Advances in Carbohydrate Chemistry and Biochemistry, Volume 45

Advances in Carbohydrate Chemistry and Biochemistry Volume 45

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Advances in Carbohydrate Chemistry

and Biochemistry Editors

R. STUART TIPSON DEREK HORTON

Board of Advisors LAURENS ANDERSON STEPHENJ. ANGYAL HANSH. BAER CLINTON E. BALLOU JOHNs. BRIMACOMBE

GUYG. S. DUTTON BENGTLINDBERG HANSPAULSEN NATHAN SHARON ROY L. WHISTLER

Volume 45

ACADEMIC PRESS, INC. Harcourl Brace jovanovich, Publishers

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COPYRIGHT @ 1987 BY ACADEMIC PRESS. INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM O R BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY. RECORDING. OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM. WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

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87 88 89 90

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CONTENTS .............................................

PREFACE .

vii

Burckhardt Helferich. 1887-1982 HERMANN STETTER

..................................

Text . . . . . . . . . . .

1

Francisco Garcia Gonzhlez. 1902-1983 ANToNlO

G6MEZSANCHEZ A N D JOSE FERNANDEZBOLAAOS

Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7

F.a.b.-Mass Spectrometry of Carbohydrates

ANNEDELL I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. F.a.b.-Mass Spectrometry: Theory and Practice . . . . . . . . . . . . . . . . . . . . . 111. F.a.b.-M.s. of High-Molecular-Weight Samples . . . . . . . . . . . . . . . . . . . . . . IV . Interpretation of F.a. b.-Mass Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Structure Assignment by F.a.b.-M.s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Future Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 24 34 41 45 54 71

The Circular Dichroism of Carbohydrates

W . CURTISJOHNSON. JR. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1. Measuring the Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill . Unsubstituted Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1v. Substituted Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73 76 78 92

Proton Spin-Lattice Relaxation Rates in the Structural Analysis of Carbohydrate Molecules in Solution PHOTlS DAISA N D ARTHURs. PERLIN

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. Spin-Lattice Relaxation Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Stereochemical Implications of Relaxation Rates . . . . . . . . . . . . . . . . . . . . . V . Limitations and Relative Merits of Relaxation Methods . . . . . . . . . . . . . . . .

125 128 138 147 163

CONTENTS

vi

"C-Nuclear Magnetic Resonance-Spectral Studies of Labeled Glycophorins

KILIANDILL 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . General Background Information about Glycophorins . . . . . . . . . . . . . . . . . 111. Labeling Studies of Glycophorin A by Way of the Reductive [ ' T I Methylation Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Labeling Studies of Glycophorin B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Conclusions and Prognosis for Further Studies . . . . . . . . . . . . . . . . . . . . .

.

170 172 175 195 197

The Chemistry and Biochemistry of the Sweetness of Sugars

CHEAYGKUAILEE 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Stereochemistry of Sweetness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Bitterness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Biochemistry of Sweetness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Methodology of Measurement of Sweet Taste . . . . . . . . . . . . . . . . . . . . . . .

199 201 310

AUTHORINDEXF O R V O L U M E 4 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECT INDEX FOR VOLUME 45 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C1IMUI.ATIVE AUTHORINDEX FOR VOLUMES 41-45 . . . . . . . . . . . . . . . . . . . . . . . . . CUMUI.ATIVE SURJECT INDEX FOH VOLUMES 41-45 . . . . . . . . . . . . . . . . . . . . . . . . .

353 369 394 396

325 349

PREFACE This volume pays tribute to two carbohydrate pioneers. H. Stetter highlights the life and work of Burckhardt Helferich, one of the last of Emil Fischer’s protCgCs and a pioneer in protective-group strategy and glycosidic coupling. The scientific career of Francisco Garcia Gonztilez, presented by his students, A. G6mez-Stinchez and J. Ferntindez-Bolaiios (Seville) provides a sensitive account of the contributions of Spain’s leading carbohydrate chemist whose work emphasized especially the reactions of monosaccharides that generate aromatic heterocycles. In line with the policy of Advances to provide periodic coverage of major developments in physical methodology for the study of carbohydrates, A. Dell (London) here surveys the use of fast-atom-bombardment mass spectrometry in application to carbohydrates. This technique has achieved rapid prominence as the “soft” ionization technique of choice for structural investigation of complex carbohydrate sequences in biological samples. The author’s extensive personal involvement in this field makes her chapter a critical, state-of-the-art overview for the specialist, as well as a valuable primer for the reader unfamiliar with this technique. In contrast to the rapid rise of f.a.b.-mass spectrometry, the use of circular dichroism in the carbohydrate field, here surveyed by W. Curtis Johnson, Jr., (Corvallis, Oregon), has evolved more slowly and been less widely appreciated. Instrumental limitations have hampered the routine use of circular dichroism; most sugars are transparent in the 1000-190-nm wavelength region of c.d. instruments, and the technique has thus invited less use for them than for nucleic acids and proteins, which show strong absorption in this region. However, as Johnson points out, c.d. is a potentially poweful tool for investigation of the stereochemistry of monosaccharides, of intersaccharide linkages, and most importantly, of the secondary structure of polysaccharides. Many valuable applications of this technique evidently lie in the future. The significance of n.m.r. spectroscopy for structural elucidation of carbohydrates can scarcely be underestimated, and the field has become vast with ramifications of specialized techniques. Although chemical shifts and spin couplings of individual nuclei constitute the primary data for most n.m.r.-spectral analyses, other n.m.r. parameters may provide important additional data. I? Dais and A. S. Perlin (Montreal) here discuss the measurement of proton spinlattice relaxation rates. The authors present the basic theory concerning spin-lattice relaxation, explain how reliable data may be determined, and demonstrate how these rates can be correlated with stereospecific dependencies, especially regarding the estimation of interproton distances and the implications of these values in the interpretation of sugar conformations. Specific applications of carbon-13 n.m.r. spectroscopy to the glycophorins, an important family of glycoproteins present in the human erythrocyte membrane, are discussed by K. Dill (Clemson), who demonstrates the value of ”C-n.m.r. spectra for the structural mapping of glycoproteins. vii

viii

PREFACE

Finally, C. K. Lee (Singapore) contributes an extensive article surveying the chemistry and biochemistry of the phenomenon of sweet taste. The sweetness of sugars has been of interest since ancient times, but even now a total understanding of this gustatory response remains elusive. The editors note with regret the passing of Laszl6 Mester on February 23, 1986. Mester worked extensively on hydrazine derivatives of sugars, and he contributed a notable article on the formazan reaction in Volume 13 of this series. Kensington, Maryland Columbus, Ohio August, 1987

R . STUARTTIPSON DEREKHORTON

Advances in Carbohydrate Chemistry and Biochemistry Volume 45

1887-1982

1902-1983

BURCKHARDT HELFERICH

1887-1982

On July 5, 1982, Burckhardt Helferich died in Bonn shortly after his 95th birthday. With his death, we have lost one of the last protagonists of the classical era of carbohydrate chemistry. The son of a Professor of Surgery at the University of Greifswald, Privy Councillor Heinrich Helferich, and his wife, Natalie, Burckhardt Helferich was born on June 10, 1887, in Greifswald. He went to the classical “Gymnasium,” first in Greifswald, and, from 1899, in G e l , where he obtained his “Abitur” leaving-certificate in 1906. He then began the study of geology at the University of Lausanne, being interrupted after only one semester by his call-up for military service. In the autumn of 1907, he started to study chemistry at the Institute chaired by Adolf von Baeyer at the University of Munich, where he passed the first “Verband’s” examination after three semesters. In 1909, he continued his studies at the University of Berlin, gaining his doctorate in 1911 under Emil Fischer. The subject of his doctoral dissertation was “Syntheses. of Certain New Glucosides.” The tremendous personality of Emil Fischer left its mark on Helferich for the rest of his life. In conversation with Helferich, one was often aware of the great veneration he always felt for his tutor and mentor. After his doctoral graduation, Helferich became Emil Fischer’s personal assistant for two years, and, from 1913 onwards, a teaching assistant. Together with Fischer, he published a series of papers on glycoside synthesis during this period. His scientific career was then interrupted by the First World War, in which he served as an officer throughout. In 1919, he resumed his position as assistant in the Berlin Institute and, in 1920, obtained his “Habilitation” with a thesis on the ring-chain tautomerism of y- and 6-hydroxyaldehydes. In 1922, Helferich was called to the position of Departmental Head at the Kaiser Wilhelm Institute for Fibre Chemistry in Berlin-Dahlem. However, he never actually occupied this position, for, in the autumn of that year, he accepted a personal chair in organic chemistry at the University of Frankfurt in the Institute headed by Julius von Braun. 1

Copyright 0 1987 by Academic Ress, Inc. All rights of reproduction in any form reserved.

2

HERMANN STEITER

In 1922, Helferich married Hildegard Kohlrautz. Five children were born of this very happy and harmonious marriage. In the spring of 1925, Helferich accepted the offer of the Chair in Chemistry and the Directorship of the Chemical Institute at the University of Greifswald, becoming the successor to Pummerer. In the summer semester of 1930, he accepted the offer of the Chairmanship of the Chemical Institute at Leipzig as the successor to Hantzsch. The outbreak of the Second World War in 1939 made work at Leipzig extremely difficult. Many students and postgraduates were called up for military service. In the further course of the war, the Institute suffered much damage from bombing. In spite of all these difficulties, Helferich attempted to maintain the functions of the Institute until, finally, in 1945, after 15 very successful years, he was evacuated to the Western Zone by the American forces of occupation. In 1945, Helferich began work as a guest professor at the University of Bonn, where, in 1947, he accepted the offer of the Chair of Chemistry and Directorship of the Bonn Chemical Institute as the successor to Paul Pfeiffer. Although the Bonn Institute had also suffered much war damage, Helferich, with his energy and enthusiasm, was able, within a short space of time, to create the necessary conditions for a resumption of teaching and research. A great personal tragedy for Helferich was the loss of his only son in the war, to be followed by the premature death of his beloved wife. During the course of his academic career, Helferich occupied many important offices. In the academic year 1951-1952, he was Dean of the Faculty of Mathematics and Natural Sciences, and, in the academic year 1954- 1955, Vice-Chancellor of the Rhenish Friedrich Wilhelm University of Bonn. From 1953-1955, he was a member of the council of the Gesellschaft Deutscher Chemiker, and was president of this society from 1956 to 1957. He made a major contribution to the rapid restoration of the scientific and personal links with foreign chemists that had been broken by the war. In 1951, he received the Emil Fischer Medal of the Gesellschaft Deutscher Chemiker, and in 1957, the “Grosse Verdienstkreuz” (Grand Service Cross) of the Federal Republic of Germany. The Technische Hochschule in Stuttgart conferred upon him the honorary doctorate of Dr. ing. h.c. The Saxon Academy of Science and the Leopoldina in Halle, East Germany, elected him an honorary member. His scientific work, which found expression in 328 publications and 16 patents, is characterized by originality and a comprehensive command of experimental method. In his first independent work, Helferich prepared y-hydroxyvaleraldehyde (4-hydroxypentanal) by reduction and ozonolysis of methylheptenone, readily available from citral by a retro-aldol reaction. He was able to show that, similarly to the saccharides, this hydroxyaldehyde exists in

OBITUARY-BURCKHARDT

HELFERICH

3

the cyclic-hemiacetal form. He also observed the same behavior with Shydroxyaldehydes. From these experimental results, Helferich drew a most important conclusion for that period, namely, that, in cyclic-hemiacetal formation by saccharides, the pyranose form must also be considered, as well as the furanose form. Up to that time, chemists had assumed that the cyclic forms of the saccharides existed exclusively in the 1,Cring (furanose) form. Some time later, Haworth and others proved that the majority of carbohydrates adopt the pyranose forms. The results on cyclic-hemiacetal formation by hydroxyaldehydes were communicated in eight papers.' Formation of a cyclic hemiaminal was observed with the corresponding acetamidoalde hydes.* Helferich's work on ethers of triphenylmethanol began as early as his time in Berlin.3 During the Frankfurt period, Helferich showed that use of the triphenylmethyl (trityl) group permits the specific etherification of terminal hydroxyl groups. As cleavage of the trityl group is achieved under mild conditions, this has become one of the most important and specific protecting groups in carbohydrate c h e m i ~ t r yHelferich .~ also extended the use of the trityl protecting group to hydroxy carboxylic acids and amino acids.' This protecting group still plays a major role in peptide synthesis. The use of the trityl protecting group permitted Helferich to carry out the first targeted synthesis of a disaccharide glycoside6 and, finally, that of a free disaccharide, namely, gentiobiose. This work, coauthored with Karl Bauerlein and Friedrich Wiegand,7 was published in 1926, and is still regarded as one of the milestones in carbohydrate chemistry. Originally, the trityl group was removed by hydrogen chloride in methanol. As the was acetyl group at 0 - 1 of the 1,2,3,4-tetra-0-acetyl-6-0-trityl-~-glucose also cleaved under these conditions, an alternative route was chosen, using 2,3,4-tri-O-benzoyl-6-0-trityl-~-glucosyl fluoride. Subsequently, they were able to remove the trityl group very gently by treatment with hydrogen bromide in glacial acetic acid at 0". The route was then open to synthesize (1) B. Helferich, Ber., 52 (1919) 1123-1131, 1800-1812; B. Helferich and 0. Lecher, ibid.,

(2) (3) (4) (5) (6) (7)

54 (1921) 930-935; B. Helferich and M. Gehrke, ibid., 54 (1921) 2640-2647; B. Helferich and T. Malkornes, ibid., 55 (1922) 702-708; B. Helferich and H. Koster, ibid., 56 (1923) 2088-2094; B. Helferich and W. Schafer, ibid., 57 (1924) 1911-1917; B. Helferich and F. A. Fries, ibid., 58 (1925) 1246-1251. B. Helferich and W. Donner, Ber., 53 (1920) 2004-2017. B. Helferich, P. E. Speidel, and W. Toeldte, Ber., 56 (1923) 766-770; B. Helferich, Angew. Chem., 41 (1928) 871-875. B. Helferich and H. Koster, Ber., 57 (1924) 587-591. B. Helferich, L. Moog, and A. Jiinger, Ber., 58 (1925) 872-886. B. Helferich, and J. Becker, Justus Liebigs Ann. Chem., 440 (1924) 1-18. B. Helferich, K. Bauerlein, and F. Wiegand, Justus Liebigs Ann. Chem., 447 (1926) 27-37.

4

HERMANN STETTER

1,2,3,4-tetra-0-acetyl- glucose from 1,2,3 $tetra- 0-acetyl-6- 0-trityl-Dbromide led glucose. Coupling with tetra-0-acetyl-a-D-glucopyranosyl directly to octa-0-acetylgentiobiose. In the following period, numerous diand tri-saccharides were assembled according to the same principle.* During his time at Leipzig, Helferich developed a new procedure for the preparation of phenyl glycosides. This involved the reaction of the peracetate of a reducing sugar with a phenol in the presence of zinc chloride or p-toluenesulfonic acid.' By careful choice of reaction conditions, it is possible to influence the ratio of a and /3 anomers of the corresponding glycosides. The development of a synthesis of ascorbic acid, which was exploited industrially for a time, also occurred during this period." The application of esters of methanesulfonic acid in carbohydrate chemistry were also investigated. A series of significant advantages emerged over the p-toluenesulfonates that had been introduced by Freudenberg." In contrast to the p-toluenesulfonates, it was found possible to introduce several methanesulfonyl groups very readily into a sugar molecule. Numerous halogeno-carbohydrates were accessible by exchange reactions involving the use of methanesulfonates and trityl ethers.I2 The first free fluoro-carbohydrate was also prepared on this basis.I3 The work on the halogeno-carbohydrates also led to the d i s c ~ v e r yof ' ~the so-called "glycoseenes" (1,5-anhydroald-l-enitols). During his Berlin period, Helferich had already begun to examine the glycoside-cleaving enzymes, the glycosidases, and this work was intensified at Leipzig. Particular attention was paid to the emulsin of sweet almonds. Separation of the P-glucosidase from the enzyme mixture was achieved," but this emulsin turned out to be a most complex mixture of enzymes.I6 Much new information was gained about the specificity of the glycosidases, and glycosidases from other sources were also studied."

(8) e.g., B. Helferich and W. Schafer, Jusrus Liebigs Ann. Chem., 450 (1926) 229-236; B. Helferich and H. Rauch, Ber., 59 (1926) 2655-2657; Jusrus Liebigs Ann. Chem., 455 (1927) 168-172. (9) B. Helferich and E. Schmitz-Hillebrecht, Ber., 66 (1933) 378-383. (10) B. Helferich and 0. Peters, Ber., 70 (1937) 465-468; Ger. Pat. 637,448 (29, 10, 1936); Chem. Absrr., 31 (1937) 709'; Br. Pat. 2,068,453 (19,1,1937); Chem. Abstr., 31 (1937) 18259. (11) B. Helferich and A. Gnuchtel, Ber., 71 (1938) 712-718. (12) B. Helferich and M. Vock, Ber., 74 (1941) 1807-1811. (13) B. Helferich and A. Gnuchtel, Ber., 74 (1941) 1035-1039. (14) B. Helferich and E. Himmen, Ber., 61 (1928) 1825-1835. (15) B. Helferich and 0. Lang, J. Prakt. Chem., 132 (1932) 321-334. (16) B. Helferich, Ergeb. Enzymforsch., 7 (1938) 83-104. (17) B. Helfereich, W. Klein, and W. Schafer, Ber., 59 (1926) 79-85; B. Helferich and J. Goerdeler, Ber. Verh. Saechs. Akad. Wiss.Leipzig Math. Phys. K l . , 92 (1940) 75-106.

OBITUARY-BURCKHARDT

HELFERICH

5

During the Bonn period, the work on glycoside and oligosaccharide syntheses was further developed. Replacement of silver oxide by mercuric cyanide in nitromethane constituted a major step forward in the reaction of acetohalogeno-carbohydrates." In this way, it was possible to obtain mainly the a-glycosides. Remarkable progress was also made in the synthesis of N-glycosyl compounds. For example, penta- 0-acetyl-D-glucose was observed to furnish with benzylamine an adduct that was converted into 2,3,4,6-tetra-O-acetyl-~-glucose on exposure to acid. At higher temperatures, the adduct formed tetra-0-acetyl-N-benzyl-D-glucosylamine, from which tetra-0-acetyl-D-glucosylamine was obtained upon catalytic hydrogenoly~is.'~ In the case of 1-amino-1-phenylethane, the reaction also makes possible the separation of racemates as only the dextrorotatory form furnishes a well crystallizing adduct.20 A specific protecting group was discovered for the 1,2-hydroxyl groups in carbohydrates. The protecting group is prepared by cycloaddition of phenanthrenequinone to a glycal, to afford a dioxene. Disaccharide syntheses were carried out with the aid of this protecting group, which is removed by ozonolysis.21 The first peptide syntheses were also initiated in Bonn.22In the later years of his research activity, Helferich concentrated particularly on the chemistry of the sulfonamides. The chemistry of the sultams was of particular interest to him.23 As a spin-off from this work he developed a psychotropic drug, namely, Ospolot. His work on enzymes was continued during the Bonn period. Studies on acid phosphatases were carried out that made a major contribution to our knowledge of these enzymes.24The isolation of a crystalline P-glucosidase from the emulsin of sweet almonds may be regarded as the crowning achievement of his work on glyco~idases.~~ It is not possible to give here a complete appreciation of his research results, impressive as they are in their abundance and scope. In many (18) B. Helferich and K. Wedemeyer, Justus Liebigs Ann. Chem., 563 (1948) 139-145; Chem. Ber., 83 (1950) 538-540. (19) B. Helferich and W. Portz, Chem. Ber., 86 (1953) 604-612. (20) B. Helferich and W. Portz, Chem. Ber., 86 (1953) 1034-1035. (21) B. Helferich and E. von Gross, Chem. Ber., 85 (1952) 531-535. (22) B. Helferich, P. Schellenberg, and J. Ullrich, Chem. Ber., 90 (1957) 700-71 1; B. Helferich and H. Boshagen, ibid., 92 (1959) 2813-2827. (23) B. Yelferich and K. G . Kleb, Justus Liebigs Ann. Chem., 635 (1960) 91-96; B. Helferich, R. Dhein, K. Geist, H. Jiinger, and D. Wiehle, ibid., 646 (1961) 32-44; 45-48. (24) B. Helferich and H. Stetter, Jusrus Liebigs Ann. Chem., 558 (1947) 234-241; 560 (1948) 191-200; Naturwissenschaften, 34 (1947) 278-279. (25) B. Helferich and T. Kleinschmidt, Hoppe-Seyler's 2.Physiol. Chem., 348 (1967) 753-758.

6

HERMANN STETTER

instances, they went far beyond the realms of carbohydrate chemistry, as is shown by a series of papers on inorganic topics.26 Helferich was active in research to almost the very end of his life. His love for experimentation and his enthusiasm for research were the driving forces for his extremely successful scientific work. He also had a supreme gift for infecting his students with his own enthusiasm for chemistry. His lectures, with their masterly experimental demonstrations, were some of the most popular in chemistry. His great personal interest in research was also demonstrated by his twice daily visits to the laboratory benches of his Diploma and Ph.D. students. All who worked with him were impressed by his personality. The benefit of his advice and generous practical assistance was always available to his students and coworkers, and he was an example to them at all times. His students have always regarded it as a special distinction to be able to call themselves Helferich students. Helferich was tall and slender, with blue eyes and darkish-blond hair, and was a very sociable person. An enthusiastic participant in the festivities of the Institute together with his students and colleagues, he also enjoyed the Carnival celebrations, which play such a prominent role in Bonn. He was particularly fond of playing skittles (ninepins) with his Ph.D. students. With Helferich's death, we have lost a great scientist and teacher who had a substantial influence on developments in chemistry. His memory will remain fresh with all those who had the good fortune to be his students and colleagues. The literature references are a selection of his most important publications. A complete list of Helferich's 328 publications and 16 patents is to be found in Chem. Ber., 118 (1985) viii-xix. HERMANN STETTER

(26) e.g., B. Helferich and K. Lang, 2.Anorg. Allg. Chem., 263 (1950) 169-174.

FRANCISCO C A R C ~ AGONZALEZ

1902-1983

The death of Francisco Garcia Gonzilez, Emeritus Professor of Organic Chemistry at the University of Seville, Spain, deprived carbohydrate chemistry of a long-lived and enthusiastic researcher. Professor Garcia Gonzllez was a pioneer in carbohydrate research in Spain, and a leader for many years of an active school of research that has now spread to several universities and research centers in that country. Don Francisco, as he was affectionately called by his students and associates, was born on July 22, 1902, in Fuente Vaqueros, a small village situated in the middle of a rich table-land west of Granada. This is one of the most beautiful areas of Southern Spain, from which Don Francisco, in his boyhood, could see the white city of Granada dominated by the red walls and towers of the Arab palace of Alhambra, and behind, the impressive blue, snow-capped mountains of Sierra Nevada. He belonged to a close-knit family of prosperous farmers, and at one time, his father, Francisco Garcia Rodriguez, was secretary of the village council. His early years were spent in the midst of a large and lively group of relatives which included his great-uncle Baldomero Garcia, a bohemian folk-poet and flamenco-singer of local fame, and his first cousins Federico Garcia Lorca, who was to become of the greatest Spanish poets and playwrights of the present century, and Francisco Garcia Lorca, a future diplomat and Professor of Spanish Literature at Columbia University in New York City. This pleasant atmosphere made Don Francisco's childhood a happy one, and he often spoke of this period of his life and of his family with great affection and pride. Don Francisco's mother, Carmen Gonzilez, died young, and, to complete his primary education, he was sent away to Almeria to study with a private tutor, and, for his secondary education, he attended the State High School in Milaga. During these years, he looked forward to his periodic returns during the holidays to Fuente Vaqueros, where he worked in the fields, performing a variety of different farming chores, especially at harvest time. From these experiences, he developed a taste for farming, and, perhaps more important, an enduring and deep love for Nature. 7

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

8

A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~~OS

Don Francisco’s generation was the first in his family to receive a higher education. In 1920, he entered the University of Granada, where he graduated with degrees in Chemistry and Pharmacy in 1925. Upon graduation, he received a scholarship from the same University to do research in organic chemistry under the guidance of Professor Gonzalo Gallas. They studied the addition of hypochlorous acid to a,P-unsaturated ketones, and the displacement reactions of the resulting chlorhydrins with amines. This work was published in Anales de la Sociedad Espaiola de Fisica y Quimica. Granada had at that time a fairly rich cultural and artistic life, especially stimulated by the composer and musician Manuel de Falla, then living in the town, Federico Garcia Lorca, who had already shown signs of his genius, and some other young people of talent who were to become preeminent in Spanish culture. Don Francisco mixed with these circles and from these years, he acquired a genuine taste for music and literature. However, the inadequate research facilities prevailing at the University of Granada at the time convinced him to leave the city and, indeed, Spain. In the summer of 1927, he used his meager savings, earned by private tutoring, to travel to Germany as a tourist and to see what was going on in the field that we now call bio-organic chemistry, a blend of organic chemistry and biochemistry which was particularly appealing to him. After he had spent a short period in Dortmund, learning the rudiments of the German language, he moved to Berlin, where he visited the Chemical Institute of the University and persuaded Professor Heinz Ohle to take him as a student. The research effort to elucidate the mechanism of glycolysis and alcohol fermentation was at a peak in Germany at that time, and, in order to gain some insight into these processes, Ohle had undertaken a systematic investigation of the chemical oxidation of some 0-protected sugar esters. The study of these model systems had led him to suggest that the hexose undergoing the breakdown into two three-carbon compounds was a D-fructose phosphate. Crystalline D-fructose phosphates were needed in order to continue these quantitative oxidation experiments, and Garcia Gonztilez was given the task of studying the phosphorylation of 2,3:4,5-di-O-isopropylidene-P-~fructopyranose. He succeeded in obtaining crystalline tris(2,3:4,5-di-Oisopropylidene-P-D-fructopyranose-1-yl) phosphate, and, by hydrolysis of this triester with sodium hydroxide, the crystalline sodium salt of bis(2,3:4,5di-0-isopropylidene-P-D-fructopyranose-1-yl) phosphate. He also found that the permanganate oxidation of this salt takes place with concomitant, benzilic acid rearrangement, to yield (2-carboxy-2-deoxy-3,4- O-isopropylidene-L-threo-pentonic acid) 5-phosphate; this compound is unstable and is broken down in dilute acid solution into methylglyoxal, glycolic acid, and carbon dioxide.

OBITUARY-FRANCISCO G A R C ~ AGONZALEZ

9

In order to establish the role of the phosphoric ester function in the oxidative splitting of the sugar, he also studied the oxidations of other D-fructose derivatives having, at C-1, polar groups other than phosphate, and established the structures of the products. Likewise, he investigated the activating capacity of an ester group at C-3 of a hexose; for example, he 3found that the oxidation of 1,2-O-isopropylidene-a-~-glucofuranose sulfate gave (1,2-0-isopropylidene-a-D-xylofuranuronicacid) 3-sulfate. These studies were described in three articles coauthored with H. Ohle, published in 1931 issues of the Berichte. During the time that Garcia Gonzilez spent in Ohle’s laboratory, he had a grant from the “Notgemeinschaft der Deutschen Wissenschaft,” and, subsequently, another one from the “Junta de Ampliaci6n de Estudios e Investigaciones Cientificas,” which was at that time the Spanish Government agency to promote scientific research. The latter grant was fairly generous, and, at the going rates of exchange, allowed him to concentrate on his work and to pursue a less ascetic living style. The four years spent in Berlin were decisive in Garcia Gonzilez’s career. His collaboration with Ohle, and the scientific atmosphere prevailing at the Chemical Institute, where such notables as E. Bergmann, H. Pringsheim, and H. 0. L. Fischer’ were still working, made him devoted to carbohydrate chemistry, and this remained his main field of research during the rest of his life. These years were also important from the personal point of view. The German capital was at that time a pole of attraction to many young scientists and artists, people he loved to mix with. He became a close friend of the Hungarian L k l 6 Vargha? then a student of Ohle’s, and shared a room for a period with Francisco Ayala, another young man from Granada, who later became a well-known novelist and taught Spanish Literature at Princeton University, U.S.A. The devaluation of the Spanish peseta in 1931 made him a pauper overnight, and in order to survive, he had to take odd jobs, such as acting as an extra in a play directed by the famous Max Reinhardt at the Deutsches Theater. He finally returned to Spain at the end of 1931. With the experimental results accumulated during his stay in Berlin, Garcia Gonzilez prepared two doctoral dissertations, entitled “New Crystalline Phosphoric Esters of D-Fructose” and “Tests on Some Assumed Phases of Alcoholic Fermentation,” which he presented in order to receive his doctorates in Chemistry and in Pharmacy, respectively, at the University of Madrid in 1932. Armed with these two degrees, he decided to pursue an academic career in his own country. His early training in a provincial (1) For an obituary, see Adu. Carbohydr. Chem., 17 (1962) 1-14. (2) For an obituary, see Adu. Carbohydr. Chem. Biochern., 28 (1973) 1-10.

10

A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~OS

university, and his long stay abroad, had kept Garcia Gonzilez out of touch with the scientific life in Spain and, particularly, the influential academic circles of Madrid. Fortunately, his work on sugar phosphates and his strong personality impressed a distinguished organic chemist, A. Madinaveitia, who was Professor of Organic Chemistry of the Faculty of Pharmacy at the University of Madrid. Madinaveitia acted as patron of Garcia Gonzilez’s doctoral theses and, more important, gave him the opportunity to work at the National Institute of Physics and Chemistry, recently created in Madrid by the Junta de Ampliacidn de Estudios e Investigaciones Cientificas with the support of the Rockefeller Foundation. The new Institute was quite well equipped, and a select group of young people had gathered there to work under the direction of such internationally reputed scientists as Blas Cabrera (in spectroscopy and electromagnetism) and Enrique Moles (in physical chemistry). Madinaveitia, who was in charge of the Organic Chemistry Section, was mainly interested in the chemistry of natural products and of compounds of pharmaceutical interest, but, instead of integrating Garcia Gonzilez into his research team, he encouraged him to develop his own ideas. Two subjects were appealing to Garcia Gonzilez. The first was the synthesis of the newly discovered vitamin C, but after discussing the project with Madinaveitia, they decided not to investigate it, because it was “too competitive,” and it was known that some strong research teams abroad were working on it. The second subject was the interaction between sugars and ketonic compounds, responsible for the antiketogenic action of the former. Shaff er (1921) had suggested that D-glucose may be antiketogenenic in human metabolism, because it combines with acetoacetic acid, or other ketonic molecules, to form compounds that are more readily oxidized than the unchanged ketonic substances. To test this hypothesis, West ( 1927) caused D-glucose to react with ethyl acetoacetate, and obtained a beautifully crystalline compound that he tentatively formulated as a cyclopropene derivative. To explain the properties of this compound in acid solution, West considered that it was in equilibrium with a 2-C-(glycosyl)acetoacetic ester. Intrigued by these unusual formulations, Garcia Gonzilez set out to study the new compounds, and demonstrated that the condensate of D-glucose and ethyl acetoacetate has the furan structure 1. This compound and its parent acid /COICIHS HC-C

II

H H H O C HOH2C-C-C-C/ ‘O’ HOOH H 1

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OBITUARY-FRANCISCO

G A R C ~ AGONZALEZ

11

readily undergo a dehydration reaction; he investigated the structure of the resulting products, and proposed a tentative formulation that he corrected in subsequent work. Garcia Gonzllez also obtained a compound, tentatively formulated as the pyrrole derivative isologous to the furan 1, by the reaction with ethyl acetoacetate. This compound had of 2-amino-2-deoxy-~-glucose been prepared, probably in an impure form, by H. Pauly and E. Ludwig (1922), who hypothesized that the natural pyrrole pigments could be formed in vivo by reaction of amino sugars with a physiological 1,3-dicarbonyl compound. This work was published in the Spanish Andes in 1934. Don Francisco spent two years at the National Institute of Physics and Chemistry, and during that time, he had a scholarship from the University of Madrid. As a consequence of his research achievements, Garcia Gonzllez was elected to the Chair of Organic Chemistry at the University of La Laguna in Tenerife (Canary Islands) at the end of 1934. He spent almost two years there, and he devoted most of this time to organizing a modest research laboratory. There he got his first collaborators (T. Quintero and R. Trujillo), and with their help, he pursued his researches. They obtained evidence of the pyrrole structure of the reaction product of 2-amino-2-deoxy-~-glucose with ethyl acetoacetate, and developed a simple procedure by which to prepare 0-isopropylidene derivatives of D-gluconic acid, starting from its calcium salt. In June of 1936, Don Francisco was appointed Professor of Chemistry of the Medical School of the University of Seville, in CBdiz. His return to Southern Spain, to which he had been looking forward, was intended to be followed by his marriage, planned for that summer, to Amalia Olmedo, a pretty, young school-teacher and musician from Granada, but suddenly, the happy panorama changed. Social and political unrest had been growing in Spain during those months, and in July, the Spanish Civil War broke out, bringing tragedy and ruin to the country and to many families. Academic life stopped, and Don Francisco was conscripted as a pharmacist by the Nationalist Army. His already famous cousin Federico Garcia Lorca and other members of the family were, under the most dramatic circumstances, killed in Granada in the first days of the war; others had to rush into exile, and a period of silent mourning and'sorrow followed. This was somehow relieved by his wedding to Dofia Amalia, which at last could be celebrated in May of 1937. This marriage constituted the beginning of a life-long partnership, marked by the deepest mutual devotion and understanding. In the ensuing years, two sons were born to the Garcia Gonzllezes: Francisco (in 1938, who graduated as a chemist and biochemist, and is Professor of Biochemistry at the Technical University of Madrid), and Bernard0 (in 1940, who became a physicist, and teaches as Professor of Physics at the University of Granada).

12

A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA$JOS

With the end of the war, in 1939, the university activities started again, but new difficulties were ahead. Although never involved in politics, Don Francisco had inherited from his family a liberal and open-minded approach to life and society which was out of tune with that prevailing in the official Spain of those days. This attitude and his detachment from the new regime made him suspected of disloyalty, and provoked an official investigation of his possible political activities and connections. This lasted for two years, after which time a resolution from the judge in charge of the investigation cleared him of all charges. His transfer, in 1943, to the Department of Organic Chemistry of the University of Seville, marked the beginning of a quiet and most fruitful period in his career. The university laboratories in Seville were rudimentarily equipped, but this was compensated for by the dedication and enthusiasm of the small group of students that gathered around him. A new research body, the “Consejo Superior de Investigaciones Cientificas,” was created after the war by the new government, and this organization included an Institute of Chemistry (the “Instituto Alonso Barba”) which had sections in different universities. Garcia Gonzilez headed the Organic Chemistry Section of the University of Seville, and this situation enabled him to pursue his studies more systematically, with some degree of financial support. Several lines of research were developed by Garcia Gonzalez and his collaborators in Seville, all of them dealing with the formation of heterocyclic compounds from monosaccharides. One of these lines was the extension of the reaction of ethyl acetoacetate with D-glucose to other 1,3-dicarbonyl compounds, and to other aldoses (pentoses, hexoses, and heptoses) and to ketoses (D-fructose and D-sorbose). The reaction, originally performed by heating the reactants with zinc chloride, or other metallic salts, in a nonaqueous medium, was also shown to occur under “physiological conditions,” that is, in the absence of catalysts, in water at room temperature and neutral pH. A variety of (alditol-1-yl)furans, 2 and 3, having alditolyl 0

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chains of different lengths and configurations, were thus obtained, and their structures were established. Oxidation with the then-new reagents lead tetraacetate or periodic acid gave a series of furanaldehydes, which were subsequently used as starting materials for the synthesis of furylalanines,

OBITUARY-FRANCISCO G A R C ~ AGONZALEZ

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furylpyruvic and furylthiopyruvic acids, and other furan derivatives. Such simple a-hydroxyoxo compounds as D-glyceraldehyde, glycolaldehyde, and 1-hydroxy-2-propanone were shown to react with 1,3-dicarbonyl compounds in the same way, yielding furan derivatives. This is, therefore, a general reaction of a-hydroxyaldehydes and a-hydroxyketones, including the monosaccharides, often referred to as the “Garcia Gonzilez reaction,” which provides one of the simplest procedures for the formation of the furan ring. Efforts were made by Garcia GonzAlez and his coworkers to elucidate the mechanism of this reaction. In one of the working hypotheses, it was considered that the aldehydo form of the sugar and the 1,3-dicarbonyl compound undergo an aldol reaction to yield a 2-C-(alditol-l-yl)-l,3-dicarbony1 compound, which is then dehydrated to form the furan. This hypothesis was supported by the isolation of the aldol-addition product of 2,3-0-isopropylidene-~-glyceraldehyde and ethyl acetoacetate. Acid hydrolysis of this compound set free the hydroxyl groups, with concomitant ring-closure to the anticipated furan. These studies, most of them carried out with the valuable collaboration of F. J. L6pez Aparicio, were reviewed in the article “Reaction of Monosaccharides with beta-Ketonic Esters and Related Substances,” published in this S e r i e ~ . ~ A second main subject of research by Garcia Gonzilez, mainly with the collaboration of A. G6mez-Sinchezywas the reaction of amino sugars with 1,3-dicarbonyl compounds. It was also found to be a general reaction of 2-amino-2-deoxyaldoses, 1-amino-1-deoxyketoses, and their N-monosubstituted derivatives, which produces the (alditol-1-y1)pyrroles 4 and 5, respectively. Using 1,3-~yclohexanediones,this reaction provides an easy 0

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route to 2- and 3-(alditol-l-yl)-4,5,6,7-tetrahydroindol-4-ones and other indoles, As with the non-nitrogenous monosaccharides, the reaction proceeds through an aldol reaction as the first step, but in this case, the isolation of N-(2-acylvinyl) derivatives of amino sugars, such as 6, which could be

(3) F. Garcia Gonzilez, Adu. Carbohydr. Chem., 11 (1956) 97-143:

14

A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~IOS

CHZOH

HOQ

O

H

subsequently transformed into the pyrroles, suggests that the aldol reaction could also be an intramolecular process. Aldosyl- and ketosyl-amines, and the N-monosubstituted derivatives, also react with 1,3-dicarbonyl compounds, to yield the pyrroles 5 and 4, respectively; these products are probably formed through p-( N-glycosylamino) a,p-unsaturated esters or ketones, which, in several instances, were isolated. These studies were reviewed in a second article, “Reactions of Amino Sugars with P-Dicarbonyl Compounds,” coauthored with A. G6mez-SlnchezYpublished in this Series? Many (alditol-1-yl) heterocyclic compounds are dehydrated under very mild conditions. This reaction, first observed by West (1927) in the furoic ester 1, was studied in detail by Garcia Gonzllez and his collaborators. The dehydration product of 1 and of its parent acid, were shown (F. Garcia GonzQlezand C. Sequeiros, 1945; F. Garcia Gonzllez, J. L6pez Aparicio, and A. Vlzquez Roncero, 1948) to be 2-C-erythrofuranosylfurans,whose p-Danomeric configuration and formation mechanism were subsequently established (A. G6mez-Slnchez and A. Rodriguez RoldQn, 1972). The reaction was extended to 2- and 3-(alditol-l-yl)pyrroles (4 and 5), the dehydration of the compounds having a pentitolyl chain giving rise to mixtures of C-glyco-furanosyland -pyranosyl derivatives of the heterocycle. On the other hand, other (alditol-1-yl) heterocyclic compounds are more reluctant to undergo the dehydration reaction. For example, 2-( D-urubinotetritol-1-y1)quinoxalineis dehydrated only by the action of strong acid, producing 2-(2-furyl)quinoxaline and 2-(3-hydroxy-2-furyl)quinoxaline (A. G6mez Sinchez, M. Yruela, and F. Garcia Gonzllez, 1954). Another main subject of research of Garcia Gonzllez, with the collaboration of J. Fernlndez-Bolafios, was the reaction of amino sugars with isothiocyanic acid derivatives. 2-Amino-2-deoxy-~-glucose hydrochloride and potassium thiocyanate were shown to give rise to 4-(~-arubino-tetritol-l(4) F. Garcia Gonzilez and A. GBmez SBnchez, Ado. Carbohydr. Chem., 20 (1965) 303-355.

OBITUARY-FRANCISCO G A R C ~ AGONZALEZ

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yl)-lH-imidazole-2-thiol (7), while the reaction of the amino sugar with alkyl and aryl isothiocyanates produces bicyclic compounds, first considered to be glucopyranoimidazolidine-2-thiones(195 l), but subsequently shown (C. J. Morel et al., 1968; F. Garcia Gonzllez, J. Fernlndez-Bolaiios, et al., 1974) to have structure 8. R’

I

HOCH

I

HCOH

I

HCOH

I

’S

CHiOH 7

8

The reaction of 1-amino-1-deoxyketoses, and their N-alkyl and N-aryl derivatives, with alkyl or aryl isothiocyanates (Huber et al., 1960) was studied in more detail, and new 4 4 alditol- 1-yl)-1-alkyl(aryl)-3-alkyl(ary1)1,3-dihydr0-2H-imidazole-2-thiones were obtained. These compounds were used as starting materials for the synthesis of DL-histidines, ~~-histidine-2thiol, and other imidazole derivatives of biological interest. The results obtained by Garcia Gonzllez and his coworkers attracted interest abroad, and it was a source of great satisfaction for him to receive, in 1953, an invitation from Professor M. L. Wolfrom to write the first of his articles in this Series. A further international recognition as a carbohydrate chemist ensued in 1965, when he was invited to serve as a member of the Editorial Advisory Board of the new international journal Carbohydrate Research. In 1975, he was asked to contribute with a lecture, subsequently p ~ b l i s h e d on , ~ “Synthesis of Polyhydroxyalkyl Heterocycles,” to a Symposium on New Synthetic Methods for Carbohydrates organized by the American Chemical Society to commemorate the 100th anniversary of the Society (New York, 1976). Although Don Francisco was rather reluctant to get involved in the formal activities of academies, he accepted membership in the Royal Academy of Sciences of Spain. He was not an outstanding speaker, but his straightforward manner and direct approach to the subject he was dealing with made (5) F. Garcia Gonzilez, J. Fernindez-Bolafios, and F. J. L6pez Aparicio, ACS Symp. Ser., 39 (1976) 207-226.

16

A. GOMEZ-SANCHEZ AND J. FERNANDEZ-BOLA~OS

his lectures most attractive. As a teacher, he was very conscientious, and conducted his classes in an informal and very relaxed manner which awakened responsiveness in the students. He was able to transmit to his graduate students his interest in the research problem at hand, and shared with them the delights of their experimental successes. His scientific stature made him highly respected; however, his excellent critical sense, which made him speak his mind regardless of the consequences, kept him out of the “corridors of power.” This did not help either his career or the development of his school. It was, therefore, very gratifying to him to see how some of his former students were pursuing research in carbohydrate chemistry in Seville, and in other universities and research centers in Spain, thus assuring the continuity of the task he had initiated. Don Francisco retired from the chair in 1972, but he stayed at the Department for six additional years as Emeritus Professor supervising research work. During his stay in Seville, he went back to farming as a hobby, as he acquired an orange grove on the outskirts of Seville, where he managed the efficient production of bitter oranges, sugar-beets, and potatoes. Although in a comfortable economic situation, he and his wife carried on a simple life-style: he never drove a car, very seldom took a taxi, and walked back and forth to the farm several times a week. He was fond of reading and of classical music and for a time was a member of the board of the Concert Society of Seville. Don Francisco went back to Granada to spend his final years. There, he still kept an interest in a fraction of his father’s land which he had inherited. In 1981, his health visibly declined, and cancer of the intestines was diagnosed. He had to suffer two major operations which were of no avail, and finally he accepted his fate with great fortitude and dignity. Don Francisco spent the following trying months surrounded by his closest relatives, receiving the most devoted care from his wife. He died on November 19, 1983. In recognition of the outstanding services rendered to the University of Seville by Professor Garcia Gonzhlez as an inspiring teacher and investigator, the Government Council of the University decided that the Department of Organic Chemistry that he headed for many years should bear his name. This decision was announced by the Rector of the University during the multitudinous homages paid to Don Francisco on the occasion of the first anniversary of his death, by the University of Seville, and by his former colleagues and students. Don Francisco was a man of many virtues. He was warm and affectionate, and was interested in everything happening around him. He had a quick and penetrating mind, and could perceive almost at first sight the intricacy of a problem or the quality of a person. He was extremely honest, both as

OBITUARY-FRANCISCO G A R C ~ AGONZALEZ

17

a man and as a scientist. It was impossible for all who had the privilege to work with him not to respect, appreciate, and admire him most deeply. ANTCNIOGOMEZ-SANCHEZ JOSE FERNANDEZ-BOLAIQOS*

In addition to those mentioned in the text, Professor Garcia Gonzilez coauthored articles with the following scientists: F. Alcudia Gonzilez, C. Alvirez Gonzilez, F. Ariza Toro, J. Bello GutiCrrez, R. Castro Brzezicki, R. Enriquez Berciano, J. FernPndez Jiminez, J. Fernhndez Garcia-Hierro, J. Fiestas Ros de Ursinos, J. Fuentes Mota, J. Galbis Phrez, J. Gasch G6mez, M. G6mez GuillCn, M. I. Goiii de Rey, A. Heredia Moreno, M. L6pez Artiguez, N. L6pez Partida, R. Maestro Durin, G. Martin Jiminez de la Plata, M. Martin Lomas, D. Martinez Ruiz, M. MenCndez Gallego, S. Muiioz Guerra, M. Ortiz Rizzo, A. Paneque Guerrero, A. M. PCrez de Guzman, M. A. Pradera de Fuentes, M. Repetto JimCnez, L. Rey Romero, J. Rodriguez Gonzhlez, I. Robina Ramfrez, E. Romin Galin, J. Ruiz Cruz, F. Sinchez LaulhC, M.Tena Aldave, and M. Trujillo PCrez-Lanzac.

* The authors express their gratitude to

Professor Francisco Garcia Olmedo, who kindly cooperated in the preparation of this tribute to his father.

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ADVANCES I N CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL. 45

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

BY ANNE DELL Department of Biochemistry. Imperial College of Science and Technology. London SW72A.7, U.K.

I . Introduction ............................. 1. Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . 2. Purpose of This Article ....................... I1. F.a.b.-Mass Spectrometry: Theory and Practice . . . . . . . . . . . . . . 1. Principles of the Technique ..................... 2 . Choice of Supporting Matrix and Matrix Additives . . . . . . . . . . . 3 . Characteristics of F.a.b.-Mass Spectra . . . . . . . . . . . . . . . . . 4 . Sample Purity and the Analysis of Mixtures .............. 5. Choice of Derivatives . . . . . . . . . . . . . . . . . . . . . . . . 6 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. F.a.b.-M.s. of High-Molecular-Weight Samples . . . . . . . . . . . . . . 1. Definition of High Mass . . . . . . . . . . . . . . . . . . . . . . . 2. Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Practical Aspects of Analysis at High Mass . . . . . . . . . . . . . . . 4 . F.a.b. "Mapping" of Permethylated, High-Mass Samples . . . . . . . . . IV. Interpretation of F.a.b.-Mass Spectra . . . . . . . . . . . . . . . . . . . 1. Molecular-Weight Assignment .................... 2. Fragmentation Pathways . . . . . . . . . . . . . . . . . . . . . . . V. Structure Assignment by F.a.b.-M.s. ................... 1. Analysis of Underivatized Samples .................. 2. Analysis of Derivatized Samples ................... 3. Monitoring Chemical and Enzymic Reactions by F.a.b.-M.s. ....... 4. Linkage Assignment . . . . . . . . . . . . . . . . . . . . . . . . . 5. Acyl-Group Location ........................ VI . Applications ............................. 1. Glycolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Glycoproteins ........................... 3. Bacterial Polysaccharides ...................... 4 . Plant Cell-Wall Polysaccharides . . . . . . . . . . . . . . . . . . . . 5. Cyclic Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . 6. Miscellaneous ........................... VII . Future Developments .........................

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Copyright @ 1987 by Academic Ress Inc. All rights of reproduction in any form reserved.

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ANNE DELL

I. INTRODUCTION 1. Historical Perspective In July 1980, at a Symposium on Soft Ionization Biological Mass Spectrometry held at Imperial College, London, the international mass-spectrometric community was first introduced to a new ionization technique for the analysis of involatile substances that was being developed by Barber and his colleagues at the University of Manchester Institute of Science and Technology.' Within a year, the new technique,* which was christened fast atom bombardment (f.a.b.), was revolutionizing mass-spectrometric studies of b i o p ~ l y m e r s . ~The - ~ reasons for this were simple: for the first time, it was possible to obtain long-lasting spectra of high quality from complex, polar molecules with relative ease; furthermore, the new f.a.b. sources were soon fitted to the high-field, mass spectrometers that had been developed some years earlier for high-mass work: and it was immediately apparent that the f.a.b.-high-field magnet combination would permit the analysis of much larger molecules than had previously been amenable to mass spectr~metry.~" Mass spectrometrists who had been using field desorption (f.d.)9 to define the molecular weights of complex carbohydrates and glycoconjugates were immediately attracted to the new technique." The first f.a.b. experiments' had shown that such small, underivatized oligosaccharides as raffinose gave spectra that were remarkably similar to f.d. spectra but which were much longer lasting. Experimentally, f.a.b. promised to be a much less demanding technique than f.d. The latter required the use of fragile wires, upon which R. S. Bordoli, and R. D. Sedgwick, in H. .R.Moms (Ed.), S o t Ionization Biological Mass Spectrometry, Heyden, London, 1981, pp. 137-152. M. Barber, R. S. Bordoli, R. D. Sedgwick, and A. N. Tyler, J. Chem. SOC., Chem. Commun.,(1981) 325-327. H. R. Moms, M. Panico, M. Barber, R. S. Bordoli, R. D. Sedgwick, and A. N. Tyler, Biochem. Biophys. Res. Commun., 101 (1981) 623-631. D. H. Williams, C. Bradley, G. Bojesen, S. Santikam, and L. C. E. Taylor, J. Am. Chem. SOC.,103 (1981) 5700-5704. H. R. Morns, A. Dell, A. T. Etienne, M. Judkins, R. A. McDowell, M. Panico, and G. W. Taylor, Pure Appl. Chem., 54 (1982) 267-279. K. L. Rinehart, Jr., L. A. Gaudioso, M. L. Moore, R. C. Pandey, J. C. Cook, Jr., M. Barber, R. D. Sedgwick, R. S. Bordoli, A. N. Tyler, and B. N. Green, J. Am. Chem.

(1) M. Barber, (2) (3) (4) (5)

(6)

(7) (8) (9) (10)

SOC.,103 (1981) 6517-6520. H. R. Moms, A. Dell, and R. A. McDowell, Biomed. Mass Specrrom., 8 (1981) 463-473. A. Dell and H. R. Moms, Biochem. Biophys. Res. Commun.,106 (1982) 1456-1462. H.-R. Schulten, In?. J. Mass Spectrom. Ion Phys., 32 (1979) 97-283. M. Linsheid, J. D'Angona, A. L. Burlingame, A. Dell, and C. E. Ballou, Proc. Natl. Acad. Sci. USA, 78 (1981) 1471-1475.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

21

samples were coated, and these had a habit of disintegrating in the high fields of the ion source just as the sample was ionizing. No such problems were encountered with f.a.b., because samples were ionized from a robust, metal surface. In the early stages of carbohydrate f.a.b.-m.s. comparative studies of f.d. and f.a.b. revealed that f.a.b. was the preferred technique.",'* A very striking demonstration of the superiority of f.a.b. was provided by data" obtained from a mycobacterial 0-methyl-D-glucose polysaccharide (MGP; l), the structure of which was being investigated by f.d.-m.s. at the time when f.a.b. was introduced. Some ten years earlier, Ballou and coworkers had proposed for MGP a structure that incorporated 18 glycosyl residues and a unit of glyceric acid.I3 Subsequent n.m.r.-spectral experiments had indicated that an additional glucosyl residue might be present, and it was considered possible that f.d. would resolve the problem by defining the molecular weight of MGP. Unfortunately, MGP had proved totally intractable to f.d.-m.s., and experiments on a smaller relative, namely, AGMGP, produced by enzymically removing the first four glycosyl residues from MGP, were equally unsuccessful. Weak f.d. spectra were eventually obtained from a sample of permethylated MGP. Clusters of ions in the region of m/z 4200 provided the first evidence for the presence of two, rather than one, additional glycosyl residues in MGP. For a short time, these results were very impressive, despite the somewhat scrappy nature of the mass-spectral traces, as no other biological compounds had previously given f.d. spectra above mass 4000.The f.d. data were soon, however, to be overshadowed by those afforded by the new f.a.b. technique. The very first time that MGP and AGMGP were introduced into the f.a.b. source of a high-field-magnet, mass spectrometer they afforded spectra of almost unbelievable quality.1','4~15No derivatization or special sample manipulations were necessary in order to produce spectra that were fully countable right u p to the molecular ions, which, for MGP, were present at rn/z3537 [M+Na]+ and 3553 [M+K]+ in the positive mode and at m / z 3513 [M - HI- in the negative mode. These data defined a molecular weight of 3514 for MGP, and showed that it did, indeed, contain 20 glycosyl residues and that one of the additional residues was methylated.

( 1 1 ) L. S. Forsberg, A. Dell, D. J. Walton, and C. E. Ballou, J. Bid. Chem., 257 (1982) 3555-3563. (12) Y. Kushi, S. Handa, H. Kambara, and K. Shizukuishi, J. Biochem. (Tokyo),94 (1983) 1841-1850. (13) C. E. Ballou, Fure Appl. Chem., 53 (1981) 107-112. (14) A. Dell and C. E. Ballou, Biomed. Mass Spectrom., 10 (1983) 50-56. (15) A. Dell and C. E. Ballou, Carbohydr. Rex, 120 (1983) 95-111.

CO,H CHIOH

I--

!

CHIOH

7

CH20Me7

CH,OMe

\

CHIOH

I

/ H

b

V OH

OCH CH,OH I

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

23

The analysis of MGP and AGMGP was a landmark in the development of f.a.b.-m.s. for the determination of carbohydrate structure. The study highlighted both the advantages and disadvantages of f.a.b.-m.s. and was a guide to the strategies most appropriate for fully exploiting the potential of the technique for the study of unknown compounds. On the positive side, the extraordinary quality of the data that could be obtained from such complex molecules encouraged the immediate application of f.a.b.-m.s. to a wide range of biological problems. Thus, there was no significant lag-time while such relatively trivial standard compounds as di- and tri-saccharides were being exhaustively analyzed. On the negative side, the fragment-ion data from both AGMGP and MGP carried a warning message. Superlicially, the results looked very good indeed; cleavages had obviously occurred at every glycosidic linkage, producing abundant “sequence ions” throughout the spectra. On more detailed analysis, however, it became apparent that these were not true sequence-ions, as many were the products of multiple cleavages and could theoretically have arisen from a variety of parent structures. It was clear that f.a.b. would not be a reliable procedure for sequencing unknown oligosaccharides, particularly those that were branched, unless the problem of ambiguous fragmentation-data could be resolved. In later Sections of this Chapter, current solutions to the problems of sequencing by f.a.b.-m.s. are described. As a general rule, unambiguous sequencing requires the study of derivatives. This is somewhat ironical, when it is considered that the major impetus for the original f.a.b. development was the provision of an ionization method that would not require that samples be derivatized. Derivatization does, however, have a lot to offer in the f.a.b.-m.s. of carbohydrates, as will be seen later. 2. Purpose of This Article

The main purposes of this article are to familiarize those carbohydrate chemists who are not specialists in mass spectrometry with the experimental aspects of f.a.b.-m.s., and to give an indication of the types of problems that can now be solved by using the technique. F.a.b.-m.s. currently plays two major roles in carbohydrate-structure analysis. These are ( i ) molecularweight determination, for which it has superseded f.d.-ms., and, to a large extent, chemical ionization,I6 and ( i i ) sequence assignment, where it has very largely replaced direct-probe, electron-impact mass Spectrometry, particularly for high-molecular-weight compounds. On its own, f.a.b.-m.s. cannot solve the complete structure of a carbohydrate, and it should always be incorporated into experimental programs (16) V. N. Reinhold and S. A. Cam, Mass Spectrom. Rev., 2 (1983) 153-221

24

ANNE DELL

that include the well established methods of methylation analysis, enzymic digestion, chemical degradation, and n.m.r. spectroscopy. Many of these procedures have been the subject of recent review^.'^-'^ 11. F.A.B.-MAss SPECTROMETRY: THEORYAND PRACTICE 1. Principles of the Technique

The main of f.a.b.-m.s. are shown schematically in Fig. 1. The hardware consists of ( i ) an atom gun (or ion gun, see later) which is either mounted on the source housing of the mass spectrometer or, if small enough, inside the housing on the source itself, ( i i ) a sample probe to the end of which is attached a small metal target onto which the sample is loaded, and (iii) suitable source-optics for the efficient extraction of ions into the analyzer of the mass spectrometer. In the f.a.b. experiment, an accelerated beam of atoms (or ions, see later) is fired from the gun towards the target, which has been preloaded with a viscous liquid (referred to as the matrix) containing the sample to be analyzed. When the atom beam collides with the matrix, kinetic energy is transferred to the surface molecules, many of which are sputtered out of the liquid into the high vacuum of the ion source. A significant number of these molecules are ionized during the sputtering process. Thus, gas-phase ions are generated without prior volatilization of the sample. The concept of ionization by means of sputtering processes was not new when Barber and his colleagues began their f.a.b. experiments. Secondaryion mass spectrometry (s.i.m.s.) was a well established technique for analyzing metal surfaces:' and it had also been used to produce spectra from solid organic samples coated on metal surfaces.22 In the s.i.m.s. experiment, beams of high-velocity ions effected ionization by sputtering the sample directly from the solid phase. The resulting mass spectra were weak and transient, and s.i.m.s. had never been used as a routine analytical tool. Barber and coworkers made radical changes to the s.i.m.s. technique. They replaced the ion gun with an atom gun, redesigned the ion source to (17) C. C. Sweeley and H. A. Nunez, Annu. Rev. Biochem., 54 (1985) 765-801. (18) J. F. G. Vliegenthart, L. Dorland, and H. van Halbeek, Adu. Carbohydr. Chem. Biochem., 41 (1983) 209-374. (19) K. Bock and C. Pedersen, Ado. Carbohydr. Chem. Biochem., 41 (1983) 27-66. (20) M.Barber, R. S. Bordoli, G. J. Elliott, D. Sedgwick, and A. N. Tyler, Anal. Chem., 54 (1982) 645A-657A. (21) A. F. Dillon, R. S. Lehrle, and J. C. Robb, Adu. Mass Spectrom., 4 (1968) 477-490. (22) A. Benninghoven, D. Jaspers, and W. Sichtemann, Appl. Phys., 11 (1976) 35-39.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

25

FIG.I.-Schematic Representation of a F.a.b. Source.

make it compatible with high-voltage, double-focusing mass spectrometers of the type used in biopolymer analysis, and finally, and most importantly, they decided to bombard a solution of the sample in glycerol, rather than the solid sample itself. The results were spectacular. F.a.b.-mass spectra lasted for minutes, and sometimes hours, in contrast to seconds in s.i.m.s. experiments. Initially, it was assumed that the use of neutrals rather than ions was an essential factor for producing high-quality spectra. It soon became clear, however, that the glycerol was the real key to the success of f.a.b. When s.i.m.s. experiments were performed with a liquid matrix, the results were identical to those obtained23by f.a.b. Herein, no distinction is therefore made between f.a.b. studies and so-called “liquid s.i.m.s.” experiments. Both positive and negative ions are produced during the sputtering process, and either can be recorded by an appropriate choice of instrumental parameters. Positive ions are the result of protonation, [M+ HI+,or cationization, [M + cation]+, whereas negative ions are preponderantly [M - HI-, but can also be formed by the addition of an anion, that is, [ M +anion]-. The type of pseudomolecular ion produced is governed by the chemical nature of the sample and by the composition of the matrix from which it is ionized. 2. Choice of Supporting Matrix and Matrix Additives

The correct choice of matrix is fundamental to successful f.a.b.-m.s., and the solubility of the sample in the matrix is a prime consideration. Glycerol (2) is the matrix most commonly used in f.a.b. experiments, and it is ideal (23) W. Aberth, K. M. Straub, and A. L. Burlingame, Anal. Chem., 54 (1982) 2029-2034.

ANNE DELL

26

for such polar compounds as underivatized carbohydrates and glycopeptides. It is not, however, a suitable matrix for samples that are very hydrophobic, for example, permethylated or peracetylated derivatives (the latter CH,OH

CH,SH

I CHOH I

CHOH

CH,OH

CHzOH

2

3

I I

give f.a.b. spectra from glycerol if the target is heatedz4but this is a procedure that we no longer recommend), or those that are inclined to form aggregates when dissolved in a polar liquid (for example, glycosphingolipids). For this type of compound, 1-thioglycerol (3) is an excellent matrix. 1-Thioglycerol is, unfortunately, considerably more volatile than glycerol and may evaporate completely before the spectra of high-molecular-weight samples have been fully recorded. If this occurs, more 1-thioglycerol may be added to the target to “revive” the sample, and the complete f.a.b. spectrum is then pieced together from several overlapping scans.z5A mixture of glycerol and 1-thioglycerol may be used in order to ensure that the sample does not dry out completely in the ion source.z6 In the author’s laboratory, it has never been found necessary to resort to matrices other than glycerol or 1-thioglycerol for the analysis of saccharides and glycoconjugates. Nevertheless, alternative matrices are often equally effective, and, in some laboratories, they are preferred. The most widely used include tetraethyleneglycol (4) and its higher-molecular-weight relatives, the poly(ethyleneglyc~l)s,~~~~~ and such basic matrices as N, N ’ bis(2-aminoethy1)ethylenediamine (“triethylenetetramine,” 5), 2,2’iminodiethanol (“diethanolamine,” 6), and 2,2’,2”-nitrilotriethanol (“triethanolamine,” 7) (basic matrices are frequently chosen for negative-ion HO-(CH,),O(CH,),O(CHZ)~~(CH,),-OH 4

H,N-(CH,),NH(CH,),NH(CH,),-NH, 5

HO-CH,-CH,-NH-CH,-CH,-OH 6

(24) A. Dell, H. R. M o m s , H. Egge, H. von Nicolai, and G. Strecker, Carbohydr. Res., 115 (1983) 41-52. (25) J. E. Oates, A. Dell, M. Fukuda, and M. N. Fukuda, Carbohydr. Res., 141 (1985) 149-152. (26) A. Dell, J. E. Oates, H. R. M o m s , and H. Egge, In?. 1. Mass Specrrom. Ion Phys., 46 (1983) 415-418. (27) M. E. Hemling, R. K. Yu, R. D. Sedgwick, and K. L. Rinehart, Jr., Biochemistry, 23 (1984) 5706-5713. (28) K . L. Rinehart, Jr., Science, 218 (1982) 254-260.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

27

f.a.b.).27,29Arita and coworkers3’ introduced a mixed matrix composed of triethanolamine, 1,1,3,3-tetramethylurea (8) and triethylenetetramine for analyzing glycosphingolipids in the negative-ion mode; this gives results similar to those obtained with l-thi~glycerol.~~ HO-CHz-CHz-N-CH,-CH,-OH

I CHZ I

CHZOH 7

H3C

0

II

/

/N-c-N H3C

\

\

CH,

CH,

8

The type of data produced in a f.a.b. experiment is affected by the pH and ionic strength of the matrix. The former may be controlled either by the addition of acids or bases, although, in practice, it is usually preferable to keep the matrix acidic. The ionic strength is partly dictated by the purity of the sample (many biological compounds are still contaminated with salts, even after extensive purification) and partly by exogenous additives. Three additives are especially useful for carbohydrate work. They are as follows. (i) Dilute aq. HCI. Addition of 1 pL of 100 or 200 m M HCl to the matrix improves the quality of the data obtained from certain types of carbohydrate. Both positive and negative studies may be assisted by acidification. In the positive-ion mode, sensitivity is enhanced if basic functional groups are present, for example, the amino group of glycopeptides. The low pH ensures that the sample is “pre-ionized,” and loss of sample by the sputtering of neutrals is minimized.20 Molecules containing several carboxyl groups, for example, gangliosides having three, or four, sialic acid residues3’ or oligogalactosiduronic give excellent, negative f.a.b. spectra when they are in an acidified matrix. The acid protonates the carboxyl groups, and discourages the formation of a plethora of molecular-ion species having various numbers of carboxylate salts. Fig. 2 shows the effect of acid-dosing on the spectrum of an oligogalactosiduronic acid. acetate. In their positive-f.a.b. studies of (ii) Sodium glyc~sphingolipids,~’~~~~~~ Egge’s group routinely add a 0.1% solution of sodium acetate in methanol to the target prior to the addition of the matrix. (29) K. Harada, M. Suzuki, and H. Kambara, Org. Mass Spectrom., 17 (1982) 386-391. (30) M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, J. Biochem. (Tokyo),93 (1983) 319-322. (31) H. Egge, J. Peter-Katalinic, G . Reuter, R. Schauer, R. Ghidoni, S. Sonnino, and G . Tettamanti, Chem. Phys. Lipids, 37 (1985) 127-141. (32) E. A. Nothnagel, M. McNeil, P. Albersheim, and A. Dell, Plant Physiol., 71 (1983) 916-926. (33) K. R. Davis, A. G . Darvill, P. Albersheim, and A. Dell, Plunt Physiol., 80 (1986) 568-577. (34) H. Egge, J. Dabrowski, and P. Hanfland, Pure Appl. Chem., 56 (1984) 807-819. (35) H. Egge and J. Peter-Katalinic, in A. L. Burlingame (Ed.), Symp. Muss Spectrom., Health and Life Sci., in press.

ANNE DELL

28

IM +3Na-&H 1[M+ZNa-3HIIM+Na-2HI-

IM+&Na-SHl-

[M-HI-

IM+SNa-6HI[M+6Na-7HI‘

A IM-HI-

IM+Na-2Hl-

FIG. 2.-Molecular-ion Regions of the Negative F.a.b. Mass Spectra of AGalA-GalA-GalAGalA-GalA-GalA-GalA. [The upper trace was obtained after loading 1 FL of a solution of the sample in 5 % aq. acetic acid (5 pg/p,L) into glycerol. The lower trace was recorded after the further addition of 1 pL of 100 m M aq. HCI. (AGalA is a dehydrated galacturonic acid residue formed when a lyase cleaves a galacturonic acid polymer; GalA is a galacturonic acid residue.)]

Consequently, the only pseudomolecular ions produced are [M + Na]+ species. These are usually abundant, permitting the o b ~ e r v a t i o nof ~~ molecular ions up to at least 6000. (iii) Ammonium thiocyanate. Dosing with an ammonium salt gives abundant [M + NH,]+ ions in the spectra of some types of permethylated oligosaccharides. Curiously, there is very little effect on the spectra of molecules containing amino sugars, but, for h e ~ o s e ~polymers ~ . ’ ~ and plant cell-wall oligosa~charides,~~ the improvements in sensitivity are dramatic. Some ammonium ions are always present in commercial 1-thioglycerol, but optimal results require a supplement of 1 p L of a 100 mM solution of an ammonium salt. Various salts may be used, but ammonium thiocyanate permits the recording of good negative-ion spectra containing intense signals for [M + SCNI-, and its chaotropic properties probably assist desorption. 3. Characteristics of F.a.b.-Mass Spectra All f.a.b. spectra are characterized by ( a ) abundant, pseudomolecular ions for both the sample and the matrix, and (b) a relatively high level of chemical “noise,” resulting in a signal at every mass number up to the (36) A. Dell, W. S. York, M. McNeil, A. G. Darvill, and P. Albersheim, Carbohydr. Res., 117 (1983) 185-200. (37) L. D. Melton, M. McNeil, A. G. Darvill, P.Albersheim, and A. Dell, Carbohydr. Rex, 146 (1986) 279-305.

F.A.9.-MASS SPECTROMETRY OF CARBOHYDRATES

29

molecular-ion region. The background ions are derived from both the matrix and the sample, and are probably formed from surface molecules that have disintegrated after receiving a direct hit from an accelerated atom; they permit the manual counting of spectra up to m/z -4000, and their appearance is a good guide as to how well the recording is proceeding. If the background signals at m/z > 1000 are weak or nonexistent, it is very unlikely that good molecular-ion signals will be present at high mass. In addition to pseudomolecular i m s and background ions, two other types of signal may be present in the f.a.b. spectrum, namely those of cluster ions and fragment ions. Most cluster ions are matrix-derived. Glycerol, for example, gives peaks at mass numbers corresponding to (92x + 1)+ and (92x - 1)-, where 92 is the mass of glycerol and x is the number of glycerol molecules in the cluster. The most abundant clusters occur below mass 1000, but x can be as high as 15. 1-Thioglycerol gives fewer cluster ions than glycerol and, when present in a mixed matrix with glycerol, it suppresses the glycerol spectrum. The glycerol clusters are absent when the sample is sufficiently surface-active and in high enough concentration to form a complete monolayer at the surface of the matrix. Such dimer ions as [2M+ HI+, [2M+ Na]+, and [2M - HI-,derived from the sample, are an infrequent phenomenon in carbohydrate f.a.b.-m.s.; they are most likely to be found in the spectra of such low-molecular-weight, polar molecules as phosphorylated oligosaccharides. Fragment ions are important for sequencing; they are discussed in Section IV,2. F.a.b. spectra may be recorded with either a data system or an oscillograph. In principle, the method of recording the spectra should not affect the results but, in practice, it does. The oscillograph produces a faithful record of the spectrum, with peak shapes, noise, and any spurious spikes remaining intact and recognizable. In contrast, the computer ignores everything below a pre-set, threshold intensity, and reports all signals as lines. Genuine, but weak, high-mass signals are usually easier to recognize in an oscillographic trace. At m / z >2000, mass assignments differ in the two types of spectra. The data system calibrates the spectrum by using the known, accurate masses (based on C 12.00, H 1.01, 0 15.99, and so on), whereas oscillographic charts are counted manually, with nominal mass assignments given to each of the signals. For an average carbohydrate, nominal masses are lower than calculated masses by about half a mass unit for each 1-thousand mass-unit increment. Pseudomolecular ions do not appear as single, “clean” signals in f.a.b. spectra. Instead, clusters of signals are always present, partly because of the presence of molecules containing the I3Cisotope, the natural abundance of which is 1.1%, and partly because oxidations and reductions can occur in the matrix during the f.a.b. experiment. For example, underivatized

30

ANNE DELL

carbohydrates frequently exhibit an intense “minus 2” signal as a result of oxidation. The most abundant molecular ion is always derived from genuine molecules of the sample, not from those of by-products. However, if the molecule contains more than 90 carbon atoms, the ‘3C-isotope peak is the most intense signal. Fig. 3 shows the type of molecular-ion cluster normally observed in three different regions of the mass spectrum. 4. Sample Purity and the Analysis of Mixtures

The fast-atom beam will desorb only those molecules that are present at the surface of the matrix. Hence, selective ionization occurs from mixtures of compounds having different surface a~tivities.~ If a carbohydrate fails to give a mass spectrum, the most likely cause is contamination with compounds that are more surface-active than the sample. Salts and detergents are frequently present in biological samples, and should be rigorously removed prior to f.a.b.-m.s. Three warning signs that impurities are present are ( i ) intense, sodium-adduct ions for each matrix cluster, ( i i ) a series of signals 44 mass units apart (from detergents), and ( i i i ) a very high level of background signals up to well over 1000 mass units, but no obvious sample or matrix peaks above mass -500. F.a.b.-m.s. is a powerful technique for examining mixtures of carbohydrates. Many examples of such analyses are given in Sections V and VI. Unless the components have very different chemical structures, all will give molecular ions. However, the relative abundance of the ions will not necessarily reflect the relative concentrations of the components. Furthermore, if more than one class of carbohydrate is present, different pseudomolecular ions may be produced for each class. An example of such a phenomenon is given in Fig. 4.

5. Choice of Derivatives Derivatives play an essential part in almost all f.a.b.-m.s. studies of carbohydrates. They facilitate spectral interpretation (see Sections IV-VI), improve sensitivity (see Section 11,6), permit the analysis of salty samples (see later), allow unambiguous sequencing (see Section V,2), confirm the presence of cyclic structures (see Section VI,5), enable spectra to be obtained from very large molecules (see Section 111,4), and help in the location of 0-acylated residues in oligosaccharides (see Section V,5). Fortunately, derivatization for f.a.b.-m.s. makes no special demands on the carbohydrate chemist. The best derivatives are those that have been used for a very long time in carbohydrate work, namely, the per-0-acetyl and the per-0-methyl. Thus, f.a.b.-m.s. can be readily accommodated into existing structural programs.

F.A.B.-MASS SPECTROMETRY O F CARBOHYDRATES

31

FIG.3.-Schematic Representation of the Appearance of Typical, Molecular-ion Clusters. [(a) Near mass 500, (b) near mass 1500, and (c) near mass 3000.1

FIG.4.-Part of the Positive F.a.b.-Mass Spectrum of a Permethylated Sample Containing a Mixture of Man,-,GlcNAc, and Glc,_, . [The high-mannose carbohydrates were obtained by hydrazinolysis of a glycoprotein, followed by N-reacetylation and reduction, and the f.a.b. spectrum shows that only a portion of the molecules were reduced (for an explanation of this, see Section V,3). The glucose polymers are contaminants that were introduced during column purifications. Each of the GlcNAc-containing compounds gives two [M + H]+ molecular-ions, corresponding to the unreduced and reduced molecules, respectively; these are 16 mass units apart. The hexose polymers give [M + NHJ+ and [M + Na]+ molecular-ions. These form pairs 5 mass units apart. Major signals not assigned on the spectrum are 1052 (1084 minus methanol), 1070 (undermethylated 1084), 1248 (I280 minus methanol), 1266 (undermethylated 1280), 1274 (undermethylated 1288), 1280 (Hex,HexNAc+), 1452 (1484 minus methanol), 1470 (undermethylated 1484), 1478 (undermethylated 1492), 1484 (Hex,HexNAc+), 1688 (Hex,HexNAc+), and 1747 (undermethylated 1761). Forthe origin of Hex,-,HexNAc+, see Section IV,2.]

32

ANNE DELL

The choice of derivative is dictated by the exact problem under study. Frequently, it may be necessary to prepare both types of derivative in order to obtain the maximum structural information. The following factors need to be taken into account when choosing the most appropriate derivative. a. Permethylation.-( i ) This gives the smallest increase in the molecular weight of the sample, but it is, experimentally, a “dirty” procedure, and chromatography must be conducted prior to analysis. ( i i ) Most permethylated glycoconjugates fragment very selectively, resulting in a limited number of sequence ions that are easy to assign, but fragmentation may be so selective that not enough sequence information is present. b. Peracety1ation.-( i ) This gives a large increase in molecular weight, but is experimentally “clean,” and spectra can be acquired within 30 min of the start of the acetylation. ( i i ) Fragmentation pathways are less specific than for permethylated samples and may give more structural information, but spectra can be more difficult to interpret. As a general rule, peracetylation is most useful for compounds below M , 2000, particularly those that have been reduced with sodium borohydride and still contain some salt. The best procedure for peracetylation is based on the method of Bourne and Samples are dissolved in 2: 1 (v/v) trifluoroacetic anhydride-acetic acid and the solutions kept for -10 min at room temperature. Reagents are removed under a stream of nitrogen, and a solution of the product in chloroform is washed with water to remove salts, and dried; the peracetylated sample is dissolved in methanol for the f.a.b. analysis. Less widely used, but valuable for particular types of problem, are derivatives prepared by causing a nucleophile to react with the reducing residue of an oligosaccharide. In one very interesting study:’ a strategy was devised for the characterization of oligosaccharides that had been reductively aminated with aniline (9), p-aminoacetophenone (10) or ethyl p-aminobenzoate (11).The chromophore permits sensitive, U.V. monitoring during 1.c. purification, and the nitrogen atom can be readily protonated, resulting in high f.a.b. sensitivity. Oximes constitute another type of reducing-end derivative that have been shown to give good f.a.b. data.26 For example, use of the (pentafluorobenzy1)oxime derivative 12 assisted the analysis of oligogalactosiduronic acids3’ (38) E. J. Bourne, M. Stacey, J. C. Tatlow, and J. M. Tedder, 1.Chem Soc., (1949) 2976-2979. (39) A. Dell, J. E. Oates, and C. E. Ballou in M. A. Chester, D. Heinegard, A. Lundblad, and S. Svensson (Eds.), Glycoconjugates, Roc. Int. Symp. Glycoconjugates, 7th, LundRonneby, Sweden, 1983, pp. 137-138. (40) W. T. Wang, N. C. LeDonne, Jr., B. Ackerman, and C. C. Sweeley, Anal. Biochem., 141 (1984) 366-381.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

33

R-C H=N-O--CHz

c=o

c=o

CH3

OCH1CH3

10

11

I

9

Fs

I

12

6. Sensitivity

Sensitivities achieved in f.a.b. analysis are both operator- and sampledependent. Experienced mass spectrometrists working with well purified samples use between 0.1 and 5 pg of sample when analyzing derivatives, and between 1 and 10 pg when analyzing native compounds. The higher the molecular weight, the greater the quantity of sample needed. Correctly loading the sample into the matrix is one of the most critical steps in f.a.b. analyses. Poor data are inevitable if the sample has been loaded in such a way that it cannot readily be present in high concentration at the surface. Samples should not, therefore, be dried on the target prior to addition of the matrix. In the author’s laboratory, the following experimental protocol has been found to give good results. ( i ) Smear 1-2 pL of glycerol or 1 :1 (v/v) glycerol-thioglycerol on the metal target by using a 5-pL micropipet. ( i i ) Dissolve the sample in either 5% aq. acetic acid (underivatized samples) or methanol (derivatives) to such a concentration that 1 p L contains the desired amount of sample for the f.a.b. experiment. ( i i i ) Using a 5-pL micropipet, carefully blow 1 pL of the sample solution onto the surface of the matrix. ( i u ) Introduce the probe into the high vacuum of the source housing for -30 s in order to pump off most of the solvent (the solvent should not be exhaustively removed, because evaporation of the last traces almost certainly assists in the subsequent f.a.b. analysis). ( u ) Remove the probe in order to check that the volume of the matrix has not significantly decreased. This is necessary because coevaporation of the solvent and the matrix can occur. If necessary, carefully replenish the matrix with a small drop of 1-thioglycerol. ( u i ) Fully insert the probe into the f.a.b. source, until it is about 1 cm from its operating position. ( u i i ) Switch on the high voltages and, while observing the repetitive scanning of a selected region of the spectrum on the visual-display unit, push the probe fully into place. The mass range selected for observation

ANNE DELL

34

will depend on prior knowledge of the type of compounds being examined. It may be possible to select the predicted molecular-ion region, or areas where key fragment-ions are expected. For complete unknowns, it is usually appropriate to choose a region of the spectrum near m / z 1000, as the intensities of background signals at this mass will reflect the likelihood of sample signals being present at higher masses. The appearance of the spectra produced in the first few seconds of atom impact will dictate subsequent steps. If the signals on the oscilloscope are reasonably intense, a full spectrum is recorded immediately; delays result in poorer quality data. If the visual display indicates that the sample is not working well, it may be necessary to retune the source rapidly or to add a little more 1-thioglycerol, or, perhaps, a matrix additive. It may even be necessary to purify the sample by, for example, preparing the per- 0-acetyl derivative. It is now well established that acetylated and permethylated samples can be analyzed at higher sensitivity than their underivatized counterparts, despite the increase in mass upon derivati~ation.’~’~ Minor components in mixtures are often only revealed after derivatization. For example, negative f.a.b.-m.s. of an oligosaccharide mixture isolated from an erythrocyte glycoprotein4’ showed only the major component (see Fig. 5a). After peracetylation, signals from higher-molecular-weight, minor components were clearly present in the spectrum (see Fig. 5b). Similarly, the largest of the cyclic p - ( 1 + 2 ) - g l ~ c a n sand ~ ~ also the largest of the cyclic, Enterobacteriacae-common antigens4’ (see Section VI,5) were detected only after the samples had been permethylated. The higher the molecular weight of the sample, the greater the difference in minimum sample-loadings needed in order to produce spectra from native samples and their derivatives. If M,is significantly >4000, the difference becomes almost infinite, because underivatized polysaccharides and glycoconjugates of such a size rarely give spectra, whereas permethylated samples as large as 20,000 have been shown to be amenable to f.a.b.-m.s. (see Section 111,4). 111. F.A.B.-M.s.

OF

HIGH-MOLECULAR-WEIGHT SAMPLES

1. Definition of High Mass

Ten years ago, it was easy to give a working definition of high mass. At that time, most mass spectrometers had a mass range of less than 1000 at (41) M. Fukuda, A. Dell, J. E. Oates, and M. N. Fukuda, J. Bid. Chem., 259 (1984) 8260-8273. (42) A. Dell, J. E. Oates, C. Lugowski, E. Romanowska, L. Kenne, and B. Lindberg, Curbohydr. Res., 133 (1984) 95-104.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

35

FIG. 5.-(a) Molecular-ion Region of the Negative F.a.b.-Mass Spectrum of a Sample of Oligosaccharides Isolated from the Band 3 Lacto~aminoglycan~' of Human Erythrocytes After Treatment with Endo-0-D-galactosidase. [No significant ions were present above mass 950. The [M - HI- signal corresponds to the composition NeuAc,HexNAc,Hex,. The minor signals at 857, 893, 915, and 937 are adduct ions from contaminating NaCl and have compositions [M+Na-2H]-, [M+NaCl-H]-, [M+Na+NaCI-2H]-, and [ M+ 2 N a+ N aC1 - 3 HI-, respectively.] (b) Molecular-ion Regions of the Negative F.a.b.-Mass Spectrum of the Sample Shown in (a) After Conversion into the Peracetyl Derivative. [The major ions at 1339 (monounderacetylated 1381) and 1381 are afforded by the component observed in the spectrum of the underivatized sample. Molecular ions for minor components are present at 1956, 2243, and 253 1, corresponding to compositions NeuAc,HexNAc,Hex, ,NeuAc,-HexNAc,Hex, , and NeuAc,HexNAc,Hex,, respectively. Each is accompanied by a signal 42 mass units lower, corresponding to one degree of underacetylation.]

full sensitivity, and this was a natural boundary between low- and high-mass work. High-mass m.s. was nonroutine and frequently difficult; few biological molecules had been analyzed at high m a ~ s . 4For ~ practical reasons, it is still useful to differentiate between low- and high-mass experiments. The distinction is somewhat subjective but, for the purposes of this article, it is convenient to consider the upper end of the 3000-4000-mass range as the start of high-mass f.a.b.-m.s. (43) A. Dell and G. W.Taylor, Mass Spectrorn. Rev., 3 (1984) 357-394.

36

ANNE DELL

There are three main reasons for this choice. Firstly, it becomes more and more difficult to obtain recordable, molecular-ion signals from underivatized carbohydrates as their M, increases significantly above 3000. Secondly, the mass spectrometers that have been used in all high-masscarbohydrate studies published at the time of writing this article are not capable of very sensitive analysis above -3800 mass units (see later). Thirdly, at masses >4000, it is usually not practicable to work at the resolution necessary for adjacent peaks to appear as separate signals in the spectrum. To do so would require that the source and collector slits be narrowed to such a degree that there would be an unacceptable loss in sensitivity. Thus, spectra acquired at mass >4000 are usually composed of unresolved clusters. 2. Instrumentation

To date, all high-mass-carbohydrate f.a.b.-m.s. has been conducted by using high-voltage, double-focusing, sector mass spectrometers. Alternative instruments capable of analyzing high-mass ions, such as time-of-flight@ or Fourier-transform mass spectrometers,4’ do not, therefore, fall within the scope of this article. The maximum mass range of a magnetic-sector, mass spectrometer is governed by the fundamental, mass-spectrometric equation m /Z(Y (B 2 R 2 ) /V, where B is the magnetic-field strength, R is the radius of the magnetic sector, and V is the accelerating voltage. High-mass ions can only be focused at high field-strengths or with large-radii magnets, unless they are analyzed at low accelerating voltages. The last option is frequently not practicable, because a lower value of V means lower sensitivity. During the 19703, high-field-magnet mass spectrometers were designed to meet the needs of biopolymer They were fitted with magnets containing pole tips made of Permendur, a high-saturation alloy of cobalt, iron, and vanadium, capable of sustaining magnetic fluxes of up to 2.3 teslas. By the time f.a.b.-m.s. was introduced, two such instruments were commercially available, the Kratos HF-MSSO and the VG Analytical ZAB-HF mass spectrometer, both having a mass range of just over 3000 at maximum accelerating voltage (8 kV). The latter has proved the more popular for carbohydrate f.a.b.-m.s. Operation of the high-field ZAB spectrometer at 7-kV accelerating voltage extends the mass range to nearly 3800 without a significant loss in sensitivity. Above that point, there is a steady decline in sensitivity. When V is lessened (44) R. D. Macfarlane and D. F. Torgerson, Science, 191 (1976) 920-925. (45) C. L. Wilkins and M. L. Gross, Anal. Chem., 53 (1981) 1661A-1668A.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

37

to below 4 kV in order to allow the detection of ions above mass 6600, the resulting sensitivity is very poor indeed. The only successful f.a.b. work performed above 6600 has been in the protein area.46s47 The high-field ZAB and MS50 mass spectrometers have now been superseded by a new generation of extended mass-range instruments that are capable of operation at full sensitivity (that is, at full accelerating voltage) Several laboratories involved in up to masses in e x c e ~ s ~of * *10,000. ~~ carbohydrate work are installing these instruments, and it will be interesting to see what effect this new generation of mass spectrometers will have on the ability to obtain data on compounds having M , values well in excess of 4000.

3. Practical Aspects of Analysis at High Mass Three types of carbohydrate sample have been reported to give data above mass 4000, namely, permethylated polysaccharides,26 permethylated glyco~phingolipids,~~ and naturally acylated forms of a mycobacterial 0methyl-D-glucose polysaccharide.’’ All are hydrophobic, and desorption is probably facilitated by their inability to form strongly hydrogen-bonded aggregates, either with themselves or with the matrix. High-mass samples are usually analyzed at low resolution (see Section III,l), and the resulting spectra contain unresolved clusters having a characteristic, Gaussian appearance (see Fig. 6). Mass assignments are made by using either a mass marker (for chart paper) or a data system. Cesium iodide is normally used for calibrating the mass marker and data system; it gives abundant cluster-ions at intervals of 360 mass units, the mass of CsI, throughout the entire working mass range’ of f.a.b.-m.s. The mass assigned to the centre of each unresolved, sample peak corresponds to its chemical molecular weight. The quality of data recorded from weak, high-mass-ion beams can usually be improved by the use of signal a~eraging.~’The computer software necessary is now available from the mass spectrometer manufacturers. Although signal averaging has not yet been applied to high-mass-carbohy(46) M.Barber, R. S. Bordoli, G. J. Elliott, N. J. Horoch, and B. N. Green, Biochem. Biophys. Res. Commun., 110 (1983) 753-757. (47) A. Bateman, A. Dell, and H. R. Moms,J. Appl. Biochem., 7 (1985) 126-132. (48) J. S. Cottrell, L. C. E. Taylor, and S. Evans, Proc. Meet. Br. Muss Spectrom. Soc, 14rh, 18-21 Sept. 1984, pp. 127-129. (49) B. N. Green and R. S. Bordoli, in S. J. Gaskell (Ed.), Muss Specrromerry in Biomedical Research, Wiley, New York, 1986, pp. 235-250. (50) M. Barber, R. S. Bordoli, A. N. Tyler, J. C. Bill, and B. N. Green, Biomed. Muss Speclrom., 11 (1984) 182-186.

ANNE DELL

38

4660

4700

4740 “/z

FIG. 6 . 4 n e of the Molecular-ion Clusters Obtained from a Sample of Deuteropennethylated, Cyclic p( 1 + 2)-Glucans (see Section VI,5). [All high-mass samples give unresolved clusters of this type if the mass spectrometer is operated at low resolution. The peak is -6 mass units wide at half height. The mass is assigned by using the mass marker, which gives marks every 4 mass units, as shown. The center of the peak corresponds to the chemical molecular weight of an [ M + NH4]+ species.]

drate problems, it will almost certainly play a vital role in this area in the future. 4. F.a.b. “Mapping” of Permethylated, High-Mass Samples From of Band 3, one of the cell-surface glycoproteins of human erythrocytes, a rapid f.a.b. procedure called “mapping” has been devised for analyzing the types of nonreducing structures present in veryhigh-molecular-weight glycoconjugates of the lactosaminoglycan type.z5 These contain long, carbohydrate chains composed of many repeats of the N-acetyllactosamine unit, /&Gal-(1+ 4)-GlcNAc, which may be modified by sialylation, fucosylation, branching, and so on, to afford determinants for a variety of such well recognized antigens as the ABH blood-group system.’* Two properties make this type of molecule an ideal candidate for f.a.b.-m.s. Firstly, as permethylated derivatives, they fragment in a reproducible and predictable way (see Section V,2), to give a diagnostic spectrum or “map” of fragment ions in which is contained information on all of the nonreducing structures present in the intact glycoconjugates. Secondly, good f.a.b. spectra may be obtained on very large molecules indeed. Spectra have been recorded from permethylated lactosaminoglycans as large as 20,000, and it is anticipated that much larger molecules will be amenable to this type of analysis. A tremendous advantage of the mapping procedure for the analysis of high-molecular-weight samples is that it makes no high-mass demands on the instrumentation. Only fragment ions are recorded, and these fall within the high-sensitivity range of high-field instruments, that is, less than 4000 mass units. (51) M. Fukuda, A. Dell, and M. N. Fukuda, J. Bid. Chern, 259 (1984) 4782-4791. (52) T. Feizi, Trends Biochem. Sci., 6 (1981) 333-335.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

39

The following protocol has been proposed2’ for mapping high-molecularweight glycoconjugates. (i) The intact glycoconjugate (or mixture of glycoconjugates) is converted into its permethylated derivative(s). (ii) Positive f.a.b. spectra are acquired in the 3000 mass range by overlapping several shorter scans, using the procedures described in Section 142. (iii) The number of sialic acid, fucose, hexose, or hexosamine residues contributing to each fragment ion is then detefmined by a simple calculation. Each of these residues has a unique mass, and it is possible to assign a unique composition to every fragment ion. Thus, the fundamental, nonreducing structure Hex-HexNAc+ occurs at rn/z 464, and increments of 174, 361, 391, and 449 mass units are added to this for additional fucose, N-acetylneuraminic acid, N-glycolylneuraminic acid, or N-acetyllactosamine moieties, respectively. F.a.b. mapping may be applied to mixtures of glycoconjugates. No prior knowledge of their size, structure, or complexity is needed. Thus, one useful application is the rapid screening of the total lactosaminoglycan fractions isolated from the surface of a cell. The f.a.b. maps are a useful guide to the types of nonreducing structures present, and may reveal the presence of cell-specific antigens. This type of analysis is illustrated in Figs. 7 and 8. Fig. 7 shows part of the f.a.b.-mass spectrum obtained from a lactosaminoglycan sample isolated from chronic myelogenous leukemia (CML) cells.” After an identical analysis, normal granulocytes yielded a sample that gave the spectrum shown in Fig. 8. Most of the fragment ions are obviously present in both spectra, albeit with intensity differences. However, there are two very significant differences. Firstly, the signal at rn/z 999, which is fairly prominent in the spectrum of the CML sample, is very weak indeed in that of the granulocyte sample. Secondly, there is a signal at rn/z 1622 in the CML spectrum that is absent from the spectrum obtained from granulocytes. These ions have compositions NeuAc, Fuc,Hex,HexNAc, and NeuAcl Fuc2Hex2HexNAc2,respectively. Their complete structures, determined by using other methods of carbohydrate analysis, are 13 and 14.

NeuAc a ( 2 + 3) Gal p ( 1 + 4) GlcNAc p( 1 + 3) Gal p ( 1 + 4) GlcNAc p( 1 +

r:.

r:.

Fuc

Fuc

14 (53) M. Fukuda, B. Bothner, P. Ramsamooj, A. Dell, P. R. Tiller, A. Varki, and J. C. Klock, J. Biol. Chem., 260 (1985) 12,957-12,967.

40

ANNE DELL

FIG. 7.-Part of the F.a.b. “Map” of a Lactosaminoglycan Sample Isolated from CML Cells.53 [Signals at 913, 999, 1087, 1261, 1274, 1362, 1448, 1536, 1622, 1710, and 1723 are A,-type ions resulting from cleavage at HexNAc residues. They have the compositions Hex,HexNAc,, NeuAc,Fuc,Hex,HexNAc, , Fuc,Hex,HexNAc,, Fuc,Hex,HexNAc,, NeuAc,Hex,HexNAc,, Hex,HexNAc,, NeuAc,Fuc,Hex,HexNAc,, Fuc,Hex,HexNAc,, NeuAc,Fuc,Hex,HexNAc, , Fuc,Hex,HexNAc, , and NeuAc, Hex,HexNAc, , respectively. The signal at 1029 is an A,-type of ion resulting from cleavage at Hex, and has the composition NeuAc,Hex,HexNAc, . The signal at 1242 is the result of methanol loss from 1274.1

FIG. &-Part

of the F.a.b. “Map” of a Lactosaminoglycan Sample Isolated from Normal

granulocyte^.^^ (See the legend to Fig. 7 for peak assignments.)

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

41

The f.a.b. mapping procedure is applicable to all permethylated polysaccharides and glycoconjugates that fragment readily. Current knowledge indicates that the main requirement is the presence of amino sugar residues at fairly regular intervals. Preliminary work54 suggested that the method could be useful for rapidly defining the type of structure present in bacterial polysaccharides whose repeating units contain amino sugars. OF F.A.B.-MAss SPECTRA IV. INTERPRETATION

1. Molecular-weight Assignment

With the special exception of “maps” (see Section 111,4), all f.a.b. spectra contain one or more pseudomolecular-ion signal(s). These afford the molecular weight of the oligosaccharide or glycoconjugate, and thus define its composition in terms of both the type and number of sugar constituents, the type of aglycon, and the type and number of such substituents as acetyl or sulfate groups. F.a.b.-m.s. cannot distinguish between isomeric monosaccharides, and compositions are given in terms of hexose, pentose, deoxyhexose, hexosamine, uronic acid, and so on. The pseudomolecular-ion region may be complex if several types of ion are present, but the spectra can usually be assigned without difficulty. In the positive-ion mode, four species are commonly present (although not usually in the same sample). They are, in rising order of molecular weight, [M + HI+, [M + NH4]+, [M + Na]+, and [M K]+. If more than one signal is present, the mass differences between the signals (see Fig. 9) usually indicate the nature of the cationizing species. For example, two pseudomolecular-ion signals separated by five mass units must be [ M + NH4]+ and [ M + Na]+. Samples that are heavily contaminated with Na+ or K+, or exist naturally as Na+ or K+ salts, give ions of the type [M - x H + ( x + l)Na]+. The addition of acid to the matrix (see Section II,2) enhances the abundance of [ M + H]+ ions, and this often helps to confirm tentative

+

n [M+HI*

-22--17

I M + N w + [M+Nal+

---+

53-16

[M+KI* [M-H+2Nal*

-c6+

FIG. 9.-Schematic Representation of Molecular-ion Signals That May Be Formed in the Positive-ion Mode, Showing Commonly Observed Mass Differences. (54) A. Dell, unpublished results.

ANNE DELL

42

FIG. 10.-Molecular-ion Region of a Permethylated Glycosphingolipid Sample. [The component having the saturated, 24-carbon fatty acid (Cer2.,:J gives the [M+H]* signal at 1345, and no adduct ion. The component having the unsaturated, 24-carbon fatty acid (Cerz4,) gives a minor [M+H]+ signal at 1343, and a major signal for the 1-thioglycerol-adduct ion at 1451.1

assignments. Some types of carbohydrate frequently form adduct ions with the matrix, giving such signals as [M+ (glycerol), + HI+. These signals are easy to recognize, because they appear at defined mass-increments above the genuine molecular ions. They are rarely more intense than the molecular ions, with one important exception. Glycosphingolipids having an unsaturated, 24-carbon fatty acid group in the ceramide moiety always give [M + 1-thioglycerol H]+ as the dominant, molecular-ion This phenomenon is illustrated in Fig. 10. In the negative-ion mode, [M -HI- is usually observed. Molecules that cannot lose a proton, such as permethylated saccharides, give negative spectra if anions are present in the matrix; for [M+Cl]- or [ M + SCNI-. Underivatized samples do not normally add anions unless the matrix is very and, at such high salt-levels, the data are usually poor. Samples that exist naturally in a salt form at neutral pH retain their Na+ or K+ counter-ion on most of the anionic groups, unless the matrix is acidified (see Section 11,2). If the pK values of the anionic, functional groups are very low, molecular-ion clusters of the general composition [M (x - l)Na - x HI- dominate the spectra, unless strong acids can be added, and this may not be possible if acid-labile groups are present in the sample.

+

+

(55) M. N. Fukuda, A. Dell, J. E. Oates, P. Wu. J. C. Klock, and M. Fukuda, J. Biol. Chem., 260 (1985) 1067-1082. (56) D. Prome, J. C. Prome, G . Puzo, and H. Aurelle, Carbohydr. Res., 140 (1985) 121129.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

43

2. Fragmentation Pathways

In this Section, fragmentation pathways that appear to be common to all classes of polysaccharides and glycoconjugates are d e s ~ r i b e d . ~ ~ ~ ~ ~ * ~ ' * ~ ~ Cleavages that occur only in specific types of molecules, for example, glycosphingolipids, are discussed in Section VI. An arbitrary code-letter is given to each pathway in order to facilitate its citation in later sections. For convenience, a pictorial representation is given for each pathway. These Schemes are speculative, because no rigorous studies, using isotopically labelled compounds, have been conducted to confirm cleavages and hydrogen shifts. Nevertheless, they provide a useful visual guide to fragmentation mechanisms. It should be noted that only the charged product is shown in each scheme. Furthermore, the choice of 4-linked pyranoses was made solely for convenience.

Pathway A Description: glycosidic cleavage to form an oxonium ion; charge retained on nonreducing end; positive-ion mode only; often referred to as A,-type cleavage, because of similarity to one of the cleavages seen in electron impact-mass spectrometry.

G2b0/"1' - y.4 CHiOH

-0

CHzOH

CHZOH

+ H

HO

OH HO

OH

(63) (64) (65)

(66)

OH

K.Yu, M. M. Rapport, and K.Suzuki (Eds.), Ganglioside Structure, Function and Biochemical Potential, Plenum, New York, 1984, pp. 55-63. M. Iwamori, M. Arita, T. Higuchi, Y. Ohashi, and Y. Nagai, Jeol News, 20A (1984) 2-9. M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, Jeol News, 19A (1983) 2-6. M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, J. Biochem. (Tokyo),94 (1983) 249-256. M. Iwamori, Y. Ohashi, T. Ogawa, and Y. Nagai, Jeol News, 21A (1985) 10-14. J. P. Kamerling, W. Heerma, J. F. G. Vliegenthart, B. N. Green, I. A. S. Lewis, G. Strecker, and G. Spik, Biomed. Mass Spectrorn., 10 (1983) 420-425. D. Abraham, W.F. Blakemore, A. Dell, M. E. Herrtage, J. Jones, J. T. Littlewood, J. E. Oates, A. C. Palmer, R. Sidebotham, and B. Winchester, Biochem. J., 221 (1984) 25-33. H. Egge, A. Dell, and H.von Nicolai, Arch. Biochem. Biophys., 224 (1983) 235-253. H. Egge, J. Dabrowski, P. Hanfland, A. Dell, and U. Dabrowski, in A. Makita, S. Handa, T. Taketomi, and Y. Nagai (Eds.) New Vistas in Glycolipid Research, Plenum, New York, 1982, pp. 33-40. M. Barber, R. S. Bordoli, R. D. Sedgwick, and J. C. Vickerman, J. Chern. SOC.,Faraday Trans. I , 7 8 (1982) 1291-1296.

(57) H. Egge, J. Peter-Katalinic, and P. Hanfland, in R. W. Leeden, R.

(58) (59) (60) (61) (62)

HO

ANNE DELL

44

Pathway B Description : glycosidic cleavage with a hydrogen transfer; charge retained on reducing end; positive- and negative-ion modes; often referred to as /3-cleavage. -+o*-

-0

/"* + o r - H 0

oo/"* + or -

CHzOH I

HO

+or-H

HO

OH

-+or-

-0

/"* + o r - H

I

0

CH20H I

Pathway D Description: ring cleavage; charge retained on reducing end; ions are 28 mass units heavier than those formed in Pathway B; positive- and negativeion modes.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

4s

-+or-

,--. + o r - H

1

0

-0

HO

OH HO

OH

+ or -

CH2OH I

Pathway E

Description: ring cleavage; charge retained on nonreducing end; infrequent pathway in the positive-ion mode, but a major pathway in the negative mode, because a stable, enolate anion results from loss of the enolic hydrogen atom; ions are 42 mass units higher than those formed in Pathway C. -+or-

CH2OH -0

CHZOH

&o&o,--.+or-H HO

OH HO

OH

V. STRUCTUREASSIGNMENT BY F.A.B.-M.s. 1. Analysis of Underivatized Samples

It is usually desirable to define molecular weights by analyzing underivatized samples, because chemically labile functional groups will then be observed. Unfortunately, underivatized oligosaccharides often give ambiguous sequence-data. The reasons for this may be deduced from a

46

ANNE DELL

consideration of the fragmentation pathways shown in Section IV,2. In an underivatized, unreduced oligosaccharide, pathways B and C are not distinguishable, and it is not possible to establish whether the resulting fragmentions are derived from the nonreducing or the reducing end of the molecule. A further complication is the presence of ions resulting from “double cleavages,” for example, a combination of pathways A and B operating at different glycosidic linkages, which give apparent nonreducing-end sequence ions that are not, in fact, derived from the nonreducing terminus. The abundance and number of fragment ions afforded by an underivatized carbohydrate vary considerably, depending on the structure of the molecule, its purity, the amount of sample loaded, and the nature of the matrix used. Sometimes, no fragment ions are present, despite molecular-ion signals of reasonable quality. Sometimes, one or more linkages are particularly labile. On rare occasions, fragmentation is extensive. A final point to note is that fragmentation pathways may differ according to the nature of the pseudomolecular ion, that is, whether it is [M HI+, [M + Na]+, and so on, because of differing internal energies. In summary, fragmentation data from an underivatized sample need to be interpreted with caution, and the primary objective for analyzing underivatized samples, unless they belong to a well characterized family such as the glycosphingolipids, should be molecular-weight assignment. Sequencing should be carried out by using derivatives.

+

2. Analysis of Derivatized Samples

Per-0-acetyl and per-0-methyl derivatives are used extensively for sequence analysis and for providing molecular-weight information at very high sensitivity. Derivatization resolves the problems of ambiguity referred to in Section V,1, because a true, nonreducing residue will be fully substituted, whereas a nonreducing residue resulting from mass-spectrometric cleavage will carry a free hydroxyl group. Derivatization has the added advantage of directing fragmentation along a limited number of well defined, fragmentation pathways, and the fragmentation of peracetylated and permethylated derivatives is particularly reliable and predictable. The major ions formed from both the peracetyl and the permethyl derivatives are derived from a single, Pathway A cleavage; less abundant ions arise from P-cleavage (Pathway B), together with a Pathway A cleavage farther along the chain. The latter can be readily identified, because they lack an acetyl or methyl group on the nonreducing residue. Thus, the hypothetical sequence M-N-P-Q-R (where the letters refer to fully methylated or acetylated, unspecified sugar residues) is expected to fragment to give sequence ions of composition M+, MN+, MNP+, and MNPQ+, but

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

47

may also give such minor ions as NP+ and NPQ+, in which N bears a free hydroxyl group resulting from a “double cleavage.” This hypothetical sequence is assumed to be hexosamine-free, because a very interesting and important phenomenon is observed when HexNAc residues are present in the sequence. Cleavage then occurs predominantly at each hexosamine Thus, were the hypothetical sequence M-N-HexNAc-Q-R the major fragment ion would be M-N-HexNAc+. In permethylated samples, the HexNAc cleavage may be exclusive, particularly if the molecule is large. In peracetylated samples, it is always the dominant cleavage. Branching patterns can be readily established in N-acetyllactosaminecontaining molecules, because of the specific cleavages that occur at each HexNAc residue. For example, a simple, linear sequence of three lactosamine repeats will give fragment ions of composition Hex,HexNAc:, Hex,HexNAc:, and Hex,HexNAc:, whereas the branched sequence (15) will not give a Hex,HexNAc: fragment-ion. Hex-HexNAc Hex-HexNAc

\

/

Hex-HexNAc-

15

The molecular ions observed in the f.a.b. spectra of peracetylated and permethylated samples are often very important for structural assignment, particularly if f.a.b. spectra were not acquired prior to derivatization. Usually, their interpretation is routine, but the fact that the exact nature of the molecular ion may vary from sample to sample needs to be taken into account. For example, it may be difficult to permethylate some structural types, for example, those containing acetamido and the major molecular-ions from these samples will be one or more multiples of 14 mass units (depending on the number of methyl groups that are absent in the majority of molecules) less than expected. Peracetylated samples frequently lack an acetyl group on the reducing group, and molecular ions of structures 16 and 17 may be present, instead of, or in addition to, 18. It has been observed6’ that peracetylated samples prepared by using acetic anhydrideCH~OAC

RO& > O H + H +

CH~OAC

Ro&>

Ro/QoAc+H+ OAc

OAc 16

CH~OAC

17

(67) C. E. Ballou and A. Dell, unpublished results.

OAc 18

ANNE DELL

48

pyridine and those prepared using trifluoroacetic anhydride-acetic acid often differ significantly in the extent of acetal acetylation.

3. Monitoring Chemical and Enzymic Reactions by F.a.b.-M.s. F.a.b.-m.s. is an ideal procedure for monitoring the progress of many of the chemical and enzymic reactions commonly used in carbohydrate chemistry. It can reveal minor by-products, as well as provide data that are often very useful in structural assignments. Some types of reactions, for example, hydrolyses and methanolyses, are conveniently monitored by loading an aliquot of the reaction mixture directly into the matrix. Prior processing is necessary only if the reaction medium is incompatible with f.a.b.-m.s. or if derivatives need to be prepared after the enzymic or chemical digestion. In this Section, several studies that highlight the types of procedures that can be readily monitored by f.a.b.-ms., have been selected for discussion. Other examples are given in Section VI. The complete structure determination of the blood-group B-active glycosphingolipid shown in Fig. l l was achieved by using a combination of m.s. and n.m.r. The composition and sequence were defined by the f.a.b.-mass spectrum of its permethylated derivative (see Fig. 11). Many of the linkage assignments could be determined from n.m.r. spectra of the intact molecule, but some ambiguities were resolved only when n.m.r. spectra were obtained from truncated molecules lacking the two galactose residues on each branch. The strategy designed for removal of these residues was digestion with (Y-D-galactosidasefollowed by Smith degradation. F.a.b.-m.s. proved to be a very useful method for monitoring the progress of each of these reactions. After treatment of the native glycosphingolipid with (Y-Dgalactosidase, the molecular ion of the permethylated derivative shifted to

668 Gal - Gal - G I cNA

1770 7 ‘Gal-GlcNA7

Gal -Gal-GlcNAc\

e

2_872

‘%a1 -GltNAc

668 ‘.i Gal-Gal-GlcNAc

664

I’

;‘,

d

3914

Gal-GltNA,.‘

Gal-Gal - G l r N A a

668

,?(, Gal-GlrNAc -Gal-GlrtCer

q Gal-Gal -GlcNA&

4

658

668

FIG.11.-Sequence of a 25-Sugar Residue Glycosphingolipid Isolated from Rabbit Erythrocyte membrane^.'^ (Cleavage points, and the masses of fragment ions of the permethylated derivative, are shown. No fragment-ions were observed above 4000, because of the poor sensitivity at high mass.)

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

49

46 4 Gal-G I c N A c T 1362 Gal -G lcNAc',\'Gal -GlcNAc2260 y'ha, - G i c N A 7 3158 Gal-GlcNAc Gal-GlcNAz

L64

.\

264

\I!

.";

Gal-GlcNAb

4 64

Gal-GlcNA&

..(

Gal-GlcNAc -Gal -GlciCer L*

658

464

FIG.12.-Sequence of the Glycosphingolipid Shown in Fig. 11, after Enzymic Degradation with cr-D-Galactosidase. (Cleavage points, and the masses of fragment ions of the permethylated derivative, are shown.)

5163, and major fragment-ions appeared at 464, 1362, 2260, and 3158 (see Fig. 12), consistent with the loss of five terminal galactose units. The f.a.b. spectrum also showed, by virtue of a weak signal at 668 for Gal-GalGlcNAc+, that some molecules were incompletely digested. The expected product of the Smith degradation is shown in Fig. 13, together with the predicted fragment-ions for the permethyl derivative. F.a.b.-m.s. confirmed that all of the terminal galactose residues had been removed, because 464 was absent, and 260 had appeared as a strong signal. However, the other predicted fragment-ions shown in Fig. 13 were either absent or of low abundance. Instead, another ion-series was present at m / z 709, 1403,2097, 2971, and 2995, indicating that one of the GlcNAc residues had unexpectedly been degraded during the oxidation with metaperiodate. This information helped in the analysis of the n.m.r.-spectral results. The use of f.a.b.-m.s. to monitor hydrolysis and acetolysis reactions is illustrated by studies67on a yeast high-mannose oligosaccharide containing two phosphoric esters (19). The molecular-ion region of the negative f.a.b. 260

2342




260

3_036

Gal-GlcNAc ;Gal- .Glc:;Cer

\/

4

GICNAL 260

FIG.13.-Predicted Sequence of the Smith-Degradation Product of the Glycosphingolipid Shown in Fig. 12. (Predicted Fragment-ions for the permethylated derivative are shown.)

ANNE DELL

50

Man a(1 + 6 ) Man a ( l - 6 ) Man cr(l+6) Man p ( 1 + 4 ) GlcNAc

r:.

Man

t :.

t:.

Man

t:.

Man-P

Man-P

I Man 19

r:. Man r.:

I Man

Man

spectrum of the underivatized molecule showed an [M - HI- signal at 2324, consistent with the expected composition. Following mild, acid hydrolysis, this signal shifted to 2000, confirming that the two mannosyl phosphate linkages had been hydrolyzed. The relative locations of the two phosphate groups were then investigated by partial acetolysis, which is known to effect selective cleavage of the (1 + 6) linkages. The composition of each acetylated product in the acetolysis mixture was determined from the masses of their molecular ions in the f.a.b.-mass spectra. A characteristic molecular-ion was observed at 2192, consistent with the composition Man,HexNAcP, (where P is phosphate), confirming that both phosphate groups are so positioned that they can be retained on a Man6HexNAc fragment produced by partial cleavage of (1 + 6) linkages. Other molecular-ions were present at 1003 (Man,P), 1291 (Man,P), 1578 (Man,HexNAcP), 1579 (Man,P), 1867 (Man,P), 2154 (Man,HexNAcP), 2442 (Man7HexNAcP), 2480 (Man7HexNAcP,), 2688 ( Man8HexNAcP), and 2768 ( Man8HexNAcP2). All of these signals were accompanied by equally prominent signals that were 102 mass units higher. A plausible structure for this “plus 102” species is structure 20. This type of acyclic molecule is known to exist in acetylation reactions. CHzOR P O A c

AcO

doAc + H+

OAc OAc

20

Acetolysis together with f.a.b.-m.s. has been applied to the preliminary screening of glycoproteins for sugar type.68 In this procedure, -100 p,g of intact glycoprotein is subjected to an acetolysis time-course. At suitable intervals, aliquots are removed, quenched with water, and the peracetylated oligosaccharides that have been released from the glycoprotein are extracted into chloroform and analyzed by f.a.b.-m.s. without further purification. The molecular weights of the acetolysis fragments are diagnostic of the type (68) S. Naik, J. E. Oates, A. Dell, G. W. Taylor, P. M. Dey, and J. B. Pridham, Biochem Biophys. Res. Commun., 132 (1985) 1-7.

F.A.B.-MASS SPECTROMETRY OF CARBOHYDRATES

51

of glycan present, for example, high-mannose, complex, lactosaminoglycan, and so on. Sialic acid linkages are surprisingly resistant to acetolysis, and the molecular weights of oligosaccharides that still contain sialic acid residues allow the assignment of N-acetyl- or N-glycolyl-neuraminic acid. Methanolysis-f.a.b.-m.s. is probably the most elegant of the conjoint chemical-f.a.b.-m.s. procedures, because the reagents form a perfect solvent for adding the sample to the matrix, the acidic conditions enhance sensitivity, and results can be obtained in a very short time. A study of one of the glycolipids isolated from a human, embryonal Carcinoma cell-line illustrates how useful and how easy the technique can be.69 Earlier work on the lactosaminoglycan-containing glycoproteins of the same cell-line had revealed significant proportions of the disialyl moiety, NeuAc-NeuAc, and it was of interest to establish whether the same structure was present in any of the glycolipids. A novel, disialyl glycolipid was finally isolated, its presence being suggested by a characteristic signal at 1186 in the f.a.b. spectrum of its permethylated derivative. This signal confirmed that a structure of composition NeuAc,Hex,HexNAc, was present at the nonreducing end, but the f.a.b. data did not reveal whether the NeuAc residues were directly linked as NeuAc-NeuAc or whether they were separately attached to the N-acetyllactosamine. In principle, methylation analysis could have solved this problem. In practice, it was easier to use f.a.b.-m.s. As soon as the f.a.b. spectrum had been recorded, a small amount of methanolic HCl was added to the small quantity of sample remaining in the tube (from which aliquots had been taken for the f.a.b. experiment). After warming gently for 15 min at -40°, the mixture was loaded onto the f.a.b. target, and a second f.a.b. spectrum was recorded. The new spectrum contained a much diminished 1186 signal, and a new signal had appeared at 436 that corresponded to a Hex-HexNAc+ fragment-ion containing two free hydroxyl groups. Thus, within 15 min of the first f.a.b. result, it was clear that the glycolipid was not the one being sought, because the two NeuAc residues had to be separately attached in order to generate two hydroxyl groups upon their removal by the methanolic HC1. The final example selected for discussion demonstrates how f.a.b.-m.s. can show what problems may occur during procedures used for isolating or characterizing oligosaccharides. The behavior of N-glycosylically linked glycans during the reaction sequence of hydrazinolysis, N-reacetylation, and reduction with sodium borohydride has been investigated” by using (69) M. N. Fukuda, B. Bothner, K. 0. Lloyd, W. J. Rettig, P. R. Tiller, and A. Dell, 1. Biol. Chem., 261 (1986) 5145-5153. (70) J.-C. Michalski, J. Peter-Katalinic, H. Egge, J. Paz-Parente, J. Montreuil, and G. Strecker, Carbohydr. Res., 134 (1984) 177-189.

ANNE DELL

52

the two model compounds Man,GlcNAc,Asn and Man,GlcNAc( Fuc)GlcNAcAsn. The product mixtures obtained after the reaction sequence was completed were separated by liquid chromatography, and negative-ion f.a.b.-m.s. was used to define the molecular weight of each component. In addition to the desired products, namely, Man,GlcNAcGlcNAcol and Man,GlcNAc( Fuc)GlcNAcol, at least 7 other compounds were formed from each model compound, including unreduced molecules, hydrazones, and the products of osazone-type rearrangement and Wolff -Kishner reduction. 4. Linkage Assignment

In most studies, f.a.b.-m.s. does not reveal linkage positions. There is, however, one well-documented exception where linkage-site-specific fragmentation is observed, namely, permethylated molecules containing HexNAc r e s i d ~ e s . ~ ~ , It ~ ~was , ’ ’ shown earlier (see Section V,2) that such compounds are cleaved specifically at the HexNAc glycosidic linkages, to give abundant A,-type sequence ions. Not mentioned earlier is the fact that these ions may be accompanied by related ions that give information on the substitution pattern of the HexNAc residue. Provided that the sequence ions are lo. The resonance for N2,N2-di['3C]methylleucine moves upfield as the pH is increased, with a pK, of 7.4. The major resonance of the N-terminal N2,N2-di[13C]methylserine residue behaves unusually as the pH is increased. It may readily be seen that the dimethylated lysine species and (6Sa.b) K. Dill, R. D. Carter, and A. R. Katritzky, Int. J. Bid. MacrornoL, 8 (1987) 318-320.

I3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS

183

the dimethylated N-terminal species may be differentiated on the basis of pH studies. Further evidence for these assignments was educed with the aid of partial-methylation studies, the results of which are shown in Figs. 5 and 6. Partial-methylation studies of glycophorins AMNand AM,using limited proportions of formaldehyde, are depicted. Clearly, the resonances pertaining to the N-terminal dimethyl resonances appear first in the 13C-n.m.r.

p.p.m.

from

Me4Si

FIG. 5.-Proton-decoupled, "C-N.m.r. Spectra (at 22.5 MHz) of the Partial-reductive, [13C]MethylationStudies of 1.5 mM Glycophorin A M N in H20at 30".[Spectra of methylated samples were recorded at a sample pH of -7.3, and typically required 10.000-30,000 accumulations. Time-domain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 2.8 Hz was applied during the processing of the data. The n.m.r. traces in this Figure relate to the following ratios of forma1dehyde:glycophorin in the reductive methylation reaction: 0.0, 2.0, 3.0, 4.6, 5.4, and 50. The peak at 34.5 p.p.m. represents monomethylated lysine. (Taken from Ref. 56.)]

-

184

KILIAN DILL

I

I

91

Qo

I . 3

I

aa

-

46

40

p.p.m.

p.p.m.

from

a6

from M e 4 9

Me4Si

FIG. 6.-Proton-de~oupled,'~C-N.m.r. Spectra (at 22.5 MHz) of the Partial-reductive, ["CIMethylation Studies of 1.2 mM Native Glycophorin AM in H,O at 30" and of -1.5 mM Deglycosylated Glycophorin AM in H 2 0 at 30".[Spectra of methylated samples were recorded at a sample pH of 7.3,and typically required 15,000-60,000accumulations. Time-domain data were collected in 8192 addresses, with a recycle time of 1 s. A digital broadening of 2.8Hz was applied during the processing of the data. The peak at 34.5p.p.m. in the spectra of traces (A) and (B) represents monomethylated lysine. (A) Native glycophorin AM; the n.m.r. traces in the Figure relate to the following molar ratios of formaldehyde: glycophorin in the reductivemethylation reaction: 2,0, 8.0,14.0,24.0, and 74.0. (B) Deglycosylated glycophorin AM; the n.m.r. traces in the Figure relate to the following molar ratios of formaldehyde :glycophorin in the reductive-methylation reaction: 2.0, 4.0,8.0,16.0,and 75.0.Reproduced from Ref. 58, by permission of the publishers, Butterworth & Co (Publishers) Ltd. @ 1983.1

spectrum. The N6,N6-di["C]methyllysine resonances appear in the spectrum only after larger proportions of formaldehyde are added. The peak at -35 p.p.m. in these spectra represents N6-mono[ '3C]methyllysine. The work dealing with the reductive [ 13C]methylation studies of glycophorin glycopeptides (see later) fully corroborated these assignments. In order to obtain a preliminary view on how the oligosaccharides may influence the structure about the N-terminus, the 13C-n.m.r.spectra of the N-terminal proteins of glycophorins AMNand AM in various degrees of glycosylation were recorded; see Figs. 7 and 8.

'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS

185

n 4.0

7.3

9.0

FIG.7.-A Portion of the Aliphatic Region of the Proton-decoupled, '3C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively ['3C]Methylated Glycophorin AMN,Asialoglycophorin AMN, and Deglycosylated Glycophorin AMN,at pH Values of 4.0, 7.3, and 9.0, Respectively. [Time-domain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 3.0 Hz was applied to the data. All glycophorin samples were -1.2 m M (in H,O), and required 10,000-20,000 accumulations. (Taken from Ref. 57.)]

I3C-MN Native

1, 4.0

7.3

9.0

FIG.8.-A Portion of the Aliphatic Region of the Proton-decoupled, I3C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively ['3C]Methylated Glycophorin AM, Asialoglycophorin AM, and Deglycosylated Glycophorin AM, at pH Values of 4.0, 7.3, and 9.0, Respectively. [Timedomain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 3.0 Hz was applied to all data. All glycophorin samples were 1.2 m M (in H,O), and required 10,000-20,000 accumulations. Reproduced from Ref. 58, by permission of the publishers, Butterworth & Co (Publishers) Ltd. @ 1983.1

-

186

KILIAN DILL

On a gross, structural basis, it would appear that removal of the (Y-DNeuAc groups does not substantially perturb the structure about the Nterminus. However, deglycosylation of almost the entire molecule seems to have a profound effect; this structural effect about the N-terminus appears to be heavily associated with glycophorin AM (see Fig. 6 and laters8). 4. Work with Glycopeptides Derived from Glycophorin A, and with Some Related Peptides and Glycopeptides

One way in which to determine whether one part of the molecule may influence the structure about the N-terminus, or whether the assignments of the [13C]methyl resonances in the 13C-n.m.r.spectra of fully reductively [13C]methylatedglycophorins AMand ANare correct is to isolate the various glycophorin glycopeptides that have been produced by enzymic or chemical means. Fig. 9 shows a portion of the aliphatic region of the I3C-n.m.r. spectra of full reductively [13C]methylated glycophorins AM and AN, fully reductively [13C]methylatedtryptic glycopeptides from glycophorins AMand AN, and fully reductively [13C]methylated N-terminal glyco-octapeptides AM and AN. The tryptic glycopeptides were obtained by treatment of glycophorins AMand AN with tryp~in.~’ They contain a mixture of glycopeptides having amino acid residues 1-31 and 1-39. In both cases, three of the five lysine residues have been removed, and the I3C-n.m.r. spectra are indicative of this fact. The ratio of the integrated intensities of the N 6 ,N6di[ 13C]methyllysine residues to those of the N-terminal N2, N2di[13C]methylated residues is 2: 1, as expected. The I3C-n.m.r. spectrum of the fully reductively [ 13C]methylated tryptic glycopeptide AMindicated that this species still appears to contain a “minor” structural component. The AM and ANglyco-octapeptides were obtained by treatment of reductively [13C]methylated, intact or tryptic glycopeptides AM and AN with cyanogen bromide. This procedure cleaves the peptide bond on the Cterminal side of the protein, and converts methionine into homoserine.60 These glyco-octapeptides should contain no lysine residues, and this was confirmed by the spectra of the [ 13C]methylated glyco-octapeptides AMand AN shown in Fig. 9C. The resonances for NZ,N2-di[13C]methylleucine and N 2 ,N2-di[’3C]methylserine residues of the glyco-octapeptides resonate at the same position as their counterparts in the spectra of the reductively [13C]methylated, intact glycophorins AM and AN, except that some of the resonances are now narrower, as expected, due to the decrease in the size of the molecule. It should be noted that the “minor” component of glycophorin AM has now been lost. This result again indicated that it may result from a cross-linking of the glycoprotein.

I3C-N.M.R.-SPECTRAL STUDIES O F GLYCOPHORINS LYS 44.1

187

LYs 44.1

FIG.9.-A Portion of the Aliphatic Region of the Proton-decoupled, "C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively [13C]Methylated Glycophorins AM and AN, Tryptic Glycophorin Glycopeptides AM and AN, and Glycophorin Glyco-octapeptides AM and AN (All in H,O at 30"). [Time-domain data were accumulated in 8192 addresses, with a recycle time of 1-1.5 s. A digital broadening of 2.8 Hz was applied. (A) 1.6 mM reductively [13C]methylated glycophorin ANat pH 7.3, after 14,208 accumulations, and 1.5 mM reductively [13C]methylated glycophorin AM at pH 7.3 after 17,847 accumulations; (B) 1.6 mM reductively [13C]methylated, tryptic glycopeptides AN at pH 7.2 after 6600 accumulations, and 2.0 mM reductively [13C]methylated, tryptic glycopeptides AM at pH 7.4 after 6999 accumulations; (C) 0.5 mM reductively ['3C]methylated glyco-octapeptide AN at pH 7.2 after 27,948 accumulations, and 0.5 mM reductively [13C]methylated glyco-octapeptide AM at pH 7.2 after 31,985 accurnulations. Reproduced from Ref. 60 by permission of the publishers, Butterworth & Co (Publishers) Ltd. @ 1984.1

The pH dependence of these various glycopeptides should probably provide some insight as to their structural surroundings; such information is provided, and discussed, in Section 111,s. 5. pH-Titration Results Involving Reductively [ "CIMethylated Glycophorin, Glycophorin Glycopeptides, and Related Peptides and Glycopeptides

In order further to determine whether the reductive, ['3C]methylation technique possibly perturbs the structure of these glycoproteins and glycopeptides, natural-abundance '3C-n.m.r.-spectral data were obtained for unmodified, and for reductively ['3C]methylated, compound 10 (for struc-

KILIAN DILL

188

TABLEI Selected, I3C-N.m.r., Chemical-shift Data" for Glycopentapeptide 10 and Its Reductively ['3C]Methylated Derivative6' Chemical shift

Carbon atom

Glycopeotapeptide

Reductively ['3C]methylated glycopentapeptide

1'

100.6 99.9 99.3 51.2 70.0 69.2 72.8 62.6 23.5 54.9 56.4 68.5 58.8 58.4 77.0 78.8 19.5 19.1 44.8

100.6 99.9 99.2 51.2 70.0 69.6 72.6 62.7 23.6 54.8 60.1 68.5 60.5 58.8 58.5 77.1 79.0 19.6 19.2 -b

2'

5' 6' CH,(Ac-2') Ser C-2

Ser C-3

Thr C-2 Thr C-3

Thr C-4 Gly C-2

Not all of the spectral data are given. The "C-enriched-methyl resonance of the di["C]methylSer residue overlaps with this resonance.

ture; see Table 11). These data are tabulated in Table I. From the chemicalshift data, it was apparent that no great structural perturbation of the glycopentapeptide had occurred as a result of the reductive ['3C]methylation. One way in which to probe the structural surroundings of a protein is to monitor the pH behavior of specific carbon sites of the 13C probes. pHtitration studies, of given resonances, had previously been used for probing of the protein structure, because they are known to provide information concerning electrostatic (salt-bridging) interactions in the protein, neighboring group-ionizations, and local environment^.^^'^^'^' (66) L. R. Brown, A. DeMarco, R. Richarz, G. Wagner, and K. Wiithrich, Eur. 1.Biochem., 88 (1978) 87-95. (67) J. S. Cohen, L. J. Hughes, and J. B. Wooten, in J. S. Cohen (Ed.), Magnetic Resonance in Biology, Wiley, New York, 1983, pp. 130-247.

TABLEI1

13C Chemical-shift Data and Titration Data" for the N-Terminal Di['3C]methylamino Groups of Glycophorins, Glycophorin Glycopeptidg and Related Peptides and Glycopeptides Titration parameters

Compoundb

PK

Intact glycophorin AN (3) Intact glycophorin AM (4) Glyco-octapeptide AN (5) Glyco-octapeptide AM (6) Asialoglyco-octapeptide AN (7) Asialoglyco-octapeptide AM (8) Glycogho:in N! ( 9 ) Ser-Sy-Tty-Tkr-Gly (10) Ser-Sef-Th;-Th;-Glu (11) Leu-Ser-Thr-Thr-Glu (12) Leu-Ser-Thr-Thr-Glu (13) Ser-Ser-Thr-Thr-Glu (14) Ser-Ser-Thr-Thr-Gly (15) Leu-Ser-Thr-Thr-Gly (16) (16) D20f Leu-Ser-Thr-Asn-Glu (17) Ser-Ser-Ser (18) (18) D 2 0 J Ser-Ala (19) Ser-Gly (20) (20) D20' Ser-Tyr (21) Ser-Met (22) Szr (23) Ser (24) Hse (25) ThrCOOCH, (26) Thr-Val-Leu (27) Tlk-Thr (28) Ala-Ser (29) Val-Thr (30) Val-Gln (31) Val-Gly (32)

7.4, 7.7, 7.4, 7.8, 7.4, 7.5, 7.4, 7.4, 7.4, 7.g8 7.5, 7.4, 7.1, 9.3, 8.7, 9.3,

Hill coefficient (nr 0.97 0.85 0.80 0.87 1.13 0.88 2.00 1.66 1.08 1.35 0.93 1.41 0.86 -

Ad

6,

Chemical shift'

0.52

42.4 42.3 42.3 42.3 42.1 42.1 43.0 43.1 42.2 42.1 42.1 43.0 43.0 43.2 42.8 42.3 42.4 42.1 42.0 41.9

42.7 43.3 42.7 43.3 42.6 43.3 42.7 43.1 43.1 42.5 42.6 43.1 43.2 42.6 42.5 42.6 43.1 43.1 43.0 43.1 43.1 42.7 43.1 43.3 43.3 43.8 42.7 42.3 42.9 42.6 42.8 42.7 43 .O

-

0.87 0.73 0.74

0.59 0.56 0.59 0.62 0.68 0.12 0.13 0.65 0.50 0.71 0.12 0.12 0.18 0.46 0.47

-

-

-

8.4, 8.0, 7.g3 8.0,

1.05 0.91 0.81 0.98

0.20 0.83 1.03 1.13

-

a Titration data for the intact glycophorins and some related glycopeptides were taken from Ref. 64. * Indicates point of 0-glycosylation by an a-D-GalNAc group. ' Indicates point of 0-glycosylation by an a-D-Gal group. In cases where A is very small, the titration parameters may be error-prone (especially n ) . A represents the chemical-shift difference between the di['3C]methylamino group in the protonated and nonprotonated forms. Chemical shifts are given for the resonances at pH 7.3. In the case where the resonance titrates as a function of pH, the chemical-shift value given is determined from the theoretical fit of the data. Titration of some samples was performed in D,O, with a limited number of titration points.

'

KILIAN DILL

190

Because of the potential of gaining structural information for reductively [13C]methylatedglycophorins AM and AMby pH-titration studies, the pHtitration behavior of reductively, di[’3C]methylated glycophorins AM and AN, and 28 related, reductively di[13C]methylated glycoproteins, glycopeptides, and peptides in H 2 0 and D20were investigated. These results are presented in Table 11, and Figs. 10 and 11. The pH-titration data for the N-terminal N2,N2-[13C]dimethylamino species were analyzed for the best pK, values and Hill coefficients ( n ) by using the following equation. A~o~(PK-PH) + 10n(pK-pH)’

In this case, ST is the best, theoretical, I3C chemical-shift value, SB is the chemical shift of the di[ ‘3C]methylamino group in the nonprotonated form, and A is the chemical-shift difference between the di[ 13C]methylamino group in the protonated and nonprotonated forms. The best fit was obtained when Ci [ST(i)- 6,,b~(i)]~ was minimized; Sobs is the chemical-shift value observed at that given pH value.

2

3

4

5

6

7

8

9

10

11

12

PH FIG.10.-pH-Dependence of the 13C Resonances for the Di[”C]methylated N-Terminal Amino Groups of Tri-L-Ser(A, 18). Glyco-octapeptide AN ( 0 , 5 ) ,and Asialoglyco-octapeptide AN (0, 7). (Taken from Ref. 61.)

'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS

191

44

42

2

3

4

5

6

7

8

9

1

0

1

1

1

2

PH FIG. 11.-pH-Dependence of the 13C Resonances for the Di[13C]methylated N-Terminal Amino Groups of Glyco-octapeptide AM (0,6), Asialoglyco-octapeptide AM (0,S), and glycopentapeptide 10. (Taken from Ref. 61.)

Several results are quite apparent from the data shown in Table 11. It is evident from the pentapeptide model compounds that substitution of amino acid residues at positions 4 and 5 does not significantly affect the structure about the N-terminus. This observation corroborated earlier work6* from agglutination-inhibition assays, which demonstrated that the nature of the amino acid at position 4 of the peptide (or glycopeptide) is not a requirement for specificity. The results also showed that glycosylation at amino acid positions 2, 3, and 4 appears to influence the structure about the N-terminus. The structural influence of neighboring glycosylation had been observed by Dill and coworkers.69 Therefore, if the structure about the N-terminal NH2 group is primarily responsible for the display of the MN blood-group antigens, the carbohydrate residues of the glycoprotein must play a role in the antigenicity. This phenomenon may be enhanced when bulkier oligosaccharide units (1) (68) J. P. Cartron, B. Ferrari, M. Huet, and A. A. Pavia, Exp. Clin. Imrnunogener., 1 (1984) 112-1 16. (69) K. Dill, R. E. Hardy, M. E. Daman, J. M. Lacombe, and A. A. Pavia, Curbohydr. Rex, 108 (1982) 31-40.

192

KILIAN DILL

are attached to amino acid positions 2, 3, and 4 of the glycoproteins, especially in glycophorin AM. Many of these results were corroborated, as discussed in the next Section, dealing with N2-mono[13C]methylspecies. The results in Table I1 also indicated that, from the I3C-n.m.r. titration data for the glycoprotein and glycopeptide species that contain an N 2 ,N2di[13C]methylSerresidue, all exhibit unusual pH behavior, and A is either very small, or zero. This may be explained on the basis that some unusual steric or hydrogen-bonding phenomena are associated with the serine residues. In fact, a number of possible hydrogen-bondings schemes have been associated with serine (and threonine) resid~es.''-'~ One that is particularly relevant to the present case deals with a strained, internal hydrogenbond between N2-H and 0-4. The results in Table I1 suggest that the titrations of the N-terminal N2,N2-di['3C]methylserine residues are influenced by the solvent ( H 2 0 or D,O); compound 18 titrates (A+0.12) in H20, and also titrates in D20, but then A is zero. These results indicate that, in glycophorin AM,a weak, internal hydrogen-bond may influence the structure about the N-terminus.

6. pH-Titration Studies Involving M~no['~C]methylatedGlycopeptides and Peptides Related to the N-Terminus of Glycophorins Another way in which to gain structural information concerning the N-terminal residue of glycophorins AM and AN is to study the N-terminal, m~no['~C]methyl derivatives; these are produced by using limited amounts of [ '3C]formaldehyde. There are distinct differences between the N2,N2di[13C]methylamino and N2-mono['3C]methylamino species: ( i ) a significant, chemical-shift difference exists between the N-terminal dimethyl ; of the 13Cresonances and monomethyl species (43 and 34 ~ . p . m . )(~i i~) all of the N-terminal dimethyl species move upfield as the pH is increased (if they move at all), whereas all of the I3C resonances of the N-terminal, monomethyl species move downfield as the pH is increased64; and ( i i i ) A for the N-terminal monomethyl species tends to be much larger than that for the N-terminal dimethyl species.64 Point ( i i i ) would tend to indicate that it may be more advantageous to study the N-terminal monomethyl species. However, because of allowable protein concentrations, detection limits on available instruments, and technical difficulties, it has thus far (70) R. E. London, J. M. Stewart, R. Williams, J. R. Cann, and N. A. Matwiyoff, J. Am. Chem. Soc., 101 (1979) 2455-2469. (71) A. Aubry, N. Ghermani, and M. Marraud, Int. J. Pept. Protein. Ree, 23 (1983) 113-122. (72) M. Marraud and A. Aubry, Int. 1. Pept. Protein Rex, 23 (1983) 123-133. (73) D. Peters and J. Peters, J. Mol. Srrucr., 90 (1982) 305-320. (74) D. Peters and J. Peters, 1. Mol. Struct., 90 (1982) 320-334.

'3C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS

193

been found difficult to study I3C-labeled, intact glycophorin, N-terminal monomethyl labels. Table I11 gives the titration parameters for the I3C resonances of the N-terminal m~no['~C]methyl labels of a variety of peptides and glycopeptides related to the N-terminus of glycophorins AM and AN. The titration TABLE111 '3C Chemical-shift Data and Titration Data for the N-Terminal M~no['~C]methylamino Groups of Glycophorin-related Glycopeptides and Peptidesu Titration parameters Hill coefficient Compound"

PK,

(n)

A*

6,

Chemical shift'

Ser s::-T$-~$-cI~ (11) Leu-Ser-Thr-Thr-Glu (12) Leu-Ser-Thr-Thr-Glu (13) Ser-Ser-Thr-Thr-Glu (14) Ser-Ser-Thr-Thr-Gly (15) Leu-Ser-Thr-Thr-Gly (16) (16) D20d Leu-Ser-Thr-Asn-Glu (17) Ser-Ser-Ser (18) (18) D,Od Ser-Ala (19) Ser-Gly (20) (20) D20d Ser-Tyr (21) Sy-Met (22) Ser (24) Hse (25) ThrCOOCH, (26) Thr-Val-Leu (27) fir-Thr (28) Ala-Ser (29) Val-Thr (30) Val-Gln (31) Val-Gly (32)

7.80 7.8, 8.1, 7.7, 7.7, 8.1, 8.22 8.1, 7.53 7.7, 7.9, 7.9,

0.97 1.05 1.00 0.93 1.04 1.09 0.97 1.02 1.02 1.12 0.98 1.04 0.95 1.00 1.09 1.14 1.03 0.76 0.92 1.16 1.13 0.98 1.07 1.12

-1.57 -1.28 -1.19 -1.51 -1.37 -1.13 -1.19 -1.19 -1.49 -1.49 -1.43 -1.49 -1.55 -1.32 -1.49 -1.31 -1.19 -0.98 -1.44 -1.19 -1.56 -1.19 -1.07 -1.01

34.6 34.2 34.2 34.5 34.5 34.2 34.1 34.2 34.4 34.3 34.4 34.5 34.4 34.2 34.5 34.4 34.7 35.2 34.9 34.9 34.1 35.0 34.9 34.8

33.7 33.1 33.2 33.4 33.5 33.2 33.1 33.2 33.5 33.2 33.2 33.3 33.1 33.2 33.4 33.1 33.5 34.8 34.0 34.1 33.7 34.0 33.8 33.8

8.0, 7.7, 7.7, 8.9, 9.6, 7.14 7.4, 7.5, 8.43 8.0, 8.1, 8.4,

* Indicates a point of 0-glycosylation by an a-D-GalNAc group. Indicates the point of 0-glycosylation by an a-D-Gal group. A represents the chemical-shift difference between the mono[ 13C]methylamino group in the protonated and nonprotonated forms. Chemical shifts are given for the resonances at pH 7.3. In the case where the resonance titrates as a function of pH, the chemical-shift value given is determined from the theoretical fit of the data. Titration of some samples was performed in D,O, with a limited number of titration points.

194

KILIAN DILL

results seem to be in agreement with the conclusions drawn from the N-terminal di[ "Clmethylated species discussed in Section 111,5, although some of the results are much clearer. The data in Table 111 also indicate that titration of the N-terminal, mon~['~C]rnethyl label may be influenced by the nature of the adjacent, amino acid residue. 7. Implications of These Results in Regard to the Display of the MN Blood-group Determinants It has already been indicated that three functional groups on glycophorins AMand ANare involved in the display of the MN blood-group determinants: the lysine residues, the N-terminal amino groups, and the carbohydrate residues. From the work in the preceding Sections, and from the data given in the next Section, a number of facts concerning the structures of the MN blood-group determinants may be obtained. It may definitely be concluded that one (or more) of the lysine residues plays a small role in determining the N-terminal structure of the glycophorins. This may best be judged by the work on glycophorin AM (see the preceding) and glycophorin BN (see later), and supports earlier work indicating that the lysine residues may be important in the display of the MN blood-group determinants. The other two functional groups that play a crucial role in the make-up of the MN blood-group determinants are the carbohydrate residues and amino residues 1 and 5 in the protein sequence of glycophorins AM and AN.Various chemical modifications and enzyme digests, along with hemaglutination assays, led to the conclusion that the N-terminal NH2 group and the a-D-NeuAc groups are indeed important. However, it must be pointed out that the various immunoglobulins and lectins may be specific for the N-terminal amino group or the carbohydrate residues. Certain evidence indicates that structural differences do exist between glycophorins AMand AN that lack the a-D-NeuAc groups, and also that these may not necessarily involve the N-terminal amino g r o ~ p s . ' ~ - ~ ~ The results concerning the N-terminal structures of glycophorins AMand AN were based on the labels that were placed on the crucial, N-terminal amino group, and they clearly showed that the amino acid residues at position 5 may play only a minor, if any, role in determining the structure of the MN blood-group determinants. The carbohydrate residues appear (75) W. J. Judd, P. D. Issitt, B. G. Pavone, J. Anderson, and D. Aminoff, Transfusion, 19 (1979) 12-18. (76) Y.Ochiai, H. Furthrnayr, and D. M. Marcus, J. ImmunoL, 131 (1983) 864-868. (77) W.L. Bigbee, M. Vanderleen, S. S. N. Fong, and R. H. Jensen, MoL Immunof., 20 (1983) 1353-1362.

'-'C-N.M.R.-SPECTRAL STUDIES OF GLYCOPHORINS

195

to play a crucial role in the structure about the N-terminus; the disaccharide may be more important than the cy-D-NeuAcgroups. The hydrogen bonding that may occur intra- or inter-molecularly in glycophorin AM also appears to be particularly important. IV. LABELINGSTUDIESOF GLYCOPHORIN B 1. Relationship of the Results to Those Obtained for Glycophorin A

As mentioned earlier, glycophorin B carries the N and the Ss blood-group antigens. It is known that the first 26 residues of the amino acid sequence are identical to those in the N-terminal portion of glycophorin AN. Moreover, relative to glycophorin A, it has a shortened amino acid chain, comprising -35 amino acid residues at the C-terminus. It is also known to contain -4 lysine residues. Fig. 12 shows a portion of the aliphatic region (30-50 p.p.m.) of the proton-decoupled, 13C-n.m.r.spectra63of fully reductively [I3C]methylated glycophorin AMNand glycophorin BN. Glycophorin B was isolated from heterozygous, red-blood cells, and was then separated from glycophorin AMNby gel-filtration chromatography on A m m ~ n y x - L o . ~ Clearly, . ~ ~ the

A

B

FIG. 12.-A Portion of the Aliphatic Region of the Proton-decoupled, "C-N.m.r. Spectra (at 22.5 MHz) of Fully Reductively Methylated Glycophorin AMN and Glycophorin BN, in H,O at 30". [Time-domain data were accumulated in 8192 addresses, with a recycle time of 1 s. A digital broadening of 3.0 Hz was applied: (A) 1.5 mM reductively, ['-'C]methylated glycophorin AMN, at pH 7.2, after 12,815 accumulations; (B)0.3 mM reductively, [*-'C]methylated glycophorin BN, at pH 7.1, after 67,874 accumulations. (Taken from Ref. 63.)]

KILIAN DILL

196

spectrum of glycophorin BN is considerably different from that of glycophorin AMN,indicating that the separation of glycoproteins was successful. The spectrum of glycophorin BN lacks the resonance of N 2 , N 2 di['3C]methylserine that is associated with glycophorin AM. However, it does contain the resonance at 42.8 p.p.m. that is associated with N 2 , N 2 di['3C]methylleucine, which is expected, because glycophorin BN contains leucine as N-terminal amino acid residues. In order to determine the number of lysine residues present in glycophorin B, the resonance for N6,N6-di['3C]methyllysine (44.1 p.p.m.) was integrated relative to the resonance for N2,N2-di[13C]methylleucine (42.8 p.p.m.). In this case, no differential Tl values or n.0.e. values should be observed for these resonances. A value of 3-4 lysine residues was obtained from integration studies63;higher accuracy could not be achieved because of the signalto-noise of the spectrum. However, these results corroborated the earlier finding that glycophorin B contains a smaller number of lysine residues than glycophorin A. 2. pH-Titration Studies of Glycophorin B. Relationship of the Results to Those Observed for Glycophorin AN and Glyco-octapeptide A N

Fig. 13 shows the effects of pH on the N-terminal N2,N2-di[13C]methylleucine resonances of glycophorin AN, glyco-octapeptide AN, and glycophorin BN. The titration parameters are listed in Table 11. The titration of N2,N2-di['3C]methylleucine of glycophorin B N resembles that of N 2 , N 2 -

43.2

-

I

I

I

I

I

I

I

I

-

c

fc-

42.8

-

-

.O 42.6

-

-

5

0

k

L

0

42.4

-

42.2

-

I 4

I

5

I

6

I

7

I

8 9 PH

I

1

I

I

0

1

1

FIG. 13.-pH-Dependence of the I3C Resonances for N-Terminal di[13C]methylleucine of Glycophorin AN, Glycophorin BN, and N-Terminal Glyco-octapeptide AN [(GO(N)]. (Taken from Ref. 63.)

I3C-N.M.R.-S PECTRAL STUD1ES 0 F G LYCOPHORI NS

197

di['3C]methylleucine of glycophorin AN and glyco-octapeptide AN. In fact, the titration curve for the methylated glycophorin BN is more similar to that of ['3C]methylated glyco-octapeptide AN than to that of [ I3C]methylated intact glycophorin AN. The results just mentioned have several implications. One is that some other portion of the intact glycophorin AN molecule seems to influence the structure of glyco-octapeptide AN, which results in different titrationpatterns for the N-terminal N2,N2-di['3C]methylleucineresidues of reductively methylated, intact glycophorin ANand glyco-octapeptide AN.Another interesting feature of Fig. 13 is that the titration curve for the N 2 , N 2 di[13C]methylleucine residue of reductively [ I3C]methylated glycophorin BNresembles that of the N2,N2-di[13C]methylleucineresidue of the reductively ['3C]methylated glyco-octapeptide AN more than that of reductively [I3C]methylated, intact glycophorin AN, indicating that some portion of the glycophorin B molecule does not influence the structure about the Nterminus which the intact glycophorin AN molecules seem to have. This difference about the N-terminus may result from the fact that glycophorin A severely aggregates in solution. For glycophorin B, on the other hand, the mode, and, perhaps, the degree, of aggregation may be slightly different.

V. CONCLUSIONS, A N D PROGNOSIS FOR FURTHER STUDIES The possible role that the lysine residues, N-terminal amino acid residues, and carbohydrate residues may play in the display of the MN blood-group determinants by glycophorins AM, AN, and BN has been investigated. Assuming that the N-terminal amino acid in each of these glycoproteins plays a prominent role in the display of the M N blood-group determinant, 13 C labels were placed on the N-terminal amino acid residues of the glycoproteins. As a result of the work, a number of conclusions may be drawn concerning the display of the MN blood-group determinants. (i) A portion of the glycoprotein, not near the N-terminus (possibly lysine residues) may influence the structure about the N-terminus. (ii) Amino acid substituents at position 4 or 5 of the glycophorin AM and AN sequence play only a little, or no, role in determining the structure about the N-terminal amino acid residue. (iii) Titration studies of the various glycopeptides indicated that the carbohydrate residues, especially D-Gal and D-GalNAc appear to influence the structure about the N-terminus significantly. There are a number of studies that need to be made in order to provide further information about the structure of the N-terminal amino acid residues. One crucial set of experiments revolves around determination of the exact structure of the minor component observed in reductively

198

KILIAN DILL

[ '3C]methylated glycophorin AM;work in this laboratory indicates that it results from head-to-head dimerization of glycophorin AMmolecules. This phenomenon could be studied by isolation of the cross-linked product (intact, or enzymically digested). These studies could provide information about the nature of the glycophorin A aggregates in aqueous solution.

ACKNOWLEDGMENTS The author thanks Alicia Brown for typing this article. He also acknowledges the financial support of DHEW (BRSG grant), the Research Corporation, and the South Carolina affiliate of the American Heart Association.

ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY. VOL. 45

THE CHEMISTRY AND BIOCHEMISTRY OF THE SWEETNESS OF SUGARS

BY CHEANG-KUAN LEE Department of Chemistry, National University of Singapore, Kent Ridge, Singapore 051 1

I. Introduction ............................................................ 11. Stereochemistry of Sweetness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Early Theories ....................................................... 2. Fundamental Structural Requirements for Sweetness . . . . . . . . . . . . . . . . . . . . . . 3. Sweetness-Structure Relationship for Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Bitterness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The AH,B Concept for Bitterness.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Lipophilicity and Bitterness. . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Bitterness-Sweetness Relationships . . . . . . . . . . . . . .................... IV. Biochemistry of Sweetness . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. The Sensory System .................................................. 2. The Peripheral Mechanisms in Taste. 3. Taste-receptor Binding . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The Quality of Sweetness.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Methodology of Measurement of Sweet Taste.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

199 201 202 207 238 310 312 318 320 325 325 326 328 339 349

Science at its best provides us with questions, not absolute answers. Norman Cousins (1976)

I. INTRODUCTION Although always a matter of popular fascination, the sense of taste was once only a subject of academic interest, but it is now of great practical and economic importance. Deep esthetic enjoyment is experienced through the sense of taste; we eat food, not nutrition, and food will preferably be eaten only if it is palatable and attractive. In civilized countries, it is far from true that a hungry man will eat anything. Commercial interest in the sensory properties of foods during the past decade has led to a great increase in basic knowledge of the phenomenon. 199

Copyright @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

200

CHEANG-KUAN LEE

However, as Lord Zuckerman’ pointed out “our understanding of taste physiology (and of flavour technology) is abysmal.” Sweetness is a gustatory response evoked by most sugars, and is relished by man as well as by many other organisms. The desire for sweetness appears to be universal and undisputed. Today, sugar, or one of its synthetic substitutes, appears in more articles of the diet than any other food ingredient, except, perhaps, common table salt.2 Sweetness is a quality that defies definition, but whose complexity can be appreciated merely by examining the molecular structures of those compounds that elicit the sensation. They come in all molecular shapes and sizes, and they belong to such seemingly unrelated classes of compounds as aliphatic and aromatic organic compounds, amino acids, peptides and proteins, carbohydrates, complex glycosides, and even certain inorganic salts. The phenomenon of sweetness has been of interest from the time of the Ancient Greeks. Theophrastus (372-287 B.C.), who succeeded Aristotle at the Lyceum in Athens, wrote a review3of the subject in his book De Sensibus. Since then, many attempts have been made4 to correlate chemical structure with sweet taste, but most of them are of limited value. These will, therefore, be discussed only briefly in the present article. A major advance in the evolution of taste theory came with understanding that the primary event in the initiation of a taste response involves the interaction of a stimulant molecule with a receptor located at the taste-cell plasma-membrane. Further progress in our understanding of the phenomenon, although slow, has been significant. Considerable interest in the mechanism of taste perception was stimulated by the Shallenberger AH,B hypothesis proposed’ in 1967. A comprehensive discussion will be provided of the influence of structure on the taste of sugars and sugar derivatives, and the effect of the structure of some high-intensity, noncarbohydrate sweeteners on taste will also be considered where it helps to elucidate the mechanism involved. Because many sweet compounds also taste bitter, a brief risumC of the role of structure on bitterness will also be incorporated. The strengths and deficiencies of the various hypotheses will be highlighted, and critically interpreted.

(1) Lord Zuckerman, British Nutrition Foundation Annual Lunch, 1975. (2) R. M. Pangborn, in G . E. Inglett (Ed.), Symposium: Sweeteners, AVI, Westport, CT, 1974, pp. 23-44. (3) Theophrastus, D e Sensibus, G . M. Stratton (Ed.), Bonset-Schippers, Amsterdam, 1964. (4) R. W. Moncrieff, The Chemical Senses, 3rd edn., CRC Press, Cleveland, Ohio, 1967. (5) R. S. Shallenberger and T. E. Acree, Nature, 216 (1967) 480-482.

CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS

201

One reason for the seemingly slow progress of understanding is the interdisciplinary nature of sweetness research.6 The conclusions that can be drawn, from, for example, physiological and psychophysical experimentation¶ must be related to what is known of the structural chemistry of the stimulus and how it may interact at the molecular level. All too often, it is not appreciated that one particular line of experimentation cannot be viewed in isolation, but must relate to other disciplines. Only by fully understanding all of the associated events leading to sweetness perception shall we understand the mechanism of sweetness perception itself. The theories that attempt to describe the initial event in sweet-taste stimulation will be discussed, as will some of the practical attempts to isolate sweet-receptor molecules. Relevant, behavioral data will be examined, particularly where the effects of molecular structure on behavior responses are being evaluated. The evaluation of sweet compounds by taste panels is crucial to the development of worthwhile structure-taste relationships. Many structuretaste studies have employed dubious, taste-panel techniques. Therefore, a critical examination of structure-taste data in the light of this observation is relevant, as are recommendations for a consistent approach in utilizing taste techniques.* Observations that have been made will be critically discussed in the light of their relevance to other approaches that attempt to elucidate mechanistic features of this complex phenomenon. No claim is made for a complete coverage of the literature, but an attempt has been made to collate all relevant information and to discuss the results that best illustrate the principles involved. OF SWEETNESS 11. STEREOCHEMISTRY

Interest in the chemical senses dates back to the earliest records of philosophic speculation. Theophrastus3 referred to the work of Democrites, who suggested that “Sweetness consists of atomic figures that are rounded and not too small; wherefore, it quite softens the body by its gentle action, and unhesitatingly makes its way throughout. Yet it disturbs the other savours, for it slips in among the other atomic figures and leads them from their accustomed ways and moistens them.” This is probably the earliest attempt to explain the phenomenon of sweetness. We have progressed a (6) M. G . Lindley, in G. G. Birch and K. P. Parker (Eds.), Sugar: Science and Technology, Applied Science, London, 1979, pp. 403-413.

* In this article, all numerical comparisons of relative sweetness are given on a molar basis.

202

CHEANG-KUAN LEE

long way since then, but our world is so predominantly visual that we tend to belittle the importance of the chemical senses, perhaps the oldest and most widely distributed of the senses in both vertebrates and non-vertebrates. This is evident from the fact that, despite the naked exposure of the taste buds in the tongue of man and other vertebrates, as well as on the fins of fishes, we have not been able to learn more about how a taste-receptor cell functions than just that it responds to chemical stimuli and transmits impulses to the brain in its associated nerve fiber. Sadly, it is probably true that our modern understanding of the phenomenon of sweetness, although more completely described and more accurately measured, is only a little nearer to an absolute explanation. 1. Early Theories

The most comprehensive attempt to relate chemical structure to taste is that of C ~ h n In . ~his classic book Die organischen Geschmacksrofe, published in 1914, he proposed that all sweet and bitter stimulants contain more than one functional group in their molecules. A multiplicity of hydroxyl groups gave sweetness, and these and a-amino acid groups and other sweet-eliciting or “sapophoric” groups were termed “glucogenes.” He also noted that these groups usually occur in pairs (see Table I). Cohn also pointed out’ that the increase of molecular weight as a homologous series is ascended is often accompanied by a gradual change in the taste of the members from sweet to bitter; for example, for the glycols, ethylene glycol, sweet; trimethylene glycol, sweet; 1,2-propyIene glycol, sweetish; tetramethylene glycol, less sweet; and hexamethylene glycol, bitter. It was suggested that the size of the sapophoric, taste-inducing group is important in lower members of the series. In the higher members, however, having larger molecules, that group is now relatively small and becomes less significant. By this, Cohn probably implied steric effects attributable to the rest of the molecule. He also noted the diminished solubility of the higher members (tastelessness eventually resulting when the compound becomes insoluble) but he was, however, unable to arrive at a hypothesis relating the changes in taste to the chemical structure. Following the success of the chromophore-auxochrome theory of dyeing during the latter half of the 19th century, Oertly and Myers’ proposed that a sweet-tasting substance must possess an “auxogluc” and a “glucophore.” This is an extension of Cohn’s observation that sapophoric groups occur in pairs. The glucophore was defined as a group of atoms having the power to form a sweet compound when combined with any, otherwise tasteless, ( 7 ) G . Cohn, Die Organischen Geschmackmfe, Siemenroth, Berlin, 1914. (8) E. Oertly and R. G . Myers, J. Am. Chem. SOC.,4 (1919) 855-867.

203

CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS

TABLEI Cohn’s Sapophoric Groups Group (OH),

Category

Taste

polyhydroxy

sweet

a-amino acids

sweet

=N--O-CH,CO,H

oximacetic acids

sweet

-N =N+= N-

azides

sweet

=N-OH

oximes

sweet

-CN

nitriles

sweet

a-ketocarboxylic acids

sweet-bitter

polynitro

bitter

nitrosulfonic acids

bitter

tertiary amines

bitter

quaternary ammonium salts

bitter

betakes

bitter

-SH

thiols

bitter

-S-

sulfides

bitter

-s-s-

disulfides

bitter

thioamides and thioureas

bitter

NH2

/

-C

\

CO, H

0

\/ I C

‘C02H

/

-N

\ I

S CS of

II

-C

-NH-

204

CHEANG-KUAN LEE TABLEI1

Oertly and Myers' Auxogluc and Glucophore Components of Sweet-tasting Compounds Compound

Formula

Aux ogI uc

Glucophore -CH-CH-

I

-H

I

OH O(H) -CH3

-CH-CH,

I

I

-CH,CHJ

OH OH -CH-C02H

-CH,CH,CH,

I

NH

0 +/

-CH,-O-N

\0-

/

-C

H34 -CH,OH

\ CL H3.n

I I

OH

H2.m

c-c-

I 1

I

CH3-CH-

x,

X"

--CH2--CH,OH OH

I

-(CH2),-CHzOH Ethylene glycol

H2C-CH-

H2C-CH,

I

I

1

Glycerol

1

I

HO OH OH D-Glucose

I

I

I

I

I

H

/

HO OH

H H OHH H2C-C-C-C-C-C

I

-CHZOH

H2C-CH-

H2C-CH-CH2

I

-H

I

HO OH

HO OH

/

0

0

I

HOHO O H H OH \ H

-c-c

Glycine

H2C-CO2H

-CH--CO,

NH2

NH2

Chloroform

CHCI,

I

-CHZOH

I

OH \ H

I

-CCI,

H

-H

-H

0 Ethyl nitrate

H3C-CH2-0-N

+ /

No-

-CH3

CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS

205

auxogluc. Restricting their efforts to sweet aliphatic compounds, they identified six glucophores and nine auxoglucs (see Table 11). The theory succeeded in bringing some order to the mass of empirical data, but it was inadequate in many respects. In particular, it could not account for the sweetness of such nonhydroxylated, but intensely sweet, compounds as saccharin (1,2-benzisothiazolin-3-one 1,l-dioxide) and dulcin [( p ethoxypheny1)ureal. Furthermore, it offered no consideration of the effects of stereoisomerism. For example, many compounds, such as amino acids, are sweet in one optically active form, and less sweet, tasteless, or even bitter in the other. Therefore, it would appear that taste is dependent not only upon the nature and number of groups in the molecule, but also their molecular geometry. All of the sweet compounds that contain Cohn’s sapophoric groups and Oertly and Myer’s glucophores contain hydrogen as an auxogluc. Substitution of this hydrogen atom often affects the taste. This led Kodama’ to propose the “vibratory hydrogen” hypothesis. Kodama’s vibratory hydrogen atom was viewed as being a form of tautomerism, and the transposition of the hydrogen atom to afford the different tautomeric forms would result in “electronic vibrations.” He therefore concluded that the taste of organic compounds is due to electronic vibrations of the sapophoric hydrogen atom. The taste of various amino acids, sugars, and aliphatic nitro compounds was studied, and it was concluded that the distance over which this hydrogen atom migrates, to give a second tautomeric form, determines the sweetness. In the case of saccharin, the sweetness was explained as due to two tautomeric forms.

Kodama’ proposed three additional rules governing the relation between taste and structure. These were (1) optical isomers have different tastes; (2) substitutions always affect the taste; and (3) the taste of electrolytes is due to the sum of the tastes of the molecular electrolyte, the anions, the cations, and complex ions. The taste of saccharin was further studied,” and it was found that (1) the alkaline-earth-metal salts are sweet, whereas the heavy-metal salts are astringent; (2) the sweet taste is lost if the sulfimide ring is cleaved, or if (9) S. Kodama, J. Tokyo Chem. SOC.,41 (1920) 495-534. (10) A. F. Hollerman, Red. Trav. Chim. Pays-Bas, 42 (1923) 839-845.

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CHEANG-KUAN LEE

the sulfimide nitrogen atom is alkylated or acylated; (3) substitution in the benzene ring diminishes the sweet taste, and introduces a bitter taste; and (4) the substitution of a halogen atom on C-6, that is, para to the carbonyl group, results in a steady decrease in sweetness, with concomitant increase in bitterness. The effect of halogen increased with its atomic weight. These early successes generated considerable interest in the relationships between taste and chemical composition. Attempts were made to relate various physical, chemical, and physicochemical properties of compounds to their sweet taste. Beck” reported that the relative sweetness of sugars could be correlated with the “contraction coefficient,” that is, the ratio of the sum of the atomic volumes to the molecular volume of the sugar. In a closely related series of compounds, such as a group of sugars, some relationship seems to exist between sweet taste and s~lubility.’~*’~ In acids and esters, a decrease in taste was observedI4 to accompany an increase in molecular weight. The taste of isomeric esters and acids was, however, irregular. In such compounds as the halogenated aminonitrobenzenes, the melting points correlated excellently with s ~ e e t n e s s . ’This, ~ however, fails with other groups of sweet compounds. The melting point of a crystal or the solubility of such compounds as sugars, is indicative of nonbonded, chemical interactions, but the theory of the existence of weak, nonbonded, chemical interactions was just becoming established at the time when these observations were made. Barral and Rand6 however, stated that there was no general law by which the taste of any compound could be predicted. Finizi and Colona” came to the same conclusion in the late 1930’s, after a very critical survey of the taste of aromatic compounds. They stated that sweet taste depends not on any factor, such as a sapophoric group, but on the entire chemical structure of the particular compound. These statements were of particular significance, because this was the first acknowledgment of the fact that the earlier theories of sweetness were gross oversimplifications of an extremely complex situation.

( 1 1 ) G.Beck, Wien. Chem. Zrg., 46 (1943) 18-22. (12) N. E. Loginov, Pishch. Promsr., 1 (1941) 32.

(13) H. T. Andersen, M. Funakoshi, and Y. Zotterman, in Y. Zotterman (Ed.), Oljacrion and Tasre, I, Pergamon, Oxford, 1963, pp. 177-192. (14) Y. Renqvist, S h n d . Arch. Physiol., 38 (1919) 97-201; 40 (1920) 117-124. (15) J. J. Blanksma, W. J. van den Broek, and D. Hoegen, Recl. Trau. Chim Pays-Bas, 65 (1946) 329-332. (16) F. Barral and A. Ranc, Rev. Sci., 56 (1918) 712-723. (17) C. Finizi and M. Colonna, Garz. Chim. Iral., 68 (1938) 132-142.

CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS

207

2. Fundamental Structural Requirements for Sweetness

By 1938, one fact was clearly established. Sweet compounds, unlike salty and sour compounds, are found in all classes of chemical compounds, including such inorganic salts as beryllium (“glucinium”) and lead salts. They are also found among compounds of all molecular shapes and sizes, and stereochemical changes may result in a very dramatic change in the taste, as seen in the gustatory differences between enantiomorphs. Kaneko” pointed out that stereo-structure was an obvious clue to this behavior. He emphasized that, whereas D-amino acids are usually sweet, the L enantiomers are generally tasteless or bitter; their tastes are not related to their dextro- or levo-rotatory power, but rather to the result of certain molecular configurations. A series of a-amino acids was synthesized, and the tastes were studied.” The results (see Table 111) showed that the D-amino acid of the general formula RR’C(CH2)C02H,will taste sweet only when R is a hydrogen atom or any homolog of the methyl group, and R is smaller than a propyl group. The great difference in taste between the D- and L-amino acids clearly confirmed that taste depends not only on the groups present but also on their arrangement in space. The importance of configuration in the sugar series’’ in determining the differences in taste was also recognized, but, in this class of compounds, there are several asymmetric centres in the molecule, each of which may or may not affect the overall taste-properties. Early studies” implicating the configuration of carbon atoms bearing hydroxyl groups (of sugars) with taste properties led to the generalization that the a is a more effective stimulant than the @ anomer. However, the results2’ obtained since then definitely show that this generalization is not only a gross oversimplification, but is basically untrue, in that the anomeric configuration alone does not directly affect the sweetness of the molecule. For example, @-D-glucose is perceived to be significantly sweeter than a-D-glucose when the crystals are allowed to dissolve in the mouth,21 whereas @-lactose is nearly twice as sweet as a-lactose.2”22It was e ~ t a b l i s h e dthat ~ ~ it is only because of changes in the bitterness of sugar molecules resulting from a change in the anomeric configuration that the overall sweetness of a sugar molecule might in turn be enhanced or depressed. T. Kaneko, Nippon Kagaku Zasshi, 59 (1938) 433-439; 60 (1939) 531-538. A. R. Lawrence and L. N. Ferguson, Narure, 183 (1959) 1468-1471. G. G. Birch, Crif.Rev. Food Sci. Nufr., 8 (1976) 57-95. R. S. Shallenberger and T. E. Acree, in Handbook of Sensory Physiology. IV; Chemical Senses, 2; Tasre, Springer Verlag, Berlin, 1971, pp. 221-277. (22) R. S. Shaltenberger, 1. Food Sci., 28 (1963) 584-589. (23) G . G. Birch, N. D. Cowell, and R. H. Young, 1.Sci. Food Agric., 23 (1972) 1207-1212. (18) (19) (20) (21)

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CHEANG-KUAN LEE

TABLEI11 Taste of D- and L-Amino Acids” Taste Amino Acids Me

L

Form

D

Form

NH,

\c/ Me’

\CO,H

Et

NH, \C /

tasteless-bitter

sweet

tasteless- bitter

sweet

tasteless-bitter

sweet

tasteless-bitter

sweet

trace bitter

trace bitter

sweet

tasteless

sweet

sweet

sweet

sweet

bitter

bitter

-\CO,H

Et’

Me

NH, \C/

Ph

NH, \C/

H” \CO,H Me

NH, \C/

H’

\CO,H

Me

NH,

\ / C Et’

\CO,H

CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS

209

Lawrence and Ferguson” also studied a number of physical properties of sweet-tasting compounds in an attempt to find the common property responsible for their sweet taste. They reported that all of the sweet-tasting compounds that they studied had structures that formed hydrogen bonds with water. They also studied the melting points of wet, sweet compounds, working on the theory that the wet melting-point depression, as compared to the dry melting-point, is a method of detecting intermolecular hydrogenbonding in the presence of intramolecular h y d r ~ g e n - b o n d sIn . ~ some ~ cases, limited trends were observed. The influence of surface tension was also studied, as it was argued that surface tension might affect the penetration of the molecule into the taste buds, and thus affect the overall taste. The latter property would also indicate whether an enzymic reaction was involved, and, if it was a redox enzyme-system, the ease of reduction or oxidation of a compound might have a bearing on its taste. However, on the whole, no relationship could be derived between these properties and the sweetness of a compound. a. Receptor Mechanism of Taste.-A major advance in the stereochemical basis of taste came with the understanding that the primary event in the initiation of taste involves the interaction of a stimulant molecule with a receptor located at the taste-cell plasma-membrane. As early as 1848, it had been suggested that sensory receptors transduce only one sensation, independent of the manner of stimulation. Behavioral experiments2’ tend to support this theory. In 1919, Renqvist14 proposed that the initial reaction of taste stimulation takes place on the surface of the taste-cell membrane. The taste surfaces were regarded as colloidal dispersions in which the protoplasmic, sensory particles and their components were suspended in the liquor or solution to be tested. The taste sensation would then be due to adsorption of the substances in the solution, and equal degrees of sensation would correspond to adsorption of equal amounts. Therefore, the rate of adsorption of taste stimulants would be proportional to the total substances adsorbed. The phenomenon of taste differences between isomers was partly explained by the assumption that the mechanism of taste involves a three-dimensional arrangement; for example, a layer of fatty acid floating on water would have its carboxylic groups anchored in the water whereas the long, hydrocarbon ends would project upwards.

(24) L. N. Ferguson, J. Chem. Educ., 9 (1958) 436-444. (25) R. Bernard, in D. A. Denton and J. P. Coghlan (Eds.), OIfaction and Taste, V, Academic Press, New York, 1975, p. 68.

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CHEANG-KUAN LEE

Lasareff26put forward a chemical theory in which each receptor was responsive to only a single taste, and that applied stimuli caused the decomposition of a material within the cell. This decomposition produced ions which then excited the nerve endings in the papillae, the concentration of the ionized products determining the magnitude of the neural activity. There are, however, a number of criticisms of these theories. Beidler27 argued that, as Renqvist14 had assumed that the magnitude of response is proportional to the amount of stimulant adsorbed per unit time, it is evident that, at equilibrium, the net velocity of adsorption is zero. It would follow that taste intensity should be zero, and the receptors completely adapted. However, Beidler showed28that the receptors do not adapt completely, but reach a steady level of response that is consistent for the duration of stimulation. Therefore, he concluded that human taste-adaption is dominated by events in the central nervous system, and not by the peripheral receptor. The same facts also prove Lasareffs assumption to be incorrect, as his experimental data also depended on a change in adaption that is not seen at the receptor level. The speed with which taste stimulation occurs, coupled with the fact that stimulation with toxic substances does no damage to the receptors, led Beidler29to suggest that taste stimulus need not enter the interior of the taste cell in order to initiate excitation. Because a taste cell has been shown to be sensitive to a number of taste qualities, and to a large number of chemical ~ t i m u l i , ~he” ~and ~ his coworkers concluded that a number of different sites of adsorption must exist on the surface of the cell. Therefore, they assumed that taste response results from adsorption of chemical stimuli to the surface of the receptor at given receptor sites. This adsorption is described by a monomolecular reaction similar to that assumed by Renqvi~t,’~ Lasareff,26and H a h r ~but , ~ ~with a difference. From the fact that each type of chemical-stimulus compound has a unique level of saturation of the taste receptor, it was concluded32that the magnitude of the response is dependent on the initial reaction with the receptor, and not on other, subsequent receptor-reactions that are common to all types of receptor stimulation. Therefore, it was assumed that the magnitude of neural response is directly proportional to the number of sites filled, the maximum response occurring when all of the sites are filled. Beidler29derived a fundamental (26) (27) (28) (29) (30) (31) (32)

P. Lasareff, Pjluegers Arch. Gesamte Physiol. Menschen Tiere, 194 (1922) 293-297. L. M. Beidler, Prog. Biophys. Biophys. Chem., 12 (1962) 107-151. L. M . Beidler, J. Neurophysiol, 16 (1953) 595-607. L. M. Beidler, J. Gen. Physiol., 38 (1954) 133-139. K. Kimura and L. M. Beidler, Am. J. Physiol., 187 (1956) 610-615. H. Tateda and L. M. Beidler, J. Gen. PhysioL, 47 (1964) 479-486. H. Hahn, Klin. Wochschr., 15 (1936) 933; Physiol. Absfr., 22 (1937) 212.

CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS

211

equation, analogous to the Michaelis-Menten equation employed to study enzyme-substrate reactions, relating the magnitude of response to the concentration of the saporous compound, namely,

C / R = C / R,+ 1/ K R , , where C is the concentration, R is the magnitude of response, R , is the maximum response, and K is the equilibrium constant. The law of mass action has been successfully applied to many drug dose-response relationships since the early work of Clark.33The systematic relation between the dose of a drug and the magnitude of its response is based on three assumption^^^: (1) response is proportional to the level of receptor occupancy (occupancy theory), (2) one drug molecule combines with one receptor site, and (3) a negligible fraction of total drug is combined with the receptors. These assumptions must also apply to Beidler’s equation. But how does the mere adsorption of a chemical stimulus to the surface of a receptor stimulate the fiber innervating the taste cells? A plausible e ~ p l a n a t i o nwas ~ ~ that there would be a difference in the concentration of ionic constituents between the cell interior and exterior when adsorption occurs. It was shown36that the cells of taste buds are normally electrically charged, the interior being negative with respect to the environment, as are most receptors. When an electrolyte or nonelectrolyte is adsorbed on the receptor surface of the taste bud, a slight change in spatial configuration of the receptor molecule may result, such that a hole is formed that is large enough for certain ionic species (probably K+) contained within the cell to escape to the exterior, thus decreasing the potential across the receptor membrane. This could stimulate the innervating fiber, either by chemical or electrical means, such that the frequency of nerve impulses generated would be proportional to the magnitude of receptor depolarization?6 It was further proposed3’ that nonionic stimuli were adsorbed to the microvillus membrane by way of hydrogen bonds, as thermodynamic calculations showed that the binding energy is of the order of only -8 kilojoules per mole, which is about the magnitude of that for hydrogen-bond formation. The events that follow the initial adsorption of the taste stimulus to the receptor matrix of the taste-cell membrane are conformational changes in (33) A. J. Clark, The Mode of Action of Drugs on Cells, Williams and Wilkins, Baltimore, 1933. (34) A. Goldstein, L. Aronow, and S . M. Malman, Principles of Drug Action, 2nd edn., Wiley, New York, 1974. (35) L. M. Beidler, Annu. Rev. Physiol., 12 (1961) 363-388. (36) K. Kimura and L. M. Beidler, J. Cell. Comp.Physiol., 58 (1961) 131-140. (37) L. M. Beidler, in Ref. 13, pp. 133-148.

CHEANG-KUAN LEE

212

A

I

B

I C

FIG. 1 .-Diagrammatic Representation of the Three Steps in the Taste-cell Transdu~tion.'~ Step 1, interaction of stimulus (S) with membrane-bound receptor (R) to form stimulusreceptor complex (SR); step 2, conformational change (SR) to (SR)', brought about by interaction of S with R (this change initiates a change in plasma-membrane conformation of taste cells, probably below the level of the tight junction); and step 3, conformational changes of the membrane result in lowered membrane resistance, and the consequential influx on intracellular ionic species, probably Na+. This influx generates the receptor potential which induces synaptic vesicular release to the innervating, sensory nerve, leading to the generator potential.

the receptor rnatri~.~' These initiate changes in the cell-membrane structure at sites distant from the receptor matrix and changes in ionic permeability of the taste-cell plasma-membranes, resulting in generation of the receptor potential. The receptor potential presumably then initiates the release of synaptic vesicles that generate the neural-spike potential3'" (see Fig. 1). Sat0 and Beidler's has been questioned by various investigators. It was that the correlation of concentration with response reflects some aspect of the neuromechanism, rather than receptor binding, but, in general, the theory has been widely a~cepted.~'.~' Dzendolet4* pointed out that the property common to all sweet compounds is that of being a proton acceptor. Based on electrophysiological (38) J. G. Brand, in J. H. Shaw and G. G. Roussos (Eds.), Sweeteners and Dental Caries, Information Retrieval, Washington, D.C., 1978, pp. 13-32. (38a) T. Sato and L. M. Beidler, J. Gen. Physiol., 66 (1975) 735-763. (39) J. D. Watson, Molecular Biology ofthe Gene, 3rd edn., Benjamin, Menlo Park, CA, 1976. (40) S. Price and J. A. DeSimone, Chern. Senses Flaoor, 2 (1977) 427-433. (41) D. R. Evans and D. Mellon, Jr., J. Gen. PhysioL, 45 (1962) 487-500. (42) F. Dzendolet, Percept. Psychophys., 2 (1967) 519-520.

CHEMISTRY AND BIOCHEMISTRY OF SWEETNESS

213

data obtained by Beidler,28he suggested that the initial taste-response is induced as the receptor sites are evacuated, and not at the initial adsorption stage, that is, it is one of dissociation (removal of a proton from the gustatory receptor-sites) rather than of adsorption. The various sweetnesses of compounds was viewed as resulting from the distribution of electrons in a molecule as a function of substitution of various groups, and it was considered that inductive and electrostatic effects alter the facility with which a proton is exchanged. However, Shallenberger21.43argued that the ease with which compounds of differing sweetness exchange protons is probably not sufficiently different to account for their varied sweetness. The energy required to exchange the proton of a very sweet compound, such as chloroform, was not consistent with the calculated activation energy of the sweet-taste response, unless the proton of chloroform was also hydrated. Furthermore, the Dzendolet concept also did not need as strict a steric interpretation as does the AH,B proposal (see Section 11,2,b), and investigators are now well aware that steric features are probably the most critical features in structure-sweet-taste activity relationships. In essence, Dzendolet's hypothesis is of such a general nature that no predictive power can be gained by its application. Shallenberger and Acree21thus suggested that the mechanism could best be described as a proton exchange between the receptor site and the sweet-tasting compound. That the initial event of taste stimulation takes place on the cell surface of the taste receptor is now universally accepted. In addition, accumulated evidence strongly suggests that taste-bud stimulation is extracellular in nature. For example, (1) the sweet-taste response is both rapid and reversible, (2) the intensely sweet proteins monellin" and t h a ~ m a t i ncould ~ ~ not possibly penetrate the cell, because of their size, and (3) miraculin, the taste-modifying glycoprotein, having a molecular weight of 44,000 would also be too large to penetrate the taste ell.^^,^' Understanding of receptors was further extended by Kaneko's earlier findings" and, subsequently, those of Solms and coworkers? that certain (43) R. S. Shallenberger, in G. Ohloff and A. Thomas (Eds.), Gustation and Olfaction, Academic Press, New York, 1971, pp. 126-132. (44) J . A. Morris and R. H. Cagan, Biochim. Biophys. Acra, 261 (1972) 114-122; R. H. Cagan, Science, 181 (1973) 32-35; R. H. Cagan and J. A. Moms, Proc. SOC.Exp. Bid. Med., 152 (1976) 635-640. (45) 2. Bohak and S.-L. Li, Biochim. Biophys. Acta, 427 (1976) 153-170; H. van der We1 and K. Loeve, Eur. J. Biochem., 31 (1972) 221-225. (46) (a) K. Kurihara, Y. Kurihara, and L. M. Beidler, in Olfaction and Tasre, Proc. Int. Symp., 3rd, 1968, Rockefeller University Press, New York, 1969, pp. 450-469; (b) K. Kurihara and L. M. Beidler, Science, 161 (1972) 1241-1243; (c) Nature, 222 (1961) 1176-1179. (47) J. Solms, L. Vuataz, and R. H. Egli, Experientia, 21 (1965) 692-694.

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CHEANG-KUAN LEE

D-amino acids are sweet whereas their L enantiomers are not. Therefore, it appears that the receptor is chiral in nature. The tastes of D and L sugars are, however, not significantly but this does not cast any doubt on those findings, as the spatial barrier does not come into play, because of the relatively small size of sugar molecules, and they could, therefore, interact equally effectively (see Section 11,2,a). In the case of amino acids, Lawrence and Ferguson” showed that R in RR’C(NH,)CO,H must not be larger than an ethyl group, which means that the spatial area is only a short distance from the carbon atom carrying the AH,B unit. This will be discussed in Section II,3,a.

b. The AH,B Hypothesis.-The bulk of the evidence of the early work suggested that the initial excitation of the taste receptor by sweet-tasting compounds involves neither enzymic nor strong chemical forces. Rather, the forces operative appeared to be rather weak bonds, although clearly dependent on chemical structure4; this has since been c ~ n f i r r n e d . “ ~ - ~ ~ However, none of the early theories offered a unified, structural answer as to why some compounds taste sweet. As early as 1943, ReinickeS3had observed that the sweetness of sucrose is due to at least “two oxygen tetrahedra in para position with two intervening carbon tetrahedra.” By this, he was probably referring to ethylene glycol, which is known to be rather The dominating physical property of sugar molecules is their hydrogenbonding capacity. With a clearer understanding of hydrogen-bond theorySsss6Shallenberger” suggested that hydrogen bonding might explain the various sweetness of sugars. He noted that the different sweetnesses encountered in the sugar series appears to be related to the degree to which the hydroxyl groups of the sugars might participate in intramolecular hydrogen-b~nding.~~.~~ Vicinal hydroxyl groups, as in an ethylene glycol unit, have conformational attributes that can be described by using acyclic orientational notation (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) (58)

R. S. Shallenberger, T. E. Acree, and C. Y. Lee, Nature, 221 (1969) 555-556. F. R. Dastoli and S. Price, Science, 154 (1966) 905-907. R. H. Cagan, Biochim. Biophys. Acta, 252 (1971) 199-206. C. K. Lee, S. E. Mattai, and G. G. Birch, J. Food. Sci., 40 (1975) 390-393. M. G. J. Beets, Structure-Actioity Relationship in Human Chernoreception, Applied Science, London, 1978. R. Reincke, Zuckerindustrie, 1 (1943) 79-82. C. J. Carr, F. F. Beck, and J. C. Karantz, Jr., 1. Am. Chem. SOC.,58 (1936) 1394-1395. L. Pauling, The Nature of the Chemical Bond, and Structure of Molecules and Crystals, IBH Publishers, Oxford, 1960. G. C. Pimentel and A. L. McClellan, The Hydrogen Bond, Freeman, San Francisco, 1960. R. S. Shallenberger, New Sci., 23 (1964) 569-570; Agric. Sci. Rev., 2 (1964) 11-20. R. S. Shallenberger, Roc. Symp. Front. Food Res., Cornell University, 1966, 45-62.

CHEMISTRY A N D BIOCHEMISTRY OF SWEETNESS

eclipsed (0”)

gauche (60”)

FIG. 2.-Some

anticlinal (120”)

215

antiperiplanar (180”)

Orientations of Rotamers of a n a-Glycol.

(see Fig. 2). The stereochemical arrangement of vicinal hydroxyl groups is important as some a-glycol groups are incapable of eliciting the sweet sensation. For hydrogen-bond formation,5s*56a hydrogen atom must be attached, through a single covalent bond, to an electronegative atom, A (such as oxygen or nitrogen), and must have a second electronegative atom, B (such as oxygen or nitrogen), or an electronegative center (such as an unsaturated carbon-to-carbon bond), within a distance of 250-280 pm. According to Pauling,ss hydrogen atoms of alcohols and of sugar hydroxyl groups may participate in an intramolecular hydrogen-bond when the oxygen-oxygen distance lies between 285 and 251 pm. The distance between the centers of vicinal oxygen atoms in sugar a-glycol groups was calculated by Reevess9 (see Table IV). Thus, in the eclipsed orientation (which is normally only encountered in certain derivatives of the furanose forms of sugars), the interatomic, oxygen-oxygen distance is well within the intramolecular hydrogen-bonding distance, so that a “short” bond is formed that has appreciable covalent character.” The anticlinal arrangement, on the other hand, has an oxygen-oxygen distance well beyond that for hydrogen-bond formation.60 In the pyranoses in the chair conformations, the vicinal hydroxyl groups can exist only in the anticlinal or gauche orientations. The gauche arrangement of a-glycols is normally encountered as diequatorial or axialTABLEIV The Oxygen-Oxygen Distance of a-Glycol Units5’ Projected angle (degrees)

Distance (pm)

0 60 120 180

25 1 286 345 371

(59) R. E. Reeves, J. Am. Chem. Soc., 71 (1949) 2116-2119. (60) R. S. Shallenberger, T. E. Acree, and W. E. Guild, J. Food Sci, 30 (1965) 560-563.

216

CHEANG-KUAN LEE

equatorial dispositions. The oxygen-oxygen distance is 286 pm, and this is just beyond the distance at which an intramolecular hydrogen-bond may form. L. P. Kuhn6‘ pointed out that, if hydroxyl groups of an a-dihydroxy compound are sufficiently close, they will form an internal hydrogen-bond. H

RC-

CR

For compounds in which the O-H...O distance is 5.4 W/amino acid residue) are generally bitter, whereas those of low, average hydrophobicity are not. This generalization does not, however, hold true for many peptides containing glycine; these have average hydrophobicity, but are Although the average hydrophobicity is generally considered to reflect lipoid solubility, Beets52 suggested that it may be a measure of the availability of a particular group for hydrogen bonding to the receptor site. However, in general, the thresholds (expressed as the logarithm of the reciprocal of threshold concentration in mol/liter) and hydrophobicity are, significantly, linearly related ( r = 0.88, significant at the p‘