Advances in Carbohydrate Chemistry and Biochemistry, Volume 49

Advances in Carbohydrate Chemistry and Biochemistry

Volume 49

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Advances in Carbohydrate Chemistry and Biochemistry Editor DEREK HORTON

Board of Advisors LAURENS ANDERSON J. GRANTBUCHANAN J. ANGYAL STEPHEN GUYG. S. DUTTON HANSH. BAER BENGTLINDBERG CLINTON E. BALLOU HANSPAULSEN JOHN S. BRIMACOMBE NATHANSHARON ROY L. WHISTLER

Volume 49

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9 8 7 6 5 4 3 2 1

CONTENTS PREFACE................................................................

vii

R e d Bognpr. 1913-1990 ANDRASLIPTAK.PALNANAsI. AND FERENC SZTARICSKAI Text

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

3

Jean Emile Courtois. 1907. 1989 FRANCOIS PERCHERON Text

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11

The Composition of Reducing Sugars in Solution: Current Aspects STEPHEN J . ANGYAL I. Introduction ........................................................ I1. Methods for Studying the Composition of Sugars in Solution ............... 111. Relative Stabilities of the Various Forms ................................ IV. Composition in Aqueous Solution: Aldoses .............................. V. Composition in Aqueous Solution: Ketoses .............................. VI. Composition in Aqueous Solution: Substituted and Derived Sugars .......... VII. Solutions in SolventsOther Than Water................................. VIII. TabulatedData .....................................................

19 20 22 25 27 28 31 32

Radical-Mediated Brominations at Ring Positions of Carbohydrates U S ZSOMSAK L ~ AND ROBERTJ. FERRIER

I. I1. 111. IV.

Introduction ........................................................ Radical-mediated Brominations........................................ The Regio- and Stereo-chemistry of the Reactions......................... Reactions of the Bromine-containing Products ........................... V . Conclusions ........................................................ VI . Addendum ......................................................... V

37 41 67 75

91 91

vi

CONTENTS 1. 4 . 3. 6.Dianhydrohexitols

PETERSTOSSAND REINHARD HEMMER I. I1. Ill . IV. V. VI .

Introduction ........................................................ 93 Nomenclature ...................................................... 96 Spectroscopic Properties. Structural Aspects. and Analytical Detection........ 99 Preparation of the Parent Compounds .................................. 119 Derivatives......................................................... 125 Applications........................................................ 158

Enzymic Methods in Preparative Carbohydrate Chemistry

SERGE DAVID.CLAUDINE AuGB. AND CHRISTINE GAUTHERON I. I1. 111. IV . V. VI . VII . VIII. IX .

Introduction ........................................................ Immobilization ..................................................... Aldol Additions and Other C-C Bond-forming Reactions .................. Phosphorylations.................................................... Glycosylations with Transferases ....................................... Transfer Reactions Catalyzed by Glycosidases............................ Miscellaneous Syntheses in Aqueous Solution ............................ Enzymes in Organic Solvents.......................................... Addendum .........................................................

176 180

189 207 218 231 234 235 236

Structure of Collagen FibriLAssaciated. Small Proteoglycans of Mammalian Origio HARIGARGAND NANCYLYON Introduction ........................................................ Structure of Different Glycosaminoglycans............................... Carbohydrate- Protein Linkage Regions ................................. Isolation and Fractionation of Small Proteoglycans........................ M, of Small Proteoglycans. Their Protein Cores. and Glycosaminoglycan Chains ............................................................ VI . N-Terminal Sequence of Small Proteoglycans ............................ VII. Amino Acid Sequence. Analysis of the Small Proteoglycan Core Protein. Deduced from Cloned cDNA .......................................... VIII. Biosynthesisof Small Proteoglycans .................................... IX . Biological Roles of Small Proteoglycans ................................. X . Addendum .........................................................

239 240 240 243

........................................................... AUTHORINDEX

263

SUBJECTINDEX...........................................................

279

I. I1. I11. IV . V.

244 251 254 256 258 260

PREFACE Tribute is paid here to the contributions in the carbohydrate field of two notable figures, Rezsi3 Bognk and Jean Emile Courtois, in articles respectively furnished by A. Liptik, P. NhnBsi, and F. Sztaricskai (Debrecen), and by F. Percheron (Pans). Analysis of the tautomeric compositions of reducing sugars in solution by classical polarimetric methods has inherent limitations, but n.m.r.-spectroscopic methods have greatly enhanced our ability to monitor and quantitate such mobile interconversions of sugars. An excellent overview of developments in this field was presented by s. J. Angyal (Kensington, N.S.W., Australia) in Volume 42. However, the rapid progress of new research, with the advent of more sophisticated spectrometers and techniques of data manipulation, has provided the motivation for a supplement, prepared again by Angyal, which updates and complements his earlier chapter and is to be used in conjunction with it. The synthetic proceduresavailable to the carbohydrate chemist have been largely dominated by standard reactions proceeding by heterolytic processes within a chiral matrix. The preparative utility of radical-mediated reactions has, however, been amply demonstrated in recent years. The chapter contributed here by L. Somsik (Debrecen) and R. J. Femer (Wellington), on bromination reactions of carbohydrates proceeding by radical processes integrates the literature related to Femer’s pioneering work in this area and underscores its excellent potential in synthesis. Continuing in the synthetic vein, S. David, C. AugC, and C.Gautheron (Paris) present a practical overview of the potential of enzymes as synthetic tools for the general organic chemist. Their chapter, with a well-selected variety of examples, should help the bench-level organic chemist to overcome the classic preconception that enzymes are exclusively the domain of the biochemist working with nanomolar amounts of material. The David AugC contribution should materially help in opening up the way for enzymes, both free and immobilized,to be used advantageously for preparative access to important and useful sugars and metabolic intermediates. P. Stoss (Dottikon, Switzerland) and R. Hemmer (Senden, Germany), in their articleon the 1,4 :3,6-dianhydrohexitols, provide the perspective of the industrial chemist and bring up to date a subject that was treated by Wiggins in Volume 5 of this series and by Soltzberg in the tabular material contributed in Volume 25. Theseanhydridesare ofconsiderable theoretical interest, but much of the rapidly burgeoning related research is recorded in the patent literature because of the wide practicalpotential manifested by these bicyclic diols.

...

vlll

PREFACE

Although the classic proteoglycans of cartilage tissue are now well characterized, considerably less is known concerning the “small proteoglycans” containing only one or two glycosaminoglycan chains on the protein core; their structures and biological roles are surveyed here by H.Garg and N. Lyon (Boston). It is with great regret that I record the passing on July 13, 1991 of R. Stuart Tipson in his 85th year. Dr. Tipson was a contributor to the first volume in this series and a member of the editorial team beginning with Volume 8 in 1954 until his retirement from the editorship at the completion of Volume 48 in 1990. A fuller survey of his life and scientific work is scheduled for an upcoming volume.

Columbus, Ohio August I991

DEREK HORTON

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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 49

R E Z S ~BOGNAR 1913- 1990

An extremely rich and comprehensive life and career ended in Debrecen, Hungary, on the evening of Sunday, February 4th, 1990, when Rezsd Bognir, Professor Emeritus of the Lajos Kossuth University of Debrecen passed away at the age of 77. Despite knowing for almost a year that he had been stricken with an incurable cancer, he went to work in his office until the very last days, making plans and engagingin organizational activities,as well as to learn. He spent the last days of his life in a guest-house and he carried his French language text-book there as he wanted to improve his French in the last months. This episode was characteristic of his whole life, but not of a man who takes leave of his life, a life that he could organizewith an imposing sense, leaving time for almost everything he deemed important. Rezd Bognk was born on March 7th, 1913 in the town of Hodmeziivasarhely, the capital of the poverty-stricken South-Eastem part of Hungary, the so-called Viharsarok (the “Stormy Corner”). This town used to be the center of the masses of poor peasantry fighting for work and a living. Although the Bogndr family never suffered from bread-and-butter worries, the solicitude of his father, Rezsd Bognar, a Presbyterian schoolmastercantor, and his mother Klara Hegedus, ensured an unclouded childhood to the little boy Rezsii, and the puritan life-style and the understanding and espousal of the problems of poor people were characteristic of the whole Bognar family, including Professor Bognk. He finished his elementary and secondary school studies in his home-town and obtained the certificate of final examination, required for attending university, in 193 1. His parents wanted the young Rezsd to stay close to home, and so they enrolled him to the University of Szeged (20 km away from the home-town) to learn to become a pharmacist. However, these studiesdid not satisfy the young man, who was primarily interested in technical and practical problems, and in the next year he moved to Budapest to continue his studies at the J6zsef.Nador University ofTechnica1and Economic Sciences,where he graduated in 1936 as a chemical engineer. 3

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LIPTAK et ai.

GCza Zemplen, the famous professor of this university, soon recognized the talent of his student, and invited him to join the Department of Organic Chemistry. However, the young Rezsii decided he would spend at least a short period of time in industry, and he worked for the Guttapercha Cs Gumi factory, but, one year later, he returned to the University and became the private assistant of Professor Gkza ZemplCn, the man who represented Organic Chemistry in Hungary in the first part of the 20th century. This outstanding scientist (see Vol. 14, p. I), who had extraordinary human features, had studied and practised organic chemistry in the laboratory of Emil Fischer in Berlin, and had obtained particularly important results in the field of the chemistry of carbohydrates. Rezsd Bognk proved to be an excellent student and disciple, and he made the most of his outstanding preparative capabilities. “Everything” crystallized in his hands, and this was especially important in those days, before the introduction of chromatographic techniques. Their firstjoint paper appeared in 1939,on the synthesis of primeverose and its derivatives [Ber. Deutsch. Chern. Ges., 72 (1939) 47-491,and it was followedby 2 1 papers up to 1944,primarily from the field of flavone and anthraquinone glycosides. Naturally, these glycoside syntheses required the preparation of numerous oligosaccharides. These studies comprised the basis of the Ph.D. thesis of Rezsii Bog&, presented in 1941 , in which the definitive syntheses of linarin (5-hydroxy-4’-methoxy-7-Prutinosyloxyflavone) and pectolinarin (5-hydroxy-6,4’-dimethoxy-7-~-rutinosyloxyflavone)were described. He lived in Budapest through the fighting and ravages of World War I1 and, after the march from the front, the restoration work of the Department of Organic Chemistry of the Technical University started under his leadership. Besides this work, most of the organizing and educational duties in the Department weighed heavily on Rezsii Bogn6r because of the foreign visiting-professorship, and later, due to the advanced stage of sickness of GCza Zempltn. Despite these manifold activities, the scientific career of Rezsii Bognar proceeded unbroken; in 1946,he qualified as privat-docent (after habilitation) and appointed a university professor in 1949.Together with G6za Zemplen, he was in 1948awarded the Kossuth Prize in the company of such world-famous persons as the composer ZoMn Kodily and the Nobel Prize laureate Albert Szent-Gydrgyi. The general scientificpublic considered Rezs6 Bognk to be the successor of G k a Zempltn at the Technological University in Budapest, and thus it is remarkable and surprising that he accepted the invitation of the Lajos Kossuth University of Debrecen to establish the Chair of OrganicChemistry, to organize the related educational duties, and to start scientific research. During the four decades of his activities in Debrecen he performed significantorganizing work in both education and research, was the Rector of the University for two election-cycles,and was appointed the Secretary-General of the Hungarian Academy of

R E Z S ~B ~ G N A R

5

Sciences. He also served as President of the Debrecen Local Committee of the Hungarian Academy of Sciences from its foundation in 1976 until his death in 1990. In Debrecen, he continued research on flavonoid compounds and carbohydrates, but with significantlychanged thematics. Completely new fields of natural products research were involved, such as isolation and chemical modification of opium alkaloids, isolation and structure elucidation of steroid- alkaloid glycosides, as well as research on antibiotics. In the field of the opium alkaloids, he made considerable efforts for the isolation of the accompanying minor alkaloids (codeine, thebaine, narcotine, narcotoline, and papaverine) in the form of industrially utilizable preparations. The partial hydrogenation of thebaine to dihydrothebaine allowed the preparation of the medicinally important dihydrocodeine. Detailed studies were performed on narcotine and narcotoline, and the total synthesisof narcotine was also elaborated. In the case of narcotoline, the elimination of the phenolic hydroxyl group was studied in particular. A new method was worked out for the synthesis of phthalidoisoquinoline alkaloids. With the morphine compounds, nucleophilic substitution of the 6-alkyl ethers and arylsulfonic esters of the ring-carbon atom and numerous derivativeshaving outstanding biological activity were synthesized.He also performed important studies in the field of steroid- alkaloid glycosides, including the isolation and structure elucidation of several new glycosides isolated from numerous Sofunurnspecies. Tometidenol, isolated in considerable quantities from Sofunurndufcurnaru L., proved to be a useful precursor for the synthesis of steroid derivatives having industrial importance. The research on flavonoids, started together with Zemplh, was continued in Debrecen, the primary aim being to modify the carbon skeleton, and the isolation of rutin on an industrial scale was also elaborated in Debrecen. From the synthetic studies on flavonoid compounds, the most important results were the separation of, and assignment of the absolute configuration to, the 3-bromoflavone isomers, obtained upon bromination of flavanone, and related studies, including conformational investigations, on the isomeric 3-hydroxyflavanone, 4-hydroxyflavone, 3-aminoflavanone, and 4aminoflavane.Procedures for the resolution of 4-aminoflavane and flavanone were also elaborated. He and his coworkers performed detailed investigationson the preparation and chemical transformations of the epoxides and aziridines derived from chalcones. His group also performed pioneering work in the field of the synthesis of flavonoids containing nitrogen and sulfur in the heterocyclic ring. In the second half of the 19503, his attention turned more and more to the antibiotic substances, and, in 1960, he founded the Antibiotic Research Group of the Hungarian Academy of Sciences. The first studies in the antibiotics field aimed at the synthesis of chloroflavonineand several analogs of

6

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novobiocin. The isolation and structural investigation of a few antibiotics (including desertomycin and flavofungin), that originated in Hungary, are also linked with the name of Rezsd BognSr. In the case of flavofungin, the assignment of a new structural unit (a pentaene chromophore conjugated with a lactone-carbonyl group) led to the recognition of a novel sub-groupof the pentaene macrolides. The zenith of the antibiotic research of his group was the elucidation of the structure of the glycopeptide antibiotics actinoidins A and B, and rktomycins (ristocetins)A and B. He and his associates were the first to isolate ristosamine, an important representative of the 3amino-2,3,6-trideoxyhexoses.This amino sugar and its stereoisomers (acosamine, daunosamine, and D-ristosamine)were synthesized by the BognSr group by the application of several methods, and the intermediates for these syntheseswere used for transformation into cyclitol derivativesby means of the Ferrier ring-transformation reaction. The amino sugars thus prepared were also utilized for the preparation of semisyntheticanthracycline glycoside antibiotics (such as daunomycin and carminomycin), as well as for aminocyclitolantibiotic analogues. In connection with the research on antibiotics, Professor Bognar returned in the last decade of his scientificactivity to one of the topics of his youth, namely, to the synthesisof oligosaccharides. He and his collaborators synthesized ristobiose (2-O-a-~-mannopyranosylD-glucose), ristotnose (0-a-L-rhamnopyranosyl-(1 3 6)-O-[a-~-mannopyranosyl-(1 2)]-~-glucose),ristriose [0-a-D-arabinofuranosyl-(1 2)0-a-D-mannopyranosyl-(1 + 2)-~-glucose],and a derivative of acobiose [2-0-(3-amino-2,3,6-~deoxy~-~-urabino-hexopyranosyl~~glucose]. Of these research topics, the most beloved one for Professor Bognhr was still the chemistry of carbohydrates. He was extremely productive in this field, and so, only the most important results of his contribution to carbohydrate chemistry can be discussed here. In the fifties, it was not entirely clear whether the secondary glycosylamines possess a glycosylic or a Schiff-basestructure. On the other hand, the simple preparation of such compounds offered the possibility of transforming sulfonamide derivatives having low water-solubility into more-soluble, and pharmacologically more effective, glycosylamine analogs. Rezsd BognSr connected the solution of the theoretical-structural problem with the demands of practice. By using p-aminosalicylic acid (PAS) and p-aminobenzenesulfonamide(PAB) as aglycons, the Bogniir group obtained glycosylamines having high water-solubility. Moreover, by extension of these studies to other aromatic amines, it was unequivocally proved that the derivatives produced were of glycosylic structure. In experiments with acetylated and methylated pyranoid derivatives the anomerscould be separated and isolated pure, and, upon U-deacetylation of the individual anomers, the a-and B forms of the unprotected glycosylaminescould be prepared.

-

-

RE&

BWNAR

7

Efforts to obtain glycofuranosylamineswere successful only in the case of methyl ether derivatives. It was also recognized that, in solution, theglycosylamines are always present in the form of anomeric mixtures, and the chance of obtainingone of the pure anomers by crystallizationis always determined by their physical characteristics. Systematic research on glycosylaminesled to observation of occurrence of the so-called transglycosylation reaction, and the mechanism of this transformation was studied and explained through the followingexamples, which have practical utility:

+

+ + g l y ~ ~ y l ‘ - O H glyc~~yl’-NH-R+ glyc~~yl-OH GIY~OSYI-NH-R GIYcosYI-NH-R + gly~o~yl’-NH-R’ glyco~yl-NH-R‘+ gly~~yl’-NH-R

-

GIYCOS~I-NH-RHZN-R’ 4 glyc~~yl-NH-R‘ H,N-R

+

Similar reactions could also be performed with the acetylated and the methylated derivatives. The real transglycosylation character of the proton-catalyzed process was unequivocally proved by demonstrating the intermediacy of a glycosylium ion. Studies on these very fast reactions allowed Rezsii Bognir to display his outstanding preparative skill; by proper choice, and change, of the experimental conditions, the equilibrium system could be completely shifted towards one direction, affording almost quantitative yields of the desired products. The Bognir team synthesized numerous glycosylated carbonic acid derivatives, of which the bis-glycosylcarbodiimidesare the most important. He was concerned with the reaction of sugars and amino acids for decades and investigated the structure of the products and the mechanism of the reactions. The most significant field of this research was the preparation of new thiazolidine and benzothiazoline derivatives, carrying a C-2 polyhydroxyalkyl side-chain, by means of the condensation of aldehydo sugars with mercapto-amino acids (L-cysteine and D-penicillamine) and o-aminobenzenethiol. The transformation of pentoses and hexoses into 2-furaldehyde and 5 4 hydroxymethyl)-2-furaldehyde,respectively, by the action of acids is a well-known reaction. Professor Bognar was long interested in ascertaining whether this reaction is reversible. With both a theoretical and a practical goal, the Bognar group then synthesized the DL forms of several important monosaccharides (xylose, ribose, and arabinose) from the aforementioned furan derivatives. By investigating the reaction of &,a-dihalo ethers with peracetylated sugars and acetylated glycosides, Rezsii Bognir recognized that these halogenating agents are extremely suitable for the synthesis of 0-acylglycosyl

8

ANDRAS LIPTAK et

a/.

halides, permitting the isolation of both anomers of the 1-halides. The reagents could also be applied for the selectivesplittingof oligosaccharide-type glycosides. As an example, from peracetylated rutin, the disaccharide component could be isolated in the form of acetyl-a-rutinosylchloride. During the last few years, this procedure has emerged as one of the most popular general methods for obtaining glycosyl halides, so much the more because benzylated or allylated sugars also readily give the sensitive, otherwise difficultly accessible 1-halides. The Bognar group successfully applied glycosyl cyanides for the synthesis of C-glycosyl heterocycles (C-nucleosides).During related studies, numerous 5-glycosyltetrazolederivatives were prepared, and, by means of their ring-transformation reactions, C-nucleoside-type 1,3,4-oxadiazoles and condensed heterocyclic compounds (triazolopyridines and triazolopyrimidines) were obtained. The latter derivatives are synthetic analogs of the antibiotic formicin. In recognition of his scientific activities, Professor Bognar was elected to membership on the editorialboard of severaljournals: Journal ofAntibiotics (from 1968), Organic Prep. Proc. International, Acta Chimica Hungarica, Magyar Kimiai Folybirat, and of the series Recent Developments in the Chemistry of Natural Carbon Compounds. Professor BognSlr’s contribution to the scientific literature totaled more than 400 publications, 30 patents, and several monographs. He worked as Visiting Professor for long periods at the universitiesof Dublin (Ireland) and Kiev (USSR). Many honors were conferred on him both in Hungary and abroad. He was awarded the Kossuth Prize twice (1948 and 1962), and honorary titles and medals, such as the JSlnos Kabay medal (1956),Purkyne medal (Czechoslovakia, 1964), Cyril1 and Method medal (Bulgaria, 1970), the Gold Medal of the Hungarian Academy of Sciences( 1982),and the GCza ZemplCn medal ( 1985). He was elected first corresponding member ( 1948) and then ordinary member (1953) of the Hungarian Academy of Sciences, member of the Bulgarian Academy of Sciences (1952) and the German Academia Leopoldina of Halle (1970). An honorary Doctor’s degree was conferred upon him by the University of Kiev (USSR, 1967)and the Lajos Kossuth University of Debrecen (Hungary, 1988). Professor Bognar was a well-known and prominent character at international scientific conferences. His kind and informal personality, great knowledge, and well considered, but never aggressive, logical arguments brought international recognition, not only to himself, but also to Hungarian carbohydrate chemists in general. Many of his former students and collaborators declared, and still declare, themselves disciples of the “Bognir-school.” He was very proud of his best students and coworkers, and he always helped and supported them, both in their scientific careers and pri-

REZSO BOGNAR

9

vate lives. The recognition and affection of his friends and collaboratorswere truly a life-giving support for Professor Bognhr. He enjoyed scientific successes, but never monopolized them. In 1962, he divided the money-prize of his second Kossuth Prize between his associates, saying that the reasons behind the high prize were the results they produced together. He always felt at home in Debrecen, and was able to resist invitationsto the beloved capital of Budapest, despite the many attractions of that metropolis. He was a warm, friendly, informal, and loveable man with ash-blue eyes and a youthful appearance, or as many of his friends recalled him, an altogether charmingperson. He bravely endured the ordeal of his last weeks with endless patience, and, instead of complaining, he still planned and thought of the future. In Professor RezsB Bognhr's person, the international scientific community has lost a scientistwith a wide intellectual horizon, who was also a great humanist.

ANDRASLIPTAK PAL NANASI FERENCSZTARICSKAI

ADVANCES IN CARBOHYDRATE CHEMISTRY A N D BIOCHEMISTRY, VOL. 49

JEAN EMILE COURTOIS 1907- 1989

Born in Pans on March 6th, 1907, Jean Emile Courtois belonged to a family that had practised the pharmaceutical profession for three generations in Saulieu. It was in this small town ofthe Burgundian Morvan, not far from Dijon, that he attended Junior High School. After some delay in which to prepare for the entrance examination for the French Colonial School, he finally chose to undertake a pharmaceutical education, a decision which gave great satisfaction to his family. This education began with a one-year introductory course in a pharmaceutical dispensary, which in his case was the family one in Saulieu. Here, the young student had a rigorous initiation into the art, and learned the conscientiousnessof pharmaceutical practice, thanks to the kindly but firm solicitude of his father and his grandfathers, all of whom were pharmacists. The high concept of these practitioners of their mission towards their patients and the public was determining for J. E. Courtois, who, the next year, attended the FacultCde Pharmacie de l’Avenue de I’Observatoire in Paris. He was a brilliant pupil who, in 1930, was graduated as a pharmacist and simultaneously as Bachelor of Science in the FacultC des Sciences. In the same way, J. E. Courtois had undertaken a hospital career: received in 1927as an Interne in Pharmacy, he was named in 1932, after competitive examination, Pharmacist of the Paris Hospitals. At that time, these functions included the direction ofboth the pharmaceuticaldispensary and the clinical chemistry laboratory of a hospital. He continued in these functions until his retirement in 1978. It must be observed here that, in France, hospital functions may be associated with an academic position. Consequently, J. 8. Courtois, who was attracted to biological chemistry, entered the Faculty of Pharmacy in the laboratory headed by Paul Fleury, his master with whom a fruitful collaboration became established that was to last for many years. His academic career proceeded harmoniously: beginning as a practical instructor, he was later to become Head of Practical Training, Associate 11

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Professor, and Professor,and he eventuallyreplaced P. Fleury in the Chair of Biological Chemistry in 1955. This long career allowed Professor Courtoisto live alongsidethe development of modem fundamental biochemistry, as well as the applications of biology to medical diagnosis, from the ancient simple manual techniques to the use of the most sophisticated devices. His double career was interrupted twice: in 1939- 1940, during the Second World War, in which he served as “auxiliary pharmacist,” and from November 1944 to October 1945 when, as Captain Pharmacist in the Forces Fran@ses de I’IntCrieur, he finished his service in the war as a volunteer. During the German Occupation period,he took into his home, in Paris, some members of the “Rbistance” who were wanted by the Gestapo. In spite of his heavy professional occupations,Courtois established a very successful career as a researcher. This activity began in 193 1; he wrote a university thesis on the adsorption of sugars by metallic hydroxides in 1932. He obtained the Doctorat es Sciencesd‘Etat in 1938, with a thesis devoted to a kinetic study of some plant phosphatases. These enzymes retained his attention for some years, but the carbohydrates,from the chemical as well as the enzymic point of view, quickly became the favorite research topic of Professor Courtois. The chemical researches were directed towards three main aims. The first dealt with periodic acid oxidation. In 1928, L. Malaprade, at the University of Nancy, hoping to specify the effect of D-mannitol upon the acidity of periodic acid, observed that the carbon-carbon linkages ofthe polyol were cleaved, and showed that this was a general feature of the specificreaction of periodic acid with a-glycols. Then, P. Fleury had the premonition that this acid should be an invaluable reagent for analytical purposes. He described the utilization and determination of this remarkably selective oxidant, working under mild conditions of pH and temperature. Then began, and continued for more than twenty years, a long series of analytical and structural researches on carbohydrates by P. Fleury, J. E. Courtois, and their coworkers. It may be recalled that sodium periodate was not then readily available and, especially during the sad years of the Second World War, had to be prepared in the laboratory. Their main results may be summarized as follows. The periodic acid oxidation of polyols afforded a method for quantitative determination of these compounds, and it was demonstrated that the first reaction products are carbonyl compounds, themselves in turn degraded from their reducing end. After complete oxidation, it is possible to make an estimate of the consumption of oxidant, as well as of the formic acid and formaldehyde that are produced. The monosaccharides are attacked preferentially at the neigh-

JEAN EMILE COURTOIS

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boring reducing groups: sequentially, the aldoses give rise to their lower homologs, whereas, for ketoses, the oxidation can begin on either side of the carbonyl group, and proceeds along the carbon chain. Very successful experimentswere carried out on the oxidation of sucrose, one mole of which needs three moles of periodic acid, with the formation of one mole each of formic acid and a tetraaldehyde. The latter is oxidized by bromine to a tetracarboxylic acid; subsequent acid hydrolysis of the oxygen bridges affords a mixture of acids, all of which were isolated and identified. These results brought in 1943 a confirmation of the structure of sucrose which was discussed, and which gained the approval of C. S. Hudson who, before that, was a little doubtful about the furanoid form of the D-fructosyl group. Similar work was done later with trehalose. In the same way, J. 8. Courtoisobtained confirmation of the structure of rafhnose, and established that of stachyose. Applying periodic acid oxidation to reducing di- and oligo-saccharides having (1 -4) linkages, J. E. Courtois observed the “overoxidation” phenomenon, which was further extended by study of the oxidation of malonic, malic, and citric acids. Two heteroside structures, amygdaloside and vicianoside, were also studied with this reagent. The second topic examined by Professor Courtoisin carbohydratechemistry concerned the isolation and structural determination of a number of plant oligosaccharides in the series of the sucrose D-galactosides. The raffinose-stachyose family was completed by the isolation from Verbascum thupsiforme of the higher homologs, verbascose and ajugose, followed by a hepta- and an octa-saccharide. Ajugose had previously been described in Japan, but with an erroneous structure. The correct one was established by Courtois, and confirmed by using an Ajugu species cultured in Paris from seeds of Japanese origin. The botanical family of Caryophyllaceae was the subject of extensive research, leading to the discovery of other types of sucrose D-galactosides: the lychnose series, where the chain of D-galactosyl residues is linked at C-1 of the D-frUCtOSY1 moiety of sucrose, and that of isolychnose, where the oligogalactosidic chain is substituted at C-3 of the D-fructose. The compounds isolated contain up to five D-galactosyl units. The comparison of these results with those from the studies of phytophysiologists led to the conclusionthat the D-galactosidesof sucrose play an important role in plants, firstly as reserve carbohydrates, readily mobilized if needed, and secondly, the accumulation in plants of these highly soluble products of low molecular weight may favor their resistance to freezing. Also may be cited the isolation, from ViCia seeds, of galactinol, a galactoside of inositol, previously known only in beetroots; it is accompanied by a higher homolog, a digalactosyl-inositol. It has since been shown elsewhere

14

FRANCOIS PERCHERON

that galactinolis the first galactosederivativeappearing after photosynthesis, and that it seems to be a transient donor for the biosynthesis of sucrose D-galactosides. Polysaccharideswere the third subject of the chemical interest of Professor Courtois, in the field of D-mannose-containing glycans: P-D-(1 +4)-mannans from palm-tree seeds; orchid-tuber glucomannans, either from Syrian salep or from wild species in France, which appeared to have mainly P-D( 1 +4) linear structures, with D-glucosyl residues inserted among residues of the main D-mannan chain; and galactomannans from various leguminous seeds. The major structural data were obtained by classical determinations, namely, methylation and periodic acid oxidation. It may be recalled that, for the first time, the non-regular repetition of the a-D-galactosylresidues, substituted at 0-6 of the D-mannan backbone of the galactomannans, was demonstrated, using the enzymic reagents a-D-galactosidaseand P-D-mannanase. Confirmationwas afforded by more-sophisticated chemical means in other countries. With this utilization ofenzymes in structural studies, we arrive now at the second major subject of Professor Courtois’s activity in the carbohydrate field, namely, the glycosidases. Several cr-D-galactosidaseswere the subject of extensive studies which led to the demonstration of transglycosylation reactions. With the enzyme from coffee-bean, using phenyl a-D-galactoside as the donor, transfer was observed of the a-D-galactosylgroup to many hydroxylated acceptors, such as methanol, free sugars, and oligosaccharides.The rate of reaction was found to depend on the structure of the acceptor, and the transfer to occur preferentially on a primary alcohol, less usually on a secondary one. Thisdiscovery permitted the first in vitro biosynthesis of ra!€inose. The seeds of Pluntugo ovata contain two a-D-galactosidaseshaving different specificity. Using sucrose as the acceptor, one enzyme transfers the D-galactosyl group to the primary alcohol group of the D-glucosyl moiety, the other one to the primary alcohol on C-6 of the D-fructosyl moiety leading to planteose. The coffeebean a-D-galactosidase, using cellobiose as the acceptor, catalyzes three transglycosylation reactions, respectively to the hydroxyl group on C-6 or C-3 of the nonreducing unit, and C-3 of the reducing one. Analogous reactions catalyzed by the almond P-D-glucosidase allowed Courtois to suggest a generalization concerning the catalytic action of glycosidases, which is always a transfer reaction, hydrolysis occurring when the acceptor of the glycosyl group is the hydroxyl group of water. Such studies were extended to a-D-galactosidasesof various origins: intestinal bacteria, Penicillium species, germinated legume seeds (Vicia, Medicugo, and Trigonellum), molluscs, and mammalian kidney.

JEAN EMILE COURTOIS

15

The a,a-trehalase from various sources retained the attention of Courtois for many years. Specimens of this enzyme were purified from bacteria (Pseudomonas), insects (may-bug), porcine gut and kidney, and human kidney, in order to compare their properties. Trehalase is always a very specific enzyme, showing no transglycosylation activity, unusual properties for a glycosidase, and, up to 1975, its only known substrate was a,&trehalose. D-GIUCOS,as well as D-gluconicacid, does not inhibit its activity, but sucrose is a potent inhibitor for mammalian a,a-trehalases. This glycosidase is a most widely distributed enzyme that may play a primary metabolic role in organismsthat use a,a-trehalose as a reserve carbohydrate, such as insects. Vertebrate trehalases are strongly inhibited by phloridzin and phloretol; it is possible that this inhibition is involved in the renal diabetes induced by the injection of phloridzin, trehalase being implicated in the active transport or renal resorption of D-glucose. Professor Courtois and his coworkers also discovered trehalase activity in human serum. This activity decreases after kidney removal, as well as in liver cirrhosis, an argument for the renal, and mostly hepatic, origin of the enzyme of the serum. Another original research contribution of J. 8. Courtois dealt with the glycosidasesand glycanases from xylophagic insects; such insects are serious predators in the forests of many countries. This work was initiated after 1955, when many sprucesin the Morvan area were destroyed after a massive infestation by a coleopteron, Ips typographus.It was observed that this insect possesses an exceptional variety of enzymes, namely, oligosaccharidasesable to hydrolyze the intracellular or sap oligosaccharides,and a wide selection of glycanases allowing the hydrolysis of most of the polysaccharides entangled in the bark and the wood. This enzymic mix was studied at different stages of development of the insect (larvae, pupae, young adult, and adults), and this revealed a positive correlation between the enzymic activities and the nutritional activity. Such studies were also carried out on eighteen other insect species that are parasites of conifers, poplars, or oaks. He then attempted to ascertain if the glycosidases are synthesized by the insect itself, or are due to the presenceof micro-organismsfrom the intestinal flora or of symbiotic mycetomae. Indeed, it was possible to observe, in the digestive tract of the larvae of a specific parasite of a coniferous species (Halobius abietis), the presence of mycetomae from which was isolated the yeast Candida brumptii. Similarly, two bacteria (Achromobacter) and a Candida were identified in the digestive tract of Ips sexdentatus. These micro-organisms always revealed enzymic activities less elevated and less varied than those of the host insects. Moreover, breeding of several insect specieson wood or bark impregnated with antibacterial and antifungal drugs showed the disappearance of the micro-organisms, the enzymic activities

16

FRANCOIS PERCHERON

remaining unmodified. It was then possible to assess that the enzymic activities permitting the attack on trees were essentially due to the enzymes of the insect, the digestive flora being only a minor component. Besides his fundamental research in the carbohydrate field, the functions of Courtois as the head of a hospital laboratory for many years led him to publish a number of papers dealing with clinical chemistry, among which may be cited: determination of ethyl alcohol, proteins, acidic phosphatases, and trehalase in blood determination of the basic groups of proteins by phytic acid; study of the phytosoluble glycoproteins in biological fluids; and identification and determination of scyllitol in urine. Under the aegis of the International Pharmaceutical Federation, he participated in the standardization of the methods proposed for the assay of such enzymes as cellulases and hemicellulases. In all fields, these researches benefited from the remarkable qualities as an analyst, acquired by Courtois with P. Fleury, to which was added his acute faculty of interpretation. This intense activity materialized in about three hundred original papers and a hundred general reviews. Professor Courtois was a real head of a school; he contributed to the professional development of a great number of students. Some of them turned towards various aspects of the pharmaceutical profession, whereas many others succeeded in an academic career in France as well as in foreign countries, or at the Centre National de la Recherche Scientifique. They all kept a great attachment to him, and often became real friends with him. It is necessary to recall the major role played by Courtoisin the Soci6t6 de Chimie Biologique: being an active member of this society since 1930, he became in 1953 the General Secretary, a very time-consuming charge he assumed and continued to 1969, before becoming President in 1972. He was there faced by multiple tasks, particularly the organization of many meetings, colloquia, or congresses. Thus, he was in touch with the international elite in biochemistry, among whom he gained a great number of friends. This untiring activity led Professor Courtois to become involved with many international authorities where he worked at the highest level: these included the International Unions of Pure and Applied Chemistry, and of Biochemistry, the International Federation of Clinical Chemistry (of which he was the president from 1964 to 1968), the Federation of European Biochemical Societies (taking part in its foundation), and International Commissions of Nomenclature. In all these authorities, his experience, his common sense, and his characteristic optimism were greatly appreciated. It was the same in France, where multiple commissionsappealed to J. 8. Courtois: the Comites Nationaux de Chimie, de Biochimie, and de Biophysique, and commissions of the Centre National de la Recherche Scientifique. He was one of the founder members of the Groupe FranCaisdesGlucides. He

JEAN BMILE COURTOIS

17

sat for many years on the Commission Permanente de la Pharmacop6e,and served as an expert at the European Pharmacopoeia. Such important activities, not only strictly scientific,but also in the service of scientific communities, were rightfully recognized by many honors and distinctions in France and elsewhere. J. E. Courtois was an officer of the Ugion d‘Honneur and of the Ordre National du MCrite, commander of the Palmes Acadtmiques, commander of the Spanish Order of Alphonso X el Sabio, member of the AcadCmie National de Pharmacie since 1945, and he was elected to the Academie Nationale de Mtdecine in 1967. He was a foreign member of the Sciencesand Letters Academy of Oslo, a corresponding member of the Real Academia de Farmacia in Madrid, Doctor honoris CUUSQ of the universities of Madrid and Ghent, and this is an incomplete list! It may be mentioned that Professor Courtois’ reputation led him to many teaching assignments (Saigon, Hanoi, Montreal, Algiers) and to answer multiple invitations to give lectures in about sixty cities all over the world. Quite obviously, such success in various fields was not a matter of chance or of gratitude, but reflected the qualities of the man. To exceptional gifts of acute intelligence, J. 8.Courtoisadded, during his whole career, his working capacities, his analytical and also synthetic mind, his enthusiasm, and his taste for human relations; in a word, hisjoie de vivre,as well as his ability in any case to give preference to the pleasant aspects. Thanks to all these qualities, he counted only friends everywhere. The culture of J. 8. Courtois was not restricted to the scientific field. His erudition was extended to history and to all forms of art, ancient and contemporary. After 1978, being retired, he worked and published in archeology, under the aegis of the AcadCmie du Morvan and of the Societt des Archtologuesde 1’Yonne.Just as in his scientificwork, we find again here the qualities of Professor Courtois, his interpretive ability, and a taste.for unexpected and sometimes surprisingcomparisons,which constituted one of the attractions of his conversation. His love for archeology led him to study in the field the artifactsof Persian, Greek, or Roman people, from Persepolisto Delphi, from Agrigente to Leptis magna, not to forget the early Christian churches in Yugoslavia or in Soviet Armenia. Going through a museum or a monument with him was a real pleasure, because he was a reliable guide, able to correct the professional ones! This inclination for art was fulfilled by his numerous trips, where, besides biochemistry, were always added characteristic visits in each city, and his memory firmly retained everything. Those who had the privilege to live close to him know his unconditional attachment to Burgundy, sometimes marked with a little bias against the historical enemies of Burgundians. One could not forget his passion for shooting, which he was pleased to share with foreign colleagues, and for sports in general, especially for rugby. For a long time, he was a regular

18

FRANCOIS PERCHERON

spectator, sometimes noisy, of major competitions upon which he later commented vigorously. J. 8. Courtois personified humanism, and represented a type of personality, with vast erudition, that is rarely encountered in this day and age. J. E. Courtois mamed Gilberte Quinque, who was herself a pharmacist. They brought up five daughters, Micky, Marielle, Chantal, Marie-Aleth, and Isabelle, who were respectively graduated in pharmacy, law, history, medicine, and mathematics, and who gave them 13 grandchildren. The year 1989was marked by an exceptionally cruel ordeal, which Professor Courtois bore with exemplary courage: the tragic and unexpected death of his eldest daughter. Severelyattacked himselfby disease in August, he died on December 9th of that year. He now rests close to his daughter and to his ancestors, as he had wished, in his dear Burgundy in the cemetery of Saulieu, leaving to all those who knew him the memory of an outstanding and warm personality.

FRANCOIS PERCHERON

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL 49

THE COMPOSITION OF REDUCING SUGARS IN SOLUTION: CURRENT ASPECTS BY STEPHEN J . ANGYAL School of Chemistry. University of New South Wales. Kensington. N.S.W. 2033. Australia I . Introduction ......................................................... I1. Methods for Studying the Composition of Sugars in Solution ................ 2 Nuclear Magnetic Resonance Spectroscopy............................. 4 Other Methods .................................................... I11. Relative Stabilities of the Various Forms ................................. 1 The Pyranose Form ................................................ 2 The Furanose Form ................................................ 4 The aldehydo and k t o Forms........................................ 5 Hydrated Carbonyl Forms........................................... 6 Variation of the Composition with Temperature ........................ IV. Composition in Aqueous Solution: Aldoses ............................... 1 Aldohexosesand Aldopentoses ....................................... 2 Aldotetroses and Related Sugars...................................... V Composition in Aqueous Solution: Ketoses ............................... 1 Hexuloses and Pentuloses ........................................... 2. Heptuloses........................................................ VI. Composition in Aqueous Solution: Substituted and Derived Sugars ........... 1 Partially @Substituted sugars........................................ 2 AminoSugars ..................................................... 3 Thio Sugars ....................................................... 4 Branched-chain Sugars.............................................. 5 . Sugars Having Fused Rings .......................................... VII. Solutions in Solvents Other than Water .................................. VIII . Tabulated Data ......................................................

. . . . . . .

.

. . . . . . .

19 20 20 21 22 22 23 24 25 25 25 25 26 27 27 28 28 28 29 30 30 31 31 32

I . INTRODUCTION A chapter' in this Series. published in 1984. summarized o w knowledge of the composition of reducing sugars in solution. and tabulated results collected up to the end of 1983. Since then. data on this subject have been (1) S.J . Angyd. Adv. Carbohydr. Chem. Biochem.. 42 (1984) 15-68 .

20

STEPHEN J. ANGYAL

published at an increasing rate, presumably for two reasons. First, authors of research papers have become conscious of the importance of these data, and it has become increasingly common to describe not only the chemical shifts and coupling constantsin the spectra of reducing sugars but also the proportion of the various forms. Secondly, improvements in the methods and techniques used have made it easier to determine such composition data from n.m.r. spectra and, occasionally, by other methods. Increased interest in the subject has alsogiven rise to severalpapers in which the variation of the composition with the change of temperature or solvent was systematically investigated. Hence, it appeared worthwhile to bring the original chapter up to date by reviewing the recent advances and gathering the new data into additional Tables. This chapter is supplementary to the original one: it does not stand on its own. The same section headings and section numbers have been used (even though there have been no additions to several sections). The Tables are also numbered in the original way. In the Tables, sugars originally shown are listed only if additional or more accurate data have become available; these sugars are marked with an asterisk. Those not thus marked appear here for the first time. References to the original article’ are shown in square brackets, as in [p. 221. 11. METHODS FOR STUDYING THE COMPOSITION OF SUGARSIN SOLUTION

2. Nuclear Magnetic Resonance Spectroscopy Practically all of the new data have been obtained by nuclear magnetic resonance (n.m.r.) spectroscopy. It was stated’ in 1984 that this method cannot detect components that occur in equilibrium in very small proportions (< l%), such as the free and the hydrated carbonyl forms. This is no longer true: n.m.r. spectrometershave been considerably improved, and two important advances now allow the detection and measurement of components that occur in the range of 0.01 -0.1%. Allerhand and coworkers*developed an “ultra-high resolution” methodology by which, with some modification of the instrument and the usual operating technique, very small signals can be detected. Using 13C-labelled~-glucose,they determined’s4 the proportion of the furanose forms, and the free and the hydrated aldehyde form at six temperatures between 27 and 82”. The smallest of these values (2) A. Allerhand, R. E. Addleman, and D. Osman,J. Am.Chem.Soc., 107 (1985) 5809-5810; A. Allerhand and C. H. Bradley,J. Mum.Reson., 67 (1986) 173- 176. (3) C. Williams and A. Allerhand, Curbohydr.Rex, 56 (1977) 173- 179. (4) S. R. Maple and A. Allerhand, J. Am. Chem. Soc., 109 (1987) 3168-3169.

COMPOSITION OF REDUCING SUGARS

21

was only 0.0024%. The method is difficult to apply and is demanding of instrument time; for example, for the spectrum at 67”,75,200 scans were averaged. The method is suitable for any sugar, but has so far only been applied to ~-glucose. On the other hand, the use ofspecifically 13C-labelledsugars, developed by Barker and Serian~~i,~-” has been applied to many sugars; it is particularly useful when the label is in position 1. Labelling results in an 100-fold increase of the 13Csignal of the labelled carbon atom, making it possible to detect components occurring in very small proportions, down to 0.0 1%; for example, for riboset0at 25 0.05% of free aldehyde. These results are discussed in Sections II1,4 and 111,s. The composition of many aldoses and two ketoses has been determinedt4 by 13C-n.m.r.spectroscopy;the results agreed well with those from previous determinations made from IH-n.m.r. spectra.

-

O,

-

4. Other Methods

Gas - liquid chromatography(g.1.c.)of trimethylsilyl derivativeshas again been used to determine the composition of some dozen sugars in water and in pyridine.15 In the latter solvent, the results agreed well with previous determinations;in aqueous solution, however, some of the values for furanoses proved to be too high, and the values for idose (presumably mutarotating rapidly [p. 231) differ considerably from those obtained by n.m.r. spectroscopy. G.1.c. of the trimethylsilyl derivativeswas also used for studying16 the mutarotation of D-fructose. The composition data obtained for the major components agreed well with those given by n.m.r. spectro~copy,~~ but those for the a-pyranose (0.4-0.82% between 10 and 5 5 O ) are much (5) R. Barker and A. S. Serianni,Acc. Chem. Res., 19 (1986) 307-313. (6) A. S. Serianni, J. Pierce, S.-G. Huang, and R. Barker, J. Am. Chem. Soc., 104 (1982) 4037-4044. (7) J. R. Snyder and A. S. Serianni, J. Org. Chem.,5 1 (1986) 2694-2702. (8) J. R. Snyder and A. S. Serianni, J. Am. Chem. Soc.,11 1 (1989) 2681 -2687. (9) J. R. Snyder and A. S. Serianni, Curbohydr.Res., 163 (1987) 169- 188. (10) M. J. King-Moms and A. S. Serianni, J. Am. Chem. Soc..109 (1987) 3501 -3508. (1 1) J. R. Snyder and A. S. Serianni, Carbohydr.Res., 166 (1987) 85-99. (12) J. Wu, T. Vuorinen, and A. S. Serianni, Curbohydr. Res., 206 (1990) 1 - 12. (13) J. R. Snyder and A. S. Serianni, Carbohydr.Res., 210 (1991) 21-38. (14) R. Rrez-Rey, H. VClez Castro,J. Crernata Alvarez, L. Fernindez Molina, and J. Hormaza Montenegro,Rev.Cienc.Quim., 16(1985)225-227 [Chem.Abstr.. 107 (1987)237,141]. (1 5) M. Paez, 0. Martinez-Castro,J. Sam, H. Olano, A. Garcia-Rasz, and F. aura-Calixte, Chromatographia,23 (1987) 43-46. (16) M. Cockman, D.G. Kubler, A. S. Oswald, and L. Wilson, J. Curbohydr. Chem.,6 (1987) 181-201. (17) F. W. Lichtenthaler and S. Renniger, J. Chem. Soc., Perkin Trans, 2, (1990) 1489- 1497.

22

STEPHEN J. ANGYAL

smaller than the figures obtained by several authors from n.m.r.spectral data. G.1.c. also showed the presence ofthe keto form but the proportion thus obtained (0.22-0.36%) may also be too small. Working at low temperatures (0-4"), h.p.1.c. on a cation-exchange resin in the calcium form will separate the pyranose anomers of most of the aldo-hexoses and -pentoses16; under these conditions, mutarotation is slower than separation. The furanoses are not separated, because they interconvert too rapidly, but, at -25 to -45 the two furanose forms of D-galactose and L-fucose have been ~eparated.'~ Attempts to separate the various forms of sugars on a preparative scale [ p. 241 have not succeeded so far.2o O,

111. RELATIVE STABILITIES OF THE VARIOUS FORMS

1. The Pyranose Form

Further attempts have been made to explain and predict the proportions of the pyranose forms in solution. It is not difficult to calculate, by various methods, the relative free energies in vacuum or in inert solvents; it is not, however, easy to take the effect of solvation into account. Clearly, solvation has a substantial effect on the composition, and the variation ofthe dielectric permittivity between different solvents does not fully account for this effect. Tvarogka and KoZirzl developed a method whereby solvation is considered in calculating the Gibbs energy which encompasses electrostatic, dispersion, and cavity terms. The composition of g glucose in each of eleven solvents was calculated. In only three solvents were experimentaldata available, and these agreed reasonably well with the calculated figures. The variation of the composition with the change in temperature in aqueous solution was also well accounted for by the results of these calculations. AM 1 semi-empirical molecular-orbital calculationshave been carried out on several sugars in order to establish energy minima and favored conformations.22However, TvaroSka and Carterz3showed that this method does not provide the correct energy differences between anomers. The difficulty lies in the comparatively small energy differences (- 1 -2 W/mol) between anomers; much-more refined calculations are necessary for this to emerge (18) S. Honda, S. Suzuki,and K.Kakehi, J. Chromatogr., 291 (1984) 317-325. (19) M. Monyasu, A. Kato, M. Okada, and Y. Hashimom, Anal. Left.,17 (1984) 689-699, 1533- 1538.

(20) S. J. Angyal, unpublished results. (21) I. TvaroHka and T. KoZiir, Theor. Chern.Acta, 70 (1986) 99- 114. (22) R. J. Woods,W. A. Szarek,and V. H. Smith, Jr., J. Am. Chem. Soc.. 112 (1990) 47324741. (23) I. Tvaroska and J. P. Carver,Abstr. Pap. Znt. Carbohydr. Symp., 15th. Yokohama (1990) c 001.

COMPOSITION OF REDUCING SUGARS

23

from the background, statistical noise of the data. Similar calculations have not been carried out for furanoses, which constitute a much more difficult problem owing to the small energy-differencesbetween conformers and the small barrier between them. Most of these calculations have focused on water as a solvent; they have been summarized in a detailed review.” It seemsthat hydration is stereospecifi~,2~ and there appears to be strong support for Kabayama and Patterson’s original proposalM[p. 241 that equatorial hydroxyl groups are more strongly solvated,and therefore stabilized,than axial ones. An interesting example is &D-ribopyranose, which, in aqueous solution, exists as a mixture (- 1 :5 ) of the ‘C,and the 4CIforms. Increasing the temperature causes a lessening of the 4C1form, while the proportion of the ‘C4form does not alter.*’ Similarly, changing the solvent from water to dimethyl sulfoxide causes a substantial diminution of the proportion of the ,C,form, while that of the lC4 form actually increases. The 4C1form has three equatorial hydroxyl groups, whereas the ‘C4form has only one. An ingenious examination of the “hydrophilicity” of the eight aldohexoses (that is, their hydrophilic volume in water) allowed a rationalization of their a :p pyranose ratios in aqueous solution.** The hydration characteristics of carbohydrates in aqueous solution provide an intriguing and challenging problem.” Future research will need to explore further the specificity and the thermodynamics of the solvation of carbohydrates. Another approach, now being actively pursued,6 1 9 121329,30 targets on mechanistic investigations. In particular, overall and unidirectional rate-constants of anomeric changes are measured, and related to the structure of sugars and to solution conditions. Eventually, such information should shed light on the molecular factors affecting ring formation and ring-opening reactions, and, hence, on their equilibria. 9

9

9

2. The Furanose Form

Another example of the observation that the side chain attached to the anomeric carbon atom in ketoses affectsthe proportion of one furanose form [p. 291 is afforded by the series 1-deoxy-D-fructose:N-substituted l-amino(24) F. Franks and J. R. Grigera, WaferSci. Revs., 5 (1990) 187-289. (25) M. J. T i t , A. Suggett, F. Franks, S. Ablett, and M. J. Quickenden, J. Solution Chem., 1 (1972) 131-151. (26) M. A. Kabayama and D. Patterson, Can. J. Chem., 36 (1958) 563-573. (27) F. Franks, P. J. Lillford, and G . Robinson, J. Chem. SOC.,Faraday Trans. I, 85 (1989) 24 I 7 2426. (28) M. D. Walkinshaw, J. Chem. SOC.,Perkin Trans. 2, (1987) 1903- 1906. (29) J. Pierce,A. S. Serianni, and R. Barker, J. Am. Chem. Sot.., 107 (1985) 2448-2456. (30) J. R. Snyder and A. S. Serianni, J. Am. Chem. Soc., 1 I I (1989) 2681 -2687.

-

24

STEPHEN J. ANGYAL

1deoxy-~-fructoses~~: Dfructose: and 1,2-dideoxy-~-urubino-3-heptulose3*;as the side chain is increased from CH3to CH2NR, to CH20H, to CH2CH3,the proportion ofthep-furanose increases from 9 to 10- 14,to 20, to 31%.

4. The uldehydo and &to Forms

The proportion of the aldehydo and keto forms obtained by n.m.r.-spectral studies agrees well, in most cases, with those obtained earlier by circular dichroism [p. 211. The proportion of the acyclic form in equilibrium is to a large extent governed by the Thorpe- Ingold effect: the presence of substituents favors the formation of rings. One manifestation of this effect is the greater tendency of secondary, rather than primary, alcohols to form cyclic acetals [p. 351; the second alkyl group attached to the secondary alcohol becomes a ring substituent on acetal formation. Illustrations of this effect are the 0.2% aldehyde content at 30” of 3-C-rnethyl-~~-erythrose, compared with 2% for D-erythrose, and the 0.3% of 3-C-methyl-~-threose,compared with 0.96% for ~-threose.’~ It is even lower (0.1%) in 3,3-di-C-methyl-~~-glycerotetrose. (Data for these equilibria have also been determined at 60”.) Removal of the hydroxyl group from C-2 increases the aldehyde content at removal of the hydroxyl equilibrium: 3.1% for 2-deoxy-D-glycero-tetroseL3; group from C-3 has little effect. The ratio at e q ~ i l i b r i u m between ~~ 5-hydroxypentanaland tetrahydro-2hydroxypyran in D20at 43.7” is 4 :96 [in contrast to what was predicted on p. 301. However, hydroxyketones cyclize to a lesser extent. At 25”, in to tetrahydro- 1aqueous solution, the ratio34of 1,6-dihydroxy-2-hexanone (hydroxymethy1)pyranis 60 :40. This example illustrates the effect, on hemiacetal formation by the keto group, of a hydroxyl group on a neighboring carbon atom, because 6-hydroxy-2-hexanone is found only in the acyclic form [p. 301.There are now sufficient examples to illustrate how the accumulation of hydroxyl groups favors the cyclic forms: the keto content at equilibrium of “1 ,3,4,5-tetradeoxyhexuloseyy is 100%; that of “2,3,4-trideoxyhexulose”is 60%,of the 1deoxyhexuloses, 6%, and of the hexuloses, -0.3%.However,in dimethyl sulfoxide,the presence of a hydroxyl group on C-1 seems to have much less effect: there is 55% of the acyclic 1,6-dihy-

-

(31) A. G6mez-Shchez and M. de Gracia Garcia Martin, Carbohydr.Res.. 149 (1986) 329345. (32) I. I. Cubero and M. T. Plaza Lbpez-Espinosa, Carbohydr. Res., 173 (1988) 41 -52. (33) J. Buddrus, M. Jablonowski, and H. Brinkmeier, Justus Liebigs Ann. Chem., (1987) 547 - 548. (34) W. A. Szarek, D. R. Martin, R. J. RaIka, and T. S. Cameron, Can. J. Chem., 63 (1985) 1222- 1227.

COMPOSITION OF REDUCING SUGARS

25

droxy-2-hexanonein equilibrium, the same proportion as for 6-hydroxy-2hexanone. It appearsthat the presence of a hydroxymethyl group adjacent to the anomeric center in ketoses favors the cyclic forms in aqueous solution, probably by favorable solvation. 5. Hydrated Carbonyl Forms When there is a branch at C-3 in an aldose, the aldehydo form is hydrated to a much lesser extent than in an unbranched sugar: the branching causes a 1,3-parallel interaction with one of the hydroxyl groups of the gem-diol. Whereas the ratio of aldehydrol to aldehyde is 10: 1 for threose and 5 : 1 for erythrose, it is only 1.7: 1 and 1.5: 1, respectively, for their 3-C-methyl derivatives, and 0.4 : 1 for 3,3-dirnethyl-~~-glycero-tetrose.'~ When there is a keto group in a ring, for example, in the aldopyranose and aldofuranose forms of aldosuloses (see Section V,l), it is extensively hydrated unless hydration gives rise to a syn-axial O / / Ointeraction (for a discussion, see Ref. 35). In the aldofuranoseformsofpentos-2-~loses,~ the keto group is almost completely hydrated; in their aldopyranose forms, it is hydrated to a considerableextent. In this case,the tendency to form a hydrate is reinforced by the inductive effect of the neighboring anomeric center. The various forms of ~-ribo-3-hexosuloseare hydrated to a lesser extent.37

6. Variation of the Composition with Temperature The results of a detailed study of D-fructose and five O-glucosyl-substituted mfructo~es'~ confirmed that increasing the temperature increases the proportion of the furanose forms. This effect is much more noticeable in water and in pyridine than in dimethyl sulfoxide. In other ketoses, the proportion of the keto form also increases with increasing temperature.**

Iv. COMPOSITION IN AQUEOUSSOLUTION: ALDOSES 1. Aldohexoses and Aldopentoses The composition of D-glucose has been determined over a wide range of temperature by Franks and coworkers27and by Maple and Allerhand4(see Table 11). Both sets of data are self-consistent, but the a:ppyranose ratio recorded by Maple and Allerhand is considerably higher, for example, 39.4 k 0.8% of a-pyranose versus 35.5 k 1% at 37".These authors added 1 1% of 1,Cdioxane to the solutions they used for recording the I3C-n.m.r. (35) S. J. Angyal, D. Range, J. Defaye, and A. Gadelle, Curbohydr.Rex, 76 (1979) 121 - 130. (36) T. Vuorinen and A. S. Seriami, Curbohydr. Res., 207 (1990) 185-210. (37) P. E. Moms, K. D. Hope,and D. E. Kiely, J. Curbohydr. Chem.. 8 (1989) 515-530.

26

STEPHEN J. ANGYAL

spectra. Dais and Perlin3*showed that addition ofas little as 10%ofdimethyl sulfoxide to an aqueous solution of D-fructose considerably lowers the proportion of the p-pyranose form. Addition of 1,4-dioxane would probably have the same effect; hence, Maple and Allerhand's a-pyranose values are too high. In an earlier paper by Williams and Allerhand,3values were obtained which were about half-way between the aforementioned two sets of values (37.3 f 1.O% at 4 1 "); an unspecified proportion of 1,4-dioxane was used as the internal standard. This case should serve as a warning that compoundsadded as internal standardsor as deuterium locks should be kept to the minimum, so as not to affect noticeably the composition of sugars in the solution. 2. Aldotetroses and Related Sugars

During an investigation of the properties of furanoses, Serianni and coworkers6 studied the composition of sugars which cannot form pyranoses. Starting with the aldotetrose9 [p. 361, they studied the 5-deoxypentoses~ some 5-0-substituted p e n t o ~ e s ,and ~ . ~ the ~ 2-pent~loses.'~ All these compounds will be discussed in this Section. In the aldoses, the furanose having 0-1 and 0-2 cis is the less stable anomer, except for the xylu compounds where the trans form slightly preponderates. The ratio of anomers is large in erythrose and its homologs and small in threose and its homologs, owing to the cis arrangement of 0-1 and 0 - 3 in the less-stableisomer. This applies to the 5-deoxypentoses(Table 11); they are similar in the pentose 5-phosphates and the 5-0-methyl-pentoses. The proportion of the acyclic forms, however, is smaller for the pentoses than for the tetroses, because ring closure in the latter occurs on primary hydroxyl groups. As found typically for glucose and idose, nearly equal proportions of the two furanose forms were observed39for 5-deoxy-5-fluoro-~-glucose (45 :5 5 ) and -L-idose (47 :53). In the pentuloses,12the anomers with 0-2 and 0 - 3 cis are the more stable anomers, since in that case 0-3 and the side chain are trans. In both pentuloses (Table IV), the proportion of the anomers is about the same (3: 1) because 0-4 is cis to a substituent on C-2 in both. The proportion of the acyclic form (which is higher in ketoses than in aldoses in any case) is much higher because the ring is formed through a primary hydroxyl group. By comparison, the homomorphous 6-0-methyl-~-fructos~~ has only 3.6%of the ketu form in equilibrium at 40". (38) P. Daisand A. S. Perlin, Curbohydr.Res., 136 (1985) 215-223; 169 (1987) 159-169. (39) R. Albert, K. Dax, S. Seidl, H. Sterk, and A. E. Stiitz, J. Curbohydr. Chem.. 4 (1985) 5 13- 520.

COMPOSITION OF REDUCING SUGARS

27

V. COMPOSITION I N AQUEOUS SOLUTION: KETOSES 1. Hexuloses and Pentuloses

The compositions of solutions of D-fructose" and of I-, 3-, 4-, 5-, and 6-O-a-ghcopyranosy~-D-fmctoses in water, pyridine, and dimethyl sulfoxide were determined at several temperatures; all these data cannot be reproduced here. Similar, although not so extensive, data were obtained by Jaseja and coworkers,'" using two-dimensional n.m.r. spectroscopy;they did not observe the presence of any of the a-pyranose form. As in other instances,increasingthe temperature was found to favor the furanose forms, as does change to organic solvents. As noted previously [ p. 391, substitution has little effecton the composition, except at the 3 position, where it lessens the proportion of thep-pyranose form substantially.The curious behavior of the 3-0-glucosyl isomer, turanose, in pyridine is discussed in Section VII. If a sugar has two carbonyl groups, each can form pyranose or furanose rings; bicyclic forms sometimes result. An example is ~-threo-3,4-hexodiulose4*;because this compound is symmetrical, and pyranoses are not possible, only two forms are present at equilibrium. In aqueous solution, and also in dimethyl sulfoxide, 72% of the a,a- and 28% of the p,P-difuranose were found at 27". The a$ form, which would have two trans-fused five-membered rings, was not observed. In dimethyl sulfoxide, there is also a small proportion of the monocyclic furanose form present. If a sugar contains both an aldehyde and a ketone function, the aldehyde will form rings mainly; the composition can be quite complex. Eight monomeric forms (out of a possible 18)were identified in the spectra ofeach of the pentos-2-ulo~es.~~ The erythro isomer was found to consist, at 23" in D,O, of 4.5% of the a-aldopyranose and 20.5%of its hydrate, 4.5% of the p-aldopyranose and 38.8%of its hydrate. 10.9%of the a-aldofuranose hydrate, 14.290 of thep-aldofuranosehydrate, 5.2%ofthe a-ketofuranose hydrate, and I. 1% of the /?-ketofuranosehydrate. For the threo isomer, the composition, in the sameorder,wasfoundtobe0.9,59.8,1.0,29.1, 1.5,1.3, l.O,and5.4Yo.These compositions were also determined at 80". There are at least ten forms in an aqueous solution of ~-ribehexos-3of which eight have been identified. Most forms have a free or a hydrated keto group: at 23', there is 44Yo of the a-furanose and 1.5% of its hydrate; 22%of the p-pyranose and 12%of its hydrate; 590of the a-pyranose and 2% of its hydrate, 1% of a bicyclic p-furanose, and 8% of a dimer. The (40) B. Schneider, F. W. Lichtenthaler, G. Steinle, and H. Schiweck, Justus Liebigs Ann. Chem., (1985) 2443-2453. (41) M. Jaseja, A. S. Perlin, and P. Dais, Mum.Reson. Chem., 28 (1990) 283-289. (42) S. J. Angyal, D. C. Craig, and J. Kuszmann, Carbohydr. Rex, 194 (1989) 21 -29.

STEPHEN J. ANGYAL

28

n.m.r. spectra are complex and it is not certain that the minor components have been correctly assigned. The major components in the equilibrium of 6-deoxy-~-xylo-hexos-5u10se~~ are the a-aldofuranose (36%), the P-aldofuranose (28%), and the pyranose involving both the aldehyde and the ketone group (36%). The ketone group in the furanoses is not hydrated; the configuration of the pyranose is p-1,a-5(1R,5R).The same type of pyranose is the main component (67%)in an aqueous solution of ~-x~~o-hexos-5-ulose." The pentuloses are discussed in Section IV,3. 2. Heptuloses

has been deterThe composition of the four 1,2-dideoxy-3-heptuloses mined.32These compounds are similar to the hexuloses, the hydroxyl group on C- 1 having been replaced by a methyl group; their composition should be similar to those of the corresponding hexuloses, and, in most cases, this is true. For 1,2-dideoxy-3-lyxo-heptulose, however, and for its lower homolog, 1-deoxytagatose,the a-furanose form (in which 0-3 and the side chain are cis) is more stable than the P-furanose (with 0-2 and 0-3 cis). A study of the conformationscan rationalize this observation: in the p-furanose, there is a quasi-syn-axial interaction between 0-2 and 0-4 which is aggravated by the side chain, particularly if bulky. There is a similar effect in the P-pyranose, which also becomes a minor constituent of the equilibrium mixture. The composition values previously reported [p. 421 for 3-deoxy-~manno-2-octulosonic acid (Kdo) are incorrect. The correct values45in a 0.72 M solution of the ammonium salt at 20" are 64: 6 : 10 :20 and, in a 0.18Msolution, 60: 11:9:20. VI. COMPOSITION IN AQUEOUSSOLUTION: SUBSTITUTED AND DERIVED SUGARS 1. Partially 0-Substituted Sugars

Another example of substitution increasing the ratio of a-to P-pyranoses

[ p. 451 is 3-O-methyl-~-dtrose~: 25% a-and 32%P-pyranose, 43% a -tp-

furanose. (43) D. E. Kiely, J. W. Talhouk, J. M.Riordan, and K. Gray, J. Curbohydr.Chem., 2 (1983) 427-438. (44) J. M.Riordan, R. E. Harry-Okuru, J. W. Talhouk, and D. E. Kiely, Abstr. Pup. fnt. Curbohydr. Symp., 12th, Utrecht (1984) A 1.57. (45) P.A.McNicholas,M.Batley,andJ. W.Redmond, Curbohydr.Res.,146(1986)219-231. (46) J. J. Patroni, R. J. Stick, D. M.G. Tilbrook, B. W. Skelton, and A. H. White, Aust. J. Chem., 42 (1989) 2127-2141.

COMPOSITION OF REDUCING SUGARS

29

Several methyl ethers of D-glucose were studied by Reuben:' who found that the a : p ratio increases from 36 : 64 to 60 :40 for the 2,3,6-trimethyl ether. A carboxymethylgroup as substituentwas found to cause less change than a methyl group. Substitution on the 2-position caused greater change than in any other position. The same effect was observed in the 0-D-~~UCOsyl-D-glucoses: their composition was similar to that of glucose, except for the disaccharidelinked in the 2-position which has more a-than p-pyranose in equilibrium.48Similar results were obtained with some methyl ethers of ~-galactose?~ All the derivatives studied had a methyl group on 0-4 in order to preclude formation of the furanose forms. 4-O-Methyl-~-galactosehas 35% of the a-pyranose in equilibrium; the 2,4-dimethyl ether has 5 5 % the 3,4-dimethyl ether, 35%;and the 4,6-dimethylether, 39%.Further methylation was found to have very little effect. Replacement of a hydroxyl group by fluorine in the 2-position in glucose, but not in mannose, increases the proportion of the a-pyranose.50 Fructoses 0-substituted by glucosyl in various positions are discussed in Section V, 1. The presence of substituents outside the ring usually does not affect the composition, Thus, D-allose 6-phosphate, D-altrose 6-phosphate, and Dmanno-heptulose 7-phosphate have practically the same composition as the parent sugar^.^' 2. AminoSugars

4-Amino-4,6-dideoxy-~-mannosehydrochloride (perosamine) forms an

-45 :55 mixture of the a-and p-pyranose forms in solution.52For 2-amino-

2,6-dideoxy-~-glucose6-sulfonic acid, the ar:p pyranose ratio is 68 :32 at 20°, indicating [p. 471 that, in solution, the sugar is zwitterioni~.~~ Four 1-amino- 1-deoxy-D-fructoses,each having a different substituenton the nitrogen atom, were ~tudied.~' The nature of the substituent makes little difference to the composition, but, in each case, in contrast to D-fructose, there was no great preponderance of thep- over thea-furanose form; in some cases, the a-furanose was preponderant. Changing the solvent from D20to

(47) J. Reuben, Carbohydr. Res., 184 (1988) 244-246. (48) T. Usui, M. Yokoyama, N. Yamaoka, K. Tuzimura, H. Sugiyama, and S. Seto, Carbohydr. Res., 33 (1974) 105- 116. (49) E. B. Rathbone and A. M. Stephen, S. Afi. J. Sci., 69 (1973) 183. (50) N. Satyamurthy, G. T. Bida, H. C. Pudgett, and J. R. Barrio, J. Curbohydr. Chem., 4 (1985) 489-512. (51) F. P. Franke, M. Kapuscinski, and P. Lyndon, Carbohydr. Rex, 143 (1985) 69-76. (52) M. J. Eis and B. Ganem, Carbohydr. Res., 176 (1988) 316-323. (53) J. Fernandez-Bolaiios, I. M. Cadla, and J. Fernandez-Bolaiios Guzman, Curbohydr. Res., 147 (1986) 325-329.

30

STEPHEN J. ANGYAL

dimethyl sulfoxide causes a much larger increase of the P- than of the a-furanose form.

3. Thio Sugars Further data have been reported on the composition of thio sugars. For 5-thio-~-altrose,an a :P-pyranose ratio of - 2 :3 was founds4;for 5-thio-~all~se,~' - 1 : 1; and for 5-thio-~-rnannose,~~ 94 :6. For 4-thio-~-galactose~~ and for 6-deoxy-4-thio-~-galactose,~~ an a- to P-furanose ratio of 2 : 1 was found, confirming again that pyranoses do not normally occur in the equilibria of 4-thioaldoses. It was reporteds9that 5-thio-a-~-lyxoseand 5-thio-PD-arabinose do not mutarotate, and therefore constitute the main form in solution; however, it is well knowna that 5-thio sugars mutarotate very slowly at pH 4.4 and lower, but rapidly at pH >6.5, and it is not evident whether the pH was checked or controlled. Mutarotation was reported for 5-thio-a-~-fucose,but the change in rotation was small, and only the signals of the a-pyranose were detected in the n.m.r. spectrum.61 4. Branched-chain Sugars

The composition of D-apiose [p. 541that was 13C-labelledinthe 1-position was studied in detail'l: at 25 it was found to be 26% Ofa-D-c?@hrU-, 44% of P-D-erythro-, 16%of a-D-threo-. 14%ofp-D-threo-furanose,5 0.0 1Yo of aldehyde, and -0.1% of aldehydrol. Several other 3-C-(hydroxymethy1)aldoses have been investigated.62Like apiose, they can give rise to four furanose forms but, pyranoses also being possible, there are altogether six cyclic forms in equilibrium. At 27", 3-C-(hydroxymethy1)- glucose consists of 19% of a-pyranose, 32% of P-pyranose, 17Yo of a-(1,4)-furanose,2 1Yo ofp-(1$)-furanose, 3%ofa-( l ,3l)-furanose, and 8%ofp-( l ,3l)-furanose; the composition of 3-C-(hydroxymethyl)-~-xyloseis 35.5, 10.5, 15, 15, 8.5, and 15.5%;that of 3-C-(hydroxymethyl)-~-lyxose, 57, 13, 20, 6, 2.5, and 1.5; and that of 3-C-(hydroxymethyl)-~-ribose, 18.5, 70.5, 3.5, 4.5, 1, 1.5%, respectively. The compositions, at 30°,of 3-C-methyl-~~-erythrose (30.1% a-furanose, 69.4% P-furanose, 0.3% aldehyde, and 0.2% aldehydrol), 3-C-methyl-~~O ,

(54) (55) (56) (57) (58) (59) (60) (61) (62)

N. A. L. Al-Masoudi and N. A. Hughes, Carbohydr. Rex, 148 (1986) 39-49. N. A. L. Al-Masoudi and N. A. Hughes, Carbohydr. Res., 148 (1986) 25-37. R. J. Capon and J. K. MacLeod, Chem. Commun., (1987) 1200- 1201. 0. Varela, D. Cicero, and R. M. de Lederkremer, J. Org. Chem., 54 (1989) 1884- 1890. D. Cicero, 0. Varela, and R. M. de Lederkremer, Tetrahedron,46 (1990) 1131 - 1144. N. A. Hughes and N. M. Munkombwe, Carbohydr. Res.. 136 (1985) 397-409. C. J. Clayton and N. A. Hughes, Carbohydr. Res., 4 (1967) 32-41. H. Hashimoto, T. Fujimori, and H. Yuasa, J . Carbohydr. Chem., 9 (1990) 683-694. S. J. Angyal, Carbohydr. Res., 216 (1991) 171-178.

COMPOSITION OF REDUCING SUGARS

31

threose (55.0 :44.3 :0.5:0.3), and 3,3-dimethyl-~~-glycer&tetrose (29.0 :7 1.O :0.04 :0.1) were determined.13 They do not differ substantially from those of the parent, unbranched tetroses, except for the much lessened proportion of the acyclic forms (see Sections II1,4 and 111,5). The compositions have also been determined at 60 . The composition of 5-C-methyl-~-glucose~~ at 37" is 6.5 :92.0:0.7 :0.8%. O

5. Sugars Having Fused Rings

The composition of 2-C-spirocyclopropyl-2-deoxy-~-arabinose was found& (by 13C-n.m.r.spectroscopy)to be pyranoses, furanoses and acyclic forms in the ratios of 10.6:2.9: 1.0. This is, actually, not a sugar having a fused ring but one with a spiro structure. The proportion of the acyclic form is surprising; apparently the spiro arrangement introduces strain into both the pyranose and the furanose forms. IN SOLVENTS OTHERTHAN WATER VII. SOLUTIONS

A detailed studyI7of D-fructose and five a-mglucopyranosyl-substituted fructoses showed that, in pyridine, the proportion of the furanose forms is higher, and in dimethyl sulfoxide much higher, than in water. A curious exception is turanose (3-O~-~-glucopyranosyl-D-Fructose), which has a slightly lower proportion of the /3-pyranose form in dimethyl sulfoxide but a much higher one (88.3% at 20") in pyridine. Neither the other isomers, nor 3-O-methyl-~-fructose,show such behavior. The high stability of the /3-pyranose form may be due to a hydrogen bond between the two sugar moieties. In fact, in the crystal structure ofturanose,there is a hydrogen bond between 0-2of the glucose and 0-4of the fructose componenP; it is possible that this bond persists in pyridine but not in water or dimethyl sulfoxide. Idose is an exception to the rule that the proportion of the pyranoses is lower in organic solventsthan in water. In 1 : I dimethyl sulfoxide-acetone, the /3-pyranose content is much higher (Table VII) than in water,& for reasons as yet unknown. It is still not clear why the furanose content is generally higher in organic solvents than in water. The effect on solvation of the water structure [p. 241 has been proposed as an explanation; it seems to explain the interesting fact38 that addition of even a small proportion (

OH

11

10

b i

13

12

0,NO

HO

ON02

% % ONO,

bN02

15

14

ON02

( 1 1) A. Jacquet, R. Audinos, M. Delmas, and A. Gaset, Biomass, 6 (1985) 193-209. (12) R. E. Barton and L. D. Hayward, Cun.J. Chem., 50 (1972) 1719- 1728. (13) L. D.Hayward and S. Claesson, J. Chem. Soc.. Chem. Commun.. (1967) 302-304.

100

PETER STOSS A N D REINHARD HEMMER

The corresponding isohexide dinitrites in solution in acetonitrile give more-complexcircular dichroism spectra. Here, from the R configurationof the nitrito group, there is observed14a positive c.d. band: for isosorbide dinitrite at 322,331,337,342,348,356,369,385, and 398 nm, for isomannide dinitrite at 310,320,330,337,342,349,357,370, and 385 nm, and for isoidide dinitrite, at 337, 348,360,375, and 391 nm. In the presence of isosorbide, and of isomannide, as a chiral environment, optical rotation is induced in symmetric carbonyl and nitro compounds, where it can be detected as circular dichroism (here the n-D* transition) in the ultraviolet spectra of the appropriate solutions. The rotational strengths of the induced c.d. dependI5on the solvent, the temperature, and the concentration.

b. Infrared Spectra.-The infrared spectra of unsubstituted isohexides show only a few characteristicabsorption bands. Complete spectra have not been presented in the literature. The unsubstituted parent compound cis-2,6-dioxabicyclo[3.3.0]octane (6) was characterized,16without assignment, by five absorption bands at 1 1 10,1060, 1040, 1020,and 900 cm-l (in CC1,). The monoketone 8 and the diketone 9 are characterized" by a carbonyl stretching-vibrationband at 1765 cm-I (KBr and acetonitrile). The diazides of four isohexides, namely, those of D-isosorbide (17) (2110 cm-'), D-isomannide (18) (2110 cm-'), L-isomannide (19) (2120 cm-l), and L-isoidide(20)(2100cm-I ) differ slightlyl'in the position of their azido group vibration (KEir). The infrared spectra (benzene solution)of the isohexide nitrates, as well as of their mixed nitric and p-toluenesulfonic esters, are well established. The positions of the v, and the vsnn bands for the nitrato group are remarkably constant, at 1645 f 3 cm-I and 1282 f 1 (endo), and 1274 k 1 cm-I (14)L.D. Hayward and R. N. Totty, J. Chem. Soc., D, (1969) 997-998. (15) L. D. HaywardandR. N. Totty, Can.J. Chem., 49 (1971)624-631. (16) M. L. J. Mihailovic, S. Konstanntinovic, and S. Dokic-Mazinjanin, Glus. Hem. Drus., Eeogrud, 41 (1976) 281-285. (17) J. Kuszmann and G. Medgyes,Curbohydr. Res., 85 (1980) 259-269.

1,4:3,6-DIANHYDROHEXITOLS

18

19

101

20

17

(exo). The vNo band is observed, for all compounds so analyzed, at 843 f 3 cm-'. The ratios of these bands are 1.5 : 1.O: 1.O. No shift occurs on changing the solvent. The constant position ofthe vm vibration band can be used1*to decide whether the ex0 or endo position is filled. The two isomers (10 and 11) of isosorbide mononitrate may also be distinguished by the different strength of the hydrogen bridges from the 5-endo- or 2-exo-OH group to the oxygen atom in the opposite ring. The 2-exo-mononitrate (free 5-OH group) shows Av = v(OH),-v(OHLd = 148 cm-', whereas the Av for the corresponding 5-endo-mononitrate is onlyL982 cm-l (spectra recorded for 5 mM solutions in CCl,). The strong TABLEI1 Infrared Data for Isohexide Derivatives

NO* Free Bonded Asym. Sym. OH

Compound

3625 Isosorbide 2-0-acetyl2-0-acetyl-5-0-mesyl2-chloro-2deoxy2-O-acetyl-5-chloro-5-deoxy- 3688 2-nitrate 3688 5-nitrate 2J-dinitrate Isomannide 2(5)-0-acetyl2(5)-0-acetyl-5(2)-0-mesylIsoidide 2(5)-0-acetyl-5(2)-chloro5( 2)deoxy2(5)-chloro-2(5)deoxy-

3620

3562 3555

c1

-

-

3555

680-755 685-755

-

-

3540 3605

1645 1645 1650

-

Others

References

-

3560 3560 -

-

680 755 680 - 755

21 21 21 21 21 19 19 20 21 21 21 21 21

( 1 8) L. D. Hayward, D. J. Livingstone, M. Jackson, and V. M. Csizmadia, Can. J. Chem.. 45

(1967) 2191 -2194. (19) M. Anteunis, G. Verhegghe, and T. Rosseel, Org. Mugn. Reson., 3 (1971) 693-701.

PETER STOSS AND REINHARD HEMMER

102

sharp band of the nitric ester group in isosorbide 2,5-dinitrate (14), at - 1645 crn-l, is suitable for detection and quantitative determinationof this compound in pharmaceutical formulations.20 The carbonyl vibration, at 1735 cm-l, of 2-0-acetylisohexide derivatives turns out to be insensitive as regards other substituents in these molecules.21 Without assignment, the infrared data for D-isomannidemonooleatewere reportedz2 in a patent: 1020m, 1060m, 1080m, 1 120m, 1 170m, 1240m, 1470m, 1650vw, 1740s, 2850s, 2920s, 3000m and 3444m. All relevant infrared data are summarized in Table 11.

c. Nuclear Magnetic Resonance Spectra.- H-N.m.r. spectroscopicdata for the three unsubstituted i s o h e x i d e ~ , 2their ~ ~ ~ *diacetates," ~~ dimesylate^,^^,^^ diammoniumdideoxy salts,23dito~ylates,~~ dideoxydiazide~,~~,~~ and other a~ylates:~as well as some mixed 2,5-0-dis~bstituted~'*~~~~ and 2(3-0or 2(5)-deoxy monosubstituted isohexides,21have been reported. Fewer such compounds have thus far been characterized by 13C-n.m.r. spectroscopy.Thus, data were reported for isosorbide, 1,21*27isomannide, ls2* the three isohexide dirnesylate~,2'2~ the 2-deoxy-2-iodo 5-mesylate,2' the dideoxydiiodides of isosorbide and isoidide,21*m isomannide ditosylate, and bis(2,5-dideo~y-2,5-diphenylphosphino)isoidide,~~ of mixed substituted acetates, and of monochloromonodeoxy compounds.21 Most of these investigations dealt with the elucidation of stereochemical relationshipsof this ring system,17,19~21~24~26,29 the examination of substitution p a t h ~ a y s , ~and ' , ~the ~,~ complexation ~ behavior toward alkali-metal ions,26 some different ammonium i0ns,2~.~' and such lanthanide chelates217as Gd(dmp), and Eu(fod), . (i) 'H-N.m.r. Spectra.-It has been shown by 'H-n.m.r. spectroscopy that only the cis isomer of 2,6-dioxabicyclo[3.3.O]octane (6) is formed during the treatment of 2-tetrahydrofuraneethanolwith lead tetraacetate.l6 D. Woo, J. K. C. Yen, and P. Sofronas, Anal. Chem., 45 (1973) 2144-2145. J. C. Goodwin, J. E. Hodge, and D. Weisleder, Carbohydr. Res., 79 ( I 980) 133 - 14 1. R. J. Tull (Merck & Co., Inc.), DE 2,249,831 (1972); Chem. Absrr., 81 (1974) 13,752. J. Thiem and H. Lueders, Makromol. Chem., 187 (1986) 2775-2785. F. J. Hopton and G. H. S. Thomas, Can. J. Chem., 47 (1969) 2395-2401. J. Thiem and H. Lueders, Staerke, 36 (1984) 170- 176. J. C. Metcalfe, J. F. Stoddart,G. Jones, T. H. Crawshaw,A. Quick, and D. J. Williams, J. Chem. Soc., Chem. Commun.,(1981) 430-432. (27) J. A. Peters, W. M. M. J. Bovee, and A. P. G. Kieboom, Tetrahedron,40 (1984) 2885-

(20) (2 1) (22) (23) (24) (25) (26)

2891. (28) (29) (30) (3 1 )

J. Bakos, B. Heil, and L. Marko,J. Organomet. Chem., 253 (1983) 249-252. P. Sohar, G. Medgyes, and J. Kuszmann, Org. Magn. Reson., 11 (1978) 357-359. J. M. Sugihara and D. L. Schmidt, J. Org. Chem., 26 (1961) 4612-4615. J. C. Metcalfe, J. F. Stoddart,G. Jones, T. H. Crawshaw, E. Gavuzzo,and D. J. Williams, J. Chem. Soc., Chem. Cornmun.,(1981) 432-434.

1,4 :3,6-DIANHYDROHEXITOLS

103

The protons on C-1 and C-5 (S4.56, sym. multiplet) are equivalent. For the methylene groups at C-3 and C-7, the signal at S 3.80 occurs as a triplet, whereas the neighboring groups at C-4 and C-8 show a symmetrical multiplet splitting at S 2.01. The following coupling constants"j were measured: 3J8,,= 3J4,5 = 3.2 Hz, and 3J3,,= 3J,,8 = 6.8 Hz. The conformations of D-isosorbide (21), D-isomannide (22), and L-isoidide (23), and of their diacetyl and dimesyl derivatives, have been studied in great detail.24

21

22

23

a X=H b X=OCOCH, c X=OS02CH,

The two molecules (22 and 23) having a C2 axis through the C-3-C-4 bond constitute an ABXYY' spin system, whereas the protons in the isohexide 21 occur as an ABXYZ spin system. In 22 and 23, the H-2 and H-5 atoms couple with those of the neighboring hydroxyl groups. For the endo-OH, this coupling is twice that observed for the exo-OH group. The magnitude of 3J depends on the orientation of the hydrogen atom of the hydroxyl group to the vicinal hydrogen atom on C-2 or C-5. Between H-2 and H-4, as well as between H-3 and H-5, a small, long-range coupling is observed in 21c between H-3 and one ofthe geminal protons (namely, H- 1 ,). The split signal of this H- 1 atom occurs at a field lower than that of the geminal neighboring atom H- 1 ., Based on these observations, the assignment of H-1 A and H- 1 in similar molecules can be carried out in the same way. The geminal coupling constant between H-1, and H-1, is sensitive to substitution effects. Replacing the hydroxyl group by an acetoxy or a mesyloxy group causes a diminution in Jm . The magnitude of change is greater when the substituent is in the ex0 than when it is in the endo position. From the data, especially the coupling constants given in the original paper, it was concluded that only two distinct conformations are acceptable for either ring. The first of these is the envelope conformation, Structure 1, where C-3 is displaced from plane I, and C-4 from plane 11. The second is the twist (T) conformation, in which C-2 and C-5 are symmetrically displaced

,

,

PETER STOSS A N D REINHARD HEMMER

104

STRUCTURE 1

below and above plane I, defined by C- 1 ,(2-4, and the oxygen atom between them. Similarly, C-4 and C-5 stand out from plane 11, as shown in Structure 2. However, the coupling constants estimated for the compounds showed" that a mixed conformation between the envelope and the twist form for the two rings is present here. The use of a generalized Karplus relation was the basis for comparison between calculated and experimental vicinal proton - proton coupling con-

STRUCTURE 2

stants of isosorbide (in acetone-d6). The significant deviation between calculated and experimentally obtained values for couplings between H-5 and B Hz H-6,, (7.3 Hz measured versus 10.7 Hz calc.) and H-5and H - ~ (6.4 measured versus 8.4 Hz calc.) was explained by assuming displacement of C-5 from the plane C-4-C-3 -0-6-C-6 in the endo direction, thus diminishing the vicinal coupling27between H-5 and H-6* and H - ~ BTable . I11 presents the coupling constants between protons in the three i~ohexides.~~ The stereochemical behavior of the 2,5-diazido-2,5-dideoxyisohexides 17-20 was also derived from their 'H-n.m.r.-spectral data (CDCI,). From them, conclusionsregarding their conformationswere drawn. In accordance with its symmetry and the lack of any coupling between H-2 and H-5, H-3 H-4 in 20 appears as a sharp singlet at 6 4.55 (CDCI,), (4.24 in2, C6D6). A double envelope structure having two axial azido groups was derived. For 2,5-diazido-~-and -L-isomannide(18and 19),an opened twist conformation, leadingto two quasi-diequatonallyoriented azido groupswas

+

1,4 :3,6-DIANHYDROHEXITOLS

105

TABLE III Proton Coupling Constantsn of the IsohexldeP C o u p l i const.nts(Hz)Between protoas Compound

1,lB

1,2

1 ~ 2 23

34

45

56,

56.

6,6,

Isosorbide

-8.5

3.4 3.3 2.9 5.1 3.8

0.5 dodecanoate > oleate. The poor reactivity of isohexides towards epichloroHO

65

67

SCHEME 13 (167) J. W. Le Maistre and E. C. Ford (Atlas Chem. Ind., Inc.), U.S. Pat. 3,225,067 (1962); Chem. Abstr., 64 (1966) 9,904. (168) J. D. Zech and J. W. Le Maistre (Atlas Chem. Ind., Inc.), U.S. Pat. 3,272,845 (1963); Chem. Abstr., 65 (1966) 20,205. (169) S. Ropuszynski and W. Jasinski, Przegl. Nauk. Znst. Technol. Org. TworzywSztucznych Politech. Wroclaw, 3 (1971) 15-38; Chem. Abstr., 76 (1972) 47,613.

1,4:3,6-DIANHYDROHEXITOLS

137

hydrin has been confirmed. It turned out to be advantageous to apply a two-step sequence for the synthesis of 72 from 71 by reaction with allyl bromide (73)and subsequent epoxidation of the allyl intermediate 74 with rn-chloroperoxybenzoic acid (75). 0-(2,3-Epoxypropyl)isohexides(72) were

m

RO

I

:

6 L O W

68 R = octadecanoyl 69 R=dodecanoyl 70 R = oleoyl

then used 170~171to generate the so-called “p-blocker side-chain” in compounds 76 containing different amine residues (see Scheme 14). R’0

R’0

H N R ~ R ~

i-l

-

71 OH

$4 72 R’O

73

R = H, NO,

0 74

76

OH

SCHEME 14

Epoxides 72 may also act as starting materials for hybrid structures 77 containing an isohexide and a glyceryl moiety, with nitric ester functions at different positions.172 Additional monoethers of isosorbide 2- and 5-nitrate 78 were synthesized by reaction of the free hydroxyl group with any of several alkyl iodides in the presence of freshly prepared silver oxide. The yields were low, as u ~ u a l . Amongst ’~ a larger series of different alcohol (170)P.Stoss and M. Leitold (Heinrich Mack. Nachf.), DE 3,421,072(1984);Chem. Abstr., 106 (1987)18,986. (171)P. Stoss, M. Leitold, and R. A. Yeates (Heinrich Mack Nachf.), EP 319,030 (1987); Chem. Abstr., 112 (1990)210,988. ( 172) P.Stoss, G.Schlueter, and R. Axmann, Arzneim. Forsch., 40 ( 1990) 13- 18.

PETER STOSS AND REINHARD HEMMER

138

77

78

nitrates, three benzyl ether derivatives of isosorbide 5-nitrate were mentioned.'& A different approach was used by messing and Chatte~jee.'~~ Isosorbide 5-methanesulfonate (79) reacted with 4-chlorophenol, affording the Walden-inverted isoidide derivative 80, which was transformed into the appropriate nitrate 81 (see Scheme 15).

-

-

OH

-

m - cq 0-CgHqC1-P

0-C6H4 C1-p c

ONOZ

OH 80 SCHEME15

79

81

In the course of the synthesis173of oxaprostaglandinsfrom 1,4: 3,6-dianhydro-D-glucitol, the latter was first monotosylatedat the 5-position and the ester benzylated, to afford 82. Elimination of the tosyl oxy group under special conditions yields the enolethers 83 and 84 (see Scheme 16) as a 2 : 1 mixture which can be separated by column chromatography. OTs

Me3COK

-

Me2S0

OCH2C6H5

82

OCHZC6 H5 83 SCHEME16

OCH2C6H5 84

(173) J. Thiem and H. Lueders, Justus Liebigs Ann. Chem., (1985) 2151 -2164.

I ,4 :3,6-DIANHYDROHEXITOLS

139

Another series of monoakylated isohexides (87) has been prepared as nucleoside analogs, with the bicyclic carbohydrate being linked like a glycoside, replacing the normal sugars D-ribose or 2-deoxy-~-erythro-hexose.174,175 For their preparation, isohexide monoacylates (85), previously synthesized with high regioselectivity,'04 were chloromethylated to 86, and subsequently reacted with numerous pyrimidine and tnazole bases to yield 87 (see Scheme 17).

86

85

87

SCHEME 11

Etherification of isohexides with substituted-benzylchloride in aqueous sodium hydroxide, or by means of sodium hydride in dimethyl sulfoxide, yields mixtures of mono- and bis-ethers, which can be conventionally separated by distillation or by column ~hromatography.'~~ The preparation of some phenyl ethers was also described, using the tosylate-phenoxide exchange reaction. Monoethers (88)synthesized in this way were transformed into carbamates (89) by reaction with sodium cyanide- trifluoroacetic acid (see Scheme 18). RO

RO

%

OH

OCONH~

89

88 R = subst. phenyl or subst. benzyl SCHEME18

(174) P. Stoss and E. Kaes (Heinrich Mack. Nachf.), DE 3,606,634 (1986); Chem.Abstr., 108 (1988) 38,315. ( 175) P. Stoss and E. Kaes, Nucleos. Nucleot., I ( 1988) 2 13 - 225. (176) J. W.LeMaistreandT.P.Mori(ICIAmericas,Inc.),U.S.Pat.4,169,152(1977);Chem. Abstr., 92 (1980) 94,676.

PETER STOSS AND REINHARD HEMMER

140

For lower dialkyl isohexides (go), especially 2,5-di-O-methylisosorbide, which is used as solvent for organic reactions or for pharmaceutical dosage formulations, several manufacturing processes have been reported. The most widely applied method, consisting of the reaction of the freediols (65) with dimethyl sulfate or methyl iodide, was improved by employing special conditions (see Scheme 19). Thus, acetone in the presence of 50% aqueous RO L

R2S04 65

or RI

SCHEME 19

sodium hydroxide was employed177as a solvent in the production of 90 (R = CH,) by methylation of isosorbide with dimethyl sulfate. An improved yield was claimed for use of tert-butanol as the solvent for isosorbide and simultaneous addition of aqueous sodium hydroxide and dimethyl sulfate.17* The application of phase-transfer conditionsfor the successfulsynthesisof di-0-methylisosorbide was demonstrated.179 The same group investigated the alkylation of isosorbide with a series of alkyl bromides, involving a solid-liquid phase transfer in weakly hydrated organic mixtures.lsO A different approach for the synthesis of di-0-methylisosorbideand other lower di-0-alkylated derivatives, using chloromethane in different solvent systems, with or without the aid of additional phase-transfer catalysts, was the subject of a patent application.18* In addition,a one-vessel dehydrationmethylation reaction starting from D-glucitol was mentioned. Mixtures of mono- and di-0-methylisosorbide resulted on alkylation of the diol with

(177) R. L. Hillard and I. D. Greene (American Cyanamid Co.), US.Pat. 4,322,359 (1981); Chem. Abstr., 97 (1982) 6,732. (1 78) M. Maurer, W. Orth, and W. Fickert (RuetgerswerkeAG), DE 3,521,809 (1 985); Chem. Abslr., 106 (1987) 120,177. (179) D. Achet, D. Rocrelle, I. Murengezi, M. Delmas, and A. Gaset, Synthesis, (1986) 642643. (180) D. Achet, M. Delmas, and A. G w t , Biomass, 9 (1986) 247-254. (181) W. M. Kruseand J. F. Stephen(IC1Americas, Inc.), EP92,998( 1982);Chem.Abstr., 100 (1984) 103,815.

1,4:3,6-DIAMiYDROHEXITOLS

141

dimethyl carbonate in the presence of a base as catalyst.1822,SDi-O-pentylisosorbide, characterized as a liquid by an optical rotation value, was mentioned in a contribution183;however, its preparation was referred to an unpublished paper. A Russian group described an example of an unsaturated ether formed by treating isosorbide (3)and isomannide (4) with acetylene. By hydrogenationofthe vinyl ethers(91), 2,5-di-O-ethylisohexides (92) were obtained'" (see Scheme 20).

3,4

OCH=CH 2

-

OEt

HCzCH

KOH-1,4-Dioxane

~CH=CH~

OEt

91

92 0

0-CH=CH-0-C-C, If //CH2

A COZH

CH3

91 2

%

JI

0-CH=CH-O-C-C

93

4'CHJ 3 2

0

SCHEME20

Reaction of 91 with methacrylic acid gives rise to double unsaturated side-chain compounds (93);these were subjected to polymerizati~n.~~~ Subjecting isosorbide to etherification with a,w-dihaloalkanes, mono- and dialkyl derivatives were obtained. These could not be transformed into polyethers. The use of trans-l,.l-dichlorobutenelead to oligomers up to the (182) J. N. Greenshields (ICI Americas, Inc.), U.S. Pat. 4,770,871 (1987); Chem.Absfr., 110 (1989) 63,514. (183) V . Vill, F. Fisher, and J. Them, 2. Nufudorsch..TeilA, 43 (1988) 1119- 1125. (184) B. I. Mikhant'ev, V. L. Lapenkov, and A. I. Slivkin, Zh. Obshch. Khim., 42 (1972) 2302-2303; Chem. Abstr., 78 (1973) 72,485. (185) V. L. Lapemkovand A. I. Slivkin,Monomery Vysokomol.Soedin.,(1973) 73-77; Chm. Abstr., 81 (1974) 37,869.

PETER STOSS AND REINHARD HEMMER

142

heptamer.lESa The dipicrate (94) of isomannide was first described IE6 in 1971. Pentafluorophenyl ethers 95 and 96 were formed by reaction of the diols with hexafluorobenzene.lE7

:xz

R’O

OZN

\

OR2

95

R’ = H, R2 = C,F,

Finally, isosorbide was tritylated to afford both of the monotrityl isomers, which were separated chromatographically.The 2-0-trityl derivative97 was then used for the preparation of isosorbide 5-nitrate (11) by way of intermediateI5*98 (see Scheme 21). Earlier reports on both of the 2,5-di-O-trityl OH

@ -

OTr

OTr 97

98 SCHEME 21

derivatives of isosorbide and isomannidewere corrected on repetition of the synthesis under more appropriate conditions, and unambiguous characterization of these compounds188was achieved. As they are diols, isohexides can be used to act as starting materials for crown ether derivatives. This type of application was first reported in 1981, when the isomannide compound 28 was prepared and investigated for its conformational behavior26and complexing properties.” During investigation of chiral crown ethers and podands containing one or two isomannide moieties, the alkylation behavior of this bis-endo-oriented diol was studied in (185a) J. Thiem, T. Hiirining, and W. A. Strietholt, Starch/Sidrke 41 (1989) 4- 10. (186) M. L. Sinnott and M. C. Whiting, J. Chem. Soc., B, (1971) 965-975. (187) A. H. Haines and K. C. Symes, J. Chem. Soc.. Perkin Trans. 1, (1973) 53-56. ( I 88) P. A. Finan and J. P. Reidy, J. Chem. Rex, Synop., (1989) 69.

1,4 : 3,6-DIANHYDROHEXITOLS

143

more detail. Bulky substituents seem to impede dialkylation because of steric hindrance. By using a superbasic medium, these difficulties could be overcome.189*190 Alkylating isomannide with 2-(2-bromoethoxy)tetrahydropyran under these conditions, followed by deprotection, treatment with thionyl chloride, and condensation of the product with isomannide or 8-hydroxyquinoline yields compounds 99-102,and other derivativeswere prepared in this way. Following this, additional podands were prepared,'* and investigations of the complexation ability of 101 with different chiral ammonium salts were performed.IwbSeveral congeners of the podands 100 display affinity to Na and Li cations, but scarcely bind K ions.'*

99

A novel approach to the production of chiral, polymeric, crown ethers incorporating isomannide was developed by a Japanese group. The optically active divinyl ether 103 was polymerized with cationic catalysts to afford 104, consisting of only cyclic constitutional units.lgl In addition, another crown ether (lOS),containing five ethylenedioxy moieties, was prepared. It is worth mentioning that the crystal and molecular structure of the known intramolecular ether 1,4 : 2,5 :3,6-trianhydro-~-mannitol(33), consisting of three fused furanoid rings, was the subject of a

(189) E. A. El'perina, R. 1. Abylgaziev, M. I. Struchkova, and E. P. Serebryakov, Zm.Akad. Nauk SSSR, Ser. Khim., (1988) 627-632; Chem. Abstr.. I 1 1 (1989) 58,204. (190) E. A. El'perina, R. I. Abylgaziev, and E. P. Serebryakov, In. Akad. Nauk SSSR. Ser. Khim., (1988) 632-637; Chem. Abstr.. 11 I (1989) 58,205. (190a) E. A. El'perina, E. P. Serebryakov, and M. I. Struchkova, Heterocycks 28 (1989) 805812. (190b) M. I. Struchkova, E. A. El'perina, R. I. Abylgaziev, and E. P. Serebryakov, Izv. Akad. NaukSSSR,Ser Khim. (1989) 2492-2500; Chem. Abstr., 112 (1990) 217,371. ( 1 90c) M. I. Struchkova, E. A. El'perina, L. M. Suslova, R. I. Abylgaziev, and E. P. Serebryakov, I n .Akad. Nauk SSSR, Ser. Khim. (1989) 2501-2504; Chem. Abstr., 112 (1990) 166,236. (191) T. Kakuchi, T. Takaoka, and K. Yokota, Mucrornol. Chem., 189 (1988) 2007-2016.

144

PETER STOSS AND REINHARD HEMMER

b

100

c

R =

R , R =

101

n n m

oh

~owoL/o

0

102

103

I ,4 :3,6-DIANHYDROHEXITOLS

145

105

b. Silyl Ethers.-To date, there is only one report involving the preparation of silyl ethers ofadianhydrohexitol.Compounds106 and 107 have been RO

-

R = H,COCH,

107

PETER STOSS A N D REINHARD HEMMER

146

obtained by treatment of isomannide or its diacetate with diethoxydimethylsilane.lg2During gas chromatography- mass spectrometricdetermination of isosorbide 5-mononitratein human serum, a silylation reaction was used. The presence of pyridineand heating at 80' lead to formation of a trimethylsilyl ether not only at C-2, where a free hydroxyl is present, but also at C-5, where the nitro group is r e p 1 a ~ e d . l ~ ~ 3. Deoxy Derivatives

a. Mono- and Di-unsubstituted.-Only a few articles on mono- and dideoxyisohexides, saturated as well as unsaturated, have appeared in the literature. As the earlier work was not fully covered by Soltzberg's article$ it seemed reasonable to include the few missing papers in the present article. A small amount of an unsaturated amine derivative 110 was isolated by Cope and Shenlg3;it was probably formed as aresult ofthe elimination of the less reactive tosylate group of isosorbide 2,5-ditosylate(108)under the influence of the dimethylamine reagent at 120" (see Scheme 22).

OTs

109 SCHEME 22

108

110

At 165 both tosyloxy groupsin 108were replaced with inversion, and the bis(dimethylamin0)-D-ghcitol derivative was obtained. On heating isosorbide (3)or its diacetate (111)in the presence of such dehydrating agents as aluminum oxide in a Pyrex-glass tube above 400°, the doubly unsaturated compound 112 is formed in - 50% yield1%(see Scheme 23). A further example of an unsaturated isohexide was publishedL73 in 1985. When 2-0-benzyl-5-0-tosylisosorbide(82) was subjected to elimination by potassium tert-butoxide, a 2 : 1 mixture of the corresponding benzylated O,

(192) B. Pavare, 0. Lukevica, and L. Maijs, Latv. PSR Zinat. Akad. Vestis,Kim. Ser., (1973) 234-238; Chem. Abstr., 79 (1973) 137,228. (192a) P. Zuccaro, S. M.Zuccaro, R. Pacifici, S. Pichini, and L. Boniforti,J. Chromatogr. 525 (1990) 447-453. (193) A. C . Cope. andT. Y. Shen, J. Am. Chem. Soc., 78 (1956) 3177-3182. (194) H. Hopff and A. Lehmann (DEGUSSA), DE 952,092 (1955); Chem. Abstr., 53 (1959)

2,2526.

1,4:3,6-DIANHYDROHEXITOLS

147

a- 1 OAc

Ac20

3

400"

-

\

500'

112

C

SCHEME 23

enol ethers83 and 84 was obtained (see Scheme 17). Experimentsto demonstrate the isomerization of compound 84, which is of limited stability even at -20°, failed. Also, as a result of a partial elimination of the exo-tosylate 108 by azide, which is a sterically hindered reaction, the unsaturated azido isohexide 113 was ~ b t a i n e d * (see ~ J ~Scheme ~ 24).

-

_N3

NaN3

108

113 SCHEME 24

The synthesis of the unsubstituted dideoxyisohexide parent compounds was performed by Cope and Shen.196J97 Isomannide dichloride (114) has been converted into ~-cis-2,6-dioxabicyclo[3.3.O]octane (115)by hydrogenolysis. The L enantiomer 117 was obtained by reaction of D-1,6-diacetoxy3,4-hexanediol ditosylate (116) with sodium methoxide (see Scheme 25). Presumably, compound 115 has also been isolated, as an extremely volatile liquid, in 15%yield from a complex mixture of other bicyclic ethers by oxidation of 1,6-hexanediol (118) with lead t e t r a a ~ e t a t e ~(see ~ ~ Scheme ,'~

(1 95) H. Lueders, Ph. D. Thesis, University ofHamburg, Federal Republic ofGermany (1984). (196)A.C.CopeandT.Y.Shen, J. Am. Chem. Soc., 78(1956)5916-5920. (197)A.C.CopeandT.Y.Shen,U.S.Pat.2,932,650(1960);Chem.Abstr., 54(1960)24,7996. (198)V.M. Micovic, S.Stojcic, S.Mladenovic, and M. Stefanovic, TetrahedronLett., (1965) 1559-1563. (199)V.M. Micovic, S.Stojcic, M.Bralovic, S.Mladenovic, D. Jeremic, and M. Stefanovic, Tetrahedron, 25 (1969)985-993.

PETER STOSS AND REINHARD HEMMER

148

a H

‘b

*

H2 Ni Raney

tl

H

114

115

H NaOCH3 Ac 0

OAc

H

116

117 SCHEME 25

HO

Pb ( O A c ) -

.

=

P b (OAc)

*-

115

/

OH

118

119 SCHEME 26

26). The authors confirmed the structure of a cis-lY4-dioxaperhydropentalene on the basis of the IH-n.m.r. spectrum, without mention of the D or L configuration. A similar approach was used by Mihailovic and coworkers.16When 2-tetrahydrofuranethanol(ll9) was treated with lead tetraacetate, an intramolecular ring-closure occurred, to give a 45% yield of (R,R)-czs-2,6-dioxabicyclo[3.3.0]octane (115), together with seven other compounds in minor proportions. Compound 115 was prepared from D-mannitol by following the established procedure of Cope and Shen,193and was used as an intermediate for the first synthesis of thiacy~lodeca-4,7-diene.~ Finally, several OH H O - 0 I

HO

\

OCHZR

- 4 4

9 Hd

bCHZR

-a ‘r

OCH2R

121

120

122

SCHEME 27 (200) V. Cere,E. Dalcanale, C.Paolucci, S. Pollicino, E. Sandri, L. Lunazzi, and A. Fava, J. Org. Chm.,47 (1982) 3540-3544.

1,4:3,6-DIANHYDROHEXITOI-S

149

monodeoxy derivatives(122) have been synthesized,for use asherbicides, by a ring-closure reaction starting from substituted diols 121 preparedz0' in a multi-step sequence from tetrahydrofurandiols 120 (see Scheme 27).

b. Halogens.-Examples of the direct halogenation of 1,4: 3,6-dianhydrohexitols during the reported period are rather rare. More frequently, nucleophilic displacement of such other substituents as mesylates or tosylates by halogens have been applied. An interesting contribution using the Arbusov reaction appeared from a Russian groupmz;they studied 1,4 :3,6dianhydro-D-glucitol 2,5-bis(tetraethylphosphorodiamidites) as examples for replacement of secondaryhydroxyl groupsby halogens in carbohydrates. When the appropriate isomannide analogue of (57) reacted with benzyl chloride at 130°, a 79% yield of the dichloro-L-isoidide (123) resulted (see Scheme 28). In contrast, no dihalogeno derivative was isolated from the isosorbide derivative. c1

67b*

C6H5CH2C1

t

Cq

130"

Cl ' bis(endo)isomer

123 SCHEME28

During gas-liquid chromatographic- mass spectrometric analysis of the acid-catalyzed dehydration reaction of D-mannitol, 1,4 :3,6-dianhydro-2chloro-2-deoxy-~-man~tol was found among the reaction products.3sCon~ according to which 2,5-di-endo trary to the postulated S N mechanism, oriented leaving-groups are substituted by different nucleophiles, resulting L-iditol), an isomerhation takes in 2,Sdi-exo derivatives(D-mannitol place in the presence of sodium iodide.203Reaction of 1,4 :3,6-dianhydro2,5-di-O-mesyl- (124) and -tosyl-D-mannitolwith sodium iodide gave a 1 : I mixture of 2,5-dideoxy-2,5-diiodo-~-iditol (126) and -D-glucitol (128). 1,4 :3,6-Dianhydro-2-deoxy-2-iodo-5-O-rnesyl-~-gluc~tol (125) and the corresponding D-mannitol derivative 127 are formed as intermediates (see Scheme 29). This unusual isomerization reaction is restricted to starting

-

(201)K.M.Sun (Shell Int. Res.), EP 264,978(1987);Chem. Abstr., 109 (1988)68,856. (202)E. E. Nifant'ev, M. P. Koroteev, and N. S. Rabovskaya, Zh. Obshch. Khim.,43 (1973) 1806- 181 1; Chem. Abstr., 79 (1973)137,414. (203)J. Kuszmann and G. Medgyes, Curbohydr. Rex. 64 (1978)135- 142.

PETER STOSS AND REINHARD HEMMER

150

OMS

tqNa I

-

I 125

't

T

OMS

cq

124

I

127 SCHEME 29

materials having the manno (bis-endo) configuration. Other nucleophiles, such as benzoate, bromide, phthalimide, or thiobenzoate, behave normally. The configuration of these isomers was determined unambiguously by l3C-n.m,r. spe~troscopy.~~ Configurational inversion in chlorine displacement of methanesulfonatesof isomannide and isosorbide, affording chlorodeoxyisosorbide and chlorodeoxyisoidide was confirmed by "C-n.m.r. investigations.*' A process for the chlorination of alcohols by causing the alcohol to react with triphenylphosphaneoxide and thionyl chloridewas also demonstrated for isosorbide among other substrates.2wIt is worth mentioning that, on brief treatment of 1,4-anhydro-~-glucitoland -D-mannitol with and -manhydrogen bromide in acetic acid, only 2,5-di-O-acetyliso-sorbide nide could be isolated, whereas, after prolonged reaction, only brominated ring-opened products result. No 1,4 :3,6-dianhydro-bromohexitolswere detected.l A study of alkyllithium-promotedring fissions of dideoxydihaloisohexides,as a source for several homochiral synthons, was published.205* 121205

c. Amines.-The exchange of the sulfonyloxy groups of sulfonylated 1,4 :3,6-dianhydrohexitolsby nucleophiles, including amines, was discussed

briefly in an earlier article.' It is known that a considerable difference in the ease of reaction exists between endo and e m displacement,and attention has (204) E. A. OBrien, T. OConner, M. R. J. Tuite, and L. High (McNeil Lab., Inc.), GB 2,182,039 (1985); Chem. Absfr., 108 (1988) 187,199. (205) K. Bock, P. Gammeltoft, and C. Pedersen, Acta Chem. Scand. Ser. B, 33 (1979) 429432. (205a) V. Cere, C. Paolucci, S. Pollicino, E. Sandri, and A.Fava, Tetrahedron Lett. 30 (1989) 6737 -6740.

1,4:3,6-DIANHYDROHEXITOLS

151

to be paid to possible Walden inversion. In the meantime, a large number of different amino-substituted isohexides have been synthesized, albeit described in only a few publications. The aim of most of this work was the generation of potential new drugs, or monomers for synthesizing polymeric compounds. The last-mentioned application was the aim of a patent206dating from 1971. 4,8-Diamino-2,6-dioxabicyclo[ 3.3.0]octanes, endo-endo as well as endo-em isomers, were used as starting materials for polyamides. First, the diamines 129 were transformed into their salts with various dicarboxylic acids, and these were polymerized to compounds 130 containing acylated amino moieties as monomeric building blocks (see Scheme 30).

-+

NN-CO- ( C H 2 j n - C 0 . .

COZH

$ 1

NH2

+

(CH214 CQ2H

-

formation

.

M-cc- ( c H 2 ) n - C 0 .

129

..

130

SCHEME 30

A similar approach was used by Thiem and L ~ e d e r s ~ ' for . ~ ~generating J~~ polyurethanes 132 and 133 from 129 or aliphatic diamines and 1,4:3,6dianhydrohexitol-derivedbis(ch1oroformates) 131. A preparation for 129, starting from mesylates and proceeding by way of azides, was provided in this connection (see Scheme 3 1). On evaluating the behavior of 1,4 :3,6-dianhydro-2,5-di-O-mesyl-~mannitol(l24) towards different nucleophiles, the doubly exchanged product 134 was mainly obtained,2o3in addition to a small proportion of the D-glucitol derivative 135 (see Scheme 32). Mesylates and tosylates of isosorbide and isomannide undergo nucleophilic displacement with a number of primary and secondary amines, as described in a pharmaceutically oriented p~blication.'~' Several patents describe the preparation of numerous aminodeoxyisohexide derivatives bearing an additional nitric ester group at the 2 or 5 position of the isohexide ring-~ystem.'~~144,207 Among these, purines and purine-alkylamines were (206)L. P.Friz, G. Anzuino, and D. Schiattarella(Montedison Fibre S. p. A.), D E 2,262,319 (1971);Chem. Abstr., 79 (1973)137,869. (207) K.Klessing (Dr.Willmar Schwabe GmbH), EP 44,932 (1980); Chem. Abstr., 97 (1982) 110,337.

PETER STOSS A N D REINHARD HEMMER

152

1,

NH-c-0

mOCOCl

tI

0

132

&OCl

5

131

0

0-C-NH-

..

(CHZ),-"El.

H2N- (CH21,-NHZ

SCHEME 31

124

PhthNk

-

N-Phth

OMS

& j + -N-Phth

134

-

N-Phth

135

SCHEME 32

used as amine moieties. They were synthesized for their potential application as cardiovascularagents. The same applies to a large number of piperazine-substituted deoxyisohexide nitrates, which have recently been synthesized via mesylates.wIO (207a) F. Suzuki, H. Hayashi, T. Kuroda, K. Kubo, and J. Ikeda (Kyowa Hakko Kogyo Co.) EP 393574 (1989); Chem. Abstr.: no reference up to Vol. 115 (1991) No. 2.

1,4:3,6-DIANHYDROHEXITOLS

153

d. Thio Derivatives.-Among the few examples in the literature, there are no unsubstituted mercaptans and no S-alkyl derivatives; only acylated thio derivativeshave been described. From I ,4:3,6-dianhydro-~-mannitol octaethyldiamidophosphite(bis(endo)isomer of 57b), by reactionwitb benzyl bromide, the diphosphonium salt 136, a compound that can be transformedm into 1,4 :3,6-dianhydro-2,5-dideoxy-2,5-(dithiocyano)-~-iditol (137) was obtained (seeScheme 33).

SCN

PhCH2Br 67b'

*

-

0-P-NEt2

SCN

1

CH2 Ph

137

136 * bis(endo)isomer

SCHEME 33

1,4:3,6-Dianhydro-2,5-di-i-O-bemoyl-2,S-dithio-~-iditol (138), together with 1,4:3,6d~nhydro-2-9benzoyl-5-~-methylsulfonyl-2-thio-~-glucitol (139) was preparedm3from isomannide 2,5-dimesylate (124) (see Scheme 34). On use of a longer reaction time, compound 138 was the sole product. OMS

SBZ c C6H5COSK

124

SBZ 138

SBZ 139

SCHEME 34

(208)

N. K. Kochetkov, E. E. Nibt'ev, and M.P.Koroteev, D&. A M . Nauk SSSR,194 (1970)587-590;Chem. A h . , 74 (1971)76,608.

PETER STOSS AND REINHARD HEMMER

154

e. Azides. -In 1980, all three 2,Sdiazides of 1,4 :3,6-dianhydro-2,5dideoxyhexitols were prepared for the first time.17JwReaction of 124 or the appropriate ditosylate With sodium azide for 2 h at 120” afforded the 2,5diazido-L-isoidide derivative 20. When the 2,5-di-O-mesyl- or -tosyl-D-isosorbide derivative (tosyi derivative 108)was similarly treated, the reaction temperature had to be increased to the boiling point of the N,N-dimethylformamide used as the solvent, and a reaction time of 4.5 h was needed in order to complete the replacement of both ester groups to afford 17. Attempts to replace the 2,5-situated mesyloxy groups in the L-isoidide derivative by azide were unsuccessful, as the starting material remained unchanged. Therefore, a “reversed” synthesis was carried out for 18, by first introducing the azido groups at C-2 and C-5 in properly substituted, acyclic hexitols 140, and then closingthe anhydro ring. Both the D and L diazidoisomannides were synthesized by a similar strategy (see Scheme 35). Compounds 20 and 17 were later prepared by an identical reaction, and 18 was also obtained from the ditosylate of ~-isoidide.~’J~~ These azides were then submitted to hydrogenolysis to afford the corresponding diamines.

NaN3

124

& -

2 h / 120°

NaN3

ioa

L

4.5 h / 160”

N3

17 MsO

N3

to N3

H O / ”

OH NaOCH3-

N3

OMS

A

N3

ia

140 SCHEME 35

(209) J. Kuszmann,G. Medgyes, F. Andrasi, and P. Berzsenyi (GyogyszerkutatoIntenet), HU 20,368 (1980); Chem. Absfr., 96 (1982) 200,099.

1,4:3,6-DIANHYDROHEXITOLS

155

f. Phosphanes. -Only one example of the preparation of a phosphorussubstituted isohexide derivative has been published since 1970.The chiral 27 has been prediphosphane of 1,4 : 3,6-dianhydro-2,5-dideoxy-~-iditol pared28from isomannide by way of its ditosylate 141 (see Scheme 36). LLP ( P h z )

*

27

b OTs

141 SCHEME36

g. C-Nitro Compounds. -The first example of dideoxyisohexideC-nitro compounds, in addition to the widely known nitrate esters, have been prepared recently. exo-4,8-Dinitro-, exo-4,4,8-trinitro-, and exo-4,4,8,8-tetranitro-2,6-dioxabicyclo[3.3.O]octanes resulted from reaction of 20 via diamine 129 (exu-amino bonds). All of the nitro derivatives had explosive properties. However there was no indication concerningtheir phannacological behavior.209a 4. Oxidation Products

The first example of an oxidation reaction with defined isolated products in the 1,4 :3,6-dianhydrohexitol series was reported210in 1963. Catalytic oxidation with oxygen in the presence of Adams' catalyst transformed and -D-glucitol(3) into the correspond1,4 : 3,6-dianhydro-~-mannitol(4) ing monoketones (8a) and (8b), respectively, whereas the L-iditol epimer (5) remained unchanged. As in the case of 3, only the monoketone was observed,this type of reaction turned out to be stereospecific, affecting only endo-disposed hydroxyl groups. This conclusion was substantiated by a longer reaction time for 4, which contains two endo hydroxyl groups, affording the diketone 9 (see Scheme 37). In addition to the parent ketones, some derivatives(such as the 2,4-dinitrophenylhydrazonesand ptolylsulfonylhydrazones) have been prepared by the same authors, and this work was covered by a patent?" A more detailed investigation of the major reaction parameters influencing the platinum-catalyzed oxidation of isosorbide (3)was undertaken2I2in (209a) T.G . Archibald and K. Baum, Synth. Commun. 19 (1989) 1493- 1498. (210) K. Heyns, W.-P. Trautwein, and H. Paulsen, Chem. Ber., 96 (1963) 3195-3199. (21 I ) Atlas Chem. Ind., Inc., FR 1,426,204(1963); Chem. Absfr.,65 (1966) 15,490. (212) F. Jacquet, C. Granado, L. Rigal,and A. Gaset, Appl. Cutul., 18 (1985) 157-172.

PETER STOSS A N D REINHARD HEMMER

156

HO

to A

OH

4

HO

tq OH

3

rn

HO c

-

02/Pt

-

no r e a c t i o n

OH 5

SCHEME 37

1985. The reaction was then optimized for maximum yield ofthe monoketone21h 8b. In contrast to those results, no useful discrimination between endo and ex0 hydroxyl groups could be found when employing ruthenium tetraoxide as an oxidant?” Compounds 4,3, and 5 each affords the diketone 9 exclusively; this was isolated as the bis(2,4-dinitrophenylhydrazone). During a search for additional synthetic possibilities, a French group an electrochemical oxidation of isosorbide to provide the monoketone 8b,accompanied by small proportions of the diketone 9. This greater susceptibility of the endo hydroxyl function to anodic oxidation was confirmedZLS by demonstrating that isomannide (4) was transformed into 8a and 9. (212a) F. Jacquet,L. Rigal, and A. Gaset, J. Chem. Techno[.Biotechnol.48 (1990) 493-506. (2 13) P.M. Collins,P. T. Doganges, A. Kolarikol, and W.G. Overend, Carhhydr. Res.. 1 1 (1969) 199-206. (2 14) F. Jacquet, A. Gaset,J. Sirnonet, and G.Lacoste,Elektrochim. Aaa, 30 ( 1985) 477-484. (215) G. Fleche, A. Gaset,and F. Jacquet (Roquette FreresS. A.), ElJ 125,986 (1983); Chm. Absrr., 102 (1985) 69,340.

1,4 :3,6-DIA"YDROHEXITOU

157

Monoketones containing additional ex0 or endo nitric ester groups have been synthesized.'" Starting from the appropriate nitrates 11 and 10, pyridinium chlorochromate as the oxidizing agent has been used for their preparation. The endo and the ex0 hydroxyl function were oxidizedin comparable yield. The opposite reaction sequence, by nitration of the monoketones 8a and 8b, was also successfully employed (see Scheme 38).

-

HNO) 8a \\ 0

OH 11

142

HO

to

-

-

0

HNOj

-

8b

;NO2

ON02

10

143

SCHEME 38

Compounds 142 and 143 could be transformed into a variety of derivatives, such as acetals, oximes, semicarbazones, and hydrazones.l' Other types of monoketonederived products, especially oxime ethers 144 containing a 3-amino-2-(hydroxypropyl)side-chain, have been synthesized and evaluated as potential drugs216(see Scheme 39).

1 42,

143

-

ONOZ

to

\\N - O T N /

OH

1

'R2

144

SCHEME 39

(216)P.StmandM.Leitold(HeinrichMackNachf.),DE3,704,604(1987);Chem. Abstr., 110 ( 1989) 75,47 1.

PETER STOSS AND REINHARD HEMMER

158

On submitting D-isomannide 2,5-dinitrate (15) to flash vacuum thermo(145) was obtained as the main lysis, 1,4 :3,6-dianhydro-~-mannopyranose product (75 - 80%yield), accompanied by 5 - 10%ofthe monoketone 8a. In contrast, when D-isosorbide 2,5-dinitrate (14) was treated under the same conditions, a 6 :3 : 1 mixture consisting2”of 8a, 145, and its D-gluco epimer 146resulted (see Scheme 40). These resultscould be explainedby a restricted number of rearrangements ofintermediateradicals, with inversion or retention of configuration, respectively. By reduction with sodium borohydride, 8a was stereoselectively converted into D-isomannide (4). ON02

tQ

< -

0 400-450’

c 6 . 7 Pa

OH

+

aa

ON02

15

145

400-450’ c

+ & h + 1 4 5 +

6 . 1 Pa

146

14

SCHEME 40

VI. APPLICATIONS 1. Chemical Uses

Under mild conditions, isosorbide (3)is converted into 1,6-dichloro-1,6dideoxy-D-glucitol (147) by reaction with boron trichloride (Scheme 4 1). Compound 147, which was not isolated in pure form, was allowed to react with benzaldehyde to afford 2,4-O-benzylidene- 1,6-dichloro-1,6ddeoxyD-glucitol(148)and 2,4 :3,5-di-O-benzylidene-1,6-dichloro-1,6-dideoxy-~glucitol(l49). By this method, the two tetrahydrofuran rings are cleaved in a very mild manner.218 (217)J. G.Batelaan,A. J. M.Weber, and U. E. Wiersum, J. Chem. Soc.,Chem. Commun., ( 1987) 1397- 1399. (218)M.A. Bukhari, A. B. Foster, and J. M. Webber, Carbohydr. Res., 1 (1966)474-481.

1,4 :3,6-DIANHYDROHEXITOLS

I59

-

to -

'CHZCl

OH

OH

C6 H CHO

BClj

C H 2 C 1 2 , 30 h at - 8 0 " -rt

b H CH2C1

3

147

CH2Cl

C1-CH2

148

149

SCHEME 41

Isosorbide (3)as a starting material from the "chiral pool" is the educt for the ten-step enantioselective ~ynthesis''~of 11deoxy-8-epi-1 1-0xaprostaglandin Fa (150a) and its (15R)diastereoisomer (150b) (see Scheme 42). (R,R)-cis-2,6-Dioxabicyclo[3.3.0]octane (115) was the starting compound

w

--

OH

OH

10 steps

OH

3

150

a:

R'

=

b:

R'

= OK, R2 = H SCHEME 42

H, R~ = OH

PETER STOSS AND REINHARD HEMMER

160

151

115

SCHEME 43

for the c h i d (E,E)-thiacyclodeca4,7diene(151a)and its 3-methyl derivative (151b);the latter hasm a helical shape (see Scheme 43). Isosorbide (3)and isomannide (4)act as chiral auxiliaries for the sodium borohydride reduction of some prochiral ketones; optical yields of up to 20% were achieved. It seems that the isohexides form chiral complexes with sodium borohydride, whereby the chiral information is transferred to the ~ubstrate.2~~ Optical active alcohols were obtained by reduction of appropriate ketones with sodium or lithium borohydride in the presence of isosorAsymmetric reduction of propiophenone using sodium borohydride, modified with (+)-camphoric acid and isosorbide, resulted in (S)-phenylethylcarbinolin 35% enantiomeric excess.219b Similar results were obtained with lithium aluminum hydride, using 4 as the c h i d ligand, by complexing the dihydridoaluminate- ketone adduct in the transition state, as shown*%in Structure 9. In this case, the optical yield stays below 5%. In all cases described, the S isomer is formed in excess.?20

STRUCTURE 9

The chiral crown ethers 101,102,104,105, and 152,and podands 100and 153 were formed from isomannide by introducing ethylene glycol units as (219) A. Hirao, H. Mochizuki, S. Nakahama, and N. Yamazaki, J. Org. Chem., 44 (1979) 1720- 1722. (219a) Teijin Ltd. JP 80,120,526 (1979); Chem. Abstr., 94 (1981) 156,521. (219b) H. H. zoorOb, Egypt. J. Chm. 29 (1986) 333-338. (220) N. Baggett and P. Stribblehill, J. Chem. Soc.,Perkin Trans. I , (1977) 1123- I 126.

1,4:3,6-DIANHYDROHEXITOLS

161

152 153

N &

bridges over one molecule, or between two molecules of i~omannide,3~J~~191 or between isomannide and other heterocyclic end-groups.IE9 Compounds 28 (ref. 31), 105 (ref. 19l), and 104were found to be capableof chiral recognition of saltsof racemic primary amines” and a-amino acids,191 forming host -guest complexes, mainly hydrogen bridges between the ammonium group and the lone pairs of the surrounding ether oxygen atoms. An isohexide monoester (not specified) has been used as an adjuvant for the synthesis of organic sulfides and oligosulfides.221 2. Pharmaceutical Uses

The continuous worldwide application of isosorbide dinitrate and, since the early eighties, of isosorbide 5-mononitrate also, has given rise to a very great number of publicationson different aspects of these drugs. It would be beyond the scope of this report to include all of the work on these two important compounds. The present synopsis is therefore restricted to other novel isohexidederivativesand to some new pharmaceuticalapplicationsof known compounds. Because of the known vasodilating activity of isohexide nitric esters, several attempts were made to improve or modify these compounds by introducing additional substituents at the second hydroxyl group. On the one hand, this type of molecular modification would influence the polarity and (221)

R. Kolta, K.Mihalszky, I. Cseko, G.Lelki, P. Szalay, and D.Fazekas (Herceghalmi Kiserleti Gazdasag), HU 39,423(1984);Chem. Absfr.,107 (1987) 58,501.

162

PETER STOSS AND REINHARD HEMMER

lipophilicity of the derivativesand could therefore cause a different biological response. On the other hand, certain new substituents could themselves contribute to the biological activity and thus generate drugs having a new profile of action. With this intent, the acetates (R = COCH,, 78a), carbamates (R = CONH2, 78b), sulfamates (R = S02NH2, 78c), and ethyl ethers (R = C2H,, 78d) of isosorbide 2- and 5-nitrate were investigated, and found to be similar in activity to the parent compounds.146Several isohexide nitrates further substituted by purine bases 154 were prepared as potential cardiovascular agents.143

R-

ONOp 154

Additional aliphatic amine derivatives (155), including*42the so-called “/3-blockerside-chain”of aryloxypropanolamines(156), and also those having purinalkylamines as substituents144 (157), exhibit useful therapeutic activities. One compound in this series has undergone advanced clinical investigations under the international nonproprietary name (INN), “teopranitol.” Nicotinic esters (R= 3-pyridylcarbony1, 78e), 4-chlorophenyl ethers (R = 4-C1-CaH4,78f), and mesylates (R = S02CH,, 78g) of all three epimeric isohexides have been the subject of a patent appli~ati0n.l~’

155

1,4:3,6-DIANHYDROHEXITOLS

163

OH 157

156

An interesting acyl moiety containing the well known calcium channel blocking dihydropyridine structure was introduced into isohexides (R = H) and their nitrates (R = NO,), giving rise129to compounds of type 158. Somidipine(INN) (31)is one derivativeof this seriesthat is at present under clinical investigation.u2 Identical structureswere later used by other groups as intermediates to generate optically active dihydropyridinecalcium antagonistsImby way of transesterification of compound 158.It has been demonstrated that pharmaceuticals consisting of compounds 158 and congeners,

H 158

0-NH 159

combined With isohexidemononitratesor glycerol nitrates, exhibit favorable effectsagainst angina pectoris.2228 Among a number of other compounds, a few isosorbide sydnonimine derivatives 159 appeared in a cardiovascularoriented report.223 (222) P.Stoss, R.Hemmer, and P.Memth (Heinrich Mack Nachf.), DE 3,906,267(1989); Chew.Abstr., 114 (1991)81,807. (222a) P. Stoss, M. Leitold (H. Mack Nachf'.) EP 361,156 (1988);Chm. Abstr., 114 (1991) 240,625. (223)K.Schoenalinger, R. Beyerle, H. Bohn, M. Just, P. Martorana, and R. E. Nitz (Cassella AG), DE 3,526,068(1985);Chem. Absfr., 106 (1987)144,012.

164

PETER STOSS A N D REINHARD HEMMER

Using isosorbide 5-nitrate (11) as part of such special amino-substituted novel /?-blocker structures, compounds 160 were claimed to be useful for treatment of heart and circulatory disorders.u4 In contrast to the foregoing

compounds of an acylated isosorbide, isohexides were ether-linked and U S ~ ~ ~ ~ as O Jphenoxy ' ~ equivalentsin oxypropanolamine-likederivatives76.

Isosorbide 2- and 5-nitrates substituted at the second hydroxyl group by a number of aliphatic, aromatic, and cinnamic acids (78, R = various acyl groups) have been claimed to be useful in vasodilating therapy."' However, it was confirmed by the authors that there seems to be no direct correlation between lipophilicity and therapeutic activity, and that structure-activity relationships in the isosorbide nitrate area are more complex than that. An oxidized stage of isohexide nitrates, wherein the remaining hydroxyl group is transformed into the ketone, gives rise to a number of derivatives 161, such as oximes, semicarbazones, acyclic and cyclic acetals, and hydrazones. lS4 Among them, 3-amino-2-hydroxypropyl-substituted oxime ethers

161

OH 162

162 exhibit interesting hybrid properties as organic nitrates and /?-blocking

In an application dealing with antithrombotic and antihypertensivecompositions, the disulfite 163 ofisosorbidewas mentioned as one ofthe possible active ingredients.225Cardiovascularactivities were also claimed for a large (224) H. Simon, H. Michel, W. Bartsch, and K. Strein (Boehringer Mannheim GmbH), DE 3,512,627 (1985); Chern. Abstr., 106 (1987) 49,604. (225) B. K. Martin (T and R Chemicals,Inc.), EP 113,235 (1983); Chem. Abstr., 101 (1984) 157,677.

1,4 :3,6-DlANHYDROHEXITOLS

165

O-S-OH

0 163

number of piperazine-substituteddeoxyisohexide nitrates,ma and for three isosorbide 5-nitrate benzyl ether derivatives.146o In addition to the cardiovasculararea, to which all of the aforementioned derivativesbelong, isohexides have also been elaborated for other pharmaceutical indications. Several mono- and di-0-alkylisohexides, as well as monoalkylisohexide carbamates, were tested for anticonvulsant activity. 176 During an investigation of the hypnotic properties of 2,5-diazido-2,5-dideoxyisohexides (17-20), the L-isomannide derivative turned out to be comparable to the known gluthethimide, whereas the D-antipode was completely inactive. The D-glucitol compound showed significant hypnotic activity, but the L-isoidide derivative, none.”J@’ Among 138 examples of various sulfamoyl compounds, three isohexide derivativeswere mentioned. Their preparation started from corresponding free hydroxyl compounds by reaction with sulfamoyl chloride, which itself had been generated in situ from chlorosulfonyl isocyanate. They were claimed for treatment of chronic arthritis and osteoporosis.225a A number of isohexide mono- and di-amines (164) failed to exhibit antitumor activity. A few of them are weak antiflammatories, albeit rather X

0’

-NR’R* 154

t 0 ~ i c . l Potential ~’ antitumor and antiviral properties were the aim of the nucleoside-like compounds 87 containing several pyrimidine, triazole, and (225a) Y. S. Lo,J. C.Nolan,D. A. Walsh, and W. J. Welstead Jr., (A. H. Robins Co.) EP 403,185 (1989). Chem.Abstr.: no reference up to Vol. 115 (1991) No. 2.

166

PETER STOSS AND REINHARD HEMMER

165

imidazole nucleobases.174~175Some monodeoxyisohexide ethers 165 substituted with phenyl groups (R) were found to be useful as herbicides.m1 Nitroxide radical-forming agents, for example, all seven isohexide monoand di-nitrates, were claimed as ingredientsin topical formulations for stimulation of hair The widespread applicability of di-0-methylisosorbide as a medium for chemical reactions or as a solvent for pharmaceutical formulations is well documented. In some cases, an additive synergism of the solvent and the solutewas observed. Sometypical examples mentioned include that it acts as a solvent for muscle-relaxant drugs, which are otherwise difficultly soluble,226and is used for topical and other types of pharmaceutical formulati0ns,227J28 transdermal controlled-release films229and tapes:w anthelmintic sol~tions,2~* antimycotic emul~ions,2~~ and for the treatment of skin disorders, such as eczema.233 The antifungalactivity of thiabenzazoleagainst Penicillium digitatum was found to be enhanced by adding various carbohydrate esters of fatty acids. Among them, isosorbide monododecanoate was moderately active?” Isomannide mono-oleate has frequently been applied to generate highly stable water-in-oil type emulsions which could act as useful adjuvants for vaccines to enhance the efficacy of incorporated antigen^.^^^-*^^ Isosorbide mono- or (225b)P. H.Proctor (P. H. Proctor) EP 327,263(1988);Chem. Abstr., 112 (1990)204,461. (226) R. 0.Beauchamp,Jr., J. W. Ward, and B. V. Franko(A. H. Robins, Co.,Inc.), US.Pat. 3,699,230(1971);Chem. Abstr., 78 (1973)20,197. (227) J. L. Chen and J. M. Battaglia (E. R. Squibb and Sons, I c . ) , U.S. Pat. 4,082,881 (1976); Chem. Abstr., 89 (1978)117,860. (228) J. C.Dederen, Expo.-Congr.Int. Technol.Pharm., 3rd, (1983)335-336;Chem. Abstr., 103 (1985)76,165. (229) M.Dittgen and R. Bombor (Ernst-Moritz-Amdt-UniversityGreifswald), D D 217,989 (1983);Chem.Abstr., 103 (1985)147,168. (230)Y.Ito, T.Horiuchi, and S . Otsuka (Nitto Electric Ind. Co., Ltd.), Jpn. Pat. 86,221,121 (1985);Chern.Abstr., 106 (1987)107,914. (231) M.R.Clark and A. Lewis (May and Baker Ltd.), DE 3,442,402(1983);Chem.Abstr., 103 (1985) 183,558. (232)M.Wischniewski and L. Feicho (Kali-Chemie Pharma GmbH), DE 3,600,947(1986); Chern.Abstr.. 108 (1988)26,961. (233) L.A.LuzziandJ.K.Luzzi,U.S.Pat.4,711,904(1986);Chem.Abstr., 108(1988)82,146. (234) Y.Nishikawa and M. Ohkawa, Chem. Pharm. Bull., 36 (1988)3216-3219. (235) R. J. Tull (Merck and Co.,Inc.), DE 2,249,831(1927);Chem. Abstr., 81 (1974)13,752.

1,4 :3,6-DIANHYDROHEXITOLS

167

di-alkyl ethers, or mixtures thereof, with preference for the dimethyl compound, were claimed as being useful for dentifrice formulations.182*240 It is worth mentioning that the explosiveproperties of the vasodilatordrug isosorbide 2,5-dinitrate can be overcome by forming its 1 : 1 complex with cyclomaltoheptaose.21

3. Technical Applications a. Food. -The sensory properties(sweetnessand bitterness)of isosorbide and isomannide, among those of other carbohydrate derivatives, have been discussed on a molecular basis.242 Isosorbide dimethyl ether ( M a ) is used as an ingredient in the manufacture of chewing gums, chewable tablets, hard candies, and nougat products.240

Isosorbide dipropanoate (166b) is used as an effectivesoftening agent, as well as a fungistat, when incorporated into bakery products.122Ethoxylated fatty acid esters of isosorbide are used as conditioners in bread making.243 Isosorbide acts as an all-purpose, plastic, shortening material in the manufacture of cakes, icings, and cream fillings, producing excellent moisture retention and aeration properties.244A process for preparing benzaldehyde and acetaldehydetakes place in presence of water and a nonionic emulsifier. The latter containing mixtures of “sorbitan-” and isosorbide-fatty acid esters2*”.Moderate surface activities were reported on perfluoroalkylated mono and di-esters of isomannide and isosorbide.112b (236) Merck and Co., Inc., Jpn. Pat. 74 72 285 (1972); Chem.Abstr., 85 (1976) 21,766. (237) A. F. Woodhour and M. R. Hilleman (Merck and Co.,Inc.), US. Pat. 3,983,228 (197 1); Chem.Absrr., 85 (1976) 182,397. (238) M. Midler, Jr. and E. Paul (Merck and Co., Inc.), U.S. Pat. 4,073,743 (1975); Chem. Absrr., 88 (1978) 197,630. (239) B. Brancq and L. De Philippe (ProduitsChimiques de la Montagne Noir), FR 2,501,526 (1981); Chem. Abstr., 98 (1983) 59,877. (240) M. J. Lynch (ICI Americas Inc.), U.S.Pat. 4,585,649 (1984); Chem. Abstr., 105 (1986) 66,28 I . (241) M. Low,L. Kisfaludy, A. Vikman, J. Szejtli, I. Stadler,1. Gemesi, I. Kolbe, G. Hofhann, M. Gyannathy, and G. Hortobagy (Richter Gedeon), HU 37,801 (1984); Chem.Abstr.. 106 (1987) 143,984. (242) C. K. Lee and G. G . Birch, J. Food Sci., 40 (1975) 784-787. (243) R. K. Langhans (ICI Americas Inc.), U.S. Pat. 3,859,445 (197 I); Chem.Absrr., 82 (1 975) 154,058. (244) D. T. Rusch (AtlasChem. Ind., Inc.), U.S. Pat. 889,005 (1970); Chem.Abstr., 75 (1971) 117,364. (244a) A. 0.Pittet, R. Muralidharaand A. L. Liberman (Internat. Flavorsand Fragrances I c . ) U.S.Pat. 4,683,342 (1987); Chem. Absfr., 1 10 (1989) 22,530.

168

PETER STOSS AND REINHARD HEMMER

Isosorbide mono(tetradecanoate) (166d) prevents the denaturation of ground fish during freezing.z45Mixed ether-ester-substitutedisohexides (especially those of isosorbide) are used as flavor enhancers.z46

R20

b OR'

166

a R'=R2=CH 3 b R1 = R2 = COCH,CH, c R' or R2 = C0(CH2)&H3 d R' or R2 = CO(CH2)12CH3 e R' = R2 = COCH(C2HS)C4H, f R' = R2 = C0(CH2)&H3 g R' = R2 = CO(CH2)&H3 h R1= R2 = CO(CH2)&H3

i R' = H, RZ= Co(CH2),CH/3-~&1cpNAc-(l+3)-~-Gal (54)d &LAMP-( 1 +4)-/3-~-GlcpNAc-(1+6)-~-Gal(53)d

fi~-Galp(I+4)-,T-~-GlcpNAc-(l+Z)-a-~Manp-(l +OMe) (57)d 1+4)-/3-~-Gl~pNAc-(l+~)-[BD-G~c~NAc-( 1+3)]-/3DGalp-(1+4)-,T-~-Glcp-(l+OMe) (55)d fidrmp( 1+4)-/3-~-GlcpNAc-(I +3)-[Bdrmp( 1 + 4 ) - f i ~ GlcpNAH 1 +6)]-/3-~-Galp-(l+4)-/3-L&lcp(l +OMe) (56)d fioGalp(l+4)-fi~-GlcpNAc-(l + A m ) (58)d

34 2 0.5 0.5

GT: 1; E:1; UP:1.2; M:3; IP:3.6; PK:4

85 30 70 70

GT:4; E:4; UP:+ IP:80; PK: 140 GT:4; E:4; UP:+ IP:80; PK: 140

0.05

36

GT :24; E :30; UP:46; PK: 290, IP:350

19

0.07

44

GT:24;E:30;UP:46;PK:290;IP:350

19

0.13

26'

GT:29; E:19; UP: 17; IP:100, PK:90

80

23 75 79 79 15

Unless otherwisestated, the galactose precursorwas Dglucosyl phosphate,the phosphorylatingagent was ATP, and the source ofenergy was enolpyruvatephosphk. Enzymes immobhed on PAN gel; Mucose 6-phosphate as precursor. Enzymes immobilized on silica gel-glutamldehyde. Enzymes immobilized on agarose. Isolation &cult.

ENZYMIC PREPARATION OF CARBOHYDRATES

225

good, inexpensive source,87where it is fairly abundant (60 U/kg). A homogeneous enzyme is not necessary for sialylation.From a suitable concentrate, the enzyme may be immobilized on agarose in good yield, after addition of 0.5 mg of bovine serum albumin per mL of extract, and dialysis against the immobilization buffer (0.1 M phosphate, pH 7.8; 25% of glycerol). Such preparations are stable for at least 5 months at 4",and may be utilized at least three times without noticeable loss of activity.l5 The second transferase (STB; see Table I) is also commercially available, and is still more expensive. It catalyses the transfer of N-acetylneuraminic acid to 0-3 of D-galactose in the terminal residue &~-Galp(1+3>wGalN A C .The ~ ~ third one (STC), so far does not appear to be at all easily available. It catalyzes the transfer of a N-acetylneuraminicacid residue to 0-3 of D-galactose in a B-~-Galp-[143(4)]-/3-~-GlcpNAcresidue. Most sialylations so far reported have been achieved with solubletransferases, and seldom on a more than 20-pmol scale (see Table IX),with the intention to prepare and describe sequences present in glycoproteins and glycolipids. Trisaccharide a-~-NeuSAc-(2 +3)-8-~-Galp( 1 ~)-P-DGlcpNAc-( 1 +OMe) was prepared with two different transferases, STB and STC.In our view, the greater efficiency of STB in this preparation deserves further investigation, as the reverse observation might have been expected in view of the known specificities of these enzymes. The one glycopeptide in Table IX, namely, 60 (Ref. 80), was prepared from the known922-acetamido- 1-N-(~-aspart-4-oyl)-2deoxy-~-~-~ucopyranosylamineby two enzymic steps, a D-galactosylation, to give intermediate 58, followed by sialylation.mIt is interesting that neither the wboxylate nor the amino group of the L-aspartamide moiety was inhibitory in these reactions. Compound 60 appears identical with a glycopeptideisolated from the urine of a patient suffering from aspartylglu~osaminuria.~~ In Table X are reported three syntheses with immobilized transferase STA.15 Comparison with the reactions of the same enzyme in Table IX outlines the advantages of immobilization: the scale has been raised, and much less activity is necessary. It is possible to work with a mole to mole ratio 18pg9

(87) D.H.vandenEijndenandW.E.C.M.Schiphorst,J.Bid. Chem.,256(1981)3159-3162. (88) J. E. Sadler, J. I. Rearick, J. C. Paulson, and R. L. Hill, J. Bid. Chem., 254 (1979) 4434 -4443. (89) J. Weinstein, U. de Souza-e-Silva,and J. C. Paulson, J. Biol. Chem., 252 (1982) 13,84513,853. (90) S. Sabesan and J. paulson, J. Am. Chem. Soc, 108 (1986) 2068-2080. (91) K. G. I. Nilsson, Curbohydr. Res., 188 (1989) 9- 17. (92) H.G. Garg and R. W.Jeanloz, Adv. Curbohydr. Chem. Biochem.,43 (1985) 135-201. (93) J. F.G. Vliegenthart, L. Dorland, and H. van Halbeek,Adv. Curbohydr. Chem.Biochem., 41 (1983) 209-374.

TABLEIX Siylations with Soluble Transferases product

Oligosacchnrides a-mNeuSAc-(2+6)-&~-GaIp(1-rOMe)

a-mNeuSA~-(2+6)-fi~-Galp-(l-r4)-&~-Glcp( 14OMe) a-~-NeuSAc-(2+6)-&~-Galp-( 1+4)-~-GlcNAc a-~-Neu5Ac-(2+6)-fioGalp( 1+4)-BDGlcpNAo(l +OMe)

N OI N

a-~-NeuSAc-(2+6)-&oGalp( 1-r4)-&~-GlcpNAc-(l+3)-fi~-Galp( 1 -4)-~-Glc a-~-NeuSAc-(2+3)-&ffialP( 1-+3)-a-~-GalpNAc-OR R = Et R = CH,CH,Br R = (CH,),CO,Me ~ - D - N ~ U ~ A C - ( Z + ~ ) - ~ ~ ~ + ~ ) - ~ ~1+OMe) D-G~C~NAC-(

Scale (Pol) 7 9 47 20 14

64 8 13 17 7 6 a-~NeuSAc-(2+3)-&~-Galp-(1+4)-&ffilcp(l +OMe) 9 a-~-Neu5Ac-(2+3)-&~-GaIp(l+4)-&ffilcpNAc-( 1 +OMe) 9 a-~-NeuSAc-(2+3)-fioGalp(l~4)-fi~-GlcpNA~l~3)-fiD-Galp(l+4)-~-Glc 7 a-~-NeuSAc-(2+3)-fioGalp(l~3)-&~-GlcpNAc-(l+3)-&D-GaIp(l+4)Glc 50 Clycopeptide a-~-Neu5Ac-(2+6)-fi~l+4)-/3-~-GlcpNAo(l +N)Asn (60) a

Yield with respect to the substrate. Yield with respect to cytidine monophosphat&”lneuramjnic gland.

Yield I”

Yield IIb

(%)

(%I

1 1 57 48 74

33 42 47 96

64 95 32 23 18 0.7 22 47 35 38

64 59 32 52 18 28

35

45

22 17 38

Units/ mmol

References

STA:32 STA:24 STA:53 STA:12 STA:21

90 90 75 90 90

STBc STB:2.5 STB:1 STB:2.3 STC:7 STC:8.5 STC:5 STC:6 STC:7 STA:lO

91 91 90 91 90 90 90 90 90 80

acid. A preparation correspondingto 65 g of porcine

TABLEX Sialyhtionswith I m m o b i i Sialyltransferases~ ~-

Nucleotide-sugar

R 4

c ~ Neu5Ac p (49)

CMP-Neu5,9Ac2(50)

Product

a-~-NeuSAc-(2--r6)-S~-Gallp(l+4)-/3-~Gl~pNAc-( 1-+2)-ol-~-Manp (1 4 O M e )(61) a-~-NeuSAc-(2+6)-/3-D-Galp(l -+4)-/3-~-GlcpNAc-(l+3)-[/3-&alp-( 1 - 4 ) /~-DCIC~NAC-( 1 --r6)]-B.~-Galp-(l-*4)-~~-Cilcp(l-.OMe) (62) a-~-Neu5,9Ac~-(2+6)-&DGalp( 1+4)-~-GlcpNAc(63)

Scale @mol)

Yield* (%)

Units/ mmol

References

100

46

4c

15

45

34

8.9

95

160

65

2.5'

15

~~

CMP NeuSAc :&&alp( 1 - + 4 ) - ~ l c p N A c a ~ 2 - 6 > y l t r a n s f ~ . Equimolecularamountsof substrate and coenzyme were used. After these couplings, the ~ ered enzyme preparation retained full activity.

~ o y -

228

SERGE DAVID et al.

HO 60

of precursor oligosaccharide to CMP-NeuSAc. Furthermore, it should not be forgotten that the enzymic gel may be used again at least three times, so that, in principle, a scale three times as high is within reach. The synthesis of tetrasaccharide-glycoside61 involved first the preparation¶by organic chemistry methods, of the known glycoside&D-GlcpNAe (1 +2)-ct-~-Manp( 1+OMe), followed by enzymic D-galactosylation to give 57, and then sialylation. Its sequence is a common feature of a class of glycoproteins. The free tetrasaccharide has been prepared by organic glycosidic coupling.94 Heptasaccharide 62 was obtained from hexasamharide 56 (see Section V,2). As in the case of D-galactosylation, enzymicsialylation turned out to be highly regioselective, leading to a single compound, whereas each galactose

(94) T. Kitajima, M. Sugimoto, T. Nukada, and T.Ogawa, Carbohydr. Res., 127 (1984) cl -c4; H.Paulsen and H. Ti&, ibid. 144 (1985) 205-229.

ENZYMIC PREPARATION OF CARBOHYDRATES

229

Ho

62

63

residue could theoretically be substituted. Heptasaccharide 62 corresponds to the product of sialylation of the D-galactose residue on the p-( 1-3) branch.9sIt is the methyl glycoside of a sialylhexasaccharide isolated from human milk.% The preparation of trisaccharide 63 illustrates the activation and enzymic coupling of the 9-acetate of N-acetylneuraminic acid. This involves the utilization of enzymes in a cascade of reactions which probably do not occur in cells: (a) synthesis of Neu5,9Ac2from the 6-acetate of N-acetylmannosamine with the catabolic sialyl aldolase, (b) activation with CMPNeuSAc synthetase,and (c) coupling. Acetylation in cells seems posterior to coupling. Terminal nonreducingN-acetyl-9-0-acetylneuraminicacid residues appear (95) C. Augk and C. Gautheron, unpublished results. (96) M. T. Tarrago, K. H. Tucker, H. van Halbeek, and D. F. Smith,Arch. Biochem. Biophys., 267 (1988) 353-362.

H

NeuSAc

+

g 0

P2°7H4

x \

o

'aoNHAC

H % ~ , ~ ~

/

\r

CMP-Neu5Ac CMP-Neu5Ac

Enolpyruvate phosphate

CDP

CMP

A

\

CHzOH

Enolpyruvate phosphate

Pyruvate

I

Ac OH SCHEME 21.-Cycle for Enzymic Sialylation which Should Allow in situ Regeneration of CMP-N-AcetylneuraminkAcid.

ENZYMIC PREPARATION OF CARBOHYDRATES

23 1

fairly widespread as part of the oligosaccharide epitope of some important antigens (see, for instance, Ref. 97). These reactions correspond to the right-hand side of Scheme 2 1. The complete Scheme would correspond to the overall synthetic route to sialosides, with the following balance: Neu5Ac

+ 2 CH2

-

+

C(OPO,H,)--CO,H R-OH sialoside f 2 CH$OCOZH P207H4

+

-B

It would be elegant to bring together the broken ends of the cycle, and make the four immobilized enzymeswork together in one vessel, so that the part played by CMP would be only catalytic,but such a cycle has not yet been reported. The difficulties come from problems of inhibition: sialyltransferases are inhibited by CTP and CDP (K, = 2. 10-5M).86 However, this inhibition was reported89to be relieved by the addition of MnCl,, and so the in situ regeneration of CMPNeuSAc should be feasible. 4, Glucosylation

Stoichiometric quantities of “uridine diphosphate glucose” were used, in the presence of a transfer enzyme, sucrose synthetase, in the soluble state (extraction given). Coupling with modified D-fructose gave sucroses modified on the D-fructosyl group, on the 1- 3-mmol scale. Thus were prepared 1I-deoxy- 1’-fluoro- (59%),98 4’-deoxy-4’-fluoro- ( 16%), and 1I-azido-1Ideoxy-sucrose ( 15%).’l 6-Deoxy-6-fluoro-~-glucosewas isomerized to 6deoxy-6-fluoro-~-fructosewith isomerase, and gave 6’deoxy-6’-fluorosucrose. VI. TRANSFER REACTiONS CATALYZED BY GLYCOSIDASES Glycosidases catalyze the hydrolysis of glycosidic bonds D-OZ

+ H20

+

D-OH

+ Z-OH,

D being a glycosyl group. The reaction occurs in two main steps, formation of a glycosyl enzyme, and transfer of the glycosyl group to a water molecule. D-0-Z

D-E

+ EH

+ HO-H

-P

+

+ ZOH D-OH + EH DE

(97) D. C. Gowda,G.Reuter, A. K. Shukla, and R. Schauer,Hoppe-Styler’s Z. Physiol. Chem., 365(1984) 1247-1253.G. N. Rogen,G. Herrler,J.C. Paulson,andH. D. Klenk,J. Eiol. Chem., 261 (1986) 5947-5951. (98) P. J. Card and W. D. Hitz, J. Am. Chem. Soc., 106 (1984) 5348-5350.

SERGE DAVID et al.

232

Other hydroxylated derivatives (A-OH), such as alcohols or sugars, are also possible acceptors: D-E

+ A-OH

-L)

D-0-A

+ EH,

so that the overall reaction is the transfer of the glycosyl group D from the donor molecule D-0-Z to one oxygen atom of the acceptor A-OH. D-0-Z+A-O-H~D-O-A+Z-OH

There are presumably two configurational inversions in such a mechanism, so that the anomeric configuration of the newly formed glycosidic bond is the same as that of the donor. Now that some glycosidasesare common and inexpensive,this scheme of glycosylation looks very attractive. Furthermore, if the acceptor is a polyhydroxylatedderivative, the reaction is regioselective, and there is a measure of control over that regioselectivity. However, yields are small,rarely exceeding 30% with respect to the donor molecule, and generally inferior, and the acceptor is added in 2-20-fold excess. At first sight, this would appear of little importance for practical purposes, for the starting sugars are often less expensive than other chemicals, such as solvents or chromatographyadsorbents, and it should not be forgotten that nonenzymic oligosaccharidesynthesis, with its many steps in the present state-of-the-art, gives small overall yields. In our view, the problem lies elsewhere: the simple derivatives obtained with glycosidaseswill be interestingas starting compoundsfor further syntheses, and thus needed in relatively large quantities. Then, separation from a great excess of sugars with like properties may be very expensive. Money and labor saved on one side may be wasted on the other. Thus, it is to be expected that these promising routes will achieve popularity when cheap separation procedures are evolved. Some significantresults are reported in Table XI. Lactose, a by-product of the dairy industries having a negligible value, acts as a source ofpD-galactopyranosyl groups in the presence of #b-galactosidase. a-D-Galactopyranosyl and a-D-mannopyranosylgroups are transferred from the corresponding pnitrophenyl glycosides in the presence of a-glycosidases. Such systems allow, for instance, a remarkably quick preparation of derivatives of the disaccharide a-D-Galp-(1 --* 3)-mGal, a sequence present in blood-group B substance, and not readily available because of its 1,Zcislinkage. (99) K. G. I. Nilssoo, Curbohydr. Res.. 167 (1987) 95- 103. (100) K. G.I. Nilssoo, Curbohydr.Res., 180 (1988) 53-59. (101) F. Bjarkliog and S.E.Godtfredsen, Tetrahedron,44 (1988) 2957-2962. ( 102) P.0.Larsson, L. Hedbys, S.Sveossoo,and K.Mosbach,Methods Enzymol., 136 (1 987) 230-233.

TABLEXI Pyranosyl Transfer with Glycosidases" Yield Acceptor

Product

(a) System:pnitrophenyl a-D-galactopyranoside a-mgalactosidase a-~-Galp(1+3)-a-~-G&ll +OC6H4N02-d4)1 a-D-Galpl1+OC,H4N0,-

d411

a-D-GalP(1+3)a-~-Galp( 1+OCHZCH=CHZ) (1 +OCHZCH=CHz) a-o-Galp(l-3)-a-~-Galp(l+OMe) a-PGalp(1+OMe) /3-~-Galp41+OMe) a--dmh(l+3)-/3-oGalp(l+OMe) (b) System: o-nitrophenyl j?-D-galactopyranoside, /?-D-galactosidase 1+OMe) /3-~-CAp-(l+3)-/3-~-Galp(l+OMe) 2,3-Epoxypropanol 1-~/3-~galactopyranosy1-[2(R,S),3-epoxypropol] (c) System: lactose,j?-D-galactosidase CH,=CH-CH,OH bDGalp(l+OCH,--CH=CH,) PhCH,OH bo-Galp(1+OCH,Ph) GalNAc /3-D-Galp( 1+6)~-GalNAc (d) System: pnitrophenyl a-D-mpnnopyranoside, a-mmannosidase a-~-Manp(1+2)-a-~-Manp( 1+OMe) cr-~-Manp(1+OMe) a-DGalp

a

Soluble enzymes, unless stated otherwk. Enzyme immobilized on tresyl-activated Sepharose.

Scale

(%)

U/mmol

12

60

1

99

18

45

0.5

100

28 17

6 28

2 0.5

99 99

17 28

400

1.5 5

101

31 14 25

17 240 7ob

100 15 1

100 100 102

15

37

10

99

10

References

(-01)

99

234

SERGE DAVID ef al.

VII. MISCELLANEOUS SYNTHESES IN AQUEOUS SOLUTION

Some lipases catalyze the selective hydrolysis of the anomeric acetate in peracetylated sugars. Thus were prepared 2,3,4,6-tetra-O-acetyl-~-galactopyranose, -D-glucopyranose, and -D-mannopyranose, 2-acetamido-3,4,6tri-O-acetyl-2-deoxy-~-glu~p~nose and -mmannopyranose, 2,3,4-tri-Oacetyl-L-rhamnoseand -L-fucose, and 2,3,5-tri-O-acetyl-~-ribofuranose and -D-xylofuranose on the 2-mmol scale, generally in good yields.lo3 These reactions were conducted in a 1 :9 mixture of N,N-dimethylformamideand 0.1 M phosphate buffer (pH 7) by stirring at room temperature in the presence of the lipase, and adjusting the pH to 7.0 with 1.0 M NaOH. Under similar conditions, methyl glycosides are 0-deacetylated on the primary position, affording, inter a h , methyl 2,3di-O-acetyl-cu-~ribofmnoside, P-D-ribofuranoside,and a-~-arabinopyranoside,~~~ and methyl 2,3,4-tri-0pentanoyl-cx-D-ghcopyranoside.104 Treatment of 3,6-di-O-butanoyl-~-glucose with the lipase from Candida cylindracea gave 3-O-butanoyl-~-glucose in 85% yield.lo5More information on acylation and deacylation, but this time in organic media, will be found in Section IX. We finish this Section with enzymic conversions that are difficult to classifyelsewhere:Takasweet, a commercialvariety of immobilizedglucose-isomerase, converts 6-0-methyl-~-fructoseand 6-deoxy-~-fructoseinto the gluco isomers in not very satisfactory yield.” A mixture of catalase (75 U/mmol) and glucose oxidase (80 U/mmol) oxidizes xylitol to ~-xylosein 5040 yield, on the 100-pmol scale.lMThe enzyme cyclodextrin a-(1 4 4 ) glucosyltransferase ( 1000 U, immobilized on silica gel-glutaraldehyde) preparation of cyclomaltohexaose (0.3 g), cyclomaltoheptaose

64 (103) W. J. Hennen, H. M.Sweers, Y.F. Wan& and C. M.Won& J. Org. Chem.. 53 (1988) 4939- 4945. (104) H.M. Sweers and C. H. Won& J. Am. Chem. Soc.,108 (1986) 6421-6422. (105) M. Thtrisod and A. M. Klibanov, J. Am. Chem. SOC.,109 (1987) 3977-3981. (106) R. L. Root,J. R. Dunwachter, and C. H. Won& J. Am. Chem. Soc.,107 (1985) 29972999.

ENZYMIC PREPARATION OF CARBOHYDRATES

235

(0.38 g), and malto-oligomers from a-D-glucopyranosyl fluoride (1 g).Io7 The same enzyme allowed the preparation of 12 mmol of the glycoside64 of ldeoxynojirimycin in 25% yield.’@

VIII. ENZYMESIN ORGANIC SOLVENTS*

Klibanov has summarized the principles of the technique in a short survey.lWEnzymes still work albeit, sometimes, in a different way, if a layer of “essential water” is somehow localized and kept on their surfaces, and the bulk water is replaced by an organic solvent. Thus, the enzymesare generally freeze-dried, and the solids suspended in an organic solvent, and then traces of water are added to ensure maximum activity. In this state, they show high conformational rigidity: heat-induced unfolding (denaturation)is hindered, and some are stable for hours at 100”.Because of this rigidity, they keep a “memory” of their previous state in water: freeze-drying in the presence of active-sitedirected molecules may yield more active conformations. The ionization state corresponding to the pH of the aqueous solution, which should be optimal, is also retained. The absence of water may have other advantages: for instance, lipases may act as esterification catalysts, a property obscured in water solution by the reverse, common hydrolytic reactions. However, it seems that empirical trials are still neceSSary in order to achieve a successful synthesis: several enzymes from different natural sources should be tested, and even enzymeshaving different specificities. For laboratory-scale preparations, the cost of such enzymes as the lipases from porcine pancreas (PPL), Cundida cylindruceu (CCL), and Chromobucterium viscosurn (CVL), and Protease N (Ammo) is negligible. Subtilisin, a protease, is much more expensive. Transesterificationof sugars and derivatives with such “active” esters as the acetate, butanoate, decanoate, or dodecanoate of 2,2,2-trichlorethanol allowed selective acylation. In this first way, ~-glucose,D-galactose, and D-mannose, in multigram quantities, gave the primary acylate in fair yield,Il0in pyridine solution, in the presence of PPL (70,000 U). This type of reaction was also selective with di- and tri-saccharides: thus, in

* The authorsare much indebted to Dr.Michel Thi5rkx.I for the preparation of this Section. (107) W. Treder, J. Thiem, and M. Schlingmann, Tetrahedron Left.,27 (1986) 5605-5608. (108) Y. Ezure,Agric. B i d . Chem., 49 (1985)2159-2165. (109) A.M. Klibanov, TIBS, (1989) 141-144. ( 1 10) M. Thinsod and A. M. Klibanov,J. Am. Chem. Soc., 108 (1986) 5638-5640.

SERGE DAVID et al.

236

N,N-dimethylformamide solution, in the presence of subtilisin, maltose, cellobiose, lactose, and (remarkably) maltotriose, gave, on the gram scale, fair yields of the primary butanoate in the nonreducing unit. Sucrose (1 3 g) gave, in 60%yield, the primary butanoate of the ghca moiety in the presence of the very cheapti1Protease N; transestefication from 2,2,2-trifluoroethyl acetate, in oxolane solution, in the presence of PPL, likewise gave the primary acetate of some methyl pentofuran~sides.~~~ Degueil-Castaing and coworkers1**introduced enol esters in transesterification, and these were also used in the carbohydrate field: N-acetylmannosamine and isopropenyl acetate in N,Ndimethylformamidesolution gave 2-acetamid0-6-O-acetyl2-deoxy-~-mannose, in the presence of protease N (1 g/mmol)." This acetate is the precursor of an important sialic acid." In a study of the selective esterifidon of hexoses already substituted at the primary alcoholic function, Th6risod and K l i b a n o ~ observed '~~ very suggestive differences in selectivity,according to the origin of the lipase. All these reactions were transesterification with 2,2,2-trichlorethyl butanoate, with 100,000 U of lipase per gram of substrate, in oxolane as the solvent. With 6-O-butanoyl-~-glucose as the substrate, lipase CVL catalyzed transesterification to 0-3, to give an 80% yield of 3,6-di-O-butanoyl-~-glucose, while PPL directed the reaction to 0-2, giving a 50% yield of 2,6-di-O-butanoyl-D-glucose. The 0-3 atom was only marginally favored over 0-2 with 6-O-butanoyl-~-galactoseand CVL, but a yield of 55% of 3,6di-O-butanoyl-D-mannose could be achieved from 6-O-butanoyl-~-mannose.Remarkably, 6-O-trityl-~-glucosewas a substrate of lipase CVL (only 20,000 U/gof substrate). Transesterification, followed by detritylation, gave a 90% yield of 3-O-butanoyl-~-glucose.

-

IX. ADDENDUM A large-scaleenzymic synthesis of the trisaccharidea-~-NeuSAc-(2+3)gal-( 1+3)-~-GlcNAc (65), the tumor-associated carbohydrateantigen CA 50, has been achieved.' l3 This is a further illustration of the cross reactivity and efficiency of STB. This sialyltransferase, like STA, was partially purified from porcine liver, accordingto a modification of Conradt's procedure.Ii4The initial rate measured for p-~-Gal-(1*3)-~-GlcNAcat saturating concentration was 18%of the one measured for the real substrate B-D(1 11) S. Rim, J. Chopineau, A. P.G. Kieboom, and A. M.Klibanov, J. Am. Chm. Sot., 1 10 (1988) 584-589. (1 12) M.Degueil-Castaing, B. De Jew, S. Drouillard, and B.Maillard, Tetrahedron Lett., 28 (1987) 953-954.

ENZYMIC PREPARATION OF CARBOHYDRATES

231

Gal-(1+3)-~-GalNAc. By using 0.7 U of STB, as a soluble preparation readily obtained from 300 g of porcine liver, the sialylation of p-D-Gal( 1 -3)-~-GlcNAc was performed on a one-mmol scale and sialylated trisaccharide 65 was obtained in 2 1% isolated yield. In this respect, the purification of reaction mixturesis still troublesome, especially because of the presence of Triton X-100; from our experience, the use of immobilized enzymes, eliminating the need for detergent, greatly facilitatesthe purification procedure. Concerning aldolases,the cloning of enzymesis becoming more and more common. Thus the bacterial fuculose-1-phosphate aldolase (EC 4.1.2.17) and 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) have been recently overexpressed in E. coli and their synthetic use has been e ~ a m i n e d . ~ ~ ~ . ~ ~ ~ ACKNOWLEDGMENTS This work was supported by the Centre National de la Recherche Scientifique and the Universiti de Paris-Sud at Orsay.

( 1 13) C. Augi, P. Francois, and A. Lubineau, Jacques Monod Conference on Chemistry, Bio-

chemistry and Molecular Biology of Glycoconjugates,Aussok, France, October 22- 21, 1990. (1 14) H. S. Conradt, K. Hane, and M. Mom, Proc. JapaneseGerman Symp. Sialic Acids,

Berlin, F.R.G., may 18-21 (1988) 104- 105. (1 15) A. Ozaki, E. J. Toone, C. H. von der Osten, A. J. Sinskey, and G. M. Whitesides, J. Am. Chem. Soc., 112 (1990) 4910-4911. (1 16) C. F. Barbas, Y-F.Wang, and C-H. Wong, J. Am. Chem. Soc., 112 (1990) 2013-2014.

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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 49

STRUCWRE OF COLLAGEN FIBRILASSOCIATED, SMALL PROTEOGLYCANS OF MAMMALIAN ORIGIN BY HARIG. GARG*AND NANCYB. LYON#

* Department of Biological Chemistry and Molecular Pharmacology and # Department of Dermatology, Harvard Medical School at the Massachusetts General Hospital, and the Shriners Burns Institute, Boston, Massachusetts 02114 I. Introduction.. ....................................................... 11. Structure of Different Glycosaminoglycans. ............................... 111. Carbohydrate-Protein Linkage Regions. ................................. IV. Isolation and Fractionation of Small Proteoglycans. ........................ V. M, of Small Proteoglycans, Their Protein Cores, and Glycosaminoglycan Chains VI. N-Terminal Sequence of Small Proteoglycans ............................. VII. Amino Acid Sequence Analysis of the Small Proteoglycan Core Protein, Deduced fromClonedcDNA ................................................. VIII. Biosynthesis of Small Proteoglycans ..................................... 1. PrimaryCulture. .................................................. 2. Explant Culture ................................................... IX. Biological Roles of Small Proteoglycans .................................. X. Addendum. .........................................................

239 240 240 243 244 253 254 256 256 257 258 260

I. INTRODUCTION** All mammalian tissues contain proteoglycans (PGs), which consist of a single protein core containing one to >200 glycosaminoglycan chains attached through 0-P-D-xylopyranosyl-(1+3)-~-serine/~-threoninelinkage(s). The various types of PGs present in different tissues can be divided into two categories, namely, (1) cell-associated PGs and (2) extracellularmatrix PGs. Of the extracellular-matrix PGs, the cartilage PGs have been

** Articles on this subject that have appeared since this text was completed are listed in the Addendum starting on p. 260.

239

Chynghl 8 1991 by Academic Press, Inc. All rights of nproductionin m y form -&.

240

HARI G. GARG AND NANCY B. LYON

extensively discussed in Cell-associated PGs have also been described in comprehensive During the past decade, collagen fibrilassociated PGs have begun to be characterized. These PGs have been isolated from normal bone, cartilage, scar, skin and other connective tissues, and have been shown to contain a protein core to which 1 or 2 glycosaminoglycan (GAG) chains are attached. Their molecular weights, in distinct contrast to the high density cartilage PG, range from 70 to 140 kDa, and for this reason they are generally referred to as “the small PGs.” The small dermatan sulfate collagen fibril-associatedPGs from interstitialmammalian tissuesare the subject of this Chapter. 11. STRUCTURE OF DIFFERENT GLYCOSAMINOGLYCANS Glycosaminoglycans (GAGS) are unbranched chains having repeating disaccharide units, which, with the exception of keratan sulfate, contain an acid and a base. The KS disaccharide unit consists of a hexose and a base. The structures of the different classes of GAG disaccharide units are given in Fig. 1, and summarized in Table I. The GAG chains of chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and heparin (HP) also contain one molecule of D-xylose and two molecules of D-galactose per molecule, along with acidic (Dglucuronic/L-iduronic) and basic (D-galactosamine/D-glucosamine)sugars. The migration of the GAG chains on cellulose acetate plates stained with Alcian Blue occursgin the following order HP < DS < HS < HA < CS. 111. CARBOHYDRATE - PROTEINLINKAGE REGIONS

The carbohydrate-protein linkages of chondroitin sulfate (CS) and dermatan sulfate (DS) in collagen-associated PGs, haveI0J1the general struc-

A.

(1) D. HeinegArd and Oldberg. FASEB J., 3 (1989) 2042-2051. (2) L. C. Rosenbergand J. A. Buckwalter, in K. E. Kuettner,R. Schleyerbach,and V. C. Hascall (Eds.), Articular Cartilage Biochemistry, Raven Press, New York, 1986, pp. 39-57. (3) K. E. Kuettner and J. H. Kimura, J. Cell Biochem., 27 (1985) 327-336. (4) A. R. Poole, Biochem. J., 236 (1986) 1- 14. (5) C. J. Handley, D. A. Lowther, and D. J. McQuillan, CellBiol. Znt. Rep., 9 (1985) 753-781. (6) V. C. Hascall, in V. Ginsburgand P. Robins (Eds.), Biology ofCarbohydrates, Wiley, New York, 1981, pp. 1-49. (7) E. Ruoslahti, Annu. Rev. CellBiol., 4 (1988) 229-255. (8) E. Ruoslahti, J. Biol. Chem., 264 (1989) 13,369- 13,372. (9) D. A. Swam, H. G. Garg, W. Jung, and H. Hermann, J. Invest. Dermatol., 84 (1985) 527-53 1. (10) L.-.k Fransson, Biochim. Eiophys. Ada, 150 (1968) 31 1-316. (1 1) F. Akiyama and N. %no, Biochim. Biophys. Acta, 674 (1981) 289-296.

COLLAGEN FIBRIL-ASSOCIATED SMALL PROTEOGLYCANS

r

CH,OSO,-

H o $

0

-

HO

OH

J n

Hyaluronan (HA)

L

NHCOCH,

OH

Keratan sulfate

(KS)

NHCOCH, OH

OH

-

i n

Chondroitin 4-sulfate (CS-4s)

Dennatan sulfate ( DS)

r CH,OSO,-

I

OH

OS0,1

Heparan sulfate (HS)

L

Heparin (HP)

FIG.1. -Structure of Merent Disaccharide Units of Glycosaminoglycans (GAGS).

HARI G. GARG AND NANCY B. LYON

242

TABLE I General Composition of the GlycosaminoglycansHaving the General Formula (A -B),, Glycosaminoglycan

Hyaluronan Chondroitin 4-sulfate Chondroitin 6-sulfate Dermatan sulfate Heparan sulfate Heparin Keratan sulfate cartilage cornea

A

B

Sulfate (moles/ disaccharide)

GlcA

GlcNAc

0

GlcA

GalNAc

GlcA

Linkage IaID

Mrmge

B

(degrees)

(kDa)

/3-( 1-3)

/3-( 1 4 )

-70

60- 10,000

0.2- 1.0

/3-(1-3)

/3-(14)

-30

5-50

GalNAc

0.2- 1.3

/3-(1+3)

/3-(14)

-19

5-50

IdoA (GlcA)

GalNAc

1.0-2.0

(~-(143)

/3-(14)

-59

15-40

IdoA (GlcA) IdoA (GlcA)

GlcNAc

0.2-3.0

( ~ - ( 1 4 ) a-(1+4)

+SO

5- 12

GlcNAc

2.0-3.0

a-(14)

(~-(14)

+48

7- 16

Gal Gal

GlcNAc GlcNAc

1.1-1.8 0.9-1.7

/3-(14)

/3-(1+3)

+45

8-12 4- 19

A

YH

OH OH

GlcA

Gal

Gal 1

COLLAGEN FIBRIGASSOCIATED SMALL PROTEOGLYCANS

243

ture D-G~cA-D-Gal- &Gal - D-Xyl- L-Ser/L-Thr (of protein), depicted in 1. However, other glycosaminoglycan (GAG)-protein linkages have also been found. The DS.GAG chain in calf ligamentum nuchae is attached to a L-lysine residue in the protein core.12 In adult human skin, the DS.GAG chain is linked by a D-xylosyl-L-alanine bond involving the C-terminus carboxyl group of L-alanine.I3 IV. ISOLATION AND FRACTIONATION OF SMALL PROTEOGLYCANS The general scheme of isolation and fractionation of small PGs from various mammalian tissues is summarized in Scheme 1. In addition to the extraction and purification procedure outlined in Scheme 1, the following buffers have also been used for extraction of PG

Mammaliantissue

c c

(1) Chopped ground in Wiley mill (under liquid N2)

(2) Extracted with 4 M guanidinium chloride

containing proteinase inhibitors

c

Supernatant fraction

4 (3) DEAE-celluloseion-exchangechromatography

c

Small PG fraction

I

7 (4) Purified by one or more of the following procedures (i) Cesium chloride density gradient, (ii) Differential ethanol precipitation, or (iii) Molecular-sieve chromatography

SCHEME I. -Isolation and Fractionation of Mammalian, Small Proteoglycans.

(12) M. 0.Longas and K. Meyer, Proc. Natl. Acud. Sci. USA., 79 (1982) 6225-6228. (13) M. 0. Longas and D. R. Azulay, Proc. Int. Symp. Clycoconjugates, 8th, (1985) V-32.

HARI G. GARG AND NANCY B. LYON

244

from tissue: 2 M calcium c h l ~ r i d e , ~ phosphatebuffered ~J~ saline,I60.05 M sodium acetate," 2-3 M magnesium chloride,18J90.15 -0.45 M sodium chloride,20-226 M urea,23and EDTA.24In certain cases, PGs were isolated from the extracts by precipitation with organic

V. U,OF SMALLPROTEOGLYCANS, THEIRPROTEIN CORES,AND GLYCOSAMINOGLYCAN CHAINS Two different species of PGs, namely, PG-I and PG-11,were first identified25in bovine cartilage by preparative SDS-PAGE. The two forms of PG were subsequentlyisolated26from bovine fetal skin and calf articular cartilage. Separation was achieved by using molecular-sieve chromatography methods: (a) Sepharose CL4B chromatography in 0.5 M sodium acetate, 0.02% sodium azide, pH 7 buffer (associative conditions), or (b) octyl-Sepharose chromatography, which separates the two types of PG based upon differences in the hydrophobic properties of the protein cores. Amino acid compositions of bovine, human, and other species of PG-I1are given in Tables 11, 111, and IV,respectively. The PG-Iamino acid compositions are given in Table V. Although PG-Iand PG-I1differ in amino acid content, both are high in L-leucine, le as par tic, and ~-glutamicacids. The structures of PG-Iand PG-11from different tissues are given in TablesVI and VII. The protein cores of PG-I1from human skin and scars, and fetal rat, are of small size. ( 14) T. H. M. S. M. Van Kuppevelt, H. M. J. Janssen, H. M. Van Beuningen,K . 4 . Cheung, M.

M. A. Schijen, C. M. A. Kuyper,and J. H. Veerlamp,Biuchim. Biophys.Acra, 926 (1987) 296- 309.

-

( 15) J. A. Purvis, G. Embery, and W. M. Oliver, Arch. Oral Biol., 29 ( 1984) 5 13 5 19.

(16) A. Oohira, F.Matsui, M. Matsuda, Y. Takida, and Y. Kuboki, J. Biol. Chem.,263 (1988) 10,240- 10,246. (17) S. P. Damle, L. CBster, and J. D. Gregory,J. Biol. Chem., 257 (1982) 5523-5527. (18) S. Onodera, Chem. Pharm. Bull.. 36 (1988) 4881-4890. (19) N. Fujii and Y. Nagai, J. Biochem. (Tokyo), 90 (1981) 1249-1258. (20) T. Nakamwa, E. Matsunaga and H. Shinkai, Biochem. J., 213 (1983) 289-296. (21) B. P. Toole and D. A. Lowther, Biochim. Biophys. Actu, 101 (1965) 364-366. (22) R. Fleischmajer, J. S. Perlish, and R. L. Bashey, Biochim. Biophys. Acta, 279 (1972) 265-275. (23) B. P. Toole and D. A. Lowther, Arch. Biochem. Biophys., 128 (1968) 567-578. (24) S. Sato, F. Rahemtulla,C. W. Prince, M. Tomana, and W. T. Butler,Cow. TissueRes..14 (1985) 65-75. (25) L.C. Roseuberg, H. U.Choi, L.-H. Tang, T. L. Johnson, S. Paul, C. Webber, A. Reiner, and A. R. Poole, J. Biol. Chem.. 260 (1985) 6304-6313. (26) H. U. Choi, T. L. Johnson, S. Pal, L.-H. Tang, L. Rosenberg, and P. J. Neame, J. Biol. C h . , 264 (1989) 2876-2884. (27) E. Matsunaga, H. Shinkai, B. Nusgens, and C. M. Lapiere, Collugen Rel. Res.. 6 (1986) 467-479.

TABLE II Amino Acid Composition(ResiauC/lOOO) of Bovine PG-II

12927 52 69 108 60 76 63 19 61 18 52 I13 23 32 71 29 25

124’* IW 51 42 60 67 105 100 12 17 93 72 49 53 14 9 58 58 13 12 55 63 115 121 24 28 31 33 66 77 24 28 46 30

* Loar M,

Fraction 10. “D not determined ’T = traa.

High M,

105.u 47 82 168 105 121 56 11 44 11 51 70

131m 45

ND”

ND”

22 57 24 25

35 77 26 27

-

71 104

76 73 51 10 57 10 79 129

12629 132% 39 38 68 75 108 85 69 83 63 81 49 43 13 0 59 53 9 14 6 0 4 8 122 147 34 29 33 33 80 51 21 30 28 32

123m 49 68 122 14 84 54 9 59 7 55 115 15 34 76 25 32

13% 46 68 98 81 68 51 12 59 12 67 135 30 33 52 30 32

123” 44 11 96 15 92 51 NDd 61

142” 58 99 148 61 I32 57 3 30

8

Tc

m w

53 131 30 33 57 28 49

38 70

33 112 24 30 61 26 21

Ty

24 67 30 35

145= 46 108 108 82 102 63

NDd 33

14532 50 98 I09 89 89 61 NDd 37 35 113 23 30 69 21 27

6832 63 I25 163 95 122 66 NDd 61 NDd 40 75 13 29 20 28 32

116“ 42 69 130

90 79 61 NTY 57 9 51 128 I3 32 60 28 35

1w33

86 98 141 92 68 61 5 54 11 31 85 20 31 48 25 31

12P 36 74 108 61 80 49 NDd 58 9 57 123 29 33 75 27 31

TABLE In Amino Acid Composition (Residue/1000) of Human PG-II !scar@

Amino Uterine acid ceniU”

Articular

-es

Placenta#

ASP

130

w

73

Thr Ser

45

ND

79 I10 77 85 53 17 50 3 43 122 20 33 68 25 41

ND ND ND ND

59 73 150 77 64 56

Glu

Pro GlY

Ala CYSas Val Met Jleu Leu Tyr Phe J-YS

His Arg a

ND ND

ND ND ND ND ND

ND ND

ND ND

ND = not determined. * T = trace

46 67 13 36 83 28 37 29 34 75

Fetal membranen

108 60 77 101

72 103 62 11 66 14 46 99 17 34 62 34 34

Gingival= 110

EpitheliumY Epidermis”

46 90 194 50 I19 79

113 52 66 129 86 1 20 77

145 50 87 I09 73 84 49

ND

ND

ND

47 3 31 62 21 27 53 10 38

48

58 11 45 121 12 41 67 26 36

Tb

43 93 20 21 63 I1

52

Dermis” 170 36 91 I38 124 107 56 3 63 1 3 45 61 5 21 8 39

Normal Hypertrophic Bone“ 112 46 72 104 97 88 68 12 64 20 32 99 16 37 65 24 44

107 40 76 114 88 109 65 14 47 9 40 90 22 46 66 23 44

133 52 75 122 70 94 50

ND 51

ND 42 109

10 29 54 20 42

COLLAGEN FIBRIL-ASSOCIATED SMALL PROTEOGLYCANS

247

TABLEIV Amino Acid Composition of Non-Human PG-I1 (Residue/lOOO) ~

Skin

ASP Thr Ser Glu

Pro GlY Ala CYSO.5 Val Met Ile

Leu TYr Phe LYS His Arg

(28) (29) (30) (31)

Skeletal Muscle

Rat'*

Pig"

RabbiP

152 46 64 111 81 63 46 24 44 21 61 116 31 27 63 20 31

128 42 65 104 72 76 49 15 66 6 56 127 23 29 87 23 32

83 35 82 97 49 156 55 3 36 9 28 67 22 18 39 23 28

E. Matsunaga and H. Shinkai, J. Invest. Dermatol., 87 (1986) 221 -226. C. H. Pearson and G. J. Gibson, Biochem. J., 201 (1982) 27-37. L. Coster and L.-A. Fransson, Biochem. J., 193 (1981) 143- 153. L. W. Fisher, J. D. Termine, S. W. Dejter, Jr., S. W. Whitson, M. Yanagishita, J. H. Kimura, V. C. Hascall, H. K.Kleinman, J. R. Hassell, and B. Nilsson, J. Biol Chem., 258

(1983) 6588-6594. (32) K. G.Vogel and D. HeinJ. Eiol. Chem., 260 (1985) 9298-9306. (33) T. R. Oegema, Jr., V. C.Hascall, and R. Eisenstein, J. Biol. Chem., 254 (1979) 13121318. (34) N. Uldbjerg, A. Malmstrom, G. Ekman, J. K. Sheehan, U. Ulmsten, and L. Wingerup, Biochem. J., 209 ( 1983) 497 - 503. (35) L. de 0. Sampaio, M. T. Bayliss, T. E. Hardingham, and H. Muir, Biochem.J., 254 (1988) 757-764. (36) M. Isemura, N. Sato, Y. Yamaguchi, J. Aikawa, H. Munakata, N. Hayashi, Z. Yosizawa, T. Nakamura, A. Kubota, M. Arakawa, and C.-C. Hsu, J. Riol. Chem., 262 (1987) 89268933. (37) M.J. Brennan, A. Oldberg, M.D. Pierschbacher, and E. Ruoslahti, J. Eiol. Chem., 259 (1984) 13,742- 13,750. (38) P. M.Bartold, 0.W. Wiebkin, and J. C. Thonard, Biochem. J.,21 1 (1983) 119- 127. (39) H. G.Garg, D. A. R. Burd, and D. A. Swann, Biomed. Res., 10 (1989) 197-207. (40) D. A. Swann, H. G. Garg,C. J. Hendry, H. Hermann, E. Siebert, S. Sotman, and W. Stafford, Collagen Rel. Res., 8 ( 1988) 295 - 3 13.

HAM G. GARG AND NANCY B. LYON

248

TABLE

v

Amino Acid Composition of Bovine and Human PG-I (Residue/lOOO)

Asp Thr Ser Glu Pro GlY Ala CYS0.5 Val Met Ileu

Leu Tyr Phe LYS His A%

137 40 61 94 75 65 54 11 53 14 41 165 32 42 43 24 41

137 36 61 85 83 60 43 0 51 14 45 174 34 33 50 30 51

116 46 77 114 67 15 47

ND 56

ND 46 120 27 31 70 51 42

ND = not determined.

In general, small PGs have low M, values compared to the high-density the GAG cartilage PG,65and, contrary to the high-density cartilage PG,66*67 chain in small PGs is attached to the NH2-terminusof the protein core, not the C02Hterminus. In PG-I, two DS.GAG chains are attached to the L-serine/L-threonine residues at the 5 and 1 1 positions,whereas, in PG-11, a single ~

(41) L. W. Fisher, G. R. Hawkins, N. Twos, and J. D. Termine, J. Biol. Chem., 262 (1987) 9702-9708. 88 (1980) 1793- 1803. (42) I. Miyamoto and S. Nagase, J. Biochem. (Tokyo)). (43) N. Parthasarathy and M. L. Tanzer, Biochemistry, 26 (1987) 3149-3156. (44) P. J. Roughley and R. J. White, Biochem. J-. 262 (1989) 823-827. (45) B. obrink, Biochim. Biophys. Acta, 264 (1972) 354-361. (46)H. Habuchi, K. Kimata, and S. Suzuki, J. Bid. Chem.. 261 (1986) 1031 1040. (47) H. G. Garg, E. P. Siebert, and D. A. Swann, Carbohydr. Res.. 197 (1990) 159- 169. (48) J. McMurtrey, B. Radhalrrishnamurthy, E. R Delferes, Jr., 0.S. Berenson, and J. D. Gregory, J. Bid. Chem., 254 (1979) 1621: 1626. (49) R.Kapoor,C.F.Phelps,L.Wer,andL.-A.Fransson,Eiochem.J..197(1981)259-268. (50) B. Radhakrishnamurthy, N. Jeansonne, and G. S. Bereuson, Biochim. Biophys.Acza, 882 (1986) 85-96. (51) B. G, J. Salisbury and W. D. Wagner, J. Biol. Chem., 256 (1981) 8050-8057. (52) J. D. Gregory, L. C&ster,and S. P. Damle, J. Biol. Chem., 257 (1982) 6965-6970.

-

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS TABLEVI of PG-I from Hum.a and Bovine Tissues

SN I-

W(kD.) PG

249

Core protein

GAG

GAG No.

IdoA (% of total uronic acid)

(%,I w/w)

Protein

2

81-84

31

26

44 44

2

25-29

29

2

25,“ 26 44

46

2

41

References

Skin Bovinefetal Articular cartilage Bovine calf Human

148

150 200

Bone Human

350

40 ~

a The data

are taken from the reference in bold print.

GAG chain is a t t a ~ h e d ~at* *the ~ ~4 position of the NH2-terminus. The number of GAG chains in small PGs vanes from one to nine, but, in general, PG-I1 contains only one to two GAG chains. Small PGs also contain Nlinked oligosaccharides,which are 1iberated’O by hydrazinolysis. These oligosaccharides are composed of di-and tri-antennary oligosaccharide struo tures of the complex type. In bovine articular cartilage PG-I, two N-linked oligosaccharides have been reported, in comparison to” PG-11. There is evidence72suggesting that the addition of free dermatan sulfate chains enhances multimerization of PG-11. (53) J. D. Gregory,S.P. Damle, H.I. Covington, and C. Cintron, Invest. Ophthulmol. Vis. Sci.. 29 (1988) 1413-1417. (54) J. R. Hassell, D. A. Newsome, and V. C . Hascall, J. Biol. Chem., 254 (1979) 12,34612,354. (55) L. Cbster, L.-A. Fransson, J. Sheehan, I. A. Nieduszynski, and C. F. Phelps, Biochem. J., 197 (1981) 483-490. (56) K. Murata and Y. Yokoyama, Biochem. Inf., 15 (1987) 87-94. (57) P. J. Roughley and R. W. White, Biochim. Biophys. Acta, 759 (1983) 58-66. (58) T. Honda, K.Katagiri, A. Kuroda, E. Matsunaga, and H.Shinkai, Collagen Rel. Res., 7 (1987) 171-184. (59) K. G. Vogel and S. P. Evanko, J. Biol. Chem.. 262 (1987) 13,607- 13,613. (60) M. Yanagishita, D. Rodbard, and V. C. Hascall, J. Eiol. Chem., 254 (1979) 91 1-920. (61) A. Oldberg, E. G. Hayman, and E. Ruoslahti, J. Biol. Chem.. 256 (1981) 10,847- 10,852. (62) T. Shinomura, K. Kimata, Y. Oike, A. Noro, N. Hirose, K.Tanabe, and S.Suzuki, J. Biol. Chem., 258 (1983) 9314-9322. (63) A. F&n and D. HeinBiochem. J.. 224 (1984) 47-58. (64) P. G. Scott, T. Nakano, C. M. Dodd, G.A. Pringle, and I. M. Kuc, Matrix, 9 (1989) 284 292. (65) V.C. Hascall and S. W.Sajdera, J. Biol. Chem., 245 (1970) 4920-4930. (66) D. Heinegiird and V. C. Hascall, Arch. Biochem. Biophys.. 165 (1974) 427-441.

-

HARI G. GARG AND NANCY B. LYON

250

TABLE VII Structure of PG-I1 Isolated from Different Tissues

w (a) Tissue

PC

GAG IdoA % (of Protein GAG Core protein No. total uronic acid) (% w/w) References0

Skin pis (i) (ii) Rat Fetal rat

Calf

2900 70 36 111-200 112

15

26 23

41-44

1

85

58 60 46

55

20

56

17-18

55

79-92

27

46 46

90

16

53-55 20 43

130 66

17.5 17.5

45 14, 21.5

20

Dermatosparactic

calf Newborn calf (i) large (ii) small cow Hum (i) Epidermis (ii) Dermis Human scar Normal Hypertrophic

0) (ii) Ligament cow

COW

75

61

28 28 29

90 90

32 18

39 39

22-17

90

23

40

23.5 29

22-17 22-16

90 13

23

40 47

30 16

60-70

48

29 11

50

23

136

39 78 I20

calf Heart valves cow Aorta

100-200

1

85

25

80

Human Cervix Human

Cornea Rabbit Monkey Sclera Bovine (i) large (ii) small Cartilage Bovine (i) large (ii) small

45 17 42 46 18, 19,20

42,4840 51

50-70 73 100-150

47 55

2-3

50

30

34

35

32 70

52,53 54

45 59

30,55 30,55

1

80-85

100 46

165-285 87- 120

44 47 - 44

20 52

25 25

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS

25 I

TABLEVII (continued) M, (a)

Human Tendon Bovine Fetal bovine Fetal membrane Human Folliculpr fluid fig Yolk sac tumor Rat Embryo

Chick

70-80

GAG

120

48 45

1-2

45

2

56

40 250-500

52

Lug 15-35 Bovine Skeletal muscle 95 21 Rabbit Bone 25 - 35 Bovine compact 80- 120 Fetal calf 80- 120 Growing rat 80- 120 Human fetal Bovine mature Temporomandibular joint disk Bovine >56

Protein

50

40-42 37

IdoA % (of

43

35,44,56,57

73

32,58 58,59 37

9

20

60

10

5

61

4-9

62 14

91

43

50

45 40

40

40 40 46

24 31 31 31,41 63

79

Whenever more than one reference is given, the data are taken from the reference in bold print.

(67) J. W. Stevens, Y. Oike, C. Handley, V.C. Hascall, A. Hampton, and B.Caterson, J. Cell. Biochem., 26 (1984) 247-259. (68) C. H. Pearson, N. Winterbottom, D. S.Fackre, P. G. Scott, and M. R.Carpenter, J. Biol. Chem., 258 (1983) 15,101-15,104. (69) R. K . Chopra, C. H. Pearson, G. A. Pringle, D. S.Fackre, and P. G. Scott, Biochem.J., 232 (1985) 277-279. (70) H. Shinkai, T. Nakamura, and E.Matsunaga, Biochem. J., 213 (1983) 297-304. (71) P. J. Name, H. U. Choi, and L. C. Rosenberg, J. Biol. Chem., 264 (1989) 8653-8661. (72) L.-A. Fratwon, L. Cbster, A. Malmstr6m, and J. K. Sheehan,J. Biol. Chem., 257 (1982) 6333-6338.

64

TABLEWI NH,-Tcrminrrl Amiao Acid Sequences of B o v h and Human PG-II Humpn

Bovine ResidueNo. Cprtilage” &lentn

SCaP

Skinu Tendo$*

Dermis” Epidermis” Normal Hypertrophic Fetalmembranem Bone41 CprtiLge”

ASP

ASP

Asp

Asp

Asp

ASP

A S P A S P

Glu Ala

Glu

Ala

Glu Ala

Glu Ala

ob

ob

ob

ob

GlY

Glu Glu A l a A l a A l a Ser Ser X“ Gly Gly Gly

G~Y

GlY

GlY

GlY

Glu Ala ? GlY

Glu Ala Xa Gly

Ile

Ile

Ile

ne

Ile

lle

Ile

Ile

Ile

Ile

GlY

Gly Pro Glu Glu

Gly Pro Glu Glu

Gly Pro Glu Glu

GlY

GlY Pro Glu

GlY Pro Glu

Ala

GlY

Glu

GlY Pro Glu

Glu

Glu

Val

Val

Val

Pro

Pro ASP

Pro

Val Pro ASP ASP

Val Pro

Val Pro

Asp

Asp

ASP

ASP

1

ASP

Asp

2 3 4 5 6 7

Glu Ala

Glu

Xa

ASP

8 9 10

Pro GlU Glu

11 12

Xa Phe

13

Pro

H i s H i s H i s Phe Phe Phe P r o P r o P r o

14 15 16 17 18 19 20 21 22 23

Glu

Glu

Val Pro Glu

Glu Val Pro Glu

ne

De

Glu Pro

Glu Pro

Glu

Pro

ASP Asp Arg

Asp Arg Asp(?)

Asp

ASP

Pro

Glu

Ala Xb Gly

Pro

Arg

Arg

Arg

ASP Phe

ASP Phe

Pro

Arg Asp

Glu Pro

Glu Pro

Pro Phe

GlU

Glu

Pro

ser(?)

ser

?

Pro

ser

Leu

Leu GlY

Leu GlY

Ser

Leu

Pro Val

Pro Val

AsPo Phe

Phe

Glu Pro

Glu

Phe

COLLAGEN FIBRIGAssoclATED SMALL PROTEOGLYCANS

253

VI. N-TERMINAL SEQUENCE OF SMALL PROTEOGLYCANS

A comparisonof the amino acid sequencingof the NH,-terminal regions of the core proteins of PG-I1 from bovine articular cartilage, sclera, skin, tendon, human skin and post-bum scars, fetal membrane, bone, and cartilage are shown in Table VIII. The A, -A, amino acid sequences for bovine and human tissue are identical, and thereafter, the sequences within the TABLEIX NH, -Terminal Amino Acid Sequences of Bovine Cartilage and Human Bone and cprtilaee PG-I

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

ASP Glu Glu Ala

Xa GlY Ala Glu Thr

Thr X" GlY Ile pro ASP Leu ASP Ser

Leu Pro Pro Thr Tyr Ser

ASP Glu Glu Ala Xa GlY Ala ASP

Thr X GlY Val Leu ASP Pro Asp

ser

Val Thr

pro

Ala Met

X = blank cycle. (73) L. Cbster, L. C. Rosenberg, M. Van der Rest,and A. R. Poole, J. Biol. Chem.,262 (1987) 3809-38 12.

254

HARI G. GARG AND NANCY B. LYON

bovine species remain identical; but in the human species, the NH,-terminus bone sequence has only some similaritiesin comparison to human-skin, scar, cartilage, and fetal-membranePG-I1protein cores. The glycosaminoglycan chain is attached& to an L-serine residue at position 4. The NH,-terminal sequences of PG-I derived from human bone and cartilage, and bovine are given in Table IX. The A,-A, amino acid sequencesofthe protein core of human bone and cartilage PG-I are identical to that of bovine cartilage; thereafter, the sequences of the next 12 amino acids are different for bovine PG-I compared to human PG-I. The glycosaminoglycan chain in PG-IappearsMto be attached at position 5 of the protein core. On the whole, there is some homology in the primary structure of the core proteins of PG-I and PG-I1 from different species, but there are also some striking differences, which may be related to their biological activities.

VII. AMINOACID SEQUENCEANALYSIS OF THE SMALL PROTEOGLYCAN CORE-PROTEIN, DEDUCED FROM CLONED cDNA The core protein of small PGs has been cloned74from a Agt 1 1 fibroblast cDNA library. Protein sequences of human bone PG-I and PG-I1 (Ref. 75) and bovine bone PG-I1 (Ref. 76) have also been deduced by the aforementioned approach. Comparison of the NH,-terminal amino acid sequence (obtained by using a gas-phase sequencer) of bovine skin and human skin/ bone PG-I1 revealed complete homology with the total protein core sequences of the same species. The derived protein-core sequences of human PG-I (biglycan)showed sufficient homology with the PG-I1(decorin);that is, 55Yo of the amino acids are identical, with others involving chemically similar amino acid substitution. These data suggest that the two protein cores may be the result of a gene duplication. Comparison of human PG-I and PG-I1 amino acid sequences (see Fig. 2) shows that PG-I and PG-I1 contain 368 and 359 amino acid residues, respectively. The PG-I and PG-I1 protein cores contain75a series of 10- 12 tandem repeats of 24 amino acid residues rich in L-leucine or L-leucine-like amino acids. Similar structural characteristics had been r e p ~ r t e d ~ for~ several - ~ ~ unrelated non-PG proteoglycan proteins. (74) T. Krusius and E. Ruoslahti, Proc. Nut/. Acad. Sci. USA, 83 (1986) 7683-7687. (75) L. W. Fisher, J. D. Termine, and M. F. Young, J. Biol. Chem., 264 (1989) 4571 -4576. (76) A. A. Day, C. I. McQuillan, J. D. Termine, and M. R. Young, Biochem. J., 248 (1987) 801-805. (77) T. Kataoka, D. Broek, and M. Wigler, CeN, 43 (1985) 493-505. (78) N. Takahashi, Y. Takahashi, and F. W. Putnam, Proc. Natl. Acud. Sci. USA, 82 (1985) 1906-1910. (79) M. Handa, K. Titani,L. Z. Holland,J. R. Roberts,and Z. M. Ruggen,J. Biol. Chem.. 26 1 (1986) 12,579- 12,585.

255

COLLAGEN FIBRIGASSOCIATED SMALL PROTEOGLYCANS

I MWPLWRLVSLLALSQALPFEQRGFWDFTLDDGPFMMNDEEASGADTSGVL

I I

I

I I I I I l l I I I I .........SGIGPEVP I

IIMKATIILLLLAQVSWAGPFQQRGLFDFMLEDEA

50 41

DPDSVTPTYSAMCPFGCHCHLRWQCSDLGLKSVPKEISPDTTLLDLQNN 100

I

I

Ill I 1111111111111 Ill

1l111111111

DDRDFEPSLGPVCPFRCQCHLRWQCSDLGLDKVPKDLPPDTTLLDLQNN

91

DISELRKDDFKGLQHLYALVLVNNKISKIHEKAFSPLWQKLYISKNHL 150

1 1

I l l I 1 I1 I I I I I I I I

11 11

II 111 I

KITEIKDGDFKNLKNLHALILVNNKISKVSPGAFTPLVKLERLYLSKNQL 141

VEIPPNLPSSLVDVRIHDNRIRKVPKGVFSGLRNMNCIEMGGNPLENSGF 200

1 1

I I

I I I I I I I I l l I 1 1 1 1 1 1 II

KELPEKMPKTLQELRAHENEITKVRKVTFNGLNaMIVIELGTNPLKSSGI 191 EPGAFDGL .~NYLRISEAKLTGIPKDLPETLNELHLDHNKIQAIELED~ 249

11111 IIIII

I II II I IIIII Ill

I

ENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAASL 241

LRYSKLYRLGLGHNQIRMIENGSLSFLPTLRELHLDNNKLARVPSGLPDL 299

I I11 I 1

Ill1 I I I I I I I I I I I I I l l I I

KGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEH 291 KLLQVVYLHSNNITKVGVNDFCPMGFGVKRAYYNGISLFNNPVPYWEVQP 349

1 IllllT Ill II IIII I

1 I 1 1 111 111 I 1 1 II

KYIQWYLHNNNISWGSSDFCPPGHNTKKASYSGVSLFSNPVQY'WEXQP 341 ATFRCVTDRLAIQFGNYKK

IIIIt 1 111 1 1 1 1 STFRCVYVRSAIQLGNYK.

368 359

FIG.2.-Compariwn of Human PG-I and PG-I1 Protein Core Amino Acid Sequences(data taken from Ref. 75). Abbreviations:A, Ma; C, Cys;D, Asp; E, Glu; F, Phe; G, Gly; H, His;I, Ile; K, Lys; L, Leu; M, Met; N,Asn; P,Pro;Q, Gln; R, Arg; S, Ser; T, W,V, Val; W,Trp; Y,Tyr.

HARI G. GARG AND NANCY B. LYON

256

VIII. BIOSYNTHESIS OF SMALL PROTEOGLYCANS 1. Primary Culture

Synthesis of PGs by primary culture from guinea-pig skinya3human skin,84-8ahuman dermals9and human gingival fibroblastsmhas been thoroughly investigated. In this system, both large and small PGs are biosynthesized. The newly synthesized PG obtained by using ~-['H]glucosamine precursor yields PG containing low proportions of radioactivity:' whereas the use of [35S]-S04precursor results in [35S]-PGsthat contain higher proportions of [35S]-radioactivity?1The majority of the [35S]-incorporation takes placea7in small PGs (mainly found in cell-culture media). When cells are cultured on collagen gels, the radioactivity is also incorporated in the m a t r i ~ . ~Disease-related ~.~* alterations in the synthesis of PG macromolecules have been r e p ~ r t e d . Influences ~ ~ - ~ ~ of such reagents as chlorate%and lipopolysaccharidewon PG biosynthesis have also been reported. Chlorate treatment leads to the formation of GAG chains having a widely varying degree of sulfation. Glycosaminoglycan synthesis, in culture, using fibroblasts from human human normal and hypertrophic scarywand embryonic chick (80) K. Titani, K. Takio, M. Handa, and Z. M. Ruggeri, Proc. Nutl. Acud. Sci. USA, 84 (1987) 56 10- 56 14. (81) C. Hashimoto, K.L. Hudson, and K. V. Anderson, Cell, 52 (1988) 269-279. (82) R. Reinke, D. E.Kwntz, D. Yen, and S. L. Zipursky, Cell, 52 (1988) 291-301. (83) T. Honda, E. Matsunaga, K. Katagiri, S. Fujiwara, and H. Shinltai, Eiomed.Res., 8 (1987) 175 183. (84) I. Carlstedt, L. CBster, and A. Malmstdm, Biochem. J., 197 (1981) 217-225. (85) L. Caster, I. Carlstedt, A. M a l m m , and B. Smstrand, Biochem. J., 222 (1984) 575582. (86) H. Larjava, J. Heino, T. Krusius, E. Vuorio, and M.Tammi, Eiochem. J., 256 (1988) 35-40. (87) J. GlW, M.Beck, and H. Kresse, J. Biol. Chem.,259 (1984) 14,144- 14,150. (88) W.Truppe and H. Kresse, Eur. J. Eiochem., 85 (1978) 351-356. (89) I. A. Schafer, L.Sitabkha, and M.Pandey, J. Eiol. Chem.,259 (1984) 2321-2330. (90) P. M. Bartold and S. J. Millar, Idect. Immun., 56 (1988) 2149-2155. (91) T. Nalcamura and H. Shinkai, J. Dermatof., 12 (1985) 489-497. (92) J. T. Gallagher, N. Gasiunas, and S . L. Schor, Eiochem. J., 215 (1983) 107- 116. (93) A. Eigavish and E. Meezan, Bimhem. Biophys. Res. Commun.,152 (1988) 99- 106. (94) Y. Shishiba, M. Yanagishita, and V. C. Hascall, Connect. Tiss.Res., 17 (1988) 119- 135. (95) S.Fukui, H. Yoshida,T. Tanaka, T.Sakano, T. Usui, and I. Yamashina, J. Eiol. Chem., 256 (1981) 10,313-10,318. (96) H. Greve, Z. Cully, P. Blumber& and H. Kresse, 3. Eiol. Chem.. 263 (1988) 12,88612,892. (97) J. E.Silbert,M.E. Palmer, D. E. Humphries,andC. K. Silbert, J. Eiol. Chem.,261 (1986) 13,397- 13,400. (98) I. Sj&berg,I. Carlstedt, L. Cbster, A. MalmstrBm, and L.-A. Fiansson, Eiochem. 3.. 178 (1979) 257-270.

-

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS

257

skinla fibroblasts, shows differencesin the proportions of incorporation of the radiolabel into different types of GAG. Several cytokines have been shown to modulate the synthesis and dejyadation of various connective-tissue components, including GAGS.Treatment of cultured human dermal fibroblasts with different human interferons (INF)resultslO1 in specific synthetic responses. INF a and p lead to concentrationdependent decreases in GAG production and collagenase production, with no effect on fibronectin synthesis. Human interferon gamma, on the other hand, leads to concentrationdependent increases in GAG and fibronectin, as well as in collagenase. Cultured human skin fibroblasts have also been treated with interleukin (IG1) a! and /3 and tumor necrosis factor (TNF)a and p, which, under the isolated in vifroconditions, lead to concentrationdependent increases in collagen, GAG, and collagenase, with inhibition of fibronectin.lo2However, the maximum increases in GAG synthesis stimulated by IG 1 and TNF may be overshadowed by catabolic effects. Several cell types, including synovial cells, dermal fibroblasts, and chondrocytes, have been stimulated by mononuclear-cellderivedIG 1 to produce high levels of proteogly~anase.~~~J~ 2. Explant Culture

Biosynthesis of small proteoglycans in organ culture from rat skin using [3sS]-S04(Ref. 46), bovine aorta using [35S]-S04and ['H]glucosamine (Ref. 105), and bovine tendon using [35S]-sulfate(Ref. 106) have been investigated, and the characteristicsofthe GAG chainsand protein cores have been studied. Retinoic acid has been used on cultured human skin explants, which respond by accumulating HA between keratinocytes.lm-'@ The dermis of skin explants also responds to retinoids with an apparent increase (99)K.Savage and D.A. Swann, J. Invest. Dermatol., 84 (1985)521 -526. (100)R.Evangelisti, G.Stabelhi, E. Becchetti, and P. Carinci, Cell Biol. Int. Rep., 13 ( 1989) 437- 446. (101)M.R.Duncan and B. Beman,Arch. Dennutof. Res., 281 (1989)1 1 - 18. (102) M.R.Duncan and B. Berman, J. Invest. Dermatol., 92 (1989)699-706. (103)M.Gowen, D.D.Wood,E. J. Ihrie, J. E. Meats,and R. G. G. Russell, Biochim.Biophys. Ada, 797 (1984)186- 193. (104)E. E. Golds, V. Santer, J. Killackey, and P. J. Roughley, J. Rheumaro[., 10 (1983) 861-871. (105)A. Schmidt, M.Prager, P. Selmke, and E. Buddecke, Eur. J. Biochem., 125 (1982) 95-101. (106)T. J. Koob and K. G. Vogel, Biochem. J., 246 (1987)589-598. (107) R. Tammi and M. Tammi,J. Ceff.Physiol., 126 (1986)389-398. (108)I. A. King, Biochim. Biophys. Acta, 674 (1981)87-95. (109) R. Tammi, J. A. Ripellino, R. U. Margolis, H. I. Maibach, and M.Tammi, J. Invest. Dermato[.,92 (1989)326-332.

258

HARI G. GARG AND NANCY B. LYON

in ground substance. This has been demonstrated using electron microscopy, and accompanies hypermetabolioappearing fibroblasts and other connective-tissue changes.II0 IX. BIOLOGICAL ROLESOF SMALLPROTEOGLYCANS Small proteoglycans are widely distributed throughout fibrous interstitial tissues, suggesting crucial roles in connective tissues. They are dynamic molecules that, through their abilities to interact with other major molecules such as collagen, appear to have critical roles in developrnentl1' and tissue modeling, and may play a role in the pathophysiology of disease processes. The binding characteristicsof small proteoglycans are multifaceted. Ionic interactions with other molecules are facilitated by way of the highly negatively charged glycosaminoglycan chains. This may permit electrostatic interactions with L-lysine and targuinine on collagen and also with such counter ions, as sodium, which then create an osmotiopressure gradient to draw and hold water molecules in tissues. GAG chains are also responsible for the ability of some DSPGs to self-associate to form multimeric PG comp l e x e ~The . ~ ~ protein core is also capable112of specific molecular interactions. The DSPG protein sequence shows homology with several nonconnee tive tissue proteins from several different species. Repeatingsequencesin the PG-I and PG-I1 protein core75are characterized by several conserved L-leucine residues and L-leucine-like amino acids found at locations that had previously been described for a highly varied group of non-proteoglycan proteins, including von Willebrand Factor-binding protein of the platelet membrane and yeast adenylate cyclase, where the repeating domain is thought to bind the enzyme to the cell membrane. Two Drosophilu proteins, chaoptin and toll, also exhibit these repeating domains, which are thought to interact with the plasma membrane and influence, respectively, morphogenesis of photoreceptor cells and dorsal-ventral pattern formation in the embryo.81s82A common theme in molecular interactions appears to be emerging in organisms as diverse as yeasts and mammals. It now appears that several non-proteoglycan proteins, which also have binding functions, contain the L-leucine-richtandem repeatsthat are also found in DSF'GS."-~~ These macromolecules may play a role in collagen fibril organization.ll*The core protein of PG-I1 binds collagen type I and type I1 in vitro and (110) L. H.Kligman, J. Am. Acud. Dermatol., 15 (1986) 779-785. (1 1 1) S. Vainio, E. Lehtonen, M. Jalkanen, M. Bernfield, and L. Saxen, Develop. Biol., 134 (1989) 382-391. ( I 12) T.R. Oegema, Jr., J. Laidlaw, V. C. Hascall, and D. D. Dziewiatkowski,Arch. Biochem. Biophys., 170 (1975) 698-709.

COLLAGEN FIBRILASSOCIATED SMALL PROTEOGLYCANS

259

affects the rate and diameter of fibril formation.ImFibromodulin, a 59 kDa protein, isolated from many connectivetissues, is structurally related to PGs and also contains similar L-leucine-rich tandem repeats.121 - lZ3 Like PG-11, fibromodulin delays collagen fibril formation and leads to fibrils with a thinner diameter. The binding to collagen seems to be at different locations, since PG-I1 and fibromodulin together have additive effects in the collagen fibrillation studies.123 The protein core appears to have very specific interactions with collagen. IZ4 Electron-microscopicstudies have revealed that DSPG-collagen interactions occur specificallyat the d- and e-bands in collagen.124The interaction depends on intact disulfide bridges on the protein core and, in collagen fibrillationstudies, is independentof the DS -GAG chain."' Biomechanical strength of collagen fibers probably also depends upon the PG-type I collagen interaction.' The physiology of reproduction may be associated with proteoglycan changes.''*'26 A role for DSPGs in dilation of the rat uterine cervix is suggested by a fourfold increase in DSPG levels in pregnancy, which decrease rapidly within the first postpartum day.127 PGs appear to play a rolegin wound healing and scar formation. PG levels fluctuate during wound healing. An initial increase in HA is replaced by increased levels of DSPG as the wound ages. DSPGs from articular cartilage have been shown to bind fibronectin non-covalently and to inhibit attachment and spreading of fibroblasts.129DSPG, the major PG in scars,is increasedgin hypertrophic scars and keloids. Many phases of wound healing and tissue remodelling may be affected by alterations in proteoglycans. (113) D.A.D.Pamy,M.H.Flint,G.C.Gflard,andA.S.Craig, FEBSLett., 149(1982) 1-7. (114) J. M.Snowden and D. A. Swann, Biopolymers, 19 (1980)767-780. (115)J.E.ScottandM.Haigh, Biosci.Rep., 5(1985)71-81. (116) J. E. Scott and C. R. Orford, Biochem. J., 197 (1981)213-216. ( 1 17) P. G.Scott, N. Winterbottom, C. M. Dodd, E. Edwards, and C. H. Peamn, Biochem. Biophys. Res. Commun., 138 (1986)I348- 1354. (118)A.K.Garg,R.A.Berg,F.H.Silver,andH.G.Garg, Biomuteriuls, 10(1989)413-419. (1 19) N. Uldbjerg and C. C. Danielsen, Biochem. J.. 251 (1988)643-648. (120) K.G. Vogel, M. Paulsson, and D. Heineghi, Biochem. J., 223 (1984)587-597. (121)D.Heinegfird, T.Larsson, Y. Sommarin, A. FranzCn, M. Paulsson, and E. Hedbom, J. Biol. Chem., 261 (1986)13,866-13,872. (122) A. Oldberg, P. Antonsson, K.Linblom, and D. Heinegilrd, EMBO J., 8 (1989)2601 2604. (123) E. Hedbom and D. Hein-, J. Biol. Chem., 264 (1989)6898-6905. (124)J. E. Scott, in E. Evered and J. Whelan, Eds., Functions of the Profeoglycuns,(Ciba Found. Symp. 124)Wiley, Chichester, 1980,pp. 104- 116. (125)C.A. Stephk and P. A. A m Comp. Biochem. Physiol.. 84B (1986)29-35. (126) N.Uldbjexg, Actu. Obstef. Gynecof. Scand., Suppl., 148 (1989)1-40. ( 127) R. Kokenyesi and J. F.Woessner, Jr., Biochem. J., 260 ( 1989)413-4 19. (128)S. A. Alexander and R. B. Donof, J. Surg. Res.. 29 (1980)422-429.

260

HARI G.G A R G AND NANCY B. LYON

X. ADDENDUM The following articles on the subject have appeared since this text was completed.

(1 30) G. Westergren-Thorsson, P. Antonsson, A. Malmstrtjm, D. Heinegiird, and A. Oldberg, The synthesis of a family of structurallyrelated proteoglycans in fibroblasts is differentiallyregulated by TGF-p, Matrix, l l (1991)177-183. (131) H.G. Gar&E. W. Lippay, D. A. R. Burd, and P. J. Neame, Purification and characterization of iduronic acid-rich and glucuronic acid rich proteoglycans implicated in human post-bum keloid scar. Carbohydr. Rex, 207 (1990)295-305. (132)G.M. Cerhchi, R. Coinu, P. Demuro, M. Formato, G. Sanna, M. Tidore, M. E. Tira, and G. Deluca, Structural and functional modifications of human aorta proteoglycans in atherosclerosis.Matrix, 10 ( 1990)362- 372. (1 33) M.C. Lane and M. Solursh, Primary mesenchyme cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan. Devel. Biol., 143 (1991)389-397. (1 34) C.K.Silbert, D. E. Humphries,M. E. Palmer,and J. E. Silbert,Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblast from normal and diabetic individuals. Arch. Biochem. Biophys., 285 (1 991) 137- 141. ( 1 35) K.Schwa, B. Breuer, and H.Kresse, Biosynthesisand propertiesofa further member of the small chondroitin/dermatansulfate proteoglycan family. J. Biol. Chem., 256 (1990)22,023-22,028. (1 36) M.R.Shetlar, C. L. Shetlar, C. W. Kischer, and J. Pindur, Implantsof keloid and hypertrophicscarsinto the athymic nude mouse: Changes in the glycosaminoglycans of the implants. Connect. Tissue Res., 26 (1991)23-36. (1 37) T. -K. Yeo,L. Brown, and H. F. Dvorak, Alterationsin proteoglycan synthesiscommon to healing wounds and tumors. Am. J.Pathol., 138 (1991)1437-1450. ( 138) D. T.Simionsescu and N. A. Kefalides, The biosynthesis of proteoglycans and interstitial collagens by bovine pencardial fibroblasts. Exp. CellRes., 195 (1991)171-176. (139) R.Fleischmajer, L.W. Fisher, E. D. MacDonald, L. Jacobs, Jr., J. S. ( 129) L. C. Rwnberg, H. U.Choi, A.R.Poole, K.Lewandowska, and L. A.Culp, in E.Evered

and J. Whelan Eds.,Functions of the Proteoglycans (Ciba Found. Symp. 124) Wdq, Chichester, 1980, pp. 47 - 68.

COLLAGEN FIBRIL-ASSOCIATED SMALL PROTEOGLYCANS

261

Perlish, and J. D. Termine, Decorin interactswith fibrillar collagen of embryonicand adult human skin. J. Sfnrct.Biol., 106 (1991) 82-90. (1 40) G. Stocker,H. E. Meyer, C. Wagner, and H. Greiling, Purificationand N-terminal amino acid sequence of a chondriotin sulfate/dermatan sulfate proteoglycan isolated from intima/media preparations of human aorta. Biochem. J.,276 (1 99 1) 4 15 -420. (141) K. Takigaki, T. Nakamura, A. Kon, S.Timura, and M.Endo, Characterization of /l-D-xyloside-induced glycosaminoglycans and oligosaccharides in cultured human skin fibroblasts.J. Biochem. (Tokyo), 109 (1991) 514-519. (142) T. C. Register and W. D. Wagner, Heterogeneity in glycosylation of

dermatan sulfate proteoglycan core proteins isolated from human aorta. Connect. Tissue Res., 25 (1990) 35 - 38. (143) V. Vilim and J. Krajickova, Proteoglycans of human articular cartilage. Identification of several populations of large and small proteoglycansand of hyaluronicacid-bindingproteinsin successivecartilage extracts. Biochem. J., 273 (1991) 579-585. (144) A. Schmidtchen, I. Carlstedt, A. Malmstriim, and L. -A.Fransson, Inventory of human skin fibroblasts proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulfate proteoglycans. Biochem. J., 265 (1990) 289-300. (1 45) S.Inerot and I. Axelsson, Structure and composition of proteoglycans from humans annulus fibrosus. Connect. Tissue Res., 26 ( 1991) 47 63. (146) H. G. Garg, E. W. Lippay, E. A. Carter, M. B. Donelan, and J. P.

Remensnyder, Proteoglycan synthesis in human skin and bum explant cultures. Burns, 17 ( 199 1) in press. (147) H. G. Garg, E. W.Lippay, and P. J. Neame, Proteoglycansin human bum hypertrophic scars fiom a patient with EhIers-Danlos Syndrome. Carbohydr.Res.. in press. ACKNOWLEDGMENTS

This work was supported by research funds fiom the Shriners Hospitals for Crippled Children of North America. The authors gratefully acknowledge the editorial work of Sarah Niemczycki.

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AUTHOR INDEX Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A

Abbas, S. A., 126 Ablett, S.,23 Abylgaziev, R. I., 142, 143, 161(189, 190) Achet, D., 140 Adalsteinsson, O., 2 1 I Addleman, R. E.,20 Adelhorst, K., 53 Agapova, N., 117 Ahnhog M., 118 Aikawa, J., 247 Aisaka,K, 176, 195(1,2) Akiyama, F., 242 A l - M ~ u d i N. , A. L.,3 1 Albert, R., 27 Albonico, S. M., 119 Album, H.E., 133, 134(146), 137(147), 162(146) Aldinucci, D., 206 Alexander, S.A., 259 Ali, M. H.,39 Allerhand, A., 20,25(27), 27(3) Amano, H., 168 A ' .m P.A.,259 Anderson, K. V.,254,258(81) Andrasi, F., 154, 165(209) Andrzejewski, D., 114 Angius, A., 107, 130(32), 13432) Angyal, S.J., 19,20(1), 22,25,28,31,35(20) Anteunis, M., 101, 102(19), 106(19), 117(19), 134(19) Antonsson, P., 258 Anzuino, G., 151, 172(206) A A , Y., 176, 195(2) Arakawa, M., 247 Amy% V.P., 130(131), 131, 151(131), l65( 131) Archibald, T. G., 155 Arena, B. J., 1 19 A&, K.,169 Aspro-Nicholas Ltd., I29

Atlas Chem. Ind., Inc., 155 Audinos, R., 99, 102(1I), 110(1 l), 113(1 I), 114(1I), 156(11) Aug&,C., 183, 184, 195(20), 197, 198(20), 199(42,44),200(20, 38), 201(38, a), 201(48), 202(20), 203(43,44), 204(41), 21 1(39), 215(15,39,69), 216,218(69), 219(15), 223(79), 225(15, 80), 229, 236(40,48), 237 Avenel, D., 32 Axen, R., 181 Axmann, R., 137 Azulay, D. R., 243

B Baggett, N., 160 Bajza, I., 58,78(56), 89(56) Bakos, J., 102, 107(28), 155(28) Baltes, W., 118 Barad, U. G., 118 Barbas, C. F., 237 Barbier, J., 124 Barker, R.,2 1,23(6), 27(6,29), 119(93), 120, 122(93),219,220 Barnes, J. M.,168, 172(253) Barnes,M.,168 Barnes, M. J., 168 Barrio, J. R., 30 Bartold, P. M.,247,254(90), 255,256(90) Barton, R. E., 99, 150(12) Bartsch, G., 1 18 Bartsch, W., 164 Bashey, R. L.,244 Batelaan, J. G., 158 Batley, M., 29 Batta, G., 58, 59(54),60(54), 75(54), 78(56), 83,89(56) BatJ. M.,166

263

264

AUTHOR INDEX

Baughn, R. L., 186,208,21 1 Baum, K., 155 Baumann, W.,200 Bayliss, M.T.,247 Beauchamp, R.O.,Jr., 166 Becchetti, E.,256 Beck, M.,254(87), 255,256(87) Beckwith, A. L.J., 38 Bednarski, M. D.,188, 191, 193,206

Beilstein,95 BeMiller, J. N.,59,64(59) Berenson, G.S.,247 Berg, R.A., 258,259(118) Berman, B., 256 Bernfield, M.,257 Bemenyi, P.,154, I65(209) Beta,W.,168 Beyerle, R.,163 Bhalla,H.L., 117, 118 Bida, G.T.,30 Bignall, J. C., 1 12 Binkley, R. W.,38,42 Birch, G.G.,167 Bischofberger, N.,19 1 Bjbrkling, F.,232 Blanken, W.W.,223 Blattner, R.,45,47(33), 49,60(33), 65, 75(33), 85,87,88(33,36,90) Blumberg, P.,256 Blumenfeld, H.,186 Bock, K.,53, 120, 121(94),125,129, 150 Bogaert, M.G.,118 Bohn, H.,130, 163(130) Boh, P. A., 130 Boitiaux, J.-P., 124 Bolte, J., 204 Bombor, R.,166 Bongiovanni, G.,118(74),119 Boniforti, L., 146 Bonn,R., 110 Boullanger, P., 126 Bouxom, B., 197, 199(42,44),203(44) Bovee, W.M.M. J., 102, 104(27), 109(27) Boyd, G.W.,112 Bradley, C. H.,20 Bralovic, M.,147 Brancq, B., 167 Brandner, J. D.,119 Brard, L.,41,60(25),61(25),62(25), 84(25), 89(25)

Bratin, K., 1 17 Bravo, P., 35 Brennan, M. J., 247 Brinkmeier, H., 24 Broek, D.,254,258(77) Bron, J., 133, 136(146a), 165(146a) Brossmer, R., 200 Brown, D.M.,129 Brown, E. D.J., 183 Brumley, W. C.,114 Buck, K.W.,128 Buckwdter, J. A., 239 Buddecke, E.,257 Buddrus, J., 24 Bukhari, M. A., 258 Burd, D.A. R., 247,252(39) Butler, W.T.,244 Buu-HoI, N.P., 40

C

Camera, E., 107, 130(32), 134(32) Camerman, A., 114,115(39) Camerman, N.,114, 115(39) Cameron, T.S., 24 Cano, J. P., 1 18 Capon, R. J., 31 Card, P.J., 213,231(71) Carinci, P., 256 Carlson, M., 117 Carlstedt, I., 254(84, 85),255,256 Carpenter, M.R.,249,252(68) Carver,J. P., 22 Castilla, I. M.,30, 125 Caterson, B., 248(67), 249 Cavay6, B., 184, 195(20),197, 198(20),

199(42,47),200(20) Cekovic, A., 128 Cekovic, Z.,124 Cere,

V., 148, 150, 160(200)

Chana, J. S., 39 Charpiot, B., 129 Chasseaud, L. F., 110, 113(34),118 Chatterjee, S.S., 133, 134(142, 143, 144),

138(147), 151(142, 143, 144), 162(142, 143, 144, 147) Chaumette, P., 124 Chen, J. L., 166 Chenault, M.K.,188

AUTHOR INDEX Cheung, K-S., 243 Chiarelli, S.N., 119 Chiba, T., 43, 76(30), 77(30) Chid, P., 129 Chistotinova,L. T., 118(77), 119 chizhov,0.S.,110, 112(33) Choi, H. U., 244,248(26), 249,253(26), 259 Chopineau, J., 236 Chopra, R.K.,249 Chou, C. H., 133 Cicero, D., 3 1 Cintron, C., 248 Cirelli, H. F., 35 Clark, M.R.,166 Clayton, C. J., 3 1 Cockman, M., 21 Coffey, S.,95 Cohen, A,, 124 Collins, P. M.,39, I56 Conley, D. L., 206 Conradt, H. S.,237 b k , G. M.W., 171 Cope, A. C., 146, 147, 148(193) Cordes, G., 119 Cortes, S. J., 35 Cossum, P. A., 112 CBster, L., 243,244,247( 17), 248,249, 252(73), 254(84,85), 255,256 Cottier, L., 38 Courtaulds Ltd., 129 Covington, H. I., 248 Coxnon, B.,35 Craig, A. S.,258 Crans, D. c., 28, 190,193(30),206,208,210 Crawshaw, T.H.,102, 107(26), 109(31), 116(26), 117(31), 142(31), 161(31) Cremata Alvarez, J., 2 1 Cseko, I., 161 Csizmadia, V. M., 101, 117(18) Cuko, I. I., 24,29(32) Cully, Z., 256 Culp, L. A., 259 Cuny, E., 82 Curran, D. P.,38 Czaja, R.F., 129

265

Damle, S.P., 243,247( 17), 248 Daniels, L., 190 Danielsen, C. C., 258 Darragh, A., 110 Date, T., 64 David, S., 184, 195(20), 197, 198(20), 199(44), 200(20,38), 201(40), 202(20), 203(43,44), 211(39), 215(39), 219, 223(79), 236(40) Davidson, I. W. F., 118 Davis, N. W., 112 Dax,K.,27 Day, A. A., 254 De Angelis, N. J., 119 De Jew, B.,236 de Lederkremer, R. M., 35 De Lucchi, O., 107, 134, 130(32), 134(32) De Philippe, L., 167 de Souza-e-Silva, U., 184, 225(18), 231(89) Dederen, J. C., 166 Defaye, J., 25, 119 Degueil-Castaing, M.,236 Dejter, S. W., Jr., 244 Delferes, E. R.,Jr., 247 DelM.,99, 102(1l), 1 lO(1l), 113(1I), 114(11), 140, 156(11) Demerseman, P., 40 Demeter, L., 133 Demuynck, C., 204 Descotes, G., 38,41,60(25), 61(25,61), 62(25), 84(25), 89(25), 126 Dhein, R.,130, 172(127) Dicarlo, F. J., 118 Dicosimo, R., 206 Dimov, N., 117 Dimova, N., 118 Ding, X.D., 117 Dirlikov, S. K, 172 Dittgen, M., 166 Dodd, C. M.,249,258,259( 117) Doganges, P. T., 156 Dokic-Mazinjanin,S., 100, 102(16), 148(16) Donoff, R. B., 259 Dorland, L., 225 Doyle, E., 1 18 Driver, G. E., 32 Drouillard, S.,236 heckhammer, D.G.,190, 193(30), 2 13, 234(34) Du Mortier, C., 35

AUTHOR INDEX

266

Duncan, M. R., 256 Dupuis, J., 72, 73 Durda, W., 126 Durette, P. L., 74 Dumachter, J. R., 190, 193(30), 194(35), 234(34)

Duxbury, J. M., 128 Dvonch, W.,133, 134(146), 137(146), 162( 146)

Dziewiatkowski, D. D., 258

E Edwards, E., 258, 259(117) Effenberger, F., 206 Ehler, D. S.,35 Einstein, F. W. B., 114, 116(41), 143(41) Ek, M. J., 30 Eisenstein, R., 244 Ekman, G., 244 Elgavish, A., 256 El'perina, E. A., 142, 143, 161(189, 190) Embery, G., 243 Emeury, J. M., 134 Endele, R., 110 Endo, S., 204 Engler, D. A., 203 Emback, S., 18 1 Esser, F., 133 Evangelisti, R., 256 Evanko, S. P., 248 Ezure, Y.,235

F

Fackre, D. S., 251, 252(68) Farkas, I., 58, 59(54), 60(54), 75(54), 78,83 Famia, F., 39 Fassbender, F., 168 Fava, A., 148, 150, 16q200) Fay, L., 1 18 Fazekas, D., 161 Feicho, L., 166 Feizi, T., 22 1 Feldmann, J., 1 19(92), 120, 135 Femandez, R., 183 Femandez-Bolaiios,J., 30

Femandez-BolaiiosGuzman, J., 30 Fernbdez Moha,L., 2 1 Femer, R. J., 40, 41(21), 42(21, 28), 43(21, 29, 36), 45,47(27, 33), 48(26), 49, 50(35), 51,52(40), 60(33), 62(63), 63, 64(20, 63), 65(20, 38), 75(33), 76, 79(21,26,28), 80(27,38), 82(40), 85, 87(27), 88(33,36, W),89(93), 91(63) Fessner, W.D., 191 Fickert, W., 140 Fdipuzzi, F., 107, 130(32), 134(32) Finan, P. A., 142 Fine, D. H., 117, 118(75), 119 Fischer, F., 141, 169, 171(183) Fischer, H., 72 Fisher, G. R., 247 Fisher, L. W.,244,248(41), 252(41), 253(41), 254, 258(75)

Fitchett, M.,38 Flanigan, I., 206 Fleche, G., 95, 156 Fleischmajer, R., 244 Flint, M. H., 258 Forcier, G., 117 Ford, E. C.,136, 171(167) Forrest, M. E., 110 Foster, A. B., 128, 158 Franke, F. P., 30,206 Franko, B. V.,166 Franks, F., 23,25(27) Franois, P., 237 Fransson, L.-A., 242,244,247,248, 249,256 FranzCn, A., 249,258 Fraser-Reid, B., 124 Freidenreich, J., 200 Friebolin, H., 200 Frigerio, M., 35 Friz, L. P., 15 1, 172(206) Frize, J., 133 Fronza, G., 35 Frost, J. W., 206 Fuji Photo Film Co., 169 Fujii, N., 243 Fujimori, T., 3 1 Fujiwara, S., 254 Fukai, M., 169 Fukui, S., 256 Fumeaux, R. H., 40,41(21), 42(21,28), 51, 52(40), 64(20), 65(20), 79(21,28), 82(40)

AUTHOR INDEX

G Gabard, B. L., I34( 142) Gabe, E. J., 47,50(35) Gadelle, A., 25 Gainsford, G. J., 47,50(35) Gallagher,J. T., 256 Gallardo Carrera, A., 133 Gammeltofi, P., 150 Ganem, B., 30 Ganesan, V., 119, 171(82) Garcia Martin,M.de G., 24,30(3 1) Garcia-Rasz, A., 2 1 Garg, A. K., 258,259( 1 18) Garg, H. G., 225, 242, 247, 25 1(40), 252(39), 258,259(9, 118) Gaset, A., 95, 99, 102(I I), 110( 1I), 113(1 I), 114(11), 140, 155, 156(11),201(40) Gasiunas, N., 256 Gautheron, C., 183, 184, 195(20), 197, 198(20), 199(42,44),200(20,38). 201(48), 202(20), 204(41), 211,215(15, 69), 216,218(69), 219(15), 223(79), 225(15,80), 229,236(40,48) Gavuzzo, E., 102, 109(31), 117(31), 142(3 l), 16I( 3 1) Gemesi, I., 167 Geria, N., 168 Giani, C., 118(74), 119 Gibson, G. J., 244 Gielsdorf, W., 118 Giese, B., 38,72,73(6), 77(76) Gilbert, B. C., 38 Gillard, G. C., 258 Ginsburg, V., 221,239 Gizur, T., 133 Glaesson, S., 99 Gli)ssl, J., 254(87), 255,256(87) GinneZ-Sinchez, A., 24,30(31) Godtfredsen, S. E., 232 Goebbeler, K.H., I18 GOB,E. U., 117, 118(75),119 Golds, E. E., 257 Golovkina, L. S., 110, 113(33) Goodwin, J. C., 102, 109(21), 110(21), 15q21) Gorrichon, J. P., 95 Gowda, D. C., 231 Gowen, M., 257

267

Granado, C., 155 Gray,K., 29 Greene, I. D., 140 Greenshields, J. N., 140 Gregory, J. D., 243,247(17), 248 Greiche, Y., 168 Greiner, J., 129 Grelewicz, J., 133 Grenier-Loustalot, M.-F., 38 Greve, H., 256 Grigera, J. R., 23, 118(77) Grigor’ev, A. B., 119 Grtjninger, K. S.,73 Gromadzinska, E., 120, 133, 135 Gyarmathy, M.,167

H Habuchi, H., 247 Haigh, M., 258 Haines, A. H., 95, 125(8), 126, 141 Haines, S. R., 47,49, 50(35), 65(38), 80(38), 88, 89(93) Hajek, M.,119, 168(85) Halkiewicz, J., 118 Hamptom, A., 248(67), 249 Handa, M.,254,258(79) Handley, C., 248(67), 249 Handy, C. J., 239 Hane, K., 237 Hanessian, S., 39,201 Hardingham, T. E., 247 Hiiring, T., 141 Harry-Okuru, R. E., 29 Harsanyi, K., 133 Hartmann, L. A., 119 Hartmann, P., 168 Hascall, V. C., 239,244,248(65,66,67), 249,256,258 Hashimoto, C., 254,258(81) Hashimoto, H.,3 1,64 Hashimoto, Y.,22 Hassell, J. R., 244,248 Hawkins, G. R., 247,248(41), 252(41), 253(41) Hayashi, H., 152, 165(207a) Hayashi, N., 247 Hayday, K., 72 Haydon, D. A., 171

268

AUTHOR INDEX

Hayman, E. G., 248 Haynie,S. L., 188,208,211(63), 219(23) Hayward, L. D., 99, 100, 101, 117(18), 133, 150(112) Hedbom, E.,258 Hedbys, L., 232 Hehemann, D. G., 42 He& B., 102, 107(28), 155(28) Heineg;Zrd,D., 239,244, 248(66), 249, 252(32), 258 Heino, J., 254(86), 255 Helferich, B., 64 Hemmer, R., 114, 116(42), 163 Hempe, W., 55,56(50), 57(50) Hendry, C. J., 247,25 l(40) Hennen, W. J., 200,234,236( 103) Hermann, H., 242,247,251(40), 259(9) Herder, G., 23 1 Heyns, K., 155 Hicks, D. R., 124 Hieke, E., 118 Higa, H. H., 183,223(16) High, L., 150 HiU, R. L,, 220,223,225 Hdard, R. L., 140 Hilleman, M. R., 167 Hirao, A., 160 Hirose, N., 249 Hirschbein, B. L., 210 Hitz, W. D., 213,231(71) Hodge, J. E., 102, 109(21), 150(21) Hoffmann, G., 167 Holland, L. Z., 254,258(79) Holm, G., 118 Honda, S., 22 Honda, T., 248,254(83) Hooghwinkel, G. J. M., 223 Hope, K. D., 25,28(37) Hop!€, H.,146 Hopton, F. J., 102, 104(27) Hori, H., 51, 52(42), 54,78(42) Horilri, H., 51,52(41), 78(41) Honto, S.,64 Horiuchi, T., 166 Hormaza Montenegro, J., 21 Hortobagy, G., 167 Horton, D., 74 Hough, L., 95 HSU,C.-C., 247 Huang, S.G., 21,23(6), 27(6)

Huchette, M., 95

Hudson,K.L., 254,258(81) Hughes,F. A., 169 Hughes,N.A., 3 1 Humphries, D. E.,256 I

Ianni, A., 35 Ichikawa, Y.,44 M e , E.J., 257 Ikeda,J., 152, 165(207a) Inch, T. D., 203 Ingold, K. U., 49,68(37), 72(37) Innocenti, F., 1 18(74), 119 Inoue, S.,204 Inoue, Y.,204 Irie, T., 135 Isemura, M., 247 Ishibashi, K., 133, 135(149) Ishiguro, S., 133, 135(149) Ito, T., 133 Ito, Y., 166 Iwamura, H., 72 Iwasaki, M., 204 J

Jablonowski, M., 24 Jackson, M., 101, 117(18) Jaquet, A., 99, 102(11), l l q l l ) , 113(11), 114(11). 156(11) Jwuet,F.,.iss, is6 Jaeger, H., 1 18 Jalkanen, M., 257 Janousek, Z., 38,40,69(23), 70 Jansen, J. C., 114, 115(40) Janssen, H. M. J., 243 Jaquet, F., 95 Jarglis, P.,55,56(48,49,40), 57(50), 58(48, 49) Jaseja, M.,28 Jasinski, W.,95, 129, 131, 136, 137, l69( 117), 171(169) Jeanloz, R. W., 225 Jeansonne, N., 247 Jeremic, D., 147 Jeroncic, C. O., 35 John, B.A., 110, 1L3(34)

AUTHOR INDEX Johnson, R. N., I18 Johnson, T. L., 244,248(26), 253(26) Jones, G., 102, 107(26, 31), 116(26), 117(31), 142(31), 161(31) Jones, J. B., 177 Jung, W., 242, 259(9) Just, M.,163

K Kabayama, M. A., 23 Kaes, E., 139 Kahne, D., 74 Kaji, E., 56, 83(52, 53) Kakebi, K., 22 Kakuchi, T., 143, 161(191) WmBn, 59,83 Kaminsky, W., 169 Kapoor, R., 247 Kapuscinski, M.,30,206 Karelson, M.M.,70 Kasper, M.,114, 116(42) Katagiri, K., 248, 254(83) Kataoka,T., 254, 258(77) Kato, A., 22 Katritzky, A. R.,70 Kawa,M.,64 Kazlauskas, R.J., 210 Kean, E.L.,183,216(17) Khanolkar, J. E., 1 17, 1 18 Kho, B. T., 118 Khudyntsev, N. A., 132 Kieboom, A. P.G., 102, 104(27), 109(27), 236 Kiegel, E., 133 Kiely, D.E., 25, 28(37), 29 Killackey, J., 257 Kim, M. J., 191,200 Kimata, K., 247,249 Kimura, J. H., 239,244 King, 1. A., 257 Kim-Moms, M.J., 21 Kisfaludy, L., 167 Kishi, H., 51, 52(41), 78(41) Kitajima, K., 204 Kitajima, T., 228 Klaus, N., 173 Kleiner, F.,169 Kleinman, H. K., 244 Klenk, H. D., 23 1

269

Klessing, K, 133, 134(142, 143, 144), 138(147), 151(142, 143, 144), 162(142, 143, 144, 147) Klibanov, A. M.,234,235,236( 105) Kligman, L. H., 257 Knightly, W. H., 129 Kobata, A., 22 1 Kochetkov, N. K., 153 Koebemick, H., I19(92), 120, 135 Kohler, J., 168 Kohn, J., 183 Kokenyesi, R.,259 Kolarikol, A., 156 Kolbe,I., 167 Kolta, R., 161 Konstanntinovic, S., 100, 102(16), 103(16), 148(16) Koob, T. J., 257 Koroteev, M. P.,149, 153 Korth, HA.,72,73 Kossmehl, G., 173 KO&, T., 22 Krantz, D. E.,254, 258(82) Krauze, S., 120, 133, 135 Krempl, E., 168 Kresse, H., 254(87,88), 255,256(87) Krull, I. S., 117 Kruse, W. M.,140 Krusius, T., 254(86), 255 Kubler, D. G., 2 I Kubo, K., 152, 165(207a) Kuboki, Y.,243 Kubota, A., 247 Kuc, I. M.,249 Kuettner, K. E.,239 Kulczycki, E., 133 Kuroda, A., 248 Kuroda, T.,152, 165(207a) Kuszmann, J., 28, 100, 102(17), 105(17), 106(29), 109(29), 110(29), 149, 150(29), 151(203), 153(203), 154(17), 165(17,209) Kuyper, C.M. A., 243 Kuzuhara, H.,44

L Lacmte, G., 156 Ladner, W. E., 188, 21 l(24) Laidlaw, J., 258

AUTHOR MDEX

270

Lambe, R. F.,110 Langhans, R.K., 167 Lapenkov, V. L.,141 Lapiere, C. M., 244 Larjava, H., 254(86), 255 Larsso, T.,258 Larsson, P. O., 232 Lauer, K., 133 Laufen, H., 118 Lawston, I. W.,203 Le Blanc, M., 129 Le Lem,G., 126 Le Maistre, J. W., 136, 139, 165(176), 171(167)

Lee, C. K., 167 Lees, W., 191 Lehmann, A., 146 Lehtonen, E.,257 Leising, M., 73 Leisung, M., 73 Leitold, M., 130, 133, 137, 157(154), 163(129), 164(170, 171)

Lelki, G., 161 Leloir, L. F., 2 18 Lenfant, M., 135 Lenkiewicz, R.S., 118(80), 119 Leproq, S., 124 Lei, S., 117 Lewandowska, K., 259 Lewis, A.,166 Lewis, P. A., 118 Libeman, A. L., 167 Lichtenthaler, F.W., 2 I, 25( 17), 28, 32( 17), 55,56(48,49,50), 57(50), 58(48,49), 82, 83(52, 53) Lillford, P.J., 23,25(27) Lim, J. J., 74

Limura, T.,35 Linblom, K.,258 Lindner, H.J., 73 Liu, W.Y., 117 Livingstone, D. J., 101, 117(18) Lloyd, J. B. F., 117, 118(47)

Lo,Y.S.,165 Loesel, W.,133 Longas, M. O., 242,243 Low, M., 167 Lowary, T.L., 124 Lowther, D. A.,239,244 Lubineau, A., 221,237

Lueders, H., 102, 105(23), 130(23,25), 138, 146(173), 147(23), 151(23,25, 195), 154(23, 195), 159(173), 173(25, 128) Lukevica, O., 146 Lunazzi, L., 148, 160(200) Lundt, I., 125, 129 Lutz, D., 118 LuWi, J. K., 166 Luzi, L. A., 166 Lynch,M. J., 167 Lyndon, P., 30

M Ma&, M., 118(74), 119 MacLeod, J. K., 31 Maddock, J., 1 18 Miidler, H., 169 Maibach, H. I., 257 Ma$, L.,146 Maillard, B., 236 Major, R. M., 110, 113(34) Malatesta, V.,49,72(37) Malbica, J. O., 118 Malleron, A., 184, 195(20), 197, 198(20), 199(44), 200(20), 202(20), 203(43,44)

Malmstrom, A., 49,68(37), 244,249, 254(84, 85), 255,256

Manfredi, A., 129 Manro, A., 39 Maple, S.R.,20, 25(4) March, J., 5 1 Margok, R. U.,257 Marko, J., 102, 107(28), 155(28) Martin, B. K., 164 Martin, D. R.,24 Martinez-Castro, O.,21 Martorana, P., 130, 163(130) Martorana, P. A., 130, 163(130) Mathieu, C., 184,219(15), 223(79) Matsuda, M., 243 Matsui, F., 243 Matsunaga, E., 244, 248,249,254(83) Matyschok, H., 126, 129 Maurer, M., 140 Mazenod, F. P.,210 McCurry, S.D., 210 McKelvey, R. D., 72 McLeod, J. K., 206

AUTHOR INDEX

McMurtrey, J., 247 McNicholas, P. A., 29 McQuillan, C. I., 254 McQuillan, D. J., 239 Meats, J. E., 257 Medem, H., 130, 172(127) Medgyes, G., 100, 102(17), 105(17), 106(29), 109(29), 110(29), 150(29), 151(203), 153(203), 154(17), 165(17), 165(209) Meezan, E., 256 Mega, T. L., 35 Meguro, H., 5 1 52(4 1,42), 54,76(46), 78(41,42,43), 79 Menezo, J.-C., 124 Merck and Co., Inc., 167 MCrienne, C., I84,2 19(19) MerCnyi, R., 38,40,69(23), 70 Merrath, P., 114, 116(42), 128, 135(104), 139(104), 163 Meshreki, M. H., 170 Metcalfe, J. C., 102, 107(26), 109(31), 116(26), 117(31), 142(31), 161(31) Metras, F., 38 Meyborg, H., 119(91),120,168(85), 172(253) Meyer, K., 242 Michel, G., 1 18 Michel, H., 164 Micovic, V. M., 147 Midler, M., Jr., 167 Mihailovic, M. L. J., 100, 102(16), 103(16), 148(16) Mihalszky,K., 161 Mikhant'ev, B. I., 141 Millar, S. J., 254(90), 255, 256(90) Miller, R., 74 Mills, J. A., 94 Minet, E., 118(74), 119 Misawa, T.,54,76(46), 79 Miyamoto, I., 247 Mizuno, N., I17 Mladenovic, S., 147 Mocali, A,, 206 Mochizuki, H., 160 Modena, G., 107, 130(32), 134(32) Mohan, V. K., 119, 171(82) Monson, K., 118 Montassier, C., 124 Moran, I. R., 2 13

27 1

Mori, K., 169 Mori, T. P.,139, 165(176) Morita, E., 117 Moriyama, A., 169 Moriyasu, M., 22 Mom, M., 237 Moms, P.E., 25,28(37) Mosbach, K., 186,232 Mubarak, A. M., 129 Muenchow, L. H., 59,64(59) Muir, H., 247 Munakata, H., 247 Munkombwe, N. M., 31 M u d , B. K. M., 119, 171(82) Muralidhara, R., 167 Murata, K., 248 Murengezi, I., 140 Myers, G. S., 133

N Naadano, D., 204 Nagai, Y.,243 Nagase, S.,247 Nakahama, S.,160 Nakajima, T., 5 1, 52(42), 78(42), 256 Nakamura, A., 168 Nakamura, T., 244,247,249 Nakano, T., 249 Nara, T., 135 Neame, P. J., 244, 248(26), 249,253(26) Nec, R., 135 Negoro, K., 131, 168(132) Neilson, K., 118 Newman, A., 323 Newsome, D. A., 248 Nieduszynski, I. A,, 248 Nifant'ev, E. E., 131(134), 132, 149, 153 Nihon Surfactants Industry Co., 168, 169 Nilsson, B., 244 Nilsson, K., 186 Nilsson, K. G. I., 225,232 Nishida, Y.,51, 52(42), 54, 78(42,43) Nishikawa, Y., 166 Nishimiya, Y ., 135 Nkhino, T., 56,83(52) Nitz, R. E., 130, 163(130) Nix, M., 73 Nolan, J. C., 165

AUTHOR INDEX

272

Noro, A., 249 Nouvertne, W., 130, 172(127) Nozaki, K.,193,234(34) Nukada, T., 228 Nunez, H. A., 219 Nusgens, B.,244 0

Oberhauser, A., 168 O'Brien, E. A., 150 (Ibrink, B., 247 Ochiai, M., 168 OConner, T., 150 Oegema, T. R., Jr., 244,258 Ogawa, T.,228

Oh, K., 135 Ohkawa, M., 166 Ohrui, H.,5 1, 52(4 1,42), 54, 76(46), 78(4 1, 42,43), 79 Oike, Y.,248(67), 249 Ojrzanowski, J., 120, 133, 135 Okada, M., 22 Olano, H., 2 1 Oldberg, A., 239,247,248,252(37), 258 Olejnicak, E., 133 Oliver, W. M., 243 Olsen, K. w., 220 Onodera, S., 243 Oohira, A., 243 m - M ~ a hE. , C., 39 Orford, C. R., 258 Orth, W., 140 Osman, D., 20 Oswald, A. S., 21 Otagiri, M.,135 Otsuka, S.,166 Overend, W. G., 156 Ozaki, A., 237 Ozawa, T., 168

P Pacifici, R., 146 Paez, M., 21

paguaga,E.. 74 Pal, S.,244,248(26), 253(26) Palmer, M. E., 256 Pandey, M., 254(89), 255

Pandraud, H. G., 32 Paoletti, F., 206 Paolucci, C., 148, 150, 160(200) PArkhnyi, A., 83 P&khnyi, L., 59 Parry, D. A. D., 258 Parthasarathy, N., 247 Pasto, D. J., 70 Patroni. J. J.. 29 Pa&n, D., 23

Paul,E., 167 Paul, S.,244 Paulsen, H., 155,228 Paulson, J. C., 183, 184,223(16), 225, 231(89)

Paulson, M.,225( 18), 258 Paulsson, M., 258 Pavare, B., 146 Peacock, D. J., 39 Pearson, C. H., 244,249,252(68), 258, 259( 117)

Pedersen,C., 119, 120, 125, 129, 150 Pedersen, H., 53, 121(94) Pederson, R. L., 190, 193(30) Pentel Co., 169 pkez-Rey, R., 2 1 Perka, J., 129 Perlin, A. S.,27, 28, 32(38), 35(38) Perlish, J. S.,244 Perry, A. R., 128 Persson, B., 118(78), 119 Peters, J. A.. 102, 104(27), 109(27), 114, 115(40)

Petrov, K. A., 13I( 134), 132 Petter, R. C., 32 Phelps, C. F.,247,248 Pichini, s., 146 Pierce, J., 21,23(6), 27(6,29) Pierschbacher, M. D., 247,252(37) Pittet, A. O., 167, 168 Pizzorno, M.T., 119 Plaza Upez-Espinosa, M.T., 24,29(32) Plessas, N. R., 39 Plucinski, J., 126 Pogliano, L., 118(74), 119 Polievktov, M.K., 118(77), 119 Poll,H. G., 173 Pollak, A., 186,208 Pollicino, S., 148, 150, 160(200) Pommier, F., 118

AUTHOR INDEX Pompliano, D.L., 206 Poole, A. R., 239, 244, 252(73), 254, 259

Popuszynski, s., 95 Pora, H., 219,225(80) Porath, J., 181 Poutsma, M. L., 67 Power,M., 112 Powers,D. G.,32 F'rager, M., 257 M y , J.-P., 38,40,60(25), 61(25,61), 62(25), 84(25), 89(25) Prasit, P., 49,88(36) Prince,C. W., 244 Pringle, G. A., 249 Proctor,P. H., 166 Prost, M., I18 h e , D.G., 118 Pudgett, H. C., 30 PuMs, J. A., 243 Putnam, F.W.,254,258(78)

Q Quick, A., 102, 107(26) Quickenden, M. J., 23

R Rabovskaya, N. S., 149 Racker, F., 204 Radhakrishnamurthy, B., 247 Rafka, R. J., 24 Rahemtulla, F., 244 Ramaiah,M., 38 Range, D., 25 Rao, K.B., 119, 171(82) Rasper, J., 1 18 Rathbone, E. B., 30 Rearick, J. I., 223, 225 Redmond, J. W., 29 Redwood, W.R., 171 Refh, S.,53 Reidy, J. P., 142 Reimer, L.M., 206 Reiner, A., 244 Reinke, R., 254,258(82) Reinking, K., 169

273

Remy, G., 38 Renner, R., 85 Resnati, G., 35 Reuben, J., 30, 32 Reuter, G., 231 Richards, G. N., 124 Richardson, A. C., 95 Richter, K., 125 Riess, J., 129 Riess, J. G., 129 Rigal, L., 155, 156 Riordan, J. M.,29 Ripellino, J. A., 257 Ripp, K. G., 213,231(71) Riva, S.,236 Roberts, J. R., 254,258(79) Roberts, M. S., 112 Robins, P., 239 Robinson, G., 23,25(27) Rocrelle, D.,140 Rodbard, D., 248 Rogers, G. N., 23 I Rolland, P. H., 118 Ranniger, S.,21,25(71), 35 Roos, O., 133 Root, R. L., 234 Ropenga, J., 135 Ropuszynski, S., 126, 129, 131, 136, 169(117), 171(169) Roseman, S., 183,216(17) Rosen, L., 118(78), 1 19 Rosenberg, L., 244,248(26), 253(26) Rosenberp, L. C., 239,244,249,252(73), 254,259 Rosevear, P. R., 2 1 Rowel, M. T., 118 Rosseel,T., 101, 102(19), 106(19), 117(19), 134(19) Rossi, M. T., 119 Rougbley, P. J., 247, 248, 252(44), 253(44), 257 Rudolph, H., 130, 172(127) Ruegge, D.,72 Ruggeri, Z. M.,254,258(79,80) Rullmann, K.H., 64 Ruoslahti, E.,241,247,248,252(37), 254 Rusch, D. T., 167 Ruseva, N.,118 Russell, R. G. G., 257 Rzepka, M.,126

274

AUTHOR INDEX S

Sabesan, S., 225 SadIer, J. E., 225 Saheki, Y., 131, 168(132) !hito, T., 130, 168(132), 191 Saito, Y., 64 Sajdera, S.W., 248(65), 249 Sakano,T., 256 Salisbury, B. G. J., 119(91), 120, 248 %burg, H., 119, 120, 168(85), 169, 172(253) Samaki, H.,204 Sampaio, L. de O., 247 Sandri,E., 148, 150, 160(200) Sanol Schwarz-Monheim, 133, I34 Santer, V., 257 Santoni, Y., 118 Sam, J., 2 1 Sibstrand, B., 254(85), 255 Sasaki, T., 131 Sato, N., 247 Sato, S., 244 Satyamurthy, N., 30 Saura-Calixte, F.,2 I Savage, K., 256 Sawicki, W., I18 Saxen, L., 257 Schaefer, H.,173 Schafer, I. A., 254(89), 255 Scharpf, F., I 18 Schauer, R., 194,202(37), 231 Schiattarella, D., 151, 172(206) Schijen, M. M. A., 243 Schiphorst, W.E. C. M.,225 Schiweck, H., 28 Schleyerbach,R., 239 Schlingmaan, M.,234(107), 235 Schlueter, G., 137 Schliilter, G., 128, 135(104), 139(104) Schmidt, A., 257 Schmidt, D. L., 102 Schneider, B., 28 Schneider, C.J., 172 Schneider, G., 118 Schneider, M. P.,177 Schoenafinger,K., 128, 130, 133, 134(107), 163(130) Schor, S. L., 256 Schreckenberg, M., 130, 172(127)

Scott, J. E., 258,259 Scott, P. G., 249,252(68), 258,259( 117) Snepanik, B., I19 SeidI, S., 27 Selavka, C., 1 17 Selmke, P.,257 Senn, M.,110 Seno,N., 242 Serebryakov, E. P.,142, 143, 161(189, 190) Sekc A. S., 21,23(6, 12, 13), 25(12, 13), 27(6,9, 12,29), 28(36), 32(13) Servadio, V., 129 Seto, S., 30 Settlage, J. A., 118 Shah,B. A,, 118 Shaper, M.,220 Shchegolev, A. A., 131( 134), 132 Sheehan, J., 248 Sheehan, J. K., 244,249 Shen, T. Y., 146, 147, 148(193) Shibaev, V. N., 177 Shimada, F., 133, 135(149) Shimizu, C., 1 17 Shingbal, D. M.,1 18 Shinkai, H., 244,248,249,254(83), 255 Shinkuma, D.,117 Shinomura, T., 249 Shishiba, Y., 256 Shukla, A. K., 231 Siebert, E., 247,25 l(40) Siebert, E. P., 247 Silbert, C. K., 256 Silbert, J. E., 256 Silver, F. H., 258, 259( 1 18) Silveri, L. A., 119 Silvestri, S., 118(79), I19 Simon, E. S.,188, 191,206 Simon, H.,164 Sirnonet, J., 156 Sinaj, P., 43,45(30), 76(30), 77(30) Sinicka, S.,126 Sinnott, M. L., 141 Sinskey, A. J., 237 Siooufi, A., 1 18 Sipursky, S.L.,254 Sitabkha, L., 254(89), 255 Sjaberg, I., 256 Skelton, B. W.,29 Slessor, K. N., 114, 116(41), 143(41) slivkin, A. r., 141

AUTHOR INDEX Smith, D. F., 229 Smith, J. H., 170 Smith, V.H., Jr., 22 Snatzke, G., 59 Snell, R. P., 117 Snowden, J. M., 258 Snyder, J. R.,21, 23(6, 13), 25(13), 27(9), 32( 13) Sofronas, P., 102, 118(20) Sohar, P., 102, 106(29), 109(29), 1 lO(29) Soltzberg, S., 94,95(4), 146(4), 150(4) Sommarin, Y.,258 Somogyi, A., 83 Somdc, L., 41,45,47,48(34), 58, 59(54), 60(54), 62,63(62), 75(54), 77(32), 78(56), 83, 84, 87(32), 89(56,94) Sotman, S., 247,251(40) Spirov, G., 1 18 Spohn, J. A., 114 Sprissler, R., I18 Stabellini, G., 256 Stadler, I., 167 Stafford, W., 247,251(40) Staub, A., 206 Stefanovic, M., 147 Stein, P. D., 206 Steinle, G., 28 Stella, L., 40,69(23) Stephen, A. M., 30 Stephen, J. F., 140, 170 Stephens, C. A., 259 Sterk, G. J., 133, 136(146a) Sterk, H., 27, 133, 165(146a) Stevens, J. D., 32 Stevens, J. W., 248(67), 249 Stick, R.J., 29 Stoddart, J. F., 102, 107(26, 31), 116(26), 117(31), 142(31), 161(31) Stojcic, S., 147 Stoss, P., 114, 116(42), 128, 130, 133(105), 134(105), 135(104), 137, 139(104), 157(154), 163(129), 164(170, 171) Strein, K., 164 Stribblehill, P., 160 Strietholt, W. A., 141 Struchkova, M. I., 142, 143, 161(189) Stuehler, H., 168 Stiitz, A. E., 27 Suami, T., 35 Suggett, A., 23

275

Sugihara, J. M., 102 Sugimoto, M., 228 Sugiyama, H., 30 Sun, K. M., 149, 17q201) Suslova, L. M., 143 Sustmann, R.,72,73 Suzuki,F., 152, 165(207a) Suzuki, S., 22,247,249 Svensson, S., 232 Swann, D. A., 242, 247,251(40), 252(39), 256, 258,259(9) Sweers, H. M., 200,234,236( 103) Sweers, H. W., 193 Symes, K. C., 141 Synder, J. R.,21,23 Szabii, I. F., 59, 84 Szabo, E. I., 118 Szafranek, J., 110, 118(35), 149(35) Szalay, P., 161 Szarek, W. A., 22, 24 Szeja, W., 128 Szejtli, J., 167

T Tadano, K., 35 Tait, M. J., 23 Takahashi, N., 254,258(78) Takahashi, Y.,254, 258(78) Takaoka, T., 143, 161(191) Takida, Y.,243 Takio, K., 254,258(80) Talhouk, J. W., 29 Tammi, M., 254(86), 255, 257 Tammi, R., 257 Tamura, S., 176, 196(2) Tanabe, H., 35 Tanabe, K., 249 Tanaka, T., 256 Tang, L.-H., 244,248(26), 253(26) Tamer, M. L., 247 Tao Eiyo Kagaku Kogyo Co., 133, 134(152), 135, 142(152) Tarcsa, E., 47,48(34), 62,63(62) Tarrago, M. T., 229 Taylor, A. R., 171 Taylor, T., 1 18 Tedder, J. M., 38 Teijin Ltd., 160 Telschow, J. E., 203

AUTHOR INDEX

276

Ter-Ovanesyan, M. R., 132 Tennine, J. D., 244,247,248(4 l), 252(4 l), 253(41), 254(75) Th6risod, M., 234,235,236( 105) Thiem, J., 102, 105(23), 130(23, 25), 138, 141, 146(173), 147(23), 151(23,25), 154(23), 159(173), 169, 171(183), 173(25, 128), 216,234(107), 235 Tbgersen, H., 120, 121(94) Thomas, G. H. S., 102, 104(24) Thompson, R. D., 1 17 Thonard, J. C., 247 Tietz, H., 228 Tilbrook, D. M. G., 29 Timmerman, H., 133, 138(146a), 165(146a) T i p a H. P., 118 Tipson, R. S., 124 Titad, K., 254,258(79,80) Tokic, Z., 128 Tomana, M., 244 Toole, B. P., 244 Toone, E. J., 237 Totty, R. N., 100 Touet, J., 183 Toupet, L.,41,60(25), 61(25), 62(25), 84(25), 89(25) Trautwein, W.-P., 155 Treder,W., 216,234(107), 235 Trotter,J., 114, 115(39) Truppe, W.,254(88), 255 Tucker, K. H., 229 Tuite, M.R.J., 150 Tull, R.J., 102, 129, 166 Turner, N. J., 194 Turner, W. R., I18(80), 119 Tuross, N., 247,248(41),252(41), 253(41) Tuseev, A. P., 132 Tuzimura, K., 29 Tvaroska, I., 22 Tyler, P. C., 41, 42,43(26, 29), 47(27), 48(26), 62(63), 63,64(63), 76(26,63), 79(26), 80(27), 87(27), 88(90), 91(63)

U Uekama, K., 135 Uldbjerg, N.,244,258,259 Ulmsten, U., 244 Unkefer, C.J., 35

Usui, T., 30,256 Uwajima, T., 176, 195(1,2)

V Vainio, S., 257 Van Beuningen, H. M., 243 van den Eijnden, D. H., 2 15,223,225 Van der Rest,M.,252(73), 254 vanderWerf,J.F., l33,138(146a),165(146a) van Dijk, W., 215 Van Etten,R.L.,35 van Halbeek, H., 225,229 Van Koningsveld, H., 114, 115(40) Van Kuppevelt, T. H. M.S., 243 Varela, O., 31 Vat*le, J. M., 201 Vedejs, E., 203 Veerkamp,3. H., 243 Vlez Castro, H., 2 1 Verhegghe, G., 101, 102(19), 106(19), 117(19), 134(19) VeyreBrew, A., 197,201(40) Viehe, H. G., 38,40,69(23), 70 Vietmeier, J., 173 Vikar, J., 133 Vikman, A., 167 Vill, V., 141, 169, 171(183) Vincze, Z., 133 Vliegenthart, J. F. G., 225 Vogel, K. G.,244,248,252(32), 257,258 von der Osten, C. H., 237 von Sonntag, C.,38 Vul'son, N.S., 110, 1 13(33) Vuorinen, T., 21, 23(12), 25,27(12), 28(36) Vuorio, E., 254(86), 255 Vuorio, E., 254(86), 255

W Waechter, W., 119 Wagner, K., 168, 172(253) Wagner, W. D., 248 Waldmann, H.,1s Waldmann, H. J., 193 Walker, T. E., 35 Walkinshaw, M.D., 23 Walsh, D. A., 165 Walton, J. C., 38

AUTHOR INDEX

Wang, Y. F., 234,236(103) W a g , Y-F., 237 Ward, J. W., 266 Warren, L., 198 Watanabe, M., 78 Wax, M., 186 Webber, C., 244 Webber, J. M., 128, 158 Weber, A. J. M., 158 Weinstein, J., 184, 225(18), 231(89) Weisleder, D., 102,109(21), 110(21), 15q21) Weisshaar, G., 200 Welstead, W. J., Jr., 165 Weprek, S.,56, 82 Wheetall,H. H., 188 White, A. H., 29 White, R. J., 247, 252(44), 253(44) White, R. W., 248 Whitesides,G. M., 177, 186, 188, 191, 193(31), 194,206,208(31), 210, 211(24,63), 213, 219(23), 237 Whiting, M. C., 141 Whitson, M., 244 Wiebkin, 0. W., 247 Wiener, C., 168 Wiersum, U. E., 158 Wiggins, L. F., 94 Wigler, M., 254,258(77) Wilchek, M., 181 Williams, C., 20, 27(3) Williams, D. J., 102, 107(26), 109(31), 117(31), 142(31), 161(31) Williams, J. F., 206 Wilson, L., 2 1 Wimmer, E., 126, 134 Wingerup, L., 244 Winterbottom, N., 249,252(68), 258, 259( 1 17) Wischniewski, M., 110, 149(35), 166 Wiseniewski, A., 110, 118(35) Woelk, H. U., 119(92), 120, 135 Woessner, J. F., Jr., 259 Wong, C. H., 177, 188, 190, 191, 193(31), 194(35), 200, 208(31), 210,211(63), 213,219(23) Wong, C. M., 234,236( 103) Won& C-H., 237 Woo, D., 102, 118(20) Wood, D. D., 257 Wood, S.G., 110, 113(34)

277

Woodhour, A. F., 167 Woods, R. J., 22 Woodward, A. J., 118 Wright, L. W., 119 Wu, J., 21,23(6), 25(12), 27(12) Wulff, G., 173

Y Yagi, T., 169 Yamada, Y., 169 Yamaguchi, Y., 247 Yamanaka, Y., 117 Yamane, A., 56,83(52) Yamaoka, N., 30 Yamashina, I., 256 Yamazaki, N., 160 Yanachkov, I., 117 Yanagishita, M., 244,248,256 Yang, D., 74 Yeates, R. A., 137, 164(171) Yen, D., 254,258(82) Yen, J. K.C., 102, 118(20) Yokota, K., 143, 161(191) Yokoyama, M., 30 Yokoyama, Y., 248 Yoshida, C., 168 Yoshida, H., 256 Yoshimura, J., 64 Yosizawa, Z., 247 Young, M. F., 254,258(75) Young, M. R., 254 Yu, W. C., 117, 118(75), I19 Yuasa, H., 31 Yurovska, M., 118

2

Zarif, L., 129 Zavalishina, A. I., 132 Zech, J. D., 136 Zen, S.,56, 83(52) Zerner, M. C., 70 Ziegler, T., 206 Ziemann, H., 119(91), 120 Zipursky, S. L., 254,258(82) Zoorob, H. H., 160 Zuccaro, P.,146 Zuccaro, S. M.,146

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SUBJECT INDEX Azides, 1,43,6-dianhydmhexitols,I54

A ( lS,2S,3R,4S,5S,7R)- 1-Acetoxy-2,3,4-tetra-

~benzoyloxy-7-bromo-6sxabicyB clo[3.2. lloctane, synthesis, 65 N-Acetylmannosamine 2,4-O-Benzylidene-l,6-dichloro-1,6-difunctional derivatives, 195, 197-199 deoxy-D-glucitol, 158- 159 synthesis of derivatives, 20 1- 203 Bis-( 1,43,6dianhydro-~-rnnit010)-30N-Acetylneuraminicacid crown-10, 107- I08 biosynthesis, 194- 195 Bis(2,4dinitrophenylhydrazone), 156 synthesis, 200 m k ,RezsB, 3-9 1-0-AcetyI-2,3,5,6-tetra-O-benzoyl-4academic career, 4 bromO-gD-galactOse, synthesis, 49 antibiotic research, 5-6 I -O-Acetyl-2,3,4-tn-o-benzoyl-4-fluoro~~ C-nucleoside synthesis, 8 ribose, synthesis, 80- 8 1 glycosylamine research, 6 - 7 Acylation, isosorbide, 126- 127 honors, 8 Agarose, immobilization on, I8 1 - 186 reaction of cr,adihalo ethers, 7 - 8 Aldohexoses, 25-26 research on tlavonoid compounds and Aldol reaction,carbohydrates, 189- 190 carbohydrates, 5 Aldopentoses, 25-26 Branched-chain sugars, in solution, 30-31 Aldoses, in aqueous solution, 25-26 Brominations, see Radical-mediated broAldotetroses, 26 minations Alkyl ethers, 1,43,6-dianhydrohexitols, I-Brorno-D-glycosyl cyanides, synthesis, 58 135-145 Amino acids PG-I C composition, 248 Carbohydrate-protein linkage regions,proteNH,-terminal sequences, 253 PG-I1 oglycans, 242 - 243 Carbon radical stabilization Eactors, radicalcomposition, 244-247 NH,-terminal sequences, 252 mediated brominations, 70-7 1 sequence analysis, proteoglycans, 254- 255 Carboxylic acid, 1,4:3,6dianhydrohexitls 3-Amin~2-hydmxypropyl-substituted esters, 125-130 oxime ethers, 164 C-C bond-forming reactions, see Enzymic Amino sugars, in solution, 29- 30,34 methods I ,6-Anhydro-2-O-benzoyl-3,4-O-iwpropyli- Chiroptical properties, 1,43,64anhydrodene-/?-D-galactose, photobromination, hexitols, 99- 100 53-54 'T-N.m.r. spectra, 1,43,6-dianhydmhexitols, 109- 11 1 1,6-Anhydrohexopyranosederivatives, radiCollagen fibrils, proteoglycans role in orgacal-mediated brominations, 5 1 -54 nization, 258-259 1,5-Anhydmpentohranose derivatives, radiCosmetics, I ,4:3,6-dianhydrohexitolsapplical-mediated brominations, 54 Antitumor agents, isohexides, 165- 166 cations, 168 Aryloxypropanolamines,8-blocker sideCourtois, Jean Emile, 1 1 - 18 chain, 162 academic career, 11- 12 ATP regeneration, sugar phosphates, 208 archeological work, 17 I 8 210 glycosidase research, 14- 15

-

279

SUBJECT INDEX

280

glycosidases and glycanasesfrom xylophagicinsects, 15-16 honors and distinctions, 17 international organizations, 1 6- 17 periodic acid oxidation research, I2- 1 3 plant oligosaccharide research, 13- 14 role in Societk de Chimie Biologique, 16 Cytidine monophosphate N-acetylneuraminic acid enzymic synthesis, 21 5 -21 6 reaction catalyzed by, 2I4- 21 5 Cytidine triphosphate, enzymic preparation,

211,213

D 1 1 -Deoxy-8-epi- 1 1 -oxaprostaglandinFa, 159 3-Deoxy-~-arabino-2-heptulosonic acid 7phosphate, 206-207 3-DeOXy-D-gUfaCto-nOUulOsoniC acid, 202204 3-Deoxy-D-manno-2-octuctulosonic acid 8phosphate, synthesis, 204,206 ~ - D ~ o x ~ P ~ u27 ~os~s, Dialysis bags, immobilization in, 188

1,4:3,6-Dianhydro-2,5-O-benzoyl-2,5dithio-L-iditol, 153 1,43,6-Dianhydro-~-glucitol, 96-97,1 17 1,43,6-Dianhydro-2,5dideoxy-2,5-(dithiocyano)-L-iditol, 153 2,3:4,5-Dianhydro-~-iditol, 124- 125 1,4:3,6-Dianhydro-~-mannitol,96 1,4:3,6-Dianhydro-~-mannopyranose, 158 1,43,6-Dianhydrohexitols,93- 173 dkyl ethers, 135-145 j?-blocker side-chain, 137 bis-glycidyl ethers, 136 crown ethers, 143- 144 2,5diGethylisohexides, 141 isohexide etherihtion, I39 isosorbide 5-nitrate, 142 oxaprostaglandins, 138 pentafluorophenyl ethers, 141- 142 analytical behavior, detection, and determination, 117- 119 Chemical Abstracts references, 94 chemical uses, 158-161 cosmetics use, 168

deoxy derivatives amines, 150- 152 azides, 154 C-nitro compounds, 155 halogens, 149- 150 mono- and di-substituted, 146- 149 oxidation products, 155 - 158 phosphanes, 155 thio derivatives, 153 esters with carboxylic and sulfonic acids,

125-130 of nitric acid, 133- 135 with phosphoric acid, 130- 132 ethoxides, 171 food applications, 167- I68 as herbicides, 170 nomenclature, 96-98 bridged systems, 97-98 fused systems, 98 sugar-derived names, 96- 97 parent compound preparation, 119- 125

2,3:4,5dianhydro-~-iditol, 124- 125 isohexides, 1 22- I24 protonation, 120 ( 1R)-[1 -ZH]isomannide,1 20- 122 pharmaceutical uses, 161 - 167 antitumor agents, 165- 166 aryloxy propanolamines, I62 di-0-methylisosorbide, 166 isohexide nicotinic esters, 162 isohexide nitric esters, 16 1 - 162 isosorbide dinitrate, 16 1 isosorbide disulfite, I64- 165 isosorbide 5-mononitrate, 16 1 isosorbide sydnonimine derivatives, 163 somidipine, 163 as plasticizers, 168- 169 polymers containing isohexide moieties,

171-173 polyurethanes, polycarbonates and polyamides, 172- 173 preparation survey, 95 silyl ethers, 145 146 spectroscopic properties, 99- I 14 chiroptical properties, 99- 100 infrared spectra, 100- 102 mass spectra, 110, 112- 114 n.m.r. spectra, 102- 1 1 1 ultraviolet spectra, 99- 100

-

SUBJECT INDEX structural aspects, 114- 117 1,43,6-Dianhydro-2-S-benzoyI-5-0-methylsulfonyl-2-thio-~-glucitol, I53 1,4:3,6-Dianhydro-2-~brornophenylsulfOnyl)-D-glUcitOl 5-nitrate, 1 14-1 15 1,43,6-Dianhydro-~-iditol, 96 2,5-Diazido-2,5-dideoxyisohexides, 10 1, 104- I05 hypnotic properties, 165 1 ~-Dibenzoyl-2’,3’,5’-tri-O-benzoyl-4’-bromoadenosine, synthesis, 65 2,43,5-Di-0-benzylidene-1,6-dichloro-l,6dideoxy-D-glucitol, 158 1 ,CDichlor~1,6diideo~y-~-gl~cit0l,I58 159 Dichloro-L-isoidide, 149 2,5-Diideo~y-2,5-diiodo-Dglucitol 149- 150 2,5-Dideoxy-2,5aiiodo-~-i~tol, 149- I50 2,5-Di-O-ethylisohexides,141 1,2-Diideo~y-3-he~t~lo~e~, 28 5,7-Dideoxy-~-xylo-heptulose, synthesis, 194 Dideoxyisohexide C-nitro compounds, 155 Differential scanning calorimetry, 1,43,6dianhydrohexitols, 1 19 5-[1,4-Dihydro-3-(rnethoxycarbonyl)-2,6-dimethyl-4-(2-nitrophenyl>5-pyridylcarbonyllisosorbide, 1 15 - 1 I6 1,3-Dihydroxyacetonephosphate, reactions with aldolase, 192 Di-0-methylisosorbide, 140, 166 (RJ)-cis-2,6-Dioxabicyclo[3.3.O]octane, 147- I48 2,6-Dioxabicyclo[3,3,9]octaneframework, 97 DS-GAG chain, 242-243

28 1

glycosylations with transferases, see Glycosylation immobilization, 180- 189 agarose, 181-186 dialysisbags, 188 poly(acry1amide) gels, 186- 188 silica gel-glutaraldehyde, 188- 189 interest in, 176- 177 in organic solvents, 235-236 phosphorylations, 207-218 nucleotides, 210-213 “nucleotide-sugars”, 213 -218 sugar phosphates, 207-210,212 syntheses in aqueous solution, 234-235 transfer reactions, catalyzed by glycosi-

dases,231-233 Ethoxides, 1,43,6-dianhydrohexitols,171 Ethyl tetra-0-acetyl-a-bidopyranoside, synthesis, 76

F Food, 1,43,6-dianhydrohexitolsapplications, 167- 168 D-FIU~~OS~ from ~~-2,3,dihydroxypropnal, 193- 194 D-glucose conversion, 180 D - F I U ~ ~1,6-bisphosphate, OS~ reactions with aldolase, 192 Furanose derivatives, radical-mediated brominations, 49- 5 1 Fused rings,sugars, in solution, 3 1

C

E Elimination reactions,radical-mediated brominations, 85-91 Enzymic methods, 175-237 C-C bond-forming reactions, 189-207 aldol reaction, 189- 190 miscellaneous reactions, 205 syntheses with glycolysis aldolase, 190-

194 syntheses with sialyl aldolase, 194-204 tnlnsketolase, 204 207 definitions and abbreviations, 177

-

Galactosylation, with transferases, 219- 224 Galactosyltransferase, 220- 221 Gas-liquid chromatography I ,4:3,6dianhydrohexitols,1 18 sugars in solution, 21 -22 &D-Glucopyranosides, synthesis, 74

D-Glucose conversion into mhctose, 180 methyl ethers, 29 Glycerol kinase, S.cerevisiae, 208 N-Glycolylneuraminic acid, synthesis, 201 Glycolysis aldolase,syntheses, 190- 194 Glycopeptide, synthesis, 221

SUBJECT MDEX

282

Glycosaminoglycans composition, 24 1 Structure, 24 1-242 di~aCCharideunits, 240 24 1 synthesis, 256 G1ycosidases pyranosyl transfer with,232-233 transfer reactions catalyzed by, 231 -233 Glycosulose derivatives, radical-mediated brominations, 54 -57 Glycosylations, with transferases, 2 18-23 1 galactosylation, 2 19- 224 glycosylation, 23 1 sialylation, 223-23 1 C-Glycosylbenzeneesters, radical-mediated brominations, 59-60 Glycosyl cyanide esters, radical-mediated brominations, 57- 59 Glycosyl halide esters, radical-mediated brominations, 60 -6 1 C-Glycosylheterocycleesters, radical-mediated brominations, 59-60 Glyculose derivatives, radical-mediated brominations, 54 - 57

-

H Halogenation, 1,43,6-dianhydrohexitok, 149- 150 'H chemical shifts, isohexide derivative ring system, 108- 109 Heptasaccharide, synthesis, 228- 229 Heptuloses, 28 Herbicides, 1,43,6-diauhydrohexitok as, 170 Hexolrinase, immobilization, 186- 187 Hexopyranose esters, 5-bromides from, 48 Hexopyranoide esters, radical-mediated brominations, 62 - 64 Hexuronic acid derivatives 5-bromides from, 43-44 radical-mediated brominations, 42 -45 'H-n.m.r. spectra, 1,4:3,6-dianhydrohexitols, 102- 109 H.p.l.c., sugars in solution, 22 Hydrocarbon films, 1,43,64anhydrohexitols, 171 Hydrogen, substitution reactions,radicalmediated brominations, 75 -79 Hydrogen atom abstraction, radical-mediated brominations

regiochemistry, 67-68 stereochemistry, 7 1 - 72 3-Hydroxybutanal, condensation, 194 I

Idose, in solution, 31, 35 Infrared spectra, 1,4:3,6-dianhydrohexitols, loo- 102 Isohexide amino-substituted, 150- 15 1 derivatives 'H chemicalshifts, ringsystem, 108- 109 infrared data, 101 dialkyl, 140 esters, 129-130 etherification, 139 fragmentation, 1 12- 11 3 monoalkylated, I39 mono- and di-nitrates, 133- 134 nicotinic esters, 162- 163 phosphorus-substituted, 155 as plasticizers, 168- 169 preparation, 122- 124 proton coupling constants, 105 unsubstituted azido, 147 Isohexide mono- and di-amines, 165 Isohexide nitrates, 118 Isohexide nitric esters,vasodilation, 161- 162 ( 1R)1-%-Isomannide, 120- I22 Isosorbide, 126 acylation, 126 127 bis(tetramethy1phosphoroicdiamide), 132 diesters, cosmetics use, 168 disulfite, 164- 165 ethoxylated monoesters, 171 platinum-catalyzed oxidation, 155- 156 proton-proton coupling constants, 104 syndnonimine derivatives, 163 Isos~rbide2-acetate, 128 Isosorbide S-acylates, 128 Isosorbide di(docosanoate), 168 169 Isosorbide diheptanoate, 168- 169 Isosorbide dimethyl ether, 167 168 Isosorbide dinitrate, 1 17 pharmaceutids, 161 Isosorbide &(-oak), 168- 169 Isosorbide dipropanoate, 167- 168 Isosorbide mono(truns-docosenate),168,171 Isosorbide mononitrates

-

-

SUBJECT INDEX

‘H-n.m.r. data, 106 pharmaceuticals, 16I Isosorbide mono-oleate, 168- 169, 17I Isosorbide mono(tetradecanoate), 168 Isosorbide 2-nitrate, 134 Isosorbide 5-nitrate, 99, 117, 142 fragmentation, 1 I3 - I 14 Isosorbide phosphinite, monosubstituted, 132

283

N-terminal sequence, proteoglycans, 25 1 253 Nuclear magnetic resonance spectroscopy 1,4:3,6dianhydrohexitols,102- I10 sugars in solution, 20 -2 1 Nucleophilic substitutions, radical-mediated brominations, 79 - 84 Nucleotides, phosphorylation, 2 10-2 13 “Nucleotide-sugars”, phosphorylation, 2 13218

K Karplus relation, 104 Ketoses in aqueous solution, 28-29 preparations, 19 1 - 192

M &D-Mannopyranosides, synthesis, 82 D-M~UUOPYIZ~UOS~I radicals, 72-73 Mass spectra, 1,4:3,6dianhydrohexitols, 110, 112-114 Methyl 5-acetoxy-tetra-0-acetyla-L-idopyranuronate, synthesis, 79 - 80 Methyl fiisomaltoside, synthesis, 53 Methyl 4-0-(2-acetamido-2deoxy-8-D-mannopyranosyl)-a-D-glucopyranoside, synthesis, 83 S-Methyl-2-@etra-O-acetyl-I -bromo-fiD-galactopyranosy1)- I ,kxadiazole, synthesis, 59 Methyl(SR>tetra-O-acetyI-S-bromo-fiD-glucopyranuronate, synthesis, 43 Methyl tetra-0-acetyl-fiD-glucopyranuronate, synthesis, 87 Methyl tetra-0-acetyl-fiL-xylo-hexulopyranosonate, synthesis, 79-80 Methyl tri-0-acetyla-L-xyh-hexulopyranosylate bromide, synthesis, 42 -43 Methyl tetra-0-acetyla-L-idopyranuronate, photobromination, 45 Molecular-orbital calculations, pyranose forms of sugar in solution, 22-23 Monobenzoylated isohexides, fragmentation, 113

N Nitric acid, 1,43,6dianhydrohexitol esters, 133- 135

0

Oligosaccharides sialylations, 225 - 226 synthesis, 22 1 Oxaprostaglandins, I38 Oxidation products, 1,4:3,6dianhydrohexitols. 155-158

P PAN gels, cross-linked, 186- 187 Penta-0-acet yl-S-brorno-~~glucopyranose, synthesis, 45 - 49 Pentose, 5-0-substituted, 26 Pentose phosphates, preparation, 2 10 2-Pentuloses, 26, 34 Peracylated aldoses furanose derivatives, 49 - 5 1 pyranose derivatives, 45 - 49 radical-mediated brominations, 45 - 5 1 PG-I amino acid composition, 248 NH,-terminal amino acid sequences, 253 protein core amino acid sequences, 255 structure, 249 PG-I1 amino acid composition, 244- 247 NH,-terminal amino acid sequences, 252 protein core amino acid sequences, 255 structure, 250-251 Pharmaceuticals, 1,4:3,6dianhydrohexitols US, 161-167 Phenyl tetra-O-acetyla-L-idopyranoside, synthesis, 76 Phenyl 1-thiohexopyranosideesters, radicalmediated brominations, 64 -65 Phosphoric acid, 1,43,6-dianhydrohexitols esters, 130- I32

SUBJECT INDEX

284

Phosphorylations,enzymic methods, see Enzymic methods Poly(acry1amide) gels, immobilization on,

186-188 Polyamides, 1,4:3,6-dianhydrohexitols,

172- 173 Polycarbonates, 1,4:3,6-dianhydrohexitols,

172-173 Polyoxyethylene isosorbide, 136 Polyurethanes, 1,4:3,6-dianhydrohexitols,

172- 173 PrOteoglycanS, 239-259 amino acid sequence analysis, 254-255 biological roles, 257-259 biosynthesis explant culture, 257 primary culture, 254,256-257 carbohydrate-protein linkage regions,

242-243 isolation and fractionation, 243-244 M,values, 248-249 N-terminal sequence, 25 1-253 Proton coupling constants, isohexides, 105 Pyranose derivatives, radical-mediated brominations, 45-49 Pyranosyl transfer, with glycosidases, 232-

233

R Radical intermediate stabilization, radicalmediated brominations, 68- 71 Radical-mediated brominations, 37-9 1 1,6-anhydrohexopyranosederivatives,

51-54 1,s-anhydropentofuranosederivatives, 54 carbon radical stabilization factors, 70- 7 1 elimination reactions, 85-9 1 C-glycosylbenzene and C-glymsylheterocycle esters, 59-60 glycosyl cyanide esters, 57-59 glycosyl halide esters, 60- 61 glyculose and glycosulose derivatives, 5457 hexopyranoside esters, 62- 64 hexuronic acid derivatives, 42-45 hydroxyl group protection, 42 introduction at C-5,39 miscellaneous compounds, 65 peracylated aldoses, 45- 5 1

phenyl 1-thiohexopyranosideesters, 64-

65 reaction conditions and suitable compounds, 4 1 -42 regiochemistry, 67- 71 hydrogen atom abstraction, 67- 68 radical intermediate stabilization,68- 7I stereochemistty, 7 1- 75 hydrogen atom abstraction, 7 1- 72 products, 73-75 radical intermediate conformation, 72-

73 substitution reactions, 75-85 nucleophilic substitutions, 79- 84 radical reactions leading to, 84-85 substitution by hydrogen, 75- 79 Radical reactions, leading to substitutions, radical-mediated brominations, 84-85 Reducing sugars, in solution, 19- 35 aldohexoses and aldopentoses, 25-26,33 aldotetroses and related sugars, 26, 34 amino sugars, 29- 30,34 branched-chain sugars, 30- 31 furanose content in organicsolvents, 31,32 gas-liquid chromatography, 2 1 - 22 heptuloses, 28 hexuloses and pentuloses, 27-28 h.p.l.c., 22 n.m.r. spectroscopy, 20-21 partially O-SUbStitUted sugars, 28-29 relative stability aldehyde and keto forms, 24-25 composition vaxiation with temperature, 25 furanose form, 23-24 hydrated carbonyl forms, 25 pyranose form, 22-23 in solvents other than water, 31, 32,35 sugars having fused rings, 31 thio sugars, 30

Regiochemistry, radical-mediated brominati on^, 67- 71

S Sepharose, mechanism of activation, 181-

182 Sialic acids, naturally occumng, 195- 196,

200- 203

Sialosides,synthesis, 23 1

SUBJECT INDEX

285

Tetra-O-acetyl-5-bromo-~~-glucopyranosyl Sialyl aldolw, syntheses with, 194-204 chloride. synthesis, 6 1 N-acetylneuraminic acid, 194- 194 N-acetylmannosamine derivatives, 195, Tetra-0-acetyl-1-bromo-/h-glucopyranosyl 197- 199 fluoride, synthesis, 6 1 M t U d y OCCUhg *C acids, 195Tetra-O-aoetyl-5-bromo-/h-glucopy1anosyl fluoride, synthesis, 6 1 196,200-203 3deoxy-~-glycero-~-gagalact~nondosonic Tetra-Oacetyl-5-bromo-/h-xylopyranose, acid, 202 -204 47-48 elimination reactions,87 Sialylation, with transferases, 223, 225-23 I Tetra-Oacetyl-D-glucopyranosylradical, 72 heptasaccharide synthesis, 228 -229 immobilizedsialyltransfm, 225,2273Cretra-O-aoety1~-D-gl~PWWlW panonitrile, synthesis, 73- 74 228 sialoside synthesis, 230 -23 1 Tetra-O-benzoyl-2-bromo-P.glucono-13soluble transferases,225-226 lactone, synthesis, 62-63 tetrasaccharide-glycosidesynthesis, 228 2,3,4,6-Tetra-~benz0~1-5-by~~~-B-D-gl~trisaccharidesynthesis, 229 cose, synthesis, 79 Silica gel-glutataldehyde, immobilization Tetrasaccharide-glycoside,synthesis, 228 Thiabenzazole, antifungal activity, 166 on, 188-189 Silyl ethers, 1,4:3,6dianhydrohexitols,145- (E,E)-Thiacyclodeca-4,7-diene, 160 Thio sugars, in solution, 3 1 146 Sodium isosorbide 5-nitrate, 134- 135 Thorpe-Ingold e&ct, 24 L-Sorbose, from ~~-2,3,dihydroxypropanal, Transesterification,sugars, 235-236 193- 194 Transferases, glycosylations with, see GlycoSornidipine, 163 sylations Stereochemistry, radical-mediated brominaTransfer reactions,catalyzed by glycositions,7 1 -75 dase~,23 1-233 Substitution reactions,radical-mediated Transketolase, synthesis, 204-207 (6s?-2,3,4,Tri-O-acetyL1,6-anhydro-6brominations, 75 - 85 Sugar phosphates, 207 - 2 10,212 bromo-@-glucose, synthesis, 5 1 Tri-Gacetyl-1,5-anhydro-2deoxy-~-araenzymes for phosphorylation,208 ATF' regeneration, 208 - 2 10 bino-hex- lenitol, synthesis, 85 pentose phosphate preparation, 2 10 24Tri-0-acetyl-1-bromcwr-DarabinopyranSugars,See also Reducing sugars osyl)-5-(triauoromethyl)-1,3,4-oxadiatransesterification, 235- 236 zole, synthesis, 60 (SRtTn-O-acetyl-5-bromo-1-thio-fiD-gluSulfonic acid, 1,43,6dianhydrohexitols esters, 125-130 copyranosid)uronate,40 2,4,6-Tn-O-acetyl-I-thio-~-e~f~ro-hex1-enopyranosid-3-ulose, 39 1,42,5:3,6-Trianhydro-~-isomannide, 116 T Tri-Obenzoyl-5-bromo-6dmxy-gL-xyloTetra-0-awl- 1,5-anhydro-~-arabinoexhex-4-ulopyranose, synthesis, 55 lenitol, synthesis, 85 2,4,6-Tri-O-benzoyl-3deoxy-psrythro-hex2-(Tetra-&mtyl- l-bmmo-j3-D-galactopy2enono-1,5-lactone, synthesis, 90-91 ranosyl)benzothiazole, synthesis, 60 Tri-O-benzoyl-cu-~-arabino-hex-2-ulopyranTetra-0-acetyl-l-bromc+D-glucopyranosyl osyi bromide, synthesis, 55 Tributylstannane, 76-77 chloride elimination reactions, 89-90 Tnose phosphate isomerase, equilibrium catalyzed by, 191 synthesis, 6 1 ~ i & , 221,229,236-237 T e t r a - O - ~ l - l - b r o ~ o - j 3 - ~ U ~ p ~ oTsryi l~ ~ ~ h a synthesis, Turanose, 31 cyanide, synthesis, 58

SUBJECT INDEX

286 U

Ultraviolet spectra, 1,43,6dianhydmhexitols, 99- 100 Uridine diphosphate glucose, preparation, 213

V Vasodilation, isohexide nitric esters, 161 162 Vicinal diol, D-threo configuration, 19 1