ADVANCES IN CARBOHYDRATE CHEMISTRY

ADVANCES IN CARBOHYDRATE CHEMISTRY VOLUME 13

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Advances in Carbohydrate Chemistry Editor MELVILLE L. WOLFROM Associate Editor R. STUART TIPSON Board of Advisors C. B. PURVES J. C. SOWDEN ROYL. WHISTLER

HERMANN 0. L. FISCHER R. C. HOCKETT W. W. PIQMAN

Board of Advisors for the British Isles E. L. HIRST

STANLEY PEAT

MAURICE STACEY

Volume 13

1958

ACADEMIC PRESS INC., PUBLISHERS NEW YORK, N. Y.

Copyright@,

1958, Academic Press Inc.

ACADEMIC PRESS INC. 111 FIFTH AVENUE NEW YORK3, N. Y. (London) LTD.,PUBLISHERS 40 PALL MALL,LONDON, S. W. 1 ALL RIGHTS RESERVED NO PART O F T H I S BOOK MAY B E REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMIBSION FROM T H E PUBLISHERS.

Library of Congress Catalog Card Number: .#-11361

ACADEMIC PRESS INC.

PRINTED I N THE UNITED $TATEP,

OF AMERICA

LIST OF CONTRIBUTORS

J. N, BEMILLER,Department of Biochemistry, Purdue University, Lafayette, Indiana GEORGEV. CAESAR,Starch Consultant, Harbor Beach, Michigan JAMESD. CRUM,Department of Chemistry, The Ohio State University, Columbus, Ohio IRVINGGOODMAN, Wellcome Research Laboratories, Tuckahoe, N . Y . ROGERW. JEANLOZ, Massachusetts General Hospital, Boston, Massachusetts

L. MESTER,The Technical University, Budapest, Hungary* F. F. NORD, Department of Organic Chemistry and Enzymology, Fordham University, New Yo&, N . Y . F. SHAFIZADEH, Department of Chemistry, The Ohio State University, Columbus, Ohio

JOHNC. SPECK,JR., Department of Chemistry, Michigan State University, East Lansing, Michigan LEONARD STOLOFF, Seaplant Corporation, New Bedford, Massachusetts ROYL. WHISTLER, Department of Biochemistry, Purdue University, Lafayette, Indiana M. W. WHITEHOUSE, Department of Biochemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

F. ZILLIKEN,Department of Biochemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

* Present address: Centre National de la Recherche Scientifique, Universitk de Paris, Facult6 de Pharmacie, Paris, France.

V

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PREFACE Volume 6 of this series contained a review of the pioneer work on sugarring conformational analysis carried out by Richard E. Reeves. This general topic has received considerable attention in later volumes and is herein extended by Shafizadeh to the formation and cleavage of the sugar oxygen rings. Current emphasis on sugar-alkali reactivity is reflected in three chapters-by Speck, by Crum, and by Whistler and BeMiller. Crum has summarized the life-work of the late J. W. E. Glattfeld (and associates) on the four-carbon saccharinic acids. These outstanding researches of Glattfeld, a Nef student, have not hitherto received the recognition they merited. Application of the long-known formazan reaction to sugar hydrazone structures is detailed by Mester, whose review, incidentally, serves to emphasize that the acyclic forms of sugar derivatives exist and cannot be ignored. Our series of chapters on the methyl ethers of the monosaccharides is continued with a contribution by Jeanloz on such derivatives of the amino sugars. The current interest in topics related to nucleic acid chemistry is reflected in a discussion of glycosyl ureides by Goodman. The chemical nature of the sialic (nonulosaminic) acids is a t last becoming clarified and this subject is summarized by Zilliken and Whitehouse. Stoloff contributes a chapter on some aspects of the polysaccharide hydrophilic colloids, and Caesar has written a fascinating account of the development of starch nitrate as an explosive. Finally, the pioneer biochemist, Carl Neuberg,-who, indeed, coined the term “biochemistry”-is memorialized in an obituary by one of his students, Professor F. F. Nord. The editors finally admit defeat in their struggles to delineate sex by elaborating the first names of female scientists. As one of our esteemed reviewers has noted, this effort has been laudable but has led to a number of interesting sex reversals; thus, what American editor could be sure of the sex indicated by many of the Scandinavian or Hungarian given names, or would know that “Evelyn” is an established name for a British male? Accordingly, only initials are employed in this volume. M. L. WOLFROM R. STUART TIPSON

Columbus, Ohio Washington, D. C.

vii

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CONTENTS CONTRIBUTORS TO VOLUME 13... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

PREFACE.. ...................................................................

vii

CARLNEUBERG. .............................................................

1

Formation and Cleavage of the Oxygen Ring in Sugars

F. SHAFIZADEH I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Formation and Hydrolysis of Aldonolactones . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Formation and Hydrolysis of the Aldosides.. . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Nitroil8 Acid Deamination of Amino Sugars.. . . . . . . . . . . . . . . . . . . . . . . . . . .

9 10 24 43

The Lobry De Bruyn-Alberda Van Ekenstein Transformation

JOHN C. SPECK, JR. J. Introduction.. . . . . . . . . . . ........................................ 11. Scope . . . . . . . . . . ...................... ........................ 111. Side Reactions. ...................... ........................ IV. Catalysis, , , . , , ................................................... V. Use of the Transformation for Synthesis.. . . . . . . . . . . . . VI. Investigations of the Mechanism. . . . . . . . . . . . VII. Present Status of the Mechanism.. . . . . . . . . . . ..................

63 65 73 79 99

The Formazan Reaction in Carbohydrate Research

L. MESTER I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . .......................... 105 11. Preparation and Structure of Sugar Formazans.. ....................... 109 111. Identification of Aldoses in the Form of Formazans.. . . . . . . . . . . . . . . . . . . . 113 IV. Preparation of Sugar Tetrazolium Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . 115 V. Preparation of Aldothionic Acid Phenylhydrazides. . . . . . . . . . . . . . . . . . . . . . 118 VI. Structure of Sugar Phenylhydrazones.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 VII. Structure of Sugar Phenylosazones . . . . . . . . . . . . . . . . 129 VIII. The Formazan Reaction of Periodate-oxidi IX. The Formazan Reaction of Oxidized Polysaccharides . . . . . . . . . . . . . . . . . . . 154 X. Preparation and Use of the Tetrazolium and Carbothionic Acid Phenylhydrazide Derivat,ives of the Oxidiz ides and the Metal Complexes of their Formazans.. . . . . ix

X

CONTENTS

The Four-Carbon Saccharinic Acids

JAMES D. CRUM

I. Introduction.

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

169

Derivatives. ...............................

The Methyl Ethers of 2-Amino-2-Deoxy Sugars

ROGER W. JEANLOZ I, Introduction.. ..... .......................... 189 11. The Met,hyl Ethers 111. The Methyl Ethers IV. The Methyl Ethers of D-Galactosamine,. . . . . . V. The Methyl Ethers VI. The Methyl Ethers of D-Altrosamine.. . . . . . . . . VII. Analytical Properties . . . . . . . . . . . . . . . . . . . . . . . . VIII. Tables of Properties of the Methyl Ethers of 2-Amino-2-deoxy Sugars.. 203 Glycosyl Ureides IRVING GOODMAN

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215

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

227

VII. Tables of Glycosyl Ureides and Related Compounds

The Nonulosaminic Acids Neuraminic Acids and Related Compounds (Sialic Acids) F. ZILLIKENA N D M. W. WHITEHOUSE I. Introduction.. ............................... 11. Occurrence and Distribution, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Detection and Determi IV. Isolation and Characte V. Chemistry and Structu VI. Neuraminolactose.. . . . . . ........................ VII. Biochemistry. . . . . . . . . VIII. Conclusion.. .. .........................

241

263

Polysaccharide Hydrocolloids of Commerce LEONARD STOLOFF I. Introduction.. ................................ 11. Terminology and Definitions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... 111. Polysacolloids in Commercial Use. . IV. Market Summary.. ................................... V. Usage Trends ...............................................

266 267

287

xi

CONTENTS

Alkaline Degradation of Polysaccharides ROY L. WHISTLERand J. N. BEMILLER I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Action of Alkali on Reducing End-units .................... 111. Effects of Alkali on Hydroxyl Groups. . .................... IV. Effects of Linkages on Alkaline Degradation.. .......................... V. Products from Polysaccharide Degradation. . . . . . . . . . . . . . . . . VI. Effects of Alkali on Oxidized Polysaccharides. . . . . . . . . . . . . . . VII. Other Glycosides which are Degraded by the beta-Alkoxy Carbonyl Mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Alkaline Fragmentation Reactions of Carbohydrates. . . . . . . . . . . . . . . . . . . . IX. Action of Unusual Bases on Sugars.. . . . . . . . . . . . . . . . . . .

289 291 294 296

323 326 328

Starch Nitrate GEORGEV. CAESAR .....................................

331

AUTHORINDEX FOR VOLUME 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECTINDEX FOR VOLUME 13... . . . . . . . . . . . . . . . . . . . . . . . . . . . CUMULATIVE AUTHORINDEX FOR VOLUMES 1-12., . . . . . . . . . . . . . . . . . . . . . . . . . . . . CUMULATIVE SUBJECT INDEX FOR VOLUMES 1-12.. . . ERRATA.....................

376

. . . . . . . . . . . . 381

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

387

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CARLNEUBERG 1877-1956 The chemical reactions occurring in living organisms were studied in the 19th century primarily by chemists and physiologists; the identification and synthesis of some of the compounds formed represent victories of organic chemistry. After von Liebig, the greatest name in this field is that of Emil Fischer, who became successively professor of chemistry at Erlangen, Wiirzburg, and Berlin. While investigating hydrazine, he embarked upon a study of the carbohydrates and synthesized many, identified them and, in numerous cases, prepared their stereoisomeric forms. Difficulties observed in fermentation studies turned Fischer’s attention to the chemical action of enzymes. An offspring of this scientifically and humanly unparalleled era (uia his teacher A. Wohl, who later became professor of chemistry a t the Institute of Technology in Danzig) was Carl Neuberg. He was born a t Hannover, Germany, on July 29, 1877, and died after a prolonged illness on the 30th of May, 1956, in New York City. Neuberg was a complex personality. The strict framework of Imperial Germany, with its class consciousness and its military code of honor, provided a strange arena for the ingenuity, ambition, and drive of the young Carl Neuberg. He studied Chemistry in Wurzburg and Berlin, and obtained his Ph. D. in 1900. At this time, biochemistry in Germany was represented by a sole chair occupied by Roehmann at the University of Breslau. Neuberg became, then, an instructor in the Division of Chemistry of the Pathological Institute of the University of Berlin under Virchow and, later, section leader in the Institute of Animal Physiology of the same University under Salkowski. In 1906, he received the title of Professor; and he was invited, in 1913, to head the Division of Biochemistry of the newly established Kaiser Wilhelni Institut fur experimentelle Therapie, the Director of which was August von Wassermann. The Institute, which was opened by the Emperor, had its origin in the centenary celebrations of the University of Berlin in 1910, when A. von Harnack, a farseeing and highly influential man, stressed the need for supplementing university studies in Germany by means of richly endowed research institutes, each directed by an eminent man of science with complete freedom in his work and choice of staff. The originally small division grew with the increasing 1

2

OBITUARY

significance of the accomplishments of Neuberg’s laboratory, which, upon the death of Wassermann in 1925, became the Kaiser Wilhelm Institut fur Biochemie, with Neuberg as Director. He remained in this position until 1934. At this time, he was prevented from continuing his activities in this Institute. He emigrated in 1938 to Palestine, after having worked before that in the Netherlands, and he settled in the United States in 1940. His retirement took him out of broad and fruitful work which had spread the fame of his Institute over all the world. Although the scope of Neuberg’s work was immense, it is possible to recognize one broad line in his field of interest, a line which was characteristic for his creativity. Glyceraldehyde (glycerose) had already become his pet subject at the time when he studied with his teacher Wohl. Was it an accident that he did so much outstanding work in the field of the threecarbon compounds, and through investigation of their nature and properties made the first wedge into an understanding of the processes which occur during the dissimilation of o-glucose? In this (structural) frame of mind, he built in his methylglyoxal (pyruvaldehyde) diagram of fermentation which was discarded only after about 25 years, when the role of phosphoglyceric acid was discovered by Nilsson. In 1908, he had already become interested in the problem of heterolysis and, simultaneously with Freund and Kaminer in Vienna, he made the important discovery that cancer cells suspended in the serum of normal men undergo dissolution, whereas they remain unchanged in the serum of tumor-bearing humans. After Neubauer had suggested pyruvic acid as the transitory intermediate in yeast fermentation, Neuberg segregated the most important member of Buchner’s zymase complex and laid the cornerstone for studies of the phase sequence of carbohydrate degradation. I n fact, Neuberg made his greatest scientific contributions to our understanding of fermentation and glycolysis. The cleavage of pyruvic acid into acetylphosphate and formic acid is a recent example of a general reaction, first demonstrated by Neuberg, in which a-keto acids are split into a fatty acid and formic acid. He introduced the method for the trapping of transitory intermediaries, enabling him to interpret correctly some phases in the mechanism of alcoholic fermentation and of the so-called glycerol fermentation introduced by Connstein and Ludecke in 1915. From these studies emerged the first indications of the mechanism of the cleavage of the Harden-Young hexose diphosphate into two sub-units later identified as triose phosphates. Neuberg’s four forms of fermentation provided the first attempt a t an integrated picture of D-glucose breakdown in which the dynamics of fermentation was explained by the competition

C. NEUBERG

3

of hydrogen acceptors. The observation that substances extraneous to fermentation (caused by yeasts and other microorganisms) may act as hydrogen acceptors and that, thereby, a vast number of aliphatic, aromatic, and heterocyclic aldehydes may be reduced to the corresponding alcohols, led t o a broad application of the concept of phytochemical reduction. These experiments regained their momentous importance in our day, when the biological oxidation and reduction of steroids became practicable. The formation of mercaptan is doubly important because of its relation to phytochemical reduction processes. The reaction is analogous to the formation of ordinary alcohols from aldehydes, and it also indicates how the plant is capable of synthesizing intensely odorous substances from aldehydes and hydrogen sulfide in a simple manner. The isolation of ethanethiol was the first case in which this substance was unequivocally prepared by a fermentation process. The significance of the ability of enzymes, such as transketolase, to give rise to carbon-carbon linkages was foreshadowed in studies on acetoin fermentation and by discovery of the enzyme carboligase. As happens so very often, especially in evaluating matters of the Arts and Sciences, we are expected to reflect upon the lasting creation(s) of a man. I n the case of Neuberg, we know that he was a master of the scientific stage, and it will, therefore, be worth while to dwell upon the details of the mechanism of glycerol (glyceritol) fermentation. Ethyl alcohol and carbon dioxide do not entirely account for the carbon utilized during the dissimilation of D-glucose, for other metabolic products, such as glycerol, are also produced. Normally, this polyhydroxy alcohol is formed to the extent of 3 % , but Connstein and Ludecke observed that, by varying the experimental conditions, the yield of glycerol could be considerably increased. Thus, if the pH of the medium is increased by adding alkaline salts (such as ammonium carbonate, sodium bicarbonate, sodium acetate, or disodium phosphate), the final glycerol content rises from 3 to 9-16%. On the other hand, an increase in bacterial contamination is also obtained under these conditions, resulting in erratic yields. These difficulties were obviated by substituting sodium sulfite for the alkaline salts, since, in this case, not only were greater quantities of glycerol obtained (23-27 %), but the salt itself acted as an antiseptic. These workers utilized their findings in the commercial manufacture of glycerol. Investigations, especially by Neuberg and his associates, concerning the mechanism of this change have revealed that there are essentially three different mechanisms for explaining the increasing yields of glycerol, depending on whether sulfite salts, alkali, or neutral solutions are present. According t o Xeuberg’s classification, considering ordinary alcoholic fermentation as the first form, these are known as the second, third, and fourth forms of fermentation, respectively.

4

OBITUARY

When sodium suljite is present in the culture medium, the Gay-Lussac expression CeH12Oe + 2 CsHbOH

+ 2 COz

is no longer valid, since glycerol and acetaldehyde accumulate a t the expense of the ethanol. The acetaldehyde is not present as such, but is trapped as the bisulfite addition product. Further studies indicated that equimolar quantities of glycerol and acetaldehyde are formed during the fermentation period. During the course of a normal fermentation, D P N H {reduced codehydrogenase I ; formed during the oxidation of 3-0-phosphoglyceraldehyde (3-0-phosphoglycerose) to 3-0-phosphoglyceric acid (3-0-phosphoglyceronic acid)] is reoxidized when acetaldehyde is reduced to ethanol. However, in the presence of sodium suljite, the acetaldehyde can no longer serve as the hydrogen acceptor for DPNH, since it combines with the sulfite salt to yield an addition compound. Under these altered conditions, dihydroxyacetone phosphate (1 ,3-dihydroxy-2-propanone phosphate) can become a suitable substrate for DPNH, resulting in the formation of glycerol phosphate, which is then converted into glycerol. CHzOH

CH20H

I c=o

+ DPNH + H@

I

CH20POaHz Dihydroxyacet one phosphate

CHzOH

I

---f

CHOH

phosphatase

'

I CHoH

CHzOP03Hz

CHzOH

Glycerol phosphate

Glycerol

The reason more glycerol is not produced under standard fermentation conditions is that acetaldehyde has a much greater affinity for D P N H than has dihydroxyacetone phosphate. The over-all relationships may be expressed as follows. CsHizOe + CHzOHCHOHCHzOH

+ CH3CHO + COz

The total equation, including the sodium sulfite, then becomes :

+

CeHlzOe NazSOa

+ HzO +. CH20HCHOHCH:OH + CHaCH(0H)SOaNa + NaHC03 .

The more acetaldehyde is trapped by the sodium sulfite, the greater is the quantity of glycerol found, but the yeast can tolerate only a certain percentage of this salt before it exerts a toxic effect. Sodium sulfite can be replaced by the corresponding magnesium, zinc, or calcium salts without any noticeable difference being observed. It is reasonable to assume that other metabolically active substances such as D-glucose and pyruvic acid can also react with sodium sulfit,e.

5

C. NEUBERG

Investigations have substantiated this, hut, in the case of D-glucose, the addition compound is unstable in aqueous solution and dissociates into its original components; whereas, with pyruvate, the reaction product is easily fermented by yeast. Since the principle involved in increasing glycerol production is to prevent acetaldehyde from being reduced to ethanol, any fixing agent which will trap the aldehyde and, at the same time, not inhibit the in vivo enzymic reactions should lead to the same result. The following substances have been found to fulfil these requirements and can be used for increasing the yields of glycerol: dimedone (5,5-dimethyl-l,3-cyclohexanedione), thiosemicarbazide, charcoal, various hydrazides, and phenylhydrazine oxalste. If the pH of the culture medium is raised into the alkaline region, the course of the yeast fermentation is changed, for, under these conditions, ethanol, acet,ic acid, glycerol, and carbon dioxide are produced. The mechanism of this reaction has been studied in detail. Instead of being reduced to ethanol, the acetaldehyde undergoes a dismutation in the presence of an aldehyde mutase, to form alcohol and acetic acid. 2 CHICHO

+ Hn0

-+

CnHsOH

+ CHICO~H

The reactions that follow are analogous to those described for the second form of fermentation. That is, since the acetaldehyde is unavailable as an acceptor for DPNH, the latter can be used in reducing dihydroxyacetone phosphate to glycerol phosphate, which firially yields glycerol. The total equation can he expressed as follows. 2 C6Hi&e

+ HzO -+

2 COz

+ CH3C02H + CnHsOH + 2 CHnOHCHOHCHzOH

Occurrence of this type of fermentation is caused by the alkalinity of the medium, since various salts or hydroxides can be used with practically the same result. I t is interesting to note that a yeast, Zygosaccharomyces acidifaciens, has been observed to bring about this third form of fermentation as its normal glycolytic process. A fourth type of fermentation has been demonstrated t o occur under special experimental conditions, for, in this case, no formation of carbon dioxide or ethanol takes place, but, instead, equimolar amounts of pyruvic acid and glycerol are produced. C6H12O6 +

CHaCOCOzH

+ CHnOHCHOHCHnOH

Neuberg’s studies of the enzymic fermentation of lactic acid resulted in the introduction of its salts as brake fluids in German artillery pieces during World War I. During the last 30 years, Neuberg’s interest was attracted to the mech-

6

OBITUARY

anisms of solubilization (hydrotropy) of insoluble matter in Nature. He explored the effects of organic phosphates and polyphosphates arising in carbohydrate metabolism in the solubilization of insoluble salts, observations which up to now have not made their full impact on biological thought. This short outline of some of Neuberg’s main contributions to biochemistry can give but a meager impression of his over-all influence in the field. It does not give an account of his discovery of a host of enzymes, of his studies of the structure of natural products, or of his synthesis of phosphorylated intermediates of carbohydrate metabolism. His work was particularly characterized by a multitude of methodological and chemical observations, and many of the chemical tools of our laboratory were discovered and developed by Neuberg and his pupils. He contributed not only some of the concepts but also the techniques to his own work. The enormous amount of work accomplished by Neuberg and his numerous pupils has been set down in about 900 publications from his laboratory. The Kaiser Wilhelm Institute of Biochemistry in BerlinDahlem gave, under his leadership, opportunity and inspiration to generations of biochemists from Europe, Asia, the Western Hemisphere, and Australia : “Wer zahlt die Volker, nennt die Namen, Die gastlich hier zusammen kamen?” His influence stemmed not only from his original contributions but also from his editorship of the “Biochemische Zeitschrift” (which he founded in 1906, at the age of 28); from his six books, one of which, “Der Harn, sowie die ubrigen Ausscheidungen und Korperflussigkeiten von Mensch und Tier” (Berlin, J. Springer, 1911) found wide distribution; and also from his membership in a number of the highest scientific councils in Germany. After he came to this country, he continued to work, often under adverse conditions, and the uninterrupted flow of publications up to the last month of his life attested to a productivity and energy unaffected by age and change of environment. He was an unusually hard worker, expecting his associates to be the same. Besides, he was amazingly well versed in classical literature and history, he read Greek and Latin as well as Hebrew, and loved hiking in the Alps. He liked to follow the wise slogan for success in scientific work formulated by P. Ehrlich: “Geld, Geduld, Geschick und Gluck.” He was an excellent conversationalist with a marvelous sense of humor and, t o the joy of many, could tell innumerable jokes in a short time. But, above all, he loved Germany in her former greatness, he upheld her in defeat, and admired her nostalgically in her miraculous resurgence after the second world conflict.

C. NEUBERG

7

Neuberg early became a widower after his marriage to Helene Lewinski. He leaves two daughters, Mrs. Irene S. Forrest of Pembroke, Massachusetts, and Mrs. Marianne Lederer of Los Angeles, California, and three grandchildren. Neuberg was one of the most bemedalled scientists of his time. Moreover, besides being the first Carl Neuberg Medalist of the American Society of European Chemists, he was a foreign member of the Academies of Sciences of Copenhagen, Gottingen, Helsinki, Leningrad, Lisbon, Lund, Munich, and Uppsala. In 1954, he received the Great Cross of Merit from West Germany’s President Heuss. Between 1920 and 1934, he was several times proposed for the Nobel Prize. Neuberg was both a man of science and of art, keenly aware of the mysteries surrounding us. He, the scientist and the artist, constantly strove to reconcile and harmonize the new with the familiar, struggling to establish partial order in total chaos. Throughout his work and his life he helped himself, helped others, and helped all men.

F. F. NORD* * Grateful acknowledgment is made t o Dr. Irene S. Forrest, Dr. E. Schwenk, Dr. W. J . Schubert, and my wife for their kind assistance and rectifications.

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FORMATION AND CLEAVAGE OF THE OXYGEN RING IN SUGARS BY F. SHAFIZADEH Department of Chemistru, The Ohio State University, Columbus, Ohio I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Formation and Hydrolysis of Aldonolactones. , .................... 1. Formation of Aldonolactones.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Bromine-water Oxidation .................................... b. Other Oxidation Methods.. . . . . . . . . . . . . . . . . . c. Theoretical Considerations. . . . . . . . . . . . . . . . . . 2. Hydrolysis of Aldonolactones . . . . . . . . . . . . . . . . . . 111. Formation and Hydrolysis of the Aldosides . . . . . . . 1. The Factors Influencing the Itate of Hydrolysis a. Steric Strain and Constitution of the Sugar. . . . . . . . . . . . . . . b. Nature of the Glycosidic G r o u p . . . . . . . . . . . . . c. Substitution of the Glycosides . . . . . . . . . . . . . . 2. Reaction Mechanism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Nitrous Acid Deamination of Amino Sugars., . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cyclic Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Pyranose Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Furanose and Non-pyranose Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Acyclic Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Amino Aldonic Acids. . . . . . . . . . ...................... b. Amino Alditols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9 10 10 11

37 43 45 51 54 54

57

I. INTRODUCTION Modern concepts of orgnuic chemistry have provided interpretation and explanation for a variety of supposedly anomalous and unusual rcactions of cnrhohydrate compounds. These concepts have often been derived from :L study of relatively more simple substances in which there has been litt.le complication resulting froin interfering and conflicting factors. Consequently, direct applicat,ion of these concepts to the more complex carbohydrate compounds without due consideration of alternative possibilities may result in oversimplification and a false picture. Despite such inherent hazards, it is believed that a useful purpose can be served by the correlation and discussion of the carbohydrate reactions with direct reference to analogous properties of simpler organic compounds. For this objective, we can define the acyclic forms of the aldoses as polyhydroxyaldehydes, and their cyclic forms as polyhydroxy-cyclohemiacetals. In these compounds, besides the additive, inductive effect of the hydroxyl 9

10

F. SHAFIZADEH

groups on the reactive function, we should also consider the more complicated steric effects. A consideration of deoxy sugars often facilitates comparison between the fully hydroxylated carbohydrates and their nonhydroxylated aliphatic relatives by providing a link or intermediate between these two classes of compound. Since the hemiacetal linkage of the reducing sugars is extremely labile, the reactions involving the carbonyl function may proceed either through the acyclic or the cyclic form. In this respect, the course of some reactions, such as the bromine oxidation of the aldoses, has been shown to proceed through the cyclic form. On the other hand, that of other reactions, for instance, the formation and hydrolysis of methyl aldosides, still remains controversial and debatable. Another interesting aspect of these reactions is the higher reactivity of tho,se compounds having an exposed glycosidic group (usually the 0 anomer). In general, the stability of the ring structure and the reactivity of a cyclic sugar are materially affected by the steric strain of its molecule. On the other hand, the formation of cyclic compounds from those acyclic carbohydrate derivatives having a number of competing hydroxyl groups is likewise of considerable interest. In the reversible reactions of this nature, a n ultimate equilibrium is established which results in the predominant formation of the less strained, more stable ring-structures. In the irreversible reactions, such as the deamination of acyclic amino sugars with nitrous acid, the resulting cyclic products usually contain the strained, nearly planar, five-membered ring-structure. This discussion will deal with some aspects of the interconversion of the cyclic forms of a carbohydrate to t)heir acyclic forms and to each other. Since a topic of this nature cannot be well covered in a single Chapter, the discussions will be limited to the consideration of a cross-section of closely related reactions which include the formation and hydrolysis of aldonolactones and aldosides and the deamination of amino sugars. 11.

FORMATION AND

HYDROLYSIS O F ALDONOLACTONES

1. Formation

OJ

Aldonolactones

Oxidation of aldoses to the corresponding aldonic acids is a fundamental feature of carbohydrate chemistry which has found extensive and frequent application. Most of the reactions resulting in the oxidation of a- and 0-D-glucose have been well investigated and are now known to be stereospecific. They often proceed through the cyclic hemiacetal rather than through the acyclic, aldehyde form. In these reactions, the 0-D anomer, which in the more stable chair conformation ( C l ) l carries an equatorial hydroxyl group a t C1, is oxidized more rapidly than the a-D anomer in which the reactive hydroxyl group a t C1 is in a less exposed, axial position (1) R. E . Reeves, Advances in Carbohydrate Chem., 6 , 107 (1951).

FORMATION A N D CLEAVAGE O F OXYGEN RINGS I N SUGARS

11

(see page 17). The following is a brief discussion of certain aspects of these reactions and their theoretical interpretation. An interesting and elaborate article on the halogen oxidation of carbohydrates and the preparation of aldonic acids has appeared in an earlier volume of this series.2 a. Bromine-water Oxidation.-Oxidation of the aldoses to the corresponding aldonic acid with bromine-water is a classical reaction which dates back t o Hlasiwetz3 and Kiliani4 and has been basic to many of the subsequent developments in carbohydrate chemistry. The reaction proceeds very rapidly under mild conditions and is retarded by the resultant accumulation of hydrogen bromide.6 If it is allowed to continue, particularly under buffered conditions, it will result in the formation of a 5-keto acid from a hexose.6 (The ketoses are resistant to the action of this reagent.‘) Consequently, the reaction has been considered to be evidence for the aldehyde structure of glucose and other aldoses and, as pointed out by Isbell and Hudson,B its mechanism has been explained by the theory that, in aqueous solutions, the small amount of the acyclic aldehydo form (in equilibrium with the cyclic, hemiacetal isomers) reacts with the reagent to give the free acid; and thus the reaction proceeds through the aldehydo f o r r n ~lo , ~as~ shown by the following equation. HOCHz-(CHOH),-CHO

+ Br2 + HzO

-+

HOCH2-(CH0H),-C0~H

+ 2 HBr

Isbell and Hudson,8 however, observed that, on oxidation of a buffered solution of D-glucose with bromine, the optical rotation first increases to a point which approximately corresponds to the rotation of D-glucono-1,5lactone and then follows a course similar to that resulting from the hydrolysis of this lactone. Similar changes were also noted in the aqueous bromine oxidation of n-galactose, L-arabinose, and n-xylose, respectively, in slightly acidic acetate buffer. These observations led to the conclusion that, contrary to the then-prevailing concepts, the pyranose sugars are directly oxidized to 1,5-1actones. Further evidence in support of this theory has been provided by Isbell,” who utilized the fact that, in the (2) J. W. Green, Advances i n Carbohydrate Chem., 3, 129 (1948). (3) H. Hlasiwetz, A n n . , 119, 281 (1861). (4) H. Kiliani, A n n . , 206, 182 (1880); H . Kiliani and S. Kleemann, Ber., 17, 1296 (1884). ( 5 ) H. H. Bunzel and A. P. Mathews, J. A m . Chem. Soc., 31, 464 (1909). (6) J . P. H a r t and M. R. Everett, J . A m . Chem. Soc., 61, 1822 (1939). (7) H. Kiliani and C. Scheibler, Ber., 21, 3276 (1888). ( 8 ) H . S. Isbell and C. S. Hudson, J . Research Natl. Bur. Standards, 8 , 327 (1932). (9) E. F. Armstrong, “The Carbohydrntes and Glycosides,” Longmans, Green and Co., London, 1924, p. 67. (10) W. N . Haworth, “Constitution of Sugars,” Edward Arnold and Co., London, 1929. (11) H. S. Isbell, J . Research N a t l . Bur. Standards, 8,615 (1932).

12

F. SHAFIZADEH

presence of barium carbonate, any free acid formed will be converted to the barium salt, while the lactones will remain unaffected. Oxidation of the anomeric pairs of 01-1)- and P-r>-mannopyranose and a-L- and p-Lrhamnopyranose with aqueous bromine, saturated with carbon dioxide and containing barium carbonate (pH 5-6), produced only 1 or 2 % of the barium salt. The rest of the oxidation products were neutral compounds which, on standing in aqueous s o l u t h , developed acidity arid displayed a change in their optical rotation similar to those exhibited by l15-lactones. The percentage of the barium salt resulting from the oxidation of the disaccharides a-lactose and p-cellobiose was, however, as high as 25 %. Application of the above method to the anomeric 0-D-and a-D-glucopyranose indicated that, at 0", the P-D is oxidized 39 times faster than the a - anomer. ~ Oxidation of the former was 50% complete in about three minutes, whereas, under identical conditions, the latter requires approximately 130 minutes; and, in both cases, u-glucono-1 ,5-lactone was the predominant product. Under the above conditions, mutarotation of u-glucose is very slow, but the oxidation is conveniently fast. Consequently, the reaction of an equilibrated solution of D-glucose progresses very rapidly until the bulk of the p-D anomer present is consumed; oxidation of the remaining a-D form then continues a t a lower rate.'* Taking into consideration the amount of the 0-Dform produced during the couree of the reactioii by mutarotation of the a-D form, it was calculated that the p-D is in fact oxidized 53 times more rapidly than the a-u anomer. Plotting of the percentage of uiioxidized sugar in the equilibrated mixture against time gave a curve, the latter part of which was a straight line representing oxidation of the a-Dform. Extrapolation of this part of the curve t,o zero time indicat,ed the amount of the a-1) anomer in the original equilibrated solution. The values obtained in this may, 36% of a-D and 64% of p-D were in good agreement with those obtained by the optical-rotation method. The aqueous-bromine oxidation rate was determined for a number of other anomeric pairs of a l d o ~ c s l4' ~and ~ it was found that, in general, the p forms (see page 17) react faster. This phenomenon has also been observed for a-D-and p-D-galactopyranuronic acid ; oxidation of these compounds provides a mixture of optically active lactones of galactaric (mucic) acid. This constitutes interesting evidence for the direct oxidation of the cyclic modifications (pyranose and furanose forms), since the oxidation of (12) H. S. Isbell and W . W. Pigman, J . Research N a l l . B u r . Standards, 10, 337 (1933). (13) H. S. Isbell and W. W. Pigman, J . Research Natl. B u r . Standards, 18, 141 (1937). (14) H.S. Isbell, J . Research Natl. B u r . Standards, 18, 505 (1937).

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

13

the open-chain form would yield gnlactaric acid, a meso conipound which should give rise to racemic 1act0nes.l~Furthermore, it has been shown that oxidation of ~-D-mannopyranurono-6,3-lactone gives D-mannaric 1,4 :6,3dilactone.lB The compound D-mannose CaC12.4HzO has a furanose structure arid gives a mixture of 1)-mannono-1,4-lactone and n-mannono-1 ,5lactone. The latter lactone is the oxidation product of the pyranose modification derived from the structure change of the labile furanose form, and, after five minutes of oxidation, it constitutes 24 % of the mixture.” A series of experiments on the oxidation of a-D-glucopyranose with various amounts of bromine and barium bromide were interpreted as showing that the oxidizing agent is free bromine.z’K , l2 Furthermore, molecular chlorine is considered18 to be t>heoxidizing agent at pH 2.2 and a t pH 3. The rate of oxidation by halogens appcars to be affected by the nature of the neighboring group. 2-Deoxy-1)-“galactose” (-Zyzo-hexose) is oxidized, both with unbuffered brominelg and with alkaline iodine solution,2° more rapidly than is D-galactose. It is interesting t o note that, according to Lippich,21the reaction of reducing sugars with hydrogen cyanide proceeds through the aldehydo form, and the instantaneous uptake of the cyanide is greater for the mutarotatirig solution of a-D sugars. b. Other Oxidation Methods.-Oxidation of D-glucose with alkaline solutions of iodine in which the oxidizing agent is the hypoiodite is similar to bromine-water oxidation with respect to the reactivity of the anomeric forms. Under certain conditions (pH about 9.2), the p-D form is oxidized approximately 28 times faster than the CY-Danomer. However, the rate of mutarotation of the free sugars is greatly enhanced in alkaline solutions, and, beyond the p H range of 11.8, becomes much faster than oxidation, the result being that, under these circumstances, both forms are oxidized a t the identical rate.23 It was reported that, a t pH 10.15 t o 13.10, the configurationally related sugars D-glucose and D-xylose are oxidized a t an approximately equal rate. The similarly related sugars u-galactose and L-arabinose showed the same phenomenon, but u-mannose was less reactive; the ratio between these rates22was 1 :1.36: 0.24. (15) (16) (17) (18) (19) (20) (21) (22) (23)

H . 8 . Iabell and H. L. Frnsh, J . Research Natl. Bur. Standards, 31, 33 (1943). H . S.Isbell and H . I,. Frush, J . Research Natl. Bur. Standards, 37, 43 (1946). H . S . Isbell, J . A m . Chem. Soc., 6 6 , 2166 (1933). N . N . Lichtin arid M. H . Saxe, J . Am. Chem. SOC.,77, 1875 (1955). W . G. Overend, F. Shafiaadeh and M. Stacey, J. Chem. SOC.,2062 (1951). Idern., unpublished work. F. Lippich, Biochern. Z . , 248. 280 (1932). 0. G . Ingles and G. C. Israel, J . Chem. Soc., 810 (1948). K . D. Reeve, J. Chern. SOC.,172 (1951).

14

F. SHAFIZADEH

Kuhn and Wagner-JaureggZ4have measured the time (minutes) required by 5 g. of hexose (or the equivalent of other sugars) t o reduce a set quantity of buffered (pH 2.3), dilute permanganate solution. They obtained the following values for equilibrated solutions: maltose 9.0, fructose 9.5, arabinose 10, galactose 13.5, lactose 34.0, mannose 21.0, and glucose 30.5 The p-D sugars reacted faster than their CY-D anomers, with the ratio being 1.7 for glucose, galactose, and mannose, 1.25 for lactose, and 1.0 for fructose. Methyl P-D-glucopyranoside has been oxidized with a saturated solution of chlorine a t room temperature. The reaction is very slow and provides D-gluconic acid, which has been isolated after 14 and “2-keto-~gluconic acid,” which appears to be B secondary product.6 It has been shown that, under the employed conditions, the glycoside is not affected by the maximum amount of hydrogen chloride which can be produced by the reaction. Thus, oxidation cannot proceed through the acid hydrolysis of the glycoside (see page 26). This is of considerable interest, in view of the fact that the reaction is much slower with methyl a-D-glucopyranoside and that a similar difference in the reactivity of the CY-Dand p-D anomers has been noted for the methyl galactosides, mannosides, and xylosides.26 These compounds preferably assume the usual chair conformation ( C l , see page 17). Methyl a- and p-D-arabinopyranosides, which are more stable in the reverse chair conformation ( I C ) , exhibit the reverse order of anomeric reactivity.*’ Oxidation of aldoses by chlorites also results in the formation of the corresponding aldonic acids. The aldopentoses react faster than aldohexoses, and monosaccharides are oxidized more rapidly than are the disaccharides. The ketoses remain unaffected unless drastic conditions are employed. The principal reaction has been shown t o be as follows. RCHO

+ 3 HC10z

--t

RCOsH

+ 2 ClOz + HC1 + HzO

With chlorous acid as the main oxidizing agent, the reaction is slow in neutral solution and becomes faster under acidic conditions.28A solution containing equimolar amounts of sodium chlorite and acetic acid affords tt slightly faster rate of oxidation for a-D-glucopyranose than for the p-D anomer. Thus, the reaction appears to follow a different path from that of the bromine-water and the hypoiodite oxidations, and to be similar to that of cyanohydrin formation?l (24) (25) (26) (27) (28)

R. Kuhn and T. Wagner-Jauregg, Ber., 68, 1441 (1925). A. Dyfverman, B . Lindberg and D. Wood, Acta Chem. Scand., 6 , 253 (1951). B. Lindberg and D. Wood, Acta Chem. Scand., 6 , 791 (1952). R. Bentley, J . Am. Chem. SOC.,79, 1720 (1957). A . Jeanes and H. S. Isbell, J . Research Natl. Bur. Standards, 27, 125 (1941).

FORMATION AND CLEAVAGE OF OXYGEN RINGS IN SUGARS

15

u-Glucose is quantitatively oxidized by the enzyme glucose oxidase to D-gluconic acid. This enzyme was discovered in the cultures of certain molds29and was subsequently isolated as the antibiotic notatin from the It ~is a flavoprotein having an culture medium of Penicillium n o tc~ tu m.~ alloxazine-adenine dinucleotide3l as the prosthetic group. Unless the enzyme is accompanied by an additional component which is capable of catalyzing mutarotation, the catalytic oxidation by this enzyme is specific for the P-D a n ~ m e r After . ~ ~ correction for the mutarotation of the a-D anomer under the influence of the buffer solution employed, the ratio between the rate of reaction of the two forms a t 20" has been shown to be @:a = 156:l. The enzyme has no effect on the anomers of allose, gulose, idose, and talose. Msnnose, altrose, and galactose are oxidized only very slightly, with ratios of 0.98, 0.16, and 0.14, respectively, as compared to 100 for 0-D-glucopyranose. The reaction has been shown to proceed through the transfer of hydrogen from D-glucose to oxygen, which results in the formation of hydrogen peroxide and D-glucono-1,5-lactone, and this organic product is subsequently hydrolyzed to u-gluconic acid, as in the following equation.33 CeHizOe

+ + Hz0 0 2

-+

+ HzOz

C~Hiz07

From the livers of various animals has been isolated an enzyme named glucose dehydrogenase which, with di- or tri-phosphopyridine nucleotide its coenzyme, reversibly converts u-glucose to D-gluconolactone. It appears to be rather specific for p-D-glucose, although P-D-xylose can also be oxidized (at a lower 36 c. Theoretical Considerations.-The differences observed in the rate of bromine oxidation of their anomers led Isbell to believe that there is an important structural difference between the a- and 0-pyranoses, and that the pyranose ring is not coplanar, since in this conformation the a- and P-positions are symmetrically It was pointed out that the difference in the a and fi positions with respect to the carbon-oxygen ring would cause differences in the reactivity of the a and fi modifications, and that a comparison of the reaction rates for the LY and fi pyranoses should (29) D. Muller, Enzymologia, 10, 40 (1941). (30) C. E. Coulthard, R. Michaelis, W. F. Short, G . Sykes, G . E. H. Skrimshire, A. F. B . Standfast, J . H . Birkinshaw and H. Raistrick, Nature, 160, 634 (1942); Biochem. J . , 39, 24 (1945). (31) D. Keiliri and E. F. Hartree, Nature, 167, 801 (1946). (32) D. Keilin and E. F. Hartree, Biochem. J . , 60, 331 (1952). (33) R. Bentley and A . Neuberger, Biochem. J., 46, 584 (1949). (34) D. C . Harrison, Biochem. J.,26, 1016 (1931); 27, 382 (1933). (35) H. J. Strecker and S.Korkes, J. Biol. Chem., 196, 769 (1952). (36) H. S. Isbell, Ref. 14, p. 523.

16

F. SHAFIZADEH

provide ' information concerning the conformation of the pyranose ring. Considering free bromine to be the oxidizing agent?' 5 , 12, l8 it was suggested that the reaction may proceed to I1 through the intermediate addition products I and 111, shown for a- and p-D-glucopyranose, in which the relative proximity or non-proximity of the hydrogen atom a t C1 to the ring oxygen can facilitate or retard the reaction. 0

H

\

I1

/OH

3

CH,OH

CH,OH I

II

I

CHiOH

m At that time, unfortunately, very little was known about the conformations of the sugars, and the attempts to correlate conformation, reactivity, and optical rotattion were necessarily limited.13-14, 37 It has recently been suggested by Isbell that the addition of bromine to the pyranose anomers provides the intermediate bicyclic structures IV and VI. The difference in the rate of reaction is related to the free energy of these intermediates and t o the relative position of the two rings38-with the glycosidic group (37) H. S . Isbell, J. Research NatZ. Bur. Standards, 67, 171 (1956). (38) H. S . Isbell, Abslracts Papers A m . Chem. Soc., 132, 1 D (1957); oral presenta-

tion.

FORMATION AND CLEAVAGE OF OXYGEN RINGS I N SUGARS

17

being axial in IV and equatorial in VI. According t o the present concepts of the conforInat,iorial analysis of carbohydrates," 39 it appears that those aldoses having :m exposed equatorial glycosidic group are, in general, more reactive40 than those which have riot (see pages 28 and 48); this is in analogy with the properties of equutorially-substituted cyclohexane rings.41

Q

; :0 ;

H

/

Q+

H Br

or

'0

Q0Fr

H.G

+0

Br

IV

V

VI

The above category should includc the /3-r)-aldopyranoses (and their

/3-L enmtiomorphs) , which preferably assume the normal chair conformation (C"142; VII), as well as t,he a-D sugars (and their enantiomorphs), such as a-D-arabinose, which are more stable in the reverse chair conformation 1C (VIII).

H

o

b

HO

OH

H

H

VII p-D- Glucopyranose

N

H

H

O

H

OH

OH

H

HO

VIII a-u-Arabinopyranose

I t should he noted that the differences in the ratcs of reaction and in physicd properties~7~ 4 3 , 44 of the anomeric compounds originate in the steric position of thc glycosidic group arid this is a function of the configuration ax well as of the conformation of the molecule. Therefore, a categorical corrclatiori of these properties with the anomeric configuration of the compound (a-D,P-D; a-L, p-L) without considering the favored (39) J. A. Mills, Advances in Carbohydrate Clwm., 10, 1 (1955). (40) R. Bentley, Nature, 176,870 (1955). (41) D. H. R . Barton, Ezpericntia, 6, 316 (1950). (42) In the terminology suggested by Reeves (see Ref. l ) ,the mirror image of V I I , p-L-glucopyranose, has the f C conformation. The designation Cf (italics) is not t o be confiised with C1 (no italics) for carbon atom number one of a sugar chain. (43) D. H. Whiffen, Chem. & Znd. (London), 964 (1956). (44) S. A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, J . Chem. SOC., 171 (1954).

18

F. SHAFIZADEH

conformation of the molecule may be misleading and inaccurate. An interesting example is that of the supposedly anomalous properties of the L-arabinose anomers, which are more stable in the reverse chair conformations (IX and X). This was the cause of certain differences of opinion on TABLE I Rate of Oxidation of Aldoses with Buffered, Aqueous Bromine Solution at 0"

Aldose

kIkIa

Azial substifrenlsb,inf k e chair conformalson NormalC

p-D-Xylose anomer &D-Glucose a anomer D-glycero-8-L-gluco-Heptose a anomer 6-L-Arabinose anomer P-D-Galactose a anomer D-glycero-&D-galacto-Heptose (Y anomer n-glycero-&L-galacto-Heptose a anomer P-D-Lyxose a anomer j3-L-Rhamnose a anomer 8-D-Mannose a anomer D-glycero-j3-L-manno-Heptose a anomer j3-D-Gulose a anomer D-glycero-j%D-gulo-Heptose anomer j3-D-Talose 0 anomer D-g~~CeTO-~-D-idO-HeptOSe a anomer

52 2.8 39 1.0 53 O.Bd 3 52 50 1.3 58 1.3 56 1.1 14 4.9 24 2.8 24 1.6 56d 2.3 13 2.2 12 1.4d 26d 2.4 11 0.9

Reverse"

18.6 39 66 0.05 38 46 52 2.9 8.6 15 25 6 9

11 12

Rate constant for the oxidation of aldoses ( k ) divided by t h a t of a-D-glucopyranose (k' = 32 X 10P min-1). b The numbers represent the position on which the substituent is axial. c In the terminology suggested by Reeves, normal chair conformation is denoted by C1 for D sugars and 1C for their enantiomorphs. d T h e value has been calculated from the rate of oxidation of an equilibrated solution of the aldose. (1

FORMATION AND CLEAVAGE OF OXYGEN RINGS IN SUGARS

19

the assignment of the a- and p - s t r u ~ t u r e s ,until ~ ~ the currently accepted nomenclature was established by Jackson and Hudson46 through their periodate-oxidation studies.

IX aY-rA-Arabinopyranose

X 0-L-Arabinopyranose

In Table I, the differences in the rates of oxidation of anomeric aldoses13, 14 have been compared with the instability factors of the two possible chair conformations'; these include the axial substituents and the presence of the A2 arrangement (see page 28). The above data have been discussed by I ~ b e l l ~149~ 36-38 v and by Bentley.27940 I n general, the ratio of the rates for the anomeric pairs appears to be related to the conformational stability of the aldoses. The ratio is greater for the heptoses, in which the normal conformation (C1 for D sugars and 1C for L sugars) is stabilized37by a large equatorial residue (-CHOH-CH20H) a t C5, and smaller for the pentoses, in which the lack of a large residue facilitates the interconversion of the two chair forms. According to this theory, in the absence of barriers to the interconversion of the two possible chair conformations, both anomers can readily assume the more reactive conformation (in which the glycosidic hydroxyl group is equatorial) and, consequently, the large differences between their rates of reaction will disappear. Since, a t a low temperature (0"), the rate of mutarotation and interconversion of the chair conformations is diminished, the reaction in certain instances exhibits a stereospecificity comparable with the specificity of enzyme systems. Further elaboration, and consistent interpretation of the above data, is a t present hampered by R lack of understanding of the differences in the mutarotation of the free sugars. 2. Hydrolysis of Aldonolactones As noted before, the direct oxidation of aldoses results in the initial formation of the corresponding 1,5-1actones. Despite this, only 1,4sldonolactones were known until the year 1914. These l14-lactones were isolated after heating concentrated solutions of the aldonic acids. In 1914, Nef4' suggested that, simultaneously with the formation of 1,4-lactones1 (45) H . S. Isbell, J. Chem. Educ., 12, 96 (1935). (46) E. L. Jackson and C . S. Hudson, J. Am. Chenz. Soc., 69, 994 (1937). (47) J . U . Nef, Ann., 403, 204 (1914).

20

F. SHAFIZADEH

other lactones of a less stable character were also produced (in varying but much smaller quantity). By prolonged heating of the aldonic acids a t low temperatures or by desiccation under vacuum, he succeeded in isolating two new compounds (D-glucono-1,5-lactoiie and wmannono-1 ,5-lactone) which he termed “0-gluconolactone” and “/3-mannonolactone.” Nef justifiably regarded the isolatioii of these compounds as evidence for the possible presence of structural rather than coiifigurational differences between the “a- and 0-methylglucosides” (see page 2 5 ) . This inference was strongly resisted by Emil F i s ~ h e rand , ~ ~so the real significance of the new lactones was obscured. Here, it should be noted that, prior to the development of Hudson’s lactone in 1910 and the subsequent methylation studies of Sir Norman Haworth, Hirst, and associates,1° the assignment of a five-membered ring structure to the lactones as well as to the aforementioned “a- and 0-methylglucosides” was based on considerations arising from the Baeyer strain theory.60According to this theory, it was assumed that all rings must he plaiiar. Therefore, it was considered that, by analogy with cyclopentane, a five-niembered ring would involve the least amount of strain in the sugar molecule and would thus be more likely to be formed.61Conclusive chemical proof for the ring structure of the lactones as well as for those of the glycosides was derived from the classical methylation studies of Haworth and his associates. These investigations demonstrated that an aldono-1 ,blactone is much more resistant toward hydrolysis (to the aldonic acid) than is its corresponding six-membered ring isomer. This difference in stability was used as a criterion for the assignment of the respective ring structure to the lactones.lOz62-67 A brief summary of the data obtained by the above authorss6 is given in Table 11. These data indicate that the rate of hydrolysis of the lactones is also, to a considerable extent, affected by the stereoisomerism of the molecule. For instance, the lactones of D-mannonic acid are more stable than those of D-gluconic acid, from which they only differ in the steric position of the hydroxyl group a t C2. A mixture of D-glucono-l,4-lactone and D-mannono1,4-lactone can be fractionated by the absorption, on an anion-exchange (48) E. Fischer, Ber., 47, 1980 (1914). (49) C. S. Hudson, J . A m . Chem. Soc., 32, 338 (1910). (50) A. Baeyer, Bet-., 18, 2269, 2277 (1885). (51) W. Charlton, W. N. Haworth and S. Peat, J. Chem. Soc., 89 (1926). (52) W. N. Haworth, Nature, 116, 430 (1925). (53) W. N. Haworth and G. C. Westgarth, J . Chem. Soc., 880 (1926). (54) W. N. Haworth and V . S. Nicholson, J. Chem. Soc., 1899 (1926). (55) H. D . K. Drew, E. H. Goodyear and W. N . Haworth, J. Chem. SOC.,1237 (1927). (56) W. N. Haworth and C. R. Porter, J . Chem. SOC.,611 (1928). (57) W. N . Haworth, IIeZv. Chim. Acta, 11, 534 (1928).

21

FORMATION AND CLEAVAGE OF OXYGEN RINGS IN SUGARS

resin, of the free acid produced by aqueous hydrolysis. Under suitable conditions, the former lactone is hydrolyzed (and absorbed) to the extent of 94 %, as compared with only 6.5 % for the latter.G8 Haworth and his associates were mainly concerned with methylated lactones, derived from the oxidation of methylated sugars or hy the direct methylation of the lactones, and thus there was seldom any complication resulting from the interconversion of the two lactone forms. Simultaneously, Levene arid S i m m investigated ~~~ the lactonization of 0.25 molar solutions of D-gluconic acid, D-galactonic acid, D-niannonic acid, and D-glycero-Dgulo-heptonic acid a t 25'. They concluded that each aldonic acid forms two lactones-a 1,5-1actone reaching an equilibrium value of 20 t o 30 % in a few hours, and a 1,4-lactone attaining a higher equilibrium value of 75 to T A B L EI1 Rate of Hydrolysis and of Equilibration of Methylated Aldonolactones Time in hr. for 25% hydrolysis

Equilibrium of I .4-lacfones

Equilibrium of I I 5-lacloncs

A ldonolatlones rime in hl.

Tetramethyl ether of D-Gluconolactone D-Galactonolactone D-Mannonolactone Trimethyl ether of D-Xylonolactone D-Arabinonolactone

%.

%

4 crd Laclone

Time in hr.

___

~

520

%

Laclone

89

5 18 140

95 5 98.5 1.5 64 36

69 55

70 4

65 35 99.5 0 . 5

75 600 not reached

-

_

-

>900

11

430 60

>500 >500

31 45

87

%

Acid

_____

13

80% after several hundred hours. The initial speed of the former reaction is 8 times that of the latter, and the various aldonic acids behave in a similar manner. The latter generalization was disputed by Haworth and associates,66 who showed that the rates of hydrolysis and formation of the lactones are considerably aff ected by the configuration of the monosaccharide. The above studies were based on the observed changes in optical rotation of the reaction mixture in a relatively concentrated solution. Isbell and associates adopted an alternative approach, based on the polarographic investigation of 0.01 molar solutions of the lactones in the presence of an inert salt.60Their detailed investigation, although complicated by certain (58) J. V . Karabinos, Euclides (Madrid), 14, 263 (1954); Chent. Abstracts, 49, 4533 (1955). (59) P . A. Levene and H. S. Simms, J. Biol. Chein., 66, 31 (1925); 68, 737 (1926). (60) H. Matheson, H. S. Isbell and E. R . Smith, J . Research Null. Bur. Standards, 28, 95 (1942).

22

F. SHAFIZADEH

limitations of the method employed, revealed that the aldono-1 ,5-lactones are reduced a t a potential of -2.2 to -2.3 volts, whereas the reduction potential of the aldono-1 ,4-lactones is usually somewhat higher and varies, according to their configuration, between -2.2 to - 2.7 volts. Furthermore, under the experimental conditions utilized, a given quantity of an aldono1,5-1actone is completely hydrolyzed to the acid within a period of several hours; in contrast, the hydrolysis of aldono-1,4-lactones is very slow in gradually approaching a state of equilibrium. Besides the size of the ring and the configuration of aldonolactones, the nature of the subslituents in the ring can also affect the rate of hydrolysis. The effect thus exerted by the presence of a methylene (deoxy) moiety (-CH-) in the ring is comparable to that resulting from a variation in the ring size. The abnormal stability, in aqueous solutions, of 2-deoxy-~-glucoheptonolactone (possibly the 1,5-lactone) and its fully acetylated derivative led Wolfrom and coworkers to predict that the behavior of 2-deoxyaldonic acids might be found to differ markedly from those of the normal sugars.6* Subsequent investigations have shown that 2-deoxyaldono-l , 4-lactones are highly stable, and in aqueous solutions remain unchanged for several days. Related compounds investigated include: 2-deoxy-di-O-methyl-~-erythropentono-1 ,4-lact0ne,~~ 2-deoxy-~-lyxo-hexono-l, 4-lactone and its trimethyl ether,Baand 2-deoxy-~-arabino-hexono-l, 4-lactone and its trimethyl An enhanced stability is also shown by 2-deoxy-di-O-methyl-~-erythropentono-1 ,5-lact0ne,~~ 2-deoxy-tri-O-methyl-~-lyxo-hexono1,5-la~tone,~~ - 1 , 5- l a ~ t o n e which , ~ ~ are hyand 2-deoxy-tri-O-methyl-~-arabino-hexono drolyzed a t a much lower rate than the corresponding methylated “normal” (non-deoxy) aldono- 1,5-1actones. The “saccharinic acids,” a group of 3-deoxy aldonic acids, 2-C-methyl aldonic acids, and 3-deoxy-2C-(hydroxymethyl) aldonic acids, afford remarkably stable 1,4-1actones and are generally isolated as such.66 Schnelle and Tollensaa have noted that “a-D-glucosaccharinolactone,” (2-C-methyl-n-ribo(?)-pentonolactone) undergoes very little hydrolysis during the course of 11 days. The properties of deoxy and C-methyl aldonolactones thus conform with the observed resistance of normal aliphatic esters and lactones toward uncatalyzed hydrolysis. It should be noted that the introduction of alkyl substituents in the closely related 1 ,li-butyrolactone and 1,5-valerolactone (61) M. I,. Wolfrom, S. W . Waisbrot and R. L. Brown, J . A m . Chem. Soc., 64, 1701 (1942). (62) R. E. Deriaz, W. G. Overend, M. Stacey and L . F. Wiggins, J . Chem. SOC., 2836 (1949). (63) W . G . Overend, F. Shafizadeh and M. Stacey, J . Chem. Soc., 671 (1950). (64) I . W . Hughes, W. G. Overend and M. Stacey, J . Chem. Soc., 2846 (1949). (65) J . C. Sowden, Advances in Carbohydrate Chem., 12, 35 (1957). (66) W. Schnelle and B. Tollens, A n n . , 271.61 (1892).

FORMATION AND CLEAVAGE O F OXYGEN RINGS IN SUGARS

23

decidedly favors the formation and stability of the lactone 1ing.~7The rates of hydrolysis and formation of these lactones in N nitric acid, as well as the composition of the equilibrated solutions, also confirm the conclusion that, in general, the five-membered ring lactones are more stable than the sixmembered ring lac tone^.^^^ 6E This is in contrast to the higher stability of the “normal” six-membered rings (those which have no carbonyl or other trigonal atoms) as compared with that of the corresponding “normal” fivemembered rings.6gCertain properties of five-membered and six-membered rings have been reviewed and rationalized by Brown and coworker^.^^ According to these authors, in the idealized, planar-ring structure of cyclepentane (XI), there is a substantial strain resulting from ten bond-oppositions; introduction of a carbonyl group, which assumes a staggered conformation (XII) with respect to the neighboring bonds, stabilizes the ring by

XI (10-bond opposition; 10 Kcal. strain)

XI1 (6-bond opposition; 6 Kcal. strain)

diminishing the number of bond oppositions to six. Conversely, in the cyclohexane ring, which normally assumes a staggered conformation free of bond opposition (XIII), a carbonyl group introduces two bond-oppositions and a certain amount of strain, as shown in XIV. These considerations also

XI11

XIV

apply t o the lactones, since, in these compounds, the two unshared electrons of the ring oxygen produce a conformational effect similar to that of the carbon-hydrogen b ~ n d s . To ~ Qexplain why a l14-iactonewith six bond(67) W. Hiickel, “Theoretische Grundlagen der organischen Chemie,” Akademische Verlagsgesellschaft M. B . H., Leipaig, 2nd edition, 1935, Vol. 2, p. 260. (68) C. W. Matusaak and H. Shechter, Abstracts Papers Am. Chem. SOC.,132, 12P (1957). (69) H. C . Brown, J . H. Brewster and H. Shechter, J . Am. Chem. Soc., 7 6 , 467 (1954).

24

F. SHAFIZADEH

oppositions is more stable than the corresponding 1,5-1actone with two bond-oppositions, it may be assumed that the former compound resists the formation of cyclic, intermediate products. Unfortunately, very little is known about the mechanism and kinetics of the hydrolysis of sugar lactones, except for the fact that the reaction proceeds with the retention of configuration. I n the unhydroxylated aliphatic series, the hydrolysis of 1,blactones is catalyzed by acid and by alkali, but there is no detectable rcaction with water alone.’O In both cases, the reaction resembles that of simple esters and proceeds with acyl-oxygen cleavage. The following mechanism has been ~uggested7~ for the acid-catalyzed reaction.

11

H20

The highly strained 1,3-1actones give, in addition to the usual acid- and base-catalyzed hydrolysis typical of ordinary esters, a n added reaction with water. This reaction proceeds through alkyl-oxygen scission and is accompanied by inversion as shown below. a3

H HzO

OHz

+ CH3--b--CHz I

I

0-c=o

-+

I CH3--C--CH2-cOOe I H

OH -+

I I

-CHa-c--CHr--CO2H H

111. FORMATION AND HYDROLYSIS OF THE ALDOSIDES The reversible reaction responsible for the formation and hydrolysis of the glycosides has been a subject of lasting interest to carbohydrate chemists, in part because of its relation to the structure and fundamental prop(70) A. A. Frost and R. G. Pearson, “Kinetics and Mechanism,” John Wiley and Sons, Inc., New York, N . Y., 1953,p. 265.

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

25

erties of the glycosides and partly for the possibility of its correlation with the corresponding enzymic reactions. When methyl p-D-ghcopyranoside (“P-methylglucoside”) was first prepared by Alberda van Ekenstein, in 1894, the rate of the acidic hydrolysis of the new compound was compared with that of the already known isomer, “a-niethylglucoside,” obtained by the reaction of D-glucose with methanol in the presence of hydrogen chloride,?1-74to emphasize the difference between the two compounds. Twenty years later, Emil Fischer isolated a third isomer from the same reaction.48 This compound, now know to be methyl a ,p-wglucofuranoside, could be hydrolyzed very readily arid was provisionally designated a t the time as “y-methylglucoside.” The advancing of what proved to be controversial formulas for these isomers stimulated an extensive study of the constitutions and cyclic structures of these carbohydrate derivatives (see page 20). During the course of these investigations, Haworth and Hirst utilized the differences between the rate of acidic hydrolysis of furanosides and pyranosides as supplementary proof for their co n ~ lu s io n sMore .~ ~ recently, much attention has been focused on this subject as a means of correlating the structure and conformation of the cyclic compounds with their chemical reactivity. Apart from the steric strain imposed by the respective ring-structure, the differences in the rates of acidic hydrolysis of glycosides can be related to such factors as the polar effect of the substituents (either in the aglycon or in the sugar moiety) and the shielding effect of the -NH3’ group in amino sugars. The following is an elaboration of these contentions; the experimental details for the isolation of the glycosides have been discussed in a previous volume of this series76and need not be repeated here. 1. The Factors InJluencing the Rate of Hydrolysis of the Aldosides

a. Steric Strain and Constitution of the Sugar.-The sources of steric strain and the differences in the free-energy content of the common five-, six-, and seven-membered rings have been discussed and reviewed by a number of authors.’, 3 g , 6 9 77-79 The five- and seven-membered rings are strained be(71) W. Alberda van Ekenstein, Rec. Irav. chim., 13, 183 (1894). (72) E. Fischer, Rer., 26, 2400 (1893). (73) E. Fischer, Ber., 28, 1145 (1895). (74) W. Koenigs and E. Knorr, B e r . , 34, 957 (1901). (75) W. N. Haworth and E. L. Hirst, J . Chem. Soc., 2615 (1930). (76) J. Conchie, G. A. Levvy and C. A. Marsh, Advances i n Carbohydrate Chem., 12, 157 (1957). (77) W. G. Dauben and K . S. Pitzer, in “Steric Effects in Organic Chemistry,” M. S. Newman, ed., John Wiley and Sons, Inc., New York, N. Y., 1956, p. 1. (78) H. C. Brown, J . Chem. SOC.,1248 (1956); Abstracts Papers A m . Chem. S O C . , 132, 3D (1957). (79) W. Klyne, in “Progress in Stereochemistry,” W. Klyne, ed., Butterworths Scientific Publications, London, 1954, Vol. 1, p. 72.

26

F. SHAFIZhDEH

cause of (a) the distortion of the tetrahedral valency angle or (b) the presence of eclipsed bonds; whereas the strain in the well-staggered, chair conformation of six-membered rings originates from the non-bonded interactions of the axial groups. In general, it is apparent that the five- and seven-membered ring compounds are more strained and have a higher content of free energy than the corresponding six-membered ring derivatives; and, in the favored chair conformation of the six-membered ring compounds, the strain and free-energy content increase with the number of axial groups. The available data on the rates of hydrolysis of the aldosides indicate a direct relationship between the rate of acidic hydrolysis and the strain (or free energy) associated with the molecule. A good example of this relationship is provided by the work of Micheel and SuckfiiPO which demonstrates that, with 0.01 N hydrochloric acid a t 95", the rate of hydrolysis of methyl a-D-galactoseptanoside (XV; lc = 2 X min-') (which is like that of the furanosides; XVII) is about 100 times greater than that of methyl a-Dgalactopyranoside (XVI; lc = 2.3 X min-l). The septanosides are not generally available and have not been well studied; however, extensive in-

Ir-

HCOMe

HCOMe

HCOH

HCOH

I I HOCH I HOCH I HCOH I HzCO-

xv

I

I

HOCH

I I HCO-I

HOCH

HzCOH XVI

1 HCOMe I I HOCH I -0CH I HCOH I HCOH

HzCOH XVII

vestigations by Haworth and ass0ciates7~have shown that the aldofuranosides are, in general, hydrolyzed much faster than the corresponding pyranosides. The results of these investigations (see Table 111) indicate that the same relationship also holds for the methylated glycosides, although the latter compounds appear to be generally more stable. I n the aldopyranoside series, extensive data are available on the rates of hydrolysis of a large variety of methyl glycosides. The systematic work on this subject was initiated by E. F. Armstrong during the course of his studies on the parallel biological effect of the glycoside-splitting enzymes.81 He compared the rates of hydrolysis of anomeric pairs of methyl D-glucopyranosides (XVIII and XIX) and methyl D-galactopyranosides (XX and (80) F. Micheel and F. Suckfull, Ber., 66, 1957 (1933). (81) E.F. Armstrong, Proc. Roy. SOC.(London), 74, 188 (1904).

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

27

TABLE111 Hydrolysis of Aldopyranosides and Aldofuranosides with 0.01 N Hydrochloric Acid at 95-100" k X

A ldoside

Methyl a-a-glucopyranouide tetramethyl ether Methyl j9-D-glucopyranoside tetramethyl ether Methyl a-D-mannopyranoside tetramethyl ether Methyl a-D-galactopyranoside tetramethyl ether Ethyl 8-D-glucofuranoside tetramethyl ether Methyl u-D-mannofuranoside tetramethyl ether

~ 0 % ~

H

OMe

H

H

XIX Methyl 8-D-glucopyranoside

Hm

HO

xx

m

0Me

XVIII Methyl a-D-glucopyranoside

H

2.5 0.4 3.0 1.0 1.0 0.4 2.3 0.4 530 140 150 25

H

HO

m HHO H

104(~&-l)

HO

HO

OMe

H

OMe

H

XXI Methyl 8-D-galactopyranoside

Methyl a-u-galactopyranoside OH

-O@OH - C3

H

XXII

H

XXI). The results indicated a considerable variation arising from differences in the configuration a t C4 (as well as of those of the glycosidic centers). More extensive investigations by Riiber and S@rensena2 and by Isbell (82) C. N. Itiiber and N . A. S@rensen,Kgl. Norske Videnskab. Selskabs Skrifter,

No. 1, 1 (1938); Chem. Abstracts, 83, 4962 (1939).

28

F. SHAFIZADEH

and FrushS3 confirmed the existence of differences between the rates of hydrolysis of the various methyl aldosides. Furthermore it was shown that ~ a-L glycosides are not necessarily more stable than their p-D all the a - or and p-L anomers. Tables IV and V provide a comparison between the rates of hydrolysis of the above methyl glycopyranosides and the degree of instability or strain in the chair conformations of their pyranose rings.84Using the Reeves method,' the instability factors are denoted by the position of the axial substituents and by that arrangement in which an axial hydroxyl group on C2 bisects the oxygen valences of C1 (XXII),S6denoted by A2. These data extend to the pyranoside series the relationship between steric strain and reactivity, and indicate that the conformational instability and non-bonded interaction of the axial substituents in the favored chair conformation result in a higher rate of hydrolysis. Another aspect of these theoretical considerations is that the methyl 0-D and 0-Laldopyranosides are only hydrolyzed faster than their corresponding a anomer when the normal chair conformation4* is favored, and thus, when the glycosidic group of the p anomer assumes an exposed, equatorial position (see page 17). It must be noted that the activation energies of the glycosides may differ, even for the closely related compounds. Consequently, the ratio of the rates provides only a qualitative comparison, and values obtained at one temperaturc inay not hold a t other temperat~res.~~*87 For instance, the ratio between the rates of hydrolysis of methyl a- and (3-D-glucopyranosides has been variously quoted as 3 down to 1.79, partly for this reason.87 However, these changes are not expected to provide exceptions to the general rules, within the experimental conditions, particularly with regard to the large observed differences between the rates of hydrolysis of aldofuranosides and aldopyranosides.86 However, these generalizations are based on the data obtained from the aldoside series and cannot be directly applied to the ketosides. For the fructosides, the difference between the rate of hydrolysis of the furanosides and pyranosides is relatively small, and both ring isomers exhibit only a slight resistance to hydrolytic cleavage of the glycosidic group.88, 89 In the bicyclic compounds, the strains in the pyranose and furanose (83) H. S. Isbell and H. L. Frush, J . Research Natl. Bur. Standards, 24,125 (1940). (84) F. Shafizadeh and A. Thompson, J . Org. Chem., 21, 1059 (1956). (85) The relative positions of C1 and C2 are shown according t o M. S. Newman, J . Chem. E ~ u c .32, , 344 (1955). (86) L. J . Heidt and C. B. Purves, J . Am. Chem. SOC.,66, 1385 (1944). (87) E. A. Moelwyn-Hughes, Trans. Faraday SOC.,26, 503 (1929). (88) C . B. Purves and C. S. Hudson, J . A m . Chem. Soc., 69, 1170 (1937). (89) C. B. Purves, J . Am. Chem. SOC.,66, 1969 (1934).

FORMATION AND CLEAVAGE OF OXYGEN RINGS IN SUGARS

29

TABLE IV Hydrolpis82*8 3 of Methyl Hexo- and Pento-pyranosides with 0.6 N Hydrochloric Acid at iVQ Aldose

Axial subslilucnls in the chair conformationb

klk'

NormalC

a-D-Glucose p anomer a-D-Mannose p anomer a-D-Galac tose p anomer a-D-Gulose p anomer ru-D-XylOSe p anomer a-L-Arabinoae p anomer a-D-Lyxose p anomer

1.0 1.9 2. 4 5. 7 5.2 9.3 58.1 19.0 4.5 9.0 13.1 9. 0 14.5 46. Id

RcverscO

1

A2,3,4,5 1,293,495 3,4,5 1,3,4,5 A2,3,5 1,2,3,5 A2,5 1,2,5 A2,3,4 1,2,3,4 4 1,4 3,4 1,314 Ratio of the rate constant ( k ) for the hydrolysis of the methyl aldopyranoside to that of methyl a-D-glucopyranoside (k' = 1.98 X lo-' min-I). The numbers represent the position OII which the substituent is axial. In the terminology suggested by Reeves, the normal chair conformat,ion is denoted by CZ for D sugars and 1C for L sugars. d Indirectly obtained value.

TABLE V Hydr0lysis8~of Methyl Heptopyranosides with 0.06 N Hydrochloric Acid at 98" Heplose

kfk"a

-

Axial substikcnls in lhe chair conformolionb

NormalC

ReverseC

D-glycero-a-L-gluco-Heptose 0.24 1 A2,3,4,5 ~-glycero-a-~-~nar~no-Heptose 0.55 1,2 3,415 p anomer 1.25 A2 1,3,4,5 D-glycero-a-D-gulo-Heptose 7.0 1,3,4 A2,5 p anomer 3. 2 3,4 1,2,5 a Ratio of the rate constant ( k ) for the hydrolysis of the methyl heptopyranoside to t h a t of methyl a-D-mannopyranoside (k" = 6.9 X lo-' min-') under the above reaction conditions. b The numbers represent the position on which the substituent is axial. c In the terminology suggested by Reeves, the normal chair conformation is denoted by C1 for D sugars and 1C for L sugars.

rings are strongly modified by the steric requirements of the second ring, so that a situation may arise in which a furanose structure is very definitely more stable arid less strained than the corresponding pyranose isomer. A well established instance is the behavior of the 3,6-anhydrohexosides, which is discussed later (see page 39).

30

F. SHAFIZADEH

b. Nature of the Glycosidic Group.-The knowledge and conclusions derived from the well investigated series of methyl glycosides are not necessarily applicable to glycosides having a different aglycon. Variations in the activation energy and the rate of hydrolysis of anomeric pairs of methyl, benzyl, and phenyl D-glucopyranosides have been investigated by Heidt and Purves,86 who have shown that the activation energy decreases] and the rate of hydrolysis increases, in the above order. Many authors have been interested in the acid hydrolysis of the naturally occurring glycosides and oligosaccharides, mainly for comparison with the parallel enzymic reactions. The literature on this subject dates back to the early work of von Sigmondgoand Armstrong,81 which culminated in the more comprehensive investigations of Moelwyn-H~ghes.~7* 9 l A summary of the results obtained by the latter author is given in Table VI. TABLE VI Hydro1ysis of Natural G1ycosides (including Disaccharides) at 60" Glycoside

Methyl a-D-glucopyranoside tetramethyl ether Methyl 8-D-glucopyranoside Mandelonitrile 8-D-glucopyranoside a-Hydroxy-o-tolyl 8-D-glucopyranoside (salicin) Hydroquinone 8-D-glucopyranoside (arbutin) Phloretin 8-D-glucopyranoside (phloridain) a-n-GlucopyranosyI a-o-glucopyranoside (a,a-trehalose) 8-D-Fructofuranosyl a-D-glucopyranoside (sucrose) 3-O-a-~-Glucopyranosyl-~-fructose (turanose) 4-O-a-~-Glucopyranosyl-~-g~ucopyranose (maltose) 4-O-p-~-G~ucopyranosyl-~-g~ucopyranose (cellobiose) 4-O-~-~-Galactopyranosyl-~-glucopyranose (lactose) 6-~-p-~-Galactopyranosyl-~-glucose (melibiose) 6-0-p-~-G~ucopyranosyl-~-glucose (gentiobiose)

k/aH@" X 10%

1.46 4.09 3.86 1.07 1.80 43.4 116 0.864 14600 11.9 16.8 5.89 16.6 15.5 1.24

38,190 19,840 33,730 34,040 31,630 30,760 22,920 40,180 25,830 32,450 30 970 30,710 26,900 38,590 33,390

a k / a H e represents the rate constant in sec-l a t unit activity of the hydrogen ions in solution. b Calories per mole.

These and other investigations indicate that there is no simple correlation yet evident between the rates of acidic and enzymic h y d r o l y ~ i s . ~ ~ - 9 ~ It has been noted that methyl 6-D-glucopyranoside (XIX) is hydrolyzed (90) A. von Sigmond, 2. physik. Chem. (Leipzig), 27, 385 (1898). (91) E.A. Moelwyn-Hughes, Trans. Faraday SOC.,24, 309 (1928);26, 81 (1929). (92) R. Kuhn and H. Sobotka, 2. physik. Chem. (Leipaig), 109, 65 (1924). (93) B.Helferich, H. E. Scheiber, R. Streeck and V. Vorsate, A n n . , 618,211 (1935). (94) W.W.Pigmttn, J . Research Null. Bur. Standards, 26. 197 (1941).

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

31

faster than methyl a-D-glucopyranoside (XVIII); this is presumably because, in thc favored conformation, the aglycon of the former compound assumes the more exposed equatorial position. The above investigations indicate that this logic does not apply to phenyl p-D-ghcopyranoside and cellobiose, which are more stable than phenyl a-D-glucopyranosidess~96 and maltose.87 For the phenyl glycosides, this phenomenon has been attributed, in part, to the nature of the phenolic bond.g6However, it seems more reasonable to assume that the relative instability of these a-Danomers may result from the presence of large, axial substituents (phenyl and glycosyl rings) in the otherwise quite stable C l conformation. A further kind of modification that may be considered is the replacement of the glycosidic oxygen atom by other elements, notably sulfur and nitrogen. A variety of synthetic 1-deoxy-1-thioglycosideshave been prepared and have been shown to be extremely stable toward acid hydrolysis.S7-100 Phenyl 1-deoxy-1-thio-0-D-glucopyranoside and phenyl l-deoxy-l-thiop-lactopyranoside were prepared by Fischer and Delbriick in 1909 through the reaction of poly-0-acetylglycosyl bromides with benzenethiol in the presence of sodium h y d r o ~ i d e .This ~ ~ reaction was later employed by Schneider and associates for the synthesis of methyl, ethyl, and benzyl l-deoxy-l-thio-~-~-glucopyranosides~~ and by Purves for the synthesis of phenyl l-deoxyl-l-thio-~-cellobioside.gg The deoxythioglycosides prepared in this manner are very stable under acid conditions and the hydrolysis of the disaccharide derivatives results in the cleavage of only the disaccharide linkage and the liberation of phenyl 1-deoxy-1-thio-P-D-glucopyranoside. 9s A series of isomeric methyl, ethyl,lol propyl, and benzylgsdeoxythioglucosides was also prepared by Schneider and associates, through the treatment of u-glucose dialkyl dithioacetals with mercuric chloride. These rompounds were found to be much more labile than the previous products, synthesized from poly-0-acetylglucosyl bromides. Despite that observation, they were (erroneously) designated as the a-D anomers. Further investigations by Green and Pacsu102indicated that the new isomers are in fact deoxythioglucofuranosides which, under acidic conditions, are (95) C. A. Bunton, T. A. Lewis, D. R. Llewellyn and C. A. Vernon, J . Chem. SOC., 4419 (1955). (96) J . T. Edward, Chem. & Znd. (London), 1102 (1955). (97) E. Fischer and K. Delbruck, Ber., 42,1476 (1909). (98) W. Schneider, J. Sepp and 0. Stiehler, Ber., 61, 220 (1918). (99) C. B. Piirves, J . A m . Chem. Soc., 61, 3627 (1929). (100) E. Pacsu and E. J. Wilson, Jr., J . A m . Chem. SOC.,61, 1450, 1930 (1939). (101) W. Schneider and J. Sepp, Ber., 49,2054 (1916). (102) J. W. Green and E. Pacsu, J . A m . Chem. Soc., 69, 1205 (1937).

32

F. SHAFIZADEH

partly hydrolyzed and partly converted to the stable pyranoside derivatives.100 Thus, it was shown that the hydrolysis of ethyl l-deoxy-l-thioa-D-glucofuranoside with 0.01 N hydrochloric acid a t 100” gives a 9.5% yield of D-glucose a t the point when the change in optical rotation indicates an 85 % completion of hydrolysis. Other products formed are ethyl l-deoxyl-thio-cr-D-glucopyranosideand its p-D anomer, as well as a small proportion of the less stable ethyl l-deoxy-l-thio-/3-n-glucofuranoside. A general comparison between the rates of hydrolysis of the “O-glycosides” and ‘(N-glycosides” is complicated by the tendencies of some of the latter compounds to undergo rearrangement. Stacey, Overend, and associates have shown that N-phenylglycosylamines (“anilides”) are easily hydrolyzed by N sulfuric acid a t 18”, and that the corresponding deoxy sugar derivatives exhibit a further, enhanced lability (see page 33).lo3 c. Substitution of the G1ycosides.-The substitution of the glycosides either in the aglycon or in the sugar moiety can result in considerable differences in the rates of hydrolysis. These differences may be attributed to several factors: polar effect of the substituents, modifications of the strain and conformational stability of the molecule, and the masking of the reactive center. I t is often difficult to establish that any particular one of these effects is the predominant factor. It has been shown by Nath and Itydonlo4that, in general, the acid hydrolysis of substituted phenyl P-~)-glucopymnosidesis facilitated by any electron-repelling properties of the substituents, and there seems to be a direct relationship between the Hammett substitution constant u and the velocity constant of the reaction, especially for the meta- and para-substituted derivatives. For instance, with 0.1 N hydrochloric acid a t 60°, the rate of hydrolysis of the p-cresyl (k = 2.32 X min-’, u = -0.170) was twice that of the corresponding phenyl (Ic = 1.15 x 10-4 min-1, u = 0.00) and seven times that of the p-nitrophenyl derivative ( k = 0.33 X min-’, u = +1.27). The differentiation between the polar and the stcric effects of the srtbstituents in the glycosyl moiety (und the aliphatic compounds in general) is more complicated. However, it appears that the glycosides are stabilized by virtue of the polar effect of the hydroxyl groups. Much light has been thrown on this subject by the recent work of Kreevoy and Taft,lo5 especially with regard to the hydrolysis of diethyl acetals. Their results are expressed (103) K . Butler, S. Laland, W. G . Overend and M. Stacey, J . Chem. Soc., 1433 (1950). (104) R. L. Nath and H . N . Rydon, Biochem. J.,67, 1 (1954). (105) M. M. Kreevoy and R. W. Taft, Jr., J . .4m. Chem. Soc., 77, 5590 (1955).

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

33

by the following modification of the Hammett equation, log(k/k,)

= (Zu*)p*

+ (An)h

in which k, is the rate constant for acetone diethyl acetal, (Za*) is the sum of the polar effects of the substituents, p* is a parameter characteristic of the reaction series, ( A n ) is the difference in the number of a-hydrogen atoms (which tend to stabilize the transition states by hyperconjugation), and h is an empirical constant. It is particularly interesting to note that the reported rate constants for glycolaldehyde diethyl acetal [HOCHzCH * (OEt)z; k = 8.47 X lop4]and acetaldehyde diethyl acetal [CH&H(OEt)z; Ic = 0.2481 differ by a factor of 3.4 x lOW. The additive polar effect of

H

H

H XXIII

the hydroxyl groups in the glycosides can be demonstrated by a comparison between the hydrolysis of 2-methoxytctr:~hydropyran '(methyl trideoxy-a ,@pentopyrunoside ; XXIII), which is reported to proceed rapidly under the influence of dilute acids a t room temperature,'06 and that of the methyl pentopyranosidcs, which are hydrolyzed by dilute acids a t elevated temperatures (see Td)le IV). The above considerations offer :m explanatiorP4 for the (well established) higher rates of hydrolysis of the deoxyglyc0sides.~3~ l o 3 , 107-112 The hydroxyl group a t C2, bciiig closer to the reactive center, exerts the largest influence; those a t C?i and CB have a lesser but nevertheless significant effect. This is shown by the differences in the stability of the compounds listed in Table VII toward acidic hydrolysis. Ij'rom these data, it also appears that the stabilizing effect of the hydroxyl group at C2 is independent of the (106) (1933). (107) (108) (109) (110) (111) (112)

R. P:iul, Bull.

SOC.

chinz. France, [5] 1, 971 (1934); Compt. rend., 197, 1652

M. Bergmmn, H. Schotte and W. Lechinsky, Ber., 66, 158 (1922). M. Bcrgmann, Ann., 443, 223 (1925). M. Bergniann and W. Breuers, Ann., 470, 38 (1929). F. Shufizudeh and M. Stacey, 6.Chem. Soc., 4612 (1957). W. G. Overend, M. Stacey and J. StanBk, J. Cheni. SOC.,2841 (1949). G. N . Itichurds, Chern. & Ind. (London), 228 (1955).

TABLEV I I Coinparative Rates of Hydrolysis of Deoxy Glyeosides Condilions

-

nydrolysis

P

Glycoside

c

A cid

Tmfi., “C.

__

Ethyl 2,3-dideoxy-j3-~-erythro- 0.001 N HCl hexopyranoside (XXIV) a anomer (XXV) 0.001 N HCI Methyl 2-deoxy-a-~-arabino- 0.1 N HC1 hexopyranoside Methyl a-D-glucopyranoside 0.1 N HC1 (XVIII) Ethyl 2,3-dideoxy-a-~-erylhroN HCI hexopyranoside (XXV) Ethyl 2-deoxy-cy-~-arabino-hexo- N HC1 pyranoside (XXVI) Ethyl a,@-D-glucopyranoside N HCl Methyl 2-deoxy-a-~-arabino-hex- N HC1 opyranoside Phenyl2-deoxy-a-~-arabino-hex- N HCl opyranoside Methyl 2-deoxy-a-~-1yro-hexoN HC1 pyranoside 0.02 N HCl Methyl 2-deoxy-aY,~-~-lyxo-hexof uranoside Methyl 2-deoxy-a-~-erythro-pen- D.005 N HCl topyranoside 8 anomer 3.005 N HC1 Methyl 2-deoxy-a,p-~-erythro- 3.005 N HC1 pentof uranoside Methyl 3-deoxy-a-~-ribo-hexoN HzSO4 pyranoside Methyl a-D-glucopyranoside N HzS04 Methyl 3-deoxy-a-~-arabino-hex- N HzS04 opyranoside (XXVII) Methyl a-D-mannopyranoside N HzSOi (XXIX) Methyl 6-deoxy-j3-~-mannopy- 3.01 N HCl ranoside (XXVIII) a anomer 3.01 N HCl 1.01 N HC1 Methyl 8-D-mannopyranoside a anomer (XXIX) 3.01 N HCl Methyl 2,3-dideoxy-4-0-(@-~- 3.001 N HC1 glucopyranosy1)-a-D-erythrohexopyranoside (“a-methyl 2’3 dideoxycellobioside”) Methyl 2-deoxy-4-0-(8-~-gluco- 1.001 N HCl pyranosyl) -a,p-D-arabino-hexopyranoside (“a,D-methyl2-de oxycellobioside ’ I ) Methyl 4-0-~-~-glucopyranosyl- 1.1 N HC1 8-D-glucopyranoside (“8-methylcellobioside”) 34

96

Exlenf,

%

u

Time, mvn.

-100

8 a: -

8

108

96 100

-100 95

20 5

108

100

5

30

07

161

03

7,500

.03

18

-100

18

107

18 18

03 10

18

10

17

63

17

63

100

100

17.! 11

100 100

100 100

15 3

11 11

100

12

100 100

12 12

100

12

100

83

100 100 100 95

85

55

83 83 83 09

100

50

30

09

95

40

510

09

-

FORMATION AND CLEAVAGE OF OXYGEN RINGS IN SUGARS

35

nature of the aglycon and of the constitution of the glycosyl moiety. The suggested correlations between this effect and the conformation of the relevant pyranose ringg6,113 are doubtful, in view of the fact that the same effect is shown by the furanosides and that, furthermore, the differences in the rates of hydrolysis which result from the modifications in the conformational features of the pyranose ring are relatively small. The additive nature of the polar effect of the hydroxyl groups is demonstrated by the gradual increase in the stability, toward acid hydrolysis, of compounds X X I V to XXIX and by the fact that the relative stabilities of the various 2-deoxyglycosides are the same as those of the fully hydroxylated glycosides discussed in this article.

H H

O

OEt WH

H

H

""y& xxv

XXIV

H

H

Et

H

XXVI

OMe

XXVII

@&

Me 0

H OH

H0

H

xxvm

H

OMe

XXIX

Scattered information on the effect of other substituents and modifications of the glycosyl moiety is to be found in the literature. Ethyl WDerythro-hexopyranos-his-eenide (XXX) is very readily hydrolyzedlo8 by 0.0001 N hydrochloric acid a t 96" or by water a t 100". The fully methylated glycosides have a lower rat,e of hydrolysis than their unmethylated 1113) G . Huber, Helu. Chim. Aclo, 38, 1224 (1055).

3G

F. SHAFIZADEH

I I CH II CH I HCOH I HCOI HCOEt

CHzOH

xxx analogs (see Table 111). The data obtained by Newth, Overend and Wiggins114 indicate that methyl 3-chloro-3-deoxy-a-~-glucopyranoside and methyl 3-bromo-3-deoxy-a-~-glucopyranosideare more stable toward acidic hydrolysis than is methyl a-D-glucopyranoside. Irvine and Hynd116J1s noted t h i t the methyl glycoside of D-glucosamine (methyl 2-amino-2-deoxy-~-glucopyranoside) is 100 times more resistant to acid hydrolysis than methyl a-mglucopyrunoside and remains essentially unchanged on heating at 100" with 5 % hydrochloric acid. They suggested116* ll6 that this stability may be due to the presence of a betaine ring structure (XXXI).Subsequently, Moggridge and Neuberger117 showed that this compound has a true glycosidic structure (XXXII) and that t,he unusual resistance toward acid hydrolysis is caused by the presence of the positively charged, amino group (-NH3*) which decreases the concentration of hydronium ions in the immediate vicinity of the glycosidic group. This resistance is countered by the blocking of the amino function with acetyl

I

HC-0

I 1 I HOCH I HCOI HCOH I

HC-NH2Me

CHzOH XXXI

(114) (115) (116) (117)

I

CH-OMe

I I HOCH I HCOH I HCOI

HC-NHz

CHzOH XXXII

F. H . Newth, W. G. Overend and L. F. Wiggins, J. Chem. SOC.,10 (1947). J. C. Irvine and A. Hynd, J. Chem. SOC., 101, 1128 (1912). J. C. Irvine and A. Hynd, J . Chem. SOC.,106, 698 (1914). R. C. G. Moggridge and A . Nenberger, J. Chem. SOC.,745 (1938).

FORMATION A N D CLEAVAGE OF OXYGEN RINGS I N SUGARS

37

or benzyloxycarbonyl groups.”7. us It is of some interest to note that the well established stability of methyl 2-amino-2-deoxy-a- and p-D-glucopyranosides does not extend to the methyl 6-amino-6-deoxy-~-glucoside prepared by Fischer and Zachllg or to the methyl 3-amino-3-deoxy-~altroside, “methyl-epiglucosamine,” which on hydrolysis gives the corresponding 1 ,6-anhydro derivative.l20,121 However, a siinilar masking effect is responsible for the resistance of the glycosidic linkage occurring between the N-methyl-L-glucosamine and the streptose moieties in streptomycin .I22

2. Reaction Mechanism According to Bunton and associate^,^^ the hydrolysis of the methyl and the phenyl a- and 0-D-glycopyranosides proceeds through the unimolecular decomposition of the glycoside’s conjugate acid (protonated glycoside). This conclusion is based on the linear relationship found between the firstorder rate constant and the Hammett acidity function.123Furthermore, it has been shown that, in an aqueous solution containing H2Ol8 the hydrolysis of the above glycosides results in the cleavage of the hexose-oxygen bond, as shown below, and provides unlabeled phenol and unlabeled meth-

Ha --

HO

OR

H

OH

+

H,O’*

-

(Fp,8H +

HOR

HO

H

OH

anol. These experimental observations (and the analogy with the well established mechanism of the acid-catalyzed hydrolysis of the acetsls106* 124-128) eliminate the likelihood of a bimolecular attacks6 on C1. Thus, the rate-determining, unimolecular step should involve the formation of a (118) A. B. Foster, D. Horton and M. Stacey, J . Chem. Soc., 81 (1957). (119) E. Fischer and K. Zach, Ber., 44, 132 (1911). (120) P. A. Levene and G. M. Meyer, J . Biol. Chem., 66,221 (1923). (121) L. F. Wiggins, J . Chem. Soc., 18 (1947). (122) R . U. Lemieux and M. L. Wolfrom, Advances i n Carbohydrate Chem., 3 , 337 (1948). (123) L. P. Hammett, “Physical Organic Chemistry,” McGraw-Hill Book Co., Inc., New York, N. Y., 1940, p. 267. (124) J . M. O’Groman and H. J. Lucas, J . Am. Chem. Soc., 72, 5489 (1950). (125) I>. McIntyre and F. A . Long, J . A m . Chem. Soc., 76, 3240 (1954). (126) J. N. Brbnsted and W. F. K. Wynne-Jones, Trans. Faraday Soc., 26.59 (1929). (127) W. J. C. Orr and J. A. V. Butler, J . Chem. Soc., 330 (1937). (128) M. M. Kreevoy and R. W. Taft, Jr., J . A m . Chem. Soc., 77,3146 (1955).

38

F. SHAFIZADEH

XXXIV

XXXVlll

- ROH11+ ROW

1-

XXXV b t

HOH~

(fast)

XXXVa

HOH

XXXVI

’

‘y-

XXX I X a + ROH] ( + HOH

4

XLI I

XXXVll

(slow)

XXXlXb

H@

-H

FIQ.I.-Postulated

11

XL

XLI

Intermediates in the Hydrolysis and Formation of Glycouides.

cyclic (XXXV) or of an acyclic (XXXIX) carbonium ion-with rapid, subsequent stages, as shown in Fig. 1. At present, there is no general or conclusive proof in favor either of the cyclic or of the acyclic pathways. However, there is much circumstantial evidence indicating the cleavage of the oxygen ring and the formation of an acyclic intermediate, particularly for the reactions involving the interconversion of furanosides and pyranosides. This mechanism accounts for the formation of ethyl l-deoxy-lthio-a-D-glucopyranoside (XLVI), ethyl 1-deoxy-1-thio-P-D-glucopyranoside (XLVII), the reducing sugar (XLIV), and a lesser amount of ethyl 1-deoxy-1-thio-0-D-glucofuranoside(XLV) from the hydrolysis of ethyl 1-deoxy-1-thio-a-D-glucofuranoside (XLIII) as described previously (see page 31). A further example is the conversion of methyl 3,6-anhydro-a-~-glucopyranoside (XLVIII) into methyl 3 ,6-anhydro-a-D-glucofuranoside (XLIX) merely on treatment, with dilute sulfuric acid, a reaction accom-

FORMATION AND CLEAVAGE OF OXYGEN RINGS I N SUGARS

39

CH20H

OH

H

H CHzOH o L a E t OH

H O CH,OH

H

H OH

H

XLV

t

on

H

H

b

0 ti

SEt OH

XLVI

HO H

OH

XLVII

panied by only a slight hydrolysis of the glycosidic 130 It is to be noted that methyl 3,6-anhydropyranoside derivatives of D-galact o ~ e , ' 132 ~ ~1)-glucose,~~9 . and ~-rnannose132are highly strained compounds and can be readily isomerized, or converted into aldehydo or dimethyl

qyMe 0.1 N H,SO,

HO H

OH

(room temp.)

OH

XLVlll

k

OH

Reducing product

XLlX

(129) (130) (131) (132) (1954).

W. N . Haworth, L. N . Owen arid F. Smith, J . Chem. SOC.,88 (1941). S. P e a t , Advances in Carbohydrate Chem., 2 , 37 (1946). W. N . Haworth, J. Jackson and F. Smith, J . Chem. SOC.,620 (1940). A. B. Foster, W. G. Overend, M. Stacey and G. Vaughsn, J. Chem. SOC.,3367

40

F. SHAFIZADEH

acetal derivatives. It appears that methyl 3 ,G-anhydro-2,4-di-O-methyl-aD-mannopyranoside is for the same reason hydrolyzed more rapidly than is the corresponding f u r a n o ~ i d e . ~ ~ ~ It has been shown that the acetolysis of methyl tri-0-acetyl-p-D-arabinopyranoside, when catalyzed with zinc chloride or 0.lG % sulfuric acid, provides a mixture of the two isomeric penta-0-acetyl-D-arabinose methyl hemiacetals in good yield.133This reaction provides good evidence for the cleavage of the cyclized bond in preference to that of the glycosidic group. The formation of an acyclic intermediate during the process of mutarotation was long ago postulated by Lowry,134-137 and has recently been confirmed by isotopic138and p o l a r o g r a p h i ~ ~investigations ~~-~~~ of aqueous solutions, and by kinetic studies with non-aqueous s o l ~ e n t s . ~ The ~~-~~~ small amounts of the reactive aldehydo forms in equilibrium, which are directly related to the configuration and structure of the sugars,13Bhave been estimated by alternative methods.21*13g In the reversible process of glycoside formation (see page 25 and Fig. l), the free aldoses, normally existing in the pyranose forms (XXXVII), provide a mixture of aldopyranosides (XXXIII) and aldofuranosides (XLII) in which the proportion of the less stable aldofuranosides initially present gradually decrease^^^^-^*^ as the reaction proceeds toward the establishment of an equilibrium. It has also been shown that, before the occurrence of equilibration, the glycosidic group is trans to the hydroxyl function at C2. Thus, this reaction should involve the cleavage of the oxygen ring and should proceed through the acyclic mechanism (with the postulated intermediates XXVIII to XLI). The principle of microscopic reversibilitylKO (133) E. M. Montgomery, R. M. Hann and C. 5. Hudson, J. Am. Chem. SOC.,69, 1124 (1937). (134) T. M. Lowry, J. Chem. SOC.,83, 1314 (1903); 86,1551 (1904). (135) G. F. Smith and T. M. Lowry, J. Chem. Soc., 665 (1929). (136) T. M. Lowry, J. Chem. SOC.,127, 1371 (1925). (137) H. Hudson, M. L. Wolfrom and T. M. Lowry, J. Chem. SOC.,1179 (1933). (138) K. Goto and T. Titani, Bull. Chem. SOC.Japan, 16, 403 (1941). (139) S. M. Cantor and Q. P. Peniston, J. Am. Chem. SOC., 62,2113 (1940). (140) J. M. Los, L. B. Simpson and K. Wiesner, J . A m . Chem. SOC., 78,1564 (1956). (141) W. G. Overend, A. R . Peacocke and J. B. Smith, Chem. & Ind. (London), 1383 (1957). (142) T. M. Lowry and I. J. Faulkner, J. Chem. SOC.,127,2883 (1925). (143) C. G. Swain and J. F. Brown, J r . , J. Am. Chem. SOC.,74, 2534 (1952). (144) A. M. Eastham, E. L. Blackall and G. A. Latremouille, J . A m . Chem. SOC., 77, 2182 (1955). (145) E. L. Blackall and A. M. Eastham, J. Am. Chem. SOC.,77,2184 (1955). (146) P. A. Levene, A. L. Raymond and R. T. Dillon, J.Biol.Chem., 96,699 (1932). (147) D . F. Mowery, Jr., and G. R. Ferrante, J. Am. Chem. Soc., 76,4103 (1954). (148) D . F. Mowery, Jr., J. Am. Chem. SOC.,77, 1667 (1955). (149) D. F. Mowery, J r . , Abstracts Papers Am. Chem. SOC.,190,9D (1956). (150) Reference 70, p. 202.

41

FORMATION AND CLEAVAGE OF OXYGEN RINGS I N SUGARS

indicates that the hydrolysis of glycosides should also proceed through the same mechanism. Further support for the above mechanism can be educed from the behavior of the dimethyl acetals of D-glucose and D-galactose under hydrolytic and glycoside-forming conditionPl, lSz; this indicates a very rapid, initial reaction due to hydrolysis of the acetal (a methoxyl group) followed by the formation of unstable glycosides (furanosides) which, in turn, are slowly converted into the stable pyranosides.161 It is interesting to note that the dimethyl acetals had long ago been postulated by E. Fischer as intermediates in glycoside formation.'* The initial formation of furanosides has several counterparts in the irreversible reactions of ~arbohydrtltes.8~ The deamination of 2-amino-2-deoxy aldonic acids and of 1-amino-1-deoxy alditols results in the formation of intermediate, acyclic, diazonium ions which give a 2,5-(or 1,4-)anhydro derivative (see page 54). Further examples are the formation of glycofuranosides (LI) by the reaction of aldose dialkyl dithioacetals (L) with mercuric chloride in the presence of mercuric oxide (to remove the resulting hydrochloric acid) and traces of water,100, 1028 153* 154 as shown below, and the formation of 1,4-anhydro 8

H C (SR)2

I

HCOH

I

HOCH

I

HCOH

+ HgClz _Hgq

I

HCOH

I

CH2OH L

HOCH

I

HCO

-I- RSHgCl

+ HCl

I I

HCOH CH20H LI ~~

(151) M.L.Wolfrom and S. W. Waivbrot, J. A m . Chem. Soc., 61, 1408 (1939). (152) H.A. Campbell and K. P. Link, J . B i d . Chem., 122,635 (1938). (153) J. W. Green and E. Pacsu, J . A m . Chem. Soc., 69, 2569 (1937). (154) J. W.Green and E. Pacsu, J . A m . Chem. Soc., 60,2056, 2288 (1938).

-

42

F. SHAFIZADEH

derivatives from alditols, as discussed later (see page 60). The theoretical aspects of ring closure in carbohydrates have been discussed by Mills.39 The feasibility of the steric requirements (suitable arrangement of C l to C4) for the formation of a five-membered ring in the xylo configuration is illustrated in Fig. 2 by the C2-C3 r o t a m e r ~ The . ~ ~ more stable, six-membered ring requires specific alignment and orientation of C5, as well as of the preceding carbon atoms. Thus, the formation of six-membered rings is less probable than that of five-membered rings, particularly a t lower

HOH @ OH

HO

FIG. 2.-Formation

und Cleavage of Furaiiose Rings.

temperatures (wherc the potential-energy barriers to free rotation become more effective). These theoretical considerations also provide an explanation for the observed relationship between the rates of hydrolysis and the steric strains of the glycosides discussed before. In the hydrolysis of aldopyranosides, the crucial stage is the formation of a high-energy, acyclic carbonium ion (XXXIX); and the rate of reaction is to a large extent a function of the energy difference, herein designated AE, , between this ion and its cyclic precursor (XXXVIII). This assumption also applies to the hydrolysis of aldofuranosides, the rate of hydrolysis of which is a function of the corresponding factor AEf The free energy associated with a five-membered

.

FORMATION AND CLEAVAGE OF OXYGZN RINGS IN SUGARS

43

ring is generally greater than that of a six-membered ring. Since both ring isomers provide the same carbonium ion, AEp is greater than AE, and, consequently, the aldopyranosides are more stable than the aldofuranosides. For the same reason, the strain associated with the ring structure of the septanosides, the bicyclic glycosides, and the stereoisomeric aldopyranosides with axial substituents will enhance the rate of hydrolysis. Interest in this subject has been stimulated by several other explanations proposed for the variation in the rate of hydrolysis of pyranosides and furanosides, based on the assumption that the reaction proceeds through the cyclic intermediates XXXIV to XXXVII. These explanations have attempted to correlate the rate of hydrolysis with the postulated conformation and stability of the intermediate^.^^^ 96, 113* It has been assumed that the initial formation of furanosides results from the greater reactivity of the furanose sugars present in the equilibrium m i ~ t u r e . ~In g this respect, it should be noted that, although the furanose sugars are certainly derived from the equilibration of pyranose forms, these speculations have not considered the excellent likelihood of the furanosides’ being formed from the transitory precursor of the furanose sugars in equilibrium. The assumption that formation and hydrolysis of the glycosides proceeds through the cyclic intermediates may be supported by analogy with the anomerization of acetylated alkyl glycosides and the dissociation of the C l acetoxy group of l12-truns-sugar acetates. It has been suggested that these reactions take place through the participation of the C2 acetoxy lS6-lb8 group, without cleavage of the oxygen The resistance of large equatorial substituents a t C5 to the postulated conformational transformations of the above cyclic intermediates readily accounts for the decreasing rates of hydrolysis of 6-deoxy hexosides, hexosides, and heptosides, as compared with those of pentosidesge*113, 167 (see Tables IV, V, and VII). Nevertheless, there is a strong possibility that this decrease in the rates of reaction may be due to the respective polar effects of the -CH3, -CH20H, and -CHOH--CHzOH substituents, as noted for methyl 6-deoxy-a- and @-L-mannopyranoside. IV. NITROUSACID DEAMINATION OF AMINOSUGARS Nitrous acid deamination of simple aliphatic primary amines involves, according to Ingold and c ~ w ~ r k ean r ~intermediate ,~ ~ ~ diazonium ion; (155) A. B. Foster and W . G. Overend, Chem. & I n d . (London), 566 (1955). (156) R. U. Lemiaux, Advances in Carbohydrate Chem., 9, 1 (1954). (157) R. U. Lemieux and C. Brice, Can. J . Chem., 33, 109 (1955); 34, 1006 (1956). (158) R. U. Lemieux, C. Brice and G. Huber, Can. J . Cheni., 33, 134 (1955). (159) 1’. Brewster, F. Hiron, E. D. Hughes, C. K. Ingold and P. A. D. S. Rao, Nature, 166, 170 (1950); C. K. Ingold, “Structure and Mechanism in Organic Chemistry,’’ G. Bell and Son, Ltd., London, 1953, p. 396.

44

F. sHAFIZADEH

this decomposes to a carbonium ion, which affords an alcohol of predominantly inverted configuration plus some olefins. The reaction proceeds through the SN1mechanism, and complete racemization is prevented by a masking effect of the departing group.

+ HONO

L-RNHz

4

L-RN@=N4 I P -+ D-ROH

+ DL-ROH+ olefins

Whitmore and Langlois have shown that the deamination of butylamine in the presence of chloride ion provides butenes, 1-butanol, and 2-butanol as the major products, and 1-chlorobutane and 2-chlorobutane as minor products.lG0The formation of these compounds was explained on the basis of an intermediate, carbonium ion. In more complicated compounds, variations in the steric configuration of the molecule and the nature of the neighboring groups may lead to the rearrangement of carbon bonds as well as to elimination and displacement products. It has been postulated that the "branching point" in the competing reactions is the intermediate diazonium ion, which may react according to the following mechanisms.l6' H

I I I I

Displacement

\ / C=C / \

Elimination

-C-C-

R

S

R

I I

H

H

I I

I 1

R

R R

R

Nz"

I I -c-cI I

\ 6 3

-+

/

H

H .I

-c-c-

I

R

NQ@ I.

I

/ \

-C-C@

C-C-

I

H -+

\@

/

C-C-

I

I

Carbonium-ion formation

Hydrogen migration

H

I

Carbon rearrangement

R

(160) F.C. Whitmore and D. P. Langlois, J. A,m. Chem. SOC.,64, 3441 (1932). (161) A. Streitwieser, Jr., J . Org. Chem., 22,861 (1957).

FORMATION AND CLEAVAGE OF OXYGEN RINGS I N SUGARS

45

The above mechanisms provide a basis for rationalizing the nitrous acid deamination of amino sugars, a reaction which yields a variety of products and often proceeds with the formation of a new ring-structure. Many of these reactions are profoundly affected by the nature of the neighboring group and by the configuration and conformation of the molecule. 1. Cyclic Reactions

a. Pyranose Sugars.-The mechanism of deamination of pyranose amino sugars can be explained on the basis of the well established, semipinacolic rearrangement of 2-amino-cyclohexanols. The work of McCasland162and of Curtin and S ~ h m u k l e r on ' ~ ~this subject, and other related investigations in aliphatic chemistry, have been discussed by K l ~ n e ~ ~ and by others.77It has been shown that t,he course of the reaction depends on the group which is antiparallel to the nitrogen atom and also trans to the Cl-C2 bond as shown for the following formulas. Stiuclure

LII LIII LIV LV

Configuration

cis

cis trans tram

Conformalion

NHI NH2 NH2 NH2

equat. axial equat. axial

Group antiparallel to N

C6 R C6 OH

Expected Product

LVI LVII LVI LVIII

cis-2-Aminocyclohexanol, which can assume conformations L I I and LIII (R = H), gives a mixture of cyclopentanecarboxaldehyde (LVI; R = H) and cyclohexanone (LVII) on deamination with nitrous acid; whereas the trans isomer [which is more stable as LIV (R = H), having both of the substituents in equatorial positions, than as LV (R = H)] g i v e P a high yield of cyclopentanecarboxaldehyde (LVI; R = H). Curtin and S ~hr nukl e r~have ~ 3 shown that trans-2-amino-1-phenylcyclohexanol gives products which are presumably derived from the epoxide LVIII (R = CaHb), whereas the cis isomer gives 99% of cyclopentyl phenyl ketone (LVI; R = CeH6).The reason for this is that the phenyl substituent tends to assume an equatorial position; consequently, the trans isomer reacts in conformation LV (R = C6&), and the cis isomer in the conformation LII (R = CeHS). (162) G. E. McCasland, J. Am. Chem. SOC., 73, 2293 (1951).

(163) D. Y. Curtin and S. Schmnkler, J. Am. Chem. SOC.,77, 1105 (1955).

46

F. SHAFIZADEH

It

(=o

O LVll

R L V Ill

It is to be noted that, when the amino group is equatorial in the above conformation, the reaction results in the formation of a five-membered ring. This generalization also applies to 2-amino-2-deoxy-a- and p-Dglucopyranose, which are stabilized in LIX and LXI (R = H) conformations by a number of equatorial substituents. In these conformations, the moieties antiparallel to the equatorial amino group are the ring oxygen atom and C4. However, the reaction proceeds through the cleavage of the oxygen ring instead of by rupture of the C4-C3 bond, and the main endproduct is 2,5-anhydro-~-mannose(chitose; LX).ls4-16* This is a sirup which and which, on treatment can be reduced to 2,5-anhydro-~-mannitol~~~ with bromine, gives 2, S-anhydro-~-mannonic acid. Further oxidation (with nitric acid) provides 2,5-anhydro-~-mannaricacid.lB6 Certain aspects of the above reaction, particularly the ingenious and unique method employed by Levene and LaForgelBs* for deducing the configuration of 2,5-anhydro-~-mannose,“chitose,” from the properties (164) (165) (166) (167) (168) (169) (170)

B. C. Bera, A. B. Foster and M. Stacey, J . Chem. Soc., 4531 (1956). A. B. Grant, New Zealand J . Sci. Technol., B37, 509 (1956). €3. Fischer and F. Tiemrtnn, Ber., 27, 138 (1894). L. Kueny, 2. p h p i o l . Chem., Hoppe-Seyler’s, 14, 330 (1890). G. Ledderhose, 2. physiol. Chem., Hoppe-Seyler’s, 4, 139 (1880). P. A. Levene and F. B. LaForge, J . Biol. Chem., 21,351 (1915). P. A. Levene, J . Biol. Chem., 36.89 (1918).

FORMATION AND CLEAVAGE OF OXYGEN RINGS IN SUGARS

47

of the related 2 ,5-anhydrohexaric acids, have been discussed by Peat in an early volume of this series130(see page 54).

LIX

LX

LXI

It should be noted that there has been some controversy regarding the structure of ‘(chitose.” Fischer and Andreael7I obtained D-glucose phenylosazone from the products of this deamination reaction, and indicated that it originated from undeaminated D-glucosamine. Neuberg and associates propo~ed’7~ a usual hexose structure for “chitose,” a conclusion which was ~ isolated a different osazone from the supported by A m b r e ~ h t , ”who deamination products; this was later shown to be D-glucose phenylosazone contaminated with D-arabinose phenylo~azone,”~ and the anhydrohexose structure was confirmed by the isolation of a crystalline diphenylhydrazone.176Further proof of the structure and configuration of 2 ,5-anhydroD-mannose has been provided by Bera, Foster, and S t a ~ e y ’and ~ ~ by Grant.lB6According to the last author,166the product readily affords crystalline derivatives with 1-benzyl-1-phenylhydrazine,(p-nitropheny1)hydrazine, and (2 ,4-dinitropheny1)hydrazirie. It reduces Fehling solution a t room temperature, but does not give a positive test with Schiff reagent; thus, it probably contains a I14-hemiacetal structure. It has been stated that the formation of the small, equilibrium amounts of the furanose or acyclic forms of D-glucosamine may account for some or all of the traces of other products formed in the above reacti0n.1~4 The mechanism suggested by Peat,130prior to the development of the present concepts on conformation and reaction mechanism, still remains valid. This mechanism is in contrast176with the rearrangement of the pyranose ring through a bivalent radical as suggested by Mat~ushimal7~ (see page 60). The reaction is not prevented by the blocking of the glycosidic group and it proceeds with both anomers of methyl 2-amino-2-deoxy(171) E.Fiecher and E. Andreae, Ber., 36,2587 (1903). (172) C.Neuberg, H.Wolff and W. Niemann, Ber., 36, 4009 (1902). (173) W. Ambrecht, Biochem. Z . , 96, 108 (1919). (174) L. Zechmeister and G. TGth, Ber., 66, 522 (1933). (175) P.Schorigin and N . N . Makarowa-Semljanskaja, Ber., 68,965 (1935). (176) A. B.Foster, Chem. & Ind. (London), 627 (1955). (177) Y.Matsushima, Bull. Chem. SOC.Japan, 24, 144 (1951).

48

F. SHAFIZhDEH

D-ghcopyranoside (LIX and LXI; R = CH,). The p form, with an equatorial glycosidic group, reacts faster178 than the a (see page 17). Desulfated heparin,’?Qwith alternating a-D-glucosamine and u-glucuronic acid units, and chitosanl73-175, 178. l80, I81 (N-deacetylated chitin) react similarly and break down to reducing constituents. In the latter example, 2,5-anhydroD-mannose diphenylhydrazone has been isolated from the product after adding (1,l-diphenyl)hydrazine.180Thus, the reaction provides an interesting method for the degradation of mucopolysaccharides, provided that they can be N-deacetylated without concomitant, drastic decomposition. N-Deacetylation of chondroitin is, however, not readily achieved, since strongly alkaline conditions lead to extensive decomposition, presumably by stepwise formation of saccharinic acidsa5*1B2 from the reducing endgroup. This peeling process can sometimes be averted by reducing the terminal unit. An alternative possibility is N-deacetylation with hydrazine.lEO The principal, amino sugar constituent of chondroitinsulfate is 2-amino2-deoxy-~-galactose. Both anomers of this sugar would be expected to assume preferentially the conformation in which the amino group takes up an equatorial position (LXII). Consequently, deamination with nitrous acid proceeds through the mechanism described for n-glucosamine, and affords 2,5-anhydro-~-talosewhich, on oxidation with bromine, gives lE4). 2,5-anhydro-u-talonic acid (obtained as a crystalline brucine salt183* More drastic oxidation with nitric acid provides crystalline 2,5-anhydro-utalnric acid (LXIIIa). Nitrous acid deaminntion, and subsequent oxidation, of 2-amino-2-deoxy-~-mannose(LXIV) under the above conditions provides D-glucaric acid (LXV) and not the 2,5-anhydro derivative expected by analogy with previous reactions.186This observation can be explained by the fact that, in 2-amino-2-deoxy-~-mannose,the configuration of the amino group is opposite to that of 2-amino-2-deoxy-~-g~ucoseand of 2-amino-2-deoxy-~-ga~actose. Consequently, in the favored chair conformation (LXIVa), the amino group assumes an axial position and the (178) A. B. Foster, 13. F. Martlew and M. Stacey, Chem. & Znd. (London), 825 (1953). (179) A. B. Foster and A. J. Huggard, Advances i n Carbohydrate Chem., 10, 336 (1955). (180) Y. Matsushima and N . Fujii, Bull. Chem. SOC.Japan, 30,48 (1957). (181) K . H. Meyer and H. Wehrli, Helv. Chim. Acta, 20, 353 (1937). (182) R. L. Whistler and J. N. BeMiller, Adoances i n Carbohydrate Chem., 13, 289 (1958). (183) P. A. Levene, “Hexosamines and Mucoproteins,” Longmans, Green and Co., London, 1925. (184) P. A. Levene, J . Biol. Chem., 31.609 (1917). (185) P. A. Levene, J . Biol. Chem., 39,69 (1919).

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

49

nitrous acid deamination, usually conducted a t room temperature, does not follow the previous pattern. However, it is interesting to note that deamination of 2-amino-2-deoxy-~-mannose, by heating with mercuric oxide, provides 2 ,5-anhydro-~-glucose(LXVI) as a crystalline compound which does not m u t a r ~ t a t e This . ~ ~ ~apparent anomaly is not totally unexpected. At elevated temperatures, the two chair conformations (LXIVa and LXIVb) are readily interconvertible and the surface reaction with mercuric oxide takes place through the less stable conformation (LXIVb) having the amino group in an exposed equatorial position. This behavior is in accordance with the theory that the molecule can react in the less stable conformation, particularly when the energy barrier is less than the activation energy of the reaction.lE6 2 ,5-Anhydro-~-glucose, unlike the corresponding D-mannose derivative, cannot form a 1,4-hemiacetal; this explains the lack of m ~ t a r o t a t i o n . ' ~ ~ These considerations throw a new light on the Dische test for hexosamines, which is based on the formation of a furan ring on nitrous acid

LXIVb (186) ltefererice 77, pp. 18 and 44.

LXVI

50

F. SHAFIZADEH

deamination of them.ls7 It appears that 2-amino-2-deoxy-~-mannosemay not provide a strong color unless the reaction conditions are modified. According to Irvine and Hynd,l16 the deamination of methyl 2-deoxy-2(dimethylamino)-n-glucopyranosidewith a hot solution of barium hydroxide provides methyl D-glucoside. The effect of conformation on the nitrous acid deamination of amino hexoses is further illustrated by the reaction of methyl 3-amino-4 ,6-O-benzylidene-3-deoxy-a-~-altropyranoside (LXVII) which, on deamination with nitrous acid, provides a quantitative yield of methyl 2 ,3-anhydro-4 ,6-O-benzylidene-cu-~-mannoside (LXVIII).lw The deamination of methyl 3-amino-3-deoxy-@-~-altropyranoside,121“methyl epiglucosamine,” gives a sirupy mixture.189 Similar treatment of methyl 2-amino-4,6-O-benzylidene-2-deoxy-a-~-altroside U

H

0 H5c+0%

H HJoH I

NHZ

H

H H

bMe

LXVII

H

LXVIII

7

LXIX

LXX H

n

H5c+%

H OH I

ti ti OMe

LXXI ~~

(187) Z.Dische and E. Borenfreud, J . B i d . Chem., 184,517 (1950). (188) L.F. Wiggins, Nature, 167, 300 (1946). (189) E.W. Bodycote, W. N. Haworth and E. L. Hirst, J . Chem. Soc., 151 (1934).

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

51

(LXIX) gives methyl 2 , 3 -anhydro-4 ,S-0-benzylidene-a-D-alloside (LXX).'8s The parent compound, methyl 4,6-O-benzylidene-a-~-altropyranoside, having two trans, fused, six-membered rings, has been shown's lgo to exist in the conformation LXXI. By analogy, the corresponding 2-amino-2-deoxy and 3-amino-3-deoxy derivatives should also have the same conformation. This indicates that, in the above compounds (LXVII and LXIX), the substituents a t C2 and C3 assume axial and antiparallel positions (with respect to each other) and the reaction follows the general rule outlined by K l ~ n e (see ' ~ page 45, reaction S). It is interesting to note that treatment of the epoxide (LXVIII) with ammonia gives 92.3% of the original amino sugar (LXVII) and some of the addition product of the two compounds.lZ1This phenomenon has been explained on the postulation that nucleophilic attack on epoxides takes place through the axial p o ~ i t i o n . 'l g~2 ~ ~ Moving clockwise around the pyranose ring, the next example is provided by the deamination of 4-amino-l,6-anhydro-4-deoxy-/3-~-mannose (LXXII), which gives 1,6:3,4-dianhydro-P-~-talose (LXXIII).lgs~ Ig4 There is some evidence indicating that the parent compound 1,6-anhydroP-D-mannopyranose, which cannot adopt the usual chair conformation ( C I ) , favors the reverse chair conformation ( I C ) to the alternative boat form (SB).' Thus, we can assume that the above derivative also favors the reverse chair conformation LXXII and that the anhydro sugar is formed through the mechanism involving axial and antiparallel substituents. 0

I

"27

HONO

H

LXXII

H

LXXIII

b. Furanose and Non-pyranose Sugars.-Deamination of 5-amino-5(LXXIV) in acetic acid deoxy-1 ,2-O-isopropylidene-a-~-xylofuranose (LXXV; X = solution provides 1,2-O-isopropylidene-au-~-xy~ofuranose OH). The same reaction performed in presence of chloride ion gives 5(190) (191) (192) (193) (194)

Reference 39, p. 37. E. L. Eliel, Reference 77, p. 132. W. G. Overend and G. Vaughan, Chem. d% Ind. (London), 995 (1955). V. G. Bashford and L. F. Wiggins, Nature, 166, 566 (1950). S. P. James, F. Smith, M. Stacey and L. F. Wiggins, J . Chem. Soc., 625 (1946)

52

F. SHAFIZADEH

chloro-5-deoxy-l , 2-O-isopropy~idene-ar-~-xy~ofuranose.'g" These products may have been derived from the displacement reaction or from the cleavage of an intermediate 3 ,5-anhydro compound. According to Ohle and Lichten~tein,~9~ the nitrous acid deamination of 6-amino-6-deoxy -l ,2-O-isopropylidene-~-~-idofuranose(LXXVI) results in the formation of 3,6-anhydro-l, 2-0-isopropylidene-0-L-idofuranose (LXXVII) which, on subsequent hydrolysis, provides 3,6-anhydro-~-

HONO HX

H2NH2c$o>

H H

0-C:

CH3 CH3

x H z ~ ~ o > H

O-pH3 C ' H,

LXXIV

LXXV

LXXVI

LXXVII

idose. This provides a contrast with the formation of 5,6-anhydro-l,2-0isopropylidene-a-D-glucofuranose (LXXIX) from 6-amino-B-deoxy-l , 20-isopropylidene-a-D-glucofuranose (LXXVIII), as reported by Bashford and Wiggins.lg3 The reaction of 6-amino-6-deoxy-l,2: 3,4-di-0isopropylidene-a-D-galactose, in which all of the hydroxyl groups are blocked, gives the expected 1 , 2 :3,4-di-O-isopropylidene-a-~-galactopyranose,lg7which can be reconverted to the initial amino sugar.

LXXVIII

LXXIX

(195) S. Akiya and T. Osawa, Yakugaku Zasshi, 76, 1280 (1956); Chem. Abstracts, 61, 4284 (1957). (196) H. Ohle and R. Lichtenstein, Ber., 63, 2905 (1930). (197) K. Freudenberg and A . Doser, Ber., 68, 294 (1925).

FORMATION A N D CLEAVAGE O F OXYGEN RINGS I N SUGARS

53

Despite the fact that the investigation of the deamination reaction of cyclic amino sugars has been rather limited and sporadic, the aforementioned reactions provide interesting examples of the formation of the various groups of theoretically expected products (see page 45), with the notable exception of the ketoses, which are of considerable potential interest. However, in the cognate field of the polyhydroxycyclohexanes (inositols), it has been reported that the nitrous acid deamination of aminodeoxy-meso-inositol (LXXX) gives some scyllitol (LXXXI) plus a sirupy tetrahydroxycyclohexanone which affords the osazone of DL-cyclose (LXXXII and LXXXIII).198

OH HO

HO

H

LXXX

LXXXI

H

H

H

J LXXXII

OH

OH

HO

H

H

LXXXIII

The deamination reaction of 2-amino-2-deoxy aldonolactones is also of interest. 2,5-Anhydro-~-mannaricacid has been obtained from the treatment of 2-amino-2-deoxy-~-mannonolactone, “epichitosaminic lactone,” with nitrous acid and subsequent oxidation of the product with nitric acid. This indicates that the reaction proceeds with a net retention of configuration, as for the corresponding free acid discussed later (p. 56). Similar treatment of 2-amino-2-deoxy-~-idonolactone, “d-dextro-xylohexosaminic lactone,” provides 2 ,5-anhydro-~-gularicacid, which is the epimer of the product derived from the free acid.109* The conversion of 1-amino- 1-deoxy-D-fructose, “isoglucosamine,” to sirupy D-fructose (reported by Fischer and Tafe11B9)may also take place (198) T. Posternak, Helv. Chini. A d a , 33, 1597 (1950). (199) E. Fischer and J. Tafel, Ber., 20,2566 (1887).

54

F. SHAFIZADEH

through the cyclic form. This reaction may be of potential value for the synthesis of other ketoses. 2. Acyclic Reactions The nitrous acid deamination of the acyclic amino alditols and amino aldonic acids yields five-membered ring compounds and acyclic products. The formation of five-membered rings may be regarded as the result of an intramolecular, substitution reaction in which a suitably placed hydroxyl group acts as the substituent. A considerable distinction can be made between this reaction and the formation of 2,5-anhydro sugars from the cyclic 2-amino-2-deoxy-hexopyranoses. I n the more stable conformations of the latter compounds, the ring oxygen which acts as a substituent is situated in a well defined and favorable position; whereas, in the acyclic compounds, the favorable conformations of the molecules are less well defined and are much more numerous. Despite that situation, the random participation of the various hydroxyl groups on the carbon chain, as substituents, is restricted by the larger strain and free-energy requirements of the three- and four-membered rings and by the smaller possibility for the formation of six-membered rings, as discussed previously (see page 42). a. Amino Aldonic Acids.-The pioneer work of Tiemann and HaarmannZo0and of Fischer and Tiemann,lB6followed by the extensive investigations by Levene and a s s o ~ i a t e s ~ lS3* ~ ~201-4 ~ indicated that deamination of 2-amino-2-deoxy-hexonic acids in general provides the corresponding 2,5-anhydro-aldonic acids. The above amino aldonic acids were prepared through the successive treatment of the pentoses with ammonia and hydrocyanic acid. The deamination products have been isolated as the crystalline 2 ,5-anhydro-hexaric acids derived by subsequent oxidation with nitric acid. The following formulas illustrate the conversion of 2-amino-2-deoxyD-gluconic acid (LXXXIV) to 2,5-anhydro-~-glucaricacid (LXXXVI), originally reported by Fischer and Tiemann.lGBThe intermediate 2,5anhydro-D-gluconic acid (LXXXV), “chitaric acid,” has been obtained in crystalline form.16* A summary of the above investigations is presented in Table VIII. Isolation of the same product from “d-dextro-ribohexosaminic acid” and “d-dextro-lyxohexosaminic acid” indicates that the common product must be 2,5-anhydro-~-talaricacid (LXIIIa) which is the same as 2,5-anhydroD-altraric acid (LXIIIb) and is derived from the oxidation both of 2,5anhydro-D-altronic acid (LXXXVII) and of 2,5-anhydro-~-talonicacid (200) (201) (202) (203) (204)

F. Tiemann and R. Haarmann, Ber., 19, 1257 (1886). P. A. Levene, J . Biol. Chem., 36, 73 (1918). P. A. Levene and F. B. LaForge, J . Biol. Chem., 20,433 (1915). P. A. Levene and F. B. LaForge, J . Biol. Chem., 22,331 (1915). P. A. Levene and E. P. Clark, J . Biol. Chem., 46, 19 (1921).

55

FORMATION AND CLEAVAGE O F OXYGEN RINGS IN SUGARS

TABLE VIII Nitrous Acid Deamination of 8-Amino-l-deozy-hexonic Acids Amino aldonic acid

Deamination groduct, a f t n ozidulion

Original designations

References

Sfruclure

Chitosaminic acid

2-Amino-2-deoxy D-gluconic acid Epichitosaminic acid 2-Amino-2-deoxy D-mannonic acid d-Chondrosaminic acid 2-Amino-2-deoxyD-galactonic acid d-Epichondrosaminic acic 2-Amino-2-deoxy (d-Dextro-lyxohexosD-talonic acid aminic acid) d-Dextro- ribohexosaminic 2-Amino-2-deox yacid D-altronic acida d-Levo-ribohexosaminic 2-Amino-2-deoxyacid D-allonic acid0 d-Levo-xylohexosaminic 2-Amino-2-deoxy acid D-gulonic acid0

d-Dextro-xylohexosaminic 2-Amino-2-deoxyacid D-idonic acida

2,5-Anhydro-~glucaric acid 2,5-Anhydro-~mannaric acid 2,5-Anhydrogalactaric acid 2,5-Anhydro-ntalaric acid;

166, 169, 202

2,5-Anhydro-~altraric acid 2,5-Anhydroallaric acid 2,5-Anhydro-~gularic (-L-glucaric) acid 2, b-Anhydro-~idaric acid

204

i

170

202, 203 170

204 170

170

a These structures are assigned by analogy with those of the other hexosaminic acids.

(LXXXVIII). This work also proves the configuration of the optically inactive galactaric and allaric isomers derived from “d-chondrosaminic C OzH

I

HCNHi

I

HOCH HCOH

I

HCOH

-

mH2cqo~~2H

H o 2 ; q y 2 H

__t

H

HO

H

HO

H

I

I

HaCOH LXXXIV

LXXXV

LXXXVI

acid” and “d-levo-ribohexossminic acid,” respectively. The configurations of the other four 2,5-anhydrohexaric acids have been similarly proved by the isolation of the enantiomorphs of 2,5-anhydro-glucaric acid from “chitosaminic acid” and from “d-levo-xylohexosaminic acid,” respec~ 9 , It has been noted that the deamination and subsequent tively.’3Q~ oxidation of chitosamine and chondrosamine provides 2,5-anhydro-D-

56

F. SHAFIZADEH

mannaric acid and 2,5-anhydro-~-talaric acid, respectively. However, since (at that time) the C2 configurations of the starting materials were

not known, these investigations could not clarify whether the deamination of the amino aldoses or the corresponding reaction of the amino aldonic acid proceeds with inversion of the C2 configuration. The D-glucose configuration of chitosamine and the D-galactose configuration of chondrosamine were later proved by unequivocal methods.206Consequently, it is now perfectly apparent that the deamination of the 2-amho-2-deoxy-~aldonic acids is accompanied by a net retention of the configuration, This conforms with the corresponding reactions of the aliphatic amino acids,169. 176, 206 reactions that have been explained by assuming the formation of a transitory a-lactone with one inversion (see reaction 6 , page 45), followed by the cleavage of the lactone with a second 161 The experimental attempts of Levene and LaForge to prove the mechanism of this reaction led to their isolation of a unique diazo compound from the nitrous acid treatment of the benzylidene derivative of ethyl chitosaminate.207Subsequent investigations have shown that this starting material (LXXXIX).208 was ethyl 2-amino-4,6-0-benzylidene-2-deoxy-~-gluconate The diazo derivative could be reconverted to the latter substance, and transformed to a variety of interesting compounds, including the 2-chloro, 2-bromo, 2,3-anhydro, and 2-deoxy derivative^.^^^^ 210 The structure assigned by Levene to the diazo group (XC), should result in the formation of C2 epimers. This leaves some grounds for the consideration of alternative formulas and of their possible bearing on the mechanism of the related reactions. Another interesting approach lay in the nitrous acid deamination of the 3-amino-3-deoxyheptonic acids (XCI and XCIII) derived from 2-amino-2deoxy-D-glucose and 2-amino-2-deoxy-~-galactose. The resulting products (205) (206) (207) (208) (209) (210)

A. B. Foster and M. Stacey, Advances i n Carbohydrate Chem., 7, 247 (1952). F. Fisher and A. Skita, 2. phpiol. Chem., Hoppe-Seyler’s, 33, 177 (1901). P. A. Levene and F. B. LaForge, J.B i d . Chem., 21.345 (1915). P. Karrer and J. Mayer, Helv. Chim. Acta, 20, 407 (1937). P. A. Levene, J. Bio2. Chem., 63,449 (1922). P. A. Levene, J . B i d . Chem., 64,809 (1922).

FORMATION AND CLEAVAGE OF OXYGEN RINGS I N SUGARS

57

COzEt

I

COzEt

I I

HCNHz HOCH

I

H Y o I I. I HCOH

HzCO

LXXXIX

f

CHCsHa

HZ

I

HOCH H

I

C

O

I HCOH

v

1

CHCeH6

1 2

HzCO

xc

could not be obtained in a satisfactory physical condition. Subsequent oxidation with nitric acid gave calcium salts having the empirical formulas211 of pentaric acids (XCII). These unexpected results were further complicated by the differences in the optical rotations of the products, which should have been identical, as may be seen from the formulas (see p. 58). A possible explanation is that the differences in the specific rotations may have derived from small amounts of impurities, and that the deamination reaction gives products XCIV and XCV, as in the case of 2-amino-2deoxy-D-glucitol discussed Inter. b. Amino Aldito1s.-Further instances of the formation of five-membered rings through the deamination of amino sugars are provided by the con(XCVI) and 1-amino-l-deoxy-Dversion of 1-amino-1-deoxy-D-mannitol glucitol to 1,4-anhydro-~-mannitol(XCVII)1e3and 1,4-anhydro-~-glucito1,212 respectively. From the deamination product of 2-amino-2-deoxyD-glucitol, Bashford and Wiggins isolated a tetraacetate which was tentatively assumed to be a 2,5-anhydro derivative.lB3Also, the treatment of 1-amino-1-deoxy-D-ribitol(XCVIII) with aqua regia (nitrosyl chloride) providesZ131,4-anhydro-~-ribitol(CI). An interesting counterpart of the above reaction has been found by Baddiley and n~sociates~1~ in the hydrolysis of L-ribitol 1-phosphate (which is the same as D-ribitol 5-phosphate) which, under acid conditions ( N hydrochloric acid), results in the formation of 1,4-anhydro-~~-ribitol. (211) (212) (213) (214)

P. A. Levene and I. Matsuo, J . Biol. Chem., 39, 105 (1919). V. G. Bashford and L. F. Wiggins, J. Chem. SOC.,299 (1948). R. Kuhn and G. Wendt, Ber., 81, 553 (1948). J. Baddiley, J . G. Buchanan and B . Cams, J . Chem. Soc., 4058 (1957).

58

F. SHAFIZADEH

COzH I I HCOH

COzH

I

HCOH

----I - _ _ _ _ HCNHz

HNOn HNOI

I HOCH I HCOH I HCOH I

HCNH~

HNOt HNO:

I

HzCOH XCI

1

HCOH = HOCH

I I

HNOn

HCOH

C 0zH

COaH

I

XCIIa

co

I I

I I

HCOH COzH

C OzH

I

XCIIb

co

HOCH

HOCH

I I HCOH I

HNOa

HNOa

HCOH

HOCH

' I HCOH I

HzCOH

HzCOH

XCIV

xcv

HzCNHz

I

HOCH

I HOCH

I HCOH

I

HCOH

HOH2C

- voy I

HOCH

HN02

H

H

I HiCOH I

HzCOH XCVI

XCVII

H

FORMATION AND CLEAVAGE OF OXYGEN RINGS I N SUGARS

59

The racemization apparently takes place before hydrolysis. Similar results have been obtained with D-xylitol5-phosphate and D-mannitol6-phosphate. 1,4-Anhydro-ribitoI is aIso formed from the treatment of ribitol with dilute mineral acid. However, since the anhydro compounds are produced more readily from the phosphates than from the unsubstituted alditols, it has been suggested that the latter compounds are not formed as intermediate products. The mechanism of this reaction has been explained by assuming protonation of the ester oxygen atom, with subsequent intraOH I

HO-P.0 I@ ~ 0 - H I \

H

XClX

H

/

XCVlll

.o

OH

H

HO HO CI

C

60

F. SHAFIZADEH

molecular substitution (XCIX). A similar mechanism may be also responsible for the formation of 1,Qanhydro derivatives from the alditols (C), a reaction which takes place less readily. In this case, the initial stage should The analogy between be the protonation of a primary hydroxyl the deamination of 1-amino-1-deoxy-D-ribitoland the above reactions is indicated by the following representations. The cyclization of alditols, under acidic conditions, to anhydro compounds having a five-membered ring216-21e appears to be a general reaction which can be educed as further experimental evidence in support of the theoretical conclusions discussed on pages 42 and 54. The intramolecular substitution thus far considered does not exclude the possibility of the occurrence of competing or alternative reactions. The deaminntion of 2-amino-2-deoxy-~-ghcitol (CII) provides, according to Matsushima,1772-deoxy-~-(‘glucose’’ (CIV) which can be isolated as the crystalline diphenylhydrazone derivative. The same reaction has been reported for the less readily available mixture of 2-amino-2-deoxy-~-arahinitol and 2-amino-2-deoxy-~-ribitol, which provides 2-deoxy-~-(‘ribose,’’ isolated as the crystalline benzylphenylhydraxone in small yield.220 It has been suggested by Matsushima that the reaction proceeds through an intermediate bivalent radical CV. More feasible explanations can be offered by analogy with the elimination or hydrogen-migration mechani~ms17~ outlined before (see page 44, reactions 2 and 4). If the reaction proceeds H

\

H-C=O

HiCOH

I HCNHa I HOCH I HCOH I HC OH I H2C OH CII

I I

HC-Na@

C Hz

I

HNOs ___t

HOCH

I HCOH I HCOH I

HzCOH CIII

HOCH

I

HCOH

I I

HCOH

.

HzCOH CIV

(215) L. F. Wiggins, Advances i n Carbohydrate Chem., 6 , 191 (1950). (216) G. Bouchardat, Ann. chim. el phys., [5] 6 , 100 (1875). (217) F. Grandel, U. S. Pat. 2,375,915 (1945); Chem. Abstracts, 40,89 (1946). (218) J. F. Carson and W. D. Maclay, J. Am. Chem. SOC.,67, 1808 (1945). (219) J . Baddiley, J. G. Buchanan and B. Carss, J. Chem. SOC.,4138 (1957). (220) Y. Matsushima and Y. Imanttga, BuZl. Chem. Soc. Japan, 26, 506 (1953); Nature, 171, 675 (1953).

FORMATION AND CLEAVAGE O F OXYGEN RINGS I N SUGARS

- HzCOH I‘

HOCH

’

61

HCOH-

II I HOCH I

CH

HCOH

HCOH

- HzCOH

I I HzCOHHCOH

through the elimination mechanism, in preference to the hydrogen migration (CIII), the enol form of 2-deoxy-~~‘glucose” (CVI) should be formed as an intermediate product.176In general, it is often difficult t o determine which of the following mechanisms may be operative.221 Another possibility that may be considered is the substitution of the amino group by a hydroxyl group. 1-Amino-1-deoxy-D-galactitol, treated with nitrous acid, provides a small amount of crystalline galactitol plus a sirup which as yet remains unidentified.222 (221) D. J . Cram, Reference 77, p. 267. (222) M. L. Wolfrom, F. Shafieadeh and P. McWnin, unpublished work.

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THE LOBRY DE BRUYN-ALBERDA VAN EKENSTEIN TRANSFORMATION BY JOHNC. SPECK,JR. Department of Chemistry, Michigan State University, East Lansing, Michigan

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . .............. 11. Scope.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63 65 65

1. Carbohydrate Transformations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Transformations of Noncarbohydrate a-Hydroxy Aldehydes and Ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 111. Side Reactions .............................. 73 1. Dehydration ............................................... 73 ............ 2. The Aldol Condensation. . . . . . . . . . 77 3. Miscellaneous Reactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 IV. Catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 1. Acid and Base Catalysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 2. Metal-ion Catalysis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 V. Use of the Transformation for Synthesis.. . . ........... 82 V I . Investigations of the Mechanism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 1. Experiments with 0-Methyl Sugars.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 2. Experiments Involving Deuterium and Tritium Exchange . . . . . . . . . . . . . 90 a. Deuterium Exchange Reactions of D-Glucose ....................... 90 b. Deuterium and Tritium Exchange Reactions Catalyzed by Triose Phosphate Isomerase, Phosphomannoisomerase, and Phosphogluco94 isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Kinetic Investigations. ............................................... 96 VII.Present Status of the Mechanism.. ...................................... 99 Addendum.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

I. INTRODUCTION The Lobry de Bruyn-Alberda van Ekenstein transformation has now been known for some 60 years. Thus, its reactions are among the oldest discovered during the period of modern carbohydrate chemistry. These reactions are very simple and have usually been imagined as proceeding via enolization. However, here, enolization leads to the unique possibility of the enediol intermediate, and the patent simplicity of this pathway has been matched by an elusiveness of mechanism which has intrigued three generations of carbohydrate chemists. These are also remarkable reactions if for no other reason than their apparent indispensability for life. The chemistry of the transformation has developed steadily since its 63

64

J. C. SPECK, JR.

first recognition, one or more of its reactions having been observed for nearly all of the known reducing sugars and for many noncarbohydrates as well. Moreover, progress continues, if one may judge by recent developments in elucidation of the mechanisms of these reactions and of their metabolic roles. In connection with the latter, it is interesting to note that, during the past 8 years, 11 new, enzymic, Lobry de Bruyn-Alberda van Ekenstein reactions have been reported. Although this transformation is well known, the Dutch chemists for whom it is named, and the circumstances which led to their collaboration, seem less so today. This situation, together with the coincidence of the centennials of the births of Lobry de Bruyn and Alberda van Ekenstein with the writing and the publication of this Chapter, make the inclusion of short biographical sketches of these men especially appropriate. Cornelis Adriaan Lobry van Troostenburg de Bruyn was born on January 1, 1857, in Leeuwarden, Holland. He was the son of Nicholas Lobry van Troostenburg de Bruyn, who was a physician, and Maria Agneta Isabella Bergsma. In 1875, Lobry de Bruyn entered the University of Leiden, where he became an assistant to Franchimont and, in 1883, obtained his doctorate. After spending a few months in Paris in the laboratories of Friedel and of Wurtz, he returned to Leiden in 1884. I n the following year, he was appointed Chemist to the Government Department of Marine, a position he held until 1896, when he succeeded Professor Jan Willem Gunning as Professor in the University of Amsterdam. Although he was offered a chair in the University of Vienna in 1901, he remained at Amsterdam until his untimely death a t the age of 47 on July 27, 1904. Lobry de Bruyn was a prolific worker whose scientific interests ranged over more than one field of chemistry. Although 29 of his scientific papers concern carbohydrates, these represent only a quarter of his published work. Many of his investigations had to do with nitrogen compoundsboth organic and inorganic (he was the first to prepare hydroxylamine and hydrazine in anhydrous form). In his last years, he turned more and more to physicochemical investigations and the examination of organic reaction mechanisms. Lobry de Bruyn’s collaborator in most of his carbohydrate investigations, Willem Alberda van Ekenstein, was born on March 28, 1858, in Groningen. His father was Eiso de Wendt Alberda van Ekenstein, and his mother, Anna Catherina Woldringh. Alberda van Ekenstein entered the Polytechnic School in Delft in 1876, and obtained a diploma in technology in 1879. He then became an assistant to Gunning a t Amsterdam. Following this, he supervised a small sulfuric acid plant for a time and then acted for a brief period as assistant to P. C. Plugge, Professor of Pharmacy and Toxicology in the University of Groningen. In 1884, on the advice of Gunning, he was appointed Chemist in the Government Sugar Laboratory

LOBRY DE BRUYN-ALBERDA VAN EKENSTEfN TRANSFORhIATfON

65

which was being established in Amsterdam by the Ministry of Finance. In 1904, he was given the title of Director, His new position and his collaboration with Lobry de Bruyn, which began in 1895, seemed to stimulate his interest in research. In the period from 1894 to 1914, he was either author or coauthor of 50 scientific papers, nearly all of which had to do with carbohydrates. He also became a master a t the isolation and purification of sugars, and obtained several crystalline for the first time. In recognition of his achievements, the Senate of the University of Groningen awarded him an honorary doctorate in 1926. Alberda van Ekenstein died on May 10, 1937.’ The collaboration of Lobry de Bruyn and Alberda van Ekenstein appears to have been occasioned by Lobry de Bruyn’s interest in those reactions of the sugars brought about by alkalis. It is interesting to note, however, that their joint efforts were not limited to this investigation, which terminated in 1899, and that they continued to publish other work throughout the remainder of de Bruyn’s life. There has in the past been some confusion about the names “Lobry de Bruyn” and “Alberda van Ekenstein.” Actually, these were family names and were the surnames which they used in their publications. It is then, entirely appropriate to identify by the name “Lobry de BruynAlberda van Ekenstein” the transformation which they first observed. The name is, however, too cumbersome to use in continual reference to this transformation and its reactions; hence, the author will often refer t o it simply as “the transformation.”

11. SCOPE 1. Carbohydrate Transformations The Lobry de Bruyn-Alberda van Ekenstein transformation has usually been considered to embrace both epimerization of aldoses and ketoses and aldose-ketose isomerization. Actually, Lobry de Bruyn and Alberda van Ekenstein observed all three reactions, so that an experimental basis for defining the transformation has existed from almost the time of its first recognition. The experimental limits of the transformation have never been extended beyond those established in the original investigations.2*3 - 3a Thus, for ex(1) Most of this biographical material was taken from obituary notices appearing in Ber., 37, 4827 (1904), J . Chem. Soc., 87, 570 (1905), and Chem. Weekblad, 34, 614 (1937). (2) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. chim., 14, 203 (1895). (3) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. chim., 16, 262 (1897). (3a) Some modification of these experimental limits of the transformation now seems to be in order, in view of the very recent findings of J. C. Sowden and R. R.

66

J. C . SPECK, JR.

ample, the transformation of an aldose (I or 11) may proceed with (1) significant epimerization a t C2, (2) considerably more isomerization to the corresponding 2-keto sugar, and (3) slight C3-epimerization of this HC=O

CHzOH

I I c=o HCOH I = I HCOH HCOH I I R

R

1% J/' HC=O

I I

HOCH HCOH

I

R

CHzOH

=

I

1 c=o

HOCH

I

R

HC=O

I HCOH I = HOCH I

I

CHzOH

I c=o

c=o

HOCH

I

R

II Y\

CH2OH

R

I

= I

HCOH

I

R

J/' HC=O

I I

HOCH HOCH

I

R

ketose. There is ample evidence, including some from recent paper-chromatographic separations of Lobry de Bruyn-Alberda van Ekenstein reaction mixtures,4, for the simultaneous occurrence of these three reactions during many such transformations. However, the difficulty of separating these mixtures seems to have discouraged attempts a t isolation of the products, so that all three have never yet been obtained in a single experiment. Table I comprises nonenzymic Lobry de Bruyn-Alberda van Ekenstein transformations of the sugars. In order to abbreviate it somewhat, only those experiments are listed in which the transformation products, or their derivatives, were obtained in reasonably pure states and in appreciable yields. Also, lactaldehyde has been arbitrarily placed with the noncarbohydrates for the convenience of discussing its isomerization with that of mandelaldehyde in the next Section. Even so, Table I includes most of the known reducing sugars and several of their derivatives, and it gives a fair idea of the present status of the transformation (as catalyzed other than by enzymes). Table I1 lists the enzyme-catalyzed reactions. These now embrace aldose-ketose isomerizations both of phosphorylated and of nonphosThompson [J.Am. Chem. Soc., 80, 1435 (1958)l. Thus, although 3-keto sugars have never been convincingly demonstrated in Lobry de Bruyn-Alberda van Ekenstein reaction mixtures, Sowden and Thompson's data from experiments on the degradation of ~-glucose-l-C'4by a strong-base resin [Amberlite IRA-400 (OH)] indicate tha t the carbonyl group actually migrates under these conditions from C1 t o C5. (4) G. Malyoth and H. W. Stein, Angew. Chem., 64, 399 (1952). ( 5 ) F. Schneider and G. A. Erlemann, Naturwissenschaflan, 39, 160 (1952).

TABLEI Nonenzymic Lobry de Bruyn-Alberda van Ekenstein Transformations of Sugars Starling sugai

Producls isolated

Catalyst

pyridine calcium hydroxide pyridine pyridine L- Arabinose

pyridine pyridine

Cellobiose D-Fructose

L-Fucose D-Galactose

L-Galactose D-Glucose

sodium hydroxide calcium hydroxide potassium hydroxide sodium hydroxide sodium hydroxide sodium phosphate buffer (pH 6.69) tartaric acid pyridine potassium hydroxide calcium hydroxide pyridine pyridine calcium hydroxide calcium hydroxide sodium hydroxide pyridine quinoline pyridinec calcium hydroxidedzd

calcium hydroxide-

Referenus

D-psicose 6 D-psicose 7 D-erythro-pentulose (“D-ribu8 lose”) D-erythro-pentulose o-nitro9 phenylhydrazone L-erythro-pentulose, L-ribose 10 p-bromophenylhydraeone L-erythro-pentulose (“L-ribu8 lose”) L-ribonic acid phenylhydrazide‘ 11 4-O-~-~-glucopyranosyl-~-man- 12 nose methyl a-D-mannopyranoside, 2 D-glucaric acidb D-glucose, D-mannose phenyl13 hydrazone D-glucose 2 D -mannose phen ylhydraz one 14 D-glucose diethyl dithioacetal 6-deoxy-~-tagatose D-tagatose, D-sorbose

15 16 3

D-talonic acid,. n-tagatose, D-sorbose D-tagatose L-tagatose D-fructose, methyl a-D-mannopyranoside D-mannose phenylhydrazone D-mannose phenylhydrazone, D-fructose o-fructose o-fructose D-fructose, D-mannose o-mannose-1-d phenylhydrazone, D-mannose-1, 2 4 2 phenylhydrazone, D-fructoseI-d, D-fructose-1, 1-dz D-fructose-1-d phenylosazone

17 18, 19 !O, 21 2 22 13 23 23 23 24

25

dzd

sulfuric acid sodium acetateacetic acid ammonia strong base resind 67

D-fructose D-fructose methylphenylosazone

26 27

D-psicose D-mannose phenylhydrazone

28 29

J. C. SPECK, JR.

68

TABLEI-Continued Starting sugar

D-Glucose-I-d

calcium hydroxide

uL-Gl ycerose D-glycero-D-galactoHeptose

pyridine barium hydroxide

D-gEyCerO-D-gU~OHeptose

pyridine calcium hydroxide calcium hydroxide

D-Gulose D-Idose Lactose Maltose D-Mannose

Products isolalcd

Caldysl

barium hydroxide pyridine barium hydroxide calcium hydroxide aqueous ammonia calcium hydroxide aqueous ammonia sodium hydroxide sodium hydroxide

Meli biose

ammonia

3-0-Methyl-D-glucose D-Sorbose

calcium hydroxide potassium hydroxide calcium hydroxide

References

D-fructose-1-d phenylosazone, D-mannose-1-d phenylhydrazone di hydroxyacetone D-manno-heptulose, D-gluco-hep tulose D-manno-heptulose D-gluco-heptulose 32,

25

D-ghco-heptulose, calcium D-g~~cero-D-gulo-heptonatea D-sorbose D-sorbose D-sorbose lactulose 37, lactulose malt ul ose maltulose D-glucose, D-fructose methyl a-D-glucopyranoside, D-fructose, D-glucaric acid6 melibiulose, 6-O-a-~-galactopyranosyl-8-D -mannopyranose 3-O-methyl-~-fructose

34

D-galactose N l-methyl-N1phenylhydrazone

30 31 31 33

35 36 35 38 28 39 28 13

2 40

41 3

2,3,4,6-Tetra-0methyl-D-glucose calcium hydroxide 2,3,4,6-Tetra-Omethyl-D-mannose barium hydroxide

2,3,4,6-tetra-O-methyl-~-man- 22

calcium hydroxide

43

2,3,4-Tri-O-methylD-xylose D-X ylose

calcium hydroxide pyridine pyridine

L-X ylose

pyridine pyridine

nose

2,3,4,6-tetra-O-methyl-~-glucose 2,3,4,6-tetra-o-methyl-~-glucose 2,3,4-tri-O-methyl-~-lyxonic acid phenylhydrazide“ n-lyxonic acid phenylhydrazide D-threo-pentulose (“D-xylulose”) D-threo-pentulose, D-lyxose (as p-bromophenylhydrasones) n-threo-pentulose, D-lyxose L-threo-pentulose (“L-xylulose”)

42 42

43 44 8 10 45

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

69

TABLEI-Continued Aldoses oxidized with bromine. b Aldaric acid obtained after nitric acid oxidation. Pyridine contained water. d Reactions carried out in deuterium oxide. Amberlite IRA-400 (OH). (6) M. Steiger and T. Reichstein, Helv. Chim. Acta, 19, 184 (1936). (7) F. W. Zerban, L. Sattler, G. Rosenthal and A. Glaubach, Sugar, 47, No. 2, 33 (1952). (8) 0. T. Schmidt and K. Heintz, Ann., 616, 77 (1934). (9) C. Glatthaar and T. Reichstein, Helv. Chim. Acla, 18, 80 (1935). (10) P. A. Levene and R. S. Tipson, J. Biol. Chem., 116, 731 (1936). (11) W. Alberda van Ekenstein and J. J . Blanksma, Chem. Weekblad, 10, 213 (1913). (12) S. N . Danilov and P. T. Pastukhov, Zhur. Obshchet Khim., 16, 923 (1946). (13) J. C. Sowden and R . Schaffer, J. A m . Chem. Soc., 74, 499 (1952). (14) H. A. Spoehr and H. H. Strain, J. Biol. Chem., 86, 365 (1929). (15) F. Petiiely, Monatsh., 84, 298 (1953). (16) J. Barnett and T. Reichstein, Helv. Chim. Acta, 20, 1529 (1937). (17) J. U. Nef, Ann., 403, 204 (1914). (18) T. Reichstein and W. Bosshard, Helv. Chim. Acta, 17, 753 (1934). (19) Y. Khouvine, G. Arragon and Y. Tomoda, Bull. soc. chim. France, [5] 6, 351 (1939). (20) C. Glatt>haarand T . Reichstein, Helv. Chim. Acta, 20, 1537 (1937). (21) K. Iwadare and €3. Kubata, Sci. Papers Inst. Phys. Chem. Research (Tokyo), 34, 183 (1938); Chem. Abstracts, 32, 3344 (1938). (22) M. L. Wolfrom and W. L. Lewis, J. A m . Chem. Soc., 60,837 (1928). (23) S. N . Danilov, E. Venus-Danilova and P. Shantarovich, Ber., 63, 2269 (1030). (24) J. C. Sowden and R.Schaffer, J . A m . Chem. Soc., 74, 505 (1952). (25) Y. J. Topper and D. Stetten, Jr., J. Biol. Chem., 189, 191 (1951). (26) H. Ost, Angew. Chem., 18, 1170 (1905). (27) P. A. Ashmarin and A. D. Braun, Byull. Eksptl. Biol. Med., 4, No. 4, 374 (1937); Khim. Referat. Zhur., 1, No. 8-9, 27 (1938). (28) L. Hough, J. K. N . Jones and E. L. Richards, J. Chem. SOC.,2005 (1953). (29) J. C. Sowden, J. A m . Chem. Soc., 76, 4487 (1954). (30) H. 0. L. Fischer, C. Taube and E. Baer, Ber., 60, 479 (1927). (31) E. M. Montgomery and C. S. Hudson, J. A m . Chem. Soc., 61, 1654 (1939). (32) W. C. Austin, J. A m . Chem. SOC.,62, 2106 (1930). (33) Y. Khouvine, Compl. rend., 199, 869 (1934). (34) J . Pratt, N. K. Richtmyer and C. S. Hudson, J. A m . Chem. SOC.,74, 2210 (1952). (35) W. Alberda van Ekenstein and J. J . Blanksma, Rec. trav. chim., 27, 1 (1908). (36) K. Gatzi and T. Reichstein, Helv. Chim. Acta, 21, 456 (1938). (37) E. M. Montgomery and C. S. Hudson, J. A m . Chem. Soc., 62, 2101 (19301. (38) J. P. L. Bots, Rec. trav. chim., 76, 515 (1957). (39) S. Peat, P. J . P. Roberts and W. J. Whelan, Biochem. J.,61, xvii (1952). (40) L. Hough, J. K. N . Jones and E. L. Richards, J. Chem. Soc., 295 (1954). (41) D. J. Loder and W. L. Lewis, J. A m . Chem. SOC.,64, 1040 (1932). (42) R . D. Greene and W. 1,. Lewis, J. A m . Chem. Soc., 60, 2813 (1928). (43) C. E. Gross and W . L. Lewis, J. A m . Chem. SOC.,63, 2772 (1931). (44) 0. T. Schmidt and R . Treiber, Ber., 66, 1765 (1933). (45) L. von Vargha, Ber., 68, 18 (1935). (I

70

J. C. SPECK, JR.

TABLE I1 Enzyme-catalyzed Lobry de Bruyn-Alberda van Ekenstein Reactions Reaction

D-Arabinose

Enzyme

D-erythro-pentulose

L-Fucose $ B-deoxy-~-tagatose D-Xylose

D-threo-pentulose

arabinoisomerase arabinoisomerase xyloisomerase

Enzyme source

Rejercnces

Escherichia coli

46

Escherichia coli

46, 47

Lactobacillus pento

48

sus

D-Mannose G D-fructose

mannoisomerase

D-Lyxose

mannoisomerase

D-Rhamnose

D-threo-pentulose

6-deoxy-~-fructose mannoisomerase

D-Sedoheptulose S D - g l ycero-D- mannoisomerase manno-heptose (?) D-Glucose 2 D-fructose glucoisomerase D-Glucose 6-phosphate D-fructose 6-phosphate D-Mannose 6-phosphate S D-fructose 6-phosphate D-Glycerose 3-phosphate 1,3-dihydroxy-2-propanone phosphate &Ribose 5-phosphate D-erythropentulose 5-phosphate

phosphoglucoisomerase" phosphomannoisomerase triose phosphate isomerase" phosphoriboisomerase

D-erythro-Pentulose 5-phosphate D-lhreo-pentulose 5-phosphate

phosphoketopentoepimerase

*

Pasturella pestis Pseudomonas hydro phila Pseudomonas saccharophila Pseudomonas saccharophila Pseudomonas saccharophila Pseudomonas saccharophila Pseudomonas hydro phila muscle

49 50, 51 52 52 52 52 53 54

muscle

55

muscle

56

yeast alfalfa spinach muscle Lactobacillus pen-

57 58 59 60, 61 62

tosus

spleen

63

Reference given only to original identification of enzyme. (46) S. 5. Cohen, J. Biol. Chem., 201. 71 (1953). !47) M. Green and S. S. Cohen, J . Biol. Chem., 219, 557 (1956). (48) S. Mitsuhashi and J. 0. Lampen, J . Biol. Chem., 204, 1011 (1953). (49) M. W. Slein, J. Am. Chem. SOC., 77, 1663 (1955). (50) R . M. Hochster and R . W. Watson, J. Am. Chem. SOC., 76, 3284 (1953). (51) R. M. Hochster and R . W. Watson, Arch. Biochem. Biophys., 48, 120 (1954). (52) M. J. Palleroni and M. Doudoroff, J. Biol. Chem., 218, 535 (1956). (53) R. 0. Marshall and E. R. Kooi, Science, 126, 648 (1957). (54) K. Lohmann, Biochem. Z., 262, 137 (1933). (55) M. W. Slein, J. niol. Chem., 186, 753 (1950). (56) 0. Meyerhof and K. Lohmann, Biochem. Z., 271, 89 (1934).

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

71

phorylated sugars, as well as the C3-epimerization of a ketose, D-threopentulose 5-phosphate. Enzymic, aldose epimerization at C2 has yet to be observed. 2. Transformations of Noncarbohydrate and a-Hydroxy Aldehydes Ketones Nefs* apparently observed the first such isomerizations of noncarbohydrates on attempting to prepare lactaldehyde and mandelaldehyde by hydrolyzing their acetates in boiling aqueous solutions. From these reaction mixtures, Nef isolated acetol and 2-hydroxyacetophenone, respectively, and he concluded that neither lactaldehyde nor mandelaldehyde could exist. Later, Wohl and Langes6prepared crystalline lactaldehyde by hydrolyzing its diethyl acetd with dilute sulfuric acid at “ordinary temperature.” However, Evans and Parkinsonss found that the hydrolysis of mandelaldehyde diethyl acetal also results in its complete isomerization to 2-hydroxyacetophenone-even when the reaction is carried out a t 0”. It is worth noting a t this point that equilibria in which the concentration of one of the isomers is hardly measurable are not uncommon among these noncarbohydrate isomerizations. Favorskiis’ reported the conversion of 3-hydroxy-4-heptanone to 4-hydroxy-3-heptanone, as well as analogous transformations of 2-hydroxy-3These reactions were pentanone and 2-hydroxy-4,4-dimethyl-3-pentanone. carried out at elevated temperatures, in ethanol solutions containing a small proportion of sulfuric acid. At the same time, Favorskii reported the isomerization of 2-hydroxypropiophenone to 1-hydroxy-1-phenyl-2-propanone under the conditions of yeast fermentation-a claim which the evidence given hardly justified and which von Auwers and Mausss* disputed. However, Temnikovase reported that this conversion does take place in both acid and alkaline media, and the list of what appear to be (57) B. L. Horecker, P . 2. Smyrniotis and J. E . Seegmiller, J . B i d . Chem., 193, 383 (1961). (58) B. Axelrod and R. Jang, J . Biol. Cheni., 209, 847 (1954). (59) J . Hurwitr, A. Weissbach, B. L. Horecker and P. 2. Smyrniotis, J . Biol. Chem., 218, 769 (1956). (60) F. Dickens and D . H. WilliamBon, Nature, 176, 400 (1955). (61) P. A. Srere, J. Cooper, V. Klybas and E. Racker, Arch. Biochem. Biophys., 69, 535 (1955). (62) P. K . Stumpf and B. L. Horecker, J . B i d . Chem., 218, 753 (1956). (63) G . Ashwell and J. Hickman, J . Biol. Chem., 226, 65 (1957). (64) J. U. Nef, Ann., sS6, 247 (1904). (65) A. Wohl and M. Lange, Ber., 4,3612 (1908). (66) W. L. Evans and C. R. Parkinson, J . Am. Chem. Soc., 36, 1770 (1913). (67) A. Favorskii, Bull. 8oc. chim. France, [4] 39, 216 (1926). (68)K . von Auwers and H. Mauss, Biochem. Z., 1M, 200 (1928). (69) T. I. Temnikova, Zhur. Obshchei Khim., 10, 468 (1940).

72

J. C. SPECK, JR.

Lobry de Bruyn-Alberda van Ekenstein isomerizations between related compounds has since been expanded to include isomerizations of 2-hydroxybutyrophenone,lO l-(4-chlorophenyl)-l-hydroxy-2-propanone,” and 2-hydroxy-l-phenyl-l,4-pentanedioneJ~ Some interesting examples of Lobry de Bruyn-Alberda van Ekenstein transformations have been observed among the steroids, Reichstein and von Euw14 isomerbed A4-pregnene-17,20-diol-3-one-21-al (111) to A4 pregnene-l7,21-diol-3,2O-dione(IV) in pyridine. Borgstrom and Gal lagher’s investigation of the transformation of 3(a) ,12(P)-dihydroxy-l1 HC=O CH2OH I I 0

’*

&

0 I11

IV

ketocholanic acid (V)lKis outstanding, inasmuch as all three of the expected products, 3(a) ,ll(a)-dihydroxy-12-ketocholanic acid (VI), 3(a),12(a)dihydroxy-11-ketocholanicacid (VII), and 3(a),11(@)-dihydroxy-l2-ketocholanic acid (VIII), were isolated from the reaction mixture.

VII

:

(1.1 %)

VIII (trace)

(70) T. I. Temnikova and E. F. AfanaR’eva, Zhur. Obshchet Khim., 11, 70 (1941). (71) T . I. Temnikova and E.I. Kulachkova, Zhur. Obehcher Khim., 10, 1324 (1949). (72) T. I. Temnikova and E. S. Kmito, Sbornik Stater Obehche’l Khim., Akad. Nauk S.S.S.R., 2, 869 (1953);Chem. Abstracts, 40, 6877 (1966).

LOBRY D E BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

73

111. SIDEREACTIONS 1. Dehydration Reactions

The formation of deoxyosones, such as, for example, the 3-deoxyosones, appears to be the most important of the dehydration reactions which may take place during Lobry de Bruyn-Alberda van Ekenstein transformations. This type of reaction, which NeP6 first proposed in suggesting mechanisms for saccharinic acid formation, is diflicult to study because the products are seldom stable in the reaction mixtures in which they are formed. Nevertheless, several different lines of evidence now indicate that reducing sugars undergo primary dehydrations of this kind, and that deoxyosones do indeed mediate in saccharinic acid formation in basic solutions, as well as in production of 2-furaldehyde and its derivatives in acidic media. A dehydration of this type has actually been observed as a side reaction of a Lobry de Bruyn-Alberda van Ekenstein transformation in a very simple system. Thus, in experiments with the DL-glycerose-1 ,3-dihydroxy-2-propanone isomerbation in acetate, formate, and trimethylacetate buffers, pyruvaldehyde appeared in the reaction mixtures!? (The formation of pyruvaldehyde from l ,3-dihydro~y-Z-propanone-~~ and DLglycerose-mineral acid mixtures?@ had been observed much earlier.) Since these experiments in acidic buffers established that this reaction is subject to general acid and base catalysis, pyruvaldehyde must be formed in alkaline mixtures also. The results of Wohl’sSoand Evans and Hass’sB1 experiments with m-glycerose in alkaline solutions containing phenylhydrazine, in which pyruvaldehyde phenylosazone was isolated, support this view. HC=O

HC=O

I

CHOH

I

CH20H m-Glycerose

- HIO

I

c=o

I

CK pyruvaldehyde

CHiOH

- HI0

I

c=o I

CHzOH

1,3-dihydroxy2-propanone

(73) M. Henae, 2.physiol. Chem., Hoppe-Seyler’s, 232, 117 (1935). (74) T. Reichstein and J. von Euw, Helu. Chim. Acta, 25, 1268 (1940). (75) E. Borgstrom and T. F. Gallagher, J . BioE. Chem., 177, 961 (1949). (76) J. U. Nef, Ann., 567, 214 (1907). (77) J. C. Speck, Jr., A. A. Forist and D. S . Miyada, Abstracts Papers Am. C h m . SOC.,127, 6D (1955). (78) G. Pinkus, Ber., 51, 31 (1898). (79) C. Neuberg and E. Kansky, Biochem. Z . , 20, 450 (1909). (80)A. Wohl, Biochem. Z . , 6, 45 (1907). (81) W. L. Evans and H. B. Hass, J . A m . Chem. SOC.,48, 2703 (1926).

74

J. C. SPECK, JR.

Wolfrom and coworkerss2 proposed this kind of primary dehydration for D-glucose and D-xylose, in order t o explain the behavior of these substances in aqueous solution. On boiling these solutions in Pyrex vessels (some of the D-glucose solutions were first slightly acidified with hydrochloric acid), two maxima, one at about 230 mp and the other in the neighborhood of 280 mp, appeared in their ultraviolet absorption spectra. The 230-mp maxima developed more rapidly near the beginning of the reactions, suggesting the presence of a conjugated diene or an enal. Later, the spectra of these D-xylose and D-glucose mixtures became identical with the spectra of 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde, respectively, for which principal maxima lie in the 280-mp region. The relative rates at which these two maxima developed were independent of indicating that the reaction mechanism the initial pH of the was the same over this range of experimental conditions. Wolfrom and coworkers proposed that the diene type of spectra arose from a certain accumulation of the enols (IX) of the 3-deoxyosones (X). The latter were HC=O

I CHOH I CHoH - H & - I I

HC=O

I COH II

CH

HC=O

I c=o I

= I

CHz

CHOH

CHOH

CHOH

CHOH

I CHOH

R

R

I

I

R

I

I

rx

CHOH

I

HC=O

- HO

I c=o

I II

CH 2 7

CH

I

CHOH

I

R X

XI HC=O

I COH I\

HC=O

I

- HO

CHO

I

R

C-

II I CH II coI CH

R

(82) M. L. Wolfrom, R. D. Schuetz and L. F. Cavalieri, J . Am. Chem. SOC.,70, 514 (1948). (83) M. L. Wolfrom, R. D. Schuetz and L. F. Cavalieri, J . Am. Chem. SOC.,71, 3518 (1949). (84) The initial pH was varied only in the D-glucose experiments.

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

75

presumed to have undergone further dehydration to either 2-furaldehyde or 5-(hydroxymethyl)-2-furaldehyde,as Hurd and IsenhouF had suggested earlier. It should be pointed out that there is reason to doubt that the equilibrium between IX and X favors IX sufficiently for it to become apparent in the spectra of these mixtures. This judgment is based on the behavior of pyruvaldehyde, the ultraviolet spectrum of which shows little, if any, enol content.77On the other hand, compound IX would undoubtedly mediate in the formation of X, and its rate of conversion to X may not be rapid enough for it to escape detection. A more likely contributof12to the 230-mp maxima in the early stages of these reactions is the type of intermediate represented by XI. Wolfrom and coworkerss2suggested XI as an intermediate in these transformations; however, they did not favor this over X as the kind of entity responsible for the 230-mp absorption. It should be pointed out that mediation by XI in these reactions was indicated in the results of earlier experiments.*6e The reactions of certain sugar ethers and disaccharides in alkaline solution86bnow appear to be analogous to the deoxyosone formation by dehydration discussed above. Those methyl ethers in which a methoxyl group is either actually or potentially bela to a carbonyl group are more labile to alkali than are the free sugars, and they initially lose methanol instead of water. Disaccharides in which the glycosyl group is attached at the same position (as the methyl group mentioned above) behave analogously.86bExamples of such reactions taken from the recent literature are , turanose (XIII)?? those of 3-O-methyl-~-glucose,8~ laminaribiose (XII)and in which the end products are the metasaccharinic acids, 3-deoxy-~“mannonic acid” (XIV), and 3-deoxy-~-“ghconic acid” (XV). [The conditions used by Kenner and his coworkers are unusual in employing highly dilute solutions, low alkalinity (about 0.04 N ) , a low temperature, long time-periods, and an extremely high molar ratio of base to sugar.] Direct evidence for the involvement of 3-deoxy-~-‘~glucosone” has not as yet been obtained for these reactions either. Nevertheless, this mechanism conforms with the outcome of alkaline degradations of similar sugars and sugar derivatives, and other pathways appear unlikely. It is interesting that good yields of the metasaccharinic acids could be obtained in these experiments under such mild conditions. For example, Kenner and RichardsE6found that a reaction mixture consisting of 11.36 grams of 3-0(85) C. D. Hurd and L. L. Isenhour, J . A m . Chem. SOC.,64, 317 (1932). (85a) See Ref. 115. (85b) R. L. Whistler and J. N . BeMiller, Advances i n Carbohydrate Chem., 13, 289 (1958). (86) J . Kenner and G . N. Richards, J . Chem. SOC.,278 (1954). (87) W. M. Corbett and 3. Kenner, J . Chem. SOC.,3274 (1954).

76

J. C. SPECK, JR.

FHZOH

k

CHZOH

AH XI1

H < O ~ OH H HO.. H OH

H H

O HO

~

~

~

HOHzC H XI11

methyl-D-glucose in 3 liters of oxygen-free lime-water (0.0412 N ) gave a 96% yield of the crude metasaccharinic acids (isolated as the calcium salts) when it was allowed to stand a t 25" for 170 hours. XI1

I-

HC=O

I HCOH I

C&OCH

I

HCOH

I

HCOH

I

CH~OH

XI11 D-G~u-

1-

D-GIUCGW

HC=O

C OzH

I c=o

-CHIOH

,

I

CHP

I

HCOH

I HCOH I

CH~OH

+Hi0

,

COaH

I HOCH I

CH2

I

HCOH

I I

HCOH

+

I

HCOH

I

CH~OH XIV

I

CHe

HCOH

I I

HCOH CHiOH

xv

Aldoses having methoxyl groups at both C2 and C3 (they may be further methylated, also) do not undergo degradation by alkali to saccharinic acids. Instead, they appear to lose methanol in the manner described above, with the formation of methyl ethers of enols of 3-deoxyosones-which are stable in alkaline media, but which are transformed either to 2-furaldehyde or its derivatives by acids. (See Section VI, Part 1, of this Chapter for further discussion of these reactions.) These reactions of methyl ethers and disaccharides, besides clarifying somewhat the nature of initial dehydrations of the reducing sugars, are in themselves possible

H

LOBRY D E BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

77

side reactions in the Lobry de Bruyn-Alberda van Ekenstein transformation. Two other dehydration reactions, reversion and anhydride formation, may possibly complicate the transformations in acidic solutions. However, they seem to be generally unimportant. The reactions leading to the saccharinic acids and to 2-furaldehyde and its derivatives do not seem a t this point to warrant further consideration as side reactions, since they have already been mentioned in connection with primary dehydration and related transformations. 2. The Aldol Condensation Both aldolization and dealdolization become significant side-reactions in alkaline reaction mixtures because of the pronounced catalysis of the aldol condensation by hydroxide ion. (Except for ammonia and compounds possessing a primary or secondary amino group, which exert a special catalytic effect, other bases do not seem to catalyze the aldol condensation in aqueous solution.s*) Aldolizations of trioses, with formation of hexoses, have been observed in several inve~tigations.89-~~ Meyerhof estimated that the triose-hexose equilibrium mixture from the condensation of DL-glycerose with 1,3dihydroxy-2-propanone (in trisodium phosphate solution) contained 92 % of hexose. Berl and F e a ~ e lin , ~their ~ kinetic examination of this aldolization in sodium hydroxide solution, were unable to detect any triose by paper chromatography at the end of the reactions. Pyruvaldehyde formation complicates any glycerose or 1 ,3-dihydroxy-2-propanonereaction in alkaline medium, and this fact probably accounts for some of the disappearance of triose from these mixtures. Nevertheless, aldolizations of these short-chain sugars are side reactions to be reckoned with, whenever circumstances permit their occurrence. Nonenzymic dealdolization of higher sugars has yet to be demonstrated directly by isolation of short-chain sugars. However, recent investigationsespecially those of Wolfrom and Schumachers4 and of Blair and S o ~ d e n , ~ ~ having to do with alkaline degradations of D-fructose and D-glucose, respectively-have afforded strong evidence for occurrence of this kind of (88) L. P . Hammett, “Physical Organic Chemistry,” McGraw Hill Book Co., Inc., New York and London, 1940, p. 344. (89) A. Wohl and C. Neuberg, Ber., 33, 3095 (1900). (90) E. Schmitz, Ber., 46, 2327 (1913). (91) H . 0. L. Fischer and E. Baer, Helv. Chim. Acta, 19, 519 (1936). (92) 0. Meyerhof and W. Schulz, Biochem. Z., 289, 87 (1937). (93) W. Berl and C. E. Feazel, J. Am. Chem. Soc., 73, 2054 (1951). (94) M. L. Wolfrom and J. N . Schumacher, J. A m . Chem. Soc., 77, 3318 (1955). (95) M. G. Blair and J. C. Sowden, J. Am. Chem. SOC.,77, 3323 (1956).

78

J. C. SPECK, JR.

+

scission. The mixture, (D DL)-sorbose, was isolated in both Wolfrom and Schumacher's and Blair and Sowden's experiments, and its formation was explained as having been brought about by dealdolization-aldolization, rather than by wandering of the carbonyl group. The results of Gibbs' experimentsss on the degradation of ~-glucose-l-C~~ and of D-glucose3,4-C214 to lactic acid also indicate cleavage of this type. The following scheme shows the theoretical posibilities of aldolization-dealdolization in the fructose-sorbose system if dealdolization is limited to triose formation and if only glycerose undergoes epimerization." It should be pointed out, however, that Sowden and Thompson's findingssa indicate that this explanation for the conversion of D-glucose to D- and L-sorbose is no longer valid. CHzOH

I I

c=o CHzOH

I

c=o

I I HCOH I HCOH I

HOCH

CHzOH

I I

CHzOH

c=o

1,3-Dihydroxy-Zpropanone 7

+ HC=O

-

L

I

I HCOH I

CHzOH

I I

CHzOH

D-Glycerose

D-Sorbose

CHzOH

I c=o I HCOH I HOCH I HOCH I

HOCH HCOH

CHzOH

D-Fructose

HC OH

CHzOH

I

lt3-Dihydroxy-2-propanone 7

+

HC=O

I I

HOCH

L 7

c=o I

HOCH

I

HCOH

I

HOCH

I

CHzOH

CHzOH

CHaOH

L-Fructose

L-Glycerose

L-Sorbose

(96) M. Gibbs, J . Am. Chem. Soc., 72, 3964 (1950). (97) SchmitP found that the aldolieation of DL-glycerose in barium hydroxide solution, which is accompanied by isomerieation of the DL-glycerose to 1,3-dihydroxy-2-propanone, gave DL-fructose and DL-sorbose. Similarly, Fischer and B a e P

LOBRY D E BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

79

Although the aldol condensation is catalyzed by acids, it has not seemed to be of any significance as a side reaction in Lobry de Bruyn-Alberda van Ekenstein transformations which have been carried out in acidic media. 3. Miscellaneous Reactions

The formation of reductones and rearrangements of the carbon chain are possible side reactions which should be considered. Reductones appear to be formed in only small concentrations during Lobry de Bruyn-Alberda van Ekenstein transformations, and their occurrence is restricted to alkaline reaction mixtures. Nevertheless, their formation is of some significance, since, in the past, they may have been confused with the hypothetical enediols of the sugars (for further discussion of this point, see Section VII of this Chapter). Rearrangements of the carbon chain have been observed with noncarbohydrate a-hydroxyketones, but, apparently, only where the alcohol group was tertiary.67 Bothner-By and Gibbsss examined the transformation of Dglucose-l-C14 in calcium hydroxide solution and found no evidence for rearrangement in the products. As far as is known, rearrangements of the carbon chain are not involved in any Lobry de Bruyn-Alberda van Ekenstein reactions of the carbohydrates, although they may occur during aldolization-dealdolization and in the formation of saccharinic acids.

IV. CATALYSIS 1. Acid and Base Catalysis The Lobry de Bruyn-Alberda van Ekenstein transformation is usually, if not traditionally, placed in the category of alkaline isomerizations. This preoccupation with a base catalysis is somewhat surprising, for, although most Lobry de Bruyn-Alberda van Ekenstein transformations have been carried out in basic media, these reactions have long been considered to proceed via enolization which is subject to general acid and base catalysis. Moreover, the first report of one catalyzed by an acid dates back almost to the time of Lobry de Bruyn and Alberda van Ekenstein’s original experiments. Thus, in 1905, Ost26reported the isomerization of D-glucose in dilute sulfuric acid a t room temperature. The reaction was allowed to proceed under these conditions for 4 months, and, at the end of this time, D-glucose, isomaltose, and D-fructose (as the calcium D-fructosate) were isolated from the reaction mixture. found that, in the condensation of D-glycerose and 1,3-dihydroxy-2-propanonein 0.01 M barium hydroxide, only D-sorbose and D-fructosewere formed. Thus, aldolization of the trioses seems to favor trans configurations at C3 and C4 of the hexose products. (98) A. A. Bothner-By and M. Gibbs, J . Am. Chem. Soc., 73, 4805 (1950).

80

J. C. SPECK, JR.

Several other investigations of the transformation have been carried out under acidic conditions. Spoehr and Strain1*observed the conversion of D-fructose to D-glucose plus D-mannose at 37" in slightly acidic phosphate buffer (initial pH, 6.69). Similarly, Englis and Hanahange found that autoclaving of D-glucose in acidic phosphate buffers produced conlol residerable concentrations of ketose. Ashmarin and coworkersloO. ported the isomerization of D-glucose to n-fructose, as well as the reverse reaction, in formate, acetate, and succinate buffers at pH 4.2. 5-(Hydroxymethyl)-2-furaldehyde was also formed in these reaction mixtures. Petuely16 reported that tartaric, acetic, and citric acid bring about the conversion of D-fructose to D-glucose and 5-(hydroxymethyl)-2-furaldehyde, and that both of these reactions, and the isomerization of D-glucose to D-fructose, occur in a variety of acidic buffers. These experiments employing acidic media were at best only semiquantitative, and none comprised a systematic investigation of the catalysis. Nevertheless, they placed the transformation in a new light and broadened the opportunities for investigating its mechanism. From the dependence of the reactions on the concentrations of formate, acetate, or succinate ions, Ashmarin'"' lo1 argued for a general acid and base catalysis. The results of Petuely's experiments16implied the same kind of catalytic effect, and Petuely wrote the reaction as an enolization catalyzed by acids and bases. The simplicity of the DL-glycerose-1 ,3-dihydroxy-2-propanoneisomerization in acidic media, together with the application of the newer analytical techniques, has made possible a kinetic examination of this reaction (see Section VI, Part 3 of this Chapter for further discussion of these experiments).'? In this study, the magnitudes of the catalytic effects caused by such species as acetic acid and acetate ion were determined. These data confirmed previous reports of catalysis produced by Bronsted acids and bases for this kind of reaction. They also indicated the operation of general acid and base catalysis in all Lobry de Bruyn-Alberda van Ekenstein transformations. A number of hydroxides and carbonates of the alkali and alkalineearth metals have been used as catalysts for these reactions. Lobry de Bruyn and Alberda van Ekenstein considered the hydroxide ion to be responsible for their effect. Michaelis and Ronalo2 examined the transformation of D-glucose in several alkaline buffers and found that the re(99) D.T.Englis and D. J. Hanahan, J . A m . Chem. SOC.,67, 51 (1945). (100) P.A. Ashmarin and Y. S. Belova, Arch. sci. b i d . (U. S. S. R.),42, No. 3, 53 (1936). (101) P. A. Ashmarin and A. D. Braun, Arch. aci. bioZ. (U. S. S. R.), 42, No. 3, 61 (1936). (102) L. Michaelis and P. Rona, Biochem. Z . , 47, 447 (1912).

LOBRY DE BRUYN-ALBERDA

VAN EHENSTEIN TRANSFORMATION

81

action rates are proportional to the concentrations of hydroxide ion. The magnitude of the base catalysis by hydroxide ion in aqueous solutions must be relatively large, since, in aqueous systems, hydroxide ion is the strongest base. However, this does not exclude the possibility of effects caused by metal ions. The question as to whether certain metal ions catalyze these reactions is considered in the next Section. Pyridine has found some use as a catalyst for the transformationespecially in applications to ketose synthesis. The base has usually been employed as both catalyst and solvent (in anhydrous form). Under these conditions, it appears to act sluggishly-a conclusion which, since anhydrous pyridine solutions of a reducing sugar contain no acid except the sugar itself, is in accord with the idea that the transformation requires an acid as well as a base for its catalysis. The possibility of increasing the reaction rates by incorporating an appropriate acid catalyst, such as phenol, into these pyridine mixtures should be explored. Other nitrogenous bases which have been reported as catalysts for the transformation are amrnonialz8quinoline, and quinaldine.lO* Catalysis of Lobry de Bruyn-Alberda van Ekenstein reactions by a strongly basic, ion-exchange resin [Amberlite IRA-400(OH)] has now been Io6 Duff106has reported that alkaline impurities present in paper brought about such transformations of several sugars when paper chromatograms thereof were dried at 110'. Wolfrom and Shilling1OBBhave observed that water is an extremely poor catalyst for these reactions. Thus, it was necessary to heat an 80% solution of D-fructose at 113' for 16 hours in order to produce a 0.1% yield of D-glucose (isolated as 1,2,3,4,6-penta-O-acetyl-~-g~ucose). This conversion may have been aided by a slight oxonium-ion catalysis, since, although the initial pH of the reaction mixture was 6.9, the final pH was 2.7. l o 4 3

2. Metal-ion Catalysis The first intimation of a metal-ion effect appeared in the third of the original papers on the transf~rmation.'~~ In this article, the action of lead hydroxide on D-mannose, D-glucose, D-fructose, and D-galactose was compared with that of potassium hydroxide. Although D-glucose and D-mannose (103) H. Midorikawa, Kagaku Kenkyusho HGkoku, 26, No. 34, Chem. Sect., 33 (105) (1949); Chem. Abstracts, 46, 5110e (1951). (104) D. B . Buhler, R. C. Thomas, B . E. Christensen and C. H. Wang, J . A m . Chem. SOC.,77, 481 (1955). (105) C. N . Turton and E. Pacsu, J . A m . Chem. SOC.,77, 1059 (1955). (106) R. B . Duff, Chem. & Ind. (London), 898 (1953). (106a) M. L. Wolfrom and W. L. Shilling, J . Am. Chem. Soc., 75. 3557 (1951). (107) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. chim., 16, 92 (1895).

82

J. C. SPECK, JR.

were observed to undergo some change under the influence of lead hydroxide, the fall in optical rotation of these mixtures was not as great as it was in the presence of potassium hydroxide. For D-galactose, lead hydroxide produced the greater negative rotational change. The constancy of the rotations of the D-glucose- and D-mannose-lead hydroxide mixtures on varying the temperature at the end of the reactions was taken to indicate that no D-fructose had formed. Lead hydroxide also brought about alteration of D-fructose without seeming to produce either D-mannose or D-glucose. Lobry de Bruyn and Alberda van EkensteinSmlO8 later employed lead hydroxide to form the yeast-unfermentable mixtures108awhich they called “glutose” and “galtose”-the former from D-glucose, D-mannose, or D-fructose, and the latter from D-galactose. But, beyond this, they did not attempt to elucidate these effects by lead ion, and the problem has since remained virtually untouched. It has recently become clear that some of the alkaline-earth metal ions catalyze the transformation. Speck and coworkers” observed catalysis by calcium and barium ions for the DL-glycerose-1 ,3-dihydroxy-2-propanone interconversion in acidic buffers, and evaluated the magnitudes of these effects. Sowden and SchaffeP found that D-mannose disappears more rapidly in calcium hydroxide solution than it does when the transformation is catalyzed by barium hydroxide, and that the effect caused by barium hydroxide is greater than that by sodium hydroxide. Sowden and Schaffer observed another interesting phenomenon in these experiments. For the D-mannose-sodium hydroxide mixture, the rotation became more negative as the reaction proceeded. However, in the first 11 hours of the reaction in calcium hydroxide solution, the change in rotation was positive. Whether these alkaline-earth metal ions are capable of directing the course of the transformation is at present unclear. KusinloBreported that, when D-glucose reacts in calcium hydroxide solution at 25’, no D-fructose appears, whereas sodium hydroxide brings about formation of n-fructose under the same conditions of time and temperature. However, this claim is contrary to the findings of Lobry de Bruyn and Alberda van Ekenstein? Sowden and Schaffer,24 and Topper and Stetten,26 all of whom isolated D-fructose from the reaction of D-glucose with calcium hydroxide under comparable conditions.

V. USE OF

THE

TRANSFORMATION FOR SYNTHESIS

The Lobry de Bruyn-Alberda van Ekenstein transformation usually gives mixtures of aldoses and ketoses, as well as the products from the (108) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trau. chim., 16, !274 (1897). (1OSa) See L.Sattler, Advances in Carbohydrate Chem., 8, 113 (1948). (109) A. Kusin, Ber., 69, 1041 (1936).

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

83

various side-reactions. In general, the ketoses are much more easily isolated from these mixtures. It is, therefore, hardly surprising that the transformation has been employed almost exclusively for synthesis of ketoses. The aldose-ketose mixtures have usually been separated by oxidizing the aldoses with hypobromite. Occasionally, an aldose has been removed by fermentation-as in the isolation of D-tagatose from a D-tagatose-DLevene and Tipsonlo separated the mixtures galactose mixture.'*, 19, from the respective transformations of D-xylose and of L-arabinose by forming and fractionally distilling the isopropylidene derivatives. The reported yields in these ketose preparations range from less than 10% to around 50%. Nevertheless, this is the method of choice for preparing certain ketoses, especially where the starting aldose can be sacrificed. For some unknown reason, the best yields have consistently been obtained in the formation of D-gluco-heptulose from ~-g~ycero-~-gulo-heptose.~~-" The procedure of Pratt, Richtmyer, and Hudsona4for preparation of this substance is given here. One hundred grams of the aldoheptose in 1 liter of lime-water was kept a t 35" for 7 days, the [a]:' value changing t o f37" (in agreement with the two values +35" and +40" reported by Austins*). To this mixture, a t room temperature, were then added 100 g. of calcium carbonate and 14 ml. of bromine; the flask was shaken a t intervals during the day. The next morning, any excess bromine was expelled by aeration, and the solution was filtered, deionized by passage through Amberlite IR-120 and Duolite A-4 ion-exchange columns, and concentrated under diminished pressure. The rotation of the partially concentrated solution corresponded to a D-gluco-heptulose content of 59 g., in good agreement with Austin's estimation of 60% of ketose in the original equilibrium mixture. The solution was now concentrated t o a moderately thick sirup that was taken up in 200 ml. of methanol and allowed to crystallize. The average yield, including material from combined mother liquors, was 50 g. of D-glucoheptulose of [a]:'+65" t o +67" (in water). (If i t is desired t o recover the aldoheptonic acid also, the aqueous solution, prior to its deionization, is concentrated to 200 ml. and most of the calcium D-glycero-D-gulo-heptonateis precipitated by the slow addition of 600 ml. of 95% ethanol.)

No method has yet been devised for bringing about formation of a single product from a nonenzymic Lobry de Bruyn-Alberda van Ekenstein transformation. However, this feat should be possible, since the reactions involved are reversible. Yields can be improved by choosing catalysts which limit side-reactions. Thus, a low concentration of hydroxide ion minimizes the aldol condensation, the seriousness of which, as a side reaction, is sufficient reason for avoiding even moderate concentrations of hydroxide ion. It is interesting to note that lime-water and anhydrous pyridine, which have been the most used as catalysts in sugar preparations by these reactions, both meet this requirement. The hydroxide-ion concentrations of the former solution have usually been low, and the calcium (110) J. V. Karabinos, Advances i n Carbohydrate Chem., 7 , 113 (1952).

84

J. C. SPECK, JR.

ion probably provides an extra catalytic effect. Anhydrous pyridine contains no hydroxide ion; moreover, since this base is a tertiary amine, it should not catalyze the aldol condensation. OF THE MECHANISM VI. INVESTIGATIONS The history of the mechanism of the Lobry de Bruyn-Alberda van Ekenstein transformation begins with the first description of its reactions. Thus, Lobry de Bruyn and Alberda van Ekenstein2 proposed that the transformation might take place by intramolecular transfer of hydrogen in

HC=O

I HCOH I

--

CHOH

I'

HC

I

CHzOH

, o = I c=o I

-

. \ T O H CH

I

-

T--

HC=O

I

HOCH

1

hemiacetal intermediates which they considered were formed from the aldoses by addition and removal of water. Soon after this, Wohl and Neuberg"' suggested the enediol intermediate for aldose-ketose isomerization. The enolization mechanism apparently won nearly immediate acceptance and it has since dominated the field. The first 30 years of the transformation seem to have been marked more by speculations on the mechanism than by experiments designed to elucidate it. Nef was apparently inspired by the concept of sugar enolization. Nevertheless, most of the numerous investigations of sugar transformations in alkaline media which Nef and his students carried out were intended to test the reasonableness of enediol intermediates in cleavage reactions and in the formation of the saccharinic acids.l12 Few, if any, were directed to determining the intimate mechanisms of the Lobry de Bruyn-Alberda van Ekenstein reactions. Up to the time of the first experiments with 0-methyl sugars, which, incidentally, were partly designed to test Nef's hydration4ehydration scheme for sugar enolization,?s the outstanding investigation of these mechanisms was that of Michaelis and Rona.lWIn this work, both the catalytic effect by hydroxide ion and the ionization of D-glucose were measured. Michaelis and Rona's mechanism, involving epimerization and enolization through removal of an a-hydrogen as a proton, is almost identical with that which is currently accepted.

1. Experiments with 0-Methyl Sugars Most of the experiments having t o do with Lobry de Bruyn-Alberda van Ekenstein transformations of 0-methyl sugars were carried out during (111) See Ref. 89,page 3099. (112) For reviews of this early work, see W. L. Evans, Chern. Reus., 6. 281 (1929); 91, 537 (1942).

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

85

a period some 25 to 30 years ago by W. Lee Lewis and his students. These experiments led to the significant discovery of the possibility of epimerization of tetra-0-methylaldohexoses and tri-0-methylaldopentoses, both being types having an 0-methyl group112"at C2,as well as to the collection of evidence for what is now recognized as concomitant elimination of p-methoxyl groups from certain of these substances. In the first of these investigations, Wolfrom and Lewis22examined the under conditions favoring behavior of 2,3,4,6-tetra-O-methyl-~-glucose a Lobry de Bruyn-Alberda van Ekenstein transformation, and found that hie-water converts this sugar to 2,3,4,6-tetra-O-methyl-~-mannose, without formation of a ketose. Moreover, a substance, or substances, appeared which had the ability to absorb iodine, so that the results obtained in the determination of aldoses by means of alkaline iodine were approximately 40% higher than that calculated as corresponding to the expected aldose concentration. On acidifying these alkaline mixtures, the iodine values gradually fell to that corresponding to 100 % of aldose. Abnormally high iodine titers were not obtained with reaction mixtures in which D-glucose was the starting sugar. In view of these findings, Wolfrom and Lewis proposed that 2 , 3 , 4 ,6-tetra-O-methyl-~-glucose(XVI) proceeds to 2,3,4,6-tetra-O-methyl-~-mannose (XVIII) via the enediol monomethyl ether (XVII). That this enediol monomethyl ether was responsible for the high iodine values observed for these mixtures was also suggested, and the implication was made that it owed its existence in alkaline solution to the stabilizing effect of the rnethoxyl group a t C2. The almost identical in lime-water, as observed behavior of 2,3,4,6-tetra-O-rnethyl-~-mannose lent support to this mechanism. by Greene and HC 08

HC=O

CHsOCH

I

HCOCH,

I

HCOH

I

CHzOCHs XVI

HC=O

It

I HCOCHs I

1

CHsOCH

COCHs

I

bane ' acid

~

CH30CH

I

HCOCH,

I

HCOH

I

C H2 0 C Ha XVII

-

I

acid , ' base

CHoOCH

I

HCOCHs

I

HCOH

I

CHzOCHs XVIII

Nevertheless, later examination of the reactions of 2,3,4-tri-O-methyl(112a) A related example, in which the compounds have a N-acetyl group at C2, involves the interconversion of N-acetyl-D-gluoosamine and N-acetyl-D-mannosamine IS. Roseman and D. G. Comb, J . Am. Chem. Soc., 80, 3166 (1958)l. See F. Zilliken and M. W. Whitehouse, this volume, p. 237.

86

J. C. SPECK, JR.

~ - x y l o s eand ~ ~ 2,3,4-tri-O-methyl-~-arabinose~l~ in lime-water revealed a deviation from the previous pattern. Epimerization and high iodine values were both observed, as before. But methanol appeared in the alkaline reaction mixtures (none had been found in the 2,3,4,6-tetra-O-methylD-glucose experiments), and, on acidifying these mixtures, more methanol was released and 2-furaldehyde was formed. As shown in Table 111, the methanol produced after acidification was nearly twice that found in the alkaline solutions. Assuming the formation of 3 moles of methanol for one of 2-furaldehyde, and allowing for some destruction of 2-furaldehyde in the acid solutions, the yields of 2-furaldehyde agreed roughly with the total yields of methanol. The yields of 2-furaldehyde also paralleled the concentrations of the hypothetical, enediol, monomethyl ether, as deduced from the abnormal, iodine titers. Finally, it was found that neither the

Moles of Volatile Substances Formed pn Mole of Penlosc Sugar

Before acidification

I

~ftn acidijcation

~~

2,3,4-Tri-O-methyl-~-xylose 0.359, 0.342 0.30 2,3,4-Tri-O-methyl-~-arabinose

0.0006, 0.0006 0.790 0.07 0.051

0.178 0.167

methylated pentoses, 2,3,4-tri-O-methyl-~-xylose and 2,3,4-tri-O-methylL-arabinose, nor the corresponding free sugars form 2-furaldehyde at appreciable rates under conditions of acidity and temperature comparable to those employed for “de-enolizing” the alkaline reaction mixtures. These observations of demethylation and 2-furaldehyde formation led ineluctably to the conclusion that lime-water had caused these methylated pentoses to form (by probable loss of 1 mole of methanol per mole) substances which were exceedingly easily converted, by further loss of 2 moles of methanol per mole, to 2-furaldehyde in acid solution. Accordingly, Lewis proposed the following scheme [shown with 2,3,4-tri-O-methyl-~xylose (XIX) as the starting substance] for this sequence of reactions. The final experiments in this series by Lewis and coworkers were carried out with the hexose ether, 3-O-methyl-n-gluco~e.~~ This substance underwent conversion in lime-water a t 35” to 3-O-methyl-n-fructose, which was obtained crystalline in 29 % yield. The remainder of the mixture appeared (113) H.T.Neher and W. L. Lewis, J . Am, Chem. Soe., 63, 4411 (1931).

LOBRY DE BRWN-ALBERDA

HC=O

I HCOCHa I CHaOCH I HCOCHI I

CHzOH XIX

87

VAN EKENSTEIN TRANSFORMATION

HCOH

II I CHsOCH I

COCHa

&

- CHOH

A

HCOCH,

I

CHzOH

alkaline solution [Ca(OH)21

HCOH

1I

CCHaOCH I

1

--f

I HCOCHa I CHzO

HC=O

C H a t ! q HCOCHa CHzO acid solution

HC=O

I

i.] CH

CHO

to consist almost entirely of 3-O-methyl-~-glucose, only about 3.5 % representing saccharinic acids. These reaction mixtures did not give high iodine values. However, methanol was found in the alkaline solutions. The apparent yields of methanol were small under these relatively mild conditions; at higher temperatures and in more strongly alkaline mixtures, production of methanol and saccharinic acid was marked. The normal iodine values were attributed to the absence of a methoxyl group at C2. This behavior was also cited as evidence that 3-O-methyl-~-glucosedoes not form a 2 3-enediol monomethyl ether. These investigations by Lewis and his students left some important mechanistic questions unanswered, not the least of which concerned the nature of the intermediates in these transformations. Wolfrom sought to clarify this problem by synthesising114and examining the properties of 2 3,4,6-tetra-O-methyl-~-glucosene-l, 2 (XX)*16 which had been suggested by Raymond116 as possibly mediating in the interconversion of 2 3 , 4 6tetra-0-methyl-D-glucose and 2 3 4 6-tetra-O-methyl-~-mannose. It was found that the 2 )3 4 6-tetra-O-methyl-~-glucosene-l, 2 absorbed 4 atoms )

)

)

)

(114) M. L. Wolfrom and D. L. Husted, J . Am. Chem. Soc., 69, 2559 (1937). (115) M. L. Wolfrom, E. G . Wallace and E. A. Metcalf, J . Am. Chem. sbc., 64, 265 (1942). (116) A. L. Raymond, i n “Organic Chemistry,” H. Gilman, ed., John Wiley and Sons, New York, 1938, Vol. 11, p. 1512.

88

J. C. SPECK, JR.

of iodine per mole and that, when it was treated with acid, the iodine absorption gradually diminished to about 1.4 atoms per mole. However, acid converted this substance to 5-(methoxymethyl)-2-furaldehyde (XXI1)-apparently through the intermediate osone (=I)--instead of to 2,3,4 ,6-tetra-O-methyl-~-glucoseand 2,3,4 ,6-tetra-O-methyl-~-mannosc. The iodine absorption data from Wolfrom and Lewis’s investigation 2 been responsible indicate that, had 2,3,4,6-tetra-O-methyl-~-glucosene-l, HC=O

COCHa

C HHCOCH, aI l : g

I

HCO

I

CH2OCHI

xx

I c=o I CH +2Ha - 8 CHaOH ’ 11 CH I HCOH

I

CH2OCHa XXI

HC=O

I II - Ha0 CH ’ I CH II C-

co-

I

CH2OCHs XXII

for the high values, its concentration would have been 0.4 molar, and it would have been necessary for most of this substance to have been conand 2,3,4,6-tetra-O-methylverted to 2,3,4,6-tetra-O-methyl-~-glucose D-mannose on acidification, in order to account for the high recoveries (85% as the purified and separated anilides) of the latter substances in 2 these earlier experiments. Thus, 2,3,4,6-tetra-O-methyl-~-glucosene-l, was ruled out, not only as an intermediate in this Lobry de Bruyn-Alberda van Ekenstein transformation, but also as a major contributor to the phenomenon of high iodine absorption. Recent experiments on alkaline degradations of sugars, which have demonstrated the facile loss of a methyl or glycosyl group beta to the carbonyl group, provide a reasonable explanation for the demethylations observed in Lewis’s work. Of these investigations, one of the most pertinent for present consideration was carried out by Kenner and Richards”’ on 2,3-di-O-methyl-~-glucose (XXIII). This substance gave no saccharinic acids in oxygen-free, calcium hydroxide solution at 15 to 20”. Instead, it lost methanol and formed another substance, which underwent transformation to 5-(hydroxymethyl)-2-furaldehyde in acid solution. Although this acid-labile intermediate to 5-(hydroxymethyl)-2-furaldehyde was not isolated in pure form, the substance could be concentrated and ozonized. The results obtained from its oaonolysis indicated that it was the methyl ether of the enol of 3-deoxy-~-“g~ucosone” (XXIV). Thus, analogous structures, represented by XXV, should probably be written for the acid-labile (117) J. Kenner and G . N. Richards, J . Chem. SOC.,2921 (1956).

LOBRY D E BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

89

products from the alkaline demethylations of 2,3,4-tri-O-methyl-~xylose and 2,3,4-tri-O-methyl-~-arabinose. Unfortunately, Kenner aiid Richards did not examine the action of alkaline iodine solutions on their acid-labile intermediate to 5-(hydroxymethyl)-2-furaldehyde. The identities of the acid-labile substances responsible for the high iodine values in Lewis’ investigations remain unknown. Substances HC=O

I

COzH

1 HC=O

I

Ca(OH)s (-CHsOH)

I

HCOH

’ +

HC=O

I HCOCHs CH,OCH

osonolyuia

I

I

HCOH

I

CHyOH

HC=O

I COCHs II CH I HCOH I HCOH I

I I HCOH I HCOH

CHzOH

HC=O

CHzOH I

XXIII

I

acid

C-

XXIV

I1 I CH II coI

CH

CHzOH HC=O CHOCHs

I

CHOCHI

HC=O

HC=O

I

Ca(0H)z

I COCH, II

I

CH

I

I

I

CHOCHs

CHOCHs

CHzOH

CHzOH

I

acid

-,

CH CHO

xxv represented by XXV, or their analogs, appear to be good possibilities provided that those derived from the tetra-0-methylaldohexoses take up large amounts of iodine-in the neighborhood of 10 atoms per mole in order to conform with the high recoveries of the aldoses in these experi-

90

J. C. SPECK, JR.

ments and the apparent absence of methanol in the 2,3,4,6-tetra-Omethyh-glucose mixture. However, it should be pointed out that, since appreciable yields of methanol were obtained from the alkaline 2,3,4-tri0-methyl-D-xylose and 2 ,3 ,4-tri-O-methyl-~-arabinosemixtures, it would appear that equivalent concentrations of the intermediates represented by XXV should have accumulated. Moreover, if their iodine absorptions were as large as 10 atoms per mole, the iodine values for these mixtures should have been much higher than were actually observed. A physicochemical investigation of the reaction of 2,3 ,4 ,6-tetra-Omethyl-D-glucose in lime-~ater~~'" failed to shed any more light on this problem, 2. Experiments Involving Deuterium and Tritium Exchange

a. Deuterium Exchange Reactions of D-Glucose.-The well known exchange of carbon-bound hydrogen for deuterium, which occurs when enoliaation and related reactions are carried out in deuterium oxide, has served as an important tool for investigating reaction mechanisms. It was first applied to examination of the Lobry de Bruyn-Alberda van Ekenstein mechanism by Fredenhagen and Bonhoeffer,ll*who carried out isomerhations of D-glucose and 2 ,3 ,4 ,6-tetra-O-methyl-~-glucosein alkaline deuterium oxide. Both calcium hydroxide and potassium hydroxide were employed as catalysts, saturated solutions of the former being used for most of the experiments. Fredenhagen and Bonhoeffer reported that appreciable quantities of carbon-bound deuterium (0.26 to 1.73 atoms per mole of D-glucose) appeared in the mixtures of products from the D-glucose reaction when it was carried out at about 40". 2 , 3 , 4 , 6-Tetra-O-methyL~glucose was reported as giving similarly (at 42") a mixture of substances which contained up to 1.08 atoms of deuterium per mole. On the other hand, no significant incorporation of carbon-bound deuterium into the mixture from the D-glucose isomerization was observed at 25", despite the observation in parallel experiments of approximately a 27 % conversion of D-glucose into ketoses. It was only when excess calcium hydroxide, instead of a saturated solution,11ewas used for the D-glucose reaction at 25" that deuterium exchange (1.57 atoms per mole) was found to take place. The behavior of 2 ,3 ,4 ,6-tetra-O-methyl-~-glucoseat 25" was not described. In view of these data, Fredenhagen and Bonhoefferl**assumed two different pathways for the transformation. They accepted an enolization (117a) J. H. Simons and H. C. Struck, J. Am. Chem. Soc., 66, 1947 (1934). (118) H. Fredenhagen and K. F. Bonhoeffer, 2. p h y s i k . Chem. (Leipzig), A181, 392 (1938). (119) Aqueous sugar solutions dissolve considerably more calcium hydroxide than can be dissolved by water. The saturated calcium hydroxide solutions referred to are presumed to have been made up with water.

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

91

mechanism for the reactions a t higher temperatures and (in the presence of excess calcium hydroxide) at lower temperatures, since these were the conditions under which exchange of deuterium occurred. But, for the isomerization at lower temperatures in the absence of an excess of calcium hydroxide, they proposed the dimer (XXVI) as an intermediate. This, according to R HC=O

I

HOCH

I I HCOH + O=CH I R

R

I

HOC-0-CH

+

I I HC-0-COH I R

CHzOH

I I

2 CEO

R

XXVI

their view, should permit of intramolecular hydrogen migration and no exchange with the solvent. Later, Goto120published data for the behavior of D-glucose at 25" in saturated, calcium hydroxide solutions (enriched with deuterium oxide) which, he claimed, confirmed Fredenhagen and Bonhoeff er's observations. Goto pointed out, however, that the mechanism involving the dimeric intermediate lacked kinetic proof and permitted no known role for the catalyst. Thus, the first experiments employing deuterium exchange cast considerable doubt on the concept of an obligatory enolization for the Lobry de Bruyn-Alberda van Ekenstein transformation. Confidence in the older, enol mechanism has only recently been restored by the work of Sowden and SchafferZ4and of Topper and Stetten.26Since these later investigations differ in approach and in some of the conclusions arrived at, they will be discussed separately. Topper and Stetten's workz6involved: (1) the reaction of D-glucose-1-d in ordinary water saturated with calcium hydroxide, and (2) the reaction of D-glucose in deuterium oxide saturated with calcium hydroxide-&. These isomerizations were carried out a t both 25" and 35". In the experiments with D-glucose-1-d a t 35", the D-mannose isolated (as the phenylhydrazone) contained 44 % of the deuterium in the starting substance, all of which was retained a t C l , whereas the D-fructose isolated (as the phenylosazone)121 retained 94% of the deuterium. A similar result (100% retention of deuterium) was reported for the D-fructose isolated from the reaction at 25". These figures for D-fructose were based on the assumption that 50% of the (120) K. Goto, Nippon Kagaku Zasshi, 63, 217 (1942); Bull. Chem. Soe. Japan, 18, 174 (1943). (121) The isolation procedure for D-fructose, in which D-glucose was removed by iodine oxidation, was checked for D-glucose contamination by adding ~ - g l u c o s e - C ~ ' ~ t o the mixture, before isolation, and examining the product for radioactivity.

92

J. C. SPECK, JR.

deuterium attached at C1 is lost on conversion to the phenylosaxone. In the second series of experiments, which were carried out with D-glucose in deuterium oxide, the D-fructose isolated contained 1.68 and 1.55 atoms of deuterium from the reactions at 35" and 25", respectively. Since the reaction times were nearly the same as those in Fredenhagen and Bonhoeffer's and Goto's investigations, the results obtained in deuterium oxide completely contradicted those of the earlier work. Topper and StettenZ6interpreted the data from their experiments with D-glucose-1-d in the following way. First, they assumed an enediol mechanism for the transformation. Then, since the D-mannose obtained from D-glucose-1-d contained only about half as much deuterium as did the D-glucose-1-d, whereas the D-fructose was supposed to retain nearly all of the deuterium originally present in this starting substance, they reasoned that the D-mannose was formed from D-fructose (with random exchange of one hydrogen or deuterium atom in the process) instead of directly from D-glucose, which would require retention of all deuterium bound at C1. In order to account for this they proposed a trans-enediol intermediate in the transformation of D-fructose into D-glucose, and a cis-enediol for the conversion of D-fructose into D-mannose. HC=O

I HCOH I HOCH I HCOH I HCOH I

CHzOH D-~~UCOSe

HOCH

CHzOH

II

COH

I

-

4

HOCH

I

HOCH

-

2

HCOH

I I

HCOH CHzOH trans-enediol

HCOH

I

HCOH

I I

HCOH CHzOH D-fructose

HC=O

II

I c=o I

I

HOC

HOCH

HOCH

HOCH

I

I

=

I

-

2

HCOH

I I

HCOH CHzOH cia-enediol

I

HCOH

I I

HCOH CHzOH D-mannose

In explaining the results of the second series of experiments, in which the D-fructose from the isomerization of D-glucose in deuterium oxide was found to contain 1.55 to 1.68 deuterium atoms per mole, Topper and Stetten found it necessary to assume that deuterium exchange had taken place at some other position, in addition to that occurring at C1, since a maximum exchange of one hydrogen atom at C1 with the solvent is implied in the preceding argument. They suggested that the additional exchange occurred at C3. Sowden and Schaffer24also examined the reaction of D-glucose in saturated deuterium oxide-calcium hydroxide-& at 25". All three of the principal components of these reaction mixtures, D-glucose, D-mannose, and D-fructose, were isolated in these experiments and their content of deute-

LOBRY D E BRWN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

93

rium determined. The concentrations of D-glucose and D-fructose in the mixtures were estimated by radioisotope dilution analysis-that of D-mannose was obtained by correcting the weight of the phenylhydrazone isolated. Sowden and SchafferZ4 also determined the distribution of deuterium along the carbon chain of D-fructose by converting it to 2 , 3:4,5-di-O-isopropylidene-D-fructopyranoseand oxidizing this derivative to 2 ,3 :4,5-di-O-isopropylidene-2-keto-~-"gluconic"acid. The deuterium contents of both of these isopropylidene derivatives were measured and are shown, together with the data for the unsubstituted hexoses, in Table IV. Sowden and Schaffer's data24 for the deuterium content of D-fructose agree quite well with the value obtained by Topper and Stetten.26Moreover, the deuterium content observed for D-mannose further contradicts TABLEIV Sowden and Schaffer's Data24 for the Isomerization of D-Glucose in Deuterium Ozide-Calcium Hydroxide-d2 at $6"" Concentrotion Substance

D-glucose concentration) ~

66.5 D-Glucose D-Fructose 28.6 0.8 D-Mannose 2,3:4,5-Di-O-isopropylidene-~-fructopyranose Potassium 2,3 :4,5-di-O-isopropylidene-2-keto-~"gluconate" 0

0.13 1.74 f 0.1 1.39 f 0.1 1.63 z!= 0.1 0.14 f 0.01

Reaction time = 21 days.

the claim that no exchange occurs under these conditions. However, as pointed out by Sowden and Schaffer,24these data are inconsistent with Topper and Stetten's assumption26 of an exchange of only one hydrogen atom a t C1, as well as with their mechanism which assigns to D-fructose the role of a necessary intermediate in the transformation of D-glucose to D-mannose. The deuterium contents of potassium 2 ,3 :4,5-di-O-isopropylidene-2-keto-~-"gluconate" and of 2 , 3 :4 ,5-di-O-isopropylidene-~-fructopyranose clearly indicate that nearly all of the deuterium in the D-fructose isolated from the mixture was a t C1. The relative proportions of deuterium in the D-fructose and the D-mannose show that not all of the D-mannose could have been formed directly from D-fructose, for such a course would have required that D-mannose have a deuterium concentration at least as high as that of the D-fructose. Since the implications contained in the earlier work employing deuterium exchange, particularly that of Fredenhagen and Bonhoeffer,118would appear

94

J. C. SPECK, JR.

to have been quite widely accepted, comment on the discrepancies between the data from the older and the more recent investigations seems appropriate. Fredenhagen and Bonhoefferl'* carried out their experiments on a very small scale and, instead of isolating the products of the reaction before analyzing for deuterium, they determined carbon-bound deuterium for the entire mixture. Examination of their data indicates a low order of reproducibility. Furthermore, it is difficult to account for the deuterium exchange reported as occurring at 25" in the presence of excess calcium hydroxide when none was obtained in saturated calcium hydroxide solution. Examination of Goto's data, which were obtained by carrying out the reactions on a somewhat larger scale in dilute deuterium oxide and by determining deuterium in both the water and the mixture of products, reveals an even lower order of reproducibility. It appears, therefore, that the more recent investigations invalidate the concept, for the Lobry de BruynAlberda van Ekenstein transformation in lime-water at 25O, of any pathway which does not permit exchange of carbon-bound hydrogen with the solvent. b. Deuterium and Tritium Exchange Reactions Catalyzed by Triose Phosphate Isomerase, Phosphomannoisomerase, and Phosphog1ucoisomerase.-It has recently been shown that three enzymes which catalyze aldose-ketose isomerizations also bring about exchange of carbon-bound hydrogen with the solvent. Rieder and RoselZ2observed what is undoubtedly a stereospecific incorporation of tritium (from tritium-enriched water) into 1 ,3-dihydroxy-2-propanone phosphate under the influence of triose phosphate isomerase. These investigatorslza had already found that muscle aldolase catalyzes replacement of hydrogen by tritium at the carbinol carbon atom of lf3-dihydroxy-2-propanonephosphate. The stereospecificity of the exchange at this carbon atom, as catalyzed by both muscle aldolase and triose phosphate isomerase, was established in the following manner: 1,3-dihydroxy-2-propanone phosphate, which had been labeled with tritium (T) in the presence of muscle aldolase, was incubated with triose phosphate isomerase. Also, 1,3-dihydroxy-2-propanonephosphate, which had been labeled by the action of triose phosphate isomerase, was treated with muscle aldolase. No significant lowering of the specific activity of the labeled 1,3dihydroxy-2-propanone phosphate occurred in either of these experiments, indicating that neither enzyme produces random labilization (or displacement) of hydrogen, and that different hydrogen atoms are affected by the different enzymes. lz6observed that D-glucose 6-phosphate incorporates approximately 1 atom of deuterium per mole when it is allowed to react with (122) S. V. Rieder and I. A. Rose, Federation Proc., 16, 337 (1956). (123) I. A Rose and S. V. Rieder, J . Am. Chem. SOC.,77, 5764 (1955) (124) Y. J Topper, Federation Proc., 16, 371 (1956). (125) Y. J Topper, J . Biol. Chem., 226, 419 (1957).

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

CHiOP

CHIOP

H

H

I

95

I

l13-Dihydroxy-2-propanone phosphate

II

CHzOP

T (where P denotes the phosphate group)

phosphoglucoisomerase in deuterium oxide. By converting the labeled Dglucose 6-phosphate to the phenylosazone, which was deuterium-free, this incorporation was established as having taken place at C2. Topper126also H

I I HOCH I HCOH I HCOH I

I I c=o I HOCH I HCOH I DCOH

DC=O HCOH '

(phoaphogz~oi 8 o ~ a a sH , a)

CHzOP XXVII DC=O

I

. cis-enediol

HCOH

I

CHzOP D-Fructose-1-d &phosphate D

I

HOCH

HCOH

HOCH

c=o

I

HCOH

I

CHzOP D-Mannose-I-d 6-phosphate

(pliosplionurnno-

Momaclae,

I

HCOH

I I

HCOH CHzOP D-Fructose-1-d 6-phosphate

deuterium exchange

96

J. C. SPECK, JR.

reported that phosphoglucoisomerase and phosphomannoisomerase affect different hydrogen atoms at C1 of D-fructose 6-phosphate. When D-glucosel-d 6-phosphate (XXVII) was incubated in water with phosphoglucoisomerase, no loss of deuterium to the solvent occurred. On the other hand, a mixture of phosphoglucoisomerase and phosphomannoisomerase brought about an exchange of deuterium (D) for hydrogen. Topper again decided that cis- and trans-enediols are the actual intermediates in these isomeriaations. 3. Kinetic Investigations Nearly all of the investigations of the kinetics of Lobry de Bruyn-Alberda. van Ekenstein reactions have failed because of the complications imposed by side-reactions. It has only recently been found that good kinetic data can be obtained for the DL-glycerose-1 ,3-dihydroxy-2-propanone intercon~ e r s i o n ?This ~ Section mainly concerns the work with this simple system, which contains implications for the mechanisms of all Lobry de BruynAlberda van Ekenstein reactions, and the attempts to extend it to the higher sugars. The m-glycerose-1 ,3-dihydroxy-2-propanoneisomerization was found to proceed at measurable rates at 50" in acidic buffers. Moreover, under these conditions, the irreversible dehydration to pyruvaldehyde appeared to be the only side-reaction of any consequence. It was actually fortunate that pyruvaldehyde formation occurred, since this permitted examination of the isomeriaation and dehydration reactions within a single system. Periodate oxidation of these reaction mixtures made possible a quantitative determination of all three components-glycerose, 1,3-dihydroxy2-propanone1and pyruvaldehyde-at any time. Since each mole of glycerose reduces 2 moles of periodate, whereas 1,3-dihydroxy-2-propmone and pyruvaldehyde each reduce only 1 mole per mole, the change in periodate consumption by such a mixture is 1 mole for each mole of glycerose which disappears. Similarly, when 1,3-dihydroxy-2-propanoneis the starting substance, the change in periodate consumed equals the increase in glycerose concentration. In either circumstance, the rate of change in periodate consumption expresses the rate of change in the glycerose concentration, and so periodate titers were employed to this end in these experiments. The total of the glycerose and 1,3-dihydroxy-2-propanoneconcentrations was conveniently measured by determining the formaldehyde liberated during periodate oxidation. The pyruvaldehyde concentration was obtained from the difference between this total (of the glycerose and 1,3-dihydroxy-2propanone concentrations) and the initial triose concentration. Good first-order plots were obtained with DL-glycerose as the starting substance; these were especially valuable for investigating the catalysis of

LOBRY D E BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

97

these reactions. In acetate buffers, the observed rates were directly proportional to the first power of the concentrations of acetic acid and acetate ion and there was no measurable catalysis by the solvent (water) or by oxonium ion. Similar results were obtained in formate and trimethylacetate buffers. Calcium and barium ions increased the rate when they were introduced into the acetate buffers at constant ionic strength, thus definitely establishing catalysis by these species in a Lobry de Bruyn-Alberda van Ekenstein reaction. This effect, which was also first-order in respect to the metal ion, depended on the presence of such a species as acetate ion. TABLEV Variation i n the Ratio of the Initial Concentration of I,$-Dihydrozy-P-propanoneto that of Pyruvaldehyde as Formed from m-Glycerose i n Different Acidic Bu,fers1z6 BUfftT

Acetate

0.20 0.30 0.10 0.30 0.133 0.40

Formate

0.20 0.133

0.20 0.40

Trimethylacetate

0.05 0.05

0.20

0.20 0.30

0.10

4.6 4.8 4.8 5.0 average for 8 determinations = 4.8 f 0.2 3.5 4.4 average for 6 determinations = 3.2 & 0.7 4.3 6.8 average for 4 determinations = 5.4 j = 1.1

The following expression sums up the catalytic effects by acetate buffers, for which the most data were obtained, and by calcium ion: -dlQl/dt

=

(k11HOAcl

+ kz[OAce] + k~[HOAclLOAce]+ k&2a@@l[OAcel)[03. 6

In this equation, [GI = the m-glycerose concentration, kl = 1.0 X 10mole-' minute-', kz = 49 x 10-6 moles-' minutes-', kt = 1.0 x mole-2 minute-', and kq = 57 X lC4molesL2minutes-l. Ratios of the initial rate of formation of 1,3-dihydroxy-2-propanoneto that of pyruvaldehyde from glycerose were determined over a considerable range of conditions and found to be nearly constant,lZ8as shown in Table V. These data were interpreted as indicating that pyruvaldehyde and 1,3dihydroxy-2-propanone are formed from a common intermediate. An estimate of the equilibrium constant for m-glycerose-1 ,3-dihydroxy2-propanone interconversion was also made in this investigation by start(126) A different mechanism appeared t o prevail under very strongly acidic conditions, since pyruvaldehyde was the only product detected from the reaction of glycerose in M perchloric acid a t 50".

98

J. C. SPECK, JR.

ing with 1,3-dihydroxy-2-propanone(which the equilibrium favors) and following the ratio of the concentrations of 1 ,3-dihydroxy-2-propanoneto DL-glycerose until this had become nearly constant. This is shown in Fig. 1. The precision obtained with periodate oxidations in following the triose reactions led to attempts to apply the procedure to hexose transformations. It is theoretically possible to follow Lobry de Bruyn-Alberda van Ekenstein isomerizations of higher sugars by periodate oxidimetry on account of the difference in periodate uptake by the aldose and ketose moieties. Thus, in

3

6

9

12

MINUTES X

16'

15

18

FIG.1.-Change in Ratio of Concentration of 1,3-Dihydroxy-2-propanone(D) to DL-Glycerose (G) with Time, Showing Extrapolation to an Equilibrium Value of Approximately 17.

the D-glucose-D-fructose-D-mannose system, D-glucose and D-mannose each consume about 5.0 moles of periodate per mole, and each mole of D-fructose reduces approximately 4.7moles. The exact amounts are easily determined empirically for a given set of conditions. If the optical rotations of such sugar mixtures are also measured, it becomes possible to determine the concentrations of all three components. The actual application of this scheme to following the transformation of n-glucose in very dilute, oxygen-free, sodium hydroxide solutions proved, nevertheless, disappointing.'*' The apparent D-mannose concentrations became negative soon after the reaction had begun, indicating the formation (127) D. S. Miyada, Ph. D. Thesis, Michigan State Univ., East Lansing, 1953.

LOBRY D E BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

99

of products other than D-mannose and D-fructose. Hence, it was not possible to investigate even the early stages of these reactions in this manner. A similar analytical scheme for following the transformation of 3 , 4 ,6tri-0-methyl-D-fructose gave somewhat better results.lZ8I n these experiwas determined ments, the disappearance of 3,4,6-tri-O-methyl-~-fructose by measuring the formaldehyde released on periodate oxidation. The periodate consumption by these mixtures served as a check on the formation of products other than 3 ,4,6-tri-O-methyl-~-glucoseand 3 ,4,6-tri-0methyl-D-mannose, since each of the 3 ,4 ,6-tri-O-methylhexoses consumes 1 mole of periodate per Actually, the periodate titers increased approximately 20 % during the time-period that the apparent concentration of 3 ,4,6-tri-O-methyl-~-fructosediminished to about 60 % of the initial concentration (when the reaction was carried out in sodium hydroxide solution). The change in periodate consumed was attributed to occurrence of demethylation. When the transformation was carried out in either calcium hydroxide or barium hydroxide solutions, the periodate consumption increased markedly. Hence, calcium and barium ions appear to catalyze this side r e a ~ t i 0 n . I ~ ~ Although examination of the kinetics of Lobry de Bruyn-Alberda van Ekenstein reactions for the higher sugars has thus far proved unfruitful, some reliable data have been obtained for the progress of such reactions under conditions less rigidly controlled than a kinetic investigation would require. For example, Sowden and Schaffer's experiments on the transformations of D-glucose, D-mannose, and ~-fructose,'~ in which the products and reactants were isolated and their concentrations (except for those of D-mannose) determined by radioisotope-dilution analysis, have undoubtedly afforded the best information available concerning the rates of these particular reactions.

STATUS OF VII. PRESENT

THE

MECHANISM

The evidence from experiments involving deuterium and tritium exchange for hydrogen, as well as that from investigations of its catalysis, (128) J. C. Speck, Jr., and D. S. Miyada, Abstracts Papers A m . Chem. SOC.,128, 20D (1955). (129) It was found t h at 3,4,6-tri-O-methyl-~-fructose consumed the theoretical quantity of periodate in 8 t o 12 hours in bicarbonate buffer, as determined by the procedure of P. Fleury and J. Lange, J . pharm. chim., 17, 107 (1933).Also, at somewhat higher concentrations of reagent, the theoretical quantity of formaldehyde was obtained in 2 hours from this sugar by the procedure of J. C. Speck, Jr., and A. A. Forist, Anal. Chem., 26, 1942 (1954). These results are higher than most of those reported by W. E. A. Mitchell and E. E. Percival, J . Chem. SOC.,1423 (1954) for longer oxidation times. (130) J. Kenner and G . N. Richards, J . Chem. SOC.,3019 (1957),have recently reported the same kind of catalytic effects in the formation of metasaccharinic acids from 3-O-methyl-~-glucose.

100

J. C. SPECK, JR.

now demands that all such transformations, both enzymic and nonenzymic, proceed by an enolization type of mechanism. Whether or not any one of these reactions requires actual enol (enediol) formation is a question which will be considered. Thus far, neither the existence of an enediol intermediate in these reactions nor the necessity for its involvement has ever been convincingly demonstrated. Kusin’s claim to having obtained evidence for such enediols in alkaline D-glucose mixtures10ehas been refuted by Petuely and Kunssberg,lsl who have presented evidence indicating that the substances in these mixtures which reduce Tillmans’ reagent (2,6-dichlorophenolindophenol) are quite stable after acidification. Petuely and Kunssberg pointed out that the substances which exhibited this strong reducing action were not sugar enediols, but were probably reductones, such as triose reductone (2,3-dihydroxy-2-propenal).Topper and Stetten’s mechanismz6for the C2epimerization of D-glucose via a cis-enediol-D-fructose-trans-enediolschemezb is without justifiation, as the earlier demonstration of C2-epimerization of 2,3,4,6-tetra-O-methyl-~-glucose by Wolfrom and Lewiszz clearly indicates. Neither are there grounds for assuming that either epimerizations or isomerizations, under the influence of enzymes, require these cis- and trans-enediol intermediates as TopperlZ6has postulated. Although it is true that all the known, enzyme-catalyzed, aldose C2-epimerizations proceed through the corresponding ketose (and require two different enzymes), the direct C3-epimerization of the ketoses, D-threo-pentulose 5-phosphate and D-erythro-pentulose 5-phosphate, is now well known. It seems probable that, in time, aldose epimerases will also be discovered. isomKinetic evidencefrom the DL-glycerose-1 ,3-dihydroxy-2-propanone eriaation has indicated that aldose-ketose isomerization and formation of a 3-deoxyosone proceed through a common intermediate. Ashmarin and coworkers’ loo*lol and Petuely’s observations,16which indicate general acid and base catalysis of 2-furaldehyde formation, tend to support such a mechanism, since 2-furaldehyde and its derivatives may indeed be formed from 3-deoxyosones. The following scheme, in which Ae is a Bronsted base and HA is its conjugate acid, is in harmony with these facts for nonenzymic epimerization (shown only at C2), aldose-ketose isomerization, and the dehydration to a 34eoxyosone. Here, aldose-ketose isomerization is shown as progressing through the hybrid anion (XXVIII), through the enediol, and, finally, through the hybrid anion (XXIX).18zThe hybrid anion (XXVIII) is also (131) F. Petuely and U. Kunssberg, Monatsh., 84,116 (1963). (132) Other possible mechanisms for formation of the enediol are those involving either a simultaneous attack by HA and Ae (on the aldoses or the ketose) or an initial attack by HA, instead of Ae.

HC=O

I I

+Ae

HOCH CHOH I

R

11 HC=O

I I CHOH I

WOH HC=O

I I

HCOH

II

R

HCOH

+Ace

CHOH

+

5:

HA

+

HCOe

RI

II I CHOH I

COH

I CHOH I

+ Ae

R

COH

R XXVIII

I HC=O

I

c=o I 4- Ae + H20

CHz

I

R XXX

HC=O

*

I 11 CH I

COH

+ Ae + HzO

R

HCOH

II c o* I CHOH I

CHzOH

I

c=o

I

CHOH

+ Ae+

R

1 A

HCOH

I c=o I

CHOH

I

R XXIX 101

+ HA

102

J. C. SPECK, JR.

proposed as an intermediate for the formation of the deoxyosone (XXX). An interesting variation of this mechanism is one which Berl and Feazelg3 suggested (but did not favor over mediation by the enediol). I n this mechanism, the hybrid anions (XXVIII and XXIX) resolve into a single resonance hybrid (XXXI) which, because of hydrogen bonding, allows exchange of hydrogen between oxygen atoms at C1 and C2, with bypassing of the enediol. The catalytic role of metal ions is probably one of facilitating removal of a proton from either an aldose or a ketose, by forming a complex such as that shown for calcium ion (XXXII). Thus, by either of the alternative routes mentioned above, such an ion is a t once a catalyst for all of the TABLEI VI Equilibrium Constants for Aldoae-Ketose Iaomerizationa6g

IK

Rcactwlrs

I

= [kclose]/[aldose]

+

D-Arabinose D-erythro-pentulose L-Fucose 6-deoxy-~-tagatose D-Xylose D-threo-pentulose D-fructose D-Mannose D-Lyxose D-lhreo-pentulose D-Rhamnose 6-deoxy-~-fructose

Refermtccs

0.18 0.14 0.19 2.45 0.39 0.58

* * *

46 46 51 52 52 52

e

HC=O.

1;

HC-0

ec-0

I

CHOH

I

R

t ,

HC-Oe

1

\Ei c=o

1 j

c-0

tf

I I

I I

t ,

Hro\H ,/

C-00

I I

CHOH

CHOH

CHOH

R

R

R

XXXI

I

R

\

H

XXXII Lobry de Bruyn-Alberda van Ekenstein reactions and for dehydration to a 3-deoxyosone. On account of side reactions, equilibrium constants are very difficult to

LOBRY DE BRUYN-ALBERDA

VAN EKENSTEIN TRANSFORMATION

103

evaluate for most nonenzymic Lobry de Bruyn-Alberda, van Ekenstein reactions. However, the specificity of enzyme catalysis eliminates these complications, and the equilibrium constants of several such reactions of nonphosphorylated sugars have recently been determined enzymically.62These are shown in Table VI. It will be interesting t o ascertain relationships between various equilibria and configurations and conformations of the sugars, at3 more of these data become available.

ADDENDUM The epimerization of 2,4,6-tri-O-metyl-~-glucoset o 2,4,6-tri-O-methylD-mannose, reported by Prentice, Cuendet and Smith'33 should have been included in Table I. This reaction apparently gives good yields, since, on a small scale, approximately 30 % of crystalline 2,4,6-tri-O-methyl-~-mannose was isolated after treatment of 2,4,6-tri-O-methyl-~-glucosewith 0.035 N barium hydroxide a t 35' for 8 days. It should also be noted that this reaction is another example of a C2 epimerization which cannot proceed through a ketose intermediate. (133) N. Prentice, L. S. Cuendet and F. Smith, J. Am. Chem. SOC.,78, 4439 (1956).

This Page Intentionally Left Blank

THE FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

BY L. MESTER* The Technical University. Budapest. Hungary I . Introduction ........................................................... 106 1 . Mechanism of the Reaction; Modern Syntheses ...................... 106 .... 107 2. Structure Determination ......... . . . . 108 3. Preparation and Application of th 4. Preparation of Tetrazolium Compounds and Their Use as Biological Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 5. Tetrazolium Compounds in the Determination of Reducing Sugars . . . . . 109 . . 109 6 . Action of Light on Formazan and Tetrazolium Compounds . I1. Preparation and Structure of Sugar Formazans . . . . . . . . . . . . . . . . . 109 I11. Identification of Aldoses in the Form of Formazans ..................... 113 IV . Preparation of Sugar Tetrazolium Compounds .......................... 115 V . Preparation of Aldothionic Acid Phenylhydrazides ...................... 118 VI . Structure of Sugar Phenylhydrazones ................................... 123 VII . Structure of Sugar Phenylosazones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 . . . . . . . . . . . . . . . . . . 131 1 . Preparation of “D-Glucosazone Formazan” . . -chain Structure of “D-Glucose” Phenyl....................................... 137 osazone . . . . . . . . . elate Structure of “D-Glucose” Phenyl3. Evidence in Fav .................................................... 138 Mixed A and Mixed B Osazones . . . . . . . . 139 5 . The Structure of Diels’ Anhydro-‘%glucose” Phenylosazone ......... 141 6 . Mutarotation of Sugar Osazones ..................................... 144 VIII . The Formazan Reaction of Periodate-oxidized Sugar Derivatives ........ 148 1 . 1,2-0-Isopropylidene-~~~-glucofuranose.............................. 148 2. Methyl a-D-Glucopyranoside ...................................

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

152 IX . The Formazan Reaction of Oxidized lysaccharides ................... 154 1 . Oxidation with Periodic Acid . . . . ................................ 154 2. Oxidation with Ozone ............................................... 155 3. Oxidation by Other Means ........................................... 158 X . Preparation and Use of the Tetrazolium and Carbothionic Acid Phenylhydrazide Derivatives of the Oxidized Polysaccharides and the Metal Complexes of their Formazans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 1 . Tetrazolium Derivatives . . . . . s .....................

* Present address: Centre National de la Recherche Scientifique, Universit6 de Paris, Facult6 de Pharmacie, Paris, France. 105

106

L. MESTER

2. Thionic Acid Phenylhydrazide Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Metal Complexes.. . . . . . . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. The Formaaan Reaction as a Means for Establishing the Structure of Polysaccharides. . . . . . . . . . . . . . . , . . . . , . , , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. Tables . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . ...

159 160 162 163

I. INTRODUCTION Formazan chemistry is generally regarded as beginning in 1892, when von Pechmann' and Bamberger and Wheelwright2 independently prepared the first representatives of this group of compounds. Some formazans had been prepared earlier by Friese,3 F i ~ c h e r ,Fischer ~ and Besthorq6 and Heller,s but their formazan structure was only established subsequently. By the turn of the century, many formazan compounds were known and many of their interesting reactions had been discussed, yet the practical significance of the group was in no way recognized. The foundations of modern interest, both theoretical and practical, in the formazans were laid in the early forties by the highly significant work of Kuhn and his coworkers; this interest has become ever-widening. Extensive studies have now been carried out in a number of important areas of formazan chemistry. These may be briefly summarized as follows. 1 . Mechanism of the Reaction; Modern Syntheses

The reaction mechanism of formazan formation has been studied by many researchers. The fundamental work of Busch and his coworkers'-9 and the studies by Hunig and Boes,lo and by Hauptmann and Periss6,ll merit special mention. A host of other authors have endeavored to widen the sphere of application of the formazan reaction, and have extended it successfully to the 4,4-N-diarylsemi~arbazones~~ and guanylhydra~ones,'~-'~ respectively. (1) H. von Pechmann, Ber., 26, 3175 (1892). (2) E.Bamberger and E. W. Wheelwright, Ber., 26, 3201 (1892). (3) P. Friese, Ber., 8, 1078 (1875). (4) E.Fischer, Ann., 190, 67 (1878). (5) E. Fischer and K. Besthorn, Ann., 212, 316 (1882). (6) G. Heller, Ann., 263, 269 (1891). (7) M.Busch and H. Pfeiffer, Ber., 69, 1162 (1926). (8)M.Busch and R. Schmidt, Ber., 63, 1950 (1930). (9) M. Busch and R. Schmidt, J . prakt. Chem., [2] 131, 182 (1931). (10) S. Hunig and 0. Boes, Ann., 679, 28 (1935). (11) H. Hauptmann and A. C. de M. PerissB, Chem. Ber., 89, 1081 (1956). (12) W. Ried and H. Hillenbrand, Ann., 681, 44 (1953). (13) F. L. Scott, D. A. O'Sullivan and J. Reilly, J . Chem. Soc., 3508 (1951). (14) F. L.Scott, D. A. O'Sullivan and J . Reilly, Chem. & Znd. (London), 782 (1952). (15) F. L.Scott, D. A. O'Sullivan and J. Reilly, J . A m . Chem. Soc., 76,5309 (1953). (16) E. Wedekind, Ber., 30, 444 (1897).

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

107

A significant new method for preparing formazans is that of Ried and Hoffschmidt,'7 which makes the heterocyclic formazans easily accessible by way of substituted esters of pyruvic acid. In his excellent general survey of the chemistry of formazans and of tetrazolium salts, Ninehamla mentioned some 500 formazans and over 200 tetrazolium compounds, but, because of the great interest in this group, their number has since increased, 2. Structure Determination

The structures of the formazans have been of interest to research workers from the outset. Kuhn and coworkers,lSHunter and Roberts,z0and Ragno and coworkersz1*2z have dealt with this question. When different groups, R' and R", are substituted in the classical spatial arrangement of a forN=N-R' R-C

/ \\

R-C

//N-NHR'

N-NHR"

(A)

(B)

mazan, the molecule permits the existence of two isomers, A and B. On the other hand, von Pechmannz3a24 observed that formazans prepared in two ways have the same melting point and that their mixed melting point exhibits no depression. He called attention to the fact that two forms would be predicted but that only one is obtained. As will be developed later, it is now known that the formazans possess a chelate structure. R'

/

/"'

...

."/

H

R-C

\

N-N

/ \

R"

f+

R-C

HN-"\ \

N=N

H

.....,'.

\

R" ~

(17) (18) (19) (20) (21) (22) (23) (24)

W. Ried and R. Hoffschmidt, Ann., 681, 23 (1953). A. W. Nineham, Chem. Revs., 66, 355 (1955). I. Hawser, D. Jerchel and R . Kuhn, Chem. Ber., 82, 515 (1949). L. Hunter and C. B . Roberts, J . Chem. Soc., 820, 823 (1941). M. Ragno and A. Bellomo, Gazz. chim.ital., 78, 45 (1948). M. Ragno and S. Bruno, Gazz. chim.ihZ., 76, 485 (1946). H. von Pechmann, Ber., 27, 1679 (1894). H. von Pechmann, Ber., 28, 869 (1895).

108

L. MESTER

3. Preparation and Application of the Metal Complexes of Fomnazans The formazans readily react with salts of heavy metals to give darkcolored, well-crystallizing complex compounds. Hunter and Roberts,2O Jerchel and Fischer,ls Wizinger and Bir6,la and Seyhan27have dealt with their preparation and structures. Owing to their ability to form complexes, a few formazan compounds have found application in analytical chemistry; thus, the so-called diphenylthiocarbazone or "dithizone," long known and used (which, rearranged to its tautomeric form, reacts as C-mercaptoN,N'-diphenylformazan2*~ 2*), is useful. Its oxygen 31 as well as C-ozy-N ,N'-diphenylformazanlaZhas likewise found analytical application. A new field in which to apply formazan complexes has been opened up by a patenta2"which utilized, in wool dyeing, the copper complexes of formazans containing hydroxyl or carboxyl groups in the ortho position.

4. Preparation of Tetrazolium Compounds and Their Use as Biological Indicators In the course of their research on invert soaps, Kuhn and JercheP have prepared tetrazolium compounds, which, in addition to having bactericidal action, were observed to display an interesting property, namely, that processes of biological reduction retransform them to red f ~ r m a z a n s . ~ ~ Starting with this observation, the efforts of numerous groups of workers resulted in the elaboration of a method suitable for indicating the presence of biological reduction processes. Of the many tetrazolium preparations, triphenyltetrazolium chloride (T.T.C.), first prepared by von Pechmann and Rungea6in 1894, has gained the widest application. This compound is a colorless, fairly water-soluble compound which, by the effect of reduction processes, is readily converted to the bright-red triphenylformazan, which is insoluble in water. (25) D.Jerchel and H. Fischer, Ann., 563, 200 (1949). (26) R. Wizinger and V. Bir6, Helv. Chim. Acta, 82, 901 (1949). (27) M.Seyhan, Chew Ber., 87, 396, 1124 (1954). (28)H.Irving and C. F. Bell, J . Chem. Soc., 3538 (1953). (29) H.Irving, S.J. H . Cooke, S. C. Woodger and R. J. P. Williams, J . Chem. Soc., 1847 (1949). (30) P.Cazeneuve, Bull. soc. chim. France, [3]29, 693 (1900). (31) P.Cazeneuve, Compt. rend., 130, 1478 (1900). (32) L.M.Kul'berg and A. M. Lendeva, Zhur. Anal. Khim., 2 , 131 (1947); Chem. Abstracts, 43, 6696 (1949). (32a) Ciba Ltd., Swiss Patent 246,475 (1947);Chem. Abstracts, 43, 5198 (1949). (33) R. Kuhn and D. Jerchel, Chem. Ber., 74, 941 (1941). (34) R.Kuhn and D. Jerohel, Chem. Ber., 74, 949 (1941). (36) H.von Pechmann and P. Runge, Ber., 27, 2920 (1894).

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

Reduction

109

,

‘ Oxidation

5. Tetrazolium Compounds in the Determination of Reducing Sugars

The tetrazolium compounds have come to play a major part in the determination of reducing sugars. Mattson and Jenseng6have elaborated a method which they based upon the colorimetric determination of the formazans that arise by the reducing action of sugars. Wallenfels and coworkers3’ have developed procedures for the determination of reducing sugars by paper chromatography and for the microdetermination of blood sugar, both based on the reduction of tetrazolium compounds. 6. Action of Light on Formazan and Tetrazolium Compounds

Hausser, Jerchel and Kuhn’v observed that, in benzene solution, the red triphenylformazan is transformed into a yellow form which is stable only while irradiated and which, in darkness, is retransformed to the red formazan. In their view, the phenomenon is attributable to the cis-trans isomerism of the formazans. These authors have also investigated in detail the action of light on triphenyltetrazolium chloride; they found that, in aqueous solution, this compound is partly transformed into formazan and partly into 2,3diphenylene-5-phenyltetrazolium chloride, the so-calledm “Photo T.T.C.” Reduction of this substance leads to a free radical that can be isolated in the solid state. It seems surprising that, during the 60 years which had elapsed since the first compounds in this group were reported, and despite the work of many investigators having a variety of objectives in formazan chemistry, no attempts to prepare the formazans of sugars had been described.

11. PREPARATION AND STRUCTURE OF SUGARFORMAZANS When allowed to react in pyridine solution with cold solutions of diazonium compounds, aldose phenylhydrazones yield brilliant-red, readily (36) A. M. Mattson and C. 0. Jensen, Anal. Chem., 22, 182 (1950). (37) K.Wallenfels, E. Bernt and G. Limberg, Angew. Chem., 86,681 (1953). (38) I. Hausser, D. Jerchel and R. Kuhn, Chem. Ber., 82, 195 (1949).

L. MESTER

110

crystallizable sugar f o r m a z a n ~ . By ~ ~ this - ~ ~method, it is possible to prepare the readily crystallized formazans of D-glucose, D-galactose, D-mannose, L-rhamnose, and other aldoses. The reaction proceeds as follows.

H C=N-NH-

1 (CHOH), I CHiOH

Aldose phenylhydrazone

[ a N E N l m pyridine

+

CH~OH Aldose diphenylformazan

Acetylation of hexose formazans with a mixture of pyridine and acetic * 42 anhydride affords readily crystallized, red pentaacetate~.~~ By starting with uronic acids, novel sugar formazans are obtained. For instance, from the barium salt of the phenylhydrazone of D-galacturonic acid, a formazan was obtained in bright-red needles. Owing to the presence of the carboxyl group, this new formazan dissolves more readily than the formazans of simple sugars and it therefore promises to be suitable, for the preparation of new nitrogen-containing sugar derivatives2', favorably soluble in water. Acetylation of the formazan of D-galacturonic acid46leads to a lactone acetate which has three acetyl groups and is an appropriate starting material for the preparation of the corresponding tetrazolium compound. These sugar compounds show all the characteristic properties of the formazans: a brilliant red color, complexing with salts of heavy metals, and ready oxidation to colorless compounds. Nevertheless, further evidence was sought, in order to prove that they really belong to the formazan group. According to all current views on the mechanism of formazan phenylhydrazones, such as a 2-methyl-2-phenylf o r m a t i ~ n , ~secondary -'~ (39) G. Zemplh and L. Mester, Magyar Tudomdnyos Akad. K6m. Tudomdnyok OszMZycfnak Klizlemhnyei, 1, 1 (1951). (40) G.Zemplh and L. Mester, Acta. Chim Acad. Sci. Hung., 2 , 9 (1952). (41) L. Mester and A. Major, Magyar T u d d n y o s Akad. K8m. T u d d n y o k Oszthlydnak Klizlembnyei, 6 , 189 (1955). (42) L. Mester and A. Major, J . Am. Chem. SOC.,77, 4279 (1955). (43) G. Zemplbn and L. Mester, Magyar Tudomdnyos Akad. K8m. Tudomdnyok OszMlyrZnak Klizlemhnyei, 3, 7 (1953). (44) G . Zemplh, L. Mester and E. Eckhart, Chem. Ber., 86, 472 (1953). (45) G.Zemplh, L.Mester and A. Messmer, Chem. Ber., 86, 697 (1953). (46) L. Mester and E. M6czBr, J . Chem. Soc., 1699 (1958).

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

H

C

\

[ a N E N ] ' pyridine

H

II \a

/

I HCOH I

111

'

N-N

/

HCOH

(CHEOhO, pyridine

HOCH

HOCH

I HOCH

I I HCOH I

HOCH

I

HCOH

I

COzH D-Galacturonic acid phenylhydrae one

COzH D-Galacturonic acid diphenylformaaan

N=N

I

C

/

L

\

/

N-N

H

HCOAc

I

D-Galacturonic 6,3-lactone diphenylformaaan 2,4,5-triacetate

hydrazone or 2,2-diphenylhydrazone, cannot possibly give formazans. This was found to hold true for the secondary phenylhydrazones of sugars as well. For example, D-galactose 2-methyl-2-phenylhydrazone and Larabinose 2,2diphenylhydrazone fail t o yield formaeans because of the substitution of the mobile hydrogen of the nitrogen atom in the a-positi0n.~7,48 (47) G . Zemplh and L. Mester, Magyar Tudom4nyos Akad. K6m. Tudomctnyok Oszfhlyctnak Kdzlemhnyei, 1, 73 (1952). (48) G . ZemplBn, L. Mester, A. Messmer and E. Eckhart, Acta Chim. Acad. Sci. Hung.. 2, 25 (1952).

112

L. MESTER

As has been pointed out in Section I, 2, when R' and RN differ, the two isomers (A and B) permitted by the classical formulation cannot be isolated (because of the chelated structure of the formazans). This phenomenon, so very characteristic of the formazans, is also encountered in the formaeans of sugars. The mono-p-bromo derivative of D-galactose diphenylformazan was prepared by two routes: one was by causing D-galactose p-bromophenylhydrazone to react with diazotized aniline; the other, by coupling D-galactose phenylhydrazone with diazotized p-bromoaniline as follows. N-NH---B~

//

-

//

C

N

-

N

H~ -

B

C

HCOH

I

HOCH

HOCH

I

I HOCH I HCOH I

HOCH

I I

HCOH

CH2 OH

HCOH

I HOCH I HOCH I

HCOH

I

CHsOH

CHsOH

[Br-eNEN]'

>

HbOH

I I HOCH I HCOH I HOCH

CHiOH

Both routes were found to give the same product: their melting points were identical and mixed melting-point determinations gave no depression of the melting point. From this, it follows that, by the modern conception of formaaan structure, the structure can be expressed by the following chelate. Accordingly, the identity of the two products affords decisive evidence for the formaean structure of the new sugar derivatives.4'e

~

113

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

/ D --

1

C\ N=N

HCOH

I I

HOCH HOCH

I I

B

r

1

*./ H

a

\/

/a -

B

\/

\

r

C‘N-”;=,

\

HCOH

c)

I

HOCH

I I HCOH I

HOCH

HCOH CHzOH

111. IDENTIFICATION OF ALDOSES IN

CHsOH

THE

FORM OF FORMAZANS

Because of their melting points and characteristic crystalline former, these new, nitrogenous, sugar derivatives can be used to advantage in identifying aldoses (see Table I).49,60 Since they contain no active methine groups,l#lothe ketose phenylhydrazones do not yield formazans; this lack of reaction is therefore suitable for distinguishing ketoses from aldoses. It is more specific than the osazone test, frequently used in the identification of sugars, for it yields a different formazan from each aldose, whereas the osazone test does not differentiate between epimers. For identifying aldoses, this reaction has been simplified. The sugar phenylhydrazone (derived from the reaction of the sugar with phenylhydrazine) is not isolated, but, after addition of pyridine, is immediately coupled to give the formazan. On standing, sugar phenylhydrazone solutions suffer a considerable diminution in their capacity for coupling, a phenomenon connected with optically observable changes in structure. Obviously, two processes are taking place in the reaction of the sugar with phenylhydrazine-processes which, from the point of view of formazan formation, have opposing effects: (i) the formation of the phenylhydrazone and (ii) the transformation of this into a form unsuitable for coupling (see Section VI). To decide as to the usefulness of the simplified reaction for aldose identification, formazan formation from the corresponding aldoses has (49) L. Mester and A. Major, Magyar Tudomhnyos Akad. Kbm. T u d d n y o k Oszthlycinak Kazlembnyei, 7. 345 (1956). (50) L. Mester and A. Major, J . Am. Chem. Soc., 78, 1403 (1956).

L. MESTER

114

been studied as a function of time, For each sugar, the reaction with phenylhydrazine has been examined in three different solvents, namely, water, 50 % aqueous alcohol, and pyridine. After identical periods of time, each solution was coupled in pyridine (to yield the formazan) by the TABLE I Melting Point and Crystal Form of the Diphenylformazans of Some Sugars Diphmyl/ormoean o/

I

D-Glucose o-Galactose D-Mannose L-Arabinose L-Rhamnose D-XylOSe

~ . p . oc. ,

177-178 167-1 68 174-1 75 173-174 175-176 123-124

80

8

I

Crystal jorm

red needles, frequent rosettes bronze-red tablets bunches of microscopic, russet needles bunches of fine, bright-red needles brilliant red needles lanceolate, red needles

c

60-

=

L

f

40-

20

-

I

i 1

20

40 Time

60

80

100

, br.

FIQ. 1.-Formation of D-Galactose N , N’-Diphenylformazan a t Various Time in water, ----.-.- in Slyoethanol, . . -... in 90% pyridine). Intervals (--

addition of diazotized aniline. The data in Figs. 1 to 5 reveal that, on the whole, the yield was highest in the pyridine solution (between the 24th and 48th hour). With D-mannose, the nearly insoluble phenylhydrazone soon separates; hence, in this case, the formazan reaction is applied to the separated phenylhydrazone and so this reaction shows no deviation as a function of time. Experiments on the separation of mixtures of sugar formazans by means of chromatography are in progress.

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

115

IV. PREPARATION OF SUGAR TETRAZOLIUM COMPOUNDS With the objective of obtaining a biological indicator less toxic than T.T.C., and in view of a new and gradually increasing interest in tetrazolium compounds, attempts have been made to prepare them by the oxidation of sugar formazans."

80

-

6o 6 0 -

0 8

.o -

,,#? , , 8 ,

8

8

0)

40-

/*

'b

-

.'

, b

,

T i m e , hr. Fro. 2.-Formation of D-Glucose N,N'-Diphenylformaran at Various Time Intervals (--in water, -.-.-.- in 50% ethanol, . in pyridine).

- -.

40

$

.-0> 0)

i

Time , hr. FIQ.3.-Formation of D-Xylose N , N'-Diphenylformazan at Various Time Intervals (+--

in water, -.-.-.- in 50% ethanol,

. . - -.in pyridine).

L. MESTER

116

40

-

20

-

a9

c

0 al

.>

I

20

I

I

40

60

80

100

Time ,hr. FIQ. 5.-Formation of L-Arabinose N,N’-Diphenylformazan at Various Time Intervals (in water,-----in 50% ethanol, ...... in pyridine).

lost their color, but failed to yield the desired reaction product; instead, partial or complete oxidation of the sugar formazan molecule occurred. For instance, oxidation of D-mannose diphenylformaean with amyl nitrite gave D-mannonic 1,4-1actone in good yield. Similar difficulties were encountered on employing lead tetraacetate aa the oxidant.

FOItMAZAN REACTION I N CARBOHYDRATE RESEARCH

117

Next, the sugar portion sensitive to oxidation was protected by means of acetylation, and the penta-0-acetyl-D-galactose diphenylfonnazan was oxidized with lead tetraacetate. By the action of the calculated amount of the oxidant, penta-0-acetyl-D-galactose diphenyltetrazolium chloride (G.T.C. acetate) was obtained as characteristically shaped, colorless prisms upon addition of an appropriate quantity of hydrochloric acid. Owing to its ionic structure, this compound is quite soluble in water and is readily reduced to the corresponding acetylated formazan, which is red. Saponification of the penta-0-acetyl-D-galactose diphenyltetrazolium N

1

//

-

N

H

~

N C

C

HCOH

I HOCH I HOCH I HCOH I

‘

-

//

N H

HCOAC

acetylation

I I AcOCH I HCOAc I

NaOCHa

AcOCH

CHsOH D-Galactose diphenylformazan

CH~OAC

Penta-0-acetyl-D-galactose diphenylformazan

l

Pb(CH:COO)r

C

HCOH

I HOCH I HOCH I HCOH I

CHnOH n-Galactose diphenyltetrazolium chloride

~

yzibic

C

NaOCHa acetylation

’

HCOAc

I I AcOCH I HCOAc I AcOCH

CH~OAC

Penta-0-acetyl- galactose

diphenyltetrazolium chloride

c10

118

L. MESTER

chloride with sodium methoxide, followed by addition of the theoretical amount of hydrochloric acid, results in a pale-yellow, hygroscopic powder, which will keep well when it is carefully sealed or is in aqueous solution. Proof of its structure was afforded, in addition to the analytical data, by its reduction by L-ascorbic acid (in a medium containing sodium methoxide) to D-galactose diphenylformazan. Thereby, the circle of reactions was demonstrated as shown on the preceding page. Direct conversion of sugar formazans to the corresponding sugar tetrazolium compounds61has been made possible by using N-bromosuccinimide, as recommended by Kuhn and Munzing.62 Polarographic studies on D-galactose diphenyltetrazolium chloride (G.T.C.) and its acetate have revealed that the reduction proceeds analogously to that of T.T.C.6S The mechanism of reduction of the sugar derivatives is, in many respects, analogous to that of T.T.C.; the changes in the number of electrons, the characteristics of the polarographic steps, and a number of other features, are identical; yet essential differences prevail in the reduction potentials and their dependence on pH. This is attributable to the introduction of the sugar residue into the molecule, and has a bearing on the use of the n-galactose derivatives as redox indicators. On the evidence from biological the sugar tetrazolium derivatives are less toxic than is T.T.C. ; consequently, they can be used to greater advantage in living tissues.

V. PREPARATION OF ALDOTHIONIC ACIDPHENYLHYDRAZIDES Reductive decomposition of sugar formazans with hydrogen sulfide leads to sugar derivatives containing nitrogen and A 1% solution of n-galactose diphenylformazan in alcohol is red; when treated with hydrogen sulfide, it is rapidly decolorized and, after a few days, gives D-thiogalactonic acid phenylhydrazide, which crystallizes in colorless needles. Phenylhydrazine, a byproduct of the decomposition reaction, may be isolated from the mother liquor. A freshly prepared solution of n-thiogalactonic acid phenylhydrazide gives no reaction either with sodium nitroprusside or with lead acetate, but, when allowed t o stand for a considerable time, both reactions are positive, presumably in consequence of decomposition. Boiled for an hour in aqueous solution, D-thiogalactonic acid phenylhydrazide is transformed into D-galactonic phenylhydrazide. Aldothionic acids and their derivatives were unknown until 1953. De(51) L. Mester and A. Messmer, J . Chem. SOC.,3802 (1957). (52) R. Kuhn and W. Munzing, Chem. Ber., 86, 858 (1953). (53) B. J&mborand L. Mester, Acta Chim. Acad. Sci. Hung., 6 , 263 (1955).

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

S

//

119

+ HzN-NH-

C

I

HCOH

I I

I

HCOH

I I

HOCH

HOCH

HOCH

HOCH

I

I

HCOH

I

CHzOH

I

I

HCOH

I

CHzOH

composition of formazans with hydrogen sulfide is also a newly detected reaction, although it had long been used in the decomposition of other (similar) compounds; for instance, by Bernthsen, for substituted amidines, as when thiobenzoic acid anilide is formed from N ,N'-diphenylbenzamidine by the action of hydrogen sulfide.64 In order to gain insight into the mechanism of the reduction, the changes in optical rotation were observed66in an alcoholic solution of D-galactose diphenylformazan, as a model compound, decolorized by the action of hydrogen sulfide; the resulting curve showed a negative minimum point and a slow logarithmic rise (see Fig. 6). The results of catalytic hydrogenation of the same model compound afforded an interpretation of the minimum point on the curve. It is known from the investigations of Jerchel and Kuhn66 that catalytic hydrogenation of formazans gives hydrazidines, when one mole of hydrogen per mole has been taken up, and that these colorless compounds revert with extraordinary ease to red formazans-even mere contact with air sufficing to bring about their retransformation. The same results are obtained with D-galactose diphenylformazan. The optical rotation of this formazan solution, decolorized by catalytic hydrogenation, shows fair agreement with the minimum optical rotation of a solution reduced with hydrogen sulfide. This fact, and the results of the afore-described experiments which led to the formation of D-thiogalactonic phenylhydrazide, constitute evidence that the first step in the reduction with hydrogen sulfide is hydraxidine formation. The ascending portion of the mutarotation curve asymptotes a t a value (54) A. Bernthsen, Ann., 1%, 1 (1878).

(55) A. Messmer and L. Mester, Chem. & Ind. (London), 423 (1957). (56) D.Jerchel and R. Kuhn, Ann., 668, 185 (1950).

120

L. MESTER

which corresponds to the optical rotation specific for pure D-thiogalactonic phenylhydrazide. The logarithmic portion, showing fair agreement between the velocity constants, points to a process of the first order. When the reaction mixture is processed, it is found that, in this partial process of the first order, phenylhydrazine is formed (along with the D-thiogalactonic phenylhydrazide) , The experimental data allow the following interpretation of the mechanism. By the action of hydrogen sulfide, the sugar formazan (I) is reduced to a sugar hydrazidine derivative (11),which becomes protonized (111); in the course of a slow SNl process, the hydrazidine ion splits off phenylhydrazine, whereafter the arising carbonium ion (IV) unites in a rapid ( t = 2 5 fl%.,abs. ethanol)

0

00

FIG. 6.-Mutarotation Hydrogen Sulfide.

160

240

320 4 0 0 Time ,hr.

400

560

640

720

of D-Galactose N,N'-Diphenylformaran Reduced with

elementary step with the HSe anion to give the aldothionic phenylhydrazide (V). Similar results obtained with other model compounds (for example, u-galactose mono-p-bromodiphenylformazan) preclude any nucleophilic addition of hydrogen sulfide a t the hydrazidine, and a similar experiment

N-N

//1\

C R

N-N"

\

H' rapid

HtS

H

,...'.

a

\/ I

\

I1

'

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

N-NH-D // I 1 -

C

I

//

N

-

N

+

HI~--NEI--m

(8x1) slow

\@

H-a

121

R

-

rapid

I11

IV

I\

R

SH

V

carried out with penta-0-acetyl-D-galactose diphenylformazan shows that the alcoholic hydroxyl groups of the sugar moiety do not participate in the process. A number of other nitrogenous and sulfur-containing sugar derivatives can be prepared by a reaction that Wuyts and Lacourt recommended in the early thirties for the identification of aldehydes and s* The principle underlying this reaction is that, with aldehydes and ketones, the thionic phenylhydrazides give thiadiazoline derivatives with extraordinary ease. The reaction proceeds as follows. r------i

- HZ0 NII

-

N

S

I - ~ -

In the presence of hydrochloric acid in alcohol solution as catalyst, the reaction takes one or two minutes to proceed to completion; the product crystallizes with ease, and a characteristic color reaction greatly facilitates its recognition : with concentrated nitric acid, or in concentrated sulfuric acid plus hydrogen peroxide, thiadiazoline displays a bright bluish-green color. D-Thiogalactonic phenylhydrazide, treated according to the procedure of Wuyts and Lacourt for one or two minutes with benzaldehyde, yields the following, readily crystallized, markedly dextrorotatory product. (57) H. Wuyts and A. Lacourt, Bull. classe sci. Acad. roy. Belg., [5] 20, 156 (1934). (58) H. Wuyts and A. Lacourt, Bull. soc. chirn. Belg., 42, 376 (1933).

122

L. MESTER

S

On the evidence of analysis and of the color reaction, the product obtained is a thiadiazoline derivative. The structural isomer of this compound, with the D-galactose residue at C2 and one of the phenyl groups a t C5, had already been prepared by W ~ y t s , ~8og ,who allowed thiobenzoic acid phenylhydrazide to react with D-galactose. Accordingly, in his case, the sugar residue was the aldehydic component, whereas, in the afore-described experiment, it participated in the reaction in the form of thionic phenylhydrazide. In this reaction, a new asymmetric carbon atom makes its appearance during ring closure and gives rise to diastereoisomers conspicuous for their extraordinarily powerful optical rotation. The antipodes of these diastereoisomersareformed when L-galactose is employed for the reaction. For some sugars (for example, L-arabinose), Wuyts and Verstraeten prepared all four isomers and detected the antipodes.81In the afore-said reaction it was possible to isolate, by means of crystallization, a markedly dextrorotatory product, and the mother liquor was pronouncedly levorotatory. The fact that, during the reaction of D-thiogalactonic phenylhydrazide with benzaldehyde, there arise diastereoisomers of powerful optical rotation constitutes further evidence for occurrence of thiadiazoline ring formation, and this, a t the same time, furnishes proof of the structure of the D-thiogalactonic phenylhydrazide used as the starting material. It should be mentioned that D-thiogalactonic phenylhydrazide has been found to possess antituberculous action, both in vitro and in vivo. (59) H.Wuyts, Compt. rend., 196, 1678 (1933). (60) H. Wuyts and R. Verstraeten, Bull. classe sci. Acad. roy. Belg., [5]20, 168 (1934);21, 415 (1935). (61) H. Wuyts and R. Verstraeten, Bull. SOC. chim.Belg., 46, 65 (1936).

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

123

VI. STRUCTURE OF SUGARPHENYLHYDRAZONES When Emil Fischer,62some 70 years ago, prepared sugar phenylhydrazones, he represented them as open-chain compounds. The conception of ring ~tructure6~-~6 arose later to account for mutarotational effects in solution. The question is still a moot one, although many workers have attempted to clarify the point. Fr&rejacquea7found that definitive evidence could not be obtained from a study of hydrolysis rates and rotational values of the hydrolyzate. Later, Stempel,6* on the basis of similar experiments, came to the same conclusion. I n earlier work, attempts had been made to elucidate the structure of the sugar phenylhydrazones on the basis of their mutarotations, but the later work of Butler and CretcheF and of Stempe168 revealed that, although mutarotational studies permitted many interesting observations, they were of no use in correlating structure to mutarotation. There were a few isolated cases in which the products obtained by mild acetylation of sugar phenylhydrazones were compared successfully with acetates obtained by other means. Early in the century, Behrend and Reinsberg,?O using this method, found evidence of ring structure in the so-called D-glucose ‘(a”-phenylhydrazone, the first-known sugar phenylhydrazone in which the cyclic structure was definitely established. Again, by the same method, Wolfrom and Christman’l proved the aldehydo structure of Dgalactose phenylhydrazone. To solve this problem, the formazan reaction was applied, in which only real Schiff systems, aldehydo or acyclic phenylhydrazones, participate. When subjected to the mild conditions of the formazan reaction (coupling in pyridine with a solution of diazotized aniline a t a temperature below -So), the structures of the phenylhydrazones do not undergo any change. For example, the known aldehydo-D-galactose phenylhydrazone produces D-galactose diphenylformazan in 86 % yield, but D-glucose “a”-phenylhydrazone of recognized ring structure does not react at all.“, 48 I n the case of sugar phenylhydrazones of unknown or questionable (62) (63) (64) (65) (66) (67) (68) (69) (70) (71)

E. Fischer, Ber., 20, 821 (1887). H. Jacobi, Ann., 272, 170 (1893). R. Behrend, Ann., 363, 106 (1907). R. Behrend and F. Lohr, Ann., 362, 78 (1908). A. Hofmann, Ann., 366, 277 (1909). M. Frhejacque, Cornpt. rend., 180, 1210 (1925). G. H. Stempel, J . Am. Chem. Soc., 66, 1351 (1934). C. L. Butler and L. H . Cretcher, J . Am. Chem. Soc., 63, 4358 (1931). R. Behrend and W. Reinsberg, Ann., 377, 189 (1910). M. L. Wolfrom and C. C. Christman, J . Am. Chem. Soc., 63, 3413 (1931).

124

L. MESTER

structure, application of the formazan reaction enables us to find out if they are cyclic or acyclic. For example, two of the three modifications of D-glucose phenylhydrazonea3-6S~ do not undergo formazan reaction, but the third does react, indicating that the former two are cyclic and the latter is acyclic (see Table 11). The formazan reaction permits an interpretation of the mutarotation 4 2 , 6 6 , 60 curves of the sugar phenylhydra~ones.~~~ TABLE I1 Physical Properties of Some Phenylhydrazones of D-Glucose Modijcalion

D-Glucose "a"-phenylhydrazone D-Glucose "p"-phenylhydrazone Skraup's D-glucose phenylhydrazone

40

80

M , p . , "C.

[aloei t 8 W d W

Pmmaran* %

169-160 140-141 116116

-87 * -50" -2-+-50' -7 * -49"

0 67 0

120

160

Time, hr. FIG.7.-Upper Curve: Mutarotation of D-Mannose Phenylhydrazone [c 0.9, pyridine-ethanol (9:1 by vol.)]; Lower Curve: D-Mannose Diphenylformazan Formation.

Earlier workersa6*68-70 had concluded from the nature of the curves that there are at least three modifications participating in the mutarotation, yet it remained undetermined until the present whether they were all cyclic, or whether the aldehydo form was represented among them, and if so in what proportion. This point can now be clarified. At certain critical points during mutarotation, the phenylhydrazone solutions are coupled to form formazan, and the yields a t the different rotational values indicate the proportion of phenylhydrazone present in the aldehydo form. These data are presented in the lower curves in Figs. 7 to 10. (72) H.Skraup, Monatsh., 10, 406 (1889). (73) C. L. Butler and L. H. Csetcher, J . Am. Chem. SOC.,61, 3161 (1929).

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

125

It thus appears that the formazan reaction offers a method of tracing and interpreting optically-observable changes in structure. The formazan values of the three modifications of D-glucose phenylhydrazone (each with a different melting point) observed during muta-

160 240 320 Time, hr. FIG.&-Upper Curve: Mutarotation of L-Rhamnose Phenylhydraaone [c 1.27, pyridine-ethanol (1: 1 by vol.)]; Lower Curve: L-Rhamnose Diphenylformaaan Formation. 80

40

80 120 160 Time, hr. FIG.9.-Upper Curve: Mutarotation of D-Galactose Phenylhydrazone (c 5.4 pyridine) ; Lower Curve: D-Galactose Diphenylformaxan Formation.

rotation clearly show, as indicated in part in Table I, that the so-called “0” form reacts and therefore has an aldehydo structure, whereas the other two do not react, and hence are two phenylhydrazones having ring structures. Henceforth, it might be expedient if D-glucose (‘a”-phenylhydrazone having a more negative rotation were designated ‘(@”; and Skraup’s hydrazone, which rotates to a lesser degree in the negative direction, were called “a.”

L. MESTER

126

The formazan reaction, carried out at certain critical rotational values, also permits the interpretation of the structural changes observable during mutarotation of D-galacturonic acid phenylhydra~one~~ (see Fig. 11). A comparison of their curves shows that mutarotation and formazan formation are parallel processes and that, consequently, it is not the lactonization of the carboxyl group, but the aldo-cyclo isomerization that plays the decisive role. 40

-40

80 160 240 320 Time, hr. FIG. 10.-Upper Curves: Mutarotation of D-Glucose Phenylhydrazones [c 5.4, pyridine-ethanol (1:1 by vol.)]; 0 , “~”-D-glucosephenylhydrazone; 0 , Skraup’s D-glucose phenylhydrazone; 0 , “jY’-D-glucose phenylhydrazone. Lower Curve : D-Glucose Diphenylformazan Formation in a Mutarotating Solution of ddehydo-DGlucose Phenylhydrazone. (No formazan was produced by the cyclic D-glucose phenylhydrazones during mutarotation.)

Time, hr.

FIG. 11.-Lower

Curve: Mutarotation of D-Galacturonic Acid Phenylhydrazone (pyridine-ethanol, 1:l by vol.) ; Upper Curve: D-Galacturonic Acid N,N’-Diphenylformazan Formation.

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

127

The formazan reaction has proved to be equally useful in disclosing the structure of metplated sugar phenylhydrazones. D-Galactose phenylhydrazone pentaacetate coupled with diazotized aniline solution produced the same D-galactose diphenylformazan pentaacetate as was obtained by acetylation of D-galactose diphenylformaznn. This proves the open-chain structure of D-galactose diphenylformazan pentaacetate and is in agreement with the findings of Wolfrom and Christman."

/a N-N-O HN-"\

/ / H C

HCOAc

I

ACOCH

I I HCOAc I

AcOCH

C

\

H

[ O N E N ] " , pyridinc

I ACOCH

I

AcOCH

I

HCOAc

I

CH~OAC

D-Galactose phenylhydrazone pentaacetate

r

CHaOAc Penta-0-acetyl-D-galactose diphenylformazan

I

pyridine AcaO

+

N-N

/ / \

C

H

HCOH

I

HOCH I

HOCH

I

HCOH CHiOH D-Galactose diphenylformazan

128

L. MESTER

I

I

CH

AcOCH

II

I

N

CH

\

/I

\

/HCOAc

4

,I I

I

+

'[NGN-O]

>

No reaction

CHzOAc H70Ac

Anhydro-D-mannose phenylhydrazone tetraacetate

+

'[NLN-O] +

Formazan

[HCOIrAc,

I

CH~OAC Acetylated aldehydo-Dmannose phenylhydrazone (amorphous)

C

/ / \

\

r N=N

/a

N-N

N-N H

AcOCH I

I

AcOCH

I I HCOAc I HCOAc

CH~OAC

D-Mannose diphenylformazan pentaacetate

C

/ / \

\

AonO c--

r

H

HOCH II HOCH

I

HCOH I

I

HCOH

I

CHzOH D-Mannose diphenylf ormazan

The method has also been applied to the two D-mannose phenylhydrazone acetates described in the literature. The fact that anhydro-D-man-

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

129

nose phenylhydrazone tet,raacetate does not undergo the formazan reaction supports the pyrazoline structure proposed for this substance by Wolfrom and Blair.74 This structure is an aldehydo form but has no h i n o hydrogen; i t therefore fulfils only one of the two prerequisites for occurrence of the formazan reaction. On the other hand, the amorphous u-mannose phenylhydrazone acetate described by Hofmann,76and more recently by Stepanenko and coworker,?6 underwent reaction but failed to produce the readily crystalline D-mannose diphenylformazan pentaacetate obtained by the acetylation of n-mannose diphenylformazan. The amorphous, acetylated D-mannose phenylhydrazone has a wide melting-point range and appears to be a mixture of partially acetylated phenylhydrazones in which the presence of the aldehydo form can be readily demonstrated by the formazan reaction. Summarizing, the formazan reaction may be used for distinguishing the open-chain from the ring structure in the free sugar phenylhydrazones and also in their acetates. The reaction can also be used for estimating the relative quantities of aldehydo and ring forms present a t any time during the mutarotation of sugar phenylhydrazones.

VII. STRUCTURE OF SUGAR PHENYLOSAZONES The structure of sugar phenylosazones, derivatives first prepared by Fischer62.77 in 1884, has remained undefined. Besides Fischer’s open-chain structure, a tautomeric azo-hydra~one7~ structure and ring structures66* 79have been proposed. Although work in the last two decades involving ultraviolet spectra studiess1’82 and examination of the acetylatedS3-84 and methylated86-8s phenylosazones appears to favor the acyclic structure proposed by Fischer, the problem cannot be considered solved. (74) M. L. Wolfrom and M. G. Blair, J . Am. Chem. Sac., 68, 2110 (1946). (75) A. Hofmann, Ann., 366, 315 (1909). (76) B . N . Stepanenko and V . A. Ignatyuk-Maistrenko, Doklady A k a d . N a u k . S. S . S . R . , 73, 1251 (1950). (77) E. Fischer, Ber., 17, 579 (1884). (78) E. Zerner and R. Waltuch, Monatsh., 36, 1025 (1914). (79) E. C. C. Baly, W. B . Tuck, E. G. Marsden, and M. Gazdar, J . Chem. Sac., 109, 1572 (1907). (SO) W. N . Haworth, “The Constitution of Sugars,” Edward Arnold & Co., London, 1929, p. 7. (81) L. L. Engel, J . Am. Chem. Sac., 67, 2419 (1935). (82) V. C. Barry, J . E. McCormick and P. W. D. Mitchell, J . Chem. Sac., 222 (1955). (83) E. E. Percival and E. G. V. Percival, J . Chem. SOC.,1320 (1937). (84) M. L. Wolfrom, M. Konigsberg and S . Soltzberg, J. Am. Chem. SOC.,68, 490 (1936). (85) S. Akiya and S . Tejima, Yakugaku Zasshi, 73, 894 (1952); Chem. Abstracts, 47, 6351 (1953).

130

L. MESTER

The acyclic structure does not explain how the reaction stops a t C2 instead of continuing to C3, and why the imino hydrogen of only one phenylhydrazone group is methylatable when each of the imino groups apparently should have the same value. In an attempt to explain the first of these observations, Baly, Tuck, Marsden and Gazdar,7@ as early as 1907, advanced the assumption (openchain form presumed) that, in the sugar phenylosazones, the two conjugated double bonds form a “condensed system” because of their inner linkage, which causes the reaction to stop at C2. Fieser and Fieser,89 on theoretical grounds, proposed for the structure of the phenylosazones the chelate tautomers VI and VII of the acyclic

N

N

//L

HC

H

VI

VII

N

/ \

HC

N

H

\ / N

VI‘

VII”

compound, these being stabilized by their ability to exist in the resonance forms VI’ and VII”. They considered that the formation of a stable ring (86) S. Akiya and S. Tejima, Yakugaku Zasshi, 73, 1574 (1952); Chem. Abstracts,

47, 9275 (1953). (87) E. G. V. Percival, J . Chem. SOC.,1770 (1936). (88) E.E.Percival and E. G. V. Percival, J . Chem. Soc., 1398 (1935).

(89) L. F . Fieser and M. Fieser, “Organic Chemistry,” D. C. Heath and Co., Boston, Mass., 1944,p. 351.

131

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

on C1 and C2 excluded the further electron displacement necessary to osazone formation according to all modern conceptions of its mechanism. Perciva1,gO in his comprehensive studies on the structure of phenylosazones, could not justify the views of the Fiesers because of lack of experimental evidence. Hardegger and S~hreier,~’ the latest workers to deal with the problem, considered that any attempt to define precisely the structure of the phenylosazones, on the basis of information at hand, would be immature. For this reason, insofar as possible, they used Fischer’s open-chain formula for the phenylosazones and their derivatives. Until the fonnazan reaction was applied to this field, the finer structure of the sugar phenylosazones, and, indeed, whether they are cyclic or acyclic, was uncertain. 1. Preparation of “D-Ghcosazone Formazan”

It is known that the formazan reaction depends upon two conditions?, The first is the presence of an aldehydo Schiff base (-CH=N-), a criterion which is fulfilled in aldehydo-phenylhydrazones,but not in keto-phenylhydrazones or in phenylhydrazones of the cyclic hemiacetal forms of the aldoses. The second condition is the presence of free imino hydrogen in the phenylhydrazone group. If Fischer’s open-chain structure is correct, the phenylhydrazone group at C1 satisfies both conditions, and the formazan reaction would be expected to proceed according to the following equation. 9 , lo

C/‘ = N - N H - ~

C=N-NH-/H -

C=N-NH-

I

R

I CI I

= N - N H - ~ -

R

c=o

I

R (90) E. G. V. Percival, Advances in Carbohydrate Chem., 3, 23 (1948). (91) E. Hardegger and E. Schreier, Helv. Chim. Acta, 35, 232 (1952).

132

L. MESTER

The name phenylosazone formazan is suggested for this group of compounds, of which a few simple representatives have been known for some time, such as those where R = CHa or R = COzH; however, these were not made from osazones, but by treatment of the corresponding a-keto-formazans with phenylhydrazine (as shown in the lower left of the above equation). For example, pyruvaldehyde phenylosazone formazan (C-acetyl-N, N’-diphenylformasan phenylhydrazone) was prepared by Bamberger and Lorenzeng2as early as 1892 by condensation of acetylformazan with phenylhydrazine. The violet or brownish-black color characteristic of all known compounds of this group can be attributed to the three chromophoric groups present in the structure. Coupled with diazo compounds in pyridine-alcohol solution, the sugar osazones fail to yield formasans. This may be due to one or both of the following reasons. 1. Instead of a phenylhydrazone group of Schiff structure, C l of “D-glucose” (D-arabino-hexose) phenylosazone may have a ring or azo structure unsuitable for formazan reaction. In this connection, Hardegger and Schreiergl have stated that, if all the structural possibilities are taken into account, their number may be as high as one hundred. In the case of “Dglucose” phenylosazone, these may be represented by nine fundamental compounds, of which the formulas IX, XI, XII, XV, and XVI-and all of the isomers (sun, anti, a, and p ) derivable from them-are unsuitable for formazan reaction. 2. The “D-glucose’) phenylosazone structure may lend itself to coupling (VIII, X, XIII, and XIV), but may contain no free imino hydrogen atom. It has long been established that, if the free imino hydrogen atom of the phenylhydrazone is replaced by an alkyl or aryl group, no formazan reHC=N-NHL=NN - H-.

I I HCOH I

HOCH

HCOH

I

CH~OH VIII Fischer (1887)

a ,‘

-

CHZ-N=N-’

a

HC=N-NH-

I

b _ N - N H - D-

I I HCOH I

HOCH

HL--N=N--HOCH

HCOH

I

CH~OH

I I HCOH I HCOH

CH~OH IX X Baly, Tuck, Marsden and Gazdar (1907)

(92) E. Bamberger and J. Lorenzen, Ber., 26, 3539 (1892).

a

133

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

HC-NH-NHII

CH-N=N-

I

1I

C - N H - N H O

C-N=N-

I HOCH I HCOH I HCOH I

CHnOH

CHzOH

XI Zerner and Waltuch (1914) HC=N-NH-

I

-

I HOCH I HCOH I HCOH I

0

--C-NH-NH-

I HOCH I HCOH I HCOH I

XI1 Zerner and Waltuch (1914) HC-N-NH-

II

-C-NH-NH-

I 1 HCOH I

HOCH

-0CHz

CHpOH --

XI11 Percival and Percival (1935) H -C-NH-NH

I I

C=N-NH-

I

XIV Percival and Percival (1935) H -C-NH-NH-

I

I I

HOCH

HOCH

I I

HCOH

CHyOH XV Haworth (1929)

CHaOH XVI Haworth (1929)

134

L. MESTER

action will g - lo The most recent investigationss3~ 94 reveal that chelation of the imino hydrogen atom produces a similar result. It is possible that the structural formula proposed by Fieser and Fie~er,8~ in which the imino hydrogen atom of the phenylhydrazone on C1 takes part in chelation, would explain the lack of formazan reaction. In order to find the reason for the failure of formazan reaction, a model experiment was set upgs using 2-quinolinecarboxaldehyde phenylhydrazoneg0whose structure, if the nitrogen of the quinoline be considered, is like that of a phenylosazone, with the six-membered chelate ring in it as proposed by Fieser and Fie~el.8~ for sugar osazones. ‘[NEZN-]

/

pyridine

‘[N=N-a]

I H-N

/N

I

alc. NaOH

c()

’ No reaction \

\N’\

/

N = N - a

C

In the model reaction carried out with this compound, it was found that, in pyridine solution, the chelate N -+ H completely inhibited formazan reaction, which indicates that, in “D-glucose” phenylosazone, similar chelation may be responsible for the failure to form a formazan. On replacing the pyridine with alkaline ethanol (often used to catalyze the formazan reaction), the chelate ring was loosened and coupling occurred to give N ,N’-diphenyl-C-(2-quinolyl)formazan. Analogous to the formazans obtained by loosening the chelate ring in the above manner, the sugar osazones of supposedly chelate structure were found to undergo reaction in alkaline alcohol and to give %-glucose” phenylosazone formazan in dark-violet needles.96~97 Mild acetylation produces the black tetraacetate of this compound. (93) G. Zempldn, L. Mester, A. Messmer and A. Major, Magyar Tudodnyos Akad. K i m . Tudodnyok Osztttlyctnak Kdzlembnyei, 6 , 303 (1955). (94) G . Zempldn, L. Mester, A. Messmer and A. Major, Acta Chim. Acad. Sci. Hung., 7, 455 (1955). (95) L. Mester, J . Am. Chem. SOC.,7 7 , 4301 (1955). (96) V. von Miller and J. Spady, Ber., 18, 3402 (1885). (97) L. Mester, Magyar Tudonuinyos Akad. Khm. Tudodnyok OszMly&nak K d z l embnyei, 6 , 201 (1955).

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

135

H I CI = N - N H - ~

1

-

I I HCOH I

I I HCOH I

HCOH

HCOH

CHsOH

CH20H

“D-Glucose’’ phenylosazone (D-arabino-hexose phenylosasone)

‘LD-Glucose”phenylosazone formasan (D-arabino-hexose phenylosasone N , N’-diphenylformasan)

ACOCH

I I HCOAc I HCOAc

CHpOAc

Tetra-O-acetyl-“D-glucose” phenylosazone formazan (tetra-O-acetyl-D-arubinohexose phenylosazone N, N’-diphenylformasan)

The structure of “D-glucosazone formazan” is also proved by the synthesis starting from D-glucosone 1-(N2-pheny1)hydrazone. Coupling thisg8 with diazotized aniline in cold pyridine yields ‘(D-glucosone formazan” [N ,N’-diphenyl-C- (D-arabino-tetrahydroxypentanoyl)formazan] as red needles. Condensation of this compound with phenylhydrazine leads to (98) G. Henseke and M. Winter, Chem. Ber., 89, 956 (1956).

136

L. MESTER

the same violet needles of “D-glucosazone formazan” as are obtained on coupling “D-glucose” phenylosazone directly.99 N

=

/

N

-

C

H C=N-NH-=

I

I

-

C=N-NH-= I

I

-

‘[NEN-Q] alo. NaOH

’

I I I HCOH I HCOH I

HOCH

HOCH

I I HCOH I HCOH

CHeOH i‘D-Glucosazone formazan”

CHzOH “D-Glucose” phenylosazone

I

H , N - N H ~

+

pyr idine

N = N - D C

H c = N - N H ~

/

-

I

I co I HOCH I

-

CO

‘“EN-] pyridine

f

I

HOCH

I

HCOH

HCOH

I I

I

1

HCOH

HCOH

I

CHzOH D-Glucosone 1-(N*-phenyl)hydrazone

CHzOH N ,N’-Diphenyl-C-(u-arabinotetrahydroxypentanoy1)formazan

The identity of the two products not only proves the structure of “Dglucosazone formazan,” but leaves no doubt that, in the D-glucosone 99 the phenylhydrazine residue phenylhydrazone prepared by Henseke,B8* is attached to C1. (99) L. Mester and

A. Major, J . Chem. Soc., 3227

(1956).

~

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

137

2. Evidence in Favor of the Open-chain Structure of ‘(D-Glucose” Phenylosazone

The conditions governing the preparation of “D-glucosazone formazan,” and the properties of the product obtained, permit certain conclusions. From the fact that “D-glucose” phenylosazone gives no reaction in pyridine but undergoes reaction in alkaline alcohol, it is obvious that the compound must have a structure in which the breaking of the chelation produces a Schiff-base system on C1 which is suitable for the formazan reaction. Such a structure can only be possible with formulas VIII, X, XIII, and XIV. Structure X must be discarded for lack of stability, as it presupposes isolated double bonds, whereas systems containing conjugated double bonds are known to be much more stable. Modern studies on the mechanism of osazone formation show that it is the formation of this stable, conjugated system which is the driving force1@’ behind the formation of osazones. On the other hand, structure X would yield, on application of the formazan reaction, a product containing the azo group assumed in formula X, whereas ultraviolet spectra studies definitely excluded its presence. The investigations of EngeP on free “D-glucose” phenylosazone, those of Wolfrom and coworkers84on tetra-0-acetyl-“D-glucose” phenylosazone, and those of Akiya and Tejimas6,88 on methylated “D-glucose” phenylosazone are all in agreement with the acyclic structure. This would appear to exclude the ring structure XIII, proposed by Percival and PercivaP and the ring structure XIV. Hardegger and Schreierg’ make no use of them in their work. The data which follow are definitely in favor of the open-chain structure postulated for the “D-glucose” phenylosazones by Fischer, and against the ring structures proposed by other workers. (a) In structures XI11 and XIV, the compounds produced upon coupling should contain two chromophoric groups of the simple formazans (CH=N, N=N), which should result in the usual red color. However, the dark-violet color of the %-glucose phenylosazone formazan” obtained is similar to that of pyruvaldehyde phenylosazone formazan (which contains three chromophoric groups). For Percival’s structures XI11 and XIV, the formation of these three chromophoric groups is out of the question. This view is fully supported by the ultraviolet spectrum of “D-glucose phenylosazone formazan” (maxima at 335 and 410 m@),which coincides with that of the indisputably acyclic pyruvaldehyde osazone formazan (maxima at 335 and 410 mp) and differs substantially from the ultraviolet spectrum of the simple D-glucose diphenylformazan (maxima at 255 and 425 mp) (see Fig. 12). (100) C. R. Noller, “Chemistry of Organic Compounds,” W.B. Saunders & Co., Philadelphia and London, 1952, p. 359.

138

L. MESTER

(b) The product obtained upon mild acetylation of “D-glucose phenylosazone formazan” contains four 0-acetyl groups, which is consistent only with an acyclic structure. The possibility that a ring structure may have been opened during the mild acetylation is emphatically refuted by the fact that the ultraviolet spectra of the free and acetylated %-glucose phenylosazone formazan” are almost identical (maxima at 335 and 410 mw), whereas such a ring opening would result in the disappearance of one chromophoric group. Formulas X, XIII, and XIV are thus eliminated, leaving only Fischer’s acyclic structure VIII.

2.0

ai

1.o

750

FIG. 12.-Ultraviolet Spectra in Ethanol. 1. “D-Glucose” Phenylosazone Formazan; 2. “D-G1UCOEe” Phenylosazone Formaean Tetraacetate; 3. Pyruvaldehyde Phenylosazone Formaean; 4. D-Glucose Diphenylformaaan.

3. Evidence in Favor of the Chelate Structure of “D-Glucose” Phenylosazone (a) Since D-glucosazone fails to react in pyridine but undergoes reaction in alkaline alcohol, the experience gained with the phenylhydrazones of in~particular with 2-quinolinecarboxaldehydephenylchelate s t r u c t ~ r eg4, ~ ~ hydrazone, permits the conclusion that, in D-glucosazone, the aldehydophenylhydrazine group must participate in a chelate structure. (b) The ultraviolet spectra of “D-glucose” phenylosazone (maxima at 256, 310, and 396 mp) and of %-glucose” 1-(2-methyl-2-phenyl)-2-phenylosazone (maxima at 258, 305, and 390 mp), which also has a structure capable of chelate-ring formation, are very similar but differ markedly from that of “D-glucose” 2-methyl-2-phenylosazone (maxima at 264 and 355 mp) in which there is no possibility of chelate-ring formation. This absence of the chelate structure, and not the presence of the methyl group, is evidently responsible for the spectral difference, since in the mixed osazone A , with a structure capable of chelate formation, the introduction of a methyl group made no spectral difference.

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

139

(c) In evaluating the ultraviolet spectrum of “D-glucose” phenylosazone, ~ others a hydruzolol some of the earlier investigators favored an ~ z o 7and structure. This apparent contradiction is removed if one considers that the phenylosazones have a chelate structure. Polarographic studies have shown that ‘%glucose” phenylosazone possesses a conjugated, chelate structure corresponding to two double bonds of identical character”J2;this can be represented by the structural formulas VI and VII (see p. 130). (d) The chelate structure of the osazones also explains why only one of the two imino hydrogen atoms of the sugar osazones is capable of being methylated. Obviously, the free imino hydrogen atom lends itself more readily to methylation than the one in the chelate ring, and this is why mild methylation of %-glucose” phenylosazone always leads t o a mono-Nmethyl derivative, and why, even upon treatment vigorous enough to methylate the hydroxyl groups, again only one of the imino hydrogen atoms 86 is replaced by a methyl (e) Methylation of “n-glucose” phenylosaxone invariably yields “D-glu(derivable from checose” 1-(N2-methyl-N2-pheny1)-2-(N2-pheny1)osazone late structure VI) or some derivative thereof, further methylated on the sugar part. For this reason, and because it has so far not been possible to prepare “D-glucose” phenylosazone derivatives having chelate structure VII, chelate structure VI is to be preferred. On the strength of the foregoing evidence, to “n-glucose” phenylosazone is assigned the open-chain structure proposed by Fischer, with the addition that its actual state is best approached by the chelate structure VI postulated by Fieser and Fieser?O

4. The Structure of the So-called Mixed A and Mixed B Osazones The formazan reaction offers a means for clarifying the structure of the mixed A and mixed B osaxones. The former was described by VotoEek and VondraEeklo3as acyclic “D-glucose” 1-(N2-pheny1)-2-(N2-methyl-N2phenyl)osazone, and the latter as acyclic %-glucose” 1-(N2-methyl-N2pheny1)-2-(N2-pheny1)osazone.The Percivalslo4assigned the same “D-frucstructure t o topyranose” 1-(N2-pheny1)-2-(N2-methyl-N2-pheny1)osazone the compounds, but differentiated them as probable syn and anti forms. Of these proposed structures, only that of the ‘%-glucose” 1-(W-methylN2-pheny1)-2-(N2-phenyl)osazone, suggested by VotoEek and VondraEek for mixed osazone B, is theoretically unsuited for formaxan formation since (101) (102) (103) (104)

P . Grammaticakis, Compt. rend., 225, 1139 (1946). B. Jhmbor and L. Mester, Acta Chim. Acad. Sci. Hung., 9, 485 (1956). E. VotoEek and R. VondraEek, Ber., 57,3848 (1904). E. E. Percival and E. G. V. Percival, J . Chem. Soc., 750 (1941).

140

L. MESTER

H IC = N - k O

HC=N-NH-

I

I

I

, = N - N - D

I HOCH I HCOH I

I

CHI

c = N - N H - ~

-

HCOH

-

I HOCH I HCOH I HCOH

I

I

CH~OH

CHaOH

A “D-Glucose” l-(Wphenyl)-2(P-methyl-Ns-phenyl) osazone

B “D-Glucose”1-(N*-methyl-N*phenyl)-2-(N*-phenyl)osazone

Structure of the mixed A and mixed B osazones according to Voto6ek and VondraEeklW H H

wn

anti

%-Fructopyranose” l-(N~-phenyl)-2-(N*-methyl-N*-phenyl)osazone

Structure of the mixed A and mixed B osazones according to Percival and PercivaP the imino hydrogen atom of the phenylhydrasone group on C1 is replaced by a methyl group. However, since mixed osazone A undergoes no formazan reaction, even in alkaline solution, it would appear that the “D-glucose” 1-(N2-methylN2-pheny1)-2-(N*-pheny1)osazonestr~cture,~7* lo4hitherto thought to be incapable of coupling, should be assigned to this substance (instead of to the mixed osazone B ) . This view is fully confirmed by recent work of Henseke and Hautschel.lo6 Concerning the finer structure of the mixed osazone A , the following points merit mention. (a) Akiya and TejimaS6* found that mixed osazone A is identical with the mono-N-methyl derivative of “D-glucose” phenylosasone obtained by (105) G . Henseke and H. Hautschel, Chem. Ber., 87,477 (1954).

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

141

mild methylation of “D-glucose” phenylosazone, and that even vigorous methylation would not replace the second imino hydrogen atom with a methyl group. (b) The ultraviolet absorption spectrum of the mixed osazone A (maxima at 256,310, and 396 mp) agrees with that of %-glucose” phenylosazone with the chelate structure (maxima at 256, 310, and 396 mp), but differs (maxsubstantially from that of “D-glucose” N2-methyl-N2-phenylosazone ima at 264 and 355 mp), in which there is no possibility of a chelate ring formation. On the basis of these experimental facts and the above considerations concerning the structure of ((D-glucosellphenylosazone, the chelate ring form VI of Fieser and FieseP appears to describe the finer structure of the mixed osazone A . In contrast to mixed osazone A , mixed osazone B undergoes fonnazan reaction with ease, to give the same I‘D-glucose phenylosazone formazan” as “D-glucose” phenylosazone itself. Further investigation showed that mixed osazone B is (b-glucose” phenylosazone contaminated with mixed osazone A , and that careful purification always yields LLD-glucose” phenylosazone. Since these findings have been confirmed by recently published work of Henseke and Hautschello6and Henseke and Bautzel1O6the so-called “mixed osazone B” appears to have lost any further justification for continuation in the literature.

5. The Structure of Diels’ Anhydro-“D-qlucose’)Phenylosazone This compound was isolated by Diels and Meyerlo7on boiling “D-glucose” phenylosazone in methanol containing traces of sulfuric acid. They believed it to be identical with 3,6-anhydro-“~-glucose”phenylosazone, which had previously been prepared by other means.lo8,log After observing differences in the physical properties of the two compounds, they suggested formula XVII, which they modified a year laterlog”by proposing the pyrazole structure XVIII. This was found to be inadequate for several reasons, and the bicyclic formula XIX, or XX, was put forward by Percival.ll0 After additional experimental work, Hardegger and SchreierQ’proposed formula XXII , observing that Fischer’s original structure for the osazones was retained in it since exact structures of osazones had not yet been unequivocally established. They also pointed out that there was only indirect (106) G.Henseke and M. Bautze, Chem. Ber., 88, 62 (1955). (107) 0.Diels and R. Meyer, Ann., 619, 157 (1935). (108) E.Fischer and K . Zach, Ber., 46,456 (1912). (109) H. Ohle, L.Vargha and H. Erlbach, Ber. 81, 1211 (1928). (109a) 0.Diels, R.Meyer and 0. Onnen, Ann., 629.94 (1936). (110) E.G.V. Percival, J . Chem. SOC.,783 (1945).

142

L. MESTER

-C=N-NH-

I

N=CH

cI = N - N H ~ -

I

H O ~ H

I I HCOH I

I

I

HCOH

HCOH

I I

HCOH -CHz

XVII Diels and Meyer (1935)

CHzOH XVIII Diels, Meyer and Omen (1936)

HC=N-NH-

I

C-NH

I I

HOCH HCOH

I

-0CHa

H

xx

XIX Percival (1945) N=CH D

~

C

I

Percival (1945)

I

NH-NH-NH-

\ / HCOH

I

HCOH

HCOH

I CHzOXXI

Hardegger and Schreier (1952)

I

CHzO XXII Hardegger and Schreier (1952)

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

143

evidence against Diels’ formula XVIII and the spirocyclic formula XXI, which they themselves had proposed, and that neither could yet be discarded. The formazan reaction, again, was found suitable for clarifying these points. Diels’ anhydro-“D-glucose” phenylosazone, like “D-glucose” phenylosazone, does not react in pyridine solution, while in an alkaline ethanol medium an osazone formazan is obtained which crystallizes as black needles.”’ Mild acetylation of this compound yields the black diacetate of Diels’ anhydro-%-glucose phenylosazone formazan.” Analysis showed both acetyl groups to be oxygen-linked. H C=N-NH--=

I I

1

C=N-NH-

I HCI I HCOH HcoH! CHoO I

0 /

‘[NCN-]) d c . NaOH

Diels’ Anhydro-%-glucose” phenylosazone

I

I I HCOAc I HCOAc I HC-

‘

HCOH

I

1J

CH20

(Diels’) Anhydro-“D-glucose phenylosazone formazan”

CHzODi-0-acetyl-(Diels’) anhydro“D-glucose phenylosazone formazan”

(111) (a) L. Mester and A. Major, Magyar Tudomdnyos Akad. K6m. Tudomdnyok Osztdlydnak K&dembnyei, 6 , 217 (1955); (b) J . Am. Chem. SOC.,77,4305 (1955).

L. MESTER

144

The fact that the formazan is formed eliminates structures XVII, XVIII, and XXI, which do not meet the conditions for this reaction. This furnishes direct evidence in place of the indirect evidence on which Hardegger and Schreierglhad discarded structures XVIII and XXI. The bicyclic structure XIX, and XX, proposed by PercivaPo can be dismissed on comparison of theultraviolet spectra.This compound,after coupling,shouldcontain the two chromophoric groups of the simple formazans, whereas osazone formazans contain three of them. Anhydro-‘%-glucose phenylosazone formazan” and its diacetate display practically the same maxima (maxima at 335 and 405 mp; and 340 and 405 mp, respectively) as does the indisputably-acyclic pyruvaldehyde phenylosazone formazan (maxima at 335 and 410 mp), and these peaks differ substantially from the values for the simple sugar for-

260

500

760

X. my.

FIG.13.-Ultraviolet Spectra in Ethanol. 1.Diels’ “Anhydro-D-glucose Phenylosazone Formazan”; 2. Diels’ Di-O-acetyl-anhydro-“D-glucosePhenylosazone Formaz m ” ; 3. Pyruvaldehyde Phenylosazone Formazan; 4. D-Glucose Diphenylformazan.

mazans, for example, for u-glucose diphenylformazan (maxima at 255 and 425 mp); see Fig. 13. Formazan formation, the presence of two 0-acetyl groups in the acetylation product of the osazone formazan obtained, and the comparative study of the ultraviolet spectra, are all in harmony with formula XXII proposed by Hardegger and Schreiergland afford evidence of its correctness. As in the case of “u-glucose” phenylosazone, these experimental findings make necessary the assigning to the finer structure of Diels’ anhydro-“Dglucose” phenylosazone the chelate-ring structure postulated (without definitive experimental evidence) by Fieser and Fieser.8O

6. Mutarotation of Sugar Osazones Mutarotation of a sugar osazone was first observed by Levene and coworkers,l12*113 but they did not attempt to give an explanation of the (112) P. A. Levene and W. A. Jacobs, Ber., 42,3249 (1909). (113) P. A. Levene and F. B. LsForge, J . Biol. Chem., 20,430 (1915).

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

145

phenomenon. Zerner and Waltuch78 suggested that it should be ascribed to the tautomeric change of the bishydrazone form (XXIII) into the azohydrazone form (XXIV) . Haworthsobelieved he had found the explanation in the cyclic form (XXV). c /H = N - - N H - ~ -

1

=

,

,

H

a

-

XXIII

-C-NH-NH

II

xxv Discarding all earlier views, EngeP endeavored to show that mutarotation was due to hydrolysis, with consequent scission of phenylhydrazine. Since the use of the formazan reaction had confirmed both the open-chain structures2* 97 and the presence of the earlier-postulated chelate ring for the sugar phenylosazones, it appeared necessary to revise the then-current interpretations of their mutarotation.114 It was found114that the mutarotation of the sugar phenylosazones indicates a reaction of the first order, as shown in Fig. 14. With the solvent carefully evaporated in vacuo, the osazone is recovered unchanged. Repeated mutarotation takes practically the same course. As regards the ultraviolet spectra of the phenylosazones during mutarotation, a slight shift of the characteristic maximum at about 395 mp toward shorter wavelengths and a gradual disappearance of the minimum around 345 mp are distinctly noticeable (see Fig. 15); the same holds true in respect to the recovered material. Reaction of the “D-glucose’’ phenylosazone remaining, in samples taken from the mutarotating solution after increasing periods of time, with diazo solution, yields less and less osazone formazan (see Fig. 16). This is obviously attributable to the same structrual change as that which is optically (114) L. Meeker and

A. Major, J . Am. Chem. SOC.,79,3232

(1957).

L. MESTER

346

perceivable during mutarotation. Repeated mutarotation produced the same yields. Since mutarotation, spectral displacement, and formazan formation after evaporation of the solvent are, in the redissolved substance, repetitive proc-

2o

0

0

t

I

I

5

10

5

10

15

20

25

15

20

25

Time, hr.

FIQ.14.-Upper Curves :Mutarotation of “D-Glucose” Phenylosazone in Pyridinealcohol (1: 1 by vol.); Lower Curves: Mutarotation of “D-Ghcose” l-(NZ-Methyl-Nzphenyl)-2-(N*-phenyl)osazone in Pyridine-alcohol (1: 1 by vol.) . -[ Initial substance, .. . . recovered substance.

-

esses, Engel’s that mutarotation depends on the hydrolysis of osazones, seems to be excluded. Since Haworth’s cyclic formula XXV is unable to yield a formazan, the transformation of the one anomer into the other cannot possibly be regarded as the cause of mutarotation, in this case. The open-chain compound XXIII cannot change into Haworth’s cyclic form XXV, for, if it could, owing to the disappearance of one chromophoric group, the maximum of the ultraviolet spectrum would have to show a substantial alteration of its

147

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

E

2.0

-

1.0

-

300 400 5 00 600 mp FIG.15.-Ultraviolet Absorption Spectrum of %-Glucose” Phenylosaaone in Ethanol (1, immediately on dissolution; 2, 1 hour after dissolution; 3, 24 hours after dissolution). [Exactly the same changes were obtained for ‘b-glucose” I-(N2-methyl-N2phenyl)-2-(N2-phenyl)osazone.]

0‘

5

10

15 Time

20

25

, hr.

FIG. 16.-Upper Curves :Mutarotation of “D-Ghcose” Phenylosaaone in Pyridineethanol (1: 1 by vol.) ; Lower Curves, “D-Ghcose Phenylosazone Formasan” Formainitial substance, . * . . . recovered substance). tion (-

-

148

L. MESTER

The observations of Weygand and coworkers116on the mutarotation of 5 6-di-O-methyl-~-arabo-hexosep-bromophenylosazone, and those of

Jones and coworkerP on the mutarotation of L-glycero-tetrulose phenylosazone and its derivatives, leave no room for a concept which holds that mutarotation might be caused by cyclization starting at C2. Finally, the mutarotation of tetra-0-acetyl-D-arabino-hexosephenylosaand of tetra-0-acetylof 4-deoxy-~-glycero-tetrosephenylo~azone,~~7 D-lyxo-hexose phenylosazone can in no way be explained by cyclization, since in these cases there is definitely no possibility for ring formation to take place. It also seems impossible that a stable, conjugated system (XXIII) of double bonds should change into an unstable compound (XXIV) having isolated double bonds, as has been suggested by Zerner and Waltuch.?8 Nevertheless, on the above evidence, it would appear that the real cause of the mutarotation is to be sought in the electron displacement which takes place in the chelate structure of the osazones upon the action of an alkaline solvent (e.g., pyridine), and which, amongst other things, is revealed in a displacement of the ultraviolet spectra and in a decrease in the coupling capacity. Concerning 1-phenylazo-2-naphthol and 1 ,2-naphthoquinone 2-phenylhydrazone, the chelate structure of which can be influenced by the solvent, reference is made to the work of Burawoy and coworkers.lls On the other hand, there are several articles in the literature pointing out that an a ,,%unsaturated azo structure is formed in the p-nitrophenylhydrazones of unsaturated ketone^,^^^-^^^ and that the rearrangement of the hydrazone of the compound of azo structure takes place upon the action of a solvent, and finally that this is a somewhat slow process causing changes in the optical rotation.lZ2 REACTION OF PERIODATE-OXIDIZED VIII. THE FORMAZAN

SUQARDERIVATIVES 1. 1 ,%0-Isopropylidene-a-D-glucofuranose

On oxidation with periodic acid, 1 ,2-O-isopropylidene-a-~-glucofuranose128+ 12*yields 1 ,2-O-isopropylidene-~-xylo-pentodiose. If this is caused to (115) F. Weygand, H.Grisebach, K. D. Kirchner and M. Haselhorst, Chem. Ber.,

88,487 (1955). (116) P.A. J. Gorin, L. Hough and J. K. N. Jones, J . Chem. SOC.,2699 (1955). (117) J. Fried, D. E. Walz and 0.Wintersteiner, J . Am. Chem. Soc., 68,2746 (1946). (118) A. Burawoy, A. G . Salem and A. R. Thompson, J . Chem. Soc., 4793 (1952). (119) F. Ramirez and A. F. Kirby, J . Am. Chem. SOC.,76,6026 (1953). (120) J. van Alphen, Rec. trau. chim., 64,305 (1945). (121) R. J. W. Le FBvre, M. F. O’Dwyer and R. L. Werner, Chem. & Znd. (London), 378 (1953). (122) W. F. McGuckin and E. C. Kendall, J . Am. Chem. Soc., 74, 5812 (1952). (123) J. C.Sowden, J . A m . C h m . SOC.,73, 5496 (1951).

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

149

react with phenylhydrazine, it gives the mono-5-(phenylhydrazone), but if first hydrolyzed, it affords the 1,5-bis(phenylhydraaone) of xylopentodiose.lZ6 These compounds had been obtained earlier by using lead tetraacetate as the oxidant.12e-12*

1 HCol

HCO1

HCO

HCO

I

I

I

:Me2

I I HCOI

I I HCOI

HOCH

HOCH

HaN-NH-4

c=o \

HCOH

I

CHzOH

+

1,2-O-IsopropylideneD-glucose

H

1,2-O-IsopropylideneD- zylo-pentodiose

I

I

HCOi

1

JMes

HCOT

1

;Me2

JMez

HCO

HCO

HOCH

HOCH

I

I

HCO-

I

[a-N=Nlm

'

I

HCO-

(124) J. C. Sowden, J . Am. Chem. SOC.,14, 4377 (1952). (125) L. Mester and E. M6cz&r,J . Chem. Soc., 3228 (1956). (126) V. Brocca and A. Dansi, Ann. chim. (Rome), 44, 120 (1954). (127) J. M. Grosheintz and H. 0. L. Fischer, J . Am. Chem. SOC.,70, 1476 (1948). (128) K . Iwadare, Bull. Chem. SOC.Japan, 16, 40 (1941); Chem. Abstracts, 36, 4740 (1941).

150

L. MESTER

/ C

/”

‘.., H

C=N-NH--

I HCOH I

2 [=NENle

HOCH

b

I HCOH

HOCH

H

zylo-Pentodiose 1,6bis(phenylhydrazone1

zylo-Pentodiose 1,5bis-

( N ,N’-diphenylformal;an)

Reaction of the mono-5-(phenylhydrazone) with diazotized aniline in pyridine-ethanol produced 1 2-O-isopropylidene-~-xylo-pentodiose 5-(N ,N’-diphenylformazan),and the l15-bis(phenylhydrazone)gave xylopentodiose 1 5-bis(N N’-diphenylformazan) .126 The former product is the first sugar derivative to contain the formazan group at the highest-numbered carbon atom; the latter belongs to a novel class of sugar formazans. 2 . Methyl a-n-Glucopyranoside The structure of the product obtained by the oxidation of methyl a-Dglucopyranoside with periodic acid has been controversial for a long time.lZ8 In the literature, six formulas (XXVI--1) are suggested. The (hydroxymethy1)-methoxy-diglycolaldehyde structures XXVI, XXVII, and XXVIII show the presence of two free aldehydo groups, wherefore they should yield a bis(formazan) compound. The “hemialdal” and “hemialdal” hydrate structures XXX and XXXI contain no free aldehydo group and are therefore not suitable for yielding a formazan. This is borne out by the fact that the periodate-oxidized 3 6-anhydro-“~-glucose” phenylosotriazole of known “hemialdal” structure, prepared by Schreier, Stohr and Hardegger,’So failed to produce a formazan. Finally, the cyclic (129) C. D.Hurd, P. J. Baker, R. P. Holys and W. H. Saunders, J . Org. Chem., 18, 186 (1953). (130) E.Sohreier, G . Stohr and E. Hardegger, Helu. Chim. A d a , 37,574 (1954).

151

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

I

c=o I CHaOCH I

Hc°CHa I c / \ 0

H

H

\

//O

C

I

0

I

HCCHzOH

I c=o

‘I \ I

HCO

CHzOH XXVI

c=o H

\ I

I

CHaOH

H XXVIII OCH,

H

I

I

HC-CH

CBO-CH 0I

H

O

O=CCH

XXVII

I HCOH

I I

CHaOCH

/

I

\

I

/ O

I

\

0 0

HO CHB-C-CH

O=C-CH

I

H

LCH-!H

I

CHzO

OH

OH

I

CHz

xxx

XXIX

OH

/

XXXI

hemiacetal structure XXIX contains an aldehydo group and is thus suitable for yielding a mono-(fonnazan). Q-NH-NH~

It 0

I

I OH

pyridiae

I

152

L. MESTER

The fact that, from periodate-oxidized methyl a-D-glucopyranoside, a mono-(formazan) could be prepared,’*’ constitutes evidence in favor of the latter’s hemiacetal structure (or the hydrate form) as expressed in the preceding formula. This finding is in full agreement with the results obtained by Smith and coworkers132in their latest selective-reduction studies. 3. Sucrose

Barry and MitchelP were the first to oxidize sucrose with periodic acid, separating the product as a phenylhydrazone. On finding that, after oxidation, each of the two monosaccharides of the sucrose reacted with only one molar proportion of phenyhydrazine, these workers attributed to the oxidized sucrose not a dialdehyde, but the following “hemialdal” [bis(hemiacetal) hydrate] structure. CHsOH CH

0-c

I 1

CH-OH

lY7 \

/

CH-OH

I CHOCHiOH I

0

/

CH-OH

I I

CHO-

CH40H

Reaction of the phenylhydrazone (in pyridine solution) with diazotized aniline yielded a bright-red formazan, which, calculated on the oxidized sucrose, contained two formazan g r 0 ~ p s .This l ~ ~ permits the conclusion that the structure of oxidized sucrose is neither “hemialdal” as postulated by Barry and Mitchell,133nor dialdehyde as proposed by other authors, but predominantly aldehydo-hemiacetal (or the hydrate form) (see p. 153).

4. Nucleosides On oxidation with periodic acid, nucleosides which contain a n-ribofuranosyl group, for example, adenosine, yield a monoformazan (131) L.Mester and E. M6cz&r, Chem. & Znd. (London), 761 (1957). (132) J. E.Cadotte, G. G. S. Dutton, I. J. Goldstein, B. A. Lewis, F. Smith and J. W. van Cleve, J . Am. Chem. SOC.,79, 691 (1957). (133) V. C. Barry and P. W. D. Mitchell, J . Chem. SOC.,4020 (1964). (134) L. Mester and E. M6cz&r,Chem. & Znd. (London), 764 (1957). (135) L. Mester and A. Major, unpublished results.

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

t

153

154

L. MESTER

this permits the conclusion that such oxidized nucleosides, are likewise, predominantly of the aldehydo-hemiacetal structure.

IX. THEFORMAZAN REACTION OF OXIDIZED POLYSACCHARIDES 1 . Oxidation with Periodic Acid

When coupled, with cooling, with phenylhydrazine, the periodate-oxidized polysaccharidescontaining two or more adjacent hydroxyl groups (for example, cellulose, starch, dextrin, and dextran) yield bright-yellow phenyl136, 137 hydrazones within a few Dissolved or suspended in ice-cold pyridine-ethanol and coupled with diazotized aniline solution, the phenylhydrazones of the oxidized polysaccharides lead to their vividly red diphenylformazans.138 From the formazan formation, it is possible to conclude the structure of the oxidized polysaccharides*36-139, and their phenylhydrazones. In polysaccharides oxidized with periodic acid, one molar proportion of each individual, oxidized monosaccharide reacts with only one mole of

XXXII

XXXIII

XXXIV (136) (137) (138) (139) (140) (1951).

E. L. Jackson and C. S. Hudson, J. Am. Chem. SOC.,69,2049 (1937). G. Jayme and M. Satre, Ber. 7 7 , 242, 248 (1944). L. Mester, J. Am. Chem. Soc., 77, 5452 (1955). H. J. Mitchell and C. B. Purves, J . Am. Chem. SOC.,64, 589 (1942). J. W. Rowen, F. H. Forsiati and R. E. Reeves, J. A m . Chem. Soc., 73, 4484

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

155

amine;’41-142 (for example, of phenylhydra~ine’~~), but this is a fact inconsistent with the dialdehyde structure hitherto assigned to oxidized polysac137 On this basis, formulas XXXII, XXXIII and XXXIV are charide~.’~~, suggested in the literature for the phenylhydra~ones.’~~ Compound XXXII is an aldehyde-hemiacetal phenylhydrazone; XXXIII is a hemiacetal-aldehydo phenylhydrazone; and XXXIV is a socalled “hemialdal” structure. Of them, only XXXIII is suited to formation of a formazan. The successful formazan reaction furnishes proof of the predominance of this hemiacetal-aldehydo phenylhydrazone structure. On these grounds, it is safe to assume that, to the oxidized polysaccharides themselves, this aldehyde-hemiacetal structure (or the hydrate form) is to be assigned. The sequence of reactions (p. 156) is suggested for the formation of the formazan of oxidized starch. For potato starch oxidized with 5 % sodium metaperiodate for twentyfour hours at room temperature, two of the three monosaccharide residues are transformed to formazans. By regulating the rate of oxidation it is possible to determine how many formazan groups form per monosaccharide unit. For instance, in a cellulose wad oxidized as prescribed by Jayme and Satre,137for 120 hours, statistically every third monosaccharide is converted into a formazan; and in a wad oxidized for four hours, every sixteenth monosaccharide. Provided that the rate of oxidation is not too high, the formazan obtained retains its original fibrous structure. The transformation into formazan can be regulated in all the other polysaccharides in the same way. With the soluble polysaccharides (for example, dextran) , oxidation is a much quicker process. By coupling the phenylhydrazones of oxidized polysaccharides with diazotized m-, 0-,or p-aminobenzoic acid, polysaccharide formazans containing the carboxyl groups in the corresponding position can be prepared.144 3

2. Oxidation with Ozone

Harries and Langheld146 were the first to show that, on oxidation with ozone, the primary alcoholic hydroxyl groups of the sugar derivatives are transformed to aldehyde groups. (141) V. C. Barry, J. E. McCormick and P. W. D. Mitchell, J . Chem. SOC.,3692 (1954). (142) 2. A. Rogovin, A. G . Jasunskaja and B. M. Bogoslovskij, Zhur. Priklad. Khim., 23, 631, 665 (1950); Chem. Abstracts, 46, 4235 (1952). (143) V. C. Barry and P. W. D. Mitchell, J . Chem. SOC.,3631 (1953). (144) L. Mester and E. M6cz&r,unpublished results. (145) C. Harries and K. Langheld, 2.physik. Chem. (Leipzig), 61, 373 (1907).

156 i

?

L. MESTER

\

w

w

c,

\Q e

I

0

T 0

a

6

U

B

0, a@

I

a

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

0

p

:

ow

"t"1 3

.SI u

0:

157

158

L. MESTER

This is now confirmed by the formazan reaction. From ozone-oxidized D-mannitol, D-mannose can be isolated in the form of its N , N’-diphenylformazan. Applying this reaction to ozone-oxidized polysaccharides (for 147 example, cellulose or starch), their bright-red formazans are Polysaccharides not containing primary alcoholic hydroxyl groups (for example, xylan) fail to give formazans on oxidation with ozone. I n agreement with the observations made in connection with ozone-oxidized alditols,146this finding seems to indicate that the formazan group takes the place of the primary alcoholic group. The preceding formulas show the formazan reaction for ozone-oxidized cellulose.

3. Oxidation by Other Means The formazan reaction has also proved suitable for demonstration of the presence of aldehyde groups in polysaccharides oxidized by other means, even where these groups are limited in number or are the products of some side reaction. Yackel and K e n y ~ nand ’ ~ ~Maurer and Reiff149 were the first to oxidize cellulose with nitrogen dioxide to obtain a poly(g1ycosiduronic acid). In addition to the principal reaction, their investigations pointed to the possibility of a number of side reactions.160 The formazan reaction revealed the presence of aldehyde groups in cellulose wads transformed to

L. Mester, Collecled Papers First Intern. Ozone conf., 1956 (in preparation). L. Mester and A. Major, Chem. & Ind. (London), 469 (1967). E. C. Yackel and W. 0. Kenyon, J . Am. Chem. Soc., 64, 121 (1942). I(.Maurer and G . Reiff, J . makromol. Chem., 1.27 (1943). 0. Pfeiffer and D. Kruger, “Beitriige zur Oxydation von Cellulose mit Stickatoffdioxyd,” Verlag Chemie, Weinheim, 1949, p. 38. (146) (147) (148) (149) (150)

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

159

poly(g1ycosiduronic acid) ; following application of the formazan reaction, the wads turned a vivid red.lbl The bright-red formazans of the compounds obtained from cellulose and starch by oxidation with dichromate were also prepared.

X. PREPARATION AND USE OF THE TETRAZOLIUM AND CARBOTHIONIC ACID PHENYLHYDRAZIDE DERIVATIVES OF THE OXIDIZED POLYSACCHARIDES AND THE METALCOMPLEXES OF THEIR FORMAZANS From the formazans of oxidized polysaccharides, new nitrogenous polysaccharide derivatives and the metal complexes of their formazans can be prepared. Since the new reactive groups take the place of the formazan functions, their positions in the molecule depend on the method of oxidation by which the formazan groups were formed. 1. Tetraxoliurn Derivatives

Dehydrogenation with N-bromosuccinimideS162 transforms the formazans of oxidized polysaccharides into their tetrazolium derivatives.lS2In an alkaline medium, these colorless or cream-colored compounds readily revert to the bright-red formazans, as with the simple tetrazolium compounds. The formazans of oxidized, soluble polysaccharides (for example, dextran, or soluble starch) yield soluble tetrazolium derivatives, and those of oxidized, insoluble polysaccharides (for example, cellulose, or xylan) give insoluble compounds. The formulas on page 160 illustrate the transformation of the formazan of periodate-oxidized cellulose to the corresponding tetrazolium compound. Polysaccharide tetrazolium compounds, particularly tetrazolium papers, seem to hold some prospect for use for analytical purposes or as biological indicators. 2 . Thionic Acid Phenylhydrazide Derivatives

Reductive decomposition of the formazans of oxidized polysaccharides, by means of hydrogen sulfide, as with the simple sugar formazans, yields sulfur-containing, nitrogenous polysaccharide derivatives which contain thionic acid phenylhydrazide g r 0 ~ p s . lFor ~ ~ example, when a pyridineethanol solution of oxy-starch formazan or oxy-dextrin formazan is saturated with hydrogen sulfide, the vividly red solution turns colorless and a yellow precipitate, which gives the Wuyts reactions7s 58 characteristic of the thionic acid phenylhydrazides, separates. (151) L. Mester and E. M6cz&r,Chem. & Ind. (London), 823 (1956). (152) L. Mester and E. M6cz&r,Chem. & Ind. (London), 848 (1956). (153) L. Mester and E. M6cr&r,Chem. & Znd. (London), 874 (1956).

160

L. MESTER

The formulas on the opposite page illustrate the reductive decomposition of oxystarch formazan by the action of hydrogen sulfide. 3. Metal Complexes

Like the simple formazans, the formazans of oxidized polysaccharides form complexes with heavy metal with ease. On warming the formazans of oxidized cellulose, starch, dextrin, dextran, or inulin with copper, cobalt, nickel, or uranium salt solutions, their characteristicallycolored metal complexes are obtained. The presence of carboxyl groups in the polysaccharide formazans frequently facilitates complex formation.166 Until the structure of the metal complexes of the polysaccharide formazans is more exactly known, structural formulas are suggested’4’J~1 5 4 , 166 in (164) L. Mester, “Die Formasan Reaktion der mit Perjodsiiure oxydierten Polysaccharide.” Meeting of the Deutsche Chemische Gesellschaft, Leipaig, 1956. (165) L. Mester, J . Polymer Sci., So, 239 (1958).

FORMAZAN REACTION I N CARBOHYDRATE RESEARCH

161

I" I

CH21

which one complex-forming metal reacts with two formazan groups, as for the metal complexes of the simple formazans.20s26 For example, the structure of the dark-violet, copper complexes obtainable from cellulose formazan is illustrated by the following formula.

162

L. MESTER

Particularly fine colors are displayed by the copper, nickel, cobalt, and uranyl complexes of cellulose formazan obtained from viscose (“artificial silk”) textiles oxidized with periodic acid or ozone; these represent a new type146,lK4of the so-called “chemically colored threads.”la, 166, 167 Under the electron microscope, the uranyl complexes of the oxy-polysaccharide formazans show a distinct shading effect which imparts a markedly dark appearance to these polysaccharide 164, 168 It is hoped that this will lead to the elaboration of a new method for the study of the structures of polysaccharides.

XI. THE FORMAZAN REACTIONAS A MEANS FOR ESTABLISHING THE STRUCTURE OF POLYSACCHARIDES In combination with other methods, use of the formazan reaction facilitates the study of polysaccharides of unknown structure and determination of the linkages in them. For example, if, from water-soluble polysaccharides, water-insoluble, highly nitrogenous formazans are prepared after oxidation with periodic acid, the estimation of the results of the periodate oxidation is greatly facilitated, since the precipitate is free from interfering, non-transformed byproducts. Similar favorable features were observed by Barry and Hirstl69, 160 on isolating phenylhydrazones and other nitrogenous condensation products of periodate-oxidized polysaccharides. After oxidation with periodic acid, isolation of the vividly red, waterinsoluble formazanlsl of an immunospecific polysaccharide obtained from Bacillus anthracis proved possible. [This polysaccharide is built up from equimolecular quantities of D-galactose and N-acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose).la2] Analytical results showed that (a) only every second monosaccharide unit had been transformed into a formazan, and (b) the N-acetyl-D-glucosamine was present in practically unchanged quantity. Accordingly, periodate oxidation and formazan formation must have taken place on the D-galactose units. From these considerations, and on the evidence of additional data obtained in the course of oxidation with periodic acid, a structure is to be assigned t o this immunospecific polysaccharide in which the N-acetyl-D-glucosamine units are present in the usual (1 -+ 4) linkage, while half the D-galactose units are (1 6) linked, and the other half occur in (1 t 4) or (1 --+ 2) linkages, or both. These details (156) D.N.Kursanov and P. A. Solodkov, Zhur. Priklad. Khim., 16,351 (1943). (157) L.H.Bock and P. L. de Benneville, U.S. Patent 2,498,874(1950);Chem. Ab-

-

stracts, 44, 7562 (1950). (158) L.Mester and F. Guba, unpublished results. (159) E. L.Hirst, J . Chem. Soc., 2976 (1955). (160) E.L.Hirst, “Some Recent Developments in the Chemistry of the Polysaccharides.” Lecture, Miinster, February 3, 1955;see Angew. Chem., 67, 283 (1955). (161) L.Mester and G. Ivtinovics, Chem. & Znd. (London), 493 (1957). (162) G.Iv&novics, 2. Immunitatsforsch., 97, 404 (1940); 98,373 (1940).

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

163

of structure appear to offer an explanation for the immunospecific properties of this p o l y s a ~ c h a r i d em4 , ~and ~ ~ ~there is reason to hope that the formazan reaction will equally facilitate structural studies on other immunospecific and bacterial polysaccharides.

XII. TABLES The following Tables record information relevant to the discussions in this Chapter. TABLB 111 Sugar Formazans and Their Derivatives ComPound

D-Glucose N ,N’-diphenylformazan

kf.#., ‘C.

A ppcarance

177-178 red needles, frequent rosettes 117-118 pale-red prisms pentaacetate 167-168 bronze-red tablets D-Galactose N ,N‘-diphenylformazan pentaacetate dark-red needles 142 D-Galactose N-phenyl-N’-(p-fluoropheny1)- 174 bronze-red tablets formazan D-Galactose N-phenyl-N’-(p-chloropheny1)- 171 dark-red prisms formazan bronze-red, fine D-Galactose N-phenyl-N‘-(p-bromopheny1)- 162 needles formazan D-Galactose N-phenyl-N’- (p-iodophenyl) - 151-153 bunches of dark-reg formazan crystals D-Mannose N ,N’-diphenylformazsn 174-175 bunches of minute, russet needles pentaacetate red prisms 95 173-174 bunches of fine, L-Arabinose N ,N’-diphenylformazan bright-red needles 175-176 brilliant-red L-Rhamnose N, N’-diphenylformazan needles 123-124 lanceolate, red D-Xylose N , N‘-diphenylformazan needles

Rcfmcnccs

50 165 50

48 166

166 166 166 50 42 50 50

50

(163) M.Heidelberger, M. L. Wolfrom, W. B. Neely and Z. Dische, J. Am. Chem.

Sac., 77, 3511 (1955).

(164) M. Heidelberger, J. A m . Chem. Sac., 7 7 , 4308 (1955). (165)L.Mester, unpublished results. (166) G. Zemplen, L.Mester, A. Messmer and E. Eckhart, Magyar Kbm. Folydirat, 69. 206 (1953).

164

L. MESTER

TABLE111-Continued

u.p.,"C.

Compound

1,2 - 0 - Isopropylidene - D - xylo - pentodiose N,N'-diphenylformazan monoacetate xylo-Pentodiose bis (N, N'-diphenylformazan) triacetate D-Galacturonic acid N, N'-diphenylformazan triacetate, lactone D-Galactose N-phenyl-N'- (2-carboxyphenyl)formazan pentaacetate Penta-0-acetyl-D-galactoseN-phenyl-N'-(4carboxypheny1)formazan D-Glucosone N , N'-diphenylformazan D-arabino-Hexose phenylosazone N , N'-diphenylformazan tetraacetate 3,6-anhydride diacetate

Appearance

Rcferenccs

-

202

red needles

152 181-18:

red crystals bunches of darkred needles 163-164 dark-red needles 151-15: red crystals red prisms 187 195 red needles 180 red crystals 182-184 bunches of red crystals 166 red needles 204-201 violet-black needles 160-161 black prisms 179-W black needles 169-17( black needles

125 125 125 125 46 46 144 144 144 99 97 97 111 111

TABLEIV Sunar Tetrazolium Compounds and Their Derivatives References

Compound

Appearance

D-Galactose diphenyltetrazolium chloride pentaacetate D-Galactose diphenyltetrazolium bromide pentaacetate Penta-0-acetyl-D-galactose N-phenylN'- (2-carboxyphenyl)tetrazolium bromide

yellowish-white, hygro. scopic powder colorless prisms pale-yellow oil

44

pale-yellow crystals bunches of colorless crystals

51 144

201 233-234 198-200

44

51

165

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

TABLEV Thioaldonic Acid Phenylhydrazides and Their Derivatives M . p . , OC.

Compound

I

D-Thiogalactonic acid phenylhydrazide 175 n-Thiogluconic acid phenylhydrazide 178-179 2,3-Diphenyl-5-(D-galacto-pentahydroxy- 163-164 pentyl) -l13,4-thiadiazoline 2,3-Diphenyl-5-(D-gluco-pentahydroxy17&171 pentyl) -1,3,4-thiadiazoline

A ppcorance

I

i colorless needles '

References .-

colorless needles colorless crystals

45 45 45

colorless crystals

45

TABLBI VI Formazans of Periodate-oxidized Sugar Derivatives N.N'-Di)henylformaean from

Oxidized methyl a-D-glucopyranoside, ClaHzlNIOl monoacetate, C Z O H ~ ~ N , ~ ~ Oxidized sucrose, C3LH33N308 triacetate, C41H38N3011 Oxidized adenosine, C22Hz4NO03

red red red red red

powder powder powder powder powder

132 132 134 134 135

166

L. MESTER

TABLEVII* Formarans of Periodate-oxidized Polysaccharides and Their Derivatives ComPound

N,N‘-Diphenylformazan from starch Cu complex UOn complex N-Phenyl-N‘-(2-carboxyphenyl)formazanfrom starch N-Phenyl-N’-(3-carboxyphenyl)formazanfrom starch N-Phenyl-N’-(4-carboxyphenyl)formazanfrom starch N ,N‘-Diphenylformazan from cellulose Cu complex Ni complex Co complex UOZ complex N-Phenyl-N’-(2-~arboxyphenyl)formazan from cellulose UOz complex N , N’-Diphenylformazan from dextran UOz complex N-Phenyl-N’- (2-carboxypheny1)formazanfrom dextran UOz complex N , N‘-Diphenylformazan from inulin UOa complex N-Phenyl-N’-(2-carboxyphenyl)formazanfrom inulin UOz complex N,N’-diphenylformazan from xylan N,N’-diphenylformazan from glycogen Cu complex UOn complex

N ,N’-Diphenylformazan from the oxidized polysac-

Appearance

References

red powder violet powder orange-red powder red powder

138 155 155 144

red powder

144

red powder

144

red violet orange-yellow gray orange-red red

138 155 155 155 155 144

orange-red red powder orange-red red powder

155 138 155 144

orange-yellow red powder orange-yellow red powder

155 138 155 144

orange-yellow red powder orange-yellow pow. der violet powder orange-yellow pow. der red powder

155 138 138 155 155 162

charide of Bacillus anlhracis _________~

* The tables marked with an asterisk list only the principal types of compounds prepared, since, depending on the degree of oxidation, many derivatives of differing composition proved t o be preparable.

167

FORMAZAN REACTION IN CARBOHYDRATE RESEARCH

TABLE VIII* Formazans of Ozone-oxidized Polysaccharides and Their Derivatives Compound

N,N’-Diphenylformazan from starch UOZ complex N , N’-Diphenylformazan from cellulose Cu complex Co complex UOZ complex

Appearance

References

red orange-yellow red violet grayish orange-yellow

146, 146, 146, 146, 146, 146,

147 155 147 155 155 155

* See footnote t o Table VII. TABLEIX* Formazan of Cellulose Oxidized with Nitrogen Dioxide

I

Compound

N,N’-Diphenylformazan from poly-(D-glucosiduronic acid)

Appearance

I

red

1

Reference

I

151

1

References

* See footnote t o Table VII. TABLEX* Tetrazolium Derivatives of Oxidized Polysaccharides

1

Compound

Diphenyltetrazolium bromide from periodate-oxidized cellulose from ozone-oxidized cellulose from periodate-oxidized starch from ozone-oxidized starch Phenyl-(2-carboxyphenyl)tetrazolium bromide from periodate-oxidized starch Phenyl-(3-carboxyphenyl)tetrazolium bromide from periodate-oxidized starch Phenyl- (4-carboxyphenyl)tetrazolium bromide from periodate-oxidized starch

Appearance

pale-yellow powder pale-yellow powder pale-yellow powder

152 146 152 146 144

pale-yellow powder

144

pale-yellow powder

144

* See footnote to Table VII. TABLE XI* Thionic Acid Phenylhydrazide Derivatives from Oxidized Polysaccharides Tbionic acid pbenyldrazidc derivatives

From periodate-oxidized starch From periodate-oxidized dextrin From ozone-oxidized starch

* See footnote

t o Table VII.

A ppearance

References

yellow powder yellow powder yellow powder

153 153 146

This Page Intentionally Left Blank

THE FOUR-CARBON SACCHARINIC ACIDS

BY JAMES D. CRUM Department of Chemistry, The Ohio Slate University, Columbus, Ohio I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... 11. Synthesis of the Four-Carbon Saccharinic Acids 1. 3,4-Dihydroxybutanoic Acids. . . . . . . . . . . . . . . . 2. 2,4-Dihydroxybutanoio Acids. . . . . . . . . . . . . . . . 3. 2,3-Dihydroxybutanoic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. 2,3-Dihydroxy-2-methylpropanoicAcids.. . . . . 5. 3-Hydroxy-2-(hydroxymethyl)propanoicAcid. ......................... 111. Tables of Properties of the Four-Carbon Saccharinic Acids and their Denvatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................

169 172 176 184 187

I. INTRODUCTION When an aqueous solution of a carbohydrate is treated with a base, carbohydrate substances known as saccharinic acids are produced. This perplexing and intriguing saccharinic acid formation has been studied by many workers, and its history and mechanism are the subject of a review in this series.’ Since the discovery of the first saccharinic acid by Peligot? chemists have used the term saccharinic acid to denote not only deoxyaldohexonic acids but all monocarboxylic acids of the general formula CnHZnO,, (isomeric with the sugars of origin)-obtained not only from the hexoses but from all the reducing sugars by the action of alkali. Although this definition is clear, it has the drawback inherent in all functional definitions, namely, that it restricts application of the term to such compounds as are actually obtainable under a given set of laboratory conditions. In order to achieve a more satisfactory definition of a saccharinic acid, Glattfeld and Millera studied the various usages of the term employed in the literature and then formulated a definition, the final form4 of which appeared in 1925, as follows: “by ‘sac-

H

chnrinic acid’ (1) (2) (3) (4)

. . . is meant any acid which would result if the -C=O

J . C. Sowden, Advances i n Carbohydrate Chem., 12,36 (1957). E. Peligot, Compt. rend., 7, 106 (1838). J . W. E. Glattfeld and G. E. Miller, J . A m . Chem. SOC.,42, 2314 (1920). J . W. E. Glattfeld and L. P . Sherman, J . Am. Chem. SOC.,47, 1742 (1925) 169

170

J. D. CRUM

group of a hydroxy-aldehyde of the formula C,H2,0, ,in which each carbon atom except the one in the aldehyde group carries one hydroxyl group, were

I

oxidized to carboxyl, at the expense of one of the -COH

groups which

1

I

1

would be reduced to a -CH

group.”

I

’

In publishing this definition, Glattfeld and Sherman4were careful to state that it was purely formal and was not to be considered a statement of their idea as to the theory underlying the actual formation of the saccharinic acids from the sugars. Whether or not these acids are really produced by such a direct internal oxidation-reduction as is implied in the definition, they may nevertheless be dejined as the products of such a reaction. The definition is not, perhaps, entirely satisfactory. It is arbitrary and formal. It does not include those acids of formula C,H2,0, which contain a quaternary carbon atom, because the hydroxy-aldehyde of like general formula required by the definition is structurally impossible. I n other words, such acids could not be formed by the imaginary reaction which is the basis for the defintion and they would, therefore, not be called saccharinic acids. On the other hand, the definition does have the advantage of perfect clarity. Furthermore, it establishes a uonnection between the saccharinic acids and the aldoses. Finally, it escapes the drawback of a functional definition, because it depends, not on an actual, but on a theoretically possible, number of isomers. The term saccharinic acid will be used in this article to denote only those compounds encompassed by the GlattfeldSherman definition. The saccharinic acids formed from some of the pentoses and hexoses have been the objects of study by Nef6 and his students. Glattfeld and Hanke6 reported in 1918 that, during the oxidation of maltose in alkaline solution, an acid had been produced whose phenylhydrazide had an analysis agreeing perfectly with that calculated for a four-carbon saccharinic acid. Furthermore, the properties of the free acid were those which would be expected of one of these acids. Its configuration could not, however, be reported a t that time because of the absence of data as to the properties and constants of the four-carbon saccharinic acids. Nef had also referred to the handicap which this lack of information had imposed on the work with sugars in alkaline solution. Consequently, with this in mind, Glattfeld began in 1920 the systematic synthesis of the four-carbon saccharinic acids. In accordance with the definition previously stated in this article, five different, positional isomers are possible. Furthermore, taking into consider(5) J. U. Nef, Ann., 376.1 (1910);J. U.Nef, 0. F. Hedenburgand J. W. E. Glattfeld, J . A m . Chem. Soc., 39, 1638 (1917);J. W.E.Glattfeld, Am. Chem. J . , 60,135 (1913). (6) J. W.E.Glattfeld and M. T. Hanke, J . Am. Chem. SOC.,40, 973 (1918).

171

FOUR-CARBON SACCHARINIC ACIDS

ation the asymmetric carbon atoms present in these positional isomers, ten stereoisomers (5 DL pairs ; Ia-Vb) and one optically inactive, positional isomer (VI) should be capable of existence. That is to say, there are eleven saccharinic acids which have four carbon atoms.' COzH

I CHz I HCOH I

CHnOH Ia

COzH

C OzH

COzH

I I CHz I

I HCOH I CHz I

I CHz I HOCH I

HOCH

CHzOH

CHzOH

CHiOH

IIa

Ib

2-Deoxy-~-glycero- 2-Deoxy-~-glytetronic acid cero-tetronic acid

IIb

3-Deoxy-~-glycero- 3-Deoxy-~-glytetronic acid cero-tctronic acid

I

3,4-Dihydroxybutanoic Acids

COzH

I I HCOH I HCOH

CI4

2,4-Dihydroxybutanoic Acids

COzH

COZH

I I HOCH I

I I HCOH I

HOCH

COzH

I I

HCOH

HOCH

CHI

HOCH

I

CHa

CHa

IIIa

IIIb

IVa

IVb

4-Deoxy-~erythronic acid

4-Deoxy-~erythronic acid

4-Deoxy-~threonic acid

4-Deoxy-~threonic acid

"

2,3-Dihydroxybutanoic Acids

COaH

I I

CHz-COH CHZOH

CO2H

COzH

HOC-CHI

HC-CH2

I I

I I

OH

CH*OH

CHzOH

Va

Vb

VI

2-Methyl -D glyceronic acid

2-Methyl-~glyceronic acid

2-Deoxy-2- (hydroxymethy1)glyceronic acid; 3-hydroxy-2-(hydroxymethy1)propanoic acid

I

2,3-Dihydroxy-2-methylpropanoicAcids

(7) The nomenclature used in this article is in conformity with the Rules of Carbohydrate Nomenclature [Chem. Eng. News, 31, 1776 (1953)l and therefore differs from t h a t used in the earlier literature. The term tetronic acid is likewise employed according t o this modern, carbohydrate usage, and should not be confused with the terminology sometimes employed by organic chemists t o designate certain substances derived from the enolic form of 2,4(3H,5H)-furandione.

172

J. D. CRUM

11. SYNTHESIS OF

THE

FOUR-CARBON SACCHARINIC ACIDS

1. 3,4-DihydroxybutanoicAcids

These 3,$-dihydroxybutanoic acids or 2-deoxytetronic acids were first prepared in the racemic form (Iab) by Hanriot8 in 1879. The synthesis was later repeated by Nef,6 who studied the racemic acid in more detail and reported a few of its properties, as well as those of its phenylhydrazide and brucine salt. The constitution of the DL acid was proved by Nef by the method of synthesis, as well as by oxidation to the known 2-deoxy-~~glycero-tetraric acid (m-malic acid). Glattfeld and Millers prepared the racemic acid, in accordance with the CN CHaOH

I CHOH I

CHaOH VII

CHaCl

$Lo+

I I

CHOH CHzOH VIII

KCN

h

I CHI I CHOH I

$2

CHzOH IX

f

COzH

COzH

CHaOH Iab

CHaOH X

I I CHI CH I 1I CHOH + CH I I

procedures of Hanriots and Nef,6 from 1-chloro-1-deoxy-DL-glyceritol(glycerol a-chlorohydrin; VIII). Treatment of the DL form of VIII with potassium cyanide yielded 2-deoxy-~~-glycero-tetrononitrile(IX) which, upon hydrolysis, produced a mixture of the desired 2-deoxy-~~-g.?ycero-tetronic acid (Iab) and y-hydroxyisocrotonic acid (4-hydroxy-cis-2-butenoic acid; X). In order to find out if the saccharinic acid obtained was actually the desired product, a saccharinic acid was also prepared by a different route. Vinylacetic acid (XIII) was prepared according to the method of Houbeng by first reacting ally1 bromide (XI) with magnesium to produce allylmagnesium bromide (XII). Treatment of this Grignard reagent with carbon dioxide, followed by hydrolysis, gave vinylacetic acid (XIII) ; this was oxidized with potassium permanganate according to the method of Penschuck,lO and a saccharinic acid (Iab) was obtained whose phenylhydrazide was identical with that of the acid prepared from l-chloro-l-deoxy-mglyceritol (VIII) by the method of Hanriot.* Fichter and SonnebornlOshad also prepared the saccharinic acid (Iab) from vinylacetic acid by the above method, but this work was repeated as these investigators had claimed the identity of their acid with that of Hanriot, solely on the barium content of its barium salt. Since this content would be the same for all of the barium (8) Hanriot, Ann. chim.et p h y s . , [5]17, 62 (1879).

(9) J. Houben, Ber., 30, 2897 (1903). (10) M.Penschuck, Ann., 283, 109 (1894). (lOa) F. Fichter and F. Sonneborn, Ber., 86, 938 (1902).

173

FOUR-CARBON SACCHARINIC ACIDS

CHzBr

CHz-Mg-Br

I

I

XI

XI1 ICO, ; H l o

COzH

I 'HZ - KMuOI I I CHOH I CHzOH Iab

COzH

I

CHz CH

II

CHz XI11

salts of the four-carbon saccharinic acids, they obviously had not proved the identity of the acids from the two sources; and Glattfeld realized that, in order to make the proof of the identity absolute, he should prepare the acids from both sources and, with definitive crystalline derivatives, prove them to be identical. Resolution of the DL acid (Iab) was attempted with brucine, cinchonine, quinine, and strychnine, and it was found that brucine gave superior results in comparison with the other alkaloid bases tried. I n this way, the most insoluble brucine salt in the resolution experiments was found to yield, on liberation of the free acid from the salt, a pure levorotatory isomer (Ia). Oxidation of this isomer with nitric acid gave levorotatory 2-deoxy-~-glycero-tetraric acid [( -)-~-malic acid; XIV] identical with the natural form. From the mother liquor of the resolution with brucine, the other isomer as the free acid (dextrorotatory ; Ib) was obtained. This isomer, however, was acid [ (+)-Dnot converted with nitric acid into 2-deoxy-~-glycero-tetraric malic acid; XV]. COzH

CHzOH

I

I

CHz

1

HCOH

I

CHzOH

HOCH

I

CHZ

I

COzH

Ia Z-Deoxy-(-)-D-glycero-tetronic acid

COzH HNOl

I i CHz I

HOCH

COzH XIV 2-Deoxy-~-glycerotetraric acid [(-)-L-Malic acid; natural form]

174

J. D. CRUM

COzH

I CHz I

CHzOH

COzH

1

HOCH

I

HOCH

I I CHi I

HCOH

HNOb ---

CHz

I

I

CHzOH

COzH

COzH

xv

Ib 2-Deoxy-(+)-~-glycero-tetronic acid

2-Deoxy-~-glycerotetraric acid [(+)-D-Malic acid]

Although Nef6 had prepared the DL form of this saccharinic acid and had converted it to 2-deoxy-~~-glycero-tetraricacid (m-malic acid), Glattfeld and Miller3 succeeded in separating the DL compound into its enantiomorphous forms and thus established the absolute configuration of the optical isomers. The preparation of the DL acid was later improved by Glattfeld and (glycidol; XVI) as an inKlaas" with the use of 1 ,2-anhydro-~~-glyceritol termediate. In this procedure, glyceritol (VII) was converted with hydrochloric acid into 1-chloro-1-deoxy-DL-glyceritol(glycerol a-chlorohydrin; CHiOH

I CHOH I

CH~OH VII

CHzCl HCI HOAc'

I

CHOH

Na ether

)

I CH~OH

THZ-J CHO

I

CHzOH XVI

VIII

[€EN

CO2H

CN

I

I

'HZ

I

Ba(0H)a

CHz

I

CHOH

CHOH

CHzOH

CHzOH

Iab

IX

I

I

VIII) which yielded, on treatment with sodium in ether, 1,2-anhydro-~~glyceritol (glycidol; XVI). Reaction of the latter with hydrogen cyanide (IX). Hydrolysis of the nitrile produced 2-deoxy-~~-glycero-tetrononitrile (IX) with barium hydroxide afforded the desired saccharinic acid (Iab). (11) J. W.E.Glattfeld and R. Klaas, J . Am. Chem. Soc., 66, 1114 (1933).

FOUR-CARBON SACCHARINIC ACIDS

175

2. 9 ,.4-Dihydroxybutanoic Acids 2,4-Dihydroxybutanoic acid, a 3-deoxytetronic acid, is the only theoretically possible four-carbon metasaccharinic acid ; it was isolated by Nef6 in the DL form (IIab) in the course of his work on the action of sodium hydroxide on D-arabinose. It therefore constitutes the only four-carbon saccharinic acid isolated to date from a sugar-alkali reaction. Resolution of this DL acid (IIab) was accomplished by Nef,b who showed that the dextrorotatory acid (IIa) upon oxidation gave rise to 2-deoxy-~-gZycero-tetraricacid [(-I-)+ malic acid; XV]. During the course of this work, several derivatives of the L and DL saccharinic acids (IIb and IIab) were prepared, but only the brucine salt of the D acid (IIa) was reported. The DL acid (IIab) and its calcium salt had been prepared by Raskel* in the course of an investigation on the action of potassium cyanide on chlorinated aldehydes. From 3-chloro-2,3-dideoxy-glycerose(3-chloropropanal; XVII) plus potassium cyanide, 4-chloro-3,4-dideoxy-n~-gZycero-tetronic acid (4-chloro-2-hydroxy-~~-butanoic acid ; XVIII) was obtained COzH CHo

I I

CHz CHzCl XVII

NHdCl 1.)KCN 2.) HCl

I

CHOH

' CI HZ I

CHzCl XVIII

COzH

I I CHz I

CHOH

CHaOH IIab

and this, on boiling with water, gave 3-deoxy-~~-glycero-tetronicacid (IIab). Since neither method was suitable for making available the large quantities of acid necessary for purposes of resolution, Glattfeld and Sander13 synthesized the DL acid (IIab) by a method similar to Raske's. The initial step was the conversion of glyceritol (VII) into acrolein (XIX); hydration of this yielded 2-deoxy-glycerose (3-hydroxypropanal; XX) which, on treatment with cyanide, gave the desired nitrile (XXI). Hydrolysis of acid (IIab). this nitrile afforded 3-deoxy-~~-glycero-tetronic The racemic 3-deoxytetronic acid (IIab) thus produced was resolved with brucine, the more insoluble brucine salt yielding, on liberation by means of barium hydroxide, a dextrorotatory acid (IIa) which gave, on oxidation with nitric acid, a product identical with 2-deoxy-~-glycero-tetraricacid [( +)-D-malic acid; XV]. Similarly, the levorotatory acid (IIb), obtained from the mother liquor of the resolution with brucine, was oxidized with nitric acid to 2-deoxy-~-glycero-tetraricacid [( - )-L-malic acid; XIV]. The (12) K. Raske, Ber., 46,726 (1912). (13) J. W. E. Glattfeld and F. V. Sander, J . Am. Chem. SOC.,43, 2675 (1921).

176

J. D. CRUM

CHzOH

I I CHzOH

CHOH

CHO

CHO

I cH

anh d MgJO;,

I I

CHz

II

CHzOH

CHz XIX

VII

xx

1

HCN

COzH

CN

I

I

CHOH

I

CHz I CHnOH IIab

HC1

CHOH

H a

CHz I I CHaOH XXI

- I

configurations of the two enantiomorphous forms of this 3-deoxytetronic acid must, therefore, be structurally represented as IIa and IIb. COzH

I HCOH I

COB HNOI

CHa

I

CHzOH IIa 3-Deoxy-(+)-~-glycerotetronic acid COzH

I HOCH I I

CHa CH20H IIb 3-Deoxy-( - ) -L-gZycerotetronic acid

I I CHz I

HCOH

COzH

xv 2-Deoxy-~-gZycerotetraric acid [(+)-D-Malic acid]

COzH

HNO A

I I CHa I

HOCH

COzH XIV 2-Deoxy-~-gZycerotetraric acid [(-)-~-Malicacid; natural form]

3. 2 ,3-Dihydroxybutanoic Acids

Prior to 1927, none of the work recorded in the literature of this subject had as its direct object the study of the two theoretically possible racemic

177

FOUR-CARBON SACCHARINIC ACIDS

acids (IIIab, IVab) of 2,3-dihydroxybutanoic acid. Their preparations had been reported incidentally to other work, and, consequently, no thorough comparative study of the acids had been made. The literature therefore left the properties, and even the separate identities, of the two racemic 2,3-dihydroxybutanoic acids in doubt. One plan of attack by early workers14 was the preparation of the “methylglycidic acids” (XXIV) and “methylglyceric acid” (IIIab) from the chlorohydroxybutanoic acids (XXIII) which resulted from the addition of hypochlorous acid to crotonic acid (trans-2-butenoic acid; XXII) and isocrotonic acid (cis-Zbutenoic acid). With crotonic acid (XXII) as the starting material, Melikoffl4 obtained crystalline 2-chloro-3-hydroxybutanoic acid (XXIII), crystalline “8-methylglycidicacid” (XXIV), and crystalline “P-methylglyceric acid” (IIIab). Isocrotonic acid (the cis isomer of XXII), under similar treatment, afforded three chlorohydroxybutanoic acids; that having m.p. 80” gave a liquid “P-methylisoglycidic acid” COzH

I CH 11 HC I

CHs XXII

COzH

COzH

I

=I

CHCl CHOH

I

CHa XXIII

I

KOH

-I

C H i CHJ

1

o +

CHs XXIV

COzH

I

HCOH

I

HCOH

I

CH:

CH2H

I + I HOCH I

HOCH

CHI

IIIab

which, in turn, yielded a “P-methylisoglyceric acid” of m.p. 45”. Apart from an accountl8 of the preparation of the ethyl ester of “p-methylglyceric acid” and an abstract of a paper by Melikoff:6 no other work was available on the dihydroxybutanoic acids produced by the hypochlorous acid method. One other important method of preparation had been used by two groups of workers; it involved the direct oxidation of the crotonic acids by means of barium permanganate. Fittig and Kochs’? obtained by this process an anhydrous, crystalline, racemic dihydroxybutanoic acid (IVab) of m.p. 74-75” from crotonic acid, and a liquid dihydroxybutanoic acid (IIIab) from isocrotonic acid. In 1904, Morrell and Hansonl* resolved the ‘‘Bmethylglyceric acid” (IIIab) of Fittig and Kochd7 with quinidine and (14) 0. von Faber and B. Tollens, Ber., 32, 2589 (1899);P.Melikoff, ibid., 16, 2586 (1882);16, 1268 (1883);C. Kolbe, J . prakt. Chem., 26, 369 (1882);P. Melikoff and P. Petrenko-Kritschenko, Ann., 266,358 (1891). (15) P.Melikoff and N. Zelinsky, Ber., 21.2052 (1888). (16) P.Melikoff, Zhur. Russ. Fiz.-Khim. Obshchestva, 16, 517 (1884);Ber., 17(R), 420 (1884). (17) R. Fittig and E. Kochs, Ann., 268, 1 (1892). (18) R.S.Morrell and E. K . Hanson, J . Chem. Soc., 86, 197 (1904).

178

J. D. CRUM

found that the levorotatory form melted at 74-75'. It can therefore be presumed that four different optically active 2,3-dihydroxybutanoic acids (IIIa, IIIb, IVa, In) had been reported in the literature. Since, however, neither Melikoff nor Kochs had recorded the yields they obtained, and as Melikoff did not give the quantities of the materials involved in the syntheses, it was necessary for Glattfeld and to reconsider the past synthetic efforts. COzH

I cH

It

HC

COiH HOCl

- I

COzH

I

HCOH

CHOH

HCOH

I

I

I

I

CH, XXII

I

COzH

I cHC1 AgOH,

CHI XXIII

I

+

HOCH

I

HOCH

I

CHI

CH, IIIab

COzH

I

BaMnO,

+

_:Hj%:"

HCOH HO CH I

I

CHa

CHa IVab

The acids (IVab) produced from both the barium permanganate reaction and also from treatment with the oxidizing agent osmium tetroxide were found to be identical, as were their phenylhydrazides; and these, in turn, were found to differ from the acids (IIIab) formed by the hypochlorous acid method. These acids (IIIab, IVab) were therefore considered by Glattfeld and Woodrufflg to be the two theoretically possible DL dihydroxy acids of the 2,3-dihydroxybutanoic acids. The trans (threo) structure (IVab) was assigned to the lower melting (74-75') and the cis (erythro) structure (IIIab) to the higher melting (81.5") isomer as a result of the work of G. Braun,2'Jdepicted in Fig. 1 , as carried out in the laboratory of Prof. Glattfeld. This proof of the configuration of the two racemic acids (IIIab, IVab) is based on the fact that trans-4,4,4trichloro-2-butenoic acid (XXIX) may be converted with sulfuric acid into the trans-dicarboxylic acid, fumaric acid (XXVIII), which yields, upon oxidation with potassium chlorate-osmium tetroxide, DL-threaric acid (XXVI). Treatment of trans-4,4,4-trichloro-2-butenoic acid (XXIX) with zinc and acetic acid produced trans-4,4-dichloro-2-butenoicacid (XXX); further reduction, with hydrogen in the presence of palladium (19) J. W. E. Glattfeld and S. Woodruff, J . Am. Chem. SOC.,49,2309 (1927). (20) G. Braun, J . Am. Chem. SOC.,62, 3176 (1930); 64, 1133 (1932).

179

FOUR-CARBON SACCHARINIC ACIDS

COzH

COzH

I

HC

II CH

I

' H L

CHCli

COzH

I HC II CH I

I II cH I

HC HzSO,

COzH

ccl3

xxx

XXIX

XXVIII KCIOI,

Pd-Hi J.

J.

COzH

I

Xa-Hg

HC

11 I

CH CHzCl

COzH

I HCOH Ba(C1O:)z , 0.30, I HOCH I

HOCH

I I

>

.__J

COzH

COzH

I

HC AgC1ol osoc b

1 HCOH I HOCH I

I

HOCH

I

HCOH

I

XXXII

COzH

I HCOH I HOCH I

INOa

COzH

I I HCOH I

HOCH

CHiOH

CHa

C Ha

CHa

XXVI

7

COzH

I

"

L

Agio

Pd-Hz

I

HCOH COzH

I

~

CHzCl

I

I

HOCH

HOCH COzH

HCOH

Pd-H,

II I

HNOI

COzH

I HCOH 1

xxv

I

CH

COzH

I

CHzC1

XXXI

-

COzH

CHiOH

XXVII

IVab 1

COzH

I I

HCoH

I

COzH XXXIII

+------

COzH

I I

HCOH HOCH

I

I

CHz

CH3 IIIab

'

AgClOa 080,

CH

I

CH3 XXII

FIG.1.-Proof of Configurations of the Racemio 2,3-Dihydroxybutanoic Acids (IIIab and IVab).20

catalyst, gave tmns-4-chloro-2-butenoic acid (XXXI). Oxidation of the latter (-1) with a barium chlorate-osmium tetroxide mixture yielded 4-chloro-4-deoxy-~~-threonic acid (XXV). Treatment of this compound (XXV) with silver oxide produced DL-threonic acid (XXVII) which was

180

J. D. CRUM

oxidized with nitric acid to DL-threaric acid (XXVI). [By reaction with nitric acid, 4-chloro-4-deoxy-~~-threonic acid (XXV) could also be converted directly into DL-threaric acid (XXVI).] Reduction of trans-4-chloro2-butenoic acid (XXXI) with hydrogen in the presence of a palladium catalyst produced crotonic acid (XXXII).Treatment of the latter (XXXII) with a silver chlorate-osmium tetroxide mixture produced 4 - d e o x y - ~ ~ threonic acid (IVab), a compound which could also be prepared from 4chloro-4-deoxy-~~-threonic acid (XXV) by reduction. This reaction seCO2H

I HocH I

C OrH

'HNOa

HCOH

I CHs

I HaNCH I

HCOH

COIH HI P '

I I CH1 I

HsNCH

I

IVa

CHs XXXIV

(Zevo)

(Zeuo)

CHa

xxxv

1

ohloramine-T

CHO

I

HCOH

I C)4 XXXVI

COaH

I

HCOH

I CHs XXXVII (Zeuo)

FIQ.2.-Configurational Correlation of (-)-4-Deoxythreonic Acid (IVa) with (-)-Threonine (XXXIV) .22

quence therefore establishes the configuration of the lower melting (74-75") isomer 'as the trans (threo) compound (IVab). This saccharinic acid (IVab) could also be produced by the reaction of isocrotonic acid (XXII) with perbenzoic acid, but treatment of isocrotonic acid (XXII) with a silver chlorate-osmium tetroxide mixture gave the higher melting (815")isomer (IIIab). This latter compound (IIIab) could also be produced from crotonic acid (XXXII) by reaction with perbenzoic acid. Therefore, the high melting isomer (81.5') must possess the erythro configuration and have the configuration of erythraric acid (mesotartaric acid; XXXIII). Glattfeld and Chittum2I resolved the 4-deoxy-~~-threonic acid (IVab) both with brucine and with quinidine, and obtained the levorotatory acid (21) J. W. E. Glattfeld and J. W. Chittum, J . Am. Chem. Soc., 66,3663 (1933).

FOUR-CARBON SACCHARINIC ACIDS

181

(IVa) as a crystalline substance whose configuration was designated D through correlationz2with D-( - )-threonine (XXXIV), as shown in Fig. 2. D-( - )-Threonine (XXXIV) (whose configurational assignment is based on its C3 hydroxyl group) gave, upon reduction with hydriodic acid and red +)-a-aminobutyric acid (XXXV).z3On the other hand, phosphorus, I,-( degradation of the original amino acid (XXXIV) with “chloramine-T” by Meyer and Rosezzled to the formation of S-deoxy-~-glycerose[D-(-)-lactaldehyde; XXXVI] which, on oxidation with bromine water, gave 3deoxy-D-glyceronic acid [D-( - )-lactic acid, XXXVII]. Furthermore, deamination of the amino acid (XXXIV) with barium nitrite and sulfuric acid gave a sirupy, levorotatory dihydroxybutanoic acid (IVa). The structural assignment was made on the assumption that no Walden inversion had occurred during the deamination reaction.24The phenylhydrazide of the latter acid (IVa) was therefore prepared and found to be identical with the phenylhydrazide of the levorotatory acid obtained by Glattfeld and Chithumz1by resolution of IVab.

4. 2 ,S-Dihgdroxy-2-methylpropanoicAcids 2,3-Dihydroxy-2-methylpropanoicacid (Vab), a saccharinic acid which was obtained in the racemic form in 1886 by MelikoP6 from 3-chloro-2-hydroxy-2-methyl-~~-propanoic acid (XLIII), was synthesized as follows from citric acid (XXXVIII). Distillation of anhydrous citric acid gave citraconic acid anhydride (XXXIX) which, on reaction with water, yielded citraconic acid (XL). Treatment of this product with hydrogen bromide gave the compound known as “citrabromopyrotartaric acid” (XLI) which, on reacd tion with sodium carbonate, produced 2-methylacrylic acid (XLII) ; thisi on treatment with hypochlorous acid, gave 3-chloro-2-hydroxy-2-methylDL-propanoic acid (XLIII). This was treated with alcoholic potassium hydroxide to yield “potassium methylglycidate” (XLIV). The opening of the epoxide ring with aqueous sulfuric acid gave the desired racemic saccharinic acid (Vab). Also reported a t this time were the potassium, silver. and amorphous calcium salts of the saccharinic acid (Vab). In 1909, Kay26reported the preparation of a small quantity of the levorotatory potassium salt of this saccharinic acid from ‘(d-a-methylisoserine” (XLV). Treatment of “d-a-methylisoserine” (XLV) with nitrosyl bromide acid (XLVI). The latter, on1 gave 3-bromo-2-hydroxy-2-methylpropanoic (22) C.E.Meyer and W. C. Rose, J . Biol. Chem., 116, 721 (1936). (23) E.Fischer and A. Mouneyrat, Ber., 33,2383 (1900);S . Oikawa, Japan J . Med. Sci., 1, (II), 61 (1925),Chem. Abstracts, 20, 1671 (1926). (24) E. Fischer, Ber., 29, 1377 (1896);P.A. Levene, Chem. Revs., 2, 179 (1926). (25) P.Melikoff, Ann., 234, 197 (1886). (26) F.W.Kay, J . C h e w SOC.,96, 560 (1909).

182

J. D. CRUM

CHS

I

CHz-COzH

c-c=o

heat

I HOC-COzH I

CHz-COzH XXXVIII

CHI

Hz0

11)-

__f

0zH

C-C

III

HC-(3-0 XXXIX

HC-C0zH XL lABr

CH3

-

CH3

I C(OH)-C02H I

a

CHzCl XLIII

I C-COZH II

Narc01

CH3

I I

C(Br)-COZH CHz-C0zH

CHI XLII

XLI

[KO.

EIZH,)-CO,K

COzH HIS04

>

I

C(OH)-CHs

I

CH~OH Vab

XLIV

treatment with alcoholic potassium hydroxide, yielded “potassium methylglycidate” (XLIV) which gave, upon hydrolysis, the potassium salt of the desired saccharinic acid (V). (An optical rotation was reported for the potassium salt, but the free acid was presumably not prepared.) COzH

COzH

I C(OH)-CHI I

C(OH)-CH,

CHzNHz XLV COzK

I

C(OH)-CHs

I

I I

CHzBr XLVI

/g;g*ic + HIO LI~HJ-COZK

CHzOH

V (levo K salt)

XLIV (levo)

Since the impractical feature of Melikoff’s synthesis of the saccharinic .acid (Vab) was the long and time-consuming preparation of 3-chloro-2-

183

FOUR-CARBON SACCHARINIC ACIDS

hydroxyS-methyl-~~-propanoic acid (XLIII), a better method for the preparation for this compound was sought by Glattfeld and Sherman! It was found that Bischoff’s method,27modified according to Ult6e,28 is easy to execute and gives good results; it consists in the addition of hydrogen cyanide to the carbonyl group of 1-chloro-2-propanone (XLVII) to (XLVIII), hydrolysis give 3-chloro-2-hydroxy-2-methyl-~~-propionon~tr~le

CHzCl XLVII

CN

COzH

CHzCl XLVIII

CHzCl XLIII

of which then gives the desired halogen acid, namely, 3-chloro-2-hydroxy-2methyl-m-propanoic acid (XLIII). Melikoff’s synthesis (XLIII + Vab) was now conducted, but it still afforded poor yields. The saccharinic acid was next prepared in 32% yield from the halogen acid (XLIII) by treatment with silver oxide, but this approach was abandoned when good results were obtained2gfrom the “acetol-form-ester.” Reaction of 1-chloro-&propanone(XLVII) with sodium formate produced the monoformate of 1-hydroxy-2-propanone (XLIX) which, through the addition of hydrogen cyanide and hydrolysis of the resulting cyanohydrin of 1-hydroxy-2-propanone (L), yielded the desired racemic saccharinic acid (Vab)

.

0

II

CH~O-CH

CHzCl

I

c=o I I

CHs XLVII

HCOtNe

I c-,o I

I

CH, XLIX

CHzOH

I C(0H)-CN I

COIH

I a C(0H)-CH, I

I

I

CH3 L

CHzOH Vab

With sufficient quantities of the DL saccharinic acid thus available, many salts, as well as the crystalline phenylhydrazide, were prepared. All attempts a t the resolution of this racemic acid by means of strychnine, quinine, or brucine failed to give, on hydrolysis of the alkaloid salts, an optically active acid. However, Glattfeld and Sherman4 were inclined to believe that resolution had actually been accomplished by means of (27) C. Bischoff, Ber., 6 , 863 (1872). (28) A. J. UltBe, Rec. trav. chim., 28, 1 (1909); C h e m Zentr., 80, (I), 1538 (1909). (29) L. Henry, Bull. acad. roy. m6d. Belg., 6 , 445 (1902); Chem. Zentr., 73, (II), 928 (1902).

184

J. D. CRUM

brucine and that subsequent heating of the aqueous solution of the resulting active acid had caused complete re-racemiiation. These investigators stated their intention to study this point more fully, but no further work on it was published. 5. 3-Hydroxy-2-(hydroxymethy1)propanoicAcid

Of the five positional isomers of the four-carbon saccharinic acids theoretically possible, 3-hydroxy-2-(hydroxymethyl)propanoicacid (VI) is the only one that lacks an asymmetric carbon atom and is optically inactive and unresolvable. The first attempt a t its synthesis, by Glattfeld and coworkers,aowas by a direct method. The reaction of 2-chloro-2-deoxyglyceritol (LLI) [prepared from ally1 alcohol (LI) by reaction with hypochlorous acid] with cyanide ion and subsequent hydrolysis of the anticipated nitrile (LIII) should have produced the desired 3-hydroxy-2-(hydroxymethyl)CHsOH

I

CH

CHzOH

CHaOH HOCl

___f

I

CHCl

I

CH-CN

NaCN

I

I1

CHzOH LII

CH2 LI

CHXOH LIII

low CH&l

I

CHOH

I

CHiOH VIII

- YH3 lcNe HCl

CH

I

CHiOH XVI

CN

CH90H

I

CH-CO&J

I

CHzOH VI

COzH

I

CO2H

I

I

CH2

I

%

CHz

I

CHOH

CHOH

CHzOH

CH2OH Iab

I

Ix

I

+

HC

II I

HC CH~OH X

(30) J. W. E. Glattfeld, G. Leavell, G.E. Spieth and D. Hutton, J . Am.Chem. Soc.,

6S. 3164 (1931).

185

FOUR-CARBON SACCHARINIC ACIDS

propanoic acid (VI). Instead of the expected acid (VI), there were obtained 2-deoxy-~~-glycero-tetronicacid (Iab) and an acid characterized as y-hydroxyisocrotonic acid (X). The formation of these unexpected products indicates that the reaction between the chlorohydrin [either 1-chloro-(VIII) or 2-chloro-deoxy-~~-glyceritol(LII)] and potassium cyanide does not involve a simple replacement of halogen by cyanide but probably proceeds instead through initial formation of the epoxide, 1,2-anhydro-~~-glyceritol (“glycidol,” XVI), which then accepts cyanide ion, to form the nitrile of the 3,4-dihydroxy acid (IX). As a further attempt to synthesize the desired saccharinic acid (VI), the experiments were redesigned, using mercuric and cuprous cyanide, and experiments were attempted avoiding the use of alkali, but the experimental results remained unaltered. 1,3-Di-O-acetyIwere next employed, but and 1 ,3-di-0-benzoyl-2-chloro-2-deoxyglyceritol these blocked compounds proved to be too unreactive to afford appreciable yields of product; even metallic sodium failed t o remove chlorine from the 1,3-dibenzoate, after boiling in benzene for fifteen hours. The 2-deoxy-2iodo derivative, when treated with cyanide ion, likewise afforded only the 3,4-dihydroxy derivative (IX) instead of the desired nitrile (LII1).l1 A. Just31had earlier shown that 2-methylpropanoic acid (LVI) can be produced by the oxidation of 1,3-dihydroxy-2,2-dimethylpropane(LIV), dimethylmalonic acid (LV) being a probable intermediate. Glattfeld and CHzOH

I Ha C-C-CHZ I

C OzH

KMnO4

CHzOH LIV

I H3C-C-CH3 I

COzH

I

+ COP

HaC-C-CHs

COzH LV

I

H LVI

Klaas” therefore reasoned analogously that pentaerythritol (LVII) should produce bis(hydroxymethy1)malonic acid (LVIII) and, ultimately, 3-hyCH,OH

I

HOHzC-C-CHzOH CHzOH LVII

C OzH

-)1 +

I HOHzC-C-CHzOH I COzH LVIII

COzH

I

HOHiC-C-CHnOH

I

H LIX

droxy-2-(hydroxymethyl)propanoicacid (LIX) ; however, the only product isolated from oxidation with either barium permanganate or potassium permanganate in either acid or neutral media was carbon dioxide. Gault and R ~ e s c hhave ~ ~ recently shown that bis(hydroxymethy1)malonic acid I

(31) A. Just, Monatsh., 17, 76 (1896). (32) H.G m l t and A. Roesch, Bull. soc. chim. France, [5] 4, 1429 (1937).

186

J. D. CRUM

(LVIII) loses carbon dioxide and formaldehyde when heated, and gives a white solid product, probably of a polymeric nature. A dibromohydrin of pentaerythritol (LX) was synthesized according to Zelinsky and K r a w e t ~and, ~ ~on being subjected to permanganate oxidation, gave as the product 2 ,2-bis(bromomethyl)-3-hydroxypropanoicacid (LXI). Attempts a t further oxidation led only to the complete destruction of the compound.s4A diethyl ether of pentaerythritol was also made from the corCHzOH

CHzOH

I

HBr

HOHzC-C-CHzOH

I

I

KMnOi

BrHzC-C-CH*Br

I

CH~OH LVII

CH~OH LX COOH

I

KMnO4

BrHl C-C-CH2Br

’ coz

I

CHsOH LXI

responding dibromohydrin (LX), but this compound was more easily atttacked by the oxidizing agent than pentaerythritol itself. 1,3-Dichloro-1,3-dideoxyglyceritol (glycerol dichlorohydrin; LXII) was therefore prepared from glyceritol (VII), and oxidized to 1,3-dichloro-2propanone (LXIII) which, upon addition of hydrogen cyanide and hydrolysis of the resulting nitrile (LXIV), gave a quantitative yield of 3-chloro-2(chloromethyl)-2-hydroxypropanoicacid (LXV). This chloro acid (LXV) CHzOH

I CHOH I

CHzCl

HCl

, CHOH I

CHzCl

NarCrlOi

I

VII

HCN

I

CHzCl LXII

CHiOH

, c=o I

CHzC1

I I

HOC-CN CHzCI LXIV

CHzCl LXIII

1

HCI

COzH

I

H-C-CHzOH

I

CHzOH VI

+ HI

COzH

I H-C-CH21 I

COzH

I - HO C-CHz HI

I

CHeI

CHZCl

LXVI

LXV

C1

(33) N. Zelinsky and W. Krawetz, Ber., 46, 163 (1913). (34) J. W.E.Glattfeld and J. M. Schneider, J . Am. Chem. Soc., 60,415 (1938).

187

FOUR-CARBON SACCHARINIC ACIDS

was then converted to the diiodide (LXVI); treatment of the latter with water at reflux temperature for eight hours apparently gave a solution of the desired saccharinic acid (VI), since, from this aqueous solution, the diiodide (LXVI) could be regenerated. Attempted isolation of the saccharinic acid from the aqueous solution resulted only in the formation of an amorphous material. This product might have arisen from the dehydration of the saccharinic acid (VI) to a (hydroxymethy1)acrylic acid-which could then polymerize to give the amorphous product actually isolated. A last attempt at synthesis was madea6by the preparation of 2-(hydroxybut this product methy1)glyceronic acid from 1 ,3-dihydroxy-2-propanone, has not to date been transformed into the desired saccharinic acid (VI). Thus, this optically inactive acid has not been obtained in definitive form, nor have any of its derivatives been reported in the literature.

111. TABLES OF PROPERTIES OF THE FOUR-CARBON SACCHARINIC ACIDSAND THEIR DERIVATIVES The following Tables give the melting points and optical rotations of the four-carbon saccharinic acids and their derivatives thus far prepared. TABLEI Properties of the 3, Q-Dihydroxybulanoie Acids Substance

Acid (Ia) barium salt brucine salt calcium salt phenylhydrazide L Acid (Ib) barium salt brucine salt phenyl hydrazide DL Acid (Iab) amide brucine salt lactone phenylhydrazide D

[Q]D. ,degrees & "C.I.# , (water)

sirup

109 sirup

sirup gum sirup 90.7 170 22.5-26 100-101

-8.29 +1.48 -29.42 +2.47 +1.71 +8.00 -1.48

References

3 3 3 3 3, 30 3 3 3 3 5, 8, 10a 36 3, 5 30 3, 5

(35) H. M. Coleman and J. W. E. Glattfeld, J . A m . Chem. SOC.,66, 1183 (1944). (36) J . W. E. Glattfeld and D. MacMillan, J . Am. Chem. SOC.,66, 2481 (1934).

I 88

J. D. CRUM

TABLE I1 Properties f the b,&-Dihydro rbutanoic Acids u.p.,'C.

Substonce

Acid (IIa) brucine salt calcium salt quinine salt L Acid (IIb) brucine salt calcium salt quinine salt DL Acid (IIab) amide brucine salt lactone phenylhydrazide

alo , degrees (woter)

Ref erences

+14.97 -20.79 +17.08 -106.4 -14.86 -32.67 -17.33 -122.9

5, 13 5, 13 13 5, 13 5, 13 5, 13 13 5, 13 5, 12, 13 36 5, 13 5, 13 5, 13

sirup 169

D

149 sirup 169 149 118.5-119.5 188-190 sirup 130

-26.73

TABLEI11 Properties of the 8.3-Dihydroxybutanoic Acids Substonce

4-Deoxy-~~-erythronic Acid (IIIab) phenylh ydrazide o-tolylhydrazide I-Deoxy-~-threonicAcid (IVa) brucine salt 4-Deoxy-~-threonicAcid (IVb) brucine salt 4-Deoxy-~~-threonic Acid (IVab) brucine salt phenylhydrazide quinidine salt o-tolylhydrazide

Substoncs

Acid (Vab) phenylhydrazide

DL

di.p.,

"c.

81.5 123.5 103 73.5-75

73.5-74.5 232-234 132 159-162 111.5

u.p.,'C. 104 107

[U]D

, dCg*CCS

(lU4kY)

-15.0 -34.6 $15.1 -20.7 -+ -22.7

+147.5

References

14, 17, 19 19 19 19, 21 19,21 19, 21 19, 21 17, 19 21 21 21 19

Refumces

4, 25

4

THE METHYL ETHERS OF 2-AMINO-2-DEOXY SUGARS

BY ROGERW. JEANLOZ Massachusetts General Hospital, Boston, Massachusetts,

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 11. The Methyl Ethers of D-Glucosamine. . . . . . . . . . . . . . . . . . . . . . . . . . . 191 1. 3-Methyl Ether. . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 4-Methyl E th er. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 3. 6-Methyl Ether. . . . . . . . . . . . . . . . . . . . . . . . . . . 4. 3,4-Dimethyl Ether.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. 3,g-Dimethyl Ether.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 6. 4,6-Dimethyl E th er. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 7. 3,4,6-Trimethyl E th er, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 8. 3,4,5,6-Tetramethyl E th er. . . . . . . . . . . . . . 111. The Methyl Ethers of L-Glucosamine., . . . . . . . . . . . . . IV. The Methyl Ethers of D-Galactosamine.. . . . 1. 3-Methyl E th er. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 .............................. 199 2. 4-Methyl E th e r. . . . . . . . . . . . . . . . 3. 6-Methyl E th er. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 4. 3,4-Dimethyl E t h e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. 4,6-Dimethyl E t h e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. 3,4,6-Trimethyl E th er. . . . . . . . . . . . . . . . . . . . . . . . . . V. The Methyl Ethers of D-Allosamine . VI. The Methyl Ethers of D-Altrosamine VII. Analytical Properties. . . . . . . . . . . . . . . . . . . . . VIII. Tables of Properties of the Methyl Ethers of 2-Amino-2-deoxy Sugars.. . 203

I. INTRODUCTION Interest in the preparation of methylated derivatives of 2-amino-2deoxy sugars, especially of u-glucosamine (2-amino-2-deoxy-~-glucose)and D-galactosamine (2-amino-2-deoxy-~-ga~actose), arose from the desire to apply the methylation procedure of structural determination to oligo- and poly-saccharides containing aminodeoxy sugars. The number of reported isolations of this type of substance has been increasing at a very fast pace during recent years.' However, the isolation, elucidation of structure,2 (1) See M. Stacey, Advances i n Carbohydrate Chem., 2 , 161 (1947); M. Stacey and P. W. Kent, ibid., 3,311 (1948) ;R. U.Lemieux and M. L. Wolfrom, ibid., 3,337 (1948); H. G. Bray and M. Stacey, ibid., 4, 37 (1949); A. B. Foster and M. Stacey, ibid., 7 , 247 (1952); A. B. Foster and A. J. Huggard, ibid., 10,335 (1955). (2) E. E. van Tamelen, J. R. Dyer, H. E . Carter, J. V. Pierce and E. E. Daniels, J . Am. Chem. Soc., 78,4817 (1956).

189

190

R. W. JEANLOZ

and synthesis3 of a new “2-aminosugar,” D-gulosamine (2-amino-2-deoxyn-gulose) and the probable isolation of another, D-talosamine (2-amino-2deoxy-~-talose),4 from biological substances, is an indication of the difficulties to be expected in the study of the structure of complex carbohydrates. The methods of synthesis of methylated derikatives of amino sugars are, in general, the same as those used for the preparation of methylated derivatives of other monosaccharides and are described in previous articles of this Series. The solubility of the starting material has governed the choice of the methylating agent, and has prompted some modifications of the classical procedures; for example, the use of dimethyl sulfate plus sodium hydroxide in dioxane solution,s and of methyl iodide plus silver oxide in N ,N-dimethylformamide.6 Limited trials of newer reagents, notably those using liquid ammonia with an alkali metal plus methyl iodide, or thallous hydroxide plus dimethyl sulfate, were not encouraging.’ Most syntheses in the D-glucosamine and D-galactosamine series, using glycosides, have been carried out with the a anomers. The preparation of these derivatives is simple, requiring only N-acetylation or total acetylation of the amino sugar hydrochlorides followed by glycosidation, whereas formation of the p anomers requires the preparation of the unstable 0acetylglycosyl halides as intermediates. Although, in the D-glucosamine series, the resulting methyl 2-acetamido-2-deoxy-c~-~-glucopyranosideis always contaminated by approximately 20% of its p anomer, it is easily purified through its 3,4,6-tri-O-acetyl? 4,6-0-ben~ylidene,~ or 6-O-triphenylmethyl (6-0-trity1)Sderivatives. Amide formation, using acetic acid, adequately protects the amino group during the methylation procedure, and no evidence for the occurrence of N-methylation is then encountered. This stability is not so evident during subsequent esbrijkation, and displacement by benzoyl’O’looor p-tolylsulfonyl (tosyl)” groups occurs. The preparation of benzyl ethers with (3) Z. Tarasiejska and R. W. Jeanlos, J. A m . Chem. Soc., 79, 2660, 4215 (1957); R. Kuhn, W. Kirschenlohr and W. Bister, Angew. Chem., 69,60 (1957). (4) H.Muir, Biochem. J., 66.33 P (1957);M.J. Crumpton, Nature, 180, 605 (1957). (5) A. Neuberger, J . Chem. Soc., 50 (1941). (6) R.Kuhn, Angew. Chem., 67, 32 (1955). (7) R. W.Jeanlos, D. M. Schmid and P. J. Stoffyn,J . A m . Chem. Soc., 79, 2586 (1957). (8) R. Kuhn, F. Zilliken and A. Gauhe, Chem. Ber., 86, 466 (1953). (9) R.W.Jeanloz, J. Am. Chem. Soc., 74,4597 (1952). (10) R.W.Jeanlos, unpublished. (108) An addendum will be published in a subsequent volume of this series, giving the complete citations for References 10,13,14, 16,24,46,47,49, and 50. (11) R. W. Jeanlos, J . Am. Chem. Soc., 76, 555 (1954).

METHYL ETHERS OF 2-AMINO-2-DEOXY SUGARS

191

solid potassium hydroxide and benzyl chloride results in the formation of by-products containing a N-benzyl group.12 Added to the fact that benzyl derivatives of D-glucosamine are difficult to crystallize, this observation contraindicates the use of these otherwise valuable intermediates. A relationship between the reactivities of the secondary hydroxyl groups toward methylation and the conformation of the molecule has been observed in only a limited number of cases. For example, the hydroxyl group a t C3 of 2-acetamido-l , 6-anhydro-2-deoxy-/3-~-galactosehas a n axial conformation; i t is quite close to the oxygen atom linked to C1 and C6, and hydrogen bonding is likely to occur. Methylation of the hydroxyl group a t this position is negligible, compared to the methylation of that a t the vicinal 4-position possessing an equatorial conformation. Another example is found on comparing the results of the methylation of methyl 2-acetamido2-deoxy-cy-~-glucopyraiioside and methyl 2-acetamido-2-deoxy-cr-~-galactopyranoside, with those of the methylation of their 6-trityl ethers. In the first two compounds, the two chair forms (“Cl” and “1C” of Reeves) inter-equilibrate easily and the hydroxyl groups a t C3 and C4 of the amino sugar have approximately equal chances of being in the equatorial position in both the ~-glucosamineand the D-galactosamine compound. It is found that both compounds easily give a 3,4,6-trimethyl ether. I n the second case, however, the presence of a bulky trityloxy group a t C6 has a strong tendency to stabilize the molecule in the “C1” chair form, resulting in an equatorial conformation for the hydroxyl groups a t both C3 and C4 of D-glucosamine, and in an equatorial and axial conformation, respectively, for the corresponding hydroxyl groups of D-galactosamine. Experimentation showed that equal amounts of the 3-, 4-, and 3,4-substituted N acetyl-6-0-trityl-~-glucosamineswere i s ~ l a t e d whereas, ,~ with N-acetyl-6O-trityl-D-galactosamine, only a small proportion of the 3 ,4-dimethyl ether was obtained, together with a large proportion of the 3-monomethyl ether.13 The ionic influence of the acetamido group on the process of methylation seems negligible, as shown in the reaction of methyl 2-acetamido-2-deoxy6-O-trityl-a-~-glucopyranoside, the hydroxyl groups of which (at C3 and C4) are equally reactive.9

11. THE METHYL ETHERS OF

D-GLUCOSSMINE

1. 3-Methyl Ether

The synthesis of the 3-methyl ether of 2-amino-2-deoxy-~-g~ucose was accomplished in 1941 by Neuberger.6 The hydroxyl groups a t C4 and C6 (12) (13)

R. W. Jeanloz and D. A. Jeanloz, Chimia, 7,233 (1953). R. W. Jeanloz, D. K. Stearns and R. G. Naves, unpublished.

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of methyl 2-acetamido-2-deoxy~-~-glucopyranoside 'were first masked by condensation with benzaldehyde; recent work has shown that the recrystallized starting material still contained up to 20 % of the /3 anomer.* The resulting methyl 2-acetamido-4 6-0-benzylidene-2-deoxy-c~-~-glucopyranoside is, however, obtained optically pure, and its fi anomer can be recovered from the mother liquors by chromatography.'* In dioxane solution, methylation a t C3 proceeded smoothly. The protecting benzylidene group was removed by means of 60% acetic acid, and subsequent action of hydrochloric acid gave the crystalline a! anomer of 2-amino-2-deoxy-30-methyl-D-glucose hydrochloride. A crystalline 2-amino-2-deoxy-3-0methyl-D-gluconic acid was formed by oxidation with yellow mercuric oxide, and a crystalline N-acetyl derivativel6P and a crystalline Schiff base (obtained by condensation with 2 - hydroxynaphthaldehyde) are formed.l4 In the course of the synthesis of 2-amino-2-deoxy-3,4-di-O-methyl-~g l u c ~ s e ,the ~ 3-methyl ether of methyl 2-acetamido-2-deoxy-6-0-trityl-aD-glucopyranoside was obtained as a secondary product on methylation with Purdie's reagents. Elimination of the trityl group afforded the same glycoside as that obtained by Neuberger. Proof of the location of the methoxyl group a t C3 was established by application of periodate oxidation t o methyl 2-acetamido-2-deoxy-3-0methyl-cu-D-glucopyranosideand to 2-amino-2-deoxy-3-O-methyl-~-gluconic acid. The first compound was resistant to oxidation, whereas the second consumed two equivalents of periodate per mole and released one mole of formaldehyde and one mole of formic acid per mole, but afforded no ammonia.'' The 3-methyl ethers of the 4,6-O-benzylidene and 4 )6-0-ethylidene derivatives of methyl 2-(N-benzyloxycarbonyl)amino-2-deoxy-a-~-glucopyranoside have been prepared by a sequence of reactions paralleling Neuberger's synthesis. The p anomer of the latter compound was obtained directly by methylation of 2-(N-benayloxycarbonyl)amino-2-deoxy-4~6-0ethylidene-D-glucosewith dimethyl sulfate and alkali. Both anomers were transformed into the free amines by catalytic hydrogen~lysis.'~~ 2. 4-Methyl Ether A 4-methyl ether of a 2-amino-2-deoxy-~-g~ucose derivative was first obtained as a by-product in the methylation of methyl 2-acetamido-2deoxy-6-0-trityl-cu-~-glucopyranoside with Purdie's reagents. )

(14) R. W. Jeanloa, unpublished. (15) R. Kuhn, A. Gauhe and H. H. Baer, Chem. Ber., 87, 1138 (1954). (16) R. W. Jeanloz and M. TrBmBge, unpublished. (17) A. Neuberger, J . Chem. Soc., 47 (1941). (17a) S. Akiya and T . Oaawa, Yakugaku Zasshi, 76, 1276 (1956); Chem. Abstracts, 61. 4284 (1957).

METHYL ETHERS OF 2-AMINO-2-DEOXY SUGARS

193

In order to ascertain the location of the methoxyl group (at C4), various attempts were made to methylate intermediates possessing masked hydroxyl groups at C3 and C6. Conclusive results were obtained using the 3 ,6-di-p-toluenesulfonate ester1*; this was prepared from methyl 2acetamido-2-deoxy-cr-~-glucopyranoside,by first protecting the hydroxyl groups at C4 and C6 with the benzylidene residue, then introducing a p-tolylsulfonyl group on the hydroxyl group at C3, removing the benzylidene residue, and finally introducing a p-tolylsulfonyl group at C6 under controlled conditions. After methylation with methyl iodide in the presence of silver oxide, the masking groups were removed by hydrogenolysis, to afford a methyl 2-acetamido-2-deoxy-4-O-methyl-cu-~-glucop~anoside identical with the one previously obtained from the 6-trityl ether. The stability of the pyranose ring during methylation was ascertained on preparing the known 3,4,6-trimethyl ether. Hydrolysis of the 4-0-methyl glycoside with hydrochloric acid gave a sirupy hydrochloride, the base of which was characterized by the properties of its crystalline N-acetyl and N-(2-hydroxy-l-naphthylmethylene)derivatives. 3. 6-Methyl Ether Two different paths were used in the synthesis of this sugar.1e Both started from methyl 2-acetamido-2-deoxy-6-O-trityl-cr-~-glucopyranoside, readily obtained in pure form from impure methyl 2-acetamido-2-deoxy-crD-glucopyranoside.OIn one synthesis, the hydroxyl groups at C3 and C4 were protected with benzoyl groups, the trityl group was removed by hydrolysis, and methylation of the resulting free hydroxyl group at C6 was achieved with methyl iodide and silver oxide. In the second synthesis, the protection was obtained with benzyl ether groups, and the methylating agent employed was dimethyl sulfate. A drawback to this route was the concomitant formation of some of the N-benzyl derivative.12The blocking groups were easily eliminated, by saponification in the first case, and by reduction in the second, to give an identical product, methyl 2-acetamido-2deoxy-6-0-methyl-cr-~-glucopyranoside. Its oxidation by periodate was consistent with the location of the methoxyl group a t C6, one mole of oxidant per mole being consumed; formation of the known 3,4 ,6-trimethyl ether was sufficient proof of the stability of the pyranose ring. Hydrolysis resulted in the isolation of the crystalline hydrochloride of the (Y anomer. The free base was further characterized by means of the crystalline SchifT base (azomethine) formed with 2-hydroxynaphthaldehyde,and, later, by means of a crystalline N-acetyl derivative.16 2-Amino-2-deoxy-6-0-methyl-cu-~-glucose hydrochloride was isolated (18) R. W. Jeanloz and C. Gansser,J . A m . Chem. SOC.,79,2583 (1957). (19) R. W. Jeanloz, J . A m . Chem. SOC.,76, 558 (1954).

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R. W. JEANLOZ

from the hydrolyzate of methylated lacto-N-fucopentaose I1 and identified by infrared absorption spectrum and x-ray diffraction pattern.lga 4. 3,d-DimethyE Ether The key intermediate in the synthesis of the 3,4-dimethyl ether of 2-amino-2-deoxy-~-glucose is methyl 2-acetamido-2-deoxy-6-O-trityl-c~-~glucopyran~side.~ Location of the trityl group at C6 was tentatively assumed, by analogy with the structure of certain other trityl ethers of carbohydrates,2Oand was verified by the preparation of the 3 ,Cdibenzoate ester and the 3,4-dibenzyl ether.’$ The trityl ether was methylated with methyl iodide and silver oxide to give, in addition to the 3,4-dimethyl ether, two monomethyl ethers which were identified as the 3-methyl ethers and the 4-methyl ether.ls The same dimethyl ether was obtained from methyl 2-acetamido-2-deoxy-3-0-methyl-6-0-trityl-cu-~-glucopyranoside, which had been prepared from methyl 2-acetamido-2-deoxy-3-0methyl-a-D-glucopyranoside.6This synthesis constitutes further evidence for the location a t C3 of one of the methoxyl groups. Graded hydrolysis with dilute acetic acid released methyl 2-acetamido2deoxy-3,4-di-O-methyl-c~-~-glucoside, from which the known 3,4,6-trimethyl ether was prepared as evidence for the stability of the pyranose ring during the methylation procedure. The crystalline hydrochloride of the a anomer of the free sugar was obtained after hydrolysis with hydrochloric acid. Its free base was further characterized by crystalline N-acetyl, N - (benzyloxycarbonyl), and N - (2 - hydroxy - 1-naphthylmethylene) derivatives. 5. 3,G-Dimethyl Ether During the elucidation of the structure of “mannosidostreptomycin” by methylation, Fried and StavelyZ1isolated two derivatives of a dimethyl ether of N-methyl-L-glucosamine. In order to compare their physical constants with those of a known compound in the D-series, Fried and Wa1zZzprepared derivatives of 2-deoxy-3,6-di-0-methyl-2-methylamino-~glucose. The addition of methylamine and hydrogen cyanide to 2,5-diO-methybarabinose (prepared by the periodate oxidation of 3,6-di-0methyl-D-glucose) was followed by hydrolysis, and yielded a mixture of a dextro- and a levo-rotatory acid.2s The dextrorotatory product was con(19a) R. Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 91,364 (1958). (20) B. Helferich, Advances i n Curbohydrute Chem., 3, 79 (1948). (21) J. Fried and H. E. Stavely, J . Am. Chem. SOC.,74, 5461 (1952). (22) J. Fried and D. E. Walz, J . A m . Chem. SOC.,74, 5468 (1952). (23) See F. A. Kuehl, Jr., E. H. Flynn, F. W. Holly, R. Mozingo and K. Folkers, J . A m . Chem.Soc., 69, 3032 (1947);M. L. Wolfrom, A. Thompson and I. R. Hooper, iWd., 88, 2343 (1946).

METHYL ETHERS OF 2-AMINO-2-DEOXY SUGARS

195

verted to the lactone, reduced, and acetylated. Purification by chromatography on alumina resulted in partial deacetylation, and two crystalline products were isolated. On the basis of their elementary analysis and chemical properties, they were assigned the structures of 4-0-acetyl-2and its l-acedeoxy-3 ,6-di-0-methyl-2-(N-methylacetamido)-a-~-glucose tate ester. From the mother liquors of the dextrorotatory acid, the levorotatory compound, 2-deoxy-3 ,6-di-0-methyl-2-methylamino-~-mannonic acid, was crystallized. was The synkhesis of 2-amino-2-deoxy-3,6-di-0-methyl-~-glucose~~ started from the known methyl 2-acetamido-2-deoxy-3-0-methyl-6-0-trityla-~-ghcopyranoside.~ The hydroxyl group a t C4 was masked by esterification with benzoyl chloride, the trityl group was removed by graded hydrolysis, and the resulting free hydroxyl group a t C6 was methylated by means of Purdie’s reagents. Alkaline hydrolysis then afforded methyl 2-acetamido-2-deoxy-3,6-di-O-methyl-a-~-glucopyranoside. The stability of the pyranose ring was shown by conversion to the known 3 ,4,6-trimethyl ether. Evidence for the location of the 0-methyl groups is based on the known structure of the starting material and on the parallelism of the synthesis with that leading to the 6-monomethyl ether. I n addition, the final product was different from either the 3,4-dimethyl or the 4,6-dimethyl ether. Treatment of the glycoside with hydrochloric acid resulted in a sirupy hydrochloride. Its free base was characterized by means of its crystalline N-acetyl and N-(2-hydroxy-l-naphthylmethylene)derivatives. 6. 4,6-Dimethyl Ether The first preparation of a 4 ,6-dimethyl ether of 2-amino-2-deoxy-~glucose was reported by Haworth, Lake, and Peat.26Action of ammonia on followed by Nmethyl 2,3-anhydro-4,6-di-O-methy1-/3-~-mannoside, acetylation of the product, led to the formation of methyl 3-acetamido-3deoxy-4,6-di-O-methyl-/3-~-altroside (in high yield) and of methyl 2-acetamido-2-deoxy-4,6-di-O-methyl-/3-~-glucoside (in a 9.4 % yield), Methylation of the latter compound yielded a 3 ,4 ,6-trimethyl ether identical with the one prepared from natural D-glucosamine.26 Synthesis of the dimethyl ether, starting from 2-amino-2-deoxy-~glucose, was accomplished using either of two different blocking groups on the hydroxyl group at C3, the benzoyl and the p-tolylsulfonyl.l1 Both were obtained by suitable esterification of methyl 2-acetamido-4,6-0bemylidene-2-deoxy-c~-~-glucoside,followed by graded hydrolysis of the benzylidene residue. After methylation by means of Purdie’s reagents, R. W. Jeanloz, J . A m . Chem. Soc., unpublished. W. N . Haworth, W. H. G. Lake and S. Peat, J . Chem. Soc., 271 (1939). (26) W.0.Cutler, W. N. Haworth and S. Peat, J . Chem. Soe., 1979 (1937). (24) (25)

196

R. W. JEANLOZ

alkaline hydrolysis in the one synthesis, and reduction in the other, eliminated the blocking group, to give the same methyl 2-acetamido-2-deoxy4 ,6-di-O-methyl-cw-~-glucoside.Methylation of this derivative gave the known 3,4 ,6-trimethyl ether, proving the stability of the pyranose ring; whereas hydrolysis of it afforded a sirupy hydrochloride whose free base was characterized by means of a crystalline S c h 8 base with 2-hydroxynaphthaldehyde, and, later, by a crystalline N-acetyl derivative.IB 2-Amino-2-deoxy-4,6-di-O-methyl-~-glucosehas been isolated from the hydrolyzate of methylated hyaluronic acid, and characterized by means of the N-(2-hydroxy-l-naphthylmethylene)derivative and the methyl cr-~-glycoside.~~ A sirup, consisting for the most part of this dimethyl ether, was obtained after fractionation of the hydrolyzate of methylated lacto-N-fucopentaose I.27a

7 . S,Q,G-Trimethyl Ether This compound was isolated after permethylation of the mixture resulting from the hydrolysis of methylated polysaccharides containing 2-amino-2-deoxy-~-glucose.2~ It had previously been prepared (for the first time) by Cutler, Haworth, and Peat.2BPreliminary attempts to protect the amino group during the methylation procedure-by precondensation with benzaldehyde, o-hydroxybenzaldehyde, and p-methoxybenzaldehyde, respectively-were not successful. The desired effect was obtained by acetylation of the amino group. Action of dimethyl sulfate and alkali, at room temperature, on a carbon tetrachloride solution of 2-acetamido-1,3 ,4 ,6-tetra-O-acetyl-229 or of 2-acetamido-2-deoxy-~-glucose,~~ afforded deoxy-cu,@-D-glucoseZ6~ methyl 2-acetamido-2-deoxy-3,4,6-tri-0-methyl-@-~-glucoside.The same compound resulted from the action of dimethyl sulfate and alkali at 50" on methyl 2-acetamido-3,4 ,6-tri-0-acetyl-2-deo~y-@-~-glucoside.2B Final proof of the structure was established by comparing it with the product obtained by methylation of methyl 2-acetamido-2-deoxy-4,6-di-O-methylp-D-glucoside,26 and with that resulting from the action of ammonia on followed by methyl 3 ,4 ,6-tri-0-methyl-2-0-p-tolylsulfonyl-@-~-glucoside acetylation.*l The tri-o-methyl-@-D-glucosideis stable in alkali, and is (27) R. W. Jeanloz, Chimia, 7, 292 (1953); Proc. Intern. Congr. Biochem., 3rd

Cmgr., Brussels. 1866, 65 (1956). (27a) R.Kuhn, H.H. Baer and A. Gauhe, Chem. Ber., 89, 2514 (1956). (28) M.Stacey and J. M. Woolley, J . Chem. SOC.,184 (1940); 550 (1942); W. N. Haworth, P. W. Kent and M.Staoey, ibid., 1220 (1948). (29)P.A. Levene, J . Biol. Chem., 157.29 (1941). (30) T.White, J . Chem. SOC.,428 (1940). (31) W. 0.Cutler and El. Peat, J . Chem. Soc., 782 (1939).

METHYL ETHERS OF 8-AMINO-2-DEOXY SUGARS

197

converted into the crystalline 2-amino-2-deoxy-3,4, G-tri-0-methyl-P-Dglucose hydrochloride by warm, aqueous hydrochloric acid. In a 2 % solution of hydrogen chloride in methanol, isomerization takes place, and the a! anomer may be isolated in excellent yield.26The same anomer results from the direct methylation of methyl 2-acetamido-2-deoxy-a!-~-g~ucopyranoside with dimethyl sulfate plus sodium h y d r o ~ i d eand , ~ ~ is converted by hydrolysis with aqueous hydrochloric acid to the same hydrochloride previously described.20Additional evidence for the location of the ring and of the methoxyl groups was educed by degradation of the hydrochloride or with chloramine-T,28 to with N-chloro-l-naphthalenesulfonamide,32 2 ,3,5-tri-O-methyl-~-ara,binose.The latter compound was identified by oxidation, and conversion of the product to the crystalline 2,3,5-tri-0methyl-~-arabinonamide.~~ Oxidation of the hydrochloride with mercuric oxide led to a crystalline 2-amino-2-deoxy-3,4, G-tri-O-methyl-n-gluconic A 7 % solution of hydrogen chloride in methanol isomerized methyl 2-acetamido-2-deoxy-3,4,6-tri-O-methyl-/3-~ -glucoside to the a! anomer, with simultaneous deacetylation.26 Transformation of the P into the a anomer under the influence of alcoholic hydrogen chloride was also observed with the methyl N-benzoyl, benzyl N-acetyl, and benzyl N-benzoyl derivatives of 2-amino-2-deoxy-3,4, G-tri-O-methyl-/3-~-glucoside.~~ I n contrast, both anomers of methyl 2-deoxy-3,4,6-tri-O-methy1-2-trimethylamino-D-glucoside hydriodide were stable to acid in methanolic solution (as well as to alkali). The P anomer of this product had been obtained by the exhaustive methylation of methyl 2-amino-2-deoxy-3,4,6-tri-O-methyl8-D-glucoside hydrobromide by the Purdie method, whereas the a! anomer was derived from methyl 3 ,4,6-tri-0-acetyl-2-amino-2-deoxy-a-~-gluco~ide hydrobromide. Distillation of the hydriodide of the a! anomer yielded methyl 2-deoxy-2-dimethylamino-3,4,6tri-0-methyl-a-~-glucoside,which proved to be resistant to alkali, thus differing from the non-methylated, parent compound.34The above-described methyl 2-amino-2-deoxy-3,4,6tri-0-methyl-a-n-glucoside has also been obtained by catalytic hydrogenation of methyl 2 - ( N - benzyloxycarbonyl)amino - 2 - deoxy -3 ,4 ,G - tri - 0 methyl+-D-glucoside and gave a 2-sulfoamino derivative by the action of sulfuric anhydride in pyridine.84" The characterization of 2-amino-2-deoxy-3,4 ,G-tri-0-methyl-0-D-glucose was accomplished by transformation into the crystalline N-acetyllls, 30 (32) A. Neuberger, J. Chem. Soc., 29 (1940). (33) W. 0. Cutler and S. Peat, J. Chem. Soc., 274 (1939). (34) J . C. Irvine and A. Hynd, J. Chem. Soc., 101, 1128 (1912). (34a) M. L. Wolfrom, R. A. Gibbons and A. J. Huggard, J . Am. Chem. Soc., 79, 5043 (1957).

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R. W. JEANLOZ

N-(2-hydro~y-l-naphthylmethylene),2~ and N-benzoyl derivatives, the last being remarkably resistant toward oxidants.s2 Synthesis of the 3 4 6-trimethyl ether of 2-deoxy-2-methylamino-~gluconic acid was carried out along lines similar to those of the synthesis of the 3,6-dimethyl etherF2By the addition of methylamine and hydrogen cyanide to 2,3,5-tri-O-methyl-~-arabinose, followed by hydrolysis, only one compound was formed, and this was related to D-glucosamine on the basis of its optical rotation. An attempt to obtain the aldose derivative by reduction with sodium amalgam was not successful, probably because of the impossibility of 8-lactone formation. 8. 3 4,6,6’-TetramethyE Ether

The sirupy diethyl dithioacetal N-methyl derivative of this compound was obtained by methylation of 2-amino-2-deoxy-~-glucose diethyl dithioacetal with dimethyl sulfate in dioxane solution.36

111. THEMETHYLETHERS OF L-GLUCOSAMINE Methyl ethers of L-glucosamine were isolated during the work21 on the elucidation of the structure of “mannosidostreptomycin.” Methylation of N-acetyl-“dihydromannosidostreptomycin,” followed by hydrolysis, acetylation, and purification of the resulting mixture by chromatography on alumina, led to the isolation of two crystalline products. These were shown to be derivatives of a dimethyl ether of L-glucosamine, and one was found to derive from the other by a deacetylation occurring during chromatography. Location of the methoxyl groups at C3 and C6 was based on the results of periodate oxidation of the starting material, “dihydromannosidostreptomycin,” and on the assumption of a pyranose ring. Final proof was obtained by comparing the melting points and optical rotations of the two derivatives with those of the synthetic 3,g-dimethyl ether of the D-series.22 Evidence for a pyranose ring in the above-described compounds was obtained by the results of methylation of N-acetyldihydrostreptomycin, followed by hydrolysis. After acetylation, purification afforded the crystalline 1-0-acetyl-2-deoxy-3,4,6-tri-O-methyl-2-(N-methylacetamido)-a-~glucose, which was converted by hydrolysis into the 1,2-deacetylated product, isolated as the crystalline hydrochloride. Identical products were synthesized by methylation of a mixture of the a and @ anomers of methyl 2-deoxy-2-(N-methylacetamido)-~-glucopyranoside, followed by acetylation and hydrolysis. As further proof, the hydrochloride was degraded by periodate ion and the resulting pentose was oxidized with bromine to (35) M. W. Whitehouse, P. W. Kent and C. A. Pasternak, J . Chem. Soe., 2315

(1954).

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199

give 2 ,3 ,5-tri-O-methyl-~-arabinonic acid, identified by means of its crystalline amide.

IV. THEMETHYLETHERS OF D-GALACTOSAMINE 1 . 3-Methyl Ether

The synthesis of the 3-methyl ether of 2-amino-2-deoxy-~-galactose~~ followed the same sequence used in the 2-amino-2-deoxy-~-g~ucose series. The hydroxyl groups a t C4 and C6 of methyl 2-acetamido-2-deoxy-~y-ogalactopyranoside were blocked by condensation with benzaldehyde. The remaining hydroxyl group was then methylated with dimethyl sulfate in dioxane solution, and the benzylidene residue was removed by hydrolysis topyranoside. to give methyl 2-acetamido-2-deox y -3-0-methyl-cy-~-galac The location of the methoxyl group was assumed to be at C3, from the negligible oxidation of this derivative by periodate, from its conversion into the known 3,4,6-trimethyl ether, and from the improbability of a substitution on the hydroxyl groups at C3 and C6 of D-galactose by a benzylidene residue. Hydrolysis with hydrochloric acid afforded a sirupy hydrochloride. Its free base was characterized by means of a crystalline Schiff base with 2-hydroxynaphthaldehyde. 2. 4-Methyl Ether In the synthesis of this compound, the methyl group was introduced first, followed by the amino group.” The starting material, 1,6: 2 ,3-dianhydro-P-D-talopyranose, was methylated with Purdie’s reagents, and the location of the methoxyl group a t C4 was confirmed by its conversion into the Reaction with am4-methyl and 2 ,4-dimethyl ethers of ~-galactose.~* monia,3g followed by acetylation, gave (in preponderant yield) 2-acetamidoTreatment with 3-0-acetyl-1 ,6-anhydro-2-deoxy-4-O-methyl-~-~-galactose. hydrochloric acid now led to simultaneous scission of the 1,6-anhydro ring and hydrolysis of the acetyl groups. A crystalline hydrochloride was isolated, and its free base was characterized by conversion to the crystalline Schiff base with 2-hydroxynaphthaldehyde. Further characterization was obtained by peracetylation followed by glycosidation with boiling, methanolic hydrogen chloride, to give the crystalline methyl 2-acetamido2-deoxy-4-0-methyl~-~-galactopyranoside. Later, a crystalline N-acetyl derivative of the free base was prepared.le (36) P. J. Stoffyn and R . W. Jeanloz, J . Am. Chem. Soc., 76.561 (1954). (37) R.W.Jeanloz and P. J. Stoffyn, J . Am. Chem. Soc., 76, 5682 (1954). (38) R.W.Jeanloz, J . Am. Chem. Soc., 76,5684 (1954). (39) See S.P.James, F. Smith, M. Stacey and L. F. Wiggins, J . Chem. Soc., 625

(1946).

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R. W. JEANLOZ

3. 6-Methyl Ether

The 6-methyl ether of D-galactosamine has been isolated from the hydrolyxate of methylated @-heparin (chondroitinsulfuric acid B).40 Its synthesis was started from methyl 2-acetamido-2-deoxy-a-~-galactopyranoside, using two different sets of intermediate^.^^ In one procedure, the hydroxyl group at C6 was protected by preparation of the trityl ether, and the hydroxyl groups at C3 and C4 were benzoylated. The trityl group was then removed and the resulting, free hydroxyl group was methylated. After alkaline hydrolysis of the benzoyl groups, crystalline methyl 2-acetamido2-deoxy-6-0-methyl~-~-galactopyranoside was isolated. The same compound was prepared in a much shorter way by blocking, with an isopropylidene residue, the hydroxyl groups at C3 and C4 of the starting material, methylating the free hydroxyl group at C6, and removing the blocking group by hydrolysis. Transformation into the known 3,4,6-trimethyl ether confirmed the stability of the pyranose ring, and hydrolysis of the glycosidic linkage and of the acetyl residue with hydrochloric acid gave the crystalline hydrochloride of the (Y anomer. Characterization of the free base was accomplished through the crystalline N-acetyl ,N-(2-hydroxy-l-naphthylmethylene), and N-acetyl-1,3,4-tri-O-acetyl derivatives.

4. 3,Q-DimethylEther Preparation of this derivative7 was started from (a) 2-acetamido-l , 6anhydro-2-deoxy-P-~-galactopyranose, derived from an intermediate in the synthesis of D-galactosamine from lactosela8and (b) its 4-methyl ether, an intermediate in the synthesis of 2-amino-2-deoxy-4-0-methyl-~-galact ~ s eThe . ~ ~hydroxyl group a t C3 proved to be quite resistant to most of the agents usually used for methylation, and so the 3,4-dimethyl ether was isolated in relatively low yield. Scission of the 1,6-anhydro ring took place simultaneously with splitting of the N-acetyl group. The vicinal free amino group thus liberated enhanced the stability of the glycosidic bond to acid hydrolysis, and a large proportion of the crystalline hydrochloride of the anhydro compound was isolated, besides the sirupy hydrochloride of the free sugar. The base of the latter compound was characterized by means of its crystalline N-acetyl and N-(Zhydroxy-1naphthylmethylene) derivatives, and by formation of the crystalline methyl 2-acetamido-2-deoxy-3,4-di-O-methyl-cw-~-galactopyranoside, through the N , 1,6-triacetyl derivative. Some /3 anomer was also isolated from the mixture obtained by glycosidation, showing the influence of the methyl ether groups on the equilibrium of the anomers. (40) R. W. Jeanloz, P. J. Stoffyn and M. TrBmBge, Federktion Proc., 18, 201 (1957). (41) P. J. Stoffyn and R. W. Jeanloz, J . Am. Chem. Soc., 80 (1958), in press.

METHYL ETHERS OF 2-AMINO-2-DEOXY SUGARS

201

5 . 4,6-Dimethyl Ether The synthesis of this dimethyl ether proceeded smoothly, along the same path of reactions followed in the 2-amino-2-deoxy-~-glucoseseries.42 The hydroxyl groups a t C4 and C6 of methyl 2-acetamido-2-deoxy-a-~galactopyranoside were masked by condensation with benzaldehyde, the hydroxyl group at C3 was blocked with a p-tolylsulfonyl group, and the hydroxyl groups a t C4 and CG were then unmasked by hydrolysis and were methylated. Removal of the p-tolylsulfonyl group by the action of sodium amalgam afforded methyl 2-acetamido-2-deoxy-4,6-di-O-methyl-aD-galactoside. This was shown to contain a pyranose ring by transformation into the 3,4,6-triniethyl ether. Subjected to acid hydrolysis, it gave the crystalline hydrochloride of the a anomer, and the free base was characterized by means of its crystalline N-acetyP and N-(2-hydroxy-1na~hthylmethylene)~~ derivatives. The 4,6-dimethyl ether of D-galactosamine has been isolated from the hydrolyznte of methylated, desulfated B-he~arin.4~" 6. 3 , 4 ,&Trimethyl Ether The synthesis of the trimethyl ether of 2-amino-2-deoxy-~-galactopyranose was reported by L e ~ e n e .43~ Methylation ~. of a ,p-D-galactosarnine pentaacetate afforded n sirupy methyl 2-acetamido-2-deoxy-3,4,6-tri-Omethyl-a ,P-D-galactoside, from which the @ anomer was isolated in crystalline form. The same compound was later prepared by S t a ~ e ythrough ,~~ the methylation of pure 2-acetamido-l , 3 ,4 ,G-tetra-O-acetyl-2-deoxy-t9-~galactose. Boiling with 1% methanolic hydrogen chloride converted the @-D-glycosideinto the a anomer. The latter compound was also synthesized by methylation of pure 2-acetamido-1 ,3 , 4 ,6-tetra-O-acety1-2-deoxy-a-~galactose, as well as of methyl 2-acetamido-2-deoxy~-~-galactopyranoside. The @-D-glycosidewas converted, by the action of 10% methanolic hydrogen chloride, into methyl 2-amino-2-deoxy-3 ,4 ,6-tri-O-methyl-a-~galactoside hydr0chloride,4~whereas hydrolysis of the a-D-glycoside with hydrochloric acid gave the crystalline hydrochloride of the a anomer of 2-amino-2-deoxy-3 ,4 ,6-tri-O-methyl-~-galactose. Its free base has been characterized by means of its crystalline N-acetyl and N-(2-hydroxy-lnaphthylmethylene) derivatives.lB The location of the methyl groups was conclusively established by oxidation (with mercuric oxide) to a tri-0methyl+-galactosaminic acid, followed by degradation (with chloramine-T) into the known 2 ,3 ,5-tri-O-methyl-~-lyxose.~~ (42) P . J. Stoffyn and R . W. Jeanloz, J . Am. Chem. SOC.,76,563 (1954). (42a) R . W. Jeanloz and P. J. Stoffyn, Federation Proc., 17, 249 (1958). (43) P . A. Levene, J . Biol. Chem., 133, 767 (1940). (44) M. Stacey, J. Chem. Soe., 272 (1944).

202

R. W. JEANLOZ

V. THE METHYLETHERS OF D-ALLOSAMINE Methyl ethers of 2-amino-2-deoxy-~-allose~~ have been prepared in order to be subsequently degraded into methyl ethers of D-ribose. Methylation of methyl 2-acetamido-4,6-0-benzylidene-2-deoxy-a-~-alloside~~ gave the 3-methyl ether, which, in turn, was hydrolyzed to afford methyl 2-acetamido-2-deoxy-3-O-methyl-cu-~-allopyranoside. After partial methylation, a dimethyl ether, probably the 3,6, was isolated, whereas exhaustive methylation afforded the 3,4,6-trimethyl ether. An identical trimethyl ether was isolated after methylation of methyl 2-acetamido-3,4,6-tri-Oacetyl-2-deoxy-cu-~-alloside.Hydrolysis of the methyl glycosides of both the 3-methyl and the 3,4,6-trimethyl ethers gave crystalline hydrochlorides, and the base of the former ether was characterized by means of its crystalline Schiff base with 2-hydroxynaphthaldehyde. Walden inversion, at C3, of methyl 2-acetamido-2-deoxy-4-O-methyl3 6-di-0-methylsulfonyliu-~-glucoside took place on heating it with sodium acetate in 2-methoxyethanol (methyl Cellosolve) solution; the product afforded, after acetylation, a sirupy methyl 2-acetamido-3 6-di-0-acetyl2-deoxy-4-O-methyl-a-~-alloside,*~ Removal of the 0-acetyl groups, followed by methylation, gave the same 3,4,6-trimethyl ether previously reported. )

)

VI. THEMETHYLETHERS OF D-ALTROSAMINE Methylation of methyl 2-acetamido-4 6-0-benzylidene-2-de0xy-w~altroside, obtained by hydrolysis of the 3-acetate ester,@gave a 3-methyl ether.49 Hydrolysis of the benzylidene group, and tritylation of the resulting glycoside, led to crystalline derivatives. )

PROPERTIES VII. ANALYTICAL All the hydrochlorides of the methyl ethers of 2-amino-2-deoxyhexoses so far synthesized give a positive color reaction with ninhydrim60Reaction of the hydrochlorides of all known methylated 2-amino-2deoxyhexoses with 2,4-pentanedione, followed by condensation with the Ehrlich reagent (p-dimethylaminobenzaldehyde) , gives a purple coloration (the ElsonMorgan reaction).60Foster, Horton, and Stacey,61using a modification of the method,62found that the 4- and 6-methyl ethers of 2-amino-2-deoxyD-glucose give a color identical with, but less intense than, that given by (45) (46) (47) (48) (49) (50) (51) (52)

R. W. Jeanloz, J . A m . Chem. SOC.,79, 2691 (1957). R. W. Jeanlor and M. TrBmBge, unpublished. R. W. Jeanlos and C. Bothner-By, unpublished. S. Peat and L. F. Wiggins, J . Chem. SOC.,1810 (1938). R. W. Jeanlor and D. M. Schmid, unpublished. R. W. Jeanlor and M. TrBmBge, unpublished. A. B. Foster, D. Horton and M. Stacey, J . Chem. SOC.,81 (1957). R. Belcher, A. J. Nutten and C. M. Sambrook, Analyst, 79,201 (1954).

METHYL ETHERS OF 2-AMINO-2-DEOXY SUGARS

203

the parent amino sugar, with maximal absorption at 511 mp; on the other hand, the 3-methyl ether gives another color, with maximal absorption at 503 mp. Using a different procedure, Cifonelli and D ~ r f m a nobserved ~~ a shift of the maximal absorption, from 540 mp to 510 mp, when the hydroxyl group at C3 was methylated. In the reaction of N-acetyl derivatives of methylated 2-amino-2-deoxy sugars with alkali, followed by condensation with the Ehrlich reagent, the amount of color obtained is found to depend upon the position of the methyl ether grouping, no coloration being observed with 4-methyl ethers.lKS64 When sodium carbonate66 is used, the color formation is enhanced by a methyl ether at64C3, whereas no difference in the color obtained is observeds0with the use of borate.67 The micromethod of Perlin@has been employed in the determination of the formic acid produced by the periodate oxidation of the methyl ethers of 2-amino-2-deoxy-~-g~ucoseand 2-amino-2-deoxy-~-galactose.~~ With most derivatives, especially those possessing a methoxyl group a t C4, the amount of formic acid liberated does not reach the theoretical value. It is evident that most derivatives react in the cyclic form, and that the formate ester formed a t the hydroxyl group of C5 by splitting of the Cl-C2 linkage is stable, or only very slowly hydrolyzed, a t the pH used, namely, 5.7. The infrared absorption spectra of some derivatives of the 3-methyl, the 4,6-dimethyl, and the 3,4,6-trimethyl ethers of 2-amino-2-deoxy-~glucose have been recorded,69and the paper-chromatographic separation (in two different sets of solvents) of the 3-methyl, 6-methyl, 3,4-dimethyl, 4,6-dimethyl and 3,4,6-trimethyl ethers of 2-amino-2-deoxy-~-glucose has been recently OF PROPERTIES OF THE METHYL ETHERS VIII. TABLES OF 2-AMINO-2-DEOXY SUGARS

The following Tables record the melting points and specific rotations of the methyl ethers of 2-amino-2-deoxy sugars and their derivatives. In those cases where two or more references are given for one compound, the physical constants reported are taken from the first paper, and are only cited from other papers when they differ significantly. (53) J . A. Cifonelli and A. Dorfman, J . B i d . Chem., 231, 11 (1958). (54) R. W. Jeanloz and M. TrBmBge, Federation Proc., 16,282 (1956). (55) D. Aminoff, W. T. J . Morgan and W. M. Watkins, Biochem. J . , 61,379 (1952). (56) J . A. Cifonelli and A. Dorfman, J . B i d . Chem., 228, 547 (1957). (57) J . L. Reissig, J. L. Strominger and L. F. Leloir, J . B i d . Chem., 217,959 (1955). (58) A. S. Perlin, J . A m . Chem. Soc., 76, 4101 (1954). (59) S. A. Barker, E. J. Bourne, M. Stacey and D. H. Whiffen, J . Chem. Soc., 171 (1954). (59a) S. Akiya and M. Tomoda, Yakugaku Zasshi, 77, 697 (1957); Chem. Abstracts, 61, 15338 (1957).

TABLEI Tlre Monomethyl Ethers of d-Amino-2-deoxy-D-ghcose Derivative of 2-AminoZdwxy-D-glucose

3-0-Methyla anomer, hydrochloride N-acetylN- (2-hydrox y -l -naphthylmet hylene) 4,6-O-ethylidene-, methyl a-D-pyranoside, hydrochloride j3 anomer N-acetyl-, methyl a-D-pyranoside 4-0-beneoyl4-O-benz yl4,6-di-0-acetyl4,6-di-0-benzoyl6-0-trityl4-0-acetyl4-0-benzoyl4-O-benz yl4,6-0-benzylideneN-(benzyloxycarbony1)-, methyl a-D-pyranoside 4,6-0-beneylidene4,g-O-ethylidenej3 anomer Methyl 4-0-benzyl-2-(N-benzylacetamido) -2-deoxy-3-0methyla-D-glucopyranoside 6-0-trityl2-Amino-2-deoxy-3-0-methyl-~-gluconic acid

Melting goint, "C.

215 (dec.) 183-185 195198 203-204 (dec.) 231 (dec.)

[ab, degrees

+123

+

Rotation solvent

+91.3

+33 (equil.) +270 (equil.) at 97

+

-19 +116 +20 +lo1

HzO

5 15 16 14 17a

Hz0 HzO CHC13 CHCla CHClz

17a 5 24 24 24 24

CHCli CHCl3

9 9 24 24 5 175. 17a 17a 17a 24

24 5

A6461

232 (dec.) 211 143-147 198-200 167-168 123-125 206-207 228-229 192-194 105-107 277-279 139-140 225-227 167 197 116118

+51 - 12 +117

CHC13 CHClz C2H60H CHCls CHCla CHCl, CHC13

127-130 230 (dec.)

+-7912

CHC13 570 HC1

+82

+53

+76 +73 +39 +74 +54

Rejerences

~

3 cl

M

zR+-

4-0-Methylhydrochloride N-acetyl-, a anomer N- (2-hydroxy-1-naphthylmethy1ene)N-acetyl-, methyl a-D-pyranoside 3,6-di-O-acetyl3,6-di-O-mesyl3,6-di-O-tosyl6-0-trityl3-0-acetyl6-0-Methyla anomer, hydrochloride N-acetyl-, a anomer N- (2-hydroxy-1-naphthy1methylene)N-acetyl-, methyl a-D-pyranoside 3 ,Cdi-O-acetyl3,4-di-O-benzoyl3,4-di-O-benzyl-

liquid 211-215 (dec.) 218-219 (dec.) 232-233 liquid 157-159 liquid 212-213 182

+90 (equil.) +79 + +69 +305 (equil.) at x 6 4 6 1 157 79 107 +75 +59 +71

Ht0 Hz0 CH aOH CHtOH CHCls CHClr CHCla CHCla CHCla

18 18 18 18 18 47 18 9 9

185-195 (dec.) 224-225 205-2417 18!3-191 126-127 liquid 197-198

+92 + +68 +74 + +48 +m (equil.) at A6461 143 +97 -8

HsO HtO CHaOH CH ,OH CHCl p CHCla CHCl a

19 16 19 19 19 19 19

+ + +

+

+99

z

1 1 4

?I

M

8

r

5Z

P

TABLEI1 The Dimethyl Ethers of f?-Amino-&-deoxy-D-glucose Derioative of 2-Amino-2-dwxy-D-glucose

Melting goinl, 'C.

[ a l ~degrees ,

3,4-Di-O-methylLY anomer, hydrochloride

20&205; 215-22. +I21 -+ +115 (dec.) N-acetyl-, a anomer 173-175 +64 +48 N- (benzyloxycarbony1)146-148 N- (2-hydroxy-1-naphthy1methylene)198-200 (dec.) t348 (equil.) X s r c N-acetyl-, methyl a-D-pyranoside 192-193 152 6-0-acetyl171 123 232-233 6-0-trityl88 2-Benzamido-2-deoxy-3,5-di-O-methyl-~-glucono-l, 4186 t-76.9 --t t 7 0 lactone Ethyl 2-benzamido-2-deoxy-3,5-0-di-O-methyl-~-gluco(do 1.5480) nate 3,6-Di-O-methylhydrochloride liquid +84 (equil.) 232-233 N-acetyl-, a anomer +90 + +37 215-218 N-(2-hydroxy-l-naphthylmethylene)+305 (equil.) a t -+

+ +

+

Rolation solvent

References

H20

9

HzO

9 9 9 9 9

CH 30H CH30H CHCl3 CHC1, HzO-CtHbOH (1:1)

+129

3

95

60 60

H2 0 Hz0 CH30H

24 24 24

A5461

N-acetyl-, methyl a-D-pyranoside 4-O-acet yl4-0-benzoyl4-0-Acetyl-2-deoxy-3,6-di-0-methyl-2(N-methylacetamido) -a-D-glucose 1-0-acetyl-

P

161-162 163-164 139-140 162.5-163.5

+116 +48 +71 -+ +31

CH 30H CHCl3 CHCla H20

24 24 24 22

140-141

+I10

CHCl3

22

*

5

2-Deoxy-3,6-di-O-methyl-2-methylamino-~-glucon~c acid 4,6-Di-0-methylhydrochloride N-acetyl-, a anomer N- (2-hydroxy-1-naphthylmethglene)-

dec. 178 liquid 227-228 192-194 (dec.)

+2.8

+88 (equil.) +88 ---t +68 +296 (equil.) a t

Hz0

22

Hz0 HzO C H 30H

11 16 11

CHaOH CH3OH CHCl3 CHC13 CHCl3

11 25 11 11 11

A5461

N-acetyl-, methyl a-D-pyranoside 6 anomer 3-0-acetyl3-0-benzoyl3-0-tosyl

199-m 187 10%110 115-116 liquid

+150 -21.5 102 +63 69

+

+

TABLEI11 The Tri- and Tetra-methyl Ethers of 8-Amino-8-deozy-~-g~uco~e Dniwlive of Z-Ani~-Zdwsy-D-glucosc

3,4,6-Tri-O-methylp anomer, hydrochloride

[a$,degrccs

dec. 210

+49.2

+

h3

R o l a l h solvcnt

+99.4

N-acetyl-, a anomer

224

N-benzoyl-, a anomer N- (2-hydroxy-1-naphthy1methylene)-

213 170-172

References

26

26 29 30 15 32 24

+56.8

210 234

+18+44.8 +75 +

+79 + +48.5 +124 + +lo5 +400 (equil.) at

?d

h 6 l

N-acetyl-, benzyl a-D-pyranoside

138

4-118.2

anomer N-benzoyl-, benzyl a-D-pyranoside p anomer methyl a-D-pyranoside

174 184 180

-36.2 +123.2 -21.75 +169.8

9,

hydrochloride N -acetyl-

p anomer

(b.p. 85'/0.004 mm.) (n21.6 1.4555) dec. 237

225-238 (dec.) 150 153-154;166-167 195 190

+129.6 +113.6 147 +104.3 +135.0 +120.0 127 -29.0 -13.1 +19.6

+

+

0.0

8

2% of HC1 in CsH6CHsOH CHCl 8 CHCl s CHCls CHsOH

33

HtO CHiOH HsO H2O CHIOH CHCl I CHCl I HzO CHIOH CHCls CHsOH

26 26

r

G

3

3

P

3315 33 g 26

345

26 26 26 9

26 26 26 29 ___

T-LE III-Continued N-benaoyl@ anomer N-(benayloxycarbonyl)N ,N-dimethylN , N ,N-trimethyl-,hydriodide fl anomer N-sulfo-,sodium salt 2-ihino-2-deoxy-3,4,6-tri-O-methyl-~-glucon~c acid N-methyl2-Deoxy-3,4,5,6-tetra-O-methyl-2-methylamino-~glucose diethyl dithioacetal

162 198 119-121 (b.p. 160"/0.03 mm.) (n: 1.4530) soft. 45 145 77 178-179 zo6-m.5

liquid

CHCl3 CHCls CHCla

+122.8 +29.6 +98.5

33 33

34a 33

+119.1 -12.9 +98.2 +10.5 +9.5 + +8.2 -15.8

CHCla CHCl3

E

#rc

33 33

HzO

34a

CHaOH HzO

2

%35

CHCl3

9

3

Bg

TABLEIV Methyl Ethers of 2-Amino-2-deoxy-L-glucose -4

Rotation solvent

4-0-Acetyl-2-deoxy-3,6-di-O-methyl-2-(N-methylacetamido)- 162.5-163.5 a-L-glucose 139.5-140 1-0-acetyldec. 160 2-Deoxy-3,4,6-tri-0-methyl-2-methylamino-~-glucose hydrochloride 118119 l-O-Acetyl-2-deoxy-3,4,6-tri-O-methyl-2(N-methylacetamido)a-L-glucose

-70

+

-29

I

-79 -107

- 108 - 142

CHCla

21 21 21 21

!a ra

t o

8

TABLEV The Monomethyl Ethers of 2-Am~no-B-deoxy-~-ga~actose Derioaliue Df P-At~'ino-2-dcoxy-D-golactose

3-0-Methylhydrochloride N- (2-hydroxy-l-napht,hylmethylene)l,&anhydride, 3-O-mesyl-, B anomer N-acetyl-, methyl a-o-pyranoside 4,6-di-O-acetyl6-0-Lrityl4-0-acetyl4-0-benzoyl4,6-0-benzylidene4-0-Methyl a anomer, hydrochloride N-acetyl-, a anomer N-(2-hydroxy-l-naphthylmethylene)-,a anomer 1,6-anhydride,3! , anomer N-acetyl3-0-acetyl3-0-mesylN-acetyl-, methyl a-D-pyranoside 3 ,g-di-O-acetyl-

Melting point. "C.

[a]=.degrees

Rofaiion soloenf

References

liquid 205-207 184-186 194-196 137-139 236-237 23&239 27S281 284-285

+119 (equil.) +132 (equil.) a t A 5 1 6 1 -55 183 +136 +75 55 93 172

Hz0 CH 3OH CHCla CH30H CHC13 CHCl3 CHC13 CHC13 CHC13

36 36 61 36 36 13 13 13 36

dec. 178 197-199 207-209 (dec.)

+lo0 +125 $102 + +82 +168 a t A 5 4 8 1 +187

95-96 122-123; 152-153 231-233 la142 24-242 114-115

+

+ +

+

37 16 37

-+

-+

-37 -53 -57 -41 147 82

+

+

H20 CHCl3 CHCla CHCl3 CHIOH CHCl3

37

7 37 61 37 37

9

3 4

M

*

3

8

6-0-Methyla anomer, hydrochloride N-acetyl1,3,4-tri-O-acetyl-,a anomer methyl a-D-pyranoside 3,4-di-O-acetyl3,4-di-O-benzoyl3,4-O-isopropylidene acetal N-(2-hydroxy-l-naphthylmethylene) derivative, a anomer

190-195 (dec.) 165-168 219-220 207-208 83-85 liquid 159-160 189-191

+lo7

+92

+92 +101 --f

+164 101 192 +153 +280 -+ +258 at

+ +

A6461

HzO HzO CHCl3 CH 30H CHC13 CHCla CHC13 CH30H

TABLEVI

The Dimethyl Ethers of d-AminoJ-deozy-D-ga~aetose Derivative of Z-Amino-Zdcozy-D-galactose

3,4-Di-O-methylhydrochloride N-acetyl-, a anomer N-(2-hydroxy-l-naphthylmethylene)-, @ anomer l,g-anhydride, @ anomer hydrochloride N-acetylN-acetyl-, methyl a-D-pyranoside 6-0-acetyl6-O-trit yl@ anomer 6-0-acety 14,6-Di-O-methyla anomer, hydrochloride N-acetyl-, a anomer N-(2-hydroxy-1-naphthylmethy1ene)N-acetyl-, methyl a-D-pyranoside 3-0-acetyl3-0-tosyl-

Melting poist. "C.

[ab,degrees

liquid 199-200 (dec.) 203-'204 (dec.)

108 +114 + +92 +I07 + +332 at A6461

dec. 250 109-111 219-220

203-204 222-224 247-249 247-248

dec. 190 221-223 183-186 22-229

111-1 12 106-108

+

-26 -87 +I46 +la

+66 -18 0

+lo7 + . 91 +I16 + +90 +223 (equil.) at A6461 +141 +I06

+94

Rotation soluml

HnO H20 CHaOH

7 7 7

HsO CHCla CH30H CHCla CHClo CHaOH CHCla

7 7 7 7 7 7 7

HzO HzO CH 3OH CHoOH CHCls CHClr

42 16 42 42 42 7

a

3 5*

3

TABLE VII The Trimethyl Ethers of 8-Amino-8-deozy-D-galactose Dnioatioe of 2-Amino-Zdeoxy-D-galoclorc

Rotation solvent

Melting point, "C. ~~

3,4,6-Tri-O-methyla anomer, hydrochloride N-acetyl-, a anomer N- (2-hydroxy-l-naphthylmethylene)-, j3 anomer methyl a-D-pyranoside, hydrochloride N-acetyl-, methyl a-D-pyranoside

6 anomer

178 197-199 194-197 227 185 191-192 223

232 187 2-Amino-2-deoxy-3,4,6-t~-o-methyl-~-galacton~c acid

+152.5 + +lo5 +121 + +97 +188 + +294 at A6461 +150.3 +121 +142 -19.5 +7 +7.0

H20 Hz0 CHaOH CHsOH CHCla CH oOH ClsCH-CHC12 CHCls CH30H

TABLEVIII Methyl Ethers of .%?-Amino-.%?-deoxy-D-allose, 2-Am~no-2-deoxy-~-a~trose, and 2-~mino-2-deoxy-~-mannose Comtound

Melting )oint, "C.

2-Amino-2-deoxy-3-0-methyl-a-~-allose hydrochloride (2-hydroxy-l-naphthylmethyleneamino)-3-O2-Deoxy-2-

169-170 (dee.) 178-179 (dec.)

methyl-@-D-allose Methyl 2-acetamido-2-deoxy-3-O-methyl-a-~-allopyrano 169-170 side 114-116 4,6-di-O-acetyl218-219 4,6-O-benzylideneliquid Methyl 2-acetamido-3,6-di-0-acetyl-2-deoxy-4-O-methyl a-D-alloside Methyl 2-acetamido-2-deoxy-3,6-di-O-methyl-a-~-allo- 122-123 pyranoside 165-170 (dec.) 2-Amino-2-deoxy-3,4,6-tri-O-methyl-@-~-allose hydrochloride 99-100 Methyl 2-acetamido-3,4,6-tri-O-methyl-a-~-alloside 149 Methyl 2-acetamido-3-O-methyl-a-~-altropyranoside 6-0-trityl200 liquid 4-0-acetyl4,6-0-bensylidene189-194 185-186 2-Deoxy-3,6-di-0-methyl-2-methylamino-~-mannonic acid (60) D.B. Hope and P. W. Kent,J . Chem. SOC.,1831 (1955). (61) R. W. Jeanloe,J . Am. Chem. SOC.,unpublished.

[a]~ degrees ,

4-43 +37

-+

-+

+58

+17 at h 4 6 1

+79 45 +68

+

4-30 --t +45 +89 +25 68 +71 +93 -3.8 -i -5.8

+

Rofation soloenl

tejwences

HzO CHaOH

46 46

CHiOH

46

CHCl3 CHCla CHC13

46 46 47

CH3OH

46

HzO

46

CHCl3

46 49 49 49 49 22

CH 30H

CHC13 CHCl3

CHC1, Hz0

3 4

3g

GLYCOSYL UREIDES BY IRVINGGOODMAN Wellcome Research Laboratories, Tuckahoe, N . Y .

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Nomenclature... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Preparation .................... 1. Acid-catalyzed Condensation of Sugars With Urea. . . . . . . . . . . . 2. Amination of Glycosyl Isocyanates and Isothiocyanates.. . . . . . 3. Condensation of Glycosylamines With Isocyanates. . . . . . 4. Miscellaneous Preparations, . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ IV. Physical Properties. . . . . . . V. Structure and Chemical Pr ............................ VI. General.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Tables of Glycosyl Ureides and Related Compounds.. . . .

215 216 217 217 220

227 228 232

I. INTRODUCTION Over half a century ago, Schoorl’ noted that C. A. Lobry de Bruyn had been unable to achieve reproducible analyses for lactose in urine, either by conversion to galactitol or to galactaric acid.2 On the basis of this observation, it appeared likely that lactose might exist in the urine in chemical combination with some other urinary constituent, such as urea. I n attempting to confirm this hypothesis, Schoorl did a doctoral thesis on the nature of the reactions het,ween sugars and urea. This extensive study has become the “cornerstone” of a challenging but little explored class of compounds, the glycosyl urcides. From their discovery, the glycosyl ureides have been of interest from a biochemical standpoint. The fact that D-glucose and urea occur in human blood in approximat,ely equimolar concentrations (about 0.006 M ) has led to speculation and to some investigation of the metabolic role of these compounds. M a ~ e r in , ~ a series of metabolic experiments with dogs, attempted to determine whether 1-D-glucosylurea is a natural constituent of riormal or of pathological blood or urine. (1) M. N. Schoorl, Rec. trav. chim., 19, 398 (1900). (2) Recent analytical methods have made i t possible to do routine determinations of urinary lactose quite readily; F. V. Flynn, C. Harper and P. De Mayo, Lancet, 266, 698 (1953). (3) P. Mayer, Biochem. Z . , 17, 145 (1909).

215

216

IRVING GOODMAN

Neuberg and Neimann? in 1905, as part of a general study of the properties of D-glucuronicacid, synthesized l-ureido-D-glucuronicacid by Schoorl’s method1 and suggested the possible occurrence of this compound under normal physiological conditions. Hynd6 reported a series of experiments also initiated to determine the physiological role of the glycosyl ureides. Fischere extended the field of glycosyl ureides to include the thioureides, and prepared derivatives belonging to both classes. In 1938, Johnson and in Bergmann’ reported the preparation of 3-cyanoacetyl-l-~-glucosylurea an unsuccessful attempt to synthesize pyrimidine nucleosides from glycosyl ureides (by the methods of Traube*and of Rupe and coworkerssfor pyrimidine formation). Goodmanloin 1956 described the synthesis of some new pyrimidine nucleosides from the glycosyl ureides. These compounds thus provide a basis for further study and speculation as possible intermediates in the biosynthesis of nucleic acids. Aside from their biochemical implications, the glycosyl ureides have found application in the paper,” textile,11-13and foodL4industries. The cyclitol derivatives of streptomycin (streptidine and strepturea), although not strictly glycosyl ureides, are mentioned here since they are close relatives of the series.16

11. NOMENCLATURE Schoorl’s major publicationls in 1903 was entitled “Les ureides (carbamides) des sucres.” The compound l-D-glucosylurea was referred to alternatively as “l’ureide du glucose” and “la carbamide du glucose.” Neuberg and Neimann4 used the term “ureidoglukuronsaure,” and Fischera identi(4) C. Neuberg and W. Neimann, 2.physiol. Chem., Hoppe-Seyler’s, 44.97 (1905). (5) A. Hynd, Biochem. J . , 20, 195, 205 (1926). (6) E. Fischer, Ber., 47, 1377 (1914). (7) T. B. Johnson and W. Bergmann, J. Am. Chem. SOC.,60,1916 (1938). (8) W. Traube, BeT., 33, 1371 (1900). (9) H. Rupe, A. Metzger and H. Vogler, Helu. Chim. Acta, 8 , 848 (1925). (10) I. Goodman, Federation Proc., 16, 264 (1956). (11) H. W. J. H. Meijer, U. 5. Pat. 2,596,268 (1952); Chem. Abstracts, 46, 10636 (1952). (12) H. W. J. H. Meijer, U. S. Pat. 2,612,497 (1952); Chem. Abstracts, 47, 1402 (1953). (13) K. Quehl, U. S. Pat. 2,116,640 (1938); Chem. Abstracts, 32, 500 (1938). Al-

though this reference has been cited in relation to the glycosyl ureides [W. W. Pigman, “The Carbohydrates,” Academic Press Inc., N. Y . , 1957, p. 4151, the products described are merely physical mixtures of urea and D-glucoce in the form of “molecular compounds.” (14) J. J. Opplt, U. S. Pat. 2,694,719 (1954); Chem. Abstracts, 48, 2639 (1955). (15) R. L. Peck, C. E. Hoffhine, Jr., E. W. Peel, R. B. Graber, F. W. Holly, R. Mozingo and K . Folkers, J . Am. Chem. SOC.,68,776 (1946). (16) M. N. Schoorl, Rec. trau. chim., 22. 1 (1903).

GLYCOSYL UREIDES

217

fied one of his products as “glucose carbamid.” Helferich and Kosche17 employed the designation “d-glucose-harnstoff” in one case, but for the corresponding methylurea derivative, “d-glucose-monomethylureid.” Ellis and Honeymanlahave listed a number of glycosyl ureides as “N-carbamoylglycosylamines.” Requirements of simplicity and uniformity in nomenclature suggest that with the Rules of the International Union of Pure and Applied Cherni~try’~ regard to simple urea derivatives and to carbohydrates be applied to the glycosyl ureides. Thus, in this review the “N-glycosides” of urea, thiourea, and guanidine are included, with generic classifications as follows. H

X

I II

Rf-N-C-N 1

2

/Rff 3\

Rflf

I

where R’ = glycosyl; R” = H, glycosyl, alkyl, or aryl; and R’” = H, glycosyl, alkyl, or aryl. X

Generic name 2Q

-0

Glycosyl ureide Glycosyl thioureide Glycosylguanidine

=s

= N - H

The compound l-~-g~ucosyl-3-methylurea (11), listed in Table I11 is assigned the following structure.21

HC 3

HOH-C

H-CO -N H c H

HO

It 111. PREPARATION 1. Acid-catalyzed Condensation of Sugars with Urea Schoorl’s chief criterion’ for establishing the occurrence of a reaction between D-glucose and urea was the gradual decrease in optical rotation (17) B. Helferich and W. Kosche, Ber., 69, 69 (1926). (18) G. P.Ellis and J. Honeyman, Advances in Carbohydrate Chem., 10, 95 (1955). (19) J . Chem. Soc., 5064 (1952). (20) The prefix “ureido” should be used in the case of the uronic acid derivatives. (21) This compound is almost certainly a 8-D-glucopyranosyl derivative; however, since (for most members of these series) neither the ring structure (furanose or pyranose) nor the configuration a t the anomeric carbon atom has been definitely established, these characteristics are not specified in the Tables of glycosyl ureides in the present Chapter.

218

IRVING GOODMAN

of the reaction mixture. However, great difficulty was encountered in the isolation of the product, which was finally obtained (in crystalline form) only after the unreacted D-glucose had been destroyed by fermentation. Since that time, there have been some improvements in the method of synthesis of 1-D-glucosylurea, but Schoorl's original approach involving the acid-catalyzed condensation of D-glucose with urea is still the basis for the most convenient methods of preparation, such as that of Hynd.6 The Hynd modification6 of Schoorl's method is given in detail since this is basically the method of choice for the preparation of most of the known glycosyl ureides. The following is the procedure6 for synthesizing l-D-glucosylurea. D-Glucose (27 g.) is dissolved in 125 ml. of water. Urea (27 g.) is added and the resulting solution is filtered. T o the clear filtrate is added 5.6 ml. of 25% sulfuric acid and, after thorough mixing, the solution is kept a t 50" for 7 days. The pale, greenishyellow solution is then concentrated under diminished pressure a t 37" t o a thin sirup. After standing overnight in the cold, the resulting colorless crystals are removed and drained (by suction filtration) and are washed with 90% ethanol. The product is practically pure urea adduct of l-D-glucosylurea (A); yield, 31 g (73% of the theoretical); mp., 167". This product may be recrystallized by dissolving i t in an equal weight of boiling water; upon slow cooling, crystals are formed; m.p., 17c172"; [ a ] -18.18' ~ (c 2.5856, in water). The 1:l additive compound (A) of l-u-glucosylurea and urea may be converted to pure l-D-glucosylurea as follows. The urea ureide (70 9.) is boiled for three days with several fresh 200-ml. portions of absolute ethanol. The yield of pure l-n-glucosylurea is practically theoretical; m.p., 208" (dec.); [ a ] -23.45' ~ (c 2.1532, in water).

With minor variations, application of this general method has resulted in the successful preparation of the majority of the known glycosyl ureides. SchoorP described the synthesis of the ureides of u-glucose, D-galactose, D-mannose, and lactose, and of the D-glucosyl derivatives of methylurea, 1,l-dimethylurea, and phenylurea (see Table 111).On the basis of changes in optical rotation (see Table I), he concluded that D-lyxose, D-xylose, L-arabinose, and maltose also react with urea to form ureides, and that benzylurea, thiourea, biuret, and urethan react with D-glucose to form the corresponding N-D-glucosyl derivatives, although he did not succeed in isolating these products. Using Schoorl's method,lB Neuberg and Neimann4 prepared the ureide of D-glucuronic acid (see Table 111). In this case, as in Schoorl's experiments, the progress of the condensation of the urea with the u-glucuronolactone was followed by observing changes in optical rotation of the remtion mixture. Two and a half months of incubation (at 40" in 5 % sulfuric acid) were required to reach equilibrium. The product was isolated, after considerable difficulty, as the barium salt.

219

GLYCOSYL UREIDES

TABLEI Changes i n Optical Rotation of Carbohydrates upon Incubation with Ureas, Amides, and Amino Acidsa Reageirts

D-Glucose and: Urea Met hylurea A~etamide22-~~ A1anine26 Benzamide22 Benz ylurea Biuret 1,3-Diethylurea 1,l-Dimet hylurea 1,3-Dimethylurea 1,l-Diphenylurea 1,3-Diphenylurea Glycine2E-z8 Guanidinc Leucine Phenylurea Taurine Thiourea Urethan Urea and: L-Arabinoi3e D-GalactoHe D-Fructosf! D-Lyxose D-Mannosc! L-Sorbose D-Xylose Lactose Maltose

Change in Optical Rolation of Mixture (Alter 14 days a6 50°C.)

Decrease Decrease No changeb No changeb No changeb Decrease Decrease No change Decrease No change No change No change No changeb No changebNo change Decrease No change Decrease Decrease Decrease Decrease No change Decrease Decrease No change Decrease Decrease Decrease

a D a t a from Schoorl.1f bMore recently, some of the amido and amino derivatives which Schoorl, on the bmis of these observations of changes in optical rotation, believed not to have been formed, have been prepared's by other methods. e See page 227.

(22) V . Deulofeu and ,J. 0. Deferrari, J . Org. Chem., 17, 1087 (1952). (23) C. Niemann and -1. T . Hays, J . A m . Chem. SOC.,67, 1302 (1945). (24) R. C. Hockett and L. B. Chandler, J . Am. Chem. S O C .66,957 , (1944). (25) M. L. Wolfrom, It. D. Schuetz and L. F. Cavalieri, J . A m . Chem. SOC.,71, 3518 (1949). (26) F. Micheel and A. Klemer, Chem. Ber., 86, 1083 (1952). (27) W. W. Pigman, E. A . Cleveland, D. H . Couch and J. H. Cleveland, J . Am. Chem. SOC.,73, 1976 (1951). (28) J . C. Irvine and A. Hynd, J . Cheni. Soc., 99, 161 (1911).

220

IRVING GOODMAN

Helferich and Kosche,’? by a modified Schoorl procedure, isolated certain products whose existence had been demonstrated earlier (see page 218) on the basis of optical-rotation studies. These included 1-L-arabinosylurea, 1,3-di-~-xylosylurea, and l-~-glucosyl-2-thiourea. They also prepared 1-D-glucosylurea, l-~-glucosyl-3-methylurea, and some derivatives (see Table 111), and demonstrated the formation of a molecular compound (1:1) of 1-D-glucosylurea with urea, as independently reported by Hynd6 at about the same time. Erickson and Keps,29 using the acid-catalyzed condensation of D-glucose and D-galactose, respectively, with n-dodecylurea, prepared the corresponding glycosylurea derivatives. 2. Amination of Glycosyl Isocyanates and Isothiocyanates

Emil Fischer,6 as part of a study of a series of “S- and N-glucosides,” prepared 1-D-glucosylureaand 1-D-glucosylthioureafrom the corresponding isocyanate (IV) and isothiocyanate (V), respectively. These intermediates were obtained by the condensation of tetra-0-acetyl-a-D-glucopyranosyl bromide (111) with silver cyanate or thiocyanate in anhydrous xylene. The

Hu CH20Ac

CH20AC

_WNO

AgCNS-

nco

H

m

OAc

H

m

OAC

AcO H Q H

OAC

P NH3 MeOH

t Cb0H H O H H - CO -N H2 HO OH

rmr (29) J.

plf

G.Erickson and J. 8.Keps, J . Am. Chem. Soc., 76,4339 (1953).

GLYCOSYL UREIDES

22 1

ureide (VII) and thioureide (VIII) were readily formed by treating the isocyanate and isothiocyanate with ammonia. When IV and V were refluxed with anhydrous ethanol, the urethans I X and VI were formed. Fischer30 also prepared 6-deoxy-6-isothiocyano-~-glucose and the corresponding 6-deoxy-6-thiourethan by a similar method. Fischer’s preparations of glycosyl ureides and thioureides, illustrated by the following reactions, have served as starting points for a number of interesting derivatives.308 Johnson and Bergmann3’ applied Fischer’s glucosyl isocyanate synthesis30 to the preparation of 1,3-di-~-glucosylurea,l-~-arabinosylurea,’and 1,3di-~-xylosylurea.~ Starting with Fischer’s method, Haring and Johnson32 prepared tetra-0-acetyl-D-glucopyranosyl isothiocyanate (V), from which they synthesized D-glucosylhydantoic acid (XIV), D-glucosylthiohydantoic acid (XVIII), and derivatives, as illustrated by the following reactions. Johnson and Bergmann,? as well as Helferich and Kosche,17 noted the possibility that the glycosyl ureides might be utilized as intermediates for an improved method of synthesis of pyrimidine nucle~sides.~~ Fischer’s method30 was used for obtaining 1-D-glucosylureaas well as 1-L-arabinosylurea and 1 ,3-di-~-xylosylurea. Micheel and Schmidt34utilized a method similar to that of Fischer in preparing a number of interesting glycoprotein derivatives for immunological studies. Tetra-0-acetyl-u-glucopyranosyl cyanate was refluxed with gelatin in dry benzene giving a product which, on hydrolysis, yielded wglucosylureido-gelatin. Pseudoglobulin, treated with the same acetylated u-glucosyl cyanate, yielded the analogous u-glucosylureido protein. Micheel and B r ~ n k h o r s tprepared ~~ other derivatives in this series by the same general method. To illustrate, tetra-0-acetyl-D-glucopyranosyl isothiocyanate (V), when treated with DL-alanine methyl ester (XIX) yielded the D-ghcosykhiourea derivative of DL-alanine, namely, 1-(tetra-0-acetyl~-glucopyranosyl)-3-(~~-alanyl methyl ester)S-thiourea (XX). The struc(30) E. Fischer, Ber., 63, 884 (1920). (30a) The reaction of free D-glucose with potassium thiocyanate results in a totally different type of compound, a D-glucoxazoline. [See w. H . Bromund and R . M. Herbst, J. Org. Chem., 10, 267 (1945).] (31) T. B. Johnson and W. Bergmann, J . Am. Chem. SOC.,64,3360 (1932). (32) K. M. Haring and T. B. Johnson, J. Am. Chem. SOC.,66, 395 (1933). (33) The first synthesis of pyrimidine nucleosides had been reported earlier [G. Hilbert and T. B. Johnson, J . Am. Chem. SOC.,62,4489 (1930)l by means of a condensation of 2,4-diethoxypyrimidine with tetra-0-acetyl-o-glucopyranosylbromide, but the yields were poor and the scope of the reaction was limited. (34) F. Micheel and K. Schmidt, Makromol. Chem., 3, 210 (1949). (35) F. Micheel and W. Brunkhorst, Chem. Ber., 88, 481 (1955).

N N N

cw H n - C S - N H - C H 2

HCI (cold)

-CQK HCI (cold)

1

H

H

a -CS-NH-CH2-C02H

HO H

S

XYIt

OH

Ha-CO-NH-CH2-CQK

I HO

41 0 0

F

223

GLYCOSYL UREIDES

tures of a few other members of the series prepared by this method are as follows. CH2OAC H

y43 I H-CS-NH-CHI C02Me

AcO

H

OAc

H

xx

XIX

Y

CH20AC H

a

t

OAc

OH

C+OAc

-

i -CS N

- H C O ~ E +~

H

ACO

i

-

-CS-NHOCQH

AcO OAc

W

H

OAC

xgn:

XXI

1-(Tetra-0-acetyl-D-gluco pyranosyl) -3-(4-carboxy-3hydroxyphenyl)-2-thiourea

1-(Tetra-0-acetyl-D-glucopyranosy1)-3-(4-~arbethoxypheiiyl)-2-thiourea

From some of the thioureides, Micheel and coworkers prepared the corresponding glycosylguanidines by treatment with an aqueous suspension of mercuric oxide,3Sa followed by reaction with the appropriate amine. CH20AC

xa

9%

NH-CO-NHoC02Et AcO H

OAc

XHII:

(35a) Treatment with mercuric oxide under anhydrous conditions should yield a

224

IRVING GOODMAN

Muller and Wilhelmsae applied the Fischer glycosyl thiocyanate method to the preparation of 6-deoxy-6-thiocyano-~-glucose and further explored These workers have examined the the synthesis of l-~-glucosy~-2-thiourea. question of the isomerization of thiocyanates and isothiocyanates, and have made some observations which should prove exceedingly useful.

m

"J_

He3

ACO

=OAc N"T

1

A

OH

m carbodiimide which would be an excellent intermediate for various interesting derivatives; see H. G. Khorana, Chem. Revs., 63, 145 (1953). (36) 5.Muller and A. Wilhelms, Bey., 74, 698 (1941).

225

GLYCOSYL UREIDES

3. Condensation of Glycosylamines with Isocyanates

With the improved availability of glycosylamines, the Fischer30 isocyanate synthesis of glycosyl ureides was modified to allow these sugar amines to react with alkyl or aryl isocyanates and isothiocyanates. Schmuck3’ treated D-glucosylamine with phenyl isocyanate and obtained 1-D-glucosyl3-phenylurea (XXVIII). Similarly, the use of phenyl isothiocyanate resulted in the formation of l-~-glucosyl-3-phenyl-2-thiourea (XXX). Helferich and M i t r o ~ s k yprepared ~~ the same compounds in a slightly different manner, by starting with tetra-0-acetyl-D-glucopyranosyl bromide (111). Shaw and WnrreneP have recently reported a new synthesis of 2,3,5tri-O-benzoyl-2’-thiouridine (XXXIV) by a method which probably passes through a glycosylthiourea intermediate stage (XXXIII). 2,3,5-Tri-00

BzoH 2

+

EtO-CH-CH-CO-NCS

h

H

H2

H OBZ

OBZ

OBZ

Hz) HoH2cc.9:

002

0

0

NaOH

t-

H

bH

OH

OBz

OBz

(37) A. A. Schmuck, Zhur. Russ. Fiz.-Khim. Obshchestva, 61, 1759 (1929); Chem. Zentr., 101, I , 3173 (1930). (38) B. Helferich and A. Mitrowsky, Chem. Ber., 86, 1 (1952). (39) G. Shaw and R. N. Warrener, Proc. Chem. SOC.(London), 351 (1957); 81 (1958). See also, R . K . Ralph and G . Shaw, J. Chem. Soe., 1877 (1956); G. Shaw, R. N. Warrener, M . H . Maguire, and R. K . Ralph, ibid., 2294, 2299 (1958); G. Shaw and R. N. Warrener, ibid., 153, 157 (1958).

226

IRVING GOODMAN

benzoyl-D-ribosylamine (-1) was condensed with 3-ethoxypropenoyl isothiocyanate (XXXII) in ethyl acetate as solvent. Upon debenzoylation and desulfurization, uridine (XXXV) was formed. Thymidine was also prepared by this method.

4. Miscellaneous Preparations Micheel and coworkers,40 in further efforts to prepare glycoproteins resembling those which occur in Nature, synthesized a series of glycosylguanidine derivatives by a novel approach. l-~-Glucosyl-2-thiourea(VII) was treated with ethyl bromide, resulting in the formation of 2-ethyl-l-~glucosyl-2-pseudothiourea hydrobromide (XXXVI). When XXXVI was CH,OH

A

OH

apamr

treated with sodium hydroxide plus glycine, N-(wglucosylguany1)glycine (XXXVII) was formed. By this method, analogous derivatives of lysine, histidine, and cystine were prepared. More recently, Micheel and Heesing,41 by using this method, prepared 6-( l-~-~-glucosylguanyl)caproicacid (XXXVIII) and 1-D-glUCOSY1-3methylguanidine. When XXXVI was treated with various protein solu-

~

(40) F. Micheel, W. Berlenbach and K. Weichbrodt, Chem. Ber., 86, 189 (1952). (41) F. Micheel and A. Heesing, Ann., 604, 34 (1957).

GLYCOSYL UREIDES

227

tions, glycoprotein derivatives were formed in which the glycosyl residue A? was linked to the protein through a guanidine Only a few examples of direct "gumidination" of monosaccharides have been reported. Morrell and B e l l a r ~treated ~~ D-glucose, D-fructose, and D-mannose, respectively, with guanidine in ethanol. The glycosylguanidines isolated were hygroscopic and had various molar ratios of carbohydrate to guanidine. Danilov and L i ~ h a n s k iattempted i~~ a series of direct guanidinations of derivatives of D-glucose, D-allose, arid waltrose, but succeeded only in linking guanidine to methyl 2,3-anhydro-4,6-0-benzylidene-a-~-alloside, to produce methyl 4,6-O-benzylideiie-2-deoxy-2-guanidino-a-~-altroside. IV. PHYSICAL PROPERTIES

As a representative member of the glycosyl ureides, the physical properties of l-u-glucosylurea16are listed here in detail. Melting point Specific gravity Index of refractioii Molecular volume Molar heat of combustion Specific rotation

208" ( dec.) 1.480 (25") 1.56 150 8307 Cal. [a]: -23.5" with no mutarotation (c 10, in water)

l-D-Glucosylurea consists of colorless, odorless, rhombic crystals with a slightly sweet taste. It crystallizes with one mole of urea of crystallization per mole6, 17 and is very readily soluble in water. It is slightly soluble in methanol (0.215 %) and in 86 % ethanol (0.72 %). It is very slightly soluble in absolute ethanol (0.042 %), and virtually insoluble in n-amyl alcohol, ethyl ether, ethyl acetate, n-hexane, benzene, chloroform, and acetone. In general, the physical properties of the glycosyl ureides and thioureides and glycosylguanidines resemble those of l-u-glucosylurea. However, the properties of 3-N-substituted glycosyl ureides are markedly modified by the 3-N-substituent. Modifications of the glycosyl residue (for example, acylation) also result in drastic changes in the physical properties of the molecule. (42) F.Micheel and E. Dinkloh, 2. physiol. Chem., Hoppe-Seyler's, 293,183 (1953); Chem. B e r . , 84, 210 (1951). (43) F.Micheel arid B. Herold, 2. physiol. Chem., Hoppe-Seyler's, 293, 187 (1953); Chem. Ber., 86, 189 (1952). (44) It. S. Morrell and A . E. Bellars, J . Chem. Sac., 91, 1010 (1907). (45) 8. N. Danilov and I. S. LishanskiI, Zhur. ObshcheZ Khim., 26, 2106 (1955); Chem. Abstracts, 60, 8462 (1956).

228

IRVING GOODMAN

V. STRUCTURE AND CHEMICAL PROPERTIES Various structures have been proposed for the glycosyl ureides, but these may be resolved into two alternative forms: the straight-chain structure as advanced by SchoorP (XXXIX) and the cyclic form as proposed by Hynd and Macfarlane,46 Helferich and Kosche'? (VIII), and others. C = N - CO - N H 2 I

HCOH I HOCH

I HCOH I

HCOH I C$OH

XlDXc

CH20H H

O -CO-NHz

HO H

OH

nm.

Although a rigorous proof of structure has not yet been reported, the known chemical properties of 1-D-glucosylurea are in harmony with the cyclic structure (VIII). These properties are summarized in Table 11, and are illustrated in the following reaction scheme. It appears ahnost certain that the structures assigned to 1-D-glucosylurea (VIII) and 1-D-ghcosylthiourea (VII) are correct, and that the more precise nomenclature for these compounds is 1-p-D-glucopyranosylurea and 1-p-D-glucopyranosylthiourea, respectively. (There is less basis for certainty with regard to the corresponding D-glucosylguanidine.) In support of these structures are the following observations. 1. The glycosyl ureide and thioureide'78 a. obtained through the isocyanate and isothiocyanate routes were prepared from tetra-0-acetyl-a-Dglucopyranosyl bromide. The glycosyl ureides derived from this source are identical with those prepared by the acid-catalyzed condensation of D-glucose with urea17 and with thiourea. 2. Fischer30 converted D-glucopyranosylthiourea to 1-D-glucosylurea by treatment of the thioureide with an aqueous suspension of mercuric oxide. The product was identical with that of Schoorl.16 3. 1-D-Glucosylurea exhibits no mutarotation, and thus it retains a stable configuration. Since, in general, the nucleophilic replacement of the halogen atom in the poly-0-acylglycosyl halides occurs with inversion of configuration a t C l , it may be assumed that the ureide and thioureide of D-glucose are the p anomers. This conclusion is supported by optical rotation data (see Table 111). 4. Complete acetylation of 1-D-glucosylurea yields a pentaacetate. This tends to eliminate the possibility of an open-chain structure (as originally assigned by SchoorllB) which would require a hexaacetyl derivative. In (46) A. Hynd and M. G. Macfarlane, Biochem. J . , 20, 1264 (1926).

229

GLYCOSYL UREIDES

TABLEI1 Chemical Properties of 1 -D-Glucosylureaa Procedure

Test with Fehling solution Barfoed's testb Treatment with cold conc. HzS04 Phenylhydrazine Attempted salt formation

Reflux with 10% NaOH Reflux with 0.1 N HzSOt Water a t 25" to 50" Water (reflux) NaOBr a t 15" N203at 0" Brz and PbC03

HzOzplus Fe(OAc)3 N a amalgam Incubation with yeast Incubation with ureasec NaIO, a t 25°C. for 24 hoursd Acetic anhydride and ZnClze Acetic anhydride and pyridinee Benzoyl chloride and NaOH

Result

Solution reduced, but much more slowly than by free D-glucose Negative after boiling for 30 minutes Dissolves with slight elevation of temperature; no loss of NH3 or COz No osasone is formed (unless the mixture is heated long enough t o hydrolyze the ureide) No salts formed with HNO,, oxalic acid, opianic acid; no precipitates with Hg(NOa)z, P ~ ( C Z H Z O Zor) ZPb, (0H)OAc Dissociation, with liberation of NH3 and D-glucose Complete hydrolysis to D-glucose and urea No reaction Hydrolysis, with formation of N H 3 , CO, , and D-glucose Slow evolution of Nz Slow evolution of Nz Oxidation takes place, but products were not identified Oxidation takes place, but products were not identified No reduction, either in acid or base No reaction No reaction 5 moles of periodate consumed per mole Forms a pentaacetate Forms a tetraacetate A tetrabenzoate is formed

Barfoed's test involves the rea Data from Schoorl,16 unless otherwise noted. duction of cupric acetate. The reagent contains 7% of Cu(OAc)2 and 1% of acetic acid in HzO. This test allows the rapid determination of the presence of free reducing sugars as contaminants in glycosyl ureides; using 0.1 g. of ureide and 1 ml. of reagent, as little as 0.5% of contamination by D-glucose can be determined. c S. Bieber and I. Goodman, unpublished data. I. Goodman and F. B. Hayes, unpublished data. This reaction was repeated by Helferich and Kosche.*7 6

addition, the pyridine-catalyzed acetylation of 1-D-glucosylurea results in a tetraacetate ester (XLII) which may be converted either to a pentaacetate (XLIII) or to 1-(2,3,4,6-tetra-O-acetyl-~-glucosy~)-3-benzoy~urea (XLVII) (see pages 230 and 231).

lG

w

0

HCI

,D -glucose

NH3

+

H2N -GO-NH2

CH20 H H a : -

GS-NH2

HO

H

BZO

a

H

. - GS- NH - 0 2

HgO_

HQHH-CO-

NH-Bz

Bz 0 H

002

k

OAc

I

Ac20 C6H5N

BZCl %H5N

1 CHzOAc H

a H-GO-NH-BZ

AcO

A

oez

h

bAc

H

OAC

Xm

H

OAc

X m

232

IRVING GOODMAN

Further confirmation of the structures assigned is nevertheless desirable, especially for those ureides whose detailed chemical properties are only little known. Since the periodate-oxidation method (which has been used so effectively by Todd and coworkers47.48 in the determination of glycosyl ring structures) was of little value in this case (see Table 11), some other approach t o this problem, such as methylation followed by hydrolysis, should be investigated. The earliest attempt to apply the glycosyl ureides in the synthesis of N-glycosylpyrimidines was that of Helferich and Kosche,17 who treated (2,3,4 ,6-tetra-O-acetyl-~-glucosyl)urea with diethylmalonyl chloride in pyridine, and obtained a product believed49to be 1-(2 ,3 ,4,6-tetra-0-acetyl~-glucosyl)d, 5-diethylbarbituric acid (XLIX) (see page 231). Johnson and Bergmann7 with a similar objective in view, proceeded to the synthesis of 1-(2,3 ,4,6-tetra-O -acetyl-D -glucosyl) -3- (2-cyan0acetyl)urea. However, their efforts a t cyclization of this compound, either by the method of Traubes or of Rupe and coworkers,9 were unsuccessful. Goodmanlo repeated the synthesis of XLV and also prepared 1-(2 ,3 , 4 ,6-tetra-O-acetyl-~glucosyl)-3-(2-cyanopropionyl)urea.These were cyclized by a modification of Traube’s method and, upon deacetylation, yielded the synthetic nuand 6-amino-l-(~cleosides 6-amino-l-(~-glucosy1)-2,4-pyrimidinedione glucosyl)-5-methyl-2 ,4-pyrimidinedione (XLVIII), respectively (see page 231).

VI. GENERAL Whether the glycosyl ureides play a role in normal metabolic processes remains a challenging question. The fact that the chemical synthesis of pyrimidine nucleosides from glycosyl ureides has been demonstrated should serve to stimulate further investigation of this problem, particularly with the application of some of the more recent, sensitive techniques including radioactive tracers and paper chromatography.60 Although the current popularity of the nucleic acids and their derivatives tends to focus attention first in that direction, other promising avenues of biosynthesis involving the glycosyl ureides have been suggested. Neuberg (47) J. Davoll, B. Lythgoe and A. R. Todd, J. Chem. SOC.,833 (1946). (48) G. A. Howard, G. W. Kenner, B. Lythgoe and A. R. Todd, J. Chem. SOC., 801 (1946). (49) Unsuccessful attempts to obtain this barbiturate derivative by the method of Helferich and Kosche have been reported (P. Newmark, “Pyrimidine Structure as Related to Chemical and Biological Activity,” Ph. D . Thesis, Univ. of Colorado, 1952). (50) A chromatographic method for the study of 1-D-glucosylthiourea has recently been reported [K. Maekawa and K . Ishimoto, Nippon Kagaku Zasshi, 77, 999 (1956)l.

GLYCOSYL UREIDES

233

and Neimann’s suggestion4that ureido-wglucuronic acid might be a normal metabolite deserves re-examination. Micheel,61 in a very stimulating review, has described the wide occurrence of carbohydrate-linked proteiiis (glycoproteins) in Nature. The exact chemistry and structure of these compounds is virtually unknown, but it appears reasonable that the glycosylurea-, glycosylthiourea-, and glycosylguanidine-linked proteins synthesized by Micheel and might resemble the natural forms in some degree (see pp. 226 and 227). Further work on chemical structure and reactivity might well include an examination of the possibility of occurrence of an Amadori rearrangement in the case of the glycosyl ureides.K2In addition, studies of the tautomerism of glycosyl ureides under various conditionsK3of pH and of the neighboringgroup effect64would be of value in the interpretation of many of the seemingly unorthodox chemical reactions of these compounds. The pharmacological and biochemical aspects of the alkyl-, aryl-, and aralkyl-ureides, -thioureides, and -guanidines have been very extensively investigated, and hundreds of compounds of these series have been described in the literature. Many of them have been found to possess marked biological activity. They have found use, for example, as diuretic agents, analgesics, antidiabetic compounds, bacteriostatic agents, antithyroid drugs, and in many other ways. It seems reasonable to suppose that an exhaustive investigation of the glycosyl ureides and related compounds might result in the discovery of a wide range of biological activity in this realm.

VII. TABLES OF GLYCOSYL UREIDESAND RELATED COMPOUNDS The following Tables record the melting points and rotations of glycosyl ureides and related compounds. (51) F.Micheel, Angew. Chem., 69A, 212 (1947). (52) Although there has been no example of the Amadori rearrangement reported for glycosyl ureides, the reaction has been described for N-glucosyl derivatives of amino acids [J.E. Hodge and C. E. Rist, J. A m . Chem. SOC.,7 6 , 316 (1953)],and i t is possible that occurrence of this reaction might account for the low yields generally obtained in the acid-catalyzed synthesis of the glycosyl ureides. [For a review of the Amadori reaction, see J. E. Hodge, Advances i n Carbohydrate Chem., 10, 169 (1955)l. (53) C. A. Grob and B. Fischer, Helv. Chim. Acta, 38, 1794 (1955). (54) F. L.Scott, R. E. Click and S. Winstein, Experientia, 13,183 (1957).

234

IRVING GOODMAN

TABLEI11 Urea Derivatives of the Hexosesa Compound

Melting point, "C.

1-D-Galactosylurea amorph. 3-Dodecyl-l-~-galactosylurea 165-188 (dec.) l-D-Glucosylurea 208 (dec.) 1,3-Di-~-glucosylurea 235-245 l-~-Glucosyl-3,3-dimethylurea 157 (dec.) l-~-Glucosyl-3-octadecylurea 181-189 (dec.) l-~-Glucosyl-3-phenylurea 223 (dec.) l-D-Glucosyhrea ureide 171-172 1a-Glucosylureido-gelatin amorph. l-(Tetra-O-acetyl-D-glucosyl)urea 100 Tetra-O-acetyl-2-deoxy-2-ureido-~-190-191 glucose 1,3-Bis(tetra-O-acety1-~-glucosyl)- 164 urea 3-Acetyl-1- (tetra-0-acetyl-D-gluco- 200 sy1)urea l-~-Glucosyl-3-methylurea 215 (dec.) l-(Tetra-0-acetyl-~-glucosyl)-3- 211-212 benzoylurea l-(Tetra-0-acetyl-~-glucosyl)-3(2135 cyanoacety1)urea ~-(Tetra-0-acety~-~-glucosy~)-3(2179-180 cyano-2-nitrosoacety1)urea l-(Tetra-0-acetyl-n-glucosyl)-3110 (dec.) phen ylurea l-(Tetra-O-acetyl-~-glucosyl)-3-(4-114 carbethoxypheny1)urea 14Cb150 l,3-Bis (tetra-0-benzoyl-D-glucos yl) urea 1-(Tetra-0-benzoyl-D-glucosy1)urea 118 l-D-Mannosylurea 188 1-ureido-D-glucuronic acid amorph. Barium 1-ureido-D-glucuronate amorph.

:a]~ degrees .

+15.0 -23.5 +4.2 -33.0 -55.0 -18.2

-

-16.9 +24.5 -3.7

Rolalion solvent

references

16 29 1, 5, 16 17, 31 16 29 16, 38 5 34 16, 17 30a 31

-15.9

16, 17

-31.8 -29.7

16, 17 17

-

6, 7

-

7

-22.0

38

-27.0

35

+19.1

7, 17

-45.8 ca. -21 -15.8

16 16 4 4

See Tables of Compounds in Ellis and HoneymanI8 for data on glycosyl isocyanates and isothiocyanates, and glycosyl-urethans, -thiourethans, -hydantoins, -thiohydantoins, and related compounds.

235

GLYCOSYL UREIDES

TABLEIV Thiourea Derivatives of the Hexoses Compound

Rotation soloent

Melting goint. "C.

l-~-Glucosyl-2-thiourea 215-216 (dec.) 2-Ethyl-1- (~-glucosyl)-2-thioimpure pseudourea, hydrobromide 3- (4-Carbethoxyphenyl)-l-~-glu14&150 cosyl-2-thiourea l-o-Glucosyl-3-pheny1-2-thiourea 121 1-(~-Glucosyl)-2-methyl-2-thioimpure pseudourea.H&Oa 1-(Tetra-~-acetyl-~-glucosyl)-3175 (4-carbethoxyphenyl)-2-thiourea 1-(Tetra-O-acetyl-~-glucosyl)-2sirup methyl-3-(4-carbethoxyphenyl)2-thiopseudourea 1-(Tetra-0-acetyl-D-glucosyl)-3115-116 (4-carboxy-3-hydroxyphenyl) -2thiourea 1- (Tetra-O-acetyl-~-glucosyl)-3177-178 (4-carboxy-3-hydroxyphenyl)-2thiourea, methyl ester 1 - (Tetra-O-acetyl-~-glucosyl)-3135 phenyl-2-thiourea 3-Benzoyl-1- (tetra-0-benzoyl-D205 ylucosyl)-2-thiourea

References

-35.7 -

6, 17, 40 40

-44

35, 40

-

38, 51 40

-30

35

-47

35

-29

35

-32

35

-

37, 38

+45.0

17

TABLEV Urea Derivatives of the Pentoses Deriualive of Urea

Melling point, "C.

KID, degrees

Rotation soloenl

References

HzO HzO H2 0 CsHsN

55 7, 17 17 17

~1 - D - Arabinosyl-

1-L-Arabinosyl1,3-Di-~-arabinosyl1,3-Bis(hexa-0-benzoyl-L-arabinoSY1)1-(Tri-0-acetyl-D-arnbinosyl) 1-(Tri-0-acetyl-L-arabinosy1)l-(Tri-O-acetyl-D-arabinosyl)-3-(2cyanoacety1)-

185-187 192 ca. 227 260-261 (dec.)

-48.0 +51.5 $62.0 +163.0

215 (dec.) 210-212 (dec.) 108

-53.0 +45.9 -

(55) I . Goodman and L. Salce, unpublished data.

C ~ H K N 55 17 CKHhN

-

55

236

IRVING GOODMAN

TABLE VI G1ycos ylguanidine Derivatives Compound

Melting Poinl, "C.

1-n-Fructosylguanidine

90

1-u-Glucosylguanidine

94 (dec.)

l-n-Ghcosylguanidine. HBr 108-109 N- (D-Glucosylguanyl)glycine 208 (dec.) N-[N- (~-Glucosylguanyl) glycyl]180 (dec.) glycine N , N-Bis(D-glucosylguanyl) -Lhistidine.0.5 HzSO, N*,NE-Bis(D-glucosylguanyl)-DL178 (dec.) lysine 6-(l-~-Glucosylguanidino)caproic 164-166 (dec.) acid l-~-Glucosyl-3-methylguanidine 169-170 (dec.) I-D-Mannosylguanidine 80 (dec.)

[do,degrees

-50.6 -4.3 +32.0 -0.6 -62.4

References

-+

H2O

43

-+

H2

26,43

H2

-

-

0

41,43 40 40

0

-

40 40 -66.9

H a0

41

-78.0

HzO

41 43

f10.0 +

-0.7 Methyl 4,6-0-benzylidene-2-deoxy-2-guanidino-a-~-altroside Disodium [ N ,N'-bis (D-glucosylguanyl) -L-cystinate] N-(Tetra-o-acetyl-D-glucosyl) N' ,N"-bis (4-carbethoxyphenyl)guanidine N- (Tetra-~-acetyl-D-glucosyl) -N'(4-~arbethoxyphenyl)-N"-[o, L2- (carbomethoxy)ethyl]guanidine N- (Tetra-0-acetyl-D-glucosyl) N'-(4-~nrbethoxyphenyl)-N"methylguanidine .HC1 N- (Tetra-0-acetyl-D-glucosyl)N'- (4-cnrbethoxyphenyl)-N"phenylguanidine

Rotation solvent

HtO

14b147

44

220 (dec.)

40 -42

CHaOH

35

+7

CHaOH

35

-24

CHaOH

35

-54

CHaOH

35

TABLEV I I Lactos ylurea Compound

Lactosylurea

1

I

Melting point, "C.

230-240 (dec.)

I

I

[a]D,

degrees

+2.1

I

1

Rotation solocnl

HzO

1

I

Refcremes

16, 46

THE NONULOSAMINIC ACIDS Neuraminic Acids and Related Compounds (Sialic Acids)

BY F. ZILLIKEN AND M. W. WHITEHOUSE Department of Biochemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Note on Nomenclature., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Occurrence and Distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. I n Mucolipids.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. I n Mucoproteins.. . . .......................................... 3. In Oligosaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Detection and Determination. . . . . . . . . . . . . . . . . . 1. Color Reactions.. . . . . . . . . . . . a. Direct Ehrlich Reaction., ........................ b. Bial's Orcinol Reaction., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . e . Diphenylamine Reaction. . . . . . .............................. d. Tryptophan-Perchloric Acid Re . 2. Paper Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . ................ 3. Histochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Isolation and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Methoxyneuraminic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. N-Acetylneuraminic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' ' ' ' ' ' ' ' 3. N-Glycolylneuraminic Acid (Porcine Sialic Acid). . . 4. N-Acetyl-0-acetylneuraminicAcids (Bovine and Equ Sialic Acids). V. Chemistry and Structure,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Derivation of Empirical Formulas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Degradation, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Stereochemistry. ................................ ................... 4. Synthesis., , . , , , VI. Neuraminolactose.. . . . . . . ................... 1. Isolation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Structure.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Biochemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1. Biosynthesis. . . . . . . . . . . . ...................................... 2. Interaction between Viruses and Mucoproteins; "Neuraminidase". . . . 3. Enzymic Degradation. .................... VIII. Conclusion. . . . . . . . . . . . . . . ................................. "

"

'

~

"

"

"

"

~

'

~

"

"

'

"

"

'

241 24.4

246 247 247 248 248 248 250 250 251 251 252 252 252

258 259 259 259 263

I. INTRODUCTION Although the aminohexoses D-glucosamine (2-amino-2-deoxy-~-glucose) and D-galactosamine (2-amino-2-deoxy-~-galactose)have been known for 237

238

F. ZILLIKEN AND M. W. WHITEHOUSE

many years as components of mucopolysaccharides and mu~oproteins,1-~ and, more recently, aminopentoses4 and a number of other aminohexoses6 have been isolated from antibiotics, only within the last decade has it been realized that there also exists in Nature a family of amino sugars containing 9 or more carbon atoms. The members of this group, neuraminic acid and its higher acylated derivatives, the so-called “sialic acids,” are found widely distributed throughout the animal kingdom in mucopolysaccharides, mucoproteins, and lipopolysaccharides (muco1ipids)-very often in association with D-glucosamine and D-galactosamine. Generically, they may be designated nonulosaminic acids; or, more precisely, amino-carboxy-deoxynonuloses, since they possess a keto group in addition t o a n amino and a carboxyl group. By virtue of the latter, they might also be described as polyhydroxy &amino acids. Herein lies one source of interest in these compounds, for, theoretically a t least, they are able to enter into combination with other sugars and amino acids through both glycosidic and peptide bonds, thus affording a chemical bridge between polypeptides and polysaccharides. Some such union between these biopolymers must be present in mucoproteins and in those mucopolysaccharides which contain a peptide moiety. It is, perhaps, significant that the nonulosaminic acids are found intimately associated with hexoses and amino acids in a large number of mucoproteins of biological interest. The first isolation of a compound of this type was made in 1900 by who described a product “paramucosin” with the composition C12H23N010 , obtained by brief acid hydrolysis of a mucosubstance from ovarian cysts.7 He was able to characterize paramucosin by its reducing properties and its ease of humin formation with acids, and he suggested that it represented an amino sugar bound to a “reduced” sugar acid. Subsequently, Levene and coworkers89 and Walz10 reported the presence, in (1) M. Stacey, Advances in Carbohydrate Chem., 2, 161 (1946). (2) A. B. Foster and M. Stacey, Advances i n Carbohydrate Chem., 7 , 247 (1952). (3) P. W. Kent and M. W. Whitehouse, “Biochemistry of the Aminosugars,” Academic Press Inc., New York, N. Y., 1955. (4) B. R. Baker and R. E. Schaub, J . Am. Chek. Sac., 76, 3864 (1953). (5) E. H. Flynn, M. V. Sigal, Jr., P. F. Wiley and K . Gerzon, J . A m . Chem. SOC., 76, 3121 (1954) ; E. E. van Tamelen, J. R. Dyer, H. E. Carter, J. V. Pierce and E. E. Daniels, ibid., 78,4817 (1956); H. Brockmann, H . B. KBnig and R. Oster, Chem. Ber., 87, 856 (1954); H . Brockmann and E. Spohler, Naturwissenschaften, 43, 154 (1955); F. A. Hochstein and P. P. Regna, J . A m . Chem. Sac., 77,3353 (1955). (6) J. B. Leathes, Arch. exptl. Pathol. Pharmakol., Naunyn-Schmiedeberg’s, 43, 245 (1900). (7) K. Mitjukoff, Arch. GynakoZ., 49, 278 (1895). (8) J. A. Mandel and P. A. Levene, 2. physiol. Chem., Hoppe-Seyler’s, 46, 386 (1905). (9) P. A. Levene and K. Landsteiner, J . Biol. Chem., 76, 607 (1927). (10) W. Walz, 2.physiol. Chem., Hoppe-Seyler’s, 166, 210 (1927).

THE NONULOSAMINIC ACIDS

239

the lipid fraction of liver, spleen, kidney, and brain, of a compound which gave a characteristic, purple color-reaction with orcinol and hydrochloric acid (Bial’s reagent). I n 1936, Blix” was able to isolate a crystalline, reducing acid, the socalled “acid carbohydrate I,” from the mucoproteins of the bovine submaxillary gland; i t had a molecular composition approximating to C12HzoN09 to C14HNNOll. The crystals gave a color reaction directly upon addition of an acidic solution of the Ehrlich reagent (p-dimethylaminobenzaldehyde), and readily formed humin with mineral acids. Further analysis indicated the presence of two acetyl groups in the molecule, whilst the failure to give a ninhydrin reaction indicated that the nitrogen was present in some form other than as a primary amino group. In the course of studies on the chemical composition of lipids in certain pathological conditions (“lipidoses”), Klenk noted the same type of substance in the brain of a n infantile, amaurotic idiot. Following methanolysis of the brain gangliosides, he isolated in 1941 a crystalline monomethoxy derivative of this compound and designated it neuraminic acid.12 In addition to giving a purple, Bial color-reaction, this acid gave a brilliant, red coloration with the Ehrlich reagent and had a composition approximating to CloHlgNOs or CllH21N09 . The presence of a free amino group and a readiness t o form humin with dilute mineral acids were also noted. A little later, Blix13 l4 called attention to the close similarity in certain qualitative reactions between his product from submaxillary mucin and the acid component of brain gangliosides. This view was further supported by the isolation of methoxyneuraminic acid from a methanolyzate of submaxillary mucin.16 Subsequently, Blix and his coworkersls termed their product from submaxillary mucin “sialic acid” (Greek, a t d o s = spittle) and further characterized it by observing (a) the liberation of carbon dioxide on heating with 12 % hydrochloric acid and (b) the development of a purple coloration with Bial’s reagent. They pointed out that neuraminic acid might result from deacetylation of sialic acid during the isolation procedure employed by Klenk, for they found that the sialic acid as isolated from bovine submaxillary mucin contains both a N-acetyl and a labile 0-acetyl grouping, whereas neuraminic acid has no acetyl groups. Employing a milder isolation procedure, Klenk and Faillard” were then able to obtain, directly from the 8

(11) G. Blix, 2. physiol. Chem., Hoppe-Seyler’s, 240, 43 (1936). (12) E. Klenk, 2. physiol. Chem., Hoppe-Seyler’s, 268, 50 (1941); 273, 76 (1942). (13) G. Blix, Skand. Arch. Physiol., 80,46 (1938). (14) G . Blix, L. Svennerholm and I. Werner, Acta Chem. Scand., 4, 717 (1950). (16) E. Klenk and K. Lauenstein, 2. physiol. Chem., Hoppe-Seyler’s, 291, 147 (1952). (16) G. Blix, L. Svennerholm and I. Werner, Acta Chem. Scand., 6 , 358 (1952). (17) E. Klenk and H. Faillard, 2.physiol. Chem., Hoppe-Seyler’s, 298, 230 (1954).

240

F. ZILLIKEN AND M. W. WHITEHOUSE

mucin, N-acetylneuraminic acid which, upon treatment with methanolic hydrogen chloride, yielded the characteristic methoxy derivative of neuraminic acid. Subsequently, a number of other compounds isolated from natural products were related to neuraminic acid by formation, from them, of its monomethoxy derivative. These included prehemataminic acid from erythrocytes,l*gynaminic acid from human milklQand meconium,2°lactaminic acid TABLEI Relationship . of . the Various Nonulosaminic Acids to Neuraminic Acid Trivial name

Neuraminic acid methoxyPrehemataminic acid Hemataminic acid Gynaminic acid Lactaminic acid methoxySerolactaminic acid methoxyOvine sialic acid Porcine sialic acid Equine sialic acid Bovine sialic acid

1

~

Relation to neumminic acid

-

methyl glycoside

Molecular composition

Melting point, “C.

-

-55.0

ca. 200 (dec.)

Refermces

15,23 18

identical methyl glycoside N-acetyl deriv. N-acetyl deriv. methyl ester of N acetyl deriv. N-acetyl deriv.

-54.2 -32.0 -32.0 -35

176-178 181 183-185

18 20 22 21

methyl glycoside (?) of N-acetyl deriv. N-acetyl deriv. N-glycolyl deriv. N-acetyl-0-acetyl deriv. N-acetyl-0-acetyl deriv.

-20

183-185

24

-31 -32 -59

185-187 185-187 183-187

23 23 23

+8

134-137

23

from cow colostrUm,21~ a2 the “sialic acids’) from the submaxillary mucins of a number of animal speciesz3[differing from bovine sialic acid in compo(18) T. Yamakawa and S. Suauki, J . Biochem. (Tokyo), 38, 199 (1951);Chem. Abstracts, 46,9085 (1951);ibid., 39, 175 (1952);Chem. Abstracts, 46,10213 (1952). (19) J. R. E. Hoover, G. A. Braun and P. Gyljrgy, Arch. Biochem. Biophys., 47, 216 (1953). (20) F. Zilliken, G. A . Braun and P. Gyijrgy, Arch. Biochem. Biophys., 64, 564 (1955);63, 394 (1956). (21) R.Kuhn, R.Brossmer and W. Schula, Chem. Ber., 87, 123 (1954). (22) R.Kuhn and R. Brossmer, Chem. Ber., 89, 2013 (1956);Angew. Chem., 68, 211 (1956). (23) G.Blix, E.Lindberg, L. Odin and I.Werner, Nature, 176,340 (1955); A d a SOC. Med. Upsaliensis, 61, 1 (1956).

T H E NONULOSAMINIC ACIDS

24 1

sition and physical properties (notably x-ray powder diffraction patterns)], and serolactaminic acidz4 from serum mucoproteins. The relationship of these various nonulosaminic acids t o neuraminic acid is indicated in Table I. 1 . Note on Nomenclature

I n the literature, there has been much confusion concerning the nomenclature of these nonulosaminic acids. Thus, the name neuraminic acid was first given to the monomethoxy compound, isolated after the methanolysis of gangliosides, and was then subsequently transferred to the methoxyl-free, parent acid.12 It should here be noted that neuraminic acid has not been isolated and characterized as such. The name “sialic acid” has been given to the several nonulosaminic acids isolated from the submaxillary mucoproteins, although the chemical composition of these varies with the species source.23Svennerholm26has proposed that the name sialic acid be restricted to the basic structure common t o all these various “sialic acids”; thus ovine “sialic acid” would be designated N-acetylsialic acid. However since this basal component, here termed sialic acid, is identical with neuraminic acid, it would seem more appropriate to adopt the name neuraminic acid for the parent acid and to retain the name “sialic acid” t o denote the various mono- and di-acyl neuraminic acids of iiatural occurrence in mucoproteins, regardless of their source. This is the convention which has been used in referring to the various nonulosaminic acids in Table I and thoughout this article.26a

11. OCCURRENCE AND DISTRIBUTION Like other naturally occurring amino sugars, the nonulosaminic acids are not present in Nature in the free state to any appreciable extent. They are found, together with hexoses, combined in mucolipids and mucoproteins from animal sources. These mucosubstances may also contain an appreciable content of u-glucosamine and D-galactosamine. Several investigators have observed that, in many instances, it has been possible to isolate only a small fraction of these 2-amino-2-deoxyhexoses from tissues and other sources for which a far larger content of aminodeoxy sugars had been indicated by the Elson-Morgan color reaction.26This discrepancy may be due, in part at least, to the presence in these sources of one or more of the nonulosaminic acids. These would react with the Ehrlich reagent regularly employed in the aminodeoxyhexose determination3 (see Section 111) and give a (24) T. Yamakawa and S. Suzuki, J . Biochem. (Tokyo), 43, 727 (1955). (25) L. Svennerholm, Actu Soc. Med. Upsaliensis, 61, 75 (1956). (25a) This nomenclature has been independently recommended for general adoption. See (F.) G. Blix, A. Gottschalk and E. Klenk, Nature, 179, 1088 (1957). (26) See Ref. 3, p p . 181 ff.

242

p. ZILLIKaN AND i d W. WHITEHOUSE

measure of the total aminodeoxy sugar content; this could result in falee values for the aminodeoxyhexoses. The many reports concerning the distribution of the latter, based entirely on the Elson-Morgan color reaction, should be reinvestigated regarding the possible co-occurrence of a nonulosaminic acid. The acids are also constituents of certain oligosaccharides in milk. A dialyzable form of a nonulosaminic acid has been detected in cerebrospinal fluid2’; this compound may be such an oligosaccharide. The distribution of the acids does not appear to be confined solely to the animal kingdom, for Barry and Goebe128have reported the isolation of colominic acid, a polymerized form of N-acetylneuraminic acid, from culture filtrates of Escherichiu coli. It remains to be established whether other bacteria and microorganisms possess the capacity to synthesize and utilize these acids. It is perhaps worth noting that D-galactose has been found in all those mucosubstances and oligosaccharides of animal origin which contain nonulosaminic acids. 1. In Mucolip‘ds

Nonulosaminic acids are found as constituents of certain water-soluble, lipid fractions (glycolipids) of animal tissues, being particularly associated with gangliosides as well as with more complex lipopolysaccharides. The detection and characterization of the nonulosaminic acid component in these mucolipids has been based upon one or more of the color reactions (see below) applied to the macromolecular material, followed by isolation therefrom of methoxyneuraminic acid after methanolysis. The question of whether or not one or more acylated forms of neuraminic acid are actually 29 has present in the mucolipid has still to be resolved. SvennerholmZ6~ reported the isolation of N-acetylneuraminic acid from brain gangliosides. Gangliosides (see Fig. 1) are sphingolipids, closely related to the cerebrosides, but distinguished from the latter by their content of glycosidically bound nonulosaminic acid@). Originally, they were so named on account of their relative abundance in the ganglion cells of the nervous system,12but gangliosides are found in most parenchymatous organs (for example, spleen and liver). In some pathological conditions, the so-called “lipidoses” (for example, Tay-Sachs and Niemann-Pick diseases), the nerve cells and other tissues may be considerably distended by a high content of ganglio(27) L. L. Uzman and M. K. Rumley, Proc. SOC.Ezptl. Biol. Med., 93, 497 (1956). (28) G. T. Barry and W. F. Goebel, Nature, 179, 206 (1957); G. T. Barry, J. Exptl. Med., 107, 507 (1958). (29) L. Svennerholm, Nature, 177, 524 (1956); Acta Chem. Scand., 10, 694 (1956).

243

THE NONULOSAMINIC ACIDS

side material .30 These diseases thus represent a disorder of sphingolipid metabolism, resulting in an enhanced production of the nonulosaminic acids. F a t t y acid

nonulosaminic acid

I

I

sphingosine- hexose-hexose-aminohexose

FIG.1.-Structure

of a Ganglioside.

Those of “rain have been the gangliosides most intensivs&jinvestigated to date.12*14, 2 g , 31-33 S ~ e n n e r h o l mhas ~ ~ separated, by chromatography, a t least three such ganglioside fractions from human brain. In a parallel study on ox-brain lipids, Rosenberg and C h ~ g a f fhave ~ ~ isolated, in addition to simple gangliosides, a complex mucolipid which contains a nonulosaminic acid and a polypeptide moiety. Gangliosides are found together with a cerebroside in horse erythrocytes, but erythrocytes from man and cattle TABLEI1 Nonulosaminic Acid Content of Some Mucolipids Material

Crystalline ganglioside Ganglioside Mucolipid Ganglioside containing amino sugar Amino sugar-free ganglioside Ganglioside

Source

human brain ox brain ox brain horse erythrocytes horse erythrocytes bovine spleen

%‘

Pefevences

24.1 24 26 15 24 20

29 32 34 35 35 36

* Measured as neuraminic acid or sialic acid. contain cerebrosides This observation is of interest, indicating a relationship between the occurrence and species source of a nonulosaminic acid and suggesting that the comparative biochemistry of these acids may well have a greater compass than the pathological implications associated with the lipidoses. (30) E. Klenk, “Biochemistry of the Developing Nervous System,’’ H. Waelsch, ed., Academic Press Inc., New York, N. Y., 1955, p. 397. (31) G. Biix and L. Odin, Acta Chem. Scand., 9, 1541 (1955). (32) A. Rosenberg, C. Howe and E. Chargaff,Nature, 177, 234 (1956). (33) C. Chatagnon and P. Chatagnon, Compt. rend. S O C . biol., 147, 1992 (1953). (34) A. Rosenberg and E. Chargaff, Biochim. et Biophys. Acta, 21, 588 (1956). (35) E. Klenk and K. Lauenutein, 2. physiol. Chem., Hoppe-Seyler’s, 296, 164 (1953). (36) E. Klenk and F. Renkamp, Z . physiol. Chem., Hoppe-Seyler’s, 273, 253 (1942).

244

F. ZILLIKEN AND M. W. WHITEHOUSE

2. I n Mucoproteins Present in animal secretions are viscosity-raising agents, amongst which is a class of mucoproteins containing appreciable proportions of one or more of the nonulosaminic (sialic) acids. These have been designated sialoproteins,3~for the convenience of distinction from other glycoproteins. Sialoproteins are widely distributed in the secretions of higher animals: tears38; urinel6’38-40; the epithelial mucins of the bronchial, gastro-intestinal, and genital tracts3?,41; allantoic,98 cyst,4l and cerebrospinal fluids2’*40; exudates40;milk1@‘ 40; and blood serum.20,40-4E Other sources of sialoproteins 41 colostrum,21 2 2 , erythrocyte^,^^ include liver,47ov~ mu cin ,~ meconium,2°~ * and nervous tissue.29These sialoproteins (see Table 111) commonly contain D-mannose and L-fucose in addition to D-galactose, D-galactosamine, and D-glucosamine, which latter hexose derivatives are also found in gangliosides. I n some, if not all of these sialoproteins, the nature of the nonulosaminic acid appears t o vary with the species. The mucin extracted from submaxillary glands is a common source for isolation of the Human saliva affords N-acetylneuraminic acid,63 which is also present in human milk, human-brain gangliosides, and human-serum proteins. Analysis of the component serum albumins and globulins indicates that iionulosaminic acids are associated only with the globulin fractions,4°*44 the cul-acid glycoprotein ( o r o s o m ~ c o i d )having ~~ the highest content (10 %). The low isoelectric point of this sialoprotein is attributable to the nonulosaminic acid content.42* 64 In pathological states in humans, the values for nonulosaminic acid in the serum are significantly increased40- 46 from a normal value of about 65 mg. per 100 ml. I n other animals, the @

(37) I. Werner, Acta SOC.Med. Upsaliensis, 68, 1 (1953). (38) L. Odin, Nature, 170,663 (1952). (39) A. J. Anderson and N. F. Maclagan, Biochem. J . , 69, 638 (1955). (40) P. Bbhm and L. Baumeister, 2. physiol. Chem., Hoppe-Seyler’s, 306, 42 (1956). (41) L. Odin, Acta Chem. Scand., 9, 714, 862, 1235 (1955). (42) L. Odin and I. Werner, Acta SOC.Med. Upsaliensis, 67, 227 (1952). (43) C. Chatagnon and P. Chatagnon, Compt. rend. SOC.biol., 148, 1226 (1954). (44) L. L. Uzman and H. Rosen, Science, 120,1031 (1954). (45) P. Bbhm and L. Baumeister, 2. physiol. Chem., Hoppe-Seyler’s, 900,153 (1955). (46) R. J. Winder, Methods of Biochem. Anal., 2, 279 (1955). (47) A. Martinsson, A. Raal and L. Svennerholm, Biochim. et Biophys. Acta, 23. 652 (1957). (48) E. Klenk and G. Uhlenbruck, 2. physiol. Chem., Hoppe-Seyler’s, 306,224 (1956). (49) E. Klenk and W. Stoffel, 2. physiol. Chem., Hoppe-Seyler’s, 303, 78 (1956). (50) J . Cravioto-Munoz, B. Johansson and L. Svennerholm, Acta Chem. Scand., 9, 1033 (1955). (51) G. B. J. Glass, M. Rich and L. Stephanson, Federation Proc., 16, 46 (1957).

245

THE NONULOSAMINIC ACIDS

normal serum values vary, according to species, from 44 mg. per 100 ml. in rabbits to 94 mg. per 100 ml. in mice.40

3. I n Oligosaccharides Milk contains a number of oligosaccharides, including some which contain a nonulosaminic acid glycosidically linked. These are discussed TABLEI11 Nonulosaminic Acid Content of Some Sialoproteins Sialoprulein source

Ovomucin Casein Cervical mucin Seminal mucin Ovarian cyst fluids normal pseudomyxamatous Submaxillary mucins

SPecier

%’

hen egg human cow human

6.0 0.75 0.45 7.4 12.0 14.7

hog

Meconium Colostrum Gastric mucin Tracheobronchial mucus Nasal mucus Orosomucoid (plasma)

human human bovine ovine porcine equine human cattle human human human human

Fetuin Brain

calf human

2 10 10-15 10-15 10-15, 2 5-7, 2.2 5.0 2.9 1.5, 4.3 3.8 2.4-4.4 9.1 10.6 14.6 6 .O 0.9

References

41 50 50 41 37 41 41 41 23 23 23, 48 23, 48 41 48 51, 37 37 37 40 41 51 52 29

* As sialic or neuraminic acidfi. further in Section VI. Human milk has a much higher content of these oligosaccharides and sialoproteins than has cow’s milk.20-40 The content of nonulosaminic acid in the colostrum of a species is even higher than that of the regular 22 (52) E. Klenk and W. Stoffel, Z . physiol. Chem., Hoppe-Seyler’s, 302, 286 (1955). (53) L. Svennerholm, Biochem. J . , 64, 11P (1956). (54) E. A. Popenoe and R. M. Drew, Federation Proc., 16, 233 (1957).

246

F. ZILLIKEN AND M. W. WHITEHOUSE

111. DETECTIONAND DETERMINATION 1. Color Reactions

Qualitatively, the presence of the nonulosaminic acids is best indicated by the brilliant-red coloration formed on addition of an acidic solution of p-dimethylaminobenzaldehyde (the so-called “direct Ehrlich reaction”), and by the bright-purple coloration which develops upon boiling with Bial’s reagent for several minutes a t 100’. I n addition, the nonulosaminic acids give a blue-violet coloration with Dische’s diphenylamine reagents6 for deoxypentoses and a positive reaction in the tryptophan-perchloric acid test.66Since no single one of these color reactions is absolutely specific for a nonulosaminic acid, it is advisable to carry out at least two of these for a qualitative analysis. For quantitative determinations, all four reactions have been employed, using either N-acetylneuraminic acid (m. p., 183-185”; [a]:’ -32.0’) or methoxyneuraminic acid [m. p., 200’ (dec.); [a]:5- 55.0’1 as colorimetric standards. a. Direct Ehrlich Reaction.-Blix was the first to note that his “acid carbohydrate I” (sialic acid) gives a purple color with an acidic solution of Ehrlich’s reagent.” Odin later recognized3*that a number of mucoproteins also give this coloration, which he attributed to the presence of sialic acid. Subsequently, Werner and Odin6’ elaborated a quantitative determination for nonulosaminic acids on this basis. Under standard conditions, a 5% solution of p-dimethylaminobenzaldehyde in mineral acid is added to the sample, and the mixture is heated for 30 min. a t 100”. The developed coloration, with Am, a t 530 and 565 mp, is then read a t 565 mp; i t is stable for several hours. The method will give incorrect results in the presence either of pyrrole derivatives or of alkali-treated mucopolysaccharides.

As regards the “direct Ehrlich chromogens,” a distinction must be made between those which give this color reaction immediately upon addition of the Ehrlich aldehyde and those which exhibit this reaction only after heating with the reagent for 2-30 minutes at 100’. The first group is formed from N-substituted 2-amino-2-deoxy sugars by treatment with alkali a t elevated temperature^.^ Chemically, they are derivatives of S-acetamidof ~ r a n ,6o~ whereas ~, the slower forming chromogens are derived from the nonulosaminic acids. The chemical structures of these latter chromogens are not known. (55) (56) (57) (58) (59) (60)

Z. Dische, Mikrochemie, 8, 4 (1930). S. S. Cohen, J . B i d . Chem., 166, 691 (1944). I. Werner and L. Odin, Acta SOC.Med. Upsaliensis, 67, 230 (1952). W. T. J. Morgan and L. A. Elson, Biochem. J . , 28,988 (1934). R. Kuhn and G. Kruger, Chem. Ber., 89, 1473 (1956) ; 90,264 (1957). F. Zilliken and E. Stevenson, Arch. Biochem. Biophys., 67, 243 (1957).

THE NONULOSAMINIC ACIDS

247

b. Bial’s Orcinol Reaction.-Klenk and Langerbeins first applied Bial’s orcinol reagent6’ to a quantitative determination of the neuraminic acid in nerve tissue and other biological secretions,62 and studied some of the factors influencing the formation of color. The method was slightly modified by Werner and Odin,67inasmuch as corrections were made for interference by several, naturally occurring carbohydrates. Whereas the original procedure required heating of the mixture for 5 min. a t 142”, Bohm and his coworkers63 used lower temperatures and corrected for a possible error caused by the presence of accompanying aldohexoses. Since aldopentoses develop a blue-green coloration with A,, a t 670 mp, they may seriously interfere with the determination of the nonulosaminic acids. Furthermore, the presence of ketopentoses or 2-furaldehyde (which develop colorations with an additional A, at 540 mp, and a t 570 and 670 mp, respectively) may also interfere with the determination. Fructose and sorbose in amounts less than 0.1 mg. yield a red-violet color which is practically indistinguishable from that given by a nonulosaminic acid.64 Thus, a violet coloration with Bial’s reagent, even though it exhibits a A,, at 570 m p , is not of itself proof of the presence of a nonulosaminic acid. The same holds true for the direct Ehrlich reaction. As the result of a very thorough study, Svennerholm26, 64 has considerably revised the method. The major modifications lie in the application of resorcinol, ferric ammonium sulfate, a heating time of 15 minutes a t 1 0 6 O , and a differential reading of the coloration at 575 and at 680 mp. This procedure has given highly accurate, reproducible results in the writers’ laboratory, when N-acetylneuraminic acid and methoxyneuraminic acid have been used as standards. c. Diphenylamine Reaction.-In applying Dische’s diphenylamine reagents6 to the determination of deoxypentoses in serum, Niazi and Statee6 noted that a purple color developed. Werner and Odin42,67 demonstrated that the ultraviolet absorption spectrum of the solution obtained from sialic acid in this reaction was identical with that from the reaction with serum and other mucoproteins. Subsequently, the method was used by Ayala and coworkers66 and has been intensively investigated by Coburn and coworker^.^^-^^ With slight modifications, Winzler has employed the (61) H. Bial, Deut. med. Wochschr., 28, 253 (1902). (62) E. Klenk and H. Langerbeins, Z. physiol. Chem., Hoppe-Seyler’s, 270, 185 (1941). (63) P. Bohm, S. Daubner and L. Baumeister, Klin. Wochschr., 32,289 (1954). (64) L. Svennerholm, Arkiv Kemi, 10, 557 (1956). (65) 5. Niazi and D . State, Cancer Research, 8, 653 (1948). (66) W. Ayala, L. V. Moore and E. L. Hess, J . Clin. Invest., 30, 781 (1951). (67) A. F. Coburn and J. Haninger, Trans. Assoc. Am. Physicians, 66, 308 (1953). (68) A . F. Coburn and J. Haninger, J. Exptl. Med., 99, 1 (1954).

248

F. ZILLIKEN AND M. W. WHITEHOUSE

method for a number of seromucoprotein~~~ and for the determination of “sialic acid” in normal and pathological sera. Orosom~coid7~~ 72 and sialic acid have been used as a standard in these serum determinations. d. Tryptophan-Perchloric Acid Reaction.- Odin and Werner& found that bovine sialic acid gives a red-brown coloration in the tryptophan-perchloric acid reaction described by Cohen for deoxypentoses,66and that the resulting ultraviolet absorption spectrum is very similar to that obtained” on applying this reagent to whole serum. The method has been employed by Winz1er7O for the determination of “sialic acid” in biological specimens. There have been a number of reports on the elevation of the tryptophan-perchloric acid chromogen in the serum of patients having a variety of pathological conditions,’O but whether this is due solely to an increase in the content of nonulosaminic acid remains to be determined. 2 . Paper Chromatography The paper-chromatographic analysis of the nonulosaminic acids has been hampered by their lability toward acid and alkali. This holds particularly for the higher acylated sialic acids. The mobilities of N-acetyl-O-acetylneuraminic acid, N-acetylneuraminic acid and its methyl ester, and N-glycolyl- and methoxy-neuraminic acid are given in Table IV. For their detection, the papers may be sprayed with reagents commonly used for the detection of keto and aininodeoxy Those of particular value include the direct Ehrlich reaction, the orcinol-trichloroacetic acid the chlorine-benzidine reagent for the detection of -NH-COgroupings,’6. ’7 and the Bial reaction as modified by Bohm und Baumeister.?* The limit of detection with the orcinol-trichloroacetic acid reagent is reported to be 5 bg. of N-acetylneuraminic acid.26 For the detection of methoxyneuraminic acid, ninhydrin may also be used. 3 . Histochemistry

The Bial color reaction has been utilized for the detection of nonulosaminic acids in defatted histological sections. This technique has (69) A. F. Coburn, L. V. Moore and J . Haninger, A.M.A. Arch. Internal Med., 92, 185 (1953). (70) R. J. Winder, Methods of Biochem. Anal., 2 , 297 (1955). (71) K. Schmid, J . Am. Chem. SOC.,72,2816 (1950). (72) H. E. Weimer, J. Mehl and R. J. Winder, J . Biol. Chem., 186, 561 (19501. (73) F. B. Seibert, M. L. Pfaff and M. V. Seibert, Arch. Biochem., 18, 279 (1948). (74) R. J. Block, E. L. Durrum and G. A. Zweig, “Manual of Paper Chromatography and Paper Electrophoresis,’’ Academic Press Inc., New York, 1955. (75) R. Klevstrand and A . Nordal, Acta Chem. Scand., 4, 1320 (1950). (76) H. N. Rydon and P. W. G. Smith, Nature, 169, 922 (1952). (77) S. C. Pan and J. D. Dutcher, Anal. Chem., 28, 836 (1956). (78) P. BBhm and L. Baumeister, Z . physiol. Chem., Hoppe-Seyler’s, SOO, 153 (1955).

TABLEIV Mobilities of the Nonulosaminie Acidp N-O-

Di-tyL, Soled r a w

1-Butanol-acetic acid-water 1-Butanol-pyridine-water 1-Butanol-pyridinewater 2-Butanol-acetoneacetic acid-water 2-Butanol-acetic acid-water Ethyl acetate-pyridine-water Ethyl acetate-acetic acid-water Ethyl acetate-pyridine-acetic acid-water

4:1:5 9:5:8 3:2:1.5 3:3:1.5:2.5 4:1:5 2:1:2 3:1:3 5:5:1:3

N-Acefylneuraminic acid

RF

RF

RF

RF

0.45 0.32 -

0.09 0.16 0.20 0.27 0.58; 0.66 0.16 0.15 Rlact 0.75b

0.39 0.59

0.19

acid

0.25 0.33

N-A cefylncuramin;c methyl eder

NGlywly!ncIIramtnlcMethoxynmrraminic N-AcetY1nmramiwacid acid

ncuramrnrc

lactose

-

0.14 0.64; 0.69 0.75; 0.85 -

-

0.14 0.97 ~~

0

Compiled from references 20, 22, 23, 25, and 78.

Rlset

refers t o mobilities relative to lactose.

RF

Rlact

250

F. ZILLIKEN AND M. W. WHITEHOUSE

revealed a wide distribution of gangliosides and sialoproteins in animal tissues, including muscle and hyaline connective tissue?g

IV. ISOLATION AND CHARACTERIZATION The nonulosaminic acids are very water-soluble, polyhydroxy &amino acids which are readily separated and purified by means of ion-exchange chromatography. Anion exchangers, in the formate or acetate form, have proved to be most convenient for separating the N-substituted acids (for example, N-acetyl- and N-glycolyl-neuraminic acids) on gradient elution with formic and acetic 'l' " Under these conditions, however, higher acylated forms (such as N-acetyl-0-acetylneuraminic acid) are not isolated, since 0-deacylation takes place readily. The completely deacylated methoxyneuraminic acid, which bears a free amino group in addition to the carboxyl grouping, may also be isolated with the aid of a strongly acidic cation-exchanger (for example, Dowex 50) .so Since the nonulosaminic acids occur naturally in a bound form, a mild hydrolysis, ranging from boiling with water to treatment at pH 1-2 at 40-80", must necessarily precede application of ion-exchange chromatography. 2os

1 . Methoxyneuraminic Acid

Although methoxyneuraminic acid is a degradation product of the sialic acids, it is the most stable and readily accessible derivative of a nonulosaminic acid yet known. Thus, its isolation from a natural source as a well characteized crystalline product [having ["ID -55.0' (in water); m. p., 200" (dec.)] provides unambiguous evidence for the presence of nonulosaminic acids. Klenk" s1 originally isolated methoxyneuraminic acid following the methanolysis of brain gangliosides with 5 % methanolic hydrogen chloride for three hours a t 105", with subsequent fractionation by means of barium hydroxide. By use of a slight modification of the method,16 the yield was later improved to about 30% of the theoretical. Crystalline methoxyneuraminic acid has been isolated from a number of sialoproteins,l6, 62 and from erythrocytes,'Sv 49 liver:', 82 serum:' and milk.lg A further modification of the methods0 employs aqueous ammonia instead of the strong-alkali treatment, thus ensuring a minimal decomposition of the methanolysis products. These latter include both the methyl glycoside (methoxyneuraminic acid) and its methyl ester. After separation on a cation exchanger (Dowex 50), the crystalline material is obtained in yields of 60% or higher. 8

(79) (80) (81) (82)

P. B. Diezel, Naturwissenschajten, 42, 487 (1955). F. Weygand and H. Rinno, 2. physiol. Chem., Hoppe-Seyler's, 306, 173 (1957). E. Klenk, 2. physiol. Chem., Hoppe-Seyler's, 288, 216 (1951). E. Klenk and H. Faillard, 2. physiol. Chem., Hoppe-Seyler's, N O , 191 (1955).

THE NONULOSAMINIC ACIDS

251

The empirical formula was finally establishedsnto be CioHlgNOs , as had been previously inferred.l2,83, 84 2. N-Acetylneuraminic Acid N-Acetylneuraminic acid was first obtained as a byproduct in the methanolysis of bovine submaxillary mucin and was separated by ionexchange c h r o m a t ~ g r a p h y . ~ ~ Prior to this, its methyl ester (although it was not recognized as such) had been isolated from dialyzed, hydrolyzed cow’s colostrum, and designated21“methoxylactaminic acid.” Independently, the free acid was also isolated from both the dialyzable and the non-dialyzable carbohydrate fractions of human milk, and described under the name2n“gynaminic acid.” With the development of improved procedures of isolation, particularly recrystallization from acetic acid and water to obtain the alkyl-free acids, it was shown that gynaminic, lactaminic, and ovine sialic acids are identical with N-acetylneuraminic acid. The final identification was made by comparison of their infrared absorption spectra and x-ray powder diffraction patterns, which were respectively identical. N-Acetylneuraminic acid has an empirical composition of C11H19NOg, and [0(]E5 -32.0’ (in water); m. p. 183-185”. On heating with methanolic hydrogen chloride, it is readily converted to monomethoxyneuraminic acid with concomitant loss of its reducing properties. Boiling the compound with alkqli yields 2-pyrrolecarboxylic The acid is most conveniently obtained, in 25 of 40-60%, from milk, colostrum, submaxillary mucin, and meconium by hydrolysis of the constituent sialoproteins and oligosaccharides with 0.01 N sulfuric acid for one hour a t 70”. It may also be obtained from these sources by enzymic digestion with neuraminidase (see Section VII) . 3. N-Glycolylneuraminic Acid (Porcine Sialic Acid)

From hog-submaxillary mucin, Blix and his first isolated a nonulosaminic acid with the elementary formula C11HIgNO10 ; it had m. p. 185-187” (dec.), [a];’ -31 f 2’. The acid is liberated both by mild hydrolysis and by enzymic degradation of the porcine submaxillary m u c o p r ~ t e i n .s6~ ~ This acid has also been found (together with N-acetylneuraminic acid) in certain gangliosides and sialoproteins of other animal species, for ex(83) A. Gottschalk, Australian J . Sci., 18, 178 (1956). (84) F . Zilliken, Conf. o n Polysuccharides in Biol., Trans. 2nd Conf., 1966, 9 (1957). (85) E. Klenk and H. Faillard, 2. physiol. Chem., Hoppe-Seyler’s, 298, 230 (1954). (86) E. Klenk and G. Uhlenbruck, Z. physiol. Chem., Hoppe-Seyler’s, 307, 266 (1957).

252

F. ZILLlKEN AND M. W. WHITEHOUSE

ample, those from horse horse kidney, and beef serum?6b Glycolic acid was isolated after hydrolysis, and identified either by the x-ray diffraction pattern of the calcium s a l P or by its color reaction with 2,7-dihydroxynaphthalenein concentrated sulfuric acid.86 4. N-Acetyl-O-acetylneuraminic Acids (Bovine and Equine Sialic Acids)

The isolation and chemical identification of higher acylated and different nonulosaminic acids from mucinous secretions of epithelial origin has been mainly the subject of work by Blix and his associates.23 The method' of isolation consists essentially in boiling the pre-purified mucin with water, evaporating the extract to dryness, and extracting the dry residue with methanol a t a low temperature. Crystallization from aqueous methanol is achieved by the addition of ether and petroleum ether. Unfortunately, this method of isolation has led to crystalline preparations of varying composition because of the extreme lability of the products, particularly toward acids. The bovine and equine acids, with their identical molecular composition of C13H21N010are distinguishable from each other by virtue of their melting points, optical rotations, and x-ray diffraction patterns. Bovine ' (in water), and [MI, sialic acid has m. p. 134-137", [a]:' +8 f 2 2,808 f 702,whereas equine sialic acid has m. p. 183-187",[a]:' -59 f 2" (water), and [MI, -20,709 f 702. Since both acids may be degraded to N-acetylneuraminic acid,23 they evidently differ as regards the position of the O-acetyl group. The possibility that they are anomers is indicated on comparing their molecular rotations with that of N-acetylneuraminic acid ([MI, - 9,888)and applying Hudson's isorotation rules.

+

v.

CHEMISTRY

AND STRUCTURE

1. Derivation of Empirical Formulas A comparison between certain chemical characteristics of the sialic, N-acetylneuraminic, and methoxyrieuraminic acids, especially their functional groups and the color reactions afforded with Ehrlich's and Bial's reagents, suggested a close relationship between these nonulosaminic acids and indicated that methoxyneuraminic acid might be the methyl glycoside of a completely deacylated sialic acid. This concept was substantiated with the conversion of N-acetylneuraminic acid into methoxyneuraminic (86a) T. Yamakawa, J . Biochem. (Tokyo), 43, 867 (1956); Chem. Abstracts, 61, 4451 (1957).

(86b) A. Martensson, A. Raal and L. Svennerholm, Acta Chem. Scad. , 11, 1604 (1957).

THE NONULOSAMINIC ACIDS

253

acid, the transformation of bovine sialic acid into N-acetylneuraminic acid, and the conversion of both the bovine and the equine sialic acid into Klenk's methoxyneuraminic acid.20's6 However, microanalytical difficulties, particularly in obtaining satisfactory and consistent values for elemental composition, made it difficult to express these results in the form of molecular relationships until quite recently. With the introduction of more refined methods of separation and purification (see Section IV), the relationships and identities between the various nonulosaminic acids were established, although only after a multitude of carefully performed analyses in the laboratories of Blix, Kuhn, and Zilliken had resolved former apparent discrepancies. The chemical relationship of the nonulosaminic acids is set out below.

+

Bovine sialic acid, [a10 8" (N-acetyl-0-acetyl) CisHniNOio (H")

I

(minus 0-aeetyl)

N-Acetylneuraminic acid, [a],- 32" CiiHisNOo ------A

I

I

(5% methanolic HC1 at 100'; Ba(0H)z)

I

(minus N-aeetyl)

I

I

I

(minus N-aeetyl PlUS 4 H z )

I

I

(PIUS-CHI)

[CoHi7NOsJ - 3 ~ q ~ - ++ z aCioHisNOs Neuraminic acid Methoxyneuraminic hypothetical parent compound acid, [a],-55"

2. Degradation The results of earlier attempts18-8' t o assign a structural formula t o the neuraminic acids were not substantiated by the chemical characteristics of the chromatographically homogeneous substances. The first clue to their true structure arose from the work of Hiyamass and of GottschalkB9~ on the composition of the carbohydrate prosthetic group of homogeneous urinary and submaxillary sialoproteins. These workers isolated 2-pyrrolecarboxylic acidg0in very small yield (0.05 %) from alkaline hydrolyzates of the sialoproteins. The 2-carboxypyrrole was not pre-formed but resulted from the action of alkali upon a nonulosaminic acid. The range of possible precursors was greatly reduced when Gottschalk foundg1that the product liberated during (87) A. Gottschalk, Nature, 167, 845 (1951). (88) N. Hiyama, T6hoku J . Exptl. Med., 61, 319 (1948). (89) A. Gottschalk, Nature, 170, 662 (1952); 172, 808 (1953). (90) A. Gottschalk, Biochem. J . , 61, 298 (1955). (91) A. Gottschalk, Nature, 174, 652 (1954).

254

F. ZILLIKEN AND M. W. WHITEHOUSE

the interaction of influenza virus and the submaxillary or urinary sialoproteins (see Section VII) also formed 2-pyrrolecarboxylic acid on treatment with 0.1 N carbonate a t 100" for 20 minutes; and Klenk and Faillardg2 reported that N-acetylneuraminic acid behaved identically under similar conditions. Since glutamine, glutamic acid, 5-pyrrolidine-2-carboxylic acid, proline, hydroxyproline, and N-acetyl-D-glucosamine all failed to form 2-pyrrolecarboxylic acid under these conditions of alkaline degradation, it was concludedgo~ that the labile 4-hydroxypyrroline (I, 11) is the actual precursor.

However, Blix and his demonstrated that bovine sialic acid, C13H21N010,contains both a N-acetyl and an 0-acetyl group, and has a total of five acetylatable hydroxyl groups (one of them a primary alcoholic group, and another in the a position to the carboxyl grouping) ; they also educed evidence for the presence of one reducing group and the absence of a glycosidic linkage. This was clearly at variance with Gottschalk's former hypothesis and led hima3, 93 t o propose the following structures for bovine sialic acid, N-acetylneuraminic acid, and methoxyneuraminic acid.

(92) E. Klenk and H. Faillard, 2.physiol. Chem., Hoppe-Seyler's, 298, 230 (1954). (93) A. Gottschalk, Nature, 176, 881 (1955).

255

T H E NONULOSAMINIC ACIDS

CHOH

/ \

HCNHz

I

HC

CH1

I

OCH,

c< COzH

0 1 '

I

CHOH

I I

CHOH CH,OH Methoxyneuraminic acid

Thus, the noriulosaminic acids were formulated as aldol condensation products of a hexosamine with pyruvic acid. This speculative concept received its first support with the isolation of N-acetyl-D-ghcosamine as a degradation product of N-acetylneuraminic acid. These degradations employedg4*9 6 nickel acetateg6 plus pyridine or mild alkali for ten minutes. Besides N-acetyl-D-ghcosamine and a number of unidentified degradation products, pyruvic acid was produced and was isolated as its (2,4-dinitrophenyl) hydrazone. In the light of all these findings N-acetylneuraminic acid may be formulated as follows (I11 and IV). No.of C atom

1 c=o I /SC*H, F\SC,H, FH2

-0CH

I

7 - COCH, HOCH H NH

I

I

HCOH I CH,OH

I

HCOH I CH,OH

111 IV J-Acetarnid0-3,5-dideoxy-D -glyc?ro-p- D- idononulopyranos-l-onic acid [p-D-( - )-N-Acetylneuraminic acid]

HCOH

I

H(ioH CH,OH V

5-Acetarnido-3,5-dideosyD-glycero- D-ido- nonulosono1 ,blactone diethyl dithioacetal ID-(-)-N-Acetylneurnminic lactone diethyl dithioacetal]

(94) R. Kuhn and R . Brossmer, Chem. Ber., 89, 2471 (1956). (95) F. Zilliken and M. C. Glick, Naturwissenschajten, 43, 536 (1956). (96) G. Zweifel and H . Deuel, Helv. Chim. Acta, 2Q, 662 (1956).

256

F. ZILLIKEN AND M. W. WHITEHOUSE

Roseman and Combg6*have recently demonstrated that both N-acetylD-glucosamine and N-acetybmannosamine separately undergo epimerization in pyridine and nickelous acetate, under conditions previously employed for the degradation of N-acetylneuraminic acid,94yielding in each instance a mixture of N-acetyl-D-glucosamine and N-acetyl-D-mannosamhe in the ratio of 8:2. These observations, together with their enzymic degradation of N-acetylneuraminic acid to N-acetyb-mannosamine plus pyruvic acid, have led these investigators to conclude that the hexosamine moiety in neuraminic acid is D-mannosamine. This finding awaits confirmation by other methods. 3, Stereochemistry

The degradation of N-acetylneuraminic acid to N-acetyl-D-ghcosamine plus pyruvic acid accounts for all the carbon atoms in the molecule and establishes the configuration at C5, C6, C7, and C8. The configuration of C4 was proved by the formationg7 of a diethyl dithioacetal lactone of N-acetylneuraminic acid (V), which can only be the y-lactone. The high levorotation ([olID = -83", in methanol) indicates, according to Hudson's lactone rule, that the hydroxyl group on C4 is trans to the acetamido group. Dissolved in dimethyl sulfoxide, N-acetylneuraminic acid mutarotates from [aID - 115' (7 min.) to +24" (24 hr.). The direction of mutarotation indicates that the crystalline acid is the P anomer (at C2) (IV). Inspection of molecular models of the different nonulosaminic acids indicates why these 4-hydroxy acids fail t o form a l14-lactone. Stericdly, they resemble m-hydroxycyclohexanecarboxylic acids, only the cis isomer of which yields a lactoneS8(at 130"). 4. Synthesis

The first synthesis of N-acetylneuraminic acid has been reported by Cornforth, Firth and Gottschalk.99 When N-acetyl-D-glucosamine (I) and oxalacetic acid (11) were kept in aqueous solution for 2-3 days a t pH 11, material giving a positive Bial test was formed, corresponding to an 8.8 % yield of N-acetylneuraminic acid (111). After ion exchange followed by charcoal chromatography, crude N-acetylneuraminic acid was obtained in a much smaller yield (only 1.7 %). It is probable that other nonuloses were also formed under these conditions and contributed to the Bial coloration. The recrystallized acid was found to be identical in its decomposition temperature, x-ray diffraction pattern, (96a) S. Roseman and D. G. Comb, J . Am. Chem. Soc., 80,3166 (1958). (97) R. Kuhn and R. Brossmer, Angew. Chem., 69, 534 (1957). (98) W. H. Perkin, Jr., and G . Tattersall, J. Chem. Soc., 91, 488 (1907). (99) J. W. Cornforth, M. E. Firth and A. Gottschalk, Biochem. J . , 66, 57 (1958).

257

THE NONULOSAMINIC ACIDS

infrared absorption spectrum, and chromatographic behavior with authentic N-acetylneuraminic acid from natural sources. COzH

-

c=o

I

CHz-C OzH I1

+ CHO

I I

-

S

I

c=o

I

-co

[

HOCH

I I

HC-NH-COCHI

-

COtH

I c=o I

CH-COZH

HC-NH-COGHI

R

COzH

-

R

CHz

41 HOCH I HC-NH-CO I

CH3

R

VI. NEURAMINOLACTOSE

1 . Isolation Trucco and Caputtol“, lol first isolated from the mammary glands of lactating rats a nitrogen-containing acidic “trisaccharide” which, on mild acid hydrolysis, yielded lactose plus a compound with the same chromatographic and colorimetric properties as a nonulosaminic acid. The newly isolated product was designated “neuraminolactose.” Components of similar chromatographic behavior were also found in the mammary gland of guinea lo3 Independently, a similar product was isolated from lactating-rat, mammary glands by Heyworth and Bacon.lo4 Neuraminolsctose is apparently only one of a series of oligosaccharides, containing nonulosaminic acids, which are produced in the mammary gland and may be derived from higher oligosaccharides by fragmentation. It was subsequently shownz0that the nondialyzable fraction of human milk contains, in addition to neutral, nitrogen-containing carbohydrates, at least two acidic oligosaccharides which, on hydrolysis, yield L-fucose, D-glucose, D-galactose, N-acetyl-D-glucosamine, and N-acetylneuraminic acid. The dialyzable carbohydrate fraction may contain as many as five (100) R. Caputto and R. E. Trucco, Nature, 169, 1061 (1952). (101) R . E. Trucco and R . Caputto, J. Biol. Chem., 206, 901 (1954). (102) F. H. Malpress and A. B. Morrison, Nature, 169, 1103 (1952). (103) F. J. Reithel, M. G. Horowita, H. M. Davidson and G. W. Kittinger, J . Biol. Chem., 194, 839 (1952). (104) R . Heyworth and J. S. D . Bacon, Biochem. J . , 68, xxiv (1954); 66, 41 (1957).

258

F. ZILLIKEN AND M. W. WHITEHOUSE

nonulosaminic acid-containing oligosaccharides, two of which are neuraminolactose .Io6 2. Structure

The structural analysis of neuraminolactose was greatly stimulated by the isolation of N-acetyl-0-acetylneuraminolactose from cow’s colostrum.22 Sixteen liters of colostrum yielded 3.6-4.G g. of the very labile, acidic “trisaccharide.” Due to its own acidity, a 1 % aqueous solution of the substance has a half-life of only 48 hours and it spontaneously hydrolyzes to lactose, N-acetylneuraminic acid, and acetic acid; whereas, at pH 6, there is no appreciable hydrolysis. Dilute alkali and acid degrade the acidic “trisaccharide” as shown below.

+

N-Acetyl-0-acetylneuraminolactose ( C ~ ~ H ~ I N O [a], Z O , 16”)

I

0.02N NsOH (cold)

CHaCOzH

+ N-Acetylneuraminolactose

1

(CZ~H~~NOI~

0.01N HCl (cold)

N-Acetylneuraminic acid, [aI0-32’

+ lactose

!,H,OH

Methyl N-acetylneuraminate

Chromic acid oxidation of one mole of the original, acidic “trisaccharide” yields 2 moles of acetic acid, isolated in the form of crystalline sodium acetate, which indicates the presence of two acetyl groups in the molecule. One of these must be an 0-acetyl group, since the neuraminic acid is N-acetylated. N-Acetyl-0-acetylneuraminolactose reduces alkaline copper reagents on heating, but its extreme alkali-lability minimizes the value of its reducing properties as a guide to t,he structure of the whole molecule. l o 6 , 1°7 The reducing group was located in the lactose Enzymic degradation of N-acetylneuraminolactose with neuraminidase (see Section VII) is accompanied by the liberation of free N-acetylneuraminic acid.22,Io7 The position of the glycosidic linkage from neuraminic acid to the lactose moiety was located a t C3 of the D-galactose (see Fig. 2).Io6 Hydrolysis of completely methylated neuraminolactose gave 2 ,3,6tri-0-methybglucose and 2 ,4 ,6-tri-O-methyl-~-galactose.Since the orig(105) F. Zilliken and T. G. Martinez, unpublished results. (106) R. Kuhn and K. Brossmer, Angew. Chem., 70, 25 (1958). (107) A. Gottschalk, Biochim. et Biophys. Acta, 23, 645 (1957).

259

THE NONULOSAMINIC ACIDS

irial “trisaccharide)’ has a low positive optical rotation configuration was assigned to the ketosidic linkage.

([a],+16’),

the a

VII. BIOCHEMISTRY 1. Biosynthesis

The ease of their chemical degradation to D-glucosamine plus a threecarbon fragment, and the ready synthesis of N-acetylneuraminic acid from N-acetyl-D-glucosamine) suggest that the nonulosaminic acids may be biogerietically derived from an aminodeoxyhexose (or a precursor CHzOH

CHZOH

newaminiduse----

---I----

HCOH

I

HCOH

I

FIG.2.-Structure

CHZOH of N-Acetylneuraminolactose.

thereof) by condensation with pyruvic acid. Some evidence has been presented that wglucose may function as a direct precursor of neuraminic acid.108,108a It is known that D-glucose is converted to D-glucosamine in animal tissues, probably via D-fructose 6-phosphate.log 2. Interaction between Viruses and MucoproteinsllO; “Neuraminidase)’

It has been found that the initiation of infection by influenza viruses involves their interaction with mucosubstances a t the surface of the susceptible cell.lL1 Human, pig, and fowl erythrocytes, although not (108) K. Lauenstein and K. I. Altmann, Nature, 178, 917 (1956). J. Biol. Chem., 230, 381 (1958). (109) L. Hough and J. K. N. Jones, Advances in Carbohydrate Chem., 11,243 (1956). (110) A. Gottschalk, Physiol. Revs., 37, 66 (1957). (111) F. M. Burnet, Ann. Rev. Microbiol., 6, 229 (1952).

(10%) H. Bost,rom, L. Roden and I. Yamashina,

260

F, ZILLIKEN AND M. W. WHITEHOUSE

susceptible to infection, possess similar surface components which adsorb a virus, allowing the formation of “bridges” between the cells and resulting in hemagglutination. It was first shown by G. K. Hirst112 that this is, essentially, part of an enzymic reaction, the enzyme being identified with the actual virus particle. Agglutination therefore represents the formation of an enzyme-substrate complex which, on incubation at 37” eventuates in destruction of the substrate and subsequent elution of the virus. The liberated virus retains its hemagglutinating activity, but the incubated cells then lose their power to adsorb the virus further. Preheating the virus, before incubation with the erythrocytes, destroys the enzymic activity but does not impair the ability of the virus to be adsorbed onto the erythrocytes and to agglutinate them. Thus, the ability of the viral enzyme to combine with its substrate is retained, although its activity is lost on heating. The net result of this enzymic activity is a liberation of the virus from the “hemagglutination complex.” Many animal viruses may bring about hemagglutination, but only those associated with influenza, mumps, Newcastle disease, and fowl plague cause hemagglutination with spontaneous elution of the virus. Hemagglutination by heat-treated, influenza virus is inhibited by a number of sialoproteins present in serum and animal secretions.38’4 6 , 7 O , 1061 1 l o ~ 11* These are distinguishable from the thermolabile inhibitors of serum (such as properdin) by their relative stability to heat. The capacity of these sialoproteins to inhibit hemagglutination is rapidly lost on incubation with the virus, with the simultaneous release of a low molecular-weight compound-the so-called “split product.” Evidently, the inhibitors are capable of acting as substrates (for the viral enzyme) alternative to the natural cell receptors. Gottschalkllo was able to characterize the “split product” formed by the action of influenza virus on ovomucin, and on the sialoprotein inhibitor from urine, by its ease of humin formation and its ability to give the Morgan-Elson color reaction for N-acylated 2-amino-2-deoxyhexoses. Accordingly, he assigned the structure of a N-substituted D-fructosamine to this product. OdirP and Klenk,16 independently, both suggested that this split product is closely related to a nonulosaminic acid, and noted its ability to give a “direct Ehrlich” color reaction. Klenk and his coworkers114 were then able to establish unequivocally that the principal product1l6 (112) G.K.Hirst, J . Ezptl. Med., 76, 49 (1942);76, 195 (1942). (113) F.Zilliken, G.H. Werner, R. K. Silver and P. GyBrgy, Virology, S, 464 (1957). (114) E.Klenk, H. Faillard and H. Lempfried, 2.physiol. Chem., Hopper Seyler’s, 801, 235 (1955). (115) It should be noted that nonulosaminic acids may not be the sole products of the viral degradation of sialoproteins.

THE NONULOSAMINIC ACIDS

261

obtained on viral degradation of the urinary sialoproteins is N-acetylneuraminic acid. It is of interest that a nonulosaminic acid is found in all the mucoprotein inhibitors so far tested and also in a mucoprotein present in erythroc y t e ~116 , ~although ~~ it is not yet clear what other chemical and physical relationships niay exist between the alternative viral substrates, the inhibitors, and the natural, cell receptors. No quantitative relationship has been noted between the capacity to inhibit hemagglutination and the content of nonulosaminic acid in the individual, inhibitory sialoproteins.as The possible role of a polypeptide or protein moiety in conferring the inhibitory properties is suggested by the observation that a mucolipid fraction (from ox brain), which contains both a peptide and a nonulosaminic acid, will inhibit viral hemagglutination.92 Destruction of the peptide component by tryptic digestion results in loss of the inhibitory function. The viral enzyme does not release all of the nonulosaminic acid present in the inhibitor^,^^, l I 7 but the readiness with which a considerable fraction of the total nonulosaminic acid is released from ovomucin and from the urinary and submaxillary sialoproteins would indicate that this fraction is terminally bound in these substrates. Gottschalk117has shown that the bovine, submaxillary sialoprotein contains sialic acid bound by a linkage which is labile t o both acid and alkali; this is inferred to be a glycosidic bond. Accordingly, he has classified the viral enzyme as a glycosidase, conferring118on it the name ‘(neuraminidase.” The action of neuraminidase is one of the few enzymic “activities” which has been characterized with certainty as a function of a whole virus. The neuraminidase of influenza virus is capable of cleaving the milk neuraminolactoses to lactose plus the corresponding nonulosaminic acidlzz* lo7 thus functioning as an a-ketosidase. In these oligosaccharides, the nonulosaminic acid is linked to D-galactose,106~ 1°7 whereas, in the sialoproteins, the acids may be linked glycosidically to D-galactosamine. D-Galactose appears to be almost absent from certain sialoprotein substrates of n e ~ r a m i n i d a s e .Evidently, ~~~ neuraminidase has no absolute specificity for the monosaccharide linked glycosidically to the nonulosaminic acid. The findingzsof colominic acid, produced by phage-resistant Escherichia coli, prompts speculation as to whether or not this phage-resistance is, in (116) (1957). (117) (118) (119)

E. Klenk and H. Lempfried, 2. physiol. Chem., Hoppe-Seyler’s, 307, 278 A. Gottschalk, Biochim. et Biophys. Acta, 20, 560 (1956). Alternatively described as “sialidase.” See Ref. 2%. A. Gottschalk and G. L. Ada, Biochem. J . , 62, 681 (1956).

262

F. ZILLIKEN AND M. W. WHITEHOUSE

part, due to an inhibition of a hydrolytic, enzymic function of bacteriophage, akin to the neuraminidase of certain animal viruses. Neuraminidase has also been obtained from a number of bacteria.120.121 From cultures of Vibrio cholerae, an enzyme has been extracted which destroys both the virus receptor site(s) of erythrocytes and the viruscombining capacity of inhibitory sialoproteins (resulting in loss of their hemagglutination-inhibitor activity). This enzyme, the receptor-destroying enzyme (RDE), has been shown to liberate either N-acetylneuraminic acid or N-glycolylneuraminic acid from various sialoproteins,86,ll3*l l B #lP1,122 and to cleave neuramin01actose~~1°7 as does viral neuraminidase. A variety of other bacterial-enzyme systems (for example, from hemolytic streptococci and from pneumococci) will likewise destroy the receptor sites, but no definite, neuraminidase activity has yet been indicated. An enzyme, closely related to neuraminidase, which degrades orosomucoid (al-acid globulin) with liberation of a nonulosaminic acid, It is has been obtained from culture filtrates of Clostridium perfringen~.~4 noteworthy that neuraminidase does not appear to be a component of any animal-enzyme system. 3. Enzymic Degradation

Two reports on the bacterial degradation of N-acetylneuraminic acid have been made. Preparations of receptor-destroying enzyme (RDE) from Vibrio cholerae were claimed123 to effect the cleavage of the C3-C4 linkage in N-acetylneuraminic acid, to form N-acetyl-D-glucosamine plus pymvic acid, although this could not be substantiated in at least one instance.lZ4By contrast, culture filtrates of Clostridium perfringens cleave the molecule to produce pyruvic acid and a N-acetylhexosamine believed to be D-mannosamine.126The same enzyme preparation will also bring about the reverse reaction, namely, condensation of N-acetyl-D-mannosamine with pyruvic acid to yield N-acetylneuraminic acid. The possibility that such an enzyme system simultaneously effects inversion of the configuration at C2 of the hexosamine cannot be discounted, particularly in view of the wide distribution of other racemases in bacteria. Certainly, much caution must be exercised in assigning absolute con(120) M. Shilo, Biochem. J . , 66, 48 (1957). (121) P. BUhm, J. Ross and L. Baumeister, 2.physiol. Chem., Hoppe-Seyler's, 308, 181, 284 (1957). (122) H. Faillard, 2.physio2. Chem., Hoppe-Seyler's, 306, 145 (1956); 307, 62 (1957). (123) R. Heimer and K . Meyer, Proc. Natl. Acad. Sci. U.S., 42, 718 (1956). (124) E. Klenk, Ciba Foundation Symposium, Chemistry and Biology of Mucopolysaccharides, 1958, 296. (125) D. G. Comb and S. Roseman, J . Am. C'hem. SOC.,80, 497 (1958).

THE NONULOSAMINIC

ACIDS

263

figurations t o the nonulosaminic acids on the basis of enzymic degradations alone. VIII. CONCLUSION This article must, by its very nature, be regarded as no more than an interim report. Clearly, there are still many features of the chemistry of these compounds which require further elucidation, as, for example, the behavior of the compounds as carboxylic acids, their analogy with the uronic acids, and their ease of lactonization. To date, progress has been greatly hindered because of the difficulties encountered in assigning the correct formulas and structures to these various acids, but it is to be hoped that further research will soon lead to a further characterization of these compounds and then to their complete, chemical synthesis. The biochemistry of the acids has really yet to be explored. However, certain aspects of their comparative biochemistry have been noted already, by implication rather than by discussion. It is evident that the variations observed, not only in the nature of the acid actually present but also in the quantities found in any one given source, reflect at a biochemical level an aspect of diflerentiation between animal species. Other such instances which might be cited are rather few, but include the nature of the bile acids,lZ6phosphagens, and visual (126) G. A. I). Haslewood, Physiol. Revs., 36, 178 (1955). (127) G. Wald, in “Trends in Physiology and Biochemistry,” E. S. G. Barron, ed., Academic Press Inc., New York, 1952, p. 337.

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POLYSACCHARIDE HYDROCOLLOIDS OF COMMERCE BY LEONARDSTOLOFF Seaplant Corporation, New Redford, Massachusetts

I. Introduction. . . . . . . . . . . . . . . . . . ..................................... 11. Terminology and Definitions. . . 111. Polysacolloids in Commerci 1. Classification. . . . . . . . . . 2. History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................... 3. Functions and Properties. IV. Market Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Usage Trends. ...... ..........................................

265

277 287

I. INTRODUCTION Advances in our knowledge of the chemistry of the polysaccharides have followed from improved techniques for examining macromolecules and their fragments. Chromatographic techniques, in particular, have permitted the separ:ition and characterization of interrelated compounds. A closer association of physicists and chemists in studying that diffuse boundary region of particle size where their two disciplines meet and become indistinguishable has pushed outward the frontiers of our understanding of macromolecular behavior. Art and empirical knowledge are now giving way to understanding and to an association of behaviors with structures. Ever so gradually, this advancing tide is making itself felt in that stronghold of art and the trade secret-the water-soluble gums and mucilages. Efforts to date have been random, with but little concern for systematization and coordination. The compilation by Whistler and Smart’ attempts to find some system in the chemistry of these substances and follows the reviews by Hirst,2 Hirst and Jones,3 and Jones and Smith.4 With the object of assisting future efforts at organization along lines that may have some practical value, this article will endeavor t o correlate the history, structure, function, botany, and economics of these compounds (1) R. L. Whistler and C. L. Smart, “Polysaccharide Chemistry,” Academic Press Inc.,New York, 1953. (2) E.L. Hirst, Endeavour, 10, 106 (1951). (3) E.L.Hirst and J. K . N. Jones, Research (London), 4, 411 (1951). (4) J. K.N. Jones and F. Smith, Advances i n Carbohydrate Chem., 4, 243 (1949).

265

266

LEONARD STOLOFF

so far as present knowledge permits, and to place in a new perspective those arbitrary divisions that have tended to separate starches, cellulose derivatives, sea-plant colloids, gums, and mucilages into different categories.

11. TERMINOLOGY AND DEFINITIONS Terminology which was vague though colorful is currently yielding to more precise designations. A gum has the subjective attribute of being gummy, a mucilage of being mucilaginous. The terms gum and mucilage have often been used interchangeably, although the pharmacist generally considers a mucilage as being a solution of a gum. All commercial gums are of plant origin, so that there is really no gain in speaking of “plant” gums. There is, however, a necessary distinction in the appellation of “water-soluble” gums, to distinguish them from similar-appearing treeexudates, the resins, which are insoluble in water but soluble in organic solvents. There is good general agreement“ 6 , 6 , ‘I that water-soluble gums are polysaccharidic materials which are more or lesa soluble in water to-form a viscous solution, a gelatinous paste, or a jelly (depending on the concentration employed) and which are insoluble in most organic solvents. This definition includes many substances that do not have the outward appearance of gums (for example, starches, pectin, and sea-plant hydrocolloids) . Outward appearance here has no value as a definitive criterion. Further use of the subjective terms gum and mucilage as group designations can scarcely be justified, since application of the terms is usually haphazard and arbitrarily restrictive. For instance, the endosperm polysaccharides (galactomannans) of the carob and guar seeds are called “gums,” but the endosperm polysaccharides (starches) of corn (maize) and wheat are not generally considered t o be “gums”; yet starches and galactomannans are similar in origin, structure, behavior, and utilization. A more generally inclusive and descriptive name for this group of substances would be polysaccharide hydrocolloids. (Following the modern trend, this name will be condensed to “polysacolloids.”) The terms gum and mucilage will be used, in this article, only insofar as they are commonly employed for specific polysacolloids, with no generic connotation. Individual products were originally given trade designations referring to their geographical source or to the trading areas whence they came-of which gum Arabic (gum from Arabia) and carrageenan (polysacolloid of sea plants marketed from Carrageen, Ireland) are vestiges. Sometimes, (5) D. C. Beach, Gums and Mucilages, i n “Encyclopedia of Chemical Technology,” Interscience Encyclopedia, Inc., New York, 1951. (6) F. N . Howes, “Vegetable Gums and Resins,” Chronica Botanica, Waltham, Massachusetts, 1949. (7) C. L. Mantell, “The Water-Soluble Gums,” Reinhold Publishing Corp., New York, 1947.

POLYSACCHARIDE HYDROCOLLOIDS OF COMMERCE

267

native descriptive terms were employed; for example, tragacanth (goat’s horn), tapioca (juiceless pith), and agar (jelly). Botanical-source designations are more commonly used for the starches and other seed polysaccharides. Abbreviated chemical terminology and related trade names are currently popular for naming polysaccharide derivatives. 111. POLYSACOLLOIDS IN COMMERCIAL USE Polysacolloids abound in Nature as structural, storage, vascular, or functional components of plant and animal tissues, but only a limited number, all from plant sources, are of commercial importance. The economic justification for the production and utilization of each is complex and varies from one geographical area to the next. The economic sidelights considered here will, therefore, be primarily pertinent t o the United States and not necessarily valid for other countries. For example, the role of corn starch in the U. S. A. would be taken by potato starch or wheat starch in Europe, by tamarind-seed polysacolloids in India, and by seaplant polysacolloids in Japan. Since the trends involving the use and movement of these products, their economics, and their history are not usually found in published studies, but do exist in the minds and thoughts of the men close to the market place, a large portion of the material presented here was obtained through discussions with those active in the trading centers. My acknowledgment of their assistance is now formally made, and my sincere expression of appreciation for their cooperation is accorded, without my holding any particular person responsible for any particular statement. My thanks are gratefully tendered to H. Goldfrank, P. Kaplan, E. Alter, and P . Littenberg of Stein, Hall and Company, to J. Johnson and M. Pellet of T. M. Duche, Inc., to D. C. Beach of S. B. Penick and Company, and t o J. Snoop of Morningstar, Nicol, Inc. 1. CZussiJication

The commercially important polysacolloids are listed in Table I according to their botanical source and function; in Table 11, according to their electrolyte nature; and, in Table 111, according to their composition and structure (following the system of Whistler and Smart’). Details of structure have already been well (8) A. Jeanes, “Dextran-A Selected Bibliography,” Northern Regional Research Laboratory, Peoria, Illinois, 1952. (9) C. A. Brautlecht, “Starch,” Reinhold Publishing Corp., New York, 1953. (10) R . W. Kerr, “Chemistry and Industry of Starch,” 2nd Edition, Academic Press Inc., New York, 1950. (11) Z. I. Kertesz, “The Pectic Substances,” Interscience Publishers, Inc., New York, 1951. (12) T. Mori, Advances i n Carbohydrate Chem., 8, 315 (1953).

268

LEONARD STOLOFF

TABLEI Arrangement of Commercially Important Polysacolloids According to their Source and Function

Sea Plant Hydrocolloids Structural Agars Alginates Carrageenans Gelans Land Plant Hydrocolloids Structural Pectins Celluloses Storage Seed Starches and Flours Corn (maize) Wheat Rice Waxy maize Sorghum Waxy sorghum Gums and Flours Carob (Locust bean) Guar (Guaran) Tarn Tuber Starches and Flours Arrowroot Potato Tapioca Pith Starch and Flour Sago Seed Coat Psyilium (Plantago) Quince Exzcdate Arabic (Acacia) Karaya Tragacanth Almond tree Ghatti Mesquite Shiral; Talha Sapote Bacterial Dextrans

POLYSACCHARIDE HYDBOCOLLOIDS OF COMMERCE

TABLEI1 Arrangement of Commercially Important Polysacolloids According to their Electrolyte Nature Electrolytes (polyanionic) Active radical: sulfate Carrageenans Gelans Agars? (see text) Active radical: carboxyl Alginates Pectins (Carboxy)cellulose derivatives Carboxy carob gum derivatives Psyllium-seed mucilage (Plantago) Quince-seed mucilage Arabic Karaya Tragacanth Almond tree Ghatti Mesquite Shiraz Sapote Active radical: phosphate Starches Arrowroot Potato Tapioca Sago Non-electrol ytes Starches and Starch Alkyl Ethers Corn (maize) Wheat Rice Waxy maize Sorghum Waxy sorghum Seed Gums and their Alkyl Ethers Carob Guaran Tara Cellulose Ethers Methyl Hydroxyethyl

269

Dextrans

*

1

c

1

r

-1,3-1,3. -1,3

-1

Guaran

-1

(?) Tara

-1

-1,4

Unknown Uronic Acid

D-Mannuronic Acid

D-Galacfuronic Acid

D-Ghuuronic Acid

L-Rhamnose

1+4

2. Diheteroglycans A. Linear

Agars (?) Carrageenans, K fraction (?) Gelans B. Branched Carob gum

D-Mannose

3 &Anhydrot-gahtoee

3,6-Anhydro-Dgalaclose

1+

r r r r

1. Homoglycans A. Linear Starch amyloses Cellulose derivatives Carrageenans, X-fraction Pectins Alginates B. Branched Starch amylopectins

r +

Polysaccharide

TABLEI11 2ortant Polysacolloids According to Their Composition and Slructure" D-Galactose

D-G~UCOSC

Arrangement of Commercially i

3. Triheteroglycans A. Probably branched Karaya Ghatti Mesquite

++ ++ -3 1 4 6

Sapote Quince-seed mucilage 4. Polyheteroglycans A. Probably branched Arabic Psyllium-seed mucilage (Plantago) (cold-water fraction) Tragacanth Almond

+-t

++

+ +

++b

++

+ ++

-1

1-2

+

++ ++

+t

+-t

+t + ++ + +-t ++ +-t ++ ++ + +-t +-t ++ - --

+-t

+

x

”

-

-

8o

Numbers and arrows indicate the glycoside linkages involved. b From associated cellulose. The following is the key employed: X = present; = present, linkages unknown; = major components, linkages unknown.

+

++

30

ss tr

cn 0 q d

s5 B0 m

272

LEONARD STOLOFF

The information in Table I11 has been taken from those reviews, except for new information on agar,13*l4 carrageenan,16-17and sapote gum1*that has since become available. A book, soon to be published,’O will bring these details up to date. No published determination of composition or structural information could be located on shiraz and talha gums. Based on their source and properties, both are probably polyglycosiduronic heteroglycans. One point requires emphasis. Most polysacolloids are complex mixtures which are difficult to separate. Intermolecular linkages involve not only other polymers but low molecular-weight substances as well. Precipitations and solvent separations that are clean-cut for mixtures of compounds in the low molecular-weight range are usually ineffective in the presence of polymers. In addition, there is no criterion of purity for the recovered parts to be analyzed. The presence of all the moieties indicated within one structure is, therefore, always open to question, particularly when the complexity exceeds two components or when a part is found in relatively small proportion. Gum tragacanth is known to consist of two components, a soluble “tragacanthin” and an insoluble “bassorin,” but insufficient work has been reported on the components to permit detailing them separately here. Some progress in separation may be at hand through the use of selective precipitations based on immunological techniques.*O (The plural name forms are used intentionally, to indicate that significant variations are found within each name designation.) The interchangeable use of the terms flours and starches for many of the storage hydrocolloids listed in Table I is a result of the separation difficulty. A flour is the crude, milled portion of that plant structure rich in starch. Starches are the granules, predominantly carbohydrate, which are separated from the storage structure. Some separations are relatively effective, as with corn (maize) and potato starches. Wheat starch cannot be separated as cleanly as corn starch, but sufficiently so that a distinction may be made between the starch and the flour. For tapioca, sago, arrowroot, and rice, the dividing line is hazy. 2. History The first technologies in foods, textiles, paper, and pharmacy must have required the use of polysacolloids. Many of those substances currently (13) C. Araki, Mem. Fac. Ind. Arts Kyoto Tech. Univ. Sci. Technol., 6, 21 (1956). (14) C.Araki, Bull. Chem. SOC.Japan, 29, 543 (1956). (15) A. N . O’Neill, J . Am. Chem. SOC.,77, 2837 (1955). (16) A. N . O’Neill, J . Am. Chem. SOC.,77, 6324 (1955). (17) S.T.Bayley, Biochim. et Biophys. Acta, 17, 194 (1955). (18) E.V. White, J . Am. Chem. Soc., 76, 267 (1953). (19) R. L. Whistler, “Industrial Gums: Chemistry and Technology,” Academic Press Inc., New York, to be published 1959. (20) M.Heidelberger, J. Adams and Z. Dische, J . Am. Chem. Soc., 78, 2853 (1956).

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employed have always been accessible, even though in a cruder form than is a t present available. The utilization of various sea plants as textile sizes in the Orient is still in a primitive form. Starches and flours from grains, legumes, and tubers have been used from before the dawn of recorded history. Gum exudates, both water-soluble and resinous, were prized articles of commerce in Biblical times. Indeed, there is evidence that gum Arabic was used about 2000 B. C. by the Egyptians as a textile adhesive and in embalming fluids. No-one knows precisely when mucilaginous seed-coats became part of the stock in trade of the Medicine Man. The commercial history of the various polysacolloids is mostly a story of slow evolution. Commercial knowledge of the exudate gums was brought t o this country through Europe, by way of the pharmaceutical and textile technologies of the Levant. The seed-coat mucilages were also part of British and European pharmaceutical lore. I n all this time, there has been little change either from the original products or in the techniques of producing and merchandizing them. The history of starch closely parallels that of the grains and tubers, and is thus more closely associated with food production than is the history of the exudate gums. The first starch was probably made from wheat, the first extensively grown cereal crop. Egyptian records of wheat starch date back to before 3000 B. C. Potato starch, a relative newcomer, was available in Europe about 1700 A. D. Tapioca starch was known and used by the Aztecs. The manioc plant, from whose root this starch is obtained, was transplanted to the Dutch East Indies, Java, and Sumatra, whence the major part of the world supply had been coming until the recent resurgence of Brazilian production. Arrowroot starch has a similar history, arrowroot being native to the West Indies but now being produced in South Africa, Australia, and the East Indies. Corn (maize), the principal source of the starch crop of the United States, is another native American plant. Sago and rice are the original oriental starch sources. Sorghum, waxy sorghum, and waxy maize starches are relatively recent innovations. The waxy starches are the result of breeding attempts made with the objective of obtaining a separate, amylopectin fraction. More recently available are chemically separated amylose and amylopectin from potato starch. Starch history parallels not only food production but also textile and paper technology. Starch has been, and still is, the cheapest and most abundant source of polysacolloid for sizing and coating. Because of their abundance, economic value, relative purity, and relative simplicity, starches have been the polysacolloids most thoroughly studied from the agronomic, technological, chemical, and physical standpoints.

274

LgONARD STOLOFF

Agar is an Oriental article of commerce, emanating from the Malay area before the Japanese monopolized its production. It was introduced to the Occident through Koch’s discovery of its value in preparing “solid” media for growing microorganisms. Attempts to compete with Japan in the production of agar, except for the manufacture of a small amount of high-quality product in the United States, were unsuccessful until World War I1 stopped this trade altogether. Cessation of hostilities found established agar plants operating in Italy, New Zealand, Portugal, Spain, and the United States ready to compete with the “cottage industry” of Japan on the basis of quality; the low wage-scales of prewar Japan that had discouraged competition are apparently now a thing of the past. As the sea-plant varieties and ecologies available to the different producing areas have changed, so have the agar properties-a matter of minor significance in some fields of application, but technologically important in others. Some of the properties (for example, gel strength and gelling temperature) are easy t o measure and compare. Others are more subtle, involving colloidal behavior difficult to measure but no less valuable and important. Carrageenan also has a commercial history with its roots lost in the past. It had for long been an unpublicized article of commerce, being exported from France and Ireland in the form of the bleached, whole plant. Users prepared their own decoctions, or employed the finely ground, whole plant. The earliest references to the material deal with its pharmaceutical value. Its introduction and use in this country followed the usual path from England and Europe and, although domestic raw material was found here in abundance at an early date, imports from Europe continued to provide the major supply until World War I1 interrupted this trade. The loss of these imports not only provided a stimulus to domestic harvesting in this country and Canada but encouraged production of the refined hydrocolloids. Improvements in quality, together with improvements in uniformity and reliability, have expanded the area of usage. Closely related sea-plants, previously of minor commercial value, have been brought into use, providing new carrageenans with a wider range of properties. The search for polysacolloid substitutes that followed the disruption of trade by World War I1 brought to light the gelans,21 seaplant polysacolloids having structures and properties intermediate between those of agars and carrageenans. An attempt at production, in the United States, from sea plants found along the North Carolina coast did not reach commercial fruition. A vigorous Danish industry, based upon a more easily harvested sea-plant form, is supplying some European markets. Its extensive use in this country is limited by the ready availability of carrageenan (21) L. Stoloff and P. Silva, Econ. Botany, 11(4), 327 (1957).

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and agar and by the differences in performance required by the United States consumer. The commercial history of the alginates is a study in persistence and planning. The perseverance of E. C. C. Stanford, who first isolated the material in 1883, studied its properties, and developed methods for its commercial production, did not suffice. The present scale of alginate usage is the result of the availability of low-cost raw material, efficient production methods, and effective merchandizing, coupled with pertinacity. This combiiiation epitomizes the Pacific Coast alginate industry in the United States, which has blazed the trail for the other producing areas both here and abroad. After several false starts, commercial success began on the West Coast in 1035, with the promotion of a mass market for sodium alginate as a stabilizer for ice cream. The large volume of business created then provided the economic leverage and a pattern for developing one mass market after another, the low cost of the raw material and of its production providing powerful assistance in this effort. The first imports of carob gum into this country were made in the late 1920’s, although this gum had been used as a thickener for textile printing pastes in Germany prior to World War I and, for some time prior to that, in Spain and Portugal as a starch replacement and as a size for paper and textiles. From Biblical times, the pods had been used in the Mediterranean countries for human and animal feed and as a source of fermentable carbohydrate. It is probable that the independent value of the bean as a source of gum was unearthed by the pharmacists, for one of the early uses of the flour in Europe was as a bulk laxative. Expansion of the American market has stimulated production of seed in Cyprus, Greece, Italy, Portugal, and Spain. Some of the seed is milled in those countries, as well as in England, Holland, Switzerland, and the United States. When World War I1 disrupted normal trade, the search for carob-gum replacements led to guar gum, guar being a forage crop and one of the oldest legumes known to man. It had been introduced into the United States for cultivation in semi-arid regions, having come from Central Africa by way of India and Egypt. Although carob gum had become available again before any significant production of guar gum had been achieved here, this new gum was found to have distinctive properties which made i t of value in its own right. Markets, and technological knowledge on its production and application, have been developed by using seeds grown in Texas (with supplies bolstered by imports from Pakistan). Additional tonnage is now being imported from India. One of the two domestic producers is protecting himself against the American farmer by setting up milling facilities iu Pakistan. Pakistani and Indian guar

276

LEONARD STOLOFF

is also arriving in this country by way of the European carob-millers, who have recognized the independent value of this polysacolloid. Endosperm of tara seed is another material that has been investigated as a replacement for carob gum. Its polysacolloid is also a galactomannan. The seed is available in Peru as a by-product of the tara pods used in tanning. The tara gum is not being marketed as such; the seeds, however, are being imported, and the gum is probably being mixed with carob and guar gums for various industrial uses. Exactly when making of jelly first became a household art is not known. As early as 1750, instructions were published for the preparation of apple, currant, and quince jellies. As the art developed, concentrates were made from pectin-rich sources, starting in the early 1900’s with the use of apple pomace in Germany and of citrus wastes in Italy. The industry soon spread to the United States, with the production of pectin concentrates from apple pomace for use in industrial and household production of jellies. By the middle 1920’s, the citrus producers had entered the field, competing with the dry pectins now available from apple pomace. Competition, and multiple sources of raw material, led to the production of pectins with various jelling properties. Increased knowledge, plentiful raw material, and substantial financial support from the citrus industry eventuated in the currently available modifications of the natural product, low ester pectins (“low methoxyl”), pectates, and pectinates. Bacteria producing slime have plagued the wine and sugar industries for years, and the slimes themselves have been the subject of academic interestn from the earliest days of bacteriology and organic chemistry. Their polysaccharidic nature and their general mode of synthesis from sucrose by exoenzymes of the associated bacteria were known from the late 1800’s. Interest in plasma expanders during the early days of World War I1 led Swedish workers to investigate certain of these polysaccharides, already known as “dertrans.” Their work, and patents derived from it, stimulated activity in this country, both government sponsored and private. The cessation of hostilities, with its attendant slump in plasma requirements, and the simultaneous development of more effective extenders, left this country with a wealth of capacity and knowledge of productionand no market. Most of the dextran-producing equipment has now been dismantled or diverted to other ends. Dextran is being offered on the market in various grades, but the competitive situation is difficult at present and, as yet, no distinctive applications have been developed that would warrant expansion of the present limited capacity. The possibility of the formation of cellulose ethers was first proposed (22) T. H. Evans and H. Hibbert, Advances in Carbohydrate Chem., 2, 203 (1946).

POLYSACCHARIDE HYDROCOLLOIDS OF COMMERCE

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in 1905, but the first practical reactions were not described until 1912, when a patent race ensued. Of the ethers of commercial importance at present, the methyl ether was the first to be described and extensively studied. I n a resumption of patent activity, the production of carboxymethyl cellulose [0-(carboxymethyl)ceIlulose] was described in 1918, and that of hydroxyethylcellulose [0-(2-hydroxyethyl)cellulose] in 1920. These products were of no commercial significance until shortly before World War 11, when the detergency-improving properties of carboxymethylcellulose2a were discovered in Germany. The trade restrictions in force immediately prior to and during that war, with the attendant shortage of fats and, consequently, of soaps, accelerated the development of non-soap detergents which required the presence of “aids” t o prevent soil redeposition. Developments in this country proceeded simultaneously during the war period, with applications other than those related to detergents in view. When the cessation of hostilities permitted revelation of the value of carboxymethylcellulose as an aid to non-soap detergents, production knowledge was sufficiently far advanced to speed the development of a “mushrooming” detergent industry in this country. The solid, economic base of a large-volume, recurrent use encouraged search for new commercial outlets. The persistence of one company in particular is noteworthy in regard to developing its utilization in food and in pharmaceutical applications. This company’s progress in making a purified product for these purposes preceded, and still exceeds, such developments elsewhere. The commercial success of carboxymethylcellulose, and the general awakening of interest in colloidal phenomena, has encouraged production and offerings of methylcellulose and hydroxyethylcellulose, both of which seem to be finding areas of application for their own peculiar characteristics.

3. Functions and Properties Polysacolloids find use because of the physical properties they exhibit when they are dissolved and when they are dried. Sols of polysacolloids are viscous, and have rheological properties that help provide stability in suspensions, emulsions, and foams. These same rheological properties have subjective attributes which provide pleasure to the sight and feel. Sols of some of the polysacolloids can be transformed into rigid structures called gels, some of which have a thermal sol-gel reversibility, whereas others require chemical action. When the sols are dried, the polysacolloid is left as a continuous, amorphous film. Many of these films have value as adhesives, sizes, and coatings. For such applications, a low, natural viscosity or an intentionally caused (23) J. V. Karabisos and M. Hindert, Advances in Carbohydrate Chem., 9, 285 (1951) 7

278

LEONARD STOLOFF

degradation is often necessary, in order to give desirable sol concentrations within workable viscosity limits, or to alter the appearance and properties of the film. For many uses, a number of the polysacolloids are interchangeable. For most applications, there is usually one polysaccharide that provides a combination of physical attributes that cannot be duplicated or even approached by any of the others. Sometimes, these physical differences are of such technological value that major economic differences may be needed in order t o effect a replacement. In other cases, the price is a n effective deterrent to use on the basis of desirable performance in determining polysacolloid selection. The polysacolloids perform in situations where the properties are those of aggregates rather than of individual molecules. The size, shape, continuity, degree and strength of bonding, and degree of solvation of the structures formed, determine the eventual physical properties of the material. For achievement of some fine point in performance and technology, combinations of polysacolloids are frequently used, borrowing “a leaf from Nature”-where combinations are the rule. This helps to add to the complexity, for most polysacolloids are heterogeneous substances to start with, the assigned structures really being simplifications of the facts or applicable t o only a portion of the substance. Polysacolloids function in fields where the important concept is the surface. Viscosity, gelation, stability (of suspensions, emulsions, and foams), adhesion, cohesion, repulsion, light transmission and reflection, are all surface phenomena. The interplay of the polysacolloid surface with the particle surfaces in emulsions, suspensions, and foams influences the viscous and gel behavior. The strength, appearance, and feel of threads, yarns, fabrics, papers, and boards are based on the interplay between polysacolloid and fiber surfaces. The physiological attributes of emolliency and demulcency involve tissue surfaces. These are all fields wherein empirical art still holds sway and where measurements and observations are, to a great degree, subjective. Although one may speak of the water-soluble gums, solubility as strictly defined is not applicable here. What is usually meant, in speaking of gums, is a description of the conditions for, and rates of, hydration. If a solution is regarded as a dispersion to the ultimate particulate entity, “solutions” of none of the polysacolloids could, in this regard, conform except a t infinitely low concentrations. A practical definition of dissolution, for gums, is the disaggregation of the solid particles to a size that will pass through commercial filters. This admittedly leaves a broad, hazy, borderline area in which one becomes the more involved, the larger the polymer molecule and the higher the concentration,

POLYSACCHARIDE HYDROCOLLOIDS OF COMMERCE

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Keeping the above stipulations in mind, the polysacolloids can be grouped into (1) those that swell but never dissolve and (2) those that can be made to “dissolve,” the latter group being subdivided according to the conditions required for dissolution. The swelling polysacolloids are karaya, almond-tree gum, the bassorin fraction of tragacanth, some forms of ghatti, quince-seed mucilage, and psyllium-seed mucilage; also, alginic acid, alginates of divalent metals, pectinic acid, and divalent pectinates, which can be made to dissolve in water by changing the counter ion. Although hydrated to the extent that there is no differential in refractive index to permit of distinguishing the particles from the medium in which they are dispersed, the particles can be sufficiently concentrated (by moderate sedimentation or filtration forces) t o demonstrate areas of sharp concentration-gradient. Complete swelling of these materials can be attained without applying heat. All the “soluble” polysacolloids pass initially through a swelling stage, the eventual dissolution occurring more readily with some than with others. Aqueous solutions can be prepared fairly readily a t room temperature with alginates of monovalent metals, alginate esters, some carrageenans, pectins, salts of low-methoxyl pectins with monovalent metals, sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, dextrans, gum arabic, mesquite gum, sapote gum, talha gum, most forms of shiraz gum and ghatti gum, and the tragacanthin portion of gum tragacanth. The rate of dissolution increases with rise in temperature, except for methylcellulose where the reverse is true. Considerable swelling in water a t room temperature is encountered with some carrageenans, gelans, gelatinized starches, and guaran. Swelling t o a lesser degree is exhibited by agar, raw starches, carob gum, and tara gum. These all dissolve when their aqueous suspensions are heated. The degree of swelling which occurs at room temperature is a rough index of the amount of heat that will be required in order to prepare a solution. The foregoing can be regarded as only a rough guide to the solubility characteristics of gums. Besides the particle size and the history of the polysacolloid specimen itself, each material responds in different degree to the presence of salts, specific cations, such hydrophilic liquids as acetone, glycerol, glycols, and other alcohols, sugars, and other hydrocolloids. A necessary prerequisite to dissolution is dispersion of the individual particles of the polysacolloid in the dissolving medium. Difficulty in achieving dispersion, or “lumping,” increases with (a) a decrease in the particle size (because of the increased surface), (b) an increase in the rate of dissolution, and (c) an increase in the viscosity. Lumping can be overcome or avoided by suitable manipulation of the foregoing factors, by the use of adequate agitation, and by a prior dispersion among non-lumping particles

280

LEONARD STOLOFF

or in such non-solvating liquids as monohydric or polyhydric alcohols or an oil. Surfactants are effective in preventing lumping of the swelling polysacolloids. The rheological properties of polysacolloid dispersions and “solutions” are never Newtonian and are seldom simple functions of the polysacolloid concentration. The other dissolved and dispersed ingredients of a given mixture have a marked influence on the net result, both qualitatively and quantitatively. Nevertheless, a rough comparison of viscous behavior can be made for average, undegraded polysacolloids. In the high-viscosity class (test concentrations, 1-2 %) are alginates, carrageenans, pectins, cellulose derivatives, carob and tara gums, guaran, quince and psyllium mucilages, and karaya, tragacanth, and almond-tree gums. In the moderateviscosity class (test concentrations, 2-5 %) are agars, gelans, ghatti gum, and starches. In the low-viscosity class are dextrans, gums arabic, mesquite, shiraz, talha, and sapote, and all the fragmented modifications of the preceding two classes. An extreme of the tendency toward aggregation is the ability to form rigid structures or gels. A gel may be defined as a structure s u m e n t l y rigid to support its o m weight or having a yield value greater than its own weight. This is admittedly a loose definition, since it does not stipulate the size of the structure, but this must, as a practical matter, vary with each mass considered. One other property that distinguishes a gel from a thixotropic sol is the irreversibility of the fracture. A structure that fulfils the second requirement but not the first could be called an incipient gel. The term gel has been used loosely by the pharmacists, colloid chemists, and physicists as applying to thixotropic structures having a high yield value. This may be attributable to the fact that thixotropic flow is characteristic of all gel structures that have been mechanically disrupted; but thixotropic flow is also observed with non-gelling materials. A polysacolloid gel can be considered to be a highly hydrated and crosslinked precipitate, and gelation may be regarded as a form of precipitation. The formation of separate precipitates rather than gels can actually be demonstrated under suitable conditions; these conditions are most easily defined and produced with chemical “sets” such as are obtained with alginates and low-methoxyl pectins, the conditions for each covering a fairly broad range. Because precipitates have effects (on the solvent medium) markedly different from those of sols and, in turn, are differently affected by such changes in the solvent as freezing, the distinction between sols and gels has practical significance. Thermally reversible gels can be prepared with agars, carrageenans, gelans, and such high-amylose starches as those of corn (maize) and wheat. Agar gels are distinctive in having a wide differential between their setting and

POLYSACCHARIDE HYDROCOLLOIDS OF COMMERCE

28 I

melting temperatures, and in being very little affected by the presence of other solutes or by the agar concentration. Agars vary in their characteristic setting and melting temperatures, but most commercial agars form gels that setz4a t about 40°C. and melt a t about 90°C. For occurrence of gelation: carrageenans and gelans require the presence of specific cations, the gel characteristics-strength, elasticity, gelling temperature, and melting temperature-varying both with the cation type and with its ionic concentration. Starch-gel characteristics are relatively unaffected by the presence of other solutes, but the setting and melting temperatures do increase with increase in starch concentration. Algins and low-methoxyl pectins can form gels by slow precipitation, usually as the calcium salt, through the controlled release of calcium from sparingly water-soluble salts or chelated systems. These gels do not melt, but they can be dissolved after conversion into the monovalent-cation form. Polysacolloids with cis-hydroxyl groups-carob gum, guaran and tara gum (galactomannans), and algins-are cross-linked by borax under alkaline conditions, forming tough, rubbery gels. Gelation can be reversed by neutralizing the alkalinity. Pectin gels are a special case involving hydrogen bonding, and they require closely controlled conditions of sugar concentration and pH for their formation. The rate of set and the ultimate gel-strength vary with the temperature, but gelation itself is not temperature-dependent. I n general, weakened gel structures result from mixing gel systemsexcept where starches are involved, probably because the dominant starch component is amylopectin and not amylose. Modifications of gel structure and characteristics often result on admixture of gelling with non-gelling systems. One of the most marked effects is the increase in gel elasticity and gel strength which results from mixing carrageenans or gelans with carob gum. The properties of suspensions, einiilsions, and foams are to some extent predictable from the rheological properties of the polysacolloids involvedbut only to a minor degree, since the polysacolloids usually function most effectively when they form bonds of some nature a t the surface of the dispersed phase, thereby changing the rheological pattern. The electrolyte polysacolloids are most likely to be involved in such attachments, but configuration, a s in immunochemistry, is of as great an importance as the presence of active groups. Also, the significance of the hydroxyl hydrogen atom as an active group has not, in many cases, been taken into account. Dextrans could be particularly pertinent in this respect. (24) L. S. Stoloff and C. F. Lee, U.S. Fish Wildlife Serv., Fishery LeafEet No. 335

(1949).

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LEONARD STOLOFF

Adhesive properties are most evident in the starches, exudate gums, and dextrans, all of which make excellent sizes. The reverse property or “antitack” is characteristic of agars, carrageenans, gelans, and the seed-coat mucilages. Other properties which are of commercial value but are peculiar to more limited groups are as follows: (a) the ability of cellulose derivatives, particularly carboxymethylcelluloseto adsorb onto cotton fibers, forming a barrier repellent to dirt; and (b) the ability of carrageenans and quinceseed mucilage to alter the aggregation of casein in milk. The last two polysacolloids are the only ones that seem to be absorbed by skin tissues, making them particularly effective in cosmetic and pharmaceutical preparations for topical application. IV. MARKETSUMMARY Starches as a group, and the three principal “volume” starches, individually, are by far the most important polysacolloids on both a weight and a money basis. They are also the cheapest polysaccharides in most areas of the world, the major exception being the Orient, where starch crops are more valuable as food and so are replaced, in other usages, by less easily assimilated polysacolloids. Corn (maize) is the most important starch crop in this country (accounting for over 90 % of the total production of starch), followed by potatoes and wheat; whereas, in Europe, the production of potato and wheat starch far exceeds that of corn. Because of its low price, corn starch enjoys a huge market wherein the unit cost is the major consideration. It has certain distinctive properties shared by wheat starch, namely, a high gelatinization-temperature, an ability to gel, and its accompanying liability, retrogradation. The presence of a minute, commercially inseparable proportion of oil sometimes creates difficulty because of ensuing rancidity. Very large amounts are used in oil-well drilling muds, textile sizes, sizes and coatings for paper and board, adhesives, and foods. Sorghum starch is similar to corn starch. The little that is produced is converted primarily into sirup, or is exported. Some small quantity is employed as a replacement for corn starch. Both waxy maize and waxy sorghum are varieties recently developed; they have a high content of amylopectin and find their major application in the food industry, in competition with tapioca and potato starches. Although wheat is grown for its starch in some European countries, wheat starch is, in the United States, a byproduct in the separation of gluten. The weight of this produced has been increased since a market for L-glutamic acid has been developed. It is usually dearer than corn starch. Because of its residual protein, the elimination of which is not practical,

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it has a mealy flavor and odor and a tendency to foam. Its major use is in making wallpaper pastes. Potato starches occur in almost endless variety, according to the potato used. I n the United States, only potatoes of poor sizes or quality (“culls”) or overproduced food-potatoes are employed, the States of Idaho and Maine furnishing the major supply. I n Europe, potatoes are intentionally selected and grown for their starch content, particularly in Holland and Ireland. Potato starch is similar to tapioca starch in many respects, and competes directly with i t in use for foods (gelatinized starches), paper, and textiles. Potato starch seems to be preferred as a warp size in cotton mills; it is an excellent flocculating agent for clays. Relatively pure amylose and amylodextrin from potato starch are currently being made available on a commercial scale a t prices competitive with those of other starch modifications. The amylose, consisting primarily of linear molecules, has a high film-strength and a high gelatinizing-temperature (5 min. a t 325°F.). The amylopectin, comprised primarily of branched molecules, shows strong complexing potentialities, and has many of the properties of waxy-grain starches. Tapioca starch of the highest quality now comes from Brazil, competing with the product of the organized plantations of Java and Sumatra. Sources of medium-grade starch are Madagascar, Thailand (Siam), and Togoland. Of the raw starches, tapioca starch has the lowest gelatinization-temperature (about lGO°F.), and forms pastes that do not retrograde. It contains no difficulty-causing proteinaceous or oil residue a s do wheat starch and corn starch, respectively. Large uses are in textile sizes, adhesives, and foods. Tapioca starch is frequently employed as a replacement for corn or potato starches, as the economics of the moment dictates. Arrowroot starch is used almost exclusively in baking, for the preparation of biscuits (“cookies”) alleged to have special dietary significance for infants and invalids. In its general properties, it is similar to tapioca starch. The major supply for the U. S. comes from St. Vincent, a British island in the Caribbean. Rice starch, because of the small size of its granules, is preferred for preparing cosmetic dusting-powders and in the binding of pharmaceutical tablets. Imports into the U. S. are principally from Belgium and Egypt. Sago starch is a diminishing import from Thailand (Siam). It produces a tough, horny film which finds specialized application in textiles and adhesives, particularly as a size for carpet backs (a field in which rubber latexes are increasingly being used). The uses of starch apply not only to the raw starches but to the endless variety of starch modifications achieved through gelatinization, wet and dry dextrinization, and derivatization (the formation of ethers and

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esters). The starch ethers and esters are more widely employed in Europe than in the United States, including their use in foods. They have a viscosity lower than that of the unmodified starches but a more rapid hydration and a smaller loss of water on freezing and thawing of the sols. The weight of gum arabic used each year in the United States is roughly 5 per cent of that of corn starch. This figure includes the best, pharmaceutical grades called “acacia” and the low-grade adulterants and substitutes for gum arabic, talha and mesquite gums. Most of the gum arabic comes from the country formerly known as the Anglo-Egyptian Sudan. The original trade-routes to Europe passed through Arabia, and hence the designation “gum arabic.” Mesquite gum, which comes from Mexico, has properties similar to those of gum arabic, but it has a lower viscosity and a high tannin content, which lower its value in many applications. Talha gum, like gum arabic, comes from the Sudan, but it is the exudate of Acacia seyal (not of Acacia senegal). The Acacia species grow in contiguous areas, not intermixed, so that the generally dirty character of the gum talha of commerce is most probably due as much to human carelessness as to natural causes. The originally large use of gum arabic as an adhesive has dwindled, giving way to the more varied and cheaper starchdextrins. The largest proportion of it is at present formulated into cough drops and similar rubberlike confections. Other large uses are in textile printing-pastes and finishing sizes, in pharmaceutical emulsions, for holding sensitizing agents onto lithographic plates, in ceramic glazes, and in the recently developed “dry flavors.” From two fifths to a half of the karaya gum harvest of India is exported to the United States. Other Indian gums with similar properties are gum ghatti and gum shiraz. These are, however, usually of a quality inferior to that of karaya gum, and they are used as cheap substitutes or adulterants for it. Most of the shiraz gum is exported through Iran (Persia). Some almond-tree gum from Portugal and Spain is used as a substitute for karaya gum in pharmaceutical applications. Because karaya gum swells without dissolving, its properties depend to a great extent on its particle size. However, karaya gum contains a labile acetate radical which is readily hydrolyzed from the dry gum at a rate dependent on the exposed surface, thereby endowing powdered karaya gum with its typical, acetic aroma. This characteristic also requires that karaya gum should not be ground too far in advance of its use. The largest proportion of karaya gum is employed in the formulation of a’ temporary adhesive for denture plates while they are in use. Other large applications are as a bulk laxative and as a thickener for textile printing-pastes. The amount used in foods is small. Gum tragacunth, imported from Iran, is subject to wide fluctuations in

POLYSACCHARIDE HYDROCOLLOIDS OF COMMERCE

285

price, because of erratic supplies occasioned by unpredictable crop failures. The cleanest, and therefore the dearest, gums are used in pharmaceutical preparations and in foods, the lower grades in textile printingpastes. Gum tragacanth was for a long time the favorite stabilizer for emulsions, particularly the acidic types, and was the favorite bodying agent for toothpaste, but it has lost a great portion of all these markets to other, more dependable, polysacolloids. Carob gum and guaran find application in the same fields, as they have closely similar properties and price. Guaran might, in many instances, be considered a more readily soluble form of carob gum. There are, however, differences in detail that often result in a preference for one over the other. The textile and paper industries use large quantities for thickening, sizing, and coating. Other large applications are in water-base paints, latex emulsions, asphalt emulsions, ceramics, and foods (particularly dairy applications). Ethers and esters of both carob gum and guaran are currently available, finding more ready acceptance in the European market than in the U. s. Small quantities of tara gum are produced here (from imported seed) t o be blended with the other galactomannans for use in the paper industry. Both psyllium and quince seeds are imported into the United States for limited, specialized uses. Almost all the psyllium seed arriving from India proceeds to pharmaceutical houses for use in laxative preparations utilizing the mucilage-containing seed husk. Quince seeds from Iran are purchased by cosmetics houses, principally for use in hand-lotions. Each user prepares his own mucilage extract directly from the seeds, as needed. Seed supplies are restricted, so that any unusual demand is reflected in increases in price. Pectin is the only polysacolloid sold to any extent in liquid form, and this use a t present constitutes a dwindling market. The major use for pectin is still in the preparation of fruit jellies and related products in the baking and confectionery industries. Some pectin is used as a stabilizer in sherbets and water ices. There are no other major industrial uses, and other food and pharmaceutical uses consume but a minor amount. With the preserves industry as a firm business foundation, such modifications of pectin as low-methoxyl pectin, pectinic acid, and pectinate salts are now being presented to the trade for gradual development of their use. Although low-methoxyl pectin and the pectinates have properties similar to those of the algins, they are sufficiently distinctive to warrant some independent applications. This comment also applies to pectins from different sources. Apple and citrus pectins normally have significantly different gelling properties (with regard to tolerances, setting rates, and optimum conditions for set and gel characteristics). These proper-

286

LEONARD STOLOFF

ties can be varied not only by appropriate selection of the raw material but also by changes in the methods of manufacture. By far the largest amount of cellulose derivatives appears in the form of industrid-grade sodium carboxymethylcellulose, for use in detergent compositions and in oil-well drilling muds. Considerable amounts of “Na CMC” are also used for thickening latex emulsions and water-base paints. The largest use in foods is in compositions for ice-cream stabilizers. There are numerous other, miscellaneous, food and pharmaceutical applications, including that as a dental-cream binder. Methylcellulose and hydroxyethylcellulose find a variety of outlets in pharmaceutical and cosmetic compoundings and in polymer emulsions for paints, and paper and textile coatings. Agar has been facing a declining market because of its high price and its unpredictable quality. Supplies for microbiological purposes in the United States now come mostly from domestic production. This use actually represents but a small quantity, as compared to that needed for bakers’ icings and materials for dental impressions. For flat icings, it is an unexcelled stabilizer, but it has been meeting stiff competition from algins in the preparation of dental-impression compoundings, where the contest for the dentists’ favor is between isothermal and thermally-reversible gels. The algins enjoy a large market in paper and paper-board production, where they are used for body sizes and coating, and in the textile industry as a thickener for printing-pastes. The food industry employs large quantities in formulating ice-cream stabilizers, salad-dressing emulsions, and miscellaneous, bakers’ icings, toppings, and fillings. The cosmetics and pharmaceutical trades use fairly large quantities for making miscellaneous emulsions and suspensions and as tablet disintegrants (calcium alginate and alginic acid). Major outlets for carrageenans are in the food, pharmaceutical, and cosmetics industries. The unique ability of carrageenans to modify casein agglomerates in milk has resulted in its extensive utilization by the dairy industry in such products as chocolate-flavored drinks and frozen confections; puddings and desserts containing carrageenans are growing items in the market. Dental creams and hand lotions account for a large portion .of the consumption in cosmetics. Most applications of carrageenans involve use of some distinctive property related to their “milk reaction,” to their strong, anionic, polyelectrolyt,e nature, or to their peculiar gelling action and gel texture. The gelans which have the same kind of gelling properties as the carrageenans, compete with the carrageenans in Europe for a share of the market in puddings and other desserts.

POLYSACCHARIDE HYDROCOLLOIDS O F COMMERCE

287

Whether or not dextran can survive, to compete with the other polysacolloids in the market, depends on the ability of industrial chemists to find a unique application for it which would result in its consumption on a very large scale. The solution may well hinge on the results of current tests on oil-well drilling muds, particularly those in which the driller encounters salt-water flows.

V. USAGETRENDS The trends in the polysacolloid market are already well established.26*28 Uniformity and reliability of supply are important considerations in the selection of products. The costlier the item, the greater the tendency to select replacements for it (and, therefore, the greater the chance of finding an effective replacement). Gum tragacanth, a prime example of this thesis, has been steadily losing ground in the world of commerce; gum karaya is another example, on a lesser scale. Gum arabic, the cheapest of the exudate gums, is barely holding its own in commerce. Attempts by the import houses to blend and to control uniformity has had only a minor effect on this trend. According to some importers, rising standards of living in the countries of origin can only hasten the change by increasing the prices for these three key polysacolloids (gums tragacanth, karaya, and arabic), because, in these countries, there seems to be no cooperative effort aimed a t reversing this trend through the introduction of technological improvements. The concentrated, economic responsibility involved in the production of the other commercial polysacolloids has required research into raw materials, processes, and finished products. This is already proving rewarding in improvements in quality and in a better knowledge of those properties and applications facilitating the empirical task of selection. (25) Anon., U . S. Dept. Commerce, C h e w & Rubber Booklet, 2(6), 29 (1955). (26) B. Idson, Chem. Week, 57 (July 21, 1956).

This Page Intentionally Left Blank

ALKALINE DEGRADATION OF POLYSACCHARIDES

BY ROY L. WHISTLERAND J. N. BEMILLER Department of Biochentistry, Purdue University, Lafayette, Indiana

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Effect of 2-0-Substitution. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . 298

4. Effect of 4-0-Substitution. . . . . . . . . 5. Effect of 6-0-Substitution. . . . . . . . .

V. Products from Polysaccharide Degradation.. . . . 1. Effects of Alkali on the Main C h a i n . . ..................... 3. Degradation Procedure. ............................ 4. Cellulose., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . 310

. . . . . . . . . . . 312 ran. . . . . . . . . . .

. . . . . . . . . . . . 313

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

323

I. INTRODUCTION Chemists have long been aware that alkaline solutions effect structural changes in carbohydrates. As a consequence, although such solutions can be used effectively for the dissolution of most carbohydrates, including polysaccharides, contact of carbohydrates with them is usually avoided. Dread of their use as solvents for carbohydrates arose principally because early 289

290

R. L. WHISTLER AND J. N. BEMILLER

workers did not realize the importance of the exclusion of oxygen from such alkaline solutions, for, in the presence of oxygen, carbohydrates in alkaline solution undergo both oxidation and alkaline degradations to produce complex mixtures of products which are extremely difficult to analyze.’ These alkaline degradations can cause a loss of up to 25% (usually 10-20 %) of alpha-cellulose during alkaline refining of pulps. In the early years of carbohydrate chemistry, many chemists studied the effects of alkaline solutions on carbohydrates. Most investigations were made in the presence of oxygen or some other oxidant, and many different mechanisms for the formation of the observed products were proposed. Early investigations are summarized in a review by Evans.2 As stated by Kenner13it is unfortunate that alkaline degradations were not scrutinized further, for it has recently been demonstrated that the process of alkaline degradation in the absence of oxygen can be used in determining structures of carbohydrates. Had it been realized earlier that such degradation may be employed as a tool in structural determinations, the structures of many carbohydrates could have been determined before the development of the procedures of methylation analysis. Recently, the study of alkaline degradation has been re-opened, with the objective of using it as a means of determining the structures of oligoand polysaccharides and of oxidized polysaccharides. That interest has indeed been rekindled is attested by the recent reviews on saccharinic acids: on the modification of monosaccharides in alkaline solution,6 and on the hot alkali stability of chemically-treated, cellulose fibers.6 Ether derivatives of monosaccharides, oligosaccharides, and alkali-sensitive glycosides’ have been used as models in determining the effect of alkalis on oxygen-free solutions of polysaccharides and oxidized polysaccharides. In view of the large number of mechanisms which have been proposed over the years and which have been reviewed recently,4 a complete study of the effects of alkalis on carbohydrates will not be presented here. Only the 0-alkoxy carbonyl rnechani~rn’~ of degradation, which has now achieved general acceptance, will be discussed in detail. Use of the alkaline-degradation technique for the exploration of the chain structure of polysaccharides was first proposed by Corbett, Kenner, (1) J. U. Nef, Ann., 403, 204 (1914). (2) W. L. Evans, Chem. Revs., 31, 537 (1942). (3) J. Kenner, Chem. & Ind. (London), 727 (1955). (4) J. C. Sowden, Advances i n Carbohydrate Chem., 12, 35 (1957). (5) R . Pieck, Ind. chim. belge, %l, 1029 (1956); Chem. Abstracts, 61, 4033 (1957). (6) A. Meller, Australian Pulp & Paper Ind. Tech. Assoc. Proc., 7. 263 (1953); Chem. Abstracts, 49, 5829 (1955). (7) C. E. Ballou, Advances i n Carbohydrate Chem., 9, 69 (1951). (7a) H . S. Isbell, J . Research Natl. Bur. Standards, 32, 45 (1944).

ALKALINE DEGRADATION OF POLYSACCHARIDES

291

and Richards.* Whistler and Corbettg suggested that alkaline degradation could be used for determining branching in a polysaccharide. The degradation encountered is a peeling process (by glycoxy-anion elimination) which starts at the reducing end and proceeds through the chain, liberating saccharinate molecules (to which branches may be attached). Information obtained from the characterization of the fragments produced by alkaline degradation can then be used to indicate or to confirm structural assignations.

11. ACTIONOF ALKALION REDUCING END-UNITS Alkaline degradation of polysaccharides begins, in general, a t the reducing end of the molecule and proceeds in a stepwise manner through the anhydroglycose chain (for exceptions, see p. 314). Consequently, an understanding of the effects of aqueous, alkaline solutions on the reducing end-group (or on modifications of it) is essential to comprehension of the mechanism of alkaline degradation. Reducing glycose units of polysaccharide chains will be transformed, in part, to their C2-epimers in alkaline solution. The classical transformation of Lobry de Bruyn and Alberda van EkensteinlO is a base-catalyzed enolization giving an enediol (11) which may either revert to the starting aldose or be converted to epimers of the original aldose (see Fig. 1); they showed that the main product is the ketose. However, the ions (I and 111) may also be in equilibrium by prototropy, without transition through an enediol (11). Support for this mechanism is given by (a) the observation of acquisition of carbon-bound deuterium when the reaction takes place in deuterium oxide,ll. l2 (b) the fact that D-fructose has been shown not to be a necessary intermediate in the conversion of D-glucose to D-mannose,’2 and (c) a n indication of the occurrence of a common intermediate which is a singlycharged, enolate ion.*3Evidence for an enediol form of sugars in alkaline solution is afforded by the ability of alkaline sugar solutions to take up large quantities of iodine,14 by the observation that sugars are oxidized (8) W. M. Corbett, J. Kenner and G. N. Richards, Chem. & Znd. (London), 462 (1953). (9) R. L. Whistler and W. M. Corbett, J . A m . Chem. SOC.,78, 1003 (1956). (10) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. chim., 14, 156, 203 (1895); 16,92 (1896); 16, 257, 262, 274, 282 (1897); 18, 147 (1899); 19, 1 (1900). See J. C. Speck, J r . , this volume, page 63. (11) Y. J. Topper and D. Stetten, Jr., J . Biol. Chem., 169, 191 (1951). (12) J. C. Sowden and R. Schaffer, J . A m . Chem. SOC.,74,505 (1952). (13) C. H. Bamford and J. R . Collins, Proc. Roy. SOC.(London), A228, 100 (1955). (14) (a) M. L. Wolfrom and W. L . Lewis, J . A m . Chem. SOC.,50, 837 (1928); (b) J . H. Simons and H. C. Struck, ibid., 66, 1947 (1934).

292

R. L. WHISTLER AND J. N. BEMILLER

with cleavage between C1 and C2 in alkaline solutions,', 1 6 and by studies of the ultraviolet absorption spectra (which show a maximum, characteristic of dicarbonyl compounds, attributedl6 to an enediol). Further evidence for the formation of an enediol is furnished by the observed ability of alkaline solutions of carbohydrates to decolorize solutions of 2,6H

H

I

t--*

R

R

R

' +H@JI-+

H

I

COH

II

COH

I

R (cis or Irans)

+H@

L

-H@

R

H

I

HCOH

I I

c=o R

I11 FIQ.1 .-The Classical Base-catalyzed Transformation of an Aldose (Lobry de Bruyn and Alberda van Ekenstein).

dichlorophenolindophenol16"and by the results of conductometric measurements which show that reducing sugars in alkaline solution behave as weak, dibasic acids." However, conductometric and polarographic measure(15) (a) J. U. Nef, Ann., 367, 214 (1907); (b) 0.Spengler and A. Pfannensteil, Z . Ver. deut. Zucker-Znd., 86, 546 (1933);Z . Wirtschaftsgruppe Zuckerind., Tech. TI., 86, 546 (1935). (16) F.Petuely and N . Meixner, Chem. Ber., 86, 1255 (1953). (16a) A. Kusin, Ber., 69, 1041 (1936);Biokhimiya, 1, 75 (1936). (17) P. Hirsch and R . Schlags, 2.physik. Chem. (Leipzig), A141,387 (1929).

ALKALINE DEGRADATION OF POLYShCCHARIDES

293

ments indicate acidity and complex formation in nonreducing sugars.*8 Here no enol is possible, so the observed acidic properties might be occasioned by permanent polarization; and this might serve to explain the observed high reactivity of the hydroxyl group on C2 in alkaline solutions of sugars (see p. 295). Additional evidence is afforded by (a) the identity of the products formed, (b) the fact that 2,3,4,6-tetra-O-methyl-~-glucose~~(~) and 2,3,4,6-tetra-O-methyl-~-mannose~~ reach the same equilibrium point when placed in alkaline solution, and that each takes up one atom of deuterium per molecule in alkaline deuterium oxide solutionlZ0(c) the successful epimerization of 2,3,4-tri-O-niethyl-~-xylose,~~ and (d) an observed chemical combination with reagents, such as ninhydrin,22 specific for enediols. I n alkaline solutions of D-glucose, the comparative rate of reaction, as well as the preferred order of formation of its C2-epimers, is in the order D-fructose > n-glucose > manno nose.^^ Universal applicability of the epimerization reaction to sugars may be demonstrated by a few examples additional to those already mentioned. In dilute bases, lactose forms l a c t ~ l o s e ,D-glycero-D-gluco-heptose ~~ yields I)-gluco-heptulose,26D-glycero-D-manno-heptose affords u-manno-heptulose,26 and 1,3-dihydroxy-2-propanone is formed from ~-glycerose.~' Even alkaline impurities in the paper of a chromatogram can cause (at least, several) sugars to be partly converted to their corresponding epimers.28However, it has been stated that mixtures of D-glucose and D-mannose can be separated by paper electrophoresis in a buffer of pH 9.2, although having been heated in it for three h o ~ r s . ~When g the hydroxyl group on C2 is blocked, no ketose is formed. Epirnerization reactions show cationic dependence. For example, it is reported that u-glucose in dilute solutions of calcium hydroxide is transformed in part to D-mannose but only slightly to D-fructose, whereas, in (18) P. M. Strocchi and E. Gliozzi, Ann. chim. (Rome), 41, 689 (1951); Chem. Abstracts, 46, 4826 (1952). (19) R . D. Greene and W. L. Lewis, J. Am. Chem. Soc., 60, 2813 (1928). (20) H . Fredenhagen and K . F. Bonhoetfer, 2. physik. Chem. (Leipzig), A181, 392 (1938). (21) C. E. Gross and W . 1., Lewis, J . A m . Chem. SOC.,63, 2772 (1931). (22) E. S. West and R . E. Rinehart, J . Biol. Chem., 146, 105 (1942); M. C . Nath, V. K. Sahu and R. M. Behki, Metabolism Clan. and E z p l l . , 6, 18 (1956). (23) J . C . Sowden and R. Schttffer, J. A m . Chem. SOC., 74,499 (1952). (24) E. M. Montgomery and C. S. Hudson, J . A m . Chem. SOC.,6 2 , 2101 (1930). (25) W. C. Austin, J . Am. Chenz. SOC.,63. 2106 (1930). (26) E. M. Montgomery and C. S. Hudson, J . A m . Chem. SOC.,61, 1651 (1939). (27) H . 0. L. Fischer, C. Taube and E. Baer, Ber., 60, 480 (1927). (28) It. B. Duff, Chem. d% Znd. (London), 898 (1953). (29) n. R . Briggs, E. F. Garner, R. Montgomery and F. Smith, Anal. Chem., 28, 1333 (1956).

294

R. L. WHISTLER AND J. N. BEMILLER

dilute solutions of sodium hydroxide, D-fructose but almost no D-mannose is formed.lea Other observations of cationic dependence have also been recorded. However, it is reported that different bases and temperatures affect the velocity of the reaction but not the nature or proportion of the products. The chief factor governing the extent of formation of ketoses and saccharinic acids is the proportion of base in solution.30These rate differences may be due partly to the divalent nature of the calcium ion, which would enable it to react with adjacent hydroxyl groups, or partly to a more rapid conversion of ketose in calcium hydroxide due to stimulation of carbonyl reactivity3' by (CaOH)@comparable with that effected by lithium ions on the interaction of diazomethane and acetone.32 Aldonic acids, although more stable than their corresponding aldoses, also undergo epimerization a t higher t e r n p e r a t ~ r e s . ~ ~Epimerization . can occur even though the C2 hydroxyl group is blocked with an alkyl gr0up.~4I n alkaline solutions, galactaric acid is transformed to DL-talaric acid.36 I n all cases studied, lactones of sugar acids epimerize more readily than salts of these acids because of the more acidic nature of the hydrogen atoms on the alpha carbon atom of esters as compared with that of carboxylate anions. Greater ease of removal of protons (by bases) from the alpha carbon atom of esters as compared with that in carboxylate anions is attributed to resonance in the carboxylate anion, which dissipates the charge so that the carbonyl group loses some of its identity. As a consequence, the carboxylate group's electron-withdrawing properties are diminished. By continued enolizations, additional isomerizations are possible, but there is little evidence for enediols other than the 1,2-enediol and the 2,3-enediol (see, however, page 328). Alditols do not epimerize in oxygen-free, dilute alkaline solutions a t temperatures used for alkaline-degradation reactions.

111. EFFECTS OF ALKALION HYDROXYL GROUPS Naturally occurring polysaccharides might be affected by alkaline solutions in places other than the reducing end. Other possible sites of reaction include (a) glycosidic Einkages, which are stable to aqueous alkalis at the temperatures ordinarily used for alkaline degradation reactions [as can (30) J. B. Gottfried and D. G, Benjamin, Znd. Eng. Chem., 44, 141 (1952). (31) J. Kenner and G. N. Richards, J. Chem. SOC.,278 (1954). (32) H. Meerwein and W. Burneleit, Ber., 61, 1840 (1928). (33) E. Fischer, Ber., 23, 799 (1890); 0. F. Hedenburg and L. H. Cretcher, J. Am. Chem. SOC.,49,478 (1927); H. T. Bonnett and F. W. Upson, ibid., 66, 1245 (1933). (34) W. N. Haworth and C. W. Long, J . Chem. SOC.,345 (1929). (35) T. Posternak, Naturwissenschaften, 23, 287 (1935).

ALKALINE DEGRADATION OF POLYSACCHARIDES

295

be established by the fact that, in general, polysaccharides in oxygenfree, alkaline solutions are not hydrolyzed or do not undergo rapid diminutions in molecular weight (see p. 323)], (b) uronic acid groups or their esters [the effects on which will be discussed later (see p. 313)], and (c) hydroxyl groups. Effects of alkalis on the various hydroxyl groups found in polysaccharides have been mainly studied by investigating the reactivity of the various hydroxyl groups in etherification reactions which take place in alkaline solutions. Reactivities can be determined because substitutions, under the conditions employed, proceed according to the laws of chance, and the three kinds of hydroxyl groups available are substituted to an extent (relative to one another) that is determined by the relative reactivities of the groups and the nature of the reaction.36 Many investigations of this type have been but the limited scope of this review does not permit complete coverage of all the available information. Starch and cellulose have been used for most of these studies, but similar results have been obtained with guaran.” In general, the C2 hydroxyl group is more reactive than the C6 hydroxyl group, and both the C6 and the C2 hydroxyl groups are much more reactive than the C3 hydroxyl group. In other cases, the C6 hydroxyl group is more reactive than the C2 hydroxyl group. In all instances, the C6 and the C2 hydroxyl groups are the most reactive, and differences between these two are often small and dependent on reaction conditions and structural variation. Of course, in permutoid reactions of cellulose where the crystalline structure influences the distribution of groups, the primary hydroxyl group on C6 does not participate, and its unavailability has been attributed to its strong hydrogen-bonding ~apability.~~ However, all the free hydroxyl groups of cellulose are weakly ionizable, as is shown by the complete exchange of hydrogen for deuterium which takes place when it is immersed in deuterium oxide.40The more acidic properties of the C2 hydroxyl group might be attributable to a permanent polarization of hydroxyl groups adjacent to a carbonyl or potential carbonyl group (see p. 293). The type of reaction between alkalis and hydroxyl groups of polysaccharides has also been thoroughly ~tudied.4~ An examination of the (36) H. M. Spurlin, J . A m . Chem. Soe., 61.2222 (1939). (37) J. M. Sugihara, Advances i n Carbohydrate Chem., 8 , 1 (1953). (38) 0. A. Moe, S. E. Miller and M. I. Buckley, J . A m . Chem. SOC.,74,1325 (1952). (39) A. G. Assaf, R . H. Haas and C. B. Purves, J . Am. Chem. Soc., 66, 66 (1944). (40) G . Champetier and R. Viallard, Compt. rend., 206, 1387 (1937); Bull. SOC. chim. France, [5] 6 , 1042 (1938). (41) See W. D. Nicoll, N. L. Cox and R. F. Conaway, in “Cellulose and Cellulose Derivatives,” E. Ott, ed., Interscience Publishers, Inc., New York, N. Y., 2nd Edition, 1954, p. 825.

296

R. L. WHISTLER AND J. N. BEMILLER

exchange of 0 1 8 between water and alkali-cellulose established that a molecular complex is formed between the hydroxyl groups of cellulose and sodium h y d r ~ x i d e .Indirect ~~ evidence for complex formation is afforded by the observation that true alkoxides formed in liquid ammonia are decomposed by m0isture.4~ Again, cationic differences are evident. For instance, potassium hydroxide is more effective in methylation (with dimethyl sulfate) than sodium hydroxide.44 I n alkali-cellulose, addition products are formed by the interaction of alkalis with hydroxyl groups, and the tendency for alkoxide formation increases in the order LiOH < NaOH < KOH < RbOH < CsOH < organic quaternary bases.46 OF LINKAGES ON ALKALINE DEGRADATION IV. EFFECTS

As stated previously, the alkaline degradation of polysaccharides proceeds by a peeling process in which the reducing end-group is liberated from a chain by elimination of the rest of the chain as a glycoxy anion. Elimination takes place when the chain is in the position beta to a carbonyl group of the reducing end-unit.'" Eliminations of this type can be exemplified by the action of alkali on 4-ethoxy-2-butanone (IV) to form463-buten-2-one (V). O H II I CH3-C-C-CH2-

+.

O-CHa-CH,

IV 10.

+ O H [CH-,(!;&

O0

CH,f)O-CH2-CH,

+--+

CH~-C-C=CH~ V

CH3-C=C€I-CH2I

O-CH,-CH,

1

+ ~o--cH~--cH~

Elimination of alkoxy anions takes place under alkaline conditions not only when there is a carbonyl group in the position beta to the alkoxide (42) I . A. Makolkin, Zhur. Obshchei Khim., 12, 365 (1943); Chem. Abstracts, 57, 3418 (1943). (43) Ref. 41, p. 873. (44) H. A. Hampton, W. N . Haworth and E. L. Hirst, J . Chem. SOC.,1739 (1929). (45) S. V. Bleshinskii and S. F. Loeitskaya, Trudy Khim. Inst. Kirgiz. Filial Akad. Nauk S. S. S. R . , 4, 73 (1951) ; Chem. Abstracts, 49, 14315 (1955). (46) A. Treibs, Angew. Chem., A60, 289 (1948).

297

ALKALINE DEGRADATION OF POLYSACCHARIDES

group but a t any time when there is an easily removable proton on the carbon atom in the position alpha to the alkoxide group. For example, ethylmagnesiuin iodide reacts with 2-methoxypropionitrile (VI) to acrylonitrile (VII). EtMgI

CH3-CH-CH2-CN

f

I

+ CHaOH

CHa-CH=CH-CN

0 CH3 VI

VII

An alkoxy or glycoxy anion is more easily eliminated by the ionized enediol than is a hydroxyl ion, and the pyrariose rings of the released glycoxy anions will open readily because of the tendency of negatively charged oxygen to form a double bond with carbon. The released end-group forms an a-dicarbonyl structure which rearranges by an intramolecular, Cannizzaro type of reaction to yield saccharinates. Intramolecular reactions of the Cannizzaro type can occur not only with dialdehydese but also** with a-ketoaldehydes and a-diketones; ("benzilic acid rearrangement") ; they can be exemplified by the base-catalyzed rearrangement of phenylglyoxal (VIII) [or of (2,4 ,6-trimethylpheny1)g l y ~ x a lto60 ~ ~ ]the salt of inandelic acid (IX) (or of 2,4,6-trimethylmandelic acid). 0

0

0

I II (I)-C-C-H

+

fast

OH"

0"

III 0-C-C-OH

H

..

cii :';:2"

'$-c-c-OH

VIII

slow

OH

0" 0

I II I

a)-C-C-OH H

fast

'

I

@-C-C0P

I

H IX

No carbon-bound deuterium is found when the reaction takes place in (47) P. Bruylants and L. Mathus, Bull. classe sci. Acad. roy. Belg., 151 11, 636 (1925) ; Chem. Abstracts, 20, 1785 (1926). (48) J. Thiele and 0. Giinther, A n n . , 347, 106 (1906); E. M. Fry, E. J. Wilson, Jr., and C. S. Hudson, J . Am. Chem. SOC.,64, 872 (1942). (48a) F. H. Westheimer, J . Am. Chetn. Soc., 68. 2209 (1936); I. Roberts and H. C. Urey, ibid., 60,880 (1938). (49) A. R. Gray and R. C. Fuson, J . Am. Chem. Soc., 66, 739 (1934). (50) E. R. Alexander, J . Am. Chem. SOC.,69, 289 (1947).

298

R. L. WHISTLER AND J. N. BEMILLER

deuterium oxide solution,61and the hydrogen atom (not the phenyl group) migrates.s1-s2 Cannizzaro reactions are best catalyzed by calcium hydroxide solutions.6s Successive elimination of an alkoxy group adjacent to an active hydrogen atom and intramolecular Cannizzaro reactions in alkaline solution, such as are found in saccharinate formation, are demonstrated by the conversion of 2-hydroxy-3-methoxy-3-phenylpropiophenone(X) to a s a l P of 2,3diphenyllactic acid (XI). 0

OHOCHi

II I

I

I

I

OH

H 6

I I Cb

X

XI

0-C-C-C-H

0139

.

eOOC-C-CHe+

1. Efect of 1-0-Substitution

Acetal linkages of 1-0-substituted aldoses are ordinarily stable in oxygenfree, alkaline solutions (see p. 323). However, substitution on the C l hydroxyl group of D-fructose causes the formation of large proportions of (XII). D-GlucosacDL-lactate and of some ‘La”-~-glu~~~a~~har~nates6s charinates are formed either by recombination of three-carbon fragmentss6 or by the anion-elimination scheme (see Fig. 2). The predominance of lactates over D-ghcosaccharinates may be due to a more facile, reversealdolization reaction in comparison with an elimination of an anion.

2. Eflect of 6-0-Substitution Saccharides substituted on the C2 hydroxyl group cannot form saccharinates in dilute alkalis6’because they are unable to form the necessary carbonyl group in the position beta to the substituted hydroxyl group. Neither are saccharinates formed from 2,3-di-O-methyl-~-glucose (XIII) which, in alkaline solution, has an absorption band corresponding to that of an a ,p-unsaturated carbonyl derivativeu (XIV). The trisaccharide, a-~-fucopyranosyl-(l-t2)-~-~-galactopyranosyl-(l~4)-~-glucose, (51) W. von E. Doering, T. I. Taylor and E. F. Schoenewaldt, J . Am. Chem. SOC., 70, 455 (1948). (52) 0. K. Neville, J . Am. Chem. SOC.,70, 3499 (1948). (53) E. Pfeil, Chem. Ber., 84, 229 (1951). (54) B. H. Nicolet, J . Am. Chem. Soc., 63, 4458 (1931). (55) J. Kenner and G. N. Richards, J . Chem. Soe., 1784 (1954). (56) J. C. Sowden and D. J. Kuenne, J . Am. Chem. Soc., 76,2788 (1953). (57) (a) R. L. Whistler and W. M. Corbett, J . Am. Chem. SOC.,77, 3822 (1955); (b) 77, 6328 (1955). 2921 (1956). (58) J. Kenner and G. N. Richards, J . Chem. SOC.,

299

ALKALINE DEGRADATION OF POLYSACCHARIDES

H

H

HCOR

I c=o HOC.\H I (

c=o

+He -H'

HOC:. I

t)

I HCOH I HCOH I

HCOH

I HCOH I

CHzOH

H

H

I HCOR I-

I

CHzOH

I HCOR I C-oe II HOC I HCOH I HCOH I

I

HCOR

I

COH

HCOH

I I

HCOH CHzOH

CH20H

/f

H

H

I

HCOR

I

qy? c-0 :

I -. HCOH I HCOH I

H

I HCOR *hOH t)

CH,OH

I II COH I C=O I HCOH I HCOH I HC

I c=o I

+Roe+

HCOH

I I

HCOH

CHzOH

CHzOH

H

I

HCH

OH

I I

HCOH

I

HCOH

I

CHzOH

OH

I 9.

I c=o

Hn C-C-0 +OHe,

' -0He

IA

LC=O

I

:

+ H3C-C-Oe

HCOH

I

HCOH

I

I c=o I

CH~OH

09

+

I

HCOH

I I

I I

c=o

HCOH CH~OH

C(CH8)(OH)

I

HCOH

I

HCOH

I

CHzOH XI1

FIQ.2.-The Anion-elimination Scheme for the Formation of D-Glucosaccharinates (where R = the remaining portion of the polysaccharide molecule).

300

R. L. WHISTLER AND J. N. BEMILLER

is degraded in alkaline solution to form a disaccharide of L-fucose with Dgalact~se.~o After elimination of the reducing end-unit, a 4-O-substituted CHO

CHO

I I

MeOCH

I HCOH I HCOH I

CHzOH

XI11

I I1 C-H I HCOH I HCOH I

C-OMe

HCOMe

o ~ e >

CHzOH

XIV

D-glucose (see p. 302), the remaining disaccharide is relatively stable to alkali because it is 2-0-substituted. Likewise, L-galactopyranosyl-(1--14)-~xylopyranosyl-( l--t2)-~-arabinoseis stable to lime ~ a t e r ~ "a t~ 25". ) 3. Efect of 3-0-Substitution

Saccharides substituted on the C3 hydroxyl group form metasacchurinates. Degradation of 3-O-methyl-~-glucose,~~ 3-0-methyl-~-fructose,~~ and 3 ,6-anhydro-D-glucoseGo affords D-glucometasaccharinates (XV), 3-deo~y-'~~-glucontlte,'~ and 3-deoxy-"D-mannonate" (see Fig. 3). Lamintirihiose (3-O-~-~-glucopyranosyI-~-glucose) and turanose (3-0-aD-glucopyrsnosyl-r+fructose) have about equal rates of so there must be common anions involved-anions which are the singly charged, enolate anions in equilibrium with each other. Degradations of 3-O-methyl-~-fructoseand 3-O-methyl-~-glucoseproceed a t virtually the same rate.31However, the degradation of 3-O-methyl-~-fructoseis slightly faster than that of 3-O-methyl-~-glucose,in agreement with the fact that D-fructose ionizes more rapidly than ~ - g l u c o s e . ~ ~ Lability to 0.05 N sodium carbonate solution also is shown by ~ - O - D galactopyranosyl-D-glucose and 3-O-~-galactopyranosyl-~-fructose.~~ From 3-0-methyl-n-xylose are formed the two analogous five-carbon metasaccharinates. The main component of the reaction mixture is 3-deoxyD-"lyxonate," although small amounts of 3-deoxy-~-"xylonate'~ are also found." Furthermore, 3-O-c~-~-xylopyranosyl-~-arabinose is degraded, in lime water, to an acid salt and ~ -x y lo se.~ 7(~ Alkaline ) degradation of 3-0(59) R. Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 88, 1135 (1955). (60) W. M. Corbett and J. Kenner, J . Chem. SOC.,927 (1957). (61) W. M. Corbett and J. Kenner, J . Chem. SOC.,3274 (1954). (62) R. Kuhn, H. H. Baer and A. Gauhe, Chem. Ber., 87, 1553 (1954). (63) W. M. Corbett, G. N. Richards and R. L. Whistler, J . Chem. SOC.,11 (1957).

H

H

H

H

I

I

I H Yj C O I c=o I ROCH I HCOH 1 HCOH I

c: YE c=o I I HCOH I HCOH I

-H" ,

ROCH

7-

+H"

COH

II

C-oe

I

c-)

I I HCOH I HCOH

CHsOB

CHzOH

CHzOH

ROCH

11 H

H

c=o

c=o

I H-COH I ROCH I HCOH I HCOH I

.

c=o COH I II

+ CH I

II c COH I ROCH I HCOH I HCOH I

c)

I

HCOH HCOH

I

CHzOH

CHzOH OH

H

I ?. ..

I

c=o c=o I

+

I

H-C-O:e

Lb=O +OHe,

HCH

I

' -OHe

l-

HCH

HCOH

HCOH

I HCOH I

I HCOH I

HCOH

I I

CHzOH

CHzOH

OH

00

1 c=o

I c=o I C O 3 (OH) I

I I HCH I HCOH I HCOH I

HC-Oe

CHzOH

-+

+

II

HCOH

CHzOH

:e

*.

ROCH

I

R

c-0

I

CHzOH H

ROe Roe

I ?.

I

I

-

H

HCH

I I HCOH I

HCOH

CHzOH XV

FIQ.3.-The Alkaline Degradation of (1+3)-Linked D-Glucoglycans (where = the remaining portion of the polysaccharide molecule). 301

302

R. L. WHISTLER AND J. N. BEMILLER

substituted sugars has been used for the synthesis of compounds which would be difficult to prepare by other mean^.^^-^^ 4, E$ect of 4-0-Substitution Alkaline degradation of 4-0-substituted sugars affords isosaccharinates (see Fig. 4). Both 4-O-methyl-~-glucose,which is first transformed to 4-0-methylD-fructose, and 4-O-methyl-~-fructose yield the same D-glucoisosaccharinates” (XVI). Other saccharides which are degraded with the formation of D-glucoisosaccherinates are maltose,68 maltulose,68(d) lactulose,@(0)cellobiose,7° cellobiulose,’o(b)cellotetraose,7°(b)partially hyDegradation of drolyzed cellulose,” and 4,6-O-benzylidene-~-glucose.~~ 4-0-substituted sugars is also indicated by the lability of a-L-fucopyranosyl(l+2)-~-~-galactopyranosyl-(l+4)-~-glucose,~~ D-Glucose, liberated by 4-0the action of alkalis on maltose, can epimerize to ~-mannose.7~ Methyl-D-fructose, as expected, is degraded faster than 4-O-methyl-~glucose, but both are degraded more slowly than the corresponding 3-0substituted sugars. The same relationship is observed with the aldose-ketose pairs, maltose-maltulose and lactose-lactulose. However, both members of the latter pair react more slowly than either maltose or maltulose. Cellobiose is also degraded much more slowly than maltose and at about the same rate as lactose. In the three mechanisms given (see Figs. 2, 3, and 4), only those ions necessary for the alkoxy-anion elimination have been presented. However, it must be remembered that a ketose can afford two ions, one of which is (64) J. Kenner and G . N . Richards, J . Chem. Soc., 3277 (1954). (65) W. M. Corbett, J. Kenner and G. N. Richards, J . Chem. SOC.,1709 (1955). (66) J. Kenner and G. N. Richards, J . Chem. SOC.,2916 (1956). (67) J. Kenner and G. N. Richards, J . Chem. SOC.,1810 (1955). (68) (a) A. P. Dubrunfaut, Monit. sci Docteur Quesneville, [3] 12, 520 (1882); (b) W. L. Lewis, Am. Chem. J . , 43,301 (1909); (c) W. L. Lewis and S. A. Buckborough, J . Am. Chem. SOC.,36,2385 (1914); (d) W. M. Corbett and J. Kenner, J . Chem. SOC., 1789 (1954). (69) (a) M. L. Cuisinier, Monit. sci. Docteur Quesneville, [3] 12, 520 (1882); Bull. soc. chim. (France), [2] 38,512 (1882); (b) H. Kiliani, Ber., 42,3903 (1909); (c) W. M. Corbett and J. Kenner, J . Chem. SOC.,2245 (1953). (70) (a) S. V. Hintikka, Ann. Acad. Sci. Fennicae, 9, 3 (1922) ; Chem. Zentr., 94.1, 296 (1923); (b) W. M. Corbett and J. Kenner, J . Chem. SOC.,1431 (1955); (c) J. W. Green, J . A m . Chem. SOC.,78, 1894 (1956). (71) (a) J. J. Murumow, J . Sack and B . Tollens, Ber., 34, 1427 (1901); (b) C. G . Schwalbe and E . Becker, J . prakt. Chem., 100, 19 (1920); (c) J. Palm&, Finska Kemislsamfundets Medd., 38, 106 (1929); Chem. Abstracts, 24, 1625 (1930). (72) C. A. Lobry de Bruyn and W. Alberda van Ekenstein, Rec. trav. chim., 18, 147 (1899); L. Kolb, Biochem. Z., 63, 1 (1914).

H

H

I c=o I

H - ~ O H

I

-He

HOCH

. +H'

I HCOR I HCOH I

H

1c=o I f :COH I HOCH I HCOR I HCOH I

CHzOH

I

C-oe

-

II I

COH HOCH

I

HCOR

I I

HCOH

CHzOH

CHzOH

11 H

H

II

HCOH

H

HCOH

I

c=o HOCFH I .f

-

[HO HCOR I:)

I HCOR

I I

I I

HCOH

HCOH

CHzOH

CHzOH

H

I I Roe + C=O I HOC II HC I HCOH I

I HCOH 1 C-oe II HOC I HCOR I HCOH I

1

CHzOH

H

I I c=o I c=o

HCOH

OH ~n I ** HOHzC-C.--O :e

HCOH

--I

L cI = o.*

+OHa

I HCH

7

I-

'-OHe

I

I HCOH I

CHzOH

+

HCH HCOH I

I

CHzOH

CHzOH

OH

Oe

c=o

c=o

I

I

I

HOH~C-C-O~

I

+

I I HCOH I

C(OH)(CH,OH)

I I HCOH I

HCH

HCH

CHzOH

CHzOH XVI

FIG.4.-The Alkaline Degradation of (1-+4)-LinkedD-G~UCOand D-Mannoglycans (where R = the remaining portion of the polysaccharide molecule). 303

304

R. L. WHISTLER AND J. N. BEMILLER

not essential for the reaction. The various ions are in equilibrium with each other, and the rate of conversion of one ion into another is not the final rate-controlling step but may be the rate-controlling step in the very early stages of the rea~tion.~a Initial rates for the formation of metasaccharinates (see Fig. 3) and isosaccharinates (see Fig. 4) are explained by the relatively slow conversion of an aldose anion (XVII) to a ketose anion (XVIII), whereas the opposite reaction is somewhat more rapid.2a HC-00

II COH I

R XVII

HCOH slow

' mpid

II I

C-00

R XVIII

In the degradation of either the 3-0-substituted sugars or the 4-0-substituted sugars, there may occur side reactions through the elimination of a hydroxyl ion (see p. 308). Xylobiose and xylotriose are degraded by alkaline solutions to give the corresponding five-carbon xyloisosaccharinates, (%)-2,4-dihydroxy-2(hydroxymethyl)butanoates.g 74 Degradation of 4-0-substituted sugars has been used in syntheses.66 5 . Efect of B-O-Substitution

It is reported that substitution of the primary hydroxyl group has an effect on the formation of saccharinates and, therefore, must have an effect on the ionic species which are formed by the action of dilute alkalis.66.76 Sugars substituted only on the C6 hydroxyl group cannot eliminate an alkoxy anion directly because of their inability to enolize to form a carbony1 group at C4. However, substitution of the C6 hydroxyl group could result in a more pronounced attack by alkali on the C4 hydroxyl group. Ionization of the C4 hydroxyl group would cause a reverse aldolization reaction (see p. 326) giving 1 ,3-dihydroxy-2-propanone,which would rearrange to DL-lactate, and a 3-0-substituted glycerose, which would be subject to further attack.66 Thus, one mole of melibiose (6-0-a-~-galactopyranosyl-D-glucose) yields two moles of lactate plus one mole of D-galacDL-Lactate, the tose or one mole of ~-ga~actopyranosy~-saccharinates.~~ main product of the alkaline degradation of 6-0-substituted sugars, is ~ ~ B-O-methyl-~-gluproduced in yields of about 80% from r n e l i b i ~ s eand cose,66as compared to 67% under similar conditions from D-glucose. The (73) W.M.Corbett, personal communication. (74) G. 0. Aspinall, M. E. Carter and M. Los, Chem. & Znd. (London), 1553 (1955); J . Chem. SOC.,4807 (1956). (75) W.M. Corbett and J. Kenner, J . Chem. SOC.,3281 (1954).

ALKALINE DEGRADATION O F POLYSACCHARIDES

305

effect of substitution in the 6-0-position is also seen in the more rapid attack by lime water on 3,6- and 4,6-di-O-methyl-~-glucose,respectively, than on 3- and 4-O-methyl-~-glucose,respectively. Most of the other acid salts which are formed are of the metasaccharinate type. The influence of 6-0-substitution on ionization has been proposed as the reason for the formation of metasaccharinates.66 Lactate is also formed from (a) 2,3,4,6-tetra-0-acetyl-/3-~-glucosyl(1- 4 - 2, 3, 6 - tri-O-acetyl-@-r,-glucosyl- (1-6) - 1,2,3,4-tetra-0-acetylP-D-glucose, (b) 2,3,4,6-tetra-O-acetyl-cr-~-glucosy~-(1-+4)-2,3,6-tri-Oacety~-~-~-glucosyl-(l-6)-1,2,3,4-tetra-0-acetyl-~-~-g~ucose, (c) 2 , 3 , 4 , 6- tetra -0 -acetyl - p - D -glucosyl- (1-4) -2 ,3 ,6 - tri- 0- acetyl-8 - D - glucosyl(1+6)-1,2,3,4-tetra-O-acetyl-P-~-mannose, and (d) 2,3,4,6-tetra-Oacety~-a-~-g~ucosyl-(l-4)-2,3,6-tri-O-acetyl-~-~-g~ucosyl(1-+6)-1,2,3,4tetra-0-acetyl-b-wrnannose as expected, in yields which are about twice as great as those from u-glucose or D-mannose 6. Efect of Structure of Glycosyl Units on Saccharinic Acids Produced Presence of an 0-alkyl or an 0-glycosyl group on the hydroxyl group a t C3 or C4 determines the type of acid produced. Therefore, formation of the ordinary, the meta, and the is0 type of D-glucosaccharhate is diagnostic of 1-0-substituted D-fructose, 3-0-substituted D-glucose or D-fructose, and 4-0-substituted D-glucose or n-fructose derivatives, respectively, if the identity of the hexose is known. Saccharinates derived from other substituted glycoses can be used as reference compounds, although some duplication will occur. For example, the same metasaccharinates will be derived from 3-0-substituted u-glucose, u-fructose, D-allose, D-mannose, and D-altrose; and all D-hexoses will produce D-isosaccharinates. Degradation of substituted 2-acetamido-2-deoxy-~-glucoses has been only cursorily investigated. In dilute bases, unsubstituted N-acetyl-Dglucosamine yields* a heterocyclic ring compound (glucoxaeoline).77The free amino group in D-glucosamine stabilizes the molecule by its ionic nature, so that it cannot be methylated by the Haworth reagent^.'^ I n 0.05 N sodium carbonate, 2-acetamido-2-deoxy-3-O-(~-~-galactopyr~nosyl) - D -glucose is easily degraded to D -galactose (and to the isomeric D-tagatose, a t high temperatures) and an unidentified product. Under the (76) 8. H.Nichols, Jr., W. L. Evans and H. D. McDowell, J . Am. Chem. Soc., 62, 1754 (1940). * But me S. Roseman and D. G. Comb, J . AWL.Chem. SOC.,80,3166 (1958). (77) T. White, J . Chem. Soc., 428 (1940);W. H.Bromund and R. M. Herbst, J . Org. Chem., 10, 267 (1945). (78) W. 0. Cutler, W. N . Haworth and 8. Peat, J . Chem. Soc., 1979 (1937);M. Stacey and J. M. Woolley, i bi d., 184 (1940).

306

B. L. WHISTLER AND J. N . BEMILLER

same conditions, 2-acetam~do-2-deoxy-4-0-(~-~-galactopyranosyl)-~-g~ucose is stable.@

V. PRODUCTS FROM POLYSACCHARIDE DEGRADATION 1 . E$ects of Alkali on the Main Chain

a. Glycans linked (2 -+ 1) and composed of ketose units, such as i n ~ l i n , ? ~ should be degraded with the formation of ordinary saccharinates.66 b. Glycans linked (1 -+ 2), such as crown-gall polysaccharide,80 should be stable67to dilute alkali a t 25”. c. Glycans linked (1 -+ 3), such as 1aminaran,81lichenan,82and certain galactanss3 and xylan~,8~ should be degraded with the production of metasaccharin ate^.^^ 6 0 , 63 These glycans require no rearrangements before chain elimination can proceed. d. Glycans linked (1 -+ 4), such as amylose,86 cellulose,8e ~ y l a n , ~and ’ mannans,88 should be degraded with the formation of isosac~harinates.~ 67-71-74 These glycans must first rearrange to a ketose form, prior to chain elimination. e. The behavior of 5-0-substituted sugars in alkaline solution is unknown. However, (1 5)-linked glycans, such as galactocarolose,8Q~ would probably be resistant to saccharinate formation. f , Glycans linked (1 6), such as p u s t ~ l a nbarley-root ,~~ g l y ~ a n and ,~~ dextrans from Leuconostoc rnesenteroide~,~3 should be relatively stable in dilute alkaline solutions a t 25”, but should be degraded very slowly, with the formation of some lactate, a t higher concentrations of alkali and higher 9

--f

--f

7 6 , 76

(79) R. L. Whistler and C. L. Smart, “Polysaccharide Chemistry,” Academic Press Inc., New York, N . Y . , 1953, p. 285. (80) Ref. 79, p. 380. (81) Ref. 79, p. 350. (82) Ref. 79, p. 335. (83) Ref. 79, p. 224. (84) V. C. Barry and T. Dillon, Nature, 146, 620 (1940); E. G. V. Percival and S. K. Chanda, ibid., 166, 787 (1950). (85) Ref. 79, p. 245. (86) Ref. 79, p. 66. (87) Ref. 79, p. 134. (88) Ref. 79, p. 152. (89) P. W. Clutterbuck, W. N. Haworth, H. Raistrick, G. Smith and M. Stacey, Biochem. J . , 28, 94 (1934). (90) W. N. Haworth, H. Raistrick and M. Stacey, Biochem J . , 31, 640 (1937). (91) B. Drake, Biochem. Z., 313, 388 (1943). (92) W. Z. Haasid, J . Am. Chem. Soc., 61, 1223 (1939). (93) Ref. 79, p. 375.

ALKALINE DEGRADATION OF POLYSACCHARIDES

307

2. E$ects of Branching on Alkaline Degradation a. Branches on C2 of a (1 -+ 3)-linked main chain will be liberated and be terminated in a stable, 2,3-unsaturated, sugar unit derived from the sugar unit in the main chain to which the branch is attached.68 A branch on C2 occurring in a (1 -+ 4)-linked main chain will stop the peeling process. b. Branches on C3 would be liberated from the main chain, and the main chain would be terminated by a stable metasaccharinate end-unit derived from that glycose unit in the main chain to which the branch is attached. The main chain would then be stable and would not be degraded further. Liberated branches would be degraded further, depending on the type of linkages in the branch. c. Branches on C4 of (1 + 3)-linked main chains would be liberated and be terminated by a stable metasaccharinate end-unit derived from that glycose unit in the main chain to which the branch is attached. These liberated branches would then be stable to dilute alkaline solutions. Branches on C4 of main chains linked (1 5) or (1 + 6) would not be liberated, because the main chains would be stable to dilute alkalis and would not undergo the peeling process. d . Branches on C6, such as are found in g ~ a r a n would , ~ ~ be liberated and be terminated with either a stable iso- or a stable meta-saccharinate end-unit derived from that glycose unit in the main chain to which the branch is attached; they would not be subject to further degradation. The type of saccharinate which terminates the branch is, of course, determined by the linkages in the main chain. --f

3. Degradation Procedure

A polysaccharide can be conveniently degraded for purposes of structural determinations in a rather simple way. The polysaccharide to be examined is dissolved in an excess of an oxygen-free solution of a base, usually saturated lime-water, and allowed to stand a t 25-37' for several months. Cations are then removed from the solution with a suitable cation-exchange resin. Residual polysaccharide may be precipitated with three volumes of ethanol, and the degradation products separated by cellulose-column chromatography, or by fractional reprecipitation of their calcium salts.a1 Degradation occurs more readily in lime-water than in a potassium hydroxide solution of similar n ~ r m a l i t y and , ~ ~ lime-water directs the degradation of (1 -+3)- and (1 + 4)-linked glycans toward saccharinates which contain the same number of carbon atoms as the sugar units involved in the parent glycan. Alkaline degradation of polysaccharides usually stops before the degra(94) Ref. 79, p. 296.

308

R. L. WHISTLER AND J. N. BEMILLER

dation is complete. Termination of the peeling process may be caused by (a) an alkali-resistant linkage in the polysaccharide, (b) the formation of stable, metasaccharinate end-groups in (1 + 4)-linked glycans (see Fig. H

H+COH

I I HCOR I HCOH I

HOCH

H

H

c=o

c-o:e

I

I c=o I -H'

,

HOCH

' SH'

f-+

I I HCOH I

HCOR

CHeOH

I c=o I COH II