Advances in Carbohydrate Chemistry and Biochemistry, Volume 24

Advances in Carbohydrate Chemistry and Biochemistry

Volume 24

1900- 1967 From portrait archives of Gemllschaft Deutscher C h e d e r (Photograph by Tita Binz, Mannheim).

Advances in Carbohydrate Chemistry and Biochemistry Editors MELVILLE L. WOLFROM and R. STUART TIPSON Assistant Editor DEREK HORTON

Board of Advisors W. W. PIGMAN

S. ROSEMAN

WILLIAMJ. WHELAN

ROY L. WHISTLER

Board of Advisors for the British Commonwealth A. B. FOSTER

SIR EDMUNDHIRST

J. K. N. JONES

MAURICESTACEY

Volume 24

ACADEMIC PRESS

New York and London

1969

COPYRIGHT @

1969, BY ACADEMICPRESS, INC.

ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

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United Kingdom Edition

Published b y ACADEMIC PRESS, INC. (LONDON) LTD. BERKELEYSQUAREHOUSE,LONDON W1X 6BA

LibrarV of Congress Catalog Card Number: 45-11351

PRINTED IN THE UNITED STATES OF AMERICA

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the Authors’ contributionsbegin.

G. 0. ASPINALL,Department of Chemistry, Trent Uniuersity, Peterborough, Ontario, Canada (333)

HANS H. BAER, Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada (1,67) D. H. BALL, Pioneering Research Laboratory, U . S . Army Natick Laboratories, Natick, Massachusetts (139) R. J. FEWER, Department of Chemistry, Birkbeck College, Uniuersity of London, London, England (199) HORACE S . ISBELL,* National Bureau of Standards, Washington, D.C. (13)

J. KISS,Chemical Research Department, F. Hofmann-La Roche G Co., Ltd., Basle, Switzerland (381) E . H . F. MCGALE, North Stafordshire Hospital Centre, Renal Unit, Princes Road, Stoke-on-Trent, England (435)

F.W . PARFUSH, Pioneering Research Laboratory, U . S . Army Natlck Laboratories, Natick, Massachusetts (139) WARDPIGMAN,Biochemistry Department, New York Medical College, New York, New York (13)

D. A. REES, Chemistry Department, University of Edinburgh, Edinburgh, Scotland (267)

*Present address: Chemistry Department, American University, Washington, D.C. V

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PREFACE

In the untimely death of Professor Melville L. Wolfrom on June 20, 1969, the world of carbohydrate chemistry lost a great and inspiring scientist, teacher, and writer. He was an Editor of Advances in Carbohydrate Chemistry from its inception in 1945 until his demise, except for 1950-1951 when he served on the Board of Advisors. On the death of Claude S . Hudson in 1952, he became the Editor, with the writer acting successively as Assistant Editor (1953-1954),Associate Editor (1955-1966), and Editor (1967 on). Upon assuming this responsibility, Professor Wolfrom made a firm resolve to continue to adhere to the high standards of scholarship and writing that had been insisted upon by Professor Hudson. The merits of this policy are attested to by the succession of laudatory reviews that every volume of this serial publication has earned. Because of this demonstrated success, it is the intention of the Editor that this policy shall be continued, even though, as Professor Wolfrom dryly remarked in the Preface to Volume 8, “the enforcement of such a policy is not without attendant difficulties.” In every volume of this serial publication, the nomenclature of carbohydrates has presented problems. In their solution, the leadership that from 1951 Professor Wolfrom so ably provided as Chairman of the Carbohydrate Nomenclature Committee (of the Division of Carbohydrate Chemistry of the American Chemical Society) was also of inestimable value in ensuring the use of correct nomenclature in all succeeding volumes of Advances in Carbohydrate Chemistry. In September of 1968, Professor Wolfrom appointed Professor Derek Horton to the position of Assistant Editor, with the understanding that his association with this publication was to commence with Volume 24 (1969). This appointment was made with the intent of preserving continuity as regards the goals and standards already mentioned. At about the same time, he decided to amend the title of the publication in order to make clear that topics in biochemistry are, as indeed they always have been, subjects of discussion herein. Finally, because of the resignation of Dr. R. C. Hockett from the Board of Advisors and the death of Professor S. Peat (a member of

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the Board of Advisors for the British Isles), he decided to appoint, as replacements, the scientists whose names now appear on the title page and to widen the scope of the latter Board by renaming it the Board of Advisors for the British Commonwealth. Kensington, Maryland R. STUARTTIPSON September, 1969 In this volume, each of two long and authoritative articles started in Volume 23 is concluded: these contributions are by Isbell and Pigman (Washington and New York) on the mutarotation of sugars in solution and by Ball and Parrish (Natick) on sulfonic esters of carbohydrates. In a masterly account of the nitro sugars, Baer (Ottawa) extends and updates Sowden's related chapter that appeared in Volume 6. Although only four years have elapsed since Ferrier (London) discussed the unsaturated sugars (Volume 20), progress in this field has been so great that he devotes an entire chapter to advances made in the interim. Rees (Edinburgh), in a stimulating discussion of the formation of polysaccharide gels and networks, develops recent ideas on conformations of macromolecules that may well serve to direct the trend of future work in this area. Aspinall (Peterborough) provides a detailed insight into the structure of gums and mucilages concerning important correlations that have been made since the subject was examined in Volume 13. Finally, two topics not hitherto covered are reviewed. Kiss (Basle) contributes a comprehensive summary of the present status of knowledge of the glycosphingolipids, carbohydrate compounds of considerable complexity and biological importance. McGale (Stoke-on-Trent) gives us a necessarily brief description of a little-explored group of substances, namely, the protein-carbohydrate compounds in human urine. An obituary of Richard Kuhn is provided by Baer (Ottawa), a former student of Kuhn's. The Subject Index was compiled by Dr. L. T. Capell. Kensington, Maryland Columbus, Ohio November, 1969

R. STUARTTIPSON DEREKHORTON

CONTENTS LISTOF CONTFUBUTORS .......................................... PREFACE.......................................................

......

v

......

1

...... vii

Richard Kuhn (1900-1967) HANSH . BAER Text ............................................................ Mutarotation of Sugars in Solution: Part I1 Catalyt. -. Processes. Isotope Effects. Reaction Mechanisms. ant Biochemical Aspects HORACEs . ISBELL AND WARD PICMAN VIII . Catalysis by Acids and Bases ........................................... 14 28 IX . Isotope Effects in Mutarotation Reactions ............................... X . Mechanisms for Mutarotation of Sugars in Aqueous Solution . . . . . . . . . . . . . 35 43 XI . Mutarotation of Certain Sugar Derivatives ............................... XI1. Equilibria and Thermodynamics of Mutarotation Reactions . . . . . . . . . . . . . . . 51 XI11. Mutarotase and the Biochemical Significance of Mutarotation . . . . . . . . . . . . 63 The Nitro Sugars HANSH . BAER

I . Introduction ......................................................... 67 70 I1. Synthesis ............................................................ 109 I11. Reactions That Alter or Remove the Nitro Group ........................ IV. Reactions That Proceed with Retention of the Nitro Group ...............115 Sulfonic Esters of Carbohydrates: Part I1 D . H . BALLAND F. W. PARRISH VII . Displacement Rdactions at Isolated Sulfonyloxy Groups ..................139 VIII . Displacement Reactions Involving Participation ........................ 167 Unsaturated Sugars R .J . FERRIER I . Introduction .......................................................... I1. Glycals ............................................................... I11. 2-Substituted Glycals .................................................. IV . 2.3-Unsaturated. Cyclic Compounds ..................................... V. 3.4-Unsaturated. Cyclic Compounds .................................. VI . 4.5Unsaturated. Cyclic Compounds ....................................

199 200 219 226 246 250

CONTENTS

X

VII . 5.6-Unsaturated. Cyclic Compounds .................................... 255 VIII . Other Unsaturated. Cyclic Compounds .................................. 260 IX. Unsaturated. Acyclic Compounds ....................................... 260 X Nuclear Magnetic Resonance Features of Unsaturated Sugars . . . . . . . . . . . . .265

.

Structure. Conformation. and Mechanism in the Formation of Polysaccharide Gels and Networks D . A . REES I . Introduction and Scope ............................................... 267 I1. The Nature of Gels and the Contemporary Problems ...................... 268 I11. Structure and Conformation of Selected Gel-forming Polysaccharides . . . . . 270 IV. Characterization of Junction Zones ..................................... 303 V. Polymer-Polymer Interactions in Junction Zones ......................... 313 Gums and Mucilages G. 0. ASPINALL I. Introduction .......................................................... I1. Fractionation and Isolation of Structurally Homogeneous Polysaccharides I11. Methods of Structural Investigation .................................... IV. The Galactan Croup of Polysaccharides ................................. V. The Glucuronomannan Group of Polysaccharides ....................... VI . The Galacturonorhamnan Group of Polysaccharides ...................... VII . The Xylan Group of Polysaccharides ................................... VIII . The Xyloglucan Group of Polysaccharides .............................. IX. Other Polysaccharides ................................................ X. Conclusions., .........................................................

333

. . 334 337 341 354 361 371 372 375

376

Glycosphingolipids (Sugar-Sphingosine Conjugates)

J . KISS

. Introduction .........................................................

I I1. 111: IV

382 Nomenclature of Glycosphingolipids .................................... 383 Some Stereochemical Aspects of Sphingosines and Phytosphingosines ..... 384 Natural and Synthetic Sphingosines. Dihydrosphingosines. and Phytosphingosines of Different Chain-lengths ................................ 390 V . Biosynthetic Pathway of Sphingosines and of Glycosphingolipids ......... 394 VI. Ceramide Mono- and Oligo-saccharides ................................ 395 VII. Sulfatides ............................................................ 403 VIII. Glycosphingolipids of Plant Materials and Micro-organisms (Phyto- and Myco-glycosphingolipids) 408 IX. Gangliosides ........................................................ 413

.

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CONTENTS

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Protein-Carbohydrate Compounds in Human Urine

E. H . F. h'fCCALE I . Nomenclature ........................................................ 435 I1. Urinary Protein-Carbohydrate Compounds .............................. 436 111. Urinary Protein-Carbohydrate Compounds of Low Molecular Weight ..... 444 IV. Conclusion. .......................................................... 451 AUTHORINDEX FOR VOLUME 24 ............................................. 453 SUBJECT INDEX FOR VOLUME 24 ............................................. 475 CUMULATIVE AUTHOR INDEX FOR VOLUMES 1-24 .............................. 501 CUMULATIVE SUBJECT INDEX FOR VOLUMES 1-24 ............................... 509 ERRATUM AND ADDENDA ..................................................... 520

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Advances in Carbohydrate Chemistry and Biochemistry

Volume 24

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RICHARD KUHN 1900-1967 Richard Kuhn was born in Vienna, Austria, on December 3rd, 1900. He was the younger of his parents’ children, the first being a daughter, Angelika. Richard Clemens Kuhn, his father, was a hydraulics engineer and senior civil servant with the Austrian Government; he planned and executed such projects as canals and harbor facilities, including the modernization of the Adriatic seaport of Trieste, which at the time lay in the realm of the Austro-Hungarian Empire. Richard Kuhn’s mother, Angelika (nke Rodler), a professional schoolteacher, obtained permission to tutor her son privately at home in lieu of grade school; only once every year was Richard obliged to report for official tests which would ascertain his good progress, and by such expedient and effectual instruction the boy was able to enter high school-the Doblinger Gymnasium -before he had even reached the age of nine. Here he was to develop a life-long friendship with a congenial classmate, Wolfgang Pauli, of later fame in theoretical physics. Gifted teachers, a serene domestic atmosphere in which learning was valued and standards were set by educated parents, and close contact with family acquaintances from among the Viennese university faculty, provided the setting where, in the midst of the cultural metropolis, Kuhn grew through boyhood in carefree, prewar years. He was early initiated to the marvels of chemistry by a friend of the family and noted scholar, Professor Ernst Ludwig, who occupied the chair of medicinal chemistry. Having grown fond of the young adept, Ludwig invited him frequently to watch and assist with the preparations for lecture experiments. Ludwig’s textbook of chemistry for aspirants of pharmacy, a present from the author, was the teenager’s first, avidly perused, source of the science over which he was to gain such unexcelled mastery in years to come. However, difficult times lay ahead and had to be negotiated with that assiduous dedication which marked Kuhn’s entire life before the seeds sown in those formative days could bring forth the wealth of bloom that has so enriched the garden of the natural sciences. World War I and ensuing defeat, revolution, collapse of traditional values and institutions, and economic turmoil with scarcity of means, 1

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galloping inflation, and political disorders of all kinds, held sway during Kuhn’s final high-school years and on through university. The seventeen-year old found himself conscripted into the army signals corps. Of robust if portly stature, Kuhn was by no means averse to the outdoors; he played tennis well, loved mountain climbing and, in later years, derived great enjoyment from joining his assistants in ball games upon the Institute lawn. Nevertheless, for those who have known his dignified comportment and civil manners, devoid of any martial and heroic traits, it is a rather incongruous thought to visualize him donning military garb and moving about in a crude barracks milieu that must have been utterly alien to his nature. Good fortune spared him major front-line action, and upon his discharge in November, 1918, he wasted no time in hurrying back to the sources of learning. He speedily managed to be enrolled at the University of Vienna and to be assigned laboratory space which was hard to obtain under the crowded and uncertain conditions of the times. For two semesters, he studied in his home town under W. Schlenk, R. Wegscheider, and A. Franke, whose lectures and courses much impressed him and evoked a lasting high regard for these teachers. However, from across the border beckoned the climaxing fame of Richard Willstiitter, Nobel laureate, who had succeeded to the Munich chair of Adolf von Baeyer and was carrying on the great chemical tradition that had sprung up in the Bavarian capital under Justus von Liebig half a century before. Richard Kuhn moved to Munich, completed with dispatch all undergraduate requirements in three more semesters, and, in 1921, was accepted for doctoral work with Willstatter, whose favorite pupil and assistant he soon became. In November, 1922, a fortnight before his twenty-second birthday, he was awarded the degree of Doctor of Philosophy, summa cum laude, having submitted a dissertation “On the specificity of enzymes in carbohydrate metabolism.” Willstiitter had at the time turned from his classical investigations on the structure and synthesis of natural products toward enzyme chemistry, and Kuhn is credited with having contributed vastly to the master’s pioneering studies in this newly developing field. It was Kuhn who introduced into Willstiitter’s laboratory the concepts and techniques of physical chemistry pertaining to accurate kinetic measurements of enzyme activities, and these were essential for guidance in the processes of isolation and purification. Willstiitter quickly realized that here was a man of rare talents, and he invited him to stay on in preparation for an academic career, lending his invaluable support in many ways. The brilliant young scientist was enabled to assert leadership by being allowed

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to direct students of his own at an unusually early stage. Displaying an enormous propensity for recognizing problems and for solving them with originality and extraordinary experimental skill, he shortly amassed a great many results by independent efforts (although within the general strategy promulgated by Willstiitter), and he thus met the rigorous demands attached to his “habilitation”’ which took place in March, 1925. The thesis was entitled “The mechanism of action of amylases; a contribution to the problem of the configuration of starch.” Less than two years later, Richard Kuhn was called to the Eidgenossische Technische Hochschule in Zurich as full professor of general and analytical chemistry, a post which he occupied for three years (1926-1929). Among the students of pharmacy who took his courses there was one who was, it seems, impressed by more than his prowess at lecturing alone; she was Daisy Hartmann, who married her professor in 1928. The happy union was blessed with six children two boys and four girls. Of them, Richard Kuhn often spoke with great affection and pride, although otherwise, in his rather introverted manner, he was not given to baring personal feelings too readily. Towards the end of 1929, Kuhn accepted an invitation to become Head of the Department of Chemistry in the newly founded and extremely well-appointed Kaiser Wilhelm Institut fur Medizinische Forschung2 at Heidelberg, where he remained for the rest of his life. He was named Director of the Institute upon the death of its founder, Ludolf von Krehl (1937). He was associated with the University of Heidelberg, first as Honorary Professor in the Faculty of Science, and, from 1950, as Professor of Biochemistry in the Faculty of Medicine. To gain some appreciation of Richard Kuhn’s scope, one may well go back in mind to the day of his habilitation at Munich, in 1925, which appears to have been a highlight in the annals of that great university, forecasting with some poignancy the diversity of potential that became his hallmark. The presentation of a formal lecture on “The position of theory in organic chemistry” was followed by an oral defense, before the faculty, of twelve propositions that covered an amazing range of topics. Among them, of special interest to carbohydrate chemists, were a challenge of the Tollens sugar formula, for (1) The procedure by which, in German universities, a scholar is accorded the privileges and duties associated with a lectureship. (2) The Institute originally consisted of departments of chemistry, physiology, physics, and pathology. It was renamed Max Planck Institut in 1950. Otto Meyerhof the biochemist, and Walter Bothe the nuclear physicist, both Nobel prize winners, headed departments for many years.

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which Kuhn maintained that proof was lacking, and a criticism of Baeyer’s strain theory - sacrosanct in the Munich of the early twenties -which he denounced as having decisively impeded progress in the field of higher alicycles. Other features of the debate included the crystal structure of phosphorus molybdic acids, the magnetic state of chlorine dioxide, aspects of reaction kinetics, displacement mechanisms, heterogeneous catalysis, enzyme-coenzyme dissociation, the action of insulin, and the significance of lactic acid in muscle physiology! Kuhn’s lifetime work, documented in more than seven hundred publications, is far-ranging indeed. It stretches over the fields of amino acids, proteins, and enzymes; aromatic and aliphatic hydrocarbons, including carotenoids as well as synthetic polyenes, cumulenes, highly acidic hydrocarbons, and radicals; vitamins, growthfactors, and inhibitors; such heterocyclics as the flavins, tetrazolium salts, and the newly discovered verdazyls and other nitrogen radicals; natural quinones; alkaloids; gangliosides; and glycosides and sugars of manifold descriptions. Inspired by the great paragon, J. H. van’t Hoff, Kuhn was particularly attracted by problems of stereoisomerism, and it was in this area that he made some of his early contributions of major significance. He was equally fascinated by the relations between structure and physical properties, especially light absorption. Much of his research was prompted by an almost playful joy in color phenomena, whether these occurred in Nature or in the test tube; and close collaboration with his physicist colleagues in the Heidelberg institute, Professor K. W. Hausser and his wife, Isolde Hausser, brought forth important contributions to spectroscopy and photochemistry. In whatever field he embarked upon exploration, he applied himself unreservedly; but his steadiest love, yielding the highest rewards over all, although some bitter disappointments too, was the interplay between structural organic chemistry and biological functions. Guided by a profound knowledge of principles and a fantastic memory for details, cognizant of all current trends in science, often stimulated by artistic intuition, Richard Kuhn moved at ease in orbits traversing nearly all of chemistry and encompassing the border precincts of physics, biology, and medicine. A large number of students and postdoctoral assistants flocked to him, each contributing with enthusiasm his share to the team’s prolific work, and from his school emerged many who later attained professorships and other creative positions throughout the world. T. Wagner-Jauregg, A. Wassermann, A. Winterstein, H. Brockmann, E. Lederer, C. Grundmann, E. F. Moller, H. Rudy, L. Birkofer, F. Weygand, P. Desnuelle,

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N. A. Sorensen, F. Giral, G. Wendt, 0.Westphal, T. Wieland, and D. Jerchel, among many others, collaborated during the first great period of Kuhn’s productivity, which extended into the early forties and culminated in the award of the Nobel prize in chemistry3 for 1938. H. Beinert, H. J. Bielig, G. Quadbeck, F. Zilliken, H. A. Staab, F. Drawert, Herb. Fischer, and others, joined later. With exceptions to be mentioned, the work with these associates was largely outside the field of carbohydrate chemistry and cannot be given proper appreciation in these pages.4 By early 1927, at about the time of his moving to Zurich at the age of twenty-six, Richard Kuhn had sixty publications to his credit, being co-author with Willstiitter of eight, and single or senior author of the rest. That work comprised extensive research on carbohydrate enzyme specificity, mainly regarding the action of sucrases, maltase, raffinase, amylases, and emulsin upon various substrates. It was recognized that yeast invertase contains two separable enzymes which can hydrolyze sucrose; they are now referred to as a-D-glucopyranosidase and p-D-fructofuranosidase. Important contributions were made on the structure of starch, turanose, melezitose, and amygdalin, and the kinetics of sugar mutarotation and of permanganate oxidation were studied (with H. Sobotka, G. E. von Grundherr, and others). Keenly interested in general problems of stereochemistry, Kuhn investigated the mechanism of the Walden inversion, the stereoisomerisms in chloromalic acid and cyclopropanedicarboxylicacid, and, particularly, the molecular asymmetry that causes rotational isomerism in orthosubstituted biphenyl derivatives (with F. Ebel, T. Wagner-Jauregg, and 0. Albrecht). He later coined the term “atropisomerism” for stereoisomerism that is based on restricted rotation about single bonds, clearly realizing the stereochemical significance of this feature at a time when the concept of conformation had not yet been established. Following his moves to Zurich and Heidelberg, Kuhn’s interest in carbohydrates was temporarily overshadowed by his rapidly developing polyene work. However, sugar studies were resumed in connection with the structural elucidation and synthesis of riboflavine, which had been isolated in 1933 in collaboration with P. Gyorgy and T. Wagner-Jauregg. Riboflavine [vitamin BB,6,7-dimethyl-9-(~-~ibo(3) The citation honored Richard Kuhn’s work on carotenoids and vitamins. Like other German Nobel laureates in that era, he was forced by the National Socialist regime to decline the prize, but, after the war, the Nobel Committee ruled the refusal null and void, and the honors were conferred. (4) For an excellent account, see 0. Westphal, Angew. Chem., 80,501 (1968).

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2,3,4,5-tetrahydroxypentyl)isoalloxazinel was synthesized, starting from D-ribose and 3,4-dimethylaniline (with F. Weygand); the 5’phosphate (flavine mononucleotide) was then prepared, and combined with the protein moiety of Warburg’s yellow enzyme, in what was the first partial synthesis of an active enzyme (1934-1936, with H. Rudy). Many glycosylamines and aminodeoxyalditols were prepared, and their reactions were studied concomitant with, and following, these achievements; one of the results was the realization (with A. Dansi) that the nonhydrolyzable, strongly reducing products that arise from aldosylamines under certain conditions are 1-amino-1-deoxyketoses. The now-familiar name “Amadori rearrangement” was proposed for this transformation by Kuhn and Weygand; actively participating in these investigations (1936- 1938) were R. Strobele and L. Birkofer. Two decades later, Kuhn was to return to the study of the Amadori rearrangement (with F. Kruger and Annemarie Seeliger), making use of it, for instance, in a simple synthesis of lactulose. Other research during the earlier period led to elucidation of the structure of picrocrocin, an alkali-labile glycoside constituting the bitter principle of saffron. In the 1940’s, Richard Kuhn’s interest in carbohydrates was sustained, and it gradually rose to become his dominant (though by no means exclusive) preoccupation during the last fifteen years of his life, a period that was also marked by a second peak of his immense productivity. He had the good fortune of being able to rely on three trusted and extremely competent associates who served with him faithfully through three decades until his death, doing carbohydrate research with dedication and providing an element of continuity in ever-changing complements of students and assistants. They were Drs. Irmentraut Low and Adeline Gauhe, and Mr. Heinrich Trischmann. The direction of work first turned toward the isolation and biological evaluation of plant glycosides. Flavonol glycosides were identified, and aglycons (rhamnetin and rhamnazin) were synthesized. A large area of study then opened up in steroid-related alkaloid glycosides that occur in the leaves of potato and tomato plants and other Solanum species. These attracted Kuhn’s interest in connection with phenomena of biological resistance which, in their diversified manifestations, had always intrigued him. Why are certain Solanaceae resistant against the potato beetle (Leptinotarsa decemlineata Say), whereas others are beset by this pest, which has blighted many a potato crop? It was discovered that certain alkaloid glycosides, namely, tomatine from tomato leaves, demissine from Solanurn demissum, and leptine from Solanum chacoense (both of which are wild potatoes,

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native to Central and South America), act as beetle repellents, rendering the leaves resistant, whereas the long-known solanine and the newly isolated chaconine, which are present in the common potato plant, are inactive. The latter two glycosides were found to contain the same aglycon, solanidine, but to differ in their branched, trisaccharidic sugar moieties. Solatriose was demonstrated to be O - ~ - L rhamnopyranosyl-( 1 -+ 2)-[O-P-D-glUCOpyranOSyl-(1 -+ 3)]-D-galactose, and chacotriose was shown to be 2,4-di-O-a-~-rhamnopyranosyl-~glucose. Tomatine and demissine were revealed to differ in their aglycons, but to contain the same sugar moiety, called lycotetraose. The latter was established to be O-P-D-xylopyranosyl-(1 -3)[O-P-D-glucopyranosyl-(1 + 2)]-O-/3-D-glucopyranosyl-(1 + 4)-D-galactose. Two highly active leptines were found to resemble solanine and chaconine, respectively, in regard to their sugar units; the activity was associated with an ester acetyl group in the aglycon (work with I. Low and, in part, H. Trischmann and A. Gauhe). Considerable improvements in the technique of sugar methylation were elaborated in connection with these studies, and have since enjoyed frequent application. In 1952,problems of biological resistance prompted Richard Kuhn to return to research on milk. It had become apparent that human milk, as opposed to cow’s milk, enhances resistance in infants against bacterial and viral infections. Collaboration with P. Gyorgy, now at Philadelphia, was resumed some twenty years after their joint investigations on lactoflavine, and a large-scale program concerning the physiology and chemistry of milk was launched. The growth-promoting activity of human milk for Lactobacillus bifidus, the normal intestinal flora that is beneficial to the baby’s health, was recognized to reside in the carbohydrate fraction; part of it is due to nitrogencontaining oligosaccharides. In the transatlantic co-operation that developed, microbiological aspects were pursued in Philadelphia, while the chemical studies mainly took place in Heidelberg. Hundreds of gallons of human milk were procured-no mean feat in research logistics -and the carbohydrate fractions were passed through batteries of chromatography columns. With A. Gauhe and H. H. Baer, about a dozen native oligosaccharides were isolated, and many additional ones were obtained by partial hydrolyses in the course of subsequent structural work. Occupying a central position among these sugars, “lacto-N-tetraose” was shown to be O-P-D-galaCtO1-+ 3)pyranosyl-(1+ 3)-0-2-acetamido-2-deoxy-~-~-glucopyranosyl-( O-P-D-galactopyranosyl-(1+ 4)-a-D-glUCOSe. Several penta- and hexasaccharides proved to be derived from the tetraose by attachment of

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L-fucose residues and N-acetylneuraminic acid residues at various positions. Furthermore, trisaccharides and tetrasaccharides composed of lactose plus one or two L-fucose residues, and of lactose plus one or two N-acetylneuraminic acid residues were found to occur in human milk.5 In elucidating the oligosaccharide structures, considerable attention was paid to alkaline degradation and its dependence on the type of linkage, especially in conjunction with the MorganElson color reaction for N-acetylhexosamines. The mechanism of the latter reaction was studied (with G. Kruger) and found to involve the formation of 3-acetamidohran derivatives as chromogens. Insights thus gained have contributed a great deal to later structural work by others in such important areas as blood-group substances, whose carbohydrate components resemble the milk oligosaccharides in many ways, Several of the di- and tri-saccharides described for the first time by Kuhn and his coworkers as hydrolytic fragments of higher saccharides have since been encountered as building units elsewhere in Nature. In concurrent investigations with R. Brossmer, crystalline 3’-0(N-acetylneuraminy1)lactose was isolated from cow colostrum and demonstrated to be a substrate for influenza virus enzyme and for the “receptor-destroying enzyme” of Vibrio cholerae. The structure of N-acetylneuraminic acid, then still a matter of contention, was clarified by chemical degradation, and the configuration at C-4 that had remained unknown was established. Finally, the acid was synthesized in good yield (with G. Baschang). Another extensive program, begun in the early sixties, was directed at the chemical exploration of the brain gangliosides. These compounds, which are glycolipids consisting of fatty acid, sphingosine, and an oligosaccharide moiety, are presumed to play an important role in the physiology of nervous tissue. With H. Wiegandt and H. Egge, several gangliosides were structurally elucidated. The carbohydrate parts attached were demonstrated to contain, as the central core, a tetrasaccharide (“ganglio-N-tetraose”), namely, 0-p-D-galactopyranosyl-(1 --* 3)-0-(2-acetamido-2-deoxy-/3-~-galactopyranosy1)(1--* 4)-O-~-D-galactopyranosyl-( 1 --* 4)-D-glucose,to which are bound N-acetylneuraminic acid residues in various numbers and at various sites. The close analogy to the milk oligosaccharides is obvious, although 2-amino-2-deoxy-~-galactose takes the place of 2-amino-2deoxy-D-glucose, and L-fucose is absent. Synthetic work included the preparation of several disaccharides for comparative and systematic purposes. Thus, 2-acetamido-2-deoxy(5) See F. Zilliken and M. W. Whitehouse, Advan. Carbohyd. Chem., 13,237(1958).

OBITUARY-RICHARD KUHN

9

lactose, 2-acetamido-2-deoxyallolactose, 3-O-P-D-galactopyranosylD-glucose, and 3-O-P-D-galaCtOpyranOSyl-D-fructose, among others, were synthesized chemically (with H. H. Baer and W. Kirschenlohr). However, the emphasis rested on the synthesis of monosaccharidic amino sugars. The method of catalytic hemihydrogenation of aaminonitriles, first elaborated with W. Kirschenlohr, and then investigated further and comprehensively employed by G. Baschang, Waltraut Bister, H. Fischer, J. C. Jochims, and D. Weiser, proved extremely versatile in the synthesis of 2-amino-2-deoxyaldoses (1956-1961). Aldoses were condensed with hydrogen cyanide and an amine (aniline, benzylamine, or ammonia) according to the pattern of Emil Fischer’s synthesis of 2-amino-2-deoxy-~-glucose, but the a-aminonitriles so obtained were then hydrogenated catalytically, the reduction being arrested at the aldehyde stage. (All eight 2-amino2-deoxy-~-hexosesand many other amino sugars, such as 2-amino-2deoxypentoses, 2-amino-2-deoxytetroses, and 6-deoxy derivatives, have thus been prepared, some for the first time.) The Kuhn synthesis (the first) of 2-amino-2-deoxy-~-arabinose still stands as the best method for obtaining this sugar. In addition, extremely interesting studies concerning epimerizations, tautomerizations, rearrangements, and dehydrations in aminodeoxyaldononitriles, aminoaldonic acids, and amino sugars were performed, and various biochemical experiments relating to amino sugar metabolism were carried out. With close to a hundred articles published in the period 1952-1967 on nitrogencontaining carbohydrates alone, Richard Kuhn certainly ranks high among those who have made the most significant contributions to this field. In between the researches sketched above, numerous reactions in the general chemistry of carbohydrates were studied. As one example may be mentioned an investigation with W. Bister on the peroxy acid oxidation of sugar dithioacetals to mono- and bis-sulfoxides and on the fission of these oxidation products. Richard Kuhn’s extraordinary personality could not fail to impress anyone who came in contact with him. Time and time again, his coworkers would stand in amazement at his ingenious deductions and often unexpected suggestions, which flowed from an inexhaustible fund of knowledge and from analogies that were not readily available or obvious to his lieutenants. Spellbound lecture audiences would hear his resonant, melodious voice expound problems and results of research, forcefully and with didactic discipline, often revealing striking interrelations between matters seemingly apart, and always heading for a finale that would open grand vistas of potential future developments. Nevertheless, however synoptic his views, it was not at all beneath Richard Kuhn to attend painstakingly to small details

10

HANS H. BAER

in the daily routine. He put great stock in all things analytical. Himself an accomplished microanalyst, he used to perform with his own hands the microanalyses for his coworkers in the days before reliable services became generally available. Or, sitting in a blacked-out room, he would patiently await light-adaptation before assisting a student with a feeble polarimeter reading. Every new compound was eyed critically under the microscope before it was accorded the attribute “crystalline” and tentatively considered respectable. This writer vividly recalls one occasion when der Chef, spatula in hand, placed several samplings of a newly isolated and as yet unelucidated oligosaccharide upon various areas of his tongue and announced, smacking his lips with delight, that it tasted similar to, but somewhat less sweet than, milk sugar. Thus expired about half the yield from several weeks of chromatographic labor! Technical details in the operation of the Institute, such as the tactical placement of apparatus, the arrangement of journals in the great library, or even the wattage of light bulbs, were given careful deliberation. One winter night, late after hours, der Chef and his assistant left the Institute. Groping for the staircase light-switch in the dark hallway, the pupil caused some fumbling delay. When light finally came, Kuhn remarked with visible pride, and quite unaware of some slight embarrassment: “The switches are located very conveniently, aren’t they? I designed the circuits myself.” In the execution of research, the senior assistants were given much leeway. Richard Kuhn was no taskmaster. He would appear in the Institute at irregular hours, walking the mile from his house with a striding gait, and he did not mind if scientific staff set their own schedules. Yet he extracted a due share of work performance from everyone, mostly by his own stimulating enthusiasm, and sometimes by gentle prodding couched in mock astonishment at sluggish chemical reactions. Occasionally, when things were unsatisfactory, he would walk off, silent, wearing a vaguely pained expression, but harsh words were never heard. Kind at heart and deeply sensitive, Kuhn shunned open confrontations. Faced with demands that he deemed unreasonable and had to decline or defer, he loathed to hurt the petitioner’s feelings by an outright rejection. His gaze would wander out through the window and rest for a minute or two upon the distant Odenwald hills, whereafter would issue the verdict, softly, in some such form as “It’s a very fine day today.” The message, however, was clear. Contrasting with his imposing physique and resounding oratory, Richard Kuhn’s mental attitude was anything but pugnacious. He preferred quiet diplomacy, which he exercised diligently on behalf of his Institute and his country’s scientific institu-

OBITUARY-RICHARD KUHN

11

tions at large, especially under the duress of wartime and postwar adversities. His stand has been interpreted unjustly in some quarters that were barred a full comprehension of the troubled waters he was at times compelled to navigate. Richard Kuhn’s health began to fail in 1965. Ironically, medical science to which he had contributed so much was unable to keep his illness in check, and he soon came to realize that there was no hope. But with great willpower he combated his fate for nearly two years, carrying on his work and his duties, and providing for the future of the Institute in progressively agonizing hardship. He died of cancer on July 31,1967. Richard Kuhn’s excellence in achievement and his relentless efforts on behalf of science, its establishments, and its due position in society have commanded wide recognition by his peers, and earned him, in rich measure, tokens of respect from many distinguished bodies of culture and learning at home and abroad. He served as President of the Deutsche Chemische Gesellschaft (1938-1945) and of the Gesellschaft Deutscher Chemiker (1964-1965),as Vice President of the International Union of Chemistry (1938) and of the Max Planck Gesellschaft (from 1955), and he was active in other councils and on boards. He was awarded more than a score of prizes and medals, including national and international distinctions of the highest standing, and twenty academies and learned societies across the world elected him to honorary membership. Honorary doctorates were bestowed upon him in Vienna, Munich, and Heidelberg, the places of his birth, education, and fulfilment. These outward signs betoken the greatness of a true architect of science, but his edifice will be appreciated, in its entire import, only in years to come. Those who were privileged to know Richard Kuhn are grievously aware that, with his passing, Chemistry has lost a genius. HANSH. BAER* APPENDIX

The following scientists have been co-authors with Professor Richard Kuhn on publications dealing with carbohydrates or carbohydrate derivatives.

F. Bar, H. H. Baer, G. Baschang, Waltraut Baschang-Bister, G. Bechtler, L. Birkofer, R. Brossmer, W. Dafeldecker, A. Dansi, F. Ebel, H. Egge, ‘The kind assistance of Dr. Adeline Gauhe is gratefully acknowledged.

12

HANS H. BAER

D. Ekong, H. Fischer, Gertrude Fischer-Schwarz, Adeline Gauhe, Johanna Graser, H. Grassner, G. E. von Grundherr, P. Gyorgy, H. J. Haas, F. Haber, R. Heckscher, K. Henkel, M. Hoffer, J. R. E. Hoover, K. Hummeler, P. Jacob, W. Jahn, J. C. Jochims, H. Kaltschmitt, W. Kirschenlohr, P. Klesse, F. Kohler, Leonore Kohler, A. Kolb, G. Kriiger, H. J. Leppelmann, W. Lochinger, Irmentraut Low, P. Lutz, D. L. MacDonald, F. Moewus, H. Muldner, H. Munch, C. Oppenheimer, H. G. Osman, G. Quadbeck,K. Reinemund, E. Rohm, Catherine S. Rose, H. Rudy, H. W. Ruelius, H. H. Schlubach, W. Schulz, Annemarie Seeliger, H. Sobotka, R. Strobele, H. Tiedemann, H. Trischmann, Dorothea Tschampel, T. Wagner-Jauregg, K. Wallenfels, D. Weiser, G. Wendt, F. Weygand, H. Wiegandt, R. Willstiitter, A. Winterstein, W. Ziese, and F. Zilliken.

MUTAROTATION OF SUGARS IN SOLUTION*: PART 11"" CATALYTIC PROCESSES, ISOTOPEEFFECTS,REACTION MECHANISMS,AND BIOCHEMICALASPECTS BY

HORACEs. ISBELL""" AND WARD PIGMAN

National Bureau of Standards, Washington, D. C . , and Biochemistry Department, New York Medical College, New York, New York VIII. Catalysis by Acids and Bases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Historical Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . 2. Evaluation of the Catalytic Coef6cients . . . . . . . . . . . . . . . . . . . . , . . . . . . IX. Isotope Effects in Mutarotation Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Basis for Effects of Water and Deuterium Oxide . . . . . . . . . . . . . . . . . . . . 2. Effect of Deuterium Oxide and Water on Mutarotation Reactions . . . . . . 3. Use of Isotope Effects for Determination of Mechanisms of Reaction . . . X. Mechanisms for Mutarotation of Sugars in Aqueous Solution.. . . . . . . . . . . . 1. Overall Reaction Mechanisms.. . . . . , . . . . . . . . , . . . . . . . . , . . . , . . . . . , . 2. Specific Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . XI. Mutarotation of Certain Sugar Derivatives . . . . . . . , , . , . . . . . . , . . . , . . . . . . . 1. Anomerization by Way of a Cyclic Carbonium Ion . , . . . . . . . . , . . . . , . , , . 2. Anomerization by Way of a Bimolecular Replacement Reaction . . . . . . . 3. Ring Contraction and Expansion . . . , . , . . . . . . . , , , . . . . . . . . . . . . . . . . . 4. Glycosylamines.. . . . . . . . . . . . .. . . , . . .. . . ......................... 5. Thio Sugars.. . . . . . . . , , . . . . . . . . . . . . . ............................ XII. Equilibria and Thermodynamics of Mutarotation Reactions . . . . . . . . . . . . . 1. Activation Energies . . . , . . . . . . , , . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . 2. Changes in Free Energy, Enthalpy, and Entropy. , , . . . . . . . . , , . . . . . . XIII. Mutarotase and the Biochemical Significance of Mutarotation . . . . . . . . . .

14 14 16 28 28 31 32 35 35 38 43 43 45 46 47 49 51 51 54 63

'The authors acknowledge the assistance of Dr. Anthony Herp and Mr. Guy Lytle, 111, in the preparation of this article, which was supported by a grant (AM-04619)from the National Institutes of Health, U. S. Public Health Service. The authors thank Dr. R. S. Tipson for constructive criticism and helpful editorial review of the manuscript. Mrs. Hilda Hams, of the library of the University of Alabama Medical Center, Birmingham, was of great help in the literature search. "Part I appeared in Aduan. Carbohyd. Chem., 23, 11-57 (1968).All numbers for Equations, Figures, References, Sections, and Tables in Part I1 continue the sequence established in Part I. ""Present address: Chemistry Department, American University, Washington, D. C.

13

HORACE S. ISBELL AND WARD PIGMAN

14

VIII. CATALYSIS BY ACIDS AND BASES 1. Historical Background The mutarotations of all reducing sugars are catalyzed by acids and bases.z18Since the mutarotations of a- and P-D-glUCOSe have been investigated more extensively than those of other sugars, they will be considered in detail. Hudson and Dalezo1found that the mutarotation constant (k, k,) is the same for a-and P-D-glUCOSe at temperatures for ranging from 0 to 40".Differences in the ionization a- and P-D-glucose, and possible differences in the transition states for the anomers, make detailed investigations of the individual velocity constants k, and k, desirable. However, in the present discussion, attention will be directed largely to the velocity constant (k, kz) for the overall reversible reaction:

+

+

a-D-glucopyranose

ki

P-D-glucopyranose.

k, Unless otherwise stated, the overall constant will be expressed in common logarithms and minutes. The acceleration of mutarotation by acids was first reported by Erdrnann,2l9and its catalysis both by acids and bases was described by UrechzzOin 1882. The early workers attributed the catalytic effect to acids and bases, but later work revealed that catalysis of mutarotation is not the exclusive property of hydrogen ions and hydroxyl ions. Hudsonzz1showed that the mutarotation is catalyzed by water molecules, and represented the mutarotation constant by the equation:

(k,+ k,) = k,,,+A[H@]

+ B[OH@l,

where the symbols in brackets represent the activities of the hydrogen and hydroxyl ions. Nelson and Beegle115 showed that plots of the mutarotation constants against pH give curves having the form of a catenary. The curves11z(see Fig. 4) representing the mutarotation Correction to Part I

Vol. 23, p. 33, line 14. For D-mannUrOnO-1,4-laCtOneread ~mannurono-6,3-lactone. (217) H. S. Isbell, H. L. Frush, and J. D. Moyer, J . Res. Nat. Bur. Stand., 72A, 769 (1968). (218) W. Pigman and H. S. Isbel1,Aduan.Carbohyd. Chern., 23,11(1968). (219) E. D. Erdmann, Ber., 13,218 (1880). (220) F. Urech, Ber., 15,2130 (1882). (221) C. S. Hudson,]. Arner. Chern. Soc. 29,1571 (1907).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

15

0.20

-

E 0.16 0

v)

f 0.12

--

0 ._ 0

0.08

c

3

.3

0.04 0.00

1

2

3

5

4

6

7

8

PH

FIG. 4.-Relative Catalytic Effects of Hydrogen and Hydroxyl Ions on Rapid and Slow Mutarotation Reactions.

constants for D-ghCOSe and for the slow phase of the mutarotation of D-galactose show long horizontal sections, from pH 2 to 6, with steep slopes below pH 2; the curves representing the mutarotation constants for D-fructose and for the fast reaction of D-galactose (pyranosefuranose interconversions) rise more rapidly on each side of the region of minimum rates. Lowry and coworker^^^^^^^^ studied the mutarotation of tetra-0methyl-a-D-glucopyranose, and found that the rate of reaction is low in dry pyridine or in dry cresol, but high in a mixture of the two solvents or in either solvent when moist. Lowry and Smith5' concluded that the mutarotation requires an acid catalyst and a base catalyst, and that amphoteric solvents are complete catalysts for the process, whereas aprotic solvents are not. They also showed that molecules of undissociated acids, cations of weak bases, and anions of weak acids have catalytic properties. Much the same concept was developed ~ ~ *came ~ ~ ~ to be independently by Bronsted and G ~ g g e n h e i m , ' and known as generalized acid and base catalysis. It was found that the rate of mutarotation of a sugar in the presence of a mixture of several catalysts may be represented by an equation of the type:

(222) T. M. Lowry and I. J. Faulkner,j. Chem. SOC., 127,2883(1925). (223) J. N.Bronsted, Trans. Faraday SOC.,24,630 (1928).

16

HORACE S. ISBELL AND WARD PIGMAN

where the symbols in brackets represent the concentrations (activities) of the catalysts, and the coefficients k, kBnrepresent the catalytic activity of the acid and base catalysts, respectively. Many attempts have been made to ascertain whether the acid- and base-catalyzed reactions are concerted or stepwise. In the concerted process, both the acid catalyst and the base catalyst take part in the transition state, with addition of a proton at one point in the molecule and with elimination of a proton at another point. Theoretically, the rate constant for a concerted reaction involving several acid-base combinations yields an equation of the form:

k =z z kHj J n

kB,, [HA,]

[BJ.

(10)

The velocity of the concerted reaction does not depend on the sum of the separate velocities of the acid and base catalysts, but on the product of the two velocities. This is not the case for the stepwise mechanism. In a stepwise process, the acid catalyst and the base catalyst act separately, and give rise to rate equation 9. Numerous workers have examined the rate constants for the mutarotation of D-glucose in the presence of acetic acid and sodium acetate in an attempt to ascertain whether equation 9 or 10 applies. Although the two equations differ widely, it is not easy to distinguish experimentally between them. It is probable that the concerted mechanism is of no significance in the mutarotation of sugars in aqueous solution, with the possible exception of reactions catalyzed by the water molecule, as will be discussed later. In solvents of low dielectric constant, the formation of ionic intermediates becomes less favored, and the concerted mechanism may apply. Some of the methods that have been used for studying these reactiofls and for determining the catalytic coefficients will be considered next. 2. Evaluation of the Catalytic Coefficients

To evaluate the catalytic coefficients for a specific reaction, the rate expression, equation 9, is set up for the known catalysts. Then, a series of measurements is made under such conditions that certain terms in the equation are negligible and the contribution of the catalyst under study becomes important. Under these conditions, the effect of the concentration of the catalyst on the rate constant may be determined. For example, to determine the catalytic coefficients for the mutarotation of D-gh.ICOSe in an aqueous sodium acetateacetic acid solution, the following equation is set up:

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

k = kHrO [HZO]

17

kHaOo [H30@]4-kHoAc [ HOACI k,Ac, [ O A C ~ ] + koH, [OH@]4- kG, [ G o ] ,

+

where k is the mutarotation constant ( k , kz),the symbols in brackets represent the concentrations (activities) of the catalysts, and kHzO, kH30a, kHOAc, koACO,and kGOare the catalytic coefficients of the substances indicated by the subscripts, G @being the D-glucosate ion. The term kHIO [H,Ol represents the contribution of the water molecules. Since [H,O] varies only slightly from 55.6 M (moles per liter) in aqueous systems, it is usually included in the kHeOterm, as in Hudson's original equation. As will be discussed later (see p. 22), this term can be broken into two parts representing the acidic and basic characteristics of water. a. Catalysis by the Water Molecules and Hydrogen Ions. -To evaluate kHZO and kHamin the rate expression ( g ) , a series of measurements is made in nonbuffered, acid solutions. In solutions of such acid strength that the influence of the hydroxyl ions upon the rate can be neglected, a linear relationship exists between the hydrogen-ion activity and the rate of mutarotation, and, when the mutarotation constants from a series of measurements are plotted against the activity of the acid, there is obtained a straight line whose intercept on the k axis is kHzOand whose slope is kHsOo.In the absence of other catalysts,

k = kHzO + kH,a [H@@I. The constant kHzOin the rate expression accounts for most of the velocity in the pH range 4 to 6; it represents the combined action of the water molecule as an acid and as a base, a subject that will be considered on p. 22. Some values of kHZOand kHaOereported in the literature for the mutarotation of D-glucose are summarized in Table VII. b. Catalytic Coefficients for Weak Acids. -The catalytic coefficient for an undissociated, weak acid may be determined from a series of mutarotation measurements by using various concentrations of the acid in question and a fixed concentration of the corresponding salt. If the experiment is restricted to acidic conditions, the catalytic effects of the hydroxyl ion and the sugar anion may be neglected, and the mutarotation constant may be represented by the equation: To evaluate kHA for a weak acid, a series of measurements is made of the mutarotation of a sugar in the presence of a fixed concentration of

HORACE S. ISBELL AND WARD PIGMAN

18

TABLE VII Catalytic Constants for Water and Hydronium Ions in the Mutarotation of D-Glucose" Temperature, degrees 0 3 14 18 18 20 20 20 25 30

kH.0 7 x 10-4 1 X 6.1 X 4.9 X 5.3 x 6.3 x 10-3 6.3 x 6.1 X 10.6 X low3 16.7 X

hso@

References

0.024 0.13 0.12 0.145 0.21 0.16 0.17 0.26 0.45

201 Trey, see Ref. 201 Lowry, see Ref. 201 Meyer, see Ref. 201 189 224 Lowry and Smith, see Ref. 224 Levy, see Ref. 201 225 201

"(k, + k,) = kH& + kHIoo [H30@](expressed in common logarithms).

sodium acetate and a variable proportion of the acid, in the pH range 3.5 to 6.5. When the mutarotation constant, k, is plotted against the concentration of the weak acid, a straight line is obtained, for which the slope is kHA and the intercept on the k axis is the sum of kHpOand the catalytic effect of the salt k,, [A@].When two sets of experiments are made with the same acid, but at different concentrations of salt, two parallel lines are obtained whose intercepts on the k axis correspond to the two different concentrations of the anion of the weak acid. When necessary, a correction can be applied for catalysis by the hydrogen ion, but ordinarily this correction is not required. Some catalytic constants for the mutarotation of D-glucose, taken from the work of Bronsted and G ~ g g e n h e i m ,are ' ~ ~given in Table VIII. c. Catalytic Coefficients for Anions of Weak Acids. - Determination of the catalytic activity of the anion (k,,) of a weak acid requires adjustment of the pH of the solution so as to keep the concentration of hydroxyl ion low, because the catalytic activity of the hydroxyl ions resulting from hydrolysis of the salt is so great that it masks the smaller catalytic effect of the anion. Usually, sufficient acid is added to the salt solution to bring the pH to values between 4.5 and 6.0. In this range, catalysis by the hydrogen and hydroxyl ions is negligible, and the mutarotation constant may be assumed to be k = kHzO kHA [HA] -t k,, [A@].

+

(224) H. S. Isbell and C. W. R. Wade,J. Res. Nat. Bur. Stand., 71A,137 (1967). (225) C. S. Huds0n.J. Amer. Chem. SOC., 29,1571 (1907).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

19

Then, with a fixed concentration of the acid (HA) and various concentrations of the corresponding salt, a plot of the mutarotation constants against the concentrations of the salt gives a linear curve. The slope of the curve gives kAo, and the intercept on the k axis is kHpO kHA [HA]. A plot from the work of Bronsted and G ~ g g e n h e i r n ' ~ ~ is shown in Fig. 5. The catalytic coefficients of strong bases and strong acids are difficult to measure, but they may be estimated, or calculated from the Bronsted relation to be considered next.

+

d. Bronsted Relationship Between Catalytic Activity and Dissociation Constants.-Acid catalysis involves the transfer of a proton from the catalyst to the substrate, and base catalysis involves the transfer of a proton from the substrate to the catalyst. Bronsted and Pedersen226pointed out that the catalytic activity of a catalyst, kA@ (or kB), in a given reaction depends on the readiness with which the proton transfer takes place, and is related to the acid (or base) strength TABLEVIII Catalytic

for the Mutarotation of D-Glucose by Weak Acids and Their Anions at 18"

pK

Acid 2.2-Dimethylpropani3ic Propionic Acetic Phenylacetic Benzoic o-Toluic Glycolic Formic Mandelic Salicylic o-Chlorobenzoic Monochloroacetic Cvanoacetic

Acid Anion catalytic catalytic constant, constant, Calculated catalytic constants"

(-log KA) kA

5.00 4.85 4.74 4.30 4.22 3.89 3.85 3.67 3.37 3.00 2.89 2.82 2.46

X

2.0 2.1 2.4 2.8 -

-

5.6 4.6 5.7

-

6.8

-

103 k,

X

103

31.4 28.1 26.5 20.0 15.2 12.2 13.7 16.5 10.8 4.6 6.4 5.4 3.8

kA

X

103

2.0 2.2 2.3 3.2 3.3 4.2 4.3 4.9 6.2 7.7 8.4 8.8 11.3

"Calculatedfrom log kA= 0.3 log KA- 1.21, and log k, = 0.34 log KB- 3.20.

(226) J. N. Bronsted and K. Pedersen, Z. Physik. Chem., 108,185 (1924).

kB x 103 31.5 28.0 26.5 18.0 17.0 13.0 13.0 11.0 8.0 6.5 6.5 5.5 4.0

HORACE S. ISBELL AND WARD PIGMAN

20 11

10

9 m '0

;;e c

7 6 I

I

1

0.05

0.10

I

0.15

0.20

1

0.25

Conc. of anion of weak acid

FIG. 5. -Catalysis by Anions.

of the catalyst. In accordance with this concept, they found, for acid catalysts, that the catalytic coefficient is given by: kA =

GAK i ,

(11)

where G A is a constant for the reaction considered, KA is the dissociation constant of the acid, and x is a factor having a value between 0 and 1.For a base catalyst, the coefficient is given by:

k~ = & KE,

(12)

where GB is a constant that is characteristic of the reaction and conditions, KB is the basic dissociation constant (that is, the reciprocal of K A for the corresponding conjugate acid), and y is characteristic of the type of reaction and the catalyst. Equations 11 and 12, expressed on a logarithmic basis, are:

113) and

(14)

For a series of isotypic catalysts, a plot of log kA (or log kB) against log KA (or log K B ) gives a straight line having a slope of x (or y). A typical plot from the work of Bronsted and G ~ g g e n h e i mis ' ~shown ~ in Fig. 6. The data for the acid-catalyzed and base-catalyzed reactions given in Table VIII give rise to the rate expressions: log k A = 0.3 log K A - 1.21, and

log k B = 0.34log K

B

- 3.20.

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

21

Considerable variation exists in the experimental data, but the results clearly show a correlation between catalytic activity and acid, or base, strength. For determination of the catalytic activity of an acid of known strength from equation 11, it is only necessary to know the value of the exponent x, the catalytic activity kA, and the dissociation constant KA of a reference acid HA of the same type as HA'. Thus, Likewise, for base catalysts:

Bronsted and G ~ g g e n h e i mstudied '~~ the catalytic activity of a wide variety of acids and bases. They found substantial differences in the relationship between catalytic activity and acid strength for catalysts of different types. Thus, the catalytic effect of a base having two negative charges is considerably greater than that of a base of the same strength having one negative charge. An excellent review of the subject is given by Bell,227who points out: "We might expect also to find -1

m x P

-0

-3 '

2

I

1

1

3

4

5

log K,=-log

K,

FIG. 6.-Dependence of Basic Catalysis on the Strength of the Basic Anion. T, 2,2dimethylpropanoate; Ph, phenylacetate; G, glycolate; S, salicylate; C, cyanoacetate; P, propionate; B, benzoate; F, formate; o-C, ochlorobenzoate; A, acetate; o-T, otoluate; M, mandelate; Ca, monochloroacetate. (227) R. P. Bell, "Acid-Base Catalysis," Oxford University Press, London, 1941. p. 82.

22

HORACE S. ISBELL AND WARD PIGMAN

a relation between the reaction velocity and the acid-base strength of the substrate when the catalyst remains the same and a series of similar substrates is investigated.” Data for typical acid and base catalysts of various types are given in Table IX.

e. Catalytic Activity of the Water Molecule -The water molecule is both an acid and a base. In the pH region of 4.5 to 6.0, and in the absence of buffers, substantially all of the catalysis of the mutarotation reaction is caused by water molecules. Hudson221and subsequent workers represented the catalytic effect of the water molecule by a single term, kHzO,and did not attempt to separate the acid and base functions. The situation is complicated by the possible existence of a concerted mechanism in addition to the acid- and base-catalyzed, stepwise process. Hence, it is of interest to consider the two types of reaction, and the acid and base functions, separately. In a stepwise process, the catalytic effect of the water molecule may be represented by:

+ ~ B ( H ~ o[H2OI, )

(17) where kA(HzO) is the catalytic activity of the water molecule as an acid, k B ( H 2 0 ) is the catalytic activity of the water molecule as a base, and the concentration of the water molecules in solution is 55.6 moles per liter. If, in the mutarotation, the parent sugar is denoted by HS, and the product by SH, the acid behavior of water may be represented bylag: ~ H Z O

= ~ A ( H ~ o[H@I )

O H @ + Hj S + H

i OH+H,O+SH+OH@.

The coefficient for the water molecule acting as a proton donor (that is, as an acid catalyst) may be calculated from equation 12 by using data for the mutarotation of D-glUCOSe at 18”, with acetic acid as the reference point. From equation 15, ~A(H,o) = k ~ o , ,

( K A ( H ~ O ) / ~= H2 ~A x ~ ) ~ ’ [~10-is/1.8x = 8.4 x 10-7.

io-5]0’3

The contribution, to the rate constant, of the water molecule as an acid catalyst is 55.6 k A ( H 2 0 ) or 4.7 X This value is about 1% of the catalytic activity of the water molecule. Base catalysis by the water molecule may be represented as: H2O

+ HS + H300

H3CP

+ SH + H2O.

may be calcuThe coefficient for the base-catalyzed reaction, kB(HnO),

TABLEIX

f C

Comparison of Various Types of Acid and Base Catalysts in the Mutarotation of D-Glucosen

1

> Acid

Base

H,go HzO

HzO OH0

Mandelic acid Acetic acid Pyridinium ion

anion anion pyridine

[Co(NH,),OHl~

[Co(NH,),OH]~

Ammonium ion

ammonia

HSO,G Hz0

so,*0 OH@

a-D-Glucose

P-D-glucose anion

P-D-Glucose

a-D-glucose anion

KA

5.6 X 1 x 4.3 X 1.8 x 3.5 x 1.6 x 3.2 x 1.2 x

10 10-16 10-5 10-6

k.4

1.4 x 10-1 9.5 x 10-5 6 X lo-, 2 x 10-3

K B

1.4 x 10-1

1O-Io

10-2

1.49 X 0.89 X lo-’, 0.36 X 2.92 X 1.59 X lo-’, 0.57 X

“Converted into decimal logarithms from the authors’ data for natural logarithms.

k, 9.5 x 6.0 x 1.1 x 2.7 x 8.3 x 7.8 x 3.2 4 x 2.17 x 1.06 x 1.74 X 1.95 X 0.58 X 0.84 X 1.49 X 0.46 X 0.71 X

103 10-2

10-I 10-3 103 103

lo2 lo2 lo2 10 lo2

lo2 10

D

Temperature, degrees

References

18 25 18 18 18 18 25 18 25 15 0 25

189 189 189 189 189 189 189 189 199 199 199 199

15 0 25 15 0

199 199 199 199 199

3+

8z $ v)

C

$ D

v)

5

B 8z E C

d c (

M

w

HORACE S . ISBELL AND WARD PIGMAN

24

lated from the data of Bronsted and G ~ g g e n h e i mby ' ~ ~means of equation 16,by using the acetate anion as a reference point. Thus: k B ( H z O ) = koAce (KB(HzO&Ace)'

= 2.7 X

lo-'

(0.018/5.56 X

lo4)'.

The value assigned to y greatly influences the value obtained for kB(HzOp For catalysis of the mutarotation of D-glucose by any of a large group of base catalysts, Bronsted and Guggenheimle9found y to be 0.4;for the catalysts listed in Table VIII, they found y to be 0.34. Use of 0.34for y in equation 16 gives a value for kB(Hzo) [HzOl that is considerably greater than the observed catalytic effect of the water molecule; use of 0.4 gives a value of kB(HzO) [HzOl corresponding to 72% of the minimum mutarotation constant, and use of 0.378for y in equation 16 makes the calculated activity equal to the total, observed, catalytic activity of the water molecule. These considerations show that, in neutral solutions, the basic catalytic function of the water molecule predominates, and that the catalytic effect observed is of the expected order of magnitude for an amphoteric substance, having the observed dissociation constant of water, and acting as a typical acid catalyst and as a typical base catalyst.

f. Catalytic Activity of the Hydroxyl Ion and the Sugar Anions.Determination of the catalytic activity of the hydroxyl ion is difficult, because the mutarotation becomes too rapid for accurate measurement when the concentration of hydroxyl ions rises above 10 pmolar. With a minute concentration of hydrogen ions, accurate measurements require rigorous exclusion of carbon dioxide and other impurities. Evaluation of the rate constant is also complicated by ionization of the sugar, a subject that was not considered by the early workers. From Osaka's measurements for D-ghCOSe in dilute, aqueous ammonia, Hudsonzz1calculated koHe to be 22,300 at 25". Bronsted and Guggenheimle9recalculated the data with allowance for the catalytic effect of the aqueous ammonia. Lowry and WilsonZzestudied the mutarotations of D-glucose and lactose, and calculated values of koH, on the assumption that, in unbuffered alkaline solutions, k = kHzO 4-koH, [OH@],and that [OH@][sugar] / [sugar anion] = KH, [Ha@] [sugar anion] / [sugar] = K A , and

K A

X

KH

= Kw.

(228) T. M. Lowry and G . L. Wilson, Trans. Faraday SOC., 24,683 (1928).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

25

As pointed out by Smith,229the values of koHO thus obtained are necessarily incorrect, because the catalytic effect of the sugar anions had not been taken into account. Smith extended Lowry and Wilson's measurements, and determined the catalytic effect of the D-glucosate anion (GO) at 0, 5 , 10, and 15". He assumed that, in unbuffered alkaline solutions, k = kH.0 koH,[OH 01 kG [G 01, and he calculated values of k,, from the relationship (k - k,,o) = k ~ 0 - Kt h X ko~o/[GH].&, is [GH] [OH@]/[Go] and is 55.6 times the equilibrium constant for the reaction [H20][GO] [GH] [OH@]. In these experiments, the contributions of the D-glucosate ion to the total rate-constant ranged from 18 to 41 %, depending on the concentration of the sugar. In a subsequent inve~tigation,2~O the study was extended, and catalytic coefficients and heats of activation were determined for catalysis of the mutarotation of D-glUCOSe in water by each of three acids and four bases. The values of k,, and koHe, obtained as outlined above, are subject to another correction. Los and S i m p s ~ n ' ~found s that the ionization constants of a- and p-D-glucopyranose differ. Because p-D-glucopyranose is the stronger acid, the pH decreases during mutarotation whenever the equilibrium is approached from the a-Danomer. Thus, the system is complicated by a change in pH as the proportions of the anomers change during the mutarotation. Experimental determination of koH, is difficult, because small variations in the hydroxyl-ion concentration cause large differences in the reaction rates. Los and Simpson Igg took into account the difference in the ionization constants of the a - and ~ p-D anomers. They postulated the equilibria:

+

+

Ga@+ H2O and

e GHa + O H @

Gp@+H,OeGHp+OH@.

It follows that the hydrolysis constants are: Khu

=

[GHa] [OH@] [&@I

and (229) G.F.Smith,J. Chem. SOC., 1824 (1936). (230) G.F.Smith and M. C . Smith,J.Chem. SOC., 1413(1937).

26

HORACE S. ISBELL AND WARD PIGMAN

By a mathematical analysis of the system and successive approximations, catalytic coefficients were calculated from the relationship:

The values obtained for the catalytic coefficients, the hydrolysis constants, and the ionization constants at absolute temperature, T, are given by the equations:

+

log koHo = log T - 3498*1 - 13.007, T

+

log Kha = - 1107*3 2.184, T

and

+

771*8 0.766, T

log&,

=--

log&

2002'4 = - --

T

5.746,

log&@ =-- 2329*3-4.359. T

At 25", the ionization constant (&a) for a-D-glucose (3.42 X is approximately half that (&,) for P-D-glucose (6.72 X 10-13). For D-glucose in equilibrium at 25", Urban and ShafFer152found an ionization constant of 8.13 x whereas the ionization constant calculated from the data of Los and Simpson for D-glucose in equilibrium is 5.5 x 10-13. Measurement of ionization constants of the anomers in alkaline solutions is difficult, because the mutarottation reaction is very rapid. Hence, relatively large errors are to be expected; nevertheless, the work clearly shows that significant differences in the degree of ionization of the anomers exist. The data for the different temperatures suggest that P-D-glUCOSe is a stronger acid than a-D-glucose by virtue of the fact that the entropy change on ionization is much larger for the P-D anomer. The temperature coefficients indicate that, at -38", the two anomers are acids of equal strength.

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

27

g. Bifunctional Catalysts. -After establishing that the mutarotation reaction requires both an acid and a base catalyst, LowryZ3* proposed that mutarotations take place by a concerted mechanism involving simultaneous transfer of a proton from an acid catalyst to the sugar and transfer of a proton from the sugar to a base catalyst. Termolecular collisions, necessary for the reaction by a ternary mechanism, are rare, compared with bimolecular collisions; hence, it would be expected that the termolecular reaction would be relatively slow. However, by use of a catalyst capable both of transferring a proton to the sugar molecule and of removing simultaneously a proton from the sugar, the process becomes bimolecular. Swain and Brownz32found that 2-pyridinol can function in this way and that it is a very effective bifunctional catalyst for the mutarotation of tetra-0-methyl-a-D-glucopyranose in aprotic solvents. 2-Pyridinol, however, does not accelerate the mutarotation of sugars in water. It is only 1/10,00Oth as strong a base as pyridine and 1/1OOth as strong an acid as phenol, yet, at 1 mM concentration in benzene, it gives a rate constant 7,000 times that calculated for an analogous concentration of pyridine or phenol. The striking difference in the catalytic effects of the mixture and of the bifunctional catalyst is explained by a concerted mechanism in which the bifunctional catalyst serves simultaneously as proton donor and proton acceptor in opening the pyranose ring.

As pointed out by Swain and coworkers,233carboxylic acids may also be effective catalysts for mutarotation, because they may react by a mechanism in which the acid molecule is hydrogen-bonded at two points to the D-glucose molecule as follows:

+ RCO,H V

k

b c = o V

(231) T. M. Lowry,J . Chem. SOC., 2554 (1927). (232) C. G . Swain and J. F. Brown, Jr.,J . Amer. Chem. SOC., 74,2534,2538 (1952). (233) C. G. Swain, A. J. Di Milo, and J. P. Cordner, J . Amer. Chem. SOC., 80, 5983 (1958).

28

HORACE S. ISBELL AND WARD PIGMAN

A somewhat similar mechanism can be envisaged for catalysis by dimeric water molecules. A bifunctional mechanism for the reaction with water seems relatively unimportant, however, because the catalytic coefficient for water molecules is in approximate agreement with the base catalysis predicted by the Bronsted equations, as described earlier (see p. 24). In contrast, the catalytic effect of 2-pyridinol is far greater than would be expected from its acid and base dissociation constants.

h. Catalytic Effect of Salts. -Ordinarily, salts of strong acids and of strong bases have little influence on the rate of m ~ t a r o t a t i o n . ' ~ ~ ~ ~ ~ ~ Thus, Pasteuf observed little difference in the mutarotations of D-glucose and D-glucose sodium chloride. Kuhn and found that the rate of mutarotation Of D-glUCOSe is retarded slightly by sodium chloride or lithium chloride over the pH range of 1to 7.7. Under some conditions, however, large effects are observed. Thus, Eastham and found that the mutarotation of tetra-0-methyl-D-glucopyranose in pyridine or nitromethane is slow, but that it is enhanced by a factor of ten on addition of 2 mM lithium perchlorate. It has been suggested that the salt stabilizes the transition state by formation of an ion-pair complex. Ix. ISOTOPE EFFECTSIN MUTAROTATIONREACTIONS 1. Basis for Effects of Water and Deuterium Oxide

Differences in the rate of mutarotation of sugars in water and in deuterium oxide provide a valuable means for studying mutarotation reactions.135,224*233,237,238 The difference in rates arises from a combination of kinetic and solvent isotope-effects, and is usually expressed as a ratiO,kH/kD, called the isotope effect. Kinetic isotopeeffects are caused by differences in the energy required for alteration of the normal and the isotopic bonds in the corresponding transition states; solvent isotope-effects can exist when the isotopic compound is used both as a reactant and as a solvent. The observed isotope-effect,kH/kD,may have values smaller than, equal to, or greater than unity. A kinetic isotope-effect is large when (234) T. M. Lowry and G. F. Smith,J. Chem. SOC.,2539 (1927). (235) R. Kuhn and P. Jacob,Z. Physik. Chem., 113,389 (1924). (236) A. M. Eastham, E. L. Blackhall, and G. A. Latremouille, I. Amer. Chem. SOC., 77,2182 (1955). (237) E. Pacsu,]. Amer. Chem. SOC., 55,5056 (1933);56,745 (1934). (238) F. A. Long and J. Bigeleisen, Trans. Faraday SOC., 55,2077 (1959).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

29

the bond joining the isotopic atom to the substrate is formed, or is broken, in the rate-determining step of the reaction. In general, the stronger the altered bond, the greater is the isotope effect. Ordinarily, the heavier isotope gives the lower reaction-rate, and, hence, values of kH/kDgreater than unity are designated as normal isotope-effects, and values less than unity as inverse isotope-effects. Solvent isotopeeffects frequently (but not always) give values of kH/kDthat are less than unity. Change of the solvent from HzO to D,O causes changes in the degree of ionization and solvation, both of the reactants and the catalysts. Thus, at 20°, the ion product of deuterium is 8.91 X whereas that of waterZ4Ois 6.76 X l O - I 5 . Because of this difference in base strength, reactions that involve proton transfer from the catalyst to the substrate prior to the rate-determining step are faster in deuterium oxide than in water. Examples of this type of reaction include: the acid hydrolysis of sucrose,241the base-catalyzed enolization of acetone,242the acid-catalyzed decomposition of ethyl d i a ~ o a c e t a t e , ~ ~ and of indolyl gly~opyranosides,2~~ and the acid anomerization of acetylated s ~ g a r s . ’ These ~ ~ , ~ reactions ~~ are known to be subject to specific acid catalysis, and the isotope effects, kH/kD, range from 0.39 to 0.69. The mechanisms involve a rapid, pre-equilibrium, proton transfer from the catalyst to the substrate (HS), followed by a ratedetermining step in which protons are not transferred. The reactions involve formation of an intermediate, conjugate acid (HSH@in water, and HSD@ in deuterium oxide). The concentrations of the intermediates depend on the following equilibria: HS

HS

+ H30@* HSH@+ H,O

+ D30@* HSD@+ D,O.

More HSD@is formed in D,O than HSH@in HzO, because D30@is a stronger acid than [email protected], reactions that take place by this mechanism are faster in deuterium oxide than in water. (239) A. K. Covington, R. A. Robinson, and R. G. Bates,]. Phys. Chem., 70,3820 (1966). (240) W. F. K. Wynne-Jones, Trans. Faraday Soc., 32,1397 (1936). (241) (a) E. A. Moelwyn-Hughes and K. F. Bonhoeffer, Naturwissenschaften, 22, 174 (1934). (b) P. Gross, H. Steiner, and H. Seuss, Trans. Faraday Soc., 32,883 (1936).(c)W. H. Hamill and V. K. La Mer,]. Chem. Phys., 4,294 (1936). (242) 0.Reitz and J. Kopp, Z. Physik. Chem., A184.429 (1939). (243) (a) P. Gross, H. Steiner, and F. Kraus, Trans. Faraday Soc., 32, 877 (1936); 34,351 (1936).(b) J. D. Roberts, C. M. Regan, and I. Allen,]. Amer. Chem. Soc., 74,3679 (1952). (244) J . P. Horwitz and C. V. Easwaran,]. Org. Chem., 33,3174 (1968). (245) (a) J. T. Edward, Chem. Ind. (London), 1102 (1955). (b) W. A. Bonner, ]. Amer. Chem. Soc., 83,962,2661 (1961).

HORACE S. ISBELL AND WARD PIGMAN

30

In marked contrast to the reactions subject to specific acid-catalysis, the decomposition of n i t ~ a m i d e the ,~~~ mutarotation reactions of sugars,224*237 (see Table X),and the hydrolysis of acetamide by strong acidsz4'proceed more rapidly in water than in deuterium oxide. These reactions are subject to general acid-catalysis, and the ratio k,/k, is greater than unity. For base catalysis in water and in deuterium oxide, isotope effects are ordinarily greater than unity. Thus, the ketonization the hydrolysis of the enol of 3-methyl-2,Cpentanedione by of acetamide by base,247and the reaction of phenyl acetate with glycine or with ammonia249give isotope effects that range from 1.1to about 1.6. TABLEX Ratios of Catalytic Coefficients for Mutarotation of Sugars in H20and in D 2 0

Sugar a-D-Glucopyranose

a-D-Galactopyranose a-D-Xylopyranose P-D-Fructopyranose

Temperature, degrees

25 20 25 20 25 25 24 24 24 20 20 20 20

Species h 3 0 0 IkD,oo kHaOo

IkDaOo

kHzOlkDsO

kH,olko,o

HOAclDOAc OAc@/OAc@( DzO)

klka kHdkDsO

klka kHzO@ IkDaO@

kH,olkrm kH,o" /kD,o@ k"*OlkD*O

Ratio

References

1.37 1.39 3.80 3.87 2.60 2.38 1.73-2.12 3.10-3.76 1.12- 1.47 1.40 3.65 1.39 3.87

135 245 243 245 243 244 260 260 260 245 245 245 245

"Ratio of rates in H 2 0 and DzO for mutarotatase-catalyzed a-p-pyranose interconversion.

For elucidation of the course of mutarotation reactions, the timing of the addition and elimination of the proton is important. If the proton transfer occurs after the rate-controlling step, it will have no primary kinetic consequence. If the proton transfer occurs during the rate(246) (a) V. K. La Mer and T. Greenspan, Trans. Faraday SOC., 33, 1266 (1937). (b) S . Liotta and V. K. La Mer,]. Amer. Chem. Soc., 60,1967 (1938). (247) 0.Reitz, Z . Elektrochem., 44,693 (1938). (248) F. A. Long and D. Watson,]. Chem. SOC.,2019 (1958). (249) W. P. Jencks and J. Carriuolo,]. Amer. Chem. SOC., 82,675 (1960).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

31

controlling step, a large, kinetic isotope-effect will be found. If proton addition occurs before the rate-controlling step, and no proton transfer occurs in the transition state, the reaction is subject to hydroniumion catalysis, and an inverse isotope-effect (arising from the difference in the basicity of HzO and DzO)will be found. Thus, the magnitude of the isotope effect, and the way in which this varies with the experimental conditions, provide means for ascertaining the reaction mechanism. 2. Effect of Deuterium Oxide and Water on Mutarotation Reactions

Numerous studies have been made of the mutarotation of sugars in ~ ~ ' that the deuterium oxide and in water (see Table X). P a c s ~ found rate of mutarotation of a-D-ghcopyranose is higher in water than in deuterium oxide. Later, Hamill and La Mer135found that the rate of mutarotation is higher in water than in deuterium oxide, regardless of the catalyst, and that the value of k,/kD is dependent on the strength of the catalyst. Thus, they found k,/kD to be 1.37 for catalysis by H,O@ and 3.80 for catalysis by water molecules. Nicolle and Weisof a large number of b ~ determined ~ h the~ rate ~of mutarotation ~ sugars, and found that the value of the ratio k,/k, lies in the range of 3.0 to 3.8; however, the values were not related to pH or to the type of reaction. Challis, Long, and Pockerzn9found that the values of k H / k Dfor the mutarotation of tetra-0-methyl-a-D-glucopyranosein water and in deuterium oxide do not differ widely from those found for a-D-glucopyranose in either solvent. From these studies, they concluded that the acid-catalyzed mutarotation involves a rapid, equilibrium exchange between the deuterium or the hydrogen atoms of the solvent and the anomeric hydroxyl groups, followed by rupture of the anomeric 0 - H bonds in water, and of the 0 - D bonds in deuterium oxide, with simultaneous ring-openings in both cases. Rupture of the anomeric 0 - H bond is effected in the transition state by the base catalyst, usually the conjugate base of the acid catalyst. Bentley and Bhate14')conducted a meritorious study of the mutarotations of D-glucose and D-galactose in the presence and absence of the enzyme mutarotase from Penicillium notatum under a variety of conditions. For measurements of the water-catalyzed and mutarotase-catalyzed reactions at 23-24", they found the following (250) J . Nicolle and F. Weisbuch, Compt. Rend., 240,84 (1955).

32

HORACE S . ISBELL AND WARD PIGMAN

values for kH/kD:D-glucose-I-H, 3.56 (water), 1.86 (mutarotase); Dglucose-1-d, 3.78 (water), 1.80 (mutarotase); D-galactose (fast reaction), 3.76 (water), 0.64 (mutarotase); and D-galaCtOSe (slow reaction), 3.10 (water), 1.12 (mutarotase). The values of kH/kD for the mutarotase-catalyzed anomerizations (slow reactions) are close to those found by other authors for acid catalysis, but the corresponding ratio for the fast reaction for D-galactose is exceptional in being less than unity. This low value suggests that this reaction, which arises from a pyranose-furanose interconversion, may operate by a fundamentally different mechanism. The data require checking, however, because of the experimental difficulties and errors involved in measuring the fast reactions. The unusual values of kH/kDobtained for mutarotase led the authors to study the catalytic action of histidine and histidylhistidine, on the basis that these peptides might be a part of the active site of the enzyme. Previously, We~theimel-2~~ had shown that certain amino acids have strong catalytic action on mutarotations, and pointed out that histidine, at pH 6.0-6.5, is a more efficient catalyst than the same concentration of a strong acid. For the mutarotation of D-glucose with histidine or histidylhistidine as the catalysts, Bentley and BhateI4O reported that the values of the ratio k H / k Dwere 3.68 and 3.85, respectively. Further information on mutarotase is given later in this Chapter (seep. 63). Isbell and Wade2%found that the isotope effects for the mutarotation of a-D-frUCtOSe, a pyranose-furanose interconversion, and for the mutarotation of a-D-ghCOSe, an a=P-pyranose anomerization, are much alike. Presumably, in both instances, the overall mutarotation arises from concurrent reactions involving acid and base catalysts. With both reactions, the isotope effects increase from about 1.3 in highly acid solutions to about 4 in slightly acid solutions, and then decrease slightly in highly alkaline solutions. The gradual change in the isotope effects is in accordance with the view that, under the conditions studied, several acid- and base-catalyzed mechanisms operate concurrently, as will be discussed later (see p. 35).

3. Use of Isotope Effects for Determination of Mechanisms of Reaction Several interpretations have been advanced to explain why the ratio k H / k Dfor mutarotation reactions is greater than unity. For the (251) F.H.Westheimer,J. Org. Chem.,2,431(1937).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

33

acid-catalyzed reaction, Bonhoeffer and c o ~ o r k e r s , ' Bell,253 ~ ~ , ~ ~and ~ P ~ r l e esuggested '~~ a slow protonation of the ring-oxygen atoms with simultaneous ring-opening (see Fig. 7). It is supposed that, prior to the rate-determining step, an intermediate is formed that is ruptured in the transition state, with cleavage of the HA bond in water and the DA bond in deuterium oxide. The bonds are stronger for weak acids than for strong acids and, hence, kH/kDis larger for weak than for strong acids.

FIG. 7. - Ring-opening by a General Acid Catalyst.

Challis and c o w o r k e r ~ , 2as~ ~well as Long and B i g e l e i ~ e n ,sug~~~ gested a different interpretation. They considered that a preequilibrium proton-transfer occurs, giving a greater concentration of the conjugate acid in D 2 0 than in H 2 0 . Were this the only effect, the kH/kD ratio would be less than unity; but, as in D,O the hydrogen atoms of the hydroxyl groups of the sugar are replaced almost instantly by deuterium, the bonds broken in the subsequent slow step (see Fig. 8) are the C - 0 bonds in both systems, the 0 - H bond in water, and the 0 - D bond in deuterium oxide. Possibly, the observed isotopeeffect is the resultant of both mechanisms proceeding concurrently.

FIG. 8.- Ring-opening with Prior Protonation of the Ring-oxygen Atom. (252) K. F. Bonhoeffer, Trans. Famday SOC., 34,252 (1938). (253) See Ref. 227, p. 151.

34

HORACE S. ISBELL AND WARD PIGMAN

The high value of kH/kD(3.5to 4.0) for the water-catalyzed reaction may arise from a substantial contribution to the overall mutarotation of the concerted mechanism proposed by Lowry. In the transition state for the concerted mechanism (see Fig. 9), an acid catalyst releases a proton to the ring-oxygen atom, and a base catalyst removes a proton from the anomeric hydroxyl group; the isotope effect is high, because two proton-transfers occur in the transition state.

I

H

FIG.%-Concerted Reaction with H,O as Catalyst.

A possible variation of the concerted mechanism includes a dimeric water molecule as a bifunctional catalyst (see Fig. 10).Another concept concerning the role of water molecules has been developed by SchmidaZs4He suggested an activated complex of the type (BH@... H,O ... GO), where B is a base, and GO is a D-ghcosyloxy anion. In the hydrogen-ion-catalyzed mutarotation of D-glucose in aqueous solutions, H,O acts as a base to remove H30@,and the activated complex is D-glucoseo...H,O ...H@@. To account for the relatively high catalytic effect of carboxylic acids, it has been suggested that they also act as bifunctional ~ a t a l y s t s , ~ ~ ~ , ~ ~ as discussed earlier (see p. 27). n I

B

rapid

I

H

H

FIG. 10.- Dimeric H,O as a Bifunctional Catalyst. (254) (a) H. Schmid, Monatsh., 94, 1206 (1963); 95, 454, 1009 (1964). (b) H. Schmid and G . Bauer, ibid., 95,1781 (1964); 97,866 (1966).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

35

x. MECHANISMS FOR h‘iUTAROTATION OF SUGARS IN AQUEOUS SOLUTION 1. Overall Reaction Mechanisms The mutarotation of a sugar in aqueous solution involves transfer of a proton from an acid catalyst to the sugar, and the transfer of another proton from the sugar to a base catalyst. The reaction starts with attack on the cyclic sugar by either an acid or a base catalyst, followed by a slow rupture of the ring. In the resulting intermediate, reaction of the hydroxyl group on C-5 of aldopentoses and higher monosaccharides yields the anomeric aldopyranoses, whereas reaction of the hydroxyl group on C-4 yields the corresponding anomeric aldofuranoses. The sequence and the timing of the addition and elimination of the protons give rise to several reaction-paths. For years, carbohydrate chemists have ascribed a major role to an acyclic aldehydo or keto intermediate in the mutarotation reaction. However, it seems difficult to reconcile this concept with the paucity of evidence for such intermediates in the equilibria of reducing sugars, despite the rapidity of mutarotation reactions and the formation of a variety of aldehydo derivatives. Regardless of the cause, the low proportion of the acyclic modification of the sugar in the equilibrium mixture shows that ring closure is very much more rapid than ring opening. In a suggested mechanism,255the behavior of the sugars in mutarotation and related reactions is ascribed to the formation of “pseudo-acyclic” intermediates. Thus, it is envisaged that, in the mutarotation reaction, the sugar ring is opened momentarily, forming a pseudo-acyclic intermediate having a conformation resembling that of the parent sugar. It is supposed that this intermediate then passes through characteristic transition-states to the a- and /3-pyranose and the a-and p-furanose modifications of the sugar. According to the reaction-rate theory,256the rates of interconversion of the anomers, as well as the rates of ring isomerization, should depend on the differences in free energy between the reactants in the ground state and in the transition states. One curve of Fig. 11 depicts a pseudo-acyclic intermediate on each side of the transition state (B) governing the ae&pyranose interconversion. The a-pyranose may be converted into a furanose form through a pyranose-furanose transition state (D)with less activation energy than that required for the (255) H. S. Isbell, H. L. Frush, C. W. R. Wade, and C. E. Hunter, Carbohyd. Res., 9, 163 (1969). (256) S. Glasstone, K. Laidler, and H. Eyring, “The Theory of Rate Processes,” McCraw-Hill Book Co., New York, 1941, pp. 153-201.

HORACE S. ISBELL AND WARD PIGMAN

36

Equilibrium with othdr acyclic and ring modifications

B

r”\ 2

a-Pyranose

8-Pyranose

a-1 ranose a-Funnose a-Furanose

13-Furanoso

Reaction coordinates

FIG. 11.-Hypothetical Role of Pseudo-Acyclic Intermediates in Mutarotation Reactions. [The curves qualitatively represent the changes in free energy of a sugar for which the P-pyranose is more stable than the a-pyranose (curve 1). The pyranosefuranose interconversion (curve 2) is faster than either the a-P-pyranose anomerization (curve 1) or the a-p-furanose anomerization (curve 3). The relative stabilities of the isomers are represented in the decreasing order: P-pyranose > a-pyranose > p-furanose > a-furanose.]

a-P-pyranose interconversion. Likewise, the transition state for the a-p-furanose interconversion (F)may be at a higher energy-level than that for the pyranose-furanose ring-isomerization. Another set of intermediates may be envisaged for pyranose-furanose interconversions involving the p anomers. The structures of the intermediates are hypothetical, but they must resemble those of the parent sugars. Thus, HammondZs7points out that: “Zf two states, as, for example, a transition state and an unstable intermediate, occur consecutively during a reaction process and have nearly the same energy content, their interconversion will involve only a small reorganization of the molecular structures.” Hence, it would be expected that the hypothetical intermediates would resemble the parent cyclic sugar in conformation, even though the ring may be opened momentarily. (257) G. S. Hammond,J. Amer. Chem. SOC., 77,334 (1955).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

37

The possibility of the existence of pseudo-acyclic intermediates is in accord with (a) the substantial difference in the entropy of ionization of a- and P-D-glUCOSe, noted by Los and S i m p s ~ n , '(b) ~ ~the high, negative entropy of activation for the water-catalyzed mutarotation noted by S ~ h m i d and , ~ ~(c) ~ the absence of a substantial proportion of a true acyclic form of the sugars in the equilibrium solution. The fact that the rate of exchange of l80in the anomeric hydroxyl group of the sugar is lower than that of the mutarotation reaction strongly suggests that the free aldehyde form is not an intermediate (see p. 44). On the basis of thermodynamic considerations, A ~ r e suggested e ~ ~ ~ that the mutarotation reaction proceeds through a high-energy, acyclic form which is stabilized by formation of a complex with the solvent molecules. The susceptibility of the atoms of sugar molecules to attack by acid and base catalysts may be associated with electronic density, as well as with stereomeric factors. Zhdanov and coworkers259have calculated the electronic charges of the atoms of various types of carbohydrate molecules by an adaptation of the molecular-orbital method. The charge distributions calculated for aldopentopyranose and aldopentofuranose molecules are as follows:

(258) T. E. Acree, "The Tautomerism of D-Glucose, D-Mannose, and D-Galactose," Ph.D. Thesis, Cornell University, 1968. (259) Yu. A. Zhdanov, V. I. Minkin, Yu. A. Ostroumov, and G . N. Dorofeenko, Carbohyd. Res., 7,156 (1968).

HORACE S. ISBELL AND WARD PIGMAN

38

The results of the calculations indicate that the electronic charges on the hydroxyl groups on the secondary carbon atoms do not differ significantly. The acidic nature of the hemiacetal hydroxyl group is in accord with the lower electron-density of the hemiacetal oxygenatom, in comparison with that of the other hydroxyl groups. The electron density of the ring-oxygen atom is even lower. Hence, in acid media, a proton should attack the anomeric oxygen-atom, in preference to the oxygen atom of the pyranoid or furanoid ring, as, for instance, in the anomerization of ethyl D-xylopyranosides in ethanol containing hydrogen chloride.260 With sugars in aqueous solutions, however, coordination of the hydrogen atom of the anomeric hydroxyl group with water further increases the electron density of the anomeric oxygen atom, and ultimately leads to transfer of electrons to the ring-oxygen atom with formation of an acyclic sugar. The several mechanisms for mutarotation reactions are sometimes characterized as acid-catalyzed or base-catalyzed. This terminology is convenient, but misleading, because both types of catalysis are involved in each instance. Hammett261used the term acid catalysis for the reaction involving the mobile and reversible addition of a proton to the ether oxygen atom followed by a rate-determining reaction with a base, and the term base-catalysis for the reaction involving mobile, reversible removal of a proton followed by a ratedetermining reaction with an acid. Isbell and FrushZs2reversed this terminology in order to emphasize (a) the function of the catalyst in the rate-determining step, and ( b ) that the high rate of mutarotation of glycosylamines in the presence of weak acids arises from acid catalysis. 2. Specific Mechanisms

a. Mobile and Reversible Addition of an Acid Catalyst to the RingOxygen Atom of the Sugar, Followed by Slow Rupture of the Ring with Transfer of a Proton to the Sugar. -The mechanism is represented by the following equations, in which HS is a sugar, HA is a catalyst, and p refers to the acyclic intermediate.

+ HA e HSaHA, equilibrium HSpaHO + A @g HSppH@+ A@e HSpHA

HSa HSaHA

(260) R. J. Femer, L. R. Hatton, and W. G. Overend, Carbohyd. Res., 8,56 (1968). (261) L. P. Hammett, “Physical Organic Chemistry,” McGraw-Hill Book Co., New York, 1940,p. 337. (262) (a) H. S. Isbell and H. L. Frush, J . Res. Nat. Bur. Stand., 46, 132 (1951);(b)]. Org. Chem.,23,1309(1958).

MUTAROTATION OF SUGARS I N SOLUTION: PART I1

11

39

(shift of C-4)

All acyclic forms + HA

/Lok H RCOH

n

-

RCOH rapid

+HA

slow

e

P

t"" H RCOH

+ A@ C -H

OH

OH HSaf

HSaf HA

@AH HSflaf H@

FIG. 12.-Acyclic Mechanism for Mutarotation Reactions.

The reaction velocity is k [HSal [HA]. As indicated in Fig. 12, the reaction sequence starts with the rapid, reversible addition of the acid, HA, to the ring-oxygen atom. This addition is followed by a series of reactions that result in a pseudoacyclic intermediate (HSaHA) which establishes equilibrium with

40

HORACE S. ISBELL AND WARD PIGMAN

other acyclic forms. In the activated complex formed initially, movement of electrons from the anomeric hydroxyl group releases the ringoxygen atom to form the pseudo-acyclic intermediate HSpaHo. Rotation of C-1 of the intermediate through 120"gives the pseudo-acyclic intermediate, HSppHo, and ring closure leads to the anomer HSP. The hypothetical transition-states for formation and cleavage of the a and p anomers differ from one another in the arrangements of the atoms in the vicinity of the anomeric center. The pseudo-acyclic intermediates account for rapid reconversion of the sugar into cyclic forms, and explain the lack of aldehydic properties. Presumably, the activated complex formed by addition of the acid catalyst to the ringoxygen atom takes part directly in the cleavage reaction. The ratedetermining step involves rupture of the oxygen ring, with release of the anion A@. In the transition state, the H-A bond is stronger for weak acids than for strong acids; this accounts for the fact that the isotope effect is higher for weak than for strong acids. With hydronium ion as the acid catalyst, cleavage of the complex in the transition state yields the acyclic cation and a molecule of water. Interaction of the solvent (HzOor DzO)with the proton of the anomeric hydroxyl group aids in rupture of the ring, but this factor is of no kinetic importance, because the bond joining the proton to the anomeric oxygen atom is not altered in the transition state. Presumably, the anomeric hydroxyl group is coordinated with the solvent throughout the process. b. Mobile and Reversible Addition of an Acid Catalyst to the Sugar Anion, Followed b y Slow Rupture of the Ring with Transfer of a Proton to the Sugar Anion.-This mechanism differs from that in subsection (a) (shown in Fig. 12) merely in that the mutarotation begins with the anion of the sugar, instead of that of the free sugar. The water-catalyzed mutarotation of D-glucose by this mechanism is depicted by Boeseken conformational formulas in Fig. 13. The difference in the entropies of activation for the ionizations of a- and p-Dglucose supports the concept of differences in the nascent, acyclic intermediates derived from the two anomers. Aside from the involvement of pseudo-acyclic intermediates, this mechanism is the same as the base-catalyzed mechanism suggested by HammettZB1and B0nh0effer.l~~

+ +

+

H S a OH 0 Sa@ H,O (equilibrium) Sa@ HA e Sa@HA(equilibrium) Sa@HA SpaH + A @ s SppH A @ Sp@HA

+

The reaction velocity is k [HSa] [OH@][HA].

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

/o

CH,OH

CH,OH

-

:' HO HO

HO

41

OH

~

b

H

H

a-o-Glucopyranose anlon

Pap

H

11 H

H CqOH

HO

OH +OHB

O

B

~

C

~

OH

-

¶ , D-

H

Hw

-

HO HO H

O

H

Glucopyranoae anion

c

' , ,>

H,.OH

2

0

HO

7

OH

HO H

H

H

H

HO

HO

OH HH

H

+OHB

uo P

FIG. 13.-Hypothetical Intermediates in the Interconversion of a- and P-D-CIUCOpyranose in Alkaline Solution. [See Fig. 11,curve 1.1

c. Mobile and Reversible Addition of a Proton to the Ring-Oxygen Atom, Followed by Addition of a Base Catalyst to the Hydrogen Atom of the Anomeric Hydroxyl Group, Followed by Slow Rupture of the Ring and Transfer of the Anomeric Proton to the Base.-Hammett261 considered this mechanism the most plausible one for general acidcatalysis. Challis and cow0rkers~~9 and Long and B i g e l e i ~ e nex~~~ plained the isotope effect for the mutarotation of D-glucose in HzO and D20 by this process. The rate-determining step of the reaction involves transfer of a proton (or a deuteron) from the anomeric hydroxyl group to a base catalyst. Involving the reaction with water as catalyst, this process yields a hydronium ion, as depicted in Fig. 14.

+ H30@ HSaH@+ H,O (equilibrium) HSaH@+ B Z H Sp a + HB@ HSpP + HB@Z HSPH@+ B HSa

The reaction velocity is k [ H S a ] [H30@][B] = k' [HSal [HB@l.

HORACE S. ISBELL AND WARD PIGMAN

42

FIG. 14.-General, Base-catalyzed Mutarotation, with Water as the Base.

d. Concerted Mechanism: Addition of a Proton to the Ring-Oxygen Atom and Transfer of a Proton from the Anomeric Hydroxyl Group to a Base in the Transition State. -The concerted reaction is discussed on p. 16. B

+ H S ~ +HA s BH@+ H S ~ A~@+e BH@+ HS&?+ A@ e B + HSP + HA

The reaction velocity = k [HSal [Bl [HA]. Sugar + H ~ O +

FIG. 15.-Hypothetical, Specific, Acidcatalyzed Mutarotation.

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

43

e. Specific, Hydrogen-Ion Catalysis. - When the ring-oxygen atom is protonated prior to ring cleavage, the reaction is subject to specific, hydrogen-ion catalysis (see Fig. 15). Specific acid-catalysis differs from general catalysis in that the H-A bond is broken prior to the ratedetermining step, and not in the transition state. For this reason, no primary isotope-effect exists, but a solvent isotope-effect occurs, because the concentration of the sugar cation is higher in deuterium oxide than in water. The solvent isotope-effect causes k,/k, to be less than unity (an inverse isotope-effect), as in the acid hydrolysis of sucrose.241Experimentally, no inverse isotope-effect has been found for the mutarotation reaction under any conditions (except, perhaps, for the mutarotase-catalyzed, rapid reaction of D-galactose; see p. 32), and specific hydronium-ion hydrolysis plays, at most, only a minor role in the overall mutarotation. XI. MUTAROTATIONOF CERTAIN SUGAR DERIVATIVES 1. Anomerization by Way of a Cyclic Carbonium Ion This process is the main course for the acid-catalyzed anomerization of sugar acetates, methyl glycosides, and related compounds in nonIn many instances, formation of the aqueous solvents.1"~244~245~260*z63 cyclic carbonium ion is assisted by participation of a neighboring acetyl group,IMbut this process is not a necessary feature. Anomerizatakes place tion of tetra-O-acetyl-2-deoxy-cr-~-urubino-hexopyranose through a cyclic carbonium ion as d e p i ~ t e d . ~ ~ ~ ' ~ '

-

(H,SO,+ HOAC) AcO C-H I OAc

AcO

//

+0 OAc

(H,SO,

AcO H

(263) J. N. BeMiller, Adoan. Carbohyd. Chem., 22,25 (1967).

CIH

0

+ HOAc)

44

HORACE S. ISBELL AND WARD PIGMAN

The cyclic mechanism for the anomerization of sugar acetates has been fully substantiated by studying isotope effect^.^^^'^' The slow step in the process is cleavage of the anomeric oxygen bond; this step is preceded by protonation of the anomeric oxygen atom. In a deuterium oxide system, this process would yield a higher concentration of the conjugate acid than would be present in a dihydrogen oxide system. Inasmuch as the rate of reaction is proportional to the concentration of this intermediate, the value of the ratio k,/kD should be less than unity, as it is in the anomerization of sugar acetates. Thus, B ~ n n e r , * ~found ( ~ ) that k,/kD = 0.6 for the acid-catalyzed anomerization of penta-0-acetyl-D-glucopyranosein 1:1AcOD-Ac,O containing DzS04and in 1:l AcOH-Ac,O containing H,S04. In striking contrast, as we have already seen (see p. 30), the values of kH/kDfor the mutarotations of the sugars in D,O and H,O are greater than unity under all conditions. Hence, the mutarotation of the sugar in water does not take place by cleavage of the anomeric hydroxyl group, as is the case for the cyclic mechanism. The behavior of sugars labeled with l80at the anomeric hydroxyl group also shows that the cyclic mechanism does not prevail under ordinary conditions. If the reaction involved elimination and addition of a hydroxyl group at the anomeric carbon atom, the rate of exchange of l80in D - g l u c o ~ e - l - ~should ~o correspond to the rate of mutarotation. Experimentally, the rate of oxygen exchange at 30" is only 5% of that of m~tarotation.'~'The small proportion of 180-exchange may occur by reversible formation of a gem-diol of an intermediate, acyclic sugar. The observation that exchange of l80is relatively slow indicates that the nascent carbonyl group of the acyclic intermediate reacts faster, intramolecularly, with neighboring hydroxyl groups than, intermolecularly, with hydroxyl ions of the solvent. In this connection, Rittenberg and Graff39found that the relative rate of exchange of l80with respect to the rate of mutarotation is minimal at pH 4, and rises rapidly below pH 3 and above pH 6. The effect of pH on the rate of the exchange reaction suggests that two independent reactions exist, one catalyzed by bases and the other by acids. The rate-determining step for the exchange reaction is not the same as that for the mutarotation, because the activation energies are different. At pH 7, the energy of activation of the exchange reaction is 23,400cal, whereas that for mutarotation is 17,200cal. The cyclic and the acyclic mechanisms differ fundamentally. In the cyclic mechanism, the acid catalyst attacks the anomeric oxygen atom, and electrons move from the anomeric carbon atom to the

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

45

anomeric oxygen atom; occurrence of the reaction requires a strong, electrophilic catalyst and a good leaving-group. Aprotic solvents are more favorable than water for this type of reaction, because of combination of the water with the acid. In the acyclic mechanism, the acid catalyst attacks the ring-oxygen atom, and electrons move from the anomeric oxygen atom to the anomeric carbon atom and from the anomeric carbon atom to the ring-oxygen atom. With glycosyl acetates, a shift of electrons to the anomeric acetoxyl group (the cyclic mechanism), and addition of a proton, give acetic acid, whereas a shift of electrons in the opposite direction (the acyclic mechanism) would result in a highly unstable, acetoxyl oxonium ion. Several comprehensive studies have been made of the anomerization and isomerization reactions of methyl glycosides under a wide variety of conditions, and the reaction rates have been correlated with configurations and conformations. The work is not discussed here, because it has been covered in several excellent With a free sugar, formation of a cyclic carbonium ion would involve elimination of a hydroxyl ion, a reasonable step, but another pathway is more plausible because the anomeric hydroxyl group has a greater tendency to ionize as an acid. Ionization of the proton of the anomeric hydroxyl group releases electrons to the anomeric carbon atom, which, in turn, reIeases the ring-oxygen atom. The distinctive feature of the reaction is the capacity of the anomeric hydroxyl group to supply a pair of electrons and, thereby, to initiate ring cleavage. Other sugar derivatives that have this capacity tend to react by the acyclic mechanism, whereas compounds that draw electrons from the anomeric carbon atom react through a cyclic carbonium ion.

2. Anomerization by Way of a Bimolecular Replacement Reaction This type of reaction may be illustrated by the reaction of tetra-0acetyl-a-D-glucopyranosylbromide in acetonitrile with silver fluoride, ~~~*~~~ to give tetra-O-acetyl-~-D-ghcopyranosyl f l ~ o r i d e .Application of a similar process to the anomerization of a free sugar in water would (264) G. A. Howard, G. W. Kenner, B. Lythgoe, and A. R. Todd, /. Chem. Soc., 855 (1946). (265) J. Conchie, G. A. Levvy, and C. A. Marsh, Advan. Carbohyd. Chem., 12, 157 (1957). (266) J. W.Green, Advan. Carbohyd. Chem.,21,95(1966). (267) See Ref. 94,p. 716. (268) B.Helferich and R. Gootz, Ber., 62,2791(1929). (269) (a) F.Micheel and A. Klemer, Chem. Ber., 85,187(1952);(b)90,1612(1957).

46

HORACE S. ISBELL AND WARD PIGMAN

result in the exchange of hydroxyl ions with the solvent. The observation that the rate of oxygen exchange with D-glucose-l-'*O is much lower than the mutarotation reaction indicates that the latter does not take place to any appreciable extent by exchange of hydroxyl ions. In general, nucleophilic attack on a six-membered ring is a slow process and is not common.

3. Ring Contraction and Expansion By transannular attack on the anomeric center by neighboring hydroxyl groups, changes in ring structure may be envisaged. Ordinarily, the geometry of the pyranose and furanose rings does not permit the overlapping of molecular orbitals required for this type of change. In some instances, an oxirane (ethylene oxide) ring may serve as the intermediate in a stepwise shift of ring structure, as in the formation of certain anhydro sugars. The formation of methyl 3,6-anhydro-a-~was glucopyranoside from methyl 2,3-anhydro-a-~-allopyranoside one of the first examples of a change from one ring structure to another by an opposite-face, neighboring-group attack.270 Although formation of anhydro sugars is not considered to be part of the mutarotation reaction, formation of anhydro sugars accompanies the many mutarotation r e a ~ t i o n s . ~ In ~ * the - ~ ~ aldohexopyranoses, ~ exocyclic hydroxyl group on C-6 is favorably situated for anhydride formation, and 1,6-anhydrohexopyranosesare formed by acid catalysis, concurrently with the mutarotation reaction. At loo", the proportion of the 1,6-anhydrohexoses at equilibrium ranges from 0.7% for Dglucopyranose to 80% for D-idopyranose. For the heptulopyranoses, the proportion of the 2,7-anhydroheptulopyranosesat equilibrium is even greater. 273*274 (270) (271) (272) (273) (274)

H. S . Isbell, Ann. Reo. Biochem., 11,65 (1940). L. C. Stewart, E. Zissis. and N. K. Richtmyer, Chem. Ber., 89,535 (1956). F. B. LaForge and C. S . Hudson,]. Biol. Chem., 30,61(1917). N. K. Richtmyer and J. W. Pratt,]. Amer. Chem. SOC., 78,4717 (1956). E. Zissis, L. C. Stewart, and N. K. Richtmyer, J . Amer. Chem. SOC., 79, 2593 (1957).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

47

4. Glycosylamines The mutarotation reactions of glycosylamines are important, not only in relation to the mutarotation of the sugars, but also with reference to the inversion reactions of nucleosides and other important biological compounds. Pioneer work by Kuhn and BirkofeP showed that, for certain nucleosides and by Todd and and other glycosylamines, long known to be sensitive to acid catalysis, both a-and P-pyranose anomerizations and pyranose-furanose interconversions take place. The mutarotation of the glycosylamines occurs in much the same way as for the sugars,262but often involves other reactions, such as h y d r o l y ~ i s . ~Usually, ~ ~ ' ~ ' hydrolysis is slow in either highly alkaline or highly acid solutions, but the rates vary widely ~~'~' according to the character of the glycosylamine. T i p s ~ n ~pointed out the exceptionally high stability of the nucleosides of cytosine and of uracil, and the fact that these compounds are readily hydrolyzed after hydrogenation. The mutarotations of glycosylamines are extremely sensitive to acid catalysts, but not to base catalysts; in striking contrast, the mutarotations of sugars are extremely sensitive to base catalysts, and not so sensitive to acid catalysts. The difference is illustrated in Table XI, which gives the catalytic coefficients for the mutarotation of Larabinopyranosylamine and a-D-glucopyranose in aqueous ammonium ,chloride solutions. It may be seen that the most active catalyst for the mutarotation of the glycosylamine is the hydronium ion, whereas, for TABLEXI Catalytic Coefficientsa of Mutarotation Reactions at 20" ~~

L- Arabinosyl-

Catalyst

HzO H@ OH @ NHY

Symbol k",Ob

kH kOH

kmi.

amine <7 x 10-6 6.9 X 108 4 x 10-2 1 x 10-2

D-Glucose 5 x 10-6 3.6 X lo-' 8 x103 1.2 x 10-3

"Data from Reference 262. The values given in the reference are for the combined constant, kHIo [H,O], but they have been changed here to kHIO.

(275) R. Kuhn and L. Birkofer, Ber., 71,1535 (1938). (276) (a) W. Pigman, E. A. Cleveland, D. H. Couch, and J. H. Cleveland, /. Amer. Chem. Soc., 73, 1976 (1951). (b) R. S . Tipson, Aduan. Carbohyd. Chem., 1, 193 (1945).

HORACE S. ISBELL AND WARD PIGMAN

48

the mutarotation of D-glucose, the most active catalyst is the hydroxyl ion. The surprising inertness of the glycosylamines to mutarotation by a basecatalyzed mechanism arises from the small tendency of an amino nitrogen-atom to release a proton. At high alkalinities, the basecatalyzed reaction takes place to a small degree with glycosylamines of primary and secondary a m i n e ~ . ~With " those of tertiary amines, there is no proton to be released, and the mutarotation reaction must take place by acid catalysis (see Fig. 16). The high sensitivity of the

H

-

Chon

CbOA

7

no

OH

OH H

H

H

H

H

H

8- D- Glucopyranoaylamine

WSP

FIG. 16.- Mutarotation of D-Glucopyranosylamine.

mutarotation of the glycosylamines to acid catalysis arises from the fact that the nitrogen atom reverts (by an electron shift) to an iminonium structure, with release of the ring-oxygen atom. The oxygen atom of the anomeric hydroxyl group of the sugar undergoes similar transformation, but the oxonium intermediate is not formed so readily as the (277) H. Simon and G .Philipp, Carbohyd.Res., 8,424 (1968).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

49

iminonium intermediate. The high sensitivity of the mutarotation of the glycosylamines to acid catalysts, and their inertness to basic catalysts, support the mechanisms given in Figs. 12 and 14, for the acid- and base-catalyzed mutarotations, respectively, of the sugars.

5. Thio Sugars

In view of the key roles that the ring-oxygen atom and the anomeric hydroxyl group play in anomerization reactions, it is of interest to consider the reactions of the sulfur analogs. Two types of such sulfur derivatives exist: those in which a sulfur atom takes the place of (a) the ring-oxygen atom or (b) the oxygen atom of the hemiacetal hydroxyl group. Sugars having a sulfur atom in the ring are derivatives of thiols, whereas the glycosyl sulfides (or 1-thioaldoses) are thiohemiacetals. The sulfhydryl group is much more nucleophilic than the hydroxyl group; hence, in forming the cyclic sugar, the carbon-sulfur bond is formed more readily than the carbon-oxygen bond.278With 4-thioaldoses and 5-thioaldoses, 4-thiofuranoses and 5-thiopyranoses are formed, respectively, in much the same way as with the oxygen analogs. Schwarz and Yule279found that the mutarotation of 5-thio-aD-xylopyranose at pH 4.4 is relatively slow (half-life, 10 hr), but that, at pH 6.6, it is very much faster (half-life, 10 min). Schwarz280noted that the mutarotation is extremely slow in acid solution, and little or no difference was found for the rates for solutions in 10 mM and 200 mM aqueous hydrochloric acid. Adley and Owen2s1also commented on the slowness of the mutarotation in acid solutions. Clayton and Hugheszs2 found similar properties for 5-thio-~-ribopyranose.The best sample of the Q-D anomer gave the following results: [a]: 127 (2 min.) --* +25" ( c 0.6, water); (k, k,) = 0.022 min-' (water, at 23"); 0.00043 min-' (1 mM potassium hydrogen phthalate, pH about 4.4, at 21"); 0.042 min-' (50 pM potassium hydrogen phthalate, pH about 6.6, at 21"). Thus, the mutarotations of the 5-thioaldopyranoses are catalyzed by bases, but not by acids. The low sensitivity of the reaction to acid catalysis presumably arises from the low basicity of sulfur, and,

+

(278) D.Horton and D. H. Hutson, Adoan. Carbohyd. Chem., 18,123(1963). (279) J. C. P.Schwarz and K. C. Yule, Proc. Chem. SOC., 417 (1961). (280) J. C. P. Schwarz, Personal communication. (281) T. J. Adley and L. N. Owen, Proc. Chem. Soc., 418 (1961). (282) C. J. Clayton and N. A. Hughes, Carbohyd. Res., 4.32 (1967).

+

HORACE S. ISBELL AND WARD PIGMAN

50

consequently, the lack of formation of the conjugate acid required as an intermediate in the acid-catalyzed mutarotation. The base-catalyzed reaction of the 5-thioaldopyranoses presumably proceeds through such pseudo-acyclic intermediates as those postulated for the base-catalyzed mutarotations of the sugars. The strong nucleophilic properties of the sulfhydryl anion favor rapid ring-closure. The 5-thioaldopyranose ring is exceptionally stable, even for sugars of relatively low conformational stability. Thus, Clayton and Hughes282 found that 5-thio-D-ribopyranose has little tendency to give furanoid or aldehydo forms, in contrast to D-ribopyranose. In aqueous solution, the thiopyranose ring must be opened to only a very minor extent, because 5-thio-D-ribopyranose reacts only very slowly with aqueous iodine, and then only after the mixture has been heated. The 4-thiofuranose ring is much less stable than the 5-thiopyranose ring; thus, on treatment with iodine at room temperature, 4-thio-~-ribofuranose is quickly oxidized to the d i s ~ l f i d e . ~ ~ ~ * ~ ~ Whistler and coworkers285compared the rates of hydrolysis of 5thioxylopyranosides with those of xylopyranosides. They found that glycosides having the sulfur atom in the ring are hydrolyzed 10 to 20 times faster than their oxygen analogs, but that compounds having the sulfur atom in the glycosidic linkage are hydrolyzed much more slowly, at rates that suggested that the hydrolysis proceeds by a rate-determining step leading to a cyclic carbonium ion. Little tendency exists for the reaction to proceed by the acyclic mechanism (see p. 441, because the glycosidic sulfur atom has little tendency to provide electrons for rupture of the oxygen ring. The mutarotations of the 1-thioaldoses are surprisingly slow, both in acid and alkaline solutions.28B1-Thio-P-D-ghcopyranose forms a sodium salt that does not exhibit mutarotation. The lack of mutarotation shown by the salt indicates that the sulfhydryl anion has little or no tendency to release the ring-oxygen atom and to form an acyclic intermediate. However, 1-thio-P-D-glucopyranose in acid solution shows mutarotation. Thus, the following mutarotation is reported for 1-thio-P-D-glucopyranosein 2 mM hydrochloric acid (c 0.9712), [a]g 17.0" (initial), +24.7" (3hr), +47.4" (23hr), +54.6" (40 hr), +58.7" (64 hr), and +58.7" (88 hr). Increase of the acid concentration to 4 mM

+

(283) H. Paulsen, Angew. Chem., 5,495(1966). (284) R. L. Whistler, M. S. Feather, and D. L. Ingles, J . Amer. Chem. SOC., 84, 122 (1962). (285) R. L. Whistler and T. Van Es,J. Org. Chem.,28,2303(1963). (286) W.Schneider, R. Cille, and K. Eisfeld, Ber., 61,1244(1928).

MUTAROTATION OF SUGARS I N SOLUTION: PART I1

51

did not substantially increase either the rate of mutarotation or the final optical rotation. On acetylation, the material from the equilibrated solution gave a mixture of anomeric pentaacetates of 1-thi0-D-glUC0pyranose, but the sodium salt gave only one pentaacetate. Some cleavage (to form hydrogen sulfide) took place, but the main reaction was the conversion of the P-D into the a-D-pyranose. The faster reaction in acid solution suggests operation of an acidcatalyzed mechanism, but the subject needs further study. 1-Thioglycosides are not affected by mild alkali, but phenyl l-thioP-D-glucopyranosides and similar compounds give, like the corresponding oxygen analogs, 1,6-anhydro-~-~-glucopyranoses. Apparently, anhydride formation for 1-thio sugars having relatively unstable conformations has not been studied, but elimination of the sulfhydryl group by an opposite-face attack of a neighboring hydroxyl group would be expected to give the same anhydro compounds as those given by the oxygen analogs. AND THERMODYNAMICS OF MUTAROTATION XII. EQUILIBRIA

REACTIONS 1. Activation Energies Hudson38 measured the temperature coefficient for the interconversions of a- and P-lactose, and Lowry and Smith57 calculated activation energies from Hudson’s data by means of the integrated Arrhenius equation:

where k, and k, are the velocity constants at the absolute temperatures Tl and T z , R is the gas constant, and E is the activation energy. (This energy of activation differsza7from the heat of activation, H t ,by about 0.6 kcal.) By the use of similar equations, Euler and Hedelius288 calculated activation energies from Osaka’s data. Isbell and Pigman”* showed that the activation energies for a=@pyranose anomerizations are usually higher than those for pyranosefuranose interconversions. Thus, the values of E given in Table VI (see page 53 of Part I) range from 18.6 to 14.2 kcal. mol-’ for the a-P-pyranose anomerizations and from 15.8 to 10.7 kcal. mol-’ for (287) E. S . Gould, “Mechanism and Structure in Organic Chemistry,” Henry Halt and Co., New York, 1959,p. 179. (288) H.von Euler and A. Hedelius, Biochem. Z., 107,150(1920).

52

HORACE S. ISBELL AND WARD PIGMAN

the pyranose-furanose interconversions. It now appears that the marked difference in the activation energies for the two types of reaction arises, in large measure, from differences in the change of entropy for the two types of reaction. The large change in entropy for the pyranose-furanose interconversions (see Table XIV,p. 56) may arise from solvent effects or from a sterically restricted, highly organized, transition state. Kendrew and Moelwyn-Hugheszw represented the effect of temperature on the mutarotation rate by the equation: l n k = C + RI l n T - EA RT' where C,J, and E are characteristic for each sugar. EA was called the apparent activation energy, and the true activation energy ( E ) was calculated from the relationship EA = E +JT. For the mutarotations of D-xylose, D-mannose, D-glUCOSe, and lactose, values of EA were found to be 16,245,16,375,16,945,and 17,225cal. mol-I, respectively. Later, Dyas and Hill, and C O W O ~ pointed ~ ~ ~ out that EA is the same as AG$. They calculated AG$ and ASS from the reaction constant (k)by the Eyring equation:

where kb is the Boltzmann constant, h is the Planck constant, T is the absolute temperature, AH* is the enthalpy of activation, and AS1 is the entropy of activation. The results obtained for the mutarotation of cY-D-ghCOSe in water or methanol-water, and with certain catalysts, are summarized in Table XII. Activation energies reportedzzO*zsl~zsz for mutarotations of D-glucose catalyzed by the water molecule, the hydrogen ion, the hydroxyl ion, and the D-glucosate anion show no consistent differences in the values corresponding to mutarotations accelerated by these catalysts. Smith and SmithZ3Ocompared the activation energies for catalysis of the mutarotations by the water molecule, and b y each of three acids and four bases, by use of the equation k = PZe-E'RT.The values of E found for the various catalysts differed only slightly, and the observed (289) H. E.Dyas and D. G . Hill,]. Amer. Chem. SOC., 64,236(1942). (290) D. G.Hill and B. A. Thumm,]. Amer. Chem. SOC., 74,1380(1952). (291) P.Johnson and E. A. Moelwyn-Hughes, Trans. Faraday SOC., 37,289(1941). (292) G.Kilde and W. F. g.Wynne-Jones, Trans. Faraday SOC., 49,243(1953).

S

~

~

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

53

TABLEXI1 Free Energies, Heats, and Entropies of Activation for the Mutarotation of D-CluCOSe in Water and Aqueous MethanolPw Temperature, degrees

MeOH, %

HsO

AcOH

H,OQ

Ad)@

AGt =Free Energy of Activation (kcal.mo1-I) 35

45

0 40 60 75 0 40 60 75

40

0 40 60 75

40

0 40 60 75

24.9

20.15 19.99 19.87 19.74 25.2 22.9 20.15 19.99 19.87 19.74 AH#=Heat of Activation (kcal.mo1-I) 16.7 15.40 18.0 18.1 18.1 18.3 AS$ =Entropy of Activation (kcal.mo1-I) -27 -24 -7 -6 -6 -5

-

22.7

21.08 20.99 20.92 20.85 21.24 21.17 21.01 20.95 17.3 17.6 17.7 17.9 -12 -11 -10 -10

variation in k was considered to be due mainly to changes in P, the probability factor. Several workers have sought to correlate catalytic activities with changes in enthalpy, free energy, and entropy by application of the theory of absolute reaction-rates. Los and SimpsonlgBevaluated the enthalpy and entropy changes during the mutarotations of a- and p-D-glucose by means of the Eyring equation (20). Values for the energy (or enthalpy) of activation and the entropy of activation were obtained for the mutarotations catalyzed by k,, , , and kcso, using the conventional procedure.293The results showed that the greater acid character noted for p-D-glucose (in comparison with a-D-glucose) arises from a difference in the entropy change upon ionization of the two isomers. Schmid and coworkers254have reported values for the free energy of activation, enthalpy of activation, and entropy of activation for the (293) L.L.Schaleger and F. A. Long,Adonn. Phys. Chem., 1,7 (1963).

54

HORACE S. ISBELL AND WARD PIGMAN

mutarotations of a-D-glucose catalyzed by water, certain acids and bases in water, and in deuterium oxide. The authors postulated molecular complexes of the type D-glucose@.. . . H20. . . . H,O@, and suggested that the sugar anion and the hydronium ion have an orienting effect on the solvent dipoles. The thermodynamic data are summarized in Table XI11 (see p. 55) with additional data from a thesis by T. E. A ~ r e e . ~ ~ * The enthalpy of activation for the deuterium oxide-catalyzed reaction is less than that for the water-catalyzed reaction. Were this the predominating factor, the D20-catalyzed reaction would be expected to be more rapid than the H 2 0reaction, which is not the case. The difference in the reaction rates arises from the entropies of activation. The entropy of activation for the reaction in D20is more negative than that for the reaction in HzO, and this effect outweighs the small difference in the enthalpy of activation. The activation energies show a decrease in entropy (in forming the transition states) that Acree ascribed to participation of solvent molecules in the transition state. He suggested that the a+P-pyranose anomerization and the pyranose-furanose interconversion proceed through a common, high-energy, open-chain form which is stabilized by a complex of sugar and solvent molecules. The pseudo-acyclic intermediates envisaged earlier in the present article (see p. 35)may also be stabilized by coordination with solvent molecules. The high negative values for the entropies of activation indicate a high degree of order in the transition states. This high state of order could arise from a complex intermediate, such as that proposed for our suggested pseudo-acyclic intermediates.

2. Changes in Free Energy, Enthalpy, and Entropy Values for the changes in enthalpy during mutarotation reactions have been obtained by direct determination of the heats of combustion, corrected for heats of s o l u t i ~ n The . ~ ~values ~ ~ ~ thus ~ obtained may be in error due to uncertainty in the heats of solution and of hydration. Enthalpy values may also be calculated from changes of the equilibrium constants with temperature,203*28S~zSo but, at present, the values may be in error, because of uncertainty as to the proportions of the constituents in the equilibrium. Hence, it is not surprising that, in some instances, there are substantial differences in the values for the thermodynamic constants obtained by the two methods. Some of the most reliable data are reported in Table XIV (see p. 56). The energy changes for several sugars were interpreted by Kaba-

TABLE XI11 Activation Energies for Mutarotation of Sugars in Water at 25”

Reaction ki

a-DGlucopyranose

P-D-glucopyranose

kz k,

Exptl. method“

References

Catalyst

AGIb

mt’

0.r.

HzO

24.66

17.24

-24.9

254

DzO

25.46

16.43

-30.3

254

AStd

K

a-DGlucopyranose

= P-Dglucopyranose k,

0.r.

a-DGlucopyranose

& P-D-glucopyranose kz

0.r.

20.42

17.23

-10.7

254

P-D-glucopyranose

0.r.

22.08

16.09

-19.8

297(b)

P-D-glucopyranose

g.1.c.

22.11

18.01

-13.5

297(b)

a-D-glucopyranose

0.r.

22.78

16.81

-19.6

297(b)

g.1.c.

22.78

17.57

-17.1

297(b)

CI

P-D-mannopyranose

ox.

21.92

17.75

-14.22

297(b)

ul

P-D-mannopyranose

g.1.c.

21.89

17.04

-16.49

297(b)

0.r.

21.43

17.43

-13.66

297(b)

g.1.c.

21.44

16.85

-15.66

297(b)

--k

a-D-Glucopyranose --!+ a-DGlucopyranose P-D-Glucopyranose P-DGlucopyranose a-DMannopyranose a-DMannopyranose

k

k2

k a a-D-glucopyranose k

k

k

P-D-Mannopyranose 4 a-Dmannopyranose k

p-DMannopyranose 4 a-Dmannopyranose k

P-D-galactopyranose

g.1.c.

22.12

16.68

-18.6

297(c)

/3-D-GalactopyranoseA a-Dgalactopyranose

g.1.c.

22.52

16.72

-19.8

297(c)

a-D-Galactopyranose-

P-D-galactofuranose

g.1.c.

22.41

15.64

-23

297(c)

P-DGalactofuranose

a-D-galactopyranose

g.1.c.

20.92

7.71

4 5

297(c)

a-D-Galactopyranose-

-

“g.1.c. = gas-liquid chromatography; 0.r. = optical rotation. bValuesin kcal.mo1-’, calculated from the Eyring equation (22).CAH$was assumed to be equal to E,. dASt = (E, - AGt)/T, expressed in cal. deg.? mol-I.

z

s

cn cn

TABLE XIV Thermodynamic Constants for Mutamtation of Sugars in Water

Mutarotation

----------

8-pyranose a-DXylopyranose a-pyranose pCellobiose 8-pyranose a-DGlucopyranose 8-pyranose a-Lactose H,O a-pyranose gMaltose * H,O 8-pyranose a-DLyxopyranose 8-pyranose a-D-Glucopyranose 8-pyranose a-D-Glucopyranose 8-pyranose a-D-Glucopyranose p-pyranose a-DMannopyranose 8-pyranose a-BMannopyranose fi-pyranose a-DMannopyranose 8-pyranose a-D-Galactopyranose a-DGalactopyranose pfuranose a-D-Galactofuranose p-pyranose

-

Measurement

Temp erature, degrees

AH (cal.deg.-' mol-I)

AG

AS

(cal.mo1-I)

(e. u.)

of combustion (one mole of a + one mole of 8) thermal -535 -379 -0.52 thermal -438 -352 -0.29 -270 -327 thermal +0.19 thermal -270 -297 +0.08 thermal 2s -126 -335 +0.70 Values based on equilibrium constants (one mole of (I + equil. mixture) 0.r. 0-20 +374 0.1. 0-20 -59 0.r. 25-35 -59 -313 +0.84 g.1.c. 25-35 -50 -310 +0.87 0.r. 0-20 +517 0.r. 15-25 +313 +480 -0.57 g.1.c. 15-25 +254 +440 -0.64 g.1.c. 25-35 -25 -410 +1.3 g.1.c. 25-35 +7,520 +1,590 +20 g.1.c. 25-35 -9,OOO -m +28 Values based on heats 25 25 25 25

References

197 197 197 197 197

5

$ F v) M

W

r

299 299

* z e g

258

U

297(b) 29704 297(b) 297(b) 297(c) 297(c) 258

2

U

c)

5Z

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

57

yama and Pattersonlo5on the basis that equatorially attached hydroxyl groups are more strongly hydrated than axial ones, and that hydration results in a more negative value of AH. The authors suggested that the entropy changes arise, in large measure, from changes in the frequency of torsional oscillation of the reducing group, with changes in configuration, and, finally, that the changes may be correlated with the conformational and configurational changes. In an elegant study, Angyal and coworker^^^*^^ sought to evaluate the conformational factors that affect configurational equilibria and chemical reactivity. The proportion of each component of an equilibrium mixture of a reducing sugar depends on its free-energy content. If the free energies of the components of the system are known, the composition at equilibrium can be calculated, and the chemical properties of the sugar can be explained and predicted. In some instances, the changes of free energy involved in the mutarotation reaction may be calculated from interaction energies. To obtain these, the equilibrium proportions of the conformers have been measured by nuclear magnetic resonance (see p. 43, Part I) or otherwise, and differences in free energy have been calculated from the equation AG = -RT In K.

The calculated free-energies given in Table XV are relative to an imaginary hexopyranose that has no non-bonded interactions. The work provides a basis for predicting the stabilities of aldohexopyranoses in conformations that may apply to certain reaction intermediates, as well as to sugars in solution. Other workers have studied conformational and configurational equilibria by nuclear magnetic resonance spectroscopy. Horton and C O W O ~ ~ ~ ~ S found ~ ~ that ~ ( ~P-D-ribopyranose ) ~ ~ ~ ~ ( ~ ) tetraacetate exists in acetone-d6 at room temperature in the CE and CA conformations, in rapid equilibrium, in the ratio of 9:11.At -&I the ", ratio of the CE and CA conformers was 2:1;at -60", the rate of interconversion of the two conformers was found to be 130 times per second. The equilibrium (294) S. J. Angyal, Aust. J . Chem., 21,2737 (1968);S. J. Angyal and K. Dawes, ibid., 21,2747(1968). (295) (a) N. S . Bhacca and D. Horton,J. Amet. Chem. Soc., 89,5993 (1967).(b) P. L. Durette, D. Horton, and N. S. Bhacca, Carbohyd. Res., 10, 565 (1969).(c) P. L. Durette and D. Horton, Chem. Commun., 338 (1969).(d) C.V. Holland, D. Horton, and J. S. Jewel1,J.Org. Chem., 32,1818(1967); N.S.Bhacca, D. Horton, and H. Paulsen, ibid., 33, 2484 (1968).(e) C.B. Anderson and D. T. Sepp, Tetrahedron, 24, 1707 (1968).(f) R. U. Lemieux and A. A. Pavia, Can.J . Chem., 46, 1453 (1968).

HORACE S. ISBELL AND WARD PIGMAN

58

TABLEXV Relative Free-Energies of Aldohexopyranosesa Conformation sugar

CA

CE

a-D-Allose P-D-Allose a-D-Ahrose p-D-Altrose a-D-Galactose P-D-Galactose a-D-Glucose p-D-Glucose a-D-Gulose P-D-Gulose a-D-Idose P-D-Idose a-D-Mannose P-D-Mannose a-D-Talose P-D-Talose

4.2 2.85 4.4 3.85 3.2 2.85 2.3 1.95 4.75 3.4 5.95 5.4 2.85 3.3 4.75 5.2

6.65 6.45 4.3 4.9 5.9 6.7 7.1 7.9 5.1 4.9 3.75 4.35 5.1 6.7 4.9 6.5

Calculated free-energies, bal.mol-l Sugar in aqueous solution

4.2 2.85 3.95 3.75 3.2 2.85 2.3 1.95 4.5 3.35 3.75 4.25 2.85 3.3 4.4 5.15

Equilibrium mixture (a P )

+

2.8 3.45 2.6 1.7 3.25 3.55 2.65 4.25

"Datafrom Ref. 93,pp. 362-432,especially Table 111, p. 360,and Table VII, p. 403.

for P-Dxylopyranose tetraacetate at room temperature showed, for the all-axial (CE) conformer, only 1 part in 5. At -84", the CE conformer was not detected. Durette and H ~ r t ~examined n ~ ~ ~eight ~ ~ ) aldopentopyranose tetraacetates in acetone-d6 at 20 to 28". The ratios of the conformers (CA/CE) and the values of AGO for the equilibrium CE CA for the tetraacetates having the configurations listed are as follows: a-D-ribo, 4.0 (-0.83 k0.36), p-D-ribo, 1.2 (-0.11 kO.09); a-D-arabino, 0.23 (+0.87 20.36); a-D-xylo, > 50 (> -2.5); p-D-xylo, 4.0 (-0.82 k0.14); a-D-lyxo, 2.9 (-0.63 k0.28); and P-D-~Yxo,0.67 (+0.24 20.29). The measurements were made at 28", except for the tetraacetate of P-D-ribopyranose at 20" and the tetraacetate of p-Dxylopyranose at 25". Other revealed that tri-0-acetyl-P-Dxylopyranosyl chloride exists in various solvents in the CE conformation, and that the pentaacetate of a-D-idopyranose in acetone and chloroform exists in the CA conformation, despite the presence of four large axial substituents. Anderson and Sepp,2s5te) by using nuclear magnetic resonance spectroscopy and vapor-phase chromatography, measured the conformational equilibria of a number of 2-substituted tetrahydropyrans in

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

59

several solvents. Except for the derivatives having a free hydroxyl group on C-2, the chief anomer was the trans form (which is not the one expected on the basis of energy considerations). A comparison with the anomeric equilibria of a number of D-xylose and Dglucose derivatives showed compositions similar to those for the conformational equilibria for the tetrahydropyran-2-01s. The major controlling factor for anomeric equilibria of pyranoses was considered to be the anomeric effect (see Part I, p. 29). Methyl 3-deoxy-P-~-erythro-pentopyranoside was shown by Lemieux and by nuclear magnetic resonance studies, to exist, in DzO, mainly in the CE conformation. In deuterated water, acetone, methyl sulfoxide, and other solvents, the compound showed specific rotations (D line) lying between +95 and +140°.A comparison of the optical rotation with the coupling constant for H-1 and H-2 gave a straight line. The anomers of 2-deoxy-~-erythro-pentopyranoside were similarly studied. The a-Lanomer showed a wide variation of optical rotation with solvent, whereas the p-Lanomer showed very little. A solvent-induced shift of the conformational equilibrium was thus evident for the a but not for the p form. The p-L anomer was primarily in the CA form. These differences in the behavior of the anomers were ascribed to differences in the type of solvation. Kabayama and Patterson1osregarded the same influence as being the main determinant of the conformation assumed (see Part.1, p. 29). Durette and H ~ r t ~ nstudied, ~ ~ ~ (by~ nuclear ) magnetic resonance spectroscopy, the anomeric equilibria at 27"of the aldopentopyranose tetraacetates in 1:l acetic anhydride-acetic acid that was 0.1 M in perchloric acid. They found the following values for the ratios of the anomeric tetraacetates (@/a)at equilibrium, with the free energy AGO values for the a /3 equilibrium (in parentheses): D-ribopyranose, 3.4 (-0.73 k0.03); D-arabinopyranose, 5.4 (-1.01 k0.03); D xylopyranose, 0.23 (+0.89 k0.03); and D-lyXOpyranOSe, 0.20 (+0.98 k0.05).

Lemieux and coworkers296investigated the mutarotation of 2-deoxyp-D-erythro-pentopyranose in deuterium oxide at 0" by nuclear magnetic resonance measurements and optical rotations. The optical mutarotation had a rapid and a slow phase. The components at equilibrium were the p-pyranose (44%),the a-pyranose (41%), and the aand p-furanoses (15%). The rate of formation of each component was determined. Bentley and coworkers170showed that mufarotations may be studied (296) R.U. Lemieux,L. Anderson, and A. H. Connor, Personal communication.

60

HORACE S. ISBELL AND WARD PIGMAN

by preparation of the per(trimethylsily1) ethers, and separation of

these derivatives by gas-liquid chromatography. Several workers have applied this method for determining the tautomers involved in mutarotation reactions, especially Shallenberger and coworkers.297 A ~ r e found e ~ ~ that ~ a 10% aqueous solution of D-galactose in equilibrium at 25" contains 32.0% of a-pyranose, 63.9% of P-pyranose, 1% of a-furanose, and 3.1% of P-furanose; whereas, a solution of D-glucose in equilibrium at 25" contains 37.2% of a-pyranose and 62.8%of P-pyranose; and a solution of D-mannose in equilibrium contains 68.0% of a-pyranose and 32.0% of P-pyranose. Connor and Anderson298also studied the equilibria of sugars by this method; they found that a solution of L-arabinose in equilibrium in water at 25" contains 57 % of a-pyranose, 30 % of P-pyranose, 8 % of a-furanose, and 5% of P-furanose.29g P-D-Glucose in the solid state has been shown by a number of workers300*301 to be converted into a-~-glucose.H~O. Sega1301 postulated the formation of @-D-glucose.H,O as the intermediate product. a-D-Lyxose stored for fourteen years was converted into a form more levorotatory than its equilibrium mixture.300Examination of other sugars has revealed many instances in which less stable crystalline sugars are converted in the course of time into more stable As discussed on page 33 of Part I, numerous studies have revealed variations in the equilibrium proportions of sugars in different solvents. Kuhn and Grassner117found that the equilibrium for reducing sugars in N,N-dimethylformamide is quite different from the equilibrium of these sugars in water, and that the activation energies for the mutarotations of D-fructose and D-glUCOSe in N,N-dimethylformamide are 6 to 8 kcal.mol-' larger than those for aqueous solutions. They found that a solution of D-fructose in N,N-dimethylformamide in equilibrium at 50"contains about 80%of the furanose form. The composition was determined by methylation at 0". The marked effect of the two solvents on the equilibrium is shown by the optical rotations given in Table XVI, and by the change in optical rotation (see Fig. 17) which takes place when a solution of D-fructose in NJV-dimethylformamide is diluted with water. (297) (a) R. S. Shallenberger and T. E. Acree, Carbohyd. Res., 1,495(1966).(b)C. Y. Lee, T. E. Acree, and R. S. Shallenberger, {bid.,9, 356 (1969).(c) T.E.Acree, R. S. Shallenberger, C. Y. Lee, and J. W. Einset, ibld., 10,355(1969).(d) H.C. Curtius, J. A. Vollmin, and M. Miiller, Z. Anal. Chem., 243 (1968). (298) A. H.Connor and L. Anderson, Personal communication. (299) See Ref. 59,p. 449. (300) H. G.Fletcher, Jr., Methods Carbohyd. Chem., 1,78(1962). (301) L.Segal, Appl. Spectr., 18,107(1964). (302) H.S.Isbell and R.S. Tipson, Unpublished results.

MUTAROTATION O F SUGARS IN SOLUTION: PART I1

61

TABLEXVI Mutarotation of Sugars in N,"-Dimethylformamide (DMF)"' at 22" Equilibrium rotation

Half-reaction time

sugar

Water

DMF

Difference

(hours)

P-L-Arabinose a--Galactose 8-DFructose a-L-Sorbose D-Ribose a-D-Xylose P-D-Mannose a-D-Glucose 8-D-Glucose

+105.5 +81.0 -92.3 -43.3 -23.7 +19.0 +14.2 +52.5

+38.7 +28.7 -22.4 -51.3 -26.2 +33.1 +39.9 +63.3

-66.8 -52.3 +69.9 -8.0 -2.5 +14.1 +25.7 +10.8

35.0 19.5 2.4 14.0

176.0 6.4 228.0 130.0

Several workers have noted that the equilibria for sugars in pyridine are quite different from those for sugars in aqueous solution. Acree, Shallenberger, and Mattick303found that an equilibrium solution of D-galaCtOSe in pyridine at 80" contains 31.7, 31.2, 13.7, and 23.4% of

15

30

45 60 Time (rnin)

75

90

105

FIG. 17.- Mutarotation of -Fructose in NJ-Dimethylformamide- Water Mixtures at 24". [(l)Change following addition of an equal volume of water to a previously equilibrated solution of Dfructose in N,N-dimethylformamide; (2)change following addition of an equal volume of water to a freshly prepared solution of crystalline D-fructose in NJ-dimethylf~rmarnide.~~~]

(303) T. E. Acree, R. S. Shallenberger, and L. R. Mattick, Carbohyd. Res., 6, 495 ( 1968).

HORACE S. ISBELL AND WARD PIGMAN

62

the a-pyranose, P-pyranose, a-furanose, and p-furanose tautomers, respectively; at 25", the distribution of the tautomers, in the same order, was 33.8, 49.0, 5.1, and 12.1%. In water at 25", the distribution of tautomers, in the same order, was 32.0, 63.9, 1.0, and 3.1%. The high proportion of the P-pyranose tautomer in the aqueous solution suggests stabilization of this structure by water molecules. The effect of the solvent on the proportions of the anomers for sugars in solution has also been studied by Perlin and coworkers by nuclear magnetic resonance measurements (see p. 46, Part I). These workers suggested that, in aqueous solutions of D-fructose, the pyranose form is stabilized by hydrogen-bonding with water molecules. Additional data supplied by Perlin are given in Table XVII. TABLEXVII Composition of Equilibrium Solutions of Sugars in Various Solvents at Ambient Temperature, as Determined by Nuclear Magnetic Resonance Measurements" Sugar D-Mannose

Solvent

DzO CD3COZD CD3OD pyridine methyl sulfoxide L-Arabinose D,O CD3COzD pyridine DRibose D,O CD&O,D pyridine pyridine (70") methvl sulfoxide

Furanose ( %)

a-Pyranoseb ( %)

p-Pyranoseb ( %)

C

C

C

C

35 29 19 18 14 35 42 25 56 (6.4) 54 (4.6) 62 (4.6) 48 (4.4) 51 (5.3)

C

C

C C

C

trace 8 35

24 18 21 30 39

C

65 48 40 20 (2.2) 28 (2.2) 17 (2.4) 22 (2.4) 10 (2.1)

"The authors thank Professor A. S. Perlin for his kindness in supplying them with the data given in this Table, and for advance copies of some of his work in press. *Figures in parentheses are the coupling constants observed. CValuesnot determined. Tetra-Omethyl-Dmannopyranose was all in the (Y-Dform in methyl sulfoxide.

Perlin304also noted that: (1)the equilibrium composition for D-glucose is relatively unaffected by the solvent; (2) substituents at C-2 affect the D-mannose equilibrium noticeably; (3) tetra-0-methyl-D-mannopyranose in methyl sulfoxide exists almost exclusively in the a-pyranose form; and (4) changes in solvent substantially afFect the pyranose-furanose equilibrium of D-ribose, as well as the conforma(304) A. S.Perlin, Personal communication.

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

63

tional equilibrium for the @-pyranosemodification. The latter effect was shown by differences in the coupling constants (given in parentheses for the pyranose modifications of D-ribose in Table XVII). Applications of modem methods of analysis are rapidly providing information concerning the equilibria existing in sugar solutions. This knowledge will ultimately provide a sound basis for predicting the behavior of sugars in chemical and biological systems.

XIII. MUTAROTASEAND THE BIOCHEMICALSIGNIFICANCEOF MUTAROTATION Keston305-307 showed that the kidney, liver, and lens tissues of many animals contain an enzyme that catalyzes the mutarotation of LY- and P-D-glucose and other sugars; it was called m u t a r o t a ~ e . ~ ~ ~ ’ ~ ~ This, or a similar, enzyme had earlier been found in preparations of D-glucose oxidase from Penicillium notatum by Bentley and NeubergeFO and Keilin and Ha~?ree.~O~ The enzyme was extensively purified by Bentley and Bhate.140 The enzyme is also widely distributed in plant tissues.311 The effect of a crude preparation of P . notatum mutarotase on the mutarotation of several sugars is given in Table XVIII, taken from the work of Levy and Cook.lS0@-Cellobiose,D-galactose, and D-glyceroD-galacto-heptose are also substrates. The enzyme shows specificity, and acts primarily on sugars having the D-ghC0 or D-gUlUCtO configuration. The presence of an enzyme, instead of an acidic or basic catalyst, is shown by the specificity, as well as by the kinetics of the reaction; the enzyme is thermolabile, and shows reaction rates dependent on the concentration of D-glucose. The anomers are affected to somewhat different extents.30sMany sugars and derivatives thereof act as inhibitors. Keston305 reported a similar anomer-specificity for rat-kidney mutarotase. Chase and coworker^^^^^^^^ purified the mutarotase from A. S. Keston, Science, 120,356(1954). A. S.Keston, Arch. Biochem, Biophys., 102,306(1963). A. S. Keston, in “Handbuch der Physiologisch und Pathologisch-Chemischen Analyse,” (Hoppe-Seyler-Thierfelder,ed.) 10th Ed., Vol. 6C,1966. J. M.Bailey and P. G. Pentchev, Proc. SOC.E x p . Biol. Med., 115,796(1964). D.Keilin and E. F. Hartree, Biochem. 1..50,341(1952). R.Bentley and A. Neuberger, Biochem. J.. 45,584(1949). J. M. Bailey, P. H. Fishman, and P. G . Pentchev, J . Biol. Chem., 242, 4263

(1967).

A. M. Chase, S . L. Lapedes, and H. C. V. Meier, J . Cell. Comp. Physiol., 61, 181 (1963). L.-K. Li, A. M. Chase, and S. L. Lapedes,]. Cell. Comp. Physiol., 64,283(1964).

HORACE S. ISBELL AND WARD PIGMAN

64

TABLEXVIII Catalysis of the Mutamtatiod of Several Sugars by P. notatum Mutarotaselm

k (min-') sugar

a-D-Clucose p-DFructose a-DXylose a-D-Mannose 2-Amino-2-deoxy-~-glucose a-DGalactose p-Maltose p-Lactose

Enzyme absent

Enzyme present

0.025 0.17 0.079 0.065 0.028 0.028 0.021 0.018

0.13 0.18 0.11 0.065 0.028 0.166 0.027 0.042

"Sugarsolutions (100 mM) in 5 mM phthalate buffer, pH 4.7, with 0.06%of &glucose oxidase at 25".

hog kidney, and studied its kinetics; the reaction constant varies with the initial concentration of a-D-glUCOSe, and follows MichaelisMenten kinetics. Although phosphate ion is a catalyst for the mutarotation of sugars generally, it inhibits the activity of mutarotase. A pH of 5.3 was found to be optimal for the separation of the enzymecatalyzed mutarotation from the nonenzymic, mutarotation reaction. The enzyme from plants also follows Michaelis-Menten kinetics.311 The biological function of mutarotase is, presumably, the enhancement of the interconversion of sugars, especially of the D-glucose anomers. Many biological reactions of D-glUCOSe undoubtedly proceed directly from the ring forms, with a different reactivity for the anomers; this has been shown for the D-glucose 6-phosphate dehydrogenase of yeast, by which the p-D anomer (as for other sugar phosphates) is rapidly oxidized, in comparison with the CY-Danomer.311,314-316 The dehydrogenase of human erythrocytes has similar specificity?16 D-Glucose oxidase also shows a greater rate of oxidation of the p- than of the CY-Da n ~ m e r . ~ "Yeast * ~ ~ hexokinase,917 glucok i n a ~ e , ~and ' ~ pyrophosphate-D-glucose pho~photransferase~l~ show no specificity for a- and P-D-ghCOSe. On the other hand, D-glucose 6-phosphate isomerase from yeast appears to act directly on the (314) (315) (316) (317) (318)

S. P. Colowick and E. B. Coldberg, Bull. Res. Counc. Israel, 11A 4,373 (1963). M. Salas, E. Viiiuela, and A. Sols,]. Biol. Chern., 240,561 (1965). J. E. Smith and E. Beutler, Proc. Soc. E x p . Biol. Med., 122,671 (1966). A. Sols and R. K. Crane,]. Biol. Chem., 210,581 (1954). J. M. Bailey, P. G. Pentchev, and P. H. Fishman, Fed. Proc., 26,854 (1967).

MUTAROTATION OF SUGARS IN SOLUTION: PART I1

65

acyclic forms.315In the major metabolic cycles for D-glucose, however, anomerization does not seem to be a limiting factor.31s The various forms of the sugars must also be considered in such processes as transport through membranes. If one form is exclusively (or even preferentially) transferred, the concentrations on each side of the membrane will not be expressed by the total concentrations (as usually measured), especially if the rates of mutarotation are different on both sides of the membrane. Keston has explained “active” transport of sugars across the membranes of the kidney and intestine on this basis, but assumed that an active form (the a2dehydo form) is present in only a small p r o p o r t i ~ n . The ~ ~ ~concept , ~ ~ ~ seems theoretically sound under non-equilibrium, steady-state conditions, but it cannot be applied to the reversal of the concentration gradient of a particular anomer. The nature of the action of the mutarotase from P. notaturn has been investigated extensively by Bentley and Bhate,lm and compared with the acid-, base-, and solvent-catalyzed reactions (see also, p. 31). Through use of l80on C-1, it was shown that dehydrogenations do not occur on C-1. In addition, no dehydrogenation occurred at carbonbound hydrogen atoms; a single-displacement mechanism was thus eliminated. The enzyme probably transfers a proton, in a process similar to that usually involved in nonenzymically catalyzed mutarotations.

(319) J. M.Bailey, P. H. Fishman, and P. G. Pentchev,J. B i d . Chem., 243,4827 (1968). (320) A. S.Keston, Science, 143,698 (1964).

This Page Intentionally Left Blank

THE NITRO SUGARS BY HANSH. BAER Department of Chemistry, University of Ottawa, Ottawa, Onturio, Canada

I. Introduction,. ......................................................... 67 1. Nomenclature and Scope.. .......................................... 67 2. Historical., ......................................................... 68 11. Synthesis.. ............................................................ 70 1. 1-Deoxy-1-nitroalditols ............................................... 70 72 2. Derivatives of 2-Deoxy-2-nitro Sugars .................................. 78 3. Derivatives of 3-Deoxy-3-nitro Sugars .................................. 94 4. Derivatives of 4-Deoxy-4-nitro Sugars ................................. 98 5. Derivatives of 6-Deoxy-6-nitro Sugars ................................. 99 6. Derivatives of Other Deoxynitro Sugars. .............................. 7. Deoxynitro Cyclitols ................................................. 100 8. Miscellaneous ....................................................... 105 9. Stereochemical Considerations ....................................... 106 109 111. Reactions That Alter or Remove the Nitro Group ......................... 1. Catalytic Hydrogenation.. ........................................... 109 2. The Nef Reaction. .................................................. 111 3. Miscellaneous Reactions ............................................. 113 IV. Reactions That Proceed with Retention of the Nitro Group ................. 115 1. Acid-catalyzed Reactions ............................................. 115 2. Base-catalyzed Reactions ............................................. 120

I. INTRODUCTION 1. Nomenclature and Scope

In carbohydrate chemistry, the notation deoxynitro signifies that the molecule contains a nitro group attached to a carbon atom of the chain and thought of as replacing a hydroxyl group of the parent carbohydrate. In systematic nomenclature, the term deoxy may become divorced from the term nitro according to the requirements of the alphabetization, but it always forms an essential part of the name. The term deoxy is frequently dispensed with for the sake of convenience, but remains implied, nevertheless, in such generic expressions as nitro sugars, nitro alditols, nitro glycosides, and the like. When a nitro 67

68

HANS H. BAER

group is attached to an alcoholic oxygen atom and so constitutes part of a nitric ester grouping, the compound is a nitrate and is not regarded as a nitro compound in this sense, although, for some nitrates, incorrect terminology (“nitrocellulose” and “nitroglycerin”) has frequently been used in the past. This review will consider deoxynitro carbohydrates only. Sugar’ and starch2 nitrates have been dealt with earlier in this Series. The development of the chemistry of nitro sugars through 1951 has been comprehensively discussed in a previous Volume,3 and in that connection, ample reference has also been made to the fundamental reactions, rooted in general aliphatic chemistry, upon which were based the syntheses and transformations of nitro sugar derivatives accomplished by then. The present Chapter can therefore be limited largely to work that has appeared subsequently. Still, a brief historical review appears to be desirable. 2. Historical

The origin of nitro sugar chemistry dates back almost half a century. Pictet and Barbier4 conceived the idea of applying the then already well-known, base-catalyzed addition reaction between nitromethane and aldehydes (the Henry reaction5ls)to glycolaldehyde, DL-glyceraldehyde, L-arabinose, and D-glucose, in attempts at lengthening the carbon chains of these compounds. The authors4 did not, however, isolate any nitro alcohols that they probably had had in their hands; they subjected the reaction mixtures to reduction, followed by deamination with nitrous acid, in order to produce the corresponding nitrogen-free alcohols, and obtained, in very low yields, glycerol from glycolaldehyde and a heptitol from D-glUCOSe. The identification of the heptitol as the alditol that is now called D-glycero-D-gdo-heptitol was later questioned, and the view has been expressed3 that the experiments “did not demonstrate unequivocally that a nitromethanealdose sugar condensation had been achieved.” The concept of introducing a nitro group into a carbohydrate molecule in this way fell dormant for more than twenty years, but it was revived and brought to (1) J. Honeyman and J. W. W. Morgan, Aduan. Carbohyd. Chem.,12,117(1957). (2) G.V.Caesar, Aduan. Carbohyd. Chem., 13,331(1958). (3) J. C. Sowden, Aduan. Carbohyd. Chem., 6,291(1951). (4) A. Pictet and A. Barbier, Helu. Chim.Acta, 4,924(1921). (5) L.Henry, Compt. Rend., 120,1265(1895). (6) (a) H.B. Hass, Znd. Eng. Chem., 35, 1151 (1943);(b) G.A. Shvegheimer, N. F. Piatakov, and S. S.Novikov, Usp. Khim., 28,484 (1959).

THE NITRO SUGARS

69

great fruition by H. 0.L. Fischer and Sowden in pioneering investigations commen~ing'.~ in 1944. In that year, the first crystalline nitro sugar derivative, namely, 1-deoxy-1-nitro-D-mannitol, was p r e ~ a r e d , ~ and thereafter, the field developed at a rapid pace, gaining major significance especially from the viewpoint of the synthesis both of sugars that were nitrogen-containing and that were nitrogen-free.3 In summary, the main emphasis during that period lay, firstly, on the addition of nitromethane (and to a smaller extent, nitroethanol) to aldoses to yield deoxynitroalditols; secondly, on the dehydroacetylation (the Schmidt-Rutz of acetylated deoxynitroalditols to give nitro-olefinic alditols and thence, by selective hydrogenation, dideoxynitroalditols; and thirdly, on the removal of the nitro group from the alditols to produce reducing sugars and deoxy sugars (the Nef reacti~n'~*'~). These processes, outlined schematically here, have, as their most important feature, an ascension of the sugar chain that rivals in versatility, and, in several instances, indeed surpasses in practical application, the cyanohydrin ~ y n t h e s i s of ' ~ Emil Fischer and Kiliani. Justifiedly, the method is referred to as the Fischer-Sowden nitromethane synthesis.

++ '

(7) See also, the obituary of Hermann Otto Laurenz Fischer by J. C. Sowden, Aduan. Carbohyd. Chem., 17.1 (1962). (8) See also, the obituary of John Clinton Sowden by S. M. Cantor, Aduan. Carbohyd. Chem.,20,1(1965). (9) J. C. Sowden and H. 0.L. FischerJ. Amer. Chem. Soc., 66,1312 (1944). (10) E. Schmidt and G. Rub, Ber., 61,2142 (1928). (11) V. V. Perekalin, "Unsaturated Nitro Compounds," Goskhimizdat, Leningrad, 1961. (Translated by L. Mandel, Israel Program for Scientific Translation, Ltd., Jerusalem, 1964.) (12) J. U. Nef, Ann., 280,263 (1894). (13) W. E. Noland, Chem. Reu., 55,137 (1955). (14) C. S. Hudson. Aduan. Carbohyd. Chem., 1,1(1946).

70

HANS H. BAER

More recently, the chemistry of nitro sugars has experienced considerable expansion in various directions, and again, much credit is due H. 0. L. Fischer, who initiated further researches and provided the stimulus for their continuation. However, many valuable contributions emanated from other quarters, too, as will be seen in the following pages. To date, only one nitro sugar has been encountered in Nature. Obtained as a hydrolytic fragment from everninomicin antibiotics, it was named evernitrose and shown to be 4-O-methyl-3-C-methy1-3nitro-2,3,6-trideoxy-~-ribo(or ~ - a r a b i n o ) - h e x o s eThe . ~ ~ pharmacolog~ ical properties or other biological potentialities of the nitro sugars have yet to receive exploration. Nonetheless, they command great interest as synthetic intermediates for rare, and otherwise less readily accessible, compounds of known or potential biological or medicinal importance, especially in the realm of amino sugars. 11. SYNTHESIS U p to the present, nitromethane has remained the principal vehicle for introducing the nitro group into sugars and cyclitols, although the reagent has been complemented by higher nitroalkanes. Inorganic nitrite and dinitrogen tetraoxide have thus far been used in only a few instances, but they show considerable promise for future applications. In the present article, the position of the nitro group in the sugar chain, not its mode of introduction, has been adopted as the principle of ordering.

1. 1-Deoxy-1-nitroalditols

In the base-catalyzed addition of nitromethane to aldehydes (the Henry r e a ~ t i o n ~ .the ~ ) , methanenitronate anion attacks the electrondeficient carbonyl carbon atom. Such derivatives of aldehydes as the hydrates and hemiacetals react readily, owing to the presence of the carbonyl compound in mobile equilibrium, and consequently, aldoses are amenable to the reaction, which generally proceeds rapidly, giving good yields at or below room temperature. Usually, t h e stoichiometric amount of an alkali is employed for promoting the reactior . and the nitro alcohols are then obtained as nitronate salts and may

(144 A. K. Ganguly, 0. Z . Same, and H . Reimann, J . Amer. Chem. SOC., 90, 7129

(1968).

THE NITRO SUGARS

71

thereafter be liberated15 by cautious acidification with cold, dilute acid or by cation exchange. The first successful syntheses of l-deoxy1-nitroalditols were conducted in methanolic medium in the presence of sodium m e t h ~ x i d eBecause .~ of their higher solubility, benzylidenated aldoses were originally preferred, but it soon transpired that unsubstituted aldoses can be used just as well, even though the system may remain heterogeneous throughout the reaction. Addition of nitromethane to 2,4-O-benzylidene-~-erythrose, 2,4-O-benzylidene-L-xylose, 4,6-O-benzylidene-~-glucose, D-eIythrOSe, D- and Larabinose, and D-mannose yielded an isolable deoxynitroalditol in each instance. D-Ribose, D-xylose, and D-glUCOSe were shown to react, but they did not furnish the nitro alcohols in pure condition; however, the reaction mixtures could be utilized for preparing nitroalditol olefins3(see also, Section IV, 2c, p. 127).Later on, analogous syntheses, also using methanolic reaction media, were performed with D-glyceroD-ga2acto-heptose7" D-altrose," D-talose,'s D-glucOSe,lsa L-idose,lsb and D-lyxo~e.'~ Difficulties experienced because of insufficient solubility of certain sugars prompted the examination of other solvents. Sowden and coworkers, employing aqueous solutions that contained the reactants and sodium hydroxide, were able to prepare nitro alcohols, not only from D-erythrose,20D-arabinose,20s"'D-mannose,20and D-glycero-D-galacto-heptose"in yields comparable to those obtained previously, but also from D-ga1actose2' and Derythro-L-manno-oct o ~ eAqueous .~~ barium hydroxide, which had served in the internal nitroalkane addition of 6-deoxy-6-nitro sugars (see Section 11,7, p. loo), was usedz4successfully in adding nitromethane to D-lyxose, Dglucose, and D-rhamnose. Methyl sulfoxide has been employed as the (15) Although loss of nitrous oxide (the Nef reactionla) occurs with great ease in the acidification of certain nitronates [see N. Kornblum and G. E.Graham, ]. A m r . Chem. Soc., 73, 4041 (1951)],no serious difficulties have been encountered in the chemistry of nitro sugars. For an investigation of the course of the nitronate acidification in dependence on the pH, see J. Armand, P. Souchay, and S . Deswarte, Tetrahedron, 20,Suppl. 1,249(1964). (16) J. V.Karabinos and C. S . Hudson,]. Amer. Chem. SOC., 75,4324(1953). (17) R.K. Hulyalkar, J. K. N. Jones, and M. B. Perry, Can.]. Chem.,41,1490(1963). (18) R. Young and G. A. Adams, Can.].Chem.,44,32(1966). (18a)D. T. Williams and M. B. Perry, Can.]. Chem., 47,2763(1969). (18b)H.H. Baer and J. Kovhi, unpublished results. (19) M. B. Perry and A. C. Webb, Can.].Chem., 46,2481(1968). (20) J. C.Sowden and R. R. Thompson,]. Amer. Chem. SOC., 80,2236(1958). (21) J.C.Sowden and R. R. Thompson,J. Amer. Chem. Soc., 77,3160(1955). (22) J. C.Sowden and D. R. Strobach,]. Amer. Chem. SOC.,82,954(1960). (23) J. C.Sowden and D. R. Strobach,]. Amer. Chem. SOC.,82,956(1960). (24) J. Yoshimura and H. Ando, Nippon Kagaku Zasshi, 85, 138 (1964);Chem. Abstracts, 61,16140(1964).

72

HANS H. BAER

solvent for the reaction of Dgalactose in the presence of sodium methoxide and anhydrous calcium sulfate.2s The addition of nitromethane to an aldose creates a new asymmetric center, so that a pair of epimeric nitro alditols is produced. Although the stereoselectivity is not very great, one of the epimers is, in some cases, isolated in an appreciable preponderance. Thus, the main ?,~~ D-mannOSe,2°*26 products obtained from D - ~ ~ U C O S C D-galaCtOSe,22*25 and D-talOSe" are those having the D-glyCeTO-D-gdO, D-glyCer0-Lrnanno, D-glyCerO-D-gUlUCtO, and D-glyCerO-L-dtrO configurations, respectively, rather than the corresponding 2-epimers. D- or L-Arabinose, on the other hand, appears to give the two epimers in equal amount^.^.^^ For a further discussion of these stereochemical aspects, see Section II,9 (p. 106). 2. Derivatives of 2-Deoxy-2-nitro Sugars

The addition of nitroethanol to aldoses constitutes a chain prolongation by two carbon atoms to furnish, theoretically, four epimeric 2deo~y-2-nitroalditols.~~ This synthesis has been studied comparatively little. No individual nitroalditols have been characterized, but the various epimeric sodium nitronate mixtures that were obtained were subjected directly to the Nef reaction, to afford the corresponding ketoses in poor to moderate overall yields. As in the reaction with nitromethane, C-1 of D-arabinose, on reaction with nitroethanol, adopts, without much selectivity, both of the configurations possible, as revealed by a subsequent Nef reaction which produced similar amounts of D-manno-heptulose and ~ - g l u c o - h e p t u l o s eDLyxose .~~~~~ gave D-galacto-heptulose and D-talo-heptulose in the ratio of 9 2 , but the combined yield wasz9less than 10%. The corresponding heptuloses having the D - u Z ~ ~ O and ~ - i d oconfigurations arose from Dribose and D-xylose, respectively, although the products were actually isolated as 2,7-anhydro sugars.2s A synthesis of sedoheptulose 7phosphate has been described30that involves addition of nitroethanol to D-ribose 5-phosphate, followed immediately by a Nef reaction and chromatographic separation of the desired ketose phosphate. Several octuloses and nonuloses have been synthesized in similar fashion. (25) L. Hough and S. H. Shute,J. Chem. SOC., 4633 (1962). (26) (27) (28) (29) (30)

J. C. Sowden and R. Sch&er,J. Amer. Chem. SOC., 73,4662 (1951). J. C. Sowden and H. 0.L. FischerJ. Amer. Chem. SOC., 69,1963 (1947). J. C. Sowden,J.Amer.Chem. S O C . , ~ 3325 ~ , (1950). J. C. Sowden and D. R. StrobachJ. Amer. Chem. SOC.,80,2532 (1958). B. A. McFadden, L. L. Barden, N. W. Rokke, M. Uyeda, and T. J. Siek,J. Amer. Chem. SOC., 87,5505 (1965); Carbohyd. Res., 4,254 (1967).

THE NITRO SUGARS

73

Thus, D-gulose afforded D-g~yce~o-L-ga~acto-octulose~~ in 9 % yield, and the D-glycero-L-tdo epimeP2 in about 4 % yield; D-glyCeT0-Dgulo-heptose gave low yields of the D-erythrO-L-gUlUCto-33 and D-e~ythro-L-ta~o-nonuloses.~~ A variant of the nitroethanol synthesis consists of a stepwise reaction of the aldose, first with nitromethane and then with f ~ r m a l d e h y d eIn . ~ ~this way, the 1-deoxy-l-nitro-DglyCeTO-D-gUlUCtO- and Dglycero-D-talo-heptitols obtainable from Dmannose were hydroxymethylated at C-1, and a subsequent Nef reaction gave the octuloses having the corresponding configurations in 3.5 and 9% yields.32 Lemieux and his associates have provided a novel avenue to nitro sugars by the action of dinitrogen tetraoxide upon acetylated glycals. It had previously been shown that nitrosyl chloride adds across the double bond in acetylated D-arabi~~al?~ D-glUCal?5*36 D-gdactal?' and D - ~ y l a l .3,4,6-Tri-O-acetyl-D-glucal ~~ (l), for example, affords (in over 90% yield) 3,4,6-tri-0-acetyl-2-deoxy-2-nitroso-~-~glucopyranosyl ~ h l o r i d e (2), ~ ~ .isolated ~~ as a colorless, crystalline d i m e P (3). When the acetylated glucal 1was allowed to react in ether solution at 0" with dinitrogen tetraoxide, an analogous dimer (5) of 3,4,6-tri-0acetyl-2-deoxy-2-nitroso-a-~-glucopyranosy~ nitrate (4) was obtained in high yield.37On the other hand, when the reaction was conducted in dichloromethane at -70°, the product was not the dimeric nitroso nitrate (5), but (amorphous) 3,4,6-tri-O-acetyl-2-nitro-D-ghcal (6) which was isolated in quantitative yield.37 The chemical behavior of dinitrogen tetraoxide is best explained by assuming existence of an equilibrium between the structural isomers 7a and 7b.It may be considered that each form has a strongly electrophilic character and that either may enter preferentially into reaction, depending on such environmental factors as the temperature and the polarity of the solvent. Thus, attack of a nucleophile (Nu) on the nitrosyl nitrate form (7b) can be expected to lead to nitrosation, with nitrate ion as the leaving group, in analogy to nitrosation by nitrosyl chloride (where chloride ion is the leaving group). If attack on the dinitro form (7a) occurs, the result will be nitration, with nitrite ion as the leaving group. (31) H.H.Sephton and N. K. Richtmyer,]. Org. Chem.,28,1691(1963). (32) H.H.Sephton, personal communication. (33) H. H. Sephton and N. K. Richtmyer, Carbohyd. Res., 2,289(1966). (34) J. K. N. Jones,]. Chem. SOC., 3643 (1954). (35) W.J. Serfontein, J. H. Jordaan,andJ. White, Tetrahedron Lett., 1069(1964). (36) R. U. Lemieux, T. L. Nagabhushan, and I. K. O'Neill, Tetrahedron Lett., 1909 (1964). (37) R. U. Lemieux, T. L. Nagabhushan, and I. K. O'Neill, Can. ]. Chem., 46, 413 (1968).

74

HANS H. BAER

-

___c

AcO

Hence, in one instance, the glucal (1)affords the nitroso nitrate (4) (which subsequently dimerizes to 5). In the other, the glucal might be expected to yield a nitro nitrite (8), but the latter, if indeed it is formed as a primary adduct, would certainly be prone to loss of nitrous acid by elimination and thereby to produce the nitro-olefin (6) that

75

THE NITRO SUGARS

has been found as the only product. In fact, the nitroso nitrate ( 4 e 5) is also very unstable, and it tends to decompose with formation, at least in part, of the same nitro-olefin (6).This decomposition must be due to elimination of nitric acid, with concomitant oxidation of the nitroso substituent. Moreover, the analogous nitroso chloride (2 e 3) readily suffers dehydrochlorination. The resultant, nonisolable ni-

- HCl

Q

ACO

76

HANS H. BAER

troso-olefin (9) is trapped in the presence of alcohols or acetic acid by adding these molecules and subsequently tautomerizing to the oxime form (9 + 10;9 + 11).However, compound 9 apparently may undergo oxidation when nucleophilic addends are absent, for the nitro-olefin (6) has been encountered as a transformation product of 2 also. However, the formation of 6 in the low-temperature addition of dinitrogen tetraoxide need not necessarily proceed by way of a nitro nitrite (8). An alternative path is c ~ n c e i v a b l ein~which ~ the only intermediate is a dipolar ion (12)which suffers loss of the proton at C-2, perhaps in a concerted mechanism as illustrated. At any rate, it seems to be a fact that, although 6 is engendered in decompositions of the nitroso compounds 2 and 4, nevertheless, low-temperature nitration of the glucal 1 involves direct introduction of a nitro group at C-2. In this process, the nitroso nitrate (4) is not an intermediate, as it was converted into 6 to only a minor extent when it was subjected to treatment with dinitrogen tetraoxide in dichloromethane at -70". Similar reactions using dinitrogen tetraoxide were performed with 3,4,6-tri-O-acetyl-D-galactaland 3,4-di-O-acetyl-D-xylal, and analogous results were observed, except that the dimeric nitroso nitrate from D-Xyhl was too unstable to allow its isolation, and only the acetylated nitrO-D-Xylal was obtained. A remarkable feature of all of these reactions is their extraordinary stereospecificity. The dimeric nitroso nitrates (and also the corresponding nitroso chlorides) arise solely by way of cis addition, and, of the two possible cis configurations attainable in theory from a given glycal (for example, a-D-gluco and P-~-manno),only one is present in the product. For an explanation, Lemieux and coworkers37 made reference to earlier studiesS*that concerned the halogenation of glycals and that showed comparable results. Furthermore, nucleophilic additions to nitro glycals, as well as to (intermediary) nitroso glycals, proceed stereospecifically. Thus, compound 6 primarily reacts with alcohols so as to give3' P-D-glucosides (13).According to nuclear magnetic resonance data, compound 6 exists in the half-chair conformation 6b, not in the conformation 6a. The latter is evidently disfavored because of a powerful A"*2' effecP0s4*(the nonbonded interaction between the nitro and 3-acetoxy substituents), and nucleophilic attack at the anomeric center in 6b is most likely to occur axially from the p-side (38) R. U. Lemieux and B. Fraser-Reid, Can../.Chem., 43,1460 (1965). (39) R. U. Lemieux and I. K. O'Neill, personal communication. (40) F. Johnson and S. K. MalhotraJ. Amer. Chem. SOC., 87,5492,5493 (1965). (41) R. Caple and W. V. Vaughan, Tetrahedron Lett., 4067 (1966).

77

THE NITRO SUGARS

of the ring.39*42 On the other hand, the nitroso glucal9, believed to be an intermediate in the glycosidation of 3, adds alcohols to furnish a-D-ghcosides (10) in a highly directed way, and, from this, the inference has been drawn that it exists in the half-chair conformation 9a, with the conjugated nitroso-olefin system in cis orientation, so as to avoid interaction with the 3-acetoxy

(Qa)

(10)

(42) R. U. Lemieux, T. L. Nagabhushan, and S. W. Gunner, Can.J . Chern., 46.405

(1968).

78

HANS H. BAER

It is obvious that, for several reasons, the research thus far discussed possesses major significance within the framework of the chemistry of nitrogenous carbohydrates. R e d u ~ t i o n of ~ ~the ,~~ tetra-O-acetyl-2oximino sugars of type 11has provided a new, high-yielding, preparative route43to 2-amino-2-deoxy sugars that include those having the D-gluco, D-manno, D-galacto, and D-talo configurations. The stereospecific f ~ r m a t i o n of ~ ~alkyl . ~ ~ and aryl 2-oximino-cu-D-glycosides of type 10, which can also be reduced to the amine stage,45or which can be deoximated (preferentially, with levulinic and subsequently reduced with b~rohydride,~’ is of special importance in view of the scarcity of general syntheses for a-D-glycosides. The nitro glycals (6 and analogs) and alkyl2-deoxy-2-nitro-glycopyranosides(13)represent the first cyclic carbohydrates bearing a nitro group on (2-2, and they will undoubtedly become subjects of interesting future studies to complement the knowledge already accumulated in the areas of 3and 6-nitro sugars.

3. Derivatives of 3-Deoxy-3-nitro Sugars Glycosides of 3-deoxy-3-nitroaldoses are readily available by the cyclization of sugar “dialdehydes” with nitromethane. Introduced in 1958 by Baer and Fischer,4*the method has been widely employed, and some aspects have been r e ~ i e w e d . ~ The ~*~O term sugar “dialdehydes” refers to products of glycol cleavage of g l y c o ~ i d e sThe . ~ ~formation, structure, conformation, and reaction of such “dialdehydes” have been discussed in detail in previous chapters of these Volu m e ~ . The ~ ~ -compounds ~ ~ exist in aqueous and alcoholic solutions as solvated, cyclic forms that are internal hemialdals or hemiacetalsSJ4 However, under the conditions of the nitromethane cyclization, mobile equilibria are rapidly shifted from such cyclic products toward carbonyl species, and it is, therefore, permissible and customary in (43) R. U. Lemieux and T. L. Nagabhushan, Can.]. Chem., 46,401 (1968). (44) R. U. Lemieux, S. W. Gunner, and T. L. Nagabhushan, Tetrahedron Lett., 2149 (1965). R. U. Lemieux and S. W. Gunner, Can.]. Chem., 46,397 (1968). C. H. DePuy and B. W. Ponder,J. Amer. Chem. Soc., 81,4629 (1959). R. U. Lemieux, R. Suemitsu, and S. W. Gunner, Can.]. Chem., 46,1040 (1968). H. H. Baer and H. 0.L. Fischer, Proc. Not. Acad. Sci. U.S . , 44,991 (1958). H. H. Baer, Tetrahedron, 20, S u p p l . 1,263(1964). F. W. Lichtenthaler, Angew. Chem. Intern. Ed. Engl., 3,211 (1964). E. L. Jackson,Org. Reactions, 2,341 (1944). J. M. Bobbitt,Aduan. Carbohyd. Chem., 11,1(1956). A. S. Perlin, Aduan. Carbohyd. Chem., 14,1(1959). R. D. Guthrie,Aduan. Carbohyd. Chem., 16,105 (1961).

THE NITRO SUGARS

79

this context to depict the dialdehydes as the acyclic structure 14. The nomenclature adopted in this Chapter derives from this

C&NOP

R =

For the purpose of synthesis of nitro sugars, the dialdehydes (14) are usually engendered by oxidation of methyl glycosides with sodium metaperiodate, but dialdehydes obtainable in the same way from other glycosides, and such related compounds as nucleosides and 1,8anhydro sugars, can be used as well, and have served to extend the scope of the synthesis considerably. Treatment of 14 or analogous dialdehydes with nitromethane and alkali results in a twofold addition reaction that joins both of the carbonyl carbon atoms with the same nitromethane molecule and produces a stereoisomeric mixture of 3-deoxy-3-aci-nitropyranoside salts (15). Subsequent deionization gives stereoisomeric, free nitro glycosides (16). The cyclization takes place with great facility and appears to be the favored process, even if an excess of nitromethane is present. It is certainly conceivable that side reactions could be occasioned by independent additions of nitromethane to each of the two carbonyl groups, or by intermolecular polyadditions. However, no products other than the nitro glycosides have thus far been identified, and side reactions seem to be negligible. The reaction is performed in methanolic or aqueous solution, is promoted by stoichiometric amounts of sodium methoxide, sodium hydroxide, or (occasionally) other basic agents, and goes to completion at 0-25" within a period of several hours to less than one hour. Preparative problems may arise in the separation of the products. Because two new asymmetric centers are formed in the step 14 + 15, and (55) In this notation, the glycol fission-products of glycosides are regarded as derivatives of diglycolaldehyde, OCH-CH2-O-CH2-CH0, in which one of the methylene carbon atoms is substituted by an alkoxyl group representing the aglycon, and in which the other methylene carbon atom carries the substituent (if any) that constituted the exocyclic part of the original sugarchain. Configurations are given as D' and L' for the alkoxylated methylene group, and as D and L for the second methylene group (if substituted).

80

HANS H. BAER

a third one is formed in the step 15+ 16,four stereoisomeric nitronates and eight stereoisomeric free nitro glycosides can theoretically be expected, barring additional isomers that may possibly be engendered by epimerization at other sites of the molecule. However, in practice, the reaction always exhibits a marked stereoselectivity, with one or two isomers of 16 preponderating and another one or two being present in small proportion in the final product. Since nitronates (15) formed in kinetically controlled cyclizations readily epimerize in alkaline solution to give thermodynamically controlled equilibria of isomers, the composition of the nitronate mixtures obtained may vary with the reaction time and the solvent. Some of the stereochemical factors that influence the product composition are discussed in Sections I1,9 (see p. 108)and IV,2a (see p. 121). Occasionally, a single nitronate will crystallize directly from the alkaline reaction mixture. Fractional recrystallization, column chromatography, and derivatization have all been successfully employed for separating free nitro glycosides (16),and where these have failed, separation and characterization of products have been possible after reduction to the amine stage. In fact, the synthesis of 3-amino-3-deoxy sugars, which commands great interest in connection with the chemistry of antibiotic substances,w was the original objective of the sugar dialdehyde-nitromethane cyclization, and, in much of the early work, this aspect was stressed more strongly than the study of the nitro sugars themselves. a. Methyl 3-Deoxy-3-nitropentopyranosides.-L'-Methoxydiglycolaldehyde (17),which was prepared by periodate oxidation of methyl P-D-xylopyranoside (but which is also derivable from any of the other methyl p-D-or a-L-pentopyranosides), gave, on treatment with nitromethane and sodium methoxide in aqueous alcohol, a crystalline salt, namely, methyl 3-deoxy-3-aci-nitro-~-~-erythro-pentopyranoside sodium salt (18),in 40% ~ i e l d . ~The ~ . ~results ' of later investigations in which the reaction conditions were slightly modified indicated that 18 actually arises to a much larger extent, but, also, that small proportions of the a-L-threo (19)and P-D-threo (20)isomers are formed.58No evidence was obtained for formation of the fourth possible isomer, the a-L-eythro salt (21). Acidification of compound 18 produced5' crystalline methyl 3(56) J. D. Dutcher. Aduan. Carbohvd. Chem., 18,259(1963). (57) H. H. Baer and H. 0.L. Fischer,]. Amer. Chem. SOC., 81,5184(1959). (58) H.H. Baer and A. Ahammad, Can.]. Chem., 41,2931(1963).

THE NITRO SUGARS

81

deoxy-3-nitro-p-~-ribopyranoside (22) as the preponderant product, together with a small proportion of the D-X& isomer (23).The latter could not be isolated, but its presence in the mother liquor of 22 was demonstrated by the isolation of the corresponding amine after catalytic hydrogenation. Hydrogenation of 22, followed by acid hydrolysis, provided a convenient route to 3-amino-3-deoxy-~-ribose, the sugar moiety of puromycin. Acidification of cyclization mixtures containing 19 and 20 affordeds8the methyl 3-deoxy-3-nitro-a-~-and -P-D-arabinopyranosides, 24 and 25, which were each isolated, by fractional recrystallization, in yields of about 5 %. Upon hydrogenation and hydrolysis, they gave the 3-amino-3-deoxy-~-and -D-arabinoses, respectively. experiment^^'.^^ in which the enantiomeric dialdehyde, namely, D'-methoxydiglycolaldehyde (26), was used led to the enantiomers of the nitro and amino sugars just mentioned.

x->l" f

4H

O

-G

H

HO

H Na0,N

l

OH

Na0,N

HO

OH

Na0,N

Na0,N

HO

O

P

82

HANS H. BAER

b. Methyl 3-Deoxy-3-nitropentofuranosides. -The nitromethane cyclization of sugar dialdehydes has the inherent structural limitation of providing pyranoid compounds only. However, an avenue to 3-deoxy-3-nitropentofuranosides has now been opened.59 Methyl 6-deoxy-6-nitro-c~-~-glucopyranoside (27) (see Section 11,5; p. 99) was oxidized with periodic acid to give the dialdehyde 28. Without being isolated, compound 28 was induced to undergo an internal Henry reaction by adjustment of the solution to pH 7.5, and the resulting solution of methyl 3-deoxy-3-nitro-pentodialdo-1,4-furanosides (29) was immediately treated with sodium borohydride. There was obtained, in 26% overall yield, a crystalline mixture of furanosides that furnished, by fractional recrystallization, methyl 3-deoxy-3-nitro-P-~ribofuranoside (30)(the major product) and methyl 3-deoxy-3-nitro-PL-arabinofuranoside (31)(the minor product). Catalytic hydrogenation of each provided the corresponding amino furanosides.

/

pH 7.5

OHCG

M

e

OH

t HO&CO NO, O HOM

e

+

HO&CG

O

M

OH (SO)

(91)

(5Q) H.H. Baer and I. Furid,]. Org. Chem.,33,3731 (1968).

e

THE NITRO SUGARS

83

This conversion of a hexopyranoside into pentofuranosides constitutes a degradation of the sugar chain “from within,” severance of the glycosidic bond being avoided, and it formally represents a transfer of a terminal nitro substituent into the ring. Since the reaction involves a rotation, about the bond to the ring oxygen atom, of the carbon atom whose configuration determines the configurational series, P-L-furanosides arise from an a-D-pyranoside. This deduction presupposes that the determinant carbon atom does not suffer basecatalyzed epimerization at any stage of the reaction sequence -for example, in the highly reactive intermediates 28 or 29. The inference can be drawn that such epimerization was absent to the extent that compounds 30 and 31 were actually isolated, but an examination of the mother liquors might well reveal the presence of hitherto undetected products resulting from epimerization at C-5. c. Methyl 3-Deoxy-3-nitrohexopyranosides.- Nitromethane cyclization of D-(hydroxymethy1)-D’-methoxydiglycolaldehyde(32)(obtainable from methyl a-D-hexopyranosides or methyl a-D-pentofuranosides) aEorded,gOin high yield, an amorphous mixture of sodium nitronates (33).The mixture of free methyl 3-deoxy-3-nitro-a-~-hexopyranosides that was liberated by treatment of 33 with potassium hydrogen sulfate or, more simply, with a cation-exchange resin, was not resolved, but was shown by catalytic hydrogenation to consist chiefly of the D-ghco (34)and D-manno (35)isomers; a small proportion of the ~ - t &isomer (36)was also found.60s61Although compound 34 preponderated (at an estimated SO%), the method was found to be particularly suited to the preparation of methyl 3-amino-3-deoxy-a-Dmannopyranoside hydrochloride, which crystallized readily from hydrogenated nitro glycoside mixtures in yields of 30-36%. The 3-amino-D-mannoside prepared in this way has served62as the starting material for the synthesis of mycosamine, a sugar component of several macrolide antibiotic^.^^ Isolation of the aminoglucoside on a preparative scale was also found feasible, but required column chromat~graphy.~’.~~ Crystalline methyl 3-deoxy-3-nitro-a-~-glucopyranoside (34)and derivative were obtained from crude its 2-0-acetyl-4,6-O-benzylidene nitro glycoside mixtures in poor to moderate yield^.^^,^^ Preparative (60) H. H. Baer and H. 0.L. Fischer,]. Amer. Chem. Soc., 82,3709 (1960,. (61) H. H. Baer,J. Amer. Chem. SOC., 8 4 8 3 (1962). (62) M. H. von Saltza, J. Reid, J. D. Dutcher, and 0. Wintersteiner,]. Amer. Chem. SOC.,83,2785 (1961);J.Org. Chem., 28,999 (1963). (63) S. Inouye, Chem. Phann. Bull. (Tokyo), 14,902 (1966). (64) H. H. Baer, F. Kienzle, and T. Neilson, Can.J. Chem.,43,1829 (1965). (65) H. H. Baer and F. Kienzle, Can.J . Chem., 45,983 (1967).

HANS H. BAER

access to derivatives of 3-deoxy-3-nitro-~-glucose is more convenient in the p-Dseries (see later). Performance of the cyclization of compound 32 in methanolic solution in the presence of sodium methoxide, and in aqueous solution in the presence of potassium hydrogen carbonate (followed, in both cases, by cation exchange), gave similar results in regard to the isomeric-product composition.61However, in the course of four days, an aqueous solution of a sodium nitronate mixture (33)underwent epimerization (see Section IV,2a; p. 120), giving a different mixture (37)which, on de-ionization, gave rise to the D-talo (36),D-gUlUCtO (38),and D - W M Z ~ ~ (35) O glycosides. These glycosides failed to crystallize, but their presence in the approximate ratios of 4:3:1 was established by isolation of hydrogenation products having the corresponding configurations. The process proved useful for the preparation of crystalline methyl 3-amino-3-deoxy-a-~-talopyranoside hydrochloride, even though column chromatography of the amino sugars was necessarya613-Amino-3-deoxy-~-galactoseis more conveniently accessible as its 8-D-glycoside (see later). A crystalline 4,6-benzyli-

THE NITRO SUGARS

85

dene acetal of 36 has been isolated,s5in poor yield, from deionized 37. The nitromethane cyclization of D-(hydroxymethy1)-L'-methoxydiglycolaldehyde (39),the periodate oxidation product of methyl P-D-hexopyranosides, represents one of the most satisfactory examples of these syntheses. The reaction afforded methyl 3-deoxy-3-nitro-P-Dglucopyranoside (40), -P-D-galactopyranoside (41), and -P-D-mannopyranoside (42), all in crystalline form and isolable without recourse to chromat~graphy.~~*~' When performed in methanolic medium, the cyclization produced 40 as the main isomer, in yields of 40% or higher;66 in aqueous solution in the presence of sodium hydroxide, the yield of 40 was lowered to about 25%, but 41 could then be isolated in 34% yield.67The isomer 42 was obtaineds7in small proportion from the mother liquors or, alternatively, it was isolated as the 4,6-benzylidene acetaLs4 Interestingly, the D - ~ U ~ Oconfiguration has not been encountered in the p-Dseries.

In addition to providing a synthetic pathwayss to kanosamine, a component of kanamycin, the convenient availability of 40 and 41 has stimulated a great deal of research that will be discussed in subsequent Sections. Acid hydrolysis of 40 furnished 3-deoxy-3-nitroD-ghCOSe, the only reducing 3-deoxy-3-nitroglycose known so far (see Section IV,lc; p. 118). It may be noted that the dialdehyde 39 can be prepared from any (66) H. H. Baer, Chem. Ber., 93,2865 (1960). (67) H. H. Baer and F. Kienzle, Can.]. Chem., 41,1606 (1963). (68) H. H. Baer,]. Amer. Chem. SOC., 83,1882 (1961).

86

HANS H. BAER

methyl P-D-pentofuranoside, as well as from methyl P-D-hexopyranosides. A case in point was the usern of methyl p-D-ribofuranoside as an incidental alternative to the use of methyl P-D-glucopyranoside in a preparation of 40. The significance of such an alternative is more obvious in syntheses of the members of the enantiomeric p-L series. L-Hexoses not having been readily available on a practical scale as starting materials, the optical antipode of 39, namely, L-(hydroxymethyl)-D’-methoxy-diglycolaldehyde (43),was prepared by periodate oxidation of methyl p-L-arabinofuranoside, and nitromethane cyclization then afforded the P-L-hexopyranosides 44, 45, and 46, although the latter two were isolated only as their 4,6-benzylidene a c e t a l ~ . ~ ~

. 1

OaN

‘OH

(43)

Synthesis of a hexopyranoside from a pentofuranoside in the way just described constitutes a chain elongation “from within,” concomitant with introduction of a nitrogen-containing substituent. This principleJ7 has been successfully applied in the chemical modification of nucleosides. Pursuant to preliminary reports on such modifications by and Li~htenthaler~l and their associates, a considerable body of detailed work in that direction has appeared.72-80 (69) H. H. Baer and F. Kienzle, Can./.Chem.,43,3074(1965). (70) K.A. Watanabe and J. J. Fox, Chem. Pharm. Bull. (Tokyo), 12,975(1964). (71) F. W. Lichtenthaler, H. P. Albrechf and G. Olfermann, Angew. Chem., 77, 131 (1965). (72) K. A. Watanabe, J. Berhnek, H. A. Friedman, and J. J. Fox, 1.Org. Chem., 30, 2735 (1965). (73) J. Bednek, H. A. Friedman, K. A. Watanabe, and J. J. Fox,]. Heterocycl. Chem., 2,188(1965). (74) K.A. Watanabe and J. J. Fox,/. Org. Chem., 31,211(1966). (75) (a) F. W. Lichtenthaler and H. P. Albrecht, Chem. Ber., 99,575(lass).(b) F. W. Lichtenthaler, P. Emig, and D. Bommer, ibid., 102,971(1969). (76) R.J. Cushley, K.A. Watanabe, and J. J. Fox,/. Amer. Chem. SOC., 89,394(1967). (77) H. A. Friedman, K.A. Watanabe, and J. J. Fox,/. Org. Chem., 32,3775(1967). (78) F. W. Lichtenthaler, T. Nakagawa, and J. Yoshimura, Chem. Ber., 100, 1833 (1967). (79) F. W. Lichtenthaler and H. P. Albrecht, Chem. Ber., 100,1845(1967). (80) F. W. Lichtenthaler and H. P. Albrecht, Angew. Chem., 80, 440 (1968);Chem Ber., 102,964(1969).

87

THE NITRO SUGARS

Ribonucleosides (47) or synthetic analogs (48) containing a p-Dglucopyranosyl, instead of the p-D-ribofuranosyl, residue are amenable to periodate cleavage of their sugar moieties, to give “nucleoside dialdehydes” (49). Nitromethane cyclization of such dialdehydes proceeds well, and affords stereoisomeric mixtures of (3-deoxy-3nitro-P-D-hexopyranosy1)pyrimidines or (3-deoxy-3-nitro-P-~-hexopyranosy1)purines (50).

HO

OH

(49)

I

(47)

I a

I b

NHCOPh

%OH

NI&,

(50)

Me I

On several occasions, individual stereoisomers of 50, or their triacetates, were isolated in crystalline form. Catalytic reduction of the nitro group furnished a large number of the corresponding 3‘-amino3’-deoxynucleosides. Configurational assignments were based on nuclear magnetic resonance data, as well as on chemical degradations that usually involved acid-catalyzed, solvolytic removal of the aglycon base, and identification, in the form of suitable derivatives, of the

88

HANS H. BAER

amino sugars that were released. It transpired that, in the products (SO), the D-gluco configuration in general preponderates, with the D-ga~actoand (or) m man no configurations being present to an appreciable extent. No other isomers have thus far been found, and so the pattern is qualitatively the same as that in the synthesis of methyl 3-deoxy-3-nitro-~-~-hexopyranosides. It must be borne in mind that, in both series, the observed ratios of isomeric products have been based on the preparative yields of crystallizable materials, instead of on a quantitative analysis of the total mixture of products that were formed but invariably escaped complete isolation. As might be expected, variations in reaction conditions can lead to different yield ~ D-galacto , ~ ~ , ~ isomer ~ ( ~ has ) been isoratios. Thus, in three c ~ s ~ s , ~the lated in amounts equal to, or slightly larger than, that of the D-glum isomer. Pyrimidine nucleosides that have been employed in these ine' ~ ~ N4-benzoylvestigations include ~ r i d i n e ~ ~(47a), . ~ ~~*y ~t i~d (i n~(47b), (48a);purine ~ y t i d i n e(47c), ~ ~ and l-P-D-gluc~pyranosylthymine~~ ~ ~ 7-p-Dnucleosides used include adenosine73 (47d),i n ~ s i n e(47e), gluc~pyranosyltheophylline~~ (48b), and 6-(dimethylamin0)-9-p-~ribofuranosylpurineEo(470.

d. Methyl 3,6-Dideoxy-3-nitrohexopyranosides. -L '-Methoxy-Lmethyldiglycolaldehyde (51),which is best prepared from methyl a-L-rhamnopyranoside, gave with nitromethane a syrupy mixture of cyclization products. Catalytic hydrogenation thereof furnished in a crystalline methyl 3-amino-3,6-dideoxy-c~-~-glucopyranoside yield of 30% (based on the L-rhamnoside).81By use of the enantiomer of 51, the same procedure afforded methyl 3-amino-3,6-dideoxy-a-~glucopyranoside.s2 Each of these amino glycosides was N,N-dimethylated, and the product was hydrolyzed to 3,6-dideoxy-3-(dimethylamino)-L-glucose and -D-glucose, respectively; the latter sugar was found to be identical with mycaminose, a magnamycin fragment whose stereochemistry was thereby established.8z Methyl 3,6-dideoxy-3-nitro-a-~-glucopyranoside (52) has since been induced to crystallizem from the syrupy cyclization mixture from 51,and column chromatography of the mother liquors on silicic acid yielded,E4in addition, the c r - ~ m a n n o(53), a - ~ - t a l o(54), and a-L-galacto (55) isomers in crystalline form. It was foundE4that, when the cyclization is performed at room temperature in methanol solution with a reaction (81) A. C. Richardson and K. A. McLauchlanJ. Chem. Soc.,2499 (1962). (82) A. C. Richard:on,J. Chem. Soc., 2758 (1962). (83) K. Capek, J. Stefiqva, and J. Jar$, Coll. Czech. Chem. Commun.,31,1854(1966). (84) H. H. Baer and K. Capek, Can.]. Chem., 47,99 (1969).

THE NITRO SUGARS

89

time of 40 min, it affords a total yield of 75% in nitro glycosides isolated, with 52, 53, 54, and 55 occurring in the approximate ratios of 18:8:3:1. Shortening of the reaction time to 20 min lowered the total yield to 55% at the expense of the L-gluco isomer (52),the ratio of products then being about 10: 10:3:2. No additional isomers could be identified, although small proportions (less than 1%) of unknown substances were formed. Configurational assignments to 53-55 were made through catalytic hydrogenation to known amino glycosides.

e. 1,6-Anhydro-3-deoxy-3-nitro-~-~-hexopyranoses. - Derivatives of 3-deoxy-3-nitrohexoses differing configurationally from those described in Section II,3c and 3d (see pp. 83-89) were produced when cis-1,3-dioxolane-2,4-dicarboxaldehyde (56)(periodate-oxidized levoglucosan) was cyclized with nitromethaneas5 One isomer, namely, 1,6-anhydro-3-deoxy-3-nitro-~-~-gulopyranose (57),crystallized in 13.5% yield, whereas the isomers having the D-UZ~TO (58)and ~ - i d o(59)configurations were shown to be present by the isolation, in yields of 15 and 7.5%, respectively, of amine derivatives following catalytic hydrogenation. The presence of a fourth isomer was indicated by paper chromatography; it was presumed to possess the D - u Z Z ~ configuration (60) and was believed to have constituted a considerable, if not the main, part of the reaction mixture not accounted for by isolated products. Rigorous configurational proof was provided for 57, 58, and 59 by a series of interconversions (at the acetamido stage) involving methylsulfonyloxy displacement reactions, with neighboring-group p a r t i c i p a t i ~ n . ~ ~ (85) A. C. Richardson and H. 0.L. Fischer,J.Amer. Chem. SOC.,83,1132 (1961).

90

HANS H. BAER

An interesting modification of the reaction just described led to a triamino sugar.8s Interaction of 56, nitromethane, and benzylamine in the molar ratios of 1:2:4 gave, in 53% yield, 1,6-anhydro-2,4-bis(benzylamino)-2,3,4-trideoxy-3-nitro-/3-~-idopyranose (61). Catalytic hydrogenation over palladium resulted in reduction of the nitro group and simultaneous hydrogenolysis of the benzyl groups, to give the corresponding 2,3,4-triamino sugar which was characterized as the trihydrochloride and as the tri-N-acetyl derivative.sE It remains to be established whether the formation of 61 takes place by way of a Mannich-type, twofold aminoalkylation of nitromethane pursuant to an addition of benzylamine to the aldehyde groups, or whether the primary step is cyclization to one or several of the nitrodiols 57-60, which then undergo, twice (successively), dehydration and amine addition (see also, Section IV,2d; pp. 135-136).

(86) F. W.Lichtenthaler and T. Nakagawa, Chem. Ber., 101,1846 (1968).

THE NITRO SUGARS

91

f. Methyl 3-Deoxy-3-nitro-heptoseptanosides. -One of the relatively few syntheses of glycosides that contain a septanoside ring was accomplishedE7by periodate oxidation of methyl 4,6-O-ethylidenea-D-glucopyranoside followed by nitromethane cyclization of the dialdehyde 62. The product, isolated in 41 %yield, was methyl 3-deoxy5,7-O-ethylidene-3-nitro-D-gZyce~o-a-~-manno-heptoseptanoside (63). Reduction, N-acetylation, and hydrolysis afforded 3-acetamido-3deoxy-D-glycero-D-manno-heptose.

Wolfrom and

applied the method to methyl 4,6-0-

benzylidene-a-D-glucopyranoside,and obtained four isomeric 3deoxy-3-nitro-heptoseptanosides. It is reported87b that dialdehyde-cellulose incorporates nitro-

methane (and other nitroalkanes), to give polysaccharides containing 3-deoxy-3-nitroheptoseptanose residues. g. 3-Deoxy-3-C-methyl-3-nitro Glycosides. - Substitution of nitroethane for nitromethane in the cyclization of some sugar dialdehydes has led to branched-chain nitro glycosides. Thus, the dialdehyde 51 was cyclized to a mixture of products from which was isolated, after acetylation, a crystalline methyl 2,4-di-O-acetyl-3,6-dideoxy-3-Cmethyl-3-nitrohexopyranoside(64) in 12% yield.@Nuclear magnetic resonance studies allowed definite assignment of the configuration at C-1, C-2, C-4, and C-5, and showed that the compound belonged to the a-L-gZuco or the a-~-aZZoseriesEEThe chemical shift of the (87) G. Baschang, Ann., 663,167 (1963). (87a) M. L. Wolfrom, U. G. Nayak, and T. Radford, Proc. Nut. Acad. Sci. US.,58, 1848 (1967). (87b) Z. I. Kuznetsova, V. S. Ivanova, and N. N. Shorygina, Zzu. Akad. Nauk S S S R , Otd. Khim. Nauk, 2087 (1962), 1686 (1963), 2232 (1964);Chem. Abstracts, 58, 7023 (1963),60,755 (1964),62,7990 (1965). (88) S. W. Gunner, W. G. Overend, and N. R. Williams, Chem. Znd. (London), 1523 (1964).

HANS H. BAER

92

acetamido methyl protons in the corresponding 3-acetamido derivative has been interpretedE9as evidence in favor of the former series.

In the cyclization of the dialdehydes 32 and 39 with nitroethane, an unexpected epimerization involving the carbon atom bearing the hydroxymethyl group was e n c o ~ n t e r e dThe . ~ ~ dialdehyde 32 gave, in 20 % yield, a crystalline methyl 3-deoxy-3-C-methyl-3-nitrohexopyranoside (65) ([a]D 79.3') which was revealed by nuclear magnetic resonance spectroscopy to consist of two components in the ratio of 1:l; these could not be separated, and were considered to be present as a molecular-addition complex. Catalytic hydrogenation produced two separable amino glycosides, 66 and 67, which were characterized further by acetylation. Physical data indicated that the amino glycosides were diastereoisomers (as had been expected), but the reducing amino sugar hydrochlorides that were formed on acid hydrolysis turned out, surprisingly, to be a pair of enantiomers, namely, 68 and 69. When 65 was hydrogenated and the resulting solution hydrolyzed without prior separation of 66 and 67, the product was a crystalline racemate of 68 with 69. The dialdehyde 39, which is a diastereoisomer (not an enantiomer) of 32, furnished, in like yield, a crystalline nitro glycoside (70) ([(.ID -78") which evidently contained, as a molecular complex, the enantiomers of the two components in 65. Hydrogenation afforded 71 and 72 (that is, the enantiomers of 66 and 67), and hydrolysis then yielded the same pair of enantiomeric sugars, namely, 68 and 69. Compounds 65-72 are depicted as D- and L-glucose derivatives. These structures are based on unambiguous configurational assignC-4, ments, by nuclear magnetic resonance spectroscopy, for C-1, (2-2, and (2-5. As regards the configuration at C-3, chemical-shift values indicated that the acetamido groups in the four amino glycoside tetra-

+

(89) F. W. Lichtenthaler and P. Emig, Tetrahedron Lett., 577 (1967). (90) H.H.Baer and G. V. Rao, Ann., 686,210(1965).

THE NITRO SUGARS

93

O=CH 0%

H

I \

acetates possessed the identical orientation. Equatorial orientation (hence, the D- or L-gluco configuration) was considered the more probable of the two possible, but a definite decision was not made.g0 Data since gathered on C-methyl-branched acetamido cyclitols having equatorially attached acetamido groups support the assignment of the D- and L-gluco configuration^.^^

94

HANS H. BAER

+

Inspection of the sequence 32 + 65 + 66 67 shows that part of the dialdehyde reacts in the expected manner, without involvement of the carbon atom bearing the hydroxymethyl group, while another part suffers epimerization at that carbon atom. The latter event presumably takes place either before the nitroethane molecule is added or in an intermediary, acyclic adduct, but it is considered unlikely to occur in the final, cyclized product. The P-L-glycoside so formed then cocrystallizes with an equal amount of the a-Dform, to give 65. The composition of the mother liquor has not yet been examined, and so the extent of epimerization is unknown. From the yield of crystalline 65 (20%), it merely follows that the extent must lie within the limits of 10 and 90%. (The same conclusion applies to the reaction of the dialdehyde 39.)An epimerization of this kind had not previously been observed in cyclizations of 32 and 39 with nitromethane (see Section II,3c; p. 83).However, a nitromethane cyclization of 32 has since been reporteds3 in which 1%of a P-L-glycoside was found, in addition to 53% of a-D-glycosides isolated. It appears, therefore, that epimerization at C-5 in reactions with different nitroalkanes is a matter of degree, probably related to various levels of alkalinity during the cyclization. No epimerization at C-5 was observeds1 in a nitroethane cyclization of the nucleoside dialdehyde from uridine (47a). The dialdehyde 17 gave, with nitroethane, a crystalline methyl 3-deoxy -3- C -methyl -3-nitropentopyranoside of as - yet-undisclosed configurati~n.~~~ 4. Derivatives of 4-Deoxy-4-nitro Sugars A novel way of introducing the nitro group into sugar molecules has been devised by Szarek and coworkers.g1bNitryl iodide (N0,I) was found to add across the olefinic double bond of benzyl2-O-benzyl3,4-dideoxy-c~-~-gEycero-pent-3-enopyranoside. The nitro group entered at C-4, and the iodine atom, at (2-3. The adduct readily underwent dehydroiodination to give a nitro-olefin whose double bond could be hydrogenated selectively with sodium borohydride. The reduced product was benzyl 2-0-benzyl-3,4-dideoxy-4-nitro-/i?-~threo-pentopyranoside. No other 4-deoxy-4-nitro aldose derivatives are known to date. (91) F. W. Lichtenthaler and H. Zinke, Angew. Chem. Intern. Ed. Engl., 5,737 (1966). (91a) H. H. Baer and K. S.Ong, unpublished work; K. S. Ong, Ph.D. Thesis, University of Ottawa, 1968. (91b) W. A. Szarek, D. G. Lance, and R. L. Beach, Chem. Commun., 356 (1968).

THE NITRO SUGARS

95

Several 4-deoxy-4-nitro ketose derivatives have been synthesized. Three crystalline 2,7-anhydro-4-deoxy-4-nitro-~-~-heptulopyranoses arose by nitromethane cyclizatione2 of the dialdehyde (74) produced by periodate oxidation of sedoheptulosan (73), and were assignede3 the D - U ~(75), D-gdo (76), and D-dtTO (77) configurations by nuclear magnetic resonance spectroscopy of the corresponding, peracetylated amino sugars. When the cyclization of 74 was performed in methanolic medium, a solid mixture of stereoisomeric nitronates was obtained in almost quantitative yield, and it could be partially separated into the individual nitronates, which furnished 75 and 76. The isomer 77

crystallized from de-ionized, mixed fractions, and was reconverted into its aci-salt by sodium methoxide. Cyclization of 74 in aqueous medium in the presence of sodium hydroxide, followed by de-ionization without prior isolation of nitronates, gave mainly 77 and 76, and 75 appeared to be a minor product, detected by chromatography only. Formation of the aci-salt of 75 was apparently not favored thermodynamically; in methanolic medium, too, it constituted only 16%of the total product, but its low solubility promoted its spontaneous crystallization from the reaction solution. Of the other two isomers, (92) H. H. Baer,]. Org. Chem.,28,1287 (1963). (93) H. H. Baer, L. D. Hall, and F. KienzleJ. Org. Chem., 29,2014 (1964).

96

HANS H. BAER

q 0, HO

HO HO

HO

0-

0

OH

(78)

Ic; CH,OH

HO

HO

I,OH

H,OH

I

OH

OH

02N.

(81)

(80)

/

MeOH $!H20H

HOQ

o

M

e

H'

+

Me OH (83)

THE NITRO SUGARS

97

77 seemed to preponderate somewhat over 76, but no accurate ratio configuration has as yet been established. An isomer having the ~ - i d o was not found, although it might have been expected to arise, in view of the analogous formation of 59 from 56 (see Section II,3e; p. 89). However, an unidentified amine was indicated chromatographically to be a minor by-product from the catalytic hydrogenation of 75, and this result possibly signifies some epimerization of 75 to the ~ - i d o isomer. Oxidation of sucrose with a limited proportion of lead tetraacetate causes favored glycol-cleavage in the D-fructofuranose ring, and affords%the dialdehyde 78. Cyclization of 78 with nitromethane furn i ~ h e din , ~58% ~ yield, a mixture of disaccharidic nitronates (79) and, thence, a mixture of nitro disaccharides (80). Catalytic hydrogenation of 80 gave crystalline a-D-gluCOpyranOSyl 4-amino-4-deoxy-P-~gluco-heptulopyranoside hydrochloride (81), the first disaccharide containing a seven-carbon amino sugar. Methanolysis of the mixture 80 produced methyl a-D-glucopyranoside (82) and a nitro glycoside mixture from which crystalline methyl 4-deoxy-4-nitro-a-D-gZ~coheptulopyranoside (83) was obtained after purification by way of its 173:5,7-dibenzylideneacetaLg5 The dialdehyde (84) obtained from benzyl P-D-fructopyranoside yielded, with nitromethane, a mixture of benzyl4-deoxy-4-nitrohexulopyranosides,= one of which, shown to have the a-~-xyloconfiguration (85), was obtained crystalline (36%)and was converted by transglycosidation and hydrogenation into methyl 4-deoxy-4-nitro-aL-sorbopyranoside and the corresponding amine.97Acetylation of the mixture of benzyl glycosides afforded the triacetate of 85 (60%) and the triacetate of a minor isomer (4%) having the P-D-lyxo configuration (86). Catalytic reduction and debenzylation of the latter, followed tetraby acetylation, gave 4-acetamido-4-deoxy-P-~-tagatopyranose a~etate.~'

H OlN (84)

(94) (95) (96) (97)

(85)

(86)

A. K. Mitra and A. S. Perlin, Can.J. Chem., 37,2047 (1959). H. H. Baer and A. Ahammad, Can.J.Chem., 44,2893 (1966). F. W. Lichtenthaler and H. K. Yahya, Tetrahedron Lett., 1805 (1965). F. W. Lichtenthaler and H. K. Yahya, Chem. Ber., 100,2389 (1967).

98

HANS H. BAER

5. Derivatives of 6-Deoxy-6-nitro Sugars 6-Deoxy-6-nitro-D-glucose (90) and 6-deoxy-6-nitro-~-idose(91) .~~ were the first reducing nitro sugars to be s y n t h e s i ~ e dNitromethane addition to 1,2-O-isopropylidene-cr-~-xylo-pentodialdo-l,4-furanose (87) produced a mixture of 6-deoxy-l,2-O-isopropylidene-6-nitro-a-~glucofuranose (88) and -p-L-idofuranose (89). These C-5 epimers could be separated by reaction with acetone, whereby 89 gave a 1,2;3,5-di-isopropylideneacetal more readily than did 88. Acid hydrolysis of the acetone groups then afforded 90 and 91.

I

OR

bR

(92) R = Ac

(94) R = Ac

(98) J. M.Grosheintz and H. 0.L. Fischer,J.Amer. Chem. SOC., 70,1476 (1948).

THE NITRO SUGARS

99

Alternative approaches to derivatives of 90 have now been devised. Nucleophilic displacement by sodium nitrite of the iodine in methyl 2,3,4-tri-0-acetyl-6-deoxy-6-iodo-a-~-glucopyranoside~(92), and in the analogous 2,3,4-tri-O-(tetrahydropyranyl) derivativelOO(93), led to the corresponding 6-nitro glycosides (94 and 95). Hydrolytic removal of the tetrahydropyranyl protecting groups from 95 gavelo0 amorphous methyl 6-deoxy-6-nitro-a-~-glucopyranoside (96). In an improved procedure that used 4:1 methyl sulfoxide-N,N-dimethylformamide as the solvent, 94 was obtained from 92 in excellent yield; removal of the acetyl groups was effected by methanolysis catalyzed by methyl p-toluenesulfonate, and gave 96 in crystalline Addition of nitryl iodide to 3-0-acetyl-5,6-dideoxy-1,2-0-isopropylidene-a-~-xylo-hex-5-enofuranose, followed by dehydroiodination of the product with sodium hydrogen carbonate in refluxing benzene, furnishedglb the same 5,6-unsaturated 6-nitro hexose derivative (182, see Section IV,2c, p. 127)that had been synthesized previously by sequential acetylation and dehydroacetylation of 88. Borohydride reduction of 182 gaveglb 5,6-dideoxy-1,2-O-isopropylidene-6-nitro-a-~-xylo-hexofuranose. 6. Derivatives of Other Deoxynitro Sugars

1,2;3,4Di -0 -isopropylidene-a-D-guZacto-hexodialdo-1,5-pyranose (97) and nitroethane gave a mixture of adducts, from which three of the four stereoisomers possible were isolated as acetates (98,6-0-acetyl-7,8-dideoxy-l,2;3,4-di-O-isopropylidene-7-nitro-octopyranose~).~~~

H

c=o 1. C,€&NO,

(99) J. M. Sugihara, W. J. Teerlink, R. Macleod, S. M. Dorrence, and C. H. Springer, J . Org. Chem.,28,2079 (1963). (100) B. Lindberg and S. Svensson, Acta Chem. Scand., 21,299 (1967). (101) G. B. Howarth, D. G . Lance, W. A. Szarek, and J. K. N. Jones, Can. J . Chem., 47,75 (1969).

100

HANS H. BAER

7. Deoxynitro Cyclitols The nitroalkane-aldehyde reaction is an excellent tool for the synthesis of alicyclic polyhydroxynitro compounds; these, in turn, are useful intermediates for the preparation of biologically interesting amino cyclitols. Grosheintz and Fischer, after having synthesizedss 6-deoxy-6-nitro-~-glucose(90) and -L-idose (91), found1O2 that, in alkaline medium, these sugars undergo an intramolecular Henry reaction that results in optically inactive deoxynitroinositols. They established that a mixture of three stereoisomers was formed, and that the composition of such mixtures depends upon whether (barium) nitronates were allowed to crystallize from the reaction solution, or whether the (sodium) nitronates were allowed to remain in solution, prior to acidification. Under identical conditions, 90 and 91 gave the same nitroinositol stereoisomers, and it was realized that mutual interconversion of isomers can occur in alkaline medium. Fractional recrystallization, acetylation, and acetonation served to separate the mixtures, at least in part, but no configurational assignments could initially be made.’02.’03 This was later achieved by other workers, who ~ o r r e l a t e d ’ ~ the - ’ ~ nitroinositols, ~ after catalytic hydrogenation, with inosamines of independent origin, and ~tudied’~’ several derivatives by nuclear magnetic resonance spectroscopy. The deoxynitroinositols designated’02 “I,” “11,” and “111” have thus been convincingly shown -~ (Ref. 107), muco-3 (101) (Ref. 107), and to possess the D L - ~ Y O(100) scyllo (102) (Ref. 105) configurations, respectively. By use of nitromethane-14C, radioactively labeled 90 and 91 and, thence, 102, were prepared, and the latter was converted by acetylation, reduction, and deamination (with inversion) into’0smyo-inosit01-2-’~C. The reactivity of 90 and 91 is so pronounced that cyclization occurs, although slowly, in neutral or even slightly acidic solution. Baer and Rank’”s observed that the nitro L-idose 91 is stable at pH 4,but cyclizes, within 24 hr, to the extent of 10%at pH 5 and to the extent of 90% at pH 6. At pH 7 and 9, a 90% cyclization was noted within 4 and 2.5 hr, respectively. The nitro D-ghCOSe (90) was found to react more slowly than 91 at pH 6 and 7, but more rapidly than 91 at pH 9. (102) (103) (104) (105)

J. M.Grosheintz and H. 0.L. Fischer,]. Amer. Chem. SOC., 70,1479(1948). B. Iselin and H. 0.L. Fischer,]. Amer. Chem. SOC., 70,3946(1948).

G.E. McCasland,J.Amer. Chem. SOC., 73,2295(1951). T.Posternak, H e h . Chim. Acta, 34, 1600 (1951);T.Posternak, W.H. Schopfer,

and R. Huguenin, {bid.,40,1875(1957). (106) G. I. Drummond, J. N. Aronson, and L. AndersonJ. Org. Chem.,26,1601(1961). (107) F. W.Lichtenthaler, Chem. Ber., 94,3071(1961). (108) H.H.Baer and W. Rank, Can.]. Chem., 43,3462(1965).

THE NITRO SUGARS

101

OH

OH nL-myo -1

muco-3

scyllo

(100)

(101)

(102)

The greater reactivity of 91 as compared with 90 (at low pH) is reminiscent of the difference between the idoses and the glucoses in regard to the ease of 1,g-anhydride formation, and is probably a reflection of the conformational instability inherent in D-or L-idopyranose structures. By applying the principle of internal Henry addition to a derivative Wolfrom and coworkers10s accomof 2-amino-2-deoxy-~-glucose, plished the first synthesis of di-N-acetyl-tetra-O-acetylstreptamine (108). Ethyl 2-acetamido-2-deoxy-l-thio-a-~-glucofuranoside (103) was oxidized with lead tetraacetate to the partially protected dialdose (104), which, with nitromethane, gave a mixture of the 5-epimeric 6-nitro thioglycosides (105). The 2-acetamido-2,6-dideoxy-6-nitrohexoses (106) produced therefrom by desulfurization with mercuric chloride were not separated, but were cyclized immediately, in the presence of barium hydroxide, to a mixture of l-acetamido-1,3-dideoxy-3-nitroinositol salts (107). Acidification, hydrogenation, and acetylation then afforded 108 (and a stereoisomer). The reaction sequence of Grosheintz and F i s ~ h e r , ~namely, ~*'~~ 87 + 88 89 + 90 91 + 100 101 102, constitutes a stepwise, nitromethane cyclization of xylo-trihydroxyglutaric dialdehyde (99) (xylo-pentodialdose), a partially protected derivative of which is 87.

+

+

+

+

(109) M. L. Wolfrom, S. M. O h , and W. J. Polglase, J . Amer. Chem. SOC., 72,1724 (1950).

102

HANS H. BAER

An analogous consideration applies to the synthesis of streptaminejust described. However, free 1,5-dialdehydes can themselves be directly cyclized with nitroalkanes to give six-membered alicycles. Thus, the dialdehyde 99, obtainable by acid hydrolysis of 87, has been to yield 100-102 on direct cyclization with nitromethane, and a similar reaction with nitroethane led112to l-deoxy-l-C-methyl-1nitro-scyllo-inositol (102, with Me replacing H on C-1). Starting from the 5-acetamido-3-cyclopentene-l,2-diols(109 and 110), Hasegawa and Sable113synthesized, in several steps, the three partially blocked acetamidocyclopentanetetrols 111, 112, and 113, and from these obtained by periodate cleavage the dialdehydes 114, 115, and 116, respectively. Nitromethane cyclization led to mixtures of acetamidonitro cyclitols. Without isolation of individual isomers, the mixtures were reduced with Raney nickel, deacetonated (where applicable), and the products acetylated. Chromatography through alumina then furnished numerous crystalline di-N-acetyl-tetra-0-acetylinosadiamines. Altogether, ten isomers (nine different ones) were characterized, and the configurations 117,a-d, 118,a-c, and 119,a-c were assigned on the basis of chemical-shift data for the acetoxy and acetamido substituents. Performance of the reactions with nitromethane14Cgave specifically labeled inosadiamines. (110) V. Brocca and A. Dansi, Ann. Chim. (Rome),44,120 (1954). (111) F. W. Lichtenthaler,Angew. Chem. Intern. Ed. Engl., 1,662 (1962). (112) F. W. Lichtenthaler and H. K. Yahya, Carbohyd. Res., 5,485 (1967). (113) A. Hasegawa and H. Z. Sable,J. Org. Chem.,33,1604 (1968).

THE NITRO SUGARS

- +?””H C=O I HCO

(1111

AcO

Acok

AcHNYH

F0

4 Products:

a. my0 -4,6 b. DL-eM-2,6

c . DL-allo-l,3 d. n e o - l , 3 or DL- cht~0-2,4

AcO

(114)

(117)

H ’i=0 (112)

103

3 Producte: a, ~ ~ - e p i - 2 , 6 b. DL-myO-1,5 c. ept-1,5 or

&,OciH

~~-epi-l,S =O

AcO

H

(115)

NHAc (118)

3 Products:

HE=O I OAc (113)-

AcOCH H NHAc TCO H

i:

(116)

a. scyllo -1,3 b. myo-1,) c. DL- cMro-l,S

-AcoQc

AcO (119)

HANS H. BAER

104

When glyoxal is allowed to react with two moles per mole"' or, better, with a large excess115of nitromethane, one molecule of the latter combines with each aldehyde group, to yield 1,4-dinitro-2,3butanediols (the meso and DL formslla). However, when equimolar amounts of the reactants are employed in aqueous sodium carbonate solution, the main product is a stereoisomeric mixture of 1,li-dinitrocy~lohexanetetrols.~'~ One uniform, sparingly soluble isomer, 1,4dideoxy-1,4-dinitro-neo-inositol (120) was isolated; the configuration was deduced from the nuclear magnetic resonance spectra of the tetraacetate and of the corresponding hexaacetylinosadiamine, and from the fact that 120 gave an isopropylidene acetal, indicating the presence of cis hydroxyl groups (see also Section IV,lb; p. 117). LichtenthalePO explained the cyclization as proceeding by way of intermediary 3-nitrolactaldehyde (121) which, when synthesized in an alternative way1lSand rendered alkaline, is said to give 120, also. Two molecules of 121 would combine with each other, head to tail; or, alternatively, one molecule of 121 could combine with glyoxal, to form the intermediate 2,4-dihydroxy-3-nitroglutaricdialdehyde (122), which would cyclize with nitromethane to give 120. In either event, the reaction would proceed by way of the intermediate 3,6-dideoxy3,6-dinitrohexoses (123).

+ OCH-CHO + 4NCH,

CHW"

+

OCH-CHO

-

+ OCH-YOH 4N\% CHOH-CHO

CHOH-CHO

(114) H. Plaut, U.S.Pat. 2,616,923(1952);Chem. Abstracts, 49,11701(1955). (115) S. S. Novikov, I. S. Korsakova, and K. K. Babievskii, Zzu. Akad. Nauk SSSR, Otd. Khim. Nauk, 944 (1960);Engl. translation, Bull. Acad. Sci. USSR Diu. Chem. Scd., 882 (1960). (116) F. I. Carroll,]. Org. Chem., 31,366(1966). (117) F. W. Lichtenthaler and H. 0.L. Fischer.]. Amer. Chem. SOC., 83,2005(1961). (118) H.0.L. Fischer, E. Baer, and H. Nidecker, Helo. Chim. Acta, 18,1079(1935).

THE NITRO SUGARS

105

In analogy to the nitromethane cyclization of pentodialdoses already discussed, a derivative of meso-tartraldehyde, namely, the hemialdal (124) of 2,3-O-cyclohexylidene-erythro-tetrodialdose,has f u r n i ~ h e d " a~ mixture of stereoisomeric 2,3-O-cyclohexylidene-5nitrocyclopentane-l,2,3,4-tetrols(125).

8. Miscellaneous Because of their kinship to syntheses of nitro sugars, some syntheses of simpler, cyclic, aliphatic nitro compounds will be mentioned. Tetrahydropyran-2-01, which is in tautomeric equilibrium with 5-hydroxypentanal(126), was found to undergo nitromethane addition catalyzed by sodium hydroxide to give120tetrahydro-2-( nitromethy1)pyran (128). Undoubtedly, the first reaction-product is the diol nitronate (127), which, under the conditions of processing (acidification and steam distillation), forms the cyclic ether 128.

OH

H h

(130)

(129)

(119) S. J. Angyal and S. D. Ger0,Aust.J. Chem., 18,1973 (1965). (120) J. Cologne and P. Corbet, Bull. SOC. Chim. France, 283 (1960).

A (131)

106

HANS H. BAER

Cyclization of glutaraldehyde (129) with nitromethane, which, when first attempted, seemed unprofitable,121 has been so elaborated122J23 as to provide, in 66 % yield, truns,truns-2-nitro-l,3-~yclohexanediol (130, R = H), the thermodynamically most stable of the three possible isomers (one DL and two meso forms). The reaction has been extended to include n i t r ~ e t h a n e , ' ~nitroethanol,126 ~~'~~ ethyl nitroacetatelZ5[which gave, preponderantly, 130 (R = Me, CH,OH, and CO,Et)l, l - n i t r o p r ~ p a n e , 'and ~ ~ phenylnitromethane (a-nitrotoluene)lZ4[which yielded products 131 (R= Et and Ph) that had the D L - C ~ ~ - ~ T Uconfiguration]. ~M 9. Stereochemical Considerations As mentioned in Sections II,1 and II,2 (see p. 72), some degree of stereoselectivity is observed in the formation of epimeric deoxynitroalditols by addition of nitromethane or nitroethanol to aldoses. Unfortunately, systematic studies concerning the factors that govern the stereochemistry of the reactions are still lacking, the research objectives having largely been of a more preparative nature. It is difficult to assess the extent to which any observed ratios of epimers reflect the rates of the primary addition reactions, and the extent to which such ratios are conditioned by secondary, thermodynamically controlled equilibrations. Obviously, such fortuitous phenomena as the solubility of the products and their tendency to crystallize have often played a role in determining the preparative yields of epimers obtained. The reaction conditions employed in individual experiments differ considerably, so that meaningful correlations between the ratios of epimeric products and the configuration and conformation of the starting sugars cannot readily be made. At best, an attempt to correlate some of the preparative results qualitatively may be made under the assumption that they carry kinetic significance. For instance, in the nitromethane addition to hexoses it might, at first sight, have been expected that the configuration at C-2 would exert a direc(121) G. E. McCasland, T. J. Matchett, and M. Hollander, J . Amer. Chem. SOC., 74, 3429 (1952). (122) F. W. Lichtenthaler, Chem. Ber., 96,945(1963). (123) F. W. Lichtenthaler, T. Nakagawa, and A. El-Scherbiney. Chem. Ber., 101, 1837 (1968). (124) F. W. Lichtenthaler, H. Leinert, and U. Scheidegger, Chem. Ber., 101, 1819 (1968). (125) S. Zen, Y. Takeda, A. Yasuda, and S. Umezawa, Bull. Chem. SOC.l a p . , 40,431 (1967). (126) F. W. Lichtenthaler and H. Leinert, C h m . Ber., 101,1815 (1968).

THE NITRO SUGARS

107

tive influence on the configuration to be engendered at the neighboring aldehyde group. However, a comparison of the reactions with D - g l u ~ o s e ,D~-~g a l a c t o ~ e ,D-mannose20s26 ~~~~~ and D-talose’* does not substantiate this expectation. The preponderant products obtained from the first two sugars are 2,3-cis-heptitols (having the D-glyCeroD-gdo and D-glycero-L-manno configuration), whereas the preponderant products from the last two sugars are 2,3-tmns-heptitols (having the D-glyCerO-D-gUlUCtO and D-glyCeTO-L-UhO configuration); in all four examples, the new asymmetric carbon atom assumes the same configuration, irrespective of that of its neighbor. A rationale for this fact will have to take into consideration the favored conformation of the aldehydo form of the sugar in the transition state (during the approach of the nitromethane anion). If it is assumed that, for the four hexoses mentioned, the conformation of the transition state resembles the C1 (D) conformation of the original pyranose and that, furthermore, the rotational orientation of the carbonyl group is similar in each of them (see Fig. l), equatorial attack by the nucleophile from the less-hindered (“front”) side will generate identical configurations at the carbonyl carbon atom, regardless of the position of the neighboring hydroxyl group. The opposite configuration would arise through a transition state resembling the other chair conformation, 1C (D), and the degree of stereoselectivity in the reaction should

D-GlucoBe, galactose

2,s- d s Products

D-MBnnOBe, D-WOBe

2,s-trans Products

FIG. 1. -Formation of Preponderant Epimers of 1-Deoxy-1-nitroheptitolsfrom the Reaction of Hexoses with Nitromethane.

108

HANS H. B U R

therefore be expected to be linked with the conformational stability of the particular pyranose. L-Idose gavelsb the 2,3-cis- and 2,3tmns-heptitols in a ratio of about 2: 1,which, on the basis of this hypothesis would imply a preponderant participation of the inverted chair form, c1 (L). In evaluating, from the conformational viewpoint, the sugar dialdehyde-nitromethane cyclizations, due reservations must also be made regarding the comparative significance of isomer ratios found in different experiments, for the same reasons that apply to the aldosenitromethane addition. However, in a few cases, the kinetically favored isomer has been unambiguously established; this was done by arresting the reaction after the elapse of various lengths of time (short of completion of the reaction), and by studies revealing the course of thermodynamically controlled interconversions of isomers (see also Section IV,2a; p. 120). Thus, the kinetically favored nitronates arising from the dialdehydes 32,39,and 51 have been found to possess tmns relationships between the glycosidic methoxyl group and the 2-hydroxyl group, as well as between the 4-hydroxyl group and the exocyclic, C-6 group.61.67*84 Cram’s rulelZ7has been invoked8’ to account for these observations. However, the dialdehydes were regarded as acyclic compounds, with the tacit implication that reaction may occur at each carbonyl group, without reference to the overall, conformational disposition of the molecule. Although the experimental results in the three cases cited (and, possibly, in others, too) are in formal agreement with the rule, it is questionable whether this result is more than a coincidence. Surely, for any prediction to be useful, it would be necessary to cope, first, with conformational ambiguities connected with the various cyclic forms in which a particular “dialdehyde” can exist prior to addition of the nitromethane molecule (see pp. 127 and 128 in Ref. 54), and second, with the conformation adopted by the primary adduct prior to final ring-closure. To evaluate these factors, it would be necessary to know at which of the two aldehydic (or potentially aldehydic) carbon atoms the primary-bond formation occurs, but in no case has this as yet been determined. Although reasonable assumptions in that regard may in some instances offer themselves on consideration of the cyclic structure (if known) of a given dialdehyde, uncertainties will arise in other instances. It does not therefore appear profitable at present to expound stereochemical generalizations on the formation of glycoside nitronates (127) D.J. Cram and F. A. Abd Elhafez,]. Amer. Chem. SOC., 74,5828 (1952); E.L. Eliel, N. L. Allinger, S. J. Angyal, and G . A. Morrison, “Conformational Analysis,’’ Interscience Publishers, New York, N.Y., 1965, p. 32.

THE NITRO SUGARS

109

(14 + 15) beyond stating the empirical fact that marked stereoselectivity does exist. Another selective principle operates in the step 15 + 16. As a rule, on being liberated from the aci structure, the nitro group will assume an equatorial position. In certain cases [a-D-and L-hexopyranosides, 1,6-anhydrohexoses, and 2,7-anhydroheptuloses (see Section II,3c-e; pp. 83-89,and Section 11,4; p. 94)], an axially attached nitro group would encounter serious steric hindrance from an axial substituent on the same side of the ring. However, in the absence of such interference, the product having an equatorial nitro group is still formed, to the exclusion of its epimer.128For an explanation of the general course of the reaction, it may be considered that axial approach of the protontransferring species to the nitronate carbon is more favored sterically than approach from the opposite direction, which is hindered by two vicinal, axial bonds (see Fig. 2).lZ9

FIG. 2. -Favored Pathway of Protonation of Cyclic Nitronates.

111. REACTIONS THATALTER OR REMOVE THE NITROGROUP

1. Catalytic Hydrogenation As has been pointed out repeatedly in the preceding Sections, catalytic hydrogenation of nitro sugars is of great importance in configurational studies, as well as for the preparation of amino sugars. Hydrogenation generally proceeds well, and rarely, if ever, have serious difficulties been encountered. Platinum, palladium-on-carbon, (128) One exception is seen in the results of acidification of the nitronate 18, which gave both of the epimers 22 and 23. The fact that 22 preponderates is an apparent anomaly that can be explained on the basis of the inverted-chair conformation that 18 has been found to favor (J. KovG and H. H. Baer, unpublished results). (129) This explanation has been offered by Johnson and Malhotra'" for the favored axial protonation of substituted cyclohexane nitronates.

110

HANS H. BAER

and Raney nickel are the preferred catalysts. Room temperature and pressure are usually adequate, and the yields are excellent, although moderate forcing-conditions have occasionally been needed in order to overcome sluggish reduction. There is really only one point that requires special attention, namely, the fact that certain nitro glycosides and related nitro alcohols readily undergo base-catalyzed epimerization (see Section IV,2a; p. 120). Hence, it is only safe to assume that configurational changes do not take place during the hydrogenation if the medium is prevented from becoming basic owing to (a) the amine formed and (b) alkaline contaminants present in some catalysts. Therefore, whenever retention of configuration is essential, as in structural analysis, it is imperative to hydrogenate in the presence of sufficient acid to keep the medium slightly acidic until the nitro compound has been completely reduced.130For preparative purposes, on the other hand, this precaution may not always be necessary, especially if the nitro compound to be reduced happens to be a thermodynamically stable isomer, or if epimerization is slow and can be tolerated in limited measure. A case in point was the preparation of methyl 3-amino3-deoxy-~-(andL-)ribopyranosides by platinumcatalyzed hydrogenation of nitro pentoside mixtures (see Section II,3a; p. 80), where yields were not impaired when acid was omitted, although the proportions of the corresponding amino xylosides, isolated as minor by-products, did increase slightly under alkaline condit i o n ~ Similarly, .~~ Raney nickel, which is cheaper and more effective than platinum, has been used to advantage in neutral media, and, in some cases where both catalysts have been employed, the results were In quite comparable with regard to the products ~btained.~"."~.'~~ hydrogenations of 3'-deoxy-3'-nitronucleosides,the choice of catalyst must take into account the reactivity of the aglycon. Thus, 1-(3deoxy-3-nitro-/3-D-g~ucopyranosyl)uracil was, in the presence of Raney n i ~ k e l , ' ~reduced ,~~ at the nitro group only, whereas p l a t i n ~ m ~ ~ . ~ ~ caused hydrogenation in the pyrimidine ring also, giving the 3'-amino5,6-dihydro derivative. Other 3'-deoxy-3'-nitronucleosideshave been or palladium-onreduced selectively by the use of Raney nicke173*77 charcoal in acetic a ~ i d . ~ ~ - * ~ Sugar derivatives containing an a-nitroalkene grouping can usually be saturated, without affecting the nitro group, by brief hydro(130) N. Kornblum and L. Fishbein, /. Amer. Chem. SOC., 77,6266 (1955); F; G. Bordwell and R. L. Arnold, J . Org. Chem., 27, 4426 (1962). An acidic medium suppresses formation of the nitronate ion that is an intermediate in epimerization, not only at the nitro carbon center but also at adjacent carbinol carbon atoms. (131) A. C. Richardson,/. Chem. SOC., 373 (1962).

THE NITRO SUGARS

111

genation with palladium in absolute ethanol3 or ethyl acetate.132However, in the nitro-olefin 132, this selectivity was diminished owing to Although the compound steric hindrance from the methoxyl became, in part, saturated to give 133, it was, in part, reduced to a (hydroxy1amino)alkene which tautomerized to the oxime 134. The a-D-the0 isomer of 132 (179 on p. 125) behaved similarly, whereas no oximes were found in selective, palladium-catalyzed hydrogenations of the corresponding unsaturated P-D-glycosides.

2. The Nef Reaction The Nef r e a c t i ~ n ’ ~is” ~the conversion, by treatment with mineral acid, of a nitronate into an aldehyde or ketone. Early applications of this reaction to deoxynitroalditols have proved extremely useful in sugar synthesis, and have been r e ~ i e w e d .The ~ general utility of the reaction is emphasized by several more-recent syntheses of ale s, ,~~k~e~~t ~o ~s ~e ~~ -, ~and ~~~- ~ disacchar~ d o ~ e ~ , ~2 -~ d -e o~ ~~y a~l d , o~s ~ (132) It has been claimed3 that no selectivity is observed in ethyl acetate, but, in recent work (see Section IV,2c, p. 128) selective hydrogenations have been achieved by using this solvent. Sodium borohydride in ethanol has also been found8’”to reduce the double bond of nitro-olefinic sugars selectively. (133) W. W. Zorbach and A. P. Ollapally,J. Org. Chem., 29,1790 (1964). (134) M . B. Perryand J. FurdovP, Can../. Chem., 46,2859(1968). (135) M. B. Perry and A. C. Webb, Can.J. Chem., 47,1245 (1969).

112

HANS H. BAER

ides.'35aApplication to 2-acetamido-l,2-dideoxy-l-nitroalditols, which are readily accessible starting-materials (see Section IV,2d, p. 133),has opened a novel and convenient avenue to 2-amino-2-deoxyaldoses that complements such other general syntheses as the hemihydrogenation of am in on it rile^'^^ and the more recent glycal method.43 Amino sugars obtained by the Nef reaction (and isolated as N-acetyl derivatives or hydrochlorides) include 2-amino-2-deoxy-~-g~ucose and - D - m a n n o ~ e , ~ ~-D-gUlOSe ' - ~ ~ ~ and - ~ - i d o s e , ' ~-D-galactose ~ * ~ ~ ~ and -Dt a l o ~ e , 'and ~ -D-allose and -D-altrOSe,134and six different 2-acetamido2-deoxyhepto~es.'~*~~ The Nef reaction is reputed to give poor results with sterically hindered, secondary nitro compound^.'^ It has been reported to fail with deoxynitroin~sitols,~~~ and difficulties may well be anticipated with 3-deoxy-3-nitro-glycosides and similar cyclic nitro sugars. Even in some of the conversions of nonrigid 2-deoxy-2-nitroalditols into ketoses (see Section 142; p. 72), the yields were less than satisfactory. Systematic studies aimed at making improvements on, or finding alternative ways for, the denitration of nitro sugars would certainly be desirable. Possibly, the permanganate oxidation of nitronates in buffered, weakly alkaline solution'44 will prove of value. By this method, truns,trans-1,3-bis(acetamido)-2-nitrocyclohexanehas been converted in good yield into cis-2,6-bis(acetamido)cyclohexanone,1~ and some 1-deoxy-1-nitroalditols have been oxidized to aldoses.laa An interesting study has been made of the photolysis of a nitroolefinic sugar.'45b Irradiation, at 253.7 nm, of an acetone solution of cis-6,7,8-trideoxy-1,2;3,4-di-0-isopropylidene-7-nitro-cr-~-guZuc~o-oct6-enose (135)gave a complex mixture from which there were isolated the tmns isomer 136 and the cis and trans isomers of 6,gdideoxy1,2;3,4-di-O-isopropylidene-c~-~-guZucto-oct-5-enos-7-ulose (137 and 138). Insofar as the formation of the two ketones involves the loss of (135a) I. Furda and M. B. Perry, Can.]. Chem., 47,2891 (1969). (136) R.Kuhn,Angew. Chem., 6 9 , B (1957). (137) A. N. O'Neill, Can.]. Chem., 37.1747 (1959). (138) J. C.Sowden and M. L. Oftedahl,]. Amer. Chem. SOC., 82,2303 (1960). (139) C.Satoh and A. Kiyomoto, Chem. Pharm. Bull. (Tokyo), 12,615 (1964). (140) S. D. Gero and J. Defaye, Compt. Rend., 261,1555 (1965). (141) J. C.Sowden and M. L. OftedahlJ. Org. Chem., 26,2153 (1961). (142) C.Satoh and A. Kiyomoto, Carbohyd. Res., 7,138 (1968). (143) C.F. Gibbs, D. T. Williams, and M. B. Perry, Can.]. Chem., 47,1479 (1969). (144) H. Shechter and F. T. Williams,]. Org. Chem., 27,3699 (1962). (145) H. H. Baer and M. C. Wang, Can.]. Chem., 46,2793 (1968). (145a) M.B. Perry, personal communication. (145b) G. B. Howarth, D. G. Lance, W. A. Szarek, and J. K. N. Jones, Con.]. Chem., 47,81(1969).

THE NITRO SUGARS

113

one atom each of hydrogen, nitrogen, and oxygen, this photolysis bears a formal resemblance to the Nef reaction.

3. Miscellaneous Reactions

Treatment of methyl 4,6-0-benzylidene-3-deoxy-3-nitro-~-~-glucopyranoside (139) or its CPD anomer (140) with basic aluminum oxide in boiling toluene furnished,lMas the major reaction product, 4,6-0benzylidene -2,3-dideoxy -D-erythro - hex-2 -enono- 1,s-lactone (144). The first step in these transformations is a reversible dehydration to the nitro-olefinic glycosides 141 and 132, respectively; use of crystalline 141 and 132 as starting compounds also leads to 144, and to partial hydroxylation back to 139 and 140, respectively. It is believed that the nitro-olefins suffer loss of their anomeric hydrogen atoms (which are allylic in these compounds) to give an intermediate ketene acetal (142) that is hydrolyzed to form the p-nitro lactone 143. There was chromatographic evidence that suggested that 143 is formed, but this compound was not isolated. &Elimination of nitrous acid from 143 would then give the product 144. The same reactions (146) H. H. Baer and W. Rank, Can./. Chem., 47,2811(1969).

114

HANS H. BAER

performed with the P-D-galacto isomer of 139 and with the corresponding nitro-olefin yielded 4,6-0-benzylidene-2,3-dideoxy-~threo-hex-2-enono-l,5-lactone (145).

In the course of the elucidation of the structure of evernitrose (see Section I,2, p. 70), p-elimination of nitrous acid was effected'" by treatment of the corresponding hexonolactone with methanolic potassium acetate. Another reaction involving loss of this acid will be described in Section IV,2c (see p. 129). The epimeric addition products (146) obtained from the tetraacetate of l-nitro-~-arabino-l-hexene-3,4,5,6-tetro1 and aniline (see Section IV,2d; p. 136) lost two molecules of water per molecule on treatment14' with acetic anhydride in pyridine, and gave 3,4,5,6-tetraO-acetyl-B-deoxy-2-(pheny1imino)-D-arabino-hexononitrile( 147). The (147) J. C. Sowden, A. Kirkland, and K 0.Lloyd,J. Org. Chern., 28,3516(1963).

THE NITRO SUGARS

115

latter was degraded by aqueous sodium hydroxide to D-arabinonic anilide (148).

Iv. REACTIONS THATPROCEED WITH RETENTION OF THE NITROGROUP 1. Acid-catalyzed Reactions

a. Acylation. -Acetylation of deoxynitroalditols is a straightforward operation that is generally performed with acetic anhydride and a catalytic amount of sulfuric acid.3 Fully acetylated deoxynitroalditols serve as synthetic intermediates for 1,e-unsaturated and 2-C-substituted derivatives (see Section IV,2c and d; pp. 127, 130, 133).Nitro cyclitols,'03~'07~117 as well as a few n i t r o g l y c o s i d e ~and ~ * ~nitronucleo~ side^,^"^^ have been acetylated in the same way, In order to minimize such possible side-reactions as anomerization of glycosides and acetolysis of acetal structures, alternative techniques of acetylation have been studied. Whereas basic catalysis leads to severe complications in certain nitro sugar derivatives (see Section IV,2a and c; pp. 124, 129), boron trifluoride etherate has proved to be an excellent agent for satisfactory acetylation of glycosides, O-benzylidenated glycosides, and disaccharides that contain a deoxynitro functi~n.'~~*'~~ An attempt at preparing an "acetobromo" derivative of 3-deoxy-3nitro-D-glucose by the direct method150r e s ~ l t e d ' ~in' the formation, in high yield, of 3-deoxy-3-nitro-~-~-glucopyranose tetraacetate; however, subsequent treatment of the tetraacetate with hydrogen bromide in glacial acetic acid did furnish the desired 2,4,6-tri-O-acetyl-3deoxy-3-nitro-a-~-g~ucopyranosy~ bromide. With silver carbonate as (148) (149) (150) (151)

H. H. Baer, F. Kienzle, and F. Rajabalee, Can.]. Chem., 46,80 (1968). H. H. Baer and F. Kienzle, Can.]. Chem., 47,2819 (1969). M. BBrczai-Martos and F. Kbosy, Nature, 165,369(1950). H. H. B u r , F. Kienzle, and W. Rank, unpublished results.

116

HANS H. BAER

the acid acceptor, the bromide gave 2,4,6-tri-O-acety1-3-deoxy-3nitro-P-D-glucose in the presence of water, and the corresponding methyl P-Dglucopyranoside in the presence of methanol.

b. Acetalation. -Benzylidenation and acetonation of nitro sugars offer no particular problems. Thus, the methyl 3-deoxy-3-nitrohexopyranosides 34, 36, 40, 41, 42, 45, and 46 gave well crystallized 4,6-benzylidene acetals by the usual procedure with benzaldehyde and zinc c h l ~ r i d e . ~ ~The ~ ~heptuloside * ~ ~ * ' ~ ~83 was purified by way of its 1,3;5,7-dibenzylidene a ~ e t a l . ~ ~

Both 6-deoxy-1,2-0-isopropylidene-6-nitro-cr-~-glucofuranose (88) and -p-L-idofuranose (89) yield a 1,2;3,5-diisopropylidenederivative; the L-idose derivative (150) is formed more rapidly than the D-glUCOSe derivative 149, so that a separation can be effected of the mixture of 88 and 89 that is produced in the nitromethane synthesisss (see Section 11,5; p. 98). The slower 3,5-acetonation of 88 has been in terms of diaxial substituent interaction that arises at the m-dioxane ring in 149, regardless of the chair conformation it adopts. That a greater strain is present in 149 is also borne out by the difference in behavior of 149 and 150 toward alkali (see Section IV,2b; p. 126). Another factor that might influence the rates of acetonation could be hydrogen bonding between the 3-hydroxyl group and the nitro group, which, according to molecular models, would make the 5-hydroxyl group favorably disposed in 89, but not in 88, for ring formation. However, the actual conformations of these compounds have not as yet been determined. Of the three deoxynitroinositols 100-102, only one, namely, the muco-3 isomer (101) forms a diisopropylidene acetal (151), whereas the others do not react with acetone under ordinary conditions.102,'01 The boat form depicted for 151 appears reasonable, and is supported (152) H. H. Baer and T. Neilson, Can.]. Chem., 43,840 (1965). (153) (a) J. A. Mills, Aduan. Carbohyd. Chem., 10, 1 (1955). (b)S. J. Angyal and L. Anderson, {bid.,14,135 (1959).

THE NITRO SUGARS

by nuclear magnetic resonance data.lo7 The myo-1 isomer

117

(loo),

which possesses one cis-glycol grouping, might have been expected to give a monoacetal (152). However, myo-inositol itself is also known to be reluctant to form an acetone derivative, and it does so only under drastic conditions. Steric hindrance in the system of three contiguous cis-hydroxyl groups has been suggested as an explanation for this f a ~ t . ' ~By ~ ' substituting ~' a nitro group for the third hydroxyl group, that reasoning could be extended to explain the resistance of 100 to acetonation. On the other hand, 1,4-dideoxy-1,4-dinitro-neo-inosito1(120) does form the monoacetall53, despite the presence of an adjacent, cisnitro group. The second cis-glycol grouping does not appear to react, probably because the diacetal 154 is energetically unfavored (as it would require a boat conformation having axial attachment of the second dioxolane ring and a flagstaff position for a nitro group)."'

(101)

118

HANS H. BAER

As only a few reducing nitro sugars are known to date, experiences in direct, acid-catalyzed glycosidation have been very limited indeed. It was shownlW by paper chromatography that 6-deoxy-6-nitro-~idose (91) is converted into unequal proportions of two glycosides by refluxing it in 1% methanolic hydrogen chloride for 5 hr.

c. Hydrolysis and Methanolysis. - Free 6-deoxy-6-nitro-~-glucose (90) and -L-idose (91) have been obtained by hydrolysis of their 0-isopropylidene derivatives with sulfuric acid and appear to be stable under the hydrolytic condition^.^^ However, they are extremely prone to cyclization in alkaline mediumlo2and, to a lesser extent, even in neutral to slightly acidic mediumlo8(see Section 11,7; p. 100).This behavior is particularly noticeable with 91, so that processing that involves neutralization of the acid catalyst may be accompanied by production of deoxynitroinositols to a marked degree. Hydrolysis of isopropylidene acetals that was promoted by a cation-exchange resin likewise led to extensive cyclization of 91, whereas, for 90, this side reaction was negligible.lo8 Methanolysis of 88 and 150 afforded the methyl glycopyranosides of 90 and 91 as syrupy, anomeric mixtures.154 The D-ghco mixture was acetylated, whereupon the product crystallized, but preparation of the pure anomers proved tedious, and it is better accomplished by an alternative synthesis (see Section 11,5; p. 99). Deacetylation of methyl 2,3,4-tri-O-acetyl-6-deoxy-6-nitro-a-~glucopyranoside (94) posed a problem, because the use of alkaline conditions seemed undesirable for the reasons discussed in Section IV,2b (see p. 125); however, methanolysis of the acetyl groups in the presence of a catalytic amount of methyl p-toluenesulfonate was suc~ e s s f u l . It ~ *is believed that the sulfonate, by slowly alkylating the solvent methanol, engendered sufficient sulfonic acid to catalyze the transesterification without affecting the glycosidic bond. When free ptoluenesulfonic acid or an excess of the sulfonate was employed, the deacetylation was accelerated, but the yields of glycoside 96 declined. Free 3-deoxy-3-nitro-~-glucosewas obtained151by hydrolysis of its methyl P-D-pyranoside (40) with 5 M hydrochloric acid for 90 min at 100". These conditions appear to be somewhat more strenuous than those required for hydrolysis of ordinary hexopyranosides. On the other hand, debenzylidenation of 2-O-a~ety1,~ 2 - d e 0 ~ y , ~ *2-~ * ~ ~ * ~ acetamid0-2-deoxy~~~~ and 2-0-alky114g*156 derivatives of methyl 4,6-0(154) H. H. Baer and W. Rank, Can.]. Chem.,43,3330(1965). (155) H. H. Baer and T. Neilson,]. Org. Chem., 32,1068(1967). (156) H.H. Baer and F. Kienzle,]. Org. Chem.,32,3169(1967).

THE NITRO SUGARS

119

benzylidene - 3- deoxy - 3-nitrohexopyranosides proceeded satisfactorily on short heating in 70% acetic acid or on refluxing in aqueous methanol with a cation-exchange resin.

d. Internal Anhydridization.-Boiling of 1-deoxy-1-nitroalditols in 1% sulfuric acid results in the production of cyclic anhydrides. A pair of epimers, namely, 1-deoxy-1-nitro-D-mannitol (155)and -D-glucitol (156), gave the same product, 2,6-anhydro-l-deoxy-l-nitro-~-mannitol (157); similarly, 1-deoxy-1-nitro-D-gulitol (158) and -D-iditol (159) 2,6-anhydro-l-deoxy-l-nitro-~-gulitol (160). Apparently, the process involves carbonium-ion formation formation at (2-2, and the favored configuration arising on ring closure is that which allows the product to exist in the more favored chair conformation. CH,NO,

I

I

HCOH

or

I HFOH CQOH

YOH C%OH

(155)

p N 0 1 HCOH I HCOH I HOCH I HCOH I C&OH

HCOH I HOCH I

(156)

or

(158)

Acid is not indispensable for effecting such cyclodehydrations. In fact, anhydrides are formed even more readily by boiling a neutral, aqueous solution of a nitroalditol, and they also arise on melting, or on prolonged storage of the ~ y r u p s . ~ ~ Furthermore, ’ ~ ~ ” ~ ~ cyclodehydration has been observed to occur under alkaline conditions. It is likely that, in these instances, the reaction proceeds by way of an a-nitroolefin formed by p-elimination of water.25The ring-closing step would (157) J. C. Sowden and M. L. Oftedahl,]. Org. Chem., 26,1974 (1961). (158) J. C. Sowden, C. H. Bowers, L. Hough, and S. H. Shute, Chem. Ind. (London), 1827 (1962).

120

HANS H. BAER

then be an internal, nucleophilic alkoxylation of the nitroalkene grouping. Thus, 1-deoxy-1-nitro-D-glycero-L-gluco-heptitol (161) and its D-glycero-L-munnoepimer (162) were, by boiling in 10% aqueous solution for 24 hr, preponderantly converted into 2,Sanhydro-ldeoxy- 1-nitro-D-glllcero-L-munno-heptitol (163) (“p-0-gdactopyranosylnitromethane”). Minor products were the corresponding 2epimer and two furanoid anhydrides. The same products were obtained by sodium methoxide-catalyzed deacetylationz5 of the pentaacetate of l-nitro-l-heptene-~-gulucto-3,4,5,6,7-pento~ (164).

fim

I

CH

HCOAc I AcOCH I AcOCH I HCOAc I ChOAc (164)

(major product)

2. Base-catalyzed Reactions a. Epimerization at &Carbon Atoms. - In their investigations on the synthesis of deoxynitroinositols from the 6-deoxy-6-nitrohexoses 90 and 91 (see Section II,5 and 7; pp. 98, loo),Grosheintz and Fischerio2 realized that identical mixtures of epimeric inositols are formed from either hexose, and furthermore, that the products are interconvertible in alkaline solution. Although the configurations were not elucidated at the time, the authors correctly concluded that epimerizations can readily occur at the asymmetric centers adjacent to the nitronate carbon atom. It has since been establishedlMthat, in epimeric equilibrium, the D L - ~ U O (100) - ~ and scyllo (102) isomers exist in com-

THE NITRO SUGARS

121

parable proportions, whereas the muco-3 (101) isomer is strongly disfavored. Originally, it had been suggestedlo2that configurational randomization occurs by way of a reversed (“retro”) Henry reaction. However, there is no evidence for such a mechanism, and no wellsubstantiated analogy appears in the literature on aliphatic nitro alcohols. A retro reaction in alkaline medium would have to be initiated by ionization of an alcoholic hydroxyl group concomitant with protonation of the nitronate anion, a process that seems highly improbable in view of the large difference in their pK values:15s

It is considered more plausible that epimerization proceeds through an intermediate nitroalkene by reversible elimination of hydroxyl ion:180

a

b

C

d

e

This assumption is fully borne out by the results of investigations on the behavior of methyl 3-deoxy-3-nitroglycopyranosidesin aqueous, alkaline solution. The glycoside nitronates e ~ h i b i P ’ .“mutaro~ tation,” which was shown to be due to epimerizations at C-2 and C-4.58*61J61 Any member of a given family epimerizes, at room temperature, to the same equilibrium mixture, Thus, the pentoside nitronates 18, 19, and 20 e q ~ i l i b r a t eto~ ~ a mixture containing about 85% of 18 and 15%of 19. The P-D-hexopyranoside nitronates 165, 166, and 167 give a mixture consisting mainly of 165 and 166 (the former preponderating), but with an appreciable proportion of 167; no 168 has been f ~ u n d . ~ ‘ . In ’ ~ ~the a-D-hexopyranoside series, epimerization (159) Nitro alcohols of the type HOCH,-C(N0,)R’R” do undergo basecatalyzed cleavage (“demethylolation”), but here, the (tertiary) nitro group cannot compete in anionization. (160) 2-Methyl-l-nitro-2-propano1, the product expected from the basecatalyzed addition of nitromethaneto acetone, spontaneously loses water to give 2-methyl1-nitro-1-propene as an intermediate which, with excess nitromethane, undergoes a Michael reaction to yield 2,.%dimethyl-1,3-dinitropropane;see Ref. qa). (161) H. H. Baer and F. Kienzle, Ann., 695,192 (1966).

122

HANS H. BAER

gave6’ a mixture containing 169,170, and 171 in the ratios of approximately 4:3:1,together with a very small proportion of 172.

The rate of “mutarotation” of 165 was found161to be pH-independent in the range of pH 11 to 13 where, according to the ultraviolet spectrum (nitronate chromophore with,,,A 250 nm, E 9 X lo4),the compound exists entirely in the salt form. The rate decreases somewhat as the proportion of free nitro glycoside 41 is increased, in an acidbase equilibrium, by lowering the pH to 10. In buffered solutions at pH 9-7, where the predominant species is the free nitro compound 41 (absence of the absorption at 250 nm and a different rotation at zero time), the epimeric changes become very slow and are pHdependent, and at even lower pH there is no spontaneous epimerization. These observations are consistent16’ with the elimination mechanism already mentioned. When a solution of a is rendered alkaline (pH > lo), the nitronate b is formed almost instantaneously, and the rate-determining step in the epimerization that follows is the elimination b --* c, which is independent of the concentration of hydroxyl ion. The olefin c is converted rapidly into d, which accumulates. At lower pH, the dissociation a + b, which is pH-dependent, becomes the rate-determining process; the olefin c is still converted rapidly into d, which, however, no longer accumulates but is protonated to e. During the course of epimerization in a sufficiently alkaline medium, the nitronate absorption at A,, 250 nm remains virtually unchanged. However, on prolonged standing, the epimeric “equilibrium” mixtures undergo a slow, secondary, chemical change.l6l This change is

THE NITRO SUGARS

123

reflected in further (slow) "mutarotation" and, very strikingly, in decrease of the peak at 250 nm, with concurrent appearance of a new peak (at 296 nm) which reaches the maximum intensity (E 1.4 x 104) within about two weeks at 25" (or after 15 min at 98"), when the original peak has disappeared. The reaction product formed from (equilibrated) 166 was isolated in 70% yield, and shown to possess the allylic nitronate structure 173. An analogous compound (174) was obtained from 18. It is considered that these dehydration products are engendered by proton abstraction from (allylic) C-5 in the anitro-olefins previously presumed to be in equilibrium with the epimerized nitronates.I6' The formation of 173 and analogous, unsaturated nitronates may be taken as direct proof of the existence of such intermediate a-nitro-olefins. R

R

Several related nitro glycosides, including 4-deoxy-4-nitro-gl yculopyranosides, undergo the same kind of dehydration to allylic nitronates, but the 2,7-anhydro-4-deoxy-4-nitroheptulopyranoses (75-77) do not. Although the latter are capable of epimerization at their carbon atoms that are in the /+position to the nitro group, as evidenced by mutarotation in alkaline solution, the intermediate anitroalkene is not transformed into an allylic nitronate, because this would involve double-bond formation at a bridgehead.161 A greatly accelerated formation of an allylic nitronate is observed

124

HANS H. BAER

when methyl 2,4,6-tri-0-acetyl-3-deoxy-3-nitro-~-~-glucopyranoside is treated with aqueous alkali.'& This behavior is explained in terms of the superior leaving-group character of acetoxyl over hydroxyl, at (2-4, and by inductive assistance to proton abstraction from (2-5, rendered by the acetoxymethyl group. A methyl group at C-5 has a retarding influence.'62 Compounds of the type of 173 and 174 are stable only as salts, depicted with mesomeric anions; upon acidification, the nitronic acids liberated have a spectroscopically observable half-life of about 20 min and suffer complex decomposition.lsl The preparative utility of nitronate epimerizations has been referred to in Section I1,3 (see p. 84). It is interesting to compare the relative thermodynamic stabilities of configurations in the glycosides already mentioned. However, rationalizations in terms of steric and polar effects must await reliable determination of the conformation in which each of these compounds exists. Epimerizations are not limited to hydroxylic reaction-media. Thus, methyl 4,6-0-benzylidene-3-deoxy-3-nitro-~-~-mannopyranoside (175) is rapidly transformed into the P-D-gluCo isomer (139) by refluxing in benzene in the presence of a trace of solid potassium hydroxide;14eand, when 175 is subjected to the treatment that results in elimination of nitrous acid (see Section 111,3;p. 113;139 + 144),

it epimerizes in part to 139, too.146 Furthermore, acetylation of 175 with hot acetic anhydride and sodium acetate gives the 2-acetate of 139, whereas, in cold pyridine, the 2-acetate of 175 is produced.w These epimerizations doubtless proceed by way of the intermediate nitro-olefin 141. The latter, which is known in crystalline form, has been shown to add water14eand acetic respectively, with ease, (162) H. H. Baer and C. W.Chiu, unpublished results.

THE NITRO SUGARS

125

the substituents at C-2 and C-3 assuming the equatorial position. Interestingly, the course of sodium acetate-catalyzed acetylation of benzylidenated nitro glycosides of the type just mentioned is governed by the configuration at C-4. The P-D-gdacto (176)and CX-D-~UZO (178)isomers suffered dehydration to the corresponding olefins (177 and 179),and no 2-acetates were ~ b t a i n e d . ~ . ~ ~

b. Cleavage of p-Nitro Acetals. -The powerful activating effect of the nitro group that makes possible the P-hydroxyl eliminations discussed in the preceding Section is responsible also for alkaline fission of acetal linkages. Helferich and Hasel" reported that basecatalyzed deacetylation of 2-nitroethyl P-D-glucopyranoside tetraacetate was accompanied by fission of the glycosidic bond. Baer and Rankl5* demonstrated that glycoside cleavage by alkali can also occur when the nitro group is linked in one of the P-positions to the ring oxygen atom: the methyl glycopyranosides (180) of 6-deoxy-6nitro-D-glucose and -L-idose are cleaved by heating (20 min at 98", or 7 hr at 60") in the presence of 1equivalent of 10 mM sodium hydroxide, whereby the free nitro sugar formed cyclizes immediately to deoxynitroinositols (100and 102). Glycosides carrying a nitro group (163) B. Helferich and M.Hase, Ann., 554,261 (1943).

HANS H. BAER

126

on the ring, in @-positionto the ring oxygen atom, may be predicted to be liable to similar fission.

OMe

-

OMe

OH

OH

t

OH

OH

Nitronates of (100) and (102)

In 150 mM sodium methoxide solution, 6-deoxy-l,2;3,5-di-Oisopropylidene-6-nitro-a-~-glucofuranose (149) loses its 3,5-0-isopropylidene group quantitatively within 30 min at room temperature.lW The L-idose isomer (150) is stablela under those conditions, but reactP4 completely within 3 hr at 50". This markedly greater stability is believed to reflect a smaller steric strain in 150 (see Section IV,lb, p. 116). The reaction is depicted as a @-elimination giving the intermediary nitro-olefin (181),which undergoes rapid methoxylation to the final products, the 5-epimeric ethers 183 and 184. The intermediacy of 181 received support from a study154of alkali-catalyzed addition of methanol to its independently synthesized 3-acetate (182). In the benzylidenated nitro glucoside1g1J8J139,and especially in the nitro g a l a c t o ~ i d e ~ 176, ~ * ' ~the ~ benzylidene group likewise is susceptible to eliminative cleavage in polar solvents, and this behavior may limit its usefulness as a protecting group. Nevertheless, acetals of this type have been found to survive carefully controlled (164) (165) (166) (167)

H.0.L.Fischer and H. H. Baer, Ann., 619,53(1958). H. H. Baer, T. Neilson, and W. Rank,Can.]. Chem.,45,991(1967). H.H.Baerand K. S. Ong, Can.]. Chem., 46,2511(1968). H. H. Baer and K. S. Ong,]. Org. Chem., 34.560 (1969).

THE NITRO SUGARS

127

I

+H@ -Me,CO

(183) (184)

R = OMe, R' = H R = H, R' = OMe

(181) R = H (182) R = Ac

reactions in basic media, and have served well in various syntheses (see Section IV,2d; pp. 131-137). c. Dehydroacetylation. - By refluxing of P-nitroalkyl acetates in a dry, inert solvent (usually benzene) in the presence of sodium hydrogen carbonate, elimination of acetic acid is caused, and anitroalkenes are produced. Known as the Schmidt-Rutz this dehydroacetylation has been applied successfully to acetylated deoxynitroalditols, as reviewed3 previously. 2-Epimeric l-deoxy-lnitroalditols yield identical polyacetoxy-1-nitro-1-alkenes,as the asymmetry at the carbon atoms involved in the formation of the olefin is lost. Selective reduction of the olefinic double bond in the presence of a palladium catalyst affords a 1,2-dideoxy-l-nitroalditolacetate, which can be saponified and the product converted into the 2-deoxyaldose by the Nef r e a ~ t i o nApplications .~ of this sequence have furnished 2-deoxy-~-ribo-hexose,'~~~'~~ 2-deoxy-~-xyZo-hexose,'~~ and 2-deoxy-~-gaZacto-heptose.'~ 182, the analogous The protected, 5,6-unsaturated nitro 1,2-~yclohexylidene a ~ e t a l , ~and * ~ the protected, 6,7-unsaturated nitro sugars'O' 135 and 136 were prepared by dehydroacetylation of the corresponding p-acetates. In the last-mentioned work,lol the usual (168) M. B. Perry and A. C. Webb, Can.]. Chem., 46,789 (1968). (169) H. Paulsen,Ann.,665,166(1963).

128

HANS H. BAER

procedure with sodium hydrogen carbonate proved unsatisfactory, but the olefins were obtained (remarkably, as separable cis and truans isomers) by employing triethylamine in boiling benzene. A SchmidtRub reaction applied to methyl 2-0-acetyl-4,6-0-benzylidene-3deoxy-3-nitro-cr-~-glucopyranoside and its anomer provided methyl 4,6-0-benzylidene-2,3-dideoxy-3-nitro-a-~-er~~hro-hex-2-enopyranosideaa (132) and its a n ~ m e r l(141) ~ ~ in excellent yields. (For the isomers having the D-threo configuration, namely, 177 and 179, see Section IV,2a; p. 125). Selective hydrogenation in ethyl acetate in the presence of palladium readily produced methyl 4,6-O-benzylidene2,3-dideoxy-3-nitro-~-~-urubino-hexopyranoside~~~ from 141, and the corresponding P-D-~YXO isomeP from 177. As already mentioned (see Section 111,l;p. lll),similar hydrogenation of the 2,3-unsaturated a-Dglycosides 132 and 179 furnished the expected, saturated derivatives in lesser yields, with simultaneous formation of oximino derivatives owing to partial reduction of the nitro function. The 2,Sdideoxy3-nitro glycosides are useful intermediates for the synthesisss~1s2~170 of di- and tri-deoxy derivatives of amino sugars. Of especial interest is the extremely facile dehydroacetylation of structures that contain a nitro group flanked on both sides by acetoxyl groups. Deoxynitroinositol pentaacetates (185 and epimers) and 1,4dideoxy-l,4-dinitro-neo-inositol tetraacetate (187) readily lose three molecules of acetic acid per molecule by the action of pyridine, to form 5-nitroresorcinol diacetate (186) and 2,8dinitrophenyl acetate (188),r e ~ p e c t i v e l y . ~ ~ ~ * ~ ~ ~

AcO

OAc

(170) H. H. Baer, K. Capek, and M. C. Cook, Can.]. Chern.,47,89 (1969).

THE NITRO SUGARS

129

Although, for 185 and 187, aromatization provides an increased driving-force for the elimination process, there is a general tendency, with nitro sugars, for unsaturation and ensuing complications148to be induced by pyridine-acetic anhydride, so that this system could be employed for acetylation in only a few favorable cases.~~148,152,15*16* Baer and Kienzle'" studied the dehydroacetylation of methyl 2,4,6tri-O-acetyl-3-deoxy-3-nitro-~-~-glucopyranoside (189) obtained by boron trifluoride-catalyzed acetylation of 40. When 189 was subjected to the Schmidt-Rutz reaction with sodium hydrogen carbonate in boiling benzene, elimination of two molecules of acetic acid per molecule occurred. An extremely reactive, nonisolable diene was formed which, under the reaction conditions, underwent racemization followed by self-addition of the racemate 190 to give a crystalline, optically inactive, tricyclic, Diels-Alder product (191). By fusion with acetamide for a few minutes, a molecule of 191 lost its oxygen bridge, a molecule of nitrous acid, and the remaining methoxyl group. The structure of the crystalline, aromatic product (192), a derivative of 7-nitroisochromene, was proved by chemical degradation. When the dehydroacetylation of 189 was performed with potassium carbonate at -70 to -5", racemization at the diene stage was partially suppressed, and an optically active stereoisomer of 191 was isolated, in addition to 191. The p-~-munnoand / 3 - D - g U h C t O isomers of 189 yielded the same Diels-Alder products.

(171) H.H.Baer and F.Kienz1e.J.Org. Chem., 33,1823(1968).

HANS H. BAER

130

d. Nucleophilic Additions and Elimination- Additions. -The electron-withdrawing effect of the nitro group in a-nitroalkenes permits facile additions of nucleophiles across the olefinic bond, resulting in &substituted nitroalkanes. a-Nitroalkenes, therefore, are versatile intermediates for a great many synthetic purposes that include, among others, amination, alkoxylation, carbon-chain extension, and introduction of chain branching." It is often unnecessary for such syntheses to employ the nitro-olefin in isolated form; frequently, although not always, the olefin may be engendered from an acetylated @nitro alcohol or other suitable precursor by the action of the nucleophile, which subsequently adds itself across the double bond. In nitro sugar chemistry, the first example, based on this principle, of formation of a methyl ether was the reaction 149+ 181+ 183 184 (see p. 126).lUThis work was later supplemented by a spectroscopic and chromatographic study154of the alkaline methoxylation of the 3acetate 182 that revealed, incidentally, that addition of methanol to the olefinic double bond was considerably faster than the concurrent deacetylation at 0-3. The action of sodium methoxide on the tetraacetate of l-nitro-lhexene-~-urubino-3,4,5,6-tetrol (193) and the triacetate of l-nitro-lpentene-D-erythro-3,4,5-triol(195) gave, as preponderant products, l-deoxy-2-O-methy~-l-nitro-~-mannitol( 194)and 1-deoxy-2-0-methyl1-nitro-D-ribitol (196), re~pectively."~As illustrated for the latter example, the configuration of the favored product is that which is expected in terms of approach of the addend from the less-hindered side (Cram's rule).

+

OAc

bH

(172) J. C.Sowden, M. L. Oftedahl,and A. KirklandJ. 0%.Chem., 27.1791 (1962).

THE NITRO SUGARS

131

Preparation of 2-0-alkyl derivatives (197, R = Me, Et, or CH,Ph) has been achieved in a number of ways.185Methanol and ethanol were added by the nitro-olefin 141 at reflux temperature within two hr, even without external catalyst;173benzyl alcohol in toluene solution reacted also, although more sluggishly. A catalytic amount of sodium alkoxide promoted almost quantitative addition of alcohol within five minutes (prolonged action caused partial debenzylidenation). Excellent yields of 197 (R = M e or Et) were obtained by refluxing the 2acetate 198 in the respective alcohol in the presence of sodium acetate, and it was found possible to produce 197 by refluxing the 2hydroxy compound 139 in toluene in the presence of the alcohol and basic aluminum oxide (compare, however, the course of reaction in the absence of a nucleophile; see Section 111,3; p. 113).Under all of the conditions investigated, the 2,Sdiequatorial arrangement of substituents resulted, apparently with an extreme degree of stereoselectivity, and the same was true for most of the nucleophilic additions to be discussed subsequently; exceptions will be mentioned. It remains to be determined whether this tendency is due primarily to stability of the products or to stereoelectronic parameters for the substrate.

-

PhCH

OR

0S.N

(141)

.

(197) R = Me, Et, or CH,Ph

OR (198) R = Ac (139) R = H

,COzCHMe,

(199) R =

fb

Me \C'Me (200) R = CO,CHMe,

(173) Alcohols should, therefore, be avoided as solvents for the recrystallization of nitro-olefinic sugars. The high reactivity of 141 may, perhaps, be ascribed to an inductive effect of the acetal grouping at C-1; it is known that 3,3,3-trichloro-1nitropropene adds alcohols without the aid of a catalyst [I. Thompson, S. LOUloudes, R. Fulmer, F. Evans, and H. Burkett, J . Amer. Chem. SOC.,75, 5006 (1953)l.

HANS H. BAER

132

Another application of alkoxylation of nitro-olefins is in the synthesis of positional isomers of muramic acid.l= Addition of isopropyl L-lactate or isopropyl D-lactate to 141 led to the side-chain epimers 199 and 200, respectively, in yields of over 80%. When isopropyl DLlactate was used, 199 and 200 were obtained as a mixture in which, interestingly, the former preponderated. Removal of protecting groups, reduction, and derivation furnished various derivatives of 3-amino-2-O-[D(and ~)-l-carboxyethyl]3-deoxy-~-glucose (201). A novel synthesis14nof disaccharides was elaborated on the same (202)and principle. Thus, 2,3,4,6-tetra-O-acetyl-/3-~-glucopyranose the nitro-olefin 141 afforded the methyl /3-D-glycoside (203)of a (1 +. 2)-linked disaccharide; similarly, reaction of 202 with 132 gave the methyl a-D-glycoside (204)of the same disaccharide.

m(QI

&

R”

frH,OAc

ACO

I

OAc (202)

t

0,N (141) R‘ = OMe, R” = H (152) R’ = H, R” = OMe

A

c

V OAc

(209) R’ = OME, R” 6 H (204) R’ = H, R” = OMe

Equimolar mixtures of 139 and 141,140 and 132,and 141 and 140 produced the intermolecular anhydrides 205, 206, and 207. An optically inactive meso compound having the same structure was obtained14@ by combination of 141 with the L-enantiomer of 139. It may be recalled at this point that cyclodehydrations of deoxynitroalditols (see Section IV,ld; p. 119)have been presumedm to take place by way of intermediate nitro-olefins. Under conditions of base catalysis, the reactions would be internal, nucleophilic alkoxylations. In view of the great facility with which base-catalyzed alkoxylations of nitro-olefinic sugars tend to occur, it might be suspected that side reactions involving the formation of methylated derivatives might accompany nitromethane cyclizations of sugar “dialdehydes” in methanolic medium. There is as yet no evidence for this, but special

THE NITRO SUGARS

133

efforts at clarifying the point have not been reported. However, when a methyl 5,7-0-benzylidene-3-deoxy-3-nitro-heptoseptanosidewas allowed to stand in methanolic solution for several months at room temperature (or when the solution was refluxed for 12 hours), a crystalline 2,Cdimethyl ether was formed in good yield.87a Addition and elimination-addition reactions with ammonia have become of considerable preparative importance. Action of this base on polyacetoxy-l-nitroalkenes'34~'95~'37~138~140~141~143 or their precursors, the acetylated l-deoxy-l-nitroalditols,'9J39J"readily yields 2-amino-1,2dideoxy-1-nitroalditols which, because of 0 + N acyl migration, emerge as N-acetyl derivatives. Pairs of 2-epimers are obtained, the preponderant product being that which carries the acetamido group cis to the 3-hydroxyl group. The favored formation of 2,3-cis products may be rationalized in the same way as has been done for the synthesis of 1-deoxy-2-0-methyl-1-nitroalditols (see p. 130).As with most acyclic systems, the degree of stereoselectivity differs in different examples; for instance, it is high in the formation of 2-acetamido-l,2dideoxy-l-nitro-D-mannitol'5Band -D-gUlitOl,'ss medium for the corresponding D-talito1 derivative,lg and moderate for the D-dlitOl derivative.'"

HANS H. BAER

134

As mentioned earlier (see Section 111,2; p. 112),the acetamidonitroalditols can conveniently be transformed into amino glycoses by the Nef reaction. The sequence is illustrated by the examplelg of the nitrogalactitol 208, which afforded the D - ~ U ~ (209) O and D - g U l U C t O (210) acetamidonitroalditols in yields (after purification) of 44.5 and 23.5%; 209 and 210 were converted into 2-acetamido-2-deoxyD-talose (211) and 2-acetamido-2-deoxy-~-galactose (212), either individually, or without prior separation. In the latter instance, yields based on 208 were 55 and 3670, respectively.

t

I

:+ ywo*

HCOAc

TWJOa AcHNCH I HOCH

H OAc

I CHaOAc

I

C%OH

ywo*

+

HCNHAc I HOCH I

HOCH I HCOH CH,OH I

H

AcHNCH I

HoYH HoYH H OH ZH,OH

C=O I HCNHAc I HOCH I

HO(iH HCOH

I CH,OH

Satoh and coworkers142*174-176 have measured the optical rotatory dispersion and circular dichroism of a large number of l-deoxy-lnitroalditols, including several 2-acetamido derivatives, and have established that the signs of the Cotton effect and of the ellipticity are governed by the absolute configuration at C-2, and are independent of the substituent at that carbon atom and of the configurations at the other asymmetric centers. Baer and associates have prepared vicinal nitroamines from protected aldoses carrying a nitro group on C-3. The action of concentrated, aqueous ammonia on both the olefin 141 and the 2-acetate 198 furnished,155in high yields, methyl 2-amin0-4~6-0-benzylidene2,3-dideoxy-3-nitro-/3-~-glucopyranoside (213), along with a small proportion of the / 3 - ~ - m a n n disomer ~ ~ (214). Analogous results were obtained17*with the a-anomers of 141 and 198. When appliedlB7to the /3-D-galacto series, the same procedure caused complications, owing to a more pronounced tendency for derivatives of 176 to undergo basecatalyzed debenzylidenation (see Section IV,2b; p. 126). However, on brief fusion with a dry mixture of ammonium acetate and acetamide, the nitro-olefin 177 did yield a nitroamine. The isolated product (215) (174) C. Satoh, A. Kiyomoto, and T. Okuda, Chem. Phamt. Bull. (Tokyo), 12, 518 (1964). (175) C. Satoh and A. Kiyomoto, Carbohyd. Res., 3,248 (1966). (176) C. Satoh, A. Kiyomoto, and T. Okuda, Carbohyd. Res., 5,140 (1967).

THE NITRO SUGARS

135

carried an N-benzylidene group, obviously stemming from benzaldehyde that must have arisen by acetal cleavage of part of the primary adduct with ammonia.167The aminonitro compounds 213 (and its aanomer), 214, and 215 were converted by straightforward methods into derivatives of 2,3-diamino-2,3-dideoxy-~-glucose,~~~*~~~ -D m a n n o ~ e ,and ' ~ ~-D-galaCtOSe.167 (141) or

-

phc' ,*cs

(177)

.--C

(198)

N

Ns Major product

(213)

II

Minor product

CHPh (215)

(214)

Treatment, with ammonia, of the 1,2-O-cyclohexylidene analoglB8 of the 6-nitro hexose derivative 182,and of the 7-nitro octose derivativeio198, led to amination at C-5 and C-6, respectively. The action of ammonia on t~ans,trans-2-nitro-l,3-cyclohexanediol diacetate (216) and on deoxynitroinositol pentaacetates (185)furnished vicinal 2-nitro-1,3-diamines which were isolated as di-Nacetyl derivatives in high yields.145,17s*180 Two successive eliminationaddition processes evidently take place in these systems, as depicted for the reaction145216 ---* 217. In tetraacetoxy-l,4-dinotrocyclohexane (187),however, elimination of acetate and addition of ammonia were followed by aromatization to 2,5-dinitroaniline.ls0 OAc I

O

N (216)

O

a (217)

t

[GNozaNo2 -@] J,

(177) (178) (179) (180)

H. H. Baer and F. KienzleJ. Org. Chem.,in press. H. H. Baer and F. Rajabalee, Carbohyd. Res., in press. F. W. Lichtenthaler, P. Voss, and N. Majer, Angew. Chern., 81,221 (1969). F. Rajabalee, Ph.D. Thesis, University of Ottawa, 1969.

136

HANS H. BAER

The diamination was also a p ~ l i e d ' 7to~ methyl 2,4,6-tri-O-acety1-3deoxy-3-nitro-P-~-glucopyranoside (189) andlsl to some analogous nucleosides (see Section II,3,c; p. 87), and the diaminonitro compounds obtained were converted into 2,3,4-triacetamido-2,3,4trideoxy-P-D-glucopyranosides. The corresponding methyl a-Dglucopyranoside was prepared178from the N-acetylated a-anomer of 213 by sequential debenzylidenation, 0-acetylation, treatment with ammonia, hydrogenation, and N-acetylation. One of the intermediates, namely, methyl 2,4-diacetamido-2,3,4-trideoxy-3-nitro-a-~-glucopyranoside, served for a synthesis of a tetraamino sugar.182Mesylation of the nitro compound, followed by a displacement reaction with sodium azide, gave the 6-azido-6-deoxy derivative in which the nitro and azido groups were then simultaneously reduced to the amino stage by catalytic hydrogenation. The interaction between nitro sugars and a number of aliphatic, cycloaliphatic, and aromatic amines has been found to proceed, in principle, in the way described for ammonia, although gradual differences, connected with various basicities and, presumably, steric requirements of the amines, are observed. Sowden and associates147 studied the addition of each of seven amines to the tetraacetate of l-nitro-l-hexene-~-arubino-3,4,5,6-tetrol (193), and, for six, they isolated only one of the two adducts possible, in yields of 44-75%. The configurations were not determined. Aniline was the only base that gave both epimeric adducts (146).Addition of methylamine to the D-xylo isomer of 193, and a Nef reaction of the adduct, furnished 2-deoxy-2-(methylamino)-~-gu~ose, a component of streptothricin-like antibiotics.'= Baer and coworkers'65 obtained N-alkyl derivatives of 213 by refluxing the 2-hydroxy compound 139 in toluene in the presence of an amine (diethylamine, piperidine, or ethyl aminoacetate) and aluminum oxide; all of the products had the D-glUC0 configuration and no evidence for the formation of stereoisomers was found. Addition of the a-anomer of the aminonitro compound 213 across the double bond of the nitro-olefin 132 gave a bis(methy1 glycosid-2y1)amine having a structure analogous to 206 (with NH replacing the ether oxygen atom).'@ Lichtenthaler and coworkers'" treated 2nitro-1,3-cyclohexanediol(130,R = H)with a variety of amines, and when these were of sufficient basicity ( ~ K ~ 2 . 8 - 5ready ), l,&diamina(181) F.W.Lichtenthaler, G. Trummlik, and H. Zinke, Tetrahedron Lett., 1213 (1969). (182) H. H.Baer and M. Bayer, unpublished results. (183) Y. Ito, Y. Ohashi, and T. Miyagishima, Carbohyd. Res., 9, 125 (1969);10,468 (1969). (184) H. H. Baer and F. Rajabalee, Can.J . Chem., in press.

THE NITRO SUGARS

137

tion occurred. The products obtained with benzylamine or dimethylamine also arose when glutaraldehyde (129) was cyclized with nitromethane in the presence of those bases; it is thought'= that the process may be regarded as sequential nitromethane-dialdehyde cyclization, dehydration, and amine addition (see also, the reaction 56-61 on p. 90). Similarly, benzylamine was used18' in aminations and diaminations of 3'-deoxy-3'-nitronucleosides. Kien~le"~ discovered a remarkable behavior of the system consisting of the nitro-olefin 141 and anthranilic acid. When 141 and a two-molar excess of the amino acid were refluxed in benzene with a catalytic amount of solid potassium hydroxide, the product obtained (218) had the D-gluco configuration. Omission of the catalyst led to a mixture of 218 and the D - ~ W ~isomer O (219), and use of equimolar amounts of reactants without a catalyst gave mainly 219. The latter crystallized in a lemon-yellow form, but was colorless in solution. Both of the acids 218 and 219 gave methyl esters, 220 and 221, with diazomethane, and the D - ~ U M O ester, also, was yellow in the solid state. Addition to 141 of methyl anthranilate gave only 220, regardless of the conditions, and addition 185 of m- or p-aminobenzoic acid furnished the m- or p-amino isomers of 218, but not of 219. Similarly, anthranilic acid with the nitro-olefin 132 (the a-anomer of 141) to give a D - g h C O adduct only. The reasons for the preponderant formation of the D - W Z U ~ ~ compound O (219) under a specific set of conditions, and for its yellow color in the solid state, are not yet clear; however, the reaction has provided a practical approach to 2,3diamino-2,3-dideoxy-~-mannose, the methyl P-glycoside of which was obtained without difficulty from 219 by acid debenzylidenation, followed by catalytic hydrogenation of the nitro group with simultaneous hydrogenolysis of the carboxyphenylamino substituent.

anthranilic acid 2 moles/mole (+ KOH)

(141)

anthranilic acid 1 rnole/rnole

H N - n

C4R (218) R = H

(220) R = Me

-

(219) R H (221) R = Me

Michael reactions between nitro sugars and reactive-methylene compounds have afforded branched-chain sugars.1B8Thus, in the (185) H.H.Baer, F.Rajabalee, and F. Kienzle, J . Org. Chem., in press.

138

HANS H. BAER

presence of triethylamine, diethyl malonate reacts with the 2-acetate 198 and with the nitro-olefin 177 to give the adducts 222 and 223, respectively. Nitroalkanes as addends furnished branched-chain dinitro sugars, namely, 224 and 225. In all of these reactions, only products that had the 2,3-diequatorial arrangement of substituents were encountered. However, there is an additional possibility of epimerism in those adducts that contain an asymmetric side-chain. Such side-chain epimers have, in fact, been shown to be formed in some of these reactions.

SULFONIC ESTERS OF CARBOHYDRATES: PART II* BY

D. H. BALL AND F. W. PARRISH

Pioneering Research Laboratory, U . S . Army Natick Laboratories, Natick, Massachusetts VII. Displacement Reactions at Isolated Sulfonyloxy Groups ..................139 1. Reactions of Primary Sulfonates ...................................... 143 148 2. Reactions of Secondary Sulfonates .................................... VIII. Displacement Reactions Involving Participation ......................... 167 1. Oxide-ring Formation ............................................... 167 177 2. Anhydronucleosides ................................................ 3. Ring Contractions During Sulfonate Displacements .................... 188 4. Alkoxyl-group Participation .......................................... 193

VII. DISPLACEMENT REACTIONSAT ISOLATED SULFONYLOXY GROUPS~" The two major factors influencing reactivity in reactions at isolated sulfonyloxy groups' are the geometry of the molecule (which will be considered in the following Sections) and the nucleophilicity of the attacking group. The relative efficiencies of different sulfonates as leaving gr6upsz3 is of secondary importance, although, in some cases, yields have been improved by judicious selection. The p-tolylsulfonyloxy group is known to be somewhat more reactive than the methylsulfonyloxy group in displacement reactions,240and the p"Part I appeared in Aduan. Carbohyd. Chem., 23, 233-280 (1968). All numbers for Figures, Formulas, References, and Sections in Part I1 continue the sequence established in Part I. References common to Parts I and I1 are listed for the convenience of the reader. (238) Elimination reactions of sulfonates have been discussed in reviews by R. J. Ferrier [Aduan. Carbohyd. Chem., 20, 67 (1965);and 24, 199 (1969)l; only examples relevant to the displacement reactions considered here will be mentioned. (1) R. S. Tipson, Aduan. Carbohyd. Chem., 8, 107 (1953). (239) M. S. Morgan and L. H. Cretcher, /. Amer. Chem. Soc., 70, 375 (1948). (240) Ref. 1, p. 185. 139

140

D. H. BALL AND F. W. PARRISH

nitrobenzenesulfonate ion has been found to be superior to p-toluenesulfonate as a leaving The use of a p-bromobenzenesulfonate instead of a p-toluenesulfonate gave an improved yield in a shorter reaction time in a displacement at C-4 of a D-glUCOSe derivative.242 Limitations to the use of the better leaving-groups have been found: attempts to use the p-nitrobenzenesulfonic ester of 1,2:5,6-di-0isopropylidene-a-D-glucofuranosein a displacement reaction with ammonia resulted in extensive decomposition, and 1,2:5,6-di-Oisopropyhdene-a-D-glucofuranosewas the only product isolated.243 The use of p-bromobenzenesulfonic esters in displacement reactions can result in substitution in the aromatic ring.244The precipitate formed during displacement of a methanesulfonate with sodium iodide is a double salt, 4NaOMrNa1, and yields based on the assumption that the precipitate is sodium methanesulfonate are therefore i n c o r r e ~ t . ~ Sodium ~J p-toluenesulfonate does not form a double salt with sodium iodide. Bimolecular, nucleophilic, substitution (SN2)reactions of the type y@+R-X+YS-. *.R...X6-+Y-R+X0

are, in general, much faster in dipolar, aprotic solvents, as, in them, most anions are less solvated, and, presumably, more reactive, than in protic solvents; this is especially true for the smaller anions. Larger, polarizable, charged transition-states are generally more solvated in aprotic and this circumstance has been shown to be the factor that lowers the activation enthalpy of a Menshutkin reaction conducted in N,N-dimethylformamide instead of methanol.248These effects, and many synthetic applications of dipolar aprotic solvents, have been reviewed."' N,N-Dimethylformamide has been particularly useful as a solvent for nucleophilic displacements in carbohydrate chemistry: for (241) P. W.Austin, J. G. Buchanan, and R. M. Saunders,J . Chem. SOC. (C),372 (1967). (242) A. F.Cook and W. G. OverendJ. Chem. SOC.(C), 1549 (1966). (243) B. Coxon and L. Hough,J. Chem. SOC., 1643 (1961). (244) D. Horton, J. S. Jewell,and H. S. Prihar, Can.J. Chem., 46,1580(1968). (2.45) A. Cohen and R. S. Tipson, J . Med. Chem., 6, 822 (1963);A. C. Richardson, J . Chem. SOC., 5364 (1964). (245a)J. Miller and A. J. Parker,J.Amer.Chem. SOC., 83,117(1961). (246) P.Haberfield, A. Nudelman, A. Bloom, R. Romm, H. Ginsberg, and P. Steinherz, Chem. Commun., 194 (1968). (247) A. J. Parker, Quart. Reo. (London), 16, 163 (1962);Adoan. Org. Chem., 5, 1 (1965);see also Ref. 295.

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

141

example, with b e n ~ o a t e , ~ 4 8a ,~~i ~d ~e , ~ t~hOi o ~ y a n a t e ,thiomethox~~~ ide,252thiolacetate,293and t h i o s ~ l f a t e ~ and ~ ~a; comparative study has been made of the results obtained by use of N,N-dimethylformamide or liquid ammonia for thiolate displacements of p-tolylsulfonyloxy groups in sorbose derivatives.255Methyl sulfoxide has been used as the solvent in displacements with azideZJ6and and Nmethyl-2-pyrrolidinone has been used for benzoate displacement^.^^^ Hexamethylphosphoric triarnide has been used in azide and benzoate d i s p l a ~ e m e n t s ~these ~ ~ * were ~ ~ ~ ;achieved at lower temperatures and were accompanied by less coloration than in corresponding reactions in N,N-dimethylformamide.26’.2s2 A practical disadvantage from the use of N,N-dimethylformamide, methyl sulfoxide, and hexamethylphosphoric triamide is that isolation of the product is sometimes complicated by the necessity of removing these high-boiling solvents (especially hexamethylphosphoric triamide) and, where a facile displacement is involved (as with most primary sulfonates), the use of such lower-boiling, aprotic solvents as acetone, butanone, or acetonitrile may be advantageous. Another factor that affects the rate of a displacement reaction is the solubility of the nucleophile. Tetraalkylammonium salts have been used to increase the concentration of nucleophile for displacements at (248) E. J. Reist, L. Goodman, and B. R. Baker,]. Amer. Chem. Soc., 80,5775 (1958). (249) E. J. Reist, R. R. Spencer, and B. R. Baker,]. Org. Chem., 24,1618 (1959). (250) E. J. Reist, R. R. Spencer, B. R. Baker, and L. Goodman, Chem. Ind. (London), 1794 (1962). (251) (a) J. Hill, L. Hough, and A. C. Richardson, Proc. Chem. Soc., 346 (1963); (b) Carbohyd. Res., 8,19 (1968). (252) J. Baddiley,]. Chem. Soc., 1348 (1951). (253) T. J. Adley and L. N. Owen, Proc. Chem. Soc., 418 (1961);J . Chem. Soc. (0, 1287 (1966). (254) J. C. P. Schwarz and K. C. Yule, Proc. Chem. Soc., 417 (1961). (255) K. Tokuyama, M. Kiyokawa, and M. Katsuhara,]. Org. Chem., 30,4057 (1965). (256) S. Hanessian and T. H. Haskell,]. Org. Chem., 28,2604 (1963). (257) C. L. Stevens, G. E. Gutowski, K. G. Taylor, and C. P. Bryant, Tetrahedron Lett., 5717 (1966). (258) (a) N. A. Hughes and P. R. H. Speakman,]. Chem. Soc., 2236 (1965);(b) Carbohyd. Res., 1,341 (1966). (259) J. J. Delpuech, Tetrahedron Lett., 2111 (1965);Bull. Soc. Chim. Fr., 1624 (1966). (260) Y. Ali and A. C. Richardson, Chem. Commun., 554 (1967);I. Chem. Soc. ( C ) , 1764 (1968). (261) A. C. Richardson, personal communication (1967). (262) For a recent review of the chemistry of hexamethylphosphoric triamide, see H. Normant, Angew. Chem. 79,1029 (1967).

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D . H. BALL AND F. W. PARRISH

C-17 in steroids,263and, in the carbohydrate field, have been used in displacements of sulfonyloxy groups by benzoate2%and It has been observed that the products from a given substrate and nucleophile are, in some cases, dependent on the solvent and the temperature, although no systematic studies have as yet been reported for carbohydrate sulfonates. This dependence is especially true when substitution and elimination mechanisms are both sterically feasible. Whereas a C-17 p-tolylsulfonyloxy group in the D-ring of a steroid underwent SN2displacement with tetrabutylammonium chloride in boiling butanone, the same reactants in N-methyl-2-pyrrolidinone at 160” gave 67% of the chloro derivative and 30% of the ~ l e f i n ? ~ Participation by the “inert” solvents p-dioxane and acetone in solvolyses of 2 - 0 4 sulfonates was demonstrated by Weiner and Sneen,26sand this possibility must be considered when such solvents are used. The influence of environmental factors on rate and mechanism has been discussed by Banthorpe.266 Potassium fluoride dihydrate in methanol at 150”was found to be superior to potassium fluoride in boiling N,N-dimethylformamide for the displacement of the methylsulfonyloxy group from methyl 2,3-0isopropylidene-5-O-(methylsulfonyl)-~-ribofuranoside.~~~~~~ Small proportions of methyl ether were formed when methanol was used, but this could be avoided by use of ethylene glycol or glycerol.” In reduction of some sterically hindered, primary sulfonates, notably D-galactose derivatives, with lithium aluminum hydride the use of ether-benzene as the solvent gave the deoxy compound (presumably by way of hydride attack on the carbon atom), whereas, in tetrahydrofuran, the product was mainly the hydroxyl compound (resulting from 0 - S c l e a ~ a g e ) . ’ For ~~,~ 2,3:4,5-di-0-isopropylidene-l-O-p-tolylsul~~ fOnyl-D-fruCtOpyranOSe, the hydroxyl compound is formed, even in ether.2 (263) H. B. Henbest and W. R. Jackson,/. Chem. SOC., 954 (1962). (264) K. W. Buck, A. B. Foster, R. Hems, and J. M. Webber, Carbohyd. Res., 3, 137 (1966);A. B. Foster, R. Hems, and J. M. Webber, ibid., 5,292 (1967). (265) H. Weiner and R. A. Sneen,/. Amer. Chem. SOC.,87,287 (1965). (266) D. V. Banthorpe, “Reaction Mechanisms in Organic Chemistry,” Elsevier Publishing Co., New York, N.Y., 1963, Vol. 2. (267) N. F. Taylor and P. W. Kent,/. Chem. Soc., 872 (1958). (268) H. M. Kissman and M. J. Weiss,/. Amer. Chem. SOC., 80,5559 (1958). (194) J. H. Westwood, R. C. Chalk, D. H. Ball, and L. Long, Jr., I. Org. Chem., 32, 1643 (1967). (269) See Part I, p. 270. (2) H. Schmid and P. Karrer, Helo. Chim. Acta, 32, 1371 (1949).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

143

1. Reactions of Primary Sulfonates The well known difference between galactose-6-sulfonates and most other primary sulfonates in reactivity toward n u c l e o p h i l e ~ ~ ~ ~ has received further attention. The resistance of 1,2:3,4-di-O-isopropy~idene-6-O-sulfony~-~-ga~actoses toward displacements, initially observed with iodide, has also been found with f l ~ o r i d e , meth~~~'~~~ oxide,la4and t h i ~ l a c e t a t e . ~However, ' ~ , ~ ~ ~ 1,2:3,4-di-O-isopropylidene6-0-p-tolylsulfonyl-D-galactose (9, R = Ts) reacts with potassium Me

Me

Me

Me

(9)

thiolacetate in N,N-dimethylformamide at 100" to give the 6-S-acetyl derivative,z73and with sodium azide in N,N-dimethylformamide at '~ in reactivity to 120" to give the 6-azido d e r i v a t i ~ e . ~Differences iodide displacement have also been observed between 1,2,3,4tetra-0-acetyl-6-0-p-tolylsulfonyl-aand -p-D-galactopyranose and the corresponding D-glucose or 2-amino-2-deoxy-D-glucosederivat i v e ~It. was ~~~ observed that, for each pair of anomers, the a-Dacetate reacted slightly faster than the p; and the retarding influence of a P-substituent at C-1 was further demonstrated with a series of p-Dglucosides. These results are contrary to a previous suggestion.z75 A low reactivity toward iodide displacement has also been found with the 1-0-(phenylsulfonyl) derivatives of 2,4:3,5-di-O-rnethyleneDL-Xylitol (10) and 2,4:3,5-di-0-benzylidene-~-xylitol~~~ (11). The

(270) (271) (272) (273) (274) (275) (276)

Ref. 1, p. 181 N. F. Taylor, Nature, 182, 660 (1958). M . Akagi, S. Tejima, and M. Haga, Chem.Pharm. Bull. (Tokyo), 11,559 (1963). J . M. Cox and L. N. Owen,]. Chem. SOC. (C), 1121 (1967). W. A. Szarek and J. K. N. Jones, Can.]. Chem. 43,2345 (1965). Ref. 1, p. 185. J. M. Sugihara and W. J. Teedink,]. Org. Chem., 29,550 (1964).

144

D. H. BALL AND F. W. PARRISH R

R

(10) R = H, L form (11) R = Ph

sulfonyloxy groups in these compounds and in 9 are in similar steric environments.277Methyl 2,3,4-tri-0-methyl-6-o-~-tolylsulfonyl-c-Dgalactopyranoside reacts with sodium iodide in acetone or N,Ndimethylformamide at twice the rate of 9 (R = Ts), and 1,2:3,4di-O-methylene-6-O-p-to~ylsulfony~-D-ga~actopyranose shows an intermediate reactivity.278Replacement of the 3,4-O-isopropylidene group of 9 (R = Ts) with an 0-ethylidene group (stereochemistry not defined) did not affect the rate of reaction. The small variation in rates found with these compounds indicates that the size of the substituent attached to 0-4is of secondary importance. As displacements of sulfonyloxy groups from 9 seem to proceed under milder conditions with electrically neutral nucleophiles (for example, ammonia or hydrazine), it was concluded that the rate is very sensitive to the presence or absence of charge on the n ~ c l e o p h i l eThis . ~ ~ ~conclusion led to the explanation that the lower reactivity of galactose derivatives was due to “an electronic field effect of the lone pairs of electrons of the ring oxygen and the axial C-4 oxygen, which tend to repel negatively charged nucleophiles approaching the rear side of the sulfonate ester group in its most stable conformation.” It seems likely, however, that the most stable conformation of the sulfonate and the direction of approach of the nucleophile may have little relevance to the transition-state energy in displacements of primary sulfonates. The higher energy of the transition state may well be due to unfavorable, dipole interactions during SN2displacements with compounds having the galacto type of stereochemistry. Inspection of models indicates that, for SN2displacements at C-6 of hexopyranoses, it is possible for the transition state to adopt a conformation in which the dipoles due (277) Although it has been shown by C. Cone and L. Hough [Carbohyd. Res., 1, 1 (1965)l that the conformation of 9 is more accurately described as the skew S: depicted (than as a chair form), the steric and electronic environments at C-6 are not greatly affected by this modification. (278) S. Nadkarni and N. R. Williams, J . Chem. SOC., 3496 (1965).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

145

to the incoming and departing groups are perpendicular to the dipole due to the ring-oxygen atom, which approximately bisects the C-1-0-C-5 angle. However, with anionic nucleophiles, such a conformation would give rise to very unfavorable interactions with an axial oxygen atom on C-4 (galacto stereochemistry), but not with an equatorial C-4-oxygen bond (gluco stereochemistry). Conformations that minimize this interaction necessitate unfavorable dipolar interactions due to the ring-oxygen atom. With neutral nucleophiles, the polarity of the new bond in the transition state is reversed, leading to an attracting interaction and a lower energy. In general, the difference in reactivity between primary and secondary sulfonates permits selective displacement of the former. For example, treatment of 3-O-acetyl-1,2-O-isopropylidene-5,6-di-O-ptolylsulfonyl-D-glucofuranosewith potassium thiolacetate in boiling acetone gave2793-O-acetyl-6-S-acetyl-1,2-O-isopropylidene-6-thio-50-p-tolylsulfonyl-a-D-glucofuranose in 91% yield; and the same compound with sodium benzoate in N,N-dimethylformamide at 95-100" gave a 64 % yield of 3-0-acetyl-6-0-benzoyl-1,2-0-isopropylidene-5O-p-tolylsulfonyl-D-glucofuranose.280 (At the boiling point of the solvent, replacement of the secondary p-tolylsulfonyloxy group also occurred, to give a 5,6-di-O-benzoyl-~-idosederivative.) Methyl 2,6di-O-p-tolylsulfonyl-a-D-galactopyranosidereacts with sodium azide in methyl sulfoxide or N,N-dimethylformamide at 100" to give a good yield of methyl 6-azido-6-deoxy-2-O-p-tolylsulfonyl-a-D-galactopyranoside,281and methyl 2,3,5-tri-O-p-tolylsulfonyl-/3-~-ribofuranoside can be converted, in 90% yield, into methyl 5-azidod-deoxy2,3-di-O-p-tolylsulfonyl-/3-~-ribofuranoside.~~~ Selective displacement of the primary methylsulfonyloxy group of methyl 2,6-di-O-(methylsulfony1)-a-D-glucopyranoside was readily effected with thiosulfate

Methyl 2,3-di-O-benzyl-4,6-di-O-(methylsulfonyl)-cy-~-glucopyranoside gave the corresponding 6-deoxy-6-iodo derivative in 85% yield on treatment with sodium iodide in b u t a n ~ n eand , ~ ~selective ~ displacements of the 6-methylsulfonyloxy group in this compound A. M. Creighton and L. N. Owen,]. Chem. Soc., 1024 (1960). D. H. Buss, L. D. Hall, and L. Hough,]. Chem. Soc., 1616 (1965). S. Hanessian and T. H. Haskell,]. Org. Chem. 30,1080 (1965). J . Hildesheim, J. ClBophax, S. D. GBro, and R. D. Guthrie, Tetrahedron Lett., 5013 (1967). (283) D. H. Ball, R. C. Chalk, and L. Long, Jr., unpublished observations. (284) C. L. Stevens, P. Blumbergs, and D. H. Otterbach, J . Org. Chem., 31, 2817 (1966); C. L. Stevens, P. Blumbergs, D. H. Otterbach, J. L. Sbominger, M. Matsuhashi, and D. N. Dietzler,]. Amer. Chem. SOC.,86,2937 (1964).

(279) (280) (281) (282)

146

D. H. BALL AND F. W. PARRISH

have also been achieved with azideZs5and t h i o ~ y a n a t e . ~With ~~'~) methyl 2,3-di-0-benzoyl-4,6-di-O-p-tolylsulfonyl-a-~-galactopyranoside, Stevens and coworkers were unable to effect a selective iodide displacement at C-6, and they suggested that the reactivities at C-4 and C-6 of D-galactopyranose should be of comparable magnitude.z8s Treatment of methyl 2,4-di-O-methyl-3,6-di-O-(methylsulfonyl)P-D-galactopyranoside with boiling M sodium hydroxide gave methyl 3,6-anhydro-2,4-di-O-methyl-P-~-galactopyranoside in 39% yield, suggesting that the 3-O-(methylsulfonyl) group is saponified at least as rapidly as thatz4at C-6 (but the possibility of a concerted reaction cannot be excluded, see p. 173). The resistance to iodide displacement of 1-0-sulfonyl derivatives of D-fructose and D-sorbose is well known,z87and selective displacement at C-6 of 2,3-O-isopropylidene-l76-di-O-p-tolylsulfonyl-~-~ctofuranose by sodium a-toluenethioxide in methanol gave 6-S-benzyl2,3-O-isopropylidene-6-thio-l-O-p-tolylsulfonyl-~-fructofuranose in 74% yield.z16 Primary sulfonyloxy groups have been displaced by methoxide ion in derivatives Of D-glUCOSeeoand D-arabinose,288by sulfite ion in 1,2-0isopropylidene-6-O-p-to~ylsulfonyl-~-g~ucofuranose,2*~ and by thiosulfate i ~ n . ~ The ~ ~reaction , ~ ~ of. ~1,2-O-isopropylidene-5-O-p~ tolylsu~fonyl-a-D-xylofuranose with sodium thiosulfate in aqueous N,N-dimethylformamide led to the first example of a pyranoid sugar containing sulfur in the ring,z54Other workersz53independently obtained the same compound by way of a thiolacetate displacement; and t h i o ~ y a n a t eand ~ ~ thiolbenzoatezsl ~ displacements were also effected with this compound. Displacements of primary p-tolylsulfonyloxy groups by thiolacetate have also been used to prepare 5-thio-Dribopyranose,z92 6 - t h i o - D - g ~ u ~ o 2-amino-2-deoxy-6-thio-~se~~~~~~~~~~~ (285) J. Hill, L. Hough, and A. C. Richardson, Carbohyd. Res., 8.7 (1968). (2.86) C. L. Stevens, P. Blumbergs, F. A. Daniher, D. H. Otterbach, and K. G. Taylor, J . Org. Chem., 31,2822 (1966).and preliminary accounts cited therein. (24) R. C. Chalk, D. H. Ball, and L. Long, Jr.,J. Org. Chem., 31, 1509 (1966). (287) Ref. 1, p. 190. (216) M. S. Feather and R. L. Whistler, J . Org. Chem., 28, 1567 (1963). (90) A. K. Mitra, D. H. Ball, and L. Long, Jr., J . Org. Chem., 27, 160 (1962). (288) S. C. Williams and J. K. N. Jones, Can. J . Chem., 43,3440 (1965). (289) M. Miyano and A. A. Benson, J . Amer. Chem. SOC., 84,59 (1962). (290) L. Lifland and E. Pacsu, Textile Res. J . , 32, 170 (1962). (291) R. L. Whistler, M. S . Feather, and D. L. Ingles,J. Amer. Chem. SOC., 84, 122 (1962);D. L. Ingles and R. L. Whistler, J . Org. Chem., 27,3896 (1962). (292) C. J. Clayton and N. A. Hughes, Chem. Znd. (London), 1795 (1962);Carbohyd.

Res., 4, 32 (1967). (293) M. Akagi, S. Tejima, and M. Haga, Chem. Pharm. Bull. (Tokyo), 10,562 (1962).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

147

glucose,294and 6-thio-~-galactose~~~ derivatives; and the displacement of sulfonic ester groups by thiolacetate has been discussed, and several other examples given, by Horton and H u t s ~ n It. ~was ~ ~shown that sodium thiolbenzoate in liquid ammonia is an effective reagent for displacement of hindered sulfonyloxy groups in D-fructose and Dsorbose derivatives.255 Azide displacement of primary sulfonyloxy groups, first reported by Cramer and coworkers in the synthesis of 6-amino-6-deoxy-~-gluCOS~ has , ~been ~ utilized in the preparation of several aminodeoxy several 5sugars. These include 6-amino-6-deoxy-~-galactose,2~~ amino-5-deoxypentose d e r i v a t i ~ e s , ~ 2,6-diamino-2,6-dideoxy-~~~*~~~ m a n n o ~ e ,2,6-diamino-2,6-dideoxy-~-allose,~~ ~~ 3,6-diamino-3,6-dideoxy-D-idose,281 2,3,4,6-tetraamino-2,3,4,6-tetradeoxy-~-ga1actose and -D-idose,260and aminodeoxy derivatives of maltose, sucrose, and t r e h a l ~ s e .In ~ ~view of the results of Lemieux and Barrette,70 it is used by probable that the penta-0-acetyltri-0-p-tolylsulfonylsucrose Umezawa and was that isomer having the p-tolylsulfonyl groups on 0-l‘,0-4, and 0-6’ (not 0-1’,0-6, and 0-6’);ifthis is so, the azide displacement described would afford 2,3,6-tri-O-acetyl-4-azido-

4-deoxy-a-~-galactopyranosyl3,4-di-O-acetyl-1,6-diazido-1,6-dideoxyp-D-fructofuranoside. Further work is needed, in order to clarify the structures of this triazidotrideoxy compound and its transformation products. The use of ammonia or hydrazine to introduce a nitrogenous function by way of displacement of a sulfonyloxy group has disadvantages, owing to the possibility that secondary amines might be formed.297 However, these neutral nucleophiles have advantages in displacements of hindered, secondary sulfonates (see p. 148); and they have been usedzs8 for some primary sulfonates, although azide displacements are here preferable. Although most reactions of 2‘,3’-O-isopropylidene-5’-O-p-tolylsulfonyladenosine (12, R = R’ = Me) involve participation by the basic (294) W. Meyer zu Reckendorf and W. A. Bonner, J . Org. Chem., 26,5241. (1961). (295) D. Horton and D. H. Hutson, Adoun. Carbohyd. Chem., 18, 123 (1963);see p. 168. (52) F. Cramer, H. Otterbach, and H. Springmann, Chem. Ber., 92,384 (1959). (59) M. L. Wolfrom, P. Chakravarty, and D. Horton, J . 0%.Chem., 30,2728 (1965). (296) P. H. Gross, K. Brendel, and H. K. Zimmennan, Jr., Ann., 683, 179 (1965). (64) S. Umezawa, T. Tsuchiya, S. Nakada, and K. Tatsuta, Bull. Chem. SOC./ u p . , 40, 395 (1967). (70) R. U. Lemieux and J. P. Barrette, J . Amer. Chem. Soc., 80,2243 (1958). (297) Ref. 1, p. 175. (298) M. L. Wolfrom, F. Shafizadeh, R. K. Annstrong, and T. M. Shen Han, J . Amer. Chem. SOC., 81, 3716 (1959).

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D. H. BALL AND F. W. PARRISH

aglycon (see p. 185), treatment of 12 (R = R' = Me) with potassium tert-butoxide in tert-butanol resulted in elimination, and the fonnation of 9-(5-deoxy-2,3-0-isopropylidene-~-~-erythro-pent-4-enofuranosy1)adenine (13, R = R' = Me).299This reaction was also successful with the corresponding 2',3'-0-(ethoxymethylidene)derivative (12, R = H, R' = OEt), and the product (13, R = H, R' = OEt), unlike the 0-isopropylidene derivative, could be converted by dilute acid into 9-(5-deoxy-~-~-erythro-pent-4-enofuranosyl)adenine.

where B = adenin-9-yl.

2. Reactions of Secondary Sulfonates

a. &-Fused Ring Systems.-Many of the examples of isolated secondary sulfonates occur in compounds of this type, and the classical example is 1,2:5,6-di-O-isopropylidene-3-O-p-tolylsulfonyl-~D-glUCOfuranOSe (14). That the products from the reaction of 14 with ammonia or hydrazine'O were D-allOSe derivatives (15 or 16) was suspected by Cope and Shen,sooand first proved by Lemieux and Chu.loa Hydrazinolysis of 14 gives a 60 % yield of 3-deoxy-3-hydrazino-l,2:5,6di-0-isopropylidene-a-D-allofuranose(16), which is readily hydrogenated to the amino derivative (15) by use of Raney nickel catalyst.10a*301 Ammonolysis of 14 gives 15 directly, but in much lower yield.302Partial hydrolysis of the N-acetyl derivative 17, followed by N-acetylation, gave a crystalline 3-acetamido-3-deoxy-1,2-0-isopropylidene-D-hexofuranose. This was oxidized with periodate, the product was reduced, and the reduction product was acetylated. Hydrolysis of the crystalline acetate with M hydrochloric acid at 100" gave 3amino-3-deoxy-~-ribose hydrochloride, confirming the D-UZZO configurationloaof 16. (299) J. R. McCarthy, Jr., M. J. Robins, and R. K. Robins, Chem. Commun., 536 (1967). (10) K.Freudenberg and F. Brauns, Ber., 55,3233(1922). (300) A. C. Cope and T. Y. ShenJ. Amer. Chem. SOC., 78,3177(1956). (10a) R. U.Lemieux and P. Chu,J. Amer. Chem. SOC., 80,4745(1958). (301) M. L.Wolfrom, F. Shafizadeh, and R. K. Armstrong,]. Amer. Chem. SOC., 80, 4885 (1958). (302) K.Freudenberg,0.Burkhart, and E. Braun, Ber., 59,714(1926).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

149

I ‘ (15) R = H (16) R = NH, (17) R = COCH,

The p-toluenesulfonic ester 14 is an example of an exo-sulfonate in a cis-fused, five-membered ring-system, a system which also occurs in some dianhydrohexitols. Although they did not identify the products, Matheson and Angyallp3 compared the reactivity of the three 1,4:3,6-dianyhdro-2,5-di-O-p-tolylsulfonylhexitols18, 19, and 20

o-mam (18)

L

-id0

(20)

(R = R’ = OTs) to iodide displacement. They showed that, for the manno no isomer (18, R = R’ = OTs), both p-tolylsulfonyloxy groups were displaced; for the D-ghco isomer (19, R = R’ = OTs), only one p-tolylsulfonyloxy group was displaced; and for the ~ - i d oisomer (20, R = R’ = OTs), both groups were unreactive. Mills303rationalized these results, and Cope and Shen300examined the reactions of the three di-p-toluenesulfonic esters with tetraethylammonium acetate ~ C J (18, in acetone, and identified the products. The D - ~ U ~ isomer R = R’ = OTs), containing two endo-p-tolylsulfonyloxy groups, readily afforded a diacetate shown to be the L-id0 isomer (20, R = R’ = OAc). Under the same conditions, the endo-p-tolylsulfonyloxy group on C-5 of the D-gZUCO isomer (19, R = R’ = OTs) was displaced, to give the acetoxy p-toluenesulfonate (20, R = OTs, R’ = OAc); the ~ - i d odi-ptoluenesulfonate (20, R = R’ = OTs), containing two em-p-tolylsulfonyloxy groups, was unreactive. Evidently, under these conditions, endo, but not exo, p-tolylsulfonyloxy groups undergo SN2 displacement. Access to the rear of the carbon atom bearing an exo-p-tolylsulfonyloxy group is hindered by the ring system, whereas the (163) N. K. Matheson and S. J. Angyal,]. Chem. Soc., 1133 (1952). (303) J. A. Mills, Adoan. Carbohyd. Chem., 10,1 (1955),c$ p. 46.

D . H. BALL A N D F. W. PARRISH

150

approach of a nucleophile to the rear of an endo-p-toluenesulfonate is relatively ~ n i m p e d e d . ~ ~ It was established3"0 that the reactions of the three di-p-toluenesulfonates with a m m ~ n i a ~and ~ * amines ~ ' ~ also occur with inversion and, in these reactions with uncharged nucleophiles, endo and exo sulfonyloxy groups can both be displaced. With dimethylamine, selective displacement of the endo-p-tolylsulfonyloxy group of the D-gluco isomer (19, R = R' = OTs) could be effected, both groups being displaced under forcing conditions. From the ~ - i d oisomer (20, R = R' = OTs), a tricyclic imine (21) was 0btained.3'~ After displacement of one of the exo-p-tolylsulfonyloxy groups by ammonia, the second undergoes intramolecular displacement. The D - ~ U ~ di-p-toluenesulfonate (18, R = R' = OTs) reacts30swith hydroxide, alkoxide, or hydride ions to give the ~-rnunnodianhydride (18, R = R' = OH). Thus, even when steric hindrance is relatively unimportant, these strongly basic nucleophiles cause 0-S cleavage (SN2Sreactions).

~ C I

The endo-methylsulfonyloxy groups of 1,4:3,6-dianhydro-2,5-di-O(methylsulfony1)-D-mannitol(18, R = R' = OMS) are readily displaced by sodium benzoate in N,N-dimethylformamide to give an L-iditol and by potassium thiolacetate in ethanol3'' at 115". As suggested by Cope and Shen,3"Othe thiolacetate formed very probably has ~ - i d stereochemistry o (20, R = R' = SAC),and is not the D - T T Z U ~ ~isomer O originally proposed.

(22)

(23)

(304) R. Montgomery and L. F. Wiggins,]. Chem. SOC., 393 (1946). (305) V. G. Bashford and L. F. Wiggins,]. Chem. SOC., 371 (1950). (306) A. C. Cope and T. Y. Shen,]. Amer. Chem. SOC., 78,5912 (1956). (307) P. Bladonand L. N. Owen,]. Chem. SOC., 585 (1950).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

151

In contrast with the reluctance of 1,2:5,6-di-O-isopropylidene-3-0p-to~y~su~fonyl-cu-D-glucofuranose (14) to undergo displacement reactions, the isomeric 1,2:5,6-di-O-isopropylidene-3-O-p-tolylsulfonyl [or (methylsulfonyl)]-D-allofuranose (22) (containing an endo-sulfonyloxy group) reacts readily with tetrabutylammonium fluoride in acetonitrile,2w and with sodium benzoate308or sodium azide308-s10in N,N-dimethylformamide. In each reaction, s N 2 displacement gave good yields of the corresponding D-glUCOSe derivatives. The endo-ptolylsulfonyloxy group of 1,2:5,6-di-O-isopropylidene-3-O-p-tolylsulfOnyl-a-D-gUlOfuranOSe (23)is also readily displaced by benzoate,311 azide,311or fluoride,312to give D-galaCtOSe derivatives, although, with (strongly basic) tetrabutylammonium fluoride in acetonitrile, elimination between C-3and C-4 competes with the s N 2 displacement. This elimination reaction is probably facilitated by the trans relationship between the sulfonyloxy group and H-4, as it was not observed in a supposedly similar reaction with the D-allOSe derivative 22.

cis-Fused, five-membered ring-systems also occur in the 1,2-0isopropylidenepentofranoses 24, 25, and 26. The reactions of these compounds (R = OTs) with tetrabutylammonium benzoate in Nmethyl-2-pyrrolidinone were studied.258'b'In all of these reactions, the primary p-tolylsulfonyloxy groups were readily displaced, and, for the ~ - r i b oisomer (24), the endo-sulfonate group underwent much slower displacement, to give 3,5-di-0-benzoyl-1,2-O-isopropylidenea-D-xyloluranose. The lower rate of displacement of the endo-p-

(308) D. T. Williams and J. K. N. Jones, Can.]. Chem., 45,7 (1967). (309) W. Meyer zu Reckendorf,Angew. Chem., 18,1023 (1966). (310) J. S. Brimacornbe, J . G . H. Bryan, A. Husain, M. Stacey, and M. S. Tolley, Carbohyd. Res., 3, 318 (1967). (311) J. S. Brimacornbe,P. A. Gent, and M. Stacey,]. Chem. SOC. (C), 567 (1968). (312) J. S. Brimacombe, A. B. Foster, R. Hems, and L. D. Hall, Carbohyd. Res., 8, 249 (1968).

152

D. H. BALL AND F. W. PARRISH

tolylsulfonyloxy group of 24 (R = OBz) compared with that of the endo-sulfonyloxy groups of 18 (R = R’= OTs) was attributed to steric hindrance by the trans group on (2-4. It may also be significant that the 1,Sdioxolane ring of these isopropylidenepentofuranoses contains one ring-oxygen atom more than the dianhydrohexitol systems mentioned, and it would be of interest to examine the rates of displacement of sulfonyloxy groups in 1,2-O-isopropylidene-3-0sulfonylerythrofuranoses. It was also found2sB‘b’that 5-deoxy-1,2-O-isopropylidene-3-O-ptolylsulfonyl-a-D-ribofuranose(24,R = H) undergoes displacement at a rate comparable with that of the 5-benzoate (24,R =OBz), indicating the absence of neighboring-group participation. The exo-p-tolylsulfonyloxy groups in the ~-arabinoand D-X&J isomers could not be displaced under these conditions. Wolfrom and coworkersm8were, however, able to displace the exo-p-tolylsulfonyloxy group of the DxyZo isomer (26,R = OTS) by using hydrazine, a neutral nucleophile, although the yield (after hydrogenolysis) of the diamino-D-ribose derivative was low. It has been shown that the exo-sulfonyloxy group of 1,2-0-isopropy~idene-3-O-p-to~y~su~fony~-a-D-xy~ofuranose (26, R = OH) can be displaced by potassium thiocyanate in N,N-dimethylf ~ r m a m i d e .This ~ ~ ~ displacement could not be effected with the 5-deoxy derivative (26,R = H), and the authors suggested that a hydrogen bond between the 5-hydroxyl group and a sulfonate oxygen atom would facilitate development of a negative charge on the sulfonate in the transition state. Another example of a cis-fused, five-membered ring-system occurs in 3,6-anhydrohexofuranose derivatives, and 3,6-anhydro-1,2-0isopropylidene-5-O-.p-tolylsulfonyl-a-~glucofuranose (27) contains an endo-sulfonyloxy group which can readily be displaced by sodium azide in N,N-dimethylformamide, and by hydrazine314 or sodium benzoate in N,N-dimethylf0rmamide,3~~ to give L-idose derivatives. Hydrazine has proved effective in displacing sulfonyloxy groups in other cis-fused ring-systems. The p-tolylsulfonyloxy group in methyl 3,4-0-isopropylidene-2-O-p-tolylsulfonyl-~-~-arabinopyranoside (28)can be considered to be em, and treatment of 28 with hydrazine, followed by hydrogenolysis and treatment with salicylaldehyde, gave a fair yield of methyl 2-deoxy-3,4-0-isopropylidene-2-(salicylideneamino)-/3-D-ribopyranoside.29B The 4-methylsulfonyloxy group in (313) J. Defaye and J. Hildesheim, Carbohyd.Res., 4,145 (1967). (314) M. L. Wolfrom, J. Bernsmann, and D. HortonJ. Org. Chem.,27,4505 (1962). (315) J. G. Buchanan and J. Conn, J. Chem. SOC., 201 (1965).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

153

b?m0: d e ,

O'CMe,

MsO Y

O

M

e

Me&/

OTs (29)

(30)R = H, R ' = OMe (31) R = OMe, R ' = H

methyl 2,3-O-isopropylidene-4-0-(methylsulfonyl)-a-~-rhamnoside (29) undergoes SN2 displacement by hydrazine to give, after hydrogenation, a 27 % yield of methyl 4-amino-4,6-dideoxy-2,3-0-isopropylidene-a-~-talopyranoside.~~~ Attempted displacements with azide give ring-contracted products mainly (see p. 190).Methyl 3,5-0isopropylidene-2-O-p-to~y~sulfonyl-c~-~-xylo~ranoside (30) was converted, in about 30 % yield, into a 2-amino-2-deoxy-D-lyxosederivative by boiling with hydrazine followed by reduction, but most of the p-D anomer (31) was recovered ~ n c h a n g e d . ~The ~ ' exo-p-tolylsulfonyloxy group of the a-Danomer (30)can be displaced by hydrazine (but probably not by a charged nucleophile), but, for the p-Danomer (31),the additional hindrance of the methoxyl group prevents nucleophilic attack at C-2.

b. Bridged-ring Systems.- In the bicyclo[3.2.1] system of methyl 3,6-anhydro-2,4-di-O-p-tolylsulfonyl-a-~-glucopyranoside (32), neither sulfonyloxy group could be displaced by hydrazine, and the lack of reactivity was attributed to steric and electronic interactions between the axially attached oxygen atom at C-3 and the incoming (316) J. Jary, P.No&, Z. Ksandr, and Z . Samek, Chem. Znd.(London), 1490 (1967). (317) D. Horton, M.L. Wolfrom, and A. Thompson,J. Org. Chem., 26, 5069 (1961).

154

D. H. BALL AND F. W. PARRISH

ro

n u ~ l e o p h i l e That . ~ ~ hindrance to the approach of a nucleophile is not the only factor to be considered is evident from recent results of Horton and coworkers with related bicyclo[3.2.l] systems which also contained cis-fused isopropylidene rings.318They showed that the four isomeric 1,6-anhydro-O-isopropylidene-O-p-tolylsulfonylhexopyranoses 33-36 are all resistant to displacement by sodium azide in boiling N,N-dimethylformamide. The lack of reaction would, perhaps, be expected for 1,6-anhydro-2,3-0-isopropylidene-4-O-p-tolylsulfonyl-p-D-mannopyranose (33)and 1,6-anhydro-3,4-0-isopropylidene20-p-tOlylSUlfOnyl-p-D-g~aCtOpyranOSe (M), as approach of the nucleophile is strongly hindered in these compounds. However, for 1,6-anhydro-2,3-0-isopropylidene-4-O-ptolylsulfonyl-P-D-talopyranose (35)and 1,6-anhydro-3,4-0-isopropylidene-2-O-p-tolylsulfonylfl-D-talopyranose (36),no groups impede the approach of a nucleophile, and the lack of reactivity was attributed to the inability of these rigid structures to attain SN2transition states having five atoms around one carbon atom318(see also, p. 162).

(33) R = H,R' = OTs (35) R = OTs, R ' = H

(34) R = H,R ' = OTS (36) R = OT8, R ' = H

(65) M. L. Wolfrom, Y.-L. Hung, P. Chakravarty, G. U. Yuen, and D. Horton,]. Org. Chem., 31,2227 (1966). (318) A. K. Chattejee, D. Horton, and J. S. Jewell, Carbohyd.Res., 7,212 (1968).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

155

The reaction of the methylsulfonyl analog of 34 with potassium fluoride dihydrate in methanol at 13W, previously believed to give 1,6-anhydro-3,4-0-isopropylidene-2-O-methyl-~-~-talopyranose,~~~ has now been shown to involve participation by the oxygen atom of the anhydro ring320(compare, p. 197). c. Exocyclic Sulfonyloxy Groups.-The first example of the displacement of a secondary sulfonyloxy group by benzoate ion was achieved by B. R. Baker and coworkers, who obtained methyl 5-0benzoyl-6-deoxy-2,3-O-isopropylidene-cY-noside (38)in 77% yield from the reaction of methyl 6-deoxy-2,3-0-isopropylidene-5-0p-tolylsulfonyl-p-D-allofuranoside(37) with sodium benzoate in boiling N,N-dimethylformamide.za Under more basic conditions (potassium tert-butoxide in boiling tert-butanol), a concerted elimination (E2)reaction occurred, to give the olefin 39 in 40% yield, together with smaller proportions of the 5-hydroxy compound and the terminal ~ l e f i n The . ~ ~ probable ~ identity of the propenyl ether 39 with the olefin (previously thought to be the 5,6-ene) obtained by the action

(319) P. W. Kent, D. W. A. Farmer, and N. F. Taylor, Proc. Chem. Soc., 187 (1959). (320) N. A. Hughes, Chem. Commun., 1072 (1967). (321) H. Anoumanian, E. M. Acton, and L. Goodman, J . Amer. Chem. Soc., 86, 74 (1964).

156

D . H. BALL AND F. W. PARRISH

of aqueous methanolic potassium hydroxide on methyl 6-deoxy-2,3-0isopropylidene-5-O-p-tolylsulfonyl-a-~-mannofuranoside~~~ (40) was also demonstrated. In the related D-glucofuranose derivative, namely, 3-0-benzyl-1,2O-isopropylidene-5-O-p-to~y~su~fony~-6-O-tri~~-a-D-g~ucofuranose, displacement with hydrazine occurred, to give a 75% yield of 3-0benzyl-5-deoxy-5-hydrazino-1,2-0-isopropylidene-~-~-idofuranose.~~~ Under conditions milder than those used for compounds 37 and 40, base-catalyzed elimination of the 5-sulfonyloxy group from compounds in this series (for example, 6-O-benzyl-1,2-0-isopropylidene5-O-p-tolylsu~fony~-a-D-glucofuranose324) gives the 5,6-ene. Whistler and coworkers325examined the action of sodium methoxide in chloroform-methanol at room temperature on several pairs of derivatives of 1,2-0-isopropylidene -a-D-glucofuranose 5-p-toluenesulfonate, one member of each pair having an alkali-stable group on C-3; they concluded that a free hydroxyl group at C-3 is essential in order that the p-elimination reaction may give the enol ether. The two mechanisms suggested as possible were criticized by Buchanan and Oakes,326who proposed that the alkoxyl ion at C-3 acts as a base for the removal of a proton from C-6 by way of a six-membered, cyclic transition state (41) to give the trans enol ether 42.

(41) R = Me, ChPh, or Tr

(42) R = Me, CbPh, or Tr

(322) I. E. Muskat,J. Amer. Chem. SOC., 56,2653 (1934);P. A. Levene and J. Compton, ibid., 57,2306 (1935). (323) R. E. Gramera, R. M. Bruce, S. Hirase, and R. L. Whistler, J . Org. Chem., 28, 1401 (1963). (324) R. E. Gramera, T. R. Ingle, and R. L. WhistlerJ. Org. Chem.,29,878 (1964). (325) R. E. Gramera, T. R. Ingle, and R. L. Whistler, J . Org. Chem., 29, 1083, 2074 (1964). (326) J. G. Buchanan and E. M. Oakes, Carbohyd. Res., 1,242 (1965).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

157

Isolated, secondary, sulfonyloxy groups of pentitols and hexitols undergo S,2 displacements; sodium benzoate in boiling N,N-dimethylformamide has been frequently used. The diisopropylidene acetal of L-rhamnitol was to be the 1,2:3,4-isomer by conversion of its monomethanesulfonate (43) into a 6-deoxy-~-gulitol derivative (44), and this exchange reaction was also used in determining the structures of some di-O-is~propylidenepentitols.~~~ Me Me

Me Me

Me Me

O K Me Me

The methylsulfonyloxy group of 3-0-benzoyl-1,2:5,6-di-O-isopropy~idene-4-O-(methy~sulfony~)-D-mannitol (45) was readily dis-

placed by nucleophilic reagents, and the mechanism of the reaction was shown to be dependent on the relative strengths of the anchimeric benzoyloxy group and the attacking n u ~ l e o p h i l e .With ~~~a strong nucleophile, such as azide ion, the S,2 reaction predominated (givine30 46), whereas, with the (weakly nucleophilic) acetate ion, the anchimeric reaction predominated.329With sodium benzoate in N,N-dimethylformamide, displacement occurred by both mechan i s m ~ When . ~ ~ ~the benzoyl group of 45 was replaced by an alkyl group, such as methyPZ9(48) or tetrahydropyranyP2 (49), the bimolecular reaction occurred exclusively, even with acetate ion. The lack of participation by a neighboring ether group was also (327) M. A. Bukhari, A. B. Foster, J. Lehmann, and J. M. Webber,]. Chem. Soc., 2287 (1963). (328) M. A. Bukhari, A. B. Foster, J. Lehmann, J. M. Webber, and J. H. Westwood, J . Chem. Soc., 2291 (1963); K. W. Buck, A. B. Foster, B. H. Rees, and J. M. Webber, Chem. Znd.(London), 1623 (1964). (329) B. R. Baker and A. H. Haines,]. Org. Chem., 28,438 (1963). (330) B. R. Baker and A. H. Haines,]. Org. Chem., 28,442 (1963). (331) M. A. Bukhari, A. B. Foster, J. Lehmann, M. H. Randall, and J. M. Webber, J . Chem. Soc., 4167 (1963). (332) B. R. Baker and H. S. SachdevJ. Org. Chem., 28,2132 (1963).

D. H. BALL AND F. W. PARRISH

158

demonstrated when 1,2:5,6-di-O-isopropylidene-3-O-methyl-4-0(methylsulfonyl)-D-glucitol (47) reacted with sodium benzoate in Me Me

OMS (45) R = Bz

Me Me

(48) R = Me (49) R = tetrahydro2 If-pyran-2-yl

(47)

(46)

boiling N,N-dimethylformamide to give 4-0-benzoyl-1,2:5,6-di-Oisopropylidene-3-O-methy~-~-ga~actitol.~~~ An unexpected elimination reaction occurred when 1,2:4,5-di-O-isopropylidene-3,6-di-O-(methylsulfony~)-D-mannitol(50) or the corresponding 6-benzoate (51) was treated with sodium benzoate in boiling N,N-dimethylformamide.333The crystalline enol ether formed was shown to be 6-0-benzoyl - 3-deoxy - 1,2:4,5-di - 0- isopropylidene-D-threo -hex-3-enitol (52). Participation by the primary benzoyloxy group was suggested as a possible explanation for the elimination between C-3 and C-4, although the transition state would be a seven-membered, cyclic, carbonium ion; this would give the stereoisomer shown (52a), whereas elimination by way of attack of benzoate ion at H-4 would give the alternative isomer.333It would be of interest to examine the reaction of analogous compounds having nonparticipating functions at C-6.

HYO’ FO‘CMe, H,~OR (50) R = ME (51) R = Bz

%COR

(52)

(333) M. A. Bukhari, A. B. Foster, J . M. Webber, and J. Lehmann, Carbohyd. Res.,l, 485 (1966).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

159

d. Sulfonyloxy Groups on Pyranoid Rings. -It was first shown that sodium benzoate-boiling N,N-dimethylformamide could displace a sulfonyloxy group on a pyranoid ring when methyl tetra-o-benzoyla-D-glucopyranoside (54) was obtained249from the action of these reagents on methyl 2,3-di-0-benzoyl-4,6-di-O-p-tolylsulfonyl-aD-galactopyranoside (53). Sodium azide in N,N-dimethylformamide also effects SN2 displacement of an axially attached sulfonyloxy at C-4, and this reaction was used by C. L. Stevens and co(viosamine) workers in the synthesis of 4-amino-4,6-dideoxy-D-glucose and its N-methyl and N,N-dimethyl derivatives (bamosamine and amosamine, respectively).286The methylsulfonyloxy group in methyl 2,3,6-tri-0-benzoyl-4-O-(methylsulfonyl)-a-~-galactopyranoside can be displaced by potassium thiocyanate in N,N-dimethylformamide at 140" for 46 but, rather surprisingly, the corresponding p-D anomer was recovered in 879'0 yield after similar treatment.Is6 For compounds lacking benzoyl groups [methyl 2,3-di-O-methyl4,6-di-O-(methylsulfonyl)-~-~-galactopyranoside and methyl 6-deoxy2,3-di-0-methyl-4-0-(methylsulfonyl)-~-~-galactopyranosidel, displacements at c-4 are readily effected.'%

(334) S. D. GPro, Tetrahedron Lett., 3193 (1966); S . D. GBro and R . D. Guthrie, J. Chem. SOC. ( C ) ,1761 (1967). (196) L. N. Owen and P. L. Ragg,J. Chem. SOC.(C), 1291 (1966).

160

D. H. BALL AND F. W. PARRISH

h i d e displacement at C-4 of methyl 2,3-di-O-benzoy1-4-0p -t o l y l ~ u l f o n y[or l ~ (~rnethyl~ulfonyl)~~~] ~ -p -L -arabinopyranoside gave good yields of methyl 4-azido-2,3-di-O-benzoyl-4-deoxy-a-~-xylopyranoside, and, thence, a convenient route to 4-amino-4-deoxy-Dxylose and its derivatives. Displacements of an equatorially attached sulfonyloxy group from C-4 of a D-glucopyranose derivative were achieved by the reaction of methyl 2,3-di-0-benzoyl-4,6-di-O-(methylsulfonyl)-a-~-glucopyranoside (55) in N,N-dimethylformamide with sodium benzoate, sodium azide, or potassium thiocyanate, to give the corresponding D-galactose derivative^^^^*^*^ 56-58. The equatorially attached 4sulfonyloxy group of the D-glucoside undergoes sN2 displacement almost as readily as the corresponding axial group of the galactopyranosides. Kinetic studies employing methyl 2,3-di-O-benzy1-4,6-dideoxy4-iodo-a-~-gluco-and -galactopyranosides showed that the D-galact0 isomer reacts only 2 to 3 times as fast as the D-ghC0 isomer with iodide in acetone.337

(56) R = OBz (57) R = N, (58) R = SCN

h i d e displacements of C-4 sulfonates of D-glUCOSe derivatives were used in synthesis of 4-amino-4,6-dideoxy-~-galactose and its N-methyl and N,N-dimethyl and of 2,3,4,6-tetraamino2,3,4,6-tetradeoxy-~-galactosederivatives.2B0It was also found that the 4-methylsulfonyloxy group of benzyl 3-0-acetyl-2-[ (benzyloxycarbonyl)amino]-2-deoxy-4,6-di-O-( meth ylsulfony1)-a-D-glucopyranoside338(or of the corresponding 2-acetamido-2-deoxy derivative339) undergoes sN2 displacement by acetate ion, to give derivatives of 2(335) E. J. Reist, L. V. Fisher, and L. Goodman,/. Org. Chem., 32,2541 (1967). (336) A. J. Dick, Ph.D. Thesis, Queen’s University, Kingston, Ontario, Canada, 1967. (337) C. L. Stevens, K. G. Taylor, and J. A. Valicenti, /. Amer. Chern. SOC., 87, 4579 (1965). (338) P. H. Gross, K. Brendel, and H. K. Zimmerman. Jr.,Ann., 683,182 (1965). (339) P. H. Gross, F. du Bois, and R.W. Jeanloz, Corbohgd. Res., 4,244 (1967).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

161

amino-2-deoxy-D-galactosein about 50 % yield. The stereochemistry of forosamine (an aminodeoxy sugar component of the spiramycin antibiotics) was proved to be 2,3,4,6-tetradeoxy-4-(dimethylamino)D-erythro-hexose by a synthesis involving acetate and azide displacements of 4-sulfonyloxy ~ q o u p s . ~ ~ ' Selective displacement of 4-sulfonyloxy groups has been achieved with methyl tetra-O-(methy~su~fony~)-a-D-glucopyranoside,340~341 triO-(methylsulfonyl) derivatives of methyl a- and p-D-XylOpyranO~ i d e and ~ methyl ~ ~ ~a- and , ~ P-~-arabinopyranosides,~~~ ~ ~ and methyl 2-0-benzoyl-3,4-di-0-p-tolylsulfonyl-~-~-~abinopyranoside.~~~ The structurally analogous 5-methylsulfonyloxy groups of methyl 1,3-0benzylidene-4,5-di-O-(methylsulfonyl)-a-~-sorbopyranoside (59) and -p-D-fructopyranoside (60) were selectively displaced by sodium azide in boiling N,N-dimethylf~rmamide.~~~ OMe

(59)R = OMS, R ' = H (60) R = H, R ' = OMS

The methylsulfonyloxy group at C-4 of methyl 2,3,6-tri-O-benzoyl4-O-(methylsulfony~)-a-D-mannopyranoside was found to be resistant to nucleophilic di~placement,3~~ probably due to the p-trans axial group at C-2 which hinders approach of the nucleophile to (2-4. This reasoning was used to explain the observation that, whereas 1,2,4,6tetra-O-benzoyl-3-O-p-to~ylsulfonyl-~-~-glucopyr~ose (61) undergoes benzoate displacement at C-3 to give penta-O-benzoyl-8-D(No displacement was allopyranose, the a-Danomer (62) does observed with either of the corresponding furanose anomers.)

(340) A.J. Dick and J. K. N. Jones, Can.J.Chem.,43,977(1965). (341) A.J. Dick and J. K. N. Jones, Can.J.Chem.,44,79(1966). (342) E. J. Reist, D. E. GuefFroy, R. W. Blackford, and L. Goodman, J . Org. Chem., 31,4025(1966). (343) D.MurphyJ. Chem.Soc.(C),1732(1967). (344) A.C.Richardson and J. M. Williams, Chem. Commun., 104 (1965).

162

D. H. BALL AND F. W. PARRISH

Benzoate displacements were effected345with both anomers of methyl 2,4,6-tri-0-acetyl-3-O-p-tolylsulfonyl-~-glucopyranoside (63 and 64), although the p-D anomer 63 reacted faster than the a - D anomer (64)(ratio of reaction rates, about 8:1).The methyl glycosides gave better yields of allose derivatives, and less decomposition was observed; and this was attributed to the stability of methyl glycopyranosides to the reaction conditions, which were shown to decompose sugar esters.345Thus, a p-trans axial substituent strongly inhibits, but does not necessarily preclude, displacement of a sulfonyloxy group. The facile displacement by azide (in N,N-dimethylformamide) of the 4-methylsulfonyloxy group of methyl 3-acetamido-2-azido-2,3dideoxy-4,6-di-O-(methylsu1fonyl)-a-D-altropyranoside(65) suggested that the reaction proceeds by way of the alternative chair conformation (65a).260

An a-cis axial substituent also gives rise to unfavorable steric and dipolar interactions in the transition state, and this explains why, for example, methyl 4,6-dichloro-4,6-dideoxy-~-~-glucopyranoside 2,3bis(chlorosulfate), but not the corresponding D-ga~actoisomer, undergoes SN2 displacement by chloride ion346,347 at C-3. This may also be (345) R. Ahluwahlia, S. J. Angyal, and M. H. Randall, Carbohyd. Res., 4, 478 (1967). (346) H. J. Jennings and J. K. N. Jones, Can.J.Chem., 43,2372 (1965);A. G. Cottrell, E. Buncel, and J. K. N. Jones, ibid., 44,1483 (1966). (347) L. Hough and A. C. Richardson, in “Rodds Chemistry of Carbon Compounds,” s. Coffey, ed., 2nd Edition, Elsevier Publishing Co.,New York, N.Y., 1967, Vol. IF, Chapter 23.

SULFONIC ESTERS O F CARBOHYDRATES: PART I1

163

a contributing factor to the inability of the bridged-ring sulfonates 35 and 36 to undergo azide displacement. The electron-withdrawing effect of the anomeric center, which inhibits displacements of primary sulfonyloxy groups at C-1 of fructose and sorbose, strongly hinders displacements at C-2 of aldopyranose derivatives. An alternative or additional effect, proposed by Hough and Richard~on,~~' may well consist in unfavorable dipolar interactions which would arise in the formation of the SN2 transition state. The three major factors that inhibit nucleophilic displacement reactions in pyranose rings appear34*to be: (i) a 1,3-diaxial interaction between the nucleophile and a ring substituent, (ii) a cis axial group adjacent to the sulfonyloxy group, and (iii) the electron-withdrawing effect, or unfavorable dipolar interactions at carbon atoms adjacent to the anomeric center. The only methyl 4-azido-4-deoxy-2,3-di-O-(methylsulfonyl)-~pentopyranoside (obtained by selective azide displacement at C-4) containing a sulfonyloxy group not subject to the above factors was the P-D-XIJZO isomer, and this could be further substituted by azidepresumably341at C-3. If the carbon atom adjacent to that bearing the sulfonyloxy group is not bonded to oxygen or other electronegative substituents (for example, a methylene carbon atom), displacement of the sulfonyloxy group is more readily achieved. Although direct comparisons were not made, it is evident that azide displacement of the methylsulfonyloxy group of methyl 4,6-0-benzylidene-2-deoxy-3-O-(methylsulfony1)-a-D-ribo-hexopyranoside (66) (boiling N,N-dimethylformamide for 15 minutes)349was more readily achieved than with methyl 2-azido-4,6-0-benzylidene-2-deoxy-3-O-(methylsulfonyl) -a-D-akropyranoside (67) (boiling, 4:1 N,N-dimethylformamide-p-dioxanefor 40 Azide and acetate displacements of 4-sulfonyloxy and -threegroups from ethyl 2,3,6-trideoxy-4-O-sulfonyl-a-~-erythrohexopyrano~ides~~' also appear to have occurred more readily than similar displacements with compounds containing electronegative groups on C-3. An azide displacement at C-2 of an aldopyranoside was achieved by the reaction of methyl 4,6-0-benzylidene-3-deoxy-2-O-ptolylsulfonyl-a-D-ribo-hexopyranoside (68) with sodium azide in Attempted azide displacements boiling N,N-dimethyIf~rmamide.~~~ (348) (39) (350) (351)

A. C. Richardson, Ann. Repts. Progr. Chem., 62, 371 (1965), 63,493 (1966). A. C. Richardson, Carbohyd. Res., 4,422 (1967); Chem. Commun., 627 (1965). R. D. Guthrie and D. Murphy,]. Chem. Soc., 6956 (1965). M. Nakajima, H. Shibata, K. Kitahara, S. Takahashi, and A. Hasegawa, Tetruhedron Lett., 2271 (1968).

164

D. H. BALL AND F. W. PARRISH

with methyl 3- azido -4,6- 0- benzylidene - 3- deoxy -2 - 0 - (methylsulfony1)-a-D-ghcopyranoside(69) were unsuccessful.350

The allylic, secondary methylsulfonyloxy groups of methyl 2,3dideoxy-4,6-di-O-(methylsulfonyl)-a-~-e~~throand -threo-hex-2enosides undergo SN2 displacement on treatment with sodium benzoate in N,N-dimethylformamide, and are displaced more readily than the primary sulfonyloxy groups.352The isolated, axial, sulfonyloxy group on C-4 of methyl 2,3-di-O-benzyl-6-deoxy-4-0-(methylsulfony1)a-D-galactopyranoside readily undergoes base-catalyzed elimination in methyl sulfoxide with methoxide ion at 70°, to give a mixture of the two olefins Under the same conditions, isolated equatorial sulfonyloxy groups unexpectedly gave methyl ethers, with retention of c ~ n f i g u r a t i o n . ~ ~ ~ e. Sulfonyloxy Groups in Furanoid Rings. -Until recently, very few examples of unassisted displacements on oxolane (hranoid) rings had been reported, although, by analogy with cyclopentyl and cyclohexyl derivatives, these should, in general, be somewhat (352) D. M. Ciment, R. J. Ferrier, and W. G. Overend,J. Chem. SOC. (C),446 (1966). (353) E. D. M. Eades, D. H. Ball, and L. Low, Jr., unpublished results. (354) E. D. M. Eades, D. H. Ball, and L. Long, Jr., J. Amer. Chem. SOC., 86, 3579 (1964);J.Org. Chem., 31,1159 (1966).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

165

easier than displacements on pyranoid rings.355A synthesis of 6-deoxy2,5-di-O-methyl-~-altrosewas achieved 356 by the reaction of methyl 2,5-di-0-methyl-3-0-(methylsulfonyl)-a-~-rhamnofuranoside (70)with sodium benzoate in boiling N,N-dimethylformamide for 6 hours; a 58 % yield of methyl 3-O-benzoyl-6-deoxy-2,5-di-O-methyl-a-~altrofuranoside (71)was obtained. Steric hindrance by the &trans

Me

4

MH eOMe w

o

M

e

H OMe

OMe

methoxyl group on C-1to nuclzophilic attack at C-3is certainly less than that of a p-trans axial group on a six-membered ring, and the relative ease of this displacement is not surprising. The 3-methylsulfonyloxy group of methyl 2-0-benzyl-5-deoxy-3-0-(methylsulfony1)a-D-xylofuranoside (72)was much more difficult to displace by benzoate ion,357and, in this reaction, hindrance to the approaching nucleophile is probably only slightly less (due to greater ring flexibility) than in the 1,2-O-isopropy~idene-3-O-p-to~y~su~fony~-D-xy~ose derivatives (26). T

MQoMe

OCqPh

s

O

TsO

C

V

OTe

The 5-sulfonyloxy group of methyl 2,3,5-tri-0-p-tolylsu~fonyl-p-Dribofuranoside (73)can be selectively displaced by sodium azide in N,N-dimethylformamide at 120" (30 minutes).282Treatment of the monoazide (or of 73)with the same reagents at 145"for 2 hours gave

(355) E. L. Eliel, N. L. Allinger, S. J. Angyal, and G. A. Morrison, "Conformational Analysis," Jnterscience Publishers, Inc., New York, N.Y., 1965,p. 204. (356) A. B. Foster, J. Lehmann, and M. Stacey,J. Chem. SOC., 4649 (1961). (357) K. J. Ryan, H. Arzoumanian, E. M. Acton, and L. Goodman, J . Amer. Chem. SOC.,86,2497(1964).

D. H. BALL AND F. W. PARRISH

166

methyl 3,5-diazido-3,5-dideoxy-2-O-p-tolylsulfonyl-~-~-xylofuranoside in 46 % yield.358 Nucleophilic displacement of the p-tolylsulfonyloxy group of 2’deoxy-3‘-O-p-tolylsulfonyladenosine(74) was achieved35nwith sodium ethanethioxide in ethanol at 80”; a 25 % yield of 6-amino-9-(2-deoxy3-~-ethyl-3-thio-~-D-threo-pentofuranosy~)purine (75) was obtained, and this was desulfurized to 2’,3’-dideoxyadenosine. It was later shown that the major product from 74 and sodium ethoxide in boiling ethanol is 9-(2,3-dideoxy-/3-~-gZycero-pent-2-enofuranosyl)adenine (76), together with lesser proportions of the 3’,5‘-0xetan.~~O The com-

I

TeO (14)

where B = adenin-9-yl.

peting SN2reaction observed in the presence of ethanethioxide ion is facilitated by the lack of an oxygen function on the adjacent carbon atom (C-2’), and it would probably not occur with ribose or arabinose derivatives. The unsaturated nucleoside 76 was also obtained from the reaction of 74 with sodium methoxide in N,N-dimethylformamide at room temperature, and an E2 mechanism (instead of intermediate oxetan formation) was suggested, as 2’,5’-dideoxy-3’-0-p-tolylsulfonyladenosine also gave the corresponding unsaturated nucleoside under the same conditions.361 A 2’,3’-unsaturated derivative of uridine was prepared by basecatalyzed elimination of the methylsulfonyloxy group of l-[e-deoxy3-O - ( m e t h y ~ s u ~ f o n y ~ ) - 5 - O - t r i ~ l - ~ - ~ - ~ ~ r e o - p e n t o ~ r a n o s y l ] u r a c The cis relationship between the aglycon and the sulfonyloxy group precluded occurrence of intramolecular displacement. J. Clbophax, S. D. Gbro, and J. Hildesheim, Chem. Commun., 94 (1968). M. J. Robins and R. K. RobinsJ. Amer. Chem. Soc., 86,3585 (1964). J. P. Horwitz, J. Chua, and M. Noel, Tetrahedron Lett., 1343 (1966). J. R. McCarthy, Jr.. M. J. Robins, L. B. Townsend, and R. K. Robins,J. Amer. Chem. Soc., 88,1549 (1966). (362) J. P. Horwitz, J. Chua, M. A. DaRooge, M. Noel and I. L. KlundtJ. Amer. Chem. SOC., 86,1896 (1964);J.Org. Chem.,31,205 (1966).

(358) (359) (360) (361)

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

167

VIII. DISPLACEMENT REACTIONS INVOLVING PARTICIPATION

The past fifteen years have seen rapid developments in the understanding and synthetic utility of reactions that simultaneously involve sulfonate groups and other functions in the molecule. New examples of three-, four-, five-, and six-membered oxide-ring formation have been recorded, and many examples of internal displacements leading to nitrogen- and sulfur-containing rings are now well known. The major development in participation reactions is undoubtedly the application to carbohydrates (primarily by B. R. Baker, L. Goodman, and their coworkers) of the studies of Winstein and coworkers with trans-2-acetoxycyclohexylsulfonates363 and of M ~ C a s l a n d ?and ~ ~ Winstein,3s5 and their coworkers with trans-2-benzamidocyclohexylsulfonates. Participation in sulfonyloxy-group displacement by oxygen or nitrogen functions in the aglycon of nucleosides has been of great value in structural manipulations. Goodman has reviewed neighboring-group participation in Volume 22 of this and this subject will therefore not be discussed comprehensively in the present article. However, Goodman specifically excluded from his review the formation of anhydro sugars by oxide ion displacements, and also the formation of anhydronucleosides (“cyclonucleosides”). These topics will therefore be discussed, together with ring contractions and alkoxyl participations, because most of the literature on these subjects has not been reviewed previously. 1. Oxide-ring Formation Hartman and Barker have examined intramolecular displacements of p-tolylsulfonyloxy groups by oxide ions, and have concluded that (363) S. Winstein, H. V. Hess, and R. E. Buckles,]. Amer. Chem. Soc., 64,2796(1942); S. Winstein, C. Hanson, and E. Grunwald, ibid.,70,812(1948);S. Winstein, E. Grunwald, R. E. Buckles, and C. Hanson, ibid., 70,816 (1948);S. Winstein, E. Grunwald, and L. I. Ingraham, ibid., 70,821 (1948);S. Winstein and R. Heck, ibid.,74,5584 (1952);R. M. Roberts, J. Corse, R. Boschan, D. Seymour, and S. Winstein, ibid., 80,1247(1958). (364) G. E. McCasland, R. K. Clark, Jr., and H. E. Carter, J . Amer. Chem. Soc., 71, 637(1949). (365) S. Winstein, L. Goodman, and R. Boschan,]. Amer. Chem. Soc., 72,2311(1950); S. Winstein and R. Boschan, ibid.,72,4669(1950). (366) L.Goodman,Aduan. Carbohyd. Chem., 22,109(1967).

168

D. H. BALL AND F. W. PARRISH

a primary p-tolylsulfonyloxy group is displaced by alkoxide ions derived from hydroxyl groups in the following order of reactivity367: p-y-OH > sec-y-OH = sec-a-OH > p- &OH. A secondary p-tolylsulfonyloxy group is displaced most readily by an alkoxide ion derived from a primary a-OH. The determination of optimal reaction-conditions (greatly facilitated by the use of thin-layer chromatography) is often critical, as initial products may undergo further reactions; this was demonstrated in syntheses of 2,3:4,5-dianhydro-~-iditol from 3,4-di-O-sulfonyl-~mannitol derivatives .368,369 Although most of the following subsections on oxide-ring formation deal with bi- and tri-cyclic systems which impose additional, and often overriding, steric constraints, cognizance should be taken of these fundamental considerations. a. Epoxides. -Examples, from the carbohydrate field, of epoxide synthesis by intramoleculzr displacement of sulfonyloxy groups are too numerous for tabulation here. In most cases, the epoxide is an intermediate in a synthesis; however, some examples relevant to the mechanism of the reaction will be discussed. The steric requirements for epoxide formation from a sulfonic ester are well established and have been discussed p r e v i o ~ s l y . ~ , ~Although ’ ~ * ~ ~ ’ quantitative data are lacking, the ease of formation of an epoxide can be approximately correlated with the degree of difficulty of attaining the coplanar transition-state essential for maximum participation. In addition to 0

+

RSOP

unfavorable, 1,3-diaxial interactions in the transition state, “passing interactions” encountered in the conformational changes necessary to an attainment of the transition state have also been p r o p o ~ e d .The ~.~~~ relative importance of the latter effect seems to rest on somewhat (367) F.C. Hartman and R. Barker,]. Org. Chem.,28,1004(1963); 29,873(1964). (368) R. S.Tipson and A. Cohen, Carbohyd. Res., 7,232(1968). (369) M.Jarman and W. C. J. Ross, Carbohyd. Res., 9,139(1969). (3) F.H.Newth, Quart. Reu. (London), 13,30(1959). (370) S. Peat, Aduan. Carbohyd. Chem., 2,37(1946). (371) L.F.Wiggins, Aduan. Carbohyd. Chem.,5,191(1950).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

169

tenuous grounds. For example, although 1,5-anhydro-4,6-0-benzylidene-2-O-p-tolylsulfonyl-~-glucitol (77) can be converted1I6into the D - ~ U W ~ epoxide O with sodium methoxide at 0”,and the corresponding reaction with methyl 4,6 - 0- benzylidene - 2 - 0 - p - tolylsulfonyl -a- D glucopyranoside (78) was performed in boiling methanol,lo5it does not appear to have been established that the latter compound requires this reaction temperature. It would be of interest to compare the rates of epoxide formation from 77 and 78 under the same conditions.

0 RO

(77) R = H (79) R = Ts

Ts

(78)R = H (80) R = Ts

Knowledge of the factors controlling the mechanisms and rates of formation of epoxides from disulfonates, discussed by N e ~ t h also ,~ suffers from a lack of quantitative and coniparative data. It appears that initial sN2s cleavage of one of the sulfonyloxy groups is followed by an intramolecular displacement of the other, and, if a primary sulfonate is involved, this is cleaved first and the stereochemistry of the reaction is predictable. When two trans, secondary, sulfonyloxy groups are involved, the reaction mechanism is not so well understood. Angyal and Gilham372proposed that the “more accessible” sulfonyloxy group will be removed first, and that this reaction will be facilitated by the inductive effect of the other sulfonyloxy group. The inductive effect appears not to be a major consideration, however, as 1,6anhydro-3,4-di-O-p-tolylsulfonyl-~-~-altropyranose is not affected by In boiling in 2.5 M sodium methoxide in methanol for 20 contrast, epoxide formation (and, presumably, initial sN2s cleavage) occurs at 0” with methyl 4,6-O-benzylidene(or isopropylidene)-2,3-diO-p-tolyls~lfonyl-a-D-glucopyranoside~~~*~~~ (80). It is of interest that (116) (105) (372) (113) (373) (374)

F. H. Newth,]. Chem. SOC., 2717 (1959). G. J. Robertson and C. F. Griffith,]. Chen. Soc., 1193 (1935). S. J. Angyal and P. T. Gilham,]. Chern. SOC.,3691 (1957). F. H. Newth, J . Chem. Soc.,441(1956). N . K. Richtmyer and C. S. Hudson,]. Amer. Chem. SOC.,63,1727 (1941). J . G. Buchanan and R. M. Saunders,]. Chern. S O C . , 1796 (1964).

D. H. BALL A N D F. W. PARRISH

170

the sulfonyl group preferentially cleaved is often on the oxygen atom preferentially sulfonylated with sulfonyl chlorides in pyridine, and this behavior has facilitated synthesis of both epoxides in several studies. Examples of this reaction have been found with the monoand di-0-p-tolylsulfonyl derivatives of methyl 4,6-O-benzylidene-a-Dgluc~pyranoside’~~ (78 and 80), 1,5-anhydro-4,6-0-benzylidene-~glucito1116(77 and 79), and 1,2:3,4-di-O-isopropylidene-epi-inosito1372 (81 and 82). Further work is needed in order to elucidate the structural O--CMe,

I

Ro.

\

(81) R = H (82) R =

Ts

and electronic factors that determine the “accessibility” of a sulfonyloxy group to SN2S cleavage. An intermolecular transsulfonylation mechanism suggested341 would seem rather unlikely tobe operative in methanol. Newth discounted a concerted mechanism: because some monosulfonate was found in a reaction mixture; however such a mechanism may well operate in some reactions.

(83)

r

Trot&?

OM6

H

T

(84)

o

t

&

c

OR (85)

where R = benzyl.

~

H

@

TrOt&C

H

@

R

OH (86)

OR (87)

SULFONIC ESTERS O F CARBOHYDRATES: PART I1

171

Epoxide formation has been postulated375 as the initial stage in the reaction of 2,3 - 0 - isopropylidene - 5- 0 - p - tolylsulfonyl -Lrhamnofuranose (83) with sodium methoxide in methanol to give (84) in 60 % methyl 6-deoxy-2,3-0-isopropylidene-~-~-allofuranoside yield.375,37s 2,3- Di-O-benzyl-5-O-(methylsulfonyl)-6-O-trityl-~-glucofuranose (85)reportedly undergoes a similar reaction with methoxide, (86).An to give methyl 2,3-di-0-benzyl-6-0-trityl-~-altrofuranoside~~~ indication that the anomeric hydroxyl group may participate in the displacement of the 5-methylsulfonyloxy group was afforded by the isolation of a crystalline compound for which the structure 1,5anhydro-2,3-di-O-benzyl-6-O-trityl-a-~-idofuranose (87) (that is, 1,4anhydro-2,3-di-O-benzyl-6-O-trityl-~-~-idopyranose) was suggested. 2,3:6,7- Di-O-isopropylidene-5-0-p-tolylsulfonyl-P-D-g~ycero-~gulo-heptofuranose (88) reacted in an analogous way with methoxide.378The rate of reaction was lower than that observed with 83, and this observation was rationalized on conformational grounds, because the intermediate state for epoxide formation from 88 (88a) appears energetically less favorable than that (83a) from the LH

c- 3

H

H

rhamnose derivative 83 (or from the D-glucose derivative 85). It was also noted that the specific rates of epoxide formation from 88a and 83a are unlikely to be the same. Methyl 2,3:6,7-di-O-isopropylideneP-D-glycero-L-tab-heptofuranoside (89) was obtained from 88 in 28 % yield, and a crystalline anhydride was isolated in 15% yield and to be 1,5-anhydro-2,3:6,7-di-O-isopropylidene-P-~-gZycero-~do-heptofuranose (90). It is interesting that the configuration of 90 is obtained by inversion at C-4 of 88, and the authors suggested that (375) E. J. Reist, L. Goodman, R. R. Spencer, and B. R. Baker,]. Amer. Chem. SOC., 80,3962 (1958). (376) P. A. Levene and J. Compton,J. Biol. Chem., 116,169 (1936). (377) T. Iwashige and H. Saeki, Chem. Pharm. Bull. (Tokyo), 15,132 (1967). (378) J. S. Brimacombe and L. C. N. Tucker,]. Chem. SOC.(C), 562 (1968).

D. H. BALL AND F. W. PARRISH

172

intramolecular transtosylation (to give the 4-0-sulfonyl pyranoid isomer of 88) preceded anhydro-ring formation. In view of these results, the stereochemistry of 87 should be determined, since, were a similar mechanism operative, 87 would have the D-gUlUCtO configuration (and not the ~ - i d configuration o shown).

(88)

I

HCO, where R = H,CO

b. Oxetans. - In 1933, Levene and Raymond reported the first synthesis of a carbohydrate oxetan (92) by treatment of 1,2-O-isopropylidene-5-O-p-tolylsulfony~-a-D-xy~ofuranose (91) with sodium meth-

oxide in methanol at room t e m p e r a t ~ r e . ~Similar '~ reactions in the nucleoside field have led to synthesis of 1-(3,5-anhydro-2-deoxy-PD-threo-pentofurano~yl)thyrnine~~~ (93), 1-(3,5-anhydro-P-~-xylof~ranosy1)uracil~~~ (94), and l-(3,5-anhydro-~-~-lyxofuranosyl)uracil~~~ (95). Several derivatives of 4,6-anhydr0-2,3-0-isopropylidene-a-~xylo-hexulofuranose (-L-sorbofuranose) (96) were prepared independently by F6her and Vargha3= and Hough and Otte$@ by base(379) P.A. Levene and A. L. Raymond,J. B i d . Chem., 102,331(1933). (380) J. P. Horwitz, J. Chua, J. A. Urbanski, and M. Noe1,J. Org. Chern.,28,942(1963). (381) I. L. Doerr, J. F. Codington,and J. J. FoxJ. Org. Chem., 30,467(1965). (382) J. F.Codington, I. L. Doerr, and J. J. FoxJ. Org. Chem., 30,476(1965). (383) 6.FBher and L. Vargha,Acta Chtm.Acad. Sci. Hung., 50,371(1966). (384) L. Hough and B. A. Otter, Carbohyd.Res., 4,126(1967).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

173

R. (93) R = Me, R'= R " = H (94)

R = R ' = H, R = OH R " = H, R' = OH

(95) R =

catalyzed, intramolecular displacements. Both groups established that several L-sorbose derivatives, previously thought to contain 1,Panhydro rings,385are, in fact, the 4,6-anhydrides. It is of considerable interest that 3,5-anhydro-1,2-0-isopropylidenea-D-xylofuranose (92) was obtained in 85% yield by treatment of 1,2O-isopropylidene-3,5-di-O-(methylsulfonyl)-a-~-xylofuranose with potassium hydroxide in ethanol at room t e m p e r a t ~ r e The . ~ ~ high yield could be explained should the 3-0-methylsulfonyl group be very much more susceptible to SN2S attack than the 5-0-methylsulfonyl group, which seems unlikely. An alternative mechanism would involve initial hydrolysis of the 5-0-methylsulfonyl group, followed by an intramolecular transmethanesulfonylation. It would, therefore, be of interest to examine the behavior of 1,2-O-isopropylidene-3-0(methylSUlfOnyl)-a-D-XylOfuranOSeunder these conditions. With the evidence at present available, a concerted mechanism, similar to that mentioned, but rejected, by NewthS for epoxide formation from di-0-sulfonyl compounds, seems a strong possibility. A similar reaction with 2,3-O-isopropylidene-1,4-6-tri-O-(methylsulfonyl)-a-~xylo-hexulofuranose gave the corresponding 4-6-anhydro derivative in 38% yield.3"

(385) K.Tokuyama, M.Kiyokawa, and N. Hoki, Bull. Chem. Soc.Jap., 36,1392(1963); K.Tokuyama, Ibid., 37,1133 (1964). (386) B.Helferich and M. Burgdorf, Tetrahedron, 3,274(1958).

D. H. BALL AND F. W. PARRISH

174

Internal displacement of a secondary sulfonyloxy group occurred during base-catalyzed elimination of the 5-p-tolylsulfonyloxy group from 3-O-acetyl-1,2-0-isopropylidene-5-O-p-tolylsulfonyl-6-O-trityla-D-glUCOfuranOSe (97);3,5-anhydro-1,2-0-isopropylidene-6-O-tritylL-idofuranose (98)was formed as a minor

9-(3,5-Anhydro-2-deoxy-P-~-threo -pentofuranosyl) adenine was formed360as the minor product from the reaction of base with 2’deoxy-3’-O-p-tolylsulfonyladenosine(74)(see p. 166). 1,3-Anhydro-2,4-0-methylene-~~-xylitol (loo), an oxetan cis-fused to a six-membered ring, was prepared by Hudson and by treatment of 2,4-O-methy~ene-~-O-p-toly~su~fony~-DL-xy~ito~ (99) with warm, aqueous sodium hydroxide. A related oxetan (102) was formed when 5,6-anhydro-2,4-0-benzylidene-l-O-p-tolylsulfonyl-~glucitol (101) was heated with 100 mM sodium Treatment of methyl 2,3-di-0-benzyl-6-0-(methylsulfonyl)-a-~-galacto-

(99) R = H, R ’ = CH,OH (D

enantiomorph)

(100) R = H, R ’ = CH,OH

(D enantiomorph)

(387) R. M. Hann, N. K. Richtmyer, H. W. Diehl, and C. S. Hudson,J. Amer. Chem. SOC., 72,561 (1950).

(388) E. Haslam and T. Radford, Chem. Commun., 631 (1965); Curbohyd. Res., 2, 301 (1966).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

175

pyranoside with sodium methoxide in methanol gives the corresponding 4,6-anhydride, which is a further example of a cis-fused 4,6-ring system.389 c. Five- and Six-membered Oxide Rings. -Zinner and coworkers390 found that, although dithioacetals of D-arabinose can be selectively sulfonylated at the primary hydroxyl group,218attempted monosulfonylation of D-ribose and D-XylOSe mercaptals gives dithioacetals of 2,5-anhydro-~-ribose (103) and 2,5-anhydro-~-xylose (104). 2,5Anhydro-D-lyxose diisobutyl dithioacetal (105, R = isobutyl) was

similarly obtained.s1 Analogous, anhydro-ring formation from 5-0sulfonyl-D-arabinose dithioacetals would lead to the remaining isomer (having all three substituents on the same side of the ring). Displacement of the 5-0-sulfonyl group presumably requires somewhat higher energy in this example, and permits isolation of the sulfonate. Contrary to a previous rep0rt,3~' selective p-toluenesulfonylation of Dgalactose dibenzyl dithioacetal does not afford a 6-O-p-tolylsulfonyl derivative: intramolecular displacement occurs, to give the 3,6anhydride (106) as the only product i ~ o l a b l eThe . ~ ~ ready displacement is predictable by analogy with the behavior of the D-lyXOSe derivative (105). Attempted mono-p-toluenesulfonylation of 2,3,5-tri0-benzyl-D-arabinitol leads393to the 1,4-anhydride (107), which also has the (favorable) trans-trans configuration. (389) D. H. Ball, unpublished results. (390) H. Zinner, H. Brandhoff, H. Schmandke, H. Kristen, and R. Hann, Chem. Ber., 92,3151 (1959). (218) H. Zinner, K. Wessely, and H. Kristen, Chem. Ber., 92,1618(1959). (61) J. Defaye, Bull. SOC.Chim. Fr., 2686 (1964). (391) J. Fernindez-Bolafios and R. Guzman de Femandez-Bolafios, Anales Real SOC. Espaii. Fis. Quim. (Madrid),54B,303 (1958). (392) J. M. Coxand L. N. Owen,J. Chem. SOC.(C), 1121 (1967). (393) Y. Rabinsohn and H. G . Fletcher, Jr.,J. Org. Chem.,32,3452 (1967).

176

D. H. BALL AND F. W. PARRISH

HO

HO

I

H

RO

3,6-Anhydrides of pyranoses and furanoses are usually formed by intramolecular displacements of 6-sulfonyloxy g r o ~ p s .These ~ ~ ~re,~~ placements often occur very readily (for example, during methylat i ~ nand , ~ during ~ attempted solvolysis of 3-O-acetyl-1,2-O-isopropylidene - 5,6 -di- 0- p -tolylsulfonyl -a-D-glucofuranose280). 3,6 -Anhydro rings have been introduced into amylose ~ h a i n s , Band ~ *2-amino-3,6~~~ anhydro-2-deoxy-~-mannosederivatives have been prepared.3w That the bridged-ring system of 3,6-anhydrohexopyranoses(structurally analogous to bicyclo[3.2. lloctane) is more strained than the cis-fused, five-membered, ring system of the corresponding furanoses has been discussed by Mills.303An example of a bridged-ring system analogous to bicyclo[2.2. llheptane is methyl 2,5-anhydro-a-~arabinofuranoside (108), which was prepared in 26 % yield by treatment of methyl 5-O-p-tolylsulfonyl-a-~-arabinofuranoside with 200 mM sodium methoxide in methanol at room t e m p e r a t ~ r e . 3Similar ~~ displacements have afforded pyrimidine nucleosides containing the 2,5-anhydro-~-arabinofuranosyl ( and 2,5-anhydro-~-lyxofuranosyl group (l10).s8zIt is of interest that 110 is formed [instead of the oxetan (95)l by treatment of ~-[5-0-(methylsulfony~)-~-D-~yxofuranosyl]uracil with a base.

p a H

(108) where B = uracil-1-yl.

(109)

(110)

(394) Ref. 1, p. 171. (68) R. L. Whistler and S. Hirase,]. Org. Chem., 26,4600(1961). (395) B. J. Bines and W. J. Whelan, Chem. Ind. (London), 997 (1960);M. L. Wolf'rom, M. I. Taha, and D. Horton, ibid.,28,3553(1963). (396) M. L. Wolfrom, P. Chakravarty, and D. Horton,]. Org. Chem., 31,2502(1966). (397) M. Cifonelli, J. A. Cifonelli, R. Montgomery, and F. Smith,]. Amer. Chsm. SOC., 77,121(1955).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

177

The second example of a 2,6-anhydrohexopyranose was prepared by an intramolecular displacement of a methylsulfonyloxy group on the ring; methyl 2,6-anhydro-3,4-di-O-methyl-a-~-mannopyranoside (112) was obtained in 47% yield by heati11g3~*a dilute solution (in methyl sulfoxide) of methyl 3,4-di-O-methyl-2-O-(methylsulfonyl)-6-0-sodioa-D-glucopyranoside (111) at 85”. ,-0 0Na0

2. Anhydronucleosides

Although anhydronucleosides have been prepared by Other methods,399intramolecular displacement of a sulfonyloxy group has been the synthetic route most often employed. With pyrimidine nucleosides, displacement is almost invariably effected by the ion derived from the oxygen function at C-2 of the pyrimidine base, whereas, with purine nucleosides, participation by N-3 is the usual route, although preparation of several purine anhydronucleosides has involved displacement by oxygen or sulfur functions at C-8. a. Pyrimidine Anhydronucleosides. -Todd and coworkers found that, when the 5’-O-p-tolylsulfonyl derivatives of 2’,3’-O-isopropylidene-cytidine400 (113) or -2’-deo~ycytidine~~l are heated in acetone, an intramolecular displacement occurs to give the p-toluenesulfonate (salt) of an anhydronucleoside, such as 114. The salt is too unstable for isolation from the reaction of the 2’-deoxycytidine derivative. 5‘-0Sulfonyl derivatives of thymidine were found to be relatively stable in E. D. M. Eades, D. H. Ball, and L. Long, Jr.,]. Org. Chem., 30,3949 (1965). For earlier reviews, see J. J. Fox and I. Wempen, Aduan. Carbohyd. Chem., 14,283 (1959);T. Uedaand J. J. Fox, ibid.,22,307 (1967); A. M. Michelson, “The Chemistry of Nucleosides and Nucleotides,” Academic Press Inc., New York, N.Y., 1963. V. M . Clark, A. R. Todd, and J. Zussman,]. Chem. SOC., 2952 (1951). W. Andersen, D. H. Hayes, A. M. Michelson, and A. R. Todd, J. Chem. SOC., 1882 (1954).

178

D. H. BALL AND F. W. PARRISH

weak base, but 3’,5’-di-O-(methylsulfonyl)thymidine(115) readily cyclizes when it is treated with ethanolic ammonia at room temperature, to give402the 2,3’-anhydride (116).Confirmation of the structure of 116 was obtained by Fox and who also showed that the 2,3’-anhydride is an intermediate in the reaction of 115 with sodium benzoate in N,N-dimethylformamide to give 2-deoxy-~-erythro-pentosyl derivatives. The acid liberated in the initial displacement catalyzes the ring-opening reaction, probably by protonation of the basic part of the molecule, and this facilitates nucleophilic at (2-3‘. Displacement reactions with 3’-O-(methylsulfonyl)-5’-O-tritylthymidine (117) and sodium iodide or lithium bromide in acetone,402and sodium benzoate,403potassium thiolbenzoate, or potassium phthalimide405in N,Ndimethylformamide, all occurred by way of the 2,3‘anhydride (118) to give, by double inversion, derivatives having the 2-deoxy-~-e~ythro-pentofuranose configuration. The unexpected reactions of 3’-O-p-tolylsulfonyl derivatives of thymidine under Oldham-Rutherford condition^^^ were thus rationalized. Alkaline hydrolysis of the 2,3’-anhydro ring occurs by aryl-oxygen fission,380*402,403 and the participation reaction thus provides a route to nucleosides having inverted configuration at C-3’. In addition to the thymidine derivatives, the reaction has been applied to a 2’-deoxy-5fluorouridine derivativem3 (119), to 2‘-deoxy-3’-O-(methylsulfonyl)5 ’ - 0 - t r i t y l ~ r i d i n e(120), ~ ~ ~ and to 3‘-O-(methylsulfonyl)-2 ’,5’-di-Otrityl~ridine.~~’ Alkaline hydrolysis of 2,3’-anhydronucleosides having a sulfonyl substituent on 0-5’ leads to 3’-5’-anhydrides (oxetans).s80 (402) (403) (404) (405) (406) (407)

A. M. Michelson and A. R. Todd,J. Chem. Soc., 816 (1955). J. J. Fox and N. C. Miller,]. Org. Chem.,28,936 (1963). K. C. Murdock and R. B. Angier,]. Amer. Chem. Soc., 84,3748 (1962). N. Miller and J. J. Fox,]. Org. Chem.,29,1772 (1964). P. A. Levene and R. S. Tipson,]. B i d . Chem., 109,623 (1935). N. C. Yung and J. J. Fox,]. Amer. Chem. SOC., 83,3060 (1961).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

179

MSb

(115) R = Me, (117) R = Me, (119) R =

R’= OM8 R’= OTr

(116) R = Me, R’= OMS (118) R = Me, R’= OTr

F, R’= OTr

(120) R = H, R ’ = OTr

Sodium tert-butoxide in N,N-dimethylformamide at 100” was found to be very effective in promoting intramolecular displacements408and 2,5‘-anhydrothymidine was obtained directly from 5’-0-(methylsulfony1)thymidine with these reagents. The same conditions have also been used to synthesize the anhydronucleosides 121and 122 from a pseudouridine derivative409and from a D-ribosyl derivative of 2,4q u i n a z ~ l i n e d i o n e .The ~ ~ ~ same reagents with 3’-O-p-tolylsulfonyluridine gave mainly l-(2,3’-anhydro-~-~-xylofuranosyl)uracil.~~~

O\

/O CMe,

Except for the 2,s’-anhydrocytidine derivative 114, initial attempts to prepare other 2,5‘- (Ref. 401) and 2,3‘- (Ref. 411) anhydrides from cytidine sulfonic esters were unsuccessful, although indications of (408) R. Letters and A. M. Michelson,]. Chem. Soc., 1410 (1961). (409) A. M. Michelson and W. E. Cohn, Biochemistry, 1,490 (1962). (410) M. G . Stout and R. K. Robins,]. O g . Chem., 33,1219 (1968).

D. H. BALL AND F. W. PARRISH

180

their formation as unstable intermediates were obtained. The conditions used for the attempted cyclizations (for example, boiling p dioxane, acetone, or 2,4-pentanedione at 1000)would have given ionic forms, such as 114, having a positive charge on the aglycon. It has been pointed that this charge decreases the stability of the glycosidic linkage, and evidence for this theory was the liberation of the a g l y ~ o n . ~Successful ~ ~ , ~ ~ ~syntheses of 2,3’-anhydrides were achieved by treatment of 3’-O-(methylsulfony1)cytidine(123) or 3’O-(methylsulfonyl)-2’,5’-di-O-tritylcytidine (124) with 1.5 equivalents of sodium tert-butoxide in N,N-dimethylformamide. Good yields of l-(2,3’-anhydro-~-~-xylofuranosyl)cytosine (125) and the corresponding ditrityl ether (126) were obtained.412

ROC*

MsO

(123) R = H (124) R = Tr

(125)R = H (126)R = Tr

The first example of a 2,2’-anhydronucleoside was prepared by Todd and coworker^'^ during the deacetylation of 3’,5’-di-O-acetyl2‘-O-p-t0lylsulfonyluridine (127) with methanolic ammonia. A crystalline anhydronucleoside was obtained, and it was shown to be 1-(2,2’-anhydro-~-~-arabinofuranosyl)uracil (2,2’-anhydrouridine) (128); the anhydride was also prepared from 5‘-O-acetyl-2’-O-p-tolylsulfonyluridine (129). Acid hydrolysis afforded l-p-D-arabinofuranosyluracil(130), identical with spongouridine. l-p-D-Arabinofuranosylthymine (131)was synthesized concurrently by a similar procedure, and was shown to be identical with s p o n g ~ t h y m i d i n e . ~ In~ *this ~~~ synthesis, methanesulfonylation of 5-methyl-5‘-O-trityluridine gave (411) (412) (78) (82) (413)

E. Benz, N. F. Elmore, and L. Goldman,]. Org. Chem.,30,3067 (1965). Y. Mizuno and T. Sasaki, Tetrahedron Lett., 4579 (1965). D. M. Brown, A. R. Todd, and S. Varadarajan,]. Chem. Soc., 2388 (1956). J. J. Fox, N. Yung, and A. Bendich,]. Amer. Chem. Soc., 79,2775 (1957). J. J. Fox and N. Yung, Fed. Proc., 15,254 (1956).

S U L F O N I C ESTERS OF CARBOHYDRATES: PART I1

A

c

O

RO

C

OTs

e

H

O

HO

C

V

H

O

C

181

e

HO

(127) R = Ac

(130) R = H

(129) R = H

(131) R = Me

an amorphous product giving a low analysis for sulfur, and it was later suggested that this product was mainly the 2,2’-anhydronucleoside.414With the 2-thio analog (132), methanesulfonylation at room temperature gave the 2,2’-anhydro-2-thio derivative (133) directlyq414

l-[2-O-(Methy~sulfonyl)-~-~-xylofuranosyl]uracil gave a 2,2’-anhydride on treatment with one equivalent of dilute alkali,407and ~-P-Dxylofuranosylthymine gave415a 3’,5’-O-isopropylidene-2’-O-(methylsulfonyl) derivative (134) which was more resistant to internal displacement than, for example, 127 or 129. However, treatment of 134 with boiling, dilute sodium hydroxide gave the 2,2’-anhydride (135) in 78% yield.416A somewhat similar displacement to give the dianhydronucleoside 136 was effected with sodium tert-butoxide in N,N-dimethylf~rmarnide.~~* (414) G. Shaw and R. N. Warrener, J . Chem. SOC.,50 (1959); Proc. Chem. SOC.,81 ( 1958). (415) J. J. Fox, N. Yung, J. Davoll, and G. B. Brown,J. Amer. Chem. SOC., 78, 2117 (1956). (416) J. J. Fox, J. F. Codington, N. C. Yung, L. Kaplan, and J. 0. Lampen, /. Arner. Chem. SOC., 80,5115(1958).

D. H. BALL AND F. W. PARRISH

182

Treatment of 2‘,3’,5’-tri-0-(methylsulfonyl)uridine (137) with one equivalent of sodium hydroxide in aqueous ethanol gave high yields of the 2-2’-anhydride (138), demonstrating the favored formation of the cis-fused, five-membered ring-~ystem.~~’ When aqueous solutions of 138 or of the corresponding S’-O-benzoyl (139) or 5‘-deoxy (140) derivatives were boiled for one hour, good yields of the D-lyxofuranosyluracil derivatives 141, 142, and 143 were obtained.418The mechanism of this reaction involves initial hydrolysis of the 2,2’anhydro bond, intramolecular displacement at C-3’, and, probably, rearrangement to the 2,2’-anhydride prior to hydrolysis.382

M

s

O

C

g

R

V

R

HC HO

e

MsO

MS

ME

C

(138) R = OMS

(137)

(139) R = OBz (140) R = H

(141) R = OMS (142) R = OBz (143) R = H

The favored, intramolecular displacement at C-3’ in the presence of a 5’-methylsulfonyloxy group, observed402with 3’,5’-di-O-(methylsulfony1)thymidine (115), was also evident in the formation,382from 1-[2,3,5-tri-O-(methy1sulfonyl)-~-~-arabinofuranosyl]uracil (144), Of 2,3’ - anhydro - 1- [2,5-di - 0 (methylsulfonyl)-~-~-lyxofuranosylluracil (145). (417) (418)

-

J. F. Codington, R.Fecher, and J . J. Fox,J.Amer. Chem. SOC., 82,2794 (1960). R.Fecher, J. F. Codington, and J. J. FoxJ. Amer. Chem. SOC., 83,1889 (1961).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

183

I

MsO (144)

For pyrimidine nucleosides, the ease of anhydro-ring formation by intramolecular displacement is in the order 2,2‘ > 2,3’ > 2,5’. Anhydrocytidine derivatives are formed more readily than the corresponding anhydrouridines, and N4-acylation further enhances the nucleophilicity of the cytosine residue.419N-Acyl-2,2’-anhydro-l-/3D-arabinofuranosylcytosines are also more susceptible to hydrolysis at pH 7 than either 2,2’-anhydrouridine or 2,2’-anhydrocytidine. The product isolated after p-toluenesulfonylation of a mixture containing N-acetyl-3’-5’-di-O-acetylcytidine(146) was N-acetyl-l-(3,5-di-Oacetyl-p-D-arabinofuranos yl)cytosine (149). The 2 ’-0-p-tolylsulfonyl intermediate (147) presumably cyclizes, to give the 2,2’-anhydride (148), which is rapidly hydrolyzed4lgto 149.

a HSJAc

0

(147)

R=H R = OTs

I

/

AcOCH,

ys/ AcO

AcO (146)

HNAc

NAc

(148)

The first 2,2’-anhydronucleoside containing a hexopyranosyl ring was prepared by treatment of 1-[3-acetamido-4,6-0-benzylidene3deoxy-2-0-(methylsulfonyl)-/3-~-glucopyranosyl] uracil (150) with sodium ethoxide. Intramolecular displacement of the methylsulfonyloxy group by the uracil residue (instead of by the truns3’-acetamido (419) H. P. M.Fromageot and C . B. Reese, Tetrahedron Lett., 3499 (1966).

D. H. BALL AND F. W. PARRISH

184

group) gave420the 2,2'-anhydronucleoside 151. When 1-[2-deoxy-3,4di-0-(methylsulfonyl) -P-D-erythro-pentopyranosyl] thymine was treated with one equivalent of potassium tert-butoxide in N,N-dimethylformamide at loo", intramolecular displacement of the 3'-methylsulfonyloxy group occurred, to give a 2,3'-anhydron~cleoside.~~~

(151)

The first example of a pyrimidine anhydronucleoside containing an imino bridge was prepared by treatment of 2,5'-anhydro-3'-0(methylsulfony1)thymidine (152) with liquid ammonia at room temp e r a t ~ r e . *Under ~~ these reaction conditions, displacement of the 3'-methylsulfonyloxy group of the presumed isocytidine intermediate (153) gave 2,3'-imino-l-(2-deoxy-~-~-thTeo-pentofuranosyl)~ymine (154). Unlike the oxygen-bridged analog, 154 could not be hydrolyzed by alkali.

,s

Me

"OC@ I

MSb

MsO (152)

(1 53)

(420) K. A. Watanabe and J. J. FoxJ. Org. Chem., 31,211 (1966). (421) G. Etzold, R. Hintsche, and P. Langen, Tetrahedron Lett., 4827 (1967). (422) I. L. Doerr, R. J. Cushley, and J. J. Fox,J. Org. Chem., 33,1592 (1968).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

185

b. Purine Anhydronucleosides. - Intramolecular displacements of sulfonyloxy groups in purine nucleosides are usually effected by N-3, and the ease of such displacements has been related to the basicity of the heterocyclic ring.400Todd and coworkers found that p-toluenesulfonylation of 2’,3’-O-isopropylideneadenosine(155) gives a mixture of the 5’-O-p-tolylsulfonyl derivative (156) and an ionic p toluenesulfonate (157) (formed by intramolecular displacement of the 5’-p-tolylsulfonyloxy They also showed that 156 is converted into 157 by heating in acetone (68% yield after 1.25 hours at 100”) or p-dioxane (an almost quantitative yield after boiling for 1 hour), and that attempted iodide displacements with sodium iodide in acetone give the iodide (salt) (158) of the anhydronucleoside. The

(155) R = H (156) R = Ts

(157) X = OTs

(158) X = I

structure of the anhydronucleoside was established by x-ray diffraction, and its formation afforded further confirmation (on stereochemical grounds) of the P-D-linkage between the D-ribofuranosyl group and the base. 3’-O-Acetyl-2’-deoxy-5’-O-p-tolylsulfonyladenosine also gave a good yield of the 3,5’-anhydride under similar conwas cyclized in boiling d i t i o n ~ and , ~ ~5’-O-p-tolylsulfonyladenosine ~ p-Toluenesulfonylation of the (less basic) 2’,3’-O-isopropylideneinosine (159) appears to be uncomplicated by anhydronucleoside formation, and good yields of the 5’-O-p-tolylsulfonyl derivative 160 are readily obtained.424It had been reported400that 160 is resistant to anhydronucleoside formation, but Holmes and Robins have shown that a good yield of the 3,5’-anhydride 161 can be obtained by boiling (423) S. H. Mudd, G. A. Jamieson, and G. L. Cantoni, Biochim. Biophys. Acta, 38, 164 (1960). (424) P. A. Levene and R. S. Tipson,]. B i d . Chem., 111,313 (1935).

D. H. BALL AND F. W. PARRISH

186

a solution of 160 in p-dioxane for 4 h0urs,4~~ conditions somewhat more vigorous than are necessary for 156. Nucleophilic displacement of the 5’-p-tolylsulfonyloxy group of 160 by methanethioxide ion in N,N-dimethylformamide afforded a 90% yield of the 5’-methylthio d e r i v a t i ~ e , but ~ ~ ~the . ~ iodide ~~ displacement was shown to give a mixture of products, among them the a n h y d r o n u c l e ~ s i d eVery . ~ ~ ~low yields of 5‘-methylthio derivatives were initially obtained in displacement reactions with 156 and methanethioxide i0ns.426*427 This result could have been due, in part, to the use of crude preparations of 156 (containing anhydronucleoside), as well as to the reaction conditions. Several 5’-alkylthio derivatives have been prepared in good yields from 156 and alkanethioxides in liquid ammonia.428 p-Toluenesulfon ylation of 2’,3’-O-isopropylideneguanosine(162) gives only the covalent p-toluenesulfonate 163, but treatment of 163 with sodium iodide in 2b-hexanedione for 4 hours at 100-110” gave429a good yield of the anhydronucleoside 164. In the absence of sodium iodide, intramolecular displacement also occurred under these conditions, to give425the ionic p-toluenesulfonate 165. The 3,5’-anhydro (instead of the possible 2,5’-imino) structure was established by Holmes and Robins.425Intermolecular displacement of the 5’-p-tolylsulfonyloxy group of 163 was, however, achieved with sodium ethanethioxide in ethanol at room temperature.429

(159)R = (160)R = (162)R = (163)R =

(425) (426) (427) (428) (429)

R‘ = H H, R‘= T s NH2, R‘= H NH2, R’ = Ts

(161)R = H, X = OTs (164)R = N&, X = I (165)R = N&, X = OTe

R.E.Holmes and R. K. Robins,]. Org. Chem., 28,3483(1963). J. Baddiley, 0. Trauth, and F. Weygand, Nature, 167,359(1951). F.Weygand and 0.Trauth, Chem. Ber., 84,633(1951). R.Kuhn and W. Jahn, Chem. Ber., 98,1699(1965). E.J. Reist, P. A. Hart, L. Goodman, and B. R. Baker,]. OTg. Chem., 26, 1557 (1961).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

187

5‘-O-Methylsulfonyl derivatives of the aminonucleoside moiety of puromycin were found to quaternize even more readily than 5 ’ - 0 p-tolylsulfonyladenosine derivative^."^ Thus, the 2’,3’-carbonate 166 or the N-(benzyloxycarbonyl) derivative of 9-[3-amino-3-deoxy-5-0(methylsulfonyl)- p - D -ribofuranosyl] - 6 - (dimethylamino)purine cyclized in high yield when kept at room temperature, or on boiling in chloroform solution for 2 hours; the anhydronucleoside 167 was obtained in 91% yield, and this result established the P-D-glycosidic linkage .430

The anomeric configuration of tubercidin (7-deazaadenosine) was similarly shown to be p-D, as the 2’,3‘-O-isopropylidene-5’-O-ptolylsulfonyl derivative underwent intramolecular quaternization in boiling acetone.431 The use of acetic anhydride as the solvent facilitates432intermolecular iodide displacements at C-5’ of 156. Acylation of the heterocyclic base occurred at N-1 (as indicated by infrared spectroscopy) during the reaction, and this inhibited occurrence of intramolecular displacement. The electron-withdrawing effect of the acyl group presumably diminishes the nucleophilicity at N-3. N-Formyl-2’,3’-O-isopropylidene-5’-O-p-tolylsulfonyladenosinealso reacts intermolecularly with lithium chloride or sodium azide in methyl sulfoxide to give 5’-chloro and 5’-azido derivatives.432 9-[2,3-O-Isopropylidene-5-O-(methylsulfonyl)-~-~-lyxofuranosy~] adenine (168) is very resistant to nucleophilic displacements at (430) B. R.Baker and J . P. JosephJ. Amer. Chem. SOC., 77,15(1955). (431) Y. Mizuno, M. Ikehara, K. A. Watanabe, S. Suzaki, and T. Itoh,J. Org. Chem., 28,3329(1963). (432) W. Jahn, Chem. Ber., 98,1705(1965).

188

D. H. BALL AND F. W. PARRISH

C-5’, and models indicate a high degree of steric hindrance to approaching n u ~ l e o p h i l e s .The ~ ~ ~2’-3’-O-isopropylidene group must also prevent anhydronucleoside formation, as, when this group was removed (by 90% aqueous acetic acid at 100” for 18 hours), intramolecular displacement of the 5’-methylsulfonyloxy group also occurred, to give433the 3,5’-anhydride 169.

The 2’-O-p-tolylsulfonyl derivative of 8-hydroxyadenosine underwent intramolecular sulfonate displacement when heated at 100-105” with sodium benzoate in N,N-dimethylformamide, and the 2’,8anhydro structure (170)was indicated.434A derivative of 2‘,8-anhydroguanosine (171) was obtained by a similar method.435 8-Hydroxy-2’,3’-0-isopropylidene-5’-O-(methylsulfonyl)guanosine (172) and the 8-bromo analog (173) appear to cyclize mainly by N-3 parti~ipation,4~~ but treatment of 173 with thiourea in boiling p dioxane gave 5’,8-anhydro-2’,3’-0-isopropylidene-8-mercaptoguanosine43e(174).An earlier rep01-P of the formation, by similar treatment, of a 5’,8-anhydro-8-mercaptoguanosine derivative from an 8-bromodi-O-(methylsulfony1)guanosineis less well documented.

3. Ring Contractions During Sulfonate Displacements C. L. Stevens and coworkers were the first to demonstrate ring contraction during attempted displacement reactions with 4-O-sulfonyl(433) E.J. Reist, D. F. Calkins, and L. Goodman,J. Org. Chem.,32,169(1967). (434) M. Ikehara, H.Tada, K. Muneyama, and M. Kaneko, J . Amer. Chem. SOC., 88, 3165 (1966). (435) M. Ikehara and K. Muneyama,]. Org. Chem., 32,3039(1967). (436) M. Ikehara and K. Muneyama,]. Org. Chem., 32,3042(1967). (85) M. Ikehara, H. Tada, and K. Muneyama, Chem. Pharm. Bull. (Tokyo), 13,639 (1965).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

OH

189

OH

(112) R = OH (113) R = Br

D-mannose derivative^.^^' All such reactions that they examined with sulfonates of methyl 6-deoxy-2,3-0-isopropylidene-cr-~-mannopyranoside (175) resulted in the formation of furanoside products, and this result probably accounts for some of the conflicting accounts With of desulfonylation of the L enantiomorph of 175 (R = Ts).322,438 acetate ion in boiling N,N-dimethylformamide, the methanesulfonate (175, R = Ms) gave, after saponification, a 65% yield of a mixture of methyl 6-deoxy-2-3-0-isopropylidene-cr-~-talofuranoside (176, R = OH) with methyl 6-deoxy-2-3-0-isopropylidene-~-~-allofuranoside (177, R = OH) in the ratio of 7: 1. Displacements at C-5 of the sulfonylated furanose derivatives 176 and 177 (R = OMs,OTs, or OBs) (437) C. L. Stevens, R. P. Glinski, K. G. Taylor, P. Blumbergs, and F. Sirokman, J . Amer. Chem. Soc., 88,2073 (1966). (438) I. E. Muskat,J. Amer. Chem. SOC., 57,778 (1935);P. A. Levene and J. Compton, ibid., 57,777 (1935).

D. H. BALL AND F. W. PARRISH

190

Me

Me

OMe

(a-D-manno) (175) R = M s , Ts, or B s

(Q -D-td0 )

(176)

proceeded normally (SN2) without further rearrangement. Treatment of the sulfonates 175 (R = Ms or Ts) with lithium a i d e in N,Ndimethylformamide gave complex mixtures from which, after reduction, the ~ - t a Z oamine (176, R = NH,) was isolated in 25%yield. From the reaction of sodium azide in boiling N,N-dimethylformamide with the L enantiomorph of 175 (R = Ms) (that is, 29), the ~ - t a Z oazide (L enantiomorph of 176, R = N3) was isolated in 34% yield.316,439 As previously mentioned (see p. 153), 29 reacts with hydrazine to give, after reduction, a 27%yield of the SN2displacement product, namely, methyl 4-amino-4,6-dideoxy-2,3-O-isopropylidene-~-~-talopyranoside.316With yields in the region of 30%, mechanistic arguments cannot be conclusive without a knowledge of the other products, but it has been suggested318that, were a molecule of hydrazine hydrogenbonded to 0-2, 0-3, and 0 - 5 of 29, it would be ideally placed for rearside attack at (2-4. As s u s p e ~ t e d , 4 the ~ ~reaction of the L enantiomorph of 165 (R = Ts) with potassium thiolbenzoate in N,N-dimethylformamide, previously thought to give a 4-S-benzyl-4-thio talopyranose derivative,lg6was shown to give the ring-contracted product (L enantiomorph of 176, R = SCHzPh).440 The highest yield from this reaction was196,440 only 11%, and it would be of interest to characterize the other products, because it is possible that ring-contraction may not be the main occurrence in this reaction. It has been shown that thiolacetate displacements of the 4-methylsulfonyloxy group of methyl 2,3-di-O-methy1-4,6-di-O-(methylsulfony1)-P-D-galactopyranosideor methyl 6-deoxy-2,3-di-O-methyl-4-0(methylsulfonyl)-/3-D-galactopyranoside give good yields (65 and 55 %, respectively) of crystalline products having D-glucopyranoside s t r ~ c t u r e s For . ~ ~these ~ ~ ~ D-galactopyranoside ~~ methanesulfonates, ring contraction is, therefore, not a major consideration during dis(439) S. Hanessian, Chem. Commun., 796 (1966). (440) C. L. Stevens, R. P. Glinski, G . E. Gutowski, and J. P. Dickerson, Tetrahedron Lett., 649 (1967). (441) L. N. Owen, Chem. Commun.,526 (1967).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

191

placements, and this behavior has been attributed to the fact that the C-5-ring oxygen bond is not trans-antiparallel to the C-4-sulfonate bond (as is the case with D-gluco- or D-manno-pyranose 4-sulfonates439.441). However, the major products from benzoate, azide, or thiocyanate displacement reactions with methyl 2,3-di-O-benzoyl(or acetyl)-4,6-di-0-(methylsulfonyl)-a-~-glucopyranoside (55) were Dgalactopyranoside derivative^^^^,^^^,^^^ (the diazido product 57 was obtained in 70% yield), and ring-contraction, if it occurs, must also be a minor pathway in these reactions. Analysis of the solvolysis products obtained by heating a solution of methyl 4-O-(p-nitrophenylsulfonyl)-a-~-glucopyranoside in acetate buffer (pH 4-5) at 100" showed the formation of methyl a-D-glucopyranoside (50%), methyl a-D-galactopyranoside (8%), methyl p-Laltrofuranoside (8%), D-glucose (8%), and traces of other products.442 Ring contraction is, therefore, a minor pathway in this reaction, and it is interesting that, when contraction does occur, the product (methyl p-L-altrofuranoside) results from inversion at C-4 and C-5. None of the C-5 epimeric compound (methyl a-D-galactofuranoside) could be detected.442 A two-stage mechanism to explain the predominant retention of configuration at C-5 during reactions of the methyl 6-deoxy-2,3-0isopropylidene-D-mannopyranoside sulfonates (175) was suggested by Owen.441The first stage involves migration of the sulfonyloxy group from C-4 to C-5, concerted with ring-contraction. This stage may well take place through ion-pair intermediates, the furanoid and pyranoid isomers being in equilibrium as shown in Scheme 1, and this stage could involve the nonclassical ion depicted. Nucleophilic displacements at C-5 of the furanoid ion-pair would then give the major products observed. The preponderance of D-glucopyranoid and D-altrofuranoid isomers in the solvolysis products of methyl 4-0-(p-nitrophenylsulfonyI)-a-~glucopyranosideM2is probably a reflection of increased SN1character under these conditions. It may also be significant that ring contraction has, so far, only been found to be the major pathway for 6-deoxy-2,3-0isopropylidene-D-mannopyranosederivatives. Direct displacement at C-4 is hindered by the p-trans-axial at C-2, and, in addition, steric constraints imposed by the five-membered acetal ring may well favor ring contraction. Displacements of the p-tolylsulfonyloxy group in the closely related methyl 2,3-O-isopropylidene-4-O-p-tolylsul(442) P. W. Austin, J. G . Buchanan, and D. G . Large, Chem. Comrnun., 418 (1967).

192

D. H. BALL AND F. W. PARRISH

& %:

H

X Scheme 1

fonyl-a-D(or L)-lyxopyranoside (178) appear to give mainly D-ribopyranose derivative^,^^.^^^ as the physical constants reported for one of the displacement products, namely, methyl 4-S-acetyl-2,3-0-isopropylidene-4-thio-P-~-ribopyranoside,4~~ were different from those of

L

Isomer (178)

(443) E. J . Reist, D. E. Gueffroy, and L. Goodman, J. Arner. Chern. SOC., 85, 3715 (1963);86,5658(1964). (444) R. L. Whistler, W. E. Dick, T. R. Ingle, R. M. Rowell, and B. Urbas, J . Org. Chem., 29,3723(1964).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

193

methyl 5-S-acetyl-2,3-0-isopropylidene-5-thio-~-~-ribofuranoside,~~~ the product that would have resulted had ring contraction occurred. It would be interesting to examine the reactions of 178 with other nucleophiles, but, tentatively, it may be concluded that for the compounds depicted in Scheme 1, the methyl group has a stabilizing effect on the furanoid carbonium ion. With 178, the furanoid isomer would be a relatively unstable, primary carbonium ion. Ring contraction was also observed445during the solvolysis of (179) and methyl 3-0-(p-nitrophenylsulfonyl)-a-~-mannopyranoside (180). of methyl 3-O-(p-nitrophenylsulfonyl)-a-~-glucopyranoside The products, 3-deoxy-3-C-formyl-a-~-lyxofuranoside (181) and -xylofuranoside (182),were obtained in approximately 40% yields as their crystalline hemiacetals. The same products were obtained by treatment of the corresponding 3-amino-3-deoxy compounds 183 and 184 with nitrous a ~ i d , 4and ~ ~ they , ~ ~are ~ clearly the result of a WagnerMeenvein type of rearrangement.447Solvolysis of methyl 2 - 0 - ( p nitro p heny lsulfony 1)-a-D-glucopyranoside, followed by re duct ion with borohydride, gave a 26 % yield of 2,5-anhydro-~-mannitol,~~~ again showing that the main product of solvolysis is the same as that from treatment of the corresponding amino sugar with nitrous 0.

(179) R = O N s , R‘ = OH, R” = H

(181) R = OH, R’ = H

(180) R = O N s , R’ = H, R” = OH

(182) R = H, R‘ = O H

(183) R = N&, R’ = OH, (184) R = NH,,

R” = H

R’= H, R”

= OH

4. Alkoxyl-group Participation Participation of alkoxyl groups in displacements of sulfonyloxy in 1958, and groups was first discussed by Winstein and (445) P. W. Austin, J. G. Buchanan, and R. M. Saunders, Chem. Commun., 146 (1965); J. Chem. SOC. (C),372 (1967). (446) S. Inoue and H. Ogawa, Chem. Pharm. Bull. (Tokyo), 8,79 (1960). (447) Ref. 355,pp. 105 and 303. (448) A. B. Foster, E. F. Martlew, and M. Stacey, Chem. Ind. (London), 825 (1953). (449) S. Winstein, E. Allred, R. Heck, and R. Glick, Tetrahedron, 3,1(1958).

D. H. BALL AND F. W. PARRISH

194

details of their extensive investigations have now been published.450-453Participation was found to be particularly favorable when the intermediate, cyclic oxonium ion is five-membered, and it is also evident, but to a smaller extent, when a six-membered, cyclic ion is involved; almost all of the difference is the result of a more negative entropy of activation for formation of the six-membered ring.453 The first example of methoxyl participation reported in carbohydrate chemistry was the migration454of a methoxyl group from C-1 to C-4 during an attempted benzoate displacement reaction with 2,3,5tri-O-benzyl-4-O-p-tolylsulfonyl-~-ribose dimethyl acetal (185). Instead of the 4-O-benzoyl-~-lyxosederivative expected, the isomeric 1-0-benzoyl-2,3,5-tri-O-benzyl-4-O-methyl-~-lyxose methyl hemiacetal (187) was obtained, presumably by way of the cyclic, oxonium ion 186. Solvolysis of the related uldehydo sugar, namely, 2,3,5-tri-0benzyl-4-O-p-tolylsulfonyl-~-ribose, readily gives 2,3,5-tri-O-benzylL-lyxofuranose, and the reaction probably involves participation by the free aldehyde H MeOCOMe

I

HCOCH,Ph I HCOCH,Ph

I

HCOTs I H,COCH,Ph (185)

-

H C(0Me)OBz I

BzO@

--

HCOCbPh

I

F MeOCH

H OCH,Ph I

H,COC&Ph

(187)

Solvolysis of methyl 6-deoxy-2,3-0-isopropylidene-4-O-(methylsulfony1)-a-D-mannopyranoside (175, R = Ms) at 170"in 9: 1p-dioxanewater containing an excess of sodium hydrogen carbonate or sodium hydroxide gave,437as the major product, 6-deoxy-2,3-0-isopropylidene-5-O-methyl-~-talofuranose(189). The authors postulated initial ring-contraction, to give an intermediate 5-0-(methylsulfonyl)-~-~E. L. Allred and S. Winstein,]. Amer. Chem. SOC., 89,3991 (1967). E. L. Allred and S. Winstein,J. Amer. Chem. SOC., 89,3998 (1967). E. L. Allred and S. Winstein,]. Amer. Chem. SOC., 89,4008 (1967). E. L. Allred and S. WinsteinJ. Amer. Chem. SOC., 89,4012 (1967). N. A. Hughes and P. R. H. Speakman, Chem. Commun., 199 (1965);J.Chem. SOC. (C),1182 (1967). (455) N. A. Hughes and P. R.H. SpeakmanJ. Chem. SOC.(C), 1186 (1967).

(450) (451) (452) (453) (454)

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

195

allofuranoside (177, R = OMS), followed by methoxyl migration to give 189; and, in support of this mechanism, a high yield of 189 was also obtained from methyl 5-O-(p-bromophenylsulfonyl)-6-deoxy-2,30-isopropylidene-P-L-allofuranoside (177, R = OBs). The tricyclic oxonium ion 188 is, presumably, the intermediate in the methoxyl migration.

(175) R = M s

or (177) R = OBs

Methoxyl participation, by way of the oxonium ion 191, probably accounts for the ready ~ o l v o l y s i s(in ~ ~aqueous ~ acetic acid at 100" for 2.25 hours) of the p-tolylsulfonyl group from 1,2-O-isopropylidene3,5-di-0-methyl-6-0-p-tolylsulfonyl-a-~-g~ucofuranose (190). 3,5-Di0-methyl-D-glucose was obtained in only 39% yield, and a more detailed study of this reaction should prove interesting. TsO,

-

3,5-di-0methyl-D-glucose

In the preceding examples of M e 0 - 5 participation, the participating methoxyl group remains in the molecule, but, in the examples of anchimeric assistance by benzyloxy groups so far reported, the intermediate oxonium ion breaks down by benzyl-oxygen fission, to give cyclic products together with products derived from benzyl sulfonic ester. R. Barker and coworkers found that the 4-benzyloxy group of 4-O-benzyl-l-O-p-tolylsulfonyl-1,4-pentanediol (192) participates in solvolysis of the sulfonyloxy group, to give 2-methyltetrahydrofuran (193) and the solvolysis products of benzyl p-toluene~ulfonate.~~~ (456) C. R. Gray, F. C. Hartman, and R. Barker,J. Org. Chem.,30,2020 (1965).

D. H. BALL AND F. W. PARRISH

196

Three 2,3,4-tri-O-benzyl-1,5-di-O-p-tolylsulfonylpentitols [xylo, ribo, and D-arabino (~-2yxo)Ialso gave high yields of 1,4-anhydrides when boiled in 95% ethanol for 24 hours; the products and their rates of formation were rationalized on stereochemical grounds.456 C%Ph

I

CH,OTs

Me

-

CH,Ph

+

(192)

(PhCbOTs)

(193)

Solvolysis of methyl 2,3-di-O-benzyl-6-0-(methylsulfonyl)-/3-~galactopyranoside (194), by heating in aqueous methanol buffered with sodium acetate, gave crystalline methyl 3,6-anhydro-2-0-benzyl/3-D-galactopyranoside (196) in 50% yield, presumably457by way of the bridged oxonium ion 195.

Under similar conditions, 3-O-benzyl-l,2-O-isopropylidene-5,6di-0-p-tolylsulfonyl(or methylsulfony1)-a-D-ghcofuranose (197) also gave4583,6-anhydrides (199). The intermediate oxonium ion 198 is structurally analogous with 191.

R;3? @OR

? % ; R RO

RZCL?

O/CMe, (197)

R = M s or T s

(198) R =

Ms or Ts

(199)

R = Ms or Ts

(457) J. S. Brimacombe and 0. A. Ching, Carbohyd. Res., 5, 239 (1967);j . Chem. SOC. (C),1642 (1968). (458) J. S. Brimacombeand 0.A. Ching, Carbohyd. Res., 8,82(1968).

SULFONIC ESTERS OF CARBOHYDRATES: PART I1

197

Participation by the C-1-C-6 oxygen atom of the anhydro ring in the solvolysis (with potassium fluoride dihydrate-methanol at 130”for 15 hours) of 1,6-anhydro-3,4-0-isopropylidene-2-O-(methylsulfonyl)p-D-galactopyranose (200) gave mainly320the p-D anomer of methyl 2,6-anhydro-3,4-0-isopropylidene-~-talopyranoside (201),not 1,6-anhydro-3,4-O-isopropylidene-2-O-methyl-~-~-talopyranose (as previously reported3lg).

o)q - ”&1-

Me C - k 0

Me$ ‘ 0

OM8

(200)

‘,, 0

MeJ”&‘0

OMe

This Page Intentionally Left Blank

UNSATURATED SUGARS

BY R . J . FEWER Department of Chemistry. Birkbeck College. University of London. London. England

I . Introduction ......................................................... 199 200 I1. Glycals ............................................................ 1. Synthesis ....................................................... 200 2. Additions Involving Halogens and Hydrogen Halides . . . . . . . . . . . . . . . . 202 3. Addition of Nitrosyl Chloride and Dinitrogen Tetraoxide . . . . . . . . . . . . .206 209 4. Addition of Sulfur-containing Reagents ............................ 5. Methoxymercuration .............................................. 210 210 6. Hydroformylation .................................................. 7. Other Additions ................................................. 212 8. Rearrangement Reactions .......................................... 213 218 9. Other Reactions .................................................. 219 I11. 2-Substituted Glycals ................................................. 1. 2-Hydroxyglycals ............................................... 219 224 2. Other 2-Substituted Glycals ........................................ JV. 2,3-Unsaturated, Cyclic Compounds .................................. 226 1. Furanoid Derivatives ............................................ 226 231 2. Pyranoid Derivatives .............................................. V. 3,4-Unsaturated, Cyclic Compounds ................................... 246 1. Furanoid Derivatives ............................................ 246 249 2. Pyranoid Derivatives ............................................. 250 VI . 4,5-Unsaturated, Cyclic Compounds .................................. 1. Furanoid Derivatives ........................................... 250 252 2. Pyranoid Derivatives .............................................. VII . 5,6-Unsaturated, Cyclic Compounds ................................... 255 1. Furanoid Derivatives ............................................. 255 257 2. Pyranoid Derivatives ................................................ 260 VIII . Other Unsaturated, Cyclic Compounds .............................. 260 IX. Unsaturated, Acyclic Compounds ..................................... X . Nuclear Magnetic Resonance Features of Unsaturated Sugars ............ 265

I . INTRODUCTION “An area soon due for much further development” was the prophetic description of unsaturated-sugar chemistry used by the Editors in 199

200

R. J. FERRIER

the Preface to Volume 20, when this subject was last reviewed.’ The research reported in the succeeding four years has amply justified this view, and has led to appreciable developments in the understanding of this class of compounds and of their potential value in synthetic chemistry. These developments are here surveyed within the same guidelines as were used previously; that is, discrete compounds will be dealt with which have one carbon-carbon multiple bond within the main sugar-chain. As before, enols, enediols, enones, dienes, and dienones will not be considered, nor will unsaturated cyclitols or heterocyclic compounds composed partially of acyclic carbohydrates. The first derivatives having carbon substituents attached by a double bond at a branch-point have now been prepared by application of the Wittig reaction to suitably protected glycosulose compounds; these compounds are also arbitrarily excluded, as are ethers or acetals bearing unsaturated substituents. For the first time, unsaturated compounds have been derived from ketoses. These are, however, few in number and, in this Chapter, are discussed together with the structurally analogous aldosyl derivatives: a 4,5-unsaturated ketopyranosyl compound is thus, for example, treated along with the 3,4-unsaturated aldopyranoses (see p. 249). 1,2Unsaturated ketopyranosyl compounds have 5,6-unsaturated aldopyranoses as their structural analogs (see p. 257), and 2-ketopyranoses having a double bond at C-2 are relatable to the glycals or to 4,5unsaturated aldopyranoses; they are arbitrarily dealt with under the latter heading (see p. 252). Although no conventions have as yet been officially accepted for naming unsaturated sugars, the “enose” names used in the previous article’ are gaining wide acceptance and will again be employed. The terms “glycal” and “2-hydroxyglycal” are still in common usage, and will likewise be used, despite their indefensibility on systematic grounds. Several new methods, and improvements in existing methods, for introducing double bonds into carbohydrates have been reported (see pp. 201,215,219,232-234, and 255). 11. GLYCALS

1. Synthesis An additional means of synthesizing glycal derivatives has been established as a consequence of an unexpected finding made during (1) R. J. Ferrier, Aduan. Carbohyd. Chem., 20.67 (1965).

UNSATURATED SUGARS

201

work on the ring-opening of epoxides with metal alkyls,’ and details of the reaction whereby 4,6-O-benzylidine-~-allal (1, R = R’ = H) is

obtainable in 75% yield by treatment of methyl 2,3-anhydro-4,6-0benzylidene-a-D-allopyranosidewith methyllithium in ether have now been published.2 However, when this procedure was repeated by other workers, who used commercially prepared methyllithium, 4,6-0-benzylidene-2-C-methyl-~-allal (1, R = Me, R’ = H) was found to be the main product: but they confirmed that freshly prepared reagent afforded the unmethylated material (1, R = R’ = H). It was then shown that the original reaction proceeds as a result of epoxide ring-opening with lithium iodide, to give methyl 4,6-O-benzylidene2-deoxy-2-iodo-a-~-altropyranoside (2, R = I, R’ = H), followed by an

elimination p r o c e ~ s .This ~ leads to (a) an improved synthesis of 4,6-O-benzylidene-~-allal (1, R = R’ = H; 85%) involving specific preparation of the iodo intermediate (2, R = I, R’ = H) and treatment of it with methyllithium, and (b) the rationalization that the elimination proceeds by attack of nucleophilic methyl on iodine, followed by ejection of the methoxyl anion. By the same means, methyl 4,6-0benzylidene-2-deoxy-2-iodo-a-~-idopyranoside (3)gave 4,6-0-benzylidene-D-gulal (4). The nuclear magnetic resonance spectra of the unsaturated compounds produced in these studies were recorded and di~cussed.~*~ (2) A. A. J. Feast, W. G. Overend, and N. R. Williams,]. Chem. SOC., 7378 (1965). (3) M. Sharma and R. K. Brown, Can.]. Chem., 44,2825 (1966). (4) R. U. Lemieux, E. Fraga, and K. A. Watanabe, C a n . ] . Chem., 46,61(1968).

R. J. FERRIER

202

I

HO

HO (3)

(4)

A re-investigation of the reaction (conducted with methyllithium free from halide ion) which af€orded the methylated derivative (1, R = Me, R’ = H), indicated that it proceeded by way of methyl 4,6-0benzylidene-2-deoxy-2-C-methyl-a-~-altropyranoside (2, R = Me, R’ = H), because this compound [prepared by opening the corresponding 2,3-anhydroalloside with (methylsulfiny1)methylcarbanion, to give methyl 4,6 - 0- benzylidene - 2 - deoxy - 2- C - [(methylsulfinyl) methyl]-a-D-altropyranoside(2, R = MeSOCH,, R’ = H), followed by reduction with Raney nickel], on treatment with methyllithium, also gave the C-methyl compound (1,R = Me, R’ = H).5 More specifically, tri-O-aCetyl-D-gUlal has been isolated in low yield from the reaction of tri-0-acetyl-D-galactal and of 1,3,4,6-tetra-Oacetyl-2-deoxy-a-~-2yxo-hexopyranose with boiling acetic acid (see p. 214), and 4,6-0-benzylidene-3-O-methyl-~-allal has been prepared by reductive desulfurization of 2-thio derivatives (see p. 225). Wide variations in the yield of tri-O-acetyl-D-ghca1 obtained by the conventional treatment of 2,3,4,6-tetra-O-acety1-a-D-glucopyranosyl bromide with zinc dust in aqueous acetic acid have been attributed to differences in the activity of the zinc.6In some instances, pretreatment of inert samples of zinc with dilute hydrochloric acid rendered them active, but in no instance were copper or platinum salts useful promoters, as has been claimed.’ Treatment of 3,4,6-tri-Oacetyl-1,2-anhydro-~-glucopyranosewith sodium cobalt tetracarbony1 and carbon monoxide in ether gave tri-0-acetyl-D-glucal as the main product,6a but this does not offer a practical alternative synthesis of the compound.

2. Additions Involving Halogens and Hydrogen Halides

Lemieux and Fraser-Reid have extended their studies on the bromination and the chloro-, bromo-, and iodo-(methoxy1)ation of (5) M. Sharma and R. K. Brown, Can.]. Chem. 46,757 (1968). (6) C. D.Hurd and H. Jenkins,Carbohyd. Res., 2,240 (1966). (6a) A. Rosenthal and J. N. C. Whyte, Can.]. Chem., 46,2239 (1968).

UNSATURATED SUGARS

203

tri-0-acetybglucal to the chlorination of this compound, and to the investigation of these reactions with tri-O-aCetyl-D-galaCtal and 2,3-dihydro-4H-pyran.' Stereoelectronic and solvation effects operating during the reactions were discussed in detail, and the relative stabilities, in each case, of the cyclic halonium and 2-halo-C-1 carbonium ions were considered to be most significant in governing the modes of addition. In reactions involving iodine, the former are the important intermediates, and trans products consequently result; whereas, in chlorinations, the 2-substituted carbonium ions are formed more readily, and this results in formation of cis adducts. Results obtained with tri-O-aCetyl-D-glUCal are given in Table I; tri-0-acetylD-galaCtal, with minor exceptions, behaved similarly; and, consistent with these findings, iodo(methoxy1)ationof 4,6-O-benzylidene-~-allal has been shown to give methyl 4,6-0-benzylidene-2-deoxy-2-iodo-aD-altropyranoside (2, R = I, R' = H), isolable in 23% yield.4 TABLEI Percentage Yields of Products Obtained by Additions to Tri-O-acetyl-D-gluca1 Reaction Chlorination Bromination Chloro(methoxy1)ation Bromo(methoxy1)ation Iodo(methoxy1)ation

Cr-D-ghbco

P-D-glUCO

> 80

-

60 8

-

41 33 33

LY-D-maAAO

P-D-mannO

30 51 66 66

-

-

Other workers have re-examined the bromination of tri-0-acetyl-Dglucal followed by methanolysis,' and have confirmed that 2-bromo-2deoxyglycosides having the P-D-gluco and P-~-rnannoconfigurations preponderate; these compounds were obtained in crystalline form, and were characterized by chemical means8 In a similar way, the products of the chlorination of this compound have been re-examined, and the a-D-gluco adduct was isolated in 62.5% yield when carbon tetrachloride was used as the solvent.g In addition, the isomer previously assigned the ( Y - D - ~ u structurelo ~~o was obtained (yield, 12.5%), but was shown to be the p-Danomer on the basis of nuclear magnetic resonance evidence, coupled with the facts that itanomerized (7) R. U. Lemieux and B. Fraser-Reid, Can.J.Chem., 43,1460 (1965). (8) H. Nakamura, S . Tejima, and M. Akagi, Chem. Pharm. Bull. (Tokyo), 12, 1302 (1964). (9) K. Igarashi, T. Honma, and T. Imagawa, Tetrahedron Lett., 755 (1968). (10) M. S. Lefar and C. E. Weill,]. Org. Chem., 30,954 (1965).

204

R. J. FERRIER

to a mixture from which the C Y - D - ~ U ~ ~compound CJ was isolated in 90% yield, and reacted with methanol to give an a-D-glycoside derivative in 89% yield. cis-Products were, therefore, formed almost exclusively in this solvent, and were shown to be produced under kinetic control; hence, a four-centered transition-state mechanism of addition was proposed. However, when 4-methyl-l,3-dioxol-2-one was used as the solvent, truns products were mainly formed.” Chlorination with hydrochloric acid and N-chlorosuccinimide gave a mixture containing all four isomers, each of which was isolated in crystalline form by preparative, thin-layer chromat~graphy.~ Reaction of tri-O-aCetyl-D-glUCa1 with bromine in the presence of finely divided silver monofluoride gave a mixture of three products, each of which was a 2-bromo-2-deoxyglycosyl fluoride [ C X - D - ~ U ~ ~ C I (70%),a-D-gluco (9%), and P-D-gluco (21%)].12Alternatively, “BrF,” generated from N-bromoacetamide and hydrogen fluoride in ether at -80” gave the same products in 55, 9, and 30% yield, together with 7% of the 2-deoxyglycosyl a-fluoride, apparently formed by direct addition of hydrogen fluoride to the double bond. When iodine replaced bromine, analogous adducts were obtained: a-~-munno(60%), a-D-gluco (6%),and P-D-glUCO (34%),and “ClF” reacted in a similar way, but in this case, the main product was the P-D-gluco adduct.12a Most of the products were isolated in crystalline form, and were characterized chemically. l2 These findings are at variance with an earlier report’ that N-bromosuccinimide and anhydrous hydrogen fluoride in ether at low temperature cause addition, to give the cis-2-bromoglycosyl fluoride derivatives specifically. However, a re-investigation, by crystallographic methods, of the main product obtained from tri-O-acetyl-D-ghca1 by the original authors showed it to be 3,4,6-tri-O-acetyl-2-bromo-2deoxy-a-D-mannopyranosyl f l ~ 0 r i d e . lNuclear ~ magnetic resonance measurements of JFSHJ were in agreement with this structure, and confirmed that the crystalline product isolated, after deacetylation, from the corresponding reaction with di-0-acetyl-D-arabinal was 2-bromo2-deoxy-J3-~-arabinopyranosyl fluoride.14 Similarly, the crystalline, (11) K. Igarashi, T. Honma, and T. Imagawa, Abstracts Papers Amer. Chem. Soc. Meeting, 155,27c(1968). (12) L. D. Hall and J. F. Manville, Chem. Commun., 35 (1968);Can. ]. Chem., 47, 361 (1969). (12a) L.D.Hall and J. F. Manville, Can.]. Chem., 47,379(1969). (13) J. C. Campbell, R. A. Dwek, P. W. Kent, and C. K. Prout, Chem. Commun., 34 (1968);Carbohyd. Res., 10,71(1969). (14) P.W.Kent and J. E. G . Barnett,]. Chem. Soc., 6196 (1964).

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deacetylated compound obtained in 25 % yield from tri-0-acetyl-Dgalactal15 was confirmed to be the a-D-galacto isomer, but the major product isolated after treatment of tri-0-acetyl-D-glucal with Niodosuccinimide and anhydrous hydrogen fluoride in ether (initially described as the / ~ - D - T T Z U ~ ~adductl'j) O was shown to be 3,4,6-tri-Oacetyl-2-deoxy-2-iodo-a-~-mannopyranosyl f l ~ 0 r i d e .These l~ products are therefore seen to conform closely with expectations. As previously reported briefly,l on treatment of glycal acetates with lead tetraacetate and anhydrous hydrogen fluoride in dichloromethane, ring contraction occurs, instead of direct addition. Further descriptions, involving chemical, polarimetric, and nuclear magnetic resonance (19Fand 'H) evidence, of the analyses of the products have now been published; thus tri-0-acetyl-D-glucal and di-0-acetyl-Darabinal gave, it is claimed, 2,5-anhydro- 1-deoxy-1,l-difluoro-Dmannitol and -D-ribitol, re~pectively.'~However, ethyl 4,6-di-0acetyl-2,3-dideoxy-c-~-~~~~~~o-hex-2-enopyranoside (5, R = Et) also gave, after deacetylation, this D-mannitol derivative," so the configuration of C-3 in the products cannot be assigned without specific proof; and in the opinion of the writer, all of the reactions probably proceed by way of 2,3-unsaturated intermediates. The authors suggested, alternatively, that the glycoside (5, R = Et) reacted to give a glycal derivative prior to halogenation, but, in view of the known relative stabilities of glycals and 2,3-unsaturated isomers (see p. 214) in acidic media, this would seem improbable. YH,OAc

In trichlorofluoromethane at low temperatures, trifluoro(fluoro-oxy)methane (FOCF,) added to the double bond of tri-O-acetyl-D-gluca1 to give the trifluoromethyl 2-deoxy-2-fluoroglycosides formed by direct, cis addition, together with the two corresponding 2-deoxy-2(15) P.W. KentandM. R. FreemanJ. Chem. Soc. (C),910(1966). (16) K. R.Wood, P. W. Kent, and D. FisherJ. Chem. SOC. (C),912(1966). (17) P.W. Kent and J. E. G. Barnett, Tetrahedron, S u p p l . 7,69 (1966);K. R. Wood and P. W. Kent,J. Chem. Soc. (C),2422 (1967).

206

R. J. FERRIER

fluoroglycosyl fluorides. Hydrolysis of the a-D-ghco adducts afforded a means of obtaining 2-deoxy-2-fluoro-D-glucose. An anomaly' regarding the nature of addition of hydrogen bromide to tri-O-aCetyl-D-glUCal, which arose from Fischer's claim that a stable carbon-bromine bond was present in the product, has now apparently been resolved. From the reaction products obtained by conducting the addition in acetic acid, 4,6-di-O-acetyl-3-bromo-2,3dideoxy-a-D-arabino-hexose(6, X = Br) was isolated in crystalline FH,OAc

AcOO

O

H

(6)

form after chromatographic separation.18 3,4,6-Tri-O-acety1-2-deoxya-D-arabino-hexose (6, X = OAc) was also isolated from the mixture of products, indicating that direct addition to the double bond had occurred concurrently in the expected way (the labile halide had been removed during the processing), and the brominecontaining compound was, most probably, formed by addition to a 2,3-unsaturated, rearrangement product of the type known to arise from tri-0-aCety1-Dglucal in the presence of acid (see p. 214).The reaction of hydrogen fluoride with tri-O-acetyl-D-gluca1also involves an allylic rearrangement, with the formation of a 2,3-unsaturated product (see p. 216). Alternatively, when the addition of hydrogen bromide is performed in nonpolar solvents, simple addition occurs, to give 3,4,6-tri-Oacetyl-2-deoxy-~-arabino-hexopyranosyl bromide, from which 3,4,6tri-O-acetyl-2-deoxy-~-~-ambino-hexopyranosylethylxanthate has now been prepared.lg In the same way, the analogous compound was synthesized from 3,4-di-O-acety~-6-O-p-to~y~su~fony~-~-glucal~~ a new glycal derivative. 3. Addition of Nitrosyl Chloride and Dinitrogen Tetraoxide

For the addition of these reagents, which were referred to in the previous article,l to acetylated glycals, details have now been pub(17a) J. Adamson, A. B. Foster, L. D. Hall, and R. H. Hesse, Chem. Commun., 309 (1969). (18) T. Maki and S. Tejima, Chem. Pharm. Bull. (Tokyo),15,1069 (1967). (19) T. Maki, H. Nakamura, S. Tejima, and M. Akagi, Chem. Pharm. Bull. (Tokyo), 13,764 (1965). (20) T. Maki and S. Tejima, Chem. Pharm. Bull. (Tokyo),15.1367 (1967).

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lished, and the products have been utilized in highly attractive and important syntheses of a-D-glycosides and aminodeoxy-a-D-glycosides. From tri-O-aCetyl-D-glUCa1 with nitrosyl chloride, 3,4,6-tri-O-acetyl2-deoxy-2-nitroso-a-D-g~ucopyranosyl chloride (7, X = C1) is obtain-

able (in dimeric form) in 80% yield, and the factors leading to the formation of this stereoisomer are believed to be those operating during the chlorination of this compound (see p. 203). The analogous a-D-galactosyl chloride was similarly prepared from tri-0-acetyl-Dgalactal in 84 % yield, but attempts to obtain the di-0-acetyl-D-xylal adduct led to the isolation of 3,4-di-O-acetyl-2-nitro-D-xylal (8). In

related fashion, reaction of acetylated glycals with dinitrogen tetraoxide gave dimeric, acetylated 2-deoxy-2-nitroso-a-D-g~ycosy~ nitrates (for example 7, X = ONOz)when conducted in ether at 0". However, when dichloromethane at -70" was employed as the solvent, no such compounds were obtained; instead, acetylated 2-nitroglycals (not formed by way of the 2-nitroglycosyl nitrates) were the products.21 Several reports have appeared on the important application of the nitrosyl chloride adducts. On treatment with alcohols or phenols in NJ-dimethylformamide at room temperature, those obtained from tri-0-acetyl-D-gluca1 and -galactal gave the corresponding tri-0acetyl-2-oximo-a-~-hexopyranosides in high yields (the aglycon entering stereospecifically), as exemplified by the synthesis of methyl (21) R. U. Lemieux, T. L. Nagabhushan, and I. K. ONeill, Can. J . Chem., 46, 413

(1968).

208

R. J. FERRIER

2,3,4 -tri - 0-acetyl- 6 -0- (3,4,6- tri - 0-acetyl-2-deoxy-2-oximino-a-~arabino-hexopyranosyl)-/3-D-glucopyranoside(9) in 70 % yield.22

OAc

These oximino glycosides were then found to be of great value in synthetic work. Reduction in the presence of palladium catalysts and hydrochloric acid gave alkyl 2-amino-2-deoxy-a-~-hexopyranosides, and provided the first controlled synthesis of aminodeoxy-a-Dg l y c o s i d e ~ Alternatively, .~~ deoximation and borohydride reduction of the resulting glycosiduloses opened the way to an excellent synthesis of a-D-glycosides, and reduction with borodeuteride offered by means of labeling H-2 s p e c i f i ~ a l l yAcetolysis, .~~ instead of alcoholysis, of the nitrosoglycosyl chlorides afforded tetra-O-acetyl-2oximinoglycoses, reduction of which with a zinc-copper couple in acetic acid, followed by acetylation, gave acetylated derivatives of 2-amino-2-deoxyhexoseshaving the amino group equatorially attached to (2-2. Alternatively, treatment of the glycosyl chlorides with acetic acid and a base (for example, triethylamine) gave the pentaacetates of the 2-oximinohexoses, and catalytic hydrogenation of these gave the epimeric products having the nitrogenous group in the axial orientation. Deacetylation of these compounds can be accomplished without difficulty, and, from tri-O-aCetyl-D-glUCa1 and -galactal, 2-amino-2deoxy-D-glucose and -mannose, and 2-amino-2-deoxy-~-galactose and -talose, respectively, were obtained25(as their hydrochlorides), each in yields of 70430%. (22) R. U. Lemieux, T. L. Nagabhushan, and S. W. Gunner, Can. J . Chem., 46,405

(1968). (23) R. U.Lemieux and S. W. Cunner, Can.J.Chem.,46,397(1968). (24) R. U. Lemieux, R. Suemitsu, and S. W. Gunner, Can.J.Chem. 46,1040 (1968). (25) R. U.Lemieux and T. L. Nagabhushan, Can.J . Chem.,46,401(1968).

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4. Addition of Sulfur-containing Reagents

In an extension of their work on the halogenation of tri-0-acetyl-Dglucal, Igarashi and Honma26 have investigated the reaction with the pseudo-halogen thiocyanogen, and have found that, whereas in ether or carbon tetrachloride no reaction occurs, in acetic acid, a complex set of products results. Careful fractionation gave 3,4,6-tri0-acetyl-2-deoxy-2- thiocyanato -a-D-glUCOpyranOSyl isothiocyanate (lo), 3,4,6-tri-O-acetyl-2-thiocyanato-D-gluca1 (11, R = SCN), and 1,3,4,6-tetra-O-acetyl-2-deoxy-2-thiocyanato-~-hexoses having the aand P-D-glUCO and a-~-rnunnoconfigurations, all of which were characterized by chemical and nuclear magnetic resonance studies.26 C&OAc

CI&OAc

(J

AcO AcOO

N

C

S

R

SCN (11)

(10)

CH,OR'

(J

R'O

SR (12)

(13)

Free-radical additions to glycals have not yet been examined extensively, but two reports on the addition of sulfur-containing reagents by a homolytic mechanism have now appeared. 3,4,6-Tri-Oacetyl-2-S-acetyl-l,5-anhydro-2-thio-~-mannitol and -glucitol (12 and 13, R = R' = Ac; 7:3),27and 1,5-anhydro-2-S-benzyl-2-thio-~-mannitol and -glucitol (12 and 13, R = CH,Ph, R' = H; 1:1)28were the products obtained when tri-0-acetyl-D-glucal and D-glucal were treated with thiolacetic acid and a-toluenethiol, respectively. (26) K.Igarashi and T. Honma,]. Org. Chem., 32,2521(1967). (27) K.Igarashi and T. Honma, Tetrahedron Lett., 751 (1968). (28) J. Lehmann and H. Friebolin, Carbohyd. Res., 2,499(1966)

210

R. J. FERRIER

5. Methoxymercuration

Reaction of glycals or substituted glycals with mercuric acetate in methanol had previously been shown' to yield methyl 2-deoxyglycosides having mercury attached directly at C-2. A series of new compounds has now been reported20 having the P-D-glUco configuration, various aglycons (suggesting a useful, general route to 2-deoxy-~-~-glucopyranosides), and the following groups bonded to the mercury atom: C1, I, CN, NO3, PhCOO, S-isoPr, SPh, S-p-tolyl, SCH,PH. In addition, the reaction has been extended to other unsaturated carbohydrates (see p. 220). In other work, methoxymercuration has been applied in the first stage of the synthesis of L-daunosamine (14). Methoxymercuration of L-rhamnal (15), followed by reduction of the carbon-mercury bond with potassium borohydride, afforded30a route to methyl 2,6-dideoxya-L-urubino-hexopyranoside (16), the enantiomorph of methyl chromoside C.

6. Hydroformylation

to aceUnder normal conditions, application of the 0x0 tylated glycals proceeds beyond the hydroformylation stage, and two products having a hydroxymethyl group at C-1 in the two possible orientations are obtainable. Rosenthal and his coworkers have now extended their earlier work' and have shown, as was expected, that 2,6-anhydro-3-deoxy-~-guZuctoand -tuZo-heptitol, (17) and (18), are the products obtained3' (after deacetylation) from tri-0-acetyl-Dgalactal. (29) J. H.Leftin and N. N. Lichtin, ZsraelJ . Chem., 3,107(1965). (30) J. P.Marsh, C. W. Mosher, E. M. Acton, and L. Goodman, Chem. Commun., 973 (1967). (ma) A. Rosenthal, Aduan. Carbohyd. Chem.,23,59(1968). (31) A. Rosenthal and D. Abson, Can.J . Chem., 43,1985(1965).

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The analogous products from di-0-acetyl-D-xylal were finally characterized completely by specific reduction of the hydroxymethyl groups to methyl groups, followed by identification of the resultant diols with the C-glycosyl derivatives synthesized by reaction of 3,4-di-O-acetyl-2-deoxy-~-threo-pentopyranosyl chloride with methylmagnesium bromide.32

Under carefully controlled conditions in which the uptake of gas was limited to 2 moles per mole, the products of direct hydroformylation of glycal esters were found to be obtainable. Thus, from di-0acetyl-D-xylal, 4,5-di-O-acetyl-2,6-anhydro-3-deo~y-~-lyxoand -xyZohexose (19 and 20) were isolated in a combined yield of 20% (by way of hydrazone derivatives). Alternatively, these compounds were much more readily prepared by application of the Pfitzner-Moffatt oxidation reaction to the corresponding hydroxymethyl analogs. With tri-O-acetyl-D-gluca1, the addition reaction was more suitable for the preparation of the 2,6-anhydroaldose compounds, the yield33 being 70 %. The hydroformylation of glycals has been described in detai1,30a*34 and the reaction has been extended to the hydroxyglycal series (see p. 220). (32) A. Rosenthal and D. Abson, C a n . / .Chem.,43,1318 (1965). (33) A. Rosenthal, D. Abson, T. D. Field, H. J. Koch, and R. E. J. Mitchell, Can. /. Chem.,45,1525 (1967). (34) D. Abson, Dissertation Abstr., 26,4227 (1966).

212

R. J. FERRIER

7. Other Additions

A further example has been given of the synthesis of a 2-deoxyaldose by the addition of water to a glycal, but, as this reaction is known to be accompanied by competing eliminations,’ it is being replaced in synthetic work by more-specific addition procedures, for example, methoxymercuration (see p. 210). From the 4-deoxy derivative of D-glucal, prepared by standard procedures from 2,3,6-tri-0acetyl-4-deoxy-~-xylo-hexopyranosylbromide, 2,4-dideoxy-~-threohexose was obtained.35 Addition of dichlorocarbene, produced by treatment of ethyl trichloroacetate with sodium methoxide, to 3,4,6-tri-O-methyl-~-glucal gave, apparently, one product (gas-chromatographic analysis) in 82 % yield. By stereochemical analogy with epoxidations, the product was considered to be 1,5-anhydro-2-deoxy-1,2-C-(dichloromethylene)(21). Demethylation was 3,4,6-tri-O-methyl-D-glycero-~-ido-hexito1 brought about with boron trichloride at -70°, to give a trio1 from which a crystalline tribenzoate was obtained; and reaction with lithium aluminum hydride caused reductive cleavage of the carbon-chlorine bonds.36A similar addition was applied to 3,4-unsaturated furanosyl compound (see p. 247).

In a reaction analogous to one previously reported,’ di-0-acetyl-Darabinal and adenine, in methyl sulfoxide containing hydrogen chloride as catalyst, gave37 9-(3,4-di-O-acetyl-2-de~xy-P-D-erythropentopyranosy1)adenine in 7% yield. Similarly, acid-catalyzed addition of 6-chloropurine and 2,6-dichloropurine to this glycal derivative afforded other 2’-deo~ynucleosides.~~~ (35) A. F. Cook and W. G. Overend, Chem. Znd. (London), 1141 (1966). (36) J. S. Brimacombe, M. E. Evans, E. J. Forbes, A. B. Foster, and J. M . Webber, Carbohyd. Res., 4,239 (1967). (37) N. Nagasawa, I. Kumashiro, and T. Takenishi,]. Org. Chem.,32,251 (1967). (374 E. E. Leutzinger, W. A. Bowles, R. K. Robins, and L. B. Townsend, J . Amer. Chem. Soc., 90,127 (1968).

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The susceptibility of the allylic ester group in tri-0-acetyl-D-glucal to hydrogenolysis in the presence of various catalysts has been carefully studied. Platinum catalysts (with ethyl acetate as the solvent) mainly caused direct additions (85%) to the double bond, whereas the incorporation of small proportions of dimethylamine promoted hydrogenolysis, so that 4,6-di-O-acetyl-1,5-anhydro-2,3-dideoxy-~-eryt~rohexitol was obtained in 95% yield. In the absence of added base, palladium catalysts would be expected to afford uncomplicated addition.38

8. Rearrangement Reactions At the time of writing of the last article,' it had been established that, in the presence of a nucleophile, acylated glycals can undergo allylic rearrangement reactions to give 2,3-unsaturated products bearing the nucleophile at C-1, and it was believed that a direct allylic displacement mechanism operated; furanoid glycal derivatives were found to be particularly reactive. Several developments have occurred that show that this type of reaction is of common occurrence, and that it can be controlled to provide a useful synthesis of 2,3-unsaturated glycosides. Following an observation that certain esters of 2-hydroxyglycals are completely rearranged to 2,3-unsaturated isomers, either on heating in high-boiling, inert solvents, or on treatment with a small proportion of boron trifluoride in an inert solvent at room temperature (see p. 221), tri-0-acetyl-D-gluca1 was subjected to the same conditions, and was found to be isomerized partially to 174,6-tri-O-acetyl-2,3CH,OAc

OR

AcO

dideoxy-~-erythro-hex-2-enopyranose (22, R = O A C ) . ~However, ~ when higher concentrations of catalyst were used in the acid-catalyzed reaction, this isomer dimerized to give products from which crystal(38) G. R. Gray and R. Barker,]. Org. Chem.,32,2764 (1967). (39) R. J. Ferrier and N. Prasad,]. Chem. SOC. ( C ) ,581 (1969).

214

R. J. FERRIER

line 1,3,4,6-tetra-O-acetyl-2-deoxy-2-C-(4,6-di-O-acetyl-2,3-dideoxy-c~D-erythro-hex-2-enopyranosy~)-~-D-glucopyranose (23) was isolated in good yield, This dimerization was considered to proceed by attack of the stabilized, 2,3-unsaturated, glycosyl carbonium ion obtained from the acetate (22, R = OAc) at C-2 of the un-ionized species.

(-J

AcO

I(xj

AcO

In the light of these findings, it seems probable that, in acetic acid containing hydrogen bromide, tri-O-aCetyl-D-glUCa1,as well as undergoing the direct addition expected, also isomerizes to the glycosyl acetate (22, R = OAc), which then adds bromine at C-3 (see p. 206). On being heated in acetic acid, tri-0-acetyl-D-galactal (which undergoes this type of allylic-rearrangement reaction much less readily than tri-O-aCetyl-D-glUCa1)undergoes both the addition and the rearrangement reaction, and from the mixture of the products, crystalline 1,3,4,6-tetra-O-acetyl-2-deoxy-a-~-Z~xo-hexose (24), 3,4,6-tri-O-acetylD-guld (25), 1,4,6-tri-O-acetyl-2,3-dideoxy-a-~-threo-hex-2-enopyranose (26), and syrupy 2-(~-gZycero-1,2-diacetoxyethyl)furan(27, R = R' = Ac) were isolated. [The last compound was tentatively assigned a pyranoid structure, but was later shown to give a nuclear magnetic resonance spectrum identical with that of the furan compound40 (see p. 218)l. Since the products of the reaction were apparently in equilibrium with each other (or at least pseudo-equilibrium, as the furan derivative is unlikely to be a component of a true equilibrium), the saturated adduct (24) was subjected to heating in acetic acid, and the unsaturated compounds 25 and 26 were again obtained.41 (40) D. M.Ciment and R. J. Ferrier, unpublished results; Professor D. Horton kindly provided the spectrum of an authentic sample. (41) R. J. Fenier andD. M. Ciment,]. Chem. SOC.(C), 441 (1966).

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““QOAC

Tri-0-acetyl-D-gluca1 undergoes reaction with alcohols and phenols in the absence of a catalyst, to give’ unsaturated glycoside derivatives (22, R = alkyloxy or aryloxy), and it has been shown that tri-0-acetyl-Dgalactal also undergoes these reactions, but markedly less readily presumably because the ester group on C-4 cannot assist the departure of the 3-acetoxyl group (see p. 223).Nevertheless, crystalline 4,6-di-0acetyl-2,3-dideoxy-a-~-threo-hex-2-enopyranosides have been obtained (in poor yield^).^' Alcohols react with acetylated glycals under forcing conditions only (in sealed tubes) and without marked stereospecificity, but, when boron trifluoride is used as a catalyst, glycosides are formed at room temperature; with tri-O-aCetyl-D-glUCa1, the reac~ preponderate (80%). An tion is highly efficient and a - products additional advantage is that inert solvents can be employed, and the alcohol can be used in unimolar proportion, so that complex glycosides can be produced. By this means, the crystalline ethyl glycoside (22, R = OEt, a-D anomer) is readily obtained in 70% yield, whereas previous methods afforded 30 % yields, and the unsaturated glycosides (22, R = cholesteryloxy, a-Danomer) and (22, R = 6-deoxy1,2:3,4-di-0-isopropylidene-a-~-galactopyranose-6-yloxy) have been synthesized with good e f f i c i e n ~ y . ~ ~ Two groups of workers have shown that, in the presence of boron trifluoride, simple acetals do not add to the double bond of tri(42) R. J. Fernier and N. Prasad, Chem. Commun., 476 (1968); J . Chem. SOC. (C), 570,575 (1969).

216

R. J. FERRIER

0-acetyl-D-glucal to give 2-deoxyglycoside derivatives having a branched-chain at C-2 (as might have been expected), but give alkyl 4,6-di -O-acetyl-2,3-dideoxy-~-erythrohex-2-enopyranosides (22, R = a l k y l o ~ y ) .In ~ ~view , ~ ~ of the facts that a molar proportion of an alcohol can react to give these products under these conditions, and that this reaction could, consequently, have been attributable to alcohol impurities in the acetals, the reactions were repeated in benzene solution with molar proportions of each acetal, and, again, the unsaturated glycosides were formed.42 Unlike hydrogen bromide and hydrogen chloride, hydrogen fluoride does not add directly to tri-O-aCetyl-D-glUCd,but, it was first reported that, in benzene solution, it gives 4,6-di-O-acety1-2,3-dideoxy-~-erythro-hex-2-enopyranosyl fluoride (22, R = F) [which, with water, is hydrolyzed to 4,6-di-O-acetyl-2,3-dideoxy-~-erythro-hex-2enopyranose (22, R = OH)] or else gives 4,6-di-O-acety1-2,3-dideoxyD-eryth~o-hex-2-enopyranosyl4,6-di-O-acetyl-2,3-dideoxy-~-erythrohex-2-enopyranoside (28), according to the conditions used. Alcoholysis of the fluoride afforded the corresponding alkyl glycoside derivatives (22, R = alkyloxy) in the same proportions as were obtained on treating tri-O-aCetyl-D-glUCa1 directly with alcohols (for example, with methanol, L Y : ~ratio = 3:2). Tri-0-benzoyl-D-glucal, obtained as a syrup by benzoylating D-glUCal, reacted in the same way, to give 4,6-di-0-benzoyl-2,3-dideoxy-~-erythro-hex-2-enopyranosyl fluoride which, on hydrolysis, gave crystalline 4,6-di-O-benzoyl2,3-dideoxy-~-erythro-hex-2-enopyranose.~~

(43) M. E. Shostakovskii, V. M. Annenkova, E. A. Gaitseva, K. F. Lavrova, and A. I. Polyakov, l z v . Sib. Otd. Akad. Nauk S S S R , Ser. Khim. Nauk, 163 (1967);Chem. Abstracts, 67,108930(1967). (44) I. Lundt and C. Pedersen, Acta Chem. Scand., 20,1369(1966).

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Later work has, however, revealed that this reaction with hydrogen fluoride is more complex than at first appeared. In anhydrous hydrogen fluoride at -70°, for example, tri-0-benzoyl-D-glucal gives either the unsaturated glycosyl fluoride previously obtained or 3,6-di-0benzoyl-2-deoxy-c~-~-~ibo-hexopyranosyl fluoride, depending on the elapsed time allowed for the reaction to proceed, and nuclear magnetic resonance evidence was obtained to show that ions of the types 29 and 30 are involved in this reaction.45

F O ,

W

0

RC I 0'0

(29)

where R = Ac or Bz.

As reported earlier,' acetylated glycals can give unsaturated nucleoside derivatives when condensed with bases. Examples of related compounds were obtained when di-0-acetyl-D-xylal or -L-arabinal were treated with benzotriazole and its 5,6-dimethyl derivative in (45) I. Lundt and C. Pedersen, unpublished results.

218

R. J. FERRIER

ethyl acetate in the presence of trifluoroacetic acid as catalyst.45aThe anomeric configurations of these products were not determined, but the isomeric pairs, for example, 30a and 30b,were also enantiomorphs so they had the same relative stereochemistry at C-1 and C-4. Similarly, compound 30c was obtained from tri-0-acetyl-D-glucal and 2acetamid0-6-chloropurine.~~~ 9. Other Reactions

The uncertain position regarding the nature of the dienes formed on acid degradation of glycals' has been clarified by the identification of the main product (> 50 %) of the reaction of D-glud in hot, aqueous acetic acid as 2-(~-gZycero-l,2-dihydroxyethyl)furan (27,R = R' = H). Tri-0-acetyl-D-gluca1,on the other hand, on being heated in ethanolic sulfuric acid, gives several products, amongst which, 2-(l-ethoxy-2hydroxyethy1)furan (27,R = H,R' = Et), 24 172-diethoxyethyl)furan (27,R = R' = Et), and 2,6-diethoxy-4-oxohexanaldiethyl acetal (31) (all racemic) were characterized, mainly by spectroscopic methods.& H

EtOCOEt I CHOEt I

HCH

c=o I

HCH I CqOEt (31)

Oxidations catalyzed by platinum oxide are now well known in carbohydrate chemistry, and their marked specificity has been examined with profit. From the reaction with D-glucal, a mixture of products was obtained, from which 2-deoxy-~-arubino-hexoseand crystalline 2,3-dihydro-3-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (32)were ~ b t a i n e d . ~Related ' enones have been encountered among

(45a) M. Fuertes, G. GarciCMuhoz, M. Lora-Tamayo, R. Madrohero, and M. Stud, Tetrahedron Lett., 4089 (1968). (45b) E. E. Leutzinger, R. K. Robins, and L. B. Townsend, Tetrahedron Lett., 4475 (1968). (46) D.Horton and T. Tsuchiya, Chem. Ind. (London),2011 (1966). (47) K.Heyns and H. Gottschalck,Chem. Ber., 99,3718(1966).

UNSATURATED SUGARS

219

the products of oxidation of partially substituted, pyranoid compound~.~~ The mass spectra of tri-0-acetyl-D-gluca1 and other unsaturated sugars have been investigated.48a 111. 2-SUBSTITUTED GLYCALS

In this Section, recent developments in the chemistry of glycal derivatives bearing substituents at C-2 will be discussed. Since the previous article' was completed, a new class of unsaturated sugars, those produced from hexulopyranosyl halides by eliminations at C-2 and C-3 has been described; they are glycals bearing substituents on C-1. Such compounds are, however, also 4,5-unsaturated pyranoid derivatives, and are considered under that heading (see Section VI, p. 252). Apparently, products arising from the removal of the anomeric proton of a cyclic glycose have not yet been reported.

1. 2-Hydroxyglycals The mass spectrum of 2-acetoxy-tri-O-acety1-D-glucal has been studied.48a a. Synthesis. - The appreciable improvements developed by Lemieux and L i n e b a ~ kfor ~ ~the synthesis of 2-acetoxy-tri-O-acetyl-Dglucal (11,R = OAc) were noted briefly in the earlier article' and have now been applied50 to the synthesis of analogs bearing other ester groups on C-3. Efforts in this laboratory to apply these procedures (use of tetrabutylammonium bromide and diethylamine in acetonitrile to remove the elements of hydrogen bromide from poly-0(48) P. J. Beynon, P. M. Collins, P. T. Doganges, and W. G. Overend,]. Chem. Soc. (C), 1131 (1966); G. M. Cree, D. W. Mackie, and A. S. Perlin, Can. J . Chem., 47, 511 (1969). (48a) A. Rosenthal, Carbohyd. Res., 8, 61 (1968). (49) R. U. Lemieux and D. R. Lineback, Can.J. Chem., 43,94 (1965). (50) R. U.Lemieux and R. J. Bose, Can.J.Chem., 44,1855 (1966).

220

R. J . FERRIER

acetylglycosyl bromides) to the synthesis of other 2-hydroxyglycal esters have not met with the same success, and it has been found preferable to convert the poly-O-acylglycosyl bromides into iodides, by brief treatment with sodium iodide in acetone, prior to the eliminations induced by diethylamine. This procedure is rapid and convenient, and, in most instances, affords good yields of products.51 Efforts to condense tetra-O-benzyl-a-D-glucopyranosylbromide with indole in liquid ammonia failed, but resulted, instead, in the isolation of crystalline 3,4,6-tri-O-benzyl-2-(benzyloxy)-~-glucal.~~~ Although furanoid derivatives in this series have not yet been formally synthesized, it appears that 2,3,5-tri-O-benzoyl-l-deoxy-~threo-pent-l-enofurano~e~~ (33) and the D-erythro is0mel5~have been produced as by-products during the synthesis of nucleosides from the chlorides. corresponding 2,3,5-tri-O-benzoyl-D-pentofuranosyl

OBa

b. Addition Reactions. - The small number of addition reactions that have been investigated in this seriess4 has now been extended by reports on the hydroformylation and methoxymercuration of 2acetoxy-3,4,6-tri-O-acetyl-D-glucal, and on a detailed study of the hydrogenation-hydrogenolysis reaction undergone by this compound. Hydroformylation in benzene solution with hydrogen and carbon monoxide in the presence of dicobalt o ~ t a c a r b o n y l ~ afforded, "~ after with two undeacetylation, 2,6-anhydro-~-glycero-D-gulo-heptitol, identified, minor products.5s Likewise, 2-acetoxy-tri-O-acetyl-Dgalactal afforded mainly 2,6-anhydro-~-gZycero-~-manno-heptitol on similar treatment. Methoxymercuration of the D-glucal derivative gave a crystalline glycoside adduct, obtained in 18%yield, which, on (51) R. J. Ferrier and G . H. Sankey,J. Chem. SOC. (C), 2339 (1966). (51a) M. N. Preobrazhenskaya and N. N. Suvorov, Zh. Obshch. Khim., 35, 888 (1965); Chem. Abstracts., 63, 14952 (1965)." (52) T. Tkaczynski, J . Smejkal, and F. Sorm, Collect. Czech. Chem. Commun., 29, 1739 (1964). (53) M. PrystaB and F. Sorm, Collect. Czech. Chem. Commun., 33,210 (1968). (54) M . G . Blair, Adoan. Carbohyd. Chem., 9,97 (1954). (55) A. Rosenthal and D. Abson, Carbohyd. Res., 3,112 (1966).

UNSATURATED SUGARS

22 1

reductive demercuration, afforded a product the levorotation of which strongly suggested the p-Dconfiguration; the configuration at C-2 was not established, but the original product would be expected to be a D - ~ U W ~ Ocompound, as methoxymercuration normally affords trans products (formed by way of mercurinium ions).29 A close investigation of the hydrogenation of 2-acetoxy-tri-0-acetylD-glucal has yielded valuable information regarding the stereochemistry of the addition, and the extent of competing hydrogenolytic rea c t i o n ~ .Platinum ~* catalysts in methanol, acetic acid, and ethyl acetate always cause hydrogenolysis, and afford 4,6-di-O-acetyl-l,5-anhydro2,3-dideoxy-~-erythro-hexitol(34) and 3,4,6-tri-O-acetyl-1,5-anhydro2-deoxy-~-arabino-hexitol(35),in proportions varying from 2: 1 to 100:0, the latter result being achieved when dimethylamine is present (see p. 213). Palladium catalysts, alternatively, cause only minor hydrogenolysis, and give mainly tetra-O-acetyl-1,5-anhydro-~-mannito1 and -glucitol, with the former preponderating.

CH,OAc

q€&OAc

c . Rearrangement Reactions. - Like the glycal esters, esters of 2-hydroxyglycals take part in rearrangement reactions that afford 2,3-unsaturated products, and it was previously reported' that 2acetoxy-tri-0-acetyl-D-glucal can be converted into the anomers of 1,2,4,6-tetra-O-acetyl-3-deoxy-~-e~ythr0-hex-2-enopyranose (36, R = OAc, R' = Ac) either in boiling acetic acid or in acetic anhydride in the presence of an acid catalyst; other 2-hydroxyglycal esters have now been shown to undergo similar reaction^.^^ It has been considered probable that the reactions proceed by nucleophilic attack at C-1 and allylic displacement of the ester group at C-3. With the aim of investigating the mechanism of the reaction in detail, Lemieux and Base?" prepared several 2-hydroxyglucal derivatives having good leaving-groups on C-3, in the hope that such compounds would be solvolyzed with allylic rearrangement, without the presence of an acidic catalyst. Attempts to prepare 2-acetoxy-4,6-di-O-acetyl-3-O-ptolylsulfonyl[or(methylsulfonyl)]-D-glucal, by use of diethylamine in

222

R. J. FERRIER

acetonitrile and the appropriate D-glucosyl bromide derivative, also caused removal of the sulfonyloxy groups, and gave products believed to be the diethyl-(2,4,6-tri-O-acetyl-3-deoxy-~-erythro-hex-2-enopyranosy1)amines (36, R = NEt,, R' = Ac), presumably because, under these conditions, the reaction under investigation occurred very rapidly.

OR

R'O

OR'

(36)

Alternatively, 2-acetoxy-4,6-di-O-acetyl-3-0-(2,6-dichlorobenzoyl)D-glucal was prepared by way of the glycosyl bromide derivative, and was readily converted into the 2,3-unsaturated acetates (36, R = OAc, R' = Ac) by treatment with acetic acid, acetic anhydride in the presence of a strong, acid catalyst, or with potassium acetate in acetic acid at 100". With the last reagent, the reaction was approximately ten times as fast as the anomerization of the products, and, as the anomers were initially produced in equal proportions, it could be concluded that the rearrangement occurred without stereosele~tivity.~~ In a further experiment, the 2,6-dichlorobenzoate was heated with methanol in pyridine, and, although the desired reaction occurred, concurrent decompositions invalidated the procedure as a means of synthesizing 2,3-unsaturated glycosides of this series. Such compounds have, however, been prepared directly from 2-hydroxyglycal esters, and from appropriate, unsaturated glycosyl derivatives bearing good leaving-groups at C-1 (see p. 240). 2-Acetoxy-4,6-di-O-acetyl-3-0mesitoyl-D-glucal was also prepared in the course of this investigation, and was readily converted50 into the unsaturated tetraacetates (36, R=OAc, R'=Ac). During closely related work in this laboratory, 2-acetoxy-tri-Oacetyl-D-glucal was heated in boiling benzyl alcohol (bp 205O) in the hope of obtaining glycosidic products, but instead, it was found that a thermal isomerization occurs, to give 1,2,4,6-tetra-O-acetyl-3-deoxy-~D-erythro-hex-2-enopyranose (36, R = OAc, R' = Ac, p-D anomer) exc l u ~ i v e l ySubsequently, .~~ nitrobenzene was used as the solvent, and (56) R. J. Ferrier, N. Prasad, and G . H. SankeyJ. Chem. SOC.(C),974 (1968).

UNSATURATED SUGARS

223

it was found that the benzoylated glycal derivative reacts even more readily, and that, in this series and with 2-hydroxy-~-xylalesters, this procedure affords an excellent means of obtaining the p-D anomers of the 2,3-unsaturated esters. It was envisaged that an SNi'type is of reaction occurs, and that, as 2-acetoxy-tri-O-acety~-D-galacta~ stable under the conditions employed for these isomerizations, anchimeric assistance is provided for the departure of the ester group at C-3 by the trans ester group on C-4 (see p. 215). The same isomerizations occur when 2-hydroxyglycal esters are treated in inert solvents with a small proportion of boron trifluoride, but the anomerization of the products is also catalyzed, so a-Dproducts p r e p ~ n d e r a t eUnder . ~ ~ these conditions, 2-acetoxy-tri-O-acety1-Dgalactal does react (but more slowly than the D-glucal isomer), and, from the products, 1,2,4,6-tetra-O-acetyl-3-deoxy-a-~-threo-hex-2enopyranose has been isolated in high yield. Following the successful synthesis of 2,3-unsaturated glycoside derivatives from glycal esters in inert solvents by use of boron trifluoride as the catalyst (see p. 215), similar reactions were attempted with 2-hydroxyglycal esters, and were found to p r ~ c e e d . ~In ' parwas found to be suitable ticular, tri-O-benzoyl-2-benzoyloxy-~-gluca~ for this purpose, as it generally affords, in good yield, crystalline products having the a-D configuration (see p. 240). The value of the procedure in the synthesis of glycosides of nonvolatile alcohols was demonstrated by the preparation of unsaturated glycosides of sterols. /3-D Anomers in this series were alternatively prepared by use of crystalline 2,4,6 - tri -0-benzoyl-3-deoxy- 1-0-(trichloroacetyl)-a-Derythro-hex-2-enopyranose (37) (see p. 240), which was synthesized by heating 3,4,6-tri-O-benzoyl-2-benzoy~oxy-~-g~ucal with trichloroacetic acid in b e n ~ e n e . ~ '

I OBz (37)

(57) R.J. Ferrier, N. Prasad, and G. H. Sankey,J. Chem. SOC.( C ) ,587(1969).

224

R. J. FERRIER

2. Other 2-Substituted Glycals 2-Nitro- and 2-nitroso-glycals have been encountered by Lemieux and his collaborators during their investigations of the reactions of -nitrosy1 chloride and dinitrogen tetraoxide with glycal esters (see p. 207 and Ref. l), and glycals bearing other substituents at C-2 have since been encountered, several of them as by-products. 4,6-0-Benzylidene-2-C-methyl-D-allal (1, R = Me), formed by epoxide ring-opening of methyl 2,3-anhydr0-4,6-O-benzylidene-a-~allopyranoside with halide-free methyllithium has already been referred to (see p. 201). A viscous oil, presumed to be 3,4,6-tri-O-acetyl2-bromo-D-glucal (11, R = Br), has been prepared by elimination, by use of triethylamine, of the elements of hydrogen bromide from the mixture of products obtained by bromination O f tri-0-aCetyl-D-glUCa1.6 It was pointed out that this bromo derivative of D-glucal had not previously been described, although there is an erroneous reference to it in Chemical Abstracts. Treatment of the compound with an excess of butyllithium gave 1,2-dideoxy-~-arabino-hexl-ynitol (38) in

HCOH I

HCOH

I CH,OH

(38)

a reaction which parallels that of 5-bromo-2,3-dihydro-4H-pyranwith metal alkyls, and in a similar way, the corresponding pentyne derivative was obtained from di-O-acetyl-Dyxylal.eTri-O-acetyl-2-chloro-~glucal was obtained from the 1,2-dichloro adduct, and was rechlorinated to give a 1,2,2-trichloro compound which was c r y ~ t a l l i n e . ~ ' ~ Several derivatives having a sulfur substituent at C-2 are now known. Reaction of 3,4,6-tri-0-acetyl-2-0-(methylsulfonyl)-~-~-glucopyranosyl N,N-dimethyldithiocarbamate with potassium acetate in ethanolic acetone gaves 3,4,6-tri-O-acety1-2-(N,N-dimethyldithiocarbamoyl)-D-glucal (11, R = SCSNMe,), and a related compound, 3,4,6-tri-O-acety~-2-thiocyanato-D-gluca~ (11, R = SCN), was isolated in 4% yield as one of several products formed in the reaction between tri-O-acetyl-D-glucal and thiocyanogenZ6(see p. 209); however, it was' (57a) P. R. Bradley and E. Buncel, Can.J.Chem.,46,3001(1968). (58) S. Ishiguro and S.Tejima, Chem. Pharm. Bull. (Tokyo), 15,1478(1967).

UNSATURATED SUGARS

225

synthesized in good yield by elimination from the corresponding glycosyl chloride. Furthermore, on treatment with sodium in 1,2dimethoxyethane, methyl S-benzyl(or S-methyl)-4,6-O-benzylidene3-0-methyl-2-thio-a-o-altropyranoside (2, R = SCH2Ph or SMe, R’ = Me) gave 1,2-, 2,3-, and 3,4-unsaturated products, amongst which, S - benz yl(and S -methyl)-4,6-O-benzylidene-3-O-methyl-2-thio-~-allal (1, R = SCH2Ph or SMe, R’ = Me) were found.59On desulfurization with Raney nickel, these gave 4,6-0-benzylidene-3-O-methyl-~-allal (1, R = H, R’ = Me), shown to be identical with the methylation product of the 3-hydroxy compound (see p. 201). Similar results were obtained with analogous 4,6-O-ethylidene derivatives.so Acetylation of 2-acetamido-2-deoxy-~-mannose with isopropenyl acetate in the presence of p-toluenesulfonic acid afforded products of direct esterification, together with unsaturated compounds, including the 2-amino analog of 2-acetoxytri-O-acety~-D-g~uca~, namely, 3,4,6tri-O-acetyl-2-(N-acetylacetamido)-D-gluca~ (11,R = NAc2),which was isolated in 14% yield. Although this compound could not be hydrogenated in the presence of a palladium catalyst, it afforded on deacetylation 2-acetamido-~-glucal,which could be hydrogenated to give mainly 2-acetamido-1,5-anhydro-2-deoxy-~-mannitol (39, R = R’ = R” = H; 79%) together with the C-2 epimer (see p. 00). Reacetylation of the deacetylated product afforded 2-acetamido-3,4,6tri-0-acetyl-D-glucal (11, R = NHAc), which also was hydrogenated, to give mainly 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-~mannitol(39, R = R’ = H, R” = AC).~’

An investigation of the mechanism of the formation of the substituted glycal (11, R = NAc,) indicateds2 that it arises from 1,3,4,6tetra-O-acetyl-2-(N-acetylacetamido)-2-deoxy-a-~-mannopyranose (39, R = R f ’ = Ac, R’ = OAc), by a process involving initial, anchimerically (59) U. G. Nayak, M. Sharma, and R. K. Brown, Can.]. Chem., 45,481 (1967). (60) U. G. Nayak, M. Sharma, and R. K. Brown, Can.]. Chem.,45,1767 (1967). (61) N. Pravdid and H. G. Fletcher, Jr.,J. Org. Chem., 32,1806 (1967). (62) N. Pravdidand H. G:Fletcher, Jr.,]. Org. Chem., 32, 1811 (1967).

226

R. J. FERRIER

assisted removal of the ester group on C-1. This conclusion suggested that the formation of the glycal derivative would be facilitated by replacement of the 1-acetoxylgroup by a better leaving-group, and, when mixtures of the anomers of 2-acetamido-3,4,6-tri-O-acetyl-l-O-benzoyl-2-deoxy-~-mannopyranosewere treated with isopropenyl acetate and p-toluenesulfonic acid, yields of the glycal derivative (11, R = NAc2) reflected accurately the proportion of the a-Danomer (39, R = H, R‘ = OBz, R” = Ac) present. As mixtures could be prepared that contained 75% of this anomer, this route affords a means of obtaining compound 11 (R = NAc2) in high yield.

Iv. 2,3-UNSATURATED, CYCLIC COMPOUNDS 1. Furanoid Derivatives a. Alkene Derivatives.- Nucleoside derivatives having double bonds at C-2-C-3 of the sugar moiety have received considerable attention, because of their significance in biochemistry; it has been proposed that they are intermediates in the biosynthesis of 2’-deoxynucleotides, and also that the 2’,3’-dideoxynucleosides, readily obtainable from the unsaturated compounds, may serve as chain terminators in the synthesis of 2’-deoxyribonucleic acid. Furthermore, some of these compounds have been shown to exhibit interesting pharmacological properties, and the finding of a 2,3-unsaturated pyranoid nucleoside in the antibiotic blasticidin S (see p. 238) has drawn additional attention to this series of compounds. Synthetic work has utilized, in the main, the base-catalyzed eliminations noted previously,’ and a series of compounds having the general structure 40 have now been synthesized as outlined in Table 11. Attempts to prepare compounds of this group by heating the derived 2’,3’-thionocarbonates with trimethyl phosphite have not met with the same success. From 5’-0-trityluridine, the 2,2’-anhydronucleoside was produced, instead of the cyclic ester, by treatment

(63) J. P. Horwitz, J. Chua, and M. Noel, Tetrahedron Lett., 1343 (1966). (64) J. R. McCarthy, Jr., M. J. Robins, L. B. Townsend, and R. K. Robins, J . Amer. Chem. SOC., 88,1549 (1966). (65) T. A. Khwaja and C. Heidelberger,]. Med. Chem., 10,1066 (1967). (66) J. P. Horwitz, J. Chua, M. A. DaRooge, M. Noel, and I. L. Klundt,J. Org. Chem., 31,205 (1966). (67) J. P. Horwitz, J. Chua, M. Noel, and J. T. DonattiJ. Org. Chem.,32,817 (1967).

TABLEI1 Unsaturated Nucleosides (40) and Their Synthesis from 2-Deoxy-D-etythro- or -threo-pentohanosy1 Nucleoside Derivatives R Nucleoside base

(in 40)

Adenine

OH

5-Fluorouracil

OH, SEt, or H OTr or OH

Uracil

OTr OTr

Thymine

OTr OTr

Uracil Thymine Cytosine Uracil 5-Fluorouracil

OH OH OH OTr OTr or OH

Precursor D-erythro-, 3'-ptoluenesulfonate D-erythro-, 3'-ptoluenesulfonate D-erythro-, 3'methanesulfonate D-erythro-, 3'methanesulfonate D-threo-, 3'methanesulfonate D-erythro-, 3'methanesulfonate D-threo-, 3'methanesulfonate 3',5'-anhydride 3',5'-anhydride 3',5'-anhydride 2,3'-anhydride 2,3'-anhydride

Reagent

References

sodium ethoxide in EtOH

63

sodium methoxide in HCONMez

64

potassium tert-butoxide in Me2S0

65

potassium tert-butoxide in MezSO

66

potassium tert-butoxide in Me2S0

66

potassium tert-butoxide in Me2S0

66

potassium tert-butoxide in Me2S0

66

potassium tert-butoxide in Me2S0 potassium tert-butoxide in Me2S0 potassium tert-butoxide in Me2S0 potassium tert-butoxide in Me2S0 potassium tert-butoxide in Me2S0

66 66 67 66 65

C

N N

-a

228

R. J. FERRIER

with bis(imidazol-1-yl)thione,68~69 but, under suitable conditions, cyclic esters can be obtained.70These esters are, however, reported to give intractable oils on being heated with trimethyl p h ~ s p h i t e , ~ ~ but they have been converted into 2’,3‘-unsaturated compounds in poor yield on treatment with inactivated Raney Unsaturated nucleosides of this class are readily hydrolyzed by acid but, under very mild conditions, a 5’-O-trityl group can be removed without rupture of the glycosidic b ~ n d .Under ~ ~ , forcing ~ ~ conditions with strong bases, certain of these compounds undergo further eliminations to give furan d e r i v a t i v e ~ , sand, ~ # ~although ~ 2’,3’-dideoxynucleosides can be obtained on hydrogenation, hydrogenolytic cleavage of the glycosidic bond can occur during this r e a ~ t i o n . ~ ~ , ~ ~ A chromatographic spray-reagent containing molybdate is reported to be diagnostic for these 2’,3’-unsaturated n~cleosides,6~ the biological and enzyme-substrate activities of which have been given some attenti~n?~.‘~ The unsaturated derivative from 5-fluorouridine, for example, shows strong activity against mouse-leukemia cells and also against Escherichia C O E ~ . To ~ ~ help overcome the nomenclature problems in this field, trivial names based on the parent nucleosides have been p r o p o ~ e d , 6thus, ~ * ~ for ~ example, the name “uridinene” has been used for the 2’.3’-unsaturated derivative of uridine.

In studies of the fundamental properties of the carbohydrate moieties of nucleosides of this class, model compounds have been prepared from 2,5-anhydropentose derivatives and from furanosides. (68) (69) (70) (71)

J. J. Fox and I. Wempen, Tetrahedron Lett., 643 (1965). W. V. Ruyle, T. Y. Shen, and A. A. Patchett,]. Org. Chem., 30,4353 (1965). G. L. Tong, W. W. Lee, and L. Goodman,]. Org. Chern., 30,2854 (1965). A. Block, M . J. Robins, and J. R. McCarthy,Jr.,]. Med. Chem., 10,908 (1967).

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2,3-Double bonds have been introduced by treatment of appropriate 2,3-cis-di-p-toluenesulfonic esters with the Tipson-Cohen reagent, namely, sodium iodide in hot N,N-dimethylformamide in the presence of zinc dust (see p. 261), and in this way, 2,5-dihydro-2(R)-formylfuran diisobutyl dithioacetal [41, R = CH(S-isoBu),, R’ = 2(R)-benzoyloxymethyl-2,5-dihydrofuran (41, R = CH20Bz, R’ = H),74,75 2(R)benzyloxymethyl-2,5-dihydrofuran(41, R = CH20CH,Ph, R’ = H),73 2(R)-azidomethyl-2,5-dihydrofuran(41, R = CH2N3,R’ = H),73and methyl 5-0-benzoyl-2,3-dideoxy-~-~-gZycero-pent-2-eno~ranoside (41, R = OMe, R’ = CH20Bz)76have been synthesized. The last compound was shown to be identical with the unsaturated glycoside prepared by Ness and Fletcher by treatment of 3,5-di-O-benzoyl-1,2dideoxy-D-erythro-pent-1-enofuranose with methanol,’ so the anomeric configuration of this product is now established. Hydrogenation of the double bond was brought about with the use of a palladium catalyst, but, with Adams catalyst, hydrogenolysis at the anomeric

center also occurred; in addition, the compound was observed to be unstable to extended heating or to mild treatment with acid, 2(benzoyloxymethy1)furanbeing produced under both conditions. Simple elimination did not occur as in the pyranoid series (seep. 232) when 2,3-trans-sulfonic esters were treated with the Tipson-Cohen reagent (sodium iodide and zinc in N,N-dimethylformamide); instead, 2,5-anhydro-3,4-di-0-p-tolylsulfonyl-~-xylose diisobutyl dithioacetal underwent a double e l i m i n a t i ~ n ~ but ~ ; see Ref. 76a. An alternative means of preparing 2,Sdihydrofuran derivatives that has its analogy in the pyranoid series (see p. 233) is, however, the treatment of appropriate aziridines with sodium nitrite in acetic acid, and, by this means, compound 41 [R = CH(S-isoBu)z,R’ = HI has been prepared in high yield.77 An illustration of the potential value of 2,3-unsaturated furanoid compounds, particularly in nucleoside synthesis, is the preparation, by way of an epoxy intermediate, of a crystalline compound claimed to be ethyl 3-amino-3-deoxy-~~-arabinofuranoside from racemic 2-etho~y-2,5-dihydro-5-(tetrahydropyranyloxymethyl)furan (41, R = OEt, (72) J . Defaye and J. Hildesheim, Bull. Soc. Chim. Fr.,940 (1967). (73) J. CICophax and S. D. GCro, Tetrahedron Lett., 5505 (1966). (74) J. CIBophax, J. Hildesheim, and S. D. GCro, Compt. Rend., 265,257 (1967); Bull. Soc. Chim. Fr., 4111 (1967). (75) J. ClBophax and S. D. GBro, Bull. Soc. Chim. Fr., 1441 (1967). (76) J. Hildesheim, J. CIBophax, and S. D. GBro, Tetrahedron Lett., 1685 (1967). (76a) J. Defaye, Bull. SOC. Chim. Fr.,2099 (1968). (77) J. Ckophax, S.D. GBro, and R. D. Guthrie, Tetrahedron Lett.,567 (1967).

230

R. J. FERRIER

R' = C5H90-OCH2).78 Methyl 5-O-trityl-p-~-ribofuranoside was obtained with good specificity on oxidation of methyl 2,3-dideoxy5-0trityl-p-~-glycero-pent-2-enoside (41, R = OMe, R' = CH,OTr) with osmium t e t r a ~ x i d e . ' ~ ~ b. Enol Derivatives. - 3-Deoxy-2-O-methyl-~-~-erythro-hex-2-enofuranose (42, R = R' = H, p-Danomer), obtainable by mild treatment

of 2,3-di-O-methyl-~-glucose(see p. 263) with alkali, afforded, on controlled reduction with sodium borohydride followed by mild treatment with acid, 3,4-dideoxy-~-gZycero-hex-3-enulose, which has the trans orientation at the double bond, and, consequently, the acyclic structure 43. By further reduction with sodium borohydride,

y%oH 'i=O CH

II HC I HCOH I ChOH (43)

the diastereoisomeric tetrols were obtained, and, from these, 3,4dideoxy - 1,2:5,6-di-O-isopropylidene-trans-~-e~ythroand -D- threohex-3-enitol were prepared (see p. 261). The D-thTeO-tetrOl shows the C=C stretching absorption band (at 1650 cm-'), but the erythro isomer does not, so the isomers can be distinguished by infrared spectroscopy; differentiation is not possible by nuclear magnetic (78) I. Iwai, T. Iwashige, and M. Asai, Japan. Pat. 4591 (1966); Chem. Abstracts, 65, 3950 (1966). (78a) N. J. Leonard, F. C. Sciavolino, and V. NairJ. Org. Chem., 33,3169 (1968).

UNSATURATED SUGARS

231

resonance spectroscopy, which, however, was used to confirm the trans character of the compound^.^^ Studies have also been made of 3-deoxy-2,5,6-tri-O-methyl-~erythro-hex-2-enofuranose(42, R = H, R’ = Me), prepared from 2,3,5,6tetra-0-methyl-D-glucofuranose.80 The derived methyl glycosides were separated, and were found to be very acid-labile, affording black polymers under several conditions, but, on reaction with methanol, they gave the methyl 3-deoxy-5,6-di-O-methyl-~-glycero-hex-3-enosidulose dimethyl acetals which, like the unsaturated free sugar, gave 5-(~-gZycero-l,2-dimethoxyethyl)-3(2H)furanone (44) on hydrolysis.

2. Pyranoid Derivatives

a. Alkene Derivatives. -The two general methods reported earlier’ for the synthesis of compounds in this class, namely, rearrangement reactions of glycal esters and direct elimination from saturated precursors, have been developed appreciably during the past four years. In the former reactions, it has been shown that glycosides can be formed readily from glycal esters by use of boron trifluoride as the catalyst (see p. 215) or, alternatively, by the use of 2,3-unsaturated7 glycopyranosyl fluoride esters as glycosylation agents (see p. 216). In work with 4,6-di-O-benzoyl-2,3-dideoxy-~-erythro-hex-2-enopyranosyl fluoride, hydrolysis gave a hydroxy compound which is the first, well characterized, crystalline, free sugar of the “pseudoglycal” series.44

(79) E. F. L. J. Anet, Carbohyd. Res., 1,95 (1965). The physical constants given for the free trans-D-threo derivative were incorrect, but were revised in Aust. J . Chem., 19,1677 (1966); see also, Ref. 157. (80) E. F. L. J. Anet, Tetrahedron Lett., 1649 (1966); Carbohyd. Res., 2,448 (1966).

232

R. J. FERRIER

Methyl 4,6-0-benzylidene-2,3-dideoxy-~-~-erythro-hex-Z-enopyranoside (45) had previously been prepared by six different routes,' several of which were not suited to production of the compound in high yield. A systematic investigation of the synthesis of the olefin has now shown that it is most efficiently obtained from methyl 2,3anhydro-4,6-O-benzylidene-a-~-allopyranoside by treatment with sodium iodide, sodium acetate, and acetic acid in refluxing acetone to give the altro iodohydrin (2, R = I, R' = H) which, on being heated in pyridine with p-toluenesulfonic acid is converted into the olefin (45) in almost quantitative yield. It was shown that the method is generally applicable, and the p-D anomer of compound 45, and the a-and p-Dthreo isomers, were similarly prepared in high yield from the correand , P-D-talo e p ~ x i d e s . ~ sponding p - ~ - a l l o(,Y - D - ~ U ~ O The related reaction in which methyl 4-0-benzyl-3-deoxy-3-iodo-20-p-tolylsulfonyl-P-L-xylopyranoside was treated with sodium iodide in acetone' has now been shown to give methyl 4-0-benzyl-2,3-dideoxy-p-~-gZycero-pent-2-enosideat low temperatures only. At room temperature, this compound is formed together with an isomer believed to be methyl 2-0-benzyl-3,4-dideoxy-~-~-gZycero-pent-3-enoside; and, as the 2,3-unsaturated glycoside is not isomerized on heating, it was proposed that the products are formed under kinetic control. Similar elimination from methyl 4-0-benzyl-3-deoxy-3-iodo2-O-p-to~y~su~fony~-6-O-trityl-a-~-glucopyranoside required elevated temperatures, and gave a crystalline, 2,3-unsaturated product.80a Iodo compounds were utilized in a further, new synthesis of unsaturated glycosides of this series. Thus, treatment of methyl 3,4,6tri-O-acetyl-2-deoxy-Z-iodo-~-~-glucopyranoside and the C Y - D - ~ U ~ ~ O isomer with sodium cobalt tetracarbonyl and carbon dioxide in ether caused eliminations, and afforded methyl 4,6-di-O-acetyl-2,3-dideoxyp- and a-~-e~ythro-hex-2-enopyranoside, respectively.8ob Alternatively, Horton and coworkers recommend the use of 2,3disulfonic esters of methyl 4,6-O-benzylidene-a-~-glucopyranoside for the preparation of the olefin 45. Heating such compounds with potassium ethylxanthate in boiling butanol, or with the Tipson-Cohen reagent (N,N-dimethylformamide, sodium iodide, and zinc dust) afforded the product in 40-55% yield, and methyl 2,3-anhydro-4,6-0benzylidene-a-D-allopyranosideand the 2,3-epithio analog also gave this compound under the former conditions.81Applied to vicinal di(80a) S. Dimitrijevich and N. F. Taylor, in press. (80b) A. Rosenthal and J. N. C. Whyte, Can.]. Chem., 46,2245 (1968). (81) E. Albano, D. Horton, and T. Tsuchiya, Carbohyd. Res., 2,349 (1966).

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233

sulfonates, the latter conditions are apparently of less value as a general route to unsaturated sugars than the reaction involving iodohydrins, as they do not give the appropriate olefins from methyl 4,6-0-benzylidene-2,3-di-O-p-tolylsulfonyl-~-~-glucopyranoside or the a-D-galact0 i ~ o m e r .Other ~ workers have advocated the specific use of a zinc-copper couple in this reaction, and have applied itsla to the synthesis of 2,3-unsaturated compounds bearing ester groups at C-4 and C-6. An application of the Tipson-Cohen reaction to the 2,3di-O-p-tolylsulfonyl-6-O-trityl derivative of amylose gave an unsaturated polymer from which a dibromo derivative was obtained.81b Other reactions that have afforded this a-erythro olefin (45) are (i) 2,3heating of methyl 4,6-O-benzylidene-a-~-mannopyranoside thionocarbonate in trimethyl phosphite (40 % yield)81;(ii)treatment of methyl 4,6-0-benzylidene-2,3-dideoxy-2,3-epimino-a-~-allopyranoside (or the manno isomer) with sodium nitrite in aqueous acetic acid [81 and 78 %yield, respectively; by way of 2,3-(N-nitrosoepimino) corn pound^]^^; (iii) hydrazinolysis of various, vicinal, azidodeoxy-ptolylsulfonyl or -(methylsulfonyl) or diazidodideoxy derivatives (65-75%; however with compounds having the substituents at both C-2 and C-3 equatorially attached, elimination did not occur)83,84; (iv) ammonolysis of methyl 4,6-0-benzylidene-2-deoxy-3-O-(methylsulfony1)-a-D-ribo-hexopyranoside(80%; reactions with sodium azide and hydrazine gave mainly products of direct d i s p l a ~ e m e n t ) (v) ~~; treatment of various 2-deoxy-3-sulfonates or 3-deoxy-2-sulfonates with soda-lime (25-50%; however, methyl 4,6-0-benzylidene-3deoxy-2-O-p-tolylsulfonyl-a-~-arabino-hexopyranoside gave a 75 % yield)86;with potassium butoxide in methyl sulfoxide, methyl 4,6-0benzylidene-2-deoxy-3-O-(methylsulfonyl)-a-~-rib~-hexopyranoside gave a 78% yield of compound 45, whereas the a-D-arabino isomer gave mainly the 3,4-unsaturated isomer (see p. 249)x7;(vi) treatment of with thiomethyl 2,3-anhydro-4,6-0-benzylidene-a-~-allopyranoside urea gave mainly methyl 4,6-0-benzylidene-2,3-dideoxy-2,3-epithio@la) B. Fraser-Reid and B. Boctor, Can./. Chem., 47,393 (1969). (81b) D. D. Clode, D . Horton, M. H. Meshreki, and H. Shoji, Chem. Commun., 694 (1969). (82) R. D. Guthrie and D. King, Carbohyd. Res., 3,128 (1966). (83) R. D. Guthrie and D. Murphy,/. Chem. Soc.,6956 (1965). (84) R. D. Guthrie and D. Murphy, Carbohyd. Res., 4,465 (1967);R. D. Guthrie, R. D. Wells, and G. J. Williams, ibid.,10, 172 (1969). (85) J. Kova;, V. Dienstbierova, and J. Jary, Collect. Czech. Chem. Commun., 32, 2498 (1967). (86) S. McNally and W. G. Overend, J . Chem. SOC. ( C ) , 1978 (1966). (87) S. Hanessian and N. R. Plessas, Chem. Cornmun., 706 (1968).

234

R. J. FERRIER

a-D-mannopyranoside, but, also, 20% of the olefin (which did not arise from the episulfide, since this compound is stable under the reaction conditions). The elimination was consequently believed to occur from thiouronium intermediates.88 Alternatively, treatment of the isomeric D-~T~UWW epoxide with potassium selenocyanate in aqueous 2-methoxyethanol gave 81% of the olefin and no episelenide, and this reaction appears to be of appreciable interest as a general means of obtaining olefinic carbohydrate derivative8O; (vii) desulfurization of 2,Sunsaturated derivatives bearing59s87thio groups on C-2. Information on the reactions of compounds in this series has been scant,' but several investigations have now helped to make good this deficiency. cis-Hydroxylation, with hydrogen peroxide and osmium tetraoxide, of ethyl 2,3-dideoxy-a-~-erythro-hex-2-enopyranoside and its diacetate afforded D - ~ U W O products almost exclusively, the acetate being obtained crystalline in 57% yield.90 Similarly, p-nitrophenyl 4,6-di-0-acetyl-2,3-dideoxy-a-~-e~ythro-hex-2-enopyranosides' and methyl 4-0-benzyl-2,3-dideoxy-6-O-trityl-a-~-erythro-hex2-enopyranoside80agive mainly the manno no products on treatment with potassium permanganate. These results were confirmed in a study that used chromatographic methods for identifying the sugars formed by hydroxylation of the double bond followed by acid hydrolysis, and, similarly, the olefin 45 gave mainly a D - ~ U M O product. However, p-nitrophenyl4,6-di-O-acetyl-2,3-dideoxy-P-~-erythro-hex2-enopyranoside gave mainly D-allose, from which it was concluded that cis-hydroxylation occurs mainly from the side of the double bond that is trans to the aglycon. For p-nitrophenyl 4,6-di-O-acety1-2,3dideoxy-a-~-threo-hex-2-enopyranoside, addition apparently occurred with equal facility from both sides of the olefinic bond, as D-gulose and D-talose were formed in approximately equal amounts after hydrolysis.8s Predictably, on cis-hydroxylation, 6-0-(4,6-di-O-acetyl-2,3-dideoxya-D-erythrohex-2-enopyranosyl)-1,2:3,4-di-O-isopropy~idene-~-D-galactopyranose afforded an adduct from which, after removal of the protecting groups, 6-0-a-D-mannopyranosy~-D-gdactose was obtained." On epoxidation of this unsaturated disaccharide derivative with benzonitrile and hydrogen peroxide, the same stereoselectivity (88) R. D. Guthrie and D. Murphy, J . Chem. Soc., 6666 (1965);see also, M. Kojima, M. Watanabe, and T. Taguchi, Tetrahedron Lett., 839 (1968). (89) T. van Es, Carbohyd. Res., 5,282 (1967). (90) C. L. Stevens, J. B. Filippi, and K. G. Taylor,J. Org. Chem., 31,1292 (1966). (91) R. J. Ferrier, W. G . Overend, and G. H. Sankey,J. Chem. Soc., 2830 (1965).

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235

was not apparent, and a crystalline (but mixed) product was obtained from which, however, 6-O-a-D-altropyranosy~-D-galactoseand 6-0-(3, Ganhydro-a!-Dglucopyranosyl)-D-galactose were isolated after suitable alkaline and acidic hydrolysis. Similar epoxidation of methyl 4,6-di-0acetyl-2,3-dideoxy-a-~-erythTo-hex-2-enopyranoside (5, R = Me) gave methyl 4,6-di-0-acetyl-2,3-anhydro-a-~-allopyranoside and -mannopyranoside in the ratio of 2:3, but, consistent with expectations, the 2,3-anhydro-~-allosidewas the major product (aZZo:manno,3: 1)when the oxidation was performed on the unacetylated analog. From methyl 4,6-di-0-acetyl-2,3-dideoxy-a-~-threo-hexopyranoside, the D-gdo and D-~UZO epoxides were produced in the ratio of 1:2, and this ratio changed to 1:30 with the unsaturated dihydroxy g l y ~ o s i d eEpoxida.~~ tions of 3,6-dihydro-(2H)pyran derivatives indicated that allylic substituents exert powerful control over the direction of epoxidati~n.~~~ Other additions to the double bond of compounds in this series have not been studied extensively, but reports now indicate that several such additions can be accomplished efficiently. Nitryl iodide is, however, not addeds2 to the double bond of compound 45, but bromination in methanol, in the presence of barium carbonate, gives crystalline methyl 4,6-0-benzylidene-2,3-dibromo-2,3-dideoxy-a-~altropyranoside in 70 % yield.03 On treatment with potassium tertbutoxide in refluxing xylene, this compound undergoes elimination of the elements of hydrogen bromide to give methyl 4,6-O-benzylidene2-brorno-2,3-dideoxy-a-~-threo-hex-3-enopyranoside (46, R = Br) in 90% yield, Likewise, compound 45 reacts with acetyl hypobromite to give methyl 3-0-acetyl-4,6-0-benzylidene-2-bromo-2-deoxy-~-~-glu-

(91a) F. Sweet and R. K. Brown, Can.]. Chem.,46,1592,2283 (1968); V. B. Mochalin, Yu. N. Porshnev, and G. I. Samokhvalov, Zh. Obshch. Khim., 38, 85 (1968); Chem. Abstracts, 69,19457(1968). (92) W. A. Szarek, D. G . Lance, and R. L. Beach, Chem. Commun., 356 (1968). (93) E. L. Albano, D. Horton, and J. H. Lauterbach, Chem. Commun., 357 (1968); Carbohyd. Res., 9,149 (1969).

236

R. J. FERRIER

copyranoside and methyl 2-0-acetyl-4,6-0-benzylidene-3-bromo-3deoxy-a-D-altropyranoside; with methanolic mercuric acetate, it gives a crystalline adduct, and, on treatment with diiodomethane and a zinc-copper couple, a cyclopropane derivative, namely, methyl 4,6-0benzylidene-2,3-dideoxy-2,3-methylene-a-~-allopyranoside, was obtained.03 Attempted detritylation of methyl 4-0-benzyl-2,3-dideoxy-6-0trityl-a-~-erythro-hex-2-enopyranosidewith hydrogen bromide caused addition to the double bond and also 1,6-anhydride formation, to give 1,6-anhydro-4-O-benzyl-3-bromo-2,3-dideoxy-~-uru~~~oh e x o ~ e ,so~ ~that ~ the addition is related to that occurring when tri-0-acetyl-D-gluca1is treated with this reagent in acetic acid. l8 Compound 45 is markedly sensitive to acidic conditions, and even prolonged contact with silica gel leads to its decomposition. In aqueous acetic acid at 70°,it is rapidly hydrolyzed to give 2-(D-glyCerO1,2-dihydroxyethyl)furan(27, R = R’ = H),8l and, in ethanol containing sulfuric acid, the same product is initially formed, but it then reacts with ethanol to give, successively, 2-(1-ethoxy-2-hydroxyethy1)furan (27, R = H, R’ = Et), 2-(1,2-diethoxyethyl)furan(27, R = R’ = Et), and 2,6-diethoxy-4-oxohexanaldiethyl acetal (all race mi^),^^ that is, the same products as were obtained on similar treatment of tri-0-acetylD-glUCal (see p. 218).The rate of hydrolytic removal of benzaldehyde from compound 45 was found to exceed that of related saturated comp o u n d ~ alternatively, ;~~ under basic conditions, compound 45 isomerizes almost completely, to methyl 4,6-0-benzylidene-2,3-dideoxya-~-glycero-hex-3-enopyranoside (46, R = H), the change being brought about by treatment with potassium te~t-butoxide.~~ Ethyl 4,6-di-O-acetyl-2,3-dideoxy-a-~-eryth~o-hex-2-enopyranoside (5, R = Et) takes part in another ring-contraction reaction on treatment with lead tetraacetate and anhydrous hydrogen fluoride (see p. 205). On deacetylation, this type of unsaturated glycoside gives a diol having one of the hydroxyl groups in the allylic position, and the nucleophilic displacements of the sulfonyloxy groups in derived disulfonates have been investigated to determine the relative ease of their removal and the general course of the substitution at the allylic center. Methyl 2,3-dideoxy-a-~-threo-hex-2-enopyranoside (47) was selected for the study, and its dimethanesulfonate was heated with (93a) N. F. Taylor, personal communication. (94) D. Horton and T. Tsuchiya, Carbohyd.Res., 3,257 (1966). (95) J. KoGa;, F. Hanousek, and J. Jar$, Collect. Czech. Chem. Commun., 33, 630 (1968).

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237

sodium benzoate in N,N-dimethylformamide. The course of the displacements and subsequent conversions of the products is illustrated in Scheme 1, which shows the tendency of the allylic group to

MeCl, pyridine y&OH (1) NaOMe (2) M6C1,

pyridine

H o o O M e

Scheme 1

be displaced initially and by a direct mechanism involving Walden inversion. Compounds having a double bond at C-3-C-4 were not det e ~ t e dIn . ~a~less-complete investigation, ethyl 2,3-dideoxy-4,6-di-Op-tolylsu~fony~-a-~-erythro-hex-2-enopyranoside was treated with sodium iodide; it gave a di-iodo product, the structure of which was not el~cidated.~' Ultraviolet, infrared, and x-ray data on compound 45 have been reported,*' and it has been pointed out! that, in this series, the-anomeric pairs of glycosides that have so far been examined conform with Hudson's isorotation rules, unlike some 2,3-unsaturated glycosyl ester^.^^*^' Features of the nuclear magnetic resonance spectra are (96) D. M . Ciment, R. J. Ferrier, and W. G . Overend,]. Chem. SOC. (C), 446 (1966). (97) K. C . Kim, J. Korean Chern. SOC., 7 , 140 (1963); Chem. Abstracts, 61, 12072

(1964).

R. J. FERRIER

238

discussed on p. 265. Methyl 4,6-di-O-acety1-2,3-dideoxy-P-~-e~ythrohex-2-enopyranoside has been examined by mass ~ p e c t r o m e t r y . ~ ~ An interesting nucleoside derivative, namely, cytosinine (48, R = H), possessing a 2,3-unsaturated, pyranoid ring is produced on

hydrolysis of blasticidin S (48, R = X ) : * an antibiotic substance active against rice-blast disease. After reconsiderationw of the structure originally proposed,'"'' the stereochemical configurations at C-2 and C-6 of the dihydropyranyl moiety of cytosinine were established by converting the compound into the same dialdehyde (49) as is obtained on periodate oxidation of cytidine.l0'

""YOY"'" O=CH HC=O

b. Enol Derivatives. - In a continuation of previous studies on the products from the alkaline degradation of 2,3di-O-methyl sugars,l Anet has obtained crystalline compounds from 2,3,4-tri-O-methyl-Dglucose, 2,3,4,6-tetra-O-methyl-~-glucose, and 2,3,4-tri-O-methyl-Dgalactose, and, by nuclear magnetic resonance spectroscopy, has shown them to have a-D-pyranoid structures (50, R = H), (50, R = Me) and (51, R = H), respectively. Alternatively, the compound isolated from the reaction products of 2,3-di-O-methyl-~-glucosewas found to be 3deoxy-2-O-methyl-~-~-erythro-hex-2-enofuranose (see p. 230), which isomerizes in pyridine to give a mixture of the a- and p-Dfuranose forms; then, on addition of water, the a-D-pyranose modifi(98)

J. J.

Fox, K. A. Watanabe, and A. Bloch, Progr.

NucLtc A d d Res. MoZ. B i d , 5,

251 (1966). (99) J. J. Fox and K. A. Watanabe, Tetrahedron Lett., 897 (1966). (100) N. Otake, S. Takeuchi, T. Endo, and H. Yonehara, Tetrahedron Lett., 1405 (1965). (101) H. Yonehara and N. Otake, Tetrahedron Lett., 3785 (1966).

UNSATURATED SUGARS

239

cation is produced. Acetylation of the crystalline sugar with acetic anhydride in pyridine gave a mixture of three products, from which the triacetate of the six-membered isomer crystallized.lo2

After methylation of compounds (50, R = Me) and (51, R = Me) with methyl sulfate, four isomeric pyranosides were isolated by gasliquid chromatography; by detailed nuclear magnetic resonance studies, these were shown to exist in the H! conformation.lm Unlike related esters (see p. 221), but like other 2,Sunsaturated glycosides (see p. 237), these anomeric pairs of glycosides were found to conform with Hudson’s isorotation rules. In dilute acid, they are degraded to methyl 3,4-dideoxy-6-0-methyl-aand P-~-glycero-hex-3-enopyranosidulose (52); these also were characterized configurationally and conformationally by nuclear magnetic resonance methods.

For preparing compounds in this class, an alternative means, involving rearrangement reactions of hydroxyglycal esters, has already been referred to (see p. 221). Such 2,3-unsaturated glycosyl esters are obtainable by isomerization of hydroxyglycal derivatives (see p. 221), and the corresponding glycosides have been synthesized by use of analogs having good leaving-groups at the anomeric center. Thus, treatment of 1,2,4,6-tetra-O-acetyl-3-deoxy-a-(or P)-D-erythrOhex-2-enopyranose (36;R = OAc, R’ = Ac) with aluminum chloride in (102) E. F. L. J. Anet, Aust.]. Chem., 18,837 (1965). (103) E. F. L. J. Anet, Carbohyd. Res., 1,348 (1965).

240

R. J. FERRIER

chloroform gave the reactive 2,4,6-tri-O-acetyl-3-deoxy-a-~-erythrohex-2-enopyranosyl chloride (36;R = C1, R' = Ac, shown by nuclear magnetic resonance spectroscopy to be over 80 % anomerically pure), which could be reconverted into the starting materials, or be used in synthesis of g l y c o ~ i d e s For . ~ ~ the latter, the isomeric products were formed without stereoselectivity; but it has been shown that the direct reaction of hydroxyglucal esters with alcohols in the presence of boron trifluoride affords the same mixtures, with the a - anomers ~ preponderating (see p. 223). Crystalline products (36,R = alkyloxy, R' = Bz, (Y-D anomers) were generally obtained from tri-O-benzoyl-2benzoyloxy-D-glucal, and the procedure can be employed in inert solvents with equimolar amounts of complex alcohol^.^' p-DAnomers in this series (36,R = alkyloxy, R' = Bz) have alternatively been obtained by solvolysis of 2,4,6-tri-O-benzoyl-3-deoxy1-0-(trichloroacetyl)-a-~-erythro-hex-2-enopyranose (36,R = OOC-CCl,, R' = Bz), and it was found that the anomeric pairs of glycosides differ little in their optical rotation at 589 nm, but can be readily distinguished by their optical rotatory dispersion curves.57 1,2,4,6-Tetra-O-acetyl-3-deoxy-(~-~-er~thro-hex-2-enose has been examined by mass A lactone and a free sugar of this series have been obtained during investigations of nucleophilic displacements at C-5 of a hexonic acid derivative, in reactions (similar to those previously reported') by which related enamines were formed. Treatment of 2,3,4,6-tetra-Obenzyl-N,N-dimethyl-5-0-(methylsulfony1)-D-gluconamidewith potassium acetate in N,N-dimethylformamide gave 2,4,6-tri-O-benzyl-3deoxy-~-threo-hex-2-enono1,5-lactone (53,R = 0)which, with

dimethylamine, gave the aldonamide; on controlled reduction with lithium aluminum hydride, this afforded crystalline 2,4,6-tri-0benzyl-3-deoxy-~-threo-hex-2-enopyranose (53,R = H,OH) in 70 Yo yield.lo4 Enolization of keto compounds offers an alternative and largely unexploited means of introducing double bonds, but the (104) H. Kuzuharaand H. G. Fletcher, Jr.,J, Org. Chern., 33,1816 (1968).

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241

crystalline enediol derivative methyl 3-0-methyl-~-erythro-hex-2enopyranuronate was obtained by diazomethane treatment of D-ribohexos-3-ulopyranuronic acid.*05

Treatment of 1,2,3,4-tetra-O-acetyl-~-rhamnosewith theophylline in the presence of an acid catalyst gave the first known nucleoside of this class, namely 7-(2,4-di-O-acety1-3,6-dideoxy-~-erythro-hex-2enopyranosy1)theophylline (53a), which was assigned the a-L configuration on account of its negative optical r 0 t a t i 0 n . l ~ ~ ~ Nuclear magnetic resonance spectroscopy has revealed, somewhat surprisingly, that the anomers of 1,2,4,6-tetra-O-acetyl-3-deoxy-~erythro-hex-2-enopyranoses(54, 55, R = CH,OAc) and the corresponding -D-g~ycero-pent-2-enopyranoses(54,55, R = H) adopt different conformations which, from the observed J4.,=, values (a-Danomers, about 9 Hz, p-D anomers, about 1 Hz) must be close to those ~ h o ~ n Consideration . ~ ~ * ~ ~of, the ~ ~pentose ~ derivatives (54 and 55,

R = H ) led to the conclusion that the acetoxyl group at C-4 favors the quasi-axial orientation, as the p-D anomer is thermodynamically favored, and the phenomenon whereby large groups at allylic cen(105) T. Kinoshita, M . Ishidate, and Z. Tamura, Chem. Pharm. Bull. (Tokyo), 14, 991 (1966). (105a) K. Onodera, S. Hirano, F. Masuda, and T. Yajima, Chem. Commun., 1538(1968). (106) R. J. Ferrier and G. H. Sankey,J. Chem. SOC. (C), 2345 (1966).

R. J. FERRIER

242

ters on unsaturated, pyranoid rings show inverted stereochemical requirements was termed the "allylic effect" and shown to influence the thermodynamic properties of such compounds appreciably.lW The hydroformylation reaction has been applied to 1,2,4,6-tetra-Oacetyl-3-deoxy-a-~-er~thro-hex-2-enopyranose (36, R = OAc, R' = Ac, a -anomer), ~ and, from the products, crystalline penta-O-acetyl-3deoxy-3-C-(hydroxymethyl)-a-~-glucopyranose (56) was isolated after acetylation and gas-chromatographic ~eparation.'~'Structural analysis was conducted by the use of nuclear magnetic resonance methods applied to the labeled analog carrying a deuterium atom at C-2.

AcO0 C&OAc 0Ac OAc (56)

c. Compounds Having a Nitrogen Substituent on the Doubly Bound Carbon Atom. - Baer and coworkers had earlier shown that methyl 4,6-0-benzylidene-2,3-dideoxy-3-C-nitro-~-~-ervthro-and -threo-hex-2-enosides can be prepared readily by base-catalyzed elimination of acetic acid from appropriate 2-0-acetyl-3-deoxy-3-Cnitroglycopyranosides,' and they have now reported the preparation of a-D-glycosides of this type, and a series of addition reactions. From methyl 4,6-0-benzylidene-2,3-dideoxy-3-C-nitro-~-~-er~~~rohex-2-enopyranoside (57), adducts having structures 58, where

0," (57)

(107) A.Rosenthal and H. J. KochJ. Amer. Chem. SOC., 90,2181(1968).

UNSATURATED SUGARS

243

R = H (Ref. 1081, NH, (Ref. log), OMe, OEt, OCH,Ph, SCH,Ph, NEt,, NCJHIO,NHCH,CO,Et (Ref. 110) OC-CH(Me)CO,-isoPr (Ref. lll), CHR'NO, (Ref. l l l a ) , and CH(CO,Et), (Ref. l l l a ) , were obtained by appropriate addition procedures, the lactate reaction giving two diastereoisomers (having different stereochemistry at the lactyl asymmetric center) which were converted into positional isomers of muramic acid. Syntheses of the a-D anomer of compound 57 and the a-threo isomer were achieved b y analogous procedures, and these compounds were found to show markedly different reactivity from that of the P-D compounds toward hydrogen in the presence of a palladium catalyst. Whereas the p-D compound was hydrogenated to give 2,3-dideoxy-3-nitro adducts having the D - U T U ~ ~ and O D-ZYXO configurations, respectively, the CX-Danomers gave these compounds together with appreciable proportions of 2,3-dideoxy-3-oximino products.*12Dehydroacetylation of methyl 2,4,6-tri-O-acetyl-3-deoxy3-nitro-/3-~-glucopyranosidecaused the formation of a diene which then dimerized.112a A compound having a related structure, namely, methyl 4,6-0benzylidene-2,3-dideoxy-3-phenylazo-a-~-erythro-hex-2-enopyranoside, was obtained on treatment of methyl 2-0-benzoyl-4,6-O-benzylidene-a-~-ribo-hexosid-3-ulose with phenylhydrazine, followed by a molar proportion of sodium methoxide. It was found to undergo additions similar to those reported for the 3-nitro anologs.112b Alternatively, 2,3-unsaturated compounds having nitrogen-containing substituents attached at C-2 have been reported. Unsaturated products obtained by acetylation of 2-acetamido-2-deoxy-~-mannose with isopropenyl acetate in the presence of p-toluenesulfonic acid have already been discussed (see p. 225). From the products of the same reaction of 2-acetamido-2-deoxy-~-galactose,1,4,6-tri-O-acetyl2 - ( N -acetylacetamido) - 2,3 - dideoxy - ~-threo-hex-2-enopyranose (59, R = R' = Ac) was isolated, in addition to a furan derivative and peracetates of the free sugar. Unlike the C-2 oxygen analog, compound (108) H. H. Baer and F. Kienzle, Can.]. Chem., 43,3074 (1965). (109) H. H. Baer and T. Neilson,]. Org. Chem., 32, 1068 (1967); but see, H. H. Baer and K. S. Ong, ibid., 34,560 (1969). (110) H. H. Baer. T. Neilson, and W. Rank, Can.]. Chem., 45,991 (1967). (111) H. H. BaerandF. Kienzle,]. Org. Chem.,32,3169(1967). ( l l l a ) H. H. Baer and K. S. Ong, Can.J.Chem.,46,2511(1968). (112) H. H. Baer and F. Kienzle, Can.]. Chem., 45,983 (1967). (112a) H. H. Baer and F. Kienzle,]. Org. Chem.,33,1823 (1968). (112b) D. Gardiner, Ph. D. Thesis, London, 1969.

244

R. J. FERRIER

59 (R = R' = Ac) could be deacetylated to give a stable, crystalline, free sugar (59, R = R' = H).l13 The vinyl azide, namely, methyl 2azido-4,6-O-benzylidene-2,3-dideoxy-a-~-eryt~ro-hex-2-enopyranoside (60, R = N3) was isolated on base-catalyzed elimination of

methyl 2-azido-4,6-0-benzylidene-2-deoxy-3-O-(methylsulfonyl)-a-~altropyranoside. It showed a strong absorption band at 243 nm (alkyl azide < 220 nm), and, on irradiation at 300 nm, decomposed to unidentified products.*' The related, crystalline enamine, namely, methyl 4,6-0-benzylidene-2,3-dideoxy-2-pyrrolidinyl-a-~-er~thr~hex-2-enopyranoside (60, R = NC4H,) was alternatively prepared in excellent yield from the corresponding hexopyranosidulose, and has been used in the C-3 alkylation of this compound.114

d. Compounds Having a Sulfur Substituent on the Doubly Bound Carbon Atom.-As noted previously (see p. 225), pyranoid compounds having a double bond at C-1-C-2, C-2-C-3, and C-3-C-4 have been isolated from the reaction of methyl S-benzyl(or S-methyl)-4,6-0benzylidene(or ethylidene) -3-0 -methyl-2-thio-a-~-altropyranoside with sodium in dimethoxyethane; and, of these, the 2,3-unsaturated products [for example, methyl 4,6-0-benzylidene-3-deoxy-2-S-methyl2-thio-a-~-erythro-hex-2-enopyranoside (60, R = SMe)] preponderate. On desulfurization with Raney nickel, compound 60 (R = SMe) gave methyl 4,6-0-benzylidene-2,3-dideoxy-a-~-e~~thro-hex-2-enopyranoside (45), and, under the alkaline conditions in which it was formed, was isomerized partially to a 3,4-unsaturated-2-thio compound.50~s0 A more satisfactory synthesis of compounds of this type has been achieved by use of sulfonic esters. Thus, methyl 4,6-0-benzylidene-30 - (methylsulfonyl)- 2 - S - phenyl - 2 - thio -a- D - altropyranoside gave methyl 4,6 - 0 - benzylidene -3-deoxy-2-S -phenyl-2-thio-a-D-erythro(113) N. Pravdid and H. G. Fletcher, Jr., Croat. Chem. Acta, 39, 71 (1967). (114) R. F. Butterworth, W. G. Overend, and N. R. Williams, Tetrahedron Lett., 3239 (1968).

UNSATURATED SUGARS

245

hex-2-enopyranoside (60, R = SPh) in 90% yield on treatment with 1,5-diazabicyclo[4.3.O]non-5-enein methyl sulfoxide, and the product could similarly be desulfurized to givee7the olefin (45). e. Compounds Having a Chlorine Substituent on the Doubly Bound Carbon Atom -On treatment with sulfuryl chloride and pyridine followed by heating with a solution of pyridinium chloride in chloroform, methyl a-L-arabinopyranoside gave crystalline methyl 3,4dichloro-3,4-dideoxy-~-~-ribopyranoside 2-chlorosulfate which, on standing in pyridine, underwent the loss of chlorosulfuric acid to give crystalline methyl 3,4-dichloro-2,3,4-tideoxy-~-~-gZycero-pent2-enopyranoside (61, R = Me, p-D anomer). Among the products of

reaction of sulfuryl chloride with L-arabinose in pyridine was obtained a dimer to which the structure 3,4-dichloro-2,3,4-trideoxy-a-Dglycero-pent-2-enopyranosyl 3,4-dichloro-2,3,4-trideoxy-a-~-g~yceropent-2-enopyranoside was assigned. It thus bears a strong similarity to the dimer formed on treatment of 4,6-di-O-acety1-2,3-dideoxy-~erythro-hex-2-enopyranosylfluoride with water (see p. 216). Methanolysis of the dimer gave the methyl glucoside (61, R = Me, p-Danomer), with a marked decrease in optical rotation which was taken to indicate that a configurational inversion at C-1 had occurred, and, consequently, that the dimer was an a,acompound.llS However, in a fuller investigation of this series of compounds,l16methyl 3,4-dichloro-2,3,4trideoxy-a-~-gh~cero-pent-2-enopyranoside (61, R = Me, a-D anomer) was prepared; it was found to have at 589 nm an optical rotation very similar to that of the p-Danomer, and, consequently, as has been noted elsewhere (see p. 239), measurements of optical rotation are unreliable for assigning anomeric configuration to such 2,3-unsaturated compounds. Indeed, nuclear magnetic resonance measurements suggested that the dimer is 3,4-dichloro-2,3,4-~ideoxy-~-~-glycero-pent-2-en0pyranosyl 3,4-dichloro-2,3,4-trideoxy-~-~-gZycero-pent-2-enopyranoside (62). In agreement with findings for the 1,2,4-tri-O-acetyl-3(115) H. J. Jennings and J. K. N. Jones, Can.J.Chem.,43,3018 (1965). (116) B.Coxon, H. J. Jennings, and K. A. McLauchlan, Tetrahedron, 23,2395(1967).

246

u\ R. J. FERRIER

c1

0

c1

deoxy-D-glycero-pent-2-enopyranoses (54, 55, R = H) (see p. 241), the P-D-glycoside (61, R = Me, p-Danomer) was found to be more stable than the a - anomer. ~ Also, whereas the a-D anomer adopted the H8 conformation, the other anomer existed mainly in the HZ form. Detailed nuclear magnetic resonance studies were made of the two glycosides, the dimer, and 3,4-dichloro-2,3,4-trideoxy-~-~-gZyceropent-2-enopyranose (61, R = H) (obtained crystalline, and in good yield, on hydrolysis of the methyl glycosides), and the relative stabilities of the glycosides were considered in terms of van der Waals energies and dipolar interactions.ll6

f. Compounds Having a Carbon Substituent on the Doubly Bound Carbon Atom.-The first members of this series have now been reported. Methyl 4,6-0-benzylidene-3-cyano-2,3-dideoxy-cr-~-ery~hr~hex-2-enopyranoside was obtained by elimination from saturated 3-cyano-3-deoxycompounds,116aand methyl 4-0-acetyl-2,3,6-trideoxy3-C-rnethyl-~-threo-hex-2-enopyranoside was formed on treatment of methyl 4-0-acetyl-2,6-dideoxy-3-C,O-dimethyl-~-~yZo-hexopyranoside with hydrogen chloride in dichloromethane.116b

v. 3,4-UNSATURATED, CYCLIC COMPOUNDS 1. Furanoid Derivatives 3- Deoxy - 1,2:5,6-di-0-isopropylidene-a-~-eryth~o-hex-3-enofunose (63, R = H), which has previously been described,' has now been shown to be the main product formed on heating 1,2:5,6-di-O-isopropylidene-3- 0 - p -tolylsulfonyl-a-D-glucose with tetrabutylam(116a) B. E. Davison and R. D. Guthrie, Carbohyd. Res., 9,254 (1969). (116b) G . B. Howarth, W. A. Szarek, and J. K. N. Jones,]. Org. Chem., 34,476 (1969).

UNSATURATED SUGARS

247

monium fluoride in acetonitrile, but the same reagent caused a direct, nucleophilic displacement at C-3 with the corresponding D-UZZG compound."' On treatment with dichlorocarbene, compound 63 (R = H) gave crystalline 3-deoxy-3,4-C-(dichloromethylene)-lY2:5,6di - 0- isopropylidene -a-D- galactose (characterized configurationally by x-ray diffraction analysis)"* in 80% yield; and, from this compound, the chlorine atoms could be reductively removed, to give the corresponding methylene a d d ~ c t In . ~ ~a similar way, hydroboration of compound 63 (R = H), which also involves cis-addition, occurs exclusively from the "upper" side of the double bond to give 1,2:5,60-isopropylidene -a-D-galactofuranose,"" and tritioboration consequently provides a means of synthesizing D-galaCtOSe-4-t from D-glucose.120Free-radical addition of a-toluenethiol occurs by initial attack on C-3 by the sulfur radical from the upper direction, followed by nonspecific addition of hydrogen at C-4, so that 3-thiO-D-glUCOSe and 3-thio-D-galactoseproducts were obtained.121 Related studies with 3-0-acetyl-1,2:5,6-di-O-isopropylidene-a-~erythro-hex-3-enose (63, R = OAc), which can be prepared crystalline, and in good yield, from 1,2:5,6-di-O-isopropylidene-a-~-ribo-3-hexulose hydrate, have provided a useful new synthesis122of D-gUlOSe, because sodium borohydride causes efficient reduction to crystalline 1,2:5,6-di-O-isopropylidene-a-~-gulose (64). Conversely, the unsaturated derivative (63, R = H) was produced, together with the 3deoxy-3-fluoro-~-galactosecompound on treatment of 1,2:5,6-di-0isopropylidene-3 - 0-p-tolylsulfonyl -D-gulose with tetrabutylammonium fluoride in acetonitrile.122a (117) K.W. Buck, A. B. Foster, R. Hems, and J. M. Webber, Carbohyd. Res., 3, 137 (1966). (118) J. S. Brimacombe, P. A. Gent, and T. A. Hamor, Chem. Commun., 1305 (1967); 1.Chem. Soc. ( R ) ,1566 (1968). (119) H.Paulsen and H. Behre, Carbohyd. Res., 2,80(1966). (120) J. Lehmann, Carbohyd. Res.. 2,1(1966). (121) J. Lehmann, Carbohyd. Res., 2,486(1966). (122) W.Meyer zu Reckendorf, Angew. Chem. Intern. Ed. Engl., 6,177(1967). (122a)J. S. Brimacombe, A. B. Foster, R. Hems, and L. D. Hall, Carbohyd. Res., 8, 249 (1968).

248

R. J. FERRIER

p OH z ,'0 M 'Mee

HbO, ,Me I C H,CO' 'Me

In work designed to develop syntheses of 3-deoxy-D-erythro-pentofuranose compounds, Brown and JoneslZ3have studied additions to 3-deoxy - 1,2-0-isopropylidene - a - ~ - g Z y c e r-pent-3-enodialdoo 1,4furanose (65, R = H), which was obtained by base-catalyzed elimination of p-toluenesulfonic acid from the corresponding 3-0-p-tolylsulfonyl-a-D-xylo-pentose derivative. Partial, catalytic hydrogenation gave 3- deoxy- 1,2-O-isopropylidene-~-~-threo-pentodialdo1,4-furanose (66) which could, however, be isomerized, largely to the a - ~ -

erythro aldehyde, under basic conditions. Addition of methanol to the unsaturated aldehyde, followed by reduction, gave a compound that was characterized by nuclear magnetic resonance spectroscopy as 1,2-0-isopropylidene-3-O-methyl-a-~-xylofuranose; it was formed with high stereospecifi~ity.'~~ For the related a$-unsaturated ketone (65, R = Ph), prepared by heating the 3-O-p-tolylsulfonyl-D-x~zo precursor in N,N-dimethylformamide in the presence of sodium carbonate, the notable feature of its chemistry was a Diels-Alder type of dimerization reaction that afforded a solid, pentacyclic product which was structurally characterized after consideration of the Woodward-Katz principle and by nuclear magnetic resonance 3 - O - A c e t y l - 1 , 2 - O - i s o p r o p y l i d e n e - ~ - ~ - g l y n o swas e obtained by treatment of the 3-ulose derivative with acetic anhydride in p ~ r i d i n e . ' ~ ~ ~ (123) D. M. Brown and G. H. Jones,]. Chem. SOC. (C), 249 (1967). (123a) T. D. Inch and P. Rich, Carbohyd. Res., 6,244 (1968). (123b) J. M. J. Tronchet and J. Tronchet, Compt. Rend., 267C, 626 (1968).

UNSATURATED SUGARS

249

2. Pyranoid Derivatives Further examples of unsaturated compounds in this category have been prepared by direct elimination reactions, and, for the first time, allylic rearrangements have now been shown to offer an alternative means of obtaining members of this series. The known methyl 4,6-0benzylidene-2,3-dideoxy-a-~-gZ ycero-hex-3-enopyranoside(46, R =H) has thus been prepared as the main product of base-catalyzed eliminamethylsulfony1)-ation from methyl 4,6-0-benzylidene-2-deoxy-3-0-( D-urubino-hexopyranoside[the D-ribo isomer, however, gave the 2,3, ~ ~ in analogous fashion, methyl olefin (45) almost e x c l ~ s i v e l y ] and, 4,6-0-benzylidene-2,3-dibromo-2,3-dideoxy-a-~-altropyranoside gave methyl 4,6-0-benzylidene-2-bromo-2,3-dideoxy-a-~-threo-hex-3-enopyranoside (46, R = Br) in high yield.93 Alternatively, the parent compound (46, R = H) is produced on reductive desulfurization of appropriate 2-alkylthio derivatives (46, R = SMe or SCH2Ph),59,60 and a related compound, namely, methyl 1,3-O-benzylidene-4,5dideoxy-a-~-gZycero-hex-4-enulopyranoside (67) (a 4,5-unsaturated 2-ketose derivative, but classified here because of structural similarity to the 3,4-unsaturated aldopyranoses) has been prepared from both a corresponding di-p-toluenesulfonate and a 5-azidodeoxy-4-O-(methylsulfonyl) derivative by appropriate elimination proced~res.'~ An isomerization of methyl 4,6-0-benzylidene-2,3-dideoxy-a-~erythro-hex-2-enopyranoside(45) to the 3,4-unsaturated compound (46, R = H) offers a satisfactory means of preparing this compound,

as the reaction is reported to occur almost completely with potassium tert-butoxide in methyl sulfoxide8'; this is in keeping with the known stability of vinyl ethers relative to allylic ethers under these conditions, but is at variance with the report that 3-propoxycyclohexene is However, the generality not isomerized to l-propoxycyclohexene.1z4 of this isomerization for the carbohydrates is indicated by the observation that 2,3-unsaturated compounds bearing an alkylthio group on C-2 (60, R = SMe or SCH,Ph) are also isomerized to give products (124) T. J. Prosser,J.Amer. Chem. SOC., 83,1701 (1961).

250

R. J. FERRIER

having C-3-C-4 double bonds (46, R = SMe or SCH,Ph; stereochemistry at C-2 ~ n d e f i n e d ) . Somewhat ~~.~~ surprisingly, an olefin, believed to be methyl 2-0-benzyl-3,4-dideoxy-P-~-glycero-pent-3enopyranoside was produced, in addition to methyl 4-0-benzyl-2,3dideoxy-~-~-gZycero-pent-2-enoside, on treatment of methyl 4-0benzyl-3-deoxy-3-iodo-2-O-p-tolylsulfonyl-~-~-xylopyranoside with sodium iodide in acetone at room temperature, and, in this instance, the 2,3-unsaturated compound did not isomerize on being heated, so the isomers were believed to have been produced under kinetic control.*Oa Pyrolysis of the N-oxides of erythromycin A and B gave products containing dihydropyranyl residues, from which methyl 3,4,6-trideoxy-hex-3-enopyranosideswere obtained by m e t h a n o l y ~ i s . ' ~ ~ ~ VI. 4,5-UNSATURATED, CYCLIC COMPOUNDS

1. Furanoid Derivatives The recognition of decoyinine (68, R = adenin-g-yl, R' = CH,OH), a nucleoside antibiotic (angustmycin A), as a derivative of a 4,s-un-

saturated sugar has stimulated several investigations into the synthesis of model compounds in this series (and of related nucleosides), and a review has consequently appeared on these compound^.'^^ Thus, for example, Hough and Otter have prepared 3-0-acetyl5-deoxy- 1,2- 0-isopropylidene - p - ~ - t h r e o - p e n t - -enofuranose 4 (69)

(124a) P. H. Jones andE. K. Rowley,]. Org. Chem., 33,665(1968). (125) L.Hough, R. Khan, and B. A. Otter, Aduan. Chem: Ser., 74,120(1968).

UNSATURATED SUGARS

251

by treatment of 3-0-acetyl-5-deoxy-5-iodo-1,2-0-isopropylidenea-D-xylose or -P-L-arabinose with silver fluoride in pyridine, but they found that, for the 3-hydroxy-~-xylocompound, the elimination was subordinate to an intramolecular, nucleophilic, displacement reaction which gave the 3,5-anhydride. Methyl 5-deoxy-2,3-0-isopropylidene-P-D-erythro-pent-4-enoside (70)was prepared in a similar way, as a better model for unsaturated nucleosides and as a potential source of derivatives of L-lyxofuranose (since hydrogenation would occur from the “upper” side of the double bond), and the related ketose derivative 6-deoxy-2,3-O-isopropylidene-/3-D-threo-hex5-enulofuranose (71)was synthesized by the same general method as

had been used for the first ketose derivative of this series.lZsHydrogenation of compound 71 likewise occurred from the sterically accessible side, and gave the D-arabino-hexulose derivative.lZ5 5-Deoxy- 1,2-O-isopropylidene-3-O-methyl-~-~-threo-pent~-enofuranose has been prepared from the 5-bromo-5-deoxy-~-xylose derivative, and reconverted into a 5-substituted D-XylOSe compound by the addition of a-toluenethiol.126a Extension of this type of work has led to the production of related 4,5-unsaturated furanosyl nucleosides, as follows: (i) the uridine analog (68, R = uracilyl, R’ = H)lZ7(prepared by deacetylation of the 2’,3’-diacetate); (ii) the 2’,3’-isopropylidene acetal of this nucleosidelZ8(prepared by treatment of 2’,3’-O-isopropylidene-5’-O-p-tolylsulfonyluridine with potassium tert-butoxide in tert-butanol, or, in lower yield, from the 2,5’-anhydronucleoside derivative), and (iii) the (126) L. Hough and B. Otter, Chem. Commun., 173 (1966). (126a) S. Inokawa, H. Yoshidi, C . 4 . Wang, and R. L. Whistler, Bull. Chem. Soc.Jap., 41,1472 (1968). (127) J. P. H. Verheyden and J. G. Moffitt,]. Amer. Chem. Soc., 88,5684 (1966). (128) M. J. Robins, J. R. McCarthy, Jr., and R. K. Robins, J . Heterocycl. Chem., 4, 313 (1967).

252

R. J. FERRIER

adenosine analog (68, R = adenin-9-yl, R’ = H)lZ9(prepared by way of a 2’,3‘-orthoester, and shown to be active against Steptococcus faecalis). Hydrogenation of the uridine compound (68, R = uracilyl, R’ = H) afforded an excellent means of preparing the 5’-deoxy-a-~Zyxo nucleoside. lZ8 Angustmycin A (decoyinine, 68, R = adenin-9-yl, R‘ = CHzOH)has been prepared from psicofuranine (71a) by way of the 1‘,3’,4’-orthoformate. Standard elimination, and removal of the ester by partial hydrolysis with acid, gave a product which was identical with the natural antibiotic. In the course of the work, compound 68 (R = adeninyl, R’ = H) was again prepared.’29a

HO

OH

(7 1a)

Elimination from a D-sorbofuranose derivative has provided a synthesis of 2,5-anhydro-l-S-benzyl-6-deoxy-3,4-di-O-(p-nitrobenzoyl)-l-thio-~-xyZo-hex-5-enitol (72, R = p-nitroben~oyl),’~~ which can be considered to be a member of this series.

I

RO

2. Pyranoid Derivatives At the time of writing of the earlier article,’ compounds in this class had been prepared only from hexuronic acid derivatives by base- or enzyme-catalyzed eliminations; methyl (methyl 4-deoxy-P-~threo-hex-4-enopyranosid)uronate(73), for example, had been pre(129) J. R. McCarthy, Jr., M. J. Robins, and R. K. Robins, Chem. Commun., 536 (1967). (129a) J. R. McCarthy, Jr., R. K. Robins, and M. J. Robins,]. Amer. Chem. SOC., 90, 4993 (1968). (130) K. Tokuyama,Japan Pat. 18,622(1967);Chem.Abstracts, 68,59845 (1968).

UNSATURATED SUGARS

253

pared by treatment of methyl (methyl a-D-ga1actopyranosid)uronate with sodium methoxide, and a series of unsaturated disaccharides had been characterized as the products of eliminase activity on poIysaccharides containing uronic acids. Work on this type of cleavage of polymers has continued, and has led to the recognition of several unsaturated oligosa~charides,'~~ but this research will not be surveyed here, as it has been concerned more with biochemical aspects than with the chemistry of the products. An extension of these studies has led to the investigation of related,base-catalyzed, eliminative degradation of poly(g1ycosiduronic)esters.132 Me

OCH,Ph (74)

A compound of this series, methyl 2,3-di-O-benzy1-4,6-dideoxy-c~-~threo-hex-4-enopyranoside (74), which has no activating group at C-5, has now been synthesized from methyl 2,3-di-O-benzyl-4,6dideoxy-4-(dimethylamino)-a-D-idopyranoside(methiodide) and -WDaltropyranoside (N-oxide) by application of the Hoffman and Cope reactions, respectively,lm and an allylic rearrangement reaction has also been found to offer an entry to this group of compounds. Thus, treatment of methyl 6-deoxy-2,3-0-isopropylidene-4-O-(methylsul(131) S. Nasuno and M. P. Starr, Biochem.J.,104,178(1967). (132) C. W.McCleary, D. A. Rees, J. W. B. Samuel, and I. W. Steele, Carbohyd. Res., 5,492(1967). (133) C. L.Stevens and D . Chitharanjan, Abstracts Papers. Amer. Chem. SOC. Meeting, 155.12~ (1968).

R. J. FERRIER

254

fonyl)-a- D - lyxo -hex - 5- enopyranoside (75) with lithium aluminum hydride gave methyl 4,6-dideoxy-2,3-0-isopropylidene-P-~-erythrohex-4-enopyranoside (76) instead of the product of direct, nucleophilic di~p1acement.l~~ The enantiomorph of compound 76 has been prepared by treatment of methyl 6-deoxy-2,3-O-isopropylidene-4-0(methylsulfony1)-a-L-talopyranosidewith sodium azide in N,Ndimethylformamide.134a

bOMe

Elrc

hcQoMe

MsO

(75)

(76)

“Glycals” formed from 2-ketoses have now been encountered for the first time, and, because of their structural similarity to the compounds of this group, will be considered here. Treatment of 1,4,5-tri-Obenzoyl - 3 - 0 - (methylsulfonyl)- p - D - fmctopyranosyl bromide with sodium iodide in acetone gave 1,5-anhydro-2,3,6-tri-O-benzoyl-4deoxy-~-erythro-hex-4-enitoi(77), from which, 1,5-anhydro-2,3,6-triO-benzoyl-4-deoxy-~-Zyxo-hexitol was obtained on h y d r o g e n a t i ~ n . ~ ~ ~ In related fashion, 1,3,4,6-tetra-O-benzoyl-a-~-sorbopyranosyl bromide gave 1 , 5 - a n h y d r o - 2 , 3 , 4 , 6 - t e t r a - O - b e n z o y l - ~ - t h r l (78), a 2-hydroxyglycal analog, as a by-product when the bromide was employed in standard syntheses of n u ~ l e o s i d e s .Another ~~~ L-sorbose derivative, namely, 2,3,6-tri-O-acetyl-1,5-anhydro-4deoxyL-threo-hex-4-enito1, has been described, and some of its addition and acid-degradation products have been studied. 138a C\H,OBz

C,&OBz

(134) J. Lehmann, Angew. Chem. Intern. Ed. Engl., 4,874 (1965). (134a) J. S. Brimacombe, 0. A. Ching, and M. Stacey, Carbohvd. Res., 8,498 (1968); J . Chem. SOC. (C),1270 (1969). (135) R. K. Ness and H. G . Fletcher, Jr.,J. Org. Chem.,33,181 (1968). (136) H. Paulsen, H. Koeser, andK. Heyns, Chem. Ber., lOO,2f%Q(1967). (136a) M . Katsuhara, S. Wakahara, and K. Tokuyama, Bull. Chem. S o c l a p . , 41,1208 (1968).

UNSATURATED SUGARS

255

4-Deoxy sugar derivatives can be obtained by hydrogenation of 4-deoxy-4-enoses; in the hexose series, these give mixed products because of the asymmetry created at C-5. Compound 73 has thus been employed to give the methyl ester methyl glycosides of the corresponding 4-deoxyhexuronic acid^,'^' and also the methyl 4-deoxyhexopyrano~ides,'~~ each of which was obtained pure after column chromatography. The formation of compound 73 and its isomers has been studied in greater depth, and it was found that the elimination /3 to the activating group decreased in the series P-galacto, p-gluco, a-manno, a-galacto, ~ r - g l u c o . 'Hydrogenation ~~~ of the products has also received further attention.'38b VII. 5,6-UNSATURATED, CYCLIC COMPOUNDS 1. Furanoid Derivatives

Since the preparation of the last article,' 5,6-dideoxy-l,2-O-isopropylidene-a-D-rylo-hex-5-enofuranose (79, R = R' = H) has been CHR' II

synthesized by use of the Tipson-Cohen reagent (namely, sodium iodide, zinc, and N,N-dimethylformamide) acting on the corresponding 5,6-disulfonic ester,81 by application of the thionocarbonate proc e d ~ r e , and l ~ ~ simply, and in high yield, from the appropriate 5,6epoxides of D-glucose and L-idose compounds by treatment with potassium selenocyanate in methanol at room t e m ~ e r a t u r eAlternatively, .~~ elimination to give compounds of this type can be brought about efficiently by heating 5,6-orthoesters with a small proportion of a carboxylic acid, and, in this way, the ethers (79, R= M e and CHzPh, R'=H) have been prepared in high yields.lqOA C-nitro member of this series, (137) H. W. H. Schmidt and H. Neukom, Tetrahedron Lett.,2063 (1964). (138) A. F. Cook and W. G. OverendJ. Chem. Soc. (C), 1549 (1966). (138a) H. W. H. Schmidt and H. Neukom, Tetrahedron Lett.,2011(1969). (138b) H. W. H. Schmidt and H. Neukom, Carbohvd. Res., 10,361 (1969). (139) D. Horton and W. N. Turner, Carbohyd. Res., 1,444 (1965). (140) J . S. Josanand F. W. Eastwood, Curbohyd. Res. 7,161 (1968).

256

R. J. FERRIER

namely, 3-0-acetyl-5,6-dideoxy- 1,2-O-isopropylidene-6-C-nitro-a-~xylo-hex-5-enofuranose (79, R = Ac, R' = NO,) has been synthesized by two routes: (i) by base-catalyzed elimination of acetic acid from 3,5di - 0-acetyl-6 -deoxy- 1,2- 0-isopropylidene -6 - C -nitro-a -D-glucofuraand (ii) by addition of nitryl iodide to the olefin 79 (R = Ac, R' = H), followed by treatment of the products with sodium hydrogen carbonate in boiling benzene.92 On treatment with methanol under basic conditions, addition occurs to give the epimeric 5-0-methylhexose compound^,'^^ and, on reduction with sodium borohydride, selective hydrogenation of the carbon-carbon double bond occurs (together with reductive cleavage of the ester group).% 3-Amino-3-deoxy compounds related to the olefin (79, R = R' = H) have been prepared by application of the Tipson-Cohen reagent.141a The addition of phosphines to double bonds of carbohydrates has been demonstrated for the first time with the alkene 79 (R = R' = H).'" Under ultraviolet light, phosphine itself gave what was believed to be a mixture of (5,6-dideoxy-1,2-0-isopropylidene-a-~-x~Zo-hexofuranose-6-y1)phosphine and bis(5,6-dideoxy-l,2-0-isopropylidene-c~-~xylo - hexofuranose - 6 - yl)phosphine, which were characterized as oxidized products; and, under similar conditions, phenylphosphine gave phenyl (5,6-dideoxy-1,2-0-isopropylidene-a-~-xylo-hexo~ranose-6-yl)phosphine, which was isolated in 75% yield as its oxide.142 Other additions which have been carried out with compound 79 (R = R' = H) have given a 5,6-cyclopropane and a 5deoxy compound having a 1,3-dioxolane ring attachedlUbto C-6. Hydrolysis of the known 3,5-0-benzylidene-6-deoxy-1,2-O-isopropylidene-a-~-xylo-hex-5-enofuranose affords a route to the antibiotic sugar 6-deoxy-~-xylo-hexos-5-ulose;~~~ the synthesis of 5-deoxy1,2-O-isopropylidene-6-O-trityl-a-~-xyZo-hex-5-enose (79, R = H, R' = OTr) has been discussed further,lM and, on hydrogenation, the compound, affords crystalline 5-deoxy-1,2-0-isopropylidene-a-~xylo-hexo~e.~~~ Because the unsaturated functions of these compounds are in the side chains, they may be built onto basic, cyclic compounds in(141) H. H. Baer and W. Rank, Can.J. Chem. 43,3330 (1965). (141a) H. Ohrui and S. Emoto, Agr. BioZ. Chem. (Tokyo), 32, 1371 (1968); Carbohyd. Res., 10,221 (1969). (142) R. L. Whistler, C.-C.Wang,and S. InokawaJ. Org. Chem.,33,2495 (1968). (142a) D. Horton and C. C . Tindall, Jr., Carbohyd. Res., 8,328 (1968). (142b) J. S. Jewel] and W. A. Szarek, Tetrahedron Lett., 43 (1969). (143) M. Nakajima and S. Takahashi, Agr. Biol. Chem. (Tokyo), 31,1079 (1967). (144) J. G . Buchanan and E.M. Oakes, Carbohyd. Res., 1,242 (1965). (145) R. E.Gramera, T. R. Ingle, and R. L. WhistlerJ. Org. Chem., 29,2074 (1964).

UNSATURATED SUGARS

257

stead of being prepared by elimination procedures, and this approach has led to an elegant means of extending the carbon chain of sugars and producing compounds of natural interest. Thus, on treatment with Wittig reagents prepared from tridecyl and pentadecyl bromide, methyl 2-amino-2,3-N,O-benzylidyne-2-deoxy-/3-~ribo-pentodialdo-1,4-furanosideafforded methyl 2-amino-2,3-N,Obenzylidyne-2,5-dideoxy-5-C-tridecylidene(and -pentadecylidene)-/3D-ribofuranoside (80), from which N-benzoyl-C18- and -CZO-phytosphingosines were ~ y n t h e s i z e d . ' Similar ~~ procedures have led to several, related, 5,6-unsaturated, furanoid d e r i ~ a t i v e s . ' ~ ~ ~In' ~ ~ J ~ * ~ particular, compounds 79 (R = Ac, R' = H) and 79 (R = R' = H) have been prepared from the dialdose 3-acetate by use of methylenetriphenylphosph~rane.'~~~

(80)

where n = 11 or 13.

2. Pyranoid Derivatives Although compounds of this group have not yet been found in natural products, they have on several occasions been proposed as possible intermediates in biosynthesis, and their chemistry has been reviewed briefly.lz5 (146) R. Gigg, C. D. Warren, and J. Cunningham, Tetrahedron Lett., 1303 (1965); J. Gigg, R. Gigg, and C. D. Warren,]. Chem. Soc. (C), 1872 (1966). (147) J. Gigg, R. Gigg,andC. D. WarrenJ. Chem. Soc. (C), 1882 (1966). (148) Yu. A. Zhdanov, Yu. E. Alekseev, and G. N. Dorofeenko, Zh. Obshch. Khim., 36,1742 (1966);37,98 (1967). (148a) Yu. A. Zhdanov, Yu. E. Alekseev, and G. N. Dorofeenko, Zh. Obshch. Khim., 37, 2635 (1967); Chem. Abstracts, 70, 11905 (1969); Zh. Obshch. Khim., 38, 231 (1968);Chem. Abstracts, 69,27681 (1968). (148b) D. G . Lance and W. A. Szarek, Carbohyd. Res., 10,306 (1969).

258

R. J. FERRIER

These pyranoid compounds offer a means of obtaining l-deoxyketoses (see p. 259), and continue to provide a source of aldos-5-uloses; the sugar component of hygromycin A has thus been prepared by hydrolyzing methyl 6-deoxy-a-~-arabino-hex-5-enopyranoside.~~~ In related fashion, methyl 6-deoxy-2,3-0-isopropylidene-a-~-Zyxo-hex5-enopyranoside (81) was i s o m e r i ~ e d in ' ~ ~the presence of an acid to methyl 6-deoxy-2,3-0-isopropylidene-a-~-Zyxo-hexosid-5-ulose (82).

The main interest in these compounds, however, has concerned their addition reactions, and it has been observed that the direction of addition can be controlled by the conditions used. Thus, hydrogenation of 1,2,3,4-tetra-O-ace~l-6-deoxy-~-~-~yZo-hex-5-enose gave 96% of the D-glUC0 product when the reaction was conducted in the presence of palladium, but as much as 29% of the L-id0 adduct was obtained when platinum oxide was used.lZs Also, on hydroboration, methyl 6-deoxy-a-~-xyZo-hex-5-enopyranoside gave methyl a-D-glucopyranoside and methyl P-L-idopyranoside in the ratio of 25,whereas similar reaction of the 2,3,4-tris(trimethylsilyl) ether gavels1 the corresponding products in the ratio of 5:3.The latter reaction has been employed for obtaining glycosides labeled with tritium at C-5 (the D-glucoside and L-idoside coming from the aforementioned enol compound, and the D-mannoside and L-guloside being obtained from compound 81). Alternatively, tritium can be introduced at C-6 in compounds of this class by treating them with silver fluoride in pyridine in the presence of a small proportion of tritiated water.lsZ a-Toluenethiol has been added to methyl 6-deoxy-a-~-xyZo-hex-5enopyranoside under ultraviolet light to give methyl S-benzyl-6thio-a-D-ghcopyranoside (83,R = SCHzPh) in good yield,lZ1and, in a related, free-radical reaction, similar addition of sodium bisulfite occurred to give, again in good yield, sodium (methyl 6-deoxy-a-~glucopyranoside-6-y1)sulfonate(83,R = S03Na).150 (149) S.Takahashi and M. Nakajima, Tetrahedron Lett., 2285 (1967). (150) J. Lehmann and A. A. BensonJ. Amer. Chem. Soc., 86,4469(1964). (151) J. Lehmann, Carbohyd. Res., 2,1(1966). (152) J . Lehmann, Carbohyd. Res., 4,196(1967).

UNSATURATED SUGARS

259

OH

A crystalline disaccharide derivative containing a double bond exocyclic to each ring has been synthesized from sucrose, and an aryl derivative, phenyl 6-deoxy-/3-~-xyZo-hex-5-enopyranoside, has been shown to be hydrolyzed by /3-D-glucosidase, but at a rate much lower than that of phenyl ~-D-glucopyran~side.'~~ A polymer derived from cellulose and having 5,6-double bonds has been described, and aspects of its chemical reactivity reported.152a Compounds formed by eliminations between C-1 and C-2 of 2-ketosyl derivatives can be assigned to this group, and, since the last Report was prepared, the first such compounds have been described. Thus, on treatment with sodium iodide in acetone solution, 3,4,5-tri-Oacetyl-1-0-p-tolylsulfonyl-a-L-sorbosyl bromide gave1302,3,4-tri-0acetyl-l,5-anhydro-6-deoxy-~-xe/Zo-hex-5-enitol (84) which, on addition of water, methanol, and bromine, gave the corresponding free sugar, glycoside, and glycosyl bromide derivatives, re~pective1y.l~~ Furthermore, the chloromercuri glycoside obtained after methoxymercuration has shown interesting chemotherapeutic properties against cancer.lS4Hydrolysis of such compounds as 84 offers a means of preparing l-deoxy-2-ketoses, and, by this method, 1-deoxy-D-fmctose has been synthesized from 1,5-anhydro-6-deoxy-~-Zzjxo-hex-5enitol (which was obtained by a standard 5,6-elimination from 1,5anhydro-D-mannitol).155

0

H*C

AcO

OAc (84)

(152a) D. G. Dimitrov, V. B. Achval, L. S. Antonyuk, L. S. Gal'braikh, and Z. A. Rogovin, Vysokomol. Soedin., Ser. A., 10, 1372 (1968); Chem. Abstracts, 69, 59515 (1968). (153) K. Tokuyama, E. Tsujino, and M. Kiyokawa, Bull. Chem. SOC. l a p . , 38, 1344 (1965). (154) K. Tokuyama, Japan Pat. 13,469 (1967); Chem.Abstracts, 68,13322 (1968). (155) A. Ishizu, B. Lindberg, and 0.Theander, Carbohyd. Res., 5,329 (1967).

R. J. FERRIER

260

VIII. OTHERUNSATURATED, CYCLICCOMPOUNDS Application of the Wittig reaction to derivatives of cyclic dialdoses, which has already been mentioned (see p. 257), provides products having the original aldehydic group as part of a new, olefinic system. The use of unsaturated Grignard reagents in analogous reactions offers a source of related, unsaturated carbohydrates having the multiple bonds farther removed from the ring, and, besides, provides a means of obtaining extended-chain compounds having triple bonds in the sugar chain. Ethynylmagnesium bromide applied to the appropriate dialdose derivative has, in this way, afforded 7,&dideoxy1,2:3,4-di-O-isopropylidene-~-glgcero-(and -L-glyCeTO)-a-D-gUlUC~Ooct-7-ynopyranose (85) and 6,7-dideoxy-l,2-O-isopropylidene-a-~gZuco(and P-~-ido)-hept-6-ynofranose(86), which can be partially reduced to the enose compounds, and otherwise applied in synthetic work.*5sA series of 6,7-unsaturated compounds of this class has been reported, and various aspects of their chemistry, including irradiation investigations, have been described. 148b*156a CH 111 C I

CHOH

CH Ill C

I

CHOH

Qi,Me

'0

'Me

Extension of this type of approach could provide a wide range of unsaturated compounds, illustrated by an acetylenic derivative having a C,4 side chain in which the unsaturation occupies the central position.'*' IX. UNSATURATED, ACYCLICCOMPOUNDS

Alditol derivatives having terminal double bonds have frequently been prepared by treatment of vicinal, primary-secondary disul(156) D. Horton, J. B. Hughes, and J. M. J. Tronchet, Chem. Commun.,481(1965). (156a) G. B. Howarth, D. G. Lance, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 47,75,81(1969);Chem. Commun., 1349 (1968).

UNSATURATED SUGARS

261

fonates with sodium iodide in acetone solution, but this procedure fails when applied to compounds containing vicinal secondary sulfonate groups. Use of boiling N,N-dimethylformamide (as the solvent) and zinc (to remove the iodine liberated during the reaction) has provided15' a new and most convenient means of preparing both cyclic and acyclic unsaturated carbohydrates (see pp. 232, 255,256), as was illustrated by the synthesis15' of the known' 1,2:5,6-di-O-isopropylidene-3,4-dideoxy-trans-~-threo-hex-3-enitol, which has, in addition, been prepared by acetonation of one of the products of reduction of 3,4-dideoxy-~-glycero-hex-3-enulose (43) with sodium b~rohydride.'~ Alternatively, treatment of 1,2:5,6-di-O-isopropylidene-3,4-di-O-ptolylsulfonyl-D-mannitol with potassium selenocyanate in boiling N,N-dimethylformamide gave the isomeric, cis-olefin, as did treatment of the corresponding 3,4-taZo-epoxide with this reagent in methanol at room temperature or in refluxing 2-metho~yethanol.~~ In similar fashion, 1,2 -dideoxy-3,4:5,6-di - 0-isopropylidene -D-arabino-hex- 1enitol was prepared from the corresponding rnanno-epo~ide,~~ and, after selective, hydrolytic removal of the terminal acetal ring, has been converted by standard procedures into 4,5-dideoxy-~-threopent-4-enose (87), a stereoisomer of a compound ~btainable'~'by chemical degradation of coenzyme B12. H ?=0

HO~H I HCOH I CH

(87)

Treatment of compounds containing two vicinal p-tolylsulfonyloxy groups with sodium benzoate in N,N-dimethylformamide can give olefinic products,15Band isolated sulfonates also undergo elimination (instead of displacement) when subjected to such conditions. Thus, 1,2:4,5-di-O-isopropylidene-3,6-di-O-(methylsulfonyl)-~-mannitol and the corresponding 6-benzoate gave 1-0-benzoyl-4-deoxy2,3:5,6-di-O-isopropylidene-~-threo-hex-3-enitol (88) in good yield.160 (157) (158) (159) (160)

R. S. Tipson and A. Cohen, Carbohyd. Res., 1,338 (1965). R. M. Saunders and C. E. Ballou,J. Org. Chem.,30,3219 (1965). S. J. Angyal and T. S. Stewart, Aust. J . Chem., 20,2117 (1967). M. A. Bukhari, A. B. Foster, J. M. Webber, and J. Lehmann, Carbohyd. Res., 1,485 (1966).

262

R. J. FERRIER CH,OBz I Me, ,OCH C I Me’ ‘OC II CH I HCO, ,Me I CH,O/C,Me (88)

The preparation, from 2-bromoglycals, of alditols containing acetylenic groups has already been referred to (see p. 224), and other such compounds have been synthesized by use of ethynylmagnesium bromide with aldehydo aldose derivatives. Diastereoisomers are produced by this procedure, but these have been separated, characterized, and investigated chemically; on partial reduction, they afford the corresponding olefinic alditol derivatives.15BJ61-1aIn a similar way, a large number of extendedchain alditol derivatives have been prepared from aldehydo aldoses by application of vinyl Grignard r e a g e n t ~ , the ~~~ J~~ Knoevenagel reactions, or, more particularly, the Wittig reaction. In application of the last reaction, l-deoxyalditol-1-ylidene groups have been condensed with such groups as CH(CH2),&H, (Ref. 165), CHCOCH, (Ref. 166), C(OEt)CO,Et (Ref. 167), CH-CH=CH2, (Fief. 168), and lO-anthr~nylidene.’~~ Many other examples have been recorded,170 and hydroxylations of the olefinic bonds have led to syntheses of higher aldose derivatives; S-deoxyglyculosonic acids are obtainable by acid hydrolysis of the enol ether derivative^.'^^ Several applications of (methoxymethy1ene)triphenylphosphorane, and the hydrolysis of the adducts to give 2deoxyaldoses, have been reported.170a (161) (162) (163) (164) (165) (166) (167) (168) (169) (170)

D. Horton and J. M. J. Tronchet, Carbohyd. Res., 2,315 (1966). D. Horton, J. B. Hughes, and J. K. ThomsonJ. Org. Chem., 33,728 (1968). J. L. Godman, D. Horton, and J. M. J. Tronchet, Carbohyd. Res., 4,392 (1967). D. J. Walton, Can../.Chem., 45,2921 (1967);46,3679 (1968). J. Gigg and R.GiggJ. Chem. SOC. (C),1876 (1966). Yu. A. Zhdanov, G . N. Dorofeenko, and L. A. Uzlova, Materialy Vses. Konf. Prob. Khim. Obmen Ugleuodou, 3rd, Moscow, 1963,67 (1965);Chem.Abstracts, 65,3946 (1966);Carbohyd. Res., 3,69 (1966). M. N. Mirzayanova, L. P. Davydova, and G. I. Samokhvalov,Dokl. Akad. Nauk SSSR, 173,367 (1967);Chem. Abstracts, 67,54368 (1967). Yu. A. Zhdanov and V. G . Alekseeva, Zh. Obshch. Khim., 37, 1408 (1967); Chem. Abstracts, 68,22161 (1968). Yu. A. Zhdanov, L. A. Uzlova, and G . N. Dorofeenko, Zh. Vses. Khim. Obshchestua im. D. I. Men&leeoa, 10,600 (1965);Chem. Abstracts, 64,3671 (1966). N. K. Kochetkov and B. A. Dmitriev, Izu. Akad. Nuuk SSSR, Ser. Khim., 1405 (1965); Chem. Abstracts, 63, 18237 (1965);Tetrahedron, 21, 8Q3 (1965); B. A.

UNSATURATED SUGARS

263

Several further examples have been reported of acyclic compounds having a double bond in conjugation with an activating group. Treatment of 2,3,4,5,6-penta-O-rnethyl-D-glucose with lime-water, for example, gave 3-deoxy-2,4,5,6-tetra-O-methyI-~-erythro-hex-2-enose (89) which, with dilute acid, underwent further elimination to give'" 3,4-dideoxy-5,6-di-O-methyl-~-glycero-hex-3-enosulose(90).Correspondingly, 2,3-di-O-methyl-D-glucose gives a 2,3-unsaturated product which cyclizes, but which has been converted into related, unsaturated, acyclic derivatives (see p. 230). From 2,3,4,5-tetra-0methyl-D-glucose, cis- and t~ans-3-deoxy-2,4,5-tri-O-methyl-~-erytho-hex-2-enose were obtained. These exist partly in the cyclic, 3,6-anhydro f01-1n.l~~~Opening of the ring of the lactone (53,R = 0) with dimethylamine gavelM the a,p-unsaturated compound NJVdimethyl-(2,4,6-tri-O-benzyl-3-deoxy-~-th~eo-hex-2-enon)amide (91).

Dmitriev and N. K. Kochetkov, Zzu.Akad. Nauk SSSR, Ser. Khim., 2483 (1967); N. K. Kochetkov and B. A. Dmitriev, ibid., 274 (1966); Chem. Abstracts, 64, 19734 (1966); B. A. Dmitriev, N. E. Bairamova, L. V. Bakinovskii, and N. K. Kochetkov, Dokl. Akad. Nauk SSSR, 173, 350 (1967); Chem. Abstracts, 67, 54381 (1967);N. K. Kochetkov, B. A. Dmitriev, and L. V. Bakinovskii, Carbohyd. Res., 5, 399 (1967);Yu. A. Zhdanov, G. V. Bogdanova, and V. G. Zolotukhina, Dokl. Akad. Nauk SSSR, 157, 917 (1964); Chem. Abstracts, 61,16137 (1964); Yu. A. Zhdanov and L. A. Uzlova, Zh. Obshch. Khim., 36, 1211 (1966);Chem. Abstracts, 65, 18670 (1966);Yu. A. Zhdanov, L. A. Uzlova, G. N. Dorofeenko, and G . I. Kravchenko, Zh. Obshch. Khim., 36, 1025 (1966); Chem. Abstracts, 65, 12273 (1966);Yu. A. Zhdanov, Yu. E. Alekseev, and G. N. Dorofeenko, Zh. Obshch. Khim., 37,98,2635 (1967);B. A. Dmitriev, N. E. Bairamova, and N. K. Kochetkov, Izu. Akad. Nauk S S S R , Ser. Khim., 2691 (1967);Chem. Abstracts, 69,27687 (1968);B. A. Dmitriev, N. E. Bairamova, A. A. Kost, and N. K. Kochetkov, Izu. Akad. Nauk SSSR, Ser. Khim., 2491 (1967);Chem. Abstracts, 69,77662 (1968); Yu. A. Zhdanov and V. G. Alekseeva, Zh. Obshch. Khim., 38, 1951 (1968);Chem. Abstracts, 70, 29212 (1969);V. A. Polenov and Yu. A. Zhdanov, Zh. Obshch. Khim., 37,2455 (1969); Chem. Abstracts, 69, 77652 (1968);Yu. A. Zhdanov and V. A. Polenov, Zh. Obshch. Khim., 38, 1046 (1968); Chem. Abstracts, 69,97046(1968). (170a) J. M. J. Tronchet, E. Doelker, and B. Baehler, Helu. Chim. Acta, 52,308 (1969); B. A. Dmitriev, N. N. Aseeva, and N. K. Kochetkov, Zzu. Akad. Nauk SSSR, Ser. Khim., 1342 (1968);Chem. Abstracts, 69, 77644 (1968);Yu. A. Zhdanov, and V. G . Alexeeva, Carbohyd. Res., 10, 184 (1969); Zh. Obshch. Khim., 38, 2594 (1968);Chem. Abstracts, 70, 58175 (1969). (171) E. F. L. J. Anet, Carbohyd. Res., 3,251 (1966);7,453 (1968). (171a) E. F. L. J. Anet, Carbohyd. Res., 8,164 (1968).

R. J. FERRIER

264 H

c=o I

COMe II

FH

H

c=o C=O I

FH

HCOMe

HC

HCOMe I CH,OMe

HCOMe

(89)

(90)

I

I

I

ChOMe

Further examples of l-phenylazo-l,2-unsaturate~ alditols derived by acetylation of aldose phenylhydrazones have been d e ~ c r i b e d , ' ~ ~ and a dimer, namely, 1,2-bis(penta-O-acetyl-~-gluconoyl)ethylene (92), formed by way of a carbene intermediate, was reported to be C&OAc I

(CHOAc), CON(Me),

I

c=o

COC&Ph

I CH'

CH I HCOChPh

CH I

I

I1 I

It

c=o I

HOCH I C&OCI&Ph

(CHOAc),

(91)

(92)

I

CH,OAc

obtained on treatment of penta-0-acetyl- 1-deoxy-l-diazo-keto-DgZuco-heptulose with copper oxide.'73 Finally, further examples of unsaturated 1-nitroalditols have been described, and have been utilized in standard syntheses of 2-deoxyaldoses; by this means, 2-deoxy-~-ribo-hexose,'~~ 2-deoxy-D-mannoh e p t o ~ e , ' 2-deoxy-~-gaZacto-heptose,~~~ ~~ and 2,6-dideoxy-~-mannoh e p t o ~ ehave ' ~ ~ been prepared. The reaction of ammonia with 3,4,5,6tetra-O-acetyl-1,2-dideoxy-l-nitro-~-urub~no-hex-l-enitol has been further examined, and found to proceed stereospecifically to give (172) H. S. El Khadem, M. L. Wolfrom, Z. M. El Shafei, and S. H. El Ashry, Carbohyd. Res., 4,225 (1967). (173) Yu. A. Zhdanov, V. I. Kornilov, and G . V. Bogdanova, Carbohyd. Res., 3, 139 ( 1966). (174) W. W. Zorbach and A. P. Ollapally,]. Org. Chem.,29,1790 (1964). (175) M. B. Perry, Can.]. Chem., 45,1295 (1967). (176) M. B. Perry and A. C. Webb, Can.]. Chem.,46,789 (1968). (177) J. Yoshimura, H. Komoto, H. Ando, and T. Nakagawa, Bull. Chem. SOC.Jap., 39,1775 (1966).

UNSATURATED SUGARS

265

2-acetamido-1,2-dideoxy-l-nitro-~-mannitol.~~~ Similar additions to the nitro-olefins obtained from D-ribo~e,'~'~ D - x y l o ~ e , D-lyx~se,'~* '~~~ D - g a l a ~ t o s e , 'D ~ ~- m a n n o ~ e , ' and ~ ~ ~ ~ - t a l o s e 'have ~ ~ afforded syntheses of the corresponding pairs of 2-amino-2-deoxyaldoses.

x. NUCLEAR MAGNETICRESONANCEFEATURES OF UNSATURATED SUGARS Nuclear magnetic resonance spectroscopy can be of great value in conformational and configurational analysis in this area and, in particular, the isomeric conduritols, whose spectra have been carefully analyzed,179serve as useful models for unsaturated, pyranoid compounds. The well known Karplus relationship is applicable to vicinal, coupled protons bonded to sp3 hybridized carbon atoms, and may be used, for example, with 2,3-unsaturated- D -erythro - hexopyranose derivatives, for assigning conformations -a large J4,5 value indicating the diaxial relationship of H-4 and H-5, and, consequently, the H$ half-chair form. For allylic systems, analogous relationships have been developed180 which, applied to six-membered ring compounds [in which the allylic quasi-equatorial (el) and quasiaxial ( a r )bonds subtend angles of approximately 40" and 80",respectively, with the vinylic C-H bonds], give the following coupling constants (see p. 93);J l e t , n - 6 . 0 Hz; J l a l , 2 +2.7 Hz; Jlet,3 -0.3 Hz; and Jlat,3 -2.5 Hz. With an important exception (see later), determined values are in good agreement with these value^:^,^^^^^^^^^^^ for methyl 4,6-0-benzylidene-2,3-dideoxy-a-(and p-)D-erythro-hex-2-enopyranosides: for example, J2,4at are -2.2 and -2.5 Hz, respectively, whereas the corresponding J2,4e, values for the a- and p-D-threo isomers are close to zero, and, for members of the 1,2,4,6-tetra-O-acetyl-3-deoxyhex-2-enopyranose series,lWJ3.4aand J3.4e values are 2-3 and 5-6 Hz, respectively. The introduction of double bonds between interacting protons increases long-range couplings, so that five-bond, homallylic couplings, which conform quantitatively with prediction^,'^^ have been (178) S. D. GBro and J. Defaye, Compt. Rend., 261,1555 (1965). (178a) M. B. Perry and J. Fordovi, Can.]. Chem.,46,2859 (1968). (178b) M. B. Perry and A. C. Webb, Can.]. Chem., 47,1245 (1969). (1784 M. B. Perry and A. C. Webb, Can.J. Chem.,46,2481 (1968). (178d) C. F. Gibbs, D. T. Williams, and M. B. Perry, Can.]. Chem.,47,1479 (1969). (179) R. J. Abraham, H. Gottschalck, H. Paulsen, and W. A. Thomas, J . Chem. SOC., 6268 (1965). (180) E. W. Garbisch,]. Amer. Chem. Soc., 86,5561 (1964).

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R. J. FERRIER

observed for the methyl 4,6-0-benzylidene-2,3-dideoxy-~-hex-2-enopyrano~ides:~J values of 0, 1.3, and 3 Hz are expected for e’e’, e’a’, and a’a‘ related, proton pairs, respectively; andJ1,, values of <0.3, 1.5, and 2.6 Hz were observed. Introduction of electronegative substituents onto carbon atoms bearing coupled protons is well known to influence coupling constants, and the effect of the ring-oxygen atom causes anomeric protons to show “anomalous” splittings (frequently of about half the predicted values). Thus, for 2,3-unsaturated, pyranoid compounds in which H-1 is equatorially attached, JlP2 and J1,3values of 2-3.5 Hz and C0.3 Hz are reported,4’116whereas, for the anomers having H-1 axial, J1,2and are 0.7-1.3 Hz and -0.8 to -1.2 Hz, re~pectively.~ Tri-0-acetyl-Dglucal hasJ,,, 3.2 Hz (calculated, 2.7 Hz) andJ1,3,t -1.3 Hz (Ref. 181; calculated -2.5 Hz), and, therefore, also displays the H-1 “anomaly.” A similar effect has been observed for couplings of vinylic protons in cyclic carbohydrate derivatives.181aIn keeping with results obtained from aliphatic compounds, vinyl ethers (glycals, for example) show couplings of about 6 Hz, which is some 4 Hz less than the value for cis-alkenes. Little work has as yet been done on the nuclear magnetic resonance spectroscopy of five-membered, cyclic, unsaturated sugars, but it is anticipated that interpretation of resonance splittings in terms of molecular geometry (as can be done for the pyranoid derivatives) will be difficult. With unsaturated, acyclic compounds, nuclear magnetic resonance provides a powerful means of determining the configuration about the double bond, trans and cis compounds giving vinylic coupling-constants oftabout 16 and 11Hz, respectively.182 The signs of the coupling constants in various unsaturated sugars have been measured. 181~182a

(181) L. D. Hall and J. F. Manville, Carbohyd. Res., 4,271 (1967). (181a) P. M. Collins, Carbohyd. Res., 10, (1969). (182) A. H. Haines, Carbohyd. Res., 1,214 (1965). (182a) L. D. Hall and J. F. Manville, Carbohyd. Res., 8,295 (1968).

STRUCTURE, CONFORMATION, AND MECHANISM IN THE FORMATION OF POLYSACCHARIDE GELS AND NETWORKS BY D. A. REES* Chemistry Department, University of Edinburgh, Edinburgh, Scotland I. Introduction and Scope .................................................. 267 11. The Nature of Gels and the Contemporary Problems. ....................... 268 111. Structure and Conformation of Selected Gel-forming Polysaccharides ........ 270 1. Cellulose Derivatives ................................................. 271 2. Agar ................................................................. 277 3. Carrageenans and Other Natural Sulfates ............................... 279 4. Glycuronans and Derivatives .......................................... 296 IV. Characterization of Junction Zones ....................................... 303 1. First Principles ....................................................... 303 2. Methods ............................................................. 305 V. Polymer-Polymer Interactions in Junction Zones .......................... 313 1. Covalent Linkages: Sephadex and Other Examples.. .................... 313 2. Double-helix Junctions: Sulfates and Related Polysaccharides ............ 314 323 3. Microcrystallites: Clycuronans and Derivatives ......................... 4. Entanglement and Shared Counterions: 0-(Carboxymethy1)cellulose . . . . . 327 5. Micelle Junctions: 0-Methylcellulose ............................... 331

I. INTRODUCTION AND SCOPE This article is written in the belief that, if polysaccharides are ever again to make major contributions to molecular theory and molecular biology, they can only do so after their behavior in gels has been understood in molecular terms. The gel is the state most typical for polysaccharides, both in biological and artificial systems. The polymer chains usually form an interconnected network that gives rise to characteristic texture and properties, in the interstices of which are molecules of solvent and other species. To some extent, therefore, the chains are free and solvated; yet some chain sections are associated to "The author is grateful to a number of colleagues in Universities and Industry for criticism of this article in draft, particularly: Dr. F. J. Buckle, Dr. F. Franks, Sir Edmund Hirst, Mr. R. H. McDowell, Dr. T. J. Painter, Dr. E. E. Percival, Dr. D. Renn, Dr. C. Stainsby, Mr. D. J. Stancioff, Mr. N. F. Stanley, Dr. W. A. B. Thomson, Dr. J. R. Turvey, Dr. D. A. Weyl, Mr. F. B. Williamson, Dr. R. Young, and Dr. R. S. Tipson. Any controversial views are, however, his own responsibility.

267

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D. A. REES

form the cross linkages - perhaps, even combined in a crystal lattice. Fibrous and other partly crystalline polysaccharides, such as chitin, cellulose, and the starch granule on the one hand, and polysaccharides in true solution on the other, may be regarded as special instances of the gel state. The one class represents a compacted gel that has been evacuated of solvent, and the other, a disrupted gel in which there is minimal interaction between chains. Polysaccharide gels have biological functions, for example, in the wall of the young plant-cell, in animal fluids and connective tissues, and in the bacterial capsule. They also have widespread commercial uses, particularly in foodstuffs, cosmetics, paper, and textiles. In the laboratory, agar and Sephadex are familiar media for gel filtration and gel electrophoresis. Derivatives of Sephadex are also used for ionexchange chromatography, and agar gels are valuable for the culture of many micro-organisms. The gel state may thus be considered characteristic of polysaccharides in the way that polypeptides and globular proteins can characteristically occur as compact particles having a high degree of internal (intramolecular) order, or nucleic acids can occur as chain pairs in highly ordered, complementary association. Polysaccharides have a distinctive contribution to make to natural-polymer chemistry in showing, in terms of molecular structure, how chain molecules can interact in three dimensions to give the physical and biological properties of gels. Two alternatives would seem to be open in the discussion of this subject. An exhaustive account of known gelling behavior would inevitably result in a technical bias. On the other hand, it should be possible to emphasize the principles, and to illustrate the limits of present understanding by chosen examples. The choice has been made reluctantly, because both types of discussion would be timely, but it would seem more sensible to marshal our ideas about the basis of gel formation before attempting to tabulate and organize the sum of factual knowledge. The present article is written from the viewpoint of the structural chemist who wishes to see how the overall properties of the gel are an outcome of molecular structure, and who would rather have a qualitative understanding in these terms than a physical or mathematical model which, even if capable of predictions with high precision, did not start from “the molecular formula.” 11. THE NATUREOF GELSAND THE CONTEMPORARY PROBLEMS

The property of a gel that is most noticeably distinctive is that its composition can approach that of a pure liquid while, in certain other

POLYSACCHARIDE GELS AND NETWORKS

269

respects, it may resemble a solid. Agar gels, for example, may contain 99.9% of water, and yet still be capable of retaining their shape and of resisting stress when tipped out of a container. To understand such gels, we must understand how so few polymer molecules can so drastically modify the properties of so much solvent. The general explanation is that the solution (or sol) that exists before gelation is a typical solution of polymer molecules, but, that gel formation involves association of chain segments in the way shown schematically in Fig. 1, resulting in a three-dimensional framework that contains solvent in the interstices. The associated regions are known as junction zones, and may be formed from two or more chains. Gelation can occur by other mechanisms, but these are unusual for fairly dilute, polymer systems of the type that will be To account for the origin of many properties, including rigidity and swelling ability, there is now an extensive theory of gel networks. These aspects have been well covered in a number of earlier artic l e ~ . ’ -The ~ ~ challenge now to the chemist, with all his new aids for structural investigation and his new understanding of molecules, is to show the precise molecular arrangements in junction zones and the

Sol

Gel

FIG. 1. -Schematic Mechanism of Gelation by a Polymer Solution. [The fine details are not meant to apply universally; for example, the polymer need not have the randomcoil conformation in the sol, and junction zones in the gel may engage a greater part of the chain length and may also engage more than two chains.] ( 1 ) P. H. Hermans, in “Colloid Science,” H. R. Kruyt, ed., Elsevier, Amsterdam, 1949, Vol. 2, p. 483. (2) J. D. Ferry,Adoan. Protein Chem., 4, l(1948). (3) P. J. Flory, “Principles of Polymer Chemistry,” Cornell University Press, Ithaca, N. Y., 1953. (4) J . D. Ferry, “Viscoelastic Properties of Polymers,” John Wiley and Sons, Inc., New York, N. Y., 1961. (5) A. Katchalsky, Progr. B i o p h y s . B i o p h y s . Chem.,4,1(1954). (5a) A. Frey-Wyssling, “Submicroscopic Morphology of Protoplasm,” Elsevier, Amsterdam, 2nd Edition, 1953.

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D. A. REES

forces that hold molecules together in them. The problem we have to ponder is, therefore, the nature of molecular interactions between polymers; this may include those interactions known for other biological polymers as tertiary structures and is the theme of the present Chapter. Other important problems, beyond our scope at the moment, include polymer-solvent and solvent-solvent interactions in gel formation. Any formal definition of a gel has been avoided, because its formulation would be difficult on the basis of external properties. By any criterion, a 1%aqueous solution of agar does form a gel when it is cooled from 95 to 20°, whereas a 1% solution of sucrose does not; but properties may be so continuous between these extremes that any dividing line would be arbitrary. Most of this Chapter is concerned with fairly permanent network structures formed from polymer solutions. As a definition of gels, this description is incomplete. Some cellulose gels, and pastes of gelatinized, starch granules, would be excluded, because they are formed by limited dispersion of solids. In other words, an arbitrary choice will be made to focus on those gels that are dilute with respect to polymer, because the information available about them at the molecular level is more precise than for others. OF SELECTED 111. STRUCTUREAND CONFORMATION GEL-FORMINGPOLYSACCHARIDES~

Lists of gel-forming polysaccharides, with their molecular structures, are available from several source^,^-'^ and other reviews deal with structure It is only necessary here to summarize the (6) For an introduction to polysaccharide structure and stereochemistry, see D. A. Rees, “The Shapes of Molecules: Carbohydrate Polymers,” Oliver and Boyd, Edinburgh, 1967. (7) F. Smith and R. Montgomery, “The Chemistry of Plant Gums and Mucilages,” Reinhold Publishing Corporation, New York, N. Y., 1959. (7a) “Kirk-Othmer Encyclopedia of Chemical Technology,” A. Standen, ed., Interscience Publishers, Inc., New York, N.Y., 2nd Edition; see especially, H. Neukom, ibid., 14,636(1967),and K. B. Guiseley, ibid., 17,763(1968). (8) “Industrial Gums,” R. L. Whistler and J. N. BeMiller, eds., Academic Press Inc., New York, N. Y., 1959. (9) E. Percival and R. H. McDowell, “Chemistry and Enzymology of Marine Algal Polysaccharides,” Academic Press, London, 1967. (10) L. Stoloff, Aduan. Carbohyd. Chem., 13,265(1958). (11) T. Mori, Aduan. Carbohyd. Chem., 8,315(1953). (12) S. Peat and J. R.Turvey, Fortschr. Chem. Org. Naturstofe, 23,1(1965). (13) D. A. Rees, Ann. Rept. Progr. Chem. (Chem. SOC. London), 62,469(1965). (14) J. S. Brimacombe and J. M. Webber, “Mucopolysaccharides,” Elsevier Publishing Company, Amsterdam, 1964. (15) G. 0.Aspinall, Aduan. Carbohyd. Chem., 24,333(1969).

POLYSACCHARIDE GELS AND NETWORKS

27 1

background information that will later be used in discussing mechanisms of gelation; this includes molecular structure, gel characteristics, and, where possible, solid-state conformations. The emphasis will be on recent developments.

1. Cellulose Derivatives a. Preparation and Structure. -Because of the abundance and low cost of cellulose, derived products are manufactured in wide variety and have many applications.R*’0*’6-’8 They are of particular interest here, because of three types of behavior, as exemplified by O-methylcellulose, 0-(carboxymethyl)cellulose,and cellulose sulfate. 0-Methylcellulose is prepared by heterogeneous methylation: cellulose is pretreated with sodium hydroxide solution by spraying or dipping, and is then autoclaved with methyl ~ h l o r i d e ; ’ ~the ~’~ intermediate is known as alkali-cellulose. Products marketed for their gelling ability usually have 1.6 to 2.0 methoxyl groups per D-glUCOSe residue, on average. An uneven distribution of substituents is to be expected within each chain, because some parts of the fiber are more accessible than others. There are also differences in reactivity within each D-glucose residue,20in the order 0 - 2 > 0 - 6 > 0-3. As a result of degradation during manufacture, the molecular weight is lower than for native celluloses. The number-average degree of polymerization would usually seem to bez1in the range of 200 to 1,000. The striking characteristic of 0-methylcelluloses is that aqueous solutions gel when they are heated and “melt” on cooling; this is the reverse of the temperature behavior of most other gels, including those from agar and gelatin.

(15a) G. 0. Aspinall, E. Percival, D. A. Rees, and M. Rennie, in “Rodd’s Chemistry of Carbon Compounds,” S. Coffey, ed., Elsevier, Amsterdam, 2nd Edition, 1967, Vol. 1F. (16) N. M. Morss and J. Oyg, in “The Chemistry and Rheology ofwater Soluble Gums and Colloids,” (S.C.I. Monograph No. 24), Society of Chemical Industry, London, 1966, p. 46. (17) A. J . Desmarais and H. 0. Esser, in Ref. 16, p. 57. (18) A. B. Savage, A. E. Young, and A. T. Maasberg, in “Cellulose and Cellulose Derivatives,” E. Ott, H. M. Spurlin, and M. W. Grafflin, eds., Interscience Publishers, Inc., New York, N.Y., 1954, Part 2, p. 882. (19) G. K. Greminger and A. B. Savage, in Ref. 8, p. 565. (20) I. Croon, Soensk Papperstidn., 61,919 (1958). (21) W. B. Neely, J . Polym. Sci., P t . A, 1, 311 (1963); see also, W. J. Hillend and H. A. Swenson, ibid., 2,4921 (1964).

D. A. REES

272

Cellulose sulfate containing about 2.2 ester groups per D-glUCOSe residue can be prepared by treatment of cellulose with a sulfur trioxide-N,N-dimethylformamide reagent.22 Although this ester is not widely used in commerce so far, it forms strong gels in the presence of an excess of potassium ions; these “melt” on heating, and set again on cooling. 0-(Carboxymethy1)cellulose is normally used as viscous, thixotropic solutions that often have a characteristic, “lumpy” t e ~ t u r e . ’ Rigid ~~’~~~~ gels can be prepared in certain special circumstances which are described later. The manufacture of this ether consists in treatment of alkali-cellulose (cell-ONa)with sodium chloroacetate: cell-00

+ C1CH2C00@+cell-0-CH2C00Q+ Cl@

The degree of substitution is much lower than for the cellulose derivatives already mentioned, and is u s ~ a l l y ’ 0.4-0.8. ~ , ~ ~ As with O-methylcellulose, a decrease in molecular weight is likely in the preparation and handling of the alkali-cellulose, and an “unevenly patchwise” distribution of substituents is to be expected, because of the effects of accessibility and inaccessibility. Important derivatives of cellulose are also manufactured by the introduction of other substituents, either alone or in combinati~n.~.~*’~-’~ However, the basic types of behavior are well illustrated by the few examples already mentioned. Several other types are excluded by the terms of reference already laid down; these include gels formed by so-called microcrystalline cellulose, which is prepared by controlled h y d r o l y ~ i s . ~Discussion **~~ of gels formed in nonaqueous systems26 will also be omitted. b. Conformation. - Because of experimental difficulties arising from irregular substitution in these derivatives of cellulose, their conformations have to be inferred from that of cellulose itself. The “Hermans” or “bent-chain” conformation (1) is now generally (22) R.G. Schweiger, Chem. Ind. (London), 900 (1966). (23) J. B. Batdorf, in Ref. 8, p. 643. (24) J. Hermans, J . Polym. Sci., Pt. C, 2, 129 (1963);M. R. Edelson and J. Hermans, ibid., 2,145 (1963);0.A. Battista, ibid., 9,135 (1965). (25) J. Hermans,J. Appl. Polym. Sci., 9,1973 (1965). (26) M. N. Vrancken and J. D. Ferry, J. Polym. Sci., 24,27 (1957); S. Newman, W. R. Krigbaum, and D. K. Carpenter,J . Phys. Chem., 60,648 (1956); K. Ninomiya and J. D. Ferry,J. Polym. Sci., Pt. A-2,5,195 (1967).

POLYSACCHARIDE GELS AND NETWORKS

273

accepted for both of the important crystalline forms of cellulose.27Its stability can be rationalized by using the methods for conformational analysis of polysaccharides that were introduced by Ramachandran and coworkers.28Each D-glucopyranose residue is assumed to have the conformation expected from classical con~iderations,2~ and a set of atomic coordinates is derived for each residue, preferably from accurate crystal-structure determinations. If a reasonable value is assumed for the bond angle at the glycosidic oxygen atom, only two variables are necessary for specifying the chain contour; these are the angles of rotation about each bond to the glycosidic oxygen atom, marked 4 and 9 in formula 2. The D-glucose residues closer to and

farther from the reducing chain terminal are respectively designated R and N, as shown in 2. The set of coordinates for each is referred to its own set of axes, with the origin at the glycosidic oxygen atom, and Or is defined by the respective bond to this atom. The definitions of O y and Oz may be arbitrary; they specify the reference state in which 4 = 9 = 0. Coordinates that correspond to any con(27) R. H. Marchessault and A. Sarko, Adoan. Carbohyd. Chem., 22,421 (1967). (28) G . N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan, in “Aspects of Protein Structure,” G . N. Ramachandran, ed., Academic Press Inc., New York, N. Y., 1963, p. 121; V. S. R. Rao, P. R. Sundararajan, C. Ramakrishnan, and G . N. Ramachandran, in “Conformation of Biopolymers,” G. N. Ramachandran, ed., Academic Press Inc., New York, N. Y., 1967, Vol. 2, p. 721; G. N. Ramachandran, in “Structural Chemistry and Molecular Biology,” A. Rich and N. Davidson, eds., Freeman, San Francisco, 1968, p. 77. (29) E. L. Eliel, N. L. Allinger, S . J. Angyal, and G . A. Morrison, “Conformational Analysis,” John Wiley and Sons, Inc., New York, N. Y., 1965; L. Hough and A. C. Richardson, in Ref. 15a.

D. A. REES

274

formation (+,$) are readily generated by means of the standard expressions for rotation of axes in two dimensions, for example, about Ox: x = x’ y = y’ cos 6 - z’ sin 6 z = y’ sin 6 z‘ cos 6

+

or, in matrix notation:

[] [: y = 0

0 cos 6 -sin 01 si: 6 cos 6

[;:I

X’

where 6 is the angle of rotation, and x, y, and z are the new coordinates. For example, the axes for R may be rotated about 0-C-4’ through $, and then in the plane of C-1-0-C-4’ through the supplement of the bridge angle, and, finally, about (2-1-0through d. Due attention is paid to the sign of each rotation.2saThe new coordinates now represent R with respect to the axes defined for N; with the original co-ordinates for N, they give the atomic positions for a disaccharide residue in the conformation (+,$). Interatomic distances are readily calculated from the Pythagoras equation, because all coordinates are now referred to the same axes. Thus, it is possible to decide whether the conformation implies any infringement of van der Waals radii, or whether particular groups are in hydrogen-bonding positions. If the same 4 and $ occur at each linkage in the polymer, the overall conformation will, in general, be a helix. Such properties as the number of residues per helix turn, and the projected residueheight, may be calculated30 from C#I and $. Computers are readily programmed to vary the angles systematically and to repeat the calculations at each stage, thus sampling and testing all possible conformations. Application of this approach to cellulose has confirmed31that the Hermans conformation is the only possibility that is free from strong steric clashes while fitting the usual interpretation of the x-ray evidence by having two fold screw symmetry and a projected residue height of 5.15 A. It is also stabilized by a hydrogen bond between successive residues, as in the crystal structures of cellobiose and (29a) D. A. Rees and R. J. SkerrettJ. Chem. SOC. ( B ) , in press (1969). (30) C. Ramakrishnan, Proc. Zndian Acad. Sci., Sect. A, 59,327 (1964); T. Shimanouchi and S. Mizushima, J . Chem. Phys., 23, 707 (1955); T. Miyazawa, J . Polym. Sci., 55,215 (1961); S . Mizushima and T. Shimanouchi,Aduan. Enzymol., 23,1(1961).

POLYSACCHARIDE GELS AND NETWORKS

275

lactose monohydrate. Further calculations, with use of semi-empirical functions, showed that the total van der Waals energy lies close to the minimum, but is displaced slightly to form the hydrogen bond, and is raised a little above the disaccharide values to allow the two fold screw axis and thus to permit efficient packing of the polymer chains. The cellulose molecule is rather restricted in conformational scope, because calculations show that about 96% of all conformations possible would involve severe van der Waals compression, and the remaining 4 % lie together (see Fig. 2).31It is therefore concluded that the Hermans form is so stable, and other possibilities are so limited, that substituted celluloses would be expected to have similar conformations, were they to crystallize. This conclusion seems to be true for cellulose tria~etate,3~ but all caution must not be abandoned,

-180

-120

-60

0

60

I20

180

d FIG. 2.-Conformational Analysis of Cellulose.31 [& and J, are the torsion angles shown in 2 and are assumed to be the only degrees of freedom possessed by the chain. Combinations of 4 and 9 that require no steric compression are enclosed by the continuous line (“fully allowed conformations”). The broken lines enclose conformations in which there is slight steric compression (“marginally allowed conformations”). All other conformations involve bad steric clashes (“disallowed”).] (31) D. A. Rees and R. J. Skerrett, Carbohyd. Res., 7,334 (1968). (32) W. J. DulmageJ. Polyrn. Sci., 26,277(1957).

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D. A. REES

because xylan is closely related in structure to cellulose, and yet it crystallizes with a three fold, screw axis.32aThe possible shapes of other P-glucans are quite different.29aa3 The packing of cellulose chains has been discussed elsewhere in this Series:’ and a detailed depiction is not needed here. To explain the changes that occur in the x-ray diffraction diagram when cotton cellulose swells in alkali, Warwicker and WrighP4have supposed that the ribbon-like chains are laid on top of each other, in stacks, The contacts within these stacks are mostly nonpolar and, at least in the dry state, the stacks would seem to be held together largely by van der Waals attraction; they are joined to each other by hydrogen bonding between equatorial hydroxyl groups (see Fig. 3). Swelling

FIG. 3. -Diagrammatic Representation of “Stacks” of Cellulose Chains and Their Possible Aggregati~n.~‘ [Each cellulose chain is ribbon-like and approximately oval in cross-section (labeled); the view is down the ribbon. Note that the stacks are labeled as “sheets” in the drawing, after the original authors. b = fiber and chain axis, which is perpendicular to the plane of this diagram and therefore not shown; a and c are the other edges of the Meyer-Misch cell.] (32a) W. J. Settineri and R. H. MarchessaultJ. Polyrn. Scl., Pt. C . 11,253 (1965). (33) D. A. Rees and W. E. Scott, Chem. Cornrnun., 1037 (1969). (34) J . 0.Warwicker and A. Wright, J. Appl. Polyrn. Sci., 11,659 (1967).

POLYSACCHARIDE GELS AND NETWORKS

277

in sodium hydroxide is considered to be caused by the ionization of hydroxyl groups, and, hence, rupture of hydrogen bonds and electrostatic repulsion. There is separation or relative movement of the stacks, which themselves seem to remain intact. Foreign molecules, such as salts, can occupy the spaces between the stacks (see Fig. 3). It would seem likely that substituted celluloses would aggregate in a similar way, but with substituent groups taking the place of foreign molecules. 2. Agar a. Structure.-Even more so than gelatin, agar can be regarded as a prototype and model for all gelling systems. The molecular structure is simple, and yet it shows, and even exaggerates, all of the important properties. Gels can be formed from very dilute solutions (containing a fraction of one per cent of agar) and these gels are rigid, have well defined shapes, and sharp “melting” and setting points, and show, clearly, the interesting phenomena of syneresis and hysteresis (see Sections IV and V). The source of agar is a group of red seaweeds (Rhodophyceae), of which Gracilaria and Gelidium are Agar is best defined examples of commercially important genera.7.g**3 as being a family of polysaccharides, and different members are often blended together in manufacture. The component having the greatest gelling tendency has been named agar0se,3~and has been s h ~ w n ~ ~ , ~ ’ to have a simple structure (3)in which 3-linked P-D-galactopyranose

residues and 4-linked 3,6-anhydro-a-~-galactopyranose residues are arranged alternately. The disaccharide 3,6-anhydro-4-O-(P-~-galactopyranosy1)-L-galactose is known as agarobiose; various derivatives (35) C. Araki, Bull. Chem. SOC. lap., 29, 543 (1956); C. Araki, Mem. Fac. Znd. Arts, Kyoto Tech. Uniu., Sci. Technol., 5,21(1956). (36) C. Araki and K. Arai, Bull. Chem. SOC. l a p . , 40, 1452 (1967), and earlier papers in the same series. (37) C. Araki, Proc. Znt. Seaweed Symp., Sth, 1966, p. 3.

278

D. A. REES

of it have been isolated in high yield after fragmentation of agarose and its methyl ether, thus providing some of the evidence for structure 3. It is not clear whether agarose can ever be obtained as a single polysaccharide by extraction of a particular species of seaweed, and the usual isolation involves fractionation. Originally, this was accomplished by separation of the polysaccharide acetates, followed by d e a c e t y l a t i ~ n More-direct .~~ methods have now been devised to meet the need for an agarose suitable for use as a medium for chromatography and electrophoresi~.~~ For commercial material, the molecular weight has been estimated by sedimentation methods39ato be about 120,000. Another well characterized member of the agar family, namely porphyran, has a more complex structure and much weaker gelling tendency. It has the same arrangement of alternating glycosidic linkages, and may be formally derived by partial 6-0-methylation of agarose (3) together with partial replacement of the L residues by L-galactose 6-sulfate. Up to half of the residues of each type may be thus modified.40This structure has been called a masked repeating structure, because the strict alternation of D and L residues is overlaid by substitution and modification; it was proved by alkaline elimination of sulfate, with 3,6-anhydride formation, followed by complete methylation to yield a product which was apparently identical with methylated a g a r o ~ e The . ~ ~ hydrolysis fragments isolated after treatment with an enzyme which seems to show a preference for (1--* 4) bonds between D-galactose and 3,6-anhydro-~-galactosesuggest that the 6-methyl ether is distributed irregularly throughout all of the porphyran molecules.42 Other polysaccharide structures that contain agarobiose segments are known, but have been less well characterized. They are often of the masked repeating type, with variations that may be the same as, or different from, those in p ~ r p h y r a n . ~ ' ,For ~ ~ ,example, ~~ 6-0methylagarobiose dimethyl acetal has been isolated after methanolysis (38) C. Araki, Nippon Kagaku Zasshi, 58,1338 (1937). (39) S. Hjertbn, Biochim. Biophys. Acta, 62, 445 (1962); B. Russell, T. H. Mead, and A. Polson, ibid., 86, 169 (1964); J. C. Hegenauer and G. W. Nace, ibid., 111,334 (1965); J. Blethen, U. S.Pat. 3,281,409 (1966). (39a) T. G. L. Hickson and A. Polson, Biochim. Biophys. Acta, 165,43 (1968). (40) D. A. Rees and E. Conway, Biochem.1..84,411 (1962). (41) N. S. Anderson and D. A. ReesJ. Chem. Soc., 5880 (1965). (42) J. R. Turvey and J. Christison, Biochem.J.,105,311,317 (1967). (43) A. N. O'Neill and D. K. R. Stewart, Can. 1.Chem., 34, 1700 (1956); A. L. Clingman, J. R. Nunn, and A. M. StephenJ. Chem. Soc., 197 (1957). (44) C. Araki, K. Arai, and S. Hirase, Bull. Chem. Soc.Jap.,40,959 (1967).

POLYSACCHARIDE GELS AND NETWORKS

279

of Ceramium bo ydenii agar.44Similar treatment of Gelidium amansii agar yields the pyruvic acid acetal, 4,6-0-( 1-carboxyethy1idene)agarobiose dimethyl which has been assigned the configuration in which the C-methyl group is e q ~ a t o r i a l The . ~ ~ polysaccharide of Gloiopeltis furcata differs from agarose in containing a high proportion of ester sulfate4' which appears4*to be situated on C-6 of the agarobiose residues. It forms viscous solutions, but does not gel. The evidence at present available is also consistent with a masked repeating structure for the water-soluble polysaccharide of Laurencia pinnatijida, which is evidently even more complex than porphyran, because the D-residues include D-galactose, its 6-methyl ether, and its 2-sulfate, and the L-residues are 3,6-anhydro-~-galac2-O-methyl-~-galactose, tose, 3,6-anhydro-2-0-methyl-~-galactose, and L-galactose. The latter two residues probably occur as 6-sulfate, and there is some evidence for branching.49 The same 2-methyl ethers occur in polysaccharides from Grateloupia elliptica, together with residues of 4-0-methyl-D-galactose and other sugars.50 4-0Methyl-L-galactose is a hydrolysis product of Gelidium amansii agar.51 b. Conclusions about Structures. - Despite considerable variation in structure within the agar family, it would seem that agarose (3)is the key to the gel properties. It is probably the major component in commercial agar, and it certainly has the strongest gelling tendency. Increasing deviation from structure 3 appears to result in a progressively diminished tendency to gel. c. Conformations. -The solid-state conformation of agarose is not yet known, but predictions made b y analogy with other polysaccharides have been attempted, and are discussed in Section II1,3c (see pp. 294 and 321).

3. Carrageenans and Other Natural Sulfates a. Carrageenan Structures. - Carrageenans, like agars, constitute a family of polysaccharides found only in marine algae of the class (45) S. Hirase, Bull. Chem. SOC. Jap.. 30, 75 (1957);S. Hirase, Mem. Fac. Ind. Arts, Kyoto Tech. Unio., Sci. Technol., 6,17(1957). (46) P.A. J. Gorin and T. Ishikawa, Can.j. Chem.,45,521(1967). (47) S. Hirase, C.Araki, and T. Ito, Bull. Chem. Soc.Jap.,31,428(1958). (48) A. Penman, D.A. Rees, and D. J. Stancioff, unpublished results. (49) D. M. Bowker and J. R.TurveyJ. Chem. SOC. (C),983,989(1968). (50) S. Hirase, C. Araki, and K. Watanabe, Bull. Chem. Soc.Jap.,40,1445(1967). (51) C. Araki, K.Arai, and S. Hirase, Bull. Chem. Soc.Jap.,40,959(1967).

D. A. REES

280

Rhodophyceae, are characteristically built up from residues having the galacto configuration linked alternately a-(1+ 3) and p(1 + 4), and, typically, have masked repeating structures. They are distinct in two important respects: the 4-linked residue can be 3,6anhydro-D-galactose (but, apparently, not the L enantiomorph which is found in agar), and the 3-linked residues in gel-forming carrageenans are, at least partly, 4-sulfated. Their molecular weights are probably of the order of several hundred thousand.53 (i) K-Carrageenan. -This polysaccharide is isolated, from Chondrus crispus for example, by specific precipitation of the potassium salt from hot-water extracts. It has long been known52to consist largely of alternately arranged p-D-galaCtOSe.4-sulfate and 3,6-anhydro-a-~galactose residues linked (1+ 3) and (1+ 4) as in formula 4. Analysis

L

OR (4)

R=H

(5) R =

sop

shows the presence of more D-gahCtOSe and sulfuric ester than can be accommodated in this structure, and various extra features have been p r o p o ~ e d , ~but ~ * ~it* is now clear that the correct explanation lies in the presence of a masked repeating structure.55 D-Galactose 6-sulfate residues formally replace some 3,6-anhydride, and 4-linked residues of both types are partly 2-sulfated. The 4-linked 6-sulfate is recognizable by means of the alkaline borohydride reaction,56which causes an increased yield of 3,6-anhydro-(4-0-p-~-galactopyranosyl)D-galactose dimethyl acetal (carrabiose dimethyl acetal) after methan o l y s i ~ The . ~ ~ resistance of a proportion of these 6-sulfate residues to periodate is consistent with a degree of 2-sulfation. The presence of

(52) A. N. O’Neill, J . Amer. Chem. Soc., 77,6324 (1955); C . Araki and S. Hirase, Bull. Chem. Soc.Jop.,29,770 (1956). (53) D. B. Smith, W. H. Cook, and J. L. Neal, Arch. Biochem. Biophys., 53,192 (1954). (54) S. T. Bayley, Biochim. Biophys. Acta, 17,194 (1955). (55) N. S. Anderson, T. C. S . Dolan, and D. A. Rees,J. Chem. SOC. ( C ) ,596 (1968). (56) D. A. ReesJ. Chem. Soc., 5168 (1961). (57) N. S. Anderson and D. A. Rees, Proc. lnt. Seaweed Symp., 5th, 1966, p. 243.

POLYSACCHARIDE GELS AND NETWORKS

28 1

3,6-anhydro-~-galactose2-sulfate residues is indicated by the results of methylation analysis55and by comparison of sulfate absorptions in the infrared spectrum with those of synthetic esters.58It is difficult to hydrolyze methyl ethers of polysaccharides that contain 3,ganhydroD-galactose sulfate residues to a representative mixture of monosaccharide derivatives, but an approach based on oxidative hydrolysis with bromine-sulfuric acid is sati~factory.~~ The polysaccharide seems to be built up entirely from carrabiose, and modified carrabiose, segments, because, after treatment with alkaline borohydride, methanolysis gives the dimethyl acetal in a yield which, when corrected for side reactions (on the basis of model experiments), is close to the the~retical.~' Polysaccharides of the K-carrageenan type would seem to be fairly widespread in red algae,9J3,59although, from one source to another, the structure may differ in important details.58 In comparison with agarose, K-carrageenan gels are less rigid and require rather higher concentrations (in the region of one per cent) of polysaccharide for their formation. Gelation here has the added interest of showing cation selectivity, because it occurs in the presence of K@,Rb@,Cs@,or NH,@ ions, but not of Li@or NaQ. (ii) K-Furcellaran.-The chemistry of this polysaccharide has been ' reviewed e l s e ~ h e r e , ~and , ~ ~only * ~ the ~ conclusions relevant to the present theme need be stated. In structure, it is similar to K-carrageenan, except that only about half of the D-galactose residues are 4-sulfated, and the molecule might be branched. Gelation shows the same type of cation sensitivity as for K-carrageenan, but the gels are more rigid and brittle, although less so than agarose gels.

(iii) &-Carrageenan.-The structure is essentially that depicted in 5, with about 10 per cent of the 3,6-anhydro-~-galactose2-sulfate

residues replaced by D-galactose 2 , 6 - d i ~ u l f a t e . ~The l * ~ proof ~ of this structure was obtained by the same methods as had been used for K-carrageenan, including rigorous characterization of the hydrolysis and oxidative-hydrolysis products obtained after methylation, and study of the action of alkaline borohydride. This polysaccharide is (58) N. S. Anderson, T. C. S. Dolan, A. Penman, D. A. Rees, G. P. Mueller, D. J. Stancioff, and N. F. StanleyJ. Chem. S O C . (C), 602 (1968). (59) W. Yaphe, Can.J.Bot.,37,751(1959). (60) N. S. Anderson, T. C . S. Dolan, and D. A. Rees, Nature, 205,1060 (1965). (61) G. P. Mueller and D. A. Rees, Trans. Drugs Seo Symp., Unio. Rhode Island, Aug. 1967,241 (1968). (62) N. S. Anderson, T. C. S. Dolan, C. J. Lawson, and D. A. Rees, in preparation.

282

D. A. REES

readily distinguished from K-carrageenan by its physical properties, and by the pronounced band in its infrared spectrum at about 805 cm-’, which is attributed to 3,6-anhydro-D-galactose 2-sulfate, and which is weak or nonexistent in the spectrum of K-carrageenans. Eucheuma spinosum and Agardhiella tenera are examples of red seaweeds from which warrageenan may be extracted. In the presence of I@ ions, warrageenan forms gels that are much more elastic than those of K-carrageenan. The increasing content of sulfuric ester in the series agarose, K-furcellaran, K-carrageenan, and warrageenan therefore coincides with progressively increasing elasticity and decreasing brittleness in the gel state.* Karrageenan forms useful, compliant gels with Ca2@ions, in contrast to K-carrageenan, which forms rather weak, brittle gels. (iv) p-Carrageenan. -This polysaccharide occurs with A-carrageenan [see Section III,3a (v); p. 2831 and K-carrageenan, and remains in solution when the potassium salt of the last-mentioned is precipitated [see Section III,3a (i); p. 2801. In some earlier publications, it was named “third component of carrageenan.” No method has yet been found for separating it from A-carrageenan without chemical modification. After treatment with alkaline borohydride, the p component can, however, be precipitated as the potassium salt. Methylation evidence, the infrared and the qualitative and quantitative nature of the methanolysis productss7 show that the material modified with alkaline borohydride is closely similar to K-carrageenan. From this and other information, the structure of p-carrageenan is essentially that depicted in formula 6, with replacement of D-galactose 6-sulfate by 3,6-anhydro-~-galactoseto an extent

*I am grateful to Mr. D. J. Stanciofffor pointing out this relationship. (63) N. S. Anderson, T. C. S. Dolan, C. J. Lawson, A. Penman, and D. A. Rees, Carbohyd. Res., 7,468 (1968).

POLYSACCHARIDE GELS AND NETWORKS

283

that is variable and dependent on the source,63and with 2-sulfate on a small proportion of each type of 4-linked residue. It is possible to separate K-carrageenan into a series of subfractions that range in structure58from 4 to a hybrid of 4 and 6. Similarly, the precipitates, having a decreasing content of 3,6-anhydro-~-galactose, that result when potassium chloride is progressively added to solutions of certain carrageenanss4 are probably K-carrageenans of increasing p-like character. It would seem that K-carrageenan and p-carrageenan represent the extremes of a structural “spectrum” found in natural carrageenans, although all of the forms are not necessarily present in the material from any one source. Even in the presence of potassium chloride, p-carrageenan has little, if any, tendency to gel. (v) A-Carrageenan.- In the older literature, the term “A-carrageenan” referred to the entire fraction of carrageenan that is soluble in potassium chloride, but this material is h e t e r o g e n e o ~ s ,and ~ ~ its compositione5 and structuree3 may vary widely with the source. (It is true that K-carrageenan may also vary, but only in the proportions, ~*~~~~~ and not the kind, of structural units.) It is therefore p r o p o ~ e dthat the name be reserved for the component having structure 7. Occa-

where R = H for some residues, and SOP for others. (7)

sionally, such material is the sole component of the fraction soluble in potassium chloride, but it is more often contaminated with p-carraprovides a geenan. Analysis for combined 3,6-anhydro-~-galactose~’ convenient, probably reliable,s3 guide to the purity. (64) A. J. Pernas, 0. Smidsrad, B. Larsen, and A. Haug, Acta Chem. Scand., 21, 98 (1967);see also, A. S. Cerezo,]. Chem. SOC. (C), 992 ( 1 967). (65) W. A. P. Black, W. R. Blakemore, J.A. Colquhoun, and E. T. Dewar,]. Sci. Food Agr., 16,573 (1965). (66) T. C. S. Dolan and D. A. ReesJ. Chem. SOC., 3534 (1965). (67) W. Yaphe and G. P. Arsenault,Anal. Biochem., 13,143 (1965).

284

D. A. REES

Early evidence for such a structure as 7 was obtained from the relative stabilities of the sulfuric esters to acid and to alkaline borohydride, and from the characterization of various cleavage-fragments isolated before and after treatment of A-carrageenan with alkaline borohydride.68 The proof was completed by methylation analysis, before and after removal of the sulfate,66and by showing that, after treatment with alkaline borohydride, an almost quantitative yield of carrabiose dimethyl acetal could be obtained by methanolysis (the yield being corrected for side reaction^).^^ Indications that the (1--* 3)-linkage has the a-D configuration were obtained by isolation of 3-0-a-D-galactopyranosyl-D-galactose after acetolysis and deacet y l a t i ~ n .The ~ ~ homologous trisaccharide was also thought to be a product, but this supposition conflicts with structure 7, and the claim has now been refuted. All of the acetolysis products are consistent with the proposed stru~ture.'~ Aqueous solutions of A-carrageenan, and of its simple salts, are viscous, but do not gel. This behavior is still observed after conversion of each 4-linked residue to a residue of 3,6-anhydro-~-galactose by elimination with alkaline borohydride, even though the product is structurally identical to L-carrageenan, except in the position of one sulfuric ester group.

b. Conformations of the Carrageenans. (i) A-Carrageenan.-An investigation of the conformations of the carrageenans was attempted over 14 years ago, when the chemical characterization of the compounds was still at an early stage;54and, in order to interpret the x-ray data, it was necessary to make certain assumptions about the molecular structure. Unfortunately, these assumptions have turned out to be untenable. Nevertheless, the photographs obtained for A-carrageenan were of sufficiently good quality to be worth an attempt at re-interpretati~n.~~ The sodium salt shows a fiber repeat distance of 25.2 A with meridianal reflections on the third, sixth, and ninth layer lines, but not on other low orders, suggesting a three fold helix. The 050 and 080 reflections also observed (taking b as the fiber axis, after B a ~ l e yare ~ ~ explained ) by the packing of cations or water having some other symmetry. The standard method of interp r e t a t i ~ nwould ~ ~ require exploration of the arrangements in space possible for the known sequence of residues in A-carrageenan, in (68) D. A. Rees,]. Chem. Soc., 1821 (1963);Biochem.1..88,343 (1963). (69) K. Morgan and A. N. O'Neill, Can.]. Chem., 37,1201 (1959). (70) C. J. Lawson and D. A. Rees,]. Chem. SOC. (C), 1301 (1968). (71) D. A. Flees,]. Chem. SOC. ( B ) ,217 (1969).

POLYSACCHARIDE GELS AND NETWORKS

285

order to find any three fold helices having the observed repeat period, that do not violate the usual bond-lengths, bond angles, or van der Waals distances. This study was made with a computer, as described7' in Section II1,lb (see p. 272), except that four variable angles must be fixed in order to define the conformation, because there are two types of glycosidic oxygen atom present. The two constraints clearly cannot determine the values for the four variables, unless most of the formal possibilities happen to involve a considerable van der Waals overlap, and they can therefore be excluded. A fairly small group of left-handed helices was obtained, of which 8 is representative. This conformation shows several similarities with

HO

(The sulfuric ester conformations are arbitrary.) (8)

maltose residues present in known crystal structure^.^^,^^ The overall shape resembles a rather flat, extended ribbon having some bending from side to side.

(ii) K-Carrageenan and ca car rage en an^^. -With a series of univalent counter-ions, oriented fibers of these polysaccharides give similar diffraction photographs, except that &-carrageenanshows much more detail, and the fiber axis repeat-distance (13.0 A) is approximately half that of K-carrageenan (24.6 A). Meridianal reflections occur on every third layer line only, suggesting three fold helices, and there (72) A. Hybl, R. E. Rundle, and D. E. Williams, J . Amer. Chem. SOC., 87,2779 (1965); S. S. C. Chu and C. A. Jeffrey, Actu Cryst., 23,1038 (1967). (73) N. S. Anderson, J. W. Campbell, M. M. Harding, D. A. Rees, and J. W. B. Samuel, J . M o l . Biol., 45,85 (1969).

286

D. A. REES

are clear intensity-relationships between the two series if comparison is made between alternate layer lines of K-, and consecutive layer lines of L-, carrageenan. The maxima in the L-diagram were sharp enough to suggest a regular, lateral packing of the helices in a hexagonal cell of side 22.6 A, with adjacent helices separated by 13.0 A. A cell of this type, having a side of about 20 A, would also fit the requirements of the K-carrageenan photograph. There is good agreement with the measured densities, provided that the dimensions refer to doubZe helices. The different fiber axis repeat-distances, and other features, are then also explained: the L-carrageenan chain forms a single helix having a pitch of 26 A, and the second chain is parallel to it, but displaced by half this distance, so that alternate layer-lines are cancelled, to give a crystallographic repeat of 13 A. In fibers of K-carrageenan, it would seem that each chain has a conformation that is slightly contracted, but otherwise similar to that in L-carrageenan, but its partner is not exactly staggered, and might even be antiparallel, and all layer lines remain. These double helices are superficially similar to those formed by nucleic acids, except that they contain sugar sulfate residues instead of sugar phosphate residues, To complete the derivation, it is necessary to make a model that fits the proposed symmetry and dimensions, and has acceptable bond lengths, bond angles, and van der Waals distances; this can be done with physical models, or with a c o m p ~ t e r , ~ 'provided . ~ ~ . ~ ~ that each chain has a right-handed, screw sense. The single chain of L-carrageenan is shown in Fig. 4, and the corresponding double helix in Fig. 5. This model accounts satisfactorily for the intensity distributions along the layer lines.73 Specific hydrogen-bonding is revealed in both K- and L-carrageenans by infrared, deuteration-dichroism methods.73 This is thought to join 0 - 2 and 0-6 of galactose residues in different strands of the same double helix. Every unsubstituted hydroxyl group in L-carrageenan would then be engaged in hydrogen bonding (see formula 5 and Fig. 5 ) within the double helix, making the conformation very stable. Relatively sharp, diffraction photographs were obtained with the NH4@,K@, Rb@,and CSO salts of K-carrageenan, but not with the Na@ or [email protected] helices in fibers would, therefore, seem to be favored by the same cations that cause gelation of solutions (see Section III,3a (i); p. 281). The photographs for warrageenan were very diffuse, (74) J. W. Campbell, Ph.D. Thesis, University of Edinburgh, 1969.

POLYSACCHARIDE GELS AND NETWORKS

287

unless alkaline borohydride was used for converting the masked repeating structure [see Section III,3a (iii); p. 2811 into a higher degree of true alternation by the reaction 9 + 10.

or

. . . Gal - A - Gal - Gal’ - Gal - A . . . HO’YBH,O . . . Gal - A - Gal - A - Gal - A . . . + SO$@ where Gal = D-galactose 4-sulfate, A = 3,6-anhydro-~-galactose2sulfate, and Gal* = D-galactose 2,6-disulfate. Were the different position of the sulfate group on the 3-linked D-galaCtOSe residue to be ignored, A-carrageenan would be represented by

. . . Gal-Gal*-Gal-Gal’-Gal-Gal’

...

Native &-carrageenan is, therefore, the idealized polysaccharide in which an occasional Gal - A - Gal segment is replaced by a A-like segment, namely, Gal - Gal” - Gal. The effect on the overall contour of the chain can be predicted if it is assumed that the most stable conformation of each segment is found in the structure of the corresponding fiber.75 When the nonreducing terminals of models of Gal - Gal” - Gal and Gal - A - Gal are then superimposed, the relative orientations of the “reducing” D-galaCtOSe residues are 11 and 12. In the depiction, this residue of the A-segment (11)is dis-

(75) D. A. Rees, I. W. Steele, and F. B. Williamson,-/. Polurn. Sci. P t . C., 28, in press

(1969).

288

D. A. REES

FIG.4.-A Single Chain of &-Carrageenan in the Conformation ProposedT3 for the Double Helix. [Beevers, miniature, molecular models76were assembled to correspond to computed coordinates by Dr. J. W. Campbell.74 The black balls are carbon atoms, the white are hydrogen atoms, the gray are oxygen atoms, and the speckled are sulfur atoms.] FIG.5.-The &-CarrageenanDouble Helix." [Beevers, miniature, molecular models were assembled to correspond to computed coordinates by Dr. J. W. C a m ~ b e l l . ~The ' convention is the same as for Fig. 4.1 (76) C. A. BeeversJ. Chem. Educ.,42,273 (1965).

POLYSACCHARIDE GELS AND NETWORKS

289

290

D. A. REES

placed by several Angstrom units behind the plane of the page, relative to the L-residue. In addition, as the formulas show, the chain has changed direction. Relative to the idealized polysaccharide (Fig. 4, shown schematically in formula 13), the native conformation is a kinked helix, namely, 14.

The physical meaning of 13 and 14 is not that polysaccharide chains in solutions or gels are “frozen” in the form of single helices (in the way that those of polypeptides sometimes are). The prototype for ordered polysaccharide conformations in solution is the amyloseiodine complex, in which the V helix is only static because of interactions with c ~ s o l u t e sAs . ~ far ~ ~as~ is ~ known, polysaccharide conformations are dynamic, unless such intermolecular interactions exist; other examples are the carrageenan double helix and polysaccharide crystal lattices. However, polysaccharides are so restricted sterically 31*71 that it is useful to regard them, in solution, as fluctuating about conformations of minimum Often,31*71 there may be only one minimum, which can be close to the ordered conformation found in the solid state. In this sense, only, can 13 and 14 be regarded as representing carrageenan chains in solution. (77) (78) (79) (80) (81)

J. M. Bailey and W. J. Whelan,J. Biol. Chem.,236,969 (1961). F. W. Schneider, C. L. Cronan, and S. K. Podder,J. Phys. Chem., 72,4563 (1968). B. Casu, M. Reggiani, G. G. Gallo, and A. Vigevani, Tetrahedron, 22,3061 (1966). P. J. Flory, Proc. Roy. SOC. (London),Ser. A, 234,60 (1956). J. F. Foster, in “Starch: Chemistry and Technology,” R. L. Whistler and E. F. Paschall, eds., Academic Press Inc., New York,N. Y., 1965, Vol. 1, p. 349.

POLYSACCHARIDE GELS AND NETWORKS

29 1

(c) Aminopolysaccharide Sulfates and Hyaluronic Acid. -Although there have been some interesting development^,^^^^^ most of the chemistry of heparin and heparitan sulfate is excluded, because these compounds have a type of structure different from that of the main group. It is still too early to attempt to do justice to the chemistry of any of these polysaccharides in relation to their properties in networks. For the present theme, there are only a few preliminary indications to be summarized, and the discussion of structures will be brief. In the natural state, the chains are often joined by glycosidic bonds to an L-serine residue of a polypeptide backbone. Other types of linkage also occur, as in keratan sulfate.84 It is not yet known whether hyaluronic acid is bound covalently to protein. The main part of each chain approximates to a regular alternation of residues and of another sugar (often a uronic of 2-acetamido-2-deoxy-~-hexose acid), but the sequence near the L-serine residues is different. In chondroitin 4 - ~ u l f a t eit, ~is:~

. . . P-D-GpA-(1+3)-P-D-Calp-( 1-3)-P-D-Galp-(

1+4)-P-D-xylp-

L-Serine . . .

The proof of this sequence, and of the nature of the carbohydrate-peptide bond, was based on enzymic erosion to a glycopeptide fragment that was then studied by adaptations of classical methods. A similar linkage-region probably occurs in heparin,86 heparitin ~ulfate,~' dermatan sulfate88and chondroitin 6 - s ~ l f a t eIt. ~is~possible ~ that the polypeptide core itself is branched.89 (82) J. A. Cifonelli and A. Dorfmann, Biochem. Biophys. Res. Commun., 7,41(1962); L. B. Jaques, L. W. Kavenagh, M. Mazurek, and A. S. Perlin, ibid., 24,447 (1966); A. S. Perlin, M. Mazurek, L. B. Jaques, and L. W. Kavanagh, Carbohyd. Res., 7, 369 (1968); M. L. Wolfrom and P. Y. Wang, Chem. Commun., 241 (1967); M. L. Wolfrom, S. Honda, and P. Y. Wang, ibid., 505 (1968); Carbohyd. Res., 11, 179 (1969). (83) A. Linker and P. Hovingh, Biochim. Biophys. Acta, 165,89 (1968). (84) N. Seno, K. Meyer, B. Anderson, and P. Hoffman, J . Biol. Chem., 240, 1005 (1965);M. B. Mathews and J. A. Cifonelli, ibid., 240,4140 (1965). (85) U. Lindahl and L. Roden, J . B i d . Chem., 241, 2113 (1366); L. Roden and R. Smith, ibid., 241,5949 (1966). (86) U. Lindahl and L. Rod&, J. Biol. Chem., 240,2821 (1965);U. Lindahl, Biochim. Biophys. Acta, 130,368 (1966). (87) J. Knecht, J. A. Cifonelli, and A. Dorfman,J. Biol. Chem., 242,4652 (1967). (88) L.-& Fransson, Biochim. Biophys. Acta, 156, 311 (1968); see also, A. Bella and I. Danishefsky,]. Biol. Chem., 243,2660 (1968). (88a) T. Helting and L. Roden, Biochim. Biophys. Acta, 170,301 (1968). (89) P. Hoffman, T. A. Mashburn, K. Meyer, and B. A. Bray, J. Biol. Chem., 242, 3799 (1967).

292

D. A. REES

The physical properties of the complex macromolecule of the protein-polysaccharide are different from those of the isolated chain^:^.^^ and some properties of the chains themselves may be changed by anchorage to p ~ l y p e p t i d e The . ~ ~ natural complexes are considerably heterogeneousg3 and, perhaps, carry chains of different structures on the same backbone.94The polysaccharidic part can be released by digestion with enzymes or, when glycosidic linkage is involved, by base-catalyzed elimination, because the glycosyloxy function is p to the peptide carbonyl The structures and properties of these fragments are known in ~ u t l i n e . ' ~ , 'Like ~ . ~ ~the agars and carrageenans, they consist of residues linked alternately (1 + 3) and (1 + 4). Some important examples of both types are listed in Table I. Some, but not all, of these structures might very well blend into each other in Nature; for example, K- and p-carrageenan, or chondroitin and chondroitin 4-sulfate. Apart from that for the region near the linkage to polypeptide, there is no convincing evidence of deviation from the alternating arrangement of (1+ 3)-and (1 + 4)-linkages, but the distribution of sulfate58~98~101~'03~*03a and the ratio of certain monomers40~58*100 might vary. Masked repeating structures would, therefore, seem to be typical of both series. M. Schubert, Fed. Proc., 25,1047 (1966). T. C. Laurent, Fed. Proc., 25,1037 (1966). C. Woodward and E. A. Davidson, Proc. Nat. Acad. Sci. U . S . , 60,201 (1968). S. Pal and M. Schubert,]. Biol. Chem., 240,3245 (1965); S. Pal, P. T. Doganges, and M. Schubert, ibid., 241,4261 (1966). (94) P. Hoffman, T. A. Mashburn, and K. Meyer, J. Biol. Chem., 242, 3805 (1967); L. Rosenberg, M. Schubert, and J. Sandson, ibid., 242,4691 (1967). (95) B. Anderson, P. Hoffman, and K. MeyerJ. B i d . Chem., 240,156 (1965). (96) E. A. Davidson and K. MeyerJ. Biol.Chem., 211,605 (1954). (97) K. Meyer, E. A. Davidson, A. Linker, and P. Hoffman, Biochim. Biophys. Acta, 21, 506 (1956); P. Hoffman, A. Linker, and K. Meyer, ibid., 30, 184 (1958); Fed. Proc., 17,1078 (1958). (98) S. Suzuki,J. Biol. Chem., 235,3580 (1960). (99) B. Weissman and K. Meyer, J . Amer. Chem. Soc., 74, 4729 (1952); 76, 1753 (1954); K. Meyer, Fed. Proc., 17, 1075 (1958); S. Hirano and P. Hoffman,J. Org. Chem., 27,395 (1962). (100) L.-A. Fransson and L. Rod& J . B i d . Chem., 242, 4161, 4170 (1967); L.-A. Fransson, ibid., 243,1504 (1968);L.-A. Fransson, Arkiv. Kemi, 29,95 (1968). (101) V. P. Bhavanandan and K. Meyer, J. Biol. Chem., 242, 4352 (1967); 243, 1052 (1968). (102) M . J. Clancy, K. Walsh, T. Dillon, and P. S. O'Colla, Proc. Roy. Dublin Soc., A, 1,197 (1960);T. J. Painter, Can.J. Chem., 38,112 (1960). (103) S. Suzuki, H. Saito, T. Yamagata, K. Anno, N. Seno, Y. Kawai, and T. Furuhashi, J . B i d . Chem., 243, 1543 (1968). (103a) M . B. Mathews and L. Decker, Biochim. Biophys. Acta, 156,419 (1968).

(90) (91) (92) (93)

POLYSACCHARIDE GELS AND NETWORKS

293

TABLEI Basic Structures of Some Polysaccharides from Animal and Seaweed Tissues: Variations on a General Structure [A - (1+ 3) - B - (1+ 4) -1, ~~

~

Polysaccharide Chondroitin 6-sulfate

4-sulfate

Hyaluronic acid Dermatan sulfate

Keratan sulfate

Agarose Porphyran

K-Carrageenan

&-Carrageenan

A-Carrageenan @-Carrageenan K-Furcellaran

~

Residue A

~

Residue B

~

References

2-acetamido-2-deoxy96 P-D-galactopyranose 2-acetamido-2-deoxyP-D-gh2opyranuronic acid 97,98 P-D-galactopyranose 6-sulfate P-D-glucopyranuronic acid 2-acetamido-2-deoxy97,98 P-D-galactopyranose 4-sulfate 99 2-acetamido-2-deoxyP-D-glucopyranuronic acid 8-D-glucopyranose a-L-idopyranuronic acid 2-acetamido-2-deoxy97,100 and P-D-glucopyranuronic P-D-galactopyranose 4-sulfate and, someacid times, 6-sulfate 2-acetamido-2-deoxyP-D-galactopyranose 101 and its 6-sulfate P-D-glucopyranose and its 6-sulfate 3,6-anhydro-a-~P-D-galactopyranose 37 galactopyranose 3,6-anhydro-a-~P-D-galactopy ranose 40-42 and its 6-methyl galactopyranose and Lether galactopyranose 6-sulfate 3,6-anhydro-a-~P-D-galactopyranose 55 galactopyranose, its 24-sulfate sulfate, and a-D-gahCtopyranose 6-sulfate 3,6-anhydro-a-~6 1,62 P-D-galactopyranose galactopyranose %sulfate 4-sulfate and a-D-galactopyranose 2,6-disulfate a-D-galactopyranose 63,66,70 P-D-galactopyranose 2,6-disulfate and its 2-sulfate 63 3,6-anhydro-a-~P-D-galactopyranose galactopyranose and a - ~ - 4-sulfate galactopyranose 6-sulfate 3,6-anhydro-a-~60.102 P-D-galactopyranose, galactopyranose and U-Dsome of it possibly galactopyranose 6-sulfate representing branch points, and its 4-sulfate P-D-gluCOpy~anurOniCacid

294

D. A. REES

Despite the stereochemical differences that, in the animal poly) in saccharides, the 4-linked residue is commonly p-D-glum C ~ ( Dbut, the seaweeds, a-D-galacto 1C(D)or the equivalent a-L-galacto C1 (L), there is the possibility that the chain conformations are similar. This is seen on comparing suitably drawn conformational formulas of such examples as agarose (3), the carrageenans (4 and 5), chondroitin 4sulfate (15), and hyaluronic acid (16).The entire group can, therefore, be considered to be one family derived from an alternating copolymer

0% CO,H

n

n

(17)of 1,3- and 1,4-cyclohexanediol residues by insertion of heteroatoms and substitution. The conformations of the residues are usually as shown in 17, but, in each series, there is an example that might

POLYSACCHARIDE GELS AND NETWORKS

295

be derived from 18. These are A-carrageenan (7), in which the 4linked D-galactose residue would almost certainly have the Cl(D) conformation and, less probably, dermatan sulfate. The favored conformation of the L-iduronic acid residue is expected to be CI(L),~' but there is as yet no direct evidence for this. Conflicting results have been obtained by use of optical rotatory d i s p e r ~ i o n . ' ~ ~ . ' ~ If the usual assumptions are made about the stereochemistry of polysaccharides (see Sections II1,lb and 3b (i); pp. 272 and 284), any differences between the various polysaccharide conformations derived from 17 can only arise from the changed nature and disposition of the substituents (for example, 3 and 16), and, sometimes, the changed position of the heteroatom, that is, the changed optical configuration of the 4-linked residue (for example, 3 and 4). Such alterations change the interactions that control the allowed values of the four variable angles of rotation, and, hence, the chain contour. A computer analysis of simple steric interactions has shown that all of the polysaccharides are highly restricted, but those of animal origin tend to be more restricted than those from plants, because the former have more, large, equatorial groups adjacent to each glycosidic oxygen atom, and these groups hinder rotation about the C - 0 bonds.'l Otherwise, the ranges of conformations possible are quite similar, and common properties that can be related to this common element in stereochemistry might be expected, as well as specific properties of individual structures. For example, further calculations have shown that double helices that are sterically feasible can be built for hyaluronic acid, chondroitin and chondroitin sulfates, keratan sulfate, dermatan sulfate in which the L-iduronic acid residue has the CI(L) conformation, and agarose,75 as well as for the carrageenans (for which the helices have been experimentally e~tablished'~). This approach is speculative, but it is worth bearing in mind that agars and carrageenans, which are cleaner and more available than the other polysaccharides, might serve as models for certain types of behavior of aminopolysaccharides. For example, there is possibly, between Ca2@ions and pairs of chondroitin 4-sulfate chains that are aligned by anchorage to polypeptide,g2 a specific interaction that recalls the specific interactions of pairs of chains in the double helix of K-carrageenan: K-carrageenan has half the charge density, and its interactions are with univalent cations. Hybrids of 17 and 18 might also occur in each series (see Table I).

(104) E. A. Davidson, Biochfm.Bfophys. Acta, 101,121 (1965).

296

D. A. REES

Protein-polysaccharides and hyaluronic acid are important components of biological gels and l u b r i ~ a n t s , but ~~~ in' ~ i~t r odo not form gels that, at least in the absence of other tissue components such as collagen, are firm and rigid like those of agars and carrageenans. Dilute solutions do, however, show evidence of being c r o s ~ - l i n k e d , ~ ~ , ~ ~ , ~ 0 6 ~ and it is possible to prepare an interesting, gel-like material from hyaluronic acid.'O' This material has been called a "viscoelastic putty," and it can be obtained from solutions as dilute as 0.3%, at slight acidity; it is cohesive and elastic, and has some ability to maintain its shape, although viscous flow becomes apparent after a few hours. Measurements of relaxation suggested that there is no qualitative difference between the nature of the cross linkage in this state and in solutions.'08 4. Glycuronans and Derivatives

a. Alginic Acid.-Alginic acid was originally found in the brown seaweeds (Phaeophyceae), which still provide the main source for it. Similar polysaccharides have since been isolated from b a ~ t e r i a . ' ~ ~ * " ~ Although D-mannuronic acid was, for many years, thought to be the only monomer residue, it has now been established that most, if not all, alginates contain L-guluronic acid residues in addition."' After methylation, reduction of the carboxyl groups, and hydrolysis, derivatives of 2,3-di - 0-methyl - D-mannose and 2,3-di - 0-methyl - L- gulose were, apparently, the only products112from a number of ~amples,''~ including one of bacterial origin.'I4 The polysaccharide is oxidized incompletely by periodate, but this anomaly has been convincingly (105) L. Dintenfass, Fed. Proc., 25,1054(1966). (106) A.G. Ogston and J. E. Stanier, Biochem.], 49,585(1951). (106a) V. C. Hascall and S . W. Sajdera, J . Biol. Chem., 244, 2384 (1969);see also, S . W. Sajdera and V. C. Hascall, ibid., 244, 77 (1969),and J. R. Dunstone and M. D . Franek, ibid., 244,3654(1969). (107) E. A. Balazs, Fed. Proc., 25,1817(1966). (108) D. A. Gibbs, E. W. Merrill, K. A. Smith, and E. A. Balazs, Biopolymers, 6, 777 ( 1968). (109) A.Linker and R. S. Jones, Nature, 204,187(1964). (110) D. M. Carlson and L. W. Matthews, Biochemistry, 5,2817(1966). (111) F.G . Fischer and H. Diirfel, Z . Physiol. Chem., 302, 186 (1955);R. L.Whistler and K. W. Kirby, ibid., 314,46 (1959);D. W.Drummond, E. L. Hirst, and E. Percival,]. Chem. SOC., 1208 (1962). (112) E. L. Hirst and D. A. Rees,]. Chem. SOC., 1182 (1965). (113) D.A. Reesand J. W. B. Samuel,]. Chem. SOC. (C),2295(1967). (114) P. A.J.Gorin and J. F. T. Spencer, Can.].Chem.,44,993(1966).

POLYSACCHARIDE GELS AND NETWORKS

297

by the suggestion that aldehyde functions of oxidized residues form stable, six-membered, cyclic acetals with the residues adjacent, so blocking further oxidation. From this, and the evidence from partial hydrolysis (see later), it follows that the chains are linear and contain (1 + 4)-linkages, although a formal possibility remains that there is a proportion of (1 + 5)-linkages. The bacterial The anomeric configuration polysaccharide is partly 0-a~etylated."~ is generally assumed to be p-D for the D-mannose and a-L for the L-gulose residue; this would be consistent with the strongly negative optical rotation, but the only rigorous proof thus far obtained is for the linkage between the D-mannuronic acid residues, as crystalline mannobiose was isolated after partial hydrolysis of the carboxylA mixed disaccharide was also obtained, reduced poly~accharide."~ proving that the two types of residue can occur in the same chain, instead of in separable homopolymers. There can be achieved some separation into fractions that are enriched with respect to individual residues,l16 but there is no indication of the existence of homopolysaccharides, except in the rather doubtful case of one bacterial preparation."O. Information about sequence has been obtained by elegant methods based on heterogeneous hydrolysis; this reaction proceeds in two stages, indicating that some parts of the sample are more readily hydrolyzed than others."' The resistant material could be fractionated into two components having degrees of polymerization of about 20 and containing, respectively, L-guluronic acid residues and Dmannuronic acid residues as the major units. The alginic acid molecule is, therefore, representable schematically as:

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

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

in which blocks containing essentially one type of residue (continuous lines) resist hydrolysis, perhaps because they are contained in regions of crystallinity and are therefore inaccessible to reagents; they are separated b y hydrolyzable parts (dotted lines). Different disaccharides are formed by hydrolysis of the accessible parts, and of each type of resistant block. The former might, therefore, approximate (114a) B. Larsen andT. J. Painter, Carbohyd. Res., 10,186 (1969). (115) E. L. Hirst, E. Percival, and J. K. Wold,J. Chem. Soc., 1493 (1964). (116) A. Haug,Acta Chem. Scand., 13,601,1250 (1959). (117) A. Haug, B. Larsen, and 0. Smidsrod, Acta Chem. Scand., 20, 183 (1966); T. J. Painter, 0.SmidsrZd, and A. Haug, ibid.,22,1637 (1968).

298

D. A. REES

quite closely to an alternating arrangement of D-mannuronic acid and L-guluronic acid residues.”* Another interesting feature is that the rate of hydrolysis of alginic acid at low concentrations of acid is even higher than would be expected on the basis of its polyelectrolyte proper tie^."^ This result has been interpreted in terms of a type of intramolecular, general-acid catalysis, in which the glycosidic oxygen atom is protonated by the carboxyl group of the aglycon. This mechanism is supported by the dependence of the rate on the stereochemistry,120and by analogy with other systems.121 No serious attempt has as yet been made to solve, by modern methods, the crystal structure of any alginate, although x-ray difiaction data have been publishedlZ2and have been discussed elsewhere in this Series.27 It is sufficient to note that alginic acid and calcium alginate, both of which are known to form gels, are insoluble in water, and seem to pack in crystal lattices in the solid state. The conformation in salt solution is disordered, but very extended.122a Rigid gels can be formed from alginates with controlled proportions of most divalent and trivalent cations. Calcium alginate is the most common of these gels in commercial use. Precipitates are formed with larger proportions of cations, or by sudden mixing; and, to obtain a useful gel texture, it is necessary to contrive the slow addition of a controlled concentration. Dialysis, or a chemical method (such as arranging for the slow hydrolysis of D-glucono-1,4-lactone to lead to release of Ca2@,by action of the D-glUCOniC acid formed on a suitable, insoluble calcium salt) can be used. Unlike those from agar and carrageenans, alginate gels do not “melt” on heating, and gelation can be induced by H@, as well as other cations. b. Pectic Substances. -This discussion will avoid use of such older terms123,124 as “pectinic acid,” “pectinates,” and “protopectin,” beA. Haug, B. Larsen, and 0.Smidsrprd, Actu Chem. Scund., 21,691 (1967). 0.Smidsdd, A. Haug, and B. Larsen, Actu Chem. Scund., 20,1026 (1966). 0.Smidsrprd, B. Larsen, and A. Haug, Curbohyd. Res., 5,371 (1967). B. Capon, Chem. Reo., 69,407 (1969);see especially, p. 426. W. T. Astbury, Nature, 155, 667 (1945); E. Frei and H.D. Preston, ibid., 196, 130 (1962). (122a) 0.Smidsrprd and A. Haug, Actu Chem. Scund., 22,797 (1968). (123) Z. I. Kertesz, “The Pectic Substances,” Interscience Publishers, New York, N. Y., 1951. (124) J. J. Doesburg, “Pectic Substances in Fresh and Preserved Fruits and Vegetables,” Institute for Research on Storage and Processing of Horticultural Produce, Wageningen, The Netherlands, 1965.

(118) (119) (120) (121) (122)

POLYSACCHARIDE GELS AND NETWORKS

299

cause the distinctions they make are not the most useful from the present point of view. Instead, the unifying structural theme for the group of complex polysaccharides collectively called “pectic substances,” namely, a backbone of (1 -+ 4)-linked a-D-galactopyranuronic acid residues, will be the point of departure. This backbone, and structures that approximate to it in having few side-chains and insertions, will be called “pectic acid”; the salts, “pectates”; and the methyl ester, “pectin.” Some polysaccharides lacking this backbone are also classed as pectic substances (for example, pectic arabinan”’), but they will not be discussed here. Pectic substances occur widely in plants, particularly in fruit and young t i s s ~ e s . ’ Woods ~ ~ ~ ’ contain ~~ only a small proportion. The chief commercial sources are the peel of various citrus fruits, and apple pomace. Early aspects of their chemistry have been reviewed in this SeriesIz6and el~ewhere,’~’ and more recent developments have been succintly summarized by Aspinall.12EWe must emphasize that the structural chemistry is more complicated than was once thought, and that this must be recognized if the physical properties are to be fully understood. Only a few polysaccharides are known that are built up almost entirely of D-galacturonic acid residues; examples are the pectic acid from sunflower headsIz9and several subfractions of other preparation^.'^^.'^^ Most pectic substances contain a selection of the extra features listed in Table 11. In some, such as those from soyb e a n ~and ~ ~mustard ~ * ~embryos,’36 ~ ~ these dominate the structure, almost to the extent that they do in the galacturonorhamnan group of gums and mucilages (see This Volume, p. 361) with which there are clear structural relationships. Most gel-forming, pectic substances E. L. Hirst, D. A. Rees, and N. G . Richardson, Biochem. J., 95,453 (1965); D. A. Rees and N. G. Richardson, Biochemistry, 5,3099 (1966); D. A. Rees and I. W. Steele, ibid., 5,3108 (1966). E. L. Hirst and J. K. N. Jones,Aduan. Carbohyd. Chem., 2,235 (1946). H. Deuel and E. Stutz, Aduan. Enzymol., 20,341 (1958). G. 0.Aspinall, Chim. Biochim. Lignine, Cellulose Hemicelluloses, Actes Symp. Intern. Grenoble, France, 1964, p. 421. C. T. Bishop, Can. J. Chem., 33, 1521 (1955); V. Zitko and C. T. Bishop, ibid., 44,1275 (1966). V. Zitko and C. T. Bishop, Can.J.Chem., 43,3206 (1965). S . S. Bhattachaqjee and T. E. Timell, Can.J.Chem.,43,758 (1965). C . 0.Aspinall and R. S. Fanshawe,]. Chem. SOC., 4215 (1961). G. 0.Aspinall, K. Hunt, and I. M. Morrison,./. Chem. SOC. (C), 1080 (1967). G. 0.Aspinall, J. W. T. Craig, and J. L. Whyte, Carbohyd. Res., 7,442 (1968). G. 0. Aspinall, I. W. Cottrell, S. V. Egan, I. M. Morrison, and J. N. C. Whyte, J. Chem. Soc. ( C ) ,1071 (1967). S . E. B. Could, D. A. Rees, N. C. Richardson, and I. W. Steele, Nature, 208,876 (1965);D. A. Rees and N. J. Wight, Biochem.]., 115,431 (1969).

D. A. REES

300

TABLEI1 Structural Variations in Pectic Substances

Feature

. . . 2)-~-Rhap-(1 . . . insertion

in galacturonan

chaina

0-Acetylation of galacturonic acid residues “Galactan” side-chains, [. 4)-P-~-Galp-(l. . .]n Branched “arabinan” side-chains

..

Xylp-(1 . , . side-chains

P-D-Galp-(l+ 2)-D-xylp-(l . . ., probably as side chains ~Y-L-Fuc~-( 1+ e)-D-Xylp-(1 . . ., probably as side chains 1+ 4)-~-Galp-( 1 . . ., probably as &D-G~A-( side chains p - ~ - G p A - ( l +6)-D-Galp-(l . ., probably as side chains

.

p - ~ - G p A - ( l +4)-~-Fucp-(l. . ., probably as side chains D-Apiose 2-O-Methyl-~-xylose B-O-Methyl-~-fucose

1

Examples of source material

References

lucerne soybean hulls lemon peel soybean cotyledons mustard embryo apple Amibilis fir bark beet soybean cotyledons soybean hulls lucerne mustard embryo apple mountain pine pollen mustard embryo lemon peel soybean cotyledons soybean cotyledons soybean hulls soybean cotyledons soybean hulls soybean cotyledons soybean hulls soybean cotyledons lemon peel soybean hulls soybean cotyledons soybean hulls lemon peel lucerne Zosteraceae lucerne plum leaves sisal

132 133 134 135 136 137 131 138,139 135 133 132 136 137 140 136 134 135 135 133 135 133 135 133 136 134 133 135 133 134 132,141 141a 132 141b 141c

“Work with polysaccharides from soybeans has shown that these residues can occur contiguously and in regions in which they alternate with D-galacturonic acid resid u e ~ ,presumably ~ ~ ~ * in ~ addition ~ ~ to more random types of distribution.

POLYSACCHARIDE GELS AND NETWORKS

301

would seem to contain about 70-80% of D-galacturonic acid residues, and it will be shown that the properties relevant to this article arise from the D-galacturonan backbone (see Section V,3; p. 323), even if other features have important modifying influences. Fibers of pectin and sodium pectate give x-ray photographs which show a fiber axis repeat distance of about 13.0 A, with meridianal reflections on the third and sixth layer lines. A three fold helix can be built to fit this evidence.142The C.Z(D)conformation was assumed for each residue, but it has since been claimed that, in solution, certain galacturonan derivatives have their monomer residues in the ~ C ( Dcon ) for ma ti or^.'^^ The evidence was provided by the chemical shifts of methyl group signals of acetates and methyl ethers, interpreted by analogy with well known variations for simple sugar derivatives, according to whether the groups are axially or equatorially attached. 144 These effects are small, and for diffuse, polysaccharide spectra, require cautious interpretation. Whether or not this conformation exists in solution, it would seem unlikely to be present in fibers, because computer methods (see Sections III,lb, II1,Sb (i), and III,3c; pp. 272,284, and 294)show that the x-ray parameters could then be fitted only by arrangements of‘the chain in which there are strong ) leads to steric ~ 1 a s h e s .On l ~ ~the other hand, the C ~ ( Dconformation (137) A. J. Barrettand D. H. Northcote, Biochem.J.,94,617 (1965). (138) F. Ehrlich and R. V. Sommerfield, Biochem. Z., 168, 263 (1926); E. K. Nelson, J . Amer. Chem. SOC., 48,2945 (1926). (139) E. L. Pippen, R. M. McCready, and H. S. Owens, J . Amer. Chem. SOC., 72, 813 (1950). (140) H. 0. Bouveng, Acta Chem. Scand., 19,953 (1965). (141) G. 0. Aspinall, B. Gestetner, J. A. Molloy, and M. Uddin,J. Chem. S O C . (C), 2554 (1968). (141a) R. G. Odova, V. E. Vaskovsky, and Y. S. Ovodov, Carbohyd. Res., 6,328 (1968). (141b) P. Andrews and L. Hough, Chem. Ind. (London), 1278 (1956); J. D. Anderson P. Andrews, and L. Hough, ibid., 1453 (1957). (1414 G. 0. Aspinall and A. Caiias-Rodriguez,]. Chem. SOC.,4020 (1958). (142) K. J. Palmer and M. B. Hartzog,J. Amer. Chem. SOC., 67, 2122 (1945); K. J. Palmer, R. C. Merrill, H. S. Owens, and M. Ballantyne,J. Phys. Chem., 51,710 (1947); K. J. Palmer, in “High Polymer Physics,” H. A. Robinson, ed., Chemical Publishing Co., Brooklyn, N. Y., 1948, p. 42. (143) S. Hirano, M. Manabe, N. Miyazaki, and K. Onodera, Biochim. Biophys. Acta, 156,213 (1968). (144) R. U. Lemieux, R. K. Kullnig, H. J. Bemstein, and W. G. Schneider, J. Amer. Chem. Soc., 80,6098 (1958). (145) D. A. Rees and A. W. Wight, unpublished results.

302

D. A. REES

two feasible arrangements. In both, the helix is a “twisted, corrugated strip,” instead of a “stretched-out, wire spring” of the carrageenan type (see Fig. 4,p. 288). One of these, having a left-handed screw axis, appears to be the same as that illustrated by Palmer and H a r t z ~ g , but ’ ~ ~the computed coordinates show some steric compression that is probably significant. The other conformation has the righthanded screw sense (19), and seems more likely on stereochemical grounds. IHelix axis

Pectic substances form two important types of rigid gel. Pectinsugar gels, familiar in jams and jellies, are prepared at low pH, and sometimes with multivalent cations added, but neither condition is necessary if esterification is complete.146Some unesterified residues (146) H. Deuel, G . Huber, and R. Leuenberger, Helu. Chdrn.Acta, 33,1226 (1950).

POLYSACCHARIDE GELS AND NETWORKS

303

are always present in commercial pectins, and the gel properties are then very sensitive to pH and, to a lesser extent, to the concentration and nature of other solute^.'^' The second type of useful gel is made at still lower degrees of esterification, with multivalent cations (usually Ca2@)and low pH, and can tolerate lower concentrations of sucrose. Other solutes can replace sucrose in the preparation of each type (see Section V,3; p. 323).

Iv.

CHARACTERIZATION OF JUNCTION ZONES

The discussion of Fig. 1 (see p. 269) will now be resumed to enquire into the way in which the chains are held together in junction zones, and the extent to which association can be explained in terms of our knowledge of polysaccharide structures and conformations.

1. First Principles The kind of forces that are to be looked for must first be considered, The phenomenon of syneresis, exhibited by many gels (especially when the concentration of polysaccharide is low) is the spontaneous release of water, with contraction of the gel volume, that may occur on standing. Agar, carrageenans, and calcium pectate behave in this way. The process is spontaneous and constitutes a shift to a more stable state, but its initiation may not occur for several hours or days. Equilibrium is, therefore, not established when the gel sets. This conclusion means that, on the time-scale customary in the laboratory, the framework is not a dynamic structure that is continually broken and re-formed; otherwise, on the same time-scale, there would be a rapid alteration to whatever the equilibrium state really is. This does not preclude rotational and restricted translational motion by polymer segments without breakage of junctions. “Aging” effects, including syneresis itself, might reflect some making and breaking of the framework, and it would, perhaps, be incorrect to regard every junction zone as being permanent, However, the conclusion is inescapable that the shape of the gel is not maintained by dynamic forces (analogous to the attractions that, in a liquid, give rise to surface tension and thereby maintain the shape of a small droplet). A similar argument could be advanced for the different properties (for example, gel strengths) of two alginate gels, having the same composition, that have been prepared by different methods of mixing. Since they are (147) C. L. Hinton,J.Sci. FoodAgr., 1,300(1950).

304

D. A. REES

different, they cannot both represent thermodynamic equilibrium, but, even so, their properties converge too slowly for detection. Finally, the same principle has been demonstrated for gelatin by observing the dissociation of fragments of the gel network by lightscattering, and showing that the process is exceedingly ~ l 0 w . l ~ ~ Several models for the gel framework would, by their nature, require the continual breaking and remaking of junction zones; they would, therefore, seem improbable for gels having the properties described, that is, that are demonstrably in nonequilibrium states. In view of this conclusion, and much supporting evidence to be given later, the following hypotheses need not be seriously considered. (i) Simple ionic bridging, for example, of negative charges on alginate or pectate chains by such divalent cations as [email protected] first pointed out by Deuel and such a situation is, in any case, incompatible with the nature of electrolyte solutions. Electrostatic forces can stabilize gels, but not by simple attraction of two negative charges to one double-positive charge. Were these attractions as strong as has sometimes been suggested, MgS04, ZnSO,, and Fe2(S04),would be as insoluble as diamond! (ii) Hydrogen bonds between isolated pairs of hydroxyl groups. Quite apart from the argument that such systems should consist of equilibria, it should be remembered that, were carbohydrate molecules to show a strong tendency to hydrogenbond to each other in aqueous solution, simply by virtue of their hydroxyl groups, sucrose would not be the textbook example of a compound having ideal-solution behavior! (iii) Chelation of single ions (for example, Ca2@)by suitably arranged hydroxyl and carboxylate groups on each of a pair of chains. Hydroxy acids form weak chelates, especially with Ca2@,which are in rapid, reversible equilibrium with the separate ions.150For pectic substances, the view that Ca2@can be bound by chelation is refuted by the results of inspection of molecular models, and by the manner in which the stability constant for binding varies with the degree of acetylation and the degree of As with electrostatic attraction, chelation methyl e~terificati0n.l~~ may well play a part in some systems, but any model that has only one cation per junction and is unaided by other forces cannot explain the facts. Some gels do have properties (such as an ability to “heal”) that suggest that their junctions are continually breaking and re-forming, (148) H. Boedtker and P. Doty,]. Phys. Chem., 58,968(1954). (149) H.Deuel, G.Huber, and L. Anyas-Weisz, Helu. Chim. Acta, 33,563(1950). (150) R. K.Cannan and A. Kibrick,]. Arner. Chem. SOC.,60,2314(1938).

POLYSACCHARIDE GELS AND NETWORKS

305

Examples are hyaluronic acidlo’ and the galactomannan-borate sysMore commonly, polysaccharide gels would, from evidence of the type already described, appear to have junction zones having a high activation-energy for dissociation. This conclusion immediately suggests a situation in which many attractions cooperate, as in a “microcrystallite.” This theory provides a good explanation for some gels, but, as will be seen, there are other possibilities as well.

2. Methods The methods available for the characterization of junctions will be illustrated by particular examples in the following Sections, but an overall view of the type of information that may be obtained will first be attempted.

a. Optical and Related Methods. - Birefringence, observed with the polarizing microscope, may show whether regions of molecular

orientation are present in the gel.151aJ52 It is sometimes possible to observe fine structure at the level provided by the microscope or the electron microscope, but care must be taken to avoid modification of the gel structure during preparation of the s p e ~ i m e n . ’ ~ ’ ~ * ’ ~ ~ . ’ ~ ~ Light-scattering methods can give information about the size and distribution of inhomogeneities (such as aggregates, or cavities of 148.152-155

b. X-Ray Diffraction. -Apart from information about the shape and packing of chains in the true solid state (see pp. 284-286), this (1504 H. Deuel and H. Neukom, in “Natural Plant Hydrocolloids,” Aduan. Chem. Ser., 11.51 (1954). (151) R. Kohn and I. Furda, Collect. Czech. Chem. Commun.,33,2217 (1968). (151a) H. Thiele, W. Joraschky, A. Plohnke, A. Wiechen, R. Wolf, and A. Wollmer, Kolloid-Z., 197, 26 (1964) and earlier papers; H. Thiele, Discussions Faraday Soc., 18,294 (1954). (151b) E. S. Halberstadt, H . K. Henisch, J. Nickl, and E. W. White,J. Colloid Interface Sci., 29,469 (1969). (152) S. A. Glikman, L. A. Roth, L. I. Khomutov, E. N. Gubenkova, and I. I. Feigelson, J . Polym. Sci., Pt. C, 16,2001 (1967). (153) E. V. Beebe, R. L. Coalson, and R. H. Marchessault, 1.Polym. Sci., Pt. C , 13, 103 (1966). (154) F. A. Bettelheim,]. Polym. Sci., Pt. A-2,5, 1043 (1967). (154a) B. Obrink,j. Chromntogr., 37,329 (1968). (155) A. Xabudzinska, A. Wasiak. and A. Ziabicki, I . Polym. Sci., Pt. C , 16, 2835 (1967).

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method can be used to show the presence of crystallites in gels, particularly if they are stretched to induce ~ r i e n t a t i o n . ’ ~ ~However, -’~~ even if crystallites are present, the question remains whether they can be identified with the junction zones.155The same problem of interpretation is encountered when the methods listed under 2a are k e d . The dilemma is well illustrated by the long controversy about the junction zones in gelatin,’5s although it seems to the present writer that the dispute has now been settled in favor of the microcrystallite t h e ~ r y . ’ ~ ~ ~ ’ ~ ~ ~ ’ ~ ~ c. Variation of Molecular Structure.-Very often, it is possible to draw conclusions about the functional groups involved in gelation by observing changes in properties when the molecular structure is altered, either by chemical reactions designed for the purpose or by use of natural differences between one sample and another. Gel formation by pectins is inhibited if the methyl ester groups are changed to ethyl or 2-hydroxyethyl group^,'^^,'^' thus proving that the junction contacts involve the galacturonan backbone and not, at least primarily, such other features as those listed in Table I1 (see p. 300). Many other applications of this approach to polysaccharide gels are given in the Sections that follow. d. Thermodynamic Properties. -Gel behavior can often be interpreted qualitatively in terms of thermodynamic laws, to show the probable nature of the forces that hold the chains in the framework. The sharp “melting point” of gelatin points to a “phase transition,” such as those that involve crystallites or regular helical conformations [see Section V,2a(i); p. 3141, and the inverted temperature-behavior of 0-methylcellulose (see Section V,5; p. 331) points toward micelle interactions.’62 Hysteresis behavior in “melting” and setting shows that the junction zones are not a set of identical chemical linkages, but a family of associations that are probably formed by “phase transitions’’ in a mechanism that involves nucleation [see Section V,2a (ii); p. 3161. The implications of syneresis have already been considered. (156) J. R. Katz, J. C. Derksen, and W. F. Bon, Rec. Trao. Chim., 50, 725 (1931);K. Hermann, G. Gerngross, and W. Z. Abitz, 2. Physik. Chem., Abt. B , 10, 371 (1930). (157) G. Diirigand A. Banderet, Helo. Chim. Acta, 33,1106 (1950). (158) A. Veis, “The Macromolecular Chemistry of Gelatin,” Academic Press Inc., New York, N. Y., 1964. (159) J. E. Eldridge and J. D. Ferry,J. Phys. Chem., 58,992 (1954). (160) P. J . Flory and 0.K. SpurrJ. Amer. Chem. SOC., 83,1308 (1960). (161) H. Neukom, Ph.D.Thesis, Zurich, 1949. (162) F. Franks, in Ref. 16, p. 55.

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307

These and other qualitative inferences will be more fully discussed for particular examples. Quantitative treatment159*163,164 is more difficult, and there are no rigorous methods for the measurement of thermodynamic quantities associated with gel formation. This is a serious limitation, not only because such quantities could give evidence for the nature of junctions, but also because one of the most important future developments will be the computation of interaction energies in junctions from known stereochemistry, as a step to the understanding of why gels form and how they are manipulated in Nature. This approach will be hampered if there are no experimental measurements for comparison and calibration. Values can be obtained from phase transitions in solids if it can be shown that these are similar to the processes in gel “melting.”1soCalorimetric measurements could be related to the molecular changes were it known, for example, how many solvated sugar residues are transferred to a junction under given conditions. An attractive approach to the heat changes in gels that can be “melted” and re-formed by temperature changes would be to treat the formation and destruction of junction zones as being the aggregation and disaggregation of certain loci on the chains:

n loci

junction

(1)

If a meaningful equilibrium constant, K, could be written for this process, the number of junctions should change with temperature in a way that is determined by Van’t Hoff s equation:

d In Kldt = AHolRT2 By measuring the change in the number of junctions with temperature, derivation, from this equation, of AHo, the “heat of cross-linking,” should be possible. Indirect methods must be used for the measurements. Eldridge and Ferry159have made the reasonable assumption that the “melting point” of the gel corresponds to the critical gelpoint of classical network theory, that is, the point at which each chain is involved, on the average, in one ~ross-1inkage.l~~~ By making several approximations, they have shown how the variation of this temperature with concentration can be used to solve for AHo. A relationship can similarly be derived for the variation of the gel “melting point” (163) J. PouradierJ. Chim. Phys., 64, 1616 (1967). (164) W. Kauzmann and H. Eyring,J. Chem. Phys., 9,41(1941). (164a) P. J. Flory, J . Amer. Chem. SOC., 63, 3096 (1941); W. H. Stockmayer,J. Chen. Phys., 12, 125 (1944);see also Ref. 3.

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D. A. REES

with the average molecular weight.15gA further possibility is use of the rigidity of the gel as a measure of the number of junction zones present.ls3 These methods are simple and elegant, but, unfortunately, there are important reservations to be considered before any confidence can be placed in the results. Approximations are made in relating experimental measurements to the change in the number of junction zones,159J63 and Van’t Hoff methods are, in general, notoriously prone to error when such assumptions are mistaken. It must also be asked whether the process can be treated as any kind of equilibrium at all. To make this assumption for those systems that show hysteresis is tantamount to supposing that, despite the irreversibility of gel formation that is shown by hysteresis [see Section V,2a (ii); p. 3161, the “melting” step is reversible, or alternatively, that “melting” is irreversible, but setting is reversible. The first possibility is equivalent to the statement that supercooling occurs in the “setting” of individual junctions, but that superheating does not occur when they “melt.” There are many precedents for such behavior in the melting and crystallization of simple compounds; for example, in the absence of appropriate nuclei, water can be kept below its freezing point for long periods of time, but ice cannot be heated above its melting point at a given pressure. Similarly, it would now seem to be agreed that the melting of crystalline polymers can be treated as a reversible process.164bBoth analogies suggest that, despite hysteresis, it might be reasonable to consider junction-zone dissociation as an equilibrium. The validity is, however, by no means established, and the assumption must be a matter of faith, and therefore of doubt also, at the present time. Even should equilibrium laws be applicable, it remains to define the equilibrium condition. The usual assumption is that use may be made of a single equilibrium-constant of the form (see equation 1):

K = [junctions]/[loci]n This equation will not hold if the junctions are microcrystallites in which n and the length of polymer segment representing a locus are both variable. Such systems ought to be treated by other methn and o d ~ . Even ~ ~if ~ ~ *the~locus ~ ~length are unique, the apparent AHo will depend very much on the particular value that is taken for n, and an arbitrary assignment will not suffice. If other species, such as (164b) L.Mandelkern, “Crystallization of Polymers,” McGraw-Hill, New York, N. Y., 1964. (165) B. H. Zimm and J. K. Bragg,]. Chem. Phys., 31,526 (1959); B. H. Zimm, ibid., 33,1349 (1960).

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cations and water, enter into the equilibrium, they should be included in the expression for K, although, in practice, they have not been. For all but very simple systems, a number of additional assumptions and approximations are therefore needed at this stage, Application of the three Van’t Hoff methods to gelatin gels gives results that are encouragingly consistent with each other and with ~,’~ simplest ~ and most widely the form predicted by t h e ~ r y . ’ ~The used of these has been based on the variation of gel “melting point” with concentration, to give results for those junctions that are in equilibrium with free chains at the “melting point.” These zones do not necessarily represent the “average junction zones” in a typical gel. The value for n has usually been taken as being 2, but it would now seem likely that the junctions are partly re-formed, collagen triple helices, for which n = 3. Re-examination of the theory shows that the values159worked out for n = 2 should, therefore, be doubled, giving -AHo = 100 to 400 kcal/mol of cross links. The highest values are for samples, having high molecular weight, that have been allowed to gel slowly; .it is entirely reasonable that the strongest junctions should be found in these gels. Measurements on collagen fiberPohave given 1.3 kcal/peptide residue for the heat of fusion in aqueous medium, from which it may be estimated that 75-300 peptide residues are involved in the gel junctions. This value is equivalent to 8-33 turns of collagen triple helix having 3 residues per turn, in qualitative agreement with the conception of gelatin junction-zones arrived at from other evidence. This result is not a quantitative test, but, as far as it goes, it would suggest that rough but useful estimates can be obtained for systems for which the basic assumptions hold. Unfortunately, there is still no way of knowing when the assumptions are valid, and extreme caution is still necessary in using the methods and results. In the absence of a network, thermodynamic quantities can be evaluated much more reliably; at present, the best approach would therefore be to work where possible with model systems in which the molecular changes correspond to those in junction formation (see Section IV,lg; p. 312). e. Spectroscopy and Optical Rotation. - Despite their great promise, these methods have been little used so far. If gelation involves changes in the secondary and tertiary structure of the polysaccharide chains, it should be possible to follow these processes in the proton magnetic resonance spectrum, and thus to obtain information about them, as has been done for corresponding changes in other natural

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When cations are involved in the junction zone, their environment could probably be studied by other methods developed for proteins and peptides; for example, by optical rotatory dispersion if they show peaks in the visible or ultraviolet spectrum,IB6or, if they are paramagnetic, by their broadening effect on signals in the proton magnetic resonance spectrum.’67 Similarly, “Rb and Yh nuclear magnetic resonance spectroscopy168could, perhaps, be used for ascertaining whether a proportion of cations becomes firmly bound when carrageenan gels are formed. Changes in optical rotation occur in the formation of gelatin gels, and their variation under different conditions has been well characterized.2 Attempts have been made to relate the changes to molecular events by calculating the “helix content” during g e l a t i ~ n , ’ ~ ~ and by using their temperature dependence to estimateIB4a “heat of cross linking.” Similar changes in optical rotation occur during the gelation of several p01ysaccharides~~J~~; these cannot be interpreted in terms of “helix contents” in quite the way that is now standard for polypeptides, because of the absence of suitable chromophores in most polysaccharides. The empirical theory of painvise interaction~’~’ might prove useful and, indeed, there has been a discussion of gelatin in these terms.ls4 It will be important to ensure that isotropic gels are used: changes in volume on gelation could conceivably cause deformation, and, hence, orientation of the type that is well known for gels.’,* Any interpretation in terms of the conventional theory of optical rotation would then be useless. Nevertheless, it has been confirmed experimentally that shifts in optical rotation that accompany gelation can have their origin in changes in conformation in the polysaccharide backbone.”l” A relationshipl7lb is now available between the optical rotation of saccharides and the (165a) For example, E. M. Bradbury, C. Crane-Robinson, H. Goldman, and H. W. E. Rattle, Biopolymers, 6,851 (1968); J. P. McTague, V. Ross, and J. H. Gibbs, ihid., 2,163 (1964); 0.Jardetsky, Aduan. Chem. Phys., 7,499 (1964). (166) For example, R. D. Gillard, Chem. Brit., 3,205 (1967). (167) For example, A. S. Mildvan, J. S. Leigh, and M. Cohn, Biochemistry, 6, 1805 (1967). (168) H. S. Gutowsky and B. R. McGarvey,J. Chem. Phys., 21,1423 (1953). (169) J. Bello, Biochim. Biophys. Acta, 109,250 (1965). (170) D. A. Rees and I. W. Steele, unpublished results; I. W. Steele, Ph. D. Thesis, University of Edinburgh, 1967. (171) W. Kauzmann, “Quantum Chemistry,” Academic Press Inc., New York, N. Y., 1957; W. Kauzmann, F. B. Clough, and I. Tobias, Tetrahedron, 13,57(1961). (171a) A. A. McKinnon, D. A. Rees, and F. B. Williamson, Chem. Commun.,701 (1969). (171b) D. A. Rees, J . Chem. SOC. (B), in press (1970).

POLYSACCHARIDE GELS AND NETWORKS

311

conformation angles and JI (see formula 2, p. 273), and it is expected that much information will be derived in the next few years by careful application of optical rotational studies to gelling polysaccharides. A broadening of the water signal in the proton magnetic resonance spectrum of agar gels might indicate a more ordered state than that obtaining in ordinary, bulk water.172The effect is also seen in solutions of other natural polymers, including other p o l y ~ a c c h a r i d e s . ~ ~ ~ - ~ ~ ~ The effect can be studied with I7O nuclear magnetic resonance spectroscopy, which has the advantage that complications are less likely from chemical exchange reactions and from paramagnetic impurities.174aThe role of the solvent will have to be included in any complete theory of gelation, and such methods, which seem to offer the possibility of directly investigating the state of the water, promise to be very useful. The interesting result was obtained with I7O nuclear magnetic resonance spectroscopy, that, for an aqueous ribonuclease gel, the state of the water changes when protein dissolves, but there is no further change with the transition from liquid to gel.174a Some information about the state of water in gels can also be obtained by dielectric dispersion

f. Other Physical Methods. - Ultracentrifugation of gels can be used for squeezing out solution from the gel framework, so that subsequent analysis may show which species are involved in the framework and which are “guests” in the interstice^.'^^ Because most polysaccharides are polymolecular, if not heterogeneous, it would be interesting to repeat such experiments under various conditions, to establish which species become tied into the network first-for example, at a series of temperatures starting just below the setting point of a gel that may be formed by cooling. Gel filtration has been suggested as a means for characterizing the gross features of networks, b y use of a theory that treats the network as being a random collection of infinitely long fiber^."^ The fraction (172) 0. Hechter, T. Wittstruck, N. McNiven, and G. Lester, Proc. Nut. Acad. Sci. U. S . , 46, 783 (1960); C. Sterling and M. Masuzawa, Makromol. Chem., 116, 140 (1968). (173) E. A. Balazs, A. A. Bothner-By, and J . Gergely, J. Mol. Biol., 1,147 (1959). (174) B. Jacobson, W. A. Anderson, and J. T. Arnold, Nature, 173,772 (1954). (174a) J. A. Glassel, Nature, 218,953 (1968). (174b) M. Masuzawa and C. Sterling, Biopolymers, 6,153 (1968). (175) P. Johnson, Proc. Roy. Soc. (London), Ser. A, 278, 527 (1964); P. Johnson and J . C. Metcalfe, Eur. Polym. J., 3,423 (1967). (176) T. C. Laurent, Biochim. Biophys. Acta, 136,199 (1967).

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of solute that penetrates the gel during chromatography (expressed as a partition coefficient) is assumed to be determined by the space available. The theory can be tested for each gel, by comparing the results of prediction with those from experiment, for the variation of retention volume with the molecular radius of the solute, and then be used for estimating the average diameter and hydration of the constituents of the network. Results of about 50 A for the diameter, and a content of 35-50% of water, were obtained for a series of rather concentrated (4-870) agarose gels. These values agree with those obtained by a light-scattering method.154a Methods are available for following stress-strain relationships with time for all kinds of polymer systems, including gels.4 Analysis of this viscoelastic behavior can lead to useful information about the network, especially when measurements are made at a series of temperatures. Interesting results have been obtained in this way for hyaluronic acid networks,Io8 including a test of the so-called entanglement hypothesis (see Section V,4a; p. 328) and an estimate of the activation energy for relaxation. Unfortunately, the internal structures of other polysaccharide systems probably change with temperature; for example, by the formation or dissociation of junction zones. This circumstance adds another variable that cannot be allowed for in the conventional treatment,"' and any parameters that are estimated must then be suspect. A promising, qualitative method for distinguishing between different types of junction178 consists in keeping the gel stretched while the decay of birefringence with relaxation of stress is followed. If the junction zones are constantly rearranging, relaxation will occur naturally by the breaking and remaking process, and lead to an isotropic gel. Birefringence decays in parallel with stress relaxation. On the other hand, if the junction zones are permanent (as in crystallites), the distortion of the network must remain, and therefore the birefringence remains. Stress relaxation probably occurs by formation of new cross-links that tend to lock the gel in its extended state. g. Junctions in the Absence of Network. - It has in some instances been possible to find experimental conditions under which polymer chains aggregate but are prevented from fonningthe three-dimensional network. This was achieved for gelatin148,178a solutions which were

(177) Ref. 4, p. 239. (178) A. V. TobolskyJ. Phys. Chem.,59,575 (1955). (178a) P. J . Flory and E. S. Weaver,J. Amer. Chem. SOC., 82,4518 (1960).

POLYSACCHARIDE GELS AND NETWORKS

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too dilute to allow network formation, and for carrageenansI7lawhich were degraded so that the chains were too short to cross-link. If it can be shown that aggregation is of the same type as in the gel junctions, for example, from similarities in optical rotational behavior,l7Ia such useful methods of polymer chemistry as the measurement of molecular weight and hydrodynamic changes can be applied in order to characterize the kinetics and thermodynamics of junctions without the usual network complications. For melting behavior with diluent, and optical rotation, were used as “fingerprints” to show that individual junctions “melt” by essentially the same process as occurs in bulk collagen. The laws that determine the bulk behavior are so well understood that a wealth of reliable information is thereby related to the gel state.

v.

POLYMER-POLYMER INTERACTIONS IN JUNCTION ZONES

1. Covalent Linkages: Sephadex and Other Examples a. Synthetic Gels.-A great deal could be written about thermodynamic properties of gels of this type, but the emphasis of this article is on the structure and conformation, and the coverage will therefore be brief. The most familiar example is Sephadex, which is used for many important biochemical separations made on the basis of size. Sephadex is made by cross-linking the chains of partly degraded dextran by a sequence of reactions with epichlorohydrin shown 0ver1eaf.l~~ Presumably, some degree of reaction also occurs by direct replacement of the chloride as the first step, and various hydrolyses are obvious side-reactions. As with so many ideas about polysaccharide gels, the possibilities of this and other reactions for introducing covalent cross-links were perceived long ago by Deuel and Neukom. 150a330 Cellulose derivatives can be cross linked with oxalyl chloride in p-dioxane solution containing a tertiary amine as the catalyst.’*’The product from cellulose acetate swells to a gel in a variety of organic solvents. (178b) P. J. Flory and R. R. Garrett,]. Amer. Chem. Soc., 80,4836 (1958). (179) P. Flodin, “Dextran Gels and Their Applications in Gel Filtration,” Pharmacia, Uppsala, Sweden, 1962. (180) H. Deuel, Helu. Chim. Actu, 30, 1269 (1947). (181) R. Signer and P. von Tavel, Helo. Chirn. Actu, 26, 1972 (1943); A. M. Rijke and W. Prins,]. Polym. Sci., 59,171 (1962).

D.A. REES

314

+

R- OH

HO’

H&,-/CH-C&Cl 0

ROCHa-CH(OH)-CH,CI

(Dextran)

HO’

+

ROCH,-CH(OH)-CHaC1-ROCHa-CH-

ROCH,CH-CH, \ /

0

+

R-OH (More dextran)

HO’

CH,

+

C1’

+

HaO

’ 0 ‘

ROCH,-CH(OH)-C&OR (Cross-linked dextran)

b. Biological Gels. -Covalently cross-linked polysaccharide chains occur in the network structure of the peptidoglycan component of bacterial cell-walls; this material is also known as murein, glycopeptide, and mucopeptide. Its structure is formally based on chitin chains that are cross linked by peptide “bridges.” Alternate residues of the polysaccharide are present as the 3-lactyl ether, and these provide the cross linkages by amide formation with the amino terminal of the peptide.182The 3-lactylated residue is known as muramic acid. The network is currently of considerable interest, because ( a ) many important antibiotic substances function by blocking its synthesis,IH3 and (b) it is the natural substrate for lysozyme, the first enzyme to have its complete tertiary structure, and probable interactions with substrate, determined by x-ray diffraction.IR4 2. Double-helix Junctions: Sulfates and Related Polysaccharides a. Carrageenans. (i) Mechanism. -To keep this discussion concise, the starting assumption will be made that a similar mechanism will explain all gels of K- and L-carrageenan with monovalent cations. Although there are differences in some gel properties (such as strength and elasticity), this assumption is justified by overriding similarities in structure, conformation, and most gel properties, and by the reasonable hypothesis that emerges. The indications that the gel is a mestastable structure imply that the free energy of activation for de(182) For a review, see H. H. Martin, Ann. Reu. Biochern., 35,457(1966). (183) J. L. Strominger, K. Izaki, M. Matsuhashi, and D. J. Tipper, Fed. Proc., 26, 9 ( 1967). (184) C. C. F. Blake, L. N. Johnson, G . A. Mair, A. C. T. North, D. C. Phillips and V. R. Sarma, Proc. Roy. SOC. (London) Ser. B, 167,378(1967).

POLYSACCHARIDE GELS AND NETWORKS

315

struction of junction zones is high (see Section IV, 1, p. 303), that is, that many forces cooperate in a specific way to hold each junction. As pointed out for gelatin,’48 the sharpness of melting point, if the Arrhenius equation is applicable, would imply so large an activation energy that the free energy of activation would not be favorable at low temperatures, and the process could not occur unless the entropy of activation were unusually high. The hysteresis behavior (see the next subsection) indicates that gelation becomes faster with decreasing temperature; such negative temperature-coefficients of reaction rate ~ e e m to ~be ~characteristic ~ ~ * ~of ~processes ~ ~ that are initiated by nucleation. All this evidence would appear to be consistent only with the view that junction formation and destruction is a “phase change” resembling protein denaturation or the melting of crystals. Similar conclusions emerge from the effect of structural variations on gel properties. The gelling tendency is extremely sensitive to polysaccharide fine-structure, suggesting that a steric “fit” is involved. The presence of as little D-galactose 6-sulfate as 1 in every 200 residues has a dramatic effect on the gel strength,58showing that a single kink, as in 14 (p. 290),can block an appreciable length of chain; this would indicate that contacts between a number of consecutive residues on each chain are necessary for gel formation. Because of these analogies with crystallization, it is logical to attempt to determine whether a reasonable hypothesis is suggested by the known manner of chain packing in the solid state (see Section III,3b (ii);p. 285). Indeed, the fibers used for x-ray diffraction studies73were prepared by very slow drying of dilute gels in the cold, and there is every possibility that the ordered structure in them grew from microcrystallite junctions. This supposition is supported by the requirements for an ordered fiber structure, which are very similar to those for gelation [see Section 111,3b (ii); p. 2861. The mechanism shown in Fig. 6 has therefore been propo~ed.7~*75 Changes in optical rotation during gel setting suggest that the two stages shown are, to some extent, consecut i ~ eA. similar ~ ~ mechanism has been suggested for gel formation by 2’deoxyribonucleic acid.Is5 Many details of this mechanism remain to be determined, but, even in schematic form, it explains several observations. Fig. 5 (see p. 289) and the accompanying text suggest that double-helix formation, and therefore gelation, would be completely blocked by the presence of D-galactose 6-sulfate or 2,6-disulfate in place of a 3,6-anhydride (1844 L. Mandelkern, Chem. Rec., 56,903(1956). (185) V. Luzzati and A. Nicolaieff,J. M o l . B i d . , 7, 142 (1963).

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316

residue, or by 2-sulfation of the 3-linked residue. The model therefore fits the facts already mentioned [see also Section III,3a (v); p. 2841. On the other hand, irregular 4-sulfation7as in K-furcellaran, or irregular 2-sulfation of the anhydride residues, as in K-carrageenans from Cigartinu species,58would not be expected to interfere sterically with Gel I (see Fig. 6), although electrostatic complications, as well as inhibition of Gel 11, might cause some modification of gel properties; again, there is agreement.

Solution

Gel I

Gel I1

FIG. 6. -Schematic Form of the Proposed Mechanism for Gelation by L- and K-Carrag e e n a n ~ . ?[The ~ . ~ ~random-coil conformation in the sol is not a necessary assumption. For the helix geometry, see Figs. 4 and 5. In “Gel I,” the chains are cross-linked by isolated double helices, whereas, in “Gel 11,” the double helices are aggregated to an unknown extent.]

(ii) Hysteresis.-This phenomenon is exhibited in a variety of physical processes, including magnetization, adsorption of gases, response of solids to stress, and solid transitions in various crystals and alloys. As was first pointed out by Everett and Whitton,lS6there are certain thermodynamic implications common to all systems, and these can be developed into a fairly general theory.IE7 A general change of any system from state A to state B will be considered first. For molecules in equilibrium, the change A + B follows the same path as B A (principle of microscopic reversibility). For some systems, however, it so happens that B + A cannot

-

(186) D. H. Everettand W. I. Whitton, Trans. Fasaduy SOC., 48,749 (1952). (187) D. H. Everett and F. W. Smith, Trans. Furuday SOC., 50, 187 (1954); D. H. Everett, ibid., 50, 1077 (1954); 51; 1551 (1955); J. A. Enderby, ibid., 51, 835 (1955); 52,106 (1956).

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be made to follow A + B in reverse. For example, a typical agar gel will “melt” when it is warmed to about 90”, but it cannot be made to set again until it cools to about 40”.It cannot then be made to “melt” again until it is warmed to 90”. No way is known by which the forward and reverse changes can be made to occur at the same temperature. The behavior is similar, though less pronounced, in carr rage en an,^^ but short, helical segments of t-carrageenani71ado not show the effect. Such phenomena are defined as hysteresis if (i) the forward and reverse paths are different, even though the changes are carried out exceedingly slowly, and (ii) all points on each path are stable and reproducible. The latter criterion distinguishes hysteresis from supersaturation or metastability. Supersaturation phenomena are not readily reproducible in detail and cannot, for example, be stopped half way through crystallization. Hysteresis is conveniently recognized by plotting scanning curves recorded by reversal of, for example, the process A + B before it has reached completion. By some suitable method of measurement, the new path is now followed back to A, where characteristic behavior is observed.Is6 The changes in optical rotation for the formation of Gel I from K-carrageenan solutions show this type of scanning behavior.75 Another example is the aggregation and disaggregation of poly(a-Lglutamic acid) in aqueous s ~ l u t i o n . ~The ~ * ~general ’~~ explanation requires a free-energy surface of the type186shown in Fig. 7 . In discussing this Figure, pairs of polysaccharide chains in three states will be considered: the random-coil conformations (C), the double helix (D), and the transition state (A), which is visualized as a partly formed double-helix. The configurational entropies lie in the order C > A > D, and the “melting” of the gel (D + A + C) and setting (C + A + D) follow the farthest and nearest arrow, respectively, in Fig. 7 , corresponding to changes at high and low temperature. For simplicity, the free energies of C and D are shown relative to A at each temperature, and the change of free energy of A with temperature then appears as a horizontal ridge parallel to the temperature axis. At high temperatures, the TAS contribution ensures that C shall have the lowest free-energy, but this term diminishes with decreasing temperature until D represents the minimum. Thus far, the free-energy changes are analogous to those of a melt undergoing the process of crystallization. With cooling of the solution, a temperature is eventually reached at which the relative free(188) B. R. Jennings, G . Spach, and T. M. Schuster, Biopolymers, 6,635(1968). (189) G.Spach and D. Constantin, Biopolymers, 6,653(1968).

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FIG. 7. -Schematic Free-energy Surface for the Melting and Setting of Carrageenan and Agar Gels. [The system follows the path shown by the arrows. Axes are labeled G (relative Gibbs free-energy), S (entropy), and T (temperature). Note that it is not suggested that the absolute free-energy of any species increases with the temperature, nor that the entropy of the transition state is constant with changing temperature (see the text). The model was constructed by Mr. F. B. William~on.'~]

energies of C and D should allow of a distribution of molecules between the two states, but this is prevented by the height of the activation barrier. The reason for this situation is, evidently, that the transition state requires a conformation that is ordered to an extent that is impossible at that particular temperature; whereas the corresponding and greater disadvantage of the double helix is offset by

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3 19

forces of attraction, it is accessible only through the transition state. With further cooling, owing to the changing TAS term, the free energy of C approaches that of A, that is, the system follows the arrows up the “valley” on the right. A point is eventually reached at which the barrier can be surmounted, but the free-energy difference between C and D then favors D so overwhelmingly that C + D occurs to a high degree of completion. Reheating now takes the system up the “valley” on the left, but, again, the distribution between D and C might not be controlled by the relative free-energies, because of the activation barrier. The explanation is now complete, but three further points must be made for accuracy. Firstly, because of random motion and the properties of the developing network, some pairs of molecules will pass over the barrier before others; and, in the reverse process, helices of different sizes, which are tied into the network in different ways, would be expected to “melt” at different temperatures; these correspond to the different “domains” that are necessary to an explanation of the general properties of hysteresis.186Secondly, it is strictly necessary for only one of the transitions to be controlled by the activation barrier, but the conclusion cannot be escaped that at least one of them is a spontaneous process and, therefore, thermodynamically irreversible. It is likely that helix formation only is kinetically controlled, because it is essentially a one-dimensional crystallization; supercooling and supersaturation occur commonly in crystallization, and show that this type of system can persist in nonequilibrium. On the other hand, helix “melting” is a special case of polymer-crystal melting like the melting of other crystals,189ais expected to be an equilibrium process. Thirdly, the effect of changing the temperature is not merely to alter TAS $ in direct proportion, because transition-state theory shows that

kT

-AGOWIT

Rate constant = - e

h

kT -- h e

AS”1IR -AH”t/RT

e

where k = the Boltzmann constant, h = Planck‘s constant, and R = the gas constant. The explanation of hysteresis as a kinetic effect requires that the rate constant for helix formation shall increase with decrease in temperature. Were T the only variable in the equation, both of the temperature-dependent terms would produce an effect (189a) A. R. Ubbelohde, Quart. Reo. (London), 4, 356 (1950).

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opposite to that observed, from which it must follow that there are changes in AH”* and/or ASS. These occur because the helix length in the transition state, defined as the point of maximum free-energy as the helix winds up, will vary with temperature, and cause the corresponding thermodynamic quantities to alter as well. Such processes, in which molecular organization “grows” from nuclei that may themselves appear by chance, are characterized by negative temperature-coefficients of reaction rate.184aThe formation of the 2’-deoxyribonucleic acid d o u b l e - h e l i ~and ~ ~ ~the ~ collagen triplehelix,178aand the general phenomenon of crystallization, all show this behavior. Although it is known75that hysteresis is associated with the formation and dissociation of carrageenan helices, rather than their aggregation (which is a separate stage of the sol-gel transformation), it is not yet known whether the network as a whole contributes to, or modifies, the phenomenon. There is, indeed, no proof that the network is not primarily responsible, although this would seem unlikely, because it is so much easier to see how helix formation could provide the thermodynamic requirements. Hysteresis becomes less marked from agarose, through K-carrageenan, to L-carrageenan, suggesting that the coil-to-helix transition becomes less cooperative in that order; this would be consistent with the relative sharpness of the corresponding changes in optical r ~ t a t i o n . ~ ~ * ’ ~ ~

(iii) Conclusions.-The double-helix m e c h a n i ~ m ~ would ~ . ’ ~ seem to fit well with all of the known facts, but, as with mechanistic studies in almost any area of chemistry, it would be desirable to obtain further proof and to fill in more details. It is not yet known what proportion of each chain is involved in the double helix, and methods are therefore needed for measurement of polysaccharide “helix contents.” Potassium salts of sulfuric esters are, in general, more readily desolvated than sodium salts1so;the reason is not yet understood, but the behavior does suggest that cation selectivity is not to be explained entirely by the geometry of the helix. Any mechanism for “winding up” a double helix would require a free chain-end, and suggests why these polysaccharides are such spectacular gel-forming agents: if (189b) J. Marmur and P. Doty, /. Mol. Biol., 3,585 (1961);J. G . Wetmur and N. Davidson, ibid., 31,349 (1968). (190) 0. Smidsred and A. Haug, J . Polym. Sci., Pt. C , 16, 1587 (1967); T. J. Painter, PTOC.Znt. Seuweed Symp. 5th, 1966, p. 305.

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32 1

“crystallization” must start from the chain ends, a tangled network, not a precipitate, is likely to result if the solution is sufficiently concentrated. Although two chains, paired in a double helix that includes the whole length of each, could make no contribution to the network, this possibility is prevented by the presence of a few kinks in each molecule (p. 290). The requirement that chains must have the same sense means that any intramolecular, helix formation must result in a loop that would hook around other chains, instead of a polymer single-crystal or a narrow “hairpin.” The natural occurrence of 3,6-anhydrogalactose is restricted to gel-forming polysaccharides, and the residue is, indeed, the only natural sugar anhydride. It was so unexpected that some early workers were reluctant to accept it as anything other than a chemical artifact. We now suggest that it occurs to fulfill a particular biological function: only by the influence of such a fused ring can a sugar ring be so constrained as to have three equatorial C-H bonds, an arrangement that increases the flexibility of the chain’l to allow winding and unwinding of the double helix.

b. Agars. -The mechanism of gelation by agarose is as yet unknown, but there are many indications that it could be similar to that for carrageenans. The polysaccharide structures are quite similar, and conformational analysis shows that the changed configuration of the 3,6-anhydride residue need not fundamentally alter the polysaccharide stere~chemistry.~~ Model building with the computer shows that double helices can be formed.75During gelation, changes in optical rotation occur that are similar to those for the carrageenans, although, interestingly, they have the opposite sense.’70Agar gels also show similar, although more marked, hysteresis behavior. Several gel properties follow a very clear trend in the order of decrease in sulfate content: L-carrageenan, K-carrageenan, K-furcellaran, agarose (see p. 282). That agarose fits into this series with the 3,6-anhydro-~galactose polymers suggests that the mechanism is similar. Since gel rigidity is related to the proportion of cross links,2 and increases in this order, it would appear that helix formation is progressively inhibited by an increasing number of sulfuric ester groups and by their interactions with cations. The secret of helix stability must, therefore, lie elsewhere, but a detailed discussion is impossible here for lack of space. c. Amino Polysaccharides. -These networks have properties quite different from those of carrageenans; for example, they flow. The

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usual e x p l a n a t i ~ n ~in~ .terms ~~ of entanglement couplings,191 is based on the extended conformations of protein-polysaccharides and hyaluronic acid in s ~ l u t i o n . It ’ ~is~imagined ~~~ that, beyond a critical concentration, each chain is forced through the domains of others, to give a tangled network. However, solutions of hyaluronic acid do not show the viscoelastic behavior that characterizes entanglement coupling in synthetic-polymer systems, and the possibility of more specific cohesion between chains should therefore be reexamined.Io8 Double-helix cross-linkages seem feasible on stereo, ~ ~explain the network properties, these chemical g r o ~ n d s . ~ ’To linkages would have to form, and to dissociate, more readily than those in carrageenans; this might be possible if cooperative effects were less pronounced (compare p. 320). For protein-polysaccharides, it is possible to imagine interactions that involve the polypeptide, rather than the polysaccharide, chains. After isolation, protein-polysaccharides from bovine nasal cartilage undergo dissociation-reassociation reactions that probably represent the breaking and re-making of native interactions; these seem to be through linking glycoprotein molecules, and to involve disulfide bonds.Iosa

d. Cellulose Sulfate. -These gels show many similarities to those of carrageenans, including rigidity, sharp “melting points,” and sensitivity to KO, but exhibit little, if any, hysteresis. Conformational analysis3] (see Section 111,lb; p. 272) shows that the double-helix mechanism is extremely improbable, because the chain is sterically prevented from deviating far from a ribbon conformation as in 1(see p. 273). This conclusion is well shown by the excellent (and expensive!) stereoscopic illustration published in the Journal of Biological of a space-filling molecular model of a hexamer having each linkage in the conformation that has been proved for the cellobiose crystal. A tentative hypothesis is that the more regularly sulfated parts of different ribbons pack with cations, in a lattice similar to that depicted in Fig. 3 (see p. 276), to form junction zones. These regions could well approximate to complete sulfation, because the degree of substitution is so high (3 2.2) that some such regular patches must exist. An important part of the driving force would be the relief of electrostatic repulsion within each chain, achieved by (191) R. S . Porter and J. F. Johnson,Chem. Reo., 66,1(1966). (192) M. Luscombe and C. F. Phelps, Biochem.)., 102,110 (1967). (193) R. A. Harte and J. A. Rupley,]. Biol. Chem., 243,1663 (1968).

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packing with cations. The specific action of KO on this system,22as well as on K-carrageenan, would seem to indicate that the effect does not depend on a particular type of junction, but reflects a more general property of the cation.’g0

e. Biological Aspects.-There is some evidence that suggests that the blocking action of 6-sulfate residues on carrageenan helixformation [see 13 and 14 (p. 290) and the accompanying text] is part of a biological mechanism for controlling network properties. For the agar series, it is known that the natural 6-sulfate content alters in a way that can be correlated with environmental demands on the organism, and, hence, on the functions of the biological gel.40 An enzyme has been characterized that converts L-galactose 6-sulfate residues into 3,6-anhydride residues within the polysaccharide chain. A similar “de-kinkase” has been detected for the carrageenan series,lS5and the well known variations in content of 6-sulfate probably reflect end-products that have been exposed to the enzyme to different extents.58It follows that, in these systems, the polysaccharide conformation is under metabolic control. 3. Microcrystallites: Glycuronans and Derivatives

A central problem with these polysaccharides is the establishing of the nature of their interactions with divalent cations. Reasons have already been given (see Section IV,l; p. 303) for dismissing the popular hypotheses of cross linking by simple electrostatic attraction, and simple chelation; these views will not, therefore, be discussed in this Section. It is not suggested that such effects have no role whatsoever in junction zones, but merely that, as explanations, they are insuficient. The problem will be re-examined in Section V,4 (see p. 330), and reasons will there be given for the uncompromising attitude now taken. a. Alginate and Pectate Gels Having Divalent Cations.-A discussion of Ca2@gels is necessary, because they are the best characterized, and, apart from reservations to be added in Section V,4 (see p. 330), there is little reason to suspect that they are atypical. Such gross properties as strength and t e ~ t u r e ’ ~ and ~ Jappearance ~ (194) D. A. Rees, Biochern.J., 81,347 (1961). (195) C. J. Lawson and D. A. Rees, unpublished results. (196) R. H. McDowell, Ref. 16, p. 19.

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under the polarizing microscope151adepend on the method of preparation of the gel, proving that gels are not equilibrium states. This conclusion immediately suggests that the junction zones are microcrystallites or a related type (see Section IV,l; p. 303); the presence of x-ray crystallinity after gentle drying of the gel,Ig7and the persistence of the same pattern of birefringencelSladuring preparation by slow diffusion, would both be consistent with this possibility (compare, Ref. 178). Increasing proportions of Ca2@added to pectates and alginates cause contraction of the gel volume1g8or an increasing tendency to exhibit s y n e r e s i ~ , ~indicating ~J~~ that the framework becomes more and more tightly linked. The end products are the precipitated calcium salts, in which there is evidence (obfor crystallinity. The process is, tained by x-ray diffraction122,200) therefore, interpreted as being a progressive increase in the number and, perhaps, the size of microcrystallites, leading eventually to a tangled, partly crystalline mass of chains. Gels prepared by diffusion show fine structure, not only in their birefringence but also in a system of pores that runs through the gel; the pores are detectable with the microscope and by light-scattering.1s1a,1s4 They probably represent pockets of solvent that had been stripped from the chains when they packed together. The effect of structural variation is also consistent with a microcrystallite hypothesis. Gelation with Ca2@is prevented by partial acetylation of alginate201and pectate,202no doubt because it is impossible for irregularly substituted chains to pack into a regular, and therefore strong, lattice. It is concluded that the balance of evidence is overwhelmingly in favor of microcrystallite junction-zones in these gels. Whether or not they are the only type of junction will be discussed in Section V,4b (see p. 330), but, almost certainly, they are the most important type. Further advances will probably follow after solution of the crystal structures of calcium alginate and calcium pectate, or, perhaps, of related oligomers, by x-ray diffraction studies.

b. Pectin-Sugar Gels.-The possibility of a specific role for the sucrose component (such as formation of hydrogen-bonded bridges (197) C. Sterling, Biochim. Biophys. Acta, 26, 186 (1957). (198) 0. SmidsrZd and A. Haug, Acta Chem. Scand., 19,329 (1965). (199) G. L. Baker and M. W. Goodwin, Delaware Agr. E x p . Sta., Bull. 246, Techn. No. 31 (1944). (200) F. A. Bettelheim and D. H. Vo1rnan.J. Polym. Sci.,24,485 (1957). (201) R. G. Schweiger,J. Org. Chem.,27,1789 (1962);Kolloid-Z., 196,47 (1964). (202) J. Solms and H. Deuel, Helu. Chim. Acta, 34, 2242 (1951); R. G. Schweiger, J. Org. Chem., 29,2973 (1964).

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325

between pectin chains) can immediately be rejected, because other solutes, such as glycerol and ammonium ~ u l f a t e , ’ ~ ~can . ’ ~ be ’ used instead. It would, in any case, seem unlikely from first principles (see Section IV,l; p. 303). Because the effect is so general, its origin can be traced to the decreased activity of water (in the thermodynamic sense) in the presence of any dissolved substance. The water is then less able, or “less free,” to solvate the polysaccharide chains, and any tendency for them to separate from solution (to form junction zones, for example) is enhanced. The same explanation can be put in other terms. In the jargon of polymer chemistry,203aqueous sucrose is apparently a “poorer” solvent than water for pectin; or, in terms of osmosis, the network is unstable, and therefore cannot form in the absence of sucrose, because it could not withstand the osmotic pressure of the pure solvent. The Ca2@and H@that are normally added to commercial gels must play only a secondary role, because gelation can occur without them if esterification is c0mp1ete.l~~ Gels are not formed if pectin is converted into the ethyl or 2-(hydroxyethyl) e ~ t e r ,showing ~ ~ ~ *that, ~ ~in~ the junction zones, there is a steric fit between methyl D-galactopyranosiduronate residues that can be prevented by larger groups. One acetyl group on every eighth residue is sufficient to diminish the gelling ability dra~tically,’~~ and this fact suggests, as with the effect of 6-sulfate on carrageenan gels (already mentioned on p. 315), that several consecutive residues on each chain are involved in the contact. Closer analogy with carrageenan gels is unlikely, because of the different conformations of the two polysaccharides (see p. 302), although it is interesting that K@ has a gel-promoting effect on pect i n that ~ ~ is~ similar ~ to, although less pronounced than, that on carrageenans. Some type of intimate packing of two or more pectin chains is certainly involved, however, and, for want of a better word and more evidence, it is included in the vague group of “microcrystallites.” The presence of nonesterified residues would destabilize the association by electrostatic repulsion and a greater preference for solvent, unless ionization were suppressed, or suitable cations were included in the lattice. This hypothesis would partly explainzo4the role of H@,Ca2@,and other cation effects’47in commercial gels. The Ca2@need not occupy fixed sites, because examples are known (for example, 2’-deoxyribonucleic acid205)in which ordered crystallites (203) W. Banks and C. T. Greenwood, Adoan. Carbohyd. Chem., 18,357 (1963). (204) C. L. Hinton, Biochem. /., 34, 1211 (1940); H. G. Harvey, /. Sci. Food Agr., 1, 307 (1950). (205) W. Fuller, M . H. F. Wilkins, H. R. Wilson, L. D. Hamilton, and S. Amott, J . MoZ. B i d , 12,60 (1965).

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are formed from polyelectrolyte chains while the counter-ions are mobile in the interstitial water. In addition, calcium pectate junctions, and junctions formed by microcrystallites of pectic acid,lg7 are more likely to be formed as the degree of esterification diminishes. c. Biological Gels.-Pectic substances in the gel state are important components of many cell walls, from the first stages of formation to the start of secondary thickening. The rather fluid partition that first separates the daughter cells after division, known as the cell plate, would, from staining properties appear to contain pectic substances.2wThe cell wall grows from this cell plate by addition of cellulose, more pectic substances, and other polysaccharides.2w~20‘ During subsequent enlargement of the cell, the wall is stretched at a rate that is probably controlled, in part, by the texture of the pectic gel,s~2w~207 Cell-wall gels in which galacturonan microcrystallites may be detected by x-ray diffraction are found in celery-stem tissue.208 These walls have a supporting function, and the presence of a strong network is, therefore, to be expected. On the other hand, it is commonly found that the pectic substances in tissues having a potential for rapid growth have a high proportion of L-rhamnose residues in the backbone and are highly b r a n ~ h e d . ’ ~ ~ Typical *’~~*’~~ side-chain structures are shown in Table I1 (see p. 300). The L-rhamnose residues no doubt represent “kinks” that prevent alignment of chains, and regular packing is also hindered by the side chains. The pectic substances in these walls would therefore be expected to form viscous solutions (instead of firm gels), thus allowing the necessary rapid expansion of the cell volume.’3s Similar structures are found in the mucilages (see This Volume, p. 369) that occur in the slime cells of certain seeds.209It would appear that the galacturonan backbone is here modified to an even greater extent, so that it cannot form a coherent, pectic matrix in the wall; in water, it swells, disrupts, and is exuded as a slimy mucilage. The biological function is at present obscure, but it might, in part, serve as a water reservoir for the germinating seed. The association with cellulose is evidently intact. It has long been known that these (206) A. Frey-Wyssling and K. Muhlethaler, “Ultrastructural Plant Cytology,’’ Elsevier, Amsterdam, 1965. (207) K. Wilson, Intern. Reu. Cytol., 17, 1 (1964);P. A. Roelofsen, Adoan. Bot. Res., 2,69(1965). (208) P. A. Roelofsen and D. R.Kreger,]. E x p . Bot., 2,332(1951). (209) A. Tschirch, “Angewandte Pflanzenanatomie,” Urban and Schwarzenberg, Wien, 1889,Vol. I, p. 193.

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mucilages give a cellulose-like glucan on acid or alkaline hydrolysis, and that they contain microfibrillar material resembling cellulose.210 The presence of crystalline cellulose IB in mustard and cress-seed mucilages has now been confirmed by x-ray diffraction and deuteration-infrared techniques, including use of the characteristic cellulose I + cellulose I1 transition and the infrared dichroism of certain bands in the spectrumz1' of cellulose I. Electron microscopy, with negative staining, has revealed thread-like structures having apparent dimensions similar to those of cellulose microfibrils (358, diameter). Ultracentrifugation shows a main component that is sedimented as a sharp band at about the same rate as a small virus, and it can be purified in the preparative ultracentrifuge. It contains about 50 per cent of cellulose, and the acid hydrolyzate contains most of the sugar components typical of pectic substances. Reagents (such as urea and potassium thiocyanate) that are unlikely to affect any covalent bonds are able to break associations between the cellulose and the solubilizing polysaccharide. It would seem that cellulose microfibrils, singly or in small groups, are held by secondary-valence forces within a sheath of hydrophilic gel that is responsible for the solubility in water. These aggregates are units of cell-wall structure, and should be valuable models for the association between cellulose and the pectic matrix in normal cell-walls.zll Alginates and pectates in the gel phase show differences in selectivity to cations that might be related to biological function; for example, the selective binding of Ca2@ions is much more marked for galacturonic and guluronic than for mannuronic acid polymers.211a 4. Entanglement and Shared Counterions:

0-( Carboxymethy1)cellulose All of the gels so far described have involved the interlocking of chain segments in microcrystallites or similar aggregates. Such junction zones account admirably for the typical properties of gels (see Section IV,l; p. 303), but the likelihood of other possibilities will next receive attention.

(210) K. Muhlethaler,E r p . Cell Res., 1,341 (1950). (211) G. T. Grant, C. McNab, D. A. Rees, and R. J. Skerrett, Chem. Commun., 805 (1969). (211a) 0. Srnidsr@dand A. Haug, Actn Chem. Scand., 22, 1989 (1968); R. Kohn, I. Furda, A. Haug, and 0.Srnidsr@d,ibid., 22,3098 (1968).

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a. Sodium 0-(Carboxymethy1)cellulose.-The physical properties of these derivatives depend on the degree of substitution and on other variables in their structure. From the present point of view, relatively concentrated dispersions (1-4 %) of products, made by the commerical method, having degrees of substitution in the range of 0.4to 0.8 are the most interesting systems. The properties can be interpreted, some of them quantitatively,212in terms of a network in which the molecules are joined by noncovalent cross-links. The dispersions are typically thixotropic, that is, destruction and formation of the network are brought about by mechanical agitation or its absence, instead of by changes in temperature. This behavior is quantitatively characterized by measurements that show that the apparent viscosity diminishes with the rate of shear and with the length of time that shear is applied.213 Hence, the network breaks down progressively as the applied shear is increased or is continued in time; it re-forms, usually during several hours, when the shearing is stopped. These properties have beell explained213in terms of the residual crystallinity that, from x-ray diffraction s t ~ d i e s , ' ~is' known to survive the (carboxymethy1)ation of cellulose. Bundles of chains (see Fig. 8) therefore exist. It is to be expected that some chains extend through

FIG. 8.-Schematic Drawing of the Simplest Type of Gel Center in 0-(Carboxymethyl)cellulose.2'3[Chains are kept in a bundle by a crystalline region that survives from the native cellulose. Larger gel-centers may have chains that pass through more than one crystalline region and thereby link them together.] (212) J. Hermans,J. Polym. Sci., Pt. A, 3,1859 (1965). (213) E. H. deButts, J. A. Hudy, and J. H. Elliott, Ind. E n g . Chem.,49,94(1957).

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more than one bundle, and thus hold larger aggregates together. Free chains, and parts of chains, are more highly substituted than the average,I6 presumably because certain segments within the bundles are to some extent protected from substitution. It would seem that the more efficiently these bundles or “gel centers” are dispersed in the sol, the stronger is the network that is subsequently formed; this is shown by comparison of the results of different methods of agitati or^,^^^ and of the properties in mixed solvents that have been added in different sequences.214When dispersion has been particularly efficient, the gel may show a fairly distinct yield-point, and be strong enough to support its own eight.'^,^'^ The gel centers evidently have some ability to recombine after dispersion, presumably by interaction between the “loose ends” (see Fig. 8). The more efficient their dispersal, the greater the chance of formation of a continuous network, instead of lumps of insoluble material or islands of network that cohere weakly; the effect of the history of the material is, therefore, understandable. Microcrystallite formation between “loose ends” of different gel-centers would seem to be an unlikely source of cohesion, because of ( a ) the ease with which the network structure is broken by shear213 and ( b ) the weakening, not strengthening, effect of salts that should stabilize any ~ r y s t a l l i t e s .The ~ ~ ~chains are kept extended by steric forces (see Section 111,lb; p. 272) and, in the absence of added salts, by electrostatic repulsion. The “loose ends” that spread out of adjacent gel-centers are forced to interpenetrate each others’ domains (and those of free chains in solution), and the presence of a network structure shows that energy will then be needed in order to pull them apart. It is unlikely that this situation is primarily the result of attraction between the chains, because added salt, which would diminish the electrostatic repulsion (and thus enhance the resultant attraction), does not strengthen the network.214 The alternative is that the chains are difficult to pull apart because of the presence of an activation barrier that has to be surmounted: atoms and groups, some of which have like charges, must be squeezed past each other in separating the chains. This explanation would seem the most likely at present. The gradual increase in the strength of the network on standing is, then, interpreted as being the result of increasing interpenetration brought about by thermal motion. The hysteresis behavior seen during formation and destruction of the network by shear““ is further evidence for such an activation barrier (compare Section V,2a (ii); p. 316). (214) P. S . Francis,]. A p p l . Polym. Sci., 5,261 (1961).

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b. 0-(Carboxymethy1)cellulose with Multivalent Cations.-The dispersions can be made to form fairly stiff gels if such multivalent cations as Fe3@,AP@,Cu2@,and Ba2@are added.16,213*214 It is unlikely that any regions of the randomly substituted 0-(carboxymethy1)cellulose chain are sufficiently regular to form additional microcrystallites with these cations, and the effect is so general that it seems improbable that any other type of specific interaction is responsible. Arguments against formation of networks by simple electrostatic “bridges” and simple chelation have already been given (see Section IV,l; p. 303). The behavior could be explained by the polyelectrolyte character of 0-(carboxymethy1)cellulose;this arises because the covalent structure of each chain holds together anionic groups that would otherwise separate by repulsion. In a typical polyelectrolyte, the sum of such repulsions is so unfavorable that, from the point of view of free energy, it is worth while to offset it by the entropically unfavorable localization of cations within the polymer domain. Measurements show that 20-80% of the counterions may be “fixed” in this way, and that binding of di- and tri-valent cations is stronger than that of univalent cations.215In the simplest case, counterions within the domain are held only by electrostatic attraction and are in dynamic equilibrium with those outside, continually passing to and fro. In 0-(carboxymethy1)cellulose dispersions, chains that pass through each others’ domains will share a common “atmosphere” of counterions; this will add to the tendency for the chains to remain in the neighborhood of each other, especially when counterion binding is enhanced by the addition of multivalent cations. In addition, it is very probable that cations within the domain will, to some extent, exchange water of hydration for carbohydrate hydroxyl groups, and some temporary cross-linking of chains may occur by this type of “chelation.” Even though individual cations exchange constantly with bulk solution, there are so many in the “atmosphere” that enough of them remain to maintain the cohesion at any instant. No individual cation can hold the chains together permanently, because its lifetime in the polymer domain is short, and this mechanism is therefore quite different from the simple ionic bridge or simple chelation model. This distinction is insisted on, because polyelectrolyte theory can lead to important predictions concerning, for example, the effect of a change of distribution of anionic groups (215) S. A. Rice and M. Nagasawa, “Polyelectrolyte Solutions,” Academic Press Inc., New York, N. Y., 1961.

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33 1

along the polymer chain. These predictions do not emerge from the simpler theories, which are therefore sterile, as well as inexact. Polyelectrolyte effects are probably also involved in alginate and pectate gels (see Section V,3a; p. 323), but the balance of evidence is that they are less important than the formation of junction zones by microcrystallites.

5. Micelle Junctions: 0-Methylcellulose Gelation does not occur with 0-methylcellulose that has been prepared by homogeneous methylation. It would seem necessary that the substituents be distributed unevenly, as they are in the usual commercial products (see Section 111,l; p. 271). The tendency to gel is enhanced by increasing the proportion of di- and tri-0-methyl-Dglucose residues,216suggesting that highly substituted regions of 0-methylcellulose chains form the junction zones. Those parts that have relatively few substituents are, presumably, necessary in order to confer solubility in water. The important clue to the mechanism is that gels are formed on heating, and “melt” on cooling, instead of the reverse. The formation and dissociation of small aggregates show similar temperature behaviorz1; this is attributed to a weakening of the structural integrity of the solvent and of its ability to hydrate the substituted chains as the temperature is raised. Such behavior is well known for aqueous solutions of certain types of solutes known as “hydrophobic” solutes.z17They cause a structuring of liquid water which collapses when the solute aggregates or forms a separate phase, so that the overall change in entropy is positive. One example is the formation of detergent micelles from isolated chains,z18and many others are important in biological systems.z19Another mechanism could be that ether oxygen atoms in densely substituted parts of 0methylcellulose, being correctly spaced,zz0promote an ordered hydrogen-bonding in the neighboring water by participating in it. This water is more weakly hydrogen-bonded than it would be to hydroxyl groups, and is entropically unfavorable. The cellulose chain is S O stiff that conformational entropy is not an important term in favor of the solution.80The result is that the overall TAS term is against (216) (217) (218) (219) (220)

A. B. Savage, Ind. Eng. Chem., 49,99 (1957). F. Franks and D. J. G. Ives, Quart. Reo. (London),20,1(1966). E. D. Goddard, C. A. J. Hoeve, and G. C. Benson,J. Phys. Chem., 61,593 (1957). G . Nbmethy, Angew. Chem. Intern. Ed. Engl., 6,195 (1967). F. Franks, Chem. Ind. (London), 560 (1968).

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the dissolved state, and, as it is only opposed by weak hydrogenbonds, chains aggregate as the temperature is raised. In either case, the main driving-force is a tendency for the solute to be expelled from contact with water, rather than any strong attraction between the solute molecules themselves, so that a precise steric fit is not required in the aggregate; for example, the interior of a detergent micelle would appear to be liquid.221 Absolute stereochemical regularity should not, therefore, be necessary for the formation of 0-methylcellulose junctions. The replacement of an occasional methoxyl group by a hydroxyl or a hydroxypropyl group need not have the catastrophic effect that would be expected were the mechanism to involve crystallization, although interference would occur to some extent, because these groups have some degree of hydrophilic character. 0-Ethyl- or 0-methyl-0-ethylcellulose should show behavior similar to that of 0-methylcellulose. As far as is known to the writer, these predictions are confirmed by industrial experience," and it is concluded that a micelle model is appropriate for 0-methylcellulose junctions.

(221)

J. Clifford, Trans. Faraday Soc., 61,1276 (1965).

GUMS AND MUCILAGES BY G. 0. ASPINALL Department of Chemistry, Trent University, Peterhoroug.., Ontario, Canada

I. Introduction..

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

333

11. Fractionation and Isolation of Structurally

Homogeneous Polysaccharides .................................... .334 111. Methods of Structural Investigation .................................. 337 IV. The Galactan Group of Polysaccharides ............................. .341 1. Acacia Gums ...................................................... 343 349 2. Mesquite Gum ................................................... 3. Other Gums of the Galactan Group ................................ 351 V. The Glucuronomannan Group of Polysaccharides ..................... 354 VI. The Galacturonorhamnan Group of Polysaccharides ..................... 361 1. Tragacanthic Acid ................................................ 36 1 2. Khaya G u m s . . .................................................. 363 365 3. Sterculia Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Acidic Mucilages and Associated Neutral Polysaccharides . . . . . . . . . . . . 369 VII. The Xylan Group of Polysaccharides .................................. 371 VIII. The Xyloglucan Group of Polysaccharides ............................ 372 IX. Other Polysaccharides .............................................. 375 X. Conclusions ......................................................... 376

I. INTRODUCTION A Chapter in one of the early Volumes of this Series’ reviewed the chemistry of gums and mucilages from both land plants and seaweeds. A comprehensive monograph2 published in 1959 gave an excellent account of these substances, with exhaustive literature coverage up to 1957-58. A multi-author book published at about the same time reviewed the physical properties both of natural gums and gums formed by chemical modification of starch and cellulose in relation to their industrial uses.3 The present Chapter is concerned primarily (1) J. K. N. Jones and F. Smith, Aduan. Carhohyd. Chern., 4,243 (1949).

( 2 ) F. Smith and R. Montgomery, “The Chemistry of Plant Gums and Mucilages,” Reinhold Publishing Corporation, New York, N. Y., 1959. (3) “Industrial Gums,” R. L. Whistler, ed., Academic Press Inc., New York, N. Y., 1959. 333

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with developments since about 1958, and deals mainly with the exudate gums, although some consideration is given to the acidic polysaccharides and associated neutral components of the seed mucilages from higher plants. Neutral mucilages of the galactomannan and glucomannan types were discussed in reasonable detail in Smith and Montgomery’s monograph,2and, in relation to the hemicelluloses, in a previous Chapter in this Series4 The seaweed mucilages form a rather special group of polysaccharides, and a monographs has reviewed the literature up to approximately 1966. The structures of the acidic polysaccharide components of gums and mucilages are all highly complex, but, as far as possible, they will be classified on the basis of those sugar residues which comprise the interior chains of the molecular structure. In this way, polysaccharides of diverse origins may be considered together, and the structural relationship of the exudate gums and seed mucilages to other groups of plant polysaccharides may be stressed.6 At the same time, it should be borne in mind that present knowledge of the detailed structures of certain polysaccharides is not yet sufficient to permit an unambiguous structural classification of all of those discussed in this article. Furthermore, new structural groups of polysaccharides may be recognized, and entirely different bases for structural classification may be required. 11. FRACTIONATION AND ISOLATION OF STRUCTURALLY HOMOGENEOUS POLYSACCHARIDES

The general problem of isolating chemically homogeneous polysaccharides has been discussed in a previous Chapter in this Series.’ Our attention here will be directed to certain facets only of this problem. In several earlier investigations: failure to effect further changes in chemical composition and physical constants on repeated reprecipitation of polysaccharide preparations was taken as constituting sufficient evidence of chemical homogeneity to warrant starting structural investigations. In a surprising number of cases, the isolation of gum components in this way has been shown to give polysaccharides that, by more rigid criteria, actually are homogeneous. (4) G. 0.Aspinal1,Adoan. Carbohyd. Chern., 14,429 (1959). (5) E. Percival and R. H. McDowell, “Chemistry and Enzymology of Marine Algal Polysaccharides,” Academic Press, London, 1967. (6) G. 0.Aspinall, Pure A p p l . Chern., 14.43 (1967). (7) W. Banks and C. T. Greenwood, Adoan. Carbohyd. Chern., 18,357 (1963).

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Increasingly, ultracentrifugation, electrophoresis, and chromatography have been used; and, ideally, as many independent criteria as possible for homogeneity, or more strictly against heterogeneity, should be used. It may be noted that, in principle, ultracentrifugation may indicate differences in molecular size and shape of the components of a polysaccharide preparation, instead of differences in chemical structure. In practice, however, for acidic polysaccharides, the reverse problem may be encountered, as molecular aggregation may result in spuriously sharp peaks.' Although a powerful technique, boundary electrophoresis involves very expensive equipment. Provided that suitable methods are available for the detection of polysaccharides, it is possible that zone electrophoresis (for example, on cellulose acetate stripss) may prove to be equally sensitive. Ion-exchange chromatography, especially on 0-(2-diethylaminoethyl)~ellulose,~ has proved to be an extremely powerful analytical procedure for the determination of chemical homogeneity of acidic polysaccharides. In principle, this technique may be extended to preparative-scale fractionations,'0 but practical difficulties in removing large quantities of inorganic salts used in eluting buffers have so far limited its use. 0-(2-Diethylaminoethyl)-SephadexA-50 is an ion-exchanger of higher capacity than the cellulose derivative, although it suffers from the disadvantages of "bleed" from the support, and of undergoing marked changes in volume with changes in solvent or buffer concentrations. Some experiments using aqueous formic acid as the eluting solvent have indicated that the ion-exchanger may prove to be a useful support for the preparative separation, with a minimum of experimental difficulties, of acidic polysaccharides containing less than about 20 % of ionizable hexuronic acid residues."J2 Although gel-permeation chromatography has been used to provide estimates of the molecular size of polysaccharides and their derivat i v e ~ , ' no ~ examples have as yet been recorded of fractionations achieved by this procedure. Several gums and mucilages are known to contain more than one polysaccharide component. Fractionations have been achieved by fractional precipitation from aqueous solution with a water-miscible (8) C. T. Bishop and W. F. Dudman, Can./. Chem., 46,3079 (1968). (9) H. Neukom, H. Deuel, W. J. Heri, and W. Kiindig, Helo. Chim. Acta, 43, 64 ( 1960). (10) H. 0.Bouveng, Acta Chem. Scand., 19,953 (1965). (11) G. 0.Aspinall and J. P. McKenna, Carbohyd. Res., 7,244 (1968). (12) G. 0.Aspinall, J. W. T. Craig, and J. L. Whyte, Carbohyd. Res., 7,442 (1968). (13) D. M. W. Anderson and J. F. Stoddart, Curbohyd. Res., 2,104 (1966).

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non-solvent, or, more satisfactorily, by the selective precipitation of the more highly acidic component as cupric or cetyltrimethylammonium salts. Gum tragacanth14-17and Khaya senegdensis g ~ m ~ ~ * provide examples of gums that contain two structurally different types of polysaccharides. The close association of quite different polysaccharides is also encountered in some of the seed mucilages, for example, those from linseed (flax seed)20s21and cress seed^.^^.^^ In some cases, the fractionation of water-soluble polysaccharides is complicated by the presence of colloidally dispersed cellulose. In mustard-seed for example, cellulose I is present and is probably bound to associated polysaccharides by secondary valence forces rather than covalent bonds. In contrast to those gums and mucilages in which polysaccharides of entirely different structural types co-occur, Anogeissus leiocarpus (formerly A. schimperi) gum contains two distinct but structurally related polysa~charides.~~-~’ The presence of two polysaccharide components, which was not recognized in an earlier was demonstrated by chromatography on 0-(2-diethyIaminoethyl)cellulose,9 and preparative separation was effected by fractional precipitation of the more acidic polysaccharide as the cetyltrimethylammonium Other gums, such as that from Combretum Zeomay contain components that exhibit minor variations in nelt~e,”~*~O composition. These variations have been demonstrated by examination of nodules from the same gum species.31 Two polysaccharide fractions have been isolated from Combretum leonense gum by (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31)

S. P. James and F. Smith,]. Chem. Soc., 739 (1945). S. P. James and F. Smith,]. Chem. Soc., 749 (1945). G. 0.Aspinall and J. Baillie,]. Chem. SOC., 1702 (1963). G. 0.Aspinall and J. Baillie,]. Chem. Soc., 1714 (1963). G. 0.Aspinall, M. J. Johnston, and A. M. Stephen,]. Chem. Soc.,4918 (1960). G. 0.Aspinall, M. J. Johnston, and R. Young,]. Chem. SOC., 2701 (1965). A. J. Erskine and J. K. N. Jones, Can.].Chem.,35,1174 (1957). K. Hunt and J. K. N. Jones, Can.].Chem.,40,1266 (1962). J. M. Tyler,]. Chem. Soc., 5288 (1965). J. M. Tyler,]. Chem. Soc.. 5300 (1965). G. T. Grant, C. McNab, D. A. Rees, and R. J. Skerrett, Chem.Commun., 805 (1969). G. 0. Aspinall, J. J. Carlyle, J. M. McNab, and A. Rudowski,]. Chem. SOC. ( C ) , 840 (1969). G. 0.Aspinall and J. M. McNab,]. Chem. Soc. ( C ) ,845 (1969). G. 0.Aspinall and J. J. Carlyle,]. Chem. SOC. ( C ) ,851 (1969). G. 0. Aspinall and T. B. Christensen,]. Chem. Soc., 3461 (1961). G. 0.Aspinall and V. P. Bhavanandan,]. Chem. Soc., 2685 (1965). G. 0.Aspinall and V. P. Bhavanandan,]. Chem. SOC.,2693 (1965). D. M. W. Anderson, E. L. Hirst, and N. J. King, Talanta, 3,118 (1959).

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separation on O-(2-diethylaminoethyl)cellulose,but no qualitative structural differences have been detected,30 and it is possible that such gums contain a continuous spectrum of related molecular species. In this connection, it should be noted that care should be taken in ensuring that gum samples are from botanically authenticated sources. Thus, valid claims32 for the heterogeneity of commercial gum samples may mean no more than that the gum was of mixed origin.13 The term homogeneous is particularly difficult to define when applied to complex macromolecules. gibbon^^^,^^ has suggested that the term polydisperse may be properly used to describe a macromolecular preparation in which the variation of all measurable parameters is unimodal. In the case of highly branched heteropolysaccharides, such as gums and mucilages, these variations may include those of (a) monosaccharide composition, ( b ) quantitative proportions of the type of linkage in which monosaccharide units are involved, and ( c ) degrees of branching, in addition to (d) those of molecular weight. Anderson and S t ~ d d a r t ’have ~ proposed the term heteropolymolecular to describe macromolecular preparations of this type, but, in the opinion of the present author, the inference that such variations constitute heterogeneity is not a necessary conclusion. In terms of the criteria defined, a unimodally polydisperse preparation is homogeneous, in contrast to one in which one or more of the measurable parameters show a bimodal or polymodal distribution and is thus heterogeneous. INVESTIGATION 111. METHODSOF STRUCTURAL Since the pioneering investigations of E. L. Hirst and J. K. N. Jones, and F. Smith and their respective collaborators on the chemistry of gums and mucilages, advantage has been taken of substantial differences in the rates of hydrolysis of glycosidic linkages to achieve a measure of selectivity in depolymerization. The most notable examples are the much higher rates of hydrolysis of furanosidic than pyranosidic (other than of 6-deoxyhexopyranosidic) linkages, and the much lower rates of hydrolysis of glycosiduronic acid linkages. Controlled hydrolysis utilizing such differences in rates has been ex(32) M. Heidelberger, J . Adams, and Z. Dische,]. Arner. Chem. SOC., 78,2853 (1956). (33) R. A. Gibbons, Nature, 200,665 (1963). (34) R. A. Gibbons, in “Glycoproteins,” A. Gottschalk, ed., Elsevier Publishing Company, Amsterdam, 1966, p. 29.

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tensively used to form oligosaccharides and degraded polysaccharides of structure less complex than that of the parent macromolecule. It should be remembered, however, that some hydrolysis of the more stable linkages always takes place during the cleavage of the glycosidic bonds that are relatively acid-labile. The autohydrolysis of acidic polysaccharides in aqueous solution, for example, is not a magical process that contravenes the laws of physical chemistry. The utility of the partial hydrolysis, or linkage-analysis, method has, since its inception, been greatly extended by developments in chromatographic procedures for the separation of oligosaccharides, and in methods and techniques for their characterization. Adsorption chromatography on carbon-Celite, and partition chromatography on filter sheets and columns of powdered cellulose, are too well known to require further e l a b ~ r a t i o nand , ~ ~ thin-layer c h r ~ m a t o g r a p h ywill ~~ probably soon achieve the same status. Although the separation of acidic oligosaccharides by ion-exchange chromatography has been employed for several year^,^'*^* the emergence of 0-(&diethylaminoethyl)-Sephadex has provided a support that, potentially, combines the advantages of ion-exchange chromatography with those of gelpermeation chromatography. This exchanger has a relatively high capacity; and, with an appropriate choice of eluting solvents, a high degree of resolution of structurally different, acidic oligosaccharides may be a c h i e ~ e d . In ~ ~the . ~ ~characterization of oligosa~charides,~~ the use of semi-micro methods (for example, for the analysis of periodate oxidation4’ and lead tetraacetate oxidation43reactions) and of gas-liquid chromatography (for the separation of methylated and other sugar derivative^^^) has considerably lessened the quantities required for a reasonably complete structure-determination. In the near future, the use of mass ~ p e c t r o m e t r ycoupled ~~ with gas chromaW. W. Binkley, Aduan. Carbohyd. Chem., 10,55 (1955).

J. G. Kirchner, in “Technique of Organic Chemistry,” A. Weissberger, ed., Interscience, New York, N. Y., 1967, Vol. 12. B. Weissman, K. Meyer, P. Sampson, and A. Linker, J . Biol. Chem., 208, 417 (1954). R. Derungs and H. Deuel, Helu. Chim. Acta, 37,657 (1954). G . 0.Aspinall and €3. N. Fraser,J. Chem. Soc., 4318 (1965). G. 0. Aspinall, I. W. Cottrell, S. V. Egan, I. M. Morrison, and J. N. C. Whyte, J . Chem. SOC.( C ) ,1071 (1967). R. W. Baileyand J. B. Pridham, Aduan. Carbohyd. Chem., 17,121 (1962). J. M. Bobbitt,Aduan. Carbohyd. Chem., 11,1(1956). A. S . Perlin, Advan. Carbohyd. Chem.,14,9 (1959). C . T. Bishop, Advan. Carbohgd. Chem., 19,95 (1964). N. K. Kochetkov and 0.S.Chizhov, Aduan. Carbohyd. Chem.,21,39 (1966).

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tography is likely to extend even further the applicability of the methylation method. It should be borne in mind, however, that, with rare exceptions, chromatographic and spectroscopic methods do not provide evidence as to the anomeric and enantiomeric configurations. Acetolysis provides a valuable method alternative to hydrolysis for the controlled fragmentation of complex polysaccharides, as the relative rates of cleavage of glycosidic linkages are often quite different from those observed with mineral acid in aqueous solution. Thus, whereas (1 --., 6)-linkages between hexose residues are the most resistant to acid h y d r ~ l y s i s , this ~~,~ type ~ of linkage is most readily cleaved by a c e t o l y ~ i s . ~Another ~ - ~ ~ example is provided by the demonstration that, on acid hydrolysis, the (1 + 3)-linkages in desulfated A-carrageenan are much more readily split than the (1+ 4)-linkages, whereas the reverse situation holds for a c e t o l y s i ~ . ~ ~ Of particular value in work on exudate gums is the relative resistance of certain 6-deoxyhexopyranosyl linkages toward a c e t ~ l y s i s ~ex~; and amples of the use of this procedure in work on gum tragacanthic acidI6 will be described later. Caution must, however, be exercised in the assessment of the structural significance of oligosaccharides formed on partial acetolysis, because depolymerization may be accomplished by a substantial degree of anomerization of glycosidic linkages. Such artifacts appear to be minor products in the cleavage of the (1 + 4)-linked @-D-mannOpyranOSylbonds in g l u ~ o m a n n a n s ~and ~ , ~galactomannan~,~~ ~ but, in the acetolysis of those oligosaccharides carboxyl-reduced Araucaria bidwillii containing (1 + @-linkages that were isolated had the a-D configuration, whereas the corresponding oligosaccharides formed on partial hydrolysis with acid had the @-D configuration. J. R. Turvey and W. J. Whelan, Biochem. J . , 67.49 (1957). S. Peat, W. J. Whelan, and T. E. Edwards,J. Chem. Soc., 29 (1961). K. Matsuda, H. Watanabe, K. Fujimoto, and K. Aso, Nature, 191,278 (1961). I. J. Goldstein and W. J. Whelan,J. Chem. Soc., 170 (1962). S. Peat, J. R. Turvey, and D. Doyle,J. Chem. Soc., 3918 (1961). Y.-C. Lee and C. E. Ballou, Biochemistry, 4,257 (1965). C. J. Lawson and D. A. Rees,J. Chem. Soc. (C), 1301 (1968). R. Kuhn, I. Low, and H. Trischmann, Chem. Ber., 88,1492 (1955). G. 0. Aspinall, A. J. Charlson, E. L. Hirst, and R. Young,]. Chem. Soc., 1696 ( 1963). G. 0.Aspinall and R. Young,J. Chem. Soc., 3003 (1965). G . 0.Aspinall, R. B. Rashbrook, and G. KesslerJ. Chem. Soc., 215 (1958). G. 0.Aspinall, R. Begbie, and J. E. McKay,J. Chem. Soc., 214 (1962). G. 0. Aspinall and J. N. C. WhyteJ. Chem. Soc., 5058 (1964). G. 0.Aspinall, J. A. Molloy, and C. C. Whitehead, Carbohyd. Res., in press.

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Enzymic hydrolysis has been little used as a means of effecting controlled degradation of gums and mucilages. As far as the author is aware, the only instance reported of use of this procedure in leading to the characterization of oligosaccharides as products of partial hydrolysis is in the depolymerization of degraded tragacanthic acid.I6 One of the most important developments in polysaccharide chemistry in recent years has been the sequence of reactions involving periodate oxidation, borohydride reduction, and mild hydrolysis with acid, commonly referred to as the Smith degradation.60The nature of the products formed depends, of course, on the distribution in the polysaccharide of those sugar residues that are resistant to oxidative cleavage. The characterization of reaction products is simplest when (a) chromatographically separable glycosides of glycerol, erythritol, and threitol are formed from those portions of the polysaccharide structure in which single, or groups of two or three, periodate-resistant sugar residues are present, and (b) substantial blocks of such sugar residues, usually from the interior structure, lead to the formation of degraded polysaccharides. The effectiveness of the Smith degradation depends on the more ready hydrolysis of acyclic acetals than of glycosides (even of glycofuranosides) with cold, dilute acid. However, the acidic acetals derived from the reduction of periodate-oxidized hexuronic acid residues appear to be significantly more resistant to cleavage by acid,Ol and the corresponding selectivity in hydrolysis has not been achieved in such cases. This problem may be overcome by carrying out the Smith degradation on the carboxyl-reduced polysaccharide. Other types of structural modification of polysaccharides, involving oxidation to and reduction of uronic acid residues, lead to markedly different “cracking patterns” on hydrolysis with mineral acid or acetolysis. The former procedure, which has been used in studies on other types of polysaccharide (for example, rye-flour arabinoxylane2 and dextranfi3)has not yet been applied to gums and mucilages. New information on fine structure has been obtained for gum a r a b i ~ , ~ ~ . ~ ~ and the gums from Araucaria bidwillii,” Anogeissus leiocarpus,2s and Sterculia u r e n ~ , 6from ~ degradations of the carboxyl-reduced pol ysaccharides. (60) I. J. Coldstein, C. W. Hay, B. A. Lewis, and F. Smith, Methods Carbohyd. Chem., 5,361 (1965). (61) C . 0. Aspinall, V. P. Bhavanandan, and T. B. Christensen, ]. Chem. SOC., 2677 (1965). (62) C. 0.Aspinall and I. M . Cairncross,]. Chem. SOC., 3998 (1960). (63) D. Abbott, E. J. Bourne, and H. Weigel,]. Chem. Soc. (C), 827 (1966). (64) C . 0.Aspinall and C. R. Sanderson, unpublished results.

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The reduction of hexuronic acid residues to hexose residues in acidic polysaccharides from gums has been achieved by ( a ) the action of diborane on 0-acyl derivatives of polysaccharides in tetrahydrofuran or bis(2-methoxyethyl) ether (“diglyme”),65and ( b )treatment of 2-acyloxyethyl esters of hexuronic acid residues in 0-acyl derivatives of polysaccharides in tetrahydrofuran with lithium borohydride.66The effectiveness of both procedures may be limited by side-reactions (for example, the reduction of some 0-acyl to 0-alkyl groups6’ in the former reaction, and the possibility of /?-elimination of 4-0-substituted hexuronic acid esters during the formation of 2hydroxyethyl esters6*).Reduction of hexuronic acid esters with sodium borohydride in aqueous solution does not usually proceed to completion, owing to competing ester hydrolysis that gives carboxylate anions that are unreactive to the reagent. Repeated esterifications and treatments with sodium borohydride are, therefore, necessary for completion of the c o n v e r ~ i o nDifficulties .~~ are also encountered during the formation of methyl esters of hexuronic acid residues in polysaccharides. Reaction with methanolic hydrogen chloride may cause cleavage of the more acid-sensitive glycosidic linkages present in the majority of polysaccharides from gums and mucilages. An alternative procedure, involving reaction with diazomethane, suffers from the disadvantage that esterification is accompanied by etherification.68

IV. THEGALACTAN GROUPOF POLYSACCHARIDES

The largest single group of exudate gums belongs to the galactan family of polysaccharides. The group includes gum arabic, other gums from Acacia species, and mesquite gum. The polysaccharides all contain ( a ) a core of D-galactopyranosyl residues mutually joined by (1 + 3)- and (1 + 6)-linkages, ( b )residues of D-glucuronic acid or its 4-methyl ether, or both, in outer chains in terminal or nearterminal positions, and (c) outer chains of L-arabinofuranosyl residues and, in some cases, L-rhamnopyranosyl residues. In respect of core structure, and in certain aspects of peripheral structure, the arabinogalactans from coniferous woods70 are similar to the exudate gums (65) F. Smith and A. M. Stephen, Tetrahedron Lett., 17 (1960). (66) D. A. Rees and J. W. B. Samuel, Chem. Ind. (London),2008 (1965). (67) E . L. Hirst, E. Percival, and J. K. Wold,]. Chem. SOC., 1493 (1964). (67a) For an assessment of methods for the reduction of alginic acid, see J. H. Manning and J. W. GreenJ. Chem. SOC.(C), 2357 (1967). (68) D. A. Rees and I. W. Steele, unpublished results. (69) J. K. N. Jones and M. B. Perry,j. Amer. Chem. SOC.,79,2787 (1957). (70) T. E. Timel1,Aduun. Carbohydrate Chem., 19,247 (1964);20,409 (1965).

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of this group. In general, the gums contain more-highly ramified structures, but there is no rigid line of demarcation in terms of molecular ~ornplexity.~’ The first detailed evidence for an interior galactan core in gums came from Smith’s classical study of degraded gum a r a b i ~Several .~~ alternative formulas were suggested to accommodate the highly branched structure required by the methylation data, and, some years later, degradation of ( a ) the degraded gum, by Smith’s proced ~ r eand , ~ (~b ) gum arabic itself, by Barry’s procedure,74showed that the (1 + 3)-linkages are concentrated in the interior chains, and implied that the (1+ 6)-linkages preponderate in the exterior chains. More-recent studies on gum a r a b i ~ involving ,~~ successive Smith degradations, have shown that the galactan core cannot be simply regarded as comb-like, with a single, (1 + 3)-linked main-chain to which other ~-D-galactopyranosylresidues are attached in (1 + 6)linkages. Various degrees of multiple branching in the interior portions of the gum structure are implied. The galactan that is obtained from gum arabic after five Smith degradations contains chains of (1+ 3)-linked P-D-galactopyranosyl residues in which branching of (1+ 6)-linkages, or both, occur approximately every 16 units. The galactan that is formed from carboxyl-reduced, arabinose-free, degraded, mesquite gum after two Smith degradations similarly contains branching or (1 + 6)-linkages, or both, approximately every eight units.76Other gums, for example, those from Acacia u ~ u b i c a ~ ~ and lemon (Citrus possess even more ramified internal structures, and no simple arrangement of the two types of linkage can be discerned. The formation of degraded gums by controlled, acid hydrolysis of the parent polysaccharides involves the preferential scission of L-arabinofuranosyl and L-rhamnopyranosyl linkages, and implies (71) G. 0. Aspinall, Chim. Biochim. Lignine, Cellulose Hemicelluloses, Actes Symp. Intern., Grenoble, France, 1964, p. 89. (72) F. Srnith,J. Chem. Soc., 1724 (1939). (73) F. Smith and D. R. Spreistersbach, Abstracts Papers Amer. Chem. Soc. Meeting, 128,15~ (1955). (74) T. Dillon, D . F. O’Ceallachain, and P. S. O’Colla, Proc. Roy. Zrislz Acad., 55B, 331 (1953); 57B, 31 (1954). (75) D. M . W. Anderson, Sir Edmund Hirst, and J. F. St0ddart.J. Chem. Soc. (C), 1959 (1967). (76) G . 0.Aspinall, D. A. Rees, and C. C. Whitehead, unpublished results. (77) D. M . W. Anderson, Sir Edrnund Hirst, and J . F. St0ddart.J. Chem. Soc. (C), 1476 ( 1967). (78) J. F. Stoddart and J. K. N. Jones, Carbohyd. Res., 8,29 (1968).

GUMS AND MUCILAGES

343

that residues of these sugars are located exclusively on the periphery of the structure. This broad generalization, which continues to provide the skeletal framework for interpretations of many gum structures, has stood the test of time in the case of gum arabic and other structurally related polysaccharides. Some D-galactopyranosyl and L-arabinopyranosyl residues are present in the exterior chains of gums, but these residues appear to be present as isolated units flanked by L-arabinofuranosyl residues. Hydrolysis of furanosyl linkages during the formation of degraded gums results in some fragmentation of the interior molecular structure. Suggestions have been made that the marked decreases in molecular size that occur during controlled hydrolysis with acid may be due to the cleavage of occasional L-arabinofuranosyl or D-galactofuranosyl bonds in the interior chains, but no evidence for either type of linkage has as yet been brought forward. Furthermore, the remarkable suggestion has been made that certain D-galactopyranosyl linkages are unusually susceptible to acid hydrolysis. In the opinion of the present writer, there are insufficient data available on the relative rates of hydrolysis of different glycosidic linkages, even in simple glycosides, to indicate clearly that the relative rates of cleavage of L-arabinofuranosyl and D-galactopyranosyl bonds in gum arabic and related polysaccharides are markedly different from those in simple glycosides, and the case for “unusual” D-galactopyranosyl bonds must be regarded as not proven.

1. Acacia Gums The structures of gum arabic and other Acacia gums may be considered in terms of ( a ) the galactan core, ( b )the nature of the hexuronic acid residues and their modes of linkage to those of D-galaCtose, ( c ) the nature of the peripheral chains of L-arabinofuranosyl and associated sugar residues, and ( d ) (where present) the location of L-rhamnopyranosyl residues. A considerable number of Acacia gums have been subjected to chemical investigation, but attention will here be directed to those of botanically authenticated origin whose structures have been established in sufficient detail to permit reasonably full comparisons to be made. Table I shows the oligosaccharides that have been adequately characterized as products of partial fragmentation. In certain cases, the presence of linkages additional to those recognized in disaccharides may be unambiguously inferred from the identification of cleavage products from methylated, degraded polysaccharides.

TABLEI Oligosaccharides from Partial Hydrolysis of Acacia Gums

Acacia gums Oligosaccharides e-D-cdp-(1-+ 3 ) - L - h (1) &L-Arap-(1 3)-~-Ara(2) /i-L-Araf-( 1 3)-~-Ara (3) &L-Araf-( 1+ 2)-~-Ara(4) &D-Galp-(1 3)-D-Cal(5) P-D-Galp-(1 6)-~-Gal(6) /3-D-GpA-(1 -+ 6)-D-Gd(7) 4Me-@-mGpA-(1 6)-~-Gal(8) ~-D-G~A 1 - ( 4)-mGalp (9) 4-Me-e-D-GpA-(1 4)-~-Calp (10) References -+

senegal

+ +

-+

-+

-+

+ + +

pycnuntha

+ + a +

a

mearnsii

arabica

nubica

nilotica

drepanolobium

+

+ + + +

+ + + + + +

+

+a + + + + +

+ + + + + + + +

86

87-89

90.91

+ + + +

-+

-+

54.75.79-81

82-84

85

a a

a a 77

“Oligosaccharidescharacterized by paper chromatographyonly, or presence of linkage inferred from other data (for example, cleavage products from methylated gum or derivative), or both.

.r,

P

+

v)

21 z > r r

GUMS AND MUCILAGES

345

The seven Acacia gums mentioned in Table I have a number of structural features in common, but differ in other respects. For each, the structure is based on a highly branched framework of P-D-galactopyranosyl residues. Degradations of the arabinose-free, degraded A . p y ~ n a n t h aA. ,~~ polysaccharides derived from Acacia r n e a r n ~ i i ,and ~ ~ A. d~epanolobiurn~' gums by the Smith or Barry procedures have shown that (1 + 3)-linkages are concentrated in the interior regions of the galactan core of these gums. However, as already noted, further studies on Acacia senegal gum75 have indicated a degree of multiple branching in the core, so that a single main-chain having branches attached in a comb-like structure provides a too-simple model for the interior chains. Acacia arabica as mentioned previously, and A. nubica gum86are even more highly ramified in their inner structures. This aspect of the structure of Acacia nilotica gum has not yet been studied. All Acacia gums so far examined contain residues of D-glucuronic acid that are (1 + 6)-linked to D-galactose, and that give rise to an aldobiouronic acid, 6-O-(~-D-glucopyranosyluronicacid)-D-galactose (7), on partial hydrolysis with acid. However, in an increasing number of Acacia gums studied, D-glucuronic acid residues are now known to be present in more than one structural environment. a-D-Glucuronic acid residues (1 + 4)-linked to D-galactose were first shown to occur in Acacia karroo gum,g2but have now been recognized in A. nubica gumse and in a number of other Acacia gumsg3 Furthermore, Dglucuronic acid residues in both types of linkage to D-galactose may occur as the 4-methyl ether.

(79) (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) (91) (92) (93)

S. W. Challinor, W. N. Haworth, and E. L. Hirst,]. Chem. SOC.,258 (1931). J . K. N. Jones,J. Chem. SOC., 1672 (1953). F. Smith,]. Chem. SOC., 744 (1939). E. L. Hirst and A. S. Perlin,]. Chem. Soc., 2622 (1954). A. S. Perlin, Anal. Chem., 27,396 (1955). G. 0.Aspinall, E. L. Hirst, and A. Nicolson,J. Chem. SOC., 1697 (1959). G. 0.Aspinall, J. J . Carlyle, and R. Young, Carbohyd. Res., 7,421 (1968). D. M. W. Anderson and G. M. Cree, Carbohyd. Res., 6,385 (1968). R. C. Chalk, J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Can.]. Chem., 46, 2311, (1968). R. C. Chalk, Ph.D. Thesis, Queen's University, Kingston, Ontario, Canada (1966). D . M. W. Anderson and K. A. Karamalla, Carbohyd. Res., 2,403 (1966). D. M. W. Anderson and I. C. M. Dea, Carbohyd. Res., 5,461 (1967). D. M. W. Anderson and I. C. M. Dea, Carbohyd. Res., 7,109 (1968). A. J . Charlson, J. R. Nunn, and A. M. Stephen,]. Chem. SOC., 1428 (1955). D. M. W. Anderson and G. M. Cree, Carbohyd. Res., 6,214 (1968).

346

G. 0. ASPINALL

Five types of partial structure (11-15) have been established in which aldobiouronic acid units are present in Acacia gums. Nonetherified, D-glUCUrOniC acid end-groups joined to D-galactose in both types of linkage (11 and 12) are present in A . nubica gum.ss Acacia arabica gum77 possesses the same two structural units (11 and 12), together with the corresponding units ( 13 and 14) containing 4-0-methyl-D-glucuronic acid residues. Whereas 4-0-methylD-glucuronic acid residues have only been found as end groups in Acacia gums, nonterminal, 4-0-substituted D-glucuronic acid residues are frequently encountered in (1 + 6)-linkage to D-galactose (15). p - ~ - G p A - (+ l 6)-D-Galp-l a - ~ - G p A - (+ l 4)-D-Galp-l 4-Me-p-~-GpA-(1 + 6)-~-Galp-l 4-Me-a-~-GpA-( 1 + 4)-D-Galp-l -(1 + 4)-p-D-GpA-(l + 6)-D-Galp-l

The substituents attached to D-glucuronic acid are readily removed on mild hydrolysis with acid, and the sugar acid units are exposed as end groups in the arabinose-free, degraded gums. For gum arabic (see later), evidence has been obtained that L-rhamnopyranosyl residues are (1+ 4)-linked to D-ghcuronic acid residues. The evidence available does not completely further define the location of residues of D-glucuronic acid or its 4-methyl ether with respect to the galactan core, although, for gum arabic, the isolation of an aldobiouronic acid, 6-O-(/3-D-ghcopyranosyluronic acid)-Dgalactose (7), as its fully etherified derivative after partial depolymerization of the methylated, degraded gum,g4suggests that, here, the hexuronic acid residues terminate outer chains of (1+ 6)-linked P-D-galactopyranosyl residues as in formula 16. Terminal residues of , ~nubica,86 ~ and A. karroog5gums a-D-glucuronic acid in A . a r ~ b i c aA. appear to be joined (1+ 4) to unbranched D-galaCtOSyl residues, but, in these gums, there is no evidence to indicate whether these Dgalactosyl residues terminate (1-+ 6)-linked chains (as in 17) or are attached as side-chains thereto (as in 18). It may, however, be noted that the latter type of structure (namely, 18) occurs in mesquite gum, where the acid residues are present as the 4-methyl ether.76 L-Arabinose is an important constituent of all Acacia gums, and may, as in A . arabica gum,77account for as much as 57% of the sugar residues. The majority of the residues of this sugar are in the furanose form, and, as far as is at present known, occur only in the exterior (94) J. Jackson and F. Smith,]. Chem. SOC.,74 (1940). (95) A. M . Stephen and D. C. Vogt, Tetrahedron, 23,1473 (1967).

GUMS AND MUCILAGES

347

1

P-D-Galp 6

t

1

P-D-Galp 6

t

1

P-D-G~A (16)

chains of the gums. A substantial proportion of these residues occurs as L-arabinofuranosyl end-groups, but, as indicated by the characterization of cleavage products from the methylated gums and by the identification of the oligosaccharides 1-4 as products of partial hydrolysis by acid, nonterminal L-arabinofuranosyl residues are also present. Apart from the linkages of isolated units thus indicated, knowledge of the detailed sequences of sugar residues in the outer chains, of variations amongst side-chains in single polysaccharide species, and of the specific sites of attachment to D-galaCtOSe residues in the core structure, is still largely lacking. Since the majority of nonterminal L-arabinofuranosyl residues are 2-0- or 3-O-substituted, and are thus resistant to periodate oxidation, successive degradations of a gum by the Smith procedure may provide evidence for the lengths of these outer chains. Work by Anderson, Hirst, and S t ~ d d a r t ' ~ provides an interesting contrast between gum arabic and A. arabica gum. Four treatments of gum arabic result in removal of all of the L-arabinose residues, thus placing an upper limit on the length of the ~ i d e - c h a i n s .After ~ ~ a similar series of reactions, Acacia arabica furnished a degraded polysaccharide that still contained 2- and 3-0substituted L-arabinofuranosyl residues, thus pointing to outer chains that are quite extended.77

348

G. 0. ASPINALL

a-D-Galactopyranosyl and P-L-arabinopyranosyl residues occur as isolated entities in the outer chains of certain Acacia gums. These units, which differ only in the presence of the hydroxymethyl group in the former, have only been found linked (1 + 3) to nonterminal L-arabinofuranosyl residues. L-Rhamnopyranosyl residues in Acacia gums have been found only as end groups. In the first group of Acacia gums to be examined in reasonable detailY2a close correspondence was noted between the proportions of L-rhamnose and D-glucuronic acid as constituent sugars, and it was suggested that residues of the former are linked (1+ 4) to those of the latter sugar. This type of linkage has, indeed, been demonstrated in gum a r a b i ~ , where ~~,~~ it provides the only known evidence for the location of L-rhamnopyranosyl end-groups in Acacia gums. However, it is now clear that many Acacia gums contain much smaller proportions of L-rhamnose than of D-glucuronic acid.%Furthermore, residues of the latter sugar are present to various proportions as the 4-methyl ether, and probably occur as end groups. Definitive proof of the location of L-rhamnopyranosyl residues in gum arabic was obtained on acetolysis of the carboxyl-reduced polysaccharide, from which degradation, 4-O-~-rhamnopyranosyl-D-glucose (19) and 0-L-rhamnopyranosyl-( 1 + 4)-O-D-glUCOpyranOSy1(1 + 6)-D-galactose (20) were identified as products of partial L-Rhap-(1 + 4 ) - ~ - G p L-Rhap-(1 + 4)-D-Gp-(1 + 6)-~-Gal

d e p ~ l y m e r i z a t i o n . ~The ~ . ~ ~a-L configuration was assigned to the rhamnosidic linkage on the basis of the specific rotation of disaccharide 19, but this assignment cannot now be regarded as certain, in view of the observation that certain glycosidic linkages may be largely anomerized during acetoly sis. 1,59 Recent papers on Acacia gums report examination of polysaccharides from A . laetayMa A . seyalyMbA . podalyriaefoliay96c A . elatagsd gums, and further studies on A . drepanolobium gum.96d*Me (96) D. M. W. Anderson and K. K. Karamalla,]. Chem. Soc. ( C ) ,162 (1966). (%a) D. M. W. Anderson, I. C. M. Dea, and R. N. Smith, Carbohyd. Res., 7,320 (1968). (96b)D . M. W. Anderson, I. C. M. Dea, and Sir Edmund Hirst, Carbohyd. Res., 8,460 (1968). (964 P. I. Bekker, A. M. Stephen, and G. R. Woolard, Tetrahedron, 24,6967 (1968). (96d) D. M. W. Anderson and I. C. M. Dea, Carbohyd. Res., 8,440 (1968). (Me) D. M. W. Anderson and I. C. M. Dea, Carbohyd. Res., 8,448 (1968).

GUMS AND MUCILAGES

349

2. Mesquite Gum Mesquite gum was the first polysaccharide in which 4-O-methyl-Dglucuronic acid as a constituent sugar was fully characterized.2 The gum possesses a number of structural features similar to those in the Acacia gums, and a re-examination of the gum has thrown fresh light on ( a ) the nature of the galactan core, (b) the mode of attachment of units of the aldobiouronic acid, namely, 4-0-(4-0-methyl-aD-glucopyranosyluronic acid)-D-galactose (lo), to the galactan core, and ( c ) some of the sequences of sugar units in the peripheral chains.76 Early studies on mesquite gum2 by White and by Smith and his collaborators established the highly branched nature of the polysaccharide, and showed the presence of peripheral chains of 2-O-substituted L-arabinofuranosyl residues and of end groups of D-glucuronic acid or its 4-methyl ether joined to D-galactose by two, or possibly three, types of linkage. After partial hydrolysis with acid, Cunneen and Smithe7 isolated a mixture of aldobiouronic acids involving (1 + 4)- and (1 + 6)-linkages to D-galaCtOSe. Although the presence of a methyl ether of D-glucuronic acid was recognized, the exact status of the D-ghcuronic acid residues in both types of aldobiouronic acid was not established. Later, an aldobiouronic acid, acid)-D-galactose namely, 6-O-(4-O-methy~-~-D-g~ucopyranosyluronic (S), was characterized as a product of partial h y d r o l y s i ~A . ~third ~ type of aldobiouronic acid unit was indicated in studies by White,egwho assigned the structure of 2,4-di-O-methy1-3-0-(2,3,4-tri-O-methyl-Dglucopyranosyluronic acid)-D-galactose, to an acidic fragment formed on partial depolymerization of methylated mesquite gum. In common with other gums of the galactan group, controlled hydrolysis of mesquite gum with acid leads to the formation of an L-arabinose-free, degraded gum. Two successive Smith degradations of the carboxyl-reduced, degraded gum have been shown to give a ~ ~ degraded galactan in which (1 + 3)-linkages p r e p ~ n d e r a t e .This polysaccharide has a markedly diminished degree of branching, similar to that in the interior chains of gum a r a b i ~as , ~only ~ approximately one residue in eight is involved in (1+ 6)-linkages. A further study of the aldobiouronic acids formed on partial hyhas confirmed the identity of 6-0-(4-0drolysis of mesquite methyl-fi-D-glucopyranosyluronicacid)-D-galactose (S), has completed (97) J. I. Cunneen and F. Smith,J. Chem. SOC., 1141 (1948). (98) M . Abdel Akher, F. Smith, and D. SpriestersbachJ. Chem. SOC.,3637 (1952). (99) E. V. White,]. Amer. Chem. SOC., 69,2264 (1947).

350

G . 0. ASPINALL

the characterization of 4-O-(4-O-methyl-a-~-glucopyranosyluronic acid)-D-galactose (lo),and, by the isolation, in small amount, of 6-0(P-D-glucopyranosyluronic acid)-D-galactose (7), has shown that a small proportion of the D-glucuronic acid residues are not etherified. No evidence was obtained for the presence of aldobiouronic acids containing (1+ 3)-linkages. In addition, however, two acidic trisaccharides have been isolated to which the partial structures 0-(40 - methylglucopyranosyluronic acid) - (1 + 6)- 0 - galactopyranosyl(1 + 6)-galactose (21) and 0- (4- 0- methylglucopyranosyluronic acid)-(1 + 4)-0-galactopyranosyl-( 1 4 3)-galactose (22) have been 4-Me-GpA-( 1 + 6)-Galp-( 1 + 6)-Gal 4-Me-GpA-( 1 + 4)-Galp-(1 3 3)-Gal

a~signed.'~ The isolation of the former trisaccharide suggests that units of the aldobiouronic acid 6-0-(4-0-methyl-~-~-glucopyranosyluronic acid)-D-galactose (€9,terminate chains of (1+ 6)-linked p-Dgalactopyranose residues (16), as suggested for Acacia gums. On the other hand, the latter trisaccharide is more likely to originate from units of the aldobiouronic acid, 4-O-(4-O-methy~-a-D-g~ucopyranosy~uronic acid)-D-galactose (lo),attached as side-chains to the chains of (1+ 6)-/?-~-galactopyranosylresidues (as in 18), since 4-0-substituted D-galactosyl residues have not been detected as branch points. In this connection, it would now seem possible that the partially methylated, acidic oligosaccharide isolated by Whitew from methylated mesquite gum was a derivative of the trisaccharide 22, and that a 3-0-substituted D-galaCtOSe residue was, indeed, present, but as the reducing group of a trisaccharide and not a disaccharide. The outer chains of mesquite gum are more complex than previously supposed. Methylation studies have now revealed the presence of 2,3- and 2,5-, in addition to 3 5 , di-0-methyl-L-arabinose amongst the cleavage products from the methylated gum. Confirmation of the linkages thus implied has been obtained b y the characterization of the oligosaccharides 4, 2, 1, and 23-26 derived from P-L-Araf-( 1 42 ) - ~ - A r a P-L-Araf-( 1 42)-L-Araf-( 1 + 2 ) - ~ - A r a @ - h a p - ( 1 43 ) - ~ - A r a ~-Amf-(1 + I ) - ~ - A r a p a-L-Araf-( 1 3 3 ) - ~ - A r a cu-D-Galp-( 1 + 3)- L-Ara L-Araf-( 1 + 6)-D-Galp-( 1 3 3 ) - ~ - A r a

peripheral units of the gum during controlled, partial hydrolysis by acid. Although the outer chains of the gum cannot be defined un-

GUMS AND MUCILAGES

351

ambiguously, it is clear that, in the absence of evidence for branching, linear chains contain more than one type of linkage. A feature not previously recognized in other polysaccharides is the presence of isolated D-galactopyranosyl and L-arabinopyranosyl residues within chains of L-arabinofuranosyl residues. The partial formulas 27 and 28 indicate possible structures for regions in the outer chains in the vicinity of the pyranoid sugar residues. P-L-Ara$( 1+ 2)-~-Araf-( 1+ 4)-P-~-Arap-( 1+ 3)-~-Ara$l 1+ 3)-~-Ara$(1+ 3)-~-Araf-l L-Araf-(1+ 6)-a-~-Galp-(

(27) (28)

3. Other Gums of the Galactan Group Other polysaccharides that are based on a galactan core, and in which the (1+ 3)-linked 0-D-galactopyranosyl residues are concentrated in inner chains, are asafoetida gum,'"'' the minor polysaccharide component of Khaya senegalensis gum,le and the gum from Araucaria b i d ~ i Z Z i i . Lemon ~ ~ ~ ~g~ J~ ~ m ~possesses ~ * ~ many ~ of the same structural features as these gums, but the degree of branching in the interior structure is more akin to that in Acacia arabica gum.77Golden-apple gUm2,102 is another member of this group of polysaccharides, although evidence is not yet available on the detailed structure of the core. Table I1 shows the oligosaccharides that have been identified as products of partial hydrolysis of these gums. Further similarities between these gums and those from Acacia species have been indicated by methylation studies that show the presence of peripheral chains terminated by L-arabinofuranosyl groups. Lemon78and golden-apple1O2gums are unique amongst gums so far studied, in that 4-0-methyl-D-glucuronic acid end-groups are attached to L-arabinose residues in the outer chains, as well as to D-galaCtOSyl residues in the core. In golden-apple gum, the peripheral chains contain a-D-xylopyranosyl residues also, but it is not yet known if these are end groups.lo2Araucaria bidwillii gum also resembles gum arabic in that L-rhamnopyranosyl end-groups have been shown to be linked (1+ 4) to non-etherified D-glucuronic acid residues. As in the case of gum a r a b i ~ , acetolysis ~ ~ , ~ ~ of the carboxyl-reduced polysaccharide led to the isolation" of 4-O-L-rhamnopyranosyl-D-glucose (19)and O-L-rhamnopyranosyl-(1--* 4)-O-D-glucopyranosy1-(1 + 6)-Dgalactose (20). (100) J . K. N. Jones and G. H. S. Thomas, Can.J. Chem., 39,192 (1961). (101) G . 0.Aspinall and R. M. Fainveather, Carbohyd. Res., 1,83 (1965). (102) B. 0. Lindgren, Acta Chetn. Scand., 11, 1364 (1957).

TABLEI1 Oligosaccharides from Partial Fragmentation of Various Gums of the Galactan Group Gums Oligosaccharides p-L-Arap-(1+ 3)-~-Ara (2) a-L-hap-(1+ 5)-L-Araf(29) 4-Me-a-~-GpA-(1 + 4)-~-Arap(30) .i-Me-a-D-GpA-(l+ 4)-~-Arap-( 1+ 5)-~-Araf(31) a-~-Xylp-( 1+ 3)-~-Ara (32) 4-Me-a-D-GpA-(1+ 3)-~-Ara(33) p-D-Galp-(l+ 3)-D-Gal(5) p-D-calp-( 1+ 6)-D-Gal(6) p-~-GpA-(l+6)-D-Gal(7) 4-Me-p-~-GpA-(1+ 6)-D-Gal(8) 4-Me-a-~-GpA-(l+4)-D-Gal(lO) References

Khaya senegalensis

Asafoetida

Araucaria bidwillii

Lemon

Golden apple

+

+

+ ++

+ +

+ +

+ + + +

19

100

11,59,101

U

(I

U

a

+

U U

+ + + +

U

+ 78

cl

P

102

'Oligosaccharides characterized by paper chromatography only, or presence of linkage inferred from other data (for example, cleavage products from methylated gum or derivative), or both.

i%

21

z $ r

GUMS AND MUCILAGES

353

Two further types of gums may provisionally be regarded as members of the galactan group of polysaccharides. Their structures have been studied in various degrees of detail, but insufficient is as yet known to relate them to those of other polysaccharides in the group. Gum traga~anth'~ contains, as a minor polysaccharide component, an arabinogalactan in which L-arabinose is, by far, the preponderant sugar constituent. The highly ramified, outer chains of L-arabinofuranosyl residues, which are involved in several types of linkage, mask the galactan core, in which (1 + 4)-, as well as (1 + 3)- and (1 + 6)-, linkages may be present. Unlike other gum polysaccharides, this arabinogalactan contains, at most, only traces of hexuronic acid residues. Pollen from mountain pine (Pinus mugo Turra)'O*'03contains two structurally unrelated polysaccharides, a xylogalacturonan and an acidic arabinogalactan, which in many respects resemble the two main components of gum traga~anth.'~*'' The most obvious difference between the pollen polysaccharide and the arabinogalactan from gum tragacanth is the presence in the former of about 10% of P-D-glucuronic acid residues, which are linked (1 ---* 6) to D-galaCtOSe.'" ~ ~ cholla ~ , ~ ~gum ~ (Opuntia Jeol gum (Odina wodier, R o x ~ . ) and f ~ Z g i d a ) ~ J ~differ , ' ~ ~ from other gums built upon a framework of D-galactosyl residues in containing D-galacturonic acid instead of D-glucuronic acid (or its 4-methyl ether) as the acidic sugar constituent. It may be noted that these gums differ from other gums containing D-galacturonic acid (see later) in that the acidic sugar units are found as end groups or in near-terminal positions in outer chains. Both gums give rise to an aldobiouronic acid, namely, 3-O-(P-D-galaCtOpyranosyluronic acid)-D-galactose (34), on partial hydrolysis with P-D-GalpA-(1 + 3)-D-Gal

(34)

acid, and the gross structures of the degraded polysaccharides formed on controlled hydrolysis of the gums are very similar. The presence of branched structures having (1 + 3)- and ( 1 + 6)-linkages between p-D-galactopyranosyl residues is indicated for both gums, but information on the distribution of the linkages is lacking. Cholla gum possesses a variety of peripheral chains, most of which are terminated by L-arabinofuranosyl and D-xylopyranosyl residues. An insight into the structure of these outer chains has been gained (103) (104) (105) (106) (107)

H. 0.Bouveng and H. Lundstrom,Acta Chem. Scand., 19,1004 (1965). A. K. Bhattacharyyaand C. V. N. Rao, Can.J.Chem.,42,104 (1964). A. K. Bhattacharyya and A. K. Mukhejee, Bull. Chen. Soc.Jap.,37,1425 (1964). V. M . Parikh and J. K. N. Jones, Can.j. Chem.,44,327 (1966). V. M . Parikh and J. K. N. Jones, Can.J.Chem., 44,1531 (1966).

G . 0. ASPINALL

354

by the characterization of 5-O-~-~-xylopyranosyl-L-arabinofuranose (35),O-P-D-xylopyranosyl-(1 + 5)-O-a-~-arabinofuranosyl-( 1 + 3)-Larabinose (36),3-0-a-~-arabinofuranosyl-~-arabinose (25),and, possibly, P-D-galactopyranosyl-L-arabinose(37) as products of partial hydroly~is.'~' P-D-xylp-( 1 + 5)-~-Araf p-D-Xylp-( 1 + 5)-cy-~-Araf-(1 + 3 ) - ~ - A r a P-D-Galp-( 1 + 3 ) - ~ - A r a

Jeol gum is, apparently, less complex in its peripheral structure, D-xylose being absent as a constituent, but no detailed sequences have yet been established.

v. THE GLUCURONOMANNAN GROUPOF POLYSACCHARIDES The aldobiouronic acid 2-O-(~-~-glucopyranosyluronicacid)-Dmannose (38),which was first isolated as a product of partial acid P-D-C~A-( 1 + 2)-D-Man

(38)

hydrolysis of damson and cherry gums,2 is a structural fragment of many polysaccharides. Where available, evidence for the location of these fragments within the molecular structure suggests that these units occur in interior chains, but definite proof has been brought forward only in recent years. Three polysaccharides of the gum group have now been shown to contain basal chains of glucuronomannan in which 4-O-substituted D-glUCUrOniC acid and 2-O-substituted Dmannosyl residues alternate. Two aldobiouronic acids, namely, 2-O-(~-D-glucopyranosy~uronic acid)-D-mannose (38) and 6-O-(P-D-glucopyranosyluronicacid)-Dare formed from gum ghatti from Anogeissus Z~tifoZiu.~~~ galactose (7), Partial hydrolysis of the gum with acid gives rise also to two series, 39 and 40, of neutral oligosa~charides.'~~ As these oligosaccharides

constitute the longest, readily characterized sequences of sugar residues, it was at first supposed that they arise from the basal chains of the polysaccharide, and that units of the two aldobiouronic acids are attached thereto as side chains. However, two successive degradations (108) G. 0.Aspinall, E. L. Hirst, and A. Wickstr2rn.J. Chem. Soc., 1160 (1955). (109) G. 0.Aspinall, B. J. Auret, and E. I,. Hirst,J. Chern. Soc. 4408 (1958).

355

GUMS A N D MUCILAGES

of the polysaccharide by the Smith procedure led to the isolation of a degraded gum from which 3-O-~-D-ga~actopyranosy~-D-galactose (5), 6-0-P-D-galactopyranosyl-D-galactose (39, n = O), 3-O-P-D-galactopyranosyl-L-arabinose (40, n = 0), and 3-O-L-arabinopyranosyl-~mannose (41) were obtained on partial hydrolysis.61 These results ~ - A r a p -1( + 3)-D-Man

(41)

imply that D-mannOSyl residues are located in the interior of the molecular structure, and partial structure 42 was proposed, in which -(I + 6 ) - P - ~ - G a l p - (+ l 6)-p-D-Galp-(1

--.)

3)-L-Ardp-(l + S)-D-Manp-l

(42)

the chains of ( 1 + 6)-linked P-D-galactopyranosyl residues are joined through an L-arabinopyranosyl “link” unit to a D-mannosyl residue in the basal chain.61 Subsequently, an acidic oligosaccharide (at first erroneously reported to be a trisaccharide1I0)was characterizedz5 as the tetrasaccharide 0-P-D-glucopyranosyluronic acid-(1 + ~ ) - O - D mannopyranosyl-( 1 +. 4)-O-P-D-glucopyranosyluronicacid-( 1+ 2)-Dmannose (43), and this provided evidence for the sequence of the sugar residues in the inner chains. P-D-GpA-(l + 2)-D-Manp-(1 + 4 ) + - ~ - G p A - ( l

-+

2)-D-Man

(43)

The known structural features of gum ghatti are summarized in structure 44. Three additional aspects on which information is not R

R

5

5

6 6 -( 1 + 4)-P-D-GpA-(1 + 2)-D-Manp-( 1 -+ 4)-P-D-GpA-(1 + 2)-D-Manp-l 3 3

t

t

1 ~-Arap 3

1 L-Arap 3

t

t 1 [ R + 3)-P-D-GalP]. 6

1

[R + 3)-P-D-Gdp],, 6

t

t

1 P-D-G~A

1 P-D-Galp (44)

where the majority of sites carry substituents R = L-Araf-(1, or, less frequently, L-Ar;i$( 1 -+ 2)-12-Afiif-( 1 , IA-Araf-(1 -+ 3)-L-A=$( 1, or L-Araf-( 1 + 5)-~-Araf-(l. ( 1 10) C. 0. Aspinall and T. B. Christensen,]. Chem. Sac., 2673 (1965).

G . 0. ASPINALL

356

yet available deserve comment. The gum contains more than 40 % of L-arabinosyl residues, of which the majority occur as furanosyl endgroups.”’ A comparison of the cleavage products from the methylated gum and the methylated, L-arabinose-free, degraded gum indicates that most of these units must be attached as side chains to the (1-+ 6)linked chains of P-D-galactopyranose residues. Of the remainder, four types of nonterminal units have been recognized, namely, 2and 3-0-substituted L-arabinofuranosyl residues, 4-0-substituted L-arabinopyranosyl or 5-0-substituted L-arabinofuranosyl residues, or both, and 3-0-substituted L-arabinopyranosyl residues.”’ Of these types of unit, only the last-named have been placed, namely, as “link” units in the partial structure 42; the others probably occur in peripheral chains. Secondly, the gum contains a small proportion of P-D-galaCtOpyranOSyl residues mutually joined by (1 --* 3)-linkages; these have not yet been placed with respect to other portions of the structure. Thirdly, units of the aldobiouronic acid 6-O-(p-D-glucopyranosyluronic acid)-D-galactose (7) are likewise unplaced. However, since the gum contains some D-glucuronic acid end-groups, it is possible that these units terminate some of the (1 + 6)-linked chains of P-D-galactopyranosyl residues, where they are provisionally placed as in formula 44. The gum from the related species Anogeissus Zeiocarpus (formerly A. schimperi) contains structural features similar to those of gum ghatti, and gives many of the same oligosaccharides on partial hydrolysis with acid, but in different proportions.28 Further studies on this g ~ mshowed, ~ ~ ,however, ~ ~ that two distinct, but structurally related, polysaccharides are present. The major component of the gum leiocarpan A has been examined in detai1.25-27In contrast to gum ghatti, in which the glucuronomannan chain constitutes a relatively small proportion of the polysaccharide structure, the main chain of leiocarpan A accounts for over 50% of the polysaccharide, and most of the side chains consist of single sugar residues. Controlled hydrolysis of leiocarpan A with acid gives a degraded poly~accharide~~ for which the partial structure 45 has been proposed -( 1 + 4)-P-D-GpA-(1 -+

e)-a-D-Manp-( 1+ 4)-P-D-GpA-(1+ 2)-a-~-Manp-l 6

t

1

D-xylp

(45) (111) G. 0.Aspinall, B. J. Auret, and E. L. Hirst,]. Chern. SOC.221 (1958).

GUMS AND MUCILAGES

357

on the basis of methylation data and of the characterization of tetrasaccharide 43 as a product of partial hydrolysis. Detailed evidence for the nature of the basal chains of leiocarpan A has been obtained by the isolation of oligosaccharides on acetolysis of the carboxyl-reduced polysaccharide.26These oligosaccharides include 2-O-P-D-glucopyra(47), and a nOSyl-D-mannOSe (46), 4-O-cY-D-mannOpyranOSyl-D-glUCOSe series (48-52) of higher oligosaccharides containing alternating resip -~ -Gp-( 1 + Z)-D-Man

(46)

1 -+ 4)-D-Gp c~- ~ -Manp-(

(47)

Gp-(1 -+ 2)-Manp-(1

(48)

4)-Gp

Manp-(1 --* 4)-Gp-(1 + 2)-Man

(49)

Gp-(1+ 2)-Manp-(1 -+ 4)-Gp-( 1 --* 2)-Man

(50)

Manp-(1 + 4)-Gp-(1 + 2)-Manp-(1+ 4)-Gp

(51)

Gp-(1 -+ 2)-Manp-(1 -+ 4)-Gp-(1 + 2)-Manp-(1 -j 4)-Gp

(52)

dues of glucose and mannose. The basal chains of leiocarpan A therefore consist, largely if not exclusively, of alternating 4-0-substituted P-D-glucuronic acid and 2-0-substituted a-D-mannopyranosyl residues. Gum ghatti and leiocarpan A are thus the first two, clearly authenticated glucuronomannans. Further studies on leiocarpan A have permitted further partial structures (53 and 54) to be proposed for the poly~accharide.~'A substantial proportion of the L-arabinofuranosyl residues can only be accommodated as single-unit side-chains linked (1-+ 3) to D-mannopyranosyl residues, as in 53. Although D-galactose is only a minor D-xylp 1

.1 6 -(1-+ Z)-D-Manp-l 3

t

1 L-kaf (53)

constituent of leiocarpan A, residues of this sugar are present in linkages similar to those in gum ghatti. The examination of a degraded polysaccharide formed on Smith degradation of carboxyl-reduced leiocarpan A has led to the formulation of side chains attached,

358

G . 0.ASPINALL

through L-arabinopyranose “link” units, to core residues of D-mannose (54), as in gum ghatti. The galactan side-chains in leiocarpan A D-Xylp 1

.1 -(1 + 2)-D-Mmp-l 3

t

1 L-hap n

5.

t

1

-( 1 + S)-P-D-Galp-(1 + 3)-B-~-Calp-

6

6

t

t

R

1

[R + 3)-P-D-Calpln 6

t 1

P-D-G~A (54)

where R = L-Araf-( 1, or L-Araf( 1 + 3)-~-Araf-(1.

probably differ in length and in degree of branching. On the assumption that L-arabinosyl residues substituted at 0-3 by D-galactosyl are “link” units in the pyranose form, the partial characterizati~n~~ of the trisaccharide as 0-(D-glucopyranosyluronic acid)-(1+ 6)-O-D-galactopyranosyl-(1 + 3)-~-arabinose(55) suggests that, in some cases, the GpA-(1 + 6)-~-Galp-( 1 + 3)-~-Ara

(55)

side chains may contain only one D-galaCtOSyl residue which, in turn, is substituted by a terminal D-glucuronic acid residue. Other chains are longer and must carry sequences of two or more 6-0-substituted D-galactosyl residues. The further stepwise erosion of the aforementioned degraded polysaccharide by the Smith procedure results in the faster removal of the chains of (1 + 6)-linked P-D-galactopyranosyl residues than of those involved in (1 +-3)-linkages, and this result has led to the suggestion2’ that the (1+ 3)-linked chains are attached as branches to the innermost D-galaCtOpyranOSyl residues in the partial structure 54. The majority of D-mannopyranosyl residues in leiocarpan A are double branch-points, and they probably also carry single D-XylOpyra-

GUMS AND MUCILAGES

359

nosy1 residues at 0-6. The galactan side-chains carry further peripheral L-arabinofuranosyl residues, and these are provisionally located in the partial structure 54. The gum exudate of Encephalartos longifolius is the first polysaccharide reported to contain 3-O-methyl-~-rhamnoseas a significant constituent."' The polysaccharide preparation, which may contain more than one molecular species, gives rise,113on partial hydrolysis with acid, to the aldobiouronic acids 6-O-(4-O-methyl-p-D-glucopyranosyluronic acid)-D-galactose (8), 6-O-(~-D-ghcopyranosyluronic acid)-D-galactose (7), and 2-O-(~-D-g~ucopyranosy~uronic acid)-D-mannose (38). In addition, a mixture of higher oligosaccharides containing residues of D-glucuronic acid and D-mannose, and probably consisting of the tetrasaccharide 43 and higher polymer-homologs, was isolated. As preliminary methylation data112suggested that the latter fragments arise from interior chains, it is probable that this polysaccharide belongs to the glucuronomannan group. Notable amongst gums giving rise, on partial hydrolysis with acid, acid)-Dto the aldobiouronic acid 2-O-(~-~-glucopyranosyluronic mannose (38)are those from Prunus species2A series of investigations has been conducted on gums from various types of cherry t ~ e e , " ~ - " ~ and blackthorn' l 7 and apricot1lstrees. On partial hydrolysis with acid, these guns furnish tri- and higher oligo-saccharides having D-glucuronic acid and D-mannose residues, and other products. The oligosaccharides have, however, not yet been characterized in sufficient detail to define the sequences of sugar residues. Similarities to gum ghatti have been indicated in some instances by the partial characterization of neutral oligosaccharides containing (1+6)-linked~-galactosyl residues (39) and, possibly, also of some members of the oligosaccharide series (40) terminated by reducing L-arabinose residues. The gums also furnish 6-O-(P-D-glucopyranosyluronic acid)-D-galactose (7)on partial hydrolysis. Methylation data, taken in conjunction with the partial-hydrolysis results, are consistent with a distribution of the (112) M. Kaplan, A. M. Stephen, and D. Vogt, S. African Med.]., 40,702 (1966). (113) A. M. Stephen and D. C. de Bruyn, Carbohyd. Res., 5,256 (1967). (114) J. Rosik, V. Zitko, S. Bauer, and J. Kubala, Coll. Czech. Chem. Commun., 31, 1072 (1966). (115) J . Rosik, V. Zitko, and J. Kubala, Coll. Czech. Chem. Commun., 31, 1569 (1966). (116) J. Rosik, V. Zitko, S. Bauer, and J. Kubala, Coll. Czech. Chem. Commun., 31, 3353 (1966). (117) J. Rosik, M. Brutenicova-Soskova, V. Zitko, and J. Kubala, Coll. Czech. Chem. Commun., 31,3410 (1966). (118) V. Zitko, J. Rosik, M. Brutenicova, and J. Kubala, Coll. Czech. Chem. Commun., 30,3501(1965).

G . 0. ASPINALL

360

major sequences of sugar units similar to that in gum ghatti, but no information bearing directly on the nature of the connecting linkages is as yet available. The time is clearly ripe for a detailed re-examination of some of the Prunus gums. The gum from Virgilia oroboides has been examined in considerable detail by Stephen and his c ~ l l a b o r a t o r s . ~Units ~ ~ - ~of~ ~the aldobiouronic acid 2-O-(P-D-glucopyranosyluronicacid)-D-mannose (38) are probably present in the interior chains, but the units appear to be joined to D-galactose instead of being mutually linked. The aldobiouronic acids 6-O-(~-D-g~ucopyranosy~uronic acid)-D-galactose (7) and 6-O-(4-O-methyl-~-~-glucopyranosy~uronic acid)-D-galactose (8) are also formed on partial hydrolysis with a ~ i d , ' ~but, ~ , ' as ~ ~in gum ghatti, the dominant features of the polysaccharide are the chains of (1 6)-linked P-D-galactopyranosyl residues to which pentosecontaining side-chains are attached as in 56. However, in contrast to -( 1 + 6)-P-D-Galp-(1 + g)-p-D-Galp-(1 + 6)-p-D-Calp-l

3

4

t

t

R

R (56)

where R = pentose-containing side-chains incorporating units of oligosaccharides 29,57,35,25, and 58.

gum ghatti, no interior L-arabinopyranosyl residues have been detected. The relationship of the dominant chains of the gum to those containing the aldobiouronic acid units and those containing (1+ 3)linked ~-D-galactopyranosylresidues have not yet been established. Virgilia oroboides gum contains a complex array of peripheral units, and controlled, partial hydrolysis with acid affords the disaccharides 5-O-a-~-arabinopyranosyl-~-arabinofuranose (29), 5-O-a-~-arabinofuranosyl-L-arabinofuranose(57),and 5-0-P-D-xylopyranosyl-L-arabinofuranose (35), together with traces of oligosaccharides to which the structures 3-O-a-~-arabinofuranosyl-~-arabinose (25) and O-CZ-Larabinopyranosyl-(1 + 5)-O-~-arabinofuranosyl-(l+5)-~-arabinofuranose (58)have been assigned. A. M. Stephen,]. Chem. SOC., 1919 (1957). F. Smith and A. M. Stephen,]. Chem. SOC.,4892 (1961). A. M. Stephen,]. Chem. SOC.,2030 (1962). A. M . Stephen,]. Chem. SOC., 1974 (1963). J. M. Blair, A. M. Stephen, and D. H. Shaw, ]. S. Africun Chem. Inst., 18, 28 (1965). (124) A. M. Stephen, Curbohyd. Res., 5,335 (1967).

(1 19) (120) (121) (122) (123)

GUMS AND MUCILAGES

36 1

a-L-Araf-(1 + 5)-~-Araf

(57)

a-L-Arap-(1 + J)-~-Araf(1 + 5)-~-Araf

(58)

VI. THEGALACTURONORHAMNAN GROUPOF POLYSACCHARIDES The various polysaccharides that may be classified in this group contain interior chains of residues of D-galacturonic acid and Lrhamnosyl groups. The relative proportions of these two sugars in the inner chains vary, and, in the gums of the Sterculia genus and, possibly, in some of the Khaya gums, residues of D-galactose are also present. In addition to the differences in core structure, entirely different arrangements of other sugars are encountered in the outer chains of the polysaccharides. Amongst the gums, three distinct subgroups may be recognized, namely, those in which the inner chains contain D-galacturonic acid residues almost exclusively, those in which inner chains contain some blocks of D-galacturonic acid units interrupted by regions in which units of both sugars are present, and those in which substantial portions of the interior chains are composed of alternating sequences of the two sugars. Polysaccharides of the three sub-groups also differ in the exterior regions of their structures. The acidic polysaccharide components of certain seed-mucilages constitute a further group of polysaccharides, of which insufficient is yet known to relate them in detail to the gums.

1. Tragacanthic Acid Tragacanthic acid, the main component of gum tragacanth, contains interior chains that approximate most closely to those of a galacturonan.14,164-O-(a-~-Galactopyranosyluronicacid)-D-galacturonic acid (59) and the polymer-homologous trisaccharide 60 have been isolated a-D-GalpA-(1 + 4)-~-GalpA 1 + 4)-~-GalpA a-D-GalpA-(1 + 4)-~-GalpA-(

as products of enzymic hydrolysis of a degraded tragacanthic acid.I6 Only (1+ 4)-linkages have been recognized, although strict linearity of the interior chains has not been rigorously proved, and other types of linkage have not been excluded. The aldobiouronic acid 2-0(a-D-galactopyranosyluronicacid)-L-rhamnose (61) is a minor product a-D-GalpA-(1+ 2)-~-Rhap

(61)

of partial hydrolysis of the polysaccharide with acid, although only a

C . 0. ASPINALL

362

small proportion of rhamnose residues is present.lZ5The polysaccharide is highly branched, and methylation analysis and partial-fragmentation evidence indicate that the side chains contain only one, two, or, possibly occasionally, three sugar residues. L-Fucose is liberated first on mild hydrolysis of tragacanthic acid with acid, and enzymic hydrolysis of the resulting, degraded tragacanthic acid gives 3-O-~-D-xy~opyranosy~-D-galacturonic acid (62), in addition to the P-D-xylp-(1 + 3)-D-GalpA

(62)

afore-mentioned oligomers of D-galacturonic acid.ls Acetolysis of tragacanthic acid provided one of the first examples in polysaccharide chemistry of the relatively greater resistance of L-fucopyranosyl linkages to acetolysis than to hydrolysis with mineral acid; acetolysis resultsls in the liberation of 2-0-a-L-fucopyranosyl-D-xylose (63) and 2-O-P-D-galaCtOpyranOSyl-D-XylOSe (64). The partial structure 65 summarizes the main, structural features of tragacanthic acid. Tragacanthic acid

I

controlled hydrolysis with acid

Degraded tragacanthic acid

I

hydrolysis with “pectinase”

59+ 60 + 62

a-~-Fucp-( 1 + 2)-~-Xy1(63) + P-D-Galp-(1 -D 2)-D-xyl (64)

T

acetolysis, followed by deacetylation

-( 1 + 4)-a-~-GalpA-( 1 + 4)-a-D-GalpA-(1 + 4)-a-~-GalpA-( 1+ 4)-a-~-GalpA-l

3

3

3

t

t

t

1

1

1

(65)

The side chains in tragacanthic acid have subsequently been encountered in the pectin-like polysaccharides from soy-bean cotyled o n ~ and ~ ~ soy-bean hulls.lZs Very small proportions of single D(125) G . O. Aspinall, D. B. Davies, and R. N. Fraser,]. Chem. SOC. ( C ) , 1086 (1967). (126) G. 0.Aspinall, K. Hunt, and I. M. Morrison,]. Chem. SOC. (C), 1080 (1967).

GUMS AND MUCILAGES

363

xylopyranosyl residues have also been found. as side chains to the galacturonan chains in the pectins from lemon pee112 and alfalfa (l~cerne).'~' Tragacanthic acid, and these pectins, also give rise, on partial hydrolysis with acid, to traces of the aldobiouronic acids 4-O-(~-D-glucopyranosy~uronic acid)-L-fucose (66) and variously P-D-G~A-( 1 + 4)-~-Fucp

(66)

linked 0-(D-glucopyranosyluronic acid)-D-galactoses. For tragacanthic acid, the D-glucuronic acid residues are probably present as end group^,^^.^^^ and it has been suggested that a few of the 2-O-a-~-fucopyranos yl-D-xylopyranose and 2-O-P-D-galaCtOpyranOSyl-D-XylOpyranose side-chains may be further substituted by D-glucuronic acid residues . The xylogalacturonan from mountain-pine pollenlo has a marked structural resemblance to tragacanthic acid; it carries single P-Dxylopyranosyl groups as side chains linked (1+ 3) to chains of (1+ 4)linked a-D-galacturonic acid residues. More-extended side-chains (of the type present in tragacanthic acid) appear to be absent, but some preliminary evidence has been obtained for the presence of (1+ 3)linked a-D-galacturonic acid residues, possibly as single-unit side chains. 2, Khaya Gums The exudate gums from Khaya species consist of partially acetylated polysaccharides. The gums contain at least two polysaccharides, and fractionation is most conveniently effected after saponification. The major components of the gums from three species, namely, Khaya grandifoliola,12BK. senegalensis,'* and K. i v o r e n ~ i ~have , ' ~ ~ been examined in considerable detail, and it is clear that the three polysaccharides possess very similar structures. There remains some doubt, however, as to whether complete homogeneity of the polysaccharide preparations has been achieved in each case. Furthermore, the possibility that degradation occurs during saponification of the native gums has been considered, and not definitely excl~ded.'~O The nature of the interior chains in these polysaccharides has been established most fully for the polysaccharide from Khaya ~ U O (127) C . 0.Aspinall, B. Gestetner, J. A. Molloy, and M. Uddin, ]. Chem. SOC. ( C ) , 2554 (1968). (128) G . 0.Aspinall, E. L. Hirst, and N. K. Matheson,]. Chem. SOC.,989 (1956). (129) G . 0.Aspinall and A. K. Bhattachajee,]. Chem. SOC. ( C ) ,in press. (130) C . 0.Aspinall and A. K. Bhattachajee,]. Chem. SOC. (C), in press.

G. 0. ASPINALL

364

rensis gum; this has been shown to give rise, among other products, to 4-O-(cr-D-ga~actopyranosy~uronic acid)-D-galacturonic acid (59), 2-O-(cr-~-galactopyranosyluronicacid)-L-rhamnose (61), and 0-(galactopyranosyluronic acid)-(1 + 2)-O-rhamnopyranosyl-(1 + 4)-0(galactopyranosyluronic acid)-(1 + 2)-rhamnose (67) on partial hyGalpA-(1 + 2)-Rhap-(1 + 4)-GalpA-(1 + 2)-Rha

(67)

drolysis with acid. The presence of much longer sequences of (1+ 4)linked D-galacturonic acid residues is implied by the separation, during the partial hydrolysis, of a degraded D - g a l a c t u r ~ n a n The .~~~ Khaya polysaccharides give rise also to 4-0-(4-O-methyl-a-~-glucopyranosyluronic acid)-D-galactose (10) on partial hydrolysis. The recognition of this second aldobiouronic acid as a partial-hydrolysis product from Khaya grandifooliola gum128provided the first evidence for the presence of both D-ghcuronic acid and D-galacturonic acid in the same polysaccharide. Although no oligosaccharides containing residues of both hexuronic acids have as yet been isolated, their presence as constituents of the same polysaccharide is inferred from the fact that 4-O-methy~-D-glucuronicacid residues are mainly endgroups linked through D-galactose to L-rhamnosyl residues (which provide the main branching-points in the polysaccharides). These observations, together with that of D-galactopyranosyl end-groups, are best accommodated in the partial structure 68. -[(l+ 4)-cr-~-GalpA-l,(1

4)-a-~-GalpA-(l+ B)-~-Rhap-( 1 + 4)-cr-~-GalpA-(l+2 ) ~Rhap-I 4 4

t

1 D-Galp 4

t

1 D-Galp

t

1 4-Me-a-~-GpA

(68)

The major polysaccharide preparations from Khaya senegalensis18 and K . i v o r e n ~ i gums s ~ ~ ~were also shown to contain L-arabinofuranosyl end-groups and 6-0- and 3,6-di-O-substituted D-galactopyranosyl residues. These sugar units are characteristic of those present in polysaccharides of the galactan group. Such a polysaccharide is, indeed, present as a minor component of K . senegalensis gurn,lg and it is not certain that this component was entirely absent from the preparation of the major component. However, in the case of the poly-

GUMS AND MUCILAGES

365

saccharide preparation from K . ivorensis gum,'29both electrophoresis and chromatography on O-(2-diethylaminoethyl)cellulose indicated the absence of such a contaminant. The question of the homogeneity of these polysaccharide preparations cannot be regarded as finally settled.

3. Sterculia Gums The exudate gums from Sterculia species are, at first sight, rather similar to those from Khaya species. They occur naturally, as partially acetylated polysaccharides, and they contain, among other residues, residues of D-galacturonic acid and L-rhamnose in the interior chains, and side chains terminated by D-glucuronic acid (not as the 4-methyl ether) and D-galactose residues. In contrast to the Khaya gums, Sterculia gums thus far examined have been shown to contain only one polysaccharide component, and more detailed studies have indicated that there are marked differences in fine structure between gums of the two groups. Firstly, those regions of the Sterculia gums in which galacturonorhamnan chains are present consist mainly of alternating residues of the two sugars, and adjacent galacturonic acid units can occur only at infrequent intervals. Secondly, interior chains also contain D-galacturonic acid residues linked to D-galactose residues. Thirdly, the sites of attachment of residues of D-glUCUrOniC acid and D-galactose in side chains to the interior chains are quite different from those in the Khaya gums. Kutira gum from Cochlospermum gossypium, a botanical species entirely unrelated to the Sterculiaceae, has a structure essentially similar to those of the Sterculia gums, and may be considered with them. 131- 133

~~ setiEarly studies on Cochlospermum g o s s y p i ~ m , 'Sterculia g e r ~ , ' ~ and ~ . ' ~Stericulia ~ caudata (Brachychiton d i v e r s i f o l i ~ m ) ~ ~ ~ gums showed that each polysaccharide contained D-galactose, Lrhamnose, and a hexuronic acid as constituent sugars. D-Galacturonic acid was recognized as a constituent of the first two polysaccharides, and D-glucuronic acid as a constituent of the last-named. However, re-examinations of these g ~ m s , ~together ~ , ' ~ ~with studies of Sterculia (131) (132) (133) (134) (135) (136)

E. L. Hirst and S . Dunstan,]. Chem. SOC., 2332 (1953). G. 0.Aspinall, E. L. Hirst, and M. J . Johnston,]. Chem. SOC.,2785 (1962). C . 0.Aspinall, R. N. Fraser, and G. R. Sanderson,]. Chem. SOC.,4325 (1965). E. L. Hirst, L. Hough, and J. K. N. Jones,]. Chem. SOC.,3145 (1949). L. Hough and J. K. N. Jones,]. Chem. Soc., 1199 (1950). E. L. Hirst, E. Percival, and R. S. Williams,]. Chem. Soc., 1942 (1958).

366

G. 0. ASPINALL

wens ("karaya") g ~ m , ' ~ ' , ' have ~ * indicated the presence, in each, of the same four constituent sugars, including both D-galacturonic acid and D-glucuronic acid. Although the proportions of the four sugars differ slightly, the main structural features of the four polysaccharides are the same. It is of interest that the first evidence for the presence of D-glUCUrOniC acid in Cochlospermum gossypium gum'32 came from immunochemical cross-reaction with Pneumococcus Type I1 polysaccharide, of which this sugar is a c o n ~ t i t u e n t . 'The ~ ~ earlier claim that D-tagatose is a constituent of Sterculia setigera gum'34 has not been substantiated, and it seems probable that the sugar was formed as an artifact from the inadvertent, basecatalyzed rearrangement of D-galactose. Partial hydrolysis of the polysaccharides from the Sterculia and Cochlospermum gums39~133J37J38 with acid affords, in each case, the acidic oligosaccharides 2-O-(a-D-ga~actopyranosy~uronic acid)-Lrhamnose (61),4-0-(a-D-ga~actopyranosy~uronic acid)-D-galactose (69),3-O-(P-D-glucopyranosyluronicacid-D-galacturonic acid (70), and O-(P-D-glucopyranosyluronicacid)-(1 + 3)-O-(a-D-galactopyranosyluronic acid)-(1 + 2)-~-rhamnose(71).The characterization of the a-D-GalpA-(1 + 4)-D-GalpA fi-~-GpA-( 1 + 3)-D-GalpA 1 + S)-a-D-GalpA-(1 + 2)-~-Rha fi-~-GpA-(

last-named oligosaccharide provides direct evidence for the presence of residues of D-glucuronic acid and D-galacturonic acid in the same polysaccharides. A further oligosaccharide, namely, 0-(galactopyranosyluronic acid)-(1+ 4)-0-(galactopyranosyluronicacid)-(1-+ 2)-rhamnose (72),has been reported as a product of the partial hydrolysis of CalpA-(1 + 4)-GalpA-(1 + 2)-Rha

(72)

Sterculia c a ~ d a t aand ~ ~S . ~ e t i g e r a 'gums, ~ ~ and of one sample of S. wens gum,13' but in no instance has the oligosaccharide yet been fully characterized by the formation of a crystalline derivative. The characterization of these oligosaccharides as products of the partial hydrolysis of the four gums, together with the identification of largely the same cleavage products from the methylated polysacc h a r i d e ~ , ~ established ~ J ~ ~ J ~ ~the close structural similarity between the polysaccharides. More-detailed partial structures may be proposed for Sterculia wens gum in the light of the characterization of the (137) G. 0.Aspinall and NasiruddinJ. Chem. Soc., 2710 (1965). (138) C . 0.Aspinall and G. R. Sanderson, unpublished results. (139) M . Heidelberger, Fortschr. Chem. Org. Natursto$e, 18,503 (1960).

GUMS A N D MUCILAGES

367

acidic trisaccharide 0-D-galactopyranosyl-( 1 + 2)-a-D-galactopyranosyluronic acid-(1+ 4)-D-galaCtOSe (73),formed on partial a c e t o l y ~ i s , ' ~ ~

and of studies on the carboxyl-reduced poly~accharide.~~ Methylation studies on Sterculia urens gum showed that almost all of the D-galactopyranosyl residues are present as end groups or as 4-0-substituted units, and the results of hydrolysis of the periodateoxidized gum indicated that all but traces of the D-galactose residues are cleaved by the reagent. Accordingly, application of the Smith degradation to the carboxyl-reduced gum led to the isolation of fragments containing D-galactosyl residues that had originated only from residues of D-galacturonic acid. The main reaction-product, a degraded polysaccharide, was shown to consist largely of alternating 4-0-substituted D-galaCtOSyl residues (from residues of D-galacturonic acid) and 2-0-substituted L-rhamnosyl residues. Table I11 shows the oligosaccharides that have been characterized as products of partial acetolysis of (a) the carboxyl-reduced gum, and (b) the degraded polysaccharide formed from the Smith degradation. The major part of the polysaccharide from Sterculia wens gum may be represented by the two partial structures 74 and 75. L-Rham-(1 + 4)-a-D-GalpA-( 1 + 2)-~-Rhap-( 1 --* 4)-a-~-GalpA-( 1 + 2)-~-Rhap-l 4 3 4 3 t t 1 P-D-G~A

1 P-D-G~A

-( 1+ 4)-a-~-GalpA-( 1 + 4)-D-Galp-l

1 D-Calp (75)

nopyranosyl end-groups have not yet been placed, but, as most of the L-rhamnosyl residues in the interior chains carry side chains at C-4, these positions are the most probable sites for attachment. The two

m

TABLE111 Oligosaccharides from Carboxyl-reduced Sterculia wens Gum

Acetolysis of carboxyl-reduced Sterculia wens guma Gal'-(1 + 2)-Rha Rha-(1 + 4)-Gal" Gal"-(1 + 2)-Rha-(l + 4)-CalU Rha-( 1+ 4)-Gal"-( 1+ 2)-Rha

Acetolysis of polysaccharides from Smith degradation of carboxyl-reduced Sterculia wens gum Gal"-(1 + 2)-Rha Rha-( 1+ 4)-Gal' Cal"(1 + 2)-Rha-(l+ 4)-Cal" GaI"-(l + 2)-Rha-(1+ 4)-Ga1"-(1+ 2)-Rha

Rha-(l+ 4)-Ga1"-(1+ 2)-Rha-(l+ 4)-Gal"

Gal*-( 1

--j

2)-Rha-(1+ 4)-GaIu-(1+ 2)-Rha-(1+ 4)-GaI0

Gal-( 1-+ 2)-Gal" Gal'-(1 + 4)-Gal Gal41 -+ 2)-Gal"-(1 + 4 X a l "Gal' represents D-galactose residues that may b e inferred directly (from resistance to oxidative cleavage by periodate) or indirectly (from known substitution patterns or from the previous characterization of the corresponding acidic oligosaccharides) to have arisen from the reduction Of D-gahChrOniC acid residues.

0 P %

?

cr

GUMS AND MUCILAGES

369

partial structures accommodate the known branching in the gum, and it is likely that the interior chains are continuous with each other, but direct evidence on this point is not yet available. 4. Acidic Mucilages and Associated Neutral Polysaccharides The acidic polysaccharides of bark and seed mucilages are of considerable complexity, and chemical investigations have been complicated by difficulties in fractionation. Graded precipitation with cupric acetate and with ethanol has indicated the presence in several mucilages of at least two poly~accharides,'~~ and this procedure has been used for the preparative fractionation of linseed mucilage.20*21 With the exception of one of the components from linseed,21none of the acidic polysaccharides have been shown to be homogeneous. Nevertheless, further information is available on the nature of the components of linseed and cress-seed mucilage, and there is reasonable circumstantial evidence that the core structures of the acidic polysaccharides contain residues of 4-0-substituted D-galacturonic acid and 2-0-substituted L-rhamnosyl groups, with the latter providing the main branching-points. A highly branched arabinoxylan accompanies the two acidic polysaccharides of linseed mucilage. This polysaccharide contains D-xylopyranosyl end-groups,20some of which are attached to residues of L-arabinose, probably by (1 -+ 4)- or (1 + 5)-linkages. Although L-arabinosyl residues have been detected only in nonterminal positions, they are probably located in exterior chains, as the Smith degradation leads to the isolation of a degraded xylan containing only (76) has traces of L-arabinose units.214-O-~-D-Xy~opyranosy~-D-xy~ose

been isolated on partial hydrolysis of the arabinoxylan,20*21 but the afore-mentioned, degraded xylan is still highly branched, and it is apparent that the core structure is more complex than that in arabinoxylans of the hemicellulose t y ~ e . ~ Both * ~ Oof the acidic polysaccharides from linseed mucilage contain residues of L-galactose, a rare constituent of plant polysaccharides.21L-Fucose is a constituent of one polysaccharide in which residues of this sugar and of L-galactose are present solely as nonreducing end-groups.21 Homogeneous polysaccharides have not yet been obtained from (140) A. J. Erskine and J . K. N. Jones, Can.J. Cliern.,34,821 (1956).

370

G. 0. ASPINALL

cress-seed mucilage, but the isolation of apparently homogeneous, the basis for the assessmethylated p o l y s a ~ c h a r i d e shas ~ ~ *provided ~~ ment of the structural significance of oligosaccharides formed on partial hydrolysis of the unfractionated rnucilageaz2The neutral polysaccharide is the first example of a xyloarabinan in which Dxylopyranosyl end-groups are attached to an arabinan core. The characterization of 3-O-c~-D-xylopyranosyl-~-arabinose (32)and O - ~ - D xylopyranosyl- (1 + 3)-O-a-~-arabinofuranosyl-(1 + 3)-~-arabinose (77) on partial hydrolysis with acid, together with methylation data, a-D-Xylp-(1 + 3)-a-L-haf-( 1 + 3)-L-ka

(77)

is consistent with the partial structure (78) for the polysaccharide, in which the core is similar to that of mustard-seed arabinan.141J42 -( 1+ li)-a-~-Ara-( 1 + 5)-a-~-Araf-(1+ 5)-a-~-Araf-l

3

3

t

t

1 a-L-Araf 3

1 a-L-Araf 3

t

t

1 a-D-Xylp

1 a-D-Xylp (78)

Two partial structures, representing major portions of interior (79) and exterior (80) chains, respectively, of the cress-seed acidic polysaccharide, may be proposed on the basis of the partial separation of acidic oligosaccharides formed on partial hydrolysis of the methylated 4-O-a-D-XylopyranOSyl-D-galaCtOpyranOSe units poly~accharide.~~ (81) form a quantitatively less-important type of side chain, and the 1(1 + 4)-D-GalpA-(l+ 4)-~-GalpA-( 1-+ 2)-~-Rhap-l 4

1 -+ 4)-~-Galp-l 4-Me D-G~A-( cU-D-xylp-(l -+ 4)-~-Galp-l (141) E. L. Hirst, D. A. Rees, and N. G. Richardson, Biochem.J.,95,453 (1965). (142) D. A. Rees and N. G. Richardson, Biochemistry, 5,3099 (1966).

(79)

GUMS AND MUCILAGES

371

disaccharide itself has been identified on partial hydrolysis of the mucilage with acid.22The arrangement of residues of D-galacturonic and 4-0-methyl-D-glucuronic acid indicates a close structural similarity to the main polysaccharide components of the Khaya gums. Slippery-elm mucilage is a galacturonorhamnan having side chains of D-galactose and 3-0-methyl-D-galactose residues2 Studies'42ahave shown that the polysaccharide is more highly branched than previously supposed, and chains of 3-O-methyl-~-galactoseresidues are linked to L-rhamnose residues in the interior chains.

VII. THE XYLAN GROUPOF POLYSACCHARIDES Relatively few examples of gums based on inner chains of D-xylopyranosyl residues have been reported, and, of these, only two polysaccharides have been studied in any detail. These two gums are structurally related to, but are more complex than, the xylans that occur universally as cell-wall components in higher plants. In the case of sapote gum (from the Peruvian tree Sapota achrus), the relationship to the acidic xylans from woods and other plant^^.'^ is apparent from the characterization of the two trisaccharides O-(a-Dglucopyranosyluronic acid)-(1 + 2)-O-P-~-xylopyranosyl-(1 + 4)-Dxylopyranose (82) and the corresponding 4-methyl ether (83),formed CY-D-G~A-( 1 + 8)-p-D-xylp-( 1 + 4)-D-Xylp 4-Me-a-D-GpA-( 1 + 8)-p-D-xylp-(1 + 4)-D-xylp

(82)

(83)

by partial hydrolysis with A high degree of branching in the interior of the molecular structure was shown earlier144in methylation studies which established the presence of end groups of L-arabinopyranose, D-xylopyranose, and D-glucuronic acid (or its 4-methyl ether). An unusual feature of the polysaccharide is the occurrence of 2-0-substituted D-glucuronic acid units, although the nature of the substituents is not known. It seems probable, however, that the structure of the gum is more complex than is at present recognized. The polysaccharides from the corm sacs of Watsonia pyramiduta (andr.) S t a ~ f . and ' ~ ~ from the seed boxes of Watsonia v e r ~ v e l d i i ' ~ ~ (142a) R. J. Beveridge, J. F. Stoddart, W. A. Szarek, and J. K. N. Jones, Carbohyd. Res., 9,429 (1969). (143) R. D. Lambert, E. E. Dickey, and N. S. Thompson, Carbohyd. Res., 6,43 (1968). (144) E. V. White,]. Arner. Chern. SOC., 75,257,4692 (1953);76,4906 (1954). (145) D. H. Shaw and A. M. Stephen, Carbohyd. Res., 1,400 (1966). (146) D. H. Shaw and A. M. Stephen, Carbohyd. Res., 1,414 (1966).

C. 0. ASPINALL

372

appear to function as food reserves. Although the polysaccharides are not exudates in the strict sense, they exhibit gum-like properties and, on a structural basis, may be included in the xylan group. The former polysaccharide is neutral, but the latter carries glucuronic acid residues as end groups. The presence of (1+ 4)-linked chains of P-D-xylopyranosyl residues in the Watsonia pyramidata polysaccharide was shown by the isolation, on partial hydrolysis with acid, of 4-0-P-D-xylopyranosyl-Dxylopyranose (76), the corresponding xylotriose (84), higher oligo@-D-XyIp-( 1 -D 4)-p-D-xylp-(l-D 4)-D-xylp

(84)

saccharides of the same series, and an essentially linear, degraded ~ y 1 a n . lFrom ~ ~ the methylation data, it is apparent that branching occurs through C-2 and C-3 of the majority of the D-xylosyl residues. Unique structures for the side chains cannot as yet be defined. However, in view of the high proportion of L-arabinofuranosyl end-groups in the polysaccharides, and of the characterization of 3-O-a-D-galactopyranosyl-L-arabinose (1)and 0-a-D-galactopyranosyl-(1 + 3)-0-a-~arabinofuranosyl-(1 + 2)-~-arabinose (85) as further products of a-D-Galp-(1 + 3)-a-~-Araf-(1 + 2)-~-Ara

(85)

partial hydrolysis, the side chains must include, as the more important types, single L-arabinofuranose residues and units of the trisaccharide 85 having the innermost residue in the furanose form. The main features of the polysaccharide may, therefore, be included in the partial structure 86. R

R

R

J.

J.

.1

3 3 3 -(1 + 4)-p-D-xylp-(l + 4)-p-D-xylp-(1 -D 4)-p-D-xylp-l 2 2 2

t

t

R

R

t

R

(86)

where R = L-Araf-1, or a-D-Galp-(l-D3)-~-Ara$(1 + 2)-~-Araf-l.

VIII. THE XYLOCLUCANGROUPOF POLYSACCHARIDES A gel-forming polysaccharide may be isolated from the thickened cell-walls of the cotyledons of the seeds of tamarind (Tamarindus

373

GUMS AND MUCILAGES

indica) by extraction with boiling ~ a t e r . ’ ~ ’ .This ’ ~ ~ polysaccharide is the most extensively examined of a group of substances (from plant seeds149)to which the term “amyloid” has been applied to indicate a characteristic, blue stain formed on reaction with iodine. The term “amyloid” is, however, a misnomer, as the polysaccharides bear no structural resemblance to amylose. Structure 87 has been proposed for the tamarind-seed polysac-( 1 + 4)-p-~-Gp-( 1 + 4)-b-~-Gp-( 1 -+ 4)-p-~-Gp-( 1 + 4)-P-~-Gp-1

6

6

6

f

f

f

1

Cr-D-xylp 6

1

1

U-D-Xylp

O-D-xj’lp

f 1 P-D-Galp (87)

charide on the basis of methylation s t ~ d i e s ’ ~and ’ partial fragmentation.14*Partial hydrolysis with acid gives 2-0-P-D-galactopyranosylD-xylose (64), acetolysis affords cellobiose (88),and enzymic hydrolysis p - ~ - G p -1(+ 4)-D-Gp

(88)

with cellulase furnishes 6-0-a-D-xylopyranosyl-D-glucose (89).Addi-

tional evidence for a cellulose-like, glucan core was obtained by the isolation, after partial hydrolysis, of an insoluble, degraded polysaccharideI5Owhich gives an x-ray diffraction diagram almost identical with that of cellohexaose, and resembling that of cellulose 11. An examination of another sample of the tamarind-kernel polysaccharide has given markedly different results, and it must be concluded that this preparation represents a distinct poly~accharide.’~’ The structural features outlined in the partial formula 87 are clearly present, as shown in a similar series of experiments. The composition of this polysaccharide is, however, clearly different from that reported (147) (148) (149) (150) (151)

E. V. White and P. S. Rao, J . Amer. Chem. SOC., 75,2617 (1953). P. Kooiman, Rec. Truo. Chim., 80,849 (1961). P. Kooiman, Actu Botan. Neerl., 9,208 (1960). P. Kooiman a n d D. R. Kreger, Biochirn. Biophys. Actu, 26,207 (1957). H. C. Srivastavaand P. P. Singh, Curhohyd. Res., 4,326 (1967).

374

G . 0. ASPINALL

for other preparations, and L-arabinose is a constituent sugar. Residues of this sugar are present as the furanose form in end groups, and may be attached as single-unit side chains, as in the partial structure 90. Furthermore, D-galactosyl residues occur in more than one

structural environment, since lactose (91) is formed as an acetolysis product. p-D-calp-(1 + 4)-D-cp

(91)

Two further xyloglucans of the same general type have been isolated from the seeds of Annona muricata and nasturtium (TTopeoleum m a j ~ s ) . The ' ~ ~ former polysaccharide gives with iodine a brownish violet instead of a blue stain, and is soluble in dilute alkali but not in water. Chemical studies have shown that the polysaccharide is intermediate in structure between the tamarind polysaccharide and cellulose. The glucan chain carries, on approximately every fourth residue, a 2-O-~-D-galactopyranosyl-a-D-xylopyranose unit attached as in the tamarind polysaccharide. The nasturtium poly~accharide'~~ has a composition similar to that of that from ~~ tamarind, examined by White and Ra0,14' and K o ~ i m a n . ' The chemical evidence, although less complete, suggests a similar structure. Is3 Polysaccharides of the xyloglucan may be of relatively widespread occurrence, and examples have been reported from the bark of Engel'~~ compression-~ood,'~~ mann spruce (Picea e n g e l m ~ n n ) ,red-spruce ' ~ ~sycamore callusthe seeds of white mustard (Brassica ~ l b a ) ,and cells.'57 The mustardIs6 and ~ycamore'~' polysaccharides are charac(152) P.Kooiman, Phytochernistry, 6, 1665 (1967). (153) D.4. Hsu and R. E. Reeves, Carbohyd. Res., 5,202(1967). (154) K. V. Ramalingham and T. Timell, Svensk. Papperstidn., 67,512(1964). (155) H.R. Schreuder, W. A. Cote, Jr., and T. E. Timell, Suensk Papperstidn., 69, 641 (1966). (156) S. E. B. Could and D. A. Rees, unpublished results; S. E. B. Could, Ph.D. Thesis, Edinburgh, Scotland (1965). (157) G . 0.Aspinall, J. W. T. Craig, and J. A. Molloy, Can.J.Biochern., in press.

375

GUMS A N D MUCILAGES

terized by the presence of L-fucose instead of, or in addition to, D-galactose. The mustard polysaccharide gives a blue stain with iodine,156but the sycamore polysaccharide gives no c~loration.'~' These polysaccharides have, so far, been subjected to less-detailed chemical study. The sycamore polysaccharide, which is elaborated by cells grown in tissue culture, may, although of unproved homogeneity, represent a further structural type, because degradation by a Streptomyces cellulase gives both cellobiose (88) and xylobiose (76),indicating the presence of chains of both the cellulose and the xylan type.'57

IX. OTHERPOLYSACCHARIDES The majority of polysaccharides considered in previous Sections of this Chapter have been examined in sufficient detail to permit their classification, at least provisionally, in terms of basal chains of sugar residues, and hence as members of a relatively small number of families of related molecular species. The exudate gum from the Nigerian tree Combretum Z e o n e n ~ e ~has ~*" been ~ studied in considerable detail, and, on the basis of methylation and partial-hydrolysis data, the partial structures 92 to 94 have been proposed. However, the relationR

R

4

.1

3 6)-P-D-Galp-(1 + B)-P-o-Galp-(1 + 6)-p-~-Galp-l 4 3

-( 1

-+

(94) where R includes the units L-Araf-I, p-D-Galp-(l Araf-I, and -(1+ 5)-~-Araf-lor -( 1 + 4 ) - ~ - A r a p - l .

+ 3)-~-Araf-l,p-L-Arap-(l

-+

3)-L-

ship between these portions of the molecular structure has not yet been established, and it is not yet clear which units form the basal chains.

376

C . 0. ASPINALL

The Combretum and Anogeissus genera are from the same botanical family (Combretaceae),and the structures of Combretum leonense gum and gum ghatti (from Anogeissus latifo2ia) provide an interesting comparison. The partial structures 92 and 93 for Combretum leonense gum are clearly similar to areas in the structure 44 of gum ghatti. In gum ghatti, however, such units are attached, through L-arabinopyranosyl residues, to interior glucuronomannan chains. Such interior chains do not occur in Combretum leonense gum, and it is tempting to suggest that they are replaced by chains (94) in which units of the acid)-L-rhamnose aldobiouronic acid 2-O-(cY-D-galaCtOpyranOSylUrOniC (61) are present. In this connection, it may be noted that at least some of these aldobiouronic acid units, in contrast to those in the galacturonorhamnans discussed in an earlier Section (see p. 361), are flanked by D-galaCtOSyl residues as in 94. A further indication of the similarity of Combretum leonense gum to gum ghatti was provided by the isolation of 3-0-p-D-galactopyranosyl-L-arabinose (40, n = 0 ) in small proportion as a product of partial hydrolysis. However, no higher oligosaccharides of this series were detected, and no 3-0-substituted L-arabinopyranosyl residues were found of the type that, in gum ghatti, acts as “link” units between internal glucuronomannan chains and the galactose-containing sidechains. The disaccharide 40 ( n = 0), therefore, probably arises from an entirely different structural environment, namely, from 3-0-p-~galactopyranosyl-L-arabinofuranosyl units in peripheral chains. On the basis of oligosaccharides formed on partial hydrolysis with acid, Combretum leonense gum most closely resembles the complex, acidic polysaccharide isolated from beech t e n s i o n - ~ o o d .The ’ ~ ~ products of partial hydrolysis of these two polysaccharides are shown in Table IV. Although the beech polysaccharide is of unproved homogeneity, the isolation of the trisaccharide O-~-D-galactopyranosyl(1 + 6)-0-/3-D-galactopyranosyl-(1+ 4)-D-galactose (96) provides the only evidence to date for the presence of (1 + 4)- and (1+ 6)-linkages between D-galaCtOSyl residues in the same polysaccharide.

X. CONCLUSIONS The classification of exudate gums and mucilages in terms of basal chain-types provides a convenient framework for the consideration of polysaccharides of highly complex structure. Such a scheme also serves to emphasize the relationship of these to other groups of plant (158) H. Meier,Acta Chem. Scand., 16,2275 (1962).

TABLEIV Partial-hydrolysis Products from Combretum leonense Gum and Beech Tension-wood Polysaccharide

C . leonense Oligosaccharides P-L-Arap-(1 + 3)-~-Ara(2) P - ~ - G a l p1( + 3)-~-Ara(40, I I = 0) P-D-Galp-[(l + S)-P-D-Galp-I],,(I + 6)-D-Cal (39, n = 0,I, and 2) P-D-Galp(1 4 4)-D-Galp (95) P-D-Galp-(1+ 6)-~-Galp-( 1 + 4)-D-Galp (96) p-~-GpA-(l+ 6 ) - ~ - C a (7) l 4-Me-P-D-C)iA-(1 + 6)-D-Gal (8) a-D-GalpiA-(l + 2)-~-Rha(61) GalpA-(I + 2)-Rhap(l + 4)-Galp (97) Galp(1 + 4[or 3])-GalpA-(1 + 2)-Rha (98)

gum

++ + +

+ + + +

Beech polysaccharide

+ + + + +

2

5

%U K

5 P

$

E

378

G . 0. ASPINALL

polysaccharides; for example, the galactan group of gums with the galactans and arabinogalactans from coniferous woods, the galacturonorhamnans with pectins, and the xylan group of gums with the hemicelluloses of higher plants. Indeed, the recognition of “unusual” structural features in gums has, on several occasions, led to the discovery of similar units in other, apparently less complex, polysaccharides. In general, exudate gums and the acidic components of seed mucilages are structurally more complex than other groups of plant polysaccharides, and the suggestion has been made that the formation of gums involves the apposition of additional sugar residues to the outer chains of polysaccharides already present in the plant (for example, as cell-wall components) and not de m o o synthesis.6 Supporting evidence for this hypothesis is, however, lacking since no obvious examples of “precursor” polysaccharides have as yet been encountered. Arabinogalactans are characteristic components of coniferous woods, but only one example of an exudate of similar, but more complex, structure has been reported from such a tree, namely, bidwiZlii).11,s9Jo’ In this instance, examinafrom Bunya pine (Arauca~ia tion of the has failed to reveal the presence of an arabinogalactan, Conversely, gums of the galactan group are exuded by many deciduous trees for which arabinogalactans have not been detected as wood components. The ability of a deciduous wood species to synthesize a polysaccharide of this type has been demonstrated; one of the polysaccharides elaborated by sycamore callus-cells has been shown to be an arabinogalactan structurally similar to those from coniferous An insufficient number of exudate gums has been studied in detail adequate for assessment of their potential in the chemical taxonomy of plants. Nevertheless, in previous Sections, a number of examples have been mentioned of structural similarities between gums from botanically related species. The Acacia genus provides the most striking example of related species that give rise to gums of the same general type, with many structural features in common.159a At the same time, substantial differences, both in overall composition96and in detailed structure,’60are apparent. Nevertheless, it is clear that certain botanical species, regarded as closely related on the basis of simi(159) G . 0. Aspinall and J. P. McKenna, unpublished results; J. P. McKenna, Ph.D. Thesis, Edinburgh, Scotland (1967). (159a) For a discussion of chemotaxonomic aspects of Acocio gum exudates see D. M. W. Anderson and I. C. H. Dea, Phytochemistry, 8, 167 (1969). (160) M. Kaplan and A. M. Stephen, Tetrahedron, 23,193 (1967).

GUMS AND MUCILAGES

379

larities in morphological characteristics, exude gums in which those chemical features most liable to variation are essentially the same. The major polysaccharide components of the respective exudates from the Sterculia and Khaya genera have not yet been shown to differ significantly from one species to another. The situation in the latter genus is, however, complicated, in one instance at least (namely, Khaya senegalensis'8~'s)by the presence in the gum of two polysaccharide components belonging to entirely different structural groups. The gums from Prunus species differ chemically, in that certain gums only give rise to the aldobiouronic acid .%O-(p-D-glucopyranosyluronic acid)-D-mannose (38) on partial hydrolysis. Insofar as units of this disaccharide arise from the basal chains of certain polysaccharides, the presence or absence of these units may reflect deep-seated differences in fundamental structure. In this Chapter, stress has been laid on the classification of polysaccharides according to basal chain-type. It should not, however, be forgotten that other gums show marked similarities in outer-chain structures, although these chains may be attached to quite different inner chains. In some cases, the presence of similar linkages in peripheral chains (for example, 3-O-a-~-galactopyranosyl-~-arabinofuranosyl units in gum arabics1 and in the corm-sac polysaccharide ~ ~ have little significance. On the from Watsonia p y r a m i d ~ t a 'may other hand, more serious account must be taken of substantial regions of structural similarity in outer chains. An obvious example is provided by the chains of (1 + 6)-linked p-D-galactopyranosyl residues in gum ghatti'OS and Combretum Zeonense gum,29which arise from trees in related genera. The relationship between molecular structure and physical propertiesI6' of polysaccharides as highly branched as are the majority of exudate gums has not been explored in any detail, but it is quite possible that the physical properties required of gums for the exercise of their biological function are determined primarily by the structure and macromolecular conformations of the outer chains. In this event, classification of certain polysaccharides according to outer chain-type would be more appropriate than any other.

(161) D. A. Rees, Adunn. Carbohyd. Chem., 24,267 (1969).

This Page Intentionally Left Blank

GLYCOSPHINGOLIPIDS (SUGAR-SPHINGOSINE CONJUGATES)

BY J. KISS* Chemical Research Department, F. Hoffmann-La Roche 6.Co., Ltd., Bade, Switzerland

I. Introduction . . . . . . . ............................................. 382 11. Nomenclature of Glyc hingolipids ................................. 383 111. Some Stereochemical Aspects of Sphingosines and Phytosphingosines ................................................... 384 1. Configurational Correlation of C,,-Sphingosine with D-GhCOSe (D-Glyceraldehyde) and L-Serine ................................... 385 2. Stereochemical Correlation of Phytosphingosines and Dehydrophytosphingosines with C18-Sphingosine and with 2-Amino-2-deoxy-~-g~ucose and D-Galactose ............... IV. Natural and Synthetic Sphingosines, Dihydrosphingosines, and 390 Phytosphingosines of Different Chain-lengths .......................... V. Biosynthetic Pathway of Sphingosines and of Glycosphingolipids . . . . . . . 394 VI. Ceramide Mono- and Oligo-saccharides ............................... 395 1. Galacto- and Gluco-cerebrosides ................................. 395 2. Ceramide Oligosaccharides ......................................... .400 3. Synthesis of Ceramide Mono- and Oligo-saccharides . . . . . . . . . . . . . . . . .401 VII. Sulfatides.. ........................................................ 403 403 1. Ceramide Monoglycosyl Sulfates .................................. 2. Ceramide Diglycosyl Sulfates ..................................... 406 3. Synthetic Studies in the Sulfatide Group. . . . . . . . . . . . . . . . . . . . . . . . . . . 407 VIII. Glycosphingolipids of Plant Materials and Micro-organisms (Phyto- and Myco-glycosphingolipids) ....... ..................... 408 IX. Gangliosides.. ...................................................... 413 1. Isolation and Purification of Gangliosides ....................... 2. Sialic Acids of the Gangliosides ................................ 3. Linkage between N-Acylsphingosines and the Oligosaccharide 421 Moiety ........................................................... 4. Structure of the Oligosaccharide Members . . . .................... 423 5. Gangliosides Isolated from Pathological Tissues ..................... 428 6. Gangliosides of Erythrocytes and Spleen ........................... 432

*Visiting research scientist in the Max-Planck Institut fur Medizinische Forschung, Heidelberg, Germany, 1964. 38 1

382

J. KISS

I. INTRODUCTION Glycosphingolipids are a group of sphingolipids containing monoor oligo-saccharide residues. The sugar residues are in direct or indirect glycosidic conjugation with the primary hydroxyl group of the N-acylated sphingosine or dihydrosphingosine (sphinganine) molecule, as in formula 1. Most of the glycosphingolipids can be described by the general formula 1. H,CO-sugar I HCNH-fatty acid I HCOH I R Glycoephingolipid (1) where R = aliphatic chain with or without a double bond, or a hydroxylated aliphatic chain with or without a double bond.

In Nature, only a relatively small number of glycosphingolipids exist in which (a) the secondary hydroxyl group of the sphingosine moiety is in covalent conjugation with fatty acids or with fat-aldehyde residues, or (b) the sugars are connected to the long-chain, acylamido polyhydric alcohol through a phosphatidylinositol bridge. Although the first glycosphingolipids were isolated from the tissues of the central nervous system of mammals, it was recognized many years ago that these groups of substances are general constituents of other organs in other classes of the animal kingdom (for example, birds, fishes, and insects) as well. Furthermore, micro-organisms, mushrooms, and plant materials (such as seeds and leaves) also contain similar substances. The structural variation of the natural glycosphingolipids is great. First, the fatty acids, bound on the amino group of the sphingosine, may differ, for example, in chain length, in degree of unsaturation, and in a-hydroxy content. Second, the chemical structure of the longchain, amino alcohol skeleton (sphingosine and its dihydro derivative) can also be varied. To date, about 20 such natural bases have been isolated. Third, the number of kinds of sugar constituent in the zooglycosphingolipids is relatively small, but it is rather large in the phyto-sphingolipids. It is interesting that the structural alteration of the glycosphingolipids as between normal and pathological mammalian organisms seems to be a very sensitive parameter.

GLYCOSPHINGOLIPIDS

383

From the point of view of solubility characteristics, the fatty acid group and the sugar moiety in the sphingosine moiety have opposing effects. Thus, the long-chain fatty acids are extremely soluble in solvents for fats, and have a strongly hydrophobic character. On the other hand, the sugar moiety of the glycosphingolipids represents the hydrophilic part of the molecule. The solubility in water is also dependent on the number of monosaccharide residues bound in the molecule. Furthermore, such strongly anionic components as neuraminic acids, and sulfate or phosphate groups, influence the ratio of the solubility in water and fat. These two kinds of solubility characteristics are probably essential in the biological roles of the glycosphingolipids when they function as biomembrane components. On the basis of these properties, the glycosphingolipids can be classified as follows. 1. Neutral glycosphingolipids (for example, zoo- and phyto-cerebrosides, diglycosylceramides, esters of cerebrosides with fatty acids, sphingoplasmogens, and gangliosides free from neuraminic acid). 2 . Anionic (acidic)glycosphingolipids (a) Anion-center inorganic residue (for example, sulfatides having a mono- or di-saccharide moiety, and phytoglycosphingolipids having a phosphate residue). (b) anion-center organic residue (gangliosides having one or more neuraminic acid components). 3. Ambident-ion sphingolipids (sphingomyelins) containing no sugar residues. OF GLYCOSPHINGOLIPIDS 11. NOMENCLATURE

For many years, it has seemed desirable to create a generally useful nomenclature for glycosphingolipids. The old names do not always sufficiently characterize the compounds to be designated, often leading to confusing and contradictory names.' Some years ago, Celmer and Carter2proposed a rational nomenclature for cerebrosides and phosphatides, but Klenk and coworkers3 kept the old names in grouping the zoo-sphingolipids, which were denoted as: (a) cerebrosides, (b) sulfatides, and (c) gangliosides. The IUPAC-IUB Commission on Biochemical Nomenclature has proposed4 that the individual lipids be denoted. For substances belonging to the group of the glycosphingolipids, names that are (1) (2) (3) (4)

H. E. Carter, P. Johnson, and E. J. Weber, Ann. Reu. Biochem., 34,109(1965). W. D.Celmer and H. E. Carter, Physiol. Reu., 32,167(1952). E.Klenk, Biochem. Z . , 335,423(1962). E.Klenk,]. Lipid Res., 8,522(1967);Biochim. Biophys. Acta, 152,1(1968).

J. KISS

384

mainly semisystematic were given, because, in this field, completely systematic names are often very complicated. This tentative nomenclature, which is partly new, provides excellent names for the various compounds, but as these new names (for example, sphinganine for C18dihydrosphingosine, and 4-~-hydroxysphinganine for C18-phytosphingosine) are not yet well known, they will be added in parentheses after the old ones.

111. SOMESTEREOCHEMICALASPECTS OF SPHINGOSINES AND PHYTOSPHINGOSINES The fundamental skeleton characteristic of the glycosphingolipids of animal and plant origin, in which a mono- or an oligo-saccharide together with the fatty acid residues are bound covalently, are denoted ~ ~ phytosphingosine (4-~-hydroxysphinganine), as s p h i n g ~ s i n eand respectively. The first sphingosine preparation was obtained in 1880 by Thudichum5 by hydrolytic cleavage of human-brain material (mainly cerebroside mixtures). After the first structural representation of this long-chain, amino alcohol had been proved erroneous, Carter and coworkerss*’proposed the correct structure, namely, 2-amino-4-octadecene-l,O-diol(2). CH,OH

~ N H ,

I HCOH

I

HC II CH I

(pLZ

CKOH H+NH2

HCOH I

7% ?Ha (?H2)U

CH,

CH,

Sphingosine

Dihydrosphingosine

(2)

(3)

(4a) The name “sphingosine,” proposed by Thudichum? is derived from the Greek verb sphingein, to bind or squeeze. This word is also etymologically related to the name of the mysterious, archaic statue called the Sphinx, the symbolic representation in Egyptian and Greek mythology of power and law, and, furthermore, of wisdom and omniscience. (5) J. L. W. Thudichum, a pupil of J. Liebig’s, put brain chemistry, which was already in the foreground of interest in the 18th and 19th centuries, on an exact basis. See J. L. W. Thudichum, “A Treatise on the Chemical Constitution of the Brain, Based Throughout upon Original Researches,” Bailliere, Tindall, and Cox, London, 1884. Also, “Die Chemische Konstitution des Menschen und Tiere,” Verlag F.Pietzcker, Tiibingen, 1901. (6) H. E. Carter, F. Click, W. Norris, and G . E. Phillips,./. Biol. Chem., 142,449 (1942); 170, 285 (1947). See also, M. W. Goldberg and P. V. Seydel, “Zur Kenntnis des Sphingosins,” Dissertation, Eidgenossische Technische Hochschule, Zurich, 1941. (7) For a historical review of the early literature, see: H. Thierfelder and E. Klenk, “Die Chemie der Cerebroside und Phosphatide,” Verlag Springer, Berlin, 1930.

GLYCOSPHINGOLIPIDS

385

The corresponding saturated derivative, dihydrosphingosine (3) (sphinganine or 2S,3R-2-amino-1,3-octadecanediol), can be obtained from sphingosine by saturation of the ally1 double bond with hydrogen. Dihydrosphingosine exists in Nature in the form of N,1-0disubstituted derivatives (for example, dihydrocerebrosides and dihydrosulfatides). It is interesting that both of these long-chain aminodiols, and their derivatives, have a similar spatial disposition, and therefore they can build into the crystal lattices of each other, giving mixed crystals.8 The geometry of the double bond of sphingosine and of some natural sphingosine derivatives has been The diastereoisomeric relationship of the amino and 3-hydroxyl groups is erythro; this was proved by degradation and synthesis.12 Sphingosines and dihydrosphingosines do not exist in the free state in animals, plants, or micro-organisms.

1. Configurational Correlation of C18-Sphingosinewith D-Glucose (D-Glyceraldehyde) and L-Serine The degradation of N-acetyl-di-0-acetyl-Cl,-sphingosine(4) with ozone gives13J4 3-amino-2-hydroxybutyrolactone(5) and tetradecanal(6).

By a two-step, catalytic hydrogenation, lactone 5 gives ~-erythro-2aminobutane-l,,3,4-triol (7). The same product (7) was obtained by similar reduction of ~-erythro-2-amino-3-hydroxybutyrolactone (S), (8) G . Fodor and J. Kiss, Nature, 171,651(1953). (9) K. Ono, Seikagaku, 19,133(1947). (10)K. Mislow,J.Amer. Chem. Soc., 74,5155(1952). (11)G. Marinetti and E. Stotz,J.Amer. Chem. Soc., 76,1347(1954). (12)For a review on structural investigations of CIB-sphingosineand CIB-dihydrosphingosine, see: C. A. Grob, Record Chem. Progr., 18,55(1957). (13)J. Kiss, G. Fodor, and D. Binfi, Chem. Ind. (London), 517 (1954);Helv. Chim. Acta, 37,1471(1954). (14)E.Klenk and H. Faillard, Z. Physiol. Chem.,209,48(1955).

386

J. KISS

which had been synthesized from D-glyceraldehyde (9) by the methods of Fischer and Feldmanx~'~ and Hamel and Painter.16These results support the stereochemical proposals of Carter, Shapiro, and Harri~on,".'~ and also a stereospecific synthesis by Grob and

The absolute configuration of C-2 in the sphingosine molecule was also proved, because oxidation with hydrogen perioxide (following the degradation with ozone) led to L-serine. 2. Stereochemical Correlation of Phytosphingosines and Dehydrophytosphingosines with C18-Sphingosineand with 2-Amino-2-deoxy-~-glucoseand D-Galactose The structure of phytosphingosine (isolated from the hydrolysis products of the g l y c o ~ p h i n g o l i p i d of s ~yeast ~ ~ ~ ~or corn) was elucidated by Carter and H e n d r i c k ~ o nIt . ~was ~ found to be 2-amino-octadecane1,3,4-triol(lO). The mono-unsaturated aminotriol, namely, the dehydrophytosphingosine ( l l ) , was similarly obtained from soya-bean and flax phosphatides. On catalytic hydrogenation, compound 11gives phytosphingosine, and, by degradation with ozone, 7-amino-5,6,8-trihydroxyoctanal(l2) and decanal(13). H. 0. L. Fischer and L. Feldmann, Ber., 65, 1211 (1932); H. 0. L. Fischer and R. Baer, Helu. Chim. Acta, 17,622 (1934);19,532 (1936). E. E. Hamel and E. P. Painter,J . Amer. Chem. SOC.,75,1362 (1953). H. E. Carter, D. Shapiro, and J. B. Harrison,J. Amer. Chem. SOC., 75,1007 (1953). H. E. Carter and C. G . Humiston,J. BioZ. Chem.,191,727 (1951). C. A. Grob and E. F. Jenni, Helu. Chtm. Acta, 35, 2106 (1952); 36, 1454, 1936 (1953). C. A. Grob and F. Gadient, Chem. Ind. (London), 660 (1956);Helu. Chim. Acta, 40,1145 (1957). J. Kiss and F. Sirokmh, Helu. Chim.Acta, 43,334 (1960). H. E. Carter, W. D. Celmer, W. E. M. Lands, K. L. Mueller, andH. H. Tomizawa, J . Biol. Chem., 206,613 (1954). F. Reindel, Ann., 480,76 (1930). H. E. Carterand H. E. Hendrickson, Biochemfstry,2,389 (1963).

387

GLYCOSPHINGOLIPIDS

Anhydrophytosphingosine (14) is also formed in small proportions during the acid hydrolysis of the phyto-glyco~phingolipids.~~

-HO (10)

Hy

HCOH

A

Hci('iHJL!3 o CH, (14)

The structural and stereochemical correlation of C18-sphingosine with Cl,-phytosphingosine was made by Prostenik and coworkerszs and by we is^.^' Triacylsphingosine (15) was oxidized with peroxy acids, giving the corresponding epoxidez8(16).The reductive opening of the oxirane ring led to the ribo-2-amino-1,3,4-octadecanetriol derivative, which, after hydrolysis and hydrogenolysis of the protecting groups, proved to be identical with natural Cle-phytosphingosine. H,COBz

I H NHBz

Hl'iOBz HCNHBz

I

HCOBz I

tH

"'i (7Ha)ia

-

'i 'i O,yH

H OBz

'CH

--

Phytosphingosine

I

(cHa)la

CH,

6%

(15)

(16)

(25) P. W. O'Connel and S. H. Tsein, Arch. Biochem. Biophys., 80,289 (1959). (26) M. Prostenik, B. Majhofer-Orescanin, and B. Ries-Lesic, Tetrahedron, 21, 651 ( 1965). (27) B. Weiss, Biochemistry, 4,686 (1965). (28) J. Kiss, G . Fodor, and D. Binfi, Research (London), 5,536 (1952).

J. KISS

388

Gigg and GiggZghave published impressive work on the synthesis of phytosphingosines. 2-Amino-2-deoxy-D-glucose was used as the starting material for the hydrophilic, asymmetric part of the molecule; it was converted into methyl 2 - benzamido -2 - deoxy -5,6 - 0isopropylidene-3-O-(methylsulfonyl)-~-~-glucofuranoside (17),which undergoes Walden inversion at C-3, with formation of an oxazoline ring, to give 18. Hydrolysis of the isopropylidene group, followed by glycol oxidation, gives the oxazoline derivative (19). Aldehyde 19 was condensed with tridecyltriphenylphosphonium bromide to give the corresponding olefin. This was hydrogenated, and the oxazoline ring of compound 20 was opened to give the long-chain sugar (21), which was converted into the alditol (22) by reduction with sodium borohydride. The compound thus obtained proved to be identical with the N-benzoyl derivative of the natural C,,-phytosphingosine.

2 -Amino- 2 - deoxy D- glucose

------c

NHBz

-

O H=

C

- R

v

V

Wittig synthesis 0,

Rv

-.

..

H,OH

-L

HQ

9

F Ph

O'ribN Ph

(19)

(20)

NHBz

In a second synthesis, Gigg and Giggo started from 2,3,5-tri-Obenzyl-4-deoxy-4-phthalimido-~-ribose (25), which was obtained (29) J. Gigg, R. Gigg, and C. D. Warren,]. Chem. SOC.( C ) ,1872 (1966). (30) J. Gigg and R. Gigg,]. Chem. SOC. ( C ) ,1876,1879 (1966).

389

GLYCOSPHINGOLIPIDS

from D-galactose by way of 1,3,4-tri-O-benzyl-5,6-O-isopropylidene1,3,4 - tri - 0 - benzyl - 5,6 - 0 - isopropylidene - 2 - 0 (methylsulfony1)-L-galactitol + 2-azido-1,3,4-tri-0-benzyl-2-deoxy5,6-O-isopropylidene-~-talitol + 2-amino-1,3,4-tri-0-benzyl-2-deoxy5,6-O-isopropylidene-~-talitol + 1,3,4-tri-O-benzyl-2-deoxy-5,6-0isopropylidene - 2 - phthalimido - L - talitol (23). Hydrolysis of the 5,6-isopropylidene protecting group gave compound 24; this was oxidized with sodium metaperiodate, and the L-ribose derivative (25) was obtained. The condensation of 25 with a suitable phosphorane (obtained, for example, from tridecyltriphenylphosphonium bromide and phenyllithium, according to Wittig) led to the olefin (26) which, on catalytic reduction followed by hydrolysis, furnished D-ribo-2amino-1,3,4-octadecanetriol (C,,-phytosphingosine) and D-ribo-2amino-1,3,4- eicosanetriol (C,,-phytosphingosine) (27). L - galactito13' +

H,CO

Me

ChOH I

H&O>Me

I I ROCH I HCOMs I H,COR ROCH

-..

-----c

ROCH I ROCH

I PhthZN-CH I H,COR

H

-

c=o

I ROCH I ROCH I Phth=N-CH I H,COR

/

(23)

(25)

Wittig

H C=CH-(CH,)n-CH,

I I

I I HCOH I

HCOH

ROCH

-..-

7% 7%

Phth=NCH I H,COR

(CHJn

I

CH, (27)

(26)

where R =-C&Ph,

Phth =

"D

\c

a

(31) J. Gigg and R. Gigg,]. Chem. SOC. (C), 82 (1966).

, and

n = 11 or 13.

390

J. KISS

Iv. NATURALAND SYNTHETIC SPHINGOSINES, DIHYDROSPHINGOSINES, AND PHYTOSPHINGOSINES OF DIFFERENTCHAIN-LENGTHS The development of chromatography, together with use of the mass spectrometer for analysis of the long-chain, amino alcohol mixtures obtained by hydrolysis of the zoo- and phyto-sphingolipids, led to the discovery of a number of natural sphingosines. K a r l ~ s o npro~~ posed the use of the trimethylsilyl ethers of the long-chain amino alcohol mixtures of animal and plant origin for mass-spectrometric and gas-chromatographic investigations. A further method for the analysis of the homologous sphingosines and phytosphingosines was elaborated by Sweeley and Moscatelly.33 By oxidation of the vicinal amino and alcohol groups with periodate, an aliphatic aldehyde results that can be identified by gas-liquid chromatography. Thin-layer chromatographic methods have been developed for identification of the dinitrophenyl derivatives of the sphingosines and their methyl ether^?^,^^ In addition to the well known CIB-sphingosine and C,,-dihydrosphingosine, a number of similar, natural s p h i n g o s i n e ~having ~ ~ C18, C1,, CIS, Cz0, and C,, chains have been i~olated.~' The sphingosine having a chain of 21 carbon atoms was isolated from Chritidia fasciculata, and characterized by Carter and coworkers. It was found to have a branched chain, and is 19-methylsphingosine. Furthermore, on the basis of the synthesis of sphingosine by Grob and Gadient,2OJenny and D r ~ e synthesized y ~ ~ a number of sphingosine analogs having short (CJ to long (Ci8-CzJ chains, including aminodiols, the cis and truns ethylenic isomers, and the acetylene analogs, in both the ey t h r o and threo series. In the Table I are listed some sphingosines, dihydrosphingosines, phytosphingosines, and so on, that have been isolated by hydrolysis of sphingolipids of animal or plant origin. Table I1 lists some derivatives of these compounds that can be obtained from the natural sphingosines by such simple chemical transformations as acylation. In Table I11 are listed some of the synthetic sphingosine derivatives that do not exist in Nature. (32) K. A. Karlsson,Acta Chem. Scand., 19,2425(1965). (33) C. C. Sweeley and E. A. MoscatellyJ. Lipid Res., 1,40(1959). (34) K.A.Karlsson,Nature, 188,312(1960). (35) C.Michalec, Biochim. Biophys. Acta, 106,197(1965). (36) H. E. Carter, R. C. Gaver, and R. K. Yu, Biochem. Biophys. Res. Commun., 22, 316 (1966);H. E.Carter and C. B. Hirschberg, Biochemistry, 7,2296(1968). (37) E.Klenk and R. T. C. Huang, Z. Physiol. Chem., 349,451(1968). (38) E.F.Jenny and J. Druey, Helu. Chim.Acta, 42,401(1959).

TABLEI Natural Sphingosines No. of compound

No. of carbon atoms in chain

16

16 16

17 17

17 17 18 9

18

10 11

18

12

18

13 14 15 16 17 18

19 19 20

18

20 20 21

Structure 2-amino-1,3-hexadecanediol 2-amino4-trans-hexadecene1,3diol 2-amino-hexadecadiene-1,3-diol 2-amino-l,3-heptadecanediol 2-amino-4-trans-l,3-heptadecanediol 2-aminoheptadecadiene-1,3-diol 2-amino-1,3,4-heptadecanetriol ~-eythro-2-amino-l,3-octadecanediol (dihydrosphingosine,sphinganine) ~-erythro-2-amino-4-truns-octadecene-1,3-diol (sphingosine) 2-amino4,14-trans,trans-octadecadiene-1,3-diol ~-tibo-2-amino-1,3,4-octadecanetriol (phytosphingosine,4-~-hydroxysphinganine) ~-ribo-2-amino-8-trans-octadecene-1,3,4-txiol (dehydrophytosphingosine) 2-amino-1,3,4-nonadecanetriol 2-aminononadecene-l,3,4-triol D-eythro-2-amino4truns-eicosene-1,3-diol 2-amino-l,3-eicosanediol 2-amino-l,3,4eicosanetriol 2-amino-l9-methyl-l,3,4-eicosanetriol

References 39

39 40 39 39 40 40

7 7 40 24 24

40 40 42 42 43 37

0 r

80

z 821

v)

2 0

TABLEI1 Some Derivatives of Natural Sphingosine and Dihydrosphingosine No. of compound

No. of carbon atoms in chain

Structure

1 2 3

18 18 18 18 18 18 18 18 18 18 18 18 18 18

~-etythro-2-acetamido-l,3-octadecanedin1 diacetate ~-erythro-2-acetamido-1,3-octadecaneGio~ ~-erythro-2-benzamido-l,3-octadecanediol dibenzoate ~-erythro-2-benzamido-1,3-octadecanediol ~-e~ythro-2-acetamido-4-truns-1,3-octadecanedio1 diacetate D-ery thro-2-benzamido4-truns-1,3-octadecanediol D-efythro(threo)-2-acetamido-4-t7ans-1,3-octadecanediol 1-acetate3-methyl ether D-2-acetamido-l-octadecanolacetate ~-erythro-2-acetamido-4-truns-octadecene-l,3-diol diacetate 4,5-epoxide ~-ribo-2-benzamido-8-truns-octadecene-l,3,4-triol ~-ribo-2-benzamido-&truns-octadecene-1,3,4-trioI triacetate ~-ribo-2-benzamido-l,3,4-octadecanetriol 2-amino-1,4-anhydro-1,3,4-octadecanetriol (anhydrophytosphingosine) 2-benzamido-l,4-anhydro-1,3,4-octadecanetriol 3-benzoate

4 5 6

7 8 9 10 11 12 13 14

References

44 44 44 44 45 44 46 18 28 24 24 43 24 24,43

Y

E rA rA

TABLE111 Some Synthetic Sphingosines and Related, Longchain, Amino Alcohols

No. of compound

No. of carbon atoms in chain

1 2 3 4

8 8 10 12 14 16 18 18 18 18 18 18 18 18 18

5 6

7 8 9 10 11 12 13 14 15

Structure

DL-threo(erythro)-2-amino4-trans-octene-1,3-diol DL-threo(erythro)-2-amino~-cis-octene-1,3-dio~ D~-erythro( threo)-2-amino-4-trans(cis)-decene-1,3-diol DL-erythro(threo)-2-amino4-truns(cis)-dodecene-1,3-diol DL-erythro(threo)-2-amino-4-trans(cis)-tetradecene-l,3-diol DL-eryt h o ( threo)-2-amino-4-trans(cis)-hexadecene1,3-diol ~~-erythro-2-amino-1,3-octadecanediol ~~-eythro-2-acetamido-1,3-octadecanediol diacetate ~~-erythro-2-benzamido-1,3-octadecanediol DL- and (-)-threo-2-acetamido-1,3-octadecanediol D~-threo-2-acetamido4-trans-octadecne-l ,3-dioldiacetate ~~-threo-2-acetamido4-cis-octadecene-l,3-diol diacetate DL-eqthro-2-amino-4-trans-octadecene-1,3-diol D~-ery thro-2-amino-4-cis-octadecene-1,3-diol

DL-erythro-( threo)-2-amino4-octadecene-1,3-dio14,5-t2

References

38 38 38 38 38 38 47 47 47,48 19 20 20

20,49 20 50

z

$

2 !Q 2

0

E: 21

394

J. KISS

V. BIOSYNTHETIC PATHWAY OF SPHINGOSINES AND OF GLYCOSPHINGOLIPIDS are, ~ - accord~~ The precursors in the biosynthesis of s p h i n g o s i n e ~ ~ ing to Brady and associate^,^^ L-serine (28) and palmitaldehyde (29). These compounds give dihydrosphingosine (30),which is catalytically dehydrogenated by flavoprotein to give sphingosine (31).

According to Brady and coworkersss and Cleland and Kennedy,56

the biosynthetic pathway of the glycosphingolipids begins with the enzymic glycosidation of the sphingosines; for example, reaction of 31 with uridine 5’-(D-galactopyranosyl pyrophosphate) (UDP-Gal) results in D-galactosylsphingosine (psychosine, 32). Compound 32 K. A. Karlsson, Acta Chem. Scand., 18,2395(1964). K. A. Karlsson and G. A. L. Holm, Acta Chem. Scand., 19,2423 (1965). M. Majhofer-Orescanin and M. Prostenik, Croat. Chem. Acta, 33, 219 (1961). K. A. Karlsson, Acta Chem. Scand., 18,565 (1964). M. Prostenik and N. Z. Stanacev, Chem. Ber., 91,961 (1958). H. E. Carter, W. P. Norris, F. H. Glick, G. E. Phillips, and R. Harris, J . Biol. Chem., 170,269 (1947). P. A. Levene and W. A. Jacobs,]. Biol. Chem., 11,547(1912). H. E. Carter, 0. Nalbandov, and P. A. Tavormina,J. Biol. Chem., 192,197 (1951). C. A. Grob, E. F. Jenni, and H. Utzinger, Helv. Chim. Acta, 34, 2249 (1951); G. I. Gregory and T. Malkin,J. Chem. SOC.,2453 (1951). N. Fischer, Chem. Znd.(London), 130 (1952). D. Shapiro, H. Segal, and H. M. Flowers,J. Amer. Chem. Soc., 80, 1194 (1957). A. E. Gal,J. Label. Compounds, 3,112 (1967). J. Zabin and J. F. MeadJ. Biol. Chem., 205,271 (1953). J. Zabin and J. F. Mead, Fed. Proc., 12,294 (1953). D. B. Sprinson and A. Coulson,J . Btol. Chem.,207,585 (1953). R. 0. Brady, J. V. Formica, and G. J. Koval, J . Biol. Chem., 233, 1072 (1958); P. E. Braun and E. E. Smell, Proc. Not. Acad. Sci. U.S . , 58.295 (1967). R. M. Burton, M. A. Sodd, and R. 0.BradyJ. Btol. Chem., 233,1053 (1958). W.W. Cleland and E. P. KennedyJ. Biol. Chem., 235,45 (1960).

GLYCOSPHINGOLIPIDS

395

is acylated by long-chain, monocarboxylic acids (acyl-Coenzyme A; Ac-Co-A) to give5’ the cerebroside 33.

Enzymic sulfation, with 3’-O-phosphonoadenylyl sulfate, of the 3-hydroxyl group of the D-galactopyranose residue of the cerebrosides obtained led to the sulfa tide^.^^^^^ VI. CERAMIDE MONO-AND OLIGO-SACCHARIDES In natural cerebrosides, the N-acylated sphingosines are in glycosidic conjugation with sugars. The connection between the sphingosine residue and the sugar moiety is at the primary (C-1) hydroxyl group of the sphingosine molecule. To date, only cerebrosides that are hexopyranosides have been isolated. In natural cerebrosides, D-galactose or D-glucose is in direct, covalent conjugation with the sphingosines. Cerebrosides that contain more than one sugar moiety have the other sugar residues glycosidically linked to the D-galacto- or D-gluco-pyranoside group. 1. Galacto- and Glucocerebrosides

The first two galactocerebrosides were isolated by Thudichum,so in 1882, from human-brain material, and were designated phrenosin and kerasin.’ The isolation of these compounds is based on solvent extraction and fractional recrystallization,B’ although the separation of the pure compounds by these methods is very difficult. Nevertheless, the analytical characterization of phrenosin and kerasin, which was effected by hydrolytic cleavage, was correct. (57) (58) (59) (60) (61)

R. 0.Brady,]. Biol. Chem., 237, pc 2416 (1962). G. Hauser, Biochim. Biophys. Actu, 84,212 (1964). I. H. Goldberg,]. Lipid Res., 2,103 (1961). J. L. W. Thudichum,]. Prukt. Chem., 133,19(1882). 0.Rosenheim, Biochem.], 7,604 (1913); 8,110 (1914).

J. KISS

396

Phrenosin gives equimolar amounts of sphingosine, D-galactose, and 2-hydroxytetracosanoic (phrenosinic or cerebronic) acid on acid hydrolysis. Kerasin has similar constituents, but the fatty acid component is tetracosanoic acid. During the first three decades of this century, galactocerebrosides were isolated from organs other than the brain and spinal cord of mammals, and from organs of other animals, including birds, fishes, and insectse7 This group of sugar-lipid conjugates is not limited to organs of animals. For example, similar compounds are found in mushrooms and plant seeds.7 Elucidation of the structure of phrenosin was obtained by hydrolytic cleavage. Whereas alkaline cleavage gave 2-hydroxytetracosanoic acid (34) and sphingosine D-galactoside (psychosine, 35), by mild hydrolysis with acid, N-(2-hydroxytetracosanoyl)sphingosine(36)and Dgalactose were obtained.62 Phrenosin

+

+

The linkage between the sphingosine moiety and the D-galactose residue was determined by Carter and G r e e n w ~ o dCatalytic .~~ reduc(62) E. Klenk, 2.Physiol. Chem., 153,74 (1926). (63) H.E.Carter and F. L. Greenwood,]. Biol. Chem., 199,283(1952).

CLYCOSPHINCOLIPIDS

397

tion of the allylic double bond in the sphingosine moiety of acetylated phrenosin (37)was accompanied by hydrogenolysis of the acetylated allylic hydroxyl group. Thus, in addition to acetylated dihydrophrenosin (38),the acetylated sphingine D-galactoside (39)was also obtained.

On the basis of this experiment, the D-galactose residue is attached to the primary hydroxyl group of the N-acylated sphingosine. This experimental result is in accordance with the structural proposal of Nakayama,g4who isolated 3-0-methylsphingosine from the products of hydrolysis of methylated phrenosin. The configuration of the 2-hydroxytetracosanoic acid (cerebronic acid) was to be D. The anomeric configuration of the D-galactopyranosyl group on the sphingosine moiety was determined by the degradation method of (64) T. Nakayama,]. Biochem. (Tokyo),37,309 (1950). (65) K. Mislow and S. BleicherJ. Amer. Chem. SOC., 76,2825 (1954). (66) D. H. S. Horn and Y. Y. PretoriusJ. Chem. Soc., 1460 (1954).

398

J. KISS

Stofin and Mulligan:' and also by synthesis.68 According to both methods, the anomeric configuration is p. The geometry of the double bond in phrenosin was shownlOJ1to be trans; it connects C-4 and C-5 of the sphingosine moiety. This point was established by degradation with ozone: kerasin gives tetradecanal from the lipophilic part of the sphingosine moiety, and 3amino-2-hydroxybutyrolactone (5) from the hydrophilic, amino alcohol moiety. The latter compound proved to be identical with the degradation product from N-acetyl-di-O-acetylsphing~sine.~~ The method of ozone degradation of the galactocerebrosides was used by Kuhn and Wiegandt70for mild degradation of the gangliosides. Besides phrenosin and kerasin, two further cerebrosides, namely, nervon and oxynervon, were isolated from human-brain material by fractional recrystallization. These substances proved to be mixtures of many cerebrosides, and not pure compound^.^^ Klenk and Schors~h'~ have elaborated an excellent method for the separation of the individual, genuine galactocerebrosides. The crude cerebroside fraction obtained by solvent extraction of a suitable tissue is separated by chromatography on silica gel. Cerebrosides containing saturated or unsaturated hydroxy fatty acids can be separated from those containing nonhydroxy fatty acids. In both of the separated parts, there are numerous individual cerebrosides. A further method of separation involves the ability to form a complex with mercuric acetate: fatty acids and their derivatives containing a cis double bond give adducts, whereas those having saturated or trans-olefinic linkages do not. By this method, four main fractions were obtained (see Table IV). The cerebroside fractions proved not to be pure compounds, but were mixtures of compounds having similar features of chemical structure. The differences between the single compounds in each group reside mainly in the chain length of the fatty acids in amide linkage. The fatty acids obtained by hydrolysis of the single types of galactocerebrosides were analyzed by gas-liquid chromatography of, for example, their methyl esters. The main fatty acid in each group was (67) P. J. Stofin and G . D. Mulligan, Abstracts Papers Amer. Chem. Soc. Meeting, 148,7D(1964). (68) D. Shapiro and H. M. Flowers,]. Amer. Chem. Soc., 83,3327(1961). (69) J. Kiss, D.BBnfi, and J. Kbbor, Helu. Chim. Acta, 43,2198(1960). (70) R.Kuhn and F.Wiegandt, Chem. Ber., 96,866(1963). (71) Y.Kishimoto and N. S. Radin,]. Lipid Res., 1,72(1959);2,335(1961). (72) E.Klenk and E. U. Schorsch, Z. Physiol. Chem.,348,1061(1967).

GLYCOSPHINGOLIPIDS

399

TABLEIV Groups of Natural Galactocerebrosides MD,

Number

Fraction

1 2 3 4

Phrenosin Oxynervon Kerasin Nervon

degrees

+ 3.98

+ 3.80 - 3.82

- 3.70

Longchain fatty acids in amide linkage D-a-hydroxy, saturated D-a-hydroxy, cis-unsaturated saturated cis-unsaturated

found to be the C,, acid (saturated, unsaturated, a-hydroxy, or ahydroxy unsaturated). Furthermore, the CIS,C20,C22,CZ3,CZ5, and c 2 6 fatty acids having these variations were present in smaller proportions. It is interesting that the chain length and structure of the fatty acids bound in the cerebrosides proved to be a very sensitive parameter, dependent, for example, on age.73-75Moreover, under pathological conditions, the ratio of these fatty acids is found to be different from the normal r a t i ~ . ~ ~ , ~ ~ Besides the galactocerebrosides mentioned, which are built u p on a sphingosine basis, galactocerebrosides were isolated that have a dihydrosphingosine ~keleton.'~ Saturation of the double bond by catalytic hydrogenation of the galactocerebrosides led to the corresponding dihydrocerebrosides. 11.69.99 Phrenosin Kerasin

-

dihydrophrenosin dihydrokerasin

-

oxynervon nervon

The diastereoisomeric configuration of the sugar-free cerebrosides (ceramides) was determined by Carter and coworkers,80and found to be erythro, in accordance with the structure of sphingosine, which is isolated after drastic hydrolysis of sphingolipid mixtures or purified cerebrosides with acid. Y. Kishimoto and N. S. Radin,]. Lipid Res., 1,79 (1959). L. Svennerholm and S. Stillberg-Stenhagen,]. Lipid Res., 9,215 (1968). P. Lesch, S. Meier, and K. Bernhard, Helu. Chim. Acto, 49,791 (1966). G. D. Cherayil,]. Lipid Res., 9,207 (1968). C . V. Marinetti, T. Ford, and E. Stotz,]. Lipid Res., 1,203 (1960);J. L. Foote and E. Coles, ihid., 9,482 (1968). E. Okuhara and M. Yasuda,]. Neurochem., 6,112 (1960). E. Klenk and R. Harle, Z. Phgsiol. Chem., 189,243(1930). H. E. Carter, J. A. Rothfus, and R. Gigg,J. Lipid Res., 2,228 (1961).

J. KISS

400

Klenk and L o h P have reported the isolation of cerebroside esters from human-brain material. One hydroxyl group of the D-galactosyl group, or of the sphingosine residue, is in covalent conjugation with longchain fatty acids. Ceramide monohexosides having a D-ghCOpyI'anOSylg r o ~ phave ~ ~ * ~ also been isolated from human liver, spleen, and serum.84 2. Ceramide Oligosaccharides

Ceramide dihexosides and trihexosides have been isolated from normal, human organs, and from tissues of patients having TaySachs', Fabry's, or Gaucher's disease.' These glycosphingolipids have structural moieties similar to those of the ceramide monosaccharides: the sugar (oligosaccharide) residue is linked P-D-glycosidically to the primary hydroxyl group of the ceramide molecule. In the class of the ceramide dihexosides, two types have been isolated: a ceramide in glycosidic conjugation with ( a ) an O-D-galaCtOpyranosyl-(1 4)-P-D-galactopyranosyl and (b) a 4 - 0 - P - ~ galactopyranosyl-P-D-glucopyranosylresidue.@ The well characterized cytolipin H (40), isolated from human, epidermoid carcinoma by Rapport, Skipski, and Sweeley,B7belongs to type b.

+.

bH (40)

(81) (82) (83) (84) (85)

E.Klenk and J. P. Lohr, 2.Physiol. Chem., 348,1712(1967). A. Rosenberg and E. ChargafF,]. Biol. Chem., 233,1323(1958).

K. Suzuki and G. C. Chen,]. Lipid Res., 8,105(1967). E.L.Kean,]. Lipid Res., 7,449(1966). S.Gatt and E. R. Berman,]. Neurochem., 10,43(1963). (86) E. Svennerholm and L. Svennerholm, Blochim. Biophys. Acta, 70, 432 (1963). (87) M.Rapport, V. P. Skipski, and C. C. Sweeley,]. Lipid Res., 2,148(1961).

GLYCOSPHINGOLIPIDS

401

Ceramide tri- and tetra-hexosides (having amino sugar residues, as well) were isolated from human organs. These ceramide oligosaccharides proved to be mainly anomalous degradation products of gangliosides (see Section IX, p. 428). A new type of glycosphingolid was isolated from beef brain by Kochetkov, Zhukova, and Glukhoded.88 For the structure of this glycosphingolipid, a sphingoplasmogen, the cerebroside-3-en01 ether (41) was proposed.

HFO-CH=CH-R’

where R and R’ = aliphatic chains.

3. Synthesis of Ceramide Mono- and Oligo-saccharides

The first experiments in this field were conducted by Klenk and Harle.79 l-O-(/I-D-Galactopyranosy1)sphingosine (psychosine) was acylated with lignoceroyl (tetracosanoyl) chloride, giving kerasin. The total synthesis of phrenosin and kerasin was performed in 1961 by Shapiro and Flowers.68 The condensation of tetra-0-acetyl-Dgalactopyranosyl bromide (42) with 3-O-benzoyl-N-cerebronoyl(or N-1ignoceroyl)sphingosine (43) in the presence of mercuric cyanide led to the corresponding /I-D-galactopyranoside (44), which, after hydrolysis of the protecting groups, gave kerasin and phrenosin. The synthetic products obtained proved to be identical with the natural compounds isolated from nerve tissues. By the same method, the corresponding D-glucocerebroside was also synthesized. For the synthesis of cytolipin H (40), a similar method was used.89 Hepta-O-acetyllactosyl bromide (45) was condensed with 3-0benzoyl-N-stearoylsphingosine(46). After removal of the protecting

(88) N. K. Kochetkov, I. G . Zhukova, and I. S . Glukhoded, Biochim. Biophys. Acta, 70,716(1963). (89) D.Shapiro and E. S. Rachaman, Nature, 201,878 (1964).

402

J. KISS

groups, the ceramide disaccharide (40) was isolated and found to be identical with natural cytolipin H.

Cytolipin H

Flowersm used a two-step procedure for the synthesis of this ceramide disaccharide, starting with DL-dihydrosphingosine. 3-0Benzoyl-N-stearoyl-DL-dihydrosphingosine was condensed with 2,3,6tri-0-acetyl-D-galactopyranosyl bromide, resulting in the correspond(90) H. M. Flowers, Abstracts Int . Symp. Chem. Natural Products, 4th, Stockholm, 1966, p. 33; Carbohyd. lies., 4 4 2 (1967).

GLYCOSPHINGOLIPIDS

403

ing substituted ceramide P-galactopyranoside, which has a free hydroxyl group on C-4 of the acetylated D-galactopyranosyl group. This compound was condensed with tetra-0-acetyl-D-glucopyranosyl bromide or with tetra-0-acetyl-D-galactopyranosylbromide, resulting in the corresponding, acetylated ceramide disaccharides. VII. SULFATIDES 1. Ceramide Monoglycosyl Sulfates

Almost a hundred of years ago, Thudichumsl recognized the existence of sulfate-containing glycosphingolipids (cerebrosulfatides) in the lipid material of human, central-nervous tissues. The isolation of relatively pure, phosphorus-free material was reported by Levenes2 and by Landsteiner and Levene.g3Blixe4obtained, by solvent extraction and subsequent recrystallization, a compound (in the form of its potassium salt, mp 209-210”) that, on hydrolysis, gave equimolecular amounts of sphingosine, cerebronic acid (2hydroxytetracosanoic acid), D-galactose, and sulfuric acid. For this compound, Blix suggested the structure O-~-galactosyl-N-(2-hydroxytetracosanoy1)sphingosine sulfate. The proof of the linkage between the lipophilic (N-acylsphingosine) and hydrophilic (sulfated D-galactopyranosyl) moieties of the sulfatide was obtained by mild hydrolysis with acid; selective cleavage of the sulfate group resulted in p h r e n o ~ i n . ~ ~ For the position of the sulfate group, Nakayama= proposed the primary hydroxyl group of the D-galactopyranosyl group, because of the fact that the sulfatide cannot be t~itylated.~’ However, the results of methylation experiments disproved this structure. Stoffyn and Stoffyn9*and Yamakawa and coworkersw isolated 2,4,6-tri-O-methyl-~ (91) J. L. W. Thudichum, “Researches on the Chemical Constitution of the Brain,” Report of the Medical Officer of the Privy Council, London, 1874 (cited by H. Thierfelder and E. Klenk, “Die Chemie der Cerebroside und Phosphatide,” Springer Verlag, Berlin, 1930). (92) P. A. Levene,J. Biol. Chem.,13,463 (1912). (93) K. Landsteiner and P. A. Levene, J . Immunol., 10, 731 (1925);J. Biol. Chem., 75,607 (1927). (94) G. Blix, 2.Physiol. Chem.,219,82 (1933). (95) P. J. StoRyn and A. Stofin, Biochim. Biophys. Acta, 70,107 (1963). (96) T. NakayamaJ. Biochem. (Tokyo),38,157 (1951). (97) S. J. Tannhauser,J. Fellig, and G. SchmidtJ. B i d . Chem., 215,211 (1955). (98) P. J. StoEyn and A. Stofin, Biochim. Biophys. Acta, 70,218 (1963). (99) T. Yamakawa, N. Kiso, S. Handa, A. Makita, and S. Yokoyama, J . Biochem. (Tokyo), 52,468 (1962).

404

J. KISS

galactose (but no 2,3,4-tri-O-methyl-~-galactose) from the hydrolyzate of the permethylated sulfatide. Consequently, the compound has the structure shown in 47. The cerebrosulfatide cannot be oxidized with periodate; this supports assignment of the sulfate group to 0-3 (equatorial) of the Dgalactopyranosyl group.

Nevertheless, according to Davison'OO and Hakamori, Ishimoda, and Nakamura,'O' on the basis of the above experiments, the possibility of a labile, 3,6-cyclic sulfate structure cannot be excluded. The dihydrosulfatide was p-toluenesulfonylated, and the product was converted into the corresponding 6-deoxy-6-iodo derivative of the dihydrosulfatide by the method of Oldham and Rutherford.lo2 After hydrogenolysis of the iodine atom, followed by hydrolysis, D-fuCOSe was isolated (as well as dihydrosphingosine and 2-hydroxytetracosanoic acid). This result provides evidence that, in the sulfatide and dihydrosulfatide molecules, there exists neither the 3,6cyclic sulfate nor the 6-sulfate group.'03 Jatzkewitz and coworkerslWand Stoffyn and StofFyn'05have reported on the synthesis of D-galactose 3-sulfate by sulfation of 4,6-0-ethylidene-1,2-O-isopropylidene-a-~-galactose (48) to give 49, followed by hydrolysis of the protecting groups. The product obtained (50) proved to be identical (on a thin-layer plate of cellulose) with the D-galaCtOSe sulfate obtained by mild hydrolysis of the natural sulfatide. (100) (101) (102) (103) (104) (105)

A. N. Davison, Biochem.]., 9 1 , 3 (1964), ~ S. Hakamori, T. Ishimoda, and K. Nakamura,]. Biochem. (Tokyo), 52,468 (1962). J. W. H. Oldham and J. K. RutherfordJ. Amer. Chem. Soc., 54,366 (1932). J. Kiss, Helo. Chim. Acta, 50,1423 (1967). H. Jatzkewitz and G. Nowoczek, Chem. Ber., 100,1667 (1967). A. Stoffynand P. J. Stoffyn,]. Org. Chem., 32,4001 (1967).

GLYCOSPHINGOLIPIDS

405

Until about ten years ago, this N-(2-hydroxytetracosanoyl)-O-(30-sulfato-D-galactopyranosyl)-C,,-sphingosinewas the only natural sulfatide known. It can be isolated from the central-nervous material of mammals. The development of methods for separating lipid substances from natural sources has opened up new aspects in this field. ( a )The use of chromatography has led to rapid and exact separation of compounds on a preparative scale, as well as in micro quantities. (b) It was found that, in tissues other than the central-nervous material (of mammals), there exists a group of glycosphingolipids having a sulfate group. Lees and coworkerslWhave described a simple procedure for the separation of brain sulfatides; the solvent extraction is combined with chromatography on Florisil, the elution being carried out with chloroform-methanol. For the chromatographic separation, Wells and Dittmer'O' used a column of silicic acid-Hyflo Supercel. Jatzkewitz108isolated two sulfatides from pathologically changed, human-brain material (leucodystrophy, type Scholz). The only difference between these two sulfatides (51), according to the degradation experiments of Jatzkewitz, lies in the fatty acid components linked to the amino group of the sphingosine residue: one is the well known "Blix" sulfatide (N2-hydroxytetracosanoylpsychosine3-sulfate), and the other is the kerasin sulfate (N-tetracosanoylpsychosine 3-sulfate). Furthermore, Jatzkewitz108 pointed out the existence of some further sulfatides that differ only in the structure of the fatty acids. The isolation of a dihydrosulfatidelo3from beef spinalcord has been reported. (106) M. Lees, J. Folch, G. H. Sloane-Stanley, and S.Cam,]. Neurochem., 4, 9 (1959). (107) M. A. Wells and J. C. Dittmer,]. Chromatog., 18,503 (1965). (108) H. Jatzkewitz, Z. Physiol. Chem., 320,134 (1960);311,179 (1958).

406

J. KISS CH,OH I

CH, (51)

where R = H (psychosine sulfate).

Green and Robinson'og pointed out that sulfatides are present not only in the tissue of the central-nervous system, but also in other organs, namely, kidney, liver, and spleen (and in mast-cell tumor). From the point of view of physiology, a noteworthy characteristic of the sulfatides is the extremely slow turnover in the brain, in contrast to that in the other organs. Also, they accumulate continuously in the brain. Menkes, Philippart, and Concone'lo determined the relationship between the concentration of the sulfatides (and cerebrosides) of the human brain and the age of the human, and found a progressive increase from birth to maturity."' Furthermore, the proportion of the a-hydroxy fatty acids increases, together with that of the C,,-fatty acids. Bernhard and coworker^^^^*^^ developed methods for determination of the lipids and lipid components in human, cerebral white-matter and cortex of different ages. Moreover, they found a greater proportion of sulfatides in the atherosclerotic than in the normal aorta,

2. Ceramide Diglycosyl Sulfates Anomalous accumulation of sulfatides was observed in the tissues (for example, nervous tissues, liver, and kidney) of patients having metachromatic l e u ~ o d y s t r o p h y . ~Besides ~ ~ J ' ~ the "normal" ceramide monohexoside sulfate, a new glycosphingolipid sulfuric ester having a (109) J. P. Green and J. D. Robinson, JrJ. Biol. Chem.,235,1621(1960). (110) J. H. Menkes, M. Philippart, and M. C. Concone, J. Lipid Res., 7 , 479 (1966). (111) A. N.Davison and N. A. Gregson, Biochem.J.,85,558(1962). (112) L.Hausheer and K. Bemhard, 2. Physiol. Chem., 331,41(1963). (113) E.Martensson, Biochim.Biophys. Acta, 116,521(1966). (114) M . J. Malone and P. J. Stoffyn, Biochim. Biophys. Acta, 98,218 (1965).

407

GLYCOSPHINGOLIPIDS

disaccharide sulfate residue was isolated. This strongly anionic glycosphingolipid constitutes about 20-25 % of the total sulfatide fraction. According to the degradation experiments of StofFyn, StofFyn, and Martens~on”~ (oxidation with periodate, permethylation studies, and hydrolytic cleavage), which were carried out on a microscale, the structure of this sulfatide is that shown in formula 52.

\?7K3;-CO-R 0-YH,

OH

Ht ’iH

HQ

0 -8-D-Galactopyranosyl-

(1-“4)-p-D-glUCOpyranosylceramidells~lle

(CH,),,

I

CH,

OH (52)

where R = -C,,H,,

.

3. Synthetic Studies in the Sulfatide Group By sulfation of phrenosin and kerasin, the first semisynthetic sulfatide was prepared by Chargaff.l17 These sulfated cerebrosides show anticoagulant activity, in contrast to the natural, “Blix” sulfatide.Ils J a t z k e w i t P has described a similar sulfation procedure that uses ~ y r i d i n e - ~ ~ Scomplex. O, The cerebron 6-sulfate is mainly formed by this procedure. flower^"^ made a total synthesis of a dihydrosulfatide by condensation of 2,4,6-tri-O-acetyl-a-D-galactopyranosyl bromide (53) with 3(115) A. Stoffyn, P. J. Stoffyn, and E. Martensson, Biochim. Biophys. Acta, 152, 353 (1968). (116) J. Folch, J. A. Meath, and S. Bogoch, Fed. Proc., 15,254 (1956). (117) E. ChargalTJ. Biol. Chem., 121,187 (1937). (118) For a critical survey and literature, see: “Biochemistry and Medicine of the Mucopolysaccharides,” F. Egami and Y. Oshima, eds., Univ. Tokyo, Japan, 1962, pp. 264-266. (119) H. M. Flowers, Carbohyd. Res., 2,371 (1966).

J. KISS

408

0-benzoyl-N-octadecanoyl-DL-dihydrosphingosine (54), resulting in formation of 3-O-benzoyl-N-octadecanoyl-l-O-(2,4,6-tri-O-acetyl-~D-galactopyranosy1)-DL-dihydrosphingosine (55). The sulfation of compound 55 with pyridine-sulfur trioxide, followed by catalytic deacetylation, led to the racemic dihydrosulfatide (56).

where R = -(CH,),6-CHS.

VIII. GLYCOSPHINGOLIPIDS OF

PLANT hJATEFUALS AND MICROORGANISMS (PHYTO- AND h~YCO-GLYCOSPHINGOLIPIDS1'Ba)

Carter and coworkers22 reported the isolation from plant seeds (for example, soya beans) of lipid material that consists of phosphatides that contain longchain, aliphatic amino alcohol-sugar conjugates. Purification of the lipid material for degradation studies was performed by solvent extraction, followed by countercurrent distribution.'20J21The compounds obtained contained'22myo-inositol residues (Ilea) See the review by H. E. Carter, P. Johnson, and E. J. Weber, Ann. Reu. Btachem., 34,109,131-133 (1965). (120) T. A. McGuire and F. R. J. Earle,/. Amer. 011 Chemists' Soc., 28,328 (1951). (121) C. R. Cholfield, H. J. Dutton, and J. Dimler, J . Amer. 011 Chemists' Soc., 29, 293 (1952);C. R. Cholfield and H. J. DuttonJ. Biol. Chem., 208,461 (1954). (122) E. Klenk and R. Sakai, Z. Physiol. Chem.,258,33 (1939).

GLYCOSPHINGOLIPIDS

409

and phosphate groups in covalent conjugation with the acylated, longchain amino alcohol and sugars. The long-chain amino alcohol was found to be identical to that of the cerebrin base that ZellnerlZ3had isolated after hydrolysis of a mixture obtained from the mushroom Amanita muscaria L. The structure of this compound has already been discussed (see Section 111, p. 386). These fatty amino alcohol-sugar conjugates are also characteristic constituents (several percent of the total lipid-material) of plant seeds other than soya beans. Similar compounds are found in corn, cotton, flax, linseed, peanut, sunflower, wheat, and other seeds, and in yeast,lZ4 and mushrooms.2J26They are designated phytogly~olipids.’~~ This group of glycosphingolipids also contains long-chain amino alcohols (for example, phytosphingosine and dehydrophytosphingosine), fatty acids, phosphate, myo-inositol, 2-amino-2-deoxy-~-g~ucose, D-g~ucuronicacid, D-galactose, D-mannose, L-arabinose, and, in some phytoglycosphingolipids, L-fucose. These constituents are present in natural phytoglycosphingolipids in almost equimolecular proportions. The most characteristic structural feature of these compounds is that the oligosaccharide part is not directly connected to the longchain, amino alcohol moiety. The connecting group on the long-chain, amino alcohol is a phosphate group, and, on the oligosaccharide part, an inositol residue. The phosphate group is bound to the primary hydroxyl group of the phytosphingosine molecule (which is N-acylated with a long-chain fatty acid), in a manner similar to that found for the sphingomyelins (in the group of the sugar-free zoo-sphingolipids). On alkaline (barium hydroxide) degradation, phytosphingolipids give “phytoceramides,” for example, N-2-hydroxytetracosanoyl) phytosphingosine l-phosphatelZ8(57). This characteristic component of the phytosphingolipids gives L-serine phosphate on oxidation with permanganate and hydrolysis, followed by periodate oxidation. (123) J.Zellner, Monatsh., 32,133(1911). (124) F.Reindel, Ann., 480.76 (1930). (125) E.Ruppol, Bull. SOC. Chim. Biol., 25,57(1943). (126) H.Wieland and G. Coutelle, Ann. 548, 270 (1941). (127) H.E. Carter, G. S. Galanos, R. H. Gigg, J. H. Law, T. Nakayama, D. B. Smith, and E. J. Weber, Fed. Proc., 16,819 (1957). (128) H.E. Carter, R. H . Gigg, J. H. Law, T. Nakayama, andE. Weber,J. Biol. Chem., 233,1309(1958).

J. KISS

410

0

7

Hp 0-<-OK

OK

The fatty acids in amide linkage on the phytosphingosine residue in the phytosphingolipid molecule proved to be cerebronic (2hydroxytetracosanoic) acid and lignoceric (tetracosanoic) acid, and, in soya-bean phytoglycosphingolipids, palmitic and stearic acids. From the water-soluble, alkaline degradation products of the phytoglycosphingolipids, phosphate-containing and phosphate-free oligosaccharides were isolated. They were fractionated on Dowex-2 (HC0,-),and the oligosaccharides obtained were separated in the order of progressively increasing molecular weight.lz9 From the above degradation products, a myo-inositol-D-glucuronic acid-2-amino-2-deoxy-~-glucosehas been isolated crystalline; it seems to be a common unit in the molecule of the phytoglycosphingolipids of all In addition, oligosaccharides containing even more monosaccharide units were isolated by Dowex chromatography; for example, tetrasaccharides [yield 41 % (D-mannosyl)-(2-amino-2-deoxy-D-glucosyl)(D-glucosyluronic acid)-myo-inositol], and penta-, hexa-, hepta-, and octa-saccharides, with and without a phosphate group. Degradation of these oligosaccharides, and of the intact phytoglycosphingolipids, with nitrite gives 2,5-anhydro-~-mannose, together with the oligosaccharide residue, which is not cleaved. On the basis of the structure of the degradation products, the structure of the phytoglycosphingolipids is given by the following schematic representation (58). The complete structure of the tetrasaccharidic phytoglycolipid has been established130aas 2-0-a-D-man(129) H.E.Carter, B. E. Betts, and D. R. Strohbach, Biochemistry, 3,1103(1964). (130) H. E. Carter, G. Brooks, R. H. Gigg, D. R. Strobach, and T. Suami, J . Biol. Chem., 239,743(1964). (130a)H.E. Carter, D. R. Strobach, and J. N. Hawthorne, Biochemistry, 8,383(1969); H.E.Carter, A. Kisic, J. L. Koob, and J. A. Martin, ibid., 8,389(1969).

GLYCOSPHINGOLIPIDS

411

nopyranosyl-6-0-[(2-amino-2-deoxy -cf-D-glucopyranosyl)-( 1+4) -CY-Dglucopyranosyluronic acid]-myo-inositol.

'3

--

R-0-P-0-Inositol-D-glucuronic HA

acid-2-amino-2-deoxy-D-glucose

t t

t

D-MannOSe

n-Galactose

L- Arabinose

(L-Fucose) (58)

where R = N-acylated phytosphingosine (D- r i b 0 -2acylamido1,3,4-octadecanetriol), or N-acylated dehydrophytosphingosine (D-ribo-2-acylamido-8- trans octadecene-1,3,4-triol).

The ratio of phytosphingosine to dehydrophytosphingosine in the phytosphingolipids isolated from a variety of seeds has been dete~~nined.~~ Wagner and Z o f c ~ i k have ' ~ ~ described the isolation and characterization of glycosphingolipids from Candida utilis and Saccharomyces cereuisiae. Fractions containing a phosphate group are structurally similar to the phytoglycosphingolipids already described, but are simpler. On hydrolysis, CIS-(17-33940) and Cz,-phytosphingosine (56-75%), and C18-dihydrosphingosine(6-7%) were obtained. In amide linkage on the phytosphingosine moiety are saturated and unsaturated fatty acids; for example, lignoceric acid (4-8%) and cerebronic acid (36-56 %). From the water-soluble part of the hydrolysis products of these glycosphingolipids, only inositol and D-mannose were isolated. The lipophilic and hydrophilic parts of the lipid molecule are connected by a phosphate group, as in 59. (131) H. Wagner and W. Zofcsik, Blochem. Z., 346,343(1966).

J. KISS

412

HCNH-CO-R HAOH

(59)

where Me = Caz+, R = alkyl, alkenyl, or 1-hydroxyalkyl, and n = 13 or 15.

From the same sources, Wagner and Z o f ~ s i k have ' ~ ~ isolated a glycosphingolipid of a cerebroside type, namely, the dihydro-D-galactocerebroside 60.

Compound 60 has a structural similarity to the glycosphingolipids of type 61 isolated from wheat-flour lipids by Carter and coworker^.'^^*'^

where R = alkyl, and R' = (a) -(CH,),4-CH,, (b)-CH-(CI&),s-CH,,

or

OH H

H

( c ) -c-(cH,),-c=c-(cH,),-cH,

OH

H

GLYCOSPHINGOLIPIDS

413

On the other hand, glycosphingolipids of the sphingosine type were isolated from ceramides of human and animal hair, and from kidn e y ~ . ' ~ Karl~son'~' ~*'~~ suggested a metabolic relationship between phytosphingosines and sphingosines in the mammalian organism, as intermediates in the biosythetic pathway of the zoo-sphingosines.

IX. GANGLIOSIDES Glycosphingolipids containing one or more residues of acylated neuraminic acid per molecule are called gangliosides. The name was given to this group of glycolipids by Klenk138in 1942. He reported the isolation and characterization of one of these compounds, although the existence of similar sugar-lipid conjugates had been intimated some years b e f ~ r e . ~ * ' ~ " ' ~ ~ Gangliosides possess properties different from those of the other groups of glycosphingolipids (such as cerebrosides and sulfatides) already described. Members of this group of glycosphingolipids are readily soluble in water or alcohol, and insoluble in nonpolar solvents. In water (and in some organic solvent^'^^,'^^) they form micelles consisting of c ~ m p l e x e s ' ~of~high J ~ ~molecular weight. They cannot pass through semipermeable membranes. The gangliosides are mainly localized in the nerve cells (as constituents of the neuronal membranes), but they can also be obtained from red-blood-cell s t r ~ m a ' ~ ~and , ' ~ from ' ~p1een.l~~ H. Wagner and W. Zofcsik, Biochem. Z., 346,333 (1966). H. E. Carter, R. Hendry, S . Nojima, N. Z. Stanacev, and K. Ono,J. B i d . Chem., 236,1912 (1961). H. E. Carter, K. Ono, S. Nojima, T. L. Tipton, and N. Z. Stanacev,]. Lipid Res., 2,215 (1961). E. Martensson, Acta Chem. Scand., 17,2356 (1963). C. Michalec and Z. Kolman, Clin. Chim. Acta, 13,529 (1966). K. A. Karlsson,Acta Chem. Scand., 18,2397 (1964). E. Klenk, 2. Physiol. Chem.,273,76 (1942). H. Thierfelder and E. Walz, 2. Physiol. Chem., 166,217 (1927). E. Klenk, 2. Physiol. Chem.,235,24 (1935). G . BIix, S c a d . Arch. Physiol., 80,466 (1938). L. Svennerholm, Acta Chem. Scand., 10,694 (1956). E. Klenk and W. Gielen, 2.Physiol. Chem.,319,283 (1960). E. G. Trams and L. J. Lauter, Biochim. Biophys. Acta, 60,350 (1962). D. B. Gammack, Biochem.].,88,373 (1963). T. Yamakawa and G. Suzuki,J . Biochem. (Tokyo), 38,199 (1951). E. Klenk and H. Wolter, 2. Physiol. Chem.,291,259 (1952). E. Klenk and F. Rennkamp, 2.Physiol. Chem., 273,253 (1942).

414

J. KISS

The gangliosides are rich in structural variations. They form special b i ~ m e m b r a n e s ’ ~in~ the J ~ ~ganglionic cells and in the cerebral cortex, and they probably play an essential role in the functions of the nervous system (“impulse transcription”). In pathological cases (for example, Tay-Sachs’ disease and allied disorder^),'^' gangliosides having special chemical structures accumulate (for example, in the brain), and lead to l i p i d o s i ~ because ’~~ of the restricted effect of the normal enzymes on them. Elucidation of the structure of the gangliosides has been acquired during the past ten years, mainly by the classical methods of carbohydrate and lipid hemi is try.'^^*'^^ The teams of Klenk in Cologne, Kuhn in Heidelberg,’55J56McCluer in Columbus, Ohio, and S~ennerholm’~’ in Sweden have participated in this impressive work. Quick developments were possible in this field because of the fundamental researches of Carter and his associates on elucidation of the structure of the ceramide glycosides free from neuraminic acid. 1. Isolation and Purification of Gangliosides

In his early studies, Klenk’38 used the solvent-extraction method for the isolation of gangliosides; this was later combined with chr~matography.’~~ The mechanically fragmented brain or spinal cord was dehydrated with acetone, as in the isolation of cerebrosides.’ Gangliosides are present in the water-soluble fraction; they were recognized by color reactions (for example, Bial’s orcinol reaction) based on the presence of sialic acid.’50The ganglioside mixture may be purified by partition H. McIlwain, “The Chemical Exploration of the Brain,” Elsevier, Amsterdam, 1963. W. D. Stein, “The Movement of Molecules Across Cell Membranes,” Academic Press, Inc., New York, N. Y., 1967. “Cerebral Sphingolipidoses,” S. M. Aronson and B. W. Volk, eds, Academic Press, Inc., New York, N. Y., 1962. L. Svennerholm, Biochem. Biophys. Res. Commun., 9,436(1962). E. Klenk, “Ueber die Chemie und Biologie der Ganglioside,” Westdeutscher Verlag, Koln and Opladen, 1966. W. Gielen, Z.Klin. Chem., 5,97 (1967). R. Kuhn, H.Egge, R. Brossmer, A. Gauhe, P. Klesse, W. Lochinger, E. Rohm, H. Trischmann, and D. Tschampel, Angew. Chem.,72,805(1960). H.Wiegandt,Angew. Chem.,80,89 (1968). L. Svennerholm,J. Lipid Res., 5,145(1964). E. Klenk and W. Gielen, Z. Physiol. Chem., 326,144(1962). K. Landsteiner and P. A. Levene, Proc. SOC. Exp. Biol. Med., 23,343(1925).

GLYCOSPHINGOLIPIDS

415

dialysis or by formation of the lead salts. Further purification was achieved by adsorption on alumina followed by elution, to achieve separation from such compounds as phosphatides and cerebrosides. Gangliosides obtained by this method proved to be mixtures of compounds having similar structures. A further disadvantage of this method is the loss of gangliosides during the isolation procedure. Nevertheless, chemical characterization of the first gangliosides isolated by these methods led to the identification of their constituent parts. Thus, on hydrolysis, sphingosine, fatty acids, N-acylneuraminic acid, and sugars were obtained. The molecular weight was determined in a nonionic medium such as N,N-dimethylformamide, and values (about 1,500)corresponding to a monomeric structure were found.143Other methods, applied to buffered, aqueous solutions (for example, the osmotic-pressure or the ultracentrifugal method), gave erroneous values160(about 200,000); these high values are explained by association of the molecules in the ionic medium. According to Klenk, the fatty acids bound covalently to the amino group of the sphingosine moiety consist mainly of stearic acid; the sugars are D-galactose (as in the cerebrosides) and D-glucose. Application of chromatographic methods for the separation of the gangliosides led to the conclusion that more than one ganglioside exists in Nature. Svennerholm,161Folch and coworkers,lB2and Trams and L a ~ t e r purified '~~ the ganglioside fraction on a column of cellulose or silicic acid, with use of mixtures of chloroform, methanol, and water for elution. The combination of the partition technique with the chromatographic method may also be used for large-scale preparation of the individual gangliosides. Micro-scale separation and quantitative determination of the gangliosides by thin-layer chromatography on silica gel was developed by Korey and G o n a t a ~ ; it ' ~can ~ be used routinely,ls4 and has proved adequate for evaluating small changes in the composition of a ganglioside mixture. The method is fast and readily reproducible, and can be used for exact determination of the ganglioside composition in pathological cases.165The individual gangliosides obtained by chro(160) T.Yamakawa, S. Suzuki, and T. Hattori, ]. Biochem. (Tokyo), 40, 611 (1953); J. D.Karkas and E. Chargaff, Biochim. Biophys. Acta, 42,359(1960). (161) L. Svennerholm, Acta Chem. Scand., 8, 1108 (1954);J . Neurochem., 10,613 (1963). (162) J. Folch, M.Lees, and G. H. Sloane-Stanley,]. B i d . Chem.,226,497(1957). (163) S.R. Korey and J. Gonatas, Life Sci., 2,296(1963). (164) K.Suzuki,]. Neurochem., 12,629(1965). (165) H. Jatzkewitz, H. Pilz, and K. Sandhoff,]. Neurochem., 12,135(1965).

J. KISS

416

matography or by Craig counter-current distribution166-168can be visibilized by spraying with p-dimethylaminobenzaldehydein hydrochloric acid or with orcinol. Further details on the isolation and purification of gangliosides are given in Svennerholm’s excellent reviews. 157~15* The yield in the preparative isolation of the gangliosides (for example, from beef brain) is rather small, about 0.1%. Thus, from 10 kg of fresh material, about 6-8 g of a mixture of gangliosides can be obtained.lS5 The ganglioside content of microsomes proved to be 0.4%, and that of the cortex of the human brain (related to the dry about 1.5%. It has been found that the ganglioside content varies quantitatively (and, to some extent, qualitatively) with the age170of the person. Precise separation of the individual gangliosides led to the conclusion that at least seven gangliosides of different molecular compositions and structures are present in the normal, mammalian brain. The notation in the literature for the fractions separated according to these methods has not yet been made uniform, and this circumstance can cause difficulties for chemists who are not specialists in this field. In Table V are listed the notations most used for some ganglioside fractions. TABLEV Designations and Molar Ratios in Composition of Some Gangliosides

I171

1 2 3 4 5

11161

1111“

Ala Guz Go An GMl GI B, GDlaGI1 Glrr GDlb C, GT, G f v

IV163.164

C:, G, C, Cz

G,

Composition Ceramide-N-triose-NANAb Ceramide-N-tetraose-NANA Ceramide-N-tetraose-di-NANA Ceramide-N-tetraose-di-NANA Ceramide-N-tetraose-tri-NANA

Molar ratios of ceramide:hexosamine: hexose:NANAb

1:1:2:1 1:1:3:1 1:1:3:2 1:1:3:2 1:1:3:3

OTay-Sachs ganglioside. bNANA=N-Acetylneuraminic acid.

(166) L. Svennerholm, Acta Soc. Med. Upsalten.,62,1(1957). (167) H.L. Methler, J. Btol. Chem., 233, 1327 (1958);E. Klenk, W. Kunau, and L. Ceorgias, Z . Physiol. Chem., 346,236(1966). (168) L. Svennerholm, “Glycolipids and Other Amino Sugar Containing Compounds in the Nervous System,” in “The Amino Sugars,” A. A. BalPzs and R. W. Jeanloz, eds., Academic Press, Inc., New York, N. Y., 1965,Vol. IIA, p. 381. (169) N. M. Papadopulos, Anal. Biochem., 1, 486 (1960). (170) R. Landolt, H.H.Hess, and C. Thalheimer, J . Neurochem., 13, 1441 (1966); A. N. Siakotos and G. Rouser,]. Amer. Oil Chemists’ Soc., 42,913(1965). (171) E.Klenk, 2.Phystol. Chem., 348,149(1967). (172) R. Kuhn and H. Wiegandt, Chem. Ber., 96,866(1963).

GLYCOSPHINGOLIPIDS

417

The individual gangliosides separated differ mainly in the structure of their carbohydrate moieties, although the sphingosine part may also differ in length and size of the saturated or unsaturated hai in'^^-'^^ and is dependent on the age of the 0 r g a n i ~ m . l ~ ~ The fatty acid linked to the amino group of the sphingosine or sphinganine part may also differ, but, in the gangliosides isolated from nervous tissues, the main fatty acid is stearic acid (80-90%) and, in the gangliosides of spleen and erythrocyte^,'^^ tetracosanoic acid (docosanoic acid, tetracosenoic acid). An exact, quantitative determination of the components of the pure individual gangliosides has been made by Svennerholm.161 2. Sialic Acids of the Gangliosides The characteristic components of the gangliosides have proved to be the sialic acids (62b) (N-acylated neuraminic acids). In 1941, Klenk179 described the isolation of N-acetylneuraminic acid after hydrolysis of gangliosides. Neuraminic acid (62a) has not been found in the free state. In Nature, it is mostly combined with sugars. It also occurs in groups of natural products other than gangliosides (for example, mucoproteinslsO). On the basis of characteristic color-reactions for (acylated) neuraminic acids (for example, Bial’s orcinol r e a ~ t i o n ’ ~ ~and * ’ ~the @ thiobarbituric acid methodla’), quantitative methods for the determination of gangliosides have been developed (for example, on thin-layer plates157.161,184.182>. In gangliosides, the N-acylated neuraminic acids are linked glycosidically to the oligosaccharide moiety of the molecule; the latter can be cleaved by hydrolysis (for example, with dilute acid or with (173) K.Sambasivaraoand R. H. McCluer, Fed. Proc., 22.300 (1963). (174) N.Z.Stanacev and E. Chargaff, Biochim. Biophys. Acta, 98,168(1965). (175) K.A. Karlsson,Acta Chem. Scand., 18,565(1964). (176) L. Svennerholm, Biochem.]., 98,20~(1966). (177) J. Menkes, Biochem. Biophys. Res. Commun., 15.551 (1964). (178) E.Klenk and G. Padberg, Z. Physiol. Chem.,327,249(1962). (179) E.Klenk, Z.Physiol. Chem., 268,50(1941). (180) G. Blix, Z.Physiol. Chem.,240,43(1936). (181) L. WarrenJ. Biol. Chem., 234,1971(1959). (181a)According to “Specification of the Molecular Chirality,” R.S. Cahn, C. H. Ingold, and W. Prelog, Angew. Chem., 78,413(1966). (182) K.Suzuki, Life Sci., 3,1227(1964).

J. KISS

418

r4% H0,C-COH

H~OH

Rm+ OCH

I

HCOH

HAOH I

CqOH

H?oH HCOH L,OH (62)

(a) Neuraminic acid (R = H); (b) sialic acid (R = acyl)

~-~ino3,~-dideoxy-~-g~yce~o~-D-ga~acto-nonu~opyranoson~c

acid (Neuraminic acid)

neuraminidase). Neuraminidase cleaves only the (Y-Danomer of the neuraminic acid g l y ~ o s i d e s . ~ ~ ~ ~ ~ ~ The chemistry of neuraminic acid and derivatives has been reviewed by Zilliken and W h i t e h o ~ s eand ’ ~ ~by GottschalK.185 Gangliosides isolated from nervous tissues contain N-acetylneuraminic acid in covalent conjugation18B-1ss(R = Ac), and the gangliosides of spleen and erythrocytes bear N-glycoloylneuraminic acid1”JW (R =-CO-CH,OH) on a hexopyranosyl group of the oligosaccharide part of the molecule. Furthermore, as already mentioned, N-acylated neuraminic acids are present in covalent conjugation in certain types of naturally occurring compounds other than glycosphingolipids (for example, glycoprotein hormones and natural mucopolysaccharides). The R. Ledeen and K. Salsman, Biochemistry, 4,48,2225 (1965); H.Jatzkewitz and K. Sandhoff, Biochim. Biophys. Acta, 70,354 (1963). F. Zilliken and M. W . Whitehouse, Adoan. Carbohyd. Chem., 13, 237 (1958). A. Gottschalk, “The Chemistry and Biology of Sialic Acids and Related Substances,” Cambridge Univ. Press, 1960. L. Svennerholm, Acta Chem. Scand., 9,1033 (1955). G. Blix and L. Odin, Acta Chem. Scand., 9,1541 (1955). E . Klenk and G. Uhlenbruck, 2. Physiol. Chem., 311,227 (1958). G . Blix, E. Lindberg, L. Odin, and J. Werner, Acta SOC. Med. Upsalien., 61, 1 ( 1956).

E. Klenk and G. Uhlenbruck, Z . Physiol. Chem.,307,266 (1957).

GLYCOSPHINGOLIPIDS

419

special biological activities of these compounds are connected with the presence of N-acylated neuraminic acids.lgl Both of the N-acylated neuraminic acids mentioned have been synthesized. Cornforth, Daines, and Gottschalk'* made the first synthesis of N-acetylneuraminic acid. They condensed 2-acetamido-2deoxy-D-glucose with oxalacetic acid at pH 11, and obtained a low yield (about 2%). Carroll and C ~ r n f o r t h 'obtained ~~ a higher yield from 2-acetamido-2-deoxy-~-mannose and oxalacetic acid. Kuhn and BaschangIB4used the same principle for the synthesis of N-acylated neuraminic acids: by condensing the potassium salt of ditert-butyl oxalacetate (64) with 2-acetamido-2-deoxy-~-mannose (63) (or with 2-acetarnido-4,6-0-benzylidene-2-deoxy-~-glucose), they obtained the corresponding lactone (65). This was hydrolyzed with water at 90-100" to give N-acetylneuraminic lactone (66) in a yield of 34%. H

c=o I

R'HNciH HOCH

+

pc,H, C ' H 'F=O

I

I c=o

0 "Or (65)

(66)

H H where R = HOHzC-C-Cand R' = Ac. HO OH

(191) For a review of the literature, see: P. Meindl and H. Tuppy, Monatsh., 96, 803 (1965). (192) J. W.Cornforth, M. E. Daines, and A. Gottschalk, Proc. Chem. SOC., 25 (1957). (193) J. W.Cornforth, M. E. Firth, and A. Gottschalk, Biochem. J., 68,57(1958);P. M. Carroll and J. W. Cornforth, Biochim. Biophys. Acta, 39,161(1960). (194) R. Kuhn and G. Baschang, Ann., 659, 156 (1962);Chem. Ber., 95,2384 (1962).

J. KISS

420

Starting from 2-(benzyloxycarbonyl)amino-2-deoxy-~-glucose and potassium di-tert-butyl oxalacetate,'" N-(benzyloxycarbony1)neuraminic lactone (66, R' = -COO-CH,Ph) was obtained. Hydrogenolysis of the benzyloxcarbonyl group led to free neuraminic acid, which forms a pH-dependent equilibrium mixture of the free base (67) and its internal Schiff base (68).

(67)

where R = -H

(68)

or -C&,

I

and R' = HO H

ci HCOH HLOH

CHzOH

The acyclic form 67 exists only in strongly acidic media. In neutral or weakly acidic solution, the cyclic product (68) is stable. Synthesis of N-glycoloylneuraminic acid was achieved by Faillard and Blohm.lgs By the general procedure of GaultlB7and Kuhn and Baschang,'% 2-deoxy-2-glycoloylamido-~-glucosewas condensed with the potassium salt of di-tert-butyl oxalacetate. The tert-butyloxy lactone obtained was converted into N-glycoloylneuraminic lactone. In gangliosides, the N-acylneuraminic acids are in WD anomeric (69) conjugation with hexopyranosides, as already mentioned, and are split off enzymically with neuraminidase.lg8 Synthetic alkyl and aralkyl glycosides of N-acylneuraminic acid, obtained by the method of Koenigs and Knorr,'B' are split with neuraminidase, like the natural glycosides of N-acylneuraminic acid. In contrast, the /3-D anomers (70), obtained from N-acylneuraminic acid and the appropriate alcohol in the presence of hydrogen ion, are resistant to neuraminidase.'B'~'BB~z~ (195) W.Gielen, Z.Physiol. Chem., 342,170(1965). (196) H.Faillard and M. Blohm, Z . Physiol. Chem.,341,167(1965). (197) H.Gault,Ann. Chtm., 6,220(1951). (198) R.Kuhn and R. Brossmer, Angew. Chem., 70,25(1968). (199) R.Kuhn, P.Lutz, and D. L. MacDonald, Chem. Ber., 99,611(1966). (200) H. Faillard, G. Kirchner, and M. Blohm, 2. Physlol. Chem., 347, 87 (1966); J. D. Karkas and E. Chargaff,]. Biol.Chem.,239,949(1964).

GLYCOSPHINGOLIPIDS

421

Hi) Q-D

(2R)""

(69)

p-D (2s)""

I where R = alkyl and R' = HCOH I HCOH I CH,OH

(70)

3. Linkage between N-Acylsphingosines and the Oligosaccharide Moiety The conjugation between the lipophilic and the hydrophilic part of the ganglioside molecule has proved to be glycosidic. Hexopyranosyl groups are linked to the primary hydroxyl group of the acylated sphingosine or acylated dihydrosphingosine (sphinganine) molecules.zO1 The structure of the gangliosides has been proved in several ways, most of which belong to the classical methods of carbohydrate chemistry; for example, the individual gangliosides were methylated, and the products were cleaved by hydrolysis. 3-0-Methylsphingosine (or 3-0-methyldihydrosphingosine)(71, R' = CHI, R and R" = H), and 0-methylated hexoses were isolated. Klenk and coworkerszo2isolated N-stearoylsphingosine D-glucoside (71a) and N-stearoylsphingosine lactoside (71b) from the products of mild hydrolysis of the gangliosides with acid; they were identified by paper chromatography. Ozonolysis has also been used for investigation of the same problem.203By this method, a special degradation of the glycosphingolipids (but not of the saturated dihydroglycosphingolipids) can be accomplished. This method of degradation had been used earlier by Klenk and DieboldzWand others13for the mild degradation of acylated sphingosines and for the degradation of palmitoylsphingosine Dgalactosides .69 The removal of the lipid part of the ganglioside molecule (for (201) E.Klenk and W. Gielen, 2.Physiol. Chem.,326,158(1961). (202) E.Klenk, U.W. Hendricks, and W. Gielen, 2.Physiol. Chem., 330,140 (1962); 348,151(1967). (203) H. Wiegandt and G. Baschang, Z.Nutulforsch., 20B,164 (1965). (204) E.Klenk and W. Diebold, Z. Physiol. Chem., 198,25(1931).

J. KISS

422

H C13-(C&)lz-C=C-C-

H

H C- CHZ-OR H OR' NHR" (71)

p 6 CH,OH

OH

where R =

or

HO

OH (a)

Hu ;

OH

(b)

R' = H or -CH,

; and R" = -CO-(CH,),4-CH~.

example, 72) by ozonolysis followed by alkaline hydrolysis permitted isolation of the intact oligosaccharide in fairly good yield. Wiegandt'= and Wiegandt and Baschang203have proposkd the following pathway for this degradation.

bYo-sugar HCNHAc I HCOH

I HC II CH I (CH,),, AH3

H,CO-sugar

H,CO-sugar I HCNHAc

HLNHAC 0s

I

+

~

C=O H

H

c=o I

+

I I

HCOH

c=o

H

2 5%

7 5%

(y%)IZ

CH,

(72)

(73)

(74)

OH@ H, 0 sugar

'i-

CbOH

(75)

&C-y + 0

H,C-C-NH, II 0

GLYCOSPHINGOLIPIDS

423

The lipid-free oligosaccharides (73 and 74) obtained could be readily separated from the other (minor) degradation products. By this method, gangliosides containing one, two, or three N-acetylneuraminic acid residues can be fragmentedaZo3 Other methods (for example, hydrolysis with dilute mineral acids, or acetolysis) cleave the glycosidic linkages in the oligosaccharide moiety too, resulting in a greater number of products. 4. Structure of the Oligosaccharide Members

The hexopyranosyl groups in covalent linkage in the oligosaccharide moiety of a molecule of the normal-brain gangliosides are those of D-glUCOSe (1 molecule), D-galactose (2 molecules) and one of the aminohexose derivatives 2-acetamido-2-deoxy-~-galactose(1 molecule) or 2-acetamido-2-deoxy-~-glucose(1 molecule). According to the experiments of Klenk and coworkers,153the permethylated gangliosides-C, and -C, of the normal brain give, after hydrolysis, 2,3,6-tri-O-methyl-~-glucose,2,6-di-O-methyl-D-galactose, 2,3,4,6-tetra-O-methyl-~-galactose, and 2-amino-2-deoxy-4,6-di-Omethyl-D-galactose (from ganglioside-C,); from ganglioside-C3, instead of 2,3,4,6-tetra-O-methyl-D-galactose,2,4,6-tri-O-methyl-Dgalactose is obtained (besides the other methylated hexopyranoses). Furthermore, by mild hydrolysis with acid, the following oligosaccharides were isolated. Lactose Ganglio-N-biose-I Ganglio-N-biose-I1 Ganglio-N-triose-I Ganglio-N-triose-I1 Ganglio-N-tetraose

D-Galp-(1 + 4 ) - ~ - G p D-Galp-(1 + 3)-~-GalNAcp D-GalNAcp-(1 + 4)-~-Galp 1 + 4)-~-Galp D-Galp-(1 + 3)-~-GalNAcp-( 1 +4)-~-Gp D-GalNAcp-(1 + 4)-~-Galp-( D-Galp-(1 + 3)-~-GalNAcp-( 1 + 4)-~-Galp-( 1 +4)-~-Gp

The last-mentioned compound (76) can be obtained from the neuraminidase-resistant ganglioside-G, by mild hydrolysis with acid (50 mM sulfuric acid at 80"). This compound is the fundamental tetrasaccharide unit of the main gangliosides isolated from the normal human (and also beef) brain. The structure of tetrasaccharide 76 was proved by oxidation with periodate. The products obtained were reduced with sodium borohydride, and the reduction products were cleaved by hydrolysis; 2amino-2-deoxy-D-galactose, erythritol, a threitol, and glycerol were isolated.

J. KISS

424

0

-

Ganglio-N tetraose (76)

H

AcPf HCOH I

&&OH Ganglioeide-G I (77)

GLYCOSPHINGOLIPIDS

425

Ganglioside-GI was cleaved in the same way, to give D-galactose, glycerol, and erythritol, in addition to sphingosine and stearic acid. The structure of ganglioside-GI is that shown in formula 77. Ganglio-N-biose-I was isolated by Klenk and coworkers202 by hydrolysis of a mixture of human-brain gangliosides with 10 mM hydrochloric acid at 100" during 45 minutes. The crystalline disaccharide, having mp 154-15T,[aID+49.7" (in water), proved to be identical with 2-acetamido -2-deoxy -3-0-P-D-galactopyranosyl -Dgalactose (78). This disaccharide had already been isolated from blood-group substance A by Morgan and coworkers,2o5and had been found to be unstable in alkaline media.

HO OH NHAc

The linkage between the oligosaccharide part of the neuraminidaseresistant ganglioside-C, and N-acetylneuraminic acid is on the 3hydroxyl group of D-galactopyranoside ring B. According to Kuhn and Wiegandt,"* on acetolysis, 0-(N-acetylneuraminic acid)-(2 + 3)a-D-galactose and 0-(N-acetylneuraminic acid)-(2+ 3')-a-lactose (79) are obtained. Compound 79 had been isolated from human- and cattlemilk colostrum by Kuhn and Brossmer.20B

(205) T. J. Painter, I. A. F. L. Cheese, and W. T. J. Morgan, Chem. Znd. (London), 1535 (1962). (206) R. Kuhn and R. Brossmer, Chem. Ber., 92,1667 (1959).

J. KISS

426

HCOH

I

ChOH 0 -(N-Acetylneuraminic acid)-(2

+

3')-~-lactose

(79)

The structure of other natural gangliosides isolated from normalhuman and bovine nervous-tissues was determined. These individual gangliosides have, in covalent conjugation, more than one residue of N-acetylneuraminic acid (NANA) that can be split off enzymically with neuraminidase, giving20sathe fundamental ganglioside-G,.

The structural correlation of natural gangliosides (usually minor components) with the structurally well-established compounds already discussed was also realized. The work has been summarized by Klenk153and Wiegandt.Iss (206a) Notation of the gangliosidesaccording to R. Kuhn and H. Wiegandt.

GLYCOSPHINGOLIPIDS

427

A molecule of ganglioside-Gll contains two residues of N-acylneuraminic acid, one of which can be cleaved enzymically by neuraminidase, giving ganglioside-GI. D-Galp-(l + 3)-P-D-GalNAcp-(l + 4)-P-~-Galp-( 1 + 4)-p-~-Gp-( 1 + I)-P-Sph 3 3 I

t

a-2-N-Ac-NANA

t

0-2-N-Ac-NANA

I

fatty acid

where N-Ac-NANA = N-acetylneuraminic acid.

This structure was established by periodate oxidation and methylation, followed by hydrolysis, as already mentioned. The formation of 2,4,6-tri-O-methyl-~-galactose and 2,6-di-O-methyl-~-galactosegave particular support to this structural proposal. On the other hand, ganglioside-GIII is structurally similar to ganglioside-GII. By treatment with neuraminidase, one molecular proportion of N-acetylneuraminic acid is cleaved, resulting in ganglioside-GI, but, after methylation with subsequent hydrolysis, 2,3,4,6-tetra-Omethyl-D-galactose and 2,6-di-O-methyl-~-galactosewere isolated. D-Galp-(1+3)-P-~-GalNAcp-(1+4)-P-~-Galp-(1+4)-P-D-Gp-( 1+1)-/3-Sph 3

t

a-2-N-Ac-NANA 8

I

fatty acid

t

a-2-N-Ac-NANA

These structures, proposed by Kuhn and Wiegandt,207were confirmed by the experimental results of Klenk and coworkers,153who hydrolyzed the individual di- and tri-sialogangliosides with polystyrenesulfonic acid in water at room temperature, by the method of Painter,208to give dimeric bis-a-~-(2-*8)-N-acetylneuraminic acid. The structure of ganglioside GI, was similarly examined; it contains three molecular proportions of N-acetylneuraminic acid in covalent conjugation. Two of these residues can be split off by neuraminidase, giving (through gangliosides GIIand GI,,) the fundamental ganglioside GI. The structural combination of gangliosides GII and GIIIled to the structure of ganglioside GI". This structure is represented by formula 80, a stereochemical depiction of which is also given. (207) R. Kuhn and H. Wiegandt, 2. Noturforsch., 18B,541 (1963). (208) T. J. Painter, Chem. Znd. (London), 1214 (1960).

J. KISS

428

t

a-2-N-Ac-NANA

t

I

a-2-N-Ac-NANA 8

fatty acid

T.

a-2-N-Ac-NANA Ganglioside Glv (80)

Besides the above types of gangliosides bearing the characteristic ceramide-1 P-D-glucopyranoside structural unit, gangliosides also exist (only in small proportions) that contain, as their fundamental unit, the structure of a ceramide-1 P-D-galactopyranoside. From the human brain, Siddiqui and McCluefogisolated a minor ganglioside containing equimolecular proportions of sphingosine (C, : CIS= 5:3), fatty acids (normal, 2-hydroxy-, and unsaturated, respectively), sialic acid, and D-gdaCtOSe?03 5. Gangliosides Isolated from Pathological Tissues Metabolic defects cause disturbances in the metabolism of sphingolipids in some diseases (for example, sphingolipidoses, infantile amaurotic idiocy, Tay-Sachs’ disease, Gaucher’s disease, and metachromatic leucodystrophy). Klenk reported210the anomalous accumulation of some gangliosides. The unusual solubility behavior of, for example, the Tay-Sachs’ brain tissues is due to qualitative and quantitative differences in the composition of the appropriate lipid.211The isolation and purification of these anomalous gangliosides were carried out similarly to those for the gangliosides of normal tissues, for example, by the method of Folch and coworkers.162In this method, the brain tissues are extracted with chloroform-methanol-water, the extract is washed with potassium chloride solution, and the extracted material is separated on a column of silicic acid by elution with an increasing concentration of methanol in chloroform. The preliminary, structural investigations of the lipid material obtained led to the conclusion that these gangliosides have a chemical composition different from those of the gangliosides isolated from (209) B. Siddiqui and R. H. McCluer, J . Lipid Res., 9, 366 (1968); E. Klenk and W. Gielen, Z. Phvstol. Chem., 333,162 (1963). (210) E. Klenk, Z. Physiol. Chem., 262,128 (1939). (211) A. Rosenberg and E. Chargaff,J . Diseases Children, 97,739 (1959).

J. KISS

430

normal, nervous tissues. Nevertheless, these gangliosides can also be isolated in small proportion from normal-brain tissues,138from erythrocyte stroma of horses,146and from beef ~ p 1 e e n . lBoth ~ ~ the lipid and sugar constituents of these gangliosides show divergences from the normal ones. Rosenberg and Stern212reported the high proportion of Cla-sphingosine isolated from brain of humans having Tay-Sachs' disease. In the gangliosides isolated from normal-brain tissues, there is a greater proportion of C20-sphingosine.213~214 The monocarboxylic acid connected in amide conjugation to the amino group of the sphingosine residue proved to consist mainly of the C,,-fatty acid. The gangliosides of Niemann-Pick disease and metachromatic leucodystrophy differ from those of normal, infant and adult brains, in that Cz4-acidswere not detected. The development of new ultramicro methods led to the exact (differential) determination of the constituents of these g a n g l i o s i d e ~of~disease. ~~ S ~ e n n e r h o l m 'found ~~ that the molecular ratios of the lipid and sugar residues of the Tay-Sachs ganglioside are ceramide : hexose : 2acetamido-2-deoxy-~-galactose : N-acetylneuraminic acid = 1:2:1:1. One molecule of this ganglioside (81) contains one residue of hexose less than the normal monosialoganglioside; otherwise, they are structurally similar. The terminal D-galaCtOpyranOSyl group is absent. D-GalNAcp-(1+4)-P-~-Galp-(1+4)-fl-~-Gp-( l+l)-P-S ph-fatty acid 3

I

CY-Z-N- Ac-NANA

From the Tay-Sachs ganglioside (81), N-acetylneuraminic acid can be cleaved enzymically, to give the ceramide trisaccharide. By degradation with ozone, or by mild hydrolysis, a trisaccharide is obtained, namely,

This ganglioside constitutes more than 90% of the total ganglioside in the brain tissues of patients with Tay-Sachs' disease; in normalbrain tissues, it is present to the extent of only 1-5% of the total gangliosides. (212) (213) (214) (215)

A. Rosenberg and N. Stern,-!. LipidRes., 7,122 (1966). N. Z. Stanacev and E. Chargaff, Biochim. Biophys. Acta, 59,733 (1962). G. Reuser, G. Feldman, and C. Galli,]. Amer. 012 Chemists' Soc., 42,411 (1965). H. Jatzkewitz,H. Pilz, and H. Hollander,Acta Neuropath., 4,75 (1964).

GLYCOSPHINCOLIPIDS

431

On the other hand, the total amount of gangliosides also increases in sphingolipidoses. According to Jatzkewitz and coworkers,165the value can reach ten times the normal. Besides the ceramide trisaccharide and its N-acetylneuraminic acid derivative, small fragments, namely, ceramide lactoside and monosialoceramide lactoside (Hematoside), were found.216 According to Jatzkewitz and coworkers,217 the accumulation of these ganglioside derivatives in the pathological cases mentioned is localized mainly in the gray matter of the brain, whereas the sulfatides accumulate in the white matter.83*218,219 Besides the ceramide lactoside, more than 50 % of glucocerebroside (ceramide monohexoside) was also found.217The accumulation of these ganglioside fragments is due to a lack of suitable enzymes (for example, N-acetylhexosaminidase220). (216) The total synthesis of this and similar compounds, carried out by Shapiro and Flowers,Es*gO is discussed in the Section on cerebrosides (see p. 401). (217) H. Pilz, K. Sandhoff, and H. Jatzkewitz,J. Neurochem., 13,1273 (1966). (218) W. D. Suomi and B. W. Agranoff,J. Neurochem., 6,211 (1965). (219) D. A. Booth, H. Goodwin, and J. N . Cummings, J . Neurochem., 7 , 337 (1966). (220) K. Sandhoff, U. Andreae, and H. Jatzkewitz, Summary on the “Wintertagung der Gesellschaft fur Biologische Chemie,” Z. Physiol. Chem.,349.13 (1968).

432

J, KISS

The enzymic degradation of the gangliosides in normal tissues was also studied. This degradation started with cleavage of the N-acetylneuraminic acid residues, and was followed by the cleavage of the other sugars linked to the ceramide moiety.221 Starting from ceramide D-glucoside, enzymic synthesis has been studied for producing g a n g l i o s i d e ~ and ~ ~ ~for . ~ ~the ~ conversion of Tay-Sachs ganglioside into monosialoganglioside by the uridine 5'-pyrophosphate of 6. Gangliosides of Erythrocytes and Spleen

According to Klenk and coworkers's and Yamakawa and coworkers,lM gangliosides obtained from horse erythrocytes have a simpler structure than those of the central nervous system. The P-D-glucopyranosyl group is linked to the primary hydroxyl group of N-tetracosanoylsphingosine. As in the brain gangliosides and the Tay-Sachs gangliosides, the sugar residue bears, on the 4-hydroxyl group of the D-galactopyranoside residue, an N-glycolylneuraminic acid residue, as in 82.

(221) G . Basu, B. Kaufmann, and S. Roseman,J. Biol. Chem., 240, ~ ~ 4 1 (1965). 15 (222) G . Gatt,J. Biol. Chem., 238, pc3131 (1963). (223) K. Sandhoff, H.Pilz, and H. Jatzkewitz, Z . Physlol. Chem., 338, 281 (1964); J . Neurochem., 12,135(1965). (224) G . Basu and B. ICaufmann, Fed. Proc., 24,479 (1965).

GLYCOSPHINGOLIPIDS

433

Some years ago, the isolation (from beef erythrocytes and spleen) was reported225of ganglioside that contained, not only the N-acylated sphingosine, N-glycoloylneuraminic acid, and hexoses (D-glucoseand D-galactose), but also 2-acetamido-2-deoxy-~-glucose. On mild hydrolysis with acid, followed by degradation with ozone, this ganglioside gives lacto-neotetraose. Lacto-neotetraose (83) had already been isolated from human milk by Kuhn and Gauhe.226

I

NHAc

I

OH

OH

On methylation, with subsequent hydrolysis, tetrasaccharide 83 gives 2,3,6-tri-O-methyl-~-glucose, 2,4,6-tri-O-methyl-D-galactose, 2-acetamido-2-deoxy-3,6-di-O-methyl-~-glucose, and 2,3,4,6-tetra-0methyl-D-galactose. By mild hydrolysis of the lacto-neotetraose, there were obtained lacto-N-triose, lacto-N-biose, and lactose (identified by paper chromatography).

(225) R. Kuhn and H. Wiegandt, Z. Nututforsch., 19B,80 (1964). (226) R. Kuhn and A. Gauhe, Chem. Ber., 95,518 (1962).

This Page Intentionally Left Blank

PROTEIN-CARBOHYDRATE COMPOUNDS IN HUMAN URINE BY E. H. F. MCGALE* North Staffordshire Hospital Centre, Renal Unit, Princes Road, Stoke-on-Trent, England I. Nomenclature

.................. 435

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

11. Urinary Protein-Carbohydrate Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . .

436

1. General Introduction . . . . . . . . . . ................. 436 2. Carbohydrate Composition . ................. 438 3. Molecular Weight . . . . . . . . 4. Glycoproteins and Proteopol . . . . . . . . . . . . . . . . 440 443 5. Peptide-Acidic (Aminodeoxyg1yco)glycuronans ......................... 111. Urinary Protein-Carbohydrate Compounds of Low Molecular Weight ..................................................... 444 1. Carbohydrate Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 2. Peptide Composition . . . . . 445 3. Properties.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 4. O r i g i n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450 IV. Conclusion . . . . . . . . . . . . . . . 451

I. NOMENCLATURE In the study of compounds containing covalently bonded sugar and amino acid residues, the considerable diversity in the nomenclature of these compounds is, in many instances, confusing. In addition to the terms glycoprotein and glycopeptide, such terms as sialoglycoprotein, acid glycoprotein, mucin, mucoprotein, and fucopeptide have been used, often without adequate restriction in their meaning. As the present Chapter is concerned with a range of proteincarbohydrate compounds that differ considerably in their composition, properties, and origin, a defined system of nomenclature is clearly important. 'The author is grateful to Miss Jaqueline B. Weiss, Department of Medical Biochemistry, Manchester University, for valuable discussion and comment during the preparation of this Chapter.

435

436

E. H. F. MCGALE

In Table I is given the system of nomenclature that will be used in this Chapter, together with the definition of each name and alternative names in common use; the overt physical properties of the particular compound are the basis of this system.’ Thus, the term glycopeptide describes a compound of low molecular weight that is predominantly peptidic in its properties and composition; conversely, a proteopolysaccharide is a compound of high molecular weight that is predominantly polysaccharidic in its properties and composition. CARBOHYDRATE COMPOUNDS I I. URINARYPROTEIN-

1. General Introduction

The presence in normal human urine of small proportions of protein and glycoprotein was first recognized by Moernel.2 in 1895. [Acidic (aminodeoxyg1yco)glycuronans are also present.*] Not until much later was the heterogeneity of urine with respect to these compounds established, when moving-boundary electrophoresis of the nonultrafiltrable, urinary fraction separated a number of proteins and glycoproteins, some of which had electrophoretic mobilities that identified them as plasma albumins and g l o b ~ l i n s .Subsequent ~ studies of those compounds not so identified have revealed the presence, in normal urine, of a considerable number of protein-carbohydrate compounds that are generally of low molecular weight and are mainly carbohydrate in composition. These compounds of low molecular weight, principally glycopeptides and peptido-oligosaccharides, are the primary concern of this Chapter, and data regarding their composition, properties, and possible origin are presented and discussed in Section I11 (see p. 444). In the present Section, data regarding the general composition and molecular weight of urinary protein-carbohydrate compounds are presented. In addition to glycopeptides and peptido-oligosaccharides, the presence in normal urine of glycoproteins, acid glycoproteins, proteopolysaccharides, and acidic (aminodeoxyg1yco)glycuronansis well established. Generally, these compounds of high molecular weight in urine have been less extensively investigated than their counterparts of low molecular weight; exceptionally, some of them have been (1) J. B. Weiss and E. H. F. McGale, in “Chromatographic and Electrophoretic Techniques,” I. Smith, ed., Heinemann Medical Books, Ltd., London, 1968, VOl. 11, p. 55. (2) K. A. H.Moerner, Scand. Arch. Physiol., 6,332(1895). (3) D. A. Rigas and C. A. He1ler.J. Clin. Inoest., 30,853(1951).

TABLEI Nomenclaturea of Protein-Carbohydrate Compounds Group of compounds

Definition

Alternative nomenclature

Examples

substances of high molecular weight having many of the physical properties of a protein, but containing covalently bonded carbohydrate component(s)

mucoproteins, mucosubstances, mucins

ovalbumin, thyroglobulin, ceruloplasmin

Acid glycoproteins

glycoproteins containing a high concentration of sialic acid and having a low isoionic point

sialoglycoproteins

orosomucoid

Proteopolysaccharides

substances of high molecular weight having many of the physical properties of a polysaccharide, but containing covalently bonded protein component(s)

Glycopeptides

substances of low molecular weight having many of the physical properties of a peptide, but containing covalently bonded carbohydrate component(s)

mucosubstances, mucopolysaccharides, mucins, mucoproteins mucopeptides, mucins

blood-group substances, intrinsic substances muramyl peptides, products of enzymic degradation of glycoproteins

Glycoproteins

Peptidooligosaccharides

substances of low molecular weight having many of the physical properties of an oligosaccharide. but containing covalently bonded peptide component(s)

mucopeptides, fucopeptides, glycopeptides

products of enzymic degradation of glycoproteins

“In addition to this nomenclature, the term acidic (aminodeoxyg1yco)glycuronan(“mucopolysaccharide”) will be used to describe high molecular-weight polysaccharides that owe their acidity to the presence of hexuronic acid or to sulfate or sulfamido groups, or both (examples include hyaluronic acid, chondroitin sulfates, and heparin). Some of the acidic (aminodeoxyg1yco)glycuronansthat are excreted in urine have been shown to contain a covalently bonded peptide component.

c

E z

*

w !a

s2

2

n

*3

om 5

F 2

83

z2

!E

lb

0 4

438

E. H. F. McGALE

characterized with respect to their composition, origin, or biological activity, and they are briefly discussed in this Section. 2. Carbohydrate Composition

In attempting to isolate protein-carbohydrate compounds from urine, dialysis or ultrafiltration techniques have generally been used for removing the large proportions of inorganic salts and “small” organic compounds normally present in urine, and the residual, nondialyzable or nonultrafiltrable fraction has been used as the starting material for further fractionation. Waldron4 showed that proteins and carbohydrates are the major coniponents of the nondialyzable fraction of normal urine; the carbohydrate content of this fraction has been determined by several authors,5-’ and the values they obtained are within the range of 30 to 39% (wlw); as shown in Table 11, hexose, aminodeoxyhexose, and sialic acid are the major carbohydrate components, with smaller proportions of 6-deoxyhexose and hexuronic acid; the yield of solid material from this fraction corresponds to 0.3-0.9% (wlw) of the total, urinary solids. The disadvantage of using the nondialyzable fraction as the starting material for further fractionation is evident from the work of Boas,8 who found that 47 % of the total, urinary aminodeoxyhexose is dialyzable; in preliminary studies, he resolved three glycopeptide-containing fractions in the dialyzable, urinary material by means of column chromatography on cellulose; he commented on the difficulty encountered in fractionating this material, since the aminodeoxyhexose accounts for only 0.002% (w/w) of the dialyzable, urinary solids.

3. Molecular Weight Most of the constituents of normal urine are probably filtered into the kidney nephrons through the glomerulus,” selective tubular (4) D. H. Waldron,Nature, 170,461(1952). (5) D.Hamerman, F. T. Hatch, A. Reife, and K. W. Bartz, J . Lab. Clin. Med., 46, 848 (1955). (6) J. S. King, Jr., W. H. Boyce, J. M. Little, and C. Artom,]. Clin. Invest., 37,315 (1958). (7) R. Bourrillon and J. C. Kaplan, Clin. Chim. Acta, 5, 732 (1960). (8) N.F.Boas, Proc. SOC.E x p . B i d . Med., 92,122(1956). (9) G. Marcotte-Boy, R. Henry, and R. Issartel, Bull. SOC.Chim. B i d , 41, 1485 (1959). (10) C . Lovatt-Evans, “Principles of Human Physiology,” J. A. Churchill and Co., Ltd., London, 12th Edition, 1956,p. 1002. (11) L. J. Rather, Medicine, 31,357(1952).

C

Ez TABLEI1

Carbohydrate Composition (%, w/w) of the Nondialyzable Urinary Fraction ~~

~~

~~

~

Type of urine"

Yield (mglliter)

Hexose

6-Deoxyhexose

Sialic acid

Uronic acid

(%)

( %)

( %)

( %)

Adult, pooled Adult, 24-hOur Child, pooled Adult, 24-hour Adult 24-hour

151-400 287 141

15.4 16.7 14.5

2.8

9.4 9.7

1.4 -

-

-

-

2.9

-

-

-

Aminodeoxyhexose (%) (mglliter) 10.2 6.1 11.4 -

15-40 17.7 16.1 28.0 41.0

Total %

References

29Bb 32.2 38.5 -

5 6 7 8 9

"In comparing data obtained from pooled and 24-hour urine specimens, percentage values have been calculated by assuming that the average volume of a 24-hour urine specimen is'O 1.5 liters. bThis value does not include sialic acid.

z

440

E. H. F. McGALE

reabsorption following this process.12 By using dextrans of graded molecular weight, W a l l e n i ~ sfound '~ that the group having maximum molecular weight that passed through the normal glomerulus in measurable quantities was within the range of mol wt 43,000 to 49,000;he obtained the following clearance values: 90% (molecular weight, 10,000);30% (20,000);and -0% (50,000). Although comparison of dextrans with proteins and protein-carbohydrate compounds may be of limited value, these values suggest that the normal kidney exerts a molecular-sieving effect over a limited range of molecular weights. Gel-filtration on Sephadex has been usedI4 for determining the distribution of the nondialyzable, protein-containing compounds of human urine in molecular-weight groups 1,000-5,000, 5,000-10,000, and 10,000-100,000 (see Table 111), and the results indicate that approximately 68% of these compounds in the urine of adults have molecular weights of 1,000-10,000 (the decreased percentage excretion of these lower molecular-weight compounds by children is, as yet, unexplained in terms of specific compounds). The values for protein distribution in the higher molecular-weight groups may now be considered to be unrealistically high; aggregation of urinary proteincarbohydrate compounds into complexes of higher molecular weight can occur, and these aggregate groups may not be resolved by gelfiltration or dialysis; these aggregation effects, which may explain the apparently anomalous diffusion properties of glycopeptides and peptido-oligosaccharides, will be discussed more fully in Section I11 (see p. 449). 4. Glycoproteins and Proteopolysaccharides

Numerous a ~ t h o r s ~ - ~have * ' ~ -attempted ~~ isolation of these groups of compounds from normal urine. Different combinations of ion-exJ. Hardwicke and J. R. Squire, Clin. Sci.,14,509 (1955). G.Wallenius,Acta SOC. Med. Upsalien.,54, Suppl. 4 (1954). R. 0.Gale, C. E. Cornelius, and J. A. Bishop, Inu. Urol.,4 , 3 3 (1966). A. J. Anderson and N. F. Maclaglan, Biochem.]., 59,638 (1955). A. J. Anderson, M. H. Lepper, and R. J. Winder, Biochem.]., 77,581 (1960). R. Bourrillon, R. Got, and J. Michon, Clin. Chim. Acta, 6,91(1961). R. Bourrillon, R. Got, and P. Comillot, Clin. Chim. Acta, 6,730 (1961). J. S. King, Jr., W. H. Boyce, J. M. Little, and C. Artom,J. Clin. Inuest., 37, 1658 (1958). (20) Z. Dische, H. Kawasaki, C. Rothschild, A. Danilichenko, and H. H. Zinser, Arch. Biochem. Biophys., 107,209 (1964). (21) K. Kung-Ying Tan, C. A. Hizer, and T. H. McGavack, Proc. SOC.E x p . Biol. Med., 119,193 (1965).

URINARY PROTEIN-CARBOHYDRATE COMPOUNDS

441

TABLE111 Percentage Distribution, in Various Molecular-weight Groups, of the Salt-soluble; Nondialyzable Urinary Proteins14 Subject

Adult male Adult female Child, male (3 to 13 yrs.) Child, female (3 to 13 yrs.)

Number of samples

Molecular-weight groups: percentage of total (k S.D.) in 24-hour urine samples 1,0005,000

5,00010,000

10,OOO100,Ooo

Greater than

16 15 22

55.8k 2.4 55.922.5 45.8% 3.1

12.3k 3.1 13.7% 1.4 8 . 9 k 0.9

6.4& 1.3 3 . 7 k 0.3 5 . 0 k 0.8

24.3k 1.4 25.9k 1.7 41.6% 3.2

11

54.2k3.7

10.222.0

5 . 7 k 1.3

30.5k 4.2

100,000

“The salt-soluble fraction is the supernatant liquor obtained on treatment of urine with 0.58 M sodium chloride to precipitate the Tamm-Horsfall acid glycoprotein.22

change chromatography, precipitation with alcohol, gel-filtration, and zone electrophoresis have generally been used, but there appears at present to be no generally accepted methodology for this fractionation and for isolation of these compounds from urine; no two groups of workers have used comparable methods, and this variation is reflected in the carbohydrate composition of the fractions that they have isolated. Forty glycoprotein-containing fractions with carbohydrate contents which vary between 43 % and 96 % have been isolated from the non-dialyzable, urinary material by fractional precipitation with isopropyl alcohol-ether, followed by continuousflow electrophoresis; hexose, aminodeoxyhexose, and 6-deoxyhexose were the major carbohydrate components of these glycoproteins, some of which may be derivatives of pituitary hormones.20 The urinary protein-carbohydrate compound most extensively characterized is the Tamm-Horsfall acid glycoprotein,22which probably originates from the kidney; it has a molecular weight of 7 X lo6 (Ref. 23), and is too large to be filtered through the glornerulu~’~; an antigen corresponding to it has not been detected in plasma.24 The carbohydrate component of this glycoprotein is made up of sialic acid (9.1 %), 2-amino-2-deoxy-D-galactose(6.12 %), 2-amino-2deoxy-D-glucose (1.06%), D-mannose (2.7%), D-galactose (5.4%), and (22) I. Tamm and F. L. Horsfall, Jr., Proc. SOC. Exp. Biol. Med., 74, 108 (1950). (23) I. Tamm, J. C. Bugher, and F. L. Horsfall, Jr., J . Biol. Chem., 212, 125 (1955). (24) R. J. Winder, in “Polysaccharides,” G. F. Springer, ed., Macy, N.Y., 1956, p. 41.

442

E. H. F. hlcGALE

6-deoxy-~-galactose(1.1%), according to M a ~ f i e l d The . ~ ~ ability of this acid glycoprotein to cause inhibition of viral hemagglutination is associated with its sialic acid, for enzymic removal of this sugar causes complete loss of this inhibitory property.22The physiological role of the Tamm-Horsfall glycoprotein is not yet clearly understood, but it has been implicated in the formation of kidney stones.26A detailed review of the chemistry and properties of this acid glycoprotein has been p u b l i ~ h e d . ~ ~ In addition to the Tamm-Horsfall acid glycoprotein, the presence in urine of small proportions of plasma albumins and globulins has been established by means of paper e l e c t r o p h o r e ~ i sultracentrif,~~~~~~~ ~ g a t i o n and , ~ ~ immunological technique^^^,^^; the latter qualitative studies showed the presence of 14 to 20 plasma biopolymers in urine. Isolation and chemical characterization of individual, plasma biopolymers has been less successfully achieved, due to the normal excretion of only small quantities (comparison of the data for plasma32 and ~ r i n e ~indicates , ~ ~ , ~that ~ urine normally contains only 0.04 to 0.10% of plasma albumins and globulins); exceptionally, an electrophoretically homogeneous albumin has been isolated in small amount (4 to 6 mg/liter of and a proteopolysaccharide has been isolated (approximately 1 mg/liter),36the carbohydrate composition of which is similaP to that of a plasma-globulin proteopoly~accharide,~~ as is shown in Table IV. A number of biologically active glycoproteins have been identified in normal urine. A glycoprotein, possessing gonadotrophin activity, which behaved as a single component when examined by electrophoresis and ultracentrifugation, has been isolated by Got and Bourrillon3’; the presence of sialic acid, 2-amino-2-deoxy-~-galactose, (25) M. Maxfield, in “Glycoproteins,”A. Gottschalk, ed., Elsevier Publishing Co. Ltd., (London);Biochim. Biophys. Acta Library, 1966,Vol. 5. (26) W. H.Boyce and M. Swanson,]. Clin. Invest., 34,1581(1955). (27) W.H. Boyce, F. K. Gamey, and C. M. Norfleet,]. Clin. Inoest., 33, 1287 (1954). (28) E. McGarry, A. H. Sehon, and B. RoseJ. Clin. Invest., 34,822(1955). (29) J. M. Creeth, R. A. Kerwick, F. V. Flynn, H. Harris, and E. R. Robson, Clin. Chim. Acta, 8,406(1963). (30) B.-L. Dinh, A. Tremblay, and D. Dufour,]. Immunol., 95,574(1965). (31) I. Bergtirrd, Clin.Chim.Acta, 6,413(1961). (32) Q.Z.Hussain, N. S. Shash, and S. N. Chadhuri, Clin. Chirn. Acta, 6,448(1961). (33) R.Gunton and A. C. Burton,]. Clin. lnoest., 26,892(1947). (34) A. Saifter and S. Gerstenfeld, Clin. Chem.,10,321(1964). (35) P.Cornillot, R. Bourrillon, and R. Got, Compt. Rend. SOC.Biol., 254, 171 (1962). (36) I. Bergtirrd, Nature, 199,174(1963). (37) E. H.Eylar, Fed. Proc., 22,538(1963). (38) R. Got and R. Bourrillon, Biochim. Biophys. Acta, 42,505(1961).

URINARY PROTEIN-CARBOHYDRATE COMPOUNDS

443

TABLEIV Carbohydrate Composition of a Homogeneous, Urinary Proteopolysaccharide, Compared with that of a Plasma a,-Globulin Proteopoly~accharide~~ Source Urine Plasma

Yield Hexose Aminodeoxyhexose 6-Deoxyhexose Sialic acid Refer( %) ( %) (%) ences (mg/liter) (%) 1 356

15.8 15.0

16.7 18-20

1.3 0.0

28.6 26-30

36 37

2-amino-2-deoxy-D-glucose, D-galactose, D-mannose, and 6-deoxy-~galactose as the carbohydrate components of this glycoprotein (57% protein) was demonstrated by these authors, who also found that the sialic acid is essential for hormonal activity.38An acid glycoprotein (40% carbohydrate) which is a potent inhibitor of trypsin has been isolated from normal urine; the probable molecular weight is within the range of 10,000 to 30,000; the composition and properties of this acid glycoprotein (curiously, named Mingin) have been reviewed by Faar~ang.~~

5. Peptide-Acidic (Aminodeoxyg1yco)glycuronans Although the presence in urine of these compounds was first recognized by Moerner,2 only recently have different types of these compounds been identified.40Three acidic (aminodeoxyg1yco)glycuronancontaining fractions possessing weak, anticoagulant activity have been identified in the nondialyzable fraction of normal urine by paper electrophoresis and detection with Alcian Blue4'; the major acidic (aminodeoxyg1yco)glycuronan component of these fractions is chondroitin 4 - ~ u l f a t e , ~which ~ - ~ ~accounts for approximately 80 % of the with ,~~ smaller ), prototal excretion (3.0-6.0 mg per liter of ~ r i n e ~ ~ portions of heparin and dermatan sulfate.44 Traces of chondroitin, hyaluronic acid, chondroitin 6-sulfate, and a keratosulfate compound have been detected.46 The showed that the chondroitin (39) (40) (41) (42) (43) (44) (45) (46)

H. J. Faarvang, Scand.1. Clin. Lab. Znoest., 17, Suppl. 83(1965). P. Astrup, Acta Pharmacol. Toxicol.,3,165 (1947). J. F. Heremans, J. P. Vaeman, and M. Heremans, Nature, 183,1606 (1959). N . Di-Ferrante and C. Rich, Clin. Chirn. Acta, 1,519 (1956). I. BergPrrd and A. G . Beam, Arner.1. Med., 39,221 (1965). A. Linker and D. Terry, Proc. SOC.E x p . Biol. Med., 113,743 (1963). G . P. KerbyJ. Clin. Znuest., 33, 1168 (1954). D . P. Varadi, J. A. Cifonelli, and A. Dorfman, Biochim. Biophys. Acta, 141, 103 (1967).

444

E. H. F. h4cGALE

sulfates are excreted with a covalently bonded polypeptide component, the molecular weights of these compounds being within the range of 6,000 to 10,000. A heparitin-peptide compound, in which L-serine is the amino acid involved in the linkage of the heparitin to the peptide, has been isolated from urine.47 111. URINARYPROTEIN-CARBOHYDRATE COMPOUNDS O F LOW MOLECULAR WEIGHT

1. Carbohydrate Composition Protein-carbohydrate compounds of low molecular weight have been identified, and partially resolved by gel-filtrati~n~*-~~on Sephadex G-25(molecular-weight fractionation-range for peptides and globular proteins51: 1,000-5,000)or by chromatography on carbonlcelite columns52;P e ~ h a nidentified ~~ 14 urinary glycopeptides by this procedure. A number of these compounds have been isolated in sufficient quantity for chemical characterization; a summary of the methods used for their isolation is given in Table V, together with the carbohydrate (quantitative) and peptide (qualitative) composition of the resultant compounds or fractions. The data presented in Table V will be the basis for subsequent discussion in this Section. In comparing the results obtained by different workers, the carbohydrate composition is the only basis upon which comparison can be made, since the quantitative peptide composition, or the molecular weight, has, in many instances, not been determined or cannot be calculated from the given data; on this basis, it would appear that no two groups of workers have obtained comparable results. The fractions designated UF-4A and B,a (see Table V) have similar contents of hexose, aminodeoxyhexose, and 6-deoxyhexose, but are different with respect to their content of sialic acid and their amino acid composition. This variation in results is, in part, a reflection of the variation in the methods that have been used for isolating urinary glycopeptides and peptido-oligosaccharides, and, from this, it would appear that there is, as yet, no generally accepted, methodical sequence for this D. Kaplan, Biochim. Biophys. Acta, 136,396 (1967). A. Lundblad and I. BergArrd, Biochim. Biophys. Acta, 57,129 (1962). T. A. Miettinen, Clin. Chim. Acta, 8,693 (1963). I. Vermousek and Z. Brada, Clin. Chim.Acto, 13,757 (1966). “Sephadex Gel Filtration in Theory and Practice,” Pharmacia AB, Uppsala, Sweden. (52) Z. PechanJ. Chromatog., 10,104 (1963).

(47) (48) (49) (50) (51)

URINARY PROTEIN-CARBOHYDRATE COMPOUNDS

445

type of study. A combination of several techniques is clearly necessary in order to provide homogeneous fractions, and considerable volumes of urine would be required so that each fraction could be obtained in sufficient quantity for complete chemical and physical characterization; whereas the latter requirement presents no difficulty, the former appears not to have been met as yet. A second factor contributing to this lack of uniformity of results may be interference by the urinary pigments during analysis of these fractions for carbohydrate. Many of the urinary protein-carbohydrate compounds contain bound p i g m e n t ~ ~ J ~which , ~ * . ~interfere ~ with analysis for aminodeoxyhe~ose~~ and hexose, hexuronic acid, and 6 - d e o x y h e x o ~ e .The ~ ~ . ~extent ~ of this interference has been investigated by McGale and Jevons,62who found that interfering chromogens are produced from urinary pigments with sulfuric acid-containing reagents commonly used for the estimation of h e x o ~ e hexuronic ,~~ and 6-deo~yhexose~~; the contribution of these chromogens to the total color developed on treating pigmented glycopeptide-containing fractions with these reagents was found to vary between 27.5 and 57.5 %. 2. Peptide Composition

A characteristic but unexplained feature of the peptide composition of all of these glycopeptides and peptido-oligosaccharides (see Table V) is the absence, or presence in traces only, of the aromatic amino acids. The presence of hydroxy-L-proline in some of these fractions might indicate that the glycopeptide components of these fractions are derived from connective-tissue components, as this (53) S.-I. Hakomori and T. Ishimoda,]. Biochem. (Tokyo), 52,250 (1962). (54) S . 4 . Hakomori, H. Kawauchi, and T. Ishimoda, Biochirn. Biophys. Acta, 65, 546 (1962). (55) M. G . Cherian and A. N. Radhakrishnan, Biochirn. Biophys. Acta, 101, 241 (1965). (56) M. C . Cherian and A. N. Radhakrishnan, Indian]. Biochem., 3,101 (1966). (57) R. Bourrillon and J. L. Vemay, Biochim. Biophys. Acta, 117,319 (1966). (58) J . S. King, Jr.. M. L. Fielden, and W. H. Boyce, Arch. Biochem. Biophys., 90, 12 (1960). (59) A. Lundblad, Biochim. Biophys. Acta, 101,46 (1965). (60) A. Lundblad, Biochim. Biophys. Acta, 101,177 (1965). (61) R. Bourrillon, P. Cornillot, and R. Got, Clin. Chim.Acta, 7,506 (1962). (62) E. H. F. McGale and F. R. Jevons, Clin. Chim. Acta, 14,528 (1966). (63) F. K. Hartley and F. R. Jevons, Biochem.]., 84,134 (1962). (64) T. Bitter and H. M. Muir, Anal. Biochem., 4,330 (1962). (65) Z. Dische and L. B. Shettles,]. Biol. Chern., 175,593 (1948).

TABLEV Method" Symbol Yield (mgl liter) A

UGPI 20 UGPII 10 UGPIII 8

B

-

C

C4.6.2 C4.6.3

D

UF-4A

E

A3b A3c B3a

F

G

Crb

2.0 9.1 2.7 0.4 3.0 1.0 4.0 2.0 1.7 1.3 3.7 3.1 6.6 2.8 2.8 5.0 1.9 1.3 4.2

Mol. wt.

Hexose 6-Deoxy- Aminodeoxy- Sialic Uronic Total hexose hexose acid acid (%)

-

(%)

5.6 0.2 2.5

13.5 5.6 7.5

4,ooob

9.1

1,200-1,500 20.0 1,200-1,500 14.1

0.0 0.0 3.5

0.0 0.0 0.0

(%I -

1.7

5.0

10.3 26.1

( %)

-

-

11.1 4.5

13.1

-

36.9

14.2

23.9

5,840c 4,370 9,450

50.1

23.7

30.3

-

-

34.4

16.6

20.0

2,300

40.4

26.7

-

17.2 43.0 40.1 5.5 26.8 24.0 22.4 28.4 29.0 15.4 24.6 14.5 28.3 26.6 6.0 19.5 21.4 16.0 20.6

3.5 13.5 9.5 0.5 7.1 3.0 4.2 4.4 3.8 2.5 3.6 2.1 2.9 3.5 0.9 2.9 3.3 1.4 1.5

-

-

-

-

(%)

-

1

-

(%I 19.1 5.8 13.5

- 31.1 - 18.6 - 68.5 - 104.1 -

3.6

-

18.7

1

-

85.8

11.7 23.3 25.6 3.0 24.0 20.6 11.5 22.6 24.2 12.9 28.7 10.0 22.8 25.6

2.5 6.7 11.8 3.2 11.7 30.0 12.1 17.6 29.2 8.5 23.8 10.8 25.1 27.5 9.1 22.5 27.2 16.0 23.2

-

34.9 86.5 87.0 12.2 69.6 77.6 50.2 73.0 86.2 39.3 80.7 38.6 79.7 83.2 16.9 63.1 70.6 45.2 62.6

-

17.5 18.7 10.7 16.0

-

-

1.2 0.6

0.9 0.7

-

1.1 1.3

74.6

"Method: A, Urine concentrated X 10;ethanol (85to 90%, v/v) and saturated methanolic lead acetate; ppt. Dowex-1 chromatography, Sephadex G-25,paper chromatography. B, Dowex-50 chromatography; Sephadex G-25;(2-diethylaminoethyl)-Sephadex; Dowex-1 chromatography; paper chromatography. C, Dialysis of urine (nd.); ethanol (50%, vlv); ppt. Sephadex G-25;O-(2-diethylaminoethyl)cellulose; Sephadex G-50;paper electrophoresis (PH 2.4,then 6.4).D, Ultrafiltration of urine (nuf.); saturated ammonium sulfate; ppt. O-(2-diethylaminoethyl)cellulose.E, Ultrafiltration of urine (nuf.); Pevikon electrophoresis (8.6);Sephadex G-75;Pevikon electrophoresis (pH 3.7). F, Ultrafiltration and dialysis of urine (uf., nd.); Pevikon electrophoresis (pH 8.6);Sephadex G-25;Pevikon electrophoresis (pH 9.2).G, Dialysis of urine (nd.); ethanol (so%, vlv); ppt, chromatographed on 0-(2-diethylaminoethyl)cellulose; starch-gel electrophoresis (pH 3.5); where nd. = nondialyzable, urinary fraction, nuf. = nonultrafiltrable, urinary fraction, uf.,nd. = ultrafiltrable, nondialyzable, urinary fraction, and ppt. =precipitate.

446

Carbohydrate and Peptide Composition of Urinary Glycopeptides and Peptido-oligosaccharides Peptide

Amino acid compositiond

References

( %)

-

-

Asp Glu Ser Gly Ala Thre His Asp Glu Ser Gly Ala Thre His Pro Asp Glu Ser Gly Ala Thre His Pro Val Leu Hyp

53.54

-

Asp Glu Ser Gly Ala Thre

Pro Val Leu Hyp Ileu CyS Tyr

55,56

-

Asp SerGlyAlaThre AspGluSerGly Thre

Pro Pro

57

-

-

Asp Glu Ser Gly Ala Thre His Pro Val Leu

6.9 Asp Glu Ser Gly Ala Thre H i s Pro 19.2 Asp Glu Ser Gly Ala Thre H i s Pro

HYP HYP Ileu

LY s

58 59

Leu

Ileu

Lys PheAla

10.4 Asp Glu Ser Gly Ala Thre His Pro Val Leu

Ileu

Tyr Lys

-

Arg 60 61

82.4 27.2 9.5 43.5 23.0 5.0 40.0 25.0 49.4 24.9 5.0 43.4 29.7 16.0 42.9 18.7

*Approximate molecular weight, calculated from gel-filtration behavior. CMolecularweight determined by ultracentrifugation (Archibald method). dAmino acids identified by paper or column chromatography after acid hydrolysis of the glycopeptide or peptido-oligosaccharide fractions. Amino acids present in trace quantities are shown in italics.

447

448

E. H. F. McGALE

amino acid occurs only in collagen [which contains 10% (W/W) of this amino acid] and elastin (2-3%) according to Tristram and Smith.66 No evidence has as yet been presented that indicates that the urinary fractions that contain carbohydrate and hydroxy-L-proline are homogeneous, and it is possible that this amino acid is present in carbohydrate-free peptides: the presence in urine of such peptides derived from collagen has been established by Meilman and coworker^.^^ A glycopeptide-containing fraction that contains peptide-bound hydroxy-L-proline (0.5%, wlw) has been isolated. It behaved as a single component on Sephadex G-25;paper electrophoresis of this fraction showed the presence of 5 glycopeptides and 6 peptides; the hydroxy-L-proline was present only in the peptide components.6s

3. Properties a. Reducing Properties. -The three fractions isolated by Hakomori and c o ~ o r k e r s (UGP ~ ~ * ~I,~11, and 111; see Table V) were found to give the following characteristic reactions: (a) reduction of alkaline ferricyanide, Bromophenol Blue, and 2,6-dichlorophenolindophenol at room temperature; (b) a positive reaction with the L-cysteinecarbazole-sulfuric acid reagent for estimation of ketoses,6gbut with a characteristic shift, from 560 to 590 nm, of the wavelength maximum of the color obtained on treating a ketose with this reagent; and (c) a positive reaction with the indirect Ehrlich (Morgan-Elson70) and Elson-Morgan7I reagents used for estimation of acetamidodeoxyhexoses and aminodeoxyhexoses, respectively. After treatment of UGP I, 11, and I11 with sodium borohydride, these reactions were not obtained. Similar reactions were obtained with a noncrystalline preparation of l-deoxy-l-~-glycino-~-fructose.~~ On the basis of these results, Hakomori and coworker^^^,^^ proposed a l-deoxy-l-(N-peptidy1)-D-ketose structure for the linkage of the peptide and carbohydrate components of UGP I, 11, and 111: this kind of structure is identical with that of the product of the Amadori rearrangement of glycosylamines. (66) G. R. Tristram and R. H. Smith, Aduan. Protein Chern., 18,227(1963). (67) E.Meilman, M.M . Urivetzky, and C. M. Rapoport,J.Clin.Inoest.,42,40(1963). (68) E.H.F.McGale and F. R. Jevons, Clin.Chfrn.Acta,17,441(1967). (69) Z.Dische and E. Borenfreund,]. B i d . Chern., 192,583(1951). (70) W.T.J. Morgan and L. A. Elson, Biochern.]., 28,988(1934). (71) L. A. Elson and W. T. J. Morgan, Biochern.]., 27,1824(1933). (72) A. Abrams, P. Lowy, and H. Borsook, /. Arne?.Chern. SOC.,77, 4794 (1954).

URINARY PROTEIN-CARBOHYDRATE COMPOUNDS

449

Naturally occurring Amadori glycopeptides have not been identi-

fied by other workers in this field. The presence of these compounds in urine has been jointly investigated by the present author73and 590 nm) Weiss7'l; they found that (a)73the characteristic color (A,, obtained on treatment of UGP I, 11, and I11 was not obtained with but was obtained with crystalline l-deoxy-l-L-glycino-D-fructose,75 unidentified intennediate(s) in the preparation of the noncrystalline material, used by Hakomori, from L-glycine and D-ghC0Se: (b) we is^,^^ using her specific modification of the test for Amadori comp o u n d ~ 'involving ~ reduction of o-dinitrobenzene, repeated the procedure used for isolating UGP I, 11, and 111, and tested for the presence of Amadori compounds at each step of the isolation procedure. She found that the precipitate obtained on treating tenfold concentrated urine with ethanol (85-90%) and saturated lead tetraacetate in methanol gave a strong, positive test for Amadori compounds, whereas the concentrate prior to this precipitation gave no reaction, thus demonstrating the formation of Amadori glycopeptides as artifacts of the method of isolation.

b. Aggregation Effects. -Comparison of the dialysis and gel-filtration behavior of urinary glycopeptides and peptido-oligosaccharides according to their molecular weights suggests apparently anomalous diffusion properties of some of these compounds. Thus, (a) A3c and B,a (9,450)were isolated (molecular weight 4,370),A,b (5,840), from the nonultrafiltrable, urinary fraction,5g(b) C 4.6.2 and C 4.6.3 (1,200-1,500)~~ and CFb (2,300)were isolated from the nondialyzable, urinary fraction, whereas the glycopeptide isolated by Cherian and R a d h a k r i ~ h a n ~(molecular ~,~~ weight 4,000)was dialyzable, and (c) the ultrafiltrable, urinary protein-carbohydrate compounds are of apparently higher molecular weight than those that are nonultrafilt~able.~~ Two considerations may help to explain these observations. Firstly, it is evident from the studies of Craig and King7' that dialysis, like gel-filtration, is a molecular-sieve effect, and that the rate of dialysis of a particular solute depends on a number of factors, including (a) the ratio of the membrane area to the volume of the retentate, (b) the (73) E. H. F. McGale, Ph.D. Thesis, Manchester University (1967). (74) J. B. Weiss, M.Sc. Thesis, Manchester University (1966). was a kind gift from (75) A specimen of crystalline 1-deoxy-1-L-glycino-D-fructose Dr. E. F. L. J. Anet; see E. F. L. J. Anet, Aust. J. Chem.,10, 193 (1957). (76) W. R. Fearon and E. Kawerau, Biochem. J., 37,326 (1943). (77) L. C. Craig and T. P. King, Methods Biochem. Anal., 10,177 (1962).

450

E. H. F. McGALE

nature of the membrane (its thickness and porosity), (c) the temperature, (d) the effect of the solvent on the solute, (e) the effect of the solvent on the membrane, (f) the viscosity of the solvent, and (g) charge effects. Therefore, no valid comparison can be made between the dialysis behavior of a glycopeptide or a peptido-oligosaccharide in total urine and its dialysis or gel-filtration behavior in a more purified form, because the composition of the “solvent” has changed as a result of fractionation. Secondly, aggregation of groups of these compounds of low molecular weight has been demonstrated; an apparently homogeneous protein-carbohydrate fraction eluted from Sephadex G-75 with water was resolved into three compounds of lower molecular weight when 100 mM sodium chloride was used instead of water, and a single glycopeptide-containing fraction eluted from Sephadex G-25 was resolved into 5 glycopeptide and 6 peptide components by means of paper electrophoresis.88 These aggregation effects would explain the presence of protein-carbohydrate compounds of low molecular weight in the nonultrafiltrable and nondialyzable, urinary fractions, and would limit the usefulness of gelfiltration for determination of molecular weights of fractions containing several of these compounds. 4. Origin

The high content of 6-deoxy-~-galactosein UF-4a, B3a, A3b, and A3c (see Table V) indicates that these peptido-oligosaccharides are derived from blood-group substances, which typically contain 8.621.6% (wlw) of this s ~ g a r . ~When ~ J ~ these fractions were tested for serological activity, slight A- and B-group activity was found in UF-4A, whereas the last three fractions were devoid of serological activity, although slight activity was found in the nonultrafiltrable, urinary material prior to fractionation. Subsequent studies by Lundblade0 have established that the excretion pattern of these peptidooligosaccharides is individually reproducible and is characteristic of the secretor status (A, B, 0, and nonsecretor) of the urine donor. The connective-tissue components collagen and elastin are the probable source of some urinary protein-carbohydrate compounds. A glycopeptide containing approximately equimolar proportions of hydroxy-L-lysine, D-glucose, and D-galactose has been isolated from (78) R. A. Gibbons, W. T. J. Morgan, and M. Gibbons, Biochent. 1..60, 428 (1955). (79) A. Pusztai and W. T. J. Morgan, Biochern.1, 80,107 (1961). (80) A. Lundblad, Nature, 211,531 (1966).

URINARY PROTEIN-CARBOHYDRATE COMPOUNDS

451

urine; it is identical in its composition and behavior to a glycopeptide prepared by enzymic and alkaline degradation of acid-soluble collagens'; and from urine has been isolated a peptide that contains hydroxy-L-lysine, desmosine, and isodesmosine (unique constituents of elastins2)and two unknown components designateds3 Hi* and V"; subsequent studiesa4 have identified the latter as 2-amino-2-deoxyD-glucose.

IV. CONCLUSION It should be evident from the previous pages of this Chapter that a complex mixture of protein-carbohydrate compounds is normally excreted in urine. The components of this mixture show considerable, individual variation with respect to their molecular weight, composition, chemical and physical properties, and origin. Many of these compounds are of low molecular weight (less than lO,OOO), and contain considerable proportions of carbohydrate; and it has, reasonably, been assumed that they are intermediates in the biosynthesis of, or degradation products of, glycoproteins and proteopolysaccharides. Similar compounds have been isolated from proteolytic digests of glycoproteins; for example, a peptide-oligosaccharide (60%carbohydrate) has been isolated from a pronase digest of ceruloplasmin (8.1% ~ a r b o h y d r a t e ) and , ~ ~ several of these compounds, approximately 80 % carbohydrate in composition, have been isolated from the products of proteolysis of a 7S-y-globulin with pepsin and pronase.@ The naturally occurring carbohydrate-rich compounds of low molecular weight, of which urine is a unique source, may originate by comparable in vivo degradation, so that their isolation and characterization may provide valuable information in the understanding of metabolism of glycoproteins and proteopolysaccharides. As evidenced from the composition of the urinary, protein-carbohydrate compounds, the origin of some of them (from blood-group substances and the connective-tissue components collagen and elastin) has been established, but whether they are intermediates in

(81) (82) (83) (84) (85) (86)

L. W. Cunningham, J. D. Ford, and J. P. Segrest,J. B i d . Chem.,242,2570(1967). J. Thomas, D. F. Elsden, and S . M. Partridge, Nature, 200,651 (1963). J. B. Weiss and F. S . Stephen, Nature, 217,661 (1968). J. B. Weiss, personal communication. G . A. Jamieson,]. Biol. Chem., 240,2019 (1965). J. R. Clamp and F. W. PutnamJ. B i d . Chem., 239,3233 (1965).

452

E. H. F. McGALE

the biosynthesis of these compounds of high molecular weight, or are degradation products of them, has not yet been determined. The studies of Kranes7 who observed very rapid excretion of peptide bound hydroxy -L-proline-1J4C following incorporation of L-proline1-'4C into connective tissue in Paget's disease, suggest that some of the urinary biopolymers of low molecular weight are intermediates of collagen biosynthesis. Numerous attempts have been made to isolate and characterize the protein-carbohydrate compounds of low molecular weight in urine. Due mainly to the lack of effective methods for isolating homogeneous fractions, the origin of many of these compounds has not yet been determined. Fractions containing several protein-carbohydrate compounds have generally been prepared by using dialysis and paperor ion-exchange chromatographic techniques, and zone electrophoresis has been used in attempting separation of the components of these groups. For the latter purpose, several authors have found considerable advantages in using such noncarbohydrate, supporting media as Peuikon C870 (a copolymer of polyvinyl chloride and polyvinyl acetateE8)for preparative electrophoresis, and they have used this technique to isolate small quantities of proteopolysaccharides,3B peptido-oligosaccharides,5g~60 and glycopeptidess8J4from urine. Other factors may contribute to the lack of uniformity of the results obtained by different authors. Firstly, factors that may influence the excretion of protein-carbohydrate compounds have not yet been adequately investigated. Variations in their excretion with age and sex have been observed (see Table 111), but have not yet been explained in terms of individual compounds or groups of compounds. Possible variations with diet have not been investigated; the influence of physiological status has only been investigated by Lundblad,BO who found that the pattern of excretion of the fucose-rich peptidooligosaccharides is characteristic of the blood-group status of the urine donor. In the absence of such data, the normal pattern of excretion (qualitative) or the normal range of excretion (quantitative) of these compounds cannot be defined. Literature values for the excretion of nondialyzable, urinary protein-carbohydrate compounds (see Table 11) range from 141 to 400 mg per liter of urine, but the proportion of these compounds in the dialyzable, urinary fraction (which contains 47% of the total, urinary aminodeoxyhexose) has not yet been determined. (87) S. M. Krane, A. J. Munoz, and E. D. Hams, Science, 157,713(1967). (88) H. J. Muller-Eberhard, Scand.]. Clin. Lab. Inoest., 12,33 (1960).

AUTHOR INDEX FOR VOLUME 24

Numbers in parentheses are reference numbers and indicate that an author’s work is referred to, although his name is not cited in the text.

A Abbott, D., 340 Abd Elhafez, F. A., 108 Abdel Akher, M., 349 Abitz, W. Z., 306 Abraham, R. J., 265 Abrams, A., 448 Abson, D., 210,211,220 Achval, V. B., 259 Acree, T. E., 37, 54, 55(297b,c), 56(258, 297b,c), 60,61 Acton, E. M., 155,165,210 Adams, G. A., 71,72(18), 107(18),111(18) Adams, J., 337 Adamson, J., 206 Adley, T. J., 49,141,146(253) Agranoff, B. W., 431 Ahammad, A., 80, 81(58), 97, 116(95), 121(58) Ahluwahlia, R., 162 Akagi, M., 143,146,203,206 Albano, E. L., 232, 235, 236(81, 93), 237(81),249(93),255(81) Albrecht, H. P., 86, 88(75a, 79, 80), 110(75a,75b, 79,80), 115(75a,79) Alekseeva, V. G., 262(170,17Oa),263 Alekseev, Yu. E., 257,262(170),263 Allen, I., 29,30(243) Ali, Y.,141,147(260), 160(260),162(260) Allinger, N. L., 108,165,273,295(29) Allred, E. L., 193, 194 Andersen, W., 177, 179(401), 180(401), 185(401) Anderson, A. J., 440 Anderson, B., 291,292 Anderson, C. B., 57,58

Anderson, D. M. W., 335, 336, 337(13), 342, 344(75, 77, 86, 89, 90, 91), 345(75, 77), 346(77, 86), 347(77),348, 349(75),351(77),378 Anderson, J. D., 300(141b),301 Anderson, L.,59,60,100,116,117(153b) Anderson, N. S., 278, 280, 281(55, 57), 282(57), 283(58, 63), 284(57), 285, 286(73), 288(73), 292(58), 293(41, 55, 60, 62, 63), 295(73), 315(58, 73), 316(58),320(73),323(58) Anderson, W. A., 311 Ando, H., 71,72(24), 107(24),264 Andreae, U., 431 Andrews, P., 300(141b),300 Anet, E. F. L. J., 231, 239, 261(79), 263, 265(103),449 Angier, R. B., 178 Angyal, S. J., 57, 105, 108, 116, 117(153b), 149,162, 165, 169,170(372),261,273, 295(29) Annenkova, V. M., 216 Anno, K., 292,295(103) Antonyuk, L. S., 259 Anyas-Weisz, L., 304 Arai, K., 277,278,279(44) Araki, C., 277, 278(37), 279(44), 280, 293(37) Armstrong, R. K., 147,148,152(298) Arnold, J. T., 311 Amold, R. L., 110 Amott, S., 325 Aronson, J. N., 100 Aronson, S. M., 414 Arsenault, G. P., 283 Artom, C., 438,439(6),440(6) Arzoumanian, H., 155,165 Asai, M., 230

453

AUTHOR INDEX, VOLUME 24

454

Aseeva, N. N., 262(170a),263 Aso, K., 339 Aspinall, G. O., 270(15a), 271, 272(15a), 299, 300(132, 133, 134, 135, 141, 141c), 301, 326(135), 334, 335, 336, 337(30),338, 339(11, 16), 340(11, 16, 26, 54, 55), 342, 344(54, 84, 85), 345, 346(77),348(11, 54, 55, 59), 349(76), 350(76), 351(11, 19, 54, 55, 59), 352(11, 19, 59, loo), 353(16, 17), 354, 355(25, 61), 356(25, 26, 27, 28), 357(26, 27), 358(25, 27), 361(16), 362(16, 40), 363(12, 16, 18, 125), 364(18, 19, 128, 129), 365(39, 129), 366(39, 132, 133), 367(64, 138), 369(4), 371(4), 374, 375(29, 30, 157), 378(6, 11, 59, 101, 157), 379(18, 19, 29,109) Astbury, W. T., 298,324(122) Astrup, P., 443 Auret, B. J., 354,356,379(109) Austin, P. W., 140,191,193

B Babievskii, K. K., 104 Baddiley, J., 141, 186(252) Baehler, B., 262(170a),263 Baer, H. H., 71, 78, 80(48),81(57,58),82, 83, 84(61), 85(64, 651, 86(57, 66), 88, 92, 93(90),94, 95,97,99(59), 100,104, 108(18b,61, 67, 84), 110(57),111(65), 112, 113, 115, 116(64, 65, 69, 95), 118(59,64,65,69,108, 149,151,152), 121(57, 58, 60, 61, 67), 122(61, 161), 123(161), 124(64, 146, 148, 149, 161), 125(64, 65), 126(154, 161), 127(154), 128(65, 69, 152), 129(64, 148, 152, 154),130(154,164),131(165),132(149, 156), 134(155,167,178),135(145,155, 167), 136(178), 137(166), 243, 256 Baer, R., 386 Bailey, J.M., 63,64(311),65,290 Bailey, R. W., 338 Baillie, J., 336, 339(16), 340(16), 352(16, 17),361(16),362(16),363(16) Bairamova, N. E., 262(170),263 Baker, B. R., 141, 155(248),157, 159(249, 250), 171,186,187

Baker, G. L., 324 Bakinovskii, L. V., 262(170),263 Balazs, E. A., 296,305(107),311,312(108), 322(108) Ball, D. H., 142, 145, 146(283), 164, 175, 176(24),177 Ballantyne, M., 301 Ballou, C. E., 261,339 Banderet, A., 306,328(157) Banfi, D., 385, 387, 392(28),398, 399(69), 421(13,69) Banks, W., 325,334,335(7) Banthorpe, D. V., 142 Barbier, A., 68 Bhrczai-Martos, M., 115 Barden, L. L., 72, lll(30) Barker, R., 168, 195, 196(456), 213, 22 l(38) Barnett, J. E. G., 204,205 Barrett, A. J., 300(137),301 Barrette, J. P., 147 Bartz, K. W., 438,439(5),440(5) Baschang, G., 91, 108(97),419, 420, 421, 422,423(203),428(203) Bashford, V. G., 150 Basu, G., 432 Batdorf, J. B., 272 Bates, R. G . ,29 Battista, 0.A., 272,323(24) Bauer, G., 34,37(254),53(254),55(254) Bauer, S . , 359 Bayer, M., 136 Bayley, S. T.,280,284(54) Beach, R. L., 94, 99(91b), 111(91b),235, 256(92) Beam, A. G., 443 Beebe, E. V., 305 Beevers, C. A., 288 Begbie, R., 339 Behre, H., 247 Bekker, P. I., 348 Bell, R. P., 21,33(227) Bella, A., 291 Bello, J., 310 BeMiller, J. N., 43,270,271(8), 272(8) Bendich, A., 180 Benson, A. A., 146,258 Benson, G. C., 331 Bentley, R., 63 Benz, E., 179(411),180 Berhnek, J., 86,88(73), 110(72,73)

AUTHOR INDEX, VOLUME 24 Berglrrd, I., 442, 443(36), 444, 445(48), 452(36) Berman, E. R., 400 Bemhard, K., 399,406(75) Bemsmann, J., 152 Bemstein, H. J., 301 Bettelheim, F. A., 305,324( 154) Betts, B. E., 410 Beutler, E., 64 Beveridge, R. J., 371 Beynon, P. J., 219 Bhacca, N. S., 57,58(295d) Bhattachajee, S. S., 299,300(131) Bhattacharyya, A. K., 353, 363, 364(129), 365( 129) Bhavanandan, V. P., 292, 293(101), 336, 337(30), 340, 355(61), 375(29, 30), 379(29) Bigeleisen, J., 28, 33, 41 Bines, B. J., 176 Binkley, W. W., 338 Birkofer, L., 47 Bishop, C. T., 299,335,338 Bishop, J. A., 440 Bitter, T., 445 Black, W. A. P., 283 Blackford, R. W., 161 Blackhall, E. L., 28,34(236) Bladon, P., 150: Blair, J. M., 360 Blair, M. G., 220 Blake, C. C. F., 314 Blakemore, W. R., 283 Bleicher, S., 397 Blethen, J., 278 Blix, G., 403,413,417,418 Block, A., 228, 238 Blohm, M., 420 Bloom, A., 140 Blumbergs, P., 145, 146, 159(286), 160(284),189,194(437) Boas, N. F., 438,439(8),440(8) Bobbitt, J. M., 78,338 Boctor, B., 233 Boedtker, H., 304, 305(148), 306(148), 312(148),315(148) Bogdanova, G. V., 262(170), 263,264 Bogoch, S . , 407 Bommer, D., 86, 88(75b), 110(75b), 115(75b) Bon, W. F.,306

455

Bonhoeffer,K. F., 29,33,43(241) Bonner, W. A., 29, 30(245), 43(245b), 44(245b), 147 Booth, D. A., 431 Bordwell, F. G., 110 Borenfreund, E., 448 Borsook, H., 448 Boschan, R., 167 Bose, R. J., 219, 221, 222(50), 240(50), 241(50) Bothner-By, A. A., 311 Boume, E. J., 340 Bourrillon, R., 438, 439(7), 440(7), 442, 443(38), 445(18), 447(57,61) Bouveng, H. O., 300(140), 301, 326(140), 335,353(10), 363(10) Bowers, C. H., 119 Bowker, D. M., 279 Bowles, W. A,, 212 Boyce, W. H., 438, 439(6), 440(6), 442, 445,447(58) Brada, Z., 444,445(50) Bradbury, E. M., 310 Bradley, P. R., 224 Brady, R. O., 394,395 Bragg, J. K., 308 Brandhoff, H., 175 Braun, P. E., 394 Brauns, F., 148 Bray, B. A., 291 Brendel, K., 147,160 Brimacombe, J . S., 151, 171, 196, 212, 247(36), 254,270,292(14) Brocca, V., 102 Bronsted, J. N., 15,19 Brooks, G., 410 Brossmer, R., 414,416(155), 420,425 Brown, D. M., 180,248 Brown, G. B., 181 Brown, J . F., Jr., 27 Brown, R. K., 201, 202, 225, 234(59), 235, 244(59, 60), 249(59, 60), 250(59, 60), 265(3) Bruce, R. M., 156 Brutenicova-Soskova, M., 359 Bryan, J . G. H., 151 Bryant, C. P., 141,161(257), 163(257) Buchanan, J. G., 140, 152, 156, 169, 174(326), 191,193,256 Buck,K. W., 142,151(264),157,247 Buckles, R. E., 167

456

AUTHOR INDEX, VOLUME 24

Chattejee, A. K., 154,190(318) Cheese, I. A. F. L., 425 Chen, G. C., 400,431(83) Cherayil, G. D., 399 Cherian, M. G., 445,447(55,56), 449 Ching, 0.A., 196,254 Chitharanjan, D., 253 Chiu, C. W., 124 Chizhov, 0. S., 338 Cholfield, C. R., 408 Christensen, T. B., 336, 340, 355(61), 356(28) C Christison, J., 278,293(42) Chu, P., 148 Caesar, G. V., 68 Chu, S. S. C., 285 Cahn, R. S., 417 Chua, J., 166,172,174(360), 178(362,380), Caimcross, I. M., 340 226,227(63,66,67),228(66,67) Calkins, D. F., 188 Cifonelli, J. A., 176,291,443 Campbell, J. C., 204,205(13) Campbell, J. W., 285, 286(73), 288(73), Cifonelli, M., 176 Ciment, D. M., 164,214,237 295(73),315(73),320(73) Clamp, J. R., 451 Cafias-Rodriguez,A., 300(141c),30: Clancy, M. J., 292,293(102) Cannan, R. K., 304 Clark, R. K., Jr., 167 Cantoni, G. L., 185 Clark, V. M., 177, 185(400) Cantor, S. M., 69 Clayton, C. J., 49,50,146,193(292) Capek, K., 88,108(84), 128 Cleland, W. W., 394 Caple, R., 76 Clbophax, J., 145,165(282), 166,229 Capon, B., 298 Cleveland, E. A., 47 Carlson, D. M., 296,297(110) Carlyle, J. J., 336, 345, 344(85), 355(25), Cleveland, J. H., 47 Clifford, J., 332 356(25,27),357(27),358(25,27) Clingman, A. L., 278 Carpenter, D. K., 272 Clode, D. D., 233 Carr, S ., 405 Clough, F. B., 310 Carriuolo, J., 30 Coalson, R. L., 305 Carroll, F. I., 104 Codington, J. F., 172, 176(381, 382), Carroll, P. M., 419 181(382), 182(382) Carter, H. E., 167, 383, 384, 386, 390, 391(24), 392(18, 24, 44, 46), 396, 399, Cohen, A., 140,168,261 Cohn, M., 310 408,409(2),410,411(24),412,413 Cohn, W. E., 179 Casu, B., 290 Celmer, W. D., 383,3€!6,408(22), 409(2) Coles, E., 399 Cerezo, A. S., 283 Collins, P. M., 219,266 Chadhuri, S. N.,442 Cologne, J., 105 Chakravarty, P., 147,154,176 Colowick, S. P., 64 Chalk, R. C., 142,145,146(283),176(24), Colquhoun, J. A., 283 344(87,88), 345 Compton, J., 156,171,189(322) 345,344(79) Challinor, S. W., Conchie, I., 45 Chargaff, E., 400, 407, 415, 417, 420,428, Concone, M. C., 406 430 Cone, C., 144 Charlson, A. J., 339,340(54), 344(54),345, Conn, J., 152 348(54),351(54) Connor, A. H., 59,60 Chase, A. M., 63 Constantin, D., 317 Bugher, J. C., 441 Bukhari, M. A., 157,158(331),261 Buncel, E., 162,224 Burgdorf, M., 173 Burkett, H., 131 Burkhart, O., 148 Burton, A. C., 442 Burton, R. M., 394 Buss, D. H., 145,150(280), 176(280) Butterworth, R. F., 244

AUTHOR INDEX, VOLUME 24 Conway, E., 278,292(40), 293(40),323(40) Cook, A. F., 140, 191(242),212,255 Cook, M. C., 128 Cook, W. H., 280 Cope, A. C., 148, 149,150(300) Corbet, P., 105 Cordner, J. P., 27,28(233), 34(233) Cornelius, C. E., 440 Comforth, J. W., 419 Comillot, P., 440,442,445(18), 447(61) Corse, J., 167 Cote, W. A., Jr., 374 Cottrell, A. G., 162 Cottrell, I. W., 299, 300(135), 326(135), 338,362(40) Couch, D. H., 47 Coulson, A., 394 Coutelle, G., 409 Covington, A. K., 29 Cox, J. M., 143, 146(273), 147(273), 175, 195(273) Coxon, B., 140, 245, 246(116), 265(116), 266(116) Craig, J. W. T., 299, 300(134), 335, 363(12),374,375(157), 378(157) Craig, L. C., 449 Cram, D. J., 108 Cramer, F., 147 Crane, R. K., 64 Crane-Robinson, C., 310 Cree, G . M., 219,344(86),345,346(86) Creeth, J. M., 442 Creighton, A. M., 145,146(279) Cretcher, L. H., 139 Cronan, C. L., 290 Croon, I., 271 Cummings, J. N., 431 Cunneen, J. I., 349 Cunningham, J., 257 Cunningham, L. W., 451 Curtius, H. C., 60 Cushley, R. J., 86, 184

D Daines, M. E., 419 Daniher, F. A., 146,159(286) Danilichenko, A., 440,441(20) Danishefsky, I., 291

457

Dansi, A., 102 DaRooge, M. A., 166, 178(362), 226, 227(66),228(66) Davidson, E. A., 292,293(96,97), 295(92) Davidson, N., 320 Davies, D. B., 362,363(125) Davison, A. N., 404,406 Davison, B. E., 246 Davoll, J., 181 Davydova, L. P., 262 Dawes, K., 57 Dea, I. C. M., 344(90,91),345,348,378 de Bruyn, D. C., 359 deButts, E. H., 328,329(213),330(213) Decker, L. 292 Defaye, J., 112, 133(140), 152, 175, 229, 265 Delpuech, J. J., 141 DePuy, C. H., 78 Derksen, J. C., 306 Derungs, R., 338 Desmarais, A. J., 271, 272(17), 292, 332(17) Deuel, H., 299, 302, 304, 305, 306(127), 313(150a), 324, 325(127, 146), 335, 336(9),338 Dewar, E. T., 283 Dick, A. J., 160, 161, 163(341), 168(341), 170(341) Dick, W. E., 192 Dickerson, J. P., 190 Dickey, E. E., 371 Diebold, W., 421 Diehl, H. W., 174 Dienstbierova, V.,233 Dietzler, D. N., 145,160(284) Di-Ferrante, N., 443 Dillon, T., 292,293(102), 342 DiMilo, A. J., 27,28(233),34(233) Dimitrijevich, S., 232, 234(80a), 250(80a) Dimitrov, D. G., 259 Dimler, J., 408 Dinh, B.-L., 442 Dintenfass, L., 296 Dische, Z., 337,440,441(20), 445,448 Dittmer, J. C., 405 Dmitriev, B. A., 262(170a),263 Doelker, E., 262(170a),263 Dorfel, H., 296 Doerr, I. L., 172, 176(381, 382), 181(382), 182(382),184

458

AUTHOR INDEX. VOLUME 24

Doesburg, J. J., 298,299(124) Dofman, A., 443 Doganges, P. T., 219,292 Dolan, T. C. S., 280, 281(55),282,283(58, 63), 284(66), 292(58), 293(55, 60, 62, 63,66), 315(58),316(58),323(58) Donatti, J. T., 226,227(67),228(67) Dorfmann, A., 291 Dorofeenko, G. N., 37, 257,262( 170),263 Dorrence, S. M., 99 Doty, P., 304,305(148),312(148),315(148), 320 Doyle, D., 339 Druey, J., 390,393(38) Drummond, D. W., 296 Drummond, G. I., 100 duBois, F., 160 Dudman, W. F., 335 Diirig, G., 306,328( 157) Dufour, D., 442 Dulmage, W. J., 275 Dunstan, S., 365 Dunstone, J. R., 296,322(106a) Durette, P. L., 57,58,59 Dutcher, J. D., 80,83(56) Dutton, H. J., 408 Dwek, R. A., 204,205(13) Dyas, H. E., 52,54(289)

E Eades, E. D. M., 164,177 Earle, F. R. J., 408 Eastham, A. M., 28,34(236) Eastwood, F. W., 255 Easwaran, C. V., 29,30(244), 43(244) Edelson, M. R., 272,323(24) Edward, J. T., 29,30(245),43(245) Edwards, T. E., 339 Egami, F., 407 Egan, S. V.,299, 300(135),326(135), 338, 362(40) Egge, H., 414,416(155) Ehrlich, F., 300(138),301 Einset, J. W., 55(297c),56(297c),60 Eisfeld, K., 50 El Ashry, S. H., 264 Eldridge, J. E., 306, 307(159), 308(159), 309(159)

Eliel, E. L., 108,165,273,295(29) El Khadem, H. S., 264 Elliott, J. H., 328,329(213),330(213) Elmore, N. F., 179(411),180 El-Scherbiney, A., 106,136(123), 137(123) Elsden, D. F., 451 El Shafei, Z. M., 264 Emig, P., 86,88(75b),92,93(89), 110(75b), 115(75b) Emoto, S., 256 Enderby, J. A,, 316 Endo, T., 238 Erdmann, E. D., 14 Erskine, A. J., 336,369(20) Esser, H. O., 271,272(17),332(17) Etzold, G., 184 Evans, F., 131 Evans, M. E., 212,247(36) Everett, D. H., 316,317(186),319(186) Eylar, E. H., 442,443(37) Eyring, H., 35,307,310(164)

F Faarvang, H. J., 443 Faillard, H., 385,420 Fairweather, R. M., 351, 352(100), 378 (101) Fanshawe, R. S., 299,300(132) Farmer, D. W. A., 155,197(319) Faulkner, I. J., 15 Fearon, W. R., 449 Feast, A. A. J., 201 Feather, M. S., 50,146 Fecher, R., 182 FBher, O., 172,173(383) Feigelson, I. I., 305 Feldman, G., 430 Feldmann, L., 386 Fellig, J., 403 Femhdez-Bolafios,J., 175 Ferrier, R. J., 30(260), 38, 43(260), 139, 164, 200, 201(1), 202(1), 203(1), 205(1), 206(1), 210(1), 212(1), 213(1), 214, 215(1), 216(42), 217(1), 219(1), 220, 221(1, 51), 222, 223(56), 224(1), 226(l), 229(l), 232(1), 234(42), 235(42), 237(51, 91), 238(1), 240(1, 57), 241(51), 242(1, 106), 246(1), 252(l), 255(I),265(106)

AUTHOR INDEX, VOLUME 24

459

Ferry, J. D., 269, 272, 306, 307(159), Frei, E., 298,324(122) 308(159),309(159),310(2),321(2) Freudenberg, K., 148 Field, T. D., 211 Frey-Wyssling,A., 269,326 Fielden, M. L., 445,447(58) Friebolin, H., 209 Filippi, J. B., 234 Friedman, H. A., 86,88(73,77) 110(72,73, Firth, M. E., 419 77) Fischer, F. G., 296 Fromageot, H. P. M., 183 Fischer, H. 0. L., 69, 72, 78, 80(48), Frush, H. L., 14,35,38,47(262) 81(57), 83, 86(57), 89, 98, 100, 101, Fuertes, M., 218 104, llO(57, 85, 117), 112(103), Fujimoto, K., 339 115(103, 117), 116(98, 102), 117(117), Fuller, W., 325 118(98, 102), 120, 121(57, 60, 102), Fulmer, R., 131 126,128(102,117),130(164),386 Furda, I., 112,304(151),305,327 Fischer, N., 393(48) FurdovB, J., 111, 112(134), 127(134), Fishbein, L., 110 133(134) Fisher, D., 205 Furio, I. 82,99(59),118(59) Fisher, L. V., 160 Furuhashi, T., 292,295(103) Fishman, P. H., 63,64(311),65 Fletcher, H. G., Jr., 60,175,225,240,244, 254,263(104) Flodin, P., 313 G Flory, P. J., 269, 290, 306, 307(160), 309(160), 312, 313(160, 178a), 315 Gadient, F., 386,390,393(20) Gaitseva, E. A., 216 (178a), 320(178a), 331(80) Flowers, H. M., 393(49), 394, 398, 401, Gal, A. E., 393(50),394 Galanos, G. S., 409 402,407,431(68,90) Galbraikh, L. S., 259 Flynn, F. V., 442 Gale, R. O., 440 Fodor, G., 385,387,392(28), 421(13) Galli, C., 430 Folch, J., 405,407,415,428 Gallo, G. G., 290 Foote, J. L., 399 Gammack, D. B., 413 Forbes, E. J., 212,247(36) Ganguly, A. K., 70 Ford, J. D., 451 Garbisch, E. W., 265 Ford, T., 399 GarciB-Mufioz, G., 218 Fordovl, J., 265 Gardiner, D., 243 Formica, J. V., 394 Foster, A. B., 142, 151(264),157, 158(331), Garrett, R. R., 313 Garvey, M., 442 165,193,206,212,247(36),261 Gatt, G., 432 Foster, J. F., 290 Fox, J. J., 86, 88(73, 74, 77), llO(72, 74, Gatt, S., 400 77), 172, 176(381,382), 177, 178, 180, Gauhe, A., 414,416(155), 433 181(382,407), 182(382),184,228,238 Gault, H., 420 Fraga, E., 201, 203(4), 232(4), 265(4), Gaver, R. C., 390 Gent, P. A., 151,247 266(4) Georgias, L., 416 Francis, P. S., 329,330(214) Gergely, J., 311 Franek, M. D., 296,322(106a) Gemgross, G., 306 Franks, F., 306,331 Gero, S. D., 105, 112, 133(140), 145, 159, Fransson, L.-A.,291,292,293(100) 165(282),166,229,265 Fraser, R. N., 338, 362, 363(125),365(39), Gerstenfeld, S., 366(39,133) Gestetner, B., 300(141),301,363 Fraser-Reid, B., 76,203,233 Gibbons, M., 450 Freeman, M. R., 205

460

AUTHOR INDEX. VOLUME 24

Gibbons, R. A.. 337,450 Gibbs, C.F.,112,133(143),265 Gibbs, D. A., 296,312(108),322(108) Gibbs, J. H., 310 Gielen, W., 413,414,415(143),416(158), 420,421,425(202), 428 Gigg, J., 257,260(147).262,388,389 Gigg, R.,257,260(147),262,388,389,399 Gigg, R.H., 409,410 Gilham, P. T., 169,170(372) Gillard, R. D.,310 Gille, R.,50 Ginsberg, H., 140 Glassel, J. A.,311 Glasstone, S.,35 Click, F. H., 384,392(44),394 Click, R.,193 Glickman, S.A., 305 Glinski, R. P.,189,190,194(437) Glukhoded, I. S.,401 Goddard, E.D.,331 Godman, J. L., 262 Goldberg, E. B., 64 Goldberg, 1. H., 395 Goldberg, M. W., 384 Goldman, H., 310 Goldman, L., 179(411),180 Goldstein, I. J.,339,340 Gonatas, J., 415 Goodman, L., 141, 155(248), 159(250), 160,161,165,167,171,186,188,192, 210,228 Goodwin, H.,431 Goodwin, M. W.,324 Cook, R.,45 Gorin, P.A.J., 279,296,297(114) Got, R.,440,442,443(39), 445(18),447(61) Gottschalck, H., 218,265 Gottschalk, A., 418,419 Could, E. S.,51 Could, S. E. B., 299, 300(136),326(136), 374,375(156) Graham, G. E.,71 Gramera, R.E., 156,256 Grant, G. T., 327,336 Gray, G. R.,195,196(456),213,221(38) Green, J. P.,406 Green, J. W., 45,341 Greenspan, T., 30 Greenwood, C. T., 325,334,335(7)

Greenwood, F.L., 396 Gregory, G. I., 393(47),394 Gregson, N. A., 406 Greminger, G. K.,271 Griffith, C.F.,169,170(105) Grob, C.A., 385,386,390,393(19,20,47), 394 Grosheintz, J. M., 98, 100, 101, 116(98, 102), 118(98, 102), 120, 121(102), 128(102) Gross, P., 29,30(243),43(241) Gross, P.H., 147,160 Grunwald, E., 167 Gubenkova, E. N., 305 GuefFroy, D. E.,161,192 Guiselev, K.B., 270 Gunner, S.W., 77,78(42),91,115(88),208 Gunton, R.,442 Guthrie, R. D., 78, 108(54), 145, 159, 164(350),165(282),229,233,234,246, 249(84) Gutowski, G. E., 141,161(257), 163(257), 190 Gutowsky, H. S.,310 Guzman de Femhdez-Bolaiios, R., 175

H Haberfield, P., 140 Harle, R.,399,401 Haga, M.,143,146 Haines, A. H., 157,266 Hakamori, S.,404 Hakomori, S.-I., 445,447(53,54), 448(53, 54) Halberstadt, E. S.,305 Hall, L. D., 95, 145, 150(280), 151, 176(280),204,206,247,266 Hamel, E. E., 386 Hamerman, D., 438,439(5),440(5) Hamill, W.H., 29,43(241) Hamilton, L.D., 325 Hammett, L. P.,38,40,41 Hammond, G. S.,36 Hamor, T. A., 247 Handa, S.,403 Hanessian, S., 141, 145, 147(256, 281), 190, 191(439), 233, 234(87), 236(87), 244(87),245(87),249(87)

AUTHOR INDEX, VOLUME 24 Hann, R. M., 174,175 Hanousek, F.,236 Hanson, C.,167 Harding, M. M., 285, 286(73), 288(73), 295(73),315(73),320(73) Hardwicke, J., 440 Hams, E.D., 452 Harris, H., 442 Hams, R.,392(44),394 Hamson, J.B.,386 Hart, P.A., 186 Harte, R. A.,322 Hartley, F.K.,445 Hariman, F.C.,168,195,196(456) Hartree, E.F.,63,64(309) Hartzog, M.B.,301,302 Harvey, H. G.,325 Hascall, V. C.,296,322(106a) Hase, M.,125 Hasegawa, A., 102,163 Haskell, T. H., 141,145,147(256,281) Haslam, E., 174 Hass, H. B.,68,121(6a) Hatch, F.T., 438,439(5),440(5) Hatton, L.R.,30(260),38,43(260) Hattori, T., 415 Haug, A., 283, 297, 298, 320, 323(190), 324,327 Hauser, G.,395 Hausheer, L.,406 Hawthorne, J. N., 410 Hay, G. W., 340 Hayes, D. H., 177, 179(401), 180(401), 185(401) Hayworth, W.N.,345,344(79) Hechter, O.,311 Heck, R.,167,193 Hedelius, A.,51 Hegenauer, J. C.,278 Heidelberger, C.,226,227(65),228(65) Heidelberger, M.,337,366 Helferich, B.,45,125,173 Heller, C.A,, 436,442(3),445(3) Helting, T.,291 Hems, R.,142,151(264),247 Henbest, H.B.,142 Hendricks, U.W.,421,425(202) Hendrickson, H. E.,386,391(24),392(24), 411(24) Hendry, R., 412(133),413

461

Henisch, H. K., 305 Henry, L.,68 Henry, R.,438,439(9),440(9) Heremans, J. F.,443 Heremans, M.,443 Hen, W.J., 335,336(9) Hermann, K.,306 Hermans, J., 272,323(24),328 Hermans, P. H., 269,310(1) Hess,H. H., 416 Hess, H. V., 167 Hesse, R.H., 206 Heyns, K., 218,254 Hickson, T. G.L., 278 Hildesheim, J., 145, 152, 16s(282), 166, 229 Hill, D.G.,52,54(289,290) Hill, J., 141, 146(251b), 160(251, 285), 191(251,285) Hillend, W.J., 271,331(21) Hinton, C.L.,303,325(147) Hintsche, R.,184 Hirano, S., 241,292,293(99),301 Hirase, S.,156,176,278,279(44), 280 Hirschberg, C.B.,390 Hirst, E. L., 296, 297, 299, 336, 339, 340(54),341, 342,344(54,75, 77,79, 82,84),345(75, 77),346(77),347(77), 348(54), 349(75), 351(54, 77), 354, 356, 363, 364(128), 365, 366(132, 134),370,379(109) Hizer, C.A,, 440 Hjerthn, S.,278 Hoeve, C.A.J., 331 Hoffman, P.,291,292,293(97,99) Hoki, N.,173 Hollander, H., 430 Holland, C. V.,57,58(295d) Hollander, M.,106 Holm, G.A.L., 391(40),394 Holmes, R. E.,186 Honda, S.,291 Honeyman, J., 68,76(1),77(1) Honma, T.,203,204(9),209,224(26) Horn, D.H. S.,397 Horsfall, F.L.,441,442(22) Horton, D., 49,57,58(295d), 59,140(295), 147,152,153,154,176,190(318), 218, 232, 233, 235, 236(81, 93), 237(81), 249(93),255(81),256,260,262(156)

462

AUTHOR INDEX, VOLUME 24

Horwitz, J. P., 29, 30(244), 43(244), 166, 172, 174(360), 178(362, 380), 226, 227(63,66,67), 228(66,67) Hough, L., 72, 107(25), 119(25), 120(25), 132(25),l40,141,144,145,146(251b), 150(280), 160(251, 285), 162, 163, 176(280), 191(251, 285), 250, 251(125), 257(125), 258(125), 259(125), 300(141b), 301, 365, 366(134) Hovingh, P., 291 Howard, G. A., 45,47(264) Howarth, G. B., 99, 112, 127(101), 135(101),246,260 HSU,D.-S.,374 Huang, R. T. C., 390,391(37) Huber, G., 302,304,325(146) Hudson, C. S., 14, 18, 22, 24, 46, 69, 71, 111(16),169,174 Hudy, J. A., 328,329(213),330(213) Hughes, J. B., 260,262(156) Hughes, N. A., 49,50, 141, 142(258), 146, 151(258b), 152(258b),155, 161(258a), 193(292),194,197(320) Huguenin, R., 100 Hulyalkar, R. K., 71,111(17) Humiston, C. G., 386,392(18) Hung, Y.-L., 154 Hunt, K., 299,300(133),336,362,369(21) Hunter, C. E., 35 Hurd, C. D., 202,224(6) Husain, A., 151 Hussain, 0.Z., 442 Hutson, D. H., 49,140(295), 147 Hybl, A. 285

I Igarashi, K., 203,204(9), 209,224(26) Ikehara, M., 187,188 Imagawa, T., 203,204(9) Inch, T. D., 248 Ingles, D. L., 50,146 Ingle, T. R., 156,192,256 Ingold, C:H., 417 Ingraham, L. I., 167 Inokawa, S., 251,256 Inouye, S., 83,94(63), 193 Isbell, H. S., 14, 18, 28(224), 30(224), 32, 35,38,46,47(262),60 Iselin, B., 100,112(103),115(103)

Ishidate, M., 241 Ishiguro, S., 224 Ishikawa, T., 279 Ishimoda, T., 404, 445, 447(53, 54), 448(53,54) Ishizu, A., 259 Issartel, R., 438,439(9),440(9) Ito, T., 279 Ito, Y., 136 Itoh, T., 187 Ivanova, V. S., 91 Ives, D. J. G., 331 Iwai, I., 230 Iwashige, T., 171,230 Izaki, K., 314

J Jackson, E. L., 78 Jackson, J., 346 Jackson, W. R.,142 Jacob, P., 28 Jacobs, W. A., 392(45),394 Jacobson, B., 311 Jahn, W., 186,187 James, S. P., 336,361(14) Jamieson, G. A., 185,451 Jaques, L. B., 291 Jardetsky, O., 310 Jarman, M., 168 Ja;, J., 88,153,190(316),233,236 Jatzkewitz, H., 404, 405, 407, 415, 418, 430,431,432 Jeanloz, R. W., 160 Jeffrey, G. A., 285 Jencks, W. P., 30 Jenkins, H., 202,224(6) Jenni, E. F., 386,393(19,47), 394 Jennings, B. R., 317 Jennings, H. J., 162, 245, 246(116), 265(116),266(116) Jenny, E. F., 390,393(38) Jevons, F. R., 445,448,450(68), 452(68) Jewell, J. S., 57, 58(295d), 140, 154, 190(318),256 Johnson, F., 76,109 Johnson, J. F., 322 Johnson, L. N., 314 Johnson, P., 52,311,383,408 Johnston, M. J., 336, 351(19), 352(19), 363(18), 364(18, 19), 365, 366(132), 379(18,19)

AUTHOR INDEX, VOLUME 24

463

Jones, G. H., 248 Keston, A. S., 63,65(305) Jones, J. K. N., 71, 73,99,111(17,34),112, Khan, R., 250, 251(125), 257(125), 127(101),135(101), 143,146,147(274), 258(125),259( 125) 151, 161, 162, 163(341), 168(341), Khomutov, L. I., 305 170(341), 245, 246, 260, 299, 333, Khwaya, T. A,, 226,227(65), 228(65) 336, 341, 342, 344(80, 87), 345, Kibrick, A., 304 351(78), 352(78, loo), 353, 354(107), Kienzle, F., 83,85(64,65),86,95, 108(67), 365,366(134), 369(20,21),371 111(65), 115, 116(64, 65, 69), 117(64, Jones, P. H., 250 65, 69), 118(149, 151), 121(67), Jones, R. S., 296 122(161), 123(161), 124(64, 148, 149, Joraschky, W., 305,324(151a) 161), 125(64, 64), 126(161), 128(65, Jordaan, J. H., 73,78(35) 69), 129(64, 148), 132(149, 156), Josan, J. S., 255 134(177),135, 137,243 Joseph, J. P., 187 Kilde, G., 52 Kim, K. C., 237 King, D., 233 King, J. S., Jr., 438, 439(6), 440(6), 445, 447(58) K King, N. J., 336 King, T. P., 449 Kaneko, M., 188 Kinoshita, T., 241 Kaplan, D., 444 Kirby, K. W., 296 Kaplan, J. C., 438,439(7), 440(7) Kirchner, G., 420 Kaplan, L., 181 Kirchner, J. G., 338 Kaplan, M., 359,378 Kirkland, A., 114,130,136(147) Karabinos,J. V., 71,111(16) Kishimoto, Y., 398,399 Karamalla, K. A., 345,344(89) Kisic, A,, 410 Karamalla, K. K., 348 Kiso, N., 403 Karkas, J. D., 415,420 Karlsson, K. A., 390, 391(39, 40, 42), 394, Kiss, J., 385, 386, 387, 392(28), 398, 399(69),404,405(103),421(13,69) 413,417 Kissman, H. M., 142 Karrer, P., 142 Kitahara, K., 163 Katchalsky, A., 269 Kiyokawa, M., 141, 147(255),173,259 Katsuhara, M., 141,147(255),254 Kiyomoto, A., 112, 133(139,142), 134(142) Katz, J. R., 306 Klerner, A., 45 Kaufmann, B., 432 Klenk, E., 383, 384, 385, 390, 391(7, 37), Kauzmann, W., 307,310(164) 295(7),396(7),398, 399,400,401,408, Kavanagh, L. W., 291 Kawai, Y., 292,295(103) 413, 414(7), 415(143), 416(158), 417 Kawasaki, H., 440, 441(20), 445, 447(54), (140), 418, 421, 423, 425(178), 426, 427, 428, 430(138, 148), 432 448(54) Kawerau, E., 449 Klesse, P., 414,416(155) Kean, E. L., 400 Klundt, I. L., 166, 178(362),226, 227(66), Keilin, D., 63,64(309) 228(66) Knecht, J., 291 Kennedy, E. P., 394 Kbbor, J., 398, 399(69), 421(69) Kenner, G. W., 45,47(264) Kent, P. W., 142, 143(267), 155, 197(319), Koch, H. J.. 211,242 Kochetkov, N. K., 262(170a),263,338,401 204,205(13) Korasy, F., 115 Kerby, G. P. 443 Koeser, H., 254 Kertesz, Z. I., 298,299(123), 325(123) Kohn, R.,304(151),305,327 Kenvick, R. A., 442 Kojima, M., 234 Kessler, G., 339

AUTHOR INDEX, VOLUME 24

464

Kolman, Z., 413 Komoto, H., 264 Koob, J. L., 410 Kooiman, P., 373,374 KOPP,J., 29 Korey, S. R., 415 Komblum, N., 71,110 Komilov, V. I., 264 Korsakova, I. S., 104 Kost, A. A., 262(170),263 Koval, C. J., 394 KO&, J., 71,108(18b),233,236 Krane, S. M., 452 Kraus, F.,29,30(243) Kreger, D. R., 326,373 Krigbaum, W. R., 272 Kristen, H., 175 Ksandr, Z., 153,190(316) Kubala, J., 359 Kiindig, W., 335,336(9) Kuhn, R., 28,47, 112, 186, 339,398, 414, 416(155),419,420,425,426,427,433 Kullnig, R. K., 301 Kumashiro, I., 212 Kunau, W., 416 Kung-Yink Tan, K., 440 Kuznetsova, Z. I., 91 Kuzuhara, H., 240,263(104)

L Labudzinska, A., 305,306(155) LaForge, F. B., 46 Laidler, K., 35 Lambert, R. D., 371 LaMer, V. K., 29,30,43(241) Lampen, J. O., 181 Lance, D. G., 94, 99(91b), 111(91b),112, 127(101),135(101),235, 256(92), 257, 260(148b) Landolt, R., 416 Lands, W.E. M., 386,408(22) Landsteiner, K., 403, 413(93), 414, 417(159) Langen, P., 184 Lapedes, S. L., 63 Large, D. G., 191 Larsen, B., 283,297,298 Latremouille, C.A., 28,34(236)

Laurent, T. C., 292,296(91),311,322(91) Lauter, L. J., 413,415 Lauterbach,J. H., 235,236(93),249(93) Lavrova, K. F.,216 Law, J. H., 409 Lawson, C. J., 281, 282, 283(63). 284, 293(62,63,70), 323,339 Ledeen, R., 418 Lee, C. Y.,55(297b,c),56(297b,c),60 Lee, W. W., 228 Lee, Y.-C., 339 Lees, M., 405,415,428(162) Lefar, M. S., 203 Leftin, J. H., 210,221(29) Lehmann, J., 157, 158(331),165,209,247, 254,258(121),261 Leigh, J. S., 310 Leinert, H., 106 Lemieux, R. U., 57, 59,73,76(37), 77(39), 78(42), 112(43), 147, 148, 201,203(4), 207, 208, 219, 221, 222(50), 232(4), 240(50), 241(50),265(4),301 Leonard, N. J., 230 Lepper, M. H., 440 Lesch, P., 399,406(75) Lester, G., 311 Letters, R., 179 Leuenberger, R., 302,325(146) Leutzinger, E. E., 212,218 Levene, P. A., 156, 171, 172, 178, 185, 189(322), 392(45), 394, 403, 413(93), 414,417(159) Lewy, G. A., 45 Lewis, B. A., 340 Li, L.-K., 63 Lichtenthaler, F. W., 78, 86, 88(75a, 75b, 78,79,80), 90,92,93(89), 94,97, 100, 102,104,106,110(75a,75b, 78,79,80, 117), 115(75a, 75b, 79, 97, 107, 117), 116(107), 117(107), 117), 120(107), 128(117), 135, 136(123, 179), 137(123, 181) Lichtin, N. N., 210,221(29) Lifland, L., 146 Lindahl, U., 291 Lindberg, B., 99,259,418 Lindgren, B. O., 351,352(102) Lineback, D. R., 219 Linker, A., 291, 292, 293(97), 296, 338, 443

AUTHOR INDEX, VOLUME 24 Liotta, S.,30 Little, J. M., 438,439(6),440(6) Lloyd, K. O.,114,136(147) Lochinger, W.,414,416(155) Lohr, J. P.,400 LOW, I., 339 Long, F. A.,28,30,33,41,53 Long, L., Jr., 142, 146(283), 164,176(24) 177 Lora-Tamayo, M., 218 Louloudes, S.,131 Lovatt-Evans, C.,438,439(10) Lowry, T. M., 15,24,27,28 Lowy, P., 448 Lundblad, A., 444, 445(48),447(59, 60), 449(59),450,452(59,60) Lundstrom, H., 353 Lundt, I., 216,217 Luscombe, M., 322 Lutz, P., 420 Luzzati, V., 315 Lythgoe, B., 45,47(264)

465

Macleod, R., 99 McNab, C., 327, 336, 340(26), 355(25), 356(25,26),357(26),358(25\ McNally, S.,233,234(86) McNiven, N., 311 McTague, J. P.,310 Madroiiero, R., 218 Mair, G . A., 314 Majer, N., 135,136(179) Majhofer-Orescanin, B.,387,394 Maki, T., 206,236(18) Makita, A.,403 Malhotra, S.K., 76,109 Malkin, T., 393(47),394 Malone, M. J,, 406 Manabe, M., 301 Mandelkem, L., 308,315 Manning, J. H., 341 ManviHe, J. F., 204,266 Marchessault, R. H.,273,276(27),284(27), 305 Marcotte-Boy, G., 438,439(9),440(9) Marinetti, G., 385,398(11),399(11) M Marinetti, G. V.,399 Marmur, J., 320 Maasberg, A. T., 271,272(18) McCarthy, J. R., Jr., 148,166,226,227(64) Marsh, C. A,, 45 Marsh, J. P.,210 228(64),25.1,252 Martensson, E., 406,407,413 McCasland, G. E., 100,106,167 Martin, H. H., 314 McCleary, C. W.,253 Martin, J. A.,410 McCluer, R. H., 417,428 McCready, R. M., 300(139),301,325(139) Martlew, E. F., 193 Mashburn, T. A.,291,292 MacDonald, D. L., 420 McDowell, R. H.,270, 277(9), 281(9), Masuda, F.,241 Masuzawa, M., 311 283(9),323,324(196),334 Matchett, T. J., 106 McFadden, B. A., 72,111(30) 128) McGale, E. H. F., 436, 445, 448, 449, Matheson, N. K., 149,363,364( Mathews, M. B.,291,292 450(68),452(68) Matsuda, K., 339 McGarry, E., 442 Matsuhashi, M., 145,160(284),314 McGarvey, B. R., 310 Matthews, L. W.,296,297(110) McGavack, T. H., 440 Mattick, L.R., 61 McGuire, T. A.,408 Maxfield, M., 442 McIlwain, H., 414 McKay, J. E.,339 Mazurek, M., 291 Mackie, D. W.,219 Mead, J. F.,394 McKenna, J. P., 335, 339(11), 340(11), Mead, T. H., 278 Meath, J. A.,407 348(11),351(11),352(11),378(11) McKinnon, A.A, 310,313(171a),317(171a) Meier, H., 376 Meier, H. C.V., 63 Maclaglan, N. F.,440 McLauchlan, K. A., 88, 245, 246(116), Meier, S.,399,406(75) Meilman, E.,448 265(116),266(116)

466

AUTHOR INDEX, VOLUME 24

Meindl, P., 419,420(191) Menkes, J. H., 406,417 Merrill, E. W., 296,312(108), 322(108) Merrill, R. C., 301 Meshreki, M. H., 233 Metcalfe,J. C., 311 Methler, H. L., 416 Meyer, K., 291, 292, 293(96, 97, 99, 101), 338 Meyer zu Reckendorf, W., 147,151,247 Michalec, C., 390,413 Micheel, F., 45 Michelson, A. M., 177, 178, 179(401), 180(401),182(402),185(401) Michon, J., 440 Miettinen, T. A., 444 Mildvan, A. S., 310 Miller, J., 140 Miller, N. C., 178 Mills, J. A,, 116, 149,150(303),176 Minkin, V. I., 37 Mirzayanova, M. N., 262 Mislow, K., 385,397,398(10) Mitchell, R. E. J., 211 Mitra, A. K., 97,146 Miyagishima,T., 136 Miyano, M., 146 Miyazaki, N., 301 Miyazawa, T., 274 Mizuno, Y.,180,187 Mizushima, S., 274 Mochalin, V. B., 235 Moelwyn-Hughes, E. A., 29,43(241),52 Moerner, K. A. H., 436,443 Moffatt, J. C., 251 Molloy, J. A., 300(141), 301, 339, 348(59), 351(59), 352(59), 363, 374, 375(157), 378(59, 157) Montgomery, R., 150, 176, 270, 272(7), 277(7), 333, 334(2), 348(2), 349(2), 351(2),353(2),354(2),359(2),371(2) Morgan, K., 284 Morgan, M. S., 139 Morgan, W. T. J., 425,448,450 Morgan, W. W., 68,76(1), 77(1) Mori, T., 270 Morrison, G. A., 108,165,273,295(29) Morrison, I. M., 299, 300(133, 135), 326(135),338,362(40) Morss, N. M., 270(16), 271, 272(16), 329(16),330(16)

Moscatelly, E. A., 390 Mosher, C. W., 210 Moyer, J. D., 14 Mudd, S. H., 185 Miihlethaler, K., 326,327 Mueller, G. P., 281, 283(58), 292(58), 293(61),315(58),316(58),323(58) Mueller, K. L., 385,408(22) Miiller, M., 60 Miiller-Eberhard, H. J., 452 Muir, H. M., 445 Mukherjee, A. K., 353 Mulligan, C. D., 398 Muneyama, K., 188 Munoz, A. J., 452 Murdock, K. C., 178 Murphy, D., 161, 163, 164(350),233, 234, 249(84) Muskat, I. E., 156,189(322)

N Nace, G. W., 278 Nadkarni, S., 144 Nagabhushan, T. L., 73,75(37),77,78(42), 112(42),207,208 Nagasawa, M., 330 Nagasawa, N., 212 Nair, V., 230 Nakada, S., 147 Nakagawa, T., 86,88(78), 90,106,110(78), 136(123), 137(123),264 Nakajima, M., 163,256,258 Nakamura, H., 203,206 Nakamura, K., 404 Nakayama, T., 397,403,409 Nalbandov, O., 392(46),394 Nasiruddin, 366 Nasuno, S., 253 Nayak, U. C . , 91, 133(87a) 225, 234(59), 244(59,60),249(59,60),250(59,60) Neal, J. L., 280 Neely, W. B., 271,331(21) Nef, J. U., 69, lll(12) Neilson, T., 83, 85(64), 116(64), 117(64), 118(152), 124(64), 125(64), 126, 128(152), 129(64, 152), 131(165), 134(155),135(155),136(165),246 Nelson, E. K., 300(138),301

AUTHOR INDEX, VOLUME 24 Nbmethy, G., 331 Ness, R. K., 254 Neuberger, A., 63 Neukom, H., 255, 270, 305, 306, 313(15Oa),325(161),335,336(9) Newman, S., 272 Newth,F.H., 168,169,170(116),173 Nickl, J.. 305 Nicolaieff, A., 315 Nicolle, J., 31 Nicolson, A., 345,344(84) Nidecker, H., 104 Ninomiya, K., 272 Noel, M., 166, 172, 174(360), 178(362, 380), 226,227(63,66,67),228(66,67) Nojima, S., 412(133,144), 413 Noland, W. E.,69,71(13), 111(13),112(13) Norfleet, C. M., 442 Normant, H., 141 Norris, W. P., 384,392(44),394 North, A. C. T., 314 Northcote, D. H., 300(137),301 Novik, P., 153,190(316) Novikov, S. S., 68,104 Nowoczek, G., 404,407(104) Nudelman, A., 140 Nunn, J . R., 278,345

0 Oakes, E. M., 156,174(326),256 O’Ceallachain, D. F., 342 O’Colla, P. S., 292,293( 102),342 O’Connel, P. W., 387 Odin, L., 418 Odova, R. G., 300(141a),301 ubrink, B., 305,312(154a) Oftedahl, M. L., 112, 119, 130, 133(138, 141) Ogawa, H., 193 Ogg, J., 270(16), 271, 272(16), 329(16), 330(16) Ogston, A. G., 296,322(106) Ohashi, Y.,136 Ohrui, H., 256 Okuda, T., 134 Okuhara, E., 399 Oldham, J. W. H., 404 Olfermann, G., 86 Olin, S. M., 101

467

Ollapally, A. P., 111,127(133),264 O’Neill, A. N., 112,133(137),278,280,284 O’Neill, I. K., 73,76(37), 77(39),207 Ong, K. S., 94, 126, 134(167), 135(167), 137(166),243 Ono, K., 385,412(133,134), 413 Onodera, K.,241,301 Oshima, Y.,407 Ostroumov, Yu. A., 37 Otake, N., 238 Otter, B. A., 250, 251(125), 257(125), 258(125),259(125) Otterbach, D. H., 145, 146, 159(286), 160(284) Otterbach, H., 147 Overend, W. G., 30(260), 38, 43(260), 91, 115(88), 140, 164, 191(242),201, 212, 219,233,234(86), 237(91),244,255 Ovodov, Y.S., 300(141a)301 Owen, L. N., 49, 141, 145, 146(253, 273, 279), 147(273),150,159,175,190(196), 191(441),195(273) Owens, H. S., 300(139),301,325(139)

P Pacsu, E., 28,30(237),31,146 Padberg, G., 417,425(178) Painter, E. P., 386 Painter, T. J., 292, 293(102), 297, 320, 323(190),425,427 Pal, S., 292 Palmer, K. J., 301,302 Papadopulos, N. M., 416 Parikh, V. M., 353,354(107) Parker, A. J., 140 Partridge, S. M., 451 Patchett, A. A., 228 Paulsen, H., 50, 57, 58(295d), 127, 129(169), 135(169),247,254,265 Pavia, A. A,, 57,59 Peat, S., 168,176(370),270,339 Pechan, Z., 444 Pedersen, C., 216,217 Pedersen, K., 19 Penman, A., 279, 281, 282, 283(58, 63), 292(58), 293(63), 315(58), 316(58), 323(58) Pentchev, P. G., 63,64(311), 65

468

AUTHOR INDEX, VOLUME 24

Percival, E., 270(15a), 271, 272(15a), 277(9), 281(9), 283(9), 296, 297, 334, 341,365 Perekalin, V. V., 69,127(11), 130(11) Perlin, A. S., 62, 78, 97, 219, 291, 338, 344(83),345 Pernas, A. J., 283 Perry, M. B., 71, 111(17,18a), 112(19,134, 135), 127(134,135), 133(19, 134, 135), 134(19),264,265,341 Phelps, C. F., 322 Philipp, G., 48 Philippart, M., 406 Phillips, D. C., 314 Phillips, G. E., 384,392(44), 394 Piatakov, N. F., 68 Pictet, A., 68 Pigman, W., 14,47 Pilz, H., 415,430,431(165),432 Pippen, E. L., 300(139),301,325(139) Plaut, H., 104 Plessas, N. R., 233, 234(87), 236(87), 244(87),245(87),249(87) Plohnke, A,, 305,324(151a) Podder, S. K., 290 Polenov, V. A., 262(170),263 Polglase, W. J., 101 Polson, A,, 278 Polyakov, A. I., 216 Ponder, B. W., 78 Porshnev, Yu. N., 235 Porter, R. S., 322 Posternak, T., 100 Pouradier, J., 307,308(163),309(163) Prasad, N., 213,215,216(42), 222,223(56), 234(42),235(42),240(57) Pratt, J. W., 46 Pravdid, N., 225,244 Preobrazhenskaya, M. N., 220 Preston, R. D., 298,324(122) Pretorius, Y.Y.,397 Pridham, J. B., 338 Prihar, H. S., 140 Prins, W., 313 Prosser, T. J., 249 Prostenik, M., 387,391(43), 392(43),394 Prout, C. K., 204,205(13) PrystaH, M., 220 Pusztai, A., 450 Putnam, F. W., 451

R Rabinsohn, Y.,175 Rachaman, E. S., 401 Radford, T., 91,132(87a), 174 Radhakrishnan, A. N., 445, 447(55, 56), 449 Radin, N. S., 398,399 Ragg, P. L., 159,190(196) Rajabalee, F., 115, 124(148), 129(148), 134(178), 135,136(178),137 Ramachandran, G. N., 273 Ramakrishnan, C., 273,274 Ramalingham, K. V., 374 Randall, M. H., 157,158(331), 162 Rank, W., 100, 113, 115, 118(108, 151), 124(146), 126(154), 127(154), 129(154), 130(154), 131(165), 136(165),243,256 Rao, C. V. N., 353 Rao, G. V., 92,93(90) Rao, P. S., 373,374 Rao, V. S. R., 273 Rapoport, C. M., 448 Rapport, M., 400 Rashbrook, R. B., 339 Rather, L. J., 438 Rattle, H. W. E., 310 Raymond, A. L., 172 Rees, B. H., 157 Rees, D. A., 253, 270(15a), 271, 272(15a), 274, 275, 276(29a),277(13), 278, 279, 280, 281(13, 55, 57), 282(57), 283(13, 58, 63), 284(57, 66),285(71), 286(71, 73), 287, 288(73), 290(31, 71), 292(13, 40, 58), 293(40, 41,55, 60,61, 62, 63, 66, 70), 295(71, 73, 75), 296, 299, 300(136), 301, 310(75), 313(171a), 315(58,73,75), 316(58),317(75,17la), 318(75), 320(73, 75, 170), 321(170), 322(31, 71, 75), 323(40, 58), 326(6, 136), 327, 336, 341, 342, 346(76), 349(76), 350(76), 370, 374, 375(156), 379 Reese, C. B., 183 Reeves, R. E., 374 Regan, C. M., 29,30,(243) Reggiani, M., 290 Reid, J., 83 Reife, A., 438,439(5), 440(5)

AUTHOR INDEX, VOLUME 24 Reimann, H., 70 Reindel, F., 386,409 Reist, E. J., 141, 155(248), 159(249, 250), 160,161,171,186,188,192 Reitz, O., 29,30 Rennie, M., 270(15a),271,272(15a) Rennkamp, F., 413,430(148) Reuser, G., 430 Rice, S. A., 330 Rich, C., 443 Rich, P., 248 Richardson, A. C., 88, 89, 110(85), 140, 141, 146(251b), 147(260), 160(251, 260,285), 161,162(260),163, 191(251, 285,348) Richardson, N. G., 299, 300(136), 326(136),370 Richtrnyer, N. K., 46,73, 111(31,33), 169, 174 Ries-Lesic, B., 387 Rigas, D. A., 436,442(3),445(3) Rijke, A. M., 313 Roberts, J. D., 29,30(243) Roberts, R. M., 167 Robertson, G. J., 169,170(105) Robins, M. J., 148, 166, 226, 227(64), 228(64),251,252 Robins, R. K., 148, 166,179,186,212,218, 226,227(64), 228(64),251,252 Robinson, J. D., 406 Robinson, R. A., 29 Robson, E. R., 442 Rod&, L., 291,292,293(100) Rohm, E., 414,416(155) Roelofsen, P. A., 326 Rogovin, Z. A., 259 Rokke, N. W., 72, lll(30) Romm, R., 140 Rose, B., 442 Roseman, S., 432 Rosenberg, A., 400,428,430 Rosenberg, L., 292 Rosenheim, O., 395 Rosenthal, A., 202, 210, 2 (304, 2 9, 220(30a),232,238(48a), 240(48a),242 Rosik, J., 359 Ross, V., 310 Ross, W. C. J., 168 Roth, L. A., 305 Rothfus, J. A., 399

469

Rothschild, C., 440,441(20) Rouser, G., 416 Rowell, R. M., 192 Rowley, E. K., 250 Rudowski, A., 336, 355(25), 356(25), 358(25) Rundle, R. E., 285 Rupley, J. A., 322 Ruppol, E., 409 Russell, B., 278 Rutherford, J. K., 404 Rutz, G., 69,127( 104) Ruyle, W. V., 228 Ryan, K. J., 165

S Sable, H. Z., 102 Sachdev, H. S., 157 Saeki, H., 171 Saifter, A., 442 Saito, H . , 292,295(103) Sajdera, S. W., 296,322(106a) Sakai, R., 408 Salas, M., 64,65(315) Salsman, K., 418 Sambasivarao,K., 417 Samek, Z., 153,190(316) Samokhvalov,G. I., 235,262 Sampson, P., 338 Samuel, J. W. B., 253, 285, 286(73), 288(73), 295(73), 296, 315(73), 320(73),341 Sanderson, G. R., 340, 365, 366(133), 367(64,138) Sandhoff, K., 415,418,431(165),432 Sandson, J., 292 Sankey, G. H., 220, 221(51), 222, 223(56), 234, 237(51, 91), 240(57), 241(51), 242(106),265(106) Sarko, A., 273,276(27), 284(27) Sarma, V. R., 314 Sarre, 0. Z., 70 Sasaki, T.,180 Sasisekharan, V., 273 Satoh, C., 112,133(139,142),134(142) Saunders, R. M., 140,169,193,261 Savage, A. B., 271,272(18),331 Schaffer, R., 72,107(26) Schaleger, L. L., 53

470

AUTHOR INDEX, VOLUME 24

Scheidegger, U., 106 Schmandke, H., 175 Schmid, H., 34,37,53,55(254), 142 Schmidt, E., 69,127(10) Schmidt, G., 403 Schmidt, H. W. H., 255 Schneider, F. W., 290 Schneider, W., 50 Schneider, W. G., 301 Schopfer, W. H., 100 Schorsch, E. U., 398 Schreuder, H. R., 374 Schubert, M., 292,296(90), 322(90) Schuster, T. M., 317 Schweiger, R. G., 272,323(22),324 Sciavolino,F. C., 230 Scott, W. E., 276 Segal, H., 393(49),394 Segal, L., 60 Segrest, J. P., 451 Sehon, A. H., 442 Seno, N., 291,292,295( 103) Sephton, H. H., 73,111 (31,32,33) Sepp, D. T., 57,58 Serfontein, W. J., 73,78(35) Settineri, W. J., 276 Seuss, H., 29,43(241) Seydel, P. V., 384 Seymour, D., 167 Shafizadeh, F., 147,148,152(298) Shallenberger, R. S., 55(297b, c), 56(297b, c), 60,61 Shapiro, D., 386, 393(49), 394, 398, 401, 431(68) Sharma, M., 201, 202, 225, 234(59), 244(59, 60), 249(59, 60), 250(59, 60), 265(3) Shash, N.S., 442 Shaw, D. H., 360,371,372(145),379(145) Shaw, G., 181 Shechter, H., 112 Shen, T. Y.,148,149,150(280), 228 Shen Han, T. M., 147,152(298) Shettles, L. B., 445 Shibata, H., 163 Shimanouchi, T., 274 Shoji, H., 233 Shorygina, N. N., 91 Shostakovskii,M. E., 216

Shute, S. H., 72, 107(25),119(25),120(25), 132(25) Shvegheimer, C. A., 68 Shwan, J. C. P., 49,141,146(254) Siakotos,A. N., 416 Siddiqui, B., 428 Siek, T. J., 72,111(30) Signer, R., 313 Simon, H., 48 Singh, P. P., 373 Sirokman, F., 189,194(437),386 Skerrett, R. J,, 274,275,276(29a),290(31), 322(31),327,336 Skipski, V. P., 400 Sloane-Stanley, G. H., 405,415,428(162) Smejkal, J., 220 Smell, E. E., 394 Smidsdd, O., 283,297,298,320,323(190), 324,327 Smith, D. B., 280,409 Smith, F., 176, 270, 272(7), 277(7), 333, 334(2), 336, 340, 341, 342, 344(81), 345(73), 346, 348(2), 349(2), 351(2), 353(2), 354(2), 359(2), 360, 361(14), 371(2),379(81) Smith, F. W., 316 Smith, C. F., 25,28,52(229) Smith, J. E., 64 Smith, K. A., 296,312(108), 322(108) Smith, M. C., 25,52(230) Smith, R., 291 Smith, R. H., 448 Smith, R. N., 348 Sneen, R. A., 142 Sodd, M. A,, 394 Solms, J., 324 Sommerfield, R. V., 300(138),301 Sorm, F., 220 Sowden, J. C., 68, 69, 71(3), 72(3, 20, 22), 76(3), 77(3), 107(20,22,26),111(3,22, 23, 2 9 , 112, 114, 115(3), 119, 127(3), 130,133(138,141), 136 Sols, A., 64,65(315) Spach, G., 317 Speakman, P. R. H., 141, 142(258), 151(258b),152(258b), 161(258a), 194 Spencer, J. F. T., 296,297(114) Spencer,R. R., 141,159(249,250),171 Spreistersbach, D. R., 342,345(73), 349

AUTHOR INDEX, VOLUME 24 Springer, C. H., 99 Springmann, H., 147 Sprinson, D. B., 394 Spurr, 0. K., 306, 307(160), 309(160), 313(160) Squire, J. R., 440 Srivastava, H. C., 373 Stacey, M., 151,165,193,254 Stallberg3tenhagen, S., 399 Stanacev, N. Z., 391(43), 392(43), 394, 412(133,134),413,417,430 Stancioff, D. J., 279,281, 283(58),292(58), 315(58),316(58),323(58) Standen, A., 270 Stanier, J. E., 296,322(106) Stanley, N. F., 281, 283(58), 292(58), 315(58),316(58),323{58) Starr, M. P., 253 Steele, I. W., 253, 287, 295(75), 299, 300(1361, 310(75), 315(75), 317(75), 318(75), 320(75, 170), 321(170), 322(75),326(136),341 Steftkovti,J., 88 Stein, W. D., 414 Steiner, H., 29,30(243),43(241) Steinherz, P., 140 Stephen, A. M., 278, 336, 341, 345, 346, 348, 359, 360, 363(18), 364(18), 371, 372(145),378,379(18,145) Stephen, F. S., 451 Sterling, C., 311,324,326(197) Stem, N., 430 Stevens, C. L., 141, 145, 146, 159(286), 160(284), 161(257), 163(257), 189, 190,194(437),234,253 Stewart, D. K. R., 278 Stewart, L. C., 46 Stewart, T. S., 261 Stoddart, J. F., 335, 337(13), 342, 344(75, 77, 87), 345(75, 77), 346(77), 347(77), 349(75),351(77,78),352(78),371 Stofin, A., 403,404,407 Stofin, P. J., 398,403,404,406,407 Stoloff, L., 270,271(10) Stolz, E., 385,398(11),399(11) Stookmayer,W. H., 307,319(164b) Stout, M. G., 179 Strobach, D. R., 71, 72(22), 107(22), 111(22,23,29),410

471

Strominger, J. L., 145,160(284),314 Stud, M., 218 Stutz, E., 299,306(127), 325(127) Suami, T., 410 Suemitsu, R., 78,208 Sugihara, J. M., 99,143 Sundararajan, P. R., 273 Suomi, W. D., 431 Suvorov, N. N., 220 Suzaki, S., 187 Suzuki, G., 413,430(146), 432 Suzuki, K., 400,415,417(164) 431(83) Suzuki, S., 292,293(98),295(103),324(98) Svennerholm, E., 400 Svennerholm, L., 399,400,413,414,415, 416(157),416(157),417(157),418,430 Svensson, S., 99 Swain, C. G., 27,28(233),34(233) Swanson, M., 442 Sweeley, C. C., 390,400 Sweet, F., 235 Swenson, H. A., 271,331(21) Szarek, W. A,, 94, 99(91b), 111(91b), 112, 127(101), 135(101), 143, 147(274), 235, 246, 256(92), 257, 260(148b), 344(87),345,371

T Tada, H., 188 Taguchi, T., 234 Taha, M. I., 176 Takahashi, S., 163,256,258 Takeda, Y.,106 Takenishi, T., 212 Takeuchi, S., 238 Tamm, I., 441,442(22) Tamura, Z., 241 Tannhauser, S. J., 403 Tatsuta, K., 147 Tavormina, P. A., 392(46),394 Taylor, K. G., 141,143(267), 146,159(286), 160, 161(257),163(257),189,194(437), 234 Taylor, N. F., 142,143(267), 155,197(319), 232,234(80a), 236,250(80a) Teerlink, W. J., 99,143 Tejima, S., 143, 146,203,206,224,236(18) Terry, D., 443

472

AUTHOR INDEX, VOLUME 24

Thalheimer, C., 416 Theander, O.,259 Thiele, H., 305,324(151a) Thierfelder, H., 384, 391(7), 395(7), 396(7),413,414(7) Thomas, G.H. S., 351,352(100) Thomas, J., 451 Thomas, W.A., 265 Thompson, A., 153 Thompson, I., 131 Thompson, N. S., 371 Thompson, R. R., 71,72(20),107(20) Thomson, J. K.,262 Thudichum, J. L.W., 384,395,403 Thumm, B.A., 52,54(290) Timell, T. E., 299,300(131),341,371(70), 374 Tindall, C. G., Jr., 256 Tipper, D. J., 314 Tipson, R. S.,47,60,139, 140, 168, 178, 185,261 Tipton, T. L., 412(134),413 Tkaczynski, T., 220 Tobias, I., 310 Tobolsky, A. V., 312,324(178) Todd, A. R., 45, 47, 177, 178, 179(401), 180(401), 182(402),185(4OO,401) Tokuyama, K., 141, 147(255),173, 252, 254,259(130) Tolley, M. S., 151 Tomizawa, H. H., 386,408(22) Tong, G. L.,228 Townsend, L. B., 166, 212, 218, 226, 227(64),228(64) Trams, E. G., 413,415 Trauth, O.,186 Tremblay, A. 442 Trischmann, H., 339,414,416(155) Tristram, G. R., 448 Tronchet, J,, 248 Tronchet, J. M. J., 248, 260, ZSZ(l56, 170a),263 Trummlitz, G.,136,137(181) Tschampel, D., 414,416(155) Tschirch, A,, 326 Tsein, S. H., 387 Tsuchiya, T., 147, 218, 232, 236(81), 237(81),255(8l) Tsujino, E., 259 Tucker, L. C.N.. 171

Tuppy, H., 419 Turner, W. N., 255 Turvey, J. R., 270,278,279,293(42), 339 Tyler, J. M., 336,370(22,23), 371(22)

U Ubbelohde, A. R., 319 Uddin, M., 300(141),301,363 Ueda, T., 177 Uhlenbruck, G., 418 Umezawa, S.,106,147 Urbanski, J. A., 172,178(380) Urbas, B., 192 Urech, F.,14 Urivetzky, M.M.,448 Utzinger, H., 393(47),394 Uyeda, M., 72,111(30) Uzlova, L. A,, 262(170),263

V Vaeman, J. P., 443 Valicenti, J. A., 160 Van Es, T.,50,234,255(89), 261(89) Varadarajan, S., 180 Varadi, D.P.,443 Vargha, L., 172,173(383) Vaskovsky, V. E., 300(141a),300 Vaughan, W.V., 76 Veis, A., 306 Verheyden, J. P.H., 251 Vermousek, I., 444,445(50) Vernay, J. L.,445,447(57) Vigevani, A., 290 Vifiuela, E., 64,65(315) Vollmin, J. A,, 60 Vogt, D. C.,346,359 Volk, B.W.,414 Volman, D. H., 324 von Euler, H., 51 von Saltza, M. H., 83 von Tavel, P.,313 Voss, P.,135,136(179) Vrancken, M. N.,272

W Wade, C. W. R., 18,28(224),30(224), 32, 35

AUTHOR INDEX, VOLUME 24

Wagner, H., 411,412,413 Wakahara, S., 254 Waldron, D. H., 438 Wallenius, G., 440,441( 13) Walsh, K., 292,293(102) Walton, D. J., 262 Walz, E., 413 Wang, C. C., 251,256 Wang, M. C., 112,135(145) Wang, P. Y.,291 Warren, C. D., 257,260( 147), 388 Warren, L., 417 Warrener, R. N., 181 Wanvicker, J. O., 276 Wasiak, A., 305,306(155) Watanabe, H., 339 Watanabe, K., 279 Watanabe, K. A., 86,88(73,74,77), llO(72, 73, 74, 77), 184, 187, 201, 203(4), 232(4), 238,265(4),266(4) Watanabe, M., 234 Watson, D., 30 Weaver, E. S., 312, 313(178a), 315(178a), 320(178a) Webb, A. C., 71, 111, 112(19, 135), 127(135), 133(19, 135), 134(19), 264, 265 Webber, J. M., 142, 151(264), 157, 158(331), 212, 247(36), 261, 270, 292(14) Weber, E. J., 383,408,409 Weigel, H., 340 Weill, C. E., 203 Weiner, H., 142 Weisbuch, F., 31 Weiss, B., 387 Weiss, J. B., 436,449,451,452(74) Weiss, M. J., 142 Weissman, B., 292,293(99),338 Wells, M. A., 405 Wells, R. D., 233,249(84) Wempen, I., 177,228 Werner, J., 418 Wessely, K., 175 Westheimer, F. H., 32 Westphal, O., 5 Westwood, J. H., 142,157 Wetmur, J. G., 320 Weygand, F., 186 Whelan, W. J., 176,290,339

473

Whistler, R. L., 50, 146,156,176,192,251, 256,270,271(8), 272(8), 296,333 White, E. V., 349,350,371,373,374 White, E. W., 305 White, J., 73,78(35) Whitehead, C. C., 339, 342, 346(76), 348(59), 349(76), 350(76), 351(59), 352(59), 378(59) Whitehouse, M. W., 8,418 Whitton, W. I., 316,317(186),319(186) Whyte, J. L., 299,300(134),335,363(12) Whyte, J. N. C., 202, 232, 299, 300(135), 326(135), 338,362(40) Wickstrom, A., 354 Wiechen, A., 305,324(151a) Wiegandt, F., 398 Wiegandt, H., 414, 416, 418(156), 421, 422, 423(203), 426,427,428(203), 433 Wieland, H., 409 Wiggins, L. F., 150,168 Wight, N. J., 299,300(136), 301,326(136) Wilkins, M. H. F., 325 Williams, D. E., 285 Williams, D. T., 71, 111(18a), 112, 133(143), 151,265 Williams, F. T., 112 Williams, G. J., 233,249(84) Williams, J. M., 161 Williams, N. R., 91,115(88), 144,201,244 Williams, R. S., 365 Williams, S. C., 146 Williamson, F. B., 287, 295(75), 310(75), 313(171a), 315(75),317(75,17la), 318, 320(75), 322(75) Wilson, G. L., 24 Wilson, H. R., 325 Wilson, K., 326 Winstein, S., 167,193,194 Wintersteiner, O., 83 Winzler, R. J., 440,441 Wittstruck, T., 311 Wold, J. K., 297,341 Wolf, R., 305,324(151a) Wolfrom, M. L., 91, 101, 133(87a), 147, 148,152,153,154,176,264,291 Wollmer, A., 305,324(151a) Wolter, H., 413 Wood, K. R., 205 Woodward, C., 292,295(92) Woolard, G. R., 348

AUTHOR INDEX, VOLUME 24

474

Wright, A., 276 Wvnne-Jones,W. F.K., 29,52

Yule, K. C., 49,141,146(254) Yung, N. C.,178,180,181(407)

Y Z Yahya, H. K., 97,102,115(97) Yajima, T.,241 Zabin, J., 394 ZeIIner, I., 409 Yamagata, T., 292,295(103) Yamakawa, T., 403, 413, 415, 430(146), Zen, S., 106 Zhdanov, Yu. A,, 37,257,262(170a),263, 432 264 Yaphe, W., 281,283 Yasuda, A., 106 Zhukova, I. G.,401 Yasuda, M., 399 Ziabicki, A.,305,306(155) Yokoyama, S., 403 Zilliken, F.,8,418 Zimm, B.H., 308 Yonehara, H., 238 Yoshidi, H., 251 Zimmerman, H.K.,Jr., 147,160 Yoshimura. J., 71, 72(24), 86, 88(78), Zinke, H., 94,136,137(181) Zinner, H., 175 107(24),110(78),264 Zinser, H.H., 440,441(20) Young, A. E.,271,272(18) Young, R., 71, 72(18), 107(18), 111(18), Zissis, E.,46 336, 339, 34064, 55), 344(54, 85), Zitko, V., 299,359 345, 34864, 55), 351(19, 54, 55), Zofcsik, W., 411,412,413 352(19),364(19),379(19) Zolotukhina, V.G.,262(170),263 Yu, R. K., 390 Zorbach, W. W., 111,127(133),264 Yuen, G.U.,154 Zussman, J., 177,185(400)

SUBJECT INDEX FOR VOLUME 24

A

5‘-O-p-tolylsulfonyl-, displacement reaction of, 187 -, 8-hydroxy-2’-O-p-tolylsulfonyl-, Acetalation, of nitro sugars, 116 Acetals, cleavage of P-nitro, 125 displacement reaction of, 188 Acetolysis -, 2’,3’-O-isopropylidene-, p-toluof polysaccharides, 339 enesulfonylation of, 185 of tragacanthic acid, 362 -, 2’,3’-O-isopropylidene-5’-O-pAcetone, enolization of,in water and in tolylsulfonyl-, displacement reactions deuterium oxide, 29 of, 147 Acids Agar carboxylic, as catalysts for mutarotation. gelation mechanism, 321 27,34 structure of,277 catalysis of mutarotation of sugars by, 14 Agarobiose, 277 catalytic coefficients for anions of weak, -, 4,6-0-( 1-carboxyethy1idene)-, 18 dimethyl acetal, 279 for weak, 17 -, 6-0-methyl-, dimethyl acetal, 278 Acylation, of deoxvnitroalditols, 115 Agarose, 277 Adenine, 9-(3,5-anhydro-2-deoxy-P-~ gelation of agars by, 321 threo-pentofuranosy1)-, 174 Alcohols -, 9-(5-deoxy-2,3-0amino, sphingosine-related, 390-393 (ethoxymethy1idene)-P-D-etythroreaction with glycals, 215 oent-4-enofuranosyl)-, 148 Alditols, acetylenic, 262 -, 9-(5-deoxy-2,3-0-isopropylidene- -, 2-amino-1,2-dideoxy-l-nitro-, 133 P-D-erythro-pent-4-enofuranosyl)-, -, l-deoxy-2-O-methyl-l-nitro-, 133 148 -, deoxynitro-, 9-(5-deoxy-P-~-erythro-pent-4acylation of, 115 enofuranosy1)-, 148 anhydridization of, 119 ~, 9-(3,4-di-O-acetyl-2-deoxy-P-~dehydroacetylation of, 127 erythro-pentopyranosy1)-,212 ~, l-deoxy-l-nitro~, 9-(2,3-dideoxy-P-~-glycero-pent-2. optical rotatory dispersion and circular enofuranosy1)-, 166 dichroism of, 134 -, 9-[2,3-0-isopropylidene-5-0synthesis of, 70 (methylsulfony1)-~-~lyxofuranosyll-, -, 2-deoxy-2-nitro-, 72 displacement reactions of, 187 Aldohexopyranoses, free energies of, 58 Adenosine, 3’-O-acetyl-2’-deoxy-5‘-O-p- Aldopentbpyranoses, tetraacetates, contolylsulfonyl-, displacement reaction formations of,58 of, 185 Aldopyranoses, 5-thio-,mutarotation of, 49 -, 7-deaza-, configuration of, 187 Aldoses, synthesis of, 111 -, 2’-deoxy-3’-O-p-tolylsulfonyl-, -, 2-amino-2-deoxy-, synthesis of,9, displacement reaction of, 166,174 112,265 -, 2’,3’-dideoxy-, 166 -, 2,6-anhydro-, 211 -, 2’,3’-0-(ethoxymethylidene)-5’-0--, 2-deoxy-, 262,264 synthesis of, 111 p-tolylsulfonyl-, displacement reac, 1-thio-, mutarotation of, 50 tion of, 148 -, N-formyl-2’,3‘-O-isopropylidene- Aldos-Suloses, 258 475

476

SUBJECT INDEX, VOLUME 24

Alginic acid, 296 a-D, displacement reactions of, 163 Alkenes, a-nitro-, alkoxylation of, 130,132 -, methyl 4,6-0-benzylidene-2Alkoxylation, of a-nitroalkenes. 130, 132 deoxy-2-iodo-a-~-,201,203 Alkoxyl group, displacement of sulfonyl- -, methyl 4,6-0-benzylidene-2oxy groups by, 193 deoxy-2-C-methyl-a-~-,202 methyl 4,6-0-benzylidene-2,3Allal, S-benzyl-4,6-O-benzylidene-3-0- -, dibromo-2,3-dideoxy-a-~-,235 methyl-2-thio-D-, 225 Altrose, 2-amino-2-deoxy-D-, 112 -, 4,6-O-benzylidene-~-,201 -, 6-deoxy-2,5-di-O-methyl-~-, 165 iodo(methyoxy1)ation of, 203 Amadori rearrangement, 6,448 --, 4,6-0-benzylidene-2-C-methyl-~-, Amines, reaction with nitro sugars, 136 201 Amino sugars, 9 -, 4,6-0-benzylidene-3-O-methyl-~-, preparation of, 109 202,225 -, 4,6-0-benzylidene-3-O-methyl-2-Ammonia, reaction with nitroalkenes and nitroalditols, 133,135 S-methyl-%thio-D-, 225 Allitol, 2-acetamido-1,2-dideoxy-l-nitro- Amosamine, 159 Amyloid, 373 D,133 Angustmycin A, 250,252 Allofuranose, 1,2:5,6-di-O-isopropyliAnhydridization, internal, of l-deoxy-ldene-3-O-(methylsulfonyl)-~-, disnitroalditols, 119 placement reactions of, 151 -, 1,2:5,6-di-O-isopropylidene-3-0- Anhydro sugars, formation by mutarotation reaction, 46 p-tOlylSUlfOny1-D-, displacement reacAnimal tissues, polysaccharides from, 293 tions of, 151 Annona muricata L, polysaccharide from Allofuranoside, methyl 5-O-(p-bromoseeds of, 374 phenylsulfonyl)-6-deoxy-2,3-O-isoAnomerization propylidene-P-L-, solvolysis of, 195 activation energies of, 51 -, methyl 6-deoxy-2,3-0of glycosidic linkages during acetolysis isopropylidene-P-D, 171 of polysaccharides, 339 methyl 6-deoxy-2,3-0-isopropylimechanism of, 45 dene-5-O-p-tolylsulfonyl-~-~-, reof sugar acetates, by way of cyclic caractivity of, 155 bonium ion, 43 Allolactose, 2-acetamido-2-deoxy-, 9 Anthranilic acid, reaction with nitro oleAllopyranose, penta-0-benzoyl-P-D-, 161 fins, 136 Allopyranoside, methyl 2,3-anhydro-a-~-, methyl 3,6-anhydro-a-~-glucopyran- Arabinal, di-0-acetyl-D-, reaction with benzotriazole and 5,6-dimethyl derivoside from, 46 ative, 217 -, methyl 6-deoxy-2,3-0-isopropyliArabinitol, 2,3,5-tri-O-benzyl-~-, sulfonyldene-p-L-, 189 ation of, 175 Allose, 2-amino-S-deoxy-~-,112 -, 2,3,4-tri-O-benzyl-1,5-di-O-p-tol-, 2,6-diamino-2,6-dideoxy-~, 147 yhlfOnyl-D-, solvolysis of, 196 Altrofuranoside, methyl 3-O-benzoyl-6Arabinofuranose, 5-0-a-~deoxy-2,5-di-O-methyl-a-~-,165 arabinofuranos yl-L-, 360 -, methyl 2,3-di-O-benzyl-6-O-tritylL-,171 -, 5-O-a-~-arabinopyranosyl-~-, 360 Altropyranoside, methyl 3-acetamido-2-, 0-a-L-arabinopyranosyl-( 1+5)-0azido-2,3-dideoxy-4,6-di-OL-arabinofuranosyl-( 1-+5)-~-,360 (methylsulfony1)-a+-, displacement -, 1,2-O-isopropylidene-3,5-di-O-ptolylsulfonyl-L-, displacement reacreactions of, 162 tions of, 151 -, methyl 2-azido-4,6-0-benzylidene-2-deoxy-3-O-(methylsulfonyl)- -,5-O-~~-xylopyranosyl-~-,354,3~

SUBJECT INDEX, VOLUME 24

477

Arabinofuranoside, ethyl 3-amino-3B deoxy-DL-, 229 -,methyl 2,5-anhydro-a-~-,176 -, methyl 5-O-p-tolylsulfonyl-a-~-, Bamosamine, 159 Barry’s procedure, 342,345 displacement reaction of, 176 Bases, catalysis of mutarotation of sugars Arabinogalactans, 341 by, 14 Arabinonic anilide, 115 Arabinopyranoside, methyl 2-0-benzoyl- Beech, polysaccharides, 376,377 Beetles, repellents for, 7 3,4-di-O-p-tolylsulfonyl-~-~-, disBenzoic acid, m(and p)-amino-, reaction placement reactions of, 161 with nitro olefins, 136 -, methyl 2,3-di-O-benzoyl-4-0Benzotriazole, reaction with glycals, 217 (methylsulfonyl)-p-L-, displacement -, 5,6-dimethyl-, reaction with glyreactions of, 160 cals, 217 -, methyl 2,3-di-O-benzoy1-4-0-pto1ylsulfonyl-P-L-, displacement reac- Bicyclo[2.2. llheptane, carbohydrates containing ring, 176 tions of, 160 -, methyl 3,4-O-isopropylidene-2-0- Bicyclo[3.2. lloctane, carbohydrates containing ring, 176 p-tolylsulfonyl-&m, displacement Birefringence of gels, 305 reactions of, 152 -, methyl tri-0-(methylsulfony1)-a-L Blasticidin S,226,238 (and P-L)-, displacement reactions of, Blix sulfatide, 405,407 Brassica a h , polysaccharide from seed 161 of, 374 Arabinopyranos yl fluoride, 2-bromo-2Bridged-ring systems, of carbohydrate deoxy-p-m, 204 sulfonic esters, displacement reacArabinose, L-, in Acacia gums, 346 tions of, 153 -, Z-amino-2-deoxy-~-,synthesis of, Bromination, of tri-0-acetyl-Dglucal, 203 9 -, 3-O-a-~-arabinofuranosyl-~-, 354, p-Bromobenzenesulfonate, in displacement reactions of carbohydrates, 140 360 Bromo(methoxyl)ation, of glycals, 203 -, 2,3(2,5- and 3,5)-di-O-methyl-, Bronsted relationship, between catalytic from mesquite gum, 350 activity and dissociation constants, 19 -, 3-O-a-~-galactopyranosyl-~-, 354, 372 -, 3-O-~-~galactopyranosyl-~-, 355, 376 -, 0-a-mgalactopyranosyl-( 14)-0C a-~-arabinofuranosyl-( 1+2)-~-,372 -, 0-(Dglucopyranosyluronic acid)( 1+6)-O-D-galactopyranosyl-(1+3)Carbohydrate-protein compounds, in L-, 358 urine (human), 435-452 -, 3-O-a-~-xylopyranosyl-~-, 370 Carbohydrates -, O-cu-D-xylopyranosyl-(1+3)-0-(~nitrogen-containing, 9 L-arabinofuranosyl-( 1+3)-L-, 370 sulfonic esters, 139-197 ~, O-~-mxylopyranosyl-( 1+5)-0-aCarbonium ion, anomerization by way of a L-arabinofuranosyl-( 1+3)-~-,354 cyclic, 43 Arabinoxylan, 369 Carboxylic acids, as catalysts for mutarotaArrhenius equation, 51 tion, 27,34 Asafoetida gum, 351 Carrabiose, dimethyl acetal, 280 Atropisomerism, 5 &-Carrageenan,281,285,288,289 K-Carrageenan, 280,285 Autohydrolysis, of polysaccharides, 338

478

SUBJECT INDEX, VOLUME 24

A-Carrageenan, 283,2,84 p-Carrageenan, 282 Carrageenans mechanism of gelation, 314 structure of, 279-290 Catalysis Bronsted relationship between dissociation constants and, 19 of mutarotation of sugars by acids and bases, 14 Catalysts, bifunctional, in mutarotation, 27,34 Catalytic activity Bronsted relationship between dissociation constants and, 19 of hydroxyl ion and sugar anions, 24 of water molecule, 22 Catalytic coefficients for anions of weak acids, 18 evaluation of, 16 of mutarotation reactions, 47 ratios for mutarotation of sugars, 30 for weak acids, 17 Cellobiose, 373,375 Cellulose, conformation of, 272 -, 0-(carboxymethy1)-,327 with multivalent cations, 330 preparation and structure of, 271 sodium salt, 328 -, 0-(2-diethylaminoethyl)-, in ionexchange chromatography, 335 -, O-methylgelation of, 331 preparation and structure of, 271 Cellulose sulfate, 322 preparation and structure of, 271 Ceramide diglycosyl sulfates, 406 mono- and oligosaccharides, 395,400 monoglycosyl sulfates, 403 Cerebronic acid, 396,397,410 Cerebrosides, 383,395 Cerebrosulfatides, 403 Chaconine, 7 Chacotriose, 7 Chlorination, of tri-0-acetyl-D-glucal, 203 Chloro(methoxyl)ation,of glycals, 203 Cholla gum, 353 Chondroitin 6-sulfate, 291 and sulfates, in urine, 443

Chromatography gas-liquid, in mutarotation studies, 60 of polysaccharide gels and networks, 268 of polysacchaddes, 335,338 of sphingosines, 390 of urine protein-carbohydrate compounds, 441,444 vapor-phase, conformation equilibria measurement by, 58 Coenzyme BI2,degradation of, 261 Collagen, as urinary protein-carbohydrate compound source, 450 Colostrum, (N-acetylneuraminy1)lactose from cow, 8 Combretum leonense, polysaccharide from gum of, 375,377 Conduritols, conformational and configurational analysis of, 265 Configuration, of unsaturated sugars, 265 Conformation of agarose, 279 of carrageenans, 284 of cellulose, 272 equilibria of, 57 of gel-forming polysaccharides, 270 of polysaccharide gels and networks, 267-332 of unsaturated sugars, 265 Cotton effect, 134 Cram’s rule, 108, 130 Cyclitols, deoxynitro-, 100 -, nitro-, acylation of, 115 1,3-Cyclohexanediol,truns,truns-2-carbethoxy-2-nitro-, 106 -, cis, truns-2-ethyl-2-nitro-, 106 -, trans, trans-2-(hydroxymethyl)-2nitro-, 106 -, truns,truns-2-methyl-2-nitro-,106 -, truns,truns-2-nitro-, 106 -, cis,truns-2-nitro-2-phenyl-,106 Cyclohexanone, cis-2,6-bis(acetamido)-, 112 Cyclopentane-l,2,3,4-tetrol, 2,3-0-cyclohexylidene-5-nitro-, isomers, 105 Cytidine, 2,5‘-anhydro-2’,3’-0-isopropylidene-, 177,179 -, 2’-deoxy-5’-O-p-tolylsulfonyl-, displacement reaction of, 177 -, 2’,3’-O-isopropylidene-5’-O-pto1ylsulfonyl-, displacement reaction

SUBJECT INDEX, VOLUME 24

479

of, 177

of urine protein-carbohydrate compounds, 441 ment reaction of, 180 Energy of activation -, 3’-O-(methylsulfonyl)-2’,5’-di-O- of anomerization, 51 trityl-, displacement reaction of, 180 of mutarotation of D-glucose, 53 Cytolipin H, 400,401 of sugars, 55 Cytosine, N-acetyl-1-(3,5-di-O-acety1-P-~Enthalpy of activation, for mutarotation of arabinofuranosy1)-, 183 Dglucose, 53 -, 1-(2,3’-anhydro-2,5-di-O-trityl-PEntropy of activation, for mutarotation of D-xylofuranosyl)-, 180 D-glucose, 37,53 -, 1-(2,3‘-anhydro-P-~xylofurano- Enzymes, synthesis of, 6 ~ y l ) -180 , Enzymic hydrolysis, see Hydrolysis Cytosinine, 238 Epimerization, of nitro sugars, 120 Epoxides, carbohydrate, 168 D Erythrocytes, gangliosides of, 432 Erythromycin A, pyrolysis of, 250 Decoyinine, 250,252 Erythromycin B, pyrolysis of, 250 Dehydroacetylation Ethanol, nitro-, reaction with aldoses, 72 of deoxynitroalditol acetates, 127 Ethyl diazoacetate, acid-catalyzed decomby a-nitroalkenes, of P-nitroalkyl aceposition in deuterium oxide and in tates, 127 water, 29 Demissine, as beetle repellent, 6 Ethylene, 1,2-bis(penta-O-acetyI-~-gluDenitration, of nitro sugars, 109,111 conoy1)-, 264 Dermatan sulfate, 291 Evernitrose, 70,114 in urine, 443 Eyring equation, 54 Desmosine, from urine peptide, 451 Deuterium oxide, effect on mutarotation F of sugars, 28,31 Diels-Alder product, 129 Filtration, of gels, 311 Diglyme, 341 Fischer-Sowden synthesis, 69 Formamide, N,N-dimethylDinitrogen tetraoxide, reaction with glymutarotation of sugars in, 61 cals, 206 as solvent for nucleophilic displaceDisaccharides, synthesis of, 115,132 ments, 140 Displacement reactions Forosamine, 161 involving participation, 167 Fractionation, of polysaccharides, of sulfonic esters of carbohydrates,

-,

3’-O-(methylsiilfony1)-, displace-

334-337

139-197 Dissociation constants, Bronsted relationship between catalytic activity and,

19 Duanosamine, synthesis of, 210

Fructofuranose, 6-S-benzyl-2,3-0-isopropylidene-6-thio-1-0-p-tolylsulfonylD-, 146 Fructofuranoside, 2,3,6-tri-O-acety1-4azido-4-deoxy-c~-~-ga~actopyranosyl

E

3,4-di-O-acetyl-l,6-diazido-1,6-di-

Elastin, as urinary protein-carbohydrate compound source, 450 Electronic density, and mutarotation, 37 Electrophoresis of polysaccharide gels and networks,

deoxy-P-D-, 147 Fructopyranoside, methyl 1,3-O-benzylidene-4,5-di-O-(methylsulfonyl)-~-~-, displacement reactions of, 161 Fructose D, mutarotation of, 15 a-D, mutarotation of, isotope effect on,

268 of polysaccharides, 335

32

SUBJECTINDEX,VOLUME 24

480

-, -,

1-deoxy-1). 259 3-0-/3-~-galactopyranosyl-~-, 9 Fucose, Lfrom linseed mucilage, 369 from tragacanthic acid, 362 -, 4-O-(~-~-glucopyranosyluronic acid)+, 363 Furan, 2(R)-azidomethyl-2,5-dihydro-. 229 -, 2(R)-benzoyloxymethyl-2,5-dihydro-, 229

-,

Z(R)-benzyloxymethyl-2,5-dihydro-, 229 -, 2-(~-gZycero-1,2-diacetoxyethyl)-, 214 I 2-(1,2diethoxyethyl)-, 218 -, 2,5-dihydro-2(R)-formyl,diisobutyl dithioacetal, 229 -, Z-(D-glycero-l,Z-dihydroxyethyl)-, 218 2-(1-ethoxy-2-hydroxyethy1)-,218 -, tetrahydro-2-methyl-, 195 3(2H)-Furanone, 5-(~-glycero-1,2-dimethoxyethy1)-,231 K-Furcellaran, 281 ~

G Galactal, tri-0-acetyl-D addition and rearrangement reactions of, 214 halogenation of, 203 reaction with alcohols and phenols, 215 Galactan polysaccharides, see Polysaccharides Galactitol, 4-0-benzoyl-1,2:5,6-di-O-isopropylidene-3-O-methyl-~-,158 Galactocerebroside, dihydro-D-, 412 Galactocerebrosides, 395 natural, 399 Galactomannans, 339 Galactopyranose, 1,6-anhydro-3,4-0-isopropylidene-%0-(methylsulfonyl)-~D-

reactivity of, 155 solvolysis of, 197 -, 1,6anhydro-3,4-0-isopropylidene-2-O-p-tolylsulfonyl-~-~-, reactivity of, 154 -, 1,2:3,4-di-O-methylene-6-O-p-

tolylsulfonyl-D-, displacement reactions nf 144 -, 4-O-a-~-xylopyranosy1-~-, 370 Galactopyranoside, methyl 3,6-anhydro-20-benzyl-p-D-, 196 -, methyl 3,6-anhydro-2,4-di-Omethyl-&D-, 146 -, methyl 6-azido-6-deoxy-2-0-ptOlylSUlfOnyl-a-D-, 145 -, methyl 4,6-0-benzylidene-3deoxy-3-nitro-p-~,reaction with basic aluminum oxide, 113 -, methyl 4,6-0-benzylidene-2-C(dicarbethoxymethyl)-2,3-dideoxy3-nitro-D, 137 -, methyl 6-deoxy-2,3-di-O-methyl4-O-(methylsulfonyl)-~-~-, displacement reactions of, 159,190 -, methyl 4,6-diazido-2,3-di-O-benzoyl-4,6-dideoxy-~-,160, 191 -, methyl 2,3-di-O-benzoyl-4,6-di(thiocyano)-4,6-dideoxy-~-, 160 -, methyl 2,3-di-0-benzoyl-4,t-di-0p-tolylsulfonyl-a+-, displacement reactions of, 159 -, methyl 2,3-di-O-benzyl-6-deoxy4-O-(methylsulfonyl)-a-~-, displacement reacnons of, 164 -, methyl 2,3-di-O-benzyl-4,6-dideoxy-4-iodo-a-D-,displacement reactions of, 160 -, methyl 2,3-di-O-benzy1-6-0(methylsulfony1)-a-D,displacement reaction of, 174 -, methyl 2,3-di-O-benzyl-6-0(methylsulfonyl)-@-D-, solvolysis of, 196 -, methyl 4,6-dichloro-4,6-dideoxyP-D-, 2,3-bis(chlorosulfate), displacement reactions of, 162 -., methyl 2,3-di-O-methy1-4,6-di-O(methylsulfony1)-&w,displacement reactions of, 159,190 -, methyl 2,6-di-0-p-tolylsulfonyl-aD-,displacement reaction of, 145 -, methyl 2,3,4,6tetra-O-benzoyl-~-, 160 -, methyl 2,4,6-tri-O-acetyl-3-deoxy3-nitro-p-~-,dehydroacetylation of, 129

SUBJECT INDEX, VOLUME 24

-,

methyl 2,3,6-tri-O-benzoyl-4-0(methylsulfonyl)-a-D(andP-D)-,dis-

481

acid)-D, from mesquite gum, 349 3-0-/3-~galactopyranosyl-~, 355 placement reactions of, 159 -, 6-O-~-~galactopyranosyl-~-, 355 -, methyl 2,3,4-tri-O-methyl-6-O-p- -, 0-/3-D-galactopyranosyl-(h 6 ) - 0 toly lsulfonyl-a-D-, displacement reacP-D-galactopyranosyl-(1--4)-~, 376 tions of, 144 -, 0-n-galactopyranosyl-( 1+2)-0-aGalactose Dgalactopyranosyluronic acid-(1-4)D,dibenzyl dithioacetal, sulfonylation D,367 of, 175 -, 3-O-(/3-~galactopyranosyluronic effect of mutarotase on mutarotation acid)-D, 353 of, 31 -, 4-O-(a-~galactopyranosyluronic in glycoprotein of urine, 441,443 acid)-D-,366 mutarotation of, 15 -, 6-O-(~-~glucopyranosyluronic 3-sulfate, 404 acid)+-, 350,354,359,360 L-, residue in plant polysaccharides, from Acacia gums, 345,346 369 -, 4-O-(4-O-methyl-a-~glucopy6-sulfonates, displacement reactions of, ranosyluronic acid)-D, 364 from mesquite gum, 349,350 143 -, 2-acetamido-2-deoxy-~,134 -, 6-0-(4-0-methyl-~~glucopy-, 2-acetamido-2-deoxy-3-O-p-~-garanosyluronic acid)-m, 359,360 from mesquite gum, 349 lactopyranosyl-D, 425 -, 0-L-rhamnopyranosyl-(1 + 4 ) - 0 - ~ -, 0-(N-acetylneuraminic acid)glucopyranosyl-(1+6)-~-,351 (2+3)-a-D, 425 -, 0-a-L-rhamnopyranosy1-(1+2)~, 2-amino-2-deoxy-~,112,208 [O-/3-~-glucopyranosyl-( 1 + 3 ) - ~ see , derivatives, 161 in glycoprotein of urine, 441,442 Solatriose -, 6-amino-6-deoxy-~,147 -, 2,3,4,6-tetraamino-2,3,4,6-tetra-, 4-amino-4,6-dideoxy-~,160 deoxy-D, 147,160 -, 3,6-anhydro-~,321 -, 6 - t h i o - ~derivatives, , 147 -, 3,6-anhydro-(4-0-/3-~galactopy- -, 0-P-Dxylopyranosyl-( 1+3)-[ 0-pranosy1)-,dimethyl acetal, 280 Dglucopyranos yl-(1 + 2 ) 1 - 0 - g ~ -, 3,6-anhydro-4-O-(P-~galactopyglucopyranosyl-(1 + 4 ) - ~ see , Lycoranosy1)-L-,277 tetraose -, 6-deoxy-, in protein-carbohydrate Galactose-4-t, 247 Galactosyl chloride, 3,4,6-tri-O-acety1-2compounds of urine, 442,443,450 deoxy-2-nitroso-a-~-,207 ~, 3-deoxy-3,4-C-(dichloromethy1ene)-1,2:5,6-di-O-isopropylidene- Galacturonic acid, 4-04 a-Dgalactopya-D-,247 ranosyluronic acid)-D, 361,364 -, 3-deoxy-1,2:5,6-di-O-isopropyli- -, 3-O-(&~glucopyranosyluronic acid)-m, 366 dene-3,4-C-methylene-a-~,247 -, 3 - O - ~ - ~ x y l o p y r a n o s y362 l-~, -, 2,3-diamino-2,3-dideoxy-~, 135 -, 4,6-dideoxy-4-dimethylamino-~, Galacturonorhamnan, 371 Galacturonorhamnan polysaccharides, see 160 -, 4,6-dideoxy-4-methylamino-~, Polysaccharides Ganglio-N-biose-I, 425 160 -, 1,2:3,4-di-O-isopropylidene-6-0-Ganglioside GI, 423 p-tolylsulfonyl-D, displacement reac- Ganglioside GI,, 427 Ganglioside GI//,427 tion of, 143 Ganglioside GI", 427,429 -, 2,4-di-O-methyl-3-0-(2,3,4-tri-OGangliosides, 383,413-433 meth yl-~glucopyranos yluronic

-,

482

SUBJECTINDEX, VOLUME 24

brain, 8 of erythrocytes and spleen, 432 isolation and purification of, 414 oligosaccharides of, structure of, 423 from pathological tissues, 428 sialic acids of, 417 Tay-Sachs, 430 Ganglio-N-tetraose,8,423 Gelatin, junction zones in, 306 Gelation of agars, mechanism of, 321 mechanism of, 314 Gels agar, 277 alginate, 323 alginic acid, 298 biological, 314,326 birefringence of, 305 carrageenan, 281,282 filtration of, 311 hysteresis of, 306 pectate, 323 from pectic substances, 302 pectin-sugar, 324 polysaccharide, structure, conformation and mechanism in formation of, 267-332 properties of, 268 spectroscopy and optical rotation of, 309 syneresis, 303 synthetic, 313 thermodynamic properties of, 306 ultracentrifugation of, 311 x-ray diffractionby, 305 Glucal, Dacid degradation of, 218 oxidation of, 218 -, 2-acetamido-~-.225 -, 2-acetamido-3,4,6-triri-O-acetyl-~, 225 -, 2-acetoxy-3,4,6-tri-O-acetyl-~-, hydroformylation and methoxymercuration of, 220 -, 3,4-di-0-acetyl-6-0-p-tolylsulfonyl-D-, 206 -, tri-0-acetyl-Dacid degradation of, 218 halogenation of, 203 mass spectra of, 219 reaction with 2-acetamido-6-chloropurine, 218

with alcohols and phenols, 215 with sulfur-containing reagents, 209 rearrangement reactions of, 213 synthesis of, 202 3,4,6-tri-O-acetyl-2-(N-acetylacetamido)-D, 225 -,3,4,6-tri-O-acetyl-2-bromo-D-, 224 -, 3,4,6-tri-O-acetyl-2-(N.N-dimethyldithiocarbamoy1)-D-,224 -, 3,4,6-tri-O-acety1-2-thiocyanatoD-, 209,224 -, tri-0-benzoyh-, reaction with hydrogen fluoride, 216 -, 3,4,6-tri-O-benzyl-2-(benzyloxy)D-,220 Glucitol, 1,5-anhydro-4,6-0-benzylidene2-O-p-tolylsulfonyl-~-,displacement reactions of, 169 -, 5,6-anhydro-2,4-0-benzylidene-l0-p-tolylsulfonyl-D-, displacement reaction of, 174 -, 1,5-anhydro-2-S-benzyl-2-thioD.209 1,4:3,6-diaiinydr0-2,5-di-O-p-tolylsulfonyl-D-, displacement reactions of, 149 -, 1,2:5,6-di-O-isopropylidene-3-0methyl-4-O-(methylsulfonyl)-~-, displacement reactions of, 158 -, tetra-O-acetyl-1,5-anhydro-~-, 221 -, 3,4,6-tri-O-acetyl-2-S-acety1-1,5anhydro-2-thio-~-,209 Glucocerebrosides, 395 Glucofuranose, 3-O-acetyl-6-S-acetyl-l,2O-isopropylidene-6-thio-5-O-p-tolylsulfonyl-a-n-. 145 -, 3-O-acetyl-6-O-benzoyl-1.2-0isopropylidene-5-0-p-tolylsulfonylD-, 145 -, 3-O-acetyl-1,2-O-isopropylidene5,6-di-O-p-tolylsulfonyl-~-,displacement reactions of, 145 -, 3-O-acetyl-l,2-O-isopropylidene5,6-di-O-p-tolylsulfonyl-a-~-,displacement reaction of, 176 -, 3-O-acetyl-l,2-O-isopropylidene5-O-p-tolylsulfonyl-6-~-trityl-a-~, displacement reaction of, 174 -, 3,6-anhydro-l,2-O-isopropylidene-5-O-p-tolylsulfonyl-a-D-, 196 I

I

SUBJECT INDEX,VOLUME 24

483

displacement reactions of, 152 -, methyl 2-amino-4,6-O-benzyli3-O-benzyl-l,2-O-isopropylidenedene-2,3-dideoxy-3-nitro-p-~,134 5,6-di-O-p-tolylsulfonyl-a-~, solvoly- -, methyl 3-amino-3-deoxy-a-~,resis of, 196 action with nitrous acid, 193 -, 6-O-benzyl-1,2-O-isopropylidene- --, methyl 3,6-anhydro-a-~, 5-O-p-tolylsulfonyl-a-~, displaceformation from methyl 2,Sanhydro-ament reactions of, 156 Dallopyranoside, 46 -, 3-O-benzyl-l,2-O-isopropylidene- -, methyl 3,6-anhydro-2,4-di-O-p5-O-p-tolylsulfonyl-6-O-trityl-a-~, tolylsulfonyl-a-D, displacement redisplacement reaction of, 156 actions of, 153 -, 2,3-di-O-benzyl-5-0-(methylsul- ---, methyl 3-azido-4,6-0-benzylifonyl)-6-O-trityl-D, displacement dene-3-deoxy-2-O-(methylsulfonyl)reactions of, 171 a-D-,displacement reactions of: 164 -, 1,2:5,6-di-O-isopropylidene-3-0- -, methyl 2-0-benzyl-4,6-0-benzylip-tolylsulfonyl-a-D, displacement dene-3-deoxy-3-nitro-F-~-,131 reactions of, 148 -, methyl 4.6-0-benzylidene-2-, 1,2-0-isopropylidene-3,5-di-Om(and p)-carboxyphenylamino-3methyl-6-O-p-tolylsu1fonyl-a-D-,soldeoxy-3-nitro-~,136 volysis of, 195 -, methyl 4,6-0-benzylidene-2-0Glucomannans, 339 carboxyphenylamino-3-deoxy-3-niGlucopyranoctro-D-,and methyl ester, 136 a-D, mutarotation in deuterium oxide -, methyl 4,6-0-benzylidene-3and in water, 31 deoxy-2-O-ethyl-3-nitro-P-~-, 131 a-D-and p-D-,ionization constants and -, methyl 4,6-0-benzylidene-3mutarotation of, 25 deoxy-2-O-methyl-3-nitro-p-~-,131 -, 3-deoxy-3-nitro-P-~,tetraacetate, -, methyl 4,6-0-benzylidene-3115 deoxy-3-nitro-a-~(andP-D)-,reaction -, penta-0-acetyl-a, anomerization with basic aluminum oxide, 113 of, 44 -, methyl 4,6-0-benzylidene-2-C-, penta-O-acetyl-3-deoxy-3-C-(hydicarbethoxymethyl-2,3-dideoxy-3droxymethy1)-a-D, 242 nitro-D, 137 -, 1,3,4,6-tetra-O-acety1-2-deoxy-2methyl 4,6-0-benzylidene-2,3-diC-(4,6-di-O-acetyl-2,3-dideoxy-a-~ 0-p-tolylsulfonyl-a-D, displacement erythro-hex-2-enopyranosyl)-/3-~, reaction of, 169 214 -, methyl 4,6-0-benzylidene-2-O-p-, 1,2,4,6-tetra-O-benzoyl-3-O-p-tol- tolysulfonyl-a-D, displacement reacylsulfonyl-a-D (and P-D)-,displacetions of, 169 ment reactions of. 161 -, methyl S-benzyl-6-thio-a-~,258 -, tetra-0-methyl-a-D, mutarotation -, methyl 6-deoxy-6-nitro-a-~-,99 of, 15,27,28,31 -, methyl 2,3-di-O-benzoyl-4,6-di-O__, 1-thio-pa-, mutarotation of, 50 (methylsulfony1)-a-D, displacement Glucopyranoside, benzyl2-acetamido-3reactions of, 160,191 O-acetyl-2-deoxy-4,6-di-O-(methyl- -, methyl 2,3-di-O-benzyl-4,6-disulfony1)-a-D-, displacement reacdeoxy-4-iodo-a-~-,displacement retions of, 160 actions of, 160 -, benzyl3-O-acetyl-2-[(benzyl-, methyl 2,3-di-O-benzyl-4,6-di-Ooxycarbonyl)amino]-2-deoxy-4,6-di(methylsulfony1)-a-D, displacement 0-(methylsulfony1)-a-D, displacereactions of, 145 ment reactions of, 160 methyl 4,6-dichloro-4,6-dideoxy-, 2-deoxy-P-~,derivatives, 210 p-D, 2,3-bis(chlorosulfate), displace-

-,

I

3-

484

SUBJECT INDEX, VOLUME 24

ment reactions of,162 mutarotation of, 24 methyl 2,6-di-O-(methylsulfonyl)catalytic coefficients of, 16 a-D,displacement reaction of, 145 mechanism of, 41 -, methyl 2-O-(p-nitrophenyla-D effect of isotope on mutarotation of, sulfonyl)-a-D, solvolysis of, 193 -, methyl 3-O-(p-nitrophenyl32 sulfonyl)-a-D-,solvolysis of, ring conenergies, heats, and entropies of actitraction by, 193 vation of mutarotations of, 53 -, methyl 4-0-(p-nitrophenylhydrate, 60 sulfonyl)-a-m, displacement reaca-D and P-D, mutarotation of, 14 tions of, 191 _, 3-amino-2-O-[~(andL)-1-carboxy-, methyl tetra-0-benzoyl-a-m, 159 ethyl]&deoxy-D-, 132 -, methyl tetra-0-(methylsulfony1)-, 2-amino-2-deoxy-~,112,208 a-D,displacement reactions of,161 in glycoprotein of urine, 441,443 -, methyl 2,3,4-tri-O-acetyl-6-deoxy--, 2-amino-2-deoxy-6-thio-~,derivaGnitro-a-D, deacetylation of. 118 tives, 146 -., methyl 2,4,6-tri-O-acety1-3-deoxy. I 4-arnino-4,6-dideoxy-~,159 3-nitro-p-D, dehydroacetylation of, -, 2-deoxy-2-fluoro-~,206 129 -, 3-deoxy-3-nitro-~,118 -, methyl 2,4,6-tri-O-acetyl-3-O-p-, &deoxy-&nitro-D, 98,100,118 tolylsulfonyl-a-D(and&D)-,displace-, 2,3-diamino-2,3-dideoxy-~, 135 ment reaction of,162 -, 4,6-dideoxy4dimethylamino-~, -, methyl 2,3,4-tri-O-acetyl-6-0159 (3,4,6-tri-0-acetyl-2deoxy-2-ox-, 4,6-dideoxy4-methylamino-~-, imino-a-marabino-hexopyranosyl)-p159 D, 208 -, 3,5-di-O-rnethyl-s, 195 Glucopyranoside-6ylsulfonicacid, -, 2,4-di-O-a-~-rhamnopyranosyl-~, methyl &deoxy-a-m, sodium salt, 258 see Chacotriose Glucopyranosylamine, D,mutarotation -, 3-O-p-~galactopyranosyl-~, 9 of, 48 -, 0-/3-mgalactopyranosyl-(1+3)-0Glucopyranosyl bromide, tetra-o-acetyl(2-acetamido-2-deoxy-/3-~galactoa-D,anomerization of, 45 pyranosy1)-(1+4) -0-B- D-galactopy-, 2,4,6-tri-O-acety1-3-deoxy-3-nitroranosyl-(1+4)-~,8 a - ~115 , -, 0-&Dgalactopyranosyl-( 1+3)-0Glucopyranosyl chloride, 3,4,6-tri-O-ace2-acetamido-2-deoxy-/3-~glucopytyl-2-deoxy-%nitroso-a-~, 207 ranosyi-( I-, 3)-O-p-D-galactopyranoGlucopyranosyl fluoride, tetra-0-acetyl-psyl-(1+4)-a-D-, 7 D,45 -, inosito~-D-g~ucuronic acid-2Glucopyranosyl isothiocyanate, 3,4,6-triamino-2deoxy-~,410 O-acetyl-2-deoxy-2-thiocyanato-a-~, -, 4-O-a-~mannopyranosyl-~, 357 209 -, 4-O-~-rhamnopyranosyl-~, 351 Glucose -, 6-thio-D, derivatives, 146 D -, 6-O-a-~xylopyranosyl-~-, 373 acid and base catalysts in mutarotaGlucose-1-d, D,effect of mutarotase on tion of, 23 mutarotation of, 32 catalytic constants for mutarotation by Glucuronic acid, residues in Acacia gums, weak acids and anions, 19 345 effect of histidine and histidylhis-, 4-0-methyl-D, in mesquite gum, tidine on mutarotation of, 32 349 mutarotase on mutarotation of, 31 Glucuronomannan polysaccharides, see ,

SUBJECT INDEX, VOLUME 24 Polysaccharides Glucuronomannans, 357 Glycals acid degradation of, 218 allylic rearrangements of, 213 dinitrogen tetraoxide addition to, 206 halogen and hydrogen halide addition to, 202 hydroformylation of, 210 methoxymercuration of, 210 nitrosyl chloride addition to, 206 nucleosides from, 217 preparation from ketoses, 254 reaction with alcohols and phenols, 215 synthesis of, 200 Glycals, 2-hydroxy-,219-223 addition reactions, 220 rearrangement reactions of, 221 synthesis of, 219 Glycopeptides, 314 peptide composition of, 445 Glycoproteins, in urine (human), 436,440 Glycopyranosides, indolyl, acid-catalyzed decomposition in deuterium oxide and in water, 29 Glycosides methyl, acid-catalyzed anomerization of, 43 nitro, acylation of, 115 oximino, 208 2,3-unsaturated, 213 Glycosphingolipids, 381-433 biosynthesis of, 394 nomenclature of, 383 sulfate-containing, 403 Glycosylamines, mutarotation of, 47 Glycuronans, 296,323 -, (aminodeoxyg1yco)-,acidic, in human urine, 436 Grignard reagents, in unsaturated carbohydrates preparation, 260,262 Guanosine, 5',8-anhydro-2',3'-O-isopropylidene-&memapto-, 188 ___,8-bromo-2',3'-O-isopropylidene5'-O-(methylsulfonyl)-, displacement reaction of, 188 &hydroxy-2',3'-O-isopropylidene5'-O-(methylsulfonyl)-, displacement reaction of, 188 -, 2',3'-O-isopropylidene-,p-tolu-

3-

485

enesulfonylation of, 186 Gulal, 4,6-O-benzylidene-~-,201 -, tri-0-acetyl-D-, 202 -, 3,4,6-t~i-O-acetyl-~, 214 Gulitol, 2-acetamido-1,2-dideoxy-l-nitroD-,133 -, 2,6-anhydro-l-deoxy-l-nitro-~, 119 Gulose, D, synthesis of, 247 -, 2-amino-2-deoxy-~-,112 -, 1,2:5,6-di-O-isopropylidene-a-~-, 247 Gum arabic, 340,341 structure of, 343,346 Gums, 333-379 Acacia, 343-3453 Acacia arabica, 342, 345, 346 Acacia drepanolobiurn, 345, 348 Acacia elata, 348 Acacia karroo, 346 Acacia laeta, 348 Acacia mearnsii, 344,345 Acacia nilotica, 344 Acacia nubica, 345,346 Acacia podalyriaefolia, 348 Acacia pycnantha, 344,345 Acacia senegal, 344,345 Acacia seyal, 348 Anogeissus latifolia (ghatti), 354,376 Anogeissus leiocarpus, 336,340,356 apricot-tree, 359 Araucaria bidwillii, 339,340,351 blackthorn-tree, 359 cherry, 354,359 cholla, 353 Citrus limonia (lemon), 342,351 Cochlospermum gossypium, 365 Combretum leonense, 336 polysaccharide from, 375,377 damson, 354 Encephalartos longifolius, 359 golden-apple, 351 jeol, 353 Khaya, 336,351,361,363-365 Kutira, 365 leiocaipan A, 356 lemon, 342,351 mesquite, 341,349 Odina wodier, Roxb. (ieol), 353 Opuntia fulgida (cholla), 353

486

SUBJECT INDEX, VOLUME 24

Prunus, 360 sapote, 371 Sterculia, 361,365-369 Sterculia urens, 340 oligosaccharides from, 368 Virgilia oroboides, 360 Gum tragacanth, 336,353,361

Heptulopyranose, 2,7-anhydro-, formation by mutarotation, 46

-,

2,7-anhydro-4-deoxy-4-nitro-P-~allo-, 95 ~, 2,7-anhydro-4-deoxy-4-nitro-P-~altro-, 95 -, 2,7-anhydro-4-deoxy-4-nitro-P-~gulo-, 95

Heptulopyranoside, a-D-glucopyranosyl 4-amino-4-deoxy-/3-D-gluco-,97 -, methyl 4-deoxy-4-nitro-a-D-gluco-, H 97 Hept-6-ynofuranose, 6,7-dideoxy-1,2-0Halogenation, of glycals, 202 isoprop ylidene-cY-D-ghco-, 260 Halo(methoxyl)ation, of glycals, 203 -, 6,7-dideoxy-1,2-0-isopropyliHeat of activation, of mutarotation of D dene-p-L-ido-, 260 glucose, 53 Heteropolymolecular, defined, 337 Henry reaction, 121 Hexanal, 2,6-diethoxy-4-0~0-,diethyl acefor deoxynitroinositols, 100 tal, 218 for nitro-sugar synthesis, 68,70 Hex-2-enamide, N,N-dimethyl-2,4,6-triHeparin, 291 O-benzyl-3-deoxy-~threo-, 263 in urine, 443 Hex-1-enitol, 1,2-dideoxy-3,4:5,6-di-OHeparitin isopropylidene-D-arabino-, 261 peptide compound, in urine, 444 Hex-3-enitol, 1-0-benzoyl-4-deoxysulfate, 291 2,3:5,6-di-O-isopropylidene-~-threo-, Heptitol, D-glycero-D-gub, 68 261 -, 2,6-anhydro-~glycero-~-gulo-, -, 6-O-benzoyl-3-deoxy-1,2:4,5-di-O220 isopropylidene-D-threo-, 158 -, 2,6-anhydro-~-glycero-~-manno-, -, 3,4-dideoxy-1,2:5,6-di-O-isopro220 pylidene-trans-D-erythro-, 230 -, 2,6-anhydro-3-deoxy-~-galacto-, -, 3,4-dideoxy-1,2:5,6-di-O-isopro210 pylidene-trans-Dthreo-, 230, 261 -, 2,6-anhydro-3-deoxy-~-talo-, 210 -, 2,6-anhydro-l-deoxy-I-nitro-~- Hex-4-enitol,1,5-anhydro-2,3,4,6-tetra-Obenzoyl-L-threo-, 254 glycero-L-manno-, 120 1,5-anhydro-2,3,6-tri-O-benzoyl~Heptofuranose, 1,5-anhydro-2,3:6,7-di-O- -, deoxy-L-erythro-, 254 isopropylidene-P-D-glycero-D-aIlo-, -, 2,3,6-tri-O-acetyl-1,5-anhydro-4171 deoxy-L-threo-, 254 -, 2,3:6,7-di-O-isopropylidene-5-0p-tolylsulfonyl-P-D-glycero-D-gulo-, Hex-5-enitol, 2,5-anhydro-l-S-benzyl-6displacement reactions of, 171 deoxy-3,4-di-O-(p-nitrobenzoyl)-1Heptofuranoside, methyl 2,3:6,7-di-0thio-Dxylo-, 252 -, 2,3,4-tri-O-acetyl-1,5-anhydro-6isopropylidene-P-D-gl ycero-L-talo-, 171 deoxy-D-xylo-,259 Hex-2-enofuranose, 3-deoxy-2-0-methylHeptose, 3-acetamido-3-deoxy-, 112 P-D-erythro-, 238 2-deoxy-~-galacto-,127,264 ~, 3-deoxy-2,5,6-tri-o-methyl-~-ery-, 2-deoxy-D-manno, 264 -, 2,6-dideoxy-~-manno-,264 thro-, 231 Heptoseptanoside, methyl 5,7-O-benzyliHex-3-enofuranose, 3-deoxy-1,2:5,6-di-Odene-3-deoxy-2,4-di-O-methyl-3-niisopropylidene-a-D-erythro-,246 tro-, 133 Hex-5-enofuranose, 3-0-acetyl-5,6-di-

I

SUBJECT INDEX, VOLUME 24

487

deoxy-1,2-0-isopropylidene-6-C-nidene-2,3-dideoxy-a-~-erythro-, 244 tro-~l-D~ylo-, 256 -, methyl 4,6-0-benzylidene-3-cy-, 3-0-benzyl-5,6-dideoxy-1,2-0ano-2,3-dideoxy-a-~erythro-,246 isopropylidene-a-Drylo-, 255 -, methyl 4,6-0-benzylidene-3-, 5,6-dideoxy-l,2-O-isopropylideoxy-2-S-methyl-2-thio-a-~erydene-a-mxylo-, 255 thro-, 244 -, 5,6-dideoxy-1,2-0-isopropyli-, methyl 4,6-0-benzylidene-3dene-3-O-methyl~a-~xylo-, 255 deoxy-2-S-phenyl-2-thio-a-~eryHex-2-enono-l,5-lactone, 4,6-O-benzylithro-, 244 dene-2,3-dideoxy-~erythro-,113 ~, methyl 4,6-0-benzylidene-2,3-, 4,6-0-benzylidene-2,3-dideoxy-~- dideoxy-a-D(andP-D)-erythro-,232, threo-, 114 244,245,249 Hex-2-enopyranose,2-acetamido-2,3-di-, methyl 4,6-0-benzylidene-2,3deoxy-Dthreo-, 244 dideoxy-a-D(andp-D)-threo-,232 -, 4,6-di-O-acetyI-2,3-dideoxy-~-e y- -, methyl 4,6-0-benzylidene-2,3thro-,216 dideoxy-3-C-nitro-a-~erythro-, 243 -, 4,6-di-O-benzoy1-2,3-dideoxy-~- -, methyl 4,6-0-benzylidene-2,3erythro-, 216 dideoxy-3-C-nitro-p-~-erythro-,242 -, 1,2,4,6-tetra-O-acety1-3-deoxy-~-, methyl 4,6-O-benzylidene-2,3erythro-, conformations of anomers, dideoxy-3-C-nitro-a-~-threo-, 243 241 -, methyl 4,6-0-benzylidene-2,3-, 1,2,4,6-tetra-O-acetyyl-3-deoxy-j3dideoxy-3-phenylazo-cu-~erythro-, Derythro-, 222 243 -, 1,2,4,6-tetra-O-acetyl-3-deoxy-a- ~, methyl 4,6-0-benzylidene-2,3D-threo-,223 dideoxy-2-pyrrolidinyl-a-D-erythro-, -, 1,4,6-tri-O-acetyl-2-(N-acetylac244 etarnido)-2,3-dideoxy-mthreo-,243 -, methyl 4,6-di-O-acetyl-2,3-di-, 1,4,6-tri-O-acetyl-2,3-dideoxy-~deoxy-cu-D(andP-D)-erythro-,232 erythro-, 213 -, methyl 4,6-di-O-acety1-2,3-di-, 1,4,6-tri-O-acetyl-2,3-dideoxy-a-~ deoxy-a-mthreo-, 215 threo-, 214 Hex-3-enopyranoside, methyl 4,6-0-ben-, 2,4,6-tri-O-benzoy1-3-deoxy-l-Ozylidene-2-bromo-2,3-dideoxy-a-~(trichloroacety1)-a-merythro-,223 threo-, 235,249 ~, 2,4,6-tri-O-benzy1-3-deoxy-~- -, methyl 4,6-0-benzylidene-2,3threo-, 240 dideoxy-a-mglycero-,236,249 Hex-2-enopyranoside,cholesteryl4,6-di-, methyl 3,4,6-trideoxy-, derivaO-acetyl-2,3-dideoxy-a-~erythro-, tives, 250 215 Hex-4-enopyranoside, methyl 2,3-di-0-, 6-deoxy-1,2:3,4-di-O-isopropylibenzyl-4,6-dideoxy-a-~-threo-, 253 dene-a-~galactopyranos-6-yl4,6-di--, methyl 4,6-dideoxy-2,3-0-isoproO-acetyl-2,3-dideoxy-a-merythro-, pylidene-P-L-erythro-,254 215 Hex-3-enopyranosidulose,methyl 3,4-, 4,6-di-O-acetyl-2,3-dideoxy-~-ery- dideoxy-6-O-methyl-a-~-(and P-D)thro-hex-2-enopyranosyl4,6-di-Oglycero-, 239 acetyl-2,3-dideoxy-~-erythro-,216 Hex-4-enopyranosiduronicacid, methyl -, ethyl 4,6-di-O-acety1-2,3-dideoxy4-deoxy-P-~-threo-,methyl ester, 252, a-merythro-, 215 255 -, methyl 4-0-acetyl-2,3,6-trideoxy- Hex-2-enopyranosylamine,N,N-diethyl3-C-methyl-~-threo-,246 2,4,6-tri-O-acetyl-3-deoxy-~ery thro-, methyl 2-azido-4,6-0-benzyli222 __l

488

SUBJECT INDEX, VOLUME 24

ment reactions of, 163 Hex-2-enopyranosylchloride, 2,4,6-tri-Oacetyl-3-deoxy-a-~erythro-, 240 -, ethyl 2,3,6-trideoxy-4-O-sulfonyla-Dthreo-, displacement reactions of, Hex-2-enopyranosylfluoride, 4,6-di-0acetyl-2,3-dideoxy-~erythro-,216 163 -, 4,6-di-O-benzoyl-2,3-dideoxy-~ -, methyl 4,6-0-benzylidene-2erythro-, 216 deoxy-3-O-(methylsulfonyl)-a-~Hex-2-enopyranuronic acid, 3-0-methylribo-, displacement reactions of, 163 D-erythro-,methyl ester, 241 -, methyl 4,6-0-benzylidene-3deoxy-2-O-p-tolylsulfonyl-a-~ribo-, Hex-2-enose,3-deoxy-2,4,5,6-tetra-Omethyl-Derythro-, 263 displacement reactions of, 163 -, 3-deoxy-2,4,5-tri-O-methyl-~-ery-, methyl 2,6-dideoxy-a-~-arabino-, thro-, cis- and trans-, 263 210 -, 1,2,4,6-tetra-O-acetyl-3-deoxy-a- Hexopyranosyl bromide, 3,4,6-tri-O-acetyl-2-deoxy-~arabino-,206 aerythro-, 240 Hex-3-enose,3-0-acetyl-1,2:5,6-di-O-iso-Hexopyranosyl ethylxanthate, 3,4,6-tri-0acetyl-2-deoxy-/3-~-arabino-, 206 propylidene-a-D-erythro-,247 Hex-5-enose, 5-deoxy-l,2-O-isopropyli- Hexopyranosyl fluoride, 3,6-di-O-benzoyl-2-deoxy-a-~ribo-,217 dene-6-O-trityl-a-D-%yZo-, 256 Hexose, 1,6-anhydrd-O-benzy1-3Hex-2-enoside, methyl 2,3-dideoxy-4,6bromo-2,3-dideoxy-~arabino-,236 di-O-(methylsulfonyl)-a-D-ery thro-, displacement reactions of, 164 -, 2-deoxy-~-arabino-, 218 -, methyl 2,3-dideoxy-4,6-di-O2-deoxy-~ribo-,127,264 (methylSUlfOnyl)-cY-D-threO-, displace- -, 2-deoxy-D-xylo-, 127 -, 5-deoxy-1,2-O-isopropylidene-ament reactions of, 164 Hex-3-enosulose, 3,4-dideoxy-5,6-di-ODXYZO-, 256 methyl-D-glycero-,263 -, 4,5-di-O-acetyl-2,6-anhydro-3Hex-5-enulofuranose,6-deoxy-2,3-0-isodeoxy-Dlyxo-, 211 propylidene-/%Dthreo-, 251 -, 4,5-di-O-acetyl-2,6-anhydro-3Hex-4-enulopyranoside, methyl 1,3-0deoxy-wxylo-, 211 benzylidene-4,5-dideoxy-a-~-, 4,6-di-O-acetyl-3-bromo-2,3-diglycero-, 249 deoxy-a-D-arabino-,206 Hex&enulose, 3,4-dideoxy-~-glycero-, -, 2,4-dideoxy-~threo-,212 230,261 -, 1,3,4,6-tetra-O-acety1-2-deoxy-aHexitol, 1,5-anhydr0-2-deoxy-1,2-CDZYXO-, 214 (dichloromethylene)-3,4,6-tri-O-, 1,3,4,6-tetra-O-acetyl-2-deoxy-2methyl-Dglycero-Dido-, 212 thiocyanato-a-D(andp-D)-gluco-,209 -, 4,6-di-O-acetyl-l,S-anhydr0-2,3- -, 1,3,4,6-tetra-O-acety1-2-deoxy-2dideoxy-werythro-, 213,221 thiocyanato-a-D-manno-,209 -, 3,4,6-tri-O-acetyl-1,5-anhydro-2- -, 2,3,4,6-tetradeoxy-4-dimethyldeoxy-aarabino-, 221 amino-Derythro-, 161 Hexofuranose, 5,6-dideoxy-l,2-O-isopro- -, 3,4,6-tri-O-acetyl-2-deoxy-a-~ pylidene-6-nitro-a-~xylo-,99 arabino-, 206 2,3,6-trideoxy-3-C-rnethyl-4-0Hexononitrile, 3,4,5,6-tetra-O-acety1-2- -, methyl-3-nitro-~-ribo(orL-arabino)-, deoxy-2-(phenylimino)-~arabino-, 70 114 Hexopyranose, l,g-anhydro-, formation by Hexoses mutarotation, 46 aminodeoxy-, in urine (human), 438, -, tetra-O-acetyl-2-deoxy-a-~-arab439 ino-, anomerization of, 43 2-amino-2-deoxy-~,preparation of, 9 Hexopyranoside, ethyl 2,3,6-trideoxy-46-deoxy-. in urine (human), 438,439 0-sulfonyl-a-Derythro-, displacein urine (human), 438,439 -2

SUBJECT INDEX, VOLUME 24

489

Hexosid-5-ulose,methyl 6-deoxy-2,3-0Idofuranose, 1,5-anhydro-2,3-di-O-benisopropylidene-a-DZyxo-,258 zyl-6-O-trityl-a-~-,171 Hexos-5-ulose,&deoxy-Dxylo-, 256 -, 3,5-anhydro-l,2-O-isopropyliHexulofuranose, 4,6-anhydro-2,3-0-isodene-6-O-trityl-P-~-,174 propylidene-a-L-rylo-, 172 -, 3-0-benzyl-5-deoxy-5-hydrazino-, 2,3-O-isopropylidene-1,4,6-tri-O1,2-O-isopropylidene-P-~-, 156 (methylsulfony1)-a-L-rylo-,displaceIdopyranose, a-D,pentaacetate, conforment reaction of, 173 mation of, 58 Hexulopyranoside, benzyl4-deoxy-4-ni-, 1,4-anhydro-2,3-di-O-benzyl-6-0tro-a-L-xylo-, 97 trityl-P-L-, 171 Hexuronic acid Idopyranoside, methyl 4,6-O-benzyliresidues, reduction to hexose residues, dene-%deoxy-2-iodo-a-~,201 341 Idose, 2-amino-2-deoxy-~-,112 in urine (human),438 -, 6-deoxy-6-nitro-~-,98, 100, 118 3,6-diamino-3,6-dideoxy-~, 147 Hex-1-ynitol, 1,2-dideoxy-~-arabino-, 224 -, -, 2,3,4,6-tetraamino-2,3,4,6-tetraHistidine, catalytic action on mutarotadeoxy-D, 147 tion, 32 -, histidyl-, catalytic action on muta- Indolyl glycopyranosides, acid-catalyzed decomposition in deuterium oxide rotation, 32 and in water, 29 Homogeneous, defined, 337 Hyaluronic acid, 291 Inosadiamines, di-N-acetyl-tetra-0-acein urine, 443 tyl-, 102 Hydroformylation Inosine, 2',3'-O-isopropylidene-,p-tolof 2-acetoxy-3,4,6-tri-O-acetyl-~g1ucal, uenesulfonylation of, 185 220 -, 2',3'-isopropylidene-5'-O-p-tolylof glycals, 210 sulfonyl-, 185 Hydrogenation, of nitro sugars, 109 Inositol, deoxynitro-, acetalation of, 116 Hydrogen fluoride, reaction with tri-0-, muco-3-deoxynitro-,100 acetyl-Dglucal, 216 -, DL-myo-l-deoxy-3-nitro, 100 Hydrogen ions, catalysis of mutarotation -, scyllo-deoxy-1-nitro-,100 by, 17 -, 1,4-dideoxy-1,4-dinitro-neo-, 104 Hydrogenolysis, of tri-0-acetyl-Dglucal, -, 1,2:3,4-di-O-isopropylidene-epi-, 213 170 Hydrolysis -, 2-O-a-~-mannopyranosy1-6-0-[(2enzymic, of polysaccharides, 340 amino-2-deoxy-a-~glucopyranosyl)of nitro sugars, 118 (1+4)-a-~-glucopyranosyluronic of polysaccharides, 337 acid]-, 411 of 5-thioxylopyranosides, 50 Inosit01-2-'~C,myo-, 100 Hydronium ion, in mutarotation, 41,43 Iodo(methoxyl)ation, of glycals, 203 Hydroxyl ion, catalytic activity of, 24 Ionization constants, of a-and fi-DglucoHygromycin A, 258 pyranose, 25 Hysteresis, 316 Isochromene, 3,5-bis(acetoxymethy1)-7of agar gels, 277 nitro-, 129 of gels, 306 Isodesmosine, from urine peptide, 451 Isotopes, effect on mutarotation reactions, 28 I determination of mechanism of reaction, 32 Iditol, 2,3:4,5dianhydro-~.168 -, 1,4:3,6-dianhydro-2,5-di-O-p-t01J ylsulfonyl-L-, displacement reactions Jeol gum, 353 of, 149

490

SUBJECT INDEX, VOLUME 24

K

-,

2-acetamido-l,2-dideoxy-l-nitroD-, 133 Karplus relationship, 265 -, 2-acetamido-1 ,2-dideoxy-l-nitroKerasin, 395,399 L-, 265 sulfate, 405 -, 2-acetamido-3,4,6-tri-O-acetyl-1,5synthesis of, 401 anhydro-2-deoxy-~,225 Ketoses, synthesis of, 111 -, 2,5-anhydro-~,193 -, 1-deoxy-, 258 -, 1,5anhydro-2-S-benzyl-2-thioKnoevenagel reaction, 262 D-, 209 Kuhn, Richard, obituary, 1 -, 2,5-anhydro-l-deoxy-l,l-difluoroD-, 205 -, 2,6-anhydro-l-deoxy-l-nitro-~, L 119 -, 3-0-benzoyl-1,2:5,6-di-O-isoproLacto-neotetraose, 433 pylidene-4-O-(methylsulfonyl)-~, Lactose displacement reactions of, 157 mutarotation of, 24 -, l-deoxy-2-O-methyl-l-nitro-~-, thermodynamics of anomerization of a130 and @-, 51 -, 1-deoxy-1-nitro-, 69 -, 2-acetamido-2-deoxy-, 8 -, 1,4:3,6-dianhydro-2,5-di-O-(meth0-(N-acetylneuraminic acid)-(2+ ylsulfony1)-D, displacement reac3)-~-,425 tions of, 150 -, 3’-O-(N-acetylneuraminyl)-, 8 -, 1,4:3,6-dianhydro-2,5-di-O-p-tolylLacto-N-tetraose, 7 sulfonyl-D, displacement reactions Lactulose, synthesis of, 6 of, 149 Leptine, as beetle repellent, 6 -, 1,2:4,5-di-O-isopropylidene-3,6Lignoceric acid, 410 di-0-(methylsulfony1)-D-,displaceLycotetraose, 7 ment reactions of, 158 Lysine, hydroxy-L-, in glycopeptide of -, tetra-O-acetyl-1,5-anhydro-~-, 221 urine, 450 -, 3,4,6-tri-O-acetyl-2-S-acety1-1,5Lysozyme, 314 anhydro-2-thio-~,209 Lyxitol, 2,3,4-tri-O-benzyl-1,5-di-O-p-t01-Mannofuranoside, methyl 6-deoxy-2,3-0ylsulfonyl-~-,solvolysis of, 196 isopropylidene-5-0-p-tolylsulfonylLyxofuranose, 2,3,5-tri-O-benzyl-~-,194 a-L-,displacement reaction of, 156 Mannopyranose, 1,6-anhydro-2,3-0-isoLyxofuranoside, methyl 3-deoxy-3-Cpropylidene-4-O-p-tolylsulfonyl-@-D-, formyl-am-, hemiacetal, 193 Lyxopyranoside, methyl 2,3-O-isoproreactivity of, 154 pylidene-4-O-p-tolylsulfonyl-a-~- Mannopyranoside, methyl 2-amino-4,6-0(or a - ~ ) -displacement , reactions of, benzylidene-2,3-dideoxy-3-nitro-@191 D-, 134 -, methyl 3-amino-3-deoxy-a-~-, reLyxose, wan hydro-^, diisobutyl dithioacetal, 175 action with nitrous acid, 193 methyl 4,6-0-benzylidene-2-(0-, 1-0-benzoyl-2,3,5-tri-O-benzyl-4carboxyphenylamino)-2,3-dideoxyO-methyl-L-, methyl hemiacetal, 194 3-nitro-D-, and methyl ester, 136 -, methyl 6-deoxy-2,3-0-isopropylidene-4-O-(methylsulfonyl)-a-D, disM placement reactions of, 189,191 solvolysis of, 194 Mannitol, 2-acetamido-1,5-anhydro-2methyl 6-deoxy-2,3-0deoxy-D, 225 isopropylidene-4-0-p-tolylsulI

I

I

SUBJECT INDEX, VOLUME 24

491

fonyl-L-, displacement reactions of, Molecular weight, of urine constituents, 189,191 438 Monosaccharides, ceramide, 395,401 -, methyl 2,3-diamino-2,3-diMorgan-Elson color reaction, 8 deoxy-P-D-, 136 Mountain-pine pollen, polysaccharides -, methyl 3-O-(p-nitrophenylsulof, 363 fonyl)-a-D-, solvolysis and ring Mucilages, 333-379 contraction of, 193 -, methyl 2,4,6-tri-O-acetyl-3-deoxy- cress-seed, 336,369 gel structure of, 326 3-nitro-P-~-,dehydroacetylation of, linseed (flax seed), 336 129 polysaccharides from, 369 -, methyl 2,3,6-tri-O-benzoy1-4-0mustard-seed, 336 (methylsulfonyl)-a-n-, displaceslippery-elm, 371 ment reaction of, 161 Mucopeptides, 314 Mannopyranosyl fluoride, 3,4,6-tri-OMuramic acid, 314 acetyl-2-bromo-2-deoxy-a-~-,204 -, 3,4,6-tri-O-acetyl-2-deoxy-2-iodo- Murein, 314 Mustard, polysaccharide from seed of, 374 (Y-D, 205 Mutarotase, effect on mutarotation of sugMannose, D-, in glycoprotein of urine, ars, 31,63,64 441,443 -, 2-amino-3,6-anhydro-2-deoxy-~, Mutarotation catalysis by acids and bases, 14 derivatives, 176 catalytic coefficient ratios for sugars, 30 -, 2-amino-2-deoxy-~-,112,208 determination of mechanism of reaction -, 3-O-~-arabinopyranosyl-D,355 by use of isotope effect, 32 -, 2,3-diamino-2,3-dideoxy-~-,135, electronic density and, 37 136 entropy of activation in water-catalyzed, -, 2,6-diamino-2,6-dideoxy-~-, 147 37 -, 2-O-P-D-glUCOpyranOSyl-D-, 357 equilibria and thermodynamics of, 51 -, 2-O-(P-D-g~ucOpyranosy~uronic of glycoside nitronates, 121 acid)-D, 354,359,360 of glycosylamines, 47 -, O-P-D-glUCOpyranOSylU~OniC acidisotope effect on, 28 ( 1+2)-O-D-mannopyranosyl-(1+4)of, 35,38,40-42 mechanism O-~-D-g~ucopyranosy~uronic acid-( 1 determination by use of isotope ef+ 2)-D-, 355 fect, 32 Mass spectra, of tri-0-acetyl-Dglucal and mutarotase effect on, of sugars, 31,63, other unsaturated sugars, 219 64 Mass spectrometry ring contraction and expansion by, 46 of polysaccharides, 338 of sugars of sphingosines, 390 in aqueous solution, mechanism of, Mesquite gum, 341,349 35 Methane, nitro-, in nitro-sugar synthesis, in N,N-dimethylformamide, 61 68,70 in pyridine, 61 Methanolysis, of nitro sugars, 118 in solution, 13-65 Methoxymercuration, of glycals, 210,220 Mycoglycosphingolipids, 408 Methylation, of sugars, 7 Methylsulfonyloxy group, reactivity of, 139 N Methyl sulfoxide, as solvent in displacement reactions of carbohydrate sulNasturtium, polysaccharide from seed of, fonic esters, 141 374 Michael reaction, 137 Nef reaction, 69,72,111,127,134 Milk, human, 7 Nervon, 398,399

492

SUBJECT INDEX, VOLUME 24

Neuraminic acid acylated, in gangliosides, 413,415 derivatives and, 417 -, N-acetyl-, 8,417,419 -, N-glycoloyl-,418,420 Neuraminic lactone, N-acetyl-, 419 -, N-(benzyloxycarbony1)-,420 Neuraminidase, 418 Nitriles, amino, hydrogenation of, 9 Nitro group reactions that proceed with retention of, in nitro sugars, 115 in sugars, reactions which alter or remove, 109 Nitrobenzenesulfonate, in displacement reactions of carbohydrates, 140 Nitrosyl chloride, reaction with glycals, 206 Nitryl iodide, nitro sugar preparation with, 94,99 Nomenclature of glycosphingolipids, 383 of nitro sugars, 67 of nucleosides, 228 of protein-carbohydrate compounds in urine, 435,437 of unsaturated sugars, 200 Nonulopyranosonic acid, 5-amino-3,5dideoxy-Dglycero-aa-gulucto-, see Neuraminic acid Nuclear magnetic resonance, of unsaturated sugars, 265 Nuclear magnetic resonance spectros copy, conformation determination by, 57 Nucleosides acylation of nitro-, 115 anhydro-, 177-188 anomerization of, 47 from glycals, 217 purine anhydro-, 185 pyrimidine, 176 pyrimidine anhydro-, 176,177 unsaturated, 226,227 synthesis of, 250 -, 2'-deoxy-, 212 -, 3'-deoxy-3'-nitro-, hydrogenation of, 110 -, 2',3'-dideoxy-, 226 Nucleotides, 2'-deoxy-, 226

0 Obituary, Richard Kuhn, 1 Octadecane-l,3,4-triol, 2-amino-, see Phytosphingosine 4-Octadecene-1,3-dioI,2-amino-, see Sphingosine Oct-6-enose, cis( and truns)-6,7,8-trideoxy1,2:3,4-di-O-isopropylidene-7-nitrool-D-gahCtO-, 112,127 Oct-5-enos-7-ulose,6,8-dideoxy-1,2:3,4di-0-isopropylidene-a-agalacto-, cis-and cis-transisomers, 112 Octopyranose, 6-0-acetyl-7,8-dideoxy1,2:3,4-di-O-isopropylidene-7-nitro-, 99 Oct-7-ynopyranose,7,8-dideoxy-1,2:3,4di-0-isopropylidene-D-glycero(and Lglycero)-ol-Dgalacto-,260

Oligosaccharides from Acacia gums, 344 ceramide, 395,400 from gum ghatti, 354 from gums of galactan group, 352 nitrogen-containing, in human milk, 7 peptido-, peptide composition of urinary, 445 from Stercculia wens gum, 368 structure of, of gangliosides, 423 unsaturated, 253 in urine, 436 Optical rotation, of gels, 309 Oxetans, carbohydrate, 172 0 x 0 reaction, with glycals, 210 Oxygen, isotope'"0, effect on anomerization of sugars, 44 Oxynervon, 398,399

P Palmitaldehyde, as precursor in biosynthesis of sphingosines, 394 Pectic acid, 299 Pectic substances, 298 gels, biological, 326 Pectin, 299,301 1,4-Pentanediol, 4-O-benzyl-l-O-ptolylsulfonyl-, solvolysis of, 195 Pent-3-enodialdo-l,4-furanose, 3-deoxy1,2-O-isopropylidene-~-~-glycero-,

248

SUBJECT INDEX, VOLUME 24

493

Pent-1-enofuranose, 2,3,5-tri-O-benzoylPhosphine, bis(5,6-dideoxy-l,2-O-isopro1-deoxy-D-erythro-,220 pylidene-a-~-xylo-hexofuranose-6-, 2,3,5-tri-O-benzoyl-l-deoxy-~yl)-, 256 threo-, 220 -, (5,6-dideoxy-1,2-0-isopropyliPent-4-enofuranose, 3-0-acetyl-5-deoxydene-a-~-xylo-hexofuranose-6-yl)-, 1,2-O-isopropylidene-P-~-threo-, 250 256 -, 5-deoxy-1,2-O-isopropylidene-3- -, phenyl(5,6-dideoxy-1,2-0-isopro0-methyl-p-L-threo-, 251 pylidene-a-~-xylo-hexofuranose-6Pent-2-enofuranoside, methyl 5-0-benyl)-, 256 zoy~-2,3-dideoxy-P-D-glycero-, 229 Phosphines, addition to unsaturated carPent-2-enopyranose, 3,4-dichloro-2,3,4bohydrates, 256 trideoxy-P-D-glycero-, 246 Phosphoric triamide, hexamethyl-, as sol. -, 1,2,4-tri-O-acetyl-3-deoxy-~-glyvent in displacement reactions, 141 cero-, 241 Photolysis, of nitro-olefinic sugar, 112 Pent-2-enopyranoside, 3,4-dichloro-2,3,4Phrenosin, 395,399 trideoxy-P-~-glycero-pent-2-enopysynthesis of, 401,403 ranosyl3,4-dichloro-2,3,4-trideoxy-P- Phrenosinic acid, 396 D-glycero-, 245 Phytoceramides, 409 -, methyl 3,4-dichloro-2,3,4-triPhytoglucolipids, 409 deoxy-cY-D(and P-D)-glycero-,245 Phytoglycosphingolipids, 408 Pent-3-enopyranoside, methyl 2-0-benPhytosphingolipids, 382 Phytosphingosine, anhydro-, 387 zyl-3,4-dideoxy-P-~-glycero-,250 ~, dehydro-, stereochemistry of, 386 Pent-4-enose, 4,5-dideoxy-~-threo-, 261 Pent-2-enoside, methyl 4-0-benzyl-2,3~, N-2-hydroxytetracosanoyl-,1phosphate, 409 dideoxy-P-L-glycero-, 232,250 Phytosphingosines, 390 Pent-3-enoside, methyl 2-0-benzyl-3,4derivatives, 257 dideoxy-P-L-glycero-. 232 stereochemistry of, 384,386 Pent-4-enoside, methyl 5-deoxy-2,3-0synthesis of, 388 isopropylidene-P-D-erythro-,251 of CZO-, 389 Pentodialdo-1,4-furanose,3-deoxy-1,2-0Picea engelmann, polysaccharide from isopropylidene-a-D-erythro-, 248 bark of, 374 -, 3-deoxy-1,2-O-isopropylidene-PPicrocrocin, structure of, 6 L-threo-, 248 Polydisperse, defined, 337 Pentopyranose, 2-deoxy-P-~-erythro-, Polymers, netpork structures from, 270 mutarotation of, 59 Polysaccharides Pentopyranoside, benzyl2-0-benzyl-3,4amino, 321 dideoxy-4-nitro-P-~-threo-, 94 sulfates, 291 -, methyl 2-deoxy-~-erythro-,conforfrom animal and seaweed tissues, 293 mational equilibria of anomers, 59 from Annona muricata L, 374 -, methyl 3-deoxy-P-~-erythro-,confrom beech, 376,377 formation of, 59 classification of, 376 Peptides, of glycopeptides and peptidofrom Combretum leonense gum, 375, oligosaccharides of urine, 445,447 377 Peptidoglycans, 314 of corm sacs of Watsonia pyramidata, Peptido-oligosaccharides 371 peptide composition of urinary, 445 fractionation and isolation of structurin urine (human), 436 ally homogeneous, 334-337 Phenols, reaction with glycals, 215 galactan group, 341 galacturonorhamnan group, 361 Phosphatides, 408

494

SUBJECT INDEX, VOLUME 24

gels and networks, structures, conformation and mechanism in formation of, 267-332 glucuronomannan group, 354 from mountain-pine pollen, 363 from mustard (white) seed, 374 from Picea engelmann (spruce)bark, 374 protein-, 292 from red-spruce compression wood, 374 from seed boxes of Watsonia versveldii, 37 1 from soy-bean, 362 structure and conformation of gel-forming, 270 from sycamore, 374 from tamarind seed, 373 from Tropeoleum majus (nasturtium) seeds, 374 xylan group, 371 xyloglucan group, 372 Porphyran, 278 Proline, hydroxy-i-, in urine compounds, 445 Protein-(carbohydrate compounds, in urine human), 435-452 Protein-polysaccharides, 292 Proteopolysaccharides, in urine (human), 436,440 Psichosine, 394,396 -, N-2-hydroxytetracosanoyl-,3-sulfate, 405,407 -, N-tetracosanoyl-, 3-sulfate, 405 Psicofuranine, 252 Purine, 2-acetamido-6-chloro-, reaction with tri-0-acetyl-Dglucal, 218 -, 6-amino-9-(2-deoxy-3-S-ethyl-3thio-@-D-threo-pentofuranosy1)-, 166 -, 9-[3-amino-3-deoxy-5O-(methylsulfonyl)-@-~-ribofuranosyl]-6-(dimethylamino)-, cyclization of, 187 Pyran, tetrahydro-2-(nitromethyl)-,105 4H-Pyran, 2,3-dihydro-, halogenation of, 203 4H-Pyran-4-one, 2,3-dihydro-3-hydroxy-2(hydroxymethy1)-,218 Pyridine, mutarotation of sugars in, 61 2-Pyridinol, as bifunctional catalyst of mutarotation, 27 2-Pyrrolidinone, N-methyl-, as solvent in .displacement reactions, 141

R Reaction mechanism, of mutarotation of sugars in aqueous solution, 35 Reactivity, of sulfonyloxy groups, 139 Rearrangements allylic, 249 of glycals, 213 of 2-hydroxyglycals, 221 Wagner-Meenvein, 193 Reduction, of hexuronic acid residues, 341 Repellents, beetle, 7 Rhamnal, L-, methoxymercuration of, 210 Rhamnazin, synthesis of, 6 Rhamnetin, synthesis of, 6 Rhamnofuranose, 2,3-O-isopropylidene5-O-p-tolylsulfonyl-~-, displacement reactions of, 171 Rhamnofuranoside, methyl 2,5-di-0methyl-3-O-(methylsulfonyl)-a-~-, displacement reaction of, 165 Rhamnose, 2-O-(a-~galactopyranosyluronic acid)+, 361,364,366,376 -, 0-(galactopyranosyluronic acid)(1+ 2)-O-rhamnopyranosyl-( 1+4)0-(galactopyranosyluronicacid)-(1+ 2)-, 364

-,

0-(@-Dglucopyranosyluronic

acid)-(1+ 3)-O-(a-D-galactopyranosyluronic acid)-(1 + 2)-i-, 366 -, 3-O-methyl-~-,359 Rhamnoside, methyl 2,3-O-isopropylidene-4-O-(methylsulfonyl)-a-i-, displacement reactions of 153,190 Ribitol, 2,5-anhydro-l-deoxy-l, l-difluoro-D-, 205 -, l-deoxy-2-O-methyl-l-nitro-~-, 130 -, 2,3,4-tri-O-benzyl-1,5-di-O-ptolylsulfonyl-, solvolysis of, 196 Riboflavine, synthesis of, 5 Ribofuranose, 5-deoxy-l,2-O-isopropylidene-3-O-p-tolylsulfonyl-a-~, displacement reactions of 152 -, 1,2-O-isopropylidene-3,5-di-O-ptolysulfonyl-a-D, displacement reactions of, 151 Ribofuranoside, methyl 2-amino-2,3-N,Obenzylidyne-2,5-dideoxy-5-C-penta-

SUBJECT INDEX, VOLUME 24

495

decylidene-pa-, 257 in urine (human), 438,439,441 methyl 2-amino-2,3-N,O-benzyli- Silicon compounds, per(trimethylsily1) dyne-2,5-dideoxy-5-C-tridecylideneethers of sugars in mutarotation &D-, 257 studies, 60 -, methyl 5-azido-5-deoxy-2,3-di .OSmith degradation, 349 p-tolyhlfonyl-P-D-, 145 of Acacia gums, 345,347 -, methyl 2,3-O-isopropylidene-5-0- of polysaccharides, 340,342 (methylsulfony1)-D, displacement Solanidine, 7 reactions of, 142 Solanine, 7 -, methyl 2,3,5-tri-O-p-tolylsulfonyl- Solatriose, 7 p-D-, displacement reactions of, 165 Solvents -, methyl 5-O-trityl-p-~-,230 for displacement reactions of carbohyRibopyranose, &D-, tetraacetate, confordrate sulfonic esters, 140 mations of, 57 effect on mutarotation of sugars, 15,60, 62 ~, 5-thio-~-,derivatives, 146 Sorbofuranose, 4,6-anhydro-2,3-0-isopromutarotation of, 49 pylidene-a-L-, 172 Ribopyranoside, methyl 4-S-acetyl-2,3Sorbopyranoside, methyl 4-amino-4O-isopropylidene-4-thio-fl-~-, 192 deoxy-a-L-, 97 -, methyl 2-deoxy-3,4-0-isopropyl-, methyl 1,3-O-benzylidene-4,5-diidene-2-( sa1icylideneamino)-p-D-, 0-(meth ylsulfony1)-a-L-,displace152 ment reactions of, 161 Ribose, 2,5-anhydro-~-, diisobutyl di-, methyl 4-deoxy-4-nitro-a-~-,97 thioacetal, 175 Soybeans, polysaccharides of, 362 -, 2,3,5-tri-O-benzyl-4-O-p-tolylSpectroscopy, of gels, 309 SUlfOnyl-D-, dimethyl acetal, disSphinganine, 382,384,385,390-393 placement by alkoxyl group, 194 -, 4-~-hydroxy-,384 Ring contraction Sphingosine, 382 and expansion by mutarotation, 46 -, 3-O-benzoyl-N-octadecanoyl-l-Oduring sulfonate displacements, 188 (2,4,6-tri-O-acetyl-P-D-galactopyranosy1)-DL-dihydro-,408 -, dihydro-, 382,384,385,390-393 -, D-galactosyl-, 394,396 S -, O-~-galactosyl-N-(2-hydroxytetracosanoy1)-,sulfate, 403 Saffron, picrocrocin in, 6 -, N-(2-hydroxytetracosanoyl)-,396 Salts, catalytic effect on mutarotation of -, palmitoyl-, Dgalactosides, 421 sugars, 28 -, N-stearoyl-, D-glucosides and lacSchmidt-Rutz reaction, 69,127,129 toside, 421 Seaweed, polysaccharides from tissues of, Sphingosines 293 N-acyl-, linkage with oligosaccharide Sephadex, 268,313 moiety, 421 in chromatography of urine protein-carbiosynthesis of, 394 bohydrate compounds, 440,444 natural and synthetic, 390-393 Sephadex A-50,0-(2-diethylamistereochemical aspects of, 384 noethyl)-, in ion-exchange chromaSpleen, gangliosides of, 432 tography, 335,338 Spongothymidine, 180 Serine, L-, as precursor in biosynthesis Spongouridine, 180 of sphingosines, 394 Spruce, polysaccharides from, 374 Sialic acids Stereochemistry, of phytosphingosines of gangliosides, 417

-,

496

SUBJECT INDEX, VOLUME 24

and sphingosines, 384 Stereoselectivity, in deoxynitroalditol formation, 106 Steric hindrance, effect on displacement reactions, 152 Streptamine, di-N-acetyltetra-0-acetyl-, 101 Sucrose, acid hydrolysis of, in water and in deuterium oxide, 29 -, penta-0-acetyltri-0-p-tolylsulfonyl-, displacement reaction of, 147 Sugar(s) acetates, acid-catalyzed anomerization of, 43 acetylated, acid anomerization in deuterium oxide and in water, 29 amino, 9 preparation of, 109 aminodeoxy, synthesis of, 147 anhydro, formation by mutarotation, 46 anions, catalytic activity of, 24 branched-chain, 137 energies of activation for mutarotation of, 55 methylation of, 7 mutarotation catalysis by mutarotase, 63,64 catalytic coefficients, ratios for, 30 in N,N-dimethylformamide, 61 in pyridine, 61 in solution, 13-65 nitro, 67-137 acetalation of, 116 acylation of, 115 historical, 68 hydrogenation of, 109 hydrolysis and methanolysis of, 118 nomenclature of, 67 nucleophilic additions and elimination-additions, 130 reaction with amines, 136 with reactive methylene compounds, 137 thermodynamic constants for mutarotation of, 56 thio, mutarotation of, 49 unsaturated, 199-266 nuclear magnetic resonance of, 265 Sugar-sphingosine conjugates, see Glycosphingolipids

Sulfatides, 383,395,403 synthesis of, 407 Sulfonic esters of bridged-ring systems of carbohydrates, displacement reactions of, 153 of carbohydrates, 139-197 Sulfonylation, of pentose dithioacetals,

175 Sulfonyloxy groups, displacement reactions, 139 Sycamore, polysaccharide from, 374 Syneresis, 303 of agar gels, 277

T Tagatopyranose, 4-acetamido-4-deoxy-bD-, tetraacetate, 97 Tagatose, D-, 366 Talitol, 2-acetamido-l,2-dideoxy-l-nitroD-, 133 Talofuranose, 6-deoxy-2,3-0-isopropylidene-5-O-methyl-~-,194 Talofuranoside, methyl 5-O-benzoyl-6deoxy-2,3-O-isopropylidene-a-~-, 155 -, methyl 6-deoxy-2,3-0-isopropylidene-a-D-, 189 Talopyranose, 1,6-anhydro-2,3-0-isopropylidene-4-O-p-tolylsulfonyl-~-~-, reactivity of, 154,163 -, 1,6-anhydro-3,4-0-isopropylidene-2-O-p-tolylsulfonyl-P-~-, reactivity of, 154,163 Talopyranoside, methyl 4-amino-4,6-dideoxy-2,3-0-isopropy1idene-a-~-, 153,190 -, methyl 2,6-anhydro-3,4-0-isopropylidene-b-D-, 197 Talose, 2-acetamido-2-deoxy-~-,134 -, 2-amino-2-deoxy-~-,112,208 Tamarindus indica, polysaccharides from seed of, 372 Tamm-Horsfa11 glycoprotein, in human urine, 441 Temperature coefficient, for anomerization, 51 Tetracosanoic acid, 396,410

SUBJECT INDEX, VOLUME 24

497

of polysaccharides, 335 of urine protein-carbohydrate compounds, 442 Unsaturated acyclic compounds, 260 Unsaturated cyclic compounds deoxy-~-erythro-hex-2-enopyranO S Y ~ ) - , 241 2,3-, furanoid, 226 3.4-, furanoid, 246 Thermodynamics of gels, 306 4,5-, furanoid, 250 for mutarotation of sugars, 51,56 5,6-, furanoid, 255 Thiocyanogen, reaction with tri-0-acetyl 2,3-, pyranoid, 231 D-glucal, 209 3,4-, pyranoid, 249 Thio sugars, mutarotation of, 49 4,5-, pyranoid, 252 5,6-, pyranoid, 257 Thymidine, 2,5’-anhydro-, 179 Unsaturated sugars, 199-266 ~, 2,3’-anhydro-5‘-O-(methylsulUracil, 1-[3-acetamido4,6-0-benzylifony1)-, 178 -, 2,5’-anhydro-3’-O-(methylsuldene-3-deoxy-2-O-(methylsulfonyl)fony1)-,displacement reaction of, 184 @~-glucopyranosyl]-,displacement -, 2,2’-anhydro-2-thio-5’-O-trityl-, reaction of, 183 181 -, 2,3‘-anhydro-1-[2,5-di-O-(methyl-, 3’,5’-di-O-(methylsulfonyl)-, dissulfonyl)-P-~-lyxofuranosyl]-, 182 placement reaction of, 178 ,1-(3,5-anhydro-p-~-lyxofurano-, 3’-O-(methylsulfonyl)-5’-0-trityl-, ~ y l ) -172 , displacement reaction of, 178 -, 1-(2,3’-anhydro-P-~xylofurano-, 2-thio-5‘-O-trityl-, methanesul~ y l ) -179 , fonylation of, 181 -, 1-(3,5-anhydro-fi-D-xylofuranoThymine, 1-(3,5-anhydro-2-deoxy-P~~yl)-,172 threo-pentofuranosy1)-, 172 -, 1-p-D-arabinofuranosyl-,180 -, 1-p-D-arabinofuranosyl-, 180 -, 1-[2-deoxy-3-O-(methylsulfonyl)-, 1-[2-deoxy-3,4-di-O-(methylsul5-O-trityl-P-~-threo-pentofuranosyl]-, fonyl)-/3-~-erythro-pentofuranosyl]-, 166 displacement reaction of, 184 -, 1-[5-0-(methylsulfonyl)-P-D-lyxo-, 2,3’-imino-l-(2-deoxy-P-~-threofuranosyll-, displacement reaction of, pentofuranosy1)-, 184 176 -, 1-[2-O-(methylsu~fonyl)-P-~-xylo-, 1-P-D-xylofuranosyl-,displacefuranosyll-, displacement reaction of, ment reaction of, 181 181 Tipson-Cohen reagent, 229,232,255 1-[2,3,5-tri-O-(methylsulfonyl)-Pp-Tolylsulfonyloxy group, reactivity of! -, D-arabinofuranosyl]-, displacement 139 reaction of, 182 Tomatine, as beetle repellent, 6 Uridine, 5’-O-acetyl-2’-O-p-tolysulfonyl-, Tragacanthic acid, 361 enzymic hydrolysis of, 340 displacement reaction of, 180 Trisaccharides, from mesquite gum, 350 -, 2,2’-anhydro-, 180 Tropeoleurn nzuju.~,polysaccharide from -, 2,2’-anhydro-3’,5’-di-O-(methylseeds of, 374 sulfony1)-, 182 Tubercidin, configuration of, 187 -, 2‘-deoxy-5-fluoro-3’-O-(methylsulfonyl)-5’-0-trityl-, displacement reaction of, 178 U ~, 2’-deoxy-3‘-0-(methyIsulfonyl)5‘-O-trityl-, displacement reaction of, Ultracentrifugation 178 of gels, 311

~, 2-hydroxy-, 396,397,410 Tetr-3-enose, 3-O-acetyl-1,2-O-isopropylidene-a-L-glycero-, 248 Theophylline, 7-(2,4-di-O-acetyl-3,6-di-

498

SUBJECT INDEX, VOLUME 24

-,3',5'-di-O-acetyl-2'-0-p-tolylsulfonyl-, displacement reaction of, 180 3'-0-(methylsulfonyl)-2',5'-di-Otrityl-, displacement reaction of, 178 -, 5-methyl-5'-O-trityl-, methanesulfonylation of, 180 -, 2',3',5'-tri-O-(methylsulfonyl)-, displacement reaction of, 182 Uridinene, 228 Urine, protein-carbohydrate compounds in human, 435-452 Uronic acids, in urine (human),438,439 -9

V Velocity constant, of mutarotation of a-Dand /+D-glucose, 14 Viosamine, 159 Viscoelastic putty, 296

w Wagner-Meenvein rearrangement, 193 Water molecules catalysis of mutarotation by, 17 as catalyst in mutarotation of sugars, 28 catalytic activity of, 22 effect on mutarotation reactions, 31 Watsoniu pyrumidata, polysaccharides from corm sacs of, 371 Watsonia versveldii, polysaccharides from seed boxes of, 371 Wittig reaction, 260,262

x X-Ray diffraction, by gels, 306 Xylal, di-0-acetyl-D-, reaction with benzotriazole and 5,6-dimethyl derivative, 217 -, 3,4-di-O-acetyl-2-nitro-~-,207 Xylan polysaccharides, see Polysaccharides Xylitol, 1,3-anhydro-2,4-0-methyleneDL-, 174 -, 2,4:3,5-di-O-benzylidene-l-O(phenylsulfony1)-, displacement reactions of, 143 -, 2,4:3,5-di-O-methylene-l-O(phenylsulfony1)-, displacement reactions of, 143

-,

2,4-O-methylene-l-O-p-tolylsulfonyl-DL-, displacement reactions of, 174 -, 2,3,4-tri-O-benzyI-l,S-di-O-ptolylsulfonyl-, solvolysis of, 196 Xylobiose, 369,375 Xylofuranose, 3,5-anhydro-l,2-O-isopropylidene-a+-, 172,173 -, 1,2-0-isopropylidene-3,5-di-O(methylsulfony1)-a-D-,displacement reaction of, 173 -, 1,2-O-isopropylidene-3,5-di-O-ptOlylSUlfOnyl-D-,displacement reactions of, 151,165 -, 1,2-O-isopropylidene-3-O-methyla-D, 248 -, 1,2-O-isopropylidene-3-O-(methylsulfonyl)-cr-D, displacement reaction of, 173 -, 1,2-O-isopropylidene-3-O-p-tolylsulfonyl-a-D-, displacement reactions of, 152 -, 1,2-0-isopropylidene-5-O-p-tolylsulfonyl-a-D-, displacement reactions of, 172 Xylofuranoside, methyl 2-0-benzyl-5deoxy-3-0-( methylsulfony1)-am-, displacement reactions of, 165 -, methyl 3-deoxy-3-C-formyl-a-~-, hemiacetal, 193 -, methyl 3,5-diazido-3,5-dideoxy-20-p-tolylsulfonyl-P-D, 166 methyl 3,5-0-isopropylidene-2-0p-tolylsulfonyl-a-D-, displacement reactions of, 153 Xyloglucan polysaccharides, see Polysaccharides Xylopyranose, p-D-,tetraacetate, conformations of, 58

-

-,

O-(a-D-glucopyranosyluronic

acid)-(1+2)-O-~-~-xylopyranosyl(1+4)-~-,371

-,

O-(4-O-methyl-a-~-glucopyrano-

syluronic acid)-(1+2)-O-p-D-xylopyranosyl-( 1+4)-~-,371 -, 5-thio-a-~-,mutarotation of, 49 -, 4-O-P-D-xylopyranosy1-D-,372 -, O-P-D-xylopyranosyl-(1-4)-8-Dxylopyranosyl-( i+4)-D-, 372 Xylopyranoside, methyl 4-azido-4-deoxy-

SUBJECT INDEX, VOLUME 24

499

2,3-di-O-(methylsulfonyl)-fl-~-,disthioacetal, 175 placement reactions of, 163 -, 2-O-a-~-fucopyranosy~-~-, 362 -, methyl 4-azido-2,3-di-O-benzoyl- -, 2-O-P-~-galactopyranosyl-~, 362, 4-deoxy-a-D-, 160 373 -, methyl tri-O-(methylsulfonyl)-a-, 4-O-P-~-xylopyranosyl-~-, 369, D(and P-D)-, displacement reactions 375 of, 161 Xylopyranosyl bromide, tri-0-acetyl-fl-D, Z conformation of, 58 Xylose, 4-amino-4-deoxy-D-,160 Zoo-glycosphingolipids, 382 -, 2,5-anhydro-D-,diisobutyl di-

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CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24 S. A., and BOURNE, E. J., Acetals BARKER, and Ketals of the Tetritols, Pentitols and Hexitols, 7,137-207 ADAMS, MILDRED. See Caldwell, Mary L. ANDERSON, ERNEST,and SANDS,LILA,A BARNETT,J. E. G., Halogenated Carbohydrates, 22,177-227 Discussion of Methods of Value in Research on Plant Polyuronides, 1, BARRETT,ELLIOTT P., Trends in the Development of Granular Adsorbents 329-344 for Sugar Refining, 6,205-230 ANDERSON,LAURENS. See Angyal, S. J. JOHN, ANET, E. F. L. J., 3-Deoxyglycosuloses BARRY,C. P., and HONEYMAN, Fructose and its Derivatives, 7,53-98 (3-Deoxyglycosones) and the Degradation of Carbohydrates, 19, 181-218 BAYNE,S., and FEWSTER,J. A., The Osones, 11,43-96 ANGYAL,S. J., and ANDERSON, LAURENS, BEELIK, ANDREW, Kojic Acid, 11,145-183 The Cyclitols, 14,135-212 ARCHIBALD,A. R., and BADDILEY, J., The BELL, D. J., The Methyl Ethers of DGalactose, 6 , l l - 2 5 Teichoic Acids, 21,323-375 ASPINALL, G. O., Gums and Mucilages, BEMILLER, J. N., Acid-catalyzed Hydrolysis of Glycosides, 22,25- 108 24,333-379. ASPINALL,G. 0.. The Methyl Ethers of BEMILLER,J. N. See also, Whistler, Roy L. BHAT,K. VENKATRAMANA.See Zorbach, Hexuronic Acids, 9,131-148 W. Werner. ASPINALL,G. O., The Methyl Ethers of BINKLEY, W. W., Column ChromatograD-Mannose,8,217-230 phy of Sugars and Their Derivatives, ASPINALL, G. O., Structural Chemistry of 10,55-94 the Hemicelluloses, 14,429-468 BINKLEY,W. W., and WOLFROM,M. L., Composition of Cane Juice and Cane B Final Molasses, 8,291-314 BIRCH,GORDONG., Trehaloses, 18, 201BADDILEY, J. See Archibald, A. R. 225 BAER, HANS H., The Nitro Sugars, 24, BISHOP, C. T., Gas-liquid Chromatog67-138 raphy of Carbohydrate Derivatives, BAER, HANS H., [Obituary ofl Richard 19,95-147 Kuhn, 24,l-12 J. B., Oligo- BLAIR, MARY GRACE,The 2-HydroxyBAILEY,R. W., and PRIDHAM, glycals, 9,97-129 saccharides, 17,121-167 BALL,D. H., and PARRISH, F. W., Sulfonic BOBBITT,J. M., Periodate Oxidation of Carbohydrates, 11,l-41 Esters of Carbohydrates: BOESEKEN, J., The Use of Boric Acid for Part I, 23, 233-280 the Determination of the ConfiguraPart 11, 24, 139-197 tion of Carbohydrates, 4,189-210 BALLOU,CLINTON E., Alkali-sensitive BONNER, T. G., Applications of TrifluoroGlycosides, 9,59-95 acetic Anhydride in Carbohydrate BANKS,W., and GREENWOOD, C. T., Chemistry, 16,59-84 Physical Properties of Solutions of BONNER, WILLIAM A., Friedel-Crafts and Polysaccharides, 18,357-398 Grignard Processes in the CarboBARKER,G. R., Nucleic Acids, 11, 285hydrate Series, 6,251-289 333

A

501

502

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24

BOURNE, E. J., and PEAT,STANLEY, The DEBELDER,A. N., Cyclic Acetals of the Methyl Ethers of D-Glucose, 5, Aldoses and Aldosides, 20,219-302 145-190 DEITZ,VICTOR R. See Liggett, R. W. BOURNE, E. J. See also, Barker, S. A. DEUEL,H. See Mehta, N. C. BOWENG, H. O., and LINDBERG,B., DEUEL,HARRYJ., JR., and MOREHOUSE, MARGARET G., The Interrelation of Methods in Structural Polysaccharide Chemistry, 15,53-89 Carbohydrate and Fat Metabolism, 2, 119-160 BRAY, H. G., D-Glucuronic Acid in Metabolism, 8,251-275 DEULOFEU,VENANCIO,The Acylated BRAY,H. G., and STACEY,M., Blood Nitriles of Aldonic Acids and Their Group Polysaccharides, 4,37-55 Degradation, 4,119-151 BRIMACOMBE, J. S.See How, M. J. DIMLER,R.J., 1,6-Anhydrohexofuranoses, A New Class of Hexosans, 7,37-52 DOUDOROFF, M. See Hassid, W. Z. ti DUBACH, P. See Mehta, N. C. CAESAR,GEORGEV., Starch Nitrate, 13, DUTCHER,JAMES D., Chemistry of the Amino Sugars Derived from Anti331-345 biotic Substances, 18,259-308 CALDWELL,MARYL., and ADAMS,MILDRED, Action of Certain Alpha E Amylases, 5,229-268 CANTOR,SIDNEYM., [Obituary ofl John ELDERFIELD,ROBERT C., The CarboC. Sowden, 20,1-10 hydrate Components of the Cardiac CANTOR, SIDNEYM. See also, Miller, Glycosides, 1,147-173 Robert Ellsworth. Chemistry of OsaCAPON,B., and OVEREND,W. G., Con- EL KHADEM,HASSAN, zones, 20,139-181 stitution and Physicochemical ProperEL KHADEM, HASSAN, Chemistry of Osoties of Carbohydrates, 15,ll-51 triazoles, 18,99-121 CARR,C. JELLEFF,and KRANTZ, JOHNC., JR., Metabolism of the Sugar Alcohols ELLIS, G. P., The Maillard Reaction, 14, 63-134 and Their Derivatives, 1,175-192 ELLIS, G. P., and HONEYMAN, JOHN, CHIZHOV, 0.S. See Kochetkov, N. K. Clycosylamines, 10,95-168 CLAMP,JOHN R., HOUGH,L., HICKSON, JOHN L., and WHISTLER,ROY L., EVANS,TAYLORH.,and HIBBERT,HAROLD, Bacterial Polysaccharides, 2, Lactose, 16,159-206 203-233 COMPTON,JACK, The Molecular ConEVANS,W. L., REYNOLDS,D. D., and stitution of Cellulose, 3,185-228 TALLEY,E. A,, The Synthesis of CONCHIE,J., LEWY, G. A., and MARSH, Oligosaccharides, 6,27-81 C. A., Methyl and Phenyl Glycosides of the Common Sugars, 12,157-187 F COURTOIS,JEAN EMILE, [Obituary of] Emile Bourquelot, 18.1-8 CRUM,JAMESD., The Four-carbon Sac- FERRIER, R. J., Unsaturated Sugars, 20, 67-137;24,199-266 charinic Acids, 13,169-188 FEWSTER, J. A.See Bayne, S . FLETCHER, HEWITTG., JR.,The ChemisD try and Configuration of the Cyclitols, 3.45-77 DAVIES,D. A. L., Polysaccharides of FLETCHER,HEWITTG., JR., and RICHTGram-negative Bacteria, 15,271-340 MYER, NELSON K., Applications in the DEAN,G. R., and GOTTFFUED, J. B., The Carbohydrate Field of Reductive DeCommercial Production of Crystalsulfurization by Raney Nickel, 5, line Dextrose, 5,127-143 1-28

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24 FLETCHER, HEWITT G., JR. See also, Jeanloz, Roger W. FORDYCE,CHARLESR., Cellulose Esters of Organic Acids, 1,309-327 FOSTER, A. B., Zone Electrophoresis of Carbohydrates, 12,81-115 FOSTER,A. B., and HORTON,D., Aspects of the Chemistry of the Amino Sugars, 14,213-281 A. J., The FOSTER,A. B., and HUGGARD, Chemistry of Heparin, 10, 335-368 FOSTER, A. B., and STACEY,hl., The Chemistry of the 2-Amino Sugars (2-Amino-2-deoxy-sugars), 7,247-288 FOSTER,A. B., and WEBBER,J. M., Chitin, 15,371-393 FOX, J. J., and WEMPEN,I., Pyrimidine Nucleosides, 14,283-380 FOX,JACKJ. See also, Ueda, Tohru. FRENCH,DEXTER,The Raffinose Family of Oligosaccharides, 9,149-184 FRENCH,DEXTER,The Schardinger Dextrins, 12,189-260 FREUDENBERG, KARL, Emil Fischer and his Contribution to Carbohydrate Chemistry, 21,l-38 G GARC~AGONZALEZ, F., Reactions of Monosaccharides with beta-Ketonic Esters and Related Substances, 11, 97-143 GAR& GONZALEZ,F., and GOMEZS h CHEZ,A., Reactions of Amino Sugars with beta-Dicarbonyl Compounds, 20,303-355 GOEPP, RUDOLPHMAXIMILIAN,JR. See Lohmar, Rolland. COLDSTEIN,I. J., and HULLAR,T. L., Chemical Synthesis of Polysaccharides, 21,431-512 GOMEZ S ~ C H E ZA. , See Garcia Gonzilez, F. GOODMAN, IRVING,Clycosyl Ureides, 13, 2 15- 236 GOODMAN, LEON, Neighboring-group Participation in Sugars, 22, 109-175 GORIN,P. A. J., and SPENCER,J. F. T., Structural Chemistry of Fungal Polysaccharides, 23,367-417 GOTTFRIED,J. B. See Dean, G. R.

503

GOTTSCHALK, ALFRED,Principles Underlying Enzyme Specificity in the Domain of Carbohydrates, 5,49-78 GREEN,JOHNW., The Clycofuranosides, 21,95-142 GREEN,JOHNW., The Halogen Oxidation of Simple Carbohydrates, Excluding the Action of Periodic Acid, 3, 129184 GREENWOOD, C. T., Aspects of the Physical Chemistry of Starch, 11, 335-385 GREENWOOD, C. T., The Size and Shape of Some Polysaccharide Molecules, 7,289-332; 11,385-393 GREENWOOD, C. T., The Thermal Degradation of Starch, 22,483-515 GREENWOOD, C. T., and MILNE, E. A., Starch Degrading and Synthesizing Enzymes: A Discussion of Their Properties and Action Pattern, 23, 281-366 GREENWOOD, C. T. See also, Banks, W. GURIN,SAMUEL,Isotopic Tracers in the Study of Carbohydrate Metabolism, 3,229-250 GUTHRIE, R. D., The “Dialdehydes” from the Periodate Oxidation of Carbohydrates, 16, 105-158 GUTHRIE,R. D., and MCCARTHY, J. F., Acetolysis, 22, 11-23 H HALL, L. D., Nuclear Magnetic Resonance, 19,51-93 HANESSIAN, STEPHEN, Deoxy Sugars, 21, 143-207 HARRIS,ELWIN E., Wood Saccharification, 4,153-188 HASKINS, JOSEPHF., Cellulose Ethers of Industrial Significance, 2,279-294 HASSID, W. Z., and DOUDOROFF,hl., Enzymatic Synthesis of Sucrose and Other Disaccharides, 5,29-48 HASSID,W. Z. See also, Neufeld, Elizabeth F. HAYNES,L. J., Naturally Occurring CGlycosyl Compounds, 18, 227-258; 20,357-369 HAYNES,L. J., and NEWTH, F. H., The Glycosyl Halides and Their Derivatives, 10,207-256

504

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24

HEHRE, EDWARDJ., The Substitutedsucrose Structure of Melezitose, 8, 277-290 HELFERICH,BURCKHARDT, The Glycals, 7,209-245 HELFERICH,BURCKHARDT, Trityl Ethers of Carbohydrates, 3.79- 111 HEYNS,K., and PAULSEN,H., Selective Catalytic Oxidation of Carbohydrates, Employing Platinum Catalysts, 17, 169-221 HIBBERT,HAROLD.See Evans, Taylor H. JOHN L. See Clamp, John R. HICKSON, HILTON,H. W., The Effects of Plantgrowth Substances on Carbohydrate Systems, 21,377-430 HINDERT,hIARJORIE. See Karabinos, J. V. HIRST, E. L., [Obituary ofl James Colquhoun Irvine, 8, xi-xvii HIRST,E. L., [Obituary ofl Walter Norman Haworth, 6,l-9 HIRST, E. L., and JONES,J. K. N., The Chemistry of Pectic Materials, 2, 235-251 HIRST,E. L., and ROSS, A. G., [Obituary ofl Edmund George Vincent Percival, 10, xiii-xx

HODGE, JOHN E., The Amadori Rearrangement, 10,169-205 HONEYMAN, JOHN,and MORGAN, J. W.W., Sugar Nitrates, 12,117-135 JOHN. See also, Barry, C. P. HONEYMAN, JOHN. See also, Ellis, G. P. HONEYMAN, HORTON,D., [Obituary ofl Alva Thompson, 19,l-6 HORTON,D., Tables of Properties of 2Amino-2-deoxy Sugars and Their Derivatives, 15,159-200 HORTON,D., and HUTSON,D. H., Developments in the Chemistry of Thio Sugars, 18,123-199 HORTON, D. See also, Foster, A. B. HOUGH,L., and JONES,J. K. N., The Biosynthesis of the Monosaccharides, 11, 185-262 HOUGH,L., PFUDDLE, J. E., and THEOBALD, R. S . , The Carbonates and Thiocarbonates of Carbohydrates, 15,91-158 HOUGH,L. See also, Clamp, John R.

HOW, M. J., BRIMACOMBE, J. S., and M., The Pneumococcal PolySTACEY, saccharides, 19,303-357 HUDSON, C. S., Apiose and the Glycosides of the Parsley Plant, 4,57-74 HUDSON, C. S., The Fischer Cyanohydrin Synthesis and the Configurations of Higher-carbon Sugars and Alcohols, 1,l-36 HUDSON,C. S., Historical Aspects of Emil Fischer’s Fundamental Conventions for Writing Stereo-formulas in a Plane, 3,1-22 HUDSON,C. S., Melezitose and Turanose, 2,l-36 A. J. See Foster, A. B. HUGGARD, HULLAR, T. L. See Goldstein, I. J. HUTSON,D. H. See Horton, D. I ISBELL,HORACES., and PIGMAN, WARD, Mutarotation of Sugars in Solution: Catalytic Processes, Isotope Effects, Reaction Mechanisms, and Biochemical Aspects, Part II,24,13-65 ISBELL, HORACES. See also, Pigman, Ward.

J JEANLOZ, ROGER W., [Obituary ofl Kurt Heinrich Meyer, 11, xiii-xviii ROGER W., The Methyl Ethers JEANLOZ, of 2-Amino-2-deoxy Sugars, 13, 189214 JEANLOZ,ROGER W., and FLETCHER, HEWITT G., JR., The Chemistry of Ribose, 6,135-174 R. D., JEFFREY,G. A., and ROSENSTEIN, Crystal-structure Analysis in Carbohydrate Chemistry, 19.7-22 JONES,DAVIDM., Structure and Some Reactions of Cellulose, 19,219-246 JONES,J. K. N., and SMITH, F., Plant Gums and Mucilages, 4,243-291 JONES,J. K. N. See also, Hirst, E. L. JONES,J. K. N. See also, Hough, L.

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24 JONSEN, J., and LALAND,S., Bacterial Nucleosides and Nucleotides, 15, 201-234

K KARABINOS, J. V., Psicose, Sorbose and Tagatose, 7,99-136 KARABINOS, J. V., and HINDERT,MARJORIE, Carboxymethylcellulose, 9, 285-302 KENT,P. W. See Stacey, M. KERTESZ,Z. I., and MCCOLLOCH,R. J., Enzymes Acting on Pectic Substances, 5,79-102 kss, J., Glycosphingolipids (SugarSphingosine Conjugates), 24, 381433 KLEMER,ALMUTH. See Micheel, Fritz. KOCHETKOV, N. K., and CHIZHOV,0. S., Mass Spectrometry of Carbohydrate Derivatives, 21,39-93 KOWKABANY, GEORGEN., Paper Chromatography of Carbohydrates and Related Compounds, 9,303-353 KRANTZ,JOHN C., JR. See Carr, C. Jelleff.

L LAIDLAW, R. A., and PERCIVAL,E. C . V., The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose, 7, 1-36 LALAND,S. See Jonsen, J. LEDERER, E., Glycolipids of Acid-fast Bacteria, 16,207-238 LEMIEUX,R. U., Some Implications in Carbohydrate Chemistry of Theories Relating to the Mechanisms of Replacement Reactions, 9 , l - 5 7 LEMIEUX,R. U., and WOLFROM,M. L., The Chemistry of Streptomycin, 3, 337-384 LESPIEAU,R., Synthesis of Hexitols and Pentitols from Unsaturated Polyhydric Alcohols, 2, 107-118

505

LEVI,IRVING,and PURVES,CLIFFORDB., The Structure and Configuration of Sucrose (alpha-D-Glucopyranosyl beta-D-Fructofuranoside),4,1-35 LEVVY,G. A., and MARSH,C. A., Preparation and Properties of P-Glucuronidase, 14,381-428 LEVVY, G. A. See also, Conchie, J. LIGGETT,R. W., and DEITZ, VICTORR., Color and Turbidity of Sugar Products, 9,247-284 LINDBERG, B. See Bouveng, H. 0 . LOHMAR, ROLLAND, and GOEPP, RUDOLPH MAXIMILIAN, JR., The Hexitols and Some of Their Derivatives, 4.211-241

M MAHER,GEORGEG., The Methyl Ethers of the Aldopentoses and of Rhamnose and Fucose, 10,257-272 MAHER,GEORGEG., The Methyl Ethers Of D-GahCtOSe, 10,273-282 MALHOTRA,OM PRAKASH.See Wallenfels, Kurt. MANNERS,D. J., Enzymic Synthesis and Degradation of Starch and Glycogen, 17,371-430 D. J., The Molecular Structure MANNERS, of Glycogens, 12,261-298 MARCHESSAULT, R. H., and SARKO, A., X-Ray Structure of Polysaccharides, 22,421-482 MARSH,C. A. See Conchie, J. MARSH,C. A. See Levvy, G. A. MCCARTHY, J. F. See Guthrie, R. D. MCCASLAND, G . E., Chemical and Physical Studies of Cyclitols Containing Four or Five Hydroxyl Groups, 20, 11-65 MCCLOSKEY, CHESTERM . , Benzyl Ethers of Sugars, 12,137-156 MCCOLLOCH, R. J . See Kertesz, Z. I. MCDONALD, EMMAJ., The Polyfructosans and Difructose Anhydrides, 2, 253-277 MCCALE,E. H. F., Protein-Carbohydrate Compounds in Human Urine, 24, 435-452

506

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24

MEHLTRETTER, c. L., The Chemical Synthesis of D-Glucuronic Acid, 8, 231-249 MEHTA, N. c., DUBACH,P., and DEUEL, H., Carbohydrates in the Soil, 16, 335-355 MESTER,L., The Formazan Reaction in Carbohydrate Research, 13, 105-167 MESTER, L., [Obituary of] Gkza Zempikn,

NEUFELD,ELIZABETHF., and HASSID, W. Z., Biosynthesis of Saccharides from Glycopyranosyl Esters of Nucleotides (“Sugar Nucleotides”), 18, 309-356 NEWTH,F. H., The Formation of Furan Compounds from Hexoses, 6, 83-106 NEWTH,F. H. See also, Haynes, L. J. NICKERSON, R. F., The Relative Crystallinity of Celluloses, 5,103-126 14,1-8 MICHEEL,FRITZ,and KLEMER, ALMUTH, NORD,F. F., [Obituary ofl Carl Neuberg, 13,l-7 Glycosyl Fluorides and Azides, 16, 85-103 0 MILLER, ROBERT ELLSWORTH, and CANTOR,SIDNEY M., Aconitic Acid, a By-product in the Manufacture of OLSON,E. J. See Whistler, Roy L. OVEREND,W. G., and STACEY,M.,The Sugar, 6,231-249 Chemistry of the 2-Desoxy-sugars, MILLS, J. A., The Stereochemistry of 8,45- 105 Cyclic Derivatives of Carbohydrates, OVEREND, W. G . See also, Capon, B. 10.1-53 MILNE,E. A. See Greenwood, C. T. P MONTGOMERY, JOHNA., and THOMAS, H. JEANETTE,Purine Nucleosides, 17, PACSU, EUGENE,Carbohydrate Ortho30 1-369 esters, 1,77-127 MONTGOMERY, REX, [Obituary of] Fred PARRISH,F. W. See Ball, D. H. Smith, 22,l-10 H., and TODT,K., Cyclic MonoMOODY,G . J., The Action of Hydrogen PAULSEN, saccharides Having Nitrogen or SulPeroxide on Carbohydrates and fur in the Ring, 23,115-232 Related Compounds, 19,149-179 H. See also, Heyns, K . MOREHOUSE, MARGARETG. See Deuel, PAULSEN, PEAT, STANLEY,The Chemistry of AnHarry J., Jr. hydro Sugars, 2.37-77 MORGAN, J. W. W. See Honeyman, John. See also, Bourne, E . J. MORI, T., Seaweed Polysaccharides, 8, PEAT,STANLEY. PERCIVAL, E. G. V., The Structure and Re315-350 activity of the Hydrazone and OsaMUETGEERT,J., The Fractionation of zone Derivatives of the Sugars, 3, Starch, 16,299-333 23-44 MYRBACK, KARL, Products of the Enzymic Degradation of Starch and PERCIVAL,E. G. V. See also, Laidlaw, R. A. Glycogen, 3,251-310 PERLIN, A. S., Action of Lead Tetraacetate on the Sugars, 14,9-61 PERLIN,A. S., [Obituary of] Clifford Burrough Purves, 23,l-10 N PHILLIPS,C . O., Photochemistry of Carbohydrates, 18,9-59 NEELY, W. BROCK,Dextran: Structure PHILLIPS,C . O., Radiation Chemistry of and Synthesis, 15,341-369 Carbohydrates, 16,13-58 NEELY,W. BROCK,Infrared Spectra of PIGMAN,WARD, and ISBELL,HORACES., Carbohydrates, 12,13-33 Mutarotation of Sugars in Solution: NEUBERG,CARL, Biochemical ReducPart I. History, Basic Kinetics, and tions at the Expense of Sugars, 4, Composition of Sugar Solutions, 23, 75-117 11-57

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24

507

PIGMAN, WARD.See also, Isbell, Horace S. POLGLASE,W. J., Polysaccharides Associated with Wood Cellulose, 10, 283-333 PRIDDLE,J. E. See Hough, L. PRIDHAM, J. B., Phenol-Carbohydrate Derivatives in Higher Plants, 20, 37 1-408 PRIDHAM, J. B. See also, Bailey, R. W. PURVES,CLIFFORDB. See Levi, Irving.

SHAFIZADEH, F., Formation and Cleavage of the Oxygen Ring in Sugars, 13, 9-61 SHAFIZADEH, F., Pyrolysis and Combustion of Cellulosic Materials, 23, 419-474 SMITH, F., Analogs of Ascorbic Acid, 2, 79-106 SMITH,F. See also, Jones, J. K. N. SOWDEN,JOHNC., The Nitromethane and 2-Nitroethanol Syntheses, 6, 291-318 SOWDEN,JOHN C., [Obituary ofl HerR mann Otto Laurenz Fischer, 17, 1-14 RAYMOND, ALBERT L., Thio- and SelenoSOWDEN,JOHNC., The Saccharinic Acids, sugars, 1,129-145 REES, D. A., Structure, Conformation, and 12,35-79 Mechanism in the Formation of SPECK, JOHN C., JR., The Lobry de Bruyn-Alberda van Ekenstein TransPolysaccharide Gels and Networks, formation, 13,63-103 24,267-332 REEVES,RICHARD E., Cuprammonium- SPEDDING, H., Infrared Spectroscopy Glycoside Complexes, 6,107-134 and Carbohydrate Chemistry, 19, REICHSTEIN,T., and WEISS, EKKEHARD, 23-49 J. F. T. See Corin, P. A. J. The Sugars ofthe Cardiac Clycosides, SPENCER, SPRINSON,D. B., The Biosynthesis of 17,65-120 RENDLEMAN, J. A., JR., Complexes of AlAromatic Compounds from D-clukali Metals and Alkaline-earth Metals cose, 15,235-270 STACEY,At., The Chemistry of Mucowith Carbohydrates, 21,209-271 REYNOLDS,D. D. See Evans, W. L. polysaccharides and Mucoproteins, RICHTMYER,NELSON K., The Altrose 2,161-201 Group of Substances, 1,37-76 STACEY,M., and KENT,P. W., The PolyRICHTMYER,NELSON K., The 2-(aldosaccharides of Mycobacterium tuberPolyhydroxyalkyl)benzamidazoles, 6, culosis, 3,311-336 STACEY,M. See also, Bray, H. G. 175-203 STACEY,hl. See also, Foster, A. B. RICHTMYER, NELSONK. See also, FletchSTACEY,M. See also, How, M. J. er, Hewitt G., Jr. ROSENSTEIN,R. D. See Jeffrey, G . A. STACEY,M. See also, Overend, W. G . ROSENTHAL,ALEX, Application of the STOLOFF,LEONARD,Polysaccharide Hydrocolloids of Commerce, 13, 2650 x 0 Reaction to Some Carbohydrate 287 Derivatives, 23,59- 114 SUGIHARA,JAMES hl., Relative ReacROSS, A. G. See Hirst, E. L. tivities of Hydroxyl Groups of Carbohydrates, 8, 1-44 S SANDS,LILA.See Anderson, Ernest. SARKO, A. See Marchessault, R. H. SATTLER, LOUIS, Clutose and the Unfermentable Reducing Substances in Cane Molasses, 3,113-128 SCHOCH,THOMASJOHN,The Fractionation of Starch, l , 247-277 SHAFIZADEH,F., Branched-chain Sugars of Natural Occurrence, 11,263-283

T TALLEY,E. A. See Evans, W. L. TEAGUE,ROBERTS., The Conjugates of D-Glucuronic Acid of Animal Origin, 9,185-246 THEANDER, OLOF, Dicarbonyl Carbohydrates, 17,223-299

508

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-24

THEOBALD, R. S. See Hough, L. THOMAS,H. JEANETTE.See Montgomery, John A. TIMELL,T. E., Wood Hemicelluloses: Part I, 19,247-302 Part II,20,409-483 TIPSON, R. STUART,The Chemistry of the Nucleic Acids, 1,193-245 TIPSON,R. STUART,[Obituary ofl Harold Hibbert, 16,l-11 TIPSON,R. STUART,[Obituary of] Phoebus Aaron Theodor Levene, 12,l-12 TIPSON, R. STUART,Sulfonic Esters of Carbohydrates, 8,107-215 TODT,K. See Paulsen, H. TURVEY,J. R., Sulfates of the Simple Sugars, 20,183-218

U UEDA, TOHRU, and FOX, JACK J., The Mononucleotides, 22,307-419

V VERSTRAETEN, L. M. J., D-Fructose and its Derivatives, 22,229-305

WEISS,EKKEHARD.See Reichstein, T. WEMPEN,I. See Fox, J. J. WHISTLER, ROY L., Preparation and Properties of Starch Esters, 1, 279307 WHISTLER,ROYL., Xylan, 5,269-290 WHISTLER,ROYL., and BEMILLER,J. N., Alkaline Degradation of Polysaccharides, 13,289-329 WHISTLER,ROYL., and OLSON,E. J., The Biosynthesis of Hyaluronic Acid, 12, 299-319 WHISTLER, ROY L. See also, Clamp, John R. M. W. See Zilliken, F. WHITEHOUSE, WIGGINS,L. F., Anhydrides of the Pentitols and Hexitols, 5, 191-228 WIGGINS, L. F., The Utilization of Sucrose, 4,293-336 WISE, LOUIS E., [Obituary ofl Emil Heuser, 1 5 , l - 9 WOLFROM,M. L., [Obituary ofl Claude Silbert Hudson, 9, xiii-xviii WOLFROM,M. L., [Obituary of] Rudolph Maximilian Goepp, Jr., 3, xv-xxiii WOLFROM,h l . L. See also, Binkley, W. W. WOLFROM,M. L. See also, Lemieux, R. U.

W Z

WALLENFELS,KURT, and MALHOTRA, OM PRAKASH, Galactosidases, 16, ZILLIKEN,F., and WHITEHOUSE,M. W., 239-298 The Nonulosaminic Acids - NeurWEBBER,J. M., Highercarbon Sugars, aminic Acids and Related Com17,15-63 pounds (Sialic Acids), 13,237-263 WEBBER,J. M. See also, Foster, A. B. ZORBACH,W. WERNER, and BHAT, K. WEIGEL, H., Paper Electrophoresis of VENKATRAMANA, Synthetic CardenoCarbohydrates, 18,61-97 lides, 21,273-321

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24 A Acetals, cyclic, of the aldoses and aldosides, 20,

219-302 of hexitols, pentitols, and tetritols, 7,

137-207 Acetic acid, trifluoro-, anhydride, applications of, in carbohydrate chemistry, 16,59-84 Acetolysis, 22, 11-23 Aconitic acid, 6,231-249 Action pattern, of starch degrading and synthesizing enzymes, 23,281-366 Adsorbents, granular, for sugar refining, 6, 205-230 Alcohols, higher-carbon sugar, configurations of,

Animals, conjugates of D-glucuronic acid originating in, 9,185-246 Antibiotic substances, chemistry of the amino sugars derived from, 18,259-308 Apiose, 4.57-74 Ascorbic acid, analogs of, 2,79-I06 Aromatic compounds, biosynthesis of, from D-glucose, 15,

235-270

B Bacteria, glycolipides of acid-fast, 16, 207-238 nucleosides and nucleotides of, 15,

201 -234

1,l-36

polysaccharides from, 2, 203-233; 3,

unsaturated polyhydric, 2,107-1 18 Aldonic acids, acylated nitriles of, 4,119-151 Aldopentoses, methyl ethers of, 7, 1-36; 10, 257-272 Aldoses and aldosides, cyclic acetals of, 20,219-302 Alkaline degradation, of polysaccharides, 13,289-329 Altrose, group of compounds related to, 1.37-76 Amadori rearrangement, 10,169-205 Amino sugars. See Sugars, 2-amino-2deoxy. Amylases, certain alpha, 5,229-268 Analysis, of crystal structure, in carbohydrate chemistry, 19,7-22 Anhydrides, difructose, 2,253-277 ofhexitols, 5, 191-228 of pentitols, 5, 191-228 Anhydro sugars. See Sugars, anhydro.

311-336 polysaccharides of Gram-negative, 15,

271-340 Benzimidazoles, 2-(aldo-polyhydroxyaIkyl)-,6, 175-203 Benzyl ethers, of sugars, 12,137-156 Biochemical aspects, of mutarotation of sugars in solution, 24,

13-65 Biochemical reductions, at the expense of sugars, 4,75- 117 Biosynthesis, of aromatic compounds from D-glucose,

15,235-270 of hyaluronic acid, 12,299-319 of the monosaccharides, 11, 185-262 of saccharides, from glycopyranosyl esters of nucleotides, 18, 309-356 Blood groups, polysaccharides of, 4,37-55 Boric acid, for determining configuration of carbohydrates, 4,189-210

509

510

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24

Bourquelot, Emile, obituary of, 18.1-8 Branched-chain sugars. branched-chain.

See

Sugars,

C

metabolism of, 2, 119-160; 3, 229-250 orthoesters of, 1,77-127 paper electrophoresis of, 18, 61-97 periodate oxidation of, 11.1-41 the “dialdehydes” from, 16, 105-158 phenol derivatives, in higher plants, 20,

371-408 Cane juice, composition of 8,291-314 Cane molasses. See Molasses, cane. Carbohydrates, action of hydrogen peroxide on, 19,

149-179 application of reductive desulfurization by Raney nickel, in the field of, 5,

1-28 application of trifluoroacetic anhydride in chemistry of, 16.59-84 application of the 0 x 0 reaction to some derivatives of, 23,59-114 as components of cardiac glycosides, 1,

147-173 carbonates of, 15,91-158 chemistry of, Emil Fischer and his contribution to, 21,l-38 complexes of, with alkali metals and alkaline-earth metals, 21, 209-271 compounds with proteins, in human urine, 24,435-452 constitution of, 15, 11-51 crystal-structure analysis of, 19, 7-22 degradation of, 19,181-218 determination of configuration of, with boric acid, 4,189-210 dicarbonyl, 17,223-299 enzyme specificity in the domain of, 5,

49-78 formazan reaction, in research on, 13,

105-167 Friedel-Crafts and Grignard processes applied to, 6,251-289 gas-liquid chromatography of derivatives of, 19,95-147 halogen oxidation of simple, 3, 129- 184 halogenated, 22, 177-227 infrared spectra of, 12.13-33 infrared spectroscopy of, 19,23-49 mass spectrometry of derivatives of, 21,

39-93 mechanisms of replacement reactions in chemistry of, 9 , l - 5 7

photochemistry of, 18,9-59 physicochemical properties of, 15, 11-

51 radiation chemistry of, 16,13-58 and related compounds, action of hydrogen peroxide on, 19, 149-179 paper chromatography of, 9,303-353 relative reactivities of hydroxyl groups of, €41-44 selective catalytic oxidation of, employing platinum catalysts, 17,16922 1 in the soil, 16,335-355 stereochemistry of cyclic derivatives of,

10,l-53 sulfonic esters of, 8, 107-215; 23, 233-

280; 24,139-197 systems, effects of plant-growth substances on, 21,377-430 thiocarbonates of, 15,91-158 trityl ethers of, 3,79-111 zone electrophoresis of, 12,81-115 Carbonates, ofcarbohydrates, 15,91-158 Carboxymethyl ether, of cellulose, 9,285-302 Cardenolides, synthetic, 21,273-321 Catalysts, effects of, in mutarotation of sugars in solution, 24,13-65 platinum, in selective catalytic oxidation of carbohydrates, 17, 169-221 Cellulose, carboxymethyl-, 9,285-302 esters of, with organic acids, 1,309-327 ethers of, 2,279-294 molecular constitution of, 3, 185-228 of wood, polysaccharides associated with, 10,283-333 Celluloses, relative crystallinity of, 5, 103-126 some reactions of, 19, 219-246 structure of, 19,219-246 Cellulosic materials,

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24 combustion and pyrolysis of, 23, 419474 Chemistry, of the amino sugars, 14,213-281 of the 2-amino sugars, 7,247-288 of anhydro sugars, 2,37-77 of carbohydrates, applications of trifluoroacetic anhydride in, 16, 5984 Emil Fischer and his contribution to, 21,l-38 crystal-structure analysis in, 19, 7-22 infrared spectroscopy and, 19, 23-49 some implications of theories relating to the mechanisms of replacement reactions in, 9, 1-57 of the cyclitols, 3,45-77 of cyclitols containing four or five hydroxyl groups, 20, 11-65 of the 2-deoxy sugars, 8.45-105 of heparin, 10,335-368 of mucopolysaccharides and mucoproteins, 2, 161-201 of the nucleic acids, 1,193-245 ofosazones, 20,139-181 of osotriazoles, 18,99-121 of pectic materials, 2,235-251 of ribose, 6,135-174 of streptomycin, 3,337-384 of thio sugars, 18,123-199 physical, of carbohydrates, 15, 11-51 of starch, 11,335-385 radiation, of carbohydrates, 16, 13-58 stereo-, of cyclic derivatives of carbohydrates, 10,l-53 structural, of fungal polysaccharides, 23, 367-417 of the hemicelluloses, 14, 429-468 of polysaccharides, 15,53-89 Chitin, 15,371-393 Chromatography, column. See Column chromatography. gas-liquid. See Gas-liquid chromatography. paper. See Paper chromatography. Color, of sugar products, 9,247-284 Column chromatography, of sugars and their derivatives, 10, 55-94

511

Combustion, of cellulosic materials, 23, 419-474 Complexes, of carbohydrates, with alkali metals and alkaline-earth metals, 21, 209-271 cuprammonium-glycoside, 6, 107- 134 Composition, of sugar solutions, 2 3 , l l - 5 7 Configuration, of carbohydrates, determination of, 4, 189-210 of cyclitols, 3,45-77 of higher-carbon sugar alcohols, 1, 1-36 of sucrose, 4 , l - 3 5 Conformation, in formation of polysaccharide gels and networks, 24,267-332 Conjugates, of D-glycuronic acid, 9,185-246 of sugars with sphinogosines, 24, 381433 Constitution, of carbohydrates, 1 5 , l l - 5 1 Crystallinity, relative, of celluloses, 5,103-126 Crystal-structure analysis, in carbohydrate chemistry, 19,7-22 Cuprammonium-glycoside complexes, 6, 107-134 Cyanohydrin synthesis, Fischer, I, 1-36 Cyclic acetals, of the aldoses and aldosides, 20, 219302 Cyclic derivatives, of carbohydrates, stereochemistry of, 10,l-53 Cyclic monosaccharides, having nitrogen or sulfur in the ring, 23,115-232 Cyclitols, 14,135-212 chemistry and configuration of, 3,45-77 containing four or five hydroxyl groups, chemical and physical studies of, 20,ll-65

D Degradation, of acylated nitriles of aldonic acids, 4, 119-151

512

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24

glycopyranosyl, of nucleotides, 18, ofcarbohydrates, 19,181-218 309-356 enzymic, of glycogen and starch, 3,251310; 17,407-430 beta-ketonic (and related substances), thermal, of starch, 22,483-515 reactions with monosaccharides, 11,97-143 3-Deoxyglycosones. See Glycosuloses, 3nitrate, of starch, 13,331-345 deoxy-. of starch, preparation and properties 3-Deoxyglycosuloses. See Glycosuloses, of, 1,279-307 3-deoxy-. sulfonic, of carbohydrates, 8, 107-215; Deoxy sugars. See Sugars, deoxy. 23,233-280; 24,139-197 Desulfurization, Ethanol, 2-nitro-, reductive, by Raney nickel, 5,l-28 syntheses with, 6,291-318 Dextran, structure and synthesis of, 15,341-369 Ethers, benzyl, of sugars, 12,137-156 Dextrins, carboxymethyl, of cellulose, 9,285-302 the Schardinger, 12,189-260 of cellulose, 2,279-294 Dextrose, methyl, commercial production of crystalline, 5, of the aldopentoses, 7,1-36; 10,257127-143 272 “Dialdehydes,” of 2-amino-2-deoxy sugars, 13, 189from the periodate oxidation of carbo214 hydrates, 16,105-158 of fucose, 7,1-36; 10,257-272 Dicarbonyl derivatives, of D-galactose, 6, 11-25; 10, 273-282 of carbohydrates, 17,223-299 of D-glucose, 5,145-190 Difructose, of hexuronic acids, 9,131-148 anhydrides, 2,253-277 of D-mannose, 8,217-230 Disaccharides, enzymic synthesis of, 5,29-48 of rhamnose, 7,l-36; 10,257-272 trityl, of carbohydrates, 3,79-111 trehalose, 18,201-225

E F

Electrophoresis, of carbohydrates, paper, 18.61-97 zone, 12,81-115 Enzymes. See also, Amylases, Galactosidases, P-Glucuronidase, acting on pectic substances, 5, 79-102 degradation by, of starch and glycogen, 3,251-310; 17,407-430 specificity of, in the domain of carbohydrates, 5,49-78 starch degrading and synthesizing, 23, 281-366 synthesis by, of glycogen and starch, 17, 371-407 of sucrose and other disaccharides, 5, 29-48 Esters, of cellulose, with organic acids, 1,309327

Fat, metabolism of, 2,119-160 Fischer, Emil, and his contribution to carbohydrate chemistry, 21, 1-38 Fischer, Hermann Otto Laurenz, obituary of, 17,l-14 Formazan reaction, in carbohydrate research, 13,105-167 Formulas, stereo-, writing of, in a plane, 3,1-22 Fractionation, of starch, 1,247-277; 16,299-333 Friedel-Crafts process, in the carbohydrate series, 6,251-289 Fructans, 2,253-277 Fructofuranoside, a-D-ghcopyranosyl p-D-,4,l-35 Fructosans, poly-. See Fructans.

CUMULATIVE SUBJECT I N D E X FOR VOLS. 1-24 Fructose, and its derivatives, 7, 53-98; 22, 229305 di-, anhydrides, 2,253-277 Fucose, methyl ethers of, 7,l-36; 10,257-272 Fungal polysaccharides, structural chemistry of, 23,367-417 Furan compounds, formation from hexoses, 6,83-106

G Galactose, methyl ethers of D-, 6, 11-25; 10, 273-282 Galactosidases, 16,239-298 Gas-liquid chromatography, of carbohydrate derivatives, 19, 95-147 Gels, polysaccharide, 24,267-332 Glucose. See also, Dextrose. biosynthesis of aromatic compounds from D-, 15,235-270 methyl ethers of D-, 5,145-190 Glucuronic acid, D-, chemical synthesis of, 8,231-249 conjugates of, of animal origin, 9, 185-246 in metabolism, 8,251-275 P-Glucuronidase, preparation and properties of, 14, 381-428 Glutose, 3,113-128 Glycals, 7,209-245 -, 2-hydroxy-, 9,97- 129 Glycofuranosides, 21,95-142 Glycogens, enzymic degradation of, 3, 251-310; 17,407-430 enzymic synthesis of, 17,371-407 molecular structure of, 12,261-298 Glycolipides, of acid-fast bacteria, 16,207-238 Glycoside-cuprammonium complexes, 6, 107- 134 G1ycosides, acid-catalyzed hydrolysis of, 22, 25- 108 alkali-sensitive, 9,59-95 cardiac, 1,147-173

513

the sugars of, 17,65-120 methyl, of the common sugars, 12, 157-187 of the parsley plant, 4,57-74 phenyl, of the common sugars, 12, 157-187 C-Glycosides. See C-Clycosyl compounds. Glycosiduronic acids, of animals, 9,185-246 poly-, ofplants, 1,329-344 Clycosphingolipids, 24,381-433 Glycosones, 3-deoxy-. See Glycosuloses, 3-deoxy-. Glycosuloses, 3-deoxy-, and the degradation of carbohydrates, 19, 181218 Glycosylamines, 10,95-168 Glycosyl azides, 16,85-103 C-Glycosyl compounds, naturally occurring, 18, 227-258; 20,357-369 Glycosyl fluorides, 16,85- 103 Glycos yl halides, and their derivatives, 10,207-256 Goepp, Rudolph Maximilian, Jr., obituary of, 3, xv-xxiii Grignard process, in the carbohydrate series, 6,251-283 Gums (see also, Hydrocolloids), 24, 333379 commercial, 13,265-287 of plants, 4,243-291

H Halogen oxidation. See Oxidation, halogen. Halogenated carbohydrates, 22,177-227 Haworth, Walter Norman, obituary of, 6 , l - 9 Hemicelluloses, structural chemistry of, 14,429-468 of wood, 19,247-302; 20,409-483 Heparin, chemistry of, 10,335-368 Heuser, Emil, obituary of, 15, 1-9 Hexitols, acetals of. 7,137-207 anhydrides of, 5,191-228

514

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24

and some of their derivatives, 4, 21124 1 synthesis of, 2,107-1 14 Hexofuranoses, 1,6-anhydro-, 7,37-52 Hexosans, 7.37-52 Hexoses. See also, Hexofuranoses. formation of furan compounds from, 6, 83-106 Hexuronic acids, methyl ethers of, 9,131-148 Hibbert, Harold, obituaryof, 16,l-11 History, of mutarotation, 23.11-57 Hudson. Claude Silbert. obituary of, 9, xiii-xviii Hyaluronic acid, biosynthesis of, 12,299-319 H ydrazones, of sugars, 3 , s - 4 4 Hydrocolloids, commercial, pol ysaccharidic, 13, 265287 Hydrogen peroxide, action on carbohydrates and related compounds, 19,149-179 Hydrolysis, acid-catalyzed, ofglycosides, 22,25- 108 Hydroxyl groups, relative reactivities of, 8, 1-44 I

Infrared spectra, of carbohydrates, 12,13-33 Infrared spectroscopy, and carbohydrate chemistry, 19,23-49 Irvine, James Colquhoun, obituary of, 8, xi-xvii Isotopes, effects of, in mutarotation of sugars in solution, '24,13-65 Isotopic tracers. See Tracers, isotopic.

K Ketals. See Acetals. Kinetics, basic, ofmutarotation, 23,ll-57

Kojicacid, 11,145-183 Kuhn, Richard, obituary of, 24.1-12 L Lactose, 16,159-206 Lead tetraacetate, action of, on the sugars, 14,9-61 Levene, Phoebus Aaron Theodor, obituary of, 12,l-12 Lipids, glycosphingo-. See Glycosphingolipids. Lobry de Bruyn-Alberda van Ekenstein transformation, 13,63-103

M Maillard reaction, 14.63-134 Mannose, methyl ethers of D-, 8,217-230 Mass spectrometry, of carbohydrate derivatives, 21,39-93 Materials, cellulosic, combustion and pyrolysis of, 23,419-474 Mechanism, in the formation of polysaccharide gels and networks, 24,267-332 of replacement reactions in carbohydrate chemistry, 9,1-57 Melezitose, 2, 1-36 structure of, 8,277-290 Metabolism, of carbohydrates, 2,119-160 use of isotopic tracers in studying, 3, 229-250 of fat, 2,119-160 of the sugar alcohols and their derivatives, 1, 175-192 D-glucuronic acid in, 8,251-275 Methane, nitro-, syntheses with, 6,291-318 Methods, in structural polysaccharide chemistry, 15,53-89 Methyl ethers. See Ethers, methyl. Meyer, Kurt Heinrich, obituary of, 11, xiii-xviii

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24 Molasses, cane, 3,113- 128 cane final, composition of, 8,291-314 Molecular structure, of glycogens, 12,261-298 Mononucleotides, 22,307-419 Monosaccharides, biosynthesis of, 11,185-262 cyclic, having nitrogen or sulfur in the ring, 23,115-232 reactions of, with beta-ketonic esters and related substances, 11, 97-143 Mucilages (see also, Hydrocolloids), 24, 333-379 commercial, 13,265-287 of plants, 4,243-291 Mucopolysaccharides. See Polysaccharides, muco-. Mucoproteins. See Proteins, muco-. Mutarotation, of sugars in solution: Part I. History, basic kinetics, and composition of sugar solutions, 23, 11-57 Part 11. Catalytic processes, isotope effects, reaction mechanisms, and biochemical aspects, 24,13-65 Mycobacterium tuberculosis, polysaccharides of, 3,311-336

N Neighboring-group participation, in sugars, 22,109-175 Networks, polysaccharide, 24, 267-332 Neuberg, Carl, obituary of, 13,l-7 Neuraminic acids, and related compounds, 13,237-263 Nickel, Raney. See Raney nickel. Nitrates, of starch, 13,331-345 of sugars, 12,117-135 Nitriles, acylated, of aldonic acids, 4,119-151 Nitro sugars. See Sugars, nitro. Nonulosaminic acids, 13, 237-263 Nuclear magnetic resonance, 19,51-93 Nucleic acids, 1,193-245; 11,285-333

515

Nucleosides, bacterial, 15,201-234 purine, 17,301-369 pyrimidine, 14,283-380 Nucleotides, bacterial, 15,201-234 glycopyranosyl esters of, 18,309-356 mono-, 22,307-419 0 Obituary, of Emile Bourquelot, 18,l-8 of Emil Fischer, 21,l-38 of Hermann Otto Laurenz Fischer, 17, 1-14 of Rudolph Maximilian Goepp, Jr., 3, xv-xxiii of Walter Norman Haworth, 6 , l - 9 of Emil Heuser, 15,l-9 of Harold Hibbert, 16,l-11 of Claude Silbert Hudson, 9, xiii-xviii of James Colquhoun Irvine, 8, xi-xvii of Richard Kuhn, 24,l-12 of Phoebus Aaron Theodor Levene, 12, 1-12 of Kurt Heinrich Meyer, 11, xiii-xviii of Carl Neuberg, 13,l-7 of Edmund George Vincent Percival, 10, xiii-xx of Clifford Burrough Purves, 23,l-10 of Fred Smith, 22,l-10 of John Clinton Sowden, 20,l-10 of A h a Thompson, 19,l-6 of G6za ZemplBn, 14,l-8 Oligosaccharides, 17,121-167 the raffinose family of, 9, 149-184 synthesis of, 6,27-81 Orthoesters, of carbohydrates, 1,77-127 Osazones, chemistry of, 20,139-181 of sugars, 3,23-44 Osones, 11,43-96 Osotriazoles, chemistry of, 18.99-121 Oxidation, halogen, of simple carbohydrates, 3, 129-148 lead tetraacetate, of sugars, 14,9-61

516

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24

periodate, of carbohydrates, 11,l-41 the “dialdehvdes” from.. 16.. 105-158 selective catalytic, of carbohydrates, employing platinum catalysts, 17, 169-221 0 x 0 reaction, application to some carbohydrate derivatives, 23,59- 114 Oxygen ring, formation and cleavage of, in sugars, 13, 9-61

effect on carbohydrate systems, 21, 377-430 Plants, glycosides of parsley, 4,57-74 gums of, 4,243-291 mucilages of, 4,243-291 polyuronides of, 1,329-344 Platinum. See Catalysts. Pneumococcal polysaccharides, 19, 303357 Polyfructosans. See Fructans. Polyglycosiduronic acids. See GlycosiP duronic acids, poly-. Polysaccharides. See also, Carbohydrates, Paper chromatography, Cellulose, Dextran, Dextrins, of carbohydrates and related comFructans, Glycogen, Glycosiduropounds, 9,303-353 nic acids (poly-),Pectin materials, Paper electrophoresis, Starch, and Xylan. of carbohydrates, 18,61-97 alkaline degradation of, 13,289-329 Parsley, associated with wood cellulose, 10, glycosides of the plant, 4,57-74 283-333 bacterial, 2, 203-233; 15, 271-340 Participation, neighboring-group, in sugars, 22, 109blood group, 4,37-55 175 chemical synthesis of, 21,431-512 Pectic materials, fungal, structural chemistry of, 23, chemistry of, 2,235-251 367-417 enzymes acting on, 5,79- 102 gels and networks, role of structure, Pentitols, conformation, and mechanism, 24, acetals of, 7,137-207 267-332 anhydrides of, 5,191-228 hydrocolloidal, 13,265-287 synthesis of, 2,107-118 methods in structural chemistry of, 15, 53-89 Percival, Edmund George Vincent, obituary of, 10, xiii-xx muco-, chemistry of, 2,161-201 of Gram-negative bacteria, 15, 271-340 Periodate oxidation. See Oxidation, periodate. of Mycobacterium tuberculosis, 3,311Phenol-carbohydrate derivatives, 336 in higher plants, 20,371-408 of seaweeds, 8,315-350 Photochemistry, physical properties of solutions of, 18, of carbohydrates, 18,9-59 357-398 Physical chemistry, pneumococcal, 19,303-357 of carbohydrates, 15,ll-51 shape and size of molecules of, 7, 289of starch, 11,335-385 332; 11,385-393 Physical properties, x-ray structure of, 22,421-482 of solutions of polysaccharides, 18, Polyuronides, 357-398 of plants, 1,329-344 Physical studies, Preparation, of cyclitols containing four or five hyof esters of starch, 1,279-307 droxyl of P-glucuronidase, 14,381-428 . groum - 20.11-65 Plant-growth substances, Properties,

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-21

of 2-amino-2-deoxy sugars and their derivatives, 15, 159-200 of esters of starch, 1,279-307 of P-glucuronidase, 14,381-428 physical, of solutions of polysaccharides, 18, 357-398 physicochemical, of carbohydrates, 15, 11-51 Proteins, compounds with carbohydrates, in human urine, 24,435-452 muco-, chemistry of, 2,161-201 Psicose, 7.99-136 Purines, nucleosides of, 17,301-369 Purves, Clifford Burrough, obituary of, 23,l-10 Pyrimidines, nucleosides of, 14,283-380 Pyrolysis, of cellulosic materials, 23, 419-474 R Radiation, chemistry of carbohydrates, 16,13-58 Raffinose, family of oligosaccharides, 9, 149-184 Raney nickel, reductive desulfurization by, 5,l-28 Reaction, the formazan, in carbohydrate research, 13,105-167 the Maillard, 14,63-134 the 0x0, application to some carbohydrate derivatives, 23,59- 114 Reactions, mechanisms of, in mutarotation of sugars in solution, 24,13-65 of amino sugars with beta-dicarbonyl compounds, 20,303-355 of cellulose, 19,219-246 of monosaccharides with beta-ketonic esters and related substances, 11, 97- 143 Reactivities, relative, of hydroxyl groups of carbohydrates, 8, 1-44

517

Rearrangement, the Amadori, 10,169-205 Reductions, biochemical, at the expense of sugars, 4,75-117 Replacement reactions, mechanisms of, in carbohydrate chemistry, 9, 1-57 Rhamnose, methyl ethers of, 7,l-36; 10,257-272 Ribose, chemistry of, 6,135-174

S Saccharides, biosynthesis of, from glycopyranosyl esters of nucleotides, 18,309-356 Saccharification, of wood, 4,153-188 Saccharinic acids, 12,35-79 four-carbon, 13,169- 188 Schardinger dextrins, 12,189-260 Seaweeds, polysaccharides of, 8,315-350 Seleno sugars. See Sugars, seleno. Shape, of some polysaccharide molecules, 7, 289-332; 11,385-393 Sialic acids, 13,237-263 Size, of some polysaccharide molecules, 7, 289-332; 11,385-393 Smith, Fred, obituary of, 22,1-10 Soil, carbohydrates in, 16,335-355 Solutions, of polysaccharides, physical properties of, 18,357-398 of sugars, mutarotation of, 23, 11-57; 24,13-65 Sorbose, 7,99-136 Sowden, John Clinton, obituary of, 20,1-10 Specificity, of enzymes, in the domain of carbohydrates, 5,49-78

518

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24

Spectra, infrared, “Sugar nucleotides.” See Nucleotides, of carbohydrates, 12,13-33 glycopyranosyl esters of. Spectrometry, mass, Sugar products, of carbohydrate derivatives, 21,39-93 color and turbidity of, 9,247-284 Spectroscopy, infrared, Sugar refining, and carbohydrate chemistry, 19,23-49 granular adsorbents for, 6,205-230 Sphingosines, conjugates with sugars, 24, sugars, 381-433 action of lead tetraacetate on, 14,9-61 Starch, amino, degrading and synthesizing enzymes, aspects of the chemistry of, 14, 21323,281-366 28 1 enzymic degradation of, 3, 251-310; derived from antibiotic substances, 17,407-430 18,259-308 enzymic synthesis of, 17,371-407 methyl ethers of, 13,189-214 fractionation of, 1, 247-277; 16, 299properties of, 15,159-200 333 reactions with beta-dicarbonyl comnitrates of, 13,331-345 pounds, 20,303-355 physical chemistry of, 11,335-385 2-amino. See Sugars, 2-amino-2-deoxy. preparation and properties of esters of, 2-amin0-2-deoxy. 7,247-288 1,279-307 anhydro, thermal degradation of, 22,483-515 chemistry of, 2,37-77 Stereochemistry, benzyl ethers of, 12,137-156 of cyclic derivatives of carbohydrates, biochemical reductions at the expense 10,l-53 of, 4,75-117 formulas, writing of, in a plane, 3,1-22 branched-chain, of natural occurrence, Streptomycin, 11,263-283 chemistry of, 3,337-384 of the cardiac glycosides, 17,65-120 Structural chemistry, conjugates, with sphingosines, 24,381of fungal polysaccharides, 23, 367-417 433 of the hemicelluloses, 14,429-468 deoxy, 21,143-207 Structure, molecular, 2-deoxy, 8,45-105 of cellulose, 19,219-246 higher-carbon, 17,15-63 of dextran, 15,341-369 configurations of, 1.1-36 of glycogens, 12,261-298 hydrazones of, 3,23-44 of polysaccharide gels and networks, methyl glycosides of the common, 12, 24,267-332 157-187 of sucrose, 4, 1-35 neighboring-group participation in, 22, x-ray, of polysaccharides, 22,421-482 109-175 Sucrose. See also, Sugar. nitrates of, 12,117-135 enzymic synthesis of, 5,29-48 nitro, 24,67-138 structure and configuration of, 4,1-35 osazones of, 3,23-44 utilizaton of, 4,293-336 oxygen ring in, formation and cleavage sugar, of, 13,9-61 aconitic acid as by-product in manuphenyl glycosides of the common, 12, facture of, 6,231-249 157-187 Sugar alcohols, related to altrose, 1,37-76 higher carbon, configurations of, 1, seleno, 1,144-145 1-36 solutions of, mutarotation of, 23,ll-57; and their derivatives, metabolism of, 1, 24,13-65 175-192 sulfates of the simple, 20,183-218

CUMULATIVE SUBJECT INDEX FOR VOLS. 1-24

519

and their derivatives, column chroma- Trehaloses, 18,201-225 Trityl ethers, tography of, 10.55-94 of carbohydrates, 3,79-111 thio, 1,129-144 developments in the chemistry of, 18, Turanose, 2,l-36 Turbidity, 123-199 of sugar products, 9,247-284 unsaturated, 20,67-137;24,199-266 Sulfates, of the simple sugars, 20,183-218 U Sulfonic esters, of carbohydrates, 8, 107-215;23, 233- Unsaturated sugars. See Sugars, un280;24,139-197 saturated. Synthesis, Ureides, glycosyl, 13,215-236 biochemical, of monosaccharides, 11, Urine, human, 185-262 protein-carbohydrate compounds in, of cardenolides, 21,273-321 24,435-452 chemical, of D-glucuronic acid, 8,

231-249 of polysaccharides, 21,431-512 W of dextran, 15,341-369 enzymic, Wood, of glycogen and starch, 17,371-407 hemicelluloses of, 19, 247-302; 20, of sucrose and other disaccharides, 5, 409-483 29-48 polysaccharides associated with cellulose of, 10,283-333 T saccharification of,4,153-188 Tagatose, 7.99-136 Teichoic Acids, 21,323-375 Tetritols, acetals of, 7,137-207 Thiocarbonates, ofcarbohydrates, 15,91-158 Thio sugars. See Sugars, thio. Thompson, Aha, obituary of, 19,l-6 Tracers, isotopic, 3,229-250 Transformation, the Lobry de Bruyn-Alberda van Ekenstein, 13,63-103

X X-Rays, crystal-structure analysis by, 19,

7-22 Xylan, 5,269-290 Z

Zemplbn, Gbza, obituary of, 14.1-8 Zone electrophoresis, of carbohydrates, 12,81-115

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Page 65, footnotes. Insert references: (120b) M. Zief and R. C. Hockett, J . Amer. Chem. Soc., 67, 1967 (1945). (12Oc) F. Cramer, H. Otterbach, and H. Springmann, Chem. Ber., 92,384 (1959).

520