Advances in Carbohydrate Chemistry and Biochemistry, Volume 28

Advances in Carbohydrate Chemistry and Biochemistry

Volume 28

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Advances in Carbohydrate Chemistry and Biochemistry Editors R. STUART TIPSON

DEREK HORTON

L.

ANDERSON

Board of Advisors D. FRENCH w . w. PIGMAN ROY

L.

w .

J.

WHELAN

WHISTLER

Board of Advisors for the British Commonwealth A.

B. FOSTER

SIR

EDMUNDHIRST

J. K. N. JONES

MAURICE

STACEY

Volume 28

ACADEMIC PRESS

New York and London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

1973

COPYRIGHT 0 1973, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRIES, INC.

111 Fifth Avenue, New York, New York loo03

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NWI

LIBRARY OF

CONQRESS CATALOQ CARD

NUMBER:45-1 135 1

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS LIST OF CONTFUBUTORS . PREFACE . . . . . .

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vii viii

LBszl6 Vargha (1903-1971) JANOS

Text

KUSZMANN

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

1

Applications of Gas-Liquid Chromatography to Carbohydrates: Part I BY GUY G . S . DUTTON I . Introduction . . . . . . . . . . . . . I1. Hydrolysis of Polysaccharides . . . . . . 111. Volatile Derivatives . . . . . . . . . . IV. Mutarotation . . . . . . . . . . . . . V. Neutral Monosaccharides . . . . . . . VI . Methyl Glycosides . . . . . . . . . . . VII . Alditols . . . . . . . . . . . . . . . VIII . Oligosaccharides . . . . . . . . . . . . IX. Acids and Lactones . . . . . . . . . . . X. Amino Sugars and Amino Alditols . . . . XI . Anhydro Compounds . . . . . . . . . . XI1. Cyclitols . . . . . . . . . . . . . . . XI11. Polyhydric Compounds . . . . . . . . XIV. Smith Degradation . . . . . . . . . . . XV.Tables . . . . . . . . . . . . . . .

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.

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.

12 14 23 38 41 51 56 67 71 78 87 89 90 98 101

Dehydration Reactions of Carbohydrates BY

MILTON S . FEATHER AND

I . Introduction . . . . . . . . . . . . I1. Reaction Mechanisms . . . . . . . . . I11. Dehydration in Acid Solution . . . . . IV Dehydration in Alkaline Solution . . . V. Reductic Acid . . . . . . . . . . . . VI . Levulinic Acid . . . . . . . . . . . VII Analyses Involving Dehydration Reactions

.

.

JOHN

. . . .

F . HAREUS

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161 163 174 193 207 212 218

Deoxyhalogeno Sugars BY WALTERA. SZAREK

I . Introduction . . . . . . . . . . I1. Synthesis . . . . . . . . . . . 111. Reactions and Synthetic Utility . .

. . . . . . . . . . . . . 225 . . . . . . . . . . . . . 227 . . . . . . . . . . . . . . 281

V

vi

CONTENTS Glycosyl Esters of Nucleoside Pyrophosphates

BY NICOLAI K . KOCHETKOV I . Introduction

AND

VLADIMIR N . SHIBAEV

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

I1. Isolation. Characterization. and Elucidation of Structure of Natural Glycosyl

307

Esters of Nucleoside Pyrophosphates .

. . . . . . . . . . . . . .310 . .334 . . 356 . . 362 . . . . . . . . . . . . . . . . . . . . . . . . 397

111. Preparation of Glycosyl Esters of Nucleoside Pyrophosphates . . . . IV. Chemical Reactivity of Glycosyl Esters of Nucleoside Pyrophosphates V . Enzymic Reactions of Glycosyl Esters of Nucleoside Pyrophosphates .

VI . Conclusion

a-DMannosidase

BY SYBILM. SNAITHAND GUILDFORDA . LEWY

I Introduction . . . . . . . . . . . . . I1 General Properties . . . . . . . . . . . 111. a-D-Mannosidase and Zinc2+ . . . . . . IV. Changes in Activity in vivo . . . . . . V Action on Naturally Occurring Substrates .

. . . . . . AUTHOR INDEX FOR VOLUME 28 . . . . . . . . SUBJECT INDEX FOR VOLUME 28 . . . . . . . . CUMULATIVE AUTHOR INDEX FOR VOLUMES 1-28 . CUMULATIVE SUBJECT INDEX FOR VOLUMES 1-28 . ERRATA. . . . . . . . . . . . . . . . . .

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. . . . . . 401 . . . . . . 402 . . . . . . . 422 . . . . . . . 434 . . . . . . . 437 . . . . . . . 447 . . . . . . . 481 . . . . . . . 517 . . . . . . . 527 . . . . . . 540

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

GUYG. S. DUTTON,Department of Chemistry, The University of British Columbia, Vancouver 8, B. C., Canada (11) MILTONS. FEATHER, Department of Agricultural Chemistry, University of Missouri, Columbia, Missouri 65201 (161) JOHN F. HARRIS, Forest Products Laboratory, Forest Service, U. S . Department of Agriculture, Madison, Wisconsin 53705 (161) NICOLAIK. KOCHETKOV, N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences, Moscow B-334, U . S . S . R. (307) KUSZMANN,Research Institute for Pharmaceutical Chemistry, Szabadsagharcosok U . 47-49, Budapest 4, Hungary (1)

JANOS

GUILDFORDA. LEVVY,Rowett Research Institute, Bucksburn, Aberdeen AB2 9 S B , Scotland (401) VLADIMIRN. SHIBAEV, N . D. Zelinsky Institute of Organic Chemistry, Academy of Sciences, Moscow B-334, U.S . S . R. (307) SYBIL M. SNAITH, Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland (401) WALTERA. SZAREK,Department of Chemistry, Queen’s University, Kingston, Ontario, Canada (225)

vii

PREFACE In a departure from the custom of annual publication that has been traditional for this Series, this twenty-eighth volume of Advances initiates a new schedule for publication of issues, accelerated to permit timely coverage of important topics in the face of greatly increased activity and interest on various fronts of the carbohydrate field. Appearance of this Volume in mid-1973, instead of the customary late autumn, reflects a decision to augment the scope and topicality of the Series by issuing volumes at intervals of about eight months instead of twelve. This Volume continues a series of articles devoted to modern methods for the separation and characterization of carbohydrates; Part I of a Chapter by Dutton (Vancouver) that is concerned with the gas-liquid chromatography of sugars and their derivatives is presented. Although less than a decade has elapsed since Bishop’s article on this subject appeared in this Series, the literature has since become vast and frequently confusing. The present article provides a critical analysis, together with extensive tabulated information, to guide the investigator in finding the best conditions for effecting separation of sugars and their derivatives. Another Chapter of broad, general interest is the contribution of Feather and Harris (Columbia, Missouri, and Madison, Wisconsin) on the dehydration reactions of sugars. The complex, sequential transformations that occur in aqueous acid or alkali, and that proceed from initial, enediol derivatives through unstable, dicarbonyl intermediates to the myriad degradationproducts, are related to problems in many areas of carbohydrate technology on the one hand, and to such applications as colorimetric methods for analysis of carbohydrates on the other. Deoxyhalogeno sugars have been of great synthetic interest in recent years, and Szarek (Kingston, Ontario) brings up to date the progress in this area since 1967, when the subject was last treated in this Series. As a complement to the comprehensive Chapter on the biosynthesis of complex saccharides by Nikaido and Hassid in Vol. 26, where the products of biosynthesis were stressed, Kochetkov and Shibaev (Moscow) here emphasize the “nucleotide sugar” biosynthetic intermediates, themselves, in a detailed article on their chemistry and enzymology. ...

Vlll

PREFACE

ix

As part of a collection of articles focussing on individual enzymes acting on carbohydrates, this Volume features a contribution by Snaith and Levvy (Aberdeen) on a-D-mannosidase, a zinc-containing metalloenzyme. The life and work of LBsz16 Vargha, the Hungarian carbohydrate chemist, is the subject of an obituary article by Kuszmann (Budapest). The Subject Index was compiled by Dr. L. T. Capell.

Kensington, Maryland Columbus, Ohio September, 1973

R. STUART TIPSON DEREKHORTON

1903- 1971

LASZLO VARGHA 1903-1971 In 1633, a captain of the Hungarian fort of Onod was made a nobleman by King Ferdinand 11, because of his heroic behavior during the war with the Turkish invaders. This captain, named KapitBny Vargha AndrBs, was the ancestor of the Vargha family, which through the centuries has given several high-ranking ecclesiastical and civil officials to Hungary. GlmBn Vargha, the father of LBsz16 Vargha, was a priest of the Reformed Church who, with his wife Vilma Oswald, settled in the village of Berhida, some 100 km west of Budapest. After the birth of their first son, LBsz16, on the 25th of January, 1903, the family moved to another little village, Kiloz, where two girls (Ilona and JolBn) and two more boys (Bda and KAlmAn) were born. The peaceful childhood of LBszl6 Vargha was blighted by the illness of his mother, who suffered from tuberculosis, which, at that time, was an essentially incurable disease. He was in his 9th year when his mother died, and, as his father did not remarry, an aunt took care of the children. After finishing primary school, LBszl6 was sent to PBpa, partly because he could continue his study at the famous Reformed college in that provincial town, and partly because one of his aunts lived there; she, being a widow without children, was glad to have a nice, quiet, modest boy in her house to look after. From that time, LBsz16 Vargha spent only the holidays in Kdoz with his family, but, because of the severe puritanism of his father, he greatly preferred the gayer and freer life in the town, under the indulgent and loving guardianship of his aunt. Her tolerance went so far that she allowed him to carry out some chemical experiments in the kitchen, as chemistry was already one of LBszl6’s most beloved subjects. However, this experimental period did not last very long, as an unexpected explosion, which caused some serious damage to the aunt’s china, led to a strict prohibition of any further chemical experiments. Nevertheless, his devotion to chemistry proved to be serious, as, after having finished secondary school with excellent results, he was enrolled in 1920 in the Philosophical Faculty of the University of Budapest for continuation of his chemical studies.

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At that time, none of the professors in the three chemical departments of that University was an organic chemist, but E. Pacsu (later, a professor of organic chemistry at Princeton University, New Jersey), who, as an associate professor, dealt with organic chemistry, took Vargha into his laboratory. After having finished his studies at the University of Budapest, Vargha continued research work in Pacsu’s laboratories, and wrote a Ph. D. thesis dealing with the partial hydrolysis of di-0-acylprotocatechualdehydes. As a result of this dissertation and his doctoral studies, for which he chose chemistry, physics, and geology, he was awarded the Ph. D. degree “summa cum laude” in 1926. Becoming devoted to organic chemistry, he moved to the Technical University of Budapest, where, at that time, the already well-known Gkza Zemplkn was the leader of the department of organic chemistry. After working there for one year, Vargha was awarded a government Fellowship enabling him to go to the University of Berlin, where he continued his research work, at the Chemical Institute, under the leadership of Prof. H. Ohle. During his two years in Ohle’s laboratories, he became devoted to carbohydrate chemistry, and this remained his most beloved field of research during the rest of his life. Despite the fact that, later, he was several times forced by circumstances to give up carbohydrate research, he returned to it whenever it became possible, and the ample experience he gained in dealing with carbohydrates proved to be of outstanding value when he later had to deal with pharmaceutical research. Among the results he achieved with H. Ohle, the synthesis of 5 6 anhydro-1,Z-0-isopropylidene-a-D-glucofuranose by treating the corresponding 6-0-p-tolylsulfonyl derivative with sodium methoxide must be mentioned, as this new synthesis of carbohydrate derivatives containing an epoxy group proved to be of general validity and opened up a simple way of introducing this reactive group into the carbohydrate skeleton. Investigation of the ring-closure and ring-scission reactions proved to be very fruitful, as, in this way, the configuration of some carbon atoms could be inverted, offering the possibility of transforming readily obtainable derivatives into carbohydrates otherwise hard to synthesize. When his Fellowship expired, he obtained a position with Prof. A. Schonberg, with whom he worked for two years as a private assistant in the Institute for Organic Chemistry at the Technische Hochschule, Berlin-Charlottenburg, There he had temporarily to give up carbohydrate research, as Prof, Schonberg dealt with organic thio compounds. Nevertheless, as a young, diligent, and ambitious chemist,

OBITUARY-LASZLO VARGHA

3

he soon became acquainted with this field of chemistry, and, by the end of this work, he had completed six publications dealing with the synthesis and investigation of thioacid derivatives. These four years that he spent in Berlin were important not only from the professional point of view. Living in the Collegium Hungaricum, he became acquainted with several young scientists of different professions, a circumstance that widened his intellectual horizon. From these years also originated his special interest in history, which, during his later life became a real hobby. His favorite reading was always of books dealing with history, especially autobiographies of famous politicians and historical novels. There was one topic that could be discussed with him at any time, and that was the history of Europe, especially that part closely related to the history of Hungary. Besides its benefits, Vargha’s stay in Berlin had had its drawbacks too, as the intense work and the alien environment weakened his none-too-strong health, causing him to acquire a gastric ulcer. During the next two decades, this illness gave him a lot of trouble, and strongly influenced his behavior, too. In 1948, he at last decided to have the ulcer removed by an operation, and, from that time on, he felt himself another person. Returning from Berlin in 1931, Vargha met difficulties in finding a job because of the economic crisis which had already made itself felt in Hungary. He eventually succeeded in obtaining a one-year Fellowship at the Medicinal-Chemical Institute of the University of Szeged, where he became a coworker of A. Szent-Gyorgyi, who was at that time studying the problem of vitamin C. Several years before, Szent-Gyorgyi had isolated a small amount of a “hexuronic acid” from adrenal gland, but, when Vargha arrived at his Institute, he had just prepared a larger amount of this material from paprika fruits. He was convinced that the “hexuronic acid” was identical with the longsought vitamin C, but, in comparison with other vitamins, unusually large doses (1 mg/kg) were needed for proving its biological activity, and some well-known vitamin experts doubted that it really was vitamin C. Theoretically, it was possible that the vitamin C activity observed had not been displayed by the “hexuronic acid” itself, but by a minor, unknown contaminant in it that could not be removed by recrystallization. Vargha, who had already acquired excellent experience in dealing with carbohydrates, succeeded in synthesizing a crystalline mono-0-isopropylidene derivative of the “hexuronic acid,” from which “hexuronic acid” having unchanged biological activity could be recovered, making the “contamination” theory im-

4

J. KUSZUANN

probable. In addition to this work, he prepared and investigated some other derivatives of “hexuronic acid,” and the data so obtained made an important contribution to the final determination of the structure of vitamin C (L-ascorbic acid). When his Fellowship at the University of Szeged expired, he received another one-year stipend from the Biological Research Institute in Tihany, then a two-year Fellowship from the Institute for Organic Chemistry at the University of Szeged, and, finally, a oneyear Fellowship from the Physiological Institute of the University of Budapest. During all these years of the economic crisis, Vargha had to live a very modest life, as the amount of each Fellowship was hardly enough to meet the cost of living. Despite the fact that he had neither coworkers nor assistance during this period, he continued his carbohydrate research, and obtained many interesting results. In a study of the acetalation of hexoses and hexitols, he obtained partially protected derivatives by introducing the boric acid method, which permitted, for example, the direct synthesis of 1,2-0-isopropylidenea-D-glucofuranose and 1,2-O-isopropylidene-~-mannitol. He proved the structure of 2,4-O-benzylidene-~-glucitol, and used this compound for working out an elegant synthesis of ~-threo-2-pentulose (L-“xylulose”). The synthesis of 5-O-methyl-~-glucose is worth mentioning, too, as this partially methylated sugar proved very important in the determination of the structure of several derivatives of D-glUCOSe. As a consequence of these researches, which were published in 13 papers, Vargha became, in 1935,a Privat-Docent (Associate Professor) at the University of Szeged, where he gave special courses dealing with the chemistry of carbohydrates. Shortly afterwards, the pharmaceutical company Gedeon Richter (Budapest) offered him a post having the possibility for organizing a research laboratory. Vargha accepted this opportunity, despite the fact that, by becoming the leader of a research department of a pharmaceutical company, he had temporarily to give up his beloved work on carbohydrates. Nevertheless, in his new position, he had not only to face the problems of pharmaceutical research, but at the same time he was able to establish his reputation, and to obtain the assistance of several young coworkers. During this period at Gedeon Richter, Vargha became convinced that successful pharmaceutical research is impossible without well-grounded, fundamental, organic chemical research. During the four years of his leadership at the company, several new and independent syntheses of important drugs were worked out in their research laboratory, and were reported in several patents and five publications.

OBITUARY-LASZLOVARGHA

5

His strengthened financial position enabled him to start a family, as, in 1937, he had married Maria Hatz, a high-school teacher of German and Hungarian, who had finished her education at the Universities of Budapest and Vienna. During their marriage, two children were born; first, a girl (Helga, in 1943, who graduated as an organic chemist, too), and then a boy (Andris, in 1948, who became an engineer). In 1940, the Hungarian government re-established the Franz Josef University in Kolozsvir, Transylvania (now Cluj, Rumania), and appointed Vargha as Professor of Organic Chemistry. His task was not only to organize a brand-new Institute, but to teach organic chemistry and to bring up a new generation of researchers. Hardly had these tasks been fulfilled when World War I1 came to an end, and Transylvania was annexed by Rumania. The new Rumanian government established the B6lyai University in Kolozsvir, and Vargha had to organize the new Institute for Organic Chemistry. In 1950, his appointment as Professor was terminated, and he therefore returned to Budapest. Having spent almost a decade in Kolozsvir as Professor of Organic Chemistry, he was able to continue his basic research dealing mainly with carbohydrates. On investigating the acid hydrolysis of substituted 5,6-anhydro-~-glucitol,he found that the terminal epoxide was split by the intramolecular attack of the 2-hydroxyl group, and that a 2,5-anhydro ring was formed. In further studies, he investigated in detail the epimerization and isomerization of sugars and alditols via epoxides, a possibility that was first revealed by his collaborative work with Ohle. Later, he investigated the direct substitution of methylsulfonyloxy and p-tolylsulfonyloxy groups by nucleophiles, and synthesized L-idose, starting from D-glucose. L-Idose proved to be an unstable sugar that, on storage, undergoes spontaneous rearrangement to L-sorbose (~-xyZo-2-hexulose). The years in Kolozsvir brought Vargha acknowledgment not only as an organic chemist, a professor, and an organizer, but as a sportsman, too. When he was working in Tihany, which is situated on a peninsula reaching into Lake Balaton, he became an enthusiastic sailor, taking part in every sailing regatta. In KolozsvAr, however, where no sailing possibilities were available, he learned to ski, and he proved to be a skilful skier. The hilly surroundings of Kolozsvir and the southern Carpathians provided him with ideal ski-slopes. He preferred to go, in the company of his friends, on ski-tours lasting several days and, besides the sport, he enjoyed the romanticism of the stormy nights spent near the open fireplace in snow-capped huts. Returning to Budapest, Vargha became a department leader of

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the newly founded Research Institute for Pharmaceutical Chemistry. The task set this Institute in 1950 was to provide for most of the research demands of the pharmaceutical industry, affording a broad survey and central co-ordination of its resources and efforts. Vargha, who some years later (in 1957) was appointed Director of this Institute, was the right person in every respect for the job, as he had gained not only a wide reputation as an organic chemist, but a lot of experience in pharmaceutical research, and excellent connections with the leaders of the Hungarian pharmaceutical industry. Under his leadership, the Institute was not only developed into one of the best equipped and most up-to-date research centers in Hungary, but owing to the results achieved, its name became known in other countries, too. To overcome the set-back of the Hungarian pharmaceutical industry caused by World War 11, this Institute, in the first decade after its founding, developed manufacturing processes for the production of some 70 new preparations already known in other countries, and helped achieve their industrial realization. These processes -at least, in part, and from the chemical point of view-were not simple reproductions, but represented novel adaptations and improvements whenever possible, often worthy to have been patented in other countries. Among the most important drugs, manufacturing processes for the following were elaborated in the Institute: chloramphenicol, oxytetracycline, penicillin-V, streptomycin, various sulfonamides, p-aminosalicylic acid, isonicotinic acid hydrazide, D-cycloserine, Vitamins C and B1, prednisolone, and various hypotensive drugs and antihistaminics. Besides these important procedures, research to discover new, original, pharmaceutical substances was also started. The first important success came in 1954 as the fruit of research in the field of cytostatics. It was Vargha’s idea to combine cytostatically active groups with carbohydrates in the hope that, by using sugars as carriers, the high toxicity of the active groups would be decreased. This 0

CH,NH,C€I&H$l I

HOCH I

HOCH I HC OH

ZCl@

I

HCOH I

CH,NH,CH,CH,CI 0

Degranol

OBITUARY-LASZLd VARGHA

7

supposition proved correct, as, among the first hexitol derivatives synthesized for this purpose, 1,6-bis[2-(chloroethyl)arnino]-l76-dideoxy-D-mannitol dihydrochloride (named Degranol) was found to check the growth of various tumors strongly. By clinical trial, it was found that Degranol is suitable mainly for the treatment of certain malignant diseases of the human hemopoietic system, for example, lymphoid leukemia, lymphosarcoma, and lymphogranulomatosis. Based on these favorable results, intensive carbohydrate research was undertaken, leading to several new cytostatics that have been employed with success in the treatment of malignant diseases; these include 176-dibromo-1,6-dideoxy-D- mannitol, 1,6-di-0-(methylsulfony1)-D-mannitol, 2,3:4,5-dianhydro-1,6-di-O-(methylsulfonyl)-~-iditol, and 1,4-bis[2-(methylsulfonyl)oxyethylamino]erythritol.Besides these pharmaceutical achievements, very important information was obtained on structure-activity relationships. It was unambiguously proved that the cytostatic activity of carbohydrate derivatives is stereospecific and is a function of molecular configuration. Moreover, the great number of compounds investigated led to a more detailed understanding of their mode of action. It was shown that strict structure-activity relationships are valid only for carbohydrate derivatives carrying chemically similar, active groups. Consequently, no general rules can be established, as the biological activity greatly depends on the active metabolites formed in vivo. If two series of carbohydrate derivatives carrying a chemically different cytoactive group obey the same structure-activity rules, it may be taken for granted that they follow a similar metabolic pathway, forming the same active intermediates. The results of this research work were published in more than 30 papers, dealing not only with the synthesis of biologically active compounds but with carbohydrate research of a high level, too, as Vargha’s decision that pharmaceutical research has to go hand-inhand with fundamental research was most definitely adhered to in this field of chemistry. During the synthesis of especially designed carbohydrate derivatives, side reactions leading to unknown compounds, the structure of which had to be established, were frequently observed. In this way, several new reactions were discovered and later successfully employed with other carbohydrate derivatives. Vargha was very progressive as far as the application of new techniques was concerned. He aided the introduction of the various chromatographic methods and the use of infrared (i.r.) and nuclear magnetic resonance spectroscopy and mass spectrometry in solving the various problems of structure determination. Despite the fact

8

J. KUSZMANN

that he was very critical as regards the interpretation of spectra, he was the first investigator to use i.r. data in proving the structure of an anhydrohexitol (2,6-anhydro-~-glucitol). In looking for cytostatically active carbohydrate derivatives, he dealt not only with sugar alcohol derivatives acting as biological alkylating agents, but also started the synthesis of potential nucleoside antagonists. For this purpose, D-arabinose was used as the starting material, and it was converted by several steps, via 2-chloro-2deoxy-D-arabinose, into 9-(2-chloro-2-deoxy-~-arabinofuranosyl)adenine. This nucleoside was found not to be an antimetabolite of adenosine, but it could be converted into 2-deoxyadenosine by hydrogenolytic removal of the chlorine atom, affording a new synthesis of this biologically important N-glycosyl derivative. During this research work, the condensation of poly-0-acetylglycosyl halides with different salts of purine bases was also studied, leading to a better understanding of the reaction mechanism. A practical result was obtained from these experiments, also, as a new, relatively simple synthesis of 2-deoxy-D-erythro-pentose (“2-deoxy-D-ribose”) was worked out, that permitted simultaneous labeling by introduction of tritium onto C-2 of this deoxy sugar. The results obtained by Vargha and his coworkers in the field of cytostatics was followed with lively interest by several research groups abroad, and it was through these studies that the Research Institute for Pharmaceutical Chemistry became internationally known. Because of this appreciation of his work, Vargha was invited to the Xth International Cancer Congress (Houston, 1970) to attend a panel concerned with cytostatically active carbohydrate derivatives. In recognition of the outstanding results achieved by Vargha in the field of pharmaceutical chemistry, the Hungarian Government presented him with the Kossuth Award in 1958, and three further high decorations in 1961, 1963, and 1965. The Hungarian Academy of Sciences elected him in 1951 to be a corresponding member, and, in 1964, to full membership. A further international recognition of Vargha as a carbohydrate chemist ensued in 1965, when he was invited to serve as a member of the Editorial Advisory Board of the newly established international journal Carbohydrate Research. As a leader of an institute dealing with pharmaceutical research, Vargha extended his research activity beyond carbohydrates to other pharmaceutically promising compounds. In a series of 15 papers, he published the results obtained by investigating the structure and rearrangement of several furan derivatives, including the p-tolylsulfonyl oximes of their ketones. This work was originally started at the

OBITUARY-LASZLO VARGHA

9

University of Kolozsvkr, but the problem became so ramified that it took several years to clear up all the details. Another important line of research was the synthesis of 3,4,5trialkoxybenzamides, as some of these derivatives showed outstanding psychopharmacological activities. Among these compounds, 4-(3,4,5trimethoxybenzoy1)morpholine (named Trioxazin) was introduced as a minor tranquilizer, N-cyclopropyl-4-(decyloxy)-3,5-dimethoxybenzamide (Denegit) as an antiepileptic agent, and 2-(1-pyrrolidiny1)ethyl 4-b~toxy-3,5-dimethoxybenzoate (Vasopenton) as a spasmolytic, for use in clinical practice. It was characteristic of his wide-ranging interests that both chemically and pharmaceutically important results were obtained by him in such disparate territories of organic chemistry as pyridine chemistry, steroids, and synthetic penicillins. Being a member of the Academy and the Director of one of the greatest research institutes in Hungary, Vargha was in a position to emphasize constantly the necessity of fundamental research. AS a result of his efforts, the ratio of exploratory to applied research was steadily increased, enabling his coworkers to deal partly with scientific problems especially related to pure chemistry. This automatically resulted in the increase of the scientific reputation of the Institute, so that some of Vargha’s coworkers became acknowledged researchers, alike at home and abroad. Under his leadership, the Institute established strong contacts and cooperation with several Universities and other research centers, both in and outside Hungary. Despite his being overburdened by the tasks of his own Institute, Vargha always did his best to serve all of the interests of organic chemical research in Hungary. As a member of several committees, he always used his influence to the benefit of his Institute, of chemical research, and, last but not least, for the benefit of carbohydrate research in general. He was not an outstanding speaker, but, having an excellent critical sense and a straightforward character, he always spoke his opinion, regardless of the consequences. Because of this trait, his coworkers could always rely on his opinion, as he had a special ability for finding out the weak or dubious points in any work, even when it dealt with problems with which Vargha was unfamiliar. He was excellent at writing scientific papers (he wrote 100 of them), restricting the contents to the sheer facts and limiting his speculations to the minimum. As the President of the Section of Organic Chemistry of the Society of Hungarian Chemists, he took part in organizing the Congresses of organic chemistry in Hungary, and was indefatigable in keeping the old, and establishing new, scientific contacts with foreign institutions.

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J. KUSZMANN

With the increase of his managemental burden, he had, to his great regret, as he was an extremely skilful organic chemist, to give up preparative work in the laboratory. Working with carbohydrates for decades, he had become a real master in crystallizing the most hopeless syrups. Later, paying only a daily visit to the laboratories of his closest coworkers, he more than once encouraged his younger colleagues by offering a prize of a bottle of champagne for crystallizing a carbohydrate derivative that he was especially interested in. If the task was fulfilled, the bottle was emptied in his company, and such events were very useful in strengthening the human contacts in his “scientific family.” To some extent, the Institute became his second home, and the older employees, knowing him for more than 20 years, regarded him as a beloved old friend rather than as their director. He was often asked for his opinion, or for his help, in solving private problems, and he never refused to lend a helping hand if there was real need for it. He was not a man of society, and he never struggled to get into the public limelight, but he liked to take part in parties, regardless of whether they were large ones, organized officially by the Institute, or just small, familiar ones arranged by the staff of a few laboratories in honor of one of his coworkers. From the early sixties, his emphysema, first detected in 1947, gradually got worse and hindered his undertaking long journeys and participating personally in meetings and congresses arranged abroad. With the progress of his illness, every physical movement became an exertion to him. The active surface of his lungs became reduced to such an extent that even covering the distance of a few steps made him out of breath. In the last months of his life, only his almost superhuman self-command, and his love of chemistry above all else, enabled him to cany on with his everyday work. Everyone who saw his heroic struggle was immensely impressed by it, and, if it can be said of anybody, he certainly was the very man who fought until his last breath for a more healthy future for mankind. LBszlo Vargha died on the 1st of July, 1971, after sudden development of pneumonia, and in his person, Hungary lost one of its outstanding chemical and pharmaceutical researchers. Not only those who knew him personally but all carbohydrate chemists who became acquainted with his ingenious research work will remember him with admiration.

JANOS KUSZMANN” * The author expresses his gratitude to Mrs. L. Vargha and Professor V. Bruckner, who generously furnished material used in this obituary.

APPLICATIONS OF GAS-LIQUID CHROMATOGRAPHY TO CARBOHYDRATES*: PART I BY GUY G . S . DUTTON Department of Chemistry. The Unioersity of British Columbiu. Vancouver. B.C., Cunuda

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Hydrolysis of Polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Volatile Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Trimethylsilyl Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 .Acetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Trifluoroacetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Isopropylidene Acetals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 . MethylEthers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 . Butaneboronates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Mutarotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Neutral Monosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . Trimethylsilyl Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Acetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Trifluoroacetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Butaneboroqates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Methyl Glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Trimethylsilyl Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.Acetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Trifluoroacetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . MethylEthers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Alditols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Trimethylsilyl Ethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . Acetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Trifluoroacetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 . Nitriles, Dithioacetals, and Methyl Ethers . . . . . . . . . . . . . . . . . . 5 . AsAlditols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Trimethylsilyl Derivative\ . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Acetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . Trifluoroacetates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

12 14 23 23 33 36 37 37 38 38 41 41 49 50 51 51 51 54 55 56 56 57 59 65 66 67 67 68 70 70

* The author gratefully acknowledges the award by NATO of a Visiting Lectureship tenalde during 1968 at the Technical University. Lyngby. Denmark. and the Max Planck Institute for Immunobiology. Freiburg. W . Germany. where material for this article was initially gathered . 11

12

G. G. S. DUTTON

IX. Acids and Lactones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Amino Sugars and Amino Alditols . . . . . . . . . . . . . . . . . . . . . . . . . 1. Hexosamines.. . , , , . . . , . . , . . , . . , , . , . . . , . , . , , . , . , . 2. Acetamidodeoxyhexoses . . . . . . . , . . . . . . . . . . . . . . . . . . . . . 3. Methyl Acetamidodeoxyhexosides . . . . . . . . . . . , . , . . . . . . . . . 4. Acetamidodeoxyalditols . . . . . . . . . . , . . . . . , . , . . . . . . . . . . . 5. Trifluoroacetates and Other Derivatives . . . . , . . . , . . . . , , . . . . . XI. Anhydro Compounds. . . . . . . , . . . . . . . . . . . . , , . , , , , . . . . . . XII. Cyclitols , . . . , . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . XIII. Polyhydric Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIV. Smith Degradation . , . . . . . . . , , . . . . . . . . . . . . . . . . . . . . . . . XV. T a b l e s . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

71 78 78 80 82 84 87 87 89 90 98 101

I. INTRODUCTION The first report’ on gas-liquid chromatography (g.1.c.) of carbohydrates was published in 1958. In the ensuing years, there have been many developments, both in the technique and in its application. In spite of this, there is no recent article dealing specifically with the application of gas-phase chromatography to carbohydrates. Earlier reviewszw4of this field date from the period 1962-64. In 1963, Sweeley and coworkers published what must now be considered a classical paper on the application of trimethylsilyl derivatives of carbohydrates in gas-liquid chr~matography.~ The discovery that these derivatives are readily formed and that they are volatile revolutionized the separation and analysis of carbohydrate mixtures. It is an expressive tribute to Sweeley that a large proportion of the work described in this article is a direct development of the method described by him and his collaborators. Sweeley and coworkers have reviewed some of these applications,6-8 and other

(1) A. G. McInnes, D. H. Ball, F. P. Cooper, and C. T. Bishop,]. Chromatogr., 1,556 (1958). (2) H. W. Kircher, Methods Carbohyd. Chem., 1, 13 (1962). (3) C. T. Bishop, Methods Biochem. Anal., 10,95 (1964). (4) C. T. Bishop,Adoan. Carbohyd. Chem., 19,95 (1964). (5) C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells,J. Amer. Chem. SOC., 85, 2497 (1963). (6) C. C. Sweeley, W. W. Wells, and R. Bentley, Methods Enzymol., 8, 95 (1966). (7) W. W. Wells, C. C. Sweeley, and R. Bentley, in “Biomedical Applications of Gas Chromatography,” H. A. Szymanski, ed., Plenum Press, New York, N. Y., 1964, Vol. 1, p. 169. (8) C. C. Sweeley, Bull. SOC. Chim. Biol., 47, 1477 (1965).

GAS-LIQUID CHROMATOGRAPHY

13

general articles on the subject have been p ~ b l i s h e d . ~ Chambers -’~ and have discussed the determination of carbohydrates in biological materials by gas-liquid chromatography, and the use of this method in the analysis of plant materials has been r e v i e ~ e d . ’ ~ ~ . ~ Brobst and others have described typical procedures for analyzing different types of carbohydrates and cyclitols by gas-liquid chromatograph~.~~~ In examining the structure of a polysaccharide, it is convenient to consider the methods involved under the three main headings: ( a ) quantitative analysis, ( b ) methylation, and ( c ) periodate oxidation. These techniques may be supplemented by partial or enzymic hydrolysis as the circumstances indicate. Each of these aspects of polysaccharide chemistry may be aided by the application of gas-liquid chromatography, either qualitative or quantitative, or both. Thus, separations impossible by other techniques may often be achieved, and analytical data obtained in a fraction of the time demanded by other methods. The present article is concerned with the use of gas-liquid chromatography for neutral, basic, and acidic sugars and some of their simple derivatives. A succeeding article will treat thd separation of methylated sugars and their derivatives. In an attempt to make this account as complete as possible, certain related aspects, such as the hydrolysis of polysaccharides, are briefly discussed, but, in general, the only references cited are to those papers that have also mentioned use of gas-liquid chromatography. These ancillary sections must, therefore, not be considered exhaustive in treatment, but rather to be representative. The present article is written primarily from the point of view of the investigator studying polysaccharide structures. In certain cases, how-

(9) J. H. Sloneker, in “Biomedical Applications of Gas Chromatography,” H. A. Szymanski, ed., Plenum Press, New York, N. Y., 1968, Vol. 2, p. 87. (10) L. S. Ettre and Z. Zlatkis, “The Practice of Gas Chromatography,” Wiley, New York, N. Y., 1967. (11) J. M. Berry, Adoan. Chromutogr., 2, 271 (1966). (12) T. Ueno, Kuguku To Seibusu, 8, 114 (1970); Chem. Abstr., 73, 52095 (1970). (13) H. V. Street, Aduun. Clin. C h e n ~12, , 217 (1969). (13a) J. R. Clamp, T. Bhatti, and R. E. Chambers, Methods Biochem. Anal., 19, 229 (1971). (1311) P. M. Holligan, New Phytol., 70, 239 (1971). (13c) P. M. Holligan and E. A. Drew, New Phytol., 70, 271 (1971). (13d) K. M . Brobst, Methods Curbohyd. Chem., 6, 3 (1972).

14

G . G . S. DUTTON

ever, carbohydrates occur in Nature either in the free state or as compounds of low molecular weight. Such examples are included, as are papers reporting syntheses wherein the compounds prepared are naturally occurring (or closely related), and where pertinent data are given. Instances in which gas-liquid chromatography has been used merely to monitor synthetic intermediates have been excluded. For work published prior to 1964, the reader is referred to the excellent article by Bishop4 in this Series. 11. HYDROLYSIS OF POLYSACCHARIDES

Common to all methods of determining the chemical composition of a polysaccharide is an initial acid hydrolysis into constituent monosaccharides, all of which are, to some extent, degraded by acid. Thus, the conditions of hydrolysis must be carefully chosen and controlled. When different types of glycosidic linkage are present in the same molecule, together with monosaccharides having different stability to acid, no one method of hydrolysis will necessarily cleave every linkage and give each component in a quantitative yield. For example, Conrad and coworkers14calculated that the L-fucopyranosyl bond is hydrolyzed 300 times faster than the D-glucosyluronic bond in 0.5 M sulfuric acid at 100". This situation arises to a greater or lesser extent for all heteroglycans. Glycosaminoglycans are very stable to acid hydrolysis, and polysaccharides containing uronic acids are moderately so. On the other hand, 3,6-dideoxy sugars found in bacterial polysaccharides are particularly acid-sensitive and, in such cases, a two-step procedure is recommended for total Many authors have commented upon the problems associated with the hydrolytic step, which has been considered to be the main source of loss in carbohydrate analy~is.'~ The fact that, in determining uronic acids, differing results were obtained, depending on whether hydrolysis or methanolysis was employed, led othersz0to conclude that (14) H. E. Conrad, J. R. Bamhurg, J. D . Epley, andT. J. Kindt, Biochemistry, 5,2808 (1966). (15) C. G . Hellerqvist, B. Lindberg, S. Svensson, T. Holme, and A. A. Lindberg, Carbohyd. Res., 14, 17 (1970). (16) C. C . Hellerqvist, B. Lindberg, S. Svensson, T. Holme, and A. A. Lindberg, Curbohyd. Res., 8 , 4 3 (1968). (17) G . Hammerling, 0. Liideritz, and 0. Westphal, Eur. J . Biochem., 15,48 (1970). (18) M. Berst, C. G . Hellerqvist, B. Lindberg, 0. Liideritz, S. Svensson, and 0. Westphal, Eur. J . Biochem., 11, 353 (1969). (19) A. A. Lehtonen, J. E. Kirkkainen, and E. 0. Haahti, A n d . Biochem., 16, 526 (1966). (20) J. R. Clamp and J. E. Scott, Chem. Ind. (London), 652 (1969).

GAS-LIQUID CHROMATOGRAPHY

15

“this demonstrates that differential determination of uronic acids is subject to considerable uncertainties due to lack of a reliable method for complete liberation of the monomers.” Every polysaccharide represents a different situation, and the optimum conditions for hydrolysis of each component should be determined independently.21 Certain polysaccharides are normally hydrolyzed with mineral acid, usually sulfuric acid, either by direct refluxing with dilute acid or by preliminary dissolution in concentrated acid. Typical procedures have been described, and the associated problems dis~ u s s e d . Although ~ ~ * ~ ~ prior solution of the polysaccharide in 72% sulfuric acid is a standard procedure,24it has been shown that part of the carbohydrate may become sulfated? leading to erroneous res u l t ~ When . ~ ~ noncrystalline polysaccharides are being hydrolyzed, the treatment with 72% acid may be slightly modified.z6In special situations, oxidative hydrolysis, for example, of carrageenan, may be achieved by using sulfuric acid in the presence of br~rnine.~’ Nitric acid is less commonly used for hydrolysis, but, in combination with urea, has been recommended for polysaccharides containing uronic acid residuesz8 and it has been used in a study of apple pectin.z9 It is generally agreed that hydrochloric acid causes more degradation than sulfuric acid, and there is evidence to suggest that different mechanisms operate.30 Work on steroidal saponins31 clearly shows the effect of changing the acid. Hydrolysis of the glycosides in aqueous alcoholic solution with hydrochloric acid gave losses of up to 40%, even for glucose. When the hydrolysis was conducted in aqueous p-dioxane containing sulfuric acid, the recoveries were in the range of 89-95%. Similarly? hydrolysis of flavonoid glycosides with (21) W. Niedermeier, Anal. Biochem., 40, 465 (1971). (22) G. A. Adams, Methods Carbohyd. Chem., 5,269 (1965). (23) J. K. N. Jones and M. B. Perry, in “Technique of Organic Chemistry,” A. Weissberger, ed., Wiley, New York, N. Y., 1963, Vol. XI, Pt. 2, p. 707. (24) J. F. Saeman, W. E. Moore, R. L. Mitchell, and M. A. Millett, Tappi, 37, 336 (1954). (25) H. L. Hardell and 0. Theander, Soensk Papperstidn., 73,291 (1970). (26) J. D. Blake and G. N. Richards, Curhohyd. Res., 14, 375 (1970). (27) N. S. Anderson, T. C. S. Dolan, and D . A. Rees, I . Chem. Soc. (C), 596 (1968). (28) M. A. Jermyn, in “Modem Methods of Plant Analysis,” K. Paech and M . V. Tracey, eds., Springer-Verlag, Berlin, 1955, Vol. 2, p. 197. (29) A. J . Barrett and D. H. Northcote, Biochern.I., 94, 617 (1965). (30) C. K. De-Bruyne and J. Wouters-Leysen, Curhohyd. Res., 17,45 (1971). (31) M. Kimura, Y. Hattori, I. Yoshizawa, and M. Tohma, Chem. Pharm. Bull. (Tokyo), 16,613 (1968).

16

G. G . S. DUTTON

hydrochloric acid gave extra peaks on a gas chromatogram, in combut the authors claimed that these parison to those from pure extra peaks could be used as a means of identification. Hydrochloric acid is, however, commonly employed for g l y c ~ p r o t e i n s h;Y-~ ~ ~ ~ ~ ~ ~ ~ drolysis of such compounds may be facilitated by prior solubilization with For the best results, hydrolysis under two sets of conditions is often necessary.36 Hydrofluoric acid has been recommended for the hydrolysis of teichoic acid polymers,37and mM perchloric acid in tetrahydrofuran has given good results with cardiac glyco~ides.~~ Trifluoroacetic acid is volatile, and thus readily removed. This acid was used by Albersheim and coworkers for the hydrolysis of plant ~ e l l - w a l l s and , ~ ~ has since been employed for cell ~ a l l s , 4 O -plant ~~ mucilages,44 blood-group 0ligosaccharides,4~ peptidogalactomannans,46 and disaccharides in blood and ~ r i n e . 4 ~It3has ~~ also been suggested as an alternative to 6 M hydrochloric acid in the determination of amino sugars,50 and for the hydrolysis of polyalcohols produced by periodate oxidation of polysa~charides.~~’ Lee

(32) J. Kagan and T. J. Mabry, Anal. Chem., 37, 288 (1965). (33) T. Gheorghiu, K. Oette, and V. Baumann, 2. Naturforsch. B., 25, 829 (1970). (34) T. Gheorghiu and K. Oette,J. Chromutogr., 48, 430 (1970). (34a) L. J. Griggs, A. Post, E. R. White, J. A. Finkelstein, W. E. Moeckel, K. G . Holden, J. E. Zarembo, and J. A. Weisbach, A n d . Biochem., 43, 369 (1971). (35) W. Niedermeier, T. Kirkland, R. T. Acton, and J. C. Bennett, Biochim. Biophys. Acta, 237, 442 (1971). (36) M. D. G. Oates and J. Schrager, /. Claromatogr., 28, 232 (1967). (37) I. T. Forrester and A. J. Wicken, Biochem. Biophys. Res. Comrnun., 25,23 (1966). (38) M. J. Frey and G. M. Jacobson, A n d . Biochem., 36,78 (1970). (39) P. Albersheim, D. J. Nevins, P. D. English, and A. Karr, Carbohyd. Res., 5, 340 (1967). (40) K. W. Talmadge, P. Albersheim, and N. W. Earle, 1. Econ. Entomol., 63, 1712 (1970). (41) A. L. Karr and P. Albersheim, Plant Physiol., 46, 69 (1970). (42) K. W. Talmadge and P. Albersheim,J. Znsect Physiol., 15,2273 (1969). (43) C. Labarca, M. Kroh, and F. Loewus, Plant Physiol., 46, 130 (1970). (43a) A. Kivilaan, R. S. Bandurski, and A. Schulze, Plant Physiol., 48, 389 (1971). (44) G. Franz, Plantu Medica, 17, 217 (1969). (45) G. Vicari and E. A. Kabat, Biochemistry, 9, 3414 (1970). (46) K. 0. Lloyd, Biochemistry, 9, 3446 (1970). (47) U. Lindahl and 0. Axelsson,J. B i d . Chem., 246, 74 (1971). (48) H. Nakamura and 2. Tamura, Chem. Pharm. Bull. (Tokyo), 18, 2366 (1970). (49) H. Nakamura and 2. Tamura, Chem. Pharm. Bull. (Tokyo), 18, 2314 (1970). (50) Y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry, 9, 25 (1970). (50a) C. G. Fraser and K. C. R. Wilkie, Phytochemistry, 10, 1539 (1971).

GAS-LIQUID CHROMATOGRAPHY

17

and examined, as a hnction of time, the extent of hydrolysis of different glycosides in 2 M trifluoroacetic acid at 100". They showed that, whereas methyl p-D-xylopyranoside, a-D-galactopyranoside, and a-D-mannopyranoside are completely hydrolyzed in 2 hours, methyl a-D-glucopyranoside reacts to the extent of only 70%. After 3 hours, the D-glucoside was 88% hydrolyzed, but extensive decomposition of other sugars occurred when the time of reaction was further extended; they therefore cautioned against the use of long periods for hydrolysis. Albersheim and colleagues39 commented on the slow hydrolysis of D-glucopyranosides and Kivilaan and coworkers.'3a also noted the decomposition of sugars with prolonged times of hydrolysis. In studies on glycoproteins, Griggs and cow o r k e r ~found ~ ~ ~that trifluoroacetic acid gives incomplete hydrolysis, they therefore preferred to use 3 M hydrochloric acid. Hough and coworkers50ccompared the use of hydrochloric, sulfuric, and trifluoroacetic acids for hydrolysis, and confirmed that hydrochloric acid gives lower recoveries than the other two acids. Also, they recommended that hydrolyses be conducted in an atmosphere of nitrogen. Trichloroacetic acid is said to give better results with glycoproteins than either sulfuric acid or ion-exchange resins?l and the solvent properties of 50% chloroacetic acid have been utilized in the hydrolysis of a p-toluenesulfonylated d e ~ t r a n . ~ * Preliminary hydrolysis with acetic acid may be helpful in avoiding N-dea~etylation,~:~ and certain pneumococcal polysaccharides may be hydrolyzed with formic The use of ion-exchange resins for hydrolysis was introduced by Wadman,55and this method is becoming increasingly popular in the field of glycoproteins. The resin may be used in the presence of dilute sulfuric acid?' or with dilute hydrochloric a ~ i d .The ~ ~last , ~ ~ (501)) Y.-C. Lee, G. S. Johnson, B. White, and J. Scocca, Anal. Biochem., 43 640 (1971). ( 5 0 ~ L. ) Hough, J. V. S. Jones, and P. Wusteman, Carbohyd. Res., 21, 9 (1972). (51) D. Grisslin and H. Weicker, Clin. Chin&.Actu, 21, 15 (1968). (52) D. A. Rees, N. G. Richardson, N. J. Wight, and Sir E. (L.) Hirst, Carbohyd. Res., 9,451 (1969). (53) C. G . Hellerqvist and A. A. Lindberg, Curbohyd. Res., 16, 39 (1971). (54) N. Roy, W. R. Carroll, and C. P. J. Glaudemans, Carbohyd. Res., 12, 89 (1970). (55) W. H. Wadman,]. Chem. Soc., 3051 (1952). (56) C. Mund and H. Venner, Z. Physiol. Chem., 338, 145 (1964). (57) J. H. Kim, B. Shome, Ta Hsiu Liao, and J. G. Pierce, A n d . Biochem., 20, 258 (1967). (58) W. F. Lehnhardt and R. J. Winzler,]. Chromntogr., 34, 471 (1968). (59) R. Kisters and H . Greiling, Z . Anal. Chem., 243,359 (1968).

18

G. G. S. DUTTON

method has been found to give excellent results.a0 In this way, 2-amino-2-deoxy-~-g~ucose may be liberated quantitatively, without decomposition of the neutral sugars.aoaSimilar methods have been used for characterizing glycoproteins by thin-layer chromatography?' for preparing a series of oligosaccharides from ovomucoids,a2and for hydrolyzing f l a v o n o i d ~ . ~ ~ Methanolysis is an alternative to hydrolysis, and may give better results. Thus Levvy and coworkersa4found that recoveries of neutral sugars were higher by methanolysis, and it has also been shown that methanolysis is less destructive of deoxy sugars in their liberation from mycosides than is aqueous sulfuric Some sugars, such as D-glucose and D-galactose, are more readily separated as derivatives of their methyl glycosides than as derivatives of the free sugars.66 Chambers and Clampaaahave made an assessment of methanolysis as it affects the analysis of glycopeptides and oligosaccharides. They showed that, with M methanolic hydrogen chloride, the release of carbohydrate is complete within 3 hours at 85", but that severe degradation occurs when 4 M or 6 M acid is used at 100".These authorsaaa considered that methanolysis is suitable for determination both of neutral and amino sugars. They also demonstrated that, under their conditions of methanolysis and trimethylsilylation, Tris buffer gives two peaks overlapping those for the methyl fucopyranosides, whereas, in direct trimethylsilylation of a hydrolyzate, citrate overlaps with the first peak for mannose. Although glycosidation affords several peaks from each sugar, the characteristic pattern of peaks may be helpful in identifying a particular component. Furthermore gas-liquid chromatograms obtained from biological material always show a certain number of unidentified, background peaks. When a sugar is determined in a form that gives only one peak, small amounts may not be detected, or will be apparently enhanced over their true value; with multiple peaks, there (60) F. Mullinax, G. L. Mullinax, M. R. Cohen, C. L. Cromwell, and J. Deboe, Zmmunochemistry, 8, 551 (1971). (60a) J. Metz, W. Ebert, and H. Weicker, Chromatographia, 4, 345 (1971). (61) E. Moczar and M. Moczar, Bull. SOC. Chim. Biol., 49, 1159 (1967). (62) A. Adam-Chosson and J. Montreuil, Bull. Soc. Chim. Biol., 47, 1881 (1965). (63) F. Sosa and F. Percheron, Bull. Soc. Chim. B i d , 51,625 (1969). (64) G. A. L e w y , A. J. Hay, J. Conchie, and I. Strachan, Biochim. Biophys. Acta, 222, 333 (1970). (65) M. Gastambide-Odier and C. VillC, Bull. SOC. Chim. Biol., 52,679 (1970). (66) C. C. Sweeley and B. Walker, Anal. Chem., 36, 1461 (1964). (66a) R. E. Chambers and J. R. Clamp, Biochern. J . , 125, 1009 (1971).

GAS-LIQUID CHROMATOGRAPHY

19

is a better chance of obtaining at least one peak free from interfering b a ~ k g r o u n dOne . ~ ~ other advantage of methanolysis is that sialic and uronic acids are more stable as their methyl esters. The stability of D-galactose, D-glucose, and D-mannose to the conditions used for methanolysis has been investigated.68It was found that refluxing of a solution of D-galactose in a 2% solution of methanolic hydrogen chloride for 5 hours gave a recovery of 81%, but, in a 15% solution, the recovery was only 38%. Under the latter conditions, recoveries of D-glucose and D-mannose were 66 and 47%, respectively. Sinkinson and WheelocksS studied methanolysis conditions in connection with the analysis of milk glycopeptides, and examined the effect of changing the acid concentration and the reaction time. Refluxing for 24 or 36 hours gave a very uneven base line, and they adopted a 4-hour reflux period, with 0.64 M methanolic hydrogen chloride. They also noted that N-acetylneuraminic acid begins to decompose before glycosidation is complete. Salfner and Uhlenbruck have made a similar study of methanolysis conditions for serologically active g l y c o p r o t e i n ~as , ~have ~ ~ ~ Yu ~ ~ and Ledeen for brain gangliosides .71 All monosaccharides are degraded by acid to greater or lesser extents. Mention has already been made of the acid lability of 3,6-dideoxy sugar^;'^ 4-amino-4-deoxy-~-arabinose, also found in bacteria, is similarly a c i d - s e n s i t i ~ eand ~ ~ should be liberated under mild conditions, for example, by hydrolysis with 0.5 M hydrochloric acid at 37". In an investigation of the T1 lipopolysaccharide of Salmonella friedenau, a careful study of the hydrolysisI8 was made. With 0.25 M sulfuric acid at loo", all of the D-ribose was liberated in 1 hour, but only 28% of the hexose and 20% of the heptose were set free. After 12 hours, the latter two were obtained in quantitative yield, but 30% of the D-ribose was decomposed. Similar results have been observed with a fructan, where refluxing with 0.5 M sulfuric acid decomposed 66% of the D-fructose, but did not affect D-glUcOSe.7"74The D-fructose (67) T. Bhatti, R. E. Chambers, and J. R. Clamp, Biochim. Biophys. Acta, 222, 339 (1970). (68) Y. Nozawa, Y. Hiraguri, and Y. Ito,J. Chromatogr., 45,244 (1969). (69) G. Sinkinson and J. V. Wheelock,]. Duiry Res., 37, 113 (1970). (70) B. Salher and G. Uhlenbruck, Z . Klin. Chem. Klin. Biochem., 9, 95 (1971). (70a) B. Salfner, Z . Klin. Chem. Klin. Biochem., 9, 486 (1971). (71) R. K. Yu and R. W. Ledeen,J. Lipid Res., 11,506 (1970). (72) (a) W. A. Volk, C. Galanos, and 0. Liideritz, FEBS Lett., 8, 161 (1970); (b) Eur. J . Biochem., 17, 223 (1970). (73) H. Hibbert, R. S . Tipson, and F. Brauns, CanJ. Res., 4,221 (1931);J. Honeyman, Methods Carbohyd. Chem., 1, 116 (1962). (74) B. A. Lewis, M. J. S. Cyr, and F. Smith, Carbohyd. Res., 5, 194 (1967).

20

G. G. S. DUTTON

was to be quantitatively liberated by 0.05 M sulfuric acid at 80". Because of the sensitivity of D-fructose to mineral acid, oxalic acid is often used for the hydrolysis of f r u c t a n ~ . ~ ~ Some workers have performed hydrolyses in an autoclave, but it has been shown that 0.5 M sulfuric acid at 120" degrades 33% of L-arabinose and 22% of D-galactose in two hours.75 Such methods are, therefore, only suitable for qualitative analyses, unless accurate corrections are made. Similarly, 90% formic acid has been found7s to decompose 48% of D-XylOSe and 36% of D-galaCtOSe in 20 hours at 100". Gas-liquid chromatography has been used to examine the products formed by the acid degradation of In addition to decomposing carbohydrate material, acid may also convert sugars into anhydro derivatives that may have the same gaschromatographic properties as other components in a mixture. Thus, it has been found that, under conditions used to hydrolyze wood and pulp polysaccharides, D-glucose gives 0.62% of 1,6-anhydro-p-~glucopyranose, which has the same retention time as a-D-Xy10Se.78 The equilibrium between acids and their anhydro derivatives has been studied by Angyal and Dawes,'$ and a system for separating 1,6-anhydro sugars has been described'" (see Section XI, p. 87). Before concentration, acid hydrolyzates are neutralized, most commonly with barium carbonate, although such organic bases as methyldioctylamine has been used.81 This step normally causes little loss, except by adsorption on, for example, barium sulfate,8z but the following points are of interest. Neutralization with ammonia has been re~ommended?~ as the neutral solution may be evaporated directly to dryness without filtration, and the ammonium sulfate formed is insoluble in methyl sulfoxide, a solvent used for trimethylsilylation. The authors83also found that, when hydrolyzates are neutralized with ion-exchange resins, the pH of the concentrated solutions may differ by as much as 2 units of pH. D-Fructose has been found to be epimerized by barium carbonate or pyridine, and lead (75) L. Hough and J. B. Pridham, Bioche~n.J.. 73,550 (1959). (76) B. Radhakrishnamurthy, E. R. Dalferes, Jr., and G . S. Berenson, A n d . Biochem., 24, 397 (1968). (77) P. E. Shaw, J. H. Tatum, and R. E. Berry, Corhohyd. Res., 5,266 (1967). (78) K. Turunen, A. Arvinen, and J. Turunen, P a p . Puu, 53, 189 (1971). (79) S. J. Angyal and K. Dawes, Aust. J. Chem., 21, 2747 (1968). (80) J. S. Sawardeker, J. H. Sloneker, and R. J. Dimler,]. Chrontutogr., 20,260 (1965). (81) E. L. Smith and J. E. Page,]. Soc. Chem. Znd., 67,48 (1948); P.H. Rubery and D. H . Northcote, Biochim. B i o p l t y . ~Actci, . 222, 95 (1970). (82) 1. Norstedt and 0. Samuelson, Scensk Pupperstidn., 69,729 (1966). (83) M. E. J. MacMillan and D. W. Clayton, personal communication.

GAS-LIQUID CHROMATOGRAPHY

21

carbonate was preferred for the neutralization stepE4Silver acetate has been recommended for neutralization after m e t h a n o l y s i ~ . ~ ~ , ~ ~ ~ ~ Direct evaporation of hydrogen chloride has been shownSSato cause high losses of carbohydrates, and to give extra peaks on the chromatogram. Alditols appear to be absorbed on the silver salts, but this loss is reversed, in a manner not yet understood, by the N-acetylation step normally used when mixtures containing amino sugars are analyzed.SSa When cellulose was hydrolyzed and the product was reduced, the formation of some D-mannitol was attributedE7to “isomerization during hydrolysis.” In certain instances, it has been found that the use of ion-exchange resins that are not of analytical grade has given rise to many spurious g.1.c. and prewashing of the resin with methanol has been r e c ~ r n m e n d e d .In ~ ~a study concerned with the determination of carbohydrates in sweet potatoes, high losses were experienced on de-ionization with a mixed-bed resin, but this phenomenon is contrary to normal experience. Losses of hexosamine have been noted on concentration of hydrolyzates, and lyophilization in the presence of (ethylenedinitri1o)tetraacetic acid has been r e c ~ m m e n d e d .In ~ ~the special case of soil hydrolyzates, where the percentage of carbohydrate is low and of inorganic salt correspondingly high, removal of iron is necessary before concentration, as, otherwise, as much as 75% of the sugars may be lost by oxidati~n.~’ For accurate results, correction factors must be established for the decomposition of each monosaccharide under the conditions of hydrolysis used. These corrections are of special importance when reliable values for a particular monosaccharide, present in only small When polysaccharides containing proportion, are desired.24,33,34,83,92-96 uronic acids are hydrolyzed, a further ambiguity is introduced, in that only partial cleavage of the glycosiduronic acid linkages may occur. (84) H. J. Bose, Ph. D. Thesis, University of Minnesota, St. Paul, Minnesota (1964). (85) A. S. Windeler and G . L. Feldman, Lipids,4, 167 (1969). (86) J. R. Clamp, G . Dawson, and L. Hongh, Biochim. Biophys. Acta, 148,342 (1967). (87) Z. S. Krzeminski and W. H. D. Leigh,]. Chetn. Soc., 1700 (1966). (88) D . A. Rees and J . W. B. Samuel,J. Chenl. Soc. ( C ) ,2295 (1967). (89) J. K. Huttunen and T . A. Miettinen, A n d . Biochem., 29,441 (1969). (90) E. F. Hartree, Anal. Biocheni., 7, 103 (1964). (91) J. M. Oades,J. Chrotizutogr., 28, 246 (1967). (92) H. Meier and K. C. B. Wilkie, Holzjbrsclzung, 13, 177 (1959). (93) B. W. Simson and T. E. Timell, Tuppi, 50,473 (1967). (94) A. Haug and B. Larsen, Actu Chem. Scand., 16, 1908 (1962). (95) G. Wulff,]. Chromotogr., 18, 285 (1965). (96) G. 0. Aspinall, J. J. Carlyle, and R. Young, Carhohyd. Res., 7,421 (1968).

22

G. G. S. DUTTON

For example, it has been determined that the common aldobiouronic acid 2-0-(4-O-methy~-a-D-g~ucosy~uronic acid)-D-xylose is one-third cleaved under normal conditions of hydrolysis, and corrections must therefore be applied.92,97 The factors influencing the quantitative determination of uronic and aldonic acid groups have been carefully studied by Norstedt and Samuelson.82Sources of error include adsorption on the barium sulfate formed from the barium carbonate used to remove the sulfuric acid, formation of lactones (and, thus, incomplete removal from the neutral components), and reversion and decarboxylation caused by the use of hydrochloric acid as the hydrolyst. The problem of lactonization in the analysis of uronic acids has been studied by Blake and Richards;g8they have also demonstrated that there is a reversible reaction between reducing sugars and weakly basic, ion-exchange resins that may lead to inaccuracies when such resins are used for removing uronic acids from h y d r o l y z a t e ~ . ~ ~ Strongly basic resins are also known to degrade neutral sugars.Ioo Hough and investigated the neutralization of hydrolyzates with three different ion-exchange resins, with barium carbonate, and with methyldioctylamine; they found that all methods except the last showed selective losses. Montreuil and coworkersIo1have reviewed the problems associated with the determination of monosaccharides, and, although their review is not concerned with gas-liquid chromatography, many of the points discussed are pertinent. The problems inherent in the hydrolysis of methylated polysaccharides are similar to those outlined, but, in addition, the possibility of occurrence of demethylation must be considered. This matter has been studied by Croon and coworkers,1o2who found that hydrogen chloride, either in water or methanol, causes a significant amount of demethylation. Formolysis in 98% formic acid caused considerable degradation, whereas 90% formic acid or sulfuric acid gave acceptable results. The hydrolysis of a methylated dextran with 90% formic acid has been described in detail.lo3The methanolysis of a methylated (97) N. Roy and T. E. Timell, Carbohyd. Res., 6,488 (1968). (98) J. D. Blake and G. N. Richards, Carbohyd. Res., 8, 275 (1968). (99) P. T. Murphy, G. N. Richards, and E. Senogles, Carbohyd. Res., 7, 460 (1968). (100) H. Kolmodin and 0. Samuelson, Suensk Papperstidn., 74,301 (1971). (101) J. Montreuil, G. Spik, J. Dumaisnil, and M. Monsigny, Bull. Soc. Chim. Fr., 239 (1965). (102) J. Croon, G. Herrstriim, G. Kull, and B. Lindberg, Acta Chem. Scand., 14, 1338 (1960). (103) J. K. N. Jones and K. C. B. Wilkie, Can. J . Biochem. Physiol,, 37, 377 (1959).

GAS-LIQUID CHROMATOGRAPHY

23

pectin was catalyzed by 72% perchloric acid,lo4 and a methylated fructan was hydrolyzed by oxalic acid in aqueous methan01.’~ The demethylation of the glycosides of 2,3,5-tri-O-methyl-~-arabinos.e has been examined,’05 and in a study of Acuciu gums, the isolation of large amounts of 2,3-di-O-methyl-~-glucosewas consistent only with the partial demethylation of 2,3,4-tri-0-methyl-D-gl~cose.~~~ The formation of degradation products during methanolysis may also result in compounds having short retention times in g . l . ~ . ~ ’

111. VOLATILEDERIVATIVES Most carbohydrates are not sufficiently volatile to be used for gasliquid chromatography, and they must therefore be converted into volatile compounds. The most usual derivatives for this purpose are trimethylsilyl (Me,Si) ethers, acetates, and trifluoroacetates. An extensive review on the trimethylsilylation of organic compounds has been written by Pierce.’O’

1. Trimethylsilyl Derivatives Conversion of sugars into their 0-trimethylsilyl (Me,Si) derivatives is most commonly achieved by reaction in pyridine with hexamethyldisilazane (1) and chlorotrimethylsilane (2) according to equation ( I ).

3 ROH

+ Me,SiNHSiMe, + Me,SiCI 1

2

-

3 ROSiMe,

+ NH4Cl

(I)

In a typical procedure, 10 mg of a sugar is dissolved in 1 ml of pyridine, treated successively with 0.2 ml of hexamethyldisilazane and 0.1 ml of chlorotrimethylsilane, and the mixture shaken for a few seconds. Reaction is normally complete within 5 minutes. This treatment corresponds to the original procedure used by Sweeley and colleagues? and is the method used in the great majority of cases. Apart from the deliberate use of other trimethylsilylating reagents, most of the variations on this fundamental reaction have been designed to meet certain special conditions. (104) T. F. Solov’eva, L. V. Arsenyuk, and Yu. S. Ovodov, Carbohyd. Res., 10,13 (1969). (105) D. H. Shaw and A. M. Stephen, Carbohyd. Res., 1, 400 (1966). (106) P. I. Bekker, A. M. Stephen, and G. R. Woolard, Tetrahedron, 24, 6967 (1968). (107) A. E. Pierce, “Silylation of Organic Compounds,” Pierce Chemical Co., Rockford, Ill., 1968; see also, A. E. Pierce, in “Synthetic Procedures in Nucleic Acid Chemistry,” W. W. Zorbach and R. S. Tipson, eds., Wiley-Interscience, New York, N. Y., 1973, Vol. 2, p. 125.

24

G . G. S. DUTTON

Differing conditions for making 0-trimethylsilyl derivatives have been compared'O8 and the proportions of solvent and of the two reagents may be varied over wide limits. However, in an investigation on sphingolipid bases, it was found that increase in the proportion of reagents gave derivatives of improved stability.10sWhen the substrate is sparingly soluble in pyridine, as in the case of heptitols,l10 inositols,111*112 and oligosaccharides,8s~113 the reaction may with advantage be conducted at 75-85', but it should be remembered that this may change the equilibrium composition (see Section IV, p. 38). The effect of temperature on the trimethylsilylation of N-acetylneuraminic acid has been investigated by Craven and Cehrke,ll4 and it has been found particularly advantageous to conduct trimethylsilylation at an elevated temperature when the reaction is heterogeneous, as in the direct treatment of potato slices.115*116 Reaction of sparingly soluble compounds can be aided by ultrasonic agitation. With simple compounds, the trimethylsilylation reaction is rapid, but, if the reaction proceeds slowly, short reaction-times lead to incomplete substitution; this is generally manifest on the gas chromatogram because of the presence of an unexpectedly large number of peaks."' Maltose has been reported to require reaction for at least 30 minutes,118and the time needed for complete trimethylsilylation of amino sugars has been examined by Oates and S ~ h r a g e r . ~ ~ Ketoses appear to react slowly, and several authors have commented on the time required for complete trimethylsilylation of D-fructose. Thus five peaks were observed after trimethylsilylation for one hour, compared to three peaks when the reaction was allowed . ~ ~ reacts ~ - ~in ~ a similar, ~ slow manto continue for 24 ~ o u ~ sSorbose (108) P. S. Mason and E. D. Smith,J. Gas Chromatogr., 4,398 (1966). (109) H. E. Carter and R. C. Gaver,J. Lipid Res., 8,391 (1967). (110) S. David and M . - 0 . Popot, Carbohyd. Res., 8,350 (1968). (111) Y.-C. Lee and C. E. Ballou,]. Chromatogr., 18, 147 (1965). (112) R. M. Roberts, J. A. Johnston, and B. W. Fuhr, Anal. Biochem., 10, 282 (1965). (113) T. Cayle, F. Viebrock, and J. Schiaffino, Cereal Chem., 45, 154 (1968). (114) D. A. Craven and C. W. Gehrke,]. Chromatogr., 37,414 (1968). (115) G . Rumpf, 1.Chromatogr., 43, 247 (1969). (116) M. Kimura, M. Tohma, Y. Okazawa, and N. Murai,J. Chromatogr.,41,110 (1969). ( 1 17) W. E. Sabbe and G. W. Cathey, Agron. J., 62, 36 (1970). (118) T. Bhatti and J. R. Clamp, Clin. Chim. Acta, 22,563 (1968). (119) G . Semenza, H.-C. Curtius, J. KolinskL, and M. Miiller, Biochim. Biophys. Acta, 146, 196 (1967). (120) H.-C. Curtius, J. A. Vdlmin, and M. Miiller, Z. Anal. Chem., 243, 341 (1968). (121) L. E. Vidauretta, L. B. Fournier, and M. L. Burks, Anal. Chim. Acta, 52, 507 (1970). (122) L. T. Sennello,]. Chromatogr., 56, 121 (1971). (123) R. E. McDonald and D. W. Newson,J. Amer. Soc. Hort. Sci., 95, 299 (1970).

GAS-LIQUID CHROMATOGRAPHY

25

ner.lZoOkuda and KonishilZ4have shown that, in the trimethylsilylation of hexuloses and heptuloses, the reaction proceeds in two stages, and that the initial product is the Me3Si ether in which the hemiacetal hydroxyl group is free; as the reaction continues, glycosidation occurs, and this component decreases. The same authors have shownlZ5that the trimethylsilylation of ~-altro-3-heptulose(coriose) gives three components corresponding to 1,2,4,5,7-penta-O-(trimethyIsilyl)-acoriofuranose, the trimethylsilyl glycosides thereof, together with the acyclic form, namely, 1,2,4,5,6,7-hexa-0-(trimethylsilyl)-keto-coriose. Trimethylsilylation normally proceeds without any change in the carbohydrate, but "glucosinolates" have been found126to undergo desulfation during the reaction; it was also observed that, even though trimethylsilylation was conducted overnight at 105",only 80% reaction was obtained. Trimethylsilylation is adversely affected by moisture, and therefore, hydrolyzates should be evaporated to dryness as completely as possible. If trimethylsilylation is catalyzed by trifluoroacetic acid, instead of chlorotrimethylsilane, moderate proportions of water may be tolerated,117~127-129 but, even under these conditions, extra peaks may be obtained from partly trimethylsilylated derivatives.I3OCatalysis with trifluoroacetic acid is useful when aqueous aliquots from a reaction are to be trimethyl~ilylated.'~~ A further advantage of this method, which has been used in the determination of 1,6-anhydrop-D-glucopyranose in corn syrup,132for c y ~ l o a r n y l o s e s and , ~ ~ ~for a series of malto-oligosaccharides,134is that ammonium trifluoroacetate is soluble in pyridine. It is also claimed that small proportions of water do not adversely affect the trimethylsilylation when the reaction mixture is diluted with N,N-dimethylf0mamide.'3~ The author of a general procedure for analyzing aqueous samples advocated injection into a short pre-

(124) T. Okuda and K. Konishi, Chem. Commun., 796 (1969). (125) T. Okuda and K. Konishi, Chem. Commun., 1117 (1969). (125a) P. A. Seib and P. C. Wollwage, Abstr. Pupers Anzer. Chem. Soc. Meeting, 157, CARB 21 (1969). (126) E. W. Underhill and D. F. Kirkland,]. Chromatogr., 57,47 (1971). (127) K. M. Brobst and C. E. Lott, Cereal Chem., 43,35 (1966). (128) L. Marinelli and D. J. Whitney,]. Znst. Brewing, 72, 252 (1966). (129) L. Marinelli and D. J. Whitney,]. Znst. Brewing, 73, 35 (1967). (130) R. Bentley and N. Botlock, Anal. Biochem., 20,312 (1967). (131) M. H. Fischer, Carbohyd. Res., 8, 354 (1968). (132) M. S. Kheiri and G. G. Birch, Cereal Chern., 46,400 (1969). (133) J. B. Beadle,]. Chrornatogr., 42, 201 (1969). (134) J. B. Beadle, J. Agr. Food Chem., 17, 904 (1969).

26

G. G. S. DUTTON

column packed with molecular sieves.'35 Alternatively, an aqueous solution may be injected onto the column, followed by a mixture of N,O-bis(trimethylsily1)acetamide(3), (trimethylsilyl)amine, and hexamethyldisilazane. It should be noted that, with these reagents, no hydrogen chloride is p r 0 d u ~ e d . l ~ ~ OSiMe,

I

MeC=NSiMe, 3

Acids likewise inhibit the formation of trimethylsilyl derivatives, and thus the acid in hydrolyzates must be carefully neutralized. In cases where deproteinization is necessary, acidic conditions should be a v ~ i d e d . ' ~ ' .High ' ~ ~ concentrations of urea, as in urine samples, also interfere with the trimethylsilylation reaction, and should be eliminated by treatment with u r e a ~ e although , ~ ~ ~ some authors have reported successful results without use of this stepells Various reagents other than hexamethyldisilazane and chlorotrimethylsilane have been recommended for trimethylsilylation. These differ in their effectiveness as trimethylsilylating agents, their stability to water, or both. N,O-Bis(trimethylsily1)acetamide (3) is an alternative trimethylsilylating reagent that has been found to trimethylsilylate L-ascorbic acid completely,139and to react with tertiary hydroxyl groups in ster o i d ~ . ' The ~ ~ . latter ~ ~ ~ observation is of interest, in view of the isolation from antibiotic substances of branched-chain carbohydrates possessing tertiary hydroxyl groups.142Some authors have recommended that the highly reactive bis(trimethylsily1)acetamide (3) be used in the absence of pyridine, in preference to a mixture of hexamethyldisilazane and chlorotrimethylsilane in this solvent.143In the tri(135) J. E. Willet, Chem. Ind. (London), 1701 (1967). (136) G. G . Esposito, Anal. Chem., 40, 1902 (1968). (137) J. H. Copenhaver, Anal Biochem., 17,76 (1966). (138) W. W. Wells, T. Chin, and B. Weber, Clin. Chim. Acta, 10, 352 (1964). (139) M. Vecchi and K. Kaiser, J . Chromutogr., 26, 22 (1967). (140) W. J. A. vanden Heuvel, J. L. Patterson, and K. L. K. Braley, Biochim. Biophys. Actu, 144, 691 (1967). (141) J. F. Klelw, H . L. Finklieiner, and D. M . White,J. Amer. Cheni. Soc., 88, 3390 (1966). (142) D. J. Cooper, M. H. Marigliana, D. M. Yudis, and T. Traube1,J. Znfec. Dis., 119, 342 (1969). (143) G . R. Waller, S. D. Sastry, and K. Kinneberg,]. Chromutogr. Sci., 9, 577 (1969).

GAS-LIQUID CHROMATOGRAPHY

27

methylsilylation of amino sugars, 3 alone was not sufficiently effective, even at 70°, but a mixture of 1 2 3 gave144complete N - and 0-trimethylsilylation in 30 minutes at 20". In contrast, a mixture of 1 and 2 gave only 0-trimethylsilylation. A powerful trimethylsilylating reagent, selective for hydroxyl groups, is N-(trimethylsily1)imidazole (4); it is relatively tolerant to

+ +

SiMe, I

HC/"CH

II

HC-N

I1

4

moisture, and reacts completely with D-ghCOSe in 50% aqueous solution;'07 hence, it is useful when aqueous solutions are sampled or ~ *combination '~~ with comsyrups are difficult to dry ~ o m p l e t e l y . ' ~In pound 1, this reagent has been found particularly effective in trimethylsilylating syrups containing a high proportion of D-fructose.'22 Although there are several instances in which N-(trimethylsily1)imid~ e d , 4it has ~ also ~ been ~ ~reported ~ ~to ~ azole has been ~ cause ghost peaks on polyester columns; this was attributed to the cleavage of some polyester linkages under the basic influence of the imida~ole.'~' Various fluoroacetamide derivatives have been used as trimethylsilylating reagents and, of these, N,O-bis(trimethylsily1)trifluoroacetamide (5) is the best known. It has approximately the same donor OSiMe, I FsCC=NSiMe, 5

(144) J. Krirkkiiinen and R. Vihko, Carbohyd. Res., 10, 113 (1969). (145) G. van Ling, C. Ruijterman, and J. C. Vlugter, Carbohyd. Res., 4, 380 (1967). (146) G. van Ling,J. Chromutogr., 44, 175 (1969). (147) R. F. Brady, Jr., Carbohyd. Res., 15,35 (1970). (148) J. J. Dolhun and J. L. Wiebers,J. Anier. Cheni. Soc., 91, 7755 (1969). (149) W. C. Butts and R. L. Jolley, Clin. Chem., 16,722 (1970). (150) R. M. Sequeira and R. B. Lew,J. Agr. Food Chem., 18,950 (1970). (150a) B. Coxon and R. Schaffer, Anal. Chem., 43, 1565 (1971). (150b) J. T. R. Clarke, L. S. Wolfe, and A. S. Perlin, J . B i d . Chetn., 246, 5563 (1971). (151) I. Johansson and N. K. Richtmyer, Carbohyd. Res., 13, 461 (1970).

~

~

28

G. G. S. DUTTON

strength as the non-fluorinated analog 3. One of the main advantages of this reagent is that the byproducts formed are more volatile than those from 3. Compound 5 was first used for amino a ~ i d s , ' ~and ~,'~~ has since been employed for h e p t u l o ~ e s , 'gly~osphingolipids,~~~ ~~ steryl g l ~ c o s i d e s ,and ~ ~ ~N-acetylneuraminic acid.l14 This reagent, with 1% of chlorotrimethylsilane (1) has been used for trimethylsilylating h e p t o n o l a ~ t o n e s ,octulosonic ~~~ acid^,'^'.'^^ and for monitoring utilization of sugars by mi~ro-organisms.~5~ A report by Donikelso discussed the use of trifluoro-N-methy1-N(trimethylsilyl)acetamide, which was shown to have a shorter retention time than other trimethylsilylating reagents. Useful comparative retention times were givenlsO for a variety of trimethylsilylated amides measured on three different columns, but this reagent has not yet been used for carbohydrates. For the determination of neomycins, diethyl-N-(trimethylsilyl)amine,Me,SiNEt, has been recommended.lsl Chambaz and Horningls2 have discussed various reagents for trimethylsilylating steroids, and Chambers and Clamp,66a who compared the trimethylsilylation of carbohydrates with different reagents, considered that such reagents as 3, 4, and 5 are not so generally useful as compounds 1 and 2, used originally by Sweeley and his colleague^.^ A wide variety of acidic compounds has been trimethylsilylated, and the preferred procedure is to treat the sodium, calcium, or barium salt, as a suspension in pyridine, with bis(trimethylsily1)acetamide and chlorotrimethylsilane;100~163*164 this gives the trimethylsilyl ester of the 0-trimethylsilyl derivative. Lead salts may be used,ls5 or the potassium salt may be used in methyl sulfoxide, the 0-trimethylsilyl derivative becoming concentrated in the upper phase.ls6 (152) D. L. Stalling, C. W. Gehrke, and R. W. Zumwalt, Biochem. Biophys. Res. Commun., 31, 616 (1968). (153) C. W. Gehrke and K. Leimer,]. Chromatogr., 57,219 (1971). (154) C. C. Sweeley and G. Dawson, Biochem. Biophys. Res. Commun., 37,6 (1969). (155) R. A. Laine and A. D. Elbein, Biochemistry, 10,2547 (1971). (156) M. B. Perry, G. A. Adams, and D. H. Shaw, J. Chromatogr., 44, 614 (1969). (157) D. T. Williams and M. B. Perry, Can.J. Biochem., 47,983 (1969). (158) M. B. Perry and A. C. Webb, Can. J. Chem., 47,2893 (1969). (159) B. Richardson, T. F. Bobbitt, and D. M. Orcut, Biotech. Bioeng., 13,453 (1971). (160) M. Donike,]. Chromatogr., 42, 103 (1969). (161) K. Tsuji and J. H. Robertson, Anal. Chem.,41, 1332 (1969). (162) E. M. Chambaz and E. C. Horning, Anal. Lett., 1,201 (1967). (163) G. Petersson, Tetrahedron, 26, 3413 (1970). (164) L. Jan& and 0. Samuelson,J. Chromatogr., 57,353 (1971). (165) J. Fitelson and G. L. Bowden, J. Ass. Offic. Anal. Chem., 51, 1224 (1968). (166) L. A. T. Verhaar and H. G. J. de Wilt,]. Chromatogr., 41,168 (1969).

GAS-LIQUID CHROMATOGRAPHY

29

Other investigators have claimed that pyridine inhibits the fonnation of trimethylsilyl esters, and have recommended the use of petroleum ether,ls7 acetone,ls8 or carbon d i ~ u l f i d eas ' ~the ~ reaction solvent. Lactones may be trimethylsilylated in the normal way by using a solution of hexamethyldisilazane and chlorotrimethylsilane in pyridine.ls3 The rate of decomposition of 0-trimethylsilyl derivatives of a series of oligogalacturonic acids has been studied;I7O it was found that the rate increases with the degree of polymerization, but that the methyl esters decay about 55 times as slowly. Pyridine is the solvent most commonly used for trimethylsilylation, but, for compounds that are sparingly soluble, methyl sulfoxide (Me2SO)or N,N-dimethylformamide may be used; complete trimethylsilylation may then usually be obtained in 10 minutes at room temperature. Richey and have called attention to the variability in quantitative determinations, because of low solubility in pyridine. Also, pyridine may be replaced, after trimethylsilylation, by another solvent, such as h e ~ a n e , ~ carbon ~ , ~ ' ~ d i ~ u l f i d e , ' ~ ~or- ' ~ ~ c y c l ~ h e x a n e , in ' ~ ~order to minimize "tailing"; this is often desirable when components of short retention time are present.176A procedure has been described for the extraction of 0-trimethylsilyl derivatives into chloroform and washing with hydrochloric acid to lessen ( a ) "tailing" and ( b )interference by pyridine.17' Trimethylsilylation has also been conducted in methyl sulfoxide for the analysis of wood-pulp hydrolyzate~,8~ but the trimethylsilyl derivative of myo-inositol has limited stability in this solvent.178Trimethylsilylation of free hexosamines has been performed in N,N-dimethylformamide at loo", but it is also possible to effect reaction at room temperature in p ~ r i d i n e . ' ~ ~ Acetonitrile has been used as the solvent for N-acetylneuraminic acid114and other acid^,'^^-'^^ and for amino sugars.ls0 Under the nor(167) D. F. Zinkel, M. R . Lathrop, and L. C. Zauk,]. Gus Claromutogr., 6, 158 (1968). (168) J. P. Shyluk, C. G . Youngs, and 0. L. Gamborg,J. Chromatogr., 26, 268 (1967). (169) M. Rowland and S. Riegelman,Anal. Bioclaem., 20,463 (1967). (170) W. R. Raymond and C. W. Nagel, Anal. Chen1.,41,1700 (1969). (171) J. M. Richey, H. G. Richey, Jr., and R. Schraer, Anal. Biochem., 9,272 (1964). (172) P. 0. Bethge, C. Holmstrom, and S. Juslin, Scerisk Papperstidti., 69, 60 (1966). (173) R. J. Penick and R. N. McCluer, Biochini. Biophys. Actu, 116,288 (1966). (174) B. N. Bowden, Plzytocliemistry, 9, 2315 (1970). (175) Y.-C. Lee and C. E. Ballou, Biochemistry, 4, 257 (1965). (176) 0. P. Bahl,]. Biol. Chem., 245, 299 (1970). (177) R. D. Partridge and A. H. Weiss,J. Chmmutogr. Sci., 8, 553 (1970). (178) D. R. Flint, Y.-C. Lee, and C. G . Huggins, J . Anier. Oil Chem. Soc., 42, 1001 (1965). (179) J. Karkkainen, A. Lehtonen, and T. Nikkari, J. Chromutogr., 20, 457 (1965). (180) W. H. Stimson, FEBS Lett., 13,17 (1971).

30

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ma1 conditions of trimethylsilylation employed by Sweeley and coworkers,5 tertiary hydroxyl groups do not react, but they react when the reaction is conducted in methyl sulfoxide or NJV-dimethylformamide.IE1The use of these two solvents for the trimethylsilylation of monosaccharides has been carefully studied by Ellis;lE2he showed that, by use of either of these solvents, an upper layer of hexamethyldisiloxane is formed in which the trimethylsilyl derivatives have high solubility. The direct injection of solutions in N,N-dimethylformamide or methyl sulfoxide avoids much of the “tailing” characteristic of pyridine, and the chromatograms may be further improved if the reaction mixture is allowed to separate into two layers and the hexamethyldisiloxane phase is injected. Solvents that have been less extensively used are N-methyl-2pyrrolidinone and hexamethylphosphoric triamide.IE3The author of this articleIE3also discussed the purification of chlorotrimethylsilane, and the anomalous results that may be obtained from the use of impure reagent have been commented on in the case of analyses of pentaerythrit01.I~~ The various methods available for the bulk purification of methyl sulfoxide have been reviewed,lE5 and a symposium on this compound reviewed its use as a solvent in selected reactions.IE6 The trimethylsilylation of a reducing sugar gives rise to multiple peaks because of the isomeric forms present at equilibrium (see Section IV, p. 38). As already noted, this multiplicity of peaks may be a useful diagnostic feature, but it may also cause undue complication of the chromatogram. In addition to reduction to alditols, or oxidation to aldonic acids, an alternative way for minimizing the number of peaks obtained is to convert the sugar into its ~ x i m eOn . ~ trimethylsilylation, oximes may give two peaks, due to syn and anti forms, one of which usually preponderates. It has been found that O-trimethylsilyl derivatives of oximes are particularly sensitive to water and, after being prepared, are stable for less than 48 hours in pyridine. By contrast, solutions in iso-octane may be preserved for 3 to 4 weeks.IE7 Clayton and H. G . JonesIEEalso commented on the ready degradation

(181) S. Friedman and M. L. Kaufinan, Anal. Chem., 38,144 (1966). (182) W. C. Ellis,J. Chromatogr.,41, 325 (1969). (183) J. F. Klebe and H. L. Finkbeiner,J. Polym. Sci. (A),7, 1947 (1969). (184) R. R. Suchanec, Anal. Chem., 37, 1361 (1965). (185) R. Philippe and J.-C. Merlin, Bull. Soc. Chim. Fr., 4713 (1968). (186) T. L. Fletcher,Quurt. Repts. Sulfur Chem., 3, 107 (1968). (187) B. S. Mason and H. T. Slaver,]. Agr. Food Chem., 19,551 (1971). (188) D. W. Clayton and H. G. Jones, Soensk Papperstidn., 73,753 (1970).

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31

of the Me,Si derivative of D-glucose oxime, both during preparation and, possibly, also on the chromatographic column. In a similar way, carbohydrates may be analyzed as the trimethylsilyl derivatives of their 0-methyloximes by sequential reaction with methoxylamine and trifluorobis(trimethylsilyl)acetamide.18sCompounds of low molecular weight, such as glycolaldehyde and glyceraldehyde, have also been separated as trimethylsilyl o ~ i r n e s . ' ~ ~ Although several advantages of dimethylsilyl (Me,HSi) ethers have been claimed,1s1few examples of their use have been reported, except for the separation of cy~loamyloses.'~~ Thus, dimethylsilyl derivatives (which are more volatile than trimethylsilyl ethers) require lower column-temperatures and have shorter retention times; for example, for glucose, the per(trimethylsily1) derivative has a retention time of 11.4 min, and the per(dimethylsily1) derivative, 5.10 min. Both types of derivative may be analyzed on the same column, and, in certain cases, compounds inseparable as their 0-trimethylsilyl derivatives may be separated as their dimethylsilyl ethers. The potential of dimethylsilylation for converting oligosaccharides into volatile derivatives should be examined. In the steroid field, it has been shown that 0-trimethylsilyl derivatives may be converted directly into trifluoroa~etates,~~~~~~~ and the relative merits of these two derivatives for the separation of glycols have been discussed.1s4 When electron-capture detectors are used, (bromoethy1)dimethylsilyl ethers give a better response than the chloro derivatives.Is5 Although this article is primarily concerned with gas-liquid chromatography of simple carbohydrates, it is worth noting that certain polysaccharides have been trimethylsilylated. Thus, chlorotrimethylsilane in pyridine yielded fully trimethylsilylated amylose, cellulose, and pullulan, but failed with dextran and a m y l o p e ~ t i n . Cellulose, '~~ amylose, and polyvinyl alcohol have also been trimethylsilylated in molten N-(trimethylsilyl)a~etamide.~~~ As examples of a new class of (189) R. A. Laine and C. C. Sweeley, Anal. Biochem., 43, 533 (1971). (190) H. Yamaguchi, T. Ikenaka, and Y. Matsushima, J. Biochem. (Tokyo), 68, 253 (1970). (191) W. R. Supina, R. F. Kruppa, and R. S. Henly, J. Amer. Oil Chern. Soc., 44, 74 (1967). (192) J. Sjovall, Mem. Soc. Endocrinol., 16, 243 (1967). (193) C. J. W. Brooks and J. Watson,]. Chromatogr., 31,396 (1967). (194) M. K. Withers,J. Gas Chrornatagr., 6,242 (1968). (195) C. Eaborn, D. R. M. Walton, and B. S. Thomas, Cheni. Ind. (London), 827 (1967). (196) G. Keilich, K. Tihlarik, and E. Husemann, Makrornol. Chern., 120, 87 (1968). (197) K. Bredereck, K. Strunk, and H. Menrad, Makroniol. Claern., 126, 139 (1969).

32

G. G. S. DUTTON

solubilized cellulose derivatives, a variety of silylated celluloses has been prepared.lE3 In most cases, complete trimethylsilylation is desired, and with 0-trimethylsilyl derivatives, steric hindrance does not arise, but, with larger groups, such as tricyclohexylsilyl, it may be very significant.lSEThe fact that cycloamyloses yield several peaks when converted into the trimethylsilyl ethers, but only a single peak as their dimethylsilyl ethers, has been attributed to possible steric hindrance.lg5 Deliberate partial trimethylsilylation may be used as a means of distinguishing between various monosaccharides,199and the nature of minor components formed on trimethylsilylation has been studied by mass spectrometry, Trimethylsilylation has been used in the separation of deuterated monosaccharides,200-203 and such deuterated trimethylsilyl derivatives as penta-0-(trimethyl-d9-sily1)glucose have been studied by gas-liquid chromatography and mass spectromet~y.'~~ 0-Trimethylsilyl derivatives have not only the advantage of being volatile but also of being readily hydrolyzed back to the parent compound.5 In a kinetic study of the methanolysis of methyl 2,3,4,6tetra-0-(trimethylsily1)-a-D-glucopyranoside, McInnes204showed that the trimethylsilyl group at 0 - 6 is removed 25 times more readily than those at 0-2, 3, or 4. The ease of desilylation may permit the effluent to be collected and then hydrolyzed, and the product ( a ) transformed into a crystalline derivative, for example, a lactone into a p h e n y l h y d r a ~ i d e ,or ~ ~( ~b ) examined by thin-layer chromatography.206In the case of a glycoside or disaccharide, regeneration of the parent compound permits the configuration of the glycosidic linkage to be determined by enzym01ysis.~~~ In a study using 14C-labelledsugars, it was shown that the recovery of trimethylsilyl derivatives was approximately 25%, whereas only (198) S. A. Barker, J. S. Brimacombe, M. R. Harnden, and J. A. Jarvis,]. Chem. Soc., 3403 (1963). (199) S. M. Kim, R. Bentley, and C. C. Sweeley, Carbohyd. Res., 5,373 (1967). (200) D. C. DeJongh, T. Radford, J. H. Hribar, S. Hanessian, M. Bieber, G. Dawson, and C. C. Sweeley,]. Amer. Chem. SOC., 91, 1728 (1969). (201) R. Bentley, N. C. Saha, and C. C. Sweeley, Anal. Chem., 37, 1118 (1965). (202) C. C. Sweeley, W. H. Elliott, I. Fries, and R. Ryhage, Anal. Chem., 38, 1549 (1966). (203) N. C. Saha and C. C. Sweeley, Anal. Chem., 40, 1628 (1968). (204) A. G. McInnes, C a n . ] . Chem., 43, 1998 (1965). (205) M. B. Perry and R. K. Hulyalkar, C a n . ] . Biochem., 43,573 (1965). (206) I. G. Zhukova and G. P. Smirnova, Carbohyd. Res., 9,366 (1969). (207) G. G. S. Dutton and A. M. Unrau, Carbohyd. Res., 1, 116 (1965).

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33

3-14% of glucose was recovered as its trifluoroacetate.208In spite of these low recoveries, the results were reproducible, thus validating the use of internal standards for the analyses of mixtures of sugars as their Me,Si derivatives. The relationship between stereoisomeric structure and retention time of per-O-(trimethylsily1)-pentosesand -hexoses has been examined,209and the following rule stated with reference to polar columns. “Retention times of isomeric aldoses with a stable identical ring form (and with the same number of substituted hydroxyl groups) increase when the groups with equatorial position are more numerous and/or when they are located nearer to C-1.” It was found that the effect caused by the number of equatorial substituents is generally far greater than that of their position relative to C-1. A supplementary equatorial substituent may have a very different action, depending on the conformation of the molecule, for example, as between a-galactose and a-glucose, this difference is minimal, as compared to that between @galactose and p-glucose. Ferrierl’O has also discussed the influence of configuration on retention time. It has been pointed out that, if a chromatographic system contains perfluoro compounds, as in valves, seals, or separators, trimethylsilyl derivatives may react with them to give spurious peaks. One such peak was identified as that from fluorotrimethylsilane.211 2. Acetates

The acetates of monosaccharides are sufficiently volatile that they may be used for gas-liquid chromatography, but they are less readily formed than the trimethylsilyl derivatives, and still present the problem of anomeric derivatives (see Section IV, p. 38). They are, therefore, rarely used, but the method is discussed in Section V.2 (see p. 49), and the known cases are presented in Table I1 (see p. 111). Despite the many advantages of converting free sugars into their O-trimethylsilyl derivatives, the formation of anomeric and isomeric derivatives (see Section IV, p. 38) may constitute a problem, especially with complex mixtures. Much effort has, therefore, been expended in seeking carbohydrate derivatives, suitable for analysis, in which the anomeric center has been eliminated. This may conveniently be accomplished by oxidation to the acid or lactone, by (208) E. F. Jansen and N. C. Baglan, /. Chromatogr., 38, 18 (1968). (209) T. Gheorghiu and K. Oette, 2. Naturforsch., B , 26, 24 (1971). (210) R. J. Ferrier, Tetrahedron, 18, 1149 (1962). (211) R. L. Foltz, M. B. Neher, and E. R. Hinnenkamp, Anal. Chem., 39, 1338 (1967).

34

G. G. S . DUTTON

reduction to the alditol, or by conversion into the nitrile via the oxime. The volatile derivatives of acids are discussed in Section IX (see p. 71). It so happens that the great majority of compounds separated as acetates are alditols or other polyhydric compounds, and this Section is therefore concerned with the problems of reduction and acetylation. There is no a priori reason why alditols and other polyols should not be separated as their trimethylsilyl ethers, and such methods are known (see Section VII,l,p. 57; Section XIII, p. 90; Table Va, p. 119; and Table XIIa, p. 151), but experience shows that the resolution of acyclic 0-trimethylsilyl derivatives is less satisfactory than that of cyclic compounds. Preparation of the alditol is normally achieved in aqueous solution by reduction with sodium or potassium borohydride. For unsubstituted glycoses, reduction is rapid,212,213 although, in certain cases, reduction was not complete in two hours.g1It is well known that the rate of reduction is significantly lower for substituted glycoses, espe~ i a l l ythose ~ ' ~ carrying groups at C-3. This observation is of particular importance when methylated glycoses are to be reduced (see Section XXI of Part 11). In the case of lactones, the reduction may need to be repeated, because of competing hydrolysis.215It has also been shown that, with low concentrations of borohydride, lactones are converted into aldoses and not into alditols.216 Polyhydroxy compounds are known to form borate complexes that can interfere with the subsequent acetylation step. In essence, two methods of acetylation are employed. In one, the conditions are sufficiently vigorous that borate complexes are cleaved during the acetylation, and, in the second, the complexes are decomposed prior to acetylation. In one example of the former method, the crude, dry alditols were refluxed for 4 hours with a mixture containing equal amounts of acetic anhydride and pyridine (- 1 ml/100 mg), and the cooled solution was injected directly into the gas chromat~graph.~~' The effect of borate on the rate of acetylation of glucitol by this (212) M. Abdel-Akher, J. K. Hamilton, and F. Smith,]. Amer. Chem. SOC., 73, 4691 (1951). (213) T. Imanari, Y. Arakawa, and Z. Tamura, Chem. Pharm. Bull. (Tokyo), 17, 1967 (1969). (214) P. D. Bragg and L. Hough,J. Chem. SOC., 4347 (1957). (215) E. SjostrBm, P. Haglund, and J. Janson, Acta Chem. Scand., 20, 1718 (1966). (216) J.-A. Hansson, N. Hartler, I. Szabo, and A. Teder, Suensk Papperstidn., 72, 78 (1969). (217) J. S. Sawardeker, J. H. Sloneker, and A. R. Jeanes, Anal. Chem., 37,1602 (1965).

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35

method was studied in connection with the analysis of wood-pulp hydrolyzates, and it was concluded that a minimum of 10 hours of refluxing was required.218Acetylation before and after the removal of borate complexes was examined, but it was considered that the most convenient way of dealing with a large number of samples was to reflux them overnight in a 1 : 1 mixture.z18However, acetylation of galactitol in this manner at 95" gave less than 40% conversion in 50 hours, and the authors claimed that prior removal of borate is essential.26Blake and Richardsz6critically examined many of the problems inherent in analysis by gas-liquid chromatography. Wilkie and coworkersz18aalso commented on the difficulty of completely acetylating glucitol and rhamnitol. Further, they showed that different, molar-response factors must be used in quantitative work, depending on whether acetylation is performed at 100" or 120". In other cases, the borate complexes were decomposed prior to acetylation, either by distillation with acidified methanol, or with methanol after removal of cations. In a study of the former method, it was concluded that, when the excess of borohydride is decomposed with acetic acid, five evaporations with methanol are required for optimal results.3s The methanol must be added to the residue obtained on concentrating the aqueous solution to dryness, as, otherwise, trimethyl borate will not be formed. It has been stated that removal of borate is more efficient when hydrofluoric acid (instead of hydrochloric acid) is used to neutralize the excess of b~rohydride."~ Alternatively, borate complexes have been decomposed by removing cations with an ion-exchange resin, followed by distillation with methanol. Acetylation may then be conducted at 100" for 20 minutes, or at room temperature overnights1 or for shorter periods.26It should, however, be noted that, when cations were removed and the product was acetylated without prior distillation with methanol, 4 hours of refluxing was still required, and irreproducible results were obtained when this time was lessened to 30 min to 1 The difficulty of acetylating in pyridine in the presence of borate was observed by 0ades;l who used an acid-catalyzed acetylation that was insensitive to large proportions of borate. Unfortunately, this method produced artifacts that had retention times similar to those of the alditol acetates, but they could be eliminated by passage through a short column of silica gel. This treatment also removed com(218) E. P. Crowell and B. B. Burnett, Anal. Chenz., 39, 121 (1967). (218a) A. J. Buchala, C. G. Fraser, and K. C. B. Wilkie, Plzytochemistry, 10, 1285 (1971). (219) E. Sjiistriim, P. Haglund, and J. Janson, Ssensk Papperstidn., 69, 381 (1966).

36

G. G. S. DUTTON

ponents responsible for “tailing” on the gas chromatograms. A method for the analysis of wood pulps used acid-catalyzed acetylation, and extraction of the acetates from water with dichloromethane.220It has been claimed that, under these conditions, acetylation is more rapid than in pyridine, and that pouring into water decomposes the excess of acetic anhydride, which may give a peak close to that of mannose. Acid acetylation has also been used for heptitols,”I and, in the case of inositols, the reaction has been catalyzed by Dowex-50 ion-exchange resin.222 Griggs and claimed that an excess of acetic anhydride causes serious “tailing,” especially with amino sugars, and they therefore recommended that, before injection, samples be dried overnight under high vacuum.

3. Trifluoroacetates Trifluoroacetates are more volatile than acetates, but have been used by relatively few investigators, either for the separation of sugars or of alditols. For the latter, borate complexes must be decomposed for the reasons already given. Trifluoroacetylation may be achieved either with trifluoroacetic anhydride in p ~ r i d i n e or , ~ with ~ ~ sodium trifluoroacetate in acetonitrile, usually with warming,224or by dissolving the substrate and sodium trifluoroacetate in N,N-dimethylformamide and adding trifluoroacetic anhydride, whereupon a vigorously exothermic reaction occurs.225The last method appears particularly advantageous for oligosaccharides. Tamura and ImanariZZ6reported that sugars treated with trifluoroacetic anhydride in acetonitrile showed several peaks, whereas reaction in tetrahydrofuran gave only one peak; they therefore preferred tetrahydrofuran as the reaction solvent, but noted that, once formed, the trifluoroacetates were more stable in acetonitrile than in tetrahydrofuran. In each of these s t ~ d i e s , ~crystalline ~ ~ - ~ ~ sugars ~ were treated, and it appears that (220) L. G. Borchardt and C. V. Piper, Tappi, 53,257 (1970). (221) H. Onishi and M. B. Perry, Can. J. Microbiol., 11, 929 (1965). (222) W. R. Sherman, N. C. Eilers, and S. L. Goodwin, Org. Mass Spectrotn., 3, 829 (1970). (223) D. Anderle and P. KoviiE,J. Chroniatogr., 49, 419 (1970). (224) M. Vilkas, Hiu-I-Jan, G. Boussac, and M.-C. Bonnard, Tetrahedron Lett., 1441 (1966). (225) T. Ueno, N. Kurihara, and M. Nakajima, Agr. B i d . Chem. (Tokyo), 31, 1189 (1967). (226) Z. Tamura and T. Imanari, Cheni. Pharm. Bull. (Tokyo), 15,246 (1967).

GAS-LIQUID CHROMATOGRAPHY

37

trifluoroacetyl derivatives have not been prepared from mutarotated mixtures. The recovery of trimethylsilyl and trifluoroacetjyl derivatives has been examined by use of I4C-labelled sugars, and it has been shown that glucose trifluoroacetate is recoveredzo8to the extent of 3 to 5% on SF-96, or of 5 to 14% on Carbowax 20 M. The separation of alditols as their trifluoroacetates has been reported by S h a ~ i r a ~ and ~ ’ by Japanese a ~ t h o r s .The ~ ~ latter ~ * ~ com~~ pared the reaction of trifluoroacetic anhydride in acetonitrile, tetrafound that ethyl acetate is the hydrofuran, or ethyl a ~ e t a t e . ”They ~ most satisfactory, as acetonitrile causes “tailing,” and tetrahydrofuran contains an impurity, not removed by distillation, which overlaps with fucitol per(trifluor0acetate). They found that, in ethyl acetate, mannitol reacts completely in 20 minutes, in contrast to the results of Shapira, who reported that hexitols require two hours at 35” when treated with trifluoroacetic anhydride and a trace of pyridine. The Japanese workers stated that the best separations were obtained on a column of 2% XF-1105, and that other columns, such as DC-1107, SE-30, or SE-52, caused “tailing” and broad peaks. 4. Isopropylidene Acetals There is one report of the use of isopropylidene acetals, for the analysis of monosaccharides in spent sulfite l i q ~ o r These . ~ ~ ~derivatives are not suitable for routine use, because of the time that must be taken in their preparation.

5. Methyl Ethers Methyl ethers were the first volatile derivatives of carbohydrates used for gas-liquid chromatography, and, although fully methylated compounds have been proposed for the analysis of sugar mixture^,^^^,^^^ the time taken for their preparation and the lack of good resolution in g.1.c. has mitigated against their general use. Methylated carbohydrates are most commonly encountered in structural investigations, and will be discussed in Part I1 of this article. (227) J . Shapira, Nature, 222, 792 (1969). (228) M. Matsui, M. Okada, T. Imanari, and Z. Tamura, Cheni. Pharin. Bull. (Tokyo), 16, 1383 (1968). (229) G. Anderson and L. A. Boggs, Abstr. Papers Ainer. Cheni. Soc. Meeting, 152, 183 (1966). (230) H. W. Kircher, Anal. Cheni., 32, 1103 (1960). (231) Yu. S. Ovodov and E. V. Evtushenko,]. Chroincttogr., 31, 527 (1967).

38

G . G . S. DUTTON

6. Butaneboronates Two preliminary communications described the use of butaneboronic acid as a reagent for converting carbohydrates into volatile derivatives. E i ~ e n b e r g ”showed ~ that sugars, alditols, and cyclitols may be separated on a column of 3% of OV-17 after conversion into butaneboronates. (Commerical samples of the reagent butaneboronic acid may be contaminated with isobutaneboronic acid, thus giving rise to isomeric derivatives; however, the purity of the reagent may be checked by gas-liquid chromatography.) These derivatives are readily volatile, and separation of the glucitol, galactitol, and manitol butaneboronates may be accomplished in 6 minutes at 200”. The parent carbohydrate is then regenerated by hydrolysis with aqueous acid. Wood and S i d d i q ~similarly i ~ ~ ~ prepared butaneboronates of sugars, and these were then trimethylsilylated in the usual way.

IV. MUTAROTATION When aldopentoses and higher glycoses are converted into their trimethylsilyl, or other, derivatives, the possibility exists that at least four isomers will be produced, namely, the a and p anomers of the furanosides and pyranosides. In the reaction of glucose with trifluoroacetic anhydride, as many as six peaks have been observed.2273-Deoxy-D-erythro-hexosulose gave 11 peaks on t r i m e t h y l ~ i l y l a t i o n , ~ ~ ~ and the products formed from fructose on trimethylsilylation have been studied in detai1.’19~1zo In the analysis of many monosaccharide mixtures, not all peaks of all glycoses are completely separated, and it may be necessary to make certain corrections to peak areas where two (or more) components overlap. The proportions of the four isomers mentioned that are given by any one monosaccharide will depend on the conditions under which the derivatives are farmed. Sweeley and coworkers5 showed that, when crystalline monosaccharides are trimethylsilylated in pyridine, the rate of reaction is very much greater than the rate of mutarotation, with the result that essentially only one derivative is formed and thus only one peak shows on the chromatogram. On the other hand, monosaccharides such as are obtained in polysaccharide hydrolyzates normally exist as mutarotated,

(232) F. Eisenberg, Carbohyd. Res., 19, 135 (1971); 21, 500 (1972). (233) P. J. Wood and I. R. Siddiqui, Carbohyd. Res., 19, 283 (1971). (234) A. A. El-Dash and J. E. Hodge, Carbohyd. Res., 18, 259 (1971).

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39

equilibrium mixtures. For quantitative results, it is imperative that experimental methods be standarized and that the ratio of the isomers formed by any monosaccharide be known precisely. Typical results, and details of the calculations necessary, are to be found in an excellent paper by Bethge and coworker^."^ With respect to the quantitative analysis of monosaccharide mixtures, it is important to appreciate that the equilibrium proportion of each isomer is dependent on the solvent. The influence of the solvent on pyranose-furanose equilibria was studied by Kuhn and G r a s ~ n e r ~ ~ ~ and, later, by Angyal and and by Mackie and Perlin?39 The mutarotational changes of carbohydrates in mixtures of p-dioxane and water have been shown to vary in a complex manner with the solvent ~omposition,2~~ and the effect of ~ y r i d i n eon ~ ~the * equilibria has been studied. In aqueous solution, the equilibrium is dependent on the concentration of the and the equilibrium values in water and after heating in 3 M hydrochloric acid have been compared.34In addition, the equilibrium values determined by optical rotation, oxidation, and gas-liquid chromatography were tabulated.34The catalytic effect of various metal ions on the rate of mutarotation has been the mutarotation of tetra-0-methyl-D-glucose has been examined,244-247 and mutarotation of sugars has been reviewed by Pigman and I ~ b e 1 1 . ~ ~ ~ The preparation of D-threo-2-pentulose (“~-xylulose”)by refluxing D-xylose in pyridine containing 1%of and the transformation of D-arabinose into a mixture of the four D-aldopentoses and two

(235) R. Kuhn and H. Grassner, Ann., 610, 122 (1957). (236) S. J. Angyal, V. A. Pickles, and R. Ahluwalia, Carbohyd. Res., 3, 300 (1967). (237) S. J. Angyal and V. A. Pickles, Carbohyd. Res., 4, 269 (1967). (238) S. J. Angyal and K. Dawes, Aust. J . Chem., 21,2737 (1968). (239) W. Mackie and A. S. Perlin, Can. J. Chem., 44,2039 (1966). (240) A. J. Hannaford, Carbohyd. Res., 3, 295 (1967). (241) A. de Grandchamp-Chaudun, Compt. Rend., C , 262, 1441 (1966). (242) N. A. Khalturi, U. V. Moiseev, V. S. Marevtse, G. A. Kogan, andG. E. Zaikor, Izu. Akad, Nauk S S S R , Ser. Khim., 1785 (1970). (243) R. Mitzner and E. Behrenwald, Z. Chem., 11,64 (1971). (244) A. Kergomard and M. Renard, Tetrahedron Lett., 769 (1968). (245) P. R. Rony,]. Amer. Chem. Soc., 90, 2824 (1968). (246) P. R. Rony, W. E. McCormack, and S. W. Wunderly,J. Amer. Chem. SOC., 91,4244 (1969). (247) H. H. Huang, A. N. H. Yeo, and L. H. L. Chia,J. Chem. Soc. ( B ) , 836 (1969). (248) W. Pigman and H. S. Isbell, Aduan. Carbohyd. Chem., 2 3 , l l (1968);H. S. Isbell and W. Pigman, Aduan. Carbohyd. Chem. Biochem., 24, 13 (1969). (249) B. Lindberg and K. N. Slessor, Carbohyd. Res., 5, 286 (1967).

40

G . G . S. DUTTON

~ - 2 - p e n t u l o s e s ” ~ *may ’ ” ~ ~be cited as extreme examples of complex reactions occurring in this solvent. Such transformations may be of synthetic v a l ~ e , but, ~ ~ in ~ -analytical ~ ~ ~ work, the possibility of their unwanted occurrence should not be overlooked. In addition, unexpected epimerization may take place during the neutralization of acid hydrolyzatess4 or during the reduction of sugars to a l d i t o l ~ L-Idu.~~ ronic acid has been detected in the products from a phytoglycolipid and shown to arise by epimerization of D-glucuronic In analyzing polysaccharide hydrolyzates, some workers have assumed that, because mutarotation in pyridine is slow and the trimethylsilylation reaction is fast, no change in the composition occurs during trimethylsilylation. Others have equilibrated the monosacIn nonaqueous charides in pyridine, before trirnethylsilylati~n.~~~~~~~ solvents, the mutarotation may be catalyzed by lithium p e r c h l ~ r a t e ’ ~ ~ which ~ ~ ’ ” is ~ ~volatile, but does or, more conveniently, by 2 - p y r i d i n 0 1 , ~ ~ not interfere with subsequent analyses. ~ eto identify d ~ “y-D-galaC~ ~ , ~ Gas-liquid chromatography was ~ tose,” to study the composition of an equilibrium solution in pyridine,2soand to examine the isomerization of D-galacturonic acid in aqueous solution.”I The technique has also been used to study the , ~D-glucose ~~ and D-mannose,262of D-glumutarotation of D - f r u c t o ~ eof cose, D-galactose, L-arabinose, and D-xylose, and to determine muta’ ~ study ~ the mutarotation coefficients for the assay of m u t a r o t a ~ e ; to rotation of L-arabinose, D-galaCtOSe, and 2-deoxy-~-erythro-pentose;‘~~ to determine the anomeric form of D-glucose produced during enzy-

M. Fedoronko and K. Linek, Collect. Czech. Cheiir. Cominun., 32, 2177 (1967). R. S. Tipson and R. F. Brady, Jr., Corbohyd. Res., 10, 549 (1969). D. Rutherford and N. K. Richtmyer, C ~ o b o h y dRes., . 11,341 (1969). M. J. How, M. D. A. Halford, and M. Stacey, Carbohyd. Res., 11, 313 (1969). H. E. Carter, D. R. Strobach, and J. N. Hawthorne, Biochemistry, 8, 383 (1969). M. L. Laver, D. F. Root, F. Shafizadeh, and J. C. Lowe, Tappi, 50, 618 (1967). P. E. Reid, B. Donaldson, D. W. Secret, and B. Bradford,]. Chroincitogr., 47, 199 (1970). C. G . Swain and J. F. Brown,]. Amer. Chem. Snc., 74, 2538 (1952). R. S. Shallenherger and T. E. Acree, Corbohyd. Res., 1, 495 (1966). T. E. Acree, H. S. Shallenlmger, Y.-C. Lee, and J. W. Einset, C w h h y d . Res., 10, 355 (1969). T. E. Acree, R. S. Shallenherger, and L. R. Mattick, Carhohyd. Res., 6,498 (1968). B. Carlsson and 0. Samuelson, Corbohyd. Res., 11, 347 (1969). Y.-C. Lee, T. E. Acree, and R. S. Shallenberger, CarholzytE. Res., 9, 356 (1969). A. H. Connor and L. Anderson, Abstr. Papers Aiiier. Clzetti. S O C . Meeting, 157, 16 CARR (1969).

~

GAS-LIQUID CHROMATOGRAPHY

41

molysis,"6" and in similar studies on other carbohydrases;z65to follow the anomerization of D-g1ucosez66and the autocatalytic mutarotation of D-glucose in p ~ r i d i n e ; and ' ~ ~ to study thin-layer chromatography of carbohydrates at low temperature.268The mechanism of action, on D-galactose, of a mutarotase isolated from Escherichia coli has been studied in a similar way,260aand Salfner70ahas examined the equilibration of sugars in connection with glycopeptide analyses.

V. NEUTRALMONOSACCHARIDES 1. Trimethylsilyl Derivatives

The original publication by Sweeley and coworkers5 was concerned with the separation of a wide range of carbohydrates, from mono- to tetra-saccharides. Most of the subsequent publications have considered the quantitative analysis of mixtures of varied complexity, although two studies have demonstrated the separation of the protium The study of from the deuterium forms of monosaccharides.z00~201 mutarotational equilibria by gas-liquid chromatography has been discussed in Section IV (see p. 38). Sawardeker and S l ~ n e k e r 'were ~ ~ among the first investigators to apply, quantitatively, the method of Sweeley and coworker^.^ They pointed out that nonpolar, silicone phases give symmetrical peaks of 0-trimethylsilyl derivatives but incomplete resolution, whereas a polar phase, such as ethylene glycol succinate, gives better resolution, but is handicapped by adsorption effects. They found that Carbowax 20 M gave excellent quantitative results, with practically no adsorption. The column was found particularly suited to the separation of D-glucose from D-galactose and D-mannose, the anomers of all of which were well resolved, but pentoses and deoxyhexoses were incompletely resolved. This separation was further studied by Cheminat and Brini,"O who were able to resolve L-arabinose and L-rhamnose partly, as well as L-fucose and other aldoses and ketoses. (264) F. W. Parrish and E. T. Reese, C ( i d ~ ~ h yRes., d . 3, 424 (1967). (265) G. Semenza, H.-C. Curtius, 0.Haunhardt, P. Hore, and M . Miiller, Corhoh!/r/ Res., 10, 417 (1969). (266) H. Jacin, J. M . Slanski, and R . J. Moshy,]. Cliromcitogr., 37, 103 (1968). (267) A. S. Hill and R. S. Shallenl>erger, Curhohyd. Res., 11,541 (1969). (268) G. Avigad and S. Bauer, Corbohyd.Res., 5, 417 (1967). (268a) F. Hucho and K. Wallenfels, Eur. J. Biocheni., 23, 489 (1971). (269) J. S. Sawardeker and J. H. Sloneker, Anal. Chem., 37,945 (1965). (270) A. Cheminat and M . Brini, Bull. Soc. Chitn. Fr., 80 (1966).

42

G. G. S. DUTTON

An area of polysaccharide chemistry in which the quantitative estimation of monosaccharides by paper chromatography has proved tedious is the field of hemicelluloses. Rapid methods of quantitative analysis are of particular importance here, because of the need to determine accurately the composition of pulp and other wood products. It is, therefore, of historical interest that much of the present information on the separation and quantitation of monosaccharides arose from such studies. One of these, by Brower and coworkers,271described the analysis of pulp hydrolyzates by use of a column of 15% glycol succinate, with rnyo-inositol as the internal standard. Good results were obtained in about 40 minutes, but it was noted that the proportion of galactose anomers was different from that obtained by Sweeley and coworker^,^ thus emphasizing the necessity for each worker to standardize his own conditions. A similar, but more detailed, study was made by Bethge and coworker^,'^^ in which the wood hydrolyzates were equilibrated in pyridine in the presence of lithium perchlorate as a catalyst, before trimethylsilylation. These authors gave figures for the equilibrium composition of each monosaccharide studied, and clearly explained the calculations necessary for the estimation of the proportion of each component when certain peaks overlap. Several liquid phases were examined and found suitable. In order to avoid “tailing,” pyridine was evaporated off after the trimethylsilylation, and injections were made of a solution in a solvent such as hexane. The columns were used isothermally or on a program of slow heating. Similar results were later published in connection with the determination of aldoses and alditols (see Section XIII, p. 90, and Ref. 608). The method of Bethge and coworkers’7ehas been used in many studies, including those on the alkaline degradation of wood p o l y s a ~ c h a r i d e s . ~Shafizadeh ~ ~ - ~ ~ ~ and published details of a very similar separation that used a column of 5 % SE-30, programmed from 100 to 180” at l”/min; this system has also been used for studies on and It should be noted that the per(trimethylsily1) derivative of 1,6anhydro-p-D-glucopyranose has the same retention time as that of a-D-xylopyranose when they are chromatographed on a column of 3%

(271) H. E. Brower, J. E. Jeffery, and M . W. Folsom, Anal. Cheni., 38, 362 (1966). (272) J.-A. Hansson and N. Hartler, Holzforschung, 24,54 (1970). (273) J.-A. Hansson and N. Hartler, Soensk Papperstidn., 71, 358 (1968). (274) J.-A. Hansson and N . Hartler, Soensk Papperstidn., 71, 669 (1968). (275) F. Shafizadeh and W. Bukwa, Phytochemistry, 9, 871 (1970). (276) F. Shafizadeh and G. D. McGinnis, Carbohyd. Res., 16,273 (1971).

GAS-LIQUID CHROMATOGRAPHY

43

SE-52 at 160". Similarly, 1,6-anhydro-p-~-glucohranosecorresponds to p-D-xylopyranose. These anhydro compounds must be formed in the acid-hydrolysis stage, and they give incorrect values for D-xylose unless their presence is detected;78 this may be done by operating the column at 135". Although many analyses are performed on alditol acetates (see Section VII, p. 56), in order to avoid the formation of multiple peaks, such a reduction is not practical when the mixture contains ketoses, notably fructose. Such analyses are mainly encountered with medical samples and in the examination of sugars occurring free in Nature. Furthermore, the peak-area ratios may be used as a means of identification, to check on the completeness of t r i m e t h y l s i l y l a t i ~ n , ~ ~ ~ ~ ~ and, despite the complex chromatograms obtained from trimethylsilyl derivatives, they have the merit of being rapidly formed.89 For all of these reasons, improvements in the separation of monosaccharides as their trimethylsilyl derivatives continue to be of considerable importance. An article that discusses the advantages of separating the trimethylsilyl ethers of methyl glycosides (see Section VI, p. 51) also gives a very comprehensive list of retention times for the trimethylsilyl ethers of the free sugars.67A model study concerned with the determination of fructose in the presence of several aldoses recommended equilibration of the mixture in the presence of 2-pyridinol as the catalyst, as it is volatile and does not interfere with the subsequent analysis.256A similar investigation, related to the determination of sugars in potatoes, examined several column packings, of which OV-1, OV-17, and a three-component liquid phase were sati~factory.~~' Model studies related to foods,278k e r a t o s ~ l f a t e sand , ~ ~nectar ~ of flowers174have been published. Another area in which the determination of neutral monosaccharides by gas-liquid chromatography has proved useful is in the analysis of glycosaminoglycans. One typical, detailed study concerned seven different column-packings, and it was concluded that 12-20% of IA-butanediol succinate or of ethylene glycol succinate gave the best results.34 Good agreement was obtained between gas-liquid chromatographic and photometric procedures in the analysis of representative glycoproteins. This method has subsequently been used (277) R. Shaw, Atner. Potato ]., 46, 201 (1969). (278) E. Cerma, B. Staucher, and M. Chimenti, Uniu. Studi Trieste, Fac. E m t l . Commer. Zst. Merceol., No. 40, 15 (1968); Chem. Abstr., 72, 131,159 (1970). (279) C. Balduini and A. Brovelli, Ztal.]. Biochem., 17,257 (1968).

44

G. G. S. DUTTON

in several medical i n v e s t i g a t i o n ~ . 2 Another ~ ~ - ~ ~ ~study on the analysis of the neutral monosaccharides in glycoproteins is that of Kisters and Greiling,59and identification, by gas-liquid chromatography, of the sugars in urine was made by Butts and J01ley.I~~ The analysis of saliva and gastric secretions which gave, after hydrolysis, D-glucose, D-galactose, D-mannOSe, and L-fucose has been reported.la4Although not all the peaks were completely resolved, all of the sugars could be satisfactorily determined. Because of the short retention time of the L-fucose derivatives, it was found desirable to remove most of the pyridine prior to injection, and to use a solid injection Alternatively, pyridine may be replaced by a more volatile solvent.176The percentage of liquid phase on the column was found to be critical. Too little resulted in poor separation of the a - ~ glucose and a-D-galactose peaks, whereas too much brought the a-L-fucose and a-D-mannose peaks too close together. The methods described in this paperza4were subsequently extended to permit the simultaneous determination of arninoglyc~ses.~~ D-Galactose, D-mannose, and L-fucose have likewise been determined in the hydrolyzates of glycoproteins,60,286 and also in fungal polysa~charides?~~ Analysis of glycosaminoglycan hydrolyzates is often complicated by the presence of basic and acidic glycoses, in addition to neutral monosaccharides. Adsorption chromatography on silica gel was used to classify such a hydrolyzate, and the neutral fraction was shown to contain D-glucose, D-galactose, and D-mannose.288Separation on ionexchange resins may also be ~ s e d . ~ lThe * ’ ~resolution ~ of the acidic and basic fractions is discussed in Sections IX (p. 71) and X (p. 78). A detailed study has been made by Ellis 289 of the factors influencing the separation of 0-trimethylsilyl derivatives. The resolution of cer(280) T. Gheorghiu, K. Oette, H. Frotz, R. Phlippen, H. J. Klein, and M. Winterfeld, Verh. Deut. Ges. Inn. Med., 75, 619 (1969). (281) T. Gheorghiu, H. Frotz, and H. J. Klein, Verh. Deut. Ces. Inn. Med., 77, 511 (1971). (282) T. Gheorghiu, H. Frotz, H. J. Klein, and R. Phlippen, Deut. Ges. VerdauungsStoffwechselkrankh., Proc. 25th Meeting, Homburg (Sam),Sept. 1969, R. Ammon and U. Ritter, eds., Georg Thiem, Stuttgart, 1971, p. 95. (283) T. Gehroghiu, H. J. Klein, H. Frotz, and G. Hubndr, in “Peptic Ulcer,” Carl J. Pfeiffer, ed., Munksgaard, Copenhagen, 1971. (284) M. D. G. Oates and J. Schrager, Biochem. J . , 97,697 (1965). (285) E. Bailey, J . Endocrinol., 28, 131 (1964). (286) 0. P. Bahl, Fed. Proc., 25, 741 (1966). (287) R. N. Fraser and B. Lindberg, Carbohyd. Res., 4,12 (1967). (288) J. E. a r k b i n e n , E. 0. Haahti, and A. A. Lehtonen, Anal. Chem.,38,1316 (1966). (289) W. C. Ellis, J . Chromatogr., 41, 335 (1969).

GAS-LIQUID CHROMATOGRAPHY

45

tain pairs, such as D-xylose and D-mannose, that are separated with difficulty was related to the concentration of the liquid phase, whereas the separation of D-glucose from D-galactose was markedly influenced by the solid support. In certain examples, the order of elution was changed by an alteration in the concentration of the liquid phase. The most suitable column was found to be one consisting of XE-60. A total of 15 liquid phases, several at different concentrations, was examined. The data were largely presented in graphical form, and the original paper289should be consulted for details. In another model study, the use of tetra(cyanoethy1)ated pentaerythritol (TCEPE) as an efficient, polar, liquid phase was recommended.290It had earlier been shown that, as a polar phase, Carbowax 20 M is superior to ethylene glycol s ~ c c i n a t eand , ~ ~it~was suggested290 that TCEPE is an improvement on both of these. With TCEPE, there 'was less base-line drift, and such pairs as erythrose and erythritol, or L-arabinose and L-rhamnose, which were not resolved, or only poorly resolved, on Carbowax 20 M were separated. It is often wise to investigate the resolution of any particular mixture on both a polar and a nonpolar column, and, therefore, this studyzg0also included an examination of the dimethylsilicone gum OV-1 as a nonpolar phase; this material is comparable to SE-30 or SE-52, but gives a more stable base-line and can be operated at a higher temperature. It was pointed out that, on OV-1 and Carbowax 20 M, glucose has a higher retentiontime than D-galactose, whereas the reverse is true on TCEPE. This characteristic of the TCEPE column is very useful in analyzing biological materials, such as plasma, which contain both of these sugars. A similar transposition was observed with 2-deoxy-~-erythro-pentose and erythritol. The routine use of TCEPE and OV-1 for the analysis of 0-trimethylsilyl derivatives was, therefore, highly recommended by these authors.290 A wide range of sugars was investigated in connection with the analysis of alfalfa, and the routine use of both nonpolar and polar columns, for example, OV-1 and OV-225, was r e c ~ m m e n d e d . ~ ~ ' It is standard practice in gas-liquid chromatography to compare the retention time of each component to that of an arbitrary standard. The actual retention-time for any compound may differ greatly from column to column. Many authors, unfortunately, only report relative retention-times, and give no indication of the absolute time involved. Admittedly, this retention time will differ slightly according to the (290) D. Farshtchi and C. W. Moss, J. Chrornatogr., 42, 108 (1969). (291) R. A. Harnlen, F. L. Lukezic, and J. R. Bloom, Can. J. Bot., 48, 1131 (1970).

46

G. G . S. DUTTON

operating conditions, but some indication of the absolute time involved should alwuys be given. This information is particularly important when new column-packings are tested, or relative efficiencies of different columns are compared. In this connection, it has been pointed out that relative retention-times may change according to the column temperature, and that p-D-glucose is particularly sensitive in this regard.34 The determination of D-glucose and D-galactose in blood plasma by g.1.c. had previously been studied by C ~ p e n h a v e r by ' ~ ~use of a column of Carbowax 20 M. Recoveries of D-galactose ranged between 96 and 101% for samples containing more than 125 pglml. Below this value, recoveries were greater than 100%. Recoveries of D-glucose were similar (95-99%), and comparison of the g.1.c. results with those from a ferricyanide, colorimetric method gave differences of -7.7 to +5.7%. In a similar analysis of D - g h C O S e in serum,138recoveries averaging 97.1% were obtained, and the g.1.c. results were compared with those obtained by use of glucose oxidase or the Nelson-Somogyi method. This i n ~ e s t i g a t i o n also ' ~ ~ estimated D-glucose, D-mannitol, D-fructose, and rnzjo-inositol in urine. D-Glucose has been estimated in blood by using L-rhamnose as a standard.292 The difficulty of separating L-arabinose from L-rhamnose has already been noted, and, in a model study directed towards the analysis of steroidal saponins, it was found that a column of (cyanoethy1)silicone (XF-1105) was particularly suitable for resolving L-arabinose, D-XylOSe, and ~ - r h a m n o s e . ~ ~ The analysis of neutral monosaccharides as their O-trimethylsilyl derivatives has been applied under widely differing circumstances and to mixtures of various degrees of complexity, as shown in Table I (see p. 102). A brief summary of some of the applications includes the analysis of corn syrup for glucose by using D-glucitol as an internal standard,293or for D-glucose and maltose oligo~accharides'~~ in corn syrups and in beer and ~ ~ r For t . this ~ type ~ of ~ determination, , ~ ~ ~ the use of such inert standards as terphenyl has been prop0sed,2~~ and this compound has been used in the analysis of 1,6-anhydro-p-~glucose.'32 The kinetics of hydrolysis of aldobiouronic acids have been studied by measuring the D-XylOSe or D-glucose liberated, using as standards D-glucose or 3-O-methyl-D-glUCOSe,respectively.295Mea(292) J. de Neef, Clin. Chim. Actu, 26,485 (1969). (293) R. J. Alexander and J. T. Garhutt, A n d . Chem., 37, 303 (1965). (294) Y. Halpern, Y. Houminer, and S. Patai,Anulyst (London), 92, 714 (1967). (295) N. Roy and T. E. Timell, Carbohyd. Res., 7, 17 (1968).

GAS-LIQUID CHROMATOGRAPHY

47

suring the ratio of D-glucose to 4-O-methyl-~-glucosehas permitted the degree of polymerization (d.p.) of a series of 4-0-methyl-maltooligosaccharides to be determined.296D-Glucose has been determined in blood in the presence of D-glucitol and xylitol, by using dimethyl phthalate as the internal standard, and the accuracy compared favorably with that of enzymic method^.'^' Analysis of starch in methyl ~~~ sulfoxide has been checked by gas-liquid c h r o m a t ~ g r a p h y ,and D-glucose from ethyl a-D-ghcoside in sake has been similarly determined.299Gas-liquid chromatography has been used to establish the identity of D-glucose obtained from 2-amino-2-deoxy-~-mannuronic acid in studies on Micrococcus lysodeikticus,30° and of L-rhamnose from M y c ~ b a c t e r i a . ~ ~ ~ Representative substances that have been analyzed during polysaccharide structural studies include chick allantoic antigen,302loren~ ~ algae,304,305 bacterial zini jelly,303 Diplococcus p n e u r n o n i ~ egreen g l y ~ o l i p i d s ,metabolites ~ ~ ~ , ~ ~ ~ of Verticillium dahliae,308Nosema apis spore^^^^ a succinoglucan,310and galactomannan~.~~' The action of nucleoside 5'-(glycosyl pyrophosphate) epirnerases,3l2and the metabolism of sugars by bacteria159have been monitored by using O-trimethylsilyl derivatives. The identity and concentration of monosaccharides occurring free in Nature in plants, syrups, and fruits have been examined by using 0-trimethylsilyl derivatives. Many of these analyses were made on (296) J. N. BeMiller and R. E. Wing, Carhohyd. Res., 6 , 197 (1968). (297) F. D. Gauchel, G . Wagner, and K. H. Bassler, Z . Klin. Chem. Klin. Biochem., 9,25 (1971). (298) R. A. Libby, Cereal Chem., 47, 273 (1970). (299) T. Imanari and Z. Tamura, Agr. B i d . Chem. (Tokyo), 35, 321 (1971). (300) S. Hase and Y . Matsushima,J. Biochem. (Tokyo), 69,559 (1971). (301) C. VillC. and M. Gastambide-Odier, Carbohyd. Res., 12,97 (1970). (302) M. J. How and J. D. Higginbotham, Carbohyd. Res., 12,355 (1970). (303) M. J. How, J. V. S. Jones, and M. Stacey, Carhohyd. Res., 12, 171 (1970). (304) E. Percival and M. Young, Phytochemistry, 10,807 (1971). (305) E. J. Bourne, P. G . Johnson, and E. Perciva1,J. Chem. SOC. (C), 1561 (1970). (306) K. Welsh, N. Shaw, and J. Baddiley, Biochem. J., 107,313 (1968). (307) B. A. Key, G. W. Gray, and S . G. Wilkinson, Biochem. J., 120, 559 (1970). (308) Y. M . Choy and A. M. Unrau, Can. J. Biochem., 49, 894 (1971). (309) P. J. Wood, I. R. Siddiqui, J. W. Vandermeer, and T. A. Gochnauer, Curbohyd. Res., 15,154 (1970). (310) A. Misaki, H. Saito, I. Ito, and T. Harada, Biochemistry, 8, 4645 (1969). (310a) Yu. S. Ovodov, R. P. Gorshkova, and S . V. Tomshich, Zmmunochentistry, 8, 1071 (1971). (311) A. M. Unrau and Y. M. Choy, Can. J. Chem.,48,1123 (1970). (312) D. F. Fan and D. S. Feingold, Plant Physiol., 46, 592 (1970).

48

G. G. S. DUTTON

mixtures of D-glucose and D-fructose in honey,313-315wort?16 corn syrup,122 sugar-cane juice,lZ1prune juice,317fruits,318fruit tissue,319and beet molasses.320Extracts from various plant materials, such as ferns,321 onions,322 holly,323 fruit-tree cotton leaves,l17 potatoes,116 sweet potatoes,lZ3and almond have similarly been examined. D-Glucose and D-galaCtOSe have been determined in and, together with D-fructose, in serum and ~ r i n e , " ~ * in' ~ ~ mammalian nerve in addition to D-glucitol and myo-inositol, and in the roots of Rehmanniu g l ~ t i n o s a . ~ ~ ~ * ~ ~ ~ ~ Fucose from glycoproteins,86~'71~176.286.328.329 glycosaminoglycans,36.2s4. 287*330,331 urine,l18 and a range of m 0 1 d ~ and ~ ~ blood-group ~ , ~ ~ ~ substance~ has ~~ been ~ estimated. Mixtures containing many components have been analyzed in con-

(313) Y. Masada, K. Hashimoto, T. Inoue, and T. Sawada, Yakugaku Zasshi, 89, 734 (1969);Chem. Abstr., 71,69,390 (1969). (314) J. Pourtallier, Z . Bienenforsch., 9, 217 (1968); see also, I. R. Siddiqui, Aduan. Carbohyd. Chem. Biochem., 25,285 (1970). (315) T. Echigo, Tamagawa Daigaku Nogakubu Kenkyu Hokoku, 115 (1970);Chem. Abstr., 74, 21,986 (1971). (316) B. Tuning, Intern. Tijdschr. Brouw. Mout., 30, 117 (1971); Chem. Abstr., 75, 108,476 (1971). (317) C. Flynn and A. W. Wendt,]. Ass. Oflie. Anal. Chem., 53, 1067 (1970). (318) D. A. Kline, E. F. Flores, and A. R. Johnson,]. Ass. 0ffic.Anal. Chem., 53, 1198 (1970). (319) M. Tavakoli and R. C. Wiley, Proc. Amer. SOC. Hort. Sci., 92, 772, 780 (1968). (320) H. G. Walker, Intern. Sugar]., 67,237 (1965). (321) C. J. Ludlow, T. M. Harris, and F. T. Wolf, Phytochemistry, 5, 251 (1966). (322) M. de Minia, Compt. Rend. V , 270, 1583 (1970). (323) T. A. Fretz, C. W. Dunham, and C. W. Woodmansee,]. Amer. Soc. Hort. Sci., 95, 99 (1970). (324) M. W. Williams and G. C. Martin, Hort. Sci., 2, 68 (1967). (325) K. L. Mikolajczak, C. R. Smith, and I. A. Wolff, J . Agr. Food Chem., 18, 27 (1970). (326) G. A. Reineccius, T. E. Kavanagh, and P. G. Keeney, J. Dairy Sci., 53, 1018 (1970). (327) M. Tomoda, S. Katii, and M. Unuma, Chem. Pharm. Bull. (Tokyo), 19,1455(1971). (327a) M. Tomoda, M. Tanaka, and N. Kondo, Chem. Pharm. Bull. (Tokyo), 19, 2411 (1971). 106, , 523 (1968). (328) J. Schrager and M. D. G. Oates, Biochem. I. (329) M. I. Horowitz and M. R. Delman,]. Chromatogr., 21, 300 (1966). (330) A. Lehtonen, J. Kiirkkainen, and E. Haahti, Acta Chem. Scand., 20, 1456 (1966). (331) J. A. Cifonelli, A. Saunders, and J. I. Gross, Carbohyd. Res., 3, 478 (1967). (332) R. N. Fraser, S. Karhcsonyi, and B. Lindberg, Acta Chem. Scand., 21,1783 (1967). (333) H. 0. Bouveng, R. N. Fraser, and B. Lindberg, Carbohyd. Res., 4, 20 (1967). (334) S. Hakomori and G. D. Strycharz, Biochemistry, 7, 1279 (1968).

GAS-LIQUID CHROMATOGRAPHY

49

nection with an examination of the free sugars in natural glycosides,B5and fungal polysa~charides,3~~ as well as in relation to the model studies already mentioned. In an investigation of the action of chlorine dioxide on cellulose oligosaccharides, the uncommon sugars D-allOSe and D-altrose were separated, together with D-glucose and D - m a n n ~ s e . ~ ~ ~ Heptoses and higher sugars may similarly be separated, and the identity of D-erythro-L-galacto-nonulose in avocado was determined by comparison of its gas-chromatographic behavior with that of a synthetic sample.33sHigher sugars have been separated, and identified, from avocado,151P i ~ h i , 3opium ~~ p0ppy,3~~ and bacterial lipopolysaccharides .339 Mason and S10verl~~ studied the separation of monosaccharides as the 0-trimethylsilyl derivatives of the oximes, as an alternative method to analyzing for sugars in foods, and Clayton and H.G. J o n e P used trimethylsilylated D-glucose oxime. Both groups of workers referred to the instability of these derivatives, as noted in Section I11 (see p. 23). Laine and Sweeley189proposed a similar method that used the methoxime derivatives. The separation of disaccharide and higher oligomers is discussed in Section VIII (see p. 67). 2. Acetates

Like trimethylsilylation, acetylation may give more than one anomer and this possibility, coupled with the fact that acetates are, in general, of low volatility, has mitigated against their general use. Separation as acetates may sometimes be a useful alternative to other methods, as in the examination of starch pr0ducts,3~~ and the multiplicity of peaks may be used as a means of identification, as in a study of the occurrence of ~ - r h a m n o s e in ~ ~al glycan produced by Myxobacterium 402. In a similar way, the purity of a glucan from Pullularia pullulans was verified,342and, in a study of Xanthomonas campestris,

(335) E. S. Becker, J. K. Hamilton, and W. E. Luck, Tappi, 48,60 (1965). (336) H. H. Sephton and N. K. Richtmyer, Carbohyd. Res., 2,289 (1966). (337) N. K. Richtmyer, Carbohyd. Res., 12,233 (1970). (338) G. Haustveit and J. K. Wold, Acta Chem. Scand., 24,3059 (1970). (339) G. A. Adams, C. Quadling, and M. B. Perry, Can. J . Microbiol., 13, 1605 (1967). (340) H. U.Geyer, Staerke, 17,307 (1965). (341) I. M. Morrison, R. Young, M. B. Perry, andG. A. Adams, Can./. Chem., 45,1987 (1967). (342) W. Sowa, A. C. Blackwood, and G . A. Adams, Can. /. Chem., 41, 2314 (1963).

50

G . G . S. DUTTON

the homogeneity of an anhydro-octulose was checked as its peracetate.343 The separation of monosaccharides as their peracetates has usually been restricted to those instances where they have been accompanied by other compounds best separated in this form. Examples include the identification of hexose in the presence of mannitol in a glucoamylase from Aspergillus n i g e ~ , "the ~ ~separation of D-glucose from erythritol in a study on oat g l ~ c a n , and 3 ~ ~the separation of D-mannose and 4-deoxy-~-erythritol from the rhamnomannan of Ceratocystis ~ l r n iAcetates . ~ ~ ~ of monosaccharides have also been used in studies on acetoxonium-ion rearrangement^^^',^^^ the equilibria between aldohexoses and their l , g - a n h y d r i d e ~the , ~ ~synthesis of methyl a-malt ~ s i d e the ,~~ reactions ~ of glycosyl and investigations on ring structure^.^^' In work on enzymic deacetylation, a series of D-glucose derivatives acetylated at specific positions was prepared, and the 0-trimethylsilyl derivatives of these D-glucose acetates were separated by gas-liquid ~ h r o m a t o g r a p h y . ~Research ~ ~ * ~ " ~ on bacterial glycolipids required the characterization of 3- and 6-O-palmitoyl-~-glucose,the separation of which, as their 0-trimethylsilyl and acetyl derivatives, was studied.354 Monosaccharides separated as their acetates are given in Table I1 (see p. 111). The acylglucose compounds separated as their O-trimethylsilyl derivatives are included in Table I, j (see p. 110). 3. Trifluoroacetates

Replacement of an 0-acetyl group by an 0-trifluoroacetyl group greatly increases the volatility of the sugar derivative, and this behavior was first utilized by Vilkas and associates,224who studied the separation of pentoses, hexoses, aminoglycoses, methyl glycosides, (343) P. A. J. Gorin, T. Ishikawa, J. F. T. Spencer, and J. H. Sloneker, Can.]. Chem., 45, 2005 (1967). (344) D . R. Lineback, Curbohyd. Res., 7, 106 (1968). (345) C. G . Fraser and K. C. B. Wilkie, Phytochemistry, 10, 199 (1971). (346) P. A. J. Gorin and J. F. T. Spencer, Curbohyd. Res., 13,339 (1970). (347) H. Paulsen, Chem. Ber., 101, 179, 186, 191 (1968); Adoun. Carbohyd. Chem. Biochem., 26, 127 (1971). (348) H. Paulsen and C. P. Herold, Chem. Ber., 103, 2450 (1970). (349) W. E. Dick, D. Weisleder, and J. E. Hodge, Carbohyd. Res., 18, 115 (1971). (350) K. Igarashi, T. Honma, and J . Irisawa, Curbohyd. Res., 13, 49 (1970). (351) S. J. Angyal and K. James, Carbohyd. Res., 15,91 (1970). (352) A. L. Fink and G. W. Hay, C a n . ] . Chem., 47, 841 (1969). (353) A. L. Fink and G . W. Hay, Can. ]. Chem., 47, 845 (1969). (354) G. Martin and J. Asselineau,]. Chromutogr., 39, 322 (1969).

GAS-LIQUID CHROMATOGRAPHY

51

and oligosaccharides. Their article was followed by two Japanese reports, one of which was directed to the determination of sugars at the nanogram level by use of electron-capture detectors;226the other was concerned with a wide range of carbohydrates and cyclitols, such as would be found in certain antibiotic substances.225The latter method has been used in a study of sugars in blood and urine,48and in the analysis of d i ~ a c c h a r i d e sShapiraZz7 .~~ reported the separation of some deoxy sugars as their trifluoroacetates. All of these studies demonstrated that trifluoroacetates are considerably more volatile than O-trimethylsilyl derivatives. For example, Vilkas and coworkers stated that the retention times of per-trifluoroacetylated glucose are 3.9 and 4.1 minutes at 150", as compared with 25 and 35 minutes for per(trimethylsily1)ated glucose at 200".This increased volatility has the disadvantage that compounds of lower molecular weight tend to be less-well resolved (for example, arabinose and xylose), but has the great advantage of shortening the time of exposure to elevated temperatures of compounds of high molecular weight. Thus, a tetrasaccharide may be eluted in 7 minutes at 250". Only model studies have so far been reported, and the method has not yet been applied to the analysis of sugar mixtures, with the exceptions The recovery of glucose-14C trifluoroacetate from a column of Carbowax 20 M or SF-96 has been shown to be 5 to 14% and 3 to 5%, respectively."'HTable I11 (p. 112) records further details of the separation of sugars and their derivatives as trifluoroacetates. 4. Butaneboronates

Preliminary work has shown that sugars may be separated directly as their b u t a n e b o r o n a t e ~or , ~after ~ ~ subsequent trirnethyl~ilylation.~~~ In the former procedure, a column of 3% of OV-17 was used at 200", and, in the latter, a column of 3% of ECNSS-M was programmed from 100" at 2" per minute. VI. METHYL GLYCOSIDES

1. Trimethylsilyl Ethers Methanolysis of polysaccharides as an alternative to hydrolysis has been discussed in Section I1 (see p. 14). In brief, this method of depolymerization may be more convenient with glycolipids, because the fatty acids are simultaneously obtained as their methyl esters, and aminodeoxyhexoside bonds are more readily methanolyzed than hydrolyzed. An additional advantage is the increased stability of

52

G. G . S. DUTTON

neuraminic acid as its methyl glycoside, methyl e ~ t e r .It~is, ~ there,~~ fore, largely in the glycolipid and glycoprotein fields that monosaccharides are determined and identified as methyl glycosides that may be rendered volatile by transformation into their 0-trimethylsilyl, 0-acetyl, or 0-trifluoroacetyl derivatives. Of these, use of the first group is the most common. A further advantage is that certain pairs of sugars are more clearly separated as methyl glycosides than as free sugars; this is particularly evident with D-ghCOSe, D-galactose, and D-glucose, D-mannose. In an examination of glycolipids and gangliosides, Sweeley and Walkers6 were the first to report the greater ease of separation of D-glucose and D-galactose as methyl glycosides, and the method has been used in studies on g l y c o l i p i d ~ ,b~a~~~t ,e~r~i ~ a ? gangliosides ~~’~~~ in brain,’l adrenal medulla,359blood,360and urine sediments.361D-Ghcose, D-galactose, and D-mannose have been identified in this way in studies on a g l u ~ o a m y l a s eand ~ ~ ~on Trichophyton.68 Penick and M ~ C l u e r ’used ~ ~ a method similar to that of Sweeley and W a l k e P in research on gangliosides, but, in an attempt to use raffinose as the internal standard, they were unable to observe a peak corresponding to D-fructose after methanolysis. This apparent decomposition of D-fructose has also been reported in connection with methanolysis of a glucofructan from algae.363 Several model studies on the separation of monosaccharides as their methyl glycosides have been published. These include three related ~ S , ~ ~ with * ~the ~ deter~ ~ ~ papers, by Hough and C O W O ~ ~ ~ concerned mination of D-galaCtOSe, D-mannose, and L-fucose, and applied to membrane g l y c o p r ~ t e i n sTheir . ~ ~ ~ method was also used in examining

(355) R. Matalon and A. Dorfman, Science, 164, 1522 (1969). (356) B. Samuelsson and K. Samuelsson, 1. Lipid Res., 10,41 (1969). (357) G. Bagdian, W. Droge, K. Kotelko, 0. Liideritz, and 0. Westphal, Biochem. Z., 344, 197 (1966). (358) W. J. Tate, H. Douglas, A. I. Braude, and W. W. Wells, Ann. N . Y. Acad. Sci., 133, 746 (1966). (359) R. Ledeen, K. Salsman, and M. Cabrera, Biochemistry, 7,2287 (1968). (360) D. E. Vance and C. C. Sweeley,]. Lipid Res., 8, 621 (1967). (361) R. J. Desnick, C. C. Sweeley, and W. Krivit,]. Lipid Rcs., 11, 31 (1970). (362) D. R. Lineback and W. E. Baumann, Crrrbohyd. Res., 14,341 (1970). (363) Y. Tsusue and T. Yamakawa,]. Biochem. (Tokyo), 58,587 (1965). (364) C. H. Bolton, J. R. Clamp, and L. Hough, Biochem. J . , 96,5c (1965). (365) C. H. Bolton, J. R. Clamp, G. Dawson, and L. Hough, Curbohyd. Res., 1, 333 (1965). (366) J. C. McPherson, J. R. Clamp, and A. J. Manstone, Immunochemistry, 8, 225 (1971).

~

~

GAS-LIQUID CHROMATOGRAPHY

53

a,-acid glycoproteii-~?~~ the glycoprotein of TA, and collagen.369Clamp and coworker^^^^,^^ extended their study, and reported relative retention-times for the 0-trimethylsilyl derivatives of the methyl glycosides of almost all of the pentoses and hexoses, as well as of their amino and aldonic acid derivatives. These authors commented on the increased ease of separating D-galactose from D-mannose. They further stressed the advantages of methanolysis, in that the production of a characteristic pattern of peaks for each glycoside minimizes the background interference invariably present when biological samples are processed. The typical pattern associated with each glycoside was used, for example, to characterize D-glucose as being the carbohydrate moiety of steroidal g l y c ~ s i d e s . 'It~ ~should be noted that, if only one glycoside peak is used in calculating the total quantity of that sugar present, the equilibrium concentration of each glycoside under standard conditions of methanolysis must b e known. Such data were given by Clamp and coworkers,67and Salfner and U h l e n b r u ~ kalso ~ ~ gave data for equilibrium mixtures of glycosides in their model study directed to the analysis of serologically active glycoproteins. Wulffg5made a study of the separation of pentoses from hexoses as their methyl glycosides, and commented on their use for the better separation of D-glucose from D-galactose. Other investigations, more concerned with amino sugars, reported separations of methyl gly~ o s i d e s ?and ~ Sinkinson and Wheelock presented methods designed for analyzing glycopeptides in milk.69*370,371 O h a ~ h i studied ~'~ a serologically active arabinomannan from Mycobacteria, and found that the pertrimethylsilylated methyl glycosides of D-arabinose and L-rhamnose are not separated on columns of Ucon or SE-30. Trimethylsilyl ethers of methyl glycosides have been used in analyzing bacterial endotoxins that contain 3,6-dideoxyhexoses, and, because of the volatility of such glycosides, careful temperature-programming is essential.973Methyl D-glucosides and analogs have been (367) T. Sato, 2. Yosizawa, M. Masulmchi, and F. Yamauchi, Curbohyd. Res., 5, 387 (1967). (368) J. F. Codington, B. H. Sanford, and R. W. Jeanloz, J . Nut. Cancer Inst., 45, 637 (1970). (369) R. L. Katzman and R. W. Jeanloz, Biocheni. Biophys. Res. Conirnuti., 40, 628 (1970). (370) J. V. Wheelock and G . Sinkinson, Biochim. Biophys. Actu, 194, 597 (1969). (371) G. Sinkinson and J. V. Wheelock, Biochinz. Biophys. Actu, 215, 517 (1970). (372) M. Ohashi,Jup. J. Erp. Med., 40, 1 (1970). (373) K. J. Ryan, H. Arzoumanian, E. M. Acton, and L. Goodman,]. Amer. Chenz. Soc., 86,2499 (1964).

54

G. G. S. DUTTON

separated during the course of investigations on the substrate specificity of Taka Ethyl a-D-glucoside has been shown to be present in and ethyl a-D-galactoside in soybean extracts.376 The carbohydrate composition of cerebrosides from spinal fluid has been and amylose triesters by using pertrimethylsilylated methyl glycosides. The separation of methyl D-glucosides from D-galactosides was studied by Bauer and coworker~,3~~ and methyl 5-deoxypentofuranosides have been separated as their 0-trimethylsilyl derivatives,373as have the methyl glycosides of 6-deoxy-~-glucoseand 6-chloro-6d e o x y - ~ - g l u c o s e .Heptoses ~~~ may similarly be separated as their gly~osides,3~ as~ also * ~ ~ may ~ hexopyranosiduloses arising from the chlorine oxidation of cellulose.382 Trimethylsilylation has been used in studying the methanolysis of monosaccharide^^^^,^^^ and the products obtained by reduction of 4-deoxyuronic acids.3s5 Various natural phenolic glycosides have been separated as trimethylsilyl ethers,386as have glucosinolates.126 Yoshida and coworkers387have compared the retention times of 41 furanosides and pyranosides of D-glucose, D-galaCtOSe, D-mannose, and D-ghCUrOniC acid as their 0-trimethylsilyl, 0-acetyl, and O-trifluoroacetyl derivatives. 2. Acetates As with the free monosaccharides, few glycosides have been investigated as their acetates, because of their lack of volatility. Examples (374) H. Arita, M. Isemura, T. Ikenaka, and Y. Matsushima, Bull. Chem. Soc. Jap., 43, 818 (1970). (375) H. Arita, M. Isemura, T. Ikenaka, and Y. Matsushima,J. Biochem. (Tokyo), 68,91, 717 (1970). (376) D. H. Honig, J. J. Rackis, and D. J. Sessa,]. Agr. Food Chem., 19, 543 (1971). (377) Y. Nagai and J. N. Kaufer,]. Lipid Res., 12, 143 (1971). (378) G. Entlicher and J. N. BeMiller, Curbohyd. Res., 16, 363 (1971). (379) V. Bilik, 5. Bauer, I. Jeio, and M. Furdik, Chem. Zuesti, 19, 28 (1965). (380) M. E. Evans, L. Long, Jr., and F. W. Parrish, J. Chromutogr., 32, 602 (1968). (381) C. E. Davis, S. D. Freedman, H. Douglas, and A. I. Braude, A n d . Biochem., 28, 243 (1969). (382) P. S. Fredricks, B. 0. Lindgren, and 0. Theander, Cellulose Chem. Tech., 4, 533 (1970). (383) V. Smirnyagin, C. T. Bishop, and F. P. Cooper, Can. J . Chem., 43,3109 (1965). (384) V. Smirnyagin and C. T. Bishop, Can. J . Chem., 46,3085 (1968). (385) H. H. Schmidt and H. Neukom, Curbohyd. Res., 10, 361 (1969). (386) M. Bolan and J. W. Steele,J. Chromatogr., 36,22 (1968). (387) K. Yoshida, N. Honda, N. Iino, and K. Kato, Curbohyd. Res., 10, 333 (1969).

GAS-LIQUID CHROMATOGRAPHY

55

where acetates have been used include the separation of methyl D-glucoside and methyl L-idoside isolated during the course of a study of L-iduronic acid in heparin,3aaand methyl a-D-glucoside isolated in the synthesis of methyl cr-rnalto~ide.~~~ The mixture of methyl glycosides and alditol obtained by methanolysis of a disaccharide alditol is readily separated as the acetates, and thus this method is a convenient means of characterizing the reducing and nonreducing end of a disaccharide.389The identity of 3-O-methyl-~-rhamnose, found to occur naturally in mycoside G , was established through the methyl g l y c ~ s i d e . The ~ ~ , methyl ~ ~ ~ furanosides of 2-deoxy-~-a~abinohexose were separated into the a! and p anomers on a column of 3% cyclohexanedimethanol succinate (HiEff-&BP) at 200°, but, under the same conditions, methyl 2-deoxy-~-Zyxo-hexofuranosides and methyl 2-deoxy-~-arabino-hexopyranosides each gave a single peak.390 3. Trifluoroacetates

Several methyl pento- and hexo-pyranosides have been studied as trifluoroacetates in connection with mass spectrometry,391and the retention times of 41 glycosides as their trifluoroacetates have been compared with those of the 0-trimethylsilyl and 0-acetyl derivative~.~ In~ a' study on sake, the separation of ethyl D-glucosides as these three derivatives was also compared.29s For the analysis of glycolipids, Ando and Y a m a k a ~ a described ~~l~ a method based on methanolysis followed by trifluoroacetylation. They examined the effect of nonpolar columns (OV-I, SE-30, and QF-1), and of columns having medium (OV-17, OV-25) and high polarity (XE-60, ECNSS-M), on the separation of the trifluoroacetates of methyl D-glucosides and methyl D-galactosides. On columns of low polarity, the galactoside derivatives were eluted first, whereas the order was reversed on a column of XE-60. The addition, to SE-30, of a small proportion of XE-60 was found to have a profound effect on the efficiency of separation of these glycosides, and the best resolution was obtained with a column consisting of 0.03% of XE-60 and

(388) A. S. Perlin and G. R. Sanderson, Curbohyd. Res., 12, 183 (1970). (389) J. Karkkainen, Curbohyd. Res., 11,247 (1969). (389a) J. KArkkainen, Curbohyd. Res., 14, 27 (1970). (389b) J. Karkkainen, Curbohyd. Res., 17, 1 (1971). (390) S. D. Schimmel and R. D. Bevill, Anal. Biochem., 37,385 (1970). (391) G. Jung, H. Pauschmann, W. Voelter, E. Breitmaier, and E. Bayer, Chromatogruphia, 3, 26 (1970). (391a) S. Ando and T. Yamakawa,J. Biochem. (Tokyo), 70,335 (1971).

56

G . G . S. DUTTON

2% of SE-30. Such a column also gave good separations of amino sugars, neuraminic acid, and sphingosine bases. These authors found that the trifluoroacetates of methyl glycosides are more stable than those prepared from reducing sugars, and are comparable in stability to trimethylsilyl ethers.

4. Methyl Ethers

The analysis of monosaccharide mixtures as the permethylated derivatives was proposed early in the application of gas-liquid chromatography to carbohydrates, but the method has now been superseded by more convenient procedure^.^^^,^^^ There are, however, situations in which this method is useful, such as during a structural study of a polysaccharide by the methylation technique. The mixture of partially methylated monosaccharides obtained by methanolysis may then be fully methylated, and the proportions of the various monosaccharides determined. This approach has been used, for example, in studies on a g a l a c t ~ m a n n a nand ~ ~ ~on tamarind-kernel polysachar ride.^^^ Such an analysis also constitutes a useful check to ensure that no significant change in the composition of the polysaccharide occurred during methylation. Additional examples of the resolution of mixtures of methyl glycosides, as trimethylsilyl ethers, peracetates, and as O-methylated derivatives are given in Table IV (p. 115). VII. ALDITOLS

The fact that each monosaccharide may give more than one peak owing to the formation of anomeric derivatives has led to a search for means to eliminate this complication. The anomeric center may be removed either by conversion into the oxime5 or the nitrile,394s394a by oxidation followed by formation of the lactone (see Section IX, p. 71), or by reduction to the alditol. The last method is simpler than oxidation, and the separation of alditols and of aldononitriles will be discussed here; additional examples are given in Table V (see p. 119). The early work on the separation of alditols has been discussed by B i ~ h o p The . ~ necessity of decomposing borate complexes (392) H. C. Srivastava, P. P. Singh, and P. V. Subba Rao, Corbohyd. Res., 6,361 (1968). (393) H. C. Srivastava and P. P. Singh, Curbohyd. Res., 4, 326 (1967). (394) V. M. Eastenvood and B. J. L. Huff, Soensk Pupperstidn., 72,768 (1969). (3944 B. A. Dmitriev, L. V. Backinowsky, 0. S. Chizhov, B. M. Zolotarev, and N. K. Kochetkov, Curbohyd. Res., 19, 432 (1971).

GAS-LIQUID CHROMATOGRAPHY

57

after reduction with borohydride has already been mentioned (see Section 111, p. 23). Despite the simplifications in the chromatograms that are introduced by reduction, it must be borne in mind that two aldoses may give the same alditol; thus, ignoring D or L configuration, there are only three pentitols and six hexitols. It may, therefore, be impossible to determine the group configuration of the parent m o n o s a ~ c h a r i d e . ~ ~ ~ Furthermore, as the reduction of a ketose gives two alditols, the method is unsuitable for the determination of, for example, D-fruCtOSe in the presence of D-glucose; this is of particular importance in clinical studies, and in analyses of fruits and plant extracts. It is for such reasons that much effort is still directed to improvements in methods for the estimation of free reducing sugars. Often, however, the advantages of working with alditols outweigh the disadvantages.

1. Trimethylsilyl Ethers Alditols are usually separated as their acetates or their trimethylsilyl ethers. In general, resolution of the trimethylsilyl ethers of the alditols is less complete than that of the parent sugars,217,344*396 and therefore, despite their ease of formation, these derivatives have normally been used only for simple mixtures. Thus, D-mannitol in potato D-glucitol in blood,297and L-fucitol from h ~ o i d a have n~~~ been determined as their trimethylsilyl ethers. Two-component mixtures, such as L-fucitol with xylitol or with D-glucitol have been analyzed and D-mannitol,311or L-arabinitol and galactit01~~~ in this way, but the method is not satisfactory for mixtures of D-glucitol with galactit01.~~~ The method has also been used to monitor the metabolism of L-arabinitol and D-mannitol in microbial media.159Gasliquid chromatography of alditols is particularly useful in distinguishing between compounds not separable on paper as, for example, glycerol and 1,4-anhydr0ribitol.~~l The fact that the common hexitols are not readily separated as their trimethylsilyl ethers may be turned to advantage in estimating total

(395) I. M. Morrison and M. B. Perry, Can. J . Biochem., 44, 1115 (1966). (396) H. G . Jones, D. M. Smith, and M. Sahasrabudhe,]. Ass. Offic.Anal. Chem., 49, 1183 (1966). (397) K. Anno, N. Seno, and M. Ota, Carbohyd. Res., 13, 167 (1970). (398) E. Percival, Carbohyd. Res., 7, 272 (1968). (399) E. J. Bourne, P. Brush, and E. Percival, Carbohyd. Res., 9,415 (1969). (400) D . M. W. Anderson and A. C. Munro, Carbohyd. Res., 12,9 (1970). (401) N. L. Gregory, J . Chromatogr., 36, 342 (1968).

58

G . G. S. DUTTON

hexitol, as in dietetic foods.3s60-Trimethylsilyl derivatives of reducing sugars and their derived alditols offer two complementary techniques; thus, sucrose overlaps with trimethylsilylated a-maltose, but is clearly separated from per-0-(trimethylsily1)maltitol; this approach has been used in the analysis of starch syrups.113It may be noted here that, in certain cases, the material constituting the column support may influence a particular separation. Thus trimethylsilylated D-glucitol and trimethylsilylated a-D-glucose overlap when a column of Carbowax 20 M on Chromosorb W is used, but are separated402by Carbowax 20 M on Gas Chrom Q . Mixtures of aldoses with alditols are usually readily separated as the 0-trimethylsilyl derivatives, and the procedure has been used to determine D-glucitol in the presence of D-glucose in fruit.317’318*324 Similarly, the nature of the two sugars in a disaccharide alditol, and the identity of the reducing end-group, have been determined.38sWith a polysaccharide, hydrolysis after reduction yields only one mole of alditol and many moles of monosaccharide per mole. The proportions of alditol and aldose obtained may readily be determined by gasliquid chromatography, whereas, by other methods, two separate colorimetric procedures are necessary. This method has been proposed as a means of measuring the degree of polymerization of oligosaccharides, and, in model experiments, 1 part of alditol in the presence of 150 parts of aldose could be determinedS4O3 Certain synthetic heptitols that could not be separated by paper chromatography or by electrophoresis have been separated as their 0-trimethylsilyl derivatives.404The procedure of reductive, alkaline hydrolysis, much used in glycopeptide research, yields alditols that have been estimated as their trimethylsilyl e t h e r ~ , 5 ~and , ~ OD-glucitol ~ in mammalian nerve has similarly been determined.406 The multiplicity of peaks obtained on trimethylsilylation of 3-deoxy-D-erythro-hexosulose has already been mentioned (see p. 24); however, the isomeric 3-deoxyhexitols formed on reduction with borohydride were readily separated as their trimethylsilyl ethers.234 El-Dash and H ~ d g gave e ~ ~a large ~ amount of tabular and graphical data on relative retention-times and showed that, when these values (402) W. E. Dick, B. G. Baker, and J. E. Hodge, Curbohyd. Res., 6, 52 (1968). (403) G. G. S. Dutton, K. B. Gibney, P. E. Reid, and J. J. M. Rowe,J. Chromutogr., 47, 195 (1970). (404) J. L. Godman, D . Horton, and J. M. J. Tronchet, Curbohyd. Res., 4,392 (1967). (405) K. 0. Lloyd and E. A. Kabat, Curbohyd. Res., 9 , 4 1 (1969). (406) W. R. Sherman and M. A. Stewart, Biochem. Biophys. Res. Comnzun., 22, 492 (1966).

GAS-LIQUID CHROMATOGRAPHY

59

are plotted against molecular weight, alditols and w-deoxyalditols behave as one group, whereas alditols having a methylene (deoxy) group within the chain have consistently higher retention-times. Similar relationships were found for sugars and lactones. Hexa-0-(trimethylsily1)galactitol is a crystalline compound, m.p. 78”, and it has been proposed for use as a standard for separations by gas-liquid c h r ~ m a t o g r a p h y . ~ ~ ~

2. Acetates Alditols are readily acetylated, and J. K. N. Jones and C O W O ~ separated a wide range of acetates, although they were unable to resolve a mixture of D-glucitol and galactitol. Their method was used for analyzing the glucan by Myxobacterium 402, for comparing extracts of Gram-negative and for examining a polysaccharide from Serrutiu m ~ r c e s c e n s .In ~ ~the ~ last study, gasliquid chromatography was particularly useful in distinguishing D-glycero-L-manno-heptose from D-galacto~e.~”Similar methods have been used in studies on algal p o l y ~ a c c h a r i d e s . ~ ~ ~ - ~ ~ ~ Despite the results obtained by Jones and coworkers,409interest in the separation of carbohydrate acetates lapsed for several years. This was partly due to the complexity of the liquid phase used by these investigators, and partly because the acetates of certain common alditols could not be separated on the then-available column-packings. In addition, it was at just about this time that Sweeley and coworkers5 introduced the use of trimethylsilylation, and demonstrated that a wide range of carbohydrates and their derivatives could be separated by this technique. With the development of new liquid phases, interest in the separation of alditol acetates has revived, and this now may be considered the most widely used method for analyzing carbohydrate mixtures, provided that reduction does not introduce an ambiguity into the analysis; also, it must be borne in mind that the multiplicity of peaks obtained by trimethylsilylation of a free sugar or of its methyl glycosides may serve a useful purpose in characterization. (407)F. Loewus, Carbohyd. Res., 3, 130 (1966). (408)S.W.Gunner, J. K. N. Jones, and M. B. Perry, Chem. Ind. (London), 255 (1961). (409)S. W.Gunner, J. K. N. Jones, and M. B. Perry, Cun. J . Chem., 39, 1892 (1961). (410)G. A. Adams, Can. J. Biochem., 45,422 (1967). (1964). (411)H.J. Creech, E. R. Breuninger, a n d G. A. Adams, C a n . ] . Biochen1.,42,593 (412)D. M. Bowker and J. R. Turvey,]. Chem. Soc. (C), 983 (1968). (413)J. R. Allsobrook, J. R. Nunn, and H. Parolis, Carbohyd. Res., 16, 71 (1971).

~

~

~

S

60

G. G . S. DUTTON

Jeanes and coworkers217investigated several column packings, and found that an organosilicone polyester (ECNSS-M) gives good separations of acetates of common alditols ranging from glycerol to glucitol; the latter had the highest retention-time of those studied (-76 minutes). They also found that the trimethylsilyl ethers of alditols were inadequately separated. Virtually all subsequent separations of alditol acetates have used this same column packing, and, to this extent, most of the subsequent separations of alditol acetates have been in accordance with their original method. Such modifications as have been proposed relate more often to the conditions of hydrolysis and acetylation, as previously discussed (see Sections I1 and 111; pp. 14 and 23). One such widely used modification involves deionization with an ion-exchange resin, addition and distillation of methanol, and acetylation for 15 minutes at 100" with 1:l pyridine-acetic anhydride; water is then added, and the acetates are extracted into chloroform and A general procedure has been described by S l ~ n e k e r , who ~ ' ~ has also described a method for determining the cellulose and apparent hemicellulose in plant tissue.41s Although ECNSS-M may be considered to be the most generally useful column-packing for alditol acetates, it is not without certain limitations. The maximum operating-temperature is rather low, and the useful operating-life of a column is short in comparison with, for example, a column of a polyester such as butanediol succinate. A good check on the performance of an ECNSS-M column is its ability to resolve, cleanly, the three peaks for the hexaacetates of D-glucitol, D-mannitol, and galactitol. This liquid phase is usually used on a treated support, such as "Gas Chrom Q," but Shaw and Moss417examined several solid supports and showed that the surface activity of the support determines to a large extent the degree of resolution obtainable. Thus, for the acetates of D-glucitol and galactitol, the best separation was obtained on the most active support tested, which was Chromosorb W not subjected to prior deactivation. Similar observations have been made by others.418Although 3% is the loading of ECNSS-M normally used (and found satisfactory in the vast majority of cases), O a d e P preferred lo%, and reported that lower percentages (414)T.Holme, A. A. Lindberg, P. J. Garegg, and T. Onn, J . Gen. Microbial., 52, 45 (1968). (415)J. H. Sloneker, Methods Carbohyd. Chem., 6,20 (1972). (416)J. H.Sloneker, Anal. Biochem., 43,539 (1971). (417)D. H.Shaw and G. W. Moss,J. Chromatogr., 41, 350 (1969). (418)G.G. S.Dutton and R. H. Walker, Cellul. Chem. Technol., 6,295(1972).

GAS-LIQUID CHROMATOGRAPHY

61

caused excessive “tailing.” He also stated that cooling of the column below loo”, even for only a few minutes, resulted in broad peaks that only became sharp again after subsequent heating overnight at 220”. Other workers have not commented on this sensitivity to thermal shock. By contrast, NiedermeieP recommended 1% ECNSS-M, as giving sharper peaks and requiring a shorter time for conditioning. Other liquid phases have been used, as may be noted in Table VI (see p. 122). In their original paper, Jeanes and coworkers217reported that a column of XE-60 was not able to resolve the acetates of L-arabinitol and ribitol, nor those of L-fucitol and L-rhamnitol; Carbowax 20 M modified with terephthalic acid was satisfactory, except for the separation of D-glucitol from galactitol as the acetates. A combination of 1.5% of ethylene glycol succinate and 1.5% of XF-1150 has been successfully used for analyzing wood p o l y s a c ~ h a r i d e sbut , ~ ~a~related material, 5% of XF-1112, did not resolve the hexitols. For analyzing plant cell-walls, 0.2% of ethylene glycol succinate 0.2% of ethylene glycol adipate 0.4% of XF-1150 resolved the acetates of L-arabinitol, xylitol, and L-rhamnitol, in addition to those of the common hexit o P In a model study related to the analysis of glycoproteins, Lehnhardt and W i n ~ l e used r ~ ~ a column of 0.75% of HiEFF-1-BP+ 0.25% of EGGS-X 0.1% of “phenyldiethanolamine” [N,N-bis(2-hydroxyethyl)aniline] to separate the acetates of L-rhamnitol and L-fucitol, as well as those of ribitol and L-arabinitol. Such a column was, however, found less effective for a mixture of D-glucitol and galactitol, and failed entirely with the acetates of 2-deoxy-~-arabino-hexitoland 2-deoxy-~-Zyxo-hexitol.The liquid phase OV-225 has been recommended419for the separation of methylated alditol acetates, and is stable to 250”. It is likely to become the preferred liquid phase for the separation of alditol (see also, Ref. 700). For relatively simple mixtures, butanediol succinate may be ~ ~ e instead d ~of ~ ECNSS-M. The difficulties inherent in analyzing wood pulps, discussed in Section V (see p. 41), led to the publication of several procedures using alditol acetates. Sjostrom and coworkers21Sdemonstrated the importance of selecting the correct liquid phase and operating conditions, in order to effect a complete separation of D-mannitol, galactitol, and D-glucitol. Excellent results were obtained, even for components present to the extent of only 1%,and comparative values for standard pulps were given for paper-chromatographic and gas-liquid

+

+

+

(419) J. Lonngren and A. Pilotti, Actu Chem. Scund., 25, 1144 (1971). (420) P. P. Singh and G. A. Adams, C a n . J .Chem., 48,2500 (1970).

~

.

62

G. G . S. DUTTON

chromatographic analyses. This method has been used by Sjostrom and E n ~ t r o min~a~study ~ on pulps produced by different processes, and by Hansson in investigating the sorption of hemicelluloses in kraft p ~ l p i n g . ~ Sjostrom ~ ~ - ~ and ~ ~ J ~ s l i nexamined ~ ~ ~ the use of an electronic integrator in such analyses, and concluded that the precision was good, but that accuracy required perfect separation of the peaks. By using a column of 3% ECNSS-M, similar results were obtained by Crowell and Burnett,218who preferred to inject a solution of the acetates in dichloromethane. When L-rhamnitol peracetate was present, it was eluted in the pyridine “tail” when the acetylation mixture was directly injected. This effect could be overcome by temperature-programming, but reproducible results were then difficult to obtain. It was thus found preferable to remove the pyridine before the injection was performed. Borchardt and Piperzz0also preferred to inject the alditol acetates in a volatile solvent, such as dichloromethane. These authorszz0noted the formation of a by-product that gave a peak close to that of D-mannitOl h e ~ a a c e t a t e The . ~ ~ procedure of Albersheim and coworkers is but, because acetylation was catalyzed by sodium acetate, interference from pyridine was excluded. This technique has been used by Albersheim and coworkers in studies on cottonw and grasshopper~,4~*~~ and by F r a n for ~ ~plant ~ mucilages. Blake and Richardszs carefully examined all of the steps involved in the analysis of complex polysaccharides, and preferred to use the alditol acetates in studies on the classificatior~,~~~ fractionation,427and molecular aggregation428of hemicelluloses and speargrass ~ y l a n . ~ ~ ~ , ~ Many bacterial polysaccharides have been analyzed as alditol acet a t e ~ , ~ ~ ~and, ~ ~for* those ~ ~ that , ~ contain ~ , ~ 3,6-dideoxy ~ ~ , ~ ~ sugars, ~ - ~ ~ ~ (421) E. Sjostrom and B. Enstrom, Tappi, 50,32 (1967). (422) J.-A. Hansson and N. Hartler, Suensk Papperstidn., 72,521 (1969). (423) J.-A. Hansson, Holzforschung, 24,77 (1970). (424) J.-A. Hansson, Soensk Papperstidn., 73,49 (1970). (425) E. Sjostrom and S. Juslin,J. Chromatogr., 5 4 , 9 (1971). (426) J. D. Blake, P. T. Murphy, and G . N. Richards, Carbohyd. Res., 16,49 (1971). (427) J. D. Blake and G. N. Richards, Carbohyd. Res., 17,253 (1971). (428) J. D. Blake and G . N. Richards, Carbohyd. Res., 18, 11 (1971). (429) J. D. Blake and G. N. Richards, Aust. J . Chem., 23,2361 (1970). (430) J. D. Blake and G. N. Richards, Aust. J . Chem., 23,2353 (1970). (431) G. Schmidt, I. Fromme, and H. Mayer, Eur. J . Biochem., 14,357 (1970). (432) H. Bjomdal, B. Lindberg, and W. Nimmich, Acta Chem. Scand., 24,3414 (1970). (433) G . A. Adams, C. Quadling, M. Yaguchi, and T. G . Tomaben, Can. J . MicrobioL, 16, 1 (1970). (434) W. Droge, V. Lehmann, 0. Luderitz, and 0. Westphal, Eur. J . Biochem., 14, 175 (1970).

GAS-LIQUID CHROMATOGRAPHY

63

a two-step hydrolysis was preferred (see Section 11, p. 14). Synthetic t y ~ e l o s e ~and ~ * a b e q ~ o s ewere ~ ~ ~characterized as their alditol acetates, and Mayer and F r ~ m m determined e~~~ the relative retentiontimes, on ECNSS-M at 155", of all the known 3,6-dideoxyalditols. The volatility of the acetates of the alditols from these 3,6-dideoxy sugars should be particularly noted, as this property may lead to low analytical value^.'^.^^^ Data for all of the possible 4-deoxy-~-hexitol acetates were reported, in connection with the proof of the presence of 4-deoxy-~-arabino-hexose in C i t r o b a ~ t e r The . ~ ~ ~0 antigens of certain Citrobacter have been shown to contain 6-deoxy-~-talose, which was identified as the alditol In connection with the structure of glycoproteins, Lloyd and Kabat studied the separation of six alditol and Kim and cow o r k e r ~examined ~~ the separation of L-fucitol, D-glucitol, D-mannitol, and galactitol. Alditol acetates have been used in studies on (448) S. Svensson, Acta Chem. Scand., 22,2737 (1968). (449) H. F. Beving, H. B. Boren, and P. J. Garegg, Acta Chem. Scand., 24,919 (1970). (450) J. Keleti, H. Mayer, I. Fromme, and 0. Liideritz, Eur.J. Biochem., 16,284 (1970). (451) J. Keleti, 0. Liideritz, D. Mlynarci, and J. Sedlak, Eur.1. Biochem., 20,237 (1971). (452) K. 0. Lloyd and E. A. Kabat, Carbohyd. Res., 4, 165 (1967).

(435) C. G. Hellerqvist, B. Lindberg, J. Znngren, and A. A. Lindberg, Acta Chem. Scand., 25,601 (1971). (436) C. G. Hellerqvist, 0. Larm, and B. Lindberg, Acta Chem. Scand., 25,744 (1971). (437) C. G. Hellerqvist, B. Lindberg, J. Lonngren, and A. A. Lindberg, Carbohyd. Res., 16, 289 (1971). (438) C. G. Hellerqvist, B. Lindberg, A. Pilotti, and A. A. Lindberg, Carbohyd. Res., 16, 297 (1971). (439) C. G . Hellerqvist, B. Lindberg, K. Samuelsson, and A. A. Lindberg, Actu Chem. Scund., 25, 955 (1971). (440) C. G. Hellerqvist, B. Lindberg, J. Lonngren, and A. A. Lindberg, Actu Chem. Scund., 25, 939 (1971). (441) C . G. Hellerqvist, 0.Larm, and B. Lindberg, Acta Chem. Scand., 23,2217 (1969). (442) H. Mayer and I. Fromme, in press. (443) P. J. Garegg, B. Lindberg, T. Onn, and I. W. Sutherland, Actu Chem. Scand., 25, 2103 (1971). (444) P. J. Garegg, B. Lindberg, T. Onn, and T. Holme, Acta Chem. Scand., 25, 1185 (1971). (445) H. Bjomdal, C. Erbing, B. Lindberg, G. FBhraeus, and H. Ljunggren,Acta Chem. Scand., 25, 1281 (1971). (446) J. Weckesser, H. Mayer, and G. Drews, Ear. J. Biochem., 16, 158 (1970). (447) M. Berst, 0. Liideritz, and 0. Westphal, Ear. J. Biochem., 18,361 (1971).

64

G . G . S . DUTTON

blood-group g l y ~ o p r o t e i n , 4 ~a~n- t~i~g~e n ~ , 3 and ~~,~ peptidogalacto~~ mannan~.~~,~~~ Other structural studies that have relied on the separation of alditol acetates are in the areas of soil p o l y s a c ~ h a r i d e splant ~ ~ * ~gums,459-461 ~~ and hngi.462-464 Other examples are given in Table VI (see p. 122). Heptitols may also be separated as their acetates, and this may provide a convenient means of distinguishing heptoses from hexoses that migrate at the same rate on paper. For example, D-galactose and D-glycero-D-manno-heptose have the same RF value on paper chromatograms, but are readily distinguished as the alditol acetates.411 The difficulties associated with the complete hydrolysis of polysaccharides containing uronic acids have been discussed in Section I1 (see p. 14). The reduction of uronic acid groups has been studied by Manning and Green46sand by Rees and Samuel.466The latter investigators showed that lithium borohydride is a particularly efficient reductant. When the acidic polysaccharide is converted into a neutral glycan, hydrolysis followed by reduction with sodium borohydride gives a mixture of alditols that may be analyzed in the usual way. This approach has been studied by Blake and and Dutton and Kabir467have shown that mixtures of D-galacturonic acid with D-glucuronic acid and its 4-methyl ether may be analyzed in this way. The method was not suitable for a mixture of D-mannuronic acid and 4-0methyl-D-glucuronic acid, as their derived alditol acetates overlapped on both the ECNSS-M and the butanediol succinate columns (453)D. B. Thomas and R. J. Winzler, Biochem. Biophys. Res. Commun., 35, 811 (1969). (454)V. M. Hearn, S. D. Goodwin, and W. M. Watkins, Biochem. Biophys. Res. Commun., 41, 1279 (1970). (455)C . Race and W. M. Watkins, FEBS Lett., 10,279 (1970). (456)A. M. Adamamu and R. H. Kathan, Biochem. Biophys. Res. Commun., 37, 171 (1969). (457)K. 0. Lloyd, FEBS Lett., 11,91 (1970). (458)J. M. Oades, M. A. Kirkman, and G . H. Wagner, Soil Sci. SOC. Amer. Proc., 34, 230 (1970). (459)A. M. Stephen and D. C. de Bruyn, Carbohyd. Res., 5,256 (1967). (460)P.I. Bekker, S. C. Chums, A. M. Stephen, and G. R. Woolard, Tetrahedron, 25, 3359 (1969). (461)G.G.S. Dutton and S. Kabir, Carbohyd. Res., in press. (462)H. Bjorndal and B. Lindberg, Carbohyd. Res., 10,79 (1969). (463)K. Axelsson, Acta Chem. Scand., 23, 1597 (1969). (464)K. Axelsson and H. Bjorndal, Acta Chern. Scand., 23, 1815 (1969). (465)J. H. Manning and J. W. Green,]. Chem. SOC. (C), 2357 (1967). (466)D. A. Rees and J. W. B. Samuel, Chem. Ind. (London), 2008 (1965). (467)G.G.S. Dutton and S. Kabir, Anal. Lett., 4, 95 (1971).

GAS-LIQUID CHROMATOGRAPHY

65

~ ~ e dLindahl . ~ and ~ *A x~ e l~~ s ~o nsimilarly ~~ separated and identified D-glucitol and L-iditol hexaacetates from heparin, and Sjostrom and coworkers have identified 4-O-methyl-~-glucuronic acid in this way.468If the uronic acid is related to one of the hexoses in the p l y saccharide, it may be reduced by means of a complex deuteride, and subsequent mass spectrometry will differentiate between the hexitol-6,6-d2 from the uronic acid and the hexitol from the constituent hexose. Acetates of sugars and alditols are usually separable, and, therefore, the hydrolysis of a reduced oligosaccharide and examination of the products as acetates presents an alternative method of simultaneously determining the degree of polymerization and identifying the reducing end. This approach has been used for identifying oligosaccharides obtained by partial hydrolysis of slippery-elm mucilage,469and by Duckworth and T ~ r v e y ~to ~ Odetermine the d.p. of oligosaccharides, in the range of d.p. 10 to 16, obtained from porphyran. An advantage of using acetates instead of 0-trimethylsilyl derivatives403is that the hexaacetates of D-glucitol, galactitol, and D-mannitol are readily crystallized, and samples collected from the effluent of the gas chromatograph may be characterized by their melting point.

3. Trifluoroacetates Just as the trifluoroacetates of reducing sugars have relatively short retention-times, so also do these esters of alditols. This method of separation was first investigated in detail by Tamura and colleague^^^^^^^^ by using a column of XF-1105. The same method, in conjunction with a capillary column, has been used by S h a ~ i r aThe . ~ ~use ~ of alditol trifluoroacetates has been shown471to give rapid separation of the three common hexitols, but partial de-esterification on the column proved a serious disadvantage. On the other hand, Hagiwara and Yamada4?Iareported, without adverse comment, the determination of glucose, galactose, mannose, and rhamnose as the corresponding alditol trifluoroacetates.

(468) E. Sjostriim, S. Juslin, and E. Seppala, Acta Chem. Scand., 23, 3610 (1969). (469) R. J. Beveridge, J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Carbohyd. Res., 9, 429 (1969). (470) M. Duckworth and J. R. Turvey, Biochem. J., 113, 693 (1969). (471) B. Coxon, personal communication. (471a) S. Hagiwara and K. Yamada, Agr. B i d . Chem. (Tokyo), 35, 1402 (1971).

66

G . G . S. DUTTON

4. Nitriles, Dithioacetals, and Methyl Ethers

The main reason for reducing a sugar to the corresponding alditol is to obtain a compound that can give only one peak on a gas chromatogram. The same objective may be achieved by conversion into the corresponding nitrile. Reaction of a sugar with hydroxylamine in pyridine gives the oxime, which may be directly dehydrated to the nitrile and this directly acetylated to the acetate. The derivatives are isolated by the addition of water, followed by extraction with chloroform, and the extract may be directly injected. Such a procedure has who applied their method been described by Eastenvood and to the analysis of cellulosic materials. Kochetkov and coworker^^^^^ extended this method to a wide variety of sugars and, in particular, showed that the time needed for preparing the acetylated aldononitrile may be shortened to 15 minutes. In many respects, nitrile acetates, which are well characterized, crystalline compounds, may be considered alternatives to alditol acetates (see also, Table V, p. 119). The following points are, however, of importance, particularly in quantitative determinations. A sample of cotton linters was analyzed as the alditol acetates and as the acetylated nitriles, and the results were compared.394The chromatogram of alditol acetates showed ten extraneous peaks, one of which had a retention time similar to that of mannitol hexaacetate; the existence of such a peak had been noted by Borchardt and Piper.220 The chromatogram of the acetylated aldononitriles showed only three unknown peaks, all of which were eluted before D-xylononitrile acetate. It should also be remembered (see Section V, p. 41) that Turunen, Arvinen, and T ~ r u n e nhave ~ ~ drawn attention to the confusion that may arise between 1,6-anhydro-p-~-glucoseand D-xylose when cellulosic hydrolyzates are analyzed as 0-trimethylsilyl derivatives. The anhydro compound must be formed in the hydrolytic stage, and it will therefore be present no matter what analytical procedure is subsequently used. The peak that is caused by the anhydroglucose in determinations as acetates has not yet been established. Williams and J. K. N. Jones have shown that the dithioacetals of aldopentoses and aldohexoses may be separated as their acetates.472 In certain cases, however, isomers were not separated (for example, D-altrose and D-talose), but reduction of the dithioacetal to the l-deoxyalditol permitted resolution of the acetates. The method is not suitable for quantitative determinations, but it does preserve the (472) D. T. Williams and J. K.N. Jones, Can.J. Chem., 44,412 (1966).

GAS-LIQUID CHROMATOGRAPHY

67

identity of the parent ald0se,3~~ an advantage also shared by the aldononitrile method. The separation of fully methylated alditols has been investigated by Ovodov and E v t u ~ h e n k o , ~but ~ ' is not suitable for routine use, because of the time necessary for the preparation of such derivatives, and the lack of resolution. 5. As Alditols

A procedure has been reported473for the direct determination, on a column of Polypak, of polyhydric compounds without conversion into derivatives. Separation of pentitols was not complete, but the method was found excellent for determining the amount of one alditol, as in the oxidation of L-arabinitol by Acetobacter suboxydans. This rapid method of monitoring the utilization of L-arabinitol permitted the reaction to be stopped as soon as all of the substrate had been consumed, before side reactions interfered.

VIII. OLIGOSACCHARIDES The separation of disaccharides and higher oligomers is not essentially different from the separation of monosaccharides, except that the volatility rapidly decreases with increasing molecular weight. Oligosaccharides may be transformed into volatile derivatives, commonly the trimethylsilyl ethers, either directly or after reduction. Other derivatives, such as trifluoroacetates, have been used, but the acetates have low volatility. Oligosaccharides are not usually converted into their methyl glycosides prior to trimethylsilylation. Detailed examples are listed in Table VII (see p. 130). Before discussing the various derivatives that have been used, it should be remembered that oligosaccharides are often obtained by chromatography on paper or on cellulose columns, and this may cause them to become contaminated by xylan; this may be eliminated by extraction of the crude fractions with hot For similar reasons, glucose that is really extraneous may be detected in fractions separated by electrophoresis on cellulose acetate.475Gas-liquid chromatography was used to show the presence in pituitary glyco-

(473) L. Dooms, D. Declerck, and H. Verachtert,]. Chromatogr., 42, 349 (1969). (474) A. B e d i k and J. K. Hamilton, Chem. Ind. (London), 1341 (1965). (475) A. Lehtonen, J. Karkkainen, and E. Haahti, J . Chromatogr., 24, 179 (1966).

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G . G . S. DUTTON

protein of D-xylose as an artifact resulting from chromatography on cellulose ion-exchanger~.~'~ 1. Trimethylsilyl Derivatives

In their original publication, Sweeley and coworkers5 showed that the trimethylsilyl ethers of sugars up to tetrasaccharides are sufficiently volatile for use in g.1.c. Since then, this method has been applied to the determination of sucrose in wort and beer, fruits and juices,121,317,318 ferns321 and other plants,117~150,174,291.323,477 potatoes,115*116.123 beet molasses,320and ~ r i n e . " ~ .l4C-Labe1led '~~ sucrose has also been used in studying the recovery of sugars in gas chromatography.208Ness and Fletcher478found that separation of the isomers of sucrose on a column of SE-30 is impossible. Sucrose lactate has been found in Cladophordes, and it may be converted into the volatile 0-trimethylsilyl d e r i v a t i ~ e . ~ ' ~ Maltose and its homologs have been separated from beer,128*129,480.48* corn and urine,li8 in the analysis of l e v ~ g l u c o s a nand , ~ ~ in ~ enzymolysis studies.265By using a short column, Beadle134was able to separate the maltose series, up to malto-octaose; this appears to be the carbohydrate of highest molecular weight that has thus far been subjected to gas-liquid chromatography. A series of cello-oligosaccharides ranging in d.p. from 2 to 6 were checked, as their O-trimethylsilyl derivatives, for purity, but details were not gi~en.~~2,483 The analysis of corn syrups is complicated by the usual problems of multiple peaks and the overlap between per(trimethylsily1)ated sucrose and per(trimethylsily1)ated a-maltose. The latter difficulty may be obviated by trimethylsilylation after reduction with borohydride, but this treatment prevents the separate estimation of glucose and fructose. 113 In analyses of honey, the wide range of oligosaccharides encountered has been separated by gas-liquid c h r ~ m a t o g r a p h y . Similar ~~~.~~~ mixtures, including a series of fructoglucans, have been isolated from onion (476) J. G . Pierce and T.-H. Liao, A n d . Biochem., 24,448 (1968). (477) P. K. Davison and R. Young,]. Chromatogr., 41, 12 (1969). (478) R. K. Ness and H. G. Fletcher, Jr.. Carbohyd. Res., 17,465 (1971). (479) E. Percival and M. Young, Carbohyd. Res., 20, 217 (1971). (480) G. E. Otter and L. Taylor,]. Znst. Brewing, 73, 570 (1967). (481) B. Tuning, lnt. Tijdschr. Brouw. Mout., 28, 113 (1969); Chem. Abstr., 72, 41,680 (1970). (482) M. Ihnat and D. A. I. Coring, Can. J . Chem., 45, 2353 (1967). (483) A. J. Michell, Carbohyd. Res., 12, 453 (1970).

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Sucrose, maltose, and lactose were included in model glycoprotein systems,34and retention times for a wide range of naturally occurring oligosaccharides, up to pentasaccharides, were listed by Clamp and

coworker^.^^ The separation and identification of disaccharides is often an important step in the elucidation of the structure of a natural polysaccharide, and P e r ~ i v ahas l~~ published ~ useful data on the O-trimethylsilyl derivatives of a variety of disaccharides and their reduction products. In some instances, the trimethylsilyl ethers of the disaccharide alditols have lower retention times than those of the disaccharide derivatives. The per-0-trimethylsilyl derivatives of gentiobiitol and maltitol were encountered in studies on the structure of Pneumococcus Type I1 capsular p o l y s a c ~ h a r i d e . ~ ~ ~ ~ Haverkamp and coworkers485studied the separation of 23 per0-(trimethylsily1)ated disaccharides on three phases having different polarity (OV-1, OV-17, and OV-25). They concluded that OV-17 is the best liquid phase for general use, and that OV-1 and OV-17 are approximately equal in their ability to separate anomeric forms; all columns were operated by using 3% of liquid phase, at a temperature of 228". They recommended that, in the initial analysis of unknown mixtures, two columns having different polarities be employed; this is a precaution that should be exercised in all g.1.c. separations. The potential of gas-liquid chromatography in such areas is well i ~ a~ serologically ~ active arabiillustrated by the work of O h a ~ h on nomannan; partial fragmentation gave a trisaccharide fraction which, on conversion into 0-trimethylsilyl derivatives, was shown to be a mixture of two trisaccharides. The nature of the glycosidic linkage in disaccharides and disaccharide alditols may be determined from study of the nuclear magnetic resonance spectrum of the per-0-(trimethylsilyl) derivative.486When reporting new syntheses of disaccharides, certain workers have included the relative retention-time as a characteristic physical property, a trend to be encouraged.487-490,490a (484) E. Percival, Carbohyd. Res., 4, 441, (1967). (484a) 0. Larm, B. Lindberg, S. Svensson, and E. A. Kabat, Carbohyd. Res., 22, 391 (1972). (485) J . Haverkamp, J. P. Kamerling, and J. F. G. Vliegenthart,]. Chromatogr., 59, 281 (1971). (486) C. G. Hellerqvist, 0.Larm, and B. Lindberg, Actu Chem. Scand., 25,743 (1971). (487) M. Dejter-Juszynski and H. M. Flowers, Carbohyd. Res., 18,219 (1971). (488) H. M. Flowers, Carbohyd. Res., 18, 211 (1971). (489) M. Shaban and R. W. Jeanloz, Carbohyd. Res., 17,411 (1971). (490) M. Shaban and R. W. Jeanloz, Carbohyd. Res., 17, 193 (1971). (490a) J . M. Berry and G. G. S. Dutton, Can. J. Chem., 50, 1424 (1972).

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G . G . S. DUTTON

Like monosaccharides, oligosaccharides may give multiple peaks, although, with increasing molecular weight, lower resolution often affords only a single peak. In an attempt to minimize multiple peaks, Mason and Slover la' studied the separation of trimethylsilylated sugar oximes, and included maltose and lactose in their investigation. Clayton and H. G . Jones188reported the separation of cellobiose from other sugars in this way. Thus far, there are only a few examples of the use of dimethylsilyl derivatives, but cyclo-hexa-, -hepta-, and -octa-amylose have been separated from each other by using these ethers.133 2. Acetates

In view of the lower volatility of carbohydrate acetates (as compared with trimethylsilyl ethers), few oligosaccharides have been separated as the acetates, although gentiobiose and laminarabiose have been chromatographed as their octaacetate~.~~' The determination of the structure of a disaccharide by mass spectrometry is a technique of increasing importance, and will be discussed further in Part I1 of this article. Interpretation of the mass spectrum is often simplified by working with the disaccharide alditol, and, in this connection, Ktirkkainen38ghas separated the reduced, common disaccharides as their acetates.

3. Trifluoroacetates In model studies,224*225 oligosaccharides have been separated as their trifluoroacetates, and Vilkas and found that a tetrasaccharide so esterified had a retention time of six minutes at 250". Similar results were obtained by Nakamura and T a m ~ r a who ,~~ showed that, on a column of OV-1 at 220",the retention time of per0-(trimethylsily1)sucrose was 12 min, whereas, at 140", the per(trifluoroacetate) required only 7 min. Other comparative data were provided for the common disaccharide alditols. A study of the concentration of lactose in blood was made by using this method;48it was also that trifluoroacetylation of disaccharides proceeds more rapidly and satisfactorily in N,N-dimethylformamide than in ethyl acetate. The increased volatility of the trifluoroacetates of monosaccharides (compared with those of the 0-trimethylsilyl derivatives) is, in (491) G. Chihara, J. Hamuro, Y.Arai, and F. Fukuoka, Nature, 225,943 (1970).

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fact, a disadvantage, as there is little difference between their retention times. It is, however, likely that trifluoroacetates will be increasingly used for oligosaccharides, where volatility is of prime importance, and this should permit application to oligomers of high molecular weight.

Ix. ACIDS

AND

LACTONES

In the carbohydrate series, the acids encountered are aldonic, aldaric, uronic, or saccharinic acids. Often, these acids are readily transformed into their lactones or methyl esters, and it is as derivatives of these that they are commonly studied. Table VIII (see p. 136) records examples in which derivatives of such acids and lactones have been subjected to gas-liquid chromatography. The problem of multiple peaks caused by the anomeric center in free sugars may be eliminated by oxidation of a neutral monosaccharide to the corresponding aldonic acid, which is then lactonized and the lactone converted into a volatile derivative. Aldonic acids may also result from reduction of the aldehyde group of uronic acids, and Perry and Hulyalkar205proposed a method for the identification of uronic acids occurring in polysaccharides that is based upon this method; for example, appropriate reduction of D-glucuronic acid gives L-gulonic acid. In model experiments,205it was found that concentration of an aqueous solution of the aldonic acids in the presence of hydrochloric acid gives exclusively the 1,4-lactones, which, on subsequent trimethylsilylation, give only one peak on the chromatogram. This method was successfully employed for the separation of D-galacturonic, D-glucuronic, and D-mannuronic acids. Of the 1,Clactones examined, only the trimethylsilyl ether of D-mannono-~,~-~actone was obtained in crystalline form. However, all of the derivatives showed characteristic differences in their infrared spectra in the range of 1500 to 600 cm-'. When this method was applied to the determination of uronic acids in a variety of p o l y s a c ~ h a r i d e s it , ~was ~ ~ impossible to find any hydrolytic conditions under which the uronic acids were quantitatively released and then reduced, a problem experienced by other workers.20The method was, however, successful in affording a qualitative, microscale procedure for the identification of hexuronic acids (which otherwise are difficult to detect). This procedure was subsequently used by Adams and coworkers to demonstrate the presence of D-glucuronic and D-mannuronic acids

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G. G. S. DUTTON

in the cellular polysaccharides of Serrutiu r n ~ r c e s c e n s . It ~ ~was ~-~~~ also used by Percival and coworkers to determine the D-glUCUrOniC acid in a glucuronoxylofucan39E~3gg and it is of interest that this method gave a result in much better agreement with the value obtained by decarboxylation than did a colorimetric procedure. S i d d i q ~ has i~~~ similarly verified that D-mannuronic acid is the only uronic acid in the extracellular polysaccharide from Arthrobucter viscosus. The method of Perry and HulyalkarZo5was further examined by Tamura and colleagues,228who investigated four different column-packings and found that no one column gave complete separation of tetrono-, pentono-, and hexono-lactones. It was also that catalytic hydrogenation of calcium D-xy~o-5-hexulosonategives a mixture of D-gluconate and L-idonate; this was separated by conversion into the 0-trimethylsilyl derivatives of the lactones. By using QF-1 as the liquid phase, it was found that single, well-separated peaks were obtained for D-gluconolactone and L-idonolactone. On a cyanosilicone column, the 1,4- and 1,5-gluconolactones were also partially separated from each other, but, on SE-52, D-glUCOn0- and L-idono-lactones were only poorly resolved. The success in separating aldonic acids as their lactones led Morrison and Perry395to extend this method to neutral monosaccharides by separation after oxidation. In this work, it was found that D-gluconic acid always gives two peaks, corresponding to the 1,4- and 1,5lactones, although the other compounds studied, which ranged from pentoses to heptoses and included deoxyglycoses, gave only one peak. The chromatograms could, however, be simplified by the prior removal of D-glucose by treatment with D-glucose oxidase. The sugars present in an immunoglobulin were identified in this way, that is, by oxidation to the lac tone^.^^^ Heptonolactones may similarly be separated as their trimethylsilyl ethers.lS6 Samuelson and coworkers49shave also reported the separation of aldonic acids as their 0-trimethylsilyl derivatives; the results confirmed Perry’s observation that concentration in the presence of (492) C . A. Adams and R. Young, Can. J. Chem., 43,2940 (1965). (493) G. A. Adams and R. Young, Ann. N . Y. Acad. Sci., 133,527 (1966). (494) G. A. Adams and S. M. Martin, C a n . ] . Biochem., 45,477 (1967). (495) G. A. Adams and R. Young, Can. J. Biochem., 43, 1499 (1965). (496) I. R. Siddiqui, Carbohyd. Res., 4, 277 (1967). (497) Chau-Yang Chen, T. Imanari, H. Yamamoto, and T. Kwan, Cham. Pharm. Bull. (Tokyo), 16,755 (1968). (498) M. B. Peny and C. Milstein, Nature, 228,934 (1970). (499) C . Peterson, H. Riedl, and 0. Samuelson, Suensk Papperstidn., 70,371 (1967).

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hydrochloric acid gives only the 1A-lactone, but it was also shown that, in the absence of this step, certain acids give rise to 3 peaks, identified as the 0-trimethylsilyl derivatives of the 1,4- and 1,5-lactones and of the trimethylsilyl ester. The formation of three peaks may particularly occur when acids are displaced by acetic acid from ion-exchange columns. Samuelson and coworkers499investigated 11 different stationary phases, and concluded that QF-1 gave the most satisfactory separations. In a temperature-programmed experiment, there was some overlapping between D-ga~actono~actone and D-gluconolactone, but, at a constant temperature of 170°, no overlapping between these two compounds was observed. It was emphasized that the choice of temperature is critical and that the temperature chosen must be suited to the components present in the sample to be analyzed. Samuelson and coworkers50"subsequently showed that aldonolactones may be identified by the mass spectra of their trimethylsilyl ethers. It should be noted that, for the pentonic acids, the derivative of the 1,klactone has a retention time lower than that of the 1,5-lactone, whereas, for D-glUCOniC acid, the situation is reversed. It was further found that, on all stationary phases examined, lactones having their trimethylsilyl groups on 0-2 and 0 - 3 in the trans position had shorter retention times than the corresponding cis isomers. In the case of the pentonolactones, it was also found that, within each category, most of the derivatives having the trans configuration at C-3 and C-4 had shorter retention-times than those having the cis configuration. The alkaline degradation of carbohydrates yields complex mixtures of saccharinic and other hydroxy acids. The separation of such mixtures has been studied mainly by Samuelson and coworkers, who have developed analytical methods using ion-exchange columns. They have also investigated gas-liquid chromatography as a means of separating a wide range of hydroxy dicarboxylic and have used such methods in conjunction with mass ~ p e c t r o m e t r yfor ' ~ ~confirming the nature of individual fractions separated by the resin columns. In the course of these investigations, it was shown that, normally, trimethysilylation of free acids gives derivatives of the lactone, whereas trimethylsilyl esters are obtained when the sodium salt is treated.163 The separation factors of these fully trimethylsilylated trimethylsilyl esters on polar, stationary phases are far superior to those ob(500) G. Peterson, 0. Samuelson, K. Anjou, and E. von Sydow, Acta Chem. Scand., 21, 1251 (1967).

74

C . C . S. DUTTON

tained with trimethylsilyl ethers of a l d i t ~ l s . 'Four ~ ~ column packings of increasing polarity (OV-1, OV-17, QF-1, and XE-60)were used, and the relative retention-times of aldaric and deoxyaldaric acids, according to the polarity, were discussed. Per-O-(trimethylsilyl)-Dglucitol was used as the reference material, and, on columns of low polarity (for example, OV-1), the deoxyhexaric acfds were less strongly held than D-glucitol, whereas the converse was true on the more-polar phases. This behavior was ascribed to important interactions between the ester groups and the polar, stationary phases in the latter case, whereas, with a nonpolar phase, the deoxyhexaric acids are eluted first, as they have one trimethylsilyl group less. It was also demonstrated that acids having a-threo groups are strongly held. (In this context, an a-threo group is defined as a group having the threo configuration at the two carbon atoms adjacent to the carboxyl group.) Thus, the affinity for the stationary phase was found to decrease in the order galacto < gluco < manno, which have 2, 1, and 0 a-threo groups, respectively, in the aldaric acids. Further considerations allowed prediction of the relative elution-patterns of other related series of acids. The methods elaborated in these model studies have been used in investigating a variety of problems concerning the reaction of sugars in alkaline media and reactions related to cellulose technology. Examples include studies on the acids found in pulp liq~ors,5~l in polysulfide pulps,502and those formed on alkaline treatment of D-glucose and c e l l o b i ~ s e ,and ~ ~ ~h y d r o c e l l u l ~ s e .The ~ ~ ~ same methods have been used to study the products of bleaching by oxygen100,505*506 and oxidation of hydrocellulose by chlorine dioxide.507 Samuelson and co-workers have also shown that analyses of cellulosic materials may be complicated by the formation of D-glucopyranosylglycolic Lindberg, Theander, and coworkers have also examined complex mixtures of acids arising from the alkaline treatment of D-xylose and

(501) D. Monzie-Cuillemet and P. Monzie, Tech. Rech. Papet., 4 (8), 74 (1966). (502) B. Alfredsson and 0. Samuelson, Suensk Papperstidn., 72, 361 (1969). (503) 0. Samuelson and L. Thede, Acta Chem. Scand., 22,1913 (1968). (504) B. Alfredsson and 0. Samuelson, Suensk Papperstidn., 71,679 (1968). (505) H. Kolmodin and 0. Samuelson, Soensk Papperstidn., 73,93 (1970). (506) 0. Samuelson and L. Stolpe, Tappi, 52, 1709 (1969). (507) S. Pettersson and 0. Samuelson, Suensk Papperstidn., 72,261 (1969). (508) G . Peterson, S. Pettersson, and 0. Samuelson, Suensk Papperstidn., 72, 222 (1969).

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D - f r U ~ t o s e ~-arabino-hexosulose~~~ ~~~*~~~ and the oxidation of cellulose by chlorine.382 The separation of saccharinic acids as their 0-trimethylsilyl derivatives has also been used by Feather and Harris512in a study on decomposition of sugars and in studies on bacterial p o l y s a ~ c h a r i d e s . ~ ~ ~ Uronic acids may also be lactonized and then trimethylsilylated, and this technique has greatly facilitated studies on glycosaminoglycans. In one example, a mixture of such glycans was separated by electrophoresis and subsequently hydrolyzed. The amino sugars were absorbed on an ion-exchange resin and the neutral and acidic fractions were evaporated and trimethylsilylated. This permitted the distribution of D-glucuronic and L-iduronic acids in the various fractions to be and allowed the fractions to be identified as hyaluronic acid and a chondroitin sulfate. The same authors had previously carried out similar studies on dermatan sulfate and chondroitin 6-sulfate together with glycosaminoglycans of skin, aorta, and umbilical cord.lg The nature of the uronic acids in heparin has long been a debatable point, but gas-liquid chromatography has been used to demonstrate the presence of both D-glucuronic and L-iduronic acids and also to determine their relative proportions in conjunction with xylose and gala~tose.'~ In a similar manner, the uronic acid in the glycosaminoglycan from squid skin513has been shown to be D-glucuronic acid, and that from a polysaccharide isolated from Clostridium welchii to be L-iduronic Other glycosaminoglycans have also been analyzed for uronic acid in this way.515-519 The products obtained by isomeriza(509) A. Ishizu, B. Lindberg, and 0. Theander, Acta Chem. Scand., 21, 424 (1967). (510) A. Ishizu, B. Lindberg, and 0. Theander, Carbohyd. Res., 5,329 (1967). (511) B. Lindberg and 0. Theander, Acta Chem. Scand., 22, 1782 (1968). (512) M. S. Feather and J. F. Harris, Carbohyd. Res., 15,304 (1970). (513) S. R. Srinivasan, B. Radhakrishnamurthy, E. R. Dalferes, and G. S. Berenson, Comp. Biochem. Physiol., 28, 169 (1969). (514) G. K. Darby, A. S. Jones, J. F. Kennedy, and R. T. Walker,J. Bacteriol., 103, 159 (1970). (515) G. S. Berenson, H. Ruiz, E. R. Dalferes, F. A. Dugan, and B. Radhakrishnamurthy, Diabetes, 19, 161 (1970). (516) G. S. Berenson, E. R. Dalferes, H. Ruiz, and B. Radhakrishnamurthy, Amer. J . Cardiol., 24, 358 (1969). (517) E. R. Dalferes, H. Ruiz, B. Radhakrishnamurthy, R. H. Rigdon, and G. S. Berenson, Proc. SOC. Exp. Biol. Med., 131, 1382 (1969). (518) E. R. Dalferes, B. Radhakrishnamurthy, and G. S. Berenson, Proc. SOC. Exp. Biol. Med., 127,925 (1968). (519) B. Radhakrishnamurthy, S. R. Srinivasan, E. R. Dalferes, and G. S. Berenson, Comp. Biochem. Physiol., 36, 107 (1970).

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G . G . S. DUTTON

tion of D-glucuronic acid520*521 and D-galacturonic acidz6' have been examined as their 0-trimethylsilyl lactones. By using these techniques, the identity of 4-O-methyl-~-iduronicacid (isolated from a pulp hydrolyzate) was established, and the acid was shown to arise by alkaline epimerization of 4-O-methyl-~-glucuronic Uronic acids have been studied similarly in connection with pect i n ~ and ~ ~oligogalacturonic ~ - ~ ~ ~ acids,170 f r ~ i t s , 3and ~~ Neukom and coworkers have studied the behavior of uronic acid and also aldaric acid derivatives.525Ascorbic acid and its analogs52shave been separated by gas-liquid chromatography and determinedzg1in plant extracts. When polysaccharides are methanolyzed, uronic acids are liberated as their methyl glycoside methyl esters. Methanolysis is a procedure commonly used with glycoproteins, and, consequently, model studies in this field have included uronic acids among the compounds s t ~ d i e d . 3 ~ , "The * ~ ~advantage ~ of stabilizing neuraminic acid and its derivatives by methanolysis has already been m e n t i ~ n e d . ' * ~ ~ ~ ~ * ~ ~ ~ Methanolysis has also been used in the determination of natural hexuronic acids,20for a series of oligogalacturonic acids,170for 4-deoxyuronic for studying the deamination of heparin,52eand in a study of the @elimination of methyl g l y c u r ~ n a t e sNeukom . ~ ~ ~ and his colleagues have also extensively studied the reaction of D-galacturonic and D-mannuronic with acidified methanol. Tamura and Imanari have examined the 0-acetyl, 0-methyl, and 0-trimethylsilyl derivatives of various 0-,N-, and S-(methyl D-glu-

(520) B. Carlsson, 0. Samuelson, T. Popoff, and 0. Theander, Actu Chem. Scand., 23, 261 (1969). (521) B. Carlsson and 0. Samuelson, Actu Chem. Scund., 23, 318 (1969). (522) E. R. Nelson, P. F. Nelson, and 0. Samuelson, Acta Chem. Scand., 22,691 (1968). (523) R. C. Wiley and M. Tavakoli, Food Technol., 23, 167 (1969). (524) M. M. Baig, S. Kelly, and F. Loewus, Plant Physiol., 46, 277 (1970). (525) 0. Raunhardt, H. W. H. Schmidt, and H. Neukom, Helu. Chim. Actu, 50, 1267 (1967). (526) T. Anmo, M. Washitak, H. Hayaski, Y. Yamaguchi, and A. Miyano, Yukuguku Zasshi, 89, 1308 (1969); Chem. Abstr., 71, 128,792 (1969). (527) T. T. Gorovits, Khim. Prir. Soedin., 5,49 (1969); Chem. Abstr., 71, 19,411 (1969). (528) K. Hotta, H. Hamazaki, M. Kurokawa, and S. Isaka, J. Biol. Chem., 245, 5434 (1970). (529) F. Yamauchi, M. Kosakai, and Z. Yosizawa, Biochem. Biophys. Res. Commun., 33, 721 (1968). (530) H. W. H. Schmidt and H. Neukom, Tetrahedron Lett., 2011 (1969). (531) H. W. H. Schmidt and H. Neukom, Helu. Chim.Acta, 49,510 (1966). (532) H. W. H. Schmidt, Tetruhedron Lett., 235 (1967).

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cosyluronate) derivatives.533They found that acetates of the phenolic and terpenoid D-glucosiduronic acids examined had too low a volatility and were unstable at high temperatures. The 0-methyl and 0-trimethylsilyl derivatives were suitable, with the former having the shorter retention times. Details of this work were published later and the method was extended to 1-0-acylglucuronates as well as N and S-D-glucosyhronic acid derivatives.534In general the N-D-glucosyluronic acid derivatives did not possess sufficient thermal stability to be useful. The work was directed to the isolation o f ~ g l u c o s i d uronic acids from urine. A similar study utilized a combination of gas-liquid chromatography and mass spectrometry to identify ethyl a-D-glucosiduronic acid as a metabolite of Other D-glucuronic acid conjugates have been examined as the trimethylsilyl ethers of their methyl esters.536 acid in lipopolyThe occurrence of 3-deoxy-~-manno-octu~osonic saccharides has prompted its synthesis,537together with the D-gal~ c t o 'and ~ ~ the D-gluco ana10gs.l~' Although gas-liquid chromatography was successfully used to analyze the products of these syntheses, it has been reported that methanolysis of a bacterial endotoxin lipopolysaccharide failed to yield 3-deoxy-D-manno-octulosonic acid, presumably because of the lability of the latter to acid.381However, Kasai and Nowotny have reported four peaks for the O-trimethylsilyl derivatives of 3-deoxy-D-manno-octulosonic acid obtained by methanolysis of the glycolipid from a Salmonella minnesota r n ~ t a n t . 5Reduced ~~ 3-deoxy-D-manno-octulosonic acid and its methyl ester have also been analyzed successfully as their acetate^.^^^,^^^ The quantitative determination of uronic acids in polysaccharides is complicated by lactonization, and these problems have been carefully examined by Blake and R i ~ h a r d s . In ~ ~the * ~ course ~ of these studies, D-glucuronic acid was reduced to D-glucitol, which was characterized as the h e x a a ~ e t a t eAs . ~ ~uronic acid residues in a polysaccharide may be reduced relatively easily,465$466 determination as the alditol provides an alternative method of a n a l y s i ~ . This ~ ~ ~pro.~~~ cedure is most valuable when different uronic acids are present in (533) Z. Tamura and T. Imanari, Chem. Pharm. Bull. (Tokyo), 12, 1386 (1964). (534) T. Imanari and Z. Tamura, Chem. Pharm. Bull. (Tokyo), 15, 1677 (1967). (535) P. I. Jaakonmaki, K. L. Knox, E. C. Horning, and M. G. Horning, Eur. J. Pharmucol., 1, 63 (1967). (536) J. B. Knaak, J. M. Eldridge, and L. J. Sullivan,J. Agr. Food Cheni., 15,605 (1967). (537) D. T. Williams and M. B. Perry, Can. J. Biochem., 47,691 (1969). (538) N. Kasai and A. Nowotny,J. Bacteriol., 94, 1824 (1967). (539) G. A. Adams, Can. J. Biochem., 49, 243 (1971).

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the same poly~accharide.~~' Sjostrom and coworkers attempted to determine the aldonic acids in a pulp liquor by separating the O-trimethylsilyl derivatives of the lactones, but, on account of the difficulties, previously referred to, of separating D-glucono- and D-galactonolactone they elected to reduce the lactones to the alditols, which were analyzed as the acetates.*15Such a reduction normally proceeds without difficulty because of the excess of sodium borohydride used, but the aldose may result instead of the alditol if the concentration of borohydride is very low.216

x. AMINO SUGARS AND AMINO ALDITOLS The two most common, naturally occurring aminoaldoses are 2-amino-2-deoxy-D-g~ucose and 2-amino-2-deoxy-D-galactose. Conventional methods of determining these amino sugars in mixtures are tedious, and many attempts have been made to adapt gas-liquid chromatography for their identification and determination. In general, amino sugars may be per(trimethylsily1)ated directly, as their N-acetyl derivatives, or as their methyl glycosides. In addition, they may be reduced to the free amino- or acetamido-deoxyalditols. Details are recorded in Table IX (see p. 143). 1. Hexosamines

In 1964, Perry540reported that the attempted per(trimethylsily1)ation of hexosamine hydrochlorides failed to give volatile derivatives, and he therefore proposed the use of the acetamidohexoses, as will be discussed. Two years later, Radhakrishnamurthy and associates541 showed that hexosamine hydrochlorides could be fully trimethylsilylated by using the ordinary reagents hexamethyldisilazane and chlorotrimethylsilane, provided that the reaction was conducted in N,N-dimethylformamide for 2 minutes at 100". They further showed that, on a column of Apiezon M, 2-amino-2-deoxy-D-glucose and 2-amino2-deoxy-D-galactose each gave two peaks that were attributed to the anomeric forms; they used this method to analyze hydrolyzates of hyaluronic acid, chondroitin and heparin,'6 after separation of the amino sugars from neutral ones by use of an ion-exchange resin. (540) M. B. Perry, Con. I. Biochem., 42,451 (1964). (541) B. Radhakrishnamurthy, E. R. Dalferes, and G. S. Berenson, Anal. Biochem., 17, 545 (1966).

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Oates and Schrager reported a method for the determination of neutral sugars in glycosamin~glycans,~~~ and extended this procedure to include h e x o ~ a m i n e s .In ~ ~the latter study, they investigated the trimethylsilyl derivatives of hexosamines and found much smaller peaks than expected. They also noted that these peaks had retention times similar to that of per-0-(trimethylsily1)-a-D-galactose. In addition, they obtained unsatisfactory results with the acetamidohexoses and preferred to use the N-ethoxycarbonyl derivatives of the amino sugars. In their work, a system was used wherein solids were injected on glass-wool plugs.36,285 Sweeley and colleagues, in their original paper^,^,^^ had shown that per(trimethylsily1)ated hexosamines can be satisfactorily chromatographed on non-polar columns such as SE-30 and SE-52 without prior N-acetylation. These results were confirmed and amplified by Kiirkkainen and coworkers179who showed that hexosamines can be trimethylsilylated in 15 minutes at room temperature by using the customary reagents hexamethyldisilazane and chlorotrimethylsilane in pyridine, provided that the ratio of trimethylsilylating reagents to hexosamine is greater than 1 O : l . Subsequent work showed that the amino group was not trimethyl~ilylated.'~~ On a non-polar column of SE-30, 2-amino-2-deoxy-~-glucosegave two peaks, and 2-amino-2deoxy-D-galactosegave one peak, but, on a polar phase, such as QF-1, each gave two peaks. Per-0-(trimethylsily1)hexoses have retention times similar to those of the hexosamine derivatives, and therefore the latter were separated from neutral sugars by ion-exchange. This method was then used to analyze glycosaminoglycans from and hagfish (Myxine g l u t i n o s ~ )Other . ~ ~ ~ workers have used the same technique for the determination of 2-amino-2-deoxy-~glucose in al-acid glycoprotein of human plasma,542and to examine keratan sulfate fractions.331 Karkkainen and V i h k ~ have ' ~ ~ extended these studies, in connection with a mass-spectroscopic investigation, to include methyl aminodeoxyhexosides, aminodeoxyhexitols, and their N-acetylated forms. Mass spectrometry clearly showed that the normal trimethylsilylation reagents caused reaction only with the hydroxyl groups, but complete trimethylsilylation was achieved when bis(trimethylsily1)acetamide was added to the reagent mixture. Conversion into the per(trimethy1silyl) derivative was obtained in 30 minutes at room temperature by using a reagent consisting of bis(trimethylsily1)acetamide-chlorotrimethylsilane-hexamethyldisilazane-pyridine in the ratios 2:1:2:20. (542) R. W. Jeanloz,Abstr. 7th Int. Congr. Biochem., Japan, August 1967.

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On a column of SE-30, both 2-amino-2-deoxy-D-g~ucoseand 2-amino2-deoxy-~-galactosegave two peaks, with some overlap of the isomers. The retention times of the per(trimethylsily1) derivatives were greater than for those having a free amino group, and the Si-N bond was found to be very labile compared with the Si-0 bond. Some decomposition occurs during chromatography, but even keeping the compound in hexane solution for 1-2 hours causes conversion into the tetrakis(trimethylsily1)derivative. With the advent of more powerful trimethylsilylating reagents, Stimson'*ohas shown that N,O-bis(trimethylsily1)trifluoroacetamide in N,N-dimethylacetamide will completely trimethylsilylate 2-amino-2deoxy-D-glucose and 2-amino-2-deoxy-D-ga~actosehydrochlorides in 10 minutes at 100". On a column of 3% Apiezon L, each hexosamine gave two peaks. No direct evidence was given that an N,O-pentakis(trimethylsilyl) derivative was obtained, but, as this reagent is known543 to form N-trimethylsilyl derivatives with amino acids, complete trimethylsilylation was inferred. Gheorghiu and Oette34have made a very detailed study of the carbohydrate analysis of biological fluids. They used an acid hydrolytic procedure prior to trimethylsilylation, and employed polar columns such as ethylene glycol succinate. Under these conditions, neutral sugars were well resolved, but 2-amino-2-deoxy-~-g~ucose and 2-amino-2-deoxy-~-galactose gave overlapping peaks. The method is satisfactory for the determination of total hexosamine, but not for differentiating isomeric amino sugars. 2. Acetamidodeoxyhexoses

Sweeley and colleagues5 also showed that acetamido sugars can be chromatographed as 0-trimethylsilyl derivatives, and they were able to separate, on a column of SE-52, the derivatives of 2-acetamido2-deoxy-D-glucose, 2-acetamido-2-deoxy-~-galactose, and 2-acetamido-2-deoxy-~-mannose. Perry540 published simultaneously a study on the separation of 2-amino-2-deoxy-D-glucose and 2-amino2-deoxy-D-galactose7and also recommended the use of the trimethylsilylated acetamido derivatives. Each sugar gave two peaks, corresponding to the anomeric glycosides, and the trimethylsilyl 2-acetamido - 2-deoxy-3,4-6- tri- 0- (trimethylsilyl)- CX-D - glycosides of each sugar have been obtained crystalline. The procedure was tested by analyzing mixtures of uronic acid and chondroitin s~lfate.5~~ (543) C. W. Gehrke, H. Nakamoto, and R. W. Zumwalt,J. Chromatogr., 45, 24 (1969).

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The method of Perry was subsequently used to determine 2-amino-2deoxy-D-glucose in lipopolysaccharides from Serratia r n a r c e s ~ e n s , 4 ~ ~ ~ ~ ~ ~ and to determine 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxyD-galactose in other polysaccharides from the same source.493,494 It has also been used to identify 2-amino-2-deoxy-D-glucose in the glycan from Myxobacterium 402, and in studies of the carbohydrate antigens of Streptococcus ~ u l i ~ ) a r i u ~ . ~ ~ ~ ~ In a paper concerned primarily with the determination of neutral sugars, Richey and coworkers examined the 0-trimethylsilyl derivatives of 2-acetamido-2-deoxy-~-glucose and 2-acetamido-2-deoxyD-galacto~e."~Similar methods for determining these compounds have also been used in studies on human yeasts and bacteria,lI2 blood-group o l i g o s a ~ c h a r i d e and s ~ ~urine."* ~ These methods were also included in a model study on the isothermal determination of sugars.256 Kiirkkainen and V i h k ~ , in ' ~ ~their work cited earlier, also studied the 0-trimethylsilyl derivatives of acetamidodeoxyhexoses. Of the two liquid phases that they used, they found QF-1 more suitable for the separation of anomers, but SE-30 was better for separating 2amino-2-deoxy-~-glucosederivatives from those of 2-amino-%deoxy-~galactose. Oates and SchrageP reported unsatisfactory results with acetamidodeoxy derivatives, and preferred to use trimethylsilylated N-ethoxycarbonyl derivatives. They used this method to analyze glycoproteins from body r n ~ c o s a ,human ~ ~ ~ ~aliva,~46 gastric aspirate~:~' and bile.548 In work on gastric-juice glycoprotein, Mathian and associates549 have separated 2-acetamido-2-deoxy-~-glucose and the corresponding D-gulacto analog as their 0-trimethylsilyl derivatives. They found that each compound gave two peaks, the relative sizes of which depended on the conditions of trimethylsilylation. Levvy and compared the determination of neutral and amino sugars by procedures involving hydrolysis and methanolysis. By the two procedures, they obtained recoveries of92 and 100% for (544) G. A. Adams and S. M. Martin, Can. J . Biochern., 42, 1403 (1964). (544a) G. C. Kothari, J. M. N. Willers, and M. F. Nichel, J. Gen. Microbiol., 68, 77 (1971). (545) A. Kobata, V. Ginsburg, and M. Tsuda, Arch. Biochern. Biophys., 130,509 (1969). (546) J. Schrager and M. D. G. Oates, Arch. Oral Biol., 16, 287 (1971). (547) J. Schrager and M. D. G. Oates, Arch. Oral Biol., 16, 1269 (1971). (548) J. Schrager, M. D. G. Oates, and A. Rosbottom, in press. Vuez, and R. Lambert, B i d . Gastro-Enterol..2, 127 (549) R. Mathian, F. Martin, J.-L. (1969).

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G . G . S. DUTTON

neutral sugars, and about 96 and 88% for amino sugars, respectively; hydrolysis (with 2M hydrochloric acid) was, therefore, recommended, for the determination of amino sugars. In each instance, N-acetylation was recommended, and is mandatory in the hydrolytic procedure if overlap of neutral and amino sugar peaks is to be avoided. These authors proposed a method of N-acetylation resembling the RosemanLudowiegSSO procedure, involving reaction with acetic anhydride in aqueous acetone on a column of Dowex-1 resin in the carbonate form. Quantitative N-acetylation was effected in 50% aqueous acetone, and methanol was shown to be inferior to acetone in promoting acetylation. The authors used a column of 3.8% SE-30, but gave no retention times; it is assumed that 2-amino-2-deoxy-D-g~ucoseand 2-amino-2-deoxy-~-galactosewere not separated. Thus the method is only suitable for determination of total hexosamine. Retention times have been given for the 0-trimethylsilyl derivatives of synthetic acetamido d i s a c ~ h a r i d e s . ~ * ~ * ~ ~ ~ 3. Methyl Acetamidodeoxyhexosides

The difficulties inherent in hydrolyzing polysaccharides containing amino sugars have been discussed in Section I1 (see p. 14); methanolysis is usually employed in such instances. The comments of Levvy and colleaguesG4in Section X,2 should, however, be noted. The amino sugar is thus obtained as a mixture of methyl glycosides. As methanolysis causes partial N-deacetylation of the naturally occurring, acetamidodeoxy sugars, it is usual to perform N-acetylation before trimethylsilylation. This approach was used by Sweeley and WalkeP in studies on glycolipids and gangliosides. Apart from the fact that methanolysis causes deacetylation to a variable extent, N-acetylation was also desirable as the trimethylsilyl derivatives of methyl D-galactoside and methyl 2-amino-2-deoxy-~-galactoside gave overlapping peaks. These authors observed that the per(trimethy1silyl) ether of methyl 2-acetamido-2-deoxy-~-galactoside gave an unexpectedly large number of peaks. This procedure has been used to analyze ceramides in and total hexosamine in blood lipids.334 Several model investigations directed toward the analysis of glycoproteins have used methanolysis, and one of the first such model studies was the article by Clamp, Dawson, and HoughEG and their two earlier communications.3G4~365 These authors examined mixtures containing D-galactose, D-mannose, L-fucose, 2-acetamido-2-deoxy-Dglucose, and neuraminic acid. The solution, after methanolysis, was (550) S. Roseman and J. Ludowieg,J. Amer. Chem. SOC., 76,301 (1954).

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neutralized with silver acetate, and N-acetylated directly with acetic anhydride. The mixture was evaporated to dryness, trimethylsilylated, and examined on a column of SE-30, whereupon each compound except the neuraminic acid gave at least two peaks; these were attributed to the anomeric pyranosides. In some instances, a small peak for the furanoside was noted. These authors examined conditions of methanolysis and N-acetylation, and gave the equilibrium composition of mixtures of glycosides. As many glycoproteins contain less than 10% of carbohydrate, the analyses were checked in the presence of a large excess of bovine-serum albumin. This addition did not invalidate the carbohydrate analyses, and the method was used to compare gas-liquid chromatography and colorimetric procedures for the analysis of typical glycopeptides and glycoproteins; excellent correspondence was observed. This procedure was essentially that used by Jeanloz and his group to analyze connective and the glycoprotein of the TA, cell.368T ~ a i has ~ ~used l methanolysis to determine the degree of polymerization of oligosaccharides from chitin. Clamp and his colleagues have extended this method to a much wider range of carbohydrates, and have reported equilibrium percentages for methyl glycosides and retention times of the derived trimethylsilyl ethers for two acetamidodeoxypentoses, seven acetamidodeoxyhexoses, and various neutral sugars and deoxy sugar^.^^^,^^ This procedure has been used to analyze glycopeptides having antigenic activity.366A very similar method, employing a column of SE-30, has been used by Sinkinson and Wheelockes to examine glycopeptides of milk371and to identify 2-acetamido-2-deoxy-~-galactose in ~-casein.~~O A variation of the original procedure of White552was used by Salfner and U h l e n b r u ~ kin~ their ~ investigation of serologically active glycoproteins; the method was to neutralize the methanolysis solution with silver carbonate, centrifuge it, evaporate it to dryness, and N-acetylate the residue by treatment for 12 hours at room temperature with acetic anhydride in methanol. Following trimethylsilylation, the derivatives were separated on a column of OV-17. They found that certain neutral and amino glycosides overlapped if the N-acetylation step was omitted. N-Acetylation causes the retention times to be greatly increased, and allows clear separation of peaks arising from acetamidodeoxy glycosides from those of neutral glycosides. This effect is much greater144on a polar column such as QF-1. (551)C. S. Tsai, Anal. Biochem., 36, 114 (1970). (552)T. White,]. Chem. Soc., 428 (1940).

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determined amino sugars in gangliosides Mora and as the trimethylsilyl ethers of the methyl acetamidodeoxyhexosides, and stressed the importance of the N-acetylation step following methanolysis. They also found that operation with a temperature program, instead of isothermally, gives better resolution of the methyl glucoside and galactoside peaks, and also permits the elution of the sialic acid derivative. Furthermore, they recommended that the columns be kept at 100"overnight (see p. 61). Methyl aminodeoxyhexosides have also been as their per(trifluor0acetates) (see Section X,5, p. 87). 4. Acetamidodeoxyalditols

It will be recalled from Section VII (see p. 56) that Gunner, Jones, and Perry4OBwere among the first to separate alditol acetates and, in a continuation of these investigations, H. G . Jones, J. K. N. Jones, and Perry553 described the separation of acetylated amino sugars and aminodeoxyalditols. As these compounds have retention times longer than those of pentitol and hexitol acetates, it was possible to separate neutral sugars and amino sugars on the same column. Unfortunately, the packings proposed were relatively complicated; when Bishop and showed shortly thereafter that acetylated amino sugars are subject to thermal degradation, attention was turned to the newly introduced 0-trimethylsilyl derivative^.^ PerryS40had shown that amino sugars can be separated from aminodeoxyalditols; Horowitz and Delman329 developed this idea and demonstrated that 2-acetamido-2-deoxy-~-glucitol and 2-acetamido-2deoxy-D-galactitol are separable as their per-0-(trimethylsilyl) derivatives on a column of butanediol succinate. The D - ~ U ~ Z ~analog ZO has a retention time intermediate between those of the other two, and could be resolved from them. This investigation was concerned particularly with identifying the alditol formed by alkaline cleavage of oligosaccharide side-chains in glycoproteins. As 2-amino-2-deoxyD-mannose is uncommon in glycoproteins, this lack of resolution is not critical. However, a column of SE-30 did separate per-0-trimethylsilyl-D-glucitol and -D-mannitol, but failed to resolve trimethylsilylated 2-acetamido-2-deoxy-~-glucitol and 2-acetamido-2-deoxyD-galactitol. Murty and H o r o w i t ~ used ~ ~ this ~ method in a subsequent (552a) I. Dijong, P. T. Mora, and R. 0. Brady, Biochemistry, 10,4039 (1971). (553) H. G . Jones, J. K. N. Jones, and M. B. Perry, Can. J . Chem., 40, 1559 (1962). (554) C. T. Bishop, F. P. Cooper, and R. K. Murray, C a n . ] . Chern., 41,2245 (1963). (555) V. L. N. Murty and M. I. Horowitz, Carbohyd. Res., 6,266 (1968).

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investigation of ovine submaxillary mucin, and demonstrated that 78% of the sialic acid released was recovered linked to 2-acetamido2-deoxy-D-galactitol. In a similar manner, Bertolini and Pigman556 established the presence of 2-acetamido-2-deoxy-~-glucitol and 2-acetamido-2-deoxy-~-galactitol after treating mucins with alkaline borohydride. T ~ a i has ~ ~ proposed l a method for determining the degree of polymerization of oligosaccharides from chitin wherein they are reduced with sodium borohydride, methanolyzed, and the content of 2-acetamido-2-deoxy-~-glucitol determined as the trimethylsilyl ether. In a study on the alkaline stability of cellulose, Clayton and JoneslEE chromatographed the trimethylsilyl ether of 1-amino-1-deoxy-D-glucitol, and the retention time of a synthetic aminodisaccharide alditol has been given.487 All of the work so far described has been primarily concerned with the separation of 2-amino-2-deoxy-~-glucoseand 2-amino-2-deoxyD-galaCtoSe, but Perry and Webb557pointed out that 2-amino-2-deoxyD-mannose, 2-amino-2-deoxy-~-gulose,and 2-amino-2-deoxy-~-talose are known to occur naturally and other hexosamines will probably be found. It is thus of interest to develop systems whereby all of the possible hexosamines may be separated. Perry and Webb557have shown that all eight of the 2-acetamido-1,3,4,5,6-penta-O-acetyl-2deoxyhexitols may be separated from each other by the use of two column-packings. Furthermore, the same system may be used to separate neutral sugars, including aldohexoses and aldoheptoses, since the neutral alditol acetates have retention times lower than that of the first 2-acetamido-2-deoxy derivative to emerge.557The degradations previously observed during the gas-liquid chromatography of acetylated acetamidodeoxy sugarP4may be avoided if ( a )the samples are injected directly onto the column packing, ( b )discontinuities in the packing are absent, and (c) the detector is close to the end of the column packing. The columns used were 10% of neopentylglycol sebacate and a mixture of 1.5% of XF-1150 and 1.5% of neopentylglycol sebacate. The separation and determination of amino sugars as the acetylated aminodeoxyalditols have been used in studies on Neisseriu siccu,558,559 (556) M. Bertolini and W. Pigman, Carbohyd. Res., 14, 53 (1970). (557) M. B. Perry and A. C. Webb, Can. J . Biochem., 46, 1163 (1968). (558) I. J. McDonald and G. A. Adams,]. Gen. Microbiol., 65, 201 (1971). (559) G. A. Adams, M. Kates, D. H. Shaw, and M. Yaguchi, Can.]. Biochem., 46, 1175 (1968).

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N . catarrhalis,5w N . p e ~ j l a v a , 5Moruxella ~~ duplex and Micrococcus cal~o-acetic~~,443 and Escherichia Vicari and in studies of blood-group oligosaccharides, and Hellerqvist and c0lleagues,5~in an examination of the common core-polysaccharide of Salmonella typhimurium, have used similar methods. The examination of amino sugars as their peracetylated aminodeoxyalditols has also been used by Liideritz and colleagues72to establish the occurrence of 4-amino4-deoxy-~-arabinosein Salmonella lipopolysaccharides, and has been extended to aminodeoxyheptoses by Williams and Lenhardt and W i n ~ l e rhave ~ ~ described a very good method for the hydrolysis of glycoproteins, but the column used for analysis appears better suited to neutral sugars, especially 6-deoxy sugars, than to amino sugars; the peracetates of 2-amino-2-deoxy-~-glucitol and 2-amino-2-deoxy-D-galactitolwere not separated. Niedermeiel' has described a procedure for the analysis of glycoproteins, in which the alditol acetates are separated on a column of 1% ECNSS-M, that is capable of resolving the derivatives of 2-amino2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose.This method has been applied to the analysis of immunoglobulin^.^^ Niedemeierss2 has stated that a column of ECNSS-M is incapable of resolving the alditol acetates of 2-amino-2-deoxy-D-mannose and 2-amino-2-deoxyD-galactose, and, for this purpose, 3% of Poly-A 103 was preferred, although it is incapable of separating the acetates of D-glucitol and galactitol . The introduction of liquid phases having improved temperaturestability has facilitated the simultaneous determination of neutral and amino sugars as acetates. Thus, Griggs and and Metz and coworkersGoa have both used columns coated with OV-225. The first group originally used a three-component liquid phase, but subsequently found that a column of 3% of OV-225 (used with temperature programming) gave comparable results. The latter group used a column of 1%of OV-225, with a temperature program from 170-230" at 1" per min. This liquid phase does not resolve the peracetates of 2-acetamido-2-deoxy-~-galactitol and 2-acetamido-2-deoxyD-mannitol. Griggs and showed that the d e c o r n p o ~ i t i o nof~ ~the ~ peracetates of aminodeoxyalditols is minimized if the temperature of the injection port is held below 220". Furthermore, they identified the product formed from peracetylated 2-amino-2-deoxy-~-glucitol (560) G . A. Adams, T. G. Tornaben, and M. Yaguchi, Can.J. Micmbiol., 15,365 (1960). (561) D. T. Williams and M. B. Perry, Can. J . Chern., 47, 4493 (1969). (562) W. Niedermeier, personal communication.

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as 2-methy~-4-(D-urabino-tetraacetoxy)oxazoline; this may be reconverted to the parent sugar derivative by hydrolysis with aqueous acid.

5. Trifluoroacetates and Other Derivatives Vilkas and showed that carbohydrates can be separated as their trifluoroacetates, and included 2-amino-2-deoxy-~-glucose and deoxystreptamine in this study. Likewise, Ueno and coworkers225 were able to separate the trifluoroacetates of certain aminodeoxyglucoses and aminodeoxycyclitols. The separation of aminodeoxyhexitols as their trifluoroacetyl derivatives has been studied by Tamura and coworkers,563who found a column of 2% XF-1105 the most satisfactory. Ando and Y a m a k a ~ a included ~~l~ amino sugars in their study of the use of trifluoroacetates for the analysis of glycolipids. They found that the main peaks representing the per(trifluor0acetates) of methyl 2-amino-2-deoxy-~-galactopyranoside and D-ghcopyranoside were clearly separated from each other, but that the first, small peak of the former overlapped the principal peak of the latter, and that the main peak of the D-galactose derivative overlapped the second, small peak of the D-glucose isomer. For the D-galactose compound, the two peaks were in the ratio of 14.8:85.2 and 7.592.5 for the D-glucose compound. However, when Tay-Sachs ganglioside was analyzed by use of these derivatives, the peaks for 2-amino-2-deoxy-D-galactose lay between those for the neutral sugars and for N-acetylneuraminic acid, and were well separated from each group. An original approach to the analysis of amino sugars has been proposed, based on their reaction with nitrous acid and subsequent reduction of the products with b ~ r o h y d r i d e .By ~ ~ this ~ procedure, 2-amino-2-deoxy-~-glucoseand 2-amino-2-deoxy-D-galactose are converted into 2,5-anhydrohexitols, whereas 2-amino-2-deoxy-D-mannose yields D-glucitol. This mixture is readily separated after trimethylsilylation. XI. ANHYDRO COMPOUNDS

Certain carbohydrates are readily converted by acid or heat into anhydro derivatives, and this reaction may complicate both identifi(563) Z. Tamura, T. Imanari, and Y. Arakawa, Chem. Pharm. Bull. (Tokyo), 16, 1864 (1968). (564) S. Hase and Y. Matsushima,]. Biochem. (Tokyo), 66,57 (1969).

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cation and quantitative determination. Thus, part of the ribitol present in cell-wall preparations may be converted into an anhydroribitol which, in many chromatographic systems, behaves similarly to glycerol. These two compounds can, however, be readily distinguished and determined by gas-liquid chromatography of their O-trimethylsilyl derivatives.401Heating converts D-glucose partially into its 1,6anhydride, and the determination of this in corn syrups is of commercial i m p o r t a n ~ e . 'Applications ~~ of gas-liquid chromatography with anhydrides of sugars are listed in Table X (see p. 147). It was in order to provide a suitable method for the quantitative determination of 1,6-anhydro-p-~-glucopyranose that Sloneker and coworkers first investigated the separation of 1,6-anhydro sugars as their trimethylsilyl ethers.*OThey examined four column-packings and concluded that XE-60 was the most satisfactory. Turunen and associate^'^ have utilized these results to show that 1,6-anhydro-/?-~-glucopyranose and its furanose analog have, as their per(trimethylsily1) derivatives, retention times similar to those of D-xylose; erroneous results may thus be obtained in the analysis of wood pulps. This problem may be overcome by operating an SE-52 column at 135", when the anhydro derivatives are separated from the xylose derivatives, whereas at 160"the peaks overlap. 176-Anhydro-/3-~-glucose may also be prepared in methyl sulfoxide, and the proportion of furanoside and pyranoside isomers determined by using trimethylsilyl ethers.1311,6-Anhydro-p-~-glucopyranose and some of its methyl ethers have been polymerized, and the reaction monitored by determining the rate of consumption of monomer,125 and the thermal analysis of 1,6-anhydro-/3-~-glucopyranosehas been Anhydro sugars have been chromatographed as their O-trimethylsilyl derivatives in studies on 4-deoxyuronic and on the pyrolysis of carbohydrates.56s.567 The epimerization of D-glueuronic acid to L-iduronic acid has been confirmed by identifying the 1,6-anhydro-p-~-idopyranose obtained on reduction.568In addition, the equilibrium between heptuloses and their 2,7-anhydrides has been ~ t u d i e d . A ' ~ useful method (see p. 87) for the identification and determination of amino sugars involves sequential treatment with nitrous acid and sodium borohydride, and separation of the anhy(565) F. Shafizadeh, C. W. Philpot, and N. Ostojic, Carbohyd. Res., 16, 279 (1971). (565a) F. Shafizadeh, G. D. McGinnis, and P. S. Chin, Corbohyd. Res., 18,357 (1971). (566) D. Gardiner, J . Chem. SOC. ( C ) , 1473 (1966). (567) Y. Halpern and S. Patai, IsraeZJ. Chem., 7, 673, 685 (1969). (568) H. E. Carter, A. Kisic, J. L. Koob, and J. A. Martin, Biochemistry, 8, 389 (1969).

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droalditols formed.564,569 In a study of the equilibrium between aldohexoses, 3-deoxyhexoses, and their 1,6-anhydrides, it was found convenient to separate the components as their and anhydro-octuloses isolated from bacterial and algal polysaccharides have been similarly studied.570In demonstrating the existence of L-iduronic acid in heparin, Perlin and S a n d e r ~ o nseparated ~~~ 1,6-anhydrop-L-idopyranose from 2,5-anhydro-~-mannitolas their acetates. L-Gulose readily forms an anhydride, and in a study on alginic acid, methylated L-guloses were isolated as their anhydrides.*8Other anhydro sugars, such as 3,6-anhydro-~-galactoseand its 2-methyl ether, may be found in algae (see, for example, Ref. 412), and, under certain circumstances, 2,3,6-tri-O-methyl-D-galactose may form an anh~dride.5~~ XII. CYCLITOLS myo-Inositol and its isomers occur in small proportion in diverse natural sources, as, for example, plant extract^,^'^^^^^,^^^,^^^ animal tissue,178,406.572-575 and b a ~ t e r i a . " ' , " ~ ,Amino ~ ~ ~ derivatives of cyclitols may also be components of carbohydrate antibiotics.225The inositols have been separated and determined by gas-liquid chromatography, most commonly as their trimethylsilyl ethers, but also as their acebtes91,576-578 and their trifluor~acetates"~.""~ (see Table XI, p. 149). Lee and Ballou"' investigated five liquid phases for the separation of inositol trimethylsilyl ethers and concluded that QF-1 gave the best results. This column packing permitted resolution of all but the myo-cis pair of inositols. They reported that the order of appearance, regardless of the stationary phase used, is: ullo, (neo, muco), racemic, (scyllo, epi), and (myo, cis). The order within the parentheses is sometimes reversed, depending on the liquid phase used. Wells and co(569) M. Isemura and K. Schmid, Biochem. J., 124, 591 (1971). (570) P. A. J. Gorin and T. Ishikawa, Can. J. Chem., 45, 521 (1967). (571) H. R. Schreuder, W. A. CBt& and T. E. Timell, Svensk Papperstidn., 69, 641 (1966). (572) W. W. Wells, T. A. Pittman, and H. J. Wells, Anal. Biochem., 10, 450 (1965). (573) M. A. Stewart, V. Rhee, M. M. Kurien, and W. R. Sherman, Biochim. Biophys. Actu, 192, 361 (1969). (574) K. Narumi and T. Tsumita, Jap. J. E x p . Med., 39, 409 (1969). (575) K. Nammi, M. Arita, M. Kitagawa, A. Kumazawa, and T. Tsumita,Jap. J. E x p . Med., 39, 399 (1969). (576) N. Shaw and F. Dinglinger, Biochem.J., 112, 769 (1969). (577) S. J. Angyal, P. A. J. Gorin, and M. E. Pitman, J . Chem. Soc., 1807 (1965). (578) Z. S. Krzeminski and S. J. Angya1,J. Chem. Soc., 3251 (1962).

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workers572have shown that, by use of a non-polar column (SE-30) and a polar one (ethylene glycol succinate), most of the isomeric inositols may be satisfactorily resolved. For example, on a column of SE-30, the relative retention-times for neo- and mum-inositol are 1.10 and 1.18, respectively, whereas, on the polyester column, the figures are 1.51and 0.91. Gheorghiu and Oette34have also shown that five of the inositols are well separated on a column of ethylene glycol succinate. Comment has already been made in Section I11 (see p. 23) on the sparing solubility of inositols in pyridine. This factor may lead to erroneous results through under-trimethylsilylation, or may require an inconveniently long time for the trimethylsilylation reaction. One of the earliest examples of trimethylsilylation in methyl sulfoxide was in connection with the determination of myo-in~sitol.’~~ Some workers have elected to use myo-inositol as an internal standard, but its sparing solubility in pyridine may cause complications; furthermore, Oates and Schrager have shown that, under certain conditions, this standard may overlap with the 0-trimethylsilyl derivative of p - D - g l u c o ~ e .As ~ ~myo-inositol ~ is of widespread occurrence in the plant kingdom, it should not be used as a standard when investigating the carbohydrate composition of plants. Hexa-0-(trimethylsily1)-myo-inositolis a solidY1l2and this compound and the crystalline per(trimethylsily1) ethers of scyllo-inositol and myo-inosose-2 have been fully characterized by L O ~ W Uwho S,~~ has proposed these compounds as suitable internal standards. The scyllo- and myo-inositols, together with myo-inosose-2, have also been used in a study on the use of electron-capture detectors with trimethylsilyl derivati~es.5~~ COMPOUNDS* XIII. POLYHYDFUC In 1952, F. Smith and coworkers5s0proposed a method for investigation of polysaccharide .structures based on the sequence: periodate oxidation, borohydride reduction, and complete hydrolysis. By this procedure, a linear, (1 ---* 4)-linked hexoglycan gives rise to glycerol from the non-reducing end-group and to a tetritol from the internal residues of the chain. Determination of the ratio of glycerol to tetritol thus provides an alternative method to methylation for the * Included here are compounds having two to four carbon atoms. Compounds having five or more carbon atoms are included in Section VII (see p. 56). (579) W. R. Sherman and S. L. Goodwin,J. Chromatogr. Sci., 7,167 (1969). (580) M. Abdel-Akher, J. K. Hamilton, R. Montgomery, and F. Smith, J . Amer. Chem. Soc., 74, 4970 (1952).

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determination of chain length. For a (1 + 4)-linked pentoglycan, the corresponding products are ethylene glycol and glycerol. It should be noted that this reaction is referred to incorrectly by many authors as the Smith degradation. This designation should only be used when advantage is taken of the relative acid-labilities of acetal and glycosidic linkages (see Section XIV, p. 98). As the method here under discussion is an alternative procedure for end-group analysis, it may conveniently be referred to as the Smith end-group analysis. Many determinations of glycerol and related compounds have been performed by Smith and his group by using paper-chromatographic separation. However, when reducing sugars, from monosaccharide residues immune to periodate oxidation, are also present, two distinct colorimetric procedures are necessary. As both types of compound may be determined simultaneously by gas-liquid chromatography, it is surprising that this technique has not yet found the wide adoption forecast by Bishop in his a r t i ~ l eExamples .~ that have been recorded are listed in Table XI1 (see p. 151). The first application of gas-liquid chromatography to such analysis was made by Bishop and Cooper,581who examined a glucomannan from jack pine and found the ratio of glycerol to erythritol to be 1:21 (by separation of their acetates). This method had the added advantage that erythritol tetraacetate is crystalline and thus readily characterized. The same method has been used to establish the presence of glycerol and the absence of D-threitol in an investigation of the galac~ ~in tans found in the albumin glands of Biomphalaria g l a b ~ u t a , "and S trophocheilus o b l o n g ~ sA. glucan ~ ~ ~ from Microsporum quinckeanum was also to be branched, by the isolation, in the ratios 72.3: 0.6 :27.1, of glycerol, erythritol, and D-glucose as their acetates. Zitko and Bishop have used a similar method to investigate a carboxylreduced galacturonan from pectic acid, which yielded ethylene glycol, glycerol, and D-threit~l.~" Bishop and associates have also shown that polysaccharides may be oxidized in methyl sulfoxide solution, In each example, either by lead t e t r a a ~ e t a t eor~ by ~ ~ periodic the compounds obtained by subsequent reduction and hydrolysis (581) C. T. Bishop and F. P. Cooper, C a n . ] . Chem., 38, 793 (1960). (582) J. Batista, C. Corrsa, A. Dmytraczenko, and J. H. Duarte, Curbohyd. Res., 3, 445 (1967). (583) J. H. Duarte and J. K. N. Jones, Carbohyd. Res., 16, 327 (1971). (584) H. Alfes, C. T. Bishop, and F. Blank, Con. J. Chem., 41, 2621 (1963). (585) V. Zitko and C . T. Bishop, Can.]. Chem., 44, 1275 (1966). (586) V. Zitko and C. T. Bishop, Cnn. 1.Chem., 44, 1749 (1966). (587) R. J. Y u and C. T. Bishop, C a n . ] . Chem., 45,2195 (1967).

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were determined and identified by gas-liquid chromatography. It should be noted that concentrated solutions of periodic acid in methyl sulfoxide are explosive,588and other explosive reactions in this solvent have been recorded.589 Alditol acetates have also been separated during structural studies on the glucan of Phytophthora cinnana0rni,5~~ on the polysaccharide elaborated by Arthrobacter V ~ S C O S U S and , ~ ~ ~in studies on sophorosides.592Erythritol has been separated and identified as the tetraacetate in studies by Wilkie and collaborators on the structure of an oat g l ~ c a and n ~ the ~ ~hemicellulose from an aquatic moss.593HofFman and Time11,594in their work on a galactoglucomannan from red pine (Pinus resinosa), have separated glycerol and erythritol as their acetates, as also have Perlin and S a n d e r s ~ nduring ~ ~ ~ studies on heparin. Gorin and his colleagues have identified erythritol (as the tetraacetate) among the degradation products of an exocellular alginic acid from Azotobacter ~ i n e l a n d i iThe . ~ ~ same ~ group, in studies on polysaccharides containing L-rhamnose, have warned that glycerol and 1-deoxy-D-erythritol (4-deoxy-~-erythritol)are indistinguishable by paper chromatography, although they are readily separable by gasliquid chromatography of their a ~ e t a t e s . 3The ~ ~ 'lipopolysaccharide ~~~ from SaZmoneZZa friedenau T1 contains mainly D-ribose and D-galactose, and, on periodate degradation, yielded D-threitol, which was separated and identified by means of the tetraa~etate.'~.~~' Tetritols, especially erythritol, have been included in model studies on the separation of alditol acetates (see, for example, Refs. 217 and 417), and a commercial analysis of glycerol as its acetate has been proposed.597 Application of the Smith end-group analysis to xylans gives ethylene glycol, glycerol, and D-xylose (as well as acidic fragments). This

(588) J. J. M. Rowe, K. B. Gibney, M. T. Yang, and G. G. S. Dutton,]. Amer. Chem. Soc., 90,1924 (1968). (589) G. G. Allan, E. Moks, and E. N. Nelson, Chem. Ind. (London), 1706 (1967). (590) L. P. T. M. Zevenhuizen and S. Bartnicki-Garcia,Biochemistry, 8, 1496 (1969). (591) J. H. Sloneker, D. G. Orentas, C . A. Knutson, P. R. Watson, and A. R. Jeanes, Can.J. Chem., 46, 3353 (1968). (592) A. P. Tulloch, A. Hill, and J. F. T. Spencer, Can. J. Chem., 46, 3337 (1968). (593) K. C. B. Wilkie and D. S. Geddes, Carhohyd. Res., 18,333 (1971). (594) G. C. Hoffman and T. E. Timell, Tuppi, 53, 1896 (1970). (595) P. A. J. Gorin and J. F. T. Spencer, Can.J.Chem., 44,993 (1966). (596) S. S. Bhattacharjee, R. H. Haskins, and P. A. J. Gorin, Carbohyd. Res., 13, 235 (1970). (597) L. Hartman, /. Chromatogr., 16, 223 (1964).

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method was applied by Dutton and U n r a ~ r to ” ~acidic ~ xylans by using paper-chromatographic separations, and was adapted by Zinbo and Time11599for gas-liquid chromatography of the trimethylsilyl derivatives. The small proportion of ethylene glycol normally present, together with the volatility of its trimethylsilyl ether that causes it to overlap with the “tail” from the pyridine solvent, made quantitative determination difficult. Nevertheless, good agreement with colorimetric determinations was obtained, and the method was used in studies on the xylans from aspen,599eastern hernlock,6OO and Norway spruce.601Mixtures of per(trimethylsily1)ated ethylene glycol and glycerol have been separated by Siddiqui and Wood6ozin studies on rapeseed “amyloid,” and in model studies on a true Smith degradation.lgOOn account of the volatility of 1,2-bis(trimethylsilyloxy)ethane, it is not practical to evaporate off the pyridine after trimethylsilylation to permit injection in a more-volatile solvent; a short period of isothermal operation before temperature programming is, therefore, recommended. Mixtures of glycerol and erythritol have been encountered in studies on Micrococcus lysodeikticus,30° and of ethylene glycol with erythritol from a seed gala~tomannan.~~’ D-Threitol and glycerol were obtainedmzaon degradation of Cludophoru rupestris, and glycerol in admixture with erythritol from a succinoglucan.310In the latter investigation, the reaction mixture also contained D-glucitol and galactitol, and these cannot be separated as their trimethylsilyl ethers. Polysaccharides containing 6-deoxyhexoses yield 172-propanediol, and this compound may be separated from glycerol, erythritol, and D-threitol by using trimethylsilyl derivatives in conjunction with ~ ~ ~ , ~ ~and ~ Hodge have incareful t e m p e r a t u r e - p r ~ g r a m m i n g .El-Dash cluded alditols and deoxyalditols in their study, referred to in Section VII (see p. 56), on the relative retention-times o f a homologous series of polyhydroxy compounds.234 When polysaccharides contain uronic acids, it is often convenient to separate the acidic oxidation-products on ion-exchange resins and to generate the lactones, which are then reduced. Applied to

(598) G . G. S. Dutton and A. M. Unrau, Can. I. Chem., 40, 348 (1962). (599) M. Zinbo and T. E. Timell, Soensk Popperstidn., 68, 647 (1965). (600) T. E. Timell and M. Zinbo, Tappi, 50, 195 (1967). (601) M. Zinbo and T. E. Timell, Soensk Papperstidn., 70,597 (1967). (602) I. R. Siddiqui and P. J. Wood, Carbohyd. Res., 17, 97 (1971). (602a) P. G . Johnson and E. Percival, J . Chem. Soc. (C), 906 (1969). (603) M. J. How and J. D. Higginbotham, Carbohyd. Res., 16, 9 (1971).

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4-0-methyl-D-glucuronic acid (which commonly occupies a terminal position in polysaccharides), this sequence gives rise to %O-methyl-~erythritol. In some polysaccharides, this uronic acid occurs together with D-glucuronic acid; the latter gives rise to glycer01.5~~ The separation and determination of glycerol and 2-O-methyl-~-erythritolare thus of great interest. This separation has been accomplished by Kubaekovh and coworkers, who. also determined ethylene glycol, glycerol, and ~ - x y l o s e The . ~ ~ ~same authors have applied similar methods in a study of the xylan from p0plar.6~~ Mixtures of sugars and alditol derivatives arising from studies on dextrans have also been separated as their 0-trimethylsilyl derivative^.^^^.^^^ The potential of the foregoing method for investigating polysaccharide structures has led to a study of model systems, such as would arise from arabinoxylans, glucomannans, galactoglucomannans, and arabinogalactans.608This work showed that all of the possible components, even when present in only small proportion, could be determined accurately by using a column of SF-96. The poorest separation was obtained for the trimethylsilyl ethers of erythritol and D-threitol, but, even for this pair, satisfactory quantitative data were obtained. Barker and coworkers608areported that a column of SE-30 does not separate this pair of isomers. Overlapping peaks were obtained from the 0-trimethylsilyl derivatives of the hexoses, and although the problem was resolved as explained in Section V (see p. 41), it would probably be simpler to introduce a second borohydride-reduction step and analyze the mixture as the acetates (see, for example, Refs. 217,417, and 595). This procedure has, in effect, been performed by Bishop and coworkers, in a study of a polysaccharide from birch saptioS although, in this instance, erythritol was the only tetritol. When a polyalcohol resulting from the Smith procedure is hydrolyzed, C-1 and C-2 theoretically give rise to glycolaldehyde,610and the fate of this compound in the analytical procedure is of interest. (604) M. Kubackovli, S. KarAcsonyi, and J. Hrivnak, Collect. Czech. Chem. Commun., 33, 2518 (1968). (605) S. Karlicsonyi and M. KubaEkovli, Collect. Czech. Chem. Commun., 34, 2002 (1969). (606) T. Yamakawa and N. Ueta, l a p . J . E x p . Med., 34, 37 (1964). (607) A. Misaki and S. Kanamaru, Agr. Biol. Chem. (Tokyo), 32,432 (1968). (608) G . G . S . Dutton, K. B. Gibney, G. D. Jensen, and P. E. Reid,J. Chromatogr., 36, 152 (1968). (608a) S. A. Barker, M. J. How, P. V. Peplow, and P. J. Somers, A n d . Biochem., 26, 219 (1968). (609) B. Urbas, G . A. Adams, and C. T. Bishop, Can. J . Chem., 42, 2093 (1964). (610) A. M. Unrau, Can. J . Chem., 41,2394 (1963).

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A small peak, identified tentatively as the acetate of glycolaldehyde, was reported by Bishop and Cooper,581but, from the stoichiometry of the reaction, it is clear this compound is formed in amounts equimolecular to the polyols formed from carbons 3 to 6. This discrepancy suggests that glycolaldehyde is largely decomposed during the hydrolysis step, and this hypothesis is substantiated by work on model systems, with and without an acid-hydrolytic step.608a-Hydroxycarbony1 derivatives may exist as dimers whose structures have been investigated by n.m.r. spectroscopy by Gardiner:l2 and Arreguin and Taboada:I3 who separated individual components by gas-liquid chromatography. Some glycolaldehyde or glyceraldehyde may survive when very mild conditions of hydrolysis are used, and thereby complicate the analysis, as the 0-trimethylsilyl derivative of glycolaldehyde dimer has a retention time similar to that of glycerol. This behavior and similar relationships have been shown graphically by Arreguin and T a b ~ a d aBalogh . ~ ~ ~ and Kolos have also commented on the difficulties of determining a-hydroxycarbonyl derivatives because of formation of dimers.614 Such interfering compounds may be removed by reduction, for example, as described by Srivastava and Singh,393who used a colorimetric determination. In a study on acetyl-group migration, Garegg conducted similar determinations by gas-liquid c h r ~ m a t o g r a p h y The . ~ ~ ~separation of glycerol and mannose has been reported606in a study on a glycolipid, and Sat0 and coworkers have discussed the separation of glycolaldehyde, erythritol, and D-threitol as their trimethylsilyl ethers.367 They found that, at 205", these three compounds have the same retention time, but, at 160", glycolaldehyde was separable from erythritol and D-threitol, and it was also possible to determine glycerol. The problem of determining glycolaldehyde has been studied by Yamaguchi and colleagues, who used methyl /3-cellobioside616and kojibiosyl-gly~erol~~~ as model substrates. They found that glycolaldehyde cannot be determined quantitatively after direct trimethyl-

(611) L. D. Hall, Carbohyd. Res., 4,429 (1967). (612) D. Gardiner, Carbohyd. Res., 2, 234 (1966). (613) B. Arreguin and J. Taboada,J. Chromatogr. Sci., 8, 187 (1970). (614) S. Balogh and E. Kolos, Proc. Anal. Chem. Conf. 3 r d 2, 177 (1970); Chem. Abstr., 74, 38,113 (1971). (615) P. J. Garegg, Arkiu Kemi, 23, 255 (1965). (616) H. Yamaguchi, T. Ikenaka, and Y. Matsushima, J. Biochem. (Tokyo), 63, 553 (1968).

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silylation; however, this compound and also glyceraldehyde can be determined if they are converted into their oximes before trimethylsilylation. The 0-trimethylsilyl derivatives of these two oximes, and of glycerol and erythritol, were then clearly separated by gas-liquid chromatography. Reference has already been made to the formation of anhydro sugars under the influence of acid (see Section XI, p. 87). In a similar manner, alditols may be converted into anhydroalditols, and these may be difficult to distinguish from other polyhydric compounds of low molecular weight. Such a problem arises in the hydrolyzates of cell walls, where ribitol and glycerol are present. It has been shown, however, by Gregory that glycerol and an anhydroribitol may be separated readily as their trimethylsilyl ethers.401 The separations so far referred to have involved conversion of the polyhydric compounds into volatile derivatives, but polyhydric alcohols may be separated directly on a column of “ P ~ l y p a k . ”This ~~~ method has been used to determine such compounds in biological media. The catalytic hydrogenation of carbohydrates yields a complex mixture consisting mainly of a l d i t o l ~ . ’Because ~~ of the marked difference in volatility of the individual compounds, and the presence of water, it has not been found possible to analyze such reactionproducts in one operation. Accordingly, a preliminary fractional distillation was conducted to give three fractions designated (i) methanol-water, (ii) water-ethylene glycol, and (iii) glycerol-hexitol, and each fraction was then analyzed by gas-liquid chr~matography.’~~ The direct separation of polyhydric alcohols does not appear to have been used in polysaccharide structural studies, except in the case of the methylated compounds that will be discussed in Part I1 of this article. Separations of polyhydric alcohols by gas-liquid chromatography are of interest in fields other than carbohydrate chemistry, and it is convenient to make brief mention of some of these applications. Several references to the separation of polyhydric compounds without the formation of derivatives are given in the paper by Vera~htert?‘~ and similar methods related to the separation of acyclic and alicyclic diols?17 to the separation of a series of a,wdiols,6’* to the analysis of ethylene glycol, methanol, and diethylene glyc01,G~~ and to the an(617) I. L. Weatheral1,J. Chrornatogr., 26, 251 (1967). (618) K. Assmann, 0. Serfas, and G . Geppert, J. Chrornutogr., 26, 495 (1967). (619) J. D. Forlini,J. Chrornatogr. Sci., 7, 319 (1969).

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alysis of glycol ethers.620Methods for the analysis of similar mixtures have also been given by 0thers.621--624 In the analysis of such compounds having free hydroxyl groups, adsorption by the tubing material may be a significant drawback, together with problems caused by the column support-material. This difficulty has been studied by Levins and Ottenstein, who showed that aluminum adsorbed glycerol completely, stainless steel adsorbed much glycerol and some 172-propanediol,whereas glass was inertPZ5 The adsorption was decreased by coating the columns with 5% FFAP. Separations of polyhydric alcohols as their trimethylsilyl ethers have been used to study humectants in tobacco626and in oils and alkyd resins.627It has also been shown that 1% aqueous solutions of polyols may be injected onto a column of silicone grease and oncolumn trimethylsilylation achieved by the subsequent injection of “Silyl 8.” This reagent does not liberate any hydrogen chloride on timethyl~ilylation.~~~ A method for the determination, in lipids, of glycerol as its 0-trimethylsilyl ethers has been published?28 and it has been noted that, when glycerol is liberated by acetolysis from phosphatidylcholine, trifluoroacetic acid gives better recovery than sulfuric A series of polyethylene glycols has been separated as their 0-trimethylsilyl derivatives on XE-61, which may be a column packing generally useful for highly polar compounds.1g4 The consumption of erythritol in microbial media has been monitored by a method involving triniethylsilyl ethers.159 The Smith analysis discussed at the beginning of this Section gives little information on the presence or absence of (1 + 6)-linkages. If, however, the polyalcohol is methylated before hydrolysis, mixtures of mono- and di-methylglycerols and erythritols (or threitols) may be obtained.B07s630*631 This method was clearly explained by Bahl and (620) J. F. Palframan and E. A. Walker, Analyst, 92,535 (1967). (621) V. N. Balakhontseva and R. M. Poltinina, Zh. Anal. Khim., 20,739 (1965). (622) T. N. Filatova, N. A. Wasyunin, and L. A. Kuznetso, Zzo. Akad. Nauk SSSR, Ser. Khim., 2581 (1969). (623) A. 1. Novoselo, A. Afanas’ev, E. P. Kalyazin, andV. F. Zakharov,Zh. Anal. Khim., 25,386 (1970). (624) V. G. Berezkhin and V. S. Kruglikova, Zh. Anal. Khim., 24,455 (1969). (625) R. J. Levins and D. M. Ottenstein,]. Gas Chromatogr., 5,539 (1967). (626) J. M. Slanski and R. J. Moshy,J. Chromatogr., 35,94 (1968). (627) G . G . Esposito and M. H. Swann, Anal. Chem., 41, 1118 (1969). (628) A. Rajiah, M. R. Subbaram, and K. T. Achaya,]. Chromatogr., 38, 35 (1968). (629) K. S. Holla and D. G. Cornwell,]. Lipid Res., 6, 322 (1965). (630) B. A. Lewis, M. J. S . Cyr, and F. Smith, 1.Org. Chem., 33, 3139 (1968). (631) A. Misaki and F. Smith, Casbohyd. Res., 4, 109 (1967).

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Smithe32in studies on “anomalous” linkages in glycogen and amylopectin, although gas-liquid chromatography was not used. In a related paper on clam an attempt was made to separate 1,3-di-Omethylglycerol, 1,4-di-O-methylerythrito17and 1-0-methyl-L-erythritol. The latter two compounds were not separated under the conditions used, but a similar mixture has been separated in the form of the corresponding acetates.59s This extension of the Smith analysis, involving methylation of the polyalcohol, has been studied in detail by Bose, both on model systems and on the Ti f r u ~ t a nWhen . ~ ~ a fructan is used, this method may give l-hydroxy-3-methoxy-2-propanone and 1,3-dimethoxy-2-propanone, together with methylated glycerols. These compounds may react further under methanolysis conditions to yield 2,5-dimethoxy2,5-bis(methoxymethy1)-1,4-dioxaneand 173-dimethoxy-2-propanone dimethyl acetal, respectively. Several of these compounds may be obtained from sucrose by a model reaction. In view of the wide variety their separation and of hydroxy compounds obtained in this identification as benzeneboronates was investigated, and certain of the latter derivatives were purified by gas-liquid chromatography. The characterization of 1,2- and 1,3-diols by the mass spectra of their cyclic benzeneboronates has also been described.634 Methyl ethers of polyhydric compounds may also be obtained by the periodate oxidation of methylated glycoses followed by borohydride reduction; this matter will be discussed in Part I1 of this article. XIV. SMITH DEGRADATION

When an endocyclic glycol group in an oligo- or poly-saccharide is cleaved by periodate, the sugar ring is broken. This modification has the effect of changing the character of the intercatenary linkage from that of a glycoside to that of a simple acetal, and the latter is considerably more acid-sensitive. Recognition of this difference permitted F. Smith to isolate glycosylalditols the structure of which established anomeric configurations in the original polysaccharide, and indicated whether or not there existed contiguous branch-points or (1 + 3)linkages. The original results of this investigation were presented in 1959 at a meeting of the American Chemical S0ciety,6~~ but a de(632) 0. P. Bahl and F. Smith,]. Org. Chem., 31,2915 (1966). (633) 0. P. Bahl and F. Smith,J. Org. Chem., 31, 1479 (1966). (634) C. J. W. Brooks and J. Watson, Chem. Commun., 952 (1967). (635) I. J. Goldstein, G. W. Hay, B. A. Lewis, and F. Smith, Abstr. Popers Amer. Chem. SOC. Meeting, 135, 3~ (1959).

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tailed discussion has not yet been published. Some typical applications have been g i ~ e n . ~ 3 ~ In this application of periodate oxidation and borohydride reduction, it is axiomatic that partial hydrolysis is employed, in order to isolate glycosylalditols, if the structure of the original material is such that they may be formed. It is this utilization of the relative acidlability of the acetal linkage, and corresponding relative stability of the glycosidic linkage, that has become known as the Smith degradation. It has already been pointed out in Section XI11 (p. 90) that Smith had some years previously published a method whereby total hydrolysis of the polyalcohol produced by periodate oxidation and borohydride reduction gave, for example, glycerol and erythritol, from the relative proportions of which the chain-length could be Many authors refer incorrectly to this procedure as the Smith degradation. The earlier method580is analogous to endgroup analysis by methylation, and may therefore be referred to as the Smith end-group analysis. The results obtained from a Smith degradation necessarily depend on the structure of the polysaccharide, but it is convenient to consider three aspects of this reaction. In the case of a highly branched polysaccharide, only terminal residues are oxidized by periodate, with the resultant formation of a residual polysaccharide and fragments of low molecular weight, such as glycerol. This approach has been much utilized in studying the structure of gums, where attention has been concentrated on the nature of the residual polysaccharide and its further degradation-products. In the case of typical gums containing L-arabinose and D-galactose, the final polysaccharide obtained by this treatment may still contain both sugars?37or it may contain galactose only.638In the second type of application, the presence of glycosylalditols in the product may be interpreted as indicating the presence of (1+ 3)-linkages or equivalent structures in the original polysaccharide. The third application, which is the one most germane in the present context, concerns the situation where a series of glycosylalditols is produced, as observed with laminaran.636When several such compounds are obtained from a natural polysaccharide, it is probable that the anomeric linkage is the same in each instance, and the higher members may be better separated by paper or thin-layer chromatography because of their high molecular weight and lack of volatility. On the other hand, whenever a polysaccharide has a ran(636) I. J. Goldstein, G. W. Hay, B. A. Lewis, and F. Smith, Methods Carbohyd. Chem., 5, 36 1 (1965). (637) D. M. W. Anderson and G. M. Cree, Carbohyd. Res., 6,385 (1968). (638) D. M. W. Anderson, I. C. M. Dea, and R. N. Smith, Carbohyd. Res., 7,320 (1968).

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dom structure, anomeric mixtures of glycosylalditols may result, and, furthermore, there may also be structural isomers present. It has been shown that, in certain instances, hexosylglycerols and pentosyltetritols, which are isomeric, may be unresolved by paper chromatogr a p h ~ In . ~the ~ ~degradation of a synthetic g l ~ c a n , 6a~fraction ~ was obtained whose analysis clearly indicated a mixture, but separation could not be achieved until the advent of Sweeley's technique using 0-trimethylsilyl derivatives. This method was then found to permit resolution both of anomeric and isomeric compounds. Since that time, glycosylalditols from a synthetic galactan,207 rnannan,B'O rhamnan,'+'I polymalto~e,s~~ and sapote gume43have been separated in this way. The paper on mannane4Ois of particular interest in that it demonstrates some of the compounds obtainable by a Smith degradation and also illustrates that paper and gas-liquid chromatography may be complementary. The Smith-degradation fragments of highest molecular weight so far separated as their 0-trimethylsilyl derivatives are diglycosylerythritols. This limit may well be an instance where the superior volatility of trifluoroacetates might prove advantageous. The problems caused by the dimerization of glycolaldehyde are discussed in Section XI11 (see p. 90). Instead of reacting with itself, the glycolaldehyde may form an acetal, either with the sugar or with the aglycon. These side-reactions have been thoroughly investigated by F. Smith and cow~rkers.B~~ In certain examples where the Smith-degradation fragments have been separated more conveniently by paper chromatography, the structure of these fragments has been demonstrated by examining their methylation products by gas-liquid chromatography. This approach has been used in studying the fine structure of l i ~ h e n a n , B ~ ~ isoli~henan,B~~ and wheat p e n t o ~ a n . B ~ ~ Such compounds as glucosylglycerol are important in another context, apart from Smith degradations, in that they have been isolated from bacterial l i p i d ~ . B ~The ~ * ~synthesis ~* of certain of these glycosylalditols has been d e s ~ r i b e d , B and ~ ~ * retention-times ~~~ have been given. Table XI11 (see p. 156) records examples in which gas-liquid (639)G. G. S. Dutton and A. M. Unrau, Can. J. Chem., 42,2048 (1964). (640)G. G. S. Dutton and A. M. Unrau,]. Chromatogr., 20,78 (1965). (641)G. G.S. Dutton and A. M. Unrau, Can.J. Chem., 43, 1738 (1965). (642)G. G. S. Dutton and A. M. Unrau,]. Chromatogr., 36,283 (1968). (643)S. Kabir, Ph. D. Thesis, University of British Columbia, Vancouver, Canada, 1971. (644)M. Fleming and D. J. Manners, Biochem. I., 100,4P (1966). (645)M. Fleming and D. J. Manners, Biochem. J., 100,24P (1966). (646)D. G.Medcalf and K. A. Gilles, Cereal Chem., 45,550 (1968).

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101

chromatography has been applied with per-0-(trimethylsily1)glycosylalditols. XV. TABLES

Entries in the Tables commence with a single compound and proceed to mixtures of increasing complexity. Some of the larger Tables, for example, I and VI, are subdivided according to the number of components in the mixture examined. Relatively few mixtures have been separated as trifluoroacetates, and thus, all such separations are grouped in Table 111, which is subdivided by class of compound studied. The order parallels that used in the other Tables. Compounds are entered in Tables by use of abbreviations, for example, EG, ethylene glycol; G1, glycerol; Er, erythritol; and Th, threitol; together with standard ones for the pentoses and higher sugars, for example Glc, glucose; Man, mannose; and 2-deoxy-Rib, 2-deoxy-erythro-pentose. The same abbreviation is used for a free sugar or a derivative thereof. This usage is clear from the heading of the Table; thus, in Table I, Glc is glucose, but in Table VI, Glc is glucitol. In multicomponent systems, the order of entry is alphabetic for such classes as pentose, deoxypentose, aldohexose (glucose first), ketohexose, and deoxyhexose. Methylated sugars are included in the Tables if they occur naturally. Because chromatographic procedures do not, in general, differentiate between enantiomers, configurational prefixes are not given. Separations discussed fully in the text are not usually entered in the Tables. Certain of the liquid phases are designated in the Tables by abbreviations: BDS, butanediol succinate; EGA, ethylene glycol adipate; EGS, ethylene glycol succinate; NPGA, neopentyl glycol adipate; NPGS, neopentyl glycol succinate; and NPGSE, neopentyl glycol sebacate. Other abbreviations used are trade names, Capillary columns coated with a liquid phase are shown thus: BDS (cap). When a column was maintained isothermally for a specified time before a temperature program was started, the time is shown in the column headed Rate (deg. min-') thus: 120 to 200

10 min 8

This signifies that the column was kept for 10 minutes at 120",and then programmed to 200" at 8"/min. (647) N. Shaw, K. Heatherington, and J. Baddiley, Biochem. J . , 107,491 (1968). (648) N. Shaw and J. Baddiley, Nature, 217, 142 (1968). (649) D. E. Brundish and J. Baddiley, Carbohyd. Res., 8, 308 (1968). (650) D. E. Brundish, N. Shaw, and J. Baddiley,J. Chem. Soc. (C), 521 (19E6).

TABLEI 0-Trimethylsilyl Derivatives of Neutral Monosaccharides Compounds separated (a) One component Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc

Glc"; Fru" (,,C labeled) Glc; Clc-1-d; Gl~-6,6-4;Gal Glc-d,; Man-d, Ara; Gal; 2-deoxy-Rib h a ; Xyl; Glc; Gal Gal Gal Glc; Gal; FN;

Column ') temp. Cc

Rate (deg. min-I)

8% NPGA 10% XF-1150 30% XF-1150 3% SE-52 5% SE-30 5% SE-30 5 % SE-30 20% SE-52 2% SE-30 or 52 5% SE-30 3% SE-30 2% ov-1 1% OV-17 3% SE-30 10% UC-W98 2 % XF-1105 2% QF-1 2% ov-1 2% SF-96 3% ov-1 3% SE-30

140-180

4

155 180 190 160 150-250

1.5

3% SE-30 30% XF-1150 (or EGS) 4% SE-52 3% SE-30 15% EGS 15% EGS

150-180 155 145

Column of

140-275 145-170 180 170 150 210 180 140 180 170,175

152 157 145

4

Material or problem studied mutarotation mutarotation mutarotation corn syrup inert standards malto-oligosaccharides

1.6-anhydro-p-D-glucop yranose dextran corn syrup xylitol in blood clinical-analysis standard Zea mays cell-wall glucan Nocardia starch analysis sake

recovery of '4c sugars mass spectra deuterated sugars mutarotation mutarotation mutarotation mutarotase mutarotation

References

266 267 262 293 294 651 132 607 127,378 297 150a 43a 697 298 299 208 200 201 263 128 258-260 268a 119

p ? U c3

E3z

15% EGS 10% SE-30 SE-30 7.5% Apiezon K 5% Ucon LB-550 10 % Polyphenyl ether 15% Apiezon K 5% SE-52 15% EGS 1% LAC 1-R-296 5 % SE-30

155 150-240 140-210 175 205 155 155,175 175 145 149 160

3% ov-1 3% SE-53 3% ov-1

130 140-190

Glc; Glc-d,

3 % SE-52 15% Apiezon M 15% BDS 15% Polyphenyl ether 10% Polyphenyl ether 15% Apiezon K 3% SE-30

190 175 200 155 155,175 160

Glc; 3-Me-Glc

3% SE-52

Sor; Gal Gal Man Man Fuc

Fuc FN 3-Deoxy-ribo-hexose 4-Deoxy-ribo-hexose

4 1.5

blood-group oligosaccharides digalactosyl-cysteine Acetabularia crenulata glycolipid glucuronoxylofucan

405 698 699 606 398

a-L-fucosidase ring structure, mass spectra anhydride equilibrium 4-substituted maltooligosaccharides

176 652 79 651

sugar epimerases Acacia arabica microbial media Mycobacterium phlei epimerization of xylose aldobiouronic acid heparin Armillaria mellea

312 653 159 654 249 295 76 333

Fucus uesiculosus

399

(b) Two components Ara; Xyl Ara; Gal Ara; Glc Rib; 6-Me-Glc Xyl; threo-pentulose Xyl; Glc Xyl; Gal Xyl; Man Xyl; Fuc

Clc; 4-Me-Glc Clc; Gal

2% SE-30

150

2

complete sepn. a-Glc and a-Glc-d, aldobiouronic acid hydrolysis kinetics 4-0-methylmaltooligosaccharides enzymolysis

201,202 295 296 265 (continued )

TABLEI (continued) Compounds separated

Column of

Glc; Gal Clc; Gal Glc; Gal Glc; Man Clc; FN Glc; Fru Glc; Fru Glc; Fru Glc; Fru Glc; FN

20% Carbowax 20 M 1% SE-30 (5% SE-30) 5% Ucon LB-550X 3.8% SE-30 3% SE-52 3% SE-30 3% SE-30 0.5% QF-1 10% ucc-W982 3% SE-54

Glc; Fru Glc; FN Glc; Fru

5% SE-30 1.5% SE-30 15% EGS 3% SE-52 1% EGSS-X (3% SE-30,3% OV-17, 15% EGS) 4% SE-52 5% OV-17 3.8% SE-30 3.8% SE-30 3.8% SE-30

Glc; FN Glc; Glc; Glc; Glc; Glc; Glc; Glc;

Fru Fru FN FN Fru Fru Fru

Clc; Fru

4% SE-52 3% SE-30 7.5% Apiezon K

Column temp. ("c)

Rate (deg. min-')

145 130-270 170 150-350 150 152 150-310 100 to 295 160-275 150 160 170 140

6 10 2-4 10 10 min 3

Material or problem studied

References

blood plasma milk products succinoglacan phytoglycolipid wort, beer enzymolysis of sucrose enzymolysis of sucrose starch potato extracts free sugars in plants

137 326 310 568 128,129,316 264 233 340 115 477

6 beet molasses 10 min, then 6 potato tubers mammalian nerve

170-290 200 190-275 160-270 195

various

150 to 260 175 175

15 min 6

4 4

320 116 406

use of electron-capture detector

579

honey onion bulb prune juice fruits fruits and leaves honey Nosema apis spores

314 322 317 318 324 315 309

sucrose lactate in Cladophorales

479

Glc: Fru

3% SE-52

150

Glc; Fru

5% ov-101 (OV-17) 3% SE-52

165 170 120 to 228 168 110-250 180 225 200

Glc; FN Glc; FN Glc; FN Glc; Fru Glc"; Fru"

Gal; Man Gal; Fuc Gal: Fuc

Gal; Fuc Man;3-Me-Man Man; Fuc Rha; 3-Me-Rha (c) Three components Ara;Xyl;Glc Ara; Glc; Gal Ara; Xyl; Gal Am; Xyl; Gal Ara; Xyl; Apiose Ara; Glc; Man Xyl; Glc; Man

3% SE-52 3% SE-52 20% NPGS 2% SF-96 2% Carbowax 20 M 20% SE-30 10% SE-30 10% SE-30 10% BDS (4 % SE-30, 2 % NPGS) 10%Carbowax 20 M 5% Ucon 550 10% QF1 20% SF-96

7.5% Apiezon K 3% SE-30 10% Apiezon K 8% SE-30 10% SE-30 5% SE-30

190 75 to 210 125-215

2 for 12 min 15 to 280

4 min 6 5

20 min 4 4

140 205 150 190 to 230

3 min 3

177 155,180 200-350 100-180

15 1

almond hulls

150

corn syrup

122

cotton leaves

117

sugar-cane juice sweet potatoes Verticillium dahliae recovery of "C sugars

121 123 308 208

Crotalaria mucronata chick allantoic antigen Lorenzini jelly

311 302 303

glycoproteins

329

Mycobacterium phlei glycopeptide mycoside G

700 528 301

Zea mays xylans

70 1

Pseudomonas fluorescens Cladophorales Cladophora rupestris

306 304 305

pectin arabinomannan of Mycobacteria sagebrush cottonwood

655 372 275 276 (continued)

TABLEI (continued) ~~

Column temp. (“C)

Compounds separated

Column of

Xyl; Glc; Man

3% SE-52 (5% XE-60)

Xyl; Gal; Man

15% EGS

110-149 149-190

Xyl; Gal; Fuc Glc; Gal; Man Glc; Gal; Man

1 % SE-30 3 % SE-52

150 120 185

Rate (deg. min-’)

135 1 2

3 min 6

Glc; Gal; Man Glc; Gal; Man Glc; Gal; Fru Glc; Gal; Fru Glc; Gal; Fru Glc; Gal; Fru Glc; Man; Fru Glc; Gal; Fuc Glc; Gal; Rha Glc; Man; Fuc Glc; Man; Rha Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Fuc (d) Four components Ara; Xyl; Glc; Fru

SE-52 15% EGS 3% ov-1 3%SE-52 3.8% SE-30 3% SE-52 3% SE-52 10% NPGS 3% SE-52 3.8% SE-30 SE-52 3% OV-17 2.5% SE-30 1% SE-30 15% BDS 15% BDS 6% SE-52 3.8% UCC-W982 2% E-301

180 158 100 130-180 125-250 130-180 170-220 150-180 150-225 145 to 205 160 155-195

4 2.5 4 2.5 2 1 2 10 min 2 4

140 175 175 90- 190

2

140 to 290

13 min 10

Material or problem studied

References

m

78

1,6-anhydro-p-~-glucopyranose in pulp hydrolyzate buckwheat endosperm

656

Fucus vesiculosus glycosaminoglycans fungal glucoamylase

702 288 344

alkaline degradation of pine glucomannan Clostridium pesfsngens serum and urine sunflower extract Rehmunniu glutinosu holly-leaf extract Polygonatum oduratum human milk Diplococcus pneumoniae 31

272

gamma-globulin

w 0

703 138 325 327,327a 323 704 545 46 60

Pseudomonas alcaligenes model for glycosaminoglycans keratan sulfate epithelial mucin of hagfish Polyporus pinicola Armillaria mellea glycoproteins gastric-juice glycoprotein

307 70a 331 330 332 287 286 549

extra-floral nectar

174

0 0 F ~

c 4 0

Ara; Xyl; Glc; Gal Ara;Xyl;Glc;Gal Ara; Xyl; Glc; Man Ara; Xyl; Gal; Rha Xyl; Glc; Gal; Man

5% SE-30 3% E-301 as in Ref. 170

175 150

1.5% SE-30

170

Xyl; Glc; Gal; Man Xyl; Glc; Man; Fuc All; Alt; Glc; Man Glc; Gal; Man; Fuc Glc; Gal; Man; Fuc

QF-1 or DC-200 15% BDS 15% EGS 20% BDS 2% Apiezon L plus 4% NPGA 15% BDS

210 175 150 142 130-210

125-250

2.4

h a ; Xyl; Glc; Gal; Man Ara; Xyl; Glc; Gal; Man

3% silicone gum rubber 3% silicone gum rubber 2% SE-52 5% XF-1105 15% 0s-138 15% BDS 3.5% BDS 5% XF-1112 15% EGS 20% SF-96

125-250 160 122 160 142 140 150 100-180 190-220

Ara; Xyl; Glc; Gal; Man Ara; Xyl; Glc; Gal; Man

5 % SE-30 1,4, or 15% XE-60

100- 180

1

Rib; Glc; Gal; Man; Fuc Xyl; Glc; Gal; Man; Fuc Xyl; Gal; Man; Fuc; Rha Glc; Gal; Man; FN; Fuc

17% EGS

Gal; Man; Fuc; 3-Me-Gal

175

(e) Five components

Er; Xyl; Glc; Gal; Fru Ara;Xyl;Glc;Gal;Man Ara; Xyl; Glc; Gal; Man Ara; Xyl; Glc; Gal; Man

2 % SE-30 SE-30 3.8% -

1.5

659 332 335 284 36 333

free sugars in ferns

32 1

4

Acer pseudoplatanus glycoprotein model for wood pulp model for wood pulp

706 83 172

2 2

model for wood pulp model for hemicelluloses and periodate oxidation model for wood pulp HCONMez and Me,SO as silylation solvents; formation of hexamethyldisiloxane model for glycoprotein keratosulfates gl ycopeptides

27 1 608

150 150 150-220

3 19 705 273,274 657 658

fruit tissue oat glucan alkaline degradation of xylan pectin antitumor activities of pol ysaccharides Cryptococcus neoformans Polyporus pinicola cellulose bleaching gastric glycosaminoglycans model for glycosaminogl ycans Armillaria mellea

1

urine

255 182

171 279 59 118 (continued)

TABLEI (continued) Compounds separated

Column of

Column temp. (“C)

Rate (deg. min-’)

10% SF-96

190

model study with amino sugars

1.5% SE-30 5 % XF-1150 15% silicone DC oil

140 120 190

model study for steroidal saponins model for natural glycosides

15% BDS

175

Armillaria mellea

287

alfalfa

291

model for potatoes

277

model for glucose in blood

292

model study

269

model for urine

149

comparison of non-polar and polar columns, especially tetracyanoethylated pentaerythritol (TCEPE) model for glycoproteins saliva, gastric juice pancreatic juice, bile, lymph, and urine

290

Material or problem studied

References

(0 More than five components Ara; Xyl; Glc; Gal; Man; FUC Ara; Xyl; Glc; Gal; Man; Rha Am; Xyl; Glc; Gal; Man; Rha Rib; Xyl; Glc; Gal; Man; Fuc Ara; Rib; Xyl; Glc; Gal; Man; Fru Ara; Rib; Xyl; Glc; Gal; Man; Fru

Ara; Xyl; Clc; Gal;

Man; FN; Rha Ara; Rib; Xyl; Glc; Gal; Man; Fuc; Rha Ara; Xyl; Glc; Gal; Fru; Psicose; Sor; FUC Er; Ara; Rib; Xyl; 2-deoxyRib; Glc; Gal; Man; FN; Fuc Ara; Rib; Xyl; 2-deoxyRib; Glc; Gal; Man; Fru; Fuc

5 % ov-1 3% OV-225 3 % ov-1 3 % OV-17 10% Carbowax 20 M terephthalate 3.8% UC-W98

110-235 110-235 170,185 170,185 135

2 2

130-270

3

15% Carbowax 20 M

170

5% SE-30

100-325

10

15% Carbowax 20 M 3 % TCEPE 3% ov-1

145 100-140 120-190

2 3

12% EGS 15 or 20% BDS (DEGS,EGA,NPGS, NPGSE,NPGA)

100-160 100-160

5 5

256 31

95

34 34 280-283

Ara; Rib; Xyl; G k ; Gal; Man; Fru; Sor; Fuc; Rha

Ara; Rib; Xyl; 2-deoxyRib; Glc; Gal; Man; FN; Fuc; Rha Ara; Lyx; Rib; Xyl; 2-deoxyRib; Glc; Gal; Man; 2-deoxy-Glc; Fuc; Sor;

Carbowax 20 M (cap) 5% Carbowax 20 M 8 % Carbowax 20 M 6% XE-60 6 % UC-W98 20% EGS

120-220 110-220 164 100-200 150-280 70-220

3 3

1.8 2.5 15

XE-60 and many others

model for food

278

myo-inositol in yeasts and bacteria

112

effect of concentration of liquid phase and solid support on resolution

289

model

270

FN Ara; Lyx; Rib; Xyl; 2-deoxyRib; Glc; Man; FN; Sor; 6-deoxy-Glc; Fuc; Rha Ara; Lyx; Rib; Xyl; Glc; All; Alt; Gal; Gul; Man; Tal; Fuc; Rha (g) Heptoses and higher sugars L-glycewm-galacto-Heptose Dglycero-D-manno-Heptose Dtalo-Heptulose L-do-Heptulose

2-Deoxy-~-gaZac to-heptose 2-Deoxy-D-manno-heptose

~

10% SE-30 20% Apiezon L 10% SE-52 -3.5% SE-30 or UC-W98

5 % SE-30 10% NPGSE 15% NPGS 10% NPGSE 1% SE-30 10% SE-30 10% NPGSE 10% NPGSE 1.5% XF-1112 EGS

+ 1.5%

160-210 170-220 150

187 165 180 180 180 205 162 162 162

1 1

model, comparison with methanolysis

67

synthesis Gram-negative bacteria avocado

110 339 151

synthesis synthesis from 3-deoxy-D-manno-octulosonic acid (“KDO”)

660 661 662

~~

(continued )

TABLEI (continued) Compounds separated

Column of

n g l ycero-D-gulo-Heptose

10% SE-30 10% SE-52 20% Apiezon L 1,4, or 16% XE-60

Sedoheptulose Sedoheptulose; mannoheptulose Sedoheptulose; mann noheptulose; Dglyceromannoactulose Six heptuloses and their 2,7-anhydrides DgZycero-nrnannoOctulose nerythro-L-galactoNonu1ose (h) Oximes Ara; Rib; Glc; Gal; Fru Glc (i) Methoximes Glc Glc-1-d Glc-6,fj-d (j) Acylated hexoses 2;2,3-; 2,3,4; 2,3,4,60-Acetylglucose Glc 3- and 6-OPalmitoylglucose

+

* Labeled with carbon-14.

Column temp. (“C)

Rate (deg. min-’)

170 180 210

XE-60 and others 3 % SE-30 15% EGS

200 185-200

1%LAC 1-R-296

177,190

r

Material or problem studied

References

model study

270

HCONMe, and Me,SO as silylation solvents effect of liquid phases and supports opium POPPY

182

equilibrium study

5

289 338

79

Q

Q .

NPGS NPGSE 3% SE-52

1% SE-30 (1% OV-17, 3% OV-25) 10% UC-W98

75-280

170 to 270 100-250

11

38 min 4 30

Pichi

337

comparison of natural and synthetic

336

model for food

187

alkaline stability of polysaccharides

188

189

3% SE-30

190

model for mass spectrometry

5 % LAC-4R-886

180

enzymic deacylation

10% SE-30

bacterial studies

352,353

354

vl

U

C

3

0 Z

TABLEI1 Acetylated Monosaccharides

Column temp. ("C)

Compounds separated

Column of

XYl Glc Glc

1.5% LAC 1-R 296 13% QF-1 20% BDS 20% Apiezon M 3 % ECNSS-M 1.5% EGS 2.5% XE-60 or 2.5% EGS 3% HiEff 8-BP

180 190 190 200

3 YU SE-52 2% XE-60 10% NPGSE 1.5% LAC 1-R-296

200-305 180 200 274

GIc Glc Glc; Ah; Ido; Man %Deoxy-~arabino-hexose 6-0-Palmitoylglucose Fuc Rha Eight aldohexoses; 3-deoxyrylo-, urubino-, and lyro-hexoses Gal; Tal Ara; Lyx; Xyl Ara; Xyl; Glc Glc; Gal; Man Gal; Gul; Ido Glc; Altr; Ido; Man 2,7-Anhydro-l-deoxy-~glycero-p-Dgulooctulopyranose

+

170 225 205

Material or problem studied

References

ring formation Pullulariu pulluluns methyl a-maltoside

351 342 349

oat glucan glycosyl chlorides acetoxonium ions

345

%I

350 348

0

Q

synthesis of 2-deoxy-~-eythroand -D-threo-pentose model for bacteria Cundida bogoriensis Myxo buc terium 402 hexose 1,g-anhydride equilibria

390

210 210

acetoxonium ions acetoxonium ions model for starch study

347 347 340

190 210 190 208

Aspergillus niger acetoxonium ions acetoxonium ions Xunthomonus campestris

344 347 348 343

354 663 341 79

c

sU

n z

z

>

3

E cd

2.5% EGS 2.5% EGS Apiezon-BDS-silicone oil 3 % ECNSS-M 2.5% EGS 2.5% EGS or XE-60 2 % NPGS

3

I-

c

TABLEI11

*

E

Trifluoroacetyl Derivatives Compounds separated (a) Monosaccharides Glc Glc Glc; Man 2-Deoxy-Rib; 6-deoxy-Glc; Fuc; Rha Am, Rib; Xyl; Glc; Gal; Man; Fru; Sor Ara; Rib; Xyl; Glc; Gal; Man

(b) Methyl glycosides Glc Glc (Et glycoside) Glc: Gal Ara; Xyl; Glc; Man Ara; Rib; Xyl; Glc; Man; Rha

Various (41) glycosides

Column of

Column Rate temp. C'c) (deg. min-'1

2% Carbowax 20 M 2% SF-96 2% XF-1105 3% SE-52

140 110

FS-1265 (cap)

160

20% Silicone rubber 3% NGS 3% QF-1 1% CNSi 1.5% SE-52 5% DC-550 12% DC-1107 3% SE-52 2% XF-1105 0.03% XE-60 and 2% SE-30 20% Silicone rubber 10% OV-17 (10% FS-1265, 5% SE-30) SE-30, SE-52, QF-1, NPGS, BDS, UO

130,250 160 160 140 90 90 100 110 140 90-230 150 100,150

Material or problem studied

References

recovery of glu~ose-'~C

208

sake model for carbohydrate antibiotics model, mainly alditols

299 225

model

224

comparison of retention times with Me$%derivatives of Glc, Gal, Man

226

227 Q Q

In U

!

0

Z

2

sake glycolipids

225 299 391a

model mass spectra

224 391

model

387

(c) Alditols Ara; Man Am; Xyl Rib; Xyl; Gal

Xyl; Man; FUC Glc; Gal; Man Glc; Man Am; Rib; Xyl; 2-deoxy-Rib; all six hexitols Ara; Rib; Xyl; Glc, All; Gal; Ido; Man; Tal Ara; Rib; Xyl; 2-deoxy-Rib; G k ; Gal; Man; Fuc; Rha; 2-deoxy-Glc; 2-deoxy-Gal (d) Oligosaccharides Sucrose; trehalose Cellobiose; lactose, maltose; sucrose, trehalose Lactose; maltose; ra5nose; stachyose; sucrose; trehalose (e) Disaccharide dditols Cellobiitol; gentiobiitol; isomaltitol; lactitol; maltitol; melibiitol; Galpa-( l+4)-Man

2% XF-1105 2% XF-1105 10% OV-17 (10% FS-1265, 5% SE-30) 2% XF-1105

140 140 100,150

sake Plantago major mass spectra

140

stem bromelain

299 707 39 1

50

2% XF-1105 FS-1265 (cap)

140 160

Polygonatum odoratum model

47 1 704 227

2% XF-1105

140

model

152

2% XF-1105 (2% QF-1 7% DC-1107 SE-30, SE-52)

140 140

model, other columns give tailing and broad peaks

213

2 9’0 XF- 1105 (OV-1,QF-1) 3% SE-52

205

model

49

160

model

225

200,220,250

model

224

20 % Silicone rubber

2% XF-1105 (OV-1,QF-1)

80

205

model [Glcp-cu-(l+3)-Glc and Glcp-a-(1+3)-Man not separated] blood, urine

49

48

(continued)

TABLE111 (continued) Compounds separated

Column of

Column temp. (“C)

Rate (deg. min-’)

Material or problem studied

References

(f) Cyclitols

Inositols: allo-, epi-, muco-, myo-, rac-, scyllo-, and amino inositols Deoxy streptamine (g) Amino sugars and glycosides GlcN; GlcNAc; 3-amino-Glc glycoside; 6-amino-Glc and glycoside; 6-acetamido-Glc glycoside; 3-amino-Man glycoside GlcN; GalN glycosides Glc; Gal; Man; Rha (h) Amino alditols Glc; Gal; Man (i) Polyhydric compounds Er EG; G1; Er; Th

EG; G1; Er; Th

SE-30 (cap) 3% SE-52 1% QF-1 20% Silicone rubber

110 110 110 200

model for carbohydrate antibiotics

225

model

224

3 % SE-52 1% QF-1

140

model

225

0 0 F

U

C

4

0.3% EGA 1% OV-17 2% XF-1105

75 115 126

2% XF-1105 (OV-1, QF-1)

180

2% XF-1105 10% OV-17 (10% FS-1265, 5% SE-30) FS-1265 (cap)

140 100,150

4 6

model, mainly amino acids

664

Corynebacterium

471a

model, complete separation

563

sake mass spectra

299 391

227

2

z

TABLEIV

Methyl Glycosides Compounds separated (a) Trimethylsilyl derivatives 5-Deoxy-Ara; 5-deoxy-Rib; 5-deoxy-Xyl Glc Glc Glc Glc Glc (Et glycoside)

Glc (Et glycoside) 6-Chloro-Glc; 6-deoxy-Glc Glc; 6-deoxy-Glc; 3,6anhydro-Clc; 3-Me-Glc; 6-Me-Glc Glc; Gal Glc; Gal Glc; Man Gal (Et glycoside)

4-Deoxy-arabino-hexoside; 4-deoxy-xylo-hexoside Xyl; Glc Glc; Gal

Column of 20% BDS 15% Carbowax 20 M SE-30 SE-30 EGS 1% OV-17 (3%SE-30) 2 % XF-1105 2 % QF-1 2 % ov-1 7 % Carbowax 20 M 10% Carbowax 6000 5 % SE-30

Column Rate temp. ("C) (deg. min-I)

Material or problem studied

References

145

synthesis

373

160

methyl a-maltoside amylose esters steryl glucosides

349 378 155

150

Nocardia

697

180 140 180

sake

299

145 185

3 % ov-1 10% BDS

170 165

15% Carbowax 20 M 3 % ov-101 SE-52

150

2 % SE-30 or 2 % OV-1

160

vinylation synthesis Taka amylase specificity

665 380 374,375

mass spectra model crystalline per(MqSi)cyclitols soybean

200 379 407 376

4-deoxyuronic acids

385

Glcf in Echinodermata blood ceramides

206 360

(continued )

TABLEIV (continued)

Column Rate temp. (“C) (deg. mi&)

Compounds separated

Column of

Glc; Gal

3% SE-30

160

Glc; Gal Glc; Gal Glc; Gal Glc; Gal Glc; Gal Glc; Gal Glc; Gal Glc; Gal Glc; Gal Glc; FN Gal; Man Xyl; Glc; Man

6% JXR 2.5% SE-30

160

3% SE-52 5% Ucon LB-550 2.5% SE-30 2.5% SE-30 3% SE-30 3% SE-30 5% Ucon LB-550X 5% Ucon LB-500 XE-60 and others

192 160 100-210 160 200 195 205

Xyl; Man; Fuc Glc; Gal; Man

3.8% SE-30 3% SE-52

140-200 185

Glc; Gal; Man

10% NPGSE 15% polyphenyl ether 10% Carbowax 6OOO 3.8% SE-30 3% SE-52 3.8% SE-30

156 175 140 140-200 145 120 to 200 140-220 160 140-200 140-200 100-260

Glc; Gal; Man Glc; Gal; Man Glc; Gal; Man Glc; Gal; Man Glc; Gal; Fuc Gal; Man; Fuc Gal; Man; Fuc Xyl; Gal; Man; Fuc

3% SE-30 2.5% SE-30 3.8% SE-30 3% SE-30 3%0V-17 (3% SE-30)

0.5

0.5 5 min 2 1 0.5 0.5 5

c

r

Material or problem studied

References

cerebrosidal 5uid urine sediments adrenal gangliosides glycolipids and gangliosides glycolipids gangliosides Salmonella and Proteus E. coli endotoxin gangliosides cattle blood-group J. glycolipid ceramides of human kidney algal glucofructan a,-acid glycoprotein model for liquid phases and solid supports connective tissue acid stability of monosaccharides glycosidation of sugars

377 361 359 66 355 173 357 358 552a 708 150b 363 367 289

glycoprotein TA, cell glucoamylase model

368 362 64

milk glycopeptides blood-group substances model gastric mucosa a,-acid glycoprotein

0 0 v, U

C 369 68 383 384

69,370,371 357 86,364,365 709 569

4 4

0

Z

~-arabino-Hexopyranosid-2ulose; D-ribo-hexopyranosid-bulose; D-ZyZo-hexopyranosid-4dose Xyl; Glc; Gal; Man; Fuc Ara; Rib; Glc; Gal; Man; Rha Am; Xyl; Glc; Gd;FUC; Rha Xyl; Glc; Gal; Man; Fuc; Rha

Glc; Gal; Man; Rha; abequose; colitose Ara; Xyl; Glc; Gal; Man; Rha Four pentoses; seven hexoses; Fuc; Rha Various monosaccharides

L-glycero-D-manno-Heptose; D-glycero-Dmannoheptose Heptose Various (41) hexofuranosides and -pyranosides

OV-17 (cap) BDS

3 % UCW-98 5 % Ucon 10% SE-30 5% Silicone-g 30 M

160 165

140-200

170

3 % OV-17

160

(1% SE-30 2.5% EGS) 3 % SE-30

to 210 160-210 100-160 120

15% Silicone oil

190

3-3.8% SE-30 or UC-W98 SE-30 Ucon 50LB-550X NPGS 5 % Ucon LB-550

140-200

3% SE-30 SE-30, SE-52, QF-1 BDS, NPGS, Ucon oil 50LB-550X

0.5

chlorine oxidation of cellulose

382

membrane glycoprotein arabinomannan

366 372

model

527

20 min 15

model for glycoprotein

15 min

bacterial endotoxins model for natural glycosides

0.5

70

381 95

model

66a,67

model

666

192

Salmonella and Proteus

357

160

bacterial endotoxins comparison of Me3Si, CH3C0, and CF3C0

381 387

(continued)

r r 00

TABLEIV (continued) Column temp. (“C)

Compounds separated

Column of

Phenolic glycosides

0.3 % OV-1, 0.3 % OV-17 1.5% EGS,0.5 % CHDMS (cyclohexanedimethanol succinate) 0.75% SE-30 1.5% SE-52 1.5% OV-225

+

Plant glycosides Thioglycosides

(b) Acetates Glc Glc; Gal; Man

2-Deoxy-Glc; 2-deoxy-Gal Glc; Id0 Rha; 3-Me-Rha (c) Fully methylated glycosides Gal; Man A n ; Xyl; Glc; Gal 6-0-Palmitoylglucose Ara; Xyl; Glc; Gal; Man; Rha

Rate (deg. min-1)

Material or problem studied

References

natural glycosides

386

188 230 225

“glucosinolates”

126

0 0 ?-J U

20 % BDS 20 % Apiezon M 2.2% SE-30 3 % QF-1

210

methyl a-maltoside

349

212 220

methanolysis of disaccharide alditols

389

3 % XE-60 1%ov-22 3% HiEff-8-BP 10% BDS 3 % SE-30

200 195 150

furanoside and pyranoside heparin mycoside G

175 175

Sesbania grandijlora tamarind kernel model for mycoside G model

20% BDS 20% BDS 3 % SE-52 20% EGA 20% Apiezon M 20% Apiezon L

152 184 152

C

4

E3z 390 388 65,301 392 393 354 231

TABLEV

Alditols, Alditol Ethers, Aldononitriles, and Dithioacetals ~~

Compounds separated (a)

Per(trimethylsily1)alditols Rib XYl Glc Glc

Column of

Column Rate temp. (“C) (deg. min-’)

Glc Glc

3.8% SE-30

Glc Glc; 3-Me-Glc Glc; 6-deoxy-Glc Gal Man Man

1%EGSS-X 5 % SE-30 5 % SE-30 10% SE-30 15% EGS 3% SE-52

Man

3% ov-1 3% OV-17

160 170 140 to 275 190-275 160-270 195 140 185 185 150-240 158 120 to 185 185 185

3% SE-52 7.5% Apiezon K

130-280 175

2.5

3% JXR

140 to 210 120 60

2 min 3 2 l(10 min) then 4

3% SE-52 01 3% JXR

4 min 6 4 4

4 3 min 6

References

mammalian nerve

401 59 402 406

starch syrups

113

prune juice fruits apples electron-capture detector Taka amylase Taka amylase blood-group oligosaccharides human serum Aspergillus niger glucoam ylase potatoes

317 318 324 579 374 375 405 138 344

p-mannosidase on ovalbumin Rehnannia glutinosa Acetabularia crenulata

710 327 699 397 407

glycerol and anhydroribitol glycoprotein p-elimination

130 150

3.5% SE-52 2% SE-30 Carbowax 20 M 15% EGS 3% SE-52 3% SE-52

Man Man Man Fuc Ara; Xyl; Glc; Gal; Man; Rha Pentitols; hexitols and their mono- and di-deoxy derivatives

Material or problem studied

Me&-galactitol, crystalline sugar degradation products

277

232

(continued )

TABLEV (continued)

c

8

Compounds separated

Column of

Column temp. (“C)

Ara; Man Ara; Gal Xyl; Glc Xyl; Glc

Glc; Man

3% ov-1 3% SE-53 5% SE-30 15% Carbowax 20 M 3% TCEPE 3% ov-1 3% SE-30 15% Apiezon K 20% EGS

180 160 145-170 145 100-140 120-190 155 155,175 70-220

15

Gal; Man Gal; Fuc

20% SE-30 10% BDS

125-215

4

h a ; Xyl; Gal

3% SE-30 10% Apiezon K 10% SE-30 3% SE-30 15% Apiezon K 3% SE-30 or 15% Apiezon K 1.2% XE-60 5% QF-1 5% SE-30 3% SE-30

Xyl; Fuc

Xyl; Glc; Man Xyl; Glc; Fuc Xyl; Gal; Fuc Rib; Xyl; Glc; Gal; Man L-glycero-ngaluc to-Heptitol Rib; Xyl; Glc; Gal; Man; Rha; Fuc, and others (b) Acetylated aldononitriles Ara; Xyl; Glc; Gal; Man Ara; Rib; Xyl; Glc; Gal; Man; Rha; L-glyceroDmanno-heptose; D-glycero-D-gulo-heptose

5% OV-225 3% NPGS

145-257 155 155,175 155 155,175

Rate (deg. min-I)

5 4 2 3

10

187 155 220 160-230

2

Material or problem studied

References

microbial media Acacia campylacantha gum blood use of tetra(cyanoethy1)ated pentaerythritol (TCEPE) as polar phase Ascophyllum nodosum

159 400 297 290

myo-inositol in yeast and bacteria Crotolaria rnucronata aminodeoxyhexitols from glycoproteins Cladophora rupestris

112 311 329

dietetic foods Fucus vesiculosus

396 399

Fucus vesiculosus

702

study of liquid phases and solid supports synthesis Pusteurella pseudotuberculosis (incomplete sepn.)

289

cellulosic materials model

394 394a

398 .p

p v, U

305

110 310a

c 4

i3z

(c)

Butanebornnates Glc; Gal; Man; Fuc

3% OV-17

200

model

232

5% LAC-4R-886 15 % LAC-4R-886 (20%Apiezon M)

198 198 198

model

472

model

23 1

(d) Acetylated dithioacetals and 1-deoxyalditols Four pentoses; eight hexoses

(e) Methyl ethers Am; Xyl; Glc; Gal; Man; Rha

20%Apiezon L 15% EGA 10% Apiezon L

+

10% methylated cellulose

(f) Alditols Ara; Rib; Xyl; Man

Polypak 1

158,180 152 138

2

VJ

E:

ro 2 250

separation incomplete

473

U

n 31

P

0

F

c

E

TABLEVI Alditol Acetates ~

Compounds separated

Column of

Column Rate temp. (“C) (deg. mi&)

Material or problem studied

References

(a) One component

Rib SDeoxy-xylitol %Me-Xyl

2% XE-60 3% ECNSS-M

160

Glc Glc

3% ECNSS-M 10% SE-30

185 220

10% NPGSE as in Ref. 58 as in Ref. 58 3% ECNSS-M 3% ECNSS-M 3% ECNSS-M see Table Vc 3% ECNSS-M 3% ECNSS-M

203

3% ECNSS-M 3% ECNSS-M ECNSS-M

200

3% ECNSS-M 3% ECNSS-M 3% ECNSS-M 3% ECNSS-M

184 190 195 190

3% ECNSS-M

190

Glc Gal Gal Gal Man Man 1-Deoxy-alditols 6-Deoxy-D-Tal 4-Deoxy-hexitols; 3-deoxyD-xylo-hexitol Abequose Tyvelose Abequose; paratose; tyvelose (b) Two components Ara; Glc Ara; Gal Ara; Man Rib; Gal Xyl; Glc Xyl; Gal

180

175 190 190 195

155

glycosidases in clover seeds Candida bogoriensis Myrococcus fulous (D isomer) Rhodopseudomona oiridis (L isomer) oat-leaf p-glucan sophorosides of 17-hydroxyoctadecanoic acid Neisseria sicca MM antigens blood-group glycoprotein snail galactan bacterial inositol mannoside Aspergillus niger glucoamylase model 0 antigens of Citrobacter 4-deoxy-~arabino-hexosein Citrobac ter synthesis synthesis model

lactonization of uronic acids Mycobacterium phlei Salmonella friedenau T1 Klebsiella 0 group 4 lipopolysaccharide Penicillus dumetosus earthworm collagen

667 663 711 50a 592 558 456 454 583 576 344 472 451 450 449 448 17

98 668 18 712

669 670

Glc; All Glc; Gal Glc; Gal Glc; Gal

3% ECNSS-M 10% NPGSE 9.5% ECNSS-M 3% ECNSS-M 3% ECNSS-M

Clc; Id0 Glc; Man

as ref. 409 10% LAC 4-R-886

Glc; Man Glc; Man Glc; Man Glc; Man Glc; Man Clc; Rha Glc; Rha Gal; Man Gal; Man Gal; Man Gal; Fuc Gal; Fuc Gal; 3-deoxy-Gal Man; 3-Me-Man (c) Three components Ara; Rib; Glc Ara; Xyl; Glc Ara; Gal; Rha Rib; Glc; Gal Xyl; Glc; Man Xyl; Glc; Man 2-deoxy-Rib; Glc; Man

reactions of 3-hexuloses Neisseriu cuturrhulis

204 190 190 198

184

3% ECNSS-M 2% NPGS 2% LAC 1-R-296 2% QF-1 as in Ref. 409 3% ECNSS-M as in Ref. 409 2% LAC 1-R-296 as in Ref. 58 2% ECNSS-M 3% ECNSS-M 3% ECNSS-M 3% OV-225

150-200 190 180 180

1.5

3% ECNSS-M 3% ECNSS-M 3% ECNSS-M

170-200 100-200 175

2 8

7% Q F - l + 1.7% BDS Apiezon-BDS-silicone oil 3% ECNSS-M

210 200 200 190 218

195 160-217

4

Rhizobium meliloti Klebsiella 0 group 9 lipopolysaccharide oxidation of acetals sugar maple (Acer sacchurum), identification of Fru Serratiu murcescens free sugars in plants Azotobacter vinelandii Ceratocystic brunnea model Serratia marcescens Diplococcus pneumoniae I1 Dermatophytes Trichosporon fermentans phosvitin chick allantoic antigen blood-group oligosaccharides blood-group oligosaccharides Mycobucterium phlei Arthrobacter viscosus Fusicoccum amygdali Acacia gums Cussonia spicata Salmonella friedenau T1 dietetic foods starch model 3-deoxy-D-manno-octulosonate in Salmonella

67 1 560

445 713 672 673 492 477 595 346 714 676 484a 674 675 715 302 45 716,717 700 591 718 460 719 18,447 396 340 434 (continued)

TABLEVI (continued) Compounds separated

Column of

Glc; Gal; Man

2.2% SE-30 3% QF-1 3% XE-60 1% ov-22 3% ECNSS-M 2% XE-60 10% QF-1 (Gal, Man sepd.) 10% EG isophthalate 3% ECNSS-M 10% NPGSE 1.5% EGSE + 1.5% XF-1150 10% BDS 3% ECNSS-M 3% ECNSS-M 3% ECNSS-M as in Ref. 409 3% ECNSS-M 10% NPGSE 10% NPGSE 3% ECNSS-M

Glc; Gal; Man Glc; Gal; Man Glc; Gal; Man

Glc; Gal; Man Glc; Gal; Man Glc; Gal; Fuc Glc; Gal; Fuc Glc; Gal; Rha Glc; Gal; Rha Glc; Gal; Rha Glc; Man; Rha Glc; Man; Rha Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Fuc Gal; Man; Abe Gal; 2-Me-Gal; 4-Me-Gal Gal; 3-Me-Gal; Rha (d) Four components h a ; Xyl; (Glc; Gal)

3% ECNSS-M 1% ECNSS-M 3% ECNSS-M 3% ECNSS-M 20% Apiezon M 15% BDS

SE-30

Column Rate temp. (“c) (deg. min-’) 212

c

Material or problem studied

References

analysis of disaccharide alditols

389

Aureobasidium pullulans Azotobacter oinelandii birch sap

677 595 609

220 220 202 180 210

185 204 190

peptidogalactomannan Morarella duplex Micrococcus calco-aceticus

46,457 433

150

Escherichia coli lipopolysaccharide M antigens Diplococcus pneumoniae Serratia marcescens Klebsiella K-type 9 Serratia marcescens Neisseria perflava Polyporus fomentarius and P . igniarius Polyporus borealis immunoglobulins G a-L-fucosidase Salmonella typhimurium LT2 Aeodes uluoidea slippery-elm mucilage

420

180 175 180

190 204 190

190 140-230 175 190 175 260 144 to 170

3

Holcus lanatus 20 min

443,444 54 493 720 494 559 462 678 35 721 436 413 469 722

0 0 vl

U

5 3z

Ard; xyl; Glc; Gal Ara; Xyl; Glc; Man

Ara; Xyl; Glc; Man Am; Xyl; Gal; Man Rib; Glc; Rha; 3-Me-Rha Xyl; Glc; Gal; Man Xyl; Glc; Gal; Man Xyl; Glc; Gal; Fuc 2-Deoxy-Rib; 2-deoxy-Xyl; 2-deoxy-Glc; 2-deoxy-Gal

3% ECNSS-M 1.5% XF-1150 1.5% EGS 20% Versamid 900 20% SE-30 3% ECNSS-M 3% ECNSS-M 3% ECNSS-M ECNSS-M 3% ECNSS-M 3% HiEff 8-BP

+

125-280

7.5

190 175 165 155-195 200

Glc; Gal; Man; 3-Me-Man

3% ECNSS-M

190

Glc; Gal; Man; Fuc Glc; Gal; Man; Fuc Glc; Gal; Man; Fuc

3% ECNSS-M 3% ECNSS-M 3% ECNSS-M

180 190 175

Glc; Gal; Man; Fuc Glc; Gal; Man; Rha

as in Ref. 58 3% ECNSS-M

190

679 422-424

wheat pentosans kraft pulping

190 195

2

hydrogenolysis of saccharides

145

Encephalortos longifolius gum bacterial polysaccharide pituitary glycoproteins core lipopolysaccharide blood-group tetrasaccharide synthesis of 2-deoxy-~-erythropentose and 2-deoxy-~-threopentose Klebsiella 0 group 5 lipopolysaccharide Klebsiella K-type 6 Polyporus ovinus thyrotropic hormone and other glycoproteins amyloids from spleen Salmonella newport Salmonella muenster Salmonella senftenberg Salmonella newineton u Salmonella typhi and Salmonella strasbourg

459 432 476 43 1 455 390

Salmonella friedenau T1 lipopolysaccharide model for wood polysaccharides use of digital integrator

447

723 724 464 57 680 435 437 438

440 725

(e) Five components

Ara; Rib; Glc; Gal; Man

3 % ECNSS-M

Ara; Xyl; Glc; Gal; Man

1.5% EGS 1.5% XF-1150 (5% XF-1112 at 200", incomplete separation of hexitols)

+

190 180-220

0.8

219 414

(continued)

TABLEVI (continued) Compounds separated

Column of

Column Rate temp. (“C) (deg. mk-’)

Ara; Xyl; Glc; Gal; Man Ara; Xyl; Glc; Gal; Rha

3% ECNSS-M 3% ECNSS-M 3% ECNSS-M 3% ECNSS-M ECNSS-M

Xyl; Glc; Gal; Rha; 3-Me-Rha Xyl; Glc; Gal; 2-Me-Gal; 6-Me-Gal Xyl; Glc; Gal; Man; Fuc Glc; Gal; Man; Rha; Abe Glc; Gal; Man; Rha; Abe

Material or problem studied

References

190-195 185 205 185 155-165

plant tissue Auena satioa plant glycosaminoglycans Stylosanthes humilis Rhodopseudomonas capsulata

416 218a 44 696 446

20% Apiezon M

220

412

3% ECNSS-M 3% ECNSS-M 3% ECNSS-M

190 195

red alga Laurencia pinnatifidia Polyporus pinicolu Salmonella typhimurium Salmonella typhimurium core Salmonella bredeney Salmonella newport Salmonella kentucky Salmonella core Brabeium stellutijolium Salmonella hybrid (B and C,) Salmonella paratyphi durazzo Salmonella typhi and Salmonella enteritidis Myxobacterium 402

Ara; Xyl; Gal; Man; Rha Glc; Gal; Man; Rha; Par Glc; Gal; Man; Rha; Tyv

3% ECNSS-M 3% ECNSS-M

Glc; Man; Rha; 2-Me-Rha; 3-Me-Rha More than five components Er; Ara; Glc; Gal; Man; Fuc Ara; Xyl; Glc; Gal; Man; Fuc

10% NPGS

Ara; Xyl; Glc; Gal; Man; Fuc

190

1% ECNSS-M

140-230

3% ECNSS-M

205

1% ov-225

170-230

3

model for glycoproteins

1

ph ytohemagglutinin polysaccharide biosynthesis mitochondrial proteins Escherichia coli core model; erythrocyte membranes

463 16 53 44 1 15,727 442 726 728 439 681 341

35 682 683,706 684 729 60a

Ara; Xyl; Glc; Gal; Man; Rha

3% ECNSS-M (1.5% EGS 1.5% XF-1150)

185

Am; Xyl; Glc; Gal; Man; Rha Ara; Xyl; Glc; Gal; Ido; Man Ara; Xyl; Glc; Gal; Man; Rha Ara; Xyl; Glc; Gal; Man; Rha Glc; Gal; Man; Rha; Abe;

3% ECNSS-M

190

+

1.5% EGS + 1.5% XF-1150 3 % ECNSS-M

180-220

model for hemicelluloses hemicellulose classification hemicellulose aggregation hemicellulose fractionation spear grass red cotton-wood 0.8

alternative to lactones

215

180

model for wood polysaccharides

218

3% ECNSS-M

195

model for kraft pulp

220

3% ECNSS-M

190

Salmonella hybrid (B and D)

730

soil polysaccharides

731

plant polysaccharides cotton cell-wall cell-wall “modifying enzyme” grasshoppers plant mucilages model for glycoproteins

39 40 41 42 44 452

soil analysis

458

190

model study

217 415

170

Salmonella typhimurium

414

Tyv

Ara; Xyl; Glc; Gal; Man; Fuc; Rha Ara; Xyl; Glc; Gal; Man Fuc: Rha

Six hexitols Ara; Rib; Xyl; Glc; Gal; Man; Fuc; Rha Ara; Rib; Xyl; Clc; Gal; Man; Fuc; Rha

Ara; Rib; Xyl; Glc; Gal; Man; Rha; Fuc; Abe; Tyk

26 426 428 427 429,430 685

+

0.2% EGS 0.2% EGA 0.4 % XF-1150

120

3 % ECNSS-M or at 175”to separate Man and Tal 3 or 5 % ECNSS-M

200

+

3% ECNSS-M (5% XE-60, 10% Carbowax 20 M modified with terephthalic acid) 3 % ECNSS-M

120-180

10 min then 1

1-2

(continued )

TABLEVI (continued)

Compounds separated

Column of

Ara; Rib; Xyl; 2-deoxy-Rib; Glc; Gal; Man; 2-deoxyGlc; (Fuc; Rha)

0.2% EGS 0.2% EGA

+

+

1.4% XE-60 (or 3% OV-225) Er; Ara; Rib; Xyl; 2-deoxy- 0.75% HiEff-1-BP + Rib; Glc; Gal; Man; Fuc; 0.25 % EGSS-X 0.1% 144-B [N,NRha; 2-deoxy-Glc; bis(2-hydroxyethy1)2-deoxy-Gal aniline] Ara; Rib; Xyl; 2-deoxy-Rib; 10 % LAC IR-296 (EGA) 10% DD-071 (EGA) All; Alt; Glc; Gal; Ido; Man; 3-Me-Glc; Rha; Fuc 10% ECNSS-M 5% DD-071+ 10% ECNSS-M (g) Heptitols 10% NPGSE D-glIjCerO-DglUCOmeso-glycero-ido-, 10% NPGSE nglycero-nidoL-glycero-nmanno3% ECNSS-M ECNSS-M L-glycero-nmanno-

Material or problem studied

150-205

model; submaxillary mucins

34a

model for glycoproteins

58

model study for soil hydrolyzates

91

225 205

Nosema apis spores synthesis

309 221

180 165

Salmonella typhimurium 414 Salmonella core lipopoly43 1 saccharide Salmonella friedenau T1 18,447 Escherichia coli 473 (see also Refs. 15,51,53,437-440,442, 728,729) Serratia marcescens 41 1,544 Serratia marcescens 493,494 339 bacterial lipopolysaccharides synthesis of heptoses 686

+

t

o

10% BDS

D-glycero-L-mannonglycero-nmannoL-gl ycero-nmannoTen isomeric heptitols

150

10% NPGSE 10% NPGSE 9.5% ECNSS-M 10% NPGS 15% BDS 15% Apiezon M (1: 1)

+

r

Column Rate temp. ("C) (deg. min-')

204 155,191 210 205

to

References

2-Deoxy-gaEacto2-Deoxy-manno-

(h) Alditols from uronic acids Glc Gal 4-Me-Glc Glc; Gal Glc; Id0 Glc; 4-Me-Glc; Gal

10% NPGSE 10% NPGSE 1.5% XF-1112 EGS

202 202

synthesis synthesis

3% ECNSS-M 3% ECNSS-M

184 180

3% ECNSS-M

185

lactonization of uronic acids Diplococcus pneumoniae mass spectrum hemicellulose model heparin uronic acid analysis

5 % BDS

+ 1.5%

210 to 225

25 min 10

660 661

98 54

468 26 47 467

0

F c

0

5

U

n

50

54 0

!? 'd

2

TABLEVII

c

Oligosaccharides Compounds separated

Column of

Column temp. (“c)

Rate (deg. min-’)

Material or problem studied

References

(a) Per-0-(himethylsily1)sucrose

3% silicone gum rubber 3.8% SE-30 1.5% SE-30 5% SE-30 3.8% SE-30 3.8% SE-30 3.8% SE-30 5% SE-30 1% SE-30

220 150 160-275 190-275 160-270 125-250 100-325 211

3% ov-1 5% ov-1 3% SE-52 3% SE-52 3% SE-52 3% SE-52 3% SE-52

from 200 110-235 150-350 150-350 110-250 238 from 150

3% SE-52

120 to 228 170

4% SE-52 3% SE-54 10% UCC-W-982

free sugars in ferns

160 then 295 150-310

urine 10 min. then 6 potatoes 6 beet molasses 4 prune juice fruits 4 holly leaves 4 urine 10 does not separate sucrose lin kage-isomers sunflowers 4 2 alfalfa 10 wort, beer, and corn syrups 4 wort and beer sweet potatoes 5 sugar-cane juice almond hulls 2 (12 min) 15 cotton 4 min 6 honey 27 min 8 (10 min) free sugars in plants 10 min 3 potatoes 10

321 118 116 328 317 318 323 149 478 325 291 128,129 316,480 123 121 150 117 314 477 115

0 0 m

2 4 4

8

2% SF-96 2% Carbowax 20 M 0.5% QF-1 3.8% UC-W98 10% Carbowax 20 M (terephthalate) 3% ov-1 3% OV-17 3% JXR 2% E-301 3% SE-52 Sucrose lactate 3% SE-30 (b) Other per(trimethylsily1) ethers of oligosaccharides Lactose 5% SE-30 Lactose 1% SE-30 HiF'ak (5% SE-30) Lactose 3.8% UC-W98 Lactulose 20% Carbowax 20 M 5% SE-30 Maltose Maltose Maltose (methyl glycoside) Raffinose Raffinose Cellobiose; maltose (also methyl glycosides)

5% ov-1 3% ov-225 10% Apiezon L 3% JXR 5% SE-30 1% SE-30 SE-30

130-270 190

3

recovery of radioactive samples

208

starch model model potatoes

340 292 277

onion bulbs extra-floral nectar

322 174

Rehmannia glutinosa Cladophorales

327 479

urine milk

149 326

model lactosuria determination of 1,6-anhydro-p D-glucose alfalfa

292 687 132

synthesis of methyl a-maltoside

349

beet molasses trimethylsilylated carbohydrate oximes amylose triesters

320 187

280

280 140-360 140 to 290 130-280 208

5 13 min 10 2.5

100-325 130-270

10 6

130-270 220 150-250

3 1.5

110-235

2

230 215 160-275 170 to 270 160

6 30 min 4

291

378 (continued)

E

TABLEVII (continued) Compounds separated

Column of

Maltose; maltotriose

20% Carbowax 20 M 1% SE-30 3% SE-52 1.5% SE-30 3% SE-54 and others 3% ov-1 15% NPGS 10% SE-30

Maltose; maltotriose Melibiose; raffinose Raffinose; stachyose Raffinose; trehalose (a,a-) Primeverose; primeverulose Isomeric trisaccharides containing Ara and Man Lactose; cellobiose Maltose; maltotriose; maltotetraose Maltose; maltotriose; maltotetraose; and others Maltose to maltopentaose Maltose; isomaltose; maltotriose; maltotetmose Maltose; isomaltose; gentiobiose; raffinose Maltose; melibiose; gentiobiose; raffinose

Mannotriose; raffinose; stachyose Cellobiose to cellopentaose Cellotriose to cellohexaose Raffinose; stachyose; (Fru),,-sucrose

Column temp. (“C)

223 190,235 140-275 150 from 200

200 270

Rate (deg. min-’)

Material or problem studied

References

enzymolysis

265

starch syrup 6 10 min, then 6 potatoes free sugars in plants 4 sunflowers epimerization of primeverose arabinomannan

113 116 477 325 252 372

10% SE-30 2% SE-52

200-350 200,250,275

4

not separated corn syrup

372 127

3% SE-52

150-350

4

wort and beer

480

3% SE-30

100-350 350 150-350

5 30 min 10

wort and beer maltopentaose wort, beer, and corn SYNP

481

3% SE-52

starch model

340

potatoes

277

2.5

Rehmannia glutinosa

327 483

5

p.m.1. spectroscopy of cellulose oligosaccharides molecular properties onion bulbs

0.5% QF-1 10% Carbowax 20 M (terephthalate) 3% ov-1 3% OV-17 3% SE-52

3% JXR

190

280 280 130-280

140-360

128,129

482

322

G El

Maltose; melibiose; melezitose; raffinose; trehalose

4 % SE-52

Lactose; maltose; gentiobiose; cellobiose; trehalose; palatinose Gentiobiose; lactose; maltose; melibiose; trehalose; fucosyl-lactose Maltose to malto-octaose Various

3.8% SE-30

23 Disaccharides

2.2% SE-30 3 % QF-1 1% NPGS 3 % JXR 15% Apiezon K 3 % SE-30 3% OV-17 (OV-1, OV-25)

Maltose; isomaltose; maltulose; melezitose; leucrose; kojibiose; nigerose Various di- to penta3-3.8% SE-30 saccharides Cellobiose; gentiobiose; 2% ov-1 laminarabiose; 3-0-p3% SE-30 cellobiosylglucose 2% ov-1 6-0-(PO-Methyl-p-~20% SE-30 glucopyranos yl)-Dgalactose (c) Peracetylated disaccharides Gentiobiose; laminarabiose 0.75% SE-30 (d) Per(trimethylsily1)ated oligosaccharide alditols Gentiobiitol 2% ov-1 3-0-a-D-Glucopyranosyl-D3% SE-30 galactitol; &O-a-D-glucop yranosyl-D-galactitol

170 to 255 255 to 290 220

245 2 10 185 90-400 2 10 2 10 228

220,300,350 220 240 270 260

27 min 8 30 min 2

15

Iwney

314

urine

327

urinary, neuraminyl oligosaccharides

89

starch hydrolyzate models for structural studies

134 484

model

485

honey

315

model

67

Zea mays cell-wall glucan

43a

synthesis

49 1

230 220 240

490a

Zea mays glucan synthesis

43a 488

(continued )

TABLEVII (continued) Compounds separated

Column of

Maltitol ; maltotriitol

3 % SE-52

Gentiobiitol; isomaltitol Gentiobiitol; lactitol; maltitol; melibiitol

5% XE-60 3 % QF-1 1% NPGS (2.2% SE-30) 2.2% SE-30 3 % QF- 1 3% XE-60 1 % ov-22 15% Apiezon K 3 % SE-30 20% SE-30

Cellobiitol; gentiobiitol; lactitol; laminarabiitol; maltitol; melibiitol Various

&0-(4-0-Methyl-p-~ glucopyranosy1)-s galactitol (as peracetate) (e) Per(trimethylsily1ated) amino disaccharides N-Acetyl-lactosamine ; 2.2% SE-30

N-acetyl-lactosaminitol; 3% QF-1 2-acetarnido-2-deoxy-3O-p-~-1% NPG S gdactosyl-D-galactose; Zacetamido-2-deoxy-3-O-p-~galactosyl-D-galactitol N-Acetyl-lactosaminitol; as ref. 389 in (d) 4-0-(2-acetamido-2deoxy-sgalactopyranosy1)-D-galactitol ZAcetamido-%deoxy-&0-a3% SE-30 L-hcopyranosyl-Dglucitol ZAcetamido-Zdeoxy-50-a3% OV- 11 D-mannopyranosyl-Dglucitol

Column temp. (“C)

Rate (deg. min-’)

140 to 275 170 2 10 185 245 263 227 230 218 2 10 2 10 275

4 min 6

Material or problem studied

References

starch syrup

113

Diplococcus pneumoniae I1 urinary, neuraminyl oligosaccharides

484a 89

structure of disaccharides by mass spectrometry, two columns necessary for complete separation models for structural studies

389

484

synthesis

490a

r

0 0 ? U

c urinary, neuraminyl oligosaccharides

245 2 10 185

240

200-232

5

89

30 z

mass spectra

389

synthesis

487

synthesis

490

SAcetamido-Sdeoxy-GO-a3% OV- 11 D-mannopyranosyl-Dglucitol (0 Per(trimethylsilyl)ateddisaccharide oximes Cellobiose 10% UC-W98 Lactose; maltose 1% SE-30 (1% OV-17, 3%

synthesis

489

alkaline stability of cellulose model for food analysis

188 187

model

133

226

model

688

235

lipopolysaccharide

689

2 10

dextran

1% ov-22

200 236 222 194 200 197

2.2% SE-30 1% ov-22

260 265

polphynn model structure of disaccharides by mass spectrometry; greater volatility of per(Me) compared with per(Me,Si) ethers structure of trisaccharides by mass spectrometry

ov-25)

(g) Per(dimethylsily1)atedcycloamyloses Cyclo-hexa-,-hepta-, and 3% JXR -octa-amyloses (h) Fully methylated oligosaccharides Sucrose; maltose; 5% NPCS cellobiose; melibiose 2% NPCS Maltose; cellobiose 2% Carbowax 20 M Sucrose; nigerose; 10% ECNSS-M isomal tose Agarobiose 3% SE-52 Melezitose; raffinose 5% NPGS Various (8) disaccharide 2.2% SE-30 alditols 3% QF-1

3% XE-60 Various (21) trisaccharides

200-232

5

100-250 170 to 270

30 38 min

325-405

20

4

52 690 688 389a

389b

TABLEVIII Acids and Lactones Compounds separated

Column of

(a) Per(trimethylsily1) aldonolactones Er BDS 10% NPGSE Ara 3% ov-1 Glc 3% OV-17 3% ECNSS-M Glc (as acetates) 1.5% EGS + 1.5% XF-1150 Gul 15% Apiezon K Glc; Id0 3% QF-1 2 % SE-52 2% CNSi (XF-1105) Glc; Gul; Man 10% NPGSE Gal; Gul 3% XE-60 ov-1 SE-30 Man ~-lyxo-5-Hexulosonic, 3% QF-1 L-ribo-5-hexulosonic Cellobionic; gulonic, and 3% QF-1 L-ribo-4-hexulosonic Er; Rib; Ara; Glc; Man BDS Ara; Xyl; Glc; Gal; Man

5% XF-1112

Column Rate temp. ("C) (deg. min-')

saccharinic acids synthesis of lyxuronic acid model for potatoes

160 170 170,185 170,185 185 185

hemicelluloses

glucuronic acid determination idonic and gulonic acids

155,175 190 190 190 175 175

140- 170

1.67

140-170

1.67

160 155-195

Material or problem studied

0.8

Serratia marcescens stigmatic exudate acid biosynthesis model Arthrobacter cjiscosus isomerization of D-glucuronic acid isomerization of D-glucuronic acid reaction of D-arabino-hexosulose and alkali aldonic acids (Glc and Gal not sepd.)

References 510 691 277 26

398,399 497

493 43 524 407 496 520 521 511 215

Glc; Gul; 3-Me-Gul; Ido; 3-Me-Id0

Ara; Lyx; Rib; Xyl; Glc; Gal; Gul; Ido; Man Pentoses; 2-deoxy-Rib; hexoses; Bdeoxyhexoses; heptoses Tetroses; pentoses; hexoses; heptoses

3% QF-1 7% DC-560 1% EGSP-Z 1% NPGS 10% NPGSE

+

10% NPGSE 10% Carbowax 20 M

3% QF-1 (also SE-30, SE-52, XE 60, ECNSS-M, EGSS-Y, EGS, HiEff-8-BP, Carbowax 1540, NPGS, DC-560 EGSP-Z) Tetroses; pentoses, and 1.5% SE-52 hexoses 2% CNSi (XF-1105) 3% NGS 3% NGS 10% NPGSE Heptonolactones (16) 10% NPGSE 3-Deoxy-D-glycero-Dgalacto-octono-1,4(2.5% SE-30, 1.5% XF-1150 lactone; 3-deoxy-Dglycero-D-talo-octono-1,41.5% NPGSE) lactone

170 185

165 165-176 170 155 140-170

1.67

new methylated uronic acid from paper pulp

522

analysis of hexuronic acids in biological material analysis of neutral sugars immunoglobulin D

205

separation of aldonic acids (and mass spectra of Me,Si lactones)

395 498 499,500

+

+

160 170 170 160 190 212

comparison of Me,Si and CF&O

228

synthesis synthesis of 3-deoxy-D-mannooctulosonic acid (“KDO’)

156 537

(continued )

c.

w

TABLEVIII (continued) Compounds separated

Column Rate temp. (“C) (deg. min-’)

Column of

3-Deox y-D-g l y cero-n

2.5% SE-30 gu~ono-octono-1,4-lactone;1.5% XF-1150 3-deoxy-Dglycero-DidoNPGSE octono-1,4-lactone; 3-deoxy-~-glucooctulosono-1,4-lactone Reduced “KDO” (acetate) 10% NPGSE

+ 1.5%

(b) Trimethylsilyl derivatives of uronic and aldonic acids GlcA QF-1, DC-200 GlcA 10% NPGSE GalA 5% SE-30 GalA 3% ov-1 3% OV-17 GalA 3% SE-52 IdoA 10% SE-30 GlcA; GalA 5% SE-30 GkA; GalA 10% SE-30 10% SE-52 20% Apiezon GlcA, GalA 3-3.8% SE-30 GlcA; IdoA 15% Apiezon M GlcA; IdoA A11A; AltA; ManA

1% SE-30 3% QF-1

205 200

204

175 175 170,185 170,185 130-180 180 200,230 180 170 210 140-200 190 140 180

01

Material or problem studied synthesis of 3-deoxy-D-glucooctulosonic acid (“KDO” analog) D-gahCtO analog

157

Gram-negative bacteria Escherichia coli Neisseria sicca

339 662 539

Cyptococcus neoformans Serratia marcescens commercial pectins model for potatoes

2.5

0.5

References

Polygonatum adoraturn Clostridium welchii uronic acids in pectin model

model heparin for other examples, see glycosaminoglycans isomerization of nglucuronic acid

158

659 492,495

523

514 319,692 270

67 76 513,515-519 19,475 520

0

P ?

tr

c

GulA; IdoA and hexulosonic 3% QF-1 acids GalA; dimer; trimer; 0.5% SE-30 unsaturated dimer and trimer 2.5% SE-52 GlcA; GalA; GulA; ManA; also glucaric and 1% SE-30 mannaric acids 2.5% XE-60 0.5% OV-1 Oxalic acid to hexaric and deoxyhexaric acids (as 0.5% OV-17 Me3Si esters) 3% QF-1 1% XE-60 (c) Per(himethylsily1) derivatives of methyl esters Ethyl 8-D-glucosiduronic 10% DC-560 acid 5% SE-52 Methyl a- and p-Dgalactofuranosiduronic acid 2.5% XE-60 and pyranoside analog Methyl a-Dmannopyranosid- 2.5 % SE-52 uronic acid GlcA GlcA; GalA GlcA; GalA GlcA; IdoA GulA; IdoA; GlcA; GalA; ManA GlcA; GalA; CulA; ManA

5% silicone G-30M 12% EGS 3% SE-30 5% Ucon LB-550 3-3.8 70SE-30 2.5% SE-52 1%SE-30 2.5% XE-60

180 130

2 then 12

190 170 170 160 160 120

90-250

2

190 190 190 170 140 140-200 205 140-200 190 170 170

0.5 0.5

isomerization of D-galacturonic acid oligogalacturonic acids

26 1

aldonic, alduronic, and aldaric acids

525

model

164

metabolite of ethanol

535

methyl glycosidation and esterification of galacturonic acid methyl glycosidation and esterification of mannuronic acid model model model heparin model

531

aldonic, alduronic, and aldaric acids

170

532 527 34 66a 529 67 525

(continued)

$$

TABLEVIII (continued) ~

Compounds separated

Column of

GlcA; GalA; ManA; GulA; IdoA Hexuronic acids

3.8% SE-30

4-Deoxy-~-threo-hex-4enuronic acid; also L-arabino and D - X I ~ ~ O isomers GlcA; GalA; ManA; 4-MeManA and their 4,5-unsaturated analogs GalA; dimer; trimer; unsaturated dimer and trimer Glucosiduronic acids, and 1-amino-1-deoxy and 1-thio analogs Phenyl, naphthyl, and bomyl glucosiduronic acid 3-Deoxy-Dmanno-octulosonic acid (“KDO”) Reduced “KDO” (acetate)

Column Rate temp. (“C) (deg. min-’) 140-200

0.5

Material or problem studied

References

model for determination of natural hexuronic acids model

666

20

SE-30, UCON-50-LB550-X, NPGS 1 % SE-30 2.5% SE-52

170 190

4-deoxyglycuronic acids

385

5 % SE-30

170

p-elimination of methyl uronates

530

oligogalacturonic acids

170

9

0.5% SE-30

130

1.5% SE-30 1.5% SE-52 1% CNSi (XF 1105) 1 % NGS 2% or 5 % SE-30

220 220

5 % Ucon LB-550 10% NPGSE 10% NPGSE

2 min then 12

.o I”

c ’ e3

2 comparison of acetyl, methyl, and Me,Si derivatives

533,534

glucuronic acid conjugates

536

192

Salmonella minnesota glycolipid

538

201 230

Gram-negative bacteria Neisseria sicca Escherichia coli

339 539 662

2

(d) Per(trimethylsily1) . . derivatives cw-Isosaccharinic; a- and p-glucometasaccharinic; a-glucosaccharinic; 3-deoxypentonic; 3,4and 2,4-dihydroxybutyric Glucometasaccharinic; glucoisosaccharinic; glucosaccharic Glucometasaccharinic acids Glucosaccharinic

of saccharinic and related acids 15% Carbowax 20 M 170 (3% SE-52 150 15% EGS) 150

BDS

10% EGS BDS

Glycolic through C,, C,, and C, acids to gluconic acid, as the Me2Si derivatives of their lactones Fourteen lactones

1 % SE-52 PO-17 (OV-17) 3 % polyphenyl ether

Saccharinic and related acids

3 % QF-1

Saccharinic and aldonic acids Various

160

BDS

QF-I, ECNSS-M ov-1 OV-17 (cap) BDS

160

160 140-165

160 165

1.67

analysis of saccharinic acids

501

saccharinic acids from glucose, mannose, and fructose

693

decomposition of sugars bacterial lipopolysaccharides 1-deoxy-D-erythro-2,3hexodiulose acids obtained by oxygenation of hexoses in aqueous alkali

512 434 510

saccharinic acids from xylose and fructose alkali treatment of hydrocellulose oxygen bleaching of hydrocellulose action of chlorine dioxide on holocellulose chlorine oxidation of cellulose

509

166

504 506 588

453 (continued ) c.

k

w rp

TABLEVIII (continued) Column Rate temp. ("C) (deg. min-')

t s

Compounds separated

Column of

Material or problem studied

Saccharinic and aldonic acids Various (19) monoprotic acids Glucopyranosylglycolic acids

1%SE-30

polysulfide pulps

502

1% SE-30

alkaline oxidation of glucose and cellobiose formation during hydrolysis of cellulose

503

References ~

3% QF-1

180

508

TABLEIX Amino Suears Compounds separated (a) Trimethylsilyl hexosamines GlcN GlcN GlcN GlcN GlcN; GalN GlcN; GalN

Column of

Column temp. ("C)

2.5% SE-30

1.2% XE-60 5 % QF-1 3.8% SE-30 2% OV-17 1%SE-30 3% SE-30 (1%QF-1)

160 180 140 205 144

Rate (deg. min-')

Material or problem studied keratan sulfate liquid phases and solid supports phytoglycolipid Rehmannia glutinosa Myrine glutinosa glycosaminoglycans

References

331 289 568 327,327a 330 179 542

GlcN; GalN GlcN; GalN GlcN; GalN

2.2% SE-30 3% QF-1 1% SE-30 15% Apiezon M 15% Apiezon M

187 140,200 140 190 170

GlcN; GalN

3% Apiezon L

175

GlcN; GalN

GlcN; GalN 20% BDS 10% SE-30 GlcN; GalN (b) 0-(Trimethylsilyl) acetamidodeoxyhexoses GlcNAc 10% NPGSE GlcNAc 10% NPGSE

GlcNAc GlcNAc GlcNAc GalNAc GlcNAc; GalNAc

100-160 180

5

190 190

GlcNAc; GalNAc GlcNAc; GalNAc

10% SE-30 3% SE-52 10% NPGSE 3% ov-1 2.2% SE-30 3% QF-1 10% SF-96 20% EGS

150-240 170-220 190 170 187 140,200 190 70-220

GlcNAc; GalNAc GlcNAc; GalNAc GlcNAc; GalNAc GlcNAc; GalNAc GlCNAc; GalNAc

3.8% SE-30 3.8% UCC-W982 10% NPGSE 10% NPGS 10% NPGSE

150-220 160 175 191 190

4 2

15 1

mass spectra

144

glycosaminoglycans heparin hyaluronic acid, chondroitin sulfate silylation with N,O-bis(trimethy1 sily1)trifluoroacetamide not separated chick allantoic antigen

475 76 541

Serratia marcescens Serratia marcescens, Salmonella typhimurium, Escherichia coli blood-group oligosaccharides human milk Myxobacterium 402 mass spectrum mass spectra

495,544 410

model myo-inositol in yeasts and bacteria urine gastric-juice glycoprotein Serratia marcescens Serratia marcescens model, (Y anomers crystn., i.r. spectra Streptococcus sulivarius

180

34 302

405 545 341 200

144 256 112

118 549 493,494 676 540 544a

(continued)

TABLEIX (continued) Compounds separated

Column of

GlcN; GalN GlcN; GalN

1% OV-225 3.8% SE-30

GlcN; GalN GlcNAc; GalNAc

3% OV-17 3.8% SE-30

GlcNAc; GalNAc GlcNAc; ManNAc

17% EGS 10% SE-30

Column temp. (“C)

Rate (deg. min-’)

170-230 145 to 205 155-195 120-200

1 10 min 2 4 2

150 150 to 210

5 min 4

GlcN; GalN 2 % Apiezon L N-(ethoxycarbonyl) and 4 % NPGA Manpcu-(1+3>GlcNAc 3 % ov-11 200-232 Manpa-( 1+6)-GlcNAc 3 % ov-11 200-232 (c) Methyl per-0-(trimethylsily1)acetamidodeoxyhexosides GlcNAc 3.8% SE-30 120-200 GlcNAc 10% NPGSE 195 GlcNAc 3% SE-30 or 100-260 3% OV-17 GalNAc 2.5% SE-30 120-190 GalNAc 3 % SE-30

GlcNAc; GalNAc, 3-acetamido-Glc Total hexosamine GlcNAc; GalNAc GlcNAc; GalNAc

3%

ov-1

2.5% SE-30 3.1 % SE-30 3.8% SE-30

References

model; erythrocyte membranes bovine gamma-globulin

60a 60

model model, N-acetylation in aqueous acetone model synthesis of N-acetylneuraminic acid gastric secretions

70a 64

2

171 253 36 Q

5 5

synthesis synthesis

490 489

2

model chitin oligosaccharides a,-acid glycoprotein

64 551 569

gangliosides model casein milk blood mass spectra

552a 69 370 37 1 360 200

5 1

170 160 150-200 140-200

c

Material or problem studied

3 0.5

blood-group ABH and Leb model model connective tissue TA, cell antigenic glycopeptides

334 364,365 86 369 368 366

0 ?

U

C

4

8z

GlcNAc; GalNAc

3% SE-30

140-200

0.5

GlcNAc; GalNAc

10% B D S (4% SE-30,2% NPGS) 2.5% EGS 1% SE-30 3% OV-17 2.2% SE-30 3% QF-1 3-3.8% SE-30 or UC-W98

125-215

4

GlcNAc; GalNAc

GlcNAc; GalNAc (also free NHz) AraNAc; RibNAc, all 2-acetamido-2deoxyhexoses except TalNAc (d) Per-0-(trimethylsilyI)acetamidodeoxyalditols GlcNAc 10% NPGSE 10% B D S GlcNAc; GalNAc; ManNAc (4% SE-30,2% NPGS) GlcNAc; GalNAc (and 3% QF-1 free NH2) 2.2% SE-30 1-Amino-1-deoxyglucitol 10% UC-W98 FucP-~-( 1+6)-GlcNAc 3% SE-30 (e) Peracetylated aminodeoxyalditols GlcNAc 3% ECNSS-M Eight aminodeoxyhexitols 10% NPGSE 1.5% XF-1150 -t 1.5% NPGSE

100- 160 160 to 210 187 140,210 140-200

195 125-215

140-200 187 100-250 240 190 240 235

model gastric mucosa glycoprotein oligosaccharides model

66a 709 329

mass spectra

144

70

25 min 15

0.5

4

30

67

model

551 329

chitin oligosaccharides glycoprotein oligosaccharides, ManNAc not sepd. on BDS submaxillary mucins mass spectra

555,556 144

alkaline stability of cellulose synthesis

188 487

Salmonella typhimurium model Neisseria sicca Neisseria catarrhalis Neisseria perflava Moraxella duplex, Micrococcus culco-aceticus Escherichiu coli blood-group of oligosaccharides

45 557 558,559 560 559 433 420 45

(continued)

TABLEIX (continued) Column temp. (“C)

Compounds separated

Column of

AraNAc; 4-acetamido-Ara; 4-acetamido-Xyl GlcNAc; GalNAc

3 % ECNSS-M

Rate (deg. min-I)

190

0.75% HiEff-1-BP

160-210

1.3

hydroxyethy1)aniline 3 % PolyA-103 (1% ECNSS-M)

210-260 140-230

5

GlcNAc; (GalNAc; ManNAc) 0.2% EGS 0.2% EGA 1.4% XE-60 (3% OV-225) GulNAc; IdoNAc; 10% NPGSE 2-acetamido-2-deoxy-Dglyceroo-Dgulo-heptitol (0Methyl neuraminate N-Acetylneuraminic acid 2% OV-17 Methyl neuraminate, methyl 2.5% SE-30 acetal Methyl N-acetylneuraminate 3.1% SE-30 2.5% SE-30 3% SE-30

150-205

1

3% SE-30 or 3% OV-17

GlcNAc; GalNAc; ManNAc

+ 0.25% EGSS-X + 0.1% N,N-bis(2-

+

+

3

245

Material or problem studied

References

occurrence in Salmonella of 4-amino-4-deoxy-~-arabinose model, not separated

72 58

model ManNAc not separated immunoglobulin model; submaxillary mucins

562 35 21 34a

synthesis

56 1

175 160

7.9

casein and glycoproteins glycolipids and gangliosides

114 66,334

150-200 100-210 140-200

3 1 0.5

100-260

5

model gangliosides model gastric mucosa a,-acid glycoprotein

364,365 552a 66a 709 569

TABLEX: Anhydro Compounds Compounds separated (a) Per(trimethylsi1yl) ethers 1,4-Anhydroribitol

Column of 3.5% SE-52

1,6-Anhydro-pglucopyranose

5% SE-30 10% Carbowax 20 M

1,6-Anhydro-@-glucofuranose and -pyranose

3% SE-52

Column Rate temp. (“C) (deg. min-’) 130 150-250

1.5

Material or problem studied

References

distinction between glycerol and anhydroribitol determination in corn syrups thermal analysis analysis of starch pyrolysis preparation of 1,Ganhydro-bD-ghcopyranose, by using MsSO pyrolysis of cellulose polymerization study

401

567 125a

374

5% SE-30

190

5% SE-30

185

Taka amylase specificity

145 168 145 160

model for determination of 1,6-anhydro-P-~glucopyranose. Pyrolysis of starch and dextran interference in pulp analysis

1,6-Anhydro-4-deoxy-parabino-hexose 1,GAnhydro-p-idopyranose

5 % XE-60 (15% EGS 3% SE-32 10% Carbowax 20 M modified with terephthalic acid) 2.5% SE-52 1 % SE-30 3.8% SE-30

2,5Anhydromannose; 2,5-anhydromannitol

3% SE-30 3% OV-17

1,6-Anhydro-P-glucopyranose and its 2-, 3-, 4-0-methyl, 2,3-di-0methyl, and 2-deoxy derivatives Methyl 3,Ganhydroglucoside 1,6-Anhydro-p-glucofuranose and -pyranose, 1,6-anhydro-a-galactofuranose and -p-galactopyranose

190 170 170 100-260

5

141 565 565a 131

80

78

4-deoxyglycuronic acids

385

epimerization of glucuronic acid a,-acid glycoprotein

568

+ HNOZ

569 (continued)

TABLEX (continued) Compounds separated

Column of

Column temp. (“C)

2,5Anhydromannitol, 2,5anhydro-3-0-methylmannitol, 2,5anhydrotalitol Six heptuloses and their 2,7-anhydrides Mono-, di-, tri-, and polysaccharides

5% SE-30

170-190

amino sugars

1% LAC 1-R-296

177,190

equilibrium study

Acetates 1,6-Anhydro-&idopyranose 1,6-Anhydro-8-idopyranose; 2,Sanhydromannitol Eight aldohexoses, together with their 1,banhydrides 3-Deoxy-ribo-, rylo-, arabino-, lyxo-hexoses and their 1,Ganhydrides 2,8-Anhydro-1-deoxy-Dglycero-wgulo-octulopyranose and 2,7-anhydro isomer Methyl ethers 1,4Anhydro-2,3,6-tri-Omethyl-8-galactopyranose 1,6-Anhydro-2,3-di-Omethyl-8-gulopyranose, 1,6-anhydro-2,3,4tri-Omethyl-P-gulopyranose

Rate (deg. min-’)

polydecane-1,lO-diol succinate, Easiman NP-10 hexatriacontane

Material or problem studied

+ HN02

References 564

79

pyrolysis

566

568

0 0

388

In

3.8% SE-30

170

UC-W-98

220

epimerization of glucuronic acid L-iduronic acid in heparin

1.5% LAC 1-R-296

274

equilibrium study

79

1.5% LAC 1-R-296 (ribo- as Me&, others as OAc) 2% NPGS

274

equilibrium study

79

210

bacterial and algal polysaccharides

570

galactan of red-spruce compression-wood alginic acid

571

10% Apiezon M 10% EGS 3% XE-60

140-180 140-180 150,175,200

2 2

c h 01

88

U C c-l c-l 0

z

TABLEXI Cyclitols Compounds separated (a) Per(trimethylsily1) ethers myo-Inositol

Column of 5% SE-30 3.8% SE-30 1.5% SE-30 3%

ov-1

3% OV-17 3% SE-52 1% SE-30 1.2% XE-60 5% QF-1 3% SE-30

myo-Inositol, pinitol, sequoyitol dextro-, myo-, scylloInositol myo-, scyllo-Inositol; myo-inosose-2 myo-, scyllo-Inositol; myo-inosose-2 (quinic, shikimic acids)

15% EGS 3% SE-52 15% EGS 10% ucc-w-982 3% SE-30 5% SE-30 5% Ucon LB-550 (SE-30, EGS) 1% EGSS-X (SE-30, OV-17, EGS) 20% EGS 20% Carbowax 20M

Column temp. (“C)

Rate (deg. min-’)

160-275 160 150

6 10 min then 6

185

185

150 to 280 160

2(12 min) 15

145 160 170 158 150-310 175 190

10

Material or problem studied

References

sugar beet phytoglycolipid potato tubers

320 568 116

potato tubers

277

almond hulls sweet potatoes brain liquid phases and solid

150 123 573 289

0

z

0

2

U 0

178

E0

406

5

serum and urine potato extracts Acetabularia crenulata cyclitols in cycads

138 115 699 694

F ’d

5

574,575

animal tissue

140

electron-capture detector

579

myo-inositol in yeasts and bacteria

112

15

E:

supports kidney, beef brain, and tissue extracts mammalian nerve

170

70-220

F

r

b (continued)

8LS

OTZ STZ

ozz LLS 9LS 16

8’0

c LOP

Z euaJ3eq

OTZ 061 087

ozz-OLT

091 091

TI1

- o d w 30 sap!souueur

103

lapour

Z

pc

% T 10 96Z-8-1 3 V 1 %S‘T 09-3x %S’1 %a-8-1 3v1 %S‘T 96Z-8-1 3 V 7 %ST H-SSN3B %F VI-SSN33 8 0 1

OIZ u a v OPI 9PI 091

091 06I‘OLT

ZLS

OPI-001

s33 %ST oc-3s %c s33 %ZI

TABLEXII: Polyhydric Compounds Compounds separateda (a) Per(trimethylsily1) ethers EG EG; G1 EG; G1

Column of

EG; G1 EG; G1 EG; GI EG; Er EG; 2-Me-Er

3.3% XE-61 100-320 10% Apiezon M 20% Apiezon M and L 132,163 (20% Apiezon M 20% SE-30, 20% DEGS) 7% Apiezon L 90-210 7% Apiezon 90-210 20% Apiezon M and L(1:l) 20% SE-30 170 20% SF-96 120-195

EG; G1; Er

20% SF-96

EG; G1; Er

20% SE-52

EG; G1; 1,4-butanediol EG; GI; Er; Th

20 % Silicone grease 20% SF-96

EG; G1; 1,2-propanediol; 1,3- and 2,3-butanediols EG; GI; Er; 1,2- and 1,3-propanediols

Rate (deg. min-I)

References

polyethylene glycols Tsuga canadensis Populus tremuloides

194 600 599

25 25

Populus monilifera Salir alba Picea abies Crotalaria mucronata 6-0-(40-methyl-pwglucopyranosy1)D-galactose Zea mays xylans

605 604 601 336 490a

rapeseed amyloid

602

on-column trimethylsilylation model for periodate-oxidized pol ysaccharides oils and alkyd resins

127 608

model, sugar degradation products

232

cr,-acid glycoprotein model, n.m.r. and mass spectra

367 613

2 3 min 3 6 min 3

20% DC-11

130 to 220 100-300

6 min 3 6

3% SE-52 3% JXR

120 or 60

2 l(10 min) 4

160,205 145-185 (loosteps)

Material or problem studied

10

90 to 220 140 to 250

Glycolaldehyde; GI; Er; Th 5% Ucon LB-500 Glycolaldehyde dimer; 5% SE-30 glyceraldehyde dimer; 1,3-dihydroxy-2-propanone dimer ~~

Column temp. (“C)

701

627

~

’ EG, ethylene glycol; Er, erythritol; G1, glycerol; Th, threitol.

(continued)

-

TABLEXI1 (continued) Compounds separated"

Column of

Glycolaldehyde dimer; glyceraldehyde dimer; 1,3-dihydroxy-2propanone and dimer Glycolaldehyde oxime; glyceraldehyde oxime; G1: Er Glycolaldehyde; G1; 1,2propanediol; Er; Th G1 GI G1

0.2 % hexatriacontane 0.21 % polydecane1,lO-diol succinate

GI G1 ~1-*4c G1; l,&-propanediol

3.5% SE-52 2% ov-1 2% Carbowax 20 M 3% SE-30 15% Apiezon K 5% SE-30 15% EGS 10% Apiezon K 5 % Ucon LB-550 5% Ucon LB-550X 1.2% XE-60 5% QF-1 3% ov-1 10% SE-30

GI; Er G1; Er G1; Th G1; Man Er Er Er Er; Th G1; 1,2-propanedioI; 1 , s butanediol; bi- and tetra-ethylene glycol

15% EGS or 5% SE-30 10% SE-30 20% SE-52 2% SE-30 20% EGS

3% SE-30

Column temp. ("C)

Rate (deg. min-')

125 125 110

8

to 160

10

100-180 70 to 200

4 20 min 4

100-110 70-220

2 15

130 170 105,135,170 155 155,175 100-180

4

155,175 205 105-195

7

140-190 153

2.5

65 to 180

2 min 13

01

Material or problem studied

References

dimeric a-hydroxy carbonyl compounds

612

model

616

model model; chick antigen Lorenzini jelly Leuconostoc dextran lipids myo-inositol in yeasts and bacteria 1,4-anhydroribi to1 Zea mays glucan recovery of 14C compounds Ascophyllum nodosum

190 603 303 607 628 112

Micrococcus lysodeikticus Southern pine Cladophora rupestris glycoprotein succinoglucan liquid phases and solid supports microbial media separation of polyols by ion exchange tobacco humectants

300 696 602a 405 310 289

KJ

0 0

v 401 43a 208 398

159 608a

626

U

c

c3

3z

(b) Acetates EG EG; G1; Er EG; G1; Th EG; GI; Th EG; 1,2-propanediol, Cly; Er; Th EG; G1; Er; Th Glycolaldehyde; glyceraldehyde; GI; Er G1 G1 G1 G1

G1; Er G1; Er G1; Er G1: Er C1; Er GI; Er GI; Er G1; Er

3% ECNSS-M 10% NPGSE 15% polyphenyl ether 3% ECNSS-M 20% SE-30 20 % Versamide 900 20% QF-1 BDS 15% EGS 3% ECNSS-M 3% ECNSS-M 3% HiEff-8-BP

185 100,150,175 190 170 125-280 210 190 to 220 190 175 110-158 200

+

Apiezon + BDS silicone oil 20% Apiezon M 20% Apiezon M 10% NPGSE

197 197 180

10% QF-1 10% EG isophthalate 10% NPGS

200 190 200

3% ECNSS-M 5% XE-60 10% Carbowax 20 M (terephthalate)

190 160

7.5

8 min 30

4

oat glucan periodate oxidn. i n Me,SO reduced galacturonan

50a 587 585

oat glucan hydrogenolysis of carbohydrates

705 145

phytoglycolipid acetyl migration in methyl mannopyranosides B. glabrata galactan S. oblongus galactan Salmonella core synthesis of 2-deoxy-~-erythropentose and 2-deoxy-D-threopentose model for starch studies

254 615

jack pine Microsporum glucan lead tetraacetate oxidations in Me,SO Serratia marcexens birch sap

581 584 586

Phytophthora cinnamoni glucan model for alditol acetates

590

582 583 434 390

340

676 609

217

(continued)

TABLEXI1 (continued) Compounds separated GI; Er GI; Er GI; Er GI; diGl

GI; 1-Me-Er GI; Er; Th GI; Er; Th Er Er

Er Er; Th Er; Th Er; Th Er; Th Th (c) Polyols EG and diEG EG and its ethers EG; GI; 1,2-propanediol and higher glycols

Column temp. (“C)

Column of 5% SE-52 3% OV-225 0.5% SE-30 on glass beads 0.25 % silicone grease on glass beads 2% XE-60 2 % LAC-1-R-296 10% ECNSS-M 3% ECNSS-M NPGSE and EGSS-X at various loadings 0.75% HiEff-1-BP 0.25 % EGSS-X + 0.1% 144B [N,Nbis(2-hydroxyethy1)aniline] 3% ECNSS-M EGS 3% ECNSS-M 2% XE-60 LAC 728 3% ECNSS-M

+

5% Carbowax Porapak S 10% polyvinylformalpropionitrile 10% Versamide 900

150-205

Rate (deg. min-I)

1

170 180 170-220 100-200

0.8 8

160-210

1.3

210 170-200 160 225 195

2

190 200 150-290

10

130-225

3.3

Material or problem studied

References

aquatic moss red pine (Pinus resinosa) model; glycoproteins glycerol analysis

593 594 34a 597

Ustilago Ceratocystis model for soil hydrolyzates Fusicoccum amygdali model for alditol acetates

596 346 91 718 417

model for neutral sugars in glycoproteins

58

oat glucan sophorosides Arthrobacter viscosus Azotobacter uinelandii heparin Salmonella friedenau TI analysis of EG analysis of EG ethers separation of polyols

345 592 591 595 388 18,447 6 19 620 618

EG; G1; Er

Polypak 1

EG; 1,Zpropanediol; 2,bbutanediol Acyclic and alicyclic diols G-C,

15 % diglycerol

15% LAC 2R-446

250 or 150 to 250 70-135 175

5 min 4 5

polyhydric compounds in biological media hydrogenolysis of carbohydrates

473

model for diols

617

145

GAS-LIQUID CHROMATOGRAPHY 155

TABLEXI11

Per-0-(trimethylsily1)glycosylalditols” ~

Column of

Column temp. PC)

Rate (deg. min-’)

20% SF-96 20% SF-96

150-240 165

3

20% SF-96 3% SE-52 5% XE-60 20% SF-96

225 185 175 or 125-190 255

3% SE-52

255

20% SF-96 3% SE-52

165,190-250 185

6

Glc-p-(l+l)-Gl

20% SF-96 3% SE-52

165,190-250 185

6

Glc-a-(1+2)-G1 Glc-/3-(1+2)-G1 Glc-/3-(l+l)-Gl (as acetate) Glc-a-(1+ 1)-Er Glc-fi-(1+ 1)-Er Glc-a-(1+2)-Er Glc-B-(1+2)-Er

20% SF-96

190-250

6

10% SE-30 20% SF-96

150-250 190,190-250

8 6

Compounds separatedb D-Xylose Xyl-a-(1+2)-G1 Xyrf’/3-(1+2)-Er D-Galactose Gal-a-(l+l)-Gl Gal+-( l+l)-Gl Gala-( 1+2)-G1 Gal-a-(l+l)-Th Gal-P-(l+l)-Th Gal-a-(1+2)-Th Gal+-( 1+2)-Th Gal-(l+2)-Glc-a-(l+ 1)-G1 D-Glucose Glc-a-(l-t 1)-G1

Material or problem studied

References

sapote gum synthetic glucan

643 639

synthetic galactan synthesis Klebsiella 0 group 5

207 649 723

synthetic galactan

207

4

synthesis, glycolipid

647,649

synthetic glucan “polymaltose” synthesis glycolipid synthetic glucan “polymaltose” synthesis “pol ymaltose”

639 642 649 647 639 642 649 642

sophoroside synthetic glucan “polymaltose”

592 639 642

Glc-a-(1+2)-Glc-a-( 1 41)-G1 Clc-P-(1+2)-Glc-a-( 1+ l)-G1 Glc-j3-(1+2)-Glc-P-( 1- l)-G1 Glc-p-(1+3)-Glc-(1+ l)-Gl Glc-a-(1+4)-Glc-a-( 1+ l)-Gl Clc-a-(1+4)-Glc-P-( l+l)-Gl Glc-P-(1+4)-clc-p-( 1+ l)-G1 Glc-p-(1+6)-Glc-P-( 1+ l)-GI Clc-a-(1-+4)-Glc-a-(1+ 1)-Er Glc-a-(1+4)-Glc-p-( I+ 1)-Er D-Mannose Man-a-(l+l)-Er Man+-( l+l)-Er Mane-(1+2)-Er Man+-( 1+2)-Er Man-a-(1+2)-G1 L-Rhamnose Rha-P-(1+2)-l-deoxy-Er Rha-a-(1+3)-1-deoxy-Er Rha-P-(1+3)-1-deoxy-Er Rha-(1+4)-Rha-( 1+3)-l-deoxy-Er “

All sugars pyranose, except as noted.

3% SE-52

255

20% SF-96

210-265

3% SE-52

255

20% SF-96

210-265

20% SF-96

235

5% XE-60

175 or 125- 190

20% SF-96

180

10

10

4

* Er, erythritol; G1, glycerol; Th, threitol.

synthesis

649

“polymaltose”

642

synthesis

649

“polymaltose”

642

synthetic mannan

640

Klehsiella 0 group 5

723

synthetic rhamnan

64 1

158

G. G. S. DUTTON

(651) R. E. Wing and J. N BeMiller, Corbohyd. Res., 10, 371 (1969). (652) H.-C. Curtius, M. Miiller, and J. A. Viillmin, J . Chromatop., 37, 216 (1968). (653) D. M. W. Anderson, Sir E. (L.) Hirst, and J. F Stoddart,]. Chem. SOC. ( C ) ,1476 (1967). (654) M. H. Saier and C. E. Ballou, J. Biol. Chem., 243,992 (1968). (655) Yu. S. Ovodov, R. G. Ovadova, 0.D. Bondarenko, and I. N. Krasikova, Carbohyd. Res., 18,311 (1971). (656) K. Asano, M. Morita, and M. Fujimaki, Agr. B i d . Chem. (Tokyo), 34,1522 (1970). (657) Yu. S. Ovodov, L. V. Mikheyskaya, R. G. Ovodova, and I. N. Krasikova, Carbohyd. Res., 18, 319 (1971). (658) F. Fukuoka, M. Nakanishi, S. Shibata, Y. Nishikawa, T. Takeda, and M. Tanaka, Gann, 59, 421 (1968). (659) G. Gargani and C. Crasso, Boll. SOC. Ztal. Biol. Sper., 44, 2026 (1968). (660) M. B. Perry and A. C. Webb, Can.J.Chem.,46,789 (1968). (661) M. B. Perry, Can.]. Chem., 45, 1295 (1967). (662) M. B. Perry and G. A. Adams, Biochem. Biophys. Res. Commun., 26,417 (1967). (663) P. A. J. Gorin and J. F. T. Spencer, Can.J . Chem., 46,3407 (1968). (664) D. J. Casagrande, J. Chromatogr., 49,537 (1970). (665) J. T. Marvel, J. M. Berry, R. 0. Kuehl, and A. J. Deutschman, Carbohyd. Res., 9, 295 (1969). (666) M. Tomoda, Yakugaku Zasshi, 87, 1057 (1967); Chem. Abstr., 68, 65,514 (1968). (667) R. SGmme, Acta Chem. Scund., 25,759 (1971). (66s) A. Misaki, N. Ikawa, T. Kato, and S. Kotani, Biochim. Biophys. Acta, 215, 405 (1970). (669) W. Mackie, Carbohyd. Res., 9, 247 (1969). (670) L. Muir and Y.-C. Lee, J. Biol. Chem., 244,2343 (1969). (671) H. P. Humphries and 0. Theander, Carbohyd. Res., 16,317 (1971). (672) D. T. Williams and J. K. N. Jones, Can. J. Chem., 43,955 (1965). (673) S . J. Scott and G. W. Hay, Can.J. Chem., 45,2217 (1967). (674) C. T. Bishop, M. B. Perry, F. Blank, and F. P. Cooper, Can. J. Chem., 43, 30 (1965). (675) P. A. J. Gorin and J. F. T. Spencer, C a n . ] . Chem., 46,2299 (1968). (676) R. Young and G. A. Adams, Can.]. Chem., 43,2929 (1965). (677) R. G. Brown and B. Lindberg, Acta Chem. Scand., 21,2383 (1967). (678) H. Bjorndal and B. Wagstrom, Acta Chem. Scand., 23, 1560 (1969). (679) D. G. Medcalf, B. L. D’Appolonia, and K. A. Gilles, Cereal Chem., 45,539 (1968). (680) P. Binette, M. Matsuzak, E. Calkins, R. Alper, and R. J. Winder, Proc. SOC. Exp. Biol. Med., 137, 165 (1971). (681) C. G. Hellerqvist, B. Lindberg, S. Svensson, T. Holme, and A. A. Lindberg, Acta Chem. Scand., 23,1588 (1969). (682) T. Takahashi, P. Ramachandramurthy, and I. E. Liener, Biochim. Biophys. Acta, 133, 123 (1967). (683) P. J. Harris and D. H. Northcote, Biochem. I., 120,479 (1970). (684) S. S. Martin and H. B. Bosmann, Exp, Cell. Res., 66,59 (1971). (685) P. J. Garegg and M. Han, Svensk Papperstidn., 71,331 (1968). (686) R. Young and G . A. Adams, Can.J. Chem., 44,32 (1966). (687) M. Miiller, J. Walker-Smith, D. H. Shmerling, H.-C. Curtius, and A. Prader, Clin. Chim. Acta, 24,45 (1969). (688) M. Gee and H. G. Walker, Anal. Chem., 34,650 (1962). (689) A. Makita and T. Yamakawa, J . Biochem. (Tokyo), 55, 365 (1964).

GAS-LIQUID CHROMATOGRAPHY

159

(690) N. S. Anderson and D. A. Rees,J. Chem. Soc., 5880 (1965). (691) R. K. Hulyalkar and M. B. Perry, Can. J. Chem., 43, 3241 (1965). (692) R. C. Wiley, M. Tavakoli, and M. D. Moore, Proc. Amer. Soc. Hort. Sci., 89, 34 (1966). (693) A. A. J. Feast, B. Lindberg, and 0.Theander,Acta Chem. Scand., 19,1127 (1965). (694) N. V. Riggs and F. M. Strong, Anal. Biochem., 19,351 (1967). (695) C. P. Way and G . N. Richards, Aust. J . Chem., 24, 1041 (1971). (696) M. Alam and G. N. Richards, Aust. J . Chem., 24, 1049 (1971). (696a) K. D. Sears, A. Beiilik, R. L. Casebier, R. J. Engen, J. K. Hamilton, and H. L. Hergert, J. Polym. Sci., Part C, 36, 425 (1971). (697) I. Yano, Y. Furukawa, and M. Kusunose,]. Gen. Appl. Microbiol., 17,329 (1971). (698) C. J. Lote and J. B. Weiss, F E B S Lett., 16, 81 (1971). (699) E. J. Bourne, E. Percival, and B. Smestad, Carbohyd. Res., 22, 75 (1972). (700) G. R. Gray and C. E. Ballou,J. Biol. Chem., 246, 6835 (1971). (701) G. G . S. Dutton and M. S. Kabir, Phytochemistry, 11, 779 (1972). (702) R. G. S. Bidwell, E. Percival, and B. Smestad, Can. J. Bot., 50, 191 (1972). (703) H. Baine and R. Cherniak, Biochemistry, 10, 2948 (1971). (704) M. Tomoda, Y. Yoshida, H. Tanaka, and M. Uno, Chem. Phorm. Bull. (Tokyo), 19, 2173 (1971). (705) A. J. Buchala and K. C. B. Wilkie, Phytochemistry, 10, 2287 (1971). (706) M. F. Heath and D. H. Northcote, Biochem.J., 125, 953 (1971). M. Tomoda and M. Uno, Chem. Pharm. Bull. (Tokyo), 19, 1214 (1971). B. L. Slomiany and M. I. Horowitz, Zmmunochem&y, 8, 719 (1971). W. Barton, P. Brown and J. R. Clamp, Clin. Chim. Acta, 36, 262 (1972). K. Sugahara, T. Okumura, and I. Yamashina, FEBS Lett., 20, 44 (1972). J. Weckesser, G. Rosenfelder, H. Mayer, and 0. Liideritz, Eur. J. Biochem., 24, 112 (1971). H. Bjorndal, B. Lindberg, J. Lonngren, K. Nilsson, and W. Nimmich, Acta Chem. Scand., 26, 1269 (1972). B. Lindberg, J. Liinngren, and W. Nimmich, Carbohyd. Res., 23, 47 (1972). M. Brogioni and P. Damiani, Znd. Aliment. (Italy), 10, 83 (1971);Chem. Abstr., 74, 83,944 (1971). R. Shainkin and G. Perlmann, Arch. Biochem. Biophys., 145, 693 (1971). M. E. Etzler, B. Anderson, S. Beychok, F. Gruezo, K. 0. Lloyd, N. C. Richardson, and E. A. Kabat, Arch. Biochem. Biophys., 141,588 (1970). B. Anderson, E. A. Kabat, S. Beychok, and F. Gruezo, Arch. Biochem. Biophys., 145, 490 (1971). M. A. Obaidah and K. W. Buck, Biochem. J., 125,473 (1971). S. C. Churms and A. M. Stephen, Carbohyd. Res., 19, 211 (1971). B. Lindberg, J . Lonngren, W. Nimmich, and J. L. Thompson, Carhohyd. Res., 25, 49 (1972). R. B. Carlsen and J. G. Pierce,]. B i d . Chem., 247, 23 (1972). L. F. Molloy and E. L. Richards,]. Sci. Food Agr., 22, 393 (1971). B. Lindberg, J. Lijnngren, and W. Nimmich, Acta Chem. Scand., in press. B. J. Gormus and R. W. Wheat,J. Bacteriol., 108, 1304 (1971). (725) C. G . Hellerqvist, J. Hoffman, B. Lindberg, A. Pilotti, and A. A. Lindberg, Acta Chem. Scand., 25, 1512 (1971). (726) A. M. Stephen and P. van der Bij1,J. S. Afr. Chem. Inst., 24, 103 (1971). (727) C. G . Hellerqvist, J. Hoffman, A. A. Lindberg, B. Lindberg, and S. Svensson, Acta Chem. Scand., 26, 3282 (1972).

160

G. G. S. DUTTON

(728) C. G. Hellerqvist, U. Rod&, and P. H. Makela, Eur. J . Biochem., 25, 96 (1972). (729) G . Hammerling, 0. Luderitz, 0. Westphal, and P. H. Makela, Eur. J . Biochem., 22, 231 (1971). (730) M. Nurminen, C. G. Hellerqvist, V. V. Valtonen, and P. H. Makela, Eur. J . Biochem., 22, 500 (1971). (731) M. V. Cheshire, C. M. Mundie, and H. Shepherd, J . Soil Sci., 22, 222 (1971).

DEHYDRATION REACTIONS OF CARBOHYDRATES* BY MILTON S. FEATHERAND

JOHN

F. HARRIS

Department of Agricultural Chemistry, University of Missouri, Columbia, Missouri, and Forest Products Laboratory,t Forest Service, U.S . Department of Agriculture, Madison, Wisconsin

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Reaction Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Saturated Heterocyclic Compounds . . . . . . . . . . . . . . . . . . . . . . . 2. Acyclic Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Unsaturated Heterocyclic Compounds. . . . . . . . . . . . . . . . . . . . . . 4. Carbocyclic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Dehydration in Acidic Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Aldoses and Ketoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Unsaturated Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Glycuronic Acids and L-Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . IV. Dehydration in Alkaline Solution . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Aldoses and Ketoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Unsaturated Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Glycuronic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Reductic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Levulinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Analyses Involving Dehydration Reactions . . . . . . . . . . . . . . . . . . . . 1. Reactions in Concentrated Acid Solution . . . . . . . . . . . . . . . . . . . . 2. Color Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 163 163 167 171 174 174 174 182 186 193 193 203 206 207 212 218 219 220

I. INTRODUCTION The reaction of carbohydrates in alkaline or acidic aqueous solutions results in a myriad of products, many of which have been recognized for well over a century. The number of identified products has greatly increased in recent years, owing to the development of sophisticated techniques for separation and identification. With the exception of anhydro sugars and oligosaccharides, found as concentration-dependent, equilibrium constituents (reversion products) in acidic solutions, all of the products result from reactions of intermediates present in the Lobry de Bruyn-Alberda van Ekenstein transformation. * Issued as Journal Paper No. 6502 of the Missouri Agricultural Experiment Station, Columbia, Mo. t Maintained at Madison, Wis., in cooperation with the University of Wisconsin. 161

162

M. S. FEATHER AND J. F. HARRIS

Although Nef,' in 1910, recognized that the isomerization of Dglucose could explain the presence of the three types of saccharinic acid formed in basic solution, it was Evans and Benoy2who first suggested that 1,2- and 2,3-enediols are the particular intermediates involved. Not until 1944 did Isbel13suggest that lY2-enediolformation is also involved in formation of furan derivatives in acidic solution, although the @elimination of a hydroxyl group from the aldehydo form of an aldose had previously been proposed4 as the initial dehydration reaction. The inference that some of the products in acidic medium might result from the presence of a 2,Senediol was not made, and the correspondence among the precursors in acid and base solutions was not realized until after the isolation and identification5 of 2-(2-hydroxyacetyl)fran in 1952. The presence of the various enediols, principally 1,2- and 2,3-enediolsYin both acidic and basic solutions is now known to account for all dehydration products except those already noted. The dehydration reactions initiated by eliminating a hydroxyl group from an enediol are discussed in the present article. The products (usually dicarbonyl compounds) of these eliminations are normally transient intermediates, and undergo further reaction. The final products formed are determined by the carbohydrate reacting, the conditions of reaction, and the character of the medium. Except for a Section on analytical methods (see p. 218), the subject matter is restricted to aqueous acids and bases. The presence of compounds other than the carbohydrate under study has only been considered where it has helped to elucidate the mechanism involved. The approach here is critical and interpretative, with emphasis on mechanism. An attempt has been made to demonstrate how similar reactions can logically lead to the various products from different carbohydrates; a number of speculative mechanisms are proposed. It is hoped that this treatment will emphasize the broad functions of these reactions, an importance that is not fully recognized. No claim is made for a complete coverage of the literature; instead, discussion of results in the articles that best illustrate the principles involved has been included.

(1) J. U. Nef, Ann., 376, 1 (1910). (2) W. L. Evans and M. P. Benoy, cited in W. L. Evans, R. H. Edgar, and G. P. Hoff, /. AmeT. Chem. soc., 48, 2665 (1926). (3) H. S. Isbell, J. Res. Nat. Bur. Stand., 32,45 (1944). (4) C. D. Hurd and L. L. Isenhour,]. Amer. Chem. SOC., 54,317 (1932). (5) R. E. Miller and S. M. Cantor, J . Amer. Chem. SOC., 74, 5236 (1952).

DEHYDRATION REACTIONS OF CARBOHYDRATES

163

Most previous articles have been confined to areas more specific than that considered here. Pigman and Anetsa have discussed from a general viewpoint the reactions of sugars with acids and bases. Newtha reviewed the formation of furan derivatives, and Sowden,’ the formation of saccharinic acids. The function of 3-deoxyglycosuloses in dehydration reactions was discussed by E. F. L. J. Anets; the chemistry of these compounds was also included in a Chapter on dicarbonyl sugar^.^ Two important ancillary articles should be noted, namely, a comprehensive review of the Lobry de Bruyn-Alberda van Ekenstein transformation,1° which included a discussion of the acidbase-catalyzed dehydration of the enediols, and” an A.C.S. monograph entitled “The Furans.” 11. REACTIONMECHANISMS

1. Saturated Heterocyclic Compounds

In the degradation reactions of sugars, major structural differences exist between products and reactants: in acidic solution, pyranoses produce furan compounds; in basic solution, acyclic saccharinic acids are formed. Ring opening, a necessity for both reactions, precedes the elimination reactions and is part of that well known complex of reactions termed the Lobry de Bruyn-Alberda van Ekenstein transformation. The acyclic enediols produced from acyclic sugars, which are the intermediates in the isomerization, are also the reactive components that initiate the degradation reactions, and the dehydration products may be regarded as products of a side reaction of the transformation. The response of the system to changes in solvent, or to addition of reactants that are capable of aldehyde addition, can often be interpreted by the effect on the mutarotational equilibrium. The stabilities of the hemiacetal ring-structures of the reacting sugar are of obvious importance. Their participation in mutarotation (5a) W. Pigman and E. F. L. J. Anet, in “The Carbohydrates, Chemistry and Biochemistry,” W. Pigman and D. Horton, eds., Academic Press, Inc., New York, N. Y., 2nd Edition, 1972, VoI IA, chapter 4. (6) F. H. Newth, Aduan. Carbohyd. Chem., 6,83 (1951). (7) J. C. Sowden, Adoan. Carbohyd. Chem., 12, 35 (1957). (8) E. F. L. J. Anet, Aduan. Carbohyd. Chem., 19, 181 (1964). (9) 0. Theander, Advan. Carbohyd. Chem., 17,223 (1962). (10) J. C. Speck, Jr., A d u ~ nCarbohyd. . Chem., 13,63 (1958). (11) A. P. Dunlop and F. N. Peters, “The Furans,” Arner. Chern. SOC. Monograph Ser., Reinhold Publishing Company, New York, N. Y., 1953.

164

M. S. FEATHER AND J. F. HARRIS

and in the formation of acyclic components in solution has been reviewed.I2 The rate of degradation is generally dependent on the ease of ring opening that controls the rate of formation of the reactive, acyclic species, D-Glucose, the most conformationally stable aldohexose, is one of the most resistant of the aldohexoses to degradation, either in alkali or in acid, In mutarotation, bases are more effective catalysts than acids; this is also true for the dehydration reactions. The maximum stability for most sugars is on the acid side of neutrality, usually in the pH range of 3.0 to 4.0. It is not possible to make predictions that relate yields of product to the stability of the sugar conformer, as the products, especially those produced in acidic medium, are unstable, can undergo further decomposition, and can also interact with the reaction intermediates. The variation in yield resulting from a change in concentration of the reacting acyclic enediol cannot be predicted without complete knowledge of the kinetics, but it has generally been found that the yields are greatest from those sugars that are conformationally unstable. 5-(Hydroxymethyl)-2-furaldehyde is formed from D-fructose in higher yield and at a much greater rate than it is formed from Dglucose. This is particularly evident when it is prepared from sucrose; only the D-fI-UCtOSe portion of the molecule reacts, and D-glucose is recovered in almost quantitative yield. 2,5-Anhydro-~-mannose ("chitose") also reacts rapidly to give high yields of 5-(hydroxymethy1)2-furaldehyde7 but the differences (as compared with D-glucose) are not so pronounced as those between D-glucose and D-fructose. To accommodate these facts, the earliest mechanisms proposed for degradation of D-fructose assumed that it was present in the furanose form, and that the ring remained intact. It was assumed that the initial reaction was the elimination of water, to form the l,%enolic form of 2,5-anhydro-~-mannose, and that further dehydration resulted in 2-furaldehyde. The necessity for D-glucose to isomerize to D-fI-UCtOSf3was assumed to account for the much lower reaction-rate of D-glUCOSe. This mechanism does not account for the observation that 2,5-anhydro-~-mannoseis less reactive than D-fructose, nor is there any evidence that 2,5-anhydro-~-mannoseis present in reacting D-fructose solutions. Nevertheless, similar mechanisms have since been p r ~ p o s e d . ' ~ -Because '~ of the ease of mutarotation of D-fructose (12)W.Pigman and H. S . Isbell, Advan. Carbohyd. Chem., 23,11 (1968);H.S . Isbell and W. Pigman, Advan. Carbohyd. Chem. Bdochem., 24,13 (1969). (13)M. L. Mednick, J. Org. Chem., 27,398 (1962). (14)C.J. Moye and Z. S . Krzeminski, Aust. J. Chem., 16,258 (1963).

DEHYDRATION REACTIONS OF CARBOHYDRATES

165

compared to its rate of dehydration, it is much more probable that the reaction proceeds through the formation of acyclic enediols. Interestingly, the 5-(hydroxymethyl)-2-furaldehydeformed by the reaction of D-fructose in acidified deuterium oxide contained no carbon-bound deuterium on the furan ring." This result supports the hypothesis that ring opening had not occurred and that the reaction proceeds by way of direct dehydration of D-fructofuranose; however, the same product from D-glucose treated under the same conditions also showed no deuterium incorporation. These experiments therefore leave the question still unresolved. Although it is probable that D-fructose, with its labile, hemiacetal linkage, reacts through an acyclic form, the 2,5-anhydro sugars, which contain a stable ring and an unsubstituted carbonyl group, probably react with the ring intact.18 The most plausible mechanism for the degradation of 2,5-anhydro-~-mannose(1) is the following. The pelimination that results in the formation of 3 from 1occurs readily €or acyclic aldehydes, and should not be substantially impeded by the ring structure. The degradationlg of D-arabinitol in 0.1%methanolic hydrogen chloride at 65", a reaction considered to proceed by way of furfuryl alcohol (see Section VI; p. 213), is similar to the conversion of 3 into 5. Further evidence for the mechanism shown is found in a study by Ness and Fletcherz0 that involved a similar reaction; it is discussed in Section II,3 (see p. 172).

vgo

HOCIt,

0

HO

H o c P C O H

V

C

H

O

HO

HO 1

Z

2

3

(15) C. J. Moye, Aust. J. Chem., 19, 2317 (1966). (16) T. G. Bonner, E. J. Bourne, and M. Ruskiewicz, J . Chem. Soc., 787 (1960). (17) M. S. Feather and J. F. Harris, Carbohyd. Res., 15,304 (1970). (18) J. Defaye, Adoan. Carbohyd. Chem. Biochem., 25, 181 (1970). (19) R. E. Deriaz, M. Stacey, E. G. Teece, and L. F. Wiggins, J . Chem. Soc., 1222 (1949). (20) R. K. Ness and H. G. Fletcher, Jr., J . Org. Chem., 28, 435 (1963).

166

M. S . FEATHER AND J. F. HARRIS

Other 2,5-anhydro sugars also react readily in acidic medium. 2,5-Anhydro-~-idoseis reported to form 5-(hydroxymethyl)-2-furaldehyde (5) several hundred times faster than D-glucose forms it.21As with 2,5-anhydro-~-mannose,the yield reported is far from that theoretically possible, undoubtedly a result of the instability of the product. 2,5-Anhydro-~-arabinoseis converted into 2-furaldehyde by warming gently in 0.05 M sulfuric acid.22In all of these examples, there is nothing to indicate that ring opening occurs during the reaction. The necessity for the presence of an aldehyde group to produce a driving force for the @elimination of a hydroxyl group is evident, because both 2,5-anhydro-~-mannonic(“chitonic”) acid and 2,5-anhydro-~mannaric (“chitaric”) acid, obtained from the oxidation of 2,5-anhydroD-mannOSe, form the appropriate furancarboxylic acids in high yields, but only when heated in hydrogen chloride gas.23 Furthermore, 2,5-anhydro-3-deoxy-~-erythro-pentose, a compound lacking a hydroxyl group in the p-position, is unaffected by refluxing in 0.05M sulfuric acid for 2.5 hours.’* The ring structures of the glycuronic acids are even more complex than those of the sugars. Replacement of the hydroxymethyl group on C-5 with a carboxyl group introduces the probability of formation of lactone ring-structures that can exist simultaneously with pyranose and furanose rings; bicyclic forms are common, and the stable rings of the parent aldoses are replaced by entirely different structures. In aqueous solution, the preponderant species of D-glucuronic acid is probably the monocyclic, pyranose structure, but it readily equilibrates to the bicyclic structure D-g~ucofuranurono-6,3-lactone, in the a-form of which it crystallizes as monoclinic plates. D-Mannuronic acid similarly exists in a bicyclic form, but D-galaCturOniC acid, which cannot give a strainless bicyclic form, crystallizes in the pyranose structure, although a study of models indicates that the furanose structure should also be present in solution. In addition to introducing these profound changes in structure, the presence of the carboxyl group attached to C-5 and involved in an ester linkage tends to render the proton on C-5 acidic. However, the overall effect seems too small to result in carbocyclic-ring formation between C-1 and C-5, or too small to result in substantial degradation reactions. It had been reported that the epimerization of D to L acids (21) C. A. Dekker and T. Hashizume, Arch. Biochem. Biophys., 78,348 (1958). (22) M. Cifonelli, J. A. Cifonelli, R. Montgomery, and F. Smith,J. Amer. Chem. Soc., 77, 121 (1955). (23) F. Tiemann and R. Haarmann, Ber., 19, 1257 (1886).

DEHYDRATION REACTIONS OF CARBOHYDRATES

167

occurred readily in aqueous alkaline solution,24but this has not been confir~ned.~~-~’ Prey and Szabolcs,28in investigating the possibility of carbocyclic-ring formation in biological systems, found that the carboxyl group exerts only a minor activating effect. In anhydrous alkaline solution, however, the acidity of the proton on C-5 is of great importance (see Section IV, 3; p. 206). 2. Acyclic Forms

The formation of acyclic enediols is, apparently, the initial reaction that leads to dehydration products. Sugar enediols are transitory compounds that have never been isolated. However, because, when treated with either acid or base, an aldose gives rise to its 2-epimer, as well as to its 2-keto isomer, a persuasive argument is provided for the lY2-enediolas the intermediate common to each of the products. The evidence in favor of these intermediates is based primarily on isotope-exchange experiments, on reactions that involve isomerizations of 0-methyl sugars, and on kinetic measurements.1° The interconversion of aldoses and the respective 2-ketoses in alkaline solution may be somewhat more complex than originally supposed, as it has been reported that a partial transfer of hydrogen from C-2 of the aldose to C-1 of the corresponding ketose occurs during the reaction.29 This observation is not inconsistent with isomerizations that involve 1,2-enediol intermediates. The transfer could occur as a result of a rapid conversion in which some of the protons originally at C-2 of the aldose molecules are retained by the solvent cage that surrounds the intermediate l,2-enediolYand are, therefore, available for addition to C-1 of the resulting ketose. It should be noted that other interpretations, such as hydride-transfer mechanisms, are also possible. The importance of 2,3-enediol as compared to 1,2-enediol formation from a ketose presents a problem that has not yet been quantitatively investigated. Lemieux30 has pointed out that the large, non(24) F. G . Fischer and H. Schmidt, Chem. Ber., 92,2184 (1959). (25) B. Carlsson, 0. Samuelson, T. Popoff, and 0. Theander, Acta Chem. Scand., 23, 261 (1969). (26) B. Carlsson and 0. Samuelson, Actu Chem. Scund., 23, 318 (1969). (27) B. Carlsson and 0. Samuelson, Carbohyd. Res., 11,347 (1969). (28) V. Prey and 0. Szabolcs, Monatsh. Chem., 89,394 (1958);Chem. Abstr., 53,9073 (1959). (29) W. B. Gleason and R. Barker, Can. J . Chem., 49, 1433 (1971). (30) R. U. Lemieux, in “Molecular Rearrangements,” Part 2, P. deMayo, ed., Interscience Publishers, Inc., New York, N. Y., 1964, p. 744.

M. S. FEATHER AND J. F. HARRIS

168

bonded interactions in the 2,3-enediol would suppress its formation. Deuterium-exchange ~ t u d i e s , ~ as' well as the absence of substantial proportions of materials, corresponding to the original aldose, having been racemized at C-3 during the Lobry de Bruyn-Alberda van Ekenstein rearrangement, suggest that the 1,2-enediol formation predominates. The presence of the 2,3-enediol has been confirmed, however, by the isolation of minor reaction-products in several instances.30 The analogous 2,3-enediol also occurs to a measurable extent in the isomerization of D-glucuronic acidZ5sz6 and D-galacturonic The first dehydration products formed by general, acid-base catalysis are represented by the enolic forms (7, 9, and 10) of the deoxydicarbonyl sugars 7a, 9a, and 10a. The enolic compounds are formed from enediols by the removal of a molecule of water through P-elimination of a hydroxyl group. For example, from the 1,2-enediol (6) derived from D-glucose Or D-fructose, the enolic form (7)of 3-deoxyD-erythro-hexosulose (7a) is produced, whereas from the 2,3-enediol n HC-0-H HC=O HC=O I I ($-OH COH c=o

-

II CH I HCOH

-KO

HO~CH I

HCOH I

I

R

I ?Ha

-4

HCOH I

R

6

R la

1

(8) either the enolic form (9) of 4-deoxy-~-glycero-2,3-hexodiulose (9a) or the enolic form (10) of l-deoxy-~-erythro-2,3-hexodiulose ChOH I C-@-H GOH

CKOH I

-w

HC-GOH I R

I

c=o

!OH

C=O I

I

I

7%

HC I

R

8

R 9

fh

-so =

-H

COH

&o I

HCOH I

I

R

R

8

ChOH

c=o

I

where R = HCOH I

CGOH

10

.

(31) M. S. Feather, Carbohyd. Res., 7, 86 (1968).

9a

7%

c=o I c=o I

HCOH I

R IOa

DEHYDRATION REACTIONS OF CARBOHYDRATES

169

(10a) can arise. The elimination mechanisms that lead to the formation of deoxydicarbonyl sugars, their subsequent reactions, and evidence for their participation in the dehydration, have been discussed in detail by Anet.s The possibility that 3-deoxyglycosuloses can be formed from D-glucose 3-phosphate and certain Amadori products by a hydride shift has been suggested:* but the data on which this proposal was based were shown to have been incorrectly inter~reted.3~ The further demonstration that D-glucose-2-d is converted into S-(hydroxymethy1)-2-furaldehydehaving no carbon-bound deuterium also supports the view that these types of intermediates are not formed by hydride shifts6or by other mechanisms that involve intramolecular transfers. The dehydration of the enediols is a reaction subject to general acid-base catalysis. The deoxyaldosulose 7a has been isolated from 3-O-benzyl-~-glucose~~ and from D-fi~ctOSe~~ after treatment with alkali, and from D-fr~ctOSe~~ and various Amadori products* after treatment with acid. The most successful preparation of 7a has been by way of amine addition compounds; an improved procedure has been reported.36 Compound 9a has been isolated as a product of the alkaline treatment of both cellobiose3' and maltose.% The isolation of 10a has not been reported, but it has been s y n t h e t i ~ a l l yprepared. ~~ The acyclic, enolic compounds 7 and 9 may exist in either cis or trans forms. Methyl ethers of 7 have been isolated in the cis form: but it is not known whether the trans forms, which must be acyclic, exist. The relative proportion of isomers is controlled by the geometry of the parent sugar enediol. Although the acyclic forms are readily interconvertible tautomers, it appears that, in acidic medium, further reaction occurs much more rapidly than any equilibrating reactions. Compound 7 undergoes rapid elimination of a second hydroxyl group to give 11. This acyclic product, also, may exist as either a cis or a trans isomer, both forms of which have been isolated.8 The loss of a third molecule of water per molecule occurs after, or simultaneously with, the cyclization of 11 (see Section 11, 3; p. 171), and results in formation of 5-(hydroxymethyl)-2-furaldehyde (5). (32) G . Fodor and J. P. Sachetto, Tetrahedron Lett., 401 (1968). (33) E. F. L. J . Anet, Tetrahedron Lett., 3525 (1968). (34) G . Machell and G . N. Richards,./. Chem. SOC., 1938 (1960). (35) E. F. L. J. Anet, Aust.J. Chem., 18,240 (1965). (36) H. El Khadem, D. Horton, M. H. Meshreki, and M. A. Nashed, Carbohyd. Res., 17, 183 (1971). (37) R. L. Whistler and J. N. BeMiller, J. Amer. Chem. SOC., 82, 3705 (1960). (38) G . Machell and G . N. Richards, J . Chem. SOC.,1932 (1960). (39) A. Ishizu, B. Lindberg, and 0. Theander, Carbohyd. Res., 5, 329 (1967).

M. S. FEATHER AND J. F. HARRIS

170 HC=O

I

c=o

- gH I

CH

7

HOhC

I

HCOH

I

CH,OH

5

1 la

11

Although not yet experimentally demonstrated, it is presumed that 9 undergoes a similar series of reactions that lead to 2-(2-hydroxyacety1)furan (13). This product has been isolated in low yield from treatment of both D-glucose and D-fructose with a ~ i d . 5 , ~ ~ C%OH

9

-

I I

c=o F=O

CH II

C-CH.,OH

CH I C%OH

0

13

I2a

12

Compound 10 cannot undergo further dehydration without first rearranging to the 3,4-enediol (14). The reaction of 14 would yield 2-acetyl-3-hydroxyfuran (isomaltol, 16), a compound produced in low

7%

7%

c=o I

COH II

COH

- I

c=o I c=o

I HCOH I C&OH

COH II CH I C%OH

14

15

II 0 15a

J ‘OH 16

(40)K.Aso and H. Sugisawa, Tohoku J . Agr. Res., 5, 143 (1954).

DEHYDRATION REACTIONS OF CARBOHYDRATES

171

yield from ~ - f r u c t o s e ~when l it reacts in mildly acidic solution. Isomaltol has seldom been reported as a dehydration product in aqueous systems, probably because of its instabiIity in aqueous acid. Owing to its inability to dehydrate without rearrangement, and the large number of tautomeric ring-forms that 1Oa might assume, the possibility of other degradation mechanisms for 10 cannot be excluded. In alkali, the enols 7, 9, and 10 rapidly tautomerize to their dicarbony1 forms, and undergo rearrangement to form the familiar saccharinic acids. The benzilic acid rearrangement by which this conversion occurs has been intensively studied.42 This reaction predominates, although some fragmentation of the deoxyglycosuloses does occur. Compound 10a is particularly susceptible to carbon-chain cleavage in the presence of calcium h y d r ~ x i d e . ~ ~ 3. Unsaturated Heterocyclic Compounds

Deoxyaldosuloses are capable of existing in numerous ring modiEl-Dash and H ~ d g e ~ ~ fications. For 3-deoxy-~-erythro-hexosulose, found that, of the 16 possible ring-forms (excluding enolic structures), evidence could be obtained for 11, although only 6 were stable in anhydrous pyridine. These varied ring-structures, and the many acyclic forms possible, introduce alternative pathways for dehydration to the same or different products; and, where the structures are nonreactive, these forms would affect the kinetic pattern of the mechanism; thus, they would influence the reaction rate and product distribution. Fewer ring-forms are possible for the 3-deoxypentosuloses. Three possible monocyclic hemiacetal rings (17, 18, and 19), none of which

eH

HoQ

O

G

OH 17

OH

17a

Ho%=-oH

Ho-218a

OH 18

O

H

p:o HO 19

(41) P. E. Shaw, J. H. Tatum, and R. E. Berry, Carbohyd. Res., 5, 266 (1967). (42) S. Selman and J. F. Eastman, Quart. Reu. (London), 14,221 (1960). (43) A. A. El-Dash and J. E. Hodge, Carbohyd. Res., 18, 259 (1971).

172

M. S. FEATHER AND J. F. HARRIS

has conformational restrictions, may be formed. Compounds 17 and 18 can eliminate a hydroxyl group readily, but 19 must revert to the acyclic form in order to react further. Dehydration of 17 by p-elimination results in the 3,4-dideoxypent-3-enosulopyranose, a hemiacetal analog of 11, which reacts further to form 2-furaldehyde. However, the reaction of 18, by way of an elimination similar to the conversion of l l a into 5, results in the formation of a 3-furanone, a relatively stable product that cannot undergo further dehydration. Such dihydrofurans as l l a and 18 dehydrate readily in aqueous acid. Evidence for this was obtained by Ness and Fletcher?O who found that 20 is converted into 21 in anhydrous methanol at room temperature, but that, in

aqueous acetone, if acidified to -3.0 M with acetic acid, 20 affords 24 in 6 hours. It was also found that the 3(2H)-furanone 25 is formed in 70% yield from 24 under very mild ~ o n d i t i o n s . ~ ~ , ~ ~ Conversion of the acyclic 3,4-dideoxypent-3-enosuloses into furans may proceed either by the sequence 26 + 28 + 27, in which dehydra(44) E. F. L. J. Anet, Tetrahedron Lett., 1649 (1966). (45) E. F. L. J. Anet, Carbohyd. Res., 2,448 (1966).

DEHYDRATION REACTIONS OF CARBOHYDRATES

RQoH

OMe

-

24

173

“QO 25

w h e r e R = CHOMe-C%OMe,

tion occurs after ring closure, or by the sequence 26 + 26a + 27, in which dehydration is concurrent with ring closure. A third posHC=O I

CH II HCOH 26a

HC=O I

c=o

I CH II CH I

CKOH

27

26

28

sibility, discussed by Anet,46that would permit the reaction to proceed despite the presence of a blocking group at C-5, has been shown to be i n ~ p e r a t i v e The . ~ ~ six-membered, heterocyclic forms of the 3,4dideoxypent-3-enosuloses cannot be dehydrated, but rings of this type formed from 10a lead to compounds of the pyrone type, such as maltol (see Section 111,l; p. 179). (46) Ref. 8, p. 211.

M. S. FEATHER AND J. F. HARRIS

174

4. Carbocyclic Compounds

In the majority of dehydration reactions, heterocyclic compounds are formed, rather than carbocyclic compounds. Many possibilities for formation of carbocyclic compounds exist, but these are important only if (a) the heterocyclic or acyclic tautomers cannot undergo further elimination reactions, or (b) the conditions of reaction greatly favor the formation of an acyclic tautomer capable of affording only the carbocyclic compound. Both five- and six-membered carbocyclic compounds have been isolated, with reductic acid being the compound most frequently reported. Ring closure occurs by an intermolecular, aldol reaction that involves the carbonyl group and an enolic structure. Many examples of these aldol reactions that lead to formation of carbocyclic rings have been ~tudied.~’ As both elimination and addition of a proton are involved, the reaction occurs in both acidic and basic solutions. As examples of the facility of this reaction, pyruvic acid condenses spontaneously to a dibasic acid at room temperature in dilute solution, and such &diketones as 29 readily cyclize to form cyclohexenones, presumably by way of 30, either in acid or base.

OH 29

30

31

111. DEHYDRATION IN ACIDIC SOLUTION 1. Aldoses and Ketoses

One of the simplest examples of a dehydration in the sugar series involves the formation of pyruvaldehyde on treatment of DL-glyceraldehyde with mineral acid.48a48aThe pyruvaldehyde is readily

(47) R. J. Reeves, in “The Chemistry of the Carbonyl Group,” S. Patai, ed., Interscience Publishers, Inc., New York, N. Y., 1966, p. 567. (48) Ref. 10, p. 73. (48a) M. Fedor6nko and J. Konigstein, Collect. Czech. Chem. Commun., 34, 3881 (1969).

175

DEHYDRATION REACTIONS OF CARBOHYDRATES

formed, and occurs in observable concentration as it cannot react further without undergoing intermolecular condensation. The dehydration products expected from this condensation would be both furans and carbocyclic compounds, but they have not yet been investigated. Little work has been done with the aldotetroses; these could lose two molecules of water per molecule to form 34. Compound 33 could form a furanone after ring closure, but, because the con-

-

HC=O

HC=O

I

HCOH I

HCOH I CH.,OH 32

HC=O I

I

COH II CH I CH.,OH

~

c=o

I CH

8% 34

33

version of 33 into 34 is rapid by comparison, the product would be 34, with 33 never being present in significant concentration. In its acyclic form, 34 cannot be dehydrated, nor can it react intermolecularly. However, the aldehydrol form could undergo ring closure to yield 3(2H)-furanone by a mechanism similar to the conversion of HC=O & z %

c%

HC=O

HC=O I

I

HQ

c=o I

COH II CH

-

I 7%

7% 7%

~ -Ha0 -*

c=o

HCOH

I COH

I

c=o I

I HqOH CH,,OH

II P

HFOH I CKOH

38

\ // hj.2-C)

t\\ HC

II

I

0 39

0-H

H@

0

CqOH

0

0

-

OH

Ho &-CH

40

176

M. S. FEATHER AND J. F. HARRIS

54a into 25 (see p. 181). The condensation of 34 with the sugar enediol to form 35 explains the small amounts of “erythropyrone” (40) found among the acidic-degradation products of erythrose.49 The condensation product 35 can lose a hydroxyl group from either C-5, C-7, or C-8 by p-elimination, but only 36 leads to 40. This com-

plex molecule is the only dehydration product reported from a tetrose. In dilute acid solution, 2-hraldehyde is formed in high yield from all of the pentoses. It is obtained in almost quantitative yield from xylose, provided that it is removed from the solution while it is being formed, The loss incurred by leaving it in the reaction medium is not primarily related to the instability of the aldehyde, but rather to its interaction with reaction intermediates that results in polymeric solid^.^^,^^ 2-Furaldehyde arises from the pentose 172-enediol;products from the 2,3-enediol would not be furans. In relatively concentrated acid solutions, other products are formed, and, although the yield of 2-furaldehyde from D-XylOSe remains high (- 93%), it is much lower for the other p e n t o s e ~Reductic .~~ acid, which has been isolated in low yield from D-Xy10Se,53.54appears to be formed in appreciable proportions from the other p e n t o s e ~Products .~~ having low molecular weight, including formaldehyde, crotonaldehyde, and acetaldehyde, have been isolated in low yield,55but the mechanism of their formation has not yet been investigated. The formation of 2-furaldehyde is analogous to the formation of 5-(hydroxymethyl)-2-furaldehydedescribed in the following discussion. The major products formed from hexoses that react in aqueous acidic solution are 5-(hydroxymethyl)-2-hraldehyde,levulinic acid, and polymeric materials. In addition, many minor dehydration products are found. In a of D-fructose, 2-(2-hydroxyacetyl)furan(13), 2-acetyl-3-hydroxyfuran (isomaltol 16), 2,3-dihydro-3,5-dihydroxy-6methyl-4H-pyran-4-one, and 3,4,5-trihydroxy-3,5-hexadien-2-one (acetylformoin) were identified. Products not formed solely by dehydration mechanisms include acetone,56 formaldehyde, acetalde(49)F. Catala, J. Defaye, P. Laszlo, and E. Lederer, Bull. Soc. Chim. Fr., 3182 (1964). (50) Ref. 11, p. 289. (51)D. F. Root, J. F. Saeman, J. F. Harris, and W. K. Neill, Forest Prod. I., 9, 158 (1959). (52)R. W.Scott, W. E. Moore, M. J. Effland, and M. A. Millett,Anal. Biochem., 21,68 (1967). (53)T. Reichstein and R. Oppenauer, Helv. Chim. Acta, 16,988 (1933). (54)M. S. Feather, J. Org. Chem., 34, 1998 (1969). (55)F. A. H. Rice and L. Fishbein, J. Amer. Chem. Soc., 78,1005 (1956). (56)El S. Amin, Carbohyd. Res., 4, 96 (1967).

DEHYDRATION REACTIONS OF CARBOHYDRATES

177

hyde, pr~pionaldehyde,~' and 5-methy1-2-f~raldehyde.~~ The lastmentioned comDound is readily formed by dehydration of 6-deoxyhexoses, but has also been frequently observed as a product of the acid treatment and pyrolysis of D-glucose and D-glucans. General aspects of the mechanisms of sugar dehydration have been discussed in Section 11, and by AneP in an earlier Volume of this Series. Anet's scheme for formation of 2-furaldehydeYshown in the following scheme, was based on experimental evidence then availHC=O

I I HOCH P I HCOH I HCOH I C%OH

HCOH II COH

HCOH

C%OH

I

~

I I HOCH I

c=o

HOCH I HCOH I HCOH

~

P

HCOH I HCOH

CI-&OH

I C&OH

41

42

43

HC$,OH C

HC=O I

HC<',OH C

HCOH I HCOH I C&OH

HC=O I OfiC\C/H

I1 /C,H HCOH I CQOH 47

I

c=o I

p HCOH I

-

II

HNC,

HCOH I HCOH

HCOH I CH,OH

I

C&OH

HC=O

-Ho~cwc*OI

ofiC,C/H

II

H'C&OH

I

5

C&OH 48

(57) F. A. H. Rice and L. Fishbein, J . Amer. Chem. Soc., 78,3731 (1956).

178

M. S. FEATHER AND J. F. HARRIS

able that included the isolation, characterization, and further ~ t u d y 5 ~ 9 ~ ~ of compounds 45,47, and 48. Because of more recent investigations, it is apparent that the scheme requires some modification, but, in general, it is still consistent with the experimental findings. The common 1,2-enediol provides an explanation for the formation of identical products from an aldose and the corresponding 2-ketose7 and, because of the large differences in reactivity of aldoses and ketoses, its formation probably constitutes the rate-determining step. A dual pathway is proposed, as only 47, the cis form, is produced on treatment of 45 with acid,so whereas both 47 and 48 were reported to have been isolated from the reaction of D-frUCtOSe.61 Compound 45 is of considerable interest, because, although it is the stable and isolable isomer of 44 and 46, it is neither a necessary intermediate in the reaction nor is it predicted to equilibrate with its trans enolic form 46 under the conditions of formation. A kinetic evaluation of this reaction35 indicated that 38% of the reaction involves 45 as a participant in the conversion of 42 into 5; the remainder presumably proceeds, directly, through 44 and 46. Subsequent experiments designed to investigate the mechanism have involved isotope (either deuterium or tritium)-exchange studies during the conversion of the sugars into the 2-furaldehyde. A determination of the proportion and the position of the isotope incorporated into the 2-furaldehyde tests the importance of ( a ) the aldose-ketose 1,2-enediol equilibrium, and ( b )the 3-deoxyglycosuloses (45) as intermediates. The existence of the equilibrium predicts an incorporation at C-1 of the sugar, which corresponds to the a-carbon atom of 2-furaldehyde7 and a 3-deoxyglycosulose intermediate predicts an isotope incorporation at C-3 of the furan ring. With D-xylose as the model compound, the conversion into 2-furaldehyde in acidified, tritiated watePg3 involved no isotope exchange at any position. This observation indicated that no 1,2-enediol equilibrium exists during the reaction, and further supported 1,2-enediol formation as the rate-determining step for the reaction. The absence of any evidence in favor of 3-deoxyglycosulose intermediates suggests a reaction sequence involving only 42, 44, and 46, and 47 and 48. Additional support for this pathway is found in converting either (58) E. F. L. J. Anet, Aust. J . Chem., 13, 396 (1960). (59) E. F. L. J. Anet, Aust. J . Chem., 14,295 (1961). (60) E. F. L. J. Anet, Aust. J . Chem., 16,270 (1963). (61) E. F. L. J. Anet, Aust. J . Chem., 15, 503 (1962). (62) M. S. Feather, Tetrahedron Lett., 4143 (1970). (63) M. S. Feather, D. W. Harris, and S . B. Nichols,]. Org. Chem., 37, 1606 (1972).

DEHYDRATION REACTIONS OF CARBOHYDRATES

179

D-glucose or D-fructose into 5-(hydroxymethy1)-2-furaldehydein solution in acidified deuterium oxide.17 The 2-furaldehyde was isolated as 5-(hydroxymethyl)-2-furoicacid, and thus this experiment did not permit an evaluation of reversible equilibration of 1,Zenediols with the parent sugars. However, the 2-furoic acid was devoid of measurable carbon-bound deuterium, which indicated the absence of 3-deoxyglycosulose intermediates. It is also noteworthy that 3-deoxy-~-erythro-hexosulose is converted, in acidified deuterium oxide, into 5-(hydroxymethyl)-2-furaldehydewith no solvent exchanges4; this result lends further support to the conclusion that 45 does not participate in the reaction as an intermediate. The formation of isomalt01~~ (16) and 2-(2-hydroxyacetyl)furan5.40.4' (13) during the acid treatment of D-fructose indicates the formation of the 2,3-enediol, as this is the predicted precursor for these compounds (see Section 11, p. 168). The presence of primary amines promotes the formation of 1-deoxydiuloses, and the pyrone 51 has been isolated from the reaction products of D-glucose with methylamine and acetic and has been found among Maillard-reaction products.66 It has also been isolated from both acidic and basic degradations of ~ - f r u c t o s e . ~Compound ~,~' 51 is a dehydration product

F COH L=o

I HCOH

yH3

c=o LI o HCOH

HO

OH 49

10

1Oa

51

tl

50

(64) M. S. Feather and K. R. Russell, J . Org. Chern., 34,2650 (1969). (65) T. Severin and W. Seilmeir, Z. Lebensrn.-Unters. Forsch., 137,4 (1968). (66) F. D. Mills, D. Weisleder, and J. E. Hodge, Tetrahedron Lett., 1243 (1970). (67) J. H. Tatum, P. E. Shaw, and R. E. Berry,]. Agr. Food Chern., 15, 773 (1967).

M. S. FEATHER AND J. F. HARRIS

180

that originates from 49, the pyranose form of l-deoxy-~-eryth~o-2,3hexodiulose. When allowed to react in an almost anhydrous medium (the only water being that produced during the dehydration reaction), compound 51 was found to form maltol (52) and isomaltol (16) in proportions varying with the reagents.ss In aqueous acidic solution, these products and acetylformoin (53) would all be expected from 50 or 51, dependent on whether or not the ring opened and the manner in which the opening occurred. The presence of 49 accounts for compounds 51, 52, 53, and 16, that are frequently detected as products of the degradation of D-fructose, particularly when amines are present. HY@ HO

’

3-H

-

OH

@ OH

-

OH

0

50

52

€I@

HO

Q+ 0-H

7%

7%

COH II COH

COH

c=o I HCOH CI q O H

7%

c=o

II

-

1

COH COH II CI h O H

50

~

I COH I1

COH I

8% 53

7% c=o

7% c=o

q F- H@

0-H

I

COH

HO

OH

OH

50

I

HCOH ?OH I ChOH

/CHS

I

COH c=o

I1 CH I CqOH

16

The effect of alkoxyl or phosphate groups on the acidic degradation of sugars has not yet been systematically studied. The aldopyranoses substituted at 0-1, that is, the aldopyranosides, are only degraded (68) P. E. Shaw, J. H. Taturn, and R. E. Berry, Carbohyd. Res., 16, 207 (1971).

DEHYDRATION REACTIONS OF CARBOHYDRATES

181

after hydrolysis, and their reaction pattern is similar to that of the aldoses. Substitution at positions other than C-5 does not greatly affect the mechanism; no favored elimination similar to that found to occur in basic solution seems to occur. In contrast to alkaline degradationreactions, the presence of an unsubstituted hydroxyl group on C-2 is not required for acidic dehydrations, and 2-0-methylaldoses are dehydrated rapidly.69 Substitution at C-5 prevents formation of the oxolane ring between C-2 and C-5, and results in products that differ from those from the unsubstituted sugars. AneP4 found that the 2,3,5trimethyl ethers of D-xylose and L-arabinose do not yield 2-furaldehyde on treatment with acid, whereas the 2,3,4-trimethyl ethers afford large proportions thereof. The hex-2-enofuranose 54, which would be expected to be the first intermediate in the degradation of 2,3,5,6-tetra-0-methyl-~-glucose, was readily converted45 in high yield into the furanone (25). However, as furanones have not been identified among the reaction products of methylated sugars, it is possible that the competitive reaction (such as that leading to 55) occurs more rapidly than the ring formation, although these types of products have not been isolated. HC=O I

CbOMe

c=o I

CH I HCOMe

HC=O

-

I

COH I1 CH I CH II

HC=O

-

COMe I

I

I

c=o I

CH II CH I COMe

C%OMe 55

R V H

Lo

R q

RQOH

-

OH

\ I

R

q

-

OH

0

H@ 25

54a

OMe I w h e r e R = -C--CH,OMe H

.

(69) M. Stiles and A. Longroy, Tetrahedron Lett., 337 (1961).

182

M. S. FEATHER AND J. F. HARRIS

In alkaline s o l ~ t i o n , 7 ~2-amino-2-deoxy-D-glucose -~~ (“D-glucosamine”) appears to undergo the usual isomerizations and degradations, but little is known about its dehydration in acid solution, although it is generally thought that the amine group confers considerable stability on this c ~ m p o u n d .5-(Hydroxymethyl)-Zfuraldehyde ~~*~~ has been reported7sto be a product of the dehydration of 2-amino-2-deoxyD-glucose under “physiological conditions,” and, in the presence of borate ions, 2-furaldehyde has been reported to be a although the yields were not reported for either. A repetition of these experiments revealeds3 that, if 2-furaldehyde is a reaction product, it is produced in a yield of less than 0.5%. Ikawa and Niemann78performed an extensive series of spectrophotometric studies on the dehydration of sugars in concentrated sulfuric acid (see Section VII, p. 219), and reported that 2-amino-2-deoxy-D-g~ucoseproduces no 2-furaldehydesYas evidenced by spectral observations, although all other reducing sugars examined did. In later experiments, however, C i ~ e r reported a ~ ~ that both 2-amino-2-deoxy-D-glucose and 2-amino-2deoxy-D-galactose (“D-galactosamine”) are dehydrated to an ultraviolet-absorbing material having a spectrum that resembles that of 2-furaldehyde or S-(hydroxymethy1)-2-furaldehyde.Because of these conflicting reports in the literature, it may be concluded that the nature of the products of dehydration is still obscure, although it seems clear that 2-amino-2-deoxy sugars react very slowly as compared with their non-nitrogenous analogs, and that reaction products of the furan type, if produced at all, are formed in very low yield. 2. Unsaturated Sugars

The glycals (such as 56) and the 2-deoxyaldoses give similar products in acid solution. This is to be expected, as water can readily be added to the double bond of a glycal to give a 2-deoxyaldose. The glycals may also react, without ring opening and particularly readily, to yield the cyclic form of the 2-ene 57 that is the common intermediate for dehydration of both types of sugar. (70) D. G. Comb and S . Roseman, J. Amer. Chem. Soc., 80,497, 3166 (1958). (71) R. Kuhn and R. Brossmer, Angew. Chem., 69,534 (1958). (72) J. Brug and G. B. Paerels, Nature, 182, 1159 (1958). (73) J. N. BeMiller and R. L. Whistler, J , Org. Chem., 27, 1161 (1962). (74) A. B. Foster and D. Horton, Aduan. Carbohyd. Chem., 14,258 (1959); see p. 258. (75) A. Gottschalk, Biochem. J., 52,455 (1952). (76) K. Heyns, C. Koch, and W. Koch, 2.Physiol. Chem., 296, 121 (1954). (77) H. K. Zimmerman and A. Cosmatos, 2.Physdol. Chem., 326,73 (1961). (78) M. Ikawa and C. Niemann, J. Biol. Chem., 180,923 (1949). (79) J. D. Cipera, Analyst, 85, 517 (1960).

DEHYDRATION REACTIONS QF CARBOHYDRATES

R

C=O

R

HCOH

HO

I

H$!OH

R

56

c=o

COH

II

I

CH

CH I HOCH

I1

CH HOO

O

183

H

HCOH HCOH I

-

7

I

HCOH HCOH I

R

R

57

The absence of a hydroxyl group from C-2 makes elimination of the 4-hydroxyl group impossible, but two other reactions are possible for 57. The more important is, apparently, the formation, between C-1 and C-4, of a 2,Ei-dihydrofuran ring that is readily dehydrated to the furan. A competitive reaction, namely, the elimination of the 5-hydroxyl group by an extended enolization, would lead to formation of carbocyclic compounds. Evidence for the formation of furans is found in the results of treatment of D-glucal (58) either with dilute acetic acid or with 25 mM ethanolic sulfuric acid for 10 minutes at 70°,to produces0the optically active 2-(~-gZycero-l,2-dihydroxyethyl)furan (60), presumably by way of the 2-ene 59. This compound is also produced from 3,6-anhyHC=O

OH

59

60

(80) D. Horton and T. Tsuchiya, Chem. Ind. (London), 2011 (1966).

184

M. S. FEATHER AND J. F. HARRIS

dro-D-glucal by hydrolysis with acid.81 An earlier reporPla that ~-hydroxy-2H-pyran-2-methanol[3-hydroxy-2-(hydroxymethyl)-2Hpyran] is the product of the dehydration when either D-glucal or D-galactal is treated with 5% sulfuric acid for 15 hours at 0” has been shown to be erroneousao,81;the product is actually 60. Mild treatment of methyl 4,6-0-benzylidene-2,3-dideoxy-a-~-erythro-hex2-enopyranoside with acid alsoa2yields 60. Of interest is an analytical procedures3 for the determination of 2-deoxy-aldohexoses, based on treating the sugar with an acid, to produce an ultraviolet-absorbing material. The spectral properties of the product, which was not isolated, are similar to those of 60, and indicate a yield of 25% under these conditions. The analogous product from the pentose glycals and 2-deoxypentoses would be furfuryl alcohol, but, as it is unstable and is readily converted into levulinic acid under the conditions of formation, it is difficult to isolate. The spectral data52and the fact that levulinic acid is the common product from 2-deoxy-D-erythro-peniose, D-arabinal, and hrfuryl alcohol10 substantiate the supposition that the mechanism is analogous to that just described. Other (unknown) products are formed in significant yield from the dehydration of 2-deoxy-~-erythro-pentose.~~*~~ The mechanism of formation of levulinic acid is discussed in Section V (see p. 212). 1,5-Anhydro-~-urubino-hex-l-enitol (“2-hydroxyglucal”) would be expected to give rise to 5-(hydroxymethyl)-2-furaldehyde,but no studies thereon have as yet been reported. Wolfrom and coworkerss5 described the similar conversion of 2-methoxy-3,4,6-tri-O-methyl-~glucal (“tetramethyl-glucoseen”) into 5-(methoxymethyl)-2-furaldehyde. The conversion of 1,3,4,5-tetra-O-acetyl-2,6-anhydro-~-threohex-2-enitol (61) into the acetate (62) of 2-(2-hydroxyacetyl)furan (13) in 50% aqueous acetic acid during 4-5 hours at 95” is also representative of the 2-hydroxyglycal reaction.s6 It should be noted that,

-

(81) J. S. Brimacornbe, I. DaAboul, and L. C. N. Tucker, Ca~bohyd.Res., 19, 276 (1971). @la) A. S. Matthews, W. G. Overend, F. Shafizadeh, and M. Stacey, J . Chem. Soc., 2511 (1955). (82) E. L. Albano, D. Horton, and T. Tsuchiya, Curbohyd. Res., 2,349 (1966);see also, Ref. 214. (83) R. J. Doyle and M. A. Pfeifer, Microchem. J., 16, 273 (1971). (84) P. Bijvoet, Anal. Biochem., 15, 31 (1966). (85) M. L. Wolfrom, E. G . Wallace, and E. A. Metcalf, J. Amer. Chem. Soc., 64, 265 (1942). (86) M. Katsuhara, S. Wakahara, and K. Tokuyama, Bull. Chem. Soc. Jup., 41, 1208 (1968).

DEHYDRATION REACTIONS OF CARBOHYDRATES

185 C€&OAc I

c=o

ACOCH I

AcO I O

A

c

-0Ac

ChOH

61

CI€,OAc I

ac 0

-

0 II

II

-choAc

c=o I c=o I CH II

CH I

ChOH

62

for occurrence of ring opening, hydrolytic conditions are required, and that 62 was not formed in anhydrous medium. Substitution of a methoxycarbonyl group for the hydroxymethyl group brings about an interesting, but not unexpected, change in the reaction. In aqueous acetic acid, 63 forms the dihydropyran derivative H@

C-OCH,

AcO

Ac 0

OAc

AcO

OAc

J

63

65

64

65 as the major product, with only a minor proportion of 2-(methoxy~ x a l y l ) f u r a n . *The ~ ~ ~last-mentioned ~ compound is formed by the same mechanism as that for 62 from 61, whereas the formation of 65 occurs by way of 64 without ring opening, and results from elimina(87) K. Goshima and K. Tokuyama, Tetruhedron Lett., 2383 (1969). (88) K. Goshima, N. Maezono, and K. Tokuyama, Carbohyd. Res., 17, 245 (1971).

M. S. FEATHER AND J . F. HARRIS

186

tion of the 5-acetoxyl group through the participation of the ester group. As would be expected, hydrolysis of the ester proceeds much faster than elimination when the saturated-ring compound 66 reacts in aqueous acidsa8

Ac 0 66

4-Pyrones are also formed from ~-threo-2,5-hexodiulose(“5-ketoD-fkUCtOSe”) (67) when it is heated in aqueous solution.89Compounds 68 and 69 are formed in high yield, with 68 preponderating. Kojic acid (68) results from p-eliminations in which the ring remains intact, but the formation of 5-hydroxymaltol (69) requires ring opening, because of the absence of a proton on C-2.

9-

0

CqOH

H

o

~

0

HO 61

c

~

+o

HHo Q c q

0 68

OH 69

3. Glycuronic Acids and L-Ascorbic Acid On treatment with aqueous mineral acid, L-ascorbic acid?O hexuronic acids?’ and glycuronansg2undergo decarboxylation by which 1 mole of carbon dioxide per mole of acid is produced. For hexuronic acids, other detectable products include 2-furaldehyde (27) (Ref. 93), reductic acid (2,3-dihydroxy-2-cyclopenten-l-one, 75; see p. 207) (Refs. 53 and 94-96), and traces of 5-formyl-2-furoic acid

-

(89) S. Oga, K. Imada, K. Asano, K. Aida, andT. Uemura, Agr. Biol. Chem. (Tokyo), 31, 1511 (1967). (90) E. W. Taylor, W. F. Fowler, Jr., P. A. McGee, and W. 0.Kenyon,./. Amer. Chem. SOC., 69, 342 (1947). (91) K. U. LefAvre and B. Tollens, Ber., 40,4513 (1907). (92) C. M. Conrad, 1.Amer. Chem. SOC., 53, 1999 (1931). (93) R. W. Herbert, E. L. Hirst, E. G. V. Percival, R. J. W. Reynolds, and F. Smith, J . Chem. SOC., 1270 (1933). (94) T. Reichstein and R. Oppenauer, Helu. Chim. Acta, 17, 390 (1934). (95) H. Ugami, Nippon Nogei Kagaku Kafshi, 10,727 (1934);Chem. Abstr., 28, 7259 (1934). (96) K. Aso, Nippon Nogei Kagaku Kaishf,15,161 (1939);Chem. Abstr., 34,379 (1940).

DEHYDRATION REACTIONS OF CARBOHYDRATES

187

(74; see p. 191) (Ref. 97). In dilute acid solutions under reflux, compounds 27 and 75 are not produced quantitatively, and 74, which contains 6 carbon atoms and is not a decarboxylation product, is produced in such low yield that it has no measurable effect on the yields

of carbon dioxide. The kinetics of the decarboxylation reaction have been studied by a number of w ~ r k e r s D. ~ ~M.~ W. ~ ~Anderson ; and Garbuttlooshowed it to be an acid-catalyzed reaction, and theylO1found that C-6 of the hexuronic acid is the source of the carbon dioxide. A quantitative yield of carbon dioxide is obtained from D-galacturonic acid when it is treated with 3.29 M hydrochloric acid under reflux for 6 hours.90 Table I indicates the relative rates of decarboxylation of a number TABLEI Relative Rates of Decarboxylation of Sugar Acidsloo Compound D-Galacturonic acid D-Glucurono-6,3-lactone ~-xy~o-5-Hexulosonic (“L-sorburonic”) acid L-lyxo-Hexulosonicacid D-arabino-Hexulosonic acid L-Ascorbic acid

Relative rate 1.0 0.7 3.7 0.7 0.7 0.7

of sugar acids. Stutz and Deuello2 measured the yields of carbon dioxide from a number of hexuronic acids after treatment with boiling 1.75 M hydrochloric acid for 4 hours, and then with 0.01 M hydrochloric acid for 20 hours. They found that 2-amino-2-deoxy-~glucuronic acid and 2-amino-2-deoxy-D-ga~acturonic acid are even more reactive than the non-aminated acids. Little information is available on the decarboxylation of glycuronic acids, other than from those derived from hexoses. Machida103 reported that both D-xyhronic acid and D-arabinuronic acid undergo decarboxylation in boiling, 12% hydrochloric acid, but the yields of (97) E. Stutz and H. Deuel, Helu. Chim. Acta, 39, 2126 (1956). (98) D. M. W. Anderson, A. M. Bews, S. Garbutt, and N. J. King, J. Chem. SOC.,5230 (1961). (99) A. Meller, Aust. I. Chem. 7, 157 (1954). (100) D. M. W. Anderson and S. Garbutt,]. Chem. SOC.,3204 (1963). (101) D. M. W. Anderson and S. Garbutt, Talanta, 8, 605 (1961). (102) E. Stutz and H. Deuel, Helu. Chim. Acta, 41, 1722 (1958). (103) S. Machida, Nippon Kagaku Zasshi, 70, 82 (1949).

188

M. S. FEATHER AND J. F. HARRIS

carbon dioxide are less than the theoretical, and the other reaction products were not examined. L-Threuronic acid is decarboxylated much faster than D-galaCtUrOniC acid, to give theoretical yields of carbon dioxide and isolable amounts of p y r u ~ a l d e h y d e .The ' ~ ~ liberation of carbon dioxide from hexuronic acids has been used as a method for analyzing (a) hexuronic acids in the presence of neutral carbohydrates, (b) polysaccharides containing hexuronic acids, and (c) oxidized polysa~charides.'~~ For accurate measurements, it is necessary to differentiate between carbon dioxide derived from the uronic acid and that derived from neutral carbohydrates. Carbon dioxide is evolved from the neutral carbohydrates at a low, but constant, rate throughout the reaction. For correction, the evolution of carbon dioxide is plotted versus time, and is then extrapolated to "zero time"; the intercept is the amount derived from the uronic acid. Despite their shortcomings, variations on this general method are the best procedures currently a ~ a i l a b l e . ' ~An ~ , ' almost ~~ quantitative yield of carbon dioxide is observed during the pyrolysis of hexuronic acids,'08 but over-decarboxylation, and differences between the reaction rates for different carbohydrates, render pyrolysis unsuitable for analytical purposes.10s The major, monomeric dehydration-product, namely, 2-furaldehyde (27), formed during the reaction results from D-galacturonic acid in yields in the vicinity of 30% of the theoretical."O This result is in contrast to its production from D-XylOSe,'l' L-ascorbic and Darabino-hexulosonic acid54; when treated under comparable conditions (3.3 M hydrochloric acid under reflux), these give almost theoretical yields of 27. The fact that 27 is produced by dehydration both of uronic acids and of pentoses has led to the suggestioni12that pentoses may be intermediates in decarboxylation reactions of uronic acids, and that treatment of such glycuronans as pectin with strong acids results in the production of pent~sans."~ Little evidence supports this theory, be(104) M. Ikawa and K. P. Link, ]. Amer. Chem. SOC., 72,4287 (1950). (105) D. M. W. Anderson, Talanta, 2, 73 (1959). (106) R. L. Whistler, A. R. Martin, and M. Harris,]. Res. Nut. Bur. Stand., 24,13 (1940). (107) M. S. Feather and R. L. Whistler, Methods Carbohyd. Chem., 1, 454 (1962). (108) A. S. Perlin, Can. J. Chem., 30,278 (1952). (109) D. M. W. Anderson, S. Garbutt, and J. F. Smith, Talanta, 9,689 (1962). (110) M. S. Feather and J. F. Harris,]. Org. Chem., 31,4018 (1966). (111) H. G. Bott and E. L. Hirst,]. Chem. Soc., 2621 (1932). (112) H. Franken, Biochem. J., 257, 245 (1933). (113) F. V. Linggood, Biochem. ]., 24,262 (1930).

DEHYDRATION REACTIONS OF CARBOHYDRATES

189

cause careful ~ e a r c h e s ”have ~ failed to detect the presence of pentoses during decarboxylation, and, for glycuronans, the rate of h y d r ~ l y s i s ” ~ is substantially higher than the rate of decarboxylation. However, D-galacturonic acid has been reported to undergo partial decarboxylation in water in the presence of certain metal ions, with the formation of L-arabinose1Is; no 2-furaldehyde was produced. Decarboxylation is even more rapid in pyridine and in the presence of Ni2+; after treatment at loo”, crystalline L-arabinose was isolated as a reaction product.’I6 D-arubino-Hexulosonic acid (“2-keto-~-gluconicacid”) gives D-erythro-pentulose and D-arabinose when so treated.l17 5-Formyl-2-furoic acid (74) has been reported to be produced, in yields sufficiently high for chromatographic detection, from both D-galacturonic acid and D-xylo-5-hexulosonic (‘I-sorburonic”) acid by treatment with boiling, 12% hydrochloric acid.s7 Although the yields were not estimated, it is probable that they are less than 1% in each case. Stutz and D e u e P isolated 25 mg of crystalline 74 from 10 g of D-galacturonic acid by treating it with concentrated sulfuric acid for 90 seconds at 60” and, on using 10 g of ~-arubino-5-hexulosonic (“5-keto-~-galactonic”) acid as the starting material, they isolated 700 mg of 74, as crystalline methyl 5-formyl-2-furoate. The preferred method of preparing 74 for laboratory use is by the dehydration of D-xylo-5-hexulosonic (“5-keto-~-gluconic”)acid in refluxing, 5% methanolic hydrogen chloride; the crystalline, methyl ester of 74 can then be isolated in 17.4% yield.lI0 CrystalIine 74 is readily prepared from the methyl ester by saponification. Pure 74 has m.p. 200-201” and A,, = 286 nm ( E = 18,000) in water. Compound 74 undergoes decarboxylation to give 2-furaldehyde, as evidenced by the results of chromatographic experirnents.”O However, the fact that 74 is decarboxyIated102*110*1’S at less than 2% of the rate for D-galacturonic acid under comparable conditions indicates that it is essentially an end product in the reaction, and not an intermediate, either in the production of 2-furaldehyde or in the decarboxylation reaction. Although mechanistic studies have not been reported for this conversion, the obvious structural relationships between 74 and its precursors suggest that 74 is formed by mech(114) C. M. Conrad,J. Amen Chem. SOC., 53,2282 (1931). (115) V. P. Kiseleva, A. A. Konkin, and J. A. Rogovin, J . Appl. Chem. USSR (Engl. Trunsl.),27, 1073 (1954). (116) G . Zweifel and H. Deuel, HeZu. Chim. Acta, 39, 662 (1956). (117) M. Matsui, M. Uchiyama, and C. E. Liau, Agr. B i d . Chem. (Tokyo), 27, 180 (1963). (118) S. Machida, Nippon Kagaku Zasshi, 64, 1311 (1943).

190

M. S. FEATHER AND J. F. HARRIS

anisms analogous to those proposed for the production of 5-(hydroxymethyl)-2-furaldehyde from hexoses. The formation of such chromones as 3,8-dihydroxy-2-methylchromone by treating uronic acids or pentoses with dilute acid was reported by A S O ,and ~ ~ studied ~ by Popoff and Theander,lm who obtained a number of these compounds in 3.5% yield, as well as some catechols. Although nothing is yet known about the mechanism of formation of these compounds, the fact that the chromones contain 10 carbon atoms and are produced both from pentoses and uronic acids suggests that they may be derived from 2-furaldehyde or reductic acid, or produced from a decarboxylated intermediate. Investigations of the mechanism of decarboxylation of hexuronic acids have largely involved kinetic and tracer studies. When either D-xy~o-5-hexulosonicacid or D-glucuronic acid is converted into 27 in acidified, tritiated water, the resulting 27 contains 18% and 15%, respectively, of the activity of the solvent as carbon-bound tritium.21 Further degradation studies showed that the isotope is situated on the h r a n ring at either position 3 or 4, or both; these atoms correspond to C-3 or C-4 of the starting uronic acid. Although it has been proposed that the dehydration of hexuronic acids is initiated by the carbonyl group of the carboxyl group,1ooit appears more probable that the first reaction would be p-elimination of the 3-hydroxyl group. The resulting 2-ene (70) could undergo various cyclization reactions that lead to some of the products observed,12’ but, again like the sugars, it should react rapidly to form the 3,4-dideoxy-3-ene (71). Decarboxylation of 71 can then occur prior to, concurrent with, or subsequent to, ring formation; these possibilities were suggested by Isbell.121 In its acyclic form, 71 could be decarboxylated by addition of a solvent proton (a) to C-3, or (b) to the carbonyl group of C-2, or (c) by transfer of the proton of the hydroxyl group of the carboxyl group to C-3. The third mechanism is similar to that of decarboxylation of P-keto acids.122The first and third of these mechanisms result in addition of a solvent proton to C-3. However, when the reaction is performed in tritiated water, the incorporation of radioactivity at C-3 does not distinguish between the various possibilities. Neither does incorporation verify their existence, as solvent-proton addition can (119) K. Aso, TohokuJ. Agr. Res., 3, 359 (1953). (120) T. Popoff and 0. Theander, Chem. Commun., 1576 (1970);Carbohyd. Res., 22, 135 (1972). (121) H. S. Isbel1,J. Res. Nat. Bur. Stand., 33,45 (1944). (122) E. S. Gould, “Mechanism and Structure in Organic Chemistry,” Holt, Rinehart and Winston, Inc., New York, N. Y., 1959, p. 346.

DEHYDRATION REACTIONS OF CARBOHYDRATES

191

occur by the keto-enol equilibria of both 70 and 72. Both the small proportion of incorporation of tritium at C-3 that has been observed and the ease of protonation of the carbonyl group would indicate that the second mechanism is favored. The product of decarboxylation of 71, namely, 2,5-dihydroxy-2,4-pentadienal (“2-0x0-glutaraldehyde”), 72, reacts further, to afford either 2-furaldehyde or reductic acid, as described in Section V (see p. 208). HC =O

HC=O

HC=O

c=o

y HCOH

COH II YH HCOH

-+

HCOH I

HCOH I

@OH

I

I

I

HC=O I COH II CH

&=m H@ -

HC

CfiO I ‘OH

1

HC II

HCOH

C//O

co

H

7oa

70

27

71

73

72

74

The formation both of 5-formyl-2-furoicacid (74) and 2-furaldehyde could result by way of the formation of the 2,gdihydrofuran 73. Elimination of the C-5 proton and the 2-hydroxyl group by a reaction analogous to that for the formation of 5-(hydroxymethyl)-2-furaldehyde yields 74, whereas decarboxylation, as shown, results in 2-furaldehyde. Once formed, 73 would be expected to give higher yields of 74 than of 27; this implies that decarboxylation occurs prior to ring formation. In addition to suggesting 72 as a source of reductic acid, Isbel1121 proposed an alternative mechanism in which decarboxylation and carbocyclic-ring formation occur simultaneously. As only one mechanism for forming 75 from pentoses is possible, Isbell suggested that the existence of a second mechanism for the formation of 75 from hexuronic acids might account for the higher yields obtained from D-galacturonic acid. However, it is more probable that the conformations of the intermediates are controlled by the configuration of the

M. S. FEATHER AND J. F. HARRIS

192

OH Y

O

OH 71

15a

H

= 0 75

original sugar, and that the reaction conditions constitute the major factor in determining the yields. All of the possibilities mentioned are credible; perhaps each participates to some extent in the reaction. No studies similar to those with aldoses have been made of substituted uronic acids, and no intermediates have been isolated. Aso,B6 who first suggested 72 as a precursor for 2-furaldehyde and reductic acid:2 has prepared 72, but its significance in the decarboxylation reactions has not been fully examined.123 On treatment with aqueous acid, L-ascorbic acid and the related aldosulosonic acids liberate 2-furaldehydego at rates approximately the same as those observed for hexuronic acids (see Table I; p. 187).93Although not experimentally demonstrated, it is generally assumed that the origin of the carbon dioxide is the carboxyl group of the acid, that C-2 through C-6 are converted into 2-furaldehydeY and that C-2 corresponds to the aldehydic carbon atom of 2furaldehyde. Kurata and S a k ~ r a ihave l ~ ~ investigated the mechanism of dehydration of L-ascorbic acid by examining the products from it and from L-xylo-hexulosonic acid after treatment for 1 hour at 100" at pH 2.2, or with 5% sulfuric acid at 100". In both experiments, 2-furaldehyde and 3-deoxy-~-threo-pentosulose(isolated as the phenylhydrazone) were the major products; no pentose was detected. Although the authors considered both possibilities, namely, decarboxylation occurring prior to and after dehydration, the latter seems quite improbable. The 6-carbon intermediates from the dehydration of 76 would not be decarboxylated readily; they would be expected to form furans other than 2-furaldehyde. Isotope-exchange studies54confirmed the mechanism illustrated,121in which decarboxylation occurs, prior to dehydration, through the 3-keto isomer 76a. (123)K. Aso, Nippon Nogei Kagaku Kaishi, 29, 985 (1955);Chem. Abstr., 51, 8658 (1957). (124)K.Kurata and Y. Sakurai, Agr. Biol. Chem. (Tokyo), 31, 170 (1967).

DEHYDRATION REACTIONS OF CARBOHYDRATES

193

The formation of 76a is essential, as the enol 76 would not be expected to be decarboxylated. When allowed to react in tritiated water, both L-ascorbic acid and D-arubino-hexulosonic acid produced 2-furaldehyde having an activity approximately 60% of that of the solvent; most of the activity was situated at the a-carbon atom. The failure to detect any pentose is probably ascribable to the high reactivity of the l,&enediol (77).

COH II

COH

I HCOH I HOCH I C$OH

-

HCOH

=

C=‘;iH@ I

HCOH I HOCH I CbOH

76

I

HOCH I

CqOH 71

HC=O

I

c=o I

CH II CH

-

I

27

I

HCOH

76a

0‘’’ -

II

COH

HC=O I COH II

HC I HOCH I

CH,OH

CH,OH

IV. DEHYDRATION IN ALKALINE SOLUTION

1. Aldoses and Ketoses The formation of saccharinic acidslZ5isomeric with the reacting carbohydrate has long been known to occur by the following reaction (125) Saccharinic acids are (monocarboxylic) deoxyaldonic acids having the general formula C,H,.O,. Of interest here are only those that can be formed from 1,2- or 2,Senediols by p-elimination followed by benzilic acid rearrangement. They are of three types: metasaccharinic, I, saccharinic, 11, and isosaccharinic, 111. The C-2 epimers are distinguished as “a’’and “p.” COzH

I

C(OH,H)

I

COzH

I C(OH,CHA I R

CH2

I

COzH

I

C(OH,CH,OH)

I

CHz

I

R

R I

I1

111

194

M. S. FEATHER AND J. F. HARRIS

sequence: ( a ) formation of an enediol, (b) p-elimination of a functional group, (c) tautomerization of an a-dicarbonyl intermediate, and ( d ) a benzilic acid rearrangement. An extensive, historical account of developments leading to this final formulation (which was first proposed by Isbel13)was given by Sowden.’ Subsequent work has firmly established the validity of the formulation (see Section 11, 2; p. 168). The first two reactions of the sequence are similar to reactions that occur in acidic medium. The 1,2- and 2,3-enediols7 and the unsaturated elimination-products derived from them, are present both in acidic and basic solutions. In general, however, reactions in basic solution are much faster than in acidic solution, because of the greater catalytic effect of the hydroxyl ion on the transformation reactions. Mechanistic differences between the media become operative in steps c and d. In acid, further dehydration, if it is possible, occurs rapidly, before equilibrium of the deoxy-enediol with the dicarbonyl compound has been established,” and the products are furans. In alkaline solution, the rapid formation of the tautomeric dicarbonyl compound permits the benzilic acid rearrangement42 to proceed. The 6-carbon saccharinic acids derived from D-glUCOSe have been investigated,12sparticular attention being given to the proportions of the “a” and “p” isomers formed. It was known that the “a”- and “P”-metasaccharinic isomers, because they are relatively stable and have a labile proton on C-2, can isomerize reversibly if treated with alkali or heat.12’ The (new) “/3’’-D-glucoisosaccharinic acid was isolated, and characterized.lZ6It was formed, together with comparable amounts of the “a” isomer, from the alkaline degradation of 4-0-substituted D-glUCOSeS, but neither product could be epimerized under the conditions of its formation. Only “a”-D-ghcosaccharinic acid (and no “/3”-D-glucosaccharinic acid) was formed from 1-0-substituted D-fructoses. To confirm this finding, “P”-L-glucosaccharinic acid was synthesized, and, subsequently, the dicarbonyl intermediate, l-deoxy-~-erythro-2,3-hexodiulose (lOa),was synthesized and shown to afford, when treated with a base, only traces of a compound that is, apparently, the “p” The principal products were “a”-D-glUcosaccharinic acid and D-erythrOniC acid, a fragmentation product. Unlike the other dicarbonyl intermediates, compound 10a has an asymmetrically substituted carbon atom adjacent to the dicarbonyl grouping; this circumstance may account for the specificity of the (126) A. A. J. Feast, B. Lindberg, and 0.Theander,Acta Chem. Scand., 19,1127 (1965). (127) B. Alfredsson and 0. Samuelson, Suensk. Papperstidn., 65,690 (1962).

DEHYDRATION REACTIONS OF CARBOHYDRATES

195

rearrangement. This explanation is, however, weakened by the finding that, on treatment of D-XylOSe with calcium hydroxide, “a”- and “P”-D-xylosaccharinic acids are formed in comparable amounts,’28.’28aand that, on similar treatment, L-sorbose affords both “a”- and “P”-L-galactosaccharinicacid in approximately equal yields. The configurations of all of the 6-carbon saccharinic acids derived from D-glucose have now been e ~ t a b l i s h e d . ’ ~ ’ ~ ~ - ’ ~ ~ The isolation and characterization of 2-C-methyl-D-threonic and -D-erythronic acids by Lindberg and coworkers128completed the characterization of the 5-carbon saccharinic acids. All five of the acids of this group are formed when D-XylOSe is treated with calcium hydroxide. In addition, eight other acids, products of fragmentation and recombination, were isolated. In M sodium hydroxide solution, “a”- and “P”-D-xylosaccharinic acids were not formed. A similar result was obtained when D-fructose was kept in concentrated sodium hydroxide solution; no “a”-D-glucosaccharinic acid was detected, whereas it was the major product from the reaction in lime-water. This same effect, accruing from the differences in the bases employed, was also apparent in the early work of Kiliani and Nef. The principal 6-carbon product obtained from D-glucose in lime-water was “a”-D-glucosaccharinic acid, whereas no trace of it could be found when the sugar was treated with hot, concentrated sodium h y d r ~ x i d e . ~ All of the 4-carbon saccharinic acids possible have been prepared and ~haracterized.’~~ The 4-carbon metasaccharinic acid has been encountered f r e q ~ e n t l y , ’ ~ ’ and ~ ~ -a’ ~ racemic ~ mixture of the 2-methylglyceric acids (2,3-dihydroxy-2-methylpropanoic acids) was obtained from 4-O-methyl-D-threo~e.~~~ Treatment of monosaccharides with alkali usually results in a preponderance of products that are not isomeric with the original sugar. Their molecular-weight distribution indicates that they must (128) A. Ishizu, B. Lindberg, and 0. Theander, Acta Chem. Scand., 21, 424 (1967). (128a) A. Ishizu, K. Yoshida, and N. Yamazaki, Carbohyd. Res., 23,23 (1972). (129) J. C. Sowden and D. R. Strobach, J. Amer. Chem. Soc., 82,3707 (1960). (130) A. B. Foster, T. D. Inch, J. Lehmann, and J. M. Webber,]. Chem. Soc., 948 (1964). (131) M. Von Glehn, P. Kierkegaard, R. Norrestam, 0. Ronnquist, and P. E. Werner, Chem. Commun., 291 (1967). (132) J. D. Crum, Adoan. Carbohyd. Chem., 13, 169 (1958). (133) J. W. Green, J. Amer. Chem. Soc., 78, 1894 (1956). (134) J. F. Harris, Carbohyd. Res., 23, 207 (1972). (135) M. Gibbs,J. Amer. Chem. Soc., 72,3964 (1950). (136) G. N. Richards, J. Chem. Soc., 3222 (1957). (137) B. Alfredsson and 0. Samuelson, Soensk Papperstidn., 71, 679 (1968).

196

M. S. FEATHER AND J. F. HARRIS

result both from fragmentation and combination. The mechanisms are complex and poorly understood. Fragmentation is a major feature of the alkaline-degradation reactions, and may occur for the original sugar, for the reaction intermediates, or for the saccharinic acid end-products. The last-mentioned have been shown to be quite stable under the conditions of their The 3-deoxy- and 4-deoxy-glycodiuloses undergo rapid reaction to give high yields of m e t a s a c c h a r i n i ~ ~and ~ * ~isosac3~ charinic acids, respectively, with only small proportions of fragmentation products. El-Dash and H ~ d g found e ~ ~ the fragmentation products of 3-deoxy-~-erythro-hexosulose (7a) to be, primarily, 4- and 2-carbon compounds, but the yields of the fragmentation products, relative to the metasaccharinic acids formed, were not reported. The precursor of saccharinic acid, namely, l-deoxy-~-erythro-2,3-hexodiulose (1Oa; see p. 168), has been shown to undergo extensive cleavage3g to give erythronic acid, a product not reported to be formed in significant proportions by such degradation of monosaccharides. Because of these facts, it may be concluded that, under nonoxidative conditions, fragmentation occurs as an initial reaction of the sugar. Fragmentation is attributed to reverse-aldol reactions similar to that shown for a ketose and its 172-enediol.Although reaction by way CGOH I COH I1 HOCH

(138) Refs. 34 and 38.

DEHYDRATION REACTIONS OF CARBOHYDRATES

197

of the ketose would appear the more facile, presence of an oxygen atom on C-2 is not necessary; this conclusion is substantiated by the formation of lactic acid from 2-deoxyhexoses and 2-0-substituted hexoses. As the sugars undergo extensive isomerization, and as, theoretically, the enolic bond can engage any two adjacent carbon atoms in the carbon chain, cleavage of all of the carbon-carbon bonds could occur by this mechanism. It should be noted that the aldehydic carbon atom of the triose enediol released from the reducing end originates from C-3 of the hexose, whereas the carbonyl group of the D-glyceraldehyde formed from the nonreducing end originates from C-4. Thus, in the absence of isomerization, hexoses labeled at either C-1 or C-6 would mainly yield lactic-3-14Cacid. However, extensive isomerization does occur, and lactic-2-14C acid and lactic-lJ4C acid are also found in considerable proportion. The presence of lactic-2-% acid is explained by the reaction of the dicarbonyl compound 78 that undergoes a benzilic

* CH,

*C&OH

tl HCOH II COH I

CKOH

*CH, I

I

I

COH I1 HCOH

C=O

HCOH I

I

HC=O

C4H COaH

-

HC=O * I

C=O CH, I

ia

*I

/

HzY;

\

*F02H HCOH I CH,

acid rearrangement by addition of a hydroxyl group either to C-1 or C-2. With addition to C-1, the rearrangement involves a hydride-ion transfer, with no change in the carbon skeleton, whereas addition to C-2 results in the transfer of the methyl group and a reordering of the carbon atoms. The extent of methyl-group transfer is greatly dependent on the type of base employed. Sowden' reported 26% of methyl-group migration in lime-water at 25", whereas values ranging from 0 to 24% have been reported for sodium and potassium hydro~ides.'~~.'~~ Cleavage of a sugar at the C-2-C-3 bond by the reverse-aldol mechanism results in the glycolaldehyde ion (79).As glycolaldehyde cannot react by a p-elimination mechanism similar to that of glyceraldehyde, it undergoes aldol addition with itself, or with other

M. S. FEATHER AND J. F. HARRIS

198

P

HC=O

HCO’

@OH

1

I1

HCoH 79

/ \

HCOH II

HCoH

HC=O I HCOH 0

79a

reactive species in the solution. Its formation and subsequent reactions account for the presence of many products, in particular, of a 4-carbon saccharinic acid, 2,4-dihydroxybutyric acid. This acid was produced in 18% yield from D-xylose, and 90% of it originated from C-1-C-2 fragments of the original ~ - x y l o s e . ’ ~ ~ Self-addition of fragments occurs by the familiar, base-catalyzed, aldol mechanism. This reaction shows general, base c a t a l y ~ i s , ’ ~ ~ ” ~ ~ indicating that the rate-controlling step is the formation of the anion (SO). As it is this anion that is liberated in the cleavage process of the HC-0’ II

HC=O

/XR’

80

HC=O I R”

+

HC=O I

‘C R’ ‘R’

-

--t

1

HC=O RCR’ I

HCO’ I

R”

--

HC=O RLRI I

HCOH R”

alkaline reaction, subsequent condensation occurs rapidly. However, it was found that, in concentrated sodium hydroxide at high temperature, the 2-carbon fragments isomerize completely before recombining, but the 3-carbon fragments do n0t.’3~It is probable that, when reaction occurs in the presence of a complexing cation such as Ca2+, (139) C. D. Gutsche, D. Redmore, R. S. Buriks, K. Nowotny, H. Grassner, and C. W. Armbruster, J. Amer. Chem. SOC., 89, 1235 (1967). (140) D. W. Griffiths and C. D. Gutsche, J. Amer. Chem. SOC.,93,4788 (1971).

DEHYDRATION REACTIONS OF CARBOHYDRATES

199

which is capable of complexing with hydroxyl groups and alkoxide anions, isomerization is hindered.140a The formation of glucometasaccharinic and glucoisosaccharinic acids has been generally well established as proceeding by way of the Nef-Isbell mechanism, but the formation of glucosaccharinic acid is not so well understood. The absence of “a”-glucosaccharinic acid among the end products in sodium hydroxide solutions, and its large yield in calcium hydroxide solutions, may, perhaps, be attributed to a kinetic effect. However, the results of Sowden‘ with D-mannose-1J4C indicate that glucosaccharinic acid is, at least in part, formed by a mechanism that differs from that for the other acids. “ a ” - ~ - G l u ~ ~ ~ a ~ ~ acid h a r iformed n i c by the isomerization of D-mannose-l-14C in lime-water was found to be labeled at both C-2 and the methyl group, in the ratio 57:39. This result was explained by Kenner and Richards141by assuming that C-3-C-4 cleavage provided 1,3-dihydroxy-2-propanone and D-glyceraldehyde that recombined to provide hexose-l -14C and hexose-3-14C.Although this mechanism properly accounts for the position of labeling, it was rejected by Sowden and as, in their opinion, it could place a maximum of only 50% of the label at C-2. Some very unconventional mechanisms have since been postulated to explain this supposed although it is readily explained by the mechanisms of fragmentation and recombination just described. The nascent, 3-carbon anion liberated from the reducing end of D-~nannOSe-l-’~Ccan participate directly in an aldol reaction, to form hexose-3-14C,but it can only afford hexose-l -14C after isomerizing. Thus, the mixture of radioactive hexoses would be mainly C-3labeled, and would give radioactive “a”-D-glucosaccharinic acid labeled principally at C-2; this agrees with the experimental result. The formation of the anion (81) could well enhance this effect by 0

CKOH

HCOH

I

I

C-Q@

C-00

1

HC-6

$,a+

0

t-

1)

$a,+

HC-6@ 81

T O H8 C-Q.

I

.j:.”

HC-6@ 0 81a

(140a) T. Mizuno and A. H. Weiss, Aduan. Carbohyd. Chem. Biochem., 29, in press (1973). (141) J. Kenner and G . N. Richards,J. Chem. SOC.,1784 (1954). (142) J. C. Sowden, M. G. Blair, and D. J. Kuenne, J . Amer. Chem. SOC., 79,6450 (1957). (143) Ref. 30, p. 747. (144) H. S. Isbell, Nat. Bur. Stand. Tech. Note 274, 54 (1965).

200

M. S. FEATHER AND J. F. HARRIS

retarding isomerization, as well as inhibiting the participation of 81a in the addition. However, this explanation is incomplete, as it does not account for the very small proportion (-2%) of activity found in the terminal, C-3 atoms of the acid; neither would the fructose isolated from the reaction mixture be expected to be labeled almost entirely144 at C-1. These facts can, perhaps, be attributed to complexing characteristics of the calcium ion that are only vaguely understood.140aCalcium is a most complicated factor in the degradation; in addition to what has been mentioned, it is known to have an effect on the fragmentation pattern3sJ38*145 and the benzilic acid rea~~angement.'"~~ Fragmentation and recombination reactions predominate in nearneutral, alkaline solutions. Fragments from the cleavage of the carbohydrate, and from its dehydration products, undergo further dehydration, condensation, and intermolecular, Cannizarro-type reactions. The benzilic acid rearrangement, an intramolecular Cannizarro reaction, seems to be inoperative. From the base-catalyzed degradation of D-fructose (pH - 8.0),Shaw and coworkers147identified 18 compounds, none of which was ( a ) isomeric with the starting material, or ( b ) a simple dehydration product. Among the products, the hydroxy-Zbutanones and l-hydroxy2-propanone (acetol) were shown to participate in forming the carbocyclic products identified, but the mechanism of their formation was not elucidated. Several furan derivatives were isolated, but no lactic acid was isolated. In a similar study but with weak acid,41most of the products were formed by a combination of enolization and dehydration steps, with little fragmentation. Hayami and his coworkers have studied the mechanism of formation of acetol and pyruvic acid from D-glu~ose-l-~~C, -6-I4C, and -3,4-I4C2,reacting in a concentrated, phosphate buffer s o l ~ t i o n . ' ~ ~ - ' ~ ~ Their data supported the supposition that the products are formed from pyruvaldehyde by way of a Cannizarro reaction. As in the formation of lactic acid, the pyruvaldehyde can be formed either from the reducing or the nonreducing end of the D-glUCOSe molecule, and the distribution of radioactivity in the pyruvic acid and acetol (145) H. P. Humphries and 0. Theander, Acta Chem. Scand., 25,883 (1971). (146) D. O'Meara and G. N. Richards,]. Chem. Soc., 1944 (1960). (147) P. E. Shaw, J. H. Tatum, and R. E. Berry, J. Agr. Food Chem., 16,979 (1968). (148) R. Goto, J. Hayami, K. Kudo, and S. Otani, Bull. Chem. Soc. lap., 34,753 (1961). (149) J. Hayami, Bull. Chem. Soc. Jap., 34,924 (1961). (150) J. Hayami, Bull. Chem. Soc. Jap., 34,927 (1961). (151) S. Otani, Bull. Chem. Soc.Jap., 38, 1873 (1965); 39, 1164 (1966).

DEHYDRATION REACTIONS OF CARBOHYDRATES

20 1

was found similar to that for the lactic acid formed in concentrated sodium hydroxide solution.'34Very little isomerization of the pyruvaldehyde occurred; this may be due to the high concentration of phosphate ion and to the formation of ion complexes similar to those formed with calcium ion. Substitution with electron-withdrawing, alkoxyl groups has a variety of effects on both the rate and the mode of degradation in alkaline solution.'52 The acetal linkage of 1-0-substituted aldoses (glycosides) is very resistant to alkaline hydrolysis, except where instability is imparted by the aglycon, as in the phenyl glycopyranosides. Under drastic conditions, cleavage does occur, but the nature of the reaction has not yet been d e t e ~ m i n e d . ' ~ ~ However, -'~~ an alkoxyl group at C-1 of D-fructose is favored for elimination, resulting in the formation of large proportions of ''a"-D-ghcosaccharinic acid.'41 Similar, favored elimination occurs136J56-159 with alkoxyl substitution at C-3 and C-4. Phosphoric esters have been found to react in the same way as the methyl ethers.160-162 The 2-0-methylaldoses undergo p-elimination to form the 2-methyl ethers of the enol form of the 3-deoxyhexosuloses.'s3 These ethers are relatively stable; benzilic acid rearrangement is not possible, because a carbonyl group cannot form at C-2, and further dehydration by elimination of the 4-hydroxyl group, which occurs readily in acid, does not proceed in base.69Although formation of these ethers has been demonstrated in many s t ~ d i e s , ' ~ ~ -their ' ' ~ reactions after (152) R. L. Whistler and J. N. BeMiller, Adoan. Carbohyd. Chem., 13, 289 (1958). (153) B. Lindberg, Soensk Papperstidn., 59, 531 (1956). (154) E. Dryselius, B . Lindberg, and 0.Theander, Acta Chem. Scand., 11,663 (1957); 12, 340 (1958). (155) J. Janson and B. Lindberg, Acta Chem. Scand., 13, 138 (1959); 14,2051 (1960). (156) G. Machell and G. N. Richards,J. Chem. SOC., 1924 (1960). (157) J. Kenner and G . N. Richards, J. Chem. SOC.,278 (1954). (158) R. L. Whistler and W. M. Corbett,]. Amer. Chem. SOC.,78, 1003 (1956). (159) W. M. Corbett and J. Kenner, J. Chem. Sac., 1431 (1955). (160) J. B. Lee, J. Org. Chem., 28, 2473 (1963). (161) C. Degani and M. Halmann, ]. Amer. Chem. SOC., 90, 1313 (1968). (162) D. M. Brown, F. Haynes, and A. R. Todd, Chem. Ber., 90,936 (1957). (163) A. Klemer, H. Lukowski, and F. Zerhusen, Chem. Ber., 96, 1515 (1963). (164) E. F. L. J. Anet, Chem. Ind. (London), 1035 (1963). (165) E. F. L. J. Anet, h s t . J. Chem., 19, 1677 (1966). (166) E. F. L. J . Anet, Carbohyd. Res., 3,251 (1966-1967). (167) E. F. L. J. Anet, Carbohyd. Res., 7, 453 (1968). (168) E. F. L. J. Anet, Carbohyd. Res., 8, 164 (1968). (169) B. Lindberg, 0.Theander, and M. S. Feather, Acta Chem. Scand., 20,206 (1966). (170) G . 0. Aspinall, R. Khan, and Z. Pawlak, Can. J. Chem., 49, 3000 (1971). (171) J. Kenner and C. N. Richards,]. Chem. SOC.,2921 (1956).

M. S. FEATHER AND J. F. HARRIS

202

prolonged heating, or exposure to more severe conditions, are only partially known. Anetlss found that 2,3,4,5-tetra-0-methyl-~-glucose readily reacts to form a mixture of cis- and trans-3-deoxy-Derythrohex-2-enoses and two 3,6-anhydroaldoses having opposite configurations at C-3. These results are explicable by assuming the existence of the following equilibria. HCQ $+Me / CH

'\;

%

,ihOMe

OMe

HC-0' II COMe +

/o'&i

-

%c\$XiOMe

HCOH II COMe /Ox&

%'\$!HOMe OMe

OMe

This is the only report of anhydroaldoses among the alkaline degradation products of 2-O-methyl-aldoses, but similar reactions have been reported for other sugars. Overend and coworker^"^ found that the major product from the alkaline treatment of 2-deoxy-D-urubinohexose is 3,6-anhydro-2-deoxy-~-urubino-hexose. This reaction undoubtedly proceeds through the formation of the 2,3-dideoxyglyc-2enose, followed by attack at C-3 by the C-6 nucleophile. Unlike the methylated product, 2-deoxy-~-urubino-hexosecan react further, to form a stable, bicyclic pyranose that results in a single configuration for the product. Similar bicyclic (anhydro) compounds are formed from the 2-acetamido-2-deoxy-~-hexoses~~~ when they react in a sodium carbonate solution. With these compounds, both of the C-2 epimers have been found in significant yields. The 2-acetamido-2deoxy-hex-2-enose and the C-2 epimeric 2-acetamido-3,6-anhydro-2deoxy-D-hexoses would be expected to yield N - [ 5 - (1,2-dihydroxyethyl)-3-fury11acetamide [3- acetamido-5- ( 1,2-dihydroxyethy1)furanl (82) in the Morgan-Elson test. The addition of the Ehrlich reagent

NHAc 82

during the test renders the solution acidic, and results in the formation of a 2,5-dihydrofuran ring that is readily dehydrated. However, (172)R. J. Ferrier, W. G . Overend, and A. E. Ryan, J. Chem. SOC., 1488 (1962). (173)V.A. Derevitskaya, L. M. Likhosherstov, V. A. Schennikov, and N. K. Kochetkov, Cafbohyd.Aes., 20,285 (1971).

DEHYDRATION REACTIONS OF CARBOHYDRATES

203

there seems to be good evidence174that some 82 is formed under the alkaline conditions prevailing during the initial part of the procedure. If this is so, it constitutes a rare instance of the formation of a furan in alkaline solution. The 2,3-dideoxyglyc-2-enose also appears to participate in the formation of 5-oxocyclopent-1-enyl phosphate (86) found among the alkaline degradation products of apurinic and apyrimidinic acids.175 The 2,3-dideoxypent-2-enose 5-phosphate (83), formed by p-elimination either from apurinic or from apyrimidinic acid, readily isomerizes to 84, a form in which it can eliminate the 5-phosphate group or, by further rearrangement of the double bonds, cyclize to 85. The fate of 87, also an expected intermediate in the alkaline degradation of 2-deoxy-~-erythro-pentose,is unknown, but it would be expected to cyclize to form 173-cyclopentanedione. HC=O I

CH II

CH

HAOH I %COPOS% 83

-

HCOH II

CH I

CH II

COH I

%C0PO*%

HC=O

- 8%

84

85

I

CH II

CH I

COH

87

86

2. Unsaturated Sugars

Reviews176of the chemistry of the unsaturated sugars include discussions of their reactions in alkaline solution; only a brief discussion of the glycals and 2-hydroxyglycals will be given here. Treatment of tri-0-acetyl-D-glucal with boiling water results in the migration of the double bond to the 2,3-positions and the elimination (174) R. Kuhn and G. ~ g e rChem. , Ber., 89, 1473 (1956); Chem. Abstracts, 51,5747 (1957); Chem. Ber., 90, 264 (1957); Chem. Abstracts, 51, 12061 (1957). (175) A. S. Jones, A. M. Mian, and R. T. Walker, J. Chem. Soc. ( C ) , 2042 (1968). (176) R. J. Ferrier, Aduan. Carbohyd. Chem., 20,67 (1965);Aduan. Carbohyd. Chem. Biochem., 24, 199 (1969).

M. S. FEATHER AND J. F. HARRIS

204

of one acetoxyl group. On treatment with barium hydroxide, the product, 4,6-di-0-acetyl-2,3-dideoxy-~-e~ythro-hex-2-enose (di-0acetyl-pseudoglucal,” 88), undergoes further reaction to form two fully deacetylated products: “isoglucal” (91)and, in low yield, “protoglucal.” Compound 88 may be formed by (a)attack of a hydroxyl group on C-1, or ( b ) opening of the pyranose ring, with formation of the 2-deoxy-~-a~abino-hexose derivative (89) and elimination of the 3-acetoxyl group. The addition of barium hydroxide results in deacetylation, liberating 90, which readily rearranged7* to isoglucal (3,6-anhydro-2-deoxy-~-arabino-hexose, 91). Compound 90 would also be expected to undergo enolization, followed by elimination of the 5-hydroxyl group and rearrangement to 92. Compound 92 may be a precursor of “protoglucal,” the structure of which has not yet been ascertained. CH,OAc

OJL

AcO

HC=O I

7% AcOCH I

HCOAc

HC=O I

CH -

1

I1 CH

I

HCOAc I

CKOAc

HCOH CKOAc I

HCOH I

\

88

CH,CHO

HO

-

H C G

OH

HO

91

90

HC=O I CH

II CH I

CKOH 92

DEHYDRATION REACTIONS OF CARBOHYDRATES

205

The presence of the 2-hydroxyl group in the 2-hydroxyglycal series permits formation of adjacent carbonyl groups, and subsequent benzilic acid rearrangement. Thus, the mechanism of their alkaline degradation is analogous to that of aldoses and ketoses, but few members of the series have thus far been studied. Corbett and Kidd177 isolated tetrahydro-3-hydroxy-5-(hydroxymethyl)-3-furoic acid (93) after treating 2,3,4,6-tetra-O-acetyl- 1,5-anhydro-n-arabino- hex- 1enitol (2-acetoxy-tri-O-acetyl-~-glucal)with lime-water, and the same acid was formed, together with D-glUCOSe, on saponifying 2-acetoxyhexa-O-acetylcellobia1.178Formation of these products is readily explained by assuming that the 2-hydroxycellobial undergoes the following reaction sequence, the last step in which is a benzilic acid rearrangement.

p

OH

Q

HO

OH

OH

OH

C02H 93

(177)W.M.Corbett and J. Kidd,J. Chem. SOC., 1594 (1959). (178)W.M.Corbett and A. M. Liddle, J . Chem. SOC., 531 (1961).

M. S. FEATHER AND J. F. HARRIS

206

3. Glycuronic Acids

The mechanism of the aqueous alkaline degradation of uronic acids, of their lactones, and of their esters is unknown. The free acids isomerize readily; D-glucuronic acid was shown to isomerize more readily than the corresponding monosaccharide, D-glUCOSe, yielding considerable proportions of C-2 and C-3 e p i m e r ~ .Thus, ~ ~ , ~it~would be expected that alkaline degradation would be initiated from the reducing end, with the formation of products analogous to those from aldoses; some evidence for this supposition has been f o ~ n d . ' ' ~ Esterification, either internally (as in the lactones), or externally, activates the carboxyl group and opens the possibility of C-4 elimination and of decomposition from the nonreducing end. However, in aqueous solution, the hydrolysis of the ester bond appears to be much more rapid than the elimination reaction.lsO In nonaqueous medium (sodium methoxide in methanol), deesterification is replaced by transesterification that permits the C-4 elimination to proceed. Yields of 50% of methyl (methyl 4-deoxy-p-~th~eo-hex-4-enopyranosid)uronate have been obtained from methyl (methyl a-D-galactopyranosid)uronate.180*181 The same reaction has been studied by using other g l y c ~ r o n a t e s . ~ ~Evidence ~ - ~ ~ * for C-4 elimination where esterification is internal is found in the reactions

OH

0 HO

9 HOCH I

HCOH

/

J HC=O I

HCOH

c=o CH II HC CO,H

(179) R. L. Whistler and G. N. Richards, J . Amer. Chem. Soc., 80,4888 (1958). (180) H. W. H. Schmidt and H. Neukom, Carbohyd. Res., 10,361 (1969). (181) P. Heim and H. Neukom, Helv. Chim. Acta, 45, 1737 (1962);Chem. Abstr., 57, 12608 (1962). (182) J. Kiss and K. Noack, Carbohyd. Res., 10,328 (1969). (183) J. Kiss, Tetrahedron Lett., 1983 (1970). (184) J. Kiss and K.Noack, Carbohyd. Res., 16,245 (1971).

DEHYDRATION REACTIONS OF CARBOHYDRATES

207

of various lactones when treated with Fehling solution185and in the formation of the reductone, 4,5-dideoxy-~-glycer-hex-truns-4-enos-3uluronic acid, from a-D-glucofuranurono-6,3-lactone.'86

V. REDUCTIC ACID

Reductic acid (2,3-dihydroxy-2-cyclopenten-l-one, 75), a colorless,

&OH 15

0

\

0

75 b

odorless compound, is produced during the acid treatment of a number of carbohydrates. As it is carbocyclic, it is an unusual product. It can be purified by sublimation ( 150°/0.2torr) or by crystallizdtion from N,N-dimethylformamide (m.p. 211-212", AEZ 267 nm, E 13,300).53~94~110 It is a relatively strong acid; one hydrogen atom can be titrated in aqueous solution. The nuclear magnetic resonance spectrum187(DzO) shows only a singlet (T 7.50), indicating that the four methylene protons are equivalent and that the molecule probably exists, in its salts, as a resonance-stabilized, singly-charged anion (75b).Because of this, C-1 and C-3, as well as C-4 and C-5, are equivalent atoms. When treated with 5% sulfuric acid for 1.5 hours at 150°,pectin and D-galacturonic acid give reductic acid in yields of approximately 10%; it is also formed on treatment of alginic acid with a ~ i d ? ~ .The ll~ original preparation from pectin has been improved by using ionexchange techniques,"' and synthetic preparations have been developed.188 2-Furaldehyde has also been reported as a source of reductic acid,la9 and this has been verified.54The yields are, however, less than 0.5%. D-Xylose also affords small proportions of reductic a ~ i d ,but ~ ~the , ~ ~ yields are, apparently, much higher from the other pentoses when they are treated in concentrated sulfuric (185) F. Smith, Adoan. Carbohyd. Chem., 2,79 (1946). (186) M. Ishidate, M. Kimura, and M. Kawata, Chem. Pharm. Bull. (Tokyo), 11, 1083 (1963). (187) M. S. Feather and J. F. Harris, unpublished results. (188) G . Hesse, E. Bayer, and P. Thieme, Chem. Ber., 99, 1810 (1966). (189) Wessanen's Koninklijke Fabrieken N. V., Dutch Pat. 61,296 (1948); Chem. Abstr., 42,7788 (1948).

M. S. FEATHER AND J. F. HARRIS

208

AsoS6 first proposed 5-hydroxy-2-0~0-3-pentenal (94) as an intermediate in the conversion of uronic acids to reductic acid,190.191 but this proposal does not appear to have been experimentally tested, although the intermediate was prepared.Ig0 IsbelPZ1 suggested B mechanism in which the formation of reductic acid and 2-furaldehyde from pentoses and uronic acids results from the reaction of different tautomers of 94. Although other mechanisms have been suggested,100J02J15 Isbell's original scheme seems adequate to explain the experimental facts. HC=O

HC=O I

I

Hc'c=o HOH,C

HC ,c=o

I1 ,CH

I1

HC,

94a

g

ao;W

72a

O

CqOH 28

94b

OH

H

HO 75a

27

0 75

During the dehydration of pentoses in acidic solution, the cis and trans 2-enes (homologs of 44 and 46; see p. 177) react rapidly to form the 3-enes, 94a and 94b. Aneta has presented evidence for the existence of cis and trans forms of the 6-carbon 2-enes. The relative proportion of the geometrical isomers 94a and 94b is controlled by the ratio of the cis and trans forms of the reacting 2-enes and by the rate of interconversion through the tautomer, 72. The trans form (94a) is unable to cyclize, whereas the cis isomer (94b) could readily cyclize to $8 and this could be dehydrated to 2-furaldehyde (27). Formed by extended enolization of 94, the diene 72 has the electronic arrangement needed for cyclization to afford 75a, a tautomer of re(190)K. Aso, H. Sugisawa, and T. Iso, N i p p o n Nogei Kagaku Kaishi, 29, 985 (1955); Chem. Abstr., 51,8658 (1957). (191)K. Aso and H. Sugisawa, N l p p o n Nogel Kagaku Kaishi, 30, 387 (1956);Chem. Abstr., 52,9963 (1958).

DEHYDRATION REACTIONS OF CARBOHYDRATES

209

ductic acid (75).The geometrical isomer shown of 72 is one of four possible structures) of which only two could cyclize to 75a. Some stereoisomeric forms of 72 (not 72a) can undergo a heterocyclic reaction to form 2-furaldehyde. The ratio of the products is determined by (a) the kinetics of the reactions shown and ( b )the stereochemistry of the original sugar that determines the proportions of 94a and 94b generated in situ. This conclusion may explain the observed differences in behavior of the pentoses in giving different proportions of 2-furaldehyde in aqueous acidic medium. In some instances, the yields of 2-furaldehyde from D-XylOSe approach the value theoretically possible, whereas reductic acid is found in only low yield under all conditions. However, L-arabinose, D-lyXOSe, and D-ribose give much lower yields of 2-furaldehyde, and, when treated in concentrated sulfuric acid, give high yields of a compound that is, apparently) reductic a ~ i d . ~The ~ , ’distribution ~~ of product is greatly dependent on the concentration of the acid; in dilute, aqueous acid, only minor proportions of reductic acid can be detected.I’O The order of reactivity of the pentoses (ribo > xylo > lyxo > arabino)IS3differs from the order of the yield of reductic acid (lyxo > rib0 > arabino > xylo),52indicative of little, if any, relationship. The conformations of the acyclic, acetylated pentose dimethyl acetals have been determined,lg4 and a parallel was noted in the relative tendency (ribo = xylo > lyxo > arabino) for the chains to adopt bent-chain (“sickle”) conformations that could facilitate ring closure. Obviously) a similar mechanism can be presented for the formation of carbocyclic compounds from the hexoses, but less is known about the products and the variation in their distribution within the group. Most of the work with the hexoses has been conducted with D-glucose and D-fructose. It is known that D-mannose and D-galactose give significantly lower yields of 5-(hydroxymethyl)-2-furaldehydethan either D-glucose or D-fructose when treated in concentrated sulfuric but no homolog of 75 has been reported. The formation of reductic acid and 2-hraldehyde from uronic acids is believed to occur through the same intermediate (72a)that is generated on decarboxylation of 3,4-dideoxy-~-gZycero-hex-3-enosuluronic acid (71)(see Section 111,3; p. 191). However) little is (192) R. M. Love, Biochem. J., 55, 126 (1953). (193) E. R. Garrett and D. H. Dvorchik, J. Pharm. Sci., 58,813 (1969). (194) J. Defaye, D. Gagnaire, D. Horton, and M. Muesser, Carbohyd. Res., 21, 407 (1972).

M. S. FEATHER AND J. F. HARRIS

210

known about the dependence of the yield of product on the structure of the hexuronic acid. In the carbazole reaction, the yield of chromophore differs widely with the particular acid under test, and is greatly affected by the addition of complexing ions.lo5 Tracer studies have only partially confirmed the mechanisms proposed. Reductic-14Cacid having 90% of the label at C-2 and 10% at C-1 (or C-3, as these are equivalent atoms) was obtained from D-gala~turonic-l-~~C acid, whereas, from D-gala~turonic-2-'~Cacid treated under the same conditions, the distribution in the product was foundl10to be 10% at C-2 and 90% at C-1. No activity was found at the methylene carbon atoms. The major portion of the radioactivity resides where it would be anticipated, but the significant proportion of activity at a second carbon atom is inconsistent with any mechanism thus far proposed. This conclusion suggests that mechanisms other than those suggested are operative, or that a scrambling of the carbon atoms in 75 may occur after it has been formed. Scrambling seems a likely possibility, as 75 could add and eliminate hydroxyl groups by the mechanism illustrated.

0

0

HO

7s

A similar result was found with D-Xy1OSe-lJ4C and 2-furaldehydem-14C.When treated under identical conditions, each formed reductic14Cacid having 60% of the activity at C-2 and 40% at C-1. Theanderlgs reported that treatment both of methyl p-~-arabinohexopyranosidulose and its related 3-ulose isomer (95a) with 0.25 M sulfuric acid for 2 hours at 100" gives large yields of reductic acid and (195) C. A. Knutson and A. Jeanes, Anal. Biochem., 24,470 (1967). (196) 0. Theander, Acta Chem. Scand., 12, 1897 (1958).

21 1

DEHYDRATION REACTIONS OF CARBOHYDRATES

small proportions of 2-furaldehyde and D-erythro-pentulose. An unidentified compound was also formed in substantial yields. Similar treatment of p-D-urubino-hexosulose (“D-ghcosone”) was found to yield no reductic acid. The following mechanism is suggested to explain these results. The conversion of 95b to 96 with elimination of the alkoxyl radical would occur more readily than the usual hydrolysis by way of a carbonium ion. Analogous to the reaction 9 .--* 9a, 96 would be rapidly transformed into 97, instead of undergoing the heterocyclic reaction feasible between C-3 and C-6. The deformylation of 99 is made possible by the absence of a proton adjacent to the carbonyl group, and could only take place after ring closure. CH,OH

CH,OH

HF=O

COH It

FH

95b

95a

96

I

OH

0

HO

GH@

XoH75

99

-@%Ha

HC 0 H II T-2COH

HC=O I

HCOH I

CH

c=o CH c=o III

CH,

CHZ

98

’

917

The absence of reductic acid from the reaction of D-arubino-hexosulose (100) would indicate that the initial step in the reaction is the elimination of the hydroxyl group at C-4 (illustrated) or C-1,or dehydration through the acyclic form. The compounds resulting from

100

212

M. S. FEATHER AND J. F. HARRIS

such reactions would not lead to reductic acid. Similar reactions can also occur with $ 6 3 ; the hydroxyl group at C-2 or C-4 could be removed, or the methoxyl group could be eliminated by formation of the 2,3-enediol, instead of the 3,4-enediol, as illustrated. One of these competitive reactions probably accounts for the unidentified products, but, evidently, the 1-alkoxyl group is the most readily removed, as reductic acid was obtained in 22% yield from 95a. The occurrence of reductic acid among the acidic degradation products of oxidized cellulose1g7can, perhaps, be explained by the presence of carbonyl groups at C-2 and C-3 in the polymer.

VI. LEWLINICACID When heated in aqueous acidic medium, D-ghcose, 2-deoxy-Derythro-pentose, furfuryl alcohol, 5-(hydroxymethyl)-2-furaldehyde, and five-carbon glycals yield levulinic acid. Because of the importance of developing tests for 2-deoxy-~-erythro-pentose,the reactions have been extensively studied, and several mechanisms have been s ~ g g e s t e d . ~ ~For ~ ~ *converting -~~* furfuryl alcohol (101) into levulinic

(197) W. A. Sohn, Angew. Chem., 60,284 (1948). (198) Ref. 11, p. 645. (199) P. Bosshard and C. H. Eugster, Aduun. Heterocycl. Chem., 7,377 (1966). (200) N. Clauson-Kass and J. T. Nielsen, Actu Chem. Scund., 9, 475 (1955). (201) N. Clauson-Kass, Actu Chem. Scund., 6, 556 (1952). (202) K. G . Lewis, J. Chem. SOC.,531 (1957); 4690 (1961). (203) L. Birkofer and R. Dutz, Ann., 608, 7 (1957); Chem. Abstr., 52, 6307 (1958). (204) L. Birkofer and F. Beckmann, Ann., 620, 21 (1959); Chem. Abstr., 53, 20028 (1959). (205) L. Birkofer and R. Dutz, Ann., 657, 94 j1962); Chem. Abstr., 58, 1340 (1963).

213

DEHYDRATION REACTIONS OF CARBOHYDRATES

acid, Dun10p'~~ proposed the following mechanism, which assumes the formation of 103, an intermediate first suggested by Leger and Hibbert.208Compound 103 satisfactorily explains the formation of levulinic acid from 2-deoxy-~-e~ythro-pentose, 5-(hydroxymethy1)2-furaldehyde, furfuryl alcohol, and similar compounds, and accounts for the products from furfurylidene derivatives and vinylfurans.198~1ss.208 Compound 103 has never been isolated, and there is no direct experimental evidence for its existence. However, it is a logical precursor €or 2,3-dihydro-5-methyl-2,3-furandiol (107) and 4-hydroxy-3pentenal (a-hydroxy-levulinaldehyde,108), both of which have been isolated from the reaction of furfuryl

r-

HO 103

(R = H)

H+OH

HC=O

H$OH HC=O

108

108a

107

Supporting evidence for the presence of the equilibrium 101 102 is found in the work of G6mez Shnchez and R ~ l d h n who , ~ ~found ~~ that the dehydration of both ethyl and methyl 2-methyl-5-(D-UTUhinotetrahydroxybutyl)-3-furoate mainly proceeds with inversion at C-l', to afford 5-(1,4-anhydro-~-~ibo-tetrahydroxybutyl)-2-methyl-3furoate. The thermodynamically less stable 1,4-anhydro-~-urubino derivative, in which the configuration is retained, was also produced in low yield. It was further shown that the anhydro derivatives are interconvertible, that is, that the dehydration is a reversible process, although the D-ribo configuration always preponderates. Furfuryl alcohol is probably, but not necessarily, an intermediate in the formation of 103 from 2-deoxypentoses7which could also be formed from acyclic precursors via 109 as shown. Evidence for this sequence is found in the hydrolysis of 2-ethoxy-4,5-dihydro-W-

(206) R. Pummerer and W. Gump, Ber., 56,999 (1923);R. Pummerer, 0. Guyot, and L. Birkofer, ibid., 68,480 (1935). (207) H. P. Teunissen, Rec. Trav. Chim., 49, 784 (1930); 50, 1 (1931). (208) F. Leger and H. Hibbert, Can. J. Res., 16B,68 (1938). (209) R. LukeS and J. Srogl, Collect. Czech. Chem. Commun., 26, 2238 (1961). (209a) A. Gdrnez SLnchez and A. R. RoldLn, Carbohyd. Res., 22,53 (1972).

M. S, FEATHER AND J. F. HARRIS

214

HC=O I

7% HCOH I

HCOH

HC=O I CH It CH I HCOH

HCOH

- I

I

C&OH

CbOH

" Z v -

II

CH I CH I1 COH I C%OH

1

HC=O

CH

II CH I COH II

103

CH,

(R = H)

109

pyran,210which readily gives a 55% yield of 2,4-pentadienal, with no trace of the expected 5-hydroxy-2-pentenal. The absence of a 4-hydroxyl group from the supposed intermediate, 5-hydroxy-2-pentenal, precludes the formation of a 2,5-dihydrofuran, and the 5-hydroxyl group must have been removed by extended enolization followed by p-elimination. A similar reaction is indicated for the formation, from D-glUCOSe, of 110; this is more likely to be formed by further dehydration of the hex-Senosulose than by way of 5-(hydroxymethyl)-2-furaldehyde, as proposed.211The acyclic, carbonyl HC=O

HC=O

c=o

COH

I

I CH II CH I HCOH I C%OH

I

II

-

1

~

CH CH II

COH

HC=O

- tH I I

c=o

CH I COH

&&OH

HC=O I

c=o

I CH It CH

I

c=o I CH,

103

(210) G. F. Woods and H. Sanders, J. Amer. Chem. SOC., 69,2926 (1947). (211) H. Hoermann and T. L. Nagabhushan, Z. Physiol. Chem., 335,283 (1964).

DEHYDRATION REACTIONS OF CARBOHYDRATES

215

compounds 109 and 110 could form carbocyclic products, but such products have not been isolated. The degradation of furfuryl alcohol in acidified methanol has been more extensively studied than the aqueous reaction. The following compounds, in addition to the levulinic ester and several dimers and

7% c=o I

CH II CH

H&(OMe),

7% c=o I 7% CH(0Me)

7H3

7% 7(OMe),

7% 7% CqMe

7% 7% CqMe

c=o I

HA (OMe),

Med

Med

trimers, have been isolated from the anhydrous medium. These are the products to be expected from the mechanism outlined. The acyclic compounds formed by ring opening of structures analogous to 103, 105, and 107 are stabilized by formation of the acetals. The absence of the methyl ester (111) of 2-methoxylevulinic acid should be par-

y-4

c=o

yI z

CH(0Me) I

C0,Me I11

ticularly noted. Birkofer and Dutz203isolated a compound that was not characterized, but was assumed to be 111. However, it is obvious that 111 could not originate through the suggested mechanism, as its formation would involve an intermolecular, oxidation mechanism. In the absence of alkoxyl radicals and the formation of acetals, the aldehyde from the ring opening of 107 undergoes further reaction, to form furanones. Seydel and isolated a chromophore ,A( 261 nm) from the reaction of 2-deoxy-D-e~ythro-pentose in (212) J. Seydel, E. R. Garrett, W. Diller, and K. J. Schaper,J. Phamn. Sci., 56,858 (1967).

M. S. FEATHER AND J. F. HARRIS

216

aqueous acid, and identified it as 5-methyl-3(2H)-furanone (112).The formation can readily be explained by the presence of 107 and the reaction sequence indicated; it is unlikely that 107 is dehydrated without ring opening.

HopCHs HC=O

HCOH I1 COH

HAOH I COH CH II

HO

&I

COH I1 I CHS

I CH, 107

i

108a

0

HO 112

As noted, furfuryl alcohol is not a necessary precursor for 107, but it was found that the alcohol gives greater yields of 112 than are obtained from the sugar. Treated in the same way, ribitol shows no significant absorption. For both 2-deoxy-~-erythro-pentoseand furfury1 alcohol, a transient absorbance at 225 nm was noted that was tentatively ascribed to the formation of 103 (R = H). The cyclic intermediate 103 undergoes the competitive reactions 103 + 105 (via 104), 103 + 107, and ring opening to afford an unsaturated dicarbonyl compound. The ring-opening reaction appears to be minor, whereas the conversions to give 105 and 107 are both of significance. In the reaction of 2-deoxy-~-erythro-pentosedescribed,212 the respective yields of 112 and levulinic acid were 15% and 50% in 1.0 M hydrochloric acid during 10 hours at BOO, whereas Clauson-Kass and Nielsen200*201 reported 10% yields of products derived from 107, and a 28% yield of methyl levulinate under similar conditions in methanolic hydrochloric acid. In refluxing, 0.5% methanolic hydrochloric acid, tri-0-acetyl-D-gluca1gives high yields213 of 113, whereas brief treatment214of tri-O-acetyl-D-ghca1 or 2 4 ~ glycero-1,2-dihydroxyethyl)furan (114) with 25 mM ethanolic sulfuric acid at 70"resulted in the rapid formation of the racemic 1'-ether (213) M. Bergmann and H. Machemer, Ber., 66, 1063 (1933). (214) D. Horton and T. Tsuchiya, Cnrbohyd. Res., 3, 257 (1966-1967); 5, 426 (1967).

DEHYDRATION REACTIONS OF CARBOHYDRATES

217

C&OH I

7% c=o I

Yh ?It.

COaMe 113

115, which reacted further, at a much lower rate, to form 116. The diethyl ether (116) was, in turn, converted into 2,6-diethoxy-4oxohexanal diethyl acetal (117). The same conditions converted 117 OH

114

CH(OEt)C&OH

115

H

I E t y OEt CHOEt I

CH(0Et)CqOEt

?Ha

c=o

p a

116

CHaOEt 117

back into 116. The ethyl ester analog of 113 was not observed; it is probable that conditions were too mild for its formation. From the reaction of D-ribose-l-14Cwith secondary amine salts in aqueous acid, Peer and van den Ouweland21gisolated 4-hydroxy-5methyl-3(2H)-furanone (120) (11.4%), and found it to be labeled entirely at the methyl carbon atom. Thus, in the presence of amines, the formation of 120 must proceed through the 1-deoxydiulose by the mechanism described in Section I1 (see p. 168). In contrast, the reaction of D-ribose-l-14C 5-phosphate2l6 gave 120 having no radioactivity in the methyl carbon atom, from which it was concluded that the methyl carbon atom originates from C-5 of the D-ribose. The (215) H. G. Peer and G. A. M. van den Ouweland, Rec. Truu. Chim.,87, 1011 (1968). (216) H. G. Peer and G . A. M. van den Ouweland, Rec. Trav. Chim., 87, 1017 (1968).

M. S. FEATHER AND J. F. HARRIS

218 HC=O I HCOH HCOH I I HCOH I H$-0PO3“

- -“‘p c=o I c=o

-2 K O

S)PO3“

CH I

pp034

------b&‘Q

II CH I “C-OPO,Hp

OH

0 119

118

H

3

C

HO

9

0

KCQ

HO

-

“CQ

OH

HO

0

120

substitution at C-5 prevents C-2-C-5 ring-closure and promotes the formation of the furanone (119) (see Section 11; p. 173) that is converted into 120 by addition of a hydroxyl group to C-4. Addition at C-2 of 119, which would result in 2-hydroxy-levulinic acid, is prevented, because the keto tautomer is greatly favored.z17The acyclic compound 118 is also capable of forming a carbocyclic product, but this has not been reported. AneP4 found that the methyl analog of 119, namely, 5-methyl-3(2H)-furanone, is very unstable, and he reported the formation of polymers therefrom in nonaqueous solvents containing a trace of an acid. VII. ANALYSESINVOLVINGDEHYDRATION REACTIONS

It is probable that, for sugars, almost all analytical procedures that involve treatment with concentrated acid are dependent on a conversion into a furan derivative and that, in most, there occurs a reaction of the derivative with a chromogen capable of producing an absorption in the visible range. Although numerous analyses can be so classified, only a relative few have been carefully studied for the dehydration product formed and for the nature of the chromophore; these are discussed in the following Section. For a more general survey, the reader is referred to articles by Binkley and othersz1*and Stangk and coworker^.^'^ (217) A. Hofman, W. V. Philipsborn, and C. H. Eugster, Helv. Chirn. Acta, 48, 1322 (1965). (218) D. h i n o f f , W. W. Binkley, R. SchaEer, and R. W. Mowry, in “The Carbohydrates,” W. Pigman and D. Horton, eds., Academic Press, Inc., New York, N. Y., 2nd Edition, 1970, Vol. IIB, p. 739. (219) J. StanBk, M. cemy, J. Kocourek, and J. PacAk, “The Monosaccharides,” Academic Press, Inc., New York, N. Y., 1963, p. 884.

DEHYDRATION REACTIONS OF CARBOHYDRATES

219

1. Reactions in Concentrated Acid Solution

Treatment of sugars with concentrated (75-90%) sulfuric acid solution is the basis for a number of procedures for sugar analyses. Usually, the expected 2-furaldehydes are produced (sometimes, with other, unidentified, ultraviolet-absorbing compounds) in sufficient yields to result in molar absorptivities, based on the starting sugar, in the order of 8 to 20 x lo3.In most cases, the 2-furaldehydes from various classes of sugars do not have maxima sufficiently different to allow one sugar to be determined in the presence of another, but 2-ketoses can often be detected in the presence of the aldoses, because of their much higher reaction rate.78 The results of Ikawa and Niemann,78 who used 79% sulfuric acid for 15 minutes at loo", of Bath,2z0who used 90% sulfuric acid for 5 minutes at loo", and otherszz1indicated that, when so treated, hexoses, 6-deoxyhexoses, and D-xylose give rise to the appropriate 2-furaldehyde as the principal ultraviolet-absorbing product, but that other pentoses, as well as hexuronic acids, afford mixtures of compounds in which 2-furaldehyde is the major, but not the sole, product. Scott and coworkers5zhave investigated some of the parameters of the dehydration reaction in concentrated sulfuric acid solution, in order to use it as an analytical method. The concentration of the sulfuric acid was found to be the most important variable in the reaction; it influences the position and the intensity of the ultravioletabsorption maximum, the stabilities of the reaction products, and, for certain pentoses and hexuronic acids, the course of the reaction. In general, an increase in the concentration of the sulfuric acid causes a shift of the 2-furaldehyde absorption to higher wavelength, and an increase in the molar absorptivity. In the 78-92% range of concentration of sulfuric acid, the molar absorptivity of 5-(hydroxymethyl)-2-furaldehyde increases at an average rate of 4% per 1%change in the concentration of the acid; the wavelength of the absorption maximum changes from 290 to 320 nm. D-Xylose was found to yield 2-hraldehyde almost exclusively, but D-lyXOSe, D-ribose, and L-arabinose produce another, as yet unidentified, compound absorbing at 289 nm, which is the maximum absorption wavelength for reductic acid. D-Glucose, D-fructose, and sucrose give almost identical yields (- 85%) of 5-(hydroxymethyl)-2-furaldehyde, but D-galactose and D-mannose give much lower yields thereof. (220) I. H. Bath, Andyst, 83, 451 (1958). (221) T. Yamazaki and F. Goto, N i p p o n Nogei Kuguku Kuishi, 39,465 (1965).

220

M. S. FEATHER AND J. F. HARRIS

In concentrated sulfuric acid, D-glucuronic acid is dehydrated more slowly than either its 4-0-methyl derivative or D-glUCOSe, probably because of the stability of its lactone. All hexuronic acids, however, eventually produce the same characteristic absorbance that corresponds roughly to a composite of 5-formyl-2-hroic acid, 2-furaldehyde7 and reductic acid. It is interesting that a thin-layer chromatographic examination of the reaction products of Dglucuronic acid with 89% sulfuric acid at 70" revealed 5-formyl-2-furoic acid as a major product, but no evidence was obtained for the presence of reductic acid. This result is in distinct contrast to the products obtained in dilute acid solutions, in which 5-fomyl-2-furoic acid is produced in very low yield, whereas reductic acid is formed in yields in excess of 10%. This method cannot be recommended for analyses of 2-deoxyD-erythro-pentose; this compound does not give rise to 2-furaldehyde7 and it gives ultraviolet-absorbing compounds having very low molar a b s o r p t i ~ i t yHowever, .~~ a method has been deviseds3for determining 2-deoxyhexoses by treatment with 4 M hydrochloric acid for 60 minutes at 80"; the product of the reaction shows a maximum absorption at 218 nm. It is interesting that this value is identical with that reported for the maximum absorption wavelength for 2-(D-ghjcero1,2-dihydroxyethyl)hran (60; see p. 1831, which has been isolated from D-glucal and from methyl 4,5-0-benzylidene-2,3-dideoxy-a-~erythro-hex-2-enopyranoside by Horton and T s ~ c h i y a . ~The ~*~~~ formation of this compound, produced in yields of -25% from both 2-deoxy-~-arubino-hexose and 2-deoxy-D-lyxo-hexose, is explained in Section I11 (see p. 183). The use of the reaction in concentrated sulfuric acid for the determination of 2-amino-2-deoxy sugars is controversial. Although Ikawa and N i e r n a n ~ ~ claimed '~ that no 2-furaldehyde (and little other ultraviolet-absorbing material) is produced from 2-amino-2-deoxy-~glucose, Cipera'@stated that treatment of both 2-amino-2-deoxy-~-glucose and 2-amino-2-deoxy-~-ga1actosewith 91% sulfuric acid for 10 minutes at 100" gives a compound having the ultraviolet-absorption characteristics of a 2-furaldehyde. From the data presented, the yields of the 2-furaldehyde were of the order of 30%. Before amino sugars can be satisfactorily analyzed by this method, it seems obvious that further investigations are mandatory. 2. Color Reactions

a. The Anthrone Test.-The usual procedure for this analysis involves mixing of 1 ml of water containing 1 mg of the sugar with 2

-

DEHYDRATION REACTIONS OF CARBOHYDRATES

221

ml of concentrated sulfuric acid containing 0.2% of anthrone (9,lOdihydro-9-oxoanthracene).zzzUnder these conditions, the heat of dilution generates a temperature sufficiently high to complete the reaction. There results a blue-green solution having a maximal absorption at 625 nm, the intensity of which can be correlated with the amount of original carbohydrate in the solution. The procedure is usually used for determining hexoses, pentoses, hexuronic acids, heptoses, and certain deoxy sugars, and hexose mono- and di-phosphatesZz3after they have been separated from one another. Because, on treatment with the anthrone hexoses and 5-(hydroxymethyl)-2-furaldehyde give solutions having identical spectral characteristics, dehydration is indicated to be the important reaction in this analysis. This conclusion is further supported by the reported isolationzz6 of lO-furfurylidene-9,lO-dihydro-9-oxoanthracene (121) after reaction of 2-furaldehyde with anthrone, and by the fact that 121 has an absorption maximum of 600 nm, a value close to that used for pentose estimations. In similar 9,lO-dihydro10-(5-methylfurfurylidene)-9-oxoanthracene(122) was reported to have been isolated after the reaction of either L-rhamnose or 5-methyl-

121

122

2-furaldehyde with the anthrone reagents. Other, more-complex compounds that give rise to violet or red-brown colors have also been reported to be formed by the reaction of pentoses or 2-furaldehyde with anthrone. It therefore seems likely that the color measured in the anthrone test is due to a number of compounds whose spectra overlap one another. A study by Hoermann and SiddiguiZ2*of the reaction of D-fructose, (222) R. Dreywood, Ind. Eng. Chem., Anal. Ed., 18, 489 (1946). (223) L. C. Mokrasch, J. Biol. Chem., 208, 55 (1954). (224) L. Sattler and F. W. Zerban, Science, 108,207 (1948). (225) L. Sattler and F. W. Zerban, J. Amer. Chem. SOC., 72,3814 (1950). (226) R. Sawamura and T. Koyama, Yakugaku Zasshi, 84,82 (1965);Chem. Absh., 61, 1818 (1965). (227) T. Koyama and R. Sawamura, Chem. Pharm. Bull. (Tokyo), 14, 1054 (1966). (228) H. Hoermann and I. A. Siddigui, Ann., 714, 174 (1968).

222

M. S. FEATHER AND J. F. HARRIS

D-glucose, and 5-(hydroxymethyl)-2-furaldehyde with anthrone lends support to these conclusions, and further indicates the complexity of the overall reaction. In the reaction of either D-frUCtOSe or 5-(hydroxymethyl)-2-furaldehyde with anthrone, at least nine compounds were observed, three of which were condensation products of anthrone itself. The other products had absorption maxima ranging from 490 to 770 nm (in sulfuric acid solution and under the conditions of the anthrone reaction). One of the prominent pigments, having a blue 620 nm) and a postulated structure corresponding to color (A,,, compound 123, was isolated and characterized by its nuclear magnetic

I23

resonance spectrum and mass spectrum. Other compounds, such as 124 (A,,, 496 and 770 nm), were also isolated and characterized;

124

they probably likewise contribute to the overall color observed. As the ultraviolet spectrum of compound 123, either in benzene solution or in sulfuric acid solution, has a maximal absorption identical with that observed when D-glucose, D-fructose, or 5-(hydroxymethyl)-2furaldehyde is treated with anthrone, it appears likely that 123 is the major contributor to the color responsible for the anthrone reaction.

b. The Carbazole Test. -This analysis22e involves heating the carbohydrate with carbazole in 80-90% sulfuric acid for 15 minutes at 100". Evidence that the reaction involves the formation of 2-furaldehyde is indirect, and based largely on spectral data. Under condi(229) S. Gurin and D. B. Hood, J . Biol. Chem., 139, 775 (1941), and references cited therein.

DEHYDRATION REACTIONS OF CARBOHYDRATES

223

tions for the reaction, the shapes and the maxima of the ultraviolet spectral curves given by reducing sugars or by 2-furaldehyde are similar.230It has also been shown that 2-amino-2-deoxy-D-glucosedoes not react.230Of interest are reports that the absorption maximum is dependent on the concentration of the sulfuric acid, and, in 84% sulfuric acid, the absorption maxima is in the range of 540-550 nm for all sugars studied.230Thus, as when concentrated acid alone is used, it is not possible, when using this method, to differentiate clearly between the various aldoses. c. The Phenol-Sulfuric Acid Reaction. -This methodZ3l involves the reaction of a carbohydrate with 80% aqueous phenol and concentrated sulfuric acid at an elevated temperature attained by the heat of dilution of the acid, followed by keeping for 30 minutes at 25-30". The resulting solution is yellow-orange, and measurements are made at 490 nm for hexoses and at 480 nm for pentoses and hexuronic acids. In these instances, the spectra given by the sugars are identical with, and superposable on, that of the appropriate substituted 2-furaldehyde. It is also of interest that 2-deoxy-~-erythrop e n t o ~ e as , ~expected, ~~ does not react; and the absorption observed for D-XylOSe is much higher than that for other pentoses and for hexuronic acids; these results are consistent with findings reported by Scott and coworkers52 for reactions in concentrated acid alone, and suggest that a substituted 2-furaldehyde is involved in the reaction with phenol.

d. The Orcinol Reaction. -This analysis involves reaction between a sugar, orcinol, and 7:3 (vlv) sulfuric acid-water at 80" for 20 minu t e ~ . ~ All~ reducing ~ - ~ ~ sugars ~ studied show absorption spectra having a maximum in the region of 400-450 nm, and, as expected, D-gluconic acid, alditols, and 2-amino-2-deoxy-D-g~ucosedo not reBy conducting the reaction under milder conditions, D-glUCOSe reacts, and L-ascorbic acid, a compound readily converted into Z-furaldehyde, also reacts; this constitutes further supportive evidence that the fundamental reaction of this analysis is one of dehydrati0n.2~~ (230) G. Holzman, R. V. MacAllister, and C. Niemann, J. B i d . Chern., 171, 27 (1947). (231) M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith, Anal. Chern., 28, 350 (1956). (232) Z. Dische and K. Schwartz, Mikrochim. Actu, 2, 13 (1937). (233) W. R. Fernell and H. K. King, Analyst, 78, 80 (1953). (234) J. Bruckner, Biochem. J.. 60,200 (1955). (235) E. Vasseur, Actu Chem. Scund., 2, 693 (1948). (236) V. E. Levine and S. Merlis, Bull. Creighton Univ. School Med., 4, 14 (1947).

224

M. S. FEATHER AND J. F. HARRIS

e. The Dische Test for 2-Deoxy-D-erythro-pentose.- 2-Deoxy-Derythro-pentose does not undergo the dehydration reactions typical of the fully oxygenated pentoses; but it does suffer a type of dehydration reaction that provides the basis for the Dische test.237‘The test involves the reaction of a sugar with acetic acid, concentrated sulfuric acid, and d i p h e n ~ l a m i n e A . ~positive ~~ test for 2-deoxypentoses consists in an intense, blue coloration. The blue color is not observed when ketoses, aldoses, 2-furaldehyde7and many other aldehydes are used. 2’-Deoxyribonucleic acid, 2-deoxy-~(andL)-erythro-pentose, Larabinal, and furfuryl alcohol give a positive test, but levulinic acid does The reaction mechanism relating to this analysis is discussed in Section VI (see p. 213). Among the intermediates formed during the dehydration of 2-deoxypentoses and C, glycals, the intermediate apparently responsible for chromogen formation is a-hydroxylevulinaldehyde (108). This compound had been erroneously characterized as o-hydroxylevulinaldehyde,an error that still persists.

(237) Z. Dische, Mikrochernie, 8 , 4 (1930).

DEOXYHALOGENO SUGARS BY WALTERA. SZAREK Department of Chemistry, Queen's Uniuersity, Kingston, Ontario, Canada

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. S y n t h e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Displacement Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Direct Replacement of Hydroxyl Groups . . . . . . . . . . . . . . . . . . . . 3. Additions to Unsaturated Carbohydrates. . . . . . . . . . . . . . . . . . . . . 4. Reaction of 0-Benzylidene Sugars with N-Bromosuccinimide . . . . . . 5. Miscellaneous Methods, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Reactions and Synthetic Utility. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Displacement Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Elimination Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Reductive Dehalogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Miscellaneous Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

225 227 227 230 260 . 276 278 281 281 290 299 304

I. INTRODUCTION A Chapter in an earlier Volume of this Series' surveyed the chemistry and biochemistry of monosaccharides monohalogenated at carbon atoms other than the anomeric carbon atom. In the succeeding years, appreciable developments have occurred in the understanding of the chemical properties of this long-known class of carbohydrate derivatives and of their value in synthetic chemistry. For several years, there has been considerable interest in deoxyhalogeno sugars, not only because of their potential intrinsic value in biochemistry and pharmacology, but also because of their utility in the synthesis of other rare sugars, such as deoxy and aminodeoxy sugars. The search for new methods of synthesis of halogenated carbohydrates therefore continues to be an active area of investigation in several laboratories. A further stimulus has been provided by the discovery that halogenation of the carbohydrate moieties of some antibiotic substances affords products having activities superior to those of the parent compounds and having improved chemotherapeutic properties. Thus, for example, treatment of linconiycin (l),an important antibiotic compound containing an aminodideoxy-octose residue, with thionyl (1) J. E. G. Bamett, Aduan. Carbohyd. Chem., 22, 177 (1967). 225

WALTER A. SZAREK

226

chloride in carbon tetrachloride,2 or, more satisfactorily, with triphenylphosphine dichloride or triphenylphosphine-carbon tetrachloride,3 resulted in replacement of the 7-hydroxyl group of the carbohydrate moiety by chlorine, to give a significantly more active antibiotic, namely, clindamycin (2). Also, a chlorinated kanamycin derivative has been found to possess strong inhibitory activity against several kinds of bacteria? The first naturally occurring derivative of a fluoro sugar is the nucleoside antibiotic nucleocidin [4’-fluoro-5’-0sulfamoyladenosinel (3); a synthesis of this compound has now been

HO 1

I OH R = OH; R‘= H

2

R = H ; R’= C1

OH

3

achieved5 (see Section 11,3b; p. 267). There is much interest in the synthesis of halogeno sugar nucleosides; these compounds are valuable synthetic intermediates, and, as with nucleocidin and other examples,6 they may themselves be of biological importance, In addition to the previous Chapter in this Series,’ two other rev i e w ~ have ~ . ~ since outlined the various methods for the preparation of deoxyhalogeno sugars. In the present Chapter, an attempt has been made to collate new information on the synthesis and chemistry of (2) R. D. Birkenmeyer, B. J. Magerlein, and F. Kagan, Abstr. 5th Zntersci. Con$ Antimicrob. Ag. Chemother., 17 (1965). (3) R. D. Birkenmeyer and F. Kagan, J. Med. Chem., 13,616 (1970). (4)T. Tsuchiya and S. Umezawa, Bull. Chem. SOC. lap., 38, 1181 (1965). (5)I. D.Jenkins, J. P. H. Verheyden, and J. G . MoffattJ. Amer. Chem. SOC., 93,4323

(1971). (6)W. Jahn,Arch. Exp. Pathol. Pharmakol., 251,95 (1965);E.T. Reese, L. B. Townsend, and F. W. Parrish, Arch. Biochem. Biophys., 125,175(1968);P.Langen and G . Kowollik, Eur. J . Biochem., 6, 344 (1968);R. Duschinsky, H.Walker, and J. Kara, Abstr. Popers Amer. Chem. Soc. Meeting, 158,MEDI 68 (1969). (7)S. Hanessian, Advan. Chem. Ser., 74, 159 (1968). (8)P.W. Kent, Chem. Znd. (London), 1128 (1969).

DEOXYHALOGENO SUGARS

227

deoxyhalogeno sugars other than glycosyl halides and deoxyfluoro sugars, and to discuss in detail the mechanistic and conformational aspects of some selected methods and reactions considered to be of most widespread interest and utility. The value of deoxyhalogeno sugars as intermediates in the synthesis of rare sugars of biological importance has also been featured. 11. SYNTHESIS 1. Displacement Reactions Nucleophilic-displacement reactions constitute some of the earliest known methods for the preparation of deoxyhalogeno sugars. A variety of leaving groups have been used, including sulfonyloxy (usually p-tolylsulfonyloxy or methylsulfonyloxy), halide, triphenylmethoxy (trityloxy), acetoxy, phosphate, and nitrate. As Barnett has surveyed the reactions in Volume 22 of this Series,' this subject will not be discussed comprehensively in the present Chapter; instead, only some general comments will mainly be made that will also be relevant to later discussions. Most, by far, of the displacement reactions that have been used for the preparation of deoxyhalogeno sugars have involved sulfonic esters. The chemistry of sulfonic esters of carbohydrates was discussed extensively in 1953 by Tipson,Yand, more recently, by Ball and Parrish'O; the specific utility of these esters for the synthesis of deoxyhalogeno sugars has been reviewed by Hanessian.' The replacement of a sulfonyloxy group by a halogen (iodine) was first applied" to a sugar derivative in 1927. Although a large number of such reactions have since been reported, there is still a paucity of quantitative, kinetic data. Nevertheless, some generalizations can be made. The reactions proceed the most readily when the sulfonate group is on a primary hydroxyl group, except when the sulfonyloxymethyl group is adjacent to either ( a ) a cis-axial substituent on a sixmembered ring, as in the 6-sulfonates of galactopyranose derivatives, or ( b ) the anomeric center, as in 2-ketose 1-sulfonates. The resistance (to replacement reactions) of the 6-sulfonates of galactopyranose derivatives, in comparison with the corresponding glucopyranoses, can be rationalized in terms of the greater steric crowding in the (9) R. S. Tipson, Adoan. Carbohyd. Chem., 8, 107 (1953). (10) D. H . Ball and F. W. Parrish, Adoan. Carbohyd. Chem., 23, 233 (1968); Adoan. Carbohyd. Chem. Biochem., 24, 139 (1969). (11) K. Freudenberg and K. Raschig, Ber., 60, 1633 (1927).

228

WALTER A. SZAREK

transition state 4 (viewed along the C-6-C-5 bond) having an axial substituent at C-4, than in transition state 5, having an equatorial substituent.

4

5

Displacement is more difficult at secondary positions12 than at primary positions on a pyranoid ring, and normally requires the use of high-boiling, aprotic solvents of high dielectric constant. Moreover, the latter reactions depend critically upon ( a ) the position of the group, and (b) the stereochemistry and conformation of the molecule. Thus, if a sulfonate is situated at C-2 of a pyranoid derivative, nucleophilic displacement with charged nucleophiles does not normally occur. It has been suggested that the diminished reactivity is due to unfavorable, polar interactions in the transition state. Also, it has been found that the presence of a vicinal, axial substituent or of a p-trans-axial substituent on a pyranoid ring inhibits replacement of a sulfonyloxy group. A summary of the steric and polar factors involved in nucleophilic-displacement reactions of sulfonic esters of carbohydrates has been published by Ri~hards0n.l~ From a consideration of these factors, it is often possible to predict the approximate reactivity of a sulfonyloxy group on a pyranoid ring; however, it is always necessary to consider the possibility that the molecule may react in a conformation different from that of the ground state. The utility of this approach in rationalizing the reactivities, towards displacement by chloride ion, of chlorosulfonyloxy groups on pyranoid sugar molecules will be described in Section II,2a (see p. 230). Because of the limited kinetic data available on nucleophilic-displacement reactions at positions other than C-1, the approach must necessarily be regarded as qualitative only. (12) The difference in reactivity of primary and secondary sulfonyloxy groups towards replacement by iodine was originally the basis of a method for distinguishing between primary and secondary hydroxyl groups in cyclic sugar molecules [see, J. W. H. Oldham and J. K. Rutherford,J.Amer. Chem. Soc., 54,366 (1932)l. (13) A. C. Richardson, Carbohyd. Res., 10, 395 (1969).

DEOXYHALOGENO SUGARS

229

One pertinent, kinetic investigation concerned the reactions of methyl 2,3-di-0-benzyl-4,6-dideoxy-4-iodo-a-~-galactoand -glucopyranoside with radioactive iodide ion in acetone.I4 The galacto isomer was found to react 2.8 and 2.4 times faster at 62.8 and 82.0", respectively, than the gluco compound. As the transition state would be the same for the reactions of both isomers, the relative rates are determined by the ground-state energies; the results indicate that the free energy of the gulacto isomer is 0.8 kcal.mole-' greater than that of the gluco isomer. Nucleophilic-displacement reactions on furanoid rings generally occur quite readily, unless the leaving group is exo in a bicyclic system having two fused, five-membered rings. Thus, 3-chloro-3deoxy- and 3-bromo-3-deoxy-~-glucosehave been prepared15 by way of treatment of 1,2:5,6-di-O-isopropylidene-3-O-p-tolylsulfonyl-~-~allofuranose with lithium chloride and lithium bromide, respectively, in N,N-dimethylformamide in the presence of calcium carbonate. The conditions selected for the preparation of deoxyhalogeno sugars by displacement reactions are typical of those employed for bimolecular, nucleophilic, substitution (SN2) reactions. Aprotic solvents are most suitable for displacements by anions, as, in them, most anions are less solvated, and, presumably, more reactive, than in protic solvents. Acetone, butanone, and higher-boiling ketones have been extensively used; however, frequently in these solvents, displacements can only be effected at primary positions. High-boiling, aprotic solvents of high dielectric constant, such as N,N-dimethylformamide, methyl sulfoxide, and hexamethylphosphoric triamide, have become extremely useful in carbohydrate chemistry; many displacement reactions at secondary carbon atoms have been effected in these solvents. A practical disadvantage accruing from the use of high-boiling solvents is that isolation of the product is sometimes complicated by their difficulty of removal. Hexamethylphosphoric triamide has been found to solubilize lithium halides in toluene; nucleophilic displacement of some carbohydrate sulfonates with these toluene-solubilized, halide ions produced in excellent yield, with short reaction-times, the corresponding deoxyhalogeno derivatives.16 The method has been adapted for use as a routine, preparative p r 0 ~ e d u r e . lDisplacement ~ of a 6-sulfonic ester required, with (14) C. L. Stevens, K. G . Taylor, and J. A. Valicenti, J. Amen Chem. SOC., 87, 4579 (1965). (15) J. E. G. Barnett, A. Ralph, and K. A. Munday, Biochem. J., 114, 569 (1969). (16) H. B. Sinclair, Carbohyd. Res., 15, 147 (1970). (17) H. B. Sinclair and L. W. Tjarks, Carbohyd. Res., 19, 402, (1971).

WALTER A. SZAREK

230

chloride, bromide, and iodide ion, 4-14, 1, and 0.25 hours, respectively. By heating for 100 to 150 times the period needed for displacement of the 6-methylsulfonyloxy group in methyl 2,3-di-O-benzoyl-4,6di-0-(methylsulfony1)-a-D-glucopyranoside,the 4-methylsulfonyloxy group was displaced by bromide and iodide ions, but not by chloride ions; however, the displacement was incomplete, and the product was a mixture of the 4-deoxy-4-halogeno derivatives having the D-glUC0 and D-galact0 configurations.

2. Direct Replacement of Hydroxyl Groups a. Reaction with Sulfuryl Chloride. -The reaction of sulfuryl chloride (SOZCl2)with carbohydrate derivatives was investigated first by Helferich and coworkers.18-20It was found that, in the compounds studied, the reagent caused the replacement of hydroxyl groups by chlorine atoms, or the formation of intramolecular, cyclic sulfates. Thus, treatment of methyl a-D-glucopyranoside (6) with sulfuryl chloride in a mixture of pyridine and chloroform at 5" afforded a compound whose structure was established, several years later,21'22 to be that of methyl 4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside 2,3-cyclic sulfate (7).The cyclic sulfates are readily cleaved by dilute

OH

6

04-0 II 0 7

alkalis, or by methanolic ammonia, to give the salt of a monosulfate; the hemi-ester is then desulfated by acid to yield the chlorodeoxy s ~ g a r . ' It ~ ,has ~ ~been shown that both of these reactions occur with retention of configuration; thus, for example, successive treatment of methyl 4,6-O-benzylidene-a-~-glucopyranoside 2,3-cyclic sulfate with methanolic ammonia and with sulfuric acid afforded, exclusively, D-glucose.21 (18) B. Helferich, Ber., 54, 1082 (1921). (19) B. Helferich, A. Lowa, W. Nippe, and H. Riedel, Ber., 56, 1083 (1923). (20) B. Helferich, G . Sprock, and E. Besler, Ber., 58,886 (1925). (21) P. D. Bragg, J. K. N. Jones, and J. C. Turner, Can. J . Chem., 37, 1412 (1959). (22) J. K. N. Jones, M. B. Perry, and J. C. Turner, Can. J . Chem., 38, 1122 (1960).

DEOXYHALOGENO SUGARS

231

J. K. N. Jones and his colleagues have extensively studied the reaction of sulfuryl chloride with carbohydrate^.^^-^^ This work has elucidated the stereochemical principles involved in the various transformations, and has made available a convenient and effective procedure for the preparation of chlorodeoxy sugars. Several examples of the utility of such derivatives, obtained by way of a reaction with sulfuryl chloride, in the synthesis of other rare sugars are discussed in Section I11 (see p. 281). In the present Section, the studies on the reaction itself are surveyed. Jennings and Jonesz4found that, when only a minimal proportion of pyridine is employed, the reaction of sulfuryl chloride with carbohydrates produces chlorosulfuric esters, instead of cyclic sulfates; thus, methyl 4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside 2,3-di(chlorosulfate) (8) was obtained by application of the reaction to methyl a-D-glucopyranoside (6),followed by isolation of the product at room temperature. These results could be rationalized on the basis of the observation that, in the presence of an excess of pyridine, appropriately oriented chlorosulfates can be converted into cyclic sulfates. For example, compound 8 reacts with pyridine to give the 2,3-cyclic sulfate 7. Presumably, one of the chlorosulfate groups is first hydrolyzed with S - 0 bond scission and, hence, with retention of configuration; the free hydroxyl group so formed then attacks the remaining chlorosulfate group, with S-Cl bond scission,30to give the cyclic sulfate. As the two chlorosulfate groups in 8 are equatorially oriented, the spatial requirement for the latter process is a very favorable H. J. Jennings and J. K. N. Jones, Can. J . Chem., 40, 1408 (1962). H. J. Jennings and J. K. N. Jones, Can. J. Chem., 41, 1151 (1963). H. J. Jennings and J. K. N. Jones, Can. J. Chem.,43,2372 (1965). H. J. Jennings and J. K. N. Jones, Can. J. Chem.,43,3018 (1965). A. G. Cottrell, E. Buncel, and J. K. N. Jones, Chem. Ind. (London), 522 (1966). A. G. Cottrell,, E. Buncel, and J. K. N. Jones, Can. J. Chem., 44, 1483 (1966). (29) S. S. Ali, T. J. Mepham, I. M. E. Thiel, E. Buncel, and J. K. N. Jones, Carbohyd. Res., 5, 118 (1967). (30) The conversion of some carbohydrate chlorosulfates into the corresponding fluorosulfates with silver fluoride in methanol,3I and the formation of a mono(azidosulfate) derivative on treatment of methyl 4,6-dichloro-4,6-dideoxy-a-~galactowranoside 2.3-di(chlorosulfate) . . . (8) . . with sodium azide in N,N-dimethvlformamide at room temperature,32 are two other examples of reactions involving sulfur-chlorine scission. (31) E. Buncel, H. J. Jennings, J. K. N. Jones, and I. M. E. Thiel, Carbohyd. Res., 10, 331 (1969). (32) H. Parolis, W. A. Szarek, and J. K. N. Jones, Carbohyd. Res., 19, 97 (1971). (33) The presence of axially attached chlorosulfate groups can lead to the formation 1,

232

WALTER A. SZAREK

Chlorosulfate groups can be readily removed to give the corresponding hydroxyl groups, with retention of configuration, by treatment of a solution of the carbohydrate chlorosulfate in methanol with sodium iodide in aqueous methanolz4;immediate liberation of iodine and evolution of sulfur dioxide occur.34 A possible mechanism for the dechlorosulfation reaction involves displacement by iodide at the chlorine atom; the initially formed iodine monochloride would react with iodide ion to give iodine and chloride ion. Alternatively, an unstable iodosulfate could be formed as an intermediate.35

Insight into the manner of formation of chlorodeoxy sugars by the reaction of sulfuryl chloride with carbohydrates containing free hydroxyl groups was gained by the series of reactions outlined in the next Scheme.z5 As has already been mentioned, treatment of methyl a-D-glucopyranoside (6) with sulfuryl chloride and pyridine in chloroform solution, and isolation of the product at room temperature, afforded methyl 4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside 2,3-di(chlorosulfate) (8). Furthermore, it was found that isolation of the product at 0" gave methyl 6-chloro-6-deoxy-a-~-glucopyranoside 2,3,4-tri(chlorosulfate) (9), and isolation at -70" yielded methyl a - ~ glucopyranoside 2,3,4,6-tetra(chlorosulfate) (10). Moreover, compound 10 was converted into the 6-chloro-6-deoxy derivative 9 on treatment with 1 mole of pyridinium chloride per mole, and compound 9 was converted into the 4,6-dichloro-4,6-dideoxy derivative either of anhydro sugars or of complex products resulting fiom elimination reactions. It has also been found that only a mildly basic reagent, such as pyridine, can be used for the conversion of chlorosulfuric esters into cyclic sulfates; thus, methyl 4,6-O-benzylidene-a-~-glucopyranoside 2,3-di(chlorosulfate) is readily converted by pyridine at 0" into the 2,3-cyclic sulfate, whereas, with a stronger base, such as sodium methoxide, it forms methyl 2,3-anhydro-4,6-0-benzylidenec~-D-allopyranoside.~* (34)It has been found32that treatment of methyl 4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside 2,3-di(chlorosulfate) (8) with sodium bromide in N,N-dimethylformamide at room temperature affords methyl 4,6-dichloro-4,6-dideoxy-a-~galactopyranoside and its 2- and 3-mono(chlorosulfate) derivatives. These three products were also formed, in addition to a mono(azidosu1fate) derivative, when 8 was treated with sodium azide in NJV-dimethylformamide at room temperature. (35)For a review on chlorosulfates, see E. Buncel, Chem. Rev., 70, 323 (1970).

DEOXYHALOGENO SUGARS

233

OMe 6

ature

c1

1

0'

OMe

excess\ 8

OMe

/iar proportion of

C,H,%Cl@

C,HB8HC1@

OMe 9

where R = S02Cl.

8 on treatment with an excess of pyridinium chloride. Thus, the chlorodeoxy groups are formed by bimolecular displacement of certain of the chlorosulfonyloxy groups by chloride ion liberated during the chlorosulfation. It is often possible to predict the reactivity of a chIorosulfonyloxy group by a consideration of the steric and polar factors affecting the formation of the transition ~ t a t e , ~as ' , ~indicated ~ in Section II,1 (see p. 227) for nucleophilic-replacement reactions of sulfonic esters of carbohydrate derivatives. Thus, it has been found that the presence of a vicinal, axiaI substituent or of a p-trans-axial substituent on a pyranoid ring inhibits replacement of a chlorosulfonyloxy group; also, a chlorosulfate group at C-2 has been observed to be deactivated to nucleophilic substitution by chloride ion, An example of the inhibitory effect of a vicinal, axial substituent was provided by the observationz5that, whereas methyl 4,6-dichloro4,6-dideoxy-P-~-glucopyranoside 2,3-di(chlorosulfate) readily under-

234

WALTER A. SZAREK

goes displacement of the chlorosulfonyloxy group at C-3 by chloride ion, the corresponding galactopyranoside, having the chloro group at C-4 in an axial orientation, is resistant to replacement. A comparison of the two transition states 11 and 12 (viewed along the C-3-C-4

\

\

60

60

c1

c1

Galactoside

Glucoside

11

12

bond) shows that there would be greater steric and dipolar repulsions for substitution at C-3 in the galactoside; in this transition state, one of the dipoles must become aligned with the permanent dipole associated with a neighboring, axial bond.I3 The reaction of methyl a-D-mannopyranoside with sulfuryl chloride gave methyl 6-chloro-6-deoxy-a-~-mannopyranoside 2,3,4-tri(chlorosulfate); even treatment of the product with pyridinium chloride for 12 hours at 50" did not effect further s u b ~ t i t u t i o n In . ~ ~this case, the lack of displacement of the chlorosulfonyloxy group at C-4 is attributed to the presence of an axial group at C-2. In the (hypothetical) transition state 13 (viewed along the C-4-C-3 bond), there would be 0SO2Cl

,

6 0

OS0,Cl

13

considerable steric interaction between the incoming chloride ion and the chlorosulfate group at C-2; it is also possible that there would be some dipolar repulsion between the upper, polar bond of the transition state and the permanent dipole associated with the C-2OS0,Cl bond.I3 Similarly, the lack of displacement at C-3 is attributed to the presence of the axial methoxyl group on C-1.

235

DEOXYHALOGENO SUGARS

An interesting application of the reaction with sulfuryl chloride consisted in the synthesis of a fully chlorinated h e x o ~ i d eTreatment .~~ of methyl 2-chloro-2-deoxy-~-~-galactopyranoside with sulfuryl chloride and pyridine in chloroform, followed by heating of the product with an excess of pyridinium chloride, afforded a tetrachloride, presumably methyl 2,3,4,6-tetrachloro-2,3,4,6-tetradeoxy-~-~-allopyranoside. The reaction of sulfuryl chloride with reducing sugars has also been studied23;glycosyl chlorides containing both chlorosulfate and chlorodeoxy groups are obtained. Thus, the reactions with D-glucose and D-XylOSe, with isolation of the products at room temperature, afford 4,6-dichloro-4,6-dideoxy-~-galactopyranosyl chloride 2,3-di(chlorosulfate) and 4-chloro-4-deoxy-~-arabinopyranosyl chloride 2,3-di(chlorosulfate), respectively. If the product of reaction with D-XylOSe is isolated at low temperature, substitution of the chlorosulfonyloxy group at C-4 by chloride ion is inhibited, and D-xylopyranosyl chloride 2,3,4-tri(chlorosulfate)is obtained in good yield; the actual experimental conditions involved treatment of D-XylOSe with sulfuryl chloride and pyridine in chloroform solution at -70" for 2 hours, followed by allowing the temperature to rise to -lo", and then maintaining it between- 10 and 0" for 30 minutes.36Significantly, it was that, under these conditions, crystalline @-D-XylOpyranose yields crystalline fl-D-xylopyranosyl chloride 2,3,4-tri(chlorosulfate) (14), and crystalline P-D-lyxopyranose is converted into a-Dlyxopyranosyl chloride 2,3,4-tri(chlorosulfate)(15). In each case,

14

15

where R = S0,Cl.

the n.m.r. spectra of the crude and pure products were identical and did not show the presence of the other anomer; the favored conformations of 14 and 15 are as shown, namely, those having chlorine in the axial orientation. These results suggest that the replacement of the hydroxyl group at C-1 by chlorine occurs by way of an inter(36) H. J. Jennings, Can. J . Chem., 46,2799 (1968). (37) H. J. Jennings, Can. J . Chem., 47, 1157 (1969).

236

WALTER A. SZAREK

mediate chlorosulfate, resulting in an overall inversion of configuration; moreover, they show that anomerization does not occur to any significant extent under the reaction conditions, thus providing a controlled synthesis of single anomers of glycosyl chlorides, even of the thermodynamically less-stable anomers. The ready availability of chlorosulfated glycosyl chlorides, the ease of regeneration3* of a hydroxyl group with retention of configuration, from a chlorosulfate, and the nonparticipating proper tie^^^ of the chlorosulfate group should make these derivatives useful glycosylating agents. Thus, for example, p-D-XylOpyranOSyl chloride 2,3,4-tri(chlorosulfate)(14) reacted, under modified Koenigs-Knorr conditions, with 1,2,3,4-tetra-O-acety1-~-D-mannopyranose to give, after deacylation, mainly 6-O-a-D-xy~opyranosyl-D-rnannopyran0se.~~ An elegant, stereospecific synthesis of two 2-chloro-2-deoxypentoses was achieved during the course of a study of the anomerization of chlorosulfated glycosyl chlorides.40It had been found37that attempts to anomerize p-D-xylopyranosyl chloride 2,3,4-tri(chlorosulfate) (14) with 0.25 mol-equivalent of aluminum chloride afforded the crystalline a-Danomer only in low yield; formed also was another compound whose structure has been established40 to be that of 2-chloro-2-deoxy-a-~-lyxopyranosyl chloride 3,4-di(chlorosulfate) (16). The dichloro derivative 16 became the exclusive product when 1.5 mol-equivalents of aluminum chloride were used; treatment of 16 with sodium iodide yielded crystalline 2-chloro-2-deoxy-~-lyxose. Similarly, a-D-lyXOpyranOSyl chloride 2,3,4-tri(chlorosulfate)(15) afforded 2-chloro-2-deoxy-a-~-xylopyranosyl chloride 3,4-di(chlorosulfate) (17). 14-

ClS0,O m 1 5 ClSO*O

c1 16 X = C1; Y = H 17 X = H ; Y = C l

(38) As has already been indicated in this Chapter, chlorosulfate groups can be removed from chlorosulfated glycosides by treatment with sodium iodide in aqueous methanol.z4 When glycosyl chlorides containing chlorosulfate groups were treated with sodium iodide in aqueous acetone, in addition to dechlorosul~~.~ has fation, hydrolysis occurred at C-1 to give the free s ~ g a r . Jennings4' reported that treatment of a- and p-D-xylopyranosyl chloride 2,3,4-tri(chlorosulfate) and of the corresponding a - ~ o - l y x oderivative with a catalytic amount of sodium iodide in methanol, in the presence of barium carbonate, resulted in the removal of the chlorosulfate groups and rapid methanolysis at C-1; the methanolysis proceeded with predominant inversion in each reaction.

DEOXYHALOGENO SUGARS

237

The formation of 16 from 14 has been interpretedm as occurring by an intramolecular displacement of the chlorosulfate group at C-2, by participation of the chlorine atom at C-1, to give initially a chloronium ion, as shown; the departure of the chlorosulfate group is

probably facilitated by co-ordination of the oxygen atoms with aluminum chloride. Favored attack by chloride ion at the more highly reactive center (C-1) would result in a net inversion of configuration at C-2 of 14, to yield 16. a-D-Xylopyranosyl chloride 2,3,4-tri(chlorosulfate) also gave the dichloro derivative 16, presumably by way of an initial anomerization to the reactive p anomer (14) in the presence of aluminum chloride. In the reaction of a-D-lyxopyranosyl chloride 2,3,4-tri(chlorosulfate)(15) with aluminum chloride, a 1,2-cis product (17) was preponderant, whereas the proposed mechanism should yield a 1,Ztruns product; this result is, presumably, due to the anomerization of the thermodynamically less-stable p anomer of 17 to the (more stable) 1,e-ci.s product (17). It is notable that, in 14 and 15, the chlorosulfate group at C-2 is displaced exclusively, a result possibly attributable to the tendency of chlorine to migrate by way of a 1,2-cyclic Pyranoid derivatives having a chlorosulfonyloxy group in a transdiaxial relationship with a ring proton may undergo an elimination reaction to yield unsaturated corn pound^.^^,^^ Thus, on treatment with sulfuryl chloride and pyridine, followed by heating with a solution of pyridinium chloride in chloroform, methyl a-L-arabinopyranoside gave crystalline methyl 3,4-dichloro-3,4-dideoxy-P-~ribopyranoside 2-chlorosulfate (18), which, on standing in pyridine, (39) B. Coxon, H. J. Jennings, and K. A. McLauchlan, Tetrahedron, 23, 2395 (1967). (40) H. J. Jennings, Can. J. Chem., 48, 1834 (1970). (41) H. J. Jennings, Can. J. Chem., 49, 1355 (1971). (42) In contrast to these chlorine migrations, it has been found43 that treatment of methyl p-D-xylopyranoside 2,3,4tri(chlorosulfate) with aluminum chloride afforded, as one of the major products, 4-O-methyl-p-~-arabinopyranosyl chloride 2,3-di(chlorosulfate); this compound was formed as a result of the favored migration of the anomeric methoxyl group to C-4. (43) C. F. Gibbs and H. J. Jennings, Con. J. Chem., 48, 2735 (1970).

WALTER A. SZAREK

238

underwent the loss of chlorosulfuric acid to give crystalline methyl 3,4-dichloro-2,3,4-~ideoxy-~-~-glycero-pent-2-enopyranoside~~ (19). B

OMe

18

19

One of the products of the reaction of L-arabinose with sulfuryl chloride in pyridine was the “dimer” 3,4-dichloro-2,3,4-trideoxy-@-~glycero-pent-2-enopyranosyl 3,4-dichloro-2,3,4-trideoxy-p-~-glyceropent-2-enopyrano~ide~~ (20); the a,a structure had originally been

u\

c1 c1

/ c1 20

assigned26to the dimeric product. The major product of the reaction with L-arabinose was 3,4-dichloro-3,4-dideoxy-~-~-ribopyranosyl chloride 2-chloro~ulfate~~; this compound was shown to be a precursor of the dimer 20 by the observation that treatment of it with pyridine in chloroform, followed by partitioning of the product between chloroform and dilute aqueous acid, led to the separation, from the latter solution, of crystalline 20. It has been suggestedzs that the dimer 20 may be formed by way of an intermediate, pyridinium glycoside. An X-ray crystallographic analysis has been performedMon methyl 4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside prepared by way of reaction of methyl a*-glucopyranoside with sulfuryl chloride; the molecule has the CI (D) conformation, and the bond distances and valency angles are similar to those in simple sugars. (44) R. Hoge and J. Trotter, J . Chem. SOC. (A), 2165 (1969).

DEOXYHALOGENO SUGARS

239

Treatment of sucrose with sulfuryl chloride in pyridine at 50” afforded44aa complex mixture of products from which were isolated, in low yields, one tetrachloro and two pentachloro derivatives.

b. Reaction with Phosphonas-containing Reagents. -A variety of phosphorus-containing reagents have been employed for replacing hydroxyl groups in carbohydrates by halogen atoms. The reaction of 1,2:5,6-di-O-isopropylidene-a-~-glucofuranose (21) with phosphorus pentachloride was reported45in 1926 to afford a low yield of 3-chloro3-deoxy-1,2:5,6-di-O-isopropylidene-a-~-glucofuranose; however, the product has since been shown to be 6-chloro-6-deoxy-1,2:3,5-di-0isopropy~idene-a-D-gluc~furanose~~-~~ (22). An example of a rearrange-

ment with phosphorus pentachloride was observed49in the conversion of ethyl 2,3,4,6-tetra-O-acetyl-D-gluconate into an ethyl 2,3,4,5-tetraO-acetyl-6-chloro-6-deoxyhexonate. Heating of 2,3,4,5-tetra-O-benzoylmannitol in chloroform in the presence of phosphorus pentachloride afforded the corresponding 1,6-dichloro-1,6-dideoxyd e r i ~ a t i v e . ~ ~ Treatment of 1,2,3,4-tetra-O-acety1-fi-D-mannopyranose with phosphoryl chloride gives tetra-O-acetyl-6-chloro-6-deoxy-fi-~-mannopyr a n o ~ e . The ~ l reaction of 2’,3’-O-isopropylidene-inosine or -guanosine with phosphoryl chloride at temperatures below 30” has been reported51a to give the corresponding 5’-chloro-5’-deoxy derivatives. During the 1950’s, Rydon and described, for the (44a) J. M. Ballard, L. Hough, and A. C. Richardson, Chem. Commun., 1097 (1972); compare, Ref. 2 1. (45) J. B. Allison and R. M. Hixon, /. Amer. Chem. Soc., 48, 406 (1926). (46) D. C. C. Smith,]. Chem. Soc., 1244 (1956). (47) E. Hardegger, G. Zanetti, and K. Steiner, Helu. Chim. Acta, 46, 282 (1963). (48) J. Baddiley, J. G . Buchanan, and F. Hardy, J . Chem. SOC., 2180 (1961). (49) A. M. Dempsey and L. Hough, Carbohyd. Res., 16,449 (1971). (50) F. Micheel, Ann., 496, 77 (1932). (51) B. Helferich and J. F. Leete, Ber., 62, 1549 (1929). (51a) K. Kusashio and M. Yoshikawa, Bull. Chem. SOC./ u p . , 41, 142 (1968). (52) S. R. Landauer and H. N. Rydon,]. Chem. SOC., 2224 (1953);D . G. Coe, S. R. Landauer, and H. N. Rydon, ibid., 2281 (1954); H. N. Rydon and B. L. Tonge, ibid., 3043 (1956).

WALTER A. SZAREK

240

halogenation of alcohols, procedures using some phosphorus-containing reagents. Two important reagents were methyltriphenoxyphosphonium iodide (23) and iodotriphenoxyphosphonium iodides3 (24), 0

(PhO),P -Me

0

I'

(PhO),P-I

23

I'

24

respectively formed by the reaction of triphenyl phosphite with methyl iodide and with iodine. The reaction of 23 with alcohols is considered to involve nucleophilic attack on phosphorus, with expulsion of phenol and formation of the alkoxyphosphonium salt 25,

23

+

ROH

-

(PhO),P-OR 0 p r@-RI I

I

+

0 I1 (PhOhP-Me

Me 25

which then affords the alkyl iodide and diphenyl methylphosphonate. According to this mechanism, the conversion of an alcohol into the corresponding iodide should occur with inversion of configuration; however, because of the possibility of nucleophilic attack by iodide ion upon alkyl iodides, the inversion can be accompanied by some racemization. The first applications of Rydon reagents in the carbohydrate field were reported in 1960 by Kochetkov and coworkers,54and by Lee and El S a ~ iA. limitation ~ ~ of the method was immediately noted, namely, the possibility of rearrangement of acetal protecting-groups. Thus, the reaction of 1,2:5,6-di-O-isopropylidene-a-D-glucohranose (21) with bromotriphenoxyphosphonium bromides4js6 afforded 6bromo-6- deoxy- 1,2:3,5-di-O-isopropylidene-a-~-glucofurnose, and the reaction of 21 withs6 23 gave 6-deoxy-6-iodo-1,2:3,5-di-O-isopropylidene-a-D-glucohranose, not the 3-deoxy-3-iodo derivative originally55 reported. Several other examples of the reaction of carbohydrates with Rydon reagents have been provided by Kochetkov and coworker^.^^ Most (53)Compounds 23 and 24 are shown as ionic species, although the contributions of pentacovalent forms, especially in nonpolar solvents, should not be excluded. The reagents have also been referred to as triphenylphosphite methiodide [(PhO),PMeI] and triphenylphosphite diiodide [(PhO),PI,]. (54)N. K. Kochetkov, L. I. Kudryashov, and A. I. Usov, Dokl. Akad. Nauk S S S R , 133, 1091 (1960). (55)J. B. Lee and M. M. El Sawi, Chem. Ind. (London), 839 (1960). (56)N.K. Kochetkov and A. I. Usov, Tetrahedron, 19,973 (1963).

24 1

DEOXYHALOGENO SUGARS

of the iodinations were performed in hot benzene. It has been found that the reaction is controlled by steric factors. Thus, no reaction occurred between 1,2-O-isopropylidene-5,6-di-O-methyl-a-~-glucofuranose and either 23 or bromotriphenoxyphosphonium bromide, presumably because of the steric hindrance caused by the trioxabicyclo[3.3.0loctane ring-system, whereas methyl 2,5,6-tri-O-methyl/I-D-glucofuranoside reacted with 23 to give a 3-deoxy-3-iodo derivative in 31% yield. In methyl 3,4-O-isopropylidene-/I-D-galactopyranoside (26),only one of the two hydroxyl groups is replaced by iodine on treatment with 23, affording methyl 6-deoxy-6-iodo-3,4-0isopropylidene-6- D-galactopyranoside (27) in 60% yield. The

OH

OH 26

21

inertness of the hydroxyl group on C-2 can, at least partly, be attributed to steric hindrance; however, it is known (see Section 11,l; p. 227) that nucleophilic displacement with charged nucleophiles is normally difficult at C-2. The reaction of methyl 2,3-di-O-methyl-6-O-p-tolylsulfonyl-cr-Dgalactopyranoside (28)with 23 gave the iodide 29 in high yield; similar treatment of methyl 2,3-di-0-methyl-6-0-p-tolylsulfonyl-a-~glucopyranoside (30)afforded the C-4 epimer (31) 29. These FH.ms

:Hams

-

HoQoMe OMe

OMe

28

29

-

HOQOMe

IQOMe

OMe 30

OMe 31

WALTER A. SZAREK

242

results demonstrate the viability of methyl ethers and p-toluenesulfonic esters5* as protecting groups for substrates in reactions with the Rydon reagent. In another example of the reaction of 23 with a pyranoid derivative having an "isolated" hydroxyl group at C-4, a ring contraction was observed59; methyl 2,3-0-isopropylidene-a-~rhamnopyranoside afforded preponderantly methyl 5,6-dideoxy-5iodo-2,3-O-isopropylidene-/3-~-allofuranoside, accompanied by some methyl 4,6-dideoxy-4-iodo-2,3-O-isopropylidene-a-~-mannopyranoside and methyl 5,6-dideoxy-5-iodo-2,3-0-isopropylidene-a-~-talofuranoside.g0 Dithioacetal derivatives have also been employed56 in reactions with 23. The primary hydroxyl group in 2,3:4,5-di-O-isopropylideneD-galaCtOSe diethyl and dibenzyl dithioacetals is readily replaced by iodine, to give the expected 6-deoxy-6-iodo derivatives in almost quantitative yields. The secondary hydroxyl group in 5-0benzoyl-2,3-O-isopropylidene-~-arabinose diethyl dithioacetal (32) was similarly replaced by iodine to give 5-O-benzoyl-4-deoxy-4-iodo2,3-0-isopropylidene-~-xylose diethyl dithioacetal (33); however, a rearranged product, namely, 4-O-benzoyl-5-deoxy-5-iodo-2,3-0-isopropylidene-L-arabinose diethyl dithioacetal (34) was also produced. H

H

EtSCSEt HCO M"C\&

~

I

HOCH I C%OBz 32

H

EtSCSEt

EtSCSEt

+

Me&& , I HCI I CHSOBz 33

HbO Me&\& I

BzOCH I

cHar

34

(57)N.K. Kochetkov and A. I. Usov, Zzu. Akud. Nuuk SSSR, Ser. Khim., 475 (1964);see also, ibld., 492 (1965). (58)Under appropriate conditions, replacement of sulfonyloxy groups by iodine can occur with the reagent 23.Thus, treatment of methyl 3-0-methyl-2,6-di-O-p-tolylsulfonyl-a-D-glucopyranoside with 23 in benzene for 5 hours at 55" gave methyl 4-deoxy - 4-iodo-3- 0-methyl - 2,6-di- 0p - tolylsulfonyl-a-galactopyranoside in 83.5% yield; however, with a higher proportion of the iodinating reagent, reaction of the latter compound occurred, by displacement of the primary p-tolylsulfonyloxy group, to form methyl 4,6-dideoxy-4,6-diiodo-3-O-methyl-2-O-p-tolylsulfonyl-a-D-galactopyranosidein 85% yield.5' (59)(a) K. Kefurt, J. Jary, and Z. Samek, Chem. Commun., 213 (1969);Collect. Czech. Chem. Commun., 35, 2613 (1970);(b) K. S. Adamyants, A. I. Usov, and N. K. Kochetkov, Zzv. Akad. Nauk S S S R , Ser. Khim., 703 (1970);(c) N. K. Kochetkov, A. I. Usov, and K. S. Adamyants, Tetrahedron, 27,549 (1971).

DEOXYHALOGENO SUGARS

243

The formation of 34 was explained by a rearrangement of 33 by way of a benzoxonium intermediate; the ion is attacked at the less-hindered, C-5 position, to give a 5-deoxy-5-iodo derivative. The structure of 34 was confirmed by its reduction to 4-O-benzoyl-1,5-dideoxy-2,3-O-isoprop ylidene-L-arabinitol. The examples already described show that the Rydon reagent 23 has provided a valuable method for the preparation of deoxyiodo sugars; these derivatives are versatile precursors to several chemically and biologically important sugars (see Section 111; p. 282). Verheyden and Moffatte’ have extended the reaction to the nucleoside field. It has been foundsZthat reactions of the primary hydroxyl group of suitably protected pyrimidine nucleosides with 23 in N,N-dimethylformamide are very rapid and give the corresponding deoxyiodo nucleosides in high yield. For example, 2’,3’-O-isopropylideneuridine afforded, after 15 minutes at room temperature, 5’-deoxy-5’-iodo2’,3’-O-isopropylideneuridinein 96.5% yield. Similarly, 3’-O-acetylthymidine and 2’,3’-di-O-acetyluridinewere converted into the corresponding 5’-deoxy-5‘-iodo derivatives in isolated yields of 88 and 84%, respectively. A complication was encountered in the cytidine series, because of the presence of a free amino group; this group was substituted to give, presumably, the phenyl methylphosphonate derivative. However, this problem could be obviated by acylation of the cytosine amino group. Despite the fact that secondary hydroxyl groups of nucleosides also react with 23 (see later), selective iodination of only the primary hydroxyl group in some unprotected, pyrimidine nucleosides can also be achieved.s2 Thus, brief treatment of thymidine with 1.1 molequivalents of 23 in N,N-dimethylformamide gave crystalline 5‘deoxy-5’-iodothymidine in 63% yield. It was even possible to effect some selective iodination of the 5’-hydroxyl group of 2,2‘-anhydrouridine without excessive cleavage of the (quite labile) anhydro linkage. The reactions of 23 with 2’,3’-O-isopropylidene derivatives of purine nucleosides, or with free adenosine, give the corresponding N3,5’-anhydronucleosidesin high yield; a 5‘-deoxy-5‘-iodo deriv(60)Displacement

reactions of methyl 6-deoxy-2,3-0-isopropylidene-cu-D-(L)mannopyranoside 4-sulfonates with charged nucleophiles have also been found to lead to ring contraction with participation by the ring-oxygen atom [see C. L. Stevens, R. P. Glinski, K. G. Taylor, P. Blumbergs, and F. Sirokman, J. Amer. Chem. Soc. 88,2073 (1966);S.Hanessian, Chem. Commun., 796 (1966), C . L. Stevens, R. P. Glinski, G. E . Gutowski, and J. P. Dickerson, Tetrahedron

Lett., 649 (1967)l. (61)J. P. H. Verheyden and J . G. Moffatt, J . Amer. Chem. Soc., 86, 2093 (1964). (62)J. P. H.Verheyden and J. G. Moffatt,J. Org. Chem., 35,2319 (1970).

WALTER A. SZAREK

244

ative was isolated, albeit in low yield, only from 2’,3’-O-isopropylideneinosine.g2 The reagent 23 decomposes in the presence of traces of water, to release hydrogen iodide; if the iodination reaction is not rapid, the loss of acid-labile groups may be a problem. Therefore, the reaction of 23 with pyrimidine nucleosides in the presence of a base was investigated.62 It was found that iodination of 2’ 3‘-O-isopropylideneuridine could be achieved within 15 minutes in pyridine; however, with longer reaction times, a 5’-deoxy-5’-pyridinium derivative was produced. The use of a small excess of the relatively non-nucleophilic base N,N-diisopropylethylamine,instead of pyridine, afforded 2,5‘-anhydro-2’,3’-O-isopropylideneuridine, in addition to the 5’-deoxy-5’-iodo derivative. When 2,2’-anhydrouridine was treated with the Rydon reagent 23 in N,N-dimethylformamide in the presence of pyridine or N,N-diisopropylethylamine,two major products were isolated after the addition of methanol; these were shown to be the phosphorus diastereoisomers of 2,2’-anhydro-5’-deoxy-5’-iodouridine 3‘-(phenyl methylphosphonate) (38).The 3’,5’-di-O-(methyldiphenoxyphosphonium) intermediate (35)is, presumably, rapidly formed, and then undergoes displacement at C-5’ by iodide ion; attack at C-3’ by iodide ion is hindered by the presence of the 2,2’-anhydro linkage. Upon addition of methanol, phenol could be displaced from 36 with formation of a methoxyphosphonium derivative (37),which would undergo rapid dealkylation by iodide ion to give the diastereoisomer 38.

0

0

(PhO),A-Me @

:PhO),h-Me 0

35

36

lMeoH

DEOXYHALOGENO SUGARS

245

0

0

0 ‘0

PhO-P=O

PhO -P-OMe I Me

I

Me 38

37

Examples of the iodination of secondary hydroxyl groups of nucleosides with methyltriphenoxyphosphonium iodide (23)have been described by Verheyden and M ~ f f a t tIt . ~has ~ been found that the reactions of 5’-protected derivatives of thymidine with 23 in N,Ndimethylformamide at room temperature afford the corresponding 3‘-deoxy-3’-iodo nucleosides with retention of configuration. The stereochemistry observed can be explained by a mechanism for the reaction which involves formation of an intermediate 2,3’-anhydronucleoside, the anhydro ring of which is subsequently opened by iodide The reaction of thymidine itself with an excess of 23 in N,N-dimethylformamide gives 3’,5’-dideoxy-3’,5’-diiodothymidine in 76% yield63; however, the reaction in pyridine gives mainly 2,3’-anhydr0-5’-deoxy-5’-iodothymidine.~~ The isolation of the latter derivative is understandable, as nucleophilic opening of anhydronucleosides is known to be an acid-catalyzed process; the acid required for the formation of the di-iodide may result from hydrolysis of 23 in the presence of traces of water. Treatment of 5’-0-(p-nitrobenzoy1)uridine with 23 in N,N-dimethylformamide did not effect iodination of the cis-vicinal, hydroxyl groups; instead, a mixture of 2’(3’)-O-methylphosphonates was obtained. The reaction of 23 with 2’,Sf-di-O-trityluridine was studied63 in N,N-dimethylformamide at 25”, and in hot benzene. In the former solvent, the major product was 1-(3-deoxy-3-iod0-2,5-di-O-trityl-~-~xylofuranosy1)uracil;in addition, there was some selective hydrolysis of the 5’-O-trityl substituent. In benzene,64there was also an inversion (63) J . P. H. Verheyden and J . G . Moffatt, /. Org. Chem., 35, 2868 (1970). (64) See also, G . A. R. Johnson, Aust. J . Chem., 21, 513 (1968).

WALTER A. SZAREK

246

of configuration at C-3 during iodination, but a selective loss of the 2’-0-trityl group occurred during processing of the reaction mixture. Considerable interest has developed in the method of conversion of alcohols, by their reaction with carbon tetrachloride and tertiary phosphines (usually triphenylphosphine), into the corresponding alkyl chloride^^^ The reaction is considered to proceed by an ionic mechanism involving nucleophilic displacement on halogen, as shown. R,P:

@

R,PX’CX,

0

%PX ‘OR’-

-

n n + X-CX, +

R’OH

0 %PORfXo

Q

R,PX ‘CX,

-

Q

R,PX ‘OR’

-

R,P=O

+

+

HCX,

R’X

The first application of the reaction in the carbohydrate field was to the chlorination of 1,2:3,4-di-0-isopropylidene-a-D-galactopyranO S ~ ~ ~ treatment ( ~ ) ; of this compound with triphenylphosphine in carbon tetrachloride for 3 hours at reflux temperature gave the corresponding 6-chloro-6-deoxy derivativeea in good yield, and a small proportion of 6-deoxy-1,2:3,4-di-O-isopropylidene-~-~-arabino-hex5-enopyranose. In another appli~ation,6~ treatment of a-D-glucofuranose 1,2:3,5-di(benzeneboronate) with triphenylphosphine in carbon tetrachloride, followed by transesterification with 1,Spropanediol, afforded 6-chloro-6-deoxy-~-glucose in 79% yield; similarly, 6-bromo-6-deoxy-~-glucosewas obtained by way of a reaction with triphenylphosphine in bromofonn for only 30 minutes at 20”. These two s t ~ d i e s ~ ~ on( ~“isolated” ) * ~ ~ primary hydroxyl groups were extendedes to the more difficult case of the 1-position in a (65)(a) I. M. Downie, J. B. Holmes, and J. B. Lee, Chem. Ind. (London), 1900 (1966); (b) J. B. Lee and T. J. Nolan, Can. J . Chem., 44, 1331 (1966);(c) J. B. Lee and I. M. Downie, Tetmhedron, 23,359 (1967);(d)J. B. Lee and T. J. Nolan, ibid., 23, 2789 (1967);(e) J. Hooz and S. S. H. Gilani, Can. J . Chem., 46, 86 (1968); (0 I. M. Downie, J. B. Lee, and M. F. S. Matough, Chem. Commun., 1350 (1968); (g) R. G.Weiss and E. I. Snyder, ibid., 1358 (1968);(h) R. G. Weiss and E. I. Snyder,J. Org. Chem., 35, 1627 (1970). (66)6-Chloro-6-deoxy-l,2:3,4-di-0-isopropylidene-c~-~-galactopyranose has been obtained in 96% yield by treatment of 1,2:3,4-di-O-isopropylidene-6-O-p-tolylsulfonyl-a-D-galactopyranose with lithium chloride in N,N-dimethylformamide [see K. W. Buck and A. B. Foster,J. Chem. SOC., 2217 (1963)]. (67)L. G. Morgel and A. M. Yurkevich, Zh. Obshch. Kim., 40, 708 (1970).

DEOXYHALOGENO SUGARS

247

2-hexulose. Both 2,3:4,5-di-O-isopropylidene-~-fructose and 2,3:4,6-di-O-isopropylidene-~-sorbose react readily with triphenylphosphine-carbon tetrachloride to yield the corresponding l-chloro-ldeoxy derivatives; hydrolysis of these derivatives gives l-chloro1-deoxy-D-fructoseand 1-chloro-1-deoxy-L-sorbose, respectively. The replacement of a secondary hydroxyl group in a monosaccharide by chlorine by this method has also been reported. Thus, methyl 2,3-di0-methyl-a-D-ghcopyranoside gave methyl 4,6-dichloro-4,6-dideoxy2,3-di-O-methyl-a-D-galactopyranoside in almost quantitative yield,pe and, similarly, 3-chloro-3-deoxy-l,2:5,6-di-0-isopropylidene-a-~-glucose and 3-chloro-3-deoxy-1,2:5,6-di-O-isopropylidene-~-~-idose were preparedae from 1,2:5,6-di-O- isopropylidene-a-D-allose and 1,2:5,6-di-O-isopropylidene-~-~-talose, respectively. Some limitations in the application of this chlorination procedure have been noted.a8 Methyl 4,6-O-benzylidene-a-~-glucoside, 1,2:4,5-di-O-isopropylidene-P-D-fructose, and 1,2:4,5-di-O-isopropylidene-~-D-~ibohexulose, for example, appear to decompose in refluxing carbon tetrachloride containing triphenylphosphine. With 172:5,6-di-0-isopropylidene-a-D-glucofuranose,a rearrangement of the 5,6-acetal linkage accompanies chlorination at C-6, to give 6-chloro-6-deoxy1,2:3,5-di-O-isopropylidene-a-~-glucofuranose. The use of triphenylphosphine-carbon tetrachloride to convert lincomycin (1)into clindamycin (2) has already been mentioned (see Section I, p. 226); the 7-bromo and 7-iodo analogs of 2 were also prepared by treatment of lincomycin hydrochloride with triphenylphosphine and carbon tetrabromide or carbon tetraiodide, with acetonitrile as the s01vent.~ The halogenation of hydroxyl groups in the carbohydrate moieties of nucleosides has also been achieved by the reaction with carbon tetrahalides and triphenylphosphine. Thus, 5’-chloro-5’-deoxy2’,3’-0-isopropylideneinosinewas produced in high yield by a reaction, with 2’,3’-O-isopropylideneinosine, perf~rmed’~ in triethyl phosphate at 100”. The 5’-bromo and 5’-iodo analogs could be obtained in good yields by treatment of 2‘,3’-O-isopropylideneinosine with bromine, cyanogen bromide, or iodine, plus triphenylphosphine. When 5‘-O-acetylinosine was treated with triphenylphosphine and carbon tetrachloride, the 3’-chloro-3’-deoxy derivative was formed, with inversion of configuration. (68) C. R. Haylock, L. D. Melton, K. N. Slessor, and A. S. Tracey, Carbohyd. Res., 16, 375 (1971). (69) B. T. Lawton, W. A. Szarek, and J. K. N. Jones, Carbohyd. Res., 14, 255 (1970). (70) K. Haga, M. Yoshikawa, and T. Kato, Bull. Chem. Soc. l a p . , 43, 3922 (1970).

248

WALTER A. SZAREK

The results of a more comprehensive study of the synthesis of nucleosides of halogeno sugars by use of triphenylphosphine-carbon tetrahalide reagents have been described by Verheyden and M ~ f f a t t . ~ ~ The reactions were performed in N,N-dimethylformamide or N,N-dimethylacetamide. The replacement of primary hydroxyl groups by chlorine or bromine is very rapid and efficienP; for example, treatment of 2’,3’-O-isopropylideneuridine with triphenylphosphine and carbon tetrachloride in N,N-dimethylformamide at room temperature afforded the 5’-chloro-5’-deoxy nucleoside in an isolated yield of 70%, and bromination of 2’,3’-O-isopropylideneinosinein N,N-dimethylacetamide gave the 5‘-bromo-5’-deoxyderivative in 49% yield. Only a low yield of 5’-deoxy-5‘-iodo-2’,3’-O-isopropylideneuridine was obtained with carbon tetraiodide and triphenylphosphine in N,N-dimethylformamide; however, in pyridine, the iodinated nucleoside was obtained in 76% yield. Some selective halogenation at C-5’ of unprotected nucleosides was also achieved. The halogenation of secondary hydroxyl groups by means of triphenylphosphine and carbon tetrahalides normally proceeds with inversion of c o n f i g ~ r a t i o n . ~Treatment ~(~) of 5’-O-tritylthymidine with triphenylphosphine and carbon tetrachloride in N,N-dimethylformamide at room temperature afforded l-(3-chloro-2,3-dideoxy-5-Otrityl-p-D-threo-pentofuranosy1)thyminein 35% yield plus the Deythro isomer in 15% yield.71In contrast to this result, the reaction of 5’- 0-( p - nitrobenzoy1)thymidine with triphenylphosphine and iodine led to 3’-deoxy-3’-iodo-5’-O-(p-nitrobenzoyl)thymidine, with complete retention of configuration; the stereochemistry observed can be explained by intervention of a 2,3’-anhydronucleoside as an intermediate. As the chloride ion is both smaller and more nucleophilic than the iodide ion in N,N-dimethylformamide, the direct sN2 displacement of a 3’-(oxyphosphonium) function can compete favorably, in the case of chloride ion, with the intramolecular displacement by the 2-carbonyl group of the thymine moiety; thus, the chlorination reaction leads preponderantly to the “inverted,” D-thW0 compound. Pyrimidine nucleosides containing a free, cis-2’,3’-diol grouping undergo, on treatment with triphenylphosphine and carbon tetrachloride in N,N-dimethylformamide, fairly selective chlorination of the 2’-hydroxyl group, with retention of configuration. Thus, (71) J. P. H. Verheyden and J. G . Moffatt,J. Org. Chern., 37,2289 (1972). (72) Although the use of N,N-dimethylformamide permits satisfactory chlorination or bromination of primary positions at room temperature, side reactions can occur between carbon tetrahalides, triphenylphosphine, and N,N-dimethylformamide that can, in some cases, result in low yields of halogenated products (see also, Section 11, 2c; p. 230).

DEOXYHALOGENO SUGARS

249

5’-O-acetyluridine gave 5’-O-acetyl-2’-chloro-2’-deoxyuridine in 38% yield, presumably by way of a 2,2’-anhydronucleoside intermediate. have obtained, in 73% yield, the 3‘-chloroG&o and 3’-deoxy C-nucleoside analog 38b on treatment of the diol 38a with triphenylphosphine and carbon tetrachloride at reflux temperature.

Sh

Ph

/

VN OH

OH

HO 38a

38b

Ponpipom and H a n e ~ s i a nhave ~ ~ described an effective and selective method for the replacement of the primary hydroxyl group in carbohydrates by a bromine atom by treatment of the alcohol with two equivalents each of N-bromosuccinimide and triphenylphosphine in N,N-dimethylformamide or dichloromethane. A variety of h n c tional groups, including ester, amide, and acetal groups, are compatible with the reaction conditions. However, acetal migration can occur; for example, 1,2:5,6-di-O-isopropylidene-~-D-glucofuranose (21; see p. 239) affords, in high yield, 6-bromo-6-deoxy-1,2:3,5-di0-isopropylidene-a-D-glucohranose. A particularly attractive feature of the method is the possibility of selective bromination of primary hydroxyl groups in polyhydroxy compounds. Thus, methyl a-D-glucopyranoside in N,N-dimethylformamide for 1 hour at 50”afforded, after acetylation, methyl 2,3,4-tri-0-acetyl-6-bromo-6-deoxy-a-~-glucopyranoside in an overall yield of 66%. The bromination reaction has also been applied to nucleosides; for example, from 2’,3’-O-benzylideneuridine was obtained the 5’-bromo-5’-deoxy derivative in 72% yield.74 It has been found that treatment of 2’,3’-O-isopropylideneuridine with N-bromosuccininimide and triphenylphosphine in N,Ndimethylformamide containing an excess of tetrabutylammonium (72a) F. Nouaille, A. M. Sepulchre, C . Lukacs, and S. D. CCro, Compt. Rend., Ser. C, 275,423 (1972). (73) M. M. Ponpipom and S. Hanessian, Curbohyd. Res., 18, 342 (1971). is treated with N-bromosuccinimide in a chlori(74) If 2’,3’-O-benzylideneuridine nated solvent, in the ubsence oftriphenylphosphine, the product is 3‘-0-benzoyl2’,5-dibromo-2’deoxyuridine (see Section II,4;p.276).

250

WALTER A. SZAREK

iodide produces the 5’-deoxy-5‘-iododerivative, presumably by way of attack by iodide ion on a 5’-(oxyphosphonium) intermediate. It was subsequently reported74a that iodination or chlorination of carbohydrates could be efficiently achieved by treatment with triphenylphosphine and N-iodosuccinimide or N-chlorosuccinimide, respectively, in N,N-dimethylformamide. Selective chlorination or bromination of the 5’-hydroxyl group in unprotected D-ribonucleosides has been achieved75by treatment with thionyl chloride or thionyl bromide, respectively, in hexamethylphosphoric triamide at room temperature for 10-15 hours; the 5’deoxy-5’-halogeno derivatives of cytidine and adenosine have been prepared in this way. The reactions presumably occur by way of alkoxyphosphonium salts as intermediate^.'^ Other examples of the use of phosphorus-containing reagents for the synthesis of deoxyhalogeno sugars, by way of Arbusov-type reactions, are described in Section II,5 (see p. 278). c. Reaction with (Halogenomethy1ene)dimethyliminiumHalides. -N,N-Dimethylformamide has been reported77to react with halides of inorganic acids (phosgene, phosphoryl chloride, phosphorus trichloride, and thionyl chloride) to form an active intermediate, namely, (chloromethy1ene)dimethyliminium chloride78 (39). Originally, 39 was employed as a formylating agent for aromatic, heterocyclic, and ethylenic compounds. However, 39 and its bromine analog (40) have been found to be highly effective for replacing hydroxyl groups by halogen. The method involves the thermal decomposition in solution of the adduct,80such as 41, formed from the alcohol and the strongly electrophilic (halogenomethy1ene)dimethyliminium halides1;formic esters can also be produced.82 (74a) S. Hanessian, M. M. Ponpipom, and P. LavallBe, Curbohyd. Res.,24,45 (1972). (75) K. Kikugawa and M. Ichino, Tetrahedron Lett., 87 (1971). . (76) Compare, G. Gawne, G. W. Kenner, and R. C. Sheppard,]. Amer. Chem. SOC., 91, 5669 (1969). (77) A. Vilsmeier and A. Haack, Ber., 60,119 (1927); A. Vilsmeier, Chem.-Ztg.,Chem. App., 75,133 (1955); Z. Arnold, Collect. Czech. Chem. Commun., 25,1313 (1960); H. H. Bosshard and H. Zollinger, Angew. Chem., 71, 375 (1959); Helu. Chim. Actu, 42, 1659 (1959). (78) In a study” of Vilsmeier-Haack adducts, it was concluded that the adducts of N,N-dimethylformamide with phosphoryl chloride, thionyl chloride, and phosRi

gene have the structure (Me,N=CHCI)OXe, in which X is P0Cl2, SOCI, or COCI, but that, in the latter two cases, SO, or C 0 2 is readily evolved to give the same adduct, (Meg=CHC1) Cle. (79) M. L. Filleux-Blanchard, M. T. Quemeneur, and G. J. Martin, Chem. Commun., 836 (1968); see also, G. Martin and M. Martin, Bull. SOC. Chim. Fr., 1637 (1963). (80) The adduct has been represented as an imino ester salt, although such a structure could be in equilibrium with related species.

DEOXYHALOGENO SUGARS

ROH

+

8

(Me,N=CHX)X@ 39

x = c1

40 X = B r

251

-

-

RX

+

Me,NCHO

41

Hanessian and P l e s ~ a were s ~ ~ the first workers to apply the method to the synthesis of deoxyhalogeno sugars. Thus, treatment of 1,2:3,4di-0-isopropylidene-a-D-galactopyranose (42)with 39 in a chlorinated solvent, such as 1,1,2-trichloroethylene, gave the imino ester intermediate 43 which, on treatment with an aqueous solution of sodium hydrogen carbonate, afforded mainly the formic ester 44; however, heating of the reaction mixture gave 6-chloro-6-deoxy-1,2:3,4-di-0isopropylidene-a-D-galactopyranose (45) in high yield.84 H

€&coc=o I

0-CMG

42

43

Me&

l o ? O-CMe, 45

(81) H. Eilingsfeld, M. Seefelder, and H. Weidinger, Chem. Ber., 96, 2671 (1963). (82) Z. Arnold, Collect. Czech. Chern. Commun., 26, 1723 (1961). (83) (a) S. Hanessian and N. R. Plessas, Chem. Commun., 1152 (1967); (b)J. Org. Chem., 34, 2163 (1969).

252

WALTER A. SZAREK

It has been found that the reaction of 39 with carbohydrate derivatives containing cyclic acetal groupings may be accompanied by a rearrangement of these groups. Thus, when 1,2:5,6-di-O-isopropylidene-a-D-glucofuranose (21)was treated with 39 at room temperature in 1,1,2,2-tetrachloroethane,the major product obtained was the 3-formic ester of 21; however, heating of a solution containing 21 and 39 at reflux temperature afforded 6-chloro-6-deoxy-1,2:3,5-di-0isopropylidene-a-sglucohranose (22) in 70% yield. A mechanism for the conversion of 21 into 22 has been suggested by HanessianaE5 The reaction of methyl 2-azido-4,6-0-benzylidene-2-deoxy-a-~altropyranoside (46) with 39 in refluxing 1,1,2,2-tetrachloroethane afforded a product which was f o r m ~ l a t e d ~as ~ ( methyl ~) 2-azido3,4-0-benzylidene-6-chloro-2,6-dideoxy-a-~-altropyranoside (47); the n.m.r. spectrum indicated that the product was a mixture of two diastereoisomers which differed in the configuration of the carbon atom of the benzylidene acetal. A possible mechanism for the formation of 47 is outlined. (QC1

OOMe

phc: 0 @

PhC H/O Q

O

M

e

2T/o

o,

M%N=C H

MqN=C

U H

Po\

ITo, t L

~(84)Treabnent of 1,2:3,4-di-O-isopropy~idene-a-~-galactopyranose (42) with an excess of (ch1oroethylidene)dimethyliminium chloride, [ M e , h ( M e ) C l ] C l e , and heating the reaction mixture, afforded the 6-acetate and, only in low yield, the 6-chloro-6-deoxy derivative (45). The diminished yield of 45 in this case is, presumably, the result of steric hindrance to the attack by chloride ion at C-6; moreover, the extra methyl group would tend to stabilize the imino ester intermediate.

DEOXYHALOGENO SUGARS

253

The reaction of 39 with methyl 4,6-0-benzylidene-2-deoxy-2-iodoa-D-altropyranoside (48) gave a complex mixture of products, from which methyl 4,6-0-benzylidene-2,3-dideoxy-a-~-erythro-hex-2-enopyranoside (50) could be isolated. The formation of 50 was explainede3cb)by attack by chloride ion on the iodine atom in intermediate 49, followed by elimination of the substituent at C-3. Compound 50 itself reacts with reagent 39, and, therefore, prolonged reaction times led to extensive decomposition.

Dods and Rothe6 have employed the reagents 39 and 40 for halogenating the carbohydrate moieties of nucleosides. It was found that the reactions of 2’,3’-O-isopropylideneuridine or of uridine with 39 or 40 in anhydrous N,N-dimethylformamide afforded 5’-chloro(bromo)5’-deoxy-2’,3’-0-isopropylideneuridine and 5’-chloro(bromo)-5’-deoxyuridine in high yields; the specificity with respect to the 5’-position in the case of uridine is particularly noteworthy. However, Kikugawa and Ichinoe7have reported that the reaction of 39 with cytidine gives 2,2’-anhydrocytidine. In attempts to synthesize arsenic-containing nucleosides, Dods and Rothe6cb)treated 2’,3’-O-isoprspylideneuridine and uridine, respectively, with arsenic trichloride, and with arsenic tribromide, in anhydrous N,N-dimethylformamide; the 5’deoxy-5’-halogeno derivatives were again obtained, in yields of 40-62%. Because arsenic trihalides have not been known to convert alcohols into alkyl halides, it is possible that arsenic trihalides react with N,N-dimethylformamide to produce (halogenomethy1ene)dimethyliminium halides. It was also reportede6@)that treatment of uridine with arsenic trichloride in N,N-dimethylacetamide at 127” gives 3’-chloro-3’-deoxyuridine in an isolated yield of 20%; however, in a recent re-e~arnination~l of this work, it was found that the preponderant products are 5’-O-acetyluridine and derivatives of 2‘(85) Ref. 7, pp. 194-195. (86) (a) R. F. Dods and J. S . Roth, Tetruhedron Lett., 165(1969);(b)J. Org. Chern., 34, 1627 (1969). (87) K. Kikugawa and M. Ichino, Tetrahedron Lett., 867 (1970).

254

WALTER A. SZAREK

chloro-2'-deoxyuridine. The acetylated compounds are produced, presumably, by way of Vilsmeier-Haack type of adducts of N,N-dimethylacetamide with arsenic trichloride. A hrther use of reagent 39, namely, in the synthesis of deoxyhalogeno sugars by way of cleavage of oxiranes, is discussed in Section II,5 (see p. 278). The reaction of halogenotriphenylphosphonium halides (triphenylphosphine dihalides) with alcohols is a useful method for the synthesis of alkyl halides (see Section II,2b; p. 239). It has been found**that (alkoxymethy1ene)dimethyliminium halides are formed in the reactions of these reagents with alcohols in N,N-dimethylformamide; a possible mechanism is shown. Hydrolysis of the (alkoxymethy1ene)dimethyliminium halide intermediate affords a formic ester, whereas Ph,$ O

Me,N=C

P \

H

X@

P&P=O

+

8 ,OR MqN=C,

H X@

-

c;.

P&,q Me,N-C,

*dI OR H

alkyl halides are obtained by thermal decomposition. It is notable that Verheyden and Moffatt71have found that the reaction of 1,2:5,6di-O-isopropylidene-a-D-glucofuranose (21)with triphenylphosphine and carbon tetrachloride in N,N-dimethylformamide at room temperature leads to the 3-O-formyl derivative of 21 as the major product, whereas Slessor and coworkersss obtained 6-chloro-6-deoxy-1,2:3,5di-O-isopropylidene-a-D-glucofuranose (22)by the reaction of 21 in refluxing carbon tetrachloride containing triphenylphosphine. The f o r m a t i ~ n ~of~ (the ~ ) 3-O-formyl derivative of 21, by way of the reaction of 21 with (chloromethy1ene)dimethyliminiumchloride (39), has already been mentioned in this Section (see p. 252). Clearly, the use of N,N-dimethylformamide as a solvent in halogenation reactions involving triphenylphosphine can lead to side rea~tions.7~ (88) M. E. Herr and R. A. Johnson,J. Org. Chem., 37,310 (1972).

DEOXYHALOGENO SUGARS

255

d. Other Reactions. - One of the side reactions most commonly encountered during the sulfonylation of carbohydrates with a sulfonyl chloride in pyridine is formation of chlorodeoxy derivatives. The chlorinated products result from the sulfonic ester by nucleophilic attack by chloride ion obtained from the pyridinium chloride formed during the sulfonylation. Generally, chlorination is the more prevalent, the higher the temperature at which the sulfonylation is performed. Several examples of sulfonylation reactions leading to the formation of chlorodeoxy sugars have been documented by Tipsong in Volume 8 of this Series. A particularly interesting, early resultE9 was the formation of l-(5-chloro-5-deoxy-2,3-di-O-p-tolylsulfonyl-p-D-ribofuranosyl)uracil,on p-toluenesulfonylation of uridine in pyridine during 18 hours at room temperature. In this reaction, the displacement of the 5’-sulfonyloxy group is, presumably, enhanced by participation of the oxygen atom at C-2 of the pyrimidine ring, to give a 2,5’-anhydronucleoside intermediate which is then attacked by chloride ion to give the 5’-chloro-5’-deoxy derivative. Long and coworkersw have examined in detail the formation of chlorodeoxy glycosides during sulfonylation of methyl a- and p-Dglucopyranosides. It was established that the compounds formulated, several years earlier,9l as methyl 4-chloro-4-deoxy-2,3,6-tri-O.p-tolylsulfonyl-a- and -@-D-glucopyranosideare, in fact, methyl 6-chloro-6deoxy-2,3,4-tri-O-p-tolylsulfonyl-aand -p-D-glucopyranoside; these compounds were obtained by using p-toluenesulfonyl chloride and pyridine at 75”. A second component obtained from methyl a - ~ glucopyranoside was shown to be methyl 4,6-dichloro-4,6-dideoxy-2,3di-0-p-tolylsulfonyl-a-D-galactopyranoside, not the D-ghco isomer (as had been statede1).Chlorination was also observed when p-toluenesulfonylation of methyl a- or p-D-glucopyranoside was performed for 16 days at 27”. The a - anomer ~ afforded tetra-0-p-tolylsulfonyl and 6-chloro-6-deoxy-2,3,4-tri-O-p-tolylsulfonyl derivatives in yields of 59% and 37%, respectively, whereas the p-D anomer gave 90% of tetra-0-p-tolylsulfonyl derivative, and only 4% of 6-chloro-6-deoxy2,3,4-tri-O-p-tolylsulfonyl derivative. Methanesulfonylation of methyl a-D-glucopyranoside at 75” afforded methyl 6-chloro-6-deoxy-2,3,4-tri0-( methylsulfony1)-a-D-glucopyranosideand methyl 4,6-dichloro-4,6dideoxy-2,3-di-0- (methylsulfony1)-a-D-galactopyranoside; methyl p-D-glucopyranoside gave methyl 6-chloro-6- deoxy- 2,3,4- tri- 0(89) P. A. Levene and R. S. Tipson,J. Biol. Chem., 105,419 (1934). (90) F. W. Parrish, F. H. Bissett, M. E. Evans, M. L. Bazinet, W. Yeomans, and L. Long, Jr., Curbohyd. Res., 6, 503 (1968). (91) K. Hess and H. Stenzel, Ber., 68, 981 (1935).

256

WALTER A. SZAREK

(methylsulfonyl)-/3-D-glucopyranoside and methyl 4,6-dichloro-4,6dideoxy-2,3-di-0- (methylsulfonyl)-/3-D-glucopyranoside,but no methyl 4,6-dichloro -4,6-dideoxy-2,3-di-O-(methylsulfonyl)-/3-~-galactopyranoside was found. In another it was observed that methanesulfonylation of methyl 2-acetamido-3-O-acetyl-2-deoxy-a-~glucopyranoside in pyridine at room temperature yields a substantial proportion of methyl 2-acetamido-3-O-acetyl-6-chloro-2,6-dideoxy4-O-(methylsulfonyl)-a-~-glucopyranoside, in addition to the 4,6-disulfonate. Of relevance to the results just described is the observationg3 of the formation of significant proportions of 6,6’-dichloro-6,6’-dideoxysucrose hexabenzoate and a monochloro-monodeoxy-mono-0-ptolylsulfonylsucrose hexabenzoate, on treatment of 6,6’-di-O-ptolylsulfonylsucrose with benzoyl chloride in pyridine at room temperature. These products arise by nucleophilic substitution of the p-tolylsulfonyloxy groups by the chloride ion of pyridinium chloride; the isolation of a monochloro-monodeoxysucrose derivative indicated a difference in reactivity of the p-tolylsulfonyloxy groups at C-6 and C-6’. It has been founds4that the use of methanesulfonyl chloride in N,Ndimethylformamide permits selective replacement of primary hydroxyl groups in hexopyranosides by chlorine to be achieved in high yield. A possible mechanism for this reaction involves nucleophilic attack by an alcohol (ROH) on the iminium salt ( M e , N ~ C H O S O , Me)Cle, derived from methanesulfonyl chloride and N,N-dimethylformamide, to give an intermediate Me,NkCHOR, which is then attacked by chloride ion. The reaction affords a convenient, direct method for preparing, from such polysaccharides as cellulose and amylose, the 6-chloro-6-deoxy derivatives of various degrees of substitution.94a Sinclairg5has described the preparation of methyl 6-chloro-6-deoxya-D-glucopyranoside from methyl a-D-glucopyranoside by treatment with sulfur monochloride (S,Cl,) in N,N-dimethylformamide; a yield of 30-35% was obtained on separation of the reaction products on a column of Darco G-60-Celite 535. The Pinner reactione6 has been employed for the preparation of (92) J. Hill and L. Hough, Carbohyd. Res., 8, 398 (1968). (93) C. H. Bolton, L. Hough, and R. Khan, Carbohyd. Res., 21, 133 (1972). (94) M. E. Evans, L. Long, Jr., and F. W. Parrish,]. Org. Chem., 33, 1074 (1968). (944 D. Horton, A. E. Luetzow, and 0. Theander, Carbohyd. Res., 26, 1 (1973); 27,268 (1973). (95) H. B. Sinclair,]. Org. Chem., 30, 1283 (1965). (96) R. Roger and D. G. Neilson, Chem. Rev., 61, 179 (1961); see pp. 188-189.

DEOXYHALOGENO SUGARS

257

deoxyhalogeno sugars from carbohydrate derivatives containing “isolated,” primary hydroxyl groups.s7 The method involves the addition of dry hydrogen halide to a mixture of the substrate and trichloroacetonitrile in chloroform in the absence of water; an intermediate hydrohalide salt of an imino ester is formed, which then affords trichloroacetamide and the product halide, probably by a bimolecular process. Methyl 2,3,4-tri-0-acetylB-chloro-6-deoxy-a-~-

ROH

+

CC1,CN

+

HX

-

-

0 II

CCl,CNH,

+

RX

glucopyranoside, methyl 2,3,4-tri-0-acetyl-6-bromo-6-deoxy-a-~-g~ucopyranoside, methyl 2,3,4-tri-0-acetyl-6-bromo-6-deoxy-a-D-mannopyranoside, and benzyl 2-acetamido-3,4-di-O-acetyl-6-bromo-2,6dideoxy-a-D-glucopyranoside have been prepared in high yields by this method. In a related study, Klemer and M e r ~ m a n n ~ found ’ ~ that addition of carbohydrate derivatives containing an “isolated” hydroxyl group to aryl cyanates, in the presence of hydrogen chloride, led to the formation of the hydrochlorides of the aryloxycarbonimidoyl derivatives of the starting sugars; these compounds are thermally unstable and decompose to aryl carbamates and the corresponding chlorodeoxy sugars. Methyl 2,3,4-tri-0-methyl-a-D-glucopyranoside ROH

+

ArOCN

+

HCl

-[

ArOCORCl’

@hH,

1

0 -ArOCNH,

II

+

RC1

and 1,2,3,4-tetra-O-acety~-p-D-ghcopyranose afforded the corresponding 6-chloro-6-deoxy derivatives in high yields, whereas methyl 2,4,6-tri-O-methyl-p-D-glucopyranoside gave methyl 3-chloro-3-deoxy2,4,6-tri-O-methyl-~-D-allopyranoside in only 17% yield. Kent and coworkerss8 have reported that N-(2-chloro-l,l,2-trifluoroethy1)diethylamine (51) reacts with 1,2:3,4-di-O-isopropylidenea-D-galactopyranose (42) in N,N-dimethylformamide at 60°, in the presence of anhydrous potassium fluoride, to give a mixture from which 6-chloro-6-deoxy-1,2:3,4-di-O-isopropylidene-~-~-galactopyra(97) M. L. Shulrnan, V. N. Yoldikov, and A. Ya. Khorlin, Tetrahedron Lett., 2517 (1970). (97a) A. Klemer and G. Mersrnann, Carbohyd. Res., 22, 425 (1972). (98) K. R. Wood, D. Fisher, and P. W. Kent,J. Chem. SOC. (C), 1994 (1966).

WALTER A. SZAREK

258

nose (45) can be isolated. N-(2-Chloro-1,1,2-trifluoroethyl)diethylamine has been used for the conversion of alcohols,QQincluding steroidal alcohols,loOinto fluorides. However, in the work of Kent and coworkers,g8there was no evidence of the presence of 6-deoxy6-fluoro-1,2:3,4-di-O-isopropylidene-a-~-galactopyranose. In the reaction of the amine 51 with alcohols, the reactive intermediate 52 is -F@ Et,N-CF,-CHClF 51

--[ ROH

-Fa

8 Et,N=C-CHClF

t/

bR-J

52

RX

+

J

Et,N-C-CHClF II

0

consideredlooto be involved; the alkyl fluoride is then the result of an attack on 52 by the fluoride ion eliminated in the formation of 52. Chloride or bromide ions should thus compete with fluoride ion. Indeed, it has been foundlo' that the reaction of lla-hydroxy-19norsteroids with the reagent 51 in the presence of an excess of lithium chloride in tetrahydrofuran gives the 1lp-chloro derivatives in good yield; the 1lp-bromo derivatives are obtained, in lower yield, by use of lithium bromide in dichloromethane. Thus, the formation, in the work of Kent and coworkers,0s of a chlorodeoxy sugar in the presence of potassium fluoride is surprising. It was found in that study that 51 reacts, at room temperature in dichloromethane, with 1,2:5,6-di-O-isopropylidene-a-~-glucofuranose (21), 1,2:3,4-di-Oisopropylidene-a-D-galactopyranose (42), and methyl 3,4-O-isopropylidene-p-L-arabinopyranosideto give the corresponding 3-, 6-, and 2-O-(chlorofluoroacetyl) derivatives; the formation of deoxyhalogeno sugars was not observed in these experiments. It has been reportedlo2that cyanuric chloride (2,4,6-trichloro-l,3,5triazine, 53) reacts with anhydrous alcohols to produce the corre(99) N. N. Yarovenko and M. A. Raksha, Zh. Obshch. Khim., 29, 2159 (1959); Chem. Abstr., 54,9724 (1960). (100) L. H. Knox, E. Velarde, S . Berger, D. Cuadriello, and A. D. Cross J. OTg. Chem., 29,2187 (1964). (101) E. J. Bailey, H. Fazakerley, M. E. Hill, C. E. Newall, G. H. Phillipps, L. Stephenson, and A. Tulley, Chem. Commun., 106 (1970). (102) S. R. Sandier,]. Org. Chem., 35,3967 (1970); see also, Chem. Ind. (London), 1416 (1971).

DEOXYHALOGENO SUGARS

259

sponding alkyl chloride and cyanuric acid in good yields; the reaction is as shown. The reaction of 53 with some di-o-isopropylidene-

hexoses containing "isolated" hydroxyl groups has been investigated.lo3Treatment of 1,2:5,6-di-O-isopropylidene-a-~-g~ucofuranose (21), or of 1,2:5,6-di-O-isopropylidene-a-~-allofuranose, with 53 in N,N-dimethylformamide for 2 hours at -75" afforded, after the addition of water, the corresponding formic esters; from the reaction with 21 was also obtained 6-chloro-6-deoxy-1,2:3,5-di-O-isopropylidene-a-D-glucofuranose (22) in low yield. The reaction of 53 with 1,2:3,4-di-O-isopropy~idene-a-~-galactopyranose (42) in N,N-dimethylformamide for 90 minutes at 75" gave an approximately 1 : l mixture of the 6-formate (44) and the 6-chloro-6-deoxy derivative (45). The formation of formic esters in the preceding examples is not surprising in view of a reportlo4 that cyanuric chloride and N,Ndimethylformamide react at room temperature to give a crystalline adduct which, on being heated at 50-60", evolves carbon dioxide and @

Cl0. A rationalization gives the salt ( Me2N-CH=N-CH=NMe2) for the formation of formic esters is, then, as shown. 0

(MqN-CH=N-CH=NMe,)

C1°

+

ROH +HC1

ROCHO

+

+

M%N-CH=N-CH-NMq I OR

Me,N-CH=NH

+

0 0 Me2N&C1

In order to obviate the formation of a formic ester, the reaction of 1,2:3,4-di-O-isopropy~idene-a-D-galactopyranose (42) was performed in p-dioxane. The 6-chloro-6-deoxy derivative (45) was again obtained, but only in low yield; the major product was assigned the novel structure of 6-0-(4,6-dichloro-1,3,5-triazin-2-yl)-1,2:3,4-di-Oisopropylidene-a-D-galactopyranose (54). (103)A. Zamojski, W.A. Szarek, and J. K. N. Jones, Carbohyd. Res., 26, 208 (1973). (104)H.Gold, Angew. Chem., 72,956 (1960).

260

WALTER A. SZAREK

0-CMe, 54

Aliphatic primary and secondary alcohols have been foundlo5to react with N,N'-dicyclohexyl-N-methylcarbodiimidiumiodide (55), in tetrahydrofuran, benzene, or hexane at 35-50", to give the corresponding iodides in high yields; the reagent 55 is prepared by heating a mixture of N,N'-dicyclohexylcarbodiimide and methyl iodide. The iodination reaction has been extended to steroidal alcohols, but attempts with carbohydrate substrates have not yet been reported. CBH,,-N=C=N-C,H,,

Me1

C,H,,,@ Me'

N=C=N-CCBH,, I@ 55

Tabushi and coworkerslo6 have reported that dichlorocarbene reacts very readily with alcohols to give the corresponding chlorides. Dichlorocarbene was generated by using an emulsifying system; the method involves treatment of the alcohol with chloroform in the presence of an aqueous solution of sodium hydroxide and a catalytic amount of benzyltriethylammonium chloride. 3. Additions to Unsaturated Carbohydrates

The increased availability of methods for the preparation of unsaturated sugarslo7 has stimulated interest in addition reactions of these compounds; the use of unsaturated carbohydrate derivatives as precursors for specifically halogenated sugars has been widely exploited. (105) R. Scheffold and E. Saladin, Angew. Chem., 84,158 (1972). (106) I. Tabushi, Z. Yoshida, and N. Takahashi,J. Amer. Chem. SOC., 93, 1820 (1971). (107) (a) R. J. Ferrier, Adoan. Carbohyd. Chem., 20, 67 (1965);(b)Adoan. Carbohyd. Chem. Eiochem., 24, 199 (1969).

DEOXYHALOGENO SUGARS

26 1

a. To Glycals. -Addition, involving halogens, to glycals is one of the most common ways of obtaining deoxyhalogeno sugars having halogen at C-2; addition of halogen furnishes a glycosyl halide substituted at C-2, from which the halogen at C-1 is readily removed, leaving the 2-halide. Ferrier107has comprehensively surveyed the studies of additions of a wide variety of reagents to glycals in two Chapters of this Series; only some selected results will, therefore, be described in this Section. A summary of results of addition of halogen to l,2-unsaturated sugars has also been presented in the previous Chapter' on halogenated carbohydrates. The synthesis of the relatively rare sugars having gem-dihalogen substitution at C-2 can be accomplished by way of halogen additions to 1,2-unsaturated substrates. In 1963, Vargha and KuszmannlORreported the conversion of di-0-acetyl-D-arabinal into 1,3,4-tri-O-acetyl2,2-dichloro-2-deoxy-D-e~ythro-pentopyranose. Chlorination of di0-acetyl-D-arabinal gave a mixture of 1,2-dichloro derivatives which, on treatment with sodium phenoxide, followed by fractional distillation, yielded a mixture of phenyl2-chloro-2-deoxy-~-glycosides and pure, syrupy 3,4-di-O-acetyl-2-chloro-~-arabinal. Further chlorination of the latter material, and displacement of the chlorine atom at C- 1 by an acetate group, gave crystalline 1,3,4-tri-O-acety1-2,2-dichloro-2-deoxy-~-erythro-pentopyranose. Bradley and Bunce1108a have since prepared 2,2-dichloro-2-deoxy-a-~-urubino-hexopyranosyl chloride, an example of a stable, free glycopyranosyl chloride.10s Chlorination of tri-0-acetyl-D-gluca1 in cold carbon tetrachloride affordedloRa 3,4,6-tri-0-acetyl-2-chloro-2-deoxy-a-~-g~ucopyranosy~ chloride and 3,4,6-tri-O-acetyl-2-chloro-2-deoxy-~-~-mannopyranosy~ chloride in the ratio of - 4:1.Treatment of a benzene solution of these two glycosyl chlorides with diethylamine yielded unchanged 3,4,6tri-O-acetyl-2-chloro-2-deoxy-~-D-mannopyranosy~ chloride and 3,4,6tri~-acety~-2-ch~oro-D-g~ucal; the substituted mannosyl chloride was isolated by crystallization, and 3,4,6-tri-O-acetyl-2-chloro-D-glucal was obtained by fractional distillation of the residue. The facile dehydrochlorination of 3,4,6-tri-0-acetyl-2-chloro-2-deoxy-a-D-g~ucopyranosyl chloride, in contrast to the behavior of the p - ~ - m a n n o isomer, is readily explained by the fact that, in the a-D-ghco isomer, the chlorine atom at C-1 and the hydrogen atom at C-2 have a trunsdiaxial relationship, which is most favorable for normal E2 elimination in cyclic systems; in the p-~-mannoisomer, the chlorine atom at (108) L. Vargha and J. Kuszmann, Chem. Ber., 96,411 (1963). (108a) P. R. Bradley and E. Buncel, Ca9i.J. Clzern., 46, 3001 (1968). (109) M. L. Wolfrom, H. G. Garg, and D. Horton, J . Org. Chem., 28, 2989 (1963).

262

WALTER A. SZAREK

C-1 and the hydrogen atom at C-2 are diequatorial, and, hence, elimination would be a much slower process. Further chlorination of 3,4,6-tri-0-acetyl-2-chloro-D-glucal afforded crystalline 3,4,6-triO-acetyl-2,2-dichloro-2-deoxy-~-arubino-hexopyranosyl chloride. It is interesting that attempts to convert this trichloro derivative into a methyl glycoside, under Koenigs-Knorr conditions, were unsuccessh l , presumably because the inductive (-1) effect of the chlorine atoms at C-2 makes ionization of the chlorine atom at C-1 difficult. O-Deacetylation of 3,4,6-tri-0-acetyl-2,2-dichloro-2-deoxy-~-urubi~ohexopyranosyl chloride with dry, methanolic ammonia gave crystalline 2,2-dichloro-2-deoxy-a-~-arubino-hexopyranosyl chloride; the loss of the chlorine atom at C-1 during O-deacetylation is, again, prevented by the inductive effect just mentioned. Adamson and Fosterllo have also prepared 3,4,6-tri-O-acetyl-2,2-dichloro-2-deoxyD-arabino-hexopyranosyl chloride. Hydrolysis of this compound with hydrochloric acid yielded crystalline 2,2-dichloro-2-deoxy-~-arabinohexose. Full details of the chlorination of tri-0-acetyl-D-glucal (56) by molecular chlorine have now been published by Igarashi and coworkers."' It was found that the proportions of the resulting dichlorides are dependent upon the polarity of the solvent used. In such nonpolar solvents as carbon tetrachloride, diethyl ether, chloroform, dichloromethane, and 1,2-dichloroethane, cis-addition products, namely, 3,4,6-tri-0-acetyl-2-chloro-2-deoxy-a-~-glucopyranosyl chloride and 3,4,6-tri-O-acetyl-2-chloro-2-deoxy-~-~-mannopyranosyl chloride, were preponderantly obtained. In polar solvents, such as nitromethane and propylene carbonate, trans-addition products, namely, 3,4,6-tri-O-acetyl-2-chloro-2-deoxy-~-~-glucopyranosyl chloride and 3,4,6-tri- 0-acetyl-2- chloro- 2- deoxy-a - D- mannop yranosy1 chloride, were the preponderant products. The results were interpreted by a mechanism which involves rapid, reversible formation of a chlorine-alkene complex, followed by rate-determining ionization to the ion pairs, 57 and 58, in which the chloride ion is associated on the same side of the original plane from which chlorine attack first occurred. Collapse of 57 and 58 gives cis-addition products. Alternatively, 57 and 58 may rearrange to the free ions 59 and 60, respectively, in which the chloride ion is now on the opposite side of the plane; the free ions, 59 and 60, lead to trans-addition products. As charge separation is favorable in polar solvents but unfavorable in nonpolar solvents, the preponderant trans addition in polar sol(110) J. Adamson and A. B. Foster, Corhohyd. Res., 10, 517 (1969). (111) K. Igarashi, T. Honma, and T. Imagawa,]. Org. Chem., 35, 610 (1970).

DEOXYHALOGENO SUGARS

263

C%OAc

;t;B

AcO

56

\

cl--clOO

u

+

1' -

complex

-----+

CB

\

P Cl---Cl@

57

P -D-manno

59

ff- D - g h C O

60

vents and cis addition in nonpolar solvents are understandable. Igarashi and Honma'" have also studied the chlorination of tri-0acetyl-D-glucal by iodobenzene dichloride. Deoxyhalogeno sugars have been obtained from the reaction of acetylated glycals with hydrogen halides. In 1920, E. Fischer and coworker^"^ claimed that hydrobromic acid can add to tri-0-acetyl-Dglucal (56) in acetic acid to give a crystalline product having a stable carbon-bromine bond. However, attempted repetition of this work by Davoll and Lythgoe114yielded a syrup which had the reactivity expected of a glycosyl bromide. This anomaly was resolved in 1967 by Maki and Tejirna.ll5 Tri-0-acetyl-D-gluca1 (56) was treated with hydrogen bromide in glacial acetic acid for 5 hours at O", and, after processing of the reaction mixture, 3,4,6-tri-O-acetyl-2-deoxy-a-~arubino-hexose (61) and 4,6-di-O-acetyl-3-bromo-2,3-dideoxy-a-~arabino-hexose (62) were isolated, each in a crystalline state. (112) K. Igarashi and T. Honma,J. Org. Chem., 35, 617 (1970). (113) E. Fischer, M. Bergmann, and H. Schotte, Ber., 53,509 (1920). (114-)J. Davoll and B. Lythgoe, J. Chem. SOC.,2526 (1949). (115) T. Maki and S. Tejima, Chem. Pharm. Bull. (Tokyo), 15, 1069 (1967).

WALTER A. SZAREK

264

IG;,

AcO

56

?-I,OAc

61

1

1. HBr, AcOH 2. q.Me,CO AgZCO,

C&OAc

62

Compound 61 was derived from 3,4,6-tri-O-acety~-2-deoxy-~-urabinohexopyranosyl bromide, which was produced by direct addition of hydrogen bromide to the double bond. The bromodeoxy sugar 62 was, presumably, formed by addition to a 2,3-unsaturated, rearrangement product of the type known116to arise from tri-0-acetyl-D-glucal in the presence of acid. When 56 is treated with hydrogen bromide in nonpolar solvents, simple addition occurs, to give 3,4,6-tri-O-acety1-2-deoxy-~-arabinohexopyranosyl bromide."' The conversion of 3,4-di-O-acetyl-6-0-ptolylsulfonyl-D-glucalinto crystalline 4-0-acetyl-3-bromo-2,3-dideoxy6-O-p-to~ylsulfonyl-a-~-urubino-hexopyranosy~ bromide in 75% yield, on treatment with hydrogen bromide in glacial acetic acid for 3 hours at room temperature, has also been reported."* Pedersen and coworkers11ghave studied the reactions of di-0-acetylD-xylal (63) and -D-arabinal (64) with hydrogen chloride and hydrogen bromide, and results similar to those of Maki and Tejima115were obtained. Treatment of di-0-acetyl-D-xylal (63) with hydrogen chloride or hydrogen bromide in benzene gave only small proportions of 3,4-di-O-acetyl-2-deoxy-~-threo-pentopyranosyl halides (65, X = C1 or Br); the main products were 4-0-acetyl-2,3-dideoxy-3-halogeno-~threo-pentopyranosyl halides (67, X = C1 or Br) and small proportions (116) H. J. Ferrier and N. Prasad,J. Chem. SOC. (C), 581 (1969). (117) J. J. K. Novik and F. Sorm, Collect. Czech. Chem. Commun., 27,902 (1962); T. Maki, H. Nakamura, S. Tejima, and M. Akagi, Chem. Pharm. Bull. (Tokyo), 13, 764 (1965). (118) T. Maki and S. Tejima, Chem. Pharm. Bull. (Tokyo), 15, 1367 (1967). (119) K. Bock, I. Lundt, and C. Pedersen, Acta Chem. Scand., 23,2083 (1969).

265

DEOXYHALOGENO SUGARS

of the corresponding D-erythro derivatives (68, X = C1 or Br). In contrast to these results, the reaction of di-0-acetyl-~-arabinal(64) with hydrogen chloride or hydrogen bromide afforded mainly the 3,4-diO-acetyl-2-deoxy-~-erythro-pentopyranosyl halides (69, X = C1 or Br)

AcO 65

63

0.

AcO

67

/

ox

AcO

\

x

AcO

AcO 64

69

and only small proportions of 2,3-dideoxy-3-halogeno compounds. On treatment with hydrogen halide, both di-0-benzoyl-D-xylal and di0-benzoyl-D-arabinal gave, almost exclusively, 3,4-di-O-benzoyl-2deoxypentopyranosyl halides. Evidence was presented that suggested that the formation of the 2,3-dideoxy-3-halogeno derivatives (67 and

266

WALTER A. SZAREK

68) proceeds by way of the 2,3-unsaturated glycosyl halide (66). In

agreement with this mechanism, the 2,3-unsaturated methyl glycoside 70, on treatment with hydrogen halide, afforded the 3-halogeno

70

compounds in the same relative proportions as those obtained from the glycals. It was also found that the reaction of methyl 3,4-di-0acetyl-2-deoxy-~-threo-pentopyranosidewith hydrogen bromide gives, in addition to the expected 3,4-di-O-acetyl-2-deoxy-D-threopentopyranosyl bromide, a small proportion of a 4-0-acetyl-3-bromo2,3-dideoxy-~-threo-pentopyranosyl bromide (67, X = Br). The reactions of acylated 2-hydroxyglycals with hydrogen chloride and hydrogen bromide have also been investigated."" Treatment of 2,3,4-tri-0-acetyl-1,5-anhydro-~-threo-pent-l-enitol, or the corresponding D-ef'Ijthf'o compound, with hydrogen chloride in benzene afforded 2,4-di-O-acetyl-3-deoxy-~-glycero-pent-2-enopyranosy~ chloride in high yield. Similar results were observed with one molequivalent of hydrogen bromide, namely, the formation of 2,3-unsaturated glycosyl bromides; with an excess of hydrogen bromide, these products underwent further reaction, to yield bromodeoxy derivatives which were, however, too unstable to be isolated. An analogous study, with 1,3,4,5-tetra-O-acetyl-2,6-anhydro-~threo-hex-2-enitol (71), has been described.I2l Compound 71 was transformed, by way of an acid-catalyzed isomerization, into 72. The reaction of 72 with hydrogen bromide in benzene, followed by glycosidation with methanol, gave three major products, two of which were the a and /3 anomers of methyl 1,3,5-tri-O-acetyl-4-bromo-4deoxy-L-tagatopyranoside (76); the pathway for their formation was considered to be that shown (72-76). The anomeric methyl L-tagatosides were also obtained by reaction of 71 with hydrogen bromide in dry benzene, also, presumably, by way of the intermediate 74.

(120) K. Bock and C. Pedersen, Acta Chem. Scund., 24, 2465 (1970). (121) M. Katsuhara, S. Wakahara, and K. Tokuyama, Bull. Chem. SOC. Jup., 41, 1208 (1968).

DEOXYHALOGENO SUGARS

267

AcoQ~H,OAc

AocQ oA :c

AcO 71

72

CH,OAc

A Q oc:

-

74

ir

73

OAC 75

Br

OAC 76

b. To Other Unsaturated Derivatives. - Several examples have now been reported of the preparation of deoxyhalogeno sugars by addition reactions with unsaturated carbohydrate derivatives other than glycals. A well-known, unsaturated sugar is methyl 4,6-0benzylidene-2,3-dideoxy-a-~-erythro-hex-2-enopyranoside (77). Treatment of this alkene with bromine in carbon tetrachloride gave a complex, intractable mixture of p r o d ~ c t s . ' ~ ~However, *'~~ when bromine was added to 77 in methanol in the presence of silver acetate, crystalline methyl 4,6-0-benzylidene-2,3-dibromo-2,3-dideoxy-a-~altropyranoside (78) was isolated in 70% yield; the dibromide 78 was (122) J. E. Christensen and L. Goodman, J . Amer. Chem. SOC., 83, 3827 (1961). (123) E. L. Albano, D. Horton, and J. H. Lauterbach, Carbohyd. Res., 9, 149 (1969).

WALTER A. SZAREK

268

also obtained, in lower yield, when silver acetate was omitted or when sodium chloride was used instead of silver acetate.lZ3Treatment of 78 with N-bromosuccinimide in refluxing carbon tetrachloride (see Section 11,4; p. 276) gave crystalline methyl 4-0-benzoyl2,3,6-tribromo-2,3,6-~ideoxy-cr-~-altropyranoside.It was foundlZ3 that acetyl hypobromite reacts with the alkene 77 in carbon tetrachloride to give a mixture of two adducts, namely, methyl 2-0-acetyl4,6-O-benzylidene-3-bromo-3-deoxy-cr-~-altropyranoside (79) and methyl 3-0-acetyl-4,6-O-benzylidene-2-bromo-2-deoxy-cr-~-glucopyranoside (80);the diaxial adduct 79 was the preponderant product. Compound 79 was converted by N-bromosuccinimide into methyl 2-O-acetyl-4-O-benzoyl-3,6-dibromo-3,6-dideoxy-cr-~-al~opyranoside. H PhCH /

T o ,

-

\

P h‘ C0 O du bO oM Me e

B r a , MeOH B r a , MeOH BaCOs % * pAgOAc,

0

0

77

I

OMe

Br

\

78

AcOBr

Br OMe

Br 79

80

The method of Horton and coworkers,123just mentioned, for bromination in methanol in the presence of silver acetate, has also been applied to the 2,3-dideoxy-6-O-trityl-a-~-erythro-hex-2-enopyranoglycan derived from a 2,3-di-O-p-tolylsulfonyl-6-O-trityl derivative of amylose; a 2,3-dibromide having a degree of substitution of approximately 2.0 was obtained.’24 (124) D. M. Clode, D. Horton, M. H. Meshreki, and H. Shoji, Chem. Commun., 694 (1969).

DEOXYHALOGENO SUGARS

269

In another methyl 5-0-benzoyl-2,3-dideoxy-~-~-glyceropent-Zenofuranoside (81) was treated with bromine in methanol in the presence of silver acetate and barium carbonate; the two monobenzoates (shown as 84) of methyl 2-bromo-2-deoxy-~-~-xylofuranoside were obtained. A rationalization for this reaction involves formation of an intermediate bromonium ion (82), produced by attack of Br@ on the less-hindered side of the double bond, which then undergoes trans-attack by the benzoate group at C-5 to give the benzoxonium intermediate (83);the monobenzoates (84) are obtained on processing of the reaction mixture with water.

81

82

Br

Br 84

83

A vinyl ether, namely, methyl 5,6-dideoxy-2,3-0-isopropylidene/3-~-erythro-hex-4-enofuranoside, has been treated with bromine in chloroform or carbon tetrachloride126;a brominated product was not isolated, but it was observed that there was instantaneous consumption of a large excess of bromine. Presumably, a facile loss of hydrogen bromide can lead to degradation products which also can consume bromine. The action of hydrogen bromide-glacial acetic acid at 10" on methyl 4-0-benzyl-2,3-dideoxy-6-O-trityl-a-~-erythro-hex-2-enopyranoside (85) causes addition to the double bond and also 1,Banhydride formation; 1,6-anhydro - 4-0-benzyl-3- bromo-2,3-dideoxy-P-~-ribo(125) R. G. S. Ritchie and W. A. Szarek, Chem. Ind. (London), in press. (126) H. Arzoumanian, E. M. Acton, and L. Goodman, J . Amer. Chem. SOC., 86, 74 (1964); see also, I. E. Muskat, ibid.,56, 2653 (1934).

WALTER A. SZAREK

270

hexopyranose (86) was isolated in 50% yield by preparative-layer chromat~graphy.'~~

hMe HBr, AcOH

PhCKO

PhC%O

85

86

Interest has developed in the addition to unsaturated carbohydrates of reagents that have been referred to as pseudohalogens. Many of these reagents are conveniently prepared in situ by the reaction of the appropriate silver salts with iodine. Thus, for example, the reaction of silver nitrite with iodine generates the pseudohalogen, nitryl iodide (NO& which has been shown to add to some unsaturated carbohydrate derivatives under mild conditions.lZ8Addition of nitryl iodide, in solution in ether, to the terminal, unsaturated sugars 3-0-acetyl-5,6-dideoxy- 1,2-O-isopropy~idene-a-D-xy~o-hex-5-enof~ranose and methyl 5,6-dideoxy-2,3-di-O-p-tolylsulfonyl-a-~-~~~~~~~hex-Senofuranoside afforded the C-iodo-C-nitro adducts 87 (Ref. 129) and 88 (Ref. 130), respectively; the configuration at C-5 in 87 and 88

CHI

0

Q o

I I 0-CMe, 81

88

has not yet been determined. These adducts are unstable on standing -particularly in the light; synthetically useful transformations are described in Section I11 (see p. 281). It has been proposed131 that the addition of nitryl iodide to alkenes proceeds by a free-radical attack of an NOz species on the double bond; the observed regio(127) S. Dimitrijevich and N. F. Taylor, Carbohyd. Res., 20,427 (1971). (128) W. A. Szarek, D. G . Lance, and R. L. Beach, Chem. Cornmun., 356 (1968). (129) W. A. Szarek, D. G . Lance, and R. L. Beach, Carbohyd. Res., 13, 75 (1970). (130) I. Szczerek, J. S. Jewell, R. G. S. Ritchie, W. A. Szarek, and J. K. N. Jones, Carbohyd. Res., 22, 163 (1972).

DEOXYHALOGENO SUGARS

27 1

chemistry of the addition reactions leading to 87 and 88 is consistent with such a free-radical pathway. When the reaction of methyl 5,6-dideoxy-2,3-di-O-p-tolylsulfonyla-~-arabino-hex-5-enofuranoside with silver nitrite and iodine was performed in more-polar solvents, such as methanol and acetonitrile, a p-iodo nitrate was produced, in addition to the C-iodo-C-nitro adduct 88; in methanol, the ratio of the two products was13o3:2.The p-iodo nitrate was identical with the adduct obtained by the reaction of the alkene with silver nitrate and iodine in a~etonitri1e.l~~ The formation of the p-iodo nitrate in the silver nitrite-iodine reaction in polar solvents may be due to the increased solubility of the silver nitrite; the available nitrite ions could then be oxidized to nitrates. The positions of the iodo and nitrate groups in the adduct have not yet been established. The addition of the pseudohalogen iodine azide (prepared from iodine monochloride and sodium azide in acetonitrile) to methyl

5,6-dideoxy-2,3-di-O-p-tolylsulfonyl-a-~-arabino-hex-5-enofuranoside has also been achieved; a crystalline p-iodo azide was isolated, in 69% yield, that was stable in the dark, but became colored on exposure to light.130Brimacornbe and coworkers133have reported the addition of iodine azide to 5,6-dideoxy-1,2-0-isopropylidene-a-~xylo-hex-5-enofuranose; X-ray crystallographic analysis established that the product is 6-azido-5,6-dideoxy-5-iodo-1,2-O-isopropylideneP-L-idofuranose. The addition of iodine trifluoroacetate (produced by reaction of iodine with silver trifluoroacetate) to unsaturated carbohydrates has been investigated.13*Treatment of 5,6-dideoxy-1,2-O-isopropylidenea-D-xylo-hex-5-enofuranose (89) with silver trifluoroacetate and iodine in acetonitrile gave 3,6-anhydro-5-deoxy-5-iodo-1,2-O-isopropylidene-a-D-gluco(and6-L-ido)furanose (91) and 5-deoxy-5-iodo1,2-0-isopropylidene-6-O-(trifluoroacetyl)-a-~-gluco(and/or P-L-ido)furanose (92), with the former preponderating. Component 91 was converted into 3,6-anhydro-5-deoxy-1,2-0-isopropylidene-a-~-xylohexofuranose (94) by hydrogenation over Raney nickel (see also, Section 111,3; p. 299), and component 92 was converted into 5-deoxy-1,2-O-isopropylidene-cr-~-xylo-hexofuranose (95) by treat(131) A. Hassner, J. E. Kropp, and G. J. Kent,]. Org. Chem., 34,2628 (1969). (132) D. H. Ball, A. E. Flood, and J. K. N. Jones, Can. J . Chem., 37, 1018 (1959). (133) J. S. Brimacornbe, J. G. H. Bryan, T. A. Hamor, and L. C. N. Tucker, Chem. Commun., 1401 (1968);J. S. Brimacombe, J. G. H. Bryan, and T. A. Hamor, J . Chem. SOC. ( B ) , 514 (1970); see also, J. S. Brimacombe, F. Hunedy, and M. Stacey, Carbohyd. Res., 13,447 (1970). (134) R. G. S. Ritchie and W. A. Szarek, Can. J . Chem., 50, 507 (1972).

WALTER A. SZAREK

272

la F CH

q0

0-CMe,

89

I

CHI 0

Q?

I

92

93

R = Tf R=H

I

0-CMe,

O-CM+

95

where Tf = CF,CO.

ment with methanol, followed by hydrogenation. The reaction of 5,6- dideoxy - 1,2-0- isopropylidene-a-~-xylo-hex-5-enofuranose (89) with silver trifluoroacetate and iodine in acetonitrile was considered to proceed by way of a cyclic, iodonium-ion intermediate such as 90. Attack by the 3-hydroxyl group at the (less-hindered) C-6 position (route a ) would then yield a 3,6-anhydro derivative (91), whereas attack by trifluoroacetate ion (route b) would give a 5-deoxy-5-iodo6-O-(trifluoroacetyl) derivative (92). Treatment of 92 with methanol

DEOXYHALOGENO SUGARS

273

afforded the iodohydrin 93. The formation of the 3,6-anhydro component 91 as the preponderant product is probably attributable to the low nucleophilicity of the trifluoroacetate anion. The formation of a 3,ganhydro derivative could be obviated by protecting the 3-hydroxyl group in alkene 89 with a trifluoroacetyl group. Addition of iodine trifluoroacetate to methyl 5,6-dideoxy-2,3-di-O-p-tolylsulfonyl-a-~urubino-hex-5-enofuranosidesimilarly affords a 5-iodo-6-O-(trifluoroacetyl) a d d u ~ t . ' ~ ~ Some cyclic, unsaturated sugars have also been treated with pseudohalogen reagents. Thus, nitryl iodide was found to add across the alkenic double bond of benzyl 2-0-benzyl-3,4-dideoxy-a-~glycero-pent-3-enopyranoside to give a crystalline, 3-iodo-4-C-nitro a d d ~ c t . ' However, ~~ methyl 4,6-0-benzylidene-2,3-dideoxy-a-~erythro-hex-2-enopyranoside(77) was found to be resistant to the addition of nitryl iodide in ether.'29 This alkene has been reported'33 to be unreactive also toward iodine azide in either acetonitrile or N,N-dimethylformamide. Nitrosyl chloride reacted with alkene 77 below room temperature, but the product decomposed, to regenerate the starting alkene, on attempted i ~ o l a t i o n . 'Treatment ~~ of methyl 5-0-benzoyl-2,3-dideoxy-~-~-gZycero-pent-2-enofuranoside (81) with silver nitrite and iodine in dry ether afforded, exclusively, furfuryl benzoate; it was established that solely iodine in ether accomplished this c o n ~ e r s i o n .An ' ~ ~addition of nitryl iodide to tri-O-acetyl-D-ghca1 could also not be achieved; a dimeric product is formed in the presence of iodine.'30 The addition of iodine fluoride to an exocyclic vinyl ether was a key step in the synthesis5 of nucleocidin (3;see p. 226). Thus, addition of iodine to a solution of the di-N-benzoyl alkene 96 in nitromethane, in the presence of silver fluoride, afforded in high yield the epimeric 5'-deoxy-4'-fluoro-5'-iodonucleosides 97 and 98. As the iodo group in 98 could not be satisfactorily displaced by certain oxygen nucleophiles, compound 98 was treated with lithium azide in N,N-dimethylformamide for 20 hours at loo", and the resultant product was N-debenzoylated with methanolic ammonia, to give 5'-azido-5'-deoxy-~'-fluoro-~',~'-O-isopropylideneadenosine (99) in 93% yield. The primary azide was converted, by way of photolysis, into the 5'-aldehyde, which was reduced with sodium borohydride to give 4'-fluoro-2',3'-O-isopropylideneadenosine(100). Compound 100 was then converted into nucleocidin (3). Other examples of the (135) W. A. Szarek and R. G. S. Ritchie, Abstr. Papers Amer. Chem. Soc. Meeting, 163, CARB 12 (1972). (136) R. G. S. Ritchie and W. A. Szarek, Carbohyd. Res., 18,443 (1971).

WALTER A. SZAREK

274

displacement of halogeno groups in sugar derivatives by azide ion are given in Section II1,l (see p. 281).

Bz.NJ$N> N '

N I

+

Me, 98

97

3

100

99

where B = N',y-dibenzoyladenine and Ad = adenine.

The reactions of 5,6-dihydro-2-methoxy-2H-pyran (101) with 1,3dibromo-5,5-dimethylhydantoin in ether-methan01,'~' and with ethanesulfenyl have been described by Baldwin and Brown. The former reaction gave a 2:l mixture of the isomers 3pbromotetrahydro-2a,4a-dimethoxypyran(102) and 3a-bromotetrahydr0-2q4,B-dimethoxypyran (103), respectively; the structures and favored conformations of the isomers are shown. The reaction of (101) ethanesulfenyl chloride with 5,6-dihydro-2-methoxy-2H-pyran (137) M. J. Baldwin and R. K. Brown, Can. J. Chem., 47,3099 (1969). (138) M. J. Baldwin and R. K. Brown, Can. J. Chem., 47,3553 (1969).

275

DEOXYHALOGENO SUGARS

Met.-

M&O eM 0e

Me

Br methanol, ether 101

+

I02

rn +

Me0

OMe

103

gives only 4@-chloro-3a-(ethylthio)-tetrahydro-2&methoxypyran (104). A proposed138route for this highly selective reaction is shown. Because of the anomeric effect, the favored conformer of 5,6-dihydro-

K L ? O M e -

woML EtS,C1

Et

J

/ OMe

SEt 104

2-methoxy-2H-pyran is considered to be that in which the bond to the 2-methoxyl group is quusi-axial. The first step is, presumably, electrophilic attack of ethanesulfenyl chloride on the dihydropyran from the less-hindered side of the molecule, namely, tmns to the

276

WALTER A. SZAREK

2-methoxyl group, to give an episulfonium-ion intermediate. The next step is attack by the chloride ion at either C-3 or C-4, with simultaneous opening of the episulfonium ring. It is believed that the steric effect of the quasi-axial methoxyl group, and its polar repulsion for the chloride ion, would strongly inhibit attack by the chloride ion at C-3. Moreover, the electron-withdrawing effect of the two oxygen atoms at C-2, which would destabilize an incipient positive charge at C-4 less than at C-3, favors reaction of the chloride ion at C-4 rather than at C-3. Cahu and D e ~ c o t e s 'have ~ ~ also subjected 2-alkoxy-5,6-dihydro-2H-pyrans to hydrohalogenation and to h ydroxyhalogenation. A key step in a synthesis140of 2-deoxy-~-eryth~o-pentose was the addition of the elements of HOBr across the carbon-carbon double bond of an alkene on treatment with N-bromosuccinimide and water.

4. Reaction of 0-Benzylidene Sugars with N-Bromosuccinimide Cyclic benzylidene acetals have been widely employed in carbohydrate chemistry as protecting groups for certain glycol systems. The synthetic utility of such acetals has been greatly extended by the development of a ring-opening reaction with N-bromosuccinimide to give bromodeoxy sugar benzoates. The reaction was reported first in the carbohydrate field by H a n e ~ s i a n ' ~certain ~; methyl 4,6-0benzylidene-a-D-hexopyranosidesafforded the corresponding methyl 4-O-benzoyl-6-bromo-6-deoxy-a-~-hexopyranosides in good yield. Very shortly after this announcement, a similar transformation of 4,6-O-benzylidene acetals by N-bromosuccinimide in the presence of benzoyl peroxide was reported by Hullar and coworkers.142The most probable mechanism p r o p o ~ e d ~for ~ ~the - ' ~reaction ~ involves formation of a benzoxonium intermediate of the type 105, followed by

105

(139) M. Cahu and C. Descotes, Bull. SOC. Chim. Fr., 2975 (1968). (140) C. Nakaminami, M. Nakagawa, S. Shioi, Y. Sugiyami, S. Isemura, and M. Shibuya, Tetrahedron Lett., 3983 (1967).

DEOXYHALOGENO SUGARS

277

attack by bromide ion at C-6 to give the 4-0-benzoyl-6-bromo-Sdeoxy derivative. The observed dependence of the reaction on lightlll and the presence of peroxide^'^^.'^^ suggests that the first step proceeds by a free-radical process; moreover, an overall, radical type of mechanism cannot be excluded. If the benzylidene acetal ring spans the oxygen atoms of two erstwhile secondary hydroxyl groups, the reaction with N-bromosuccinimide usually affords isomeric, bromodeoxy sugar benzoates. Thus, for example, the reaction with methyl 2,3-0-benzylidene-5-0methyl-p-D-ribofuranoside (106) afforded145 an approximately 1:1 mixture of the bromides 109 and 108, presumably by way of attack on C-2 and C-3 of the benzoxonium intermediate 107 by bromide ion.

106

107

Meocw +

OBZ

BzO

109

108

If a participating group, such as an ester or hydroxyl group, is present near to the acetal ring, a rearrangement of the benzoxonium intermediate can occur; the rearranged ion can then be attacked intramolecularly or externally by a n ~ c l e o p h i l e . ' ~ ~ As investigations of the reaction of N-bromosuccininimide with (141)S. Hanessian, Carbohyd. Res., 2, 86 (1966). (142)D. L. Failia, T. L. Hullar, and S. B. Siskin, Chem. Commun., 716 (1966). (143)S. Hanessian and N. R. Plessas, J. Org. Chem., 34, 1035 (1969). (144)S. Hanessian and N. R. Plessas, J. Org. Chem., 34, 1045 (1969). (145)S. Hanessian and N. R. Plessas, J. Org. Chem., 34, 1053 (1969). (146)T.L.Hullar and S. B. Siskin,]. Org. Chem., 35,225 (1970).

278

WALTER A. SZAREK

benzylidene acetals of sugars have been succinctly outlined by P a ~ l s e n ' ~in ' Volume 26 of this Series, no further discussion of the reaction is given in this Section. The reaction has been widely applied to the preparation of aminodeoxy and deoxy sugars, and examples of this synthetic utility will be described in Section I11 (see p. 281).

5. Miscellaneous Methods A well known and effective method for introducing halogen atoms into sugar molecules is by cleavage of carbohydrate oxiranes by halogen-containing reagents. This topic has been surveyed by Barnett' in the previous article on halogenated carbohydrates; much information is also contained in the article by Williams148on oxirane derivatives of aldoses, in Volume 25 of this Series. The reagents that have been employed include halogen acids, Grignard reagents, and lithium, magnesium, and sodium salts. Deoxyhalogeno sugars have also been obtained by the cleavage of epithio and epimino sugars.' A novel method of opening of oxiranes involves the use of (chloromethy1ene)dimethyliminium chloride (39) [see Section II,2c; p. 2501, monochlorodeoxy or dichlorodideoxy derivatives are obtained, depending upon the reaction conditions employed.83 Thus, methyl 2,3-anhydro-4,6-0-benzylidene-a-~-allopyranoside (110) reacts with 39 in 1,1,2,2-tetrachloroethaneat room temperature to give, upon hydrolysis of the primary adduct 111 with an aqueous solution of sodium hydrogen carbonate, methyl 4,6-0-benzylidene-2-chloro2-deoxy-3-O-formyl-a-~-altropyranoside (112). If a solution of 39 and 110 in 1,1,2,2-tetrachloroethaneis heated at reflux temperature, methyl 3,4-0-benzylidene-2,6-dichloro-2,6-dideoxy-a-~-al~opyranoside (113) is obtained in high yield; the n.m.r. spectrum of 113, like that of 47 (see Section 11, 2c; p. 250), showed the presence of two diastereoisomers which differed in the configuration of the benzylidene-acetal carbon atom. It has been that treatment of 1,6-anhydro-2-deoxy-2-fluoroP-D-glucopyranose with hydrogen bromide in acetic acid, in the presence of acetic anhydride, affords 3,4-di-O-acetyl-6-bromo-2,6dideoxy-2-fluoro-a-~-glucopyranosylbromide. The formation of this product occurs, presumably, by an initial cleavage of the C-6-0-6 bond to give a 6-bromo derivative, which then reacts to give the dibromide. It is noteworthy that 2,3,4-tri-O-acetyl-1,6-anhydro-~-~-glucopyranose (147) H. Paulsen, Aduan. Carbohyd. Chern. Biochem., 26, 127 (1971). (148) N. R. Williams, Aduan. Carbohyd. Chem. Biochem., 25, 109 (1970). (148a) M. Cerny, V. PfikrylovP, and J. Pacik, Collect. Czech. Chem. Commun.,37,2978 (1972).

DEOXYHALOGENO SUGARS

fB

Me,N=C

279

o ,

c1@

H

110

111

/I @ H P PhC

J

OMe OCHO

0 ‘ 113

112

gave, even after 20 hours, almost exclusively 2,3,4,6-tetra-O-acetyla-D-glucopyranosyl bromide; in this case, cleavage of the C-1-04 bond is favored. The stability of the C-1-04 bond in the former example is, presumably, the result of the inductive (-1) effect of the fluorine atom on C-2. Mengel and Robins’4Rbreported that treatment of 2’,3’-0-(1-meth0xyethylidene)adenosine with pivaloyl chloride in hot pyridine gives 4(2-0-acetyl-3-chlorod-O-pivaloyl-~-~-xylofiranosyl)-6-(pivalamido) purine. The possibility of obtaining deoxyhalogeno sugars from hydrazino derivatives of carbohydrates has been investigated.’” 3-Deoxy-3hydrazino-1,2:5,6-di-O-isopropylidene-~-~-allofuranose is converted, by iodine in chloroform, into 3-deoxy-3-iodo-1,2:5,6-di-O-isopropylidene-a-D-glucofuranose in high yield. N-Iodosuccinimide in chloroform at O”, or iodine in aqueous potassium iodide, also afforded this deoxyiodo sugar, together with the gem-diiodo derivative, 3-deoxy3,3-diiodo-1,2:5,6-di-O-isopropylidene-a-~-ribo-hexofuranose; N-bro(148b) R. Mengel and M. J. Robins, Abstr. Pupers Amer. Chem. SOC. Meeting, 161, CARB 2 (1971). (149) D. M. Brown and G. H. Jones, J. Chem. SOC. (C), 252 (1967).

WALTER A. SZAREK

280

mosuccinimide gave the expected 3-bromo-3-deoxy-~-glucosederivative and only a trace of the gem-dibromide. It has been found150 that, under mild conditions, 1,4:3,6-dianhydro-D-glucitol is converted by boron trichloride mainly into 1,6-dichloro-1,6-dideoxy-~-glucitol. Similarly, the reactionI5l with methyl 3,6-anhydro-a-~-glucopyranoside afforded methyl 6-chloro-6deoxy-wD-glucopyranoside as one of the products, isolated as the 2,3,4-tri-O-benzoyl derivative. The synthesis of deoxyhalogeno sugars has been achieved, using phosphorus-containing derivatives as substrates, by way of Arbusovtype reaction^.'^^ Inokawa and have described the synthesis of deoxyiodo sugars by the reaction of cyclic phosphite or phosphonite derivatives of sugars with methyl iodide. Thus, treatment of 1,2-0-isopropylidene-a-D-glucofuranose(114) with triphenyl phosphite in N,N-dimethylformamide, in the presence of sodium ethoxide, afforded the phosphite derivative 115, which was converted, by treatment with methyl iodide in a sealed tube at lOO", into the deoxyiodo sugar 116 in 80% yield. Similarly, compound 119 CH,OH

HoLH

0

Q?

P(OPh)s

p: -

CHJ I

0 MeP'T+? 0 II

p w > , 0-CMe,

0-CMe,

114

0-CMe,

115

116

was prepared by the reaction of 1,2-O-isopropylidene-a-~-xylofuranose (117) with diphenyl phenylphosphonite, followed by treatment of the resultant intermediate 118 with methyl iodide. HOCH, 0

G).

PhP (OPh),

Phl-?

0-CMe,

0-CMe, 118

117

Me

I 1

0-CMe, 119

DEOXYHALOGENO SUGARS

28 1

Corey and J. E. Andersoni54 have described a useful method for the conversion of alcohols into iodides by employing the reagent 120, which is readily prepared from catechol and phosphorus trichloride. Reaction of 120 in ether solution with an alcohol in the presence of pyridine affords the corresponding phosphite 121. Treat-

120 121

x = c1 X

=

OR

ment of 121 in dichloromethane with iodine produces the alkyl iodide. 111. REACTIONS AND SYNTHETIC UTILITY

Although several examples of deoxyhalogeno sugars of the type discussed in this Chapter have been known for many years, the potential utility of these compounds as synthetic intermediates remained relatively unexplored until the last decade. The increased availability of methods for the preparation of deoxyhalogeno sugars has stimulated interest in the reactions of these compounds; in the present Section, developments in this area are discussed. Deoxyhalogeno sugars have been found particularly useful in the synthesis of other rare sugars, such as deoxy and aminodeoxy sugars. 1. Displacement Reactions

Deoxyhalogeno sugars are susceptible to nucleophilic attack, leading either to displacement, elimination, or anhydro-ring formation. The ease of displacement decreases in the order I > Br> C1 > F; the iodo and bromo derivatives have, therefore, been especially utilized in such reactions, although several reactions with chlorodeoxy sugars have now been reported as a result of the increased availability of these compounds. The approach delineated in Section 11,l (see p. 227) for predicting the reactivity of sulfonic esters can be expected also to be applicable, in an approximate and qualitative way, (150) M. A. Bukhari, A. B. Foster, and J. M. Webber, Carbohyd. Res., 1, 474 (1966). (151) M. A. Bukhari, A. B. Foster, and J. M. Webber, Carbohyd. Res., 4, 105 (1967). (152) See Ref. 1, pp. 182-183, for examples. (153) S. Inokawa, K. Seo, H. Yoshida, and T. Ogata, Bull. Chem. Soc. lap., 44, 1431 (1971). (154) E. J. Corey and J. E. Anderson, J. Org. Chem., 32,4160 (1967).

282

WALTER A. SZAREK

to halides. Some early examples of nucleophilic-substitution reactions with deoxyhalogeno sugars have been cited in the previous Chapter' on these compounds. Only some later studies are described in the present Section. Nucleophilic-substitution reactions of deoxyiodo sugars are well known. Kochetkov and C O W O ~ ~ ~ ~ have S ~ ~provided ( ~ ) ~ several ' ~ ~ ~ ~ ~ ~ examples of such reactions. In Section II,2b (see p. 242), it was mentioned that the reaction of methyl 2,3-0-isopropylidene-a-~rhamnopyranoside with methyltriphenoxyphosphonium iodide (23) affords methyl 5,6-dideoxy-5-iodo-2,3-0-isopropylidene-~-~-allofuranoside, methyl 5,6-dideoxy-5-iodo-2,3-0-isopropylidene-a-~-talofuranoside, and methyl 4,6-dideoxy-4-iodo-2,3-O-isopropylidene-a-~mannopyranoside. A variety of nucleophilic-substitution reactions of these isomeric iodides has been studied,5s(c)and it was found that the course of the reactions depends upon the nucleophilicity of the reagent employed. Thus, with a strong nucleophile, such as thiolbenzoate, the reactions proceed with inversion and without elimination; with azide ion, a weaker nucleophile, the reactions proceed also with inversion, but some elimination of hydrogen iodide occurs, and, in the reaction of methyl 4,6-dideoxy-4-iodo-2,3-0-isopropylidenea-L-mannopyranoside, the displacement of iodine is, to some extent, accompanied by contraction of the pyranoside ring. Treatment of methyl 5,6-dideoxy-5-iodo-2,3-0-isopropylidene-~-~-allofuranoside with sodium benzoate in N,N-dimethylformamide for 7 hours gave a mixture of unsaturated derivatives, together with methyl 5-0-benzoyl-6-deoxy-2,3-0-isopropylidene~-~-talofuranosideand its C-5 epimer. Displacements of iodo groups have been involved in certain syntheses of biologically important carbohydrate derivatives. Thus, in one ~ynthesis'~'of the antibiotic lincomycin, a key step was conversion of methyl 2,3,4-tri-0-acetyl-6-deoxy-6-iodo-l-thio-a-~-galactopyranoside into the corresponding 6-nitro derivative by treatment with sodium nitrite in N,N-dimethylformamide. A route to 2-deoxyD-erythro-pentose has been developed'58 which has the advantage of being applicable to the preparation of the sugar specifically labelled at C-1. Thus, treatment of l-deoxy-2,4-O-ethylidene-l-iodo-~-eryth-

-

(155) A. I. Usov, K. S. Adamyants, and N. K. Kochetkov, Izo. Akad. Nuuk SSSR, Ser. Khim., 2546 (1968);697 (1969); 1740 (1971). (156) N. K. Kochetkov, A. I. Usov, and K. S. Adamyants, Zzo.Akad. Nauk S S S R , Ser. Khim., 885 (1970). (157) B. J. Magerlein, Tetrahedron Lett., 33 (1970). (158) I. Ziderman and E. Dimant,J. Org. Chem., 32 1267 (1967); see also, I. Ziderman, Carbohyd. Res., 18,323 (1971).

DEOXYHALOGENO SUGARS

283

ritol with sodium cyanide in methyl sulfoxide at 37" gave 2-deoxy3,5-0-ethylidene-~-erythro-pentononitrile; reduction of this compound with an excess of Raney nickel in aqueous acetic acid, followed by acid-catalyzed hydrolysis, afforded the 2-deoxy sugar. Derivatives of 6-amino-6-deoxy-~-glucose-6-~~N have been s y n t h e s i ~ e d in ' ~ ~high yield and high chemical and isotopic purity by way of the reaction of 6-deoxy-6-iodo-1,2:3,5-di-O-isopropylidene-a-~-glucofuranose with potassium phthalimide-15N. The reaction of methyl 6-deoxy-6-iodo-2,3,4-tri-O-methyl-a-~glucopyranoside with methyl 2,3,4-tri-O-methyl-a-~-glucopyranoside 6-O-(S-sodium dithiocarbonate) (methyl 2,3,4-tri-O-methyl6-thio-a-~-glucopyranoside) (methyl 2,3,4-tri-O-methyl-a-D-glucopyranoside) 6,6'-dithiocarbonate. Several examples of displacement reactions with bromodeoxy sugars are known. One synthetic procedure of current interest is the generation of bromodeoxy groups by the reaction of 0-benzylidene sugars with N-bromosuccinimide (see Section 11,4; p. 276) and subsequent transformation of these groups. Thus, for example, Horton and LuetzowlG0achieved a facile synthesis of 6-amino-6-deoxy-~-mannose by way of treatment of methyl 2,3-di-O-acetyl-4,6-0-benzylidenea-D-mannopyranoside with N- bromosuccinimide in dry carbon tetrachloride to give methyl 2,3-di-O-acetyl-4-O-benzoyl-6-bromo6-deoxy-a-~-mannopyranoside, followed by conversion of this bromo derivative, with sodium azide in N,N-dimethylformamide, into the corresponding 6-azido derivative. In another example,'81 methyl 4-O-benzoyl-6-bromo-2,3,6-trideoxy-a-~-erythro-hexopyranoside was obtained from methyl 4,6-0-benzylidene-2,3-dideoxy-a-~-erythrohexopyranoside by a reaction with N-bromosuccinimide, and converted into the 6-iodo analog in 82% yield by treatment with potassium iodide in N,N-dimethylformamide at 58";the 6-deoxy-6-iodo compound was then readily transformed, by saponification and subsequent hydrogenation, into methyl 2,3,6-trideoxy-a-D-e~ythro-hexopyranoside (methyl a-amicetoside). A diamino sugar, 2,4-diamino-2,3,4,6-tetradeoxy-~-a~abino-hexopyranose (kasugamine), is a component of the antibiotic kasugamycin. Two key steps in a synthesisls2 of this sugar were the preparation of a bromodeoxy sugar and a subsequent displacement reaction by azide (159) B. Coxon, Carbohyd. Res., 19, 197 (1971). (159a) D. Trimnell, W. M. Doane, and C. R. Russell, Carbohyd. Res., 22, 351 (1972). (160) D. Horton and A. E. Luetzow, Carbohyd. Res., 7, 101 (1968). (161) E. L. Albano and D. Horton, /. Org. Chem., 34,3519 (1969). (162) S. Yasuda, T. Ogasawara, S. Kawabata, I. Iwataki, and T. Matsumoto, Tetrahedron Lett., 3969 (1969).

WALTER A. SZAREK

284

ion. Thus, hydroboration of 2-ethoxy-3,4-dihydro-6-methyl-2H-pyran (122), followed by treatment with chloramine, gave the amine 123, which was isolated as the acetylated derivative 124. Treatment of 124 with bromine containing hydrogen chloride yielded three bromo compounds, 125,126, and 127; both of the isomers 126 and 127 could be converted into 125. Compound 125 yielded the azide 128 on treatment with sodium azide in methyl sulfoxide at 100-105”. Catalytic hydrogenation of 128 produced the amine 129; optical resolution of the amine 129 was effected with D-threaric acid [(-)-tartaric acid]. The resolved amine was acetylated to afford ethyl 2,4-bis(acetamido)-2,3,4,6-tetradeoxy-~-~-ur~bino-hexopyranoside (130), a derivative of kasugamine.

O

O

E

t

RNH D

122

123 124

Me

Me

J

O

E

t

R=H R = Ac

/ Me

OEt

AcNH

Br

Br 125

126

127

Me

L l )

AcNH 128 129 130

R = Ns R = NH, R = NHAc

The synthesis of inosamines from bromodeoxyinositols has been achieved by way of displacement reactions with sodium azide in boiling, aqueous 2-methoxyethanol or N,N-dimethylf~rmamide.’~~

285

DEOXYHALOGENO SUGARS

Sugars containing a carbon-phosphorus bond have been prepared by application of the Michaelis-Arbuzov reaction to bromodeoxy sugars. Thus, the reaction of 5-bromo-5-deoxy-1,2-O-isopropylidene3-0-methyl-a-D-xylofuranose (131)with triethyl phosphite yields the corresponding diethyl phosphonate (132);compound 132 was employed for the synthesis of a sugar derivative having phosphorus as

0-CMe, 131

0-CMe, 132

the ring heter0at0m.l~~ Other examples of the reaction with bromodeoxy and deoxyiodo sugars have since been reported.lB5Similarly, the reaction of 5'-deoxy-5'-iodo-2',3'-0isopropylideneuridine with triethyl phosphite yields the corresponding 5' - (diethyl phosphonate).lB6In contrast to these results, the reactions, with triethyl phosphite, of the a-keto halides 3,4,5,6,7-penta-O-acetyl1-chloro-1-deoxy-D-galacto-heptulose and 3,4,5,6-tetra-O-acetyl1-homo-1-deoxy-L-fructose have been reported'66a to afford vinyl phosphates, as shown.

Only relatively few displacements of chloro groups in carbohydrate derivatives have been reported. Treatment of 6-chloro-6-deoxy-1,2:3,5di-0-isopropylidene-a-D-glucofuranose(22) with anhydrous hydrazine for 2 days at reflux temperature yielded the corresponding 6-deoxy-6-hydrazino deri~ative.~' The chloro group in methyl 6-chloro6-deoxy-a-D-glucopyranoside could be displaced by a benzoate group to afford methyl 6-O-benzoyl-a-~-g~ucopyranoside, in 74% yield, by (163) T. Suami, S. Ogawa, and M. Uchida, Bull. Chem. SOC. JaP., 43, 3577 (1970). (164) R. L. Whistler and C.-C. Wang,J. Org. Chem., 33,4455 (1968). (165) S. Inokawa, Y. Tsuchiya, H. Yoshida, and T. Ogata, Bull. Chem. Soc.Jap.,43,3224 (1970); see also, S . Inokawa, K. Yoshida, H. Yoshida, and T. Ogata, Carbohyd. Res., 26, 230 (1973). (166) A. Holy, Tetrahedron Lett., 881 (1967). (166a) L. A. Uzlova, Z. I. Glebova, and Yu. A. Zhdanov, Zh. Obshch. Khirn., 42, 483 (1972).

286

WALTER A. SZAREK

treatment with sodium benzoate in N,N-dimethylformamide at reflux temperature for 16 hours.94 Displacements by azide ion have also been achieved. Thus, treatment of methyl 2,3-di-O-acetyl-4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside with a twofold excess of sodium azide in N,N-dimethylformamide for 12 hours at 120-130" gave a syrupy product that was 0-deacetylated to afford crystalline methyl 4,6-diazido-4,6-dideoxy-ac-~-glucopyranoside in 90% yield.167 When methyl 4,6-O-benzylidene-3-chloro-3-deoxy-~-~-allopyranoside (prepared by the reaction of sulfuryl chloride with methyl 4,6-0benzylidene-p-D-glucopyranoside)was treated with sodium azide in N,N-dimethylformamide for 1 hour at 130", the displacement reaction, which afforded methyl 3-azido-4,6-0-benzylidene-3-deoxyp-D-glucopyranoside as the preponderant product, was accompanied by an elimination reaction leading to methyl 4,6-O-benzylidene-3-deoxy-p-~-erythr~-hex-3-enopyranoside.'~~~'~~ The preparation of 6-azido-6-deoxy-1,2:3,4-di-O-isopropylidene-a-~-galactopyranose, by a displacement reaction with 6-chloro-6-deoxy-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose(45), has also been reported.83The conversion of methyl 6-chloro-6-deoxy-a-~-glucopyranoside into the corresponding 6-azido-6-deoxy derivative can be effected in high yield,168a and similar transformations with the 6-chloro-6-deoxy derivatives of cellulose and amylose afford the azide analogs having the same degrees of substitutione4a;photolysis of these azides was employed as a preparative route to the corresponding monomeric or polymeric 6-aldehydes (Dgluco-hexodialdo-1,s-pyranosidederivatives). The sequence is shown for conversion of 6-chloro-6-deoxycellulose (132a)by way of the 6-azido-6-deoxy analog (132b)into the aldehydo polysaccharide 132c; the aldehyde groups in 132c actually exist largely in the form of intra- or inter-molecular hemiacetals. A synthesis of 6-amino-6-deoxy-a,a-trehalose, a positional isomer of an antitubercular antibiotic, trehalosamine, has been achievedlBSb by way of a displacement reaction of 6-bromo-6-deoxy-a,a-trehalose heptaacetate with sodium azide in N,N-dimethylformamide; the 6-azido-6deoxy derivative was obtained in 75% yield. The required 6-bromo-6deoxy-a,a-trehalose was one of the compounds formed on treatment of anhydrous a,a-trehalose with a mixture of triphenylphosphine and N-bromosuccinimide in N,N-dimethylformamide at room temperature for 43 hours. (167) €3. T. Lawton, W. A. Szarek, and J. K. N. Jones, Carbohyd. Res., 15,397 (1970). (168) E. H. Williams, W. A. Szarek, and J. K. N. Jones, Curbohyd. Res., 20,49 (1971). (168a) D. Horton, A. E. Luetzow, and J. C. Wease, Carbohyd. Res., 8, 366 (1968). (168b) S. Hanessian and P. LavallCe,J. Antibiot. (Tokyo),25,683 (1972). (168c) P. C. Srivastava, K. L. Nagpal, and M. M. Dhar, Experientia, 25, 356 (1969).

287

DEOXYHALOGENO SUGARS

' 0

-Q

(-J

NaN,

Me,SO

OH 132a

\0

OH 132b

132c

The synthesis of some dinucleoside phosphates has been achievedlssc by the reaction of 5'-chloro-5'-deoxynucleosides with nucleotide anions. In that work, the 5'-chloro-5'-deoxynucleosides were conveniently obtained by treatment of the corresponding 2',3'-O-isopropylidene-nucleosidewith thionyl chloride. If an appropriately situated, unsubstituted hydroxyl group is available, anhydro-ring formation usually takes precedence over substitution or elimination. Thus, with alkali, the bromo-, chloro-, and iodo-deoxy sugars react analogously to sulfonic ester derivative^'^^ to afford epoxides in good yield.22,1gg It has been e ~ t a b l i s h e d 'that ~~ 1,2:5,6-dianhydro-~-mannitol is formed in aqueous solutions of 1,6-dibromo-1,6-dideoxy-~-mannitol (an antitumor agent) kept at pH 8; moreover, the biological effects are consistent with occurrence of such a conversion in t.1it.10.~~~ Other than epoxy-ring formation, 3,6-anhydro-ring formation is especially favored with deoxyhalogeno sugars; in fact, the first 3,6-anhydrohexose derivative, namely, methyl 3,6-anhydro-P-~-glucopyranoside, was ~ b t a i n e d " ~ by treatment of methyl 2,3,4-tri-0-acetyl-6-bromo-6-deoxy-~-~-glucopyranoside with barium hydroxide. 2,SAnhydrides of have also been obtained by intramolecular displacement of halide ions. Thus, for example, treatment of 3,4-di-O-acetyl-2-bromo-2-deoxy-~-xylopyranose (133) with ( p-nitropheny1)hydrazine 3,4-di-O-acetyl-2,5-anhydro-~-lyxose (pnitropheny1)hydrazone (135) and 3,4-di-O-acetyl-2,5-anhydroD-xylose (p-nitropheny1)hydrazone (136). The formation of two (pnitropheny1)hydrazones can be explained by an initial displacement of the bromine atom at C-2 by 0-5, to give 3,4-di-O-acetyl-2,5(169) F. H. Newth, W. G. Overend, and L. F. Wiggins,]. Chem. Soc., 10 (1947);G. N. Richards and L. F. Wiggins, ibid., 2442 (1953);J. G. Buchanan, ibid., 955 (1958). (170) M. Jannan and W. C. J. Ross, Chem. 2nd. (London), 1789 (1967). (171) L. A. Elson, M. Jarman, and W. C. J. Ross, E u r . ] . Cancer, 4,617 (1968);see also, E. M. Acton, M. Keyanpour-Rad, J. E. Christensen, H. H. Tong, R. P. Kwok, and L. Goodman, Carbohyd. Res., 22, 477 (1972). (172) E. Fischer and K. Zach, Ber., 45, 456 (1912). (172a) J. Defaye, Aduan. Carbohyd. Chem. Biochem., 25, 181 (1970). (173) A Gerecs, Magy. Kem. Foly., 68, 211 (1962).

288

WALTER A. SZAREK

anhydro-aldehydo-D-lyxose(134), which can afford 135 directly, and 136 by way of a prior epimerization at C-2.

A

c

o

~

o

-[ H

A

CBr

AcO

c

o

r

n

AcO

o

]

CHO

133

134

+ p AcO

AcO 136

135

Lemieux and Fra~er-Reid"~ have reported that the reaction of methyl 3,4,6-tri-O-acetyl-2-deoxy-2-iodo-~-~-glucopyranoside (137) with bromine and silver acetate in acetic acid containing potassium acetate gives an almost quantitative yield of 1,3,4,6-tetra-O-acetyyl2,5-anhydro-~-mannosemethyl hemiacetal (139), obtained as an CbOAc KOAc, Br,, AgOAc AcOH

+

A

AcO c o

q

O

M

OOAc 137

138

/

CGOAC A

c

O

0

J

m

AcO

HC(OMe)(OAc) 139

(174) R. U. Lemieux and B. Fraser-Reid, Can. J . Chem., 42,547 (1964).

e

289

DEOXYHALOGENO SUGARS

equimolar mixture of the C-1 epimers. The oxocarbonium ion 138 was indicated to be a discrete reaction-intermediate, formed by participation of the ring-oxygen atom. When the same reaction was perf~rmed"~ on the epimeric iodide (140), approximately 20% of the product was 1,3,4,6-tetra-O-acetyl-2-O-methy~-~-~-glucopyranose (143), and approximately 60% was the 3,3,4,6-tetraacetate of methyl 2-brorno-2-deoxy-a-~-arabino-hexopyranosid-Sulose3-hydrate (144).

[cOApT] +

A

c

O

m

OAc

Me0

AcO

Me 143

141

iAco*

-

OMe

142

c

C qo O A cr

0 n

AcO

J

AcO

A

I

bMe

hen

144

The intervention of the 1,2-methoxonium ion (141) was postulated, leading to compound 143. However, the main course of reaction in the brominolysis of 140 was rationalized by invoking an elimination to give an intermediate enol acetate (142),which subsequently underwent acetoxybromination to form 144. Carbohydrate oxetanes have been prepared from deoxyiodo sugars without the use of strongly alkaline reagents. Thus, treatment of 5-deoxy-5-iodo-l,2-0-isopropylidene-~~-xylofuranosewith silver fluoride in cold pyridine afforded 3,5-anhydro-1,2-0-isopropylidene(175)R. U. Lemieux and B. Fraser-Reid, Can. J . Chem., 42,539 (1964).

290

WALTER A. SZAREK

a-D-xylofuranose in 85% yieldIT6(see also, Section 111,2; p. 291). This reaction has been extended to the preparation of 4,6-anhydro derivatives of ~-xylo-hexulofuranose*~~; for example, crystalline 4,6-anhydro-l-O-benzoyl-2,3-0-isopropylidene-c~-~-xylo-hexu1ofuranose was prepared by treatment of l-O-benzoyl-6-deoxy-6-iodo-2,3-0isopropylidene-a-L-xylo-hexulofuranose with anhydrous silver fluoride in pyridine. Neighboring-group participation reactions by sulfur and nitrogen functions have also been observed for deoxyhalogeno sugars. An example is the formation of the anhydride 146, which possesses an oxathiabicyclo[2.2.2]octane ring-system constrained in a boat-like conformation, on treatment of methyl 4-O-benzoyl-2-S-benzoyl-6bromo-6-deoxy-2-thio-a-~-altropyranoside (145) with methanolic r\

Br -CH, - y C / P h

OdMe

BzO

OMe

=

& k

HO

HO

OMe

I45 146

sodium methoxide."* An oxazabicyc1o[2.2.2.]octane7namely, methyl 2,6-dideoxy-2,6-imino-N-(phenylsulfonyl)-a-~-altropyranoside, was obtained by treatment of methyl 4-O-benzoyl-6-bromo-2,6-dideoxy2-(phenylsulfonamido)-a-~-altropyranoside with methanolic sodium methoxide; the bromo derivative was the product of the reaction of methyl 4,6-0-benzylidene-2-deoxy-2-(phenylsulfonamido)-a-~-altropyranoside with N-bromo~uccinimide.~~~ 2. Elimination Reactions

During the past decade, there has been intense activity in the study of the synthesis and reactions of carbohydrate derivatives containing a carbon-carbon double bond in the sugar chain. FerrierIo7 has summarized the status of knowledge in this area, up to 1969, in earlier Volumes of this Series. A wide variety of rare and modified sugars can be synthesized by way of addition reactions to unsaturated sugars; (176) L. Hough and B. A. Otter, Chem. Commun., 173 (1966). (177) L. Hough and B. A. Otter, Carbohyd. Res., 4, 126 (1967). (178) A. B. Foster, J. M. Duxbury, T. D . Inch, and J. M. Webber, Chem. Commun., 881 (1967).

DEOXYHALOGENO SUGARS

29 1

deoxyhalogeno sugars have frequently served as precursors to the unsaturated compounds. Moreover, some unsaturated carbohydrates are themselves of biological significance. Elimination reactions with carbohydrate derivatives containing primary halogeno groups have been known for several years; the resultant unsaturated sugars are either alkene or enolic derivatives. Thus, for example, treatment of suitably protected 6-bromo-6-deoxyor 6-deoxy-6-iodo-aldohexopyranose derivatives with silver fluoride in pyridine affords the 6-deoxyhex-5-enose vinyl ether^^^^^'^^; such dehydrohalogenations have also been achieved1s1*182 by means of sodium methoxide in methanol. The 5,6-unsaturated, pyranoid derivatives are precursors of aldos-5-uloses. Methyl 6-deoxy-p-~-xylo-hex5-enopyranoside7 for example, undergoes acid-catalyzed hydrolysis and subsequent rearrangement to afford crystalline 6-deoxy-~-xyloh e ~ o s - 5 - u l o s e ~the ~ ~ ;sugar component of hygromycin A has been prepared by hydrolyzing methyl 6-deoxy-a-~arabino-hex-5-enopyranoside.lS3The application of the reaction with silver fluoride to the pentofuranoid and hexulofuranoid series, to afford exocyclic vinyl ethers, was not reported 1966. Treatment of 3-O-acetyl5-deoxy-5-iodo-1,2-O-isopropylidene-cr-~-xylofuranose or -P-L-arabinofuranose with silver fluoride in pyridine gave 3-O-acetyl-5-deoxy1,2-0-isopropylidene-P-~-threo-pent-4-enofuranose (147); an unsaturated derivative was not obtained from the 3-hydroxy-~-xylo compound, but instead, an intramolecular, nucleophilic-displacement reaction occurred to afford the 3,5-anhydride. 4-O-Acetyl1-O-benzoyl-6-deoxy-6-iodo-2,3-0-isopropylidene-~-~-xylo-hexulofuranose gave a good yield of a vinyl ether which, on catalytic de-esterification, afforded crystalline 6-deoxy-2,3-0-isopropylideneP-D-threo-hex-5-enulofuranose (148). It has been reportedg3 that Mea

HZCQ?

HaOH

HacQ O-CMe, 147

HO 148

(179) B. Helferich and E. Himmen, Ber., 61, 1825 (1928). (180) B. Helferich and E. Himmen, Ber., 62, 2136 (1929). (181) K. Freudenberg and K. Raschig, Ber., 62, 373 (1929). (182) J. Lehmann and A. A. Benson, J. Amer. Chem. Soc., 86,4469 (1964). (183) S. Takahashi and M. Nakajima, Tetrahedron Lett., 2285 (1967).

WALTER A. SZAREK

292

treatment of 2,3,4,1’,3’,4’-hexa-O-benzoyl-6,6‘-dideoxy-6,6’-diiodosucrose with silver fluoride in pyridine affords the 5,5’-diene. have made a detailed study of the Lemieux and synthesis of some 2,Sunsaturated, pyranoid derivatives. They showed that methyl 4,6-O-benzylidene-2,3-dideoxy-a-~erythro-hex-2-enopyranoside (77) could conveniently be obtained from methyl 2,3-anhydro-4,6-O-benzylidene-a-~-a~lopyranoside by treatment with sodium iodide, sodium acetate, and acetic acid in acetone at reflux temperature to give methyl 4,6-0-benzylidene-2-deoxy-2-iodo-a-~-altropyranoside which, on being heated in pyridine with p-toluenesulfonic acid, is converted into the alkene 77 in high yield. It was found that the method is generally applicable, and the p-D anomer of compound 77, and the a- and p-D-threo isomers, were similarly prepared in high yield from the corresponding P-D-uZZ~,a-D-gulo, and P-D-talo oxiranes. However, Inchis5 has reported that methyl 4,6-0-benzylidene-2-deoxy-2-halogeno-a-~-altropyranosides can undergo facile, acid-catalyzed, acetal migrations to give methyl 3,4-0-(R)- and -(S)-benzylidene-2-deoxy-2-halogeno-a-~-altropyranosides. It was also found in that that, whereas reduction of methyl 3,4-0-(R)benzylidene-2-chloro-2-deoxy-6-0-ptolylsulfonyl-~-D-d~opyranoside with lithium aluminum hydride affords methyl 3,4-O-(S)-benzylidene-2,6-dideoxy-a-~-ribo-hexopyranoside, similar reduction of the corresponding 2-deoxy-2-iodo derivative affords only methyl 2,3,6-trideoxy-a-~-erythro-hex-2-enopyranoside. Some 2,3-unsaturated sugars have also been prepared by the reaction of 2-iodinated carbohydrates with sodium cobalt tetracarbonyl and carbon monoxide.ls8 Methyl 3,4,6-tri-O-acetyl-2-deoxy2-iodo-/3-~-glucopyranoside(149) in ether reacts at room temperature to give, in high yield, methyl 4,6-di-O-acetyl-2,3-dideoxy-p-~-erythrohex-2-enopyranoside (150). Under the same reaction conditions,

0 FH,OAc

AcO

VH,OAc CO, NaCo(CO),

ether, 25”

I

I 150 149

(184) R. U. Lemieux, E. Fraga, and K. A. Watanabe, Can. J . Chem., 46, 61 (1968). (185) T. D. Inch, Carbohyd. Res., 21,37 (1972). (186) A. Rosenthal and J. N. C. W-hyte, Can. J. Chem., 46,2245 (1968).

DEOXYHALOGENO SUGARS

293

methyl 3,4,6-tri-0-acetyl-2-deoxy-2-iodo-a-~-mannopyranoside failed to react, but, at loo", it was readily converted into the a anomer of 150. When methanol was used as the solvent, the elimination of iodine and an acetoxyl group was accompanied by complete removal of the remaining ester groups. No reaction occurred when methyl 3,4,6-tri-0-acetyl-2-bromo-2-deoxy-~-~-gluco(and a-D-mann0)pyranoside were treated with sodium cobalt tetracarbonyl and carbon monoxide in ether for 10 hours at 100". Dimitrijevich and Taylor1s7have found that treatment of methyl 4 - 0 - benzyl-3-deoxy-3- iodo-2-O-p-tolylsulfonyl-P-~-xylopyranoside (151)with sodium iodide in acetone at O-6affords methyl 4-O-benzyl2,3-dideoxy-~-~-glycero-pent-2-enopyranoside (152); however, at room temperature, the reaction yields the 2,3-alkene 152 and methyl 2-0-benzyl-3,4-dideoxy-~-~-glycero-pent-3-enopyranoside (153). A

I 151

152

153

where Bzl = benzyl.

possible mechanism for the formation of two isomeric alkenes involves an allylic rearrangement of the benzyl ether group in the 2,3-alkene 152, to give an equilibrium mixture of 152 and the 3,4-alkene 153. When methyl 4,6-0-benzylidene-3-chloro-3-deoxy-~-~-allopyranoside (154) was heated with sodium benzoate in tetrahydrofuran at reflux temperature, elimination occurred to give, in 85% yield, methyl 4,6-0-benzylidene-3-deoxy-~-~-erythro-hex-3-enopyranoside16s(155). A comparable elimination reaction has been reported by Horton and coworkers123; on treatment with potassium tertbutoxide in refluxing xylene, methyl 4,6-0-benzylidene-2,3-dibromo2,3-dideoxy-a-~-altropyranosideundergoes elimination of the elements of hydrogen bromide to give a 90% yield of methyl 4,6-0benzylidene-2-bromo-2,3-dideoxy-a-~-threo-hex-3-enopyranoside. Treatment of compound 155 with dilute hydrochloric acid in acetone at reflux temperature resulted in O-debenzylidenation, and elimination of water, to afford, in 71% yield, a hexopyranoside containing an a$-unsaturated, ketone grouping, namely, methyl 2,3(187) S. Dimitrijevich and N. F. Taylor, Carbohyd. Res., 11,531 (1969).

294

WALTER A. SZAREK

dideoxy-~-~-glycero-hex-2-enopyranosid-4-ulose~~~(l56). There is considerable interest in such compounds as 156, because of their potential utility as intermediates in the synthesis of other sugar derivatives, such as deoxy, branched-chain, and aminodeoxy sugars, of biological significance. Two examples of the synthetic utility of compound 156 were provided by its facile conversion into methyl 2,3,6-trideoxy-~-~-threo-hexopyranoside (methyl p-D-rhodinoside) and methyl 2,3,6-trideoxy-/3-~-erythro-hexopyranoside (methyl p-Damicetoside). A base-catalyzed, elimination reaction was a key step in a synthesis of D-ribose from L-glutamic acid.188In that work, L-glutamic acid was converted, by a series of reactions, into 5-0-ben~yl-2~3-dideoxyD-glycero-pentofuranose (157);from compound 157, a mixture of glycosides was obtained which, on treatment with bromine and calcium carbonate, gave the monobromo derivative 158 as a mixture of diastereoisomers. Base-catalyzed dehydrobromination of 158 afforded the unsaturated derivative 159. Hydroxylation of 159 with potassium permanganate or with osmium tetraoxide gave a mixture of methyl 5-O-benzyl-p-~-ribofuranosideand methyl 5-O-benzyl-a-~1yxofuranoside. Dehydrohalogenation reactions have been involved in several routes for the total synthesis, involving pyran intermediates, of

(188) K. Koga, M. Taniguchi, and S. Yamada, Tetruhedron Lett., 263 (1971).

295

DEOXYHALOGENO SUGARS

monosaccharides. Thus, for example, the synthesis of methyl 2,3anhydro-4-deoxy-6-O-methyl-a-~~-hjxo - hexopyranoside (164) has been achievedlE9from 3,4-dihydro-2-(methoxymethyl)-2H-pyran (160) in the following way. Bromomethoxylation of 160 gave a 9:l mixture of the two isomers 161 and 162. This mixture was treated with a boiling solution of sodium methoxide in methanol under reflux, to give a product that contained at least 95% of trans-5,6-dihydro-2-methoxy6-(methoxymethyl)-2H-pyran(163).rn-Chloroperoxybenzoic acid converted 163 into a mixture of methyl 2,3-anhydro:4-deoxy-6-O-methyla-DL-lyxo-hexopyranoside (164) (>95%) and methyl 2,3-anhydro-4deoxy-6-O-methyl-a-~~-~ibo-hexopyranoside (165)(>5%). In a related MeOCH,

I

I60

NH, (liquid) MeOH, Br,, 1-55" I

OMe OMe 161

162

roH NaOMe,

MeOCIt, E & O M e

'

164

+

MeOCIt,

-

0-Me

MeOCH,

163

-0Me '0'

165

296

WALTER A. SZAREK

study,lgOcompound 166 (~btained'~' by bromomethoxylation of 3,4-dihydro-3-methoxy-2H-pyran with 1,3-dimethylhydantoin in ethermethanol) was utilized to prepare methyl 4-O-methyl-a-~~-arabinopyranoside. Compound 166 was first dehydrobrominated with a boiling solution of potassium hydroxide in methanol, to give cis5,6-dihydro-2,5-dimethoxy-2H-pyran (167). Treatment of 167 with

-

M c O # M e

Br OMe 166

I61

m-chloroperoxybenzoic acid in dichloromethane afforded, almost exclusively, one epoxide, which was converted into methyl 4-0methyl-a-DL-arabinopyranoside by the action of aqueous potassium hydroxide. For the total synthesis of monosaccharides, a particularly significant development is the conversion of acrolein dimer (168) into the alkenes 6,8-dioxabicyclo[3.2.lloct-3-ene173 and 6,8-dioxabicyclo[3.2.l]oct-2-ene (174) by way of brominated intermediate~.~~2J93 Acrolein dimer was first reduced with sodium borohydride to give 3,4-dihydro-SH-pyran-2-methanol(169). When the alcohol 169 was heated in refluxing benzene containing a catalytic amount of p-toluenesulfonic acid, 6,8-dioxabicyclo[3.2.l]octane (170) was formed. Treatment of 170 with bromine in carbon tetrachloride gave a mixture of two isomeric monobromides, considered to be transand cis-4-bromo-6,8-dioxabicyclo[3.2.l]octane (171 and 172). Heating of the mixture of 171 and 172 in refluxing ethanolic potassium hydroxide gave the two alkenes 173 and 174, which were readily separated by gas-liquid chromatography; the proportions of the two isomeric alkenes obtained depended upon the proportion of base to monobromide used in the dehydrohalogenation. 6,8-Dioxabicyclo[3.2.1]oct-Sene (173) and its isomer 174 have been used for the synthesis of several monosaccharides and their derivative^.^^^^^^^^^^^ ~

(189) F. Sweet and R. K. Brown, Can. J. Chem., 46, 2283 (1968). (190) R. M. Srivastava and R. K. Brown, Can. J. Chem., 48,2341 (1970). (191) R. M. Srivastava and R. K. Brown, Can. J. Chem., 48, 2334 (1970). (192) F. Sweet and R. K. Brown, Can. J. Chem., 46,2289 (1968). (193) T. P. Murray, C. S. Williams, and R. K. Brown, J. Org. Chem., 36, 1311 (1971). (194) U. P. Singh and R. K. Brown, Can.J. Chem., 48, 1791 (1970); 49, 1179, 3342 (1971). (195) T. P. Murray, U. P. Singh, and R. K. Brown, Can. J. Chem., 49, 2132 (1971).

DEOXYHALOGENO SUGARS

168

169

297

170

Br Br 172 171

EtOH

173

174

During the 1960's, total syntheses of some sugars found in antibiotics were also achieved by way of elimination reactions of brominated intermediates. One of these syntheses was that of methyl (181). Mycaminose is a sugar component in the DL-mycamin~side'~~ antibiotics magnamycin, spiramycin, and leucomycin. Treatment of 175 with bromine in boiling methanol containing hydrogen chloride yielded three bromo compounds, namely, 176, 177, and 178. Each of these three compounds could be converted into the unsaturated derivative 179; compound 178, for example, whose n.m.r. spectrum suggested that the bromine and methoxyl groups are diaxial (oneproton doublet at ~ 5 . 3 1 , J1 Hz), readily afforded 179 on treatment with sodium azide in N,N-dimethylformamide at 120-125'. Oxidation of 179 with peroxybenzoic acid gave the epoxide 180, which afforded methyl DL-mycaminoside (181) when treated with a saturated, aqueous solution of dimethylamine.

(196) S. Yasuda and T.Matsumoto, Tetrahedron Lett., 4397 (1969).

298

WALTER A. SZAREK

The unsaturated alcohol 179 was also a key intermediate for the synthesi~'~'of methyl DL-oleandroside (182) and its C-3 epimer, methyl DL-cymaroside (183). Oleandrose is a sugar component of

(197) S. Yasuda and T. Matsumoto, Tetrahedron Lett., 4393 (1969).

DEOXYHALOGENO SUGARS

299

cardiac glycosides and of the antibiotic oleandomycin. Brominated intermediates have also been employed1gsin a synthesis of DL-desosamine (DL-picrocin).Desosamine is a component of several macrolide antibiotics, including erythromycin, oleandomycin, and narbomycin; its structure has been shown to be that of 3,4,6-trideoxy-3-(dimethylamino)-D-xylo-hexose. The p-iodo nitro adducts obtained by addition of nitryl iodide to unsaturated sugars (see Section 11,3b; p. 270) readily undergo dehydro-iodination on treatment with sodium hydrogen carbonate in boiling benzene, to give the highly reactive a-nitroalkenes. Thus, the 5-iodo-6-C-nitro adducts (87 and 88) respectively obtained from 3-0-a~etyl-5~6-dideoxy1,2-O-isopropylidene-a-~-xyZo-hex-5-enofuranose and methyl 5,6-dideoxy-2,3-di-O-p-tolylsulfonyl-a-~-urub~nohex-5-enofuranoside afforded 3-0-acetyl-5,6-dideoxy- 1,2-O-isopropylidene-6-nitro-a-~-xy~o-hex-5-enofuranose~~~ (184) and methyl 5,6-dideoxy - 6-nitro- 2,3- di - 0- p -to1yl sulfonyl-a -~-urubino-hex-5-eno f u r a n o ~ i d e(185), l ~ ~ respectively. Similarly, the 3-iodo-4-C-nitro adduct obtained from benzyl 2-0-benzyl-3,4-dideoxy-a-~-glycero-pent-3enopyranoside gave benzyl 2-0-benzyl-3,4-dideoxy-4-nitro-a-~-gZycero-pent-3-enopyrano~idel~~ (186). The electron-withdrawing effect

J- f 0-CMe, 184

CH II HCNO,

I

OBzl

OTs 185

186

of the nitro group in a-nitroalkenes permits facile additions of nucleophiles to the carbon-carbon double-bond, to give p-substituted nitroalkanes. Unsaturated, nitro sugars of this type have, therefore, proved to be versatile intermediates in a variety of synthetic reactions, such as amination, alkoxylation, carbon-chain extension, and introduction of chain branching. Examples of such reactions were discussed by Baerlg9in Volume 24 of this Series.

3. Reductive Dehalogenation The deoxy sugars200are an important class of carbohydrates that occur quite widely in Nature. Deoxyhalogeno sugars are useful (198) F. Korte, A. Bilow, and R. Heinz, Tetrahedron, 18, 657 (1962). (199) H. H. Baer, Aduan. Carbohyd. Chem. Bfochem., 24,67 (1969).

WALTER A. SZAREK

300

intermediates in the synthesis of deoxy sugars. It has been known for several years that the iodo and bromo derivatives can be reduced to form the deoxy sugars by a variety of reducing agents, including zinc in acetic acid, sodium amalgam in aqueous ether or ethanol, lithium aluminum hydride, and hydrogen in the presence of palladium-on-charcoal or Raney nickel. Hanessian200(a) has described many examples of the reductive dehalogenation of deoxyhalogeno sugars in an earlier Volume of this Series; in the present Section, only some later examples and new developments will be discussed. Prior to 1969, there were few reports of the reduction of chlorodeoxy sugar^^^'^^; however, in that year, it was reported201that these derivatives can be reduced by a particularly active form of Raney nickel catalyst. Thus, hydrogenation over Raney nickel of methyl 4,6-dichloro-4,6-dideoxy-3-O-methyl-~-galactopyranoside (188) [prepared by reaction of methyl 3-0-methyl-D-glucopyranoside (187) with sulfuryl chloride, followed by dechlorosulfation of the product by use of sodium iodide] gave a product which, on acid-catalyzed hydrolysis, afforded202D-chalcose (189), a sugar component of an antibiotic A facile synthesis of 4,6-dideoxy-~-xylo-hexose has been achievedse by an analogous route to afford an overall yield of 65% from the commercially available methyl a-D-glucopyranoside.

,

I

OH 187

OH

OH I88

189

The biologically important sugars paratose (3,6-dideoxy-~-ribohexose) and tyvelose (3,6-dideoxy-~-urubino-hexose) have also been conveniently prepared203by routes involving reductive dechlorination by hydrogenation over Raney nickel catalyst; these 3,6-dideoxy(200)For reviews, see (a) S. Hanessian, Aduan. Carbohyd. Chem., 21, 143 (1966);(b) Aduan. Chem. Ser., 74 (1968);(c) R. F. Butterworth and S. Hanessian, Aduan. Carbohyd. Chem. Biochem., 26,279 (1971). (201)B.T.Lawton, D. J. Ward, W. A. Szarek, and J. K. N. Jones, Can.J.Chem., 47,2899 (1969). (202)A synthesis of chalcose has also been achieved by way of reduction of a deoxyiodo sugar [see N. K. Kochetkov and A. I. Usov, Tetrahedron Lett., 519 (196311. (203)E. H.Williams, W. A. Szarek, and J. K. N. Jones, Can. J . Chem., 49,796 (1971).

DEOXYHALOGENO SUGARS

301

hexoses have been isolated from lipopolysaccharides elaborated by Gram-negative bacteria. One of the routes employed for the synthesis of paratose (193) also constitutes a further demonstration of the versatility of the method of preparation of chlorodeoxy sugars by use of sulfuryl chloride. Thus, methyl 4,6-0-benzylidene-3-chloro3-deoxy-/3-~-allopyranoside(154) was initially prepared by reaction of methyl 4,6-0-benzylidene-~-~-glucopyranoside with sulfuryl chloride, followed by dechlorosulfation of the product by use of sodium iodide; acid-catalyzed 0-debenzylidenation of compound 154 then gave methyl 3-chloro-3-deoxy-/3-~-allopyranoside (190). Treatment of 190 with sulfuryl chloride, followed by dechlorosulfation of the product, afforded methyl 3,6-dichloro-3,6-dideoxy-~-~-allopyranoside (191).The non-substitution by chloride ion of the intermediate chlorosulfonyloxy group at C-4 is attributed to the presence of the vicinal, axial substituent at C-3;also, a chlorosulfonyloxy group at C-2 is deactivated to nucleophilic substitution by chloride ion (see Section II,2a; p. 233). Hydrogenation of 191, in the presence of potassium hydroxide, over Raney nickel gave methyl 3,6-dideoxy-/3-~-~ibohexopyranoside (192) which, on acid-catalyzed hydrolysis, afforded paratose (193).

dl

OH

OH

C1

190 R = OH 191 R = C1

154

Me

Me

I

OH

OH 193

192

already cited, methyl In the work of Lawton, Szarek, and 4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside was converted into

302

WALTER A. SZAREK

methyl 4,6-dideoxy-a-~-xyZo-hexopyranoside by hydrogenation over Raney nickel in the presence of potassium hydroxide. However, when triethylamine was substituted for potassium hydroxide, a selective, reductive dechlorination occurred at C-4 to give methyl 6-chloro-4,6dideoxy-a-~-xyZo-hexopyranoside.~~~ Similarly, hydrogenation of methyl 3,4,6-trichloro-3,4,6-trideoxy-a-~-allopyranoside (194) over Raney nickel in the presence of potassium hydroxide affords methyl 3,4,6-trideoxy-a-~-e~yth~o-hexopyranoside (195), whereas hydrogenation in the presence of triethylamine leads to methyl 6-chloro-3,4,6trideoxy-a-~-eyth~o-hexopyranoside~~~ (196). An explanation for the

196

observed selectivity in these two cases has not yet been offered. The scope of the selective dechlorination in the presence of triethylamine should be examined, because the reaction is potentially of considerable significance in synthesis. One example of the utility of this procedure was provided by the ready synthesis of a 4-deo~yhexose.'~' Another example of a selective dehalogenation in the carbohydrate field has been reported by Hanessian and P l e ~ s a s . * ~Catalytic (~) hydrogenation of methyl 4-O-benzoyl-3-bromo-2,6-dichloro-2,3,6trideoxy-a-D-mannopyranoside over palladium-on-carbon in the presence of barium carbonate gave methyl 4-O-benzoyl-6-chloro-2,3,6(204) B. T. Lawton, W. A. Szarek, and J. K. N. Jones, unpublished results.

DEOXYHALOGENO SUGARS

303

trideoxy-a-D-erythro-hexopyranoside. The presence of a bromine atom at C-3 apparently leads to the selective reduction of the halogen atoms on C-2 and C-3, relative to the chlorine atom on C-6, because, in a precursor, namely, methyl 3,4-0-benzylidene-2,6-dichloro-2,6dideoxy-a-D-altropyranoside, the two chlorine atoms were inert to catalytic hydrogenation. have achieved the reduction of some chloroArita and deoxy sugars in high yield by means of tributyltin hydride in the presence of 2,2’-azobis(2-methylpropionitrile).The reaction with methyl 2,3-di-0-acetyl-4,6-dichloro-4,6-dideoxy-~-~-galactopyranoside at 60” gave methyl 2,3-di-O-acetyl-6-chloro-4,6-dideoxya-D-xybhexopyranoside as the main product. A free-radical mechanism has been proposedzosfor the reduction of alkyl halides by organotin hydrides. In accordance with this proposal, it was found that the presence of the radical initiator 2,2’-azobis(2-methylpropionitrile) was essential for the reduction of chlorodeoxy sugars; moreover, the relative reactivities of the two chlorine atoms in methyl 2,3-di-0acetyl-4,6-dichloro-4,6-dideoxy-a-~-galactopyranoside follow a freeradical order. Tributyltin hydride in the presence of 2,2’-azobis(2-methylpropionitrile) has been used20saalso for the reduction of the bromodeoxy sugar moieties of some pyrimidine nucleosides. An investigation of the reduction of chlorodeoxy sugars with lithium aluminum hydride has been reportedP8 In one experiment, 3-deuterio1,2:5,6-di-O-isopropylidene-a-D-allofuranose (197) was prepared, and converted into 3-chloro-3-deoxy-3-deuterio-1,2:5,6-di-O-isopropylidene-a-D-glucofuranose (198) by treatment with triphenylphosphine-carbon tetrachloride; reduction with lithium aluminum hydride gave 3-deoxy-3-deuterio- 1,2:5,6-di-O-isopropylidene-a-~ribo-hexofuranose (199),a result which established that the reduction must have occurred with- retention of configuration at C-3. Melton and Sless0l-2~~ have prepared mono-6-chloro-6-deoxy-,mono6-bromo-6-deoxy-, and mono-6-deoxy-6-iodo-cyclohexaamylose by nucleophilic displacements of the sulfonate group in mono-6-O-ptolylsulfonylcyclohexaamylose. The monoiodo and monochloro derivatives were each reduced to mono-6-deoxycyclohexaamylose over W-2 and W-4 Raney nickel catalysts, respectively. The synthesis of 5-deoxy-1,2-0-isopropylidene-a-~-rylo-hexofur(205) H. Arita, N. Ueda, and Y. Matsushima, Bull. Chem. SOC. Jap., 45, 567 (1972). (206) L. W. Menapace and H. G. Kuivila, J . Amer. Chem. SOC., 86,3047 (1964). (206a) H. Hiebabeckf, J. FarkaS, and F. h m ,Collect. Czech. Chem. Commun., 37, 2059 (1972). (207) L. D. Melton and K. N. Slessor, Carbohyd. Res., 18, 29 (1971).

WALTER A. SZAREK

304

Ph,P

HO

0-CMe,

D

197

0-CMe,

198

199

anurono-6,3-lactone has been achieved by the reaction of 5-bromo-5deoxy- 1,2-O-isopropylidene-/3-~-idofuranurono-6,3-lactone with thiolacetic acid, or a-toluenethiol, in pyridine, or by the reaction of the bromo derivative with triethyl phosphite.208 Nickel carbonyl in tetrahydrofuran has been foundzoaato reduce, under mild conditions, polyhalogenomethyl groups to di- or mono-halogenomethyl groups, a procedure employed in a synthesis of 5-bromo-5-deoxy-~~-lyxose and -xylose. 4. Miscellaneous Reactions

It is known that C-I bonds undergo homolytic cleavage by ultraviolet light, and this reaction has been used for the preparation of a deoxy sugar.z0gThus, irradiation (Pyrex filter) of a methanol solution of 6-deoxy-6-iodo-1,2:3,4-di-0-isopropylidene-a-~-galactopyranose in the presence of sodium hydroxide led to a rapid, almost quantitative conversion into 6-deoxy-1,2:3,4-di-O-isopropylidene-a-~-galactopyranose. When the irradiation is performed in tert-butyl alcohol, a poorly hydrogen-donating solvent, 6-deoxy- 1,2:3,4-di-O-isopropylidenea-D-galactopyranose is produced in only 36% yield, together with 6-deoxy-1,2:3,4-di-0-isopropylidene-~-uru~~~o-hex-5-enopyranose in (208) T. Irimajiri, H. Yoshida, T. Ogata, and S. Inokawa, Bull. Chem. SOC.Jap., 43,3242 (1970). (208a) T. Kunieda, T. Tamura, and T. Takizawa, Chem. Commun., 885 (1972); see also, T. Tamura, T. Kunieda, and T. Takizawa, Tetrahedron Lett., 2219 (1972). (209) W. W. Binkley and R. W. Binkley, Carbohyd. Res., 8, 370 (1968); 11, 1 (1969).

DEOXYHALOGENO SUGARS

305

32% yield. Photolysis of an iodo precursor has also been employed as a route to 6-deoxy-a,a-trehalose.209" In addition to undergoing reactions of the type described in Section I11 (see p. 281), halogeno groups can influence the reactivity of a carbohydrate molecule by their steric and electronic properties, Of particular interest is their effect on the rate of hydrolysis of glycosides. Barnettl has summarized the results of studies of the acidcatalyzed hydrolysis of glycopyranosides containing halogeno groups. An additional example is found in the work of Buncel and Bradley,210 who employed methyl 2-chloro-2-deoxy-~-~-glucopyranoside as the substrate. It was observed that this compound is hydrolyzed, in 2 M hydrochloric acid at 60", 35 times more slowly than methyl p-Dglucopyranoside, and more than lo4 times more slowly than methyl 2-deoxy-/3-~-urubino-hexopyranoside. Application of the Hammett criterion, and of the Bunnett criterion, indicated a unimolecular (A-1) mechanism of hydrolysis; however, the entropy of activation was considerably smaller than that observed for the hydrolysis of methyl p-D-ghcopyranoside, a result that was interpreted as being indicative of partial A-2 character. An aromatization has been observed211 with 1,3,5-tri-O-acetyl2-chloro-2-deoxy-~-arabinose; when this compound was distilled at 1500/0.5 torr, some decomposition occurred to give 2-(acetoxyme thyl)-4-chlorofuran. A branched-chain iodo sugar derivative, 1,5-anhydro-4,6-0-benzylidene-2,3-dideoxy-3-C-(iodomethyl)-~-ribo-hex-l-enitol [4,6-0-benzylidene-3-deoxy-3-C-(iodomethyl)-~-allal] (200), is one of the products formed on treatment of methyl 4,6-0-benzylidene-2,3dideoxy-a-~-erythro-hex-2-enopyranoside (77) with the SimmonsSmith reagent (diiodomethane and zinc-copper coup1e).123*212 Compound 200 displays high solvolytic reactivity, an observation that has been rationalized by supposing the formation of the highly stabilized carbonium (201). Thus, under conditions wherein methyl 2,3,4-tri-0-acetyl-6-deoxy-6-iodo-a-~-glucopyranoside required more than 24 hours to react appreciably with an excess of silver nitrate in 50% aqueous p-dioxane buffered with silver carbonate, the iodide 200 was hydrolyzed completely in less than 1 minute; the product of hydrolysis of 200 is the cyclopropyl aldehyde 202. Methanolysis of (209a) E. R. Guilloux, J. Defaye, R. H. Bell, and D. Horton, Carbohyd. Res., 20, 421 (1972). (210) E. Buncel and P. R. Bradley, Can. J. Chem., 45,515 (1967). (211) J. Kuszmann and P. Sohlr, Carbohyd. Res., 14,415 (1970). (212) B. Fraser-Reid and B. Radatus, Can. J. Chem., 47,4095 (1969). (213) B. Fraser-Reid and B. Radatus, Can. J . Chem., 48,2146 (1970).

WALTER A. SZAREK

306

200 gave a 1:l mixture of the a and /3 anomers of methyl 4,6-0-benzyl-

idene-2,3-dideoxy-2,3-C-methylene-a-~-allopyranoside (203).In basic media, compound 200 undergoes quantitative dehydro-iodination to the conjugated diene, namely, 1,5-anhydro-4,6-0-benzylidene-2,3dideoxy-3-C-methylene-~-erythro-hex-l-enitol (204). Compound 200

202

200

203

201

204

has also been transformed,214by way of a brominated intermediate, into the C-3 epimer, namely, 1,5-anhydro-4,6-0-benzylidene-2,3dideoxy-SC-(iodomethyl)-~-u~ubino-hex-l-enitol[4,6-O-benzylidene3-deoxy-3-C-(iodomethy1)-D-glucal]. (214) B. Fraser-Reid and B. Radatus, Chem. Commun., 779 (1970).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES BY

NICOLAI K. KOCHETKOV AND VLADIMIR N. SHIBAEV N . D . Zelinsky Institute of Organic Chemistry, Academy of Sciences, Moscow, U.S.S.R.

I. Introduction

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

307

11. Isolation, Characterization, and Elucidation of Structure of Natural

Glycosyl Esters of Nucleoside Pyrophosphates . . . . . . 1. GeneralMethods.. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . 310 ...... .... . .

2. Adenosine 5‘-(Glycosyl Pyrophosphates). . . . . . . . . . . . . . . . . . . . 3. Cytidine 5’-(Glycosyl Pyrophosphates). . . . . . . . . . . . . . . . . . . . . 4. Guanosine 5’-(Glycosyl Pyrophosphates) . . . , . . . . . . . . . . . 5. Thymidine 5’-(Glycosyl Pyrophosphates) . . . . . . . . . . . . . . . . . . . 6. Uridine 5’-(Glycosyl Pyrophosphates) . . . . . . . . . . . . . . . . . . . 7. Related Nucleotide Derivatives . . . . . . . . . . . . . . . . , . , . . . . 111. Preparation of Glycosyl Esters of Nucleoside Pyrophosphates . . . . . . . . 1. Enzymic and Fermentation Procedures . . . . , . . . . . . . . . . . . . . . 2. Chemical Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Chemical Modification of Natural “Sugar Nucleotides” . . . . . ... IV. Chemical Reactivity of Glycosyl Esters of Nucleoside Pyrophosphates . . . V. Enzymic Reactions of Glycosyl Esters of Nucleoside Pyrophosphates . . . . 1. Transformations of the Glycosyl Croup . . . . . . . . . . . . . . . . . . . . 2. Splitting of the Pyrophosphate Linkage . . . . . . . . . . . . . . . . . . . 3. Splitting of the Glycosyl Linkage (Glycosylation) . . . . . . . . .. VI. Conclusion.. . . . . .. .. . . .. . .. . . . .. .. ... .. . . .

.

.

. .

.

..

.

.... .

.

..

... . ..

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

310 314 315 318 322 324 332 334 335 344 354 356 362 363 388 391 397

I. INTRODUCTION

The important role of glycosyl esters of nucleoside pyrophosphates (often referred to loosely as “sugar nucleotides” or “nucleoside diphosphate sugars”) in carbohydrate metabolism is well known. Two comprehensive Chapters of this Series,’B2 as well as several surveys in other publication^^-^ have already appeared. These reviews deal mainly with the metabolism of these compounds, and (1) E. F. Neufeld and W. Z. Hassid, Aduan. Carbohyd. Chem., 18, 309 (1963). (2) H. Nikaido and W. Z. Hassid,Advan. Cusbohyd. Chem. Biochem., 26,351 (1971). (3) V. Ginsburg, Aduun. Enxymol., 26,35 (1964). (4) W. J. Kelleher,J. Pharm. Sci., 54, 1081 (1965). (5) W. Z. Hassid, in “Metabolic Pathways,” D. M. Greenberg, ed., Academic Press,

New York, 1967, Vol. 1, p. 307. 307

308

N. K. KOCHETKOV AND V. N. SHIBAEV

particularly with the synthesis of glycosides, polysaccharides, and complex carbohydrate-containing polymers. The purpose of this Chapter is to summarize the present knowledge concerning the isolation, structure, preparation, and chemical reactions of glycosyl esters of nucleoside pyrophosphates. These aspects have been considered only briefly in the articles cited. Another important topic, which has not been treated previously in a comprehensive manner from a chemical viewpoint, is the mechanism of the enzymic reactions of these compounds, and the specificity of their interaction with the corresponding enzymes. These topics will also be considered here. The first studies of “sugar nucleotides” were reported in 19491950. At that time, Park isolated from Staphylococcus aureus several complex derivatives of uridine 5’-pyrophosphate containing a residue and a peptide Simultaof 2-acetamido-2-deoxy-~-glucose neously, Leloir and coworkers isolated, from a D-galactose-adapted yeast (Saccharomyces fragilis), a nucleotide co-factor for the conversion of D-galactose derivatives into those of D-glucose. Its structure has been established as uridine 5‘-(a-D-glucopyranosyl pyrophosphate).8 Rapid progress in the identification of various glycosyl esters of uridine 5’-pyropho~phate~-’~ was followed by the isolation of different nucleoside derivative^.'^-'^ The glycosyl esters of five nucleoside pyrophosphates are now known to occur in Nature. Their typical structures are given by formulas 1-5. Derivatives of these and other nucleosides have also been prepared by chemical or enzymic methods. More than a hundred similar compounds have been described, including derivatives having the hexosyl residue (6) J. T. Park and M. Johnson,]. B i d . Chem., 179, 545 (1949). (7) J. T. Park,]. Biol. Chem., 194, 877, 885, 897 (1952). (8) C. E. Cardini, A. C. Paladini, R. Caputto, and L. F. Leloir, Nature, 165, 191 (1950); R. Caputto, L. F. Leloir, C. E. Cardini, and A. C. Paladini,]. B i d . Chem., 184, 333 (1950). (9) E. Cabib, L. F. Leloir, and C. E. Cardini, 1. B i d . Chem., 203, 1055 (1953). (10) I. D. E. Storey and G. J. Dutton, Biochem. J . , 59,279 (1955). (11) E. E. B. Smith and G. T. Mills, Biochim. Biophys. Acta, 13, 386 (1954). (12) V. Ginsburg, P. K. Stumpf, and W. Z. Hassid,]. B i d . Chem., 223, 977 (1956). (13) E. Cabib and L. F. Leloir,]. B i d . Chem., 206, 779 (1954). (14) J. Baddiley and N. L. Blumsom, Biochim. Biophys. Acta, 39, 376 (1960). (15) R. Okazaki, Biochem. Biophys. Res. Commun., 1,34 (1959); Biochim. Biophys. Acta, 44,487 (1960). (16) H. Nikaido and K. Jokura, Biochem. Biophys. Res. Commun., 6, 304 (1961). (17) V. Ginsburg, P. J. O’Brien, and C. W. Hall, Biochem. Biophys. Res. Commun., 7, 1 (1962). (18) H. Kauss and 0. Kandler, Z. Naturforsch., B , 17, 858 (1962).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

HO

R =

309

R

OH

OH

OH

H

OH

1

2

3

4

5

1

Adenosine 5’-(a-~-glucopyranosylpyrophosphate) (“adenosine diphosphate glucose”)

2 Cytidine 5’-(a-~-glucopyranosylpyrophosphate)

(“cytidine diphosphate glucose”) 3 Guanosine 5‘-(~u-~-glucopyranosyl pyrophosphate)

guanosine diphosphate glucose”) 4 Thymidine 5‘-(c~-~-glucopyranosyl pyrophosphate) (“ thymidine diphosphate glucose“) 5

Uridine 5’- (a-D-glucopyranosylpyrophosphate) (‘I uridine diphosphate glucose”)

substituted by sulfate, phosphate, or a lactic acid residue having a peptide chain attached. Analogous oligosaccharide derivatives are also known. Some comments on the nomenclature used are in order. The monosaccharide residue linked through its anomeric position with the pyrophosphate group is referred to as the “glycosyl group,” and the p-D-ribofuranosyl residue is described as the “monosaccharide of the nucleoside residue.” For numbering the atoms of the heterocyclic base, non-primed numerals are used. The atoms of the “monosaccharide of the nucleoside residue” are assigned singly-primed numerals, and those of the glycosyl group are denoted by doublyprimed numerals. It should be noted that primes are not required for a component residue in a systematic name when that component is given in parentheses in the name.

310

N. K. KOCHETKOV AND V. N. SHIBAEV

11. ISOLATION, CHARACTERIZATION, AND ELUCIDATION OF STRUCTURE OF NATURAL GLYCOSYL ESTERSOF NUCLEOSIDE PYROPHOSPHATES 1. General Methods

Dilute perchloric acid or trichloroacetic acid, or ethanol, is usually employed for extraction of the glycosyl esters of nucleoside pyrophosphates from biological material^.'^ The high lability of these compounds in acidic media (see Section IV, p. 356) leads to unavoidable losses during extraction with acids. Extraction with ethanol can lead to difficulties, as ethanol may not completely inactivate pyrophosphatases present in the tissue; the action of these enzymes may result in partial degradation of the nucleoside pyrophosphate derivatives. Such a situation has been encountered particularly with plant tissues.20 The isolation of an individual glycosyl ester of a nucleoside pyrophosphate is a rather difficult task. Ion-exchange column-chromatography and preparative, paper-chromatography have been the main methods used for solving problems in separation. Two ion-exchange chromatographic procedures have found wide application. Both make use of strongly basic resins, such as Dowex-1 or Dowex-2. Cabib, Leloir, and Cardinig suggested the use of sodium chloride in dilute hydrochloric acid for eluting nucleotides from a column of ion-exchange resin. Modifications involve gradient elution2' and the use of acid solutions of ammonium,2z lithium,14 or calciumz1 chlorides as eluants. Another procedure is based on the application of formic acid-ammonium formate mixtures.23 The ion-exchange separation usually affords individual fractions of structurally related glycosyl esters of nucleoside pyrophosphates, containing the same nucleotide residue, but differing in the structure of the glycosyl groups. Separation of the esters of N-acetylhexosamines, uronic acids, and neutral monosaccharides from one another is also usually achieved. ( 19) Detailed description of different extraction procedures and several separation

techniques for complex nucleotides have been given by J. J. Saukkonen, Chromutogr. Reo., 6,53 (1964). (20) D. F. Cumming, Biochem. 1..116, 189 (1970). (21) H. G. Pontis and N. L. Blumsom, Biochim. Biophys. Actu, 27,618 (1958). (22) E. Recondo, M. Dankert and S. Passeron, Biochim. Biophys, Actu, 107, 129 (1965). (23) R. B. Hurlbert, H. Schmitz, A. F. Brumm, andV. R. PotterJ. Biol. Chem., 209,23 (1954).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

311

Preparative, paper-chromatography is frequently used for further fractionation of the resulting mixtures. The high lability of glycosyl esters of nucleoside pyrophosphates seriously limits the choice of solvent systems. Systems used most commonly are neutral or slightly acidic mixtures of ethanol with ammonium a ~ e t a t e , ’ or ~ ’weakly ~~ acidic solvents based on 2-methylpropionic acideZ6 A solvent system containing morpholinium borate has also been found extremely Some difficulties arise in desalting the solutions of glycosyl esters of nucleoside pyrophosphates obtained by chromatography. The method developed by Leloir’s groupe involves the adsorption of the nucleotide derivatives on charcoal, followed by their elution with aqueous ethanol containing ammonia. This procedure has enjoyed wide use, although it may result in a considerable loss of nucleotide material, especially of guanosine derivatives (compare Ref. 20). Gel filtration through a column of SephadexZ8or Bio-gelz9was found advantageous in many cases. Other procedures used for desalting include the precipitation of insoluble calcium salts of glycosyl esters of nucleoside pyrophosphates, removal of volatile ammonium or triethylammonium salts in DUCUO, or extraction of suitable salts (such as lithium chloride, or ammonium acetate or 2-methylpropionate) with organic solvents. Paper chromatography may be used instead of such an extraction procedure.30 The unambiguous characterization of glycosyl esters of nucleoside pyrophosphates is difficult, as no suitable crystalline derivatives are known for them, and the hygroscopic nature of their ammonium and metal salts prevents reliable interpretation of the data of elementary analysis. Comparative identification of a glycosyl ester of a nucleoside pyrophosphate with an authentic sample is usually based on the identity of their ultraviolet spectra and by comparison of chromatographic mobilities of the samples and their degradation products. In addition to paper-chromatographic techniques, it may be of value to use paper (24) A. Paladini and L. F. Leloir, Biochem. J., 51, 426 (1952). (25) The artifacts possible with an ethanol-neutral ammonium acetate system are described by K. C. Towey and R. M. Roberts, J. Chromatogr., 47, 287 (1970). (26) R. Magasanik, E. Vischer, R. Doniger, D. Elson, and E. Chargaff,]. Biol. Chem., 186, 37 (1950). (27) H. Carminatti, S. Passeron, M. Dankert, and E. Recondo,J. Chromatogr., 18,342 (1965). (28) E. I. Budowsky and V. N. Shibaev, Vopr. Med. Khim., 13, 554 (1967). (29) M. Uziel and W. E. Cohn, Biochim. Biophys. Acta, 103, 539 (1965). (30) J. L. Strominger, J. Biol. Chem., 237, 1388 (1962).

312

N. K. KOCHETKOV AND V. N. SHIBAEV

electrophoresis,3l chromatography on thin layers of ECTEOLA-32or p ~ l y ( e t h y l e n e i m i n e ) -cellulose ~ ~ * ~ ~ powder or on poly(ethy1eneimine)treated paper,35 or gas-liquid chromatography of 0-trimethylsilyl The ability of the compound under investigation to participate in enzymic reactions specific for the authentic compound is of the utmost importance for identification and quantitative determination, Elucidation of the structures of those glycosyl esters of nucleoside pyrophosphates that are known has been based mainly on analytical data and on identification of their degradation products after treatment with acids or enzymes. A typical example of such a degradation is shown in Scheme 1 for uridine 5’-(a-D-glucopyranosyl p yrophosphate). The degradative procedures available include: (a) Mild, acidic hydrolysis (10mM hydrochloric acid or 10% acetic acid are commonly used reagents) to produce the nucleoside 5’-pyrophosphate (6) and the monosaccharide. (b) More-vigorous acidic hydrolysis to convert the pyrophosphate 6 into the nucleoside 5’-phosphate (7). (c) Cleavage of the pyrophosphate linkage in 5, by using pyrophosphatases, to give the nucleotide (7) and glycosyl phosphate (8). enzymes are usually used. Snake-venom3’ or ( d ) Conversion of the nucleoside 5’-phosphate into a nucleoside under the action of phosphate monoesterase or snake-venom 5‘nucleotidase. With the use of crude snake-venom, the degradations (c) and ( d ) proceed simultaneously. (e) Conversion of the glycosyl phosphate into the free monosaccharide by the action of phosphate monoesterase. The nucleotides resulting after degradation can readily be identified by use of ultraviolet-spectral and chromatographic techniques. (31) A. M. Crestfield and F. W. Allen, Anal. Chem., 27,424 (1955);A. B. Banerjee and N. C. Ganguli,]. Electroanal. Chem., 2, 501 (1961). (32) C. P. Dietrich, S. M. C. Dietrich, and H. G. Pontis, J . Chromatogr., 15, 277 (1964). (33) K. Randerath and E. Randerath,]. Chromatogr., 16,111 (1964);Anal. Biochem.,l3, 574 (1965). (34) D. D. Christianson, H. B. Sinclair, and J. W. Paulis, Biochim. Biophys. Acta, 121, 412 (1966). (35) H. Verachtert, S. T. Bass, J. Wilder, and R. G. Hansen,Anal. Biochem., 11, 497 (196s). (36) F. Eisenberg and A. H. Bolden, And. Biochem., 29,284 (1969).The procedure used results in a degradation of the glycosyl esters of nucleoside pyrophosphates, but the pattern obtained seems to be characteristic. (37)N. 0. Kaplan and F. E. Stolzenback, Methods Enzymol., 3, 899 (1955). (38) A. Kornberg and W. E. Priger, 1.Biol. Chem., 182, 763 (1950).

I

v)

z-io

8

o=n-o

L

O A - 0

I x I

4

j!

O=&-0

l x I

I

X

0 X

8

b

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

I0

O=&-0

I

I 0

0

I

O=&-0

/

313

314

N. K. KOCHETKOV AND V. N. SHIBAEV

Their characteristic optical rotatory dispersion or circular-dichroism curves, and their infrared spectra, rich in characteristic frequencies, may be useful. Paper chromatography p e w i t s preliminary identification of the glycosyl phosphate or monosaccharide resulting after degradation, and the specific enzymic reactions of these products are widely used to provide additional evidence. 2. Adenosine 5'-(Glycosyl Pyrophosphates)

The most common compound of this class is adenosine 5'-(a-Dglucopyranosyl pyrophosphate) (1). It has been isolated from extracts of many plants, including ripening grains of C O ~ : ~ .rice?' ~ ~ larch wood,2O and Chlorella ~ e l l s ' ~It*has ~ ~been * ~ found to be the main component among the nucleotides adsorbed on granules of potato s t a r ~ h . 4The ~ isolation of adenosine 5'-(a-D-glucopyranosyl pyrophosphate) from leaf-rust u r e d o ~ p o r e s ~loose-smut ~, chlamydospores,46 and Salmonella cells4' has been reported. As for the occurrence of the ester 1 in mammals, the situation is not quite clear; thus far, it has been identified only in one instance, in an extract of the blood cells of a single individuaL4*No enzymic reactions specific for 1 have been reported in mammals, although the rather unspecific enzyme pyrophosphorylase, widely distributed in animal tissues, is capable of catalyzing its b i o s y n t h e s i ~The , ~ ~availability ~~~ of a synthetic sample of the ester 1 (prepared from a-D-glucopyranosyl phosphate, see Section 111, p. 344), and of specific enzymes for comparE. Recondo, M. Dankert, and L. F. Leloir, Biochem. Biophys. Res. Commun., 12, 204 (1963). M. Dankert, S. Passeron, E. Recondo, and L. F. Leloir, Biochem. Biophys. Res. Commun., 14,358 (1964). T. Murata, T. Mikaminawa, and T. Akazawa, Biochem. Biophys. Res. Commun., 13,439 (1963);T. Murata, T. Mikaminawa, T. Akazawa, and T. Sugiyama, Arch. Biochem. Biophys., 106, 371 (1964). G. F. Jenner, Plant Physiol., 43, 41 (1968). M. V. Pakhomova, T. N. Zaitzeva, and 0. N. Albitzkaya, Biokhimiya, 30, 1204 (1965). G. G. Sanwal and J. Preiss, Phytochemistry, 8, 707 (1969). A. C. Cassels and M. A. Harmey, Arch. Biochem. Biophys., 126, 486 (1968). M. A. Elnaghy and P. Nordin, Arch. Biochem. Biophys., 110, 593 (1965). V. Ginsburg,J. Biol. Chem., 241,3750 (1966). M. L. Cantore, P. Leoni, A. F. Leveroni, and E. F. Recondo, Biochim. Biophys. Acta, 230, 423 (1971). (49) H. Verachtert, S. T. Bass, and R. G. Hansen, Biochim. Biophys. Acta, 92, 482 (1964). (50) H. Verachtert, P. Rodriguez, S. T. Bass, and R. G. Hansen,J. Biol. Chem., 241, 2007 (1966).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

315

ing it with samples isolated from tissues, makes it a simple matter to determine its configuration at C-1 and the absolute configuration of the glucopyranosyl group. Galactosyl and mannosyl esters of adenosine 5’-pyrophosphate have been isolated from corn grains.40 The former has also been found in extracts of Chlorella cells43and in larch wood.2O Presumably, these compounds are a-D-hexopyranosyl derivatives, as the extract of corn grains contains enzymes that can catalyze the synthesis of the respective “sugar nucleotides” from adenosine 5’-triphosphate with a-D-galactopyranosyl phosphate or a-D-mannopyranosyl phoshate.^^ The latter reaction was also detected with a pyrophosphorylase from animal tissues.50 An arabinosyl ester of adenosine 5’-pyrophosphate whose absolute configuration in the glycosyl group is unknown, has been reported to occur in Chlorella extracts.@ A 2-acetamido-2-deoxyglucopyranosylester of adenosine 5’-pyrophosphate occurs in corn grains,40and it may be prepared biosynthetically with extracts of these grainP and with extracts of Azotobacter vinelandii cells.52 The enzymic digestion of an “adenosine diphosphate sugar” fraction from larch wood leads, among other monosaccharide products, to a fructose.20This result suggests the occurrence of a corresponding fructosyl ester, but its structure remains undetermined.

3. Cytidine 5‘-(Glycosyl Pyrophosphates) The occurrence of this group of “sugar nucleotides” is limited mainly to the cells of Gram-negative bacteria. Those bacterial mutants defective in lipopolysaccharide synthesis are useful for preparative isolation of such esters, as the concentration of “sugar nucleotides” in normal cells is very low. Cytidine 5’-(cu-D-glucopyranosyl pyrophosphate) (2) has been isolated from Salmonella typhimurium strain4’ and the chlamydospores of loose smut (Ustilago No reports on its occurrence in higher plants or animals have appeared, although an unexpectedly high activity of pyrophosphorylase capable of synthesizing 2 has been detected in the endosperm of corn (maize) grai11s.5~ Three of the five naturally occurring 3,6-dideoxyhexoses responsible for the 0-antigen specificity of Gram-negative bacteria were (51) S. Passeron, E. Recondo, and M. Dankert, Biochim. Biophys. Actu, 89, 372 (1964). (52) K. Kimata and S. Suzuki,J. Biol. Chem., 241, 1099 (1966). (53) J. D. Vidla and J. D. Loerch, Biochim. Biophys. Actu, 159, 551 (1968).

N. K. KOCHETKOV AND V. N. SHIBAEV

316

isolated from bacterial-cell extracts as glycosyl esters of cytidine 5’-pyrophosphate. The derivative (9)of 3,6-dideoxy-a-~-xylo-hexopyranose (abequose) was isolated from a strain of Salmonella typhirnurium,ls that (10) of 3,6-dideoxy-a-~-ribo-hexopyranose (paratose) from Salmonella parat ~ p h i and , ~ ~a mixture of 10 and the ester (11) of 3,6-dideoxy-a-~arabino-hexopyranose (tyvelose) from Salmonella enteritidis.ls It was shown that these derivatives are formed from cytidine 5’-(cr-D-glucopyranosyl pyrophosphate) by treatment with nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of cell extracts of the respective bacterial strain. For example, formation of 9 is characteristic of preparations from Salmonella, group B,85,56or Pasteurella pseudotuberculosis, type II.56 The derivative 10 was obtained with extracts of Salmonella, group and Pasteurella pseudotuberculosis, type I and 111,56 and a mixture of 10 and 11 with those of Salmonella, group D,55-60or Pastewella pseudotuberculosis, type IV.56-5B,Bo Under similar conditions, the ester (12)of cytidine 5’-pyro-

OH

OH 9

10

OH 11

12

R = cytidine I‘-pyrophosphoryl

phosphate with 3,6-dideoxy-~-~-arubino-hexopyranose (ascarylose) is formed by the action of an extract of Pasteurella pseudotuberculosis, type V.56,61The fifth example of the antigen-specific 3,6-di(54) R. M. Mayer and V. Ginsburg, Biochem. Biophys. Res. Commun., 15,334 (1964). (55) H. Nikaido and K. Nikaido,]. Biol. Chem., 241, 1376 (1966). (56) S. Matsuhashi, M. Matsuhashi, and J. L. Strominger,]. Biol. Chem., 241, 4267 (1966). (57) A. D. Elbein, Proc. Nat. Acad. Sci. U.S . , 53, 803 (1965). (58) A. E. Hey and A. D. Elbein,]. Biol. Chem., 241, 5473 (1966). (59) S. Matsuhashi and J. L. Strominger, Biochem. Biophys. Res. Commun., 20, 169 (1965). (60) S. Matsuhashi,]. Biol. Chem., 241,4275 (1966). (61) S. Matsuhashi, M. Matsuhashi, J. G. Brown, and J. L. Strominger, Biochem. Biophys. Res. Commun., 15, 60 (1964).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

317

deoxyhexoses was isolated as a guanosine 5’-pyrophosphate derivative (see Section IT, p. 321). Determination of the absolute configurations of the 3,6-dideoxyhexoses involved their degradation, according to Scheme 2, to malic acid, and identification of the latter acid with L-malate dehydrogena ~ eThe . ~anomeric ~ configuration in 9 - 12 was assumed to be the same CHO I HCOH I

y% HOCH I

HOCH I

C H.3

COzH I HCOH

COzH I HCOH 1 ?Ha CHO

COaH I HCOH

- - NaIO

I

NaIO,

?HZ HOCH I HOCH

NaIO

I

YHz COzH

I

CHS

Degradation of ascarylose to L-malic acid Scheme 2

as in the a-D-glucopyranosyl derivative (2), as this point is not involved in the enzymic reactions leading to conversion of 2 into the 3,6-dideoxyhexosyl derivatives. Two unusual glycosyl esters of cytidine 5’-pyrophosphate were found in extracts of Azotobacter uinelandii.62The branched-chain, 0-methylated heptose called vinelose was identified as the glycosyl component, and the structures 13 and 14 were assigned to these

HO

bH

13 R = H

14 R = CH,OCH,CO

“sugar n u ~ l e o t i d e s . ” These ~ ~ . ~ ~structures are based on the results of periodate oxidation, mass spectra, and nuclear magnetic resonance (62) S. Okuda, N. Suzuki, and S. Suzuki, Biochim. Biophys. Acta, 82, 436 (1964). (63) S. Okuda, N. Suzuki, and S. Suzuki,]. Biol. Chem., 242,958 (1967). (64) S. Okuda, N. Suzuki, and S. Suzuki,]. Biol. Chem., 243, 6353 (1969).

318

N. K. KOCHETKOV AND V. N. SHIBAEV

spectra, of vinelose and its derivatives, and on the conversion of vinelosyl phosphate into L-lactic acid. The esters of cytidine 5’-pyrophosphate with 6-deoxy-cr-~-xylohexopyranos-4-ulose (15) and 3,6-dideoxy-a-~-erythro-hexopyranos4-ulose (16) are probably normal metabolites in Gram-negative bac-

Q ‘OR I

I

OH 15

OH 16

R = cytidine 5’-pyrophosphoryl

teria. These compounds have not yet been identified in bacterial extracts, but partial purification of enzymes capable of catalyzing the conversion of cytidine 5’-(cr-D-glucopyranosylpyrophosphate) (2) into 15 (Refs. 55-59,65,66) or 15 into 16 (Ref. 67) has been achieved. The glycosyl esters 15 and 16 were proved to be intermediates in the synthesis from 2 of 3,6-dideoxyhexosyl esters (see Section V,l,d, p. 384). The characteristic ultraviolet spectra of these glyculose derivatives in alkaline solution (A, near 320 nm) and their color reaction with o-phenylenediamine, were used for their identification. Catalytic hydrogenation of 15, or reduction with sodium borohydride and subsequent acidic hydrolysis, leads to a mixture of D-fucose and 6-deoxy-D-glucose, thus proving the structure of the glycosyl group. Abequose and paratose were obtained after analogous treatment of the ester 16. 4. Guanosine 5’-(GlycosylPyrophosphates)

The “sugar nucleotides” of this group are ubiquitous in Nature. Guanosine 5’-(a-D-glucopyranosylpyrophosphate) (3) has been found in extracts of mammary gland,68 in various p l a n t ~ , 2 and ~ * ~ in ~ the

(65) M. Matsuhashi, J. M. Gilbert, S. Matsuhashi, J. G. Brown, and J. L. Skominger, Biochem. Biophys. Res. Commun., 15,55 (1964). (66) S. Matsuhashi, M. Matsuhashi, J. G. Brown, and J. L. Strominger,J. Biol. Chem., 241,4283 (1966). (67) S . Matsuhashi and J. L. Skominger,J. Biol. Chem., 242, 3494 (1967). (68) D. M. Carlson and R. G . Hansen,J. B i d . Chem., 237, 1260 (1962). (69) R. R. Selvendran and F. A. Isherwood, Biochem. J.. 105,723 (1967).

GLYCOSYL ESTERS O F NUCLEOSIDE PYROPHOSPHATES

319

mycelium of Eremothecium ~ s h b y i . ’The ~ most common example of this group is guanosine 5’-(a-D-mannopyranosyl pyrophosphate) (17), the biogenetic precursor of the respective esters of various uronic acids and deoxy sugars. It was first isolated from yeast13 and ~~-~~ then identified in extracts of many m i c r o - o r g a n i ~ r n s , 4 ~ *higher plants,20.44.69,75 and animals.30,76-82

oo H,OH

HO

0 II

HO

OH

17

Both of the enantiomorphs of galactose occur as esters with guanosine 5’-pyrophosphate. The derivative (18) of a-D-galactopyranose was identified among the “nucleoside diphosphate sugars” of Chlo~ e l l u strawberry ,~~ leaves,6g and larch wood,20 and that (19) of p-Lgalactopyranose was isolated from extracts of the red alga Porphy~u pe~jGorutu,*~ and the albumen gland of the snail Helix p o m ~ t i u . ~ ~ (70) H. G. Pontis, A. L. James, and J. Baddiley, Biochem. ]., 75,428 (1960). (71) A. Ballio, C. Casinovi, and G. Serluppi-Crescenzi, Biochim. Biophys. Acta, 20, 415 (1956). (72) K. 0. U. Persson, Acta Chem. Scand., 17, 2750 (1963). (73) H. Ankel, D. G . Farrell, and D. S. Feingold, Biochim. Biophys. Acta, 90, 397 (1964) (74) S. E. Mansurova, Z. A. Shabarova, N. S. Kulaev, and M. A. Prokof’ev, Biokhimiya, 31, 1057 (1966). (75) Jean Gregoire, Jana Gregoire, N. Limozin, and L. V. Van, Cornpt. Rend., 257, 3508 (1963); Bull. Soc. Chim. Biol., 47, 195 (1965). (76) R. Denamur, G. Fauconneau, and G. Guntz, Compt. Rend., 248,2531 (1959);Reu. EspaA. Fisiol., 15, 301 (1959). (77) T. Johke,]. Biochem. (Tokyo), 54, 388 (1963). (78) M. Ramuz, C. Judes, and P. Mandel,]. Neurochem., 11,826 (1964). (79) V. I. Zhivkov, Biokhimiya, 30, 255 (1965). (80) V. I. Zhivkov and K. Chelibonova-Lorer, Ukr. Biokhim. Zh., 39, 153 (1967). (81) J. W. Donovan, J. G. Davis, and L. U. Park, Arch. Biochem. Biophys., 122, 17 (1967). (82) M. Endo and Z. Kosizawa, Arch. Biochem. Biophys., 127, 585 (1968). (83) J. C. Su and W. Z. Hassid, Biochemistry, 1, 474 (1962). (84) E. M. Goudsmit and E. F. Neufeld, Biochim. Biophys. Acta, 121, 192 (1966).

320

N. K. KOCHETKOV AND V. N. SHIBAEV

OH 18

HO 19

R = guanosine I’-pyrophosphoryl

Enzymic reactions with D-galactose oxidase or L-fucose isomerase were used for identification of the monosaccharide isolated after degradation of the esters. Another approach for determination of the absolute configuration of the monosaccharide component^^^ involves their interaction with radioactive potassium cyanide and conversion of the products into a mixture of heptonamides. Isotopic-dilution experiments showed the thus establishing the presence of D-glycero-L-manno-heptonamide, D-galacto configuration of the starting hexose. Galactose and arabinose of unknown configuration were obtained after hydrolysis of a “guanosine diphosphate sugars” fraction from pig milk.85 A similar fraction from strawberry leaves produced a considerable proportion of D-Xy10Se.6g A trace component present in a preparation of guanosine 5‘-(a-Dmannopyranosyl pyrophosphate) from yeast has been identified as a D-glycero-D-manno-heptosyl ester of guanosine 5’-pyropho~phate.~~ This compound seems to be the only known example of a “nucleoside diphosphate aldoheptose.” A fructosyl ester of guanosine 5’-pyrophosphate has been reported to occur in Eremothecium ashbyP and Candida utilis;87its structure remains undetermined. Uronic acid derivatives are also known among the guanosine 5’-pyrophosphate esters. A mixture of the a-D-mannopyranosyluronic acid ester (20) and, presumably, the p-L-gulopyranosyluronic acid ester (21)was isolated from the brown alga Fucus gardneri.88The conversion of the uronic acid in the ester 20 into D-mannitol, and that

(85) R. Denamur, G. Fauconneau, and G. Jarrige-Guntz, Ann. Biol. Anim. Biochim. Biophys.,1, 74 (1961). (86) V. Ginsburg, Biochem. Biophys. Res. Commun., 3, 187 (1960);V. Ginsburg, P. J. O’Brien, and C. W. Hall, J. Biol. Chem., 237, 497 (1962). (87) T. Saviova and J. K. Miettinen, Actu Chem. Scund., 20, 2444 (1966). (88) T.-Y. Lin and W. Z. Hassid, J. B i d . Chem., 239, 943 (1964); 241, 3283 (1966).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

321

in the ester 21 into D-ghconic acid, was used for elucidation of the structure of these derivatives. Ester 20 seems to be a normal metabolite in Arthrobacter viscosus, as its formation by enzymic oxidation of the mannose derivative 17 was demonstrated in this strain.89 Among the deoxyhexosyl esters of guanosine 5’-pyrophosphate, the most common is the P-L-fucopyranosyl ester 22. It was isolated from ~~ mammary gland,76 and extracts of Aerobacter a e r o g e n e ~ ,milk,g1 blood and was considered of probable occurrence in higher plants also (compare Ref. 92). Guanosine 5’-(a-D-rhamnopyranosyl pyrophosphate) (23) was shown to be a product of enzymic reduction of the mannosyl ester (17)in the presence of a plant extractg3or an enzyme preparation from an unidentified strain of Gram-negative b a ~ t e r i a . 9 ~In 3 ~the ~ latter example, the 6-deoxy-~-talosylester, presumably 24, is also produced.

The ester (25) of guanosine 5’-pyrophosphate with 3,6-dideoxyp-L-xylo-hexopyranose (colitose) was isolated from an extract of (89) J. Preiss, Biochem. Biophys. Res. Commun., 9, 235 (1962);J. Biol. Chem., 239, 3127 (1964). (90) V. Ginsburg and H. N. Kirkman, J . Amer. Chem. Soc., 80, 3481 (1958). (91) R. Denamur, G. Fauconneau, and G. Guntz, Compt. Rend., 246, 2820 (1958). (92) T.-H. Liao and G. A. Barber, Biochim. Biophys. Acta, 230,64 (1971). (93) G. A. Barber, Biochim. Biophys. Acta, 165, 68 (1968). (94) A. Markovitz, Biochem. Biophys. Res. Commun., 6, 250 (1961). (95) A. Markovitz,J. Biol. Chem., 239,2091 (1964).

322

N. K. KOCHETKOV AND V. N. SHIBAEV

Escherichia c ~ l i The . ~ ~mutant strain 5-5, defective in lipopolysaccharide synthesis, accumulates a considerable amount of this nucleotide and may be used for its preparative i ~ o l a t i o n . 9 ~ ~ ~ ~ The derivatives 22-25 are formed in Nature from the ester 17 (see Section V,l,c, p. 379 and V,l,d, p. 383). The 6-deoxy-a-~-lyxohexopyranosyl-4- d o s e ester (26) was demonstrated as an intermediate.92,95,99-101

25

26

R = guanosine 5‘-pyrophosphoryl

Some evidence has been presented suggesting the occurrence of esters of guanosine 5’-pyrophosphate with oligosaccharides. An oligosaccharide composed of a glucose and a mannose was obtained in low yield among the products of enzymic degradation of a “guanosine diphosphate sugar” fraction from larch wood.20 A similar fraction from pig milk gives lactose and unidentified oligo~accharides.~~

5. Thymidine 5’-(GlycosylPyrophosphates) The glycosyl esters of this series are characteristic metabolites of micro-organisms and take part in the biosynthesis of deoxy sugars and aminodeoxy sugars. Thymidine 5’-(a-D-glucopyranosyl pyrophosphate) (4) and the analogous D-galaCtOSe derivative have been isolated from extracts of Pasteurella pseudotuberczllosis.lQ2The ester of thymidine 5’-pyrophosphate with a-D-mannopyranose was found in an extract of Streptomyces g r i ~ e u s , ’ ~and . ’ ~ ~the occurrence of the D-ribosyl ester in the (96) E. C. Heath, Biochim. Biophys. Acta, 39,377 (1960). (97) E. C. Heath and A. D. Elbein, Proc. Nut. Acad. Sci. U . S . , 48, 1214 (1962). (98) A. D. Elbein and E. C. Heath,]. B i d . Chem., 240, 1919 (1965). (99) V. Ginsburg,]. Biol. Chem., 236,2389 (1961). (100) A. Markovitz, Proc. Nat. Acad. Sci. U . S . , 51, 239 (1964). (101) A. D. Elbein and E. C. Heath,]. B i d . Chem. 240, 1926 (1965). (102) S. Matsuhashi and J. L. Strominger,]. Bacteriol., 93,2017 (1967). (103) N. L. Blumsom and J. Baddiley, Biochern.]., 81, 114 (1961).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

323

same source was also reported.'04 A brief abstract reported a claim of the isolation from sugar beet of esters of thymidine 5'-pyrophosphate with D-glucose and D-galacturonic acid,'05 but a detailed description was lacking five years after the initial report. The most common derivative of this group of "sugar nucleotides" is thymidine 5'-(P-~-rhamnopyranosylpyrophosphate) (27), isolated from extracts of b a ~ t e r i a ' ~ * ' ~ and ~ ~strept~mycetes.'~ '~' The assignment

0 II

HoQ-HO

OH

HO 27

of anomeric configuration is based on the lability of the nucleotide in alkaliI4 (compare Section IV, p. 359) and on comparison of the glycosyl phosphate derived from ester 27 with synthetic a-L-rhamnopyranosyl phosphate. lo* A mutant strain of Escherichia coli Y-10, defective in L-rhamnose synthesis, accumulates'0g a considerable amount of "thymidine diphosphate sugars." Three of them were identified"O as the esters of thymidine 5'-pyrophosphate with 6-deoxy-a-~-xylo-hexopyranos4-ulose (28), 6-deoxy-~-glucose,and D-fUCOSe. The fourth component was found to be a 4-acetamido-4,6-dideoxy-~-galactose derivative11'*112 (29); it occurs also in Pasteurella pseudotuberculosis102and its

(104) J. Baddiley, N. Blumsom, A. DiGirolamo, and M . DiGirolamo, Biochim.Biophys. Actu, 50, 391 (1961). (105) R. Katan and G. Avigad, IsrueZJ. Chem., 3, llOP (1966). (106) R. Okazaki, T. Okazaki, and Y. Kuriki, Biochim. Biophys. Actu, 38, 384 (1960). (107) T. Okazaki, J. L. Strominger, and R. Okazaki,]. Bucteriol., 86, 118 (1953). (108) G. A. Barber, Biochim.Biophys. Actu, 141, 174 (1967). (109) J. L. Strominger and S. S. Scott, Biochim. Biophys. Actu, 35, 552 (1959). (110)R. Okazaki, T. Okazaki, J. L. Strominger, and A. M. Michelson,]. Biol. Chem., 237, 3014 (1962). (111) M. Matsuhashi and J. L. Strominger,J. Biol. Chem., 239, 2454 (1964). (112) C. L. Stevens, P. Blumbergs, D . H. Otterbach, J . L Strominger, M. Matsuhashi, and D. N. Dietzler,J. Amer. Chem. Soc., 86,2937 (1964).

324

N. K. KOCHETKOV AND V. N. SHIBAEV

=Q OR

0

AcHNQoR

OH 28

OH 29

R = thymidine 5'-pyrophosphoryl

enzymic synthesis (compare Section V,l,e, p. 385) was shown with extracts of other ba~teria.'~'."~ An analogous derivative isolated from strain B of Escherichia coZiloswas praved to be the 4rr-epimerl1l*ll4 of 29. Thymidine 5'-(a-D-glucopyranosyl pyrophosphate) is converted into a derivative of a 3-acetamido-3-deoxyhexosewhen incubated with an extract of Xanthomonas c u m p e ~ t r i s .Degradation ~~~ of the monosaccharide from this "sugar nucleotide" with hypoiodite and periodate (compare Scheme 2, p. 317) produces 3-hydroxyaspartic acid, thus establishing the position of the acetamido group. The more-common 2-acetamido-2-deoxyhexoses have not been found as thymidine 5'-pyrophosphate derivatives. Nevertheless, the enzymic synthesis of thymidine 5'-(2-acetamido-2-deoxy-a-~-glucopyranosyl pyrophosphate) and -(2-acetamido-2-deoxy-a-~-galactopyranosyl pyrophosphate) has been achieved with enzyme preparations from Pseudomonas aeruginosa,llsAzotobacter uinelandii,52and gastric mucosa."' 6. Uridine 5'-(GlycopyranosylPyrophosphates)

Derivatives of uridine 5'-pyrophosphate are the most common natural "sugar nucleotides," and about forty of them have been isolated. Chromatographic separation of free nucleotides from various sources usually gives the glycosyl esters of uridine 5'-pyrophosphate in three fractions, containing the derivatives of neutral monosaccharides, uronic acids, and 2-acetamido-2-deoxyglycoses, respectively. (113) M. Matsuhashi and J. L. Strominger,J. Biol. Chem., 241,4738 (1966). (114) C. L. Stevens, P. Blumbergs, F. A. Daniher, J. L. Strominger, M. Matsuhashi, D . N. Dietzler, S. Suzuki, T. Okazaki, K. Sugimoto, and R. Okazaki,J. Amer. Chem. SOC., 86, 2939 (1964). (115) W. A. Volk and G. Ashwell, Biochern. Biophys. Res. Commun., 12, 116 (1963). (116) S. Komfeld and L. Glaser,J. Biol. Chem., 237, 3052 (1962). (117) R. Kornfeld, S. Kornfeld, andV. Ginsburg, Biochem. Biophys. Res. Commun., 17, 578 (1964).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

325

Uridine 5’-(a-D-glucopyranosyl pyrophosphate) (5), first isolated from yeast extract,8prevails as a rule in the first fraction. It is scarcely feasible to record here all of the references available, but the first reports on its isolation from extracts of b a ~ t e r i a , ” ~ .higher ”~ plants,’2”20 and animal tissues,11,121 should be noted. The preponderance of the ester 5 causes difficulties in the isolation and identification of other components of this fraction. The D-glucosyl derivative 5 may be converted into the D-ghcuronic acid analog by specific enzymic oxidation (see Section V,l,a, p. 363) and the derivative so modified may then be separated by chromatographic techniques. Uridine 5’-(a-Dgalactopyranosyl pyrophosphate) (30) seems also to be a rather frequent component among the “sugar nucleotides” of bacteria,lz2 yeast,123higher plants,12 and animal^.'^^^^^^ The a-D-galactofuranosyl derivative (31)has been shown to be an intermediate in the biosynCKOH

I

OH 30

I

CH,OH 31

R = uridine 5’-pyrophosphoryl

thesis of the galactocarolose produced by Penicillium charlesii G . Smith.126Its structure was confirmed by the formation of formaldehyde upon periodate oxidation, and the easy splitting of the pyrophosphate linkage by alkali (compare Section IV, p. 358). Uridine 5’-(a-D-mannOpyranOSyl pyrophosphate) has been found in low concentration in human b l o o d - c e l l ~ . ~ ~ In addition to the esters 5 and 30, such derivatives of a-D-xylopyranose and P-L-arabinopyranose are common in the “uridine diphos(118) E. E. B. Smith G. T. Mills, and E. M. Harper,]. Gen. Microbiol., 16, 426 (1957). (119) J. A. Cifonelli and A. Dorfman,]. Biol. Chem., 228,547 (1957). (120) R. Bergkvist, Acta Chem. Scand., 10, 1303 (1956); 11, 1457 (1957). (121) W. J. Rutter and R. G. Hansen,]. B i d . Chem., 202,323 (1953). (122) H. Nikaido, Biochim. Biophys. Acta, 48, 460 (1962). (123) G. T. Mills, E. E. B. Smith, and A. G. Lochhead, Biochim. Biophys. Acta, 25,521 (1957). (124) R. G. Hansen, R. A. Freedland, and H. M. Scott,]. Biol. Chem.,219, 391 (1956). (125) K. Isselbacher,]. B i d . Chem., 232, 429 (1958). (126) A. G. Trejo, G. J. F. Chittenden, J. G. Buchanan, and J. Baddiley, Biochem.]., 117, 637 (1970);A. G . Trejo, J. W. Haddock, G. J. F. Chittenden, and J. Baddiley, ihid., 122, 49 (1971).

326

N. K. KOCHETKOV AND V. N. SHIBAEV

phate sugar" fraction from plant^.'^*'^' The first of these was found also in an extract of Cryptococcus l ~ u r e n t i iand ~ ~ in milk.12s*'29 A scrupulous analysis of the fraction from parsley showed the presence of the D-apiose derivative in minute pr~portion.'~' The isolation of a fructosyl ester of uridine 5'-pyrophosphate from several plants has been r e p ~ r t e d . ' ~ ~ , ' ~ ~ - ' ~ ~ Uridine 5'-(fi-~-rhamnopyranosylpyrophosphate) occurs in extracts of higher plant^,'^^.'^^ in the golden brown alga Ochromonus and in some b a ~ t e r i a . ~ ' "Its ~ ~ biosynthesis from malhamen~is,'~~ uridine 5'-(a-D-glucopyranosyl pyrophosphate) includes intermediate formation of the 6-deoxy-a-~-ry~0-hexopyranos-4-ulose derivative. Such a reaction has been demonstrated with enzymes from tobacco leaves'3s and Chlorella ~ e 1 l s . Extracts l~~ of Digitalis purpurea leaves contain the ester of uridine 5'-pyrophosphate with 2,6-dideoxyD-ribo-hexose (digitoxo~e).'~~ The sole component of the "uridine diphosphate uronic acid" fraction from animal ~ ~ s s uC h~l ~ rse l Z, ared ~ ~ and ~ ~ ~ ~ ,~~ ~algae,83 some b a ~ t e r i a ' ~is~ ~ "~ uridine 5'-(a-D-g~ucopyranosyluronic acid pyrophosphate) (32),whereas a similar preparation from Pneumococcus140 contains only the a-D-ga~actopyranosy~uronic acid derivative. Both esters have been shown to occur in extracts of mung bean.'41,142 The "uridine diphosphate 2-acetamido-2-deoxyglycose" fractions

(127)H.Sandermann and H. Grisebach, Biochim. Biophys.Acta, 156,435 (1968);Eur. J. Biochem., 6, 404 (1968). (128)A. Kobata and S. Ziro, Biochim. Biophys. Acta, 107,405 (1965). (129)R. Denamur and P. Gaye, Bull. SOC. Chim. Biol., 49, 1793 (1967). (130)N. S. Gonzales and H. G. Pontis, Biochim. Biophys. Acta, 69, 179 (1963). (131)E. G. Brown and B. S. Mangat, Biochim.Biophys. Acta, 148,350 (1967). (132)Y.Umemura, M. Nakamura, and S. Funahashi,Arch. Biochem. Biophys., 119,240 (1967). (133)M.M.V. Hampe and N. S. Gonzales, Biochim. Biophys. Acta, 148, 566 (1967). (134)H.b u s s , Biochem. Biophys. Res. Commun., 18,170 (1965). (135)E. E. B. Smith, B. Galloway, and G. T. Mills, Biochim. Biophys. Acta, 33, 276 (1959). (136)G. A. Barber, Arch. Biochem. Biophys., 103,276 (1963). (137)G. A. Barber and M. T. Y. Chang, Arch. Biochem. Biophys., 118, 659 (1967). (138)G. Franz and H. Meyer, Biochim. Biophys. Acta, 184,658 (1969). (139)G. 1. Dutton, Biochem. I., 71, 141 (1959). (140)E. E. B. Smith, G. T. Mills, and E. M. Harper, Biochim. Biophys. Acta, 23, 662 (1957). (141)J. Solms and W. Z. Hassid,J. Biol. Chem., 228, 357 (1957). (142)E. F. Neufeld and D. S. Feingold, Biochim. Biophys. Acta, 53, 589 (1961).

~

~

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

327

I

I

OH

OH

32

33

R = uridine 5'-pyrophosphoryl

from yeast? strept~mycetes,'~~ some b a ~ t e r i a , ~ ' ~ ' ' 'crustacean^,'^^ ~*'~~ and molluscs146have been shown to consist of uridine 5'-(2-acetamido2-deoxy-a-~-g~ucopyranosylpyrophosphate) (34) as the sole component. The preparation from animal t i s s u e ~ , 3 ~higher , ' ~ ~ plant^,^^,'^^ and some bacteria148contains a mixture of the ester 34 and the closely related 2-acetamido-2-deoxy-a-~-galactopyranosyl derivative (35).The latter can be isolated in pure form after splitting the nucleotide 34 with a specific pyropho~phorylase.'~~

NHAc 34

NHAc 35

36

R = uridine 5'-pyrophosphoryl

Uridine 5' - (2acetamido-2- deoxy-a-~-glucopyranosyluronic acid pyrophosphate) (36) was isolated from extracts of Achromobacter ge~rgiop~litanurn ' ~ ~Micrococcus ~ and l y s o d e i k t i c ~ sThe . ~ ~ester ~ ~ of uridine 5'-pyrophosphate with an unusual amino sugar, namely, a 2-acetamido-2,4,6-trideoxyhexoseof unknown configuration, has

(143) N. Akamatsu, J . Biochem. (Tokyo), 59, 613 (1966). (144) J. L. Strominger,]. Biol. Chem., 234, 1520 (1959). (145) M. R. Lunt and P. W. Kent, Biochem.]., 78, 128 (1961). (146) W. Wylie and M. Smith, Cun.J. Biochem., 42, 1347 (1964). (147) H. G. Pontis, J . Biol. Chem., 216, 195 (1955). (148) L. Glaser, Biochim. Biophys. Actu, 31, 575 (1959). (149) J. L. Sbominger and M. S. Smith,J. B i d . Chem., 234, 1288 (1959). (149a) E. J. Smith, Biochim. Biophys. Actu, 158, 470 (1968). (149b) P. Biely and R. W. Jeanloz,J. Biol. Chem., 244,4929 (1969).

328

N. K. KOCHETKOV AND V. N. SHIBAEV

been reportedI5O to appear when the ester 34 is treated with L-glutamine and pyridoxal phosphate in the presence of an enzyme preparation from Type XIV Pneurnococcus. A 2-acetamido-2,6-dideoxyhexopyranos-4-ulose derivative served as an intermediate.150 The main function of the ester 34 in bacterial cells seems to be its participation in the biosynthesis of the glycopeptide cell-wall polymer. If this process is blocked, there results the accumulation of a high concentration of "sugar nucleotide" precursors in the cell. A number of these compounds have been isolated; the simplest one is the ester of uridine 5'-pyrophosphate with N-acetylmuramic acid [2-acetamido3-0-(~-l-carboxyethyl)-2-deoxy-~-g~ucose] (37), first obtained from Staphylococcus uureus cells that had been treated with p e n i ~ i l l i n ~ J ~ ~ or Gentian Vi01et.l~~ An intermediate in the biosynthesis of 37 was (38). isolated and shown to be the 3-enolpyruvate ether152J53 CH,OH

NHAc \ CH,CHCO,H

CH,OH

\

NHAc

H,C=C--CO,H 38

37

R = uridine 5'-pyrophosphoryl

A series of derivatives of the ester 37, having amino acid or peptide residues linked through the carboxyl group of the lactic acid residue, has been described. Their exact structures depend on the growth conditions and the bacterial strain used. The chain attached may contain ~ - a l a n i n e ; ~ * ' ~ 'the ~ ' ~dipeptide ~.'~~ L-alanyl-D-glutamic ~-alanyl-y-~-glutamy1-~-lysine,'~~-~~~ L-ala ~ i d ; ~the ~ ~tripeptides , ' ~ ~ (150) J. Distler, B. Kaufman, and S. Roseman,Arch. Biochem. Biophys., 116,466 (1966). (151) J. L. Strominger, J. Biol. Chem., 224, 509 (1957). (152) J. L. Strominger, Biochim. Biophys. Actu, 30, 645 (1958). (153) K. G. Gunetileke and R. A. Anwar,J. Biol. Chem., 243, 5770 (1968). (154) P. E. Reynolds, Biochim. Biophys. Acta, 52, 403 (1961). (155) R. Plapp and 0. Kandler, Arch. Mikrobiol., 50, 171 (1965). (156) J. L. Strominger and R. H. Threnn, Biochim. Biophys. Actu, 36, 83 (1959). (157) M. Saito, N. Ishimoto, and E. Ito,J. Biochem. (Tokyo), 54, 273 (1963). (158) J. L. Strominger, R. H. Threnn, and S. S. Scott, J. Amer. Chem. Soc., 81, 3803 (1959). (159) J. L. Strominger and C. H. Birge,J. Bacteriol., 89, 1124 (1965). (160) F. C. Neuhaus and W. G. Struve, Biochemistry, 4, 120 (1965).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

329

anyl-y-D-glutamyl-meso-diaminopimelic L-alanyl-y-D-glutarnyl-~-ornithine,'~~ L-seryl-y-D-ghtamyl-L-ornithine,'66 and glycyly-D-glutamyl-L-ly~ine;~~~ the pentapeptides L-alanyl-y-D-glutamylL-ly~yl-D-alanyl-D-alanine,~"~~"~~.'~~~~~~ L-alanyl-7-D-glutamyl-mesodiaminopimeloy1-D-dany1-D-alanine,16* L-alanyl-7-D-glutamyl-L-ornithyl-D-alanyl-D-alanine,'65glycyl-7-D-glutamyl-L-lysyl-D-alanyl-D-alanine,159and g~ycy~-y-D-g~utamy~-L-homosery~-D-a~any~-D-a~an~ne,'6g or the hexapeptide L-alanyl-y-D-glutamy1-(N-L-alanyl)-L-lysyl-D-alanylD-alax~ine.'~~ The corresponding derivative of N-glycolylmuramic acid with L-danyl-y-D-glutamyl-meso-diaminopimelic acid has also been i~olated.'~~,'~' Another specific type of glycosyl ester of uridine 5'-pyrophosphate is that having a monosaccharide sulfate moiety. Such compounds have been found in appreciable amounts in hen o v i d u ~ t , ' ~where ~,'~~ synthesis of the glycosaminoglycan membranes of the egg occurs. "Uridine diphosphate sugar sulfates" that have been identified include a monosulfate of uridine 5'-(a-D-galactopyranosyl pyrophoshate)'^^ substituted at C - 2 or C - 4 (as concluded from periodateoxidation data), the 4-sulfate of the 2-acetamido-2-deoxy-a-~-galactopyranosyl derivative (39, R2 = H),81*172-'74 its 4,6"-disulfate (39, R2 = S03H),173and the 6-phosphate 4-sulfate (40) of uridine 5'-(2-acetamido-2-deoxy-a-~-glucopyranosyl pyr~phosphate).~~~ In addition to the esters of uridine 5'-pyrophosphate with monosaccharide sulfates, hen oviduct contains corresponding esters with two saccharides. The first known member of this series was a unique derivative 41, in which the monosaccharide residues are linked

(161) D. G. Comb, W. Chin, and S. Roseman, Biochim. Biophys. Acta, 46, 394 (1961). (162) R. A. Anwar, C. Roy, and R. W. Watson, Can.J.Biochem. Physiol.,41,1065 (1963). (163) R. Plapp and 0. Kandler, Arch. Mikrobiol., 50, 282 (1965). (164) T. Nakatani, Y. Araki, and E. Ito, Biochim. Biophys. Acta, 156, 210 (1968). (165) R. Plapp and 0. Kandler, Biochem. Biophys. Res. Commun., 28, 141 (1967). (166) I. Miller, R. Plapp, and 0. Kandler, Z . Naturforsch., B , 23, 217 (1968). (167) P. Mandelstam, R. Loercher, and J. L. Strominger,J. Biol. Chem.,237,2683 (1962). (168) J. L. Strominger, S. S. Scott, and R. H. Threnn, Fed. Proc., 18, 334 (1959). (169) A. N. Chatterjee and H. R. Perkins, Biochem. Biophys. Res. Commun., 24, 489 (1966). (170) K. Takayama, H. L. David, L. Wang, and D. S. Goldman, Biochem. Biophys. Res. Commun., 39, 7 (1970). (171) J. F. Petit, A. Adam, and J. Wietzerbin-Falszpan, FEBS Lett., 6, 55 (1970). (172) J. L. Strominger, Biochim. Biophys. Acta, 17, 283 (1955). (173) N. Nakanishi, H. Sonohara, and S. Suzuki, J. Biol. Chem., 245, 6046 (1970). (174) J. Picard, A. Cardais, and L. Duberhard, Nature, 202, 1213 (1964).

N. K. KOCHETKOV AND V. N. SHIBAEV

330

OOR1

H 0 3 s 0 ~ 0 R 1

HOsSO

I

I

NHAc

NHAc 39

40

R1 = uridine 5'-pyrophosphoryl

through a phosphate diester linkage.175.17s Its sulfate esterified in the D-galaCtOSe residue has also been is01ated.I~~

O Q "0

0 OH

HO

OH

41

Mild, acidic hydrolysis of the derivative 41 gave uridine 5'-pyrophosphate, 2-acetamido-2-deoxy-~-glucose6-phosphate, and D-galactose. Alkaline degradation leads to uridine 5'-(2-acetamido2-deoxy-a-D-glucopyranosyl pyrophosphate) and a-D-galactopyranosyl phosphate, among other products. A disaccharide derivative (42) of more conventional structure has also been isolated from hen 0 ~ i d u c t . Its I~~ structure was confirmed by its conversion into L-fucose and uridine 5'-(2-acetamido-2-deoxya-D-glucopyranosyl pyrophosphate) after treatment with a-L-fucosidase. Mild, acidic hydrolysis of the ester 42 produces a disaccharide whose structure was confirmed by periodate oxidation. Human milk and colostrum,178or milk and colostrum of pig,lZ8are also sources of (175) S. Suzuki,J. B i d . Chem., 237, 1393 (1962). (176) 0. Gabriel and G. Ashwell,]. Biol. Chem., 237, 1400 (1962). (177) Y. Nakanishi, S. Shimizu, N. Takanashi, M. Sugiyama, and S. Suzuki, J . Biol. Chem., 242, 967 (1967). (178) A. Kobata,J. Biochem. (Tokyo), 53, 167 (1963).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

331

“uridine diphosphate oligosaccharides.” The main component seems to be the disaccharide derivative 43 (R2 = R3 = H). The preparation from pig milk contained, in addition, a small proportion of the analogous disaccharide derivative having a 2-acetamido-2-deoxy-~galactose residue.lZ8 CqOH

0’ P

HO

HO

CHzOH

O NHAc R l

P

H

O

q

O

NHAc R 1

o

ORz 42

43

R’ = uridine 5’-pyrophosphoryl

A trisaccharide derivative (43, R2 = a-L-fucopyranosyl, R3 = H) has been isolated from pig milk.178*179 Closely related trisaccharide derivatives have been reported to occur in cow colostrum,lEOand in ewe mammary glands and colostrum.ls1 Both of these sources contain the “trisaccharide nucleotides” 43 (R2= H, R3 = N-acetylneuraminyl) and 43 (R2 = H, R3 = N-glycolylneuraminyl). In two additional, isomeric, derivatives from cow colostrum, 2-acetamido-2-deoxy-~-glucopyranosyl and D-galactosyl residues are linked through C-6. In contrast, ewe mammary gland and colostrum contain trisaccharide derivatives in which N-acetyl- or N-glycolyl-neuraminic acid residues are attached through a (2 + 6)-linkage to the D-galactose residue of uridine 5’-(2-acetamido-2-deoxy-3-O-~-D-ga~actopyranosy~-a-~-galactopyranosyl pyrophosphate).18’ Oligosaccharide derivatives of uridine 5’-pyrophosphate are probably present in higher plants. Cellobiose and a disaccharide composed of a glucose and an arabinose were identified among products of enzymic hydrolysis of a “uridine diphosphate sugar” fraction from larch wood.20 (179) A. Kobata,J. Biochem. (Tokyo), 59, 63 (1966). (180) C. W. Jourdian, F. Shimizu, and S. Roseman, Fed. Pfoc., 20, 161 (1961); G. W. Jourdian and S. Roseman, Ann. N . Y. Acad. Sci., 106, 202 (1963). (181) R. E. Denamur and P. J.-B.Gaye, E u 7 . J . Biochem., 19, 23 (1971).

332

N. K. KOCHETKOV AND V. N. SHIBAEV

7. Related Nucleotide Derivatives This sub-section is devoted to nucleotide derivatives that may participate in biosynthesis of certain polymers containing carbohy" drate. They are generally included among the sugar nucleotides," although their structures differ somewhat from those of the glycosyl esters of nucleoside 5'-pyrophosphates so far discussed. a. Esters of Cytidine 5'-Phosphatewith 3-DeoxyoctulosonicAcids. The N-acetylneuraminic acid derivative 44 is widely distributed. It was isolated from a strainlE2of Escherichia coli, and has been obtained from cytidine 5'-triphosphate and N-acetylneuraminic acid by the action of enzyme preparations from Neisseria meningitidislE3 and from animal t i s s ~ e s . ' ~ The ~ - * latter ~ ~ enzyme can also make use of N-glycolylneuraminic acid as a substrate, to give the respective cytidine 5'-phosphate derivative.

HO

OH

HCOH I

HCOH I

CH,OH 44

The structure shown was confirmed by the identification of cytidine 5'-phosphate and N-acetylneuraminic acid after mild acidic hydrolysis, and by the stability of the derivative on treatment with sodium borohydride. Comparison of the optical rotation of the starting derivativelE2(44), the products of its hydrolysis,lE2and the anomeric (182) D. G. Comb, F. Shimizu, and S. Roseman,J. Amer. Chem. Soc., 81, 5513 (1959); D. G . Comb, D. R. Warren, and S. Roseman, J. Biol. Chem., 241, 5637 (1966). (183) L. Warren and S. Blacklow,J. Biol. Chem., 237, 3527 (1962). (184) S. Roseman, Proc. Nut. Acud. Sci. U.S., 48,437 (1962). (185) M. Shoyab, T. N. Tattabiranin, and B. K. Bachhawat, J , Neurochem., 11, 639 (1964). (186) E. L. Kean and S. Roseman,J. Biol. Chem., 241, 5643 (1966).

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333

pyranoid glycosides of N-acetylneuraminic acid,18’ indicates the D-glyceTo-p-D-gnlacto (D-erythro-a-L-arabino) configuration. A similar ester was prepared from cytidine 5’-triphosphate and 3-deoxy-D-manno-octulosonic acid by use of an enzyme188 from Escherichia coli, but its purification failed because of the extreme lability of this derivative. The structure of an unusual compound isolated from spores of Ustilago has not yet been completely clarified. According to data available,189it seems to be a cytidine 5’-(D-glucosylphosphate) derivative having a peptide chain attached to a hydroxyl group of the D-ribose residue.

b. Hexitol and Pentitol Esters of Nucleoside 5’-Pyrophosphates.The main component of the “adenosine diphosphate sugar” fraction from a Salmonella typhimurium strain was unexpectedly found to be adenosine 5’-(D-mannitol l - p y r o p h o ~ p h a t e )(45). ~ ~ ~ Upon treatment with acid or with snake-venom pyrophosphatase, it produces adenosine 5’-phosphate and D-mannitol l-phosphate; these observations confirm the structure assigned.

II I

HOYH

HCOH I

I

HO

1

OH

HYOH H,COH

45

As early as 1954, cytidine 5’-(~-ribitoll-pyrophosphate) (46) was found in extracts of Lactobacillus a r u b i n o s u ~ ,and ~ ~ ~its structure was soon established.’g2J93 It has been isolated from many other (187) R. K. Yu and R. Ledeen,J. B i d . Chem., 244, 1306 (1969). (188) M. A. Ghalambor and E. C. Heath, Biochem. Biophys. Res. Commun., 10, 346 (1963);J. Biol. Chem., 241, 3216 (1966). (189) M. A. Elnaghy and P. Nordin, Biochem. Biophys. Res. Commun., 18,501 (1965). (190) B. M. Scher and V. Ginsburg,J. Biol. Chem., 243, 2385 (1968). (191) J. Baddiley and A. P. Mathias,]. Chem. Soc., 2723 (1954). (192) J. Baddiley, J. G. Buchanan, B. Carss, and A. P. Mathias, J . Chem. Soc., 4583 (1956). (193) J. Baddiley, J. G. Buchanan, and B. Carss,J. Chem. SOC., 1869 (1957).

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N. K. KOCHETKOV AND V. N. SHIBAEV

b a ~ t e r i a . ' ~ ~ 'Its ~ ~biosynthesis -'~~ has been demonstrated not only with enzymes of bacterial origin, but also with preparations from yeast and C h l o r e l l ~ . ' ~ ~ H&OR

I

HOCH I HOCH I HOCH I &COH

H,COR I HCOH

46

47

I

H,COH

R = cytidine 5'-pyrophosphoryl

c. Esters of Nucleoside 5'-Pyrophosphates with Trioses and Glycerol.-Resembling the L-ribitol derivative (46), cytidine s'-(D-glycerol l-pyropho~phate)'~~ (47) is common in bacteria, where it participates in the biosynthesis of teichoic acids and also in lipid metabolism. First isolated from Lactobacillus ~ ~ ~ b i n 0 s u sit, ~ ~ ~ 3 ~ ~ occurs in many other ~ t r a i n s l ~and ~ , ' has ~ been prepared biochemi~ally.'~~~~~~ The isolation of uridine 5'-(1,3-dihydroxy-2-propanonepyrophosphate) from a Pneurnococcus strain has been reported.202 111. PREPARATION OF GLYCOSYL ESTERSOF NUCLEOSIDEPYROPHOSPHATES

The preparative isolation from natural sources of glycosyl esters of nucleoside pyrophosphates is rather laborious, and such isolative procedures are usually applied only until more convenient, synthetic procedures become available. However, the natural source (194) H. P. Clark, P. Clover, and A. P. Mathias, J. Gen. Microbiol., 20, 156 (1959). (195) J. J. Saukkonen, Nature, 192, 816 (1961). (196) L. Glaser,]. Biol. Chem., 239, 3178 (1964). (197) D. R. D. Shaw, D. Milerman, A. N. Chatterjee, and J. T. Park,]. Biol. Chem.,245, 5101 (1970). (198) D. R. D. Shaw, Biochem.]., 82, 297 (1962). (199) The nucleotide esters 46 and 47 are often referred to as D-ribitol5-pyrophosphate and L-glycerol3-pyrophosphate derivatives. (200) J. Baddiley, J. G. Buchanan, A. P. Mathias, and A. R. Saunderson,]. Chem. SOC., 4186 (1956). (201) M. Burger and L. Glaser,]. Biol. Chem., 239, 3168 (1964). (202) E. E. B. Smith, B. Galloway, and G. M. Mills, Biochem. Biophys. Res. Commun., 5, 148 (1961).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

335

can compete quite effectively if starting material having a high content of “sugar nucleotides” is available. Examples of such direct procedures include the simultaneous isolation of uridine 5’-(a-Dglucopyranosyl pyrophosphate), uridine 5’-(2-acetamido-2-deoxya-D-glucopyranosyl pyrophosphate), and guanosine S’-(a-D-mannopyranosyl pyrophosphate) from yeast,203 and the preparation of guanosine 5’-(a-D-mannopyranosyl pyrophosphate) and uridine 5‘- (2-acetamido- 2- deoxy-4-O-sulfo-a-~-ga~actopyranosyl pyrophosphate) from egg albumen.81 1. Enzymic and Fermentation Procedures a. D-Glucose Derivatives. -The enzymic preparation of nucleoside 5‘-(a-D-ghcopyranosy~pyrophosphates) is based on the interaction of the appropriate nucleoside 5’-triphosphate and D-glUCOsy1 phosphate ( S ) , as shown in Scheme 3.

0

+

OH I

OH

II HO - P - 0 I OH

0 I1

0 II

-P -0-P-OR I I OH OH

OH R = nucleoside-5’-yl

Scheme 3

The reaction is cataIyzed by specific enzymes, usually termedm4 “sugar nucleotide pyrophosphorylases.” The equilibrium constant is not far from unity, and addition of inorganic pyrophosphatase, an enzyme that converts inorganic pyrophosphate irreversibly into (203) H. G . Pontis, E. Cabib, and L. F. Leloir, Biochim. Biophys. Acta, 26, 146 (1957). (204) The systematic name for these enzymes is “nucleoside triphosphate: a - D glycosyl phosphate-nucleotidyl transferases” (E.C. 2.7.7 group).

N. K. KOCHETKOV AND V. N. SHIBAEV

336

inorganic phosphate, is of general use in synthetic procedures to favor formation of the “sugar nucleotide.” The pyrophosphorylase of uridine 5’-(a-D-glucopyranosy1pyrophosphate), first detected in yeast,205is widely distributed in Nature, and purified preparations of the enzyme have been obtained. With such a preparation from mammary gland, the phosphate 8 gave uridine 5’-(a-D-glucopyranosyl pyrophosphate) in about 95% yield.206 Enzymic synthesis catalyzed by pyrophosphorylases proceeds satisfactorily only on a small scale, and its main application consists in the preparation of radioactive “sugar nucleotides.” Radioactive starting materials more accessible than a-D-glucopyranosyl phosphate may be used for this purpose. Uridine 5’-(a-D-glucopyranosy1-’4C pyrophosphate) was obtained from labeled D-glucose 6 - p h o ~ p h a t e , ~ ~ ~ D-ghcose208-210*210a or a D-glucose-D-fructose mixture,211with the use of a partially purified pyrophosphorylase preparation from yeast. The incubation mixture contained the reactants and enzymes necessary for the transformations shown in Scheme 4.

adenosine B‘-triphosphate

CH L O .

adenosine 5‘-pyrophosphate 1

\

CH,OPO,H,

hexokinase I

OH

OH

phosphoglucoisomerase ; a-D-glucose 1,6-diphosphate YH,OH

OH

Scheme 4

(205) A. Munch-Petersen, H. M. Kalckar, E. Cutolo, and E. E. B. Smith, Nuture, 172, 1037 (1953);A. Munch-Petersen, Actu Chern. Scund., 9, 1523 (1955). (206) D. K. Fitzgerald and K. E. Ebner, Anal. Biochem., 15, 150 (1966). (207) L. Glaser,J. Biol. Chem., 232, 627 (1958).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

337

In some procedures, reactants were added to permit regeneration of the nucleoside 5’-triphosphate through the enzymic reaction of the nucleoside 5’-pyrophosphate with enolpyruvate phosphatezogor 3-0phosphonoglycerate.210a,211 The most convenient procedure seems to be that of Wright and Robbins,zll in which uridine 5’-triphosphate was used as the single nucleotide and a preparation from yeast contained all of the enzymes required, except for hexokinase. The yield of the “sugar nucleotide” was about 60%, and could be enhanced further by using a pure pyrophosphorylase preparation.210 The same procedure was appliedZlzfor the preparation of uridine 5’-(a-D-glucopyranosyl pyrophosphate)-3”-t, -4”-t, or -5”-t. A cell-free extract of Phytophthora cinnamoni was used for the synthesis of the 6”-t derivative,z13as well as yeast p y r o p h o s p h o r y l a ~ eAn . ~ ~enzyme ~~ from Salmonella typhimurium was found quite satisfactory214for obtaining this and other labeled “nucleotide sugars.” A large-scale preparative procedure for uridine 5’-(a-D-glucopyranosyl pyrophosphate) has been described.z15Uridine 5’-phosphate is converted into this “sugar nucleotide” in a yield of 50-60% when it is incubated with D-glucose, inorganic phosphate, magnesium sulfate, and a crude yeast-enzyme preparation. It is possible to isolate more than 20 g of “sugar nucleotide” from a single incubation. The pyrophosphorylase procedures have been applied widely for preparation of various naturally occurring a-D-ghcopyranosyl esters of nucleoside pyrophosphates. For synthetic purposes, it is common to use crude or partially purified enzyme preparations, which may be a mixture of individual, specific enzymes. The synthesis of adenosine 5r-(a-D-glucopyranosyl pyrophosphate) was achieved with pyrophosphorylases from Arthrobacter uiscosus,216~z17 Azotobacter ~ i n e l a n d i i , ~ ~

(208) R. E. Trucco, Nature, 174, 1103 (1954). (209) E. P. Anderson, E. S.Maxwell, and R. M. Burton, J. Amer. Chem. Soc., 81, 6514 (1959). (210) J. A. Thomas, K. K. Schlender, and J. Lamer, Anal. Biochem., 25, 486 (1968). (210a) A. V. Morozova and B. N. Stepanenko, Biokhimiya, 35, 117 (1970). (211) A. Wright and P. W. Robbins, Biochim. Biophys. Acta, 104, 594 (1965). (212) J. S. Schutzbach and D. S. Feingold, J. Biol. Chem., 245, 2476 (1970). (213) M. C. Wang and S. Bartnicki-Garcia, Anal. Biochem., 26, 412 (1968). (213a) J. G. Schiller, A. M. Bowser, and D. S.Feingold, Carbohyd. Res., 21,249 (1972). (214) H. Nikaido and M. Sarvas, J . Bacteriol., 105, 1073 (1971). (215) T. Tochikura, H. Kawai, S. Tobe, K. Kawaguchi, M. Osugi, and K. Ogata, Hakko Kogaku Zasshi, 46,957 (1968). (216) L. Shen and J. Preiss, J. Biol. Chem., 240, 2334 (1965). (217) J. Preiss, L. Shen, E. Greenberg, and N. Gentner, Biochemistry, 5, 1833 (1966).

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N. K. KOCHETKOV AND V. N. SHIBAEV

wheat flour,21B rice grains,41and rat mammary g l a n d ~ ; ~ that ~ * ~of O cytidine 5’-(a-D-glucopyranosyl pyrophosphate) with enzymes from Arthrobacter viscosus,21sAzotobacter vinelandii,52 and different Salmonella strain^.^^.^^,^^,^^^,^^^ Guanosine 5’-(a-D-glucopyranosyl pyrophosphate) was prepared by using enzymes from Arthrobacter,216 Eremothecium ashbyi,221 Hansenula holstii,222 and animal tissue^.^^*^^ Bacterial pyrophosphorylases were used for enzymic synthesis of thymidine 5’-(a-D-glucopyranosyl The lack of absolute substrate specificity with the sugar nucleotide pyrophosphorylases allows the preparation of some analogs of the natural derivatives. The yeast pyrophosphorylase effective for synthesis of uridine 5’-(a-D-glucopyranosyl pyrophosphate) was used for synthesis of the analogous derivatives of pseudouridine228(48) and 5 - h y d r o ~ y u r i d i n e(49). ~~~ Inosine 5’-(a-D-glUCOpyranOSylpyrophosphate) (50) was obtained by use of the pyrophosphorylases, effective for the respective guanosine derivative, from animal tissues,5O pea,223or Arthrobacter visC O S U S . ~The ~ ~ enzymic synthesis of the a-D-glucopyranosyl pyrophosphate esters of 2 ’ - d e o ~ y u r i d i n eand ~ ~ ~2 ’ - d e o ~ y a d e n o s i n ehas ~ ~also ~ been described. Even some modifications in the glycosyl group are possible. Incubation of yeast with 2-deoxy-~-arabino-hexose leads to the corresponding glycosyl esters of uridine 5 ’ - p y r o p h o ~ p h a t and e ~ ~guano~ sine 5‘-pyropho~phate.~~l Pyrophosphorylase-catalyzed synthesis has also been reported for producing the 2-amino-2-deoxy-~-glucopyranosyl esters of uridine 5 ’ - p y r o p h o ~ p h a t e , adenosine ~ ~ , ~ ~ ~ 5’-pyro(218) J. Espada,]. B i d . Chem., 237, 3577 (1962). (219) R. M. Mayer and V. Ginsburg,]. B i d . Chem., 240, 1900 (1965). (220) R. D . Bevill, Biochem. Biophys. Res. Commun., 30, 595 (1968). (221) H. G . Pontis and S. M. E. Pontis, Biochim. Biophys. Acta, 89, 554 (1964). (222) R. K. Bretthauer, D. R. Wilken, and R. G. Hansen, Biochim. Biophys. Acta, 78, 420 (1963). (223) G. A. Barber and W. Z. Hassid, Biochim. Biophys. Acta, 86, 397 (1964). (224) S. Kornfeld and L. Glaser, Biochim. Biophys. Acta, 42,548 (1960);J . Biol. Chem., 236, 1791 (1961). (225) 0. Gabriel and G. Ashwell,]. Biol. Chem., 240, 4123 (1965). (226) J. H. Pazur and E. W. Shuey,]. Biol. Chem., 236, 1780 (1961). (227) R. L. Bernstein and P. W. Robbins,]. Biol. Chem., 240, 391 (1965). (228) M. Rabinowitz and I. H. Goldberg,]. Biol. Chem., 238, 1801 (1963). (229) P. Roy-Burman, S. Roy-Burman, and D. W. Visser, J . Biol. Chem., 243, 1692 (1968). (230) P. Biely and S. Bauer, Biochim. Biophys. Acta, 121, 213 (1966); Collect. Czech. Chem. Commun., 32, 1588 (1967). (231) P. Biely and S. Bauer, Biochim. Biophys. Acta, 156, 432 (1968). (232) F. Maley and H. A. Lardy, Science, 124, 1207 (1956).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

/

339

\

Y HO

OH

phosphate,52cytidine 5’-pyropho~phate,5~ and thymidine 5’-pyrophosphosphate used phate.52*11s The 2-amino-2-deoxy-a-~-glucopyranosy~ as starting material is evidently a close enough analog of a-D-ghcopyranosyl phosphate to enter into the reaction.

b. D-Mannose Derivatives. - Guanosine 5’-(a-D-mannopyranosyl pyrophosphate) pyrophosphorylase catalyzes the reaction between guanosine 5’-triphosphate and a-D-mannopyranosyl phosphate in a manner similar to that shown in Scheme 3. The enzyme, first found in yeast,233is of common occurrence; preparations from Arthrobacter ~ i s c o s u and s ~ ~from ~ animal t i s s ~ e swere ~ ~ *used ~ ~ for the synthesis. To obtain the radioactive “sugar nucleotide” from D - m a n n o ~ e - ~it~ c , is essential to employ an enzyme preparation free of D-mannose phosphate isomerase, which catalyzes the conversion of D-mannose 6-phosphate into D-fructose 6-phosphate that subsequently participates in various metabolic transformations. It is convenient to use an extract from a mutant strain of Salmonella typhimurium that lacks mannose phosphate i ~ o m e r a s e ?or ~ ~a partially purified preparation . ~ ~ ~synthesis of adenosine 5’-(a-D-mannopyfrom A r t h r o b a ~ t e rThe ranosyl pyrophosphate) has been achieved by use of anima150 and plant51pyrophosphorylases. (233) A. Munch-Petersen, Actu Chem. Scand., 10,928 (1956). (234) J. Preiss and E. Wood,]. Biol. Chem., 239, 3119 (1964). (235) S. M. Rosen, L. D . Zeleznick, D. Fraenkel, I. Weiner, M. J.Osborn, and B. L. Horecker, Biochem. Z., 342,375 (1965). (236) J . Preiss and E. Greenberg, Anal. Biochem., 18,464 (1967).

N. K. KOCHETKOV A N D V. N. SHIBAEV

340

The D-mannose “nucleotide sugars” derived from 2’-deoxyguano ~ i n and e ~ ~ ~ were prepared by using enzymes that catalyze the synthesis of guanosine 5’-(a-D-mannopyranosyl pyrophosphate). A fermentation procedure has been d e ~ c r i b e d ~for ~ ’ large-scale ,~~~ production of the latter “sugar nucleotide.” Bakers’ yeast transforms guanosine 5’-phosphate into the D-mannosyl pyrophosphate ester in 45% yield when a mixture with D-glucose, potassium phosphate, and magnesium sulfate is incubated. c. D-Galactose Derivatives. - Pyrophosphorylases are known that act in the manner just described, and catalyze the reaction of a - ~ galactopyranosyl phosphate with adenosine 5’-triphosphate?l thymidine 5 ’ - t r i p h o ~ p h a t e , ~ and ~ ~ - uridine ~~l 5 ’ - t r i p h o ~ p h a t e . ’These ~~,~~~ reactions have occasionally been used for obtaining the respective nucleoside 5’-(a-D-ga~actopyranosy~ pyrophosphates). Another process, depicted in Scheme 5, is frequently employed for synthesis of the uridine derivative 30; it comprises the reaction of !?

C H,OH

Qo-

:H,OH

+

HO

I

OH

OH HO

51

OH

5

FH,OH

+

II

OH

OH 8

HO 30

OH Scheme 5

(237) T. Tochikura, K. Kawaguchi, T. Kano, and K. Ogata, Hakko Kogaku Zasshi, 47, 564 (1969). (238) K. hwaguchi, K. Ogata, and T. Tochikura, Agr. Biol. Chem. (Tokyo), 34, 908 (1970).

GLYCOSYL ESTERS O F NUCLEOSIDE PYROPHOSPHATES

341

uridine 5’-(a-D-glucopyranosylpyrophosphate) (5) and a-D-galactopyranosyl phosphate243(51). The equilibrium may be shifted to favor the formation of uridine 5’-(a-D-galactopyranosyl pyrophosphate) (30) by enzymic removal of a-D-ghcopyranosyl phosphate (8)as 6-O-phosphono-D-gluconate by the action of phosphoglucomutase and D-glucose 6-phosphate dehydrogenase. Preparative procedures have been described that employ the enzymes from the yeast Saccharomyces f r a g i l i ~and ~ ~ ~calf liver.244 Fermentation procedures useful for the production of uridine 5‘-(a-D-galactopyranosyl pyrophosphate) involve the cultivation of bacterial mutant-strains that are deficient in the 4-epimerase for 30 (see Section V, l,b, p. 369) in D-galactose-containing or by incubating Tordopsis candida cells with uridine 5’-phosphate, D-galactose, potassium phosphate, and magnesium sulfate.248 The analog of this “sugar nucleotide” lacking the hydroxyl group pyroat C - 2 , namely, uridine 5’-(2-deoxy-a-~-lyxo-hexopyranosyl phosphate) is formed in admixture with uridine 5’-(2-deoxy-a-Darabino-hexopyranosyl pyrophosphate) when yeasts249 or higher plantsZ5Oare treated with 2-deoxy-~-lyxo-hexopyranose.

d. 2-Acetamido-2-deoxy-~-glycosyl Derivatives. - Radioactive urpyrophosphate) has idine 5’-(2-acetamido-2-deoxy-a-D-glucopyranosy~ been prepared by using specific pyrophosphorylases from yea~t’~~,’~’ and from liver h o m o g e n a t e ~ .Corresponding ~~~ pyrophosphorylase-

(239) J. H. Pazur and J. S . Anderson,]. Biol. Chem., 238, 3155 (1963). (240) E. F. Neufeld, Biochem. Biophys. Res. Commun., 7,461 (1962). (241) R. B. Frydman, E. F. Neufeld, and W. Z. Hassid, Biochim. Biophys. Acta, 77, 332 ( 1963). (242) E. F. Neufeld, V. Ginsburg, E. W. Putnam, D. Fanshier, and W. Z. Hassid, Arch. Biochem. Biophys., 69, 602 (1957). (243) The corresponding enzyme should be referred to as “uridine 5’-(a-Dglucopyranosyl pyroph0sphate):a-D-galactopyranosyl phosphate uridylyl transferase” (E.C. 2.7.7.12). (244) E. S. Maxwell, J . Biol. Chent., 229, 139 (1957). (245) H. Wiesmeyer and E. Jordan, Anal. Biochem., 2, 280 (1961). (246) A. E. Shedlowsky, H. A. Boye, and S . Brenner, A n d . Biochem., 8, 362 (1964). (247) N. Morikawa, Y. Imae, and H. Nikaido, ]. Biochem. (Tokyo), 56, 145 (1964). (248) T. Tochikura, K. Kawaguchi, H. Kawai, Y. Mugibayashi, and K. Ogata, Hakko Kogaku Zasshi,46,970 (1968). (249) W. Fischer and G. Weidemann, Biochim. Biophys. Acta, 93, 677 (1964). (250) W. Fischer and G. Weidemann, 2. Physiol. Chem., 336, 206 (1964). (251) L. Glaser and D. H. Brown, Proc. Nat. Acad. Sci. U . S . , 41, 253 (1955). (252) R. R. Wagner and M. A. Cynkin, Anal. Biochem., 25,572 (1968).

N. K. KOCHETKOV AND V. N. SHIBAEV

342

catalyzed reactions were also employed for synthesis of the thymidine52,116*117 and a d e n o ~ i n eanalogs. ~~,~~ Animal tissues have been reported to contain the pyrophosphorylase of uridine 5’-(2-acetamido-2-deoxy-a-~-galactopyranosyl pyrowhich may be used for the synthesis of this “sugar nucleotide”; it has also been prepared254according to Scheme 6, 0

I

52

HO

I

OH

53

CH,OH I

NHAc HO

OH

35

Scheme 6

by exploiting the lack of absolute specificity of uridine 5’-(a-D-glucopyranosyl pyrophosphate): a-D-galactopyranosy1 phosphate uridylyl transferase (compare the preceding Sub-section). 2-Amino-2-deoxya-D-galactopyranosyl phosphate (52) participates in the enzymic reaction instead of the a-D-galactopyranosyl derivative, and the ester (53) of uridine 5’-pyrophosphate is thereby produced. The latter was N-acetylated with acetic anhydride in aqueous methanol, giving the desired acetamido ester (35). (253) T. Sawicka and T. Chojnacki, Bull. Acad. Pol. Sci., Ser. Sci. Biol., 18,125 (1970). (254) F. Maley, Biochem. Biophys. Res. Commun., 39,371 (1970).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

343

Fermentation procedures for preparing uridine 5’-(2-acetamido2-deoxy-a-D-glucopyranosyl pyrophosphate) have also been reported. One of them255involves the cultivation of Helminthosporium sativum in the presence of 2-amino-2-deoxy-D-glucose and the antibiotic polyoxin; the latter is an inhibitor of chitin biosynthesis. The 0thel-2~~ utilized the incubation of yeast cells with uridine 5’-phosphate in the presence of an excess of 2-amino-2-deoxy-D-glucose and inorganic phosphate.257 The intravenous injection of 2-amino-2-deoxy-~-g~ucose-’~C into rats, and subsequent isolation of the “uridine diphosphate N-acetylhexosamine” fraction, has been suggested as a fast and convenient method for preparing small amounts of a mixture of radioactive uridine 5’-(2-acetamido-2-deoxy-a-~-glucopyranosyl pyrophosphate) and uridine 5’- (2-acetamido - 2- deoxy- a - D - galactopyranosyl p yro phosphate). e. Derivatives of Other Monosaccharides. - Pyrophosphorylases are known that are specific for some other glycosyl phosphates that occur naturally. Plant extracts contain enzymes capable of catalyzing the reaction of uridine 5’-triphosphate with a - D - x y l o p y r a n 0 s y 1 , ~ ~ ~ ~ ~ ~ ~ /3-~-arabinopyranosy1,242 a-D-glucopyranosyluronic a ~ i d , and ~ ~ ~ * ~ ~ a-D-ga~actopyranosy~uronicacidZ6l phosphates. Guanosine 5’-(/3-~fucopyranosyl pyrophosphate) was prepared from guanosine 5’-triphosphate and the glycosyl phosphate by the action of a pig-liver enzyme.262 Several cases have been documented in which a different, naturally occurring, glycosyl phosphate substitutes for the true substrate of the pyrophosphorylase. Adenosine 5’-(a-D-xylopyranosyl pyrophosphate) was prepared by the action of the pyrophosphorylase of adenosine 5’-(a-D-glucopyranosylpyrophosphate) from wheat flour,218 (255) A. Endo, M. Kakiku, M. Hore, H. Abe, and T. Misato, Biochem. B i o p h y s . Res. Commun., 39, 718 (1970). (256) T. Tochikura, H. Kawai, and T. Gotan, Agr. Biol. Chem. (Tokyo),35, 163 (1971). (257) P. J. O’Brien and E. F. Neufeld, Biochim. Biophys. Actu, 83, 352 (1964). (258) V. Ginsburg, E. F. Neufeld, and W. Z. Hassid, Proc. Nut. Acud. Sci. U . S . , 42,333 (1956). (259) D. S. Feingold, E. F. Neufeld, and W. Z. Hassid, Arch. Biochem. Biophys., 78, 401 (1958). (260) P. K. Wong and Y. K. Lau, Biochim. B i o p h y s . Actu, 220,61 (1970); R. M. Roberts and K. M. K. Rao, Fed. Proc., 30, 1117 (1971); R. M. Roberts,J. Biol. Chem., 246, 4995 (1971). (261) E. F. Neufeld, D. S. Feingold, S. M. Ilves, G. Kessler, and W. Z. Hassid,J. Biol. Chem., 236, 3102 (1961). (262) H. Ishihara and E. C. Heath,J. Biol. Chem., 243, 1110 (1968).

344

N. K. KOCHETKOV AND V. N. SHIBAEV

and guanosine 5’-(a-~-rhamnopyranosyl pyrophosphate) was produced

by use of the mung-bean pyrophosphorylase of guanosine 5’-(a-~mannopyranosyl p y r o p h o ~ p h a t e ) . ~ ~ Enzymic transformations of the glycosyl group in readily accessible D-glucosyl and D-mannosyl esters of nucleoside 5’-pyrophosphates are of great utility for the preparation of various “sugar nucleotides.” These reactions are discussed in detail in Section V,l (see p. 363); the following transformations, in which glycosyl esters of nucleoside pyrophosphates serve as analogs of the monosaccharide substrate of the enzymes, provide some examples from the later literature. The strain of Agrobacterium turnefaciens capable of oxidizing D-glucose derivatives at C-3 was found to convert uridine 5’-(a-Dghcopyranosyl pyrophosphate) into the a-~-ribo-hexopyranosyl-3ulose derivativezs4(54). Experimental conditions have been found for oxidation of uridine 5’-(a-D-galactopyranosylpyrophosphate) with D-galactose o x i d a ~ e . ~ ~ ~ The reaction product is the a-D-galacto-hexodialdosederivative (55). Analogous treatment of uridine 5’-(acetamido-2-deoxy-a-~-galactopyranosyl pyrophosphate), and subsequent reduction of the product with sodium borohydride-t, affords a convenient synthesis of the 6-tritiated d e r i ~ a t i v e . ~ 6 ~ ~

54

55

R = uridine B‘-pyrophosphoryl

2. Chemical Synthesis The only general approach to the chemical synthesis of glycosyl esters of nucleoside pyrophosphates thus far found useful involves

(263) G . A. Barber, Biochemistry, 8, 3692 (1969). (264) S. Fukui, Agr. Biol. Chem. (Tokyo), 34, 321 (1970). (265) G . L. Nelsestuen and S. Kirkwood, Fed. Proc., 30,1117 (1971);l.Biol. Chem.,246, 3828 (1971). (265a) G. L. Nelsestuen and S. Kirkwood, Anal. Biochem., 40, 359 (1971).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

+ OH

345

0

RO-P-x II

(or x@)

I 00

I O@

56

t +

X’

(or X)

OH 57

Scheme I

the interaction of an activated derivative (56) of the nucleoside 5’-phosphate with the glycosyl phosphate, as shown in Scheme 7. Nucleoside 5’-phosphates are employed as starting materials to prepare the activated derivatives (56). Their synthesis has been discussed in a Chapter in this Series.z66The methods used most frequently for preparing glycosyl phosphates involve the interaction of protected glycosyl bromides with diphenyl phosphate and subsequent removal of the protective g r o u p ~ , ~or~ ’by fusion of the peracetylated monosaccharides with anhydrous phosphoric acid.z6s a. Phosphoramidate method. -A zwitterionic phosphoramidate derivative (56, X@= @NHRIRz,where R1 and Rz are alkyl groups) is most probably a reactive intermediate in the reaction between a nucleoside 5’-phosphoramidate and a glycosyl phosphate. The pyrophosphate 57 and the corresponding amine are the products. The first application of this reaction for the synthesis of sugar nucleotides was reported in 1958, when Moffatt and K h ~ r a n a ~ ~ ~ prepared uridine 5’-(a-D-glucopyranosyl pyrophosphate) (5) in 59% yield from uridine 5’-phosphoramidate (58). Other examples of similar (266) T. Ueda and J. J. Fox, Aduan. Cnrbohyd. Chem., 22,307 (1967). (267) T. Posternak,]. Amer. Chem. Soc., 72, 4824 (1950). (268) D. L. MacDonald, J . Org. Chem., 27, 1107 (1962). (269) J. G . Moffatt and H. G. Khorana,]. Amer. Chem. Soc., 80,3756 (1958).

346

N. K. KOCHETKOV A N D V. N . SHIBAEV 0

FH,OH

OH

OH

HO

R

YH,OH

0

OH

HO

OH

5

syntheses are known, including the use of 5’-phosphoramidates derived from various other nucleosides,103~270-272 and such N-substituted analogs as guanosine 5’-phosphorocyclohexylamidate273.274 (59), adenosine 5’-phosphoropiperidateZ2(60), nucleoside 5’-phosphoroimidazolidate~,~~~*~~~ and adenosine 5’-phosphoro(P + N)phenylalanineZ7’ have been reported. However, the most popular modification278has been the one that exploits nucleoside 5’-phosphoromorpholidates (61). (270) T. Ueda, Chem. Pharm Bull. (Tokyo), 8,464 (1960). (271) M. Honjo, Y. Furukawa, K. Imai, H. Moriyama, and K. Tanaka, Chem. Pham. Bull. (Tokyo), 10, 225 (1962). (272) M. Honjo, Y. Furukawa, and Y. Kanai, Biochim. Biophys. Actu, 91, 525 (1964). (273) T. Ueda and E. Ohtsuka, Chem. Pharm. Bull. (Tokyo), 7,935 (1959). (274) J. Baddiley, N. A. Hughes, and A. L. James,J. Chem. Soc., 2574 (1961). (275) F. Cramer, H. Neunhoeffer, K. H. Scheit, G. Schneider, and J. Tennigkeit, Angew. Chem., 74, 387 (1962). (276) V. N. Shibaev, G. I. Eliseeva, and N. K. Kochetkov, Izo. Akad. Nauk S S S R , Ser. Khim., in press. (277) V. G . Shestakov, Z. A. Shabarova, and M. A. Prokof’ev, Biokhimiya, 29, 690 (1964). (278) S.Roseman, J. J. Distler, J. G. Moffatt, and H. G. Khorana,J. Amer. Chem. SOC., 83, 653 (1961).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

347

<exNH* (6 Iril 0

0

0

C,HI,NH-P--OCH2

:@

HO

c N - ! ; O C V

OH

HO

59

OH

60

n f : HO

OH

61

The choice of these derivatives, namely, those formed.from amines of moderate strength, appears to be the best compromise for the synthesis to be effective. Although the susceptibility of phosphoramidates to attack by nucleophiles increases with enhancement of the basic strength of the parent amine,279,280 the reverse trend is observed for the yields of nucleoside 5'-phosphoramidates when they are prepared from nucleoside 5'-phosphates by condensation with the appropriate amines through the action of N,N'-dicyclohexylcarbodiimide.279

0 II. RO-P-OH

I 00

+

HNR'R'

C,HIIN=C=NC,Hl,

0 II RO-P-NR'R' I O@

(279) J. G. Moffitt and H. G. Khorana,]. Arner. Chem. SOC., 83, 649 (1961). (280) This conclusion has been drawn by Moffatt and Kh~rana,''~based on limited data. A subsequent, more detailed study [R. K. Ledneva, N. N. Preobrazhenskaya, Z. A. Shabarova, and M. A. Prokof'ev, Molek. Biologiya, 5, 264 (1971)l clearly shows an unexpected decrease in the rate of the acid hydrolysis of the phosphoramidates derived from strong amines. If a similar order of reactivities exists for the pyrophosphate synthesis, the phosphomorpholidate derivative seems close to being the most active.

N. K. KOCHETKOV AND V. N. SHIBAEV

348

The morpholidate procedure was found to be efficient for preparing the “sugar nucleotide” derivatives of uronic a ~ i d ~ , 22-acet~ ~ , ~ ~ ~ * ~ amid0-2-deoxyglycoses,~~~,~~~-~~~ d e o x y g l y c ~ s e s , ~a ~hexopyranos~-~~~ 4 - ~ l o s eand , ~ ~a ~d i~s a c ~ h a r i d e In . ~ ~addition ~ to the synthesis of the glycosyl esters of naturally occurring nucleoside 5‘-pyrophosphates, the method allows preparation of a number of their analogs having a modified nucleoside residue, such as derivatives of the methylated uridines299-301 and a d e n o s i n e ~ ,4~-~t h ~ i. o~ ~~ r~i d i n e (62), ~ ~ ~2-thio,~~~ ~ r i d i n e ~ O (63), ’ ” ~ ~i~s o ~ y t i d i n e ~(64), ~ ~ .6~-~a ~ z a ~ r i d i n e ~(65), ~ ~ 9in~~~ osine,50,216,302,308 and x a n t h o ~ i n e (66). ~~~*~~~ S

I

I R

R

62

63

I

NHZ

1

R

R 64

65

R= I R

H 66

I

HO

I

OH

T.-Y. Lin, A. D. Elbein, and J. C. Su,Biochem. Biophys. Res. Commun., 22, 650 (1966). C. L. Villemez, A. L. Swanson, and W. Z. Hassid, Arch. Biochem. Biophys., 116, 446 (1966). D. M. Carlson, A. L. Swanson, and S. Roseman, Biochemistry, 3, 402 (1964). J. E. Silbert,J. Biol. Chem., 239, 1310 (1964). E. A. Davidson and R. W. Wheat, Biochim. Biophys. Acta, 72, 112 (1963). T. Y. Kim, Hwahak Kwa Kongop Ui Chinbo, 4, 149 (1964); Chem. Abstr., 62, 13219b (1965). E. Harel, J. E. Silbert, and N. Sharon, Arch. Biochem. Biophys., 117,296 (1966). W. L.Salo and H. G. Fletcher, Jr., Biochemistry, 9, 878 (1970). H. Heymann, R. Turdiu, B. K. Lee, and S. S. Barkulis, Biochemistry, 7, 1393 (1968). G. A. Barber, Biochem. Biophys. Res. Commun., 8, 204 (1962). J. H. Nordin, W. L. Salo, R. D. Bevill, and S. Kirkwood, Anal. Biochem., 13, 575 (1965). N. K. Kochetkov, E. I. Budowsky, N. D. Gabrieljan, Yu. Yu. Kusov, and V. N. Shibaev, Carbohud. Res., 5, 367 (1967). (293) N. K. Kochetkov: E. I. Budowsky, V. N. Shibaev, and Yu. Yu. Kusov, Izv. Akad. Nauk SSSR, Ser. Khim., 1136 (1969).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

349

Several papers describe the preparation of analogs that differ from naturally occurring “sugar nucleotides” in the anomeric configuration of the glycosyl r e s i d ~ e . ~Furthermore, ~ ~ ~ ~ ~ it~has * ~ been ~ ~possible * ~ ~ ~ to obtain an analog (67) of uridine 5’-(a-D-glucopyranosyl pyrophos-

HO

OH

61

(294) N. K. Kochetkov, E. I. Budowsky, T. N. Druzhinina. N. D. Gabrieljan, I. V. Komlev, Yu. Yu. Kusov, and V. N. Shibaev, Carbohyd. Res., 10, 152 (1969). (295) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and Yu. Yu. Kusov, Zzu. Akad. Nauk S S S R , Ser. Khim., 404 (1970). (296) V. N. Shibaev, Yu. Yu. Kusov, I. V. Komlev, E. I. Budowsky, and N. K. Kochetkov, lzu. Akad. Nauk S S S R , Ser. Khim., 2522 (1969). (297) F. Schanbacher and D. R. Wilken, Biochim. Biophys. Acta, 141, 646 (1967). (297a) G. L. Nelsestuen and S. Kirkwood,J. Biol. Chem., 246,7533 (1971). (298) A. H. Olavesen and E. A. Davidson, J. Biol. Chem., 240, 992 (1965). (299) N. K. Kochetkov, E. I. Budowsky, and V. N. Shibaev, Biochim. Biophys. Acta, 32, 415 (1961); Izu. Akad. Nauk S S S R , Otdel Khim. Nauk, 1035 (1962). (300) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, G. I. Eliseeva, M. A. Grachev, and V. P. Demushkin, Tetrahedron, 19, 1207 (1963). (301) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and G. I. Eliseeva, Zzu. Akad. Nuuk S S S R , Ser. Khim., 1779 (1966). (302) N. D. Gabrieljan, E. B. Lapina, S. M. Spiridonova, V. N. Shibaev, E. I. Budowsky, and N. K. Kochetkov, Biochim. Biophys. Acta, 158,478 (1969). (303) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and S. M. Spiridonova, Zzu. Akad. Nauk S S S R , Ser. Khim., 2514 (1969). (304) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and M. A. Grachev, Biochim. Biophys. Acta, 59, 747 (1962); 120. Akad. Nauk S S S R . Ser. Khim., 1592 (1963). (305) N. K. Kochetkov, E. I. Budowsky, and V. N. Shibaev, Khim. Prir. Soedin., 409 (1965). (306) N. K. Kochetkov, E. I. Budowsky, and V. N. Shibaev, Khim. Prir. Soedin., 328 (1965). (307) E. I. Budowsky, V. N. Shibaev, G. I. Eliseeva, and N. K. Kochetkov, Zzu. Akad. Nauk S S S R , Ser. Khim., 1491 (1964). (308) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and S. M. Spiridonova, lzu. Akad. Nuuk S S S R , Ser. Khim., 2775 (1969). (309) I. Das, M. A. Wentworth, H. Ide, H. G. Sie, and W. H. Fishman, Biochim. Biophys. Acta, 201, 375 (1970).

350

N. K. KOCHETKOV AND V. N. SHIBAEV

phate) that is derived from 5'-deoxyuridine 5'-phosphonate and contains a C-P bond.310 In all of the foregoing examples, an activated derivative of the nucleotide has been employed for generating the pyrophosphate link. An attempt to use an activated glycosyl phosphate failed;311the only product identified from the reaction between b-D-glucopyranosyl phosphoramidate (68) and uridine 5'-phosphate was the cyclic phosphate 69. The ease of participation of the sterically accessible, C-2 hydroxyl group probably accounts for this result. The usual procedure

for synthesis by the morpholidate method consists in the interaction of the 4-morpholine N , N ' -dicyclohexylcarboxamidinium nucleoside 5'-phosphoromorpholidate with a 2-4-fold excess of the trioctylammonium glycosyl phosphate in anhydrous pyridine, for several days at room ternperat~re,2'~or for several hours at an elevated temp e r a t ~ r e .The ~ ~ yields ~ , ~ ~of~ the desired products are generally high (65-70%), although decreased yields are encountered with guanosine derivatives (because of their low solubi1ity),3l2and also when the reactants are used in equimolar ratio.312Presumably, one of the side reactions proceeding under the conditions of the pyrophosphate synthesis involves degradation of the glycosyl ester of the nucleoside pyrophosphate, by a process similar to that taking place in alkaline solution (see Section IV; p. 358). The higher yields of a-D-mannopyranosyl derivatives obtained, compared with the yields of a - ~ glucopyranosyl derivatives under identical conditions, support this idea;291small proportions of compounds chromatographically identical with the monosaccharide cyclic phosphates were found in the reaction mixtures.313 Dramatic decreases in yield are encountered in micro-scale adapta(310) P. C. Bax, F. Morris, and D. H. Rammler, Biochim. Biophys. Acta, 201,416 (1970). (311) T. Ueda, Chem. Phamn. Bull. (Tokyo), 8, 459 (1962). (312) A. D. Elbein, Methods Enzyrnol., 8, 142 (1966). (313) V. N. Shibaev, unpublished observation.

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

351

tions of the synthesi~,2~’ and may be ascribed to hydrolysis of the nucleoside 5’-phosphoromorpholidate by traces of water. Several modifications of the procedure have been suggested to overcome this d i f f i ~ ~ l t y one ; ~ ~of~themzg1 * ~ ~ ~involves , ~ ~ ~ the use of a vacuum system for drying and mixing of the starting materials, and highly reproducible yields were obtained when methyl sulfoxide was used as solvent.314 The triethylammonium salts of both of the reactants are preferable in this procedure, and yields are quite high, even when an excess of the nucleoside 5’-phosphoromorpholidate is used. The use of trimethyl phosphate as a solvent has been recommended in a patented procedure.315

b. Mixed Anhydride Method.-The reaction shown in Scheme 7 (see p. 345) can be achieved with the use of a mixed anhydride derivative (56, where X is an acyl residue). The resulting pyrophosphate (57) is obviously a derivative of similar structure, and attack by the anion X@ should lead back, as a result of the reverse reaction, to the original mixed anhydride (56) and glycosyl phosphate. The direction of the process is under thermodynamic control, and thus the more stable anion should preponderate at equilibrium. An efficient synthesis of the pyrophosphate diester (57) may be accomplished if the mixed anhydride (56) is derived from an acid that is a stronger one than the nucleoside 5’-phosphate and the glycosyl phosphate. The first application of this method, for the synthesis of uridine 5’-(a-D-glucopyranosyl pyrophosphate) and uridine 5’-(a-D-galaCtOpyranosyl pyrophosphate), involved the use of 2’,3’-di-O-benzyluridine 5‘-(benzyl pho~phorochloridate)~~~ (70). The desired glycosyl

PhCqO 70

OCqPh

PhCH,O

OCH,Ph

71

(314) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and K. S. Lebedeva, Izu. Akad. Nauk S S S R , Ser. Khim.,897 (1969). (315) Y. Fujimoto, Ger. Patent 1,918,282 (1969); Chem. Abstr. 7 2 , 4 4 0 7 6 ~(1970). (316) A. M. Michelson and A. R. Todd, J. Chem. SOC., 3459 (1956).

N. K. KOCHETKOV AND V. N. SHIBAEV

352

esters were obtained in 15% yield after hydrogenolysis of the protected intermediates [for example, (71)] over a palladium catalyst. A convenient procedure for synthesis of pyrophosphates by this method was developed by M i c h e l s ~ n . ~ ~ It’employs . ~ ~ ~ mixed anhydrides (72) formed from nucleoside 5’-phosphates and diphenyl 0 II RO-P-0’ I

00

0

+

II

C1-P-OPh I OPh

(C,%),N

0 0 II I1 RO-P-0-P-OPh I I 0’ OPh 72

phosphate; these anhydrides can be prepared readily from the nucleotide and diphenylphosphorochloridate in p-dioxane or p-dioxane-N,N-dimethylformamide solution in the presence of tributylamine.319Under these conditions, the mixed anhydrides 72 are quite stable, although they are readily susceptible to nucleophilic attack in pyridine solution; with glycosyl phosphates, they give the esters of nucleoside 5’-pyrophosphates. The reaction is rapid and reaches completion within an hour at room temperature. Yields as high as 90% have been r e p ~ r t e d ,and ~ ~only ~*~ a slight ~ ~ excess of the glycosyl phosphate is required. Traces of water do not affect the yields to any appreciate extent, and decreased yields are encountered only when more than 5% of water is present.318 This promising method has so far been used mainly for the synthesis of common hexosyl derivatives, but it has been extended to uronic and deoxy sugaP3derivatives and found to be efficient. Nucleoside 5’-phosphorothioates have also been employed as activated nucleotide derivatives for synthesis of p y r o p h o s p h a t e ~ . ~ ~ ~ The interaction of tributylammonium 2’,3’-di-O-benzoyluridine 5’phosphorothioate (73) with silver a-D-glucopyranosyl and a-D-galactopyranosyl phosphates in pyridine solution, with subsequent debenzoylation, gave the corresponding glycosyl esters in 60-70% yield. This procedure can probably be classified as a variant of the mixed-anhydride method, the driving force of the reaction being the formation of insoluble silver sulfide.

(317) A. M. Michelson, Chem. Ind. (London), 1267 (1960). (318) A. M. Michelson, Biochim. Biophys. Acta, 91, 1 (1964). (319) A. M. Michelson,J. Chem. SOC., 1957 (1958). (320) A. M. Michelson and F. Wold, Biochemistry, 1, 1171 (1962). (321) T. Hata and I. Nakagawa,J. Amer. Chem. SOC., 92,5516 (1970).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

353

0 II

S

I

BzO

OBz 13

c. Carbodiimide and Related Methods. -The active intermediate (56) in the pyrophosphate synthesis (see Scheme 7, p. 345) may

contain a grouping that can undergo protonation and electronic shift as shown. <-C*-

-

O=C-NI

I

H0

I

H

An unstable derivative of this type is believed to result when phosphoric monoesters are treated with carbodiimides. The interaction of 0

II

R’O-P-OH I

00

+

RaN=C=NR2

-

0 N-Ra II II R’O-P-0-C I I 0 ’ HN-R2

uridine 5’-phosphate, a-D-glUCOpyranOSyl phosphate, and N,N’-dicyclohexylcarbodiimide was used in 1954 for the first synthesis of uridine 5’-(a-D-glycopyranosyl p y r o p h ~ s p h a t e )This . ~ ~ ~result was of great significance at that time, even though the yield obtained was only 3.5%. A related method, of preparative value, was subsequently devel0ped.3~~ It makes use of the 2-hydroxypyridyl esters (74), of nucleoside 5’-phosphates, which can readily be obtained from nucleoside 5’-phosphates and 2-pyridinol. The reaction of the derivative (74) with glycosyl phosphates in anhydrous pyridine gives rise to the

(322) G . W. Kenner, A. R. Todd, and R. F. Webb, J . Chem. SOC., 2843 (1954). (323) W. Kampe, Chem. Ber., 99, 593 (1966).

354

N. K. KOCHETKOV AND V. N. SHIBAEV

HO

OH

I

1

C,H,,N=C=

I

NC,Hll

I

OH

HO 74

glycosyl esters of nucleoside pyrophosphates in yields of 60-70%. The procedure has been applied in the synthesis of a-D-glucopyranosyl esters of adenosine, guanosine, thymidine, and uridine 5’-pyrophosphates. 3. Chemical Modification of Natural “Sugar Nucleotides”

The high lability of glycosyl esters of nucleoside pyrophosphates strongly limits the possibilities for their chemical modification. However, some conversions may be accomplished on a preparative scale. Hydrogenation of uridine 5’-(a-D-ghcopyranosyl pyrophosphate) over rhodium on alumina resulted in the 5,6-dihydrouridine derivative229*324 (75). Several modifications in the heterocyclic base of adenosine 5’-(a-D-ghcopyranosyl pyrophosphate) have been described. 9

OH I

HO

I

OH

7s

(324)N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and G . I. Eliseeva, Zzo. Akud. Nuuk SSSR, Ser. Khim., 914 (1965).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

355

Treatment with methyl sulfate in aqueous solution at pH 7 leads to the 1-N-methyladenosine derivative325(76),and bromination under controlled conditions converts it into the 8-bromoadenosine analog‘26 (77).No-Hydroxyadenosine and No-methoxyadenosine derivatives (78)were obtained after reaction with hydroxylamine or methoxyamine .327

76

78

77

(R’ = H, Me)

R =

HO

OH

Various transformation of the glycosyl group have also been reported. Specific acetylation of the amino group in uridine 5’-(2amino-2- deoxy-a-D-galactopyranosylp y r ~ p h o s p h a t e )has ~ ~ ~been mentioned in Section III,l,d (p. 342). Treatment of uridine 5’-(a-Dgalactopyranosyluronic acid pyrophosphate) with diazomethane led to the corresponding methyl D-galacturonate derivative.282A similar transformation was effected for uridine 5’-(2-acetamido-2-deoxy-a-~glucopyranosyluronic acid p y r o p h ~ s p h a t e ) , ’ and ~ ~ ~ the resulting methyl ester was converted into the 2-acetamido-2-deoxy-~-glucose derivative by reduction with sodium borohydride. (325) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and S. M. Spiridonova.Khirn. Prir. Soedin., 123 (1968). (326) E. I. Budowsky, V. N. Shibaev, S. M. Spiridonova, and N. K. Kochetkov, Zzo. Akad. Nauk S S S R , Ser. Khim., 1280 (1971). (327) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and S. M . Spiridonova, Izw. Akad. Nauk S S S R , Ser. Khim., in press.

356

N.

K. KOCHETKOV A N D V. N. SHIBAEV

Oxidation of uridine 5’-(a-D-glucopyranosy1pyrophosphate) with oxygen over a platinum catalyst afforded the derivative of a-D-glucopyranosyluronic acid pyrophosphate in about 40% The conversion of thymidine 5’-(6-deoxy-a-~-xylo-hexopyranosyl4-ulose pyrophosphate) into thymidine 5’-(D-fucosyl and 6-deoxyD-glycosyl pyrophosphates) was accomplished by reduction with sodium b o r ~ h y d r i d e . The ~ ~ ~reaction . ~ ~ ~ products were separated by paper chromatography in a borate-containing solvent-system.

Iv. CHEMICAL REACTIVITYOF GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES Glycosyl esters of nucleoside pyrophosphates undergo rapid degradation in dilute acids, leading to the nucleoside 5‘-pyrophosphate and the monosaccharide (see Section 11,l; p. 312). This reaction was first observed during studies on the chemical structure of uridine 5’-(a-D-glucopyranosy~pyrophosphate)? and was subsequently found to be general. Between pH 2.5 and 3.5, the rate of hydrolysis was observed to be proportional to the hydrogen-ion ~oncentration.3~~ A high energy of activation (38 kcal. mole-’) and an entropy of activation having a high positive value are characteristic for this reaction.331 Although no mechanistic studies of this hydrolysis have been reported, the data available suggest that the mechanism is similar to that proposed for the hydrolysis of a-D-glUCOpyranOSylphosphate in weakly acidic namely protonation of the glycosyl pyrophosphate derivative and slow heterolysis of the resulting monoanion (79) to produce a cyclic carbonium ion (SO), a species considered to be an intermediate in the hydrolysis of glycosides (for a review, see Ref. 333). The dependence between the reactivity of “sugar nucleotides” and the structure of the glycosyl group appears similar to that observed in simple glycosides, although the information available is very limited. For example, the a-D-galaCtOpyranOSyl and p-D-glucopyranosyl esters of adenosine 5‘-pyrophosphate are cleaved more (328) B. Jacobson and E. Davidson, Nature, 189, 663 (1961). (329) S.-F. Wang and 0. Gabriel,]. Biol. Chem., 244,3430 (1969). (330) H. Zarkowsky, E. Lipkin, and L. Glaser, Biochem. Biophys. Res. Commun., 38, 787 (1970). (331) E. I. Budowsky and V. N. Shibaev, Khim. Prir. Soedin., 233 (1968). (332) C. A. Bunton, D. R. Llewellyn, K. G. Oldham, and C. A. Vernon,]. Chem. Soc., 3588 (1958). (333) B. Capon, Chem. Reo., 69,407 (1969).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

357

OH 19

A

0

Monosaccharide

II

RO-P-0-P-OH

c-

oo I

0

I1

oo I

OH 80

rapidly than adenosine 5’-(a-D-glucopyranosyl pyrophosphate)22 (at pH 3 and loo”, the time of half-life is about 5 min for the first two, and 11 min for the third). The degradation of 3,6-dideoxyhexopyranosyl esters of cytidine 5’-pyrophosphate proceeds faster than that of the a-D-glucopyranosyl ester.56 The enhancement of hydrolytic rate is most pronounced for the 2-deoxyhexosyl derivatives, which release monosaccharide readily,230even at pH 7.5. Introduction of an acetamido group in place of a hydroxyl group at C-2” resulted in stabilization of the glycosyl linkage: the time to half hydrolysis for adenosine 5’-(2-acetamido-2-deoxy-a-~-glucopyranosyl pyrophosphate) isz2 15 min at pH 3 and 100”. A substantial effect in the same direction is observed when a carboxyl group replaces the hydroxymethyl group on C-5. Uridine 5’-(2-acetamido-2-deoxy-a-D-glucopyranosyluronic acid pyrophosphate) requires incubation for 40 min for complete hydrolysis under standard conditions (10 mM hydrochloric acid at 100°),149b whereas treatment for 15 min is sufficient to cleave most of the normal “sugar nucleotides.” If an amino group is introduced at C-2”, the rate of hydrolysis is diminished considerably. Treatment of uridine 5’-(2-amino-2-deoxy-a-D-glucopyranosyluronic acid pyrophosphate) with M hydrochloric acid at 100” results in only 30-50% hydrolysis after 3 h0urs.3~~ High lability in acid is very characteristic of the ester of cytidine (334) J. E. Silbert and E. F. X. Hughes, Biochirn. Biophys. Acta, 83, 355 (1964).

N. K. KOCHETKOV AND V. N. SHIBAEV

358

5‘-phosphate with N-acetylneuraminic acid; its time to half hydrolysis is about 3 hours at pH 5.25 and 37O.lS2 Methanolysis of the glycosyl linkage in “sugar nucleotides” take place335when they are treated with 0.1 M hydrogen chloride in 50% aqueous methanol for 2 min at 100”. Under these conditions, uridine 5’-(2-acetamido-2-deoxy-a -D-glucopyranosyl pyrophosphate) produces methyl 2-acetamido-2-deoxy-/3-~-glucopyranoside, and uridine 5’-(a-D-glUCOpyranOSylpyrophosphate) gives methyl /3-D-glUCOpyranoside together with an unidentified product. Alkaline hydrolysis of uridine 5’-(a-D-glucopyranosyl pyrophosphate) results in the formation of uridine 5’-phosphate and a-D-glucopyranose 1,2-cyclic phosphatez4 (81). The reaction reaches completion after 30 min at 0” in concentrated aqueous ammonia, or after 2 min at 100” and pH 8.5. Partial conversion of the cyclic phosphate (81) into a-D-glUCOpyranOSyl phosphate and D-glUCOSe 2-phosphate occurs under conditions of elevated temperature.

+

0 II RO-P-0’ I

0 ’

OH 81

The ability of the glycosyl esters of nucleoside pyrophosphates to undergo such degradation is strongly dependent upon the stereochemistry and conformation of the glycosyl group. Furanosyl derivatives split in the manner described only if they have a cis-disposition of the substituents at C-1” and C-2”. This reaction may be applied for determining the anomeric configuration in these derivative^.^^^^'^^ In pyranosyl derivatives, the steric requirements for formation of the cyclic phosphate include an equatorial-equatorial or equatorialaxial arrangement of the corresponding substituents. These conditions are fulfilled in a-D-glucopyranosyl derivatives having the normal (C1)conformation336(82), and in other glycosyl esters having an analogous spatial disposition of the C-1” and C-2” s ~ b s t i t u e n t s . ~ ~ ~ The same is true for /3-D-glucopyranosyl derivatives (83), and they undergo cleavage in alkali, as shown for the adenosine 5’-pyrophosphate esters.z2 (335)D. Brooks and J. Baddiley, Biochem. I., 115, 307 (1969).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

359

oQ I 83

82

Among D-mannopyranosyl derivatives, cyclization should be possible for p anomers and prohibited for the a anomers, as the latter have both substituents in axial orientation in the normal ( C 1 ) conformation, as shown in 84. Accordingly, stability toward alkali is a characteristic of the a-D-mannopyranosyl esters of nucleoside 5'pyroph~sphates.~~~~~~~~~ Esters of p-D-mannopyranosyl pyrophosphates have not yet been prepared, and consequently their behavior in alkaline solution is unknown. However, thymidine 5'-(p-~-rhamnopyranosylpyrophosphate), in which the steric disposition of groups should be similar in the normal (1C) conformation of the glycosyl group (85), is cleaved readily under alkaline condition^.'^^

oo I 84

OH

00 I 85

Glycosyl esters of nucleoside 5'-pyrophosphates that have a hydrogen atomZ3Oor an acetamido groupg instead of a hydroxyl group at C - 2 are stable in alkali. Facile cleavage under alkaline conditions has also been observed for ribitollgZ and glycerolzm esters of cytidine 5'-pyrophosphate. Cytidine 5'-(3,6-dideoxy-a-~-a~abino-hexopyranc>syl pyrophosphate) (336) The normal, or C ~ ( D )conformation , of the glycosyl group was deduced (see Ref. 337) from the splittings in the nuclear magnetic resonance spectra of a - D - g ~ u ~ o p y r a n o s ycr-D-gala~topyranosy1,~~~ ~~'~~~~~ 2-acetamido-Z-deoxy-a-~g l u c o p y r a n o ~ y l , 2and ~ ~ ~a-D-glucopyranosyluronic ~~~ esters of uridine 5'pyrophosphate and guanosine 5'-(a-D-mannopyranosylpyr~phosphate)."'.~~' (337) K. Onodera and S. Hirano, Biochem. Biophys. Res. Commun., 25, 239 (1966).

360

N. K. KOCHETKOV AND V. N. SHIBAEV

has been reportedBoto undergo degradation to produce free monosaccharide upon brief treatment with 10 mM aqueous alkali. This unexpected reaction has not been observed with other, similar esters of 3,6-dideoxyhexopyranoses.A free monosaccharide is one of the products formed, in about 25% yield, when cytidine 5’-(6-deoxya-D-xylo-hexopyranosyl-4-dose pyrophosphate) is subjected to alkaline treatment,gs and two unidentified products are also produced. It remains unclear whether or not the rate of cleavage of the glycosy1 bond in “sugar nucleotides” depends upon the structure of the nucleotide residue, and the same uncertainty is true for other reactions that affect the glycosyl group; no systematic kinetic studies have been reported. However, it may be noted that there appears to be no essential difference between the rates of acidic hydrolysis of the 5’-(cr-D-ghcopyranosy1pyrophosphates) of uridine and N3rnethyl~ridine.~~~ On the other hand, the presence of a glycosyl group attached to the nucleoside pyrophosphate has been found to influence the reactivity of the heterocyclic base. Thus, the catalytic hydrogenation of uridine and 6-azauridine 5’-(a-D-glucopyranosyl pyrophosphates) to the 5,g-dihydro derivatives proceeds more slowly than that of the respective nucleoside 5’-phosphates or 5’-pyropho~phates.~~*~~~~ Such differences have not been observed in comparisons of analogous derivatives of 2’-deoxyuridine, N3-methyluridine, and cytidine. A similar effect occurs in the reaction of some pyrimidine nucleoside derivatives with hydroxylamine. Studies of the mechanism of this reaction with uridine derivatives340shows that the initial point for nucleophilic attack is at C-6, and the resultant 5,6-dihydro-6-(hydroxyamino)uridine derivative (86) is an intermediate in the conversion of the uridine derivative into the ribosylurea derivative 87 and 2-isoxazolin-5-one (88),as shown in Scheme 8. Uridine 5’-phosphate or 5’-pyrophosphate and 2’-deoxyuridine 5’-phosphate react with hydroxylamine approximately 1.5 times faster than the related 5’-(a-D-glucopyranosyl pyrophosphates), whereas the velocities are the same for reactions of the corresponding

(338) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and G. I. Eliseeva, Dokl. Akad. Nauk S S S R , 159,605 (1964). (339) E. I. Budowsky, T. N. Druzhinina, G . I. Eliseeva, N. D . Gabrieljan, N. K. Kochetkov, M. A. Novikova, V. N. Shibaev, and G. L. Zhdanov, Biochim. Biophys. Acta, 122, 213 (1966). (340) E. I. Budowsky, V. D. Domkin, and N. K. Kochetkov, Dokl. Akad. Nauk S S S R , 190,99 (1970).

CLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

ROCH

-

CY I HO

HOHN L

NH OH 2 ROC&

0

I OH

,A

361

O

0 0

I

1

OH

HO 86

yHz ,C=O

k@go

+

H

I

1

HO

OH 88

87

Scheme 8

derivatives of N3-methyluridine and i ~ o c y t i d i n e . ~ ~ Thus, @ . ~ ~a ~decrease in the reactivity of the heterocyclic base appears to depend specifically on the nature of the nucleoside residue, and similar specificity is observed for two different reactions involving attack at the double bond of the pyrimidine nucleus. The dependence of the effect upon the structure of the glycosyl group seems to be unspecific. The rate of the reaction with hydroxylamine was found to be diminished in the cy~lohexy1,3~~ p-Dg l u c ~ p y r a n o s yand l ~ ~ 2-deoxy-a-~-arabino-hexopyranosyl~~~ ~ esters of uridine 5’-pyrophosphate, as compared with that for uridine 5’-phosphate or 5’-pyrophosphate. The first two derivatives also show a decrease in their rates of h y d r o g e n a t i ~ n . ~ ~ ~ The observed difference between some nucleoside 5’-pyrophosphates and their glycosyl esters in the reactivity of the base has been interpreted339 as indirect evidence for intramolecular interaction (341) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and C. I. Eliseeva, Dokl. Akod. Nauk S S S R , 172,603 (1967). (342) N. K. Kochetkov, E. I. Budowsky, V. N. Shibaev, and G. I. Eliseeva, in preparation.

(343) V. Farkai, P. Biely, and

s. Bauer, Biochim. Biophys. Acta, 165, 63 (1968).

362

N. K. KOCHETKOV AND V. N. SHIBAEV

between the heterocyclic base and the glycosyl group in “sugar nucleotides” (compare also, Section VI; p. 398). It was suggested that the presence of the -N3H group in the base structure is an essential requirement for such interaction, and removal of the hydroxyl group for C-2’ weakens the forces of interaction, leading to the disappearance of the effect in the hydrogenation reaction, but not in the reaction with hydroxylamine. Such an interpretation is favored by the finding that the decrease in the rate of reaction with hydroxylamine in the case of uridine 5‘-(a-D-glucopyranosyl pyrophosphate) depends upon a change of the entropy of and also by the results of a study of this reaction in the presence of methyl a - D - g l ~ c o p y r a n o s i d e ? ~ Under ~.~~~ the latter conditions, the reactivity of the D-glucosyl ester changes only slightly, whereas that of uridine 5‘-phosphate decreases considerably, with the result that the difference practically disappears. The detection of conformational transitions344in solutions of “sugar nucleotides” is also in accord with this explanation. V. ENZYMIC REACTIONSOF GLYCOSYLESTERSOF NUCLEOSIDE PYROPHOSPHATES

“Sugar nucleotides” can participate in many enzymic reactions, and these may be divided into three separate groups. The first one includes transformations of the glycosyl group, conversions that are very diverse from the chemical viewpoint. These reactions result in transformation of the “primary” glycosyl esters of nucleoside pyrophosphates, namely, those formed from naturally occurring glycosyl phosphates, into other derivatives of similar structure, which may be termed “secondary sugar nucleotides.” The second group of reactions includes those resulting in splitting of the pyrophosphate linkage in glycosyl esters of nucleoside pyrophosphates. The pyrophosphorylase reactions responsible for the biosynthesis of “primary sugar nucleotides” belong to this group. Another important reaction is transfer of the glycosyl phosphate residue, a process that takes place in the biosynthesis of some complex, carbohydrate-containing polymers. Finally, the reactions of the third group involve splitting of the glycosyl linkage in the glycosyl pyrophosphate esters and transfer of the glycosyl group to an acceptor. These reactions are essential for the biosynthesis of glycosides, polysaccharides, and polymers of more-complex structure. To avoid overlap with the article by Nikaido and Hassid,2 the (344) S. Hirano, Biochem. Biophys. Res. Comrnun.,43, 1219 (1971).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

363

emphasis in this Chapter is mainly on the enzymic reactions of the first group, although a brief discussion of some of the other reactions will also be presented.

1. Transformations of the Glycosyl Group a. Conversion of Glycosyl Pyrophosphate Esters into Glycosyluronic Acid Pyrophosphate Esters. - Several naturally occurring glycosyl pyrophosphate esters are capable of participating in enzymecatalyzed, irreversible oxidation, as shown in Scheme 9. Two moles of CH,OH I

CONH,

+ I R'

H,O

Y

NAD@ Nicotinamide adenine dinucleotide

89

NADH Nicotinamide adenine dinucleotide, reduced form

90

Y =

HO

HO a

OH

OH

series, R1 = OH, R2 = H, Rs = uridine 5'-pyrophosphoryl

b series, R1 = NHAc, Ra = H, Rs = uridine 5'-pyrophosphoryl c

series, R1 = H, R2= OH, Rs = guanosine 5'-pyrophosphoryl Scheme 9

364

N. K. KOCHETKOV AND V. N. SHIBAEV

“nicotinamide adenine dinucleotide” (NAD @)per mole of the sugar nucleotide are reduced during the reaction. Three dehydrogenases of this type34s have been partially purified, and they catalyze the oxidation of uridine 5’-(a-D-glucopyranosyl pyrophosphate) (89a), uridine 5’-(2-acetamido-2-deoxy-cw-D-glucopyranosyl pyrophosphate) (89b), and guanosine 5’-(a-~-mannopyranosylpyrophosphate) ( 8 9 ~ ) . The first of these enzymes has been studied the most thoroughly. Its activity has been detected in many sources, and purified preparations have been obtained from calf and beef rat tiss u e ~ : hen ~~~ oviduct,3s0 pea seedlings,3s1Cryptococcus l a u ~ e n t i i , ~ ~ ~ and Aerobacter ~ e r o g e n e s .Extensive ~~~ purification of the liver enzyme was achieved8“ The stereochemistry of hydrogen transfer has been studied with the liver enzyme by using the labeled NAD@ derivative354(91). The reduced pyridine nucleotide was shown to have the structure 92, thus indicating that both hydride ions that are transferred from 89a

Y

Y 91

92

attack on the same (“B”) side of the pyridine nucleus. This conclusion has been confirmed by experiments with uridine 5’-(a-Dglucopyranosyl-6-t p y r o p h o ~ p h a t e ) Quantitative .~~~~ transfer of the (345) These enzymes are systematically named “nucleoside 5’-(a-D-glycopyranosyl pyrophosphate): NAD oxidoreductases” (E.C. 1.1.1 group). It should be noted that only a few enzymes are known, except for the ones just mentioned, that catalyze oxidation of a primary carbon atom to a carboxylic acid group. (346) J. L. Strominger, E. S. Maxwell, J. Axelrod, and H. M. Kalckar,J. Biol. Chem., 224, 79 (1957). (347) F. J. Ballard and I. T. Oliver, Biochim. Biophys. Acta, 71,578 (1963). (348) D. Wilson, Anal. Biochem., 10,472 (1965). (349) J. Zalitis and D. S. Feingold, Arch. Biochem. Biophys., 132,457 (1969). (349a) R. J. Molz and I. Danishefsky, Biochim. Biophys. Acta, 250, 6 (1971). (350) A. Bdolah and D. S. Feingold, Biochim. Biophys. Acta, 159, 176 (1968). (351) J. L.Strominger and L. W. Mapson, Biochem. I.,66, 567 (1957). (352) H. Ankel, E. Ankel, and D. S . Feingold, Biochemistry, 5, 1864 (1966). (353) A. Bdolah and D. S. Feingold,J. Bacteriol., 96, 1144 (1968). (354) G. Krakow, J. Ludowieg, J. H. Mather, W. H. Normore, L. Tosi, S. Udaka, and B. Vennesland, Biochemistry, 2, 1009 (1963).

365

GLYCOSYL ESTERS O F NUCLEOSIDE PYROPHOSPHATES

C-6 hydrogen atoms to NAD@has been contrary to the earlier claim that a considerable proportion of the label is exchanged with the medium.213 The intermediate formation of an aldehyde derivative (93) has been suspected for many years, but early attempts to trap it were u n s ~ c c e s s f u l ?Only ~ ~ the a-D-glucopyranosyl and a-D-glucopyranosyluronic acid derivatives were detected when the dehydrogenation reaction was conducted in the presence of hydroxylamine or thiosemicarbazide, although these reagents were found to inactivate the enzyme, An intermediate of unusual structure was claimed356 to accumulate in the reaction mixture after reduction of 1 mole of NADQ per mole, but this result could not be confirmed by other investigator~.2~~,~~‘ Later, Nelsestuen and K i r k ~ o o dwere ~ ~ ~able to prepare the aldehyde derivative (93), and they showed that it is a

OH 898

OH

OH 93

9oa

probable intermediate as, in the presence of the dehydrogenase, it may be oxidized with NAD@or reduced with NADH. However, it seems that, under the usual conditions of the reaction, it is not present in appreciable concentration; possibly, it is involved only as a component of a complex with the enzyme. of the Studies of product i n h i b i t i ~ n ~ and ~ ’ , ~initial ~~ dehydrogenase reaction are consistent with a “hexa uni ping-pong” which requires that an irreversible step of the process should occur in the reaction sequence prior to addition of the second molecule of NAD@to the enzyme. The irreversible step is, most probably, formation of the uronic acid (90a) from the aldehyde 93, and several mechanisms may be written to explain the kinetics ob(355)E. S. Maxwell, H. M. Kalckar, and J. L. Strominger, Arch. Biochem. Biophys., 65, 2 (1956). (356)P. C. Simonart, W. L. Salo, and S. Kirkwood, Biochem. Biophys. Res. Commun., 24, 120 (1966). (357)J. Zalitis and D. S . Feingold, Biochem. Biophys. Res. Commun., 31,693 (1968). (358)E. F. Neufeld and C. W. Hall, Biochem. Biophys. Res. Commun., 19,456 (1965). (359)V. N. Shibaev and T. N. Druzhinina, Molek. Biologiya, 6,471 (1972).

N. K. KOCHETKOV AND

366

V. N. SHIBAEV

served. One of these, shown in Fig. 1, postulates that the a-D-glucopyranosyl ester (89a) interacts with an enzyme-NAP complex containing the pyridine nucleotide molecule that had been added prior to liberation of the D-glucuronic acid derivative (90a). NADH

89s

-

NAD@ 90s

*

<

NADH

- E 9 0 a ,

(p

NADH FIG. 1.-Possible Mechanism for the Uridine 5’-(a-D-Glucopyranosyl Pyrophosphate) Dehydrogenase Reaction. [The dashed line shows the reaction in the presence of the aldehyde 93.1 N\,Q

High specificity towards the structure of the glycosyl residue of the substrate is characteristic of uridine 5’-(cu-D-glUCOpyranOSyl pyrophosphate) dehydrogenase. Inversion of configuration at C-2”, or substitution of an acetamido group for the hydroxyl group at this position in the substrate molecule, produces inactive derivat i v e ~ . ~ ~On* ~the~ other * ~ ~ hand, ’ the 2-amino-2-deoxy-a-~-glucopyranosyl ester may participate in the reaction.334The corresponding derivatives of a - ~g a- l a c t o p y r a n o ~ eand ~ ~4-deoxy-a-~-xylo-hex~~~~~ ~ p y r a n o s e ~were @ ~ found not to be substrates, but were competitive inhibitors of the reaction with respect t 0 ~ 89a, ~ ~the1 inhibition ~ ~ ~ constants for the liver enzyme being respectively 7 and 25 times the K,,,(Michaelis constant) for 89a. The ester of uridine 5’-pyrophosphate with a-D-gulucto-hexodialdose(55) participates in the direct reaction with NAD@,but not in the reverse reaction with NADH.265 Strong inhibition of the reaction was observed, with enzymes of different origin, in the presence of uridine 5’-(a-D-xylopyranosy1 (360) G. Salitis and I. T. Oliver, Biochim. Biophys. Acta, 81, 55 (1964). (361) N. K. Kochetkov, V. N. Shibaev, T. N. Druzhinina, Yu. Yu. Kusov, and M. M.

Mazhul, in preparation.

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

367

pyrophosphate).349a*350,352,353*358 In all examples investigated, except , ~ ~inhibition ~ was found for the enzyme from Aerobacter a e ~ o g e n e sthe to be non-linear, and was interpreted as being an allosteric effect.358 esters The P-~-arabinopyranosyl~~~ and 6-deoxy-a-~-glucopyranosyl~~~ are similar, although much less efficient, inhibitors. Several reports have dealt with the specificity of the liver enzyme towards the structure of the nucleoside residue in the “sugar nucleotide.” It has been found that substitution at C-5 and C-6 of the base residue does not prevent the compound from participating in the reaction; the a-D-glucopyranosyl pyrophosphate esters of 5 , 6 - d i h y d r o ~ r i d i n e , ~6-azauridine,339,362a363 ~~~~~~ 5-methyl~ridine:~~ 5 - h y d r o ~ y u r i d i n e ,5~-~f~l u~o~r~o~~ r i d i n e , ~ 5-br0mouridine,3~~ ~~ and 5-iod0uridine~~~ are all substrates for uridine 5’-(a-~-glucopyranosyl pyrophosphate) dehydrogenase. Analysis of the pH-dependence of the kinetic r a t e - c o n s t a n t ~led ~ ~to ~ the conclusion that the un-ionized form of the CONHCO group of the uracil base is essential for binding the “sugar nucleotide” to the enzyme. In accordance with this conclusion, the substrate properties are lost in the N3-methyluridine,339~363 i~ocytidine,3~~ and ~ y t i d i n e ~derivatives, ~ ~ , ~ ~ whereas ~ , ~ ~ ~the pseudouridine derivative, which possesses the CONHCO group, can participate in the reaction.228However, some changes in this grouping are possible: 2-thiouridine 5’-(a-D-glucopyranosyl pyrophosphate) and, to a lesser extent, 4-thiouridine 5’-(a-D-glucopyranosylpyrophosphate) can act as substrates for the d e h y d r ~ g e n a s e . ~ ~ ~ The substitution of a 2-deoxy-P-~-erythro-pentofuranosylgroup for the /3-D-ribofuranosyl group in the nucleoside residue is permissible; thus, the 5’-(a-D-glucopyranosyl pyrophosphates) of 2‘-deoxyuridine239,384 and t h ~ m i d i n e ~are ~ ’ .substrates ~~~ for the enzyme, although their apparent KM values are about 15-fold higher than those for the uridine and 5-methyluridine derivatives, and the maximum velocities are diminished. The data available on the significance of the functional groups in the substrate 89a, in relation to its ability to interact with the liver dehydrogenase, are summarized in the following formula. Enclosed (362) N. D. Goldberg, J. L. Dahl, and R. E. Parks, J . B i d . Chem., 238, 3109 (1963). (363) N. D. Gabrieljan and A. V. Venkina, Dokl. Akad. Nauk S S S R , 156, 1379 (1964). (364) N. D. Gabrieljan, T. N. Druzhinina, G . I. Eliseeva, E. B. Lapina, K. S. Lebedeva, and V. N. Shibaev, Biokhimiya, 34,235 (1969). (365) N. K. Kochetkov, V. N. Shibaev, G. I. Eliseeva, T. N. Druzhinina, and M. M. Mazhul, in preparation.

(366) Zalitis and F e i n g ~ l d ~were ~ ’ able to observe a trace of activity with the ~ ’ - ( ( u - D glucopyranosyl pyrophosphates) of cytidine, guanosine, and adenosine in reaction with the liver dehydrogenase. The products have not been identified.

368

N. K. KOCHETKOV A N D

V. N. SHIBAEV

....#

0

0

. "...... ra . nu.

within dashed lines are groups that may not be changed without loss of substrate properties, probably as a consequence of a marked decrease in binding to the enzyme, or an essential increase of nonproductive binding. It is possible to substitute chemically-simiIar groups for groups enclosed within dotted lines (for example, -OH+ NH2,=O + =S), but changes that are more radical may result in loss of substrate properties. The shaded areas indicate the sites at which groups unrelated to those replaced may be introduced without dramatic loss of binding ability; these sites seem to be of the least significance for enzyme-substrate interaction. The correct anomeric configuration of the a-D-glucopyranosyl pyrophosphate ester is essential (the p-D-glucopyranosyl ester does not but the exact distance between the nucleoside moiety and the glycosyl group seems to be a less critical factor. Thus, the phosphonate analog 67 can serve as a substrate.210 A uridine 5'-(2-acetamido-2-deoxy-a-~-glucopyranosy1 pyrophosphate) dehydrogenase has been obtained in partially purified form from extracts of a strain of Achromobacter georgi~politanurn.~~~ The product 90b was found to be a competitive inhibitor of the reaction when the concentration of the substrate (89b) was varied; such inhibition should occur if the kinetic mechanism of the reaction is similar to that of the dehydrogenase of 89a. Substitution of 89a for 89b, or of NAD@ phosphate ( N A D P ) for N A P , is possible, but results in a 10-fold decrease of the reaction rate. A preparation of guanosine 5'-(a-D-mannopyranosyl pyrophosphate) dehydrogenase was obtained from cells of Arthrobacter v ~ s c o s u s . ~ ~ , ~ ~ ~ The 2'-deoxyguanosine derivative participates in the reaction, and D.-F. Fan, C. E. John, J.

Zalitis, and D. S. Feingold, Arch. Biochern. Biophys., 135, 49 (1969). (368) J. Preiss, Methods Enzymol., 8, 285 (1966).

(367)

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

369

its apparent K M value is 5-fold higher than that for the guanosine derivative. Removal of the amino group from C-2 of the heterocyclic base results in a loss of substrate properties: inosine s’-(a-D-mannopyranosyl pyrophosphate) is inactive. Also inactive as substrates are the a-D-mannopyranosyl pyrophosphate esters of adenosine, cytidine, and thymidine, and the a-D-glucopyranosyl and a-D-galactopyranosyl esters of different nucleoside 5’-pyrophosphates. “Sugar nucleotide” dehydrogenases specific for adenosine or thymidine pyrophosphate esters are not known. Extracts of sugar beet have been reported360to catalyze the interaction between thymidine 5’-(a-D-glucopyranosyl pyrophosphate) and NADP leading to formation of thymidine 5’-(a-D-galactosyluronicacid pyrophosphate), but attempts to purify individual enzymes from these extracts have been unsuccessful.

b. Inversion of Hydroxyl Groups in the Glycosyl Group.-(i) Epimerization at C - 4 . Reversible transitions between glycosyl esters O L-arubino conhaving the D-ghco and D - g d a c t o , or the D - X ~ ~ and figurations, are known for a series of uridine and thymidine derivatives. The examples that have been reported are shown in Scheme 10. R’

R’

RZ

94

95

series, R’ = CH,OH, R2 = OH, Rs = uridine 5’-pyrophosphoryl series, R1 = C&OH, R2 = OH, Rs = thymidine 5’-pyrophosphoryl series, R1 = H, R2 = OH, R3 = uridine 5‘-pyrophosphoryl series, R1 = C02H, R2 = OH, Rs = uridine 5’-pyrophosphoryl series, R1 = C&OH, R2 = NHAc, Rs = uridine B’-pyrophosphoryl series, R’ = CH,OH, Rz = NHAc, R3 = thymidine 5’-pyrophosphoryl Scheme 10

(369) R. Katan and G . Avigad, Biochem. Biophys. Res. Commun., 24, 18 (1966).

370

N. K. KOCHETKOV AND V. N. SHIBAEV

Conversion of the a-D-glucopyranosyl derivative (94a) into the a-D-galactopyranosyl ester (95a) was demonstrated370in 1951 as the first example of an enzymic reaction of a "sugar nucleotide." The enzyme that catalyzes this reaction, namely, uridine 5'-(a-D-ghcopyranosyl pyrophosphate) 4"-epimera~e,~~l is common in Nature. Purified preparations have been obtained from yeast,372,373 Escherichia coZi,374-376 mung-bean wheat germ?'" and animal tissues .244,379,380

It seems certain that intermediate oxidation of uridine 5'-(a-Dglucopyranosyl pyrophosphate) with NAD@ occurs during the reaction, and that the resulting hexosyl-4-ulose derivative is then reduced stereospecifically. The epimerases from animal tissues and wheat germ require addition of NAD@for the reactions, whereas the enzymes from other sources already contain the pyridine nucleotide firmly bound. Ultraviolet-spectral change characteristic of conversion of approximately 20% of the enzyme-bound N A W into NADH occurs when uridine 5'-(a-D-glucopyranosyl pyrophosphate) is added to a solution of Escherichia coli e p i m e r a ~ eand , ~ ~complete ~ reduction of by prolonged inthe pyridine nucleotide has been cubation with an excess of the "sugar nucleotide." Treatment of E . coli epimerase or yeast epimerase with sodium borohydride also leads to of the enzyme-bound NAD@,as well as incubation of these enzymes with uridine 5'-phosphate and L-arabinose or related mon~saccharides.~*~-~"~ The reduced epimerases

(370) L. F. Leloir, Arch. Biochem. Biophys., 33, 186 (1951). (371) This is a systematic name for the enzyme (E.C. 5.1.3.2.).Other enzymes discussed in this subsection belong to the same group. (372) E. S. Maxwell and H. de Robichon-Szulmajster,J. Biol. Chem., 235, 308 (1960). (373) R. A. Darrow and R. Rodstrem, Biochemistry, 7 , 1645 (1968). (374) D. B. Wilson and D. S. Hogness,J. Biol. Chem., 239,2469 (1964). (375) Y. Imae, N. Morikawa, and K. Kurahashi, J. Biochem. (Tokyo), 56, 138 (1964). (376) D. B. Wilson and D. S. Hogness,J. Biol. Chem., 244, 2132 (1968). (377) T. N. Druzhinina, M. A. Novikova, and V. N. Shihaev, Biokhimiya, 34,108 (1969). (378) D.-F. Fan and D. S. Feingold, Plant Physiol., 44, 599 (1969). (379) C. M. Tsai, M. Holmberg, and K. E. Ebner, Arch. Biochem. Biophys., 136, 233 (1970). (380) P. Rodriguez and 0. Bello, Arch. Biochem. Biophys., 143, 579 (1971). (380a) Y. Seyama and H. M. Kalckar, Biochemistry, 11, 40 (1972). (381) G. L. Nelsestuen and S . Kirkwood, Fed. Proc., 29, 337 (1970). (382) C. R. Creveling, A. Bhaduri, A. Christensen, and H. M. Kalckar, Biochem. Biophys. Res. Commun., 21, 635 (1965); A. U. Bertland, B. Bugge, and H. M. Kalckar, Arch. Biochem. Biophys., 116, 280 (1965). (383) A. U. Bertland and H. M. Kalckar, Proc. Nat. Acad. Sci. U. S . , 61, 629 (1968).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

371

lose their enzymic activity,297a,381*384 but activity may be restored by spontaneous oxidation in air or by treatment with the hexosulose derivative (compare the following discussion). As shown in Fig. 2, two mechanisms involving an intermediate oxidation may be written for the epimerization at C-4. In the first one (A), the oxidation results in an a-D-xybhexopyranosyl-4-dose derivative (96), which is then attacked by a hydride ion from the opposite side of the carbonyl group; a change in conformation of the enzyme-intermediate complex seems necessary for such a process. The mechanism depicted under ( B ) postulates oxidation at C-Y, and the resulting hexopyranosyl-3-ulose derivatives (54 and 97) then achieve equilibrium through the common enediol intermediate (98) before undergoing reduction at C-3”. Compound 98 may also be formed from the hexopyranosyl-4-ulose ester 96, and in such a manner, both of the pathways may be linked. Isotopic studies have shown that there is no incorporation of radioactivity into the products from tritiated NAD@,NADH, or ~ a t e r . ~ ~ ~ , Moreover, during epimerization of the D-glucose-4-t derivative, the label was found to be retained completely at C - 4 of the thus indicating that the same hydrogen atom that is removed from the substrate is added back to the intermediate when the product is formed, without exchange with the medium. This transfer of hydrogen is strictly i n t r a m o l e c ~ l a r , 2as ~ ~indicated ~ . ~ ~ ~ by an experiment on the simultaneous epimerization of the labeled and non-labeled substrates. When E . coli epimerase that had been reduced with sodium borohydride-t was incubated with thymidine 5’-(6-deoxy-a-~-xylohexopyranosyl-4- ulose pyrophosphate), this “sugar nucleotide” (which probably behaves as an analog of the 4-ulose derivative 96) is converted into a mixture of the thymidine 5’-pyrophosphate esters of 6-deoxy-~-glucose-tand D - f i ~ 0 S e - t . An ~ ~ ~analogous result has been obtained with the uridine derivative.297aMore-direct evidence in favor of mechanism A has been presented by Maitra and Anke1,387a who showed incorporation of tritium at C - 4 of the reaction products

~~

~

~~

(384) H. M. Kalckar, A. U. Bertland, and B. Bugge, Proc. Nut. Acad. Sci. U . S., 65, 1113 (1970). (385) A. U. Bertland, Y. Seyama, and H. M. Kalckar, Biochemistry, 10, 1545 (1971). (386) R. D. Bevill, F. Smith, and S. Kirkwood, Biochem. Biophys. Res. Commun., 12, 152 (1963). (387) L. Glaser and L. Ward, Biochim. Biophys. Acta, 198, 613 (1970). (387a) U. S. Maitra and H. Ankel, Proc. Nut. Acad. Sci. U . S., 68,2660 (1971).

N. K. KOCHETKOV AND V. N. SHIBAEV

372 FH,OH

YH,OH

YH,OH

I

I

I

OH

OH

94a

OH 95a

96

tl HOQOR

-

SIrZ??

Ho

OH

OH

" O D O R 0

OH

98

54

97

,'

94a

I

t

I

E,"4DH 96 95a

NAD' 94a

*

54 I,

-

NAD@

FIG. 2.-Possible Mechanisms for the Uridine 5'-(u-D-Clucopyranosyl Pyrophosphate) 4'-Epimerase Reaction. [E and E' denote different conformers of the enzyme.]

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

373

obtained after incubation of the E . coli epimerase with uridine 5’-(a-D-galactopyranosylpyrophosphate) and treatment of the reaction mixture with sodium borohydride-t. The isotope effect in the epimerization of uridine 5’-(a-D-glucopyranosyl-4-t pyrophosphate) is consistent with transfer of H - 4 in a rate-limiting step.388A large isotope-effect for the D-glucose-3-d derivative and none for the 4-d and 5-d derivatives have been reported389in the reduction of E . coli epimerase with uridine 5’-phosphate and D-glucose. This result suggests transfer of H-3 of the monosaccharide in the reaction, and it was claimed that mechanism B is operative in the epimerase reaction.389However, more-recent studies show a significant mechanistic difference between the epimerization of a “sugar nucleotide” and the reduction of the enzyme-bound NAD@ 3 ~particular, ~~~~~~~ with uridine 5’-phosphate and a r n o n o s a ~ c h a r i d e . In an aldonic acid was shown to be the reaction product under the latter conditions .389a The uridine 5’-(a-D-glucopyranosyl pyrophosphate) 4-epimerase shows high specificity with respect to the structure of the glycosyl group of the substrate. Inversion of the hydroxyl group at C - 2 , or its replacement by an acetamido group, are not permissible for the yeast enzyme.390The effect of replacing the C-2” hydroxyl group by hydrogen has not been investigated with isolated enzymes, but and 2-deoxyinterconversions of 2-deoxy-~-urubino-hexopyranose D-lyxo-hexopyranose in yeasPo and higher plants249,391 suggest that the epimerases do interact with the 2-deoxyhexosyl esters of uridine 5’-pyrophosphate. The hydroxyl group in proper orientation at C - 3 seems absolutely essential for substrate properties. Neither a-Dallopyranosyl nor 3-0-methyl-a-D-glucopyranosyl esters can participate in the reaction with yeast e p i m e r a ~ eThe . ~ ~ same ~ is true for derivative (99) with respect to the 3-deoxy-a-~-ribo-hexopyranosyl the epimerases from liver and from mung beans.392This compound was found to be a competitive inhibitor, having a K i value higher

(388) G. L. Nelsestuen and S. Kirkwood, Biochim. Biophys. Acta, 220,633 (1970). The value previously reported for yeast epimerase [R. D. Bevill, E. A. Hill, F. Smith, and S. Kirkwood, Can. J. Chem., 43, 1577 (1965)l was found to be erroneous. (389) L. Davis and L. Glaser, Biochem. Biophys. Res. Commun., 43, 1429 (1971). (389a) Y. Seyama and H. M. Kalckar, Biochemistry, 11, 36 (1972). (390) W. L. Salo, J. H. Nordin, D . R. Peterson, R. D. BevilI, and S. Kirkwood, Biochim. Biophys. Acta, 151, 484 (1968). (391) J. Kocourek, M. Tichii, V. JirAEek, and J. KoBtii?, Biochim. Biophys. Acta, 71, 497 (1963). (392) T. N. Druzhinina, M. A. Novikova, and V. N. Shibaev, Biokhimiya, 34,518 (1969).

374

N. K. KOCHETKOV AND V. N. SHIBAEV

than the apparent KM value of the D-glycosyl ester, by a factor of 40 with the liver enzyme, and 10 with the mung-bean enzyme.392 The contribution of the hydroxyl group at C - 4 in substrate binding to the enzyme is not very large, as the K ivalue for the 4-deoxy-a-~xylo-hexopyranosyl derivative (100) is only twice that ofthe apparent KM value of uridine 5’-(a-D-galactopyranosylpyrophosphate) with mung-bean enzyme.392 CH,OH

HOO

O

R

O

O

OH 99

R OH

100

R = uridine 5‘-pyrophosphoryl

The significance of a substituent at C-5” was found to differ according to the source of the epimerase. A preparation of yeast enzyme catalyzes epimerization of a-D-XylOpyranOSyl, p-L-arabinopyranosyl, and a-D-fucopyranosyl esters of uridine 5’-pyropho~phate,3~~ but the data obtained suggested participation, in this reaction, of an additional enzyme that is present in admixture. The purified wheat-germ enzyme does not interact with the a-Dxylopyranosyl derivati~e:’~ but the enzyme from E , coli epimerizes the latter esteP3 and also that of a-~-galacto-hexodialdo-1,5-pyranose.~~~ Uridine 5’-(6-deoxy-a-~-glucopyranosyl pyrophosphate) is not a substrate for the liver epimerase, but interacts, albeit slowly, with the mung-bean e n ~ y r n e . 3It~was ~ found to be a competitive inhibitor, inhibition constants being close to that of the ester 99. It seems, therefore, that the hydroxyl group at C - 6 is essential for interaction with the liver enzyme. The effect, on substrate properties, of structural changes in the nucleoside residue, as studied with uridine 5’-(a-~-glucopyranosyl pyrophosphate) 4-epimerases from liveP3g.3s4*394 and mung bean,364*377 is qualitatively similar to that just discussed for uridine s’-(a-D-glucopyranosyl pyrophosphate) dehydrogenase. The enzymes tolerate various substitutions at C-6 and C-5 (such as those resulting in derivatives of 5,6-dihydrouridine, 6-azauridine, and 5-methyluridine) (393) H. Ankel and U. S . Maitra, Biochem. Biophys. Res. Commun., 32, 526 (1968). (394) T. N. Druzhinina, M. A. Novikova, and G . L. Zhdanov, Dokl. Akad. Nauk SSSR, 164, 1175 (1965).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

375

or substitution of sulfur for oxygen at C-2. Substrate properties are lost upon methylation at N-3 or upon transition from uridine derivatives to those of cytidine and isocytidine. The a-D-glucopyranosyl pyrophosphate esters of cytidine, adenosine,395 guanosine, and inosine cannot participate in the reaction with the liver e p i m e r a ~ e . ~ ~ ~ On the contrary, the first two derivatives are epimerized by the E . coli enzyme.297a The hydroxyl group at C-2’ of the nucleoside seems not to be significant for interaction of uridine 5’-(a-D-glUCOpyranOSylpyrophosphate) with liver and mung-bean epimerases, as 2 ‘ - d e o ~ y u r i d i n e ~ ~ ” ” ~ ~ ~ and t h ~ m i d i n derivatives e~~~ are efficient substrates for these enzymes, . ~ ~epimerase ~ although they are not substrates for yeast e p i m e r a ~ eThe from E . coli reacts with the thymidine pyrophosphate derivative.297a Uridine 5‘-(~-D-g~ucopyranosyl p y r o p h o ~ p h a t e )and ~ ~ the phosphonate derivative31067 do not undergo epimerization with the yeast enzyme. Little is known at present concerning other epimerases that may participate in the reactions shown in Scheme 10 (see p. 369). Thymidine 5’-(a-D-glucopyranosyl pyrophosphate) 4-epimerase activity was detected in b a ~ t e r i a l ~and ~ ~ plant240,241.378 -~~~ extracts, but, in most instances, it is difficult to decide whether this activity is connected with the presence of an enzyme specific for the thymidine derivative (94b) or whether it results from some unspecificity of the epimerase for the uridine pyrophosphate ester (94a). A case seems to be proved for a specific epimerase in extracts of Pasteurella and wheat germ,378whereas, in a mung-bean pseudotuberculo~is~~~ epimerase preparation, a single enzyme seems to interact with both of the sugar n u c l e o t i d e ~ Uridine .~~ 5’-(a-D-xylopyranosylpyrophosphate) (94c) 4-epimerase activity has been found in plant and in a preparation of the epimerase for 94, from yeast.390The enzyme has been purified from wheat germ,40° and

(395) One preparation of the liver epimerase was shown to interact with adenosine 5’(a-D-ghcopyranosyl pyrophosphate) [T. N. Druzhinina and N. D. Gabrieljan, Dokl. Akad. Nauk SSSR, 178,603 (1968)l.This reaction seems to be a property of this particular preparation, and was not found in the others (T. N. Druzhinina, personal communication).

(396) R. Tinelli, A. M. Michelson, and J. L. Strominger,]. Bacteriol., 86, 243 (1963). (397) J. H. Pazur, K. Kleppe, and A. Cepure, Biochem. Biophys. Res. Commun., 7, 157 (1962). (398) W. L. Adair, R. W. Gaugler, and 0. Gabriel, Fed. Proc., 30, 1117 (1971). (399) E. F. Neufeld, D. S. Feingold, and W. Z. Hassid,]. Biol. Chem., 235,906 (1960). (400) D.-F. Fan and D. S. Feingold, Plant Physiol., 46,592 (1970).

376

N. K. KOCHETKOV AND V. N. SHIBAEV

it seems specific for 94c; it does not interact with 94a or 94d. The 4-epimerization of the a-D-glucopyranosyluronic acid derivative 94d has been demonstrated in Type I pneumococcus401 and in p l a n t ~ . 4M ~ ~i c-r~o -~~~r g a n i s m s ~ and ~ ~ -animal ~’ t i s s ~ e s ~contain ~ ~ , ~ ~ ~ - ~ uridine 5’-(2-acetamido-2-deoxy-a-~-glucopyranosyl pyrophosphate) (94e) 4-epimerase activity, and activity for the thymidine derivative 94f occurs in extracts of Pseudomonas aeruginosa.l10 No mechanistic studies on these epimerases have been reported. Some of them require NAD@for activity, but the question remains open as to whether those enzymes lacking this requirement contain the pyridine nucleotide firmly bound. (ii) Epimerization at C - 2 . An enzyme that catalyzed a change of configuration of the glycosyl group, from a-D-ribo to a-D-arabino, in cytidine 5’-(3,6-dideoxyhexopyranosylpyrophosphates) was obtained in partially purified formso from Pasteurella pseudotuberculosis type IV. This epimerase, which requires NAD@,is highly specific with respect to the glycosyl group of the “sugar nucleotide”: introduction of hydroxyl groups at C-3” and C-6”, or inversion of the hydroxyl group at (2-4, are not permissible. Interconversions of thymidine 5’- (a-D - mannopyranosyl pyrophosphate) and 5’-(a-D-glucopyranosylpyrophosphate) have been reported to be catalyzed by extracts of Streptomyces g r i s e u ~ . ’ ~This ~ * ’interest~~ ing reaction requires further investigation. Inversion of the acetamido group at C - 2 , with simultaneous hydrolysis of the glycosyl linkage, occurs when uridine 5’-(2-acetamido2-deoxy-a-~-g~ucopyranosyl pyrophosphate) (34) is treated with a rat-liver e n ~ y r n e . ~The ’ ~ . products ~~~ are uridine 5’-pyrophosphate (6) and 2-acetamido-2-deoxy-~-mannose(101), and they are also formed from uridine 5’-(2-acetamido-2-deoxy-a-~-mannopyranosyl pyrophos(401)E. E. B. Smith, G. T. Mills, H. P. Bernheimer, and R. Austrian, Biochim. Biophys. Acta, 29, 640 (1958);E. E. B. Smith, G. T. Mills, R. Austrian, and H. P. BernheimerJ. Gen. Microbiol., 22,265 (1960). (402)D. S. Feingold, E. F. Neufeld, and W. Z. Hassid,]. Biol. Chem., 235,910 (1960). (403)E.F.Neufeld, Methods Enzymol., 8, 276 (1966). (404)H.Ankel and R. G . Fisher, Biochim. Biophys. Acta, 178,415(1969). (405)L.Glaser,J. B i d . Chem., 234, 1801 (1959). (406)C. E. Cardini and L. F. Leloir, J. Biol. Chem., 225, 317 (1957). (407)S. M. Gompertz and W. M. Watkins, Biochem. J.. 88, 6P (1963). (408)F. Maley and G. F. Maley, Biochim. Biophys. Acta, 31,577 (1959). (409)B. Jacobson and E. A. Davidson, Biochim. Blophys. Acta, 73, 145 (1963). (410)R. L. Perlman, A. Tesler, and A. Dorfman, J. B i d . Chem., 239, 3623 (1964). (411)D. G. Comb and S. Roseman, Biochim. Biophys. Acta, 29,653 (1958). (412)L. Glaser, Biochim. Biophys. Acta, 41,534 (1960).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

377

phate) (102)with the same enzyme413without the intermediate appearance of 34. The reverse is also true: the ester 102 is not an intermediate in the reaction with its epimer 34.

0 II

0 II

NHAC 102

34

101

6

R = uridyl-5’

Two mechanisms have been suggested for this reaction. One of them4I3 postulates the irreversible glycosylation of the enzyme by 34, leading to an intermediate 103, which is then converted into the enzyme-bound product 104 (as shown in Scheme 11)through oxida$!H,OH

Q FH,OH

Po\

OEnz

HO

H 0o-H i H A c

NHAC

103

104

Scheme 11

(413)W. L.Salo and H. G . Fletcher, Jr., Biochemistry, 9,882 (1970).

378

N. K. KOCHETKOV AND V. N. SHIBAEV

tion at C - 3 by NAD@(compare Fig. 2B, p. 372, for a similar mechanism in related epimerizations at C-4”).The glycosyl-enzyme intermediate 104, which may also be formed by glycosylation of the enzyme by 102, is then hydrolyzed to give 2-acetamido-2-deoxy-D-mannose (101). According to the second mechanism,414 formation of the intermediate 2-acetamidoglycal (105) from the “sugar nucleotide” is an irreversible step of the process. This derivative then undergoes further stereospecific hydration, leading to the product 101.

,

NHAc

105

Both mechanisms seem to be consistent with incorporation of a label from tritiated water into 101 (at C-2),4I2but not into the unreacted glycosyl pyrophosphate esters,413 and also with kinetic evidence414for liberation of the pyrophosphate 6 prior to release of 101. No requirement for added NAD@has been reported, and a search for pyridine nucleotide firmly bound to the enzyme may lead to conclusive evidence for the mechanism.

(iii) Epimerization at C - 5 . Incubation of uridine 5’-(a-D-ghCOpyranosyluronic acid p y r o p h o ~ p h a t e )with ~ ~ a rabbit-skin enzyme preparation and NAD@, followed by acidic hydrolysis, leads to L-iduronic a ~ i d . This ~ ~ observation ~ * ~ ~ ~ suggests the formation of the corresponding pyrophosphoric ester (106), although the latter

-

HoQoR

HOQOR

bIi

OH 32

106

R = uridine I’-pyrophosphoryl

(414) D. B. Ellis and K. M. Sommar, Fed. Proc., 30, 1117 (1971). (415) R. Jacobson and E. A. Davidson,J. Biol. Chem., 237,638 (1962).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

379

has not as yet been isolated and characterized. A 4”-ulose derivative seems to be a probable intermediate in this reaction. No incorporation of radioactivity from tritiated water into the reaction product was observed, and the a-D-glucopyranosyl derivative failed to react under similar conditions. (iv) Simultaneous Epimerizations at C - 3 and C - 5 . Extracts from the albumen gland of Helix pomatia have been found to catalyze the conversion of guanosine 5’-(a-~-mannopyranosylpyrophosphate) into guanosine 5’-(P-L-galactopyranosyl pyr~phosphate);~’~ the reaction requires NAD@. c. Conversion of Hexosyl Pyrophosphate Esters into 6-Deoxyhexosyl-4-ulose Pyrophosphate Esters. - Scheme 12 shows an irreversible conversion that is known for a series of “primary” hexose nucleotides. It consists of reduction at C-6and simultaneous oxidation at C - 4 , and the process results in the loss of a water molecule.417

I

I

R ‘

R’ I 07 a

series, R’

b series, c

=

108

OH, Rz = H, Rs = thymidine 5’-pyrophosphoryl

R’ = OH, R* = H, R3 = cytidine 5’-pyrophosphoryl

series, R’ = H, Rz = OH, RS = guanosine 5’-pyrophosphoryl

d series, R’ = OH, R2 = H, RB= uridine 5’-pyrophosphoryl

Scheme 12

The reaction of thymidine 5’-(a-~-glucopyranosylpyrophosphate) (107a) has been studied the most thoroughly; the enzyme catalyzing it has been detected in extracts of many bacteria (compare the following sub-section). Partially p ~ r i f i e d , 6and, ~ , ~ ~later, ~ highly329*419 purified, (416) E. M. Goudsmit and E. F. Neufeld, Biochem. Biophys. Res. Commun., 26, 730 (1967). (417) The trivial name “oxidoreductase” is often used for enzymes that catalyze this reaction, but the systematic Enzyme Nomenclature includes them in the hydrolyase group (E.C.4.2.1) and not in the oxidoreductase one. (418) J. M. Gilbert, M. Matsuhashi, and J. L. Strominger,J. B i d . Chem., 240, 1305

(1965). (419) H. Zarkowsky and L. Glaser,]. BioZ. Chem., 244,4750 (1969).

380

N. K. KOCHETKOV AND V. N. SHIBAEV

enzyme preparations have been obtained from Escherichia coli B. This enzyme contains one mole of NAD@per mole of protein. The pyridine nucleotide can be released by the action of p-chloromercuribenzoate, and the enzyme activity is lost by this treatment. The reverse process occurs418when the inactivated enzyme is incubated with cysteine and NAD@,and this procedure has been used to bind radioactive NAD@to the enzyme.330A requirement for NAD@has been demonstrated for various similar enzymes from other source^.^^^^^^ The evidence available is consistent with mechanism A depicted in Fig. 3. Oxidation of the a-D-glucopyranosyl pyrophosphate derivative (107a) at C-4” by NAD@is followed by irreversible elimination of water from the p-hydroxy ketone 109, with subsequent reduction of the conjugated ketone (110) by NADH. Only the NAD@ complexes can release the “sugar nucleotide,” and, consequently, the intermediates 109 and 110 are not present in free form, uncomplexed with enzyme-NADH. The formation of enzyme-bound NADH during the reaction is supported329by the appearance of characteristic fluorescence when the purified enzyme is treated with 107a. Under these conditions, 4-5% of the bound NAD@is converted into NADH, as concluded from experiment^^^^*^^^ with the enzyme complexed with radioactive NAD@. Treatment of the reaction mixture with sodium borohydride-t followed by acidic hydrolysis led to major fractions of 6-deoxyD-glucose-t and D-fuCOSe-t, together with a minor fraction of D-galact o ~ e - 4 - tThis . ~ ~ fact ~ indicates that 109 is formed as an enzyme-bound intermediate, and allows the exclusion of a possible, alternative mechanism, shown in Fig. 3 as B, for the conversion of 107a into 108a through an intermediate glycos-5-ene derivative 111. The ease with which the hexosyl-4-ulose derivative 109 undergoes dehydration was demonstrated in a non-enzymic, model reaction. Oxidation of methyl p-D-galactopyranoside with oxygen and a platinum catalyst, followed by hydrogenation over the same catalyst, resulted in a 35% yield of methyl /3-D-fucopyrano~ide,4~~ presumably formed through reactions analogous to those in Fig. 3. During the conversion of thymidine 5’-(a-glucopyranosyl-4-tpyrophosphate)424or the 4 - d the label was found to be trans(420) L. Glaser and S. Kornfeld,J . Biol. Chem., 236, 1795 (1961). (421) H. Zarkowsky, E. Lipkin, and L. Glaser,J. Biol. Chem., 245, 6599 (1970). (422) J. Lehmann and E. Pfeiffer, FEBS Lett., 7,314 (1970). (423) 0. Gabriel, Carbohyd. Res., 6, 111 (1968). (424) 0. Gabriel and K. C. Lundquist, J . Biol. Chem., 243, 1479 (1968). (425) A. Melo, W. H. Elliott, and L. Glaser, J . Biol. Chem., 243, 1467 (1968).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

381

FIG.3. -Mechanism for the Conversion of Thymidine 5'-(ol-~-Glucopyranosyl Pyrophosphate) (107a) into Thymidine 5'-(6-Deoxy-a-~-ry~o-hexopyranosyl-4-ulose Pyrophosphate) (108a).

382

N. K. KOCHETKOV AND V. N. SHIBAEV

ferred quantitatively to C - 6 in 108a, the process being strictly intrarnole~ular.4~~ In accordance with the mechanism just discussed, during the reaction, the label at C - 5 of 107a is and incorporated425from D,O at C - 5 of 108a. In the reaction with the 3 - t derivative of 107a, the product retains almost all of the label at C-3, but a small fraction was found to be transferred to C-6" in an unexpected way.427A considerable isotope-effect has been observed with the 5 - t ~ i t i a t e dand ~ ~4-deuterated4,I ~ derivatives of 107a. The stationary concentration of enzyme-bound NADH increased to 20% in the latter example, suggesting that the dehydration or reduction steps are rate-limiting. The enzyme is highly specific towards the structure of the substrate; in particular, it can not use the uridine pyrophosphate ester as a substrate. Thymidine 5'-(6-deoxy-cx-~-glucopyranosyl pyrophosphate) was f ~ ~ nto dundergo ~ ~ slow ~ . conversion ~ ~ ~ into 108a, and in this case, the reaction rate is limited by the slow liberation of product from the EY,$,tHcomplex. As a result, the 6-deoxy-cu-~-glucopyranosyl ester strongly inhibits329the conversion of 107a. This inhibition is not observed with the analogous D-fucopyranosyl derivative. Enzymes that catalyze the dehydration of cytidine 5'-(a-D-glucopyranosyl pyrophosphate) (107b), as shown in Scheme 12 (p. 379), have been partially purified from Salmonella typhP8 and Pasteurella p s e u d o t u b e r ~ u l o s i sTheir . ~ ~ ~mechanism ~~ of action is probably similar to that observed with the thymidine derivative. In fact, the requirement58*65,66 for NAD@and the quantitative transfePO of the label from C - 4 in 107b to C - 6 in 108b have been demonstrated. The enzyme from P . pseudotuberculosis does not tolerate inversion of the hydroxyl groups at C - 2 or C-4",nor the substitution of other heterocyclic bases for the cytosine residue, but substitution of the C-2' hydroxyl group by hydrogen is possible without loss of substrate properties.66 A purified preparation of the enzyme specific for guanosine 5 ' 4 ~ ~ mannopyranosyl pyrophosphate) (107c) was obtained from the J-5 mutant strain of Escherichia coli.'O' The enzyme was found to contain firmly bound NAD@. The analogous conversion of the uridine derivative 107d has been observed in extracts of tobacco leaves'36 and Chlorella cells,137although the enzyme has not yet been purified. The enzymic reactions described here are frequently used for preparation of the 6-deoxyhexosyl-Culose esters of nucleoside pyrophosphates that are inacces(426) K. Hermann and J. Lehmann, Eur. I. Biochem., 3,369 (1968). (427) 0. Gabriel and G. Ashwell,]. Biol. Chem., 240, 4128 (1965). (428) S.-F. Wang and 0. Gabriel,]. Biol. Chem., 245, 8 (1970).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

383

sible by other methods, and it is common practice to employ crude enzyme preparations for this purpose.

d. Conversion of 6-Deoxyhexosyl-4-ulosePyrophosphateEsters into 6-Deoxyhexosyl and 3,6-Dideoxyhexosyl Pyrophosphate Esters. -The rather complicated reactions of this type involve stereospecific reduction at C - 4 that is accompanied in many instances by epimerization at C - 3 and C-5, or by substitution of hydrogen for the hydroxyl group at C - 3 , or both. Several enzymes are likely to participate in these processes, but they have not been adequately characterized yet.

(i) Reduction at C-4. The only reaction of this type known is the conversion of guanosine 5'-(6-deoxy-a-~-lyro-hexopyranosyl-4-ulose pyrophosphate) into the 6-deoxy-a-~-mannopyranosyl ester by an enzyme preparation from Leucaena g l a u c ~ or , ~into ~ a mixture with the 6-deoxy-a-~-talopyranosylester by an enzyme from non-identified Gram-negative b a ~ t e r i a . ~The ~ , ~reductants ~ , ~ ~ ~ ~may be NADH or NADPH, the latter being the more efficient. (ii) Reduction at C - 4 with Epimerizations at (2-3'' and C-5. The conversion of thymidine 5'-(a-D-ghcopyranosyl pyrophosphate) into - hexopyranosyl-4-ulose pyrophosthymidine 5' - (6-deoxy-a-~-xylo phate), discussed in the previous Sub-section, is the first step in the biosynthesis of thymidine 5'4 P-L-rhamnopyranosyl pyrophosphate) (27). The complete process has been observed in extracts of Streptococcus faecalis,226Pseudomonas aeruginosa,420Escherichia coli,'07*110*396 Salmonella strains,lo7 and Streptomyces griseus.lo4The second step involves reaction of the 6-deoxyhexosyl-4-ulose ester 28 with reduced NADP as shown.

o

o

o

R

+NADPH

+

HQ

HO

OH 28

OH

+

NADp

27 R = thymidine Bf-pyrophosphoryl

The reaction seems to be i r r e v e r ~ i b l e , 4and ~ ~ ,two ~ ~ ~different protein fractions are required for the process.430One of them catalyzes the (428a) M. W. Winkler and A. Markovitz, J. Biol. Chem., 246,5868 (1971). (429) L. Glaser, Biochim. Biophys. Acta, 51, 169 (1961). (430) A. Melo and L. Glaser, J. Biol. Chem., 243, 1475 (1968).

384

N. K. KOCHETKOV AND V. N. SHIBAEV

incorporation of label from D,O at C - 3 and C-5”of the substrate, probably through keto-enol equilibration. Labeling at C - 3 proceeds faster than at C - 5 , but attempts to demonstrate intermediate products have so far proved unsuccessful. Hydrogen from labeled pyridine nucleotide is transferred to C-4” of the A strong isotopeeffect was observed431when the substrate was tritiated at C-3”, and this derivative undergoes practically no conversion under the usual conditions. A similar reaction with uridine derivatives was demonstrated with plant extract^,^^^,^^^*^^^ although it has been studied to a lesser degree. Enzyme preparations that can convert a-D-mannopyranosyl or 6-deoxy-a-~-~yxo-hexopyranosyl-4-u~ose esters of guanosine 5’-pyrophosphate into the p-L-fucopyranosyl ester were obtained from Aerobacter a e r o g e n e ~ Escherichia , ~ ~ ~ ~ ~ ~c ~ l i , Salmonella ’~~ strains,433 higher plants,s2 and animal tissues.434Further purification of these enzymes will be necessary for mechanistic studies.

(iii) Formation of 3,6-Dideoxyhexosyl Derivatives. The conversion pyrophosphate) of cytidine 5’-(6-deoxy-c~-~-gluccpyranosyl-4-ulose (15) into 3,6-dideoxyhexopyranosyl esters involves at least three different protein^.^^,^^^ Two of them are required for the following reaction.

Although the reaction mechanism remains unclear, it seems not to be related to the one suggested for conversion of ribonucleotides into 2’-deo~yribonucleotides.~~~ Further reduction of 16 is catalyzed by the third protein fraction. (431) 0. Gabriel, J . B i d . Chem., 241, 924 (1966). (432) V. Ginsburg,J. Biol. Chem., 236, 2196 (1960). (433) R. H. Kornfeld and V. Ginsburg, Biochim. Biophys. Acta, 117, 79 (1966). (434) D. W. Foster and V. Ginsburg, Biochim. Biophys. Acta, 54, 376 (1961). (435) H. Pape and J. L. Strominger,J. Biol. Chem., 244, 3598 (1969).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

385

The origin of this fraction seems to determine the stereochemistry at C-4" and C - 5 in the reaction product (see Section 11,3, p. 316). Similar 3,6-dideoxyhexosyl-4-ulose derivatives probably intervene in the conversion of guanosine 5'-(6-deoxy-a-~-lyxo-hexopyranosyl4-ulose pyrophosphate) into the colitose ester, a reaction is catalyzed by an enzyme from Escherichia c0liaS7 e. Formation of Aminodeoxyhexosyl Pyrophosphate Esters. -The pyrointeraction of thymidine 5'-(6-deoxy-a-D-g~ucopyranosyl-4-u~ose phosphate) with L-glutamic acid in the presence of pyridoxal phosphate and bacterial enzymes gives rise to 4-amino-4-deoxyhexopyranosyl derivatives."' The stereochemistry of the product depends upon the enzyme source. A preparation from Escherichia coli B produced the D-ghco isomer (112),whereas formation of the D-galacto isomer (113) was observed with enzymes from E . coli Y-10 and

OH

+

'

O

O

R

C0,H I CHNH, I

(y%)z

-

CO,H I

co

~

I

(

C0,H

p

112

+

2

CO,H

OH H2NQoR OH

R = thymidine 5'-pyrophosphoryl

113

many other bacterial strains.l13 The equilibrium constant113is about 0.5. Partial purification has been reported for the enzyme preparations from E . coli B113and Pasteurella p s e u d o t u b e r ~ u l o s i s . ~ ~ ~

f. Decarboxylation of Glycosyluronic Acid Pyrophosphate Esters. The only known substrate for enzymic decarboxylation is uridine 5'-(a-D-glucopyranosyluronicacid pyrophosphate). A simple reaction of this type, leading to the a-D-xylopyranosyl ester, has been ob(436) H. Ohashi, M. Matsuhashi, and S. Matsuhashi,J. Biol. Chem., 246, 2325 (1971).

N. K. KOCHETKOV AND V. N. SHIBAEV

386

served in extracts of higher plants,402 green algae,437and animal t i s s ~ e s ; ~partially ~*,~~~ purified enzyme-preparations have been obtained from Cyptococcus laurentii and wheat germ.441The reaction with the yeast enzyme requires NAD@and is strongly inhibited by the addition of NADH.440When the reaction is conducted in tritiated water, the label is incorporated and the a-D-xylopyranosyl-5-t derivative is A similar, but not identical, product was obtained212after enzymic decarboxylation of the substrate labeled at C-5. The difference between these products is connected with the stereochemistry at C-5, as the radioactive glycolic acids isolated after degradation of the a-D-xylopyranosyl-5-t esters by successive treatment with snake-venom pyrophosphatase, sodium periodate, and sodium hypoiodite are unlike. That obtained from the radioactive substrate loses the label completely when treated with glycolic acid dehydrogenase, but no loss of label occurs with the other product. This result indicates that inversion of configuration at C - 5 occurs during the decarboxylation step. An isotope effect was observed in the reaction with the a-D-glucopyranosyluronic acid derivative labeled at C-4”, but not with the C-3”- and C-5-tritiated substrates.212 The mechanism suggested243includes successive, intermediate formation of the 4”-ulose ester 114, the carbanion 115, and the 4”-ulose 116, all of which are firmly bound to the enzyme-NADH complexes. The sequence for the 5-labeled substrate is given in Scheme 13. COnH

OH HO

OR I

OH

OH

OH

114

115

OH 116

R = uridine 5’-pyrophosphoryl Scheme 13

387

GLYCOSYL ESTERS O F NUCLEOSIDE PYROPHOSPHATES

A more-complicated reaction has been observed with enzyme preparations from p a r ~ l e and ~ ~Lemna ~ * ~minor.442-444 ~ ~ ~ The D-apiosyl ester (117) was found to appear, together with the a-D-XylOpyranOSyl ester, after decarboxylation of uridine 5’-(a-D-g~ucopyranosy~uronic acid pyrophosphate). The formation of 117 was suggested as occurring through rearrangement of the intermediate a-L-threo-pentopyranosyl4-ulose ester (116a), followed by reduction of the aldehyde445118.

o

-

qOR OH

QOR-QoR HO

116a

OH

HO

118

OH 117

R = uridine 5’-pyrophosphoryl

Transfer of hydrogen from C-4’’ of uridine 5‘-(a-D-glucopyranosyluronic acid pyrophosphate) to C-3’ of the apiosyl group in the ester 117 has been demonstrated.445aThe conversion of 116a into 118 was hypothesized446to involve aldol cleavage, isomerization of the resulting a-hydroxy aldehyde, and intramolecular, aldol reaction as shown. 116

-

O=C

hR

r0 \

HOH.$

CHO

HO
118

CHO

(437) H. Ankel, E. Ankel, D. S. Feingold, and J. S. Schutzbach, Biochim. Biophys.Acta, 136, 172 (1967). (438) A. Bdolah and D. S. Feingold, Biochem. Biophys. Res. Commun., 21,543 (1965). (439) J. E. Silbert and S. DeLuca, Biochim. Biophys. Acta, 141, 193 (1967). (440) H. Ankel and D. S. Feingold, Biochemistry, 5, 182 (1966). (441) H. Ankel and D. S. Feingold, Biochemistry, 4, 2468 (1965). (442) H. Sandemann, Jr.. G. T. Tisue, and H. Grisebach, Biochim. Biophys. Acta, 165, 550 (1968). (442a) E. Wellmann, D. Baron, and H. Grisebach, Biochim. Biophys. Acta, 244, 1 (1971). (443) H. Sandennann, Jr., and H. Grisebach, Biochim. Biophys. Acta, 208, 173 (1970). (444) D. L. Gustine and P. K. Kinde1,J. Biol. Chem. 244, 1382 (1969). (445) H. Grisebach and U. Dobereiner, Biochem. Biophys. Res. Commun., 17, 737 (1964). (445a) W. J. Kelleher and H. Grisebach, Europ. J. Biochem., 23, 136 (1971). (446) J. M. Picken and J. Mendicino,]. Biol. Chem., 242, 1629 (1967).

388

N. K. KOCHETKOV AND V. N. SHIBAEV

The ratio of a-D-xylopyranosyl to D-apiosyl derivatives in the reaction products with the enzyme from L. minor seems to remain constant after efficient purification of the enzyme447or under treatment with a variety of inhibitors, but the ratio is affected by the ammonium-ion c ~ n c e n t r a t i o nThis . ~ ~ ~suggests that the same enzyme, or closely related ones, participate in both of the reactions. A similar observation has been reported for the parsley enzyme.447a

g. Other Conversions.-The enzyme-catalyzed N-acetylation of 2-amino-2-deoxy-a-D-g~ucopyranosy1116 or 2-amino-2-deoxy-a-~-galactopyranosy1116 esters of thymidine 5’-pyrophosphate and their 4”-substituted counterparts”’ has been observed in the presence of acetylcoenzyme A and bacterial enzyme-preparations. From hen oviduct, enzymes have been isolated and purified that catalyze sulfation of glycosyl esters of nucleoside Spyrophosphates by the action of 3‘-0-phosphono-5’-adenylyl hydrogen s ~ l f a t e . ~ ~ * . ~ ~ ~ Purification of some enzymes participating in the biosynthesis of glycopeptide cell-wall precursors has also been achieved. Examples include preparation of the enzymes necessary for synthesis of the enol ether 38 (see Section 11,6, p. 328),152.153*450 reduction of the enol ether to the muramic acid and the addition to the latter of amino a ~ i d s or ~ the ~ ~dipeptide - ~ ~ ~D-alanylD-alanine.1s0’457’458 2. Splitting of the Pyrophosphate Linkage

Several enzymic reactions that result in splitting of the pyrophosphate linkage and nucleophilic substitution at the phosphorus atom (447) E. Wellmann and H. Grisebach, Biochim. Biophys. Acta, 235, 389 (1971). (447a) D. Baron, E. Wellmann, and H. Grisebach, Biochim. Biophys. Acta, 258, 310 (1972). T. Harada, S. Shimizu, Y. Nakanishi, and S. Suzuki,]. Biol. Chem., 242, 2288 (1967). M. Tsuji, S. Shimizu, Y. Nakanishi, and S. Suzuki,]. Biol. Chern., 245, 6039 (1970). K. G.’Gunetileke and R. A. Anwar, J . Biol. Chem., 241,5740 (1966). A. Taku, K. G. Gunetileke, and R. A. Anwar,]. Biol. Chem., 245, 5012 (1970). E. It0 and J. L. Strominger, J . Biol. Chern., 237, 2689 (1962). E. It0 and J. L. Strominger,]. Biol. Chem., 239, 210 (1964). S. G. Nathenson, J. L. Strominger, and E. Ito,J. Biol. Chern., 239, 1773 (1964). E. Ito, S. G. Nathenson, D. N. Dietzler, J. S. Anderson, and J. L. Strominger, Methods Enzymol., 8, 324 (1966). Y. Mizuno and E. Ito, J . Biol. Chem., 243, 2665 (1968). D. G. Comb,]. Biol. Chem., 237, 1601 (1962). E. Ito and J. L. Strominger, J . Biol. Chem., 237,2696 (1962).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

389

are known for “sugar nucleotides.” The general process is represented by Scheme 14. It seems probable that the intermediate 119 (R’ = nucleoside, R2 = glycosyl, R3 = H or an electron-accepting group of the enzyme) intervenes, as trisubstituted pyrophosphates are known to be attacked readily by nucleophiles, and such attack results in substitution at the phosphorus atom of the phosphate monoester group (compare Section 111,2,b, p. 352).

119

Scheme 14

Reactions of this type described in the literature include participation of various nucleophiles, such as water, inorganic phosphate, phosphate monoester, or inorganic pyrophosphate. Hydrolysis of “sugar nucleotides” with unspecific pyrophosphatases has already been mentioned (Section ILl, p. 310). A similar reaction is catalyzed by a bacterial enzyme specific for adenosine 5’-(a-~-glucopyranosylp y r o p h ~ s p h a t e ) .The ~ ~ ~ specific conversion of uridine 5’-(a-D-glucopyranosyl pyrophosphate) into a-Dglucopyranosyl phosphate, uridine, and inorganic phosphate was observed with an enzyme from Escherichia coli;459,460 a preparation from Bacillus subtilis can act in a similar mannelAgl on different “sugar nucleotides.” Phosphorolysis of the glycosyl esters of sugar nucleotides leads to the corresponding nucleoside 5’-pyrophosphate and glycosyl phosphate. A wheat-germ enzyme specific for adenosine 5’-(a-D-ghcopyranosyl pyroph~sphate),~~? and a yeast enzyme specific for guanosine 5’-(a-D-mannopyranosyl p y r o p h o ~ p h a t e ) , 4are ~ ~ known. ,~~~ (459) A. Melo and L. Glaser, Biochem. Biophys. Res. Commun., 22, 524 (1966); L. Claser, A. Melo, and R. J. Paul,]. Biol. Chem., 242, 1944 (1967). (460) H. F. Dvorak, Y.Anraku, and L. A. Heppel, Biochem. Biophys. Res. Commun.,24, 628 (1966); H. C. Neu and L. A. Heppel, J . B i d . Chem.,240, 3685 (1965); 242, 3898 (1967);Biochemistry, 7, 3766 (1968). (461) J. Mauck and L. Glaser, Biochemistry, 9, 1141 (1970). (462) M. Dankert, I. R. Goncalves, and E. Recondo, Biochim. Biophys. Acta, 81, 78 (1964). (463) H. Carminatti and E. Cabib, J. B i d . Chem., 240, 2110 (1965). (464) E. Cabib, H. Carminatti, and N. M. Woyskovsky,J. Biol. Chem.,240,2114 (1965).

390

N. K. KOCHETKOV AND V. N. SHIBAEV

The exchange reaction between uridine 5’-(cu-D-glucopyranosyl pyrophosphate) and a-D-galactopyranosyl phosphate has been described in Section III,l,c (see p. 340). One of the most impressive findings has been the discovery of lipid intermediates in the biosynthesis of polysaccharides (see Refs. 2 and 465.) At least two structurally different types of these compounds exist: the intermediate may be an isoprenoid alcohol ester of the glycosyl pyrophosphate or the analogous derivative of the glycosyl phosphate. Derivatives of the first type are formed by reaction between the “sugar nucleotide” and the alcohol phosphate, for example, undecaprenyl phosphate (120), which participates in the biosynthesis of Salmonella lipopoly~accharide.~~~

Unlike the examples previously discussed, this interaction leads to splitting of the pyrophosphate linkage in the “sugar nucleotide,” with the liberation of nucleoside 5’-phosphate. Such reactions have been demonstrated for cr-D-galactopyrano~y1,4s”-~~~ 2-acetamido2-deoxy-a-D-g~u~opyranosy1,3~~~~~~ and pentapeptidyl-m~ramyl~’~~~~~ esters of uridine 5’-pyrophosphate. Pyrophosphorolysis of sugar nucleotides leading to nucleoside 5’-triphosphates and glycosyl phosphates is the reverse of the enzymic synthesis of “sugar nucleotides” (compare Section III,l, p. 335). Many purified preparations of pyrophosphorylases have been described, and high, although not absolute, specificity towards the “sugar nucleotide” structure is characteristic of most of them. For example, purified calf-liver uridine 5’-(a-D-glucopyranosylpyrophos(465) L. Rothfield and D. Romeo, BucterioZ. Rev., 35, 14 (1971). (466) A. Wright, M. Dankert, P. Fennessey, and P. W. Robbins, PTOC.Nut. Acud. Sci: U.S . , 57, 1798 (1967). (467) M. Dankert, A. Wright, W. S. Kelley, and P. W. Robbins, Arch. Biochem. Biophys., 116,425 (1966). (468) M. J. Osborn and R. Yuan Tze-Yuen, J. B i d . Chem.,243,5145 (1968). (469) F. E. Frerman, F. A. Troy, and E. C. Heath,]. B i d . Chem., 246, 118 (1971). (470) L. J. Douglas and J. Baddiley, FEBS Lett., 1, 114 (1968); H. Hussey and J. Baddiley, Biochem. J., 127, 39 (1972). (471) J. S. Anderson, M. Matsuhashi, M. A. Haskin, and J. L. Strominger,]. B i d . Chem., 242, 3180 (1967). (472) M. G . Heydanek, W. G. Struve, and F. C. Neuhaus, Biochemistry, 8,1214 (1969).

CLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

391

phate) pyropho~phorylase~’~ catalyzes the slow splitting (at a rate about 3% of that for the true substrate) of thymidine 5’-(a-D-glupyrocopyranosyl pyrophosphate), uridine 5’-(a-D-ga~actopyranosy~ phosphate), and uridine 5’-(a-D-xy~opyranosylpyrophosphate); and the very slow cleavage (at a rate of less than 1%)of cytidine 5’-(a-Dglycopyranosyl pyrophosphate). A calf-liver enzyme that catalyzes the pyrophosphorolysis of guanosine 5’-(a-D-mannopyranosyl pyrophosphate) and guanosine 5’-(a-D-glucopyranosyl pyrophosphate) shows essential specificity: the apparent KM value for the D-glucosyl ester is about 6000 times that for the D-mannosyl

3. Splitting of the Glycosyl Linkage (Glycosylation) Many enzyme-catalyzed glycosylation reactions are known, and participation in these reactions seems to be the main function in Nature of glycosyl esters of nucleoside pyrophosphates. In these derivatives, nucleophilic attack at the anomeric carbon atom should be greatly facilitated if the negative charges on oxygen atoms of the pyrophosphate group are removed. In consequence, intermediates such as 121, where R’ = nucleoside, and R2= hydrogen atoms or electrophilic enzyme groups, are probable intermediates in these reactions.

AH

0

0

ORa

OR2

121

A wide variety of nucleophilic reagents can participate in enzymic reactions and act as acceptors of the glycosyl group. a. Water. -A highly specific enzyme from yeast catalyzes the conversion of guanosine 5’-(a-D-glucopyranosyl pyrophosphate) into D-glucose and guanosine 5’-pyropho~phate.~’~

(473) G . J. Albrecht, S. T. Bass, L. L. Seifert, and R. G . HansenJ. Biol. Chem., 241, 2968 (1966). (474) S. Sonnino, H. Caminatti, and E. Cabib, J. Biol. Chem., 241, 1009 (1966);Arch. Biochem. Biophys., 116, 26 (1966).

392

N. K. KOCHETKOV AND V. N. SHIBAEV

b. Alcohols.-Numerous reactions of this type are well documented (see Ref. 2 for details). Known examples include the glycosylation of alcoholic hydroxyl groups in monosaccharides, glycosides, oligosaccharides and polysaccharides, alditols and their derivatives, lipids, terpenoids, steroids, serine or threonine residues in proteins, and the (hydroxymethy1)cytosine base in deoxyribonucleic acid. The reaction may occur either with inversion or with retention of configuration at the anomeric center. c. Hemiacetals.-The reactions known include the synthesis of sucrose and sucrose phosphate by plant enzymes, a,a-trehalose 6-phosphate with yeast, Mycobacteria, Streptomyces, and insect enzymes (compare Refs. 1 and 2), and, probably, P,P-trehalose with pneumococcal enzymes.475 d. Phenols. - Simple phenols and more-complex derivatives (such as quercitin and apigenin) have been reported to interact with uridine 5’-(a-D-ghcopyranosyl pyrophosphate) in the presence of enzyme preparations from plants or invertebrates, and with uridine 5’-(a-Dglucopyranosyluronic acid pyrophosphate) in the presence of enzymes from mammals (compare Refs. 2, 4, 5, and 476). All known examples involve inversion of configuration at C-1”. e. Carboxylic Acids. -The formation of 1-0-acyl-D-glucuronic acids has been demonstrated with a n t h r a n i l i ~ ?0-aminobenzoic,4~~ ~~ and r e t i n o i ~acids ~ ~ ~as acceptors. Several examples of the synthesis of 1-0-acyl-D-glucoses are also known.479*480

f. Thiols. - Enzymic S-glucosylation has been observed for benzenethi01,~~’ 5-mercapto~racil,~*’ and phenylacetothiohydroxamate.48z g. Amines. -Aromatic amines are converted into N-(D-glucosyluronic acid) derivatives by a liver and into N-(D-glucosyl derivatives by a soybean preparation.484 (475) E. E. B. Smith and G. T. Mills,J. Gen. Microbiol., 39, 255 (1963). (476) A. Kleinhofs, T. A. Haskins, and H. J. Gorz, Phytochemistry, 6, 1313 (1967). (477) G. J. Dutton, Biochem.J.,64,693 (1956);G. J. Dutton and I. H. Stevenson, Biochim. Biophys. Acta, 31, 568 (1959). (478) K. Lippel and J. A. Olson,J. Lipid Res., 9, 168 (1968). (479) G. Jacobelli, M. J. Tabone, and D. Tabone, Bull. S O C . Chim. Biol., 40,955 (1958); M. J. Tabone, G . Jacobelli, and L. Dluzniewicz, Ann. Inst. Pasteur, 104, 771 (1963). (480) J. J. Comer and T. Swain, Nature, 207, 634 (1965). (481) T. Gessner and M. Acara,J. Biol. Chem., 243,3142 (1968). (482) M. Matsuo and E. W. Underhill, Biochem. Biophys. Res. Commun., 36,18 (1969); Phytochemistry, 10, 2279 (1971).

GLYCOSYL ESTERS O F NUCLEOSIDE PYROPHOSPHATES

393

h. Phosphoric Monoesters. - The interaction of isoprenoid alcohol monophosphates with uridine 5’-(a-D-glucopyranosyl pyropyrophosp h o ~ p h a t e ) ~or~ guanosine ~ , ~ ~ ~ -5’-(c-w-D-mannopyranosy~ ~~~~ phate)487-489,489a may result in formation of the nucleoside 5’-pyrophosphate and an isoprenoid alcohol ester of the glycosyl phosphate. Inversion of configuration at C-1 was shown in reactions with the D-ghcosyl derivative. The glycosyl transferases (E.C. 2.4 group) usually show high specificity towards the structure of the glycosyl acceptor. Furthermore, the enzyme specificity seems to determine which of the available nucleophilic acceptor-groups participates in the reaction, and also determines the stereospecificity of the glycosylation. The inversion of configuration at the anomeric center is readily predictable on the basis the “simple displacement mechanism,” whereas retention of configuration may be the consequence of “frontal displacement,” or of “double displacement” with intermediate formation of a covalent, glycosyl-enzyme comp0und.4~~ No evidence for the latter type of mechanism has yet been obtained for glycosylation reactions involving sugar nucleotides.” In particular, kinetic studies491show that different mechanisms seem to operate in the synthesis of sucrose (122) from uridine 5’-(cY-D-glUCOpyranOSyl pyrophosphate) on the one hand, and from a-D-glucopyranosyl phosphate on the other; the latter reaction is a well known example of glycosylation by the “double-displacement mechanism.”492 “

(483) J. Axelrod, J. K. Inscoe, and G. M. Tomkins, Nature, 179, 538 (1957). (484) D. S. Frear, Phytochemistry, 7, 381 (1968). (485) N. H. Behrens and L. F. Leloir, Proc. Nut. Acud. Sci. U. S . , 66, 153 (1970). (486) A. Wright, Fed. Proc., 28,658 (1969). (486a) K. Nikaido and H. Nikaido,]. B i d . Chem.,246,3912 (1971); I. C. Hancock and J. Baddiley, Biochem. ]., 127, 27 (1972). (487) H. Kauss, FEBS Lett., 5, 81 (1969). (488) M. Scher, W. J. Lennarz, and C. C. Sweeley, Proc. Nut. Acud. Sci. U.S . , 59, 1313 (1968). (489) M. Lahav, T. H. Chiu, and W. J. Lennarz, J. Biol. Chem., 244, 5890 (1969). (489a) N. H. Behrens, A. J. Parodi, L. F. Leloir, and C . R. Krisman, Arch. Biochem. Biophys., 143, 375 (1971); J. B. Richards, P. J. Evans, and F. W. Hemming, Biochem. ]., 124, 957 (1971). (490) D. E. Koshland, in “Mechanism of Enzyme Action,” W. D. McElroy and B. Glass, eds., Johns Hopkins Press, Baltimore, Maryland, 1954, p. 608. (491) N. D. Gabrieljan, R. L. Komaleva, and V. N. Shibaev, Molek. Biologiyu, 7,337 (1973). (492) M. Doudoroff, H. A. Barker, and W. Z. Hassid,]. B i d . Chem., 168,725 (1947); H. Wolochow, E. W. Putnam, M. Doudoroff, W. Z. Hassid, and H. A. Barker, ibid., 180, 1237 (1949); R. Silverstein, J. Voet, D. Reed, and R. H. Abeles, ibid.,242, 1338 (1967); J. G. Voet and R. H. Abeles, ibid., 245,1020 (1970).

N. K. KOCHETKOV AND V. N. SHIBAEV

394

q CH,OH

iL;; CH,OH

HO

OR

OH

HO

+

"

O

C

OH p CH,OH

_t

HO I

HO 122

Recognition of the glycosyl-donor structure by the transferase enzyme also shows high specificity. Uridine 5'-pyrophosphate esters act as glycosyl donors in most glycosyl transfer-reactions known at present;2 these include transfer reactions of monosaccharides biogenetically related to D-glucose or 2-acetamido-2-deoxy-~-glucose. Thymidine derivatives may effectively replace the uridine analog in some reactions, such as in the synthesis of sucrose,493-496 D-galacturonan,281and glycogen;497 p-D-glucosides of simple p h e n o l ~ , 3 q~~*e~r c~i~t i n , 4and ~ ~ triterpene alcohols;500and N-glycosyl derivatives of aromatic a m i n e ~In . ~the ~~ first two examples, a very slow reaction was also observed with the cytidine derivative^.^^^.^^^ More-detailed studies of enzyme specificity towards the structure of the nucleoside residue have been performed with the sucrose synthetase of pea ~ e e d l i n g ~ ,and 3 ~with ~ ~wheat-germ ~ ~ ~ * ~arbutin ~ ~ ~ ~ ~ ~ synthetase.339*364-503 Neither enzyme can use the ~ y t i d i n e isocyti,~~~ dine, or N3-methyluridine derivatives as substrates. On the other hand, the a-D-glucopyranosyl pyrophosphate esters of 4-thiouri-

(493) Y. Milner and G. Avigad, Nature, 206,825 (1965). (494) W. J. Grimes, B. L. Jones, and P. Albersheim, J. Biol. Chem., 245, 138 (1970). (495) E. Slabnik, R. B. Frydman, and C. E. Cardini, Plant Physiol., 43, 1063 (1968). (496) C. E. Cardini and E. Recondo, Plant Cell Physiol. (Tokyo), 3,313 (1962). (497) R. Komfeld and D. H. Brown,]. Biol. Chem.,237, 1772 (1962). (498) R. J. Goncalves, Enzymologia, 26, 287 (1963). (499) G . A. Barber, Biochemistry, 1,463 (1962). (500) C. T. Hoi, Y. Umemura, M. Nakamura, and S. Funahashi,]. Biochem., (Tokyo), 63, 351 (1968). (501) N. D. Gabrieljan, M. A. Novikova, and G . L. Zhdanov, Dokl. Akad. Nauk S S S R , 151, 1453 (1963).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

395

dine, 2-thiouridine, S76-dihydrouridine, 6-azauridine, 5-methyluridine, and 2’-deoxyuridine may participate in glycosylation. Thus, the structural requirements for these enzymes appear similar to those of uridine 5’-(a-D-glucopyranosylpyrophosphate) dehydrogenase and 4”-epimerase. Some glycosyl transferases show an unusual “double” substrate specificity; that is, they strongly favor as substrates a pair of unrelated “sugar nucleotides,” such as adenosine and uridine derivatives.505 Adenosine 5’-(a-D-glucopyranosyl pyrophosphate) is the unique l 4)-glucan catalyzed by glycosyl donor in the synthesis of a - ~ - (+ certain soluble enzymes from bacteria506and On the other hand, plant enzymes bound to the starch granules may also use the uridine derivative quite effi~iently.~lO-~l~ Disintegration of the granules results in a change of the enzyme specificity, which becomes close to that of the soluble enzymes.513Similar enzymes from yeast, liver, and muscle favor the uridine pyrophosphate ester as a substrate, but can also use the adenosine derivative.514 Adenosine 5’-(a-D-glucopyranosyl pyrophosphate) was found to participate efficiently in the synthesis of sucrose under catalysis by various different enzymes.494,496~515*516 A study of the substrate specificity of the trehalose phosphate

(502) N. D. Gabrieljan and N. B. Kozlova, Biokhimiya, 31, 760 (1966). (503) N. D. Gabrieljan and A. V. Venkina, Dokl. Akud. Nauk SSSR, 165, 439 (1965). (504) Sucrose synthetase from pea seedlings seems to be more specific than similar enzymes horn other plants. Unlike them, it cannot accept thymidine 5‘-(a-Dglucopyranosyl pyrophosphate) as a substrate.364 (505) E. Recondo and L. F. Leloir, Biochem. Biophys. Res. Commun., 6, 85 (1961). (506) E. Greenberg and J. Preiss, J. Biol. Chem., 240, 2341 (1965); J. Preiss and E. Greenberg, Biochemistry, 4, 2328 (1965). (507) R. B. Frydman and C. E. Cardini, Biochem. Biophys. Res. Commun., 14, 353 (1964); 17,407 (1965);Biochim. Biophys. Acta, 96, 294 (1965); Arch. Biochem. Biophys., 116, 9 (1966); R. B. Frydman, B. C. DeSouza, and C. E. Cardini, Biochim. Biophys. Actu, 113, 620 (1966). (508) J. Preiss and E. Greenberg, Arch. Biochem. Biophys., 118, 702 (1967). (509) H. P. Ghosh and J. Preiss, Biochemistry, 14, 1354 (1965). (510) P. Viswanathan, Indian J. Biochem., 5, 188 (1968). (511) Y. Tanaka and T. Akazawa, Plant Cell Physiol. (Tokyo), 9, 405 (1968). (512) R. B. Frydman, Arch. Biochem. Biophys., 102, 242 (1963). (513) R. B. Frydman and C. E. Cardini,J. Biol. Chem., 242, 312 (1967). (514) S. H. Goldemberg, Biochirn. Biophys. Acta, 56, 357 (1962). (515) M. A. R. de Fekete and C. E. Cardini, Arch. Biochem. Biophys., 104, 173 (1964). (516) R. N. Shukla and G. G. Sanwal, Arch. Biochem. Biophys., 142, 303 (1971).

396

N. K. KOCHETKOV AND V. N. SHIBAEV

synthetase from M y c o b ~ c t e r i aled ~ ~to ~ an interesting finding. This enzyme preparation can use either uridine or guanosine derivatives as the glycosyl donor. Further fractionation of the proteins gave a fraction for which the guanosine ester was a much more efficient substrate than the uridine analog. This relationship was found to be reversed upon addition of another fraction that, alone, was devoid of enzymic activity. The effect seems to be connected with the presence of ribonucleic acid in the second fraction, as the effect was lost when the fraction was treated with ribonuclease, and restored when poly(uridylic acid) was added. The significance of structural variation in the adenosine residue for interaction of adenosine 5’-(a-~-glucopyranosylpyrophosphate) with enzymes has not been studied in detail. Evidently, the hydroxyl group at C-2’ may be removed, as the 2‘-deoxyadenosine derivative was found to be quite acceptable as a substrate for the synthesis of sucrose302,515 and a - ~ 1 - (+ 4)-linked g l ~ ~ aSubstrate n ~ . proper~ ~ ~ ~ ~ ~ ties for a sucrose synthetase in thepea are lost in some analogs having a modified adenine nucleus,302 such as the 5’-(a-D-glucopyranosyl pyrophosphates) of N1-methyladenosine, N6-methyladenosine, N6,N6-dimethyladenosine, inosine, and xanthosine. Adenosine 5’-(p-~-glucopyranosylpyrophosphate) has been reported to interact with soluble D-glucosyl transferases from sweet corn and from potato,507but the reaction products have not been identified. There is some information on the specificity, towards the structure of the glycosyl group, of D-glucoSyl transferases that use uridine 5’-(cY-D-glUCOpyranOSyl pyrophosphate). The hydroxyl group at C-2” was found to be non-essential for the sucrose synthetase of the pea,343 as the 2-deoxy-a-D-arabino-hexopyranosyl ester is a good substrate. This glycosyl group may be incorporated instead of D-glucose into a , a - t r e h a l o ~ e , ~ ~maltose,51s * * ~ ~ ~ other o l i g o s a ~ c h a r i d e s ,and ~ ~ ~ the glycogen of yeast.521On the other hand, the presence of the hydroxyl group at C - 3 seems to be significant. Uridine 5’-(3-deoxy-a-~-ribohexopyranosyl pyrophosphate) does not serve as a substrate for the sucrose synthetase of the pea, nor for wheat-germ arbutin synthetase,

(517) G. L. Liu, B. W. Patterson, D. Lapp, and A. D. Elbein,]. Biol. Chem., 244, 3728 (1969); D. Lapp, B. W. Patterson, and A. D. Elbein, ibid., 246, 4567 (1971). (518) V. Farkas, S . Bauer, and J. Zemek, Biochim. Biophys. Acta, 184, 77 (1969). (519) W. Fischer and G. Weidemann, Z. Physiol. Chem., 346, 171 (1966). (520) J. J. Kriegelstein and W. Fischer, Z. Physiol. Chem., 3r8, 1256 (1967). (521) P. Biely, V. FarkaS. and 5. Bauer, Biochim. Biophys. Acta, 158, 487 (1968).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

397

or liver-glycogen s y n t h e t a ~ e The . ~ ~ ~substrate analogs devoid of a hydroxyl group at C - 4 or C - 6 react with sucrose synthetase, although the former reaction is of low efficiency. Only the 6-deoxy-a-~-glucopyranosyl ester serves as an alternative substrate in the synthesis of arbutin and glycogen.522

VI. CONCLUSION The specific arrangement of monosaccharide residues is now believed to determine the biological functions of many different carbohydrate-containing polymers. The specificity of biosynthesis of these polymers is controlled by many factors, but among them the recognition of the glycosyl esters of nucleoside pyrophosphates by the enzymes seems to be of paramount importance. Such recognition is essential for all stages of the process, namely, the synthesis of primary sugar nucleotides” with pyrophosphorylases, conversion of the “primary sugar nucleotides” into the “secondary” derivatives, and the various glycosylation reactions. The diversity of the structures of naturally occurring “sugar nucleotides” and of their enzymic reactions has been described in this Chapter. The question now arises as to whether the recognition processes are unique for each enzyme-substrate pair, or whether there exist some common features between different enzymes in this respect. The latter possibility seems more attractive and, more importantly, it is supported by some experimental evidence. From studies on enzyme specificity towards the structure of uridine 5’-(a-D-ghcopyranosy~ pyrophosphate) and its analogs, as described in the previous Section, it is possible to distinguish three classes of molecular sites. (a) Universally essential sites, which are necessary for all of the enzymes studied. These include a hydroxyl group at C - 3 and an acylamido grouping (CONHCO) of the uracil base, although possibly only the N-H group between two electron-deficient carbons is of real importance. ( b ) Universally non-essential sites, in which functional groups may be removed or changed without loss of substrate properties. These include the double bond of the heterocyclic base and the hydroxyl groups at C-2’ and C - 2 . (c) Specific sites essential for some enzymes and non-essential for others; for example, hydroxyl groups at C - 4 and C - 6 . It may be “

(522) N. D. Gabrieljan, E. B. Lapina, Yu. Yu. Kusov, V. N. Shibaev, and N. K. Kochetkov, Dokl. Akad. Nauk S S S R , 199,962 (1971).

398

N. K. KOCHETKOV AND

V. N. SHIBAEV

significant that various naturally occurring glycosyl esters of uridine 5’-pyrophosphates differ in structure at these points. It appears, therefore, that the enzyme may use the recognition of (u)-type sites for differentiating uridine 5’-(glycosyl pyrophosphates) from other “sugar nucleotides,” and recognition of (c)-type sites for selecting the necessary glycosyl derivative from among other uridine 5’-pyrophosphate esters. Tests of the validity of this conclusion require further investigations of enzyme specificity by use of specially designed substrate analogs. It would be very interesting to test whether there exist, for other naturally occurring uridine 5’-(glycopyranosyl pyrophosphates), structural sites of types (a) and ( b )that are similar in function. The recognition mechanism suggested appears to require participation of a specific conformer of the “sugar nucleotide” in interaction with the enzyme. that folded conformations are characterisIt has been tic of “sugar nucleotides” in solution and are essential for their interaction with different enzymes. In contrast to this statement, other s ~ g g e s t i o n s have ~ ~ ~ ,assumed ~~~ that only in the particular case of the uridine 5’-(a-D-glucopyranosyl pyrophosphate) 4-epimerase reaction is there an interaction between the uracil and D-glucose residues. However, the first h y p o t h e s i ~ ,invoking ~~~~~~~ the existence of a fraction of the folded Conformations in solutions of “sugar nucleotides,” does explain the universality of the recognition of these derivatives by different enzymes. Intramolecular hydrogen-bonding was proposed as stabilizing such conformations, before data concerning universally non-essential sites became available. By use of molecular models, all groups common to the “sugar nucleotides” derived from the same nucleoside were tested for their ability to participate in intramolecular hydrogenbonding. The number of conformations possible was found to be limited. Among these, the conformations having maximal intramolecular hydrogen-bonding were suggested as the most probable. For example, the folded conformation of uridine 5’-(a-D-glucopyranosyl pyrophosphate), as shown in Fig. 4, is fixed by three hydrogen bonds: (i) 0-2...H-0-2’, (ii) 3-N-H-..0-3, and (iii) 0-4...H-O-2 . Present knowledge of enzyme specificity is consistent with this model

(523) N. K. Kochetkov, E. I. Budowsky, and V. N. Shibaev, Biokhimiya, 28,741 (1963). (524) H. M. Kalckar, Adoan. Enzymol., 20, 111 (1958). (525) H. de Robichon-Szulmajster, J. Mol. B i d , 3, 253 (1961).

GLYCOSYL ESTERS OF NUCLEOSIDE PYROPHOSPHATES

399 ,OH

FIG. 4. -The Folded Conformation of Uridine 5’-(a-D-Glucopyranosyl Pyrophosphate).

if it is assumed that elimination of hydrogen bond (ii) results in destabilization of the folded conformation, and loss of the hydrogen bonds (i) or (iii) affects the stability of the folded conformation to a lesser degree. Direct experimental evidence for the existence of an ordered conformation of “sugar nucleotides” in solutions has been reported by Hiran0,3~~ who observed characteristic optical-rotatory changes for a series of these compounds upon transition from water to concentrated urea solutions. The structural requirements for such an ordered conformation are still not clear. However, data at this point, based on indirect kinetic evidence from hydrogenation and hydroxylaminolysis reactions (see Section IV, p. 360), seem to accord with the hypothetical model just described. Further studies on the conformations of “sugar nucleotides” in solution are highly desirable.

This Page Intentionally Left Blank

BY SYBIL M . SNAITH

AND

GUILDFORD A . LEVVY

Rowett Reseurch Institute. Bucksburn. Aberdeen. Scotland I . Introduction., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. General Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. DistributioninNature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

401 402 402 2 . Subcellular Localization in Mammalian Tissues . . . . . . . . . . . . . . . . 405 3 . Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408 4 . Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 412 5 . pHandActivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. pHandstability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 7. Inactivation by Heat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416 8. Activation and Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416 420 9 . Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. a-D-Mannosidase and Zinc2+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422 1. The Effect of pH on the Stability of Metal Chelates . . . . . . . . . . . . . . 422 2 . The Classification of Metal-dependent Enzymes . . . . . . . . . . . . . . . . 423 3. The Effects of Zinc2+and Other Cations . . . . . . . . . . . . . . . . . . . . . 424 4 . The Effect of Chloride Ion on Limpet a-D-Mannosidase Activity . . . . . . 431 5 . a-D-Mannosidase as a Metalloenzyme . . . . . . . . . . . . . . . . . . . . . . 433 IV . Changes in Activity in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434 V . Action on Naturally Occurring Substrates . . . . . . . . . . . . . . . . . . . . . . 437 1. Ovalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437 2 . Other Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

I . INTRODUCTION There are two attributes of a-D-mannosidase (EC 3.2.1.24) that receive particular attention in this article. namely. its behavior as a zinc-containing metalloenzyme. and its action on naturally occurring. D-mannose-containing molecules . However. such more-systematic considerations as the kinetics of action and the distribution of the enzyme in Nature are not overlooked . Early work on a-D-mannosidase of plant origin. notably in almond emulsin. has been described.’ There has been a revival of interest in the enzyme from all sources. and an extensive survey of plant (1) “The Carbohydrates. ” W . Pigman. ed., Academic Press. New York. N . Y., 1957. 40 1

402

S . M. SNAITH AND G . A. LEVVY

materials was made by Levvy and McAllan2 in 1962. It has been known for some time that a-D-mannosidase can be found in moll u s c ~ . In ~ , ~1956, the unexpected observation was made that a - ~ mannosidase is ubiquitous in mammalian tissue^.^ By this time, it was becoming evident that D-mannose is a common component of glycoproteins, but the anomeric configuration of the sugar residue was still in doubt. The wide occurrence of a-D-mannosidase as compared to that of p-D-mannosidase suggested5 that, so far as animal tissues were concerned at least, most of the D-mannose in glycoproteins would prove to have the a-Dconfiguration. From subsequent work, it appears that this is to a large extent true. In addition to the glycosidase a-D-mannosidase, there is another enzyme, having overlapping specificity, that has sometimes been loosely referred to as a-mannosidase, namely, exo-a-D-mannanase. Both glycosidases and exo-glycanases remove terminal glycosyl residues, with some overlap in their action, but the former mainly act on short, and the latter on long, sugar chains. Endo-glycanases hydrolyze internal bonds in polysaccharides. The hypothesis has been p r o p o ~ n d e d ~that - ~ glycosidases can be distinguished from exo-glycanases, which in many respects they resemble, in that they act with retention of configuration of the glycosyl bond, whereas the latter cause inversion thereof. Those endo-glycanases that have been studied appear to act with retention of configuration. a-D-Mannosidase displays its maximum activity at a slightly acid pH and, like most other glycosidases, is specific for hydroxyl ion under ordinary conditions. The discovery that, in its catalytically active form, the enzyme contains zinc ion lends additional interest to its action.1° 11. GENERALPROPERTIES 1. Distribution in Nature

Since Levvy and coworkers5 first showed that a-D-mannosidase occurs in mammals, its presence has been detected in virtually every (2) G . A. Levvy and A. McAllan, Nature, 195, 387 (1962). (3) T. Nagaoka, Tohoku J . Erp. Med., 51, 131 (1949). (4) J. Conchie, G . A. Levvy, and C. A. Marsh, Biochem.J., 62, 2 4 (1956). . (5) J. Conchie, J. Findlay, and G . A. Levvy, Nature, 178, 1469 (1956). (6) F. W. Parrish and E. T. Reese, Carbohyd. Res., 3,424 (1967). (7) E. T. Reese, A. H. Maguire, and F. W. Parrish, Can. J. Biochem., 46, 25 (1968). (8) D. E. Eveleigh and A. S. Perlin, Carbohyd. Res., 10, 87 (1969). (9) J. E. G . Barnett, Biochem. J., 123, 607 (1971). (10) S . M. Snaith and G . A. Lewy, Biochem. J . , 106, 5 3 (1968). ~

(u-D-MANNOSIDASE

403

TABLEI a-D-Mannosidase Activity" of Certain Mammalian Tissues Tissue Epididymis Liver Kidney Spleen Pancreas Small intestine Prostate

Rat

150,0OOz6 6,40W6 3,800b12 1,80015 5,8OOz7 6,20W5 2,500b12

Pig

Mouse

16,000b'2 5,40015 7,30015 4,70015

-

1,100b'2 32012

15,00@13 3,00025 4,6OOz5 1,800'5 1,lOOe'~ 1,7OOz5 -

-

Rabbit

4,20025 3,00P5

-

52OZ5 4,4OOz5

-

"Results are expressed as pg of p-nitrophenol liberated per g of moist tissue from 6 mM p-nitrophenyl a-D-mannoside in 1 hr at 37" and p H 5. bAssayed at 2 mM substrate concentration and pH 5. 'Assayed at 2 mM substrate concentration and p H 4.6.

mammalian tissue or fluid that has been e ~ a m i n e d . ~ ~Levels l l - ~ ~ of a-D-mannosidase have been measured in human blood,21,22 in cow's milk,23in semen from a number of different species,24and in different parts of the alimentary tract.25Table I gives figures for the activity for a few of the richer tissues. Some of the assays were made at a substrate concentration of 2 mM, a concentration at which the enzyme displays no more than half of the activity obtained with 6 mM substrate, the concentration used in later measurements. a-D-Mannosidase activity is particularly high in the epididymis, and the level in this tissue has been shown to increase with the age of the mammal, and to be under the influence of androgens.26.28 (11)J. Conchie and G . A. Levvy, Brit. J . Cancer, 11, 487 (1957). (12) J. Conchie, J. Findlay, and G. A. Levvy, Biochem.J., 71, 318 (1959). (13) J. Conchie, J. Findlay, and G. A. Levvy, Nature, 183, 615 (1959). (14) G. A. Levvy, A. McAllan, and A. J. Hay, J. Endocrinol., 23, 19 (1961). (15) J. Conchie and A. J. Hay, Biochem. J.. 87, 354 (1963). (16) P. A. Ockerman, Lancet, 2,239 (1967). (16a) B. Hultberg, Stand.]. Clin. Lab. Znuest., 26, 155 (1970). (17) T. J. Langley and F. R. Jevons, Arch. Biochem. Biophys., 128, 312 (1968). (18) T. Okumura and I. Yamashina,]. Biochem. (Tokyo), 68, 561 (1970). (19) H. B. Bosmann and B. A. Hemsworth, Biochim. Biophys. Acta, 242, 152 (1971). (20) T. Sukeno, A. L. Tarentino, T. H. Plummer, and F. Maley, Biochemistry, 11, 1493 (1972). (21) J. M. J. Tronchet, Compt. Rend., 256, 1395 (1963). (22) J. E. Courtois and M. Mangeot, Compt. Rend., 270,2727 (1970). (23) A. Mellors and V. R. Harwalkar, Can. J. Biochem., 46, 1351 (1968). (24) J. Conchie and T. Mann, Nature, 179, 1190 (1957). (25) J. Conchie and D. C. Macdonald, Nature, 184, 1233 (1959). (26) S. M. Snaith, A. J. Hay, and G. A. Levvy,J. Endocrinol., 50,659 (1971). (27) S. M. Snaith, unpublished results. (28) J. Conchie and J. Findlay, J . Endocrinol., 18, 132 (1959).

S. M. SNAITH AND G. A. LEVVY

404

Ovariectomy caused a pronounced fall in the a-D-mannosidase activity of mouse uterus, and the normal level was restored by administration of estrone.28Although the ovary is not particularly rich in a-D-mannosidase, the greater proportion of the activity in cattle, pig, and human ovaries was found to be concentrated in the corpus l ~ t e u m . ' ~ , ~ ~ The activity of the enzyme in cancer tissue has been measured," and changes in its activity in vaginal fluid have been examined as a possible diagnostic aid in cervical cancer.29 Low levels in human tissues have been associated with a storage disorder resembling Hurler's syndrome,16,16a It has been known for a long time that a-D-mannosidase occurs in plant seeds2,30-38; Table I1 gives some activity values. It would appear that, in certain instances at least, the activity increases on geimination31,35-38;in Phaseolus vulgaris, the specific activity increased 35-f0ld.~~ Germinated seeds of Vicia sativa and Trigonella foenum graecum have been used as sources of the enzyme in studies of its proper tie^.^^*^* From Table 11, it would appear that emulsin might TABLEI1 a-D-Mannosidase Activity" of Certain Plant Seeds Material

Activity

References

Jack-bean mealb Almond emulsin' Almond emulsind French bean (Phaseolus vulgaris) Lettuce Lucerne (alfalfa) Ryegrass

750,000 60,000 3,700,000 6 1,000 40,000 25,000 8,000

39 40 27 2 2 2 2

aResults are expressed as pg of p-nitrophenol liberated per g of seed from 6 mM p-nitrophenyl a-D-mannoside in 1hr at 37" and pH 5. *Coarsely ground beans. 'Defatted sweet-almond meal. d A commercial P-D-ghcosidase preparation purified from emulsin. a-D-Mannosidase activity is given per g of purified material. (29) J. G. Lawson,J. Obstet. Gynaec. Brit. Emp., 67, 305 (1960). (30) H. Hbrissey, Compt. Rend., 172, 766 (1921). (31) K. Hill, Ber. Verhandl. Sachs. Akad. Wiss. Leipzig, Math.-Phys. Klasse, 86, 115 (1934);Chem. Abstr., 28, 5843 (1934). (32) B. Helferich and S. Winkler, Z. Physiol. Chem., 209, 269 (1932). (33) B. Helferich and A. Iloff, Z. Physiol. Chem., 221, 252 (1933). (34) Y.-T. Li,J. Biol. Chem., 241, 1010 (1966). (35) R. Goldberg, Compt. Rend., 264, 1036 (1967). (36) K. L. M. Agrawal and 0. P. Bah1,J. Biol. Chem., 243, 103 (1968). (37) S. Beaugiraud, F. Percheron, J. E. Courtois, and C. Lanchec, Bull. SOC. Chim. Biol., 50, 621 (1968).

a-D-MANNOSIDASE

405

be a useful source of a-D-mannosidase; in particular, the commercially available, partially purified, P-D-glucosidase preparation. Since 1949, when Nagaoka3 showed that a-D-mannosidase occurs in snails, it has been found in several other m011uscs,4~~~-~~ and some representative figures are given in Table 111.With marine-molluscan a-D-mannosidase, it was found necessary to have chloride ion present ~~,~ in~the case of the limpet, in order to achieve full a ~ t i v i t y , and, Patella ~ u l g a t a(see ~ ~ Section IIS; p. 412), it has been shown that zinc ion is also required. a-D-Mannosidase has also been reported insects?* and earthworm^.^^ Ba~terial~O.~~ to be present in helminth~,~’ and fungal extract^^^-^^ have been used for splitting off terminal, D-mannose residues from polysaccharides, but the enzymes responsible are probably more correctly regarded as exo-glycanases (see Section 11,9; p. 420).

2. Subcellular Localization in Mammalian Tissues The intracellular distribution of a glycosidase in a mammalian tissue can be studied by centrifugal fractionation of a homogenate made in “isotonic” (0.25 M ) sucrose solution. The “membranes” of subcellular particles are preserved in this medium and, provided that there is no leakage of the enzyme, its partition between the different sizes of particle can be measured. Before the enzyme is assayed, it may be necessary to destroy the structure of the particles, in order to make the enzyme completely accessible to the substrate.

(38) F. Petek and E. Villarroya, Bull. SOC. Chim. Biol.,50, 725 (1968). (39) S. M. Snaith and G . A. Levvy, Biochem. J., 110, 663 (1968). (40) J. Conchie, personal communication. (41) J. Conchie and G . A. Levvy, Biochem. J., 65,389 (1957). (42) J. E. Courtois, F. Petek, and T. Dong, Bull. SOC. Chim. Biol., 44, 11 (1962). (43) T. Muramatsu,Arch. Biochem. Biophys., 115, 427 (1966). (44) T. Muramatsu,J . Biochem. (Tokyo), 62, 487 (1967). (45) T. Muramatsu and F. Egami,J. Biochem. (Tokyo), 62,700 (1967). (46) S. M. Snaith, G . A. Levvy, and A. J. Hay, Biochem. J., 117, 129 (1970). (47) R. H. Nimmo-Smith and J . E. D. Keeling, E x p . Parasitol., 10, 337 (1960). (48) J. E. Courtois, C. Chararas, and M.-M. Debris, Compt. Rend., 252, 2608 (1961). (49) Y.-T. Li and M. R. Shetlar, Comp. Biochem. Physiol., 14,275 (1965). (50) G . H. Jones and C. E. Ballou, J . Biol. Chem., 244, 1043 (1969). (51) P. A. J. Gorin, J. F. T. Spencer, and D. E. Eveleigh, Carbohyd. Res., 11, 387 (1969). (52) D. J. D. Hockenhull, G. C. Ashton, K. H. Fantes, and B. K. Whitehead, Biochem. J., 57, 93 (1954). (53) J. K. Baird and W. L. Cunningham, Biochem. J., 120, 18P (1970). (54) K. L. Matta and 0. P. Bah1,J. Biol. Chem., 247, 1780 (1972).

TABLE111 a-DMannosidase Activity" in Molluscs Assay conditions

Material

Activity

References

Substrate concentration (mM)

PH

C1- concentration (mM)

ZnZ+concentration

(mM) 9

z

Patella vulgata (visceral hump)

330,000

46

6

3.5

100

0.1

38,000

45

4.6

4.0

370

0

14,000

44

4.6

4.0

370

0

Turbo cornutus (liver)

Charonia lampas (liver)

aResults are expressed as pg of p-nitrophenol liberated per g of moist tissue from p-nitrophenyl a-D-mannoside in 1 hr at 37" under the conditions stated.

U

407

a-D-MANNOSIDASE

This is commonly done by adding a nonionic detergent, Triton X-100, which does not react with the enzyme. Centrifugal fractionation of a homogenate made in a sucrose medium yields four main fractions: I, nuclei (contaminated with unbroken cells); 11, large cytoplasmic granules (mitochondria); 111, small cytoplasmic granules (microsomes); and IV, the optically clear, supernatant liquor (extragranular cytoplasm). The objective in preparing the sucrose homogenate is to achieve maximum rupture of cells, with minimum release of particulate enzyme into fraction IV. Table IV shows the distribution of a-D-mannosidase in different TABLEIV Distribution of a-D-Mannosidase Activities” in Sucrose Homogenates of Mammalian tissue^'^ Fraction Tissue Mouse liver kidney spleen

T2146 tumor Rat liver spleen

I

I1

111

Iv

Recovery (%)

1 11 9 13

37 41 20 15

47 44 32 34

14 16 44 44

112 105 106

1 10

41 27

50 50

8 0

100 87

99

_______

“Results are expressed as percentages of the activity of the whole sucrose homogenate. All assays were done in the presence of Triton X-100, with p-nitrophenyl a-n-mannoside as substrate. Fractions: I, nuclei and unbroken cells; 11, mostly mitochondria; 111, mostly microsomes; and IV, final supernatant liquor.

tissues homogenized in sucrose solution under standardized conditions. Because other glycosidases in the preparations did not give similar patterns of distribution, differences between tissues for the partition of a-D-mannosidase cannot be regarded merely as artifacts. In mouse liver and kidney and in rat liver, a-D-mannosidase activity appeared to be equally distributed between the two cytoplasmic-granule fractions. With mouse spleen and cancer tissue, a considerable proportion of the enzyme was found free in the cytoplasm. Rat spleen, on the other hand, lacked this cytoplasmic fraction. Inasmuch as the enzyme within the cytoplasmic granules was not fully active in a sucrose homogenate until the membranes had been disintegrated, a-D-mannosidase conforms to the definition of a “lysosomal” hydrolase.

408

S. M. SNAITH A N D G . A. LEVVY

The theory has been advanced that there exists in the cell a particulate structure somewhat smaller than the mitochondrion, the “lysosome,” that contains certain autolytic enzymes in a latent situation. The lysosome is very largely based upon measurements made in sucrose homogenates of rodent liver. Although the results for a-D-mannosidase in this tissue (see Table IV) are not incompatible with the theory, the results for other tissues do not always conform to it. In particular, the contrast between mouse and rat spleen argues against a universal single particle to which a-D-mannosidase is confined. Apart from the results quoted in Table IV, not much work has been done on the intracellular location of a-D-mannosidase. Although the lysosome theory has stimulated much work in cytology, certain reservations remain with regard to the biochemical observations upon which it ultimately rest^.^^,^^ 3. Assay

The chromogenic substrate p-nitrophenyl a-D-mannopyranoside is now used almost exclusively in measuring a-D-mannosidase activity. The p-nitrophenol liberated is determined colorimetrically. Unlike reducing-sugar methods of assay, this colorimetric method can be used for enzyme preparations at all stages of purity, including homogenates of the starting material. It is sensitive and highly specific, and permits immediate distinction of a-D-mannosidase from /3-Dmannosidase and other glycosidases. For the assay of a-D-mannosidase, the incubation mixture employed in our laboratory contained 0.5 ml of it4 acetate buffer at a pH appropriate for the particular enzyme preparation, 1.5 ml of 16 mM p-nitrophenyl a-D-mannopyranoside, 1.5 ml of water (which could be replaced by other additives as required), and 0.5 ml of suitably diluted, enzyme preparation. Affer 1 hour at 37”, the reaction was terminated, and the color was developed by adding 4 ml of 0.4M glycine-sodium hydroxide buffer, pH 10.5. The mixture was centrifuged for 15 minutes at 1500 g, and the color intensity of the liberated p-nitrophenol (25-150 pg) in the supernatant liquor was measured on a Spekker photoelectric absorptiometer, with use of Ilford No. 601 violet filters having maxima1 transmission at 430 nm, and a 1-cm light path. Separate control-experiments for enzyme and sub(55)C. deDuve, in “Subcellular Particles,” American Physiological Society Symposium, T. Hayashi, ed., Ronald Press, New York, N. Y., 1959,p. 128. (56)C. deDuve, Ciba Found. Symp. Lysosomes, 1 (1963). (57)J. Conchie and G . A. Levvy, Biochem. Soc. Symp., 23, 86 (1963). (58)G.A. Levvy and J. Conchie, Progr. Btophys., 14, 105 (1964).

CY-D-MANNOSIDASE

409

strate were performed. Variations of the same basic procedure have been used by several other authors. With a-D-mannosidase preparations in a high state of purity, the addition of 0.01% of bovine albumin to the assay mixture may lead to a small increase in activity (not more than lo%), probably by lessening denaturation of the enzyme. a-D-Mannosidase from marinemolluscan sources is, to a considerable extent, activated in the assay by chloride ion, and, to some extent, by certain other anions. Maximum activity is displayed by the enzyme from the limpet, €'. uulgata, when 0.1 M sodium chloride is included in the incubation mixture46 (see Section 11,5; p. 412). Chloride ion has no effect on the activity of jack-bean or rat-epididymal a-D-mannosidase. Although a-D-mannosidase from mammalian, plant, and molluscan sources is dependent upon zinc for its catalytic activity, the addition of this ion has a marked effect in the enzyme assay only at those pH values where the active, protein-metal complex dissociates appreciably despite the presence of substrate. (Dissociation, which is greater at lower values of pH, is lessened in the presence of substrate.) The presence of zinc ion in the assay (0.1 mM) is thus of particular importance in the case of the limpet enzyme, where the pH of optimal activity is 3.5. Jack-bean and rat-epididymal a - ~ mannosidase are both assayed at pH 5, and up to 10% activation may be observed with zinc. The effects of albumin, Zn2+,and C1- are different in character and, therefore, when they are evident, they are all additive in an assay. With preparations from all sources inactivated (see Section 111,3; p. 424) with (ethylenedinitri1o)tetraacetate (EDTA), addition of Zn2+ in the assay causes an immediate and complete restoration of activity. 4. Purification

For use as a carbohydrate reagent, a-D-mannosidase has to be freed from other glycosidases. For instance, wherever it occurs, this enzyme appears to be accompanied by comparable levels of 2-acetamido2-deoxy-~-~-glucosidase (EC 3.2.1.30) and p-D-galactosidase (EC 3.2.1.23), both of which could interfere with its action on glycoproteins, as well as by lower levels of many other glycosidases. During early attempts at purification, the instability of a-D-mannosidase was a major obstacle. Progress was difficult until the need for zinc was recognized.'O We have now obtained the enzyme, free from other glycosidases, from three sources, one plant, one mammalian, and one molluscan. Zinc ion must always be added at some stage in the purification, to maintain the enzyme protein in its catalytically active

410

S. M. SNAITH AND G. A. L E V "

form, or the preparation must be conducted under such conditions that sufficient zinc is retained by the enzyme. Jack-bean meal is a rich and convenient source of a-D-manno~ i d a s e and , ~ ~the enzyme displays zinc-dependence in characteristic fashion.39At pH values below neutrality, the enzyme is unstable unless a zinc salt is added, and the instability is particularly marked during dialysis or gel chromatography. In our first method of purification,39the later stages were performed at pH 5 in the presence of 100 /AMzinc sulfate. Table V gives a brief outline of the purification procedure. TABLEV Purification of a-D-Mannosidase from jack-bean Meal"

Recovery (%) 1. Acetone-dried powder 2. Aqueous extract 3. 45-60% satd. ammonium sulfate solution 4. Dialysis 5. Pyridine treatment 6. Gel chromatography

Specific activity units" per mg of proteinb

100 67

2,900 3,200

61 61 50 47

24,000 31,000 250,000 440,000

=One unit of a-D-mannosidase liberates 1 pg of p-nitrophenol from 6 mM p-nitrophenyl a-D-mannoside in 1 hr at 37" and pH 5. *Protein was determined by the method of Lowry and coworker~,5~ with bovine albumin as the standard. Zn2+was added to the preparation after Stage 3.

The jack-bean meal was converted into an acetone-dried powder, and this was extracted with water. (Preliminary treatment with acetone leads to sharper fractionation with ammonium sulfate.) After fractionation with ammonium sulfate, the enzyme was dialyzed against a buffered solution of zinc sulfate at pH 5, and then incubated in 2 M pyridine solution for 20 minutes at 37". (Treatment with pyridine destroys all of the 2-acetamido-2-deoxy-~-~-glucosidase and p-D-galactosidase present, and precipitates a great deal of inactive protein.) Finally, the preparation was passed through a column of Sephadex G-100, to give a single peak for protein that paralleled the a-D-mannosidase activity. The product, which could be stored at pH 5 in the presence of zinc ion, appeared to be homogeneous on the ultracentrifuge. No separation of the protein band was achieved by electrophoresis. (59) 0.H. Lowry, N. J. Rosebrough, A. L. Fan, and R. J. Randall,J. Biol. Chem., 193, 265 (1951).

(u-D-MANNOSIDASE

411

The purified, a-D-mannosidase preparation was found to be free from several other glycosidases, besides 2-acetarnido-2-deoxy-p-~glucosidase and p-D-galactosidase. Other enzymes whose presence could hinder the use of a-D-mannosidase as a carbohydrate reagent include p-D-mannosidase (EC 3.2.1.25) and the glycoaspartamidase that cleaves the glycosylamine linkage in 2-acetamido-N-~-aspart4-oy~-2-deoxy-p-D-glucopyranosylamine (see Section V,1; p. 437). Unlike some other sources of a-D-mannosidase, jack-bean meal lacks P-D-mannosidase and the glycoaspartamidase. Rat epididymis is also a good source of a-D-mannosidase, but it is not so readily available, in quantity, as jack-bean meal. A procedure similar to that just described was employed to obtain the enzyme from rat epididymis60 in the same state of purity as the jack-bean a-D-mannosidase, including freedom from p-D-mannosidase and glycoaspartamidase. A somewhat different procedure was needed for purifying the limpet enzyme to the same The activity of a-D-mannosidase from this source was rapidly destroyed by pyridine. Selective inwas, therefore, activation of 2-acetamido-2-deoxy-~-~-glucosidase effected by warming the preparation in 60% ethanol. P-D-Galactosidase was removed in the course of fractionation. At pH values above neutrality, jack-bean a-D-mannosidase, unlike the enzyme from the other two sources, is stable, and the native, protein-metal complex does not dissociate. By working at pH 8, it is possible to purify the enzyme from jack-bean meal without addition of zincs2' The procedure shown in Table V was followed with only slight modification (see Table IX, Section 111,5; p. 433). After precipitation with ammonium sulfate at the natural pH of the aqueous extract (pH 6.3), as shown in Table V, the enzyme was dissolved in glycine-sodium hydroxide buffer at pH 8, and the solution dialyzed against the same buffer. The preparation was briefly brought to pH 5 before treatment with pyridine. After the enzyme had been precipitated with acetone, it was taken up in the glycine buffer at pH 8, and the solution was dialyzed as before. Finally, it was passed through Sephadex G-100 in the glycine buffer. The concentrated a-D-mannosidase preparation can be stored in this buffer. It appears probable that the success of this procedure depends partly upon the sequestering action of the glycine buffer towards toxic cations (see Section 111,3; p. 424). Whereas, at pH 5, it was necessary to add sodium chloride to keep the enzyme in solution during the later stages of purification, this was not necessary at pH 8. (60) S. M. Snaith and C . A. Levvy, Biochem. j . , 114, 25 (1969).

412

S . M. SNAITH AND G. A. LEVVY

Earlier methods for the purification of a-D-mannosidase from jackbean meal were given by Y.-T. Li,34,61and other plant preparations that have been subjected to purification include those from 2'. foenum gruecum,3' V. ~ u t i v uand ,~~ Chromatographic separation of the different glycosidases, including a-D-mannosidase, in P. vulguris has been and the glycosidases in jack-bean meal have been separated by isoelectric focusing,63 The enzyme from P. vulguris has been subjected to extensive p u r i f i ~ a t i o n . ~ ~ a-D-Mannosidase has been purified from the marine molluscs Churonia lampusMand Turbo C O T ~ U ~ Ubut, S , ~ for ~ the former at least, the final preparation was still contaminated with other, potentially interfering, glycosidases in significant proportions. Okumura and Yamashina18 have extensively purified a-D-mannosidase from pig kidney, and have freed it from other important glycosidases. 5. pH and Activity

With the exception of the enzyme from the limpet, P. vulgatu, a-D-mannosidase from most of the important sources shows optimal activity at pH values lying between 4 and 5. For the enzyme from jack-bean and that from rat epididymis,6O we employed a pH of 5 for routine assays. If this is not the actual optimum, it is close to it on the broad pH-activity curves, and, at this pH, the addition of Zn2+and other cations has relatively little effect in the assay, thus simplifying the study of the various metal complexes that can be formed by the enzyme protein. The position with regard to the limpet enzyme is more compli~ a t e dAlthough .~~ zinc has little effect in the assay at pH 5, this pH is so far removed from the sharp optimum at pH 3.5 (Cl- present) that the enzyme displayed less than 20% of its activity. Furthermore, a large proportion of the enzyme seemed to be firmly complexed with toxic cations derived from the limpet, and these were not displaced by addition of an excess of zinc during an assay at pH 5 (see Fig. 1). At its relatively low pH of optimal activity, the limpet protein-metal complex was readily dissociable, even in the presence of substrate, and, consequently, on assay at this pH, Zn2+seemed to displace toxic cations, allowing the enzyme to display its full, potential activity. The dissociation of the active protein-Zn2+ complex at low pH can be seen from the pH-activity curves shown in Fig. 1. This Figure also shows the effect of C1-. Chloride ion not only accelerates hy(61) Y.-T. Li,J. Biol. Chem., 242, 5474 (1967). (62) M. Saita, T. Ikenaka, and Y. Matsushima, J . Biochem. (Tokyo), 70, 827 (1971). (63) Y.-T. Li and S.-C. Li, J . Biol. Chem., 243,3994 (1968). (64) E. Paus and T. B. Christensen, Eur. J . Biochem., 25,308 (1972).

413

2

3

4

5

6

PH

FIG.1.-Hydrolysis at Different pH Values, in Acetate Buffer, of6 mMp-Nitrophenyl a-D-Mannoside by Limpet a-D-Manno~idase.~~ [A, no addition; in the presence of A. 0.1 mM ZnSO,; 0, 0.1 M NaCl, and 0 , O . l M NaCl+ 0.1 mM ZnSO,. The a-D-mannosidase activity is expressed as a percentage of the maximum value obtained in the presence of Zn*+and C1-.I

drolysis at all values of pH, but also causes a shift in the pH optimum. As already mentioned, added Zn2+has very little effect on the activity shown by the enzyme at pH 5, but it has a marked effect at the pH optimum, and this effect is additive to the effect of C1-. Maximum activity was observed at pH 3.5 with C1- at 100 mM and Zn2+at 100 pM (that is, there was a 1000-fold difference in the effective concentrations of the two ions), and we have normally assayed limpet a-D-mannosidase under these conditions. The effect of sodium chloride on the activity of a molluscan a-D-mannosidase preparation was first observed by M ~ r a m a t s u .The ~ ~ distinction between the effects of Zn2+ and C1- on limpet a-D-mannosidase is more fully considered later (see Section 111,4; p. 431). 6. pH and Stability

In this Section, it is necessary to consider, in some detail, the behavior of a-D-mannosidase as a zinc-dependent enzyme. The effect

414

S. M. SNAITH AND G . A. LEVVY

of varying the pH on the stability of the enzyme in the absence of substrate is markedly influenced by the addition of Zn2+ or of a chelating agent, such as EDTA. Results obtained with the purified, jack-bean enzyme are shown in Table VI. Above pH 7, the enzyme was quite stable, but, as the pH TABLEVI Residual Activity" of Purified, Jack-bean a-~-Mannosidase*'after Incubation for 3 Hours at 37" at Various pH Values Residual activity (%)

PH

No addition

4 5 6 7 8 9 10

37 57 84 105 93 96

7

1 mM ZnSO,

1 mM EDTAb

104 97 86 91 106 106 105

3 34 80 86 108 109 104

"a-D-Mannosidase was assayed at p H 5, with p-nitrophenyl a-D-mannoside as the substrate. The samples were incubated beforehand in acetate (pH 4 to 7)or phosphate (pH 8 to 10) buffer, in the presence of 0.01% of albumin. The results are expressed as percentages of the initial activity of the enzyme preparation. * (Ethylenedinitri1o)tetraacetate.

was lowered, it became progressively less stable unless Zn2+ was added to the preparation. The effect of lowering the pH was very pronounced when EDTA was added. These results suggest that the enzyme is a protein-metal ion complex that is readily dissociated at low pH, but is quite stable above neutrality. Albumin was always included in the incubation mixture in these experiments, in order to lessen the irreversible inactivation of the dilute, enzyme protein that may occur at all pH values (see Section 111,3; p. 424). Table VI explains the fact already noted (see Section 11,4; p. 409) that addition of Zn2+is necessary during dialysis of the jack-bean enzyme at pH 5, but not at pH 8. Although rat-epididymal a-D-mannosidase is not so convenient as the jack-bean enzyme for use as a carbohydrate reagent, it may be of particular interest in mammalian physiology. It differs from the plant enzyme in that the protein-Zn2+ complex is more readily dissociable, there being no pH at which the purified epididymal enzyme is completely stable (see Table VII). Greatest stability was displayed between pH 5 and 6, but inactivation was still marked unless Znz+

(Y-D-MANNOSIDASE

415

TABLEVII

Residual Activity" of Purified, Rat-epididymal a-D-MannosidasesO after Incubation for 3 Hours at 37"at Various pH Values Residual activity (%) PH

No addition

1 mM ZnSO,

4.5 5.0 5.5 6.0 6.5 7.0

45 61 69 70 58 37

80 92 90 91 76 33

"The experiment was performed as in Table VI.

was added. Irreversible inactivation was evident at pH 7 , despite the presence of albumin and zinc. Crude, rat-epididymal preparations were found to be much more stable than the purified enzyme,60and, in the presence of Zn2+,there was no loss in activity in 3 hours at 37" at values of pH between 5 and 6. EDTA accelerated the inactivation at acid pH, for both crude and purified preparations. The effect of Zn2+and EDTA on the stability of purified, limpet a-D-mannosidase at different pH values is shown in Table VIII. This TABLEVIII

Residual Activity" of Purified, Limpet a-D-Mannosidaseafter Incubation for 3 Hours at 37"at Various pH Values Residual activity (%) PH

No addition

1 mM ZnSO,

1 mM EDTA

3.0 3.5 4.0 4.5 5.0 6.0 7.0

67 51 62 69 81 93 30

31 107 100 101 97 94 28

3 5 34 41 67 75 51

"a-D-Mannosidase was assayed at pH 3.5 in the presence of 0.1 M NaCI, with p-nitrophenyl a-D-mannoside as the substrate. Zinc sulfate (0.1 mM) was also included in the enzyme-assay medium, except for the results shown in the last column. The samples were incubated beforehand in acetate buffers in the presence of 0.01% of albumin. The results are expressed as percentages of the initial activity of the enzyme preparation, assayed with or without Znz+as appropriate.

416

S . M. SNAITH AND G. A. LEVVY

preparation was like the purified epididymal enzyme, in that there was no pH at which it was completely stable in the absence of added Zn2+.The stability was highest at pH 6. Once again, EDTA accelerated reversible inactivation at acid pH. In the presence of Zn2+,the limpet preparation, unlike the purified mammalian enzyme, was completely stable over the pH range of 3.5to 5. Irreversible inactivation became marked at pH 7, despite the presence of albumin. The firm binding of toxic cations by limpet a-D-mannosidase at pH 5 has already been mentioned (see Section 11,5; p. 412).This fact is not evident on assay at pH 3.5,because, at that pH, immediate exchange occurs with Zn2+ in the assay medium. On assay at pH 5 (used to arrest any cation exchange), an enzyme preparation may exhibit only about one-quarter of its potential activity at this pH. It is possible to accomplish replacement of endogenous, toxic cations by Zn2+,either directly by incubation with this cation, or indirectly, after incubation with EDTA (see Section 111,4; p. 431). Subsequent assay at pH 5 then reveals activation. The lower the pH of incubation, the faster the removal of toxic cations.46

7. Inactivation by Heat Despite the instability that is associated with loss of zinc, a-D-mannosidase in a crude, rat-epididymal preparation was relatively h e a t - ~ t a b l e , sand ~ * ~was ~ inactivated more slowly than the accompanying 2-acetamido-2-deoxy-~-~-glucosidase and p-D-galactosidase when incubated at 65" and pH 6.5. The greater heat-stability of a-D-mannosidase (compared with that of other glycosidases) has also been observed with preparations from T. c o r n ~ t u s beef , ~ ~ liver," pig kidney,18 and hen oviduct.20In the last two preparations, heat treatment was used during purification of the enzyme, in order to remove interfering glycosidases.

8. Activation and Inhibition The affinity of an enzyme for its substrate is usually considered to be the reciprocal of &,, the Michaelis constant, but it is, in fact, the reciprocal of &, the dissociation constant of the enzyme-substrate complex. It is seldom that K, can be measured directly, but K,,,normally provides a good approximation. The latter constant can be found experimentally: it is the substrate concentration at which the enzyme displays half-maximal velocity. In the reaction,

E +S

kl k, * ES -+ E + products, ke

+

where E is the enzyme and S the substrate, K, = k,/k,, and K,,,= ( k , k,)/k,. The maximum velocity of hydrolysis attained when the enzyme is fully saturated with sub(65) ]. Conchie and A. J. Hay, Biochem. ]., 73, 327 (1959).

(Y-D-MANNOSIDASE

417

strate, V,, is proportional to k3.Although V, is measured at the same time as K,, the two constants vary independently with different substrates. The affinity of an enzyme for an inhibitor (I) is the reciprocal of &, the dissociation constant of the enzyme-inhibitor complex, which can be directly measured. Enzyme inhibition (or activation) can often be regarded as either essentially competitive or non-competitive in character. A competitive inhibitor causes an increase (or a competitive activator causes a decrease) in K,. According to theory, the inhibitor makes a given concentration of substrate less effective by competing at the active center in the enzyme; Vmdoes not alter. Ki = [I1 KmI(K:m - Km), where K;, is the apparent Michaelis constant in the presence of the given concentration of inhibitor. The value of K, is then the concentration of inhibitor needed to double the value of K,. Effective concentrations of substrate and competitive inhibitor can b e compared by means of these two constants. As a rule, competitive inhibitors bear, in their chemical structure, a resemblance to the substrate, and they tend to be much more specific in their action than non-competitive inhibitors. It is evident that a small value of Kt relative to Km denotes an efficient, competitive inhibitor. A non-competitive inhibitor causes an apparent fall in the amount of enzyme present, irrespective of the concentration of the substrate. In purely non-competitive inhibition, Ic, does not alter.

K j = [I] u ’ / ( u- d), where u’ and u are the velocities of reaction in the presence and absence of the inhibitor. At a fixed concentration of substrate, Kt is thus the concentration of inhibitor needed to halve the activity of the enzyme. Non-competitive inhibitors are usually of less theoretical interest than competitive, and can be of a very diverse nature.

Activation of a-D-mannosidase from the limpet by Zn2+ and C1provides a particularly good example of the ways in which the kinetics of hydrolysis may be altered. Fig. 2 shows the effect of Zn2+,C1-, or both, on the velocity of hydrolysis of substrate at varying concentration. Inspection of the curves reveals that Zn2+increases the affinity of the enzyme for the substrate (competitive type of effect), whereas the main effect of C1- is to increase the rate of hydrolysis (non-competitive effect). The values of K,,, and V, were derived by the method of Lineweaver and Burk,B6as shown in Fig. 3. In the presence of both Zn2+and C1-, K , had a value of 4.5 mM. When Zn2+was omitted, K,,, rose to 12 mM, but there was no change in V,. When C1- was omitted, K , was 1.6 mM, and V,,, fell by 75%. It seems that the addition of Zn2+increased kl,whereas C1- increased k3, and that k, was large enough (compared with k,) for K , to alter. In many enzyme reactions, k, is negligible compared with ktr and it can then alter without any perceptible change in K,. (66) H. Lineweaver and D. Burk, J. Amer. Chem. SOC., 56, 658 (1934).

418

S . M. SNAITH AND G . A. LEVVY

1

2

3 4 5 6 7 8 Concentration of substrate (mM)

9

10

FIG. 2.-Effect of Concentration of Substrate on the Rate of Hydrolysis4sof p-Nitrophenyl a-D-Mannoside in Acetate Buffer, pH 3.5, at 37” by a Crude Preparation of Limpet a-D-Mannosidase in the Presence of 0 , O . l mM ZnSO, and 0.1 M NaC1; A, 0.1 M NaCl only; and 0 , O . l mM ZnSO, only.

The results in Fig. 3 may be considered according to Frieden’s treatments' of the action of “modifiers” on single-substrate enzymes. This approach calls for no assumptions as to whether the modifier is an inhibitor or an activator, or whether it acts at the active center in the enzyme or not. It can be depicted as follows. 1. E + S = E S 2. E + M e E M 3’ ES + 4. E M + S = E M S

k. +

E +products

-%

EM

+ products

As will be seen later (see Section 111,3; p. 424), a-D-mannosidase freed from Zn2+by the action of EDTA cannot combine with the substrate. The results for Zn2+,shown in Fig. 3, fit Frieden’s formula for this limiting case, in which reaction 1 and, hence, reaction 3 do not occur. When C1- is regarded as a modifier of limpet a-D-mannosidase, (67) C. Frieden, J . B i d . Chern., 239, 3522 (1964).

CY-D-MANNOSIDASE 50

-1.0

-0.5

419

r

0

0.2

0.4

0.6

0.8

1.0

[sl-’ FIG.3.-Derivation

of K , and V, for Limpet a - ~ - M a n n o s i d a s eActing ~~ on p-Nitrophenyl a-D-Mannoside in the Presence of: 0 , 0.1 mM ZnSO, and 0.1 M NaCI; A, 0.1 M NaCl only; and 0 , O . l mM ZnSO, only. ([S] is the concentration of the substrate, and u the rate of reaction; the intercept on the vertical axis is l/V,,,, and on the horizontal axis, - l&.)

it best fits Frieden’s case of activation in which EMS is much more rapidly hydrolyzed than ES (that is, kb is much greater than ka), but the modifier has a slightly higher affinity for E (reaction 2) than for ES (reaction 3). Competitive inhibition of a-D-mannosidase is very well illustrated by the action of D-mannono-1,5-lactone. p-D-Glycosidases are powerfully and selectively inhibited by the aldono-1,5-lactones of configuration corresponding to that of the sugar residue in the substrate, and the effect is always competitive. In certain instances, of which a-D-mannosidase is one, inhibition extends to the a-D-glycosidase (see Ref. 69). D-Mannono-1,5-lactones8gave a value of 0.07 mM for K, with ratepididymal a-D-mannosidase, compared with 12 mM for K , for p-nitrophenyl a-D-mannopyranoside,68 and 57 mM for phenyl a-Dmannopyrano~ide.~5With jack-bean a-D-mannosidase, the values (68) G . A. Levvy, A. J. Hay, and J. Conchie, Biochem.J.,91, 378 (1964).

420

S. M. SNAITH AND G.

A. LEVVY

were 0.12 mM and 2.5 mM for the lactone and the p-nitrophenyl glycoside, respectively,6l and with hen-oviduct a-D-rnannosidase:O 0.11 mM and 4.5 mM. Comparable data are not available for the limpet enzyme, but a crude preparation was inhibitedsa to the extent of 50% by 0.1 mM D-mannono-~,~-~actone at a concentration of p-nitrophenyl a-D-mannopyranoside of 6 mM: the measurement was made at pH 5 in the absence of added C1- and Zn2+. An inhibitor can react at the groups that constitute the active center of an enzyme, or at the groups that determine the specificity. As shown earlier,6O a 1,5-lactone fulfils both of these requirements. Although a great deal of work has been done using aldono-1,4-lactones as inhibitors, it appears probables0 that these produce inhibition only insofar as they are converted into the 1,Slactones in aqueous solution. D-Glucal ( 1,5-anhydro-2-deoxy-~-u~ubino-hex1-enitol) has been shown to be a competitive inhibitor of a-~-rnannosidase,'~ but it has less than one-tenth of the inhibitory power of D-mannono-1,5-lactone. D-Glucal is less specific than ~-mannono-l,5-lactone,because it also inhibits a- and @-D-gluco~idases.~~ a-D-Mannosidase is inhibited or inactivated in the assay by certain heavy-metal ions (for example, Hg2+ and Ag+) and by iodoacetate ion,20~34~39*44~45~60~65 Their action is probably non-competitive, but the effects of these ions on the kinetics of hydrolysis have not yet been investigated. Other heavy-metal ions, such as Co2+ and Cd2+, can displace Zn2+from a-D-mannosidase with concomitant loss of activity, but only in the absence of s ~ b s t r a t e .These ~ ~ * ~metals ~ are, to some extent, selective in their action, as they affect only a limited number of enzymes.

9. Specificity a-D-Mannosidase catalyzes hydrolysis of alkyl and aryl a-D-mannopyranosides. The enzyme from most sources is also able to cause hydrolysis of small oligosaccharides containing a-D-( 1+2)-, a - ~ 1+3)-, -( a - ~ 1+4)-, -( and a - ~ 1+6)-linked -( D-mannOpyranOSe residue^.^^,^^* 61*70~72 The enzyme can cause removal of terminal, nonreducing a-D-mannOpyranOSyl groups from glycoproteins and glycopeptides (see Section V; p. 437). P-D-Mannosidase is not so widely distributed as the a-enzyme, but known molluscan a-D-mannosidase preparations do contain p-Dmannosidase in relatively high a ~ t i v i t y . ~It, ~has ~ ,been ~~,~ suggested ~ (69) G. A. Levvy and S. M. Snaith, Adoan. Enzyrnol., 36, 151 (1972). (70) J. Schwartz, J. Sloan, and Y. C. Lee, Arch. Biochem. Biophys., 137, 122 (1970). (71) E. T. Reese, F. W. Parrish, and M. Ettlinger, Carbohyd. Res., 18, 381 (1971). (72) K. Sugahara, T. Okumura, and I. Yamashina, FEBS Lett., 20, 44 (1972).

CY-D-MANNOSIDASE

421

(see Section V; p. 437) that the trace of p-D-mannosidase activity (about 1% of the a-D-mannosidase) that occurs in crude, rat-epididyma1 preparationsl2Smis important in the degradation of g l y c ~ p r o t e i n s . ~ ~ P-D-Mannosidase has also been shown to be present in hen oviduct, at an activity comparable to that of the a-D-mannosidase.20Although, in certain instances, an aldon0-1~5-lactonepermits rapid distinction between an a-D-glycosidase and the corresponding p-D-glycosidase (through its much greater inhibitory action on the latter), this is not true of a- and p-D-mannosidase. However, it is quite easy to distinguish between the two enzymes when chromogenic substrates are employed. Distinction of a-D-mannosidase from exo-a-D-mannanase is not so straightforward. As noted in the Introduction (see p. 401), exoglycanases remove terminal residues from sugar chains, with inversion of configuration at the glycosyl bond, whereas glycosidases act on smaller molecules, with retention of configuration It has also been suggested7s71that exo-p-D-glucanase can be distinguished from p-D-ghcosidase, in that the former is more feebly inhibited than the latter by D-glUCOnO-1,5-laCtOne. There was, however, no marked difference in this respect between a-D-glucosidase and exo-a-D-glucanase. It seems probable that the bacterial “a-mannosidase” of G. H. Jones and B a l l o ~ , 5 ~ having * ~ ~ ,a~pH ~ optimum lying between 6.5and 7, is in fact an exo-a-D-mannanase, because it causes inversion at the glycosyl bond.51 The purified enzyme, which is activated50 by Ca2+, acts on yeast a-D-mannan by removing D-mannose residues from the (1+2)- and (1+3)-linked side-chains, leaving the (1+6)-linked mainchain intact. It will, however, remove D-mannose residues from (1+6)linked oligosaccharides derived from the main chain. This enzyme has very little action on the glycosides normally employed as substrates for a-D-mannosidase. On the other hand, a true a-D-Inannosidase, despite its action on the small oligosaccharides prepared from yeast D-mannan, had little action on the parent polysaccharide.s1,70The effect of D-mannono-1,5-lactone on exo-a-D-mannanase has not yet been examined. In addition to the usual a-D-mannosidase having a pH optimum between 4 and 5, a similar enzyme (optimal activity at pH 6.5)has been observed in rat liver by Marsh and G ~ u r l a yand , ~ ~in pig kidney, (73) T. Sukeno, A. L. Tarentino, T. H. Plummer, and F. Maley, Biochem. Biophys. Res. Commun., 45, 219 (1971). (74) G. H. Jones and C. E. Ballou,]. Biol. Chem., 243, 2443 (1968). (75) G. H. Jones and C. E. Ballou,J. Biol. Chem., 244, 1052 (1969). (76) C. A. Marsh and G. C. Gourlay, Biochim. Biophys. Acta, 235, 142 (1971).

S . M. SNAITH A N D G. A. LEVVY

422

by Okumura and Yamashina.18 It would be interesting to know whether the enzyme having its pH optimum at 6.5 is a true a-D-mannosidase or an exo-a-D-mannanase. The enzyme from rat liver was as strongly inhibited by D-mannono-175-lactone as the accompanying a-D-mannosidase having a pH optimum of 5. It has also been reported76a that an a-D-mannosidase showing optimal activity near neutrality is present in ram epididymis, testis, and semen. Rat epididymis lacks this activity,sObut it is present in rat testis.27In every case, the neutral a-D-mannosidase activity is inhibited by the addition of Zn2+to the assay, in agreement with the results for rat liver.76 (Two enzymes that have been found to remove terminal 6-deoxya-L-galactopyranosyl groups provide another interesting example in which classification of one enzyme as a glycosidase and the other as an exo-glycanase would explain the overlapping specificity in their action on glycosides, oligosaccharides, and polymer^.'^) 111. a-D-MANNOSIDASE AND ZINC^+ 1. The Effect of pH on the Stability of Metal Chelates In order to understand the interactions of proteins with metals, it is necessary to ~ ’ ~following, simplified consider the effect of pH on the stability of metal c h e l a t e ~ . ’ ~The treatment illustrates the equilibria that can occur between a metal ion, a chelating agent (or other ligand), and hydrogen ion. Although it is almost impossible to apply the equations quantitatively to metalloenzymes, they do illustrate the interactions of the different variables. For a metal M and a ligand A, the stability constant (K) for the complex MA is given by

K=-

[MI[Al‘

In a metal buffer (that is, a given mixture of cation and chelating agent)

where the total concentration [M,] of metal is much greater than the concentration [MI of free metal, [MA] approximates to [M,] and [A] approximates to [A,] - [M,], [&I and [A] being the concentrations of the total and free ligand, respectively. The value of [MI is then given by [Mf]/K([At]- [MJ). This equation applies to any medium wherein the pH is high enough to avoid association between A and hydrogen ion. At lower pH values, the term [A] in the equation for K must be corrected to take protonated forms of A into consideration. [A,] = [MA] + [A] + [HA] + [H,A] + ........ + [H,AI .’. [At1 - [MA] = [A1 + [HA] + [HZAI + **......+ [H,A] = CK

[A]

For a given pH, (Y is constant and indicates the ratio of the total amount of chelating agent not bound to metal to the amount of the completely dissociated, chelating (76a) S . Bullock and B. Winchester, Biochem. Soc. Trans., 1, 491 (1973).

(77) G. A. Levvy and A. McAllan, Biochern.J., 80,435 (1961). (78) J. Raaflaub, Methods Biochern. Anal., 3,301 (1956). (79) S . Chaberek and A. E. Martell, “Organic Sequestering Agents,” Wiley, New York, N. Y., 1959.

a-D-MANNOSIDASE

423

anion. The apparent stability-constant of MA a t a given pH, KUH,is given by K,, = [MA]/[M][A]a = K / a , where K is the true stability constant. The greater the value of a (that is, the lower the pH), the smaller is K u H . At a given pH, [MI can be found, as before, by rearranging the equation for KpHto give [MI = [M,]/K,,([A,I - [M,]). In the case where the ligand is in 100% excess, the expression simplifies to [MI = l/KpH.For a metalloenzyme, the chelating agent is the enzyme protein. As the enzyme is active only in the form of the protein-metal complex, the stability constant for the enzyme is the reciprocal of the concentration of free metal at 50% activity ([MA] = [A]). If another chelating agent (such as EDTA) is added to the medium, it will compete with the enzyme for the cation. Inactive proteins that are present in an impure, enzyme preparation may interfere in a similar way.

2. The Classification of Metal-dependent Enzymes

Metal-dependent enzymes have been divided into two group^,^^-^^ the “metalloenzymes” and the “enzyme-metal-ion complexes.” Metalloenzymes are those that contain one or more functional metal atoms per enzyme molecule. The metal is firmly bound to the protein, and the enzyme can be purified without any loss in activity. The content of functional metal in the preparation approaches a limiting value during purification. Enzyme-metal-ion complexes are more readily dissociable than metalloenzymes. It is necessary to add the functional, metal ion during or after purification, in order to maintain or restore full catalytic activity. In practice, there is no rigid distinction between these two groups of enzymes. Although it was once considered that the metal could not be removed from a metalloenzyme without breakdown of the protein molecule, it has now been found that, in nearly every case, the metal can, reversibly, be detached by working at a more acid pH or by the use of a suitable chelating agent. Reversible dissociation of the metal, with concurrent changes in enzyme activity, can be a useful indication that the metal is, indeed, functional. At least one enzyme is known that is a well defined metalloprotein (glutamate dehydrogenase, EC 1.4.1.3),but from which the zinc can be removed without any loss in catalytic activity.n4 The division of the metal-dependent enzymes into two groups, depending upon the ease of dissociation of the complex, obscures a more fundamental distinction based on the mode of action of the metal, and that is, classification as the metal-containing enzymes and (80) B. L. Vallee, Adoan. Prot. Chem., 10, 317 (1955). (81) B. G . Malmstriim and A. Rosenberg, Aduan. Enzymol., 21, 131 (1959). (82) B. I,. Vallee and J. E. Coleman, in “Comprehensive Biochemistry,” M. Florkin and E. H. Stotz, eds., Elsevier, Amsterdam, 1964, Vol. 12, p. 165. (83) B. L. Vallee and W. E. C. Wacker, in “The Proteins,” H. Neurath, ed., Academic Press, New York, N. Y., 2nd Edition, 1970, Vol. 5. (84) R. F. Colman and D. S. Foster, J . B i d . Chem., 245, 6190 (1970).

424

S. M. SNAITH AND G. A. LEVVY

the metal-activated enzymes. The metal-containing enzymes may or may not be dissociable at their pH of action, but the metal is an integral part of the enzyme in its catalytically active form. Metalactivated enzymes, a large number of which are known, are those in which the cation merely acts to increase the rate of reaction, without affecting the structure of the enzyme. The effect is analogous to that already discussed for chloride ion acting on limpet a-D-mannosidase. Activation is instantaneous, and depends directly upon the concentration of the ion that is added to the enzyme-assay mixture. Metal chelates are less readily dissociated at an alkaline pH than at an acid pH, as shown in the previous Section, and, on the whole, the well characterized metalloenzymes are stable and catalytically active in neutral or slightly alkaline solution. As already seen (see Section 11,5; p. 412), a-D-mannosidase is active at a slightly acid pH, and most of the work on the purification of the enzyme has been conducted in this pH region. We found it necessary to add zinc ion in order to maintain activity during the purification (see Section 11, 4; p. 409) of a-D-mannosidase from jack-bean rat epididymis,60 and the limpet.46It was subsequently found possible to purify a-D-mannosidase from jack-bean meal without adding zinc by working at an alkaline pH, although the enzyme is inactive in this pH region.27 Other partial purifications of the enzyme from various sources have met with some ~ ~ c c e s s , ~ although ~ - ~ ~ ,the ~ , ~ ~ , ~ ~ possibility that the enzyme may contain a dissociable metal ion was not considered. The enzyme from P. vulgaris has been purified without the addition of zinc ion by working at a suitably high pH.64 3. The Effects of Zinc2+and Other Cations

In this Section, a-D-mannosidase from jack-bean m e a P will be treated as typical of the enzyme from the three sources that we have studied, with only brief mention of the enzyme from rat epididymis and the limpet. We have also obtained evidence that a-D-mannosidase from one of the oldest known sources, namely almond emulsin, is a zinc-dependent enzyme, but we have not studied it in any detail.B0 As discussed under “Purification” and “pH and Stability” [see Sections II,4 (p. 409) and II,6 (p. 413)], a-D-mannosidase is unstable at low pH values unless Zn2+ is added. In the following experiments employing a partially purified, jack-bean meal preparation (see Table V, stage 3; p. 410) to which Zn2+ had not been added, the enzyme was pre-incubated at 37” and pH 5 before assay at the same pH with p-nitrophenyl a-D-mannopyranoside as the sub-

CPD-MANNOSIDASE

425

-

I

I

0

I

I

I

I

1

2

3

I

4

Period of incubation (hr)

FIG. 4.-Effect of' Zn2+ and EDTA on a-D-Mannosidase Activitye5 in a Partially Purified, Jack-bean Meal Preparation Incubated for Various Periods of Time at 37" and pH5. [a, Enzyme alone; W , enzyme 1 mM ZnSO,; and 0, enzyme+ 1 mM EDTA. The results are expressed as a percentage of the activity in the unincubated, enzyme preparati~n.~]

+

strate. Fig. 4 shows the effect of incubating the enzyme for various periods of time by itself, or in the presence of zinc sulfate or EDTA. After 4 hours, the activity of the control had fallen to 60%, whereas, in the presence of 1 mM EDTA, only 20% of the activity remained. However, 1 mM zinc sulfate maintained the activity at a level slightly higher than the initial value of the control. No other cation was found to behave like Zn2+in this experiment. One of the most striking indications of the importance of Zn2+for a-D-mannosidase activity was obtained with preparations that had been inactivated by incubation with EDTA. On addition of an excess of Zn2+to the assay mixture, complete activity was regained instantaneously, regardless of the extent of prior inactivation. (When the EDTA-inactivated enzyme described in Fig. 4 was assayed in the presence of Zn2+,the points followed the line for the Zn2+-stabilized enzyme.) Again, no other cation that we have examined can replace Zn2+,leaving little doubt as to the identity of the activating cation in the original material. It also follows that EDTA must withdraw Zn2+ from the protein-metal complex. Had EDTA merely formed a

S . M. SNAITH AND G. A. LEVVY

426

0

1

2

3

4

5

6

Period of incubation (hr)

FIG.5.-D;cay in the a-D-Mannosidase Activityg5of a Partially Purified, Jack-bean Meal Preparation During Incubation at 37" and pH 5, and the Restoration of Activity by Zn2+. [ZnSO, (final concentration 1 mM) was added to a sample of the incubation mixture after 3 hr, and incubation was continued. 0 , Enzyme alone; and 0,enzyme 1 mM ZnSO,. The results are expressed as a percentage of the activity in the unincubated, enzyme p r e p a r a t i ~ n . ~ ~ ]

+

ternary complex with the enzyme protein and Zn2+,it should have been possible to produce reactivation by the addition of cations other than Zn2+having a high affinity for EDTA. Zinc not only provides protection, but can gradually reverse spontaneous inactivation, as illustrated in Fig. 5. The enzyme was incubated at 37, and zinc sulfate was added after 3 hours to cause a progressive recovery of activity. The slow reactivation is in contrast to the instantaneous effect of Zn2+ on an EDTA-treated, enzyme preparation. In a preparation that had lost activity during storage without the addition of Zn2+,the gradual increase in activity on incubation with Zn2+ frequently led to a value greater than that of the unincubated control. The bivalent ions of cadmium, cobalt, and copper, in concentrations of the order of 10-100 pM, accelerate inactivation in experiments of the type shown in Figs. 4 and 5. An excess of ZIP+ prevents, or reverses, inactivation by these ions.

427

a-D-MANNOSIDASE

All of these results can be explained if it is assumed that spontaneous inactivation is due to gradual displacement of Zn2+ from combination with the enzyme protein by traces of toxic cations present in the preparation. Replacement of toxic cations by Zn2+is, in turn, equally slow. EDTA sequesters all cations, including Zn2+, to make all of the enzyme protein immediately available for reaction with added Zn2+. Spontaneous inactivation of the enzyme can be prevented by EDTA present at a suitably low concentration, presumably through removal of endogenous, toxic cations. This behavior is illustrated in Fig. 6, which shows the residual activity in an a-D-mannosidase

0. I

I

10

100

1000

Concentration of EDTA ( p M )

FIG. 6.-Residual a-D-MannosidaseActivityss in a Partially Purified, Jack-beanMeal Preparation After Incubation with Various Concentrations of EDTA for 3 hr at 37" and pH 5. [The concentration of EDTA is shown on a logarithmic scale, and the results are expressed as a percentage of the original activity in the enzyme preparati~n?~]

preparation incubated for 3 hours at pH 5 with various concentrations of the chelating agent. The enzyme was completely protected from inactivation by 1 pM EDTA. As the concentration of EDTA was increased, removal of Zn2+ from the enzyme became evident from the loss in activity. The metal-free enzyme protein was remarkably stable46 and could be stored for quite long periods at 0" in EDTA solution, provided that albumin was also included to prevent irreversible inactivation (see later). (The metal-free protein from the limpet, but not that from rat epididymis, was also stable under these conditions.1

S. M. SNALTH AND G. A. LEWY

428

Antagonism between an inactivating cation and Zn2+ could be demonstrated by incubating the jack-bean enzyme with mixtures of the two cations in different proportions. Experiments were performedS0with Cu2+,CdZ+,and Co2+. Fig. 7 shows the effect of an

r

140

Concentration of variable component ( p M )

FIG.7.--Residual a-D-Mannosidase A~tivity9~ in a Partially Purified, Jack-bean Meal Preparation After Incubation with Different Two-component Mixtures for 3 hr at 37" and pH 5. [A,10 pLM ZnS04 increasing concentrations of CuSO,; and A, 10 p M CdS04 increasing concentrations of ZnS04. The concentration of the variable component is shown on a logarithmic scale, and the results are expressed as a percentage of the initial activity of an untreated control; the value of this control after 3 hr at 37" is shown by the broken line.]

+

+

increasing proportion of Cu2+ in overcoming the protective action of Zn2+, and of an increasing proportion of Zn2+ in diminishing inactivation by Cd2+. Similar experiments were conducted in which the enzyme was incubated with a mixture of EDTA and either Zn2+ or a toxic cation in various proportions. The results confirmed the suggestion already made that, depending upon the concentration, EDTA either inactivates the enzyme by removing Zn2+ or protects it against inactivation by certain other cations. Although there was little dissociation of the active, protein-zinc complex above neutrality

429

~-D-MANNOSIDASE

(see Section 11,6; p. 413), it was still possible to demonstrate antagonism between a toxic cation and Zn2+at pH 7 and 8. It was found that the purified, jack-bean enzyme (see Table V,

stage 6; p. 410) undergoes spontaneous inactivation much more slowly than the crude preparation, although EDTA and Zn2+acted as before. This result suggested that endogenous, toxic cations were largely removed during the purification, perhaps during the gel chromatography. Accelerated inactivation of a purified, enzyme preparation could be achieved by adding a trace of a toxic cation [for example, 5 pM copper(I1) sulfate], to approximate to the rate for a crude preparation. A new factor was encountered with the purified enzyme, namely irreversible inactivation (or denaturation) due to the high dilution of protein employed. This effect, which was superimposed upon the Zn2+-reversible inactivation, could be arrested by the addition of albumin to the system. The two types of inactivation could be distinguished as shown in Fig. 8. As a matter of routine,

*O

r

50

40 30

17

18

19

20

''

41

Total period of incubation (hr)

FIG.&-Effects of Albumin and Zn2+on the Activity of a - ~ - M a n n o s i d a s e .[A ~ ~purified, jack-bean meal preparation was incubated beforehand at 37" and p H 5 in the presence, and absence, of 0.01% of albumin. After 17 hr, ZnSO, (final concentration, 1mM) was added to a sample of each solution, together with albumin where appropriate to maintain an 0.01% concentration thereof, and incubation was continued. 0 , Enzyme alone; 0, enzyme + ZnSO,; A,enzyme albumin; and A, enzyme albumin + ZnSO,. The results are expressed as a percentage of the activity in the unincubated, enzyme preparation.]

+

+

S. M. SNAITH AND G . A. LEWY

430

albumin was present in all incubation mixtures and assays using the purified enzyme. Thus far, the action of an excess of zinc in stabilizing a-D-mannosidase preparations has been considered. The experiments next discussed are concerned with its action, if any, in the enzyme assay. Fig. 9 shows the effects of Zn2+, Cd2+, and EDTA on the rate of

"

1

2

3

4

Period of incubation (hr)

FIG.9.-Hydrolysis of6 mM p-Nitrophenyl c ~ - ~ - M a n n o s i dby e ~a~Partially .~~ Purified, Jack-bean Meal Preparation for Various Periods of Time at 37" and pH 5 . [ 0 , No addition; in the presence of 0 , 1 0 0 pM ZnSO,; A, 100 pM EDTA; and A, 10 pM CdSO,.]

hydrolysis of the substrate by a partially purified, jack-bean meal preparation. In the absence of Zn2+,slow inactivation became evident when the period of incubation was prolonged beyond one hour. When Zn2+was added, the rate of hydrolysis was linear for at least 4 hours. Addition of either Cd2+ or EDTA accelerated the fall in velocity. A comparison of the rates of inactivation of the enzyme in the presence and absence of substrate indicated that the proteinzinc complex was stabilized by the substrate39 [compare Fig. 4 (p. 425) and Fig. 91. Purified, jack-bean a-D-mannosidase did not re(85) S. M. Snaith, Ph. D. Thesis, University of Aberdeen (1968).

(Y-D-MANNOSIDASE

431

quire the addition of Zn2+ in order to maintain a constant rate of hydro1y s is. Substrate not only arrested spontaneous inactivation of the enzyme, but prevented the restoration of activity by Zn2+ in a preparation that had already undergone spontaneous decay. It was concluded that, regardless of whether it contains Zn2+or a toxic cation, the substrate so combines with the protein-metal complex as to prevent dissociation, and, hence, inactivation or reactivation as the case might be. On the other hand, the addition of Zn2+caused immediate reactivation of an EDTA-treated preparation, even in the presence of substrate, suggesting that the substrate cannot combine with the metal-free, enzyme protein. In its behavior with Zn2+,other metal ions, and EDTA, rat-epididymal a-D-mannosidase closely resembles the jack-bean enzyme. The greater ease of dissociation of the epididymal protein-zinc complex has been discussed under “pH and Stability” (see Section 11,6; p. 413). Apart from the effects of the strongly bound, toxic cations in the native preparation (see Section 11,5; p. 412), limpet a-D-mannosidase also resembles the jack-bean enzyme in its zinc-dependence. Fortunately, at the relatively low pH optimum (3.5) of the enzyme, the metals are freely dissociable, and toxic cations can be displaced by the addition of Zn2+. Full activity is only shown by the limpet enzyme at the pH optimum when an excess of Zn2+is present in the assay medium [see Sections II,3 (p. 408) and II,5 (p. 412)]. Many of the effects of metals on a-D-mannosidase from different sources, reported by other authors, are merely examples of activation or inhibition by the metal in the assay; they do not illustrate metaldependence of the type just discussed. However, a-D-mannosidase from pig kidney,I8 soy bean,G2and P . vulgarisa4has also been shown to be completely stabilized by the addition of Zn2+.Activity lost by the action of EDTA on rat-liver a-D-mannosidase was reversed by the addition of Zn2+to the assay mixture.7GWe have made a similar observation with the enzyme from rat liver.27 4. The Effect of Chloride Ion on Limpet a-D-Mannosidase Activity

This Section presents direct evidence for the conclusions arrived at indirectly from kinetic data (see Section 11,8; p. 416), namely, that C1- accelerates the hydrolysis of the substrate by limpet a-Dmannosidase, whereas Zn2+is an essential component of the enzyme in its catalytically active form.

S . M. SNAITH AND G. A. LEWY

432

300

c I/-

IL

\-

50

0

I

2

3

Period of incubation (hr)

FIG.10.-The Effect? of EDTA, ZnSO,, and NaCl on the Activity of Limpet a-D-Mannosidase When Incubated for Various Periods of Time at 37" and pH 5. [Enzyme 1 mM EDTA: 0 , assayed without addition of C1- or Zne+; 0, assayed in the presence of 100 mM NaCl. Enzyme 1 mM EDTA 1.1 mM ZnSO,: A,assayed without addition of C1-; and A, assayed in the presence of 100 mM NaCl. Enzyme 1mM EDTA 100 mM NaCl: W, assayed without addition of ZnZ+;and 0, assayed in the presence of 1.1 mM ZnS04. The results are expressed as a percentage of the activity in the unincubated, enzyme preparation assayed without addition of C1- or ZnZ+.]

+

+

+

+

+

Unlike Zn2+,C1- acts only during the enzyme assay. Fig. 10 shows the effects of Zn2+,C1-, and EDTA on the enzyme during incubation at pH 5 . Subsequent assay was conducted at pH 5 in order to retard cation exchange. The unincubated control showed an increase of -100% in the rate of reaction when C1- was added to the assay medium. This difference was maintained in parallel assays, with and without C1-, throughout the course of the experiment. EDTA caused inactivation that was not counteracted by adding C1- during the pre-incubation. Zn2+not only prevented inactivation by EDTA, but produced some activation, presumably due to displacement of endogenous, toxic cations. This factor was also evident when the enzyme, pre-incubated with EDTA in the presence of C1-, was reactivated by Zn2+in the assay. Unlike Zn2+,C1- did not prevent inactivation of the limpet enzyme by added cations (such as Cd2+),but produced the usual increase in the rate of hydrolysis.

433

5. a-D-Mannosidase as a Metalloenzyme

From the evidence presented in the preceding Sections, it may be seen that a-D-mannosidase from all the sources that have been intensively examined requires Zn2+ in order to maintain it in the catalytically active form. Apart from its ease of dissociation, the complex behaves as a typical metalloenzyme. The enzyme from different sources differs in the ease of dissociation at a given pH and, hence, in the susceptibility to interference by contaminating cations. In certain preparations, the activity of the enzyme can be lowered virtually to zero by sequestrating Zn2+ with EDTA, and yet the original activity in the preparation is restored by adding Zn2+.No other cation examined does this, suggesting that, in the native enzyme, Zn2+must be the activating ion. With the possible exception of the enzyme from the limpet, it seems that, in tissue preparations, the enzyme protein is fully activated by combination with Zn2+. Although most other cations have little effect on the activity of a-D-mannosidase, certain bivalent cations, notably Cu2+,CdZ+,and Co2+,combine firmly with the enzyme, displacing Zn2+and causing inactivation in every case.39,46,60 Unlike the metalloenzyme carboxypeptidaseS6 (EC 3.4.2. l), a-D-mannosidase in the metal-free state cannot combine with substrate so as to prevent subsequent restoration of activity by the metal; metal-free preparations are immediately activated by Zn,2+ even in the presence of s u b ~ t r a t e . ~On ~ *the ~~*~ other hand, substrate combines so firmly with metal complexes of a-D-mannosidase, regardless of whether the metal ion is Zn2+or an inactive cation, that it lessens dissociation (and, consequently, metal exchange) to small proportions. To establish that a-D-mannosidase is a metalloenzyme, the proportion of bound zinc in the active molecule must first be determined. This experiment has been performed for the enzyme from jack-bean Two types of preparation were employed. The first was purified, throughout, at pH 8 without any addition of Zn2+ (see Section 11,4; p. 409). The second enzyme preparation was purified in the presence of added Zn2+at pH 5 up to stage 5 of the original procedure (see Table V; p. 410). It was then freed from unbound Zn2+ by dialysis against glycine buffer of pH 8, and passed through a column of Sephadex G-100 in the same buffer. As may be seen from Table IX, the final specific activity was, in each instance, slightly less than that shown in Table V. (86) J. E. Coleman and B. L. Vallee,]. Biol. Chem., 237, 3430 (1962).

434

S. M. SNAITH AND G. A. LEVVY

Table IX gives typical results of analysis for zinc and other metals at the various stages of purification at pH 8, and also for material purified initially at pH 5 and then freed of excess Zn2+at pH 8 in the last stage. The final value for zinc in the protein was the same for both types of preparation. Other metals were present in the purifiedenzyme preparation in relatively small proportions. The molecular weight of the enzyme was estimated by ultracentrifugation to be 230,000. This value is in reasonable agreement with estimates of the molecular weight of a-D-mannosidase from P . vulgmis6*(200,000)and soybeans2 (180,000). For a molecular weight of 230,000, the zinc content given in Table IX corresponds to two gram-atoms per mole of protein. It is not sufficient to show that the active protein contains zinc, in order to classify the enzyme to be a metalloenzyme. It must be proved that the zinc in the metalloprotein is functional. This point was examined2' with preparations similar to the final products listed in Table IX. Although the enzyme was completely resistant to the action of EDTA at pH 8 (see Table VI; p. 414), the preparations were still inactivated by the chelating agent at pH 5. Activity was restored by Zn2+,but by no other cation examined. Replacement of Zn2+by Cu2+,with a concomitant loss in activity, could be demonstrated in these preparations at pH 8, as it could at pH 5 (see Section 111,3; p. 424). This process was prevented, or reversed, by the addition of an excess of Zn2+. Cation exchange seemed to occur more rapidly at the higher pH value. As the Zn2+required for catalytic activity could be retained during purification, a-D-mannosidase may be classified as a metalloenzyme. The enzyme seems to be completely specific for Zn2+as the activating cation. IV. CHANGESIN ACTIVITY in vivo Although the a-D-mannosidase activity of several different organs in mammals has been found to alter under a variety of conditions (see Section 11,l; p. 402),11,'2*'4*2s,2e the most striking changes are those produced by sex hormones in the epididymis and uterUs.26*z8 In rats and mice, a-D-mannosidase activity in the epididymis increases with the age of the animal, up to maturity: orchidectomy causes a dramatic fall in the enzyme activity of the adult tissue (up to 100-fold), but activity can be partially restored by the administration of testosterone. Ovariectomy results in a 10-fold fall in the activity of a-D-mannosidase in mouse uterus, and this is completely reversed by estrone.

TABLEIX Metal Analysis of Fractions during Purificationz7 of Jack-bean a-D-Mannosidase

Stage Meal Aqueous extract Purified at pH 8 . 4560% satd. ammonium sulfate solution Pyridine treatment Gel chromatography Purified at pH 5 and freed from excessd of ZnZ+ Gel chromatography

Recovery of Specific activity Metal contentc ( p g per g of protein) of a-mmannosidase a-D-mannosidase Recovery of Zn (units" per g of meal) (pg per g of meal) (units per mg of proteinb) Zn Cu Fe Mn N i Cd Co 750,000 660,000

650,000 430,000 440,000

400,000

19 10

2.6 1.1 0.72

0.58

3,000 4,600

27,000 140,000 340,000

360,000

76 71

32 34

106 110 360 90 560 60

520

40

100 30 83 36

37 27 90

40

36 55 30

12

20 26

1 1

2 3

45 60 55

2 4 <2

1 0 -

<2

-

30

"One unit of a-Dmannosidase liberates 1 pg of p-nitrophenol from 6 mM p-nitrophenyl a-Dmannoside in 1 hr at 37" and pH 5. bProtein was determined by the method of Lowry and coworkers,59with bovine albumin as the standard. cMetal analysis was performed by atomic absorption spectrophotometry. dThe enzyme preparation was purified to stage 5 of the original procedure (see Table V) and dialyzed at pH 8 to remove the excess of Znz+; finally, gel chromatography was conducted at the same pH.

$ z z

p

K

436

S. M. SNAITH AND G . A. LEVVY

One of the most interesting aspects of the changes in a-D-mannosidase activity known to occur in vivo is the possible relationship with changes in zinc concentration in the tissues. In the case of rat epididymis, changes in enzyme activity have been monitored in parallel with measurements of zinc content.2s There was a positive correlation between the two variables, as both increased with age. The drop in a-D-mannosidase activity resulting from orchidectomy was accompanied by a 4-fold fall in the zinc content of epididymis, but the restoration of enzyme activity produced by subsequent injection of testosterone was not reflected in a detectable rise in the proportion of zinc. In zinc-deficient rats, the zinc content of the epididymis was only about half the normal v a l ~ e , ~ and ~ . ~ the ‘ level of a-D-mannosidase activity was also little over half the value usually observed.26However, the zinc concentration of the tissue was still in vast excess over that required for stoichiometric combination with the enzyme protein, calculated on any probable estimate of its specific activity and molecular weight (see Section 111,5; p. 433). Although a-D-mannosidase appears to be under endocrine contro1,26*28 its natural function remains obscure. It is probable that the enzyme is important in the catabolism of D-mannose-containing glycoproteins,12.16*88 and, therefore, that the important trace element zinc could be indirectly implicated in this process. It is well known that zinc deficiency has an adverse effect on fertility in the male,B9,90 and it could be argued that the changes observed in a-D-mannosidase activity in uterus and epididymis indicate that this enzyme is important in reproduction. It is scarcely justifiable to ascribe a causeand-effect relationship to the correlations observed between the zinc content and the a-D-mannosidase activity in epididymis. All that can be said with any certainty is that, under certain circumstances, the two variables respond together to a common stimulus. In a tissue having a high a-D-mannosidase activity, such as the epididymis, zinc may be needed in great excess to lessen dissociation of the catalytically active protein-zinc complex and the consequent displacement of Zn2+by other bivalent cations (such as Cd2+)to yield

M. J. Millar, M. I. Fischer, P. V. Elcoate, and C. A. Mawson, Can. J . Biochem. Physiol., 36, 557 (1958). J. D. Hocking, R. D. Jolly, and R. D. Batt, Biochem. J., 128,69 (1972). T. Mann, “The Biochemistry of Semen and of the Male Reproductive Tract,” Methuen, London, 1964. (90) E. J. Underwood, “Trace Elements in Human and Animal Nutrition,” Academic Press, New York, N. Y., 3rd Edition, 1971.

437

a-D-MANNOSIDASE

inactive complexes. It is known that cadmium has harmful effects on reproductive processes, in both males and fern ale^.^^,^^ a-D-Mannosidase is not unique among glycosidases in its response to steroid hormones.28 Such changes, first observed with p-D-glucosiduronase (EC 3.2.1.31), were later shown to extend to 2-acetamido2-deoxy-p-~-glucosidaseand p-D-galactosidase, as well as to a-D-mannosidase. Although it was at one time considered that there was no evidence for conjugation of steroid hormones and their metabolites with any sugar other than D-glucuronic acid, this concept is no longer held, at least as far as 2-acetamido-2-deoxy-~-glucose is concerned.93-g6

v. ACTIONON NATURALLYOCCURRING

SUBSTRATES

1. Ovalbumin

a-D-Mannosidase has become widely accepted as a structural reagent in the glycoprotein field. In this respect, there seems to be no difference in the action of the enzyme from mammalian, plant, and molluscan sources. It is employed in conjunction with other suitably purified glycosidases, notably 2-acetamido-2-deoxy-~-~-glucosidase.s7*s8The use of a-D-mannosidase is best illustrated with ovalbumin. This glycoprotein is not only the one most extensively studied in this connection, but it is one in which the composition of the sugar moiety and its linkage to the protein have been well characterized c h e m i ~ a l l y .The ~~~ molecule ’~ contains only one small oligosaccharide residue, consisting of the two sugars D-mannose and 2-acetamido2-deoxy-~-glucose,which is linked to L-asparagine via 2-acetamido2-deoxy-~-glucose. In examining the action of a-D-mannosidase on ovalbumin and ovalbumin glycopeptides, it is necessary also to consider the in(91) J. Parizek,J. Endocrinol., 15, 56 (1957). (92) V. H. Fern and S. J. Carpenter, Nuture, 216, 1123 (1967). (93) D. S. Layne, Endocrinol., 76, 600 (1965). (94) M. Arcos and S. Lieberman, Biochemistry, 6,2032 (1967). (95) H. Jirku and M. Levitz, J . Clin. Endocrinol. Metub., 29, 615 (1969). (96) D . G. Williamson, D. C. Collins, D . S. Layne, R. B. Conrow, and S. Bemstein, Biochemistry, 8, 4299 (1969). (97) R. D. Marshall and A. Neuberger, Aduan. Carbohyd. Chem. Biochem., 25, 407 (1970). (98) D. H. Leaback, in “Metabolic Conjugation and Metabolic Hydrolysis,” W. H. Fishman, ed., Academic Press, New York, 1970, Vol. 2, p. 443. (99) A. Neuberger and R. D. Marshall, in “Glycoproteins,” A. Gottschalk, ed., Elsevier, Amsterdam, 1966, p. 299. (100) A. Neuberger, A. Gottschalk, and R. D . Marshall, in “Glycoproteins,” A. Gottschalk, ed., Elsevier, Amsterdam, 1966, p. 273.

438

S. M. SNAITH AND G. A. L E V "

dividual actions of the enzymes 2-acetamido-2-deoxy-/3-~-glucosidase, p-D-mannosidase, and glycoaspartamidase (see Section 11,4; p. 409). With certain other glycoproteins, neuraminidase (EC 3.2.1.18), a-D-galactosidase (EC 3.2.1.22), p-D-galactosidase, a-L-fucosidase, and P-D-xylosidase (EC 3.2.1.f) must also be taken into account. The first evidence for the presence of an a-D-mannosyl link in ovalbumin glycopeptides was provided by the action of almond emulsin,101~102 although the absence of /3-D-mannosidase from the enzyme preparation was not confirmed until later.lo3The presence of 2-acetamido-2-deoxy-~-~-glucosidase in emulsin left doubt as to the terminal position of all of the D-mannOSe released. The carbohydrate in ovalbumin is linked to the protein by a glycosylamine bond, as in the compound 2-acetamido-N-~-aspart-4-oyl2-deoxy-~-D-g~ucopyranosylamine. Glycoa~partamidase~~~ (2-acetamido-l-aspartamido-l,2-dideoxy-~-~-glucose amidohydrolase) splits off L-aspartic acid from this type of molecule, and ammonia is liberated spontaneously from the resultant glycosylamine at neutral or acid pH. This enzyme does not act on ovalbumin, and its action on a glycopeptide is very slow until practically all of the amino acids, except L-asparagine, have been removed by p r o t e o l y s i ~ The . ~ ~ ~size of the carbohydrate component does not appear to affect enzymic hydrolysis, and it is possible to obtain the intact oligosaccharide free from amino acids, that is, without change in the sugar composition. The whole glycopeptide fraction from ovalbumin has usually been foundss to have D-mannose:hexosamine:L-asparagineratios of 53:1. It was at first believed that this represented a molecule of fixed composition, but it is now generally accepted that it is a statistical average for a group of glycopeptides containing not less than two hexosamine residues per molecule. Early work with a purified 2-acetamido-2-deoxy-~-~-glucosidase preparation showed that one hexosamine residue can be released102*106 from a glycopeptide molecule having a mean D-mannose:hexosamine ratio of 53. Provided that all of the hexosamine in excess of two residues per molecule is terminal, there is no difficulty in explaining the loss of one molecule of hexosamine from a mixed glycopeptide having a mean (101) A. P. Fletcher, G. S. Marks, R. D. Marshall, and A. Neuberger, Biochem.J., 87, 265 (1963). (102) J. R. Clamp and L. Hough, Biochem. J., 94,502 (1965). (103) J. Conchie, A. L. Gelman, and G. A. Lewy, Biochem. J., 106, 135 (1968). (104) J. Conchie and I. Strachan, Biochem. J.. 115,709 (1969). (105) J. Conchie, A. J. Hay, I. Strachan, and G. A. Levvy, Biochem. J., 115, 717 (1969). (106) H. H. Kaufman and R. D. Marshall, Proc. Intern. Congr. Biochem., 6th, New York, 159 (1964).

(Y-D-MANNOSIDASE

439

sugar composition of 5:3. Experiments with an emulsin preparation suggested that some of the D-mannose was also in a terminal position*101,102 This observation was confirmed with purified a-D-mannosidase preparations from jack-bean C. l ~ r n p u s and , ~ ~ T. cornutus.45 A somewhat more-detailed studylo5was made with an ovalbumin preparation that had an initial D-mannose:hexosamine ratio of 5:4. The total glycopeptide isolated after proteolysis had the same ratio. It could be separatedio7on Sephadex G-25 into a range of fractions having integral D-mann0se:hexosamine ratios as far apart as 5:5 and 5:2. The D-mannose:amide nitrogen ratio remained approximately constant, at 5:l. Certain of these glycopeptides, containing L-asparagine as the only amino acid, and the oligosaccharides derived from them by the action of glycoaspartamidase, as well as ovalbumin itself, were subjected to hydrolysis by a-D-mannosidase and 2-acetamido2-deoxy-p-~-glucosidase. When the ovalbumin was incubated with purified, jack-bean a - ~ mannosidase, at least two molecules of D-mannose per molecule were released, suggesting that these residues are a-D-linked in a terminal position. Two glycopeptides having different contents of hexosamine were subjected to the action of purified 2-acetamido-2-deoxy-P-~glucosidase, and, in each case, two hexosamine residues per molecule were either inaccessible, or resistant to attack by the enzyme. From the separation on Sephadex, a glycopeptide fraction having a D-mann0se:hexosamine ratio of 50:37 was incubated with a mixture of purified a - D-mannosidase and 2- acetamido-2- deoxy - p- D- glucosidase. The experiment was continued, with addition of fresh enzyme mixture to the residual glycopeptide, until liberation of reducing sugars had almost stopped. In this type of experiment, any a-D-linked D-mannose residues shielded by p-D-linked 2-acetamido-2-deoxyD-glucose will become open to enzymic attack, and the reverse is also true. Approximately four residues of D-mannose and 1.5 of hexosamine per molecule were released. Apparently, two residues of hexosamine and one of D-mannose were either inaccessible, or insusceptible to attack. When the results are compared with those obtained by the action of the individual enzymes on ovalbumin itself, it is seen that the action of 2-acetamido-2-deoxy-/3-~-glucosidase had apparently made two more D-mannose residues available for hydrolysis by a-D-mannosidase. However, a-D-mannosidase had not increased the amount of hexosamine susceptible to hydrolysis by 2-acetamido-2-deoxy-~-~-glucosidase. These results are consistent (107) G. A. Levvy, J. Conchie, and A. J. Hay, Biochim. Biophys. Acts, 13% 150 (1966).

440

S. M. SNAITH AND G. A. LEWY

with structure 1 for the glycopeptide (ignoring possible branching in the D-mannose side-chain). (p - D - G l c N A c ~ p - D - G l c N A ~ ~ - D - M a n - t a - D - M a n ~ D - G l c N A ~ D - G l c N A ~ L - A s n

(y-D-Manjcr-D-ManjD-~an 1

Results of an experiment with an oligosaccharide free from amino acid were less conclusive, but were not incompatible with the structure shown. The oligosaccharide was prepared from a separated glycoprotein fraction having a D-mannose:hexosamine ratio of 5:3. a-D-Mannosidase and 2-acetamido-2-deoxy-/3-~-glucosidase were used alternately. Initially, a-D-mannosidase removed about 1.5 D-mannose residues per molecule. Subsequent treatment of the residual oligosaccharide with 2-acetamido-2-deoxy-/3-~-glucosidase released rather less than one proportion of hexosamine residue, and then another 1.5 residues of D-mannose became open to attack by a-D-mannosidase. R. Montgomery and his colleagues1o8fractionated, on Dowex 50, the “a~partamidoglycan~’ from ovalbumin. The five components obtained were each subjected to exhaustive hydrolysis with a-D-mannosidase and 2-acetamido-2-deoxy-/3-~-glucosidase, used separately. The results are shown in Table X.One way in which to explain these results would be to change the position of the extra hexosamine residue in 1, to give the alternative structure 2.

-

p-~-GlcNAc+a-D-Man- cY-D-Man- D-G~cNAc+D-G~cNAc+L-Asn

t

P-D- (GlcNAc) +a-D- M an +a-D- Man +D-Man

2

To account for fractions A, C, and D, the presence of another WDlinked D-mannose residue as an additional branch must be postulated, and, in fraction A, it must be assumed that the fifth hexosamine residue (which, again, is p-D-linked) does not block this particular D-mabnOSe residue. Yamashina and coworker^^*^'^ isolated, on Dowex 50, the total “aspartamidoglycan” from a proteolytic digest of ovalbumin. It had mean D-mannose: hexosamine: L-asparagine ratios of 5:3:1. Purified a - ~ mannosidase liberated exactly half the D-mannOSe from the glycopeptide. The residue was subjected to chromatography on Bio-Gel (108) C.-C. Huang, H. E. Mayer, and R. Montgomery, Curbohyd. Res., 13, 127 (1970).

TABLEX Composition of Different “Aspartamidoglycans” Obtained from Ovalbumin, and the Effect of Enzymic Hydrolysis108

Fraction

A B C D E

Molar percentage of isolated “aspartamidog1ycan”a

5 12

36 27 20

No. of sugar residues per asparagine residue

-

1

~

Mannose

6 5 6 6

5

Hexosamine

a-D-Mannosidase

2-Acetamido-Zdeoxy-fi-~glucosidase Mannose Hexosamine

5

1

3

5 4

0

3 2 0 0

2 2

Sugar residues remaining (by difference)

Sugar residues hydrolyzed by

1 5 4

5 5 5

2 2 2 2 2

1 1 ~

~~

~

“These fractions, altogether, account for only 30% of the mannose in the ovalbumin digest before separation by ion-exchange chromatography.

Ip

t

442

S. M. SNAITH AND G. A. LEVVY

P-4, and gave two products, in approximately equimolar amounts, having D-mann0se:hexosamine ratios of 5:4 and 1:2, respectively. The first was considered to have resulted from the action of a-D-mannosidase on Montgomery’s fractions A, B, and C (see Table X),but, theoretically, it should only be produced from fraction C. The second product was attributed to the action of a-D-mannosidase on fractions D and E. Thus, three groups of workers, each using a slightly different approach, have found that ovalbumin glycopeptide has an inner core composed of one D-mannose and two hexosamine residues, linked to L-asparagine through hexosamine. It would appear that the D-mannose residue has an “anomalous” linkage, resistant to a-D-mannosidase. It has been suggested that this residue is /3-~-linked.~~*’~ P-D-Mannosidase preparations from several different sources have now been shown to release D-mannose from the resistant core.20~72~73~109 Sugahara, Okumura, and Y a m a ~ h i n ahave ~ ~ purified p-D-mannosidase from a molluscan source. It had no action on the unfractionated “aspartamidoglycan” obtained from ovalbumin, and, similarly, the glycopeptide having D-mann0se:hexosamine:L-asparagineratios of 5:4:1 was resistant to attack by this enzyme. However, from the product having one D-mannose and two hexosamine residues to one L-asparagine residue, p-D-mannosidase released the remaining D-mannose. Further evidence for the presence of p-D-linked D-mannose in the inner core of ovalbumin was provided by the results obtained on use of purified enzymes from hen oviduct.20a-D-Mannosidase released four residues of D-mannose from the molecule of a glycopeptide having D-mannose:hexosamine:L-asparagine ratios of 5:2:1, whereas P-D-mannosidase had no effect thereon. P-D-Mannosidase did, however, remove the remaining D-mannose residue. It would appear that the resistant core has structure 3. P-D-Man-* D - G ~ C N AD-G~cNAc-. C~ L-Asn.

3 After removal of all of the D-mannose, one more hexosamine residue could be hydrolyzed off by 2-acetamido-2-deoxy-~-~-glucosidase, indicating that this residue, also, is p - ~ - l i n k e d . ~ ~ R. Montgomery and coworkerslo8and Yamashina and coworkers7z advocated the general structure 4 for the group of “aspartamidoglycans” obtained after proteolysis of ovalbumin. (109) Y.-T. Li, S.-C. Li, and Y. C. Lee, Fed. Proc., 31,466 (1972).

(Y-D-MANNOSIDASE

443

(D-GlcNAc)o,l,2+D-Man+D-GlcNAwD-GlcNAc+L-Asn

t D-Man0,,-+(D-Man),

t

D-Man+@-GlcNAc),,, 4

This gives rise to structure 5 for the resistant core. D-G~cNAc- D-G~cNAc+L-Asn

t

P-D-Man 5

It is difficult to reconcile general structure 4 with the results of Conchie and C O W O ~ ~ already ~ ~ S mentioned, ~ ~ ~ * ~in particular ~ ~ with the action of a mixture of a-D-mannosidase and 2-acetamido-2-deoxy-p-~glucosidase that gave a final product containing two hexosamine residues as well as one D-mannose residue per molecule. On the other hand, structure 3 for the resistant core is consistent with all of the results discussed. More-recent work on the sequence of sugar residues in this core, employing an endo-2-acetamido-2-deoxyhexosidase, has provided convincing evidence that 3 is the correct structure.l1° A slightly larger fragment containing five D-mannOSe and two hexosamine residues per molecule seems to be common to all of the glycopeptides that have been isolated from ovalbumin. A useful step forward would be to ascertain whether this fragment has an invariant structure. 2. Other Glycoproteins

The other glycoproteins now to be mentioned are all believed to have the 2-acetamido-2-deoxy-~-glucosyl-asparagine bond, although the evidence for this is not usually so strong as for ovalbumin. Again, as with ovalbumin, these glycoproteins often display microheterogeneity, that is, variability in the degree of completion of the carbohydrate residues in otherwise homogeneous glycoproteins. The glycosylamine linkage is, of course, not the only one encountered in glycoproteins; there are others in which the carbohydrate is linked glycosidically to a hydroxy amino acid."' All of the glycoproteins (110) A. L. Tarentino, T.H. Plummer, a i d F. Maley,J. B i o l . C h o n . , 247, 2629 (1972). (111) H.G . Spiro, Ann. Rec;. Biocheni., 39, 599 (1970).

444

S. M. SNAITH AND G.

A. LEWY

that are believed to contain the L-asparaginyl bond have been shown to contain D-mannose. a. Ribonuclease B. -An aspartamido-oligosaccharide 112 isolated from ribonuclease B by proteolytic digestion contained six D-mannose and two 2-acetamido-2-deoxy-~-glucose residues per molecule. Jackbean a-D-mannosidase released five D-mannOSe residues per molecule. The sixth D-mannose residue was removed from the residual glycopeptide by periodate oxidation, whereupon 2-acetamido-2-deoxyp-D-glucosidase released one hexosamine residue. As the original workers suggested, the inner core of this glycopeptide appears to have structure 3 (shown for the product from ovalbumin). L-ASparagine could be removed from the glycopeptide (containing one D-mannose and two hexosamine residues) with glycoaspartamidase. On using a p-D-mannosidase preparation from hen oviduct, the D-mannose residue was removed from the same glycopeptide, suggesting the presence of a p-D-li~~k.'~ b. Pineapple Stem Brome1ain.-Another glycoprotein that is believed to have the same type of carbohydrate-protein linkage as ovalbumin is br~melain.'~J'~-"~ The composition of the carbohydrate moiety is one molecular proportion of L-fucose, one of D-xylose, two of 2-acetamido-2-deoxy-~-glucose, and two or three of D-mannose. Exhaustive treatment of the glycopeptide with a-D-mannosidase set free all but one D-mannose residue per molecule. D-Xylose and L-fucose residues could also be removed by the use of the appropriate glycosidases. The last D-mannose residue could be removed by treatment with periodate. When all of the neutral sugars had been removed, 2-acetamido-2-deoxy-~-~-glucosidase released one hexosamine residue per molecule. Once again, the glycopeptide seems to have a core of one D-mannose and two hexosamine residues per molecule, resistant to the action of a-D-mannosidase. c. Takaamylase.-Takaamylase contains six D-mannose and two 2-acetamido-2-deoxy-~-glucose residues in the carbohydrate chain. At least four D-mannose residues per molecule can be removed by treatment of the glycopeptide with a-~-mannosidase.l~*~~.~~."~,'~~ It (112) A. L. Tarentino, T. H. Plummer, and F. MaleyJ. Biol. Chem., 245, 4150 (1970). (113) J. Scocca and Y. C. Lee, J. Biol. Chem., 244,4852 (1969). (114) Y. Yasuda, N. Takahashi, and T. Murachi, Biochemistry, 9, 25 (1970). (115) Y. C. Lee, Fed. PTOC.,30, 1223 (1971). (116) H. Yamaguchi, T. Mega, T. Ikenaka, and Y. Matsushima,]. Biochem. (Tokyo),66, 441 (1969).

a-D-MANNOSIDASE

445

has been ~ u g g e s t e d ~ ~that J l ~the inner core of the molecule has structure 3, and it has been reported that the final D-mannose residue can be removed by the p-D-mannosidase from pineapple.10g

d. Further Examples.-a-D-Mannosidase, in conjunction with other glycosidases, has also been used in structural studies on the carbohydrate moieties of glycopeptides from human yG immunoglobuli n ~ , "human ~ yM immunoglobulins,11shuman o r o s o m u ~ o i dhuman ,~~~ chorionic gonadotrophin,lZ0rat-liver microsomes,lZ1and thyroglobulin.1229123 Purified a-D-mannosidase has been claimed to liberate D-mannose from o v o m u ~ o i dand ~ ~ from a glycopeptide from soy-bean hemagg1~tinin.I~~ Apart from those examples discussed in which purified a-D-mannosidase was employed, numerous other studies have been made of D-mannose-containing glycoproteins by purely chemical methods; for reviews, see articles by Marshall and Neuberge? Spiro,"' and Pazur and A r o n ~ o n . ~ ~ ~

(117)R. Kornfeld, J. Keller, J. Baenziger, and S. Kornfeld, J. Biol. Chem., 246, 3259 (1971). (118)S. Hickrnan, R. Komfeld, C. K. Osterland, and S. Kornfeld,J. Biol. Chem., 247, 2156 (1972). (119)P. V. Wagh, I. Bomstein, and R. J. Winzler, J . Biol. Chem., 244, 658 (1969). (120)0. P. Bahl, 1.Biol. Chem., 244,575 (1969). (121)Y.-T. Li, S.-C. Li, and M. R. Shetlar,J. Biol. Chem., 243, 656 (1968). (122)M.Fukuda and F. Egami, Biochem.]., 123,415 (1971). (123)T. Arima and R. G . Spiro,J. Biol. Chem., 247, 1836 (1972). (124)H.Lis, ZsraelJ. Chem., 6, 114P (1968). (125)J. H.Pazur and N. N. Aronson, Jr., Aduan. Carbohyd. Chem. Biochem., 27,301 (1972).

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AUTHOR INDEX FOR VOLUME 28 Numbers in parentheses are reference numbers and indicate that an author’s work is referred to, although his name is not cited in the text. A

Alfes, H., 91, 153(584) Alfredsson, B., 74, 141(504). 142(502), Abdel-Akher, M., 34, 90, 99(580) 194, 195, 196(127) Ali, S. S., 231 Abe, H., 343 Allan, G. G., 92 Abeles, R. H., 393 Acara, M., 392 Allen, F. W., 312 Achaya, K. T., 97, 152(628) Allison, J. B., 239 Acree, T. E., 40, lOZ(258, 259, 260, 262) Allsobrook, J. R., 59, 124(413) Acton, E. M., 53, 54(373), 115(373),269, Alper, R., 125(680),158 287 Amin, El S., 176 Acton, R. T., 16, 86(35), 124(35), 126(35), Aminoff, D., 218 146(35) Anderle, D., 36 Adair, W. L., 375 Anderson, B., 123(716,717), 159 Adam, A., 329 Anderson, D. M. W., 57, 99, 103(653), Adamamu, A. M., 64, 122(456) 120(400), 158, 187, 188, 190(loo), Adam-Chosson, A., 18 208(100) Adams, G. A., 15,28,29(156), 49, 59(341), Anderson, E. P., 336(209), 337, 341(209) 61,62(420), 72, 77(339), 81(341, 410), Anderson, G., 37 85, 86(420, 559), 94, 109(339, 662), Anderson, J. E., 281 111(341,342),122(558),123(492,560, Anderson, J. S., 340(239), 341, 367(239), 676), 124(420, 423, 475, 494, 559, 388,390 609), 125(341), 128(339, 411, 493, Anderson, L., 40, 102(263) 494, 544, 686), 136(493), 137(156), Anderson, N. S., 15, 135(690), 159 138(339,492,539, 662), 140(339,539, Ando, S., 55, 84(391a), 87, 112(391a) 662), 143(341,410,493,494,495,544, Anet, E. F. L. J., 163, 169, 172, 173(44), 177, 178(35), 181(45), 196(59), 201, 676), 145(420, 433, 558, 559, 560), 153(609,676), 158 202, 218 Adamson, J., 262 Angyal, S. J., 20, 39, 50(79), 88(79), Adamyants, K. S., 242, 282(59) 89(79), 103(79), 110(79),111(79, 351), Afanas’ev, A., 97 148(79), 150(577, 578) Agrawal, K. L. M., 404, 412(36), 424(36) Anjou, K., 73, 137(500) Ahluwalia, R., 39 Ankel, E., 364, 367(352), 386(437), 387 Aida, K., 186 Ankel, H., 319, 326(73), 364, 367(352), Akagi, M., 264 371,374, 376, 386(437, 440,441), 387 Akamatsu, N., 327 Anmo, T., 76 Akazawa, T., 314, 338(41), 395 Anno, K., 57, 119(397) Anraku, Y., 389 Alam, M., 126(696), 152(696), 159 Albano, E. L., 184, 220(82),267, 268(123), Anwar, R. A., 328, 329, 388(153) 273(123), 283, 293(123), 305(123) Arai, Y., 70, 133(491) Albersheim, P., 16, 17, 35(39), 61(39), 62, Arakawa, Y., 34, 37(213), 65(213), 87, 127(39, 40, 41, 42), 394, 395(495) 113(213),114(563) Albitzkaya, 0. N., 314, 315(43) Araki, Y., 329 Albrecht, G. J., 391 Arcos, M., 437 Alexander, R. J., 46, 102(293) Arima, T., 445 447

448

AUTHOR INDEX, VOLUME 28

Baine, H., 106(703), 159 Baird, J. K., 405 Baker, B. G., 58, 119(402) Balakhontseva, V. N., 97 Balduini, C., 43, 107(279) Baldwin, M. J., 274, 275(138) Ball, D. H., 12, 227, 271 Ballard, F. J., 364 Ballard, J. M., 239 Ballio, A., 319 Ballou, C. E., 24, 29, 89(111), 103(654), 105(700), 123(700), 150(111), 158, 159,405, 421 Balogh, S., 95 Bamburg, J. R., 14 Bandurski, R. S., 16, 17(43a), 27(43a), 102(43a),133(43a),152(43a) Banerjee, A. B., 312 Barber, G. A., 321, 322(92), 323,326, 338, 344, 348, 352(263), 382(136, 137), 383(93), 384(92,136, 137, 290), 394 Barker, H. A., 393 Barker, R., 167 Barker, S. A., 32,94, 152(608a) Barkulis, S. S., 348 Barnett, J. E. G., 225, 226(1), 227(1), 229, 278(1), 281(1), 305, 402 B Baron, D., 387, 388 Bachhawat, B. K., 332 Barrett, A. J., 15 Backinowsky, L. V., 56, 66(394a), 120 Bartnicki-Garcia. S., 92, 153(590), 337, 365(213) (394a) Baddiley, J., 47, lOO(647, 648, 649, 650), Barton, W., 116(709),146(709), 159 101, 105(306), 156(647, 649), 157 Bass, S. T., 312, 314,315(50), 338(49, 50), (649), 239, 308, 310(14), 319, 320 339(50), 340(50), 348(50), 391 (70), 322, 323(14), 325, 333, 334 Bassler, K. H., 47, 57(297), 102(297), (191), 344(103), 346, 358(126), 359 120(297) (103, 192, 200), 376(103, 104), 383 Bath, I. H., 219 Batista, J., 91, 153(582) (104), 390(335),393 Batt, R. D., 436 Baenziger, J., 445 Baer, H. H., 299 Bauer, k., 41, 54, 115(379),338, 357(230), Bagdian, G., 52, 54(357), 116(357), 359(230), 361,396(343) Baumann, V., 16, 21(33) 117(357) Baglan, N. C., 33, 37(208), 51(208), Baumann, W. E, 52, 116(362) 68(208), 105(208),112(208), 131(208), Bax, P. C., 350, 375(310) Bayer, E., 55, 112(391), 113(391), 152(208) 114(391),207 Bahl, 0. P., 29, 44(176), 48(176, 286), 98, 103(176),106(286),404, 405, 412(36), Bazinet, M. L., 255 Bdolah, A., 364, 367(353), 386(438), 387 420(54), 424(36),445 Baig, M. M., 76, 136(524) Beach, R. L., 270, 273(129), 299(129) Bailey, E., 44, 79(285) Beadle, J. B., 25, 31(133), 68, 70(133), Bailey, E. J., 258 133(134), 135(134)

Arita, H., 54, 115(374,375), 119(374,375), 147(374),303 Arita, M., 89, 149(575) Armbruster, C. W., 198 Arnold, Z., 250(82), 251 Aronson, N. N., Jr., 445 Arreguin, B., 95, 151(613) Arsenyuk, L. V., 23 Arvinen, A,, 20, 43(78), 88(78), 106(78), 147(78) Arzoumanian, H., 53, 54(373), 115(373), 269 Asano, K., 106(656), 158, 186 Ashton, G. C., 405 Ashwell, G., 324, 330, 338, 382 Aso, K., 170, 186, 190, 192, 207(119), 208 Aspinall, G. O., 21, 201 Asselineau, J., 50, 110(354), 111(354), 118(354) Assmann, K., 96, 154(618) Austrian, R., 376 Avigad, G., 41, 323, 369, 394 Axelrod, J., 364, 366(346), 392(483), 393 Axelsson, K., 64, 125(464),126(463) Axelsson, O., 16, 65, 129(47)

AUTHOR INDEX, VOLUME 28 Beaugiraud, S., 404, 412(37), 424(37) Becker, E. S., 49, 107(335) Beckmann, F., 212, 213(204) BeBlik, A., 67, 159 Behrens, N. H., 393 Behrenwald, E., 39 Bekker, P. I., 23, 64,123(460) Bell, R. H., 305 Bello, O., 370 BeMiller, J. N., 47, 54, 102(378, 651), 103(296, 651), 115(378), 131(378), 158, 169, 182, 201 Bennett, J. C., 16, 86(35), 124(35), 126(35), 146(35) Benoy, M, P., 162 Benson, A. A., 291 Bentley, R., 12, 23(5), 25, 28(5), 30(5), 32(5), 38(5), 40(130), 41(5), 42(5), W5), 59(5), 68(5), 79(5), 80(5), i00(5), 102(20l), 103(201) Berenson, G. S., 20, 75(76), 78(76), 103(76), 138(76, 513, 515, 517, 518, 519), 143(76, 541) Berezkhin, V. G., 97 Berger, S., 258 Bergkvist, R., 325 Bergmann, M., 216, 263 Bernheimer, H. P., 376 Bernstein, R. L., 338 Bemstein, S., 437 Berry, J. M., 13, 69, 115(665), 133(490a), 134(490a),151(490a),158 Berry, R. E., 20, 171, 176(41), 177(41), 179(41), 180, 200(41) Berst, M., 14, 19(18), 62(18, 447), 63, 92(18, 447), 122(18), 123(18, 447), 125(18, 447), 128(447), 154(18, 447) Bertland, A. U., 370(384, 385), 371 Bertolini, M., 85, 145(556) Besler, E., 230 Bethge, P. O., 29, 39, 40(172), 42, 107(172) Beveridge, R. J., 65, 124(469) Bevill, R. D., 55, 111(390), 118(390), 125(390), 153(390), 338, 348, 350 (291), 351(291), 371, 373, 382 (220) Beving, H. F., 63, 122(449) Bews, A. M., 187 Beychok, S., 123(716,717), 159 Bhaduri, A., 370

449

Bhattacharjee, S. S., 92, 98(596), 154(596) Bhatti, T., 13, 19, 21(67), 24, 26(118), 43(67), 48(118), 52(67), 53(67), 68(118), 69(67), 76(67), 81(118), 83(67), 107(118), 109(67), 117(67), 130(118), 133(67), 138(67), 139(67), 143(118),145(67) Bidwell, R. G. S., 106(702), 120(702), 159 Bieber, M., 32, 41(200), 102(200), 115(200),143(200),144(200) Biely, P., 327, 338, 353(149b), 357(149b, 230), 359(230),361, 396(343) Bijvoet, P., 184 Bilik, V., 54, 115(379) Bilow, A., 299 Binette, P., 125(680), 158 Binkley, R. W., 304 Binkley, W. W., 218, 304 Birge, C. H., 328, 329(159) Birkenmeyer, R. D., 226, 247(3) Birkofer, L., 212,213(204) Bishop, C. T., 12, 14, 25, 46(132), 54, 68(132),76(384), 84,86(554), 88(132), 91, 94, 95, 102(132), 116(383, 384), 123(674),124(609),131(132),153(581, 584, 585, 586, 587, 609), 158 Bissett, F. H., 255 Bjomdal, H., 62(432, 445), 63, 64, 122 (712), 123(445), 124(462, 678), 125 (432,464), 158, 159 Blacklow, S., 332 Blackwood, A. C., 49, lll(342) Blair, M. G., 199 Blake, J. D., 15, 22, 35, 62, 64, 65(26), 77(98), 122(98),127(26,426,427,428, 429, 430), 129(26, 98), 136(26) Blank, F., 91, 123(674),153(584), 158 Bloom, J. R., 45, 68(291), 108(291), 130(291), 131(291) Blumbergs, P., 242(60), 243, 323, 324 Blumsom, N. L., 308, 310(14), 322, 323(14), 344(103), 359(103), 376(103, 104), 383(104) Bobbitt, T. F., 28, 47(159), 57(159), 97(159), 103(159), 120(159), 152(159) Bock, K.,264, 266 Boggs, L. A., 37 Bolan, M., 54, 118(386) Bolden, A. H., 312 Bolton, C. H., 52, 82(364, 365), 116(364, 365), 144(364, 365), 146(364, 365),

450

AUTHOR INDEX, VOLUME 28

256, 291(93) Bondarenko, 0. D., 105(655),158 Bonnard, M.-C., 36, 50(224), 70(224), 87(224), 89(224), 112(224), 113(224), 114(224) Bonner, T. G., 164(16),165 Borchardt, L. G., 36,62, 66, 127(220) Boren, H. B., 63, 122(449) Bornstein, I., 445 Bose, R. J., 21, 40(84), 98(84) Bosmann, H. B., 126(684),158, 403 Bosshard, H. H., 250 Bosshard, P., 212, 213(199) Botlock, N., 25, 40(130) Bott, H. G., 188 Bourne, E. J., 47, 57, 72(399), 103(399, 699), 105(305), 119(699), 120(305, 399), 136(399),149(699),159,164(16), 165 Boussac, G., 36, 50(224),70(224), 87(224), 89(224), 112(224), 113(224), 114(224) Bouveng, H. O., 48, 103(333), 107(333) Bowden, B. N., 29, 68(174), 106(174), 131(174) Bowden, G. L., 28 Bowker, D. M., 59, 126(412) Bowser, A. M., 337, 364(213a),365(213a) Boye, H. A., 341 Bradford, B., 40, 43(256), 81(256), 108(256),143(256) Bradley, P. R., 261, 305 Brady, R. F., Jr., 27, 40 Brady, R. O., 84, 116(552a), 144(552a) 146(552a) Bragg, P. D., 34, 230, 231(21) Braley, K. L. K., 21 Braude, A. I., 52, 54, 77(381), 116(358), 117(381) Brauns, F., 19, 20(73), 23(73) Bredereck, K., 31 Breitmaier, E., 55, 112(391), 113(391), 114(391) Brenner, S., 341 Bretthauer, R. K.,338 Breuninger, E. R., 59, 64(411), 128(411) Brimacombe, J. S., 32, 184, 271 Brini, M., 41, 109(270), 110(270), 138(270) Brobst, K. M., 13, 25, 46(127), 68(127), 102(127), 132(127),151(127) Brogioni, M., 123(714), 159

Brooks, C. J. W., 31, 98 Brooks, D., 358, 390(335) Brossmer, R., 182 Brovelli, A., 43, 107(279) Brower, H. E., 42, 107(271) Brown, D. H., 341,394 Brown, D. M., 201,279 Brown, E. G., 326 Brown, J. F., 40 Brown, J. G., 316, 318, 360(66), 379(65), 380(65), 382(65, 66) Brown, P., 116(709),146(709), 159 Brown, R. G., 124(677),158 Brown, R. K., 274, 275(138),295(189), 296 Bruckner, J., 223 Brug, J., 182 Brumm, A. F., 310 Brundish, D. E., lOO(649, 650), 101, 156(649), 157(649) Brush, P., 57,72(399), 103(399), 120(399), 136(399) Bryan, J. G. H., 271 Buchala, A. J., 35, 107(705), 126(218a), 153(705), 159 Buchanan, J. G., 239, 325, 333, 334, 358(126), 359(192, 200) Buck, K. W., 123(718), 154(718), 159, 246 Budowsky, E. I., 311, 348(294, 295, 296, 299,300,301,302,303,304,305,306, 307, 308), 349, 350(299), 351, 354, 355,356,360(331), 361(339),362(341, 342), 366(294), 367(339), 368(342), 374(295), 375(339), 394(339), 396 (302), 398(339) Bugge, B., 370(384), 371 Bukhari, M. A., 280(150, 151), 281 Bukwa, W., 42, 105(275) Bullock, S., 422 Buncel, E., 231, 232, 233(27, 28), 235(27), 261,305 Bunton, C. A., 356 Burger, M., 334 Buriks, R. S., 198 Burk, D., 417 Burks, M. L., 24, 48(121), 68(121), 105(121), 130(121) Burnett, B. B., 35, 62, 127(218) Burton, R. M., 336(209), 337, 341(209) Butterworth, R. F., 299(200), 300 Butts, W. C., 27, 44, 68(149), 108(149), 130(149), 131(149)

AUTHOR INDEX, VOLUME 28 C

451

Chau-Yang Chen, 72, 136(497) Chelibonova-Lorer, K., 319 Cabib, E., 308, 310, 311(9), 319(13), 327 Cheminat, A., 41, 109(270), 110(270), (9), 335, 359(9, 13), 389, 391 138(270) Cabrera, M., 52, 76(359), 116(359) Cherniak, R., 106(703),159 Cahir, M., 276 Cheshire, M. V., 127(731),160 Calkins, E., 125(680), 158 Chia, L. H. L., 39 Cantor, S. M., 162, 170(5),179(5) Chihara, G., 70, 133(491) Cantore, M. L., 314, 321(48), 325(48) Chimenti, M., 43, 109(278) Capon, B., 356 Chin, P. S., 88, 147(565a) Caputto, R., 308, 325(8), 356(8) Chin, T., 26, 46(138), 48(138), 106(138), Cardais, A., 329 119(138),149(138) Cardini, C. E., 308, 310, 311(9), 325(8), Chin, W., 329 327(9), 356(8), 359(9), 376, 394, Chittenden, G. J. F., 325, 358(126) 395(496),396(507, 515) Chiu, T. H., 393 Carlsen, R. B., 124(721),159 Chizhov, 0. S., 56, 66(394a), 120(394a) Carlson, D. M., 318, 338(68), 339(68), Chojnacki, T., 342 348,391(68) Choy, Y. M., 47, 57(311), 93(311), Carlsson, B., 40, 76(261), 136(520, 521), 105(308, 311), 120(311) 138(520), 139(261), 167, 168(25, 26, Christensen, A,, 370 27), 206(25, 26, 27) Christensen, J. E., 267, 287 Carlyle, J. J., 21 Christensen, T. B., 412, 424(64), 431(64), Carminatti, H. 311, 389, 391 434(64) Carpenter, S.J., 437 Christianson, D. D., 312 Carroll, W. R., 17, 124(54),129(54) Chums, S. C., 64, 123(460, 719), 159 Carss, B., 333, 359(192) Cifonelli, J. A., 48, 79(331), 106(331), Carter, H. E., 24, 40, 88, 104(568), 142(331), 166, 325, 326(119), 327 142(568), 147(568), 148(568), 149 (119) (568), 153(254) Cifonelli, M., 166 Casagrande, D. J.. 114(664), 158 Cipera, J. D., 182, 220(79) Casebier, R. L., 159 Clamp, J. R., 13, 14, 18, 19, 21(66a, 67), Casinovi, C., 319 24, 26(118), 28, 48(86, 118), 52, 53, Cassels, A. C., 314 68(118), 69, 71(20), 76(20, 66a, 67), 81(118), 82, 83(67, 366), 107(118), Catala, F., 176 Cathey, G. W., 24, 25(117), 48(117), 109(67), 116(86, 364, 365, 709), 68(117), 105(117),130(117) 117(67, 366), 130(118), 133(67), Cayle, T., 24, 35, 58(113), 68(113), 138(67), 139(67), 140(20), 143(118), 119(113),132(113),134(113) 144(86, 364, 365, 366), 145(67), Cepure, A., 375 146(364,365,709),159,438,439(102) Cerma, E., 43, 109(278) Clark, H. P., 334 Cern>;,M., 278 Clarke, J. T. R., 27, 116(150b) Chaberek, S., 422 Clauson-Kass, N., 212, 216 Chambaz, E. M., 28 Clayton, D. W., 20, 21(83), 29(83), 30, 49, Chambers, R. E., 13, 18, 19, 21(66a, 67), 70, 85, 107(83), 110(188), 135(188), 28,43(67), 53(66a, 67), 69(67), 76(67), 145(188) 83(67), 109(67), 117(67), 133(67), Clode, D. M., 268 138(67),139(67),145(67) Clover, P., 334 Chang, M. T. Y., 326, 382(137), 384(137) Codington, J. F., 53, 83(368), 116(368), Chararas, C., 405 144(368) Chargaff, E., 311 Coe, D. G., 239 Chatterjee, A. N., 329,334 Cohen, M. R., 18, 27(60),44(60), 106(60),

452

AUTHOR INDEX, VOLUME 28

144(60) Cohn, W. E., 311 Coleman, J. E., 423, 433 Collins, D. C., 437 Colman, R. F., 423 Comb, D. G., 182, 329, 332, 358(182), 376, 388 Conchie, J.. 18, 53(64), 81(64), 82(64), 116(64), 144(64),402, 403(5), 404(11, 28, 40), 405(4), 407(15), 408, 416, 419(65), 420(41, 65, 68), 421(12), 434(11, 12, 28), 436(12, 28), 437(28), 438,439( 103,104,105),443(105,107) Connor, A. H., 40, 102(263) Conrad, C. M., 186, 189, 192(92) Conrad, H. E., 14 Conrow, R. B., 437 Cooper, D. J., 26 Cooper, F. P., 12, 54, 84, 86(554),91, 95, 116(383), 123(674),153(581), 158 Copenhaver, J. H., 26,46, 104(137) Corbett, W. M., 201, 205 Corey, E. J., 281 Comer, J. J., 392 Cornwell, D. G., 97 C o d a , C., 91, 153(582) Cosmatos, A., 182 C M , W. A., 89, 148(571) Cottrell, A. G., 231,233(27,28),235(27) Courtois, J. E., 403, 404, 405, 412(37), 424(37) Coxon, B., 27, 65, 102(150a), 113(471), 236(39), 237, 238(39), 283 Cramer, F., 346 Craven, D. A., 24, 28(114), 29(114), 146(114) Cree, G. M., 99 Creech, H. J., 59, 64(411), 128(411) Crestfield, A. M., 312 Creveling, C. R., 370 Cromwell, C. L., 18, 27(60), 44(60), 106(60), 144(60) Croon, J,, 22 Cross, A. D., 258 Crowell, E. P., 35, 62, 127(218) Crum, J. D., 195 Cuadriello, D., 258 Cumming, D. F., 310, 311(20), 314(20), 315(20), 318(20), 319(20), 322(20), 33l(20) Cunningham, W. L., 405

Curtius, H.-C., 24, 25(120), 38(119, 120), 40(119), 41, 68(265), 102(119), 103(265, 652L 131(687), 132(265), 158 Cutolo, E., 336 Cynkin, M. A., 341 Cyr, M. J. S., 19,20(74),97, lOO(630)

D DaAboul, I., 184 . Dahl, J. L., 367 Dalferes, E. R., Jr., 20, 75(76), 78(76), 103(76), 138(76, 513, 515, 516, 517, 518,519), 143(76,541) Damiani, P., 123(714),159 Daniher, F. A., 324 Danishefsky, I., 364, 367(349a) Dankert, M., 310, 311, 314, 315(40), 339(51), 340(51), 342(51), 346(22), 357(22), 358(22),359(22), 389, 390 D’Appolonia, B. L., 125(679), 158 Darby, G. K., 75, 138(514) Darrow, R. A., 370 Das, I., 349 David, H. L., 329 David, S., 24, 109(110),120(110) Davidson, E. A., 348(298), 349, 356, 376, 378(409) Davis, C. E., 54, 77(381), 117(381) Davis, J. G., 319, 329(81),335(81) Davis, L., 373 Davison, P. K., 68, 104(477), 123(477), 130(477),132(477) Davoll, J., 263 Dawes, K., 20, 39, 50(79), 88(79), 89(79), 103(79), 110(79),111(79), 148(79) Dawson, G., 21, 28, 32, 41(200), 48(86), 52(86), 82(86), 102(200), 115(200), 116(86, 365), 143(200), 144(86, 200, 365), 146(365) Dea, I. C. M., 99 Deboe, J., 18, 27(60), 44(60), 106(60), 144(60) Debris, M. M., 405 de Bruyn, D. C., 64,125(459) De-Bruyne, C. K., 15 Declerck, D., 67, 96(473), 121(473), 128(473), 155(473) deDuve, C., 408 Defaye, J., 165, 166(18), 176, 209, 287, 307

AUTHOR INDEX. VOLUME 28

453

de Fekete, M. A. R., 395, 396(515) Dobereiner, U., 387 Dods, R. F., 253 Degani, C., 201 Dolan, T. C. S., 15 d e Grandchamp-Chaudun, A., 39 DeJongh, D. C., 32, 41(200), 102(200), Dolhun, J. J., 27 Domkin, V. D., 360 115(200),143(200), 144(200) Dejter-Juszynski, M.,69, 85(487), 134 Donaldson, B., 40, 43(256), 81(256), (487), 145(487) 108(256),143(256) Dekker, C. A., 166, 190(21) Dong, T., 405 Delman, M. R., 48, 84, 105(329), Doniger, R., 311 120(329),145(329) Donike, M., 28 DeLuca, S., 386(439), 387 Donovan, J. W., 319, 329(81), 335(81) de Minia, M., 48, 68(322), 104(322), Dooms, L., 67, 96(473), 121(473), 128 131(322), 132(322) (473), 155(473) Dempsey, A. M., 239 Dorfman, A., 52, 116(355),325, 326(119), Demushkin, V. P., 348(300), 349 327(119),376 Denamur, R. E., 319, 320, 321(76), Doudoroff, M., 393 322(85), 326, 331 Douglas, H., 52, 54, 77(381), 116(358), de Neef, J., 46, 108(292), 131(292) 117(381) Derevitskaya, V. A., 202 Douglas, L. J., 390 Deriaz, R. E., 165, 184(19),224(19) Downie, I. M., 246 de Robichon-Szulmajster, H., 370, 371 Doyle, R. J., 184, 220(83) (372), 398 Drews, G., 62(446),63, 126(446) Descotes, G., 276 Dreywood, R., 221 Desnick, R. J., 52, 116(361) DrGge, W., 52, 54(357), 62, 116(357), DeSouza, B. C., 395, 396(507) 117(357), 123(434), 141(434), 153 Deuel, H., 187, 189(102),208(102) (434) Deutschman, A. J., 115(665). 158 Druzhinina, T. N., 348(294), 349, 360, de Wilt, H. G. J., 28, 141(166) 361(339), 365, 366(294), 367(339, Dhar, M. M., 286, 287(168c), 293(168c) 361), 370, 373, 374(339, 364, 377, 294(168c) 392), 375(339, 364, 394), 394(339, 364), 395(364),398(339) Dick, W. E., 50, 55(349), 58, 111(349), 115(349), 118(349), 119(402), 131 Dryselius, E., 201 Duarte, J. H., 91, 122(583), 153(582, 583) (349) Duberhard, L., 329 Dickerson, J. P., 242(60), 243 Dubois, M., 223 Dietrich, C. P., 312 Duckworth, M., 65 Dietrich, S. M. C., 312 Dugan, F. A., 75, 138(515) Dietzler, D. N., 323, 324, 388 DiGirolamo, A., 323, 376(104), 383(104) Dumaisnil, J., 22 DiGirolamo, M., 323, 376(104), 383(104) Dunham, C. W., 48, 68(323), 106(323), Dijong, I., 84, 116(552a), 144(552a), 130(323) Dunlop, A. P., 163, 176, 212, 213(198) 146(552a) Dimant, E., 282 Duschinsky, R., 226 Dimitrijevich, S., 270, 293 Dutton, G. G. S., 32, 58, 60, 61(418), 64, Dimler, R. J., 20, 88(80), 147(80) 65(403, 467), 69, 77(461), 78(467), 92, 93, 94(598), 95(608), 100(207), Dinglinger, F., 89, 122(576), 150(576) 105(701), 107(608), 129(467), 133 Dische, Z., 223, 224 (490a), 134(490a),141(588), 151(490a, Distler, J. J., 328, 346, 350(278) Dluzniewicz, L., 392 608, 701), 152(608), 156(207, 639, Dmitriev, B. A., 56, 66(394a), 120(394a) (642), 157(640,641, 642), 159 Dutton, G . J.. 308, 326(lo), 392 Dmytraczenko, A., 91, 153(582) Dutz, R., 212 Doane, W. M., 283

454

AUTHOR INDEX, VOLUME 28

Duxbury, J. M., 290 Dvorak, H. F., 389 Dvorchik, D. H., 209

E Eabom, C., 31, 32(195) Earle, N. W., 16, 62(40), 127(40) Easterwood, V. M., 56, 120(394) Eastman, J. F., 171, 194(42) Ebert, W., 18, 61(60a), 86, 126(60a), 144(60a) Ebner, K. E., 336, 370 Echigo, T., 48, 68(315), 104(315), 133 (315) Edgar, R. H., 162 Effland, M. J., 176, 184(52), 207(52), 209(52), 219(52),220(52), 223(52) Egami, F., 405, 406(45),412(45), 416(45), 420(45), 424(45), 439(45), 444(45), 445 Eilers, N. C., 36 Eilingsfeld, H., 250(81), 251 Einset, J. W., 40, 102(259) Eisenberg, F., 38, 51(232), 119(232), 121(232),151(232),312 Elbein, A. D., 27, 53(155), 115(155),316, 318(57, 58), 322, 348, 350, 351(312), 382(58, 101), 385(97), 394(281), 396 Elcoate, P. V., 436 El-Dash, A. A., 38,58(234),93,171,196 Eldridge, J. M., 77, 140(536) Eliseeva, G. I., 346, 348(300, 301, 307), 349, 354, 360, 361(339), 362(341, 342), 367(339), 368(342), 374(339, 364), 375(339, 364), 394(339, 364), 395(364),398(339) El Khadem, H., 169 Elliott, W. H., 32,103(202),380,382(425) Ellis, D. B., 378 Ellis, W. C., 30, 44, 45(289), 107(182), 109(289), 110(182, 289), 116(289), 120(289),142(289),149(289),152(289) Elnaghy, M. A., 314, 315(46),333(46) Elson, D., 311 Elson, L. A., 287 Endo, A., 343 Endo, M., 319 Engen, R. J., 159 English, P. D., 16, 17(39),35(39), 61(39), 62(39), 127(39) Enstrom, B., 62

Entlicher, G., 54, 102(378), 115(378), 13l(378) Epley, J. D., 14 Erbing, C., 62(445), 63, 123(445) Espada, J., 338, 343(218) Esposito, G. G., 26, 97(136), 151(627) Ettlinger, M., 420, 421(71) Ettre, L. S., 13 Etzler, M. E., 123(716),159 Eugster, C. H., 212, 213(199),218 Evans, M. E., 54, 115(380), 225, 256, 286(94) Evans, P. J., 393 Evans, W. L., 162 Eveleigh, D. E., 402, 405, 421(8, 51) Evtushenko, E. V., 37, 56(231), 67, 118(231), 121(231)

F FBhraeus, G., 62(445), 63, 123(445) Failia, D. L., 276(142), 277 Fan, D. F., 47, 103(312), 368, 370, 374(378), 375(378) Fanshier, D., 340(242), 341, 343(242), 375(242) Fantes, K. H., 405 Farkai, J., 303 FarkaH, V., 361, 396(343) Farr, A. L., 410, 435(59) Farrell, D. G., 319, 326(73) Farshtchi, D., 45, 108(290), 120(290) Fauconneau, G., 319, 320, 321(76), 322(85) Fazakerley, H., 258 Feast, A. A. J., 141(693), 159, 194, 196(126) Feather, M. S., 75, 141(512), 165, 168, 176, 178, 179(17), 188(54), 192(54), 194(17),201, 203(54), 207 Fedorohko, M., 40, 174 Feingold, D. S., 47, 103(312), 319, 326(73), 337, 343, 364(213a), 365 (213a), 366(349), 367(350, 352, 353, 357), 368, 370, 374(378), 375(378), 376, 386(212, 437, 438, 440, 441), 387 Feldman, G. L., 21 Fennessey, P., 390 Fern, V. H., 437 Fernell, W. R., 223 Ferrier, R. J., 33, 202, 203, 204(172), 260,

AUTHOR INDEX, VOLUME 28

455

261, 264, 290 Fraga, E., 292 Filatova, T. N., 97 Franken, H., 188 Filleux-Blanchard, M. L., 250 Franz, G., 16, 62, 126(44), 127(44), 326 Findlay, J., 402, 403(5), 404(14, 28), Fraser, C. G., 16, 35, 50, 92(345), 421(12), 434(12, 14, 28), 436(12, 28), 111(345), 122(50a), 126(218a), 153 437(28) (sOa), 154(345) Fink, A. L., 50, lll(352, 353) Fraser, R. N., 44, 48(287), 49(332), Finkbeiner, H. L., 26, 30, 32(183), 147 103(333), 106(287, 332), 107(332, 333), 108(287) (141) Finkelstein, J. A., 16, 17(34a), 36(34a), Fraser-Reid, B., 288, 289, 305, 306 61(34a), 86(34a), 90(34a), 128(34a), Frear, D. S., 392(484),393, 394(484) 146(34a), 154(34a) Fredricks, P. S., 54, 75(382), 117(382) Fischer, E., 263, 287 Freedland, R. A,, 325, 326(124) Fischer, F. G., 167 Freedman, S. D., 54, 77(381), 117(381) Fischer, M. H., 25, 88(131), 147(131) Frerman, F. E., 390 Fischer, M. I., 436 Fretz, T. A,, 48, 68(323), 106(323), Fischer, W., 341, 373(249, 250), 396 130(323) Fishbein, L., 176, 177 Freudenberg, K., 227, 291 Fisher, D., 257, 258(98) Frey, M. J., 16 Fisher, R. G., 376 Frieden, C., 418 Fishman, W. H., 349 Friedman, S., 30 Fitelson, J., 28 Fries, I., 32, 103(202) Fitzgerald, D. K., 336 Fromme, I., 62(442), 63, 122(450), Fleming, M., 100 125(431), 126(442),128(431) Fletcher, A. P., 438, 439(101) Frotz, H., 44, 108(280,281, 282, 283) Fletcher, H. G., Jr., 68, 130(478), 165, Frydman, R. B., 340(241), 341, 375(241), 172, 348, 377, 378(413) 394, 395, 396(507) Fletcher, T. L., 30 Fuhr, B. W., 24,81(112), 89(112), 90(112), Flint, D. R., 29, 89(178), 90(178), 109(112), 120(112), 143(112), 149 149(178) (112), 152(112) Flood, A. E., 271 Fujimaki, M., 106(656), 158 Flores, E. F., 48, 58(318), 68(318), Fujimoto, Y.,351 104(318), 119(318), 130(318) Fukuda, M., 445 Flowers, H. M., 69, 85(487), 133(488), Fukui, S., 344 134(487),145(487) Fukuoka, F., 70, 107(658), 133(491), 158 Flynn, C., 48,58(317), 104(317),119(317), Funahashi, S., 326, 394 Furdik, M.,54, 115(379) 130(317) Fodor, G., 169 Furukawa, Y., 102(697), 115(697), 159, Folsom, M. W., 42, 107(271) 346, 349(271) Foltz, R. L., 33 Forlini, J. D., 96, 154(619) Forrester, I. T., 16 Gabriel, O., 330, 338, 356, 375, 379(329), Foster, A. B., 182, 195,246, 262, 280(150, 380(329), 382(329),384 Gabrieljan, N. D., 348(294, 302), 349, 151), 281, 290 Foster, D. S., 423 360, 361(339), 366(294), 367(339), Foster, D. W., 384 374(339, 364), 375(339, 364), 393, Fournier, L. B., 24, 48(121), 68(121), 394(339, 364, 502, 503), 395(364), 105(121), 130(121) 396(302), 397, 398(339) Fowler, W. F., Jr., 186, 187(90), 192(90) Gagnaire, D., 209 Fox, J. J.. 345 Galanos, C., 19, 86(72), 146(72) Fraenkel, D., 339 Galloway, B., 326, 334

456

AUTHOR INDEX, VOLUME 28

Gamborg, 0. L., 29 Ganguli, N. C., 312 Garbutt, J. T., 46, 102(293) Garbutt, S., 187, 188, 190(100), 208(100) Gardiner, D., 88, 95, 148(566), 152(612) Garegg, P. J., 60, 62(443, 444), 63, 86(443), 95, 122(449), 124(443, 444), 125(414), 127(414, 685), 128(414), 153(615),158 Garg, H. G., 261 Gargani, G., 107(659),138(659), 158 Garrett, E. R., 209, 215, 216(212) Gastambide-Odier, M., 18, 47, 55(65, 301), 105(301), 118(65), 123(301), 143(301) Gauche], F. D., 47, 57(297), 102(297), 120(297) Gaugler, R. W., 375 Gaver, R. C., 24 Gawne, G., 250 Gaye, P. J.-B., 326, 331 Geddes, D. S., 92, 154(593) Gee, M., 135(688), 158 Gehrke, C. W., 24, 28(114), 29(114), 80, 113(152),146(114) Gelman, A. L., 438, 439(103) Gentner, N., 337 Geppert, G., 96, 154(618) Gerecs, A., 287 GBro, S . D., 249 Gessner, T., 392 Geyer, H. U., 49, 104(340), 111(340), 123(340), 131(340), 132(340), 153 040) Ghalambor, M. A., 333 Gheorghiu, T., 16, 21(33, 34), 29(34), 33, 39(34), 43(34), 44, 46(34), 69(34), 76(34),80, 108(34,280,281,282,283), 139(34),143(34),150(34) Ghosh, H. P., 395, 396(509) Gibbs, C . F., 237 Gibbs, M., 195, 197(135) Gibney, K. B., 58, 65(403), 92, 94, 95(608), 107(608),141(588),151(608), 152(608) Gilani, S. S. H., 246 Gilbert, J. M., 318, 379(65), 380(65, 418), 382(65) Gilles, K. A., 100, 125(679),158, 223 Ginsberg, V., 81, 106(545),143(545),307, 308, 314, 315(47), 316, 319(47), 320,

321, 322, 324, 325(12), 326(12, 47), 333, 338(17), 340(242), 341, 342(117), 343(242), 375(242),384(99) Glaser, L., 324, 327, 334, 336, 338, 339(116), 341, 342(116), 356, 371, 373, 376(116), 378(412), 379, 380 (330), 382(421, 425), 383(420), 384 (429), 388(116),389 Glaudemans, C. P. J., 17, 124(54), 129(54) Gleason, W. B., 167 Glebova, Z. I., 285 Glinski, R. P., 242(60), 243 Gochnauer, T. A., 47, 104(309), 128(309) Godman, J. L., 58 Gold, H., 259 Goldberg, I. H., 338, 367(228) Goldberg, N. D., 367 Goldberg, R., 404 Goldemberg, S. H., 395 Goldman, D. S., 329 Goldstein, I. J., 98,99 G6mez SBnchez, A., 213 Gompertz, S. M., 376 Goncalves, I. R., 389, 394 Gonzales, N. S . , 326, 327(130) Goodman, L., 53, 54(373), 115(373), 267, 269, 287 Goodwin, S. D., 64, 122(454) Goodwin, S. L., 36, 90, 104(579), 119 (579), 149(579) Gorin, P. A. J., 50, 89, 92(346), 98(596), lll(343, 663), 122(663),123(346,595, 675), 124(595), 148(570), 150(577), 154(346, 595, 596), 158, 405, 421(51) Goring, D. A. I., 68, 132(482) Gormus, B. J., 125(724),159 Gorovits, T. T., 76, 117(527), 139(527) Gorshkova, R. P., 47, 120(310a) Gorz, H. J., 392 Goshima, K., 185, 186(88) Gotan, T., 343, 359(256) Goto, R., 200, 219 Gottschalk, A., 182, 437 Goudsmit, E. M., 319,379 Gould, E. S., 190 Gourlay, G. C., 421, 422(76), 431(76) Grachev, M. A., 348(300, 304), 349 Grasslin, D., 17, 44(51) Grassner, H., 39, 198 Grasso, C., 107(659), 138(659), 158 Gray, G. R., 105(700), 123(700), 159

AUTHOR INDEX, VOLUME 28 Gray, G. W., 47, 106(307) Green, J. W., 64, 77(465), 195 Greenberg, E., 337, 339,395, 396(506) Gregoire, Jana, 319, 327(75) Gregoire, Jean, 319, 327(75) Gregory, N. L., 57, 88(401), 96, 119(401), 147(401), 152(401) Greiling, H., 17, 44, 58(59), 86, 107(59), 119(59) Griffiths, D. W., 198 Griggs, L. J., 16, 17, 36, 61(34a), 86, 90, 128(34a),146(34a), 154(34a) Grimes, W. J., 394, 395(494) Grisebach, H., 326, 358(127), 387, 388 Gross, J. I., 48, 79(331), 106(331), 142 (331) Gruezo, F., 123(716, 717), 159 Guilloux, E. R., 305 Gump, W., 212(206), 213 Gunetileke, K. G., 328, 388(153) Gunner, S. W., 59, 84 Guntz, G., 319, 321(76) Gurin, S., 222 Gustine, D. L., 387 Gutowski, G. E., 242(60), 243 Gutsche, C. D., 198

H Haack, A., 250 Haahti, E. O., 14, 44, 48, 67, 75(19, 475), 79(330, 475), 106(228, 330), 138(19, 475), 142(330), 143(475) Haarmann, R., 166 Haddock, J. W., 325, 358(126) Hammerling, G., 14, 63(17), 122(17), 126(729), 160 Haga, K., 247 Hagiwara, S., 65, 114(471a) Haglund, P., 34, 35, 61(219), 78(215), 125(219), 127(215),136(215) Hakomori, S., 48, 82(334), 144(334), 146(334) Halford, M. D. A., 40, 144(253) Hall, C. W., 308, 320, 338(17), 365, 367(358) Hall, L. D., 95 Halmann, M., 201 Halpern, Y., 46, 88, 102(294), 147(567) Hamazaki, H., 76, 105(528) Hamilton, J. K., 34, 49, 67, 90, 99(580), 107(335), 159, 223

457

Hamlen, R. A., 45, 68(291), 108(291), 130(291),131(291) Hamor, T. A., 271 Hampe, M. M. V., 326 Hamuro, J., 70, 133(491) Han, M., 127(685),158 Hancock, I. C., 393 Hanessian, S., 32, 41(200), 102(200), 115(200), 143(200), 144(200), 226, 227, 242(60), 243, 249, 250, 251, 252(83), 253(83), 254(83), 276(141, 143, 144, 145), 278(83), 286(83), 299(200),300, 302 Hannaford, A. J., 39 Hansen, R. G., 312, 314, 315(50), 318, 325,326(124), 338(49,50,68),339(50, 68), 340(50), 348(50), 391(68) Hansson, J.-A., 34, 42, 62, 78(216), 106(272),107(273,274), 125(422,423, 424) Harada, T., 47,57(310), 93(310), 104(310), 152(310),388 Hardegger, E., 239, 285(47) Hardell, H. L., 15 Hardy, F., 239 Harel, E., 348 Harmey, M. A., 314 Harnden, M. R., 32 Harper, E. M., 325, 326(118),327(118) Harris, D. W., 178, 182(63) Harris, J. F., 75, 141(512), 165, 176, 179(17), 188, 194(17), 195, 197(134), 198(134),201(134),207 Harris, M., 188 Harris, P. J., 126(683),158 Harris, T. M., 48, 49(321), 68(321), 107(321), 130(321) Harder, N., 34, 42, 62, 78(216), 106(272), 107(273,274), 125(422) Hartman, L., 92, 154(597) Hartree, E. F., 21 Hanvalkar, V. R., 403 Hase, S., 47, 87, 89(564), 93(300), 148(564), 152(300) Hashimoto, K., 48 Hashizume, T., 166, 190(21) Haskin, M. A., 390 Haskins, R. H., 92, 98(596), 154(596) Haskins, T. A., 392 Hassid, W. Z., 307,308,319,320,325(12), 326(12, 83), 338, 340(241, 242), 341,

458

AUTHOR INDEX, VOLUME 28

343(242), 348, 362, 375(241, 242), 376, 386(402), 390(2), 392(215), 393, 394(2) Hassner, A., 270(131),271 Hata, T., 352 Hattori, Y., 15, 46(31), 108(31) Haug, A,, 21 Haustveit, G., 49, llO(338) Haverkamp, J. P., 69, 133(485) Hawthorne, J. N., 40, 153(254) Hay, A. J.. 18, 53(64), 81(64), 82(64), 116(64), 144(64), 403, 404(14, 26), 405, 406(46), 407(15), 409(46), 411(46), 412(46), 415(46), 416(46), 418(46), 419(46, 65), 420(65, 68), 424(46), 432(46),433(46),434(14,26), 436(26),438,439( 105),443(105,107) Hay, G. W., 50, 98, 99, llO(352, 353), 123(673),158 Hayami, J., 200 Hayaski, H., 76 Haylock, C. R., 246(68), 247, 254(68), 303(68) Haynes, F., 201 Hearn, V. M., 64,122(454) Heath, E. C., 322, 333, 343, 382(101), 385(97) Heath, M. F., 107(706),126(706),159 Heatherington, K., 100(647), 101, 156 (647) Heim, P., 206 Heinz, R., 299 Helferich, B., 230, 239, 291, 404 Hellerqvist, C. G., 14, 17, 19(15, 18), 60(16), 62(15, 16, 18, 53, 435, 436, 437, 438, 439, 440, 441), 63, 69, 86, 92(18), 122(18), 123(18), 125(435, 436, 437, 438, 440, 725), 126(15, 16, 53,439,441, 681,727, 728), 127(730), 128(18),154(18),158, 159, 160 Hemming, F. W., 393 Hemsworth, B. A., 403 Henly, R. S., 31 Heppel, L. A., 389 Herbert, R. W., 186, 192(93) Hergert, H. L., 159 Hbrissey, H, 404 Hermarm, K., 382 Herold, C. P., 50, lll(348) Herr, M. E., 254 Herrsbom, G., 22

Hess, K., 255 Hesse, G., 207 Hey, A. E., 316, 318(58), 382(58) Heydanek, M. G., 390 Heymann, H., 348 Heyns, K., 182 Hibbert, H., 19, 20(73), 23(73), 212(208), 213 Hickman, S., 445 Higginbotham, J. D., 47, 64(302), 93, 105(302), 123(302), 143(302), 152 (603) Hill, A., 92, 122(592), 154(592), 156(592) Hill, A. S., 41, 102(267) Hill, E. A., 373 Hill, J., 256 Hill, K., 404 Hill, M. E., 258 Himmen, E., 291 Hinnenkamp, E. R., 33 Hiraguri, Y., 19, 52(68), 116(68) Hirano, S., 358(337), 359, 362, 399 (344) Hirst, Sir E. (L.), 17, 103(653), 135(52), 158, 186, 188, 192(93) Hiu-I-Jan, 36, 50(224), 70(224), 87(224), 89(224), 112(224), 113(224), 114(224) Hixon, R. M., 239 Hockenhull, D. J. D., 405 Hocking, J. D., 436 Hodge, J. E., 38, 50, 55(349), 58(234), 93, 111(349), 115(349), 118(349), 119 (402), 131(349),171, 179, 196 Hoermann, H., 214,221 Hoff, G. P., 162 Hoffman, G. C., 92, 154(594) Hoffman, J., 125(725),126(727), 159 Hofman, A,, 218 Hoge, R., 238 Hogness, D. S., 370 Hoi, C. T., 394 Holden, K. G., 16, 17(34a), 36(34a), 61 (34a), 86(34a), 90(34a), 128(34a), 146 (34a), 154(34a) Holla, K. S.,97 Holligan, P. M., 13 Holmberg, M., 370 Holme, T., 14, 19(15), 60(16), 62(15, 16, 444), 63, 124(444), 125(414), 126(15, 16, 681), 127(414), 128(414), 158 Holmes, J. B., 246

AUTHOR INDEX, VOLUME 28 Holmstrom. C., 29, 39(172), 40(172), 42(172), 107(172) Holi, A., 285 Holzman, G., 223 Honda, N., 54, 55(387), 112(387), 117 (387) Honeyman, J., 19, 23(73) Honig, D. H., 54,115(376) Honjo, M., 346, 349(271) Honma, T.,50, 111(350),262, 263 Hood, D. B., 222 Hooz, J., 246 Hore, M., 343 Hore, P., 41, 68(265), 103(265), 132(265) Horecker, B. L., 339 Homing, E. C., 28, 77, 139(535) Homing, M. G., 77, 139(535) Horowitz, M. I., 48, 84, 105(329), 116 (708), 120(329), 145(329, 555), 189 Horton, D., 58, 169, 182, 183, 184, 209, 216, 220(214), 256, 261, 267, 268, 273(123), 283, 286(94a), 293(123), 305(123) Hotta, K., 76, 105(528) Hough, L., 17, 20, 21, 22, 34, 48(86), 52, 82, 116(86, 364, 365), 144(86, 364, 365), 146(364, 365), 239, 256, 290, 291(93, 176), 438, 439(102) Houminer, Y., 46, 102(294) How, M. J., 40, 47, 64(302), 93(303), 94, 105(302, 303), 123(302), 143(302), 144(253),152(303,603, 608a) Hiebabeckf, H., 303 Hribar, J. H., 32, 41(200), 102(200), 115(200),143(200), 144(200) Hrivnak, J., 94, 151(604) Huang, C.-C., 440, 441(108), 442(108) Huang, H. H., 39 Hubndr, G., 44, 108(283) Hucho, F., 41, 102(268a) Huff, B. J. L., 56, 66, 120(394) Huggins, C. G., 29, 89(178), 90(178), 149(178) Hughes, E. F. X., 357, 366(334) Hughes, N. A., 346 Hullar, T. L., 276, 277, 290(146) Hultberg, B., 403, 404(16a) Hulyalkar, R. K., 32, 71, 72, 136(691), 137(205),159 Humphries, H. P., 123(671), 158, 200 Hunedy, F., 271

459

Hurd, C. D., 162 Hurlbert,R. B., 310 Husemann, E., 31 Hussey, H., 390 Huttunen, J. K., 21, 24(89), 43(89), 133(89), 134(89) I Ichino, M., 250, 253 Ide, H., 349 Igarashi, K., 50, 111(350),262, 263 Ihnat, M., 68, 132(482) Iino, N., 54, 55(387), 112(387), 117(387) Ikawa, M., 182, 188, 219(78), 220(78) Ikawa, N., 122(668), 158 Ikenaka, T.,31, 54,93(190), 95, 115(374, 375), 119(374, 3751, 147(374), 152 (190, 616), 412, 431(62), 434(62), 444 (62), 445(62) Iloff, A., 404 Ilves, S. M., 343 Imada, K., 186 Imae, Y.,341, 370 Imagawa, T., 262 Imai, K., 346, 349(271) Imanari, T.,34, 36, 37(213), 47, 51(226), 54(299), 55(299), 65(213, 228), 72(228), 77, 87, 102(299), 112(226, 299), 113(213, 299), 114(299, 563), 115(299), 136(497), 137(228), 140 (533, 534) Inch, T.D., 195, 290,292 Inokawa, S., 280(153),281,285,304 Inoue, T., 48 Inscoe, J. K., 392(483),393 Irimajiri, T.,304 Irisawa, J., 50, 111(350) Isaka, S., 76, 105(528) Isbell, H. S., 39, 162, 164, 190, 191, 192(121), 194, 199, 200(144), 212(3) Isemura, M., 54, 89, 115(374, 375), 116 (569), 119(374, 375), 144(569), 146(569), 147(374,569) Isemura, S., 276 Isenhour, L. L., 162 Isherwood, F. A., 318, 319(69), 320(69) Ishidate, M., 207 Ishihara, H., 343 Ishikawa, T.,50, 89, 111(343), 148(570) Ishimoto, N., 328 Ishizu, A., 75, 136(510), 141(509, 510),

460

AUTHOR INDEX, VOLUME 28

169, 171(39), 194(39), 195, 196(39), 109(151) 200(39) Johke, T., 319 Iso, T., 208 John, C. E., 368 Isselbacher, K., 325, 326(125) Johnson, A. R., 48, 58(318), 68(318), Ito, E., 328, 329, 388 104(318), 119(318),130(318) Ito, Y., 19, 52(68), 116(68) Johnson, B. A., 254 Iwataki, I., 283 Johnson, G. A. R., 245 Johnson, G. S., 17 J Johnson, P. G., 47,93, 105(305), 120(305), Jaakonmaki, P. I., 77, 139(535) 152(602a) Jacin, H., 41, 102(266) Johnston, J. A., 24, 81(112), 89(112), Jacobelli, C., 392 90(112), 109(112),120(112),143(112), Jacobson, B., 356, 376, 378(409) 149(112), 152(112) Jacobson, G. M., 16 Jokura, K., 308, 316(16) Jacobson, R., 378 Jolley, R. L., 27, 44, 68(149), 108(149), Jahn, W., 226 130(149),131(149) James, A. L., 319, 320(70),346 Jolly, R. D., 436 James, K., 50, 1.11(351) Jones, A. S., 75, 138(514),203 Jansen, E. F., 33, 37(208), 51(208), Jones, B. L., 394, 395(494) 68(208), 102(208),105(208),112(208), Jones, G. H., 279,405, 421 131(208), 152(208) Jones, 13. G., 30, 49, 57, 58(396), 70, 84, Jansen, L., 28, 73(164), 74(164), 139(164) 85, 110(188), 120(396), 123(396), Janson, J., 34, 35, 61(219), 78(215), 135(188), 145(188) 125(219),127(215),136(215),201 Jones, J. K. N., 15, 22, 59, 65, 66, 84, 91, Jaman, M., 287 121(472), 122(472, 583), 123(672), Jarrige-Guntz, G., 320, 322(85) 124(469), 153(583), 158, 230, 231(21, Jarvis, J. A., 32 22), 232(24, 25), 233(25, 27, 28), Jar);, J.. 242 234(25), 235(23, 27), 237(24, 26), Jeanes, A. R., 34, 57(217), 60, 61, 92, 238(26), 247, 259, 270, 271(130), 123(591), 127(217), 153(217), 154 273(130), 286, 287(22), 299(130), (591), 210 300(69), 301, 302(167) Jeanloz, R. W., 53, 69, 79, 82(489, 490), Jones, J. V. S., 17, 22(50c), 47, 93(303), 105(303), 152(303) 83(368, 369), 116(368,369), 134(490), 135(489),142(542),144(368,369,489, Jordan, E., 341 490), 327,353(149b),357(149b) Jourdian, G. W., 331 Jeffery, J. E., 42, 107(271) Judes, C., 319 Jenkins, I. D., 226, 273(5) Jung, G., 55, 112(391),113(391), 114(391) Jenner, G. F., 314 Juslin, S., 29, 39(172), 40(172), 42(172), Jennings, H. J., 231, 232(24, 25), 233(25), 62, 65, 77(468), 107(172), 129(468) 234(25), 235(23), 236(36, 37, 39, 40), K 237(24, 26), 238(26) Jensen, G . D., 94, 95(608), 107(608), Kabat, E. A., 16, 58, 63, 69, 81(405), 86, 151(608),152(608) 103(405),119(405),123(45,484a, 716, Jermyn, M. A,, 15 717), 127(452), 134(484a), 143(405), Jevons, F. R., 403, 416(17) 145(45), 152(405),159 Jewell, J. S., 270, 271(130), 273(130), Kabir, M. S., 105(701),151(701),159 299(130) Kabir, S., 64, 65(467), 77(461), 78(467), JeZo, I., 54, 115(379) 100,129(467), 156(643) Jirii6ek, V., 373 Kagan, F., 226, 247(3) Jirku, H., 437 Kagan, J., 16 Johansson, I., 27, 28(151), 49(151), Kaiser, K., 26

AUTHOR INDEX, VOLUME 28 Kalckar, H. M., 336, 364, 365, 366(346), 370(384, 385), 371, 373(380a),398 Kalyazin, E. P., 97 Kamerling, J. P., 69, 133(485) Kampe, W., 353 Kanai, Y., 346 Kanamaru, S., 94, 97(607), 102(607), 152(607) Kandler, O., 308, 314(18), 328, 329(155) Kano, T., 340, 359(237) Kaplan, N. O., 312 Kara, J., 226 Karksonyi, S., 48, 49(332), 94, 106(332), 107(332), 151(604,605) Kkkkainen, J. E., 14, 27, 29, 44, 48, 55, 58(389), 67, 70, 75(19, 475), 79(330, 475), 81, 83(144), 106(288, 330), 118(389), 124(389), 134(389), 135 (389a, 389b), 138(19, 475), 142(179, 330), 143(144,475),145(144) Karr, A., 16, 17(39),35(39), 61(39), 62(39, 41), 127(39,41) Kasai, N., 77, 140(538) Katan, R., 323, 369 Kates, M., 85,86(559),124(559),145(559) Kathan, R. H., 64, 122(456) Kato, K., 54, 55(387), 112(387), 117(387) Kato, S., 48, 106(327), 119(327), 131(327), 132(327),133(327),142(327) Kato, T., 122(668),158, 247 Katsuhara, M., 184, 266 Katzman, R. L., 53, 83(369), 116(369), 144(369) Kaufer, J. N., 54, 116(377) Kaufman, B., 328 Kaufman, H. H., 438 Kaufman, M. L., 30 Kauss, H., 308, 314(18), 326, 393 Kavanagh, T. E., 48, 104(326), 131(326) Kawabata, S., 283 Iiawaguchi, K., 337, 340, 341, 359(215, 237, 248) Kawai, H., 337, 341, 343, 359(215, 248, 256) Kawata, M., 207 Kean, E. L., 332 Keeling, J. E. D., 405 Keeney, P. G., 48, 104(326), 131(326) Kefurt, K., 242 Keilich, C., 31 Keleti, J., 63, 122(450,451)

461

Kelleher, W. J., 307, 387, 392(4) Keller, J., 445 Kelley, W. S., 390 Kelly, S., 76, 136(524) Kennedy, J. F., 75, 138(514) Kenner, G. W., 250,353 Kenner, J., 199,201 Kent, G. J., 270(131), 271 Kent, P. W., 226,257,258, 327 Kenyon, W. O., 186, 187(90), 192(90) Kergomard, A., 39 Kessler, G., 343 Key, B. A., 47, 106(307) Keyanpour-Rad, M., 287 Khalturi, N . A., 39 Khan, R., 201, 256, 291(93) Kheiri, M. S., 25, 46(132), 68(132), 88(132), 102(132),131(132) Khorana, H. C., 345, 346, 347, 348(279), 350(278) Khorlin, A. Ya., 257 Kidd, J., 205 Kierkegaard, P., 195 Kikugawa, K., 250, 253 Kim, J. H., 17, 63(57), 125(57) Kim, S. M., 32 Kim, T. Y., 348 Kimata, K., 315,324(52), 337(52), 338(52), 339(52), 342(52) Kimura, M., 15, 24, 46(31), 48(116), 68(116), 89(116), 104(116), 108(31), 130(116),132(116), 149(116),207 Kindel, P. K., 387 Kindt, T. J., 14 King, H. K., 223 King, N. J., 187 Kinneberg, K., 26, 32(143) Kircher, H . W., 12, 37 Kirkland, D. F., 25, 54(126), 118(126) Kirkland, T., 16, 86(35), 124(35), 126(35), 146(35) Kirkman, H . N., 321 Kirkman, M. A., 64, 127(458) Kirkwood, S., 344, 348(297a), 349, 350(291), 351(291), 365, 366(265), 370(297a), 371(297a, 381), 373, 395(297a) Kiseleva, V. P., 189, 208(115) Kisic, A., 88, 104(568),142(568),147(568), 148(568), 149(568) Kiss, J., 206

462

AUTHOR INDEX, VOLUME 28

Kisters, R., 17, 44, 58(59), 86, 107(59), Korte, F., 299 Kosakai, M., 76, 139(529) 119(59) Kitagawa, M., 89, 149(575) Koshland, D. E., 393 Kivilaan, A., 16, 17, 27(43a), 102(43a), KGsizawa, Z., 319 133(43a),152(43a) KGstiF, J., 373 Klebe, J. F., 26, 30, 32(183), 147(141) Kotani, S., 122(668),158 Klein, H. J., 44, 108(280, 281, 282, 283) Kotelko, K., 52, 54(357), 116(357), Kleinhofs, A., 392 117(357) Kothari, G. C., 81, 143(544a) Klemer, A,, 201, 257 KovPE, P., 36 Kleppe, K., 375 Kline, D. A., 48, 58(318), 68(318), Kowollik, G., 226 Koyama, T., 221 104(318),119(318),130(318) Kozlova, N. B., 394(502),395 Knaak, J. B., 77, 140(536) Krakow, G., 364 Knox, K. L., 77, 139(535) Knox, L. H., 258 Krasikova, I. N., 105(655), 107(657), 158 Knutson, C. A,, 92, 123(591), 154(591), Kriegelstein, J. J., 396 Krisman, C. R., 393 210 Kobata, A,, 81, 106(545), 143(545), 326, Krivit, W., 52, 116(361) Kroh, M., 16, 136(43) 330(128),331(128, 178) Koch, C., 182 Kropp, J. E., 270(131),271 Koch, W., 182 Kruger, G., 203 Kochetkov, N. K., 56,66(394a), 120(394a), Kruglikova, V. S., 97 202, 240, 241(56, 57), 242(56), Kruppa, R. F., 31 282(59), 300, 346, 348(294, 295, 296, Krzeminski, Z. S., 21, 23(87), 40(87), 89, 150(578), 164 299, 300,301, 302, 303,304,305,306, 307, 308), 349, 350(299), 351, 354, KubaEkovl, M., 94, 151(604,605) 355, 356, 360, 361(339), 362(341, Kudo, K., 200 342), 366(294), 367(339, 361), Kudryashov, L. I., 240 368(342), 374(295, 339), 375(339), Kuehl, R. O., 115(665),158 394(339),395(302),397, 398(339) Kuenne, D. J., 199 Kocourek, J., 218,373 Kuhn, R., 39, 182, 203 Konigstein, J., 174 Kuivila, H. G., 303 Koga, K., 294 Kulaev, N. S., 319 Kogan, G. A,, 39 Kull, G., 22 KolinskL, J,, 24, 38(119), 40(119), 102 Kumazawa, A., 89, 149(575) Kunieda, T., 304 (119) Kolmodin, H., 22, 74(100) Kurahashi, K., 370 Kolos, E., 95 Kurata, K., 192 Komaleva, R. L., 393 Kurien, M. M., 89, 149(573) Komlev, I. V., 348(294, 296), 349, Kurihara, N., 36, 51(225), 70(225), 366(294) 87(225), 89(225), 112(225), 113(225), Kondo, N., 48, 106(327a),142(327a) 114(225) Konishi, K., 25, 88(125) Kuriki, Y., 323, 324(106) Konkin, A. A,, 189, 208(115) Kurokawa, M., 76, 105(528) Koob, J. L., 88, 104(568), 142(568), Kusashio, K., 239 Kusov, Yu. Yu., 348(294, 295, 296), 349, 147(568),148(568), 149(568) Komberg, A., 312 366(294), 367(361),374(295), 397 Kusunose, M., 102(697),115(697),159 Kornfeld, R., 324, 342(117),394, 445 Komfeld, S., 324, 338, 339(116), 342(116, Kuszmann, J., 261, 300(108),305 117), 376(116), 380, 383(420), 388 Kuznetso, L. A., 97 (116), 445 Kwan, T., 72, 136(497)

AUTHOR INDEX, VOLUME 28 Kwok, R. P., 287

463

Lehnhardt, W. F., 17, 61, 128(58), 146(58), 154(58) L Lehtonen, A. A., 14, 29, 44,48, 67,75(19, Labarca, C., 16, 136(43) 475), 79(179,330,475), 106(288,330), Lahav, M., 393 138(19, 475), 142(179,330),143(475) Laine, R. A., 28, 31, 49,53(155), 110(189), Leigh, W. R. D., 21, 23(87), 40(87) 115(155) Leimer, K., 28 Lambert, R., 81, 106(549), 143(549) Leloir, L. F., 308, 310, 311, 314, 315(40), Lance, D. C., 270, 273(129),299(129) 319(13), 325(8), 327(9), 335, 356(8), Lanchee, C., 404, 412(37), 424(37) 358(24), 359(9, 13), 370, 376, 393, 395 Landauer, S. R.,239 Lemieux, R. U., 167, 168(30), 199, 288, Langen, P., 226 289,292 Langley, T. J., 403, 416(17) Lennarz, W. J., 393 Lapina, E. B., 367, 374(364), 375(364), Leoni, P., 314, 321(48), 325(48) 394(364), 395(364), 397 Levene, P. A., 255 Lapp, D., 396 Leveroni, A. F., 314, 321(48),325(48) Lardy, H. A., 338, 341(232) Levine, V. E., 223 Lam, O., 62(436, 441), 63, 69, 123(484a), Levins, R. J,, 97 124(436), 126(441), 134(484a) Levitz, M., 437 Levvy, G . A., 18, 53(64), 81, 82, 116(64), Lamer, J., 336(210), 337, 368(210) Larsen, B., 21 144(64), 402, 403(5), 404(2, 11, 26, Laszlo, P., 176 39), 405(4), 406(46), 408, 409(10, 46), Lathrop, M. B., 29 410(39), 411(46), 412(39, 46, 60), Lau, Y. K., 343 415(46, 60), 416(46, 60), 418(46), Lauterbach, J. H., 267, 268(123), 273 419(46), 420(39, 40, 60, 68), 421(12, (123), 293(123), 305(123) 60), 422(60), 424(39, 46, 60), 425(39), Lavalke, P., 250, 286 426(39), 427(39), 428(39), 429(39), Laver, M. L., 40, 42(255), 107(255) 430(39), 432(46), 433(39, 46), 434(11, Lawson, J. G., 404 12, 26), 436(12, 26), 438, 439(103, Lawton, B. T., 247, 286, 300(69), 301, 105), 443(105, 107) 302(167) Lew, R. B., 27, 48(150), 68(150), 89(150), 105(150), 130(150), 149(150) Layne, D. S., 437 Lewis, B. A., 19, 20(74), 97, 98, 99, 100 Leaback, D. H., 437 Lebedeva, K. S., 351, 367, 374(364), (630) 375(364), 394(364), 395(364) Lewis, K. C., 212 Ledeen, R. W., 19, 52(71), 76(71, 359), Li, S.-C., 412, 442, 445(109) 404, 405, 410(34), 412, 420(34, Li, Y.-T., 116(359),333 Lederer, E., 176 61), 421(61), 424(61), 439(34), 442, Ledueva, R. K., 347 445(34, 109) Lee, B. K., 348 Liao, T.-H., 68, 125(476), 321, 322(92), Lee, J. B., 201, 240, 246 384(92) Lee, Y. C., 17, 24, 29, 40, 89(111, 178), Liau, C. E., 189 90(178), 102(259, 262), 122(670), Libby, R. A., 47, 102(298) 149(178), 150(111), 158,420, 421(70), Liddle, A. M., 205 442,444, 445(109, 115) Lieberman, S., 437 Leete, J. F., 239 Liener, I. E., 126(682), 158 LefGvre, K. U., 186 Likhosherstov, L. M., 202 Leger, F., 212(208), 213 Limozin, N., 319, 327(75) Lehmann, J.. 195, 291,380, 382 Lin, T.-Y., 320, 348, 394(281) Lehmann, V., 62, 123(434), 141(434), Lindahl, U., 16, 65, 129(47) Lindberg, A. A., 14, 17, 19(15), 60(16), 153(434)

464

AUTHOR INDEX, VOLUME 28

62(15, 16, 53, 435, 437, 438, 439, 440), 63, 86(53), 125(414, 435, 437, 438, 440, 725), 126(15, 16, 53, 439, 681,727), 127(414),128(414),158,159 Lindberg, B., 14, 19(15), 22, 39, 40(249), 44, 48(287), 49(332), 60(16), 62(15, 16, 18, 435, 436, 437, 438, 439, 440, 441, 443, 444, 445), 63, 64, 69, 75, 86(443), 92(18), 103(249, 332), 106(287, 332), 107(332, 333), 108(287), 122(18, 712), 123(18, 445, 484a, 713, 723, 725), 124(436, 443, 444, 462, 677, 720), 125(432, 435, 437, 438, 440), 126(15, 16, 439, 441, 481, 727), 128(18), 134(484a), 136(510, 511), 141(509, 510, 693), 154(18), 156(723),157(723), 158, 159, 169, 171(39), 194(39), 195, 196(39, 126), 200(39), 201 Lindgren, B. O., 54, 75(382), 117(382) Lineback, D. R., 50, 52, 57(344), 106 (344), I11(344), 116(362), 119(344), 122(344) Linek, K., 40 Lineweaver, H., 417 Linggood, F. V., 188 Link, K. P., 188 Lipkin, E., 356, 380, 382(421) Lippel, K., 392 Lis, H., 445 Liu, G. L., 396 Ljunggren, H., 62(445),63, 123(445) Llewellyn, D. R., 356 Lloyd, K. O., 16, 58, 63, 64, 64(46), 81(405), 103(405), 106(46), 119(405, 457), 123(716), 124(46), 127(452), 143(405),152(405),159 Lochhead, A. G., 325 Lonngren, J., 61, 62(435, 437, 440),63, 122(712), 123(713), 124(720), 125 (435, 437, 440, 723), 156(723), 157 (723), 159 Loerch, J. D., 315 Loercher, R., 329 Lowa, A., 230 Loewus, F., 16, 59, 76, 90, 115(407), 119(407), 136(43, 407, 524), 150(407) Long, L., Jr., 54, 115(380), 255, 256, 286(94) Longroy, A,, 181, 201(69) Lote, C. J., 103(698),159

Lott, C. E., 25,46(127), 68(127), 102(127), 132(127), 151(127) Love, R. M., 209 Lowe, J. C., 40, 42(255), 107(255) Lowry, 0. H., 419,435(59) Luck, W. E., 49, 107(335) Ludlow, C. J., 48, 49(321), 68(321), 107(321),130(321) Ludowieg, J., 82, 364 Liideritz, O., 14, 19(18), 52, 54(357), 62(18, 447), 63(17), 86, 92(18, 447), 116(357), 117(357), 122(17, 18, 450, 451, 711), 123(18, 447), 125(447), 126(729), 128(18, 447), 141(434), 146(72), 153(434), 154(18, 447), 159, 160 Luetzow, A. E., 256,283,286(94a) Lukacs, G., 249 Lukes, R., 213 Lukezic, F. L., 45, 68(291), 108(291), 130(291), 131(291) Lukowski, H., 201 Lundquist, K. C., 380 Lundt, I., 264 Lunt, M. R., 327 Lythgoe, B., 263

M Mabry, T. J., 16 McAllan, A., 402, 403, 404(2, 14), 422, 434(14) MacAllister, R. V., 223 McCluer, R. N., 29, 52, 116(173) McCormack, W. E., 39 Macdonald, D. C., 403 MacDonald, D. L., 345 McDonald, I. J., 85, 122(558),145(558) McDonald, R. E., 24, 48(123), 68(123), 89(123), 105(123), 130(123), 149(123) McGee, P. A., 186, 187(90), 192(90) McGinnis, G. D., 42, 88, 105(276), 147 (565a) Machell, G., 169, 196, 200(138), 201 Machemer, H., 216 Machida, S., 187, 189 McInnes, A. G., 12,32 Mackie, W., 39, 122(669),158 McLauchlan, K. A., 236(39), 237, 238(39) MacMillan, M. E. J., 20, 21(83), 29(83), 107(83) McPherson, J. C., 52, 76(366), 83(366),

AUTHOR INDEX, VOLUME 28 117(366), 144(366) Maezono, N., 185, 186(88) Magasanik, R., 311 Magerlein, B. J., 226, 282 Maguire, A. H., 402, 421(7) Maitra, U. S., 371, 374 Makela, P. H., 126(728, 729), 127(730), 160 Maki, T., 263, 264 Makita, A., 158 Makita, M., 12, 23(5), 28(5), 30(5), 32(5), 38(5), 41(5), 42(5), 56(5), 59(5), 68(5), 79(5), 80(5), lOO(5) Maley, F., 338, 341(23), 342, 353(254), 376, 403, 416(20), 420(20), 421(20), 442(20, 73), 443, 444(73) Maley, G. F., 376 Malmstrom, B. G., 423 Mandel, P., 319 Mandelstam, P., 329 Mangat, B. S., 326 Mangeot, M., 403 Mann, T., 403, 436 Manners, D. J., 100 Manning, J. H., 64, 77(465) Manstone, A. J., 52, 76(366), 83(366), 117(366), 144(366) Mansurova, S. E., 319 Mapson, L. W., 364, 366(351) Marevtse, V. S., 39 Marigliana, M. H., 26 Marinelli, L., 25, 46(128, 129), 68(128, 129), 102(12% 104(128, 129), 130 (128, 129), 132(128, 129) Markovitz, A., 321, 322(95), 383(94, 95), 384(100) Marks, G. S., 438, 439(101) Marsh, C. A,, 402, 405(4), 421, 422(76), 431(76) Marshall, R. D., 437, 438(99), 439(101), 445 Martell, A. E., 422 Martin, A. R., 188 Martin, F., 81, 106(549), 143(549) Martin, G., 50, 110(354), 111(354), 118(354),250 Martin, G. C., 48, 58(324), 104(324), 119(324) Martin, G. J., 250 Martin, J. A., 88, 104(568), 142(568), 147(568), 148(568), 149(568)

465

Martin, M., 250 Martin, S. M., 72, 81, 124(494), 128(494, 544), 143(494,544) Martin, S. S., 126(684),158 Marvel, J. T., 115(665), 158 Masada, Y.,48 Mason, B. S., 30, 49, 70, 110(187), 131(187), 135(187) Mason, P. S., 24 Masubuchi, M., 53, 95(367), 116(367), 151(367) Matalon, R., 52, 116(355) Mather, J. H., 364 Mathian, R., 81, 106(549), 143(549) Mathias, A. P., 333, 334(191), 335, 359(192, 200) Matough, M. F. S., 246 Matsuhashi, M., 316, 317(56), 318(56), 323, 324(111, 112), 338(56), 357(56), 360(66), 379(65), 380(65, 416), 382 (65, 66), 385(113),390 Matsuhashi, S., 316, 317(56), 318(56, 59), 322, 323(102), 338(56), 357(56), 360(60, 66), 376(60),379(65), 380(65), 382(65, 66), 384(67), 385 Matsui, M., 37, 65(228), 72(228), 137 (228), 189 Matsumoto, T., 283, 297, 298 Matsuo, M., 392 Matsushima, Y., 31, 47, 54, 87, 89(564), 93(190, 300), 95, 115(374, 375), 119(374, 375), 147(374), 148(564), 152(190,300, 616), 303, 412, 431(62), 434(62), 444(62), 445(62) Matsuzak, M., 125(680), 158 Matta, K. L., 405, 420(54) Matthews, A. S., 184 Mattick, L. R., 40, 102(260) Mauck, J., 389 Mawson, C. A., 436 Maxwell, E. S., 336(209), 337, 341(209), 364,365,366(346), 370(244),371(244, 372) Mayer, H., 62(442, 446), 63, 122(450, 711), 125(431), 126(442, 446), 128 (431), 159 Mayer, H. E., 440, 441(108), 442(108) Mayer, R. M., 316 Mazhul, M. M., 366, 367(361) Medcalf, D. G., 100, 125(679),158 Mednick, M. L., 164

466

AUTHOR INDEX,VOLUME 28

Mega, T., 444 Meier, H., 21, 22(92) Meller, A., 187 Mellors, A., 403 Melo, A., 380, 382(425),383, 389 Melton, L. D., 246(68), 247, 254(68), 303(68) Menapace, L. W., 303 Mendicino, J., 387 Mengel, R., 279 Menrad, H., 31 Mepham, T. J., 231 Merlin, J.-C., 30 Merlis, S., 223 Mersmann, G., 257 Meshreki, M. H., 169, 268 Metcalf, E. A., 184 Metz, J., 18, 61(60a), 86, 126(60a), 144 (604 Meyer, H., 326 Mian, A. M., 203 Micheel, F., 239 Michell, A. J., 68, 132(483) Michelson, A. M., 323, 351, 352(110), 375, 383(110, 396) Miettinen, J. K., 320 Miettinen, T. A., 21, 24(89), 43(89), 133(89),134(89) Mikaminawa, T., 314, 338(41) Mikheyskaya, L. V., 107(657),158 Mikolajczak, K. L., 48, 106(325),130(325), 132(325) Milerman, D., 334 Millar, M. J., 436 Miller, I., 329 Miller, R. E., 162, 170(5), 179(5) Millett, M. A., 15, 21(24), 176, 184(52), 207(52), 209(52), 219(52), 220(52), 223(52) Mills, F. D., 179 Mills, G. M., 334 Mills, G. T., 308, 325(11), 326(118), 327(118), 376, 392 Milner, Y., 394 Milstein, C., 72, 137(498) Misaki, A., 47, 57(310), 93(310), 94, 97(607), 102(607),104(310), 122(668), 152(310, 607), 158 Misato, T., 343 Mitchell, R. L., 15, 21(24) Mitzner, R., 39

Miyano, A,, 76 Mizuno, T., 199, 200(140a) Mizuno, Y., 388 Mlynarci, D., 63, 122(451) Moczar, E., 18 Moczar, M., 18 Moeckel, W. E., 16, 17(34a), 36(34a), 61(34a), 86(34a), 90(34a), 128(34a), 146(34a), 154(34a) Moffitt,J. G., 226, 243, 244(62), 245(62), 248, 254, 273(5), 345, 346, 347, 348(279) Moiseev, U. V., 39 Mokrasch, L. C., 221 Moks, E., 92 Molloy, L. F., 124(722), 159 Molz, R. J., 364, 367(349a) Monsigny, M., 22 Montgomery, R., 90, 99(580), 166, 440, 441,442 Montreuil, J., 18, 22 Monzie-Guillemet, D., 74, 141(501) Monzie, P., 74, 141(501) Moore, M. D., 138(692),159 Moore, W. E., 15, 21(24), 176, 184(52), 207(52), 209(52), 219(52), 220(52), 223(52) Mora, P. T., 84, 116(552a), 144(552a) Morgel, L. G., 246 Morikawa, N.,341, 370 Morita, M., 106(656),158 Moriyama, H., 346, 349(271) Morozova, A. V., 336(210a),337 Morris, F., 350, 375(310) Morrison, I. M., 49, 57, 59(341), 67(395), 72, 81(341), 111(341), 126(341), 137 (395), 143(341) Moshy, R. J., 41, 97, 102(266), 152(626) Moss, C. W., 45, 108(290),120(290) Moss, G. W., 60, 154(417) Mowry, R. W., 218 Moye, C. J., 164(15),165 Miiller, M., 24, 25(120), 38(119, 120), 40(119), 41, 68(265), 102(119), 103 (265, 652), 131(687),132(265), 158 Muesser, M., 209 Mugibayashi, Y., 341, 359(248) Muir, L., 122(670),158 Mullinax, F., 18, 27(60), 44(60), 106(60), 144(60) Mullinax, G . L., 18, 27(60), 44(60),

AUTHOR INDEX, VOLUME 28 106(60), 144(60) Munch-Petersen, A., 336, 339 Mund, C., 17 Munday, K. A., 229 Mundie, C. M., 127(731), 160 Munro, A. C., 57, 120(400) Murachi, T., 16, 113(50),444 Murai, N., 24, 48(116), 68(116), 89(116), 104(116), 130(116), 132(116), 149 (116) Muramatsu, T., 405, 406(44, 45), 412(44, 451,413, 416(45), 420(44, 45), 424(44, 45), 439(45), 444(45) Murata, T., 314, 338(41) Murphy, P. T., 22 Murray, R. K., 84, 86(554) Murray, T. P., 296 Murty, V. L. N., 84, 145(555) Muskat, I. E., 269

N Nagabhushan, T. L., 214 Nagai, Y.,54, 116(377) Nagaoka, T., 402, 405, 420(3) Nagel, C. W., 29, 76(170), 139(170), 140(170) Nagpal, K. L., 286 Nakagawa, I., 352 Nakagawa, M., 276 Nakajima, M., 36, 51(225), 70(225), 87 (225),89(225), 112(225),113(225),114 (225), 291 Nakaminami, C., 276 Nakamoto, H., 80 Nakamura, H., 16, 51(48, 49), 70(48), 113(48,49), 264 Nakamura, M., 326, 394 Nakanishi, M., 107(658), 158 Nakanishi, N., 329, 330(173) Nakanishi, Y., 330, 388 Nakatani, T., 329 Narumi, K., 89, 149(574,575) Nashed, M. A., 169 Nathenson, S. C., 388 Nef, J. U., 162, 195(1) Neher, M. B., 33 Neill, W. K., 176 Neilson, D. C., 256 Nelsestuen, C . L., 344, 348(297a), 349, 365, 366(265), 370(297a), 371(297a, 381), 373, 375(297a)

467

Nelson, E. N., 92 Nelson, E. R., 76, 137(522) Nelson, P. F., 76, 137(522) Ness, R. K., 68, 130(478), 165, 172 Neu, H. C., 389 Neuberger, A., 437, 438(99), 439(101), 445 Neufeld, E. F., 307, 319, 326, 340(240, 241, 242), 341, 343(242), 365, 367(358), 375(240, 241, 242), 376, 379, 386(402) Neuhaus, F. C., 328, 388(160), 390 Neukom, H., 54, 76(385), 115(385), 139(525, 531), 140(385, 530), 147 (385), 206 Neunhoeffer, H., 346 Nevins, D. J., 16, 17(39), 35(39), 61(39), 62(39), 127(39) Newall, C. E., 258 Newson, D. W., 24, 48(123), 68(123), 89(123), 105(123), 130(123), 149(123) Newth, F. H., 163, 169(6),287 Nichel, M. F., 81, 143(544a) Nichols, S. B., 178, 182(63) Niedenneier, W., 15, 16(21), 61, 86(35), 124(35), 126(35), 146(21, 35, 562) Niemann, C., 182, 219(78), 220(78), 223 Nikaido, H., 307, 308, 316(16), 318(55), 325, 337, 338(55), 341, 362, 390(2), 392(2), 393, 394(2) Nikaido, K., 316,318(55),338(55),393 Nikkari, T., 29, 79(179), 142(179) Nilsson, K., 122(712),159 Nimmich, W., 62, 122(712), 123(713), 124(720),125(432),159 Nimmo-Smith, R. H., 405 Nippe, W., 230 Nishikawa, Y., 107(658),158 Noack, K., 206 Nolan, T. J.. 246 Nordin, J. H., 348, 373 Nordin, P., 314, 315(46), 333(46) Normore, W. H., 364 Norrestam, R., 195 Norstedt, I., 20, 22 Northcote, D. H., 15, 20, 107(706), 126(683, 706), 158, 159 Nouaille, F., 249 Novak, J. J. K., 264 Novikova, M. A., 360, 361(339), 367(339), 370, 373, 374(339, 377, 392), 375

468

AUTHOR INDEX, VOLUME 28

(339, 394), 394(339),398(339) Novoselo, A. I., 97 Nowotny, A., 77, 140(538),198 Nozawa, Y., 19, 52(68), 116(68) Nunn, J. R., 59, 124(413) Nurminen, M., 127(730),160 0 Oades, J. M., 21, 34(91), 35(91), 60, 64(91), 89(91), 127(458), 128(91), 150(91), 154(91) Oates, M. D. G., 16, 24, 44(36), 48(36, 284), 79, 81(328), 90, 107(36, 284), 130(328), 144(36) Obaidah, M. A., 123(718), 154(718), 159 O’Brien, P. J., 308, 320, 338(17),343 Ockerman, P. A., 403,404(16), 436(16) Oette, K., 16, 21(33, 34), 29(34), 33, 39(34), 43(34), 44, 46(34), 69(34), 76(34), 80, 108(34, 280), 139(34), 143(34), 150(34) Oga, S., 186 Ogasawara, T., 283 Ogata, K., 337, 340, 341, 359(215, 237, 248) Ogata, T., 280(153), 281, 285, 304 Ogawa, S., 284(163),285 Ohashi, H., 385 Ohashi, M., 53, 69, 105(372), 117(372), 132(372) Ohtsuka, E., 346 Okada, M., 37, 65(228), 72(228), 137(228) Okazaki, R., 308, 323(15), 324(106, 107), 352(1lo), 383(107, 110) Okazaki, T., 323, 324(106, 107), 352(110), 383(107, 110) Okazawa, Y., 24, 48(116), 68(116), 89 (116), 104(116), 130(116), 132(116), 149(116) Okuda, S., 317 Okuda, T., 24, 88(125) Okumura, T., 119(710), 159, 403, 412, 416(18), 420, 422, 431(18), 440(72), 442, 444(18) Olavesen, A. H., 348(298),349 Oldham, K. G., 356 Oliver, I. T., 364, 366 Olson, J. A., 392 O’Meara, D., 200 Onishi, H., 36, 128(221) Onn, T., 60, 62(443, 444), 63, 124(443,

444), 125(414),127(414), 128(414) Onodera, K., 358(337),359 Onuma, M., 48, 106(327), 119(327), 131(327), 132(327), 133(327), 142 (327) Oppenauer, R., 176, 186(53), 203(53), 207(94) Orcut, D. M., 28, 47(159), 57(159), 97(159), 103(159), 120(159), 152(159) Orentas, D. G., 92, 123(591),154(591) Osborn, M. J., 339, 390 Osterland, C. K., 445 Ostojic, N., 88, 147(565) Osugi, M., 337, 359(215) Ota, M., 57, 119(397) Otani, S., 200 Ottensteiri, D. M., 97 Otter, B. A., 290, 291(176) Otter, G. E., 68, 130(480),132(480) Otterbach, D. H., 323 Ovadova, R. G., 105(655), 107(657), 158 Overend, W. G. 184, 202, 204(172), 287 Ovodov, Yu. S., 23, 37, 47, 56(231), 67, 105(655), 107(657), 118(231), 120 (310a), 121(231), 158

P Pacak, J., 218, 278 Paerels, G. B., 182 Page, J. E.,20 Pakhomova, M. V., 314, 315(43) Paladini, A. C., 308, 311, 325(8), 356(8), 358(24) Palframan, J. F., 97, 154(620) Pape, H., 384 Parizek, J,, 437 Park, J. T., 308, 328(7), 329(7), 334 Park, L. U., 319, 329(81),335(81) Parks, R. E., 367 Parodi, A. J., 393 Parolis, H., 59, 124(413),231 Parrish, F. W., 41,54, 104(264), 115(380), 226, 227, 255, 256, 286(94), 402, 420, 421(6, 7, 71) Partridge, R. D., 29 Passeron, S., 310, 311, 314, 315(40), 339(51), 340(51), 342(51), 346(22), 357(22), 358(22), 359(22) Patai, S., 46, 88, 102(294), 147(567) Patterson, B. W., 396 Patterson, J. L., 26

AUTHOR INDEX, VOLUME 28 Paul, R. J., 389 Paulis, J. W., 312 Paulsen, H., 50, 111(347, 348), 278 Paus, E., 412, 424(64), 431(64),434(64) Pauschmann, H., 55, 112(391), 113(391), 114(391) Pawlak, Z., 201 Pazur, J. H., 338, 340(239),341, 367(239), 375, 383(226),445 Pedersen, C., 264, 266 Peer, H. G., 217 Penick, R. J., 29, 52, 116(173) Peplow, P. V., 94, 152(608a) Percheron, F., 18, 404, 412(37), 424(37) Percival, E., 47, 57, 68, 69, 72(398, 399), 93, 103(398, 399, 699), 104(479), 105(304, 305), 106(702), 119(699), 120(305, 398, 399, 702), 131(479), 133(484), 134(484), 136(398, 399), 149(699),152(398, 602a), 159 Percival, E. G. V., 186, 192(93) Perkins, H. R., 329 Perlin, A. S., 27, 39, 55, 89(388), 92, 116(150b), 118(388), 148(388), 154 (388), 188, 402, 421(8) Perlman, R. L., 376 Perlmann, G., 123(715),159 Perry, M. B., 15, 28, 29(156, 157), 32, 36, 49, 57, 59(341), 67(395), 71, 72, 77 (157, 158, 339), 78,80, 81(341),84,85, 86, 109(339,660, 661, 662), 111(341), 123(674), 126(341), 128(221, 339), 129(660,661), 136(498,691), 137(156, 205, 395, 537), 138(157, 158, 339, 662), 140(339, 662), 143(341, 540), 145(557), 146(561), 158, 159, 230, 231(22),287(22) Persson, K. 0. U., 319 Petek, F., 404(38), 405, 412(38), 424(38) Peters, F. N., 163, 176,212,213(198) Peterson, D. R., 373 Peterson, G., 28, 29(163), 72, 73(163, 499), 74, 137(499,500), 142(508) Petit, J. F., 329 Pettersson, S., 74, 142(508) Pfeifer, M. A., 184, 220(83) Pfeiffer, E., 380 Philippe, R., 30 Philipsbom, W. V., 218 Phillipps, G. H., 258 Philpot, C. W., 88, 147(565)

469

Phlippen, R., 44, lOS(280, 282) Picard, J.. 329 Picken, J. M., 387 Pickles, V. A., 39 Pierce. A. E., 23,27(107) Pierce, J. G., 17, 63(57), 68, 124(721), 125(57, 476), 159 Pigman, W., 39, 85, 145(556), 163. 164, 401 Pilotti, A., 62(438),63, 125(438, 725), 159 Piper, C. V., 36, 62, 66, 127(220) Pibnan, M. E., 89, 150(577) Pittman, T. A., 89, 90(572), 150(572) Plapp, R., 328, 329(155) Plessas, N. R., 251, 252(83), 253(83), 254(83), 276(143, 144, 145), 277, 278(83), 286(83), 302 Plummer, T. H., 403, 416(20), 420(20), 421(20), 442(20, 73), 443, 444(73) Poltinina, R. M., 97 Ponpipom, M. M., 249,250 Pontis, H. G., 310, 312,319, 320(70), 326, 327(130), 335, 338 Pontis, S. M. E., 338 Popoff, T., 76, 136(520), 138(520), 167, 168(25), 190, 206(25) Pop&, M.-O., 24, 109(IlO), 120(110) Post, A., 16, 17(34a), 36(34a), 61(34a), 86(34a), 90(34a), 128(34a), 146(34a), 154(34a) Posternak, T., 345 Potter, V. R., 310 Pourtallier, J., 48, 68(314), 104(314), 130(314),133(314) Prader, A., 131(687),158 Prasad, N., 264 Preiss, J., 314, 315(44), 319(44), 321, 326(44), 337, 338(216),339, 340(234), 348(216), 368(89), 395, 396(506, 509) Preobrazhenskaya, N. N., 347 Prey, V., 167 Pridham, J. B., 20 Priger, W. E., 312 PfikrylovA, V., 278 Prokof'ev, M. A., 319, 346,347 Fummerer, R., 212(206), 213 Putnam, E. W., 340(242), 341, 343(242), 375(242),393

Q Quadling, C., 49, 62, 77(339), 109(339),

470

AUTHOR INDEX, VOLUME 28

124(433), 128(339), 138(339), 140 (339), 145(433) Quemeneur, M. T., 250

R Raaflaub, J., 422 Rabinowitz, M., 338, 367(228) Race, C., 64, 125(455) Rackis, J. J., 54, 115(376) Radatus, B., 305, 306 Radford, T., 32, 41(200), 102(200), 115(200),143(200),144(200) Radhakrishnamurthy, B., 20, 75(76), 78(76), 103(76),138(76,513,515,516, 517, 518, 519), 143(76,541) Rajiah, A., 97, 152(628) Raksha, M. A., 258 Ralph, A., 229 Ramachandramurthy, P., 126(682), 158 Rammler, D. H., 350, 375(310) Ramuz, M., 319 Randall, R. J., 410, 435(59) Randerath, E., 312 Randerath, K., 312 Rao, K. M. K., 343 Raschig, K., 227, 291 Raunhardt, O., 41, 68(265), 76, 103(265), 132(265),139(525) Raymond, W. R., 29, 76(170), 139(170), 140(170) Rebers, P. A,, 223 Recondo, E., 310, 311, 314, 315(40), 321 (48), 325(48), 339(51), 340(51), 342(51), 346(22), 357(22), 358(22), 359(22), 389, 394, 395(496) Redmore, D., 198 Reed, D., 393 Rees, D. A., 15, 17, 21, 64, 77(466), 89 (88), 135(52,690), 148(88), 159 Reese, E. T., 41, 104(264), 226, 402, 420, 421(6, 7, 71) Reeves, R. J., 174 Reichstein, T., 176, 186(53), 203(53), 207(94) Reid, P. E., 40, 43(256), 58, 65(403), 81(256), 94, 95(608), 107(608), 108(256),143(256),151(608),152(608) Reineccius, G. A,, 48, 104(326), 131(326) Renard, M., 39 Reynolds, P. E., 328, 329(154) Reynolds, R. J. W., 186, 192(93)

Rhee, V., 89, 149(573) Rice, F. A. H., 176, 177 Richards, E. L., 124(722), 159 Richards, G. N., 15, 22, 35,62, 64,65(26), 77(98), 122(98), 126(696), 127(26, 426, 427, 428, 429, 430), 129(26, 98), 136(26), 152(696), 159, 169, 195, 196, 199, 200(138), 201(136), 206, 287 Richards, J. B., 393 Richardson, A. C., 228, 234(13), 239 Richardson, B., 28, 47(159), 57(159), 97(159), 103(159), 120(159), 152(159) Richardson, N. G., 17, 123(716), 135(52), 159 Richey, H. G., Jr., 29, 48(171), 81, 107(171), 144(171) Richey, J. M., 29, 48(171), 81, 107(171), 144(171) Richtmyer, N. K., 27,28(151), 40, 49(151), 109(151), llO(336, 337), 132(252), 151(336) Riedel, H., 230 Riedl, H., 72, 73(499), 137(499) Riegelman, S., 29 Rigdon, R. H., 75, 138(517) Riggs, N. V., 149(694), 159 Ritchie, R. G. S., 269, 270, 271(130), 273(130), 299(130) Robbins, P. W., 336(211), 337, 338, 390 Roberts, R. M., 24, 81(112), 89(112), 90(112), 109(112), 120(112),143(112), 149(112), 152(112),311, 343 Robertson, J. H., 28 Robins, M. J,, 279 Rod&, U., 126(728), 160 Rodriguez, P., 314, 315(50), 338(50), 339(50), 340(50), 348(50), 370 Rodstrem, R., 370 Ronnquist, O., 195 Roger, R., 256 Rogovin, J. A., 189, 208(115) RoldPn, A. R., 213 Romeo, D., 390 Rony, P. R., 39 Root, D. F., 40, 42(255), 107(255), 176 Rosbottom, A., 81 Rosebrough, N. J., 410, 435(59) Roseman, S., 82, 182, 328, 329, 331, 332, 346, 348, 350(278),358(182), 376 Rosen, S. M., 339

AUTHOR INDEX, VOLUME 28 Rosenberg, A., 423 Rosenfelder, G., 122(711), 159 Rosenthal, A., 292 Ross, W. C. J., 287 Roth, J. S., 253 Rothfield, L., 390 Rowe, J. J. M., 58, 65(403), 92, 141(588) Rowland, M., 29 Roy, C., 329 Roy, N., 17, 22, 46, 103(295), 124(54), 129(54) Roy-Burman, P., 338, 354(229), 365(229), 367(229) Roy-Burman, S., 338, 354(229), 365(229), 367(229) Rubery, P. H., 20 Ruijterman, C., 27, 96(145), 125(145), 153(145), 155(145) Ruiz, H., 75, 138(515,516, 517) Rumpf, G., 24,68(115), 89(115), 104(115), 130(115),149(115) Ruskiewicz, M., 164(16), 165 Russell, C. R., 283 Russell, K. R., 179 Rutherford, D., 40, 132(252) Rutter, W. J., 325 Ryan, A. E., 202, 204(172) Ryan, K. J., 53, 54(373), 115(373) Rydon, H. N., 239 Ryhage, R., 32, 103(202) S

Sabbe, W. E., 24, 25(117), 48(117), 68(117), 105(117), 130(117) Sachetto, J. P., 169 Saeman, J. F., 15, 21(24), 176 Saha, N. C., 32, 41(201), 102(201), 103(201) Sahasrabudhe, M., 57, 58(396), 120(396), 123(396) Saier, M. H., 103(654), 158 Saita, M., 412, 431(62), 434(62), 444(62), 445(62) Saito, H., 47, 57(310), 93(310), 104(310), 152(310) Saito, M., 328 Sakurai, Y., 192 Saladin, E., 260 Salfner, B., 19, 41, 53, 83, 106(70a), 117(70), 144(70a), 145(70) Salitis, G., 366

471

Salo, W. L., 348, 365, 373, 377 Salsman, K., 52, 76(359), 116(359) Samek, Z., 242 Samuel, J. W. B., 21, 64, 77(466), 89(88), 148(88) Samuelson, O., 20, 22,28,40, 72, 73(164), 74(100, 164), 76(261), 136(520, 521), 137(499,500,522), 138(520),139(164, 261), 141(504, 506), 142(502, 503, SOB), 167, 168(25, 26, 27), 194, 195, 196(127), 206(25, 26) Samuelsson, B., 52 Samuelsson, K., 52, 62(439), 63, 126(439) Sandermann, H., Jr., 326,358(127),387 Sanders, H., 214 Sanderson, G. R., 55, 89(388), 92, 118(388), 148(388),154(388) Sandier, S. R., 258 Sanford, B. H., 53, 83(368), 116(368), 144(368) Sanwal, G. G., 314, 315(44), 319(44), 326(44), 395 Sarvas, M., 337 Sastry, S. D., 26, 32(143) Sato, T., 53, 95, 116(367), 151(367) Sattler, L., 221 Saukkonen, J. J., 310, 334 Saunders, A., 48, 79(331), 106(331), 142(331) Saunderson, A. R., 334, 359(200) Saviova, T., 320 Sawada, T., 48 Sawamura, R., 221 Sawardeker, J. S., 20, 34, 41, 45(269), 57(217), 60(217), 61(217), 88(80), 127(217), 147(80), 153(217) Sawicka, T., 342 Schaffer, R., 27, 102(150a),218 Schanbacher, F., 348(297), 349 Schaper, K. J., 215, 216(212) Scheffold, R., 260 Scheit, K. H., 346 Schennikov, V. A,, 202 Scher, B. M., 333 Scher, M., 393 Schiaffino, J., 24, 35, 58(113), 68(113), 119(113), 132(113),134(113) Schiller, J. G., 337, 364(213a), 365(213a) Schimmel, S. D., 55, 111(390), 118(390), 125(390), 153(390) Schlender, K. K., 336(210),337,368(210)

472

AUTHOR INDEX, VOLUME 28

Schmid, K., 89, 116(569), 144(569), 146(569),147(569) Schmidt, G., 62, 125(431),428(431) Schmidt, H., 167 Schmidt, H. W. H., 54, 76(385), 115(385), 139(525, 531, 532), 140(385, 530), 147(385),206 Schmitz, H., 310 Schneider, G., 346 Schrager, J., 16, 24, 44(36), 48(36), 79, 81(328), 90, 107(36, 284), 130(328), 144(36) Schrarer, R., 29, 48(171) 81, 107(171), 144(171) Schreuder, H., R., 89, 148(571) Schulze, A., 16, 17(43a), 27(43a), 102 (43a), 133(43a),152(43a) Schutzbach, J. S., 337, 386(212, 437), 387 Schwartz, J., 420, 421(70) Schwartz, K., 223 Scocca, J., 17, 444 Scott, H. M., 325, 326(124) Scott, J. E., 14, 71(20), 76(20), 140(20) Scott, R. W., 176, 184(52), 207(52), 209(52),219, 220(52),223 Scott, S. J., 123(673),158 Scott, S. S., 323, 328, 329 Sears, K. D., 159 Secret, D. W., 40, 43(256), 81(256), 108(256),143(256) Sedlak, J.. 63, 122(451) Seefelder, M., 250(81),251 Seib, P. A., 25, 147(125a) Seifert, L. L., 391 Seilmeir, W., 179 Selman, S., 171, 194(42) Selvendran, R. R., 318, 319(69), 320(69) Semenza, G., 24, 38(119), 40(119), 41, 68(265), 102(119), 103(265), 132 (265) Sennello, L. T., 24, 48(122), 105(122) Seno, N., 57, 119(397) Senogles, E., 22 Seo, K., 280(153),281 Sephton, H. H., 49, 110(336), 151(336) Seppala, E., 65, 77(468), 129(468) Sepulchre, A. M., 249 Sequeira, R. M., 27, 48(150), 68(150), 89(150), 105(150), 130(150), 149(150) Serfas, O., 96, 154(618) Serluppi-Crescenzi, G., 319

Sessa, D. J., 54, 115(376) Severin, T., 179 Seyama, Y., 370(385),371, 373(380a) Seydel, J., 215, 216(212) Shaban, M., 69, 82(489, 490), 134(490), 135(489), 144(489,490) Shabarova, Z. A., 319, 346, 347 Shafizadeh, F., 40, 42, 88, 105(275, 276), 107(255), 147(565, 565a), 184 Shainkin, R., 123(715),159 Shallenberger, R. S., 40,41, 102(258,259, 260, 262, 267) Shapira, J., 37, 38(227), 51, 65, 112(227), 113(227), 114(227) Sharon, N., 348 Shaw, D. H., 23, 28, 29(156), 60, 85, 86(559), 124(559),137(156), 145(559), 154(417) Shaw, D. R. D., 334 Shaw, N., 47, 89, 105(306), lOO(647, 648, 650), 101, 122(576), 150(576), 156 (647) Shaw, P. E., 20, 171, 176(41), 177(41), 179(41), 180, 200(41) Shaw, R.,43, 57(277), 108(277), 119(277), 131(277), 132(277), 136(277), 138 (277), 149(277) Shedlowsky, A. E., 341 Shen, L., 337, 338(216),348(216) Shepherd, H., 127(731),160 Sheppard, R.C., 250 Sherman, W. R., 36, 58, 89(406), 90, 104(406,579), 119(406,579), 149(406, 573, 579) Shestakov, V. G., 346 Shetlar, M. R., 405, 445 Shibaev, V. N., 311, 346, 348(294, 295, 296,299,300,301,302,303,304,305, 306, 307, 308), 349, 350(299), 351, 354, 355, 356, 360(331), 361(339), 362(341,342),365,366(294),367(339, 361), 368(342), 370, 373, 374(295, 377), 375(339, 392), 393, 394(339), 396(302), 397, 398(339) Shibata, S., 107(658),158 Shibuya, M., 276 Shimizu, F., 331,332, 358(182) Shimizu, S., 330, 388 Shioi, S., 276 Shmerling, D. H., 131(687), 158 Shoji, H., 268

AUTHOR INDEX, VOLUME 28 Shome, B., 17, 63(57), 125(57) Shoyab, M., 332 Shuey, E. W., 338, 383(266) Shukla, R. N., 395 Shulman, M. L., 257 Shyluk, J. P., 29 Siddigui, I. A., 221 Siddiqui, I. R., 38, 47, 51(233), 72, 93, 104(233, 309), 128(309), 136(496), 151(602) Sie, H. G., 349 Silbert, J. E., 348,357,366(334), 386(439), 387 Silverstein, R., 393 Simonark, P. C., 365 Simson, B. W., 21 Sinclair, H. B., 229, 256, 312 Singh, P. P., 56, 61, 62(420), 86(420), 95, 118(392,393),124(420),145(420) Singh, U. P., 296 Sinkinson, G., 19, 53, 83(370, 371), 116(69, 370, 371), 144(69, 370, 371) Sirokman, F., 242(60), 243 Siskin, S. B., 276(142, 146), 277, 290(146) Sjostrom, E., 34, 35, 61, 62, 65, 77(468), 78(215), 125(219), 127(215),129(468), 136(215) Sjovall, J., 31 Slabnik, E., 394 Slanski, J. M., 41, 97, 102(266), 152(625) Slessor, K. N., 39, 40(249), 103(249), 246(68), 247, 254, 303(68) Sloan, J., 420, 421(70) Slomiany, B. L., 116(708), 159 Sloneker, J. H., 13,20,34,41,45(269), 50, 57(217), 60(217), 61(217), 88(80), 92, 111(343), 123(591), 126(416), 127 (217, 415), 147(80), 153(217), 154 (591) Slover, H. T., 30, 49, 70, 110(187), 131(187), 135(187) Smestad, B., 103(699),106(702),119(699), 120(702), 149(699), 159 Smirnova, G. P., 32, 115(206) Smirnyagin, V., 54, 76(384), 116(383,384) Smith, C. R., 48, 106(325), 130(325), 132(325) Smith, D. C. C., 239 Smith, D. M., 57, 58(396), 120(396), 123(396) Smith, E. D., 24

473

Smith, E. E. B., 308, 325(11), 326(118), 327(118), 334, 336, 376, 392 Smith, E. J., 327 Smith, E. L., 20 Smith, F., 19, 20(74), 34, 90, 97, 98, 99(580), 100, 166, 186, 192(93), 207, 223, 370(386),371, 373 Smith, J. F., 188 Smith, M., 327 Smith, M. S., 327 Smith, R. N., 99 Snaith, S. M., 402, 403, 404(26, 27), 405, 406(46), 409(10, 46), 410(39), 411(27, 46), 412(39, 46, 60),414(27), 415(46, 60),416(46, 60),418(46), 420(39, 60), 421(60), 422(27, 60),424(27, 39, 60), 425(39), 426(39), 427(39), 428(39), 430(39), 431(27), 432(46), 433(27, 39, 46, 69), 434(26, 27), 435(27), 436(26) Snyder, E. I., 246, 248(65h) Sohir, P., 305 Sohn, W. A., 212 Solms, J., 326 Solov’eva, T. F., 23 Somers, P. J., 94, 152(608a) Sommar, K. M., 378 Spcmme, R., 122(667), 158 Sonnino, S., 391 Sonohara, H., 329,330(173) Sorm, F., 264,303 Sosa, F., 18 Sowa, W., 49, 111(342) Sowden, J. C.,163, 194, 195(7), 197, 199, 200(7) Speck, J. C., Jr., 163, 167(10), 175 Spencer, J. F. T., 50, 92(346), lll(343, 663), 122(592, 663), 123(346, 595, 675), 124(595), 154(346, 592, 595), 156(592), 158, 405, 421(51) Spik, G., 22 Spiridonova, S. M., 348(302, 303, 308), 349, 354, 355, 396(302) Spiro, R. G., 443, 445 Sprock, G., 230 Srinivasan, S. R., 75, 138(513, 519) Srivastava, H. C., 56, 95, 118(392, 393) Srivastava, P. C., 286, 287(168c), 293 (168c),294(168c) Srivastava, R. M., 296 Srogl, J., 213 Stacey, M., 40, 47, 93(303), 105(303),

474

AUTHOR INDEX, VOLUME 28

144(253), 152(303), 165, 184(19), 224(19), 271 Stalling, D. L., 28, 113(152) StanBk, J., 218 Staucher, B., 43, 109(278) Steele, J. W., 54, 118(386) Steiner, K., 239, 285(47) Stenzel, H., 255 Stepanenko, B. N., 336(210a),237 Stephen, A. M., 23, 64, 123(460,719), 125 (459), 126(726),159 Stephenson, L., 258 Stevens, C. L., 229, 242(60), 243, 323, 324 Stevenson, I. H., 392 Stewart, M. A,, 58, 89(406), 1.04(406), 119(406), 149(406,573) Stiles, M., 181, 201(69) Stimson, W. H., 29, 80, 143(180) Stoddart, J. F., 65, 103(653), 124(469),158 Stolpe, L., 74, 141(506) Stolzenback, F. E., 312 Storey, I. D. E., 308,326(10) Strachan, I., 18, 53(64), 81(64), 82(64), 116(64), 144(64), 438, 439(104, 105), 443(105) Street, H. V., 13 Strobach, D. R., 40, 153(254),195 Strominger, J. L., 311, 316, 317(56), 318(56, 59), 319(30), 322, 323(102), 324(107, l l l ) , 327(30), 328(144), 329(151, 160), 334(144), 338(56), 352(110), 357(56), 360(66), 364, 365, 366(346, 351), 375, 379(65), 380(65), 382(65, 66), 383(110, 396), 384, 385(111, 113),388(152), 390 Strong, F. M., 149(694),159 Strunk, K., 31 Struve, W. G., 328, 388(160),390 Strycharz, G. D., 48,82(334), 144(334), 146(334) Stumpf, P. K., 308, 325(12),326(12) Stutz, E., 187, 189(102),208(102) Su, J. C., 319, 326(837, 348), 394(281) Suami, T., 284(163),285 Subbaram, M. R., 97, 152(628) Subba Rao, P. V., 56, 118(392) Suchanec, R. R., 30 Sugahara, K., 119(710), 159, 420, 440(72), 442 Sugimoto, K., 324

Sugisawa, H., 170, 179(40),208 Sugiyama, M., 330 Sugiyama, T., 314, 338(41) Sugiyami, Y.,276 Sukeno, T., 403, 416(20), 420(20), 421(20), 442(20, 73), 444(73) Sullivan, L. J., 77, 140(536) Supina, W. R., 31 Sutherland, I. W., 62(443), 63, 86(443), 124(443) Suzuki, N., 317 Suzuki, S., 315, 317, 324(52), 329, 330(173), 337(52), 338(52), 339(52), 342(52), 388 Svensson, S., 14, 19(15),60(16),62(15, 16, 18), 63, 69, 92(18), 122(18, 448), 123(18, 484a), 126(15, 16, 727), 128(18), 134(484a),154(18), 159 Swain, C. G., 40 Swain, T., 392 Swann, M. H., 97, 151(627) Swanson, A. L., 348, 353(282) Sweeley, C. C., 12, 18, 23, 28(5), 30, 31, 32(5), 38, 41(200), 42, 49, 52, 56(5), 59, 68,79, 80, 82(360), 100, l02(200, 201), 103(201), llO(l89), 115(200, 360), 116(66, 361), 143(200), 144 (200, 360), 146(66),393 Sweet, F., 295(189),296 Szabo, I., 34, 78(216) Szaboles, O., 167 Szarek, W. A,, 65, 124(469),231, 247, 259, 269,270,271( 130),273(129,130), 286, 299(129, 130), 300(69), 301, 302(167) Szczerek, I., 270, 271(130), 273(130), 299(130)

T Taboada, J., 95, 151(613) Tabone, D., 392 Tabone, M. J., 392 Tabushi, I., 260 Ta Hsiu Liao, 17, 63(57), 125(57) Takahashi, N., 16, 113(50),260, 444 Takahashi, S., 291 Takahashi, T., 126(682), 158 Takanashi, N., 330 Takayama, K., 329 Takeda, T., 107(658), 158 Takizawa, T., 304 Taku, A., 388

AUTHOR INDEX, VOLUME 28 Talmadge, K. W., 16, 62(40, 42), 127(40, 42) Tamura, T., 304 Tamura, Z., 16, 34, 36, 37(213), 47, 51(48, 49, 226), 54(299), 55(299), 65, 70(48), 72, 77, 87, 102(299), 112(226, 299), 113(48, 49, 213, 2991, 114(299, 563), 115(299), 137(228), 140(533, 534) Tanaka, H., 106(704), 113(704), 138(704), 159 Tanaka, K., 346, 349(271) Tanaka, M., 48, 106(327a), 107(658), 142(327a),158 Tanaka, Y., 395 Taniguchi, M., 294 Tarentino, A. L., 403, 421, 442(73), 443, 444(73) Tate, W. J., 52, 116(358) Tattabiranin, T. N., 332 Tatum, J. H., 20, 171, 176(41), 177(41), 179(41), 180, 200(41) Tavakoli, M., 48; 76(319), 107(319), 138(319,523, 692), 159 Taylor, E. W., 186, 187(90), 192(90) Taylor, K. G., 229, 242(60), 243 Taylor, L., 68, 130(480), 132(480) Taylor, N. F., 270, 293 Teder, A., 34, 78(216) Teece, E. G., 165, 184(19),224(19) Tejima, S., 263, 264 Tennigkeit, J,, 346 Tesler, A., 376 Teunissen, H. P., 212(207), 213 Theander, O., 15, 54, 75(382), 76, 117(382), 123(671), 136(510, 511, 520), 138(520), 141(509, 510, 693), 158, 159, 163, 167, 168(25), 169, 171(39), 190, 194(39), 195, 196(39, 126), 200(39), 201, 206(25), 210, 256, 286(94a) Thede, L., 74, 142(503) Thiel, I. M. E., 231 Thieme, P., 207 Thomas, B. S., 31, 32(195) Thomas, D. B., 64, 141(453) Thomas, J. A., 336(210), 337, 368(210) Thompson, J. L., 124(720), 159 Threnn, R. H., 328, 329 Tichi, M., 373 Tiemann, F., 166

475

Tihlarik, K., 31 Timell, T. E., 21, 22, 46, 89, 92, 93, 103(295), 148(571), 151(599, 600, 601), 154(594) Tinelli, R., 375, 383(396) Tipson, R. S., 19, 20(73), 23(73), 40, 227, 255 Tisue, G. T., 387 Tjarks, L. W., 229 Tobe, S., 337, 359(215) Tochikura, T., 337, 340, 341, 343, 359(215, 237, 248, 256) Todd, A. R., 201, 351, 353 Tohma, M., 15, 24, 46(31), 48(116), 68(116), 89(116), 104(116), 108(31), 130(116), 132(116),149(116) Tokuyama, K., 184, 185, 186(88),266 Tollens, B., 186 Tomkins, G. M., 392(483),393 Tomoda, M., 48, 106(327, 327a, 704), 113(704, 707), 117(666), 119(327), 131(327), 132(327), 133(327), 138 (704, 140(666), 142(327, 327a), 158, 159 Tomshich, S. V., 47, 120(310a) Tong, H. H., 287 Tonge, B. L., 239 Tornaben, T. G., 62, 86, 123(560), 124(433),145(433,560) Tosi, L., 364 Towey, K. C., 311 Townsend, L. B., 226 Tracey, A. S., 246(68), 247, 254(68), 303(68) Traobel, T., 26 Trejo, A. G., 325, 358(126) Trimnell, D., 283 Tronchet, J. M. J., 58, 403 Trotter, J., 238 Troy, F. A., 390 Tmcco, R. E., 336(208), 337 Tsai, C. M., 370 Tsai, C. S., 83, 85, 144(551),145(551), Tsuchiya, T., 183, 184, 216, 220(214), 226, 300(4) Tsuchiya, Y., 285 Tsuda, M., 81, 106(545), 143(545) Tsuji, K., 28 Tsuji, M., 388 Tsumita, T., 89, 149(574,575) Tsusue, Y.,52, 116(363)

476

AUTHOR INDEX, VOLUME 28

Tucker, L. C. N., 184, 271 Tulley, A., 258 Tulloch, A. P., 92, 122(592), 154(592), 156(592) Tuning, B., 48, 68, 104(316), 130(316), 132(481) Turdiu, R., 348 Turner, J. C., 230, 231(21, 22), 287(22) Turunen, J., 20, 43(78), 88(78), 106(78), 147(78) Turunen, K., 20, 43, 88, 106(78), 147(78) Turvey, J. R., 59, 65, 126(412)

U Uchida, M., 284(163),285 Uchiyama, M., 189 Udaka, S., 364 Ueda, N., 303 Ueda, T., 345, 346, 349(270), 350 Uemura, T., 186 Ueno, T., 13, 36, 51(225), 70(225), 87, 89(225), 112(225), 113(225), 114(225) Ueta, N., 94, 95(606), 103(606) Ugami, H., 186 Uhlenbruck, G., 19, 53, 83, 117(70), 145(70) Umemura, Y.,326, 394 Umezawa, S., 226, 300 Underhill, E. W., 25, 54(126), 118(126), 392 Underwood, E. J., 436 Uno, M., 106(704), 113(704, 707), 138(704),159 Unrau, A. M., 32, 47, 57(311), 93(311), 94(598), 100, 105(31l), 106(308), 120(311),156(639,642), 157(340,641, 642) Urbas, B., 94, 124(609), 153(609) Usov, A. I., 240, 241(56, 57), 242(56), 282(59), 300 Uziel, M., 311 Uzlova, L. A,, 285

V Valicenti, J. A., 229 Vallee, B. L., 423, 433 Valtonen, V. V., 127(730),160 Van, L. V., 319, 327(75) Vance, D. E., 52, 82(360), 115(360), 144(360) vanden Heuvel, W. J. A., 26

van den Onweland, G . A. M., 217 van der Bijl, P., 126(726), 159 Vandenneer, J. W., 47, 104(309), 128(309) van Ling, G., 27, 96(145), 125(145), 153(145), 155(145) Vargha, L., 261, 300(108) Vasseur, E., 223 Vecchi, M., 26 Velarde, E., 258 Venkina, A. V., 367, 394(503), 395 Venner, H., 17 Vennesland, B., 364 Verachtert, H., 67,96, 121(473),128(473), 155(473), 312, 314, 315(50), 338(49, 50), 339(50), 340(50), 348(50) Verhaar, L. A. T., 28, 141(166) Verheyden, J. P. H., 226, 243, 244(62), 245(62), 248, 254, 273(5) Vernon, C. A., 356 Vicari, G., 16, 86, 123(45), 145(45) Vidauretta, L. E., 24, 48(121), 68(121), 105(121), 130(121) Vidla, J. D., 315 Viebrock, F., 24, 35, 58(113), 68(113), 119(113), 132(113),134(113) Vihko, R., 27, 79, 81, 83(144), 143(144), 145(144) Vilkas, M., 36, 50, 70, 87, 89(224), 112(224),113(224),114(224) Villarroya, E., 404(38), 405, 412(38), 424(38) VillB, C., 18,. 47, 55(65, 301), 105(301), 118(65, 301) Villemez, C. L., 348 Vilsmeier, A., 250 Vischer, E., 311 Visser, D. W., 338, 354(229), 365(229), 367(229) Viswanathan, P., 395 Vliegenthart, J. F. G., 69, 133(485) Vlugter, J. C., 27, 96(145), 125(145), 153(145), 155(145) Vollmin, J. A., 24, 25(120), 38(120), 103(652) 158 Voelter, W., 55, 112(391), 113(391), 114(391) Voet, J. G., 393 Volk, W. A., 19, 86(72), 146(72),324 Von Glehn, M., 195 von Sydow, E., 73, 137(500) Vuez, J.-L., 81, 106(549),143(549)

AUTHOR INDEX, VOLUME 28

477

126(446), 159 Weicker, H., 17, 18, 44(51), 61(60a), Wacker, W. E. C., 423 86(60a), 126(60a), 144(60a) Weidemann, G., 341, 373(249, 250), 396 Wadman, W. H., 17 Weidinger, H., 250(81), 251 Wagstrom, B., 124(678), 158 Weiner, L., 339 Wagh, P. V., 445 Wagner, G., 47, 57(297), 102(297), Weisbach, J. A., 16, 17(34a), 36(34a), 86(34a), 90(34a), 128(34a), 146(34a), 120(297) 154(34a) Wagner, G. H., 64, 127(458) Weisleder, D., 50, 55(349), 111(349), Wagner, R. R., 341 115(349), 118(349), 131(349), 179 Wakahara, S., 184, 266 Walker, B., 18, 52(66), 79(66), 82, 116(66), Weiss, A. H., 29, 199, 200(140a) Weiss, J. B., 103(698), 159 146(66) Weiss, R. G., 246, 248(65h) Walker, E. A., 97, 154(620) Wellmann, E., 387, 388 Walker, H., 226 Walker, H. G., 48, 68(320), 104(320), Wells, H. J., 89, 90(572), 150(572) Wells, W. W., 12, 23(5), 26, 28(5), 30(5), 131(320), 135(320), 149(320) 32(5), 38(5), 41(5), 42(5), 46(138), Walker, R. H., 60, 61(418) 48(138), 52, 56(5), 59(5), 68(5), 79(5), Walker, R. T., 75, 138(514), 203 80(5), 89, 90(572), 100(5), 106(138), Walker-Smith, J., 131(687), 158 116(358), 119(138), 149(138), 150 Wallace, E. G., 184 Wallenfels, K., 41, 102(268a) (572) Welsh, K., 47, 105(306) Waller, G. R., 26, 32( 143) Wendt, A. W., 48, 58(317), 68(317), Walton, D. R. M., 31, 32(195) 104(317), 119(317), 130(317) Wang, C.-C., 285 Wentworth, M. A., 349 Wang, M. C., 337, 365(213) Wang, S.-F., 356, 379(329), 380(329), Werner, P. E., 195 Westphal, O., 14, 19(18), 52, 54(357), 382(329) 62(18, 447), 63(17), 92(18, 4471, Ward, D. J., 300 116(357), 117(357), 122(17, 181, Ward, L., 371 123(18, 434,447), 125(447), 126(729), Warren, D. R., 332, 358(182) 128(18, 447), 141(434), 153(434), Warren, L., 332 154(18, 447), 160 Washitak, M., 76 Wheat, R. W., 125(724), 159, 348 Wasyunin, N. A., 97 Wheelock, J. V., 19, 53, 83(370, 371), Watanabe, K. A. 292 116(69, 370, 371), 144(69, 370, 371) Watkins, W. M., 64, 122(454), 125(455), Whistler, R. L., 169, 182, 188, 201, 206, 376 Watson, J., 31, 98 285 White, B., 17 Watson, P. R., 92, 123(591), 154(591) White, D. M., 26, 147(141) Watson, R. W., 329 White, E. R., 16, 17(34a),36(34a), 61(34a), Way, C. P., 159 86(34a), 90(34a), 128(34a), 146(34a), Wease, J. C., 286 154(34a) Weatherall, I. L., 96, 155(617) Webb, A. C., 28, 29(158), 77(158), 85, 129 White, T., 83 Whitehead, B. K., 405 (660), 138(158), 145(557) Whitney, D. J.. 25, 46(128, 129), 68(128, Webb, R. F., 353 129), 102(128), 104(128, 129), 130 Webber, J. M., 195, 280(150, 151), 281, (128, 129), 132(128, 129) 290 Weber, B., 26, 46(138), 48(138), 106(138), Whyte, J. N. C., 292 Wicken, A. J., 16 119(138), 149(138) Weckesser, J,, 62(446), 122(711), Wiebers, J. L., 27 W

478

AUTHOR INDEX, VOLUME 28

Wiesmeyer, H., 341 Wietzerbin-Falszpan, J., 329 Wiggins, L. F., 165, 184(19),224(19), 287 Wight, N. J., 17, 135(52) Wilder, J., 312 Wiley, R. C., 48, 76(319), 107(319), 138(319,523, 692), 159 Wilken, D. R., 338, 348(297),349 Wilkie, K. C. B., 16, 21, 22(92), 35, 50, 92(345), 107(705), 111(345), 122(50a), 126(218a), 153(50a, 705), 154(345, 5931, 159 Wilkinson, S . G., 47, 106(307) Willers, J. M. N., 81, 143(544a) Willet, J. E., 26 Williams, C. S., 296 Williams, D. T., 28, 29(157), 66, 77(157), 86, 121(472), 122(472), 123(672), 137 (537), 138(157),146(561), 158 Williams, E. H., 286, 300 Williams, M. W., 48, 58(324), 104(324), 119(324) Williams, N. R., 278 Williamson, D. G., 437 Wilson, D. B., 364, 370 Winchester, B., 422 Windeler, A. S., 21 Wing, R. E., 47, 102(651), 103(296, 651), 158 Winkler, M. W., 383 Winkler, S., 404 Winterfeld, M., 44, 108(280) Winzler, R. J., 17, 61, 64, 125(680), 128(58), 141(453), 146(58), 154(58), 158, 445 Withers, M. K., 31, 97(194), 151(194) Wold, F., 352 Wold, J. K., 49, llO(338) Wolf, F. T., 48, 49(321), 68(321), 107(321), 130(321) Wolfe, L. S., 27, 116(150b) Wolff, I. A., 48, 106(325), 130(325), 132(325) Wolfrom, M. L., 184,261 Wollwage, P. C., 25, 147(125a) Wolochow, H., 393 Wong, P. K., 343 Wood, E., 339, 340(234) Wood, K. R., 257, 258(98) Wood, P. J., 38, 47, 51(233), 93, 104(233, 309), 128(309),151(602)

Woodmansee, C. W., 48, 68(323), 106 (323), 130(323) Woods, G. F., 214 Woolard, G. R., 23, 64,123(460) Wouters-Leysen, J., 15 Woyskovsky, N. M., 389 Wright, A., 336(211), 337, 390, 393 Wulff, G., 21, 49(95), 53(95), 108(95), 117(94) Wunderly, S. W., 39 Wusteman, P., 17, 22(50c) Wylie, W., 327

Y Yaguchi, M., 62, 85, 86(559), 123(560), 124(433, 559), 145(433, 559, 560) Yamada, K., 65, 114(471a) Yamada, S., 294 Yamaguchi, H., 31, 76, 93(190), 95, 152(190, 616), 444 Yamakawa, T., 52, 55, 84(391a), 87, 94, 95(606), 103(606), 112(391a), 116 (363), 158 Yamamoto, H., 72, 136(497) Yamashina, I., 119(710), 159, 403, 412, 4 W W , 420, 422, 431(18), 440(72), 442,444(18) Yamauchi, F., 53, 76, 95(367), 116(367), 139(529), 151(367) Yamazaki, N., 195 Yang, M. T., 92, 141(588) Yano, J., 102(697),115(697), 159 Yarovenko, N. N., 258 Yasuda, S., 283, 297, 298 Yasuda, Y., 16, 113(50),444 Yeo, A. N. H., 39 Yeomans, W., 255 Yoldikov, V. N., 257 Yoshida, H., 280(153),281, 285, 304 Yoshida, K., 54, 55(387), 112(387), 117(387), 195, 285 Yoshida, Y., 106(704), 113(704), 138(704), 159 Yoshida, Z., 260 Yoshikawa, M., 239, 247 Yoshizawa, I., 15, 46(31), 108(31) Yosizawa, Z., 53, 76, 95(367), 116(367), 139(529), 151(367) Young, M., 47, 68, 104(479), 105(304), 131(479) Young, R., 21,49,59(341),68,72,81(341),

AUTHOR INDEX, VOLUME 28 104(477),111(341), 123(477,492,676), 124(493), 126(341), 128(493, 686), 130(477), 132(477), 136(493), 138 (492, 495), 143(341, 493, 495, 676), 153(676), 158 Youngs, C. G., 29 Yu, R. J., 91, 153(587), 333 Yu, R. K., 19, 52(71), 76(71) Yuan Tze-Yuen, R., 390 Yudis, D. M., 26 Yurkevich, A. M., 246 2 Zach, K., 287 Zaikor, G. E., 39 Zaitzeva, T. N., 314, 315(43) Zakharov, V. F., 97 Zalitis, J., 364, 365, 36?(357), 368 Zamojski, A., 259 Zanetti, G., 239, 285(47) Zarembo, J. E., 16, 17(34a), 36(34a), 61(34a), 86(34a), 90(34a), 128(34a), 146(34a), 154(34a)

479

Zarkowsky, H., 356, 379, 380(330), 382(421) Zauk, L. C., 29 Zeleznick, L. D., 339 Zemek, J., 396 Zerban, F. W., 221 Zerhusen, F., 201 Zevenhuizen, L. P. T. M., 92, 153(590) Zhdanov, G. L., 360, 361(339), 367(339), 374(339), 375(339, 394), 394(339), 398(339) Zhdanov, Yu. A., 285 Zhivkov, V. I., 319 Zhukova, I. G., 32, 115(206) Ziderman, I., 282 Zimmerman, H. IC, 182 Zinbo, M., 93, 151(599, 600, 601) Zinkel, D. F., 29 Zitko, V., 91, 153(585, 586) Zlatkis, Z., 13 Zollinger, H., 250 Zolotarev, B. M., 56, 66(394a), 120(394a) Zumwalt, R. W., 28, 80, 113(152) Zweifel, G., 189

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SUBJECT INDEX FOR VOLUME 28 A

syl pyrophosphate), enzymic preparation of, 338 Abequose 5’-(arabinosyl pyrophosphate), occurester of cytidine pyrophosphate, isolarence of, 315 tion of, 316 5’-(a-~galactopyranosylpyrophosgas-liquid chromatography of, 63 phate), enzymic synthesis of, 340 Acetalation, of hexoses and hexitols, 4 5 ’-(galactosyl pyrophosphate), occurAcetals rence and isolation of, 315 dithio-, acetylated, gas-liquid chroma5’-(a-~glucopyranosyl pyrophostography of, 66, 121 phate), enzymic synthesis of, 337 isopropylidene, in gas-liquid chromaoccurrence and isolation of, 314 tography, 37 structure of, 309 Acetamide, N,O-bis(trimethylsily1)-, triin sucrose synthesis, 395 methylsilylation with, 26 5 ’-(Dmannitol 1-pyrophosphate), isolation of, 333 - , N-[5-(1,2-dihydroxyethyl)-3-furyl]-, formation of, 202 5’-(a-D-mannopyranosyl pyrophosphate), enzymic synthesis of, 339 -, trifluoro-N,O-bis(trimethylsily1)-,as 5‘-(mannosyl pyrophosphate, occurtrimethylsilylating agent, 27, 80 rence and isolation of, 315 -, trifluoroa-methyla-(trimethyl5’-phosphoro(P + N)phenylalanine, sily1)-, trimethylsilylation with, 28 in synthesis of glycosyl nucleoside Acetates, and gas-liquid chromatography pyrophosphates, 346 of monosaccharides, 33-36 5’-phosphoropiperidate, in synthesis Acetic acid, in hydrolysis of polysacchaof glycosyl nucleoside pyrophosrides, 17 phates, 346 -, trichloro-, in hydrolysis of glycopro5’-(a-D-xylopyranosyl pyrophosphate), teins, 17 enzymic synthesis of, 343 -, trifluoroAdenosine, 5’-azido-5’-deoxy-4’-fluorocatalytic action on trimethylsilylation, 25 2’,3‘-O-isopropylidene-, preparation in hydrolysis of plant cell-walls, 16 of, 273 Acetol, formation of, mechanism of, 200 - , 8-bromo-, 5’-(a-~-glucopyranosyl Acetonitrile, solvent for trimethylsilylapyrophosphate), preparation of, 355 tion, 29 -, 2’-deoxyAcetylation, of alditols, effect of borate -, 4’-fluoro-2‘,3’-O-isopropylidene-, complexes on, 34 preparation of, 273 Acetylformoin, from dehydration of - , 4 ’-fluoro-5’-O-sulfamoyl-, synthesis Dfructose, 176, 180 of, 226,273 Acrolein dimer, monosaccharides pre5 ‘-(a-D-glucopyranosyl pyrophosphate), pared from, 296 enzymic preparation of, 338 Adenine, 9-(2-chloro-2-deoxy-~-arabino- synthesis of, 8 furanosy1)-, synthesis of, 8 , N6-hydroxy-, 5’4a-D-glucopyranosyl Adenosine pyrophosphate), preparation of, 355 5’-(2-acetamido-2-deoxy-a~~-gluco- -, N6-methoxy-, 5’-(~y.~glucopyranosyl pyranosyl pyrophosphate), enzymic pyrophosphate), preparation of, 355 preparation of, 342 -, 2’,3’-0-(1-methoxyethy1idene)-, occurrence and biosynthesis of, 315 reaction with pivaloyl chloride, 279 5’42-amino-2-deoxy-cr-~glucopyrano- - , N’-methyl-, 5‘-(a-~glucopyranosyl

-

48 1

482

SUBJECT INDEX, VOLUME 28

pyrophosphate), preparation of, 355 rivatives, gas-liquid chromatography Albumin, effect on a-Dmannosidase acof, 156, 157 tivity, 427,429 Aldobiouronic acids, hydrolysis kinetics Alcohols of, gas-liquid chromatography of, 46 halogenation of, 240 Aldohexoses, 2-deoxy-, determination of, iodination of, 281 184 reactions with (alkoxymethy1ene)di- Aldol reactions, carbocyclic rings from methyliminium halides, 254 carbohydrates by, 174 with carbon tetrachloride and tertiary Aldonic acids phosphines, 246 analysis of, 22 with N-(2-chloro-1,1,2-trifluoroethyl)- gas-liquid chromatography of, and diethylamine, 258 lactones, 71-78 with cyanuric chloride, 258 trimethylsilyl derivatives, gas-liquid with dichlorocarbene, 260 chromatography of, 138-140 N,N’-dicyclohexyl-N-methyl- Aldonolactones, per(trimethylsily1) dewith carbodiimidium iodide, 260 rivatives, gas-liquid chromatography Aldaric acids, gas-liquid chromatography of, 136-138 of, 71-78 Aldono-1,4-lactones, as inhibitors of Alditols, crDmannosidase activity, 420 acetates, gas-liquid chromatography of, Aldono-1,5-lactones, as inhibitors of 34,59-65, 122-129 a-D-mannosidase activity, 419 and trimethylsilyl ethers, gas-liquid Aldononitriles chromatography of, 43 acetylated, gas-liquid chromatography disaccharide, trifluoroacetyl derivaof, 66, 120 tives, gas-liquid chromatography gas-liquid chromatography of, 56 of, 113 Aldopyranosides, acidic degradation of, gas-liquid chromatography of, 56-67 180 from oligosaccharides, per(trimethy1- Aldoses silyl)ated, gas-liquid chromatogdehydration in acidic solutions, 174raphy of, 133, 134 186 per(trimethylsily1) derivatives, gasin alkaline solution, 193-207 liquid chromatography of, 119, 120 -, 3,6-anhydro-, by alkaline degradatrifluoroacetates, gas-liquid chromatogtion, 202 raphy of, 37,65, 113 -, 2-deoxy-, dehydration in acid solutrimethylsilyl ethers, gas-liquid chrotion, 182-186 matography of, 57 -, 2-0-methylfrom uronic acids, acetates, gas-liquid alkaline degradation of, 201, 202 chromatography of, 129 dehydration of, 181 -, acetamidodeoxyAldosuloses, deoxy-, dehydration of, 171 gas-liquid chromatography of, 84-87 Aldosulosonic acids, dehydration of, 192 per-0-(trimethylsilyl) derivatives, gas- Aldotetroses, dehydration in acidic soluliquid chromatography, 145 tions, 175 -, aminoAlfalfa, analysis by gas-liquid chromatoggas-liquid chromatography of, 78 raphy, 45 trifluoroacetyl derivatives, gas-liquid Algae, gas-liquid chromatography of chromatography of, 114 green, 47 - , aminodeoxy-, peracetylated, gas- Alginic acid, gas-liquid chromatography liquid chromatography of, 145, 146 of, 89 -, 3,6-dideoxy-, gas-liquid chromatog- (A1koxymethylene)dimethyliminium haraphy of, 63 lides, reactions with alcohols. 254 - , glycosyl-, per-0-(trimethylsilyl) de- Alkyl chlorides, preparation of, 246,254

SUBJECT INDEX, VOLUME 28

483

Alkyl fluorides, preparation of, 258 carbon tetrachloride, 247 Alkyl halides, reduction by organotin Almond emulsin, a-D-mannosidase from, hydrides, 303 424,438 Altropyranoside, methyl 2-0-acetyl-4Alkyl iodides, preparation of, 281 Q-benzoyl-3,6-dibromo-3,6-dideoxyAllal, 4,6- O-benzylidene-3-deoxy-3-C(iodomethyl)-D-, preparation and a-D-,preparation of, 268 methanolysis of, 305 -, methyl 2-O-acetyl-4,6-O-benzyliAllantoic antigen, gas-liquid chromatogdene-3- bromo-3-deoxy-a-~-,prepraphy of chick, 47 aration of, and reaction with NAllofuranose, 3-deoxy-3-hydrazinobromosuccinimide, 268 1,2 :5,6-di-O-isopropylidene-a-~-, - , methyl 2-azido-3,4-O-benzylidene-6reaction with iodine in chloroform, chloro-2,6-dideoxy-a-~-, preparation 279 of, mechanism of, 252 - , 3-deuterio-1,2 :5,6-di-O-isopropyl-, methyl 2-azido-4,6-O-benzylidene-2idene-a-m, preparation of, 303 deoxy-a-m, reaction with (chloro- , 1,2 :5,6-di-O-isopropylidene-a-~-, methy1ene)dimethyliminium chloreaction with cyanuric chloride, 259 ride, 252 Allofuranoside, methyl 5,6-dideoxy-5- , methyl 4-O-benzoyl-2-S-benzoyl-6iodo-2,3-O-isopropylidene-g~bromo-6-deoxy-2-thio-a-~-, reaction preparation of, 242 with methanolic sodium methoxide, and iodine displacement in, 282 290 Allopyranoside, methyl 2,3-anhydro-4,6- - , methyl 4-O-benzoyl-6-bromo-2,60-benzylidene-a-Ddideoxy-2-(phenylsu1fonamido)-areaction with (chloromethy1ene)diD-, reaction with methanolic sodium methyliminium chloride, 278 methoxide, 290 with sodium iodide, sodium acetate - , methyl 4-0-benzoyl-2,3,6-tribromoand acetic acid, 292 2,3,6-trideoxy-a-D-, preparation of, - , methyl 4,6-O-benzylidene-3-chloro268 3-deoxy-p-~- , methyl 4,6-0-benzylidene-2-chloro2-deoxy-3-O-formyl-a-D-, preparation preparation of, 301 reaction with sodium azide, 286 of, 278 with sodium benzoate in tetrahydro- - , methyl 3,4-O-(R)-benzylidene-2chloro-2-deoxy-6-O-p-tolylsulfonylfuran, 293 - , methyl 4,6-0-benzylidene-2,3-didea-D-,reduction with lithium alumioxy-2,3-C-methylene-a-~and -ED-, num hydride, 292 preparation of, 306 - , methyl 4,6-0-benzylidene-2-deoxy2-halogeno-a-~-, acetal migrations - , methyl 3-chloro-3-deoxy-p-~-, prepin, 292 aration of, 301 - , methyl 3,4-0-(R)- and 3,4-O-(S)-ben- , methyl 3-chloro-3-deoxy-2,4,6-trizylidene-2-deoxy-2-halogeno-a-~, 0-methyl-p-D-, preparation of, 257 preparation of, 292 - , methyl 3,6-dichlor0-3,6-dideoxy-p-m, preparation of, 301 - , methyl 4,6-0-benzylidene-2-deoxy- , methyl 2,3,4,6-tetrachloro-2,3,4,62-iodo-a-Dtetradeoxy- p-D-, preparation of, 235 preparation of, 292 - , methyl 3,4,6-trichloro-3,4,6-trideoxy- reaction with (chloromethy1ene)dia-D-, hydrogenation over Raney methyliminium chloride, 253 - , methyl 3,4-O-(R)-benzylidene-2nickel, 302 Allose, D-, gas-liquid chromatography of, deoxy-2-iodo-6-0-p-tol ylsulfonyl-cr49 D-, reduction with lithium aluminum - , 1,2 :5,6-di-O-isopropylidene-a-~-, hydride, 292 reaction with triphenylphosphine- - , methyl 4,6-0-benzylidene-2-deoxy-

484

SUBJECT INDEX, VOLUME 28

2-(phenylsulfonamido)-a-~-, reaction with N-bromosuccinimide, 290 -, methyl 4,W-benzylidene-2,3-dibromo-2,3-dideoxy-a-~preparation of, and reaction with N-bromosuccinimide, 267 reaction with potassium tert-butoxide, 293 -, methyl 3,4-0-benzylidene-2,6-dichloro-2,6-dideoxy-a-~hydrogenation of, 303 preparation of, 278 -, methyl 2,6-dideoxy-2,6-imino-N(phenylsulfony1)-a-D-, preparation of, 290 Altrose, D-, gas-liquid chromatography of, 49 Amicetoside, methyl a-,preparation of, 283 -, methyl P-D, preparation of, 294 Amino acids derivatives with uridine 5’-pyrophosphate N-acetylmuramic acid esters, 328 trimethylsilylation of, 28 Amino sugars, see Sugars Amylose triesters, gas-liquid chromatography of, 54 trimethylsilylation of, 31 , 6-azido-6-deoxy-, derivatives, preparation of, 286 , 6-chloro-6-deoxypreparation of, 256 reaction with sodium azide, 286 Anhydro sugars acetates and methyl ethers, gas-liquid chromatography of, 148 degradation of 2,5-, mechanism of, 165, 166 formation of I,&, 20 gas-liquid chromatography of, 87-89 per(trimethylsily1) ethers, gas-liquid chromatography of, 147, 148 preparation of 2,5-, 287 Anthrone test, in sugar analysis, 220-222 Antiepileptic agents, Denegit, 9 Antigens, gas-liquid chromatography of, 47,64 Antispasmolytic agent, Vasopenton, 9

-

Apurinic acid, alkaline degradation of, 203 Apyrimidinic acid, alkaline degradation of, 203 Arabinal, di-0-acetyl-D halogen addition to, 261 reaction with hydrogen chloride or hydrogen bromide, 264 -, di-0-benzoyb, reactions with hydrogen halides, 265 Arabinitol D,degradation of, mechanism of, 165 L-, gas-liquid chromatography of, 57, 67 Arabinofuranose, 3-0-acetyl-5-deoxy-5iodo-1,2-O-isopropylidene-~-~-, reaction with silver fluoride, 291 Arabinomannan, gas-liquid chromatography of, 53 Arabinopyranose, &Lester of uridine 5’-pyrophosphate, enzymic preparation of, 343 isolation of, 325 Arabinopyranoside, methyl a+, reaction with sulfuryl chloride, 237 - , methyl 3,4-O-isopropylidene-P-~-, reaction with N-(2-chloro-l,l,2-trifluoroethyl)diethylamine,258 -, methyl 4-0-methyl-a-DL-, preparation of, 296 Arabinopyranosyl chloride, 4-chloro-4deoxy-L-, 2,3-di(chlorosulfate), preparation of, 235 Arabinose D,mutarotation in pyridine, 39 ester with adenosine 5’-pyrophosphate, occurrence of, 315 L-

gas-liquid chromatography of, 46 mutarotation and gas-liquid chromatography of, 40 reaction with sulfuryl chloride, 238 -, 2,5-anhydro-~-,dehydration of, 166 -, 4-O-benzoyl-5-deoxy-5-iodo-2,3-0isopropylidene-L-, diethyl dithioacetal, Preparation of, 242 - , 5-0-benzoyl-2,3-0-isopropylideneL-, diethyl dithioacetal, reaction with methyltriphenoxyphosphonium iodide, 242

SUBJECT INDEX, VOLUME 28

-, -,

1,3,5-tri-O-acetyl-2-chloro-2-deoxyD-, aromatization of, 305

2,3,4-tri-O-methyl-~-,dehydration of, 181 - , 2,3,5-tri-O-methyl-~-,dehydration of, 181 Arabinuronic acid, D, decarboxylation of, 187 Aromatization, of 1,3,5-tri-O-acety1-2chloro-2-deoxy-~-arabinose, 305 Arsenic compounds, of nucleosides, attempted preparation of, 253 Arsenic tribromide, reactions with uridines, 253 Arsenic trichloride, reactions with uridines, 253 Aryl cyanates, chlorination with hydrochloric acid and, of carbohydrates, 257 Ascarylose degradation of, 317 ester of cytidine pyrophosphate, isolation of, 316 Ascorbic acid and analogs, gas-liquid chromatography of, 76 decarboxylation of, 186 L,dehydration of, 188 dehydration of, mechznism of, 192 trimethylsilylation of, 26 Aspartamidoglycan, from ovalbumin, 440-442 Avocado, sugars, gas-liquid chromatography of, 49

485

Benzilic acid rearrangement in alkaline degradation, 205 in dehydration of sugars, 171 in lactic acid formation, 197 in saccharinic acid formation, 194 Benzoic acid, 4-butoxy-3,5-dimethoxy-, 2(1-pyrrolidiny1)ethyl ester, as spasmolytic agent, 9 Biological fluids, gas-liquid chromatographic analysis of, 80 Blood gas-liquid chromatography of, 45-47,81 mglucitol determination in, by gasliquid chromatography, 57 a-Dmannosidase in human, 403 Borate complexes effect on acetylation of alditols, 34 effect on trifluoroacetylation of sugars, 36 Boron trichloride, reaction with anhydro carbohydrate derivatives, 280 Bromelain, a-Dmannosidase as structural reagent for, from pineapple stem, 444 Bromination, of sugars, with N-bromosuccinimide, 249 (Bromomethy1ene)dimethyliminium bromide, in synthesis of bromodeoxy sugars, 250 Butaneboronates carbohydrate, in gas-liquid chromatography, 38,51 gas-liquid chromatography of monosaccharide, 120 C

B

Cadmium ion, effect on a-D-mannosidase Bacteria activity, 426, 428, 430, 433 gas-liquid chromatographic analysis Cancer tissue, a-Dmannosidase activity for, 81 in, 404 Gram-negative extracts, gas-liquid chro- Cannizarro reaction, with pyruvaldehyde, matography of, 59 200 Beer, analysis by gas-liquid chromatogra- Carbazole test, in sugar analysis, 222 Carbene, dichloro-, reaction with alcophy, 46 Benzamide, ~-cyclopropyl-4-(decyloxy)hols, 260 3,5-dimethoxy-, synthesis as antiepi- Carbocyclic compounds, from dehydraleptic agent, 9 tion reactions of carbohydrates, 174, 191 Benzene, iodo-, dichloride, reaction with Carbodiimide, N,N’-dicyclohexyl-, in tri-0-acetyl-Dglucal, 263

486

SUBJECT INDEX, VOLUME 28

synthesis of glycosyl esters of nucleoside pyrophosphates, 353 Carbodiimides, in synthesis of glycosyl esters of nucleoside 5'-pyrophosphates, 353 Carbodiimidium iodide, N,N'-dicyclohexyl-N-methyl-, reactions with alcohols, 260 Carbohydrates butaneboronates, in gas-liquid chromatography, 38 dehydration reactions of, 161-224 gas-liquid chromatography of, 11-160 sulfonylation of, 255 volatile derivatives for gas-liquid chromatography, 23-38 Carbon tetrachloride-triphenylphosphine, reactions with alcohols and carbohydrates, 246 Casein, gas-liquid chromatography of, 83 Cellobial, 2-acetoxyhexa-O-acetyyl-, alkaline degradation of, 205 Cellobiose dehydration of, 169 gas-liquid chromatographic separation of, 70, 74 Cellulose alkaline stability and gas-liquid chromatography of, 85 hydrolysis of, 21 in plant tissue, gas-liquid chromatographic determination, 60 trimethylsilylation of, 31 -, 6-azido-6-deoxy-, derivatives, preparation of, 286 - , 6-chloro-6-deoxypreparation of, 256 reaction with sodium azide, 286 Cerebrosides, gas-liquid chromatography of, 54 Chalcose, D,preparation of, 300 Chitaric acid, dehydration of, 166 Chitin, oligosaccharides, degree of polymerization of, 83,85 Chitonic acid, dehydration of, 166 Chitose, degradation mechanism of, 164, 165 (Chloroniethy1ene)dimethyliminium chloride, in synthesis of chlorodeoxy sugars, 250, 278

Chondroitin sulfates, gas-liquid chromatography of hydrolyzates, 78 Chromatography gas-liquid, applications to carbohydrates, 11-160 column packings for, 60 history, 12 ion-exchange and paper, in isolation of glycosyl esters of nucleoside pyrophosphates, 310 Chromone, 3,8-dihydroxy-2-methyl-, from uronic acids or pentoses, 190 Clindamycin preparation of, 226 from lincomycin, and 7-bromo and 7-iOdO analogs, 247 Cobalt ion, effect on a-D-mannosidase activity, 426,428, 433 Colitose, ester with guanosine 5'-pyrophosphate, 321 Copper ion, effect on cr-Dmannosidase activity, 426, 428, 433 Coriofuranose, 1,2,4,5,7-penta-O-(trrimethylsily1)- a-, and trimethylsilyl glycosides, 25 Coriose, trimethylsilylation of, 25 - , 1,2,4,5,6,7-hexa-O-(trimethylsilyl)keto-, 25 Corn syrups, gas-liquid chromatographic analysis of, 46,48, 68 Cotton, gas-liquid chromatographic analysis of, 62 Cyanuric chloride, reactions with alcohols and carbohydrates, 258 C yclitols acetates, gas-liquid chromatography of, 150 gas-liquid chromatography of, 89, 90 per(trimethylsily1) ethers, gas-liquid chromatography of, 149, 150 trifluoroacetyl derivatives, gas-liquid chromatography of, 114 - , aminodeoxy-, gas-liquid chromatography of, 87 Cycloamyloses determination by trimethylsilylation, 25 gas-liquid chromatographic separation of, 70 per(dimethylsilyl)ated, gas-liquid

SUBJECT INDEX, VOLUME 28

487

chromatography of, 135 5'-(~-ribitol pyrophosphate), biosynseparation as dimethylsilyl derivathesis and isolation of, 333 tives, 31 5'-(vinelosyl pyrophosphate), 317 Cyclohexaamylose, 6-bromo-6-deoxy-, -, 2,2'-anhydro-, preparation of, 253 preparation of, 303 Cytidines, iodination of, 243 -, 6-chloro-6-deoxy-, preparation of, 303 Cytostatic activity, chemical structure -, 6-deoxy-, preparation of, 303 and, of carbohydrates, 7 -, 6-deoxy-6-iodo-, preparation of, 303 Cytostatics, synthesis of, 7 Cyclohexenones, from dehydration reactions of carbohydrates, 174 D 2-Cyclopenten-l-one, 2,3-dihydroxy-, see Reductic acid Decarboxylation Cymaroside, methyl DL-, synthesis of, 298 enzymic, of glycosyluronic acid pyroC ytidine phosphate esters, 385 5'-(2-amino-2-deoxy-a-~-glucopyrano- of sugar acids, kinetics of, 186,187,190 syl pyrophosphate), enzymic synDechlorination, of chlorodeoxy sugars in thesis of, 339 presence of triethylamine, 302 5'-(6-deoxy-a-~-x ylo-hexopyranosyl-4Degradation ulose pyrophosphate), 318 of glycosyl esters of nucleoside 5'-pyro5'-(3-deoxyoctulosonic acid phosphate), phosphates, 356 332 Smith, of polysaccharides, 98-101 5'-(3-deoxy-~-manno-octulosonicacid of sugars, 163 triphosphate), 333 Degranol, synthesis and antitumor activ5'-(3,6-dideoxy-cr-~rabino-hexoity, 6 7 Degree of polymerization pyranosyl pyrophosphate), isolaof 4-0-methylmaltopolysaccharides tion of, 316 by gas-liquid chromatography, 47 5'-(3,6-dideoxy-P-~-arabino-hexo3f oligosaccharides, gas-liquid chromapyranosyl pyrophosphate), isolatography of, 58, 83, 85 tion of, 316 5'-(3,6-dideoxy-cr-~-ribo-hexopyranosyl Dehalogenation, reductive, of deoxyhalpyrophosphate), isolation of, 316 ogeno sugars, 299 5'-(3,6-dideoxy-a-~-xy lo-hexopyranos y 1 Dehydration of aldoses and ketoses in acidic solupyrophosphate), isolation of, 316 tion, 174-193 5'-(3,6-dideoxy-a-~-erythro-hexopyranosyl-4-ulose pyrophosphate), in alkaline solution, 193-207 analyses involving, 218-224 318 5'-(a-~-glucopyranosylpyrophosphate), of carbohydrates, 161-224 enzymic synthesis of, 338 of hexuronic acids, 190 occurrence and isolation of, 315 Dehydrohalogenation, of deoxyhalogeno sugars, 291, 294 structure of, 309 5'-(~-glucosyl phosphate), derivative, Deiodination, by ultraviolet irradiation, 304 from spores of Ustilago auenae, 333 Demethylation, in hydrolysis of methyl5'-(glycerol pyrophosphate), isolation ated polysaccharides, 22 of, 334 5'-(N-glycolylneuraminic acid phos- Denegit, antiepileptic agent, 9 phate), 332 Desalting, of solutions of glycosyl esters 5'-(glycosyl pyrophosphates), alkaline of nucleoside pyrophosphates obtained by chromatography, 311 hydrolysis of, 359 reaction with (chloromethy1ene)di- Desilylation, of trimethylsilyl derivatives, 32 methyliminium chloride, 253

488

SUBJECT INDEX, VOLUME 28

E Desosamine, DL-, synthesis of, 299 Dextrans Earthworms, a-Dmannosidase in, 405 gas-liquid chromatography of, 94 hydrolysis of methylated, 22 ECNSS-M, in gas-liquid chromatography, Dietetic foods, hexitols in, gas-liquid 60 chromatographic estimation of, 58 EDTA, effect on a-D-mannosidase acDiethylamine, N-(2-chloro-1,1,2-trifluorotivity, 425-431 ethyl)-, reactions with alcohols and Enzyme-metal-ion complexes, catalytic carbohydrates, 257 activity of, 423 Diethyl-N-(trimethylsilyl)amine, triEnzymes, classification of metal-depenmethylsilylation with, 28 dent, 423 Digitoxose, ester of uridine 5‘-pyrophos- Enzymic reactions, of glycosyl esters of phate, isolation of, 326 nucleoside pyrophosphates, 362-396 Enzymolysis Dimethylsilylation, of cycloamyloses, 31 configuration of glycoside linkage by, 32 Dinucleoside phosphates, synthesis of, of Dglucose and gas-liquid chromatog287 6,8-Dioxabicyclo[3.2.l]octane, prepararaphy, 40 tion of, 296 Epididymis -, cis- and trans-4-bromo-, preparation a-D-mannosidase activity in, 403 of, 296 as a-Dmannosidase source, 411 6,8-Dioxabicyclo[3.2.l]oct-2-ene,prep- Epimerases, aration of, 296 gas-liquid chromatography of, 47 6,8-Dioxabicyclo[3.2.l]oct-3-ene, prepinversion in glycosyl group by, 369-379 aration of, and isomer, 296 Erythritol, gas-liquid chromatography of, p-Dioxane, effect on mutarotation of 92 carbohydrates, 39 - , 1,4-bis[2-(methylsulfonyl)oxyethylamino]-, cytostatic activity, 7 Diplococcus pneumoniae, gas-liquid - , l-deoxy-2,4-O-ethylideneI-iodo-D, chromatography of, 47 Disaccharide alditols, trifluoroacetyl reaction with sodium cyanide, 282 derivatives, gas-liquid chromatogra- Erythromycin, sugar component desosphy of, 113 amine, 299 Disaccharides Erythronic acid, D, acetates, gas-liquid chromatography of, from l-deoxy-~eqthro-2,3-hexo70 diulose, 196 amino, per(trimethylsilyl)ated, gasformation from Dfructoses, 194 liquid chromatography of, 134 -, 2-Gmethyl- D-, isolation and characin blood and urine, trifluoroacetic acid terization of, 195 in hydrolysis of, 16 “Erythropyrone,” formation from eryperacetylated, gas-liquid chromatograthrose, 176 phy of, 133 Erythrose, dehydration in acidic solution, sulfates, esters with uridine 5’-pyro176 phosphate, in hen oviduct, 330 Ethanesulfenyl chloride, reaction with Dische test, for 2-deoxy-~eqthro-pen5,6-di hydro-2-methoxy-2H-pyran, tose, 224 274 Disilazane, hexamethyl-, trimethylsilyla- Ethylenediaminetetraacetic acid (EDTA), tion of sugars with, 23 effect on a-Dmannosidase activity, Dithioacetals, acetylated, gas-liquid 425-431 chromatography of, 121 Ethyl phosphite, reactions with deoxyhal-

SUBJECT INDEX, VOLUME 28 ogeno sugars, 285 Exo-P-D-glucanase, activity of, 421 Exo-glycanases, activity of, 402 Exo-cu-Dmannanase, activity of, 402, 421 F Flavonoid glycosides, hydrolysis of, 15 Foods, gas-liquid chromatography of, 43 Formamide, N,N-dimethyleffect on trimethylsilylation, 25 solvent for trimethylsilylation, 29 Formates, carbohydrate, formation of, 251,259 Formic acid, in hydrolysis of polysaccharides, 17, 20, 22 Fragmentation, and alkaline-degradation reactions of sugars, 196, 200 Fructans hydrolysis of, 19 of methylated, 23 Fructoglucans, gas-liquid chromatography of, 68 Fructose D-, alkaline degradation of, 200 degradation mechanism of, 164 dehydration in acid solution, 176,179 dehydration reactions of, 176 determination in presence of aldoses by gas-liquid chromatography, 43 ester of guanosine 5’-pyrophosphate, 320 ester of uridine 5’-pyrophosphate, isolation of, 326 mutarotation and gas-liquid chromatography of, 40 saccharinic acids from, 194, 195 trimethylsilylation and mutarotation of, 38 trimethylsilylation of, 24, 27 -, I-chloro-1-deoxy-D-, preparation of, 247 - , 1,2 :4,5-di-O-isopropylidene-P-~-, reaction with carbon tetrachloride-triphenylphosphine, 247 -, 2,3 :4,5-di-O-isopropylidene-~-,reaction with triphenylphosphine and carbon tetrachloride, 247 -, 3,4,5,6-tetra-O-acetyl- l-bromo-l-de-

489

oxy-L-, reaction with triethyl phosphite, 285 Fruit juices gas-liquid chromatography of, 48, 57 uronic acids, gas-liquid chromatography of, 76 Fucitol, from fucoidan, determination b y gas-liquid chromatography, 57 Fucoidan, L-fucitol determination in, by gas-liquid chromatography, 57 Fucopyranose, p-Lester of guanosine 5’-pyrophosphate, enzymic preparation of, 343 isolation of, 321 Fucose, Dester of thymidine 5’-pyrophosphate, occurrence of, 323 preparation of, 356 gas-liquid chromatography of, 48 ZFuraldehyde from dehydration of 2-amino-2-deoxy-~glucose, 182 formation from pentoses, 176 in sugar dehydrations, 176, 177 from hexuronic acids by decarboxylation, 186-193 -, 5-(hydroxymethyl)from dehydration of 2-amino-2-deoxy-~ glucose, 182 formation from D-fructose and other sugars, 164, 165 -, 5-methyl-, formation in dehydrations of hexoses, 177 Furan, 3-acetamido-5-( 1,2-dihydroxyethyl)-, formation of, 202 - , 2-(acetoxymethyl)-4-chloro-, formation from 1,3,5-tri-O-acetyl-2-chloro2-deoxy-~arabinose,305 - , 2-acetyI-3-hydroxy-, preparation of, 170, 176, 179, 180 - , 24 ~-gZycero-l,2-dihydroxyethyl)-, formation from D-glucal dehydration, 183 - , 2-(&-hydroxyacetyl)acetate, formation of, 184 from dehydration of D-frur’ x e , 176,179 isolation and identifica’ m , 162, 170 - , 2-(methoxyoxalvl’ ,rormation of, 185 3(2H)-Furanonp

490

SUBJECT INDEX, VOLUME 28

formation in dehydration reaction, 172, 175 from methylated sugars, 172, 181 Furfuryl alcohol degradation in acidified methanol, 215 levulinic acid from, 212-218 2-Furoic acid, 5-formylfrom hexuronic acids, 186, 189, 191 3-Furoic acid, tetrahydro-3-hydroxy-5(hydroxymethy1)-,isolation of, 205

G Galactal, D, dehydration of, 184 Galactitol acetylation of, effect of borate on, 35 gas-liquid chromatography as trimethylsilyl ether, 57 - , hexa-0-(trirnethylsily1)-, as standard in gas-liquid chromatography, 59 Galactocarolose, biosynthesis of, 325 Galactofuranose, a-D, ester of uridine 5'-pyrophosphate, isolation of, 325 Galactomannans, gas-liquid chromatography of, 47,56 Galactopyranose, ff-D-

ester of adenosine 5'-pyrophosphate, enzymic preparation of, 340 isolation of, 315 ester of guanosine 5'-pyrophosphate, isolation of, 319 ester of thymidine 5'-pyrophosphate, enzymic preparation of, 340 isolation of, 322 ester of uridine 5'-pyrophosphate, enzymic synthesis and fermentation production of, 340,341 isolation of, 325 oxidation with Dgalactose oxidase, 344 synthesis by mixed anhydride method, 351 PL-, ester with guanosine 5'-pyrophosphate, isolation of, 319 -, 2-acetamido-2-deoxy-a-~ ester of thymidine 5'-pyrophosphate, enzymic preparation of, 324 ester with uridine 5'-pyrophosphate, enzymic preparation and biosynthesis of, 342, 343

isolation of, 327

-, 2-amino-2-deoxy-a-~, ester of uridine 5'-pyrophosphate, preparation of, 355 - , 6-azido-6-deoxy-1,2:3,4-di-O-isopropylidene-a-D, preparation of, 286 - , 6-chloro-6-deoxy-1.2:3,4-di-0-isopropylidene-wDpreparation of, 246, 251, 252, 257, 259 reaction with sodium azide, 286 - , 6-deoxy-l,2 :3,4-di-O-isopropylidenea-D-, preparation by irradiation, 304 - , 6-deoxy-6-iodo-1,2:3,4-di-O-isopropylidene-a-D, deiodination by irradiation, 304 - , 6-0-(4,6-dichloro-1,3,5-triazin-2-yl)1,2 :3,4-di-Gisopropylidene- a-D-, preparation of, 259 - , 1,2:3,4-di-O-isopropylidene-a-~ chlorination by carbon tetrachloride and triphenylphosphine, 246 reaction with (chloromethy1ene)dimethyliminium chloride, 251 with N-(2-chloro-l,l,2-trifluoroethyl)diethylamine, 257, 258 with cyanuric chloride, 259 - , 6-0-formyl-1,2 :3,4-di-O-isopropylidene-a-D, preparation of, 251, 259 ,Galactopyranoside, methyl (Y-D, hydrolysis of, trifluoroacetic acid in, 17 - , methyl 2-chloro-2-deoxy-p-~,reaction with sulfuryl chloride, 235 - , methyl 4deoxy-4-iodo-2,3-di-Omethyl 6-O-p-tolylsulfonyl-a-D-, preparation of, 241 - , methyl 6-deoxy-6-iodo-3,4-0-isopropylidene-pa-, preparation of, 241 - , methyl 2,3-di-0-acetyl-4,6-dichloro4,6-dideoxy-a-~ reaction with sodium azide, 286 with tributyltin hydride, 303 - , methyl 2,3-di-O-benzy1-4,6-dideoxy4-iOdO-ff-D, reaction with radioactive iodine, 229 - , methyl 4,6-dichloro-4,6-dideoxy-a-~-, 2,3-cyclic sulfate, preparation of, 230, 231 2,3-di(chlorosulfate), preparation of, 231,232 hydrogenation over Raney nickel, 301 preparation of, 238

SUBJECT INDEX, VOLUME 28

491

methyl 4,6-dichloro-4,6-dideoxy-P- - , 2-amino-2-deoxy-~ dehydration in acid solution, 182 D-, 2,3-di(chlorosulfate), displacegas-liquid chromatography of, 78, 79 ment reaction, 234 -, methyl 4,6-dichloro-4,6-dideoxy-2,3- -, 6-deoxy-6-iodo-2,3 :4,5-di-O-isopropylidene-D, diethyl and dibenzyl die-methyl-a-n, preparation of, 247 dithioacetals, preparation of, 242 - , methyl 4,6-dichloro-4,6-dideoxy-2,3- , 2,3 :4,5-di-O-isopropylidene-~-,didi-0-(methylsulfony1)-a-s, preparaethyl and dibenzyl dithioacetals, retion of, 255 actions with methyltriphenoxyphos-, methyl 4,6-dichloro-4,6-dideoxy-2,3phonium iodide, 242 di-0-p-tolylsulfonyl-a-D, preparaGalactoside, ethyl a-D-,in soybean extion of, 255 tracts, 54 - , methyl 4,6-dichloro-4,6dideoxy-30-methyl+-, hydrogenation over Galacturonic acid, n Raney nickel, 300 -, methyl 2,3-di-O-methyl-6~-p-tolyldecarboxylation of, 187 dehydration of, 188, 189 sulfonyl-a-D, reaction with methylgas-liquid chromatography of, 64 triphenoxyphosphonium iodide, 241 isomerization and gas-liquid chroma-, methyl 3,4-O-isopropylidene-P-~, tography of, 40 reaction with methyltriphenoxyphos-, 2-amino-2-deoxy-n, decarboxylation phonium iodide, 241 of, 187 - , methyl 2,3,4-tri- O-acetyl-B-deoxy-6iodo-I-thio-a-D-, reaction with so- Gangliosides gas-liquid chromatography of, 84, 87 dium nitrite, 282 methanolysis of, 19 Ga1actopyranosid)uronic acid, methyl methanolysis, trimethylsilylation and (methyl, reaction with sodium methgas-liquid chromatography, 52 oxide in methanol, 206 Galactopyranosyl chloride, 4,g-dichloro- Gastric secretions, analysis by gas-liquid chromatography, 44, 81 4,6-dideoxy-~, 2,3-di(chlorosulfate), Gentiobiitol, per-0-trimethylsilyl derivapreparation of, 235 tives, gas-liquid chromatography of, Galactopyranosyluronic acid, a-D69 ester with uridine 5’-pyrophosphate, Gentiobiose, octaacetate, gas-liquid chroenzymic preparation of, 343 matography of, 70 isolation of, 326 methyl ester, ester with uridine 5 ‘-pyro- Glucal, Dphosphate, preparation of, 355 dehydration of, 183 L-Galactosaccharinic acid, a- and p-, foras inhibitor of a-Dmannosidase acmation from L-sorbose, 195 tivity, 420 “Galactosamine,” D, see Galactose, - , 2-acetoxy-tri-o-acetyl-D-,alkaline 2-amino-2-deoxy-~degradation of, 205 Galactose, - , 4,6-0-benzylidene-3-deoxy-3-C-(iodon methyl)-D, preparation of, 306 gas-liquid chromatography of, in - , 2-hydroxy-, dehydration of, 184 blood, 46 - , 2-methoxy-3,4,6-tri-O-methyl-~, demethanolysis of, 19 hydration of, 184 mutarotation and gas-liquid chroma- , tri-0-acetyl-D tography of, 40, 41 alkaline degradation of, 203 “ y ” - ~ ,gas-liquid chromatography of, chlorination of, 261, 262 40 reaction with hydrogen bromide, 263 -, 4-acetamido-4,6-dideoxy-~, ester of with iodobenzene dichloride, 263 thymidine 5’-pyrophosphate, and with nitryl iodide, 273 epimer, 323

-,

492

SUBJECT INDEX, VOLUME 28

-, 3,4,btri-O-acetyl-2-chloro-~-,prep-

pylidene-a-D preparation of, 240 reaction with potassium phthalimide"h', 283 - , bdeoxy-6-iodo- 1,2-O-isopropyliDdene-a-D, methylphosphonite, prepacetylation of, effect of borate on, 34, aration of, 280 - , 5-deoxy-5-iodo-l,2-O-isopropylidene35 determination in blood by gas-liquid 6-O-trifluoroacetyl-a-~, preparation chromatography, 57 of, 271 in mammalian nerve, determination - , 1,2:5,6-di-O-isopropylidene-a-~ by gas-liquid chromatography, 58 reaction with N-bromosuccinimide and -, 2,banhydro-~,structure of, infrared triphenylphosphine, 249 with bromotriphenoxyphosphonium study in, 8 bromide, 240 -, 5,banhydro-~,hydrolysis of, 5 with carbon tetrachloride-triphenyl-, 2,4-O-benzylidene-~,structure of, 4 phosphine. 247 -, 1,4: 3,bdianhydro-~, reaction with with (chloromethy1ene)dimethyliboron trichloride, 280 minium chloride, 252 - , 1,6-dichloro-1,6-dideoxy-~, preparawith N-(2-chloro-l,l,2-trifluoroethyl)tion of, 280 diethylamine, 258 Glucoamylase, gas-liquid chromatograwith cyanuric chloride, 259 phy of, 50 Glucofuranose, a-D, 1,2:3,5-di(benzenewith phosphorus pentachloride, 239 boronate), reaction with carbon tetrawith triphenylphosphine-carbon tetchloride and triphenylphosphine, rachloride, 254 246 -, 3-0-formyl-1,2: 5,6-di-O-isopropyli- , 1,6-anhydro-p~,-,gas-liquid chromadene-a-D-, preparation of, 254 tography of, 43 - , 1,2-O-isopropylidene-a-~ reaction with triphenyl phosphite and - , 3,6-anhydro-5-deoxy-5-iodo-1,2-0isopropylidene-a-D-, preparation of, methyl iodide, 280 synthesis of, 4 271 - , 5,banhydro- 1,2-O-isopropylidene-a- Glucofuranoside, methyl 3-deoxy-3-iodoD,synthesis of, 2 2,5,6-tri-O-methyl-p~, preparation - , 3-bromo-3-deoxy-1,2:5,bdi-0-isoof, 241 propylidene-a-D, preparation of, 280 - , methyl 2,5,6-tri-O-methyl-P-~,reaction with methyltriphenoxyphos-, 6-bromo-6-deoxy-1,2:3,5-di-O-isopropylidene-a-D, preparation of, 240, phonium iodide, 241 Glucoisosaccharinic acid 249 - , 3-chloro-3-deoxy-3-deuterio-1,2:5,6a- and p-, formation of, 194 di-0-isopropylidene-a-D, preparaformation of, 199 tion and reduction of, 303 Glucometasaccharinic acid, formation of, -, 6-chloro-6-deoxy-1,2:3,5-di-O-isopro199 pylidene-a-DGluconic acid, 2,3,4,6-tetra-O-acetyl-~, preparation of, 239,247, 252, 254, 259 ethyl ester, reaction with phosphorus reaction with anhydrous hydrazine, 285 pentachloride, 239 - , 6-deoxy-6-hydrazino-1,2:3,5-di-0Glucopyranose, A-D isopropylidene-a-D, preparation of, ester with adenosine 5'-pyrophos285 - , 3-deoxy-3-iodo-1.2 :5,bdi-0-isoprophate, 309, 314 pylidene-a-D, preparation of, 279 enzymic preparation of, 337 - , 6-deoxy-6-iodo-1,2:3,5-di-O-isoproin sucrose synthesis, 395 aration and chlorination of, 261, 262 Glucans, gas-liquid chromatography of, 50,59 Glucitol,

SUBJECT INDEX, VOLUME 28 ester of 8-bromoadenosine 5'-pyrophosphate, preparation of, 355 ester with cytidine 5'-pyrophosphate, 309,315,338 ester with 2'-deoxyadenosine 5'-pyrophosphate, enzymic preparation of, 338 ester with 2'-deoxyuridine 5'-pyrophosphate, enzymic preparation of, 338 ester with 5,6-dihydrouridine 5'-pyrophosphate, preparation of, 354 ester with guanosine 5'-pyrophosphate, 309, 338 ester of NR-hydroxyadenosine pyrophosphate, preparation of, 355 ester with 5-hydroxyuridine 5':pyrophosphate, enzymic preparation of, 338 ester with inosine 5'-pyrophosphate, enzymic preparation of, 338 ester of NG-methoxyadenosine 5'-pyrophosphate, preparation of, 355 ester of N1-methyluridine 5'-pyrophosphate, preparation of, 355 ester with thymidine 5'-pyrophosphate, 309, 338 epimerase action on, 375 isolation of, 322 mechanism of conversion of, 381 ester with uridine 5 '-pyrophosphate, 309,312, 313 carbodiimides in synthesis of, 353 degradation of, 356 enzymic preparation of, 336, 337 isolation of, 325 mechanism of epimerase reaction with, 372 synthesis by mixed anhydride method, 351 synthesis by phosphoramidate method, 345 - , 2-acetamido-2-deoxy-a-~ester of adenosine 5'-pyrophosphate, 315,342 of thymidine 5'-pyrophosphate, enzymic synthesis of, 324, 342 of uridine 5'-pyrophosphate, enzymic preparation of, 341 fermentation production and biosynthesis of, 343

493

isolation of, 327 2-amino-2-deoxy-a-~, esters of uridine, adenosine, cytidine and thymidine 5'-pyrophosphates, enzymic syntheses of, 338,339 -, 1,6-anhydro-p-~determination of, in corn syrup, 25 gas-liquid chromatography of, 42 - , 1,6-anhydro-2 deoxy-2-fluoro-p-~-, reaction with hydrogen bromide, 278 - , 1,2,3,4-tetra-O-aCetl-PD-, reaction with aryl cyanate and hydrochloric acid, 257 - , 1,2,3,4-tetra-O-acetyl-6-chloro-6deoxy-P-D-, preparation of, 257 - , 1,3,4,6-tetra-O-acetyL20-methyl-pD,preparation of, 289 - , 2,3,4-tri-O-acetyl- 1,6-anhydro-p-~-, reaction with hydrogen bromide, 278 Glucopyranose-'C, a-D, ester with uridine 5'-pyrophosphate, enzymic preparation of, 336 Glucopyranose-t, a-D, esters with uridine 5'-pyrophosphates, 337 Glucopyranoside, methyl a - ~ hydrolysis of, trifluoroacetic acid in, 17 reaction with N-bromosuccinimide and triphenylphosphine, 249 with sulfur monochloride, 256 with sulfuryl chloride, 230, 231 2,3,4,6-tetra(chlorosulfate), preparation of, 232 -, methyl a-D a n d p-D, sulfonylation of, 255 -, methyl p-D, hydrolysis of, 305 - , methyl 2-acetamido-3-O-acetyl-6chloro-2,6-dideoxy-4-0-( methylsulfonyl)-a-D-, preparation of, 256 - , methyl 2-acetamido-3-O-acetyl-2deoxy-a-D, methanesulfonylation of, 256 - , methyl 2-acetamido-3,4-di-O-acetyl6-bromo-2,6-dideoxy-a-~-, preparation by Pinner reaction, 257 - , methyl 3-0-acetyl-4,6-O-benzylidene-2-bromo-2-deoxy-a-~-,preparation of, 268 - , methyl 3,6-anhydro-a-~-, reaction with boron trichloride, 280 - , methyl 3,6-anhydro-P-~,preparation of, 287

-,

494

-,

SUBJECT INDEX, VOLUME 28

methyl 3-azido-4,6-0-benzylidenesulfuryl chloride, 300 -, methyl 2,3,4,6-tetra-O-(trimethyl3-deoxy-p-~,preparation of, 286 - , methyl 6-azido-6-deoxy-a-~, prepSilyl)-(Y-D-,methanolysis of, 32 aration of, 286 -, methyl 2,3,4-tri-O-acetyl-6-bromo-6- , methyl SO-benzoyl-a+, preparadeoxy-a-D, preperation of, 249, 257 tion from 6-chloro-6-deoxy analog, -, methyl 2,3,4-tri-O-acetyl-6-bromo-6285 deoxy-p-D, reaction with barium - , methyl 2-chloro-2-deoxy-p-~, hyhydroxide, 287 -, methyl 3,4,6-tri-O-acetyl-2-bromo-2drolysis of, 305 - , methyl 6-chloro-6-deoxy-c~-~ deoxy-P-D, reaction with sodium displacement of chloro by benzoate cobalt tetracarbonyl and carbon monoxide, 293 group, 285 -, methyl 2,3,4-tri-0-acetyl-6-chloro-6preparation of, 256, 280 reaction with sodium azide, 286 deoxy-a+, preparation by Pinner 2,3,4-tri(chlorosulfate),preparation of, reaction, 257 232 - , methyl 2,3,4-tri-O-acetyl-6-deoxy-6- , methyl 6-chloro-6-deo~y-tetra-O-piodo-a+-, hydrolysis of, 305 prepara- , methyl 3,4,6-tri-O-acetyl-2-deoxy-2tolylsulfonyl-a-D and +-D, iodo-p-Dtion of, 255 -, methyl 6-chloro-6-deoxy-2,3,4-tri-O- reaction with bromine and silver acmethyl-cY-D-, preparation of, 257 etate, 288 - , methyl 6-chloro-6-deoxy-2,3,4-tri-O- with sodium cobalt tetracarbonyl and (methylsulfony1)-a-D and -P-D-, carbon monoxide, 292 - , methyl 2,3,4-tri-O-methyl-a-~, repreparation of, 255 - , methyl 6-chloro-6-deoxy-2,3,4-tri-O- action with aryl cyanate and hydrochloric acid, 257 p-tolylsulfonyl-a-D and +-D, prep- , methyl 2,4,6-tri-O-methyl-p-~, rearation of, 255 - , methyl 4-deoxy-4-iodo-2,3-di-Oaction with aryl cyanate and hydromethyl 6-O-p-tolylsulfonyl-cz-D-, chloric acid, 257 preparation of, 241 Glucopyranosyl bromide, 3,4-di-O-acetyl- , methyl 4,6-diazido-4,6-dideoxy-a-~, 6-bromo-2,6-dideoxy-2-fluoro-a-~, preparation of, 286 preparation of, 278 -, methyl 2,3-di-O-benzoyl-4,6-di-O- - , 2,3,4,6-tetra-O-acetyl-a-~, prepara(methylsulfony1)-a-D, displacement tion of, 279 reactions, 230 Glucopyranosyl chloride, 3,4,6-tri-O-, methyl 2,3-di-O-benzyl-4,6-dideoxy- acetyl-2-chloro-2-deoxy-(~-~-,prep4-iOdO-(Y-D, reaction with radioactive aration of, 261, 262 iodine, 229 - , 3,4,6-tri-0-acetyl-2-chloro-2-deoxy-p- , methyl 4,6-dichloro-4,6-dideoxy-~D,preparation of, 262 D-, 2,3-di(chlorosulfate), displaceGlucopyranosyluronic acid, (Y-Dment reaction, 233, 234 ester of uridine 5'-pyrophosphate, de- , methyl 4,6-dichloro-4,6-dideoxy-2,3carboxylation of, 385 die-(methylsulfony1)-p-m, preparaenzymic preparation of, 343 tion of, 2,W isolation of, 326 - , methyl 2,3-di-O-methyl-a-~, reac- -, 2-acetamido-2-deoxy-(~-~ tion with triphenylphosphine-carbon ester of uridine 5'-pyrophosphate, isolatetrachloride, 247 tion of, 327 - , methyl 2,3-di-O-methyl-6-0-p-tolylmethyl ester, ester of uridine 5'-pyrosulfonyl-a+, reaction with methylphosphate, preparation and reductriphenoxyphosphonium iodide, 241 tion of, 355 -, methyl 3-O-methyl-~-,reaction with Glucosaccharinic acid, formation of, 199

SUBJECT INDEX, VOLUME 28 L-, a- and p-, synthesis of, 194 “Glucosamine,” D,see Glucose, 2-amino2-deoxy-D Glucose

D,

anomerization and gas-liquid chromatography of, 41 degradation of, 164 gas-liquid chromatography of, in blood, 46,47 I4C-labeled, alkaline degradation of, 200 methanolysis of, 19 mutarotation and gas-liquid chromatography of, 40, 41 reaction with sulhryl chloride, 235 saccharinic acids from, 194, 195 trifluoroacetylation and mutarotation of, 38 - , 2-amino-2-deoxy-~ dehydration of, 182 gas-liquid chromatography of, 78, 79 6-’SN-amino-6-deoxy-~-,synthesis of, 283 3-bromo-3-deoxy-~,preparation of, 229 6-bromo-6-deoxy-~-, preparation of, 246 3-chloro-3-deoxy-~, preparation of, 229 6-chloro-6-deoxy-~, preparation of, 246 3-chloro-3-deoxy-1,2 :5,6-di-O-isopropylidene-a-D, preparation of, 247 g - d e o x y - ~ ,ester of thymidine 5’-pyrophosphate, 323 5-0-methyl-D-, synthesis of, 4 penta-O-(trimethyl-d,,-silyl)-D-, gasliquid chromatography and mass spectrometly of, 32 tetra-0-methyl-D, mutarotation of, in pyridine, 39 2,3,4,5-tetra-O-methyI-~, alkaline degradation of, 202 2,3,5,6-tetra-O-methyl-~-, dehydration of, 181 “Glucoseen,” tetramethyl-, dehydration of, 184 Glucoside, ethyl a-D, in sake, gas-liquid chromatography and, 54, 55 -, methyl 4,6-O-benzylidene-a-D-, re-

495

action with carbon tetrachloride-triphenylphosphine, 247 Glucosiduronic acids, gas-liquid chromatography of phenolic and terpenoid, 77 Glucosinolates, gas-liquid chromatography of, 25, 54 Glucuronic acid, D

alkaline degradation of, 206 epimerization of, 40 gas-liquid chromatography of, 64 - , 2-amino-2-deoxy-~, decarboxylation of, 187 - , 4-O-methyl-~,gas-liquid chromatography of, 64,65 Glutamic acid, L-, Dribose synthesis from, 294 Glycals alkaline degradation of, 203 dehydration in acid solution, 182-186 deoxyhalogeno sugars by addition reactions with, 261 G1yceraldeh yde dehydration to pyruvaldehyde, 174 gas-liquid chromatography of, 96 separation from glycolaldehyde as trimethylsilyl oximes, 31 Glycerol ester of cytidine 5’-pyrophosphate, isolation of, 334 gas-liquid chromatography of, 57, 91 Glycoaspartamidase, activity of, 438 Glycodiuloses, 3-deoxy-, alkaline degradation of, 196 -, 4-deoxy-, alkaline degradation of, 196 Glycolaldehyde gas-liquid chromatography of, 95 separation from glyceraldehyde as trimethylsilyl oximes, 31 Glycolipids gas-liquid chromatography of bacterial, 47,50 methanolysis of, and gas-liquid chromatography, 51 trifluoroacetates, gas-liquid chromatography of, 55 Glycopeptides a-Dmannosidase action on, 420 a-Dmannosidase as structural reagent for, 437-445

496

SUBJECTINDEX, VOLUME 28

methanolysis of, 18, 19 Glycoproteins gas-liquid chromatography of, 43, 44, 48,63 hydrolysis of, 16, 17, 86 a-Dmannosidase action on, 420 a-D-mannosidase as structural reagent for, 437-445 in catabolism of, 436 methanolysis of, 19 methanolysis, timethylsilylation, and gas-liquid chromatography of, 52 pituitary, Dxylose in, gas-liquid chromatography of, 67, 68 Glycosaminoglycans gas-liquid chromatography of, 48, 49, 54, 75, 79 hydrolysis of, 14 G1ycosidases activity of, 402 effect on a-mmannosidase activity, 409, 438 Glycosides of amino sugars, trifluoroacetyl derivatives, gas-liquid chromatography of, 114 cardiac, perchloric acid in hydrolysis of, 16 chlorodeoxy, formation during sulfonylation, 255 flavonoid, hydrolysis of, 15 hydrolysis of, effect of halogeno groups on, 305 methyl, acetates, gas-liquid chromatography of, 54,118 gas-liquid chromatography of, 51-56 methylated (fully), gas-liquid chromatography of, 118 trifluoroacetates, gas-liquid chromatography of, 55, 112 trimethylsilyl ethers, gas-liquid chromatography of, 43, 51-54, 115-118 phenolic, trimethylsilyl derivatives, gas-liquid chromatography of, 54, 118 plant, trimethylsilyl derivatives, gasliquid chromatography of, 118 steroid, gas-liquid chromatography of, 53 thio-, trimethylsilyl derivatives, gasliquid chromatography of, 118

Glycosphingolipids, timethylsilylation of, 28 Glycosuloses, 3-deoxy-, intermediates in 2-furaldehyde formation in sugar dehydrations, 177, 178 Glycosylation, glycosyl esters of nucleoside pyrophosphates in, 391-397 Glycosyl esters, of nucleoside 5'-pyrophosphates, 307-399 Glycuronans decarboxylation of, 186, 188 dehydration of, 188 Glycuronic acids alkaline degradation of, 206 decarboxylation of, 186, 190 degradation of, 166,168 Gonadotrophin, a-Dmannosidase in structural studies of human chorionic, 445 Grasshoppers, gas-liquid chromatographic analysis of constituents of, 62 Guanosine 5'-(6-deoxy-a-~lyxo-hexopyranosyl-4ulose pyrophosphate), 322 5'-(2-deoxy-~-arubino-hexosyl pyrophosphate), enzymic synthesis of, 338 5'-(6-deoxy-~talosyl pyrophosphate), 321 5'-(3,6-dideoxy-P-~-xylo-hexopyranosyl pyrophosphate), isolation of, 321 5'-(fructosyl pyrophosphate), occurrence of, 320 5'-(P-~-fucopyranosyl pyrophosphate), enzymic synthesis of, 343 occurrence and isolation of, 321 5 ' - ( a - and ~ P-L-galactopyranosyl pyrophosphates), isolation of, 319 5'-(a-~glucopyranosylpyrophosphate), enzymic synthesis of, 338 occurrence and isolation of, 318 structure of, 309 5'-(~-~-gulopyranosyluronic acid pyrophosphate), isolation of, 320 5'-(~glycero-~-manno-heptosylpyrophosphate), occurrence and isolation of, 320 5'-(a-D-mannopyranosyl pyrophosphate), enzymic and fermentation production of, 339,340 occurrence and isolation of, 319

SUBJECT INDEX, VOLUME 28 5’-(a-~-mannopyranosyluronic acid pyrophosphate), isolation of, 320

497

guanosine 5’-pyrophosphate, 320 Dglycero-L-munno-, gas-liquid chro5’-(oligosaccharide pyrophosphates), matography of, 59 322 Heptoses 5’-phosphorocyclohexylamidate,in gas-liquid chromatography of, 49, 54 synthesis of glycosyl nucleoside trimethylsilyl derivatives, gas-liquid pyrophosphates, 346 chromatography of, 109 5’-(a-~rhamnopyranosylpyrophosHeptulose, 3,4,5,6,7-penta-O-acetyl-lchloro-1-deoxy-D-gulacto-, reaction phate), enzymic preparation of, 344 isolation of, 321 with triethyl phosphite, 285 Guanosine, 2‘-deoxy-, 5’-(a-~-mannopyra- 3-Heptulose, Daltro-, trimethylsilylation of, 25 nosy1 pyrophosphate), enzymic synHeptuloses, trimethylsilylation of, 25, 28 thesis of, 340 Heteroglycans, hydrolysis of, 14 - , 2‘,3’-O-isopropylidene-,reaction 3,5-Hexadien-2-one, 3,4,5-trihydroxy-, with phosphoryl chloride, 239 Gulopyranosyluronic acid, p-L-, ester of from dehydration of &fructose, 176, guanosine 5‘-pyrophosphate, isola180 tion of, 320 Hex-1-enitol, 1,5-anhydro-~-arabino-,dehydration (theoretical) of, 184 - , 1,5-anhydro-4,6-0-benzylidene-1,2,3H trideoxy-3-C-(iodomethyl)-~-uraHalogenation, of alcohols, 240 bino-, preparation of, 306 (Halogenomethy1ene)dimethyliminium - , 1,5-anhydro-4,6-0-benzylidene-1,2,3halides, in synthesis of deoxyhalotrideoxy-3-C-(iodornethyl)-~ribo-, geno sugars, 250 preparation and methanolysis of, 305 Helminths, a-Dmannosidase in, 405 -, 1,5-anhydro-4,6-0-benzylidene-1,2,3Hemagglutinin, a-Dmannosidase action trideoxy-3-C-methylene-~-ery tho-, on soybean, 445 preparation of, 306 Hemicelldoses - , 1,5-anhydro-2-deoxy-~-arubino-,as gas-liquid chromatography of, 62 inhibitor of a-Dmannosidase activity, 420 in plant tissue, gas-liquid chromatographic determination, 60 -, 2,3,4,6-tetra-O-acetyyl-1,5-anhydro-~trimethylsilylation and gas-liquid chroarubino-, alkaline degradation of, 205 matography of, 42 Hex-2-enitol, 1,3,4,5-tetra-O-acetyl-2,6Heparin anhydro-L-threo-, dehydration of, 184 reaction with hydrogen bromide, 266 deamination of, gas-liquid chromatography and, 76 Hex-5-enofuranose, 3-0-acetyl-5,6-digas-liquid chromatography of hydrodeoxy-l,2-O-isopropylidene-a-~lyzates, 75, 78 rylo-, reaction with nitryl iodide, 270, hydrolysis of, trifluoroacetic acid in, 16 299 L-iduronic acid in, 55 - , 3-0-acetyl-5,6-dideoxy-1,2-O-isoHeptitols propylidene-6-nitro-a-~-xylo-, preparation of, 299 acetates, gas-liquid chromatography of, 64,128 - , 5,6-dideoxy-1,2-0-isopropyIidene-cuacetylation of, 36 D-xylotrimethylsilyl ethers, gas-liquid chroreaction with iodine azide, 271 with iodine trifluoroacetate, 271 matography of, 58 with silver trifluoroacetate and ioHeptonolactones gas-liquid chromatography of, 72 dine, 272 trimethylsilylation of, 28 Hex-4-enofuranoside, methyl 5,6Heptose, D-glycero-D-munno-, ester of dideoxy-2,3-O-isopropylidene-~-~

-,

498

SUBJECT INDEX, VOLUME 28

Hex-5-enopyranoside, methyl 6-deoxy-aDarabino-, hydrolysis of, 291 - , methyl 6-deoxy-P-~rylo-,hydrolysis and rearrangement of, 291 Hex-2-enopyranosid-4-ulose, methyl 2,3dideoxy-P-Dglycero-, preparation of, 293,294 Hex-4-enopyranosid)uronic acid, (methyl Cdeoxy-P-~-threo-, methyl ester, preparation of, 206 Hex-2-enose, cis- and trans-d-deoxy-D, by alkaline degradation of 2,3,4,5tetra-0-methyl-D-glucose, 202 - , 4,6-di-O-acetyl-2,3-dideoxy-~ erythro-, formation and degradation of, 204 Hex-5-enulofuranose, 6-deoxy-2,3-0isopropylidene-P-Dthreo-, prepara0-isopropylidene-P-L-arabinotion of, 291 formation of, 246 Hexitols preparation by irradiation, 304 acetates, gas-liquid chromatography of, Hex-2-enopyranoside, methyl 4-0-benzyl-2,3-dideoxy-6-0-trityl-a-~-ey th127, 128 trimethylsilyl ethers, gas-liquid chroTO-, reaction with hydrogen bromidematography of, 57 acetic acid, 269 -, 4-deoxy-~,acetates, gas-liquid chro- , methyl 4,6-0-benzylidene-2,3matography of, 63 dideoxy-a-DerythroHexodialdo-l,5-pyranosides, Dgluco-, bromination of, 267 preparation of, 286 dehydration of, 184 Hexodialdose, a-D-galacto-, ester of preparation of, 253, 292 uridine 5'-pyrophosphate, preparareaction with nitryl iodide, iodine tion of, and 6-tritiated derivative, azide, or nitrosyl chloride, 273 344 with Simmons-Smith reagent, 305 - , methyl 4,6-0-benzylidene-2,32,3-Hexodiulose, 1-deoxy-Derythroalkaline degradation of, 196 dideoxy-p-D-erythro-, preparation of, from Dfructose dehydration, 179, 180 292 - , methyl 4,6-0-benzylidene-2,3preparation of, 168, 169 preparation and dehydration of, 194 dideoxy-a-D and -pDthreo-, prep- , Cdeoxy-Dglycero-, preparation of, aration of, 292 - , methyl 4,6-di-O-acetyI-2,3-dideoxy- 168, 169 PDerythro-, preparation of, and a B,SHexodiulose, Dthreo-, dehydration of, anomer, 292 186 -, methyl 2,3,6-trideoxy-a-~e~ythro-, Hexofuranose, 3,6-anhydro-5-deoxy-1,2preparation of, 292 0-isopropylidene-a-DxyZo-, preparaHex-3-enopyranoside, methyl 4,6-0tion of, 271 benzylidene-P-Deythro-, prepara- -, 3-deoxy-3-deuterio-l,2: 5,6-di-O-isopropylidene-a-D-ribo-,preparation of, tion of, 293 303 -, methyl 4,6-0-benzylidene-2-bromo2,3-dideoxy-a-~-threo-, preparation -, 3-deoxy-3,3-diiodo- 1,2 :5,6-di-O-isopropylidene-a-D-ribo-,preparation of, of, 293 - , methyl 4,6-0-benzylidene-3-deoxy279 P-D-erythro-, preparation of, 286 -, 5-deoxy-1,2-O-isopropylidene-a-~ erythro-, reaction with bromine in chloroform or carbon tetrachloride, 269 Hex-5-enofuranoside, methyl 5,6dideoxy-2,3-di-O-p-tolylsulfonyl-a-~arabinoreaction with iodine trifluoroacetate, 273 with nitryl iodide, and with iodine azide, 270, 271, 299 -, methyl 5,6-dideoxy-6-nitr0-2,3-di-Op-tolylsulfonyl-a-L-arabino-,preparation of, 299 Hex-2-enopyranoglycan,2,3-dideoxy-6-0trityl-a-oerythro-, bromination in methanol and silver acetate, 268 Hex-5-enopyranose, 6-deoxy-l,2 :3,4-di-

SUBJECT INDEX, VOLUME 28

499

- , methyl 3,4-O-(S)-benzylidene-2,6-dixylo-, preparation of, 271 Hexofuranurono-6,3-lactone,5-deoxy-1,2deoxy-a-Dribo-, preparation of, 292 0-isopropylidene-a-D-xyZo-, prepara- , methyl 6-chloro-4,6-dideoxy-a-~tion of, 303 xylo-, preparation of, 302 Hexonic acid, 2,3,4,5-tetra-O-acetyl-6- - , methyl 6-chloro-3,4,6-trideoxy-a-~chloro-6-deoxy-, ethyl ester, preparaeythro-, preparation of, 302 tion of, 239 -, methyl 2,3-di-O-acetyl-6-chloro-4,6Hexopyranose, 1,6-anhydro-4-O-benzyldideoxy-a-D-xylo-, preparation of, 303 3-bromo-2,3-dideoxy-p-~-ribo-, prep- , methyl 3,6-dideoxy-p-~-ribo-, preparation of, 269 aration of, 301 -, 2-deoxy-a-~-arabino-,ester with uri- - , methyl 4,6-dideoxy-a-~-xylo-, preparation of, 302 dine 5'-pyrophosphate, fermentation - , methyl 2,3,6-trideoxy-a-~-erythro-, production of, 341 preparation of, 283 - , %deoxy-a-D-lyro-, ester with uridine 5'-pyrophosphate, fermentation pro-, methyl 2,3,6-trideoxy-P-~-erythro-, preparation of, 294 duction of, 341 - , 2,4-diamino-2,3,4,6-tetradeoxy-~- - , methyl 3,4,6-trideoxy-a-~-erythro-, preparation of, 302 arabino-, synthesis of, 283 - , methyl 2,3,6-trideoxy-p-~-threo-, -, 3,6-dideoxy-a-~arabino-,see preparation of, 294 Tyvelose Hexopyranosid-3-ulose 3-hydrate, methyl -, 3,6-dideoxy-p-~-arabino-,see 2-bromo-2-deoxy-a-~ara bino-, Ascarylose 3,3,4,6-tetraacetate, preparation of, -, 3,6-dideoxy-a-~-ribo-,see Paratose 289 -, 3,6-dideoxy-a-~-xyZo-, ester of cytidine pyrophosphate, isolation of, 316 Hexopyranosiduloses, gas-liquid chromatography of, 54 -, 3,6-dideoxy-P-~-xyZo-,ester with guaHexopyranos-3-ulose, a-wibo-, ester of nosine 5'-pyrophosphate, 321 uridine 5'-pyrophosphate, enzymic Hexopyranoside, ethyl 2,4-bis(acetamipreparation of, 344 do)-2,3,4,6-tetradeoxy-p-~-ara bino-, Hexopyranos-4-ulose, 6-deoxy-a-~preparation of, 284 lyxo-, ester with guanosine 5'-pyro- , methyl 2,3-anhydro-4-deoxy-6-0phosphate, 322 methyl-a-DL-lyxo-, synthesis of, 295 -, 6-deoxy-a-~-xylo- , methyl 2,3-anhydro-4-deoxy-6-0ester of cytidine 5'-pyrophosphate, 318 methyl-a-DL-ribo-, preparation of, of thymidine 5'-pyrophosphate, 323 295 - , methyl 4-O-benzoyl-6-bromo-6-de- -, 3,6-dideoxy-a-~-erythro-, ester with cytidine 5'-pyrophosphate, 318 OXY-CY-D-, preparation of, mechanism Hexopyranosyl bromide, 4-0-acetyl-3of, 276 bromo-2,3-dideoxy-6-0-p-tolylsul-, methyl 4-O-benzoyl-6-bromo-2,3,6fonyl-a-D-arabino-, preparation of, trideoxy-a-wrythro , preparation of, 264 283 3,4,6-tri-O-acetyl-2-deoxy-D-arabino-, -, methyl 4-O-benzoyl-6-chloro-2,3,6preparation of, 264 trideoxy-a-D-erythro-, preparation of, Hexopyranosyl' chloride, 2,2-dichloro-2302, 303 deoxy-a-D-arabino-, preparation of, -, methyl 4-O-benzoyl-2,3,6-trideoxy-6261,262 iodo-a-D-erythro-, preparation of, 283 - , methyl 4,6-O-benzylidene-a-~-,reac- - , 3,4,6-tri-O-acetyl-2,2-dichloro-2-deoxy-D-arabino-, preparation of, 262 tions with N-bromosuccinimide, 276 - , methyl 4,6-0-benzylidene-2,3-dide- Hexosamines gas-liquid chromatography of, 78.79 oxy-a-D-erythro-, reaction with Ntrimethylsilylation of, 29 bromosuccinimide, 283

-.

500

SUBJECT INDEX, VOLUME 28

trimethylsilyl derivatives, gas-liquid Hexulofuranose, 4-0-acetyl-1-0-benzoyl6-deoxy-6-iodo-2,3-0-isopropylichromatography of, 143 dene-a-L-xylo-, reaction with silver Hexose, acetamidodeoxyfluoride, 291 alkaline degradation of, 202 - , 4,6-anhydro-l-O-benzoyl-2,3-O-isogas-liquid chromatography of, 80-82 propylidene-a-L-xy lo-, preparation of, 0-trimethylsilyl derivatives, gas-liquid 290 chromatography of, 143, 144 -, 1-0-benzoyl-6-deoxy-6-iodo-2,3-O-iso- , 2-acetamido-2,4,6-trideoxy-,ester propylidene-a-L-, reaction with silof uridine 5'-pyrophosphate, isolation ver fluoride, 290 of, 327 - , 3,6-anhydro-2-deoxy-~-u~ubino-, for- Hexulose, 1,2 :4,5-di-O-isopropylideneP-D-ribo-, reaction with carbon tetramation of, 202, 204 - , 2-deoxy-~-arabinochloride-triphenylphosphine, 247 alkaline degradation of, 202 Hexuloses, trimethylsilylation of, 25 esters of uridine 5'-pyrophosphate and Hexulosonic acid, D-urabino-, dehydration of, 188, 189, 193 of guanosine 5'-pyrophosphate, enzymic synthesis of, 338 -, ~-xylo-,dehydration of, 192 - , 4-deoxy-~-urabino-, gas-liquid chro- bHexulosonic acid, D-XY~O-, dehydration matography of, 63 of, 189 - , 4,6-di-O-acetyl-3-bromo-2,3-dideoxy-Hexuronic acids a-D-arabino-, preparation of, 263 analysis of, 188 decarboxylation of, 186, 190 - , 2,2-dichloro-2-deoxy-~-arabino-, dehydration of, 190 preparation of, 262 -, 2,6-dideoxy-~-ribo-,ester of uridine gas-liquid chromatography of, 71 Honey, gas-liquid chromatographic anal5'-pyrophosphate, isolation of, 326 - , 3,6-dideoxy-~-arabino-,see Tyvelose ysis of, 48, 68 - , 3,6dideoxy-~-ribo-,see Paratose Hormones, sex, effect on a-D-mannosi-, 4,6-dideoxy-~-xylo-,synthesis of, 300 dase activity, 434,437 - , 3,4,6-tri-O-acetyl-2-deoxy-a-~Humectants, tobacco, gas-liquid chromaarabino-, preparation of, 263 tography of, 97 -, 3,4,6-trideoxy-3-(dimethylamino)-~- Hyaluronic acid, gas-liquid chromatography of hydrolyzates of, 78 xylo-, as structure of desosamine, 299 Hydantoin, 1,3-dibromo-5,5-dimethyl-, Hexoses reaction with 5,6-dihydro-2-methoxyyacylated, gas-liquid chromatography of trimethylsilyl derivatives, 110 W-pyran, 274 Hydrazine, reaction of anhydrous, with dehydration of, 176, 209 Hexosides, methyl acetamidodeoxy chlorodeoxy sugars, 285 gas-liquid chromatography of, 82-84 -, (p-nitropheny1)-, reaction with broper-O-(trimethylsilyl) derivatives, gasmodeoxy sugar, 287 liquid chromatography of, 144,145 Hydrocellulose, gas-liquid chromatograHexosulose, 3-deoxy-~-elythrophic analysis of, 74 alkaline degradation of, 196 Hydrochloric acid, in hydrolysis of polyin dehydration of D-fructose, 179 saccharides, 15 preparation of, 168, 169 Hydrofluoric acid, in hydrolysis of structures of, 171 teichoic acid polymers, 16 trimethylsilylation and gas-liquid chro- Hydrolysis matography of, 58 of glycosyl esters of nucleoside 5'-pyrotrimethylsilylation and mutarotation of, phosphates, 356-360 38 mechanism of, of deoxyhalogeno sugHexos-5-ulose, bdeoxy-D-xylo-, preparaars, 305 tion of, 291 of polysaccharides, 14-23

SUBJECT INDEX, VOLUME 28

501

phate), enzymic synthesis of, 338 5'-(a-~-mannopyranosyl pyrophosphate), enzymic synthesis of, 340 - , 5'-O-acetyl-3'-chloro-3'-deoxy-, preparation of, 247 , 5'-bromo-5'-deoxy-2',3'-O-isopropylidene-, preparation of, 247, 248 I - , 5'-chloro-5'-deoxy-2',3'-O-isopropylidene-, preparation of, 247 Iditol, 2,3 :4,5-dianhydro- 1,6-di-O- , 5'-deoxy-5'-iodo-2',3'-O-isopropyli(methylsulfony1)-L-, cytostatic acdene-, preparation of, 247 tivity, 7 Idofuranose, 3,6-anhydro-5-deoxy-5-iodo- -, 2',3'-O-isopropylidene1,2-O-isopropylidene-P-~-, preparareaction with phosphoryl chloride, 239 tion of, 271 with triphenylphosphine-carbon tetrabromide, 248 - , 6-azido-5,6-dideoxy-5-iodo-l,2-O-isopropylidene-P-L-, preparation of, 271 Inositol, hexa-O-(trimethylsilyl)-myo-,90 -, per(trimethylsily1)-scyllo-, 90 - , 5-deoxy-5-iodo-1,2-0-isopropyliInositols, dene-6-O-trifluoroacetyl-p-~-, prepacetylation of, 36 aration of, 271, 272 gas-liquid chromatography of, 89 Idofuranurono-6,3-lactone, 5-bromo-5Inosose-2, per(trimethylsily1)-myo-,90 deoxy-l,2-O-isopropylidene-P-~-, Insects, a-D-mannosidase in, 405 debromination of, 304 Idose, L-, synthesis and rearrangement of, Iodination, of alcohols, 260 Iodine azide, reactions with unsaturated 5 carbohydrates, 271 - , 2,5-anhydro-~-,degradation of, mechIodine fluoride, reaction with unsaturated anism of, 165 sugars, 273 , 3-chloro-3-deoxy-1.2 :5,6-di-O-isoIodine trifluoroacetate, reaction with unpropylidene-P-D-, preparation of, 247 saturated carbohydrates, 271 Iduronic acid, L-, from D-glUCurOniC acid by epimeriza- Isoglucal, formation of, 204 Isomaltol tion in pyridine, 40 from dehydration of D-fI'uCtOSe, 176, in heparin, 55, 75 179, 180 Imidazole, N-(trimethylsily1)-, trimethylpreparation of, 170, 176, 179 silylation with, 27 Isosaccharinic acid, from deoxyglycodiIminium compounds uloses by alkaline degradation, 196 (alkoxymethy1ene)dimethyliminium Isotopes, effect on reactions of glycosyl halides, reaction with alcohols, 254 esters of nucleoside pyrophosphates, (bromomethy1ene)dimethyliminium 371-373 bromide, in synthesis of bromodeoxy sugars, 250 (chloromethy1ene)dimethyliminium J chloride, in synthesis of chlorodeoxy sugars, 250,278 Jack-bean meal a-D-mannosidase from, purification of, Immunoglobulin 410 gas-liquid chromatographic analysis of, residual activity of purified, 414 72,86 a-D-mannosidase in structural studies of human, 445 K Inosamines, synthesis of, 284 Kanamycin, chlorinated, 226 Inosine Kasugamine, synthesis of, 283 5'-(a-~-glucopyranosyl pyrophos-

Hydroxylamine, reactions with glycosyl esters of nucleoside 5'-pyrophosphates, 360-362 Hygromycin A, sugar component, preparation of, 291

-

-

502

SUBJECT INDEX, VOLUME 28

Kasugamycin, diamino sugar component Lyxopyranosyl chloride, a-D, 2,3,4-triof, 283 (chlorosulfate), preparation and reacKeratosulfates, gas-liquid chromatogrations of, 235-237 - , 2-chloro-2-deoxy-a-~-, 3,4-di(chlorophy of, 43, 79 sulfate), preparation of, 236 Ketoses Lyxose, 5-bromo-5-deoxy-~~-,synthesis dehydration in acidic solutions, 174182 of, 304 - , 2-chloro-2-deoxy-~-, preparation of, in alkaline solution, 193-207 trimethylsilylation of, 24 236 Kojic acid, from ~threo-2,5-hexodiulose - , 3,4-di-O-acetyl-2,5-anhydro-aldehydoby dehydration, 186 D-, and p-nitrophenylhydrazone, preparation of, 287, 288

L Lactic acid, formation from hexoses, 197 Lactones, of carbohydrate acids, gasliquid chromatography of, 71-78 Laminarabiose, octaacetate, gas-liquid chromatography of, 70 Leucomycin, sugar component mycaminose, 297 Levulinaldehyde, a-hydroxy-, in dehydration of 2-deoxypentoses, 224 Levulinic acid from glycal dehydration, 184 preparation of, 212-218 Limpet a-D-mannosidase, activation of, 417419 pH and activity of, 412 purification of, 411 Lincom ycin chlorination of, 225 reaction with triphenylphosphinecarbon tetrachloride, 247 synthesis of, 282 Lipopol ysaccharides gas-liquid chromatography of, 49 hydrolysis of, 19 Lithium aluminum hydride, reduction of chlorodeoxy sugars by, 303 Lobry de Bruyn-Alberda van Ekenstein transformation, 161, 163, 168 Lorenzini jelly, gas liquid chromatography of, 47 Lysosome theory, a-D-mannosidase activity and, 408 Lyxofuranoside, methyl 5-O-benzyl-a-~-, preparation of, 294 Lyxopyranose, p-D-,reaction with sulfuryl chloride, 235

M Magnamycin, mycaminose sugar component, 297 Maltitol, per-0-(trimethylsilyl) ether, gasliquid chromatography of, 58, 69 Maltol, from Dfructose dehydration, 180 -, 5-hydroxy-, by ~-threo-2,5-hexodiulose dehydration, 186 Malto-oligosaccharides, 4-0-methyl, degree of polymerization by gas-liquid chromatography, 47 Maltose dehydration of, 169 and homologs, gas-liquid chromatography of, 68 trimethylsilylation of, 24 a-Maltoside, methyl, synthesis, gasliquid chromatography in, 50 Mammalian nerve gas-liquid chromatographic analysis of, 48 D-glucitol in, determination by gasliquid chromatography, 58 Mammalian tissues a-Dmannosidase in, 403,407 subcellular localization of a-D-mannosidase in, 405 Mannaric acid, 2,5-anhydro-~,dehydration of, 166 Mannitol, D-

ester of adenosine 5’-pyrophosphate, 333 in potato extracts, determination by gas-liquid chromatography, 57 - , 1,6-bis[2-(chloroethyl)amino]-l,6-dideoxy-D, dihydrochloride, synthesis

SUBJECT INDEX, VOLUME 28 and antitumor activity of, 7

-,

503

methyl 2,3-di-O-acetyl-4-0-benzoyl6-bromo-6-deoxy-c~-~-,preparation of, 287 283 -, 1,6-dibromo-l,6-dideoxy-~- , methyl 2,3-di-0-acetyl-4,6-OObenzylias antitumor agent, 287 dene-a-D-, reaction with N-bromocytostatic activity, 7 succinimide, 283 - , 1,6-di-O-(methylsulfonyl)-~-, cyto- -, methyl 4,6-dideoxy-4-iodo-2,3-O-isostatic activity, 7 propylidene-a-L-, 1,2-0-isopropylidene-~-, synthesis of, preparation and iodine displacement, 4 282 - , 2,3,4,5-tetra-O-benzoyl-,reaction preparation of, 242 with phosphorus pentachloride, 239 - , methyl 2,3,4-tri-O-acetyl-6-bromo-6Mannonic acid, 2,5-anhydro-~-,dehydradeoxy-a-D-, preparation by Pinner retion of, 166 action, 257 ~-Mannono-l,4-lactone,trimethylsilyl - , methyl 3,4,6-tri-O-acetyl-2-bromo-2ether, gas-liquid chromatography of, deoxy-a-D-, reaction with sodium co71 balt tetracarbonyl and carbon mon~-Mannono-l,5-lactone,inhibition of a-Doxide, 293 mannosidase activity by, 419 -, methyl 3,4,6-tri-O-acetyl-2-deoxy-2Mannopyranose, a-Diodo-a-D, reaction with sodium coester with adenosine 5'-pyrophosphate, balt tetracarbonyl and carbon monoxide, 293 315, 339 Mannopyranosyl chloride, 3,4,6-tri-Owith 2'-deoxyguanosine 5'-pyrophosphate, enzymic preparation of, 340 acetyl-2-chloro-2-deoxy-a-~-,preparation of, 262 with guanosine 5'-pyrophosphate, - , 3,4,6-tri-0-acetyl-2-chloro-2-deoxy-~319 with guanosine 5'-pyrophosphate, enD-, preparation of, 261, 262 zymic preparation and fermentaMannopyranosyluronic acid, a-D-,ester of tion production of, 339,340 guanosine 5'-pyrophosphate, 320 with inosine 5'-pyrophosphate, enMannose, Dzymic preparation of, 340 methanolysis of, 19 with thymidine 5'-pyrophosphate, mutarotation and gas-liquid chromaisolation of, 322 of uridine 5'-pyrophosphate, 325 tography of, 40 -, 1,2,3,4-tetra-O-acetyl-P-D-,reaction - , 6-amino-6-deoxy-~-, preparation of, with phosphorus pentachloride, 239 283 - , tetra-O-acetyl-6-chloro-6-deoxy-p-~,- , 2,5-anhydro-~-, degradation mechpreparation of, 239 anism of, 164, 165 -, 1,3,4,6-tetra-O-acetyl-2,5-anhydro-D-, - , 6-O-a-~-xylopyranosy~-~-, preparamethyl hemiacetal, preparation of, tion of, 236 Mannopyranoside, methyl a-D 288 a-DMannosidase, 401-445 hydrolysis of, trifluoroacetic acid in, 17 reaction with sulfuryl chloride, 234 action on naturally occurring substrates, -, methyl 4-O-benzoyl-3-bromo-2,6-di437-445 activation and inhibition of, 416-420 chloro-2,3,6-trideoxy-a-~, dehaloactivity, in certain mammalian tissues, genation of, 302 , methyl 6-chloro-6-deoxy-a-D-, 2,3,4403 tri(chlorosulfate), preparation of, 234 changes in viuo, 434-437 -, methyl 2,3-di-O-acety1-6-azido-4-0- in molluscs, 405,406 benzoyl-6-deoxy-a-~-,preparation of, of plant seeds, 404 in sucrose homogenates of mamma283

-, 1,2:5,6-dianhydro-~-, formation of,

-

504

SUBJECT INDEX, VOLUME 28

lian tissues, 407 assay, 408 distribution in Nature, 402-405 in sucrose homogenates of mammalian tissues, 407 effect of zinc2+and other cations on activity of, 422, 424-431 epididymis, purification of, 41 1 residual activity of purified, 414, 415 history, 401 inactivation by heat, 416 jack-bean, purification of, 410-412 residual activity of purified, 414 limpet, activation of, 417-419 effect of chloride ion on activity of, 431,432 purification of, 411 residual activity of purified, 415 metal analysis of fractions during purification, 435 as metalloenzyme, 423,433 pH and activity of, 412,413 pH and stability of, 413-416 purification of, 409-412,424 residual activity of purified, after incubation, 414, 415 specificity of, 420-422 as structural reagent in ovalbumin, 437443 subcellular localization in mammalian tissues, 405-408 /%DMannosidase assay, 408 distribution in Nature, 420 occurrence and activity of, 420 Mannoside, p-nitrophenyl a - ~ hydrolysis, effect of limpet a-D-mannosidase on, 418,419 by jack-bean meal, 430 Mannuronic acid, D-, gas-liquid chromatography of, 64 Metal chelates, effect of pH on stability of, 422,424 Metalloenzymes, 423,433 Metasaccharinic acid 4-carbon, preparation of, 195 from deoxyglycodiuloses by alkaline degradation, 196 Methanesulfonylation, of hexopyranosides, 256 Methanolysis

deacetylation in, 82 of glycolipids and glycoproteins, and gas-liquid chromatography, 51 of glycopeptides and oligosaccharides, 18 of glycosyl esters of nucleoside 5'-pyrophosphates, 358 of monosaccharides, gas-liquid chromatography and, 54 of uronic acids, gas-liquid chromatography and, 76 Methoximes, of monosaccharides, gasliquid chromatography of trimethylsilyl derivatives, 49, 110 Methyl sulfoxide purification of, 30 solvent for trimethylsilylation, 29 (Methyl 2,3,4-tri-O-rnethyl-6-thio-a-~glucopyranoside) (methyl 2,3,4-tri0-methyl-a-D-glucopyranoside)6,6'dithiocarbonate, preparation of, 283 Michaelis-Arbuzov reaction, carbonphosphorus sugars by, 285 Microbial media, L-arabinitol and Dmannitol metabolism in, gas-liquid chromatography of, 57 Microsomes, a-Dmannosidase in structural studies of rat-liver, 445 Milk gas-liquid chromatographic analysis of, 48, 53, 81, 83 a-D-mannosidase in cow's, 403 Molds, gas-liquid chromatographic analysis of, 48 Molluscs, a-D-mannosidase activity in, 405,406 Monosaccharides acetates, gas-liquid chromatography of, 33,49 acetylated, gas-liquid chromatography of, 111 deuterated, separation by trimethylsilylation, 32 isopropylidene acetals, in gas-liquid chromatography, 37 sulfates, esters with uridine 5'-pyrophosphates, 329 synthesis of, 295, 296 trifluoroacetyl derivatives, gas-liquid chromatography of, 112 trimethylsilylation and methanolysis

SUBJECT INDEX, VOLUME 28 of, 54 and mutarotation of, 38 solvents for, 30 trimethylsilyl ethers, gas-liquid chromatography of, 41-49, 102-110 Morpholine, 4-(3,4,5-trimethoxybenzoy1)-, synthesis as tranquilizer, 9 Mucilages plant, gas-liquid chromatography of, 62 trifluoroacetic acid in hydrolysis of, 16 Mucins, gas-liquid chromatography of, 85 Mucopolysaccharides, analysis by gasliquid chromatography, 43, 44 Muramic acid, N-acetyl-, ester of uridine 5’-pyrophosphate, isolation of, and derivatives, 328 -, N-glycolyl-, ester of uridine 5‘-pyrophosphate, isolation of, 329 Mutarotase, mutarotation coefficients and gas-liquid chromatography in assay of, 40 Mutarotation, in gas-liquid chromatography, 38-41 Mycaminose, synthesis of, 297 Mycaminoside, methyl DL-, synthesis of, 297 Mycoside G, gas-liquid chromatography of, 55 Mycosides, methanolysis of, 18

N Narbomycin, sugar component desosamine, 299 Nectar, flower, gas-liquid chromatography of, 43 Neomycins, determination by trimethylsilylation, 28 Neuraminic acid, gas-liquid chromatography of, 56 -, N-acetylester with cytidine 5’-phosphate, 332 trimethylsilylation of, 28, 29 trisaccharide esters with uridine 5’-pyrophosphate, 331 -, N-glycolylester with cytidine 5’-phosphate, 332 trisaccharide esters with uridine 5’-pyrophosphates, 331

505

Nickel carbonyl, reduction of polyhalogenomethyl groups, 304 Nicotinamide adenine dinucleotide and phosphate, in isolation of cytidine pyrophosphate glycosyl esters, 316 in transformations of glycosyl groups, 364-369 Nitric acid, in hydrolysis of polysaccharides, 15 Nitriles, acetates, gas-liquid chromatography of, 66 Nitryl iodide, reaction with unsaturated carbohydrate derivatives, 270, 299 Nomenclature, of glycosyl esters of nucleoside pyrophosphates, 309 D-elythro-L-galacto-, gasNonulose, liquid chromatography of, 49 Nosema apis, gas-liquid chromatography of spores of, 47 Nucleocidin, synthesis of, 226, 273 Nucleosides arsenic-containing, attempted preparation of, 253 bromination by N-bromosuccinimide and triphenylphosphine, 249 5‘-(glycosyl pyrophosphate) epimerases, gas-liquid chromatography in monitoring action of, 47 5’-(glycosyl pyrophosphates), 307-399 carbodiimides in synthesis of, 353 chemical reactivity of, 356-362 chemical syntheses of, 344-356 chromatography of, 310 degradation of, 311-314,356 enzymic reactions of, 362-396 enzymic splitting of glycosyl linkage, 391-397 enzymic splitting of pyrophosphate linkage, 388 extraction of, 310 history, 308 2-hydroxypyridyl esters of nucleoside 5’-phosphates in synthesis of, 353 inversion of hydroxyl group in, 369379 isolation, characterization and structure of natural, 310-334 nomenclature, 309 preparation of, 334-356 transformations of glycosyl group, 363-368

SUBJECT INDEX, VOLUME 28

506

halogenation of, 247 halogeno sugar, synthesis of, 226 5'-phosphoroimidazolidates,in synthesis of glycosyl nucleoside pyrophosphates, 346 5'-phosphoromorpholidates,in synthesis of glycosyl esters of nucleoside 5'-pyrophosphates, 346-351 5'-phosphorothioate, in synthesis of pyrophosphates of nucleosides, 352 5'-pyrophosphates, glycerol and triose esters, 334 hexitol and pentitol esters, 333 reactions with (bromo(or ch1oro)methy1ene)dimethyliminium bromide(or chloride), 253 ribo-, chlorination or bromination by thionyl chloride or bromide and hexamethylphosphoric triamide, 250 Nucleosides, 5'-chlorod'-deoxy-, dinucleosides from, 287 - , 5'-deoxy-4'-fluorod'-iodo-, preparation of, 273 -, deoxyiodo-, preparation of, 243 of Nucleotides, pyrophosphorolysis sugar, 390 0

Obituary, LBsz16 Vargha, 1-10 Octulosonic acid, 3-deoxy-, esters with cytidine 5'-phosphate, 332 -, 3-deoxy-~-gaZacto-,synthesis of, 77 -, 3-deoxy-~-gZuco-,synthesis of, 77 -, 3-deoxy-wmannoester with cytidine triphosphate, 333 synthesis and gas-liquid chromatography of, 77 Octulosonic acids, trimethylsilylation of, 28 Oleandomycin desosamine component of, 299 sugar component oleandrose, 299 Oleandrose, in cardiac glycosides, 298 Oleandroside, methyl DL-, synthesis of, 298 Oligogalacturonic acids, 0-trimethylsilyl derivatives, decomposition rates for, 29 Oligosaccharides acetates, gas-liquid chromatography

of, 70 blood-group, trifluoroacetic acid in hydrolysis of, 16 degree of polymerization, determination by gas-liquid chromatography, 58, 83, 85 esters with uridine 5'-pyrophosphate, 331 gas-liquid chromatography of, 67-71 guanosine 5'-pyrophosphate esters, 322 malto-, determination by trimethylsilylation, 25 a-D-mannosidase action on, 420 methanolysis of, 18 methylated (fully), gas-liquid chromatography of, 135 per(trimethylsily1) ethers, gas-liquid chromatography of, 130-135 trifluoroacetates, gas-liquid chromatography of, 70, 113 trimethylsilyl ethers, gas-liquid chromatography of, 68-70 Onion bulbs, gas-liquid chromatographic analysis of, 68 Orchidectomy, effect on a-wmannosidase activity, 434, 436 Orcinol reaction, in sugar analysis, 223 Orosomucoid, a-D-mannosidase in structural studies of human, 445 Ovalbumin aspartamidoglycans from, composition and effect of enzymic hydrolysis of, 440-442 a-wmannosidase action on, 437-443 Ovariectomy, effect on a-D-mannosidase activity, 404, 434 Ovomucoid, a-D-mannosidase action on, 445 Oxalic acid, in hydrolysis of fructans, 20 Oxathiabicyclo[2.2.2]octane, sugar derivative, 290 Oxazabicyclo[2.2.2]octane, sugar derivative, 290 Oxetanes, carbohydrate, preparation of, 289 Oximes gas-liquid chromatography of, 49,70 of monosaccharides, gas-liquid chromatography of trimethylsilyl derivatives. 110 per(trimethylsily1)ated disaccharide,

SUBJECT INDEX, VOLUME 28 gas-liquid chromatography of, 135 trimethylsilylation of, 30

507

glycero-, preparation of, 293 Pent-2-enopyranosyl bromide, 2,4-di-0acetyl-3-deoxy-~-glycero-, preparation of, 266 P Pent-2-enopyranosyl chloride, 2,4-di-0Paratose acetyl-3-deoxy-~-glycero-, preparaester of cytidine pyrophosphate, isolation of, 266 tion of, 316 Pent-3-enosulose, 3,4-dideoxy-, dehydration of, 172, 173 preparation of, 300 Pectin Pentitols, gas-liquid chromatography of, dehydration of, 188 67 hydrolysis of methylated, 23 Pentofuranose, 5-0-benzyl-2,3-dideoxyreductic acid from, 207 D-glycero-, preparation of, 294 uronic acids, gas-liquid chromatogra- Pentononitrile, 2-deoxy-3,5-0-ethyliphy of, 76 dene-D-eythro-, preparation of, 283 2,4-Pentadienal, 2,5-dihydroxy-, forma- Pentopyranose, 1,3,4-tri-O-acetyl-2,2-dition of, and reductic acid from, 191, chloro-2-deoxy-~-eythro-, preparation of, 261 192 Pentaerythritol, trimethylsilylation, effect Pentopyranoside, methyl 3,4-di-O-acetylof impure solvents on analysis of, 30 &-deoxy-~-threo-,reaction with hy-, tetra(cyanoethy1)-, in gas-liquid chrodrogen bromide, 266 matography, 45 Pentopyranosyl bromide, 4-0-acetyl-3Pent-1-enitol, 2,3,4-tri-O-acetyl-l,5-anhybromo-2,3-dideoxy-~-threo-, prepdro-eeythro-, reaction with hydroaration of, 264, 266 gen chloride, 266 - , 3,4-di-O-acetyl-2-deoxy-~-eythro-, - , 2,3,4-tri-O-acety1-1,5-anhydro-~preparation of, 265 threo-, reaction with hydrogen chlo- Pentopyranosyl chloride, 4-0-acetyl-3ride, 266 chloro-2,3-dideoxy-~-threo-, preparaPent-4-enofuranose, 3-0-acetyl-5-deoxytion of, 264 1,20-isopropylidene-p-~-threo-, - , 3,4-di-O-acetyl-2-deoxy-~-eythro-, preparation of, 291 preparation of, 265 Pent-2-enofuranoside, methyl 5-0-henPentopyranosyl halides, 3,4-di-O-benzoyl-2,3-dideoxy-~-~-glycerozoyl-2-deoxy-, preparation of, 265 bromination in methanol and silver Pentosans, formation of, 188 acetate, 269 Pentose, 2-deoxy-~-eythroreaction with nitryl iodide, 273 alkaline degradation of, 203 Pent-2-enopyranoside, methyl 4-0-hendehydration of, 184 zyl-2,3-dideoxy-P-~-glycero-,prepDische test for, 224 aration of, 293 gas-liquid chromatography of, 45 - , methyl 3,4-dichloro-2,3,4-trideoxy-P- mutarotation and gas-liquid chromatogD-glycero-, preparation of, 238 raphy of, 40 - , 3,4-dichloro-2,3,4-trideoxy-P-~synthesis of, 282 tests for, 212 glycero-pent-2-enopyranosyl 3,4-dichloro-2,3,4-trideoxy-~-~-glycero-, -, 2-deoxy-~-eythro-,synthesis of, 276 preparation of, 238 Pentoses, dehydration of, 176, 188, 208 Pent-3-enopyranoside, benzyl 2-0Pentosulose, 3-deoxy-, structures of, and benzyl-3,4-dideoxy-a-~glycero-, redehydration, 171, 172 -, %deoxy-L-threo-, formation from Laction with nitryl iodide, 273,299 - , benzyl2-0-benzyl-3,4-dideoxy-4ascorbic acid, 192 2-Pentulose, D-threo-, preparation from nitro-a-D-glycero-, preparation of, 299 D-xylose, 39 -, methyl 2-0-benzyl-3,4-dideoxy-P-~-

508

SUBJECT INDEX, VOLUME 28

-, L-threo-, synthesis of, 4 Peptides, derivatives with uridine 5'-pyrophosphate N-acetylmuramic acid esters, 328 Peptidogalactomannans gas-liquid chromatographic analysis of, 64 hydrolysis of, trifluoroacetic acid in, 16 Perchloric acid, in hydrolysis of cardiac glycosides, 16 PH and activity of a-D-mannosidase, 412 effect on stability of metal chelates, 422 and stability of a-D-mannosidase, 413 Phenol-sulfuric acid test, in sugar analysis, 223 Phosphine, triphenyland N-bromosuccinimide, reactions with carbohydrates, 249 and carbon tetrachloride, reactions with alcohols and carbohydrates, 246 Phosphonium bromide, bromotriphenoxy-, as reagent for bromination of carbohydrates, 240 Phosphonium iodide, iodotriphenoxy-, as reagent for iodination of alcohols and carbohydrates, 240 -, methyltriphenoxyin displacement reactions with deoxyhalogeno sugars, 282 reactions with nucleosides, 243 as reagent for iodination of alcohols and carbohydrates, 240 Phosphoramidates, in synthesis of glycosyl esters of nucleoside 5'-pyrophosphates, 345-351 Phosphoric triamide, hexamethylas solvent for trimethylsilylation, 30 and thionyl bromide or chloride, reaction with Dribonucleosides, 250 Phosphorus compounds, in deoxyhalogeno sugar synthesis, 280 Phosphorus pentachloride, reactions with carbohydrate derivatives, 239 Photolysis, in 6-deoxy-a,a-trehalose synthesis, 305 Picrocin, DL-,synthesis of, 299 Pinner reaction, deoxyhalogeno sugars preparation by, 256 Plant cell-walls, hydrolysis of, trifluoroacetic acid in, 16

Plant extracts gas-liquid chromatography of, 48, 57 uronic acids, gas-liquid chromatography of, 76 Plant seeds, a-D-mannosidase activity of, 404 Plant tissue, cellulose and hemicellulose in, gas-liquid chromatography of, 60 Polyhydric compounds acetates, gas-liquid chromatography of, 153-155 gas-liquid chromatography of, 90-98 per(trimethylsily1) ethers, gas-liquid chromatography of, 151, 152 trifluoroacetyl derivatives, gas-liquid chromatography of, 114 Polysaccharides aldehydo, preparation of, 286 algal, gas-liquid chromatographic analysis of, 59 analysis of, 188 biosynthesis of, lipid intermediates in, 390 fungal, gas-liquid chromatography of, 44 hydrolysis of, 14-23 structural studies by gas-liquid chromatography, 47 trimethylsilylation of, 31 Polyvinyl alcohol, trimethylsilylation of, 31 Poppy, opium, gas-liquid chromatographic analysis of, 49 Potato extracts gas-liquid chromatography of, 43 Dmannitol determination in, by gasliquid chromatography, 57 &-Propanone, 1,3-dihydroxy-, ester of uridine 5'-pyrophosphate, isolation of, 334 Propionitrile, 2,2'-azobis(2-methyl-, in reduction of chlorodeoxy sugars, 303 Protocatechualdehyde, di-0-acyl derivatives, hydrolysis of, 2 Protoglucal, formation of, 204 -, di-0-acetyl-, formation and degradation of, 204 Pseudohalogens, reactions with unsaturated sugars, 270 Pseudouridine, 5'-(a-D-glucopyranosyl pyrophosphate), enzymic preparation

SUBJECT INDEX, VOLUME 28

509

of, 338 eraldehyde, 174 Pullulan, trimethylsilylation of, 31 Pyruvic acid, mechanism of formation of, Purine, 9-(2-0-acetyl-3-chloro-5-0200 pivaloyl-P-Dxylofuranosyl)-6-( piva1amido)-, preparation of, 279 R 2-alkoxy-5,6-dihydro-2H-pyran, Pyran, hydrohalogenation and hydroxy- Rearrangements benzilic acid, in alkaline degradation, halogenation of, 276 - , 3a-bromotetrahydro-2a,4P-dime205 thoxy-, preparation of, 274 in dehydration of sugars, 171 - , 3~-bromotetrahydro-2a,4a-dimein lactic acid formation, 197 in saccharinic acid formation, 194 thoxy-, preparation of, 274 - , 4p-chloro-3a-(ethylthio)tetrahydroLobry de Bruyn-Alberda van Eken2p-methoxy-, preparation of, 275 stein, 161, 163, 168 W-Pyran, cis-5,6-dihydro-2,5-dimeReductic acid thoxy-, preparation of, 296 formation of, 191 from hexuronic acids by decarboxyla- , 3,4-dihydro-2-(hydroxymethyl)-, tion, 186. preparation from acrolein dimer, 296 preparation and properties of, 207-212 - , 5,6-dihydro-2-methoxy-, reactions with 1,3-dibromo-5,5-dimethylhydan- from Dxylose and other pentoses, 176 Reductic-'4C acid, preparation of, 210 toin and with ethanesulfenyl chloReduction, of chlorodeoxy sugars, 302, ride, 274 -, truns-5,6-dihydro-2-methoxy-6-(meth303 Reductone, formation of, 207 oxymethy1)-,preparation of, 295 - , 3,4-dihydro-2-(methoxymethyl)-,bro- Reproduction, a-Dmannosidase effect on, 436 momethoxylation of, 295 4H-Pyran-2-carboxylic acid, 5,6-dihydro- Resins, ion-exchange, in hydrolysis of glycoproteins, 17, 22 3-hydroxy-4-oxo-, methyl ester, formation of, 185 Retention time - , 3,4,5-triacetoxy-5,6-dihydro-,methyl of furanosides and pyranosides, 54 ester, dehydration of, 185 in gas-liquid chromatography, 45, 69 4H-Pyran-4-one, 2,3-dihydro-3,5-dihystereoisomeric structure and, 33 of 0-trimethylsilyl derivatives, 82 droxy-6-methyl-, from dehydration of Rhamnitol, acetylation of, 35 D-fructose, 176, 179, 180 - , 3-hydroxy-2-methyl-, from D-fructose Rhamnomannan, gas-liquid chromatogradehydration, 180 phic analysis of, 50 Pyridine Rhamnopyranose, a-D-, ester of guanosine effect on mutarotation of carbohydrates, 5'-pyrophosphate, enzymic prepara39 tion of, 321, 344 as solvent for trimethylsilylation, 29 -, PLBPyridinol, esters of nucleoside 5'-phosester of thymidine 5'-pyrophosphate, phates, in synthesis of pyrophosisolation of, 323 phates, 353 ester of uridine 5'-pyrophosphate, bioPyrolysis, of hexuronic acids, 188 synthesis of, 326 Pyrophosphorolysis, of sugar nucleotides, Rhamnopyranoside, methyl 2,3-0-isopropylidene-a-L-, reaction with 390 methyltriphenoxyphosphonium ioPyrophosphorylases, sugar nucleotide, 335 dide, 242, 282 ZPyrrolidinone, N-methyl-, as solvent for Rhamnose, L-, gas-liquid chromatography trimethylsilylation, 30 of, 46, 47 Rhodinoside, methyl p-D-, preparation Pyruvaldehyde, formation from DL-&C-

SUBJECT INDEX, VOLUME 28

510

of, 294 Ribitol, L,ester of cytidine 5’-pyrophosphate, biosynthesis and isolation of, 333 - , l&anhydro-, gas-liquid chromatography of, 57 Ribofuranoside, methyl 5-0-benzyl-ED, preparation of, 294 -, methyl 2,3-0-benzylidene-5-0methyl-p-D, reaction with N-bromosuccinimide, 277 Ribonuclease B, a-Dmannosidase as structural reagent for, 444 Ribopyranoside, methyl 3,4-dichloro-3,4dideoxy-P-D, 2-chlorosulfate, preparation of, 237 Ribopyranosyl chloride, 3,4-dichloro-3,4dideoxy-ED, 2-chlorosulfate, preparation of, 238 Ribose, Dester of thymidine 5’-pyrophosphate, occurrence of, 322 synthesis from L-gluta,micacid, 294 -, e - d e o x y - ~ synthesis , of, 8 Ring structures, gas-liquid chromatography and, 50 Rydon reagents for halogenation of carbohydrates, 240 reactions with nucleosides, 243 S

Saccharinic acids formation of, 163, 193, 194 gas-liquid chromatography of, 71,73,75 per(trimethylsily1) derivatives, gasliquid chromatography of, 141, 142 preparation of, 198 Sake, ethyl a-Dglucoside in, and gasliquid chromatography, 54, 55 Saliva, analysis by gas-liquid chromatography, 44,81 Saponins hydrolysis of steroidal, 15 steroidal, gas-liquid chromatography of, 46 Semen, a-Dmannosidase in, 403 Serratia marcescens, polysaccharide, gasliquid chromatography, 59 Silane, chlorotrimethylpurification of, 30

trimethylsilylation of sugars with, 23

-, fluorotrimethyl-, effect on chromatography, 33 Silicone polyester (ECNSS-M), organo, in gas-liquid chromatography, 60 Silver trifluoroacetate, reaction with iodine on unsaturated sugars, 272 Simmons-Smith reagent, 305 Smith degradation, of polysaccharides, 98-101 Solvents for chromatography of glycosyl esters of nucleoside pyrophosphates, 311 for displacement reactions, 229 effect on mutarotation of monosaccharides, 39 for trimethylsilylation, 29 Sorbose, Lbgalactosaccharinic acids from, 195 from L-idose rearrangement, 5 trimethylsilylation of, 24 - , 1-chloro-1-deoxy-L-, preparation of, 247 - , 2,3 :4,6-di-O-isopropylidene-~-, reaction with triphenylphosphine-carbon tetrachloride, 247 Sorburonic acid, G,dehydration of, 189 Soybean extracts, ethyl a-Dgalactoside in, and gas-liquid chromatography, 54 Sphingosine bases, gas-liquid chromatography of, 56 Spiramycin, sugar component mycaminose, 297 Starch, analysis by gas-liquid chromatography, 47 Starch syrups, analysis by gas-liquid chromatography, 58 Stereochemistry cytostatic activity and, of carbohydrates,

7 effect on enzymic reactions of glycosyl esters of nucleoside pyrophosphates, 364 on hydrolysis of glycosyl esters of nucleoside 5’-pyrophosphates, 358 Steric hindrance, effect on trimethylsilylation or dimethylsilylation, 32 Steroidal saponins, hydrolysis of, 15 Steroids 0-trimethylsilyl and trifluoroacetates, 31

SUBJECT INDEX, VOLUME 28

511

trimethylsilylation of, 26, 28 Sugar nucleotides, history, 307, 308 Steryl glucosides, trimethylsilylation of, Sugars 28 2-amino-2-deoxy, dehydration of, 182 Streptamine, deoxy-, trifluoroacetate, gasamino, gas-liquid chromatography of, liquid chromatography of, 87 56, 78, 85 Succinimide, N-bromomethanolysis in determination of, 18 reactions with 0-benzylidene sugars, trifluoroacetates, gas-liquid chroma268,276-278 tography of, 87 and triphenylphosphine, reactions with trifluoroacetic acid in determination of, 16 carbohydrates, 249 -, N-chloro-, chlorination of carbohytrifluoroacetyl derivatives, gas-liquid drates with, 250 chromatography of, 114 -, N-iodotrimethylsilylation of, 24, 27, 29 trimethylsilyl derivatives, gas-liquid iodination of carbohydrates by, 250 reaction with hydrazino carbohydrates, chromatography of, 143-146 279 anhydro, acetates and methyl ethers, Succinoglucan, gas-liquid chromatogragas-liquid chromatography of, 148 degradation mechanism of 2,5-, 165, phy of, 47,93 166 Sucrose formation of 1,6-, in hydrolysis of gas-liquid chromatographic analysis polysaccharides, 20 of, 68 gas-liquid chromatography of, 87-89 per-0-(trimethylsilyl) derivative, gasper(trimethylsily1) ethers, gasliquid chromatography of, 130, 131 liquid chromatography of, 147,148 per-0-(trimethylsilyl) ether and per(trifluoroacetate), gas-liquid chromapreparation of 2,5-, 287 tography and retention times of, 70 benzylidene acetals, reactions with Nreaction with sulfuryl chloride, 239 bromosuccinimide. 276-278 synthesis of, adenosine 5'-pyrophosbromodeoxy, displacement reactions, phate a-Dglucopyranosyl ester in, 283 with carbon-phosphorus bond, prepa395 trimethylsilyl ether, gas-liquid chromaration of, 285 chlorodeoxy, preparation of, 231-239, tography of, 58 - , chlorodeoxy-0-p-toly Isulfonyl-, hexa255,278 benzoate, preparation of, 256 reduction of, 302, 303 - , 6,G'-dichloro-6.6'-dideoxy-,hexadegradation and dehydration of, 163 benzoate, preparation of, 256 deoxyhalogeno, 3,Ganhydro-ring for- , 6,6'-di-O-p-tolylsulfonyl-,reaction mation, 287 with benzoyl chloride in pyridine, displacement reactions of, 281-290 256 elimination reactions, 290 - , 2,3,4,1',3',4'-hexa-O-benzoyl-6,6'-difrom glycals, 261 deoxy-6,6'-diiodo-, reaction with silfrom hydrazino carbohydrates, 279 ver fluoride, 292 neighboring-group reactions of, 290 Sugar analyses preparation by direct replacement of anthrone test, 220-222 hydroxyl group, 230-260 preparation by displacement reaccarbazole test, 222 dehydration reactions in concentrated tions, 227-230 acid solution, 218-222 preparation by reaction with (haloorcinol test in, 223 genomethy1ene)dimethyliminium phenol-sulfuric acid reaction, 223 halides, 250, 251 preparation by reaction with phosSugar-cane juice, gas-liquid chromatographorus-containing reagents, 239phy of, 48

512

SUBJECT INDEX. VOLUME 28

250 preparation of, 257,263 reductive dehalogenation of, 299304 synthesis and chemistry of, 225-306 synthetic utility of, 281-306 deoxyiodo, carbohydrate oxetanes, preparation of, 289 preparation of, 243 reactions of, 282 synthesis of, 280 deoxynitro unsaturated, 299 3,6-dideoxy, preparation by hydrolysis, 14, 19 enediol acyclic, formation in dehydration reactions, 167-171 isothermal determination of, gas-liquid chromatography and, 81 IT-labeled, trimethylsilylation of, 32 metabolism by bacteria, gas-liquid chromatography and, 47 trimethylsilylation of, 23-33 unsaturated, alkaline degradation of, 203-205 reactions of, 260 reactions with nitryl iodide, 299 Sulfonylation, of carbohydrates, 255 Sulfuric acid, in hydrolysis of polysaccharides, 15 Sulfur monochloride, reaction with methyl a-Dglucopyranoside, 256 Sulfuryl chloride in chlorodeoxy sugars preparation, 300 reaction with carbohydrate derivatives, 230-239 T

282 Talose, 6-deoxy-w, ester with guanosine 5’-pyrophosphate, 321 - , 1,2 :5,6-di-O-isopropylidene-P-~-, reaction with triphenylphosphine-carbon tetrachloride, 247 Tamarind kernel, polysaccharide, gasliquid chromatography of, 56 Teichoic acids biosynthesis of, 334 polymers, hydrolysis of, 16 Terphenyl, in gas-liquid chromatography, 46 Tetritols, acetates, gas-liquid chromatography of, 92 Thionyl bromide, and hexamethylphosphoric triamide, bromination of D ribonucleosides by, 250 Thionyl chloride, and hexamethylphosphoric triamide, chlorination of ~ r i bonucleosides by, 250 Threitol, D,gas-liquid chromatography of, 93 D-Threonic acid, 2-C-methyl-, isolation and characterization of, 195 Threose, 4-O-methyl-~,saccharinic acids from alkaline degradation, 195 Threuronic acid, L-, decarboxylation of, 188 Thymidine 5‘-(2-acetamido-2-deoxy-a-~galactopyranosyl pyrophosphate), enzymic synthesis of, 324 5‘-(2-acetamido-2-deoxy-a-~glucopyranosyl pyrophosphate), enzymic synthesis of, 324, 342 5’-(4-acetamido-4,6-dideoxy-~galactosyl pyrophosphate), and epimer, 323

Tagatopyranoside, methyl 1,3,5-tri-Oacetyl-4-bromo-4-deoxy-a-~-and -pL-, preparation of, 266 Takaamylase gas-liquid chromatographic analysis of, 54 a-Dmannosidase as structural reagent for, 444 Talofuranoside, methyl 5-O-benzoyl-6deoxy-2,3-O-isopropylidene-a-~-, preparation of, 282 - , methyl 5,6-dideoxy-5-iodo-2,3-O-isopropylidene-a-L-, preparation of, 242,

5’-(2-amino-2-deoxy-a-~-glucopyran-

osyl pyrophosphate), enzymic synthesis of, 339 5’-(6-deoxy-~glucosyIpyrophosphate), enzymic synthesis of, 323 5’-(6-deoxy-a-~-xyloheptopyranosyl-4d o s e pyrophosphate), enzymic synthesis of, 323 5’-(~fucosylpyrophosphate), preparation of, 323,356 5’-(a-~galactopyranosylpyrophosphate), enzymic preparation of, 340

SUBJECT INDEX, VOLUME 28

513

isolation of, 322 with alcohols and carbohydrates, 258 5’-(a-~-glucopyranosylpyrophosphate), Tributyltin hydride, reduction of chloroenzymic synthesis of, 338 deoxy sugars by, 303 epimerase action on, 375 Triethylamine, dechlorination of chloroisolation of, 322 deoxy sugars in presence of, 302 mechanism of conversion of, 381 Triethyl phosphite, reactions with bromostructure of, 309 deoxy and deoxyiodo sugars, 285 glucose and Dgalacturonic acid esters Trifluoroacetates of 5’-pyrophosphate, 323 of alditols, gas-liquid chromatography 5’-(a-~-mannopyranosyl pyrophosof, 65 phate), occurrence of, 322 in gas-liquid chromatography, 36, 50 5’-(P-~-rhamnopyranosylpyrophosof oligosaccharides, gas-liquid chromaphate), hiosynthesis of, 383 tography of, 70 isolation of, 323 of sugars, gas-liquid chromatography of, 5’-(~-ribosyl pyrophosphate), occur87 rence of, 322 Trimethylsilylation, of sugars, 23-33 - , 3’-O-acetyld’-deoxy-5‘-iodo-, prep- Trimethylsilyl ethers aration of, 243 in gas-liquid chromatography of neutral - , 2,3’-anhydro-5‘-deoxy-5‘-iodo-, prepmonosaccharides, 41-49 aration of, 245 of oligosaccharides, gas-liquid chromatography of, 68-70 -, S-deoxy-5‘-iodo-, preparation of, 243 -, 3‘-deoxy-3’-iodo-5’-O-(p-nitrobenzo-Trioxabicyclo[3.3.0loctane, ring system, steric hindrance by, 241 y1)-, preparation of, 248 Trioxazin, tranquilizer, 9 - , 3’,5‘-dideoxy-3’,5’-diiodo-, preparaTrisaccharide, esters with uridine 5‘-pyrotion of, 245 -, 5’-O-(p-nitrobenzoyl)-, reaction with phosphate, 331 triphenylphosphine and iodine, 248 Tyvelose -, 5’-O-trityI-, reaction with triphenylester of cytidine pyrophosphate, isolaphosphine-carbon tetrachloride, 248 tion of, 316 1-(3-chlorc-2,3-dideoxy-5-Ogas-liquid chromatography of, 63 Thymine, bityl-D-elythro-pentofuranosy1)-, preparation of, 300 preparation of, 248 - , l-(3-chloro-2,3-dideoxy-5-O-trityl-~U D-threo-pentofuranosyl)-, preparation Ultraviolet light, cleavage of carbon-ioof, 248 Thyroglobulin, a-D-mannosidase in strucdine bonds by, 304 tural studies of, 445 Uracil, 1-(5-chlor0-5-deoxy-2,3-di-0-pTin compounds, tributyltin hydride, reto1ylsulfonyl-P-D-ribofuranosy1)-, duction of chlorodeoxy sugars by, 303 preparation of, 255 Tobacco, humectants, gas-liquid chro- , 1-(3-deoxy-3-iod0-2,5-di-O-trityl-p-~matographic analysis of, 97 xylofuranosy1)-, preparation of, 245 Urea, effect on trimethylsilylation reacTranquilizer, Trioxazin, 9 tions, 26 Trehalosamine, isomer of, 286 a,&-Trehalose, 6-amino-6-deoxy-, prep- Uridine, 5‘-(2-acetamido-2-deoxy-a-~-ga~actoaration of, 286 - , 6-azido-6-deoxy-, preparation of, 286 pyranosyl pyrophosphate), biosyn-, 6-bromo-6-deoxy-, heptaacetate, reacthesis and enzymic synthesis of, tion with sodium azide, 286 342,343 -, 6-deoxy-, preparation by photolysis isolation of, 327 of iodo precursor, 305 5‘-(2-acetamido-2-deoxy-a-~glucopyranosyl pyrophosphate), enzymic 1,3,5-Triazine, 2,4,6-trichloro-, reactions

514

SUBJECT INDEX, VOLUME 28

synthesis of, 341 fermentation production and biosynthesis of, 343 isolation of, 327 5'42-acetamido-2-deoxy-a-~-glucopyranosyluronic acid pyrophosphate), isolation of, 327 5'-(2-acetamido-2,4,6-trideoxyhexosyl pyrophosphate), isolation of, 327 5'-(N-acetylmuramic acid pyrophosphate), isolation of, and derivatives, 328 5'-(2-amino-2-deoxy-a-~galactopyranosyl pyrophosphate), preparation of, 355 5'-(2-amino-2-deoxy-a-~-g~ucopyran-

osyl pyrophosphate), enzymic synthesis of, 338 5'-(P~-arabinopyranosyl pyrophosphate), enzymic synthesis of, 343 isolation of, 325 5'-(2-deoxy-a-~-arabino-hexopyranosyl pyrophosphate), fermentation production of, 341 5'-(2-deoxy-a-~-Zyxo-hexopyranosyl pyrophosphate), fermentation preparation of, 341 5'-(2-deoxy-~arabino-hexosyl pyrophosphate), enzymic synthesis of, 338 5'-(2,6-dideoxy-~-ribo-hexosyl pyrophosphate), isolation of, 326 5'-(1,3-dihydroxy-2-propanone pyrophosphate), isolation of, 334 5'-(fructosyl pyrophosphate), isolation of, 326 5'-(a-~-galactofuranosylpyrophosphate), in biosynthesis of galactocarolose, 325 5'-(a-D-galactopyranosyl pyrophosphate), enzymic synthesis and fermentation production of, 340, 341 isolation of, 325 oxidation with Dgalactose oxidase, 344 synthesis by mixed anhydride method, 351 5'-(a-~1-galactopyranosyluronic acid pyrophosphate), enzymic synthesis of, 343 isolation of, 326

5'-(a-~-glucopyranosylpyrophosphate), carbodiimides in synthesis of, 353 chemical synthesis by phosphoramidate method, 345 degradation of, 312, 313,356 enzymic preparation of, 336, 337 isolation of, 325 mechanism of epimerase reaction with, 372 structure of, 309 synthesis by mixed anhydride method, 351 synthesis of analog with C-P bond, 349 5'-(a-~-glucopyranosy1-'4C pyrophosphate), preparation of, 336 5'-(a-~-glucopyranosy1-3"-t,-4"-t, -5"'-t and -6-t pyrophosphates), preparation of, 337 5'-(a-D-glucopyranosyluronic acid pyrophosphate), decarboxylation of, 385 enzymic synthesis of, 343 isolation of, 326 5'-(N-glycolylmuramic acid pyrophosphate), isolation of, 329 5'-(a-gaZacto-hexodialdose pyrophosphate), enzymic preparation of, 344 5'-(a-~-ribo-hexopyranos-3-ulose pyrophosphate), enzymic preparation of, 344 5'-(a-D-mannopyranosyl pyrophosphate), in human blood-cells, 325 5 '-(methyl 2-acetarnido-%deoxy-a-~glucopyranosyluronate pyrophosphate), preparation and reduction of, 355 5'-(methyl a-D-galactopyranosyluronate pyrophosphate), preparation of, 355 phosphoramidate, in nucleotide synthesis, 345 5'-pyrophosphate, disaccharide sulfate esters, in hen oviduct, 329 monosaccharide sulfate esters, in hen oviducts, 329 oligosaccharide esters, 331 trisaccharide esters, 331 reactions with (bromo(or ch1oro)methylene)dimethyliminium bromide(or chloride), 253

SUBJECT INDEX, VOLUME 28

515

rachloride, 248 5’-(P-~-rhamnopyranosylpyrophosphate), biosynthesis of, 326 Urine 5’-(a-~xylopyranosy1pyrophosphate), gas-liquid chromatographic analysis of, 325 48,81 sugars in, identification by gas-liquid enzymic synthesis of, 343 Uridine, 5‘-O-acetyl-, reaction with trichromatography, 44, 46 phenylphosphine-carbon tetrachlo- Uronic acids ride, 249 alditols from, gas-liquid chromatogra- , 5‘-0-acetyl-2’-chloro-2’-deoxy-, prepphy of acetates, 129 aration of, 249 alkaline degradation of, 206 - , 2,2‘-anhydro-5‘-deoxy-5’-iodo-, 3’-0analysis of, 22 (phenyl methylphosphonate), prepdecarboxylation and dehydration of, aration of, 244 186-193 - , 2,5’-anhydro-2’,3’-O-isopropylidene-, determination of, 14 preparation of, 244 esters of guanosine 5’-pyrophosphate, - , 2’,3’-di-O-benzyl-. 5’-(benzyl phos320 phorochloridate), in synthesis of nugas-liquid chromatography of, 64,71-78 cleoside glycosyl pyrophosphates, reductic acid from, 209 351 trimethylsilyl derivatives, gas-liquid -, 2’,3’-O-benzylidene-, bromination by chromatography of, and methyl N-bromosuccinimide and triphenylesters, 138-140 phosphine, 249 - , 2’,3’-O-benzylidene-5’-bromo-5’V deoxy-, preparation of, 249,253 -, 2’,3’-O-benzylidene-5’-deoxy-5’- Vargha, LAszlb, obituary, 1-10 iodo-, preparation of, 250 Vasopenton, as spasmolytic agent, 9 -, 5’-bromod’-deoxy-. preparation of, Verticillium dahliae, gas-liquid chroma253 tography of metabolites of, 47 - , 5’-chloro-5‘-deoxy-, preparation of, Vinelose, ester of cytidine pyrophosphate, 253 317 Vitamin C, history, 3 -, 5’-chloro-5‘-deoxy-2’,3’-O-isopropylidene-, preparation of, 248, 253 - , 2‘-deoxy-, 5’-(a-~-glucopyranosylpyW rophosphate), enzymic synthesis of, 338 Wood pulp, gas-liquid chromatography of, - , 5’-deoxy-5‘-iodo-2’,3’-O-isopropyli42, 61, 74, 78, 88 deneWort, analysis by gas-liquid chromatograpreparation of, 243, 248 phy, 46 reaction with triethyl phosphite, 285 - , 2’,3’-di-O-acetyl-5’-deoxy-5’-iodo-, x preparation of, 243 - , 5,6-dihydro-, 5‘-(a-D-glucopyranosyl Xylal, di-0-acetyl-D-, reaction with hypyrophosphate), preparation of, 354 drogen chloride or hydrogen bro-, 2‘,5’-di-O-trityl-, reaction with methmide, 264 -, di-0-benzoyl-D, reactions with hyyltriphenoxyphosphonium iodide, drogen halides, 265 245 -, 5-hydroxy-, 5’-(cu-D-glUCOpyranOSyl Xylan, speargrass, gas-liquid chromatopyrophosphate), enzymic preparation graphic analysis of, 62 Xylitol, trimethylsilyl ether, gas-liquid of, 338 -, 2’,3’-O-isopropylidene-,reaction chromatography of, 57 with triphenylphosphine-carbon tetXylofuranose, 3-0-acetyl-5-deoxy-5-iodo-

516

SUBJECT INDEX, VOLUME 28

1,2-O-isopropylidene-a-~-, reaction with silver fluoride, 291 - , 3,5-anhydro- 1,2-0-isopropylidene-aD,preparation of, 289, 290 -, 5-bromod-deoxy-l,2-O-isopropylidene-3-O-methyl-a-~,reaction with triethyl phosphite, 285 - , 5-deoxy-5-iodo-1,2-O-isopropylidene-

D

dehydration of, 176, 188 gas-liquid chromatography of, 46 mutarotation and gas-liquid chromatography of, 40 mutarotation in pyridine, 39 in pituitary glycoprotein, gas-liquid chromatographic analysis of, 67, 68 a-Dreaction with sulfuryl chloride, 235 methylphenylphosphinate, preparation saccharinic acids from, 195 of, 280 - , 5-O-benzoyl-4-deoxy-4-iodo-2,3-0reaction with silver fluolide, 289 -, 1,2-O-isopropyhdene-a-D-, reaction isopropylidene-D, diethyl dithioacewith diphenyl phenylphosphonite tal, preparation of, 242 and methyl iodide, 280 - , 5-bromo-5-deoxy-~~-,synthesis of, 304 Xylofuranoside, methyl 2-bromo-2-de-, 3,4-di-O-acety1-2,5-anhydro-~-, (p-nioxy-p-D, monobenzoates, preparatrophenyl)hydrazone, preparation of, tion of, 269 287 Xylopyranose, a-D ester of adenosine 5'-pyrophosphate, -, 2,3,4-tri-O-methyI-~-,dehydration of, enzymic preparation of, 343 181 of uridine 5'-pyrophosphate, en- - , 2,3,5-tri-O-methyl-D-, dehydration of, zymic preparation of, 343 181 DXylosaccharinic acid, a- and P-, foroccurrence of, 325 reaction with sulfuryl chloride, 235 mation from Dxylose, 195 - ,. 3,4-di-O-acetyl-2-bromo-2-deoxy-~, Xylulose, D,preparation from xylose, 39 reaction with (p-nitropheny1)hydraL-, synthesis of, 4 zine, 287 Xyluronic acid, D,decarboxylation of, Xylopyranoside, methyl p-D, hydrolysis 187 of, trifluoroacetic acid in, 17 - , methyl 4-0-benzyl-3-deoxy-3-iodo-20-p-tolylsulfonyl-P-L-, reaction with Y sodium iodide, 293 Yeasts, gas-liquid chromatographic analXylopyranosyl chloride, D, 2,3,4-triysis of, 81 (chlorosulfate), preparation and reactions of, 235, 236 - , 2-chlor0-2-deoxy-a-~, 3,4-di(chloroZ sulfate), preparation of, 236, 237 Zinc ion, effect on a-Dmannosidase, 424 Xylose,

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28 A

BARKER, G. R., Nucleic Acids, 11, 285333 ADAMS,MILDRED. See Caldwell, Mary L. BARKER, S. A., and BOURNE, E. J., Acetals ALEXEEV,Yu. E. See Zhdanov, Yu. A. and Ketals of the Tetritols, Pentitols and Hexitols, 7, 137-207 ALEXEEVA,V. G. See Zhdanov, Yu. A. ANDERSON, ERNEST,and SANDS,LILA,A BARNETT, J. E. C., Halogenated Carbohydrates, 22, 177-227 Discussion of Methods of Value in BARNETT, J . E. G., and CORINA,D. L., Research on Plant Polyuronides, 1, Sugars Specifically Labeled with Iso329-344 topes of Hydrogen, 27,127-190 ANDERSON, LAURENS. See Angyal, S. J. BARRETT, ELLIOTT,P., Trends in the DeANET,E. F. L. J., 3-Deoxyglycosuloses velopment of Granular Adsorbents for (3-Deoxyglycosones) and the DegraSugar Refining, 6,205-230 dation of Carbohydrates, 19,181-218 C. P., and HONEYMAN, JOHN, ANGYAL,s. J., and ANDERSON, LAURENS, BARRY, The Cyclitols, 14, 135-212 Fructose and Its Derivatives, 7,53-98 A. R., and BADDILEY, J., The BAYNE,S., and FEWSTER, J. A., The ARCHIBALD, Teichoic Acids, 21, 323-375 Osones, 11,43-96 BEELIK,ANDREW, Kojic Acid, 11,145-183 ARONSON, N. N., JR. See Pazur, John H. BELL, D. J., The Methyl Ethers of ASPINALL, G, O., Gums and Mucilages, 24,333-379 D-CalaCtOSe, 6, 11-25 ASPINALL,G. O., The Methyl Ethers of BEMILLER, J. N., Acid-catalyzed Hydrolysis of Glycosides, 22,25-108 Hexuronic Acids, 9, 131-148 BEMILLER, J. N. See also, Whistler, ASPINALL, G. O., The Methyl Ethers of Roy L. D-Mannose, 8, 217-230 ASPINALL,G. O., Structural Chemistry of BHAT, K. VENKATRAMANA.See Zorbach, W. Werner. the Hemicelluloses, 14, 429-468 BINKLEY,W. W., Column Chromatography of Sugars and Their DerivaB tives, 10,55-94 W. W., and WOLFROM, M. L., BINKLEY, BADDILEY, J. See Archibald, A. R. Composition of Cane Juice and Cane BAER,HANSH., The Nitro Sugars, 24, Final Molasses, 8,291-314 67-138 BIRCH, GORDONG., Trehaloses, 18,201BAER,HANSH., [Obituary of] Richard 22s Kuhn, 24,l-12 BISHOP, C. T., Gas-liquid ChromatogJ. B., BAILEY,R. W., and PFUDHAM, raphy of Carbohydrate Derivatives, Oligosaccharides, 17, 121-167 19,95-147 BALL,D. H., and PARRISH, F. W., SulBLAIR,MARY GRACE,The 2-Hydroxyfonic Esters of Carbohydrates: glycals, 9, 97-129 Part I, 23,233-280 BOBBITT,J . M., Periodate Oxidation of Part 11, 24,139-197 Carbohydrates, 11, 1-41 BALLOU,CLINTONE., Alkali-sensitive BOESEKEN, J., The Use of Boric Acid for Glycosides, 9, 59-95 the Determination of the ConfiguraBANKS, W., and GREENWOOD, C. T., Phystion of Carbohydrates, 4, 189-210 ical Properties of Solutions of PolyBONNER, T. G., Applications of Trifluorosaccharides, 18,357-398 517

518

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28

acetic Anhydride in Carbohydrate Chemistry, 16,59-84 BONNER,WILLIAMA., Friedel-Crafts and Grignard Processes in the Carbohydrate Series, 6, 251-289 BOURNE,E. J,, and PEAT,STANLEY,The Methyl Ethers of D-Glucose, 5, 145190 BOURNE,E. J . See ulso, Barker, S. A. BOUVENG, H. O., and LINDBERG,B., Methods in Structural Polysaccharide Chemistry, 15,53-89 BRADY,ROBERTF., JR., Cyclic Acetals of Ketoses, 26, 197-278 BRAY,H. G., D-Glucuronic Acid in Metabolism, 8,251-275 BRAY,H. G., and STACEY, M., Blood Group Polysaccharides, 4, 37-55 BRIMACOMBE, J. S. See How, M. J. BUTTERWORTH, ROGERF., and HANESSUN, STEPHEN,Tables of the Properties of Deoxy Sugars and Their Simple Derivatives, 26, 279-296

C CAESAR,GEORGEV., Starch Nitrate, 13, 331-345 CALDWELL, MARY L., and ADAMS,MILDRED,Action of Certain Alpha Amylases, 5, 229-268 CANTOR,SIDNEYM., [Obituary of] John C. Sowden, 20,l-10 CANTOR,SIDNEYM. See ulso, Miller, Robert Ellsworth. CAPON,B., and OVEREND, W. G., Constitution and Physicochemical Properties of Carbohydrates, 15, 11-51 CARR,C. JELLEFF,and KRANTZ, JOHN C., JR., Metabolism of the Sugar Alcohols and Their Derivatives, 1, 175-192 CHIZHOV,0. S. See Kochetkov, N. K. CHURMS,SHIRLEYC., Gel Chromatography of Carbohydrates, 25, 13-51 CLAMP,JOHN R., HOUGH,L., HICKSON, JOHN L., and WHISTLER,ROYL., Lactose, 16, 159-206 COMPTON, JACK,The Molecular Constitution of Cellulose, 3, 185-228 CONCHIE,J., LEWY, G. A., and MARSH,

C. A., Methyl and Phenyl Glycosides of the Common Sugars, 12, 157-187 CORINA,D. L. See Barnett, J. E. G. COURTOIS,JEANEMILE,[Obituary of 1 Emile Bourquelot, 18, 1-8 COXON,BRUCE,Proton Magnetic Resonance Spectroscopy of Carbohydrates, 27, 7-83 CRUM,JAMESD., The Four-carbon Saccharinic Acids, 13, 169-188 D DAVIES, D. A. L., Polysaccharides of Gram-negative Bacteria, 15, 271-340 DEAN,G. R., and GOTTFRIED,J. B., The Commercial Production of Crystalline Dextrose, 5, 127-143 DE BELDER,A. N., Cyclic Acetals of the Aldoses and Aldosides, 20,219-302 DEFAYE,J., 2,5-Anhydrides of Sugars and Related Compounds, 25, 181-228 DEITZ,VICTORR. See Liggett, R. W. DEUEL,H. See Mehta, N. C. DEUEL,HARRYJ., JR., and MOREHOUSE, MARGARET G., The Interrelation of Carbohydrate and Fat Metabolism, 2, 119-160 DEULOFEU,VENANCIO, The Acylated Nitriles of Aldonic Acids and Their Degradation, 4, 119-151 DIMLER,R. J., 1,6-Anhydrohexofuranoses, A New Class of Hexosans, 7,37-52 DOUDOROFF, M. See Hassid, W. Z. DUBACH,P. See Mehta, N. C. DURETTE,PHILIPPEL., and HORTON,D., Conformational Analysis of Sugars and Their Derivatives, 26,49-I25 DUTCHER,JAMESD., Chemistry of the Amino Sugars Derived from Antibiotic Substances, 18, 259-308 DUTTON,GUYG. S., Applications of Gas-liquid Chromatography to Carbohydrates: Part I, 28, 11-160 E ELDERFIELD,ROBERTC., The Carbohydrate Components of the Cardiac Glycosides, 1, 147-173

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28 EL KHADEM, HASSAN,Chemistry of Osazones, 20, 139-181 EL KHADEM,HASSAN,Chemistry of Osotriazoles, 18, 99-121 EL KHADEM,HASSAN,Synthesis of Nitrogen Heterocycles from Saccharide Derivatives, 25, 351-405 ELLIS, G. P., The Maillard Reaction, 14, 63-134 ELLIS, G. P., and HONEYMAN, JOHN,Glycosylamines, 10,95-168 EVANS,TAYLOR H., and HIBBERT, HAROLD,Bacterial Polysaccharides, 2,203-233 EVANS,W. L., REYNOLDS, D. D., and TALLEY,E. A., The Synthesis of Oligosaccharides, 6,27-81

519

Fox, J. J., and WEMPEN,I., Pyrimidine Nucleosides, 14,283-380 FOX,JACKJ. See ulso, Ueda, Tohru. FRENCH,DEXTER,The Raffinose Family of Oligosaccharides, 9, 149-184 FRENCH,DEXTER,The Schardinger Dextrins, 12, 189-260 FREUDENBERG, KARL, Emil Fischer and his Contribution to Carbohydrate Chemistry, 21, 1-38 G

G A R CG~O N Z ~ E F., Z , Reactions of Monosaccharides with beta-Ketonic Esters and Related Substances, 11, 97-143 CAR& GONZALEZ,F., and G ~ M E Z F SANCHEZ,A., Reactions of Amino Sugars with beta-Dicarbonyl ComFEATHER,MILTON S., and HARRIS,JOHN pounds, 20,303-355 GOEPP, RUDOLPH MAXIMILIAN,JR. See F., Dehydration Reactions of Lohmar, Rolland. Carbohydrates, 28, 161-224 GOLDSTEIN,I. J., and HULLAR,T. L., FERRIER, R. J., Unsaturated Sugars, 20, Chemical Synthesis of Polysaccha67-137; 24,199-266 rides, 21,431-512 FEWSTER,J. A. See Bayne, S. FLETCHER,HEWITT G., JR., The Chem- GOMEZSANCHEZ,A. See Garcia GonzPlez, F. istry and Configuration of the CycliGOODMAN, IRVING,Glycosyl Ureides, tols, 3, 45-77 13,215-236 FLETCHER,HEWITT C . , JR., and RICHTGOODMAN, LEON,Neighboring-group MYER, NELSON K., Applications in Participation in Sugars, 22, 109-175 the Carbohydrate Field of Reductive GOFUN,P. A. J., and SPENCER,J. F. T., Desulfurization by Raney Nickel, 5, Structural Chemistry of Fungal Poly1-28 saccharides, 23,367-417 FLETCHER,HEWITTG., JR. See ulso, GOTTFP~ED, J. B. See Dean, G. R. Jeanloz, Roger W. ALFRED,Principles UnFORDYCE,CHARLESR., Cellulose Esters GOTTSCHALK, derlying Enzyme Specificity in the of Organic Acids, 1,309-327 FOSTER,A. B., Zone Electrophoresis of Domain of Carbohydrates, 5,49-78 GREEN,JOHN W., The Glycofuranosides, Carbohydrates, 12,81-115 21,95-142 FOSTER, A. B., and HORTON,D., Aspects of the Chemistry of the Amino Sugars, GREEN,JOHNW., The Halogen Oxidation of Simple Carbohydrates, Excluding 14,213-281 the Action of Periodic Acid, 3, 129FOSTER,A. B., and HUGGARD, A. J., The 184 Chemistry of Heparin, 10,335-368 GREENWOOD, C. T., Aspects of the PhysiFOSTER,A. B., and STACEY,M., The cal Chemistry of Starch, 11,335-385 Chemistry of the 2-Amino Sugars GREENWOOD, C. T., The Size and Shape (2-Amino-2-deoxy-sugars),7,247-288 of Some Polysaccharide Molecules, FOSTER,A. B., and WEBBER, J. M., Chitin, 15,371-393 7,289-332; 11,385-393

520

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28

GREENWOOD, C. T., The Thermal Degradation of Starch, 22,483-515 GREENWOOD, C. T., and MILNE,E. A., Starch Degrading and Synthesizing Enzymes: A Discussion of Their Properties and Action Pattern, 23, 281-366 GREENWOOD, C. T. See also, Banks, W. GURIN,SAMUEL,Isotopic Tracers in the Study of Carbohydrate Metabolism, 3,229-250 GUTHRIE,R. D., The “Dialdehydes” from the Periodate Oxidation of Carbohydrates, 16, 105-158 GUTHRIE,R. D., and MCCARTHY, J. F., Acetolysis, 22, 11-23

H HALL,L. D., Nuclear Magnetic Resonance, 19,51-93 HANESSIAN, STEPHEN,Deoxy Sugars, 21, 143-207 HANESSIAN, STEPHEN.See also, Butterworth, Roger F. HARRIS,ELWIN E., Wood Saccharification, 4, 153-188 HARRIS, JOHNF. See Feather, Milton S. HASKINS,JOSEPH F., Cellulose Ethers of Industrial Significance, 2,279-294 HASSID,W. Z., and DOUDOROFF, M., Enzymatic Synthesis of Sucrose and Other Disaccharides, 5, 29-48 HASSID,W. Z. See also, Neufeld, Elizabeth F. HASSID,W. Z. See also, Nikaido, H. HAYNES,L. J., Naturally Occurring C-Glycosyl Compounds, 18,227258; 20,357-369 HAYNES,L. J., and NEWTH,F. H., The Glycosyl Halides and Their Derivatives, 10,207-256 HEHRE,EDWARDJ., The Substitutedsucrose Structure of Melezitose, 8, 277-290 HELFERICH,BURCKHARDT, The Glycals, 7,209-245 HELFERICH,BURCKHARDT, Trityl Ethers of Carbohydrates, 3, 79-111 HEYNS,K., and PAULSENH., Selective Catalytic Oxidation of Carbohydrates,

Employing Platinum Catalysts, 17, 169-221 HIBBERT,HAROLD.See Evans, Taylor H. HICKSON,JOHN L. See Clamp, John R. HILTON,H. W., The Effects of Plantgrowth Substances on Carbohydrate Systems, 21,377-430 HINDERT,MARJORIE. See Karabinos, J. V. HIRST,E. L., [Obituary of] James Colquhoun Irvine, 8, xi-xvii HIRST,E. L., [Obituary of] Walter Norman Haworth, 6 , l - 9 HIRST,E. L., and JONES,J. K. N., The Chemistry of Pectic Materials, 2, 235-251 HIRST,E. L., and ROSS, A. G., [Obituary of] Edmund George Vincent Percival, 10, xiii-xx HODGE,JOHN E., The Amadori Rearrangement, 10,169-205 HONEYMAN, JOHN,and MORGAN, J. W. W., Sugar Nitrates, 12,117-135 HONEYMAN, JOHN. See also, Barry, C. P. HONEYMAN, JOHN. See also, Ellis, G. P. HORTON,D., [Obituary of] Aha Thompson, 19, 1-6 HORTON,D., [Obituary of] Melville Lawrence Wolfrom, 26, 1-47 HORTON,D., Tables of Properties of 2-Amino-2-deoxy Sugars and Their Derivatives, 15, 159-200 HORTOND., and HUTSON,D. H., Developments in the Chemistry of Thio Sugars, 18, 123-199 HORTON,D. See also, Durette, Philippe L. HORTON,D. See also, Foster, A. B. HOUGH,L., and JONES,J. K. N., The Biosynthesis of the Monosaccharides, 11, 185-262 HOUGH,L., PRIDDLE,J. E., and THEOBALD,R. S., The Carbonates and Thiocarbonates of Carbohydrates, 15,91-158 HOUGH,L. See also, Clamp, John R. HOW, M. J., BRIMACOMBE, J. S., and STACEY,M., The Pneumococcal Polysaccharides, 19, 303-357 HUDSON,C. S., Apiose and the Glycosides of the Parsley Plant, 4, 57-74

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28 HUDSON, C. S., The Fischer Cyanohydrin Synthesis and the Configurations of Higher-carbon Sugars and Alcohols, 1, 1-36 HUDSON,C. S., Historical Aspects of Emil Fischer's Fundamental Conventions for Writing Stereo-formulas in a Plane, 3, 1-22 HUDSON,C. S., Melezitose and Turanose, 2, 1-36 HUGGARD, A. J. See Foster, A. B. HULLAR,T. L. See Goldstein, I. J. HUTSON,D. H. See Horton, D.

52I

Nucleosides and Nucleotides, 15, 201-234

K

KARABINOS, J. V., Psicose, Sorbose and Tagatose, 7,99-I36 KARABINOS, J. V., and HINDERT,MARJORIE,Carboxymethylcellulose, 9, 285-302 KENT,P. W. See Stacey, M. KERTESZ,Z. I., and MCCOLLOCH,R. J., Enzymes Acting on Pectic Substances, 5, 79-102 KISS, J.. Glycosphingolipids (SugarI Sphingosine Conjugates), 24,381433 INCH,T. D., The Use of Carbohydrates KLEMER,ALMUTH. See Micheel, Fritz. in the Synthesis and Configurational KOCHETKOV, N. K., and CHIZHOV,0. S., Assignments of Optically Active, Mass Spectrometry of Carbohydrate Non-carhohydrate Compounds, 27, Derivatives, 21, 39-93 KOCHETKOV, NICOLAIK., and SHIBAEV, 191-225 ISBELL,HORACES., and PIGMAN,WARD, VLADIMIR N., Glycosyl Esters of, Mutarotation of Sugars in Solution: Nucleoside Pyrophosphates, 28, Part 11. Catalytic Processes, Isotope 307-399 Effects, Reaction Mechanisms, and KORT, M. J., Reactions of Free Sugars Biochemical Aspects, 24, 13-65 with Aqueous Ammonia, 25,311-349 ISBELL,HORACES. See also, Pigman, KOWKABANY, GEORGEN., Paper ChromaWard. tography of Carbohydrates and Related Compounds, 9,303-353 KRANTZ,JOHNC., JR. See Carr, C. Jelleff. J KUSZMA", J ~ N O S[Obituary , ofl JAMIESON, G. A., [Obituary of] William LAsz16 Vargha, 28, 1-10 Werner Zorbach, 27,l-6 JEANLOZ,ROGERW., [Obituary of J Kurt Heinrich Meyer, 11, xiii-xviii LAIDLAW,R. A., and PERCIVAL, E. G. V., The Methyl Ethers of the AldopenJEANLOZ,ROGERW., The Methyl Ethers toses and of Rhamnose and Fucose, 7, of 2-Amino-2-deoxy Sugars, 13, 1891-36 214 LALAND, S. See Jonsen, J. JEANLOZ,ROGER W., and FLETCHER, HEWITTG., JR., The Chemistry of LEDERER,E., Glycolipids of Acid-fast Ribose, 6, 135-174 Bacteria, 16, 207-238 LEMIEUX,R. U., Some Implications in JEFFREY,G. A., and ROSENSTEIN, R. D., Crystal-structure Analysis in CarboCarbohydrate Chemistry of Theories hydrate Chemistry, 19, 7-22 Relating to the Mechanisms of ReJONES,DAVIDM., Structure and Some placement Reactions, 9, 1-57 LEMIEUX,R. U., and WOLFROM,M. L., Reactions of Cellulose, 19, 219-246 The Chemistry of Streptomycin, 3, JONES,J. K. N., and SMITH,F., Plant 337-384 Gums and Mucilages, 4, 243-291 LESPIEAU,R., Synthesis of Hexitols and JONES,J. K. N. See also, Hirst, E. L. Pentitols from Unsaturated PolyJONES,J. K. N. See also, Hough, L. hydric Alcohols, 2, 107-118 JONSEN,J., and LALAND,S., Bacterial

522

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28

LEVI,IRVING,and PURVES,CLIFFORDB., The Structure and Configuration of Sucrose (alpha-D-Glucopyranosyl beta-PFructofuranoside), 4, 1-35 LEWY, G. A., and MARSH,C. A., Preparation and Properties of P-Glucuronidase, 14,381-428 LEVVY,G. A. See also, Conchie, J. LEVVY,GUILDFORD A. See also, Snaith, Sybil M. LIGGETT,R. W., and DEITZ, VICTORR., Color and Turbidity of Sugar Products, 9, 247-284 LINDBERG,B. See Bouveng, H. 0. LOHMAR,ROLLAND,and GOEPP, RUDOLPHMAXIMILIAN, JR., The Hexitols and Some of Their Derivatives, 4,211-241 M MAHER,GEORGEG., The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose, 10,257-272 MAHER,GEORGEG., The Methyl Ethers of DGalactose, 10,273-282 MALHOTRA, OM PRAKASH. See Wallenfels, Kurt. MANNERS,D. J., Enzymic Synthesis and Degradation of Starch and Glycogen, 17,371-430 MANNERS, D. J., The Molecular Structure of Glycogens, 12,261-298 R. H., and SARKO, A., MARCHESSAULT, X-Ray Structure of Polysaccharides, 22,421-482 MARSH,C. A. See Conchie, J. MARSH,C. A. See Levvy, G. A. MARSHALL, R. D., and NEUBERGER, A,, Aspects of the Structure and Metabolism of Glycoproteins, 25,407-478 MCCARTHY, J. F. See Guthrie, R. D. MCCASLAND, G. E., Chemical and Physical Studies of Cyclitols Containing Four or Five Hydroxyl Groups, 20, 11-65 MCCLOSKEY, CHESTERM., Benzyl Ethers of Sugars, 12, 137-156 MCCOLLOCH, R. J. See Kertesz, Z. I. MCDONALD,EMMA J,, The Polyfructosans and Difructose Anhydrides, 2, 253-277

MCGALE,E. H. F., Protein-Carbohydrate Compounds in Human Urine, 24, 435-452 MCGINNIS,G. D. See Shafizadeh, F. MEHLTRETTER, C. L., The Chemical Synthesis of DGlucuronic Acid, 8, 231249 MEHTA,N. C., DUBACH,P., and DEUEL, H., Carbohydrates in the Soil, 16, 335-355 MESTER,L., The Formazan Reaction in Carbohydrate Research, 13, 105-167 MESTER,L., [Obituary of] GCza ZemplCn, 14, 1-8 MICHEEL,FRITZ, and KLEMER,ALMUTH, Glycosyl Fluorides and Azides, 16, 85-103 MILLER,ROBERTELLSWORTH,and CANTOR,SIDNEYM., Aconitic Acid, a By-product in the Manufacture of Sugar, 6,231-249 MILLS,J. A., The Stereochemistry of Cyclic Derivatives of Carbohydrates, 10, 1-53 MILNE,E. A. See Greenwood, C. T. MONTGOMERY, JOHN A., and THOMAS, H. JEANETTE,Purine Nucleosides, 17, 301-369 MONTGOMERY, REX, [Obituary of] Fred Smith, 22,l-10 MOODY,G. J., The Action of Hydrogen Peroxide on Carbohydrates and Related Compounds, 19,149-179 MOREHOUSE, MARGARET G. See Deuel, Harry J., Jr. MORGAN,J. W. W. See Honeyman, John. Mom, T., Seaweed Polysaccharides, 8, 315-350 MOYE, C. J., Non-aqueous Solvents for Carbohydrates, 27,85-125 MUETGEERT, J., The Fractionation of Starch, 16,299-333 MYRBACK, KARL,Products of the Enzymic Degradation of Starch and Glycogen, 3,251-310 N

NEELY,W. BROCK,Dextran: Structure and Synthesis, 15,341-369 NEELY,W. BROCK,Infrared Spectra of

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28 Carbohydrates, 12, 13-33 NEUBERG, CARL,Biochemical Reductions at the Expense of Sugars, 4, 75-117 NEUBERGER, A. See Marshall, R. D. NEUFELD,ELIZABETHF., and HASSID, W. Z., Biosynthesis of Saccharides from Glycopyranosyl Esters of Nucleotides (“Sugar Nucleotides”), 18,309-356 NEWTH,F. H., The Formation of Furan Compounds from Hexoses, 6,83-106 NEWTH,F. H. See also, Haynes, L. J. NICKERSON, R. F., The Relative Crystallinity of Celluloses, 5, 103-126 NIKAIDO,H., and HASSID,W. Z., Biosynthesis of Saccharides from Glycopyranosyl Esters of Nucleoside Pyrophosphates (“Sugar Nucleotides”), 26,351-483 NORD,F. F., [Obituary of] Carl Neuberg, 13, 1-7 0

OLSON,E. J. See Whistler, Roy L. OVEREND, W. G., and STACEY,M., The Chemistry of the 2-Desoxy-sugars, €445-105 OVEREND,W. G. See also, Capon, B. P

PACSU,EUGENE,Carbohydrate Orthoesters, 1, 77-127 PARRISH,F. W. See Ball, D. H. PAULSEN,H., Cyclic Acyloxonium Ions in Carbohydrate Chemistry, 26, 127195 PAULSEN, H., and TODT,K., Cyclic Monosaccharides Having Nitrogen or Sulfur in the Ring, 23, 115-232 PAULSEN, H. See also, Heyns, K. PAZUR,JOHNH., and ARONSON,N. N., JR., Glycoenzymes: Enzymes of Glycoprotein Structure, 27, 301-341 PEAT,STANLEY,The Chemistry of Anhydro Sugars, 2,37-77 PEAT,STANLEY.See also, Bourne, E. J. PERCIVAL,E. G. V., The Structure and Reactivity of the Hydrazone and

523

Osazone Derivatives of the Sugars, 3,23-44 PERCIVAL,E. G. V. See also, Laidlaw, R. A. PERLIN,A. S., Action of Lead Tetraacetate on the Sugars, 14,9-61 PERLIN,A. S., [Obituary of] Clifford Burrough Purves, 23,l-10 PHILLIPS,G . O., Photochemistry of Carbohydrates, 18,9-59 PHILLIPS,G. O., Radiation Chemistry of Carbohydrates, 16,13-58 PIGMAN,WARD,and ISBELL,HORACES., Mutarotation of Sugars in Solution: Part I. History, Basic Kinetics, and Composition of Sugar Solutions, 23, 11-57 PIGMAN,WARD. See also, Isbell, Horace S. POLGLASE,W. J., Polysaccharides Associated with Wood Cellulose, 10,283333 PRIDDLE,J. E. See Hough, L. PRIDHAM,J. B., Phenol-Carbohydrate Derivatives in Higher Plants, 20, 371-408 PRIDHAM,J. B. See also, Bailey, R. W. PURVES,CLIFFORDB. See Levi, Irving.

R RAYMOND, ALBERTL., Thio- and Selenosugars, 1, 129-145 REES, D. A., Structure, Conformation, and Mechanism in the Formation of Polysaccharide Gels and Networks, 24, 267-332 REEVES,RICHARDE., CuprammoniumGlycoside Complexes, 6, 107-134 REICHSTEIN,T., and WEISS,EDHARD, The Sugars of the Cardiac Glycosides, 17, 65-120 RENDLEMAN, J. A., JR., Complexes of Alkali Metals and Alkaline-earth Metals with Carbohydrates, 21, 209271 REYNOLDS,D. D. See Evans, W. L. RICHTMYER, NELSONK., The Altrose Group of Substances, 1,37-76 RICHTMYER, NELSONK., The 2-(aldoPolyhydroxyalkyl)benzimidazoles, 6,175-203

524

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28

RICHTMYER, NELSON K. See also, Fletcher, Hewitt G., Jr. R. D. See Jeffrey, G. A. ROSENSTEIN, ROSENTHAL, ALEX,Application of the 0 x 0 Reaction to Some Carbohydrate Derivatives, 23, 59-114 ROSS,A. G. See Hirst, E. L.

S SANDS,LILA.See Anderson, Ernest. SARKO,A. See Marchessault, R. H. SATTLER, LOUIS,Glutose and the Unfermentable Reducing Substances in Cane Molasses, 3, 113-128 SCHOCH,THOMAS JOHN,The Fractionation of Starch, l, 247-277 SHAFIZADEH, F., Branched-chain Sugars of Natural Occurrence, 11,263-283 SHAFIZADEH, F., Formation and Cleavage of the Oxygen Ring in Sugars, 13, 9-61 SHAFIZADEH, F., Pyrolysis and Combustion of Cellulosic Materials, 23,419474 F., and MCGINNIS, G. D., SHAFIZADEH, Morphology and Biogenesis of Cellulose and Plant Cell-walls, 26,297-349 VLADIMIRN. See Kochetkov, SHIBAEV, Nicolai K. SIDDIQUI,I. R., The Sugars of Honey, 25, 285-309 SMITH,F., Analogs of Ascorbic Acid, 2, 79-106 SMITH,F. See also, Jones, J. K. N. SNAITH,SYBILM., and LEVVY,GLIILDFORD A., a-D-Mannosidase, 28, 401-445 SOL,Alditol Anhydrides, 25, SOLTZBERG, 229-283 SOWDEN, JOHNC., The Nitromethane and 2-Nitroethanol Syntheses, 6,291-318 SOWDEN, JOHNC., [Obituary of] Hermann Otto Laurenz Fischer, 17, 1-14 SOWDEN, JOHNC., The Saccharinic Acids, 12,35-79 SPECK,JOHNC., JR., The Lobry de Bruyn-Alberda van Ekenstein Transformation, 13,63-103

SPEDDING, H., Infrared Spectroscopy and Carbohydrate Chemistry, 19, 23-49 SPENCER,J. F. T. See Gorin, P. A. J. SPRINSON, D. B., The Biosynthesis of Aromatic Compounds from D-Glucose, 15,235-270 M., The Chemistry of MucoSTACEY, polysaccharides and Mucoproteins, 2,161-201 STACEY,M., and KENT,P. W., The Polysaccharides of Mycobacterium tuberculosis, 3,311-336 STACEY,M. See also, Bray, H. G. M. See also, Foster, A. B. STACEY, STACEY, M. See also, How, M. J. STACEY,M. See also, Overend, W. G. STOLOFF,LEONARD, Polysaccharide Hydrocolloids of Commerce, 13, 265-287 STRAHS,GERALD,Crystal-structure Data for Simple Carbohydrates and Their Derivatives, 25,53-107 SUGIHARA, JAMESM., Relative Reactivities of Hydroxyl Groups of Carbohydrates, 8, 1-44 SZAREK, WALTERA,, Deoxyhalogeno Sugars, 28,225-306

T TALLEY,E. A. See Evans, W. L. TEAGUE,ROBERTS., The Conjugates of D-Glucuronic Acid of Animal Origin, 9, 185-246 OLOF,Dicarbonyl CarbohyTHEANDER, drates, 17, 223-299 THEOBALD, R. S. See Hough, L. THOMAS,H. JEANNETTE. See Montgomery, John A. TIMELL,T. E., Wood Hemicelluloses: Part I, 19,247-302 Part 11,20,409-483 TIPSON,R. STUART, The Chemistry of the Nucleic Acids, 1, 193-245 TIPSON,R. STUART,[Obituary of] Harold Hibbert, 16, 1-11 TIPSON,R. STUART,[Obituary of] Phoebus Aaron Theodor Levene, 12, 1-12

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-28 TIPSON, R. STUART,Sulfonic Esters of Carbohydrates, 8, 107-215 TODT,K. See Paulsen, H. J. R., [Obituary of] Stanley TURVEY, Peat, 25, 1-12 J. R., Sulfates of the Simple TURVEY, Sugars, 20, 183-218

U UEDA,TOHRU,and FOX,JACKJ., The Mononucleotides, 22,307-419

V VERSTRAETEN, L. M. J., D-Fructose and Its Derivatives, 22, 229-305

W WALLENFELS,KURT,and MALHOTRA, OM PRAKASH, Galactosidases, 16, 239-298 WEBBER,J. M., Higher-carbon Sugars, 17,15-63 WEBBER,J. M. See also, Foster, A. B. WEIGEL,H., Paper Electrophoresis of Carbohydrates, 18,61-97 WEISS,EKKEHARD. See Reichstein, T. WEMPEN,I. See Fox, J. J. WHISTLER,ROYL., Preparation and Properties of Starch Esters, 1,279307 WHISTLER,ROYL., Xvlan, 5,269-290 WHISTLER,ROYL., and BEMILLER,J. N.,

525

Alkaline Degradation of Polysaccharides, 13,289-329 WHISTLER,ROY L., and OLSON,E. J., The Biosynthesis of Hyaluronic Acid, 12, 299-319 WHISTLER,ROY L. See also, Clamp, John R. WHITEHOUSE,M. W. See Zilliken, F. WIGGINS,L. F., Anhydrides of the Pentitols and Hexitols, 5, 191-228 WIGGINS,L. F., The Utilization of Sucrose, 4,293-336 WILLIAMS,NEIL R., Oxirane Derivatives of Aldoses, 25, 109-179 WISE, LOUISE., [Obituary of] Emil Heuser, 15, 1-9 WOLFROM,M. L., [Obituary of] Claude Silbert Hudson, 9, xiii-xviii WOLFROM,M. L., [Obituary of] Rudolph Maximilian Goepp, Jr., 3, xv-xxiii WOLFROM,M. L. See ulso, Binkley, W. W. WOLFROM,M. L. See ulso, Lemieux, R. U. Z

ZHDANOV, Yu. A., ALEXEEV,Yu. E., and ALEXEEVA, V. G., The Wittig Reaction in Carbohydrate Chemistry, 27, 227-299 ZILLIKEN,F., and WHITEHOUSE,M. W., The Nonulosaminic Acids - Neuraminic Acids and Related Compounds (Sialic Acids), 13, 237-263 ZORBACH,W. WERNER,and BHAT,K. VENKATRAMANA, Svnthetic Cardenolides, 21,273-321

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CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28 A

Acetals, cyclic, of the aldoses and aldosides, 20,

219-302 of ketoses, 26,197-277 of hexitols, pentitols, and tetritols, 7,

137-207 Acetic acid, trifluoro-, anhydride, applications of, in carbohydrate chemistry, 16,59-84 Acetolysis, 22,11-23 Aconitic acid, 6,231-249 Action pattern, of starch degrading and synthesizing enzymes, 23,281-366 Acyloxonium ions, cyclic, in carbohydrate chemistry, 26,

127-195 Adsorbents, granular, for sugar refining, 6,205-230 Alcohols, higher-carbon sugar, configurations of,

1, 1-36 unsaturated polyhydric, 2,107-118 Alditols, anhydrides of, 25,229-283 Aldonic acids, acylated nitriles of, 4,119-151 Aldopentoses, methyl ethers of, 7, 1-36;10,257-272 Aldoses, oxirane derivatives of, 25,109-

Analysis, conformational, of sugars and their derivatives, 26,49-I25 of crystal structure, in carbohydrate chemistry, 19,7-22 Anhydrides, 2,5-, of sugars, 25,181-228 difructose, 2,253-277 of alditols, 25,229-283 of aldoses, 25,109-179 of hexitols, 5,191-228 of pentitols, 5,191-228 Anhydro sugars. See Sugars, anhydro. Animals, conjugates of D-glucuronic acid originating in, 9, 185-246 Antibiotic substances, chemistry of the amino sugars derived from, 18,259-308 Apiose, 4,57-74 Ascorbic acid, analogs of, 2,79-106 Aromatic compounds, biosynthesis of, from D-glucose, 15,

235-270 B Bacteria, glycolipides of acid-fast, 16,207-238 nucleosides and nucleotides of, 15,

179

201-234

Aldoses and aldosides, cyclic acetals of, 20,219-302 Alkaline degradation, of polysaccharides, 13,289-329 Altrose, group of compounds related to, 1,37-

polysaccharides from, 2,203-233;3,

311-336 polysaccharides of Gram-negative, 15,

271-340 Benzimidazoles,

76

2-(aldo-polyhydroxyalkyl)-, 6,175-203

Amadori rearrangement, 10,169-205 Amino sugars. See Sugars, 2-amino-2deoxy. Ammonia, aqueous, reactions with free sugars, 25,311-349 Amylases, certain alpha, 5,229-268

Benzyl ethers, of sugars, 12,137-156 Biochemical aspects, of mutarotation of sugars in solution,

24,13-65 Biochemical reductions, at the expense of sugars, 4,75-I17

527

528

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28

Biogenesis, and morphology, of cellulose and plant cell-walls, 26, 297-349 Biosynthesis, of aromatic compounds from D-ghCOSe, 15,235-270 of hyaluronic acid, 12, 299-319 of the monosaccharides, 11, 185-262 of saccharides, from glycopyranosyl esters of nucleoside pyrophosphates (“sugar nucleotides”), 18,309-356; 26, 351-483 Blood groups, polysaccharides of, 4,37-55 Boric acid, for determining configuration of carbohydrates, 4, 189-210 Bourquelot, Emile, obituary of, 18, 1-8 Branched-chain sugars. See Sugars, branched-chain. C

Cane juice, composition of, 8,291-314 Cane molasses. See Molasses, cane. Carbohydrates, action of hydrogen peroxide on, 19, 149-179 application of reductive desulfurization by Raney nickel, in the field of, 5, 1-28 application of trifluoroacetic anhydride in chemistry of, 16,59-84 application of the 0 x 0 reaction to some derivatives of, 23, 59-114 applications of gas-liquid chromatography to, Part I, 28, 11-160 as components of cardiac glycosides, 1, 147-173 carbonates of, 15,91-I58 chemistry of, Emil Fischer and his contribution to, 21, 1-38 the Wittig reaction in, 27, 227-299 complexes of, with alkali metals and alkaline-earth metals, 21, 209-271 compounds with proteins, in human urine, 24,435-452 constitution of, 15, 11-51 crystal-structure analysis of, 19,7-22 crystal-structure data for, 25, 53-107

cyclic acyloxonium ions, in the chemistry of, 26,127-195 degradation of, 19. 181-218 dehydration reactions of, 28, 161-224 determination of configuration of, with boric acid, 4, 189-210 dicarbonyl, 17,223-299 enzyme specificity in the domain of, 5,49-78 fonnazan reaction, in research on, 13, 105-167 Friedel-Crafts and Grignard processes applied to, 6, 251-289 gas-liquid chromatography of derivatives of, 19,95-147 gel chromatography of, 25, 13-51 halogen oxidation of simple, 3,129-184 halogenated, 22, 177-227 infrared spectra of, 12, 13-33 infrared spectroscopy of, 19,23-49 mass spectrometry of derivatives of, 21,39-93 mechanisms of replacement reactions in chemistry of, 9, 1-57 metabolism of, 2, 119-160; 3, 229-250 non-aqueous solvents for, 27, 85-125 orthoesters of, 1,77-127 paper electrophoresis of, 18, 61-97 periodate oxidation of, 11, 1-41 the “dialdehydes” from, 16, 105-158 phenol derivatives, in higher plants, 20,371-408 photochemistry of, 18,9-59 physicochemical properties of, 15, 1151 proton magnetic resonance spectroscopy of, 27, 7-83 radiation chemistry of, 16, 13-58 and related compounds, action of hydrogen peroxide on, 19,149-179 paper chromatography of, 9,303-353 relative reactivities of hydroxyl groups of, 8, 1-44 selective catalytic oxidation of, employing platinum catalysts, 17, 169-221 in the soil, 16,335-355 stereochemistry of cyclic derivatives of, 10, 1-53 sulfonic esters of, 8, 107-215; 23, 233280; 24,139-197 systems, effects of plant-growth substances on, 21,377-430

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28 thiocarbonates of, 15, 91-158 trityl ethers of, 3, 79-111 use of, in the synthesis and configurational assignments of optically active, non-carbohydrate compounds, 27,191-225 zone electrophoresis of, 12,81-I15 Carbonates, of carbohydrates, 15, 91-158 Carboxymethyl ether, of cellulose, 9,285-302 Cardenolides. See also, Glycosides, cardiac. synthetic, 21, 273-321 Catalysts, effects of, in mutarotation of sugars in solution, 24, 13-65 platinum, in selective catalytic oxidation of carbohydrates, 17, 169-221 Cellulose, carboxymethyl-, 9,285-302 esters of, with organic acids, 1,309-327 ethers of, 2,279-294 molecular constitution of, 3, 185-228 and plant cell-walls, morphology and biogenesis of, 26, 297-349 of wood, polysaccharides associated with, 10, 283-333 Celluloses, relative crystallinity of, 5, 103-126 some reactions of, 19,219-246 structure of, 19,219-246 Cellulosic materials, combustion and pyrolysis of, 23,419474 Chemistry, of the amino sugars, 14, 213-281 of the 2-amino sugars, 7,247-288 of anhydro sugars, 2,37-77 of carbohydrates, applications of trifluoroacetic anhydride in, 16, 5984 Emil Fischer and his contribution to, 21, 1-38 crystahtructure analysis in, 19, 7-22 infrared spectroscopy and, 19,23-49 some implications of theories relating to the mechanisms of replacement reactions in, 9, 1-57 the Wittig reaction in, 27, 227-299 of the cyclitols, 3,45-77 of cyclitols containing four or five

529

hydroxyl groups, 20, 11-65 of the 2-deoxy sugars, 8,45-105 of heparin, 10,335-368 of mucopolysaccharides and mucoproteins, 2, 161-201 of the nucleic acids, 1, 193-245 of osazones, 20, 139-181 of osotriazoles, 18,99-121 of pectic materials, 2, 235-251 of ribose, 6, 135-174 of streptomycin, 3, 337-384 of thio sugars, 18, 123-199 physical, of carbohydrates, 15, 11-51 of starch, 11,335-385 radiation, of carbohydrates, 16, 13-58 stereo-, of cyclic derivatives of carbohydrates, 10, 1-53 structural, of fungal polysaccharides, 23,367-417 of the hemicelluloses, 14, 429-468 of polysaccharides, 15,53-89 Chitin, 15,371-393 Chromatography, column. See Column chromatography. gas-liquid. See Gas-liquid chromatography. gel. See Gel chromatogr paper. See P a p e r h o&y. atography. Color, of sugar products, 9,247-284 Column chromatography, of sugars and their derivatives, 10, 55-94 Combustion, of cellulosic materials, 23, 419-474 Complexes, of carbohydrates, with alkali metals and alkaline-earth metals, 21, 20927 1 cuprammonium-glycoside, 6, 107-134 Composition, of sugar solutions, 23, 11-57 Configuration, of carbohydrates, determination of, 4, 189-210 of cyclitols, $45-77 of higher-carbon sugar alcohols, 1, 1-36 of sucrose, 4, 1-35 Configurational assignments, of optically active, non-carbohydrate compounds, use of carbohydrates in, 27, 191-225 Conformation,

530

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28

in formation of polysaccharide gels and networks, 24,267-332 Conformational analysis, of sugars and their derivatives, 26, 49-125 Conjugates, of D-glucuronic acid, 9, 185-246 of sugars with sphingosines, 24,381433 Constitution, of carbohydrates, 15, 11-51 Crystallinity, relative, of celluloses, 5, 103-126 Crystal-structure, analysis, in carbohydrate chemistry, 19, 7-22 data, for simple carbohydrates and their derivatives, 25,53-107 Cuprammonium-glycoside complexes, 6, 107-134 Cyanohydrin synthesis, Fischer, 1, 1-36 Cyclic acetals, of the aldoses and aldosides, 20,219302 of hexitols, pentitols, and tetritols, 7, 137-207 of ketoses, 26, 197-277 Cyclic acetoxonium ions, in carbohydrate chemistry, 26,127-195 Cyclic derivatives, of carbohydrates, stereochemistry of, 10, 1-53 Cyclic monosaccharides, having nitrogen or sulfur in the ring, 23,115-232 Cyclitols, 14, 135-212 chemistry and configurations of, 3, 45-77 containing four or five hydroxyl groups, chemical and physical studies of, 20, 11-65

D

Degradation, of acylated nitriles of aldonic acids, 4, 119- 151 of carbohydrates, 19, 181-218 enzymic, of glycogen and starch, 3,

251-310; 17,407-430 thermal, of starch, 22,483-515 Dehydration, reactions of carbohydrates, 28, 161-224 3-Deoxyglycosones. See Glycosuloses, 3-deoxy-. 3-Deoxyglycosuloses. See Glycosuloses, 3-deoxy-. Deoxyhalogeno sugars. See Sugars, deoxyhalogeno. Deoxy sugars. See Sugars, deoxy. Desulfurization, reductive, by Raney nickel, 5, 1-28 Dextran, structure and synthesis of, 15,341-369 Dextrins, the Schardinger, 12, 189-260 Dextrose, commercial production of crystalline, 5,127-143 “Dialdehydes,” from the periodate oxidation of carbohydrates, 16, 105-158 Dicarbonyl derivatives, of carbohydrates, 17,223-299 Difructose, anhydrides, 2,253-277 Disaccharides, enzymic synthesis of, 5,29-48 trehalose, 18,201-225

E Electrophoresis, of carbohydrates, paper, 18,61-97 zone, 12,81-115 Enzymes. See also, Amylases, Galactosidases, P-Glucuronidase, Clycoenzymes, a-D-Mannosidase. acting on pectic substances, 5,79-102 degradation by, of starch and glycogen, 3,251-310; 17,407-430 of glycoprotein structure, 27, 301-341 specificity of, in the domain of carbohydrates, 5,49-78 starch degrading and synthesizing, 23, 281-366 synthesis by, of glycogen and starch, 17,371-407 of sucrose and other disaccharides, 5,29-48

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28 Esters, of cellulose, with organic acids, 1, 309-327 glycopyranosyl, of nucleoside pyrophosphates, 18,309-356; 26,351483 glycosyl, of nucleoside pyrophosphates, 28,307-399 beta-ketonic (and related substances), reactions with monosaccharides, 11,97- 143 nitric, of starch, 13, 331-345 of starch, preparation and properties of, 1,279-307 sulfonic, of carbohydrates, 8, 107-215; 23, 233-280; 24, 139-197 Ethanol, 2-nitro-, syntheses with, 6,291-318 Ethers, benzyl, of sugars, 12, 137-156 carboxymethyl, of cellulose, 9,285-302 of cellulose, 2,279-294 methyl, of the aldopentoses, 7, 1-36; 10, 257-272 of 2-amino-2-deoxysugars, 13, 189214 of fucose, 7, 1-36; 10,257-272 of D-galactose, 6, 11-25; 10,273-282 O f D-glucose, 5, 145-190 of hexuronic acids, 9, 131-148 of D-mannose, 8,217-230 of rhamnose, 7, 1-36; 10,257-272 trityl, of carbohydrates, 3,79-111

F Fat, metabolism of, 2, 119-160 Fischer, Emil, and his contribution to carbohydrate chemistry, 21, 1-38 Fischer, Hermann Otto Laurenz, obituary of, 17, 1-14 Formazan reaction, in carbohydrate research, 13, 105-167 Formulas, stereo-, writing of, in a plane, 3, 1-22 Fractionation, of starch, 1,247-277; 16,299-333 Friedel-Crafts process, in the carbohydrate series, 6, 251-289 Fructans, 2,253-277

53I

Fructofuranoside, a-D-glucopyranosyl p-D-,4, 1-35 Fructosans, poly-. See Fructans. Fructose, and its derivatives, 7,53-98; 22, 229305 di-, anhydrides, 2, 253-277 Fucose, methyl ethers of, 7,1-36; 10,257-272 Fungal polysaccharides, structural chemistry of, 23,367-417 Furan compounds, formation from hexoses, 6,83-106

G Galactose, methyl ethers of D-, 6, 11-25; 10,273282 Galactosidases, 16,239-298 Gas-liquid chromatography, applications of, to carbohydrates: Part I, 28,ll-160 of carbohydrate derivatives, 19, 95147 Gel chromatography, of carbohydrates, 25, 13-51 Gels, polysaccharide, 24, 267-332 Glucose. See ulso, Dextrose. biosynthesis of aromatic compounds from D-, 15,235-270 methyl ethers of D-, 5, 145-190 Glucuronic acid, D-, chemical synthesis of, 8,231-249 conjugates of, of animal origin, 9, 185246 in metabolism, 8,251-275 p-Glucuronidase, preparation and properties of, 14, 381428 Glutose, 3, 113-128 Glycals, 7, 209-245 - , 2-hydroxy7,9,97-129 Glycoenzymes: enzymes of glycoprotein structure, 27,301-341 Glycofuranosides, 21,95-142 Glycogens, enzymic degradation of, 3,251-310; 17,407-430 enzymic synthesis of, 17,371-407 molecular structure of, 12, 261-298

532

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28

Glycolipides, of acid-fast bacteria, 16,207-238 Glycoproteins. See Proteins, glyco-. Glycopyranosyl esters, of nucleoside pyrophosphates (“sugar nucleotides”), biosynthesis of saccharides from, 18,309-356; 26, 351-483 Glycoside-cuprammonium complexes, 6, 107-134 Glycosides, acid-catalyzed hydrolysis of, 22, 25- 108 alkali-sensitive, 9, 59-95 cardiac, 1, 147-173 the sugars of, 17,65-I20 methyl, of the common sugars, 12,157187 of the parsley plant, 4,57-74 phenyl, of the common sugars, 12,157187 C-Glycosides. See C-Glycosyl compounds. Clycosiduronic acids, of animals, 9, 185-246 poly-, of plants, 1,329-344 Glycosphingolipids, 24,381-433 Glycosones, 3-deoxy-. See Glycosuloses, 3-deoxy-. Glycosuloses, 3-deoxy-, and the degradation of carbohydrates, 19, 181-218 Glycosylamines, 10,95-168 Clycosyl azides, 16,85-103 C-Glycosyl compounds, naturally occurring, 18,227-258; 20, 357-369 Glycosyl esters, of nucleoside pyrophosphates, 28, 307-399 GlycosyI fluorides, 16,85-103 Glycosyl halides, and their derivatives, 10,207-256 Goepp, Rudolph Maximilian, Jr., obituary of, 3, xv-xxiii Grignard process, in the carbohydrate series, 6, 251289 Gums (see also, Hydrocolloids), 24,333379 commercial, 13, 265-287 of plants, 4,243-291

H Halogen oxidation. See Oxidation, halogen. Halogenated carbohydrates, 22, 177-227 Haworth, Walter Norman, obituary of, 6, 1-9 Hemicelluloses, structural chemistry of, 14,429-468 of wood, 19,247-302; 20,409-483 Heparin, chemistry of, 10,335-368 Heuser, Emil, obituary of, 15, 1-9 Hexitols, acetals of, 7, 137-207 anhydrides of, 5,191-228 and some of their derivatives, 4,211241 synthesis of, 2,107-114 Hexofuranoses, 1,6-anhydro-, 7,37-52 Hexosans, 7,37-52 Hexoses. See also, Hexofuranoses. formation of furan compounds from, 6,83-106 Hexuronic acids, methyl ethers of, 9, 131-148 Hibbert, Harold, obituary of, 16, 1-11 History, of mutarotation, 23, 11-57 Honey, the sugars of, 25,285-309 Hudson, Claude Silbert, obituary of, 9, xiii-xviii Hyaluronic acid, biosynthesis of, 12,299-319 Hydrazones, of sugars, 3, 23-44 Hydrocolloids, commercial, polysaccharidic, 13, 265287 Hydrogen, isotopes of, sugars specifically labeled with, 27, 127-190 Hydrogen peroxide, action on carbohydrates and related compounds, 19, 149-179 Hydrolysis, acid-catalyzed, of glycosides, 22, 25108

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28 Hydroxyl groups, relative reactivities of, 8, 1-44

I Infrared spectra, of carbohydrates, 12, 13-33 Infrared spectroscopy, and carbohydrate chemistry, 19,23-49 Irvine, James Colquhoun, obituary of, 8, xi-xvii Isotopes, effects of, in mutarotation of sugars in solution, 24, 13-65 of hydrogen, sugars specifically labeled with, 27, 127-190 Isotopic tracers. See Tracers, isotopic.

K Ketals. See Acetals. Ketoses, cyclic acetals of, 26, 197-277 Kinetics, basic, of mutarotation, 23, 11-57 Kojic acid, 11, 145-183 Kuhn, Richard, obituary of, 24, 1-12

L Lactose, 16, 159-206 Lead tetraacetate, action of, on the sugars, 14, 9-61 Levene, Phoebus Aaron Theodor, obituary of, 12, 1-12 Lipids, glycosphingo-. See Glycosphingolipids. Lobry de Bruyn-Alberda van Ekenstein transformation, 13, 63-103

M Maillard reaction, 14, 63-134 Mannose, methyl ethers of D-, 8, 217-230 a-D-Mannosidase, 28,401-445 Mass spectrometry, of carbohydrate derivatives, 21,39-93 Materials, cellulosic, combustion and pyrolysis of, 23,419-474

533

Mechanism, in the formation of polysaccharide gels and networks, 24,267-332 of replacement reactions in carbohydrate chemistry, 9, 1-57 Melezitose, 2, 1-36 structure of, 8, 277-290 Metabolism, of carbohydrates, 2, 119-160 use of isotopic tracers in studying, 3,229-250 of fat, 2, 119-160 of the sugar alcohols and their derivatives, 1, 175-192 D-glucuronic acid in, 8,251-275 Methane, nitro-, syntheses with, 6,291-318 Methods, in structural polysaccharide chemistry, 15,53-89 Methyl ethers. See Ethers, methyl. Meyer, Kurt Heinrich, obituary of, 11, xiii-xviii Molasses, cane, 3, 113-128 cane final, composition of, 8, 291-314 Molecular structure, of glycogens, 12,261-298 Mononucleotides, 22,307-419 Monosaccharides, biosynthesis of, 11, 185-262 cyclic, having nitrogen or sulfur in the ring, 23, 115-232 reactions of, with beta-ketonic esters and related substances, 11,97-I43 Morphology, and biogenesis of cellulose and plant cell-walls, 26, 297-349 Mucilages (see also, Hydrocolloids), 24, 333-379 commercial, 13, 265-287 of plants, 4,243-291 Mucopolysaccharides. See Polysaccharides, muco-. Mucoproteins. See Proteins, muco-. Mutarotation, of sugars in solution: Part I. History, basic kinetics, and composition of sugar solutions, 23, 11-57 Part 11. Catalytic processes, isotope

534

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28

effects, reaction mechanisms, and biochemical aspects, 24, 13-65 Mycobacterium tuberculosis, polysaccharides of, 3, 311-336

N Neighboring-group participation, in sugars, 22, 109-175 Networks, polysaccharide, 24, 267-332 Neuberg, Carl, obituary of, 13, 1-7 Neuraminic acids, and related compounds, 13,237-263 Nickel, Raney. See Raney nickel. Nitrates, of starch, 13, 331-345 of sugars, 12, 117-135 Nitriles, acetylated, of aldonic acids, 4, 119-151 Nitrogen heterocycles, synthesis from saccharide derivatives, 25,351-405 Nitro sugars. See Sugars, nitro. Non-aqueous solvents for carbohydrates, 27,85-125 Nonulosaminic acids, 13,237-263 Nuclear magnetic resonance, 19,51-93 Nucleic acids, 1, 193-245; 11,285-333 Nucleosides, bacterial, 15,201-234 purine, 17,301-369 pyrimidine, 14,283-380 pyrophosphates, glycopyranosyl esters Of, 18,309-356; 26,351-483 glycosyl esters of, 28, 307-399 Nucleotides, bacterial, 15,201-234 mono-, 22,307-419 0

Obituary, of Emile Bourquelot, 18, 1-8 of Emil Fischer, 21, 1-38 of Hermann Otto Laurenz Fischer, 17, 1-14 of Rudolph Maximilian Goepp, Jr., 3, xv-xxiii of Walter Norman Haworth, 6,1-9 of Emil Heuser, 15, 1-9 of Harold Hibbert, 16, 1-11

of Claude Silbert Hudson, 9, xiii-xviii of James Colquhoun Irvine, 8, xi-xvii of Richard Kuhn, 24, 1-12 of Phoebus Aaron Theodor Levene, 12,l-12 of Kurt Heinrich Meyer, 11, xiii-xviii of Carl Neuberg, 13, 1-7 of Stanley Peat, 25, 1-12 of Edmund George Vincent Percival, 10, xiii-xx of Clifford Burrough Purves, 23, 1-10 of Fred Smith, 22,l-10 of John Clinton Sowden, 20,l-10 of Alva Thompson, 19, 1-6 of LBsz16 Vargha, 28, 1-10 of Melville Lawrence Wolfrom, 26, 1-47 of GCza Zempkin, 14, 1-8 of William Werner Zorbach, 27, 1-6 Oligosaccharides. 17, 121- 167 the raffinose family of, 9, 149-184 synthesis of, 6,27-81 Orthoesters, of carbohydrates, 1, 77-127 Osazones, chemistry of, 20, 139-181 of sugars, 3, 23-44 Osones, 11,43-96 Osotriazoles, chemistry of, 18,99-121 Oxidation, halogen, of simple carbohydrates, 3, 129-148 lead tetraacetate, of sugars, 14,9-61 periodate, of carbohydrates, 11, 1-41 the “dialdehydes” from, 16, 105-158 selective catalytic, of carbohydrates, employing platinum cadysts, 17, 169-221 Oxirane derivatives, of aldoses, 25, 109179 0 x 0 reaction, application to some carbohydrate derivatives, 23,59-114 Oxygen ring, formation and cleavage of, in sugars, 13,9-61 P Paper chromatography, of carbohydrates and related com-

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28 pounds, 9,303-353 Paper electrophoresis, of carbohydrates, 18, 61-97 Parsley, glycosides of the plant, 4,57-74 Participation, neighboring-group, in sugars, 22, 109175 Peat, Stanley, obituary of, 25, 1-12 Pectic materials, chemistry of, 2,235-251 enzymes acting on, 5,79-102 Pentitols, acetals of, 7, 137-207 anhydrides of, 5,191-228 synthesis of, 2,107-118 Percival, Edmund George Vincent, obituary of, 10, xiii-xx Periodate oxidation. See Oxidation, periodate. Phenol-carbohydrate derivatives, in higher plants, 20, 371-408 Photochemistry, of carbohydrates, 18,9-59 Physical chemistry, of carbohydrates, 15, 11-51 of starch, 11,335-385 Physical properties, of solutions of polysaccharides, 18, 357-398 Physical studies, of cyclitols containing four or five hydroxyl groups, 20, 11-65 Plant-growth substances, effect on carbohydrate systems, 21, 377-430 Plants, cell walls of, morphology and biogenesis of, 26,297-349 glycosides of parsley, 457-74 gums of, 4,243-291 mucilages of, 4, 243-291 polyuronides of, 1,329-344 Platinum. See Catalysts. Pneumococcal polysaccharides. 19,303357 Polyfructosans. See Fructans. Polyglycosiduronic acids. See Clycosiduronic acids, poly-. Polysaccharides. See also, Carbohydrates,

535

Cellulose, Dextran, Dextrins, Fructans, Glycogen, Glycosiduronic acids (poly-), Pectic materials, Starch, and Xylan. alkaline degradation of, 13,289-329 associated with wood cellulose, 10, 283-333 bacterial, 2, 203-233; 15, 271-340 blood-group, 4,37-55 chemical synthesis of, 21,431-512 fungal, structural chemistry of, 23, 367-417 gels and networks, role of structure, conformation, and mechanism, 24, 267-332 hydrocolloidal, 13, 265-287 methods in structural chemistry of, 15, 53-89 muco-, chemistry of, 2,161-201 of Cram-negative bacteria, 15,271-340 of Mycobacteriurn tuberculosis, 3,311336 of seaweeds, 8,315-350 physical properties of solutions of, 18, 357-398 pneumococcal, 19,303-357 shape and size of molecules of, 7, 289332; 11,385-393 x-ray structure of, 22,421-482 Polyuronides, of plants, 1,329-344 Preparation, of esters of starch, 1,279-307 of p-glucuronidase, 14,381-428 Properties, of 2-amino-2-deoxy sugars and their derivatives, 15, 159-200 of deoxy sugars and their simple derivatives, tables of, 26,279-296 of esters of starch, 1,279-307 of p-glucuronidase, 14,381-428 physical, of solutions of polysaccharides, 18, 357-398 physicochemical, of carbohydrates, 15, 11-51 Proteins, compounds with carbohydrates, in human urine, 24,435-452 glyco-, aspects of the structure and metabolism of, 25,407-478

536

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28

enzymes (glycoenzymes), 27, 301-341 muco-, chemistry of, 2,161-201 Proton magnetic resonance spectroscopy, of carbohydrates, 27, 7-83 Psicose, 7, 99-136 Purines, nucleosides of, 17,301-369 Purves, Clifford Burrough, obituary of, 23, 1-10 Pyrimidines, nucleosides of, 14,283-380 Pyrolysis, of cellulosic materials, 23, 419-474

R Radiation, chemistry of carbohydrates, 16, 13-58 Raffinose, family of oligosaccharides, 9, 149-184 Raney nickel, reductive desulfurization by, 5, 1-28 Reaction, the formazan, in carbohydrate research, 13,105-167 the Maillard, 14,63-134 the 0x0, application to some carbohydrate derivatives, 23, 59-114 the Wittig, in carbohydrate chemistry, 27,227-299 Reactions, dehydration, of carbohydrates, 28, 161-224 mechanisms of, in mutarotation of sugars in solution, 24, 13-65 of amino sugars with beta-dicarbonyl compounds, 20,303-355 of cellulose, 19,219-246 of free sugars with aqueous ammonia, 25,311-349 of monosaccharides with beta-ketonic esters and related substances, 11, 97-143 Reactivities, relative, of hydroxyl groups of carbohydrates, 8, 1-44 Rearrangement, the Amadori, 10, 169-205 Reductions, biochemical, at the expense of sugars, 4,75-117 Replacement reactions,

mechanisms of, in carbohydrate chemistry, 9, 1-57 Rhamnose, methyl ethers of, 7, 1-36; 10, 257-272 Ribose, chemistry of, 6, 135-174 S

Saccharides, biosynthesis of, from glycopyranosyl esters of nucleoside pyrophosphates (“sugar nucleotides”), 18, 309-356; 26,351-483 synthesis of nitrogen heterocycles from, 25,351-405 Saccharification. of wood, 4, 153-188 Saccharinic acids, 12, 35-79 four-carbon, 13, 169-188 Schardinger dextrins, 12, 189-260 Seaweeds, polysaccharides of, 8, 315-350 Seleno sugars. See Sugars, seleno. Shape, of some polysaccharide molecules, 7, 289-332; 11,385-393 Sialic acids, 13, 237-263 Size, of some polysaccharide molecules, 7, 289-332; 11,385-393 Smith, Fred, obituary of, 22,1-10 Soil, carbohydrates in, 16, 335-355 Solutions, of polysaccharides. physical properties Of, 18,357-398 of sugars, mutarotation of, 23, 11-57; 24,13-65 Solvents, non-aqueous, for carbohydrates, 27y85-125 Sorbose, 7,99-136 Sowden, John Clinton, obituary of, 20, 1-10 Specificity, of enzymes, in the domain of carbohydrates, 5,49-78 Spectra, infrared, of carbohydrates, 12, 13-33 Spectrometry, mass,

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28 of carbohydrate derivatives, 21, 39-93 Spectroscopy, infrared, and carbohydrate chemistry, 19,23-49 nuclear magnetic resonance, 19, 51-93 proton magnetic resonance, of carbohydrates, 27, 7-83 Sphingosines, conjugates with sugars, 24, 381-433 Starch, degrading and synthesizing enzymes, 23,281-366 enzymic degradation of, 3, 251-310; 17,407-430 enzymic synthesis of, 17,371-407 fractionation of, 1,247-277; 16,299333 nitrates of, 13, 331-345 physical chemistry of, 11,335-385 preparation and properties of esters of, 1,279-307 thermal degradation of, 22,483-515 Stereochemistry, of cyclic derivatives of carbohydrates, 10,l-53 formulas, writing of, in a plane, 3, 122 Streptomycin, chemistry of, 3,337-384 Structural chemistry, of fungal polysaccharides, 23,367-417 of the hemicelluloses, 14,429-468 Structure, molecular, of cellulose, 19, 219-246 of dextran, 15,341-369 of glycogens, 12,261-298 of polysaccharide gels and networks, 24,267-332 of sucrose, 4, 1-35 x-ray, of polysaccharides, 22,421-482 Sucrose. See also, Sugar. enzymic synthesis of, 5,29-48 structure and configuration of, 4,l-35 utilization of, 4,293-336 Sugar, aconitic acid as by-product in manufacture of, 6,231-249 Sugar alcohols. See also, Alditols, Hexitols, Pentitols, Tetritols. higher-carbon, configurations of, 1,

537

1-36 and their derivatives, metabolism of, 1, 175-192 “Sugar nucleotides.” See Nucleoside pyrophosphates, glycopyranosyl or glycosyl esters of. Sugar products, color and turbidity of, 9,247-284 Sugar refining, granular adsorbents for, 6, 205-230 Sugars, action of lead tetraacetate on, 14,9-61 amino, aspects of the chemistry of, 14,213281 derived from antibiotic substances, 18,259-308 methyl ethers of, 13, 189-214 properties of, 15, 159-200 reactions with beta-dicarbonyl compounds, 20,303-355 2-amino. See Sugars, 2-amino-2-deoxy. 2-amino-2-deoxy, 7, 247-288 2,5-anhydrides of, 25, 181-228 anhydro, chemistry of, 2,37-77 benzyl ethers of, 12, 137-156 biochemical reductions at the expense Of, 4,75-117 branched-chain, of natural occurrence, 11,263-283 of the cardiac glycosides, 17, 65-120 conjugates, with sphingosines, 24,381433 deoxy, 21, 143-207 tables of the properties of, and their simple derivatives, 26, 279-296 2-deoxy, 8,45-105 deoxyhalogeno, 28,225-306 free, reactions with aqueous ammonia, 25,311-349 higher-carbon, 17, 15-63 configurations of, 1, 1-36 of honey, 25,285-309 hydrazones of, 3,23-44 methyl glycosides of the common, 12, 157-187 neighboring-group participation in, 22, 109-175 nitrates of, 12, 117-135 nitro, 24,67-138

538

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28

osazones of, 3,23-44 oxygen ring in, formation and cleavage of, 13,9-61 phenyl glycosides of the common, 12, 157-187 related to altrose, 1, 37-76 seleno, 1, 144-145 solutions of, mutarotation of, 23,ll-57; 24, 13-65 specifically labeled with isotopes of hydrogen, 27, 127-190 sulfates of the simple, 20, 183-218 and their derivatives, column chromatography of, 10,55-94 conformational analysis of, 26,49125 thio, 1, 129-144 developments in the chemistry of, 18,123-199 unsaturated, 20, 67-137; 24, 199266 Sulfates, of the simple sugars, 20, 183-218 Sulfonic esters, of carbohydrates, 8,107-215; 23,233280; 24, 139-197 Synthesis, biochemical, of monosaccharides, 11, €85-262 of cardenolides, 21,273-321 chemical, of D-glucuronic acid, 8, 231249 of polysaccharides, 21,431-512 of dextran, 15,341-369 enzymic, of glycogen and starch, 17,371-407 of sucrose and other disaccharides, 5, 29-48 of nitrogen heterocycles from saccharide derivatives, 25,351-405 and configurational assignments of optically active, non-carbohydrate compounds, use of carbohydrates in, 27, 191-225

T

Tables, of the properties of deoxy sugars and

their simple derivatives, 26,279296 Tagatose, 7,99-136 Teichoic acids, 21, 323-375 Tetritols, acetals of, 7, 137-207 Thiocarbonates, of carbohydrates, 15,91-158 Thio sugars. See Sugars, thio. Thompson, Aha, obituary of, 19, 1-6 Tracers, isotopic, 3,229-250 Transformation, the Lobry de Bruyn-Alberda van Ekenstein, 13, 63-103 Trehaloses, 18,201-225 Trityl ethers, of carbohydrates, 3,79-111 Turanose, 2, 1-36 Turbidity, of sugar products, 9, 247-284

U Unsaturated sugars. See Sugars, unsaturated. Ureides, glycosyl, 13, 215-236 Urine, human, protein-carbohydrate compounds in, 24,435-452

V Vargha, LBsz16, obituary of, 28, 1-10 W

Wittig reaction, in carbohydrate chemistry, 27,227-299 Wolfrom, Melville Lawrence, obituary of, 26, 1-47 Wood, hemicelluloses of, 19,247-302; 20, 409-483 polysaccharides associated with cellulose of, 10,283-333 saccharification of, 4, 153-188

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-28

X X-Rays, crystal-structure analysis by, 19, 7-22 Xylan, 5,269-290

2 ZBmplen, Giiza, obituary of, 14, 1-8 Zone electrophoresis, of carbohydrates, 12,81-I15 Zorbach, William Werner, obituary of,27, 1-6

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ERRATA VOLUME26 Page 46, line 21. For “l-(Adenin-9-yl)-l-deoxyl-” read “1-(Adenin9-y1)-1-deoxy-.” Page 277, line 10. For “9.3” read “93.”

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