Leu–Ala)3 –(Leu–Leu–Lac) 2 –OEt has a positive maximum at 192 nm with [Θ] = 36,000 and a negative maximum at 206 nm with [ Θ ] = –25,000 and a distinct shoulder near 220 nm with [Θ] = –11,100. The CD spectra indicate that these depsipeptides assume different helix conformations. NMR spectroscopy is widely used to assign helices in peptides. The cross peak between α -proton of the ith amino acid residue and amide proton of the i + 4 amino acid residue, d αN (i, i + 4) is characteristic of α -helix, while d α N (i, i + 2) is of 3 1 0 -helix [4]. The connectivity of the cross peaks ROESY for the depsipeptides in CDCl3 is denoted (Figure 2). The ROE connectivities demonstrate that the depsipeptide Boc–(Leu–Leu–Ala)2 –(Leu–Leu–Lac) 3 –OEt assumes 3 10 -helix from the N -terminus to Leu 1 1 . In Boc–(Leu–Leu–Ala)3 – (Leu–Leu–Lac) 2 –OEt, the ROE were detected for connectivities not only d αN (i, i + 4) but also d α N(i, i + 2). The ROE connectivities indicate that Boc–(Leu– Leu–Ala) 3 –(Leu–Leu–Lac) 2 –OEt assumes 3 10 -helix as well as α -helix. These results are supported by analyses of the dihedral angle φ. These results demonstrate that the substitution of Lac9 residue with Ala residue cause an increase of α -helical conformation. It is now clear that the lack of the hydrogen bonding at the Lac9 residue is important factor for forming 3 10 -helix in the depsipeptide Boc–(Leu–Leu–Ala)2 –(Leu–Leu–Lac) 3 –OEt. References 1. 2. 3. 4.

Barlow, D.J. and Thornton, J.M., J. Mol. Biol., 201 (1988) 601. Toniolo, C. and Benedetti, E., TIBS., 16 (1991) 350 Manning, M.C. and Woody, R.W., Biopolymers, 31 (1991) 569. Wagner, G., Worgotter, D., Vasak, M., Kagi, J.R.H. and Wuthrich, K., J. Mol. Biol., 187 (1986) 131.

97 ¹H Nuclear Magnetic Resonance analysis of [Ser 6 , Ser20 ]-human-Epidermal Growth Factor as a model for the intermediate of the disulfide folding pathway H. MURAKAMI, N. KOBAYASHI, T. SHIGEMATSU, K. NIHEI and E. MUNEKATA Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Introduction Human epidermal growth factor (hEGF) has conservative three disulfide bonds between cysteine residues 6–20, 14–31 and 33–42. The three dimensional structure of the small protein has 30% of antiparallel β -sheet which has been investigated by NMR technique [1]. hEGF can refold spontaneously from reduced and denatured state. Recently, its disulfide folding pathway in vitro was elucidated by Chang et al., in which a remarkably populated intermediate (14–31, 33–42) possessed native disulfide bonds was demonstrated [2]. To understand the disulfide folding pathway, we analyzed the conformational properties of a variant, [Ser6 , Ser 20 ]-hEGF as a model for the intermediate (14–31, 33–42) by ¹H NMR in aqueous solution comparing with wild type hEGF.

Figure 1. NOESY spectra of wild type hEGF and [Ser 6 , Ser 2 0 ]-hEGF. Long range C αH-C αH connectivities in the wild type and variant hEGF are shown. The spectra were measured in 90%H 2 O/10%D2 O containing 50 mM glycine-d2 HCl pH 2.8 at a mixing time of 150 ms, 301 K.

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Figure 2. Representative plots of amide peak volume in absolute COSY spectra versus exchange time in wild type hEGF and [Ser 6 , Ser20 ]-hEGF. For the hydrogen-exchange experiments, COSY spectra in absolute mode were measured after dissolving the lyophilized peptide in 100% D 2 O containing 50 mM glycine-d 2 HCl buffer (pH 2.8) at 301 K.

Results and discussion The differences of the α-proton chemical shift between variant and wild type were below 0.10 ppm except for Tyr13 , Glu24 and Cys 31 , and most of all the long range NOE connectivities were commonly observed in wild type and variant of hEGF. Furthermore, 13 slowly exchanging amide protons existed in both proteins. These facts suggest that the variant of hEGF has a native like conformation. Their exchanging rates at 301 K were 5–10 times higher than those of wild type, indicating that the stability around the β -strands of the variant was reduced by replacing the disulfide bonds into two Ser residues. The most remarkable point is that the hydrogen-exchanging rate on the most of the amide were reduced, indicating that the hEGF molecule has a slight cooperative system on the folding like as larger globular protein. These findings also suggest that the accumulation of intermediate (14–31, 33–42) can be due to two sulfide group buried in the folded hEGF and shielded from the solvent.

References 1. Hommel, U., Harvey, T.S., Driscoll P.C. and Campbell, I.D. J. Mol. Biol. ( 1992) 227, 271–282. 2. Chang, J.-Y., Schindler, P., Ramseier U. and Lai, P.-H J. Biol. Chem. (1995) 270, 9207–9216.

98 pKa shift by NH···S hydrogen bond in the hair-pin turn structure of Cys-containing oligopeptides Norikazu Ueyama, Shino Moriyama, Takafumi Ueno, and Akira Nakamura Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560 Japan

Introduction Thiol group has been often found in the active site of various biologically important enzymes. These enzymatic reaction proceeds through thiol and its thiolate, e.g. glutaredoxin (thiol-protein oxidoreductase), papain (cysteine protease) and protein tyrosine phosphatase. The pKa value of these thiols occasionally changes during the reaction cycles. The change of the pKa value is mainly considered to be due to the location of base group near the thiol to promote the deprotonation of SH by the electrostatic interaction. Recently we found the stabilization of thiolate anion by adjacent amide NH. The NH···S hydrogen bond enables the formation of stable Fe(III) P-450 model complexes using a doubly hydrogen bonded thiolate, 2,6(t-BuCONH) 2 C 6 H 3 S– [1]. The strong NH···S hydrogen bond forms in less polar solvent like dichloromethane.

Results and Discussion Oligopeptides containing Cys thiolate anion were synthesized from Z -Cys– Pro–Leu–OMe (Z = benzoxylcarbonyl), Z -Cys–Pro–OMe, Z-Cys–Leu–Gly– OMe and Z -Cys–Leu–OMe using alkanethiolate anion without racemization. For example, (NEt 4 ) (Z -Cys(S – )–OMe) was synthesized by a reaction with (NEt 4 )(S-t-Bu) in acetonitrile at room temperature. This thiolate anion was oxidized by iodide at room temperature to give (Z-Cys–OMe)2 ( [ α] D – 7 7 . 2 ° , c 0.1, EtOH) compared with [α] D –78.0° for authentic sample under the same conditions. However, (NEt 4 )(Z-Cys(S – )–OMe) is thermodynamically unstable at room temperature. The solution structures of both thiolate anion and thiol states for these oligopeptides were determined using a simulated annealing method by 600 MHz ROESY technique. Figure 1 shows the ¹H NMR spectra of both states of Z -Cys–Pro–Leu–OMe and Z-Cys–Leu–Gly–OMe in 10 mM CD3 C N at 30°C. The large lowfield shift of amide NH ¹H NMR signal of amino acid residue at the third position from the Cys residue was observed for the thiolate anion. The shift indicates that the amide NH forms strong NH· · · S hydrogen 288 Y. Shimonishi (ed): Peptide Science – Present and Future, 288–290 © 1999 Kluwer Academic Publishers, Printed in Great Britain

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Figure 1. ¹H NMR spectra in the amide NH regions of the thiol and thiolate states for (a ) Z -Cys– Pro–Leu–OMe and (b) Z -Cys–Leu–Gly–OMe in CD 3 CN at 30°C.

bond with thiolate anion to provide a folded hair-pin turn structure as shown in Figure 2, The strong hydrogen bond leads to a folding hair-pin turn structure in these Cys-containing oligopeptide thiolate anions. The pKa values of these Cys-containing oligopeptides were obtained in aqueous micellar solution (2 mM 1-O-n-octyl- β - D -glucopyranoside) since the NH ··· S hydrogen bond forms only in less polar solvent. Water-insoluble oligopeptides exist in micelles. Fortunately, proton quickly difuses through the

Figure 2. Solution structures of thiolate anions of ( a) (NEt4 )(Z-Cys–Pro–Leu–OMe) and (b) ( N E t4 )(Z-Cys–Leu–Gly–OMe) determined using a simulated annealing method (Varian Unity 600, Felix 95.0, InsightII, Discover 3, NMRchitect, CVFF, εr = 1 ) .

290 micelles to the out-side aquous phase. Both thiols of Z -Cys–Pro–Leu–OMe and Z -Cys–Leu–Gly–OMe show pKa 9.0 at 25°C whereas Cys-containing dipeptides, Z-Cys–Pro–OMe and Z-Cys–Leu–OMe, exhibit pKa 9.5 and 11.2, respectively, under the same conditions. The lower pKa is caused by the formation of NH...S hydrogen bond in the thiolate state. The thiolate anion also plays important role for the regulation of specific enzymatic reaction in the active site of metalloenzymes. For example, the folding structure by the NH... S hydrogen bond has been found in the axial position of P-450 or chloroperoxidase. The peptide P-450 or chloroperoxidasemodel complexes having the above tripeptides similarly possess NH . . .S hydrogen bond, e.g. [FeI I I (OEP)(Z-cys–Pro–Leu–OMe)] (OEP = octaethylporphyrinato) or [Fe I I I (OEP)( Z-cys–Pro–Leu–OMe)]. The NH... S hydrogen bond contributes to the positive shift of redox potential and the thermodynamical stabilization of the Fe(III) state.

Reference 1. Ueyama, N., Nishikawa, N., Yamada, Y. Okamura, T. and Nakamura, A. J. Am. Chem. Soc., 118 (1996) 12826.

99 Effect of environment on the stability of helical Aib-model peptides H. KODAMA¹, T. HARA¹, Y. HIGASHIMOTO¹, H. YAMAGUCHI¹, M. JELOKHANI-NIARAKI², S. OSADA¹ and M. KONDO¹ ¹ Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840, Japan ² PENCE, 713 Heritage medical Research Centre, University of Alberta, Edmonton, Alberta, Canada

Introduction It is well known that peptides containing Aib residues form α - and/or 3 10 helical structures leading to voltage-dependent ion channel in lipid membranes. We have previously reported the synthesis of Aib-containing gramicidin analogs [1,2] and model-peptides, Ac-(Aib–Lys–Aib–Ala)n -NH 2 (n = 1–5, BKBA-N, N denotes the number of residues). Among the Aib-peptides, the longer peptide, BKBA-20 may aggregate and form alamethicin-like barrel-stave conformations in phospholipid bilayers [2]. In the present study, to characterize the structures and ion-selectivities of the Aib-peptides containing charged or aromatic residue, we have synthesized novel Aib-peptides containing Glu, Ser, Gly and Trp residues (Figure 1). The conformational stabilities of the synthesized peptides in aqueous and phospholipid membrane environments have been investigated by means of CD spectroscopy.

Results and discussion Peptides were synthesized by solid-phase method. The structures were confirmed by ESI-MS. The concentration of peptide solution was determined by quantitative AAA. CD spectra of BXBA-20 peptides are shown in Figure 2. In the aqueous buffer, helicities of the peptides increased with enhanced hydrophobic moments [3], but not depended on the helix propensity parameter of replaced residue, e.g., the Chou and Fasman scale [4]. The hydrophobic

Figure 1.

Structures of Synthetic Aib-Model Peptides (B = Aib),

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Figure 2 CD spectra of BXBA-20 in 50 mM phosphate buffer, pH 7.4 ( a), TFE (b), and DPPC vesicles (c).

moment measures the extent of amphiphilicity of a helix structure. Blondelle et al. reported the role of amphipathicity that is a determining feature in the self-association ability of peptides or protein segments [5]. This mention emphasized the effect of amphipathicity on helix formation, even if residues having high helix propensity such as Aib were contained in the peptides. BEBA-20 peptides exhibited multi-level channel behaviors (data not shown) on DiPhyPC membrane. This level transfer was explained as barrel-stave channel model such as alamethicin channel [6]. However, current patterns of BEBA-20 peptide were observed even at lower peptide concentration and transbilayer potential (< 20 mV) rather than the reported conditions for alamethicin channel [6]. From the present study, BXBA-20 peptides are expected to be suitable templates for intermembrane stable channel forming structures, as simple models for studying structure-function properties of more elaborate protein ion channels.

Acknowledgments The authors thank to Drs. K. Sakaguchi and E. Appella in NIH (MD, USA) for ESI-MS analysis of synthetic peptides. This study was partly supported by research grants from the Ministry of Education, Science and Culture of Japan (09780529).

References 1. Jelokhani-Niaraki, M., Kodama, H., Ehara, T. and Kondo, M., J. Chem. Soc., Perkin Trans 2 (1995) 801. 2. Higashimoto, Y., Kodama, H., Hara, T., Jelokhani-Niaraki, M. and Kondo, M., Peptide Chemistry 1996 (1997) 253.

293 3, Eisenberg, D., Nature, 299 (1982) 371. 4. Chou, P.Y. and Fasmsn, G.D., Ann. Rev. Biochem., 47 (1978) 251. 5. Perez-Paya, E., Houghten, R.A. and Blondelle, S.E., J. Biol. Chem., 270 (1995) 1048. 6. Sansom, M.S.P., Prog. Biophys. Mole. Biol., 55 (1991) 139.

100 Structural analysis of mastoparan by solid-state nuclear magnetic resonance K. FUKUSHIMA¹, S.-M. KIM¹, T. FUJIWARA¹, T. KOHNO², K. WAKAMATSU³, S.-W. KANG 4 and H. AKUTSU¹ ¹ Department of Bioengineering, Yokohama National University, Hodogaya-ku Yokohama, Japan ² Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan ³ Faculty of Engineering, Gunma University, Kiryu, Gunma, Japan 4 Department of Chemistry, Pusan National University, Pusan, Korea

Introduction Mastoparans are a class of peptide toxins from wasp venom. Amino acid sequence of mastoparan X is Ile–Asn–Trp–Lys–Gly–Ile–Ala–Ala–Met–AlaLys–Lys–Leu–Leu–NH 2 . It is mainly composed of hydrophobic and basic amino acid residues. Its C-terminus is amidized. The actions of mastoparan X appear to involve several signal transduction pathways through the activation of G protein [1]. It was suggested that the membrane-bound conformations of mastoparans are important for the efficient activation of G-protein. Mastoparan X is a peptide mimetic to the third-loop, which has been known as the G-protein binding site. Crystallization of G-protein-activating receptor and NMR structural study of the receptor are diflicult. This peptide is more suitable for NMR experiments owing to its small molecular weight. Mastoparan X complexed with lipids is too large for liquid-state NMR analysis. Thus this is a good model for the structural analysis of the G protein activator. Mastoparan X has been synthesized in two different methods. We aim to prepare fully 13 C, 15 N and selectively 1 3 C labeled peptide for the analysis by solid-state nuclear magnetic resonance. Preliminary results for solid-state NMR structural study are presented. Results and discussion A fusion gene of ubiquitin and mastoparan X was expressed in E. coli T h e fusion protein with His-tag was purified by Ni 2+ -nitrilo-tri-aceticacid affinity column chromatography. Then the mastoparan peptide was cleaved by yeast ubiquitin hydolase and purified by high-performance liquid chromatography. To avoid the oxidation of methionine, 1 mM 2-mercaptoethanol was added to the buffer solutions for the purification. We have obtained about 0.8 mg fully 13 C, 15 N labeled mastoparan X from 1L culture. We have measured solid-state cross polarization/magic angle sample spinning (CP MAS) 13 C nuclear magnetic resonance spectra of chemical synthesized 294 Y, Shimonishi (ed): Peptide Science – Present and Future, 294–296 © 1999 Kluwer Academic Publishers, Printed in Great Britain

295

Figure 1. 13 C CP-MAS NMR spectora of non-labeled Mastoparan X lyophilized from H 2O (A) and 50% TFE (B).

mastoparan X. We synthesized selectively 13 C labeled mastoparan X by solidphase chemical synthesis. CP MAS 13 C NMR spectra of the non-labeled (Figure 1) and selectively 13 C labeled (Figure 2) peptide ware obtained. The spectra were recorded on a solid-state NMR spectrometer at the 13 C resonance

Figure 2. 13 C CP-MAS NMR spectra of selectively H 2 O (A) and crystallized from 100% TFE (B).

13 C

labeled, Mastoparan X lyophilized from

296 frequency of 100 MHz. The sample was obtained by crystallization from a trifluoroethanol solution. It has been reported that the most of mastoparan X takes on α -helix in 50% trifluoroethanol solution [2]. The spectra of nonlabeled mastoparan X has characteristic 13 C chemical shifts [3]. However there was no distinct difference between spectra of mastoparan X obtained from water and TFE solutions. The chemical shift of the methyl 13 C signal of Ala10 selectively labeled suggested mastoparan X takes α-helix. The linewidth of the methyl 13 C was about 200 Hz. A T 2 measurement revealed the signal is broaden inhomogeneously. Therefore mastoparan X in the solid state did not have a single conformation. Preparation of solid mastoparan X having an unique structure is under way. Further structural analysis of this peptide in solid is going to be carried out by using fully 13 C and 15 N labeled and specifically 13 C labeled samples.

References 1. Higashijima, T., Uzu, S., Nakajima, T. and Elliott M.R., J. Biol Chem., 263 (1988) 6491. 2. Higashijima, T., Wakamatsu, K., Takemitsu, M., Fujino, M., Nakajima, T. and Miyazawa, T., FEBS Lett., 152 (1983) 227. 3. Saito, H., Magn. Reson. Chem., 24 (1986) 835.

101 Four-helix bundle formation only through special pathway via micelle state H. MORII¹ , H. UEDAIRA¹ , M. ISHIMURA¹, S. KIDOKORO², T. KOKUBU¹ and S. OHASHI¹ ¹National Institute of Bioscience and Human-Technology, Tsukuba Ibaraqi 305-8566, Japan ²Sagami Chemi cal Research Center, Sagamihara, Kanagawa 229-0012, Japan

Introduction It has been generally recognized that amphipathic peptides associate to form α-helix bundles as dimer, trimer, or tetramer according to the concentrationdependent equilibrium. While, ovine corticotropin-releasing factor (CRF), a peptide hormone of 41 residues, was observed to behave somewhat curiously. Gel filtration experiments of CRF gave two bands bearing ACTH-releasing activity [1]. Also, its circular dichroism (CD) measurements exhibited only a slight concentration dependence of molar ellipticity [2, 3]. These results suggest that the monomer-oligomer equilibrium is too much slow in the condition of low concentration. In order to reveal how the CRF oligomers are formed, the monomer and the oligomer, which would be the tetramer as estimated by sedimentation equilibrium experiment [2], were fractionated by size exclusion chromatography (SEC) and studied further in detail by spectroscopic and thermodynamic methods.

Results and discussion The CD measurements of the monomer and the tetramer revealed that their secondary structures are random coil and α -helix-rich one, respectively. The sequence of ovine CRF, which is SQEPPISLDLTFHLLREVLEMTKADQLAQQAHSNRKLLDIA with C-terminal amide group, has potentially amphipathic α-helical region (7–22) as predicted by the author’s original method [4]. Therefore, the tetramer would be 4-α-helix bundle, The simply dissolved CRF solutions contain both monomer and tetramer irrespective of the concentrations as shown by SEC. Moreover, the experiments of standing or diluting the fractionated monomer and tetramer solutions exhibited no conversion between them at least within a day. Thus, the monomer-tetramer equilibrium is too slow to be detected at the concentrations of CRF lower than 0.1 mM. On the other hand, above the concentration of 0.1 mM the high a-helicity similar to that of tetramer was observed by CD. This would be due to the 297 Y. Shimonishi (ed): Peptide Science – Present and Future, 297–299  1999 Kluwer Academic Publishers, Printed in Great Britain

298

Figure 1 Illustration of probable equilibrium above the concentration of 0.1 mM.

micelle state of CRF. The micelle state was also proved by binding ability for 8-anilino-1-naphthalene-sulfonate and the observation of endothermic enthalpy of dilution across the concentration of 0.1 mM. When the monomer solution was concentrated, the micelles of CRF appeared in the solution. While, when the micelle-rich CRF solution was diluted, it became to be the mixture of monomer and tetramer. Their molar ratio was constant even on further dilution. These results suggest that the CRF tetramer is formed only through the micelle state (Figure 1). This is inconsistent with the generally-known fact that the helix bundle tetramer is formed by the equilibrium reaction of monomer and tetramer. Measurements of differential scanning calorimetry and temperature-scanning CD have shown that the tetramer undergoes cooperative thermal transition at about 55°C (Figure 2). On the contrary, the micelle showed only gradual change into the dissociated state upon heating. Therefore, it would be concluded that the CRF micelle has some different folding characteristics from the helix bundle tetramer, which seems to have folding structure like native proteins [5]. The unique folding pathway observed in this work resembles the collapse model for thermodynamic protein folding.

Figure 2 Thermal transition curves of 0.005 mM (a) and 0.3 mM (b) CRF solutions superimposed with that of the isolated CRF tetramer at 0.02 mM (c).

299 References 1. Vale, W., Spiess, J., Rivier, C. and Rivier, J., Science 213 ( 1981) 1394. 2. Lau, S.H., Rivier, J., Vale, W., Kaiser, E.T. and Kézdy, F.J., Proc. Natl. Acad. Sci. USA 80 (1983) 7070. 3. Dathe, M., Fabian, H., Gast, K., Zirwer, D., Winter, R., Beyermann, M., Schümann, M. and Bienert, M., Int. J. Peptide Protein Res. 47 (1996) 383. 4. Morii, H., Takenawa, T., Arisaka, F. and Shimizu, T., Biochemistry 36 (1997) 1933. 5. Morii, H., Uedaira, H., Ishimura, M., Kidokoro, S., Kokubu, T. and Ohashi, S., Biochemistry 36 (1997) 15538.

102 Synthesis of peptides containing 9-aminofluorene9-carboxylic acid by the modified Ugi reaction T. YAMADA,* K. MAKIHIRA, S. SUZUKI, K. YONEDA, R. YANAGIHARA and T. MIYAZAWA Department of Chemistry, Faculty of Science, Konan University, Higashinada-ku, Kobe 658-8501, Japan

Introduction The incorporation of α,α -disubstituted glycine into peptide sequences provides an excellent tool to constrain the backbone conformation and to include welldefined secondary structures. Typically, α-aminoisobutyric acid (Aib) is a strong helix promoting residue. Recently, we could succeed in synthesizing a variety of fully protected tripeptides containing α ,α-diphenylglycine (Dph) in the central position by the modified Ugi reaction [1, 2]. A systematic structural analysis of the Dph-containing tripeptides by X-ray analysis interestingly revealed that the Dph residue adopts a folded conformation in the 310 -helical region if the following residue adopts a folded conformation of opposite handedness, though a Dph residue usually prefers a fully extended conformation [3, 4]. Now we report synthesis of peptides containing a new α,α-disubstituted glycine, 9-aminofluorene-9-carboxylic acid (Afc), which has almost the same structure as Dph, except that two phenyl groups of Dph are bound each other at their ortho-positions. Interestingly, Afc has a rigid planar side-chain in which two phenyl groups are forced to be coplanar, in contrast with Dph in which they are distorted each other.

Results and discussion First we tried to synthesize Afc itself, but we could obtained it neither by the Bücherer method via hydantoin nor by a method containing the modified Ugi reaction as a key step. Secondly, we have undertaken to synthesize a tripeptide containing Afc directly by the modified Ugi reaction using 9-fluorenonimine as a key compound, which was well prepared from 9-fluorenone and liquid ammonia [5]. Various tripeptides containing Afc (Z–AA1 –Afc–AA 3–OMe) were successfully synthesized by the modified Ugi reaction using 9-fluorenonimine, N-benzyloxycarbonylamino acid (Z–AA 1) and isocyanides derived from HCO–Gly–OMe and HCO–Aib–OMe. Removal of a Z group and hydrolysis of methyl ester of these tripeptides could be easily attained. 300 Y. Shimonishi (ed): Peptide Science – Present and Future, 300–302  1999 Kluwer Academic Publishers, Printed in Great Britain

301

Table 1.

Synthesis of Afc-containing tripeptides

AA 1

AA 3

Time (d)

Yield (%)

Mp (°C)

[α]D 25

Ala Val Leu Gly Aib Gly Aib

Gly Gly Gly Gly Gly Aib Aib

12 12 12 12 12 20 20

42 29 15 40 44 47 47

— — — 185–186 190–191 163–164 145–146

– 25.8° – 38.0° – 25.2° — — — — (c 1.0, MeOH)

Couplings of Z–Leu and Leu–OMe to N- and C-terminal of the tripeptide, respectively, with EDC–HOBt gave the corresponding tetrapeptides in good yields, e.g., Z–Leu–Ala–Afc–Gly–OMe (96%), Z–Ala–Afc–Gly–Leu–OMe (85%). Temperature dependencies of NH chemical shifts in NMR of Z –Ala– Afc–Gly–Leu–OMe both in CDCl 3 and in DMSO-d 6 indicate that it adopts the folded conformation stabilized by an intramolecular hydrogen-bond which Leu–NH participates. Thus Afc may occupy the i + 1 corner position of a β-turn. Further, some hexapeptides containing two Afc residues separated by two other amino acids residues could also be synthesized in good yields. The investigation on conformations and functions of these Afc-cocontaining peptides are now in progress. Acknowledgements This work was supported by The Hirao Foundation of the Konan University Association for Academic Research. References 1. Yamada, T., Omote, Y., Miyazawa, T., Kuwata, S., and Matsumoto, K., In Suzuki, A. (Ed.) Peptide Chemistry 1991 (Proceedings of the 29th Symposium on Peptide Chemistry), Protein Research Foundation, Osaka, 1992, pp. 367–372.

302 2. Yamada, T., Omote, Y., Yamanaka, Y., Miyazawa, T., and Kuwata, S., Synthesis, (1998) 991. 3. Pavone, V., Lombardi, A., Saviano, M., Di Brasio, B., Nastri, F., Fattorusso, R., Zaccaro, L., Maglio, O., Yamada, T., Omote, Y., and Kuwata, S., Biopolymers, 34 (1994) 1595. 4. Pavone, V., Lombardi, A., Saviano, M., Nastri, F., Zaccaro, L., Maglio, O., Pedone, C., Omote, Y., Yamanaka, Y., and Yamada, T., J. Peptide Science, 4 (1998) 21. 5. Verardo, G. and Giumanini, A.G., Synthetic Commun., 18 (1988) 1501.

103 Effects of mutations on tetramer formation of tumor suppressor protein p53 Y. HIGASHIMOTO 1, M.S. LEWIS 2, W.M. POINDEXTER KENNEDY3, A.M. GRONENBORN 3, G.M. CLORE3, E. APPELLA1 and K. SAKAGUCHI 1 1 Lab. of Cell Biology, National Cancer Institute, 2 Biomedical Engineering and Instrumentation Program, National Center for Research Resources, 3 Lab. of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National institutes of Health, Bethesda, MD 20892, USA

Introduction The tumor suppressor p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage [1, 2]. Inactivation of p53 through either deletions, mutations or interaction with cellular or viral proteins appears to be a critical step in the etiology of many cancers. Mutations in the p53 gene have been found in more than 50% of human tumors Tetramer formation is critical to p53’s ability to activate transcription from natural promoters (e.g. WAF1/CIP1). The structure of the core tetramerization domain has been solved by both NMR and X-ray crystallography [3–5]. The tetramer is best described as an antiparallel dimer of dimers and is comprised of a four helix bundle with two anti-parallel β-sheets located on opposing faces of the molecule. While the majority of p53 mutations found in human tumors are located in the site-specific DNA-binding domain, a number of missense, stop, and frame-shift mutations, are found within the tetramerization domain [6]. We have selected four tetramer domain mutants for a study of how mutations affect the tetramer formation of p53. Four mutant peptides comprising residues 319–358, L330 H, G334V, R337C and E349D, were chemically synthesized.

Results and discussion Circular dichroism and equilibrium ultracentrifugal analyses showed that all four mutants could form the wild-type tetramer conformation at low temperature. However, the G334V peptide the G334V peptide polymerized through an α – β transition conformational change as a result of increasing temperature or the addition of organic solvent with lapse of time. All mutants were less stable to temperature and guanidine hydrochloride denaturation than the wild-type peptide (WT). The order of the stability was 303 Y. Shimonishi (ed): Peptide Science – Present and Future, 303–305  1999 Kluwer Academic Publishers, Printed in Great Britain

304 Table 1.

Oligomeric state of wild-type and mutant p53 peptides at 37°C.

p53 peptide

Oligomeric state

∆G° (kcal/mol)

Kd (µ M)

319–358 (WT) L330H G334V R337C E349D

monomer-tetramer monomer-tetramer polymer monomer-tetramer monomer-tetramer

–26.6 ± – 16.6 ± – –21.6 ± –25.3 ±

0.72 1540 – 10.4 1.47

Figure 1.

0.9 0.1 0.1 0.6

CD spectra of mixed tetramer between wild-type and mutant p53 peptides at 37°C.

WT > E349D > G334V > R337C > L330 H, with the L330H mutant peptide having a tetramerization equilibrium constant more than 1000 times smaller than that of the WT (Table 1). At concentrations in the µM range, the wildtype peptide forms nearly 100% tetramer. In contrast, the mutant peptides, L330H and R337C, form almost no tetramers. Of great interest is the fact that the CD study revealed that the WT tetramers were less stable in the presence of mutant peptides. Figure 1 shows that the spectra of the two peptides mixed in the solution (experimental spectra) differ from the summed spectra of the individual peptides (theoretical noninteraction spectra). The conformation of the WT peptide was disordered by the addition of mutant peptides. Especially, L330H and G334V induced dramatic changes in the spectra of the WT peptide. Interestingly, the mixture of WT and G334V showed the spectrum of β -structure, indicating that G334V also induced structural change of the WT peptide by the interaction. This result suggests that the tetramer mutants can interact and form heterotetramers with the wild-type p53 protein. Thus, the destabilization of p53 tetramer causes loss of the sitespecific DNA binding, resulting in the inactivation of the wild-type protein. References 1. Levine, A.J., Cell, 88 (1997) 323. 2. Gottlieb, T.M. and Oren, M., Biochim. Biophys. Acta, 1287 (1996) 77.

305 3. Sakamoto, H., Lewis, M.S., Kodama, H., Appella, E., and Sakaguchi, K., Proc. Natl. Acad. Sci, USA, 91 (1994) 8974. 4. Clore, G.M., Ernst, J., Clubb, R., Omichinski, J.G., Kennedy, W.M., Sakaguchi, K., Appella, E. and Gronenborn, A.M., Nature Struct. Biol., 2 (1995) 321. 5. Sakaguchi, K., Sakamoto, H., Lewis, M.S., Anderson, C.W., Erickson, J.W., Appella, E., and Xie, D., Biochemistry, 36 (1997) 10117. 6. Beroud, C., Verdier, F., and Soussi, T., Nucleic Acids Res., 24 (1996) 147.

104 Disulfide-coupled folding of the circulating form of uroguanylin Y. HIDAKA1 , M. OHNO1, B. HEMMASI2, O. HILL3 , W.-G. FORSSMANN3 and Y. SHIMONISHI1 ¹Institute for Protein Research, Osaka University, Yamada-oka 3-2, Suita, Osaka 565-0871, Japan ²Institute of Organic Chemistry, University of Tubingen, 72076 Tubingen, Germany ³Lower Saxony Institute for Peptide Research, D-30 625 Hannover, Germany

Introduction Plasma form of human uroguanylin (guanylyl cyclase activating peptide, GCAP-II) is an endogenous ligand for a particulate guanylyl cyclase C [1]. The peptide consists of 24 amino acid residues and two disulfide linkages [1], as shown in Figure 1. Our findings indicate that a linear peptide, containing 4 cysteine residues, is thermodynamically impossible to produce a peptide with the native disulfide bond pairing as a major product by air-oxidation in vitro, and that the correct disulfide bond pairing is required for its biological activity. This finding prompted us to examine how the peptide is able to overcome this energic barrier to folding. This report describes a study of the disulfide-coupled foldings of GCAP-II and the precursor protein (proGCAP-II), in order to elucidate the folding mechanism.

Results and discussion GCAP-II was chemically synthesized by the method reported previously [2], following the formation of disulfide linkages in a stepwise manner using two kind of selectively removable protecting groups for the Cys residues. Two topological isomers (GCAP-II-N and GCAP-II-N') were obtained by the chemical synthesis of GCAP-II. The measurement of the biological activities of two isomers revealed that GCAP-II-N was the bioactive form of GCAP-II. The disulfide-coupled folding of synthetic GCAP-II was examined from the reduced GCAP-II in the presence of reduced and oxidized glutathione (GSH and GSSG, respectively). Native disulfide pairing was not achieved efficiently and the predominant pairings were non-native in the presence of GSH and GSSG (Figure 2A). This suggests that GCAP-II requires an additional factor in order

FKTLRTIANDDCELCVNVACTGCL Figure 1.

Primary structure of GCAP-II.

306 Y. Shimonishi (ed): Peptide Science – Present and Future, 306–308 © 1999 Kluwer Academic Publishers, Printed in Great Britain

307

Figure 2. HPLC profiles of refolded GCAP-II (A) and C-terminal peptide (Thr-Ile-Ala-uroguanylin) of proGCAP-II (B). (A) GCAP-II-N and GCAP-II-N' were represented as N and N ' , respectively. The refolding reaction was proceeded in the presence of 2 mM GSH and 1 mM GSSG. (B) N and N' represent the N and N' form of Thr–Ile–Ala–uroguanylin, respectively.

to achieve its native conformation. Considerable attention has recently been focused on the amino-terminal precursor sequence (the pro region) and its role in protein folding. To investigate the function of the prosequence [3] of GCAP-II in its folding pathway, we prepared proGCAP-II (the precursor polypeptide of GCAP-II containing the pro- and mature-sequence) using E. coli and examined the folding of proGCAP-II under the same conditions as that used above. proGCAP-II was expressed as an inclusion body in E. coli, and then the inactive proGCAP-II was renatured and purified. The purified GCAP-II was reduced again by dithiothreitol (DTT) and refolded in the presence of GSH and GSSG. Single peak was observed on the HPLC analysis of the reaction mixture. The C-terminal peptide from the refolded proGCAP-II was prepared by an endoproteinase Arg-C treatment and trypsin agarose column chromatography. Figure 2B shows an HPLC profile of the C-terminal fragments of proGCAP-II, corresponding to Thr–Ile–Ala–uroguanylin, by Arg-C digestion. The bioactive form (N in Figure 2B) of Thr–Ile–Ala–uroguanylin, possessing the native disulfide pairings, was predominantly observed with small amounts of the topological isomer (N' in Figure 2B) on the HPLC, while no other isomers were detected. This suggests that the prosequence of GCAP-II facilitates both the correct folding and the pairing of the native disulfide linkages of GCAP-II. References 1. Hess, R., Kuhn, M., Schultz-Knappe, P., Raida, M., Fuchs, M., Klodt, J., Adermann, K., Kaever, V., Cetin, Y., and Forsmann, W.G., FEBS Lett. 374 (1995) 34.

308 2. Shimonishi, Y., Hidaka, Y., Koizumi, M., Hane, M., Aimoto, S., Takeda, T., Miwatani, T., and Takeda, Y., FEBS Lett. 215 (1987) 165. 3. Hill, O., Cetin, Y., Cieslak, A., Magert, H.J., and Forssmann, W.G., Biochim. Biophys. Acta 1253 (1995) 146.

105 Solution structure of the extracellular domain of type-I receptor for bone morphogenetic protein-2/4 as studied by nuclear magnetic resonance spectroscopy T. YAMAZAKI¹, T. HATTA², Y. NISHIUCHI³, E. KATOH¹, T. NATSUME 4 , N. UENO5 , Y. KYOGOKU² and Y. KOBAYASHI6 ¹Structural Biology Unit, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan ²Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan ³Peptide Institute Inc., Protein Research Foundation, Minoh-shi, Osaka 562, Japan 4 Mikoshiba Project, ERATO, Japan Science and Technology Corporation, 2-9-3 Shimomeguro, Meguro-ku, Tokyo 153, Japan 5 National Institute of Basic Biology, 444 Okazaki, Japan 6 Faculty of Pharmaceutieal Sciences, 1–6 Yamadaoka, Suita, Osaka 562, Japan

Introduction Bone morphogenetic proteins (BMPs), originally identified as proteins to induce bone formation at extraskeletal sites in vivo, are the largest subfamily of the transforming growth factor-β (TGF- β) superfamily. Recent studies have shown that the BMPs are multifunctional cytokines that regulate growth, differentiation, and apoptosis of various cell types including osteoblasts, chondroblasts, neural cells and epithelial cells. In addition to the above functions, BMP-2 and BMP-4 regulate dorsal-ventral patterning during mesoderm induction. Like other members of the TGF-β superfamily, BMPs transduce signals by binding to two distinct receptors, type-I and type-II, on the cell surface. Both type-I and type-II receptors are transmembrane serine/threonine kinase receptors that form a heteromeric complex upon ligand binding. In contrast with the extensive structural information now available about the ligands, virtually no structural information is available for both type-I and type-II receptors. In order to further understand ligand-receptor complex formation and the functions of BMPs, we have determined three-dimensional solution structure of the extracellular domain (ECD) of type-IA receptor for BMP-2/4 by heteronuclear multidimensional NMR spectroscopy.

Results and discussion The soluble extracellular domain (ECD) of the mouse BMP type-IA receptor (sBMPr-1A) was prepared as a fusion protein with thioredoxin in E. coli. This unglycosylated sBMPr-1A was in monomer form in solution and bound to BMP-4 with similar affinity as a glycosylated sBMPr-1A produced in a silk309 Y. Shimonishi (ed): Peptide Science – Present and Future, 309-310 © 1999 Kluwer Academic Publishers, Printed in Great Britain

310

Figure 1 Two views of a ribbon diagram of the three-dimensional solution structure of sBMPr-1A, showing the backbone and disulfide bonds. The central core region from 25 to 102 has well defined structure while the N- and C-terminal domains are highly flexible. The position 40 is a potential glycosylation site.

worm expression system and the intact membrane-associated receptor [1]. The unglycosylated sBMPr-1A displayed essentially the same dose-dependent inhibitory effect on alkaline phosphatase activity of MC3T3-E1 cells as the glycosylated one. These results indicate that the ECD of the BMP type-IA receptor is sufficient for high-affinity binding to its ligands and that the sugar chain in the ECD is less important for the ligand recognition by the receptor. The disulfide structure of sBMPr-1A was determined as 28–49, 30–34, 43–67, 79–91 and 92–97 by the combined use of enzymatic digestion and chemical synthesis. The observed disulfide bonding patterns are similar to those of snake neurotoxins. A variety of double- and triple-resonance experiments on uniformly 15N- or 13 C/ 15 N-labeled sBMPr-1A provided essentially complete 1 H , 13 C and 15 N NMR signal assignments for the protein. With the availability of essentially complete signal assignments, three-dimensional solution structures of sBMPr-1A were determined based on NMR structural data using the hybrid distance geometry-dynamical simulated annealing method as contained in X-PLOR3.1. sBMPr-1A has a relatively flat structure composed of two layers, one formed by a three-stranded β -sheet and the other by a two-stranded β -sheet and a short helix. This is the first three-dimensional structure of the ECD of type-I receptor within the members of TGF- β receptor family that has been elucidated.

Reference 1. Natsume, T., Tomita, S., Iemura, S., Kinto, N., Yamaguchi, A. and Ueno, N., J. Biol. Chem. 272 (1997) 11535.

106 Comparison of three-dimensional solution structures of dendrotoxin I, a potassium channel blocker, and calucicludine, a calcium channel blocker, determined by nuclear magnetic resonance spectroscopy E. KATOH1 , H. NISHIO2 , T. INUI2, Y. NISHIUCHI2 , T. KIMURA2, S. SAKAKIBARA2 and T. YAMAZAKI 1 ¹Structural Biology Unit, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan. ²Peptide Institute Inc., Protein Research Foundation, Minoh-shi, Osaka 562, Japan

Introduction Dendrotoxin I (DTX-1), a 60 amino acid residue peptide isolated from the venom of the African Elapidae snake Dendroaspis polylepis polylepis (the black mamba) [1,2], has been shown to specifically target neuronal voltage-sensitive potassium channels. Calcicludine (CaC), also a 60 amino acid residue peptide with 40% sequence homology to DTX-1, was recently isolated from the venom of Dendroaspis angusticeps (the oriental green mamba) and identified to be a potent blocker of high-threshold calcium channels with high affinity for L-type channels in cerebellar granule neurons [3]. Both DTX-I and CaC have an identical disulfide structure composed of three intramolecular disulfide bonds at Cys7–Cys57, Cys16–Cys40 and Cys32–Cys53, and are structurally homologous to the Künitz-type protease inhibitor. DTX-I and CaC have no mutual interaction in spite of their structural similarity. In order to elucidate the structure-activity relationships of these homologous peptides with different biological activities, we synthesized DTX-I and CaC, and determined their three-dimensional solution structures by NMR spectroscopy.

Results and discussion DTX-I and CaC were synthesized by the solution procedure using our newly developed N in -cyclohexyloxycarbonyltryptophan suitable for Boc strategy [4,5]. The synthetic DTX-I contains the Asp residue at position 12 instead of Asn in the originally reported sequence, as we have recently revised the amino acid sequence for the natural DTX-I [6]. Sequence-specific resonance assignments of DTX-I and CaC were achieved according to the conventional method developed by Wüthrich and coworkers [7] and the structures of DTX-I and CaC were calculated using the hybrid distance geometry-dynamical simulated annealing method as contained in 311 Y. Shimonishi (ed): Peptide Science – Present and Future, 311–312 ©1999 Kluwer Academic Publishers, Printed in Great Britain

312

Figure 1. Backbone superimpositions of 27 and 29 structures resulting from simulate annealing calculations of CaC(a) and DTX-I(b).

X-PLOR 3.1. In these calculations, ensembles of 720 and 795 geometrical restraints derived from the NMR experiments were used for DTX-I and CaC, respectively. The molecular structures of DTX-I and CaC are composed of three regular secondary structure patterns (Figure 1): a short 310-helix, a hairpin β -sheet twisted by 180° and a regular α -helix. The three secondary structure motifs are joined by two disulfide bridges. The RMSD are 1.23 Å and 0.68 Å for the backbone heavy atoms of DTX-I and CaC, respectively. Although the overall conformations of DTX-I and CaC are almost similar, the structure at the N-terminus and distribution of cationic residues which may participate in the bindings to potassium and calucium channels are different between DTX-I and CaC. Detailed structure-activity relationships will be described elsewhere.

References 1. Strydom, D.J., Nature, New Biol., 243 (1973) 88. 2. Rehm, H. and Lazdunsky, M., Proc Natl. Acad. USA, 85 (1998) 4919. 3. Schweitz, H. Heurteaux, C., Bois, P., Moinier, D., Romey, G. and Lazdunski, M., Proc. Natl. Acad, Sci. USA, 91(1994) 878. 4. Nishio, H., Nishiuchi, Y., Kimura, T. and Sakakibara, S., In Nishi, N. (Ed), Peptide Chemistry 1995, Protein Research Foundation, Osaka, 1996, p. 113. 5. Nishiuchi, Y., Nishio, H., Inui, T., Kimura, T. and Sakakibara, S., Tetrahedron Letters, 42 (1996) 7529. 6. Nishio, H., Inui, T., Nishiuchi, Y., Medeiros, C.L.C., Rowan, E.G., Harvey, A.L., Katoh, E., Yamazaki, T., Kimura, T. and Sakakibara, S., J. Peptide Sci., in press. 7. Wüthrich, K., NMR of proteins and nucleic acid, John Wiley, New York (1986).

107 Sequence, conformation, and function of peptides, retro peptides and sense and antisense gene products W.L. DUAX¹, V. PLETNEV², J. BRUENN³ and D. GHOSH 1,4 ¹Hauptman-Woodward Medical Research Inst., Inc., 73 High St., Buffalo, NY 14203 USA ²Shemyakin Institute, 16/10 Miklukho-Maklay St., Moscow 117871 Russia ³SUNY Buffalo, 661 Cooke Hall, Buffalo, NY 14260 USA 4 Roswell Park Cancer Inst., · Elm & Carlton Sts., Buffalo, NY 14263 USA

Introduction KP6 is an ion channel forming toxin from U. maydis, a fungal pathogen that appears to kill cells by altering potassium and proton gradients at the plasma membrane. The KP6 preprotoxin is processed into polypeptides α (79 amino acids) and β (81 amino acids), both of which are required for toxicity. Both peptides have many features similar to those of neurotoxins and cardiotoxins including similar size, several potential disulfide bridges, a large percentage of aromatic amino acids, and prediction of β -strand secondary structure based upon sequence analysis and circular dichroism measurements.

Discussion The crystal structure determination of KP6α revealed a monomer composed of a four stranded antiparallel β -sheet with two α -helices on one side and a small α -helix segment at the N terminus. Three intermolecular salt bridges link monomers of KP6 α into trimers organized around a three-fold symmetry axis. The trimer has a funnel shape that is 7.1 Å across the mouth and narrows to a 4.2 Å opening just large enough to permit an ion to pass. The trimer has many structural features that have been postulated to be critical elements in pore domains of mammalian ion channels [1]. The 4.2 Å funnel bore is lined by six phenyl rings. These phenyl rings arise in part from a GFG sequence in KP6 α that may correspond to a G(Y/F)G sequence that is conserved in K + channels [2]. These phenyl groups in KP6α form a cage similar to one postulated to be responsible for K+ selectivity relative to Na+ on the basis of a b initio calculations [3,4]. Two potential ion binding sites in the water-filled channel include one at the center of the trimer of phenyl rings which are occupied by NH 4+ ions which are isostructural with K+ ions. In our crystals two trimers are related by a two-fold axis perpendicular to the funnel axis generating a symmetric pair of funnels. The open ends of the self-assembling ion pore (Figure 1) would lie on the bilayer surface and the narrow bore would 313 Y. Shimonishi (ed): Peptide Science – Present and Future, 313–315 © 1999 Kluwer Academic Publishers, Printed in Great Britain

314

Figure 1

be at the center of the bilayer. The salt bridges that hold the trimers together are separated by 30 Å and portions of the funnel lip would extend well out of the membrane. Sequence homology between KP6α and KP6 β, in particular the relative position of three disulfide bridges, have allowed us to model the structure of KP6β upon that of KP6α with a high degree of confidence. A puzzling similarity exists between the tertiary structure of KP6 α and a histidine containing phosphocarrier protein HPR from E. coli [5] which has a similar disposition of secondary structural elements (the four-stranded β sheet and two α helices) and identical connectivity in a reversed chain trace. This unusual structural feature may be more than a curiosity in light of the fact that some membrane active toxins having reverse (or retro) sequence are observed to have comparable activity to the native toxins [6,7]. We have constructed a computer model for the three-dimensional structure of retro KP6 α based upon the crystallographically observed structure of KP6 α. The α carbon skeletons of models of KP6α and its retromer align closely despite the

315 difference in orientation of the Cα – C β bonds. Reversing the direction of each amino acid in the sequence while retaining the disulfide bridge produces a molecule with a very similar α carbon backbone trace and differing in amino acid side chain orientations. Nevertheless, dynamic calculations and energy minimization produce a molecule that is stereochemically feasible and not unlike the original structure. Models of trimers and hexamers of retro-KP6 α have also been constructed on the basis of the KP6 α trimer and hexamer. The hourglass shaped hexamer of retro-KP6α is very similar to that of KP6 α i n overall shape but it has a larger central pore that should retain ion transport properties but lack ion selectivity. The similarity in fold of some native and retro peptides and, the similarities in fold between KP6 α and HPR when aligned with N for C reversed relative to one another, could offer provocative new insights into protein folding and peptide channel design. Supported in part by NIH grant No. TW00374.

References 1. Favre, I. et al., Biophysics J. 71(1996) 3110. 2. Kerr, I.D., Nature 373 (1995) 112. 3. Kump, R.A. and Dougherty, D.A., Science 261(1993) 1708. 4. Miller, C., Science 261(1993) 1692 5. Jia, Z., Quail, J.W., Waygood, E.G. and Delbaere, L.T.J., J, Biol. Chem. 268 (1993) 22490. 6. Bessalle, R., Kapitkovsky, A., Gorea, A., Shalit, I. and Fridkin, M., FEBS 274 (1990) 151. 7. Merrifield, R.B., Juvvadi, P., Andreu, D., Ubach, J., Boman, A. and Boman, H.G., Proc. Natl. Acad. Sci.-USA 92 (1995) 3449.

108 Ion conductance in a gramicidin channel: the righthanded double-stranded double-helical dimer B.M. BURKHART, N. LI, D.A. LANGS and W.L. DUAX Hauptman-Woodward Medical Research Institute, 73 High Street, Buffalo, NY 14203

Introduction The X-ray crystal structure of gramicidin A complexed with Cs + ions has been determined to 1.40 Å resolution and found to be a right-handed doublestranded double-helical dimer. This structure demonstrates the structural features required to insert in the lipid bilayer and to allow passage of cations. The structural details agree with the previously published experimental details from a variety of CD, NMR, and Raman experiments with regards to peptide conformation, hydrogen bonding, and ion locations relative to particular amino acid residues. Furthermore, the structure determination reveals the presence of a single Cs + ion per channel distributed over three binding sites with water molecules filling the interstitial sites. This structure provides the best candidate to date for the ion-conducting form of the gramicidin channel.

Results and discussion Commercially available gramicidin D (Sigma), a mixture of the naturally occurring isomers of gramicidin A, was crystallized from a methanol (30 mg/mL) solution containing 250 mM CsCl and 39,976 data were collected to 1.40 Å resolution on an R-AXIS II area detector preserving the anomalous signal from the bound Cs + ions (space group P212121, a = 31.06 Å b = 31.88 Å c = 52.11 Å). The structure was solved by molecular replacement using a hypothetical model and verified using the Cs+ anomalous scattering. The structure was refined against the diffraction data to a crystallographic R-factor of 15.6% for all 17,776 unique data. Surprisingly, the structure was found to be a right-handed double-stranded double helical dimer (Figure 1A) with three Cs + sites per ion channel. The dimer is approximately 26 Å in length with approximately 7.2 residues/turn and containing a 4 Å in diameter pore. An NMR structure determination of uncomplexed gramicidin A in hydrated lipid bilayers yielded the most commonly accepted model for gramicidin, a right-handed single-stranded singlehelical dimer [1] with a similar pore size and channel dimensions. Several lines of evidence demonstrate that the current structure determination is not only correct, but provides a glimpse at the ion conducting form of 316 Y. Shimonishi (ed): Peptide Science – Present and Future, 316–318 © 1999 Kluwer Academic Publishers, Printed in Great Britain

317

Figure 1. (A) Illustration of the double-stranded double-helical dimer indicating the position of Cs + sites. (B) Histogram plot of N-H dipole angles in the current structure superimposed on the 15N-NMR powder pattern (adapted from [6]) of oriented gramicidin A in lipid bilayers.

gramicidin as it is conducting Cs + ions. This structure is right handed as required by CD [2] and NMR [3] and provides a rigid channel with little inward movement of carbonyls for ion conduction as demonstrated experimentally by recent data [4]. NMR spectroscopy has been used to probe the ion 15 [5], both sites binding sites which indicate binding near D-Leu 10 and L-Trp 15 N-NMR measurements of ion binding in the current structure. Furthermore, of gramicidin A in oriented lipid bilayers have been used to determine the N-H dipole angles of the backbone amides [6,7]. Interestingly, direct measurement of these angles in the current structure closely reproduce the original 15 N NMR powder pattern and provide a clear rationale for the distribution of chemical shifts (Figure 1B) with the L-amino acids shifted upfield (~ 200 ppm) relative to the D -amino acids (~ 150 ppm) because of a systematic difference in the N-H angle relative to the dimer axis (15° vs. 35°). The Cs + ion in each channel is distributed over three binding sites in a ratio of 40%/40%/20% from top to bottom. The binding sites occur near pairs of Trp residues on the surface of the dimer suggesting a long range electrostatic effect in addition to the association between the Cs + ions and the carbonyl π electrons. There is little discernable inward tilting of the carbonyls to directly coordinate to the Cs+ ions. Water molecules occupy the space between each Cs + site, or fill the channel in the absence of Cs+ at that site, such that each Cs + is carried through single file with a pair of coordinating waters in each ‘axial’ position. Electrostatic calculations demonstrate a net negative surface on the interior of the channel as required for cation selectivity and a relatively neutral exterior such as would be required for membrane insertion.

References 1. Ketchem, R.R., Hu, W., and Cross, T.A., Science, 261 (1993) 1457. 2. Killian, J.A., Prasad, U., Hains, D., and Urry, W., Biochemistry, 27 (1988) 4848.

318 3. Nicholson, L.K. and Cross, T.A., Biochemistry, 28 (1989) 9379. 4. Tian, F.T., Lee, K.-C., Hu, W., and Cross, T.A., Biochemistry, 35 (1996) 11959. 5. Woolf, T.B. and Roux, B., Biophys. J., 72 (1997) A396. 6. Nicholson, L.K., Moll, F., Mixon, T.E., LoGrasso, P.V., Lay, J.C., and Cross, T.A., Biochemistry, 26 (1987) 6621. 7. Mai, W., Hu, W., Wang, C., and Cross, T.A., Prot. Sci., 2 (1993) 532.

109 Protein folding intermediates and Alzheimer’s disease J.P. LEE¹, S-S. ZHANG¹, C. CLISH¹, N. CASEY¹, D. HASSELL¹, E. STIMSON², W. ESLER², J. MAGGIO², Y. LU³, A.M. FELIX³ and J.W. PENG 4 ¹Department of Chemistry, Boston University, 590 Commonwealth Ave., Boston, MA 02215, USA ²Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA ³Roche Research Center, Hoffman-La Roche, Nutley, NJ, 07110, USA 4 Vertex Pharmaceuticals, Cambridge, MA 02139, USA

Introduction The small family of diseases known as amyloidoses [1] are characterized by the degenerative accumulation of proteinaceous deposits. Alzheimer’s disease (AD) is the most widespread, with the accumulation of senile amyloid deposits within the cortex of the human brain. The precise cause of AD is unknown, there are no effective treatments that slow or arrest the disease, and given the opportunity, it is one of the most fatal diseases in human society. Growing evidence supports a causal [2] relationship between the formation of amyloid deposits and AD (although perhaps not exclusively), and a clear scientific goal is understanding the molecular mechanism(s) which underlie formation of amyloid deposits. The short ubiquitous peptide Aβ is the principal component of AD amyloid deposits, and is believed to be a naturally occurring proteolytic fragment of a transmembrane protein known as the Amyloid Precursor Protein (APP). Laboratory models of Aβ self-assembly have been problematic because they rely upon monitoring high concentration (e.g. ~ 106 > physiological) increases in turbidity, a non specific chemical event. However a new radioiodination based plaque competence assay [3], which is mechanistically distinct, sheds light onto a rather compelling structure function relationship. Results include: 1 . From the amino terminus to residue 28, A β c o n g e n e r s a r e p l a q u e incompetent. 2 . A central 26-mer (residues 10–35) contains both necessary and sufficient structural information to support plaque competence. 3 . While plaque competence is pH dependent, substitution of most of the ionizable residues which react over the observed titration range (to analogs which do not ionize), results in altered but not abolished plaque competence. 4 . Several different substitutions of residues within a central hydrophobic cluster (CHC, residue 17–21), results in plaque incompetence, suggesting a critical role for this putative conformational determinant. 319 Y. Shimonishi (ed): Peptide Science – Present and Future, 319–321 © 1999 Kluwer Academic Publishers, Printed in Great Britain

320 5. A covalent linkage created between residues 16–22, (design based upon solution conformation) results in plaque incompetence, BUT with similar structure.

Results and discussion NMR CSI analysis [4] shows that both full length Aβ and the central 26 mer adopt similar conformations within their common residues, however, neither molecule contains any helical or sheet structure in water solution. CD analysis corroborates these data. Consequently the central 26 residues has been established as a structural paradigm for the plaque competence [5] behavior of A β . Using a newly developed ULTRAlinear NMR probe [6] highly accurate translational diffusion measurements show that in water solution at 200–300 µ M A β is a monomeric species. Isotopic labeling of Αβ has allowed refinement of conformational data as well as measurement of heteronuclear relaxation times, and therefore determination of order parameters (S²) using the reduced spectral density mapping approach [7]. With such biophysical data in hand, the stage was set to complete distance geometry calculations with independent insight into local ‘foldedness,’ and thereby a priori addressing some of the difficulties associated with detailed conformational analysis of short linear polypeptides. In sharp contrast to previous results observed in mixed organic solvents [8], in water solution at mid pH Aβ adopts a surprisingly stable compact structure which is completely devoid of canonical secondary structure. Conformational stability apparently results from hydrophobic condensation of flanking residues about the CHC. The entire polypeptide backbone exists as a series of loops, with four Type IV turns (22–25, 24–27, 27–20, and 30–33), all condensed upon the nucleus formed by the CHC (residues Leu 17, Val 18, Phe 19, Phe 20, Ala 21). The additional hydrophobic residues from the carboxy termini in direct contact with the CHC are Ile 32 and Met 35, while in the amino terminus Tyr 10 and Val 12 directly contact the CHC. Hydrodynamic calculations [9] identify a conspicuous hydrophobic patch with one face of the CHC at its center, forming the major contributor to the anomalous non-polar/polar surface area (~1.8). Disruption of the CHC based patch (e.g. F19T), renders Aβ plaque incompetent [10]. Based upon these results, we introduce an AMYLOIDOGENIC FOLDING HYPOTHESIS which invokes similarities between the hydrophobic collapse of protein folding and an initial event during Aβ self-association which relies upon low affinity hydrophobic docking. Presumably, similar to molten globular folding transitions, during maturation of amyloid deposits affinity slowly increases via development of new electrostatic, van der Waals, and hydrogen bonding interactions between the peptide and the deposit (template). It is predicted that there exists several discrete steps underlying the total molecular mechanism of

321 Aβ deposition. Individual steps are likely to be accessible via chemical, kinetic, and thermodynamic dissection based upon structure function studies as presented here.

References 1. Sipe, J.D. (1992) Ann. Rev. Biochem., 61, 947–975. 2. Selkoe, D.J. (1994) J. Neuropathol. Exp. Neurol., 53, 438–447. 3. Maggio, J.E., et al. (1992) PNAS. USA, 93,1125–1129. 4. Wishart, D.S. (1992) Biochemistry, 31,1647–1651. 5. Lee, J.P., et al (1995) Biochemistry, 34,5191–5200. 6. Lee J.P., et al (1995) 37th ENC, Ascilomar CA, (sub. to JACS). 7. Torchia, D.A. (1993) in NMR of Proteins, p 190–219, CRC Press, An. Arb. 8. Stricht, H., et al. (1995) Eur. J. Biochem., 233, 293–298. 9. Riseman, J. (1950) J. Chem. Phys., 18, 512–516. 10. Esler, W.P. (1996) Biochemistry, 35, 749–757.

110 Topological isomers of human uroguanylin: function and conversion T. YOSHIDA¹ , R. TANABE², S. KUBO³, N. CHINO³, T. KIMURA³, S. SAKAKIBARA³, M. NAKAZATO 4 , K. KANGAWA5 and Y. KOBAYASHI¹ ¹ Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565 Japan ² Analytical Research Laboratory, Tanabe Seiyaku Co., Ltd, Osaka, 532 Japan ³ Peptide Institute, Inc., Protein Research Foundation, Minoh, Osaka, 562 Japan 4 Third Department of Internal Medicine, Miyazaki Medical College, Miyazaki, 889-16 Japan 5 National Cardiovascular Center Research Institute, Suita, Osaka, 565 Japan

Introduction Human uroguanylin, a 16-residue peptide which has two disulfide bridges, is an endogenous activator for intestinal guanylyl cyclase [1, 2]. We found that this compound contains two conformationally distinct peptides, both of which are chemically identical. One of them shows bioactivity but the other doesn’t [3]. These isomers are interconvertible in aqueous media, but the rate constant for interconversion is sufficiently slow that we were able to determine each structure in solution by NMR. The details of structure determination are described in the following paper. We measured the rate of interconversion between these isomers at various conditions to investigate the possible mechanism of interconversion.

Results and discussion The active uroguanylin isomer adopts a right-handed spiral topology as that of a heat stable enterotoxin (ST) [4]. The inactive isomer, however, adopts a left-handed spiral. The difference in topology causes the different arrangement of side chains that comprises the putative receptor binding site, which has been shown by mutational studies of ST [5]. In particular, the side chain of Ala11, which may be crucial for the receptor binding, is buried by the side chain of Thr13 in the inactive isomer (Figure 1). Because ST is reported to bind to the GC-C receptor by hydrophobic interactions, we suggest that the binding affinity of the inactive isomer to the GC-C receptor may decrease due to the steric hindrance and the low hydrophobicity caused by the hydroxyl group of the Thr13 side chain. The interconversion between these isomers was measured by RP-HPLC as the change in purity of each isolated isomer at different pH. The acceleration was detected at low pH. The plot of the rates of interconversion versus pH has 322 Y. Shimonishi (ed): Peptide Science – Present and Future, 322–323 © 1999 Kluwer Academic Publishers, Printed in Great Britain

323

Figure 1 Surface representation of (A) the active isomer and (B) the inactive isomer of human uroguanylin.

a midpoint at pH 4.5, which may correspond to the pKa of a carboxyl group. This result implies that the transition state of the interconversion has carboxyl groups in close proximity. We found that human des-Leu16 uroguanylin interconverted at least 10 times faster than the native compound. These facts strongly suggest the interconversion mechanism in which the C terminal segment (Thr 13 –Leu 16 ) penetrates through the closed loop that consists of the residues Cys 4 –Cys 12 .

References 1. Harma, F.K., Forte, L.R., Eber, S.L., P Pidhorodeckyj, N.V., Kraus, W.I., Freedman, R.H., Chin, D.T., Tompkins, J.A., Fok, K.F., Smith, C.E., Duffin, K.L., Siegel, N.R. and Currie, M.G., Proc. Natl. Acad. Sci. USA, 90 (1993) 10464. 2. Kita, T., Smith, C.E., Fok, K.F., Duffin, K.L., Moore, W.M., Karabatos, P.J., Kachur, J.F., Harma, F.K., Pidhorodeckyj, N.V., Forte, L.R. and Currie, M.G., Am. J. Physiol., 266 (1994) F342. 3. Chino, N., Kubo, S., Miyazato, M., Nakazato, M., Kangawa, K. and Sakakibara, S., Letters in Peptide Science, 3 (1996) 45. 4. Ozaki, H., Sato, T., Kubota, H., Hata, Y., Katsube, Y. and Shimonishi, Y., J. Biol. Chem., 26 (1991) 5934. 5. Carpick, B.W. and Gariépy, J., Biochemistry, 30 (1991) 4803.

111 Topological isomers of human uroguanylin: structure determination by nuclear magnetic resonance R. TANABE¹, T. YOSHIDA², S. KUBO³, N. CHINO³, T. KIMURA³, S. SAKAKIBARA³, M. NAKAZATO4 , K. KANGAWA5 and Y. KOBAYASHI² ¹ Analytical Research Laboratory, Tanabe Seiyaku Co., Ltd, Osaka, 532 Japan ² Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka, 565 Japan ³ Peptide Institute, Inc., Protein Research Foundation, Minoh, Osaka, 562 Japan 4 Third Department of Internal Medicine, Miyazaki Medical College, Miyazaki, 889-16 Japan 5 National Cardiovascular Center Research Institute, Suita, Osaka, 565 Japan

Introduction Uroguanylin and guanylin are endogenous peptide hormones that regulate intestinal salt and water transport [1]. They stimulate cyclic guanine monophosphate (cGMP) production by activating the guanylate cyclase-C (GC-C) receptor as well as heat stable enterotoxins (ST) secreted by pathogenic bacteria [2, 3]. In the chemical synthesis of human uroguanylin by two-step selective disulfide bond forming reaction, two compounds separable by HPLC were generated [4]. Though they are inter-convertible isomers, one is active for stimulation of cGMP production in cultured T84 cells, and the other is inactive. As the rate of internal conversion was very slow, we determined the solution structure of each compound by ¹H-nuclear magnetic resonance (NMR).

Results and discussion Each compound was separated by HPLC and dissolved in phosphate buffer (10% D 2 O, pH 3.6) to make 5 mM concentration. For each form of human uroguanylin, 2D-NMR spectra (DQF-COSY, TOCSY, NOESY) were acquired on a Bruker DRX-500 spectrometer. The cross peaks were well separated and assigned in a sequential specific manner. Because of the slow rate of conversion, in the spectra of one form, there were not any signals of another form. We calculated the solution structure of each form individually by means of simulated annealing procedure using the program XPLOR with the distance restraints derived from nuclear Overhauser effects (NOES ). The resulting 10 structures of both were well defined, and the root-mean squared deviation for the backbone atoms is 0.67 Å for the active form and 0.48 Å for the inactive one (Figure 1). 324 Y. Shimonishi (ed): Peptide Science – Present and Future, 324–325 © 1999 Kluwer Academic Publishers, Printed in Great Britain

325

Figure 1.

Superimposed 10 structures of a) active form and b) inactive form of human uroguanylin.

Each structure is identical as to three loops: first is formed by backbone from N-terminal to Cys7 and disulfide bond between Cys4 and Cys 12 , second is formed by backbone from Cys 7 to Cys12 , third is formed by backbone from Cys 12 to C-terminal and disulfide bond between Cys 7 and Cys 15 . However, their orientations with respect to each other are quite different, and it is confirmed that these compounds are indeed topological isomers. As the relative orientation of three loops in the active form results in an arrangement of the backbone with right-handed spiral like ST [5], it seems that this topology is important for GC-C receptor binding.

References 1. Forte, L.R., Fan, X., Hamra, F.K., Am. J. Kidney Dis., 28 (1996) 296. 2. Currie, M.G., Fok, K.F., Kato, J., Moore, R.J., Hamra, F.R., Duffin, K.L., Smith, C.E., Proc. Natl. Acad., Sci. 89 (1992) 947. 3. Kita, T., Smith, C.E., Fok, K.F., Duffin, K.L., Moore, W.M., Karabatsos, P.J., Kachur, J.F., Hamra, F.K., Pidhorodeckyj, N.V., Forte, L.R., Currie, M.G., Am. J. Physiol., 266 (1994) F342. 4. Chino, N., Kubo, S., Miyazato, M., Nakazato, M., Kangawa, K., Sakakibara, S., Letters in Peptide Science, 3 (1996) 45. 5. Ozaki, H., Sato, T., Kubota, H., Hata, Y., Katsube, Y., Shimonishi, Y., J. Biol. Chem., 266 (1991) 5934.

112 Homology modelling of the glycoprotein hormones: examination of the receptor recognition characteristics P. GOMME¹, J. WHISSTOCK¹, P. THOMPSON¹, A. LESK² and M. HEARN 1,* ¹ Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3168, Australia ² Department of Haematology, University of Cambridge, UK * To whom correspondence should be addressed.

Intoduction The glycoprotein hormones (GPHs) are a family of structurally related proteins, which include chorionic gonadotropin [CG], follitropin [FSH], lutropin [LH] and thyrotropin [TSH]. These endocrine hormones are characterised by the specific non-covalent dimerisation of non-identical carbohydrate containing subunits, designated the alpha- (α -) and beta- (β -) subunits. The GPHs bind to G-protein receptors via a series of loci present within both the α- and β -subunits. Both subunits contain a cystine knot motif at their core, indicating that the GPHs belong to the cystine knot superfamily of growth factors (nerve growth factor, NGF; platelet derived growth factor, PDGFβ ; transforming growth factor TGF β 1/ β3, activins β A βA / βE βE , inhibins, bone morphogenic growth factor, OP1; brain-derived neurotrophic growth factor, BDNGF; Mullerian inhibitory factor, MIF; Xenopus Vg-1 gene product; Drosophilia decapentaplegic gene product and over 25 other members with growth factor or cell differentiational properties). In the current study, an homology modelling approach has been used to derive structural models for human TSH (hTSH) and FSH (hFSH) from the hCG X-ray crystallographic structure [1, 2]. In addition, the electrostatic potential surfaces of the resulting model structures were compared with information on the putative hormone-receptor (H–R) binding sites as part of our studies to identify surface motifs and loci which are likely to contribute to the binding specificity of these hormones for their specific G-protein receptors located on thyroidal or gonadal cells.

Results and discussion The primary structures of hCG, hTSH and hFSH were aligned using Clustalw 1.7 and the amino acid residues previously implicated in receptor binding (RB) were mapped onto the primary structures. The identification of these amino acid residues as contributors to the putative receptor binding sites has been 326 Y. Shimonishi (ed): Peptide Science – Present and Future, 326–329  1999 Kluwer Academic Publishers, Printed in Great Britain

327 recently detailed in our associated publications [3–5]. The results indicate that, in general, the putative receptor binding amino acid residues for these three GPHs are located in common segments within either the α -subunit (ie Loop-L2, C-terminus) or the β -subunit (ie N-terminus, Loop-L1, Loop-L3, C-terminus). The results demonstrate that for each hormone type (hCG, hTSH & hFSH), specific residues within these receptor binding loci contribute to the RB behaviour, thereby providing a level of hormone specificity. To investigate the orientation of the RB sites in the GPH quaternary structures, homology models of the hTSH and hFSH molecules were derived using the Modeller module within the Quanta97 molecular modelling suite (Figure 1). The RB residues were then mapped onto the quaternary structures of the hTSH and hFSH. The results suggest that the RB surface loci of these GPHs are predominantly generated from within the central portion of the proteins structure. Furthermore, the RB loci form distinct patches and not a single continuous binding face on the surface of the GPHs. Such multi-site loci and their potential for selective and independent interactions with the corresponding G-protein receptor sites would be concordant with previous studies [3–7] based on investigations utilising synthetic peptide fragments or site directed mutagenesis procedures. Moreover, such multisite loci would satisfy the criteria of the binary docking model of Combarnous [8] for GPH–GPH receptor recognition. It is noteworthy that the pattern of the RB loci vary between the different GPH types, reflecting the specific nature of the H–R interaction. In order to further delineate the role of some of the molecular forces associated with glycoprotein hormone-receptor interaction, the electrostatic surfaces of the GPHs were defined using the program GRASP [8] (results not shown). According to these results, the dominant feature in the predicted RB loci is a large cavity, the size and charge of which varies between the different GPHs. Thus, within the hTSH molecule the presence of a bulky side-chain at β -His 10 forces the positively charged residues α-Lys 48 to be positioned prominently at the bottom of the cavity, whilst the presence of an Ala residue in the analogous position of the hTSHβ - H i s 10 in both hFSH and hCG results in the generation of a deeper cavity. Further, these deeper cavities of both hFSH and hCG contain other charged residues that are hidden or less accessible in hTSH. In the case of hFSH, the bottom of the cavity contains a highly conserved negatively charged residue, β -Asp 13 , whilst hCG contain a negatively charged residue β -Asp 111. Because of this reversal in the direction of the charged vector associated with β -Asp 13 a n d β -Asp 111 residues will result in an orientationally dependent recognition with the corresponding region(s) of the G-protein receptor, a function alignment role in this biomolecular interaction can be evoked. Moreover, it can be noted that several of the amino acid residues within the β-subunit sequence region hTSHβ [52–78] which show prominent surface accessibility in terms of their exposure on the electrostatic surface of hTSH, have formerly been assigned on the basis of monoclonal antibody footprinting experiments or studies with chimeric synthetic peptides to contribute to the

328

Figure 1. Homology models of hTSH (I) and hFSH (II) derived from the X-ray crystal structure of hCG (III). Residues involved in HR interactions are highlighted as ball and sticks, the disulphide bonds are highlighted by open circles. The α-subunit is shown as a solid line structure and the βsubunit is shown as a dashed line structure. The stereo diagrams were prepared using a program by Dr Arthur Lesk (Department of Haematology, University of Cambridge, UK).

329 receptor binding site(s) of hTSH. For example, in previous studies we have identified the epitope sites which are recognised by two anti-hTSH monoclonal antibodies, designated mAb279 and mAb299. Based on a variety of structure/ function criteria, these epitopic sites are thought to be located in close proximity to the receptor binding loci of hTSH. Using panels of synthetic peptides, inhibition of the binding of the mAb279 and mAb299 to radiolabeled [I 125 ] bTSH- β was documented, with the peptide TSH- β [56–68] shown to specifically inhibit the binding of mAb299 to I 125-bTSH- β , whilst having no effect on mAb279 binding. This finding was consistent with other studies [9] which had localised the mAb299 epitope site to the TSH β -[56–68] region, which forms part of the loop region [L3] of the β -subunit and is held in close proximity to a second loop region [L1] and the C-terminus of the TSH β -subunit by the disulphide bonds β Cys 16 –Cys 67 and β Cys 19 –Cys 105 . In addition, chimeric linear and cyclic peptide constructs incorporating the sequences 14 K . . . L20 and 6 5 P . . . T 72 , joined by a β Ala–β Ala bridge show enhanced inhibition of the specific binding of mAb299 to hTSH. These finding are also in agreement with other studies in which the Tyr residues located at TSH β -59 and TSH β -74 have been shown to be partially protected from iodination when TSH is complexed with mAb299. Collectively, these investigations provide additional data supporting the concept that the exquisite specificity manifested by the GPHs in their interaction with their corresponding G-protein receptors is manifested through (at least) a binary mode with the receptor binding loci providing a synergistic attribute for the discrimination in terms of receptor recognition and binding afinity differences. These results thus will aid in the development of low molecular weight bioactive compounds with specific GPH activities and further our knowledge of the mechanisms involved in the GPH-receptor interaction.

Acknowledgements This work was supported by the National Health and Medical Research Council of Australia.

References 1. Lapthorn, A.J., Harris, DC., Littlejohn, A., Lustbader, J.W., and Canfield, R.E., Machin, Morgan, F.J., Isaacs, N.W., Nature, 329 (1994) 455. 2. Wu, H., Lustbader, J.W., Liu, Y., Canfield, R.E., and Hendrickson, W.A, Structure, 2 (1994) 545. 3. Fairlie, W.D., Stanton, P.G., and Hearn, M.T.W. Eur. J. Biochem., 240 (1996) 622. 4. Gomme, P.T., Thompson, P.T., Stanton, P.G., and Hearn, M.T.W. J. Pept. Res., (1997) in press, 5. Fairlie, W.D., Stanton, PG., and Hearn, M.T.W. Eur. J. Biochem., 228 (1995) 373. 6. Szkudlinski, M.W., The, N.G., Grossmann, M., Tropea, J.E., and Weintraub, B.D., Nature Biotechnol., 14 (1996) 1257. 7. Combarnous, Y., Endocr. Rev., 13 (1992) 670. 8. Nicholls, A., GRASP: Graphical representation and analysis of surface properties. Columbia University, ( 1992). 9. Gomme, P.T., Thompson, P.T., Stanton, PG., Hearn, M.T.W. J. Pept. Sci., (1998) in press.

113 Prion protein structure and pathology of transmissible spongiform encephalopathies (TSE) K. WÜTHRICH, M. BILLETER, R. RIEK, G. WIDER, S. HORNEMANN and R. GLOCKSHUBER Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule, CH-8093 Zürich

Introduction Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases for which it has been proposed that they are related to a novel infectious agent, the prion [1,2]. TSEs have been reported to occur as infectious, inherited, and spontaneous diseases. Following the ‘protein-only hypothesis’ [2,3] the causative agent is a pathogenic conformation of the prion protein (PrP). PrP is ubiquitous in mammalian cells in a benign, cellular conformation ( PrPC) . In rare cases it may be transformed into the infectious scrapie conformation ( P r P Sc ), which has been observed in the form of insoluble, protease-resistant aggregates in the brain of affected individuals [4, 5]. The most widely discussed TSEs are Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE). Other human prion diseases include kuru, the Gerstmann–Sträussler–Scheinker syndrome (GSS) and fatal familial insomnia (FFI). The ‘mad cow crisis’ in Europe has greatly added to public concern about TSEs and raised keen interest in the biological foundations of TSE pathology and TSE transmission. Thus, questions relating to the ‘species barrier’ [4, 5], i,e., the relative ease of infection between individuals of different species relative to that between individuals of the same species, have attracted intensive interest, in particular regarding possible transmission of BSE from cows to humans through the food chain [6, 7]. Another focus are the pointmutations in human PrP that have been linked with inherited prion diseases, i.e., GSS, FFI and certain forms of CJD [1]. To provide a foundation for discussions of these phenomena on the molecular level, we undertook to solve the three-dimensional structure of the cellular form of the prion protein in solution, using nuclear magnetic resonance (NMR) spectroscopy.

Results For the NMR structure determination we started out using the standard protocol [8], with modern techniques that rely on isotope labeling of the protein with 15 N and 13 C [9]. The general strategy followed is illustrated with 330 Y. Shimonishi (ed): Peptide Science – Present and Future, 330–334 © 1999 Kluwer Academic Publishers, Printed in Great Britain

331 Figure 1: The decisive breakthrough toward a structure determination came about when it was realized that the fragment of residues 121–231 of the mouse prion protein, mPrP(121–231), forms a self-folding domain with sufficient longtime stability to enable data collection for a NMR structure determination [10,11]. Subsequently, once we succeeded in expressing the full-length polypeptide chain, mPrP(23–231) [12], it turned out that knowledge of the resonance assignments and the structure of mPrP(121–231) greatly aided in the data analysis of the intact protein, since much of the work could be done by difference spectroscopy [13]. The NMR structure of mPrP( 121–231) (Figure 2) contains three α -helices and a two-stranded antiparallel β -sheet. The helices extend from residues 144 to 154, 175 to 193, and 200 to 219, and the β -strands contain the residues 128 to 131 and 161 to 164. The first turn of the second helix and the last turn of the third helix are linked by the single disulphide bond in the protein. The

Figure 1. Schematic presentation of the amino acid sequence of mammalian prion proteins, with residues 23–231. The C-terminal fragment of residues 121–231 is shaded, and the posttranslational modifications are indicated. In the N -terminal part of the molecule the octapeptide repeats are indicated, where the residues written in italics are variable.

Figure 2. Structure of mPrP(23–231). A C-terminal domain of residues 126–231 forms a globular structure with three α-helices and a short β-sheet, which was also observed in mPrP(121–231). The backbone 15 N– 1H moieties of the residues 126–230 manifest rotational correlation times of several ns, which is typical for a folded protein of this size. The residues 121–125 and the N-terminal segment 23–120 (represented by black dots) show features of a flexible, ‘random coil-like’ polypeptide, with rotational correlation times for the 15 N– 1 H groups of τc < 1 ns (adapted from ref. [13]).

332 twisted V-shaped arrangement of the helices 2 and 3 forms the scaffold onto which the β -sheet and the first helix are anchored. The regular secondary structure elements and the connecting loops are well defined, with the sole exceptions of residues 167 to 171 and the chain-terminal segments 121–125 and 220–231. The polypeptide fold is stabilized by hydrophobic interactions in a core that contains primarily side chains of the second and third helices and the β -sheet, one side chain of the first, mostly hydrophilic helix, and the side chains of the residues 134, 137, 139, 141, 158 and 198, which are located in loop regions. With the exceptions of Ile 139, Ile 184 and Val 203, the residues of the hydrophobic core are invariant in the known mammalian prion protein sequences [14]. Hydrophobic surface patches in mPrP(121–231) are located near the β -sheet and the loop preceding the first helix. The surface; of mPrP(121–231) is otherwise characterized by a markedly uneven distribution of positively and negatively charged residues [10]. Overall, although it has a polypeptide fold that is not so far represented by the protein structures in the Brookhaven Protein Data Bank [15], the polypeptide mPrP(121–231) displays typical features of a globular protein (see also [11]). The NMR structure determination of mPrP(23–231) [13] showed that the C-terminal domain of residues 126–231 has the same fold as in mPrP( 121–231) and that the N -terminal polypeptide segment 23–120 is flexibly disordered. From a technical point of view it should be emphasized that the information on the segment 23–120 of mPrP shown in Figure 2 could not have been obtained with any other technique but NMR: for each residue except the prolines the time scale for intramolecular mobility of the polypeptide backbone could be determined to be shorter than 1 nanosecond ( 10 – 9 s). In addition, for the indole rings of all tryptophan residues in the segment 23–120 similar mobility could be demonstrated, which is of particular interest for understanding possible roles of the octapeptide repeats (see Figure 1). Overall, the NMR approach shows that the structure of the C -terminal domain 126–231 is the same as in mPrP(121–231), that the N -terminal domain 23–120 forms an extended, highly flexible structure, and that the linker peptide 121–125 is also in an extended conformation, with mobility that is intermediate between that of the tail and that of the globular domain [13] (Figure 2). The results obtained for mPrP(23–231) have recently been confirmed by studies of two fragments of the prion protein from Syrian hamster, shPrP(90–231) [16] and shPrP(29–231) [17], which show coinciding NMR structures except for local differences in the least well defined molecular region at the end of helix 3.

Discussion Prior to the structure determination of mPrP(121–231) [10] discussions on structure-function correlations of the prion protein were based on structure

333 predictions [18,19] that are, at least in the case of PrP C, different from the real structure. The NMR structure for PrP C now presents, for the first time, a rational basis for such discussions. By analogy with other areas of biomedical research it can be expected that structure-based rational thinking will support future development of diagnosis and possibly treatment of TSEs. Space only permits to indicate some lines of thought based on the NMR structure of mPrP, which are elaborated in detail elsewhere. For example, the presence of an extensive polypeptide segment with the properties of a flexible extended coil in PrP C could enable structural transitions to P r P Sc aggregates that might display sizeable β -sheet content [20] without major conformational rearrangements in the C-terminal domain 126–231. In particular, the combination of the result presented in Figure 2 with earlier findings that the entire polypeptide segment 90–231 is protected against protease K digestion in PrP Sc [2] emphasizes that there is a major change in the structural arrangement of Sc the residues 90–125 between PrPC and PrP . Furthermore, the NMR structure determination has already resulted in identification of the location in the threedimensional structure of a likely contact site for a hypothetical host protein, ‘protein X’ [21,22], which may be required for the formation of PrPSc and might play an important role for the species barrier, and of the epitopes for a PrP Sc -specific monoclonal antibody [23]. Finally, the availability of the threedimensional structure enables detailed studies of possible effects of the amino acid replacements in human PrP that segregate with inherited human prion diseases [24]. Acknowledgements Financial support was obtained from the Schweizerischer Nationalfonds (Projects 31.49047.96, 438 + 0.050285 and 438 + 0.050287). S.H. is supported by a grant from the Boehringer Ingelheim Fonds. We thank MS E. Ulrich for the careful processing of the manuscript. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Prusiner, S.B., Science, 216 (1982) 136. Prusiner, S.B., Science, 252 (1991) 1515. Griffith, J.S., Nature, 215 (1967) 1043. Prusiner, S.B., Science, 278 (1997) 245. Weissmann, C., FEBS Lett., 389 (1996) 3. Collinge, J., Sidle, K.C.L., Meads, J., Ironside, J., and Hill, A.F., Nature, 383 (1996) 685. Roberts, G.W. and James, S., Curr. Biol., 6 ( 1996) 1247. Wüthrich, K., NMR of Proteins and Nucleic Acids, Wiley, New York, 1986. Bax, A. and Grzesiek, S., Acc. Chem. Res., 26 (1993) 131. Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., and Wüthrich, K., Nature, 382 (1996) 180. 11. Hornemann, S. and Glockshuber, R., J. Mol. Biol., 261 (1996) 614. 12. Hornemann, S., Korth, C., Oesch, B., Riek, R., Wider, G., Wüthrich, K., and Glockshuber, R., FEBS Lett., 413 (1997) 277.

334 13. Riek, R., Hornemann, S., Wider, G., Glockshuber, R., and Wüthrich, K., FEBS Lett., 413 (1997) 282. 14. Schätzl, H.M., Da Costa, M., Taylor, L., Cohen, F.E., and Prusiner, S.B., J. Mol. Biol., 245 (1995) 362. 15. Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.F., Brice, M.D., Rodgers, J.R., Kennard, O., Shimanouchi, T., and Tasumi, M., J. Mol. Biol., 112 (1977) 535. 16. James, T.L., Liu, H., Ulyanov, N.B., Farr-Jones, S., Zhang, H., Donne, D.G., Kaneko, K., Groth, D., Mehlhorn, I., Prusiner, S.B., and Cohen, F.E., Proc. Natl. Acad. Sci. USA, 94 (1997) 10086, 17. Donne, D.G., Viles, J.H., Groth, D., Mehlhorn, I., James, T.L., Cohen, F.E., Prusiner, S.B., Wright, P.E., and Dyson, H.J., Proc. Natl. Acad. Sci. USA, 94 (1997) 13452. 18. Huang, Z., Gabriel, J.M., Baldwin, M.A., Fletterick, R.J., Prusiner, S.B., and Cohen, F.E., Proc. Natl. Acad. Sci. USA, 91 (1994) 7139. 19. Huang, Z., Prusiner, S.B., and Cohen, F.E., Folding Des., 1(1995) 13. 20. Caughey, B.W., Dong, A., Bhat, K.S., Ernst, D., Hayes, S.F., and Caughey, W.S., Biochemistry, 30 (1991) 7672. 21. Billeter, M., Riek, R., Wider, G., Hornemann, S., Glockshuber, R., and Wüthrich, K., Proc. Natl. Acad, Sci. USA, 94 (1997) 7281. 22. Kaneko, K., Zulianello, L., Scott, M., Cooper, C.M., Wallace, AC., James, T.L., Cohen, F.E., and Prusiner, S.B., Proc. Natl. Acad. Sci. USA, 94 (1997) 10069. 23. Korth, C., Stierli, E., Streit, P., Moser, M., Schaller, O., Fischer, R., Schulz-Schaeffer, W., Kretschmar, H., Raeber, A., Braun, U., Ehrensperger, F., Hornemann, S., Glockshuber, R., Riek, R., Billeter, M., Wüthrich, K., and Oesch, B., Nature, 390 (1997) 74. 24, Riek, R., Wider, G., Billeter, M., Hornemann, S., Glockshuber, R., and Wüthrich, K., Proc. Natl. Acad. Sci. USA, 95 (1998) 11667.

114 Peptide nucleic acid (PNA) P.E. NIELSEN Center for Biomolecular Recognition, Department of Medical Biochemistry & Genetics Biochemistry Laboratory B, The Panum Institute, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark

Introduction PNA (peptide nucleic acid) is a DNA mimic in which the natural deoxyribosephosphate diester backbone of the nucleic acid has been exchanged for a pseudo peptide (amide) backbone composed of 2-aminoethyl glucine unit having the nucleobase attached to the glycine nitrogen via an ‘acetyl’ linker [l–4] (Figure 1). This backbone structure is homomorphous to that of the natural nucleic acid as indicated by chemical bond distances as well as by computer model building. PNA was originally conceived as a reagent designed to bind sequence specifically to double stranded DNA via major groove triplex recognition [1]. Quite surprisingly, however, it was discovered that binding of homopyrimidin PNA to double stranded targets takes place via a strand displacement mechanism involving an extremely stable PNA2 –DNA triplex (Figure 2) [1,5,6]. Furthermore, PNA oligomers also recognize complemen-

Figure 1.

Chemical structures of DNA and PNA. ‘B’ is a nucleobase.

335 Y. Shimonishi (ed): Peptide Science – Present and Future, 335–341 © 1999 Kluwer Academic Publishers, Printed in Great Britain

336

Figure 2. Schematic drawing of a PNA-dsDNA strand displacement complex (right) consisting of two PNA strands (composed of thymines (T) and cytosines (C)) that invade the DNA duplex and bind to the complementary DNA strand (composed of adenines (A) and guanines (G)) by Watson–Crick and Hoogsteen base pairing (left). The non-complementary DNA strand is extruded as a single stranded loop.

tary RNA, DNA or another PNA oligomer very efficiently and with high sequence specificity [3–7]. These properties of PNA combined with high biological and chemical stability have raised a considerable interest in PNAs within fields of gene therapeutic medicine, genetic diagnostics, prebiotic evolution and molecular biology.

Structure of PNA complexes The excellent DNA mimicking properties of PNA as predicted from hybridization of circular dichroism results [3] were confirmed by the NMR and X-ray crystallography structure determination of PNA-RNA [8], PNA–PNA [9] and PNA–DNA [10] duplexes and a PNA 2 –DNA triplex [11]. In particular the structure of the P N A2 –DNA triplex and the PNA–PNA duplex also showed, however, that the PNA does indeed prefer a helical conformation, the P-form (Figure 3) which is distinctly different from both the B-form preferred by DNA and the A-form preferred by RNA. Specifically, the P-form helix is much wider (28 Å versus ca. 20 Å for the B form) and with almost double the pitch ( 16–18 bases versus 10–11 for B- or A-form helices).

337

Figure 3. Structure of an 18-mer PNA duplex build from the X-ray crystallography data obtained from a selfcomplementary hexamer PNA [9]. Side (left) and end (right) – views are shown.

Gene therapeutics The very favorable hybridization properties of PNA both regarding binding to single stranded RNA and double stranded DNA combined with ease of synthesis and chemical modification as well as high chemical and in particular biological stability has made PNA a very attractive lead for the development of gene therapeutics [12]. Specifically in vitro experiments have demonstrated good antisense efficiency of PNA [13–15] and PNA triplex strand invasion complexes with double stranded DNA effectively block transcription in vitro [13,16]. Furthermore, it has been found that telomerase can be inhibited by very low concentrations of PNA targeted to the RNA moiety of this ribonucleoprotein [17], and the HIV reverse transcriptase is also blocked by PNA bound to the template RNA [18].

Genetic diagnostics A variety of PNA based techniques that are of interest for genetic diagnostics have been developed by exploiting the unique properties of PNA. Sequence specific detection by hybridization can utilize a prehybridization principle with subsequent separation of DNA bound and nonbound PNA by capillary [19]

338 or gel electrophoresis [20] since the free PNA due to its electrostatically neutral backbone will not migrate in the electric field. Furthermore, PNA has shown good promise as capture probes for sample preparation [21–22] and PNA based elecrochemical DNA sensors [23], and PNA hybridization arrays [24] are also being developed.

Modified PNA backbones The easily accessible chemistry of PNA invites to backbone modification in order to improve and/or modulalte the properties of PNA. Although most modificating so far have proven inferior to the original aminoethylglycine backbone in terms of hybridization properties [4] two recent developments are encouraging. First, the backbone can be ‘chemically functionalized’ without any major penalty in hybridization potency by using other α -aminoacids such as lysine, leucine, glutamic acid instead of glycine in the backbone [25], and secondly it was recently shown that the amide bond in the backbone may – with some reduction in hybridization efficiency – be substituted with a phosphonic ester [26] (Figure 4).

Alternative nucleobases The chemistry of PNA also allows for convenient development and testing of alternative nucleobase either for improved Watson–Crick or Hoogsteen

Figure 4. Examples of PNA oligomers containing a single phosphonic ester (PHONA) monomer or a single α-amino acid monomer (R = a side chain of the natural amino acids).

339 recognition. Such nucleobases include the pseudoisocytosine that very efficiently and without pH dependence replaces protonated cytosine for Hoogsteen recognition of guanine [27]. Recognition of thymine and cytosine by a Hoogsteen bound third strand nucleic acid (analogue) have been a longstanding and still unsolved problem, but the newly developed pyridazinone ‘e-base’ [28] (Figure 4) is an interesting step in this direction. Finally, the adenine substitute, diaminopurine which supplies an additional hydrogen bond to an A-T base pair stabilizes PNA duplex by 2–6°C per diaminopurine-thymine base pair [29]. Future prospects The results obtained so far with PNA clearly makes this type of molecules very interesting for genetic medicine and diagnostics although many issues still need to be addressed and many questions still needs answers. However, since PNA is basically a peptide that ‘thinks it is a nucleic acid’ PNAs are also interesting

Figure 5. Hoogsteen recognition of guanine by pseudoisocytosine is a ‘permanently protonated’ cytosine mimic [27] Proposed Hoogsteen recognition of thymine by the novel nucleobase ‘E’ that circumvents the sterically blocking methylgroup of thymine [28].

Figure 6. Comparison of a diaminopurine-thymine and guanine-cytosine base pair. The ‘extra’ 2-amino group of diaminopurine is shown in bold.

340 in their own right in terms of molecular recognition and supramolecular assembly, and this molecule may even tell us something about the genetic material itself, the DNA molecule and its evolutionary prebiotic origin [30].

Acknowledgements This work was supported by the Danish National Research Foundation.

References 1. Nielsen, P.E., Egholm, M., Berg, R.H., and Buchardt, O., Science 254 (1991) 1497. 2. Egholm, M., Buchardt, O., Nielsen, P.E., and Berg, R.H., J. Amer. Chem. Soc. 114 (1992) 1895. 3. Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S.M., Driver, D.A., Berg, R.H., Kim, SK., Norde αn, B., and Nielsen, P.E., Nature 365 (1993) 556. 4. Hyrup, B. and Nielsen, P.E., Bioorg. Biomed. Chem. 4 (1996) 5. 5. Nielsen, P.E., Egholm, M., and Buchardt, O., J. Mol. Recognition. 7 (1994) 165. 6. Cherny, D.Y., Belotserkovskii, B.P., Frank-Kamenetskii, M.D., Egholm, M., Buchardt, O., Berg, R.H., and Nielsen, P.E., Proc. Natl. Acad. Sci. USA. 90 (1993) 1667. 7. Jensen, K.K., Ørum, H., Nielsen, P.E., and Nordén, B., Biochemistry 36 (1997) 5072. 8. Brown S.C., Thomson S.A., Veal J.M., and Davis, D.G., Science 265 (1994) 777. 9. Rasmussen, H., Kastrup, J.S., Nielsen, J.N., Nielsen, J.M., and Nielsen, P.E., Nature Structural Biology 4 (1997) 98. 10. Eriksson, M. and Nielsen, P.E., Nature Structural Biology 3 (1996) 410. 11. Betts, L., Josey, J.A., Veal, J.M., and Jordan, S.R., Science 270 (1995) 1838. 12. Knudsen, H. and Nielsen, P.E., Anti Cancer Drugs 8 (1997) 113. 13. Hanvey, J.C., Peffer, N.C., Bisi, J.E., Thomson, S.A., Cadilla, R., Josey, J.A., Ricca, D.J., Hassman, C.F., Bonham, M.A., Au, K.G., Carter, S.G., Bruckenstein D.A., Boyd, A.L., Noble S.A., and Babiss, L.E., Science 258 ( 1992) 1481. 14. Knudsen, H. and Nielsen, P.E., Nucleic Acids Res. 24 (1996) 494. 15. Gambacorti-Passerini, C., Mologni, L., Bertazzoli, C., Marchesi, E., Grignani, F., and Nielsen, P.E., Blood 88 (1996) 1411. 16. Nielsen, P.E., Egholm, M., and Buchardt, O., Gene 149 (1994) 139. 17. Norton, J.C., Piatyczek, M.A., Wright, W.E., Shay, J.W., and Corey, D.R., Nature Biotechnology 14 (1996) 615. 18. Koppelhus, U., Zachar, V., Nielsen, P.E., Liu, X., Eugen-Olsen, J., and Ebbesen, P., Nucleic Acids Res. 25 (1997) 2167. 19. Carlsson, C., Dulay, M.T., Zare, R.N., Noolandi, J., Nordeαn, B., Nielsen, P.E., Zielenski, J., Tsui, L.-C., and Jonsson, M., Nature (corresp) 380 (1996) 207. 20. Perry-O’Keefe, H., Yao, X.-W., Coull, J., Fuchs, M., and Egholm, M., PNA Pre-Gel Hybridization, An Alternative to Southern Blotting. Proceedings of the National Academy of Sciences, USA 93, 14670–14675 (1996). 21. Ørum, H., Jørgensen, M., Koch, T., Nielsen, P.E., Larsson, C., and Stanley, C., Biotechniques 19 (1995) 472. 22. Seeger, C., Batz, H.-G., and Ørum, H., BioTechniques 23 (1997) 512. 23. Wang, J., Palecek, E., Nielsen, P.E., Rivas, G., Cai, X., Shiraishi, H., Dontha, N., Luo, D., and Farias, M.A., J. Amer. Chem. Soc. 118 (1996) 7667. 24. Weiler, J., Gausepohl, H., Hauser, B., Jensen, O.N., and Hoheisel, J.D., Nucl. Acids Res. 25 (1997) 2792. 25. Haaima, G., Lohse, A., Buchardt, O., and Nielsen, P.E., Angewandte Chemie 35 (1996) 1939. 26. Peyman, A. et al., Ange. Chem. Int. Ed. Engel. 35 (1997) 2636. 27. Egholm, M., Christensen, L., Dueholm, K., Buchardt, O., Coull, J., and Nielsen, P.E., Nucleic Acids Res. 23 (1995) 217.

341 28. Eldrup, A.B., Dahl, O. and Nielsen, P.E.. J. Amer. Chem. Soc. 119 (1997) 11116. 29. Haaima, G., Hansen, H.F., Christensen, L., Dahl, O., and Nielsen, P.E., Nucleic Acids Res. 25 (1997) 4639. 30. Böhler, C., Nielsen, P.E., and Orgel, L.E., Nature 376 (1995) 578.

115 Receptor based design, synthesis and biology of novel somatostatin analogs M. GOODMAN, X. JIANG, J. IGARASHI, H. LI, T.-A. TRAN and R.-H. MATTERN Department of Chemistry & Biochemistry, University of California at San Diego, La Jolla, CA 92093-0343, USA

Introduction Somatostatin (SRIF), a cyclic peptide originally isolated from ovine hypothalamus [1], is a potent physiological inhibitor of growth hormone (GH) (Figure 1). In addition to GH, SRIF has inhibitory effects on many other hormones including insulin, glucagon and gastric acid. The actions of SRIF are mediated by specific, high affinity membrane receptors. Up to date, five SRIF receptor subtypes, denoted sst1-5, have been identified in various species by molecular cloning [2]. They are G-protein coupled receptors (GPCRs). Development of subtype-selective SRIF analogs is a challenge which will not only facilitate the studies of pharmacological properties of individual sst receptors but also have important clinical applications in the treatment of metabolic disorders, cancer and CNS diseases. In this presentation, based on the results obtained from mutagenesis studies [3,4] we modeled sst receptors by using rhodopsin as a template and docked SRIF analogs into the binding site. Based on the structure of the receptorligand complex, we designed and synthesized novel SRIF analogs with specific subtype selectivities.

Results and discussion The sequences of human sst1-5 (hsst1-5) were analyzed by TMpred to identify the transmemberane regions. The transmemberane regions of rhodopsin were then aligned with the corresponding regions of hsst1-5. Modeling hsst2 and hsst5 were carried out by using InsightII/Discover (Biosym/MSI, San Diego) program [5]. Each TM helix was built individually SRIF Sandostatin L-363,301

Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr -Ser-Cys]-OH D-Phe-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr(ol) c[Pro 6-Phe 7-D-Trp 8-Lys9-Thr 10 -Phe 11 ]

Figure 1. Somatostatin and its synthetic analogs. The superscript numbers in L-363,301 denote the equivalent position in somatostatin.

342 Y. Shimonishi (ed): Peptide Science – Present and Future, 342–345 © 1999 Kluwer Academic Publishers, Printed in Great Britain

343 as a right-hand α -helix. The side chain orientations of every amino acid in the helix were adjusted to optimize the nonbond interactions. The backbones of minimized helices were superimposed on the corresponding backbones in rhodopsin to form the helical bundle. At this point, each helix was subjected to rotation in order to optimize the interactions with the neighboring helices and with the membrane content. The helical bundle was minimized before the extracellular loop III (ECL III) was built between TM helix 6 and 7. Finally, the simulated hsst2 and hsst5 were fully minimized. In order to investigate specific interactions between the receptor and its ligands, SRIF analogs such as L-363,301 (Figure 1) and Sandostatin (Figure 1) were docked into the binding sites of hsst2 and hsst5. The ligand in its conformation as found in solution was first manually docked into the binding site using an energy grid to monitor the nonbond interactions. The ligand was then randomly translated, rotated and energy minimized to locate its best position in the binding site. Finally, molecular dynamics (500 ps) was carried out to simulate the interactions between the ligand and its receptor. The structure of hsst2/L-363,301 complex is shown in Figure 2. The ligand was inserted into the center pole where the binding site is located. ECL III is located on the top of the binding pocket. From our previous studies, we knew that Lys 9 is critical to the bioactivity as well as the hydrophobic side chains of Phe 7, D -Trp 8 and Phe 11. Using our model of hsst2/L-363,301 complex, we defined specific interactions between the ligand and its receptor. In order to study the properties of hsst receptors further, the electrostatic potentials of the receptor in the membrane environment were calculated by using a continuum model. Upon examining the electrostatic potential surface, we found very different electrostatic properties of ECL III in hsst2 and hsst5. Therefore, we envision that subtype selective somatostatin analogs can be

Figure 2. The structure of hsst2/L-363,301 complex.

344 c[Xaa 6-Phe 7 -D-Trp 8 -Lys 9-Thr 10 -Phe 11 ] X a a = N-benzylglycine (Nphe) N-(S)- α-methylbenzyl glycine ((S)-β -Me-Nphe) N-(R)- α-methylbrnzyl glycine ((R)-β -Me-Nphe) Figure 3. Structures of peptoid containing SRIF analogs. The superscript numbers in the structure denote the equivalent position in somatostatin.

developed by modifying the corresponding residues in the ligands that interact with the binding pocket and ECL III. In hsst2, ECL III is relatively neutral. We designed and synthesized a family of peptoid containing SRIF analogs (Figure 3) in which aralkyl peptoid residues are introduced at position 6 of L-363,301 to enhance the interactions towards hsst2. On the other hand, in hsst5, ECL III is highly acidic. Based on this fact, our rationale was to incorporate basic residues such as Lys at the exocyclic position of RC-121 to promote the selectivity towards hsst5 (Figure 4). Because of its better selectivity as compared to sandostatin towards hsst2 and hsst5, we chose RC-121 as the parent compound in this study. The binding affinities of our peptoid containing SRIF analogs towards hsst15 are shown in Table 1. The parent compound L-363,301 has high affinities to hsst2 and hsst5 (0.76 nM and 7.6 nM respectively). When Nphe is introduced at position 6, its affinity to hsst2 is slightly lowered to 2.95 nM while its affinity to hsst5 is decreased by 10 fold. This binding profile shows the selectivity towards hsst2. A more dramatic effect is observed when (R) β MeNphe is incorporated into the structure. Its affinity to hsst2 is better than the Nphe analog while its affinity to hsst5 is reduced by another 4 fold to 288 nM. The (S) βMeNphe analog does not increase its selectivity to hsst2 compared to the L-363,301 and Nphe analogs. In in vivo studies, peptoid containing analogs strongly inhibit GH release as L-363,301. In contrast, they do not inhibit insulin release compared to L-363,301 [6]. These novel features both in vitro and Sandostatin RC-121 96149 Figure 4. Table 1.

D-Phe-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Thr(ol) D-Phe-c[Cys-Phe-D-Trp-Lys-Val-Cys]-Thr-NH 2 D-Phe-c[Cys-Phe-D-Trp-Lys-Val-Cys]-Lys-NH 2

Designed SRIF analogs target hsst5. Binding affinities of SRIF analogs towards hsst1–5 (Kd (nM) ± SEM)*.

L-363,301 Nphe 6 L-363,301 (R)βMeNphe 6 L-363,301 (S)βMeNphe 6 L-363,301

hsst1

hsst2

> > > >

0.76 2.95 2.04 6.17

1000 1000 1000 1000

± ± ± ±

0.05 0.71 0.75 1.34

hsst3

hsst4

hsst5

525 ± 37 282 ± 27 407 ± 50 339 ± 68

> 1000 > 1000 > 1000 > 1000

7.6 ± 1.5 69 ± 3 288 ± 7 115 ± 3

* These bioassays were carried out at Novartis GmBH, Basel, Switzerland.

345 Table 2.

Binding affinities of SRIF analogs towards hsst1–5 (Ki (nM) ± SEM)*.

Sandostatin RC-121 96149

hsst1

hsst2

hsst3

hsst4

hsst5

875 ± 180 >l000 >1000

0.57 ± 0.08 1.7 ± 0.5 115 ± 18

26.8 ± 7.7 >1000 >1000

>1000 >1000 >1000

6.8 ± 1.0 13.1 ± 1.2 26.6 ± 8.8

*These bioassays were carried out at Biomeasure Inc., Milford, USA.

in vivo confirmed our predictions of the interactions between hsst2 and the peptoid containing SRIF analogs. The binding affinities of designed hsst5 selective compounds are shown in Table 2. The parent analog RC-121 binds strongly to hsst2 (1.7 nM) compared to hsst5 (13.1 nM). Our designed analog 96149 exhibits reversed binding profile between hsst2 and hsst5. Its affinity to hsst2 is decreased nearly 100 fold compared to RC-121 while its affinity to hsst5 is only 2 fold weaker. Thus, 96149 represents our first designed hsst5 selective analog.

Conclusion In this approach, we modeled somatostatin receptor-ligand complexes. Based on the information obtained from the structures of the ligand-receptor complexes, we designed and synthesized novel somatostatin analogs with enhanced selectivities.

Acknowledgement We wish to thank Drs Barry Morgan and John Taylor at Biomeasure Inc., Dr Daniel Hoyer at Novartis and Dr Michel Afargan at Peptor Ltd. for the biological assays. We also wish to thank Dr Jim Briggs for helpful discussions. This work was supported by National Institute of Health (NIHDK-15410).

References 1. 2. 3. 4. 5.

Brazeau, P., Vale, W., Burgus, R., et al., Science, 129 (1972) 77–79. Reisine, T. and Bell, G.I., Endocr. Rev., 16 (1995) 427-442. Schertler, G., Villa, C., and Henderson, R., Nature, 362 (1993) 770. Kaupmann, K., Weber, H.P., Mattes, H., and Lubbert, H., EMBO Journal, 14 (1995) 727-735. Igarashi, J., Jiang, X., and Goodman, M., Proceeding of the First International Symposium, in this paper we show an equivalent approach for hsst3-ligand complex. 6. Tran, T.-A., Mattern, R.H., Goodman, M., et al., J. Med. Chem., 41(1998) 2679-2685.

116 NMR structure of mastoparan-X bound to G protein α subunit H. KUSUNOKI 1,2, T. KOHNO¹ , K. SATO¹, T. TANAKA² and K. WAKAMATSU 2,3 ¹ Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan ² Faculty of Engineering, Gunma University, Kiryu, Gunma, 376, Japan ³ The Institute of Physical and Chemical Research, Wako, Saitama 351–01, Japan

Introduction Mastoparans, a family of tetradecapeptides from wasp venom, are known to activate GTP-binding regulatory proteins (G proteins) [1], and have been used as low-molecular-weight mimetics of G protein-coupled receptors in the investigations of the G protein’s roles in signal transductions as well as the interaction between G proteins and receptors. To obtain insight into the G protein-receptor interactions and to design new potent and selective G protein activators, it is important to determine the detailed conformation of mastoparans bound to G proteins. Conventional two-dimensional (2D) transferred nuclear Overhauser effect (TRNOE) spectroscopies, which are quite useful for determining a peptide’s conformation bound to a large protein, however, have a serious drawback of low spectral resolution; proton resonances of free peptide molecules through which the conformation of a bound molecule is monitored, are often not well separated from each other to unambiguously assign each NOE cross peak. Here we report the precise conformation of mastoparan-X (MP-X) determined by three-dimensional (3D) heteronuclear TRNOE spectroscopies using MP-X uniformly enriched with 13 C and 15 N.

Results and discussion NMR sample preparation Non-enriched MP-X was chemically synthesized by standard solid-phase methodology. MP-X uniformly enriched with 15 N and/or 13 C was expressed in E. coli cells and purified using a ubiquitin fusion protein system as described [5]. Peptides were dissolved in an NMR sample buffer [90% H2 O/10% D2 O or 99.9% D 2 O containing 10 mM acetic acid-d 4 (pH 6.0) and 1 mM DTTd 10 ]. Sample pH (direct pH meter reading) was adjusted to 6.1±0.1. Recombinant polyhistidine-tagged Gilα (Gil α ) was expressed in E. coli cells and purified as described [2]. 346 Y. Shimonishi (ed): Peptide Science – Present and Future, 346-350 © 1999 Kluwer Academic Publishers, Printed in Great Britain

347 NMR spectroscopy All NMR measurements were performed either on a Bruker AMX-500 or ARX-400 spectrometers at 20°C. MP-X resonances were assigned by 3D HNCACB, 3D CBCA(CO)NH, 3D HNCO, 2D ¹H–13 C HSQC, and 3D 13 C edited TOCSY-HSQC spectra of uniformly 13 C /15 N-enriched MP-X and by 2D ¹H– l 5N HSQC and 3D 15 N-edited TOCSY-HSQC spectra of uniformly 15 N-enriched MP-X. TRNOE spectra (τ m = 100 ms) of MP-X (5 mM) in the presence Gilα (0.25 mM) were recorded by 2D NOESY of non-enriched MP-X, HMQC-NOESY-HMQC 3D 15 N-edited NOESY-HSQC and 3D 15 N-edited of uniformly 15 N-enriched MP-X, and 3D 13 C-edited NOESY-HMQC of uniformly 13 C/ 15 N-enriched MP-X. Distance constraints and structure calculations Inter-proton distance restraints were obtained from the homonuclear and heteronuclear NOESY spectra with a mixing time of 100 ms. All NOE data were divided into three classes: strong, medium and weak, corresponding to distance upper limits of 2.7,3.6, and 4.5 Å in the inter-proton distance restraints. Structure calculations were carried out by the X-PLOR 3.1 program with simulated annealing protocols using 151 distance constraints. Impact of isotope-enrichment of MP-X on the resolution of peptide resonances ¹H– 15 N and ¹H–13 C HSQC spectra of uniformly isotope-enriched MP-X are shown in Figure 1. A number of resonances having similar ¹H chemical shifts are well resolved in the heteronuclear dimensions, indicating that the isotopeenrichment and the edition of resonances by heteronuclear chemical shifts are very useful for resolving resonances of random-coil peptides.

Figure 1.

¹H– 15N (left) and ¹H– 13 C HSQC (right) spectra of uniformly isotope-enriched MP-X.

348 TRNOE In a NOESY spectrum of MP-X alone (τ m = 100 ms), only a few weak NOEs were observed (data not shown), being consistent with the expectation that the magnitude of NOEs is close to zero for small peptides like MP-X. In contrast, a TRNOESY spectrum of MP-X in the presence of Gil α showed many strong negative NOEs (Figure 2) in agreement with the previous report [3]. These observations indicate that these NOEs reflect the conformation of MP-X molecules in the G protein-bound state.

Assignment of NOEs 2D and 15 N-edited 3D TRNOESY spectra (amide-aliphatic region) in the presence of Gilα are compared in Figure 2. In the 2D spectrum (left), chemical shift degeneracy of amide protons prevented unambiguous identification of each NOE cross peaks, especially those involving Ala8–Ala10. However, these cross peaks are distinctly resolved in separate planes of 3D spectrum (right). NOE cross peaks in the aliphatic region, which are also severely overlapped in the proton 2D spectrum, were unambiguously assigned in a 13 C-edited TRNOESY spectrum (not shown). Taken together, by inspecting heteronucleusedited TRNOE spectra we could newly identify 23 inter-residue and 12 intraresidue NOE cross peaks.

Figure 2. 2D (left) and 3D ( τ m = 100 ms).

15N-edited

(right) TRNOE spectra of MP-X in the presence of Gil α

349

Figure 3. Best-fit 14 structures of MP-X bound to Gila calculated using 2D and 3D TRNOE data (left) and 2D TRNOE data alone (right).

Description of calculated structures Figure 3 (left) shows the final 14 structures with the lowest energies, for which none of the NOE distance violation was larger than 0.5 Å. The peptide is shown to adopt α-helical conformation except for the N-terminal two residues. For comparison, structures calculated using 2D TRNOE data alone are also shown (right). The convergence of the structures was greatly improved by incorporating 3D data, and the backbone RMSD values from the averaged coordinate positions (for Trp3-Leu14) reduced to 0.27 ± 0.07 Å (2D plus 3D) from 1.70 ± 0.41 Å (2D). The obtained α -helical structure is in good agreement with the known structure-activity relationships of mastoparans. For example, the extent of activation of G proteins by mastoparans decreases when they are designed to have low amphiphilc moments [4]. In conclusion, we have demonstrated that the heteronuclear multidimensional TRNOE experiments of peptides uniformly enriched with 13 C and 15 N are powerful for determining detailed conformations of peptides bound to large proteins. We believe that our present study will be useful for the development of peptides that specifically regulates G proteins and for the understanding of receptor-G protein interactions.

Acknowledgements This work was supported in part by Ministry of Education, Science, Sports and Culture, Japan, New Energy and Industrial Technology Development Organization (NEDO), and the Institute of Physical and Chemical Research (RIKEN).

350 References 1. Higashijima, T., Uzu, S., Nakajima, T., and Ross, E.M., J. Biol. Chem. 263 (1988) 6491. 2. Tanaka, T., Kohno, T., Kinoshita, S., Mukai, H., Itoh, H., Ohya, M., Miyazawa, T., Higashijima, T., and Wakamatsu, K., J. Biol. Chem. 273 (1998) 3247. 3. Sukumar, M. and Higashijima, T., J. Biol. Chem, 267 (1992) 21421. 4. Higashijima, T., Burnier, J., and Ross, E.M., J. Biol. Chem. 265 (1990) 14176. 5. Kohno, T., Kusanoki, H., Sato, K., and Wakamatsu, K., J. Biomol. NMR 12 (1998) 109.

117 Peptides and molecular recognition: head-to-tail selfassembly, formation of amphipathic surfaces and recognition of anionic superhelices E. GIRALT¹, I. DALCOL¹, C. FERRER¹, P. GOROSTIZA², T. HAACK¹, A.D. HAMILTON³, D. LUDEVID4 , E. NICOLAS¹, M.W. PECZUH³, X. SALVATELLA¹, J. SÁNCHEZ-QUESADA5 , F. SANZ² ¹Departament de Química Orgànica and ²Departament de Químicà Física, Universitat de Barcelona, Spain ³Department of Chemistry, University of Yale, USA 4 Departament de Genètica Molecular, CID-CSIC, Spain 5 Departamento de Química Orgánica, Universidad Autónoma de Madrid, Spain

Introduction Non-covalent interactions govern protein folding and self-assembling processes in Nature. Efficient ‘de novo’ design of homo- and heteromolecular assemblies depends especially on our ability to control non-covalent interactions such as van der Waals packing, hydrogen bonding, and electrostatic forces. Self-assembling synthetic peptides are excellent model systems for getting an insight into these phenomena.

Results and discussion Uteroglobin is a 140-residue protein made by two identical chains of 70 residues each crosslinked together in an antiparallel way via two disulfide bridges. Uteroglobin is a highly helical protein; its eight α -helix segments define a large cavity where sizeable hydrophobic compounds such as progesterone or retinol can be incorporated with high affinity. We and others have been active in the study of the conformation tendencies of different segments of uteroglobin [1–3]. In this paper, however, we will focus on the study of uteroglobin dimerization. Head-to-tail dimeric proteins, such as uteroglobin, provide an excellent model system for the investigation of self-assembly mediated by helix-helix packing interactions. We have recently found that two short cysteine-containing peptides (1 and 2 ) that mimic the primary structure of the channel-like domain of reduced rabbit uteroglobin self-assemble, inducing spontaneous selective heterodimerisation. Thus, when an equimolar mixture of 1 and 2 were air-oxidized in an aqueous buffer at pH 7.1 in the presence of 50% hexafluoroisopropanol, a single oxidation compound 3 was formed. The reaction was complete after 1 h at r.t. This behaviour contrasts with that of the 351 Y. Shimonishi (ed): Peptide Science – Present and Future, 351–356 © 1999 Kluwer Academic Publishers, Printed in Great Britain

352

Figure 1. Uteroglobin

segments.

same equimolar mixture of 1 and 2 when the oxidation reaction takes place in 100% aqueous buffer, i.e. in the absence of hexafluoroisopropanol. All these data can be interpreted on the basis that sequences 1–14 (1 ) and 48–70 (2) of uteroglobin contain all the information required to govern the head-to-tail selfassembly of the native protein. 1 and 2 span, respectively, the first and the fourth helices of the uteroglobin monomer. This means that only these two helices are sufficient to induce the antiparallel dimerization of the protein. In our model system the addition of an helicogenic solvent, such as HFIP, is required to induce helix formation and allow the two peptides to express the structural information already present in their sequence. One can speculate that in the entire protein the rest of the molecule plays a similar role to that of HFIP, either through other helix–helix contacts or, simply, by providing a more hydrophobic microenviroment. Maize γ -zein may be analyzed as consisting of three major domains: i) an N-terminal domain with a proline-rich repetitive structure ((VHLPPP) 8 ); ii) a central region whose structure is of the P-X type; and iii) a C-terminal cysteinerich domain. In maize endosperm γ -zein is found localized at the periphery of protein body organelles [4]. Geli et al. [5] have recently shown that this proline-rich domain is crucial for the targeting of the entire protein to the endoplasmic reticulum as well as for the formation of protein bodies. We have in the past applied CD as well as NMR [6,7] to the conformational analysis of this region and shown that it tends to adopt left -handed polyproline II type helices. Now, using Atomic Force Microscopy, we have found that the synthetic peptide (VHLPPP)8 self-assembles on graphite surfaces forming amphipathic monolayers with a measured height of 1.2 mm. that corresponds to the diameter of a single polyproline II helix. Organised domains of nanofibrils (5–7 mm wide) are also observed. This fibrile formation has been confirmed by Transmission Electron Microscopy. The biological relevance of these results comes from the fact that, taking into account the alternating hydrophobic character of V and L vs. H side chains, it can be easily anticipated that the observed monolayers formed by (VHLPPP) 8 must be amphipathic. This suggests a possible mechanism for the γ -zein targeting and protein body formation that would start with the bidimen-

353

Figure 2. AFM images of a proline-rich repetitive peptide (Ac-(VHLPPP) 8 -NH2 ) aggregated on a pyrolitic graphite surface.

sional aggregation of its N-terminal domain at the amphipathic surface of the endoplasmic reticulum followed by a subsequent covalent cross-linking via cysteine oxidation. The last topic from our contribution is related to our recent efforts towards the rational design of artificial receptors of protein surfaces. We have recently reported that chiral bicyclic guanidinium tetramers (4) (Figure 3) self-assemble around bivalent sulfate templates forming double strand helices [8]. The carboxylate-guanidinium interaction is one of the most efficient non-covalent interactions since it combines electrostatic attraction with the concomitant

Figure 3. Tetraguanidinium ion (4) and peptides (5–8) used on the anionic superhelix recognition experiments.

354 formation of two hydrogen bonds. We report here on the extension of our previous work on guanidinium-sulfate interactions toward the use of tetraguanidinium compounds for the molecular recognition of peptides with carboxylic groups in their side-chains [9]. Peptides 5–7 (Figure 3) were synthesized using standard Fmoc/t Bu solid-phase procedures. Conformational analysis by CD in methanol/water 9: 1 of peptides 5–7 both alone and in the presence of increasing amounts of the tetraguanidinium ions 4 leads to the following conclusions: i) peptide 5 binds strongly to 4 with a binding constant, determined by CD, of ca. 3.4 × 10 5 and with 1 : 1 stoichiometry (determined via a Job CD titration); ii) decreasing the number of aspartic residues (peptides 6 and 7 ) causes a decrease of the affinities towards tetraguanidinium receptors; iii) 2D-NMR experiments show that complexation of 5 and 4 results in better chemical shift dispersion of the protons from aspartic acid residues (Figure 4); iv) both CD and NMR data support the idea that complexation of 5 and 4 causes a significant increase of the helicity of the peptide. We have recently extended the experiments described above (performed in methanol/water 9: 1) to pure water. Again, the spectroscopic data show that for the complex between 5 and 4: i) there is complexation; and ii) there is a certain amount of helix induction when the complex is formed.

Figure 4. Expansion of a TOCSY 2D-NMR spectrum from a 1 : 1 mixture of 4 and 5 in MeOH– H2 O 9 : 1 at 288 K.

355 Our design of peptide 5 takes into account that in right-handed α -helical structures the side-chains at positions i/i + 3 /i + 6 /i + 9 define a, let us say, ‘lefthanded superhelix’. In the same manner those side-chains at position i/i + 4 /i + 8 /i + 12 will define a ‘right-handed superhelix’. Our results show that the anionic superhelix defined by the charged aspartic acid side-chains at the i + 3n positions of 5 is recognized and stabilized by the synthetic tetraguanidinium receptor. A computer-based data base search has allowed several proteins presenting the structural motif D/E-X-X-D/E-X-X-D/E-X-X-D/E in helical structures located at their surfaces to be identified. Among them, because of its great interest as a possible therapeutic and diagnostic target, we have chosen to work with the tumour suppressor protein p53. Our first approach to the molecular recognition of p53 surface using artificial receptors is based on the presence of four copies of the sequence 8 at the surface of the protein. Each of these copies adopts an α -helical structure (10). We have synthesized peptide 8 on a solid-phase using a standard Fmoc/ t Bu strategy. 2D-NMR data agree with peptide 8 adopting a nascent helical structure in methanol/water mixtures. Work on the study of recognition and/or helix induction on complexation of 8 with tetraguanidinium ions is in progress.

Acknowledgements We thank Carmen López, David Bellido, Joan Mendoza and Alejandro del Giorgio at the Serveis Científico-Tècnics of the University of Barcelona for technical assistance with transmission electron microscopy and CICYT (PB95–1131), NATO and Generalitat de Catalunya (Centre de Referència en Biotecnologia) for financial support. We also acknowledge the use of the NMR facilities of the Serveis Científico-Tècnics of the University of Barcelona.

References 1 . Nicolás, E., Bacardit, J., Vilaseca, M., and Giralt, E., ‘Peptides 1994. Proceedings of the TwentyThird European Peptide Symposium’. (H.L.S. Maia, Ed.), Escom, Science Publishers, Leiden, The Netherlands, 1995, pp. 570–571. 2 . Nicolás, E., Bacardit, J., Ferrer, T., and Giralt, E., Lett. in Peptide Science, 2, 353–362. (1995). 3 . Improta, S., Pastore, A., Mammi, S., and Peggion, E., Biopolymers 34 (1994) 773 and references therein. 4 . Ludevid, M.D., Torrent, M., Martinez-Izquierdo, J.A., Puigdomènech, P., and Palau, J., Plant Mol. Biol. 3 (1984) 605. 5 . Geli, M.I., Torrent, M., and Ludevid, M.D., Plant Cell 6 (1994) 1911. 6 . Rabanal, F., Ludevid, M.D., Pons, M., and Giralt, E., Biopolymers. 33, 1019–1028 (1993) (invited review). 7 . Pans, M., Feliz, M., Celma, C., and Giralt, E., Magnet. Res. Chem. 25 (1987) 402. 8 . Sanchez-Quesada, J., Seel, Ch., Prados, P., Mendoza, J., Dalcol, I., and Giralt, E., J. Am. Chem. Soc., 118, (1996) 277–278.

356 9 . A preliminary account on this work has been recently published: Peczuh, M.W., Hamilton, A.D., Sánchez-Quesada, J., de Mendoza, J., Haack, T., and Giralt, E., J. Am. Chem. Soc., 119 (1997) 9327-9328. 10. Clore, G.M., Omichinski, J.G., Sakaguchi, K., Zambrano, N., Sakamoto, H., Appella, E., and Gronenborn, A.M., Science 265 (1994) 386.

118 Non-immobilised ligand interaction assay (NILIA) – the CD approach G. SILIGARDI and R. HUSSAIN EPSRC Chiroptical & ULIRS Optical Spectroscopy Centres, King’s College London, Department of Pharmacy, Manresa Road, London SW3 6LX, U.K Email: [email protected].

Introduction Aromatic aa residues are commonly found at the host/ligand interface providing excellent in situ molecular probes in protein receptors, catalytic sites, peptides, substrates, cofactors, inhibitors and drugs. The CD of aromatic residues can be used, therefore, to screen qualitatively and quantitatively ligand binding in vitro as well as to monitor enzymatic catalysis. For a single binding site, the binding constant (Ka) or dissociation constant (Kd) can be determined by analysing the CD data at a single wavelength by non-linear regression method. The negative solution of the quadratic equation derived from the general equilibrium reaction in which the CD intensity is proportional to the concentration of each species is used in the analysis. Here we present examples of host/ligand complexes studied in solution by NILIA-CD. Results and discussion CD spectroscopy is a very sensitive technique to protein conformational changes. AmiC is a bacterial protein that binds acetamide (acm) and butyramide (btm) ligands. The acm and btm binding to AmiC were sensed and discriminated just by one residue (Trp285) out of 367. For a ligand:protein complex, the thermal stability represented by the melting temperature Tm correlate with the ligand binding affinity. For the amide : AmiC complexes the thermal stability and the amide binding affinity were found to be AmiC: acm > AmiC:btm > mutant AmiC: btm. The CD spectra of AmiC:btm and mutant AmiC:btm were converted to that of AmiC:acm on adding acm to the btm:protein complexes (O’Hara, Pearl, Siligardi, manuscript in preparation). Upon addition of acm to the btm and mutant AmiC:btm complexes, the btm ligand was readily displaced by acm. Competition studies are very useful because they help the spectroscopic analysis in particular the assignments of the CD bands associated with the ligand binding species. This is critical for drug:protein binding where more than one ligand species might be bound to the host protein. The binding of tri-N-acetylchitothriose inhibitor (NAG3) to hen white-egg lysozyme was detected through the changes of the CD associated with the 357 Y. Shimonishi (ed): Peptide Science – Present and Future, 357–359 © 1999 Kluwer Academic Publishers, Printed in Great Britain

358

Figure 1. CD spectra of lysozyme/NAG3 at various molar ratios. Kd = 1.3 µ M was calculated using a non-linear regression analysis of CD data at 294 nm.

tryptophan side-chain of lysozyme. From the crystal structure (1hew.pdb), three Trp residues are interacting with NAG3 (Figure 1). Kd = 1.3 µ M was calculated using a non linear regression analysis (Figure 1). The 1:1 stoichiometry of the complex was determined using a lysozyme concentration of 445 µ M in the titration with NAG3 (Figure 1). In the backbone region (data not shown) the small CD changes observed between the lysozyme and the lysozyme:NAG3 [1:3] complex were assigned to the 1Bb and 1Ba transitions of the Trp residues and not to conformational changes of the lysozyme on binding to NAG3. Conclusions NILIA-CD can be applied to any molecule that possess chromophores that absorbs light in the UV region (peptide, peptidomimetic, nucleic acid, carbohydrate). Under these conditions the ligand-host complex can be monitored without immobilising any of the components and no radio-labelling is required

359 NILIA-CD is fast and enables us to determine the binding/dissociation constant and collect conformational information that, together with structural data from other techniques, contribute significantly towards the building of the 3D model of the complex.

Acknowledgements GS would like to thank the American Peptide Society for the travel award.

119 Characterization and evolutionary aspect of a Trimeresurus flavoviridis serum inhibitor, PLI-I, against its venom basic phospholipase A 2 isozymes I. NOBUHISA1 , S. INAMASU 1, M. NAKAI 1 , A. TATSUI 1 , T. MIMORI 1, T. OGAWA 1 , Y. SHIMOHIGASHI 1, Y. FUKUMAKI 2, S. HATTORI3, H. KIHARA 4 and M. OHNO 5 1 Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan 2 Institute of Genetic Information, Kyushu University, Fukuoka, Japan 3 Institute of Medical Science, University of Tokyo, Kagoshima, Japan 4 Biotechnology Research Laboratory, Takara Shuzo Co. Ltd., Shiga, Japan 5 Kumamoto Institute of Technology, Kumamoto, Japan

Introduction The binding proteins against Trimeresurus flavoviridis (habu snake, crotalinae) venom phospholipase A2 (PLA 2 ) isozymes were fractionated from their sera on four columns each being conjugated with one of four different isozymes. PLI-I, which inhibits three basic venom PLA2 s, has been isolated and most of its amino acid sequence has been determined. The cDNA and gene encoding PLI-I were then cloned and sequenced. Based on the structures of genes of PLA 2 inhibitors, their evolutionary origins are discussed.

Results and discussion Inhibitory effect of PLI-I toward basic venom Lys-49-PLA2 isozyme (BP-I) was examined with 2-arachidonoylphosphatidylcholine-dipalmitoylphosphatidylcholine (3:1) liposomes encapsulating carboxyfluorescein (CF) as substrate. CF leaked out by BP-I decreased from 40 to 10% in the presence of 20-fold amount of PLI-I. The liberation of arachidonic acid by BP-I was also reduced to 53% in the presence of PLI-I. Myotoxicity caused by BP-I was completely inhibited in the presence of PLI-I 16 times as much as BP-I. The cDNA clone for PLI-I was isolated from T. flavoviridis liver cDNA library. It consisted of a coding region of 603 nucleotides, a 5'-untranslated region (UTR) of 39 nucleotides, and a 3'-UTR of 130 nucleotides. PLI-I cDNA encoded a protein of 200 amino acid residues, including a signal peptide of 19 amino acids. The putative primary structure from the cDNA is completely in accord with the partial amino acid sequences determined from the protein. One putative N-linked glycosylation site was predicted at Asn 157. The amino acid sequence of PLI-I was compared with those of other snake venom PLA2 360 Y. Shimonishi (ed): Peptide Science – Present and Future, 360-361  1999 Kluwer Academic Publishers, Printed in Great Britain

361 inhibitors. Although PLI-I exhibited high and low degrees of sequence identity to Crotalus neutralizing factor (CNF) (83%) from C. durissus terrificus serum and 31 kDa (64%) and 25 kDa subunits (28%) from Naja naja kaouthia serum, the 16 half-cystine residues are all conserved. In addition, since PLI-I has two repeated units composed of 90 amino acid residues, as found for CNF, it can be said to belong to CNF-like inhibitor family. On the other hand, no sequence identity was seen between PLI-I and PLI-A or PLI-B which are inhibitory serum proteins against highly lipolytic Asp-49-PLA2 from T. flavoviridis venom. The gene encoding PLI-I was isolated from T. flavoviridis genomic DNA library and sequenced. It was composed of five exons divided by four introns, being in contrast with four exons and three introns of the genes encoding PLI-A and PLI-B. CNF-like inhibitors are structurally similar to urokinase-type plasminogen activator receptor (uPAR), Ly-6, CD59, and neurotoxins which contain unit(s) composed of approximately 90 amino acid residues [1]. A similar gene structure is also found for PLI-I, Ly-6, CD59, and uPAR, that is, one unit consisting of 90 amino acid residues is encoded over two exons. However, in spite of the structural similarity of these genes, their products have various functions. The conserved positions of half-cystine residues among them may predict similar tertiary structures. Since both CD59 and neurotoxins have a three-finger motif in their three dimensional structures [2], it is likely that PLI-I has two threefinger motifs. Their inherent functions should be determined by the internal sequences of the loop regions between half-cystine residues because of low sequence similarity. Our recent studies showed that crotalinae snake venom gland PLA 2 isozyme genes have evolved via accelerated nucleotide substitutions only in the mature protein coding region to fulfill their diverse physiological functions [3]. However, occurrence of coevolution of their inhibitors is not likely because gene organizations are different between PLI-I and PLI-A (or PLI-B) and the former contains three-finger motifs while the latter has a carbohydrate recognition domain. Thus, it could be assumed that the evolutionary origin of PLI-I differs from PLI-A and PLI-B. It is likely that some of the serum proteins which had some degree of spontaneous affinity for PLA2 have mutated in an adaptive manner to the forms capable of tight binding to and inhibiting particular PLA 2 species in the venom as a requisite for self-protection.

References 1. Ohkura, N., Inoue, S., Ikeda, K. and Hayashi, K., Biochem. Biophys. Res. Commun. 204 (1994) 1212. 2. Kieffer, B., Driscoll, P.C., Campbell, I.D., Willis, A.C., Anton van der Merwe, P. and Davis, S.J., Biochemistry 33 (1994) 4471. 3. Nakashima, K., Nobuhisa, I., Deshimaru, M., Nakai, M., Ogawa, T., Shimohigashi, Y., Fukumaki, Y., Hattori, S. and Ohno, M., Proc. Natl. Acad. Sci. USA 92 (1995) 5605.

120 A 24 mer peptide with activities of discrimination and recombination of nucleic acids N. SUGIMOTO and S. NAKANO Contribution of the Department of Chemistry, Faculty of Science, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658, Japan

Introduction RecA protein (352 residues) is one of the ssDNA binding proteins concerning a homologous DNA recombination and a DNA repair (SOS response) in prokaryote [1]. This protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and then recombines these strands. Here, we synthesized a 24 mer model peptide of Ac-Ile-Arg-Met-Lys-Ile-Gly-ValMet-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-LysPhe-Tyr-NH 2 (1), which is a potential ssDNA binding site (L2 and helix G regions) of E. coli RecA protein suggested by the crystal structure [2], and investigated its discrimination property of ssDNA from dsDNA and recombination property.

Results and discussion Surface plasmon resonance (SPR) analyses showed that 1 immobilized on the sensorchip was able to bind to ssDNA of d(ACCTAGTC), but neither dsDNA of d(GTCAGGAATCTG)/d(CAGATTCCTGAC), hairpin-looped DNA of d(CGTCA 8 GACG), nor some unnatural nucleotides (Figure 1). When the peptide bound to the ssDNA, a slow α -helical perturbation around the helix G region was observed for a few ten minutes, while less conformational change of the peptide was shown in the presence of the dsDNA. On the basis of these results, we developed an ssDNA affinity column with this peptide supports. Most ssDNAs bound to the support, although no specific interaction was observed with dsDNAs [3]. The binding energy ∆ G° 20 between 1 and poly(dT) was estimated - 7.8 kcal mol- 1 in 100 mM NaCl-phosphate buffer. Poly(dT) formed an extended conformation by interacting with the peptide, so the stacking interaction between Phe locating at the center of the peptide and nucleobases are most probable. Interestingly, the peptide-ssDNA complex was able to bind to the DNA strand complementary to the bound ssDNA, which was confirmed by CD and SPR analyses. It is considered that this cDNA recognition property are due to the α -helical perturbation around the helix G region. 362 Y. Shimonishi (ed): Peptide Science – Present and Future, 362–363  1999 Kluwer Academic Publishers. Printed in Great Britain

363

Time/s Figure 1. (A) Chemical structures of octanucleotides used in this study. (B) SPR signals for 320 µM octanucleotides (ssDNA, ssRNA and 2'-5' U8 ), 480 µM neutral DNA, 640 µM hairpinlooped DNA, arid 480 µM d(GTCAGGAATCTG)/d(CAGATTCCTGAC). These were flowed across the sensorchip on which 1 was covalently bound.

Recently, a 20 residues peptide of E. Coli RecA protein (193-212), which composed of the L2 with some conserved amino acids at both L2 termini was reported to bind to ssDNA and dsDNA, and also indicated the DNA recombination property by the peptide without any cofactors [4]. Here, the peptidessDNA complex was able to bind to pBR322, resulting 1 also possesses the DNA strand recombination activity. These results suggest that this 24 mer oligopeptide has the activities of discrimination and recombination of nucleic acids similar to RecA protein.

References 1. 2. 3. 4.

Kowalczykowski, S.C., Anu. Rev. Biophys. Biophys. Comm. 20 (1991) 539. Story, R.M., Weber, I.T., and Steitz, T.A., Nature 355 (1992) 318. Sugimoto, N, and Nakano, S., Chem. Comm. (1997) 2125. Voloshin, O.N., Wang, L., and Camerini-Otero, R.D., Science 272 (1996) 868.

121 Phosphorylation of synthetic peptides containing proline homologs by cdc2 kinase or cdk5 S. ANDO 1, T. IKUHARA2, T. KAMATA3, Y. SASAKI 2, S. HISANAGA4, T. KISHIMOTO 4, H. ITO 5 and M. INAGAKI 5 1 Chemistry

Laboratory, Saga Medical School, Saga 849, Japan Science Research Center and 3 Analytical Research and Computer Science Center, Asahi Chemical Industry Co., Ltd., Fuji 416, Japan 4 Laboratory of Cell and Developmental Biology, Faculty of Bioscience, Tokyo Institute of Technology, Yokohama 227, Japan 5 Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464, Japan 2 Life

Introduction While cyclin-dependent protein kinases (cdks), including cdc2 kinase and cdk5, play critical roles in controlling the cell cycle or cell differentiation through proline-directed phosphorylation [1,2], the molecular basis for the requirement of proline as a substrate specificity determinant has remained obscure. To obtain information on the structural factors of proline required for the phosphorylation, we prepared the peptides representing the site in vimentin phosphorylated by cdc2 kinase and containing various N -methylamino acids or proline homologs in place of proline, and tested them as substrates for cdc2 kinase or cdk5. Molecular dynamics (MD) and molecular mechanics (MM) simulations for the peptides were also performed to evaluate significance of proline for steric structures at the phosphorylation site.

Results and discussion Structures of the peptides synthesized in this study are shown in Figure 1. The peptide Vim(50-60) represents the sequence around the in vivo and in vitro phosphorylation site, Ser 55, of vimentin by cdc2 kinase [3,4]. Glycine 58 was replaced by arginine in [Arg 58 ]Vim(50-60); otherwise cdk5 (which critically requires a basic amino acid at the third residue on the carboxyl-terminal side of the site [5,6]) does not phosphorylate Ser 55 in vimentin. To evaluate the significance of the ring structure of Pro 56, this residue was replaced by Sar, MeAla, MeVal, or MeLeu. To evaluate the significance of the ring size, the residue was replaced by Aze or Pip. Peptides were synthesized using Bocamino acids and MBHA-resin on an ABI 431A peptide synthesizer. After deprotection and cleavage from the resin with 1M TFMSA/TFA, peptides were purified by gel chromatography and then by RP-HPLC. Identity of the peptides was confirmed by fast atom bombardment mass spectrum measurement. Cdc2 364 Y. Shimonishi (ed): Peptide Science – Present and Future, 364–366  1999 Kluwer Academic Publishers, Printed in Great Britain

365

Figure 1. Structures of synthetic peptides. Amino acids except glycine and sarcosine are all in L-form. Aze, azetidine-2-carboxylic acid; MeAla, N -methylalanine; MeLeu, N -methylleucine; MeVal, N-methylvaline; Pip, piperidine-2-carboxylic acid; Sar, sarcosine.

kinase in a complex with cyclin B was prepared from mouse FM3A mammary tumor cells [4,5]. Cdk5 in a complex with the 23-kDa regulatory subunit was prepared from the microtubule fraction of porcine brain [6]. Peptide Vim(50-60) or [Arg58 ] Vim(50-60) was readily phosphorylated by cdc2 kinase with a K m of 0.886 mM and a V max of 0.481 µmol/min/mg, or a K m of 0.099 mM and a V max of 0.397 µmol/min/mg, respectively. Cdk5 phosphorylated [Arg 58 ] Vim(50-60) with a K m of 1.43 mM and a Vmax of 0.524 µmol/min/mg, but failed to phosphorylate Vim(50-60). The sites phosphorylated in the peptides were assigned to be Ser 55 by amino acid sequencing. The replacement of Pro 56 in the peptide Vim(50-60) or [Arg 58 ]Vim(50-60) by the N -methylamino acids increased the K m values 3- to 6-fold or 7- to 40-fold for cdc2 kinase, respectively. Thus, opening the ring structure of proline significantly increased K m values. The peptide [Pip 56, Arg 58 ]Vim(50-60) or [Aze 56, Arg 58 ]Vim(50-60) gave a 2- or 4-fold higher K m value compared to that obtained with [Arg 58 ]Vim(50-60), respectively. Thus, the ring size of proline was significant for recognizing cdc2 kinase. The replacements by the N-methylamino acids and the proline homologs decreased the V max values several folds for cdc2 kinase. Apparently, the proline homologs were preferable to N -methylamino acids. Thus, the pyrrolidine ring of proline was required to maintain a high V max value for cdc2 kinase. The effect of replacement of Pro 56 in [Arg 58 ]Vim(50-60) by Aze, Pip, Sar or MeAla on the K m value for cdk5 parallels qualitatively that found in phosphorylation of the same peptides by cdc2 kinase, suggesting that cdk5 recognized peptides with a proline specificity similar to that of cdc2 kinase. These replacements, however, led to more degenerative effects on the Vmax value for cdk5 than for cdc2 kinase. Thus, in terms of maintaining a high Vmax , cdk5 showed a stronger preference for proline over other amino acids, when compared to the preference exhibited by cdc2 kinase. MD and MM simulations were performed for Ac-Ser-X-Gly–Arg-CH 3 in which X is Pro, Pip, MeLeu or Sar. The results obtained suggest that the pyrrolidine ring of proline was optimal for inducing and stabilizing a β -turn

366 at the Ser-X-Gly–Arg sequence. Furthermore, we found that, in proportion to increase in the probability of the turn formation, the Km values of the peptides for cdc2 kinase or cdk5 decreased. Thus, the relative activities of the peptides might be related to how well each peptide could assume a β -turn at the phosphorylation site. It was, however, unknown whether the peptides bound to the catalytic site of the enzymes exactly form the turn structure. The implication of the inverse correlation observed here remained to be further clarified.

References 1. Norbury, C. and Nurse, P., Annu. Rev. Biochem., 61 (1992) 441. 2. Lew, J. and Wang, J.H., Trends Biochem. Sci., 20 (1995) 33. 3. Chou, Y.-H. et al., J. Biol. Chem., 266 (1991) 7325. 4. Kusubata, M. et al., J. Biol. Chem,, 267 (1992) 20937. 5. Ando, S. et al., Biochem. Biophys. Res. Commun., 195 (1993) 837. 6. Hisanaga, S. et al., Cell Motil. Cytoskel., 31 (1995) 283.

122 Synthesis of oligomerization domain of p53 and its analogs and their association properties in buffer solution M. MIYABE¹, S. LEE¹, G. SUGIHARA¹, H. SAKAMOTO², K. MENO³, Y. ASO³ and K. SAKAGUCHI 4 ¹ Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka 814-0180, Japan ²Department of Medical Biochemistry, School of Medicine, Kurume University, Kurume, Fukuoka 830-0011, Japan ³ Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka 812-8581, Japan 4 Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892, USA

Introduction Human p53 is a tumor-suppresser gene product associated with control of the cell cycle and with growth suppression. From chemical synthesis and analytical ultracentrifugation, the p53 protein is known to form homotetramers in solution. Recently, Sakamoto et al. have found its tetrameric oligomerization domain (residues 319 to 360) present at C-terminus of p53 [1]. The structure of the tetramerization domain was estimated by NMR and X-ray crystallography as follows. (1) the monomer, which consists of a β -strand and an α -helix, associates with a second monomer across an antiparallel β -strand and an antiparallel helix–helix interface to form a dimer (2) two of these dimers associate across a second and distinct parallel helix–helix interface to form the tetramer. Interestingly, in solution, the tetramers are in equilibrium only with monomers, i.e., neither dimer nor trimer was observed at equilibrium. Thus, to investigate the oligomerization mechanism from monomer to tetramer, we designed and synthesized six analogs of the oligomerization domain (Figure 1).

Figure 1. Primary structure of tetrameric oligomerization domain (p53, residues from 319 to 360) present at C-terminus of p53 protein and its related analogues (P53-1 ~ 6). Bold letters show substituted amino acids.

367 Y. Shimonishi (ed): Peptide Science – Present and Future, 367 – 369 © 1999 Kluwer Academic Publishers, Printed in Great Britain

368 Results and discussion The peptide of tetrameric oligomerization domain and its related analogs designed consist of 40 amino acid residues where residues 8 to 15 and 18 to 38 were found to take β - and α -helical structure, respectively, from X-ray crystallographic data. Characteristics of the designed analogs are as follows. 1) p53-1: The residues forming hydrophobic core in β- and α -helical structure of p53 oligomer are replaced by Val and Leu, respectively. 2) p53-2: The residues, Glu and Asp forming salt bridges are replaced by Gln. 3) p53-3 and 4: Hydrophobic residues participating A–C interaction and A–B in four unites (A, B, C and D) of tetramers are replaced by Leu, respectively. 4) p53-5 and 6: Only hydrophobic residues in β -structure and in α -helical structure are replaced by Val and by Leu, respectively. CD spectra of p53 and its related analogs sin buffer solution are shown in Figure 2. The results of their analytical ultracentrifugation using a Beckman XLA-TG-2 analytical centrifuge are listed in Table 1. The p53-2~4, designed

Figure 2. Circular dichroism spectra of p53 analogs. All spectra were recorded with 20 µM peptide in 50 mM sodium phosphate buffer, pH 7.5 at 20°C. a: p53, b: p53-1, c: p53-2, d: p53-3, e: p53-4, f: p53-5, g: p53-6. Table 1.

Oligomerization status and secondary structure of p53 peptides CD pattern

P53 p53-1 p53-2 p53-3 p53-4 p53-5 p53-6 N.D.: not determined

α -helix random α -helix α -helix α -helix random β-strand

oligomeric state

Kd(M)

tetramer monomer tetramer tetramer tetramer monomer monomer

4.57 × 10 – N.D. N.D. 6.30 × 10 – 85.1 × 10 – N.D. N.D.

6

6 6

369 by considering A–B, A–C and charge interactions in p53 tetramer, took α-helical and tetrameric structure, while the peptides, p53-1, 5 and 6, which altered the central hydrophobic core in α -helices and β -structure by the residues Val and Leu stabilizing their secondary structure, respectively, took random or βstructure. These results suggest that the suitable packing between hydrophobic residues in the α -helix and β -structure as well as the secondary structure of each unit in tetramer is necessary for keeping the tetrameric form, while charge, A–B, and A–C interactions between each subunit are not. It is, however, noted that the substitution of hydrophobic residues participating A–C interaction to Leu led to one order less stable state.

References 1. Sakamoto, H., Lewis, M.S., Kodama, M., Appella, E., and Sakaguchi, H., Proc. Natl. Acad. Sci. USA, 91(1994) 8974.

123 Stabilization of tyrosine-O-sulfate residues through the formation of a conjugate acid–base pair T. YAGAMI¹, K. KITAGAWA², C. AIDA², H. FUJIWARA² and S. FUTAKI³ ¹ National Institute of Health Sciences, Kamiyoga, Setagaya-ku, Tokyo 158, Japan ² Niigata College of Pharmacy, Kamishin’ei-cho, Niigata 950-21, Japan ³ Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611, Japan

Introduction The sulfation of tyrosine residues is one of the most popular post-translational modifications for peptides and proteins [1]. It has been proposed that the sulfate plays an important role in the specific protein–protein interactions during protein transport. The negative charge of a tyrosine- O-sulfate [Tyr(SO 3 H)] residue should participate in these interactions, but little is known about their detailed chemical principles. In this study, we investigated the interaction of a Tyr(SO 3 H) residue with cationic functional groups on the same peptide chain and on externally added basic peptides.

Results and discussion Based on the desulfation mechanism of sodium aryl monosulfates [2], a Tyr( SO 3 H) residue is predicted to be rapidly desulfated in acidic solutions and under non-polar conditions unless it is stabilized by forming an ion pair or a conjugate acid–base pair with cationic functional groups. Indeed, we could rationalize the characteristic mass-spectral peak patterns of multiply sulfated peptides with this desulfation mechanism [3]. Conversely, we can investigate the formation of the conjugate acid–base pair including a Tyr(SO3 H) residue by comparing the stability of the sulfate in acidic solutions and under nonpolar conditions, for example, in the gaseous phase. As peptide samples containing a Tyr(SO3 H) residue, we synthesized cholecystokinins (CCKs) and gastrins that were different not only in chain length but also in the number of cationic functional groups attached to them [4]. A series of CCK-12 derivatives that had a different type of amino acid at the fourth residue from the N-terminus were also prepared. The rates of desulfation in acidic solutions were followed by reverse-phase HPLC. The extent of fragmentation (–SO 3 ; –80 amu) in the gaseous phase was examined by liquid secondary-ion mass spectrometry (LSIMS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 370 Y. Shimonishi (ed): Peptide Science – Present and Future, 370–372 © 1999 Kluwer Academic Publishers, Printed in Great Britain

371

372 The Tyr(SO 3 H) residues became stable with the increasing number of cationic functional groups on the peptide chain. This tendency was common to the sulfated peptides in both the gaseous phase and the acidic solutions. The guanidino function of Arg and the α - and the ε -amino groups were revealed to be the strong intramolecular conjugate bases. Moreover, externally added peptides with Arg residues could stabilize the sulfate. The conjugated peptide-ion peaks without loss of the sulfate were recorded in the positive-ion MALDI-TOF-MS spectra of CCK-8 mixed with a basic peptide containing three Arg residues (Figure 1). In contrast, no conjugated peptide-ion peaks were detected in the mass spectra of the non-sulfated counterpart. Such an intermolecular acid–base pair formation was the most efficient between a Tyr(SO3 H) residue and multiple Arg residues. The intermolecular conjugation of a Tyr(SO3 H) residue with a Lys residue or an N-terminal amino group was not as strong as that of the multiple Arg residues. Our experimental results indicate that a Tyr(SO3 H) residue tends to form a stable conjugate acid–base pair with cationic functional groups, especially the guanidino function of the Arg residues, in aqueous solutions and under nonpolar conditions. Not confined only to intramolecular ion-pairing, but the intermolecular acid–base pair formation was also demonstrated. These findings are helpful to deduce the driving force of the specific protein–protein interactions promoted by a Tyr(SO3 H) residue. Such an energetically favorable conjugate acid–base pair may also be formed at the sites of the specific protein–protein interactions under biological conditions.

References 1. Niehrs, C., Beiß wanger, R. and Huttner, W.B., Chem. Biol. Interact., 92 (1994) 257. 2. Kice, J.L. and Anderson, J.M., J. Am. Chem. Soc., 88 (1966) 5242. 3. Yagami, T., Kitagawa, K. and Futaki, S., Rapid Commun. Mass Spectrom., 9 (1995) 1335. 4. Kitagawa, K., Aida, C., Fujiwara, H., Yagami, T. and Futaki, S., Tetrahedron Lett., 38 (1997) 599.

124 Circular dichroism and fluorescence study of the interaction of synthetic J protein from bacteriophage φK with DNA S. ONO¹, T. OKADA¹, S. AIMOTO², T. FUJII¹, C. SHIMASAKI¹ and T. YOSHIMURA¹ ¹ Department of Chemical & Biochemical Engineering, Toyama University, Toyama 930, Japan ² Institute for Protein Research, Osaka University, Osaka 565, Japan

Introduction Small icosahedral bacteriophages φK, α 3, G4 and φ X174 have highly basic small proteins, J proteins, which bind their circular single-stranded DNA (cssDNA). J proteins are 24–37 amino acids long and have been shown to be essential for DNA packaging in phage morphogenesis [1–3]. However there have been no detailed information on the mode of interaction between J protein and DNA. Recently, Kodaira et al. studied the interaction of synthetic φ K bacteriophage J protein (SynJ, 23 amino acids long, KKARRSPSRRKGARLWYVGGSQF) by gel retardation assay and showed that J protein binds tightly to the φ K bacteriophage css-DNA to form a compact complex in 50 mM Tris-HCl (pH 7.3) [4]. However, since proteinDNA interaction is generally dependent on ionic strength, their result observed under low salt conditions may not show exact binding property of J protein to φK css-DNA. In order to study the interaction mode of J protein with DNA, in this work, we examined circular dichroism (CD) and fluorescence spectral changes accompanied by the complex formation between SynJ and φ K cssDNA under various salt conditions [5].

Results and discussion CD spectral change upon titration of φ K css-DNA with SynJ in 50 mM TrisHCl buffer containing 200 mM NaCl (physiological conditions) was examined (Figure 1A). This ionic strength is thought to be comparable to that in E. coli [6]. As the SynJ concentration increased, a minimum (248 nm) and two maxima (219 and 277 nm) which were observed for free css-DNA became more positive and negative, respectively. Also, these three bands shifted to longer wavelengths. The plot of CD values at 277 nm versus 1/R (molar ratio of SynJ to nucleotide) showed that there are three regions with respect to the structural change of the SynJ/css-DNA complex (Proceedings of the 15th American Peptide Symposium). Below 1/R 0.065, no significant spectral change 373 Y. Shimonishi (ed): Peptide Science – Present and Future, 373–375 © 1999 Kluwer Academic Publishers, Printed in Great Britain

374 was observed and the CD change at 277 nm was also small. In the 1/R range of 0.065–0.11, the maximum and minimum (277 and 248 nm) decreased and shifted to longer wavelength rapidly. Beyond 1/R 0.11, the CD spectra showed a shallow minimum (256 nm) and two isochroic points (242 and 275 nm) without significant change, indicating that css-DNA might be saturated with SynJ at around this 1/R ratio. These CD data strongly suggests a cooperative structural change of the SynJ/css-DNA complex in the buffer containing 200 mM NaCl. In order to investigate the effect of ionic strength on the interaction between SynJ and css-DNA, CD spectra upon titration of css-DNA with SynJ in the Tris-HCl buffer without NaCl and with 1 M NaCl were measured. Under the low ionic strength conditions, a drastic CD change observed as the SynJ concentration increased, while a gentle CD change under the high inonic strength conditions (Proceedings of the 15th American Peptide Symposium). These CD spectral changes indicated that SynJ interacts with css-DNA cooperatively to form different SynJ/css-DNA complexes depending on ionic strength. Binding capability of SynJ to css-DNA was evaluated from the results of fluorescence titrations with increasing amounts of SynJ at various NaCl concentrations (0, 0.2 and 1 M) since the fluorescence of SynJ (Trp and Tyr) was quenched upon addition of css-DNA. Titration curve at 200 mM NaCl consisted of three regions, i.e. 1/R ranges 0–0.039, 0.039–0.14, and beyond 1/R 0.14 (Figure 1B). This titration pattern was different from those under low and high inonic strength conditions (data not shown). These findings indicated that cooperative binding between SynJ and css-DNA takes place depending on NaCl concentration. Moreover, the stoichiometric value n, the number of nucleotides covered by one SynJ, was evaluated to be about 10 by quantitative

Figure 1. CD (A) and fluorescence (B) titrations of css-DNA with SynJ in 50 mM Tris-HCl (pH 7.3) containing 200 mM NaCl. The 1/R value is the molar ratio of SynJ to nucleotide. The concentrations of SynJ and nucleotide for CD and fluorescence titrations were 0–25 µM and 118–97 µM, and 0–210 nM and 1.0–0.87 µM, respectively.

375 analysis of the fluorescence titration at 200 mM NaCl according to the method by Alma et al. [7]. These results demonstrated that SynJ interacts not only electrostatically but also hydrophobically with css-DNA.

References 1. 2. 3. 4. 5.

Fane, B.A., Head, S. and Hayashi, M., J. Bacter., 174 (1992) 2717. Hamatake, R.K., Aoyama, A. and Hayashi, M., J. Virol., 54 (1985) 345. Kodaira, K.-I. and Taketo, A., Mol. Gen. Genet., 195 (1984) 541. Kodaira, K.-I., Oki, M., Kakikawa, M., Kimoto, H. and Taketo, A., J. Biochem., 119 (1996) 1062. Preliminary work has been published as a communication: Ono, S., Okada, T., Aimoto, S., Fujii, T., Morita, H., Shimasaki, C. and Yoshimura, T., Chem. Lett., 1996 (1996) 1063. 6. Burgess, A.B., Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 613. 7. Alma, N.C.M., Harmsen, B.J.M., de Jong, E.A.M., Ven, J.V.D. and Hilbers, C.W., J. Mol. Biol., 163 (1983) 47.

125 Unusual collagen assembly induced by acidic biopolymers in vitro H. KITAMURA¹, C. IWAMOTO¹, T. HAYAMI¹, S. HAN¹, Y. NODASAKA², N. SAKAIRI¹, S. TOKURA¹ and N. NISHI¹ ¹ Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060, Japan ² School of Dentistry, Hokkaido University, Sapporo 060, Japan

Introduction Some kinds of biopolymers such as glycosaminoglycan are known to regulate collagen–fibrils formation [1]. In the present study, assembly of collagen molecules and the subsequent fibril-formation in the presence of acidic biopolymers were investigated by the turbidity assay and transmission electron microscopic observation.

Results and discussion Pepsin-digested type I collagen solutions (PDC, 1.0 mg/ml, pH 3.0, Koken, Co. Ltd. Japan) were neutralized with phosphate buffered saline (pH 7.4) containing double-stranded DNA (0.03 mM, 7.5 kbp, Yuki Fine Chemical Co. Ltd., Japan), phosphated chitin (P-chitin, Mw; 30,000, d.s.; 0.8) or carboxymethyl chitin (CM-chitin, Mw; 98,000, d.s.; 1.0), and incubated at 37°C for 60 min. Assembly of the collagen molecules was monitored by measuring turbidity (OD400) of the mixture. As shown in Figure 1., increase in turbidity was quickened by the

Figure 1. Turbidimetric curves of PDC in the absence (A) and presence of CM-chitin (B), P-chitin (C), and DNA (D).

376 Y. Shimnishi (ed): Peptide Science – Present and Future, 376–378 © 1999 Kluwer Academic Publishers, Printed in Great Britain

377 addition of DNA, delayed by P-chitin, and not changed by CM-chitin. The collagen–fibrillogenesis is generally delayed by proteoglycans and phosphoproteins [2]. These data suggest the specific interaction of DNA with PDC molecules. The resulted collagen fibrils were observed by transmission electron microscopy (H-800, Hitachi Co. Ltd. Japan). In general, the PDC fibrils does not show the distinct cross-bandings pattern, the characteristic feature of the native collagen fibrils [3]. However, very distinct cross-bandings pattern was observed on the PDC fibrils formed in the presence of DNA or P-chitin, and in addition, size of the fibrils became larger (Figure 2.). On the contrary, the fibrils formed with CM-chitin were not changed. The feature of fibrils obtained in the presence of DNA and P-chitin was quite different from FLS structure and SLS aggregates, induced by proteoglycans and ATP, respectively [4]. The present results would imply a specific interaction between the acidic biopolymers and collagen molecules. Regularly arranged phosphate groups in DNA and P-chitin would play key roles, such as an excellent polymer matrix, in the effect on the collagen assembly. Acknowledgment This work was supported by the Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 07555600 and No. 09876088).

Figure 2. Transmission electron micrographs of PDC fibrils in the absence (A) and presence of CM-chitin (B), P-chitin (C), and DNA (D). Magnification, × 10,000.

378 References 1. Scott, J.E., Biochem. J., 252 (1980) 313. 2. Gelman, A.R., Conn, M.K. and Termine, J.D., Biochim. Biophys. Acta, 630 (1980) 220. 3. Capaldi, M.J. and Chapman, J.A., Biopolymers, 21 (1982) 2291. 4. Gross, J., Highberger, J.H. and Schmitt, F.O., Proc. Nat. Acad. Sci. USA, 40 (1954) 679.

126 The role of nucleoplasmin – molecular chaperone protein – in the fertilization process N. NISHI, K. HOZUMI, K. IWATA, A. IIHARA, H. KAWAHARA and N. SAKAIRI Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060, Japan

Introduction Nucleoplasmin is the most abundant protein in the Xenopus laevis oocyte nucleus. It is able to assemble nucleosomes by binding histones and transferring them to DNA in vitro, and the first protein named as molecular chaperone [1]. It is an acidic, thermostable protein and forms a stable pentamer. Recently, it was reported that nucleoplasmin removed sperm nuclear binding proteins, protamines, from the sperm nuclei of Xenopus laevis and converted into nucelosome in vitro. Thus nucleoplasmin plays a leading role in the whole fertilization process and may act as both of an assembly and a disassembly factors for remodeling of sperm chromatin in vivo [2, 3]. We have studied the interaction of nucleoplasmin with DNA-salmine complex as a simple model of salmon sperm nuclei in a molecular level, and reported that a poly glutamic acid tract in the carboxyl terminus side of nucleoplasmin is mainly related to the removal of protamine from sperm nuclei [4]. In the present study, the interaction of nucleoplasmin with histone octamer, fractionated histones and also with DNA were investigated by fluorescence to obtain the further information on the mechanism of the conversion into nucleosome by nucleoplasmin.

Results and discussions Interactions of nucleoplasmin with histone octamer and fractionated histones (H1, H2A, H2B, H3, H4, H2A–H2B dimer and H3–H4 tetramer) were investigated by fluorescence. Nucleoplasmin has two tryptophan residues in the amino acid sequence and its spectrum shows fluorescence maximum near 340 nm. When histone octamer was added to the nucleoplasmin solution, the fluorescence intensity gradually decreased and the fluorescence maximum slightly blue-shifted showing the isofluorescent point (Figure 1A). Similar phenomena were observed also for the fractionated histones. Among them, influence of histone H2A on the spectrum was found to be most drastic. Further, decrease in the fluorescence maximum by the addition of H2A–H2B dimer was more 379 Y. Shimonishi (ed): Peptide Science – Present and Future, 379–380 © 1999 Kluwer Academic Publishers, Printed in Great Britain

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Figure 1. Chage in the fluorescence spectrum of nucleoplasmin by the addition of histone octamer (A) and DNA (B). [nucleoplasmin] = 0.4 µM in EM (100 µM KCl, 2 mM MgCl2 , 10 mM TrisHCl, pH 7.4). Exited at 285 nm.

than that by H3–H4 tetramer. It has been also shown by affinity chromatography that H2A–H2B dimer has the larger affinity with nucleoplasmin than H3–H4 tetramer [5]. From the present results, it can be estimated that especially histone H2A interacts strongly with the polyglutamic acid tract in the carboxyl terminus side of nucleoplasmin, covering the Trp residue near the tract. Interaction of nucleoplasmin with DNA was also investigated by fluorescence (Figure 1B). When DNA was added to the nucleoplasmin solution, the fluorescence intensity at 340 nm decreased and the fluorescence maximum slightly red-shifted. This result clearly suggests that nucleoplasmin interacts also with DNA, although it has been thought that nucleoplasmin doesn’t interact with DNA. The Lys-rich sequence in the carboxyl terminus of nucleoplasmin would be the domain to interact with phosphoric acid groups of DNA.

Acknowledgment We thank Mr. Koichi Mita, Dr. Toru Itoh and Prof. Chiaki Katagiri (Division of Biological Science, Graduate School of Science, Hokkaido University) for their valuable suggestions and discussions.

References 1. 2. 3. 4.

Laskey, R.A., Honda, B.A., Mills, A.D. and Finch, J.T., Nature, 275 (1978) 416. Ohsumi, K. and Katagiri, C., Dev. Biol., 148 (1991) 295. Itoh, T,. Ohsumi, K. and Katagiri, C., Dev. Growth Differ., 35 (1993) 59. Iwata, K., Hozumi, K., Itoh, T., Sakairi, N., Tokura, S., Katagiri, C. and Nishi, N., Int. J. Biol. Macromol., 20 (1997) 171. 5. Philpott, A.A., Leno, G.H. and Laskey, R.A., Cell, 65 (1991) 569.

127 Conformation of chick DNA topoisomerase I as determined by limited proteolysis L.M. ASSAIRI Institut de Génétique et Microbiologie, CNRS, URA 2225, Université de Paris-Sud, Centre d’Orsay, 91405 Orsay Cédex, France

Introduction DNA topoisomerases [1] are enzymes which nick and reclose the phosphodiester backbone of the double helix of DNA (type I topoisomerase for one strand cut only, type II topoisomerase for two strands cut*). DNA topoisomerase I has been found in all organisms from bacteria to eukaryotic cells and a tyrosine is the amino acid which is covalently bound to the extremity of the phosphodiester bond cut forming thus, a covalent DNA-protein intermediate. However the mechanism of action of DNA topoisomerase I has evolved from bacteria to eukaryotes. Indeed, the covalent intermediate is formed between the 5'P extremity and the tyrosine in bacteria although it is formed between the 3'P extremity and the tyrosine in eukaryotes. The eukaryotic DNA topoisomerases I have been reported to be sensitive to proteolytic degradation during their purification.

Results and discussion The DNA labelling technique mainly identifies two forms of 66 and 110 kDa with a specific affinity to the phosphocellulose resin suggesting that the enzyme is composed of two domains joined by a hinge region which is very sensitive to cellular proteases. The comparative analysis of the eukaryotic DNA topoisomerase I amino acid sequences gives some information concerning the conformation of the enzyme. A mild proteolysis of Chick DNA topoisomerase I or of covalent DNAprotein complex by using Staphylococcus aureus V8 protease which clives at the COOH extremity of the glutamic acid, leads to the production of several peptides (Figure 1). The 66 kDa product is the major proteolytic fragment obtained. Several eukaryotic genes have been cloned and sequenced [2] and the reactive tyrosine which has been determined by chemical analysis [3] is located at the COOH extremity of the protein. The V8 proteolytic fragments obtained from the chick DNA topoisomerase I are compared with the human enzyme amino acid sequence [2]. Within the COOH moiety of the molecule which 381 Y. Shimonishi (ed): Peptide Science – Present and Future, 381–382 © 1999 Kluwer Academic Publishers, Printed in Great Britain

382

Figure 1. Limited proteolysis of Chick DNA topoisomerase I A) The Chick DNA topoisomerase I was digested by Staphylococcus aureus V8 protease and analysed on 10% polyacrylamide in the presence of SDS gel. lane 1: high molecular weight markers, lane 2: low molecular weight markers, lane 3: Chick DNA topoisomerase I, lane 4: Chick DNA topoisomerase I digested by V8 protease, lane 5: V8 protease. B) The chick DNA topoisomerase I was incubated with denatured nick translated calf thymus DNA then, the covalent complex formed between DNA topoisomerase I and DNA was digested by micrococcal nuclease and then TCA precipitated. The samples were analysed by electrophoresis on 15% polyacrylamide gel in the presence of SDS.

contains the reactive tyrosine, we can also distinguish the following sequence 488 Arg··· 508 Arg–X–Glu–His which could correspond to the Arg ··· His–X–X– Arg triad which specifies the DNA integrase activity [3]. This amino acid sequence located within the COOH moiety is present in the low molecular weight (66 kDa) eukaryotic DNA topoisomerases I.

References 1. Wang, J., Ann. Rev, Biochem., 54 (1985) 665. 2. D’Arpa, P., Machlin, P.S., Ratrie, H., Rothfield, N.F., Cleveland, D., and Ernshaw, C.W., Proc. Natl. Acad. Sci., USA 85 (1988) 2543. 3. Parson, R.L., Evans, B.R., Zheng, L., and Jayaram, M.J., Biol. Chem., 265 (1990) 4527. 4. Lynn, R., Bjornsti, M-A, Caron, P., and Wang, J., Proc. Natl. Acad. Sci., USA 86 (1989) 3559.

128 The two adenosine triphosphate and nucleic acid binding sites of the calf thymus single-strand DNA-dependent adenosine triphosphatase L.M. ASSAIRI Institut de Génétique et Microbiologie, CNRS, URA 2225, Université de Paris-Sud, Centre d’Orsay, 91405 Orsay Cédex, France

Introduction A single-strand DNA-dependent adenosine triphosphate (ATPase) has been isolated from calf thymus [1]. The biological role of this enzyme is still unknown but the enzyme could be involved in the replication, the transcription or the recombination of DNA. The characteristics are well documented [2] now and can help the understanding of its mechanism of action. The enzyme hydrolyses ATP into ADP and inorganic phosphate in the presence of singlestrand DNA and the ADP produced is an efficient competitive inhibitor. The new observations presented in this communication, deal with the study concerning the disappearance of the inhibitory effect of ADP on the ATP hydrolysis upon heat-denaturation of the protein and the evidence that the two recognition sites for DNA and ATP are distinct on the protein.

Results and discussion The enzyme hydrolyses ATP as well as dATP into ADP or dADP and inorganic phosphate in the presence of single-strand DNA. The enzyme was preincubated at different temperatures 37, 45 and 50°C. An inhibition of 50% was found after 12 min of preincubation at 45°C or 5 min at 50°C. Single-strand DNA (calf thymus) protects the enzyme against the heat denaturation occurring at 45°C and ATP is an effective protector only in the presence of the magnesium ions. The N-ethylmaleimide which irreversibly blocks the sulfhydryl group of the cysteine, inhibits the ATP hydrolysis activity catalysed by the enzyme (Figure 1). The activity is inhibited at 50% in the presence of 2.5 mM of N-ethylmaleimide and no activity is recovered in the presence of 5 mM N-ethylmaleimide. However, N-ethylmaleimide has no effect on the interaction between the enzyme and DNA. The presence of ATP or single-strand DNA added to the incubation mixture previously to the N -ethylmaleimide does not protect the enzyme against its inactivation. 383 Y. Shimonishi (ed): Peptide Science – Present and Future, 383–384 © 1999 Kluwer Academic Publishers, Printed in Great Britain

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Figure 1. N-Ethylmaleimide discriminates between the two recognition sites ATP and DNA. Purified calf thymus ssDNA-dependent ATPase (26.4 pmoles) was preincubated in 10-microL of incubation mixture in the presence of various amounts of N-ethylmaleimide and 100 microG BSA at 30°C. After 15 min, 5 microL fractions (13 pmoles enzyme) were incubated in the presence of 15.5 × 10 –3 M13DNA (ratio enzyme/DNA = 15.5 × 10 –3 pmole/pmole) in a 20-microL incubation mixture (10 mM Tris-HCl pH 7.4, 1 mM DTT, 1 mMgCl 2, 1 mM ATP, [ 14 C]ATP. After 30 mins at 30°C, 5 microL fractions were used for ATPase activity and 10 microL fractions for the DNA binding.

Several enzymes require the ATP hydrolysis in order to exhibit their biological activity but the topography of their ATP recognition site has evolved. The design of new inhibitors or more efficient substrates will give information concerning the topography of the ATP binding site. Moreover the design and the synthesis of biosensors specific to the DNA-dependent ATPase could allow the measurement of the variation of inactive and active forms of the enzyme during the cell cycle for example.

References 1. Assairi, L. and Johnston, I., Europ. J. Biochem. 99 (1979) 71. 2. Assairi, L., Perspectives On Protein Engineering 1997, BIODIGM LTD. 3. Assairi, L., Protein Engineering 10 (supplement) (1997) 59.

129 Induced helical structure in highly charged peptides A. LOMBARDI, L. ZACCARO, G. ANSANELLI, C. PEDONE and V. PAVONE Centro Interuniversitario di Ricerca sui Peptidi Bioattivi & Centro di Studio di BiocristallografiaCNR, University of Naples ‘Federico II’, Via Mezzocannone 4, I-80134 Napoli, Italy

Introduction The design of short peptide able to fold into a unique three-dimensional structure represents one of the major goals of peptide and protein chemistry [1]. In fact, designed peptides with well-defined structures and properties, which mimic those of natural proteins, may offer the advantage of performing specific functions by using small molecules. Even though significant achievements in this area have been made recently, further studies are required for completely understanding the sequence–structure relationship in peptides and proteins. Helical structures are certainly important structural motifs: they not only confer structural stability to proteins, but also take part in many fundamental biological activities, such as protein-DNA interactions. In this respect, many efforts have been devoted in understanding the role of the polypeptide chain length and composition in determining the helical folding. It is widely accepted that many factors can affect the helix stability: (i) the presence of N- a n d C-terminal capping residues; (ii) specific side chain and charge-dipole interactions; (iii) the incorporation of covalent macrocycles; (iv) the different helix propensity of the amino acids, all play a central role in inducing helix formation [ 2 ] . C α,α -disubstituded glycines, such as Cα,α -dimethyl glycine (Aib) and 1-aminocycloalkane-1-carboxylic acid (Ac n c) residues, display a high helical propensity in the 310 /α -helical regions [3]. However, to the best of our knowledge, there are no examples in the literature of water soluble, highly charged helical peptides containing Cα,α -disubstituded glycines. Thus the effectiveness o f Cα,α-disubstituded glycines in inducing helical structures have been demonstrated only for hydrophobic peptides in organic solvents. We report here the synthesis and conformational analysis in solution, by nmr spectroscopy of a highly charged, 14-membered peptide, named finger 4, intended to mimic the domain 1 of the Zif 268 protein. Results and discussion Our initial design of finger 4 has evolved from a detailed analysis of the structure of the Zif 268 protein – DNA complex, as determined by X-ray 385 Y. Shimonishi (ed): Peptide Science – Present and Future, 385–387 © 1999 Kluwer Academic Publishers, Printed in Great Britain

386

Figure 1. Hypothetical mode of interaction of finger 4 with DNA.

crystallography [4, 5]. In particular, the 16–30 sequence of Zif 268 domain 1 was chosen as template for building the model peptide. The hypothetical mode of interaction between finger 4 and a polynucleotide sequence, containing the GCG recognition triplet, is schematically depicted in Figure 1. The peptide was synthesized by means of solid phase strategy, using the classical protocols of the Fmoc chemistry. Circular dichroism measurements showed the finger 4 to be highly helical in solution. It binds, with a 1: 1 stoichiometry and a dissociation constant approximately 3 µ M, a 17-mer hairpin DNA, which contains the GCG recognition triplet. On the other hand, finger 4 structurally related peptides, lacking the specificity residues (Arg³ and Arg9 ), showed a lower affinity; also finger 4 showed lower affinity for scrambled DNA sequences. Structural characterization in solution, by nmr spectroscopy, confirmed the CD results. The pattern of the NOE connectivities strongly supported the presence of a helical structure. The large number of experimental information derived from nmr spectra allowed us to easily built an approximate model for finger 4, which was refined by Restrained Molecular Dynamic (RMD) simulations. The structure derived from the RMD calculations was in good agreement with all the experimental data.

Conclusions In this paper we have reported a preliminary analysis of the functional and structural properties of a rationally designed DNA-binding peptide. Finger 4 is able to bind a DNA target sequence. More remarkably, the peptide sequence shows a high helical content, thus demonstrating the effectiveness of the Aib residue in inducing helical structures even in highly charged, water-soluble peptides.

387 References 1. Bryson, J.W., Betz, S.F., Lu, H.S., Suich, D.J., Zhou, H.X., O’Neil, K.T. and DeGrado, W.F., Science 270 (1995) 935 and references therein. 2. Scoltz J.M. and Baldwin, R.L., Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 95. 3. Toniolo, C. and Benedetti, E., ISI Atlas of Science: Biochemistry (1988) 225. 4. Pavletich, N.P. and Pabo, C.O., Science, 252 (1991) 809.

130 Nociceptin, a novel neuropeptide with multiple functions J.-C. MEUNIER Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, UPR 9062, 205 route de Narbonne, 31077 Toulouse Cedex 4, France

Introduction Homology cloning and, more recently, the sequencing of whole genomes, have identified many open reading frames encoding proteins of unknown function, in particular putative G protein-coupled membrane receptors. Identification of orphan receptors in this way has marked the advent of ‘reverse pharmacology’ to identify the corresponding physiological ligands. Along this line, the screening of cDNA libraries by hybridization at low stringency with opioid receptor cDNA probes, or with probes generated by selective amplification of genomic DNA with degenerate primers, led several laboratories to isolate a cDNA (ORL1 [1], ROR-C [2], XOR1 [3,4], LC132 [5], MOR-C [6], C3 [7], FLAT7-5EU [8], Hyp 8-1 [9], oOR [10]) encoding a protein with the primary structure of a G protein-coupled membrane receptor, analogous to those of opioid receptors. This opioid receptor-like receptor displayed no affinity for opioid ligands and remained ‘orphan’ until late 1995, when two groups reported separately the isolation of its endogenous ligand, nociceptin [11] or orphanin FQ [12], a heptadecapeptide which resembles dynorphin A, the endogenous ligand of the κ -opioid receptor. The ORL1 receptor and nociceptin are the elementary components of a new peptide-based signalling pathway in the nervous sytem. A great deal of information on all aspects of nociceptin has now accumulated, indicating that the peptide acts at the molecular and cellular levels in very much the same way as opioids do, however to produce pharmacological effects that sometimes differ from, and even oppose, those of opioids. The presently available data on the new peptide will be summarized here as a digest of a recent and extensive review [13] in which a complete set of references is to be found. The opioid receptor-like 1 (ORL1) orphan receptor Primary structure Alignment of the cDNA-deduced amino acid sequences of the ORL1, µ -, δ a n d κ -opioid receptors (Figure 1) highlights the conserved regions, especially the second (77.3% amino acid identity), third (72.7%) and seventh (75.0%) 388 Y. Shimonishi (ed): Peptide Science – Present and Future, 388–397 © 1999 Kluwer Academic Publishers, Printed in Great Britain

389

390 transmembrane helices, and the intracellular loops (59.1–85.4%). Overall, the ORL1 receptor is equally distant (63–65% homology) to the three types of opioid receptor, but its acidic second exofacial loop makes it resemble more closely the κ -(whose endogenous ligand is dynorphin A) than the µ - or δ opioid receptors. The murine ORL1 receptor (367 amino acids) is shorter than its human counterpart (370 amino acids), the difference accounted for by a three residue insertion in the N-terminal domain. Localization ORL1 receptor transcripts are particularly abundant in the cortical and corticolimbic areas, hypothalamus, brain stem, and the dorsal and ventral horns of the spinal cord [1, 2, 5, 7, 8] and in cell bodies of the dorsal root ganglion [8]. Extensive immunohistochemical mapping of the ORL1 protein in the central nervous system of the rat has yielded distributions in agreement with those obtained from in situ hybridization studies, indicating that the ORL1 receptor is predominently expressed in local-circuit (inter)neurones [14]. The wide yet discrete distribution of the ORL1 receptor mRNA and/or protein, suggested the receptor has the potential to modulate a variety of central processes, including learning and memory, attention and emotions, movement and motor processes, homeostasis, neuroendocrine secretion, and virtually every modality of sensory perception, including nociperception.

Nociceptin, the endogenous ligand of the ORL1 receptor Identification Based upon the structural analogy of ORL1 and κ -opioid receptors, the endogenous ligand of the ORL1 receptor was expected to be a dynorphin-like peptide. It was therefore sought after in acetic acid extracts from nerve tissue as a basic peptide which would inhibit adenylyl cyclase in recombinant CHO cells expressing the orphan receptor. Fractionation of rat brain and porcine hypothalamus extracts by conventional procedures ultimately led two laboratories [11, 12] to independently isolate and identify a heptadecapeptide whose amino acid sequence is indeed similar to that of dynorphin A (Figure 2). The synthetic peptide strongly inhibits forskolin-induced accumulation of cAMP in CHO cells expressing the ORL1 receptor (ED50 at about 1 nM) and,

Figure 2. Comparison of amino acid sequences of the new peptide and dynorphin A. Identical amino acid residues are shaded. The ‘message’ and ‘address’ domains of dynorphin A, as defined by Chavkin and Goldstein [60], are indicated.

391 when injected intracerebroventricularly in mice, induces hyperalgesia in the hote plate [11] and tail-flick [12] tests. The novel peptide was named ‘nociceptin’ [11], after its apparent pro-nociceptive properties, or ‘orphanin FQ' [12]. Precursor Evidence that the novel peptide must be physiologically relevant and synthesized locally was provided by the isolation, from a rat brain library, of a (partial) cDNA encoding a larger precursor polypeptide whose sequence contains one copy of nociceptin, framed by Lys-Arg convertase excision motifs [11 ] . Interestingly, the full-length cDNA was being independently isolated in Japan as an orphan cDNA whose mRNA (N23K) is transiently up-regulated during dibutyryl-cAMP-induced neurite outgrowth of mouse neuroblastoma NS20Y cells [15]. A variant form of precursor cDNA, N27K, has been identified in dibutyryl cAMP-treated NS20Y cells. It encodes a protein of 212 amino acids, in which the two last amino acids in the shorter form are replaced by a stretch of 27 amino acids [16]. The prepronociceptin (ppnoc) gene has been partially sequenced and mapped to the short arm of human chromosome 8 [17]. The amino acid sequence it encodes is highly conserved across murine and human species (Figure 3), especially the C-terminal third which hosts nociceptin itself. Moreover, the nociceptin precursor contains putative cleavage sites other than those flanking nociceptin, raising the possibility that it may be the precursor not only to nociceptin, but also to other bioactive neuropeptides. Localization Northern blot analyses have shown the ppnoc gene to be predominantly expressed, as a major mRNA species ≈1.3 kb long, in the brain and spinal cord of rat and human [17, 18]. In the human brain, expression levels are high in amygdala and subthalamic nuclei, intermediate in hypothalamus, substantia nigra, and thalamus [18]. The 1.3 kb mRNA species is also present in rat ovary [17], human spleen, leukocytes and fetal, but not adult, kidney [18]. In the mouse brain, expression of the N27K mRNA is developmentally regulated, and occurs maximally soon after birth [16]. In situ hybridization and irnmunohistochemical studies have provided pronociceptin maps that are in general agreement with the localization of the ORL1 receptor [19]. In the rat spinal cord, nociceptin-ir is particularly strong in the superficial dorsal horn, lateral spinal nucleus and the region dorsal to the central canal [20], and survives unilateral dorsal rhizotomy, indicating that it originates from central, rather than primary afferent neurones [20]. Most significantly, confocal microscopic analysis of two-colour double stained sections of the cord reveals only rare occurences of co-localization of nociceptin and opioid peptides in nerve fibers and terminals [20, 21].

392

393 Binding properties and structure-activity relationships In membrane preparations derived from CHO cells expressing the ORL1 receptor, 125 I-labeled [Tyr 14 ] nociceptin [12], and [³H] nociceptin [22] bind a single class of sites with very high affinity (KD ≈ 0.1 nM). Interestingly, binding of [³H] nociceptin to the ORL1 receptor is quite efficiently and stereospecifically competed with by the opiate lofentanil (KI ≈ 20 nM) [22], suggesting 4-anilinopiperidine as a lead for designing novel, non peptidic ligands of the ORL1 receptor. The primary structures of nociceptin and dynorphin A displaying clear similarities (Figure 2), the two neuropeptides may have a similar functional architecture [11]. However, nociceptin appears to be much more rapidly inactivated than dynorphin A upon stepwise C -terminal amino acid deletion [22–24], and the properties of nociceptin/dynorphin A chimeric peptides indicate that the peptide basic core contributes more to biological activity in nociceptin than in dynorphin A [25]. Thus, nociceptin and dynorphin A are not expected to bind their receptors at equivalent sites, congruent with the finding that an ORL1/ κ-opioid hybrid receptor binds both nociceptin and dynorphin A with very high affinity [26]. Nociceptin-induced early postreceptor events Nociceptin has been reported to (i) inhibit forskolin-induced accumulation of cAMP in recombinant CHO cells expressing the ORL1 receptor [11, 12], in neuroblastoma × glioma NG 108-15 hybrid cells [27], and in mouse brain homogenates [28], (ii) stimulate an inwardly-rectifying K + conductance in rat brain slices containing dorsal raphe nucleus [29], locus coeruleus [30] or periaqueductal gray neurons [31], and (iii) inhibit several types of voltage-gated C a ++ channels in SH-SY5Y human neuroblastoma cells [32], as well as in dissociated rat hippocampal neurons [33]. The actions of nociceptin on ion conductances are expected to reduce neuronal excitability and/or presynaptic transmitter secretion. In fact, nociceptin suppresses the K + -evoked release of glutamate from rat cerebrocortical slices [34], the light-evoked release of acetylcholine from cholinergic neurones in the rabbit eye-cup preparation [35], and the basal and electrically-induced release of enkephalin in the guinea pig myenteric plexus [36]. The peptide also depresses glutamatergic transmission in the spinal cord [37], as well as excitatory glutamatergic and inhibitory GABAergic transmission in rat periaqueductal gray neurons [31].

In vivo central effects of nociceptin Pain and analgesia Nociceptin increases reactivity to noxious stimulation (hyperalgesia) when i.c.v.injected. in mice [11, 12], or systemically in a land snail [38]. It even produces

394 allodynia upon i.t. administration in the mouse [39]. However, nociceptin has also been reported to inhibit stress- and opioid-induced analgesia, indicating that the peptide is endowed with functional anti-opioid properties [40, 41]. In the rat, i.t. nociceptin induces robust and long-lasting analgesia and, in the decerebrated spinal rat preparation, depresses a spinal nociceptive flexor reflex [42]. That nociceptin acts as a spinal analgesic is supported by a number of electrophysiological studies in the cord showing that the peptide depresses the C-fiber evoked wind-up and post-discharge of dorsal horn neurones [43], both the A and C fiber-mediated components of the glutamatergic population ventral root potential elicited by electrical stimulation of the dorsal root [39], and the NMDA- and natural stimuli-evoked activity not only of nociceptive and wide dynamic range, but also of low threshold (non nociceptive) neurones [44]. Locomotion In mice, low doses of i.c.v. nociceptin have been found to stimulate locomotor activiy and exploratory behavior [45]. At higher doses however, the peptide decreases horizontal and vertical (rearing) locomotor activities, and to induce ataxia and loss of the righting reflex [12]. In the rat, pronounced and longlasting motor impairment, including disruption of coordination, balance and muscle tone is induced upon i.c.v. injection of 10 and 100 nmol nociceptin [46]. Motivation In the anaesthetized rat, nociceptin (15–150 nmol/animal, i.c.v.) suppresses dopamine release in the nucleus accumbens [47]. Yet, the peptide (0.1–100 nmol/animal, i.c.v.) is motivationally neutral in the rat place preference/aversion test [48]. Furthermore, in contrast with one may have expected given the peptide’s supraspinal anti-opioid properties, and abundance in the locus coeruleus, nociceptin (2, 10 or 50 nmol) does not precipitate a withdrawal syndrome when i.c.v.-injected in the morphine-dependent rat [49]. Cognitive processes Microinjection in hippocampus of 10 nmol nociceptin dramatically impairs spatial learning in the rat, as assessed in the place navigation test [50]. Also, recent electrophysiological data in dentate regions of rat hippocampal slices indicate that nociceptin inhibits induction of long-term potentiation [51, 52]. Feeding Just like opioids do, especially κ -opioid receptor agonists, nociceptin stimulate food intake in the rat, whether the peptide is administered i.c.v. [53] or into the shell of nucleus accumbens or ventromedial hypothalamic nucleus [54].

395 Ex vivo and in vivo peripheral effects of nociceptin Smooth muscle Several ex vivo and in vivo studies have identified nociceptin activities in peripheral tissues, especially smooth muscle. The peptide inhibits the electrically-induced contraction of the isolated guinea pig renal pelvis (ED 5 0 ≈ 30 nM) [55], and mouse vas deferens (IC 5 0 ≈ 20 nM) [56, 57], and exerts vasorelaxation on phenylephrine pre-contracted rings from cat renal, mesenteric, carotid and femoral arteries [58]. In the rat, intravenously infused nociceptin causes a decrease in mean arterial pressure [59]. Kidney Intravenous infusion of nociceptin markedly increases urine flow whilst suppressing urinary sodium excretion in the rat [59]. Similar effects are observed upon i.c.v. administration of the peptide, raising the possibility that the peptide has the ability to cross the blood-brain barrier. Immune cells Nociceptin may play a role in immune function. ORL1 receptor mRNA is present in mouse splenic lymphocytes [10], in human peripheral blood lymphocytes and lymphocytic cell lines [9], and ORL1 receptor mRNA-specific antisense nucleotides inhibit lipopolysaccharide-induced mouse lymphocyte proliferation and production of immunoglobulins G and M[10].

Conclusion and perspectives Inverse pharmacology has proven of great heuristic value here, leading to the discovery of the ORL1 receptor and its natural ligand, nociceptin, the basic components of a new peptide-based signalling pathway in the nervous sytem. Although the discovery of nociceptin, a neuropeptide with multiple functions, does not introduce new concepts in fundamental brain mechanisms, it will certainly improve our knowledge of brain physiology, and may find therapeutical applications, for example in the management of pain or hyponatremic and water-retaining deseases. However, given the wide distribution of noc/oFQ and its receptor, the pharmacological profile of nociceptin is likely to be incomplete, and other as yet unknown functions of the peptide remain to be discovered. Most helpful in this respect will be the identification of new ligands of the ORL1 receptor, particularly antagonists. Such ligands may already exist in the vaults of the pharmaceutical industry. Others await the process of rational design, perhaps based on the structure of non peptide ligands of the ORL1 receptor, such as the opiate lofentanil [22]. If research on nociceptin carries

396 on unabated at the present pace, potentially clinically interesting new compounds could become available in the not too distant future.

Acknowledgements Research in the author's laboratory is supported by the CNRS, the Association pour la Recherche sur le Cancer, the Ministère de 1’Education Nationale, de 1’Enseignement Supérieur et de la Recherche, and the European Commission.

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397 25. Lapalu, S., Moisand, C., Mazarguil, H., Cambois, G., Mollereau, C., and Meunier, J.-Cl., FEBS Lett., 417 (1997) 333. 26. Mollereau, C., Lapalu, S., and Meunier, J.-C., in Abstracts of the 27th International Narcotics Research Conference, Long Beach (USA), 1996, no. TU4, p. 75. 27. Ma, L., Cheng, Z.-J., Fan, G.-H., Cai, Y-C., Jiang, L.-Z. and Pei, G., FEBS Lett., 403 (1997) 91. 28. Mathis, J.P., Ryan-Moro, J., Chang, A., Hom, J.S.H., Scheinberg, D.A., and Pasternak, G.W., Biochem. Biophys. Res. Commun., 230 (1997) 462. 29. Vaughan, C.W. and Christie, M.J., Brit. J. Pharmacol., 117 (1996) 1609. 30. Connor, M., Vaughan, C.W., Chieng, B., and Christie, M.J., Brit. J. Pharmacol., 119 (1996) 1614. 31. Vaughan, C.W., Ingram, S.L., and Christie, M.J., J. Neurosci., 17 (1997) 996. 32. Connor, M., Yei, A., and Henderson, G., Brit. J. Pharmacol., 118 (1996) 205. 33. Knoflach, F,, Reinscheid, R., Civelli, O., and Kemp, J.A., J. Neurosci., 16 (1996) 6657. 34. Nicol, B., Lambert, D.G., Rowbotham, D.J., Smart, D., and McKnight, A.T., Brit. J. Pharmacol., 119 (1996) 1081. 35. Neal, M.J., Cunningham, J.R., Paterson, S.J., and McKnight, A.T., Brit. J. Pharmacol., 120 (1997) 1399. 36. Gintzler, A.R., Adapa, I.D., Toll, L., Medina, V.M., and Wang L., Eur. J. Pharmacol., 325 (1997) 29. 37. Faber, E.S.L., Chambers, J.P., Evans, R.H., and Henderson, G., Brit. J. Pharmacol., 119 (1996) 189. 38. Kavaliers, M. and Perrot-Sinal, T.S., Peptides, 17 (1996) 763. 39. Okuda-Ashitaka, E., Tachibana, S., Houtani, T., Minami, T., Masu, Y., Takeshima, H., Sugimoto, T., and Ito, S., Mol. Brain Res., 43 (1996) 96. 40. Mogil, J.S., Grisel, J.E., Reinscheid, R., Civelli, O., Belknap, J.K., and Grandy, D.K., Neuroscience, 75 (1996) 333. 41. Mogil, J.S., Grisel, J.E., Zhangs, G., Belknap, J.K., and Grandy, D.K., Neurosci. Lett., 214 (1996) 131. 42. Xu, X.-J., Hao, J.-X., and Wiesenfeld-Hallin, Z., NeuroReport, 7 (1996) 2092. 43. Stanfa, L.C., Chapman, V., Kerr, N., and Dickenson, A.H., Brit. J. Pharmacol., 118 (1996) 1875. 44. Wang, X.-M., Zhang, K.M., and Mokha, S.S., J. Neurophysiol., 76 (1996) 3568. 45. Florin, S., Suaudeau, C., Meunier, J.-C., and Costentin J., Eur. J. Pharmacol., 317 (1996) 9. 46. Devine, D.P., Taylor, L., Reinscheid R., Monsma Jr, F.J., Civelli, O., and Akil, H., Neurochem. Res., 21(1996) 1387. 47. Murphy, N.P., Ly, H.T., and Maidment, N.T., Neuroscience, 75 (1996) 1. 48. Devine, D.P., Reinscheid, R., Monsma Jr, F.J., Civelli, O., and Akil, H., Brain Res., 727 (1996) 225. 49. Tian, J.-H., Xu, W., Mogil, J.S., Grisel, J.E., Grandy, D.K., and Han, J.-S., Brit. J. Pharmacol., 120 (1997) 676. 50. Sandin, J., Georgieva, J., Schoγtt, P.A., Ögren, S.O., and Terenius, L., Eur. J. Neurosci., 9 (1997) 194. 51. Yu, T-P., Fein, J., Phan, T., Evans, C.J., and Xie, C.-W., Hippocampus, 7 (1997) 88. 52. Ikeda, K., Kobayashi, K., Kobayashi, T., Ichikawa, T., Kumanishi, T., Kishida, H., Yano, R., and Manabe, T., Mol. Brain Res., 45 (1997) 117. 53. Pomonis, J.D., Billington, C.J., and Levine, A.S., NeuroReport, 8 (1996) 369. 54. Stratford, T.R., Holahan, M.R., and Kelley, A.E., NeuroReport, 8 (1997) 423. 55. Giuliani, S. and Maggi, CA., Brit. J. Pharmacol., 118 (1996) 1567. 56. Berzetei-Gurske, I., Schwartz, R.W., and Toll, L., Eur. J. Pharmacol., 302 (1996) R1. 57. Calo, G., Rizzi, A., Bogoni, G., Neugebauer, V., Salvadori, S., Guerrini, R., Bianchi, C., and Regoli, D., Eur. J. Pharmacol., 311(1996) R3. 58. Gumusel, B., Hao, Q., Hyman, A., Chang, J.-K., Kapusta, D.R., and Lippton, H., Life Sci., 60 (1997) PL 141. 59. Kapusta, D.R., Sezen, S.F., Chang, J.-K., Lippton, H., and Kenigs, VA., Life Sci., 60 (1997) PL 15. 60. Chavkin, C. and Goldstein, A., Proc. Natl. Acad. Sci. USA, 78 (1981) 6543.

131 An anti-HIV peptide T22 is confirmed to be a selective CXCR4/fusin inhibitor by comparative inhibition study of a chemokine SDF-1, synthesized by regioselective disulfide bond formation H. TAMAMURA¹ F. MATSUMOTO¹, K. SAKANO¹, A. OTAKA¹, T. MURAKAMI², H. NAKASHIMA³, M. WAKI4 , A. MATSUMOTO4 , N. YAMAMOTO² and N. FUJII¹ ¹ Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan ² Tokyo Medical and Dental University, School of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan ³ Kagoshima University Dental School, Sakuragaoka, Kagoshima 890-8544, Japan 4 SEIKAGAKU Corporation, Higashiyamoto, Tokyo 207-0021, Japan

Introduction Stromal cell-derived factor-1 (SDF-1) [1] belongs to CXC-chemokine between two subfamilies, the CXC- and CC-chemokines [2] and is a lymphocyte chemoattractant. A CXC-chemokine receptor, CXCR4/fusin, and a CC-chemokine receptor, CCR5, serve as coreceptors in association with CD4 for the entry of T-cell line-tropic (T-tropic) and macrophage-tropic (M-tropic) strains of HIV-1, respectively [3], and SDF-1 blocks infection of T-cells by T-tropic HIV-1 [4]. Thus, SDF-1 and its derivatives have the potential to become attractive candidates for the chemotherapy of HIV infection. The human SDF-1 (residues 1–67), having two intramolecular disulfide bonds [5] and one oxidation-sensitive amino acid, Trp (Scheme 1), was synthesized by Springer et al., and it was identical to the purified natural murine SDF-1 in activity [4a]. However, they employed random disulfide bond formation of the fully reduced SDF-1 ([Cys(SH) 9 , 1 1 , 3 4 , 5 0 ]-SDF-1) by air-oxidation. It would be valuable to define the activity of this peptide whose disulfide bridges are unambiguously formed. Thus, we attempted to synthesize SDF-1(1–67) by regioselective disulfide bond formation. The differential S-protection of 4 Cys-residues and a stepwise disulfide bond formation must be carried out regioselectively. Until now, several disulfide bond-forming reactions, i.e., iodine [6], thallium(III) trifluoroacetate [7], DMSO [8] and sulfoxide-silylating reagents [9], have been found to convert some S-protected Cys into cystine by us and others. However, one potential limitation with the use of these reactions is the oxidative modification of the indole ring of Trp. We have recently developed a new procedure for disulfide bond forming reaction in S -Acm-containing peptides [10] using an AgOTf-DMSO/aqueous HCl system (Figure 1) [11]. In this system S -Acm groups are removed by AgOTf from Cys(Acm)-residues [12] and subsequent DMSO/aqueous HCl 398 Y. Shimonishi (ed): Peptide Science – Present and Future, 398–402 © 1999 Kluwer Academic Publishers, Printed in Great Britain

399

Figure 1. Disulfide bond-forming reaction in S-Acm-containing peptides using the AgOTf and DMSO/aqueous HCl system

treatment conducts two reactions: the conversion of Cys(Ag)-residues into cysteine-residues with precipitation of AgCl by the action of HCl, and the disulfide bond formation of cysteine-residues by the action of DMSO in aqueous solution [13]. Under these conditions, no significant side reactions are observed with oxidation-sensitive amino acids such as Met, Tyr and Trp. Thus, we synthesized human and murine SDF-1s(1–67) by the regioselective disulfide bond formation using a combination of the AgOTf-DMSO/aqueous HCl system with air oxidation in order to investigate the feasibility of practical applications of this combination strategy to relatively large and complicated peptides. In addition, we have previously found that a tachyplesin analog, T22 ([Tyr 5,12 , Lys 7 ]-polyphemusin II) [14], has strong anti-HIV activity comparable to that of AZT, and that T22 inhibits a virus-cell fusion. In the present study, we also examined the interaction of T22 with CXCR4/fusin using the synthetic SDF-1.

Results and discussion SDF-1 has a single substitution of Ile to Val at a position 18 between mouse and human. The protected peptidyl resins of human and murine SDF-1s were constructed by Fmoc solid-phase synthesis on Wang resins (Scheme 1) [15]. Two different protecting groups, Trt and Acm, were employed for the protection of 4 side chains of the Cys residues. For the side-chain protection of the other

400 amino acid residues, the following groups were used: Trt for Asn and Gln, Boc for Lys and His, Bu t for Glu, Tyr, Asp, Thr and Ser, and Pmc for Arg. The peptidyl resins were treated with 1M TMSBr-thioanisole/TFA to cleave peptides from the resins and remove the protecting groups except for the S -Acm groups. The resulting crude 2SH, 2Acm-peptides, [Cys(SH) 9,34 , Cys(Acm) 11,50 ] SDF-1s (Scheme 1, 1a and b ), were air-oxidized to establish the first disulfide bond. After HPLC purification, monocyclic products, [Cys(Acm)11,50 ]-SDF-1s (2a and b ), were subjected to sequential treatment with AgOTf/TFA and DMSO/1 M HCl aq. (v/v) to establish the second disulfide bond. Each crude bicyclic product exhibited a sharp main peak on analytical HPLC (data not shown). HPLC purification yielded human and murine SDF-1s (3a and b ) in satisfactory yields [human: 13.8%, calculated from [Cys(Acm) 11,50 ]-human SDF-1 2a, mouse: 16.4%, calculated from [Cys(Acm) 11,50 ]-murine SDF-1 2b ]. Its disulfide-pairings proved to be identical with those of the natural peptide based on ion spray mass analysis of peptide fragments derived from the successive digestion treatment of the synthetic peptide by trypsin (TRCK-treated type XIII from bovine pancreas) and prolyl endopeptidase (from Flavobacterium) (data not shown). [Ion spray-MS (reconstructed), human: Found, 7827.5. Calc. for C 350 H 566 N 104 O 92 S4 , 7826.5; mouse: Found, 7841.0. Calc. for C351 H 568 N 104 O 92 S 4 , 7840.6. Human: [α] 28 D = –92.3° (c = 0.1, H 2O), mouse: [α] 28 D = – 84.8° (c = 0.1, H 2O).] EC 50 of the synthetic human and murine SDF-1s for 50% protection in an assay of HIV-induced cytopathogenicity were 463 nM and 201 nM, respectively, and for 50% inhibition of HIV-1 p24 antigen production were 630 nM and 750 nM, respectively. Furthermore, these peptides have ability to induce an increase in intracellular Ca 2 + mediated by CXCR4/fusin (data not shown). These activities are comparable with the reported data. In conclusion, unequivocal total synthesis of human and murine SDF-1s has been accomplished by stepwise and regioselective disulfide bond formation using air-oxidation and the AgOTf-DMSO/HCl system. This combination method has proven to be useful for synthesis of relative1y large and complicated two disulfide bridges-containing peptides, particularly when Trp residues are present. The synthetic SDF-1 was shown to increase intracellular Ca 2+ levels and inhibit infection by T-tropic HIV-1. It is used to confirm the antagonism of T22 against CXCR4 in the following experiments. Next, we report the relation of T22 with second receptors. First, we examine the effect of T22 on infection. U87MG and HOS cells, expressing CD4 and CXCR4 or CCR5, were used as target cells. Target cells were infected with T-tropic HIV-1(strains: NL432, IIIB) or M-tropic HIV-1 (strains: SF162, NL432env162). NL432env162 is a recombinant T-tropic HIV-1 clone that has the M-tropic HIV Env region. T-tropic HIV-1 infection into the cells expressing CXCR4 and CD4 was inhibited by 0.3 µM T22, but not by 3 µM a control peptide, 4Ala-T-I (Figures 2 and 3). In sharp contrast, M-tropic HIV-1 infection into the cells expressing CCR5 and CD4 was not inhibited by T22. The data

401

Figure 2.

The sequences of T22 and a control peptide 4Ala-T-I.

Figure 3. Inhibition of HIV-1 infection by T22 or 4Ala-T-I(4Ala). a, b) U87MG.CD4 transfected with CXCR4 (a) or CCR5 (b).

obtained in U87MG cells are shown in Figure 3. Similar results were obtained with HOS.CD4 cells (data not shown). These data indicate that T22 specifically inhibits the T-tropic HIV-1 infection mediated by CXCR4, but not the M-tropic HIV-1 infection mediated by CCR5. Second, to examine whether T22 inhibits the T-tropic HIV-1–cell fusion, we used a fusion assay based on α -complementation of β -galactosidase [16]. Cell fusion mediated by CXCR4 and NL432 Env was strongly inhibited by T22 in a dose-dependent manner, but not by 4Ala-T-I. In sharp contrast, T22 had no effect on cell fusion mediated by CCR5 and JRCSF Env (data not shown). Third, we investigated the effect of T22 on Ca 2+ mobilization induced by SDF-1 in order to examine whether T22 interacts with CXCR4. Addition of 3 µM T22 to CXCR4 transfectants extinguished response to a subsequent addition of 100 nM SDF-1, whereas T22 had no effect on Ca2+ induced by MCP-1. 4Ala-T-I had no inhibitory effect on Ca2+ induced by either SDF-1 or MCP-1 (data not shown). This result suggests that T22 is a CXCR4 inhibitor. In the present study, T22 was shown to be a potent inhibitor of infection by T-tropic HIV as well as Ca 2 + mobilization induced by SDF-1. Thus, T22 is a selective CXCR4/fusin inhibitor that blocks T cell-line tropic HIV-1 entry into cells. Our present results will be useful for developing agents against HIV-1 infection.

402 References 1. Nagasawa, T., et al., Proc. Natl. Acad. Sci. USA, 91 (1994) 2305; Bleul, C.C., et al., J. Exp. Med., 184 (1996) 1101. 2. Iizasa, H. and Matsushima, K., Clin. Immunol., 28 (1996) 733 (and references therein). 3. D’Souza, M.P. and Harden, V.A., Nature Medicine, 2 (1996) 1293 (and references therein). 4. a) Bleul, C.C., et al., Nature, 382 (1996) 829; b) Oberlin, E., et al., Nature, 382 (1996) 833. 5. The disulfide array of SDF-1 is deduced from that of human Interleukin 8 which belongs to the same CXC-chemokine superfamily: Clore, G.M., et al., Biochemistry, 29 (1990) 1689. 6. Kamber, B., et al., Helv. Chim. Acta, 63 (1980) 899. 7. Fujii, N., et al., J. Chem. Soc., Chem. Commun., 1987, 163. 8. Otaka, A. et al., Tetrahedron Lett., 32 (1991) 1223. 9. Akaji, K., et al, J. Am. Chem. Soc., 114 (1992) 4137; Koide, T., et al., Synlett, 1991, 345. 10. Veber, D.F., et al., J. Am. Chem. Soc., 94 (1972) 5456. 11. Tamamura, H., et al., Tetrahedron Lett., 34 (1993) 4931. 12. Fujii, N., et al., J. Chem. Soc., Chem. Commun., 1989, 283. 13. Fujii, N., et al., in G. Jung and E. Bayer (Eds) Peptides 1988, Walter de Gruyter, Berlin, 1989, vol. 1988, pp. 58–60; Tam, J.P., et al., J. Am. Chem. Soc., 113 (1991) 6657. 14. Tamamura, H,, et al., Bioorg. Med. Chem., 6 (1998) 1033 (and references therein). 15. Wang, S.S., J. Am. Chem. Soc., 95 (1973) 1328. 16. Tachibana, K., et al., J. Eep. Med., 185 (1997) 1865.

132 Isolation of the nucleoplasmin-like molecular chaperone protein from salmon egg N. SAKAI, K. HOZUMI, K. IWATA, N. SAKAIRI and N. NISHI Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060, Japan

Introduction On the process of fertilization, sperm nuclei are brokendown and decondensation of highly compact chromatin structures occurs preceding the DNA synthesis. This process is apparently coupled to the replacement of sperm nuclear binding proteins (SNBPs), like protamines, by nucleosomal histones [1]. DNA in eukaryotic chromosome is folded into a repeating chain of nucleosome subunits, each consisting of histone core and approximately 200 base pairs of DNA. Thus, the male genome can participate in the chromosomal activities in embryonic development. In the forming process of this ordered nucleosome structure, an assembly factor, nucleoplasmin, which is one of the molecular chaperone proteins and the most abundant protein in the Xenopus laevis oocyte nucleus, is required [2]. Nucleoplasmin has also decondensation activity for amphibian sperm nuclei by removing the SNBPs from DNA. Owing to these two roles of nucleoplasmin that replaces the SNBPs to histone after fertilization, this molecular chaperone protein seems to be very important material in the fertilization process. Nucleoplasmin has decondensation activity not only for amphibian sperm nuclei but also for other species such as human [3] and mussel (Mytilus calfornians) [4]. Recently, we have reported that the sperm nuclei of salmon (Oncorhynchus keta), were also decondensed [5]. Up to now, nucleoplasmin has only been found in amphibian eggs. But, in light of this view, it is expected that this kind of protein is also present in eggs of the other species. In fact, a p22 protein, that has been isolated from Drosphila embryos [6], and a nucleoplasmin-like protein isolated form Mytilus oocyte, are also able to mediate the decondensation of Xenopus sperm nuclei. These results suggest the possibility that nucleoplasmin-like molecular chaperone protein also exists in unfertilized salmon egg. In the present study, nucleoplasmin-like protein was isolated from salmon egg by modified purification method for nucleoplasmin using an open column and dialyzing procedure against water for removing the rich fatty components in the yolk of salmon egg. The nucleoplasmin-like protein from salmon egg induced not only sperm nuclei decondensation, but also reconstitution of nucleosomal structure, like 403 Y. Shimonishi (ed): Peptide Science – Present and Future, 403–406 © 1999 Kluwer Academic Publishers, Printed in Great Britain

404 nucleoplasmin. The protein isolated from salmon egg in the present study would be a new molecular chaperone protein.

Results and discussion Isolation and purification of this protein were carried out principally according to the previous improved method employed for nucleoplasmin from Xenopus laevis [5]. The unfertilized mature eggs of salmon were obtained from the Association of Hokkaido Salmon Propagation, Chitose, Hokkaido. Salmon eggs were homogenized, centrifuged at 10,000 g for 10 min at 2°C and then the supernatant was collected. To remove the rich fatty components of yolk in the salmon egg, the residue was dissolved in water, dialyzed against water, and insoluble part was removed, then dialyzed against the extraction medium (SEM: 150 mM KCl, 2 mM MgCl2 , 10 mM Tris-HCl, pH 7.4). Taking into account that nucleoplasmin is a thermostable protein, the solution was heated at 80°C for 10 min and the insoluble denatured proteins were removed. The solution was then applied for anion exchange chromatography (DEAE-Sepharose CL-6B) and gel filtration chromatography (Superdex 200 HR). Fractions containing proteins were identified by the absorbance at 285 nm, and the fractions with sperm decondensation activity were collected. It was found that the fractions had the decondensation activity not only for salmon sperm nuclei, but also for Xenopus sperm nuclei. Figure 1 (b) is a observation by fluorescence microscope for Xenopus sperm nuclei after incubation for 30 min. An extensively spread out pattern was observed compared to the state before addition of the fraction (a). This fraction was pooled, concentrated and substituted by salmon extraction medium. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the solution without 2-mercaptoethanal showed single band at molecular weight of 35,000.

Figure 1 Fluorescence microscopic observation for Hoechst 33258-stained Xenopus sperm nuclei in the absence (a) and presence (b) of the protein with the salmon sperm decondensation activity. (× 200).

405 On the other hand, it gave two bands at molecular weights of 17,000 and 19,000, when 2-mercaptoethanol was used. The present protein with the salmon sperm decondensation activity seems to be composed of two monomer units. Difference in the molecular weights between two units might be due to the deference in the degree of phosphorylation. In fact, nucleoplasmins from different origins, i.e. from oocyte and mature egg, showed different molecular weights at SDS-PAGE analysis owing to the different degree of phosphorylation. It is known that the nucleosome structure can be observed by agarose gel electrophoresis for nucleohistone that is previously digested by micrococcal nuclease, showing the ~200 bp band. It was investigated whether the present protein from salmon egg has the forming ability of nucleosome structure, like nucleoplasmin, or not. Solutions of histone and the protein from salmon egg were mixed first, DNA solution was added, and then incubated for 1hr at 37°C. The mixture was digested by micrococcal nuclease and then by trypsin in order to hydrolyze proteins. DNA was extracted with neutralized phenol solution and applied to agarose gel electrophoresis (Figure 2). When DNA and histone were incubated without the protein from salmon egg, only very dispersed pattern was observed (lane 2). While lane 1, incubated with the present protein solution, showed clear ~ 200 bp band, similarly as digested nucleohistone (lane 3), indicating the formation of the nucleosome structure. The protein isolated from unfertilized salmon egg is estimated to be a new molecular chaperone protein. We like to call this protein as salmon nucleoplasmin-like protein (SNP).

Figure 2 2% Agarose gel electrophoresis of the DNA digested by micrococcal nuclease in the various situations. Lane 1: DNA solution was added to the mixture of histone and SNP, and then digested. Lane 2: DNA solution was mixed with histone in the absence of SNP solution, and then digested. Lane 3: Nucleohistone was digested as a standard. Lane 4: DNA was digested

406

Figure 3 Amino acid compositions (mol%) of nucleoplasmin and salmon nucleoplasmin-like protein (SNP).

Amino acid composition of the acid hydrolysate of SNP was shown in Figure 3, and compared with that of nucleoplasmin. Amino acids sequence of nucleoplasmin has already been deduced by cDNA, and is known to be a Glurich, acidic protein. It has been estimated that the acidic region in nucleoplasmin interacts with basic DNA-binding proteins such as histone and protamine. On the other hand, the most major amino acid of SNP was Asp rather than Glu. Further, amount of basic amino acids (Lys, Arg) in SNP was found to be smaller than those in nucleoplasmin. Instead, amount of His in SNP is lager than that in nucleoplasmin. Both of Xenopus (amphibians) and Salmon (fish) are vertebrate, and these molecular chaperone proteins are estimated to have similar functional role for histone, protamine and DNA. Difference in the amino acid composition between nucleoplasmin and SNP is very interesting in regard to their interaction mode with the DNA-binding proteins and DNA.

References 1. 2. 3. 4. 5.

Ohsumi, K. and Katagiri, C., Dev. Biol., 148 (1991) 295. Laskey, R.A., Honda, B.M., Mills, A.D. and Finch, J.T., Nature, 275 (1978) 416. Itoh, T., Ohsumi, K. and Katagiri, C., Develop. Growth Differ., 35 (1993) 59. Rice, P., Gorduno, R., Itoh, T., Katagiri, C. and Ausio, J., Biochemistry, 34 (1995) 7563. Iwata, K., Hozumi, K., Itoh, T., Sakairi, N., Tokura, S., Katagiri, C. and Nishi, N., Int. J. Biol. Macromol., 20 (1997) 171. 6. Kawasaki, K., Philpott, A., Avillion, A.A., Berris, M. and Fisher, P.A., J. Biol. Chem., 269 (1994) 10169.

133 Study of neurotoxic peptides from Chinese scorpion Buthus martensi Karsch M.-H. LING¹, Y.-M. XIONG², Z-D. LAN¹, H. FEI¹, D.-C. WANG² and C.-W. CHI¹ ¹ Shanghai Institute of Biochemistry, Academia Sinica, Shanghai China ² Beijing Institute of Biophysics, Academia Sinica, Beijing, China

Introduction The Chinese scorpion Buthus martensi Karsch (BmK), which is widely distributed in northern china, is one of rich sources for the kind of ligands of ion channels [l–6]. After elucidated cDNA and genomic sequences of BmK α-mammalian neurotoxin-BmK M1, which inhibits inactivation of sodium channel resulting in prolonging firing of sodium current [7], using Rapid Amplification of cDNA Ends(RACE) technique, four cDNA genes, which encoded two excitatory insect neurotoxins(BmK IT1-1, BmK IT1-2), one antiepilepsy peptide(BmK AEP), and one new peptide(BmK AS) which enhances the binding of radiolabled-ryanodine on ryanodine receptor in sarcoplasmic reticulum of rabbit skeletal muscle, were cloned and sequenced. The protein sequence of BmK IT1-2 was also determined, which was consistent with that deduced by cDNA sequence, it was worth pointing out that this peptide was found to have an analgesic function.

Results and discussion The method of Rapid Amplification of cDNA Ends (RACE) is exploited for amplifying an unknown nucleic acid sequence from either its 3' or its 5'-end in two steps. cDNAs were first derived from total mRNA by RT-PCR, and then used as templates for RACE amplification. This method of amplification has been used for amplification and cloning of rare mRNAs that may be missed. It raises challenge to conventional cDNA cloning methodologies. By combining the 3'- and 5' partial sequences obtained by RACE method, the full-length cDNA sequence can be cloned. Using this method, two cDNA Sequences of excitatory insect toxins BmK IT1-1 and IT1-2 were elucidated. The open reading frames both encoded the precursor peptides with the signal peptides of 18 amino acid residues, and the mature peptides of 70 and 72 amino acid residues, respectively (Figure 1). The cDNA deduced amino acid sequence of BmK IT1-1 is homologous with its protein sequence except three residues. The cDNA deduced amino acid 407 Y. Shimonishi (ed): Peptide Science – Present and Future, 407–410 ©1999 Kluwer Academic Publishers, Printed in Great Britain

408

Figure 1. The cDNA deduced amino acid sequences of excitatory insect neurotoxins BmK IT1-1 and BmK IT1-2 via 3' and 5' RACE. The different nucleic acids are bolded. The primers of 3' and 5' RACE are underlined.

sequence of BmK IT1-2 is apparently different from that of BmK IT1, furthermore there are two extra residues in its C-terminal. Especially, this peptide was found to have an analgesic biological function. The cDNA sequences of BmK AEP and BmK AS was also determined using the RACE method. The deduced gene of BmK AEP encoded the precursor peptide with the signal peptide of 21 amino acid residues, the mature peptide of 61 amino acid residues, and additional three residues G–K–K at its C-terminal (Figure2). The precursor of this toxin might be amidated at the C-terminal. The deduced amino acid sequence was highly homologous with the depressant insect neurotoxins(date not shown). It implies that this peptide might be evolved from the original of these insect toxins. The cDNA sequence

409

Figure 2. The cDNA deduced amino acid sequence of BmK AEP. The primers of 3' and 5' RACE are underlined.

Figure 3. The cDNA deduced amino acid sequence of BmK AS. The primers of 3' and 5'RACE are underlined.

of BmK AS encoded the precursor peptide of the signal peptide of 19 amino acid residues, and the mature peptide of 66 amino acid residues (Figure 3). The deduced amino acid sequence is completely consistent with its amino acid sequence [8]. The comparison of the amino acid sequences of these toxin precursors (as predicted by cDNAs) and toxins (as determined from peptide sequencing) gives an insight into the precursor structures and the post processing to generate

410 their corresponding toxins. The post-translation processing of BmK AEP is somewhat different from that of insect toxins (BmK IT1-1 and BmK IT1-2) and BmK AS. In addition to the cleavage of the signal peptide, the extra C-terminal G–K–K residues of BmK AEP might be removed by a specific exopeptidase, followed by α -amidation. Whether this α -amidation indispensable of its biological function remaines to be clarified. In order to understand the structure-function relationship of these toxins and their transduction pathway to their corresponding receptors, all these toxins are being expressed.

References 1. Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Xu, K., and Rochat, H., Toxicon, 34 (1996) 987. 2. Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., and Chi, C.W., Toxicon, 35 (1997) 723. 3. Li, H.M., Wang, D.C., Zeng, Z.H., Jin, L., and Hu, R.Q., J. Mol. Biol., 261 (1996) 415. 4. Zhou, X.Y. and Yang, D., Biochem. J., 257 (1989) 509. 5. Ji, Y.H., Xu, K., Kawano, S., Hirayama, Y., Kiraoka, M., Kuniyasu, A., and Nakayama, H., Chinese Sci. Bulletin, 42 (1997) 147. 6. Romi-Lebrun, R., Martin-Eauclaire, M-F., Escoubas, P., Wu, F.Q., Lebrun, B., Hisada, M., and Nakajima, T., Eur. J. Biochem., 245 (1997) 457. 7. Xiong, Y.M., Ling, M.H., Wang, D.C., and Chi, C.W., Toxicon, 35 (1997) 1025. 8. Ji, Y.H., Li, Y.J., Song, B.L., Yamaki, T., Mochizuki, T., Hoshino, M., and Yanaihara, N., In Kawashima, S., Kikuyama, S. (Eds.) Advances in Comparative Endocrinology (Proceedings of the XIIIth International Congress of Comparative Endocrinology), 1997, p 629.

134 Impervious peptides as growth hormone secretagogues R. DEGHENGHI¹ , F. BOUTIGNON¹ , G. MUCCIOLI² , E. GHIGO² and V. LOCATELLI³ ¹ Europeptides, 95108-Argenteuil, France ² University of Turin, 10126-Torino, Italy ³ University of Milan, 20129-Milano, Italy

Introduction The search for orally active, selective GH secretagogues (GHS), starting from Hexarelin (His- D -Mrp–Ala–Trp- D -Phe–Lys–NH 2 , where Mrp is 2-MethylTrp) has led us to shorter ‘impervious’ peptides, so named because they are poor substrates to peptidases. We have also measured the binding affinities of our analogues, and of the known GHS MK-0677 [1], to membranes from different human tissues, including peripheral tissues (heart, adrenals), as a possible indication of selectivity of action [2]. Results and discussion Hexarelin has been downsized to a pentapeptide (EP51216): GAB- D -Mrp-D Mrp-Mrp–Lys–NH 2 (GAB = γ-aminobutyryl) and a tripeptide (EP51389): AibD -Mrp- D -Mrp-NH 2 (Aib = α -aminoisobutyryl), more potent than Hexarelin in the infant rat model [3] and orally active in the dog at 1 mg/Kg [4]. The resistance (‘imperviousness’) to peptidases and protease of Hexarelin (EP23905), the pentapeptide EP51216 and the tripeptide EP51389 was measured in vitro by incubation at 37°C for one hour in conditions that caused extensive degradation of an LHRH analogue chosen as a reference peptide. The results are summarised in Table 1. This table demonstrates the ‘imperviousness’ of EP23905 and EP51389 respectively. Not surprisingly, EP51389 is totally resistant because of its structure. The sensitivity of EP51216 to trypsin and protease is essentially due to the deamidation of the C-terminal amide. Surprisingly, EP23905 (Hexarelin) is very resistant to these enzymes, probably due to its’ ‘cyclic’ structure [5] having a protective effect. Table 1.

EP51216 EP51389 EP23905

(The percentage of degradation is calculated as: 100% of residual peptide). trypsin

chymotrypsin

pepsin

protease

37% 0% 6%

0% 0% 0%

0% 0% 0%

51% 0% 4.5%

411 Y. Shimonishi (ed): Peptide Science – Present and Future, 411–412 © 1999 Kluwer Academic Publishers, Printed in Great Britain

412 Table 2. Inhibition of 125 I-Tyr–Ala–hexarelin binding to membranes from different tissues by various compounds. The displacement of Hexarelin (100% inhibition) was used as control.

Pituitary Hypothalamus Hippocampus Heart Adrenal

Hexarelin

EP-51389

EP-51216

MK 0677

100 100 100 100 100

16 76 40 6 39

75 95 78 93 88

42 35 22 4 14

In search for more selective GHS, we have measured the binding of our analogues, and of the known GHS MK-0677 to membranes from different human tissues, including peripheral tissues (heart, adrenals) by displacement of the radioligand 125 I-Tyr–Ala–hexarelin with 1 µM of the test substance. The results are shown in Table 2. The tripeptide EP-51389 preferentially binds to hypothalamic membranes and has practically no binding (like MK-0677) to the heart. The clinical implications of these findings are presently being investigated.

References 1. Patchett, A.A., Nargund, R.P., Tata, J.R., Chen, M.-H., Barakat, K.J., Johnston, D.B.R., Cheng, K., Chan, W.W.-S., Butler, B., Hickey, G., Jacks, T., Schleim, K., Pong, S.-S., Chaung, L.Y.-P., Chen, H.Y., Frazier, E., Leung, K.H., Chiu, S.-H.L., and Smith, R.G., Proc. Natl. Acad. Sci. USA 92 (1995) 7001. 2. Muccioli, G., Ghè, C., Ghigo, M.C., Papotti, M., Arvat, E., Boghen, M.F., Nilsson, H.L., Deghenghi, R., Ong, H., and Ghigo, E., J. Endocrinol. (1998) in press. 3. Deghenghi, R., Cananzi, M.M., Torsello, A., Battisti, C., Müller, E.E., and Locatelli, V., Life Sciences 54 (1994) 1321. 4 . Deghenghi, R., Boutignon, F., Luoni, M., Rigamonti, A., Cella, S.G., Broglio, F., Arvat, E., Ghigo, E., and Locatelli, V., 2nd Int. Symposium on Growth Hormone Secretagogues, Tampa, FL. February 13–16, 1997 p.9 (abstract). 5. Momany, F.A. and Bowers, C.Y. in Bercu, B.B. and Walker, R.F. (Eds.), Growth Hormone Secretagogues, Springer-Verlag, New York, (1996), p. 73.

135 Effect of carnosine and its natural derivatives on apoptosis of neurons induced by excitotoxic compounds A. BOLDYREV 1,2, D. LAWRENCE³ and D. CARPENTER³ ¹ M.V. Lomonosov Moscow State University, 119899 Moscow, Russia ² Laboratory of clinical neurochemistry, Institute of neurology, Russian Academy of Medical Sciences, 123367 Moscow, Russia ³ Wadsworth Center for Laboratories and Research, University at Albany, SUNY, Albany 12201-0509, USA

Introduction It is well known that long lasting activation of glutamate receptors in neurons results in excitotoxic cell death (either apoptotic or necrotic). This phenomenon is accompanied by an increased generation of reactive oxygen species (ROS) inside the cells [1, 2]. Generation of ROS induced by exposure of neurons to N-methyl- D -aspartic acid (NMDA) or kainate believes to be primary reason for their excitotoxic effect resulting in cell death [2]. In attempt to find natural defence mechanism against this kind of excitotoxicity we focused attention on neuropeptide carnosine which is known as hydrophylic antioxidant protecting neurons against un-favorable conditions in vivo [3].

Results and discussion Exposure of rat neurons preliminary loaded with dichloro-dihydro-fluorescein (DCF), to NMDA or kainic acid (100 µM–1.5 mM) results in increase in DCF fluorescence because of the rise in the intracellular ROS formation. Effect of both NMDA and kainate on the DCF-supported fluorescence depends on time and concentration of the ligand. Maximal level of fluorescence is achieved at 1 mM of ligand after 20–30 min exposure. In both cases, carnosine pre-loaded neurons were characterized by much slower response to excitotoxic ligands. Washing of the neurons pre-loaded with carnosine not sufficiently weakened its effect, while addition of carnosine to the medium containing untreated cells simultaneously with NMDA or kainate was only partially effective. It was suggested that main effect of carnosine consists of rather quenching of ROS generated inside the cell than suppression of interaction of glutamate receptors with the ligands used. This is in agreement with known ability of carnosine to interact with superoxide radicals and hydroxyl radicals to neutralize their toxic effects [4]. 413 Y. Shimonishi (ed): Peptide Science - Present and Future, 413-415 © 1999 Kluwer Academic Publishers, Printed in Great Britain

414 Table 1.

Effect of carnosine on the excitotoxic death of rat cerebellum granule cells Distribution of neurons, %

Conditions

Viable

Necrotic

Apoptotic

CONTROL NMDA, 1 mM, 30 min + CARNOSINE KAINATE 1 mM, 60 min + CARNOSINE

79 ± 5 64 ± 3 75 ± 3 54 ± 4 68 ± 3

6±3 7±2 9±4 7±3 12 ± 5

15 ± 5 27 ± 5 15 ± 3 38 ± 6 16 ± 4

Suppression by carnosine of excitotoxic activation of glutamic receptors is a dose dependent and has a K 0.5 value at 0.75 mM carnosine which lies within the physiological range of carnosine content in vertebrate brain (0.7–2.5 mM). We have compared ability of carnosine and its derivatives, homocarnosine ( γ-aminobutiryl-histidine) and acetyl-carnosine to suppress kainate induced fluorescence of the neurons. High concentrations of kainate induced progressively increased cell death being either apoptotic or necrotic. We have measured the former with annexin 5 containing Apoptotic Detection Kit and the latter – by propidium iodide addition. Short-term exposure of the cells to excitotoxic ligands resulted in a decrease in amount of viable cells and subsequent increase in the amount of both apoptotic and necrotic cells. Neurons pre-loaded with carnosine were more resistant against apoptotic transformation which was in agreement with a suppression of DCF-supported fluorescence of the neurons by carnosine. In Table 1 the data are presented characterizing anti-apoptotic effect of carnosine (1 mM final concentration) under different conditions. At the same time, homocarnosine and acetylcarnosine, both demonstrating anti-radical activity close to that of carnosine were absolutely not effective in prevention of apoptosis of neurons. Thus, we conclude that other reactions behind free radical induction of apoptosis are important and these are not modifyed by carnosine related compounds but are also under control of carnosine itself.

Acknowledgements This work was supported by Fullbright Foundation (#21224, 1996–97, USA) and Russian Foundation for Basic Research (#95-04-11069 and 94-04-49078, Russia).

References 1. Patel, M., Day, B.J., Crapo, J.D., Fridovich, I., and McNamara, J.O., Neuron, 16 (1996) 345. 2. McGowan, A.J., Fernandes, R.S., Samali, A., and Cotter, T.G., Biochem. Soc. Transact., 24 (1995) 229.

415 3. Boldyrev, A.A., Dupin, A.M., Pindel, E.V., and Severin, S.E., Comp. Biochem. Physiol., 89B (1989) 245. 4. Boldyrev, A., Stvolinsky, S., Tyulina, O., Koshelev, V., Hori, N., and Carpenter, D., Cell. Molec. Neurobiol., 12 (1997) 259.

136 Morphological differences observed by the different modes of action of the antibacterial peptides, cecropin B and its analogs CB-1, CB-2 and CB-3 on bacterium and cancer cell H.M. CHEN, S.C. CHAN, W.L. YAU, D. SMITH and W. WANG Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Introduction Cecropins were originally discovered in the pupae of the cecropia moth (Hyalophora cecropia) [1] and are involved in humoral immune response against intruders such as non-pathogenic bacteria [2]. In addition to the cecropin’s anti-bacterial function, their inability to lyse healthy mammalian cells [3] while being capable of attacking transformed cells [4] may make them potentially useful as peptide anti-cancer drugs (a different strategy to the currently used chemical anti-cancer drugs). In this communication, we focus mainly on the area of morphological changes induced by cecropin B and its analogs, CB-1, CB-2 and CB-3 on the bacterium, Klebsiella Pneumoniae (KP), and the cancer cell, HL-60 leukemia (HL-60).

Results and discussion The LCs and the IC50 s of CB, CB-1, CB-2 and CB-3 were shown in Table 1. Since CB-3 has negligible effects on KP and HL-60, it was excluded from the TEM investigations. The TEM of the effects of CB, CB-1 and CB-2 on KP were shown in Figure 1. TEM micrographs were taken in the major-axis section view (MASV) and the minor-axis section view (MISV). MASV and MISV micrographs of KP normal cells (Figures 1A and 1B, respectively) show normal ellipse-like shapes. After the cells were treated with cecropin and its analogs, different shapes were observed in the bacteria. KP CB cells show a more convex Table 1.

Measurements of the LC and IC 50 for the peptides on KP and HL-60.

Peptides

LC KP (µ M)

IC50,HL-60 (µ M)

CB CB-1 CB-2 CB-3

0.26 0.39 0.36 N/A

14.1 7.5 9.2 > 50

416 Y. Shimonishi (ed): Peptide Science – Present and Future, 416–418 © 1999 Kluwer Academic Publishers, Printed in Great Britain

417

Figure 1.

TEM of KP by cecropins.

shape (Figures 1C-MASV, 1D-MISV) and KP CB-1 cells show a more concave shapes (Figures 1E-MASV, 1F-MISV). When compared to KP normal cells, K P CB-2 cells showed a similar effect to KP CB-1 cells (TEM data not shown). The TEM effects of CB, CB-1 and CB-2 on HL-60 were shown in Figure 2. A

Figure 2. TEM of HL-60 by cecropins.

418 rounded cell (inside ring area) covered by abundant of cilia and bulbous projections (outside ring area) is observed for normal HL-60 cells (Figure 2A). Section view of HL-60 cells after treatment with CB-1 and CB-2 for 15 min are shown in Figures 2B and 2C, respectively. These results show that the profiles of both the cell itself and the outer area are seriously distorted. Many white bubble-like lumps located outside in HL-60 cells were similar whether they were treated by CB-1 or CB-2. After the same incubation times, HL-60 cells treated by CB (Figure 2D) were relatively less distorted than those treated by CB-1 and CB-2. Further morphological distortions of HL-60 cells treated with CB were shown in Figures 2E and 2F after incubation for 30 min and 60 min, respectively. Both the membrane and microvilli areas of the cell have been badly distorted. For KP, the phenomena of ‘swelling’ and ‘shrinking’ during the cytolysis induced by CB and CB-1/CB-2, respectively, were observed. These appear to be caused by an influx of water into the cell membrane and the leakage of cytoplasm, respectively, due to the type of the pores formed by the peptides. The greater cytolytic capacity of CB on KP cells my be due to the influx of water being more efficient than the leakage of cytoplasm or to CB being more readily retained by the bacterial cells. On cancer cells, the greater cytolytic capacity of the more cationic CB-1 and CB-2 may be attributable to their being retained more readily on the anionic membrane. Further development of theoretical models to explain these different modes of action are proceeding.

References 1. 2. 3. 4.

Steiner, H., Bennich, H., Boman, H.G., Engstrom, A., and Hultmark, D., Nature, 292 (1981) 246. Boman, H.G., Cell, 65 (1991) 205–207. Zasloff, M., Proc. Natl. Acad. Sci. USA, 84 (1987) 5449–5453. Chen, H.M., Wang, W., Smith, D., and Chan, SC., Biochim. Biohys. Acta, 1336 (1997) 171.

137 Isolation and characterization of bioactive peptides from the sea cucumber, Stichopus japonicus M. OHTANI¹, E. IWAKOSHI¹, Y. MUNEOKA¹, H. MINAKATA² and K. NOMOTO² ¹ Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739, Japan ² Suntory Institute for Bioorganic Research, Osaka 618, Japan

Introduction In the previous study, we isolated a number of bioactive peptides from the body wall of the sea cucumber, Stichopus japonicus, and determined the primary structures of four species of the isolated peptides [1]. In the present study, we made further identification of bioactive peptides of the sea cucumber using newly prepared body-wall extract and determined the structures of 14 peptides including the four found in the previous study. Here, we report the primary structures and the actions of the 14 peptides. Results and discussion The extract of the body wall of the sea cucumber (5 kg, 60 animals) was subjected to HPLC purification, and through 5–8 steps, 14 species of bioactive peptides were purified. The purified peptides were subjected to sequence analysis and FAB-MS measurement, and their structures were predicted. Peptides with the predicted structures were synthesized, and the HPLC behavior of each synthetic peptide was compared with that of the corresponding native peptide using both reverse-phase and ion-exchange columns. The structures of the 14 peptides are shown in Table 1. The determined structures showed that any of the peptides dose not appear to be a member of any peptide family identified previously in the other phyla, though the four peptides termed holokinins seem to be significantly related to the vertebrate peptide bradykinin. It is well known that wasps (insects) have bradykininlike peptides as toxic substances. Bradykinin-related peptides may widely be distributed in the animal kingdom. Other two peptides designated stichoMFamide 1 and 2 appear to be members of SALMFamide family. Several members of SALMFamide family have previously been identified in some starfishes [2] and another sea cucumber [3]. These facts suggest that members of SALMFamide family are widely distributed in echinoderms but not in animals of the other phyla. The effects of the 14 peptides were examined on the radial longitudinal muscle (RLM) and the intestine of the sea cucumber. All the peptides were found to have 419 Y. Shimonishi (ed): Peptide Science – Present and Future, 419–420 © 1999 Kluwer Academic Publishers, Printed in Great Britain

420 Table 1. Effects of peptides isolated from the sea cucumber, Stichopus japonicus, on the radial longitudinal muscle (RLM) and the intestine Effect RLM

Intestine

SWYG-1 SWYG-2 SWYG-3

SWYGSLG SWYGTLG SWYGSLA

Inhi or Poten Inhi or Poten Inhi

No No No

Holokinin-1 Holokinin-2 Holokinin-3 Holokinin-1(3–7)

PLGYMFR PLGYM(O)FR PLGY(Br)M(O)FR GYMFR

Inhi Inhi Inhi Inhi

Cont Cont Cont Cont

NGIWYamide Stichopin Sticho-MFamide-1 Sticho-MFamide-2 KIamide-9 GLRFA GN-19

NGIWYamide DRQGWPACYDSKGNYKC GYSPFMFamide FKSPFMFamide KHKTAYTGIamide GLRFA GGRLPNYAGPPRMPWLIHN

Cont Inhi No No Inhi Poten No

Cont No Relax Relax Cont Cont Cont or Relax

-

Structure

-

Name

Inhi: inhibition of twitch contraction. Poten: potentiation of twitch contraction. Cont: contraction. Relax: relaxation. No: no effect. M(O): oxidized Met. Y(Br): (o-Br)-Tyr.

contractile, relaxing, twitch-potentiating or twitch-inhibiting effect on the RLM or the intestine or both of the sea cucumber. These effects of the peptides on the muscles of the sea cucumber are summarized in Table 1. The effects of the peptides were also examined on the anterior byssus retractor muscle of Mytilus edulis (mussel, mollusc), the heart of Meretrix lusoria (clam, mollusc), the crop and the hind gut of Gryllus bimaculatus (cricket, arthropod), the rectum and the vagina of Coturnix japonica (quail, vertebrate) and the ileum of rat. All the peptides, except holokinins, were found not to have any appreciable effect on the muscles of the non-echinodermal animals at least at low concentrations such as 10 –8 M and 10 – 7 M. Holokinins showed bradykinin-like activity on the vertebrate muscles.

Acknowledgement This work was partly supported by THE SUNBOR GRANT from the Suntory Institute for Bioorganic Chemistry.

References 1. Iwakoshi, E., Ohtani, M., Takahashi, T., Muneoka, Y., Ikeda, T., Fujita, T., Minakata, H., and Nomoto, K., Ohno, M. (Ed), Peptide Chemistry 1994, Protein Research Foundation, Osaka, 1995, pp. 261–264. 2. Elphic, M.R., Price, D.A., Lee, T.D., and Thorndyke, M.C., Proc. R. Soc. Lond., 243 (1991) 121. 3. Diaz-Miranda, L., Price, D.A., Greenberg, M.J., Lee, T.D., Doble, K.E., and Garcia-Arraras, J.E., Biol. Bull., 182 (1992) 241.

138 Purification of peptide fractions with anticomplementary activity from red ginseng (Panax ginseng, C.A. Meyer) J.S. AHN¹, T.H. Kwon², H. PARK³ and K.H. KIM 1,2 ¹ Institute of Biotechnology,² Graduate School of Biotechnology, Korea University, Seoul 136–701, Korea ³ Division of Genetics and Physiology, Korea Ginseng and Tobacco Research Institute, Dae Jon 305-345, Korea

Introduction The roots of red ginseng (Panax ginseng, C.A. Meyer) are divided into four groups by their size and quality [1] of which the latter is generally judged by color, particularly the color inside the roots. It is considered that the roots of the red ginseng (normal) have much higher pharmacological values than the white-inside (abnormal). Although the production of the white-inside depends on environmental factors such as temperature, soil content, and light intensity [2], indigenous causes are largely unknown. Special attention has been recently given to biologically active nitrogen compounds which appear to be important for medicinal efficacy of ginseng [3]. In this study tentative peptide fractions from the red ginseng roots were compared with those of the white-inside and partially purified and characterized.

Results and discussion The elution profiles of gel filtration chromatography were very similar between the extracts of the red and the white-inside ginseng roots (data not shown). The fractions showing an anticomplementary activity [4] were pooled and applied to high-pressure liquid chromatography (HPLC), – – – – using Superdex peptide HR10/30. When the HPLC elution profiles of the two were compared at 215 nm, small but different peaks were found (Figure 1) and one of them showed the highest anticomplementary activity (Table 1). Other fractions were also screened to show inhibition to the in vitro topoisomerase activity, among which the fractions #2, 4 and 5 had strong inhibition particularly in topoisomerase I (Figure 2). Those fractions with above activities were further analysed to show a single but broad band on thin layer chromatography and stained with ninhydrin solution. These fractions were also stained on 20% polyacrylamide electro421 Y. Shimonishi (ed): Peptide Science – Present and Future, 421–423 © 1999 Kluwer Academic Publishers, Printed in Great Britain

422

Figure 1. The comparison of the elution profiles of HPLC between the red and the white-inside ginseng. Numbers represent the fraction number used in the table. Table 1. Anticomplementary activities of the fractions before and after HPLC using superdex peptide column. After HPLC (Fraction number)

Test sample

Before HPLC

1

2

3

4

5

Inhibition (%)

82.2

13.8

11.7

30.1 -—

5.1

4.3

Figure 2. The inhibition of the in vitro topoisomerase activity. Numbers represent the fraction numbers used in the HPLC.

phoresis gels at low molecular weight range of less than 5,000 (data not shown). Further purification and structural studies of the putative peptide fractions are underway.

Acknowledgements We thank Dr S.R. Oh at KRIBB for his help for anticomplementary activity and are grateful to the Korean ministry of A&F (the advanced R&D grant

423 #296012–3). J.S.A was supported in part by the graduate school of biotechnology, Korea University.

References 1. Park, M.K., Korean Ginseng (1994) by Korea ginseng and tobacco research institute, p 44. 2. Lee, J.W., Shin, D.Y., and Kim, M.S. (1977) The report for the institute of tobacco technology, 783. 3. Park, H., Cho, B.G., and Lee, M.-K. Korean J. ginseng Sci. 14(2) (1990) 317. 4. Oh, S.R., Jung, K.Y., and Lee, H.K. Agri. Chem. and Biotech. 39(2) (1996) 147.

139 HIV-cell fusion inhibitor, T22 ([Tyr5,12 , Lys 7 ]polyphemusin II), and its related compounds H. TAMAMURA¹, M. WAKI², A. OTAKA¹, T. MURAKAMI³, H. NAKASHIMA4, A. MATSUMOTO²,N. YAMAMOTO3 and N. FUJII¹ ¹ Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan ² SEIKAGAKU Corporation, Higashiyamato, Tokyo 207-0021, Japan ³ Tokyo Medical and Dental University, School of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan 4 Kagoshima University Dental School, Sakuragaoka, Kagoshima 890-8544, Japan

Introduction Tachyplesin and polyphemusin, are highly abundant in the hemocyte debris of horseshoe crabs, and inhibit the growth of Gram-positive and Gram-negative bacteria and some fungi [1]. These peptides also have antiviral activity against vesicular stomatitis virus, influenza A virus and HIV-1 [2]. We have synthesized more than 100 peptide analogs of tachyplesin and polyphemusin in an effort to screen for analogs with enhanced anti-HIV activity. Among these peptides, we found a novel compound, T22 ([Tyr 5,12 , Lys 7 ]-polyphemusin II), which showed strong anti-HIV activity in vitro. EC 50 of T22 for 50% protection in an assay of HIV-induced cytopathogenicity was 2.6 nM, a value comparable to that of AZT (5.2 nM). CC 50 of T22 was 17 µM. Our previous studies showed that T22 exerts its effect on an HIV-cell fusion process, and that T22 binds specifically to both gp120 (an envelope protein of HIV) and CD4 (a T-cell surface protein) [3]. Furthermore, very recently we found that T22 exhibits anti-HIV activity through its specific binding to the second receptor for HIVentry, fusin [4]. T22, which is an 18-residue, Arg-rich peptide amide, takes an antiparallel β -sheet structure with a type II β -turn, that is maintained by two disulfide bridges [5]. Results and discussion Herein, we define pharmacophores of T22 which may aid in the design of more effective compounds. For this purpose, several analogs of T22 were synthesized in order to investigate the biological effects. These modification examined the number of Arg residues and positive charges in the N-terminal and C-terminal regions, the peptide chain length, the disulfide ring structure, the two repeated Tyr-Arg-Lys motifs, a positive charge in the turn region and the aromatic ring of Trp³ (Figure 1). The present SAR study examines the contributions of each region of T22 to activity, cytotoxicity (Table 1). In this study, several compounds possessing 424 Y. Skimonishi (ed): Peptide Science – Present and Future, 424–426  1999 Kluwer Academic Publishers, Printed in Great Britain

425

Figure 1.

Sequences of T22 and its analogs. Orn:

L -ornithine,

Nph:

L -2-naphthylalanine.

Table 1. Anti-HIV activity of T22 analogs. EC50 s are based on the inhibition of HIV-induced cytopathogenicity in MT-4 cells. CC50 s are based on the reduction of the viability of mock-infected cells. SI is shown as CC50 /EC 50 . IC50 s are based on the inhibition of viral antigen expression examined by the indirect IF method. CC 50 ratio is CC 50 of each analog/CC 50 of T22 (M/M). EC5 0 ratio, IC 50 ratio and SI ratio were also calculated similarly. N.T.: not tested. compound

CC 50 ratio

EC 50 ratio

IC 50 ratio

SI ratio

T22 T52 T54 T56 T100 T101 T102 T103 T104 T105 T111 T112 T115 T116 T117 T201 T202 T203 T204

1 3.1 0.88 0.80 3.2 1.5 5.7 1.0 1.2 5.0 0.99 0.98 0.63 0.40 4.7 0.95 1.0 0.72 0.59

1 210 40 0.20 280 4.8 2.0 6.0 20 24 160 2.2 0.40 0.20 6.3 9.1 1.1 0.69 0.066

1 N.T. N.T. N.T. N.T. 1.3 2.5 2.1 11 50 42 2.2 0.64 0.4-0 5.4 6.5 0.93 0.99 0.25

1 0.013 0.023 3.9 0.012 0.31 2.9 0.17 0.058 0.21 0.0063 0.44 1.6 2.0 0.74 0.10 0.96 1.0 8.9

426 higher SIs than that of T22 have been found. These new findings will aid in elucidating the basis for the high anti-HIV activity of T22, and may lead toward rationally designing more effective compounds containing the essential pharmacophores of T22.

References 1. 2. 3. 4. 5.

Miyata, T., et al., J. Biol. Chem., 263 (1988) 16709. Morimoto, M., et al., Chemotherapy 37 (1991) 206. Tamamura, H., et al., Biochim. Biophys. Acta, 1298 (1996) 37. Murakami, T., et al., J. Exp. Med., 186 (1997) 1389. Tamamura, H., et al., Biochim. Biophys. Acta, 1163 (1993) 209; Tamamura, H., et al., Biochem. Biophys. Res. Commun., 205 (1994) 1729.

140 Efficient analogs of an anti-HIV peptide, T22 ([Tyr 5,12 , Lys 7 ]-polyphemusin II), having low cytotoxicity H. TAMAMURA¹, M. WAKI², A. OTAKA¹, T. MURAKAMI³, H. NAKASHIMA 4 , A. MATSUMOTO², N. YAMAMOTO³ and N. FUJII 1a ¹ Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyoku, Kyoto 606-8501, Japan ² SEIKAGAKU Corporation, Higashiyamato, Tokyo 207-0021, Japan ³ Tokyo Medical and Dental University, School of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan 4 Kagoshima University Dental School, Sakuragaoka, Kagoshima 890-8544, Japan

Introduction A tachyplesin analog peptide, T22 ([Tyr5,12 , Lys7 ]-polyphemusin II), has been shown to have strong anti-HIV activity. T22, an 18-residue peptide amide, takes an antiparallel β -sheet structure that is maintained by two disulfide bridges [1]. A recent SAR study of T22 examined contributions of characteristic regions to its activity and cytotoxicity and provided some information [2]. For example, the presence of the major disulfide ring is indispensable. Thus, we have designed and synthesized several shortened analogs of T22 in order to find a more suitable lead compound, TW70 (des-[Cys8,13 , Tyr 9,12 ]-[ D -Lys 10, Pro 11 ]-T22). Herein, we also report our studies on reducing cytotoxicity.

Results and discussion TW70 is a 14-residue analog having one disulfide bridge. The anti-HIV activiy and selectivity index (SI = CC50 /EC 50 ) of TW70 (EC50 = 7.9 nM, SI = 3400) were comparable to those of T22 (EC50 = 6.0 nM, SI = 2900) [3]. The antiHIV activity of TW70 has been correlated with their conformation as analyzed by NMR (Figure 1). TW70 was found to take an antiparallel β -sheet structure similar to that of T22. This result indicates that the molecular size of T22 can be reduced without loss of anti-HIV activity or significant change in its secondary structure. T22 and TW70 are basic peptides containing 5 Arg residues and 3 Lys residues in the molecules. Thus, the number of positive charges might be related in part to relatively strong cytotoxicity of T22 and TW70. Here we synthesized several analogs, in which the number of positive charges was reduced through amino acid substitution using Glu and L -citrulline (Figure 2). As a result, several efficient compounds were found to possess a higher SI than those of T22 and TW70 (Table 1). 427 Y. Shimonishi (ed): Peptide Science – Present and Future, 427–429 © 1999 Kluwer Academic Publishers, Printed in Great Britain

428

Figure 1. Schematic representation of the structure of TW70. Arrow: NOE connectivities.

Figure 2. Amino acid sequences of Glu or L -citrulline substituted analogs. Ci: L -citrulline.

Table 1. Anti-HIV activity and cytotoxicity of these analogs. EC50 s are based on the inhibition of HIV-induced cytopathogenicity. CC 50 values are based on the reduction of the viability of mock-infected cells. SI is shown as CC 50 /EC 50 . IC50 values are based on the inhibition of viral antigen expression. NT.: not tested. Compound

Charges

SI

CC 50 (µ M)

EC 50 (nM)

IC 50 (nM)

T22 TW70 T121 T122 T125 T127 T128 T131 T132 T134 T135 T136

9 9 8 6 7 6 6 8 8 7 7 7

650 1300 2700 > 3600 3400 > 1500 > 3400 4200 14000 33000 4900 14000

16 16 89 > 210 130 > 210 > 210 84 75 120 110 88

25 12 32 57 38 130 61 20 5.4 3.7 22 6.5

39 N.T. 43 78 81 84 89 56 81 14 46 14

429 In conclusion, a reduction of the number of total positive charges was proven to be a useful strategy for developing lowly cytotoxic compounds. Furthermore, there is a positive correlation between the number of the total positive charges and CC 50 values, with total +6 or +7 charges representing a nice balance between activity and cytotoxicity. These findings will aid in the rational design of effective compounds possessing much higher potency and lower cytotoxicity.

References 1. Tamamura, H., et al., Biochim. Biophys. Acta, 1163 (1993) 209. 2. Tamamura, H., et al., Biochem. Biophys. Res. Commun., 205 (1994) 1729. 3. Waki, M., et al., Chem. Lett., 1996, 571.

141 Cyclic RGD peptides with a novel mimetic G. HÖLZEMANN 1 , S.L. GOODMAN1 , C. RÖLZ 2 and H. KESSLER2 1

Frankfurter Str. 250, Merck KG aA, D-64271 Darmstadt, Germany Institut für Organische Chemie und Biochemie der Technischen Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany 2

Introduction The integrins and their ligands regulate a range of cell-cell and cell-matrix interactions. The αv β 3 integrin is expressed on tumor-induced angiogenic blood vessels which makes inhibitors of this receptor valuable tools for therapeutic purposes [1]. A number of cyclic peptides containing the Arg–Gly–Asp(RGD) sequence were reported to be selective inhibitors of this integrin [2]. cyclo(–RGD D -Phe–Val–) [c(RGDfV)], for example, serves as a standard in many biological assays. Whereas the RGD part of the molecule is very sensitive to changes, the remaining two amino acids, D-Phe and Val, were exchanged by a variety of amino acids and building blocks without loosing affinity or selectivity for the αv β 3 integrin [3,4]. We have synthesized a substituted 2-(3-aminomethyl-phenylamino)-propionic acid building block and incorporated it into cyclic RGD peptides. The 3-aminomethyl-phenylamine (AMP) residue is similar to the Mamb (m-(aminomethyl)benzoic acid) residue described by A.C. Bach, II, and co-workers [5].

Results and discussion The synthesis of the building block involves a nucleophilic substitution of an appropriate α -bromo ester with 3-amino benzylalcohol. Here, we used the α-bromo esters derived from the amino acids phenylglycine, phenylalanine, and valine. After conversion of the benzyl alcohol into a benzyl amine and subsequent protection, the building block was ready for incorporation into the peptide. The linear peptide was build up with C terminal Gly in order to prevent racemization at the cyclization step. The diastereomeric mixtures of the peptides caused by the racemic building block were either separated after cyclization or after deprotection. To obtain the final products we had to cleave the β benzyl ester of aspartic acid and the Mtr group of the arginine side chain. Hydrogenation of the benzyl ester gave the desired peptides, whereas basic cleavage yielded rearranged products at the Asp residue [6]. 430 Y. Shimonishi (ed): Peptide Science – Present and Future, 430–431 © 1999 Kluwer Academic Publishers, Printed in Great Britain

431

3: R = Phenyl 4: R = Benzyl 5: R = Benzyl 6: R = Isopropyl 7: R = Isopropyl

Figure 1. Structures of the cyclic RGD peptides described in this work. The compounds represent diastereomeric pairs with the exception of 3.

After assignment of the resonances HMBC and ROESY spectra were recorded to obtain information on the connectivity of the Asp residue. Diagnostic cross peaks were observed between the Asp carbonyl carbons and AMP HN and Ηα protons. The assignments were supported by ROESY cross peaks between the AMP NH and Asp Hα or H β protons. To determine the chirality at the new building block, distance geometry and simulated annealing calculations were performed using NMR derived constraints. Analysis of the structures yield only hints for the S and R configuration at Phe Cα of 4 and 5, respectively. The binding affinity of the peptides was measured using immobilized integrin receptors and biotinylated soluble ligands [3,4]. Compounds 1 and 2 are almost as potent as the standard peptide c(RGDfV) on the αv β 3 receptor, and more potent on the receptor which makes them less selective. The stereochemistry at the new building block has only a small influence on the binding to the α v β3 receptor. The rearranged cyclic peptides 3–7 are app. 10-fold less active than the desired RGD analogues. In conclusion, we have obtained cyclic RGD peptides with a novel mimetic. Asp side chain rearranged peptides were unambiguously identified by NMR spectroscopy.

References 1. Strömblad, S. and Cheresh, D.A., Chem. & Biol. 3 (1996) 881. 2. Haubner, R., Finsinger, D., and Kessler, H., Angew. Chem. Int. Ed. Engl. 36 (1997) 1374. 3. Haubner, R., Gratias, R., Diefenbach, B., Goodman, S.L., Jonczyk, A., and Kessler. H., J. Am. Chem. Soc. 118 (1996) 7461. 4. Haubner, R., Schmitt, W., Hölzemann, G., Goodman, S.L., Jonczyk, A., and Kessler, H., J. Am. Chem. Soc. 118 (1996) 7881. 5. Bach, II, A.C., Eyermann, C.J., Gross, J.D., Bower, M.J., Harlow, R.L., Weber, P.C., and DeGrado, W.F., J. Am. Chem. Soc. 116 (1994) 3207. 6. Lender, A., Yao, W., Sprengeler, P.A., Spanevello, R.A., Furst, G.T., Hirschmann, R., and Smith, III, A.B., Int. J. Peptide Protein Res. 42 (1993) 509.

142 Isolation and cDNA cloning of oryctin, a novel cysteine-rich antibacterial peptide, from the coconut rhinoceros beetle, Oryctes rhinoceros. J. ISHIBASHI 1, H. SAIDO-SAKANAKA1 , H. NAKAZAWA1 , M. YAMAMOTO 2 and M. YAMAKAWA1 1 Laboratory of Biological Defense, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan 2 Pharmaceutical Discovery Research Laboratories II, Teijin, Hino, Tokyo 191-0065, Japan

Introduction Although insects do not have an immune system that involves antigen-antibody reactions, they possess efficient self-defense mechanisms against bacterial infection, for example, antibacterial peptides are known to play an important role in insect immunity. To date, over 150 insect antibacterial peptides have been isolated and their structures determined [1, 2]. Among these peptides, few antibacterial peptides except insect defensins were revealed to contain cysteine residues. Here we report a novel cysteine-rich antibacterial peptide designated oryctin, which was isolated from hemolymph of the coconut rhinoceros beetle, Oryctes rhinoceros.

Results and discussion Potent antibacterial activity was induced in hemolymph of O. rhinoceros 3rd instar larvae upon injection of bacteria. An antibacterial peptide, oryctin, was purified from hemolymph of O. rhinoceros larvae immunized with Escherichia coli by 4 steps of reversed phase HPLC using Staphylococcus aureus as indicator bacteria. Finally, about 1.0 µg of oryctin was obatained from 1.0 ml of the hemolymph. Oryctin showed antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria. The N -terminal amino acid sequence was determined by amino acid sequencer. The oryctin cDNA was cloned by 3 steps of polymerase chain reaction using fat body mRNA from immunized larvae. Deduced amino acid sequence from the nucleotide sequence indicated that oryctin contains a 85 amino acid precursor, and a mature peptide (66 amino acids) is assumed to be produced by cleavage of the signal peptide (Figure 1). Oryctin was identified to contain 6 cysteine residues, and the positions of the residues were completely different from that of the insect/arthropod defensin consensus sequence [3]. Moreover, there is no sequence homology between oryctin and any other known proteins. The calculated mass of oryctin 432 Y. Shimoniski (ed): Peptide Science – Present and Future, 432–433 © 1999 Kluwer Academic Publishers, Printed in Great Britain

433

Figure 1. Nucleotide and deduced amino acid sequences of oryctin cDNA. Amino acid sequence in bold letters represents the mature sequence. Nucleotide sequence underlined represents polyadenylation signal.

was in good agreement with measured value, suggesting that oryctin is a monomeric peptide and has neither oligosaccharide chain nor other modifications. The calculated isoelectric point of oryctin was 6.45, which is significantly lower than that of other antibacterial peptides.

Acknowledgments This work was supported by Enhancement of Center of Excellence, Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan.

References 1. Boman, H.G., Annu. Rev. Immunol. 13 (1995) 61–92. 2. Miyanoshita. A. and Yamakawa, M., Bio Industry 13 (1996) 28–39. (A review in Japanese) 3. Cociancich, S., Goyffon, M., Bontems, F., Bulet, P., Bouet, F., Menez, A., and Hoffmann, J., Biochem. Biophys. Res. Commun. 194 (1993) 275–279.

143 Eumenine mastoparans. A new class of mast cell degranulating peptides in the eumenine wasp venom K. KONNO 1, H. TAKAYAMA1 , M. HISADA 2, Y. ITAGAKI 2, H. NAOKI2, N. KAWAI3, A. MIWA4 , T. YASUHARA5 and Y. NAKATA6 1

Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan Suntory Institute for Bioorganic Research, Shimamoto, Osaka 618, Japan 3 Department of Physiolgoy, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan 4 Department of Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183, Japan 5 Department of Nutrition, Junior College of Agriculture, Tokyo University of Agriculture, Setagaya, Tokyo 156, Japan 6 Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Hiroshima, Hiroshima 734, Japan 2

Introduction Solitary wasps paralyze insects or spiders with stinging venom and feed the paralyzed victims to their larvae. Accordingly, the venoms should contain a variety of constituents acting on nervous systems [1]. However, only a few constituents of the venoms have been chemically characterized, despite that thousands of solitary wasp species inhabit on the earth [2–4]. We have been investigating the constituents of solitary wasp venoms by using the lobster leg muscle preparation as a bioassay system, and found a novel peptide neurotoxin from a spider wasp venom [5]. On the other hand, mastoparan-like peptides named eumenine mastoparans have been found in the eumenine wasp venoms. We report here the isolation, structure, and biological effects of eumenine mastoparans, a new class of mast cell degranulating peptides. Results and discussions The venom reservoirs of the female Anterhynchium flavomarginatum micado, the most abundant eumenine wasp in Japan, were dissected, and the lyophilized venom reservoirs were extracted with 1:1 MeCN-H 2 O/0.1% TFA. The extracts were subjected to reversed phase HPLC to isolate eumenine mastoparan-AF as the major constituent of this venom. The amino acid sequence was analyzed by mass spectrometry (FAB-MS/MS and MALDI-TOF-CID) together with Edman degradation, and corroborated by the solid-phase synthesis. This peptide inhibited the neurotransmission of the lobster leg muscle at 0.1–1 mM, causing membrane depolarization and muscle contraction. In a similar way, eumenine mastoparan-ER was isolated from the venom of Eumenide rubronotatus. The sequence was analyzed mainly by the ladder 434 Y. Shimonishi (ed): Peptide Science – Present and Future, 434–435 © 1999 Kluwer Academic Publishers, Printed in Great Britain

435 sequencing combined with MALDI-CID-MS [6], and corroborated by the solid-phase synthesis. Eumenine mastoparan-AF Ile-Asn-Leu-Leu-Lys-Ile-Ala- Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH 2 Eumenine mastoparan-ER Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr Mastoparan Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala -Leu-Ala-Lys-Lys-Ile-Leu-NH 2 The structures of these peptides are similar to that of mastoparan, a mast cell degranulating peptide in a hornet venom. In fact, both of them were active in stimulation of histamine release from RBL-2H3 cells; eumenine mastoparan-AF was as active as mastoparan, whereas eumenine mastoparan-ER was somewhat less than that.

Acknowledgments We thank Drs S. Yamane (Biological Laboratory, Faculty of Education, Ibaraki University) and A. Endo (Laboratory of Biology, Faculty of Science and Engineering, Ritsumeikan University) for collection and identification of wasps. Thanks are also due to Dr. T. Nakajima (Suntory Institute for Bioorganic Research) for his invaluable discussion.

References 1. Piek, T. and Spanjer, W. In Piek, T. (Ed) Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioral Aspects, Academic Press, London, 1986, pp. 169–307. 2. Eldefrawi, A.T., Eldefrawi, M.E., Konno, K., Mansour, N.A., Nakanishi, K., Oltz, E., and Usherwood, P.N.R., Proc. Natl. Acad. Sci. USA, 85 (1988) 4910. 3. Piek, T., Fokkens, R.H., Karst, H., Kruk, C., Lind, A., van Marle, J., Nakajima, T., Nibbering, N.M.M., Shinozaki, H., Spanjer, W., and Tong, Y.C., In Lunt, G.G., (Ed) Neurotox ‘88: Molecular Basis of Drug and Pesticide Action, Elsevier, Amsterdam, 1988, pp. 61–76. 4. Yasuhara, T., Mantel, P., Nakajima, T., and Piek, T., Toxicon, 25 (1987) 527. 5. Konno, K., Miwa, A., Takayama, H., Hisada, M., Itagaki, Y., Naoki, H., Yasuhara, T., and Kawai, N., Neurosci. Lett., 238 (1997) 1. 6. Hisada, M., Itagaki, Y., Naoki, H., Nakajima, T., Konno, K., and Takayama, H., will be reported in due course.

144 Direct evidence of the generation of lactoferricin from ingested lactoferrin H. KUWATA 1,2, T.T. YIP³, M. TOMITA¹ and T.W. HUTCHENS 2,3 ¹ Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., Kanagawa, Japan ² Department of Food Science and Technology, University of California, Davis, CA, USA ³ Ciphergen Biosystems, Inc., Palo Alto, CA, USA

Introduction Lactoferrin is an 80 kDa protein present in various biological fluids and in granules of neutrophils [1]. It is especially abundant in mammalian colostral whey and is thought to play an important protective role in nursing infants. However, little is known about the digestive fate of lactoferrin in the gastrointestinal tract. We have used surface-enhanced laser desorption/ionization (SELDI) in a process known as affinity mass spectrometry to evaluate the in vivo generation of lactoferricin ® (Lfcin), a peptide with potent antimicrobial and immunomodulatory properties released from the N-terminal region of lactoferrin by pepsin cleavage.

Results and discussion Surface-enhanced affinity capture (SEAC) is a method which consists of affinity capture of the analyte and ordered presentation of the molecules for subsequent analysis by laser desorption/ionization mass spectrometry [2]. As the affinity capture component of the SEAC probe, we used an immobilized terminal n-butyl group. Unlike previous efforts to detect Lfcin with antibodies, the n-butyl SEAC device distinguished Lfcin from intact lactoferrin, partially degraded lactoferrin, lactoferrin-derived peptides, and unrelated peptides. In model studies, human (Gly¹ – Ala 49 , Arg²–Ile 50 ), porcine (Ala¹–Ala 46 , Ala¹ – Ala 48 ) and bovine (Phe 17–Phe 41, Phe 17 –Ala 42 ) Lfcin generated in vitro were found to bind specifically to this SEAC probe. The level of detection of Lfcin by SELDI affinity mass spectrometry in the model mixture (i.e. serum) was typically 200 pg/mL–25 ng/mL, while by MALDI mass spectrometry under the same conditions it was more than 20 µg/mL. To evaluate Lfcin generation in vivo, the experimental design involved feeding 200 mL of 10 mg/mL (1.22 × 10 – 4 mol/L) bovine lactoferrin to an adult volunteer, and the gastric contents were recovered 10 min after ingestion. Lfcin produced in vivo was directly captured by the SEAC device (Figure 1). The amount of Lfcin in the gastric contents was determined to be 436 Y. Shimonishi (ed): Peptide Science – Present and Future, 436–438 © 1999 Kluwer Academic Publishers, Printed in Great Britain

437

Figure 1. MALDI (upper) and SELDI (lower) mass spectra of the gastric contents from a volunteer who ingested lactoferrin. Molecular ion peaks in the MALDI spectrum were not resolved because of the high complexity of the gastric contents. Identified molecular ion peaks are shown with asterisks.

16.9 ± 2.7 µg/mL (5.4 ± 0.8 × 10 –6 mol/L). However, a large proportion of the ingested lactoferrin was not completely digested. Lactoferrin fragments containing the Lfcin region were further analyzed by in situ hydrolysis with pepsin after being captured by the SEAC device (Figure 2). As much as 5.7 ± 0.7 × 10 – 5 mol/L of the partially degraded lactoferrin fragments were found to contain the Lfcin region, including peptide domains 17–43, 17–44, 12–44,9–58 and 16–79 of bovine lactoferrin. These results show that bovine Lfcin can be produced in the human stomach after ingestion of an

Figure 2. Mass spectra of gastric contents before (thin line) and after (thick line) in situ hydrolysis. Each of the spectra was normalized by the peak intensity of an internal standard.

438 infant formula supplemented with bovine lactoferrin. It is now important to determine whether Lfcin is generated in the intestinal tract of formula-fed and breast-fed infants, and geriatric patients consuming foods enriched with lactoferrin.

References 1. Masson, P.L., Heremans, J.F., and Schonne, E., J Exp. Med., 130 (1969) 643. 2. Hutchens, T.W and Yip, T.T., Rapid Commun., Mass Spectrom., 7 (1993) 576.

145 Human immunodeficiency virus type 1 Vif-derived peptides inhibit the viral protease and arrest virus production C. GILON¹, A. FRIEDLER¹, L. BARAZ², I. BLUMENZWEIG², O. NUSSINUV², M. STEINITZ³ and M. KOTLER² ¹ Department of Organic Chemistry, The Hebrew University, Givat ram, Jerusalem 91904, Israel ² Department of Moleculur Genetics and ³ Department of Pathology, The Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel

Introduction Vif is one of the auxiliary proteins expressed by Human immunodeficiency virus type 1 (HIV-1) and conserved among HIV-1 isolates and in lentiviruses. Vif is required for productive HIV-1 infection of peripheral blood lymphocytes (PBL) and macrophages in cell culture [1, 2]. Vif inhibits the viral protease (PR)-dependent auto-processing of truncated HIV-1 Gag–Pol precursors expressed in bacterial cells and efficiently inhibits the PR-mediated hydrolysis of peptides in cell-free systems. The obstructive activity of Vif was assigned to the 92 amino acids residing at the N' terminus of Vif (N' Vif) [3]. To determine the minimal Vif sequence essential for the inhibition of PR, we synthesized overlapping peptides derived from N'-Vif. These peptides were then assessed using two in vitro and two in vivo systems: (i) inhibition of purified PR, (ii) binding of PR, (iii) inhibition of the autoprocessing of the Gag–Pol polyprotein expressed by a vaccinia virus vector (vVK-1) and (iv) inhibition of mature virus production in human cells. The peptides derived from two regions of N' Vif,† encompassing residues Tyr30–Val65 and Asp78–Val98, inhibited PR activity in both the in vitro and the in vivo assays.

Results and discussion The sequences of the overlapping N' Vif derived peptides are shown in Figure 1. Peptides 4, 41, 43, 6 and 7 were found to inhibit PR in vitro. Overlapping Peptides 6 and 7, were found to be the most potent inhibitors. The interaction of Vif-derived peptides with PR was tested by ELISA using rabbit anti-PR. Microtiter plates were coated with peptides and PR was then† added with or without preincubation with the cognate peptide. Peptides 4,41, 42, 43 and 6, but not Peptide 3 significantly bound PR. Binding of PR to absorbed peptides was specifically inhibited by preincubation with the cognate peptide 439 Y. Shimonishi (ed): Peptide Science – Present and Future, 439–441 © 1999 Kluwer Academic Publishers, Printed in Great Britain

440

441 Next, we analyzed the effect of Vif-derived peptides on the processing of HIV-1 precursors expressed in eucaryotic cells by a vaccinia vector (vVK-1) [4]. Infection of human T-cell lines with vVK-1 resulted in high levels of Gag protein production within 24h, allowing the natural PR-mediated processing of viral precursors and the release of virus-like particles. Extracts prepared from vVK-1 infected cells and from the particles exported to the medium contained completely or partially processed Gag proteins, namely, p55Gag, MA-CA p41 and CA p24. Extracts from vVK-1 infected Hut 78 and CEM incubated for 24h with peptides 42 and 43 contained the same HIV-1 polyproteins present in the control vVK-1 infected cells. However, lysates of cells incubated with Peptides 41, 6 and 7 contained the unprocessed MA-CA and p55Gag polyproteins and only small amounts of mature CA protein. The effect of Vif-derived peptides on virus maturation was assessed by incubating Hut 78 cells with the peptides for 48 h 5 days post infection with HIV-1IIIB. Treatment of cells with Vif-derived peptides did not influence the autoprocessing of Gag polyproteins. On the other hand, Peptides 41, 6 and 7 but not Peptides 42 and 43, prevented the release of particles containing CA into the culture media. Peptides 41 and 6 reduced the titer of the infectious virus by three orders of magnitude, whereas Peptide 7 lowered the titer by 6 to 10 fold. Thus, we conclude that Peptides 41 and 6, and to a lesser extent Peptides 43 and 7, interfere with the autoprocessing of viral precursors in human cells infected with vVK-1 or HIV-1 and cause a significant reduction in the number of infectious virions released from the HIV-1 infected cells. Our finding that Vif, N '-Vif and Vif-derived peptides inhibited the autoprocessing of Gag and Gag–Pol polyproteins in bacteria and eucaryotic cells is consistent with the supposition that Vif is responsible for the delay in PR activation. Such vif-derived peptides provide an attractive potential therapeutic agent for inhibition of HIV PR during HIV-l infection in humans.

References 1. Gabuzda, D.H., Lawrence, K., Langhoff, E., Terwilliger, E., Dorfman, T., Haseltine, W.A., and Sodroski, J., J. Virol. 66,6489–6495 (1992). 2. Von Schwedler, U.U., Song, J., Aiken, C., and Trono, D. J. Virol. 67,( 1993) 4945–4955. 3. Kotler, M., Simm, M., Zhao, Y.S., Sova, P., Chao, W., Ohnona, S.F., Roller, R., Krachmarov, C., Potash, M.J., and Volsky, D., J. Virol. 71, 5774–5781 (1997). 4. Karacostas, V., Nagashima, K., Gonda, M.A., and Moss, B., Proc. Natl. Acad. Sci. USA 86, 8964–8967 (1989).

146 A novel cardioactive peptide of Aplysia contains a D -amino acid residue F. MORISHITA1 , Y. NAKANISHI1, S. KAKU1, Y. FURUKAWA1 , S. OHTA2, T. HIRATA3 , M. OHTANI4 , Y. FUJISAWA4 , Y. MUNEOKA4 and O. MATSUSHIMA¹ 1

Department of Biological Science, Faculty of Science, Hiroshima University, Higashi-Hiroshima, 739, Japan 2 Instrumental Center for Chemical Analysis, Hiroshima University, Higashi-Hiroshima, 739, Japan 3 Department of Chemistry, Faculty of Science, Hiroshima University Higashi-Hiroshima, 739, Japan 4 Laboratory of Physiology, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739, Japan

Introduction The cardiovascular system is essential in many aspects of physiological functions in animals. In molluscs, cardiac activation is involved in various stereotyped behaviors such as reproduction [1]. Now, the significance of the neuropeptides, as well as non-peptidic neurotransmitters such as serotonin [2], has been recognized in the regulation of cardiovascular system. However, regulatory mode and the chemical nature of the relevant peptides are not fully understood in the cardiovascular system of molluscs. One practical approach to investigate the peptidergic regulation of cardiovascular system is to purify and structurally identify cardioactive peptides from the hearts themselves, and then analyse their physiological function(s). We started such a project on the heart of an opisthobranch gastropod, Aplysia kurodai, because, this animal has many identifiable neurons in its ganglia, which provids us a suitable model system to investigate the physiological actions of neuropeptides. In this paper, we summerize the purification, structure determination and bioactivity of a novel cardio-excitatory tripeptide which contains a D -tryptophan.

Results and discussion To purify the cardioactive substances, the heart (auricle, ventricle and cristae aorta) was isolated from A. kurodai. The crude extract from 750 hearts was subjected to the sequential HPLC separation, using the reversed phase or cation-exchange columns. By testing the effect of each fraction on Aplysia heart, a cardio-excitatory substance was purified. It was sequenced by the automated Edman degradation analysis as Asn–Trp–Phe. The result of mass spectrometry 442 Y. Shimonishi (ed): Peptide Science – Present and Future, 442 – 443 © 1999 Kluwer Academic Publishers, Printed in Great Britain

443 (FAB-MS) was m/z = 465.2 [M + H+ ]. Based on these analysis, we synthesized a tri-peptide of which structure wasAsn–Trp–Phe–NH 2 . However, elution profiles of the synthetic peptide was different from those of native peptide. Possible reason for this disagreement is the presence of D -amino acid(s) in the native peptide. Among the synthetic D -amino-acid containing peptides tested, elution profiles of a peptide, Asn-D -Trp–Phe–NH 2, was exactly the same to those of native one. Thus, we concluded the structure of this peptide as AsnD -Trp–Phe–NH 2 (NdWFamide). Although some D -amino acid-containing peptides such as fulicin [3] have been known, no structually related peptide has been reported so far. NdWFamide increased the tension of heart beating and the rhythmic contraction of the anterior aorta with the threshold concentrations around 10 –11 M. NdWFamide appeared to be multi-functional peptide, because it also modified the bursting activity of R15 neuron that has been known to regulate the cardiovasclar system of Aplysia [4]. Thus, this peptide may also mediate both central and peripheral functions in A. kurodaii. NdWFamide was cardioactive on the heart of land snail, Euhadra congenita, but not on those of other four molluscan spices tested. Presumably, NdWFamide itself or its related peptide may regulate the cardiac function of pulmonates. In any experiments tested so far, the bioactivities of synthetic NWFamide which contains L -Trp were negligible. Probably, the D -configuration of tryptophan may contribute to stabilize the peptide so that it fits in the receptor. The signal transducing pathway mediating the actions of NdWFamide and the distribution of the peptide among the Aplysia tissues remaine to be elucidated.

References 1. Ligman, S.H. and Brownell, P.H. J. Comp. Physiol. 157A (1985) 31 2. Liebeswar, G., Goldman, J.E., Koester, J. and Mayeri, E. J. Neurohysiol. 38 (1975) 767 3. Ohta, N., Kubota, I., Takao, T., Shimonishi, Y., Yasuda Kamatani, Y., Minakata, H., Nomoto, K., Muneoka, Y. and Kobayashi, M. Biochem. Biophys. Res. Commun. 178 (1991) 486 4. Skelton, M.E., and Koester, J., (1992) J. Comp. Physiol. A, 171 (1992) 141

147 Purification and primary structure of four novel neuropeptides in Aplysia kwrodai K. NAKAMARU¹ , F. MORISHITA¹ , Y. FURUKAWA¹ , S. OHTA², T. HIRATA², M. OHTANI³, T. TAKAHASHI³, Y. FUJISAWA³, Y. MUNEOKA³ and O. MATSUSHIMA¹ ¹Department of Biological Science, Faculty of Science, Hiroshima University, Higashi-hiroshima 739, japan

² Instrumental Center for Chemical Analysis, Hiroshima University, Higashi-hiroshima 739, Japan ³ Physiological Laboratory, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-hiroshima 739, Japan

Introduction The opisthobranch mollusc, Aplysia californica, has been widely used for neurobiological experiments, since its central nervous system (CNS) is relatively simple in structure and comprises only several tens of thousands of neurons. Since discovery of FMRFamide in a molluscan species, important roles of neuropeptides have been delineated in regulation of physiological processes. Especially, peptidergic modulation of the accessory radula closer muscle by two identified cholinergic motor neurons, B15 and B16, has been elaborately studied in relation to feeding behaviors [1–4]. Peptidic substances involved in such modulation include myomodulins [1,2], small cardioactive peptides (SCP) [3] and buccalin [4]. We report here isolation and structural determination of novel myoactive peptides from the CNS of the closely related species, Aplysia kurodai, which show potent inhibitory effects on spontaneous contractions of isolated gizzard of the animal.

Results and discussion Aplysia kurodai were collected at the seashore in Hiroshima and Shimane Prefectures. All ganglia constituting the CNS were isolated from about 500 animals and pulverized in liquid nitrogen. Peptidic fraction was extracted by boiling the powdered CNS in 4% acetic acid for 10 min and applied to disposable C18 columns. The material retained with the columns (RM) was eluted with methanol. Biological activities of an aliquot (1/1,000) of the RM were examined on isolated preparations of different parts of digestive organs. Isolated preparations of the esophagus, gizzard and intestine exerted somewhat rhythmical spontaneous contractions. The RM showed potent inhibitory effects on contractile acitivity of all tested tissues. Then, the RM was subjected to procedures for purification of the myosuppressive substance(s), which 444 Y. Shimonishi (ed): Peptide Science – Present and Future, 444–445 © 1999 Kluwer Academic Publishers, Printed in Great Britain

445 included high performance liquid chromatography (HPLC) and bioassay with isolated gizzard. Through several steps of HPLC using reversed-phase and cation-exchange columns, four inhibitory peptides were isolated and structurally analysed by amino acid sequence analysis and FAB-MS measurement; Pro–Arg–Gln–Phe–Val–NH 2 , Ala–Pro–Gly–Tyr–Ser–His–Ser–Phe–Val–NH 2, Val–Pro–Gly–Tyr–Ser–His–Ser–Phe–Val–NH 2 and Arg–Leu–Pro–Ser–Tyr– Gly–His–Ser–Phe–Leu–NH 2.All these peptides are somewhat homologous to each other in that all these peptides have -FV(L)amide as their C -terminal structure and that the fourth residues from the C-terminus are basic. We could also isolate eight more peptides highly homologous to these four peptides by enzyme-linked immunosorbent assay (ELISA) using antibody raised against one of the Mytilus Inhibitory Peptides (MIP), GSPMFVamide, which was originally isolated from the pedal ganglia of a bivalve mollusc, Mytilus edulis [5,6]. This is probably because of the fact that the four peptides found in the present study as well as MIP have -FV(L)amide at their C -termini. Although physionogical function of these inhibitory peptides still remains to be investigated, these neuropeptides may play a principal role in regulation of gut motility and seem to constitute an important peptide family in invertebrate phyla.

References 1. Cropper, E.C., Tenenbaum, R., Kolks, M.A.G., Kupfermann, I. and Weiss, K., Proc. Natl. Acad. Sci. USA, 84(1987) 5483. 2. Cropper, E.C., Vilim, F.S., Alevizos, A., Tenenbaum, R., Kolks, M.A.G., Rosen, S., Kupfermann, I. and Weiss, K., Peptides, 12 (1991) 683. 3. Cropper, E.C., Lloyd, P.E., Reed, W., Tenenbaum, R., Kupfermann, I. and Weiss, K., Proc. Natl. Acad. Sci. USA, 84 (1987) 3486. 4. Cropper, E.C., Miller, M.W. Tenenbaum, R., Kolks, M.A.G., Kupfermann, I. and Weiss, K., Proc. Natl. Acad. Sci. USA, 85 (1988) 6177. 5. Hirata, T., Kubota, I., Iwasawa, N., Takabatake, I., Ikeda, T. and Muueoka, Y., Biochem. Biophys. Res. Commun., 152 (1988) 1376. 6. Fujisawa, Y., Zool. Sci., 13 (1996) 795.

148 Structure and function of fragments of antibiotic bactenecin 5 H. AOYAGI, M. TSUIKI, K. YOSHIDA, T. NIIDOME and T. HATAKEYAMA Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852-8521, Japan

Introduction Bactenecin 5 (Bac 5) is a cationic antibiotic peptide consisting of 43 amino acid residues. It has a repeating region of Arg–Pro–Pro–Phe (or Ile) [1]. To clarify the structure-function relationship of the repeating region in Bac 5, we previously synthesized Ac-(Arg–Pro–Pro–Phe) n –NHCH 3 ( n = 2, 4, 6, 8 and 10) as models. These peptides were supposed to have a polyproline II-like structure by CD measurement and a γ -helical structure by energy calculation. However, no antibacterial activity was found [2]. In the present study, we synthesized four peptides containing the N-terminal portion of Bac 5 and the repeating region models, i.e., H–Arg–Phe–Arg–Pro–Pro–Ile–Arg–(Arg–Pro–Pro–Phe)n– N H 2 (n = 2, 3, 5 and 7) (1, 2, 3 and 4 ) and investigated their properties and biological activities.

Results and discussion The CD measurements of the peptides were performed under various conditions. All the peptides showed a negative band at 204 nm with a slight shoulder at 227 nm in the buffer solution (pH 7.4) and in the presence of DPPC vesicles at 25 and 50°C. The CD curve resembled that of polyproline II. Similar result was obtained in the presence of DPPC/DPPG (3/1) vesicles at 25°C. However, all the peptides changed their conformations at 50°C. Since Bac 5 was reported to interact with inner membranes of sensitive bacteria and inhibit the function of membrane proteins, the membrane-perturbing activity of the peptides to leak carboxyfluorescein (CF) form CF-entrapped vesicles was examined. In the presence of DPPC vesicles, no leakage was observed at 25°C and little leakage at 50°C. Together with the result of CD study, this observation suggested that the peptides hardly interact with DPPC (neutral) lipid. They again showed negligible leakage activity for DPPC/DPPG (3/1) (acidic) lipid vesicles at 25°C, whereas strong membrane perturbation was found at 50°C and the order of activity was 1 < 2 < 3 < 4 (Figure 1a). It can be said that long chain length of peptide and electrostatic interaction of cationic 446 Y. Shimonishi (ed): Peptide Science – Present and Future, 446–447 © 1999 Kluwer Academic Publishers, Printed in Great Britain

447

Figure 1. CF leakage (a) and vesicle fusion (b and c). a: DPPC/DPPG( 3/1), [Lipid] = 70 µM, 50°C. b: DPPC; c: DPPC/DPPG( 3/1), [Lipid] = 70 µ M, 25°C. (∆ ) 1 ; (
) 2; ( ) 3; ( ◊ ) 4.

group in peptide with anionic portion in phospholipid are important for membrane perturbation. Membrane perturbation is usually accompanied by membrane fusion. In measurements of membrane-fusion activity at 25°C, the peptides showed the maximum activities of 15–20% for DPPC vesicles at 5 µ M of peptides and 100% for DPPC/DPPG (3/1) vesicles at 2–5 µ M (Figure 1b,c). However, no antibacterial activity against Gram-positive and -negative bacteria nor hemolytic activity was found. There are two different reports on the antimicrobial activity of Bac 5. One describes that the N -terminal portion is important for the activity [3], and another presents the importance of the repeating region [4]. Further conformational and biological study is necessary for explanation for the exact role of the repeating region in Bac 5.

References 1 . Frank, R.W., Gennaro, R., Schneider, K., Prizybyski, M. and Romeo, D., J. Biol. Chem., 265 (1990) 18871. 2 . Niidome, T., Mihara, H., Saiki, T., Oka, M. and Aoyagi, H., In Kitada, C. (Ed) Peptide Chemistry 1996, Protein Research Foundation, Osaka, 1997, pp. 185–188. 3. Gennaro, R., Scocchi, M., Skerlavaj, B., Tossi, A. and Romeo, D., In Hodges, R.S. and Smith, J.A. (Eds) Peptides: Chemistry, Structure and Biology, ESCOM, Leiden, 1994, pp. 441–463. 4 . Raj, P.A., Marcus, E. and Edgerton, M., Biochemistry, 35 (1996) 4314.

149 Production of adrenomedullin in macrophages A. KUBO, N. MINAMINO, Y. ISUMI, T. KATAFUCHI, K. KANGAWA and H. MATSUO National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan

Introduction We have demonstrated that cultured endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) produce and secrete adrenomedullin (AM) [1, 2]. The production and secretion of AM from ECs and VSMCs were augmented with interleukin-1 (IL-l), tumor necrosis factor α (TNF- α) and lipopolysaccharide (LPS) [3, 4]. In the in vivo study, injection of LPS into rats elevated plasma AM concentration 20 fold, and the plasma levels of AM in patients with septic shock were 40–50 times higher than those in healthy volunteer [5, 6]. Macrophages are activated by exposure to stimuli of LPS, and start to produce and secrete various cytokines, such as IL-1 and TNF-α, which are known to induce septic shock. These data suggest that macrophage may be another candidate for AM producing cell in sepsis in addition to ECs and VSMCs. To elucidate functions of AM produced and secreted from macrophage, we investigated AM production in murine monocyte/macrophage cell line (RAW 264.7) as well as mouse peritoneal macrophages.

Results and discussion AM concentrations in culture media of RAW 264.7 cells were measured after 14 h incubation by using radioimmunoassay specific for AM. We first examined effects on AM production of various substances inducing differentiation or activation of RAW 264.7 cells (Table 1). Phorbol ester (TPA), retinoic acid (RA), LPS and interferon-γ (IFN- γ ) increased AM production maximally about 6 fold. Although a basal secretion level of AM from RAW 264.7 cells was much lower than that of rat VSMCs, the stimulated secretion rate from RAW 264.7 cells was comparable to that of VSMCs. IL-1 β and TNF- α showed no effect on AM production from RAW 264.7 cells, although these substances augmented AM production in ECs and VSMCs (Table 1) [3, 4]. RA, which stimulates differentiation into macrophage, exerted synergistic effect on AM production when applied with TPA, LPS and IFN-γ, while IFN- γ completely suppressed AM production in RAW 264.7 cells being stimulated with LPS (data not shown). Dexamethasone, estradiol and TGF- β suppressed AM production (Table 1). We further investigated AM production from mouse peritoneal 448 Y. Shimonishi (ed): Peptide Science – Present and Future, 448–449 © 1999 Kluwer Academic Publishers, Printed in Great Britain

449 Table 1.

Effect of various substances on AM production in RAW 264.7 cells

Substances

Concentration

IR-AM (fmol/10 5 cells/14 h)

Control TPA RA LPS IFN-γ TNF-α IL-1β TGF-β Dexamethasone Estradiol

10 – 7 M 10 – 6 M 100 ng/ml 100 U/ml 50 ng/ml 10 ng/ml 10 ng/ml 10 – 5 M 10 –5 M

0.860 1.411 1.403 5.475 3.989 0.972 0.958 0.641 0.205 0.577

± 0.036 ± 0.108* ± 0.043* ± 0.054* ± 0.068* ± 0.029 ± 0.021 ± 0.016* ± 0.012* ± 0.014*

Each values represents mean ± SEM of six separate dishes. *) P < 0.05 vs. control. Table 2.

Production of AM in mouse peritoneal macrophages

Substances

Concentration

IR-AM (fmol/10 5 cells/24 h)

Control TPA RA IFN-γ LPS

10 – 7 M 10 – 6 M 100 U/ml 1000 ng/ml

0.20 ± 0.04 0.19 ± 0.01 0.28 ± 0.03 0.18 ± 0.02 1.83 ± 0.35*

Each values represents mean ± SEM of five separate dishes. *) P < 0.05 vs. control

macrophages. Peritoneral macrophage also secreted AM and its production was stimulated about 9 fold with LPS (Table 2). Even in the macrophages, RA exerted synergistic effect on AM production with LPS, while IFN-γ suppressed AM production stimulated with LPS (data not shown). Based on these findings, macrophage should be recognized as one of sources of circulating AM. Especially in the case of septic shock, AM production in macrophage is deduced to be highly augmented by the stimulation of LPS, and the secreted AM may cooperatively participate in the pathogenesis of sepsis with that from ECs and VSMCs.

References 1. Sugo, S., Minamino, N., Kangawa, K., Miyamoto, K., Kitamura, K., Sakata, J., Eto, T., and Matsuo, H., Biochem. Biophys. Res. Commun., 201(1994) 1160. 2. Sugo, S., Minamino, N., Shoji, H., Kangawa, K., Kitamura, K., Eto, T., and Matsuo, H., Biochem. Biophys. Res. Commun., 203 (1994) 719. 3. Sugo, S., Minamino, N., Shoji, H., Kangawa, K., Kitamura, K., Eto, T., and Matsuo, H., Biochem. Biophys. Res. Commun., 207 (1994) 25. 4. Isumi, Y., Shoji, H., Sugo, S., Tochimoto, T., Yoshioka, M., Kangawa, K., Matsuo, H., and Minamino, N., Endocrinology, 139 (1998) 838. 5. Shoji, H., Minamino, N., Kangawa, K., and Matsuo, H., Biochem. Biophys. Res. Commun., 215 (1995) 531. 6. Nishio, K., Akai, Y., Murao, Y., Doi, N., Ueda, S., Tabuse, H., Miyamoto, S., Dohi, K., Minamino, N., Shoji, H., Kitamura, K., Kangawa, K., and Matsuo, H., Crit. Care Med., 25 (1997) 953.

150 Adrenomedullin production in fibroblast cell lines Y. ISUMI, N. MINAMINO, T. KATAFUCHI, A. KUBO, K. KANGAWA and H. MATSUO National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan

Introduction Adrenomedullin (AM), a potent vasorelaxant peptide, was first isolated from human pheochromocytoma tissue [1]. Our systematic survey has demonstrated that cultured vascular smooth muscle cells (VSMC) and endothelial cells (EC) actively produce and secrete AM and that their AM gene transcription levels are much higher than that of adrenal gland in the rat [2, 3]. TNF, IL-l and LPS were found to be the most potent stimulants of AM production in VSMC and EC, suggesting that AM may participate in the induction of hypotension in septic shock [2, 4]. In fact, plasma AM concentration was markedly elevated in LPS-injected rats and in patients with septic shock [5]. In LPS-injected rats, AM mRNA levels were elevated in almost all tissues [6]. These data indicate that vascular cells are major sites of AM production, especially in the case of the septic shock. At the same time, non-vascular cells of some tissues were deduced to be able to produce AM in the septic shock.

Results and discussion Lung is one of the candidates for AM production in the non-vascular cells. We found that cultured normal human lung fibroblast (NHLF) secretes immunoreactive (IR-) AM. We further examined and found that fibroblasts (Swiss 3T3 (mouse), NHLF and Hs68 (human skin)) are generally able to produce and secrete AM. To elucidate the regulation mechanism of AM production in fibroblast, we measured IR-AM concentrations in the culture medium after 14 h incubation with stimulants by using RIA specific for AM. Among them, TNF-α, IL-1β , LPS and dexamethasone augmented AM production, while TGF- β 1, and IFN- γ potently suppressed AM production (Table 1). AM receptors were expressed on fibroblasts, and AM increased cAMP production in a dose-dependent manner. Especially, Swiss 3T3 cells have high affinity AM receptors (Figure 1). Moreover, AM stimulated DNA synthesis in Swiss 3T3 cells via the elevation of intracellular cAMP in a dose-dependent manner (Figure 2). These results indicate that AM production in fibroblast is regulated by a variety of substances in a manner quite similar to that of rat VSMC and EC. Based on the data obtained in this study, AM secreted from fibroblast is 450 Y. Shimonishi (ed): Peptide Science – Present and Future, 450–452 © 1999 Kluwer Academic Publishers, Printed in Great Britain

451 Table 1.

Effects of various substances on IR-AM secretion from fibroblasts. IR-AM (fmol/10 5 cells/l4 h)

Substance Control TNF-α IL-1β LPS TGF- β1 IFN-γ Dexamethasone

Concentration

Swiss 3T3 cells

Hs68 cells

NHLF cells

50 ng/ml 10 ng/ml 100 ng/ml 10 ng/ml 100 U/ml 10 –7 M

3.34 9.73 4.12 3.89 1.11 1.46 8.80

92.1 ± 1.4 190.5 ± 3.8 a 197.7 ± 2.5 a 184.5 ± 4.2 a 43.5 ± 0.4 a 79.2 ± 1.0 a 110.6 ± 1.9 a

95.6 ± 1.8 125.2 ± 2.4 a 143.5 ± 3 . 0 a 97.6 ± 1.9 29.3 ± 0.5 a 69.0 ± 1.4a 120.8 ± 1.6 a

Each value represents mean ± SEM of six wells.

± 0.05 ± 0.16 a ± 0.07 a ± 0.07 b ± 0.03 a ± 0.06 a ± 0.29 a a

P < 0.01 vs. control.

b

P < 0.05 vs. control.

Figure 1 Stimulation of cAMP production in Swiss 3T3 cells with AM, CGRP and AM antagonists.

Figure 2 Effect of AM in combination with insulin on DNA synthesis.

deduced to function as an autocrine and paracrine regulator of fibroblast. In the tissue, mesenchymal cells including fibroblasts are the target cells of LPS and inflammatory cytokines which stimulate AM production. The secreted AM may act on surrounding cells expressing AM receptors, indicating that AM could be a local regulator mediating inflammatory stimuli in addition to its potent effect as a vasorelaxant in the blood vessels.

452 References 1 . Kitamura, K., Kangawa, K., Kawamoto, M., Ichiki, Y., Nakamura, S., Matsuo, H. and Eto, T., Biochem. Biophys. Res. Commun., 192 (1993) 553. 2 . Sugo, S., Minamino, N., Shoji, S., Kangawa, K., Kitamura, K., Eto, T. and Matsuo, H., Biochem. Biophys. Res. Commun., 203 (1994) 719. 3 . Sugo, S., Minamino, N., Kangawa, K., Miyamoto, K., Kitamura, K., Sakata, J., Eto, T. and Matsuo, H., Biochem. Biophys. Res. Commun., 201 ( 1994) 1160. 4 . Isumi, Y., Shoji, H., Sugo, S., Tochimoto, T., Yoshioka, M., Kangawa, K., Matsuo, H. and Minamino, N., Endocrinology, 139 (1998) 2552. 5 . Nishio, K., Akai, Y ., Murao, Y., Doi, N., Ueda, S., Tabuse, H., Miyamoto, S., Dohi, K., Minamino, N., Shoji, H., Kitamura, K., Kangawa, K. and Matsuo, H., Crit. Care. Med., 25 (1997) 953. 6 . Shoji, H., Minamino, N., Kangawa, K. and Matsuo, H., Biochem. Biophys. Res. Commun., 215 (1995) 531.

151 Chemical and biological characterization of neuropeptides in the sinus glands of the kuruma prawn, Penaeus japonicus H. NAGASAWA¹, W.-J. YANG², K. AIDA² and H. SONOBE³ ¹ Department of Applied Biological Chemistry, University of Tokyo, Bunkyo, Tokyo 113, Japan ² Department of Aquatic Biosciences, University of Tokyo, unkyo, Tokyo 113, Japan ³ Department of Biology, Faculty of Science, Konan University, Kobe 658, Japan

Introduction It has been known that the X-organ/sinus gland complex in the eyestalk of crustaceans produces a number of neuropeptides, which regulate carbohydrate metabolism, molting, vitellogenesis, and body color change [1]. The amino acid sequences of some neuropeptides, hyperglycemic hormone (CHH), moltinhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH), have been shown to be similar, and they formed a peptide family designated a CHH-family. They were further classified into two types, Types I and II, according to the amino acid sequences [2]. Type I included CHH, and Type II consisted of MIH, VIH, and MOIH. However, some peptides showed more than two activities. We attempted to isolate and characterize CHH-family peptides from the marine prawn Penaeus japonicus.

Results and discussion We have reported the isolation and structural determination of six CHH-family peptides (Pej-SGP-I-VI) from the sinus glands of the kuruma prawn P. japonicus [2]. In the present experiment, a seventh peptide (Pej-SGP-VII) was purified to homogeneity by the same procedure as in the previous six peptides. This peptide was most abundant among the seven CHH-family peptides. It was sparingly soluble in aqueous solution but soluble in 30% acetonitrile containing 0.9% sodium chloride or in 2 M urea. Thus, the crude extract in 0.9% sodium chloride containing 2 M urea was applied to reverse-phase HPLC. The amino acid sequence of Pej-SGP-VII was determined by combination of aminoterminal sequence analysis of its S-carboxymethylated derivative with sequence and mass spectral analyses of tryptic and endoproteinase Asp-N fragment peptides: AAFDPSCTGVYDRELLRRLSRLCDDCYNVFREPKVAMECRSNCFFNPAFVQCLEYLIPAELHEEYQALVQTV(amide). Pej-SGP-VII was most similar to Pej-SGP-V and VI, and was classified into Type I. It consisted 453 Y. Shimonishi (ed): Peptide Science – Present and Future, 453–454 © 1999 Kluwer Academic Publishers, Printed in Great Britain

454 of 72 amino acid residues with conserved six cysteine residues and a carboxylterminal amide like other peptides except for Pej-SGP-IV. The arrangement of intramolecular three disulfide bonds was determined on Pej-SGP-III. This peptide was first digested with trypsin and the resulting large fragment was further digested with chymotrypsin, affording two fragments. One contained a disulfide bond and straightforwardly the connection between Cys 7 and Cys 43 was established. The other contained two disulfide bonds and was digested with endoproteinase Asp-N. Sequence analysis of the product established the connections between Cys23 and Cys 39 , and between Cys 26 and Cys 52 by detection of phenylthiohydantoin derivative of cystine. Pej-SGP-I, II, III, V, VI, and VII showed hyperglycemic activity in a dosedependent manner, when they were injected into eyestalk-ablated kuruma prawn, while only Pej-SGP-IV did not (partly reported in [3]). The six peptides were almost equally potent, but the efficacy of elivating glucose levels in the hemolymph of eyestalk-ablated prawns was in the following order: V, VI, VII > I, III > II. Conversely, Pej-SGP-IV inhibited synthesis and release of ecdysteroid by the Y-organ of the crayfish, Procambarus clarkii, cultured in vitro [4], but the other six peptides did not except for Pej-SGP-V and VI which showed much weaker molt-inhibiting activity than IV (Pej-SGP-VII not tested). Thus, structure-activity relationship was clear; Type I peptides had hyperglycemic activity, and a Type II peptide had molt-inhibiting activity. Comparison of the amino acid sequence of Pej-SGP-IV with those of V and VI suggested the importance of Phe 45 and Tyr 46 , which were present only in these three peptides, for molt-inhibiting activity. However, more information about threedimensional structure of these peptides and their receptors is required for definite conclusion.

References 1. 2. 3. 4.

Keller, R., Experientia, 48 (1992) 439. Yang, W.-J., Aida, K., and Nagasawa, H., Aquaculture, 135 (1995) 205. Yang, W.-J., Aida, K., and Nagasawa, H., Peptides, 18 (1997) 479. Yang, W.-J., Aida, K., Terauchi, A., Sonobe, H., and Nagasawa, H., Peptides, 17 (1996) 197.

152 Preparation of a poly(ethylene glycol) hybrid containing two active fragments of laminin M. MAEDA¹, M. KAWASAKI¹, Y. KANEDA², Y. MU² Y. TSUTSUMI², S. NAKAGAWA² and T. MAYUMI² ¹ Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Arise 518, Ikawadani-cho Nishi-ku, Kobe 651–21, Japan ² Faculty of Pharmaceutical Sciences, Osaka University, Yamadaoka 1-6, Suita-shi, Osaka 565, Japan

Introduction Since poly(ethylene glycol) (PEG) is stable, bioinert and only weakly immunogenic, it appears to be a promising drug carrier. We have prepared biologically active PEG hybrids of laminin- and fibronectin-related peptides with PEG, and have searched more convenient PEG modification for peptide synthesis. An amino acid type PEG (aaPEG) can be used to prepare a bifunctional peptide-PEG hybrid of X-PEG-Y (where X and Y are different peptides). BocaaPEG-OH is obtained by coupling poly(oxyethylene) dipropylamine with succinic anhydride and subsequently reacting the product with (Boc) 2 O [1]. In the present study, we developed a simple method for the preparation of aaPEG from poly(oxyethylene)diglycolic acid, and prepared FmocaaPEG-OH, which can be extensively used in the solid phase peptide synthesis. Laminin, a cell adhesion protein, consists of three peptide chains (A, B1 and B2), and promotes adhesion and growth of epithelial and tumor cells. The B1 chain has the sequences Pro–Asp–Ser–Gly–Arg (PDSGR) and Tyr–Ile–Gly– Ser–Arg (YIGSR), which have been found to inhibit experimental metastasis in mice [2]. The PEG-hybrids of these active peptides, PDSGR-PEG and PEG-YIGSR, also have inhibitory effects [3]. In the present study, we prepared PDSGR-aaPEG-YIGSR using the new aaPEG method and assessed its antimetastatic activity.

Results and discussion Amino acid type PEG (aaPEG) was prepared by adding excess ethylenediamine to a preactivated poly(oxyethylene)diglycolic acid with DCC (Figure 1). A range molecular weight of Poly(oxyethylene)diglycolic acid was 2500 ~ 3400. After gel-filtration on sephadex G-25, it was reacted with Fmoc-OSu, and the reaction product was purified by gel-filtration on sephadex LH-20. Thus obtained Fmoc-aaPEG-OH was used for usual solid phase synthesis. Starting 455 Y. Shimonishi (ed): Peptide Science – Present and Future, 455– 456 © 1999 Kluwer Academic Publishers, Printed in Great Britain

456

Figure 1 Preparation of Fmoc-aaPEG-OH.

from amide–Rink–Resin, H–Tyr(Bu t )–Ile–Gly–Ser (Bu t )–Arg(Pmc)–Rink– Resin was obtained using DIPC coupling followed by 20% piperidine according to the Fmoc strategy. Fmoc-aaPEG-OH was reacted triplicatedly to the protected peptide resin until Kaiser test became negative. After deprotection of the Fmoc at N -terminus, stepwise coupling was continued at N-terminus by solid phase method. At the last amino acid coupling, Boc–Pro–OH was employed and Boc–Pro–Asp(OBu t )–Ser(Bu t )–Gly–Arg(Pmc)–aaPEG–Tyr)Bu t )– Ile–Gly–Ser(Bu t )–Arg(Pmc)–Rink–Resin was prepared. The final deprotection and cleavage were achieved by TFA treatment, and the final product was purified by reverse-phase HPLC. Amino acid ratios from an acid hydrolysate of the product were: Pro 0.60, Asp 1.00, Ser 2.00, Gly 2.44, Arg 1.60, Tyr 1.17, Ile 1.23. The peptide content of the hybrid was 0.30 mmol/g. Prior to examination of the inhibitory effect of this hybrid on experimental metastasis in mice, we confirmed that PDSGR-aaPEG-YIGSR had no cytotoxicity. The hybrid and B16-BL6 melanoma cells were separately injected through the mouse tail vein. Two weeks later, the number of melanoma colonies on the lung surface was counted microscopically. PDSGR-aaPEG-YIGSR exerted inhibitory activity dose-dependently. The inhibitory effect of 375 nmol of PDSGR-aaPEG-YIGSR was more potent than that of 375 nmol of YIGSR or PDSGR alone.

References 1. Izuno, Y., Kurosaka, T., Maeda, M., Kawasaki, K., Kaneda, Y., Mu, Y., Tsutsumi, Y., Nakagawa, S. and Mayumi, T., Peptide Chemistry 1996 (1997) 265 2. Kleinman, H.K., Graf, J., Iwamoto, Y., Sasaki, M., Schasteen, C.S., Yamada, Y., Martin, G.R. and Robey, F.A., Arch. Bioehem. Biophys., 272 (1989) 39 3. Kawasaki, K., Murakami, T., Namikawa, M., Mizuta, T., Iwai, Y., Yamashiro, Y., Hama, T., Yamamoto, S. and Mayumi, T., Chem. Pharm. Bull., 42 (1994) 917

153 Modification of an anti-fungal peptide (ESF-1) for enhanced inhibitory activity and resistance to proteolysis J. HASTINGS¹, V. VADYVALOO¹, G. DYKES¹, K. TAMURA² and S. AIMOTO² ¹ Department of Genetics, University of Natal, Pietermaritzburg, South Africa ² Institute for Protein Research, Osaka University, Japan

Introduction Over the past decade many naturally occurring antimicrobial peptides have been isolated and include linear peptides (magainins, and bacteriocins such as leucocin and pediocin), disulfide linked peptides (insect defensins), lantibiotics (nisin) and synthetic antimicrobial peptides (ESF-1) [1]. ESF-1 is a synthetic peptide which mimicks the charge distribution and amphipathic α -helical structure of part of PGLa, a magainin peptide from frogs [2]. A previous study by Powell et al. [2] showed that ESF-1 and variants had particularly strong antimicrobial activity against pathogenic fungi of importance to commercial crop plants [2]. Of all the peptide variants studied, ESF-1 itself showed the greatest activity against Fusarium oxysporum, with a reported MIC of 2.5 µM (6 µg/ml) [2]. Since Fusarium oxysporum f.sp. cubense race 4 is an important pathogen of Musa sp (banana) ESF-1 respresents a possible mechanism of disease control. In this study we investigated the substitution of amino acids in ESF-1 for the purpose of enhancing its inhibition of microorganisms and resistance to proteolytic breakdown by banana plant extracellular fluid extracts.

Results and discussion ESF-1 is an amphipathic twenty amino acid peptide which adopts a typical α -helix conformation in membrane mimicking environments such as micellular SDS, while remaining randomly coiled in aqueous solution (Figure 1). ESF-1 and two variants, with substitutions at positions seven (ESF-GR7) and position three and seven (ESF-SA3) respectively, were synthesized and tested for activity against Carnobacterium mobile, a quick growing bacterial indicator of peptide activity (Table 1). The synthetic variants showed a 20 fold increase in activity using a standardised microtitre well assay as described by Hancock et al. [3]. As one of the potential applications of ESF-1 is the protection of banana plants from Fusarium oxysporum f.sp. cubense race 4, the stability of the peptide 457 Y. Shimonishi (ed): Peptide Science – Present and Future, 457–459 © 1999 Kluwer Academic Publishers, Printed in Great Britain

458

Figure 1. lar SDS.

Circular dichroism spectra of ESF-1 in aqueous solution and in the presence of micellu-

Table 1. Minimum inhibitory concentration Carnobacterium mobile DSM 4848

(MIC)

of

ESF-1

and

variants

against

MIC Peptide name

Aminio acid sequence

Carnobacteriun mobile

ESF-1 ESF-SA3 ESF-GR7 ESF-HPQ

MASRAAGLAARLARLALRAL MAARAARLAARLARLALRAL MASRAARLAARLARLALRAL HPQMASRAAGLAARLARLALRAL

50 nM 2.5 nM 2.5 nM 1 µM

in the presence of banana plant extracellular fluid was determined. The addition of banana plant extracellular fluid to the MIC assay eradicated biological activity from all peptides. HPLC analyses were conducted in the presence and absence of banana extract as well as in the presence of protease inhibitors. The proteolytic activity of the banana fluid initially appeared to be due to the action of exoproteases. However, the addition of the HPQ motif to the N-terminus of ESF-1 did not enhance its stability and resulted in a peptide with lowered activity (Table 1). Further examination of HPLC traces after treatment with banana plant extracellular fluid revealed several early eluting degradation products of ESF-1. Sites of proteolytic cleavage were determined by amino-terminal sequencing of HPLC fractions showing it occured at a specific internal amino acid motif. Degradation products were not evident after co-treatment with a proteinase inhibitor cocktail (complete , Mini (Boehringer Mannheim).

Acknowledgements This work was partially supported by grants to John W Hastings from AECI (Pty) Ltd., and the Foundation for Research Development (SA).

459 References 1. Rao, A.G., Molecular Plant-Microbe Interactions., 8 (1995) 6–13. 2. Powell, W.A., Catranis, C.M., and Maynard, C.A., Molecular Plant-Microbe Interactions, 8 (1995) 792–794. 3. Hancock, R.E.W., [WWW document] (1997, 28 October) URL http://www.interchange.ubc.ca/

154 Structure determination and cDNA cloning of paralytic peptide in Bombyx mori S.-D. HA¹, A. SUZUKI¹, S. NAGATA¹, M. TANAKA² and H. KATAOKA¹ ¹ Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ² Institute of Molecular and Cellular Bioscience, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Introduction Insect hemolymph contains various proteinaceous components essential for insect growth and development, such as transport proteins, storage proteins, peptide hormones, and defensive peptides induced by bacterial invasion or injury. In 1991, a curious fact was found that the lepidopteran larvae, Manduca sexta, were paralyzed when the hemolymph obtained from other larvae of the same species was injected [1]. Then the factor was isolated from a several kind of lepidopteran hemolymph and characterized as 23 amino acid peptides. We here report the isolation and structure determination of paralytic peptide from hemolymph of the silkworm, Bombyx mori, and the molecular cloning of the cDNA of this peptide. Results and discussion About 13 µg of paralytic peptide was purified from 19 ml of B. mori hemolymph by the procedure consisted of extraction with 50% acetone, Vydac C 4 reversed phase cartridge and 4 steps of reversed phase HPLC. The purified peptide caused rapid, rigid paralysis when injected into the fourth instar larva of B. mori at the dose of 3.7 ng/animal. The complete amino acid sequence of the paralytic peptide was determined as H-Glu-Asn-Phe-Val-Gly-Gly-Cys-AlaThr-Gly-Phe-Lys-Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-OH by sequence analysis and mass spectrometry of native and carboxymethylated peptides. The synthetic peptide with a disulfide bond in a molecule showed the same retention time on HPLC and paralytic activity indistinguishable from that of the natural peptide. The homology between Bombyx and other paralytic peptide is 70–90% (Figure 1). Although the synthetic peptide with amide at the carboxyl-terminus showed the same degree of paralytic activity as that of free-acid form peptide, truncated peptides of Glu from the amino terminus and Phe from the carboxyl terminus had no paralytic activity. To reveal a precursor structure of Bombyx paralytic peptide, cDNA was cloned and characterized. A partial nucleotide sequence encoding paralytic 460 Y. Shimonishi (ed): Peptide Science – Present and Future, 460–461 © 1999 Kluwer Academic Publishers, Printed in Great Britain

461

Figure 1 Homology of paralytic peptides.

peptide was amplyfied by RT-PCR using mRNA prepared from whole body of B. mori as a template. Then, λ gt10 cDNA library prepared from the same mRNA was screened with the PCR product as a probe. By screening of 1.5 × 105 plaques, seven positive clones were isolated. The nucleotide sequence of the positive clones revealed that the precursor protein is composed of 131-amino acid residues with a signal peptide and that the mature paralytic peptide is encoded in the carboxyl-terminal region, followed by termination codon. Since arginine is encoded just before amino terminus of the mature peptide, the paralytic peptide could be produced from the precursor by proteolytic cleavage. We also succeeded in expressing precursor protein using E. coli system. The expressed precursor protein showed no paralytic activity, and was processed to produce paralytic peptide after incubation with the hemolymph. Therefore, we concluded that the hemolymph of B. mori contains the precursor protein with no paralytic activity and that the paralytic peptide is yielded from the precursor by a processing enzyme activated by bleeding.

Reference 1. Skinner, W.S., Dennis, P.A., Li, J.P., Summerfelt, R.M., Carney, R.L., Quistad, G.B., J. Biol. Chem., 266 (1991) 12873–12877.

155 Isolation and characterization of a bacteriocin-like peptide produced by Brevibacterium linens ATCC 9172, an orange cheese coryneform bacterium C. OLIVEIRA, A. CORREIA and M.T. BARROS Department of Biology, University of Aveiro, Campus de Santiago, 3810 Aveiro, Portugal

Introduction Peptides with antimicrobial activity are produced by both prokaryotic and eucaryotic cells. The purification, characterization and study of the mode of action of peptides that can inhibit the growth of bacteria and fungi has increased exponentially during the last 5 years due to the promising uses as preservatives or therapeutic agents [1]. Bacteriocins constitute a quite heterogenous group of bacterial exoproteins, protein complexes or peptides with antibiotic effect against other than the producer strains and usually targeted toward species related to the producer strain. Little is known about bacteriocins from Gram positive bacteria and specially those produced by coryneform bacteria [2]. Members of the coryneform group, mainly classified as Brevibacterium species, play an important role on cheese ripening; untill now, most of the strains classified as Brevibacterium linens have been isolated from the surfaces of cheeses. Red smear cheese are manufactured by inoculation of the surface of cheese with a complex flora of yeasts and coryneform bacteria taken from ripened cheese. This kind of manipulation might introduce pathogens on the final product. It is therefore, of considerable interest to develop starter cultures that inhibit undesired microorganisms. Bacteriocins from B. linens are evisaged as a mean for accomplishing such an objective [3,4]. In fact B. linens strains producing bacteriocins with capability for inhibiting the growth of some of the more common pathogens found on dairy products (for example, Listeria monocytogenes ), could be valuable starters for industrial cheese ripenning.

Results and discussion The antimicrobial activity is present on the supernatants of batch cultures of B. linens ATCC 9172. For purification of the active compound, culture supernatants were submitted to dialysis against PEG 6000 followed by a sterilisation step with a 0.2 µm pore size membrane. The next step of purification was a gel filtration (G100) followed by ion exchange chromatography (DEAE). Reversed phase chromatography was used as the final purification step. Control of each purification step was performed by SDS-PAGE. The antimicrobial activity was 462 Y. Shimonishi (ed): Peptide Science – Present and Future, 462–464 © 1999 Kluwer Academic Publishers, Printed in Great Britain

463

Escherichia coli Bacillus subtilis Leuconostoc oenos Brevibacterium lactofermentum Corynebacterium glutamicum Pseudomonas aeruginosa

Figure 1. Antimicrobial activity of the peptide excreted by Brevibacterium linens against other bacterial strains.

tested on Tryptic Soy Agar, using Corynebacterium glutamicum as the indicator organism. Our results show that the antimicrobial activity is associated to a dimeric protein composed by two subunits with apparent molecular weights of 30 kDa. The purification protocol sugests that the protein is complexed with lipidic molecules. The pI value is close to 6.8. We analysed the antimicrobial activity of the purified bacteriocin produced by B. linens ATCC 9172 against representatives of Gram-positive and Gramnegative bacteria. As other previously characterized bacteriocins from Grampositive origin, this one has no action against Gram-negative bacteria; it is active against other representatives of the coryneform group, e.g., Corynebacterium glutamicum and C. xerosis and also against strains of Listeria monocytogenes isolated from dairy products. Further characterization shows that the bacteriocin molecule is highly stable, even if submited to hard conditions. The dialysed extract kept the antimicrobial activity after beeing submited to 100°C for 30 min. The bacteriocin is also stable at pH 4.0. The action of proteases and lipases was also tested. The antimicrobial activity was not altered after treatment with Pepsin A, Trypsin and Lipase PS, from Pseudomonas cepacia. We are interested on elucidation of the molecular mechanisms of expression of the bacteriocin, determining the optimal conditions for its biosynthesis and a further knowledge about its mode of action.

Acknowledgements This work was supported by the ‘Research Institute’ of the University of Aveiro (Portugal).

464 References 1. 2. 3. 4.

Nicolas, P. and Mor, A., Ann. Rev. Microbiol., 49 (1995) 277. Jack, R.W., Tagg, J.R., and Ray, B., Microbiol. Rev., 59 (1995) 171. Maisnier-Patin, S. and Richard, J., Appl. Environ. Microbiol., 61 (1995) 1847. Valdés-Stauber, N. and Scherer, S., Appl. Environ. Microbiol., 60 (1994) 3809.

156 Synthesis, conformational analysis, and biological activity of opioid peptide analogs containing side chain fluorinated amino acids D. WINKLER¹, N. SEWALD¹, M. GUSSMANN¹, M. THORMANN², H.-J. HOFMANN², N.N. CHUNG³, P.W. SCHILLER³ and K. BURGER¹ ¹ Department of Organic Chemistry, University of Leipzig, Talstr. 35, D-04103 Leipzig, Germany ² Department of Biochemistry, University of Leipzig, Talstr. 33, D-04103 Leipzig, Germany ³ Clinical Research Institute of Montreal, 110 Pine Av. West, Montreal, Quebec, Canada, H2W 1R7

Introduction Several peptides isolated from mammalian brain are endogenous agonists of the opiate receptors (µ, δ , and κ ). They are of current interest in medicinal chemistry and neurobiology because of their mediation of strong analgesic effects. New analogues of opioid peptides including dermorphins (µ -selective) as well as enkephalins like cyclo -[ D -Pen², D -Pen5 ]enkephalin (DPDPE) analogs ( δ-selective agonist) containing the partially fluorinated amino acid 4,4-difluoro-2-aminobutyric acid (DFAB) [1] were synthesized. Their opioid activities and receptor selectivities were determined in vitro. The solution conformations of the DPDPE analogs have been examined using CD and NMR in combination with molecular mechanics (MM) and molecular dynamics (MD) calculations.

Results and discussion Peptides were synthesized by solid phase peptide synthesis following a Fmoc protocol both for the linear and the cyclic analogs. D - and L -4,4-difluoro2-aminobutyric acid were synthesized according to a published procedure [1]. The linear analogs were assembled on Rink amide MBAH resin as solid support, using the Fmoc-compatible tert.-butyl ether side chain protection. 2-Chlorotrityl resin was used for the synthesis of DPDPE analogs. The last coupling was performed with Z-Tyr(Bzl)-OH. The use of Fmoc-S-benzylD -penicillamine allowed for the simultaneous reductive cleavage (Na, liquid ammonia) of all protective groups after cleavage of the linear precursor peptide from the resin at the end of the synthesis. Cyclization via side chain sulfur atoms was achieved in solution. Products, by-products, and critical intermediates were characterized by RP HPLC in combination with MALDI TOF mass spectrometry. The opioid activity profiles of the compounds were determined in the guinea pig ileum (GPI) as µ -representative and mouse vas deferens (MVD) 465 Y. Shimonishi (ed): Peptide Science – Present and Future, 465–466 © 1999 Kluwer Academic Publishers, Printed in Great Britain

466 Table 1. Summarized results of GPI and MVD assays. Compound

GPI assay # IC 5 0 [ n M ]

MVD assay # IC 5 0 [ n M ]

IC 5 0 ratio (GPI/MVD)

[ D -DFAB² , Met 5 -NH 2 ] enkephalin [D-DFAB²] TAPP [ D-DFAB²] TNPO DPDPE [ L -DFAB³] DPDPE [ D-DFAB³] DPDPE

186 ± 72 6.97 ± 0.38 75.2 ± 14.4 >10 000 >10 000 >10 000

17.5 ± 1.8 12.7 ± 0.6 105 ± 38 5.30 ± 0.59 155 ± 5.1 17.9 ± 2.1

10.6 0.78 0.71 > 1890 > 65 > 559

#

Mean of 3 determinations ± SEM.

as δ -representative assays (Table 1). Replacement of Gly in position 2 of [Met5NH 2 ]enkephalin and the two Dermorphin 1–4 analogs by D -DFAB resulted in compounds with µ and δ agonist potencies comparable to those of the reference peptides [2]. DPDPE analogs containing non-fluorinated L -configured amino acids in position 3 are more potent than their correlates with D -configured amino acids. The IC 50 values increase in the series Xaa³ = Ala < Ser < Abu [3]. The situation is reversed in the case of the [DFAB³] DPDPE derivatives: [D -DFAB³] DPDPE is not only 9 times more potent than [L -DFAB³]DPDPE, but also more δ -selective. The D - D F A B ³ analog is at least two orders of magnitude more potent and 40 times more δ selective than the D -Abu³ analog, and is also more potent and ten times more δ selective than [L -Abu³] DPDPE [4]. These unexpected results were correlated with the results of the solution conformational analysis. There are two main groups of structures. DPDPE and [D -Xaa³]DPDPE derivatives belong to the first group of conformations, while [L -Xaa³]DPDPE derivatives form the second group. This contradicts the biological results for Xaa³ = Ala, Ser, Abu and suggests a difference between the receptor bound conformation and solution conformation of DPDPE derivatives.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Innovationskolleg ‘Chemisches Signal und biologische Antwort’).

References 1. Winkler, D. and Burger, K., Synthesis, (1996) 1419. 2. Schiller, P.W., Nguyen, T.M.-D., Chung, N.N., and Lemieux, C., J. Med. Chem., 32 (1989) 698. 3. Haaseth, R.C., Horan, P.J., Bilsky, E.J., Davis, P., Zalewska, T., Slaninova, J., Yamamura, H.I., Weber, S.J., Davis, T.P., Porreca, F., and Hruby, V.J., J. Med. Chem., 37 (1994) 1572. 4. Winkler, D., Sewald, N., Burger, K., Chung, N.N., and Schiller, P.W., J. Pept. Sci., in press.

157 Structural requirement tryptophan 1,3 of tridecapeptide mating pheromone of Saccharomyces cerevisiae N.J. HONG, Y.A. PARK and J.W. LEE Department of Applied Microbiology, Yeungnam University, Kyongsan 712-749, Korea

Introduction Study of mating in Saccharomyces cerevisiae may allow insights into the mechanism of action of peptide hormones since this process is triggered by diffusible peptides. Sexual conjugation between haploid cells of Saccharomyces cerevisiae is mediated through the action of diffusible mating pheromone, two of which have been designated as a-factor and α -factor (Trp–His–Trp–Leu–Gln–Leu– Lys–Pro–Gly–Gln–Pro–Met–Tyr) [1,2]. These peptides are recognized by membrane-bound receptors on their respective target cells. It is generally recognized that the function of a peptide is coded by its primary structure and that peptide derivatives can be used to define structural domains necessary for biological activity. We generated [Trp1,3 ] amide analogs of α -factor to determine the contributions of [Trp 1,3 ] of the α-factor to its biological activity.

Results and discussion To determine the structural requirement of Trp at position 1 and 3 for α -factor activity, a 13-amino acid (WHWLQLKPGQPMY) pheromone secreted by haploid α -cell of Saccharomyces cerevisiae, we have synthesized seven α-factor amide analogs, in which Trp at position 1 and 3 were replaced or deleted; [desTrp¹,Phe³] α -factor amide, [des-Trp¹]α-factor amide, [Ala¹,Ala³] α -factor amide, [Ala¹] α-factor amide, [Ala³]α-factor amide, [D -Trp¹] α -factor amide, [ D -Trp¹, D -Trp³] α -factor amide, by solid phase methodologies on a Knorr resin. Purification of all peptides to 97% or greater homogeneity was accomplished by high-performance liquid chromatography. All peptides were characterized by amino acid analysis and fast atom bombardment mass spectrometry. The activity of these analogs was measured by a growth arrest assay in which lawns of target cells were grown in the presence of filter disks containing α -factor analogs (Table 1). Also morphological change (Shmoo assay) in target cells was observed for the biological activity of analogs. In Ala substituted analogs, [Ala¹] α -factor amide and [Ala³]α-factor amide showed 8 and 13 times lower activities than natural α -factor respectively, whereas [Ala 1,3 ] α-factor amide was inactive (Figure 1). The results showed that Trp³ is more important determinant of pheromone potency than Trp¹. [Des-Trp¹] α-factor amide was 22-fold less 467 Y. Shimonishi (ed): Peptide Science – Present and Future, 467–469 © 1999 Kluwer Academic Publishers, Printed in Great Britain

468 Table 1. Minimal concentration of synthetic α-mating factors required for biological activity by shmoo assay in S. cerevisiae 2180-1A. α-factor peptide analogs

α -factor [Ala¹] α-factor amide [Ala¹, Ala³] α-factor amide [Ala³] α-factor amide [ D -Trp¹, D -Trp³] α-factor amide [D-Trp³ ] α -factor amide [des-Trp¹] α-factor amide [des-Trp¹, Phe³]α-factor amide

Concentration (µg/ml) 0.1 0.6 25 2 5 15 5 10

Figure 1. Bioactivity of α-factor analogs by halo assay in S. cerevisiae. (left: in S. cerevisiae 2180-1A, right: in S. cerevisiae 7925) a: α-factor, b: [Ala¹] α-factor amide, c: [Ala¹, Ala³] α-factor amide, d: [Ala³] α-factor amide, e: [D-Trp¹, D-Trp³] α-factor amide, f: [D-Trp³] α-factor amide, g: [desTrp¹] α-factor amide, h: [des-Trp¹,Phe³] α-factor amide.

active than natural α-factor. This is almost as same as previous results reported by Naider et al. [3]. [Des-Trp¹,Phe³] α-factor amide, was 240-fold less active than α -factor. Although strain used was not identical, this compound showed 80-fold higher activity than C -terminal carboxylic acid analog, [desTrp¹,Phe³]α-factor [3]. In D -Trp substituted analogs, [D -Trp³] α-factor amide showed 240-fold reduced activity, whereas [D -Trp 1,3 ]α-factor amide showed 6-fold increased activity compared with [D -Trp³] α-factor amides. The competition studies showed that [des-Trp¹,Phe³]α-factor amide and [des-Trp¹]α-factor amide analogs act as an antagonist against [Ala³]α-factor amide analog. It is also suggested that C-terminal amide modification of α-factor or it’s analogs leads to slight increase or no change in activity.

Acknowledgements This research was supported by the Yeungnam University Research Grants in 1996.

469 References 1. Herskowitz, I., Microbiol. Rev., 52 (1988) 536. 2. Naider, F. and Becker, J.M., CRC Crit. Rev. Biochem., 21 (1986) 225. 3. Shenbagamurthi, P., Baffi, R., Khan, S.A., Lipke, C., Pousman, Becker, J.M., and Naider, F., Biochemistry, 22 (1983) 1298.

158 Synthesis and in vitro effects of short analogues of β-amyloid peptide on resting transmembrane potential M. ZARÁNDI¹, B. PENKE¹, G. LASKAY², E. KLEMENT¹, I. OCSOVSZKI³, K. JOST¹, J. VARGA¹, K. NAGY¹, G.K. TÓTH¹ and K. GULYA 4 ¹ Department of Medical Chemistry, Albert Szent-Györgyi Medical University, H-6720 Szeged, Dóm tér 8, Hungary ² Department of Botany, József Attila University, H-6722 Szeged, Egyetem u. 2, Hungary ³ Department of Biochemistry, Albert Szent-Györgyi Medical University, H-6720 Szeged, Dóm tér 9, Hungary 4 Department of Zoology and Cell Biology, József Attila University, H-6722 Szeged, Egyetem u. 2, Hungary

Introduction Alzheimer’s disease is a progressive neurodegenerative disorder affecting the aging population. The principal component of senile plaques is the β-amyloid peptide (β A) which contains 39–43 amino acids. β A is produced from amyloid precursor protein (APP) and induces cytotoxic actions in neural and glial cells [1,2]. The biologically active center of β A for neurotoxic effects is located in the β A[25–35] sequence. Previously, we reported the synthesis of short peptide sequences of the active center of β A in order to find a compound which might be capable to prevent the neurotoxic effects of β A [3]. The structure-inhibition relationships of these peptides were investigated by measuring the intracellular Ca2 + concentration in rat astroglial cells in vitro. Among the peptides synthesized, a tetrapeptide (Propionyl–Ile–Ile–Gly–Leu–NH 2, Pr–IIGL) was found to antagonize effectively the long-term elevation of intracellular Ca 2 + concentration induced by β -amyloid[1–42] (β A[1–42]) [3]. In other previous experiments, we had also observed that β A[1–42] induces an early hyperpolarization of the resting membrane potential in cultured neural cells (manuscript is under preparation). Based on these preliminary results, we studied whether Pr–IIGL and several other peptides of related amino acid sequence had any short-term effect on neural cells.

Results and discussion In order to evaluate the effect of systematic replacement with Ala within the sequence of Pr–IIGL, four new analogs (Pr–Ala–Ile–Gly–Leu–NH2 , Pr– Ile–Ala–Gly–Leu–NH 2 , Pr–Ile–Ile–Ala–Leu–NH 2 , Pr–Ile–Ile–Gly–Ala–NH 2) 470 Y. Shimonishi (ed): Peptide Science – Present and Future, 470–471 © 1999 Kluwer Academic Publishers, Printed in Great Britain

471 were synthesized on MBHA-resin by manual SPPS methodology using Bocchemistry. The compounds were purified by preparative RP-HPLC (>95% purity) and characterized by amino acid analysis, ES-MS, and analytical HPLC. A comparative investigation were carried out on the effect of the peptides on the resting membrane potential of cultured neural cells. The cells were derived from an immortalized human neural cell line of striatal origin. The potential-sensitive oxonol fluorescent dye DiBAC 4 was used to monitor any change in the resting transmembrane potential of the cells. Since this dye is a lipophilic anion, its accumulation in the cells is inversely related to the actual magnitude of the resting transmembrane potential, and a decrease in its uptake being indicative of a relatively higher membrane potential. After the cells were treated with β A[1–42] or its short analog peptides for 10 min, they were labelled by 2 µM DiBAC4 for 2 min. The fluorescence of the individual cells was analysed quantitatively by using a Becton Dickinson FACStarPLUS flow cytometer using 488 nm excitation and 540-nm emission wavelengths. 10,000 cells were analysed in every sample. In the experiments, β A[1–42] caused a decrease in the fluorescence intensity of the cells labelled by DiBAC4 indicating a hyperpolarization. Contrary to our expectations, Pr–IIGL also showed a slight decrease in the fluorescence intensity and caused a smaller hyperpolarization than β A[1–42]. Since this tetrapeptide shares a sequence homology with the putative active center of the original β A[1–42], it was of interest to see whether analogues of Pr–IIGL containing Ala substitutions could exert any similar action in this test. Therefore, the action of the new analogues were also investigated. None of the peptides Pr–Ala–Ile–Gly–Leu–NH 2, Pr–Ile–Ile–Ala–Leu–NH 2 , Pr–Ile–Ile– Gly–Ala–NH 2 was found to be effective in inducing a hyperpolarization. This finding indicates that i) the IIGL sequence could be an integral part of the putative active center of β -amyloid peptide and ii) substitution of Ala for Ile, Gly, or Leu in the tetrapeptide (Pr–IIGL) brings about a loss of agonistic activity in inducing hyperpolarization. These studies provide valuable information about the active center of β -amyloid peptide. However, further studies are required to test whether these short peptides are capable to antagonize the neurotoxic effects of β A . Acknowledgements This work was supported by OTKA Grants #T 022546 and #T 022542. References 1. Korotzer, A.R., Pike, C.J., and Cotman, C.W., Brain Res., 624 (1993) 121. 2. Kowall, N.W., Beal, F.M., Busciglio, J., Duffy, K.L., and Yanker, B.A., Proc. Natl. Acad. Sci. USA, 88 (1991) 7247. 3. Laskay, G., Zarándi, M., Varga, J., Jost, K., Fónagy, A., Torday, Cs., Latzkovits, L., and Penke, B., Biochem. Biophys. Res. Commun., 235 (1997) 479.

159 Macrotorials: synthesis and characterization of model compounds related to PTH (1–34) A.F. SPATOLA¹, W. MA¹, A. HARVEY², S. CHANDRASEKHAR² and J.A. DODGE² ¹ Department of Chemistry, University of Louisville, Louisville, KY 40292 USA ² Eli Lilly and Company, Lilly Research Laboratories, Indianapolis, IN 46285 USA

Introduction Macrocycles are ubiquitous in nature and represent promising opportunities for drug lead discovery and optimization. But cyclic peptides and pseudopeptides are not always easily synthesized, especially when the desired targets are combinatorial mixtures with their diversities maximized. Our systematic approach to this problem started with the preparation of small numbers of cyclic peptides and pseudopeptides [1]. Some biological targets are not activated by low molecular weight ligands. We have now introduced a new concept called macrotorials; these structures represent a class of fixed cyclic peptides with variable linear peptide or peptidomimetic appendages.

Results and discussion In order to demonstrate this approach, we began with the synthesis of both individual cyclic compounds with two linear appendages as well as libraries with mixtures of amino acids in the N- and C -terminal ‘tails’ as depicted in Figure 1. The individual compounds were composed of 24 amino acids. The

Figure 1.

Macrotorials: components and integrity.

472 Y. Shimonishi (ed): Peptide Science – Present and Future, 472–474 © 1999 Kluwer Academic Publishers, Printed in Great Britain

473 N - and C -terminal fragments correspond to the N- and C -terminal portion of parathyroid hormone (1–34) and were selected in view of the literature evidence that these regions serve as activation and binding domains, respectively [2]. Four individual PTH model compounds were prepared (Table 1). These served to verify our synthetic approach which included mid-synthesis, on-resin cyclization via acid-labile, side chain protecting groups (Fmoc-Lys(Boc) and Fmoc-Glu(O t Bu)), while the C-terminal and end terminal sequences were elongated with standard Boc chemistry. The bioassay of these compounds did not confirm their surrogacy for PTH (1–34). While two individual PTH analogs at first showed promising indications in a proteoglycan synthesis assay, these compounds did not later prove to be active in a cAMP assay or to bind to the PTH receptor. Using a comparable synthetic strategy, several macrotorial libraries were prepared. These mixtures combine elements from prior work in our laboratory (cyclic peptide libraries) [3], the Mutter group (TASP concept) (4) and Houghten’s positional scan approach [5,6]. These libraries are currently being tested in a variety of binding assays where discontinuous binding and activation elements are suspected.

Table 1. Sequence of individual PTH-related cyclic peptide analogs MW

Sequence

1) H-Ala-Val-Ser-Glu-Ile-Gln-Sar-Sar-cyclo [Glu-Pro-Ala-Lys-]Sar-Sar-Arg-Lys-LysLeu-Gln-Asp-Val-His-Asn-Phe-OH 2) H-Ser-Val-Ser-Glu-Ile-Gln-Sar-Sar-cyclo [Glu-Pro-Ala-Lys-]Sar-Sar-Arg-Lys-Lys-LeuGln-Asp-Val-His-Asn-Phe-OH 3) Ac-Ala-Val-Ser-Glu-Ile-Gln-Sar-Sar-cyclo [Glu-Pro-Ala-Lys-]Sar-Sar-Arg-Lys-Lys-LeuGln-Asp-Val-His-Asn-Phe-OH 4) Ac-Ser-Val-Ser-Glu-Ile-Gln-Sar-Sar-cyclo [Glu-Pro-Ala-Lys-]Sar-Sar-Arg-Lys-Lys-LeuGln-Asp-Val-His-Asn-Phe-OH

Calc.

Found

2603.8

2603.4

2619.8

2619.7

2645.8

2645.7

2661.8

2661.1

Acknowledgements Drs. Philip Andrews and Rachel Loo at the University of Michigan Protein and Carbohydrate Structure Facility have provided mass spectral analyses. Amino acid analyses were performed at the University of Louisville. Dr. Robert Gray assisted with the CD studies. We appreciate the financial support of NIH GM 33376.

474 References 1. Spatola, A.F. and Romanovskis, P.J., in Combinatorial Peptide and Nonpeptide Libraries, G. Jung, Ed., VCH, Weinheim, 327–347 (1996). 2. Chorev, M. and Rosenblatt, M. in The Parathyroids, J.P. Bilezikian, M.A. Levine, and R. Marcus, (Eds), Raven Press Ltd., New York, 139–156 (1994). 3. Spatola, A.F., Crozet, Y., DeWit, D., Yanagisawa, M., J. Med. Chem., 39, (1986) 3842. 4. Mutter, M. and Vuilleumier, S., Angew Chem. Int. Ed. Engl., 28, (1989) 535. 5. Pinilla, C., Appel, J.R., Blanc, P., Houghten, R.A., Biotechniques, 13, (1992) 901. 6. J. Eichler, A.W. Lucka, C. Pinilla, R.A. Houghten, R.A. Molecular Diversity, 1, (1996) 233.

160 Developments in peptide synthesis and peptidomimetic chemistry: HIV protease analogs and substrate-based inhibitors Y. KISO Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607–8414, Japan

Introduction Protection/deprotection steps and amide-forming reactions are important in peptide synthesis. We have developed new methods such as reductive acidolysis [1], 2-(benzotriazole-1-yl)-oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI) [2], and 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP) [3]. These new methods are successfully applied to the syntheses of biologically important peptides. The modern synthetic methods [4] of the peptide chemistry greatly contributed to the fields important for human being, such as AIDS research. The human immunodeficiency virus type-1 (HIV-1) codes for a virus-specific aspartic protease responsible for processing the gag and gag-pol polyproteins and for the proliferation of the retrovirus. The HIV-1 protease, a 99 amino acid residues peptide, functions in homodimeric form which is associated noncovalently. Kent et al. [5] synthesized an HIV-1 protease analog, in which the two Cys residues were replaced by the isosteric L-α-amino-n-butyric acid, whereas we replaced the two Cys residues by a natural amino acid, L-alanine. The [Ala 67,95 ] derivative of HIV-1 protease (NY-5 isolate) was synthesized by the solid-phase method based on the Boc-Bzl strategy using PAM resin [6]. Furthermore, we synthesized [Tyr 6,42 , Nle36,46, (NHCH 2 COSCH 2 CO)51–52 , Ala 67,95 ] HIV-1 protease (NY-5 isolate) using the thioester linkage chemical ligation method [7] and [NHCH2 CH 2 SCH 2 CO 51–52 , Ala 67,95 ] HIV-1 protease using thioether linkage chemical ligation method [8,9]. Interestingly, singlechain tethered dimers of HIV proteases, artificial peptides having the same topology as pepsin-like aspartic proteases, also show similar enzymatic activity and are more stable than the natural enzyme [10]. In this paper, we report the synthesis of an HIV-1 protease dimer analog which consists of two identical peptide chains covalently linked by a disulfide bridge (Figure 1) and substratebased peptidomimetic HIV protease inhibitors (Figure 2). 475 Y. Shimonishi (ed): Peptide Science – Present and Future, 475–479 © 1999 Kluwer Academic Publishers, Printed in Great Britain

476

Figure 1. An HIV-1 protease dimer analog.

Results and discussion A covalently linked dimer analog of HIV protease The chemical ligations were widely applied to syntheses of many large peptides (enzyme, vaccine, artificial peptide/protein, etc.). In the synthesis of a protease dimer analog covalently linked by a disulfide bridge, we adopted this strategy for the preparation of monomer unit. In order to make a disulfide bridge between two protease monomers, we introduced a Cys residue as a substitute for Asn98, because Asn 98 and Asn 198 are located very close in the active conformation. Cys residues having a sulfhydryl group were replaced by Ala. A monomer unit, [NHCH 2 CH 2 SCH 2 CO 51–52 , Ala 67,95 , Cys(Acm) 98 ] HIV-1 PR was synthesized employing the chemoselective reaction of a bromoacetylated peptide, BrCH2 CO-[Ala 67,95, Cys(Acm)98 ]HIV-1 PR (53–99) (MALDITOF/MS value [M + H] + : 5298.42), with a mercaptoethylamide peptide segment, HIV-1 PR (1–50)-NHCH 2 CH 2SH(MALDI-TOF/MS value [M + H]+: 5602.68), prepared by Fmoc-based SPPS. The reaction proceeded within 4h and the product (MALDI-TOF/MS value [M + H] + : 10,818.7) was easily purified by gel-filtration (yield: 55%). Then the disulfide formation was achieved using iodine (r.t., 1 h). The homogeneous dimer analog (MALDI-TOF/MS value [M + H] + : 21,494.8) was obtained by the purification on ODS-column (yield: 11%). After the folding procedure, the synthetic peptide possessed proteolytic activity comparable to the recombinant wild-type HIV-1 protease in an assay system using KARVY*Phe(NO 2 )EANle-NH 2 as a substrate (Km = 10.6 µM). Similarly to the case of monomer analog, this dimer analog had weak affinity to the Tyr-Pro-type substrate, SQNY*PIV. Consequently, the molecular recognition of the analogs differs from the wild-type enzyme. These enzyme analogs should be useful tools for evaluation of protease inhibitors. Substrate-based peptidomimetic inhibitors (Figure 2) Inhibition of HIV protease became a major target for AIDS chemotherapy. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing allophenylnorstatine [Apns; (2S,3S)3-amino-2-hydroxy-4-phenylbutyric acid] with a hydroxymethylcarbonyl (HMC)isostere[6]. Among them, the tripeptide KNI-272 was a highly selective and superpotent HIV protease inhibitor (Ki = 5.5 pM). KNI-272 exhibited

477

Figure 2. Substrate-based peptidomimetic HIV protease inhibitors.

478 potent in vitro and in vivo antiviral activities with low cytotoxicity [6]. The NMR, X-ray crystallography and molecular modeling studies showed that the HMC group in KNI-272 interacted excellently with the aspartic acid carboxyl groups of HIV protease active site [11]. These results imply that the HMC isostere is an ideal transition-state mimetic. HIV-1 develops in vitro a high level of resistance to KNI-272 by acquiring mutations in the protease-encoding gene, though it takes relatively a long time. Mutations conferring KNI-272-resistance were V32I, L33F, K45I, F53L, A71V and I84V. The EC 50 values of KNI-272 against wild-type HIV-1 and KNI-272-resistant HIV-1 were 0.04 µM and 2 µ M, respectively. The sensitivity of KNI-272 reduced 50-fold against resistant HIV-1. In order to overcome the resistance, the Apns-containing peptides were screened. Among them, KNI-241 was found to be active against both wild-type HIV-1 (EC 50 0.04 µ M) and KNI-272-resistant HIV-1 (EC50 0.04 µ M) [12]. However, the bioavailability of KNI-241 was very low. The P3 moiety of KNI-241 is interacting with both 53-Phe of wild-type protease and 53-Leu of the mutant protease. The solution, crystalline and complex structure of KNI-272 were similar except for P3 moiety [13]. Therefore, we considered that P2-P2' moiety might be core group for enzyme inhibition and studied small-sized dipeptide inhibitors as advantageous compounds. P2 was replaced by several alkyl and hydrophilic groups. Among them, KNI-549 containing a dimethyl and a carboxyl groups exhibited HIV protease inhibitory activities (HIV protease inhibition = 76.3% at 50 nM). In order to enhance the inhibitory activity, we cyclized P2 moiety in KNI-549 to obtain the rigid form. The resulting KNI-577 was a potent HIV protease inhibitor (87.6% inhibition at 50 nM) and showed remarkable anti-HIV-1 (IIIB) activity (EC50 0.02 µ M in CEM-SS cells). This antiviral activity of KNI-577 was higher than that of the tripeptide KNI-272. KNI-577 had very low cytotoxicity (CC50 > 200 µ M) and the bioavailability after intraduodenal administration in rats was 48% [14]. Furthermore, we designed considering the symmetry of HIV protease dimer and introduced 2-methylbenzylamine group in P2'. The resulting KNI-764 exhibited highly potent enzyme inhibition (Ki = 0.035 nM) and surprisingly potent anti-HIV activity (EC 50 0.007 µ M) with low cytotoxicity (CC 50 > 20 µ M). By serial passage of HIV-1 in the presence of increasing concentration of KNI-764, a variant with a 16 fold reduction in susceptibility to KNI-764 was isolated. Mutations of KNI-764-resistant HIV-1 protease were L10F, S37N, I47V and V82I. This mutation pattern was different from other HIV protease inhibitors such as saquinavir, ritonavir, indinavir or nelfinavir. In addition, KNI-764-resistant HIV-1 was sensitive to those HIV protease inhibitors. KNI-764 had a favorable oral-bioavailability (42%) and a long plasma half life (94 min) in dogs. In conclusion, this study suggests that the small-sized dipeptide HIV protease inhibitors are promising candidates for anti-HIV drugs.

479 References 1. Kiso, K., Kimura, T., Yoshida, M., Shimokura, M., Akaji, K., and Mimoto, T., J. Chem. Soc., Chem. Commun., (1989) 1511.; Kimura, T., Fukui, T., Tanaka, S., Akaji, K., and Kiso, Y., Chem. Pharm. Bull., 45 (1997) 18. 2. Kiso, Y., Fujiwara, Y., Kimura, T., Nishitani, A., and Akaji, K., Int. J. Peptide & Protein Res., 40 (1992) 308. 3. Akaji, K., Kuriyama, N., and Kiso, Y., Tetrahedron Letters, 35 (1994) 3315; Akaji, K., Kuriyama, N., and Kiso, Y., J. Org. Chem. 61 (1996) 3350. 4. Kiso, Y., and Yajima, H., In Gutte, B. (Ed) Peptides: Synthesis, Structures, and Applications, Academic Press, San Diego, 1995, pp. 39–91. 5. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B.K., Baldwin, E., Weber, I.T., Selk, L.M., Clawson, L., Schneider, J., and Kent, S.B.H., Science 245 (1989) 616. 6. Kiso, Y., Biopolymers, 40 (1996) 235. 7. Schnoelzer, M., and Kent, S.B.H., Science 256 (1992) 221. 8. Englebretsen, D.R., Garnham, B.G., Bergan, D.A., Alewood, P.F., Tetrahedron Lett., 36 (1995) 8871. 9. Nishizawa, N., Kimura, T., Matsumoto, H., Fujiwara, T., Takaku, H., Akaji, K., and Kiso, Y., In Kitada, C. (Ed.) Peptide Chemistry 1996, Protein Research Foundation, Osaka, 1997, pp61–64. 10. DiIanni, C.L., Davis, L.J., Holloway, M.K., Darke, P.L., Kohl, N.E., and Dixon R.A.F., J. Biol. Chem., 265 (1990) 17438; Bhat, T.N., Baldwin, E.T., Liu, B., Cheng, Y.S.E., and Erickson, J.W., Nature Str. Biol., 1 (1994) 552. 11. Baldwin, E.T., Bhat, T.N., Gulnik, S., Liu, B., Topol, I.A., Kiso, Y., Mimoto, T., Mitsuya, H., and Erickson, J.W., Structure, 3 (1995) 581; Wang, Y.X., Freedberg, D.I., Yamazaki, T., Wingfield, P.T., Stahl, S.J., Kaufman, J.D., Kiso, Y., and Torchia, D.A., Biochemistry, 35 (1996) 9945; Wang, Y.X., Freedberg, D.I., Wingfield, P.T., Stahl, S.J., Kaufman, J.D., Kiso, Y., Bhat, T.N., Erickson, J.W., and Torchia, D.A., J. Am. Chem. Soc., 118 (1996) 12287. 12. Tanaka, M., Mimoto, T., Anderson, B., Gulnik, S., Bhat, T.N., Yusa, K., Hayashi, H., Kiso, Y., Erickson, J.W., and Mitsuya, H., 11th Int. Conf. AIDS (1996) Abst. Tu.A.260. 13. Ohno, Y., Kiso, Y., and Kobayashi, Y., Bioorg. Med. Chem., 4 (1996), 1565. 14. Kiso, Y., Yamaguchi, S., Matsumoto, H., Mimoto, T., Kato, R., Nojima, S., Takaku, H., Fukazawa, T., Kimura, T., and Akaji, K., Arch. Pharm. Pharm. Med. Chem. 331 (1998) 87.

161 Thia zip reaction for the facile synthesis of large cyclic peptides J.P. TAM, Y.-A. LU and Q. YU Department of Microbiology and Immunology, Vanderbilt University, A5119 MCN, 1161 21st Avenue S., Nashville, TN 37232-2363, USA

Introduction This paper describes the development of a strategy for the synthesis and characterization of Circulin B which is a large end-to-end cyclic peptide containing multiple disulfide bonds. In particular, we describe a facile, end-to-end cyclization strategy for unprotected peptides, which we refer to as the thia zip reaction. The rationale for developing the thia zip reaction is based on our synthetic goals to prepare and test the biological activity of several plant-derived, endto-end cyclic peptides [1]. A typical example is Circulin B (Figure 1), which contains 31 amino acids and is believed to be the largest end-to-end cyclic peptide found naturally. Circulins [2] and their homologs, Kalata and Cyclopsychotride, are rich in disulfide bonds, cationic, and may be useful as broad-spectrum antimicrobial agents. The structure as well as the disulfide connectivity of Circulin B has not been determined. However, the 1 H-NMR structure of its homolog Kalata [3] shows three short β -strands and a disordered loop, braced by three disulfide bonds of Cys 1–4, 2–5 and 3–6 in a cystine knot motif. The synthetic effort to form this cystine-knot motif may present a challenge because of the relatively similar energetics to form all 15 possible disulfide isomers in such a closely packed environment as well as a closed structure. A two-step disulfide forming strategy [4] is also described to deal with this challenge.

Results and discussion Similar to our previous effort for the synthesis of Cyclopyschotride [1], the overall synthetic strategy to prepare Circulin B consisted of three steps (Figure 2). First, the linear peptide containing an α-Cys and a COOH-thioester peptide was prepared by a stepwise solid phase synthesis using Boc-chemistry. We used a thioester resin developed by Hojo and Aimoto [5]. After the Circulin

B

CSCKNKVCYRNGVIP–CGESCVFIPCIST–LLG

Figure 1

480 Y. Shimonishi (ed): Peptide Science – Present and Future, 480–484 © 1999 Kluwer Academic Publishers, Printed in Great Britain

481

Figure 2

assemblage of the sequence and removal of the protecting groups by HF, an α-cysteinyl unprotected peptide containing the COOH thioester was obtained. Second, the crude or purified peptide was then adjusted to pH 7.5 to permit the end-to-end cyclization. The cyclization proceeds through the formation of an α -aminothiolactone and then a spontaneous S,N -acyl migration to form the lactam. The scope and mechanism of forming a cyclic peptide from a peptide containing a cysteine at the amino terminus and a COOH-thioester have been reported recently by our laboratory [6]. The third step involving the formation of disulfide bonds was mediated by DMSO oxidation method [7] to complete the synthesis. Thus, the end-to-end cyclization and the disulfide formation are essentially a one-pot reaction with an unprotected linear precursor after its cleavage from the resin. This simplified scheme, without any protection, deprotection, and activation agents, was highly efficient. Indeed, cyclizations were completed in < 6 hr with

482 yields of 75–90%. In addition, there was little observable polymerization in this cyclization even when the reaction was performed at high mmolar concentrations. The efficiency for cyclizing the 93-member ring of Circulin B and the lack of polymerization led us to undertake further mechanistic study. To answer these questions, we designed a series of model peptides including those derived from Circulin B. Based on these experiments [8], the cyclization mechanism is found to proceed through a series of thiolactone exchanges, and we have referred to this method of cyclization as the thia zip reaction (Figure 2). The thia zip reaction contains three steps: a slow thiolactone formation under the influence of ring chain tautomerization, a slow and reversible thiolactone exchange for ring expansions to arrive at the α-aminothiolactone, and a fast and reversible S,N-acyl isomerization to give the lactam. The slow steps are conformation dependent, and their intermediates can be isolated. The S,N acyl isomerization occurring at the amino terminus via a 5-member ring is sequence independent and likely provides the driving force for the thia zip reaction. The apparent concentration-independent cyclization can be attributed to two factors. First, a highly reductive environment is used in the reaction with a large excess of a small thiol to prevent intersegmental cross-linking disulfide formation or transthioesterification. Second, with many internal thiols, the linear peptide readily undergoes the entropic-favored, ring chain tautomerization to form a thiolactone. Subsequent thiol-thiolactone exchanges will lead to the desired cyclic peptide. To support the proposed thia zip reaction proceeding the thiolactone exchange pathways, we systematically blocked off the internal thiols by the Acm groups so that the thiolactone formation or thiolactone exchanges would have to undergo larger intermediates. For example, Circulin B contains five internal thiols and the thiol-thiolactone exchanges would involve ring sizes of 12–27 atoms. Blocking two, four and five internal thiols increases the range size to 18–37, 18–45 and 94, respectively. As the ring sizes increase, the rates for the zip reaction should decrease in the absence of conformational assistance. The analog with all the internal thiols blocked by the Acm group could only undergo the unassisted, direct thiolactone formation. To remove the conformational assistance in the zip reaction, the zip cyclizations were performed in 8M urea at pH 7.5. As expected, the rates as measured by 50% conversion to the cyclized product were 0.6 and 128 hr. There is a more than 100-fold difference in the reaction rate between the zip-assisted cyclization and the unassisted directed cyclization. After the cyclization through the thia zip reaction, the third part of our synthetic strategy is the disulfide formation within a closed structure of a cyclic peptide. This type of disulfide formation radically departs from the conventional synthetic approaches as well as the natural pathways of forming disulfides in an open-chain linear structure. Thus, it is of interest to see whether there is any significant difficulty in our proposed approach of disulfide bond formation. The one-step disulfide formation of Circulin B afforded > 9 three-disulfide

483 isomers and about 2% of the desired product. Circulin B has a unique cysteine knot motif in which all three disulfides are closely packed at a sulfur-sulfurbonded core. Thus, it is not surprising that the one-step disulfide formation leads to nine disulfide isomers. To limit the number of disulfide isomers, we employed a two-step disulfide forming strategy using two orthogonal protecting groups for thiols with four Cys protected with MeBzl and two with Acm. Thus, after HF cleavage, four Cys thiols were free and could aid the zip-assisted cyclization. Oxidation by DMSO [6] at pH 7.0 would produce maximally three disulfide isomers instead of 15 in the one-step approach. Oxidative removal of the Cys-Acm by l 2, in MeOH under acidic conditions afforded the third pair of disulfide to complete the synthesis. Using the two-step approach, all three isomers of the two-disulfide Circulin B were found and the desired product was obtained in about 30% yield. The disulfide forming process was slow and required 36 hr for completion. Timecourse experiments monitored by HPLC showed the reaction after 10 hr the presence of three 2-disulfide isomers as well as several one-disulfide intermediates. We have found that strategy of protecting the Cys pair by Acm group does not appear to affect the yield of disulfide formation. Protecting the 2,5-Cys or 3,6 Cys pairs with Acm affords about 30% of the desired disulfide isomer. The yield of the disulfide isomer could be increased by recyclizing the process through reduction of the misfolded disulfide isomer by a thiol or R 3 P and treatment with DMSO. After two recyclings, the yield of the disulfide could increase to 65%. With three disulfide isomers obtained in our synthesis, it becomes necessary to develop a simple and efficient method for identifying their disulfide connectivity through fragmentation of these cyclic peptides. For this purpose, the general approach of chemical proteolysis by partial acid hydrolysis developed recently by Zhou and Smith [9] appears to be ideal for the highly constrained cyclic peptides with multiple disulfide bonds. To facilitate our work, we also studied the chemical basis of proteolysis in dilute acids using model peptides. We found that some peptide bonds are more susceptible to acid hydrolysis than others. These include peptide bonds containing Asp, Glu, Ser, Thr, His, Gly and Pro. The susceptibility of these peptide bonds can be attributed to their side chain nucleophiles which could participate in anchimeric assistance to form an anhydride, imide, or ester through N to O - or N to N -acyl shifts such as those found in Asp, Gly, Ser, Thr and His or the lack of steric hindrance such as Gly or increase of the bond strain such as Pro. A combination of two or more susceptible amino acids form a ‘hot spot’ or primary cleavage site that provides a basis for predicting the cleavage segments. In Circulin B, there are four such predicted primary sites, and we have experimentally confirmed their susceptibility through the isolation of either single or double nicked large fragments. Through partial acid hydrolysis we were able to identify the disulfide connectivity of all of the disulfide isomers. Since the disulfide connectivity of Circulin

484 B has not been determined, it is gratifying that we are able to confirm that the disulfide pairing of Circulin B is Cys 1–4, 2–5 and 3–6, similar to those of Kalata and Cyclopyschotride whose disulfide connectivity was recently confirmed by total synthesis in our laboratory [1]. The synthetic product is identical to the natural product both chemically and biochemically. It coeluted with the native Circulin B in RP-HPLC while the two misfolded disulfide isomers did not. In conclusion, an efficient orthogonal cyclization of unprotected, cysteinerich peptides via thia zip reaction has been developed. Furthermore, a twostep disulfide formation scheme increases the yield of the desired disulfide isomer when a cysteine-knot motif is present, as in the case of Circulin B. Partial acid hydrolysis combined with MS analysis provides unambiguous disulfide connectivity of highly constrained cyclic peptides. In our strategy, intramolecular acyl transfer reactions form the key elements in both the aminolysis and proteolysis involved in the chemical ligation and fragmentation of unprotected peptides in aqueous solutions.

References 1. Tam, J.P. and Lu, Yi-An, Tetrahedron Letters 38 (1997) 5599. 2. Gustafson, K.R., Sowder, II, R.C., et al., J. Am. Chem. Soc. 116 (1994) 9337–9338. 3. Saether, O., Craik, D.J., et al., Biochemistry 34 (1995) 4147–4158. 4. Yang, Y., Sweeney, W., Schneider, K., Chait, B., and Tam, J.P., Protein Science 3 (1994) 1267–1275. 5. Hojo, H. and Aimoto, S., Bull. Chem. Soc. Jpn. 64 (1991) 111–117. 6. Zhang, L. and Tam, J.P., J. Am. Chem. Soc. 119 (1997) 2363–2370. 7. Tam, J.P., Wu, C-R., Liu. W., and Zhang, J-W., J. Am. Chem. Soc., 113 ( 1991) 6657. 8. Tam, J.P., Lu, Y-A., Yu, Q., and Rao, C., J. Am. Chem. Soc., submitted. 9. Zhou, Z. and Smith, D.L., J. of Pro. Chem. 9 (1990) 523.

162 Synthesis of disulfide-bridged heterotrimeric collagen peptides. Conformational properties and digestion by matrix metalloproteinases J. OTTL, D. GABRIEL, H.Y.C.D. MÜLLER, H.-J. MUSIOL, W. BODE and L. MORODER Max-Planck-Institute of Biochemistry, Martinsried, Germany

Introduction The collagen type I mediated biological activities (cell activation and signaling, matrix metalloproteinase binding and catabolism) reside in specific loci of the rod-like triple-helical heterotrimeric molecule. To explore the structural properties of these functional epitopes synthetic heterotrimeric fragments folded into a sufficiently stable triple-helix are required. Thereby two main problems have to be resolved: i) the regioselective crosslinking of three peptide chains and ii) the induction of the one-residue shift register in the correct order. In folding processes of natural collagens selection of the α -subunits and their registration is induced by the propeptides and stabilized by complex cystine-knots. Although there are strong indications for an α1/ α2/ α 1 register in type I collagen [1], unambiguous experimental proofs are still missing. For the design and synthesis of heterotrimeric collagen peptides that mimic the interstitial collagenases (MMP1 and MMP8) binding and cleavage site as well as the cell adhesion and fibronectin binding site of collagen type I, we used this putative α1 /α2 /α 1 assembly for the fragments 772–784 of the α1 and α 2 subunits which were extended N-terminally by (GPO) n and C-terminally crosslinked with the cystine-knot shown in Figure 1 [2].

Results and discussion For crossbridging the three α -chains in the defined α 1/ α 2/ α 1 order, besides selective protection of the Cys residues, three different chains, i.e. α 1, α 2 and α1', were required (Figure 1). The single chains were synthesized in stepwise manner on solid supports by the Fmoc/tBu strategy whereby the Cys residues were spaced with one or two glycines from the resin linker to avoid undesired side reactions. All coupling steps were performed with HBTU/HOBt except for Cys residues which were incorporated via the Pfp esters to prevent racemization [4]. Even using larger excesses of acylating amino acid derivatives and double-coupling steps in the GPO(tBu) portion of the sequences, a large microheterogeneity of the crude products was observed that derived mainly 485 Y. Shimonishi (ed): Peptide Science – Present and Future, 485–489 © 1999 Kluwer Academic Publishers, Printed in Great Britain

486

Figure 1. CPK model of the (GPO) n triple helix [3] with the built-in C-terminal cystine-knot (see insert). Alignment of the α1, α2 and α1' chain peptides of heterotrimer A (n = 3), B (n = 5), C (n = 5) with Gln → Asn and Val → Phe replacements and αl and α2 acetylated, and D (n = 5) with an additional GPO repeat preceeding the cysteine-knot; O = Hyp.

from uncomplete aminoacylation of the imino acids. Extensive purification by RP-chromatography was therefore required with correspondingly low yields. Following the approach of previous syntheses of collagen peptides based on the use of Boc–GPO(Bz1)–OH [5] or Fmoc–GPO–OH [6] besides difficulties encountered in the preparation of the tBu-protected or unprotected Fmoc– GPO–OH, due to the expected diketopiperazine formation, again large amounts of microheterogeneities were observed. These derive from diketopiperazine formation and thus cleavage of the Gly-Pro dipeptide at the N-terminus of the growing peptide chains in the Fmoc-cleavage and subsequent acylation steps. The side reaction is largely prevented by the use of Boc–GPO(Bzl)–OH where N α-deprotection is performed with TFA [5]. Finally, the strategy of Sakakibara et al. [7] was applied by the use of POG derivatives. With FmocPOG–OH as synthon and Tentagel-S-PHB as solid support an optimized protocol could be elaborated which allowed to obtain the single α -chain peptides in satisfactory yields and at a high degree of homogeneity. For the regioselective interstrand disulfide-bridging of the three peptides in the preselected α 1/α 2/ α1'order, after various less satisfactory attempts [8] we succeeded to obtain the heterotrimers in high yields following the strategy outlined in Scheme 1. We made exclusive use of the thiol/disulfide exchange reaction of activated unsymmetrical disulfides in aqueous buffer (pH 4.5–5.0) under exclusion of air oxygen. Thereby previous experiences in the conversion of Cys(Acm) peptides with sulfenylchlorides into unsymmetrical disulfides were exploited to activate the disulfide linked hetero-dimers as Cys(Npys) derivatives [8]. By this procedure disulfide bridges were formed almost quantitatively, thus allowing for isolation of the heterotrimers by HPLC in good yields and as analytically well characterized compounds (HPLC, ES-MS, amino acid analysis). Upon reduction of the final products with tributylphosphine, HPLC

487

Scheme 1. Regioselective disulfide bridging of the α1, α2 and αl' peptides to the heterotrimers A, B, C and D.

served to additionally confirm their heterotrimeric composition. By reoxidation of the equimolar mixture of α1, α 2 and α 1' peptides the correct heterotrimer was not formed even in statistical amounts as monitored by comparative HPLC. All four heterotrimers showed the characteristic CD spectrum of triplehelices at 5°C with Rpn values [5] comparable to that of native collagen (Table 1). Conversely, the Rpn values of the single-chain peptides were compatible only with partial folding into poly(Pro)-II helices, thus confirming the supposedly weak triple-helix tendency of the collagenase cleavage site despite the presence of Gly residues at every third position. Thermal denaturation of the heterotrimers as monitored by CD at 221 nm indicated fully reversible unfoldings The analysis of the denaturation curves revealed i) two not well separated transitions with the average Tm values listed in Table 1, ii) the decise role of the number of consensus repeats in dictating the stability of the triplehelical fold and iii) a significant effect of minor sequence variations in the cleavage site (Gln → Asn and Val → Phe). Taking into account the strong contribution of solvation to the stability of triple-helices [3], the observed lower T m value of the heterotrimer C can reasonably be explained by the locally slightly Table 1. CD spectral data (5°C) and thermal denaturation parameters of the heterotrimers in the collagenase assay buffer (10 mM CaCl2 , 100mM NaCl, 50 mM Tris.HCl; pH 7.5). Compounds Trimer A Trimer B Trimer C Trimer D Collagen [5 ]

min. (nm) 196 198 199 198 197

cross (nm) 211 211 211 211 213

max. (nm)

Rpn

T m (°C)

222 222 222 222 222

0.120 0.125 0.115 0.126 0.130

8 34 32 35 37

488 changed side-chain hydrophobicities. Interestingly, N-terminal acetylation of two chains in the trimer C which was performed to allow for specific attachment of the collagen peptide to matrices in surface plasmon resonance (SPR) experiments, did not produce the enhanced stability of the triple-helix expected from suppression of electrostatic repulsions at the N-terminus. The collagenases process collagen type I with a single scission across all three α -chains at residues 775–776. Because of the high specificity of the cleavage site it is reasonable to assume that this recognition epitope exhibits very peculiar structural features which strongly depend upon the register of the α -chains. The heterotrimer B in its α 1/ α 2/α1' assembly was cleaved in a single cut into the two expected fragments without detectable intermediates in the time course of enzymatic processing by MMP8 (K m = 5 µM; K m = 1 µM for collagen type I). Conversely, the related α 1 peptide is digested at extremely low rates [2]. The observation that the trimer B is processed quantitatively at a single locus without release of intermediate digestion products in our opinion clearly confirms that the α 1/ α2/ α1 register represents indeed that of native collagen type I. To further analyze the ability of the synthetic heterotrimers to mimic the collagenase cleavage site of collagen type I SPR spectroscopy was performed on the immobilized trimers A and C with the inactive mutants of the collagenase (MMP1; Glu200A1a) and gelatinase A (MMP2; Glu375A1a). The collagenase binds tightly to the stable triple-helical trimer C (K d = 3–4 µM), but does not recognize the trimer A which at room temperature is unfolded and thus more gelatin-like. Conversely, the gelatinase was found to bind with a 2–3 fold higher affinity (Kd = 1–2 µM) the gelatin-like trimer A than the collagen-like trimer C. To conclude, with the synthetic strategy described we succeeded to our knowledge for the first time in the regioselective disulfide-bridging of three peptide strands and to prepare collagenous heterotrimers that fully mimic the collagenase cleavage site of collagen type I.

Acknowledgements The study was supported by the SFB 469 of the DFG. The authors thank H. Nagase (Kansas City) for the MMP1 and G. Murphy (Cambridge) for the MMP2 mutant.

References 1. Hofmann, H., Fietzek, P.P., and Kühn, K., J. Mol. Biol., 125 (1978) 137. 2. Ottl, J., Battistutta, R., Pieper, M., Tschesche, H., Bode, W., Kühn, K., and Moroder, L., FEBS Lett., 398 (1996) 31. 3. Bella, J., Eaton, M., Brodsky, B., and Berman, H.M., Science, 266 (1994) 75. 4. Musiol, H.-J., Siedler, F., Quarzago, D., and Moroder, L., Biopolymers, 34 (1994) 1553. 5. Feng, Y., Melacini, G., Taulane, J.P., and Goodman, M., J. Am. Chem. Soc. 118 ( 1996) 10351.

489 6. Fields, C.G., Lovdahl, CM., Miles, A.J., Matthias Hagen, V.L., and Fields, G.B., Biopolymers, 33 (1993) 1695. 7. Sakakibara, S., Inouye, K., Shudo, K., Kishida, Y., Kobayashi, Y., and Prockop, D.J., Biochim. Biophys. Acta, 303 (1973) 198. 8. Moroder, L., Battistutta, R., Besse, D., Ottl, J., Pegoraro, S., and Siedler, F., 1998. In Ramage, R. and Epton, R. (Eds) Peptides 1996 (Proceedings of the 24th European Peptide Symposium) pp. 59–62, Mayflower Scientific Ltd, Kingswinford.

163 Combined solid-phase and solution approach to protein synthesis Y. NISHIUCHI, J. BÓDI, B.-P. XIANG, S. ISAKA, T. INUI, H. NISHIO, T. KIMURA and S. SAKAKIBARA Peptide Institute, Inc., Protein Research Foundation, Minoh-shi, Osaka 562, Japan

Introduction Today, the chemical synthesis of protein is of great interest to the peptide chemist, and several techniques using solid phase and solution methods are available. From our extensive research into the synthesis of proteins, we developed a segment condensation method in solution for their synthesis using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and chloroform (CHL) [1]. To further refine our strategy in solution synthesis, we introduced a standard solid phase method on an N -[9-(hydroxymethyl)2-fluorenyl] succinamic acid (HMFS) linker developed by Albericio et. al. [2] for synthesizing protected peptide segments (based on the Boc/Bzl or cHx type), which is known as the time-determining step. However, several problems needed to be solved in order to synthesize homogeneous segments for using the subsequent segment coupling reaction in solution. The dehydration of the side chain amide of the Asn residue to the corresponding nitrile during stepwise elongation of the protected peptide segments on a solid support is one of the serious side reactions. This side reaction can be avoided by temporary protection using the Xan group on the Asn residues, which is quantitatively removable by a single treatment with TFA. The partial or complete cleavage of the side-chain protecting groups, such as the For group on the Trp residue and the BrZ group on the Tyr residue during detachment of the segments from the HMFS resin by treatment with 10% piperidine or 20% morpholine in DMF, could also be avoided by using of the cyclohexyloxycarbonyl (Hoc) group and the 3-pentyl (Pen) group, which were recently developed by our group. By applying our present strategy, we were able to rapidly synthesize homogeneous protected peptide segments for use in the subsequent segment coupling reaction in solution. Results and discussion Preparation of protected peptide segments on HMFS resin The HMFS linker was prepared as reported previously [2]. Loading of the resin with the first Boc-amino acid was performed either by coupling of the 490 Y. Shimonishi (ed): Peptide Science – Present and Future, 490–494 © 1999 Kluwer Academic Publishers, Printed in Great Britain

491 preformed linker-amino acid building block onto the resin (method A, Figure 1) or by coupling of the Boc-amino acid onto the linker-functionalized resin (method B). While method B has the advantage of great flexibility regarding the substrate coupled onto the resin, method A allows exact determination of the degree of amino acid substitution. Furthermore, by using method A, the risk of racemization with the first amino acid resulting from the use of N, N dimethyl-4-aminopyridine (DMAP) during the loading reaction was effectively excluded by recrystallization of the linker-amino acid building blocks. When HBTU or HATU is used as a coupling reagent to achieve a high degree of coupling efficiency, the major side reaction during the step-wise elongation of the protected peptide segments on a solid support is dehydration of the side chain amide of the Asn residue. The dehydration occurred even in the case of in-situ neutralization synthesis protocol (data not shown). To suppress this side reaction, we employed the Trt or Xan group on the Asn residue as a temporary protecting group. While the resistance of the Trt group to the TFA treatment resulted in part of the Trt group remaining even after several coupling cycles, the Xan group colud be quantitatively removed by a single TFA treatment. Cleavage of protected peptide segments from the HMFS resin The protected peptide segments from the HMFS resin were detached by treatment with 10% piperidine or 20% morpholine in DMF for 20–30 min (Figure 1). Under these conditions, the formation of aspartimide peptides was examined by evaluating their purities on HPLC using the model tetrapeptide, Boc–Ala–Asp(OcHx)–X –Ala (X = Gly, Phe or Asn); X are known to be the most susceptible amino acids to base-catalyzed conditions (e.g., piperidine and morpholine) through dehydration, transpeptidation and epimerization. In all cases, the extent of the formation of aspartimide peptides was more than 20%

Figure 1. Loading of the resin with the first amino acid and protocol for the synthesis of protected peptide segments.

492 when treated with 10% piperidine in DMF for 1 h, but was less than 0.2% when treated with 20% morpholine in DMF for 1 h. However, the aspartimide peptide formation was found to be negligible as long as morpholine was used within 30 min. Next, partial or complete cleavage of the For group on the Trp residue and the BrZ group on the Tyr residue was observed during detachment of the segments even by treatment with 20% morpholine in DMF. We, therefore, decided to employ the Hoc group for the side-chain functional group of the Trp residue. The Hoc group was completely stable under the conditions used for detachment of the segments from the HMFS resin and could be quantitatively removed by standard HF procedure without the use of thiol as reported previously [3]. In the case of the protecting group for the Tyr residue, the BrZ group is the most commonly used since cleavage of a carbonate-type protecting group generates the carbonic acid ester intermediate to suppress alkylation by carbocations formed in the HF treatment. To develop the carbonate-type protecting groups more stable against basic conditions than the BrZ group, we introduced Hoc and 2,4-dimethylpent-3-yloxycarbonyl (Doc) [4] groups into the hydroxyl group of the Tyr residue and examined the stability during treatment with 20% morpholine in DMF for 1 h. Both protecting groups tested were much more resistant to the detachment conditions than the BrZ group, but about 10% could still be removed, while the BrZ group was completely removed within 2 min. Thus, much effort has been focused on the design of phenolic ether-type protecting groups which are completely stable against basic conditions but have increased stability against repetitive TFA treatment. Decreasing the electrophilicity of carbocation generated in the HF reaction is known to decrease the formation of 3-alkylated Tyr. Based on these considerations, we introduced sec.-alkyl groups, i.e., 3-pentyl (Pen) and cyclopentyl groups, instead of halosubstituted benzyl groups to the side functional group of the Tyr residue. Loss of the Pen and cyclopentyl groups by the treatment with 50% TFA/DCM (0.03%/h) was significantly reduced when compared with that of the BrZ group (0.08%/h). Furthermore, both protecting groups were found to be stable during the morpholine treatment in DMF to detach the peptide from the resin. The amounts of alkylated side products of these protected Tyr derivatives under the standard HF procedure are summarized in Table 1, in comparison with those of the Cl 2 Bzl and BrZ groups. These

Table 1.

Formation of 3-alkylated Tyr by HF treatment Formation of 3-alkylated Tyr (%)

Condition

3-pentyl (Pen)

cyclopentyl

Cl 2 Bzl

BrZ

HF HF/anisole (9 : 1) HF/p-cresol (9 : 1)

0.8 0.5 0.5

4.2 2.7 2.3

69 23 10

0.6 0.3 0.2

The HF reactions were carried out at –5°C for 1 h.

493 results confirmed that the Pen group possessed all of the chemical properties required for our strategy. Application of the HMFS linker to practical peptide synthesis In order to demonstrate the utility of the combined solid-phase and solution approach, our present strategy was applied to the synthesis of muscarinic toxin I [5], a snake toxin consisting of 66 amino acids which acts on muscarinic acetylcholine receptors. The protected peptide was assembled in solution from seven segments, which were synthesized with an automatic synthesizer (ABI 433A or ACT model 90) using the Boc strategy on HMFS resin except for the C-terminal segment, which was prepared by the solution method. The following side-chain-protected amino acids were employed: Asp(OcHx), Glu(OcHx), Asn(Xan), Gln(Xan), Arg(Tos), Lys(ClZ), His(Bom), Cys(Acm), Ser(Bzl), Thr(Bzl), Tyr(Pen) and Trp(Hoc). Each protected peptide segment was detached from the HMFS resin by treatment with 20% morpholine in DMF for 20–30 min without any side reactions as described above. This procedure could be effectively performed even in the case of sparingly soluble segments (e.g., segment (1–11) in Figure 2) in DMF or NMP, which could be quantitatively extracted with a mixture of CHL and TFE or hexafluoroisopropanol (HFIP) (3:1). The homogeneity of each segment was checked by amino acid analysis, TLC, MALDI-TOF MS and RP-HPLC. It was found to be more

Figure 2. Synthesis of protected muscarinic toxin I (top) and HPLC profile of the crude (8 Acm)-peptide (bottom). a: TFA, b: Pac-Br/CsCO3 , c: Zn/AcOH-HFIP, d: WSCD/HOBt, e: WSCD/HOOBt

494 than 95% pure. The segment coupling reaction was carried out as shown in Figure 2 using WSCD in the presence of HOBt or HOOBt in DMF or NMP and/or CHL/TFE(3:1). In order to assess the homogeneity of the protected peptide thus obtained, all the protecting groups, except for Acm groups, were removed by treatment with HF/anisole (9:1) at – 5°C for 1 h. The homogeneity of the crude (8 Acm)peptide on HPLC is also shown in Figure 2, demonstrating that the combined solid-phase and solution approach developed in the present study could be successfully applied to the rapid synthesis of large peptides of sufficiently good quality. Using the present approach, we are now trying to synthesize green fluorescent protein (GFP) consisting of 238 amino acids.

References 1. Kuroda, H., Chen, Y.-N., Kimura, T. and Sakakibara, S., Int. J. Peptide Protein Res., 40 (1992) 294. 2. Rabanal, F., Giralt, E. and Albericio, F., Tetrahedron, 51 (1995) 1449. 3. Nishiuchi, Y., Nishio, H., Inui, T., Kimura, T. and Sakakibara, S., Tetrahedron Lett., 37 (1996) 7529. 4. Rosenthal, K., Karlström, A. and Undén, A., Tetrahedron Lett., 38 (1997) 1075. 5 . Jolkkonen, M., Van Giersbergen, P.L.M., Hellman, U., Wernstedt, C., Oras, A., Satyapan, N., Adem, A. and Karlesson, E., Eur. J. Biochem., 234 (1995) 579.

164 A simple linker for the attachment of aldehydes to the solid phase. Application to solid phase synthesis by the Multipin method N.J. EDE and A.M. BRAY Chiron Technologies Pty. Ltd., 11 Duerdin Street, Clayton, Victoria. 3168, Australia

Introduction Peptidomimetics based on C-terminal aldehydes are important tools for studying serine and cysteine proteases [1]. Although several methods for solid phase synthesis of peptide aldehydes have been reported, many of these are not well suited to multiple parallel synthesis. Semicarbazone-based linkers for amino acid aldehydes require preformation, and cleavage requires the use of formaldehyde, necessitating product purification [2]. Linkers based on the Weinreb amide [3–5] are attractive as the carboxylate substrate is easily coupled to the solid phase; however, cleavage products are contaminated with LiAlH4 and yields are generally low. Aldehyde generation via ozonolysis of a supportbound alkene has been described, but this innovative method has yet to come into routine use [6]. Acetal linkage has been used, however, this approach requires high concentrations of aldehyde and strongly acidic conditions to drive the attachment reaction to completion [7]. We have developed a simple linking strategy, which allows an aldehyde substrate to be linked directly to the solid phase via an oxazolidine. The linkage is stable to TFA and other conditions used in Fmoc peptide synthesis, cleaves with mild aqueous acid [8].

Results and discussion As presented in Scheme 1, a support-bound serine or threonine 1 is treated with a dilute solution of substrate aldehyde 2 (0.1 M in MeOH, 60°C, 2 h) to

Scheme 1. Attachment of Aldehydes to the Solid Phase via Oxazolidine Formation.

495 Y. Shimonishi (ed): Peptide Science – Present and Future, 495–497 © 1999 Kluwer Academic Publishers, Printed in Great Britain

496 Table 1. Peptide Aldehydes Prepared on Oxazolidine Linker-derivatised SynPhase ID

Sequence

Comment

5 6 7

Ac-Tyr-Ala-Phe-Val-H Ac-Leu-Leu-Arg-H Ac-Phe-Val-Arg-H

Model [9] Leupeptin Cathepsin B inhibitor [1]

Crowns.

ES-MS data (observed m/z)

ES-MS Assignment

525.2 427.4, 445.4 447.4, 465.3

[M + H] + [M + H] + , M + H 2 O + H] + [M + H] + , M + H 2 O + H] +

give a stable oxazolidine 3. The capture of aldehyde via imine formation is highly efficient, allowing for direct coupling to the solid phase. Following solid phase synthesis, the target aldehyde 4 is released from the solid phase by treatment with 5% AcOH(aq) for 30 min at 60°C. The method was developed and applied on the Multipin system [8, 9], a technique developed for solid phase library synthesis. Syntheses were performed on a hydrophilic graft polymer (MA/DMA), which is suited to aqueous chemistries. As an example, the method was applied to the synthesis of 5–7. A s presented in Table l, the product aldehydes 5–7 gave ES-MS signals consistent with the respective aldehyde ([M + H] + ) and, in the case of 6 and 7, t h e hydrated form ([M + H2O + H] + ). Leupeptin 6 has been reported to exist as an equilibrium mixture of hydrate, cyclic amino1 and aldehyde [1], and this was evident in the broad multiple peaks observed by LC-MS. The oxazolidine linker for aldehydes is a low cost linker, which is compatible with Fmoc peptide synthesis and side chain deprotection with TFA. Cleavage is performed with mild aqueous acid, which can be removed without leaving toxic residues. The linker is suited to the multiple parallel solid phase synthesis of potential protease inhibitors.

Acknowledgements The authors thank Ben Gubbins, Rachael Griffiths. Michael FitzGerald, Heather Patsiouras, Petro van Poppel and Wayne Sampson for their skilled technical support.

References 1. Otto, H.-H., Schirmeister, T., Chem. Rev., 97 (1997) 133. 2. Murphy, A.M., Dagnino Jr., R., Vallar, P.L., Trippe, A.J., Sherman, S.L., Lumpkin, R.H., Tamura, S.Y., and Webb, T.R., J. Am. Chem. Soc., 114 (1992) 3156. 3 . Nahm, S. and Weinreb, S., Tetrahedron Lett., 22 (1981) 3518. 4. Fehrentz, J.-A., Paris, M., Heitz, A., Velek, J., Liu, C.-F., Winternitz, F., and Martinez, J., Tetrahedron Lett., 36 (1995) 7871. 5. Dinh, T.Q. and Armstrong, R.W., Tetrahedron Lett., 37 (1996) 1161. 6. Pothion, C., Paris, M., Heitz, A., Rocheblave, L., Rouch, F., Fehrentz, J.A., and Martinez, J., Tetrahedron Lett., 38 (1997) 7749.

497 7. Aurell, M.J., Borx, C., Centa, M.L., Lopis, C., Tortajada, A., and Mestres, R., J. Chem. Res. (M), (1995) 2564. 8. Ede, N.J. and Bray, A.M., Tetrahedron Lett., 38 (1997) 7119. 9. Maeji, N.J., Valerio, R.M., Bray, A.M., Campbell, R.A., and Geysen, H.M., Reactive Polymers, 22 (1994) 203.

165 Stereoselective synthesis of dehydroaminobutyric acid P.M.T. FERREIRA, H.L.S. MAIA and L.S. MONTEIRO Department of Chemistry, University of Minho, Campus de Gualtar, 4710 Braga, Portugal

Introduction α, β -Dehydroamino acid are constituents of many recently discovered peptides, including several fungal metabolites with antibiotic activity [1]. Of the many methods available for the synthesis of dehydroamino acids, β-elimination processes are among the most frequently used [2,3]. However, these methods are often low yielding and multistep processes requiring tedious purifications to remove side products. In the case of dehydroaminobutyric acid the work-up procedures are complicated by the possibility of obtaining a mixture of the two stereoisomers.

Results and discussion By using our recently developed methodology described elsewhere in these proceedings i.e. the combined use of tert-butylpyrocarbonate (Boc 2 O) with dimethylaminopyridine (DMAP) in dry acetonitrile, high yields in C-protected N-acyl- ∆ Abu(Boc) were obtained from a variety of N,C-protected threonine derivatives (Table 1). Sampling the reaction mixture throughout the preparation, showed that the reaction proceeds through a three step pattern. The first step was the introduction of the tert -butyloxycarbonyl group at the amine function and this was followed by acylation of the hydroxyl group to give the corresponding tert-butylcarbonate; finally, this compound underwent β -elimination to yield the final product. NMR and chromatography studies showed only the presence of the Z-isomer. This stereoselectivity seems to result from the bulkiness of the groups introduce at the α -amine function, which would force and thus facilitate a trans E2elimination. This is in agreement with results obtained by Srinivasan et al. [4] Table 1.

Results obtained in the synthesis of dehydroaminobutyric acid derivatives (Z-isomer).

Reagent

Product

Yield/%

Tos–Thr–OMe Z(NO 2 )–Thr–OMe Boc–Thr–OMe Boc–Thr–OH

Tos–∆ Abu(Boc)–OMe Z(NO 2 )–∆ Abu(Boc)–OMe Boc– ∆ Abu(Boc)–OMe Boc– ∆ Abu(Boc)–OBu t

87 92 87 73

498 Y. Shimonishi (ed): Peptide Science – Present and Future, 498–499 © 1999 Kluwer Academic Publishers, Printed in Great Britain

499 Table 2.

Results obtained in the electrolysis of dehydroaminobutyric acid derivatives (Z-isomer).

Reagent

Product

Yield/%

Tos– ∆ Abu(Boc)–OMe Z(NO 2 )–∆ Abu(Boc)–OMe

Boc– ∆ Abu–OMe Boc– ∆ Abu–OMe

93 78

who reported that base induced β -elimination of O-tosylated N-acyl-DLThr–OMe (threo type) proceeds via a trans E2-elimination to give predominantly the Z-isomer. Full or partial deprotection of dehydroaminobutyric acid derivatives is now being tried and, so far, we have been able to remove the tosyl and the p-nitrobenzyloxycarbonyl group by electrolysis (Table 2). Now, we are attempting to remove the methyl ester from several dehydroaminobutyric acid derivatives and also the simultaneous removal of the two Boc groups from Boc– ∆ Abu(Boc)–OMe. This work shows that application to threonine of the methodology previously developed in our laboratories for the synthesis of dehydroalanine derivatives, allows the synthesis of dehydroaminobutyric acid derivatives in high yields, using mild reaction conditions and simple work-up procedures. This methodology offers the further advantage of leading to a single isomer.

Acknowledgments We wish to acknowledge the University of Minho, the Fundação da Ciência e Tecnologia and the Fundação Calouste Gulbenkian for financial support.

References 1. Jung, G., Angewan. Chemie, Int. Ed. Engl., 30 (1991) 1051. 2. Noda, K., Shimohigashi, Y., and Izumiya, N., The Peptides, Analysis, Synthesis, Biology, Academic Press, New York, 1983, p. 286. 3. Schmidt, U., Lieberknecht, A., and Wild, J., Synthesis (1988) 159. 4. Srinivasan, A., Stephesen, R.W., Olsen, R.K., J. Org. Chem, 47 (1977) 2256.

166 Efficient preparation of dehydroalanine derivatives P.M.T. FERREIRA, H.L.S. MAIA and L.S. MONTEIRO Department of Chemistry, University of Minho, Campus de Gualtar, 4710 Braga, Portugal

Introduction α, β -Dehydroamino acid residues are naturally occurring compounds frequently encountered in many biologically important peptides, which include a class of polycyclic peptide antibiotics known as lantibiotics (nisin, epidermin, subtilin) and several enzymes from plant and bacterial sources. Dehydroamino acids have also been introduced into peptides for structure-activity relationship studies, since they affect both chemical reactivity and conformation. In solid phase peptide synthesis these compounds can be used as linkers [1–4]. Several methods have been developed for the synthesis of dehydroamino acids [1,2]. β -Elimination reactions using β -hydroxyamino acid (serine and threonine) derivatives as precursors of dehydroalanine (∆ Ala) and dehydroaminobutyric acid (∆ Abu) constitutes the major method used for the synthesis of these compounds. With most of the β -elimination procedures, the side reactions that sometimes occur give rise to poor yields and formation of complex mixtures.

Results and discussion In our previous work, it was found that treatment of serine derivatives with benzyl chloroformate, DMAP and triethylamine in DMSO give dehydroalanine derivatives in fairly good yields (between 51 and 76%) [5]. In view of these results, we considered using double acylation at the α-amine group of serine in order to facilitate the β -elimination reaction. Thus, when reacting N,C-protected serine derivatives with tert-butylpyrocarbonate in dry acetonitrile using DMAP as catalyst the only product isolated was C-protected- N-acyl-N-Boc– ∆ Ala (Table 1). Due to base induced cleavage of the Fmoc group by the catalyst DMAP, an attempt to apply this methodology to an Fmoc derivative of serine failed t o g i v e t h e C-protected- N-Fmoc-N-Boc– ∆ Ala, giving instead C-protectedN,N -(Boc)2– ∆Ala. All the starting materials used in the reactions of Table 1 were both N- and C-protected, which implied two initial protection steps prior to the dehydration process. This led us to investigate the use of derivatives of serine with only Nor C -protection as starting materials. This approach led to more sluggish 500 Y. Shimonishi (ed): Peptide Science – Present and Future, 500–501 © 1999 Kluwer Academic Publishers, Printed in Great Britain

501 Table 1.

Results obtained in the synthesis of dehydroalanine derivatives.

Reagent

Product

Yield /% *

Tos-Ser-OMe Tos-Ser-OBzl Z-Ser-OMe Z(NO 2 )-Ser-OMe Z(NO 2 )-Ser-OBzl Bz-Ser-OMe Bz-Ser-OBzl Bz(NO 2 )-Ser-OMe Dpp-Ser-OMe

Tos- ∆Ala(Boc)-OMe Tos- ∆Ala(Boc)-OBzl Z- ∆ Ala(Boc)-OMe Z(NO 2)-∆ Ala(Boc)-OMe Z(NO 2)- ∆ Ala(Boc)-OBzl Bz- ∆ Ala(Boc)-OMe Bz-∆ Ala( Boc)-OBzl Bz(NO 2)-∆Ala(Boc)-OMe Dpp-∆ Ala(Boc)-OMe

99 97 85 81 91 92 92 85 88

*

crude yield, pure by TLC.

Table 2. Results obtained in the synthesis of dehydroalanine derivatives. Reagent Boc-Ser-OH H-Ser-OMe Z-Ser-OH *

Product Boc-∆Ala(Boc)-OBu Boc-∆ Ala(Boc)-OMe Z -∆Ala( Boc)-OBut

Yield /% * t

73 82 65

crude yield, pure by TLC.

reactions with slightly lower yields (Table 2), but it had the advantage of saving a reaction step in addition to the possibility of forming a tert -butyl ester by using a derivative with a free carboxyl function as starting material. A possible disadvantage of this method would be eventual diffculties in removing the N - and/or C-protecting groups. Hence, selective deprotection with the aim of obtaining H-∆ Ala–OR and R'-∆ Ala–OH is being carried out. So far, both electrochemical removal of Tos and Z(NO 2 ) and cleavage of Boc with TFA have been carried out successfully. Other cleavage methods are under investigation. The present methodology allows the preparation of dehydroalanine derivatives from serine in high yields, using mild reaction conditions leading to simple work-up procedures. Acknowledgments We wish to acknowledge the University of Minho, the Fundação da Ciência e Tecnologia and the Fundação Calouste Gulbenkian for financial support. References 1. Schmidt, U., Hausler, J., Olher, E., and Poisel, H., Fortschr. Chem. Org. Naturst., 37 (1977) 251. 2. Noda, K., Shimohigashi, Y., and Izumiya, N., The Peptides, Analysis, Synthesis, Biology, Academic Press, New York, 1983, p. 286. 3. Schmidt, U., Lieberknecht, A., and Wild, J., Synthesis (1988) 159. 4. Jung, G., Angewan. Chemie, Int. Ed. Engl. 30 (1991) 1051. 5. Ferreira, P.M.T., Maia, H.L.S., and Rodrigues, L.M., Peptides 1996 (proceedings of the 24th European Peptide Symposium, Mayflower Scientific Ltd., Kingswinford (U.K.), 1997, in press.

167 Totally stereocontrolled synthetic routes for (E )-alkene dipeptide isosteres N. FUJII, T. IBUKA, N. MIMURA, H. TAMAMURA, A. OTAKA, H. OHNO, H. AOYAMA, K. OKANO, M. HIROHASHI Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Introduction The goal of our recent research has been focused on the development of reliable and practical synthetic routes for diastereomerically pure (E)-alkene dipeptide isosteres[EADIs] in connection with the structure-based molecular design of peptide-lead drugs. The stereochemical outcomes of the organocopper-medi)- α, β-enoates 2 or γ , δ - syn-δ ated anti -S N 2' reactions of γ ,δ-cis- γ , δ epimino-(E amino- γ - mesyloxy-(E)- α, β -enoates 4 have been well documented by us [1] as depicted below. However, the obstacle associated with this strategy was clearly the shortage of efficient synthetic methods of the key intermediates 2, which gave L , L -type EADIs 3. Four diastereomixtures of γ ,δ-epimino- α, β -enoates 1 can be readily prepared by the sequence of known reactions starting from L -amino acids. Based on the a b-initio molecular orbital calculations [2], we have developed synthetically useful Pd(0)-catalyzed isomerization reactions of diastereomixtures of γ, δ-epimino- α , β -enoates 1, which predominantly give the desirable γ , δ - cis-γ , δ -epimino-( E)- α , β -enoates 2 in high yields. Furthermore, a new finding that a brief treatment of the same substrates 2 with dil. MeSO3 H quantitatively affords the regio- and stereoselectively ring-opened products 4 made it possible to synthesize the homochiral L , D-type EADIs 5 [3]. Essentially in the same manner starting from D -amino acids, stereoselective synthesis of D, D -type and D , L -type EADIs is feasible. Thus, the totally stereocontrolled synthetic routes for four sets of homochiral EADIs have been established.

Figure 1. Complete Stereocontrolled Synthetic Process for (E )-Alkene Dipeptide Isosteres.

502 Y. Shimonishi (ed): Peptide Science – Present and Future, 502–503 © 1999 Kluwcr Academic Publishers, Printed in Great Britain

503

Figure 2. Application of Diastereoconvergent Synthetic Process of (E)-Alkene Dipeptide Isosteres to Ras-Farnesyl Transferase Inhibitors.

The practical application of the above straightforward diastereoconvergent process to derivertizations of the potential peptide-lead drugs involving Ras-farnesyl transferase inhibitor and bombesin/GRP antagonist was examined. H-Cys ψ [CH 2 –NH]Val ψ [(E )CH=CH]Val–Met–OMe 6 and H-Cys ψ [CH 2 –NH]Val ψ[( E)CH=CH]Phe–Met–OMe 7 were synthesized as depicted in Figure 2. The compounds 6 and 7 exhibited weak but remarkable growth inhibitory activity against a bladder cancer cell line (T24; H-Ras mutant) and a pancreatic cancer cell line (AsPC-1; K-Ras mutant), respectively. H-D -Phe– Gln–Trp–Ala–Val–Gly–His–Leu ψ [(E)CH=CH]Leu–NH 2 (EABI-1), which was synthesized by essentially the same manner as above, exhibited strong antagonistic activity (IC 50 = 4.66 nM) for amylase release [4], but had no growth inhibitory activity against small-cell lung cancer cell lines (NCI-N417, H526) and pancreatic cancer cell lines (Pant-1, MiaPACA-2, AsPC-1) in sharp contrast to the previous reports by others [5].

References 1 . Fujii, N., Nakai, K., Tamamura, H., Otaka, A., Mimura, N., Miwa, Y., Taga, T., Yamamoto, Y. and Ibuka, T., J. Chem. Soc., Perkin Trans. I. 1995, 1359 and references cited therein. 2 . Ibuka, T., Mimura, N., Ohno, H., Nakai, K., Akaji, M., Habashita, H., Tamamura, H., Miwa, Y., Taga, T., Fujii, N. and Yamamoto, Y., J. Org. Chem., 62 (1997) 2982 and references cited therein. 3 . Tamamura, H., Yamashita, M., Muramatsu, H., Ohno, H., Ibuka, T., Otaka, A. and Fujii, N., Chem. Commun. (1997) 2327. 4 . Fujimoto, K., Doi, R., Hosotani, R., Wada, M., Lee, J.-Uk, Koshiba, T., Ibuka T., Habashita, H., Nakai, K., Fujii, N. and Imamura, M., Life Sciences, 60 (1997) 29 5 . Szepeshazi, K., Schally, A.V., Halmos, G., Lamharzi, N., Groot, K. and Horvath, J.E., Proc. Natl. Acad. Sci, USA, 94 (1997) 10913 and references cited therein.

168 Synthesis of protected 2,3-epoxy amines utilizing azaPayne rearrangement of activated 2-aziridinemethanols under basic conditions A. OTAKA, Y. UMEDA, H. TAMAMURA, T. IBUKA and N. FUJII Grduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

Introduction Protected 2,3-epoxy amines 3 are potential synthetic intermediates for preparing peptidomimetics such as hydroxyethylene dipeptide isosteres (HDI) which replace the scissile amide bond in peptidase substrate sequences. Protease inhibitors possessing HDI unit have received increasing attention as promising therapeutic drugs for AIDS [1]. Except for a few examples, generally, 3 is synthesized from α -amino acids via epoxidation of halohydrin 1 [2] or olefin intermediates 2 [3] obtained from the corresponding α-amino aldehydes. One potential limitation with use of theses reaction sequences is that the substituents in the P1 position of the HDI unit are restricted by the amino acids employed (Figure 1). Fine-tuning of a substituent in this P1 position can be one of the crucial points for achieving the optimum fit for the active center of the enzyme. For this purpose, a more flexible approach to epoxides 3 which is not limited by the amino acids employed is desired. Herein, we report a synthetic method for protected 2,3-epoxy amines 3 utilizing aza-Payne rearrangement of activated 2-aziridinemethanols [4].

Results and discussion In order to evaluate the usefulness of our method for the preparation of protected 2,3-epoxy amines, benzyl substituted epoxy amine 3, a synthetic intermediate for saquinavir 12 as an HIV protease inhibitor, was selected for

Figure 1.

General synthetic routes to protected 2,3-epoxy amines and their utilization.

504 Y. Shimonishi (ed): Peptide Science – Present and Future, 504–506 © 1999 Kluwer Academic Publishers, Printed in Great Britain

505 a synthetic target. Starting from L-serine, 1,2-amino alcohols needed for the preparation of aziridine methanols were synthesized by two different ways. Syn-1,2-amino alcohol 5 was preferentially formed by the addition of BzlMgCl in the presence of Cu(I) salt to Mts-protected serinal 4 (syn : anti = 98 : 2). On the other hand, anti -1,2-amino alcohol 8 requisite for preparation of saquinavir was synthesized with moderate diastereoselectivity (syn : anti = 3 : 7) by the following reaction sequences; reaction of Mts–Ser–OH 7 with BzlMgCl in the presence of nBuLi followed by LiBH 4 reduction and protection of primary OH group. Mitsunobu reaction of resulting anti -1,2-amino alcohol 8 yielded protected anti -aziridine methanol 9a. After removal of TBS group, aza-Payne rearrangement of aziridine methanol 9b with KH in THF gave the requisite protected 2,3-epoxy amine 3b (Figure 2). Reaction of 3b with a decahydroisoquinoline derivative 10 gave protected HDI 11a. After removal of the Mts group with 1 M TMSOTf-thioanisole in TFA, sequential condensation of two carboxylic acid components onto 11b gave saquinavir 12 (Figure 3). This procedure involves the addition of organometallics to protected serine or serinal and subsequent aziridine formation using Mitsunobu reaction followed by the aza-Payne rearrangement under basic conditions. Finally the methodology described here features that the substituent in the P1 position is depended on the organometallics used for addition reaction.

Figure 2. Synthetic routes to protected 2,3-epoxy amines utilizing aza-Payne rearrangement. i) BzlMgCl–Cu(I) salt, ii, v) Mitsunobu reaction, iii, vi) KH in THF, iv) nBuLi–BzlMgCl followed by LiBH 4 , Mts = mesitylene-2-sulfonyl, TBS = tert-butyldimethylsilyl.

Figure 3.

Synthesis of saquinavir, QuiAsn = N-quinolinyl-2-carbonyl-L-asparagine.

506 References 1. 2. 3. 4.

De Clercq, E., J. Med. Chem., 38 (1995) 2491. Parkes, K.E.B., et. al., J. Org. Chem., 59 (1994) 3656. Fässler, A., et. al., J. Med. Chem., 39 (1996) 3203. Ibuka, T., et. al., J. Org. Chem., 60 (1995) 2044.

169 Convenient preparation of phosphopeptide fragments using High TFMSA system and its application to synthesis of long chain phosphopeptides K. MIYOSHI, A. OTAKA, M. KANEKO, H. TAMAMURA and N. FUJII Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan

Introduction Protein phosphorylation provides structural and functional changes for proteins involved in intracellular signal transduction pathways [1]. Due to the important role of phosphorylation/dephosphorylation in biology, long chain phosphopeptides have received much attention as useful biological and biochemical tools to elucidate various cellular processes including signal transduction. Previously we reported a efficient synthetic methodology for phosphopeptides [2, 3]. In the present study, we tried to develop a new synthetic methodology for long chain phosphopeptides and found that partially protected phosphopeptide fragments [ClZ(2-chlorobenzyloxycarbonyl) group for lysine and methyl groups for phosphoamino acids] can be obtained by treatment of protected peptidyl resins with High TFMSA system with efficient cleavage and removal of the other protecting groups. This finding prompted us to apply the High TFMSA system to the preparations of the useful partially protected phosphopeptide fragments as the building blocks in thioester fragment condensation strategy [4]. Here, we report the convenient synthetic method of phosphopeptide fragments using High TFMSA system.

Result and discussion Initially, in order to evaluate the general applicability of High TFMSA reagent systems for the synthesis of the partially protected peptide fragments, we examined the acidity of the High TFMSA system. Using protected amino acids and protected amino acid linked resins, the usefulness of the High TFMSA reagent mixture (TFMSA : TFA : m -cresol = 1 : 9 : 1 (v/v)) for the removal of the side chain protecting groups and the release of amino acids from the resins was examined (Table 1). The amount of each amino acid in solution was quantified using an amino acid analyzer. These result indicated that, under the above deprotection condition, the C1Z group on lysine could be almost kept intact and removal of the other protecting groups and release of free amino acids from Merrifield resin could be achieved. 507 Y. Shimonishi (ed): Peptide Science – Present and Future, 507–509 © 1999 Kluwer Academic Publishers, Printed in Great Britain

508 Table 1. Deprotection of protected amino acid derivatives with High TFMSA system (TFMSA : TFA : m-cresol = 1: 9 : 1 (v/v) at 4°C). Regeneration(%) of free amino acids

Boc-Asp(OBzl)-OH Boc-Trp(Mts)-OH Boc-Ser(Bzl)-OH Boc-Tyr(Cl 2 Bzl)-OH Boc-Arg(Mts)-OH Boc-Glu(OBzl)-OH Boc-Thr(Bzl)-OH Boc-His(Bom)-OH Boc-Lys(ClZ)-OH Boc-Leu-Pam resin Boc-Glu(OBzl)-Merrifield resin

30 min

1h

2h

100 78 86 83 92 95 95 100 6 17 70

100 73 92 83 93 100 100 100 9 23 76

100 70 87 83 94 100 100 100 16 25 84

Next, We examined the efficiency of other reagent systems for synthesis of the peptide fragment. We carried out the acidolytic deprotection of FmocLys(ClZ)-OH using several TFMSA systems (Table 2). The ratio of components were estimated by comparison of the HPLC peak areas. This result demonstrated that the High TFMSA system was best condition for preparation of the partially protected peptide fragments and could be also applied to synthesis of methionine containing peptide fragment. Next, a model peptide resin 1 was prepared using standard Boc-based solidphase techniques. The treatment of the completed resin with High TFMSA system resulted in the release of the peptide from the resin with concomitant removal of the only Mts group on Arg residue (Figure 1). The analyses of TabIe 2.

Treatment of Fmoc-Lys(ClZ)-OH with several TFMSA systems. Conditions (at 4°C, (v/v))

1 . TFMSA : TFA : m-creso1 = 1: 9 : 1 2 . TFMSA : TFA : m-creso1 = 1: 9 : 1 + Met(leq.) 3 . TFMSA : TFA : p -cresol = 0.5 : 8.5 : 1 4 . TFMSA:TFA:anisole= 1:9: 1 5 . TFMSA : TFA : DMS : m-cresol = 1: 8 : 1: 1 6 . TFMSA : TFA : thioanisole : m-cresol = 1: 8 : 1: 1 Fmoc-Lys(Z)-OH 1 . TFMSA : TFA : m -cresol = 1: 9 : 1

Ratio(%) of Fmoc-Lys-OH 20 mm

60 min

N.D. 3.6 5.5 N.D. 11.5 41.9

6.7 5.8 — 9.8 31.5 83.6

60.6

86.3

Figure 1. Synthesis of peptide fragment using High TFMSA system.

509 crude deprotected peptide by HPLC and IS-MS showed that no significant side reactions were encountered.

References 1. Johnson, G.L., Vaillancourt, R.R., Curr. Opin. Cell Biol., 6 (1994) 230. 2. Otaka, A., Miyoshi, K., Kaneko, M., Tamamura, H., Fujii, N., Nomizu, M., Burke, T.R., Jr., Roller, P.P., J. Org. Chem., 60 (1995) 3967. 3. Otaka, A., Miyoshi, K., Roller, P.P., Burke, T.R., Jr., Tamamura, H., Fujii, N., J. Chem. Soc., Chem. Commun., (1995) 387. 4. Hojo, H., Aimoto, S., Bull. Chem. Soc. Jpn., 65 (1992) 3055.

170 An efficient synthesis of tryptophan-containing cystine peptide using the silyl chloride-sulfoxide system Y. FUJIWARA, T. KIMURA, K. AKAJI and Y. KISO Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan

Introduction In 1991, we developed a disulfide bond-forming reaction using silyl chloridesulfoxide and applied the method to the syntheses of biologically active peptides containing one to three disulfide bonds, such as human brain natriuretic peptide, somatostatin, β -human atrial natriuretic peptide and human insulin [1, 2]. The reaction is completed within 10–30 min without modification at the nucleophilic amino acid (Met, His, or Tyr), except for Trp. To synthesize a Trp-containing cystine peptide using the silyl chloride, the indole moiety of Trp has to be protected with formyl (CHO) group, since the chlorination of the indole ring proceeded rapidly [1, 3]. However, in the previous synthesis of somatostatin using Trp(CHO), formation of a side product, [Lys(CHO)9 ]somatostatin, derived from formyl group migration was inevitable to some extent [3]. In this paper, we describe a facile and efficient method to synthesize a Trpcontaining cystine peptide using the silyl chloride method.

Results and discussion We have developed an alternative synthetic scheme (Figure 1) using Trp(Mts)[Mts; mesitylene-2-sulfonyl] [4] instead of Trp(CHO) to circumvent the formation of [Lys(CHO)]-derivative. The Mts group was selected as a indole-protecting group, since the group were stable under 1N NaOH, hydrazinolysis, TFA, and TFA-thioanisole(10 : 1), but was easily removed by 1M trifluoromethanesulfonic acid(TFMSA)-thioanisole in TFA. We also found NinMts group was stable under 20%piperidine/DMF(24 hr at 25°C) treatment for Fmoc-cleavage and CH3 SiCl3 -PhS(O)Ph in TFA (4hr at 25°C) treatment for disulfide bond-forming reaction. In order to demonstrate the feasibility of this scheme employing successive treatment with CH 3 SiCl 3 -PhS(O)Ph/TFA and TFMSA-thioanisole/TFA, we synthesized somatostatin using Fmoc-Cys(Acm)-2-chlorotrityl(Clt) resin [5] as a starting resin (Figure 1). After the construction of the peptide-resin by standard SPPS, the resulting fully protected peptide-resin was treated with 510 Y. Shimonishi (ed): Peptide Science – Present and Future, 510– 511 © I999 Kluwer Academic Publishers, Printed in Great Britain

511

Figure 1. Synthetic scheme for somatostatin using Trp(Mts) derivative: (a) HPLC profile of crude [Cys(Acm) 3 , 1 4 , Trp( Mts) 8 ]-somatostatin; (b) HPLC profile of reaction mixture after the treatment of (a) with CH 3 SiCl 3 -PhS(O)Ph/TFA; (c) MPLC profile of crude somatostatin after TFMSA treatment.

TFA-scavengers to give the crude [Cys(Acm)3,14 , Trp(Mts) 8 ]-somatostatin. After FPLC purification, the purified peptide was treated with CH 3 SiCl 3 -PhS(O)Ph in TFA at 25°C for 15 min to form the disulfide bridge. [Trp(Mts) 8 ]- somatostatin thus obtained was then treated with TFMSAthioanisole/TFA for 10 min at 25°C to give the crude somatostatin without significant modification on the peptide chain. After purification by FPLC, the homogeneous peptide was obtained in 25% yield(calculated from the starting Fmoc-Cys(Acm)-Clt resin). In conclusion, we developed a facile and efficient method to synthesize a Trp-containing cystine peptide using Trp(Mts) derivative. These results show that the method adopted for the present synthesis (Figure 1) is suitable for the syntheses of complex cystine-peptides, especially those having Trp residues.

References 1. Akaji, K., Tatsumi, T., Yoshida, M., Kimura, T., Fujiwara, Y., and Kiso, Y., J. Am. Chem. Soc., 114 (1992) 4137. 2. Akaji, K., Fujino, K., Tatsumi, T., and Kiso, Y., J. Am. Chem. Soc., 115 ( 1993) 11384. 3. Akaji, K., Fujino, K., and Kiso, Y., In Yanaihara, N. (Ed.), Peptide Chemistry 1992, ESCOM, Leiden, 1993, p. 27. 4. Fujii, N., Futaki, S., Yasumura, K., and Yajima, H., Chem. Pharm. Bull., 32 (1984) 2660. 5. Fujiwara, Y., Yokotani, J., Akaji, K., and Kiso, Y., Chem. Pharm. Bull., 44 (1996) 1326.

171 Synthesis of an HIV-1 protease dimer analog covalently linked by a disulfide bridge M. KISHIDA, H. MATSUMOTO, N. NISHIZAWA, T. KIMURA, Y. FUJIWARA, K. AKAJI and Y. KISO Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan

Introduction HIV-1 protease, a 99 amino acid residues peptide, acts as proteolytic enzyme by forming a homodimer. Interestingly, single-chain tethered dimers of HIV proteases, artificial peptides having the same topology as pepsin-like aspartic proteases, also show similar enzymatic activity and are more stable than the natural enzyme [1]. In this paper, we report a synthesis of an HIV-1 protease dimer analog which consists of two identical peptide chains covalently linked by a disulfide bridge (Figure 1). The enzyme analog showed good stability and activity, and is expected to be a useful tool for assay of protease inhibitors.

Results and discussion The chemical ligations were widely applied to syntheses of many large peptides (enzyme, vaccine, artificial peptide/protein, etc.) and we prepared HIV-1 protease analogs employing the ligation forming thioester or thioether [2]. In the synthesis of a protease dimer analog covalently linked by a disulfide bridge, we adopted this strategy for the preparation of monomer unit. In order to make a disulfide bridge between two protease monomers, we introduced a Cys residue as a substitute for Asn 98 , because Asn98 and Asn 198 are located very

Figure 1.

An HIV-1 protease dimer analog covalently linked by a disulfide bridge.

512 Y. Shimonishi (ed): Peptide Science – Present and Future, 512–513 © 1999 Kluwer Academic Publishers, Printed in Great Britain

513

Figure 2. Synthesis of HIV-1 prstease (PR) dimer analog. HPLC conditions: YMC AM-302 (4.4 × 150 mm) MeCN (30–60%, 30 min) in 0.1% TFA, flow rate = 1 ml/min, OD = 230 nm.

close in the active conformation. Cys residues at positions 67 and 95 having a sulfhydryl group were replaced by Ala. A monomer unit, [NHCH 2 CH 2 SCH 2 CO 51–52, Ala 67,95 , Cys(Acm) 98 ]HIV-1 protease ( 3 ) was synthesized employing the chemoselective reaction of a bromoacetylated peptide ( 2 ) with a mercaptoethylamide peptide segment (1 ) prepared by Fmoc-based SPPS (Figure 2). The reaction proceeded within 4h (HPEC elution pattern of the crude sample is shown in Figure 2) and the product was easily purified by gel-filtration (yield: 55%). Then the disulfide formation was achieved using iodine (r.t., 1h). The homogeneous dimer analog was obtained by the purification on ODS-column (yield: 11%). After the folding procedure, the synthetic peptide possessed proteolytic activity comparable to rec. HIV-1 protease in an assay system using KARVY*Phe(NO 2 )EANle-NH 2 as a substrate (0.1 M MES, 5.6 mM NaCl, pH 5.5) (Km = 10 µM). References 1. DiIanni, C.L., Davis, L.J., Holloway, M.K., Darke, P.L., Kohl, N.E. and Dixon, RAF., J. Biol. Chem., 265 (1990) 17438; Bhat, T.N., Baldwin, E.T., Liu, B., Cheng, Y.-S.E. and Erickson, J.W., Nature Struct. Biol., 1 (1994) 552. 2. Nishizawa, N., Kimura, T., Matsumoto, H., Fujiwara, Y., Takaku, H., Akaji, M. and Kiso, Y., Peptide Chemistry 1996, (1997) 61.

172 Enzymatic peptide synthesis using activated esters as acyl donors in organic media T. MIYAZAWA, E. ENSATSU, K. TANAKA, R. YANAGIHARA and T. YAMADA Department of Chemistry, Faculty of Science, Konan University, Kobe 658-8501, Japan

Introduction Protease-catalyzed peptide synthesis has attracted much attention in recent years, because it has several advantages over the chemical couplings. However, the protease’s narrow substrate specificity is often regarded as a major drawback from a synthetic viewpoint, and its broadening remains a challenge. Recently, we investigated the α-chymotrypsin-catalyzed peptide synthesis using esters of N -protected amino acids or peptides as acyl donors and found that the use of the 2,2,2-trifluoroethyl ester instead of the conventional methyl ester is advantageous for the preparation of peptides containing non-protein amino acids such as halophenylalanines [1]. Furthermore, the extremely low efficiency during the coupling of an inherently poor amino acid substrate, e.g., Ala which lacks a large hydrophobic side chain, using the methyl ester as acyl donor was found to be greatly improved by the use of such esters as the trifluoroethyl or carbamoylmethyl ester. The ameliorating effect of the latter ester was especially significant [2]. In the present work, the superiority of the method using such activated esters was further ascertained in segment condensations and also in the couplings mediated by another protease.

Results and discussion When a series of N -benzyloxycarbonyl- L -Ala esters (Z- L -Ala-OR) were allowed to react with L-Leu-NH 2 in acetonitrile containing 4% Tris buffer (pH 7.8) at 30°C a rough proportionality was observed between the logarithm of the relative initial rate (v rel ) of consumption of the substrate ester and the polar substituent constant, σ * [3], of R which is a measure of its polar or inductive effect: the higher the electron-withdrawing ability of the R group, the higher the v rel value. This correlation is indicative of the predominance of the effect of the R group on the acyl-enzyme formation over the binding of the substrate ester onto the enzyme. In the case of the carbamoyl-methyl ester, however, its effect on the latter must also be relatively important, because the electronwithdrawing ability of this group should not be very large compared with other 514 Y. Shimonishi (ed): Peptide Science – Present and Future, 514–515  1999 Kluwer Academic Publishers, Printed in Great Britain

515 Table 1. Effect of donor esters on the coupling yield (%) between Z-(Gly)n -Xaa-OR and Leu–NH 2 mediated by α-chymotrypsin.

L-

(Gly) n -Xaa

Time

R = C H3

R = CH2 CF 3

R = CH 2 CONH 2

D L -Phe(2Cl)

lh lh 5min 5 min

3.2 0.8 14 15

19 14 56 62

39 25 95 88

D L -Phe(

2Br) Gly- L -Phe Gly-Gly- L -Phe

Table 2. Effect of donor esters on the coupling yield (%) between Z-L -Xaa-OR and L -Leu-NH 2 mediated by papain (at pH 8.0, after 24 h). Xaa

R=CH 3

R = CH2 CF 3

R = CH 2 CONH 2

Ala Phe

27 2.0

32 1.8

96 83

groups. In this regard, it is interesting to note that the N , N-dimethylated carbamoylmethyl ester considerably diminished the peptide yield. The superiority of the carbamoylmethyl ester over other activated esters was demonstrated by the coupling of 2-chloro- or 2-bromo-DL -Phe bearing a bulky halogen atom at the o -position as carboxyl component (Table 1). Although the peptide yield using the trifluoroethyl ester remained unsatisfactory, it was significantly improved by the use of the carbamoylmethyl ester. This ester was also applied to segment condensations (Table 1). The coupling yield between Gly- L -Phe or Gly–Gly- L -Phe and L -Leu was extremely high using the carbamoylmethyl ester as well as the trifluoroethyl ester, with no fear of racemization. In the couplings mediated by another protease, papain (Table 2), Phe was found to be a poorer amino acid substrate than Ala when their methyl esters were used as carboxyl components. The peptide yields were hardly improved by the use of the trifluoroethyl ester, while they were greatly enhanced using the carbamoylmethyl ester in the couplings of both Ala and Phe.

Acknowledgments This work was supported in part by a grant from the Hirao Taro Foundation of the Konan University Association for Academic Research.

References 1. Miyazawa, T., Nakajo, S., Nishikawa, M., Imagawa, K., Yanagihara, R., and Yamada, T., J. Chem. Soc., Perkin Trans. 1, (1996) 2867. 2. Miyazawa, T., Tanaka, K., Yanagihara, R., and Yamada, T., In Kitada, C. (Ed) Peptide Chemistry 1996 (Proceedings of the 34th Japanese Peptide Symposium), Protein Research Foundation, Osaka, 1997, p. 33. 3. Taft, Jr., R.W., In Newman, M.S. (Ed) Steric Effects in Organic Chemistry, John Wiely, New York, 1956, chap. 13.

173 Facile synthesis of the acid-labile peptide amide linker containing the 10,11-dihydro-5H dibenzo [a, d ] cyclohepten-5-yl group M. Noda Technology Research Laboratory, Shimadzu Corp. Nishinokyo, Kysto 604, Japan

Introduction In a previous paper, linkers for peptide amide formation under mild conditions during Fmoc solid phase peptide synthesis were prepared based on the 10,11-dihydro-5 H -dibenzo [ a, d ] cyclohepten-5-yl group (Sdibenzosuberyl group) and the 5 H -dibenzo[a, d] cyclohepten-5-yl group (5-dibenzosuberenyl group) [1]. The more acid-sensitive linkers derived from these groups are cleaved in high yield at lower TFA concentrations. Many peptides have been made using these linkers with excellent yields and purities. However, the preparation of 2-( m-methoxyphenethyl) benzsic acid (4 ) needing catalytic hydrogenolysis of 3-(methoxybenzylidene)phthalide using Raney Ni (4 kg/cm², 50°C), was not suitable for large scale preparation. The present paper describes the facile synthesis of 3-methaxy-10,11-dihydro-5H -dibenzo[ a , d ]cyclohepten5-one (5) prepared via m- methoxystilbene-2-carboxylic acid, and its conversion to the peptide amide linker of the 5-dibenzosuberyl group.

Results and discussion The synthesis of m-methoxystilbene-2-carboxylic acid (3), the precursor of the key intermediate, 2-methoxy-10,11-dihydro-5H -dibenzo [ a, d ] cyclohepten5-one (5), was reported by the Wittig reaction of the phosphsnium salt derived from methyl 2-methylbenzoate with m -anisaldehyde [2]. The intermediate should be amenable to the more simple one-step synthesis for large scale preparation. The fusion reaction of 2-carboxybenzaldehyde and m -methoxyphenylacetic acid in the presence of sodium acetate gave rise to m -methoxystilbene2-carboxylic acid with concomitant evolution of carbondioxide in 76% yield. The catalytic hydrogenation of 3 with 5% Pd-C gave 2-(methoxyphenethyl)benzoic acid (4), which was ringcyclized by treatment of PPA to give 5 without any regioisomers in 86% yield. Demethylation of 5 with AlCl3 afforded 6. The resulting OH group of 6 was alkylated with ethyl 5-brsmovalerate in the presence of t-BuOK followed by hydrolysis of the resulting ester with aqueous NaOH to give corresponding acid 7. The reduction of 7 with NaBH 4 516 Y. Shimonshi (ed): Peptide Science – Present and Future, 516–518  1999 Kluwer Academic Publishers, Printed in Great Britain

517

Figure 1. Synthesis of the peptide amide linker resins containing the 10,11-dihydro-5H-dibenzo[a,d]cyclopenten-5-yl group. a: CH3 CO 2 Na, 190°C; b: H2 5%Pd-C; c: PPA, 145°C; d: AlCl3 , benzene; e: t-BuOK, Br(CH2 ) 4 CO 2 Et, DMF; f: NaOH, dioxane-H2 O; g: NaBH 4 , isopropylalcohol; h: Fmoc-NH 2 , pts, AcOH; i: TGS-NH 2 , PyBOP-HOBT-IPEA, DMF.

gave the unstable alcohol (8). The easily formed alcohol cation in AcOH and a catalytic amount of p -toluenesuluphonic acid (pts) was trapped with 9-fluorenylmethoxycarbamate (Fmoc-NH2 ) to give the dibenzosuberyl linker (9) in 400% overall yield. The desired linker-resin range was achieved by coupling the polystyrenepolyethyleneglycol graft copolymer functionalized with the amino group (Tenta-Gel-S NH2 ) using PyBOP-HOBT-DIPEA in DMF. The linker-resin analogue in which the dibenzosuberyl group was coupled to the resin with one carbon unit was prepared from 2-hydroxybenzosuberone (6) [2]. In order to assess the relative rate cleavage of 10 and 11, Fmoc Val derivatives prepared from 10 and 11 were treated with 50%TFA–5%phenol in DCM as a cleavage cocktail. Half lives of Fmoc Val were ca. 3 min for 10 and ca. 12 min for 1 1. The results indicated that the Fmoc Val derivative from 10 can be rapidly cleaved at a lower concentration of TFA than that from 11.

518 References 1 . Noda, M., Yamaguchi, M., Ando, E., Takeda, K., and Nokihara, K., J. Org. Chem., 59 (1994) 7968. 2 . Ramage, R., Irving, S.L., and McInnes, C., Tetrahedron Letters, 34 (1993)6599.

174 Synthetic approach to microsclerodermins S. SASAKI 1 , Y. HAMADA 2 and T. SHIOIRI 1 1

Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467, Japan 2 Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho, Inage-ku, Chiba 263, Japan

Introduction Microsclerdermin A(R = OH) and B(R = H) have been isolated from a deep sea sponge of the genus Microscleroderma sp [1]. These 23-membered cyclic hexapeptides have intriguing antifungal activities against Botrytis cinerea, Candida albicans, Fusarium oxysporum, Helminthosporium sativum, and Pyricularia oryzae. Especially, they inhibit the growth of Candida albicans at a loading of 2.5µg/disk in the standard disk assay, but the other biological activities have not been uncovered yet. As a continuation of our studies on the synthesis of marine natural products, we have launched a total synthesis of this unique cyclic peptides.

Results and discussion Microsclerodermins contain four unusual amino acids, one of which (2S,3R, 4S, 5S , 6S ,11E)-3-amino-6-methyl-12-(4-methoxyphenyl)-2,4,5-trihydroxydodec-11-enoic acid (AMMTD) features five consecutive stereogenic centers, as shown in Figure 1.

Figure 1

519 Y. Shimonishi (ed): Peptide Science – Present and Future, 519–520 © 1999 Kluwer Academic Publishers, Printed in Great Britain

520

Scheme 1

Among the unusual amino acids contained in microsclerodermins, we tried the synthesis of AMMTD first [2]. Herein, we now succeeded in the stereoselective synthesis of AMMTD as its protected form using the Sharpless asymmetric dihydroxylation [3] and the Paterson’s anti -aldol reaction [4] as key steps. An efficient synthesis of the other amino acids, the pyrrolidinone equivalent a n d ( R )-3-hydroxy-4-aminobutyric acid (GABOB), was also achieved (Scheme 1). Now we have been trying the synthesis of the D -Trp-2-caboxylic acid and coupling of amino acids to construct the full carbon skeleton of microsclerodermins.

Acknowledgement This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

References 1. Brewley, C.A., Debitus, C., and Faulkner, D.J., J. Am. Chem. Soc. 116 (1994) 7631. 2. Sasaki, S., Hamada, Y., and Shioiri, T., Tetrahedron Lett. 38 (1997) 3013. 3. Sharpless, K.B., Amberg, W., Bennan, Y.L., Crispino, G.A., Hartung, J., and Zhang, K.S., J. Org. Chem. 57 (1992) 2771. 4. Paterson, I., Wallace, D.J., and Velazquez, S.M., Tetrahedron Lett. 35 (1994) 9083 and 9087.

175 Synthetic approach to caledonin, a natural bolaphile K. LEE and T. SHIOIRI Faculty of Pharmaceutical Sciences, Nagoya City University Tanabe-dori, Mizuho-ku, Nagoya 467, Japan

Introduction Caledonin is a modified peptide isolated from the marine tunicate Didemnun rodriguesi [1]. Caledonin, among other relevant structural features, possesses a new β -amino acid residue, a cycloguanidine group and presents strong metalcomplexing properties. Namely, caledonin is a natural bolaphile which strongly binds Zn I I and Cu I ions, and has in vitro cytotoxicity against Kb cells (85% inhibition at 10 µg/ml). Herein, we report synthetic studies on caledonin which has unique structures and significant biological activity.

Results and discussion Caledonin comprises a central L-phenylalanine residue connected via its amino g r o u p t o (S)-3-amino-5-mercaptopentanoic acid (a new sulfur-containing β-amino acid), and by its carboxy group to a six memberd cycloguanidine moiety bearing an n-octyl chain, as shown in Figure 1. We have investigated synthetic approach to Hpoi and Ampa units. The interesting cycloguanidine group in Hpoi would be formed via the HgCl 2 promoted guanylation [2] of precursor A or guanylation method reported by Overman [3]. We selected Boc-(R)-Asp(OBzl)-OH as a starting material to synthesize Ampa according to the retrosynthetic strategy shown in Scheme 1. Introduction of one more carbon atom at the α -carboxylic acid site would be achieved through the cyano derivative D, which could be prepared by the following step

Figure 1

521 Y. Shimonishi (ed): Peptide Science – Present and Future, 521–522 © 1999 Kluwer Academic Publishers, Printed in Great Britain

522

Scheme 1

: (1) reductive conversion of the ω -carboxylate group to the ether group, (2) reduction of the α -carboxyl group and transformation of the resulting alcohol to the cyanide, and (3) replacement of the N-protected group. The ether function of D would be converted to the thioether function through the iodide C. Now we have been trying to complete the synthesis of Hpoi and Ampa.

References 1. Vazques, M.J., Quinoa, E., Riguera, R., Ocampo, A., Igresias, T. and Debitus, C., Tetrahedron Lett., 36 (1995) 8853. 2. Yang, F.Y., Kowalski, J.A. and Lipton, M.A., J. Org. Chem., 62 (1997) 1540. 3. Overman, L.E., Rabinowitz, M.H. and Renhowe, P.A., J. Am. Chem. Soc., 117(1995)2657.

176 Synthetic approach to antillatoxin, an exceptionally ichthyotoxic cyclic lipodepsipeptide H. FUJIWARA, F. YOKOKAWA and T. SHIOIRI Faculty of Pharmaceutical Sciences, Nagoya City University Tanabe-dori, Mizuho-ku, Nagoya 467, Japan

Introduction Antillatoxin (1) was isolated by Gerwick and co-workers from a marine cyanobacterium, Lyngbya majuscula, and found to exhibit considerable ichthyotoxic activity (LD5 0 = 0.05 µg/ml) [1]. The structure of antillatoxin (1) shown in Figure 1 consists of the tripeptide portion and the hydroxy carboxylic acid moiety. The tripeptide portion is composed of L -alanyl- L -N-methylvalyl-glycine while the hydroxy carboxylic acid moiety contains the exo-olefin group and conjugated diene. The absolute stereochemistry was determind to be 4S,5R,2'S,5’S.

Results and discussion We have been quite interested in its activity as well as the unique structure, and launched the synthesis of antillatoxin (1). The overall retrosynthetic strategy for the synthesis of antillatoxin (1) is shown in Scheme 1. Antillatoxin (1 ) would be constructed from the lactone 5 and the tripeptide. Coupling of the tripeptide with the lactone 5 would provide the compound 4, which would further undergo deprotection and then cyclization with diphenyl phosphorazidate (DPPA, (PhO)2 P(O)N 3 ) [2] to give the compound 3. Finally the Pd catalyzed vinylation would lead to the formation of antillatoxin (1). The synthesis of the lactone 5a, one of the analogs of 5, started from the aldehyde 6 by the Evans aldol reaction [3] with 7. After protection of the resulting alcohol 8, reduction with LiAlH 4 afforded the alcohol 9. Oxidation, cis-selective Horner–Emmons reaction, followed by cyclization afforded the

Figure 1

523 Y. Shimonishi (ed): Peptide Science – Present and Future, 523–524 © 1999 Kluwer Academic Publishers, Printed in Great Britain

524

Scheme 1

Scheme 2

Scheme 3

α, β -unsaturated lactone 10. Addition of nitromethane, the Nef reaction, followed by protection of aldehyde afforded the lactone 5a. The required tripeptide was efficiently synthesized from H–Gly–OEt in a stepwise manner using diethyl phosphorocyanidate (DEPC, (EtO)2 P(O)CN) [4], as shown in Scheme 3. In summary, we have achieved the synthesis of the lactone 5a. The coupling of the lactone 5a with the tripeptide, macrolactamization of the compound 4, the Pd catalyzed vinylation of the iodide 3, and then the total synthesis of antillatoxin (1) are actively being performed in our group.

References 1. 2. 3. 4.

Orjala, J., Nagle, D.G., Gerwick, W.H., J. Am. Chem. Soc., 117 (1995) 8281. Shioiri, T., Yamada, S., Org. Synth., Coll. Vol. 7, (1990) 206 Gage, R., Evans, D.A., Org. Synth., 68 (1989) 83 Takuma, S., Hamada, Y., Shioiri, T., Chem. Pharm. Bull., 30 (1982) 3147.

177 Desulfation vs. deprotection: direct solid-phase synthesis of Tyr(SO3 H)-containing peptides using the SN1-type deprotection procedure K. KITAGAWA¹, T. YAGAMI², C. AIDA¹, H. FUJIWARA¹, S. FUTAKI³, M. KOGIRE4 , J. IDA 4 and K. INOUE 4 ¹ Niigata College of Pharmacy, Niigata 950-21, Japan ²National Institute of Health Sciences, Tokyo 158, Japan ³Institute for Medicinal Resources, The University of Tokushima, Tokushima 770, Japan 4Faculty of Medicine, Kyoto University, Kyoto 606, Japan

Introduction Recent advances in the Fmoc-chemistry made the direct synthesis of the sulfated peptide possible using the solid-phase method, however, even when the Fmocstrategy is adopted, conditions for the final cleavage/deprotection with acidic reagents becomes very crucial. During the course of our efforts to find a general synthetic method for the Tyr(SO3 H)-containing peptides, we noticed that TFA is not a destructive acid towards Tyr(SO3 H) as long as the treatment is conducted at low temperature [1]. Based on this finding, we used 90% aqueous TFA at low temperature as the deprotection system for Tyr(SO3 H)-containing peptides. In this study, we also performed kinetic studies on the desulfation reaction and the deprotection reactions to understand this deprotection system. Results and discussion Kinetic studies were performed to compare the desulfation rate from FmocTyr(SO 3 Na) with the deprotection rates of the side-chain protecting groups from Fmoc-Arg(Pbf) and Fmoc-Ser( t Bu). The Fmoc-amino acid derivatives were treated with neat TFA at a specified temperature (0°C, 18°C, and 30°C) and the degree of desulfation or deprotection was determined by HPLC. In all cases disappearance of the starting materials analyzed by HPLC followed good first-order kinetics. From these experiments, the Arrhenius plot (Figure 1) and activation parameters for each reaction in TFA (0°C) were obtained. The most striking difference between the activation parameters determined for each reaction is that the desulfation reaction possesses positive entropy terms (∆ S ‡ = + 59.6 J • K – 1 mol – 1). Also we found that the deprotection rates of Arg(Pbf) and Ser(t Bu) in TFA at 30°C were 12 times and 65 times faster than the desulfation rate of Tyr(SO3 Na), while these differences in the reaction rates were enhanced to 84 times and 182 times, respectively, at 0°C. The addition of a soft base such as thioanisole or EDT in TFA significantly accelerates the desulfation from the Tyr( SO3 H) residue as previously reported 525 Y. Shimonishi (ed): Peptide Science – Present and Future, 525–526 © 1999 Kluwer Academic Publishers, Printed in Great Britain

526

Figure 1 The Arrhenius plots for the desulfation rate of Tyr(SO3 Na) and the deprotection rates of Arg (Pbf) and Ser(t Bu). All the reactions were carried out in neat TFA.

[1]. The deprotection reaction, therefore, has to be performed as efficiently as possible without the addition of a soft base. We considered that the SN 1-type deprotection method is preferable for the deprotection of the Tyr(SO3 H)-containing peptide rather than the SN 2-type deprotection method. In this regard, TFA is a suitable deprotection reagent for the Tyr(SO3 H)-containing peptide because i) the desulfation rate in TFA is remarkably retarded at low temperature, and ii) the deprotection reaction in TFA proceeds in a S N 1-mode. Thus, by using a large difference in the reaction rates between the desulfation and the SN 1-type deprotection at low temperature, it seems possible to remove a large number of the protecting groups with minimum damage to the Tyr(SO3 H) residue. In a practical procedure, we used 90% aqueous TFA as a deprotection reagent. The deprotection rate of the Pbf group from Arg(Pbf) is retarded by the addition of H 2 0 (10%) to TFA, however, its deprotection rate is still 40 times faster than the desulfation rate in this medium. Direct solid-phase synthesis of human CCK-39 was successfully achieved by a novel approach using a 2-chlorotrityl-resin [2], in which the above described acidic deprotection system was used as the key reaction. The fully protected peptide-resin was subjected to a two-step cleavage/deprotection; first with a mixture of AcOH-CF3 CH 2 OH-CH 2 C1 2 (1: 1: 3 v/v, 25°C, 30 min) for cleavage, followed with 90% aq. TFA (0°C, 15h) for deprotection. The main peak in the HPLC chromatogram was proved to be the CCK-39 (amino acid analysis and MALDI-MS) and, after a one-step HPLC purification, a highly homogeneous product was obtained. The overall yield (8%) is judged to be acceptable for the synthesis of the nearly 40-residue sulfated peptide.

References 1. Yagami, T., Shiwa, S., Futaki, S. and Kitagawa, K., Chem. Pharm. Bull., 41 (1993) 376. 2. Kitagawa, K., Aida, C., Fujiwara, H., Yagami, T. and Futaki, S., Tetrahedron Lett., 39(1997) 599.

178 Enzymatic syntheses of oligopeptides in organic solvents Y.-H. YE, G.-W. XING, G.-L. TIAN, G. CHEN and C.-X. LI Department of Chemistry, Peking University, Beijing 100871, P.R. China

Introduction Enzymatic syntheses of peptides are stereospecific without racemization. The side chain functional groups of amino acids do not need to be protected. Most studies on enzymatic synthesis in the past were carried out in aqueous buffers or in the mixture of aqueous and organic solvents. Thermodynamically, the enzyme and the product of the reaction are more stable in organic solution, where the formation of peptide bonds is more favorable than the hydrolysis of the product. Moreover, most organic compounds are soluble in organic solvent. In the present study, we focused our research on the enzymatic syntheses of peptides in organic solvents. Although the enzymatic reaction is carried out in organic solvents, a small amount of water namely essential water is required for the reaction. Different enzymes, solvents, amount of essential water and pH etc. were studied and compared. P–DL–Ala–OY (P = Z or Boc, Y = H or Me) and P–DL–Tyr–OEt (P = Z or Boc) were coupled with Gly–NHNHPh by papain and α -chymotrypsin respectively in aqueous-organic mixed solvent or organic solvent to obtain the expected optically pure products P-L–Ala–Gly–NHNHPh and P-L–Tyr–Gly–NHNHPh [1]. The precursors of two sweeteners, N-protected aspartame P–Asp–PheOMe and a new sweetener candidate P–Asp–Ala–OcHex (P = Z or For, cHex = cyclohexyl) [2] were synthesized as model peptides by thermolysin in tert-amyl alcohol and reaction conditions were studied systematically [3]. N-protected Leu-enkephalin phenyl hydrazine, Z-Tyr–Gly–Gly–Phe–Leu–NHNHPh was synthesized successfully by thermolysin in tert-amyl alcohol. The two fragments, Z-Tyr–Gly–Gly–OEt and Boc–Phe–Leu–NHNHPh were synthesized by α -chymotrypsin and thermolysin in dichloromethane or tert-amyl alcohol respectively. The physical constants of all peptides synthesized by enzymes were identical with those of peptides synthesized by chemical method.

Results and discussion Condensation of N-protected DL-amino acids (or esters) with Gly-NHNHPh or N-protected-AspOH with DL–Phe–OMe (or DL–Ala–OcHex) in the presence of enzyme (papain, α -chymotrypsin or thermolysin) at optimum pH suc527 Y. Shimonishi (ed): Peptide Science – Present and Future, 527–529 © 1999 Kluwer Academic Publishers, Printed in Great Britain

528 cessfully formed the expected optically pure protected oligopeptides. The reactions can be carried out in an aqueous-organic mixed solvent or organic solvent. The amino acid ester as acyl donor was better than the corresponding acid. C-terminal, phenyl hydrazine was a better substrate than the corresponding ester for most enzymes. The amount of essential water needed depends on different enzyme. Synthesis of Z-Tyr–Gly–Gly–OEt catalyzed by α -chymotrypsin in dichloromethane, 0.15 ~ 0.25% water (V/V) was enough for the coupling reaction. The optimized water content was 6–8%(V/V) for the syntheses of Z-Asp–Phe–OMe and Z-Asp–Ala–OcHex catalyzed by thermolysin in t e r t-amyl alcohol. For synthesis of Boc–Phe–Leu–NHNHPh using thermolysin as a catalyst in tert-amyl alcohol, the optimized water content was 8%. Usually for hydrophobic solvent the amount of essential water was needed less than relatively polar organic solvent. There was almost no product formation when there was no essential water in organic solvents. The optimum pH value of enzymatic reaction in organic media depends on the substrate. For example, in the synthesis of N-Z-L-Ala–Gly–NHNHPh catalyzed by papain, Z-DL–AlaOH as a substrate, the optimum pH = 5.0, while Z-DL-Ala–OCH 3 as a substrate, the optimum pH increased to 8.2. In the syntheses of sweetener precursors, various amount of Et3 N was added to the thermolysin reaction in tert-amyl alcohol, the observed pH was changed from 5 to 10, the results showed that the good yield could be obtained at observed pH = 8 or 9, namely an excess of 1.6% Et3 N was good for peptide bond formation by thermolysin in tert -amyl alcohol. The isolated yield can be enhanced by increasing of the molar ratio of amino component/carboxyl component. When the ratio was 4:1, the isolated yield of Z-Asp–Phe–OMe was almost quantitative (100%). Synthesis of Leu-enkephalin and N-Acyl-enkephalin amide were reported previously [4, 5] which synthesized by enzyme in water-organic mixed solvent or organic solvent. We synthsized N-protected Leu-enkephalin phenyl hydrazine by thermolysin in tert-amyl alcohol in modest yield (42%) for the first time. Both fragments Z-Tyr–Gly–Gly–OEt and Boc–Phe–Leu–NHNHPh were also synthesized by α -chymotrypsin and thermolysin in dichloromethane and tert-amyl alcohol in good yields (71% and 67% respectively). Acknowledgement This project was supported by the National Natural Science Foundation of China. References 1. Tian, G.L., Liu, Y., Wang, H., and Ye, Y.H., Chem. J. Chinese Univ., 17 (1996) 55. 2. Zeng, G.Z., Chen, J.T., He, H.Z., Wang, Z.Q., and Yan, J.S., J. Agric. Food Chem., 39 (1991) 782.

529 3. Xing, G.W., Tian, G.L., Lu, Y., and Ye, Y.H., in Peptides: Biology and Chemistry (Proceedings of the 1996 Chinese Peptide Symposium), Xu, X.J., Ye, Y.H., and Tam, J.P. (Eds), Kluwer Academic Publishers, The Netherlands, 1998, pp. 50–51. 4. Kimura, Y., Nakanishi, K., and Matsuno, R., Enzyme Microb. Technol., 12 (1990) 272. 5. Gill, I. and Vulfson, E.N., J.Chem. Soc. Perkin Trans. 1, (1992) 667.

179 Studies of racemization in peptide synthesis for a phosphoryloxy-benzotriazin-one coupling reagent (DEPBT) H. LI¹, X. JIANG¹, Y.-H. YE², C.-X. FAN² and M. GOODMAN¹ ¹ Department of Chemistry & Biochemistry, University of California at San Diego, CA 92037, USA ² Department of Chemistry, Peking University, Beijing 100871, China

Introduction Racemization during peptide syntheses has been a major concern for all peptide chemists [1]. Much research has been carried out to develop new coupling reagents which allow rapid amide bond formation and inhibit racemization. The reagent, 3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin-4(3H)-one (DEPBT), has been recently developed [2] (Figure 1). It has been shown that this new coupling reagent, as with BOP and HBTU, is efficient both in solid phase peptide synthesis and peptide synthesis in solution. It can also be used for cyclization reactions. Analysis of DEPBT by X-ray diffraction confirms its chemical structure (Figure 1). In our laboratories, we have established a protocol to measure intrinsic rates of racemization employing the separation and quantitative analyses of enantiomeric N-protected amino acid derivatives by chiral HPLC [3]. In this presentation, we carried out racemization studies to compare DEPBT with commonly used coupling reagents such as BOP, HATU and HBTU.

Figure 1. The chemical and X-ray structures of 3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin4(3H)-one (DEPBT).

530 Y. Shimonishi (ed): Peptide Science – Present and Future, 530–533 © 1999 Kluwer Academic Publishers, Printed in Great Britain

531 Results and discussion In the general protocol developed in our laboratories, amino acids are allowed to react with the coupling reagent (the activator) to form the activated intermediate in situ. After a specific delay time during which the activated intermediate is exposed to tertiary amine, benzylamine is added to quench the reaction instantly. The resulting benzyl amide product is analyzed by chiral HPLC (Figure 2). The delay time is chosen so that typical reactions exhibit 15–30% racemization. From our previous studies, Boc–Ser(Bzl)–OH and Boc–Cys(4MB)–OH have been shown to possess high propensities toward racemization [3]. Therefore, these two amino acids derivatives were chosen in the current study for maximum sensitivity. The chiral HPLC analysis was carried out using a chiral column (‘Chiracel OD’ from Daicel Chemical Co.). Detection was based on UV absorption at 214 nm. The mobile phase was hexane : isopropanol (90 : 10). Under these conditions, baseline separation of the L and D enantiomers was obtained. For Boc–Cys(4MB)–NHBzl, the retention times for D and L isomers are 4.7 min. and 6.7 min. respectively. In the case of Boc–Ser(Bzl)–NHBzl, the retention time for D and L isomers are 5.7 min and 7.2 min. Comparison studies of different coupling reagents were carried out (Figure 3). Different activations of Boc–Ser(Bzl)–OH are studied using DIEA in DCM. In the case of DEPBT mediated coupling, even after 60 minutes delay time,

Figure 2. Table 1. OH.

The general protocol for analysis of racemization using chiral HPLC. Comparison studies of racemization of in situ activated intermediates of Boc–Ser(Bzl)–

Activation

Delay Time

L : D Ratio

PyBrop (1 eq) HATU (1 eq) HBTU (1 eq) BOP (1 eq) DEPBT (1 eq) DEPBT (2 eq)

4 min 4 min 15 min 15 min 60 min* 60 min*

65 : 35 84 : 16 79: 21 85 : 15 95 : 5 96 : 4

* Even with the much longer delay time, very little racemization is observed

Yield 81% 91% >99% 95% 70% >99%

532

Protocol for racemization studies of different coupling reagents.

Figure 3.

less than 5% D -isomer was observed. The yield was not ideal when one equivalent DEPBT was used. This may due to the fact that a small amount of coupling reagent decomposed during the treatment with tertiary base. When two equivalents of DEPBT was used, after 60 min delay, we only observed 4% D-isomer and a quantitative yield was obtained. This demonstrates that DEPBT is an efficient coupling reagent both in terms of yield of peptide coupling and inhibition of racemization. The effect of solvent on racemization and yield of DEPBT mediated coupling was also studied using Boc–Cys(4MB)–OH (Figure 4). Two solvent systems, DCM and THF, were compared (Table 2). No racemization was observed in either of the solvent. The yield for DEPBT mediated coupling is poor when the reaction is carried out in DCM. However, when two equivalents of DEPBT is used as coupling reagent in THF, quantitative yields were obtained. Since the activated intermediate formed during peptide coupling is prone to racemization, we synthesized and isolated the OAt and OOBt active esters of Boc–Cys(4MB) as crystalline materials (Figure 5). The intrinsic rates of racemization of these active intermediates were determined in DCM and THF as shown in Table 3. The results obtained are consistent with the in situ activation studies. DEPBT and its corresponding HOOBt ester efficiently inhibits racemization. In addition, THF is a better solvent than DCM for coupling reactions. The data from our studies demonstrates that DEPBT is an excellent coupling reagent. Based on our results, we proposed that the phosphorus moiety in

Figure 4.

Protocol to study the effect of the solvent on racemization and yield.

Table 2. The effect of the solvent on racemization and yield of an in situ activated intermediate for Boc–Cys(4MB)–OH Activation DEPBT DEPBT DEPBT DEPBT

(1 (2 (1 (2

Figure 5.

eq) eq) eq) eq)

Solvent

Delay Time

L: D

DCM DCM THF THF

15 15 30 30

100 : 0 100 : 0 100 : 0 100 : 0

min min min min

Protocol for racemization studies of pre-activated intermediate.

Yield 46% 73% 61% > 99%

533 Table 3. Racemization studies of pre-activated intermediates for Boc-Cys(4MB)–OAt and Boc– Cys(4MB)–OOBt. X

Solvent

Delay Time

L: D ratio

OAt OOBt OAt OOBt

DCM DCM THF THF

5 5 30 30

87: 13 98.5:1.5 95: 5 100.0

min min min min

Yield 94% 85% 98% >99%

DEPBT promotes the formation of the HOOBt active ester. The HOOBt ester in turn strongly inhibits racemization and forms the peptide bond efficiently during peptide coupling.

Acknowledgement The authors would like to acknowledge support for this research by the National Institute of Health NIHDK-15410 and NIHDA-05539.

References 1. Kemp, D.S., In Gross, E. and Meienhofer, J. (Eds.), The peptides, Analysis, Synthesis, Biology., Academic press, Orlando, 1979, Vol. 1, p. 315–383. 2. Fan, C.X., Hao, X.L., Ye, Y.H., Synthetic communications, 1996, V26, 1455–1460. 3. Romoff, T., Tran, T., and Goodman, M., Journal of Peptide Research 49, 1997, 281–292.

180 Effect of polyethylene glycol on the synthesis of oligopeptides by proteases in an organic medium Y. HIRANO, Y. NAGAO and T. TERAI Department of Applied Chemistry, Faculty of Engineering, Osaka Institute of Technology, 5-16-1 Ohmiya, Asahi-ku, Osaka 535-8585, Japan

Introduction In nature, enzymes are functional mainly in aqueous solution. However, in an organic solvent containing a small amount of water, it catalyzes peptide bond formation, the reverse of hydrolysis reaction. In this work, we discuss the effect of polyethylene glycol (PEG) on enzymatic peptide synthesis in an organic solvent, without the formation covalent bonds between PEG and the enzyme. An amino-protected amino acid was coupled with a carboxyl-protected amino acids to give the oligopeptides Z-Tyr– Arg(NO 2 )–OBzl, Boc–Val–Pro–OMe, and Boc–Gly–Asp(OBzl)–OBzl by α chymotrypsin, thermolysin and papain, respectively, in an organic solvent containing a small amount of water and PEG.

Results and discussion The relationship between the percentage of reaction (Z-Tyr–Arg(NO2 )–OBzl) and the amount of PEG added to the PBS solution is shown in Figure 1(a). In all of the systems containing PEG, the percentage of reaction was higher

Figure 1 Relationship between the percentage of reaction and amount of PEG. (a) Synthesis of :0.5 ml. (b) Synthesis Z-Tyr–Arg(NO 2 )–OBzl by α-chymotrypsin. PBS :  : 0.075 ml,  : 0.2 ml, : 0.5 ml. of Boc–Val–Pro–OMe by thermolysin. PBS :  : 0 ml,  : 0.2 ml,

534 Y. Shimonishi (ed): Peptide Science – Present and Future, 534–535 © 1999 Kluwer Academic Publishers, Printed in Great Britain

535 Table 1. Km and V m a x values determined from Lineweaver-Burk plots of Z-Tyr–Arg(NO 2 )–OBzl synthesis by α-chymotrypsin.

PEG-added system (PEG = 2 mg) PEG-free system

Km (mmol)

Vm a x (mmol/min)

38.4 37.8

0.72 0.32

than that in the PEG-free system. As shown in Figure 1(a), the amounts of PBS and PEG were important for high yields of dipeptide. Figure 1(b) shows the relationship between the percentage of reaction (Boc–Val–Pro–OMe) and the amount of PEG added to the PBS solution in the reaction catalyzed by thermolysin. This system demonstrated the same tendency as described above. In the PEG-added system, the enzyme and water were homogeneously dispersed in the organic medium, and the PEG presumably functioned as a surface-active agent. These results suggest that, in aqueous medium, α -chymotrypsin and thermolysin catalyze the hydrolytic reaction, however, in an organic solvent containing a small amount of water and PEG, proteases catalyze dipeptide bond formation, the reverse of hydrolysis reaction. Thus, the reaction changes from a hydrolytic one in the presence of high water concentration to peptide bond formation with very low water concentration. K m values obtained for both reaction systems at Z-Tyr–Arg(NO2 )–OBzl were the same (Table 1), although the addition of PEG contributed to the reaction rate by functioning to accelerate the reaction system. The Km values indicate that PEG acted as an anti-non-competitive inhibitor of α -chymotrypsin in the PEG-added system. As clearly shown in Table 1, the addition of PEG enhanced the rate of peptide synthesis. The reaction rate in the PEG-added system was approximately twice as fast as that in the PEG-free system, although the mechanism for the effect of PEG in this reaction is not clear. PEG, having both hydrophilic and hydrophobic characteristics, may be adsorbed onto the enzyme surface in the presence of a small amount of water, due to its hydrophilic nature, and partial covering of the enzyme with PEG may expose the substrate molecules to the active site of the enzyme by this hydrophobic nature. The presence of PEG did not damage the enzyme in an organic medium. Thus, the reaction rate was enhanced in the presence of PEG. Accordingly, it was possible to synthesize dipeptide using a non-modified enzyme in an organic medium by the addition of PEG. This method of peptide synthesis may be useful for obtaining large quantities of peptides.

References 1. Hirano, Y., Terai, T. and Nakajima, A. Agric. Biol. Chem., 55 (1991) 2461. 2. Hirano, Y., Nagao, Y., Tamai, N. and Goto, K. Peptide Chemistry, 1996 (1997) 69.

181 Synthesis of a cyclic tetrapeptide related to trapoxin B N. NISHINO¹, K.-Y. TOMIZAKI², M. KOMORI², K. IMA-IZUMI², Y. KOMATSU³ and T. MIMOTO³ ¹ Institute for Fundamental Research of Organic Chemistry, Kyushu University, Fukuoka 812-8581, Japan ² Department of Applied Chemistry, Kyushu Institute of Technology, Kitakyushu 804-8550, Japan ³Pharmaceuticals & Biotechnology Laboratory, Japan Energy Co., Toda 335-8502, Japan

Introduction Trapoxin B, a mammalian histone deacetylase inhibitor, is a cyclic tetrapeptide (cyclo(-Aoe–Phe–Phe- D –Pro-)) containing an unnatural amino acid Aoe (2-amino-8-oxo-9,10-epoxydecanoic acid) [1]. Cyl-1, a root elongation inhibitor is also a cyclic tetrapeptide containing Aoe (cyclo(-Aoe- D -Tyr(Me)Ile–Pro-)) [2], but the cyclic peptide framework is obviously different from trapoxin B. In the present study, we examined the amino acid sequence and concentration dependencies on the synthesis of cyclic tetrapeptide analogs, cyclo(-Lys(Z)–Phe–Phe- D –Pro-) (Figure 1A) and cyclo (-Lys(Z) - D -Tyr(Me)– Ile–Pro-) (Figure 1B) [3].

Results and discussion Synthesis of trapoxin B analog The linear peptides with four different amino acid sequences, Lys(Z)–Phe–PhePhe–Phe– D -Pro–Lys(Z), Phe-D -Pro–Lys(Z)–Phe, and D -Pro– D-Pro, Lys(Z)–Phe–Phe were prepared respectively. We chose O -(7-azabenzotriazole1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HATU), one of the fastest condensation reagent, for the cyclization reaction. The reaction mixtures of cyclization in DMF in various concentrations were analyzed by HPLC and

Figure 1. Structures of (A) trapoxin B and its analog, and (B) Cyl-1 and its analog.

536 Y. Shimonishi (ed): Peptide Science – Present and Future, 536–538 © 1999 Kluwer Academic Publishers, Printed in Great Britain

537

Figure 2. The ratios of tetra-/octapeptide in cyclization products at various concentrations for four linear peptides with different sequences. (A) trapoxin B analog, (B) Cyl-1 analog. To a solution of peptide in DMF at various concentrations were added HATU (1.5 eq) and DIEA (3 eq). After 1 hr, the reaction mixture was concentrated to syrup, then dissolved in DMF (0.5 mL) and analyzed by HPLC.

the ratios of tetra-/octapeptide were plotted against the concentrations (Figure 2). Two peptides, Lys(Z)–Phe–Phe- D - P r o , D -Pro–Lys(Z)–Phe–Phe gave the cyclic tetrapeptide as a major product at 0.1 mM. The higher concentrations increased the formation of the cyclic octapeptide. The other two sequences did not give the cyclic tetrapeptide efficiently. Synthesis of Cyl-1 analog The cyclization of four precursors of Cyl-1 analog was also carried out, the results being given in Figure 2B. D -Tyr(Me)–Ile–Pro–Lys(Z) gave the cyclic tetrapeptide as a major product. The other three peptides, Ile–Pro–Lys(Z)-D Tyr(Me), Pro–Lys(Z)-D -Tyr(Me)-Ile, and Lys(Z)-D -Tyr(Me)–Ile–Pro, did not give the cyclic monomer efficiently. Even D -Tyr(Me)–Ile–Pro–Lys(Z) could not be converted to the cyclic peptides completely (the conversion less than 50%) at 0.1 mM. Therefore, the cyclization at 0.25 mM seemed most efficient for the synthesis of the cyclic tetrapeptide. As summary, two sequences in which D -Pro was positioned at N- or C-terminal, Lys(Z)–Phe–Phe-D - P r o , D -Pro–Lys(Z)–Phe–Phe, gave the trapoxin B analog efficiently at 0.1 mM. The Cyl-1 analog could be synthesized from D -Tyr(Me)–Ile–Pro–Lys(Z) most efficiently at 0.25 mM. References 1. Kijima, M., Yoshida, M., Sugita, K., Horinouchi, S., and Beppu, T., J. Biol. Chem., 268 (1993) 22429.

538 2. Takayama, S., Isogai, A., Nakata, M., Suzuki, H., and Suzuki, A., Agric. Biol. Chem., 48 (1984) 839. 3. Miyashita, M., Nakamori, T., Akamatsu, M., Murai, T., Miyagawa, H., and Ueno, T., In Kitada, C. (Ed.) Peptide Chemistry (Proceedings of the 23th Peptide Symposium), Protein Research Foundation, Osaka 1996; pp. 137.

182 Highly efficient solid phase cyclization of peptides by methylenedithioether or disulfide bond formation M. UEKI¹, T. IKEO¹, K. NAKAMURA¹ and T. MARUYAMA² ¹ Department of Applied Chemistry, Faculty of Science, Science University of Tokyo, 1–3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan ² Torey Research Center, Inc., 1,111 Tebiro, Kamakura 248, Japan

Introduction Recently a trisulfide derivative was found in biosynthetic human growth hormone produced in E. coli [1]. This finding aroused a strong interest in activities of trisulfide analogs of biologically active peptides [2, 3]. However, the trisulfide bond is not stable at pH 7 and rapidly converted to disulfide at pH 9 [2]. In these years we have establish the very mild method for the formation of the S–CH 2 –S bond [4–6], which is corresponding to a mono-carba analog of the trisulfide bond and more stable. However, these works were so far made in solution. In this study we aimed to extend the method to highly efficient solid phase cyclization.

Results and discussion The S–CH2 –S bond formation was found to occur when the thiol protecting dimethylphosphinothioyl (Mpt) group was removed with tetrabutylammonium fluoride hydrate (TBAF • xH2 O) in CH2 Cl 2 . The S -Mpt group is an acid-stable protecting group suitable for the Boc strategy synthesis. Then, application of the S–CH 2 –S bond forming reaction to solid phase cyclization was tried by the Boc strategy at first. However, we could not obtain any reproducible results. In some cases many peaks, which did not contain that for the desired compound with S–CH2 –S bond, were observed in HPLC. A thioacetal structure is said to be stable under acidic conditions. However, stability in such a strong acid like HF, which is used in the final deprotection and release of a peptide chain from the resin support, has not been recorded to the best our knowledge. Then, stability of the S–CH2 –S bond in the strong acid media was examined. Whereas complex decomposition occurred in liquid HF, the S–CH2 –S bond was found to be quite stable in trifluoromethanesulfonic acid (TFMSA) containing proper scavengers and trifluoroacetic acid (TFA). Therefore, there must be any unknown causes in the solid matrix to prevent the S–CH2 –S bond formation. Studies to find and remove these causes are still now on. 539 Y. Shimonishi (ed): Peptide Science – Present and Future, 539–541 © 1999 Kluwer Academic Publishers, Printed in Great Britain

540 The S-Mpt group is very labile under basic conditions and its use is limited to the Boc strategy synthesis. In the 34th Symposium on Peptide Chemistry of Japan, we reported the S–CH 2 –S bond forming cyclization of penicillaminecontaining peptides [6]. For penicillamine we failed to obtain an S -Mptsubstituted building block. Instead, linear precursors with free thiol groups were used successfully to give the desired S–CH2 –S bond formed products. Through these studies optimal conditions for the highly efficient S–CH2 – S bond forming cyclization in solution were established. Then, in this study, we tried to apply these conditions to solid phase side-chain to side-chain cyclization. To accomplish selective removal of an S-protecting group on solid support, we used methoxytrityl (Mmt) group for thiol protection. A sequence of [Arg 8 ]-vasopressin was constructed on 4-methylbenzhydrylamine (MBHA) resin using Applied Biosystems 430A FastMoc program. Two S-Mmt groups were removed selectively with 1% TFA in CH2 Cl 2 /triethylsilane (95 : 5) and then the resin was treated with 4 eq. of TBAF • xH2 O in CH 2 Cl2 at RT for 3 h. Release with TFMSA/PhSMe/EDT/EtSMe/TFA (2 : 3 : 1 : 1 : 20) gave a crude peptide in 88% yield. Pure material could easily be obtained by preparative HPLC. FAB-MS: Found 1098 [M + H]+ . We have so far dealt with the S–CH2 –S bond formation between two Cys residues using TBAF • xH2 O in CH 2 Cl 2 . In the 33rd Symposium on Peptide Chemistry of Japan, we also reported the clear and rapid conversion of a Cys(Mpt) residue to a disulfide by treatment with TBAF • xH2 O in carbon tetrachloride [4]. Following the success of solid phase S–CH2 –S bond forming cyclization, S–S bond forming cyclization on solid support was also aimed. For this purpose, dilution of CCl4 , which is not a good solvent for solid phase reactions, with CH2 Cl 2 was tried. When the same resin described above was treated with 6 eq. of TBAF • xH 2 O in CCl4 /CH 2 Cl 2 (30 : 70) at RT for 3 h, highest yield of [Arg 8 ]-vasopressin was obtained. FAB-MS: Found 1084 [M + H]+ . No methylene insertion occurred under this condition. In conclusion, we could establish highly efficient methods for solid phase cyclization, in which side-chain to side-chain S–CH2 –S and S–S bonds were formed by simple treatment of a peptidyl dithiol with TBAF • xH 2 O in CH2 Cl 2

541 and CCl 4 /CH2 Cl 2 , respectively. Further studies on their applications are underway.

References 1. Jespersen, A.M., Christensen, T., Klausen, N.K., Nielsen, P.F., and Sørensen, H.H., Eur. J. Biochem. 219 (1994) 365. 2. Moutiez, M., Lippens, G., Sergheraert, C., and Tartar, A., Bioorg Med. Chem. Lett. 7 (1997) 719. 3. Chen, L., Zauliková, I., Slaninová, J., and Barany, G., J. Med. Chem. 40 (1997) 864. 4. Ueki, M., Komatsu, H., Saeki, A., and Katoh, T., In Maia, H.L.S. (Ed.) Peptides 1994 (Proceedings of the 23rd European Peptide Symposium), ESCOM, Leiden, p. 175. 5. Ueki, M., Saeki, A., and Nakamura, K. in Nishi, N. (Ed.) Peptide Chemistry 1995 (Proceedings of the 33rd Symposium on Peptide Chemistry), Protein Research Foundation, Osaka, 1996, p. 41. 4. Ueki, M. and Nakamura, K., In Kitada, C. (Ed.) Peptide Chemistry 1996 (Proceedings of the 34th Symposium on Peptide Chemistry), Protein Research Foundation, Osaka, 1997, p. 37.

183 4-(2-(1-Hydroxyethyl)-1,3-dithiacyclohex-2-yl)phenoxyacetic acid (HETPA): photolabile linker for Fmoc solid phase synthesis M. UEKI, S. ARIMOTO and R. SASAKI Department of Applied Chemistry, Faculty of Science, Science University of Tokyo, 1–3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan

Introduction Benzoin and phenacyl esters as well as o-nirobenzyl esters belong a category of photolabile protecting group. A useful phenacyl-type linker for solid phase synthesis has already been reported, however its use is limited to the Boc strategy synthesis [1]. Recently we applied the idea of protection of protecting group to phenacyl group and developed acetalized phenacyl group for carboxyl protection [2] and a dithioacetalized phenacyl linker for Fmoc/But solid phase synthesis [3]. In this study synthesis and use of a more photolabile α -methylp-alkoxyphenacyl-based linker, 4-(2-(1-hydroxyethyl)-1,3-dithiacyclohex-2-yl)phenoxyacetic acid (HETPA), were aimed.

Results and discussion Synthesis of HETPA linker was performed according to Scheme 1. Unfortunately, HETPA linker molecule was highly hydrophilic and could not be isolated from aqueous solution after alkaline hydrolysis. Instead, it was isolated in the form of phenacyl ester. Incorporation of the C-terminal Fmocamino acid was achieved using this HETPA phenacyl ester. The key point to use thioacetal as carbonyl protecting group is the oxidative method for dethioacetalization. The release of an acyl component by photolysis from a benzoin ester resin obtained from a dithian-protected benzoin ester resin was reported to depend, by unclear reasons, on the oxidation methods for dethioacetalization [4]. In peptide synthesis, presence of oxidation-sensitive amino acid residues should also be considered. Iodine oxidation was adopted in this work. Fmoc-Leu-(HETPA)-OH was incorporated onto H-Ala-4-methylbenzhydrylamine (MBHA) resin using the FastMoc cycle on Applied Biosystems 430A synthesizer. Chain elongation of a neuromedin N sequence was performed by the same program. The α -methylphenacyl function was regenerated by treating the resin with 1 M iodine solution in THF-2,6-lutidine-H 2 O (40 : 10 : 1) at RT for 3 h. Release of the peptide by photolysis (350 nm) was traced by 542 Y. Shimonishi (ed): Peptide Science – Present and Future, 542–544 © 1999 Kluwer Academic Publishers, Printed in Great Britain

543

Scheme 1.

Synthetic route to HETPA phenacyl ester.

Scheme 2.

Solid phase synthesis using HETPA linker.

HPLC. Whereas complete reaction took 30 h, amino acid ratio (Leu/Ala) of hydrolyzates of the remaining resin showed that 93% of the peptide was released. Unfortunately, isolated yield was 47% based on the amount of Leu loaded. One of the reasons of the low yield would be presence of a by-product. Since a single peak was observed in HPLC of a peptide released by fluorolysis [5], production of the by-product would be attributed to photolysis. Elucidation of this side reaction and improvements of yields are now going on.

References 1. Bellof, D. and Mutter, M., Chimia 39 (1985) 317. 2. Ueki, M., Mizuno, M., Nakata, H., Ishiwata, A., Sasaki, R., Komiya, S., and Katoh, T. In Okada, Y. (Ed.) Peptide Chemistry 1993 (Proceedings of the 31st Symposium on Peptide Chemistry), Protein Research Foundation, Osaka, 1994, p. 53.

544 3. Ueki, M., Sasaki, R., Nakamura, A., Tsurusaki, T., Okumura, J., and Arimoto, S. In Nishi, N. (Ed.) Peptide Chemistry 1995 (Proceedings of the 33rd Symposium on Peptide Chemistry), Protein Research Foundation, Osaka, 1996, p. 49. 4. Routledge, A., Abvell, C., and Balasubramanian, S., Tetrahedron Lett., 38 (1997) 1227. 5. Ueki, M. and Amemiya, M., Tetrahedron Lett., 28 (1987) 6617.

184 Kinetic study of the pseudolysin-catalyzed synthesis of Z-Ala–Phe–NH 2 S. RIVAL, J. SAULNIER and J. WALLACH Biochimie Analytique et Synthèse Bioorganique, Université Claude Bernard Lyon 1, 43 bd du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France

Introduction In recent years, enzymatic peptide synthesis, including synthesis of bioactive peptides and analogues, semi-synthetic proteins and bioactive peptides from recombinant precursors has been attracting increasing attention (for a review see [1, 2]). Pseudomonas aeruginosa elastase (pseudolysin), a thermolysin-like metalloproteinase has been shown to synthesize N-protected dipeptide amides with good yields and purities [3,4]. While both enzymes show some structural similarities [5] elastase but not thermolysin can incorporate tyrosine and tryptophane in P'1 position with high synthesis rates (S. Rival unpublished). Kinetic mechanisms of peptide synthesis by thermolysin have been already proposed [6–8]. In this work, mainly from conductimetric measurement of initial rates, we have studied the enzymatic mechanism of the pseudolysincatalyzed synthesis of Z-Ala–Phe–NH 2 and the influence of some parameters. Results and discussion The synthesis rate of N α-benzyloxycarbonylalanylphenylalaninamide from Z-Ala and Phe–NH 2 (Bachem) has been studied at 30°C in methanol: water (35:65) in presence of pseudolysin (E.C.3.4.24.26, Nagase Biochemicals). The synthesis initial rates have been calculated from RP-HPLC measurements with a Pharmacia-LKB apparatus fitted with a LKB RP18 Spherisorb ODS-2 column, the elution has been carried out with water: acetonitrile: TFA (65 : 35 : 0.05) and the absorbance changes have been monitored at 214 nm. The optimal pH value has been found around 7.3, close to the value reported for peptidase activity of the pseudolysin. The synthesis is not significantly changed for ionic strength values below 0.15. The synthesis greatly depends on the nucleophile excess. Furthermore the optimal excess is related to the nucleophile itself, e.g. it is about 2 for Phe–NH 2 and higher than 6 for Tyr–NH 2 . The pseudolysin activity is notably decreased with dimethylformamide whereas similar activities have been observed for others solvents i.e. methanol, acetonitrile and ethyle acetate. We have developed a conductimetric protocol for recording the pseudolysincatalyzed synthesis of Z-Ala–Phe–NH 2 . As expected, the conductance decreases 545 Y. Shimonishi (ed): Peptide Science – Present and Future, 545–546 © 1999 Kluwer Academic Publishers, Printed in Great Britain

546

Figure 1 Pseudolysin-catalyzed synthesis of Z-Ala–Phe– NH 2: Hanes –Woolf plots obtained for various concentrations of Z-Ala and fixed concentrations of Phe–NH2 (10–200 mM).

with time due to the decrease of charged molecules during the reaction. The calibration curves are similar for media containing 35% methanol and either water or 5–10 mM Tris and for different nucleophile excesses (S.D. between slopes is about 8%). A good correlation between conductimetry and HPLC has been shown (R = 0.99, slope = 1.03). The main advantages of the conductimetric method are the possibility to do a continuous measurement of the initial rates and the short time required for each experiment, about 12–15 min compared to 1 hour for LC. Furthermore, the accurancy of conductimetric recordings is good (S.D. is 3% for six independent assays). The conductimetric method has been used to determine the kinetic parameters of the pseudolysin for Z-Ala and Phe–NH2 . Hanes-Woolf plots indicate a rapid equilibrium random mechanism (an example is given Figure 1). The values of kinetic constants are 6.5 mM product/min/µM pseudolysin (Vm), 70 mM (K M of Z-Ala) 180 mM (K M of Phe–NH 2 ). The binding of one substrate has been found to have a negative effect on the binding of the other ( α = 20). A similar mechanism was reported for thermolysin but the α value was found close to or less than 1 [6–8]. References 1. 2. 3. 4. 5. 6. 7. 8.

Kullmann, W., Enzymatic Peptide Synthesis. CRC Press, Boca Raton, FL, 1987. Bongers, J. and Heimer, E.P., Peptides 15 (1994) 183. Pauchon, V., Besson, C., Saulnier, J. and Wallach, J., Biotechnol. Appl. Biochem. 17 (1993) 217. Rival, S., Charlieux, D., Besson, C.M., Saulnier, J.M. and Wallach, J.M. In Schneider, C.H. and Eberle, A.N. (Eds.) Peptides 1994 (Proceedings of the 23rd European Peptide Symposium) ESCOM, Leiden, 1995, p. 236. Thayer, M.M., Flaherty, K.M. and McKay, D.B., J. Biol. Chem. 266 (1991) 2864. Wayne, S.I. and Fruton, J.S., Proc. Natl. Acad. Sci. USA 80 (1 983) 3241. Riechmann, L. and Kasche, V., Biochim. Biophys. Acta 872 (1986) 269. Kunugi, S. and Yoshida, M., Bull. Chem. Soc. Jpn 69 (1996) 805.

185 Solid phase peptide synthesis using 2-(4-Nitrophenyl)sulfonylethoxycarbonyl (NSC) amino acids for Luteinizing hormone-releasing hormone (LHRH) S.H. LEE, J.I. PARK, W.U. CHWEH, Y.D. KIM and H.J. KIM Research Institute, Hyundai Pharm. Ind. Co., Ltd., Bucheon, 422231 Korea

Introduction The Nα -9-fluorenylmethoxycarbonyl (Fmoc) amino acid groups are base labile. This characteristics have been widely applied to the solid phase peptide synthesis to replace acid-labile t-butoxycarbonyl(BOC) amino protecting groups. However, the extreme base sensitivity of Nα-Fmoc-protected group unstable in neutral aprotic solvents is one of the disadvantages of Fmoc-amino acids. 2-(4-Nitrophenyl) sulfonylethoxycarbonyl (Nsc) group can be used as the appropriate protecting group for Na-amino acids in SPPS application and liquid phase peptide synthesis [1]. Base-labile Nsc- and Fmoc-protecting groups are compared as temporary Nα-amino protection in the automated SPPS of Luteinizing hormone-releasing hormone (LHRH) and ( D -Trp 6 )– LHRH on MilliGen 9050+ PepSynthesizer

The Structure of 2-(4-Nitrophenyl)sulfonylethoxycarbonyl (Nsc) amino acid.

Results and discussion LHRH and its analogue were synthesized on a PAL-PEG-PS resin (0.19 mmol/g) using Nsc and Fmoc chemistry. Nsc-amino acids were activated by BOP/HOBt in the presence of DIPEA in DMF. Deprotection of protected peptide-resin was performed with 20% piperidine, 1% DBU in DMF. To analyze the results of these experiments, the resins were identically cleaved with a cocktail of TFA/phenol/thioanisole/ethane dithiol/H2O (86/4/4/2) for 1 hr. Examination by HPLC analysis produced the following results. By hydrolysing just 10 mg of the peptides (using Nsc-amino acid and Fmoc-amino acid) in 6.0 N HCl solution for 24 h at 110°C, we were able to carry out an amino acid analysis. The peptides was composed of nine and eight amino acids respectively, LHRH; Glu, Ser, His, Gly(2), Arg, Tyr, Leu, Pro, (D -Trp 6 )–LHRH; Glu, Ser, 547 Y. Shimonishi (ed): Peptide Science – Present and Future, 547–549 © 1999 Kluwer Academic Publishers, Printed in Great Britain

548 Table 1. Characteristics of LHRH and (D-Trp6 )–LHRH synthesized by different protecting methods. Trp of (D -Trp 6 )–LHRH is D -form. a: crude yield, b: 0.1% aqueous TFA and acetonitrile containing 0.085% TFA. The sample was eluted with a linear gradient of 20% B to 70% B obtained in 30 min at a flow rate of 1.0 ml/min (216 nm). Peptides

Amino acid sequence

Amino acid

Yield, %

LHRH LHRH LHRH ( D-Trp6 )–LHRH (D-Trp 6 )–LHRH (D-Trp 6 )–LHRH

(Pyro)-EHWSYGLRPG (Pyro)-EHWSYGLRPG (Pyro)-EHWSYGLRPG (Pyro)-EHWSYWLRPG (Pyro)-EHWSYWLRPG (Pyro)-EHWSYWLRPG

Nsc Fmoc Nsc(-Opfp) Nsc Fmoc Nsc(-Opfp)

72 65 95 62 54 93

a

Purity, % b 93 87 48 62 65 67

Figure 1. HPLC on an Analytical Reversed-Phase Hypersil ODS 5 mm C 1 8 Column (4.6 × 200 mm) of the Synthetic Peptides after Purification. (a) LHRH by Nsc-amino acids, (b) (D -Trp 6 )– LHRH by Nsc-amino acids. The solvent systems were 0.1% aqueous TFA and acetonitrile containing 0.085% TFA. The sample was eluted with a linear gradient of 20% B to 70% B obtained in 30 min at a flow rate of 1.0 ml/min. The elution profile was monitored at 216 nm.

His, Gly, Arg, Tyr, Leu, Pro. FAB Mass analyses with VG70-VSEQ Mass spectrometer were obtained molecular ion, LHRH; 1183 [M + H]+ , (D-Trp 6 )– LHRH; 1312 [M + H] + . LHRH and (D -Trp 6 )–LHRH were synthesized successfully with Nsc-amino acids. Nsc-amino acids and activated ester are better reagents for solid Phase synthesis because of higher yield and higher purity than Fmoc-amino acids. New base-labile Nsc-amino acid was easily applied in normal automatic solid phase peptide synthesizer [2]. Syntheses of LHRH and (D -Trp6)–LHRH proved that Nsc-amino acids were completely interchangeable with Fmoc-amino acids as amino building blocks [3,4]. Nsc-amino acids are more stable and more economic than Fmoc-amino acids.

References 1. Samukov, V.V., Sabirov, A.N., and Pozdnyakov, P.I., Tetrahedron Letters, Vol. 35, No. 42 (1994) 7821. 2. Kim, Y.D., Cho, B.K., Park, J.I., Lee, S.H., and Kim, H.J. 5th Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries, London (in pub. 1998).

549 3. United states Patent (1996) 5,616,788. 4. Sabirov, A.N., Kim, Y.D., Kim, H.J., and Samukov, V.V. (Eds), Peptides: Chemistry, Structure and Biology (Proceedings of the 15th American Peptide Symposium), Nashiville, TN, 1997, p. 2–98, Pept. Protein. Lett. (in press).

186 Novel synthesis through repeated S-cyanocysteine mediated segment condensations O. ITO¹, Y. ISHIHAMA¹, Y. ODA¹, T. TAKENAWA², K. YOSHINARI² and M. IWAKURA²* ¹ Tsukuba Research Laboratories, Eisai Co., Ltd. 5–1–3 Tokodai, Tsukuba, Ibaraki 300–26, Japan. ² National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan. *Corresponding author. e-mail: [email protected]

Introduction Segment condensation of unprotected peptides is a very interesting approach for total synthesis of proteins [1–4] and a combinatorial synthesis. It is wellknown that the cleavage reaction of an S -cyanocysteinyl peptide generate an amino-terminal peptide and a carboxyl-terminal fragment blocked by a 2-iminothiazolidine[5,6]. We speculated that the intermolecular attack of an α -amino group on the activated carbonyl carbon atom would also occur and the binding of a S -cyanocysteinyl peptide with an α -amino group of an N-terminal free peptide would be possible. We demonstrate here that an S-cyanocysteine-mediated α-carbon activation is useful for combining two peptide segments with peptide bonding, and also propose a strategy for the repeated segment condensation using the S-cyanocysteinyl peptide as a building block.

Result and discussion The intermolecular reactions were evaluated using small synthetic peptides (Table 1). The synthetic peptides were selected by considering the minimization of the side chain effects, and having moderate retention for HPLC. Angiotensin II was also employed as a natural peptide. The standard protocol of the reaction was as follows: 200 nmol of the electrophile peptide was added to 200 µL of 0.1 M borate – 0.1 M NaOH buffer (pH 9.6) containing 400 nmol of the nucleophile. The reaction was monitored using an on-line reversed-phase HPLC/electrospray mass spectrometer (LC/MS). The condensation peptide (CS-peptide) as well as the β -elimination product (BE-peptide), the N-terminal peptide (CN-peptide) and the C -terminal peptide with a 2-iminothiazolidine4-carbonyl amino terminal (CC-peptide) were observed in the reaction products. In Table 1, the ratio of these products as well as the residual substrate are listed. The structure of the Ac-AAK lactam, which was the intramolecular reaction product, was confirmed by ¹H-NMR, MS and tandem MS. The 550 Y. Shimonishi (ed): Peptide Science – Present and Future, 550–551 © 1999 Kluwer Academic Publishers, Printed in Great Britain

551 Table 1.

1 2 3 4

Segment Condensation Reaction electrophile

nucleophile (2eq.)Solvent a

ratio of the product b CS CN BE

residual (%)

AcAAKC(CN)A AcYAACG(CN)A AcYAACG(CN)A AcYAAGC(CN)A

intramolecular-Lys none Gly [Val 5 ]-angiotensinII

A A A A

1c – 1 1

0.02 1 0.12 1.2

0.06 0.31 0.09 0.74

ND d 31.7 8.0 29.4

Ae Ae Ae Ae

1c 1c 1c 1c

10.2 2.90 0.28 0.21

1.57 0.48 0.11 0.07

15.5 31.9 30.7 11.4

Block synthesis (expected product: YAAG-AAAAG-GAAYA) AAAAGC( t Bu)A YAAGC(CN)A (A+B) ( A B + C ) YAAGAAAAGC(CN)A GAAYA (B+C) AAAAGC (CN)A GAAYA ( A + B C ) YAAGC(CN)A AAAAGGAAYA a

Solvent A: pH 9.6 (0.1 M borate – 0.1 M NaOH). The reaction was at RT. and for 3 hours. The ratios of the product were based on the absorbance at 210 nm. The ratios of the product were calculated from the intensity of the molecular ion signals from the mass spectrometer. d Not detected. e The reaction time was 18 hours. b c

structures of the C S-peptides such as Ac-YAAGGAAYA were determined by the tandem MS, whereas other CS-peptides were assigned by LC/MS. As the pH of the reaction solvent increased, the reaction rate became faster and the production of the by-products was suppressed(data not shown). Considering the pKa values of the amino group, the concentration of the nonionic form would be important for the rate and the selectivity. Among the natural amino acids as nucleophiles, only Cys did not react with the electrophile peptide(data not shown). However, this limitation could be overcome by using S -alkyl derivatives such as Met and S-t Bu–Cys. The segment condensation was yielded the expected peptides as shown in Table 1. This reaction was not limited to peptides. For example, a site specific immobilization at C -terminal of the enzyme on a solid support having free NH2 group has been accomplished (Iwakura, et al., unpublished observation). Further studies on the improvement of the reaction yield and the application to the solid synthesis are now in progress in our laboratories.

References 1. 2. 3. 4. 5. 6.

Schnolzer, M. and Kent, S.B.H., Science, 256(1992) 221. Dawson, P.E., Muir, T.W., Clark-Lewis, I. and Kent, S.B.H., Science, 266 (1994) 776. Baca, M., Muir, T.W., Schnolzer, M. and Kent, S.B.H., J. Am. Chem. Soc., 117 (1995) 1881. Liu, C., Rao, C. and Tam, J.P., J. Am. Chem. Soc., 118 (1996) 307. Catsimpoolas, N. and Wood, J.L., J. Biol. Chem., 241 (1966) 1790. Jacobson, G.R., Schaffer, M.H., Stark, G.R. and Vanaman, T.C., J. Biol. Chem., 248 (1973) 6583.

187 Total synthesis of human pleiotrophin, a 136-residue heparin-binding neurotrophic factor having five disulfide bonds T. INUI¹, M. NAKAO¹, H. NISHIO¹, Y. NISHIUCHI¹, S. KOJIMA², T. MURAMATU³, T. KIMURA¹ and S. SAKAKIBARA¹ ¹ Peptide Institute Inc., Protein Research Foundation, Minoh-shi, Osaka 562-8686, Japan. ² Laboratory of Gene Technology and Safety, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305-0000, Japan, ³ Department of Biochemistry, Nagoya University School of Medicine, Showa-ku, Nagoya 466-0065, Japan

Introduction Recently, we established a procedure suitable for the solution synthesis of large peptides using our newly developed solvent system, a mixture of trifluoroethanol (TFE) and chloroform (CHL) or dichloromethane (DCM) [1]. Using this procedure, we succeeded in synthesizing human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues [2]. Here, we report the total synthesis of human pleiotrophin (hPTN), another heparin-binding neurotrophic factor, consists of 136 amino acid residues having five intramolecular disulfide bonds (Figure 1) [3], in order to further demonstrate the usefulness of our procedure. hPTN is a MK family also participating in the tumor growth, angiogenesis and embryogenesis, and consists of two distinctive domains, i.e., the N-terminal domain difined by three disulfide bonds and the C -terminal one defined by the remaining two disulfide bonds.

Results and discussion The synthesis was carried out in solution by applying our maximum protection strategy. The following side-chain protected amino acids were used: Arg(Tos), Lys(ClZ), Asp(cHx), Glu(cHx), Ser(Bzl), Thr(Bzl), Tyr(BrZ), Trp(Hoc) and Cys(Acm). The fully protected peptide was assembled from 14 segments in the

Figure 1.

Structure of hPTN.

552 Y. Shimonishi (ed): Peptide Science – Present and Future, 552–554 © 1999 Kluwer Academic Publishers, Printed in Great Britain

553

Figure 2. Synthetic route of the protected hPTN

form of Boc-peptide-OPac except for the C-terminal segment, which was protected by the benzyl ester (Figure 2). The sequence (89–110) was originally intended to divide between the segment (89–97) and (98–110). During the synthesis of the segment (98–110: BocECQKTVTISKPCG-OPac), however, the intermediate (100–110) was insoluble in CHL/TFE or DMSO after incorporation of the Gln 100 residue. To overcome this solubility problem, the ECQ sequence (98–100) was deleted from the segment (98–110) and joined to the C-terminus of the segment (89–97). The resulting segments, Boc-(89–100)-OPac and Boc-(101–110)-OPac, were soluble in CHL/TFE. Next, each segment was coupled with WSCI in the presence of HOBt or HOOBt from the C-terminal segment to obtain two large intermediates, Boc-(1–64)-OPac and Boc-(65–136)-OBzl, corresponding to the N- and C -domains, respectively. The final coupling reaction could be carried out in CHL/TFE within 1 h. All the protecting groups including the Hoc group on the Trp residues except the Acm group were removed by the standard HF procedure in the absence of thiol. The (10 Acm)-peptide was purified by RP-HPLC followed by IEX-HPLC. After removal of the remaining Acm groups, the reduced peptide was subjected to the oxidative folding reaction at r.t for 1 day in 1M AcONH 4 buffer (pH 7.7) containing 1 mM EDTA in the presence of redox reagents. The product was isolated to homogeneity by RP-HPLC. The synthetic product was confirmed to have the same disulfide structure as that reported for hPTN by peptide mapping with digests of lysylendpeptidase followed by V8 protease and Asp-N protease [4]. Synthetic hPTN possessed the same neurite extentional activities in rat embryo brain cells as those of natural mPTN and mMK. Furthermore, synthetic hPTN was found to have the same activity in terms of plasminogen activator enhancing activity in the bovine aortic endothelial cells as that of hMK.

References 1. Kuroda, H., Chen, Y.-N., Kimura, T. and Sakakibara, S., Int. J. Peptide Protein Res., 40 (1992) 294–299. 2. Inui, T., Bodi, J., Kubo, S., Nishio, H., Kimura, T., Kojima, S., Maruta, H., Muramatsu, T. and Sakakibara, S., J. Pep. Sci., 2 (1996) 28–39.

554 3. Li, Y.-S., Milner, P.G., Chauhan, A.K., Watson, M.A., Hoffman, R.M., Kodner, C.M., Milbrandt, J. and Deuel, T.F., Science, 250 (1990) 1690–1694. 4. Fabri, L., Nice, E.C., Ward, L.D., Maruta, H., Burgess, A.W. and Simpson, R.J., Biochemistry International, 28 (1992) 1–9.

188 Engineering of snake venom cardiotoxin by chemical synthesis approach C.-Y. WU¹, S.-T. CHEN² and K.-T. WANG 1,2 ¹ Institute of Biological Chemistry, Academia Sinica, Taiwan. ² Department of Chemistry, National Taiwan University, Taiwan

Introduction Snake venom cardiotoxins (CTXs), a group of basic proteins containing 60–63 amino acids and 4 disulfide bonds, are major lethal components of elapid snake venom [1, 2]. CTXs display very diverse pharmacological effects, including lethal toxicity, hemolysis, cytolysis, muscle contractures, membrane depolarization, and activation of tissue phospholipase C [3–6]. Recently, polypeptides containing 60–80 amino-acid residues can be successfully synthesized in our laboratory by solid-phase peptide synthesizer with satisfying yields within about 10 days. The success of this methodology is a strong encouragement for us to try to synthesize CTXs and their analogues to elucidate their structure and function relationships. First, we have developed a chemical synthesis methodology to successfully produce fully active CTX II (Naja Naja Atra) [7]. Second, we have used above method to engineer the N-terminal residue of CTX II (Naja Naja Atra) and to study the effects of N-terminal substitutions on its structure and biological function [8]. Results and discussion The complete sequence of CTX II (Naja Naja Atra) is chemically synthesized by an automatic peptide synthesizer. After completion of peptide-chain elongation, crude reduced CTX sample is obtained from the peptide-resins, purified by reverse-phase high performance liquid chromatography (RP-HPLC), airoxidized, and repurified by RP-HPLC, and the overall yields of synthetic CTX II is 3.9 mg (3.9%). Physicochemical characterization of these synthetic CTX II is carried out by amino acid analysis, mass spectroscopy, capillary electrophoresis, peptide mapping, circular dichroism spectroscopy, and lethal toxicity [7]. As compared with natural CTX II, the results indicate that the synthetic CTX possesses the same physicochemical properties as those of natural one. Therefore, this synthesis method provides a direct and rapid route to obtain other snake toxins and their analogues for studying the structure/function relationship of CTXs. To study the role of N-terminal L-leucine residue in CTX II (Naja naja atra), this residue is systematically replaced with D-leucine (CTXII-L1-D-L) and 555 Y. Shimonishi (ed): Peptide Science – Present and Future, 555–577 © 1999 Kluwer Academic Publishers, Printed in Great Britain

556

Figure 1. Fluorescence spectra of ANS in the presence of various CTX samples. Curves: (1) CTX II, (2) CTXII-L1- D -L and (3) CTXL1G. The dotted line shows the fluorescence of ANS in the absence of sample. Table 1.

Biological activities of CTX II and its analogs.

Toxin

LD 5 0 (µg/g body wt)

Muscle contracture a

CTXII CTXII-L1-D-L CTXII-L1G

2.47 (2.34–2.62) 4.32 (4.11–4.53)* 6.77(6.54–7.01)*

780±130(3)* 200±10(3)* 170±40(3)*

a

Values given are meanst ± S.E.M. with the numbers of experiments in parentheses. * p < 0.05 as compared with the value of CTX II.

glycine (CTXII-L1G) by above established chemical synthesis method. Both CTX analogues are characterized by amino acid analysis, mass spectrometry and peptide mapping [8]. In the structural aspect, changing the N-terminal L -Leu by D -Leu or Gly causes a drastic alteration in the whole CTX II structure as detected by circular dichroism [8], 1-anilino-naphthalene-8-sulfonate (ANS) flourencence assay (Figure 1). In the functional aspect, both analogues are still retained substantial biological activity of native CTX II (Table 1). The results indicate that both the chirality and the side-chain of the N-terminal leucine residue of CTX II are important elements in maintaining the whole CTX II structure.

Acknowledgments Support for this research provided by Academia Sinica and the National Science Council, Taipei, Taiwan, is gratefully acknowledged.

References 1. Fryklund, L. and Eacker, D. Biochemistry 14 (1975) 2865. 2. Bougis, P.E., Marchot, P., and Rochat, H. Biochemistry 25 (1986) 7235. 3. Lee. C.Y. Ann. Rev. Pharmacol. 12 (1972) 265.

557 4. Dufton, M.J. and Hider, R.C. in Snake Venom (Harvey, A.L., ed) Pergamon Press, Inc., NY, 1991, p. 259. 5. Harvey, A.L. Handbook of Natural Toxins, 5th Ed., Marcel Dekker, NY, 1991, p.85, 6. Fletcher, J.E. and Jiang, M.S. Toxicon 31 (1993) 669. 7. Wu, C.Y., Chen, S.T., Ho, C.L., and Wang, K.T.J. Chin. Chem. Soc. 43 (1996) 67. 8. Wu, C.Y., Chen, W.C., Ho, C.L., Chen, ST., and Wang, K.T. Biocehm. Biophys. Res. Comm. 233 (1997) 713.

189 Specificity of Cardosins A and B in peptide synthesis A.C. SARMENTO², M. BARROS¹, L. SILVESTRE¹ and E. PIRES² ¹ Cellular Biology Center, Department of Biology, University of Aveiro, 3810 Aveiro, Portugal ² Center for Neuroscience and Cell Biology, Department of Biochemistry, 3000 Coimbra, Portugal

Introduction The enzymatic peptide synthesis has established itself as a productive approach for the preparation of various biological active peptides [1]. Cardosins A and B are two aspartic proteinases [2] extracted and purified from the stigmas of the cardoon Cynara cardunculus L. traditionally used in Portugal in the manufacturing of artisanal cheeses [3]. These enzymes are purified by a simple two-step procedure, that includes a gel filtration and an ion-chromatography, in just a couple of hours [2]. The aim of this work is to make a first approach on Cardosins specificity towards peptide synthesis. We have used a system developed by Barros [4] constituted by an aqueous phase containing the enzyme and an organic phase that extratcs the product. The product is purified by HPLC-RP and identified by amino acid analysis. Results and discussion The main advantage of enzymatic peptide synthesis is the high specificity of the reactions. Thus a central aspect in the development of this technology is the search for enzymes with a very well define specificity. Considering this, we had studied Cardosin’s specificity towards amino acid in P'1 position, with different P1 and P2 amino acid. The condensation of CBZ × Phe with Phe × OMe, Val × OMe or Met × OMe shows that Cardosins A and B have the same order of preference (Table 1) towards P'1 amino acid, Phe > Val > Met. If we use Cardosins not isolated (i.e. before the ion-chromatography step), the dipeptide production is, in general, lower, showing that there might be some king of interaction between both Cardosins. In comparison with Pepsin the results show different order of preference (Phe > Met > Val). For the dipeptide CBZ × Phe–Val × OMe Cardosins A and B are more efficient than Pepsin. In the case of CBZ.Tyr in P1 amino acid position, the dipeptide production by Cardosins changes, and their preference becames, for Cardosin A: Val > Met > Phe, while for Cardosin B is Val > Phe. At the same time, amino acid production decreases (with one exception). This clearly shows that the amino acids surrounding the bound are very important and clearly interact with the catalytic residues. 558 Y. Shimonishi (ed): Peptide Science – Present and Future, 558–559 © 1999 Kluwer Academic Publishers, Printed in Great Britain

559 Table 1.

Di- and tri-peptide production by Cardosin A, Cardosin B and Pepsin (in percentage). CBZ · Val · Phe

CBZ · Val · Leu

Phe · OMe Met · OMe Val · OMe

CBZ · Phe 21.6 4.3 13.8

CBZ.Tyr 0.3 0.5 4.1

1.5 0.6 0.2

0.2 0.2 0.0

Cardosin A

Phe · OMe Met · OMe Val · OMe

81.0 6.1 76.6

2.1 – 66.7

3.9 8.6 3.1

0.7 1.9 0.4

Cardosin B

Phe · OMe Met · OMe Val · OMe

43.3 8.8 15.1

0.7 1.7 4.0

1.0 1.4 0.2

0.3 0.4 0.1

A and B

Phe · OMe Met · OMe Met · OMe Val · OMe

100.0 9.6 9.6 6.3

–

–

Pepsin

–

We have, also, studied tripeptide production by these enzymes. We used two dipeptides (CBZ · Val · Phe and CBZ · Val · Leu) in order to see the influence of Valinyl in P2 position. Once again, the results show that Cardosins A and B have different preferences towards P'1 amino acid and the introduction of Val in P2 position modifies their prefence. Thus, Cardosin A preference becomes Phe > Met > Val, while Cardosin B is Met > Phe > Val. If Leu is in P1 position, instead of Phe, there is no change in these enzyme’s order of preference and they follow exactly the same preference as with Phe in P1 position. The big difference is the tripeptide production, which clearly shows, that Cardosin A and Cardosin B prefer Phe in P1 position. On the basis of the results above described, and taking in account the stability of Cardosins in organic solvents [5], as well as the simplicity of the purification procedure, these enzymes seem suitable to be considered in future development of enzymatic peptide synthesis process. In other hand, concerning industrial purposes, and knowing that more time in purification involves more money spent, these results suggest that the last purification step, in order to isolate Cardosin A from Cardosin B, becomes unnecessary. Acknowledgements The authors ackowledge the financial support given by JNICT (BD-11019/97) and Gulbenkian Foundation. References 1. Stepanov, V.M., Pure & Appl. Chem., 68 (1996) 1335–1339. 2. Faro, C., Veríssimo, P., Lin, Y., Tang, J., and Pires, E. (Eds.), Aspartic Proteinases: Structure, Function, Biology and Biomedical Implications, Takahashi, K., Plenum Press, New Yorh, 1995, p. 373. 3. Faro, C., Moir, A.J., and Pires, E., Biotech. Lett., 14 (1992) 841. 4. Barros, M., Faro, C., and Pires, E., Biotech. Lett., 15 (1993) 239. 5. Barros, M., Carvalho, M., Garcia, M., and Pires, E., Biotech. Lett., 14 (1992) 174.

190 A facile preparation of 2,2-difluoro-1,3,2-oxazaborolidin5-ones(I) and their application to side-chain protection of Asp, Glu, Ser and Thr J. WANG and W. LI Department of Molecular Biology, Jilin University, Changchun, China 130023

Introduction Recently we have reported the synthesis of 2,2-difluoro-1,3,2-oxazaborolidin5-ones(I) from anhydrous monosalt of amino acid and BF 3 E t2 O. By this method, compound I with free side-chain function groups can be transformed into side-chain protected intermediate I, then after hydrolysis, a wide variety of desired protected amino acids can be obtained, for in stance, Synthesis of Asp(OBu t ), Glu(OBut ), Ser(Bu t), Thr(Bu t), Ser(Bzl) and Thr(Bzl) [1, 2] etc., is shown in Scheme 1.

Scheme 1.

Compounds I Prepared From Amino Acid Salts.

However, a great need still exists for a versatile and facile procedure to prepare compound I. In preparation of anhydrous salts from amino acids, a high-vaccum lyophilizer is needed and for some amino acids, the salt form is insoluble in common solvents or can not give anhydrous form. Herein we would like to report a new, general and practical method for synthesis of compounds I.

Results and discussion The exchange reaction between boron trifluoride and trimethyl borate has been described previously in the literature [3]. Simply by mixing of above reagents in THF, methoxydifluoroborate can be obtained as shown in Scheme 2. 560 Y. Shimonishi (ed): Peptide Science – Present and Future, 560–562 © 1999 Kluwer Academic Publishers, Printed in Great Britain

561

Scheme 2.

Formation of methoxydifluoroborate.

Then methoxydifluoroborate generated in situ reacts with amino acid to form compounds I. It is worthy of note that BF 3 is not only a reactant but also acts as a catalyst, the molecular ratio of BF3 /B(OMe) 3 is greatly more than 2, normally ranges from 5.7–10.0 depending on the property of side-chain function groups. This indicates there are interaction of BF 3 and side chain function groups. The formation of compounds I is depicted as in Scheme 3.

Scheme 3.

Formation of compounds I in one-pot method.

A typical experiment in preparation of Ser(Bu t ) is described below. 10 mmol Ser was suspended in dry THF (20 ml) under N2 then BF 3 Et 2 O (2.4 ml) and B(OMe)3 (0.48 ml) were added, The solution was stirred at room temperature until the solid disappeared, keep stirring over night, the THF was removed in vacuo, dioxane (30 ml) and anhydrous H3 PO 4 (0.4 ml) were added, then stirred at room temperature for 10min and kept at reduced pressure(aspirator) for another 10 min. The follow procedure is similar as we described in the literature [1, 2].

Conclusion The new method for one-pot preparation of compounds I were developed. It is a simple, inexpensive and easy to scale-up procedure, and it is proved to be effective in preparation of Ser(But ), Ser(Bzl), Thr(Bu t ), Thr(Bzl), Asp(OBu t ) and Glu(OBu t). Further applications of the method are under way.

562 References 1. Wang, J. et. al. Chem. Pharm. Bull. 44(11), 2189–2191 (1996). 2. Wang, J. et. al. J. Chem. Soc. Perkin Trans(I), 1997(5) 621–624. 3. Binder, H. et. al. Z. Naturfousch, Vol. 38b, No. 5, 554–558.

191 ESR-resin solvation approach for the investigation of the highly peptide-loaded synthesis protocol E.M. CILLI¹, G.N. JUBILUT¹, S. SCHREIER² and C.R. NAKAIE¹ ¹ Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua 03 de Maio, 100, São Paulo, SP, 04044-020, Brazil ² Department of Biochemistry, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil

Introduction In spite of clear economical advantages in synthesizing peptides with highly substituted resins (lesser solvent consumption, higher amount of peptide per synthesis, etc), this protocol has been systematic avoided as it may aggravate chain interactions inside beads and therefore, to difficult coupling reaction. To better evaluate this assertion, the present report applied a solvation–electron spin resonance (ESR)-rate of coupling approach to model peptides differing in size and in polarity and synthesized comparatively in low and highly substituted resins.

Results and discussion Resins commonly used in Boc-chemistry (MBHAR, BHAR and PAMR) were synthesized in forceful conditions and substitution degrees higher than 2 mmol/g were obtained for those resins. The following peptide sequences were studied assembled in low and highly (around 0, 2 and 2.5 mmol/g,) substituted solid supports: the [ D -Phe 12 , Nle 21,38 , Ala 32 ]-CRF 12–41 analog, the ING (72–74)-acyl carrier protein fragment, free or bound to the lysyl-branched (K) 2 K-F core, the vasoactive DRVYIHPF (angiotensin II) and the aggregating VVLGAAIV octapeptide. The peptide contents inside resin beads ranged up to 83% (w/w) and to estimate the more appropriate solvent system for each synthesis, swelling properties of beads in several solvents was measured with a microscope in different positions of the growing peptide chains. For instance, Figure 1 shows solvation changes during the VVLGAAIV assembly in a 2.6 mmol/g BHAR. The correlation between solvation and polarity of media, given by the sum of electron acceptor (AN) and electron donor (DN) of solvent, proposed as a novel solvent polarity parameter [1] was also applied for these class of peptide-resins. Low peptide-loaded resins displayed maximum solvation in solvents characterized by (AN + DN) numbers around 20 and the solvation degree of highly loaded resins depend on the polarity of the sequence, including the amount and nature of protecting groups. The CRF analogue, the angio563 Y. Shimonishi (ed): Peptide Science – Present and Future, 563–565 © 1999 Kluwer Academic Publishers, Printed in Great Britain

564

Figure 1. Swelling of VVLGAAIV-BHAR (2.6 mmol/g) in different positions.

tensin II and the VVLGAAIV sequence showed better solvation in solvents with (AN + DN) values around 40. Otherwise, more polar solvents characterized by higher (AN + DN) values, were necessary for solvation of highly loaded resins containing hydrophilic sequences. As examples, the ING sequence bound directly to 2.6 mmol/g BHAR or through the lysyl-branched core-linker displayed a shift in the maximum solvation region to solvents with (AN + DN) numbers around 50. The ESR approach to estimate peptide solvation inside polymeric matrix [2] and where, the Toac spin label protected with the Fmoc group [3] is coupled to peptide-resin under study, was also applied for the investigation of solvation of some of these peptide-resins. The ESR spectra obtained showed that, as a rule, the higher the swelling of resin, the faster the molecular movement inside the bead, thus confirming the feasibility in applying ESR spectroscopy for monitoring peptide synthesis. Differently from swelling studies, the ESR method is sensitive to factors such as the viscosity of the medium or the presence of peptide chain populations with different mobility. The rate of coupling in some of these resins followed exactly the mobility degree obtained by ESR spectra. At last, using the same experimental conditions for coupling steps, low and highly peptide loading protocols were compared as to their yield of syntheses, estimated by purity degree of crude peptides in analytical HPLC. Similar results were obtained in both protocols, thus suggesting that the use of more appropriate solvent systems for coupling may in part overcome difficulties in using highly substituted resins. Second, for the same scale of synthesis, smaller amount of this type of resin is needed. This implies that lesser solvent is necessary for solvation of these resins, thus allowing comparatively higher concentration of reactants during couplings. This effect may also explain in part, the similarity in the synthesis yield observed with both protocols.

565 References 1. Cilli, E.M., Oliveira, E., Marchetto, R., and Nakaie, C.R., J. Org. Chem. 61 (1996), 8992–9000. 2. Cilli, E.M., Marchetto, R., Schreier, S., and Nakaie, C.R., Tetrahedron Lett. 38 (1997), 517–520. 3. Marchetto, R., Schreier, S., and Nakaie, C.R., J. Am. Chem. Soc. 115 (1993) 11042–11043.

192 Synthesis of α -amino phosphonic acids by catalytic asymmetric hydrogenation M. KITAMURA, M. YOSHIMURA, M. TSUKAMOTO and R. NOYORI Department of Chemistry and Molecular Chirality Research Unit, Nagoya University, Chikusa, Nagoya 464–01, Japan

Introduction α-Amino phosphonic acids such as 1 and 2 are significant elements in peptide science and future biochemical technology. The tetrahedrally arranged phosphorus atom with an sp³ hybridization and dibasic acid properties modify the size and shape of the three-dimensional structures as well as the isoelectronic points of the parent α -amino acids, influencing their chemical and biological activities. The wide range of possibilities of their applications as well as the activity dependency upon their absolute configuration has resulted in the steady growth of the demand for the asymmetric synthetic method possessing high applicability as well as the technical and economical benefits. A solution to the problem has been given.

Results and discussion The asymmetric hydrogenation of amido-functionalized olefins and ketones with BINAP–Ru(II) complexes (BINAP = 2,2 ′-bis(diphenylphosphino)-1,1′ binaphthyl) has been utilized [1, 2]. Dimethyl 1-(formamido)ethenylphosphonate (3a) was hydrogenated in methanol containing 0.5 mol% of Ru(CH 3 COO) 2 [ (S)-binap] under 4 atm of H 2 and at 30°C, giving the (R ) -4a in a quantitative yield and with 97% enantiomeric excess (ee). The R catalyst formed the S -enriched product. The ee value of 4a in the reaction at 2 and 20 atm was 97 and 83% ee, which was reduced sharply to 58% by increasing the hydrogen pressure to 100 atm. The catalytic hydrogenation method has been applied to 3b and 3c which contain a methyl or phenyl substituent at the β position. When (E)-3b with a methyl group cis to the formamide group was hydrogenated under the above conditions, (R )-4b in 98% ee was quantitatively 566 Y. Shimonishi (ed): Peptide Science – Present and Future, 566–568 © 1999 Kluwer Academic Publishers, Printed in Great Britain

567

obtained. The geometric isomer, (Z)-3b, was practically inert under the standard hydrogenation conditions. Because of the lower reactivity of Z substrate, an E/Z mixture can be used directly without separation of the stereoisomers. Thus, similarly, when a 5.4:1 mixture of (E)- and (Z)-3c was hydrogenated with S catalyst, (R)-4c was produced in 90% yield and in 98% ee together with ( Z ) 3c recovered in ca. 9% yield. This method allowed the synthesis of phosphoalanine, phosphoethylglycine, and phosphophenylalanine with high enantiomeric purity [3]. Use of configurationally labile α -amido β -keto phosphonic esters 5 as the substrates for BINAP–Ru method leads to the stereoselective synthesis of threonine-type compounds with two consecutive stereogenic centers [4]. Hydrogenation of (±)-5b in methanol at 4 atm using 0.1 mol% of RuCl 2 [(R)binap](dmf) n afforded quantitatively the protected phosphothreonine (1R,2R) 6b in > 98% ee and (1S,2R)-6b in > 90% ee in a 97 : 3 ratio. The 2-phenyl substrate (±)-5c was also hydrogenated by (R)-BINAP–Ru catalyst to give (1R,2R)-6c in 95% ee and in a 98 : 2 syn: anti selectivity. The predominant formation of (1R,2R)-6 among four possible stereoisomers is ascribed to the excellent combination of stereochemical and kinetic factors. In the presence of the (R)-BINAP catalyst, the R substrate is more reactive than the S enantiomer ( k R > k S ), while the stereoinversion of the slow-reacting (S)-5 occurs more efficiently than hydrogenation (k inv > k S ). The present method is general, chirally flexible, and very practical. A clean reaction, operational simplicity, and facile product isolation are also the benefits, providing a powerful tool for general asymmetric synthesis of α -amino phosphonic acids.

References 1. Kitamura, M., Hsiao, Y., Ohta, M., Tsukamoto, M., Ohta, T., Takaya, H., and Noyori, R., J. Org. Chem., 59 (1994) 297.

568 2. Kitamura, M., Ohkuma, T., Inoue, S., Sayo, N., Kumobayashi, H., Akutagawa, S., Ohta, T., Takaya, H., and Noyori, R., J. Am. Chem. Soc., 116 (1988) 629. 3. Kitamura, M., Yoshimura, M., Tsukamoto, M., and Noyori, R., Enantiomer, 1 (1996) 281. 4. Kitamura, M., Tokunaga, M., Pham, T., Lubell, W.D., and Noyori, R., Tetrahedron Lett., 36 (1995) 5769.

193 Process impurity identification in MSI-78 J.L. CHANG, J. BAI, J. JIANG, R. PILGRIM, W.-S. CHANG, T.O. KOLLIE, R. RASMUSSEN, E. TEWS, R.B. MILLER and J.C. TOLLE Abbott Laboratories, CAPD, D-54Z, R-13, 1401 Sheridon Road North Chicago, IL60064, USA

Introduction MSI-78 is a 22-amino acid peptide amide for treatment of infection in diabetic foot ulcers. Recent phase III clinical trials successfully demonstrated the efficacy and safety of the drug. A solution phase synthesis has been developed to prepare the peptide at the multi-kilogram scale. In response to a request by U.S. Food and Drug Administration [1], identification of all impurities at a level greater than 0.1% was pursued. H-Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-AlaPhe-Val-Lys-Ile-Leu-Lys-Lys-NH 2 · xHOAc (MSI-78 or H-1-22-NH 2 ) Results and discussion Characterization of process impurities in MSI-78 was carried out using LC-MS, and HPLC relative retention time (RRT) match with authentic samples of MSI-78 analogs, i.e. single point diastereomers, mono-deletion, mono-addition, and mono-acetylation derivatives. Impurities other than these analogs were isolated by preparative HPLC and further characterized using amino acid analysis (AAA), Edman sequencing [2], Tandem Mass Spectroscopy (MS/MS), and HPLC RRT match. Seventy-three authentic samples of potential impurities were prepared by either solid phase synthesis using Fmoc chemistry or solution phase peptide synthesis. The impurities identified are listed in Table 1. 15–20 Impurities have been observed at ≥ 0.1 pa%. The structure of O=C(7-22-NH 2 ) 2 is shown in Figure 1.

Figure 1. The structure of O=C-(7-22-NH2 ) 2 .

Acknowledgements We thank MS Sally Dorwin for amino acid analysis and Edman sequencing, Drs Alex Buko and Robert Johnson for mass spectroscopy, M S J e a n n e 569 Y. Shimonishi (ed): Peptide Science – Present and Future, 569–570 © 1999 Kluwer Academic Publishers, Printed in Great Britain

570 Table 1. Characterization of process impurities in MSI-78. Impurity Structure

RRTa

O=C(7-22-NH 2 ) 2 H-1-16-OH O=C(6-22-NH 2) 2 D-Ala 1 5 5 D-Phe additional Lys 4 H-1-22-OH additional Lys 2 2 H-1-22-OMe H-1-22 lactam 22 D-Lys Des-Lys2 21/22 Lys 2 2 (tBu) Lys 2 1 (tBu) Lys 1 0 (tBu) Lys 4 (Ac) Lys 1 1(Ac) Lys 1 4 (Ac) Lys 1 0 (Ac) Gly 1 (Ac) H-(1-22) 2 -NH 2

0.41 0.46 0.58 0.73 0.77 0.80 0.90 0.93 0.98 1.10 1.14 1.18 1.26 1.30 1.34 1.41 1.43 1.51 1.58 1.60 2.35

MS

AAA

Sequencing

MS/MS

RRT match

a HPLC conditions: Vydac C18, 5 µ colomn (4.6 mm × 25 cm). A: 0.075% TFA in H2 O; B:0.075% TFA in ACN, 20–27.5% B over 20 min, 20–27.5%B over 40 min, 32.5–55%B over 20 min. Colomn temperature at 30°C0

Cullerton, Lucy Fung, Marina Miletic, and Dr Ofelia Nawrot for some of the HPLC analysis.

References 1. Department of Health and Human Services, Food and Drug Administration, International Conference on Harmonisation; Guideline on Impurities in New Drug Substances; Availability, Federal Register, Vol. 61, No 3, Thusday, January, 1996, Notices, p. 372. 2. Edman, P. and Begg, G., Eur. J. Biochem. 1, (1967) 80–92.

194 Identification of unknown intermediate during methanolysis of Z-Lys(Boc)–OSu R.B. MILLER, J. JIANG, Y. LIU and J.C. TOLLE Abbott Laboratories, Chemical and Agriculture Product Division, D-54Z, R-13, 1401 Sheridan Road, North Chicago, IL 60064-4000, USA

Introduction Z-Lys(Boc)–OMe was required as a starting material in the preparation of a peptide containing lysine as the C-terminal residue. Z-Lys(Boc)–OSu, a known derivative of lysine [1], was chosen as the precursor of the ester (Scheme 1) . During methanolysis of Z-Lys(Boc)–OSu (1) in the conversion to the methyl ester (2), a transient observable intermediate (3) was observed by in-process HPLC. The intermediate appeared in the early stage of the reaction, with gradual disappearance until the reaction was complete. A typical time-course of the reaction is shown here (Table 1). The structure for the reaction intermediate 3 is derived from opening of the hydroxysuccinimide ring by attack of methanol on one of the ring carbonyls [2] preferentially to the ‘activated’ ester carboxyl in the lysine portion. Results and discussion A structure of the intermediate was proposed based on LC-MS analysis of the reaction mixture, which noted that the compound was +32 in mass different

Scheme 1 Table 1. Time-course of the Appearance/disappearance of Intermediate 3 in the Methanolysis of Z-Lys(Boc)–OSu. 1h Z-Lys(Boc)–OSu Intermediate 3 Z-Lys(Boc)–OMe

39.8% 2.5% 48.8%

2h 25.2% 3.6% 61.9%

5h

3h 13.2% 4.5% 72.8%

3.6% 5.5% 81.3%

571 Y. Shimonishi (ed): Peptide Science – Present and Future, 571–573  1999 Kluwer Academic Publishers, Printed in Great Britain

8h

24 h

48 h

72 h

0.9% 4.7% 84.5%

0 1.3% 88.5%

0 0.2% 89.8%

0 0 89.8%

572

Scheme 2

from Z-Lys(Boc)–OSu. Confirmation of the proposed structure was provided by synthesis as shown in Scheme 2. Succinic anhydride (4) was first converted to monomethyl succinate [3] (5), which was coupled with benzylhydroxyamine using carbonyl diimidazole to form compound (6). After hydrogenation of the benzyl group, the resulting hydroxamic acid [4] (7) is coupled with Z-Lys(Boc)-OH using DCC to afford intermediate (3). The conclusion is that the open chain anhydride 3 was found to be an intermediate formed during methanolysis of amino acid N-hydroxysuccinimide esters and still reactive in that reaction condition. No similar intermediate was found when amine nucleophiles were used. Additionally, no similar intermediate was detected when amino acid N -hydroxyphthalimide esters were used as electrophile.

Acknowledgment We thank David N. Heinrichsmeyer and professor Miklos Bodanszky for their helpful discussions and the Analytical Department of Abbott Laboratories for assistance.

573 References 1 . Anderson, G.W., Zimmermann, J.E., and Callahan, F.M., J. Amer. Chem. Soc. 85 (1963) 3039; Anderson, G.W., Zimmermann, J.E., and Callahan, F.M., J. Amer. Chem. Soc. 86, (1964) 1839. 2 . Bodanszky, M., Principles of Peptide Synthesis, 2nd edition, Springer-Verlag: Berlin, Heidelberg, New York, London, Paris, Tokyo, Hong Kong, Barcelona, Budapest, 1993, p. 34. 3 . King, F.E., Jackson, B.S., and Kidd, D.A.R., J. Chem. Soc. (1951) 243; Zuffanti, S., J. Chem. Ed. 25 (1948) 481; Cason, J., Org. Synth. 25 (1945) 19. 4 . Bauer, U., Ho, W.-B., and Koskinen, A.M.P., Tetrahedron Lett. 38 (1997) 7233; Ranganathan, D., Kurur, S., Karle, I., Tetrahedron Lett. 38 (1997) 4659.

195 Synthesis of phosphorylated polypeptide by a thioester method S. AIMOTO, K. HASEGAWA, X. LI, M. SEIKE and T. KAWAKAMI Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan

Introduction A thioester method has been successfully used in the preparation of polypeptides [1]. In order to make the method applicable to the preparation of polypeptides which contain phosphorylated amino acids, as well as aspartic acids and cysteines, we investigated a combination of protecting groups, along with segment coupling conditions which did not involve the undesirable removal of Acm group, which are used for the protection of mercapto groups. As a target peptide, we chose a phosphorylated partial sequence of the cAMP response element binding protein 1 (19–106), [Thr(PO3 H 2 )69,71 ]-CRE-BP1(19–106)N H 2 (Figure 1) [2].

Results and discussion As building blocks, three partially protected peptide segments, Boc[Lys(Boc) 23,46,48,54 , Cys(Acm)27,32 ]-CRE-BP1(19–56)-SCH 2 CH 2 CO-β-AlaN H 2 (1 ), Fmoc-[Thr(PO 3 H 2) 69,71, Lys(Boc) 77, Cys(Acm) 76 ]-CRE-BP1(57–83)SCH 2 CH 2 CO- β-Ala-NH 2 (2), [Lys(Boc)97,98,105,106 ]-CRE-BP1(84–106)-NH 2 ( 3), were prepared, using the Boc-based solid-phase method. The partially protected peptide 2 was obtained in 2% yield, based on the C-terminal Gly residue in a peptide resin, Fmoc-Pro-Ala-Arg(Mts)-Asn-Asp(OcHep)-Ser(Bzl)Val-Ile-Val-Ala-Asp(O cHep)-Gln-Thr(PO 3 cPen 2 )-Pro-Thr(PO 3 cPen 2 )-ProThr(Bzl)-Arg(Mts)-Phe-Leu-Lys(Z(2-Cl))-Asn-Cys(Acm)-Glu(OBzl)-Glu(OBzl)Val-Gly-SCH 2 CH 2 CO- β-Ala-MBHA resin, by treatment with TFMSA containing thioanisole, m-cresol, EDT and TFA for 3 h at 0°C and 1 h at 20°C, followed by the introduction of a Boc group. Boc-Thr(PO 3 c Pen 2 ) was a useful derivative for the introduction of Thr(PO3 H 2 ) residue [3]. The segment condensation was carried out in the presence of a silver salt and HOObt, and the

Figure 1 The amino acid sequence of phosphorylated cAMP response element binding protein 1(19–106)-NH 2 , [Thr(PO 3 H 2 )69,71 ]-CRE-BP1(19–106)-NH 2 . T(P) denotes phosphothreonine.

574 Y. Shimonishi (ed): Peptide Science – Present and Future, 574–575 © 1999 Kluwer Academic Publishers, Printed in Great Britain

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Figure 2. A schematic showing of the route for the preparation of [Thr(PO3 H 2 )69,71 ]-CREBP1(19–106)-NH 2 . i) AgNO3 + HOObt+ DIEA, ii) DTT, iii) piperidine, iv) AgCl + HOObt + DIEA, v) DTT, vi) 5% 1,4-butanedithiol/TFA, vii) AgNO3 + DIEA + H 2O, viii) 1M HCl.

yield at each coupling step was about 50% (Figure 2). Although silver chloride is only slightly soluble in DMSO, the coupling reaction proceeded at a reasonable rate, with no undesirable removal of Acm groups. Finally, phosphorylated CRE-BP1(19–106)-NH 2 was obtained in 12% yield, based on peptide 3.

Acknowledgments This research was partially supported by the Grant-in-Aid for Scientific Research on Priority Areas No. 06276102 from the Ministry of Education, Science, Sports and Culture.

References 1 . Hojo H. and Aimoto, S. Bull. Chem. Soc. Jpn., 64 (1991) 111; Hojo, H., Yoshimura, S., Go, M. and Aimoto, S. Bull. Chem. Soc. Jpn., 68 (1995) 330; Tamakami, T., Kogure, S. and Aimoto, S. Bull. Chem. Soc. Jpn., 69 (1996) 3331. 2 . Maekawa, T., Sakura, H., Ishii, C.K.., Sudo, T., Yoshimura, T., Fujisawa, J., Yoshida, M. and Ishii, S. EMBO J., 8 (1989) 2023. 3 . Wakamiya, T., Togashi, R., Nishida, T., Saruta, K., Yasuoka, J., Kusumoto, S., Aimoto, S., Kumagaye, K.Y., Nakajima, K. and Nagata, K. Bioorg. Med. Chem., 5 (1997) 135.

196 Synthesis of reaper protein by a thioester method using silver chloride as an activator T. KAWAKAMI and S. AIMOTO Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan

Introduction In the previous papers, we described polypeptide syntheses using a thioester method, in which peptide thioesters are used as building blocks [1]. A peptide thioester, which contained a Cys(Acm) residue, was successfully condensed in the presence of silver nitrate and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3benzotriazine (HOObt) in the synthesis of adrenomedullin [2]. Loss of the Acm groups during segment condensation was not observed in this synthesis. However, the undesirable removal of an Acm group was observed in the synthesis of reaper protein, which contains 65 amino acid residues including one Cys residue [3]. In order to overcome the problem, we searched the conditions under which an Acm group is stable and the condensation reactions proceed at an acceptable rate using model peptide thioesters [4]. In this paper, we describe the effect of silver salts and the synthesis of reaper protein.

Results and discussion Since reaper protein contains the partial sequence Thr–Ser–Cys–His, we prepared a peptide thioester, Fmoc–Phe–Thr–Ser–Cys(Acm)–His–Gly– SCH 2 CH 2 CO -β–Ala–NH 2 (1), as a model compound. The segment condensations of peptide 1 and tetrapeptide, Leu–Arg–Glu–Ser–NH 2 (2), were carried out in the presence of silver salts, HOObt, and N , N-diisopropylethylamine (DIEA) in DMSO [4]. The reactions were analyzed by reversed phase HPLC (RP-HPLC) and matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). Silver chloride promoted the condensation reaction more slowly than silver nitrate, and the condensations were complete within 4 h in the both cases. At that time, 43% of the Acm group was removed, as evidenced by HPLC peak areas, when silver nitrate was used, while silver chloride caused no removal. HPLC analysis suggested that the removal of the Acm group occurred mainly after the completion of the condensation reaction. Thus, silver ions have a higher affinity for the thioester moiety than for the mercapto group, which is protected by an Acm group. This suggests that excess of silver ions would be expected to accelerate the removal of the Acm group. 576 Y. Shimonishi (ed): Peptide Science – Present and Future, 576–578 © 1999 Kluwer Academic Publishers, Printed in Great Britain

577 In order to examine the effect of hydroxyl groups of Thr and Ser in peptide 1, a peptide thioester, Fmoc-Phe-Leu–Ala–Cys(Acm)–His–Gly–SCH 2 CH 2 COβ –Ala–NH 2 (3), was condensed with peptide 2 . When silver nitrate was used, 65% of the Acm group in the condensation product was removed after 4 h stirring. In addition, the des-Acm product was not observed after 4 h when silver chloride was used. Next, the His residue, adjacent to Cys(Acm), was replaced by Phe, thus Fmoc-Phe-Thr–Ser–Cys(Acm)-Phe–Gly–SCH 2 CH2 CO–Ala–NH 2 (4) was condensed with peptide 2. Even when silver nitrate was used, after 4 h, the desAcm product was observed in less than 1%. These results strongly suggest that the His residue is involved in the coordination of the sulfur atom on Cys(Acm) with silver ion, which is required to activate the Acm group toward nucleophilic attack. We concluded that the stability of the Acm group in the presence of silver ions is dependent on the type of the amino acid residues in the vicinity of Cys(Acm). Although silver chloride is only sparingly soluble in DMSO, the condensation reactions proceed at a reasonable rate with minimal removal of the Acm group. Using this strategy, reaper protein was successfully synthesized. The protein was divided into two segments, Boc-[Lys(Boc) 20 ]-reaper(1–32)-SCH 2 CH 2 COβ -Ala–NH 2 (5) and H-[Cys(Acm) 49 , Lys(Boc) 52, 56, 59, 62]-reaper(33–65)-OH (6 ) (Figure 1). In this synthesis, the amino acid residue at the condensation site was Trp. Peptides 5 and 6 were condensed in the presence of silver chloride

Figure 1 Segment condensation of peptides 1 and 2.

Figure 2. RP-HPLC elution profiles for the reaction mixture of the condensation of peptides 5 and 6. Peak a is the desired product 7, and b is [D -Trp 3 2 ]-7, respectively. Column: Cosmosil 5C18ARII (4.6 × 250 mm), eluent: linear increase of acetonitrile concentration from 30 to 70% in 0.1% aq. trifluoroacetic acid over 40 min at a flow rate of 1.0 mL/min.

578 (3 eq), HOObt (30 eq), DIEA (20 eq) in DMSO. The RP-HPLC elution profile of the reaction mixture after 48 h is shown in Figure 2. The desired product, Boc-[Cys(Acm) 49 , Lys(Boc ) 20,52,56,59,62 ]-reaper(1–65)-OH (7) was obtained in 60% yield with negligible removal of the Acm group.

Acknowledgments This research was partly supported by a Grant-in-Aid for Scientific research on Priority Areas No. 06276102 from the Ministry of Education, Science, Sports and Culture.

References 1. Hojo, H. and Aimoto, S., Bull. Chem. Soc. Jpn., 64 (1991) 111; Hojo, H. and Aimoto, S., Bull. Chem. Soc. Jpn., 65 (1992) 3055; Hojo, H., Kwon, Y., Kakuta, Y., Tsuda, S., Tanaka, I., Hikichi, K., and Aimoto, S., Bull. Chem. Soc. Jpn., 66 (1993) 2700; Hojo, H. and Aimoto, S., Bull. Chem. Soc. Jpn., 66 (1993) 3004; Kwon, Y., Zhang, R., Bemquerer, M.P., Tominaga, M., Hojo, H., and Aimoto, S., Chem. Lett., 1993, 881; Hojo, H., Yoshimura, S., Go, M., and Aimoto, S., Bull. Chem. Soc. Jpn., 68 (1995) 330. 2. Kawakami, T., Kogure, S., and Aimoto, S., Bull. Chem. Soc. Jpn., 69 (1996) 3331. 3. White, K., Grether, M.E., Abrams, J.M., Young, L., Farrell, K., and Steller, H., Science, 264 (1994) 677. 4. Kawakami, T. and Aimoto, S., Chem. Lett., 1997,1157.

197 Preparation of a peptide thioester by a 9-fluorenylmethoxycarbonyl solid phase method X. LI, T. KAWAKAMI and S. AIMOTO Institute for Protein Research, Osaka University, 3–2 Yamadaoka, Suita, Osaka 565, Japan

Introduction Partially protected peptide thioesters are useful building blocks for the synthesis of proteins [1]. The peptide thioester, however, could be prepared only by the tert-butoxycarbonyl (Boc) solid-phase method, because the thioester moiety is labile to amines such as piperidine, which is employed as a deblocking reagent in the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS). If a peptide thioester could be prepared, based on Fmoc SPPS, the thioester method would be a more useful and acceptable method for the preparation of proteins and cyclic peptides.

Results and discussion In order to prepare a peptide thioester by the Fmoc strategy, a deblocking reagent which can rapidly remove Fmoc group, while leaving the thioester intact, is required. For this purpose, two model peptides bearing an Fmoc group and a primary or tertiary thioester, respectively, were synthesized and the feasibility of various amines was tested using these model peptides. The basicity of amines was modified by adding 1-hydroxybenzotriazole (HOBt) or 2,4-dinitrophenol based on the fact that these reagents are known to suppress the cyclization of the aspartyl residue during base treatment [2, 3]. At appropriate concentrations, these reagents suppressed the aminolysis of thioesters and accelerated the removal of Fmoc group. A twenty percent hexamethyleneimine (HMI) solution containing 1.5 M HOBt was found to be the most satisfactory for our purposes. It completely removed the Fmoc group within twenty minutes and had the least affection on the thioester moiety. To test its feasibility as a deblocking reagent in practical Fmoc SPPS, a partial sequence of verotoxin subunit B (16–25), Fmoc-DDDTFTVKVGSC(CH3 )2 CH 2 CONH 2 , was synthesized using this reagent. The HPLC elution profile of the crude product (Figure 1A) showed that the desired peptide thioester was obtained. However, a number of defective peptide thioesters, which lacked one or two amino acids, were also observed. Under the similar conditions, the VT subunit B (26–33), Fmoc-DKELFTNR-NH 2 was synthesized, using an NMP solution containing 20% HMI and 1.5 M HOBt. This 579 Y. Shimonishi (ed): Peptide Science – Present and Future, 579–580 © 1999 Kluwer Academic Publishers, Printed in Great Britain

580

Figure 1. RP-HPLC elution profiles of crude products. Panel ( A) shows Fmoc-VT(16–25)SC(CH 3 ) 2 C H2 CONH 2 , (B): Fmoc-VT(26–33)-NH 2 ; (C): Fmoc-VT(18–25)-SCH 2 CH 2 CONH 2 , respectively. Column: Cosmosil 5C 1 8 AR (10 × 250 mm); eluent: 0.1% TFA in aq acetonitrile; flow rate: 2.5 mL/min.

peptide was successfully prepared (Figure 1B). These results suggest that the NMP solution containing 25% HMI and 1.5 M HOBt is not always strong enough to remove Fmoc groups on peptides having various sequences. As a deblocking reagent, tertiary amines were also examined, expecting the avoidance of the aminolysis of the thioester moiety. Although an NMP or DMSO-NMP (1:1) solution containing 25% 1-methylpyrrolidine satisfied our requirements in model experiments in a solution phase, this deblocking reagent did not work well in a practical Fmoc SPPS. Considering the difference between solid phase and solution phase reactions, 2% HMI was added in order to remove the Fmoc group completely; 2% HOBt was added to suppress the possible aminolysis by HMI. This reagent was then employed to synthesize a fragment of VT subunit B(18–25)-SR, Fmoc-DTFTVKVG-SCH 2 CH 2 CONH2 . The HPLC elution profile of the crude product (Figure 1C) indicated that the desired peptide thioester was obtained as the main product and almost no defective peptides were produced. Thus, it appears that a 25% 1-methylpyrrolidine NMP-DMSO (1:1) containing 2% HMI, and 2% HOBt is an effective deblocking reagent for the preparation of peptide thioesters by the Fmoc strategy.

References 1. Hojo, H. and Aimoto, S., Bull. Chem. Soc. Jpn.., 64 (1991) 111; Hojo, H., Yoshimura, S., Go, M., and Aimoto, S., Bull. Chem. Soc. Jpn., 68 (1995) 330; Kawakami, T., Kogure, S., and Aimoto, S., Bull. Chem. Soc. Jpn., 69 (1996) 3331. 2. Martinez, J. and Bodanszky, M., Int. J. Peptide Protein Res., 12 (1978) 277. 3. Lauer, J.L., Cynthia, C., Fields, G., and Fields, G.B., Lett. Peptide Science., 1 (1994) 197.

198 Chemical synthesis of barnase which possesses phosphoserine residue in place of the catalytic glutamic acid at position 73 H. HOJO 1, K. YAMAUCHI 1, M. KINOSHITA1 , M. GO 2 , T. KAWAKAMI 3 and S. AIMOTO 3 1

Department of Bioapplied Chemistry, Osaka City University, Osaka 558, Japan Division of Biological Science, Nagoya University, Nagoya 464-01, Japan 3 Institute for Protein Research, Osaka University, Osaka 565, Japan

2

Introduction We previously reported the chemical synthesis of barnase, a bacterial RNase with 110 amino acid residues, using partially protected peptide thioesters as building blocks [1]. The synthetic barnase possessed full enzymatic activity, demonstrating the applicability of the thioester method to enzyme synthesis. Based on this result, we attempted the modification of the activity of barnase by substituting one of the catalytic amino acid, Glu 73, with phosphoserine. The difference in the acidity of the carboxylic acid and phosphoric acid at the catalytic site would cause changes in some properties of barnase, such as optimum pH and the activity. These data would provide valuable information for the analysis of enzyme action as well as designing a new enzyme.

Results and discussion The preparation of the engineered barnase was carried out according to the previous synthesis, except for the preparation of peptide thioester 3, which contains phosphoserine. This peptide was prepared by the synthetic method for phosphopeptide [2], using cyclopentyl group as a protecting group for O-phosphono group [3] as shown in Figure 2. The yield was 2.5% based on the amino group on MBHA-resin. Boc-[Lys(Boc) 19,27 ]-Barnase(1–34)( 1), Fmoc-[Lys(Boc) 39,49 ]-Barnase(35–52)SC(CH 3 )2 CH 2 CO-Nle-NH 2 SCH 2 CH 2 CO-Nle-NH 2 (2 ), and [Lys(Boc) 98,108 ]-Barnase(82–110) (4) were prepared according to the previous procedure in yields of 12 to 16% [1]. Segment coupling was carried out following Figure 3. Peptides 3 and 4 (1 eq to 3 ) were dissolved in DMSO. HONSu (25 eq to 3), DIEA (8 eq to 3 ), and AgNO3 (8 eq to 3 ) were then added in succession. The solution was stirred overnight at room temperature in the dark. Piperidine (5% v/v to DMSO) was added and the solution was stood for 1 h to obtain peptide 5. According to the same procedure, peptides 2 and 5, peptides 1 and 6 were successively 581 Y. Shimonishi (ed): Peptide Science – Present and Future, 581–583 © 1999 Kluwer Academic Publishers, Printed in Great Britain

582

Figure 1. The amino acid sequence of the engineered barnase. The arrows indicate the sites of segment coupling. The asterisks show the catalytic amino acid. The Glu73 in native barnase was replaced with phosphoserine reisdue (Ser(P)).

Figure 2.

Synthetic procedure for peptide 3.

Figure 3.

Synthetic procedure for Ser(P)

73

-barnase.

condensed. The crude peptide was treated with TFA containing 5% EDT for 15 min and purified by HPLC to obtain peptide 7 in 4% yield. The amino acid composition and MALDI-TOF mass spectrometric data agreed well with that predicted.

583

Figure 4.

The hydrolytic activity of the barnases toward yeast RNA.

The synthetic barnase had a similar CD spectrum with the native one, but the intensity of the minimun at around 205 nm increased, indicating that the substitution of Glu 73 to Ser(P) might cause small structural change from the native one. The RNA hydrolytic activity of the barnases were measured according to the method by Rushizky et al. [4]. As shown in Figure 4, the native barnase had 1.6 × 10 6 unit activity at around pH 8. In contrast, the activity of synthetic barnase was negligible between pH 4 and 9. The reason for this result is now being analyzed in point of structural change and the reaction mechanism.

References 1. Hojo, H. and Aimoto, S., Bull. Chem. Soc. Jpn., 66 (1993) 3004. 2. Otaka, A., Miyoshi, K., Kaneko, M., Tamamura, H., Fujii, N. et al., J. Org. Chem., 60 (1995) 3967. 3. Wakamiya, T., Togashi, R., Nishida, T., Saruta, K., Yasuoka, J. et al., Bioorg. Med. Chem., 5 (1997) 135. 4. Rushizky, G.W., Greco, A.E., Hartley, R.W., Jr., and Sober, H.A. et al., Biochemistry, 2 (1963) 787.

199 Disulfide oxidation in water: investigation of CLEAR supports for on-resin cyclization K. DARLAK, M. DARLAK and A.F. SPATOLA Peptides International, Inc., 11621 Electron Drive, Louisville, KY 40299, USA

Introduction CLEAR (Cross-Linked Ethoxylate Acrylate Resin) was introduced by Kempe and Barany in 1996 [1] and further developed in 1997 [2]. CLEAR possesses a highly branched, highly cross-linked ethylene glycol matrix which has excellent swelling properties in a variety of solvents including water. It has outstanding potential for peptide synthesis, especially for difficult sequences; yet its hydrophilic nature is also attractive for biological applications [3]. In order to probe the versatility of CLEAR, several classic organic transformations have been previously examined [4]. These include Mitsunobu and Suzuki couplings. In most cases, CLEAR resins provided organic products with purity and yields comparable to polystyrene supports. In this report we explore the use of CLEAR supports for on-resin disulfide-forming oxidations.

Results and discussion In order to assess the possible advantages of CLEAR resin, we have utilized the aqueous compatibility of the poly(oxyethylene) backbone with common oxidizing agents used in disulfide forming reactions: K 3 Fe(CN)6 , I2 , and DMSO. As synthetic models, we have selected the peptides: an RGD cyclic hexapeptide, a cyclic dermorphin analog and oxytocin. The peptide sequences were assembled using Fmoc/t-butyl chemistries with the methoxytrityl (Mmt) moiety used for cysteine thiol protection. Condensations were performed using a 3–4 fold excess of amino acid and HBTU/DIEA as the activating agent in DMF (or NMP when using the Fastmoc protocol [5]). Following removal of the Mmt protecting groups on cysteines, oxidations were carried out either on CLEAR amide resin (Figure 1) or in solution as a positive control. K3 Fe(CN) 6 solutions used were 0.1 M–0.5 M; DMSO was used in large concentrations (50%); iodine was used both to effect removal of the Mmt groups and for disulfide oxidations (a 10-fold excess was used). A comparison of the results shows that yields and relative purities were comparable among the resin-bound methods (Table 1). In particular, K 3 Fe(CN) 6 oxidations of peptides bound to CLEAR supports produced good yields and purity, especially with an RGD sequence. With oxytocin, the best 584 Y. Shimonishi (ed): Peptide Science – Present and Future, 584–586 © 1999 Kluwer Academic Publishers, Printed in Great Britain

585

Figure 1. Table 1.

Structure of CLEAR amide resin. Summary of Results From Successful Oxidation Reactions.

Product

Oxidant

Solvent composition

Reaction time[h]

Purity [%]

Yield [%]

c(CRGDFC)-NH 2 c(CRGDFC)-NH 2 c(CRGDFC)-NH 2 c(CRGDFC)-NH 2 c(CRGDFC)-NH 2 Y-c(CFGYPC)-NH 2 Y-c(CFGYPC)-NH 2 Y-c(CFGYPC)-NH 2 Y-c(CFGYPC)-NH 2 Oxytocin Oxytocin Oxytocin Oxytocin

MeOH:H 2 O:AcOH(75:20:5) H2 O: MeOH (1.2: 1) K3 Fe(CN) 6 (0.22 M0 K3 Fe(CN) 6 (0.25 M) K3 Fe(CN) 6 (0.1 M) K3 Fe(CN) 6 (0.25 M) K 3 Fe(CN) 6 (0.22 M) K 3 Fe(CN) 6 (0.25 M) K 3 Fe(CN) 6 (0.1 M) K3 Fe(CN) 6 (0.25 M) K3 Fe(CN) 6 (0.22 M) K3 Fe(CN) 6 (0.25 M) K3 Fe(CN) 6 (0.1 M)

I 2 (10 eq) H 2 O:MeOH (4: 1) H 2 O:MeOH (1.2:1) H 2 O:DMF (4:1) H2 O:DMF (1:10) H 2 O:MeOH (4:1) H2 O:MeOH (1.2: 1) H 2 O:DMF (4:1) H2 O:DMF (1:10) H 2 O:MeOH (4:1) H2 O:MeOH (1.2:1) H 2 O:DMF (4: 1) H 2 O: DMF (1: 10)

12 24 24 24 24 24 24 24 24 24 24 24 24

57 84 80 76 76 68 53 52 37 25 15 34 28

48 40 50 29 65 22 32 31 55 43 19 37 40

results were obtained with water/DMF or water/MeOH combinations, in contrast to water alone (data not shown). DMSO oxidations were not successful with any of the three peptides tested. On-resin cyclization has been promoted by several laboratories [6,7] as a more efficient oxidation procedure, partly due to the pseudo-dilution effect of the resin environment. The use of water as a co-solvent, together with the unique CLEAR resin, are key factors making this combination compatible with inorganic oxidants such as K 3 Fe(CN) 6 . Because of the attractive swelling properties of CLEAR resin, it will be of interest to see if this support will facilitate the correct on-resin cyclization of peptide sequences with additional pairs of cysteine residues [8].

References 1. Kempe, M. and Barany, G., J. Am. Chem. Soc., 118, (1996) 7083. 2. Darlak, K., Romanovska, I., Spatola, A.F., Barany, G., and Kempe, M., Poster 141, 15th American Peptide Symposium, Nashville, Tennessee, June 14–19,1997. 3. Kempe, M. and Barany, G., Poster 191, 15th American Peptide Symposium, Nashville, Tennessee, June 14–19,1997.

586 4. Chatla, N., Darlak, K., and Spatola, A.F., 5th International Symposium on Solid Phase Synthesis and Combinatorial Chemical Libraries, London, England, Sept. 2–6, 1997. 5 . Fastmoc is a trademark of Perkin Elmer/Applied Biosystems. 6. Andreu, D., Albericio, F., Solé, N.A., Munson, M.C., Ferrer, M., and Barany, G., In Pennington, M.W. and Dunn, B.M. (Eds) Peptide Synthesis Protocols, Volume 35, Humana Press, New Jersey, 1994, pp. 91–170. 7. Lebl, M. and Hruby, V.J., Tetrahedron Lett., 25 (1984) 2067. 8. CLEAR resins are commercially available from Peptides International, Inc., Louisville, Kentucky USA.

200 Non-natural peptide modifications using sugars and lipophilic side chains H. KESSLER*, F. BURKHART, M. KOPPITZ and E. LOHOF Institute of Organic Chemistry and Biochemistry, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany

Introduction Peptides and proteins are frequently posttranslationally modified to modulate activity, metabolic stability and compartmental localization. The best described systems are molecular switches provided by phosphorylation. On the other hand glycosylation is used to decorate the surface of proteins with hydrophilic moieties which are often specifically recognized. In addition, lipomodification, e.g., farnesylation, serves to anchor the molecules at the membrane to specifically localize the peptide/protein at the interphase. Here we discuss non-natural modifications of peptides via sugar residues as well as lipophilic residues to modulate the hydrophilic/lipophilic profile of bioactive peptides and to enhance metabolic stability as well as prolong life time in vivo by hindering excretion via the kidney or bile. The direct incorporation of aliphatic side chains or sugars into the amino acids via links inert to enzymatic degradation opens new ways to improve the properties of peptides for their use as drugs.

Lipomodification Aliphatic side chains have been incorporated via alkylation of the sulfhydryl group of Cys, via N -alkylation of peptide bonds and by using α-amino fatty acids [1]. The modified peptides were used as model compounds to investigate the behavior of partially flexible peptides at the hydrophilic/hydrophobic interphase, but also to study the influence of the chemical nature of lipophilic groups on the activity of the mating pheromone hormone of the fungus Ustilago maydis [2]. (a) Lipomodification of a mating factor. The pathogenic development of the mold Ustilago maydis (corn smut disease) is initiated by the fusion of two haploid cells of different mating type, similar to the same process in yeast [3]. This process is controlled by mating pheromones, short peptides of the sequence H-G-R-D-N-G-S-P-I-G-Y-S-S-Xaa- Z and H-N-R-G-Q-P-G-Y-Y-Xaa-Z, in which Xaa is farnesylated Cys-OMe [4]. We synthesized these compounds, along with derivatives where Xaa was modified by using α -amino fatty acids 587 Y. Shimonishi (ed): Peptide Science – Present and Future, 587–594 © 1999 Kluwer Academic Publishers, Printed in Great Britain

588 of different chain length, as well as N -hexadecyl-Gly–OMe [5]. It turned out that the lipophilic character is most important for activity, and that no specific modification such as the farnesyl group is required for the biological response. The peptides did not show any conformational preferences even when studied in SDS micells. (b) Conformational changes induced by a non-isotropic medium. The interaction of peptide hormones with their transmembrane receptor is regulated via a receptor subtyp-specific lipophilic/hydrophilic profile [6]: It has been suggested that the hormone first interacts with the cell membrane and then diffuses laterally along the cell surface to the binding sites. The interphase regulates the activity via interaction with the ‘address region’ of the hormone, and modulates the activity of the ‘message region’ through a number of possible factors such as the depth of insertion, the orientation at the interphase or changes in the peptide conformation. To investigate these phenomena a number of amphiphilic cyclic peptides were synthesized which could be investigated by NMR spectroscopy in CDCl3 and in SDS micells (used as a membrane mimic). Using the NMR constraints, the peptides were then simulated at the interphase of a CCl 4/H 2 O two-phase box (also serving as a membrane mimic) [7]. Among the 8 cyclic peptides we could identify one – cyclo (– D Asp–Asp–Gly–Ahds– Ahds–Gly–) – which clearly shows different conformations in CDCl 3 and in SDS micells. This proves that an anisotropic environment can induce distinct conformational rearrangements in these peptides. The analysis of the MD trajectory in the two-phase box can answer the question about orientation and insertion in the different phases, e.g., when the structure derived from NMR data in CDCl 3 is in the MD calculations artificially incorporated into the CCl 4 phase, the peptide moves to the water phase, penetrates with the peptidic part into the water (Figure 2) and finally changes

Figure 1.

Structures of cyclo(– D Asp–Asp–Gly–Ahds–Ahds–Gly–) in CDCl3 (a) and SDS/H 2 O (b).

589

Figure 2. Snapshots of the trajectory of cyclo(– D Asp–Asp–Gly–Ahds–Ahds–Gly–) demonstrate the phase transition and orientation.

the peptide conformation as shown in Figure 1. Only the lipophilic tail remains in the CCl4 phase.

Glycomodification The most important naturally occuring glycosylations are O-glycosides in the α - or β -configuration bound to serine or threonine residues, and N-glycosides with a β - glycosidic link to the side chain of asparagine residues. Over the past few years we have developed several unnatural O-, S-, and C -glycosylated amino acids, and incorporated them into cyclic model peptides for conformational studies, as well as into bioactive peptides. 2-Deoxy- O-glycoamino acids were synthesized via the N -iodo-succinimide promoted addition of Ser or Thr to a glucal, and subsequent reductive removal of the iodine in position 2 [8]. Also, Michael addition to hex-1-en-3-uloses could be used to synthesize new O -glycoamino acids, although the yields were only moderate [9].

590 Furthermore S-glycosylated cysteines were synthesized [10]. Glycosylated cysteines are extremely rare in nature, and the incorporation of S - instead of O-glycosides is an attractive alternative for the synthesis of enzymatically stable glycopeptides. T h e s y n t h e s i s o f C -glycosylated amino acids is quite challenging. Conventional glycosylation requires the addition of a carbanion to the (cationic) anomeric carbon. However, carbanions in protected amino acids will lead to a number of undesired side reactions. Anomeric carbanions of the sugar, on the other hand, eliminate quite easy to form glycals. To prevent this undesired reaction we prepared a glycosidic dianion by leaving the hydroxyl group in position 2 unprotected, while all other hydroxyl groups are benzylated [11]. The anomeric anions turned out to be stable against inversion under the reaction conditions, leading to pure α - or β -anomers. They react with aldehydes, methyl iodide, protons and deuterons as well as with carbon dioxide. This opens a way for the synthesis of a variety of new C -glycosylated amino acids. The C -glycosylated amino acid 1 (Figure 4), containing a reversed amide bond, was prepared by addition of CO 2 to a glycosyl-dianion and subsequent coupling of the resulting heptonic acid to a diaminopropionic acid derivative [12]. The C -glycosylated amino acids containing a single carbon chain between the sugar residue and the amino acid were either prepared by the radicalic addition of glycosyl bromides to dehydroalanine derivatives (Figure 5) [13], or by the coupling of an amino acid aldehyde with a dianion of the glucosamine (Figure 4) [14].

Figure 3.

The O- and S -glycosylated amino acids synthesized in our group.

Figure 4. strategy.

The C -glycosidic analogues of N-glucoasparagine were synthesized using the dianion

591

Figure 5. The epimeric C-glycosylated amino acids 3a and b with a shortened side chain were synthesized by the addition of a glycosyl radical to a dehydroalanine.

The C-glycosylated amino acid 3b, a rhamnosylated D -cysteine and a rhamnosylated D -serine were incorporated to a buserilin-derivative (LH-RH analogue used by Hoechst in the chemical castration of prostate cancer patients) in which the O -tert.butyl- D -serine is substituted (Figure 6) [15]. The rhamnosylated derivatives have the same activity as buserilin, but a much higher solubility in water. The retroamide 1 (Figure 4) (CGaa) and the corresponding N -glycosylated amino acid (NGaa) were incorporated into a cyclic model peptide ( cyclo(– D Pro–Phe–Ala–Gaa–Phe–Phe–) 4 and 5) to determine the influence of the sugar residue and the glycosidic linkage on the peptide conformation (Figure 7). The structures determined by NMR-spectroscopy and molecular dynamic calculations for both peptides gave evidence for an interaction between the carbohydrate moiety and the peptide backbone. Carbohydrates can also be used as peptide building blocks by transforming them into sugar amino acids (SAAs) [17, 18]. They typically carry an amino and a carboxyl functional group in distinct substitution patterns, which allows them to induce significant conformational restrictions when incorporated into peptides. Figure 8 shows a series of SAAs which can be used as a construction kit for peptide conformations.

Figure 6.

Biological activity of glycosylated LH-RH-agonists.

592

Figure 7. The structures of N-glycosylated cyclic peptide 4 and the corresponding C -glycosylated analogue 5 containing a reversed amide bond in the linker determined by NMR techniques, distance geometry and restraint molecular dynamic calculations [16].

Figure 8.

SAA in a construction kit for peptide conformations.

All SAAs contain a six membered ring with all substituents in equatorial positions. In order to characterize the conformational behaviour of these peptidomimetics, several cyclic SAA peptides were synthesized, and their conformations were determined by NMR spectroscopy and molecular dynamic calculations. In these cyclic peptides, SAA 2, 3 or 4 are linked to tetrapeptides containing one D -amino acid. The D -amino acid in i + 1 position of the lower loop stabilizes

593

Figure 9.

Conformation of SAA 2, 3 and 4 (see Figure 8) in cyclic peptides.

a β II'-turn. In case of the first two peptides, SAA 2 and 3 occupy the i + 1 and i + 2 position of a pseudo β -turn of the northern hemisphere. The peptide containing SAA 4 forms a narrow loop similar to a γ -turn.

Acknowledgement Financial support by the Deutsche Forschungsgemeinschaft and the Fonds der chemischen Industrie is gratefully acknowledged.

References 1. Kessler, H., Guba, W., Mathä, B., Koppitz, M., Haeßner, R., Lutz, J., and Moroder, L., In Shimonishi, Y. (Ed.) Japanese-German Symposium on Peptide Chemistry (Proceedings of the 6th Akabori Conference), Protein Research Foundation, Osaka, Japan, 1996, pp. 172–178. 2. Koppitz, M., Haeßner, R., Spellig, T., Kahmann, R., and Kessler, H., In Kaumaya, P.T.P. and Hodges, R.S. (Eds.) Peptides: Chemistry, Sructure and Biology (Proceedings of the 14th American Peptide Symposium), Mayflower Scientific Ltd., England, 1996, pp. 201–202. 3. Xue, C.B., McKinney, A., Lu, H.F. Jiang, Y., Becker, J.M., and Naider, F., Int. J. Pept. Prot. Res. 47 (1996) 131. 4. Bölker, M., Urban, M., and Kahmann, R., Cell, 68 (1992) 441. 5. Koppitz, M., Spelling, T., Kahmann, R., and Kessler, H., Int. J. Pept. Prot. Res. 48 (1996) 377. 6. a) Schwyzer, R., Biopolymers, 37 (1995) 5. b) Schwyzer, R., J. Mol. Recognit., 8 ( 1995) 3. 7. Koppitz, M., PhD-thesis, TU-München, 1996. 8. Kottenhahn, M. and Kessler, H., Liebigs Ann. Chem. (1991) 727. 9. Michael, K. and Kessler, H., Tetrahedron Lett., 37 (1996) 3453. 10. a) Gerz, M., Matter, H., and Kessler, H., Angew. Chem. Int. Ed. Engl., 32 (1993) 269. b) Käsbeck, L. and Kessler, H., Liebigs Ann./Recueil, 1997, 165. 11. a) Wittmann, V. and Kessler, H., Angew. Chem. Int. Ed. Engl., 32 (1993) 1091. b) Frey, O., Hoffmann, M., and Kessler, H., Helv. Chim. Acta, 77 (1994) 2060. c) Hoffmann, M. and Kessler, H., Tetrahedron Lett., 35 (1994) 6067. 12. a) Hoffmann, M., Burkhart, F., Hessler, G., and Kessler, H., Helv. Chim. Acta, 79 (1996) 1519. b) Frey, O., Hoffmann, M., and Kessler, H., Angew. Chem. Int. Ed. Engl., 34 (1995) 2026.

594 13. Kessler, H., Wittmann, V., Köck, M., and Kottenhahn, M., Angew. Chem. Int. Ed. Engl., 31 (1992) 902. 14. Burkhart, F., Hoffmann, M., and Kessler, H., Angew. Chem. Int. Ed. Engl., 36 (1997) 1191. 15. Michael, K., Wittmann, V., König, W., Sandow, J., and Kessler, H., Int. J. Peptide Protein Res., 48 (1996) 59. 16. Kessler, H. and Seip, S., In Croasmun, W.R., Carlson, R.M.K. (Eds.), Two-Dimensional NMR Spectroscopy, VCH, Weinheim, 1994,619. 17. Kessler, H., Graf von Roedern, E., Lohof, E., Haubner, R., Geyer, A., and Wittmann, V., In Wünsch, E. (Ed.) German-Japanese Symposium on Peptide Chemistry (Proceedings of the 5th Akabori Conference), Max-Planck-Gesellschaft, Germany, 1994, pp. 1100–115. 18. a) Graf von Roedern, E. and Kessler, H., Angew. Chem. Int. Ed. Engl., 33 (1994) 687. b) Graf von Roedern, E., Lohof, E., Hessler, G., Hoffmann, M., and Kessler, H., J. Am. Chem. Soc., 118 (1996) 10156.

201 Diastereoselective synthesis of glycosyl- α -aminoacids and glycopeptide derivatives S. BOUIFRADEN, M. EL HADRAMI, J.-P. LAVERGNE, J. MARTINEZ* and P. VIALLEFONT Laboratoire des Aminoacides, Peptides et Protéines, CNRS-UMR 5810, Universités Montpellier I et II, 34095 Montpellier Cédex 5, FRANCE. E-mail: [email protected]

Introduction Glycopeptides constitute an important class of compounds which are widely distributed among living organisms. The sugar component can modulate the biological properties of the protein by playing an essential role in molecular recognition. The sugar group also improves protein solubility as well as bioavailability by accelerating its transport across membranes [1]. Sugar moieties are usually associated with proteins by a C–N or C–O bond. Our aim was to develop the synthesis of glycosyl-α -aminoacids and glycopeptide derivatives in which the sugar and the aminoacid are joined by a C–C bond resistant to enzyme cleavage. Results and discussion In the first part we report the synthesis of glycosyl- β -hydroxy- α-amino esters. β -hydroxy- α-amino acids are important compounds because they are constitu[3] ents of biologically active peptides [2], precursors of antibiotic β -lactams and enzymatic inhibitors [4]. Our approach is based on the condensation, on the two sugar aldehydes 1a and 1b, of glycine enolates prepared from Schiff base of 2-hydroxypinan-3-one (R,R,R) 2 [5], (Figure.1). The anion has been prepared using t-BuOK in THF. For the two sugars, 1a and 1b, a mixture of two diastereomers was obtained after condensation. The major product was isolated after chromatography column on silica-gel; 3 a arising from the ribose 1a, and 3b, coming from the galactose 1b constitute respectively 82% and 66% of the mixture. The stereochemistry of these compounds has been determined from crystallographic analysis on β -hydroxy- α amino esters 4a and 4b obtained after hydrolysis of 3 by citric acid. We observed here a double asymmetric induction; we have previously shown that alkylation of Schiff bases prepared from 2-hydroxypinan-3-one took place with excellent diastereoselectivity [6]. Various glycine enolates (prepared from aminoesters 6, 7, and 8 by treatment with LDA in THF at –80°C) were reacted with the carbonyl group of 595 Y. Shimonishi (ed): Peptide Science – Present and Future, 595–599 © 1999 Kluwer Academic Publishers, Printed in Great Britain

596

Figure 1.

1 , 2 : 5 , 6 - d i -O-isopropylidene- α - D -ribohexofuranos-3-ulose 5 [7] (Figure 2). Starting from compound 6, the reaction took place in excellent yield and with total diastereoselectivity implying a two-fold control of chirality. The afforded compound 9 contained the aminoester residue in the exo position. The structure and the stereochemistry of 9 were assigned from the spectral data and from the X-ray diffraction pattern. In the case of compound 7, the reaction occurred with lower yield and with reduced diastereoselectivity. The major isolated diastereoisomer 10 had a stereochemistry identical to that of

Figure 2.

597

Figure 3.

598 compound 9. This stereochemistry was deduced from the X-ray diffraction pattern for its spirolactone derivative 16 (Figure 3) described below. The enolate of the Schiff’s base 8 afforded a spirooxazolidine 11. The stereochemistry of compound 11 was confirmed by X-ray analysis. Hydrogenolysis of compounds 9 and 10 took place quantitatively in the presence of palladium hydroxide in a mixture of THF/ethanol affording the glycosylated aminoesters 12 and 1 3 (Figure 2) with a free amine group. We were unable to cleave the tert-butyl ester of compound 9 in the presence of TFA without altering the molecule. However, we have obtained the corresponding glycosylaminoacid 14 by saponification of the methyl ester 1 0 u n d e r n o n - e p i m e r i z i n g c o n d i t i o n s (dioxane/NaOH). The glucofuranos-3-yl-aminoester 15 with a free amino group was obtained by hydrolysis of compound 11 in the presence of 15% citric acid. Four glycosyl-α-aminoesters (or acids) of controlled chirality were thus obtained and by coupling with properly protected aminoacid derivatives can afford glycopeptides [8]. However, it should be noted that under the coupling conditions the glycosylaminoacid 14 showed tendency to lactonise readily. In the presence of BOP/DIEA in dichloromethane the spirolactone 16 w a s obtained in high yield (Figure 3). This synthon was used for the synthesis of new glycopeptides [9]. Coupling to three amino esters, H-Gly–OMe, H-L -Ala–OMe and H-L -Phe–OMe, was carried out in the presence of DMAP. With glycine methyl ester two reactions were in competition. We have isolated compound 17 arising from the O-acyl opening of the spirolactone (yield = 50%) but also the spirolactame 18 (yield = 20%) arising from the O-alkyl cleavage of the spirolactone 3 followed by an intramolecular cyclisation with nucleophilic attack of the acid function by the nitrogen atom of the intermediate (Figure 3). With alanine and phenylalanine methyl esters we have isolated the products 19 (yield = 90%) and 20 (yield = 77%), arising exclusively from the cleavage of the O-acyl bond (Figure 3). In the last case we observed an extent of racemization of 9%. These glycopeptides will be introduced in biological active peptides in order to know how they modulate the activity and in particular the biodisponibility.

References 1. Rewiews: a) Schwarz, R.T., Datema, R., Adv. Carbohydr. Chem. Biochem., 40 (1982) 287. b) Olden, K.B., Bernard, B.A., Humphries, M.J., Yeo, T., Yeo, K., White, S.L., Newton, S.A., Bauer, H.C., Parent, J.B., Trends Biochem. Sci. Pers. Ed., 10 (1985) 78. 2. Kurokawa, N., Ohfune, Y., J. Am. Chem. Soc., 108 (1986) 6041. 3. Miller, M.J., Acc. Chem. Res., 19 (1986) 49. 4. Nakatsuka, T., Miwa, T., Mukaiyama, T., Chem. Lett., (1981) 279; (1982) 145. 5. Oguri, T., Kawai, N., Shiori, T., Yamada, S., Chem. Pharm. Bull., 26 (1978) 803. 6. El Achqar, A., Boumzebra, M., Roumestant, M.L., Viallefont, P., Tetrahedron, 44 (1988) 5319.

599 7. Jones, G.H., Moffat, J.G., Methods Carbohydr. Chem., 6 (1972) 315. 8. Bouifraden, S., Lavergne, J.-P., Martinez, J., Viallefont, P., Riche, C., Tetrahedron: Asymmetry, 8 (1997) 949. 9. Bouifraden, S., Lavergne, J.-P., Martinez, J., Viallefont, P., Synth. Commun., 27 ( 1997) 3909.

202 Influence of glycation on cis/trans isomerization and tautomerization in novel morphiceptin-related Amadori compounds I . IGROVI

¹, J. KIDRI

² and

. HORVAT¹

¹ Department of Organic Chemistry and Biochemistry, ‘Rudjer Boskovic’ Institute, P.O.B. 1016, 10000 Zagreb, Croatia ² National Institute of Chemistry, P.O.B. 30, 61015 Ljubljana, Slovenia

Introduction Glycation, the non-enzymatic reaction between reducing sugars and the amino groups of amino acids, peptides and proteins, collectively known as the Maillard reaction, leads to the formation of N -substituted 1-amino-1-deoxy-ketoses or Amadori compounds [1]. Over a period of time, Amadori compounds degrade and form advanced glycation end products (AGES ), capable of cross-linking and modification of protein functions [2]. It has been suggested that the AGE S are involved in the tissue aging processes, diabetic complications, and a variety of diseases including Alzheimer’s disease. In an effort to better understand the role of Amadori compounds related to casein-derived peptides, which can be formed during the production of milk-based foods, we have prepared Amadori compounds related to morphiceptin (H–Tyr–Pro–Phe–Pro–NH 2 ), a highly selective and the most active, opioid peptide from the bovine β -casomorphin group of peptides. The aim of this work was to investigate the effect of glycation on peptide backbone cis/trans isomerization and sugar moiety tautomerization by using NMR spectroscopy.

Results and discussion The synthetic approach to protected Amadori compound 2 involved the synthesis of the isopropylidene protected Amadori compound of tyrosine 1 [3] and its coupling with the tripeptide H–Pro–Phe–Pro–NH2 (Figure 1). Deprotection of 2 in aqueous trifluoroacetic acid afforded partially and fully deprotected morphiceptin-related Amadori compounds 3 and 4. NMR spectra were recorded on a Varian Gemini spectrometer operating at 300.1 MHz and 600.14 MHz for ¹H and 75.5 MHz for 1 3 C nuclei. Experiments were performed at 25°C using 50 mM solutions in DMSO-d 6 or D 2 O. Signals 600 Y. Shimonishi (ed): Peptide Science – Present and Future, 600–602 © 1999 Kluwer Academic Publishers, Printed in Great Britain

601

Figure 1.

Synthetic approach to morphiceptin-related Amadori compounds.

were assigned by the combined use of 1D and 2D homonuclear and heteronuclear experiments. Inspection of NMR data revealed the presence of four discernible isomers in DMSO solution for Amadori compounds 2 and 3 arising from cis-trans isomerization about the Tyr¹–Pro² and Phe³–Pro4 peptide bonds. The estimated ratio of configurational isomers for compound 2, was trans,trans: cis, trans : trans, cis : cis, cis = 45 : 30 : 15 : 10. The spectroscopic data obtained for the partially protected Amadori compound 3 in DMSO solution gave the following ratio of configurational isomers: trans, trans : cis, trans : trans, cis : cis, cis = 47 : 24 : 18 : 11. These results showed that the cis trans equilibrium about the Tyr¹–Pro² and Phe³–Pro 4 peptide bonds is shifted toward the trans conformer (60–75%) in both fully and partially protected Amadori compounds. Comparison of the data obtained for Amadori compounds 2 and 3 with those obtained for morphiceptin [4] showed that the equilibrium fraction of all-trans isomers in N-glycated morphiceptin derivatives was smaller than in the parent peptide, and suggests stabilization of the cisamide bonds in 2 and 3 by hydrophobic interaction between the protected sugar moiety and the peptide chain. The NMR spectra of the unprotected morphiceptin-related Amadori compound 4 indicated the presence of multiple conformers due to the peptide backbone cis-trans isomerization as well as tautomerism of the 1-deoxy-D fructose moiety. In DMSO solution the following equilibrium composition was found: α-furanose 40%, β -furanose 28%, β -pyranose 27%, and α -pyranose 5%. The estimated equilibrium composition in water solution was β -pyranose 66%, β -furanose 27%, and α -furanose 7%.

602 References 1. 2. 3. 4.

Ledl, F. and Schleicher, E., Angew. Chem. Int. Ed. Engl. 29 (1990) 565. Vlasara, H., J. Lab. Clin. Med. 124 (1994) 19. Jakas, A. and Horvat, Š., J. Chem. Soc. Perkin Trans. 2 (1996) 789. Goodman, M. and Mierke, D.F., J. Am. Chem. Soc., 111 (1989) 3489.

203 Chemoenzymatic synthesis of glycophosphopeptides J.E. SANDER, T. POHL and H. WALDMANN Institut für Organische Chemie der Universität, Richard-Willstätter-Allee 2, 76128 Karlsruhe, Germany

Introduction The covalent modification of proteins by phosphorylation and addition of OGlcNAc residues are important regulatory processes which mediate biological signal transduction [1]. For instance, the cytosolic form of RNA polymerase II is heavily glycosylated but during its transition from an initiating to an elongating complex, the carbohydrates are removed and the protein is phosphorylated. For the study of such biological phenomena, characteristic peptides which embody both types of modifications may serve as efficient tools. However, their synthesis is complicated by their pronounced acid and base lability as well as their multifunctionality. These properties make the application of protecting groups necessary which can be removed under the mildest conditions. For the construction of such peptide conjugates, two enzyme labile blocking groups have been developed in our group: the N -terminal p -phenylacetoxybenzyloxycarbonyl(PhAcOZ) group [2] and the C-terminal choline ester [3].

Results and discussion The PhAcOZ protecting group has been shown to be removable under very mild conditions in the synthesis of glycophosphopeptides.

Figure 1.

T h e p -phenylacetoxybenzyloxycarbonyl (PhAcOZ) protecting group.

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Figure 2. Structure of the glycosylated and phosphorylated hexapeptide from the repeat sequence of RNA polymerase II.

It embodies (a) a functional group (a phenylacetate) that is recognized by the biocatalyst (penicillin G acylase) and that is bound by an enzyme labile linkage to (b) a functional group (a p -hydroxybenzyl urethane) that undergoes a spontaneous fragmentation upon cleavage of the enzyme-sensitive bond resulting in (c) the liberation of a carbamic acid derivative which decarboxylates. When this enzymatic protecting group technique was combined with classical chemical methods, a complex phosphoglycohexapeptide was built up, which embodies two glycosylated, one phosphorylated, and one underivatized hydroxyamino acid. This peptide represents a characteristic partial structure of the repeat sequence of the large subunit of RNA polymerase II which becomes glycosylated or phosphorylated while the enzyme carries out its biological functions. The conditions under which the enzymatic deprotections proceed are so mild that no undesired side reaction is observed (i.e., no rupture or anomerization of the glycosidic bonds and no β -elimination of the phosphate or a carbohydrate occur). In addition, the specificity of the biocatalyst guarantees that the peptide bonds and the other protecting groups present are not attacked either.

References 1. Hinterding K., Alonso-Diaz, D., and Waldmann, H., Angew. Chem. Int. Ed. Engl. (1998) 32 (1998) 688. 2. Pohl, T. and Waldmann, H., Angew. Chem. Int. Ed. Engl. 35 (1996) 1720; J. Am. Chem. Soc. 119 (1997) 6702. 3. Schelhaas, M., Glomsda, S., Hänsler, M., Jakubke, H.-D., and Waldmann, H., Angew. Chem. Int. Ed. Engl. 35 (1996) 106.

204 Design and synthesis of novel Src homology-2 domain inhibitors R.J. BROADBRIDGE, R.P. SHARMA and M. AKHTAR Division of Biochemistry & Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, S016 7PX, UK

Introduction Src homology-2 (SH2) domains are modules of about 100 amino acid residues found in many intracellular signal-transduction proteins that bind phosphotyrosine containing polypeptide sequences with high affinity and specificity. The protein p56lck is a Src-like, lymphocyte-specific tyrosine kinase, containing an SH2 domain, that participates in signalling pathways initiated through the T cell antigen receptor upon T lymphocytes and is required for T cell maturation. The SH2 domain is thought to be responsible for the recruitment and the regulation of p56lck kinase activity. Crystallography data [1] has indicated that up to 30% of hydrogen bonding between high affinity phosphopeptides bound to the p56lck SH2 domain can be attributed to the peptide backbone. The contacts made between the SH2 domain and the peptide backbone would be critical for specificity, and chemical alterations to the backbone of phosphopeptides could afford novel high affinity motifs. Discovery of such peptide mimetics may result in new immunosuppressants and anti-cancer agents with inherent protease resistance.

Results and discussion Previously we have reported the application of tri-tert butoxysilyl esters (TtBos) of amino acids in peptide synthesis on the solid phase from the N to C direction [2–4]. ‘One-bead–one-peptide’ combinatorial libraries (each library containing approximately 1.5 million 6-mer peptides) were generated upon TentaGel-S resin in a split synthesis manner (0.3 mmol/g, which had been previously substituted with the Fmoc-linker and β Ala–Gly spacer). The portion of the peptide synthesised in the C to N direction was by t Boc chemistry. A single urea bond (per library) was inserted into various positions with CDI, and the peptide subsequently lengthened (in the N to C direction) by T-t Bos chemistry. A single phosphotyrosine residue (per library) was incorporated into the libraries at various positions by using Nα-tBoc-Tyr(P0 3 Me2 )OH [6]. Prior to screening, the peptide side chains were deprotected (including the methyl groups of phosphotyrosine) by low TFMSA conditions (TFMSA/TFA/ 605 Y. Shimonishi (ed): Peptide Science – Present and Future, 605–606 © 1999 Kluwer Academic Publishers, Printed in Great Britain

606 DMS/meta-cresol 10/50/30 10 v/v, 4 h, 0°C). TFMSA treated beads from libraries were incubated with p56 lck SH2-GST fusion protein [5]. The beads were thoroughly washed, incubated with rabbit anti-GST, washed again, and subsequently incubated with goat anti-rabbit HRP conjugate. Beads that contained ligands that bound to the SH2 domain were visualised with Diaminobenzidine (DAB) staining. Positive beads were pooled and destained (8 M guanidine hydrochloride, pH 2, 20 min, followed by gentle sonication in DMF). The destained beads were separated, and the parent peptide (together with the ladder sequences contained with it) cleaved from the resin by treatment with diethylamine (cleavages with piperidine were faster, however piperidine adducts of the peaks were evident upon the electrospray). The cleavage mixtures were filtered, desiccated, then dissolved in water/acetonitrile 50/50, v/v, and the ligand identified by injecting the solution into an electrospray mass spectrometer (ESMS) monitoring the negative ion. The bead binding assay gave about 100 highly coloured beads. Cleavages of single destained beads (as described above) were subjected to ESMS (negative ion). Two sequences HO–Phe-ureapTyr–Gly–Phe–Phe–Pro–OH and HO–Glu-urea-pTyr–Glu–Ala–Arg–Pro– OH which gave promising results were chosen for further work which is continuing. This investigation has yielded several novel, high affinity compounds which have generated further study in our laboratory. The urea bond could give the compounds extra resistance to proteases as well as increasing the number of hydrogen bond interactions between the ligand and the SH2 domain. Investigations are currently under way to discover the effect of modifying the carboxyl of the peptides to binding affinity, and to introduce various phosphonotyrosine analogues.

References 1. Eck, M.J.,Shoelson, S.E., and Harrison, SC., (1993), Nature 362, 87–91. 2. Sharma, R.P., Jones, D.A., Corina, D.L., and Akhtar, M., In Hodges, R.S. and Smith, J.A. (Eds.) Peptides: Chemistry, Structure and Biology (Proceedings of the 13th American Peptide Symposium), ESCOM, Leiden, (1994), 127–129. 3. Sharma, R.P., Broadbridge, R.J., Jones, D.A., Corina, D.L., and Akhtar, M., In Epton, R. (Ed.), Innovation and Perspectives in Solid Phase Synthesis, 1994: Biological and Biomedical Applications, Mayflower Worldwide, (1994), Ch 52, 353–356. 4. Sharma, R.P., Broadbridge, R.J., Jones, D.A., Corina, D.L., and Akhtar, M., In Lu, G., Tam, J.P., and Du, Y. (Eds.), Peptides Biology and Chemistry. 5. Kitas, E.A., Perich, J.W., Johns, R.B., and Tregear, G.W., (1988), Tet. Let. 29, 3591–3592. 6. Payne, G., Shoelson, S.E., Gish, G.D., Pawson, T., and Walsh, C.T., (1993), Proc. Natl. Acad. Sci.USA 90, 4902–4906.

205 Chemo-enzymatic syntheses of eel calcitonin analogs having natural N -linked oligosaccharides T. INAZU1 , M. MIZUNO1 , I. MURAMOTO1 , K. HANEDA1 , T. KAWAKAMI2 , M. SEIKE 2 , S. AIMOTO2 , K. YAMAMOTO 3 and H. KUMAGAI3 1

The Noguchi Institute, Tokyo, Japan Institute for Protein Research, Osaka University, Osaka, Japan 3 Graduate School of Agriculture, Kyoto University, Kyoto, Japan 2

Introduction The growing interest in glycoprotein on cell surface as ligands of biological important response, such as cell recognition, cell adhesion, and so on, has stimulated development of new methods for the convenient syntheses of glycopeptides. Recently we have reported a solid-phase synthesis of glycopeptide containing Asn(GlcNAc) residue by the dimethylphosphinothioic mixed anhydride (Mpt–MA) method without protecting the hydroxyl functions of sugar moiety [1]. Also we have reported the transglycosylation reaction of synthetic peptide having GlcNAc residue using endo- β - N -acetylglucosaminidase of Mucor hiemalis (Endo-M) [2]. In this paper we describe the syntheses of eel calcitonin (CT) analogs having natural N-linked sugar chain, 8, 9 and 1 0.

Results and discussion First, we synthesized glycosyl eel CT containing Asn(GlcNAc) residue, [Asn(GlcNAc) 3 ]-CT (1), by the combined method of Mpt–MA and peptide thioester segment condensation [3] on solid support in good yield. Boc– Asn(GlcNAc)–OH, which is a key-compound in this synthesis, was prepared by a similar method to that described in our previous report [4]. The peptide thioester resin 3 was prepared from 2 by a Boc-strategy using the DCC-HOBt coupling method. The corresponding Mpt–MAs of Boc–Asn(GlcNAc)–OH, Boc–Ser(Bzl)–OH and Fmoc–Cys(Acm)–OH were one by one introduced into 3. The glycopeptide thioester 4 was obtained in 12% yield by treatment with anhydrous HF containing 10% anisole and reversed-phase HPLC (RP-HPLC). The other peptide segment 5 was prepared by a Boc-strategy. Glycopeptide

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thioester segment 4 and partially protected peptide segment 5 were added to a mixture of AgNO 3 , 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOObt) and DIEA in DMSO. The reaction mixture was stirred for 16 h at room temperature. Partially protected glycopeptide analog of CT was obtained in 78%. After cleavage of Boc and Fmoc groups, and RP-HPLC purification, precursor 6 was prepared. The precursor 6 was treated with AgNO3 and DIEA in aqueous DMSO, followed by 1N HCl/DMSO at room temperature to remove the Acm groups and form a disulfide bond [5]. After RP-HPLC purification, the glycopeptide analog of eel calcitonin 1 was obtained in 9% overall yield based on the amount of amino group in the starting NH2 -resin. Next, disialo and asialo complex-type oligosaccharide of human serum transferrin and high mannnose type oligosaccharide of ovalbumin were transferred to the GlcNAc moiety of 1 by Endo-M. The desired CT analogs having natural N-linked sugar chain 8 , 9 and 10 were obtained in 8.5%, 7.5% and 3.5% yields respectively. These conditions were similar as described previously [2]. Endo-M transglycosylated the sialo complex-type oligosaccharide most effectively, which was the poorest substrate for hydrolysis.

References 1. Inazu, T., Mizuno, M., Kohda, Y., Kobayashi, K. and Yaginuma, H. ‘Peptide Chemistry 1995,’ ed by N. Nishi, Protein Research Foundation, Osaka (1996), p. 61.

609 2. Haneda, K., Inazu, T., Yamamoto, K., Kumagai, H., Nakahara, Y. and Kobata, A. Carbohydr. Res., 292 (1996) 61. 3. Mizuno, M., Muramoto, I., Kawakami, T., Seike, M., Aimoto, S., Haneda, K. and Inazu, T. Tetrahedron Lett., 39 (1998) 55. 4. Inazu, T. and Kobayashi, K. Synlett, (1993) 869. 5. Kawakami, T., Kogure, S. and Aimoto, S. Bull. Chem. Soc. Jpn., 61 (1996) 3331.

206 Chemo-enzymatic synthesis of bioactive peptide derivatives containing N-linked oligosaccharides K. HANEDA 1 , T. INAZU1 , M. MIZUNO1 , R. IGUCHI1 , K. YAMAMOTO 2 , K. FUJIMORI 2 , Y. SHIMADA 2 and H. KUMAGAI2 1 2

The Noguchi Institute, Tokyo, Japan Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Introduction Recently, we reported the ‘chemo-enzymatic synthesis’ [1] of complex glycopeptide based on the chemical synthesis of glycopeptide containing N-acetylglucosamine (GlcNAc) attached to the asparagine (Asn) residue of the peptide [2] and the transglycosylation of oligosaccharide to this N -acetylglucosaminyl peptide catalyzed by endo- β -N-acetylglucosaminidase of Mucor hiemalis (Endo-M) [3]. Such complex glycopeptides as glycosylated Peptide T [4] or calcitonin [5, 6] derivatives, in which sialo complex-type asparagine (N)-linked oligosaccharides were attached to the Asn residue of the peptides, were synthesized by this method. Substance P is a neuropeptide consisting of eleven amino acids [H–Arg– Pro–Lys–Pro–Gln–Gln–Phe–Phe–Gly–Leu–Met–NH 2 ]. This peptide has no Asn residue but has two glutamine (Gln) residues at the position 5 and 6, to which biological addition of oligosaccharide is impossible. We tried to add N -linked oligosaccharides to the each Gln residue of this peptide by using our chemo-enzymatic method, in order to investigate the effect of sugar or sugar chains on the solubility, activity or biological stability of the peptide.

Results and discussion Solid-phase synthesis of glycosyl Substance P derivatives containing N-acetylglucosaminyl glutamine [Gln(GlcNAc)] residue. N α -9-Fluorenylmethyloxycarbonyl (Fmoc)-N γ -glycosylated glutamine derivative [Fmoc–Gln(GlcNAc)–OH] (1), which was an important intermediate for N -glycopeptide synthesis, was synthesized by the reaction of N -acetylglucosaminyl azide and Fmoc-glutamic acid α -ester in the presence of tributylphosphine [7]. Fmoc–Gln(GlcNAc)–OH became a good glycoside acceptor of transglycosylation reaction by Endo-M. Using Fmoc–Gln(GlcNAc)–OH instead of Fmoc–Gln–OH, the solid-phase synthesis of glycopeptide containing GlcNAc attached to the 5th or 6th Gln residue of Substance P, [Gln(GlcNAc)5 ]–SP (2) or [Gln(GlcNAc) 6 ]–SP (3), 610 Y. Shimonishi (ed): Peptide Science – Present and Future, 610–612 © 1999 Kluwer Academic Publishers, Printed in Great Britain

611 were performed by dimethylphosphinothioic mixed anhydride (Mpt-MA) method without protecting the hydroxyl functions of sugar moieties [2].

Scheme 1.

Enzymatic synthesis of Substance P derivatives containing N-linked oligosaccharides by transglycosylation reaction of Endo-M. N-linked oligosaccharides to The transglycosylation reactions of [Gln(GlcNAc) 5 ]–SP or [Gln(GlcNAc) 6 ]–SP as a glycoside acceptor were performed catalyzed by Endo-M in the reaction mixtures containing glycoside donor, glycoside acceptor and Endo-M [1]. Figure shows the synthetic procedure of the Substance P derivatives containing disialo biantennary complextype oligosaccharide attached to the Gln residue at the 5th position of the peptide (4) using [Gln(GlcNAc)5 ]–SP (1) as a glycoside acceptor. The transgly-

Scheme 2.

612 cosylation product (4) was detected by HPLC (ODS column) as a peak in front of the remaining acceptor [Gln(GlcNAc)5 ]–SP. Poor solubility of the acceptor substrates was a limiting factor of the transglycosylation reactions. [Gln(GlcNAc) 6 ]–SP also became a glycoside acceptor. Sialo or asialo complextype oligosaccharides derived from human transferrin or high-mannose type one of ovalbumin were transferred to the GlcNAc moiety of [Gln(GlcNAc) 5 ]–SP or [Gln(GlcNAc) 6 ]–SP catalyzed by Endo-M. The transglycosylation products were isolated by HPLC and identified by mass spectrometry. Synthesis of complex glycopeptide containing N -linked oligosaccharide attached to the Gln residue of the peptide is impossible biologically, but became possible by using the chemo-enzymatic method.

References 1 . Haneda, K., Inazu, T., Yamamoto, K., Kumagai, H., Nakahara, Y., and Kobata A., Carbohydr. Res., 292 (1996) 61. 2 . Inazu, T., Mizuno, M., Kohda, Y., Kobayashi, K., and Yaginuma, H., Peptide Chemistry 1995 (1996) 61. 3 . Yamamoto, K., Kadowaki, S., Watanabe, J., and Kumagai, H., Biochem. Biophys. Res. Commun., 203 (1994) 244. 4 . Yamamoto, K. et al., Carbohydr. Res., 305 (1998) 415. 5 . Mizuno, M. et al., Tetrahedron Lett., 39 ( 1998) 55. 6 . Haneda K. et al., Glycoconjugate J. 14 (1997) S113; Haneda, K., et al., Bioorg. Med. Chem. Lett., 8 (1998) 1303. 7 . Inazu, T. and Kobayashi, K., Synlett (1993) 869.

207 Optimization of N ,N'-dialkyldiamide-type phosphate protecting group for phosphopeptide synthesis M. UEKI, M. GOTO and J. OKUMURA Department of Applied Chemistry, Faculty of Science, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162 Japan

Introduction Amide-type protecting groups have recently been reported as phosphate protecting group in phosphopeptide synthesis by the Fmoc strategy. [1,2] In our communication [1] we showed preference of N , N '-dipropyl- and to N , N , N ', N '-tetramethylphosphoroN,N'-diisopropylphosphorodiamides diamide by Chao, et al. [2] However, at that time we could not observe any difference between N-propyl- and N-isopropylamides. In this study, we newly added N-isobutylamide as the third candidate and optimized the N-substituting alkyl groups.

Results and discussion Synthesis of building blocks were performed as sketched in the following scheme. Side-chain N ,N'-dialkylphosphorodiamidation of Z-Tyr-OBzl with (RNH) 2 P(O)Cl was possible using either lithium diisopropylamide (LDA) or a combination of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) and 4-dimethylaminopyridine (DMAP) as base. Catalytic hydrogenolysis of 1a–b followed by reaction with Fmoc-OSu under the usual conditions gave the corresponding Fmoc derivatives 2a–b as colorless crystals. However, in the case of N-propylamide scale-up was difficult because of presence of a closely resembling side-product. Structure elucidation of the isolated side-product revealed the reactivity of phosphorodiamidate nitrogen atom. Then, preparation of N,N'-diisobutyldiamide derivative 2c with β -branch was tried. As expected, pure material of 2c was easily obtained in high yield.

613 Y. Shimonishi (ed): Peptide Science – Present and Future, 613–614 © 1999 Kluwer Academic Publishers, Printed in Great Britain

614 All the Fmoc derivatives were quite stable and could be stored for more than a year at room temperature without change. Racemization of the tyrosine residue during N ,N'-dialkylphosphorodiamidation was not detected by the Marfey’s method. Cleavage potential of the three phosphate protecting groups were compared using the model dipeptides, Fmoc–Tyr[P(O)(NHR) 2 ]–Val-OMe (3a–c). N-propyl- (3a) and N -isobutylamides (3c) could be deprotected completely within 4h in 95% TFA solution. However, N-isopropylamide (3b) was cleaved rather slowly and took 12h for complete deprotection. Further, solid phase syntheses of a model peptide, H–Gly–Val–Tyr(P)–Ala–Ala–Ser-Gly–OH [3] were performed using 2a–c as building block. Again, incomplete deprotection of N -isopropylamide was observed. The most important feature of amide-type protection is the cleavage without generating any cationic species. Then, a methionine-containing peptide derived from the native platelet-derived growth factor-β receptor (PDGF- β ) sequence, H–Tyr(P)–Val–Pro–Met–Leu–OH [4], was synthesized using 2c on the Applied Biosystems 430A peptide synthesizer using the FastMoc cycle. The peptide was deprotected and released simultaneously by treating the resin with TFA/ H 2 O/EDT/PhSMe/PhOH (8.25 : 0.5 : 0.25 : 0.5 : 0.75 (v/v/v/v/w)) for 4 h at room temperature. The crude peptide was obtained in 67% (content 85%) and easily purified by preparative HPLC. In the case of phosphoserine, we also obtained N , N '-dipropyl-[5] and N,N'-diisobutyldiamide derivatives as colorless crystals. Whereas both compounds decomposed to some extent in organic solvents, which was much less for N-isobutyl-amide, they could successfully be utilized for solid phase synthesis of H–Arg–Ala–Ser(P)–Val–Ala–OH [6]. In conclusion we could establish N, N '-diisobutyldiamide as the optimized form of N ,N'-dialkyldiamide-type phosphate protecting groups of phosphotyrosine and phosphoserine.

References 1 . Ueki, M., Tachibana, J., Ishii, Y., Okumura, J. and Goto, M., Tetrahedron Lett., 37 (1996) 4953. 2 . Chao, H.-G., Leiting, B., Reiss, P.D., Burkhardt, A.L., Klimas, C.E., Bolen, J.B. and Matsueda, G.R., J. Org. Chem., 60 (1995) 7710. 3 . Valerio, R.M., Bray, A.M., Maeji, N-J., Morgan, P.O. and Perich, J.W., Lett. Pept. Sci., 2 (1995) 33. 4 . Ramalingam, K., Eaton, S.R., Cody, W.L., Loo, J.A. and Doherty, A.M., Lett. Pept. Sci., 1 (1994) 73. 5 . Ueki, M., Igarashi, M., Goto, M., Okumura, J., Saeki, A. and Ishii, Y., Proceedings of the 24th Symposium of the European Peptide Society, in press. 6 . Grehn, L., Fransson, B. and Ragnarsson, U. J. Chem. Soc. Perkin Trans. 1, 1 (1987) 529.

208 Solid phase synthesis of O-glycopeptide – Preparation o f O -(N-acetylgalactosaminyl)-threonine derivative and its application to peptide synthesis T. TESHIMA, T. URABE, T. YAMAMOTO, K.Y. KUMAGAYE, K. NAKAJIMA and T. SHIBA Peptide Institute, Inc., Protein Research Foundation, 4-1-2 Ina, Minoh, Osaka 562-8686, Japan

Introduction Glycopeptide is the partial structure of glycoprotein which shows important biological activity. In order to clarify the function of the carbohydrate, we need a facile procedure for synthesizing the glycopeptide. The linkage between carbohydrate and protein can be classified into two categories. One is the N-glycosidic bond linked to the Asn residue, and the other is the O -glycosidic bond to Ser or Thr residue. We have been able to successfully apply the Boc strategy to synthesize the N-linked glycopeptide with one N-acetylglucosamine unit. Moreover, as the O -glycoside bond of N -acetylgalactosamine (GalNAc) seems to be stable against HF treatment from the results of structural study of glycoprotein [1], we used one-step HF treatment for the final deprotection in the syntheses of supprescins, the O -glycopeptide containing Ser(GalNAc).

Results and discussion In this study, we tried to extend our solid phase synthetic strategy to the synthesis of the Thr(GalNAc)-containing peptide. We synthesized the Thr derivative with GalNAc whose hydroxy groups were protected by benzyl groups as shown in Figure 1. The glycosylation of Boc–Thr–OBzl(OMe) with the 1-trichloroacetimidate (1) or 1-fluoride (2 ) of azide sugar which was obtained from tri-O-benzyl galactal in three steps, gave the α -glycosyl Thr derivative (3). The azide group of compound 3 was converted to the acetylamino group by treatment with Ph3 P followed by acetylation. Boc and methoxybenzyl ester of 4 was cleaved by the treatment of TFA, and the free amino acid thus obtained was reacted with Boc2 O to give Boc–Thr(O-Bzl 3 –GalNAc)–OH (5 ) . To demonstrate the usefulness of our synthetic strategy, we chose the partial sequence of hirudin P6 as a model peptide to synthesize. Hirudin P6 is the glycoprotein composed of 63 amino acids which has thrombin inhibitory activity. All amino acids were introduced automatically except the glycosylated Thr derivative. After HF treatment, the desired glycopeptides were obtained and their structures were confirmed by NMR, MALDI-TOF mass, and amino 615 Y. Shimonishi (ed): Peptide Science – Present and Future, 615–617 © 1999 Kluwer Academic Publishers, Printed in Great Britain

616

Figure 1.

Figure 2.

Synthesis of Boc–Thr(O-Bzl 3 –GalNAc )–OH (5).

Seqences of model peptides synthesized in this study.

acid analyses. Hirudin P6 (45–52) and (46–52) were successfully synthesized by the solid phase peptide synthesis using the Boc strategy. From results obtained in our recent studies on solid phase synthesis of glycopeptide, we found that N-acetylgalactosaminylated O-glycopeptide as well as N -acetylglucosaminylated N-glycopeptide could be synthesized by the Boc strategy using one-step HF treatment for the final deprotection.

617 Acknowledgement The present work was supported in part by the New Energy and Industrial Technology Development Organization (NEDO).

Reference 1. Mort, A.J. and Lamport, D.T.A., Anal. Biochem., 82 (1977) 289.

209 Design of novel, nonpeptide oxytocin receptor antagonists R.M. FREIDINGER, M.G. BOCK, B.E. EVANS, D.J. PETTIBONE and P.D. WILLIAMS Departments of Medicinal Chemistry and New Lead Pharmcology, Merck Research Laboratories, West Point, Pennsylvania 19486, USA

Introduction There is currently a need for improved therapeutic agents for treating preterm labor [1,2]. In this regard, the hypothesis of oxytocin (OT) receptor blockade as a potentially more selective method of tocolysis has continued to gain support from results obtained in clinical studies with the peptide OT antagonist, atosiban [3]. Our laboratory has focussed on the identification of OT antagonists with properties suitable for both oral and intravenous administration. Results and discussion Initial efforts dealt with cyclic hexapeptides that were based on a bacterial fermentation product discovered by screening. A number of potent, water soluble, receptor-selective OT antagonists such as L-366,948 (Figure 1) were obtained from a medicinal chemistry program, but none had the desired property of oral bioavailability [4]. In parallel with the effort in the cyclic hexapeptide class, the screening effort identified L-342,643 as a weak active from the chemical collection at Merck

Figure 1.

Structures of oxytocin and a cyclic hexapeptide oxytocin antagonist.

618 Y. Shimonishi (ed): Peptide Science – Present and Future, 618–622 © 1999 Kluwer Academic Publishers, Printed in Great Britain

619 (Figure 2). The low molecular weight and relatively rigid structure of L-342,643 made it an attractive starting point for a medicinal chemistry effort. Chemical modifications identified camphor as an affinity-enhancing replacement for the tolyl ring, and additional modification of the camphor ring to include a carboxylic acid for water solubility produced L-366,509, the first orally active OT antagonist [4]. Additional systematic modifications in this structural class revealed that a substituent on the camphor ring at the C2-endo position containing a properly positioned hydrogen bond accepting group could improve OT receptor affinity to the low nanomolar range, and that an o-tolylpiperazine could function as an equipotent isostere for the spiroindenylpiperidine portion of the molecule. Potent OT antagonists such as L-368,899 resulted from this general design. L-368,899 in particular possessed a desirable profile including good potency as an antagonist of OT-induced uterine contractions in rats and late gestation pregnant rhesus monkeys, selectivity for binding to OT vs. AVP receptors in vitro and a good separation of OT and AVP antagonist activities in vivo in rats, oral bioavailability in several species, sufficient aqueous solubility for intravenous formulation, and an excellent overall preclinical toxicology profile. In healthy human volunteers, L-368,899 proved to be generally well-tolerated given orally and intravenously [5] and was orally bioavailable (F = 20%). L-368,899 was shown to block the uterine response to exogenous OT in women in the immediate postpartum period [6], thus establishing that the compound was indeed an OT antagonist in humans. The development of L-368,899 was discontinued, however, because of the high oral dose, 600 mg at 6 hour intervals, required to maintain trough plasma levels associated with complete block of the uterine response to OT as projected from the study in postpartum women.

Figure 2.

Progression from screening lead to development compound

620 First pass metabolism involving hydroxylation at the 5-exo position and on the C9 methyl group in L-368,899 was found to be a limiting factor for obtaining good plasma levels after oral dosing in rhesus monkeys, and incubation of L-368,899 with human liver microsomes produced hydroxylated camphor derivatives as the major metabolites. Several chemical approaches involving modification of the camphor portion of the structure in an attempt to improve overall metabolic stability were investigated. Good receptor affinity was obtained with some of these new designs, but the half-lives of such compounds in in vitro metabolism experiments and in vivo in rats were not improved over those of L-368,899. An alternative lead completely lacking the camphor moiety was discovered during evaluation of Otsuka’s orally bioavailable AVP-V1a antagonist OPC 21268 [7] in radioligand receptor binding assays at Merck. OPC 21268 was found to possess modest human OT receptor affinity, and because of a significant species difference between human and rat AVP-V1a receptors, the compound was found to be a selective human OT receptor ligand (Table 1). Systematic modification of OPC 21268 produced affinity-enhancing changes which included replacing the dihydroquinolinone ring with a benzoxazinone Table 1 Oxytocin Antagonists based on OPC 21268

K i values refer to displacement of ³H-OT (OT column) or ³H-AVP (AVP-V1a and AVP-V2 columns) from specific binding sites in the indicated tissues. AD 5 0 values refer to the intravenous (iv) or intraduodenal (id) dose required to inhibit oxytocin-induced uterine contractions by 50% in the rat.

621 ring, inclusion of an ortho-methoxy group on the benzoyl ring, and constraining the acetamidopropoxy group in the form of an N -acetyl-4-piperidine ring. Combining these modifications produced L-371,257 [4] which exhibited nanomolar level OT receptor affinity and good human OT receptor selectivity. L-371,257 proved to be a potent OT antagonist in rats and was orally bioavailable in rats (F = 39%) and rhesus monkeys (F = 13%). Solubility-enhancing modifications were then sought in order to facilitate formulation for intravenous use. Systematic structure-activity studies indicated that structural changes on the N -benzoyl-4-(benzoxazinonyl) piperidine portion of the molecule were generally deleterious to OT receptor affinity, whereas the piperidinyl ether terminus of the molecule was more tolerant to change. Removing the acetyl group in L-371,257 to reveal a basic piperidine nitrogen improved aqueous solubility but reduced OT receptor affinity. Benzylation of the piperidine nitrogen, a modification which reduces basicity and adds lipohilicity, improved receptor affinity. Affinity was further improved by incorporating a more electron-deficient pyridine ring (L-371,377). L-371,377 had good bioavailability in rats as evidenced by the favorable id/iv ratio of AD50 values in the functional assay. Methylation of the pyridine ring (L-372,237) improved in vivo potency and duration of OT antagonist activity in rats vs. L-371,377. Plasma levels of L-372,237 in rats after oral or iv administration, however, were surprisingly low and at the later time points the major species found in the circulation were two metabolites: the N -dealkylation product L-371,209 and the pyridine N-oxide L-372,662. L-372,662 was independently synthesized and was found to have a very attractive profile. L-372,662 competitively inhibited OT-induced contractions of the rat uterus in vitro (pA2 = 7.9) and was a potent OT antagonist in vivo in rats with a good duration of action. In addition, L-372,662 exhibited high affinity for human OT receptors (Ki = 4.9 nM), was a potent OT antagonist in late gestation pregnant rhesus monkeys (AD50 = 30 µg/kg iv), had excellent oral bioavailability in several species (F = 90% dog; 80% rat; 36% rhesus monkey) with an iv half-life of 2 hours, and possessed good aqueous solubility (10 mg/mL at pH 5) to allow for iv formulation. The results indicate that L-372,662 offers advantages over L-368,899 and possesses an overall profile which makes it an attractive compound for further investigation.

References 1. Fuchs, A.R., Fuchs, F., Stubblefield, P.G., (Eds.) Preterm Birth. Causes, Prevention, and Management, second edition., McGraw-Hill, New York, 1993 2. Canadian Preterm Labor Investigators Group, N. Engl. J. Med. 327 (1992) 308. 3. Goodwin, T.M., Valenzuela, G.J., Silver, H., Creasy, G., Obstet. Gynecol. 88 (1996) 331. 4. Freidinger R.M., Pettibone D.J., Med. Res. Rev. 17 (1997) 1. 5. Hatangandi, S., Grasing K., Murphy, M.G., Siebold, J.R., Barrett, J.S., Chavez, C., Constanzer, M., Vigna T, Baglivo, P., Capra, N., Gonyo, G., Knuppel, R.A., Society for Gynecologic Investigation Annual Meeting 1994 p.271.

622 6. Egerman, R., Murphy, M.G., Dougherty, E., Capra, N., Staub, T., Winchell, G., Constanzer, M.L., Chavez, C., Meyer, N., Cotroneo, M., Caritis, S., Sibai, B., Am. J. Obstet. Gynecol. 172 (1995) 414. 7. Ohnishi, A., Ko, Y., Fujihara, H., Miyamoto, G., Okada, K., Odomi, M., J. Clin. Pharm. 33 (1993) 230.

210 Development of potent non-peptide GPIIb/IIIa antagonists with tri-substituted β -amino acid derivatives as a new conformational restriction unit Y. HAYASHI, J. KATADA, T. HARADA, K. IIJIMA, A. TACHIKI, Y.TAKIGUCHI, M. MURAMATSU, H. MIYAZAKI, T. ASARI, T. OKAZAKI, Y. SATO, E. YASUDA, M. YANO, I. OJIMA¹ and I. UNO Life Science Research Center, Advanced Technology Research Laboratories, Nippon Steel Corporation, 3–35–1 Ida, Nakahara-ku, Kawasaki 211-0035, Japan ¹Department of Chemistry, State University of New York at Stony Brook, NY 11794–3400, USA

Introduction Undesired platelet aggregation and subsequent thrombosis are suspected to play an important role in various vasoocclusive diseases such as unstable angina and stroke [1]. The effective drugs to prevent such irregular platelet aggregation are in serious demand. Fibrinogen receptor, GPIIb/IIIa, has been one of the major targets for antagonist development as a new promising class of such anti-thrombotic agents, because fibrinogen binding to GPIIb/IIIa is thought to be a final common pathway of platelet aggregation through crosslinking of adjacent platelets [2]. In this binding process, it is known that the RGD sequence(s) in fibrinogen is responsible for the recognition of GPIIb/IIIa and the guanidino group of the Arg residue and the β -carboxylic acid of the Asp residue are the essential functionality in this recognition [3]. Here, we report the development of new orally active non-peptide GPIIb/IIIa antagonists.

Results and discussion 1. Lead compound generation (a combinatorial chemical approach) It has been suggested from many conformation-activity relationship studies of small RGD peptides that the spatial distance between the guanidino group and the β -carboxylic acid in the RGD sequence is a very important factor and it should be within the distance of 13~16Å to show potent GPIIb/IIIa antagonist activity [4]. Therefore, if the Gly residue functions only as a linker in the RGD sequence, we can design antagonistic non-peptide molecules only by arranging appropriate basic and acidic moiety so that the distance is within 13~16Å and by fixing the molecule to the proper conformation. Combinatorial strategy would be very helpful for such molecular design. Here, 623 Y. Shimonishi (ed): Peptide Science – Present and Future, 623–627 © 1999 Kluwer Academic Publishers, Printed in Great Britain

624

Figure 1.

Three component combinatorial strategy and discovery of NSL-95301.

we adopted three component combinatorial strategy, in which molecules were constructed from basic unit, spacer and acidic unit. Especially, to adjust the distance between both units and to reduce the conformational flexibility, the ring structure was employed in both basic unit (benzoic acid derivatives) and acidic unit (piperidine derivatives) (Figure 1). As the result of inhibitory assay for collagen-induced human platelet aggregation, the combination of 4-amidinobenzoic acid, 3-aminopropionic acid, and piperidine-4-acetic acid, i.e., [N1-S3-C2], showed the highest inhibitory activity. Thus, this was chosen as the compound for further modification regarding the spacer unit with various kinds of α and/or β substituted 3-aminopropionic acid derivatives. From this modification, NSL-95301(1), whose spacer unit is 3-amino-3-phenyl-2,2-dimethyl-propionic acid (Figure 1.), showed the potent inhibitory activity. The resolution of racemic NSL-95301 by the chiral HPLC revealed that (+)-NSL-95301 was an active enantiomer with an IC 50 value of 92 nM, which is 300 times more potent than that of (–)-enantiomer. This study is a very simple, but successful example of combinatorial chemical approach, and suggests that, when the appropriate information for the active conformation of the molecule was obtained, a lead compound with potent activity can be efficiently discovered even from a small size of combinatorial compound library with limited diversity, although the high diversity and the big number of compounds in combinatorial library are important factors in general. 2. Molecular modeling study To understand the mechanism under the potent activity of NSL-95301 (1), we carried out conformational analysis of 1 and its related less active des-phenyl analog at the β -position in the central β -amino acid derivative. From the superimposition of conformers whose steric energy was less than 10 KJ in the 10,000 Monte Carlo conformational search on Macromodel ver 5.5d, it is revealed that NSL-95301 exhibited a rigid cup-shaped conformation in spite of its linear structure as compared with des-phenyl analog (Figure 2.). This

625

Figure 2.

Superimposition studies of the low energy conformations.

cup-shaped conformation with considerable rigidity can be ascribed to restricted movements of the backbone arised from the steric repulsion among the phenyl group at the β -position, the gem-dimethyl group at the α -position and the piperidine ring. These results suggest that the substituent at the β position will considerably affect the molecular conformation and the molecular flexibility, and the potent activity of 1 is ascribed to the fixation of the molecule to its bioactive conformation preferable for the binding to GPIIb/IIIa. 3. Lead compound optimization for higher activity On the basis of above findings, we have synthesized a series of NSL-95301 analogs, in which the β -substituent of the tri-substituted β -amino acid residue is varied. This SAR study at the β -position indicates that the replacement of the β -phenyl group of NSL-95301 with an relatively small alkyl substituent retains or improves inhibitory activity for platelet aggregation, and the bulkiness of the alkyl β -substituents has rather small (less than a factor of two) effects on the biological activity. Next, we carried out further optimization of β -ethyl analog 2 (IC 50 84 nM) in order to improve anti-platelet activity and pharmacokinetic properties, since the plasma half-life of this analog (T 1/2 β 78 min) was slightly longer than that of others with comparable in vitro potency. As a result, the introduction of a fluorine atom at the m -position of the amidinobenzoyl group (3, NSL-96173) not only increases the activity (IC50 62 nM), but also prolongs the plasma half-life (T1/2 β 89 min). However, 3 showed only a limited bioavailability after p.o. administration in the guinea pig. 4. Lead compound optimization for oral administration To improve oral bioavailability of 3 , we modified both the terminal amidino and carboxyl groups in an effort to increase hydrophobicity. However, the C-terminus esterification as prodrugs brought only slight improvement in oral

626

Figure 3.

Lead compound optimization.

bioavailability regardless of the nature of the ester. Since mono-alkylation of the amidino group significantly decreased the anti-platelet activity, the effects of di-alkylation were also investigated by modifying the ethyl ester of 3. As the result, the active form, i.e., free C-terminal, of NSL-96184 (4) with a thiazolidine group at one of the amidino nitrogens not only exhibited higher activity (IC 50 43 nM) than that of 3 (IC 50 62 nM), but also 4 possessed a high C max value (1.2 µg/ml) in rats after oral administration (10 mg/kg). To evaluate pharmacological and pharmacodynamic properties of 4, ex vivo platelet aggregation was examined by the oral administration of 4 in guinea pigs. The 10 mg/kg oral administration of 4 results in the complete inhibition of ex vivo platelet aggregation over 3 h. It is noteworthy that the onset of the anti-platelet activity was very rapid and almost the maximum inhibition was observed even at the 30 min period after the administration, the earliest time point of the experiment. Since SC-54684A was reported to have an oral bioavailability of more than 20% in dogs [5], we used SC-54684A as a reference compound for evaluation of 4. Racemic SC-54684A, when administered orally at the same dose as that used for 4 , showed less than 70% inhibition for ex vivo platelet aggregation in guinea pigs at its maximum. The synthesis of individual enantiomers of the racemic 4 was performed with (R)- or (S)-3-(N-Boc)amino-2,2-dimethyl-pentanoic acid prepared by the chromatographical separation of those diastereomeric (R)-1-(1-naphthyl)ethylamine adducts. Then, it has been found that only (R)-4 is a highly potent inhibitor (IC 50 22 nM; (S)-4 : 3100 nM). In conclusion, the active form of (R)-4 is a highly potent antagonist of the platelet fibrinogen receptor, inhibiting platelet aggregation in vitro effectively. This compound is characterized by the presence of the tri-substituted β -amino acid residue, 3-ethyl-2,2-dimethyl-β -alanine, which is responsible for fixing the molecule to its active conformation. In vivo assay results show that 4 has a good oral bioavailability in the guinea pig. The onset of anti-platelet action is

627 very fast, whereas its duration of action is relatively short. These results suggest that 4 (NSL-96184) may have an excellent therapeutic potential, especially for anti-thrombotic treatment in the acute phase.

References 1. 2. 3. 4.

Falk, E., Circulation, 71 (1985) 699. Phillips, D.R., Charo, I.F., Parise, L.V. and Fitzgerald, L.A., Blood, 71 (1988) 831. Ruoslahti, E. and Pierschbacher, M.D., Science, 238 (1987) 491. Katada, J., Hayashi, Y., Sato, Y., Muramatsu, M., Takiguchi, Y.; Harada, T., Fujiyoshi, T. and Uno, I., J. Biol. Chem. 272 (1997) 7720. 5. Nicholson, N.S., Panzer-Knodle, S.G., Salyers, A.K., Taite, B.B., Szalony, J.A., Haas, N.F., King, L.W., Zablocki, J.A., Keller, B.T., Broschat, K., Engleman, V.W., Herin, M., Jacqmin, P., Feigen, L.P., Circulation 91 (1995) 403.

211 Design and synthesis of potent and selective inhibitors of the osteoclast-specific cysteine protease, cathepsin K D.F. VEBER, R.W. MARQUIS, Y. RU, D.S. YAMASHITA, H.-J. OH, S.K. THOMPSON, S.M. HALBERT, T.J. CARR, J.G. GLEASON, T.A. TOMASZEK, JR. M.A. LEVY, M. BOSSARD, D.G. TEW, I.E. JAMES, J. BRIAND, S.A. CARR, D. ZYMBRYKI, L. LEE-RYKACZEWSKI, R.L. DESJARLAIS, M.S. McQUENEY, K.J. D’ALESSIO, B. ZHAO, C.A. JANSON, S.S. ABDEL-MEGUID and W.W. SMITH SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406

Introduction Cathepsin K, a cysteine protease of the papain superfamily, has been recognized as being an important contributor to bone resorption mediated by osteoclasts. As such, inhibitors of this enzyme offer therapeutic potential for osteoporosis [1]. We have reported new classes of cysteine protease inhibitors which span the active site of cathepsin K [2, 3]. X-ray crystallographic analyses have confirmed that these peptidomimetic ketones and hydrazides bind to regions of both prime and unprime sides of the active site. They also appear to form a covalent attachment to the central, active site cysteine within the resolution of the X-ray structure. The bis(acylamino)-2-propanone, I, forms a reversible complex and shows competitive kinetics. The diacylcarbohydrazide, II, shows a single turnover on analysis of the reaction in solution and behaves as an effectively irreversible inhibitor of the enzyme, releasing one equivalent of Cbz-leucyl hydrazide [3]. Key binding to cathepsin K is seen to derive from the Cbz-leucyl groups of all three inhibitors, I, II and III. These groups may have metabolic and pharmacokinetic deficits which we have sought to overcome by systematic introduction of further peptidomimetic elements.

Results and discussion The thiazole of III is a peptidomimetic replacement of one of the acyl hydrazide groupings of II and has been shown by X-ray crystallography to bind on the prime side of the active site. It is also seen to bind with apparent carbonyl addition to the active site cysteine. Thiazole III differs from II in the binding kinetics to cathepsin K, with II being an effectively irreversible inhibitor. III 628 Y. Shimonishi (ed): Peptide Science – Present and Future, 628–630 © 1999 Kluwer Academic Publishers, Printed in Great Britain

629 Table 1.

Peptide mimetic elements in cathepsin K inhibitors.

* Inhibition of human recombinant cathepsin K as determined by the methods described in [5]. **This reversible binding constant was determined by extrapolation of the time dependent binding curves seen with this apparently single turnover inhibitor [3].

in contrast, shows apparently reversible kinetics but also shows turnover in assays at high (µM) enzyme concentrations. The exact nature of the mechanisms of inhibition is a matter of continuing study. One successful design of a peptidomimetic element for the prime side Cbzleucyl is the phenoxy-phenylsulfonamide group of compound IV which is nearly

630 10 fold more potent than I. X-ray crystallographic studies show that a related compounds having a 2-pyridyl sulfonamide or 4-pyridyl carbonyl in place of the Cbz group bind with the pyridyl groups on the prime side of the active site [2]. Cyclization of the ketone portion of I from the unprime side nitrogen to the prime side ketone C-α results in the highly potent pyrrolidone derivatives V and VI. V retains both amide groups of I but the prime-side amide is now tertiary, a feature which has been associated with improved intestinal transport because of reduced hydrogen bonding [4]. This tertiary amide is replaced by a so-called reduced amide group in VI. This tertiary amino group is also advantageous because it improves solubility in aqueous medium. Analysis of the enzyme-bound forms of V and VI by X-ray crystallography reveals that the tertiary nitrogen is bound on the unprime side of the active site. These studies give examples wherein all but one of the N–H groups of I and II can be eliminated individually while retaining high potency for inhibition of cathepsin K. Such studies are a critical step in the design of inhibitors with therapeutic potential.

References 1. Veber, D.F., Drake, F.H., and Gowen, M., Current Opinion in Chemical Biology, 1 (1997) 151–156. 2. Yamashita, D.S., Smith, W.W., Zhao, B., Janson, C.A., Tomaszek, T.A., Bossard, M.J., Levy, M.A., Oh, H.-J., Carr, T.J., Thompson, S.K., Ijames, C.F., Carr, S.A., McQueney, M., D’Alessio, K.J., Amegadzie, B.Y., Hanning, C.R., Abdel-Meguid, S., DesJarlais, R.L., Gleason, J.G., and Veber, D.F., J. Am. Chem. Soc., 119 (1997) 11351–11352. 3. Thompson, S.K., Halbert, S.M., Bossard, M.J., Tomaszek, T.A., Levy, M.A., Zhao, B., Smith, W.W., Abdel-Meguid, S.S., Janson, C.A., D’Alessio, K.J., McQueney, M.S., Amegadzie, B.Y., Hanning, C.R., DesJarlais, R.L., Briand, J., Sarkar, S.K., Huddleston, M.J., Ijames, C.F., Carr, S.A., Garnes, K.T., Shu, A., Heys, J.R., Bradbeer, J., Zembryki, D., Lee-Rykaczewski, L., James, I.E., Lark, M.W., Drake. F.H., Gowen, M., Gleason, J.G., and Veber, D.F., Proc. Natl. Acad. Sci. USA, 94 (1997) 14249–14254. 4. Conradi, R.A., Hilgers, A.R., Ho, N.F.H., and Burton, P.S., Pharmaceutical Res., 8 (1991) 1453–1460. 5. Votta, B.J., Levy, M.A., Badger, A., Bradbeer, J., Dodds, R.A., James, I.E., Thompson, S.K., Bossard, M.J., Carr, T., Connor, J.R., Tomaszek, T.A., Szewczuk, L., Drake, F.H., Veber, D.F., and Gowen, M., J. Bone and Min. Res. 12 (1997) 1396–1406.

212 Difference of structural requisite in design of inhibitor of cathepsin B and papain: X-ray crystal structures of complexes T. ISHIDA1 , A. YAMAMOTO 1 , K. TOMOO1, K. MATSUMOTO2, M. MURATA2 , K. KITAMURA2 ¹ Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences 4-20-1 Nasahara, Takatsuki, Osaka 569-11, Japan ² Research Center, Taisho Pharmaceutical Co. Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama 330, Japan

Introduction Lysosomal cysteine proteases are abundant in living cells and play important roles in intracellular proteolysis. Since the imbalance of their enzymatic activities could cause serious diseases, the development of low-molecular inhibitors that can moderately control the activity has been desired as useful therapeutic drugs. Because cathepsin B (CB) being a major component in lysosomal enzymes, previously, we designed CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline] as its potent and specific inhibitor based on the crystal structure of papain: E-64-c (a potent covalent-type inhibitor for general cysteine protease) complex [1]. In order to investigate the inhibitory mechanism of CA074 against CB at atomic level and to make clear the structural requirement between CB and papain in design of inhibitors specific for these enzymes, we determined the structures of bovine spleen CB:CA074 and papain : 1 (N-(L-3-carboxyloxirane-2-carbonyl)-L-isoleucyl-Lproline) complexes by X-ray analyses; a preliminary result for CB:CA074 complex has been reported [2]. Results and discussion The Ile-Pro sequence of CA074, which is necessary for selective inhibition of CB, was located at the Sn' (n = 3 and 2, respectively) subsites, while that of 1

Scheme 1.

631 Y. Shimonishi (ed): Peptide Science – Present and Future, 631–633 © 1999 Kluwer Academic Publishers, Printed in Great Britain

632 is positioned to the Sn (n = 2 and 3, respectively) subsites of papain (Figure 1). This is obviously due to the difference of functionality between the residues constructing respective binding pockets. As a main reason why the Ile-Pro sequence of CA074 prefers to binding to the S' subsites of CB, though CB has sufficient space to accommodate this moiety at S subsites, it could be proposed the just fit formation of double hydrogen bonds between the carboxyl oxygens of the Pro C-terminal and the imidazole nitrogens of His-110 and His-111 residues; these are missing in papain and other cysteine proteases. The propylcarbamoyl moiety of CA074 was located at the S1 and S2 sites, where the carbonyl O is hydrogen-bonded to Gly-74 NH, and the amide NH is in the range of hydrogen bond with Gly-198 O [ = 4.02 Å]. On the other hand, the Ile-Pro moiety of 1 was located at the hydrophobic S2 and S3 subsites of papain, respectively. Since similar binding has been observed for E-64-c in the complex with papain, its inhibitory activity could be closely related to the strength of S2–P2 and S3–P3 interactions. Since both of papain and CB belong to the cysteine protease family, the comparison between the binding modes of 1 and CA074 will make clear the different structural requisites that are important for effective design of (noncovalent) inhibitors specific for these enzymes. The X-ray results indicated that the Sn' (n = 1,2) subsites (Ser-105–His-111 of the occluding loop, Val-176, Leu-181, Met-196 and Trp-221) characterize the active site of CB, where the space is compact, as compared with that of papain Sn' (n = 1, 2) subsites constituted by Gln-19, Ala-137, His-159, and Trp-177. On the other hand, the structural feature of papain binding pocket is characterized at Sn (n = 2 and 3) subsites. The S2 subsite (Pro-68, Val-133, Val-157, Asp-158, Ala-160 and Ser-205) forms a compact and narrow cleft, compared with that of CB (Pro-76, Ala-173, Met-196, Ala-200 and Glu-245), and the S3 subsite (Tyr-61, Gly-66, and Tyr-67) provides an important space for the hydrophobic interaction with

Figure 1.

(A) CA074 : CB complex. (B) 1 : papain complex.

633 P3 substituent, while the S3 subsite of CB is not so strict enough to impose any specificity for the substrate. The present studies provide useful information on clarifying (i) the outline of Sn' subsites in papain, which has not so far clearly been demonstrated, and (ii) the difference of functionality between the binding pockets of papain and CB.

References 1. Sumiya, S., Yoneda, T., Kitamura, K., Murata, M., Yokoo, C., Tamai, M., Yamamoto, A., Inoue, M., and Ishida, T., Chem. Pharm. Bull., 40 (1992) 299. 2. Yamamoto, A., Hara, T., Tomoo, K., Ishida, T., Fujii, T., Hata, Y., Murata, M., and Kitamura, K., J. Biochem., 121(1997) 974.

213 Synthesis of peptides with aziridine-2,3-dicarboxylic acid as an α -amino acid equivalent building block Tanja Schirmeister Department of Pharmaceutical Chemistry, University of Freiburg, Hermann-Herder-Str. 9, D-79104 Freiburg, Germany

Introduction The reactivity of aziridines is characterised by their susceptibility to nucleophilic ring opening [1]. For this reason aziridine-2,3-dicarboxylic acid is a useful building block for peptide inhibitors of cysteine proteases the inactivation of which is caused by alkylation of the active site’s cysteine residue [2]. Different methods of peptide synthesis have been evaluated in order to synthesise new cysteine protease inhibitors [3] with peptide structures, containing (R,R)- and (S,S)-aziridine-2,3-dicarboxylic acid, respectively, as electrophilic component. Special emphasis has been placed on the following issues: 1 . Peptide coupling at one of the carboxylate functions of the aziridine. 2. N-Acylation with amino acids and dipeptides, respectively. 3 . Removal of N- and C-protecting groups, respectively. 4. Synthesis of peptides with one free carboxylate function at the N-acylated aziridine ring.

Results and discussion 1. Peptide coupling at one of the carboxylate functions of the aziridine can be carried out by the DPPA method (7a,b; 9a,b). Protection of the aziridine nitrogen is not necessary. Stepwise or fragment condensations are possible (Figure 1). 2. The method of choice for N-acylation with amino acids is the symmetric anhydride method (3a,b). N-acylations with dipeptides are possible via mixed anhydrides with isobutyl chloroformiate (5a,b; 6b) (Figure 1). 3. Removal of the BOC group can be carried out with TFA in dichloromethane (4b). Attemps to cleave this group with HCl in ethyl acetate led to polymerisation. The carboxyl protecting benzyl ester can be removed by catalytic hydrogenolysis over Pd-C (8a) (Figure 1). 4 . Since ester hydrolysis of N-acylated aziridinyl diethyl esters (e.g. 3b) by bases or hydrolases leads to preferred amide hydrolysis due to the lability of the aziridide structure, benzyl ethyl aziridine-2,3-dicarboxylate (rac. 10) [5] was 634 Y. Shimonishi (ed): Peptide Science – Present and Future, 634–636 © 1999 Kluwer Academic Publishers, Printed in Great Britain

635

Figure 1. i. LiOH, EtOH, DOWEX, 50%; ii. DCC, BOC-L-Phe, DMAP, 90%; iii. TFA/CH2 Cl 2 1/1, quant.; iv. iButOCOCl, NMM, DMF, Z-L-Ala-L-Phe (5) or Z-Gly-Gly (6), 70%; v. DPPA, TEA, L-Leu-Bzl Tos, DMF, 60%; vi. Pd-C/H 2 , MeOH, atm. press., quant.; vii. DPPA, TEA, L-Leu-L-Pro-Bzl HCl, DMF, 60%; viii. DPPA, TEA, L-Pro-Bzl HCl, DMF, 60%; compds. a are R,R configured, compds. b are S,S configured at the aziridine ring.

Figure 2. i. DCC, BOC-L-Phe, DMAP, 86%; ii. Pd-C/H 2 , MeOH, atm. press., 95%; compds. 11 and 12, respectively, are diastereomeric mixtures with R,R and S,S configured aziridines.

chosen as aziridine building block. The N-acylation of this starting material is successful with symmetric anhydrides (11a + b ). Subsequent catalytic hydrogenolysis leads to the N-acylated aziridine with one free carboxylic acid function (12a + b) (Figure 2).

Acknowledgements Financial support by the Deutschen Forschungsgemeinschaft DFG, Germany, and the Faculty of Chemistry and Pharmacy, University of Freiburg, is gratefully acknowledged.

636 References 1. Hata, Y. and Watanabe, M., Tetrahedron, 43 (1987) 3881. 2. Schirmeister, T., Arch. Pharm. Pharm. Med. Chem., 329 (1996) 239. 3. All synthesised aziridinyl peptides are selective and irreversible inhibitors of cysteine proteases. Serine, aspartate and metallo proteases are not inactivated. 4. Synthesis in 5 steps from D- or L-diethyl tartrate as described in ref. [2]. 5. Synthesis via Michael addition of diphenylsulfimide to benzyl ethyl fumarate.

214 Isolation and characterization of prolyl endopeptidaseinhibiting peptides from bovine brain S. MARUYAMA¹ and T. OHMORI² ¹ National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan ² Research and Development Center, Nippon Meat Backers, Inc., Midorigahara, Tsukuba, Ibaraki 300-26, Japan

Introduction Prolyl endopeptidase [EC 3.4.21.26] (PEP), which specifically cleaves the peptide bond at the carboxyl side of proline residues of several biologically active peptides, is widely distributed in various organs, particularly in the brain. Endogenous inhibitors of PEP have been detected in porcine pancreas (Mr = 6,500, Yoshimoto et al., 1982), rat brain (Mr = 7,000, Soeda et al., 1985) and sperm of ascidian (Mr = 8,000, Yokosawa et al., 1983), but their structures have not been clarified. We previously isolated some PEP-inhibiting peptides from bovine brain [1]. The amino acid sequence of the peptide having the highest inhibitory activity was MPPPLPARVDFSLAGALN (corresponding to the sequence 38 to 55 of glial fibrillary acidic protein, GFAP(38–55)). In the present study, the brain PEP inhibitory activity of synthetic peptides related to GFAP(38–55) and other proline-rich peptides was examined.

Results and discussion GFAP(38–55) showed a K i of 8.6 µM for bovine brain PEP (Table 1). The value is essentially the same as that of the inhibitor purified from rat brain Table 1. Peptide

Ki (µM)

MPPPLPARVDFSLAGALN (Bovine GFAP(38–55)) MPPPLPTRVDFSLAGALN (Human) MTPPLPARVDFSLAGALN (Mouse) MPPPLPA MPPPLP MPPPL PPAYPPPPVP (3 BP2–10)a EPPPPEPPPI b

8.6 4.6 8.3 73 8.8 NI 49 1400

Enzyme, PEP purified from bovine brain, Substrate, N -benzyloxycarbonyl-glycyl- L -proline p nitroanilide; NI, no inhibition. a SH3-binding peptide, b prolinr-rich peptide isolated from bovine brain

637 Y. Shimonishi (ed): Peptide Science – Present and Future, 637–638 © 1999 Kluwer Academic Publishers, Printed in Great Britain

638 (K i = 2.6 µM, Soeda et al. 1985). Homologous peptides of human type and mouse type also inhibited brain PEP. Among various synthetic fragments of G F A P ( 3 8 – 5 5 ) , o n l y M P P P L P h a d a K i nearly the same as that of GFAP(38–55). This N-terminal peptide MPPPLP and C-terminal ARVDFSLAGALN were gradually released from GFAP(38–55) during prolonged incubation with brain PEP, and the released peptide MPPPLP was not hydrolyzed further with brain PEP. On the other hand, MPPPLP was rapidly cleaved to MPPP and LP by PEP produced by Flavobacterium meningosepticum. MPPPLP was thus shown not to be an inhibitor of bacterial PEP. To examine whether other proline-rich sequences in mammalian protein inhibit PEP, various proline-rich peptides, the sequences of which resemble the N-terminal sequence of GFAP(38–55), were synthesized. Of the peptides, a SH3-binding peptide (3BP2–10) was the most inhibitory, though the extent was only 1/6 that of GFAP(38–55). Thus, GFAP(38–55) and the cleaved short peptide MPPPLP may function as PEP inhibitors in the brain. The effects of these peptides on mouse behavior are being investigated. Although PEP-inhibiting activity was rather low, a new proline-rich peptide EPPPPEPPPI was isolated from the hot water extract of bovine brain (Table 1). In connection with proline-rich peptides, we searched for a peptidase capable of hydrolyzing oligo-proline. Because, oligo-proline-hydrolyzing activity in tissues of mammals has not been reported except for aminopeptidase P that hydrolyzes oligo-proline weakly. Then, a 140 kDa protein, which hydrolyzes oligo-prolines such as Pro-Pro-Pro-Pro and Pro-Pro-Pro, was purified from the homogenate of bovine brain. However, the properties of the enzyme were found to resemble those of aminopeptidase P.

Reference 1. Ohmori, T., Nakagami, T., Tanaka, H. and Maruyama, S., Biochem. Biophys. Res. Commun., 202 (1994) 809.

215 Structure based design and synthesis of novel orally active thrombin inhibitors M. HARAMURA¹, T. SHIRAISHI¹, T. HANEISHI¹ K. KUROMARU¹, M. OHTA¹, J.-M. KIM² W.H. NAM², K.Y. JUNG², G.-H. JEON² and T. KOZONO¹ ¹ Chemistry Research Lab., Chugai Pharmaceutical Co. Ltd., 1-135 Komakado Gotemba Shizuoka 412 Japan ² C&C Research Laboratories, 146-141 Annyung-ri Taean-ub Hwasung-goon Kyunggi-do 445-970 Republic of Korea

Introduction Thrombin is a serine protease which plays an important role in blood coagulation cascade. It enzymatically converts fibrinogen to fibrin, which subsequently polymerizes to form a matrix for clot formation. Platelet activation by thrombin leads to shape change, platelet aggregation and release of secretory granules [1]. In active site of thrombin, small molecule thrombin inhibitors occupy the three active pockets of thrombin. The primary specificity site S1 has Asp residue that is positioned at the bottom of the pocket to make a salt bridge with basic P1 group of inhibitor. The thrombin specific hydrophobic pocket P is ideally suited to the binding of medium-size nonpolar groups. The hydrophobic pocket D is preferentially adept to aromatic groups that contribute considerably to the binding affinity of inhibitors [2]. A number of small molecule thrombin inhibitors are in clinical use as potential antithrombic agents, however they are limited to parenteral administration. Argatroban is one of the most selective thrombin inhibitors known, and is marketed in Japan as an intravenous agent. Argatroban is an effective anticoagulant, however, it is parenteral agent that is currently only suitable in acute care [3]. For longer term anticoagulation, an orally bioavailable thrombin inhibitor is required. Therefore, we aimed to design and develop potent and orally active thrombin inhibitors utilizing Structure Based Drug Design (SBDD) technique. Limitation on the oral bioavailability, especially oral absorption, is posed by the presence of a highly basic guanidino group, or by electrophilic functional groups in most small molecule thrombin inhibitors. Factors influencing solute absorption were pointed by Hilingers et.al. [4]. For the small molecule thrombin inhibitors, size and conformation are not permitted to change drastically because of decrease of thrombin inhibition activity. Charge and lipophilicity are key issue to improve oral absorption of the small molecule thrombin inhibitors. 639 Y. Shimonishi (ed): Peptide Science – Present and Future, 639–641 © 1999 Kluwer Academic Publishers, Printed in Great Britain

640

Figure 1. Table 1.

Structure of designed compounds. Thrombin inhibition activity of designed compounds.

Compound

R

IC 50 (nM)

1 5 7 9 10

Benzyl 1-Naphthylmethyl 3-Bromo1naphthylmethyl 3,3-Biphenyl-n-propyl 2-Phenethylbenzyl

191.4 9.0 7.3 7.2 2.7

Result and discussion Utilizing 3D structure of lead compound/thrombin complex, compound 1 was designed in consideration of, 1) increased lipophilicity which may improve permeability in intestine, and 2) binding interaction to retain inhibition activity in order to obtain thrombin inhibitors that show activity in oral administration. Compound 1 was synthesized, however, did not show anticipated thrombin inhibition activity. X-ray crystal structure of compound 1/thrombin complex was solved to examine the cause of low inhibition activity. 3D structure of compound 1/thrombin complex showed that benzyl group could not occupy D-pocket sufficiently. Therefore, compounds which were expected to have improved hydrophobic interaction with D-pocket of thrombin were designed. Based on the molecular modeling study, compound 5 which has 1-naphthylmethyl group for D-pocket was designed. Furthermore, compound 5 was synthesized and exhibited high thrombin inhibition activity. Compound 5 showed prolonged plasma coagulation time (thrombin time) in rats after oral administration. Expected binding mode of compound 5 was confirmed by X-ray crystallographic study of compound 5/thrombin complex. 3D structure of compound 5/thrombin complex exhibited that D-pocket was occupied smartly by 1-naphthylmethyl group. However, it also showed that it could let activity improve more to fill up space to still stay inconsiderably, too. Derivatives from compound 5 were designed and synthesized in order to get more active thrombin inhibitors. Compound 7,9,10 had higher thrombin inhibition activity than compound 5. X-ray crystal structure of these compounds/thrombin complexes were solved immediately. 3D structure of compound 7/thrombin complex showed that Br just filled a gap of hydrophobic pocket. On the other hand, in 3D structure of compound 9 or 10/thrombin complex, the extensive hydrophobic interaction in D-pocket was formed.

641 We have successfully designed and synthesized a potent and orally active inhibitor of thrombin by using structure based drug design technique. SBDD has contributed greatly to generate good candidates in a short research term.

References 1. 2. 3. 4.

Gould, R.J. and Shafer, J.A., Perspectives in Drug Discovery and Design 1 (1993) 419. Stubbs, M.T. and Bode, W., Perspectives in Drug Discovery and Design 1(1993) 431. Bush, L.R., Cardiovascular Drug Reviews 3 (1991) 247. Burton, P.S., Conradi, R.A. and Hilger, A.R., Advanced Drug Delivery Reviews 7 (1991) 365.

216 Design of bivalent plasmin inhibitors Y. TSUDA1, Y. OKADA 1, K. WANAKA2, M. TADA2, S. OKAMOTO 2, A. HIJIKATA-OKUNOMIYA³ and Y. KONISHI4 1 2 3 4

Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan Kobe Research Projects on Thrombosis and Haemostasis, Kobe, Japan Faculty of Health Science, Kobe University School of Medicine, Kobe, Japan Biotechnology Research Institute, National Research Council of Canada, Montreal, Canada

Introduction Plasmin [EC 3.4.21.7], a serine protease, plays an important role in the fibrinolytic system which is geared to remove fibrin from the circulation. Plasmin formed is mainly inactivated by α2-antiplasmin in human plasma. α2 Antiplasmin binds to the lysine-binding sites (LBS) on plasmin with a dissociation constant of 1.8–1.9 × 10 –10 M and then binds the active site of plasmin simultaneously [1]. Due to its bivalent inhibition, α2 -antiplasmin exhibits the high affinity to plasmin. The LBS specifically bind certain amino acids such as Lys, ε-aminocaproic acid (EACA) and trans -4-aminomethyl-cyclohexanecarboxylic acid (t -AMCHA). In this presentation, we describe design and synthesis of bivalent plasmin inhibitors which consist of the active site blocking segment, t-AMCHA-Tyr-EACA, the LBS blocking segment, Lys, and a linker connecting these blocking segments.

Results and discussion The peptides were prepared by the solution procedure using Boc-protected amino acids and the solid phase procedure using Fmoc-protected amino acids. The purity of the final peptides was established by TLC, analytical HPLC and mass spectrometry. First, an active site blocking segment was investigated. Some peptides were synthesized based on the structure of t-AMCHA-Tyr(Br-Z)-octylamide, which is a specific plasmin inhibitor directing an active site with an IC5 0 value of 0.80 µM (Y.Okada, unpublished). Among the peptides synthesized, t-AMCHATyr(Br-Z)- EACA amide exhibited comparatively a potent inhibition for plasmin with an IC50 value of 1.6 µM and high solubility. As a result, t-AMCHATyr(Br-Z)-EACA was employed as an active site blocking segment: t-AMCHATyr-EACA and Lys were connected by a linker with various chain length. The inhibitory effect of the synthetic peptides with various chain length are summarized in Table 1. The peptide with a linker of 13 atom (peptide 1) inhibited plasmin activities on D-Val-Leu-Lys-pNA (S-2251) and fibrin with IC50 values 642 Y. Shimonishi (ed): Peptide Science – Present and Future, 642–643 © 1999 Kluwer Academic Publishers, Printed in Great Britain

643 Table 1.

The inhibitory effect of t-AMCHA-Tyr-EACA-[Linker]-Lys on the plasmin activities I C5 0 (µM)

Compound

Linker

S-2251

Fibrin

1 2 3 4 5 6 Lys

-NH-(CH 2 )1 1 -CO-NH-(CH 2 )-CO-NH-(CH 2 )1 1-CO-NH-(CH 2)3 -CO-NH-(CH 2 )1 1-CO-NH-(CH 2 ) 3 -CO-NH-(CH 2 )3-CONH-(CH 2 ) 5 -CO-

5.7 14 23 110 160 100 50000

19 31 33 69 88 65 9000

of 5.7 µM and 19 µM, respectively. The peptide with longer linkers (peptide 2 and 3) also showed similar potencies with IC 50 values of 14 µM and 23 µM, respectively. The peptide with 0–11 atom linkers (peptide 4, 5 and 6) exhibited a drastic decrease in inhibitory activity (IC 50: 100–160 µM). Thus, the length of 13 atom is sutable for linking the active site and LBS binding segments. The LBS on plasmin(ogen) is contained within its kringle domains (K 1-K 5 ). Peptide 1 would bind one of them, and a peptides with much longer linker than that of peptide 3 could bind the other one to inhibit the plasmin activity. Next, the inhibition mode of peptide 1 was compared with that of α 2 -antiplasmin. In the amidolytic assay, the addition of t-AMCHA (1 mM) reduced the reaction rate between plasmin and peptide 1 to 50%. The influence of t-AMCHA on the rate of plasmin-α 2-antiplasmin was also observed. This finding suggests that peptide 1 binds to plasmin in the similar manner to α 2-antiplasmin and acts as a bivalent plasmin inhibitor. In conclusion, this work has demonstrated the plasmin inhibitor designed bivalent inhibition.

Acknowledgments The authors thank Mr.T.Yamamoto for measurements of mass spectra at Institute for Life Science Research, Asahi Chemical Industry Co., Ltd. This work was funded in part by a Grant-in-Aid for Scientific Research No.10098478 from the Ministry of Education, Science, Sports and Culture of Japan.

Reference 1. Barrett, A.J. and Salvesen, G. (eds.) Protease Inhibitors, Elsevier Science Publishers BV, 1986, p. 457.

217 Suicide-inhibition of human immunodeficiency virus type-1 protease by the gag precursor processing product (p2 g a g peptide) S. MISUMI, M. TOMONAGA, A. KUDO, K. FURUISHI and S. SHOJI* Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862, Japan

Introduction Little is known about what inhibits the proteolytic activity of HIV-1 PR after it has completed processing during maturation phase of the viral life cycle. A viral inherent HIV-1 PR inhibitor has been sought to overcome the problem of identifying an anti-HIV-1 drug that is active against the multiple drugresistant form of the virus.

Results and discussion The p2gag peptide (AEAMSQVTNTATIM) processed from HIV-1 Pr55gag b y HIV-1 PR was identified as a suicide inhibitor of the enzyme (Ki = 30.2 µM and IC 50 = 10 µM for the synthetic peptide substrate, succinyl-SQNYPIVQ), and potently inhibited the proteolytic cleavage of the viral precursor protein (Pr55 gag ) into functional structural units (p17 gag and p24 gag ) in vitro. T h e nonapeptide (AEAMSQVTN) derived from N-terminus of the p2gag peptide exhibits a potent inhibitory action on HIV-1 PR, but the other peptides (AEAMSQ, AEAMSQV, AEAMSQVT, VTN and VTNTATIM) do not (Table 1). It was determined by exclusion gel chromatography that HIV-1 PR after treatment of the synthetic p2gag peptide dissociated from the active dimeric form to an inactive monomeric form (Figure 1A, 1B). The p2 gag peptide and HIV-1 PR were also detected in HIV-1 viral particles using both matrixassisted laser desorption/ionization time-of-flight mass spectrometric (MALDI Table 1. Inhibitory activity of the synthetic p2gag peptide against HIV-1 PR. Relative inhibition is expressed as a percentage of the inhibition (100%) of HIV-1 PR at 250 µM P2 g a g peptide (AEAMSQVTNTATIM). Relative inhibition (%) Peptide sequence

10 µM

100 µM

AEAMSQVTNTATIM AEAMSQVTNSATIM AEAMSQVTN

50% 39% 76%

76% 94% 81%

644 Y. Shimonishi (ed): Peptide Science – Present and Future, 644–645 © 1999 Kluwer Academic Publishers, Printed in Great Britain

645

Figure 1. The exclusion gel chromatography of HIV-1 PR treated with the P2g a g peptide and MALDI TOF-MS spectrum for the p2g a g peptide derived from the viral particle.

TOF-MS) and western immunoblot analyses (Figure 1C). Taken together, these results suggest that the p2gag peptide is the inhibitor of preventing dimerization of HIV-1 PR, and that the enzyme activity is completely suicide inhibited with the accumulated p2gag peptide producing by the processing of Pr55gag during viral maturation. [1]

Reference 1. Misumi, S., Kudo, A., Azuma, R., Tomonaga, M., Furuishi, K., and Shoji, S., Biochem. Biophys. Res. Commun., 241(1997) 275–280.

218 Design of plasma kallikrein selective inhibitor and its application to affinity chromatography Y. OKADA¹, Y. TSUDA¹, K. WANAKA², M. TADA², Y. NAKAYA², U. OKAMOTO², S. OKAMOTO², N. HORIE³ and A. HIJIKATA-OKUNOMIYA 4 ¹ Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-21, Japan ² Kobe Research Project on Thrombosis and Haemostasis, Tarumi-ku, Kobe 655, Japan ³ Department of Food Science and Nutrition, School of Human Environmental Science, Mukogawa Women's University, Nishinomiya 663, Japan 4 Faculty of Health Science, Kobe University School of Medicine, Suma-ku, Kobe 654-01, Japan

Introduction Plasma kallikrein liberates bradykinin from high molecular weight kininogen. It was also reported that plasma kallikrein can activate factor XII, prourokinase and plasminogen. Thus, plasma kallikrein has a broad spectrum of activities, but as yet, detailed studies of the role of plasma kallikrein remain to be performed. Therefore, plasma kallikrein selective inhibitor should be a powerful experimental tool for investigating the roles of the enzyme and may also play an important role in the development of new type of clinical therapy. Previously, we developed plasma kallikrein selective inhibitor, trans4 - aminomethylcyclohexanecarbonyl- L - phenylalanine - 4 - aminophenylacetic acid (PKSI-527) [1]. As shown in Figure 1, this compound consisits of three parts, trans -4-aminomethylcyclohexanecarbonyl, L-phenylalanine and 4-carboxymethylanilide. This presentation deals with 1) the studies on the role of each moiety of this compound for the manifestation of selective inhibitory activity 2) application of PKSI-527 to affinity chromatography.

Figure 1. Structure of PKSI-527.

646 Y. Shimonishi (ed): Peptide Science – Present and Future, 646–648 © 1999 Kluwer Academic Publishers, Printed in Great Britain

647 Results and discussion Studies on the structure-activity relationship From the X-ray analysis of the complex of PKSI-527 with trypsin (instead of plasma kallikrein), it was revealed that amino group of aminomethylcyclohexanecarbonyl moiety (P 1 position) interacts with Asp189 of trypsin. The carbonyl group of the above moiety might interact with hydroxy group of Ser residue in the active center of the enzyme [2]. Therefore, reduction of the carbony group of PKSI-527 to methylene group decreased inhibitory activity dramatically. When the P 1 ' position is Phe, benzene ring of Phe can interact with some part of plasma kallikrein much more strongly than other enzymes, such as plasmin. Interestingly, the replacement of phenylalanine moiety of PKSI-527 with 4-benzyloxyphenylalanine or 4-(2-bromobenzyloxycarbonyloxy)phenylalanine afforded the compound which could inhibit not only plasma kallikrein but also plasmin fairly potently. Concerning P2 ' moiety, it was revealed that the existence of carboxyl function reduced inhibitory activity against plasmin, resulting in increasing the selectivity of plasma kallikrein inhibitor. Based on the studies on the structure-activity relationship, compounds (1 and 2, Figure 2) were designed as more potent plasma kallikrein selective inhibitors. 1 and 2 inhibited plasma kallikrein selectively with IC50 values of 0.56 and 0.71 µM, respectively. These inhibitors are valuable tools for investigating the roles of plasma kallikrein and might be candidates for therapeutic agents. Application of PKSI-527 to affinity chromatography In order to obtain pure plasma kallikrein for the study of its intrinsic roles and for preparing its complex with PKSI-527 to study the inhibitory mode of our synthetic inhibitor against plasma kallikrein, affinity chromatography having PKSI-527 as a ligand was developed. PKSI-527 was coupled with Toyopearl by water soluble DCC. The kaolin activated plasma was applied to

Figure 2.

Structure of the Designed Plasma Kallikrein Selective Inhibitors.

648 PKSI-527-Toyopearl column, equilibrated with 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM sodium chloride. After washing with same buffer, plasma kallikrein was eluted with 50 mM glycine-hydrochloric acid buffer (pH 3.0). The plasma kallikrein was purified 181-fold with a yield of 89% from kaolin activated plasma.

References 1. Wanaka, K., Okada, Y., Tsuda, Y., Okamoto, U., Hijikata-Okunomiya, A., and Okamoto, S., Chem. Pharm. Bull., 40 (1992) 1814. 2. Nakamura, M., Tomoo, K., Ishida, T., Taguchi, H., Tsuda, Y., Okada, Y., Okunomiya, A., Wanaka, K., and Okamoto, S., Biochem. Biophys. Res. Commun., 213 (1995) 583.

219 Structural essentials for novel inhibition of serine protease chymotrypsin Y. SHIMOHIGASHI, Y. YAMAUCHI, K. IKESUE, I. MAEDA, M. KAWAHARA and T. NOSE Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan

Introduction Dipeptide H- D -Leu–Phe–NHCH 2 C 6 H 4(p-F) (Figure 1) inhibits chymotrypsin strongly in a competitive manner [1]. When the complex of this dipeptide with chymotrypsin was analyzed crystallographically, a novel type of inhibitory conformation of peptide became prominent in the active center of chymotrypsin [2]. The C-terminal benzyl group fitted the S 1 substrate recognition site, where para-fluorine atom was involved in hydrogen bonding, at the bottom of the pocket, with the hydroxyl group of chymotrypsin Ser-189. The dipeptide backbone was in the hydrophobic cavity, where ordinary substrates make sequential subsite interactions. The side chains of D -Leu and L -Phe folded together, producing a so-called CH/π interaction, to from a hydrophobic core, and in this conformation the Phe-phenyl group was interacting with chymotrypsin His-57. Furthermore, the isobutyl group of D -Leu was in the interaction with

Figure 1. Three dimensional structure of dipeptide H- D -Leu–Phe–NHCH 2 C6 H4 (p-F) in the active center of serine protease chymotrypsin.

649 Y. Shimonishi (ed): Peptide Science – Present and Future, 649–651 © 1999 Kluwer Academic Publishers, Printed in Great Britain

650 chymotrypsin Trp-215 on the other side of Phe-phenyl. Thus, the hydrophobic core produced by the D -Leu-Phe dipeptide unit located in the enzyme hydrophobic cavity surrounded by His-57, Ile-99, and Trp-215. Furthermore, the π – π stacking interaction between Phe-phenyl of dipeptide and His-57-imidazole of chymotrypsin was found in this inhibitory conformation. This interaction is quite unique in terms of direct structural restriction of one of the catalytic triad by the inhibitor molecule. In the present study, we have designed and synthesized a series of dipeptides to verify the importance of these structural elements in inhibition of chymotrypsin.

Results and discussion When H- D –Phe–Leu–NHCH 2 C 6 H 5 , which has a reversed sequence of H-D Leu–Phe–NHCH 2 C 6 H 5 , was assayed for inhibition of the hydrolysis of substrate acetyl- L -tyrosine ethyl ester, the inhibitory activity was diminished to only about 8% (Table 1). Similar results were obtained for dipeptides with such combinations of amino acids as (Thr, Phe), (Arg, Phe), and (Gln, Phe) in the D–L configurational sequence (data not shown). These clearly indicated that the Phe residue at position 2 is crucially important, and thus the π – π stacking interaction between the Phe-phenyl and His-57-imidazole is definitely one of key interactions to inhibit the enzyme. A series of D -aliphatic amino acids were placed at the position 1 in order to assess the importance of side chain-side chain CH/π interaction. It was found that a stable CH/ π interaction is necessary for strong inhibition and this is substantiated by the presence of hydrogens on γ -carbon of the D -aliphatic amino acid residues at position 1 (Table 1). All these results clearly indicated that structural elements of H- D -Leu–Phe– NHCH 2 C 6 H 4( p -F) in unique conformation of dipeptide is necessary altogether for enzyme inhibition.

Table 1. Chymotrypsin inhibitory activity of a series of dipeptide NHCH(R 2 )CONHCH 2 C6 H 5 in the D-L configurational sequence. Dipeptides N H 2 CH(R 1 )CO–NHCH(R 2 )CONHCH 2 C6 H 5 R1 R2 CH 2 CH(CH 3 ) 2 CH 2C 6 H 5 CH 3 CH 2 CH 3 CH(CH 3 ) 2 C(CH 3 ) 3 CH 2 CH 2 CH 3 CH 2C(CH 3 )3

CH 2C 6 H5 CH 2 CH(CH 3 )3 CH 2C 6 H 5 CH 2C 6 H 5 CH 2 C 6 H 5 CH 2 C 6 H 5 CH 2 C 6 H 5 CH 2 C 6 H 5

NH2 CH(R 1 )CO–

Ki (µM)

Relative potency (%)

4.1 49.6 25 15 3.6 1.4 13 5.5

100 8.3 16 27 110 290 31 75

651 References 1. Shimohigashi, Y., Maeda, I., Nose, T., Ikesue, K., Sakamoto, H., Ogawa, T., Kawahara, M., Nezu, T., Terada, Y., Kawano, K. and Ohno, M. J. Chem. Soc., Perkin Trans 1 (1996) 2479. 2. Maeda, I., Kashima, A., Inoue, Y., Sugio, S., Nose, T. and Shimohigashi, Y. Peptide Chemistry 1996 (1997) 229.

220 KNI-764, A novel dipeptide-based HIV protease inhibitor containing allophenylnorstatine T. MIMOTO¹, R. KATO¹, H. TAKAKU¹, S. MISAWA¹, T. FUKAZAWA¹, S. NOJIMA¹, K. TERASHIMA¹, H. SATO¹, M. SHINTANI¹, Y. KISO² and H. HAYASHI¹ ¹ Pharmaceuticals & Biotechnology Laboratory, Japan Energy Corporation, Toda-shi, Saitama 335, Japan ² Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan

Introduction Inhibition of human immunodeficiency virus (HIV) protease is one of the most important and promising approach for the therapeutic intervention of HIV infection. Previously, we have reported a series of novel peptidomimetic HIV protease inhibitors based on the transition-state mimic concept. One of which is KNI-272 [1, 2], a highly potent HIV protease inhibitor (Ki = 0.235 nM; E C 50 = 27 nM) currently in clinical study. Although there are many drugs approved or in clinical study, more drugs having different resistance profile, better compliance, less side effects are required. As part of our continuing efforts, we prepared a number of small-sized HIV protease inhibitors containing Apns [allophenylnorstatine: (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid]Thz as the transition-state isostere (P1-P1').

Results and discussion The introduction of the 3-hydroxy-2-methylbenzoyl group as the P2 ligand to the basic structure of H-Apns-Thz-NHtBu (the P1-P1'-P2' structure of KNI-272) produced a dipeptide lead compound (KNI-576). The C2 operation along axis in the Apns-Thz site generated the 2-methylbenzylamide group as the P2’ ligand. In addition, two methyl substitution at the thiazolidine ring resulted in a highly potent HIV-1 protease inhbitor, KNI-764 (Ki = 0.035 nM). KNI-764 was highly active against wild-type HIV-1 in the absence or presence of 50% human serum, and the EC 50 values were 26 nM or 31 nM, respectively. By serial passage of HIV-1 in the presence of increasing concentration of KNI-764, a variant with a 16 fold reduction in susceptibility to KNI-764 was isolated. The amino acid mutation of KNI-764-resistant HIV-1 protease was mainly observed at position 10 (L to F), 37 (S to N), 47 (I to V), and 82 (V to I). This mutation patterns was different from the other HIV 652 Y. Shimonishi (ed): Peptide Science – Present and Future, 652–653 © 1999 Kluwer Academic Publishers, Printed in Great Britain

653

Figure 1.

Chemical structure of KNI-576 (left) and KNI-764 (right).

Table 1. Comparison of antiviral activity of KNI-764 and other protease inhibitors in the absence or presence of 50% human serum (HS). HIV-1 IIIB/CEM-SS

HIV-1 PR Ki (nM) KNI-764 KNI-272 saquinavir ritonavir indinavir nelfinavir

Table 2.

0.035 0.235 0.138 0.098 0.739 0.931

EC 50 (nM)

EC 50 + 50% HS (nM)

EC 95 (nM)

26 27 7.4 29 6.5 8.7

31 100 28 210 9.8 160

52 58 13 113 42 18

Pharmacokinetic profile of KNI-764 in dogs

route (condition)

dose

AUC

i.v. p.o (fasted) p.o. (fed) p.o. (fasted 2 hr)

25 25 25 25

3,095 1,150 1,003 1,301

F 37 33 42

Cmax 5.30 4.02 6.14

Tmax

T 1/2

Cltot

Vd(ss)

94

0.88

1.58

60 90 30

dose = mg/kg, AUC =µM · min, F = %, Cmax = µM, Tmax = min, T 1/2 = min, Cltot = 1/hr/kg, Vd(ss) = l/kg

protease inhibitors (ex. saquinavir, ritonavir, indinavir, or nelfinavir). In addition KNI-764-resistant HIV-1 was nearly completely sensitive to those HIV protease inhbitors. In pharmacokinetic study in dogs, it was found that KNI-764 had a favorable bioavailability (33–42%) and a long plasma half-life (94 min), and then highly suppressive levels (> EC 95 ) was maintained for 12–24 hours at an oral dose of 25 mg/kg. In conclusion, KNI-764 was a promising candidate for the treatment of AIDS.

References 1. Mimoto, T. et. al., Chem. Pharm. Bull., 40 (1992) 2251 2. Kageyama, S. et. al., Antimicrob. Agents Chemother., 37 (1993) 810

221 Self-association of cystatin E. JANKOWSKA, B. BANECKI¹, W. WICZK and Z. GRZONKA Faculty of Chemistry, University of ¹ Faculty of Biotechnology, University of

Sobieskiego 18, Kladki 24,

80-952, Poland 80-822, Poland

Introduction Cystatin C, a natural inhibitor of cysteine proteases, is a low molecular weight protein of 120 amino acid residues in a single polypeptide chain with two disulfide bridges in the C-terminal part. Its Leu 68 → Gln 68 mutated variant is responsible for hereditary cystatin C amyloid angiopathy, which is caused by self-association and aggregation of L68Q-cystatin C mutant in blood brain vessels [1]. In the present investigation, we have undertaken a detailed study of self-association of wild-type cystatin C using fluorescence and CD spectroscopy, and micro-calorimetry with the aim to improve our understanding this phenomenon.

Results and discussion Cystatin C was a generous gift from prof. A. Grubb (University Hospital, Lund, Sweden). CD spectra of cystatin C were recorded using Aviv 62 spectropolarimeter in phosphate buffer (pH = 6.64). The fluorescence measurements were performed using Perkin-Elmer LS-50B spectrofluorimeter. Denaturation of cystatin C was achieved by adding guanidine hydrochloride or raising the temperature. Differential scanning calorimetry measurements were performed using a Microcal MC-2 scanning calorimeter. As can be seen at the Figure 1 the process of the denaturation is complex. The first plateau is observed at a concentration of about 0.4–1.3 M of Gdn*HCl and the second at a concentration of about 1.4–3.5 M. These data suggest the formation of a dimeric form of the protein at the first concentration range, still having quite well conserved secondary structure. The second plateau indicates the existence of an extended monomeric form of cystatin C. At higher concentrations of Gdn*HCl the protein seems to have a fully denatured form. The CD pattern of the denaturation of cystatin C is similar to the published electrophoretic studies of the dimerization process of cystatin C [2]. We obtained similar results of the denaturation/dimerization process by increased concentration of Gdn*HCl using the fluorescence spectroscopy technique. The calorimetric profile of cystatin C shows four peaks centered at 19.5, 28.0, 35.7 and 67.3°C indicating that the denaturation process can not be 654 Y. Shimonishi (ed): Peptide Science – Present and Future, 654–656 © 1999 Kluwer Academic Publishers, Printed in Great Britain

655

Figure 1. Changes of the native fraction contribution during denaturation of cystatin C by guanidine hydrochloride calculated based on the CD measurements. Table 1. Thermodynamic parameters associated with the thermal unfolding of Cystatin C. (Ratio of calorimetric enthalpies (Hcal) to van’t Hoff enthalpies (vHoff) were calculated using the DA-2 software package (Microcal)). Transition point [°C]

Hcal [kcal/mol]

Hcal/vHoff

19.5* 28.0 ± 0.4 35.7 ± 0.4 67.3 ± 0.4

19.1* 6.2 ± 0.1 4.2 ± 0.1 3.8 ± 0.1

– 0.93 0.80 1.05

* values held constant during nonlinear fit

confirm as a simple two-state mechanism; the transition most likely involves partially folded intermediates (Table 1). The peak centered at about 67°C is related to the dimerization process and this process can be described as a simple two-state mechanism (Hcal/vHoff ≈ 1, see Table 1) [3]. On the other hand the changes observed for lower temperatures are more complex and difficult to interpret them univocally, not only because of the complexity of the process, but also because of lack of data at temperatures lower than 15°C. Because of the complexity of the association process of cystatin C other techniques which are able to give more informations about the changes of secondary and tertiary structure of the protein will be applied. Acknowledgements Supported by Polish Committee for Scientific Research (KBN) grants: 279/P04/97/13 and BW/8000-5-0350-7.

656 References 1. Abrahamson, M. and Grubb, A., Proc. Natl. Acad. Sci. USA, 91(1994) 1416. 2. Ekiel, I. and Abrahamson, M., J. Biol. Chem., 271 (1996) 1334. 3. Freire, E., Methods Enzymol., 240 (1994) 503.

222 The substrate specificity of microbial carboxyl proteinases B.M. DUNN¹ and K. ODA² ¹ Department of Biochemistry & Molecular Biology, Box 100245 J. Hillis Miller Health Center, University of Florida College of Medicine, Gainesville, FL32610-0245, USA ² Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto, Japan

Introduction The substrate specificity of a proteolytic enzyme is determined (in some cases) by the interaction of amino acid residues of the substrate with an extended active site cleft. The Aspartic Proteinase family represents a good example of enzymes possessing a binding cleft capable of interacting with 7–8 amino acid residues. Examples of secondary specificity and the structural basis of this has been our focus [1] The bacterial carboxyl proteases represent a new class of enzyme of unknown structure [2]. Unlike the Aspartic Proteinases, the carboxyl proteases are not inhibited by pepstatin [3], and they lack sequence homology to the former family. Curiously, however, they are able to cleave the series of oligopeptides we have prepared for the analysis of the secondary specificity of the Aspartic Proteinases. This poster compares the results of analysis of cleavage of 34 peptide substrates by Pseudomonas sp. 101 carboxyl proteinase (PCP) and Xanthomonas sp. T-22 carboxyl proteinase (XCP), in the context of the results reported for members of the aspartic proteinase family.

Results and discussion Figure 1 illustrates two widely divergent results from exploring the efffects of substitution at various positions in the octapeptide substrate, Lys–Pro–Ala–Lys–Phe*Nph–Arg–Leu, where cleavage occurs in all cases between the aromatic amino acids, Phe and Nph ( p-NO 2 Phe). Pepsin (white bars) is able to cleave most of the peptides, although with varying efficiencies. As the parent peptide was chosen because it was a good pepsin substrate, we expected derivatives of the parent sequence to be cleaved and to reveal aspects of sub-site preferences. This can be clearly seen in the P4 substitutions in Figure 1, where the parent residue, Pro, provides the best fit to the enzyme. Ala substitution in P4 is accepted with reduced 657 Y. Shimonishi (ed): Peptide Science – Present and Future, 657–659 © 1999 Kluwer Academic Publishers, Printed in Great Britain

658 enthusiasm, while the other substituents gave poorer response. In contrast, human cathepsin E (slanted stripes) does not cleave most of the peptides in this series. Only when the residue at P2, Lys in the parent peptide, is replaced by Ser, Ala, Asp, or especially Leu, do we see efficient cleavage of the resulting octapeptide. Turning to PCP, we see that this carboxyl proteinase (horizontal stripes) shows a response to the 28 substituted peptides that mirrors that seen for human cathepsin E. Only the substitution of Leu in P2 provides efficient cleavage. In contrast, in Figure 1 we see that XCP (black bars) shows a response more like pepsin, in that all the peptides are cleaved, again with varying efficiencies. In this case, substitution at P3 produces nearly as big of a response as substitution at P2. In our analysis of the classic aspartic proteinase family, we have prepared two sets of peptides, one based on the series shown in Figure 1, and a second that has the bulkier Ile in P3 and the hydrophobic Glu residue in P2. Glu can be negatively charged, but at the pH of most of our studies (3–6), we believe that Glu is mostly neutral and, hence, hydrophobic. In this second series, we have observed that for PCP the preferences for the P2 residue are roughly the same in the two series; the larger Leu substitution is favored in both series. Thus, for PCP it does not matter whether there is a small Ala or a larger Ile in the P3 position. Turning to XCP, we found that the smaller Ala and Ser are now preferred as P2 substitutions, while in the first series in Figure 1, the larger Leu was slightly better than Ala. Thus, the change from the small Ala in P3 in the series shown in Figure 1 to the larger, bulkier Ile in P3 in the second series changes

Figure 1.

659 the preference for the P2 residue. We conclude that the P3 Ile fills up the S3 pocket in XCP and limits the size of the substituent that can be accomodated in P2. From this we also conclude that the S3 pocket of PCP is much larger than that of XCP.

References 1. Dunn, B., Scarborough, P.E., Swietnicki, W., and Davenport, R., in: Peptide Synthesis and Purification Protocols, Dunn, B.M. and Pennington, M.W.(Eds), Humana Press, Clifton, NJ., (1994) pp. 225–243. 2. Oda, K. and Murao, S., in: Structure and Function of The Aspartic Proteinases: Genetics, Structures, and Mechanisms, Dunn, B.M. (Ed.), Plenum Press, New York and London (1991) pp. 185–201. 3. Murao, S., Oda, K., and Matsushita, Y., Agric. Biol. Chem., 37: 1417–1421 (1973).

223 Allophenylnorstatine containing HIV-1 protease inhibitors: design, synthesis and structure–activity relationships for selected P2 and P2' ligands A.A. BEKHIT¹, H. MATSUMOTO¹, H.M.M. ABDEL-RAHMAN¹, T. MIMOTO², S. NOJIMA², H. TAKAKU², T. KIMURA¹, K. AKAJI¹ and Y. KISO¹ ¹ Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan ² Pharmaceutical & Biotechnology Laboratories, Japan Energy Co., Niizo-minami, Todashi, Saitama 335, Japan

Introduction The human immunodeficiency virus (HIV) encodes an aspartic protease that processes polyprotein precursors into viral structural proteins and replicative enzymes. This processing is essential for the assembly and maturation of fully infectious virions. Thus the design of inhibitors of HIV protease is an important therapeutic target in the treatment of AIDS [1]. Incorporation of allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] into PhePro peptidomimetic inhibitors of HIV protease has led to the development of a novel series of highly potent antiviral compounds, represented by the tripeptide compound KNI-272 and the dipeptide KNI-727 [2,3]. The present work reports the synthesis and structure–activity relationships of dipeptide inhibitors in which the P2 and/or P2' ligands were replaced with selected moieties (Table 1).

Results and discussion Dipeptide inhibitors were synthesized by the solution method in stepwise manner. Introduction of 2,6-dimethylphenoxyacetyl group at P2 and benzylamine at P2' resulted in an active compound; KNI-901 (50 nM protease inhibition = 86.0%). The 2-fluoro derivative KNI-905 showed similar activity to KNI-901. On the other hand, the 3-fluoro derivative KNI-906 (50 nM inhibition = 73.4%) exhibited about 17% reduction in the activity. Furthermore, we replaced the dimethylphenoxyacetyl group by N-butyloxycarbonylanthranilyl moiety. The resulting compounds KNI-923, KNI-924 & KNI-925 showed considerable reduction in activity. Particularly the 3-flouro derivative had the lowest activity within this series (5 µM inhibition = 45.9%). Compounds having the 3-hydroxy-2-methylbenzoyl moiety at P2 and a heterocyclic ring system at 660 Y. Shimonishi (ed): Peptide Science – Present and Future, 660–661 © 1999 Kluwer Academic Publishers, Printed in Great Britain

661 Table 1. Structures and HIV protease inhibitory activities of dipeptides containing selected P2 and P2' ligands.

P2' possessed variable activity. The most active compound within this series was KNI-909. This study showed that optimized balance of lipophilicity and hydrophilicity of P2 and P2' ligands is essential to enhance the inhibitory activity against HIV protease.

References 1. De Clercq, E., J. Med. Chem., 38 (1995) 2491. 2. Kiso, Y., Biopolymers, 40 (1996) 235 3. Yamaguchi, S., Mitoguchi, T., Nakata, S., Kimura, T., Akaji, K., Nojima, S., Takaku, H., Mimoto, T. and Kiso, Y., Peptide Chemistry 1996 (1997) 297.

224 Allophenylnorstatine containing HIV-1 protease inhibitors: design, synthesis and structure-activity relationships for selected P2 ligands H.M.M. ABDEL-RAHMAN¹ , H. MATSUMOTO¹, A.A. BEKHIT¹, T. MIMOTO², S. NOJIMA², H. TAKAKU², T. KIMURA¹, K. AKAJI¹ and Y. KISO¹ ¹Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607, Japan ²Pharmaceutical & Biotechnology Laboratories, Japan Energy Co., Niizo-minami, Todashi, Saitama 335, Japan

Introduction The design and development of potent HIV protease inhibitors remains an attractive target for antiviral therapy. We have already synthesized a novel class of HIV protease inhibitors containing allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid]. The tripeptide KNI-272 (Figure 1) possesses excellent characteristics of protease inhibition, antiviral activity, enzyme selectivity, and low toxicity [1]. In order to improve the in vivo behavior of KNI-272, we reported dipeptide HIV protease inhibitors with potent enzyme inhibitory activity [2, 3], including KNI-577 (50 nM inhibition = 87.6%) and KNI-727 (50 nM inhibition = 95.9%) (Figure 1). In the present work we further investigated the structure-activity relationships of the

Figure 1.

Structure of KNI-272 and small-sized analogues.

662 Y. Shimonishi (ed): Peptide Science – Present and Future, 662–664 © 1999 Kluwer Academic Publishers, Printed in Great Britain

663 Table 1. Structures and HIV protease inhibitory activities of the synthesized dipeptides.

dipeptide inhibitors to obtain the optimal compromise between the HIV protease inhibitory activity and pharmacokinetic properties.

Results and discussion In an effort to increase the bioavailability of the dipeptides, we introduced heterocyclic moieties via urea linkage at P2 position, but this approach resulted in considerable reduction in the activity. This may be attributed to the increased hydrophilicity of the P2 moiety within the amphiphilic S2 subsite. On the other hand, the activity of the dipeptides was enhanced by incorporation of amide type ligands at P2 position (Table 1). The high activity of KNI-917 and KNI-920 could be referred to the improved binding of their P2 ligands due to the presence of hydrophobic interactions and the hydrogen bonding ability of these ligands. In conclusion, we synthesized new dipeptide inhibitors, and some of them showed good HIV protease inhibitory activity. However further design of compounds which have more suitable balance of lipophilcity and hydrophilicity may lead to novel inhibitors with good enzyme inhibitory activity as well as pharmacokinetic properties.

664 References 1. Kiso, Y., Biopolymers (Peptide Science), 40 (1996) 235–244. 2. Yamaguchi, S., Mitoguchi, T., Nakata, S., Kimura, T., Akaji, K., Nojima, S., Takaku, H., Mimoto, T., and Kiso, Y., Peptide Chemistry 1996, (1997) 297–300. 3 . Kiso, Y., Yamaguchi, S., Matsumoto, H., Mimoto, T., Kato, R., Nojima, S., Takaku, H., Fukazawa, T., Kimura, T., and Akaji, K., (Proceedings of the 15th American Peptide Symposium, Nashville, 14–19 June 1997, Abst. No. L023.

225 Opioid peptide analogs with novel activity profiles as potential therapeutic agents for use in analgesia P.W. SCHILLER, G. WELTROWSKA, R. SCHMIDT, T.M.-D. NGUYEN, I. BEREZOWSKA, Y. CHEN, C. LEMIEUX, N.N. CHUNG, B.C. WILKES and K.A. CARPENTER Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Que., Canada H2W 1R7

Introduction Centrally acting µ opioid receptor agonists are potent analgesics but also produce a number of side effects, including dependence, tolerance, respiratory depression, adverse gastrointestinal effects, etc. The results of recent research indicate that the development of opioid compounds with δ agonist properties or with a mixed µ agonist/ δ antagonist profile may lead to analgesics that are devoid of some of these side effects. δ Agonists are able to produce analgesic effects with decreased development of physical dependence, no depression of respiratory function and reduced gastrointestinal effects. The concept of mixed µ agonist/δ antagonists i s b a s e d o n t h e o b s e r v a t i o n t h a t c h r o n i c co-administration of morphine and the non-peptide δ antagonist naltrindole attenuated the development of morphine tolerance and dependence [1]. Another interesting concept is the development of µ opioid analgesics unable to cross the blood-brain barrier or placental barrier for use in peripheral or obstetrical analgesia. In the present paper we describe further progress made in the development of δ agonists, mixed µ agonist/δ antagonists and µ agonists with potential as obstetrical analgesics.

Results and discusion Recently, we reported that the dipeptide derivative H-Tyr–Tic–NH–(CH 2 )2 -Ph (Ph = phenyl) is a moderately potent and selective δ agonist (Table 1) and that very subtle structural modifications of this compound converted it from a δ agonist to a δ antagonist [2]. For example, replacement of the phenyl group of the phenylethyl substituent with a p-fluorophenyl or 2-pyridyl group resulted in δ antagonist properties, as did reversal of the amide bond linking the Tic residue and the phenylethyl moiety. The results of a molecular mechanics study revealed that both the δ agonist dipeptide and the various δ antagonist dipeptides were able to assume an almost identical low energy conformation (Figure 1). In the case of the agonist, this conformer showed good spatial overlap of its tyramine moiety and phenylethyl aromatic ring with the corre665 Y. Shimonishi (ed): Peptide Science – Present and Future, 665–669 © 1999 Kluwer Academic Publishers, Printed in Great Britain

666

Figure 1. Spatial overlap of low energy conformer of H-Tyr–Tic–(CH2 ) 2 -Ph (agonist) with the proposed bioactive conformation of JOM-13 (left) and of low energy conformer of H-Tyr–Tic– NH–(CH 2 ) 2 –Ph(p-F) (antagonist) with naltrindole (right).

sponding Tyr1 tyramine group and Phe3 aromatic ring of the cyclic tetrapeptide δ agonist H-Tyr-c[D-Cys–Phe-D-Pen]OH (JOM-13) in its proposed bioactive conformation [3]. The antagonist in that same conformation showed good 2 spatial overlap of its Tyr 1 tyramine portion and Tic aromatic ring with the corresponding tyramine moiety and aromatic ring (indole moiety) of the nonpeptide δ antagonist naltrindole, in agreement with a proposed model of the δ receptor-bound conformation of TIP(P) antagonists [4]. It thus appears that the subtle structural modifications discussed above do not result in a conformational change of the ligand but affect the molecule’s mode of receptor binding due to different steric interactions or altered hydrogen bonding patterns. Depending on these intermolecular interactions with receptor moieties, the dipeptide may either bind to and stabilize an active receptor conformation (in the case of the agonists) or an inactive one (in the case of the antagonists). Recently, it was found that introduction of a second phenyl group at the β -carbon of the phenylethylamine substituent contained in the parent dipeptide δ agonist resulted in a compound, H-Tyr–Tic–NH–CH2 –CH(Ph) 2 , which showed 20-fold increased δ agonist potency and somewhat higher δ receptor selectivity [5]. In an effort to further improve the δ agonist potency and/or δ specificity of this compound, additional substituents were introduced in paraposition of both phenyl rings. Both H-Tyr–Tic–NH–CH2 –CH(Ph[pNO2 ])2 and H-Tyr–Tic–NH–CH 2 –CH(Ph[ p NH 2 ]) 2 retained high δ agonist potency and showed further improved δ receptor selectivity (Table 1). In comparison with the δ agonist DPDPE, H-Tyr–Tic–NH–CH 2 –CH(Ph[ p NH 2 ]) 2 displayed 6-fold higher δ receptor affinity and 35 times higher δ receptor selectivity in the rat brain membrane receptor binding assays and about the same δ agonist potency in the mouse vas deferens (MVD) bioassay. Both diastereomers of the

667 Table 1.

In vitro opioid activities of dipeptide δ opioid agonists. MVD assay

Opioid receptor binding

Compound

IC50 [ n M ]

Κ δ i [nM]

Κµι /Κ δι

H–Tyr–Tic–NH–(CH 2 )–Ph H–Tyr–Tic–NH–CH 2 –CH(Ph) 2 H–Tyr–Tic–NH–CH2 –CH(Ph[pNO 2 ] )2 [pNH 2 ]) 2 H–Tyr–Tic–NH–CH 2 –CH(Ph H–Tyr–Tic–NH–CH 2 –CH(Ph)COQEt (I) (II) H–Tyr–Tic–NH–CH 2 –CH(Ph)COOEt H–Mmt–Tic–NH–CH 2 –CH(Ph)CQOEt (I) H–Mmt–Tic–NH–CH 2 –CH(Ph)COOEt (II) DPDPE

82.0 3.77 8.91 7.81 1.28 8.64 1.62 2.24 5.30

5.22 0.981 7.17 2.67 0.569 3.03 1.63 1.22 16.4

13.2 29.4 88.6 2000 1560 50.5 101 65.3 57.5

dipeptide H-Tyr–Tic–NH–CH2–CH(Ph)COOEt have recently been shown to be potent and selective δ agonists [5]. Replacement of Tyr1 with 2'-methyltyrosine (Mmt) in both diastereomers resulted in compounds that retained high δ agonist potency and δ selectivity. Furthermore, these compounds are more lipophilic due to the presence of the additional methyl group. The highly lipophilic character and low molecular weight of these dipeptide derivatives should facilitate penetration of the blood-brain barrier and, therefore, they have potential as centrally acting analgesics. The tetrapeptide amides H-Dmt–Tic–Phe–Phe–NH2 (DIPP–NH 2 ; Dmt = 2',6'-dimethyltyrosine) and H-Dmt–TicΨ [CH 2 NH] Phe–Phe–NH2 ( D I P P – ΝΗ 2 [Ψ]) have recently been reported to be potent mixed µ agonist/ δ antagonists with µ and δ receptor affinities in the subnanomolar range [6]. In the rat tail flick test DIPP–NH2 [Ψ] given i.c.v. produced an analgesic effect (ED50 = 0.002 µ g) comparable to that of morphine (ED50 = 0.003 µ g; i.c.v.) (M. Fundytus et al., unpublished results). Infusion of DIPP–NH 2 [Ψ] (i.c.v.) for 7 days at massive doses (15 × ED90/h or 30 × ED90/h [ED90 determined in analgesic test]) did not produce any dependence in rats, whereas administration of morphine according to the same protocol resulted in highly dependent animals. Also, rats receiving DIPP–NH 2 [Ψ] (i.c.v.) at a dose corresponding to the ED90 every hour stayed analgesic much longer (17h) than morphinetreated animals (4h), indicating that the mixed µ agonist/ δ antagonist produced less chronic tolerance than morphine. Thus, DIPP–NH2 [Ψ] turned out to be a potent analgesic producing no dependence and tolerance at a reducted level, as predicted for a compound with a mixed µ agonist/ δ antagonist profile. The Trp 3 -analog of DIPP–NH 2 , H-Dmt–Tic–Trp–Phe–NH2 , also turned out to be a potent and balanced µ agonist/ δ antagonist. Replacement of Tyr1 in TIPP– NH 2 with Nα ,2',6'-trimethyltyrosine (Tmt) resulted in a compound, H-Tmt– Tic–Phe–Phe–NH 2 , showing partial µ agonism in the guinea pig ileum (GPI) assay and potent antagonism in the MVD assay. The pseudopeptide H-Tmt– TicΨ [ C H 2 NH]Phe–Phe–NH 2 w a s f o u n d t o b e a m o d e r a t e l y p o t e n t µ antagonist/ δ antagonist.

668 Table 2.

In Vitro Opioid Activity Profiles of DALDA, Endomorphins and Analogs. Opioid receptor binding

GPI Compound

IC 5

H–Tyr–D–Arg–Phe–Lys–NH 2 ( D A L D A ) H–Mmt–D–Arg–Phe–Lys–NH 2 H–Dmt–D–Arg–Phe–Lys–NH 2 H–Tyr–Pro–Trp–Phe–NH 2 (endomorphin-1) H–Tyr–Pro–Phe–Phe–NH 2 (endomorphin-2) H–Dmt–Pro–Trp–Phe–NH 2

254 10.9 1.41 17.2 7.71 2.96

0

[nM]

K µi [nM]

K δi / K

1.69 0.222 0.143 2.32 2.06 0.416

11400 10450 14700 724 1910 15.8

µ i

The dermorphin analog H-Tyr-D-Arg–Phe–Lys–NH2 (DALDA) is a µ opioid agonist with excellent µ receptor selectivity (Table 2) [7]. Substitution of Tyr1 in DALDA with Mmt or Dmt resulted in compounds with greatly increased µ agonist potency. [Dmt1] DALDA showed picomolar µ receptor affinity (K µi = 143 pM) and unprecedented µ receptor selectivity (Kδi /Κ iµ = 14700), being a more potent and more selective µ agonist than endomorphin-1, endomorphin-2 and the Dmt1-analog of endomorphin-1. The therapeutic potential of DALDA for use in obstetric analgesia was examined in the pregnant sheep model (i.v. bolus or i.v. infusion). Pharmacokinetic studies revealed that fetal plasma levels of DALDA were less than 1% of maternal plasma levels (H.H. Szeto et al., unpublished data). This result indicates that, unlike in the case of non-peptide µ opiates (fentanyl, meperidine, morphine, etc.) currently used in obstetrical analgesia, DALDA penetrates the placental barrier only to a very minimal extent. Furthermore, DALDA did not affect the fetal cardiovascular system and respiration, and had only a minimal effect on maternal cardiorespiratory parameters. In rats, intrathecal (i.t.) administration of DALDA resulted in a potent analgesic effect (ED50 = 0.207 µ g) of long duration (4 h) in the tail flick test, whereas morphine was less potent (ED50 = 1.141 µ g) and produced analgesia lasting for less than 2 h. The long-lasting analgesic effect of DALDA may be due to its polar nature (positive charge of 3+), which may prevent its rapid diffusion out of the i.t. space. Taken together, these results indicate that DALDA given i.t. looks promising as an obstetrical analgesic.

References 1. Abdelhamid, E.C., Sultana, M., Portoghese, P.S. and Takemori, A.E., J. Pharmacol. Exp. Ther., 258 (1991) 299. 2. Schiller, P.W., Weltrowska, G., Bolewska-Pedyczak, E., Nguyen, T.M.-D., Lemieux, C., and Chung, N.N., In Ramage, R. and Epton, R. (Eds.) Peptides 1996 (Proceedings of the 24th European Peptide Symposium), Mayflower, Kingswinford, UK, 1997, pp. 785–786. 3. Lomize, A.L., Pogozheva, I.D., and Mosberg, H.I., Biopolymers, 38 (1996) 221. 4. Wilkes, B.C. and Schiller, P.W., Biopolymers (Peptide Science), 37 (1995) 391. 5. Schiller, P.W., Weltrowska, G., Berezowska, I., Lemieux, C., Chung, N.N., Carpenter, K.A., and Wilkes, B.C., In Tam, J.P. and Kaumaya, P.T.P. (Eds.) Peptides: Chemistry, Structure and

669 Biology (Proceedings of the 15th American Peptide Symposium), Kluwer, Dordrecht, 1998, in press. 6. Schiller, P.W., Weltrowska, G., Schmidt, R., Nguyen, T.M.-D., Berezowska, I., Lemieux, C., Chung, N.N., Carpenter, K.A., and Wilkes, B.C., Analgesia, 1(1995) 703. 7. Schiller, P.W., Nguyen, T.M.-D., Chung, N.N., and Lemieux, C., J. Med. Chem. 32 (1989) 698.

226 Peptide radiopharmaceuticals: studies of somatostatin receptors and ligands for tumor targeting S. FROIDEVAUX, A. HEPPELER², M. BEHE², E. JERMANN², A. OTTE², J. MÜLLER-BRAND², H.R. MÄCKE², P. HILDEBRAND, C. BEGLINGER¹and A.N. EBERLE Departments of Research and ¹ Internal Medicine and ²Institute of Nuclear Medicine, University Hospital and University Children’s Hospital, CH-4031 Basel, Switzerland

Introduction Peptide radiopharmaceuticals for cancer diagnosis have become very important in the field of nuclear oncology in the past few years [1], in particular the use of radiolabeled analogs of somatostatin such as [ 1 1 1 In]-DTPA-[D-Phe¹]octreotide (OctreoScan ® ) or [ 1 2 3I]-[Tyr³]-octreotide [2]. The advantage of small peptide ligands as compared to antibodies directed against cell surface markers is the improved tumor penetration of peptides, their relatively rapid excretion through the kindeys and their low immunogenicity. Although somatostatin analogs such as OctreoScan have become the most widely used radiopeptides in the clinic, their application is limited to the detection and visualization of sst2 somatostatin receptor expressing tumors. Therefore, a number of additional peptides are currently studied and developed as radiopharmaceuticals for in vivo targeting. Current studies of our own laboratories focus on the development of melanotropin, gastrin-releasing peptide and somatostatin ligands as radiopharmaceuticals and on the analysis of receptor expression for these peptides. This paper presents data on the in vitro and in vivo regulation of sst2 somatostatin receptors, on the in vivo biodistribution of a somatostatin analog containing a novel metal chelator, as well as its application to tumor localization in patients.

Results and discussion DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was developed as new universal chelator for diagnostic and therapeutic radionuclides such as 1 1 1 In, 6 8 Ga or 9 0Y. DOTA complexing stable isotopes of Ga 3 +, In 3 + o r Y 3 + were crystallized and the structures of the complexes determined by X-ray diffraction analysis. It was demonstrated that the coordination chemistry of DOTA complexing Ga 3 + differs markedly from the other two complexes. Following attachment of DOTA to the N-terminus of either octreotide to form the chelator-octreotide derivative DOTAOC, or [Tyr³]-octreotide to form the 670 Y. Shimonishi (ed): Peptide Science – Present and Future, 670–673 © 1999 Kluwer Academic Publishers, Printed in Great Britain

671 derivative DOTATOC (Figure 1), the conjugates were labeled with 1 1 1In, 6 7 Ga o r 9 0Y. Displacement binding analysis of these DOTATOC and DOTAOC radioligands using rat brain cortex membranes and nonlabeled octreotide showed that [ 6 7Ga]-DOTATOC has a KD of 0.47 nmol/l whereas that of [ 1 1 1In]-DOTATOC was 2.37 nmol/l. Similarly the [ 67Ga]-DOTAOC had a higher affinity than the [ 1 1 1 In]-DOTAOC (KD = 1.83 vs. 4.14nmol/l). Competition binding analysis using [1 2 5I]-[Tyr³]-octreotide as radioligand and DOTATOC complexed with stable isotopes of the metals gave IC 5 0 of 0.95 nM for Ga 3 +, 3.73 nM for In 3 + and 3.65 nM for Y 3 +. The corresponding values for DOTAOC were 2.04, 9.09 and 9.63 nM. These data illustrate that there is a correlation between the in vitro affinity of the different DOTA-conjugates and the coordination chemistry of DOTA-metal complexes. An imporant aspect for successful targeting with radiopeptides is the in vivo up- or down-regulation of the relevant receptors in tumor tissue. Table 1 shows that octreotide down-regulates sst2 receptors to about the same extent and with similar kinetics in vitro and in vivo so that in vitro studies are predictive for the situation found in vivo. Comparison of the biodistribution of three DOTATOCs labeled with 1 1 1 In, 67 Ga or 9 0Y showed that they all incorporate well into the tumor with comparatively low uptake into other organs or rapid clearance by the kidneys (Figure 2). It is particularly interesting that the low KD for [ 6 7Ga]-DOTATOC correlates well with the excellent ratio of tumor uptake to kidney uptake of this radiopeptide. [ 1 1 1In]-DOTATOC was clinically applied for the diagnosis of metastases and [9 0 Y]-DOTATOC was successfully used in several patients for tumor treatment by internal radiotherapy [3]. This demonstrated the clinical usefulness of these compounds and their superiority over DTPA-OctreoScan. As shown before with experimental animals, the ratio of tumor uptake to kidney

Figure 1. Structure of the [Tyr³]-octreotide analog DOTATOC containing the metal chelator DOTA (1 ,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid) at the N-terminus.

672 Table 1..

Comparison of homologous down-regulation of sst2 receptors in vitro and in vivo.

Maximum down-regulation in vitro a

Maximum down-regulation in vivo d

% of controls b

time (h) c

% of controlsb

time (h)

time (h)

10

0.5

10

0.5

16–24

Recovery of normal receptor level in vivod c

e

a AR4–2J

cells were preincubated for 0.5 to 48 h with octreotide and binding assays with intact cells were performed with 125 I-[Tyr³ ]-octreotide as radioligand. bResults are expressed as % of specific binding when compared to controls and represent the residual binding capacity after maximum receptor down-regulation. c Time after which maximum down-regulation was observed. d Octreotide was administered i.v. to tumor-bearing mice. Between 0.5 h and 48 h post-injection, tumors were removed and binding assays were performed using membranes isolated from the tumors. e Time interval after which the level of receptor expression returned to normal values.

Figure 2. Comparison of the biodistribution of DOTATOC labeled with different isotopes. Biodistribution (% I.D./g ± SEM, n = 4–7) at 4 hours postinjection of 5 µCi [ 67 Ga]-DOTATOC, [ 90 Y]-DOTATOC or [ 111 In]-DOTATOC alone (left panel) or in combination with 50 µg unlabeled octreotide (right panel) in Swiss nude mice bearing AR4-2J rat pancreatic tumor.

uptake in patients is also much more favorable for [ 111In]-DOTATOC than for [ 111 In]-OctreoScan (Figure 3). In conclusion, we demonstrate the usefulness of DOTA as a universal chelator for 111 In, 68 Ga and 90 Y that may be used also with other peptides. [68 Ga]DOTATOC is a potentially useful somatostatin receptor ligand suitable for PET, whereas [ 111 In]-DOTATOC appears to be superior to OctreoScan for SPECT. The new tracer [ 90 Y]-DOTATOC is a very promising radioligand for receptor-mediated radiotherapy.

673

Figure 3 Comparison of [ 111 In]-OctreoScan and [ 1 1 1 In]-DOTATOC applied to a patient with multiple metastases of a primary tumor of unknown origin. The scintigram was taken 24 h post injection. Note the considerably faster kidney clearance with [111 In]-DOTATOC.

Acknowledgments This work was supported by the Swiss Cancer League, the Roche Research Foundation and the Swiss National Science Foundation. We gratefully acknowledge the expert technical assistance of Mrs Sibylla Stutz and Mrs Maja Meier.

References 1. Thakur, M.L., Nucl. Med. Commun., 16 (1995) 724. 2. Krenning, E.P., Kwekkeboom, D.J., Bakker, W.H., Breeman, W.A.P., Kooji, P.P.M., Oei, H.Y., van Hagen, P.M., Postema, P.T.E., De Jong, M., Reubi, J.C., Visser, T.J., Reijs, A.E.M., Hofland, L.J., Koper, J.W. and Lamberts, S.W.J., Eur. J. Nucl. Med., 20 (1993) 283. 3. Otte, A., Jermann, E., Behe, M., Goetze, M., Bucher, H.C., Roser, H.W., Heppeler, A., MüllerBrand, J. and Maecke, H.R., Eur. J. Nucl. Med., 24 (1997) 792.

227 Fatty acid acylated insulins display protracted action due to binding to serum albumin I. JONASSEN¹ , S. HAVELUND¹, P. KURTZHALS¹, J. HALSTROM¹, U. RIBEL¹, E. HASSELAGER¹, U.D. LARSEN¹, J.L. WHITTINGHAM² and J. MARKUSSEN¹ ¹ Insulin Research, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark ² University of York, Heslington, England

Introduction Substitution therapy by naturally occurring hormone polypeptides are often hampered by rapid clearance after s.c. administration. The rapid clearance is a major obstacle and has received much attention over the years [1]. In the case of insulin this problem has been overcome by the classical strategy to produce long-acting insulin formulations of crystalline or amorphous suspensions, which after s.c. injection form a slowly dissolvable depot [2, 3]. These preparations have served the diabetic patient well for many years but a number of drawbacks are encountered with this strategy. Crystalline suspensions require shaking of the vial before injection and the rate of dissolution of the depot depends on the local blood flow through the capillaries which is influenced by exercise and temperature [4]. Although the crystalline formulations provide insulin with prolonged action there is a need for a product with longer duration of action and improved action profile. Attempts to overcome these problems by engineering a new insulin analogue with pI near the physiological pH facilitated preparation of an acid soluble formulation of analogue OPID 174 which formed a precipitate after s.c. injection [5]. However, this insulin inherited some of the drawbacks observed with traditional crystalline formulations. An alternative approach is an insulin which is soluble and remains in solution after s.c. injection having longer duration of action than NPH-insulin and smoother action profile. We have pursued this goal by providing serum albumin binding properties to insulin analogues. Serum albumin is the most abundant protein in the extracellular fluid and facilitate transport of a variety of ligands, e.g. fatty acids, metal ions, and numerous drugs and offers an almost unlimited capacity [6]. Acylation of insulin at position Nε B29 by fatty acids provides insulin with high albumin binding affinity. Results and discussion Acylation of insulin was achieved by acylation of the corresponding protected insulin derivative. Human insulin or des(B30) human insulin were reacted with 674 Y. Shimonishi (ed): Peptide Science – Present and Future, 674–677 © 1999 Kluwer Academic Publishers, Printed in Great Britain

675 Table 1. Binding of fatty acid acylated insulins to HSA and sIR (soluble insulin receptor) and disappearance from site of injection after subcutaneous injections in pigs.

Insulin

Binding to HSA Relative to NN304 N ε B 2 9 -tetradecanoyl B30-

Soluble human insulin N ε B29 -decanoyl N ε B29 -tetradecanoyl N ε B29 -decanoyl B30N ε B29 -undecanoyl B30N ε B29 -dodecanoyl B30N ε B29 -tridecanoyl B30N ε B29 -tetradecanoyl B30N ε B29 -hexadecanoyl B30NPH, human insulin

0 0.063 0.58 0.12 0.27 0.42 0.71 1.00 0.69 0

Binding to SIR Relative to human insulin

Disappearance in pigs T 50% (h)

1 0.76 0.41 0.76 n.d. 0.54 n.d. 0.46 0.19

2.0 5.1 11.9 5.6 6.9 10.5 12.9 14.3 12.4 10.5

All insulin analogues were synthesised from human insulin. B30-denotes Des-(B30) human insulin. N ε B29 -tetradecanoyl B30- was selected for clinical development.

di-t-butyldicarbonat in DMSO/triethylamine (20:1), and Gly A1, PheB1 di-Boc insulins were separated from the isomers by RP-HPLC. The hydroxysuccinimide esters of fatty acids were prepared from fatty acids and N-hydroxysuccinimide using dicyclohexylcarbodiimide in DMF for ester formation, and ethanol for crystallisation of the products. The ε-amino group of Lys B29 was selectively acylated by treatment of Gly A1 , PheB1 di-Boc insulin with the fatty acid hydroxysuccinimide esters in dimethylformamide/DMSO (1:7) at 15°C, using 20 equivalents of a tertiary amine, e.g. 4-methylmorpholine. The protecting groups were removed with trifluoroacetic acid and the LysB29 acylated insulins were purified by RP-HPLC. Binding studies were conducted using immobilised serum albumin and S IR (soluble, extracellular domain of the insulin receptor). The experimental conditions were further developed and optimised as described elsewhere [7, 8]. Pharmacokinetic and pharmacodynamic studies were carried out in conscious pigs 4–5 month of age and weighing 70–95 kg after being fasted overnight. From Table 1 it can be seen that there is a correlation between affinity for serum albumin and the chain length of the fatty acid used for acylation. Likewise, a correlation is also observed between affinity for HSA and the disappearance after s.c. injection of acylated insulins in pigs. Surprisingly, deletion of Thr B30 from the acylated insulin led to improved affinity for HSA compared to the full length counterpart. Despite introduction of a tetradecanoic acid ligand the affinity for S IR was 46% compared to human insulin. Thus N ∈B29 -tetradecanoyl des-(B30) human insulin appears to be a perfect choice for clinical development as no further increase in disappearance T 50% and affinity for HSA is obtained by changing the ligand from tetradecanoic acid to hexadecanoic acid. Disappearance from the site of s.c. injection is significantly slower for Nε B29 tetradecanoyl des-(B30) human insulin than for NPH insulin. The binding to

676 serum albumin leads to formation of insulin analogue – albumin complexes which diffuses slowly in the tissue and consequently leads to a long time of disappearance from the site of s.c. injection(Table 1). Fasted pigs were subjected to intravenous injections in random order of human insulin and NN304 insulin with one week interval (Figure 1). The plasma glucose profiles were monitored and significant differences were observed. Glucose clamp experiments demonstrated that NN304 displayed the same overall blood glucose lowering activity as NPH. However, the profile was smoother and more even compared to NPH which displayed a characteristic peak effect shortly after s.c. injection (Figure 2). The duration action has been extended and the NPH activity peak which is the main cause of nocturnal hypoglycaemic events, has been significantly reduced.

Conclusion The insulin example demonstrates that it is possible to alter pharmacokinetics and dynamics. By careful choice of ligand and site of substitution it is possible to provide the molecule with the desired characteristics without loosing potency. In this example the following characteristic of insulin has been obtained: 1. A prolonged acting insulin analogue which can be formulated as a neutral soluble preparation 2. Insulin has been provided with albumin binding properties 3. Improved duration of action compared to NPH insulin 4. Improved dynamic profile without sharp peeks of activity, i.e. smoother action

677

References 1. 2. 3. 4. 5.

Lee, V.H.L. Peptide and protein drug delivery, Marcel Dekker, New York (1990). Hallas-Møller, K., Diabetes 1 (1956) 7–14. Krayenbühl, C. and Rosenberg, T., Rep Steno Mem Hosp Nord Insulinlab 1 (1946) 60–73. Frid, A., Östman, J., and Linde, B., Diabetes Care 13 (1990) 473–477. Markussen, J., Hougaard, P., Ribel, U., and Sørensen, A.R., Protein Engineering 1 (1987) 205–213. 6. Peters, T., Adv.Protein Chem. 17 (1985) 161–245. 7. Kurtzhals, P., Havelund, S., Jonassen, I., Kiehr, B., Larsen, U.D., Ribel, U., and Markussen, J., Biochem. J. 312 (1995) 725–731. 8. Markussen, J., Havelund, S., Kurtzhals, P., Andersen, A.S., Halstrøm, J., Hasselager, E., Larsen, U.D., and Jonassen, I., Diabetologia 39 (1996) 281–288.

228 Melanotropin fragments containing difluoromethylornithine: synthesis and cytotoxicity H. SÜLI-VARGHA1, J. BÓDI 1, H. MEDZIHRADSZKY-SCHWEIGER1 , L. NAGY 1 , R. MORANDINI 2 and G.E. GHANEM2 1 Research Group for Peptide Chemistry, Hungarian Academy of Sciences, Eötvös University, H-1518 Budapest 112, POB 32, Hungary 2 L.O.C.E., Free University of Brussels, rue Heger Bordet 1, B-1000 Bruxelles, Belgium

Introduction For targeting melanoma previously we have synthesized and tested N-(2-chloroethyl)- N-nitrosourea congeners [1] and melphalan derivatives [2] of melanotropin (α-MSH) fragments. Although most of these conjugates exert receptor mediated in vitro cytotoxic activity, their potency does not surpass that of the parent drug. The reason for this may be due to the relative low number of the cell surface receptors whereby the toxic drug concentration can not be developed in the cell. Since enzymes essential for cell functioning can be inhibited in relatively low inhibitor concentration, we chose as drug the DLα-difluoromethylornithine (DFMO), the suicide inhibitor of the ornithine decarboxylase (ODC), which is vitally important in the polyamine biosynthesis and cell proliferation [3]. The therapeutic efficiency of DFMO in melanoma may be increased by coupling it to MSH fragments with receptor recognizing ability on the tumor cell.

Results and discussion We have synthesized DFMO–Lys–Pro–Val–NH 2 (1 ), DFMO–Gly–His-D -Phe– Arg–Trp–Gly–OMe (2), and DFMO–Glu–His–Phe–Arg–Trp–Gly–OMe (3). The peptides were prepared in solution and DFMO was coupled as δ -BocDFMO with the aid of BOP or HBTU reagents. The normal procedure for the introduction of the Boc group with Boc-pyrocarbonate, according to nmr, led to the formation of the δ -derivative of DFMO. Attaching the racemate DFMO to the N-terminus of optically pure peptide carriers results in epimeric DFMO-peptide congeners. In the case of 3 we were able to separate the epimeric peptides by HPLC on a silica gel column resulting in 3a and 3b. However, the separation of the epimers is not necessary, since both enantiomers of DFMO are active enzyme inhibitors. The elongation of the MSH fragments with DFMO does not alter their specific receptor recognising ability, since in the receptor binding assay the 678 Y. Shimonishi (ed): Peptide Science – Present and Future, 678–680 © 1999 Kluwer Academic Publishers, Printed in Great Britain

679 Table 1. IC 5 0 values of the DFMO-peptide conjugates and of DFMO alone in the uptake assay.

3

HTdR

IC 5 0 mM Compound

HBL

LND1

Mel180

FNBB

DFMO 1 2 3a 3b

> 2.7 0.6 0.5 0.2 0.2

2.5 0.5 1.1 0.3 –

2.7 > 1.1 1.1 0.3 –

> 2.7 > 1.1 > 1.1 > 1.4 –

DFMO-peptides behaved similarly to the natural hormone fragments, namely, the central DFMO-MSH sequences 2 and 3 are able, while the C -terminal DFMO-fragment 1 is unable to displace specifically the labelled melanotropin, (Nle 4 , D -Phe 7 ) α-MSH, from its receptor. In the frog skin bioassay 3a and 3b have similar activity (2 × 10 6 ) as their carrier peptide (1 × 106 units/mmol). The cytotoxic effect of the conjugates was measured in two human melanoma cell lines (HBL containing, LND1 lacking melanotropin receptors), in a carcinoma cell line (Me180) and in normal human fibroblasts (FNBB) by the tritiated thymidine incorporation assay with DFMO as control. After long (48 h) incubation the conjugates proved to be more efficient in the inhibition of cell growth than DFMO itself, the highest activity being shown by 3a and 3b (Table 1). Although DFMO-conjugates of the central MSH hexapeptide derivatives, 2 and 3 are capable of specifically binding to the hormone receptors, there was no higher cytotoxicity shown in the receptor positive HBL cells than in the other cell cultures. The cell growth inhibition is obviously due to the release of DFMO from the carrier peptide because, according to the experimentally proved ODC inhibition mechanism, a free α -carboxyl group is required on the inhibitor to achieve irreversible inhibition. On the other hand, according to our previous investigations, MSH fragments do not inhibit cell proliferation. Thus the DFMO-peptide congeners may be considered as prodrugs, from which free DFMO is released by enzymatic hydrolysis. In contrast to peptide nitrosourea congeners and to melphalan-peptides, this prodrug approach resulted in peptide–drug conjugates with higher cytotoxicity compared to that of the parent drug.

Acknowledgement This work was supported by the Hungarian National Research Fund (OTKA T 015709).

680 References 1. Jeney, A., Kopper, L., Nagy, P., Lapis, K., Süli-Vargha, H., and Medzihradszky, K., Cancer Chemother. Pharmacol., 16 (1986) 129. 2. Morandini, R., Süli-Vargha, H., Libert, A., Botyánszki, J., Medzihradszky, K., and Ghanem, G., Int. J. Cancer, 56 (1994) 129. 3. McCann, P.P. and Pegg, A.E., Pharmacol. Ther., 54 (1992) 195.

229 Synthesis of RANTES-related peptides and their effect on human immunodeficiency virus-1 infection Y. NISHIYAMA1 , T. MURAKAMI 2 , K. KURITA1 and N. YAMAMOTO2 1 Department of Industrial Chemistry, Faculty of Engineering, Seikei University, Musashino-shi, Tokyo 180, Japan 2 Department of Microbiology, Tokyo Medical and Dental University School of Medicine, Bunkyo-ku, Tokyo 113, Japan

Introduction RANTES (regulated upon activation, normal T-cell expressed and secreted), one of the CC-chemokines, is a chemotactic and activating agent for a variety of leukocytes including T-lymphocytes [1]. RANTES as well as macrophage inflammatory protein (MIP)-1α and MIP-1β has been reported to inhibit infection with human immunodeficiency virus (HIV)-1 in vitro by interacting with CC-chemokine receptor-5 (CCR-5), a coreceptor for macrophage-tropic HIV-1 [2–4]. This suggests the possibility of effective treatment of HIV-1 infected individuals, if appropriate sites of CCR-5 could be blocked with lowmolecular-weight ligands. Our attention has been focused on the structural requirements for the anti-HIV-1 activity of chemokines to develop a novel class of anti-HIV-1 agents. The first ten of 68 amino acid residues of the RANTES molecule were known to be critical for the intrinsic chemotactic activity [5], and we have reported that the synthetic peptide corresponding to this region, Ac-( 10 Ala-RANTES 1–10)-NH2 , exhibited potent anti-HIV-1 activity [6]. On the other hand, H-(RANTES 9–68)-OH, which lacks the first eight amino acids, was also reported to be a potent anti-HIV-1 peptide [7]. These results imply the existence of multiple anti-HIV-1 domains in RANTES. We report here the synthesis and anti-HIV-1 activity of a series of overlapping peptides, which scan the whole sequence of RANTES.

Results and discussion All peptides were prepared by the Fmoc-based solid-phase method, purified by reversed-phase HPLC, and characterized by FAB-MS. All the Cys residues were replaced with Ala to prevent the disulfide bond formation. Phytohemagglutinin-activated peripheral blood mononuclear cells were infected with HIV-1 J R - C S F and cultured in the presence or the absence of synthetic peptides for 7 days. The amount of soluble HIV-1 p24 in each culture supernatant was determined by enzyme-linked immunosorbent assay. 681 Y. Shimonishi (ed): Peptide Science – Present and Future, 681–683 © 1999 Kluwer Academic Publishers, Printed in Great Britain

682

Figure 1.

Anti-HIV-l activity of RANTES-related peptides

As shown in Figure 1, Ac-(RANTES 37–46)-NH 2 , Ac-(RANTES 53–62)NH 2 , and Ac-(RANTES 57–68)-NH 2 possessed potent anti-HIV-1 activity in addition to Ac-( 10 Ala-RANTES 1–10)-NH 2 and Ac-( 10 Ala-RANTES 5–14)NH 2 reported previously [6]. These results clearly indicate the existence of the multiple domains responsible for anti-HIV-1 activity. The small anti-HIV-1 peptides found here as well as the ones reported previously would be promising lead compounds for the development of coreceptor-directed anti-HIV-1 agents.

Acknowledgments We are grateful to Asahi Chemical Industry Co. Ltd. for FAB-MS measurement. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (10180229) from the Ministry of Education, Science, Sports and Culture of Japan.

References 1. Schall, T.J., Cytokine 3 (1991) 165–183. 2. Cocchi, F., DeVico, A.L., Garzino-Demo, A., Arya, S.K., Gallo, R.C. and Lusso, P., Science 270 (1995) 1811–1815. 3. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Marzio, P.D., Marmon, S., Sutton, R.E., Hill, C.M., Davis, C.B., Peiper, S.C., Schall, T.J., Littman, D.R. and Landau, N.R., Nature 381 (1996) 661–666.

683 4. Dragic, T., Litwin, V., Allaway, G.P., Martin, S.R., Huang, Y., Nagashima, K.A., Cayanan, C., Maddon, P.J., Koup, R.A., Moore, J.P. and Paxton, W.A., Nature 381 (1996) 667–673. 5. Wells, T.N.C., Guye-Coulin, F. and Bacon, K.B., Biochem. Biophys. Res. Commun. 211 (1995) 100–105. 6. Nishiyama, Y., Murakami, T., Kurita, K. and Yamamoto, N., Chem. Pharm. Bull., 45 (1997) 2125–2127. 7. Arenzana-Seisdedos, F., Virelizier, J.–L., Rousset, D., Clark-Lewis, I., Loetscher, P., Moser, B. and Baggiolini, M., Nature 383 (1996) 400.

230 The influence of structural motifs of amphipathic peptides on the permeabilization of lipid bilayers and the antibacterial and hemolytic activity M. DATHE 1 , D. MACDONALD 2, W.L. MALOY 2, M. BEYERMANN1, E. KRAUSE1 and M. BIENERT1 1 2

Institute of Molecular Pharmacology, Alfred Kowalke Str. 4, D-10315 Berlin, Germany Magainin Pharmaceuticals, Inc., 5110 Campus Drive, Plymouth Meeting, PA 19462 USA

Introduction Cationic, amphipathic, α -helical peptides are membrane active and able to disturb the barrier function of cell membranes [1, 2]. Some of the peptides possess antibacterial selectivity and are therefore of potential clinical relevance. Based on the hypothesis that the membrane activity is modulated by global structural features of the peptides [3] we investigated the role of hydrophobicity (H), the hydrophobic moment (µ ) and the angle subtended by the hydrophobic helical face on the two steps of the membrane disturbing process: binding and membrane permeabilization. The peptide-induced effect on negatively charged phosphatdylglycerol (PG) and neutral phosphatidylcholine (PC) membranes was investigated using CD spectroscopy and fluorescence methods. The permeabilization of lipid vesicles was related to the antibacterial and hemolytic effect.

Results and discussion Starting from the sequence KLALK LALKA WKAAL KLA NH 2 (KLA1; H = – 0.025, µ = 0.329, = 280°) sets of model peptides were synthesized with one of the parameters being modified whereas the others were kept almost constant. CD investigations revealed peptide helicities between 50 to 60% in the lipid bound state. All analogs induced dye release from negatively charged liposomes at sub-micromolar concentrations. The effect was dominated by a high affinity as a result of strong electrostatic interactions and scarcely influenced by modification of H and µ (Figure 1). The peptides appeared to remain at the membrane surface. However, reduction of distinctly impaired the permeabilizing effect. High activity at large is associated with a deeper insertion of the hydrophobic peptide domain into the membrane and a reduced surface area of the polar face at the membrane interface. With reduction of the negative membrane charge the structural motifs gained increasing importance for peptide-membrane interaction. Despite very low 684 Y. Shimonishi (ed): Peptide Science – Present and Future, 684–686 © 1999 Kluwer Academic Publishers, Printed in Great Britain

685

Figure 1. Permeabilizing acitivity of KLA peptides of different H, µ and ψ on POPC and POPG vesicles (EC 5 0 -concentration of the half maximal effect).

binding, the hydrophobic model peptide KLA1 was active at nanomolar concentrations in permeabilizing neutral bilayers (Figure 1). This high activity can be attributed to a substantially enhanced permeabilizing efficiency of the peptides at the neutral lipid membrane and can be explained by very effective hydrophobic interactions between the nonpolar helix face and the lipid acyl chains thus disturbing the architecture of the non-polar bilayer region. Reduction of H and µ lead to substantially reduced peptide binding and lowered the permeabilizing efficiency. Interestingly, at the neutral membrane the effect of changes of was comparably low thus highlighting the dominance of peptide hydrophobicity. The effects on charged and neutral model membranes correlated with the antibacterial and hemolytic activity, respectively. Decrease of each individual parameter caused significantly reduced hemolysis while the effect against bacteria was much less pronounced (except the loss of activity at low H), thus resulting in enhanced antibacterial specificity. In conclusion, the investigations showed: (i) The are effective modulators of peptide-membrane structural motifs, H, µ and interactions. (ii) Hydrophobic interactions, disturbing the inner membrane region are more effective in membrane permeabilization than a high peptide accumulation at the negatively charged membrane surface. With respect to cationic helical peptides (iii) the hydrophobic parameters have little influence on peptide activity against negatively charge membranes (E. coli ) but (iv) specificity seems to be achieved with peptides of moderate hydrophobicity, low hydrophobic moment and large angle of the hydrophobic domain.

686 References 1. Saberwal, G. and Nagaraj, R., Biochim Biophys. Acta 1197 (1994), 109. 2. Cornut, I., Thiaudiere, E., and Dufourcq, J., in Epand, R.M. (Ed.) The amphipathic helix, CRC Press Inc., Boca Raton, 1993, pp 173. 3. Dathe, M., Wieprecht, T., Nikolenko, H., Handel, L., Maloy, L., MacDonald, D.L., Beyermann, M., and Bienert, M., FEBS Letters 403 (1997) 208.

231 Proline at position 14 of alamethicin is essential for the hemolytic activity, catecholamine secretion from chromaffin cells and enhanced metabolic activity in endothelial cells M. DATHE¹ , C. KADUK¹ , E. TACHIKAWA² , H. WENSCHUH³ , M. MELZIG 4 and M. BIENERT 4 ¹ Institut für Molekulare Pharmakologie, Alfred Kowalke Str. 4, 10409 Berlin, Germany ² School of Medicine, Iwate Medical University, Uchimaru 19-1, Morioka 020, Japan ³ Max-Planck-lnstitut für Infektionsbiologie, Monbijoustr. 2, 10117 Berlin, Germany 4 Institut für Pharmazie, Humboldt-Universität, Goethestr. 54, 13086 Berlin, Germany

Introduction Alamethicin from the fungus trichoderma virides is an ion channel forming peptaibol with hemolytic and antibacterial activity [1]. Proline at position 14 of the sequence (AcAibProAibAlaAibAlaGlnAibValAibGlyLeuAibProValAibAibGluGlnPheol), a highly conserved structural motif in many related peptides, has been suggested to play a crucial role for the function of alamethicin [2]. Any change in its position has recently been described to distinctly influence voltage induced conductivity and single channel formation in lipid bilayers [3]. This study investigated the effect of the shift of proline 14 towards the N - and C -terminal extremity of the peptide chain on (i) hemoglobin release from human red blood cells, (ii) catecholamine secretion from bovine adrenal chromaffin cells and (iii) metabolic activity of bovine aortic endothelial cells. Additionally, the peptide induced permeabilization of lipid membranes was studied by fluorescence spectroscopic determination of dye efflux from neutral large unilamellar vesicles.

Results and discussion Alamethicin exhibits activities on different mammalian cells in the micromolar concentration range. Half maximal hemolytic activity (EC50 ) was found at 30 µM alamethicin, and total lysis was reached at 100 µM. Incubation of bovine adrenal chromaffin cells with alamethicin in the presence of extracellular Ca2 + caused peptide concentration dependent secretion of catecholamines up to 50 µM. Obviously, the effect is initiated by the influx of Ca2+ into the cells which has been described to be essential for triggering catecholamine secretion. Mild release of catecholamines in the absence of Ca2+ has to be associated with cell membrane damage. Furthermore, alamethicin transiently stimulated 687 Y. Shimonishi (ed): Peptide Science – Present and Future, 687–688 © 1999 Kluwer Academic Publishers, Printed in Great Britain

688 Table 1. Helicity of alamethicin peptides in buffer and in the presence of POPC vesicles and the permeabilizing activity on POPC bilayers. Peptide induced hemolysis of ³human red blood cells, catecholamine secretion from 4 bovine adrenal chromaffin cells (in the presence (+) and absence (–) of Ca 2+ ) and metabolic activity of 5 bovine aortic endothelial cells. (Cp -peptide concentration, + + + high effect, – no effect).

Position of Pro

Helicity (%) C p = 25 µM POPC buffer (10 mM)

Dye release (%) C p = 1.8 µM POPC (12 µM)

Hemolysis³ (%) (C p = 30µM)

Pro 11 Pro 12 Pro 13 Pro 14 Pro 15 Pro 16 Pro 17

8 18 7 7 6 8 5

– – – 50 – – –

– – – 50 – – –

52 85 27 44 24 42 38

Catecholamine secretion 4 (%) C p = 50 µM Ca(+)

Ca(–)

Metabolic activity 5

1.5 1.4 0.9 55.5 2.0 2.0 1.8

– – – 4.3 – – –

– – – + + + – – –

the metabolic rate of endothelial cells in a concentration dependent mode up to 20 µM. The observed inhibition of metabolism at higher concentrations point to a toxic effect. All analogs with proline at position 11, 12, 13, 15 and 16 were completely inactive in the biological assays. The loss of activities correlated with the reduction of channel forming properties in a lipid bilayer [3] and with the loss of lipid bilayer permeabilizing activity. Studying peptide induced dye release from phophatidylcholine vesicles, the native alamethicin was found to be most active. At the EC50 of 1.8 µM all analogs did not show any effect. The activity on the different membrane systems did not correlate with peptide helicity in the vesicle bound state but appeared to be dependent on peptide accumulation at the lipid matrix. Thus, we associate the changes in activities with modifications of membrane affinity rather than conformational changes of the peptides. In conclusion the results show that alamethicin exhibits different biological activities which are strongly related to proline at position 14 of the native sequence.

References 1. Sansom, M.S.R. in Noble, D., Blundell, T.L (Eds.) Progress in Biophysics and Molecular Biology, Pergamon Press, Oxford, 1991, pp 139. 2. Fox, F.O. and Richards, F.M., Nature 330 (1992) 325. 3. Kaduk, Ch. Duclohier, H., Dathe, M., Wenschuh, H., Beyermann, M., Molle, G., and Bienert, M., Biophys. J. 72 (1997) 2151.

232 Enkephalin metabolism in rat brain after bestatin administration N.A. BELYAEV¹, E.F. KOLESANOVA², V.Y. BARONETS¹, N.N. TEREBILINA¹ and L.F. PANCHENKO¹ ¹ State Center for Medico-Biological Problems of Narcology, 3, M. Mogilstevskii per., 119121, Moscow, Russia ² Institute of Biomedical Chemistry, 10, Pogodinskaya ul., 119832, Moscow, Russia

Introduction Bestatin, [(2S,3R)-amino-2-hydroxy-4-phenylbutanoyl]-1-leucine, is a potent inhibitor of different aminopeptidases with K i 10 –6 –10 –7 M. Since brain enkephalins are known to be at least partially catabolized with aminopeptidases [1], bestatin may affect brain enkephalin contents, as well as other parameters of enkephalinergic system. This supposition was proved indirectly by observing enkephalin-like analgetic effects after intracisternal (i.c.) and conjunctival (conj.) applications of bestatin in rats [2]. This work presents the results of the study of bestatin effects on biochemical parameters of brain enkephalinergic system.

Results and discussion Met- and Leu-enkephalin (Met- or Leu-enk) contents, [³H]-D-Ala²-D -Leu 5 enkephalin ([³H]DADL) binding, enkephalinase A, aminopeptidase M, arylamidase, and enkephalin convertase activities were determined in rat midbrains + hypothalami and striata as described earlier [1, 3]. Bestatin was either injected i.c. as a dimethylsulfoxide solution to cannulated rats (5 µ1 per animal, bestatin dose 0.7 mg/kg weight), or applied onto conjunctiva of rats as a solution in 0.9% NaCl (10 µ1 per each eye, bestatin dose 0.4 mg/kg weight). Control animals received solvent administrations. These bestatin doses were proven to cause analgesia in tail-flick tests [2]. Acute i.c. injection of bestatin resulted in reliable changes of Met-enk levels in rat brain: elevation (20 min and 24 h after the injection, and decrease at the end of the 2-h interval. No significant changes in Leu-enk contents, as well as in both enk contents in striatum was observed. Complex character of midbrain Met-enk content changes pointed out to the possible existence of compensation mechanisms which regulate this parameter afters its deviation because of bestatin effect. Studies of the dynamics of various brain enkephalinergic system parameters after conj. application of bestatin proved the existence of such mechanisms 689 Y. Shimonishi (ed): Peptide Science – Present and Future, 689–690 © 1999 Kluwer Academic Publishers, Printed in Great Britain

690 Table 1. Relative changes in enkephalinergic system parameters in rat brain regions after conj. application of bestatin (% of control values; *-p < 0.05, **-p < 0.01, ***-p < 0.001). Midbrain Parameter Met-enk Leu-enk [3H]DADL binding Enkephalinase A activity Aminopeptidase M activity Arylamidase activity Soluble enkephalin converse activity Membrane enkephalin convertase activity

0.3 h 85 85

Striatum 1.5 h

3h

24 h

0.3 h

1.5 h

75* 85

101 109

127* 124*

102 117

112 96

3h 90 47***

24 h 89 80

91

101

103

141*

115*

98

112

99

102

93

92

120

111

112

136

67

92

81*

100

110

768

90

100

87

83**

97

113

117*

127*

128**

103

105

103

108

99

91

121**

110 95

107

93

138*

more reliably. Parameter changes in the experiment in relation to control values are presented in Table 1. Moreover, bestatin application did not only change parameter values, but caused the disturbance of the normal-state diurnal dynamics of the most parameters. Reciprocal interrelations between one and the same parameter in midbrain and in striatum also differed significantly in bestatin-applicated animals than in control ones. Thus, bestatin influences the function state of enkephalinergic system in midbrain and in striatum. This may underlie the use of bestatin as a regulator of this system in the cases when its disturbances take place. However, bestatin may cause not only direct, but also indirect action on enkephalinergic system functioning, the latter including compensation mechanisms, as well as possible effects of bestatin on kytorphin and angiotensin brain systems.

References 1. Belyaev, N.A., Kolesanova, E.F., Baronets, V.Yu., Annaeva, E.R., Pozdnev, V.F., and Panchenko, L.F., Biochemistry (Russ.) 55 (1990) 1778. 2. Belyaev, N.A., Kolesanova, E.F., Rotanova, T.V., Kelesheva, L.F., and Panchenko, L.F., Byull. Eksp. Biol. Med. 110 (1990) 474. 3. Belyaev, N.A., Kolesanova, E.F., Baronets, V.Yu., and Panchenko, L.F., Biochemistry (Moscow) 58 (1993) 721.

233 Synthetic antibacterial peptides derived from insect defensin isolated from a beetle, Allomyrina dichotoma H. SAIDO-SAKANAKA¹, J. ISHIBASHI¹, A. MIYANOSHITA² and M. YAMAKAWA¹ ¹ Laboratory of Biological Defense, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan. ² Laboratory of Applied Entomology, Faculty of Agriculture, University of Tokyo, Tokyo 113-8657, Japan

Introduction Defensin from a beetle (Allomyrina dichotoma), that consists of 43 amino acid residues, is known to have antibacterial activity against Gram-positive bacteria but not against Gram-negative bacteria [1]. In addition, this peptide is shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA). It has potential as a therapeutic agent against bacterial infections. However, this antibacterial peptide is too large to be a therapeutic molecule because of its antigenicity. Short peptides having antibacterial activity are suitable for design of therapeutic agents.

Results and discussion To determine the active site, 64 overlapping, 12mer peptides with either free carboxylates or amides at their C-terminal spanning the sequence of the defensin were synthesized by multi-pin technique (Figure 1) [2]. An amidated 19L-30R

Figure 1. The overlapping synthetic peptides spanning the sequence of defensin from a beetle. 64 overlapping, 12mer peptides with either free carboxylates or amides at their C-terminus were synthesized using multi-pin technique.

691 Y. Shimonishi (ed): Peptide Science – Present and Future, 691–692 © 1999 Kluwer Academic Publishers, Printed in Great Britain

692 Table 1. Minimal inhibitory concentration (MIC) of synthetic analogues of the 19L-30R fragment. MIC was determined in liquid medium (nutrient broth, Difco) after incubation for 10 h at 37°C.

Defensin LCAAHCLAIGRR-NH 2 (19L-30R) LCAAHCLAIRRR-NH 2 LCAAHCLAILRR-NH 2 LCAARCLAIGRR-NH 2 LCAALCLAIGRR-NH 2 LRAAHRLAIGRR-NH 2 LLAAHLLAIGRR-NH 2

S.aur

MIC(µg/Ml) MRSA

E.coli

4 18 7.5 10 7.5 6 6 10

30 64< 64< 64< 64< 48 64 64
290 min vs. that of Dyn(1–8) = 47 min). HPLC analysis of the metabolites after prolonged digestion revealed that the Leu 4 –Arg 5 and/or Arg5 –Arg 6 bonds of these analogs are most labile to plasma enzymes. 701 Y. Shimonishi (ed): Peptide Science – Present and Future, 701–703 © 1999 Kluwer Academic Publishers, Printed in Great Britain

702 Table 1. Opioid receptor binding affinities and GPI potencies of Der–Dyn hybrid analogs and dynorphin(1–13). GPI

Ki (nM) 3

Peptides ( 1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

3

3

[ H] U-69593 (κ )

[ H]DAMGO (µ)

[ H]DPDPE (δ)

IC 5 0 (nM)

shift fold a

0.047 ± 0.014 1.78 ± 0.56 1.94 ± 0.26 1.88 ± 0.32 202 ± 29 502 ± 103 2.99 ± 0.38 1.58 ± 0.47 0.135 ± 0.008 0.215 ± 0.036 0.659 ± 0.247

0.813 ± 0.273 57.0 ± 5.7 115 ± 13 50.1 ± 12.7 70.6 ± 20.4 10.5 ± 2.6 45.5 ± 21.5 8.59 ± 1.10 32.9 ± 1.4 110 ± 25 75.8 ± 12.5

–

0.11 85.8 39.3 21.6 708 111 535 128 50.3 54.9 56.8

n.s. b n.s. n.s. n.s. 4.5 39.7 n.s. n.s. n.s. n.s. n.s.

786 ± 145 151 ± 52 308 ± 179 469 ± 137 162 ± 54 1045 ± 335 516 ± 151 430 ± 141 408 ± 99 962 ± 431

α Abbreviations: D-alanine; ψ , ψ (CH 2 NH) peptide bond; meR, N -methyl-arginine. a shift fold of IC5 0 in the presence of µ-antagonist CTAP (1 µM). b not significant.

Next, we synthesized several hexapeptide analogs of 3 modified mainly by peptide backbone with a reduced peptide bond (CH2 NH) or by C-terminal alkylamides. Introduction of the CH2 NH bond at Arg5 –Arg 6 ( 6 ) or Leu4 –Arg 5 (7) did not hamper the κ receptor affinity, but proved detrimental to κ receptor selectivity due to increase in µ receptor affinity (7 ). Interestingly, the C-terminal alkylation led to analogs (8 ~ 10 ) with high affinity and selectivity for κ receptor. The in vitro bioactivity of all analogs were also measured by GPI assay to estimate potency at peripheral κ receptors. The interaction of peptides with κ receptors was clarified using CTAP, a specific µ receptor antagonist [4]. Results showed that IC50 values of penta- and tetrapeptide analogs (4 , 5) shifted significantly upon addition of CTAP, indicating the involvement of µ receptors in the GPI response triggered by 4 and 5 . These results coincide well with those of binding assays. No significant shift in potency was observed with the other analogs, suggesting that the hexapeptide analogs tested in this study can specifically interact with κ receptors in the periphery. The results of 8 , 9 a n d 10, which show relatively lower GPI potencies than expected from the binding assays, also suggest that these analogs can discriminately interact with central κ receptors, whereas Dyn(1–13) can indiscriminately do with both central and peripheral κ receptors.

References 1. Corbett, A.D., Paterson, S.J., McKnight, A.T., Magnan, J., and Kosterlitz, H.W., Nature, 295 (1982) 339. 2. Corbett, A.D., Paterson, S.J., McKnight, A.T., Magnan, J., and Kosterlitz, H.W., Nature, 299 (1982) 79.

703 3. Sasaki, Y., Hosono, M., Fujita, H., Suzuki, K., Sakurada, S., Sakurada, T., and Kisara, K., Biochem. Biophys. Res. Commun., 130 (1985) 964. 4. Kramer, J.H., Shook, J.E., Kazmierski, W., Ayres, E.A., Wire, W.S., and Hruby, V.J., J. Pharmacol. Exp. Thear., 249 (1989) 544.

239 Selective cytotoxicity of dermaseptins J. GHOSH¹, I. KUSTANOVICH², Y. SHAI¹ and A. MOR² ¹ Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel ² The Wolfson Center for Applied Structural Biology, The Hebrew University of Jerusalem, Givat Ram 91904 Jerusalem, Israel

Introduction Despite a large heterogeneity in primary and secondary structures, antimicrobial peptides are active against a large spectrum of microorganisms, yet most of them are generally non-toxic toward mammalian cells [1, 2]. Illustrating this selectivity, we recently showed that the malaria parasites that live inside erythrocytes, are also targeted by the dermaseptins peptides, i.e., seconds after addition of dermaseptins, the peptides accumulated within the parasites. This sharply contrasted with the labeling patterns of non infected cells where dermaseptins bound to the erythrocytes plasma membrane only. Moreover, both dermaseptin S3 (DS3) and dermaseptin S4 (DS4) affected the viability of the intracellular parasite, but DS3 was cytotoxic only for the malaria parasite, whereas DS4 was toxic also for erythrocytes [3]. Towards understanding the molecular mechanisms involved in the selective cytotoxicity of dermaseptins, various labeled and unlabeled versions of DS3 and DS4 were investigated at three different levels: peptide interaction with erythrocytes, peptide–peptide interaction in solution and peptide interaction with model phospholipid membranes.

Results and discussion Addition of DS4 (labeled or unlabeled) to RBC resulted in instantaneous hemolysis. The ghosts were labeled exclusively at their periphery, where peptide aggregates were clearly observable. HPLC analysis of the post-hemolysis supernatant indicated that DS4 binding to RBC was linear over a wide range of concentrations. DS3 also induced hemolysis, however, the DS3 induced hemolysis was 100 fold less efficient than DS4. DS3 bound to RBC almost to the same extent as DS4 yet did not induce massive hemolysis. The aggregational properties of DS3 and DS4 in aqueous solution were determined using rhodamine labeled peptides. Although the concentrations of Rho–DS3 and Rho–DS4 were nearly equal, Rho–DS4 was less fluorescent (> pGlu1, Leu³.

References 1. Nandha, K.A. and Bloom, S.R., Biomed. Res., 14, Supplement 3, 71–76 (1993). 2. Sakura, N., Kurosawa, K., Naminohira, S., Sakai, T., and Hashimoto, T., Peptide Chemistry 1992, Yanaihara, N. (Ed.), ESCOM Science Publishers B. V., Leiden, p406–408 (1993). 3. Hashimoto, T., Kurosawa, K., and Sakura, N., Chem. Pharm. Bull., 43, 1154–1157 (1995). 4. Kurosawa, K., Sakura, N., and Hashimoto, T., Chem. Pharm. Bull., 44,1880–1884 (1996). 5. Kurosawa, K., Sakura, N., and Hashimoto, T., Peptide Chemistry 1994, Ohno, M. (Ed.), Protein Research Foundation, Osaka, p 325–328 (1995).

244 Cyclic peptides having inhibitory site of α-amylase inhibitor tendamistat T. HIRANO¹, S. ONO¹, H. MORITA¹, T. YOSHIMURA¹, H. YASUTAKE², T. KATO² and C. SHIMASAKI¹ ¹ Department of Chemical and Biochemical Engineering, Toyama University, Gofuku 3190, Toyama 930, Japan ² Deptartment of Applied Microbial Technology, Kumamoto Institute of Technology, Kumamoto 860, Japan

Introduction Tendamistat (HOE 467) is an extracellular protein from Streptomyces tendae 4158 and shows strong inhibitory activity for porcine pancreatic α -amylase (PPA) (Ki = 0.2 nM) [1]. Tendamistat is 74 amino acids long and consists of two separated cyclic parts (27 and 17 amino acids long). The 3D-structure have been studied by X-ray crystallography and NMR and consequentry the inhibitory site (Ser 17–Trp 18 –Arg 19 –Tyr 20 ) is known to exist in the small cyclic part [2–5]. In this study, to design potent and small inhibitors for α -amylase, five short cyclic peptides bridged by a disulfied bond between N- and C-terminal Cys residues, Ten(16–22), Ten(15–23), Ten(14–24), Ten(13–25), and Ten(12–26), were synthesized (Figure 1). Their inhibitory activities against PPA and circular dichroism (CD) spectra in buffer and TFE were measured.

Results and discussion All five peptides were synthesized by manual solid phase method on Fmoc chemistry. After cleavage and deprotection of the side chain protecting groups, the peptides were purified by reverse-phase HPLC followed by air-oxidation in ammonium acetate solution (pH 8.0) to give the desired products. The final products established by MALDI-TOF-MS and amino acid analysis. The inhibitory activities of the five cyclic peptides for PPA were measured using 2-chloro-4-nitrophenyl 4 4-O- β- D -galactopyranosyl- β-maltotetraoside as a substrate. Ten(15–23) showed stronger inhibitory activity than any other peptides. Ten(16–22) also exhibited potent inhibitory activity, while no inhibitory activity was observed for Ten(12–26), Ten(13–25) and Ten(14–24) under

Figure 1. Amino acid sequence of the small loop of Tendamistat.

716 Y. Shimonishi (ed): Peptide Science – Present and Future, 716–718 © 1999 Kluwer Academic Publishers, Printed in Great Britain

717

Figure 2.

Dixon plots of Ten( 15–23) for PPA.

the same conditions. As a result of kinetic analyses, it was found that Ten(15–23) and Ten(16–22) inhibit PPA competitively with Ki values of 0.29 ± 0.1 µM and 0.83 ± 0.2 µM, respectively (Figure 2). To examine the structure-inhibitory activity relationship, CD spectra for the five cyclic peptides in 20 mM Tris-HCl buffer (pH 7.0) and in TFE were compared. Ten(15–23) showed a different CD spectrum from those for other peptides under these conditions (data not shown). The CD data in the buffer and TFE suggested that Ten(15–23) exhibits a tendency to form β -structural conformation. Since TFE is known to break peptide backborn structure, TFEsensitivity of CD spectra for the cyclic peptides were examined. TFE-induced

Figure 3.

Effect of TFE on the structure of Ten(15–23).

718 CD spectra for Ten(15–23) showed a minimum at 213 nm and a shoulder at 233 nm (Figure 3). This TFE-sensitivity differed from those for other peptides, supporting the characteristic structural property of Ten(15–23). In conclusion, Ten(15–23) exhibited most potent inhibitory activity for PPA. The inhibitory activity should result mainly from a stabilization effect of the β -structural conformation due to its suitable peptide loop size. Therefore, the present results suggested that Ten(15–23) is useful for the design of small α -amylase inhibitors as a lead compound.

References 1. Vertesy, L., Oeding, V., Bender, R., Zepf, K., and Nesemann, G., Eur, J. Biochem., 141 (1984) 505–512. 2. Pflugrath, J.W., Wiegand, G., Huber, R., and Vertesy, L., J. Mol. Biol., 189 (1986) 383. 3. Kline, A.D., Braun, W., and Wuthrich, K., J. Mol. Biol., 189 (1986) 367–382. 4. Wiegand, G., Epp, O., and Huber, R., J. Mol. Biol., 247 (1995) 99. 5. Oconnell, J.F., Bender, R., Engels, J.W.K.-P., Scharf, M., and Wuthrich, K., Eur. J. Biochem., 220 (1994) 763.

245 Doxorubicin liposomal preparations coated with Laminin related peptides D. POLO, I. HARO, D. CUNILLERA and F. REIG Department of Peptide and Protein Chemistry. C.I.D.-C.S.I.C., Jordi Girona 18, Barcelona 08034, Spain

Introduction Liposomes have proved to be a good carrier system for cytostatics; doxorubicin liposomal preparations, show higher antitumoral activity and lower toxicity than free drug solutions. [1, 2]. Targeting liposomes loaded with cytostatics, to tumoral cells would improve its efficacy and reduce toxicity of conventional treatments [3,4].

Results and discussion Specific killing of cancer cells can be achieved by exploiting differences in cell surface expression of laminin receptors. Two peptides belonging to laminin active sites were synthesized: YIGSR (NH2) (Peptide 1) and GYSRARKEAASIKVAVSARKE (Peptide 2), as well as two inactive sequences: PEAGD (Peptide 3) and NIDTTDPEAGDKDTGRGLK (Peptide 4). The incorporation of these peptides to the surface of liposomes was carried out following different methodologies. Liposomes common constituents were: hydrogenated egg phosphatidyl choline (HEPC), egg phosphatidylglycerol (EPG) and cholesterol (Chol) (1: 1: 2, molar). In some cases 10% of N -glutaryl phosphatidyl-ethanolamine (NGPE) was incorporated in order to provide a carboxy terminal group to link the peptide. Polyethyleneglycol (PEG) was also added in some experiments (6%) to the lipid mixture either as monomethoxy PE-PEG or as PE-PEG-COOH. Peptide linkage to the liposomal surface was carried out using N -ethyl- N'(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (NHSS) as coupling agents. Peptide hydrophobic derivatives were prepared linking decanoic and myristic acids and cholesterol hemisuccinate, to the amino terminal end of YIGSR(NH2) . These derivatives were incorporated to liposomes either by incubation with preformed liposomes (Method A) or by mixing with lipids (Method B). Doxorubicin was the cytostatic of choice for all of these preparations. Liposomes were characterized by size, PL/Drug and PL/peptide relationship. Results obtained are summarized in Table 1. 719 Y. Shimonishi (ed): Peptide Science – Present and Future, 719–721 © 1999 Kluwer Academic Publishers, Printed in Great Britain

720 Types of liposomes: Type A: HEPC/EPG/Chol/Decanoyl-Peptide 1/DX Type B: HEPC/EPG/Chol/Myristoyl-Peptide 1/DX Type C: HEPC/EPG/Chol/Cholesteryl-Peptide 1/DX Type D: HEPC/EPG/Chol/NGPE-Peptide 1/DX Type E: HEPC/EPG/Chol/PE-PEG/NGPE-Peptide 1/DX Type F: HEPC/EPG/Chol/NGPE/DSPE-PEG-Peptide 1/DX Type G: HEPC/EPG/Chol/NGPE-Peptide 2/DX Type H: HEPC/EPG/Chol/DSPE-PEG-Peptide 2/DX Type I: HEPC/EPG/Chol/DSPE-PEG-Peptide 3/DX Type J: HEPC/EPG/Chol/DSPE-PEG-Peptide 4/DX The incorporation of hydrophobic peptides to the initial phospholipid mixtures (Method B) increases the mean diameter of samples if compared with samples prepared incubating vesicles previously formed with hydrophobic peptides (Method A). Concerning Doxorubicin entrappment, liposomes obtained following Method B give sligthly lower yields of encapsulation than the rest of preparations. Peptide incorporation to the surface of liposomes determined for hydrophobic peptides is maxima for the Myristoyl derivative in both types of liposomes. Chemical linkage of free peptides to the surface of liposomes either by NGPE or DSPE-PEG-COOH do not modify the size of liposomes. Moreover, best results are obtained using the DSPE-PEG-COOH derivative. This suggests that steric effects can play a role, being yields maxima for less hindered carboxyl groups. Table 1. Liposomes

(nm)

[PL]/[Doxo] (mg/mg) (µmol/mg)

Hydrophobic Peptide 1: Method A 112 6.8 Type A Type B 113 6.7 Type C 96 6.9

[PL]/[Pept.] (mol/mol) (µmol/mg)

Doxo %

Peptide %

8.1 8.1 8.3

22.0 14.3 34.1

29.4 17.8 32.2

85.0 86.1 83.2

43 71 39.2

Hydrophobic Peptide 1: Method B 143 8.5 10.2 Type A Type B 235 8.5 10.2 Type C 129 7.9 9.5

24.9 13.8 46.2

33.3 17.2 43.5

67.8 67.8 72.4

37.5 62.9 28.3

Peptides 1 and 2 linked by NGPE 95 8.8 10 Type D Type E 80 5.8 6.6 Type G 115 7.2 8.2

13.5 11.2 17.2

22.7 18.8 7.4

63.0 79.1 88.8

36 35 46

Peptides 1,2,3 and 4 linked by DSPE-PEG-COOH 90 5.2 5.9 9.6 Type F Type H 85 7.1 8.1 15.2 Type I 82 4.5 5.1 11.1 Type J 85 5.8 6.6 23.5

16.2 6.7 22.7 11.7

96 76 85 84

70 77 80 50

721 References 1. 2. 3. 4.

Oku, N. et al., Advanced Drug Delivery Reviews, 24 (1997) 215–223. Crommelin, D.J.A. et al., Journal of Controlled Release, 46 (1997) 65–75. Sadzuka, Y. and Hirota, S., Advanced Drug Delevery Reviews, 24 (1997) 257–263. Zhu, G. et al., Cancer Chemother. Pharmacol., 39 (1996) 138–142.

246 Importance of hydrophobic region in cationic peptides on gene transfer into cells and enhance effect of lysosome-disruptive peptide N. OHMORI 1, T. NIIDOME 1 , T. KIYOTA2 , S. LEE2 , G. SUGIHARA2 , A. WADA 3 , T. HIRAYAMA3 , T. HATEKEYAMA 1 and H. AOYAGI 1 ¹ Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852, Japan ² Department of Chemistry, Faculty of Science, Fukuoka University, Fukuoka 814-80, Japan ³ Department of Bacteriologyy, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan

Introduction Gene transfer techniques have caused an important advance in the treatment of both inherited and acquired diseases. We have previously reported that amphiphilic α-helical peptides containing cationic amino acid residues showed high gene transfer abilities into cells [1]. In the present study, in order to clarify the importance of hydrophobic region in amphiphilic structures on their gene transfer abilities, we synthesized five peptides, which have various widths of hydrophobic region and investigated the relationship between the structural features and their gene-transfer abilities. Futhermore, we also investigated the effects of addition of a lysosome-disruptive peptide, 43 E, on the transfection efficiency.

Results and discussion We synthesized five cationic Hel series peptides, which were composed of Leu and Lys residues of ratios of 13 : 5, 11: 7, 9 : 9, 7 : 11 and 5 : 13 respectively (Figure 1.). We examined the DNA binding abilities of five peptides by agarose gel electrophoresis and DNase I digestion assay. The results indicated that Hels 13–5 and 11–7 with large hydrophobic region strongly bound to plasmid

Figure 1.

The primary structures of Hel peptides.

722 Y. Shimonishi (ed): Peptide Science – Present and Future, 722–723 © 1999 Kluwer Academic Publishers, Printed in Great Britain

723

Figure 2.

The enhanced effects of transfection efficiency by adding 43 E to Hel 11–7-DNA complex.

DNA. Furthermore, the transfection abilities of the peptides were determined by the expression of luciferase from its cDNA in COS-7 cells. As the results, the order of transfection efficiencies was Hel 13–5 > Hel 11–7 > Hel 9–9 and depended on the width of hydrophobic region in amphiphilic structure. These results suggested that the hydrophobic region in amphiphilic structure of the peptide was important for formation of peptide-DNA complex and efficient gene-transfer into cultivated cells. Furthermore, we examined enhancement of the transfection efficiency by the addition of lysosome-disruptive peptide, 43 E (LAELLAELLAEL), to cationic peptide-DNA complexes [2]. We could enhance transfection efficiency of Hel 11–7-DNA complex due to the addition of 4 3 E (Figure 2). The transfection effects on the transfection efficiency of Hel 11–7 clearly increased with increasing amounts of 43 E. The enhanced efficiency by 43 E at charge ratio of 16 reached to the efficiency level of Hel 11–7 with chloroquine treatment.

References 1. Niidome, T., Ohmori, N., Ichinose, A., Wada, A., Mihara, H., Hirayama, T., and Aoyagi, H., J. Biol. Chem. 272 (1997) 15307. 2. Ohmori, N., Niidome, T., Wada, A., Hirayama, T., Hatakeyama, T., and Aoyagi, H., Biochem. Biophys. Res. Commun. 235 (1997) 726.

247 Synthesis and pharmacological properties of oxytocin antagonists containing conformationally constrained amino acids G.K. TÓTH 1, K. BAKOS1 , I. ZUPKÓ2 , F. FÜLÖP 3 , I. PÁVÓ4 and G. FALKAY 2 ¹ Department of Medical Chemistry, ²Department of Pharmacodynamics, ³Department of Pharmaceutical Chemistry, 4 Department of Endocrinology, A. Szent-Györgyi Medical University, H-6720 Szeged, Hungary

Introduction There has recently been an increasing tendency towards the rational design of highly active and selective analogues of different peptides. The small peptides are usually highly flexible molecules, and the structures observed in solution depend greatly on the environment. Local constraints are introduced, therefore, in order to restrict the conformational freedom of the parent peptide and to stabilize the desired bioactive conformation. The recently increasing interest in posttranslationally modified peptides is partly due to the search for conformationally constrained amino acids. Modulation of the flexibility of a peptide backbone from an extended conformation to a β turn structure is an important breakthrough in the rational design of highly selective and active peptide or peptidomimetic drugs.

Results and discussion Analogues of oxytocin containing D-Trp, D-Cpa (p-chlorophenylalanine), L-Dbt (dibromotyrosine), L-3Tic, 1,2,3,4-tetrahydro-norharman-1-carboxylic acid (Car) and 2-amino-2-carboxy-1,2,3,4-tetrahydronaphthalene (Atc) with R or S configurations in position 2 were synthesized. The Atc and Car containing peptides were synthesized in the solid phase by using racemic mixtures of R and S Atc and Car. After the liquid HF cleavage, the resulting crude diastereomeric mixtures were separated by means of RP-HPLC. The structure and the purity of the resulting analogues were characterized by mass spectrometry, HPLC and CZE investigations. The pharmacological properties of these oxytocin analogues were tested on an in vivo system, by their inhibition of uterine motor activity. The intrauterine pressure was measured with a Millar catheter fitted with a latex microballoon inserted into the post partum rat uterus. Atosiban was used as reference material and the results of the pharmacological measurements are shown in Figure 1. According these investigations some of 724 Y. Shimonishi (ed): Peptide Science – Present and Future, 724–725 © 1999 Kluwer Academic Publishers, Printed in Great Britain

725

Figure 1.

Representative dose response curves of the investigated peptides.

these analogues proved more potent than the reference material. The described investigations are further evidence of the crucial role of the amino acid in position two in case of these oxytocin analogues. The racemic conformationally constrained amino acids can be incorporated into the growing peptide chain and after the synthesis the diastereomeric products can be easily separated by RP HPLC. The inhibitory potency of oxytocin analogues can be increased the incorporation of some conformationally constrained bulky aromatic amino acids instead of the tyrosine in position two.

References 1. Hruby, V.J. Life Sci., 31 (1982) 189. 2. Akerlund, M., Carlsson, A.M., Melin, P., and Trojnar, J. Acta Obset. Gynecol. Scand., 64 (1985) 449. 3. Keirse, M.J., Am. J. Obstet. Gynecol., 173 (1995) 618. 4. Pavo, I., Slaninova, J., Klein, U., and Fahrenholz, F., J. Med. Chem., 37 (1994) 255. 5. Rastogi, S.N., Bindra, J.S., and Anand, N., Ind. J. Chem., 9 (1971) 1175. 6. Vejdelek, Z.J., Trcka, V., and Protiva, M., J. Med. Pharm. Chem., 3 (1961) 427. 7. Bankowski, K., Manning, M., Seto, J., Haldar, J., and Sawyer, W.H., Int. J. Peptide Protein Res., 16 (1980) 382. 8. Lebl, M., Hill, P., Kazmierski, W., Kárászová, L., Slaninová, J., Fric, I., Hruby, V., J. Int. J. Peptide Protein Res., 36 (1990) 321. 9. Manning, M., Cheng, L.L., Stoev, S., Bankowski, K., Przybylski, J., Klis, W.A., Sawyer, W.H., Wo, N.C., and Chan, W.Y., J. Pept. Sci., 1 (1995) 66. 10. Procházka, Z. and Slaninová, J. Collect. Czech Chem. Commun., 60 (1995) 2170. 11. Majer, P., Slaninova, J. and Lebl, M., Int. J. Peptide Protein Res., 43 (1994) 62.

248 Identification of the structural elements responsible for high biological activity of dimeric opioid peptide biphalin A. MISICKA1,3,6 , A.W. LIPKOWSKI1,3,5 , D. STROPOVA2 , H.I. YAMAMURA2 , P. DAVIS2 , F. PORRECA2 and V.J. HRUBY1 Departments of ¹ Chemistry and ² Pharmacology, University of Arizona, Tucson, AZ85721 (USA) 3 Medical Research Centre, Polish Academy of Sciences, 02-106 Warsaw (Poland) 4 Industrial Chemistry Research Institute, 01-793 Warsaw, (Poland) 5 Department of Chemistry, Warsaw University, 02-093 Warsaw (Poland) 6 Department of Chemistry, Warsaw University, 02-093 Warsaw (Poland)

Introduction Biphalin, a dimeric opioid peptide [1] expresses high biological activity both in vitro and in vivo study [2]. This peptide combines two tetrapeptidic opioid pharmacophores connected with a hydrazide bridge. Tyr-D-Ala–Gly–Phe–NH–NH ← Phe ← Gly ← D-Ala ← Tyr Biphalin Tyr-D-Ala–Gly–Phe–NH–NH ← Phe des (Tyr-D-Ala–Gly) biphalin (AA232) Presently, this peptide is under development as a potential analgesic [2]. Several explanations have been proposed of its high potency, including simultaneous binding to two receptor sites, active metabolites, or higher probability of interaction with one receptor of a molecule containing two pharmacophores. Most of these hypotheses assume the necessary existence of two active pharmacophores in one molecule. The study of the metabolism of biphalin indicate that des(Tyr-D-Ala–Gly)biphalin could be one of the major metabolite of biphalin. Therefore we synthesized de novo respective peptide (AA232) and its analogues for evaluation of biological activities and structural studies.

Results and discussion The sequences opioid receptor binding and biological activities [3] of new synthesized fragment of biphalin and its analogues are presented in Table 1. Surprisingly we have found that removing (possible metabolite) one pharmacophore (Tyr-D-Ala–Gly-) from dimeric biphalin does not significantly affect opioid receptor binding and biological activity profile. This suggests that the pharmacophore responsible for the biological properties of biphalin is de 726 Y. Shimonishi (ed): Peptide Science – Present and Future, 726–727 © 1999 Kluwer Academic Publishers, Printed in Great Britain

727 Table 1.

Binding affinities and bioassays of biphalin fragment and its analogues.

(Tyr-D-Ala–Gly–Phe–NH-)2 Biphalin Tyr-D-Ala–Gly–Phe–NH–NH ← Phe AA232 Tyr-D-Ala–Gly–Phe–NH–NH ← Trp AA228 Tyr-D-Ala–Gly–Trp–NH–NH ← Phe AA227

Binding Ki ± SEM (nM)

Bioassay IC 50 ± SEM (nM)

δ

MVD

µ

GPI

1.4 ± 0.4

27.0 ± 1.5

8.8 ± 0.3

14.9 ± 6.0

0.74 ± 0.32

26.8 ± 4.4

2.4 ± 0.2

46.1 ± 19.8

8.3 ± 2.2

14.6 ± 2.6

7.1 ± 1.5

29.3 ± 6.6

2.0 ± 0.1

2.6 ± 0.3

12925

26.0 ± 9.0

facto one tetrapeptide extended with a hydrazide bridge and an aromatic amino acid residue (Phe4') on the other side of this bridge. In consequence the pharmacophore of biphalin will combine one phenol and amino group of Tyr(1), and two phenyl rings of Phe(4) and Phe(4'). To compare the topographical relations of the aromatic rings, we have synthesized analogues of AA232 in which Phe(4) or Phe(4') have been replaced with tryptophan. The receptor binding and biological activities of resulted analogue with tryptophan in position 4' are similar to the parent compound AA232. The replacenent of Phe(4) with Trp resulted with ten fold decreases in both, MVD and GPI bioassays, but without significant changes in receptor binding properties. The analogues will be used for measurement of distances betwen the phenol (Tyr1) and respective idolyl rings in conformers in solutions.

Acknowledgements This work has been supported with NIDA grant (06284) and Medical Research Centre of Polish Academy of Sciences

References 1. Lipkowski, A.W., Konecka, A.M., and Sroczynska, I, Peptides 3 (1982) 697. 2. Lipkowski, A.W., Misicka, A., Hruby, V.J., and Carr, D.B., Polish J. Chem. 68 (1994) 907. 3. Misicka, A., Lipkowski, A.W., Horvath, R., Davis, P., Porreca, F., Yamamura, H.I., and Hruby, V.J., Life Sci. 60 (1997) 1263.

249 Orally active memory-enhancing peptides obtained by designing bioactive peptides derived from food proteins M. YOSHIKAWA1 , S. SONODA1 , T. MATSUKAWA1 , Y. JINSMAA1 , Y. TAKENAKA1 , M. TAKAHASHI2 , S. FUKUDOME3 , H. FUKUNAGA4 , H. KANETO4 and M. TAKAHASHI4 1 Research

Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan 3 Food Research Laboratory, Nisshin Flour Milling Co. Ltd., Ohimachi, Saitama 356–8511, Japan 4 Faculty of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852-8131, Japan 2 National

Introduction We tried to obtain orally effective memory-enhancing peptides by designing two types of bioactive peptides derived from food proteins. Gluten exorphin A5 (Gly–Tyr–Tyr–Pro–Thr) is an opioid peptide isolated from the enzymatic digests of wheat gluten [1]. The peptide is selective for δ -receptor and its IC50 value in the mouse vas deferens (MVD) assay system was 60 µM. This peptide induced grooming behaviour and potentiated learning performance after the oral administration in mice at a dose of 100–300 mg/kg. Derivatives were synthesized to obtain peptides effective at lower dose. We isolated many kinds of ileum-contracting peptides from the tryptic digests of various proteins by using the guinea-pig ileum assay system [2, 3]. Most of them were complement C3a agonist peptides having the following structure hydrophobic residue-X1 -Leu-X 2 -Arg at their carboxyl termini. Casoxin C, Tyr– Ile–Pro–Ile–Gln–Tyr–Val–Leu–Ser–Arg, a typical example of C3a agonist derived from bovine κ -casein showed anti-opioid nature such as anti-analgesic and anti-amnesic activities after the intracerebroventricular (icv) administration in mice. Pentapeptides having C3a agonist activity were synthesized to test oral effectiveness.

Results and discussion I.

Central effect of gluten exorphin A5 and its derivatives.

Gluten exorphin A5 showed no analgesic activity after the icv administration in mice. However, this peptide induced grooming behaviour after oral administration at doses of 30 and 300 mg/kg in mice. In the step-through type passive avoidance experiment, this peptide orally given to mice at a dose of 300 mg/kg immediately after the training trial improved learning performance (Figure 1). 728 Y. Shimonishi (ed): Peptide Science – Present and Future, 728–730 © 1999 Kluwer Academic Publishers, Printed in Great Britain

729

Figure 1. Effect of pre- and post-training gluten exorphin A5 on memory/learning in the stepthrough type one-trial passive avoidance learning task. Gluten exorphin A5 at doses of 30 and 300 mg/kg was administered orally 1 hr before the training trial (pre-training) or immediately after the training trial (post-training). * P < 0.05 compared with the saline group. Each column represents the median and interquartile ranges.

From alanine scanning study Tyr-Pro sequence in gluten exorphin A5 was found to be essential for the opioid activity. Among its derivatives synthesized, [D-Ala 1 ]-gluten exorphin A5 showed the highest opioid activity with IC50 value of 1 µM in the MVD assay. The IC 50 value of [L-Ala1 ]-gluten exorphin A5 in the same assay was 91 µM. [D-Ala1 ]-gluten exorphin A5 improved learning performance of mice at doses of 1 and 3 mg/kg. This means that memory enhancing activity of gluten exorphin A5 is related to its δ -opioid activity. II.

Central effect of C3a agonist peptides

We isolated many C3a agonist peptides from enzymatic digests of proteins [2, 3]. Among them, casoxin C showed anti-analgesic and anti-amnesic activities after the icv administration in mice. In fact, C3a itself also showed similar effect. This seems to be related to that C3a agonist peptides stimulate acetylcholine release. Together with the fact that the C3a receptor is found in brain (4), this means that the immunopeptide complement C3a also functions as a neuropeptide. Many pentapeptide derivatives of Tyr–Pro–Leu–Pro–Arg which corresponds to the carboxyl terminus of oryzatensin, a C3a agonist peptide derived from rice albumin were synthesized. Some of them showed anti-opioid activity after the oral administration. Thus, bioactive peptides derived from food proteins are useful as lead compounds to design orally effective neuropeptides.

730 References 1. Fukudome, S. and Yoshikawa, M., FEBS Lett., 296 (1992) 107. 2. Takahashi, M., Moriguchi, S., Ikeno, M., Kono, S., Ohata, K., Usui, H., Kurahashi, K., Sasaki, R., and Yoshikawa, M., Peptides, 17 (1995) 5. 3. Takahashi, M., Moriguchi, S., Suganuma, H., Shiota, A., Tani, F., Usui, H., Kurahashi, K., Sasaki, R., and Yoshikawa, M., Peptides, 18 (1997) 329. 4. Ames, R.S., Li, Y., Sarau, H.M., Nuthlaganti, P., Foley, J.J., Ellis, C., Zeng, Z., Su, K., Jurewicz, J., Hertzberg, R.P., Bergsma, D.J., and Kumar, C., J. Biol. Chem., 271 (1996) 20231.

250 Dimers of bradykinin and substance P antagonists as potential anti-cancer drugs J.M. STEWART 1,2 L. GERA1 and D.C. CHAN 2 ¹Department of Biochemistry and ² Cancer Center, University of Colorado Medical School, Denver, Colorado 80262, USA

Introduction Small cell lung cancer (SCLC) is of neural crest origin, and SCLC cells synthesize and express receptors for a variety of neuropeptides. Prominent among these are bradykinin (BK) and bombesin (BN). These mediators appear to be important in autocrine stimulation of cancerous growth of these cells, and several studies have reported use of antagonists of peptide mediators to inhibit growth of SCLC in vitro and in vivo. Most work has focused on some broadspectrum substance P (SP) antagonists that appear to act principally by inhibiting BN receptors. Certain of these are cytotoxic for SCLC cells, even though only few SCLC cell lines express SP receptors. We have studied a large series of BK analogs for their ability to antagonize BK-evoked elevation of intracellular free calcium in SCLC cells, and have found many potent antagonists, which, however, are not cytotoxic for cultured SCLC cells. Recently we found that dimers of our potent BK antagonists are selectively cytotoxic for several lines of SCLC, both in vitro and in vivo in nude mice bearing SCLC implants [1].

Results and discussion We now report dimers of BK and SP antagonists and heterodimers of SP and BK antagonists that are potent selectively cytotoxic agents for SCLC. Although straight-chain analogs of SP and bombesin have shown toxicity against SCLC, none of the simple BK antagonists were toxic to cells, although they were very effective for inhibition of BK-evoked elevation of intracellular free calcium in SCLC cultures. Typical of this behavior is B-9430, a very potent ‘third-generation’ BK antagonist which is active against both B1 and B2 BK receptors and shows a long half-life in vivo [2]. When this antagonist was crosslinked by suberimide at the N -terminus (B-201), potent cytoxic activity was found. Dimers of ‘first-generation’ BK antagonists, such as CP-127, were introduced by investigators at Cortech [3], and while they are quite potent antagonists in many BK assays, were not cytotoxic. When the linker in CP-127 was moved to the N -terminus of the dimer (B-197) significant toxicity was found. Even dimers of the potent ‘second-generation’ Hoechst antagonist HOE-140 [3] 731 Y. Shimonishi (ed): Peptide Science – Present and Future, 731–732 © 1999 Kluwer Academic Publishers, Printed in Great Britain

732 Table 1.

Structures and activities of peptides used in this study. ACTIVITY in vitro, IC 5 0 (µM)

B-9430 B-197 B-201 B-218

CP-127 B-237 B-240 B-215

STRUCTURE

Ca + + Flux

Growth, SHP-77

DArg–Arg–Pro–Hyp–Gly–Igl–Ser–DIgl–Oic–Arg BSH(Cys–DArg–Arg–Pro–Hyp–Gly–Phe–Ser–DPhe–Leu–Arg) 2 SUIM(–DArg–Arg–Pro–Hyp–Gly–Igl–Ser–DIgl–Oic–Arg) 2 a–DDD–(Lys–DArg–Arg–Pro–Hyp–Gly–Igl–Ser–DIgl–Oic–Arg) DArg–Arg–Pro–Hyp–Gly–Phe–Cys–DPhe–Leu–Arg BSH DArg–Arg–Pro–Hyp–Gly–Phe–Cys–DPhe–Leu–Arg DNMF–DTrp–Phe–DTrp–Leu(r)Leu–NH2 (Orosz SP antag.) α –DDD(DNMF–DTrp–Phe–DTrp–Leu(r)Leu–NH 2 ) 2 Lys–DArg–Arg–Pro–Hyp–Gly–Igl–Ser–DIgl–Oic–Arg |—SUB–DNMF–DTrp–Phe–DTrp-Leu(r)Leu–NH 2

0.1 0.05 0.02 —

>40 0.2 0.15 0.3

0.5

>40

— — —

6 40 8

2

Abbreviations: (Amino acids): DNMF, D-N-Methyl phenylalanine; Hyp, Hydroxy proline; Igl, 2-Indanyl glycine; Leu(r)Leu, Pseudo Leu(CO→ CH2)Leu; Oic, Octahydroindole-2-carboxylic acid. (Cross-linkers): BSH, bisSuccinimidohexane; DDD, Dodecanedioyl-; SUB, Suberyl-; SUIM, Suberimidyl-.

showed only low cytotoxicity against SCLC. Orosz et al. [4] reported that a pseudopeptide substance P antagonist (B-237) was active against SCLC. We confirmed this activity, and found that neither a homodimer (B-240) nor a heterodimer of this peptide with the best BK antagonist (B-215) showed increased cytotoxicity. Certain of these new dimers are toxic to SCLC lines that show multidrug resistance phenotypes, testifying to the different mechanism of toxicity of these agents. Preliminary studies indicate that these new dimers act by stimulation of apoptosis in SCLC cells. Peptide dimer B-201 inhibited the growth of SCLC cell line SHP-77 when implanted subcutaneously in athymic (nude) mice. These dimers offer a new avenue for anti-cancer drug development.

References 1. Chan, D.C., Gera, L., Helfrich, B., Helm, K., Stewart, J., Whalley, E., and Bunn, Jr., P., Immunopharmacology 33 (1996) 201. 2. Stewart, J.M., Gera, L., Chan, D.C., Whalley, E.T., Hanson, W.L., and Zuzack, J.S., Can. J. Physiol. Pharmacol. 75 (1997) 719. 3. Wirth, K., Hock, F.J., Albus, U., Linz, W., Alpermann, H.G., Anagnostopoulos, H., Henke, S., Breipohl, G., Konig, W., Knolle, J., and Scholkens, B.A., Br. J. Pharmacol. 102 (1991) 774. 4. Orosz, A., Schrett, J., Nagy, J. Bartha, L, Schon, I., and Nyeki, O., Int. J. Cancer 60 (1995) 82.

251 Ovine Leydig cell insulin-like peptide: a sheep relaxin? J.D. WADE ¹, N.F. DAWSON ¹, P.J. ROCHE 1,4 , L. OTVOS, JR.², R.J. SUMMERS³, Y.-Y. TAN³ and G.W. TREGEAR¹ ¹ Howard Florey Institute, University of Melbourne, Parkville, Victoria 3052, Australia. ² The Wistar Institute, 3601 Spruce St, Philadelphia, PA 19104, USA ³ Department of Pharmacology, Monash University, Clayton, Victoria 3168, Australia. 4 Department of Medical Laboratory Science, RMIT, Melbourne, Victoria 3001, Australia

Introduction Relaxin, a two-chain, three disulfide bonded peptide, is produced principally by the ovary during pregnancy [1]. Its principal roles include softening of the cervix and remodelling of the interpubic ligaments in preparation for birth. Despite much effort, no relaxin peptide has been identified in sheep or goat and it appears that, surprisingly, these ruminants do not produce relaxin although there is some evidence for relaxin-like responses in the reproductive tract of ruminants following administration of exogenous peptide from other species [2]. More recently, a novel gene was found in the Leydig cells of the boar testis and its expression product, known as Leydig cell insulin-like peptide (Ley I–L), shown to possess striking structural similarity to relaxin [3]. Curiously, like relaxin, the Ley I–L gene has since been shown to be expressed in the ovary of the human [4] and the pregnant sheep [5]. We sought to determine if Ley I–L may fulfil the biological role of relaxin in the sheep.

Results and discussion PCR was used to isolate and characterize Ley I–L mRNA from pregnant sheep ovary [5]. The deduced amino acid sequence of the putative expression product is shown in Figure 1. The peptide consists of two chains, A- and B-, of length 26 and 32 residues respectively. The three cystines are presumably linked in a manner similar to other members of the insulin superfamily including relaxin. There is close homology to other Ley I–Ls including those from the pig and cow.

Figure 1.

The primary structure of ovine Ley I–L.

733 Y. Shimonishi (ed): Peptide Science – Present and Future, 733–736 © 1999 Kluwer Academic Publishers, Printed in Great Britain

734 The approach employed for the successful chemical synthesis of relaxin [6] was used for the preparation of ovine Ley I–L, viz., separate assembly of each of the two chains by the continuous flow Fmoc-solid phase method followed by their combination in solution. To prevent possible base-mediated aspartimide formation of the C-terminal Asp-Gly, 6% piperazine in DMF [7] was used for N α -Fmoc deprotection during the synthesis of the B-chain. For both peptides, the use of the backbone amide bond protecting group, the N-(2-hydroxy4-methoxybenzyl) (Hmb) [8] was required for satisfactory assembly. The Hmb group prevents hydrogen bonded intramolecular association of the growing peptide chain, is stable to the conditions of Fmoc solid phase synthesis, and is removed during the final peptide-resin acidolysis. It was introduced via bisFmoc–(Hmb)–Gly–OPfp acylation. After synthesis, the two peptides were each cleaved and sidechain deprotected by treatment with TFA containing appropriate scavengers. Conventional purification by RP-HPLC on Vydac C18 supports yielded the S-reduced A- and B-chains in good yield. The two peptides were combined by aeration in CAPS buffer at high pH. The ratio of chains was 4:1 (w/w, A/B) and the reaction was carried out at 4°C and monitored by analytical RP-HPLC. After 21 h, there was no further reaction as evidenced by the absence of starting peptide and the synthetic ovine Ley I–L was isolated by preparative RP-HPLC in approximately 4% overall yield relative to the initial B-chain-resin. Chemical characterization including by CZE, RP-HPLC and MALDITOF-MS (found: MH+ 6,250.4, calc: MH + 6,249.0) confirmed the high purity of the peptide. An aliquot of the peptide was also subjected to limited tryptic digestion and analyzed by MALDITOF-MS. As shown in Figure 2, two key fragments were identified which showed the parallel alignment of the two chains to be identical to that of insulin and relaxin. The synthetic peptide in water (0.3 mg/ml) exhibited a loosened α -helical circular dichroism spectrum (data not shown). The α -helicity of the peptide increased from approximately 25% to approximately 70% following addition of TFE to 50% (v/v). A similar pattern of secondary structure was observed for human Gene 2 relaxin [9] although it assumes an increased amount of average helix contribution (35% in water) to the conformational equilibrium compared with the sheep peptide. The peptide was assayed for relaxin-like activity in the rat isolated atrial assay [10]. At high concentrations (1 µM), it did not produce positive chronotropic and inotropic responses nor significantly antagonize responses to 10 nM human Gene 2 relaxin. At this concentration, relaxin causes a maximal chronotropic and inotropic effect (Figure 3). These results, together with the finding that the synthetic peptide ≤ 1 µM did not compete with 33 P-human Gene 2 relaxin for binding to rat uterine tissue (data not shown), clearly show that, at physiological concentration, ovine Ley I–L is not a relaxin-like hormone. A recent report that Ley I–L gene knockout male mice lacked mature sperm and were sterile provides conclusive evidence for a role for Ley I–L in spermatogenesis [11]. The function of Ley I–L in the female remains unknown following

735

Figure 2. MALDITOF-MS of a tryptic digest of synthetic ovine Ley I–L. The peak with MH + 1,639.7 corresponds to A(18–26)/B(21–26) (calc. MH+ 1,638.9) and the peak with MH + 1,943.9 to A(9–17)/B(9–16) (calc. MH + 1,943.3).

Figure 3.

Rat isolated atrial assay of synthetic ovine Ley I–L.

the observation that Ley I–L gene knockout female mice had normal estrous cycles and were fertile. However, our finding that the gene is expressed specifically in the theca interna cells of the ovary suggests that the peptide may exert an autocrine or paracrine effect within the thecal layer and influence ovulation [5]. The availablity of synthetic peptide will enable an examination of this possibility.

736 The question of whether sheep produces a biologically active relaxin remains unanswered. As discussed by Hartung et al. [12], it is possible that some ruminants may have further evolved to function without the need for a relaxinlike hormone. Alternatively the levels of relaxin may be so low as to defy current detection methods, both immunological and molecular biological. Neither possibility is entirely convincing given that ruminants seemingly possess a relaxin physiology [2]. A third alternative, and perhaps the most probable, is that there exists an as yet undiscovered relaxin homologue which differs sufficiently in structure to ‘conventional’ relaxin so as not to be recognized by immunological and nucleic acid hybridization techniques. Support for this possibility is provided by the recent identification of an alternatively spliced relaxin mRNA species in human placenta [13]. The expression product is predicted to be a single chain 93 residue two cystine peptide containing the B-chain of relaxin.

Acknowledgements The work carried out at the Howard Florey Institute was supported by an Institute block grant from the NHMRC of Australia. We thank Mary Macris (Florey Institute) for both the mass spectroscopy and amino acid analyses.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13.

Bryant-Greenwood, G.D. and Schwabe, C., Endocrine Rev., 15 (1994) 5. Porter, D.G., Lye, S.J., Bradshaw, J.M.C., and Kendall, J.Z., J. Reprod. Fert., 61 (1981) 409. Adham, I.M., Burkhardt, E., Benahmed, M., and Engel, W., J. Biol. Chem., 268 (1993) 26668. Tashima, L.S., Hieber, A.D., Greenwood, F.C., and Bryant-Greenwood, G.D., J. Clin. Endocrinol. Metab., 72 (1994) 899. Roche, P.J., Butkus, A., Wintour, E.M., and Tregear, G.W., Mol. Cell. Endocrinol., 121 (1996) 171. Wade, J.D., Layden, S.S., Lambert, P.F., Kakouris, H., and Tregear, G.W., J. Prot. Chem., 13 (1994) 315. Dölling, R., Beyermann, M., Haenel, J., Kernchen, F., Krause, E., Franke, P., Brudel, M., and Bienert, M., In Maia, H.L.S. (Ed) Proceedings of the 23rd European Peptide Symposium, ESCOM, Leiden, 1995, pp. 244–245. Johnson, T., Quibell, M., and Sheppard, R.C., J. Pep. Sci., 1(1995) 11. Wade, J.D., Lin, F., Salvatore, D., Otvos, L., and Tregear, G.W., Biomed. Pept. Prot. Res., 2 (1996) 27. Tan, Y.Y., Wade, J.D., Tregear, G.W., and Summers, R.J., Brit. J. Pharmacol., 123 (1998) 762. Adham, I.M., Zimmermann, S., and Engel, W., Reprod. Dom. Animals, 32 (1997) 73. Hartung, S., Kondo, S., Abend, N., Hunt, N., Rust, W., Balvers, M., Bryant-Greenwood, G., and Ivell, R., In MacLennan, A.H., Tregear, G.W., and Bryant-Greenwood, G.D. (Eds.) Progress in Relaxin Research (Proceedings of the 2nd International Congress on the Hormone Relaxin), Global Publication Services, Singapore, 1995, p. 439. Gunnersen, J.M., Fu, P., Roche, P.J., and Tregear, G.W., Mol. and Cell Endocrinol., 118 (1996) 85.

252 Self-assembling peptides in biology, materials science and engineering S. ZHANG, M. ALTMAN, R. CHAN, P. LEE and R. MA Center for Biomedical Engineering 56–379 & Department of Biology Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307 USA

Introduction A new class of oligopeptide based biological materials was discovered from the self-assembly of ionic self-complementary oligopeptides [1–3]. This class of biomaterials has considerable advantages in a number of applications, including platforms or scaffolding for tissue engineering [4], drug delivery of protein and peptide medicine as well as surface coating [5]. There is wide variability in the design and usage of these biological materials in various areas. In most cases, the designed peptides form beta-sheet structures in aqueous solution with two distinct surfaces, one hydrophilic, the other hydrophobic. The unique structural feature of these peptides is that they can form complementary ionic bonds with regular repeats on the hydrophilic surface. The ionic complementary sides have been classified into several moduli, i.e. modulus I, II, III, IV, etc., and mixed moduli. This classification is based on the hydrophilic surface of the molecules which have alternating + and – charged amino acid residues, either alternating by 1, 2, 3, 4 and So on. For example, molecules of modulus I have – + – + – + – +, modulus II, – – + +– –+ +, modulus, IV – – – – + + + +. One of the peptides with 16 amino acids, (DAR16-IV), has a beta-sheet circular dichroism spectrum at ambient temperature but shows an abrupt transition at high temperatures to a stable alpha-helical spectrum without a detectable random coil intermediate. On cooling, it retains the alphahelical form and takes several weeks at room temperature to partially return to the beta-sheet. Similar structural transformations were observed by changing the pH. This suggests that secondary structures with specific sequences in segments of proteins may undergo such conformational transformations. These findings may provide insights into protein–protein interactions and pathogenesis of some protein conformational diseases, such as scrapie or Alzheimer’s disease. Upon the addition of monovalent alkaline cations or the introduction of the peptide solutions into physiological media, these oligopeptides spontaneously assemble to form macroscopic structures which can be fabricated into various geometric shapes [7]. Scanning EM reveals that the matrices are made of interwoven filaments with about 10–20 nm in diameter and pores about 50–100 nm in diameter [1, 4, 8]. A variety of mammalian cell types has been 737 Y. Shimonishi (ed): Peptide Science – Present and Future, 737–744 © 1999 Kluwer Academic Publishers, Printed in Great Britain

738 shown to attach to the peptide matrices. These peptide materials can also have various biological properties by incorporating different biological ligands. Furthermore, the peptides can also be designed as monolayer surface coating for specific cell pattern formation. This biological surface engineering technique will provide tools to study cell–cell communication and cell behavior [5].

Results and discussion Molecular modeling and engineering We designed many self-assembling peptides for various studies as listed in Table 1. The sequences of one of the ionic self-complementary peptides, AEAEAKAKAEAEAKAK (EAK16-II, A, alanine, E, Glutamate, and K, lysine), was originally found as a repeated segment in a yeast protein [9]. Its molecular structure and proposed complementary ionic pairings between positively charged lysines and negatively charged glutamates in a nonoverlap and an overlap arrangement are illustrated in Figure 1. This structure represents an example of this class of self-assembling beta-sheet peptides that undergo spontaneously association under physiological conditions [1, 2, 4, 7]. If the charged residues are substituted, i.e., the positive charged lysines are replaced by positively charged arginines and the negatively charged glutamates are replaced by negatively charged aspartates, there are essentially no drastic effects on the self-assembly process. However, if the positively charged resides, Lys and Arg are replaced by negatively charged residues, Asp and Glu, the peptide can no longer undergo self-assembly, although they can still form beta-sheet structure in the presence of salt. This observation is in agreement with previous reports [10–13]. Peptide materials structure Peptides form a matrix readily when exposed to salt. A close examination of these peptide materials using scanning electron microscopy (SEM) revealed that the materials were made of interwoven fibers. These fibers have relatively regular fiber diameter and enclosure. Many peptide materials that are capable of matrix-formation have been examined and they all have similar appearances [1, 4, 8]. Figure 2 presents an example of a peptide material at high resolution. Mechanical properties of peptide materials Recently, the mechanical properties for one peptide material, EFK8, has been examined [8]. This peptide material showed interesting mechanical properties. Their fiber density increases as the concentration of the peptide increases. The mechanical strength is proportional to the concentration of the peptide with a Young’s modulus of about 2 kPa and 15 kPa at 0.3% and 1% in water,

739 Table 1. Self-assembling oligopeptides studied. β , β-sheet; α, α-helix; r.c., random coil; N/A, not applicable; ND, not determined. *Both VE20 and RF20 are in β-sheet form when they are incubated in solution containing NaCl. They do not self-assemble to form macroscopic matrices. N/A, not applicable.

respectively [8]. It should be pointed out that the peptide materials had no observable swelling when the materials were delivered into a salt solution. This is likely due to the high water content of the material, in which 99% of the material is water. This is a very important factor if the materials are to be

740

Figure 1. Molecular model of self-assembling peptide EAK16-II. A) Two molecules of EAK16-II in an antiparallel type form 8 pairs of ionic bonds between Lys and Glu in a complete nonoverlap form on their hydrophilic side. The hydrophobic side has 8 Ala. B) Four EAK16-II peptides form an overlap assembly where two molecules in the inside form 4 ionic bonds on the hydrophilic side. The same molecules also form hydrophobic interactions between Ala. This type of interaction has been found in the structure of silk.

further developed as the scaffold for tissue engineering and tissue repair. This unique property of this material removes the chance of there being an unregulated expansion of the scaffold that could lead to adverse physiological effects on neighboring tissues. Peptide scaffold for cell attachment and differentiation The peptide self-assembly phenomenon was a serendipitous observation during an experiment when the cytotoxicity for tissue cells was examined using the peptides. The cells exhibited robust growth and no cytotoxicity was observed. However, a thin layer material that was attached to the cells was observed under a phase contrast microscope. This thin layer of material occurred only in the duplicated cell culture dishes where the EAK16-II peptides were added. Whereas in other dishes where different peptides were used, this phenomenon was not observed. A number of mammalian cells have been tested and all have been found to be able to form stable attachments with the peptide materials [4]. Several peptide materials have been used to test for their ability to support cell proliferation and differentiation. These results suggest that the peptide materials do not only support various types of cell attachment, but can also allow the attached cells to proliferate. For example, peptide-attached-rat PC12 cells were observed to undergo differentiation and extensive neurite outgrowth in the presence of nerve growth factor (NGF) [7]. In addition, when primary

741

Figure 2. Images of biological materials from the self-assembling peptides of RAD16-II. A) The peptide material is fabricated as a thread with a diameter 0.3 mm through a thin needle. Each scale is 1 mm. Materials of such thread with a length of greater than 20 cm has been produced from 1 ml peptide solution [4]. B) The SEM structure of RADA16-II. The material is selfassembled from individual interwoven fibers. The diameter of the fiber is about 10–20 nm and the enclosures are about 50–100 nm. The bar represents 1 micron.

mouse neuron cells were allowed to attach to the peptide material, the neuron cells projected lengthy axons that followed the specific contours of the selfassembled peptide surface (Figure 3). Structural transformation of peptides from beta-sheet to alpha-helix Most peptides studied are quite stable at various conditions. However, the data from the DAR16-IV peptide was a great surprise. When the peptide powder was dissolved in water and measured immediately by CD, it exhibited an alpha-helical structure. However, after being stored at 4°C for a few days,

742

Figure 3 The cell attachment and differentiation on the peptide material. A) Rat PC12 cells on the peptide materials without exposure to NGF. Cell density increased over time on the peptide materials. B) When the cells were exposed to NGF, they start to differentiate and project long processes over 0.5 mm. The processes followed the contour of the material – similar to roads following the contours of mountains. C) Mouse neuron cells project axons on the peptide material over 0.4 mm in few days. Because the materials do not have a flat surface, the axons on the materials is not always in focus, again, suggesting that they follow the surface contours of the peptide materials.

743 it formed a stable beta-sheet structure. Upon heating at an elevated temperature, 50°C or higher, the beta-sheet abruptly transformed into an alpha-helix again. Interestingly, when standing at 23°C for several weeks, it slowly changed back to a beta-sheet structure. It showed considerable hysteresis. The sequence of DAR16-IV has a cluster of negatively charged glutamate residues close to N-terminus and a cluster of positively charged Arg residues near C-terminus. It is well known that all alpha-helices have an intrinsic helical dipole moment from C-terminus toward N-terminus [14]. Because of the unique sequence of DAR16-IV, its side chain charges balance the helical dipole moment. Recently, several more peptides with such charge distributions have been designed and have confirmed the initial findings [Zhang, unpublished]. It would be interesting if such examples could be found in proteins. This dynamic structural behavior is quite surprising, but it forces us to reconsider the problem of protein folding and protein–protein interactions. It is believed that most secondary structures in proteins are stable once they are formed. However, proteins frequently undergo catalysis, transport, interaction with other substances and are involved in a wide range of activities. It is reasonable to think that protein secondary structures are not static, rather, they can adopt many conformations to accommodate their biological functions. From a material science and engineering stand point, these structural transformations can be viewed as possible molecular switches that can be regulated by changing temperature or pH. The molecular length accompanying the structural change is approximately 2 fold so that one form would be distinguishable from the other. Surface self-assembling peptides for cell pattern formation We recently developed peptides for biological surface engineering. We are interested in modifying surfaces to probe detailed molecular and cellular phenomena in a well controlled manner. These oligopeptides have three distinctive features: a ligand, a linker, and an anchor. All three groups can be tailored for a specific purpose. Since the motif chosen (Arg–Ala–Asp–Ser)n, or written as (RADS)n has high specificity for integrin receptors, the peptide can serve as a ligand for attaching cells to surfaces. A variety of cell types can form cell patterns as shown in this study. New and advanced technologies frequently increase our knowledge and open new avenues of research. It also provides means to enhance our understanding of extremely complex biological phenomena. The development of various selfassembling peptide systems have made it possible to dissect the complex problems of cell–cell communication, cell–cell interaction, cell–material interaction, cell migration, and cell mechanical compliance. Acknowledgment We thank Dr. Alexander Rich for generous support. Drs. Todd Holmes, Lin Yan, Michael Lassle, Xing Su for advising and carrying out some experiments.

744 We also thank Eric Chang for critically commenting and editing this paper. This work is supported in part by a grant from the Army Research Office.

References 1. Zhang, S., Lockshin, T., Cook, R. and Rich, A., Biopolymers 34 (1994) 663. 2. Zhang, S., Holmes, T., Lockshin, T. and Rich, A., Proc. Natl. Acad. Sci. USA 90 (1993) 3334. 3. Zhang, S. (1994) Perspective in Protein Engineering 1996 (CD-ROM Edition) (Geisow, M.J., Ed.), Biodigm Ltd. (UK). (Net search key words: protein engineering). . ISBN 0952901501. 4. Zhang, S. Holmes, T., DiPersio, M., Hynes, R., Su, X. and Rich, A., Biomaterials 16 (1995) 1385. 5. Zhang, S., Yan, L., Altman, M., Lässle, M., Nugent, H., Frankel, F., Lauffenburger, D., Whitesides, G., and Rich, A. (1998) Biomaterials, in press. 6. Zhang, S. and Rich, A., Proc. Natl. Acad. Sci. USA 94 (1997) 23. 7. Holmes, T., Su, X., Delacalle, S., Rich, A and Zhang, S. (1998) (Submitted). 8. León, E., Verma, N., Zhang, S., Lauffenburger, D. and Kamm, R., J. Biomaterials Science: Polymer edition 9 (1998) 293. 9. Zhang, S., Lockshin, C., Herbert, A. Winter, E. and Rich, A., EMBOJ. 11 (1992) 3787. 10. Brack, A. and Orgel, L., Nature, 256 (1975) 383. 11. Trudelle, Y., Polymer 16 (1975) 9. 12. St.Pierre, S., Ingwall, R.T., Varlander, M.S, and Goodman, M., Biopolymers, 17 (1978) 1837. 13. Osterman, D.G. and Kaiser, E. T. J. Cell. Biochem. 29 (1985) 57. 14. Hol, W., Prog. in Biophys. and Mol. Biol. 145 (1985) 149.

253 Hybridization properties of nucleic acid analogs bearing peptide backbone M. FUJII*, J. HIDAKA and T. OHTSU Department of Industrial Chemistry, Faculty of Engineering in Kyushu, Kinki University, 11-6 Mayanomori, Iizuka, Fukuoka 820, Japan

Introduction Various chemical modifications have been made on such synthetic DNAs and RNAs in order to overcome some drawbacks of them, including degradability by cellular nucleases, impermeability into a cell membrane, and low hybridization affinity caused by electrostatic repulsion between phosphate backbones. Among those, introduction of peptide backbone into such molecules seems to be attractive because peptide compounds can be expected to have such preferable properties as a nuclease resistance, membrane permeability, and a good affinity and specificity to nucleic acids. In this study, oligopeptides containing β -aminoalanine bearing a nucleobase were synthesized and hybridization properties of them were examined by Tm measurement.

Results and discussion P r e p a r a t i o n s o f N -t-butoxycarbonylglycyl- Nβ-(thymin-1-ylacetyl)- L -β-aminoa l a n i n e (7 , T * ) a n d N - t-butoxycarbonylglycyl- N β-(cytosin-1-ylacetyl)- L - β amino-alanine (8 , ZC*) were achieved as shown in Scheme 1. Cleavage and deprotection was performed with TFMSA-TFA-DMS-m-cersol (1: 5 : 3 : 1). Purification by RPHPLC (MegapakSIL C18–10, 10 mm, 10 × 250 mm) eluted

Scheme 1.

Synthesis of DNA Analogs Containing β-Aminoalanine Modified with Nucleobases.

745 Y. Shimonishi (ed): Peptide Science – Present and Future, 745–747 © 1999 Kluwer Academic Publishers, Printed in Great Britain

746 Table 1.

Double and Triple-Helix Formation by Peptide DNA Analogs P and Q.

Strand

Tm a (°C)

Target

b

P

A1 0

P P

rA1 0 5'-GCTA 1 0TCG-3'/ 3'-CGAT 1 0 AGC-5' 5'-GCTA 5 (GA) 5 TCG-3'/ 3'-CGAT 5 (CT) 5 AGC-5'

Q

26.5 36.2 c 36.4 d 21.4 19.3 22.8

∆Tm(°C)

+ 13.5 + 13.2 + 13.4 — – 0,4 + 1.5

a Measured in a buffer containing 50 mM Tris, pH 7.0, 20 mM MgCl 2 , 100 mM NaCl, [Strand] = [Target] = 0.5 mM b 140 mM NaCl, 10 mM sodium phosphate, pH 7.2, c 20 mM MgCl 2 , 1.0 M d NaCl, pH 7.0 0 M MgCl 2 , 0 M NaCl, pH 7.0.

with linear gradients of acetonitrile in 0.1% THF gave T* 10 (P ) and T * 5 (C*T*) 5 (Q). As shown in Table 1, formation of hybrid double strand by P and dA 10 was confirmed by observing hypochromic effect at pH 7.0. It is to be noted that the melting temperature (Tm) was higher than that for natural DNA double strand by 13.5°C (1.35°C/base) [1]. The stability of the hybrid was not affected by salt concentration. The peptide P also formed double strand with rA10 and the Tm was 21°C. Moreover, it was shown that P could bind to double stranded DNA by triple helix formation with comparable affinity (Tm = 19.3°C, ∆Tm = –0.7°C). Oligopeptide Q containing mixed pyrimidine bases was also shown to form triple helix with double stranded DNA (Tm = 22.8°C, ∆ Tm = + 1.5°C) [2].

Conclusions The oligopeptides P and Q were designed to have nucleobase moieties at the interval of six atoms on the backbone, which was previously demonstrated to be critical for the hybrid formation with DNA or RNA by P.E. Nielsen and his coworkers [3]. The linkage between nucleobase and the backbone in P and Q is longer than that in DNA or PNA by two atoms. It is also pointed

(a) Figure 1.

(b) Interresidue H Bonding

(c) Intraresidue H Bonding

Intramolecular Hydrogen Bonding in 1 (a) and PNA (b and c).

747 out by P. E. Nielsen’s group that the linkage is a little flexible in its length. It can be postulated that Watson-Crick base pairing in duplex and Hoogsteen base pairing in triplex by P and Q is made possible because of the favorable orientation of the base moieties caused by intramolecular hydrogen bond as shown in Figure 1 [4].

References 1. Nielsen, P.E., Egholm, M., Berg, R.H. and Buchardt, O. Science, 254 (1991) 1497. 2. Moser, H.E., Dervan, P.B. Science, 238 (1987) 645. 3. Hyrup, B., Egholm, M., Nielsen, P.E., Witung, P., Norden B. and Buchardt, O. J. Am. Chem. Soc., 116 (1994) 7964. 4. Almarsson, O., Bruice, T.C. Proc. Natl. Acad. Sci, USA, 90, 9542 (1993).

254 Copper(II) complexes of the modified ACTH active center analogue hexapeptides – investigation by means of CD spectroscopy I. VOSEKALNA 1 , B. GYURCSIK 2 and E. LARSEN 3 1 Latvian

Institute of Organic Synthesis, Aizkraukles Str. 21, LV-1006 Riga, Latvia Reaction Kinetics Research Group of Hungarian Academy of Sciences at Department of Inorganic and Analytical Chemistry, Attila Jozsef University, Dom ter 7, P.O.Box 440, H-6701 Szeged, Hungary 3 The Royal Veterinary and Agricultural University, Department of Chemistry, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 2

Introduction It is known that a part of copper(II)-ions is exchangeable in vivo and is associated with albumin and low-molecular weight peptides containing L -histidine. In recent years evidence was accumulate supporting the hypothesis that peptides may be among important mediators of human allergic disease. Structure-activity studies have been performed on series of natural and synthetic polypeptides. In order to find peptides possessing histamine-releasing activity several linear and cyclic analogues of the ACTH active center were investigated, and induction of histamine release from mast cells by cyclic analogues was observed [1, 2]. It is known that metal ions may act synergistically with peptides promoting their biological activity [3, 4]. For the reasons mentioned above we prepared and investigated a number of linear and cyclic hexapeptides which can be considered as modified analogues of the ACTH active center. In order to determine the composition and stability of the complexes formed under various conditions the equilibrium processes and solution structure of hexapeptide – copper (II) complexes were investigated by means of CD spectroscopy and potentiometric titration as well.

Results and discussion All linear and cyclic peptides investigated are found to bind copper ion in alkaline aqueous solutions. CD spectra in visible and UV part are obtained for ligand : copper(II) ratio 2:1; 1:1; 2:3; 1:2. From such experiments we are able to conclude that the cyclohexapeptides not containing histidine form only 1:1 coordination entities. Such coordination causes a complete change of conformation of the peptides. Peptide molecules containing His residue seem to have a more complicated behaviour towards Cu 2+ . The cyclic peptides form 748 Y. Shimonishi (ed): Peptide Science – Present and Future, 748–749 © 1999 Kluwer Academic Publishers, Printed in Great Britain

749 at least two different coordination compounds and the linear peptides form at least three coordination compounds. Peptides with Lys as C-terminal residue and containing also His residue have a clear tendency to show CD of three species. The conformation of peptide chain is not dramatically changed. One of the linear peptides (L -Lys-L -Leu- L -Ala-L -His- L -Phe–Gly) was chosen to determine the equilibrium constants of its proton and copper(II) complexes in aqueous medium at different ligand to metal ratios. Protonation constants were determined from five independent pH-metric titrations. The hexapeptide investigated contains four donor groups, which deprotonate with increasing pH in the pH 2–10 interval. Investigations with copper (II) ion revealed that the ligand is able to keep 2 equiv. of metal ion in solution in the whole pH interval studied. Thus the ligand to metal ratio was varied from 2 : 1 to 1 : 2 during the potentiometric titrations. In the systems with L : M = 2 : 1 and 1 : 1 the same species are formed, i.e. complexes containing the metal ion and the ligand in equivalent amounts and differing from each other only in their protonation states. It means that there is no space for two bulky ligands around the copper(II) ion. However, in the systems with L : M = 1 : 2, completely different binuclear species are formed. A very important observation was that both the electronic absorbtion and the CD spectra of the copper(II) – hexapeptide systems in L : M = 2 : 1 and 1 : 1 composition showed almost the same pattern over the whole pH region studied, irrespective of the ligand to metal ratio. As in the case of potentiometric titrations, however, a difference was detected between the latter and the L : M = 1 : 2 systems. The results show that the metal ion complexation changes the structure of the hexapeptide, which is the most important feature concerning its biological function Until the physiological pH, complexes with a macrochelate ring (including the N-terminal α-amine and the imidazole nitrogen of histidine) and increasing number of deprotonated amide nitrogens are dominant. The imidazole nitrogen leaves the coordination sphere of the metal ion only above ca. pH 8, allowing the formation of classical peptide type coordination with fused five-membered chelate rings starting from the N-terminal α -amine up to the third deprotonated amide group, as in the case of peptides with non-coordinating side chains. Irrespective of the ligand excess applied during the measurements, only mono complexes were found in different protonation states. Binuclear complexes were detected, however, in the system with ligand to metal 1 : 2 concentration ratio with relatively separated copper(II) centers. References 1. Porunkevich, J.A., Ratkevich, M.P., Kublis, G.G., Mutulis, F.K., and Chipens, G.I., Biohimija (Russ.) 55 (1990) 988. 2. Ratkevich, M.P., Porunkevich, J.A., Ancans, J.E., and Chipens G.I., Biohimija (Russ.) 54 (1989) 1611. 3. Pickart, L. and Thaler, M.M., J. Cell. Physiol. 102 (1980) 129. 4. Daignault, S.A., Arnold, A.P., Isab, A.A., and Rabenstein, D.L., Inorg. Chem. 24 (1985) 3984.

255 The structure–activity relationships of the sweet protein brazzein – roles of acidic amino acids H. IZAWA and Y. ARIYOSHI Central Research Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan

Introduction Brazzein is a thermostable, sweet-tasting protein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon [1]. Brazzein consists of 54 amino acid residues and has four intramolecular disulfide bonds [2]. It is wellknown that the carboxyl groups of sweet compounds participate in binding with the receptor. Brazzein contains five Asp residues at positions 2, 25, 29, 40 and 50, and four Glu residues at positions 9, 36, 41 and 53. The roles of these acidic amino acids were investigated by replacing each with an Ala residue by the fluoren-9-yl-methoxycarbonyl (Fmoc) solid-phase synthesis.

Results and discussion Nine brazzein analogues were synthesized by the same Fmoc solid-phase method as used for the synthesis of brazzein [3]. The peptides were synthesized by single coupling of the 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole (HBTU/HOBt) method, except for the His and Arg residues which were incorporated by double coupling of the HBTU/HOBt method. The peptide-resins were deprotected and cleaved from the resin support. The resulting peptides were treated with dithiothreitol (DTT) in 0.2 M ammonium acetate buffer (pH 8.0) containing 6 M guanidine hydrochloride to prevent polymer formation. The peptides were purified by preparative reversed phase HPLC (RP-HPLC) and characterized by analytical HPLC and electrospray ionization mass spectrometry (ESIMS), which gave satisfactory results. The reduced analogues thus obtained were folded simply by keeping them at 25°C in aqueous solutions, without any redox systems such as

Figure 1.

Structure of brazzein

750 Y. Shimonishi (ed): Peptide Science – Present and Future, 750–751 © 1999 Kluwer Academic Publishers, Printed in Great Britain

751 Table 1.

Sweetness of brazzein analogues in comparison to sucrose on a weight basis

Brazzein analogues in which an Asp residue was replaced

Sweetness in comparison to sucrose

Brazein analogues in which a Glu residue was replaced

Sweetness in comparison to sucrose

[Ala 2 ] brazzein [Ala 25 ] brazzein [Ala 29 ] brazzein [Ala 40 ] brazzein [Ala 50 ] brazzein

500 250 1000 1250 750

[Ala 9 ] brazzein [Ala 36 ] brazzein [Ala 41 ] brazzein [Ala 53 ] brazzein Synthetic brazzein

1000 times 20 times 1000 times 1250 times 500 times

times times times times times

cysteine/cystine or glutathione/oxdized glutathione (GSH/GSSG). The folding process of each reduced analogue proceeded readily similarly to the process of reduced brazzein. The peptides were purified by RP-HPLC and characterized by analytical HPLC, ESIMS, amino acid analysis and CD spectrometry with satisfactory results. Sweetness was evaluated by matching a threshold concentration of each analogue with that (0.6%w/v) of sucrose (Table 1). The threshold concentration was chosen to avoid confusion arising from the persistent lingering sweet taste of the analogues. The previously synthesized brazzein was 500 times sweeter than sucrose on a weight basis under the same conditions [3]. The sweetness of six analogues ([Ala 29 ]-, [Ala 40 ]-, [Ala 50 ]-, [Ala 9 ]-, [Ala 41 ]-, and [Ala 53 ]brazzeins) was increased. The hydrophobic groups are known to be as important as the carboxyl groups of sweet compounds for binding with the receptor. The substitution of Ala for Asp or Glu might affect the hydrophobicity of brazzein. The sweetness of two analogues ([Ala 25 ]-, and [Ala 36 ]brazzeins) was lowered, and that of [Ala 36 ]brazzein was changed to a large extent. The low potency of [Ala 36 ]brazzein suggests that the Glu 36 residue is involved in binding with the receptor.

References 1. Ming, D. and Hellekant, G., FEBS Lett., 355 (1994) 106. 2. Kohmura, M., Ota, M., Izawa, H., Ming, D., Hellekant, G., and Ariyoshi, Y., Biopolymers, 38 (1996) 553. 3. Izawa, H., Ota, M., Kohmura, M., and Ariyoshi, Y., Biopolymers, 39 (1996) 95.

256 Asymmetric synthesis of β -amino acids: dynamic kinetic resolution utilizing 2-oxoimidazolidine4-carboxylate as a chiral auxiliary K. NUNAMI¹, A. KUBO², H. KUBOTA² and M. TAKAHASHI² ¹ Lead Generation Research Laboratory, 2 Lead Optimization Research Laboratory, Tanabe Seiyaku Co., Ltd., 16–89, Kashima-3-chome, Yodogawa-ku, Osaka 532, Japan

Introduction As a part of our studies on exploiting a new category of stereoselective transformation process using 2-oxoimidazolidine-4-carboxylate as a chiral auxiliary, we reported the dynamic kinetic resolution of tert-butyl (4S)-3-[(2RS) 2-bromoacyl]-1-methyl-2-oxoimidazolidine-4-carboxylate (1 ) with an amine, which led to the synthesis of a range of optically active α -amino acids [1,2]. We expected that an extension of this novel methodology by employing carbon nucleophiles would enable the efficient synthesis of a wide variety of optically active β -amino acid derivatives. In this paper, we wish to report the first example of highly stereoselective carbon–carbon bond-forming reaction by chiral auxiliary based dynamic kinetic resolution of α -halo acid, which can be applied to the synthesis of a variety of optically active α - or β -alkyl β -amino acid derivatives [3].

Results and discussion Malonic ester enolates were selected as suitable carbon nucleophiles for the dynamic kinetic resolution of 1 because they have soft nucleophilicity sufficient for an alkylation reaction. Besides, their alkylated products were expected to be converted to chiral β -amino acid derivatives. We performed the reaction in several kinds of polar solvents, which were verified to accelerate epimerization at the asymmetric carbon attached to the bromo substituent of 1a in the presence of a base [1]. The reaction of 1a with 3 equiv of sodium dimethyl malonate in polar solvents at 25°C resulted in the predominant formation of (S , R)-2a (Table 1, entry 1–4). The stereoselectivity was greatly affected by a solvent. The best result was observed in both yield and stereoselectivity by using HMPA, in which it was ascertained that the epimerization of 1a w a s extremely fast (entry 4). The use of sodium dibenzyl malonate as a nucleophile increased the stereoselectivity affording (S,R )-2b of 88% de in 82% yield (entry 5). Variations of R¹ on the substrates gave satisfactory yields and stereoselectivity (entry 5–7). Next, we investigated the transformation of the asymmetrically 752 Y. Shimonishi (ed): Peptide Science – Present and Future, 752–754 © 1999 Kluwer Academic Publishers, Printed in Great Britain

753 Table 1.

entry

Reaction of Bromide 1a–c with Malonic Ester Enolate in a Polar Solvent

substrate

solvent

1 1a DMF 2 1a DMSO 1a NMP 3 1a HMPA 4 1a HMPA 5 6 1b HMPA 7 1c HMPA ª Determined by HPLC analysis.

time (h) 24 3 3 3 12 24 48

R¹ Me Me Me Me Me Et i-Bu

R² Me Me Me Me Bn Bn Bn

product 2a 2a 2a 2a 2b 2c 2d

(S , R):( S,S )ª yield(%) 68 : 32 77 69 : 31 97 87 : 13 90 8 8 : 12 92 94: 6 82 94: 6 80 95: 5 79

alkylated products to β -amino acid derivatives which are noticeable synthetic intermediates. Hydrogenation of ( S,R )-2b and successive decarboxylation occurred easily to afford (S,R )-3. Carboxylic acid (S,R )-3 was converted to each type of the optically active α - or β -substituted β -amino acid derivatives. Curtius rearrangement of (S,R )-3 followed by treatment with benzyl alcohol and removal of the chiral auxiliary with NaOMe afforded methyl (2 R)3-(benzyloxycarbonylamino)-2-methylpropionate [ (R)-4 ]. On the other hand, tert -butyl(3 R )-3-(benzyloxycarbonylamino)butanoate [(R)-6 ] was synthesized by Curtius rearrangement of (R)-5, which was easily prepared from (S,R )-3. In summary, we have developed highly stereoselective carbon–carbon bondforming reaction by dynamic kinetic resolution of α -bromo amide 1, and the product was easily converted to β -amino acid derivatives. This procedure would be a new entry to the synthesis of both of the optically active α - or β-alkylβ amino acid derivatives.

754 References 1 . Nunami, K., Kubota, H., Kubo, A., Tetrahedron Lett., 35 (1994) 8439. 2 . Kubota, H., Kubo, A., Takahashi, M., Shimizu, R., Da-te, T., Okamura, K., and Nunami, K. J. Org. Chem., 60 ( 1995) 6776. 3 . A part of this work has been reported in previous communication: Kubo, A., Takahashi, M., Kubota, H., Nunami, K., Tetrahedron Lett., 36 (1995) 6251.

257 The function and insertion of a agatoxin-TK

D -Serine

residue in ω -

M. KUWADA¹, T. WATANABE¹, T. TERAMOTO¹, Y. SHIKATA¹, Y. NISHIZAWA¹, K.Y. KUMAGAYE² and K. NAKAJIMA² ¹Eisai Tsukuba Research Laboratories, Tsukuba 300-26, Japan ²Peptide Institute Inc., Protein Research Foundation, Osaka 562, Japan

Introduction Multiple types of voltage-dependent calcium channels in mammalian neurons play important roles in controlling various nervous functions. There are at least six subtypes of voltage-dependent calcium channels, namely the T-type, L-type, N-type, P-type, and Q-type channels, classified on the basis of their electrophysiological and pharmacological properties. Among them, both the P-type and Q-type calcium channels have recently been reported to play a dominant role in the central nervous system [1]. In 1993, we discovered a potent and selective peptide blocker of the P-type [2–4]. a n d l o n g - l a s t i n g Q - t y p e c h a n n e l s , n a m e d ω -agatoxin-TK ω -Agatoxin-TK, also called ω-agatoxin-IVB by Adams et al. [5], is a 48-aminoacid peptide purified from the vemon of the American funnel web spider. This peptide has a unique structure profile including a high-density disulfide core formed by four disulfide bonds and the presence of D -Serine at position 46 in a hydrophobic C-terminal region. In this paper, we have reviewed the functional role and biosynthetic pathway of the D -Ser 46 in ω -agatoxin-TK.

Results and discussion A very interesting aspect of ω -agatoxin-TK is that the configuration of Ser 46 plays a crucial role in the blocking of the calcium channels. A stereoisomer of the peptide, L-Ser 46 - ω-agatoxin-TK, was also found in the venom, whereas the isomer showed only 80- to 90-fold less potent inhibition of the calcium channels compared with the D -form toxin [3,6]. Structure-function studies demonstrated that the specific conformation of the C-terminal region, together with the disulfide core, might be essential for the blocking of the calcium channels (3). The two peptide diastereomers have distinct physico-chemical properties, including solubility, chromatographic behavior [6,7], a secondary structure [7], and sensitivities to proteolytic enzymes [3,6]. The D -form toxin has a lower solubility in water than the L-form toxin at neutral pH. During reversedphase HPLC and hydrophobic chromatography, the D -form toxin was eluted 755 Y. Shimonishi (ed): Peptide Science – Present and Future, 755–756 © 1999 Kluwer Academic Publishers, Printed in Great Britain

756 more slowly than the L-form toxin under neutral conditions. These data indicated that the D -form toxin adopted a relatively more hydrophobic folding compared with the L-form toxin. Interestingly, the circular dichroism spectra of the two toxins revealed that the D -form toxin has a higher β -sheet content than the L -form toxin. These data imply that the D -Ser 46 residue may be involved in the formation of an additional intramolecular β -sheet structure in the C-terminal region, which contributes to the hydrophobic nature of the D -form toxin. Additionally, limited proteolysis of the two peptides revealed that the D -Ser toxin is more resistant than the L-form toxin to Staphylococcus aureus V8 protease and porcine pepsin. On the basis of these data, it was suggested that the configuration of the Ser 46 residue may determine the conformation and functional properties of ω -agatoxin-TK. A novel post-translational epimerization was found to involve the insertion of D -Ser 46 into ω -agatoxin-TK [8]. We have discovered a novel peptide isomerase in the spider venom which specifically inverts L-Ser 46 to D -Ser 46 of the peptide. The isomerase is a 29- kDa polypeptide which has a significant sequence homology with the catalytic site of the serine proteases. The epimerization mechanism would involve two catalytic bases of the isomerase, one of which removes an α -carbon proton from the L -Ser 46 of ω -agatoxin-TK, and the second base in the conjugated acid form delivers a proton to the α -carbon from the opposite face, leading to conversion of the configuration of the Ser 46 residue. The peptide isomerase is the first enzyme from a eukaryotic organism that converts the absolute configuration of the amino acid residue in the peptide.

References 1 . Teramoto, T., Niidome, T., Miyazawa, T., Nishizawa, Y., Katayama, K., Mayumi, T., and Sawada, K., NeuroReport., 6 (1995) 1684. 2 . Teramoto T., Kuwada, M., Niidome, T., Sawada, K., Nishizawa, Y., and Katayama, K., Biochem. Biophys. Res. Commun., 196 (1993) 134. 3 . Kuwada, M., Teramoto, T., Kumagaye, K.Y., Nakajima, K., Watanabe, T., Kawai, T., Kawakami, Y., Niidome, T., Sawada, K., Nishizawa, Y., and Katayama, K., Mol. Pharmacol., 46 (1994) 587. 4 . Teramoto, T., Niidome, T., Kimura, M., Ohgoh, M., Nishizawa, Y., Katayama, K., Mayumi, T., and Sawada, K., Brain Res., 756 (1997) 225. 5 . Adams, M.E., Mintz, I.M., Reily, M.D., Thanabel, V., and Bean, B.P., Mol. Pharmacol., 44 (1993) 681. 6 . Watanabe, T., Teramoto, T., Kuwada, M., Shikata, Y., Niidome, T., Kawakami, Y., Sawada, K., Nishizawa, Y., and Katayama, K., In ohno, M. (Eds) Peptide Chemistry 1994 (Proceedings of the 32nd Japanese Symposium on Peptide Chemistry), Protein Research Foundation, Osaka, 1995, p.253. 7 . Watanabe, T., Kuwada, M., Kumagaye, K.Y., Nakajima, K., Nishizawa, Y., and Asakawa, N.D., in Marshak, R., Techniques in Protein Chemistry VIII, Academic Press, 1997, p.543. 8 . Shikata, Y., Watanabe, T., Teramoto, T., Inoue, A., Kawakami, Y., Nishizawa, Y., Katayama, K., and Kuwada, M., J. Biol. Chem., 270 (1995) 16719.

258 Physiological role of the delta sleep-inducing peptide (DSIP) V.M. KOVALZON Severtsov Institute of Ecology and Evolution, Academy of Sciences, Leninsky Prospect 33, Moscow 117071, Russia

Introduction DSIP, a nonapeptide isolated from the rabbits cerebral venous blood by a Swiss team 20 years ago [1], is regarded as one of the most enigmatic neuropeptides: its natural occurence is still doubtful [2], and its involvement into sleep regulation remains to be elucidated [3,4]. The problem is, the main part of exogenously administrated DSIP sample could be inactivated by specific aminopeptidase just before it’s binding with putative receptor or complexing with protective carrier protein [5,6]. We tried to overcome these difficulties using a large group of artificial DSIP analogs possessing higher proteolytic resistance and optimal conformation as well. The peptides have been synthesized by I.I. Mikhaleva et al. (Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow) using both classical and solid-phase methods [7, 8].

Results and discussion Experiments were performed in rabbits with preliminary implanted electrodes and a single intracerebroventricular cannula for injection of the peptide solutions; otherwise the same animals treated with a saline were used as controls. The electrical activity of the brain (EEG) and neck muscles (EMG) was recorded continuously for at least 12 h. The signals were amplified, band-pass filtered and A/D converted. EEG power spectra were calculated over 4-s epochs for the frequency range 1.0–25 Hz by a FFT routine. The EEG and EMG signals were processed by PC and stored on a DAT tape drive. The data obtained by the paperless recording method were analyzed and scored visually. The results of the EEG spectral analysis were summarized, processed and calculated using a special software. Despite the poor effects of the parent DSIP, pronounced pulsatile changes in sleep profile distributed throughout the 12-hr day-light recording period have been found after administration of several aminopeptidase-resistant analogs. The substitution of the N-terminal Trp for D -Tyr was among the most effective. In many cases, the peak of sleep-promoting effect was delayed for 6–8 hrs from the moment of 757 Y. Shimonishi (ed): Peptide Science – Present and Future, 757–758 © 1999 Kluwer Academic Publishers, Printed in Great Britain

758 8 9 10 C 4 5 6 7 2 3 N 1 DSIP Trp ----Ala-Gly-Gly-Asp-Ala-Ser---Gly-Glu Dermorphin Tyr-- D-Ala-Phe-Gly-Tyr-Pro-SerNH 2 DLP Tyr ----Ala-Phe-Gly-Tyr-Pro-Ser---Gly-Glu-Ala Figure 1. DSIP and Dermorphins. DSIP, Delta Sleep-Inducing Peptide isolated from the rabbit blood [1]. Dermorphin isolated from the frog (Phyllomedusa sauvagei) skin [9]. DLP, DermorphinLike Peptide, liberated after enzymatic digesting of prodermorphin expressed in mammalian cells in vitro [10].

administration. Taking into account closed structural similarity between original DSIP and newly discovered dermorphins [9, 10] really containing a Tyr residue as N-terminus (Figure 1) revealed recently using a peptide bank EROPMoscow [11] (A.A.Zamyatnin, Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow), we propose that still unknown endogenous DSIP-like/dermorphin-like peptide is involved into modulation of both slow wave and paradoxical sleep, which activity might be mediated through several hypothalamic-pituitary hormones. These latter peptides (members of ACTH family, growth-related hormones, etc.) possess sleep-promoting or suppressing actions and have closed anatomical and/or functional relations with endogenous DSIP/DSIP-like peptide(s) [4, 12].

Acknowledgement Supported in parts by grants from the ISF (NEY000), ISF and the Russian Government (NEY300), and INTAS (#93–01612).

References 1. Schoenenberger, G.A. and Monnier, M., Proc. Nat. Acad. Sci. USA, 74 (1977) 1282–1286. 2. Sillard, R., Schulzknappe, P., Vogel, P., Raida, M., Bensch, K. W., Forssmann, W.G., and Mutt, V., Eur. J. Biochem., 216 (1993) 429–436. 3. Borbely, A.A. and Tobler, I., Physiol. Revs., 69 (1989) 605–670. 4. Kovalzon, V.M., J. Evol. Biochem. Physiol., 30 (1994) 195–199. 5. Bjartell, A., Delta Sleep-Inducing Peptide: a mammalian regulatory peptide. Grahns Boktryckeri, Lund, 1990. 6. Inoue, S., Biology of sleep substances. CRC Press, Boca Raton (Fl.), 1989. 7. Mikhaleva, I., Sargsyan, A., Balashova, T., and Ivanov, V. In Voelter, W., Wunsch, E., Ovchinnikov, J., and Ivanov, V, (Eds.) Chemistry of Peptides and Proteins, Walter de Gruyter, Berlin, 1982, v.1. p. 289–297. 8. Prudchenko, I.A., Stashevskaya, L.V., Mikhaleva, I.I., and Ivanov, V.T., Bioorg. Chem. 19 (1993) 43–55. 9. Montecucchi, P.C., de Casiglione, R., Piani, S., Gozzini, I., and Erspamer, V., Int. J. Peptide Protein Res., 17 (1981) 275–283. 10. Seethaler, G., le Caer, J.-P., Rossier, J., and Kreil, G., Neuropeptides, 25 (1993) 61–64. 11. Zamyatnin, A.A., Prot. Seq. Data Anal., 4 (1991) 49–52. 12. Charnay, Y., Bouras, C., Vallet, P.G., Golaz, J., Guntern, R., and Constantinidis, J., Neuroendocrinology, 49 (1989) 169–175.

259 Comparison of secondary structure and antibacterial activity of bactericidal peptides derived from bovine and Korean native goat lactoferrin K-I. SHIMAZAKI¹, M.S. NAM², T. TAZUME¹, H. KUMURA¹, K. MIKAWA¹, K-K. LEE² and D-Y. YU² ¹ Dairy Science Laboratory, Department of Animal Product Science, Institute of Agricultural Science, Hokkaido University, Sapporo, 060-8589 Japan ² Plant and Animal Cell Technology Division, Korea Research Institute of Bioscience and Biotechnology, KIST, Taeduck Science Town, Taejon 305-606, Korea

Introduction A multifunctional protein ‘lactoferrin’, that is found in milk and other secretory fluids and also in blood, is known to have roles in antimicrobial effects, modulation of inflammatory response, immune system activation, control of myelopoiesis, regulation of cell growth and many other functions. Hydrolysates produced by pepsin cleavage of human and bovine lactoferrin were found to contain a potent bactericidal peptide, named ‘lactoferricin ® ’ [1]. We have compared antimicrobial activity, secondary structure and interaction of bovine lactoferricin (LFcin B), disulfide bond cleaved and alkylated LFcin B and Korean native goat lactoferricin (LFcin G) peptide that was chemically synthesized based on the mRNA sequence analysis [2].

Results and discussion As in Table 1, antibacterial activities against E. coli O111 of 6 kinds of lactoferrin, and their hydrolysates generated by pepsin digestion were compared. Hydrolysates showed more effective than intact lactoferrin. The fraction showTable 1. Comparison of antimicrobial activities (MICa values) of intact lactoferrin, lactoferrin hydrolysate and the most active fraction against E. coli O111. Intact lactoferrin bovine goat (Saanen) sheep (Booroola Dorset) horse human KNgoat a c

c

1, 2 mg/ml > 7.5 > 7.5 > 7.5 3c 5

Lactoferrin hydrolysate

Active fraction

0.1 mg/ml 1 1 1 0.5 c nd

5.5b , 6 c µg/ml 150b 170b 250 b 100c 40 d , 30–40 b

Minimum inhibitory concentration. b The active fraction obtained by HPLC using C18 column. From reference [1]. d LFcin G peptide. nd, Not determined.

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760

Figure 1. CD spectra of LFcin B (A), disulfide cleaved and alkylated LFcin B (B) and LFcin G peptide (C) in the presence and absence of trifluoroethanol and the effects of heparin binding on the secondary structure (D) of LFcin B and LFcin G peptide. All figures are drawn in the same scale.

ing the strongest antibacterial activities of lactoferrin hydrolysates was isolated by reverse-phase HPLC. Here, amino acid sequence of LFcin B is FKCRRWQWRMKKLGAPSITCVRRAFA and that of LFcin G peptide is CYQWQRRMRKLGAPSITCIRRTS. Both peptides consist of mainly β -sheet structure (1/3 to 1/2 of the total structure) and unordered structure (ca. 1/2) and have no α -helix conformation judged by CD spectral measurements. When each peptide solution was mixed with a helix-forming solvent, trifluoroethanol, a little change of secondary structure was observed (Figure 1A, B and C). On the other hand, we found that some basic amino acid residues located in LFcin part of bovine lactoferrin are responsible to heparin binding (K.Shimazaki et al., unpublished). It may be explained that the modulating effect for inflammation by lactoferrin may be concerned with heparin-lactoferrin binding. In the presence of heparin at the weight ratio of near 2 (heparin/LFcin), a distinct spectral change due to the conformational alteration of the peptide was induced (Figure 1D) and this change was reversible in the addition of 1 M NaCl. These results suggest the possibility that lactoferrin changes its conformation to perform biological function through the interaction with cell surface substances or mucosal components.

References 1. Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K., and Tomita, M., Biochim. Biophys. Acta, 1121 (1992) 130. 2. Lee, T.H., Shimazaki, K., Yu, S.L., Nam, M.S., Kim, S.J., Lee, K.K., and Yu, D.Y., Animal Genetics, 28 (1997) 367.

260 Synthesis of enantiomerically pure non proteinogenic α-amino acids L. LECOINTE, J.-M. RECEVEUR, V. ROLLAND, M.-L. ROUMESTANT, P. VIALLEFONT and J. MARTINEZ L.A.P.P., UMR CNRS 5810, Universités Montpellier I & II, STL, Place E.Bataillon, 34095 Montpellier Cedex 05, France

Introduction Optically pure non proteinogenic α -amino acids are important as pharmaceuticals and as crop protection agents. Moreover they are synthetic precursors and are used as chiral units for synthesis of fine chemicals and for incorporation into modified biologically active peptides. We describe here the synthesis of enantiomerically pure non proteinogenic α -amino acids like 4-methylene glutamic acid derivatives [1] which are neuroactive compounds, azatyrosine which is known as an antibiotic and anticarcinogen [2], trichloro and tribromophenylalanine which are incorporated into biologically active peptides for tritiation and 7-azatryptophane which is a potential fluorescent probe [3]. Results and discussion The key step of the strategy is the diastereoselective alkylation and (or) protonation of enolates of chiral Schiff bases prepared from 2-hydroxypinan-3-one and α -amino esters. For alkylation reactions, the alkylating agents were easily prepared from commercially available materials (scheme 1) Diastereoselective alkylation For enolate formation in alkylation reactions two methods were used (Table 1): Method A: The chiral Schiff base 1 prepared from 2-hydroxypinan-3-one and tert-butylglycinate was treated with two equivalents of lithium diisopropylamide (LDA) in anhydrous THF under Argon at – 78°C.

Scheme 1.

761 Y. Shimonishi (ed): Peptide Science – Present and Future, 761–763 © 1999 Kluwer Academic Publishers, Printed in Great Britain

762

Scheme 2. Table 1. Enolate formation

R

Compound

Yield

d.e.

method B method A method B

R1 R2 R3

2a 2b 2c

60 60 70

> 98 > 98 > 98

Method B: The same chiral Schiff base 1 was treated with one equivalent of CH3 MgBr followed by one equivalent of LDA in anhydrous THF under Argon at – 78°C (Scheme 2). The Schiff bases were easily cleaved by citric acid or boric acid (70% to 80% yield) to yield the corresponding amino esters which were easily transformed into amino acids by 5 N HCl treatment. Diastereoselective protonation Racemic amino esters (azatyrosine, tribromophenylalanine methyl esters) were obtained by alkylation of ethyl N -diphenylmethylene glycinate with the bromides previously described followed by hydrolysis. For 7-azatryptophane the best method is the alkylation of diethyl N -acetylaminomalonate with azagramine in xylene using NaOH as base. Treatment of the alkylated product with NaOH followed by 5 N HCl led to the amino acid which was esterified to yield the amino ester. Chiral Schiff bases prepared from 2-hydroxypinan-3-one and these amino esters were deprotonated by tert-BuOK at –78°C in anhydrous THF and reprotonated by a saturated solution of NH4 Cl (Scheme 3). In these conditions azatyrosine and tribromophenylalanine were obtained in good yields and excellent d.e., whereas 7-azatryptophane was prepared only in 66% d.e.. This last compound was synthesized enantiomerically pure by enzymatic resolution of the N-acetyl amino acid by Aspergillus genus acylase.

Scheme 3.

763 Conclusion Diastereoselective alkylation and (or) protonation of chiral Schiff bases allowed the synthesis of enantiomerically pure azatyrosine, tribromophenylalanine and 4-methylene glutamic acid. For 7-azatryptophane these methods were unsuccessful and enzymatic resolution using an aminoacylase was the best way to prepare the enantiomerically pure compound. Moreover some of these compounds can also be considered as polyfunctionalized building blocks useful in combinatorial chemistry.

References 1. Receveur, J.M., Roumestant, M.L., and Viallefont, P., Amino Acids, 9 (1995) 391. 2. Shindo-Okada, N., Makabe, O., Naghara, H., and Nishimura, S., Molecular carcinogenis, 2 (1989) 159. 3. Négrerie, N., Bellefeuille, S.M., Whitham, S., Petrich, J.M., and Thornburg, R. V., J. Am. Chem. Soc., 112 (1990) 7419.

261 Improvements of activity and stability against proteases of biologically active peptides by coupling of a cyclic peptide derived from an endothelin antagonist RES-701-1 K. SHIBATA¹, T. SUZAWA¹, S. SOGA², T. MIZUKAMI1, K. YAMADA², N. HANAI¹ and M. YAMASAKI¹ ¹ Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194, Japan ² Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411, Japan

Introduction An endothelin (ET) antagonist RES-701–1 (1, Figure 1) has a unique cyclic structure in its N-terminal [1]. The cyclic structure of 1 is essential for its biological activity and is important for its high stability against degradation by proteases. By coupling of the cyclic moiety of 1 (2 , Figure 1), to the N-terminal of a weak ET antagonist [A15] ET-1 (12–21), the receptor binding activity has shown to be remarkably improved [2]. The linear peptide (4 ) of the same sequence as 2 -[A15] ET-1 (12–21) (3) has similar binding activity for ET B receptor [3], however, the stability against proteases has been proved to be much different from 3. Degradation of 4 by subtilisin, one of the typical protease, was much faster than 3 (Figure 2a). RES-701–1, as well as ET-1, has been reported to have a turn structure in its C -terminal, which is considered to be important for its receptor binding [4]. We have predicted from these results that the cyclic peptide 2 can stabilize the solution structure, particularly the turn structure, of the peptides coupled to the C -terminal, which results in

Figure 1. Structures of RES-701–1 (1) and biologically active peptides coupled with the cyclic moiety of 1 (2).

764 Y. Shimonishi (ed): Peptide Science – Present and Future, 764–766 © 1999 Kluwer Academic Publishers, Printed in Great Britain

765

Figure 2. Time course of degradations of peptides by several proteases at 37°C. (a) 3 (closed circles) and 4 (open circles) by subtilysin. (b) 5a (open circles) and 5b (closed circles) by trypsin. (c) 7a (open circles) and 7b (closed circles) by prolylendopeptidase.

the improvement of biological activity and stability against proteases. To evaluate this prediction, we have coupled the cyclic peptide 2 to known cell adhesion inhibiting RGD-peptides [5] and protein farnesyltransferase (FTase) inhibiting peptides [6], which have been reported to have turn structures in their sequences, and tested the biological activities and stabilities against several proteases.

Results and discussion Two typical RGD-peptides 5a and 6a, derived from the sequence of human fibronectin, have been coupled with 2 ( 5b and 6 b, respectively. Figure 1) by solid phase coupling of 2 to sidechain-protected peptides on the solid support. We have investigated the biological activity of these peptides by cell adhesion inhibition of mouse melanoma cell B16-F10 to mouse fibronectin. Peptide 5a inhibited the cell adhesion in IC50 of 1.1 mM while 5b in IC 50 of 0.29 mM, that is, the biological activity of 5a has been improved by about 4-times by coupling of 2 . Peptide 5b also showed about 4-times higher inhibiting activity on the collagen-stimulated aggregation of isolated rabbit platelet. Coupling of 2 to 5a has also resulted in the improvement of stability against trypsin as shown in Figure 2b. By coupling of 2 to 6a , cell adhesion-inhibiting activity has improved only slightly, while the stability against chymotrypsin has improved remarkably (data not shown). FTase inhibiting peptides 7a and 8a, derived from the C -terminal sequence of human N-Ras, were coupled with 2 (Figure 1). Their in vitro activities were evaluated by the inhibition of farnesylation of v-Ki-Ras protein by isolated bovine FTase. Peptide 7b showed 3-times higher inhibiting activity than 7a, IC 50 of 0.56 µM and 1.6 µM, respectively, while 8b showed slightly improved activity. Stabilities against prolylendopeptidase were remarkably improved by coupling both with 7a (Figure 2c) and 8a (data not shown). These results have been considered to indicate the validity of our prediction; the cyclic peptide can stabilize the solution structures of the peptides coupled

766 to the C -terminal. Generally, the cyclization of some peptides themselves has been reported to result in stabilizing the solution structures of the peptide backbone. Coupling of our cyclic peptide is proposed to be a novel structurestabilizing method for biologically active peptides, results in improvements of activity and stability.

References 1. Yamasaki, M., Yano, K., Yoshida, M., Matsuda, Y., and Yamaguchi, K., J. Antibiot., 47 (1994) 276. 2. Suzawa, T., Shibata, K., Tanaka, T., Matsuda, Y., and Yamasaki, M., Bioorg. Med. Chem. Lett., 7(1997) 1715. 3. Shibata, K., Suzawa, T., Tanaka, T., Matsuda, Y., and Yamasaki, M., In Nishi, N. (Eds) Peptide Chemistry 1995, Protein Research Foundation, Osaka, 1996, pp. 281–284. 4. Katahira, R., Shibata, K., Yamasaki, M., Matsuda, Y., and Yoshida, M., Bioorg. Med. Chem., 3 (1995) 1273. 5. Callahan, J.F., Bean, J.W., Burgess, J.L., Eggleston, D.S., Hwang, S.M., Kopple, K.D., Koster, P.F., Nichols, A., Peishoff, C.E., Samanen, J.M., Vasko, J.A., Wong, A., and Huffman, W.F., J. Med. Chem., 35 (1992) 3970, and references cited therein. 6. Stradley, S.J., Rizo, J., and Gierasch, L.M., Biochemistry, 32 (1993) 12586, and references cited therein

262 Structure-activity relationship of cyclic depsipeptides, AM-toxin analogs M. MIYASHITA, T. NAKAMORI, T. MURAI, H. MIYAGAWA, M. AKAMATSU and T. UENO Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Introduction AM-toxins I-III are host-specific phytotoxins of Alternaria alternata apple pathotype which cause veinal necrosis on apple leaves of susceptible cultivars such as Indo and Starking Delicious. The activity of AM-toxin II, III and I increases in that order. The toxins act on the chloroplasts and the plasma membranes, but the mechanism of action of these toxins is not completely understood. Their structures were determined by Ueno et al. [1]. The toxins are cyclic tetradepsipeptides containing two unusual amino acids and an L- α hydroxy acid (Figure 1). Previous structure-activity studies indicated that the structural modifications of L-Hmb, L-Amp and ∆ Ala residues of AM-toxin (Figure 1) drastically lowered necrotic activity. Although some analogs in which the L–X residue was modified have also been synthesized [2], the effect of the residue on the activity has not been systematically investigated. In this study, a series of AM-toxin I analogs in which the L –X residue was modified were newly synthesized, then their necrotic activity was measured. In order to examine the relationship between the structure and activity, the active conformation of some AM-toxin analogs was also analyzed. Results and discussion The Fmoc-based solid-phase peptide synthesis method with DIPCDI/HOBt was used to obtain linear depsipeptides [3]. Formation of the ester bond of

Figure 1.

Structure of AM-toxin analogs.

767 Y. Shimonishi (ed): Peptide Science – Present and Future, 767–769 © 1999 Kluwer Academic Publishers, Printed in Great Britain

768 the main chain was carried out with DIPCDI/DMAP. Cyclization of tetradepsipeptides was effected using HATU/diisopropylethylamine. The ∆ Ala was formed by the Hofmann degradation of the D -2,3-diaminopropionic acid at the final step [4]. The necrotic activity was measured using the small leaves derived from the micropropagated shoot culture of susceptible cultivar Indo. The leaves were put into 50 µl of the distilled water containing 1% agar, then 20 µl of various concentrations of toxin solutions were layered over the agar. After incubation for 48 hours in the dark, the development of necrosis along the veins was observed. The minimum concentration for the induction of necrosis was used as an index for the activity. The structures and necrotic activity of AM-toxin analogs which were synthesized in this study are shown in Table 1. All analogs having a longer alkyl side chain than that of L-Ala showed 10 times lower activity than that of AM-toxin I. But the activity of the L -Tyr analog decreased by 1000 times. This shows that the length of the alkyl side chain at the L -Ala position does not have a great effect on the activity, but a relatively bulky side chain such as that of a Tyr residue may be unfavorable to the activity. Since the Tyr side chain is likely to affect not only interaction with the putative receptor but also the conformation of the compound, molecular dynamics calculation was performed for the AM-toxin I and the L-Tyr analog. The software Insight II/Discover was used for molecular modeling, energy calculation and molecular dynamics simulation. The 2D NOESY spectrum data (500 MHz) in DMSO-d 6 was used for the distance constraints in the calculation of conformations of AM-toxin I. The result showed the lowest energy backbone conformation of the L -Tyr analog was not significantly different from that of AM-toxin I. Therefore, it suggests some steric factors of the Tyr side chain unfavorably affect its binding to the receptor, lowering the activity.

Table 1.

Necrotic activity of AM-toxin I and its analogs

necrotic activity (M) 10-7 10 -8 10 -9 10 -10 10-11

R (AM-toxin I)

+

+

+

+

–CH 2CH 3

(L-Abu)

–CH 2 CH2 CH3

(L-Nva)

+ +

+ +

+ +

– –

–CH 2CH 2 CH2 CH3

(L-Nle)

+

+

+

–

–CH 2

(L-Tyr)

+

–

– C H3

OH

–

769 Acknowledgments Computation time was provided by the Supercomputer Laboratory, Institute for Chemical Research, Kyoto University.

References 1. Ueno, T., Nakashima, T., Hayashi, Y., and Fukami, H., Agric. Biol. Chem. 39 (1975) 1115. 2. Aoyagi, H., Mihara, H., Lee, S., Kato, T., Ueno, T., and Izumiya, N., Int. J. Pept. Protein Res. 25 (1985) 144. 3. Miyashita, M., Nakamori, T., Akamatsu, M., Murai, T., Miyagawa, H., and Ueno, T., in Kitada, C., (Eds) Peptide Chemistry 1996, Protein Research Foundation, Osaka, 1997, p. 137. 4. Kanmera, T., Aoyagi, H., Waki, M., Kato, T., and Izumiya, N., Tetrahedron Lett. 22 (1981) 3625.

263 Effects of chemical modification of Ca2 + -dependent lectin CEL-III on its hemolytic and carbohydratebinding activities H. KUWAHARA, T. FUNADA, T. NIIDOME, T. HATAKEYAMA and H. AOYAGI Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki 852, Japan

Introduction CEL-III is a Ca 2 + -dependent, galactose/GalNAc-specific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). This protein shows strong hemolytic activity toward rabbit and human erythrocytes. Hemolysis is caused by colloid osmotic rupture due to ion-permeable pores formed by CEL-III oligomer in cell membrane [1]. Oligomerization of CEL-III is assumed to be triggered by the binding of CEL-III to specific carbohydrate receptors on erythrocyte membrane. The hemolytic activity drastically increases in the alkaline pH region. This suggests that amino acid residues having ionization groups may be involved in the activity. In this study, we examined the effects of chemical modification of amino and carboxyl groups on the activities of CEL-III.

Results and discussion Figure 1 shows the relationship between hemolytic activity and the average number of succinyl or glycine methylester groups introduced in CEL-III. Hemolytic activity as measured by the absorbance of released hemoglobin decreased with increasing concentration of the reagent. The introduced succinyl or glycine methylester groups were determined by MALDI-TOF mass spectrometry based on the increase of the mass of the protein. When 7 amino groups per protein were modified with succinic anhydride of 20 mM, the hemolytic activity decreased to 25% (Figure 1A). However, carbohydrate-binding and hemagglutinating activities were almost completely retained. On the other hand, When ca. 23 carboxyl groups per protein were modified with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glycine methylester, the hemolytic activity decreased to 10% (Figure 1B). Carbohydratebinding and hemagglutinating activities also decreased. 770 Y. Shimonishi (ed): Peptide Science – Present and Future, 770–771 © 1999 Kluwer Academic Publishers, Printed in Great Britain

771

Figure 1. Relationship between hemolytic activity and the number of modified residues. CEL-III was modified with succinic anhydride (panel A) or 1-ethyl-3-(3- dimethyl aminopropyl) carbodiimide (EDC) and glycine methylester (panel B).

These results suggest that amino groups are involved in hemolytic activity of CEL-III, and their modification leads to inhibition of pore-formation process without considerable reduction of the carbohydrate-binding activity. Although modification of carboxyl groups also lead to remarkable decrease in hemolysis, it may result from inactivation of carbohydrate-binding activity by the modification of carboxyl group(s) located at or near the carbohydrate-binding sites of the protein.

Reference 1. Hatakeyama, T., Nagatomo, H., and Yamasaki, N., J. Biol. Chem., 270 (1995) 3560.

264 Coacervation of elastomeric polypeptides: microscopic observations and light scattering measurements K. KAIBARA1, T. WATANABE 1, K. MIYAKAWA2, M. KONDO 3 and K. OKAMOTO 4 1

Department of Chemistry, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812, Japan 2 Department of Applied Physics, Faculty of Science, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka 814-01, Japan 3 Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840, Japan 4 Department of Biochemical Engineering and Science, Faculty of Computer Science and Systems Engineering, Kyusku Institute of Technology, Iizuka, Fukuoka 820, Japan

Introduction Elastomeric protein, elastin, is an important and major constituent of the biological elastic fiber in mammalian tissues such as aortal wall, lung, and ligament. Molecular assembly process of precursor protein, tropoelastin, in extracellular space viewed as a key step of the elastogenesis can be mimicked by the temperature-dependent coacervation of aqueous elastin-related polypeptide solutions. Characterizations of the molecular self-assembly process during liquid-liquid phase separation are required to clarify structure, mechanism, and function of elastin. Thermodynamic stability concepts of the phase behavior of elastomeric polypeptide–water system are also important to understand the structural basis of the multiple functions of elastin as an extracellular matrix.

Results and discussion Coacervation process of aqueous solutions of α -elastin derived from bovine neck ligament (0–20 mg/ml) or synthetic elastin model block copolymer with repeating pentapeptide sequences, (VPGVG) n , (0–5 mg/ml) was examined by phase contrast microscopy and light scattering photometry as a function of temperature and polypeptide concentrations. Light scattering experiments were useful to analyze early stages of phase separation process. Light scattering measurements demonstrated a role of the critical fluctuations on the condensation of polypeptides leading to supramolecular ordering and characteristic differences of critical process with off-critical process [1, 2]. Coacervation of aqueous elastomeric polypeptide solution was essentially described by a phase diagram with lower critical solution temperature. Binodal and spinodal temperatures were estimated by a light scattering experiments within the α -elastin concentration ranges 0–1 mg/ml. Microscopic observations 772 Y. Shimonishi (ed): Peptide Science – Present and Future, 772–774 © 1999 Kluwer Academic Publishers, Printed in Great Britain

773 of phase separated microcoacervate droplets under Brownian motion were useful to trace phase behavior even for systems with high peptide concentrations where photometric measurements were unavailable. Computational analyses of phase contrast microscopic images evaluating size, shape, and number of coacervate droplets were correlated with photometric measurements. Microscopic determinations of the binodal temperatures were carried out based on the droplet size histogram and droplet number-temperature relationships. At lower concentration ranges of α -elastin, 0–1 mg/ml, binodal temperatures estimated by the light scattering measurements and microscopic observations were correlated well. At around critical concentration of α -elastin, 0.11 mg/ml, droplet size was maximal and broad size distribution was observed, probably due to the critical process with large concentration fluctuation in solution phase. After initiating the phase separation, further temperature elevation caused increases in hydrodynamic size of light scattering particles and microscopic droplet size. On the contrary in the off-critical conditions, both the hydrodynamic size and visible droplet size were reduced and size distribution was narrow. The hydrodynamic size of scattering particles and microscopically visible droplet size are not necessarily comparable, however, qualitative changes as a function of temperature and polypeptide concentration were identical. One possible explanation for the smaller droplet size in off-critical situations was suggested from the light scattering experiments. Hydrodynamic size of scattering particles below the temperature at which the phase separation initiates were smaller in offcritical conditions than those in critical conditions. Localized and small concentration fluctuations of the off-critical process lead to form primary aggregates before the initiation of phase separation. During the coacervation process, these aggregates reduced their size by excluding bound water molecules based on the hydrophobic interactions. In case of the system containing polypentapeptide, (VPGVG)n ; n ≥ 40, coacervate droplet behavior was similar to that of critical conditions of α-elastin system. The droplet size distribution was broad and droplet sizes increased with elevating temperature. Droplet sizes were larger, in spite of the low molecular weight, than those of α -elastin system. It was suggested that more hydrophobic molecular characteristics and specific molecular conformation, such as β -spiral, of polypentapeptide contribute to these self-assembly process. Both in the α -elastin and polypentapeptide systems, appearances of elongated aggregates, approximately 10 µm × 1 µm dimension, were detected. Conditions to form elongated aggregates were still unknown, however, factors affecting the transformation process of spherical droplets to elongated aggregates are examined extensively. Conditions in extracellular space, where the self-assembly processes of tropoelastin proceed, seems to be quite different those in usual solution system. It is required for better understanding of physicochemical aspects of elastogenesis and elastin function to examine the effects of unusual conditions of solvent and external forces.

774 References 1. Miyakawa, K., Totoki, M., and Kaibara, K., Biopolymers 35 (1995) 85. 2. Kaibara, K., Okamoto, K., Miyakawa, K., and Kondo, M., In Nishi, N. (Ed.) Peptide Chemistry 1995, Protein Research Foundation, Osaka, 1996, p345.

265 Preparation and properties of novel gramicidin S analogs possessing a polymethylene bridge between ornithine side chains K. YAMADA, K. ANDO Y. TAKAHASHI, H. YAMAMURA, S. ARAKI, and M. KAWAI Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Showa-Ku, Nagoya 466-8555, Japan

Introduction Gramicidin S (GS) is a cyclic decapeptide with the primary structure cyclo(–Val– Orn–Leu- D -Phe–Pro–Val–Orn–Leu- D -Phe–Pro–), which shows antibacterial activity against gram-positive bacteria [1]. The charged Orn side chains are important for the antibacterial activity. The analogs possessing methylamino or trimethylammonio groups in place of the free amino groups on Orn side chains also exhibited the activity [2]. We will report the preparation of novel GS analogs in which the charged N δ atoms of the two Orn side chains are connected by a polymethylene chain (Figure 1).

Results and discussion Upon N -methylation of GS derivatives using CH 3 I–Ag 2 O, the Orn side chains protected with Boc or Cbz groups remained intact [2] due to the presence of H-bonds between the N δ H(Orn) and C=O(D -Phe) groups [3]. Since the Orn residues protected with electron-withdrawing Tfa and Tos were methylated without difficulty, we employed p-nitrobenzenesulfonyl (NBS) group as the N δ substituent for the N -alkylation [4] to construct the polymethylene-bridged analogs of GS. Reaction of bis-NBS derivative [Orn(NBS)2,2' ]GS (1) with Br(CH 2 ) n Br ( n = 3, 4 and 5) afforded the intramolecularly bridged products (2) in 57–73% yields (Figure 2). Deprotection of the NBS groups by treatment with 2-mercaptoethanol [4] furnished the bridged analogs (3) in 42–67% yield.

Figure I.

The structures of a) GS and b) intra- and c) intermolecularly bridged analogs.

775 Y. Shimonishi (ed): Peptide Science – Present and Future, 775–776 © 1999 Kluwer Academic Publishers, Printed in Great Britain

776

Figure 2.

Preparation of intramolecularly bridged analogs of GS.

1

The H NMR spectral analysis of 1, 2, and 3 in DMSO-d 6 solutions suggested that all these compounds adopt similar β -sheet conformation to that of parent GS. NBS-induced deshielding of the NH(D -Phe) was observed only in 1 but not in 2 presumably due to lack of the N δ H(Orn)...C=O( D -Phe) H-bonding in the bridged 2, and ROESY analysis indicated steric proximity of meta -H of NBS group and C α H(Pro) in 2 (n = 4 and 5). CD spectra of 3 ( n = 4 and 5) were almost superimposable to that of GS while 3 (n = 3) showed very similar spectrum but with slightly weaker intensity. Therefore, the trimethylene-bridged derivative seemed to adopt slightly different conformation from others. For the preparation of an intermolecularly bridged derivative, mono-NBS derivative [Orn(Boc) 2 ,Orn(NBS) 2' ]GS was treated with trimethylene bromide and the reaction mixture was subjected to gel filtration chromatography to afford the desired dimeric product in 66% yield, which was characterized by negative FAB-MS: C 145 H 209 N 26 O 32 S 2 ([M–H] – ). Since the location of the charged N atoms are restricted in these bridged analogs, they should be helpful in obtaining new insight into the biological activity of GS.

Acknowledgments The authors are grateful to Nikken Kagaku, Ltd. for the supply of GS • 2HCl and to Prof. R. Katakai and Ms. K. Kobayashi (Gunma Univ.) for 1 H NMR spectroscopy and to Dr. K. Kano (Tsumura, Ltd.) for FAB-MS measurement.

References 1. Izumiya, N., Kato, T., Aoyagi, H., Waki, M., and Kondo, M., Synthetic Aspects of Biologically Active Cyclic Peptides – Gramicidin S and Tyrocidines, Kodansya, 1979. 2. Kawai, M., Ohya, M., Fukuta, N., Butsugan, Y., and Saito, K., Bull. Chem. Soc. Jpn. 64 (1991) 35. 3. Kawai, M., Yamamoto, T., Yamada, K., Yamaguchi, M., Kurobe, S., Yamamura, H., Araki, S., Butsugan, Y., Kobayashi, K., Katakai, R., Saito, K. and Nakajima, T., Lett. Pept. Sci., 5 (1998) 5. 4. Miller, S.C. and Scanlan, T.S., J. Am. Chem. Soc. 119 (1997) 2301.

266 Effect of nitroprusside on amyloid β -peptide induced inhibition of MTT reduction in pheochromocytoma PC12h and C1300 neuroblastoma cells T. TAKENOUCHI and E. MUNEKATA Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Introduction Amyloid β -protein (Aβ ) which contains 39–43 amino acid residues is one of the major constituents of senile plaques in Alzheimer’s disease (AD). Aβ i s known to have a neurotoxic effect in primary hippocampal neurons and several cultured neuronal cell lines, which may include the mediation of reactive oxygen species production [1]. In this study, we have investigated the effect of Aβ peptides in rat pheochromocytoma PC12h and murine C1300 neuroblastoma cells by using MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} reduction assay, because inhibition of MTT reduction activity caused by A β peptides is known as one of the early indicators of its toxicity [2]. Also, we examined the effect of sodium nitroprusside (SNP), a spontaneous nitric oxide (NO) generator, on the actions of Aβ peptides in both cell lines.

Results and discussion Exposure of the cells to A β peptides, A β1–40 and its fragment Aβ 25–35 [3], induced a concentration-dependent inhibition of MTT reduction in both cell lines (Figure 1 A-C), and MTT-dependent LDH release due to cell lysis in

Figure 1. Effect of SNP on Aβ peptides, Aβ1–40 (A) and Aβ25–35 (B) -induced inhibition of MTT reduction in PC12h cells, and C1300 cells (C). Data shown are expressed as percentages of control values and represented by the mean ± SEM (n = 5–13).

777 Y. Shimonishi (ed): Peptide Science – Present and Future, 777–778 © 1999 Kluwer Academic Publishers, Printed in Great Britain

778

Figure 2. Effect of SNP on MTT-dependent LDH release after treatment with Aβ25–35 in PC12h cells (A). Effect of hydroxylamine (B) and 8-Br-cGMP (C) on Aβ 25–35-induced inhibition of MTT reduction in PC12h cells. Data shown are expressed as percentages of control values and represented by the mean ± SEM (n = 4).

PC12h cells (Figure 2 A) [4]. We also found that SNP significantly prevented the inhibition of MTT reduction and MTT-dependent LDH release caused by A β peptides (Figure 1 A–C and Figure 2 A), although a high concentration of SNP ( ≥ 333 µM) was remarkably toxic by itself. Since the inhibition of MTT reduction caused by Aβ is known as one of the first indicators of its toxicity, these findings suggest that Aβ peptides have a toxic effect in these cell lines, and SNP may attenuate the Aβ peptide-induced toxicity. In regard the mechanisms of the actions of SNP, hydroxylamine which also generates NO and 8-Br-cGMP, a membrane-permeable analogue of cyclic GMP (cGMP), failed to prevent the inhibition of MTT reduction caused by Aβ 25–35 (Figure 2 B, C), implying that the effect of SNP may be mediated by the NO-independent pathway. The cell death induced by other oxidative insults, such as glutamate and hydrogen peroxide (H 2 O 2 ), could not be attenuated by SNP in both cell lines (data not shown). Thus, the observed effect of SNP might not be due to its direct antioxidative action.

References 1. 2. 3. 4.

Behl, C., Davis, J.B., Lesley, R., and Schubert, D., Cell, 77 (1994) 817. Shearman, M.S., Ragan, C.I., and Iversen, L.L., Proc. Natl. Acad. Sci. USA, 91 (1994) 1470. Yankner, B.A., Duffy, L.K., and Kirschner, D.A., Science, 250 (1990) 279. Hertel, C., Hauser, N., Schubenel, R., Scilheimer, B., and Kemp, J.A., J. Neurochem., 67 (1996) 272.

267 Practical approach to enzymatic resolution of Fmocand Boc-protected amino acid racemates E. MARCZAK 1, A.W. LIPKOWSKI 1,2 , K. CZERWINSKI 1 and J. KAZIMIERCZAK 1 1 2

Industrial Chemistry Research Institute, 8 Rydygiera Str., 01-793 Warsaw, Poland Medical Research Centre Polish Academy of Sciences, 5 Pawinskiego Str., Warsaw, Poland

Introduction Enantiomers of protected non-protein amino acids are useful substrates in synthesis of peptide analogs for structure-activity relationships studies. Introduction of such amino acids to naturally occuring sequences of peptides modifies their primary properties like resistance to hydrolysis by proteolytic enzymes or/and modifies capability of binding to the receptor. The availability of non protein amino acids is the critical point in a such approach. The stereoselective synthesis and resolution (enzymatic or chemical) of racemic amino acids are two methods, most commonly used for non-protein amino acid synthesis. Although the majority of methods is mostly focused on the preparation of free amino acids, N -protected amino acids are used in the peptide synthesis. In order to obtain the N-protected amino acids used directly in peptide synthesis we have developed a method of resolution of N-protected racemic amino acids by selective enzymatic hydrolysis of their esters. In our experiments Boc- and Fmoc-protecting groups were used as the most interesting for further application in peptide synthesis. Amino acids with such N-protecting groups and their alkyl esters are very lipophylic therefore reaction should be carried out in water containing high concentration of organic solvents. We have optimized reaction conditions for hydrolysis of Fmoc- or Boc-aromatic amino acid methyl esters catalyzed with α-chymotrypsin in water/acetonitrile solutions. Enzyme can be succesfuly adsorbed on solid phase (silica gel) without visible desorption in optimal reaction conditions what makes possible to carry out this reaction as a continous process.

Results and discussion Methyl esters of Boc- and Fmoc-protected aromatic amino acids were incubated with α -chymotrypsin in water-organic medium. For compounds which have been hydrolyzed by α -chymotrypsin in solution hydrolyses with enzyme adsorbed on silica gel were done. Thus α-chymotrypsin from bovine pancreas 779 Y. Shimonishi (ed): Peptide Science – Present and Future, 779–781 © 1999 Kluwer Academic Publishers, Printed in Great Britain

780 was adsorbed on silica gel by passing its solution in Tris buffer pH 8 through a column packed with silica gel. The gel with adsorbed enzyme was washed with buffer/acetonitrile and used in further experiments. Solutions of respective substrates in water/organic media were passed through the column. Reaction products were separated and than unreacted ester of D-amino acid was hydrolyzed chemicaly. The yields were determined by HPLC. Results of hydrolysis of methyl esters of Boc- and Fmoc-aromatic amino acids with and without α -chymotrypsin (Table 1) indicate that α -chymotrypsin can hydrolyze Boc- and Fmoc-protected amino acid methyl esters, but it hydrolyzes only these phenylalanine analogs which have modified aromatic ring in position 4 and 3 ( D,L -phenylalanine, D,L -tyrosine, D,L -p-chorophenylalanine, D,L -p-fluorophenylalanine, D,L -m-fluorotyrosine). Analogs being modified in 2 position of aromatic ring (D,L - o-methylphenylalanine), rigid structure (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) or being modified in position beta of aliphatic chain (D,L -threo-3-phenylserine) have not been hydrolyzed. No hydrolysis was observed without the enzyme. These results indicate that either modification of aromatic ring in position 2 (by introduction of methyl group or by rigidization of the molecule) or modification of aliphatic chain in beta-position changes substrate molecule making it unable to effective binding during formation of enzyme-substrate complex and thus blocking hydrolysis of such substrates by α-chymotrypsin. In summary proposed method of resolution of N-protected racemic amino acids by selective enzymatic hydrolysis of their esters with α-chymotrypsin seems to be very attractive because it allows the preparation of enantiomers of N -protected amino acids which can be directly used in peptide synthesis.

Table 1. Hydrolysis of methyl esters of Boc- and Fmoc-amino acids with and without of α-chymotrypsin (Reaction conditions: temp. 25°C, pH 8.2, content of acetonitril 50% (vol.). Degree of hydrolysis, %

Amino acid substrates

Reaction time, h

α-chymotrypsin

without enzyme

Boc- D,L–Phe–OMe Boc- D,L - p-Cl–Phe–OMe Boc- D,L - p-F–Phe–OMe Boc- D,L - o -Me–Phe–OMe Boc- D,L-TyrOMe Boc- D,L - m -F-Tyr–OMe Boc-L-Tic–OMe Boc- D,L -threo-3-Ph-Ser–OMe Fmoc- D,L –Phe–OMe Fmoc- D,L - p-Cl–Phe–OMe Fmoc- D,L - o-Me–Phe–OMe Fmoc- D,L -Tyr–OMe Fmoc- L -Tic–OMe Fmoc- D,L -threo-3-Ph-Ser–OMe

96 96 96 96 48 48 96 96 48 48 96 48 48 48

50.1 35.9 44.6 0 49.0 49.05 0 0 50.2 37.8 0 50.1 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0

781 Adsorptively bound α-chymotrypsin was very stable during the tests. Enzyme desorption in water-acetonirile mixture was almost negligible.

Acknowledgements This work was supported by Polish Scientific Research Committee (Grant No. 3 T09B 04711).

268 Rapid translocation of amphipathic α-helical and β -sheet-forming peptides through plasma membranes of endothelial cells J. OEHLKE, A. SCHELLER, K. JANEK, B. WIESNER, E. KRAUSE, M. BEYERMANN and M. BIENERT Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany

Introduction Most biopolymers, such as peptides, proteins or oligonucleotides do not cross the plasma membrane of cells efficiently. However, peptides exhibiting the propensity to form amphipathic helices at water-lipid interfaces have repeatedly been shown capable of crossing the plasma membranes of mammalian cells [1–4]. Such peptides may be exploited successfully as vectors for oligonucleotides and other oligopeptides [1, 2]. The aim of the present study was to define the structural features of such peptides which are responsible for cellular uptake and to investigate the mode of translocation across the plasma membrane.

Results and discussion A set of helical model peptides I–VI (Table 1) was designed and synthesized. All peptides have a high tendency to form helical conformations in TFE-water mixtures, but vary with respect to charge and amphipathicity (hydrophobic Table 1, Quantity of internalized peptide after exposing bovine aortic endothelial cells for 30 min at 37°C to 3.9 µM of peptide I–IX.

Sequence

Charge

I II III IV V

Flu-KLALKLALKALKAALKLA-amide Flu-klaklalkalkaalkla-amide Flu-KALKLKLALALLAKLKLA-amide Flu-LKTLATALTKLAKTLTTL-amide Flu-LLKTTALLKTTALLKTTA-amide

VI

Flu-LKTLTETLKELTKTLTEL-amide

VII

Flu-LLKTTELLKTTELLKTTE-amide

5+ 5+ 5+ 3+ 3+ 3+, 3– 3+, 3–

VIII

Flu-DPKGDPKGVTVTVTVTVTGKGDPKPD-amide

IX

Flu-DPKGDPKGVTVTvtVTVTGKGDPKPD-amide

Flu = 5(6)-Carboxy-fluorescein; n.d. = not detectable.

782 Y. Shimonishi (ed): Peptide Science– Present and Future, 782–783 © 1999 Kluwer Academic Publishers, Printed in Great Britain

4+, 4– 4+, 4–

Hydrophobic moment

Uptake [pmol/mg protein]

0.334 0.334 0.003 0.275 0.000

452 ± 27 476 ± 70 n.d. 1240 ± 284 n.d.

0.197

145 ± 56

0.000

n.d. 170 ± 32 < 10

783 moment). Peptides I, IV and VI being highly amphipathic and the analogs III, V and VII are the corresponding non-amphipathic counterparts. Peptide II represents the all-D-isomer of I. The combination of a (Val-Thr) 5 domain possessing a high β -structure forming propensity with charged C- and N -terminal, non-structured sequences resulted in a water-soluble β-sheet forming model peptide VIII (Table 1) [5]. The replacement of two amino acids in the center of the (VT) 5 motif by the corresponding D-amino acid residues (Peptide IX) eliminates β -sheet formation [5]. The incubation of bovine aortic endothelial cells with peptides I, IV and VI, respectively, resulted in an extensive cellular uptake as demonstrated by confocal laser scanning microscopy. Conversely, exposure of the cells to the nonamphipathic peptides produced no or only slight fluorescence in the cell interior. The determination of peptide concentrations inside the cell was made by HPLC after treatment of the cells with non-permeable, diazotized 2-nitroaniline, thus allowing discrimination of membrane-associated and internalized peptide fractions. Comparing the uptake of peptides I–VII by aortic endothelial cells, the most striking observations are that the non-amphipathic peptides III, V and VII do not enter the cells and that the all-D-peptide II internalizes as effectively as its L-isomer I (Table 1). Although helical amphipathicity favours the translocation process, the uptake is obviously not restricted to helical peptides. Thus peptide VIII, forming amphipathic β -sheet-like structures at water/lipid-interfaces [5], exhibits extensive internalization into endothelial cells, whereas the non-structured peptide IX internalizes only weakly. The uptake of the helical, amphipathic peptide I was found to be at least in part energy- and temperature independent, pointing to a non-endocytic mechanism. In contrast, strong energy and temperature dependence was found for the internalization of the β -sheet-forming peptide VIII and a reduced internalization of I and VIII was observed at pH 6, in the presence of monensin or hyperosmolar sucrose; such effects have often been cited as being characteristic of clathrin dependent endocytosis. However, the high quantity of the internalized peptides appear hardly reconcilable with an endocytic mechanism.

References 1 . Derossi, D., Res. Neurol. Neurosci., 8 (1995) 17. 2 . Lin, Y.-Z., Yao, S., Veach, R.A., Torgerson, T.R., and Hawinger, J., J. Biol. Chem., 270 (1995) 14255. 3 . Wagner, E., Plank, C., Zatloukal, K., Cotten, M., and Birnstiel, M.L., Proc. Natl. Acad. Sci. USA, 89 (1992) 7934. 4 . Oehlke, J., Krause, E., Wiesner, B., Beyermann, M., and Bienert, M., Protein Peptide Lett., 3 (1996) 393. 5 . Krause, E., Beyermann, M., Fabian, H., Dathe, M., Rothemund, S., and Bienert, M., Int. J. Peptide Protein Res., 48 (1996) 559.

269 Peptide analogues as vaccines and immunomodulators M.H.V. Van REGENMORTEL, N. BENKIRANE, H. DUMORTIER, G. GUICHARD, C. MÉZIÈRE, S. MULLER and J.P. BRIAND UPR 9021, IBMC – CNRS, 15 rue René Descartes, 67084 Strasbourg cedex, FRANCE

Introduction The antigenic properties of proteins are defined by two types of antigenic sites or epitopes. The B-cell epitopes which are recognized by the receptors of B lymphocytes and by antibodies correspond to surface patches of the protein which can often be mimicked by linear peptides of 5–15 residues. Such peptides usually correspond to a short stretch of the protein sequence and they are able, when used for immunization, to induce antibodies that cross-react with the parent protein. Epitopes are usually classified as either continuous or discontinuous depending on whether the amino acid residues in the epitope are contiguous in the polypeptide chain or not. One method for identifying continuous epitopes in a protein consists in analyzing a set of overlapping synthetic peptides encompassing the entire sequence of the protein for their capacity to bind to the antiprotein antibodies. Another approach is to test the reactivity of sets of random peptides synthesized without any reference to the sequence of the protein antigen. Using random combinatorial peptide libraries, it is relatively easy to obtain all possible hexapeptides or heptapeptides by chemical synthesis or phage display techniques. This approach often leads to the identification of antigenically active peptides that show no sequence similarity with any part of the protein antigen. Such epitopes which are called mimotopes are usually thought to mimic discontinuous epitopes of the antigen. In order to be qualified as a mimotope, the peptide should not only bind to the antiprotein antibody, but is should also be able to elicit antibodies that react with the original antigen it is supposed to mimic. It has been shown that recombinant phages displaying mimotopes of viral epitopes on their surface are able to induce antibodies that recognize the viral antigen and neutralize viral infectivity. This has led to the suggestion that such phages may be useful as potential vaccines [1]. Synthetic peptides representing immunodominant B-cell epitopes of certain viruses, bacteria and parasites have considerable potential as synthetic vaccines [2, 3]. In spite of some encouraging results, no commercial peptide vaccine has yet reached the market place, mainly because peptides are rapidly degraded in vivo by proteases and because they tend to mimic only imperfectly the conformation of the native epitope in the infectious agent. 784 Y. Shimonishi (ed): Peptide Science – Present and Future, 784–787 © 1999 Kluwer Academic Publishers, Printed in Great Britain

785 The second type of protein epitope known as T-cell epitope corresponds to linear peptide fragments derived from the intracellular processing of the antigen which interact with MHC (Major Histocompatibility Complex) molecules and T lymphocyte receptors. Class I MHC molecules are present on the surface of all nucleated cells and present peptides from proteins of intracellular origin to CD8 + cytotoxic T cells (CTL) while class II MHC molecules are present on lymphoid cells and present extracellular antigen peptides to CD4 + helper T cells. T cell-mediated responses are triggered when T cell receptors (TCR) bind to peptide/MHC complexes and a series of intracellular signaling events are initiated which lead to the release of cytokines and to the lysis of target cells by CTLs. The ternary association between a TCR and its MHC/peptide ligand is amenable to various immune intervention strategies. The binding site of an MHC molecule can be blocked by a high affinity peptide and this may inhibit the pathogenic T cell activation observed in various autoimmune and allergic disorders [4]. Another approach is to use so-called altered peptide ligands or peptide analogues that are able to act as TCR partial agonists or antagonists for antigen-specific immunomodulation and immunotherapy [5].

Results and discussion We have shown recently that B cell epitopes can be mimicked very effectively by means of all-D-retro peptides, also known as retro-inverso (RI) peptides [6]. RI peptide analogues are obtained by assembling amino acid residues in the reverse order from that in the original sequence and replacing L- by D -amino acid residues [7]. The side chains in RI peptides are oriented as in the parent L -structure and antibodies raised to RI peptides cross-react strongly with the native protein antigen. RI peptide analogues are much more stable than natural peptides to proteolysis and have considerable potential as synthetic vaccines. RI peptides corresponding to an immunodominant epitope of foot-and-mouth disease virus (FMDV; residues 141–159 of the VP1 protein) have been found to elicit in experimental animals high levels of antibodies that neutralize the infectivity of the virus [8]. In guinea pigs, a single inoculation of only 7 µg of the RI analogue protected the animals against a challenge infection with the cognate virus [9]. Since RI peptides always present a reversal of their end-groups compared to L -peptides, it is often necessary to modify the end-groups, in order to retain biological activity. In the FMDV RI peptides, C-2 substituted malonic acid residues were introduced to mimic the C-terminus of the parent L -structure and in addition the N -terminus was blocked by amidation. In view of the crucial role played by the peptide backbone in MHC-peptide interactions, it would seem a priory unlikely that all-D-retro peptides presenting a reversed backbone with respect to the side chains would bind as strongly to MHC molecules as do their L -peptide counterparts. This is indeed what has

786 been observed in a number of studies with MHC class II molecules [10, 11]. However, in a recent study of two Th cell epitopes (residues 103–115 of protein VP1 of poliovirus and residues 435–446 from the third constant region of a mouse heavy chain IgG2a), it was found that RI analogues of these two epitopes showed a significant degree of binding to MHC class II molecules and were able to induce T cells in vivo to proliferate and secrete IL-2 in vitro in the presence of the parent peptide [12]. The observed lower affinity of the RI peptides for the MHC molecules (compared to L-peptides) was probably due to an altered hydrogen bonding pattern of peptide main chain atoms with atoms in the MHC groove. However, this appears to be compensated in vivo since a T cell response equivalent to that obtained with the cognate L-peptides was observed following immunization of BALB/c mice with the RI analogues. Apparently enough MHC-peptide complexes are formed to allow T cell activation to take place. These results illustrate the potential of RI peptides as immunogens for class II restricted T cells. In MHC class I molecules, the N- and C -termini of the peptide are positioned in two pockets at each end of the cleft through extensive hydrogen bonding to highly conserved MHC residues [13]. RI peptides are thus even less likely to bind to MHC I molecules than to MHC II molecules. Instead of using all-D-retro peptides, a series of partially modified RI analogues of a T cell epitope of influenza virus (residues 58–66 of the matrix protein) were tested for their ability to associate with MHC class I heavy chains and promote HLA-A2 assembly [14]. Considerable differences were found in the capacity of the various analogues to bind to HLA-A2. Significant HLA-A2 assembly was observed in the presence of analogues modified at the P1–P2, P4–P5, P6–P7 and P7–P8 amide bonds whereas incorporation of the RI modification between P2–P3, P3–P4, P5–P6 and P8–P9 was found to be detrimental for binding. These results confirm the major contribution in HLA-A2 binding of peptide main chain atoms at residues P2, P3, P8 and P9, as revealed by the crystal structure of HLA-A2 complexed to the L -peptide M58–66 [15]. Surprisingly, reversal of the direction of the amide bond between P1–P2 gave the most potent analogue, with an efficiency for HLA-A2 stabilization similar to that of the parent L -peptide. Since the crystal structure shows that the carbonyl oxygen and the amide nitrogen at the P1–P2 peptide bond are hydrogen bonded to residues 159 and 63 of HLA-A2, this suggests that the reversal of the amide bond at P1–P2 may lead to an alternative hydrogen bonding pattern that is also capable of stabilizing the complex. Peptide analogues with reduced peptide bonds have also been tested for their ability to bind to MHC class I molecules. Eight peptide analogues of a T cell epitope of Plasmodium berghii (residues 252–260 of circumsporozoite protein) were obtained by replacing one peptide bond at a time by a reduced peptide bond ψ (CH 2 –NH). Five of the bonds could be reduced with only a modest effect on the binding to recombinant MHC I molecules while the introduction of the reduced bond between P3–P4, P7–P8 and P8–P9 com-

787 pletely abolished binding [16]. Since peptides with reduced peptide bonds are much more resistant to proteases than L -peptides [17], such analogues may be useful as immunomodulating and immunotherapeutic agents in the treatment of cancer and allergy and of autoimmune and viral diseases.

References 1. 2 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Delmastro, P., Meola, A., Monaci, P., Cortese, R. and Galfrè, G., Vaccine, 15:11 (1997) 1276. Arnon, R. and Van Regenmortel, M.H.V., FASEB J., 6 (1992) 92. Nicholson B.H. (Ed.) Synthetic Vaccines, Blackwell Scientific Publications (1994). Guichard, G., Exp. Opin. Ther. Patents, 7 (1997) 29. Jameson, S.C. and Bevan, M.J., Immunity, 2 (1995) 1. Guichard, G., Benkirane, N., Zeder-Lutz, G., Van Regenmortel, M.H.V., Briand, J.P. and Muller, S., Proc. Natl. Acad. Sci., USA, 91 (1994) 9765. Chorev, M. and Goodman, M., Acc. Chem. Res., 26 (1993) 266. Muller, S., Briand, J.P., Benkirane, N., Guichard, G., Van Regenmortel, M.H.V., Newman, J.F.E. and Brown F., Vaccines 97, Cold Spring Harbor Laboratory Press (1997) 17. Briand, J.P., Benkirane, N., Guichard, G., Newman, J.F.E., Van Regenmortel, M.H.V., Brown, F. and Muller, S., Proc. Natl. Acad. Sci., USA, 94 (1997) 12545. Hill, C.M., Liu, A., Marshall, K.W., Mayer, J., Jorgensen, B, Yuan, B., Cubbon, R.M., Nichols, E.A., Wicker, L.S. and Rothbard, J.B., J. Immunol., 152 (1994) 2890. Hervé, M., Maillère,B., Mourier,G., Texier,C., Leroy,S. and Ménez,A., Mol. Immunol., 34 (1997) 157. Mézière, C., Viguier, M., Dumortier, H., Lo-Man, R., Leclerc, C., Guillet, J.G., Briand, J.P. and Muller, S., J. Immunol., 159 (1997) 3230. Madden, D.R., Ann. Rev. Immunol., 13 (1995) 587. Guichard, G., Connan, F., Graff, R., Ostankovitch, M., Muller, S., Guillet, J.G., Choppin, J. and Briand, J.P., J. Med. Chem., 39 (1996) 2030. Madden, D.R., Garboczi, D.N. and Wiley, D.C., Cell, 75 (1993) 693. Guichard, G., Calbo, S., Muller, S., Kourilsky, P., Briand, J.P. and Abastado, J.P., J. Biol. Chem., 270 (1995) 26057. Benkirane, N., Guichard, G., Briand, J.P. and Muller S., J. Biol. Chem., 271 (1996) 33218.

270 Combinatorial peptide libraries and molecular recognition in T-cell mediated immune response B. FLECKENSTEIN¹, K.-H. WIESMÜLLER², M. KALBUS³, R. MARTIN 4 and G. JUNG¹ ¹ Institute of Organic Chemistry, University of Tübingen, 72076 Tübingen, Germany ² EVOTEC BioSystems GmbH, Grandweg 64, 22529 Hamburg, Germany ³ Neurological Clinic, University of Tübingen, 72076 Tübingen, Germany 4 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-1400, USA

Introduction Combinatorial peptide libraries are well established tools in basic research and represent a valuable source in the screening for new lead structures [1]. Major histocompatibility complex (MHC) class II molecules are cell-surface glycoproteins that take up peptide antigens within the cell and present them to CD4 + T-cells at the cell surface. T-helper cells are part of the cellular immune system and responsible for identification of presented foreign antigens. During antigen recognition the T-cell receptor (TCR), the MHC class II molecule and the presented peptide form a ternary complex inducing T-cell activation upon a signal transduction cascade into the T-cell. The exact knowledge of the MHC class I and class II restricted sequence motifs is a prerequisite for the construction of fully synthetic vaccines and for inhibitors of T-cell reactivity in autoimmune response. Independent from the motifs of natural self peptide pools [2,3] and individual T-cell epitopes, the use of complex synthetic peptide libraries [4] allows the complete dissection of peptide-MHC binding, and anchor position as well as residues involved in T-cell receptor contact can be deduced [5].

Results and discussion In a recent study, peptide binding to the MHC class II molecule HLADRB1*1501 (DR2b), associated with the autoimmune disease Multiple Sclerosis, was characterized in detail by applying 220 undecapeptide amide sublibraries (O/X 10 ) in the positional scan format [6] in a competition assay. Sublibraries, each composed of 10 degenerated (X) positions and one defined (O) position were prepared by solid phase peptide synthesis using Fmoc/ tBu-chemistry, Rink amide-MBHA-polystyrene resin and a robot for multiple synthesis as described [7]. The amino acid composition in the randomized 788 Y. Shimonishi (ed): Peptide Science – Present and Future, 788–791 © 1999 Kluwer Academic Publishers, Printed in Great Britain

789 and defined sequence positions was determined by pool sequencing [3], electrospray mass spectrometry [4] and amino acid analysis. Binding of the fluorescence-labeled DR2b-ligand AMCA-HLA-A3(152–166) to purified DR2b-molecules was competed with the 220 O/X 1 0 sublibraries. The competition of each sublibrary was divided by the competition of the completely randomized X 11 -library resulting in 220 relative competition (rel C) values, which are summarized in a DR2b-allelespecific Activity Pattern of the undecapeptide amide library (Table 1). This method allows to analyze and to quantify the impact of all 20 L -amino acids in each sequence position of undecapeptides on the binding to HLA-DR2b. According to the rel C-values of the corresponding sublibraries, defined amino acids are classified as unfavourable (rel C < 0.7), indifferent (0.7 < rel C < 1.4) and favourable (rel C > 1.4) for DR2b-binding. In addition, the Activity Pattern is the basis for the new algorithm Actipat searching for T-cell epitopes within protein sequences [8]. Thus, new DR2b-ligands, e.g. from the measles virus nucleoprotein could be identified and binding to DR2b was confirmed experimentally. In a second study, the antigen recognition of autoreactive human CD4+ -Tcell clones (TCC) specific for myelin basic protein peptide (MBP 86–96) was dissected by the response to the 220 O/X 10 undecapeptide amide sublibraries in a ³H-thymidine incorporation assay [9]. Despite the enormous complexities of the fully randomized X 11 -peptide library (2,048 × 10 14 individuals) and of 13 individuals), some TCC showed a significant each sublibrary (1,024 × 10 proliferation resulting in stimulation indices (SI) up to 50 at peptide concentrations of 100 µg/ml. This clearly indicates that antigen recognition of these TCC has to be considered as highly degenerated, since the individual peptide Table 1. HLA-DRB1*1501-specific activity pattern of the undecapeptide amide library. In each sequence position amino acids are classified into unfavourable, indifferent and favourable ones, according to the rel C-values of the corresponding sublibraries. For clarity, only the number of indifferent amino acids is given. sequence positions 2 3 4 1

5

6

7

8

9

10

11

unfavourable amino acids: rel C < 0.7 D, E, N, Q, T, S, K 20

D, H, P D, S, D, E E, F, D, D, E D, W E, l G, P, N, E, G, E R, W, G, K, S P number of different amino acids: 0.7 < rel C < 1.4

9

10

18

B, M, V, I

V, L, I

7

15

18

14

15

16

favourable amino acids: rel C > 1.4 I

F, I, L, W, M, Y

F, Y

Y

N, S

N, S M, B

W

19

790

Figure 1. Response of TCC 5G7 to artificial undecapeptide amides, designed on the basis of the Recognition Pattern of the undecapeptide amide library. Both peptides act as superagonists, inducing stronger response of TCC 5G7 than MBP(86–96).

Figure 2. Response of TCC 5G7 to self and foreign undecapeptide amides, identified by screening protein databases according the Recognition Pattern of the undecapeptide amide library. Even ligands that share only two amino acids with the MBP(86–96) sequence act as superagonists (e.g. the human Cytomegalo-Virus-peptide).

concentration in the assay is in the attomolar range. According to the Activity Pattern of the O/X 10 -library, which describes binding to the DR2b-molecule, antigen recognition of the TCC was summarized in a Recognition Pattern containing relative stimulation indices (rel SI = SI of a given sublibrary divided by the SI of the X 11 -library). Based on these results, artificial peptide ligands for the TCC 5G7 were designed, just by combining the most effective amino acids with respect to T-cell proliferation. These ligands were shown to be more

791 potent than MBP(86–96) itself, surprisingly by a factor of 10000 and more (Figure 1). Furthernore, screening of protein databases with the library information revealed not only MBP(86–96) as a ligand for TCC 5G7, but also several peptides derived from self and foreign antigens (Figure 2). These ligands induced proliferation of TCC 5G7, and again, some of them even at lower concentrations than MBP(86–96). Thus, based on the Recognition Pattern of TCC 5G7 obtained from screening a combinatorial peptide library, about eight superagonists for TCC 5G7 could be identified, but even more than 16 peptide ligands inducing T-cell proliferation were discovered in this way. These results underline the value of combinatorial peptide libraries for characterizing peptide binding to MHC class II molecules, resulting in the prediction and design of new high-affinity ligands as potential T-helper epitopes or antagonists for autoreactive T-cells. Studying antigen recognition of T-cell, new highly potent superagonistic peptide antigens can be identified, which might be useful for the development of new therapeutical structures with respect to tolerance induction.

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Jung, G., Combinatorial Peptide and Nonpeptide Libraries. Verlag Chemie, Weinheim, 1996. Falk, K., Rötzschke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G., Nature 351 (1991) 290. Stevanovic, S. and Jung, G., Anal. Biochem. 212 (1993) 212. Metzger, J.W., Kempter, C., Wiesmüller, K.-H., and Jung G., Anal. Biochem. 219 (1994) 261. Fleckenstein, B., Kalbacher, H., Muller, C.P., Stoll, D., Halder, T., Jung, G., and Wiesmüller, K.-H., Eur. J. Biochem. 240 (1996) 71. Pinilla, C., Appel, J.R., Blanc, P., and Houghten, R.A., Biotechniques 13 (1992) 901. Wiesmüller, K.-H., Feiertag, S., Fleckenstein, B., Kienle, S., Stoll, D., Herrmann, M., and Jung, G., In Jung, G. (Ed) Combinatorial Peptide and Nonpeptide Libraries, Verlag Chemie, Weinheim, 1996, p. 203. Fleckenstein, B., Jung, G., and Wiesmüller, K.-H., In Brown, F., Burton, D., Doherty, P.C., Mekalanos, J., and Norrby, E. (Eds) Vaccines 97, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1997, p. 65. Hemmer, B., Fleckenstein, B.T., Vergelli, M., Jung, G., McFarland, H,F., Martin, R., and Wiesmüller, K.-H., J. Exp. Med. 185 (1997) 1651.

271 On the puzzle of the acute phase reactants C-reactive protein and serum amyloid A E.J. YAVIN, L. PRECIADO-PATT, H. BERGER and M. FRIDKIN Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, ISRAEL

Introduction Human C-reactive protein (CRP) and serum amyloid A (SAA) are major acute phase proteins whose blood concentration may rise up to 1000-fold over the normal level within a relatively short period following tissue injury or the onset of inflammation [1]. Although the physiological functions of CRP and SAA as intact proteins are still largely obscure, they specifically bind to human neutrophils and are subsequently degraded by neutrophilic enzymes to yield various low molecular weight peptides with most significant anti-inflammatory related activities [2–4]. This fact may be associated with the possible protective role of both CRP and SAA during the acute phase response.

Results and discussion Recently, we demonstrated that CRP derived peptides, containing the sequence VTVAPVHI (CRP 89–96 ), are potent inhibitors of human leukocyte elastase (hLE) [5,6] which is associated with the severe and permanent tissue damage observed in several devastating inflammatory diseases [7]. In contrast, CRP as a whole pentameric protein possesses no inhibitory effect on hLE [6]. Several biologically active peptides, isolated from in-vitro proteolysis of CRP by neutrophilic enzymes are contained within CRP’s single disulfide loop region i.e., residues 36–97 [2, 4, 5]. To further explore this potential regulatory domain, we synthesized and evaluated ten overlapping peptides, 15 amino acid residues in length, spanning CRP 36–97, and obtained a powerful hLE inhibitor, EILIFWSKDIGYSFT (CRP 62–76 , peptide 1 Table 1). In contrast to peptide 1, all other 15-mers yielded no significant inhibitory activity with the exception of CRP 82–96 , which yields minor inhibitory activity (K i = 55 µM) in agreement with the values described for peptides containing CRP 89–96 [6]. Furthermore, the inhibitory activity of peptide 1 is potent by more than two orders of magnitude in comparison to a peptide of similar chain length derived from the active site of hLE’s plasma inhibitor: α 1 -protease inhibitor ( α 1 PI 344–360 , K i = 40 µM) [5]. Initially, peptide 1 appeared incompatible with the known active site preferences of hLE [5]. Recently, however, using electrostatic792 Y. Shimonishi (ed): Peptide Science – Present and Future, 792–796 © 1999 Kluwer Academic Publishers, Printed in Great Britain

793 Table 1. hLE inhibition constants of peptides 1–15, determined by inhibiting the hydrolysis of methoxy-succinyl-Ala-Pro-Val-nitroanilide. n.s.i.: no significant inhibition. The Glu6 2 and Lys 6 9 amino acid residues are marked in bold . Substituted amino acids are underlined. *The spacer in peptide 11 is NH 2 –(CH 2 ) 1 1 –COOH.

potential calculations, a prominent positive pocket was illuminated on the surface of hLE which is generated by two exposed positive arginine residues. The sequence of CRP 88–96 , i.e. EVTVAPVHI, contains a glutamic acid residue which appears to bind into hLE’s positive pocket, and subsequently increase inhibitory activity. The side chain of Val 94 resides within hLE’s major hydrophobic pocket [8]. In the adaptation of our theoretical binding configuration to peptide 1 (Figure 1), the Glu 62 side chain may reside within hLE’s distant positive pocket while Lys 69 may bind to hLE’s major hydrophobic pocket, which is somewhat negatively charged [8]. Removing the Glu 62 residue from peptide 1 or substituting it with alanine decreases substantially the inhibitory activity (peptides 2, 3 ) , emphasizing the importance of Glu 62 in hLE binding. With the additional deletion of the Ile-Leu residues (peptide 4), inhibitory activity is completely lost. Peptide 1 is fairly tolerant towards C-terminus truncation (peptides 5–7 ) , again contrasting the importance of Glu 62 . With the additional deletion of Asp 70 (peptide 8), inhibitory activity is completely lost. In peptides 9–11, the

Figure 1 Schematic representation of CRP 6 2 – 7 1 interacting with hLE’s subsites. The negative side chain of CRP’s Glu 6 2 is shown to reside within hLE’s positive pocket, and the positive side chain of Lys 6 9 is shown to reside within hLE’s major hydrophobic pocket.

794 hydrophobic amino acid stretch (ILIFW or LIFW) was altered which resulted in a marked decrease, yet substantially retaining, inhibitory activity. These results indicate the important contribution of the hydrophobic subsite interactions in hLE binding. Peptides 12–14 indicate the individual importance of the Trp 67 , Ser 68 , and Lys 69 residues (alanine substitution of Lys 69 yields a highly insoluble peptide), while peptide 15 indicates the importance of residue alignment (see Figure 1). Incubation of peptide 1 with hLE did not yield any observable degradation products under similar degradation conditions in which the Val-His bond of CRP 89–96 (VTVAPVHI) is totally cleaved (observed by HPLC). Under similar degradation conditions with trypsin, rapid specific cleavage of the Lys 69 –Asp 70 bond was observed. The explanation for the remarkable stability of peptide 1 towards hLE is presently unknown. We hope that an X-ray structure which we are now attempting will help explain the peptide’s stability and illuminate our theoretical binding configuration. The potent inhibitory activity of peptide 1 suggests that CRP-derived peptides may play an important role in regulating the activity of matrix-degrading enzymes. The concealed nature of CRP-derived peptides within the intact pentamer may be beneficial in preserving their inhibitory activity during migration. Specifically, the hydrophobic nature and stability of peptide 1 may perhaps assist in its accumulation and resistant towards unwanted modifications. The co-accumulation of intact CRP and human neutrophils at inflammatory loci, the repetitive subunit of CRP, and its high evolutionary sequence conservation suggest that CRP may be degraded in-vivo to generate modulatory peptides as part of a complex protective homeostatic mechanism [5–8]. The adhesion of neutrophils to endothelial cells is an early and vital step in the inflammatory reaction, which may be affected by SAA and some of its related fragments. We have thus investigated the possibility that the neutrophilSAA encounter is receptor-mediated. SAA can specifically bind, either as a free protein or when associated with HDL 3 , to human neutrophils or to respective plasma membranes [3]. The dissociation constants are in the nM-range with about 10 5 binding sites/cell. Competition assays performed with SAA derived synthetic peptides revealed that SAA 77–104 (the C-terminus domain), inhibits completely the binding of 125 I-SAA to neutrophils similarly to recombinant (r)-SAA. This peptide may exist in sera of patients who experienced amyloid deposition (of i.e. AA 1–76 ) in various tissues. Along with its chemo-attractive effects on neutrophils and monocytes [9], SAA also induces CD 4+ or CD 8 + T cell adhesion and migration through human umbilical cord endothelial cell monolayers, without affecting the surface expression of representative cell adhesion molecules, thus acting in a similar manner to chemokines such as MIP-1β , RANTES, and IP-10. We have found that SAA, a non-chemokine protein, interacts specifically with vessel wall and extra-cellular matrix (ECM)-linked glycoprotein moieties, attaches temporarily to these matrix substrates, and provides pro-adhesive stimuli for resting CD4 +

795

Figure 2 Specific binding of 1 2 5 I-rSAA to ECM (open circles) or BSA (closed circles) determined by the differences between total and non-specific binding.

T cells. Although the binding affinity between 125 I-rSAA and immobilized ECM appears to be substantially tight, in the magnitude of 10 –9 M (Figure 2), the ECM seems to serve only as a temporary anchorage site for SAA [10]. Of the tested ECM-glycoproteins, SAA and its AA fragment appear to interact with laminin (LN) and vitronectin, but not with fibrinogen (FN) or collagen-II. The sites within SAA which both LN and ECM bind appear to reside within the amino acid sequence of the AA molecule [10]. Interestingly, the AA molecule also contains an RGD-like cell adhesive epitope, which may account for soluble AA interfering with the adhesion of activated T cells to FN, and probably also for AA inducing leukocyte migration through endothelial cell monolayers [9]. Herein, ECM or LN, to which micromolar concentrations of rSAA or AA were added and subsequently complexed, significantly enhanced (20–30% at 10–100 µM) the binding of resting CD4+ T cells. Although the mechanisms underlying the pro-adhesive properties of ECM-associated rSAA or AA is not clear, it is reasonable to assume that these immobilized mediators induce activation of β 1 -integrins. In the present study, rSAA, and its related peptides, in particular its AA fragment, but not the C-terminal portion of SAA, interacted with ECM, laminin and vitronectin, and induced the binding of resting human CD 4 + T cells to these moieties in an apparent β 1-integrin-mediated fashion. SAA and its proteolytically cleaved AA, are capable of modulating the accumulation of immune cells in ECM regions rich with such mediators. The associations /interaction of neutrophils with endothelial cells is an early and requisite event in the inflammatory reaction. It is plausible that SAA and some of its related fragments, generated from proteolysis of SAA e.g., by neutrophilic enzymes [11], may affect this process through their specific binding to neutrohpils.

References 1. Steel, D.M. and Whitehead, A.S. Immunol. Today 15 (1994) 81 2. Buchta, R., Pontet, M. and Fridkin, M. FEBS Lett. 211 (1987) 165.

796 3. Preciado-Patt, L., Pras, M. and Fridkin, M. Int. J.Pept. Prot. Res. 48 (1996) 503. 4. Shephard, E.G., Anderson, R., Rosen, O., Myer, M.S., Fridkin, M., Strachan, A.F. and De Beer, F.C. J. Immunol. 145 (1990) 1469. 5. Yavin, E.J., Yan, L., Desiderio, D.M. and Fridkin, M. Int. J. Peptide Protein Res. 48 (1996) 465. 6. Yavin, E.J., Yan, L., Desiderio, D.M., Pontet, M. and Fridkin, M. Lett. Peptide Science 4 (1997) 157. 7. Groutas, W.C. Med. Res. Rev. 7 (1987) 227. 8. Yavin, E.J., Eisenstein, M. and Fridkin, M. Biomed. Pep. Prot. Nucleic Acids 2 (1997) 71. 9. Badolato, R., Wang, J.M., Murphy, W.J., Lloyd, A.R., Michiel, D.F., Bausserman, L.L., Kelvin, D.S. and Oppenheim, J.J. J. Exp. Med., 180 (1994) 203. 10. Preciado-Patt, L., Hershkoviz, R., Fridkin, M. and Lider, O. J. Immunol. 156 (1996) 1189. 11. Preciado-Patt, L., Levartowsky, D., Prass, M., Hershkoviz, R., Lider, O. and Fridkin, M. Eur. J. Biochem. 223 (1994) 35.

272 Design of peptidic and non-peptidic CD4 inhibitors as novel immunotherapeutic agents [1–5] S. LI, T. SATOH, T.M. FRIEDMAN, J. GAO, A.E. EDLING, R. TOWNSEND, U. KOCH, S. CHOKSI, X. HAN, S. SHAN, J.M. ARAMINI, M.W. GERMAN, R. KORNGOLD and Z. HUANG* Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA

Introduction The interaction between CD4 and major histocompatibility complex (MHC) class II proteins is critical for the activation of CD4 + T cells which are involved in transplantation reactions and a number of autoimmune diseases. We identified a CD4 surface pocket as a functional epitope implicated in CD4-MHC class II interaction and T cell activation. A small synthetic cyclic heptapeptide was designed and shown by high resolution NMR spectroscopy to closely mimic a portion of proposed CD4 surface pocket. In addition to the peptide design, a computer-based strategy was used to screen ~ 150,000 non-peptidic organic compounds in a molecular database and identify a group of compounds as ligands of this CD4 surface pocket. Both the cyclic heptapeptide and organic compounds were shown to specifically block CD4-MHC class II interaction and exhibit significant inhibition of immune responses in animal models of autoimmune disease and allograft transplant rejection. A multicenter phase I clinical trial will be initiated for the CD4 cyclic heptapeptide in bone marrow transplant patients. These results demonstrate the therapeutic potential of these peptidic and non-peptidic CD4 inhibitors as novel immunotherapeutic agents. Furthermore, they illustrate a small molecule approach that may have general implications for studying other immunoglobulin superfamily structures and interactions that are involved in diverse biological functions.

Results and discussion To search for potential CD4 functional epitopes that could be targeted for the design of new inhibitors, a computer analysis was conducted for the CD4 D1 domain in conjunction with synthetic peptide mapping. This led to the identification of a surface pocket potentially involved in the CD4-MHC class II interaction. This CD4 surface pocket is formed by the FG loop (also known as the third complementarity determining region or CDR3) and the CC' loop (Figure 1). While the CDR3 has long been proposed to be involved in MHC 797 Y. Shimonishi (ed): Peptide Science – Present and Future, 797–800 © 1999 Kluwer Academic Publishers, Printed in Great Britain

798

Figure 1. The peptidic and non-peptidic organic inhibitors of CD4 (left). The binding of the organic inhibitor TJU103 to the CD4 D1 surface pocket (right).

class II interaction and recent studies with peptide mimics have confirmed their involvement, our analysis suggested that the highly protruded CC' loop may also serve as a surface epitope critical for CD4 function. Based on this finding, a cyclic peptide, cyclo (CNSNQIC), was designed as a CD4 D1 domain CC' loop mimic, incorporating a disulfide bridge to enhance the structural stability of the β -turn around NSNQ of the native CC' region. The high resolution NMR spectroscopic experiments confirmed that this peptide adopts conformations highly resembling a type I β -turn of the native CC' loop, as observed in the crystal structure of CD4 D1D2 domains. This synthetic peptide mimic of the CD4 CC' loop was shown to effectively inhibit CD4-MHC class II interaction, block CD4-dependent T cell response in vitro, and possess significant immunosuppressive activity in vivo in murine models of experimental allergic encephalomyelitis (EAE) for MS and of skin allograft transplantation These findings suggested the therapeutic potential of this CD4 peptide inhibitor and demonstrated a general approach of bioactive peptide design by the functional mimicry of protein surface epitopes to generate potential novel therapeutic agents. In addition to the synthetic petpides as described above, we are also developing small non-peptidic organic CD4 inhibitors as a different class of potential immunotherapeutics. Compared to peptides, organic inhibitors may possess several potential advantages: their non-peptidic structures are less susceptible to enzymatic degradation and thus are more stable; they could be taken through the oral route which is most desirable in terms of patient compliance; and they may be more amenable to chemical synthesis and modification. We used a computer-based screening approach to search large databases of organic com-

799 pounds for potential inhibitors of CD4 function. In contrast to the traditional random screening, which requires laborious and expensive testing of large libraries of organic compounds, this computer-based strategy provides an efficient automatic method for the initial screening of these large databases on the computer to identify a short list of possible candidate compounds for actual biological testing, thus saving an enormous amount of both time and resources. We applied this computational screening approach to search ~ 150,000 nonpeptidic organic compounds in a chemical database for potential ligands of the CD4 D1 surface pocket. This led to the discovery of several novel CD4 inhibitors. Among them, TJU103 was found to be the most promising lead compound after a series of biological evaluations (Figure 1). TJU103 was shown to be non-toxic to lymphocytes in vivo and appeared to be specific for inhibition of CD4 + T cell-mediated responses. The compound displayed significant immunosuppressive activity mediated in murine models of EAE, skin allograft rejection, and graft-versus-host disease (GVHD). These results demonstrated that the computer screening of chemical databases may represent an effective approach to study structure and function of CD4 surface functional sites and discover small molecular inhibitors with therapeutic potential. These studies may provide a paradigm for the inhibitor design of many immunoglobulin superfamily (IgSF) protein structures and interactions. As members of IgSF may have a conserved backbone folding pattern, it is likely that this generic structure provides some common scaffolds for efficient proteinprotein interactions. It is interesting to note that similar structural themes may be involved in a diversity of molecular interactions and biological functions of IgSF proteins. Therefore, it is possible that the computer screening approach developed from this study of the CD4 protein may also be applicable to the design of small non-peptidic inhibitors of other IgSF molecular interactions.

Conclusion We have proposed the CC' loop as an important functional epitope on the CD4 surface for intermolecular binding, and found that small peptide mimics of this epitope are sufficient to interrupt a larger protein-protein interface. In particular, a synthetic cyclic heptapeptide (CNSNQIC) has been shown to closely mimic the CC' surface epitope, effectively block CD4-MHC class II-dependent cell rosetting, possess significant immunosuppressive activity in vitro and in vivo, and strongly resists proteolytic degradation. These findings have demonstrated a general approach of bioactive peptide design by the functional mimicry of protein surface epitopes to generate potential novel therapeutic agents. In addition, we have also tested the hypothesis that non-peptidic organic inhibitors targeting a small functionally important surface epitope could be sufficient for the effective blockade of a protein-protein interaction with a large

800 interface. Employing theoretical analysis of protein structure and subsequent synthetic peptide mapping approaches, we have identified a surface pocket on the CD4 D1 domain as a critical functional epitope, involved either directly or indirectly in stable CD4-MHC class II interaction and T cell activation. A computer-based database screening strategy has been employed to identify a diverse group of novel non-peptidic organic ligands of this CD4 surface pocket that specifically block CD4-MHC class II cell adhesion and exhibit significant immunosuppressive activity in animal models of autoimmune disease and transplant rejection. This computer screening approach should have general implications for studying many IgSF molecules that utilize similar surface structural features for diverse intermolecular binding and biological functions. Finally, the results from this study have demonstrated that the structure-based, computer-assisted design of small non-peptidic inhibitors of large proteinprotein interfaces may indeed be an achievable goal.

Acknowledgements These studies are supported by grants from the National Institutes of Health, the American Cancer Society, the National Multiple Sclerosis Society, and the Sidney Kimmel Foundation for Cancer Research. We would like to thank members of our laboratories and our collaborators for their contribution and helpful discussion. Z.H. is the recipient of the Kimmel Scholar Award in cancer research.

References 1. Satoh, T., Li, S., Friedman, T.M., Wiaderkiewicz, R., Korngold, R., and Huang, Z., Biochem. Biophy. Res. Commun. 224 (1996) 438–443. 2. Satoh, T., Aramini, J.M., Li, S., Friedman, T.M., Gao, J., Edling, A., Townsend, R, Germann, M.W., Korngold, R., and Huang, Z., J. Biol. Chem. 272 (1997) 12175–12180. 3. Li, S., Gao, J., Satoh, T., Friedman, T., Edling, A., Koch, U., Choksi, S., Han, X., Korngold, R., and Huang, Z., Proc. Natl. Acad. Sci. USA 94 (1997) 73–78. 4. Huang, Z., Li, S., and Korngold, R., Med. Chem. Res. 7 (1997) 137–150. 5. Huang, Z., Li, S., and Korngold, R., Peptide Science in press (1997).

273 Molecular design of T cell-costimulatory peptides for vaccine development T. FUKUMOTO, N. TORIGOE, S. KAWABATA, Y. ITO and K. SUGIMURA Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

Introduction Two important signals are required for the T cell activation. One is through T cell receptors. The other signal is via the costimulatory molecule CD28 or CTLA4 which binds to CD80 or CD86 expressed on antigen-presenting cells. The engagement of CD28/CD80 or CD86 is essential for the development and maintenance of T cell responses. Recent studies have suggested that the CTLA4 delivers an inhibitory signal for downregulating lymphocyte activation. Inhibition of this pathway by antagonists may upregulate the immune responses [1–3]. Thus, the peptide antagonists are useful in vaccine development. However, it has been difficult to design such peptide antagonists from the primary amino acid sequence because the binding groove is often formed by 3D folding structure. We report here a novel approach to design the peptide antagonists with 3D-similarity by employing a phage display library and a monoclonal antibody (mAb) that recognize the conformationl binding site of ligand as a mold to copy the binding site. The phage library clones selected by the mAb may display moieties that mimic the 3D structure of ligand binding site. It is predicted that this pseudo-ligand binds to natural receptor molecules and inhibits the receptor/ligand binding. We demonstrate here a successful isolation of a peptide motif (F2) recognized by anti-CTLA4 mAb and exhibiting strong immunopotenciating activity.

Result and discussion Panning was performed using the anti-CTLA4 mAb (UC10–4F10–11, Pharmingen) as previously described [4, 5] and five motifs (F1, 2, 4, 5, 6) were identified. The F2 phage clone showed specific binding activity to recombinant CD80 but not CD86 [6]. The motif is inserted in the gene-3 proteins (g3p) which displays only 3–5 copies in contrast to 2700 copies of the gene-8 proteins (g8p) per fd phage [7, 8]. To confirm the binding activity of the F2 motif to membranous CD80 or CD86 molecules expressed on CHO cells, we purified the gene 3 protein of F2 (F2-g3p) by size-exclusion chromatography on a 801 Y. Shimonishi (ed): Peptide Science – Present and Future, 801–802 © 1999 Kluwer Academic Publishers, Printed in Great Britain

802

Figure 1. Augmenting effect of F2-g3p. Wild type fd-primed spleen cells (1.5 × 10 5 /well) were or absence of 0.15, 0.3, stimulated with 5 µg/ml of wild type fd phage in the presence 1.0, 3.0 or 10 µg/ml of g3p.

HiLoad superdex 200 (26/60) column (Pharmacia). The F2-g3p showed the binding activity to CD80 + CHO cells but not CD86 + CHO cells. The F2-g3p also showed the inhibitory activity on the interaction of CTLA4 with CD80 but not CD86 (Ref. Fukumoto, T. et al. submitted). To examine the effects of the F2 motif on the immune response, T cell proliferation activities were estimated. Balb/c mice were injected intraperitoneally with 10 µg of wild-type fd phage in phosphate buffered saline (PBS). Four weeks later, their spleen cells were stimulated with 5µg/ml of wild-type fd phage in the presence or absence of various concentration of F2-g3p. Three days later, T cell proliferation was monitored by pulsing cells with 0.5 µCi ³Hthymidine (Amersham, St. Louis, MO) for the final 18 hr. F2-g3p strongly augmented the T cell response induced by the stimulation of wild-type fd phage while the g3p derived from unrelated phage clone (K7) did not enhanced the T cell proliferation (Figure 1). These results suggested that the F2-g3p as a pseudo CTLA4 molecule might selectively block the negative signal through CTLA4/CD80 interaction, which resulted in the augmentation of T cell proliferation. These results taken together, it was demonstrated that anti-CTLA4 mAb could become a mold to copy the conformational image of the CTLA4-binding structure. This study implies that the F2 motif-positive virus particles or tumor cells may be useful in AIDS and cancer vaccine development.

References Morton, P.A. et al., J. Immunol. 156 (1996) 1047. Walunas, T.L., Bakker, C.Y., and Bluestone, J.A., J.Exp. Med. 183 (1996) 2541. Krummel, M.F. and Allison, J.P., J. Exp. Med. 182 (1995) 459. Parmley,S.F. and Smith, G.P., Gene 33 (1988) 305. Nishi, T. et al., FEBS letters 399 (1996) 237. Fukumoto, T., Torigoe, N., Kawabata, S., Kaneko, Y., Murakami, M., Uede, T., Nishi, T., Yamada, G., and Sugimura, K., Peptide Chemistry (C. Kitada. Ed) 1996 p 313. 7. Wuttke, G.G. et al., Biochimica et Biophysics Acta, 985, 239–247 (1989). 8. Crissman, J.W., Smith, G.P., Virology, 132, 445–455 (1984). 1. 2. 3. 4. 5. 6.

274 The identification of dominant epitopes of h-FKBP-12 recognized by monoclonal antibodies, 3F4 and 2C1, against h-FK-506 binding protein 12(h-FKBP-12) M. IWAI¹, N. SHINKURA², Y. YAMAOKA², K. NAKAMURA³, K. TAMURA³ and M. KOBAYASHI 4 ¹ Marine Technical College, Faculty of Liberal Arts and Science, Ashiya, 659, Hyogo, Japan ² Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, Kyoto, 606-01, Japan ³ Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Yodogawa-ku, Osaka, 532, Japan 4 Fujisawa Research Institute of America, Inc., Northwestern University, Evanston Research Park, 1801 Maple Avenue, Evanston, IL, USA

Introduction The natural product FK-506 and Cyclosporin A (CsA), two structurally different immunosuppressants bind to two different target proteins, immunophilins, FK-506 binding proteins (FKBPs) and cyclophilin, respectively. Binding to their cognate immunophilin in vivo induces a gain of function and results in the formation of the true drug entity. In the case of FKBP/FK-506 and cyclophilin/CsA complexes, this gain is suggested to correspond to modulation of the phosphatase activity of calcineurin [1, 2]. In order to investigate the cellular role of the immunophilins by immunohisto-chemical method, we have been studying the identification of dominant epitopes of FKBP-12 (107 amino acids, Figure 1) recognized by McAbs, 3F4 and 2C1. Results and discussion In this study, six fragment peptides (P-1 to P-6, Figure 1) designed from the sequence of human FKBP-12 were synthesized manually by Boc/Bzl based

Figure 1. Primary structure and synthesized peptide fragments(P-1 to P-6) of h-FKBP-12(left) and the binding of the fragments to the mouse anti-r-h-FKBP-12 McAbs, 3F4 and 2C1 (right).

803 Y. Shimonishi (ed): Peptide Science – Present and Future, 803–804 © 1999 Kluwer Academic Publishers, Printed in Great Britain

804 solid phase peptide synthesis using Merrifield resin. The acylation steps were carried out in DCM-DMF using 3 equiv. Boc-amino acid anhydride and in DMF using 3 equiv. Boc-amino acid HOBt ester in the case of the Boc-Asn, Boc-Gln and Boc-Arg(Tos). The coupling reactions were monitored by Kaisertest and repeated to completion when necessary. The peptides were removed from the resin by HF containing 10% anisole as scavenger. Deformylation was carried out using 0.1 M NaOH in the case of P-4 which contains Trp residue. The deprotected peptides were purified on a C18 reversed phase HPLC and characterized by analytical HPLC, amino acid analysis, FAB mass spectrometry and amino acid sequence analysis. Bindings of the peptides prepared to the mouse anti-r-h-FKBP-12 McAbs(3F4 and 2C1) were examined by the specific ELISA. P-4 (Lys 47 ~ Met 66 ) showed the strong binding to 3F4 and P-6 (His 87 ~ Glu 107 ) to 2C1 (Figure 1). The binding of the McAbs to FKBP-12 was not affected by the heterodimeric complex formation with FK-506, or by the heteropentameric complex formation with FK-506, Ca2+ , calmodulin and calcineurin. This is confirmed by comparing with the 3-dimensional structure of FK-506/FKBP-12 [3] and of FK-506/FKBP-12/Ca 2 + /calmodulin/calcineurin [4] complex. These McAbs with established epitope recognition should provide a way to understand the dynamic localization of FKBP-12 through the cellular response process in T-cell activation.

References 1 . Schreiber, S.L., Liu, J., Albers, M.W., Karmacharya, R., Koh, E., Martin, P.K., Rosen, M.K., Standaert,R.F., and Wandless, T.J., Transplantation Proceedings, 23 (1991) 2839. 2 . Thomson, A.W., Bonham, C.A., and Zeevi, A., Therapeutic Drug Monitoring, 17 (1995) 584. 3 . (a) Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L., and Clardy, J., Science, 251 (1991) 839. (b) Van Duyne, G.D., Standaert, R.F., Schreiber, S.L., and Clardy, J., J. Amer. Chem. Soc., 113 (1991) 7433. (c) Christopher, A.L., Thomson, J.A., and Moore, J.M., FEBS Letters, 302 (1992) 89. 4 . Kissinger, C.R., Parge, H.E., Knighton, D.R., Lewis, C.T., Pelletier, L.A., Tempczyk, A., Kalish, V.J., Tucker, K.D., Showalter, R.E., Moomaw, E.W., Gastinel, L.N., Habuka, N.,Chen, X., Maldonado, F., Barker, J.E., Bacquet, R., and Villafranca, J.E., Nature, 378 (1995) 641.

275 Contribution of peptide backbone atoms to binding of an antigenic peptide to class I major histocompatibility complex molecule N.G. SAITO and Y. PATERSON Department of Microbiology and The Eldridge Reeves Johnson Foundation for Molecular Biophysics, University of Pennsylvania School of Medicine, Room 323 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6076, USA

Introduction VSV-8, an H-2K b restricted T-cell epitope derived from the nucleoprotein of vesicular stomatitis virus [1] is an octapeptide (RGYVYQGL) recognized by Clone 33, a CD8 + cytotoxic T lymphocyte (CTL) clone derived from C57BL/6 mice [2]. There have been several informative studies to determine the contribution of peptide anchor residues to the MHC-peptide interaction, using VSV-8, SEV-9, and OVA-8 as model peptides [3–8) in which anchor and nonanchor residue side chains were modified by amino acid substitution. However, to our knowledge, only one study has fully explored the role of N- a n d C-termini to the stability of peptides on the H-2Kb molecule [9]. In that study, N-acetylation of VSV-8 decreased the affinity to the MHC molecule only by one order of magnitude, while C-amidation did not result in any observable change. In none of these studies, has the role of non-terminal peptide backbone atoms been fully explored. This endeavor requires a method to segregate the contribution of the peptide backbone from that of the termini and side chains. In order to measure the relative contributions of side chain atoms, peptide termini, and backbone atoms to the stability of the Kb -VSV-8 complex, a retroinverso analog of VSV-8 (RI VSV-8) that mimics the side chain topochemistry of the native peptide while reversing the order of backbone atoms was synthesized (Figure 1). However, the reversal of the backbone also results in the reversal of N- and C -termini. In an attempt to compensate for this structural feature, a series of RI VSV-8 analogs with C -terminal carboxamide and/or N-terminal malonyl modifications were also synthesized. In addition, C-terminal carboxamide and/or N-terminal acetyl variants of the native VSV-8 peptide were synthesized to determine the effect of the disruption of the peptide termini in the context of the native peptide backbone structure. These VSV-8 analogs were then used in in-vitro CTL assays and peptide competition CTL assays to observe the effect of disrupting the non-terminal backbone structure on binding of VSV-8 to H-2Kb [10]. 805 Y. Shimonishi (ed): Peptide Science – Present and Future, 805–807 © 1999 Kluwer Academic Publishers, Printed in Great Britain

806

Figure 1.

Structures of VSV-8 peptide and its retro-inverso analog, RI VSV-8.

Results and discussion Results from single peptide CTL assays and competition CTL assays imply that none of the RI VSV-8 analogs are capable of binding to the peptide binding groove of the H-2K b molecule. In contrast, all three of the terminally modified analogs of VSV-8 were capable of sensitizing target cells for CTL mediated lysis. To investigate the structural basis for the failure of RI VSV-8 analogs to bind to the H-2K b molecule, molecular models of each analog bound to the MHC molecule were built for analysis. These models showed no profound differences in the number of van der Waals overlaps that occur between peptide atoms and the H-2K b atoms compared to native VSV-8 for any of the analogs. In contrast, an analysis of the hydrogen bonds formed between retro-inverso VSV-8 analogs and H-2Kb showed a drastic decrease in the number of hydrogen bonds compared to that between native VSV-8 peptide and H-2Kb . The results of our study suggest that non-terminal backbone atoms of an antigenic peptide play a major role in its ability to bind tightly to the H-2K b molecule. Furthermore, our results support a hypothesis that antigenic peptides are always aligned consistently parallel to the α 1 helix through the polarity of the peptide encoded by the backbone atoms. These results are also relevant to the development of peptidomimetic vaccines for the generations of CTLs. Design of efficacious nonpeptide analogs of class I MHC binding antigens may only be possible by conserving the hydrogenbonding capacity of the backbone atoms while mimicking the overall topochemistry of the native peptides. References 1. Van Bleek, G.M. and Nathenson, S.G., Nature, 348 (1990) 213. 2. Sheil, J.M., Bevan, M.J., and Lefrancois, L., J. Immunol., 138 (1987) 3654.

807 3. Jameson, S.C. and Bevan, M.J., European J. Immunol., 22 (1992) 2663. 4. Shibata, K., Imarai, M., van Bleek, G.M., Joyce, S., and Nathenson, S.G., Proc. Natl. Acad. Sci. U.S.A., 89 (1992) 3135. 5. Saito, Y., Peterson, P.A., and Matsumura, M., J. Biol. Chem., 268 (1993) 21309. 6. Lipford, G.B., Hoffman, M., Wagner, H., and Heeg, K., J. Immunol., 150 (1993) 1212. 7. Lipford, G.B., Bauer, S., Wagner, H., and Heeg, K., Vaccine, 13 (1995) 313. 8. Neefjes, J.J., Dierx, J., and Ploegh, H.L., Eur. J. Immunol., 23 (1993) 840. 9. Matsumura, M., Saito, Y., Jackson, M.R., Song, E.S., and Peterson, P.A., J. Biol. Chem., 267 (1992) 23589. 10. Saito, N.G. and Paterson, Y., Mol. Immunol., 34 (1997) 1133.

276 B cell and T cell epitopes of Der f 2, a house dust mite major allergen of the Dermatophagoides farinae Y. HANTANI, N. UESATO, G. ITOH, T. ANDO, R. HOMMA, S. IKEDA, Y. INO and M. KURUMI Torii Pharmaceutical Co., Ltd., 1-2-1 Ohnodai, Midori-ku, Chiba-Shi, 267, Japan

Introduction Dermatophagoides farinae is an important allergen source for house dust mite allergy. The major allergen Der f 2 is a protein of 129 amino acid residues, devoid of glycosylation sites and contains three internal disulfide bridges (Cys8 Cys 114 , Cys 21 -Cys 27 , and Cys 73 -Cys 78 ) [1]. Der f 2 is a very important major allergen which is the best recognized by allergic patients (over 90% of the patients) [2]. The modulation of Der f 2 specific immunological response may lead to a therapy for house dust mite allergy. Based on this concept, specific peptide immunotherapy has been investigated using B cell epitopes or T cell epitopes of Der f 2.

Results and discussion The pin method is now a usual technique for the determination of B and T cell epitopes [3]. However, this method has the disadvantages that the purity of peptides on pins is not reliable, and disulfide bond in cystein-containing peptides can not be formed. In order to overcome these disadvantages, we chemically synthesized peptides and purified them by HPLC. Besides, we formed disulfide bond in peptides containing two cystein residues by dimethylsulfoxide oxidation. Then the peptides were bound to the pins which had been activated with 4-hydroxyphenyldimethylsulfonium methylsulfate via amino groups. Twenty-three overlappping peptides with a length of 15 amino acids that span the sequence of Der f 2 were synthesized by SPPS, and each peptide was coupled to the pin. The reactivity of A/J (H-2 a ) mice anti-Der f 2 IgG antibodies with the panel of synthetic peptides was examined by ELISA. The two regions of 18–42 and 58–82 reacted with anti-Der f 2 IgG antibodies, and these were located in the vicinity of the disulfide bridges (Cys 21-Cys 27 and Cys 73 -Cys 78 ). The sera obtained from other strains (BALB/C (H-2d ), C3H/He (H-2 k ), C57BL/6 (H-2b )) gave similar results. On the other hand, all synthetic peptides failed to bind A/J normal mice IgG antibodies. Furthermore, peptides corresponding to residues 18–32 and 63–77 inhibited the binding of anti-Der f 2 808 Y. Shimonishi (ed): Peptide Science – Present and Future, 808–809 © 1999 Kluwer Academic Publishers, Printed in Great Britain

809 IgG antibodies to recombinant (r) Der f 2-pin. These results suggest that two regions of 18–42 and 58–82 will be major B cell epitopes. A/J mice were subcutaneously immunized with rDer f 2 (0.1 mg) emulsified in complete Freund’s adjuvant (CFA) at the base of tail. Ten days later, T cell responses to overlapping peptides were determined by the index of [³H]thymidine incorporation in the lymph node cells obtained from immunized A/J mice. The high responder A/J mice recognized one region of 18–32. Since both B and T cell epitopes clustered around the loop area (Cys 21 Cys 27 ), Der f 2 peptide derivatives with a length of 10 amino acids near this area were synthesized to determine the further detailed T cell epitope. The high r e s p o n d e r A / J m i c e r e c o g n i z e d N -(3-carboxypropionyl)[2-aminobutanoic acid 21 ] Der f 2(15–24)amide (peptide 156). Peptide 156 (2 mg/day) was intravenously administered into A/J mice for 3 consecutive days before immunization of rDer f 2 (0.1 mg) in CFA. Ten days later, the lymph node cells were prepared and cultured. Thus, IL-2 and IL-4 production were assessed by ELISA. Pretreatment of peptide 156 suppressed the IL-2 (Th1 phenotype) and IL-4 (Th2 phenotype) production. Peptide 156 (2 mg) was intravenously administered into A/J mice twice (– 7 and – 3 days) before immunization of rDer f 2 (0.01 mg) adsorbed to aluminum hydroxide. IgE antibody titers were determined by passive cutaneous anaphylaxis (PCA) tests using pooled mice sera 14 days after immunization in SD rats. Immunization of rDer f 2 elicited a strong PCA reaction (anti-Der f 2 antibody titer of 1280). Pretreatment of peptide 156 suppressed the primary IgE antibody production (titer of 320). The delayed-typed allergic animal model was established in A/J mouse, in which the activation of Th1 type cytokine was thought to be involved in the pathogenesis of allergic inflammation. Six days after the subcutaneous immunization with rDer f 2 (0.05 mg) in CFA, allergic reaction was elicited by the injection of rDer f 2 (0.025 mg) with aluminum hydroxide into the footpad. The footpad swelling was reached to maximal level 24 h after this challenge. Pretreatment with intravenous injections of peptide 156 (5 mg/body/day) for 3 consecutive days before immunization strongly suppressed the footpad swelling. Our results show that peptide immunotherapy would be a potential remedy for allergic diseases.

References 1. Nishiyama, C., Yuuki, T., Takai T., Okumura, Y., and Okudaira, H., Int. Arch. Allergy appl. Immunol., 101 (1993) 159. 2. Heymann, P.W., Chapman, M.D., Aalberse, R.C., Fox, J.W., and Platts-Mills, T.A.E., J. Allergy Clin. Immun. 83 (1989) 1055. 3. Geysen, H.M., Meloen, R.H., and Barteling, S.J., Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 3998.

277 Conformational and binding studies of the peptide isolated from phage display library by anti-mouse CTLA-4 monoclonal antibody Y. ITO, Y. OOMURA, K. EZAKI, S. KOJIMA, T. FUKUMOTO, N. TORIGOE, S. HASHIGUCHI and K. SUGIMURA Department of Bioengineering, Faculty of Engineering, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

Key words: phage display library, CTLA-4, T cell activation, costimulatory signal, CD80

Introduction Initiation of T cell activation requires both the main signal through T cell receptors that recognize MHC-antigenic peptide complex and the costimulatory signal. The costimulatory molecules of CD80/86 on the antigen presenting cell (APC) deliver both the positive and negative regulatory signals through CD28 and CTLA4 on T cell, respectively [1]. The specific inhibitors for these costimulatory molecules make it possible to control the T cell activation. Peptide library-phage display system is a powerful tool to search such inhibitory peptides from a large library of random peptides (~ 10 8 ). To select the peptides that block the costimulatory signals, the phage clones were selected from the phage library displaying random 15mer peptides on the gene 3 protein (g3p) [2, 3] by panning against anti-CTLA4 mAb (UC10-4F10-11) that specifically inhibits the binding between CD80 and CTLA4. The selected clones designated as F2 and F6 actually augmented the antigen-specific T cell proliferation [4]. In this study, the conformational and binding studies of g3p and the synthetic peptides originated from these clones were investigated on the mechanism of T cell proliferation by these peptide motifs.

Results and discussion The peptide motifs on the phages selected by panning against anti-CTLA4 mAb may hold CTLA4-like structure, because the antibody possesses the complementary or a mold-like structure to CTLA4, and therefore the motif is expected to bind its ligands (CD80/CD86). To confirm this, the binding between the purified g3p of F2/F6 and CD80/CD86 recombinant proteins (CD80-Ig, CD86-Ig) were examined. The F2-g3p bound to CD80-Ig immobilized on the 810 Y. Shimonishi (ed): Peptide Science – Present and Future, 810–812 © 1999 Kluwer Academic Publishers, Printed in Great Britain

811 ELISA plate, but not to CD86-Ig, indicating that the target molecule of F2 motif is CD80. This result was confirmed by surface plasmon resonance (SPR) analysis. The F2-g3p tightly bound to CD80-Ig conjugated on the sensor chip of BIAcore with a dissociation constant (Kd) of 4.3 nM at 20°C. The F2-g3p inhibited the binding between CD80-Ig and CTLA4-Ig. These results suggested that the enhancement of T cell proliferation with F2 motif was possibly caused by the blockade of the negative-signaling pathway between CTLA4 and CD80-Ig. On the other hand, the F6-g3p bound neither CD80 nor CD86. The F2 peptide(sF2:GFVCSGIFAVGVGRCGAAGAET)bearing 22 amino acids with C-terminal flanking sequences (7 amino acids) was chemically synthesized and oxidized to form the disulfide bridge by the two Cys involved in the sequence. To examine the role of these Cys, the Ser-derivative (sF2Ser) with Ser substituted for Cys were also synthesized. The sF2 bound to antiCTLA4 mAb and inhibited the binding between anti-CTLA4 mAb and CTLA4-Ig but sF2Ser did not, indicating that this disulfide bridge is important for the binding activity of F2 motif. To further investigate this, CD spectrums of these peptides were measured (Figure 1). The spectrum of sF2 indicated a typical β sheet conformation (about 50% content in buffer), but the sF2Ser showed almost random structure. These data indicated that the disulfide bridges in the F2 motif can stabilize the β sheet structure that was necessary for the binding with mAb. The sF2 synthesized here could not bind to CD80-Ig in spite of tight binding of F2-g3p to CD80. Therefore, the mutagenesis for the affinity maturation of F2 is now performed using phage display system to improve the binding ability and specificity.

Figure 1. The circular dichroism (CD) spectrums of sF2 and sF2Ser peptides in buffer (pH 6.8) and 40% trifluoroethanol (TFE).

812 The F2 motif can augment the antigen specific T cell proliferation possibly by blocking the negative costimulatory signal via CD80/CTLA4 interaction. Such a molecule would be of value in enhancing the vaccine effect in AIDS and cancer diseases.

References 1. 2. 3. 4.

Krummel, M.F. and Allison, J.P., J. Exp. Med., 183 (1995) 2541. Nishi, T., Budde, R.J.A. et al. FEBS letters, 399 (1996) 237. Fukumoto, T., Torigoe, N. et al. Peptide Chemistry Kitada, C. (Ed.) 1996, 313. Fukumoto, T., Torigoe, N. et al. submitted.

278 Formation of DNA-anti-DNA antibody immune complex on collagen matrix M. SASABE, H. KITAMURA, K. SHINDO, N. SAKAIRI and N. NISHI Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060, Japan

Introduction Anti-double-stranded DNA (anti-dsDNA) antibodies have been found in sera of patients with systemic lupus erythematosus (SLE), and generally detected by radioimmunoassay (RIA) for the diagnosis of SLE [1]. However, detailed interaction mechanism of anti-DNA antibody with DNA in vivo is still unclear. Previously, it was shown that DNA-anti-DNA antibody immune complex deposits on the tissues which consist of collagen [2]. In the present study, binding property of anti-DNA antibodies to DNA immobilized with collagen was investigated by an enzyme-linked immunosorbent assay (ELISA) and compared with those by RIA.

Results and discussion Double-stranded DNA was immobilized on collagen-coated 96-well microtiter plates for ELISA (Corning Co. Ltd., USA) by the following procedures. 50 µl of pepsin-digested type I collagen solution (0.1 mg/ml, pH 3.0; Koken Co. Ltd., Japan) was added to the wells and completely dried at room temperature. 50 µl of DNA solution (0.1 mg/ml, Mw. ca. 7.5 kbp; Yuki Fine Co. Ltd., Japan) was added to the wells and incubated at 37°C, for 1 h. Free DNA in the supernatant was measured by the method to use Hoechst 33258, and the amount of DNA immobilized with collagen was calculated; 1.57 µg of DNA was immobilized as DNA-collagen complex on the well. Binding of the anti-DNA antibodies to the DNA-collagen complex was examined by the modified ELISA method [3]. The diluted serum containing anti-DNA antibodies (519 kIU/L, The Binding Site Co. Ltd., UK) of patient with SLE was used as a standard sample. As shown in Figure 1, amount of the anti-DNA antibodies adsorbed to DNA-collagen complex decreased gradually as the sample solutions were diluted. On the other hand, no binding of antibodies to collagen alone was detected. These results suggest that the antiDNA antibodies have an affinity with DNA immobilized with collagen as solid-phase antigen, and actually form DNA-anti DNA antibody immune complex in vitro. 813 Y. Shimonishi (ed): Peptide Science – Present and Future, 813–815 © 1999 Kluwer Academic Publishers, Printed in Great Britain

814

Figure 1.

Dose-dependent antibody binding to DNA-collagen complex

or collagen

.

Monoclonal antibody against DNA [4] was affinity-purified from culture medium of hybridoma (CAL-13) from SLE-prone MRL/Mp-lpr/lpr mice. Reactivity of the monoclonal antibody to DNA was examined by two ELISA methods using poly-L-Lys (pLys) or collagen as immobilizing reagents. The antibody actually bound both to the DNA-pLys and the DNA-collagen complexes. However, binding of the antibody to pLys was also observed in the same condition. This phenomenon is consistent with the report that nonspecific binding of anti-DNA antibody was caused by basic reagents used for immobilization of DNA [5]. In contrast, binding to collagen was scarcely detected as shown in Figure 2. These results indicate that collagen is a very useful immobilizing reagent for ELISA. Detections of anti-DNA antibodies in sample sera of patients with SLE (n = 40) were carried out by ELISA using collagen and RIA. The reactivity of antiDNA antibodies to DNA-collagen complex determined by ELISA and RIA showed poor coincidence; i.e., the correlation of R² = 0.134. It would be due to the difference between solid phase (ELISA) and liquid phase (RIA) DNAs.

Figure 2. Comparison of the anti-DNA antibody-binding in ELISA. Collagen-DNA complex , collagen , pLys-DNA complex , and pLys were used as solid phase antigen.

815 These varieties of anti-DNA antibodies shown in this study would be important for elucidation of not only diagnosis and pathogenesis of SLE but the interaction between DNA and anti-dsDNA antibodies in vivo.

Acknowledgements We would like to thank Dr E. Matsuura (Institute of molecular and Cellular Biology, Okayama University Medical School) for his helpful discussions. This work was supported by the Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 07555600 and 09876088).

References 1. Wold, R.T., Young, F.E., Tan, E.M., and Farr, R.S., Science, 161 (1968) 806. 2. Koffler, D., Schur, PH., and Kunkel, H.G., J. Exp. Med., 126 (1967) 607. 3. Kitamura, H., Matsuura, E., Nagata, A., Sakairi, N., Tokura, S., and Nishi, N., Int. J. Biol. Macromol., 20 (1997) 75. 4. Ichikawa, K., Suzuki, T., Hashimoto, Y., Sumida, T., Tomioka, H., Yoshida, S., and Koike, T., Lupus, 1 (1992) 239. 5. Kroubouzos, G., Tosca, A., Konstadoulakis, M.M., and Varelzidis, A., J. Immunol. Methods., 148 (1992) 261.

279 Natural IgM antibodies against human endogenous retroviruses HERV-H demonstrated by means of synthetic peptides R. PIPKORN 1 , A. LAWOKO2,3 and J. BLOMBERG 3 1

Deutsches. Krebsforschungszentrum., Heidelberg, Germany S. Virol., Dpt Med. Microbiol., Lund U. Sweden 3 Dpt. Inf. Dis. & Clin. Microbiol., Uppsala U. Sweden 2

Introduction Natural antibodies are antibodies which exist in most people without a clear immunisation event. They often belong to the IGM class. They tend to react with evolutionarily conserved epitopes. Recently, functions of natural antibodies, like survellance against tumor development, have been discovered. There is also evidence that natural antibodies can become aggresive autoimmune antibodies by class switsching from IgM to IgG. Knowing that humans have thousands of endogenous retroviral genes it is interesting to study to what degree they evoke an immune response. We therefore studied the occurance of IgG and IgM antibodies to synthetic peptides with sequence derived from human endogenous retriviruses. Results and discussion We initially surveyed HIV-1 seronegative sera for reactivity against a set of peptides representing conserved regions of endogenous and related exogenous RVs. Table 1.

Sequences of peptides used in the study.

MLVCAC

RTHML3_2 HUTM1: 1

TNSVMSFIWQSAPDIGRKLGRLEDLKNKTLGDLVREAEKVY NKRETPEEREERIRRETEEKEERRRTEDEQKEKERDRRRHRE MSKLL DLKDCFFTIPLAEQDCERFAFT LQNRRGLDLLTAEKGGLCIFLNEE

Table 2. Frequencies of IgG, and IgM rectivities to the peptides selected from the crosssectional study, in sera from two groups of patients. Peptide: RTML3-2 MLVCACT HUTM1:1

HIV-1 negative sera: IgG 19/102 20/102 2/102

816 Y. Shimonshi (ed): Peptide Science – Present and Future, 816–817 © 1999 Kluwer Academic Publishers, Printed in Great Britain

HIV-1 negative sera: IgM 33/98 45/98 6/98

817 Table 3. Single character representation of longitudinal study of reactivity to three peptides. HIV-1 negative individuals have the prefix ‘N’. Peptides are listed in the columns. The prefix ‘M’ is added to distinguish IgM from IgG testing. Serially taken sera are listed on the horizontal plane. Black represents strong reactivity, absorbance difference > 1.0. Grey, moderate reactivity with absorbance difference > 0.3 > 0.1. White represents nonreactivity. IgM reactivity is represented by an ‘m’ for moderate reactivity,and ‘M’ for strong reactivity.

The three peptides used in longitudinal surveys for IgG and IgM reacitity in 17 HIV-1 seronegative but iatrogenically immunosuppressed patients were: 1) RTHML3_2, a peptide derived from the pol region of a MMTV-like HERV-K human retroviral element clone3_2, 2) HUTM 1:1, a transmembrane region of env peptide derived from the MLV-like HERV-H, 3) MLVCAACT from the capsid protein of MLV To our surprise, it proved to be common that healthy persons have IgM, but much less commonly IgG, to such retroviruses.

280 Evaluation of region-specific antibodies for rat and human Chromogranin A with use of synthetic peptide N. YANAIHARA1 , Y. NISHIKAWA2 , K. IGUCHI 2 , T. MOCHIZUKI 2 , M. HOSHINO2 , S. NAGASAWA1 , L. JUN1 , Y. FUTAI 1 and C. YANAIHARA3 1

Yanaihara Institute Inc., Awakura, Fujinomiya-shi, 418, Japan Department of Pharmaceutical Sciences University of Shizuoka, Yada, Shizuoka, 422, Japan 3 Department of Pharmacy, Hyogo Medical University, Hyogo, 663, Japan 2

Introduction Chromogranin A(CgA) is a member of the secretogranin/chromogranin class of protein that occurs in secretory granules of wide variety of endocrine cells and neurons [1]. CgA is present in catecholamine storage vesicles of the chromaffin cells and sympathetic postganglion neurons and co-released with catecholamines during exocytosis. The CgA molecule contains numerous pairs of basic amino acid residues which may act as sites for specific proteolytic processing to smaller biologically active peptides and others. The aim of the present study was to evaluate multiple region-specific antibodies for rat and human CgA-related fragments for the measurement of immunoreactive(IR) CgA in rat and human tissue, plasma, urine and saliva.

Results and discussion We have previously developed four different types of region-specific radioimmunoassays(RIAs) for rat CgA using anti-rat CgA(1–28) serum, antirat CgA(94–130) serum, anti-rat CgA(359–389) serum and anti-rat pancreastatin(33–51) serum [2]. Among battery of assay systems, RIA developed with anti-rat CgA(359–389) serum was found to be useful for measurement of IR-CgA levels in rat and human plasma [3]. The sequence comparison of rat and human CgAs showed that amino acid sequence of rat CgA(359–389) was homologous to that of human CgA (344–374). Thus, synthetic peptide, which correspond to human CgA (344–374) [4] was used as antigen for producing antisera in rabbits. CgA(344–374) and the related peptide were described previously. The RIA for human CgA was developed using synthetic human CgA(344–374) as standard antigen, 125 Ihuman CgA(344–374) (approximately 5,000 cpm) as labeled antigen and antihuman CgA(344–374) serum RY76(final dilution X8,400). The minimum detection limit of the RIA was approximately 1.80fmol/tube. Dilution curves of 818 Y. Shimonishi (ed): Peptide Science – Present and Future, 818–819 © 1999 Kluwer Academic Publishers, Printed in Great Britain

819

Figure 1.

Schematic diagram of Primary Structure of Human Chromogranin A.

human plasma and urine samples as well as the extracts of human adrenal gland and pancreas were parallel to the standard curve, suggesting that the present RIA can be used for measurement of human IR-CgA levels. The fasting plasma IR-CgA levels detected by the present RIA were 305–74 fmol/ml(n = 28). These values were in the range of plasma CgA levels in the fasting normal subjects in the assays reported by other investigators. IR-CgA levels in human urine, saliva and cereblospinal fluid were 96–168 (n = 22), 33–56 (n = 8) and 18–43 (n = 5) fmol/ml, respectively. As has been reported [5], the plasma IR-CgA levels in patients with renal failure, pheochromocytoma and medullary thyroid carcinoma were significantly higher than those of the fasting normal subjects in the present RIA. The plasma IR-CgA, detected in the present study, is considered to be either the processing product of CgA containing human CgA(344–374), or part of it. The present study demonstrates clearly that the well-designed synthetic peptide is an extremely valuable antigen for production of antibody specific for macro molecular protein such as CgA.

References 1. O’Connor, D.T., Regulatory Peptides 6(1983) 263. 2. Nagasawa, S., Nishikawa, Y., Ohshima, K., Iguchi, K., Mochizuki, T., Greeley, Jr. G.H., and Yanaihara, N., Biomed. Res. 16 (1995) 83. 3. Nishikawa, Y., Iguchi, K., Mochizuki, T., Hoshino, M., Futai, Y., Abe, K., Li, J., and Yanaihara, N., Kitada, C. (Ed.), Peptide Chemistry, 1997, p. 125. 4. Konecki, D.S., Benedum, U.M., Gerdes, H.-H., and Huttner W.B., J. Biol. Chem. 17 (1987) 17026. 5. O’Connor, D.T. and Bernstein, K.N., New Eng. J. Med. 311 (1984) 764.

281 Design of synthetic antigen containing nonimmunogenic T cell epitope core and simple nonnative flanking regions F. HUDECZ1 , K.A. WILKINSON2 , M.H. VORDERMEIER2, J. KAJTÁR 3, S. JURCEVIC 2 , R. WILKINSON 2 and J. IVANYI 2 1 Research Group of Peptide Chemistry, Hungarian Academy of Science, Eötvös L. University, Budapest 112, POB 32, H-1518 Hungary 2 Tuberculosis and Related Infections Unit, MRC, Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London W12 0HS, UK 3 Department of Organic Chemistry, Eötvös L. University, Budapest, Hungary

Introduction The T cell recognition of naturally processed proteins or synthetic peptides depends not only on the sequence of the epitope core, but also on its flanking residues. Recently, the CD4 + T cell epitope core of 65 FNLWGPAFHERYPNVTITA 83 region of the 38 kDa glyco-lipoprotein, secreted constituent of M. tuberculosis was localised to amino acid residues 75 RYPNVTI 81 [ 1 ] . Peptide containing the deduced core was not immunogenic [2]. In the present study we aimed to design synthetic peptide antigens by introduction of flanking residues both at N- and C-terminal of the T cell epitope core.

Results and discussion Peptides were prepared with flanks composed of either amino acid residues from the native sequence and terminated by Ala and/or Ser residues or oligopeptides of Ala or Ser exclusively. (Table 1). The analytical RP-HPLC profiles (data not shown) demonstrated the high purity of the peptides used in these Table 1. Characteristics of synthetic peptides containing the deduced T cell epitope core of the 65–83 domain of 38 kDa protein of M. tuberculosis.

a

Determined by amino acid analysis.

820 Y. Shimonishi (ed): Peptide Science – Present and Future, 820–822 © 1999 Kluwer Academic Publishers, Printed in Great Britain

821

Figure 1. The effect of non-native flanking regions on the binding to isolated H-2-Ab . Inhibition of binding of the biotinylated CLIP peptide (0.7 mM) to H-2-Ab by peptides.

experiments. Elongation of the epitope core by four Ala at both N- and C-terminals resulted in peptide A4 (75–81)A 4 which was stimulatory for murine hybridoma T cells [38.H6] as well as for lymph node cells from 65–83 primed C57BL/10 mice [3]. Peptide A2 SA(75–81)A 4 proved to be more effective in inducing proliferation than the native 65–83 (data not shown). Peptides were tested for binding to isolated MHC II glycoprotein using peptide VSKMRMATPLLMQALP (CLIP) with high affinity for H-2-Ab . Peptides with Ala flanking regions [A 4 (75–81)A 4 and A2 (73–83)A 2 ] showed somewhat lower binding to isolated H-2-Ab than native peptide 65–83 (Figure 1), while the binding of Ser substituted A2 SA(75–81)A 4 was almost identical with that of the native peptide. These results suggest that it is possible to construct simple, synthetic CD4 + T cell stimulatory peptides with high potency from a non-stimulatory, ‘silent’ epitope core by addition of flanking residues not being part of the native sequence. Acknowledgement This work was supported by grants from WHO (T9/181/133), from the Hungarian Research Fund (T 014964) and from the Ministry of Education (0101/1997).

822 References 1. Vordermeier, H.M., Harris, D.P., Moreno, C., Singh, M., and Ivanyi, J., Int. Immunol. 7 (1995) 559. 2. Wilkinson, K.A., Vordermeier, M.H., Iványi, J., and Hudecz, F., Vaccine, 1998 (submitted). 3. Wilkinson, K.A., Vordermeier, M.H., Kajtár, J., Wilkinson, R., Jurcevic, S., Iványi, J., and Hudecz, F., Mol. Immunol., 34 (1998) 1237.

282 Immune response to newly exposed regions of Band 3 protein of the red blood cell membrane during malaria infections U.A.K. KARA¹, L.H. KWAN¹ and R.M. KINI² ¹ School of Biological Sciences and b Bioscience Centre, National University of Singapore, Singapore, Singapore 0511

Introduction Malaria is an infectious parasitic disease caused by the protozoan Plasmodium. It afflicts between 200–300 million people and results in more than 2 million deaths a year worldwide. The parasite has a very complex life cycle including mosquito stages, liver and blood stages. Control of the disease relies heavily on environmental management, vector control efforts, chemoprophylaxis and chemotherapy after early diagnosis but hopes for future immunoprophylaxis by vaccination are high. Several experimental vaccines are presently undergoing human trials. All theses vaccine constructs use parasite specific antigen material for immunisation. We looked into the possibility of inducing an antibody response against normally cryptic epitopes of host proteins that become exposed upon infection. Such epitopes would only be accessible to the immune response under disease conditions. Case in point, when the malaria parasite infects the erythrocyte host cell, the Band 3 protein which is an intergral part of the membranes of red blood cellls, undergoes conformational changes. These conformational changes lead to the exposure of new segments within the Band 3 protein that are cryptic in normal, uninfected red blood cells. A synthetic peptide corresponding to one such newly exposed segment of the mouse Band 3 protein was tested for its immunogenicity and protective function in murine malaria.

Results and discussion We used the murine malaria model of the Plasmodium berghei ANKA parasite strain in Swiss White mice to determine the immunogenicity of one particular Band 3 region and its protective function against malaria infections. Mice were immunised with three doses of a synthetic peptide corresponding to this region of Band 3. The animals were subsequently challenged by administering blood infected with the lethal murine Plasmodium berghei malaria. Red blood cells infected with the parasite undergo morphological and biochemical changes that expose normally cryptic regions of the Band 3 protein. As could be 823 Y. Shimonishi (ed): Peptide Science – Present and Future, 823–824 © 1999 Kluwer Academic Publishers, Printed in Great Britain

824 demonstrated by indirect immunofluorescence assay, immune serum from mice immunised with the synthetic peptide recognised epitopes that are present on the surface of Plasmodium infected red blood cells but are absent in uninfected erythrocytes. Immunisation with the peptide did confer partial protection against malaria. All immunised mice showed slightly slower development of parasitemia compared to control animals. Two out of six immunised mice recovered from low level parasitemia while all other animals died. The immune serum of one of the immunised and partially protected mice was used for passive immunisation studies. Mice were injected with parasites plus immune serum or with parasites alone. One out of six mice that had been immunised with the immune serum of the previously surviving mouse again recovered from low level parasitemia while all other immunised mice showed delayed onset of parasitemia compaired to control animals. A possible mechanism to explain this partially protective function of the synthetic peptide against malaria infection after active immunisation is that antibodies bind to the infection-induced exposed region of Band 3 and complement mediated killing of the infected red blood cell takes place. In the passive immunisation study, immune serum was injected into mice together with parasite-infected red blood cells thus allowing antibody-antigen formation resulting in attenuation of the parasites. In addition, antibody dependent complement activated cytotoxicity could have helped clearing parasites or at least, caused a delay in the development of parasitemia. This novel approach of using a host protein segment instead of parasite antigens as a target for immunisation may provide alternative avenues in our fight against diseases caused by intracellular pathogens. The main advantage of such an approach lies in its ability to overcome potential vaccine failures which may occur due to mutations of pathogen antigens.

Acknowledgements The authors would like to acknowledge the expertise of Dr Jill Tham in preparing this presentation and Ms Wong Tsuey Yi in helping perform the immunofluorescence assays. This work was supported by research grants awarded to Drs R.M. Kini and A.U. Kara by the National University of Singapore.

References 1. 2. 3. 4.

Crandall, I., Collins, W., Gysin, J., and Sherman, I., PNAS, 99 (1993) 4703. Crandall, I., Guthrie, N., and Sherman, I., Am. J. Trop. Med. Hyg., 52 (1995) 450. Pattarroyo, M., Amador, R., Clavijo, P. et al., Nature, 332 (1988) 158. Bittle, J., Houghton, R., Alexander, H. et al., Nature, 298 (1982) 30.

283 Peptide-induced antibodies as a tool to link genomes and proteomes T. FRÖHLICH and G.J. ARNOLD Genzentrum der Universität München, Feodor-Lynen-Str. 25, D-81377 München, Germany

Introduction Due to the intense efforts in the international genome projects, a huge number of DNA sequences and some complete genomes are presently available. However, using only DNA data, it is impossible to predict protein structure and function or to understand the intracellular network of protein interactions. Instead, the qualitative and quantitative analysis of a cells protein content under defined biological conditions is inevitable and referred to as proteome analysis [1]. Using 2D PAGE as the most powerful technique to separate and display large numbers of proteins in combination with mass spectrometry [2], individual protein spots can be linked to genetic data, if those are available for the analyzed protein fragments. We currently establish a systematic method to link existing genetic data to the corresponding proteomes. Our concept is based on synthetic peptides deduced from coding DNA sequences, which are used to generate protein-specific antibodies in mice or rats. These sensitive and specific probes facilitate the identification of proteins separated by 2D PAGE and represent powerful tools for functional and histological analysis. Results and discussion We established a procedure for a systematic DNA based genome to proteome linkage, using peptide induced mice or rat antisera in combination with 2D Western blots of tissue lysates. The general strategy is as follows: The DNA sequences are selected from DNA databases. Appropriate sequences for antigenic peptides are selected. Two independent antisera induced by different peptides are made for each analyzed gene, since only one antibody might lead to wrong identifications due to unspecific cross-reactions. Cell extracts are analyzed by high resolution 2D-PAGE and spots are detected by immunoblotting. Feasibility study using human gastric intrinsic factor (HIF) cDNA as a model system To demonstrate the feasibility of our strategy for a systematic genome to proteome linkage, our procedure was applied to the cDNA sequence of HIF, 825 Y. Shimonishi (ed): Peptide Science – Present and Future, 825–827 © 1999 Kluwer Academic Publishers, Printed in Great Britain

826 a glycoprotein specifically expressed in gastric mucosa, and responsible for the transport of vitamin B12 from the stomach to the ileum mucosa. HIF348 peptide EEAQRKNPMFKFE, corresponding to AA 348-360 in the mature HIF protein, was synthesized (Fmoc/PyBOP/tBu/Trt/Pmc strategy on AMS422), attached to thyroglobulin, inoculated in Balb/c mice, and the serum was used for immunoblotting. The silver stained 2D gel of human gastric mucosa and the 2D Western blot, using HIF348 induced antiserum, are shown in Figures 1A and 1B, respectively. Two signals specific for two HIF isoforms can be clearly identified by the peptide induced antibody. The 2D coordinates correspond to the molecular weight and isoelectric points previously reported for the HIF glycoprotein. As a control, a rabbit anti-intrinsic factor (porcine) antiserum, prepared in our laboratory, was used for 2D Western blot analysis (data not shown) detecting the same spots plus two more isoforms. Feasibility study using cDNA of mouse insulin-like growth factor binding proteins (IGFBPs) To use our strategy for the mouse genome to proteome linkage, antibodies were generated in Lewis rats using IGFBPs as a test system. IGFBPs are a family of six proteins modulating the action of the two insulin-like growth factors. IBPep1 peptide EMTEEQLLDSFHLMAPSRED, corresponding to AA 141–160 of the mature IGFBP1 protein was synthesized (Fmoc/PyBOP/ tBu/Trt/Pmc strategy on AMS422). An antiserum with titers similar to that raised in Balb/c mice was obtained in Lewis rats. The silver stained 2D gel of mouse liver and the corresponding 2D Western blot using an immunopurified anti-IBPep1-antiserum are shown in Figures 1C and 1D. The 2D coordinates determined correspond to that of 2D mapped human IGFBP1. In a control immunodetection performed with IBPep1 antiserum preincubated with IBPep1 peptide, the IGFBP1 spot disappeared, demonstrating the specificity of the reaction (data not shown).

Figure 1. (A) Silver stained 2D gel of human gastric mucosa lysate and (B) immunodetection of HIF using HIF348 induced antiserum. (C) Silver stained 2D gel of murine liver lysat and (D) immunodetection of IGFBP1 using IGPep1 induced antiserum.

827 The procedure presented here is a widely applicable approach to set up a link between genome and proteomes starting from genomic data.

References 1. Wilkins, M.R., Williams, K.L., Appel, R.D., and Hochstrasser, D.F., Proteome Research: New Frontiers in Functional Genomics. Springer, 1997, p. 1. 2. O’Connel, K.L and Stults, J.T., Electrophoresis 18 (1997) p. 349.

284 Porphyrin-peptide conjugates with stabilized helices and sheets G.R. GEIER, III, T. BAAS and T. SASAKI Department of Chemsirty, Box 351700, University of Washington, Seattle, WA 98195, USA

Introduction The design and synthesis of artificial proteins has become an increasingly valuable approach for studying protein folding, for understanding the behavior of native proteins, and for developing novel catalysts [1]. Attempts have been made to mimic protein structure using a variety of template molecules to organize short peptide sequences into defined secondary and tertiary structures [2, 3]. We describe here the design, synthesis, and charaterization of a novel porphyrin-peptide conjugate system [4].

Results and discussion Figure 1 shows the basic architecture of a porphyrin-peptide conjugate system. We designed the porphyrin-α-helix conjugate, 1-a, as a model for cytochrome P-450. The porphyrin ring functions both as a template for the organization of an α -helical peptide chain and as a catalytic site for oxidation of organic substrates. The α -helical peptide portion provides a chiral, hydrophobic substrate-binding site whose dimensions and properties may be readily varied by changing the peptide sequence. The conjugate has been synthesized by a solution phase coupling of the peptide segment to a bis-bromoacetyl derivative of tetraphenylporphyrin. We have examined the Mn(III) and Fe(III) systems

Figure 1.

The structure of porphyrin-peptide conjugates.

828 Y. Shimonishi (ed): Peptide Science – Present and Future, 828–830 © 1999 Kluwer Academic Publishers, Printed in Great Britain

829 Table 1. Mn(III) conjugate-silica oxidation experiments with PhIO in 50% isopropanol, 50% 10 mM pH 7 phosphate buffer/The values in italics are the results of the Mn(III)TPP-silica control experiments.

% Yield 77 92

Turnover 163 186

(2)

65 99

126 190

(3)

50 68

107 142

(4)

0 15

0 29

(5)

0 23

0 45

Rank (1)

Substrate

for their catalytic properties toward epoxidation of alkenes. The metalated conjugates were immobilized on a silica support which has been modified with an imidazole ligand. Steric interactions between the peptide chain and the solid surface should encourage the axial ligation from the non-peptide side, allowing for a substrate to bind to the cavity between the helix and the porphyrin ring. The immobilized conjugates catalyzed the oxidation of styrene to the corresponding epoxide by iodosylbenzene (PhIO). A series of alkenes were examined to probe the structure of the putative binding site. Table 1 shows a summary of the oxidation experiments in isopropanol-water mixture (1:1). Although oxidation was observed in good yield under a variety of conditions and a degree of substrate discrimination was observed, all reactions led to the formation of racemic epoxides. Possible explanations for the absence of enantioselectivity include the symmetric placement of 4 leucine residues on top of the porphyrin ring. We have recently synthesized the porphyrin- β -sheet conjugates, 1-b. The cisand trans-isomers of a bis-bromoacetyl derivative of tetraphenylporphyrin were synthesized, and reacted separately with Cys-containing peptides to prepare the conjugate. In this system, an antiparallel β -sheet structure is expected to form only on the cis-isomer. The characterization of the β -sheet conjugates with NMR and CD are now underway.

830 References 1. DeGrado, W.F., Raleigh, D.P., and Handel, T., Curr. Opin. Struct. Biol., 1 (1991) 984–993. 2. Lieberman, M., Tabet, M., Tahmassebi, D., Zhang, J., and Sasaki, T. Nanotechnology, 2 (1991) 203–205. 3. Altmann, K.H. and Mutter, M. Int J Biochem, 22 (1990) 947–56. 4. Geier, G.R. III. and Sasaki, T., Tetrahedron Lett., 38 (1997) 3821–3824.

285 Molecular recognition of cyclic HIV protease inhibitors: highly orally-bioavailable DMP851 P.Y.S. LAM*, J.D. RODGERS, R. LI, C.-H. CHANG, Y. RU, P.K. JADHAV, C.G. CLARK, J.A. MARKWALDER, S.P. SEITZ, S.S. KO, L.T. BACHELER, G.N. LAM, M.R. WRIGHT, S. ERICKSON-VIITANEN, P.S. ANDERSON and G.V. TRAINOR DuPont Pharmaceuticals Co., Experimental Station, P.O.Box 80500, Wilmington, DE19880-0500

Introduction Due to the long-term potential drug resistance problems associated with HIVPR mutations, a main emphasis in the search for the next generation of structurally-different HIVPR drugs is in the area of cyclic ureas [1]. Cyclic ureas tend to have low solubility due to the high rigidity of the preorganized structure and the intermolecular diol/urea bidentate chelation as seen in the crystal lattice. The low solubility resulted in low oral bioavailability in many cyclic ureas. This presentation describes the use of nonsymmetric cyclic ureas with a floppy alkyl P2 to break up the crystal packing and to increase solubility and thus improve the pharmacokinetics.

Results and discussion n-Butyl was chosen as a P2 sidechain because it has the best Ki among the symmetrical n-alkyl P2/P2' cyclic ureas. A series of cyclic ureas containing n-butyl P2 and other potent hydrogen-bond donating/accepting P2' previously synthesized in DMPC were prepared and evaluated. This leads to DMP851 [2], n-butyl P2 and 3-aminoindazole-5-methyl P2', with excellent oral bioavailability. DMP851 is currently undergoing clinical studies. Figure 1 highlights the key compounds in the cyclic urea clinical studies.

References 1. Lam, P.Y.S., Jadhav, P.K., Eyermann, C.J., Hodge, C.N., Ru, Y., Bacheler, L.T., Meek, J.L., Otto, M.J., Rayner, M.M., Wong, N.Y., Chang, C.H., Weber, P.C., Jackson, D.A., Sharpe, T.R. and Erickson-Viitanen, S., Science, 263 (1994) 380–384. 2. Rodgers, J.D., Lam, P.Y.S., Johnson, B.L., Wang, H., Li, R., Ru, Y., Ko, S. S., Seitz, S.P., Trainor, G.L., Anderson, P.S., Klabe, R.M., Bacheler, L.T., Cordova, B., Garber, S., Reid, C., Wright, M.R., Chang, C.-H. and Erickson-Viitanen, S., Chem. Bio., 5 (1998) 597–608.

831 Y. Shimonishi (ed): Peptide Science – Present and Future, 831–832 © 1999 Kluwer Academic Publishers, Printed in Great Britain

832

Figure 1. Key compounds in cyclic urea clinical studies.

Index

10,11-dihydro-5H-dibenzo[ a,d ]cyclohepten5-yl group (5-dibenzosuberenyl group) 516 1 H-nuclear magnetic resonance (NMR) 195, 263, 266, 324 2,3-epoxy amines 504–5 2-(4-nitrophenyl)sulfonylethoxycarbonyl (Nsc) 547 2-aziridinemethanols 504 2α-helix 110 2α-helix peptide 107 2D PAGE 825 3-(diethoxyphosphoryloxy)-1,2,3-benzotrazin4(3H)-one (DEPBT) 530 3 1 0 -heli structures 249 3 1 0 -helix 284 4-(2-(1-hydroxyethyl)-1,3-dithiacyclohex-2yl)phenoxyacetic acid (HETPA) 542 4-methylene glutamic acid derivatives 761 5-alkyl-3-aminobenzoic acids 92 7-azatryptophane 761–3 9-aminofluorene-9-carboxylic acid 300 A β -peptide 319 Ac-peptide 160, 162 acetyl-amino acid 160 acid hydrolysis 157, 160–1 acid–base pair 370, 372 Acm groups 576 activated esters 514–5 active carbonate 39, 40 active conformation 767 acute phase proteins 792 acyl donors 514 acylated insulins 674–5 adhesion 764–5 adrenomedullin 448, 450 affinity chromatography 646–7 agonist 202–3 Aib-model peptides 291 AIDS 652, 653 Alamethicin 67, 687–8 albumin binding 674, 676 allergy 808 allophenylnorstatine 652 α,α-disubstituted glycine 300 α,β-dehydroamino acid 500 α-amino fatty acids 587 α-amino phosphonic acid 566 α-amylase inhibitor 716 α-chymotrypsin 534–5

α-factor 467–8 α-fluoroglycine 47–8 α -helical conformation 34 α -helical peptides 684 α-helices in nonpolar media 232 α -helix 101–2, 104–5, 263 α -helix bundle 64, 66 α -lactalbumin 136 α 2 -antiplasmin 642–3 αββα -type structural motif 101 α v β 3 integrin 430 Alzheimer’s disease 319, 470 AM-toxin 767–8 Amadori 600–1 amino acid sequence and concentration dependencies 536 ammo acid type PEG (aaPEG) 455 amino alcohols 39 aminoindazole 831 amphipathic α-helical 782 amphipathic peptides 684 amphipathic surfaces 351 amphiphilic α-helical peptide 117 amphiphilic α-helix segments 78 amyloid β-protein 777 amyloid fibril 104–5 analgesia 407, 665, 668 analgesic activity 139–40 analogs 185–6 analogues of oxytocin 724 ANP receptor 163–4 antagonist 202–3, 215 antagonists for peptide receptors 26 anti-cancer drugs 731 anti-double-stranded DNA (anti-dsDNA) antibodies 813 anti-epilepsy 407 anti-HIV-1 activity 68l–2 anti-HIV-1 domains 681 anti-HIV-1 peptides 439 anti-inflammatory 792 anti-TNF peptidomimetic 61 antibacteria 416 antibacterial activity 269–70 antibacterial and hemolytic effect 684 antibacterial peptides 432–3, 691 antibiotic peptide 446 antibodies 113, 188–9, 816, 825–6 anticomplementary activity 421–2 antifungal activity 519 antifungal peptide 697–8

833 Y. Shimonishi (ed): Peptide Science – Present and Future, 833–842 © 1999 Kluwer Academic Publishers, Printed in Great Britain

834 antigen recognition 788–91 antigenic mapping 274–5 antigenic sites 784 antihypertensive peptide hormone 163 antimalarial drugs 704 antimetastatic activity 455 antimicrobial 263–4 antimicrobial peptide 88, 457, 697, 704, 707 antioxidative peptides 134 antiparallel β-strand 367 antiparallel helix–helix 367 antitumor drugs 49 Aplysia 442–4 apoptosis 49–50 application of PKSI-527 646 [Arg 8 ]-vasopressin 539–40 artificial flavo-enzymes 76 artificial functional membrane proteins 67, 69 artificial heme enzyme 80 artificial ion channels 59–60 artificial protein with designed loops 78 artificial proteins 98–100 asialoglycoprotein receptor 709–10 aspartic proteinases 558, 657 asymmetric synthesis 566–7 ATP hydrolysis 383–4 ATPase 383–4 autoimmune disease multiple sclerosis 788 automation 141, 143 AVP(4-8) 174–8 aza-Payne rearrangement 504–5 azatyrosine 761–3 aziridine-2,3-dicarboxylic acid 634 aziridines 44–5, 635 backbone 805–6 backbone amides 316–7 backbone cyclization 70 bactenecin 5 446 bacterial carboxyl proteases 657 bacteriocins 462–3 bacteriophage φ K 373 Band 3 protein 823 barnase 586–3 basic amphiphilic helices 57 benzotriazole ester 82–3 bestatin 689–90 β -amyloid peptide 470 β -elimination 498–500 β -hairpin 113 β -helix-like structure 98–99 β -isopropylphenylalanine 119 β -methylphenylalanine 119 β -sheet 30–2, 73–4, 228, 231, 424 β -sheet structure 280–1 β -sheet-forming peptides 782 β -strand 98–9, 101–2 β -structure 104–5

β -turn 249–50 β -turn mimetics 82 βαβα− structure 80 bifunctional anti-HIV compounds 41–2 BINAP–Ru(II) complex 566 binding 205–6 binding analysis 670–1 binding assay 226–7 binding-site structure 163–4 bioactive peptides 419 biodistribution 670–2 biological activity 185 biopolymers 30 biphalin 726–7 bivalent plasmin inhibitors 642 BMP type-IA receptor 309–10 Boc strategy 615–6 bombesin/GRP antagonist stereoselective synthesis 502 Bombyx mori 460 bombyxin 245–8 bone growth 711–2 bone morphogenetic proteins (BMPs) 309 bradykinin 419–20 bradykinin antagonists 731 brain 637–8 Brevibacterium linens 462–3 bridged analog 775–6 building block 550 bundled peptide 117–8 C-terminal alkylamides 701–2 C-terminal residue 571 Cl300 neuroblastoma cells 777 Ca 2+ -depleted α-LA 136–7 calcium 183 calcium channel 695–6 calcium channel blocker 311 calcium channels 271 Cα,α-disubstituded glycines 385 Cα -methyl 249 Cα -phenylglycine 249 cAMP 450–1 cAMP production 711–2 cAMP response element binding protein 1 574 cancer 188–9 cancer cells 416, 418 captopril 693 carbamate pseudopeptides 39–40 carbamoylmethyl ester 514–5 carbohydrate 770–1 carbohydrate chain 166 cardioactive peptide 442 cardiotoxin 555 carnosine 413–4, 693–4 casein 728 catalyst 828 catalytic activity 107, 109

835 catalytic hydrogenation 566 catecholamine secretion 687–8 catecholamines 818 cathepsin B 631 cathepsin K 628–30 C–C bond 595 CD 260–1, 284–5 CD spectra 367–8 CD spectroscopy 291 CD spectrum 253–4 CD28 or CTLA4 801 CD4 mimetic 121–2 CD80 810–2 cDNA sequence 407–8 CDR 113 cecropin A(1–8)-magainin 2(1–12) 269 cellular signaling 218, 220 cellular uptake 782–3 CH/π interaction 649–50 channel formation 57–8 characterization 569 charged peptides 385 chemical features 212–3 chemical ligation 475–6, 512 chemical modification 770 chemical synthesis 21, 23, 581 chemo-enzymatic 610, 612 chemokines 681 chicken crop 714–5 chimeric synthetic peptides 326–7 chiral HPLC 530–1 chitin 376–7 cholecystokinin receptor 190 cholic acid 59–60 choline 603 chorda tympani nerve 193–4 chromogranin A 818 chymotrypsin 649–50 circular dichroism (CD) 85, 232, 303, 373, 716 circulins 480 cis/trans 600 class I major histocompatibility complex 805 classification of amino acids 132 CLEAR resin 584–6 cloning 432 coacervation 772–3 coiled coil 64, 128–31 collagen 376–7, 485–8 combinatorial chemical approach 623–4 combinatorial chemical libraries 141, 143 combinatorial design 141 combinatorial libraries 123, 132, 605 complement C3a 728, 729 compound arrays 141, 143 computer biochemistry 212 condensations 550 conductimetric method 545–6

confocal microscopic analysis 699 conformation 255–8, 260–1, 271, 319–20, 322, 346, 348–9 conformational analysis 195–6, 277 conformational change 64 conformational restriction 207 conformationally constrained dipeptide 47 conformationally constrained amino acid 724–5 conotoxin 282–3 corticotropin-releasing factor 297 coryneform bacteria 462 costimulatory molecule 801 costimulatory signal 810 costimulatory signal 812 coupling reagent 530–2 Creutzfeldt-Jakob disease 330 cross-bandings pattern 376–7 cross-linked ethylene glycol matrix 584 crosslinking 223–5 crustaceans 453 cryptic epitopes 823 crystal structure 313, 631 Cs + ions 316–7 CTLA-4 810 CXC-chemokine 398, 402 CXCR4/fusin inhibitor 398, 401 cyclic depsipeptides 767 cyclic lipodepsipeptide 523 cyclic oligopeptides 92 cyclic peptides 59–60, 88, 218–9, 472, 519, 716–7, 764–6 cyclic RGD peptides 430–1 cyclic tetrapeptides 260–1, 536–7 cyclic ureas 831 cyclin-dependent protein kinases 364 cyclization 92 cyclization reaction 536 cycloguanidine group 521 cyclopropylphenylalanine 207–8 Cynara cardunculus L. 558 Cys thiolate anion 288 Cys-containing oligopeptides 288–90 cystatin C 654–5 cysteine proteases 628, 654 cysteine-rich 432 cystine knot 326 cystine-containing peptides 41–2 cystine-know motif 480 cystine-stabilized α -helix motif (CSH) 235 cytochrome P-450 828 cytostatics 719 cytotoxic T lymphocyte (CTL) 805 cytotoxicity 678–9 D -amino

acid 442–3 600–1 D -Ser 271 6 ( D-Trp )–LHRH 547–8 D-fructose

836 database 132–3 deblocking reagent 579–80 decanoyl-Glu-ONp 78 deep-seated mycoses 697 dehydroalanine 251, 500–1 dehydroaminobutyric acid 498–9 δ opioid agonists 665, 667 deltorphin 119–20 de novo design 78, 107, 110 depsipeptide 284–5 dermaseptins 704, 706 Dermatophagoides farinae 808 dermorphin-dynorphin hybrid analogs 701 Der f 2 808–9 designing new drugs 121–2 desulfation 525–6 desulfation mechanism 370 diagnostic cross peaks 430 diastereoselective synthesis 595 diethyl phosphorocyanidate (DEPC) 523 difluoromethylornithine 678 digestive fate 436 dimer analog 512–3, 775 dimeric novobiocins 210 dimerization 210–1 dimethylphosphinothioic mixed anhydride (Mpt–MA) 607 dioxopiperazine 82–3 dipeptide derivatives 545 dipeptide isosteres 502 diphenyl phosphorazidate (DPPA) 523 disulfide bonds 280–1, 266–7, 286–7, 306, 453–4, 695–6 disulfide oxidation 584 disulfide-bridging 485–6, 488, 512 dithioacetalized phenacyl linker 542 diversity 132–3 DLS 110 DMP851 831 DMSO 41–2 DNA 376–7, 379–80, 813–5 DNA gyrase 210 DNA mimic 335–6 DNA protein 381 DNA synthesis 450–1 DNA topoisomerases 381–2 DNA-binding peptide 385–6 DNA-protein-interactions 141 DOTA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid) 670–1 double-stranded DNA 362 DPDPE 465–6 drug design 1 drug discovery 797 duration of action 674, 676 ( E )-alkene 502 (E)-alkene isosteres 44 echinoderm 419–20

effective hydrophobicity 88–90 elastin 772–3 elastomeric polypeptide–water system 772 elongation of protected segments on solid support 6 encoded libraries 141 end-to-end cyclic peptide 480 Endo-M 607, 610–2 endocytosis 709–10 endothelin 266 energy calculations 82–3 enkephalin 207–8, 465 enkephalin metabolism 689 enterococcus faecalis 215 enthalpy and entropy, retro-inverso isomer 240 enzymatic activity 581 enzymatic peptide synthesis 527, 534 enzymatic protecting group 603–4 enzymatic stability 701 enzymatic synthesis 545 enzyme inhibition 649–50 enzyme-linked immunosorbent assay (ELISA) 813 epidermal growth factor receptor 200 epitopes 784–6, 803 equilibrium ultracentrifugal analyses 303 ErbB2 receptor 210 ESF-1 457–8 ESR 563–4 eumenine mastoparan-AF 434–5 eumenine mastoparan-ER 434 evolutionary origin 360–1 exocyclic peptidomimetics 61–3 extended conformation 251–4 extra-thermodynamic effects 240, 244 farnesyltransferase 764–5 fibrillogenesis 376–7 fibroblast 450–1 Fischer’s octadecapeptide 253–4 FK-506 803–4 flanking residues 820–1 flip-flop 707–8 fluorescein isothiocyanate (FITC)-labeled albumin 699 fluorescence 57–8, 85, 373–5, 654 fluorescence spectroscopic methods 277 fluorine-containing amino acids 47 Fmoc solid phase peptide synthesis 516 Fmoc solid-phase 193 Fmoc SPPS 579–80 Fmoc/Bu t solid phase synthesis 542 folding 286–7, 306–7, 319–20 folding pathway 297–8 foot-and-mouth disease virus 784–5 four α-helix bundle structure 76 FT-IR absorption 249, 251, 253

837 function groups 560–1 fungi 457 G protein 294, 346, 348–9 G protein-coupled membrane receptor 388 G protein-coupled receptor 205 G-protein receptors 326, 329 Gabriel reaction 47 galactose 709–10 gels 73 gene 357, 360–1 gene therapeutic medicine 335–6 gene transfer 709–10, 722–3 genetic diagnostics 335–7 genomes 825 GH secretagogues 411 glial fibrillary acidic protein 637 glucagon 205–6 gluten 728–9 Gly-peptide linkage 154 glycation 600 glycomodification 587 glycopeptide 607–8, 610, 612, 615–6 glycopeptide derivatives 595 glycophosphopeptides 603 glycoprotein hormones 326 glycosylated amino acids 587, 589–l GPCR 174, 177–8 Gπ Ib/IIIa 144, 623, 625 gramicidin A 54–5, 316–7 gramicidin S 775 graphite-like layers 251–2 Grb2-SH2 domain 218, 220 growth arrest assay 467 guanylation 521 guanylin 324 guanylyl cyclase 306 guanylyl cyclase (GC) C 221 guanylyl cyclase activating peptide 306 gurmarin 193–4 gut motility 444–5 h-FK-506 binding protein 12(h-FKBP12) 803 hair-pin turn structure 288–9 HATU 536–7 heat-denaturation 383 heat-stable enterotoxin (STa) 221 hEGF 286–7 helical structure 385–6 helices 828 helix bundle 297–8 helix peptide 52 helix-forming tendencies 232, 234 heme 110–2 heme proteins 80 hemolysis (hemolytic activity) 88–90, 269–70, 687, 770 heparin-binding neurotrophic factor 552

hepatoma 709–10 heterocycles 123 heterotetramers 303–4 heterotrimers 485–8 hexamethyleneimine 579 hexapeptide analogs 701–2 HF treatment 615–6 high enantiomeric purity 566–7 High TFMSA 507–8 higher through-put screening 134–5 Hirudin P6 615–6 histone 379–80 HIV 652–3 HIV protease 475–6, 478, 831 HIV protease inhibitors 475–8, 652 HIV-1 121, 141–3, 439, 441, 512–3 HIV-1 enhancer binding 141 HIV-1 PR inhibitor 439, 644 HIV-1 protease inhibitors 660, 662 HIV-cell fusion 424 Hmb 733–4 HMFS 490–3 homology modelling 326 hormone-receptor (H-R) binding sites 326 HPLC 421–2 hPTH 711–2 hPTH(1–34) analogues 711 hsst3 receptor 195–6 human CCK-39 525–6 human CgA (344–374) 818 human growth hormone (hGH) 1 human immunodeficiency virus type 1 141 human p53 367 human pleiotrophin 552 hybrid 455–6 hybridization 335–8 hydantoins 47–8 hydrolysis 154–5 hydrolysis of p-nitrophenyl esters 78 hydrophobic interactions 232, 234 hydrophobic moments 291 hydrophobic region 722–3 hydrophobicity 76–7, 240 hyperglycemic hormone 453 ichthyotoxic activity 523 identification 569 IGF-I 223 IgG 816–7 IgM 816–7 immunoglobulin superfamily 797, 799 immunomodulation 784–5 immunophilins 803 immunosuppressants 803 immunotherapy 808–9 ‘impervious’ peptides 411 impurities 569–70 inhibition of uterine motor activity 724 inhibitor 430, 631–2

838 inhibitors of cysteine proteases 634 inhibitory activity 716–8 inhibitory conformation 649–50 inositol phosphate production 190 insect defensin 691 insulin 223–5, 245–8, 674–6 insulin analogue 223, 225 insulin receptor substrate-1 (IRS-1) 200–l insulin secretion 183–4 intact cells 699–700 intermediate 286–7 intermolecular reactions 550 internal conversion 324 intracellular 215 iodine oxidation 542 ion channels 255–6, 291–2, 313 ion conductance 316 ion-channel peptide 67 ion-pairing 370, 372 iron porphyrin 80, 107 isotope-enrichment 346–7 JMV-180 190–1 J protein 373 KNI-764 652–3 κ receptor selectivity 701–2 kuruma prawn 453–4 L -362,823

195–6 dipeptides 257 lactam bridge 128–30 lactoferricin 436 lactoferrin 436–8, 697 laminin 455, 719 layer chromatography 171 lectin 770 Leu-enkephalin 527–8 leucine zipper sequence 67–8 Leydig cell 733 Leydig cell insulin-like peptide 733 ligand-binding 221–2 ligands/targets 15 ligand/receptor interaction 61 ligation of peptides 6 light scattering measurements 772–3 linkers 516 lipid membranes 707 lipomodification 587 lipophilic peptides 704 lipopolysaccharide 448 liposomes 719–20 long terminal repeat (LTR) 141 luciferase activity 190–1 luteinizing hormone-releasing hormone (LHRH) 547 lysine 571 lysine-binding sites (LBS) 642 lysosome-disruptive peptide 722–3 L -aspartyl

M. tuberculosis 820 MA proteins 70 macrophages 448–9 macrotorials 472, 474 magnesium 185–6 major histocompatibility complex (MHC) class II molecules 788 manual synthesis 134 marine natural product 519 mass spectrometry 146, 436 mast cell degranulating peptides 434 mastoparan X 294–6, 346 matrix assisted laser desorption ionization time of flight mass spectrometry (MALDOTOF-MS) 166, 644 matrix metalloproteinase 485 matrix-assisted laser desorption/ionization (MALDI) 146 MCH receptor 226 melanin-concentrating hormone (MCH) 226 melanocortin receptor antagonists 26, 28 melanotropin fragments 678 melatonin 693–4 membrane activity 684 membrane environments 232, 234 membrane fusion 446–7 membrane permeabilization 684–5, 707 membrane perturbation 117–8, 446–7 membrane proteins 57, 255 memory 728–9 mercaptoethylamide peptide 512–3 metabolic activity 687–8 metal binding 64–6 metal-complexing 521 metallopeptide 64–6 methanesulfonic acid 44, 154–5, 157–8, 160–l methanesulfonyl chloride 92–3 methanolysis 571–2 methoxydifluorobate 560 MHC II binding 820 micelle state 297–8 micelles 263–4 micro-calorimetry 654 micro-heterogeneity 152–3 microbial carboxyl proteinases 657 microscopic observations 772–3 microscopy 73 mimetic 430–l mini-hemeprotein 107 mini-proteins 121–2 mixed µ opioid agonist/δ antagonists 665 mixtures 123–6 model peptides 85–6, 362 molecular assemblies 52 molecular chaperone 379 molecular database 797 molecular diversity 171

839 molecular dynamics 282, 767–68 molecular mechanics 98, 100–l molecular recognition 1, 351, 354–5 molluscs 442 molt-inhibiting hormone 453 morphiceptin 600–1 morphology 416 MRSA 691–2 MSI-78 569–70 MTT 777–8 µ opioid agonists 665 Multipin method 495 murine malaria 823 muscarinic toxin I 490, 493 myosuppressive substance 444 myristoyl-peptide 154 N to C direction 605 N,N' -dialkyldiamide-type phosphate protecting groups 613 N-glycosylation 221–2 N-methylamino acids 364 N -succinimidyl carbonates 39–40 N -terminal residue 555 neurite extentional activities 552–3 neuromedin U 714–5 neuropeptide 388, 391, 393, 395, 444–5 neurotoxin 407–8 new molecular chaperone protein 403–5 new sulfur-containing β-amino acid 521 NH···S hydrogen bond 288–90 nicotinic acetylcholine receptor 282 NMR 228, 230, 245–6, 260–1, 280, 282, 286 NMR solution structure 223–5 NMR spectroscopy 309 NMR study 82 nociceptin 198–9, 388, 391, 393–5 non-natural amino acid 59 non-proteinic amino acids 21 non-proteinogenic α-amino acids 761 nonnatural amino acids 34–5 nonpeptide 618 Nsc-amino acids 547–8 nuclear localization signal 70, 699 nuclear magnetic resonance spectroscopy 330 nucleoplasmin 379–80 nucleosome 379, 403–5 obstetric analgesics 665 octadecapeptide 253–4 octreotide 670–2 oligomer 770 oligopeptide 212 ω -Agatoxin-TK (ω-Aga-TK) 271 ω -conotoxin 695 on-line post-column derivatization 152 on-resin cyclization 472–3, 584–5 on-resin disulfide-forming 584 one peptide on one bead 134

one-pot 41 one-pot preparation 560–l opioid 198–9 opioid agonists 139–40 opioid bioassay 726 opioid drug development 665 opioid peptide 665, 726, 728 opioid receptor binding 726 opioid receptors 207–8 OPLC 171, 173 optimization of the N-substituting alkyl groups 613 oral bioavailability 618–9, 621 orally active thrombin inhibitors 639, 641 orally-bioavailable 831 organic solvents 527–8 orientation 52–3 orphanin FQ 198 Oryctes rhinoceros 432 osteoporosis 628 overlapping 12mer peptides 691 overpressured layer chromatography 171 oxidative folding 235, 238 oxidative stress 693 oxidization of 1-alkyl-NAHs 76 oxytocin 185–6, 618, 620 oxytocin antagonists 26–7 oxytocin receptor antagonists 618 p 2 gag peptide 644–5 p-phenylacetoxybenzyloxycarbonyl(PhAcOZ) group 603 pain and analgesia 388 pancreatic islets of Langerhans 183 papain 534, 631–3 parallel synthesis 123 paralytic activity 460–1 paralytic peptide 460–l partially fluorinated amino acid 465 PC12h 777–8 PEG 455–6 pepstatin 657 peptide 421–2, 563–4, 805–6 peptide aldehydes 495 peptide amide 516–7 peptide chemistry 15–6 peptide diastereomers 88 peptide drug 21, 25 peptide family 444–5 peptide libraries 21, 24, 128, 139, 472–4, 788, 791 peptide models for folding 228 peptide nucleic acid 335 peptide radiopharmaceuticals 670 peptide scanning (PEPSCAN) 274 peptide science 15–6, 19 peptide sequencing 146–7, 150 peptide substrates 657 peptide synthesis 188–9, 475, 530, 558–9, 634

840 peptide thioesters 576, 579–81 peptide-ligand interactions 240 peptide-lipid interaction 117 peptides 416, 418, 719–20, 792–5 peptidic and non-peptidic inhibitors 797 peptidoconjugate 21, 24 peptidomimetic 475–7, 628–9, 660, 662 peptidomimetic antagonist 26, 29 peptidomimetics 49, 144, 504 peptosome 54–6 permeabilization of lipid membranes 687 peroxidase 80–1 Personal OPLC 171 PET 670, 672 pGlu-peptide 157–8 pH-dependent β-structure 85 pH-sensitive liposomes 699–700 phage display library 801, 810 phage display system 136 pheromone 215–7 phosphatidylinositol (π ) 3-kinase 200 phospholipid bilayer 85–6, 117 phosphopeptide synthesis 613 phosphopeptides 507 phosphorylated polypeptide 574 phosphorylation 364–6 phosphoserine 581–2, 613–4 phosphotyrosine 613 photoaffinity labelling 223–5 photocrosslinking 226 photoinduced electron transfer 34 photolysis 542–3 physiological functions 792 π –π stacking 649–50 plasma kallikrein selective inhibitor 646–7 plasmid 215 plasmid DNA 722 plasmid pFos-Luc 190 plasminogen activator enhancing activity 552–3 plasmodium 823 platelet aggregation 623–6 polyethylene glycol (PEG) 534 polyphemusin 424 polyphemusin II 427 polyproline II-like structure 446 porphyrin 828–9 porphyrin-binding 113 positional scanning 123–4, 126 positionally adressable libraries 141 post-source decay (PSD) 146 potassium channel blocker 311 precursor protein 460–l preterm labor 618 preventing dimerization of HIV-1 PR 644–5 prion protein 330–2 Pro residue 260 pro-sequence 266–7

process 569–70 processing enzyme 460–1 prodrugs 678–9 programmed cell death 49 proline homologs 364–5 proline-rich 351–3 proline-rich peptides 637 prolonged acting 674, 676 prolyl endopeptidase 637 prosequence 306–7 protease inhibitors 628 protease-catalyzed peptide synthesis 514 protected intermediate 560 protective role 792 protein 319–20 protein engineering 555 protein folding 228, 313, 315 protein interactions 797, 799 protein kinase 174–5 protein synthesis 490 protein-protein interactions 1 proteinomimetic 70 protein–protein interactions 274–5, 370 proteolysis 381–2 proteolytic cleavage 457–8 proteolytic enzyme 657 proteomes 825, 827 prothoracicotropic hormone 166 pseudolysin 545, 546 pseudopeptide 39–40 PTH 1–34 (parathyroid hormone 1–34) 472 pTyr peptide 218 pure plasma kallikrein 646–7 purification by HPLC 6–7 pyrenylalanine 34–5, 37 pyridylaminated sugar chain 166–7 pyroglutamic acid 157 racemic amino acids 527 racemization 530–3 Ranalexin 263–4 RANTES 681–2 RANTES-related peptides 68l–2 rapid equilibrium random bireactant mechanism 545 Ras-farnesyl transferase inhibitor 502–3 rat BKB2 receptor 277 rat CgA(359–389) 818 rat hepatocytes 200 RAW 264.7 448–9 reaper protein 576–7 RecA protein 362–3 receptor 198–9, 202–3, 205–6, 215, 221, 223–5 receptor-based ligand design 342 receptor modeling 342 receptor structures 212 recognition 383–4 recombinant synthesis 711

841 recombinant technique 163 recombination 362–3 red ginseng 421 redox potential 235–6 reducing cytotoxicity 427 regio- and stereo-selective ring-opening reactions 44 regioselective 235, 237, 485–6, 488 regioselective cleavage 154 relaxant activity 139 relaxin 733–4, 736 repression 141 RES-701-1 764 resin 563–4 retro-inverse peptides 784 retro-inverso 805–6 retro-inverso- isomers 240 retro-KP6 α 313 reverse pharmacology 388 RGD 623 RIA 818–9 RNA polymerase II 603–4 ruminants 733, 736 S -cyanocysteine 550 Saccharomyces cerevisiae 467 SALMFamide 419 salmon 403, 405 salmon nucleoplasmin-like protein (SNP) 403, 405–6 salt bridge 266–7 saquinavir 504–5 SBDD 639, 641 scorpion 407 scorpion toxins 121 SDF-1 398–402 sea cucumber 419–20 secondary specificity 657 segment 550–1 segment condensation 514–5, 576 segment condensation reactions in solution 6 SELDI 436–7 selective cleavage 160–1 selective cytotoxicity 704 selective removal 157–8 selenocysteine 235, 238 self-assembled monolayer 52–3 self-assembling ion pore 313 self-assembly 30, 73–4, 110, 112, 351–2 self-association 291–2, 654 self-replication 104 serine 500–l serum albumins 152–3 SH2 domain 605–6 sheets 828 Shmoo assay 467 side-chain 560–1 signal transduction 210 signaling pathway 174–8

silver salt 574, 576 silyl chloride method 510 single-stranded DNA 362, 373, 383 small cell lung cancer 731 small globular protein 57 smooth muscle contraction 714–5 S N1-type deprotection procedure 525 snake venom 555 sodium nitroprusside 777 software 146, 150 solid phase 188 solid phase cyclization 539–40 solid phase peptide synthesis 547 solid phase synthesis 495–6, 525–6, 615–6 solid-state Nuclear Magnetic Resonance 294 solitary wasps 434 solution conformation 465–6 solution structure 282 solution synthesis 552 solvation 563–4 somatostatin 195, 342–5, 510–1, 670, 672 somatostatin analogue 26, 28 somatostatin receptor 342, 345, 670 SPECT 670, 672 spectroscopy 30 sperm nuclei 403–4 Src homology 605 stability 764–6 stepwise affinity aklylation 163 stereoisomers 207–8 stereoselectivity 498 structural classification 212 structural transition 104 structure 442–3 structure-activity relationship 198, 202, 245–6, 646, 660, 662, 714–5, 767 structure-function 240 structure-function relationships 15, 17 structure-inhibition relationships 470 structure/function 326, 329 substance P 610–l substance P antagonists 731 substrate specificity 76, 631, 657 substrate specificity determinant 364 sugar amino acids 587, 591 suicide inhibitor 644 sulfation of tyrosine 370 sulfhydryl group 152 super secondary structures 98–9, 101–2 superagonists of T-cell response 788 superhelices 351 supramolecular assemblies 54 sweet taste 257 sweeteners 193–4, 527 sweetness-suppressing 193 synergism 707 synthesis 144–5, 610–l, 678, 761, 763 synthesis of glycosyl-α-aminoacids 595

842 synthesis of green fluorescent protein (GFP) 6 synthesis of large peptides 6, 9 synthesis of peptides 300 synthesis of proteins 579 synthetic antigens 820 synthetic peptides 816 synthetic studies 521 systemic lupus erythematosus (SLE) 813 S–CH 2 –S bond 539–40 S–S bond 539–40 T cell activation 788, 810, 820 T cell–costimulatory peptides 801 T- tBos 605 T-tropic 398, 400–l T22 398–401, 424–8 tachyplesin 424 tachystatin A 280 tapes 30, 32, 73–4 targeting 719 taurine 693, 694 TBAF · xH 2 O in CCl4/CH 2 Cl 2 (30:70) 539 tendamistat 716 tetramer domain mutants 303 thermal transition 297–8 thermolysin 534–5 thia zip reaction 480, 482, 484 thioester 607–8 thioester fragment condensation 507 thioester method 574 thioether 218 three-dimensional solution structure 309–10 three-dimensional structure 245–7, 330, 333 three-finger motif 360–l thrombin 202–3 thrombin inhibitors 639–40 thymopoietin 188 TMSCl 41–2 TNF-receptor 61–2 TNFα 6 1 – 2 TOFMS 146–7 topographical constraint of side chain groups 26, 29 topoisomerase activity 421–2 topological isomers 324–5 total synthesis 519, 523–4 toxins 271 transduction 205 transfection efficiency 722–3 transforming growth factor- β (TGF-β ) 309 transglycosylation 607

transient observable intermediate 571 translocation 782–3 transmembrane peptides 67 transmissible spongiform encephalopathies 330 trapoxin B 536–7 tri-n-butylamine 152 tri-substituted β-amino acid 623, 625–6 tribromophenylalanine 761–3 trifluoromethanesulfonic acid 510 Trimeresurus flavoviridis serum inhibitor 360 tripeptide 139–40 tripeptide libraries 134 TRNOE 346–9 tropane 144–5 Trp(Hoc) 490, 493 Trp(Mts) derivative 510–l Trp-containing cystine peptide 510–l tumor suppressor p53 303 turns 228–30 TW70 427–8 two-step disulfide forming strategy 480, 483 Tyr(Pen) 490, 493 Tyr(SO 3 H)-containing peptides 525 tyrosine kinase inhibitors 49–50 tyrosine phosphorylation 200–l Ugi reaction 300 ultracentrifugation 367–8 undesirable removal of Acm group 574–5 unnatural benzoic amino acids 92–3 unusual amino acid 132–3, 519–20 uroguanylin 324–5 uteroglobin 351–2 uterotonic activity 185–6 vaccination 823 vaccines 784–5 vasopressin 174 venom basic phospholipase A 2 isozymes 360 vesicles 54, 56 Vif protein 439 voltage-gated potassium channels 255–6 voltage-sensor 255 wasp venoms 434 X-ray crystal structure 316, 639–40 X-ray diffraction 249, 251 Z-isomer 498–9 Z-Lys(Boc)–OMe 571 Zif 268 protein 385