Advances in Carbohydrate Chemistry, Volume 21

ADVANCES IN CARBOHYDRATE CHEMISTRY VOLUME 21 Advances in Carbohydrate Chemistry Editor MELVILLE L. WOLFROM Associate...

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ADVANCES IN CARBOHYDRATE CHEMISTRY

VOLUME 21

Advances in Carbohydrate Chemistry Editor MELVILLE L. WOLFROM Associate Editor R. STUART TIPSON Board of Advisors W. W. PIOMAN

It. C. HOCKET~

ROYL. WHISTLEB

Board of Advisors for the British Isles SIB EDMIIND H I ~ T

STANLEY PEAT

MAURICE STACEY

1966

Volume 21

ACADEMIC PRESS REPLICA REPRINT

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or Haroourt Breoe Jovanovloh. Publlehere

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This is an Academic Press Replica Reprint reproduced directly from the pages of a title for which type, plates, or film no longer exist. Although not up to the standards of the original, this method of reproduction makes it possible to provide copies of books which otherwise would be out of print.

PRINTBD IN THE UNITED STATES OF AMERICA

8182

9 8 7 6 5 4 3 2

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

A. R. ARCHIBALD, Microbiological Chemistry Research Laboratory, Department of Organic Chemistry, University of Newcaslle uponTyne, England (323)

J. BADDILEY, Microbiological Chemistry Research Laboratory, Department

of Orgunic Chemistry, University of Newcastle upon Tyne, England (323)

K. VENKATRAMANA BHAT,Department of Chemistry, Georgetown University, Washington, D. C. (273) 0. S. CHIZHOV, Institute for Chemistry of Natural Products, Academy of SdenCeS, MO~COW, U. S. S. R. (39) KARLFREUDENBERG, 6800 Heidelberg, Wilckensstrasse 34, Germany (1) I . J. GOLDSTEIN, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan (431)

JOHNW. GREEN,The Institute of Paper Chemistry, Appleton, Wisconsin (95)

STEPHENHANEBSIAN, Research Laboratories, Parke, Davis & Company, Ann Arbor, Michigan (143) H. W. HILTON,Experiment Station, Hawaiian Sugar Planters’ Association, Honolulu, Hawaii (377) 0

T. L. HULLAR,Department of Medicinal Chemistry, School of P h a m c y , State University of New York at Buflalo, Bufalo, New York (431)

N . K. KOCHETKOV, Institute for Chemistry of Natural Products, Academy of Sciences, Moscow, U . S. S. R. (39) J. A. REINDLEMAN, JR., Northern Regional Research Laboratory, Northern Utilization Research and Development Division, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois (209) W. WERNERZORBACH, Department of Chemistry, Georgetown University, Washington, D. C. (273) V

This Page Intentionally Left Blank

PREFACE The editors herewith present the twenty-first volume in this serial publication. To celebrate our “coming of age,” we are proud to offer a review of the contributions of Emil Fischer to carbohydrate chemistry, by one of his students, Professor Karl Freudenberg. In translation, some of the fine expression and style of the original German may have been lost, yet the review is nevertheless an outstanding evaluation of Fischer’s contributions to the fundamentals of modern carbohydrate chemistry. Current organic chemistry is ever utilizing new instrumentations and techniques that have originated in physics and have been perfected by the modern instrument fabricator. The newest instrument to impinge upon the carbohydrate field is the mass spectrometer, and a review of current work on its use in this area is made by Kochetkov and Chizhov (Moscow). This volume contains two chapters which update topics presented in earlier onea. The chemistry of the deoxy sugars has been expanded considerably since the review in Volume 8 by Overend and Stacey (Birmingham), as is attested by the chapter by Hanessian (Ann Arbor). The article on synthetic cardenolides, or cardiac glycosides, by Zorbach and Bhat (Georgetown) is an extension of related topics previously reviewed by Elderfield (Volume 1) and by Reichstein and Weiss (Volume 17). The discussion of chemical synthesis of polysaccharides, by Goldstein and Hullar, is another contribution to those chapters on carbohydrate polymers which have appeared in this serial publication. Green (Appleton) reviews the generally neglected topic of glycofuranosides. Inorganic chemistry is included in the chapter on complexes of alkali metals and alkalineearth metals with carbohydrates by Rendleman (Peoria). Finally, two topics in biochemistry are reviewed by Archibald and Baddiley (Newcastle) and by Hilton (Honolulu), the former being concerned with the teichoic acids, and the latter with the effectsof plant-growth substances on carbohydrate systemd in plants. The Subject Index for this as well as for the preceding volume has been prepared by Dr. L. T. Capell, long associated with Chemical Abstracts and an internationally recognized authority on organic nomenclature. ColumbuR, Ohio Gaithersburg, Md. Nowmber, 1866

M. L. WOLFROM R. STUARTTIPBON

vii


CONTENTS LIST OF CONTRIBUTOR^ .................................................... PREFACE .................................................................

v vii

Emil Fiacher and Hie Contribution to Carbohydrate Chemistry

KARLFREUDENBERQ I . Introduction ........................................................ I1 Emil Fischer and His Scientific I11 The System of the Monoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V . Development and Extension of V . Ex+uensionof the System, and Tr ......... V I . Oligo- and Poly-saccharides.... ......... V I I StericSeri es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Insights into Stereochemistry and Its Relation to Biochemistry. . . . . . . . . . . IX . GeneralReferencea ..................................................

.

2

.

8

.

27 32 34

16

38

MMSSpectrometry of Carbohydrate Derivatives

.

.

. .

N K . KOCH~TXOV AND 0 S CHIZHOV

I Introduction ........................................................ I1. The Basic Principles of Mass Spectrometry of Organic Compounds . . . . . . . . I11. M w Spectra of Carbohydrate Derivatives ............................. IV Conclusion.......................................................... Addendum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

39 40 46 92

93

The Glycofuranosides

.

JOHN W GREEN

. .

I Introduction ........................................................ I1. Conformation of the Glycofuranosides................................. 111 Formation of Glycofuranosides in Acidic Methanol . . . . . . . . . . . . . . . . . . . . . . IV. Preparation of Glycofuranosidea from Dithioacetals ...................... V . General Preparative Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Structure of Glycofuranosidea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

95

96 101 112 121 127 137 140

Deoxy Sugus STEPHEN HANESSIAN

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

ix

143 144

CONTENT8

X

I11. Monodeoxy Sugars .................................................. I V Dideoxy Sugars ..................................................... V . Trideoxyhexwes..................................................... VI . Chromstog;raphy.................................................... V I I . Nuclear Magnetic Resonance Spectroscopy............................. VIII MeesSpectrometry., ................................................

.

.

145 183 196 197 201 201

Complexes of Alkali Metals and Alkaline-earth Metals with Carbohydrates

.

.

J. A RENDLEMAN. JR

. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 . Complexes of Carbohydrates with Metal Salk . . . . . . . . . . . . . . . . . . . . . . . . . .211 . Complexes from the Interactioniof Carbohydrates with Metal Bases. . . . . . 237 . Alcohohtea from Reactions, in Liquid Ammonia, of Carbohydrates with

I I1 I11 IV

Alkali Metale. Alkalinwarth Metals. and Alkali Metal Amides . . . . . . . . . 269 Synthetic Cardenolides

w . WERNER

zORBACH

AND

K

.

VENKATBAMANA

BUT'

. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !273 . General Methodology Employed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 . Synthesis of Individual Glycosides..................................... 281 . Contribution of the Carbohydrate Component to Physiological Activity . . . . 311 . Table of Synthetic Cardenolidee............... . . . . . . . . . . . . . . . . . . . . . . . . 318

I I1 I11 IV V

The Teichoic Acids

.

.

A R . ARCHIBALD AND J BADDILEY

I . Introduction ........................................................ 323 I1. Surface Structurm of Qram-positive Bacteria ........................... 324 IIX, Discovery of the Teichoic Acids ....................................... 326 I V The Hydrolysis of Eaters of Phosphoric Acid ............................ 328 V Membrane Teichoio Acids ............................................ 332 V I Wall Teichoic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 VII Teiohoic Acids of Actinomycetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 VIII . The Looation of Teichoic Acida in Relation to Cell Structure . . . . . . . . . . . . . . 365 372 IX . Biosynthesis........................................................

. .

. . .

The Effects of Plant-growth Substances on Carbahydrrte Systems

. .

H W HILTON

I. Introduction ........................................................ I1. Indole-3-acetic Acid and 1-Naphthaleneacetic Acid. ..................... I11 Plantgrowth Substances Used aa Herbicides ...........................

.

377 381 392

xi

CONTENTS

IV. Glycosidea and Other Carbohydrate Derivatives as Plant-growth Substances V. Gibberellins and Kinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI The Effects of Plant-growth Subtenca on Sugarcane.................... VII . A b h i o n a n d nipening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

408 416 421 429

Chemical Synthesis of Polysaccharides

.

I . J . GOLDSTEIN AND T. L HULLAR I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Condensation Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Addition Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . IV Methods of Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Applications of Synthetic Polysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

AUTHORINDEXFOR VOLUME 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VOLUME 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CUMULATIVE AUTHOR INDEX FOR VOLUMES 1-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CUMULATIVE SUBJECT INDEX FOR VOLUMES 1-21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EFLRATA ................................................................. STJBJsCT INDEX FOR

431 434 477 491 507 513 538 655 562 572


EMIL FISCHER AND HIS CONTRIBUTION TO CARBOHYDRATE CHEMISTRY* BY KARLFREUDENBERG Heidelberg, Gennany

DEDICATED TO THE MEMORY OF MAXBERGMANN (1886-1944) I. Introduction. ....................................................... 11. Emil Fischer and His Scientific Work. ................................. ................. 111. The System of the Monoees.. .................I. . IV. Development and Extension of the System.. ........................... 1. Beginnings: The Sugars and Phenylhydrazine. . . . . . . . . . . . . . . . . . 2. Additional Methodical Approaches. ........................... 3. Synthesis of Glucose (1890). ....................................... 4. The Configuration of Glucose.. ..................................... 5. The Configuration of Galactose. .................................... V. Extension of the System, and Transformations of the Sugars.. . . . . . . . . . . . . 1. General........................... ... 2. AminoSugere .................................................... 3. Glucal and Deoxy Sugars.. ..................... 4. Functional Derivatives and Their Application. . 5. Glycosides.. ..................................................... 6. Nucleosides and Other N-Glycosyl Compounds. . . . . . . . . . . . 7. Acyl Derivatives; Acyl M VI. Oligo- and Poly-saccharides. . . . . . . .................... 1. General ........................................................... 2. Di- and Tri-saccharides. . VII. Skric Series. ....................................................... VIII. Insights into StereochemiRtryand Its Relation to Biochemistry, . . . . . . . . . . IX. General References. .................................................

* Translated

2 2 8 10

12 13

15 16

17 21

27 32 34 38

from the German by Gerhart Schwab, with the assistance of Edward

W.Koos, Dr. J. M. Harkin, and the editors. The frontispiece is from a hitherto unpub-

lished portrait, the original of which ie to be found in the Institute of Organic Chemistry of the University of Erlangen, Germany. In this biographical notice, we shall maintain, in English translation, some of the nomenclature employed by Emil Fiwher. The names used by Fischer sufficed to meet the needs of the structures as then known, and have served as a solid basis for modern carbohydrate nomenclature, which, however, requires the definition of structural features unknown to Fischer.

2

KARL FREUDENBERQ

I. INTRODUCTION Emil Fischer himself collected, and edited, the greater part of his own work in the carbohydrate field in hie well-known book “Untersuchungen uber Kohlenhydrate und Fermente.” This volume was completed in 1908. Subsequent *ark was compiled under the same title by Max Bergmann, Fischer’s student and faithful assistant; this second volume was published in 1922. Both volumes contain a list of the titles and places of journal publication of his papers, as well as a subject index to them. The present author accordingly considers it unnecessary to cite references here. In a few instances, work is described which appeared subsequent to 1922.

11. EMILFISCHER AND HIS SCIENTIFIC WORK When Emil Fischer died in August, 1919, at the age of 67 years, he bequeathed a lifework of rare comprehensiveness. He had contributed greatly to our knowledge of the material world, and especially to that of organisms. He refocused the thinking in organic chemistry back to its starting point, the world of animated Nature. Emil Fischer was born on October 9th, 1852, and grew up in Euakirchen, near Cologne, Germany. His father was a highly successful business man, and was versatile, full of vitality and practical wisdom, and the posseasor of a cheerful outlook on life. His mother came from the Poensgen family, and was the calmer, more contemplative partner in the happy marriage; her son,inherited much of her nature. From his father, he learned to persevere in working toward his goals, to make quick decisions, and grasp good opportunities. His father first apprenticed him in the timber business, but this experiment failed because of the young apprentice’s antipathy toward this career and because of his persistent illnessdue to a chronic stomach disorder. At long last, Emil waa allowed to study. His first semesters in KekulcYs laboratory in Bonn did not satisfy the ambitious, young chemistry student. In the Fall of 1872, he transferred to Adolf Baeyer and F. Rose in Strassburg. In spite of having these excellent teachers, here as well as in Bonn, he was depressed by the abundance of disorganized material which he despaired of mastering, as he once told me in later years. The clear organization of physics, this older, more mathematical, and already better formed sister science, m taught by Kundt, made him hesitate as to which of the two discipline8 he should choose. But Baeyer won. Fischer’s doctoral work, too, was not without those crises which are often of benefit and which tend to beset primarily the seriouR student. Baeyer’s established ability to evaluate the personalities of young men-he himself possessed a magnificent, harmonious personality-caused him to recognize the worth of the young doctor. He entrusted to him the instruction of the student laboratory

EMIL FISCHER AND HIS SCIENTIFIC WORK

3

course in organic chemistry. Here, in an attempt to save a student’s unsuccessful experiment, Emil Fischer made his first discovery-phenylhydrazine. It was with a thesis on the substituted hydrazines that he qualified in 1878 for the rank of Privatdozent in Munich, whence he had followed his teacher Baeyer. In 1882, at the age of 30 years, he became full Professor and Director of the Chemical Institute at the University of Erlangen; a t the age of 33, Professor in Wtirzburg; and, in 1892, when 40 years old, Professor in Berlin. Very early, he developed his characteristic manner of working. He worked with many selected coworkers, subordinating each individual problem to the clearly envisaged general goal, and, thus, worked out the whole. Adolf von Baeyer accomplished his excellent work with fewer coworkers, whom he used to retain for many years on end; Baeyer might be compared to the leader of a reconnaissance patrol with a trained eye for the terrain and the possibilities of the pathways. In contrast, Emil Fischer was the clever tactician who proceeded on a broad front, here gaining ground very quickly, there lagging back cautiously, until that which lay behind was all in his safe possession. He knew how to make good use of the great advantages of the research program required for doctoral candidates at German universities. Teamwork, as he developed. it, demands a superior leader. It was magnificent to work under his direction and yet independently; as a research instructor, he blazed the trail for the generations succeeding him. Nevertheless, obstacles that slowed down his work arose at times. Chronic phenylhydrazine poisoning had already troubled him during his years in Erlangen. This he overcame, but it reappeared at intervals in Wurzburg and, later, in Berlin. In Wursburg, the need for a new institute caused him much extra work. For him, the practical man, the planning was easy; but he had to fight hard to obtain the requisite funds. This challenge presented new and time-consuming problems, but these were successfully mastered by the efficient businessman’s eon. Later, in Berlin, the same Ptruggle was repeated, but again he emerged the victor. In his construction of them chemistry institutes, too, he again set an example for his successors to follow. His years in Wiirzburg, although full of work and struggle, were nevertheless the happiest of his life. There, in Wurzburg, he married his beloved wife, Agnes Gerlach, who bore him three sons. In the summer of 1892, although reluctant to abandon Wiirzburg, of which he had grown so fond, he accepted an appointment to the greatest professional chair that Kaiser Wilhelm’s Reich had to offer, the chair of chemistry in Berlin, vacated by the death of August Wilhelm von Hofmann. Althoff, the director of the Universities Section in the Prussian Ministry of Education, promised the now forty-year-old Fischer a large new Institute,

4

KARL FREUDENBERG

However, it waR only after much controversy that the latter wai occupied eight yearn later. Out of this conflict with Althoff grew mutual respect and friendship. The Inetitute waR magnificently designed for the standards of that time. It was destroyed during World War 11, but was rebuilt essentially in its original form; and the design of the Institute is, today, still a practical one. Emil Fischer preferred to think synthetically, rather than analytically, Unlike 80 many of his contemporaries, he never gave unbridled rein to his synthetic efforts, nor did Be fall into the temptation of purposeless synthesis. He always remained a true scientist-a student of Nature. He thought in broad correlations, and applied his skill to fundamental problems. Whereas it was said, with some exaggeration, that the congenial Victor Meyer had work done on a different subject on each individual laboratory bench, Emil Fischer proceeded systematically toward distant goals. Theoretical questions played a minor role in his thoughts, although he proved to be a great scientific thinker when he systematically developed and consolidated the sugar series. There were many who were better read than he, but no-one who had more practical experience. He loved the intimate conversation, and I remember many a blissful hour, when he returned to the laboratory at night and sat down to chat, or when he interrupted our writing work so that we might have a simple meal in his villa on the Wannsee. Still clear to me are the memories, of the simplicity of this great man,his respect for nature, and his modesty in his efforts to understand it. The illness and early death of his wife, and the loss of two sons in the First World War, saddened the life of the now lonely man. Yet he lived to see the beginning of the successful Rcientific career of his son Hermann.1 He mastered the administrative burdens of his large Institute with typical competence. Until about 1910, he gave the introductory lectures in inorganic chemistry, with many demonstration experiments. Fischer’s lectures were distinguished by harmony between the spoken word and the experiment, and between objective earnestness and stimulating perfect diction. About that time, 1910, his duties became nearly overwhelming. I once heard him remark-although this statement wa.s not intended to be taken literally-“the move to Berlin was the mistake of my life.” The plan for the “Kaiser-Wilhelm Gesellschaft” matured. The idea was created by Fischer together with his fricnd Adolf Harnack. Fischer was the adviser to the minister Althoff, and to his successor, on questions in the area of chemistry. When World War I began, Fischer was one of tho first to point out, tho t,otdly ~nndoqunt,ceconomic! prepnrstion of Germany. He brought


5

the industrial leaders of the Ruhr district together, and showed them the seriousness of the situation. Extended travel and the chairmanship of committees on the economics of the war were too much for him. When I talked to him in the spring of 1916, during a military furlough of mine, he was aware of the impending, unfortunate outcome of the war. Knowing what ww going to occur, he lived to see the ddbbcle, and he understood the consequences for his countrymen. Nevertheless, he continued his studies with the little group of students who were left-among them, Max Bergmann-until, in August, 1919, the old ailment reappeared and brought the end. Even such a talented life of investigation has its high-lights and shadows. Periods of impetuous progress were followed by years of moderate advances, for example, at the end of the first decade of this century, when his work on the proteins came to a stop because of experimental difficulties insurmountable at that time. It is greatness, too, to bear such periods of lesser productivity calmly and without becoming disconcerted. Emil Fischer’s life wm based on responsibility: responsibility for the austerity and purity of his work and its aims-responsibility for the university as an important organ of our cultural and economic life-and responsibility for each of his students, who always found him ready for discussion and consultation, whenever a question arose concerning their professional goals. He was a civil servant, but no regulations could command the performance of duties he imposed upon himself and on which his life waa based. His influence far exceeded his scientific work and its material consequences. This great individual was a man of inflexible veracity and simplicity, fully devoted to his mission and, withal, a sensitive and shy personality, respectful but commanding respect. Only from such a man can arise the great influences which I am trying to describe. When Justus Liebig’s university studies began about 1820, only irregular courses were available. When Robert Bunsen first went to a university in 1830, he found somewhat more organization in what was presented to him. Emil Fischer registered at the University of Bonn in 1871, when he was nineteen years of age, and in contrast to Liebig and Bunsen, was confronted with chemistry as a clearly defined science, offered in well-ordered courses. This is why Emil Fischer was more influenced by the school which molded him, especially since Adolf Baeyer soon became his teacher. Fischer’s Ph.D. research was carricd out at Stramburg, on fluorescein, and brought him close to the rapidly Moomirig chcmixtry of dyes. Although the great tradition of irivcstigtttion on uric acid niid related compounds had led from .J. Lichig arid F. Wijhler through A. Rwyer to A. Strecker, Baeyer’s group was Atill well familiar with this ficld, and this apparently produced the stimulation which brought about Fischer’s decision, in 1881, to work on

6

KAHL FREUDENBERG

the purines, as he later called them. All other topics which he took up and studied, intensively originated from his own initiative. For ten years, he remained within AdoH Baeyer’s sphere of action. Many a scientist in such an environment becomes dependent on his excellent teacher and cannot fight his way to independence.It was quite the contrary with Emil Fischer. He had discovered phenylhydrazine on his own in his early years, and his thorough study of its reactions led to work on indole; his return later to the ttiphenylmethane dyes was also influenced by the discovery of phenylhydrazine. His first work on sugars, which appeared in 1884, likewise resulted from his studies on phenylhydrazine and the osazones formed by it. Here he was in new territory into which only few had ventured before him, The sugar investigations were continued in Wbzburg, and later in Berlin, with typical energy. It was not a large step from the carbohydrates, their stereochemistry, and the enzymes acting on them, to the amino acids and proteins. It is understandable that the creator of carbohydrate chemistry desired to execute equally good work with the proteins, which presented new tasks for the practised stereochemist. A chance observation on tyrosine led down a narrow path to the phenolic carboxylic acids and thence to the gallotannins, where he encountered the sugars again. One guiding principle behind all his larger projects was the aasumption of repeated interlinking of bifunctional molecules. Thie assumption was realized in the polysaccharides, the proteins, and, to some extent, the tannins. In synthesizing the galloylgallic acids, he encountered the phenomenon of acyl migration, and from this m s e a plan to investigate the fats. Only a few publications on this topic had appeared when he died. When the main fundamental work in one field was done, he moved on to a new one. Only the carbohydrates and their reciprocal relations to the enzymes, as well as the resulting stereochemical problems, fascinated him even to his last days. The greater part of Emil Fischer’s enormous work was directed to entire groups of natural compounds, which he treated fundamentally and, for his time, most thoroughly. The finding that phenylhydrazine forms readily isolable, characteristic compounds with the sugars led him into the sugar field, which had until then been confused and which confronted experimenters with unusually difficult prwtical problems. He discovered the connections between the many kinds of sugars and discovered new ones, using phenylhydrazine again and again, but also applying other methods. He utilized the stereochemistry of the sugars to develop the theory of Van’t Hoff and Le Be1 on the spatial arrangement of atoms, and this led to unanticipated consequences. Here he proved himmlf to be a scientific thinker of the highest rank. In nightly walks through old Wlirzburg, he pondered over the laboratory work of the day, creating and classifying spatial conceptions. A new


7

phase in the chemical observation of Nature was beginning. A new leader was arising to replace the aging Baeyer. In Berlin, the work on the purines was brought to a close, and that on the sugars was continued; indeed, Fischer carried it on throughout his 27 years in Berlin. At the same6:time,he turned to the then central problem of biochemistry, the proteins. On the basis of his experience in the fields of carbohydrates, proteins, and enzymes, Fischer repeatedly pointed out that synthetic chemistry should follow the lead of Nature and develop milder methods. At the same time, he laid the ground-work for a more important finding, The proteins showed very clearly the consequence of the bifunctional nature of their building units, the amino acids. The simple sugars are also bifunctional and are able to combine with themselves into larger compounds. By studying the sugars, amino acids, and proteins, Emil Fischer came to understand and to establish experimental evidence as to the manner in which natural compounds of high molecular weight are built up. This assuredly was his greatest and most important discovery. Organic chemistry developed from pharmacy 200 years ago. Carl Wilhelm Scheele, and, after him, especially French and German pharmacists, began to isolate and investigate natural compounds from plants. About 70 years later, around 1835, synthetic organic chemistry began. From then on, whatever living Nature had to offer to the organic chemist was supplemented and, from time to time, even surpassed by synthetic products, then being discovered aa one new group after another. Synthesis consequently became an end in itself in many laboratories. New methods were developed, leading to an ever-increasing command over materials. The systematic classification of organic compounds was placed in the foreground, and many an organic chemist lost contact with general problems. This course was altered by a few men in the last quarter of the nineteenth century-and a leader among them was Emil Fischer. With the aid of the abundant newly developed organic syntheses, he attacked the problems of natural compounds, not in the form of fortuitously encountered parts of the metabolism of plants or animals, but in that of the fundamental material basis of the living cell. Under his leadership, synthetic and theoretical chemistry was reunited with biochemistry, and a broad scientific basis was restored to organic chemistry. The investigation of natural compounds received an enormous impetus. Biochemistry was established in the chemical research laboratories. It would be misleading to believe that the impact of such an innovator would be limited to his own field. The organic chemical industry began its rise 100 years ago with the synthesis of dyes. There have always been mutual relationships between the laboratories of industry, directed toward production, and those of the universities, which have limited themselves

8

KARL FREUDENBEltQ

increasingly to basic research. Industry absorbs the results obtained in pure science in the universities. Consequently, the influence of such a man as Emil Fischer is not restricted to the laboratories and libraries of the learned, but rather is transferred step by step to the industrial economy, often in t i rapid, sometimes in a slow development. An example of this is encountered in Fischer’s discovery of the bifunctional interlinking of the units in polymeric compounds. Industry and economy shape the lives of individuals and peoples. We can thus appreciate the influence of a creative mind such as Emil Fischer in the scientific world, in scientific thinking, and in the education of generations of chemistry students. We detect it in the laboratories and offices of industry; and we encounter it i t hospitals, in produce markets, in agriculture, and in the household.

111. THESYBTEM OF THE MONOSES The historical aspects of Emil Fischer’s fundamental conventions for writing stereoformulas in a plane have been thoroughly delineated.2 It may be mentioned that the perspective formulas generally attributed to W. N. Haworth were actually a revival of J. B8ese!enJs cyclic formulas.* Van% Hoff had developed a method for representing a sequence of asymmetric centers, such as are uniquely encountered in the sugars, by a system involving tetrahedral geometric forms combined with an algebraic sign denoting right- or left-handedness as established by the observer’s position. This system was bssed on Van’t Hoff’s erroneous concept of optical superposition, which was corrected only in 1931.‘ Emil Fischer found Van’t Hoff’s method unsuitable for his purpose. He, therefore, daringly proceeded to picture the actual spatial models mentally, and invented the two-dimensional projection formulas based upon certain conventions.6This found immediate acceptance, and has since been employed universally. His method of distinguishing antipodes, by means of the prefixed letters d and I , originated partly from genetic relationships and partly from the measwed optical rotation, designated today by (+) and (-). Some years after Fischer’s work, d and I were replaced by the prefixes D and L in order to correlate configurations more closely in a system based relatively on his conventional standards. Emil Fischer suggested the now well-known family tree (see Fig. 1) of the D-seriee of the aldoms, based upon their relationship to n-glyaeraldehyde, despite the fact that this aldehyde and a numbq of other sugars were not known in his time. Figure 1 deviates in (2) C. 8.Hudson, This S m h , 8, 1 (1948). (3) J. BOeaeken, Bet., 46, 2612 (1913). (4) K.Freudenberg and W. Kuhn, Bet., 64, 703 (1931). (5)1 E. Fiecher, Bet., 24, 1836 (1891).

Yo ECOH I EY EOCE I

ECOH J&E

D-(+)Galactose (14)

ECO I

T

"OF" ECOH I E,COE

D-(+)-TPlo.e (15)

HCO I HOCH 1 ECOH I

ECO I I

ECOH

&OH D-(-)-ErgthrOSe

(1)

HCO I HCOH I

E&OH D-(+)-Qy

ceraldebyde (1)

ho. 1.-The

D

Series of the Aldoses. (The rotatory signs in parentheses refer to the equilibrated solutions in water.)

10

KARL FREUDENBERQ

another point from the original nomenclature of E. Fischer. He had originally assigned the prefix 1 to the gulose (12), i d o s (13), xylose ( 6 ) , threose (3) series, to which we iiow amign the prefix D. Such a change was propoaed by M. A. Ibsanoff6 who pointed out that a set of configurational relationships might be inconsistent unless a single asymmetric center was used as a reference standard, namely, that of Dglyceraldehyde. His conclusions were independent of the actual fulfilment of all the experimental steps later completed by A. Wohl and Momber and others. The correction waa accepted and was substantiated' after Wohl had prepared D-(+)glyceraldehyde. In 1891, Fischer agreed to a numbering of the carbon atoms that begins with the most highly oxidized carbon atom. In the case of the dicsrboxylic acids and sugars alcohols, which are structurally symmetrical, such numbering is not unequivocal. Fischer's acyclic formulas were later adapted to the ring forms, when the existence of these became established. In analogy to the common y-lactones, Fischer and the older chemists wrote the sugar rings as furanoid systems. It was only after Emil Fischer's death that W. N. Haworth showed that the six-membered pyranosc rings are more stable, and occur more often, than those of the fivemembered furanoses. The need for such ring formulas dates back to B. Tollens and his m u t e rotation studies. E. Fischer agreed with such cyclic representations for the glycosides, acetates, and acetohalogenoses without reservation; for the free sugars, however, he retained the right to make me of the acyclic, aldehyde formulas whenever this was sufficient to represent reactions, or whenever it was clearer, as, for example, in the representation of thr configurationd relationships of the monoses.

IV. DEVELOPMENT AND EXTENSION OF THE SYSTEM 1. Beeinnings :The Sugars and Phenylhydrazine

As remarked above, in the early eighties, the field of the sugars was not very inviting to the experimentalist, and Fischer would not have decided to enter it had it not been for his discovery of phenylhydrazine, a new and powerful tool to use in sugar chemistry. After the osazones had been discovered, their formation was investigated more closely, and it was found that the first stage was the formation of the phenylhydrazones; among these, the pheqylhydrazone of mannose waa outstanding because of its low solubility, and it was used to identify and to crystallize mannose, which could be obtaincd from it by treatment with aldehydes or acids. Manno,se is (6) M.A. Roaanoff, J . Am. Chem. Soc., 28, 114 (1906). (7) A. Wohl and K.Fraudenberg, Bet., 66, 309 (1923).

EMIL FISCHER AND HIS SCIENTI~ICWORK

11

ucacmihlc by oxidation of muiitiitol, hut it w&nmade readily avtlilable by tho tiydrolyni~of vcgctablc ivory turnings. Pentows and hcxoscn could be distinguished readily with phenylhydrazine. The compound hitherto called “isodulcitol” wae identified as a sugar by means of phenylhydrazine, and was designated ae rhamnose from that time on. Glucose and fructose (which H. Kiliani had discerned to be an aldohexose and a ketohexose, respectively) and mannose were found to give one and the same phenylosazone; the three sugars, therefore, have the same configuration at carbon atoms 3,4, and 5. On treatment with strong acid, the osazones form the osones, which were recognized aa being 2-ketoaldoses by their reaction with o-phenylenediamine. With phenylhydrazine, the osones reform osazones. Zinc dust and acetic acid converts glucosone into fructose. Generally, the aldehyde group of osones is reduced first. Thus, both glucose and mannose could be converted into fructose by this route. By utilizing previous work of H. Kiliani, Fischer oxidized aldoses to aldonic acids and, by further oxidation, to the dibaaic acids; for example, gluconic acid was converted into saccharic (glucaric) acid, and galactonic acid into mucic (galactaric) acid, both of which are tetrahydroxyadipic acids. The pentose which Kiliani recognized as arabinose yielded, by way of arabinonic acid, a trihydroxyglutaric acid. This and other acids were characterized by Fischer phenylhydrazides, from which the respective acid could be regenerated with baryta. Occasionally, the acids were also isolated as their cadmium salts. (p-Bromophenyl)hydrazine, asymmetrical l-methyl-l-phenylhydrazine, and 1,ldiphenylhydrazine were also used for the formation of hydrazides or hydramnes. 2. Additional Methodical Approaches

H. Kiliani, aa Fischer always emphatically acknowledged, discovered and developed the method of building up the aldose series by the cyanohydrin reaction to give nitriles; from the nitrile, the next higher aldonic acid could then be prepared. In 1890, A. Wohl, working in Fischer’s Berlin laboratory, elaborated the dehydration of an aldose oxime to the nitrile, from which the next lower aldose could be prepared by loss of hydrocyanic acid. Fischer exploited the possibilities of sugar extension and degradation afforded by the use of these two important methods. He himself found that aldonolactones could be reduced with sodium amalgam to the aldoses. Thus, the way was opened to proceed from any pentose to the next higher aldose, which, as he soon showed, always arose in two stereoisomeric forms because of the introduction of a new asymmetric center. Another novel method created was the epimerization of aldonic

12

KARL FREUDENBERO

acids caused by heating them in tertinry amines. This steric rearrangement results in an equilibrium involving the two steric positions of the hydroxyl group on the carbon atom adjacent to the caxboxyl group. These methods, together with the customary (but coristantly improved) methods of oxidation and reduction, comprised the preparative tools of Lsugar chemistry before the turn of the century.

3. Synthesis of Glucose (1890) I n 1861, A. Butlerow had heated polyoxymethylene with lime-water, and had obtained a sirup which had a sweet taste and showed characteristic sugar reactions. Twenty-five years later, 0. L6w improved this reaction by using formaldehyde and lime-water at room temperature; but his product, too, could not be adequately characterized by analysis. ’At this point, Emil Fischer began his work. By means of phenylosaeone formation, he demonstrated that both workers had obtained a mixture of sugars. From this mixture, he obtained a crystalline phenylosazone having properties that suggested that it could be glucose phenylosazone. The yield of this phenylosazone was remarkably improved when acrolein dibromide was used instead of formaldehyde. After careful treatment with cold baryta water, Fischer isolated a bromine-free material from which he obtained the phenylosazones of two isomeric hexoses which were named a- and ,3-acrose. Even better results were obtained from the condensation starting with “glycerose,” which is formed from glycerol by oxidation with bromine in the presence of sodium bicarbonate, and which is a mixture of glyceraldehyde and dihydroxyacetone. From the condensation mixture, he again obtained the two acroae phenylosazones. The “a-acrosazotie” obtained was, apart from its optical inactivity, strikingly similar to glucose phenylosazone. Cautious reduction of the a-acromne afforded a sweet sirup which, on further reduction, yielded a beautifully crystalline hexahydric alcohol, a-acritol, which exhibited such a striking similarity to mannitol that it could well be supposed to be its inactive form. However, one kilogram of glycerol yielded only 200 milligrams of a-acritol. Because of this low yield, another route to it, starting from the natural sugttrs, was sought. Natural Dmtbnnose is the aldehyde of natural D-mannitol, and is transformed by the action of bromine water into D-mannonic acid, which was isolated a8 its phenylhydrazide. The acid was regenerated from the phenylhydrazide and isolated as its crystalline lactone. Kiliani had obtained the enantiomorph of this lactone on applying the cyanohydrin reaction to natural barabinose. A mixture of both lactones formed a racemate. Then, by taking recourse to his newly discovered reduction of the lactones to the aldoses, a reaction which Fischer designated the most significant in the

EMlL FIBCHER AND HIS SCIENTIFIC WOltK

13

entire series, he, now secured D-, t,and Dcmaiuiose, and, from them, the corresponding mannitols. a-Acritol was shown to be DL-ni:ititiitol. .is noted above, the a-acritol arose froni a-ncrose, which, in turn, hid beeii obt:iiud by the reduction of a-acrosone. Sirice the reduction of an omne leads to a ketose, a-acrose must have been Dcfructose; and this conclusion was confirmed by the formation of levulinic acid from crude a-acrose with acids, and by the fact that cy-acrose was partially fermented by yeast, leaving dextrorotatory cfructose. Emil Fischer compared the above work with the construction of a tunnel: “If the mountain is not too broad, one can dig through in one direction. Otherwise, the engineer has to start the work from the opposite side, too. However, the engineer is fortunate, in that he can determine the point of attack by exact measurements and has the certainty of bringing both parts together in the massive interior. Our science is, unfortunately, still far from being deductive enough to permit calculations like that. The chemist can, therefore; count himself lucky if he digs his way through the material from the opposite point8 and finds the connection in the interior by several zig-zag paths.” Now the way wm paved for further work. DLMannose was oxidized to DL-mannonic acid, and its two antipodes were separated by means of brucine. The acids were rearranged to D- and L-gluconic acid by the action of a tertiary amine. D- and L-Glucose were then obtained from the lactones. Also, D-, L-, and Dcmannose too were now obtainable synthetically. The nature of /3-acrose was not explained until later; it is Dcsorbose, and is formed, together with DL-fructose, by the condensation of “glycerose,” the mixture of glyceraldehyde and dihydroxyacetone. With respect ,to the natural hexitol sorbitol, which is formed from D-glucose, D-fructose is 2-keto-sorbito1, and L-sorbose is 5-keto-sorbitol. Many decades later, H. 0. L. Fischer extended his father’s work by preparing D-fructose and D-sorbose through an aldol reaction from a mixture of D-glyceraldehyde and dihydroxyacetone? 4. T h e Configuration of Glucose

The question of the configurations of the sugars, and of methods for eqtabliahiag them, W ~ first S conceived during the course of the synthesis of glucosc described above. The work fell into two distinct divisions: the first concerned the mannitol series, to which glucose belongs; and the second, the dulcitol series, which has galactose as the central compound. In Fischer’s words: “The difference between the mannitol and the dulcitol series lies in the fact that the two innermost carbon atoms in inannose are arranged (8) €3. 0.L. Fiffiher and E.Baer, Helu. Chim. Ada, 19, 519 (1936).

KARL FRI!JUDENBDRQ

14

differently, whereas those in the dulcitol series are arranged similarly. Since, initially, all changes were performed on the outer carbon atoms, it was very difficult to get to the dulcitol series from the mannitol series.” Repeatedly, difficulties arose, because of supernumerary substances which did not fit into the system, such as isosaccharic acid and chitonic acid, which finally both proved to be furanoid derivatives. In the synthesis of glucose, only a few hints regarding its configuration were found, but the problem was completely elucidated between 1891 and 1894. The key to Fischer’s solution lay in the transformation of the monose8 into alditols or into dicarboxylic acids, thus making the ends of the carbon chains alike. Thereby, a few of the monoses lose their optical activity, and some pairs lead to identical products. He designated some of these dicarboxylic acids (or tetrahydroxyadipic acids) “mucic acids” (for example, galactaric acid as “mucic acid,” and allaric acid as “allomucic acid”) and others as “sacchazic acids” (for example, D-glucaric acid as “D-saccharic acid,” and idaric acid as “idosaccharic acid”). During the work on the Rynthesis of glucose, mannose, and fructose, so many observations had been made that the existence, and the symmetry relations, of the still unknown monoses and their acids and alcohols could be predicted with certainty. This theoretical work, based upon the theories of Van’t Hoff and Le Bel, enabled Fischer even to include in the following derivation such members of the sugar group (and their derivatives) aa were actually discovered only later. The following exposition, in contrast to the exact historical development, avoids all of the detours into which Fischer wm forced before he visualized the formulation of the pertinent questions and the definition of his objectives. In addition, Fischer’s original papers are written very concisely, and, for good comprehension, they require thorough familiarity with the subject! D-GlUCORe and its epimer, D-mannose, each give an active dicarboxylic acid, namely, Pglucaric acid and D-mannaric acid; hence, neither of these two sugars can have the configuration assigned to the two epimeric systems allose-altrose (8, 9) and galactose-talose (14,IS) [ ( 8 ) and (14)provide inactive acids]. Among the four remaining sugars (formulas 10 to 13), (11) (mannose) and (13) (idose) are out of the question as the proper configuration for glucose, since they give different dicarboxylic acids which are not formed by any other sugars. On the other hand, D-glucaric acid is formed from (10) (D-glucose), as well as from the enantiomorph of (12) (that is, L-gulose). Fischer had this gulose available. D-Glucaric acid forms a 1,Clactone which was reduced, by way of bgulonic acid, to tgulose; and, conversely, this sugar could be oxidized back to Dglucaric acid. (9) For a historical review of these researches, me C.S. Hudson, J . Chem. Edw., 18, 353 (1941).

EMIL FISCHEB AND HIS SCIENTIFIC WORK

15

I n order to make a final decision between glucose and gulose he had to examine the related pentosee, D-arabinose (5) and D-xylose (6). Arabinose-as could be shown by chain extension and degrading of glucose and mannose-forms a portion of both of these hexoses; its configuration c m be neither that of ribose, nor that of xylose, because, in contrast to arabinose, these pentoses forni inactive dicarboxyljc acids. The formula (7)(lyxose) is ruled out for arabinose, since a one-carbon addition to it leads to an inactive (galactaric) acid and an active (talaric) acid. Consequently, D-arabinose must have formula (5) ; and with that conclusion, (10)had been derived for D- (+)-glucose-but with one restriction. Fischer had to choose between (lo) and its enantiomorph; he chose (10), and wrqs aware of the arbitrary nature of this decision. The same decision fixed the form of all the other series-related sugars represented in Fig. 1. That Fischer had accidentally chosen the correct absolute configuration was not realized until after his death. 5. The Configuration of Galactose

The question of the configuration of galactose was solved in 1904. There still remained the choice between the dicarboxylic acids derived from (8) or (14)for the configuration of galactaric acid. The conversion of this acid into racemic (Dctartaric) acid favored the selection of the dicarboxylic acid derived from (14),but Fischer did not consider this observation to be definitive proof. Galactose forms two galactoheptonic acids which, on oxidation, give two optically active pentahydroxypimelic acids. This would not be the case with ( 8 ) , as one acid would be a meso form. Therefore, galactaric acid must arise from ( 14) or its enantiomorph. Natural galactose is, then, either (14)or its enantiomorph. A decision on this question was found during the work on L-rhamnose (17) (see Fig. 2),a natural C-methylpentose (Bdeoxyhexose) whose configuration at C-5 was then unknown (a circumstance which need not interfere with our considerations). Will and Peters had found (in 1889) that, on oxidation with nitric acid, this sugar loses its C-methyl group, to give the same optically active trihydroxyglutaric acid (16) that Kiliani had obtained from natural I,arabinose (the enantiomorph of 5) ; accordingly, mhamnose is either (17) or its epimer at C-5. Fischer produced an “a-” and a “&rhamnohexonic” acid by applying the cyanohydrin reaction to rhamnose; the ‘‘b” form could also be obtained by epimerization of the “cr-”acid. The “cr-”acid formed galactaric acid (19)on treatment with nitric acid. Only rhmnose of configuration (17) (or its C-5 epimer) can give a rhamnohexonic acid from which galactaric acid can be formed. Therefore, a-rhamnohexonic acid has the configuration (18)(or its C-5 epimer) . The epimer (20) gives

16

XARL FREUDENBERG

L-talaric acid, which could also arise from btaloae or caltrose. It was possible to eliminate the latter alternative, because talose was known to be the epimer of galactose. bTalose thus has formula (22), and its enantiomorph is epimeric with &galactose. Therefore, wgalactose has formula ( 14).

V. EXTIWSION OF THE SYSTEM, AND TRANSFORMATIONS OF THE SUGARS 1. General

Fischer himself considered the above synthesis of glucose, and the determination of the steric structures of glucose and galactose, to be a roundedoff piece of research. We have not shown the detours and obstacles which detain4 the experimentalist. The paths, the concepts adopted, and even the aims to be achieved could be determined only aa the work progressed. A survey will now be given of other results, obtained concurrently with the above, but this survey will necessarily be limited to the more important findings.

EMIL FISCHER AND HIS MCIENTIFIC WORK

17

The framework created for the interrelationships of the hexoses and pentoses, and their derived alcohols and mono- and di-carboxylic acids, revealed gaps which were all closed in the course of time, essentially by means of the preparative methods already described. Among these grips was &ribose, which P. A. Leveiic ltiter found in the nucleic widcr. Other natural compounds found their placrs in the system. Adonitol, found by E. Merck, proved to be the pentitol related to D-ribose; G. Bertrand isolated D-iditol, previously synthesized by Fischer, from Sorbue aucuparia. Fischer synthesized natural perseitol, discovered by Maquenne, and recognized it as one of the mannoheptitols. Another polyhydric alcohol, volemitol, found in a mushroom by E. Bourquelot, was shown by Fischer to be a heptitol. Ascending from the hexoses, he obtained heptoses, octoses, and nonoses. A. Wohl proceeded, by means of his method of degradation, from arabinose and xylose to the tetroses; he synthesized D-(+)-glyceraldehyde,1° which was the basis of the system. Rhamnonic acid was epimerized by Fischer to epirhamnonic acid, and this was reduced to cepirhamnose, which was recognized to be 6-deoxy-L-glucose. He also worked on the C-methylpentose chinovose, but this was not established11as being 6-deoxyD-glucose until after his dcath. Whereas “acetobromoglucose” and its analogs are dealt with under the acyl derivatives of the sugars, “acetodibromoglucose” (2 ,3,4-tri-O-acetyl6bromo-6deoxy-~-g~ucosy~ bromide), isolated in 1902, will be discussed here because of its involvement with C-6 of D-glucose. It is obtained by the prolonged action of hydrogen bromide on D-glucose pentaacetate. With methanol, the dibromo compound gives the acetylated methyl glycoside of the 6bromohydrin; and this, with base, affords the methyl glycoside of an anhydroglucose which shows no mutarotation and exhibits a slow, but positive, fuchsin-sulfur dioxide test. Fischer proposed the structure of a 5 , 6 or a 3,6-anhydride for this compound. The latter alternative was later proved t o be correct. The anhydro sugar forms a phenylosaxone, and can be reduced to an anhydrosorbitol. Hydrogenolysis of the bromohydrin yields a C-methylpentose (6deoxy-~-glucose)which is the enantiomorph of the G epirhamnose (obtained by epimerization and subsequent reduction of rhamnonic acid). Thus, the configuration at C-5 of natural rhamnose (6deoxy-L-mannose) was elucidated. 2. Amino Sugars

Ntrturtil glucon:iiniiic gcivc? glucLonc!phenylosazor~o.D-Arubinose treated wi 111 hydrociy:iriic! :wid rtlid nn~moniiiformcd n nitrile which could be (10) A. Wohl and F. Momlwr, Ber., 47, 3340 (1914);60, 455 (1917). (11) K.Freudenberg and K. %Whig, Ber., 82, 373 (1929).

18

KARL FREUDENBERQ

hydrolyzed to D-glucosaminic acid. Hence the glucosamine obtained from chitin is a 2-amino-2-deoxyhexos, being 2-amino-2deoxy-~-glucose,as ww found much later.I2Fischer found that nitrous acid deaminates glucosamine and its related acid, furanoid 2 ,banhydro derivatives being formed. Starting from glucal,la he also obtained an amino hexose which he called epiglucosamine. Much later, other workers established that this compound l-hino-ldeoxy- D-fructose was obtained by is 2-amino-2-deoxy-~-altrose. reduction of glucose phenylomzone. Acrose phenylosazone behaved similarly, yielding 1-amino-ldeoxy-Dbfructose. 6-Amino-gdeoxyglucose was produced by +heaction of ammonia on the methyl glucoside 6-bromohydrin.

3. Glucal and Deoxy Sugars In 1914, Fischer discovered another transformation of the sugars on treating the “twetohalogenoses” with zinc dust. Glucose thereby forms glucal (24). The reaction is general for the aldoses. One molecule of glucal

D-G~uc~~ (24)

takes up two atoms of hydrogen or bromine. The triacetate of dibromoglucal can be transformed into the monobromohydrin of a hexose, which gives glucose phenylosazone. On treatment with ozone, and subsequent deacetylation, D-glucal tiacetate yields D-arabinose. Tri-O-acetylglucal dibromide reacts with methanol and silver carbonate to give two methyl glycosides of the bromohydrin, both of which form the same methyl 2-deoxyglucoside on reduction with sodium amalgam; (12) Sibungaber. Heidelberger Akad. Wiaa., 9 Abh. (1931). Here it is shown that glucoAsminic acid exhibits optical properties corresponding to a n-a-amino acid; 0. Lutz rrnd R. R. Jirpumns, Ber., 66, 784 (1932); P. Pfeiffer and W. Christeleit, 2. Phyeid. Cham., 247, 282 (1037); W. N. Haworth, H. W. G. Lake, end S. Pqat, J . C h . Soc., 271 (1939); W. 0. Cutler and S. Peat, aBd., 782 (1939); M. L. Wolfrorn, R. U. hrnieux, and 53. M. Olin, J . A m . Chem. Soc., 71, 2870 (1949). Those lwt autliow related gluconanrine to natural ( + ) - s h i n e by methrxb not involving operations on the symmetric center bearing the amino group. (13) See next Section.

EMIL FISCHER AND HIS SCIENTIFIC W O R K

19

this glucoside is hydrolyzed neither by yeast extract nor by emulsin. However, after deacetylation and mild treatment with acid,2deoxyglucose is formed. These experiments revealed a mute to the 2deoxy monoses, substances which later became very important. No noticeable amounts of halogen can be split off from one of the above methyl glucosidebromohydrins by ammonia, even under drastic conditions. The other affords the aminohexose mentioned. Some of the experiments described above can also be effected with the corresponding chloro compounds. 4. Functional Derivatives and Their Application a. Compounds with Acetone.-With acetone and a small proportion of mineral acid, fructose and arabinose form “diacetone” (di-0-isopropylidene) compounds; rhamnose yields a monoacetone compound; and glucose gives a mixture of a mono- and a di-acetone derivative. Later, Fischer employed 8-=glucose for this reaction, as this anomer dissolves more quickly in the reaction mixture than the common a-D form. The monoacetone compound from glucose could be produced from the diacetone compound by mild, partial hydrolysiswith acid. Fischer did not speculate on the constitution of the acetone compounds. As they would not reduce Fehling solution, he assumed that their carbonyl group was involved in the condensation. Alditols, such as mannitol and erythritol, form compounds with three or two molecules of acetone; in aqueous acetone, they combine with only two or one. In all cases, the sugar or alditol could be regenerated with dilute acids. In later work, Fischer used these acetone compounds for the preparation of partially acylated sugars and alditols. b. Compounds with Benza1dehyde.-Benzylidene compounds obtained from alpitols were used for the isolation and characterization of alditole, as well as for their purification, since the parent substanceswere readily regenerated from them. With alditols containing an odd number of hydroxyl groups, one hydroxyl group always remained free; and sometimes, several did not react. Even the hydroxyl groups of alditols having an even number sometimea did not react completely with benzaldehyde. In one case, the monobenzylidene acetal of a glucoheptitol, isomerism was observed which Fischer believed might have arisen from the new asymmetric center formed on the benzylidene carbon atom. The benzylidene compounds, like the acetone compounds, served for the synthesis of partially acylated derivatives. c. Mercaptals.-Fischer found that aldoses react with alkanethiols (and also, a-toluenethiol), in concentrated mineral acid solution, to form acyclic mercaptals (dithioacetals). Cyclic mercaptals were obtained with ethanedithiol and 1,3-propanedithiol. Fischer used these derivatives for

20

KARL FREUDENBERG

the isolation of sugam, as they are formed readily and are, for the most part, easy to crystallize. Today, they form the starting point for certain acyclic derivatives of the sugars. d. Acetohalogeno Sugars; 0rthoacetates.-When Fischer entercd this field, in 1901, the common form of acetobromoglucose ([a]D +199” in chloroform) was known. Its discoverers, W. Koenigs and E. Knorr, had found that it could be converted into methyl Bglucoside and into 8-glucose pentaacetate. Fischer prepared acetobromoglucose by starting from a- and 8-D-glucose pentaacetates. He concluded that, in one of these reactions, a Wdden inversion had occurred. He greatly simplified the preparation of acetobromoglucose by treating the acetylated sugar with hydrogen bromide in acetic acid. He designated the bromo sugar derivative “&acetobromoglucose”; today, we know that a Walden inversion always occurs &.wing the reaction with alcohols, and this bromo derivative is now assigned to the a-D series. The reaction waa extended to other sugars. An analogous chloro (and bromol tetraacetyl derivative w a obtained from galactose. A crystalline hepta-0-acetylmaltosyl chloride was synthesized; the bromide did not crystallize well. By using hydrogen halide in acetic acid, acetobromolactose and acetobromocellobiose, aa well as aceto-iodoglucose and aceto-iodocellobiose were prepared. Representatives of the &acetohalogeno sugars are likewise known. Glucose pentabenzoate was synthesized, and transformed into the benzoylated bromo compound. Fischer obtained a crystalline quarternary salt from acetobromoglucose and pyridine. An anomaly appeared in the rhamnose series. When acetobromorhamnose was treated with methanol, and the product was deacetylated with alkali, a “methyl rhamnoside acetate” wm obtained which had retained an acetate group not saponifiable by alkali; Fischer named this compound y-methyl rhamnoside monoacetate. It was not until 1930 that it wm shown to be the lJ2-(methyl orthoacetate) (25). Today, more orthoaoetates of the same kind are known in the sugar series.

L-Rhamnose 1,2(methyl orthoacetate) (25)

EMIL FIBCHER AND HI8 SCIENTIFIC WOXK

21

5. Glycosides

The many natural compounds of the sugars with alcohols and phenols present a challenge to the synthetic chemist. In 1879, A. Michael had synthesized the first glucoside by allowing acetochloroglucose to react with a phenol. The course of this reaction was rather abstruse, and the @-Dglucoside waa produced in only low yield. In 1893, Fischer found a method of synthesizing alkyl glycosides by saturating a mixture of a sugar and an alcohol with hydrogen chloride. When the sugar was too difficultly soluble in the mixture, he found that he could use its acetylated or acetochloro derivative just as well. Using this reaction a t room temperature, Fischer obtained glycosides (not all crystalline) of aldoses, fructose, and glucuronic acid with different alcohols and hydroxy acids. He favored the cwrently accepted, cyclic hemiacetal structure for the glucosides, but formulated them with the furanoid ring for glucose proposed in 1883 by B. Tollens. w. N. Haworth later established that the glucose residue in such glucosides is present in the form of a pyranoid ring. In 1908, he wrote concerning the methyl at-wglucoside discovered by him in 1893 and its predicted fl anomer found a year later by Alberda van Ekenstein: “After I observed (1894)their characteristic behavior toward emulsin and yeast, I designated the two substances as a- and &methyl-& glucosides, and extended this distinction to the entire class of glucosides. Immediately after the discovery of the alkyl glucosides, I called attention to their structural relation to the two then-known glucose pentaacetates, and advanced the opinion that these are not structural isomers but stereoisomers. I extended the concept of the glucosides to the polysaccharides.” In 1895, Fischer reported a greatly improved glycoside synthesis. He heated a mixture of the sugar with the alcohol containing one percent or less of hydrogen chloride, and continued the heating until the Fehling reduction was no longer positive. Better yields were obtained by this procedure, especially with the acid-labile sugars such as fructose. Again, an anomeric mixture was obtained from glucose, together with an amorphous, readily hydrolyzable product, which he considered to be the acyclic dimethyl aoetdtl of glucose. It was not until 1914 that he succeeded in obtaining this substance in analytically pure condition (by distillation at 0.2 mm) and thus established that here, also, he had a monomethyl compound. Hc named the new glucoside “methyl y-glucoside”; in this name, the prefix had no steric or constitutional significance. About ten years later, W. N. Haworth demonstrated that this product is an anomeric mixture of methyl D-glucofuranosides. Glycosides could also be prepared from ketoses by using the new method. Fischer had also utilized the acetohalogeno sugars in a Koenigs-Knorr

22

KARL FltEUDlCNBEltO

type of condensation with alcohols, using either silver oxide or silver carbonate, and had thus obtained alkyl glycosides. For phenolic glycosides, he employed the alkali phenoxide in dry form, or in solution in water or acetone; glucosides of phenol, resorcinol, phloroglucinol, and 2,4 ,6-tribromophenol were obtained in this way. In his later years, Fischer concerned himself with improvements in this synthesis of glycosides, which had always produced the &D form mainly. By addition of quinoline to the reaction mixture, he obtained an anomeric mixture of glycosides from which the difficultly available C~-Dforms could sometimes be obtained by fractional recrystallization. In the reaction with phenols, he found that some deacetylation may occur, so that improved yields could be obtained by reacetylation procedures. Fischer’s faculty for observation, and his perseverance, led to glycoside syntheses that were more and more complicated, with respect to both the sugar and the aglycon. There is now no difficulty in transforming such disaccharides as maltose or lactose into their acetobromo compounds and, thence, into glycosides. When acetobromoglucose was dissolved in ether, and treated with an aqueous, alkaline solution of benzenethiol, phenyl 1-thioglucoside was obtained; acetobromolactose waa also utilized successfully in this type of synthesis. The cyanogenetic glycoside amygdalin (26) has long been of interest to

&H Amygdalin (a6)

chemists. It is now known to be the B-gentiobioside of the nitrile of D-( -)mandelic acid (27). In 1894, Fischer found that only one molecule of

D- (-1 -Mandel lc acid

(27)

EMIL FISCHER AND HIS SCIENTIFIC WONK

23

glucose WL~Bremoved from amygdalin by yeast extract-a surprising result, as both linkages present are now known to be Bwglucosidic. There remained, after digestion with yeast, a substance which Fischer designated as “Z-mandelonitrile glucoside.” In 1917, E. Fischer and M. Bergmanil treated ethyl dZ-mandelate with acetobromomoglucose and silver oxide, and obtained a diastereoisomericmixture of ethyl O-~-glucosyl-d-mandelate and ethyl 0-D-glucosyl-Lmandelate. This was transformed, through the acetylated amides, into the acetylated nitrile mixture, which was separated by fractional ,recrystallization into the glucosides of (+)- and ( -)mandelonitrile. The latter was identical with that obtained from amygdalin. The former corresponded to sambunigrin, which h d been found in Nature by Bourquelot and Danjou (1905) and which, on hydrolysis with acid, gave c (+)-mandelic acid. The same route was adopted in 1919 to effect the synthesis of natural linamarin, which was known to be the glucoside of acetone cyanohydrin. The simplest member of the series, glycolonitrile glucoside, was also prepared.

6. Nucleosidee and Other N-Glycosyl Compounds Stimulated by the work of P. A. Levene and W. A. Jacobs on the cleavage products of the nucleic acids, Fischer (mainly working together with B. Helferich) published, from 1914 onwards, research on wglucosyl derivatives of purines in which the sugar was attached either to a nitrogen atom or to an oxygen atom. Fischer’s experience with the purines was thereby combined with his knowledge of the sugars. His method relied on the reaction between the acetobromo sugar and salts of the purines, especially the silver salts, in nonaqueous media at elevated temperatures. The N-glucosyltheophylline prepared in this way, through the tetraacetate, had the glucose residue in the 8-D configuration linked to the nitrogen atom at position 9. Linkage to oxygen is here not possible. In contrast, the glucosyltheobromineis hydrolyzed by water at 20’. Only the pyrimidine nucleus comes into question as the position of attachment of the sugar in this compound, and for this linkage, only the nitrogen atom at position 1 or the oxygen atoms at 2 or 6 are possible; a similar situation exists for 1,3 ,’l-trimethyluric acid. Fischer and Helferioh devised a new method for the preparation of glucosyhdcniiie. Thcy started witb dic:hloroadenine, glucosylated it, and replaced the chlorine atoms by hydrogen (by the action of hydrogen iodide and phosphonium iodide). The AT-glucosyladenineso obtained had the sugar residue attached to the nitrogen atom at position 7 or 9 (it was later shown to be at 9). The dichloro-N-gluctosyladerliliewas transformed into a monochloro-N-glucosyladenine. On treatment of this compound

24

KAHL FHEUDENBERQ

with ammonia, followed by nitrous acid, a crystalline glucosylpurine, probably a derivative of guanine, wm obtained. Galactosyl and rhamnosyl derivatives of the purines were also prepared. A further step toward the synthesis of nucleotides was taken in 1914, when Fischer succeeded in phosphorylating N-glucosyltheophylline with phosphoryl chloride and pyridine. The crystalline product obtained bore the phosphate group’on the glucose portion. The substances mentioned above illustrate how Fischer repeatedly performed syntheses of glycosyl combinations wherein the aglycon moiety was varied. The number of such examples was increased in 1914.Starting from silver succinimide and acetobromoglucose, a tetrwetate was obtained which , on deacetylation with ammonia, led to N-8-D-glucosylsuccinimide. Acetobromoglucose and silver thiocyanate gave D-glucosyl thiocyanate which, with ammonia, produced N-glycosylthiourea. When the same reactions were effected with silver cyanate, a N-D-glucosylurea was obtained, which was identical with that prepared in 1903 by N. Schoorl directly from >glucose and urea. ‘7. Acyl Derivatives; Acyl Migration; Gallotannins

In the foregoing Sections, we have frequently mentioned the acetates and other esters, such as the pentaacetates of the hexoses and octaacetates of the disaccharides. It is appropriate to summarize here Fiucher’s work on the partially acylated sugars and alditols. He encountered some of these compounds accidentally; but, when their relation to natural compounds became apparent, he prepared them by intent, using constantly improved methods. When acetohalogeno sugars are treated with moist silver oxide, acetates are produced having the C-1 hydroxyl group free; some of these exhibit mutarotation. Hexose tetraacetates, rhamnose triacetate, and partially acetylated disaccharides were obtained in this manner. Among the disaccharide derivatives, maltose heptaacetate ( 1910) was outstanding, because its ease of crystallization was so great that it could be used for the identification of this disaccharide. Glucose tetrabenmate was synthesized from the benzoate analog of acetobromoglucose. In nearly all other :cases, isopropylidene derivatives were used. “Diacetoneglucose” formed a monobenzoate from which the acetone groups could be removed by dilute acids. The glucose monobenzoate so produced is isomeric with, or may possibly be identical with, a product obtained from vacoiniin, which C. Griebel had isolated in 1010 from cranberries (Vac& n h m viti8 idea). As noted previounly, one ttcetoiie residue is very readily removed from

EMIL FISCHER AND HIS SCIENTIFIC W O R K

25

diacetoneglucose; the three free hydroxyl groups (now known to be at C-3,C-5, and G O ) in the monoacetone compound were benzoylated. After removal of the remaining acetone residue, a glucose tribenzoate was obtained which, from carbon tetrachloride, crystallieed with one molecule of carbon tetrachloride. The original monoacetone tribenmate could be regenerated from this product. Fischer obtained (in 1915-1916) many other partially ,acylated products from isopropylidene derivatives of dulcitol, mannitol, and erythritol. In the reaction of acetobromorhamnose with methanol, Fischer obtained, among other products, a sirupy substance (not well defined) that he considered might have arisen by a change in ring size, with accompanying acyl migration. Acyl migration was first established (in 1911) on a sound experimental basis in his synthesis of digaJlic acid (28) , and was later recognized

HO

OH

OH m-Digallic acid (2 8)

to occur frequently with partially acylated alditols. Thus, two forms of dictcetonedulcitol were obtained, from which two different dibenzoates, which were partially interconvertible, were produced. He explained these migratione by what is now known as neighboring-group participation.

I

I

I

HCO’

I

I

HCOH

I

HCOH

‘OH

I

HC-C-R

II d0

Fischer once termed hexoses “monotonous” because all five of the available hydroxyl groups give derivatives, with little differentiation in reactivity. He broke the “monotony” mainly through use of the isopropylidene derivatives, and thereby prepaxed the way for later developments,

26

KARL FREUDENBERG

Nature provides us with many glycoside esters in which the acyl group is attached to the sugar portion; an example is thc compound vacciniin already mentioned. However, the largest group of natural acyl derivutivcs of sugars is to be found in the gallot.anninsand related substances. After Fischer had found (from 1912 onwards) that the two most iniportant types of gallotannin , namely, the Turkish and Chinese gnllotannins, contain a small proportion of glucose which is difficultly separable with acids from its combination with gallic acid, he esterified a-D-glucose with tris-0-(methoxycarbony1)galloyl chloride in quinoline. After removal of the methoxycarbonyl groups with alkali, a tannin WIM produced which consistd essentially of penta-0-galloyl-wglucose. Since the same product arose from both a- and j3-D-g1ucoseJ a tetra-0-galloyl-D-glucose was also considered as another possibility for the product. On using tri-0-acetylgal\oyl chloride in the same sequence, different products were obtained from the D-glucose anomers (1914). The synthetic tannin had roughly the same content of glucose and gallic acid as had the Turkish gallotannin (from the galls of Quercus infectoh) , However, the natural product contains several percent of bonded ellagic acid (29) and, furthermore, after methylation14 and hydrolysis, it yields,

HO

Ellagic acid (29)

beside8 tri-0-methylgallic acid , a small proportion of di-0-methylgallic acid. Thus, Turkish tannin is not a simple ester of D-glucose with gallic acid. Nevertheless, most of the hydroxyl groups of its D-glucose residue are esterified with gallic acid. For comparative purposes, the anomers of D-glucose pentakis (tri-0-methylgallate) were prepared ; the &D form crystallized. The gallotannin from Chinese leaf galls (Rhus semidata) contains 8-10 gallic acid residues for each glucose residue. A large part of the gallic acid (14) The requisite treatment with diazomethane must be done in the nbsence of methanol, as otherwise the diaaomethane catalyzes a degrdative de-esterification (1914); K.Freudenberg, “Die Chemie der netfirlichen Gerbstoffe,” Springer, 1920.


27

is esterifid, at the met&hydroxyl group,,with a second gallic acid residue. u-Gluccse was then successfully esterified with five equivalents of mdigallic acid (28), employing the methoxycarbonyl or, better, the acetyl group as the protecting group. This synthetic tannin showed great similarities to natural Chinese gallotannin, which, however, has a constitution that is not so regulax D-G~UCOS~ pentakis( penta-O-methyldigallate) was also synthesized, A tri-0-galloyl-D-glucose, prepared by way of monoacetoneglucose, showed. the properties of a tannin. It consisted, in the main, of 3,4,5-tri-Ogalloyl-D-glucofuranose.In 1918, an amorphous mono-O-galloyl-D-glucose was obtained from diacetoneglucose; it did not have tanning properties. After the elucidation of the structure of diacetoneglucose (in 1923), this compound .,odd be regarded as 3-O-galloyl-~-glucose.A mono-O-galloylD-fructose, prepared analogously, was crystalline. From acetobromoglucose and the silver salt of tri-0-acetylgallic acid, with subsequent deacetylation, there w8s prepared a crystalline D-glucosyl gallate; this did not have the properties of a tannin. Hamameli tannin and naturally occurring chebulinic acid wers found to contain sugars esterified with gallic acid (1912).

VI. OLIQO-AND POLY-SACCHARIDES 1. General Monosaccharides “give rise to all polysaccharides in one and the same way, namely, by loss of water, to form residues which condense to a larger system. In the reverse sense, all of the complicated carbohydrates can revert to the simple sugars by hydrolysis. The polysaccharides include sucrose and milk sugar (lactose), as well as the various gums and waterinsoluble materials, such as starch and cellulose.” Considering the great number of monosaccharides, the great variety found in the polysaccharides can be readily understood. Fischer’s investigations of the glucosides led “to the surprising result that, in principle, there is no difference between the glccosides and the complicated carbohydrates.’ The latter are simply to be considered as glucosides of the sugars. This is in harmony with the fact that they are cleaved by acids and enzymes, and also with the results 90 far obtained by synthesis in this difficult area.” When Fischer referred to polysaccharides, he included in that designation di- and tri-saccharides ( 1894) , The name “oligosaccharide” was introduced later, by B. Helferich. Fischer was aware of the fact that the linkage from monnse to monose is glycosidic in nature, and he consequently adopted the cyclic acetal formulas suggested by B. Tollens. He well knew that, in such tionreducing disaccharides as sucrose and trehalose, the hydroxyl group8 of the hemiacetal function are involved in the glycosidic linkage,

28

KARL FREUDENBEHO

He formulated the oxygen-containing ring in the furanoid form type, as w&s customary in those days; the predominance of the pyranose ring (W. N. Haworth, 1926) waa still unknown. As with the simple glycosides, he differentiated between u- and 8-glycosidic linkages from monose to monose. During Fischer’s lifetime, stuchyose, a tetraaaocharide, wns the highest “polysaccharide” of fairly well known structure; beyond this was unexplored territory. It was known only that cellulose, starch, and glycogen afford glucose diaaccharides and dextrins, and that they contain glucosidic linkages and have high molecular weights. Recalling the formation of his “isomaltoas” by treatment of glucose with hydrochloric acid, Fischer stated that, if this treatment is prolonged, “the synthetic process exceeds the formation of isomaltose, and more complicated compounds arise which can be compared to dextrins. The results obtained here may be poor, but suffice for demonstrating, in principle, the possibilities of the synthesis. Certainly, a long way lies ahead before starch or cellulose can be made artificially--but one can already be assured that the aim is not inaccessibly high.” It is evident that, at that time, he already considered that the true polysaccharides already mentioned are the higher members of a series starting with the di- and tri-saccharides. The situation was the same as that found later with the proteins. Starting from the amino acids, he proceeded, by way of the oligopeptides, to an octadecapeptide and thus reached the group of the peptones. For lack of experimental evidence, he wm forced to disregard whether enlargement of the “chains” (the expression was first used in this sense by Tollens), and, as a result, a large molecular size, sufEced to explain the properties of the proteins, starch, and cellulose, or whether some unknown constitutional principles were involved. The inability of starch, cellulose, and glycogen to reduce Fehling solution to a stoichioinetrically detectable extent proved, in Fischer’s time, to be an inburmountable obstacle to every speculation; and so, Fischer concentrated his attention on the oligosaccharides. 2. Di- and Tri-saccharides a. Natural Compounds.-hLaetose forms a phenylosazone and, from this, an omne. With dilute acids, the osazone yields an anhydride (1887). Hydrolysia of lactosone gives glucosone and galactose. WGlucose, therefore, constitute8 the reducing portion of lactose (1888).Five years later, Fischer assigned a ring structure to the galactose portion, and, in 1908, extended this to the glucose portion. Which oxygen atom of the glucose moiety carried the galactose residue remained an open question. Aldobjow form acetohalogeno compounds when their octaacetates are

EMIL FIYCHER A S D HIS SCIENTIFIC WORK

29

treated with hydrogen halide in glacial acetic acid. Experiments designed to produce a tetrasaccharide of the trehalose type from acetobromolactose (with silver carbonate) led to a mixture containing the desired product and lactcse (1910). In 1896, Fischer developed the “pfienylhydrazine test” for the detection of hydrolytic scission of disaccharides, especially by enzymes; this depends on the fmt that the phenylosazones of disaccharides are soluble in hot water, whereas those of the monosaccharides are not. Lactose is hydrolyzed by emulsin (1894) and by lactase; it is not fermentable by yeast, and is unaffected by invertase (1894). An extract of the small intestine of horses and cattle, especially from young animals, hydrolyzes lactose (1896). The action of enzymes on lactose allowed it to be classified, along with cellobiose and maltose, with the “normal” (and not the y-type of) methyl glucoside (1914). In the discussion of maltose, the relationship of lactose to the @-serieswill Be mentioned later. Sucrose yields glucose phenylosazone, only, after hydrolysis (1894). Fuming hydrochloric acid produces (chloromethy1)furaldehyde (1914). The hydrolysis of sucrose by acids was found to be about half as fast as that of “methyl y-glucoside,” and very much faster than the hydrolysis of the anorneric forms of the “normal” methyl glucosides. After Purdie and Irvine (in i905) had obtained from methylated sucrose the same tetramethyl ether of glucose as from the “normal” methyl glucosides, Fischer concluded (in 1914) that the similarity between sucrose and his “methyl y-glucosids” which he had noted could not reside in the glucose portion of sucrose. Sucrosein fermented by nearly all yeasts (1894,1898). Inversion precedes fermentation (1895). Of various animal secretions tried, the only one effective in cleaving sucrose was that from the mucous membrane of the small intestines of several animals (1896). Sucrose was found to be unaffected by emulsin (in 1894). An approximhtely correct formula for sucrose had been published in 1883; its shortcomings were the incorrect ring size for the glucose residue and the uncertainty regarding configurations at the interlinked carbonyl groups. Fischer performed a few experiments with the natural disaccharide trehalose. It does not react with phenylhydrazine. A diastase from .green malt has no action on it; Frohberg yeast has a weak action (1895). Carp blood hydrolyzes it rapidly, in contrast to the blood of other fishes. Extracts of the mucous membrane from the small intestines of horses and cattle are also active (1906). A. Xalanther, in Fischer’s laboratory, found, in 1898. that wine yeasts also hydrolyze this disaccharide. On the basis of reports in the literature, Fischer considered gentiobiose to be an O-&D-glucosylglucose (see cellobiose, p. 30).

30

KARL FREUDENBERG

The nctturally occurring trisaccharide rafinose (melitriose) is hydrolyzed as easily as sucrose, by nearly all yeasts, into fructose and the disaccharide melibiose (Scheibler and Mittelmeier, 1889). The fructose part appears to be combined in the same manner aa in sucrose (Fischer, 1898). Melibiose is hydrolyzed to glucose and galactose by emulsin or, before fermentation, by bottom yeast (1902). The same enzymes split melibiosone into glucosone and galactose; with phenylhydrazine, the resulting glucosone forms glucose phenylosazone in the cold, and the galactose gives galactose phenylosazone on heating (1902) ; therefore, melibiose is an O-galactosylglucose. From acetochlorogalactose, glucose, and sodium hydroxide in aqueous alcohol, a synthetic O-galactosylglucose was obtained which Fischer thought waa melibiose (1902) ; perhaps, it waa actually synthetic lactose (Schlubach and Rauschenberger, 1926). Fischer confirmed the disaccharide formulation for turanose by osazone formation (1894). Turanose is formed from the trisaccharide melezitose, which is fermentable with yeast.

b. Derived Disaccharides.-In contra& to the oligosaccharides discussed above, maltose is a product that does not occur in Nature as such, but only arises by the action of enzymes on starch or glycogen. As it is a reducing disaccharide of glucose, maltose forms both a phenylosazone and an osone. Treatment of the osone with an aqueous extract of brewers’ yeast liberates glucosone, which reacts with phenylhydrazine in the cold to produce glucose phenylosazone (1902) ; after filtration and warming, another portion of glucose phenylosaaone was obtained (from the glucose). Maltohionic acid is hydrolyzed by acids to a mixture of glucqnic acid and glucose (1889). A characteristic derivative of maltose is the readily crystallizable mdtose heptaacetate previously mentioned. Maltose is fermented by nearly all yeasts and, like sucrose, is hydrolyzed by invertase. It is unaffected by emulsin. The parallel between methyl a-D-glucoside and maltose is unmistakable (1898). Maltose and lactose exhibit differences similar to those shown between methyl a-D-glucoside and methyl p-Dglucoside (1894). Cellobicse is readily obtainable through its octaacetate, which is prepared by the aoetolysis of cellulose. It served Fiseher repeatedly as a counterpart to maltose end lactose. The acetobromo and aeetoiodo compounds crystallize weil; the former was used for the preparation of the heptaacetate and many cellobiosides, among them, those of glycolic acid and its amide and nitrile. With silver oxide, it gave the acetate of a nonreducing tetrasaccharide; but it could not be obtained in pure condition. Most of the reactions establish& for glucal could also be effected with cellobial (with G. ZemplCn, 1910).


31

Cellobiore and its &glycosides were hydrolyzed to glucose by emulsin. Cellobiosone and hydrocellobial were also cleaved by this enzyme. Extracts of Aspergillus niger and Kefir (a Caucasian micro-organism that ferments milk) hydrolyzed cellobiose; yeast extract was inactivc. Cellobiose behaves toward eniulsin and yeast extract “like ycntiobioae and (Fischer’s) ‘isomaltose’and has some similarities to lactose.” Furthermore, cellobiose, “isomaltose,” and gentiobiose have the same configuration of their glycosidic linkage; maltose has the other. Fischer expressed the imminent conclusion that cellobiose, lactose, and gentiobiose have a p-D glycosidic linkage only with reservation, because pure, individual enzymes were not at, hand (1909). c. Synthetic Disaccharide8.-Although Fischer’s isomaltose is now known to have been a mixture,16 and the name now designates a pure disaccharide prepared by other methods, there is little doubt that his preparation did contain this substance. He described a phenylosazone. He made his preparation (1890, 1894, 1895) by the action of concentrated hydrochlcric acid on glucose. Impurities that were precipitated on adding alcohol were discarded. Addition of ether to the mother liquor precipitated a mixture which was subjected to yeast fermentation. The phenylosamne was obtained from the nonfermentable residue. More directed syntheses of disaccharides were effected by treating a solution of an acetochloro sugar in ether with a solution of a second sugar in aqueous alcohol containing alkali.16 In this manner, an O-glucosylgalactose, an 0-galactosylglucose (mentioned previously), and an O-galactosylgalactose were obtained, all more or less pure. All three formed phenylosazoiies. Xone of the three was in any marked way fermented by top yeast, s3 that, in this manner, admixed monosaccharides could be removed. On the other hand, bottom yeast fermented the 0-glucosylgalactose and the 0-galactosylglucose, but not the 0-galactosylgalactose. Emulsin hydrolyzed all three. Isolactose is a disaccharide arising when a mixture of glucose and galactose is treated with Kefir extract (1902). I t is different from lactose and melibiose, and forms a crystalline phenylosazonk. When acetobromoglucose was treated with moist silver oxide, a mixture of brominefree acetates was formed from which was obtained, besides glucose tetraacetate, a small proportion of u, crystalline disaccharide octa-

(15) A. Thompmn, K. Anno, M. L. Wolfrom, and M. Inatome, J . Am. Chem. Soc., 76, 1309 (1854); A. Thompson and M. L. Wolfrom, ibid., 76, 5173 (1954). (16) “On introducing acetochloroglucose into an alkaline solution of a monosaccharide in aqueous alcohol, apart from thcir condensation, partial removal of acetyl groups, in the form of ethyl acetate, occurs.” This was the first indication of transesterification by alkoxide (1902), a reaction that later achieved great importance.

32

KARL FltEUDENBEHGI

acetate. From the octaacetate, a sirupy disaccharide (called isotrehalose) was generated. This disaccharide does not reduce Fehling solution, and, in contrast to natural trehalose, is levorotatory. After hydrolysl, it react’s with phenylhydrazine to give glucose phenylommne only. I t therefore has the constitution of trehdoae, but differs in the configuration of its glucosidic linkage. VII. STERIC SERIES

E. Fischer wrote in 1896: “The niost serious discrepancy in the stereochemical system of the sugrtr group at this time is CRW by the uncertainty concerning the configuration of dextrorotatory tartaric acid.”. Since all stereochemical considerations originate from tartwic acid, and , furthermore, since this acid bears simple relationships to malic acid (34), asparagine, etc., I have tried . .again and again to resolve the problem.” It w a successfully ~ solved with the aid of rhamnose (17).The configuration at (3-2, C-3, and C-4 of this sugar had been elucidated at that time. Rhamnose “can be degraded to a methyltetrose (30)by the excellent method of Wohl. If the methyltetrose is finally oxidized with nitric acid, d-tartaric acid (31)is formed. Since, under the same conditions, Gtrihydmxyglutsric acid @e w u referring to tarubiino-trihydroxyglutaric acid (la)] is formed from rhamnose (17),and muck (galactaric) acid (19)is formed from rhamnohexonic acid (18) ,and furthermore, since, in all cases, , . .the methyl group is lost, it seems reasonable to explain the conversion of the methyltetrose into tartaric acid in the same manner. . .,’

..

...

.

.. .

COaH I HCOH I HOCH

Aw

(+)-Tartaric acid (91)

.

CO,H I HOCH I HYOH COaH

(-)-Tartar Ic

acid

(32)

yaH HVOH

4 COSH

meso-

Z0,H

Tar-

D-(+)-

taric, acid (33)

(34)

Malic acid

It now became clear that the (+Nwtrtric acid produced by oxidation of Dsnocharic (mglucaric) acid is formed from its first four carbon atoms, and that racemic tartaric acid (31 32) is produced in the same manner, from mucic (galactaric) acid (19).The choice of formula (31) for (+)-

+

(17) Recher oalled it &tartaric acid.

EMIL FISCHEa AND HIS SCIENTIFIC WORK

33

tartaric acid (rather than 32) follows automatically from the decision already made (between enantiomorphous forms) for the D-glucose projection formula; we now know that this choice conformed to the absolute conGgunttion established later. Fischer had foreseen that the configuration of glucose could be derived from that of the tartaric acids (1894); but this prophecy was rigorously fulfilled only after his death, when M. Bergmann found, in 1921, that n-glucaroditlmide gives (-)-tartaric acid by way of the dialdehyde. Erythrib1 (which is meso) arises from &glucose by way of D-arabinonic acid and D-erythrose (0.Ruff, 1900);the latter can be oxidized to meso-tartaric acid (S.Przyhytek, 1884). (+)-Tartaricacid (31)gives information about the configurations at C-2 and C-3 of D-glucose (lo), (-)-tartaric acid (32) about those at C3 and C-4, and meso-tartaric acid (33) about those at G 4 and G5. (+)-Malic acid is formed by treatment of (+)-thrtaric acid with hydriodic acid (W. Bremer, 1876) and, hence, formula 34 was assigned to It. E. Fischer later classified D-( -)-glyceric acid in this series, and A. Wohl and F. Momber included D-(+)-glyceraldehyde. When Fischer decided to create a system for the a-substituted acids, he wittingly deviated from the convention for the system of sugm and took C-2 (C-u) of the a-hydmxy acids aa the reference carbon atom. He them fore classified (+)-tartaric acid as d (later changed into D). In doing &I, he proceeded from the consideration that ite a-carbon atom corresponds to that in gluconic acid and that, as a twofold a-hydroxy acid, it contains the group of the a-monohydroxy fatty acids twice over, as may be seen by rotating the lower half of its projection formula through 180'. In 1894, Fischer wrote concerning the programmatically demanded extension Gf the configurational theory beyond the sugars to the rr-substituted carboxylic acids and all the other aliphatic compounds containing asymmetricdly substituted carbon atoms: "A vast new field of experimental research has been operied, and since its treatment must ultimately always be connected to the sugars, it will undoubtedly furnish not only a general spreochemicd system for the optically active aliphatic compounds but also mmy new results on the chemicd transformations of the carbohydretes whioh can be of special interest for the chemistry of plants and animals." This prediction haa been fulfilled to an unexpected degree. Today,the configurational theory dominates organic chemistry. l+htural tartaric acid is dextrorotatory in water at the D-line of sodium and has twc asymmetric centers, possessing a rotating axis of symmetry, aa dso do@ mannitol; thus, positions 2 and 3 are chemically and sterically equivalent dthough apparently of opposite configurational sign. Fischer regarded (+)-tartaric acid as the very prototype for the D configuration of the arhydroxy acids. He extended the D designation to all of the a-substi-

34

KARL FREUDENBERG

tuted carooxylic acids (for example, the amino and chloro derivatives) having the configuration

This convention for such acids has been generally accepted. Only one exception has been made in recent years to this convention for denoting the ccnfigurations of a-substituted acids, and curiously, this is with the basic member of the series itself, namely (+)-tartaric acid. If this acid is considered, not as an a-hydroxy acid but, reverting to the sugars, as a sugar derivstive, then it has, of course, to be named I,-(+)-threaric acid.18 This nomenclature is currently preferred by nomenclature commissions; I suggest letting future developments make the final choice between D-(+)-tartaric acid and L(+)-threaric acid for (+)-tartaric acid. Fiscter’s statement, cited above, that aspartic acid bears a simple relationship to the a-hydroxy acids and, consequently, to the sugars, was made with the explicit reservation that no Walden inversion may occur on deaminatioii with nitrous acid (1896). This condition could not be retained. Furthermore, his conclusion that acetobromoglucose belongs to the / 3 - ~ series because it forms j3-D-glucosides is also inadmissible. It is amazing that these two cases seem to be the only ones in which he was misled by the then partially unknown, sometimes unfathomable phenomena of inversion.

VIII.

INSIGHTS INTO STEREO~HEMIGTRYAND

ITSRELATION TO BIOCHEMISTRY It was emphasized above that, during the course of his experimental work, Emil Fischcr had to create new concepts on which to base further exploratory research. A few examples of this will now be cited. In 1894, Fischer wrote: “It will probably be possible to obtain all hembers of the sugar group by a combination of the cyanohydrin reaction with the reduction of lactones, as .won as we have succeeded in finding the two optically activo forms of glyc!eraldohyde. All observations agree with the immcriamc forcseeri by Van’t Hoff, ttbove all the dieappearanca of 1:eomrrs if the molccule becomes constitutionally symmetric. This iricludea the transformation of different stereoisomers into one and the same substance if one of several asymmetric renters is abolished.” An example of this is (18) See Rule 28, J . Chem. Soc., 5117 (1952); Rule 29, J . Org. Chsm., 18, 288 (1903).

EMIL FISCHEH AND HIS SCIENTIFIC WORK

35

the forination of the same omzone from *mannose and *glucose. This proce~swaw new. In the iiiitial confusion, it was the great achievement of Emil Fischer to disengage himself from Van% Hoff ’8 method of writing configurational formulas, and to go back to the ordered tetrahedral models themselves, for which he had to invent a projection of the steric arrangement onto the plane of depiction. Initially. only the nitrile of L-mannonic acid was found on addition of hydrocyanic acid to Larabinose; this acid retained the original arabinose in the asymmetry centers 3,4,and 5.The new center of asymmetry created in this way at C-2 was first considered by Fidcher-to be racemic. This would have meant that the Gmannonic acid should be a partial racemate; however, attempts to separate it into two stereoisomers failed. The idea of a partial racemate led to the question as to whether Lmannonic acid and wgluconic acid (which are enantiomorphous on carbon atoms 3,4,and 5) could form such a partial racemate (which would still be optically active). Such a compound could not be isolated, but (‘negative results have only limited vdues as proof.” Soon afterwards, the bmannonic acid was recognized to be a homogeneous substance, and hence an unequivocal configuration had to be assigned to C-2 of its molecule. This meant that the three &symmetriccenters brought in by the arabinose had determined the configuration of the new asymmetric center produced in the cyanohydrin synthesis. In 1898, when a small amount of the nitrile of Lgluconic acid was found in the same reaction mixture, he wrote: “The simultaneous formation of two stereoisomeric products on the addition of hydrocyanic acid to aldehydes, which has been observed here for the first time, is quite noteworthy, theoretically as well as practically”; and, in 1894: ‘(These observations are, to my knowledge, the first definitive evidence that further synthesis with asymmetric systems proceeds in an asymmetric manner. Although this statement does not at all contradict theory, it is by no means a consequence of it.” Not until then could he attempt to derive the configurations of the individual sugars, “encouraged by the excellent agreement with theory of progressing observations which often appeared surprising to me.” The concepts thus obtained touch on the phenomenon of “assimilation.” Fischer wrote in 1890: “Chemical synthesis leads. . . to optically inactive acrose. In contrast to this, only active sugars have so far been found in plants. No known fact contradicts the supposition that the plant produces. . . first the inactive sugars which it then resolves, and uses the member8 of the d-mannitol series to build up starch, cellulose, inulin, and the liko, while using their optiattl i,wmers for other purposes, now unknown. . . Since then, I havo attempted it1 vain to find Z-glucose or

.

36

KARL FHEUDENBEM

Lfructom in leaves.” In 1894: “It is impossible to doubt that &glucosc and &fructose or their polysaccharides are produced predominantly, if not exclusively, by assimilation. In accord with the precepts of Pasteur, one notea a marked differencebetween the natural and the laboratory synthesis. As the latter glways yields inactive products, it may be said to proceed in a symmetrical manner. In reality, though, this is no longer true for campounds having several asymmetrically substituted carbon atoms. If the synthesis of hexoses from formaldehyde or glycerose or acrolein dibromide were to ttike place in a totally symmetrical manner, the chances of formation for each of the 16 isomeric aldoses or 8 ketoses would be equal. However, despite all efforts, I have not succeeded in finding another one of the known hexoses (naturally, in the racemic form) besides a-acrose.” A t that time, Fischer still considered the &acme (Dcsorbose) to be a sugar having an anomalous carbon chain. “It therefore, follows that particular configurations are preferred in chemical syntheses, and that there are equal chances for the mirror images only. . I have found that, once a molecule is asymmetric, its extension also proceeds in an asymmetric sense. If one should then consider that the mannononose, formed from mannosa by the asymmetric, three-fold addition of hydrocyanic acid, could be cleaved back into the ohginal hexose and the three-carbon addition product, it would be found that the latter would be an optically active system. The one active molecule would then have given rise to a second active one. It seems to me that this concept offers a simple solution for the enigma of natural asymmetric synthesis. According to the plant physiologists, carbohydrate formation takes place in the chlorophyll granule, which itself consists entirely of optically active substances. I can imagine that the formation of carbohydrates is preceded by the generation of a compound of carbonic acid or formaldehyde with those substances; and that then, since the combination is already asymmetric, the condensation to give the sugars also takes place in an asymmetric fashion. The final sugar would then be releaeed from the combination, and would later be used by the plant, as is known, to produce all the other organic components. Their asymmetry can thus be readily explained by the nature of the matwid from which they were produced. Of course, this also forms new chlorophyll nuclei which, in turn, produce active sugar. In this manner, the optical activity propagates from molecule to molecule, as life itself does from cell to cell. It iR, therefore, not necessary to attribute the formation of optically activa suhstmcas i n the plant to asymmetric foices lying outside the organism, as Pasteur had supposed. . . . This concept completely climinates the difference between natural and artificial synthesis. The advance of science has rcmoved this last chemical hiding place for the once 90 highly esteemed vis vitalia. Now we can produce active molecules

..

EMIL FISCHER AND HIS BCIENTIFIC WORK

37

artificially without the aid of an organism. Nevertheless, there is still one eamntial difference between chemical and natural synthesis. Laboratory experiments always initially produce an inactive product which must be resolved by special operations, whereas the process of assimilation leads directly to exclusively active sugars.” Emil Fischer could be highly astonished at unexpected results. This was also true of the thesis worked out by him that the configurations of individual groups in stereoisomeric substances can cause profound differences in their chemical, physical, and biochemical behavior. In contrast to all the other polyhydroxy dicarboxylic acids that he knew, mannaric acid reduces Fehling solution and shows a yellow coloration on heating with alkali. He was at first reluctant to believe that the constitution he had to assign to it was correct, although this was later established unequivocally. The differences in the chemical behavior of stereoisomers are especially striking when they react with other asymmetric systems. This applies especially to enzymes and, consequently, to biochemical reactions in general. An example is methyl 6deoxy-&D-g1ucopyranosidel which is hydrolyzed by emulsin, whereas the closely related methyl b-D-xylopyranoside is not attacked. In 1894, he wrote: ‘‘From the observations under discussicn, which hitherto could not be made to the same extent in any other group, it clearly follows that the same kind of isomerism also affects chemical transformations and brings about differences which are at least aa large as those found with unsaturated and cyclic stereoisomers.Alcoholic fermentation takes place with glucose, mannose, galactose, and fructose, which are all hexoses. However, the observation that glyceraldehyde and dihydroxyacetone, as well as mannononoseJl0can also be fermented shows the surprising fact that the most ordinary functions of a living organism depend more on the molecular geometry than on the composition of its nutrients. This represents a significant extension of Pasteur’s observation that micro-organiams alter only one of two enantiomorphs, and reminds one of a statement of Pasteur’s relating the different tastes of the two (stereo)isomeric asparagines to the asymmetry of neural substance. The action of enzymes and yeasts involves a far-reaching chemical process which takes place readily or not at all, depending on the configuration of the substrate upon which they act. Here, apparently, the geometric structure has such a profound influonce on the action of chemical affinities that it seemR to me permissible to compare the two molecules under reaction with B key and lock. If one wnrits to do justice to the kriowri fact that several yeasts can ferment a larger number of hexoses than other yeasts, the picture could be completed by the differentiation between a main key (19) Tlint thc mannononose waa fermentable was shown later to be an error.

38

KARL FIU!XJDENL)EItCf

and special keys.” Feeding experiments, carried out by others with isomeric sugars, showed that “the fermentable sugars are also the true precursors of glycogen. Moreover, it can be concluded from this that the organism can produce glucDse from its isomers fructose, mannose, and galactose. In the reverse manner, galactose, a component of lactose, is most probably produced by mammals from nutrient glucose.” A comment made by E. Fischer-probably incidentally-in 1904, is characteristic of the manner of thinking of the great synthetic chemist, and may serve as a final observation: “Only 6 of the 32 heptoses and only 2 of the 128 aonoses have been prepared. But, since these compounds have not yet been round in NaturePo and are, therefore, of only minor interest, their systematic elaboration may be left for a later period.”

IX. GENERALREFERENCES 1. E. Fischer, “Erinnerungen aus der Stramburger Studentenzeit (1872-1875),” in “Gemmelte Werke,” A. von Bseyer, ed., Vieweg und Sohn, Braunschweig, 1905, VOl. 1, p. xxi. 2. E. Fischer, “Untersuchungen ilber Kohlenhydrate und Fermente (1884-1908),” J. Springer, Berlin, 1909. 3. Fischer, E., “Untersuchungen Uber Depside und Cerbstoffe (190&1919),” J. Springer, Berlin, 1919. 4. A. von Harnack, “Grabrede flLr Emil Fischer,” 1919 (personally distributed). 6. C. Harries: B. Abderhalden, A. von Weinberg, E. Trendelenberg, and L. Lewin, “Dem Andenken an Emil Fhher,” N~um‘8amchqften,7, 841 (1919). 6. N. 0. Forster, “Emil Fischer Memorial Lecture,” J . C A n . Soc., 117, 1157 (1920).

7. K. HOWC~, “Emil Fischer, sein Leben und sein Werk,” Deutsche Chemische Geaellachaft, Berlin, Ber. (Special Issue), 1921. 8. E. Fieaher, “Aus Meinem Leben,” M. Bergmann, ed.,J. Springer, Berlin, 1922. 9. E. Fischer, “Untemchung ilber Kohlenhydrate und Fermente, I1 (1908-1919),” M. Bergmann, ed., J. Springer, Berlin, 1922. 10. M. Bergmann, “Emil Fischer,” in “Das Buch deutscher Chemiker,” 1930, Vol. 2. 11. B. Helferich, “Emil Fiaoher eum 100 Geburtetag,” Angeu. Chem., 66, 45 (1953). 12. K. Frendenberg, “Emil Fischer, ein Wegberaiter der Biochemie,” in “Foracher und Wissenechbftler im Heutigen Europa,” H. Schwerte and W. Spenpler, eds., G. Stalling, Oldenberg, 1955, Vol. 1, p. 158. 13. K. Freudenberg, “Ed1 Fischer” (Lecture), Jahresh. Heidelberger Akad. Wiss. 1945/66, 161 (1959).

14.

K. Freudecberg, “Emil Fischer,” in “Neue Deutsche Biographie,” Historkche

Kommiseion bei der Bayerischen Akademie der Wimnschaften, ed., Duncker und Humblot, Berlin, 1961, Vol. 5, p. 181. 16. B. Helferich, “Emil Fischer,” in “Great Chemists,” E. E. Farber, ed., Interecience, New York, N. Y.,1961, p. 983. (20) Later, some of the aldoheptoses (and wtuloees and nonulosrts) were actually

found in Nature.

MASS SPECTROMETRY OF CARBOHYDRATE DERIVATIVES

BY N. K. KOCHETKOV A N D 0.S. CHIZHOV Instilute for Chemistry of Natural Produck, Academy of Seiencee, Moeoow, U. S. S. R.

I. Intioduction .......................................................... 11. The Basic Principles of Maas Spectrometry of Organic Compounds.. . . . . . . . . 1. Principal Designs of Maas Spectrometer, and the Physical Principles Involved. ..................... 2. Treatment of the Mass Spectra ...................... 3. Saope and Limitations of Mass Spectrometry. The Principles of Interpretation of Mass Spectra.. ......................................... 111. Maas Spectra of Carbohydrate Derivatives. . ............. 1. General Remarks.. .................... ............. 2. Monocyclic Derivatives: Pyranoid and Furanoid . . . . . . . . . . . . . . . . . 3. Monosaccharide Derivatives Having Fused Rings. 4. Acyclic Derivatives of Monosaccharides. . . . . . . . . . . . . . 5. Mass Spectra of Miscellaneous Carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusion. . . . . . . . . . . . . . . . . . . .

39 40

43

43 46 46

90

I. INTRODUCTION The present article deals with the applications of mass spectrometry to the structural analysis of carbohydrate derivatives. The mass-spectral techniqge has now become a useful supplement to chemical methods, and provides ready solution of a variety of problems which were a stumbling block in the classical approaches. The principles of mass spectrometry of organic compounds are described in a number of excellent reviews and monographs.l-‘ Hence, we shall not attempt to present here any theory of the mass-spectral method that is more comprehensive than that necessary for understanding the discussion. The mess-spectral method was first applied to carbohydrate derivatives in 1958, when Reed and coworkers6reported the mass spectra of D-glucose, I

(1) J. H. Beynon, “MBSRSpectrometry arid its Applicatiom to Organic Chemistry,”

IClsevier, Am~terdnm,Holland, I!)On. (2) I c i s 4 l-Ca-aldofuranosides seems to hold very definitely in acid and neutral systems; the order seems to be reversed in alkaline systems. The order of stability: warabinofuranosides > D-ribofuranosides > wxylofuranosides > wlyxofuranosides, has also been demonstrated in acid systems to a certain extent; this order is not so important aa the effect of the C-1 and C-2 interactions alone. The conformations given above have only C-2 and C-3 exoplanar. This provides tho best solution for relieving interactions; very little interaction should occur between C-1 or C-4 in the plane and the ring-oxygen atom. Calculationss7 derived from x-ray diffraction data for nucleic acids and sucrose have shown that C-2 or C-3 is the exoplanar atom for all examples studied. However, where the endocyclic bonds of C-2 or C-3 are prevented (by substitution at the exocyclic bonds) from flexing, other conformations can result. Thus, in both methyl 2,3-anhydro-a- and- 8-wribofuranoside, the four ring-carbon atoms are held in a plane by the anhydro group, and the ring-oxygen atom is exoplanar ; nuclear magnetic resonancela shows no proton-proton coupling between C-1 and C-2, or between C-3 and C-4. Similar data have been reported for other 2,3-anhydroaldo-pento- and -hexo-furanosides. Whereas anglea of 0' and 120' were projected for the angles of the cisand tram- hydroxyl groups to the planar furanoid ring, probable values of 50' have been reported for cis-hydroxyl groups, and 75' or 160" for truns(17) M. Sundsralingam, J . Am. C h m . Sor., 87, 599 (1966). (18) L. L). Hall, Chem. Znd. (London), 960 (1983).

100

JOHN

W. GltEEN

hydroxyl groups; these values are for envelope (V) conformation^.^' It is considered thph a value of 75’ is more reasonable for trans-hydroxyl groups where reactions of such hydroxyl groups are slow, aa with periodate. If complete rexistance to a reagent is shown, the possibility of an angle of 160’ should be considered. An angle of 150” has been found in one of the Dfruc tof uran ose residues in 1’ ,2-anhydro-l-0- (a-wfructofuranosyl) -&Dfructofuranose : the a-D-furanoid ring is rigidly locked, and is very resistant to periodate oxidation.I6Capon and ThackerlBahave shown, for the methyl aldofurariosides of D-glucose, D-galactose, D-xylose, and carabinose, that the anomers can be clearly distinguished by their nuclear magnetic resonance spectra. The lJ2-truns compounds all have J values of 2 C.P.S. or less; the values for the 1,2-cis compounds are in the 4.04.5 C.P.S.range. In the Eormation of glycofuranosides from the aldopentoses, the oxygen atom attached to C-4, the configuration of which defines the chirality (D or L family) of the given sugar, is included in the ring; therefore, the hydroxymethyl group on C-4 will be in roughly the same position, relative to the plane of the ring, for all of the D-aldopentofuranosides and for all of the ~aldopentofuranosides.le In the aldohexofuranosides, C-5, the “configurational” or reference carbon atom, is not included in the ring, and so the bulky dihydroxyethyl (CHOH-CHaOH) group attached to C-4 does not necessarily have, for the D series MI a group, and the L series aa a group, the same position in reference to the ring. For half of the Paldohexoses, in methyl 8-D-glucofuranoside (9) and methyl a-wmannofuranoside (lo), for example, the exocyclic Wdihydroxyethyl) group is above the plane, as for the (3-4 group of the D-aldopentofuranosides. For the other half of the wddohexoseb, for example, in methyl Swgalactofuranoside (1l), this large group is below the plane, and these glycosides are 4-C-substituted taldopentofuranosides; thus, (11) resembles methyl a-barabinofuranoside (12).

The reasoning for the respective conformational stabilities assigned t o the aldopentofuranosides listed in Table I can be extended to the aldohexofuranosides and the ketohexofuranosides. The introduction of a twocarbon group instead of a one-carbon group at C-4 of the aldohexofuranosides may have some effect on the C-3, C-4 interaction, but it seems to be minor. The glycofuranosides of n-galactose and 6-deoxy-~-galactose b-fucose) may be compared with those of the carabinofuranosides, those of D-glucoRe and D-glucurono-6,%lactone with the D-xylofuranosides, and those of D-mannosc wid O-tlooxy-L-rnniitioso (L-rhnninose) with the Dnricl L-IyxcifiirnrioRitles, respectively. (18s) l.3, Ciy)oii rttid I). Thncker, Proc. ( ’ h p m . Soc., 369 (1964). ( l e ) ThiH inclunion of the oxygen atam of the “u-hydroxyl” or “lphydroxyl” group at (2-6 within the ring occurs for both sldohexo- and aldopento-pyranoeides, and 80 no difficulty occure in comparing the various inembers of these seriee,

101

THE QLYCOFURANOQDES

q

HOH,C OH

HCOH HO&L (11)

OH (12)

In the case of the methyl D-fructofuranosides, there is a relationship to the warabinofuranosides, with a different type of C-1, C-2 interaction. Here, the hydrogen atom on C-1 has been replaced by a hydroxymethyl group, and the difference in the C-2 interactions with’the C-3 hydroxyl group should be small for the anomers; the effect of the aglycon group (OMe) and the hydroxymethyl group will probably be similar. 111. FORMATION OF GLYCOFURANOSIDES IN ACIDICMETHANOL This reaction, a brief glycosidation of a sugar with methanol at room temperature, with hydrogen ion as the catalyst, is the Fischer reaction.’ For preparative purposes, it involves the isolation of kinetic products (glycofuranosides),formed at a relatively high rate, before their subsequent conversion, at a much lower rate, into the thermodynamically more stable products (glycopyranosides). The reaction ie usually performed at a low temperature for a short period of time, with a low concentration of acidic catalyst. The reaction time ia often monitored by following changes in optical rotation or decrease in reducing power, or by chromatography. Use has been msde of protecting groups suitably located to prevent the formation of the pyranosides. The presence of another ring (lactone or anhydro) often stabilizes the furanoside. 1. Isolation of Products It WBR from such n remtion mixture (obtained by shaking 20 g. of CY-Dglucose with 400 g. of methanol nontrtiriing 1% of hydrogen chloride for 15 hours at room temperature, neutralizing with silver carbonate, evaporating the solution, and extracting the resulting sirup with ethyl acetate) that Fischer isolated his methyl “gamma” D-glucoside, a nonreducing sirup that waa readily hydrolyred by dilute acids. This impure material (and

102

JOHN

W. GREEN

other "gamma" glycosides) had been methylated and converted into known, crystalline productsm before any crystalline glycofuranosides had been isolated. Hence, the ring structure of this impure gamma glucoside was demonstrated to be furanoid before fractionation into pure components had been aohieved. Levene and coworkers,a' using the ease of hydrolysis (10 minutes in 0.1 N hydrochloric acid at 100") of the aldofuranosides aa an analytical tool, studied the rate of glycoside formation (see Fig. 1) and confirmed the preliminary formation of aldofuranosides and subsequent conversion into aldopyranosides. The first crystalline furanoside waa prepared by Haworth and P0rter.l A protecting technique was used, starting with 1,2-0-isopropylidene-a-~glucofuranose; this compound was converted into the 5,6-carbonate, and the isopropylidene group waa removed with ethanolic hydrogen chloride, with formation of the two ethyl wglucofuranoaide 5 ,&arbonatee. These were separated by fractional recrystallization and each waa individually saponified to the D-glucofuranoside; the CY-D anomer was obtained aa a crystalline compound. The &D anomer was a hygroscopic solid, but it was later obtained in crystalline form by Phillips.g* (Methyl a-wglucofuranoside waa obtained in a similar manner.a*) Methyl a-wmannofuranoside wm prepared by a somewhat similar technique, starting from D-mannofuranose 2,3;5,&dicarbonate2'; the glycoside was also prepared directly, in good yield,28 by methyl glycosidation on seeding with a crystal obtained by the protecting techniq~e.~'Thus, methyl a-D-mannofuranoside may be considered to be the first ddofuranoside to be Crystallized directly from a Fischer reaction mixture. Although the f h t crystalline glycofuranoside waa prepared in ethanolhydrogen chloride, most of those subsequently prepared have been formed in acidio methanol, without the aid of protecting groups. Methyl CY-Dfructofuranoside waa prepared'd by allowing a solution of D-fructose in methanolic hydrogen chloride to reach its maximum dextrorotatory value, and then fermenting the unreacted sugar by treating the mixture with bakers' yeast. The sirupy mixture of fructosides remaining was fractionated (20) W. N. Haworth, E. L.Hirat, and E. J. Miller, J . Chem. Sw., 2436 (1927); H. D. K. Drew, E,H. Goodyear, and W. N. Haworth, ibid., 1337 (1927); C. F. Allpress, W. N. Haworth, and J. J. Inbter, ibid., 1234 (1927); W. N. Haworth end C. R. Porter, ibid., 616 (1928); P.A. Levene and Q. M.Meyer, J . Bwl. Chem., 76, 809 (1928); H. G.'Bott, E. L. Hirst, and J. A. B. Smith, J . Chem. Iqoc., 659 (1930). (21) P. A. Levene, A. L. Raymond, and R. T. Dillon, J . Biol. Chem.,96, 699 (1932). (22) D. D.Phillip, J . Am. Chsnt. floc., 76, 3598 (1954). (23) W. N. Haworth, C. R. Porter, and A. C. Waine, J . Chem. floc., 2254 (1932)(24) W. N. Haworth and C. R. Porter, J . Chem. Soc., A49 (1930). (26) W. N. Haworth, E. L. Hirst, and J. I. Webb, J . Chem. floc., 651 (1930). (26) C. B. Purves and C. 8.Hudmn, J . Am. Chem. Iqoc., 66,708 (1934).

THE OLYCOFURANOSIDES

looo-Lyxose/

103

-----I*-

--. 500

-Free wgor ----------

/@@

500

50

Time (hours)

Pyranosids Furonosib

Fro. 1.-Percentages of Free Sugar, Furanwide, and Pyranoaide during Glymide Formation at 25" in Methyl Alcohol Containing 0.5 per cent of Hydrogen Chloride.*'

with ethyl acetate, and a 10% yield of the crystalline product was finally obtained. Methyl am-arabinofuranoside was also prepared" by allowing the acidic methanol solution to reach its maximum dextrorotation; an ether extraction was used to obtain a 9% yield of product. The separation of glycosides on a cellulose column was first applied to a mixture of pyranosides**;subsequently, separations were achieved for the methyl sfructofuranosides and D-gdactofuranosides.*O This technique waa soou applied to many other mixtures; it permits not only the isolation of crystalline products, but also a more quantitative evaluation of the formation of g l y c o f u r a n o s i d e ~ . ~ ~ ~ (27) E. M. Montgomery rriitl C . S. IIutlwnri, J . Am. Chem. Soo., 69, M)2 (1937). (28) L. Trough, J. h'. N. J011ep1,atid W. 11. Wadmim, J . C h .Soc., 1702 (1950). (29) I. Auge8tad, E. Berner, and E. Weigner, Chem. Zd. (London), 376 (1953). 130) C. E. Ballou and H. 0. L. Fisoher, J . Am. Chem. SOC.,75,4605 (1953). (31) I. Augestad and E. Berner, Actu Chem. Scand., 8, 261 (1954). (32) D. F. Mowery and G. R. Ferrante, J . Am. Chena. SOC.,76, 4103 (1964). (33) G. P..Barker and D. C. C. Smith, J . Chem. h e . , 2161 (1954). (34) W. M. Watkins, J . C h . Soc., 2064 (1955).

104

JOAN W. UIEEEN

A large amourit of data has been accumulated during the preparation of the various glycofuranosides, and some of this is given in Table 11. These data are to a great extent qualitative, and the purpose of giving them here is only to show the preponderant anomer formed under certain conditions of acidic methyl glycosidation. Most of the data are derived from the results of chromatographic separations; some of them have been obtained by gas chromatography.l6 In almost every case, the preponderant anomer is the trans-l ,2-glycoside; the interaction between its aglycon group and the C-2 hydroxyl group is much leas than for the cis-1,2 anomer. Conformational stabilities for the aldopentofursnosides have already been discussed (see Section I1 and Table I). To 8 certain extent, the conformational stabilities of the aldohexofuranosides are comparable to those of the corresponding pentofuranosides; but the former have a bulkier group at C-4 to cause interactions that lower the stability. For the wfructofuranosides, there is apparently little difference between interactions of either the aglycon methyl group or the hydroxymethyl group (on C-2) with the C-3 hydroxyl group; the anomers are formed in approximately equal yields. The high yields obtained indicate the conformational stability of the D-fructofuranoside structure, which is very similar to tSat of the Darabinofuranosides. Although hydrogen chloride has been the catalyst principally employed in the Fischsr method, other acids, notably sulfuric acid, have been used, ae well as acidic, ion-exchange resins.8bag The advantage of the latter reagents is that the insoluble resin can be readily filtered from the reaction solution. It is held that the replacement of soluble acids by insoluble resins does not appreciably alter the formation of the glycofuranosides. The glycosides formed from the tetroses are, of necessity, furanosides. (36) E. M. Osman, K. C. Hobbs, and W. E. Waletan, J . Am. C h . Soc., 75,2726 (1961). (36) J. E. Cadotte, F. Smith, and D. Sprieaterbach, J . Am. C h .SOC.,74, 1601 (1962). (37) W. H. Wadman, J . Chcm. rSoc., 3061 (1962). (38) G. R. Dean and R. E. Pyle, Britiah Pat. 670,480 (1962); Chem. Abetracfs, 46, 9332 (1962). (39) D.F. Mowery, J . Ah. C h .Boc., 77, 1667 (1966). (40)J. N. B u i r and A. 8.Perlin, Can. J . C h . , 88,2217 (1980). (41) R. Barker and H. G . Fletcher, Jr., J. Org. C h m . , 26, 4605 (1961). (42) C. T. Biohop and F. P. Cooper, Can. J . Chsm., 40, 224 (1980). (43) 0. KjoelSerg and 0. J. Tjelveit, Acta Chem. Scand., 17, 1641 (1963). (44) P. W. Auetin, F. E. Herdy, J. G. Buchanan, and J. Baddiley, J . Chem. SOC.,6360 ( 1963). (46) D.F. Mowery, Jr., Method8 Carbohydrals C h . , 2, 328 (1963), (46) I). F. Mowery, Jr., J . Org. Chsm., 96, 3484 (1961). (47) J. Cf. Uarclirier and E. E. Percivnl, J . Chm. ~ o c . ,1414 (1958). (48) I. Auptad and E. Berner, Acb C h . Swnd., 10,811 (1966).

TABLE I1 Formation of Glycofuranosides in Acidic Methmol

Sugar

D-ED-Threose L-Arsbine D-Arabmaee D-Ribose D-Xylose

D-Lyxose

D-GlucOse D-Galactose

D-Mannose DFructose L-Fucose

GRhamnose ~-G~ucuron+6,3-lactone

Normality of HCI

0.6 0.6 0.01 0.a328 0.0028 0.28 [0 .18N H2SOd 0 .OO28 0.0036 0.14 0.00% 0.04 [O. 18 N Ha04 0.0037 cO.36 N H W I [DoweX-50 resin CDowex-50 resin 0.5 0.04 0.22

0.0055 0.14 [Nalcite HCIt resin

Temp., 'C.

25 25 65 35 35 25

4 35

65 25 35 65 25

65 65

65 65 25

65 15 65 25 65

Time, min.

Product formation Predicted References 0 Typeof snomer Anomer Anomer data" a

5 5

11 3

180

56 1 1

c

50 14 9 6 0 2 0 1

-

360 L

D

22

1 large 54

840 5 360@) 14 120 0.35 50 15 0.61 12 41 D 17 21 4Mw) 30 470 4200 2 180 2

-

65 2 22

0.32 3.3 16 80 1.69 45

1.70

small 3

9 54 1.05 3 0.34

45 36 44

-

62 72

A B A C C A B C A

C C A

A A

B A C A A

A A A A

B a

a a

B B B B B B a a

B B B

40

40

31 15 15 33 411 15 31 42 15 43

441 31

a

451

Q

461

B B a

29 43 47 48

B B

351

n

a Key: A, % of product isolated; B, ratio of products, determined by chromatography; C, relative rate of formation; D, reaction allowed to proceed to constant rotation.

z

M

JOHN W. GREEN

106

The ready formation of furanosides from wglucurono-6,3-lrtctone is aided by the presonce of the laotone ring. Mixtures of the anomers of glycofuranosides have often been used as starting materials for various syntheses, instead of the individual anomers. Thus, methyl a!,P-wribofuranoaide is often encountered in the literature of the nucleic acids. Such mixtures can be converted into the esters, or into the glycofuranosyl halides, and then into the individual glycofuranosides. Some of these applications will be discussed later (see pp. 121). 2. Kinetics

Several kinetic studies have been made, since the initial work of Levene and coworkers,*1which have established the importance of the anomerization of the furanosides, and the reversibility of the conversion of furanosides into pyranosides. However, this work has thus far been confined to the four aldopentoses and to two aldohexoses. With the d d of column chromatography, MoweryM studied the rate of formation of furanosides and pyranosides from Parabinose. The rate of furanoside formation ww extrapolated back to zero time, and an initial rate was obtained (see Table 111).The less stable anomer, the bfuranoeide, waa initially formed to the grertter extent; in the final equilibrium mixture, the more stable (u-D anomer waa preponderant. Tmm I11 Rats of Formation. of Aldooidea in Acidic Methanol'" Sugar

*Mannose

tArabinose

Glyaoride

Oridnal rate mnstant

Equilibrium Solution cornpodtion, time, houm

%

rrfunrnoeide @-furanosida a-pmoeide &pyrenoside

0.61 0.34 0.12 0.03

2 2 89

a-furanaside Bfuranoeide a-pyranoside ppyranoside

1.3 2 .o 0.15 0.16

23

7 8 24

45

72 72 72 72 24 24 24 24

a Theee v d u a M e r from those in Table 11, in that the rates are extrapolated back to mro time a i d do not ne-rily reprwnt the overall rate for the reaotion; nor do they represent anomerieation. b t a oonstsnte are first order, given with time (in hours) and oommon logarithms. Reaction conditions: Dowex-60 reain and methanol at 65'.

THE C)LYCOFURANOBIDES

107

Extrapolation of rates back to zero time also showed a small but definite formation of pyranosides from the free sugar, in addition to conversion from the furanoddes. The reversibility of the conversion of furanosides into pyranosides waa definitely demonstrated by obtaining the final equilibrium composition from two directions, namely, from the a-Dpyranoside and from the free sugar. A detailed study of the methyl glycosidation of the four aldopentoses has been made by Bishop and C o 0 p e r , 1 with ~ ~ ~ ~the aid of gas chromatography of the esters of the resulting pentofuranosides and pentopyranosides. Three competing reactions were established for the formation of the furanosides: (1) an irreversible formation of furanosides from the free sugars; (2) an anomerization of the furanosides; and (3) conversion of the furanosides into the pyranosides. (A fourth reaction, anomerization of the pyranosides, will not be considered here.) The kinetic data are given in Table IV, and the compositions of the equilibrium mixtures are given in Table V. I n both of these Tables, the data are given for the four pentoses in the same order as in Table I, that is, in the order of their conformational stability. The furanosides having the lowest number of nonbonded interactions are formed most rapidly, have a lower rate of anomerization, and a lower rate of conversion into the pyranosides. There is also a higher percentage of these more stable furanosides in the final equilibrium mixtures. The reverse holds for those glycofuranosides having a greater number of interactions. Thus, we find a much higher rate of anomerization for the methyl D-lyxofuranosides (in contrast to the D-arabinofuranosides), and only a small proportion of the a-wlyxofuranoside and no j3-D-lyxofuranoside in the h a 1 equilibrium mixture. In all cases, the rate of anomerization is very high, relative to the rate of conversion into pyranosides; this rate of anomeriaation is the major factor in determining the relative yields of the a- and ,9-furanosides from a reaction mixture, and not the relative proportions of these two anomers initially formed from the free sugar. The ratio of anomers waa found to be approximately constant throughout the course of the reaction (1.7:l for the j3- and a-~-xylofuranosides).~* Only with *lyxose watj there any evidence of pyranoside formation early in the reaction. A slight reverse reaction, furanoside to pentose, was also noted for this sugar. This behavior is in agreement with the low conformational stability of the D-lyxofuranosides. However, no initial formation of maxabinopyranosides was noted. The more probable formation of furanosides as compared with pyranosides, especially at lower temperatures, has been discussed by Shafizadeh.' Specific alignment and orientation of the group on C-5 is required

TABLE IV Kinetic Data for Methyl G l y d d a t i o n of Mdopentoeee"

--i

226 57 19

homerhation of fursn-osidess 4

a+8 *a

1.39 11.3 49

very malI

Conversion into pyrauusided h

Furanogide

pyranoaide *a

0 .m 0.069 0.12 0.84

5; 4.35 3 -4

29 very 0.11 0.00s 0 .o004 0.001

5.74 15.2 78

0.32 3.3 1.69

-695 740 -320

verylerge 7.9 8.8 320

6-50

-1260 -1330

-3530

-4080

The reactions were performed in methanol containing 0.0028 d l HCl at 35". * This rate constant could not be determined, because of the low solubility of ~arabimosein methanol; its value is considered to be very large, based on the amount of furanoside at equilibrium. K = v]/[a]. K = [arpyranorside] CBpyrsnoside]/[~-furanoside] C&furandde].

a P Eg

TEE ffLYCOFURANOSIDES

109

TABLE V D-AldopentofuranosideComposition at Equilibrium" Sugar"

a-Furanoside, PFuranoside,

96

%

Total furanoeide,

% . DArabinc-se D-Ribose D-Xylose D-Lyxose 3-O-Methyl-D-arabmose 20-Methyl-warabinose 2,3-Di-O-methyl-~-arabinose ;U)-Methyl-D-xylose 2-0-Methy 1-wxylose 2,3-Di-O-rnethyl-D-xylose ~

a

6.8 17.4 3.2 0

21.5 6.2 1.9 1.4

28.3 22.6 5.1 1.4

50.7

66.7 75.4 9 .o 12.8 16.4

_ _ _ _ _ _ _ ~ ~ ~ ~

Reaction conditions: 2% of sugar in 0.28 M MeOH-HCl at 35".

for the formation of pyranosides, in addition to the alignment of the carbon atoms also required for formation of the furanoside ring. The rates of methyl glycosidation are approximately proportional to the concentration of acid catalyst. In the early part of the glycosidation, a single reaction is predominant and can be characterized as first order. A cyclic, carbonium intermediate (13) wiw proposed for the mechanism of the anomeriration of the furanosides. D-XylOSe

HO

Hpy=xpH -

HOH,C,

HI

,OH

H

O

H

,

HoTQ(MF OH

Ho,c'H+

1-P

(1s)

Methyl (I-Dxy lofur anoside

C

!

~

M

OH

MeOH

+I@\ Methyl

p 8-D-

xylofuranoeide

The effect of adverse interactions is shown even more strongly in the case of the mono- and di-methyl ethers of Darabinose and D-xylose (see

~

110

JOHN W. QBIDEN

Table V). The distances between trans-hydroxyl groups (equatorialequatorial) in pyranosidos is 2.8 A.; for the truns-hydroxyl groups in furanosides, this distance is greater, namely, 3.44 A. This mems that there is a greater increase in adverse interations contributed by the bulky methoxyl groups in the pyranosides than for the furanosides. This relatively greater stability of the ethers of the furanosides is shown by their higher proportion in the final equilibrium mixtures, as compared with the unmethylated sugar. A kinetic study of the methyl glycosidation of wmannose was also made by M~wery.'~ The more stable anomer, the a-D-mannofuranoside, was formed at a higher initial rate (see Table 111); the proportions of both furanosides in the final equilibrium mixture wm too small to permit accurate comparison of isomer distribution. The conformational stability of the D-mannofuranosides may be compared with that of the wlyxofuranosides; the furanoid structures are similar, except for the bulky two-carbon group at C-4 of the hexoside. This similarity is shown in the very small proportion of wlyxofuranosides (see Table V) and of D-mannofuranosideo (see Table 111) in the final equilibrium mixtures, and also in the initial formation of wmannopyranosides" and of wlyxofuranosides.16 Overend and coworkers'@have studied the action of acidic methanol on methyl &wglucofuranoside, with two concentrations of acid catalyst (see Table VI). The rapid anomerization in methanol under the conditions studied gave only furanosides. Use of 14C-labeled methanol gave labeled furanosides, showing participation of the solvent in the anomerisation. These authors suggested the reaction (14) (15) to explain the role of methanol.

H

HO€I,C

d

q

HOMe

HOH, HdQEEe

+ MeOH

L 7

HMe

H (14)

OH (15)

The slower conversion of the anomeriaed D-glucofuranosides into wglucopyranosides waa studied with a higher concentration. of acid. The concentration of furanosides waa followed by determining the aqount of formaldehyde formed by periodate oxidation. It waa concluded that the same acyclic ion (16) is formed from both furanoid anomers, and that ring closure gives a mixture of pyranosides similar to that found in the equilibrium mixture of pyranosides. (48) B. Capon,G. W. Loveday, and W. G. Overend, Chsrn. Ind. (London), 1537 (1962).

THE QLYCOFURANOSIDES

111

TABLEVI Action of Acidic Methanol on Methyl D-GlucofuranoaidesU H e m tion

Anomerisationb Conversion into pyranosidesc

loSk (in em.-') at

Molarity of acida

0.10 2.0

25.0"

35.0"

1.05 0.748

2.88

35.2"

40.1"

2.65

3.25 10.2

45.0"

Methsuesulfonic acid. b The substrate is the &D-furanoside; the data are for 10' (kl kl), determined polarimetrically; the anomeriaed mixture contains f33% of the fl and 37% of the a form. 0 The substrate is the anomerised mixture; the data are for 106 h,the overall rate co-t, determined from the formaldehyde formed on oxidation with periodate. 0

+

The composition of the anomerized D-glucofuranosides (63% of /3: 37% of a) has exactly the same ratio (1.7:l) as that observed by Bishop and Cooper16 for the mxylofuranosides. This excellent agreement can be correlated with the structure of these hexofuranosides and pentofuranosides; presumably, their conformational stabilities are very similar.

Capon and T h a ~ k e r ' @ have ~ demonstrated the formation of aldofuranosides from acyclic acetals of D-glucose and &galactose in 0.05 M hydrochloric acid at 35'. The yields (kinetically controlled) of furanosides for the two acetals were 98 and 71%, respectively, showing the successful competition, in each case, of the C-4 hydroxyl group over the solvent. The authors have suggested a mechanism of simultaneous ring-closure and breaking of the acetal bond; the rate constant for the D-glucose acetal is much greater than that for the D-galactose acetal. This variation in the nucleophilicity of the C-4 hydroxyl group is also discussed on p. 120. (49a) B. Capon and D. Thacker, J . Am. Chem. Soe., 87, 4199 (1966). (49b) Unpubliihed work of A. A. Court (quoted in above reference).

112

JOHN W. QRPJEN

IV. PREPARATION OF GLYCOFURANOSIDEB FROM DITHIOACETALS

1. Isolation of Products This method, developed by Pacsu and Green,a consists in treating a sugar dialkyl dithioacetal (17) with niercuric chlorido and yellow iiicrcurio oxide in a chosen alcohol; the reaction is irreversible, and gives a mixture of the anomeric glycofuranosides (18) and (19). The reaction is maintained neutral, snd there is no anomeriaation end no oonversion of furanosides into pyranosides. The method has been applied to most of the common sugarB, with the exception of D-xylose. It is not applicable to =glucose, because a stable &yl l-thio-a-wglucofuranoside is formed. The reaction is valuable aa a preparative method, and products have often been crystdl i d directly from the reaation mixture. In a few cmes, acetals have been obtained instead of the glycofuranosides. H EtSCSEt

HAOH I

YHOH THOH

YOH C%OH (17)

(18)

(19)

where Y = -CHOH-CH,OH

As the glycofuranosides are the final thermodynamic products, there is no need to control the reaction conditions closely, unlike the isolation of kinetic products in the Fischer method. Constant agitation is needed, to ensure reaction of the insoluble mercuric oxide, and a desiccant (Drierite) is wed for removing the water aa it is formed. In most of the examples cited in this review, the diethyl dithioacetal is used as the Btarting material. In Table VII is given B list of products obtained by this method; the preponderant anomer obtained is the trans-1 ,2-glycofuranoside (IS), an effect similar to that noted for products formed by the Fischer method (see Table I1 on p. 105). The data in Table VII are limited in scope; the yields are mostly preparative, and do not represent quantitative recoveries. In only four of the examples cited are the yields above 50%. The 91% yield of the anomers of ethyl Grhamnofuranoside was obtained by separation on a chromatographic column, and represents the only quantitative information on this method. In all other examples, the products were isolated by crystallisation techniques, and it has often been assumed that the crystalline product isolated, often in low yield, had been the preponder-

113

THE GLYCOPCJIUSOSIDES

TABLE \.?I Glycofuranosides Formed from Dithioacetals

Glycofuranoside

Anomer

Yield, %

Predicted References anomer

GArabino furanoside methyl ethyl

a

14

a

24

a a

50 50

n-Lyxofuranoside methyl

a

61

U

51

B B

20

B B

50 3,52 53

n-Galactofuranoside methyl ethyl ethyl prowl benzyl D-Mannofuranoside methyl methyl ethyl ProPYl ieopropyl methyl 2-0-methyl-

a

B B a

B

70 5 23

12 64 13

B B B a a a

a

higha high0 higha

ff

41

a

r.,-Rhamnofuranoside ethyl ethyl

a

56 36

a

B

Methyl n-fructofuranoside

a

Methyl 2-acetamido-Zdeoxy-Dglucofuranoside

B

a

a

a a

50 50 54 54 54 54 54 55

a

5G 56

low

-

57

high"

B

58

a High yields are assumed from the contextij of the articles; no numerical data were given.

(50) J. W. Green and E. Paceu, J. Am. Chem. Soc., 60, 2056 (1938). (51) M. Nys and J. 1.' Verheijden, Bull. Soc. Chim. Belg., 69, 57 (1960). (52) J. W.Green i r r d JC. I'acsu, .I. Am. Chem. Soc., 80, 1205 (1937). (63) J. W. Gram arid E. Pwsu, J . A m . C'hem. Soc., 69, 2569 (1937). (54) A. Scut1srlr;oocI uiicl 14:. I'w~11, J . .4 v b . (.'htsm. S~JC., 62, 203 (1940). and H. M. Triater, J . A m . C/iuni. Soc., 68, 925 (1941). (55) E. PIL(:NU (56) J. D. Ueerdes, B. A. Lewis, It. Montgomery, and F. Smith, Anal. Chem., 26, 264 ( 1954). (57) E. Paceu, J. Am. Chem. Soc., 61, 1671 (1939); 60, '2277 (1938). (58) M. W. Wliitehouse and P. W. Kent, Tulruhedron, 4, 425 (1958).

114

JOHN W. GREEN

ant component in the reaction mixture, although this assumption is not necessarily valid. The formation of the trans-l,2-glycofuranosideais effected in neutral solution, where anomerization cannot occur. Treatment of 3 ,4-O-isopropylidene-2,S-di-O-methyl-~-rhamnosediethyl dithioacetal with mercuric chloride in boiling methanol gave6ga 61% yield of the crbfuranoside; no trace of the &-I, anomer was found. In this experiment, with an acidic solution (no mercuric oxide present), anomerization could have led to the formation of the more stable anomer. In the normal reactions cited, with maintenance of a neutral solution, no anomerization can occur, and yet the more stable anomer, presumably the kinetic product, is still the major anomer formed. Although dithioacetals of glucose do not form the wglucofuranosides, those of Zdeoxy-Parabino-hexose do; Stacey and coworkersm were able to convert the dibenzyl dithioacetal into a mixture of the methyl 2-deoxywarabino-hexofuranosides. Thus, the C-2 hydroxyl group has a bearing on the course of the reaction. 2. Alkyl LThioaldofuranosides

The l-thioglycosides have been reviewed in detail by Hortoh and Hutsono; the present Section deals only with the l-thioaldofuranosides, mostly in relation to their preparation from dithioacetals. The formation of these products from the dialkyl dithioacetals falls into two categories. The first group comprises compounds readily formed (or isolated), and is confined to derivatives of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-~-glucose,and wribose. The products are obtained in high yield; the formation is generally aacomplished in aqueous solution, with one mole of mercuric chloride per mole of dithioacetal, and the solution is kept neutral. The second category involves derivatives of lower stability-those of D-galactose and 6-O-benzoyl-D-arabinose ; here, similar modes of prepare tion are used, but the yields are much lower. In 1916, Schneider and Seppo1prepared ethyl l-thio-a-glucofuranoside by treating an aqueous solution of one mole of the diethyl dithioacetal with one mole of mercuric!chloride, and maintaining neutrality by neutralizing the hydrochloric acid formed with aqueous Rodium hydroxide. The product wm regarded by them as a “normal” ( L e . , pyranoid) l-thioglyB.Foster, J. Lehman, and M. Stacey, J . Chem. sbc., 4649 (1961). (60)W. G. Overend, M. Stacey, aid J. StanCk, J . Chem. Soc., 2841 (1049). (61) W. ScLeider and J. Sepp, Ber., 49, 2054 (1916). (MI) A.

THE GLYCOFURASOSIDES

115

cwide,O although they remarked that it was more readily hydrolyzed than the BD anomer prepared from tetra-0-acetyl-a-mglucopyranosyl bromide. The furanoid nature of the former compound was shown60 by its eaw of acid hydrolysis and isorotation values, and was further confirmed by periodate oxidation.ea A standard method of preparation of this l-thioaldofuranoside, and of others, was established by Pacsu and Wilson'J'; it consists of performing the reaction (20)-421) in aqueous solution, with H EtSCSEt I HCOH I CHOH

LHOH I CHOH I CH,OH

(20)

0 . 5 HgCI,

+ 0 . 5 HgO

Y,H

qsEt + HgClSEt + 0.5 H,O

H, OH

HO

(21)

where Y = -CHOH- CH,OH

0.5 mole of mercuric chloride per mole, and excess of mercuric oxide to

maintain a neutral solution (and to generate another half mole of mercuric chloride). In contrast to the preparation of the aldofuranosides, the alkyl group of the 1-thioaldofuranoside is the alkyl group of the original dialkyl dithioacetal. As the solvent does not participate in the formation of the 1-thioaldor'uranoside, it does not have to be nucleophilic; thus, acetone has been used."s Cadmium carbonate may also be employed, instead of mercuric oxide, to maintain neutralitye6; here, one mole of mercuric chloride per mole of dithioacetal is needed. The methyl, propyl, and bengl 1-thio-a-wglucofuranosides were preparede' by the original method (with neutralization by sodium hydroxide). Use of the method of Pacsu and Wilsone4gave the methyl, ethyl, propyl, and isopropyl 1-thio-a-D-ribofuranosidesin yields ranging from 65 to 80%. Sodium (methyl 1-thio-a-D-g1ucofuranosid)uronate and the ethyl and propyl analogs were obtained6' similarly from the sodium salt of the (62) It is iiiteresting that Schneider and Seppal gave the correct formula for their product,

and showed a furanoid ring; it waa not until a decade later that the pyranoid ring was accepted for the "normal" glycosidea. (63) M. L. Wolfrom, S. W. Waisbrot, D. I. Weisblat, and A. Thompson, J . Am. Chem. Soc., 66, 2063 (1944). (64) E. Pacsu and E. J. Wilson, Jr., J . Am. Chem. Soc., 61, 1450 (1939). (65) E. J. Reist, P. A. Hart, L. Goodman, and B. R. Baker, J . Am. Chem. Soc., 81, 5176 (1959). 166) H. Zinner, A. Koine, and H. Nima, Chem. Ber., 93, 2705 (1960). (07) Y, Nitta and A. Momoee, Yakugaku Zasshi, 82, 574 (1962); Chem. Ab&ack, 68, 4631)(1963).

116

JOHN W. ORIOEN

corresponding dithioacetal. The anomers of ethyl 2-acetamido-2-deoxyl-thio-D-gldcofuranoside were also prepared,(n the CY-D anomer in 56% yield and the BDanomer in lower yield (as the triacetate). Wolfrom and coworkers~+71were able to prepare various l-thio-a-P galactofuranoeides, generally isolated as the acetates, after purification by column chromatography. Thus, the Paceu arid Wilson method gave sirupy ethyl l-thio-a?.D-galactofuanoside,and a crystalline acetate. This product was also obtained by treatment of the dithioacetal with dilute hydrochloric acid and then mercuric oxide. Ethyl Zacetamido-2-deoxy-l-thio-~-~galactofuranoside w&s prepared in 32% yield, and the P-D anorner in 3% yield. Whereaa ethyl l-thio-/3-D-arabinofuranosidecannot be prepared directly, the 5-0-benzayl diethyl dithioacetal gave 38% of ethyl 5-O-benzoyl-l-thio&r+arabinofuranoside, which was debeneoylated to the desired product. The &benzoate of ethyl l-thio-a-D-ribofuranosideWBB similarly prepared.w Two other compounds, ethyl 2-tlcetamido-2-deoxy-l-thio-8-c~abinofuranoside" and ethyl 2-acetamido-2-deoxy-l-thio-cr~-xylofurq~ide,7~ were prepared from the corresponding D-galacto and Dgluco analogs by periodate oxidation, and subsequent borohydride reduction of the product. Zinner and coworkers studiedw the possibility of formation of l-thioaldofuranosidss from other sugar dithioacetals, and noted that, both with Pmannose dithioacetals and D-xylose dithioacetals, only the free sugar waa formed ;no thiofuranosides were obtained. With Plyxoae dithioacetals, both the free sugar and the l-thiofuranoside were formed: the latter, detected in only small amounts (on a paper chromatogram), w w not iaolated. All of the major products obtained, starting from the dithioacetals, have a cis relationship of the alkylthio group and the hydroxyl (or 2-acetamido-2deoxy) group at C-2. This formation of a atable, cis derivative is reminiscent of the similar relative stability of certain mylglycosyl halides." A corresponding effect has been noted for the l-thioaldopyranosides. Ethyl 2-acetarnido-3,4,6-tri-O-acetyl-2-deoxy1-thio-a-D-glucopyranoside is resistant to the action of mercuric chloride in hot, neutral, methanol solution, whereas the B-D anomer is readily converted into the 8-wgluco(68) M. L. Wolfrom, S. M. O h , and W.J. Polglase, J . Am. Chem. Soc., 73, 1724 (1960). (69) M. L. Wolfrom, Z. Yoaiiawa, and B. 0. Julieno, J . Org. Chem., 34, 1629 (1969). (70) M. L. Wolfrom, P. McWain, R.Pqnucco, and A. Thompon, J . Org. Chem., 39, 464 (1964). (71) M. L. Wolfrom and 2.Yo&awa, J . A m Cheni. Soc., 81, 3474 (1969). (72) M. L. Wolfrom and Z. Yodaawa, J . Am. Chern. Soc., 81, 3477 (1969). (73) M. t. Wolfrom and K. Anno, J . Am. Chem. Soe., 76, 1038 (1953). (74) L. J. lluyne~find F. €1. Newth, Aduaw. Carhh&de Chem.,10, 207 (1956).

THE GLYCOFURANOSIDES

117

pyranoside?6 Again, ethyl l-thio-p-Pmannopyranoside has been sh0w11~~ to be resistant to the action of mercuric chloride in methanol. Both of these nonreactive 1-thioaldopyranosides have this cis relationship. Conversely, whereaa dithioacetals of wribose afford the l-thio-D-ribofuranosidesreadily, those of 2-deoxy-Dglthro-pentose do not, but give the free sugar and unchanged dit hioacet a1 instead .m It is interesting that such resistance is encountered in a compound having a cis relationship and the alkylthio group; the latter is a leaving group which leaves readily, whose displacement should be facilitated by mercuric chloride. This effect will be discussed in more detail in the following Section. 3. Mechanism

A series of possible pathways for the formation of aldofuranosides (25) and of l-thioaldofuranosides (26) from dithioacetals (22) is shown on p. 118. Pathway AC leads to the aldofuranoside through an acyclic monothioacetal (23); the latter can also afford the diacetal (24) by pathway B . Pathway D leads directly from the dithioacetal to the l-thioaldofuranoside. A third pathway, EF, in aqueous solution, leads to the free sugar (28). Pathways C' and F' show alternative formations of the aldofuranoside and the free sugar, respectively, starting from the l-thioaldofuranoside. PacsuS7had proposed the possibility of an acyclic intermediate, a monothioacetal (23), in the formation of aldofuranosides and l-thioaldofuranosides; this suggestion was based on the isolation of acetals as minor products formed from the dithioacetals. Wolfrom and c~workers,?~ starting with such a monothioacetal, prepared from unrelated systems, showed that such a pathway is valid for &galactose. A very high yield (85-9001,) of ethyl 8-Pgalactofuranoside was obtained from wgalactose diethyl monothioaeetal; the yield was much higher than that obtaineds2directly from the dithioaeetal. For &glucose, the formation of ethyl l-thio-a-D-glucofuranoaide from a S-ethyl-O-methyl monothioacetal was not demonstrated; instead, a 95% yield of methyl &D-glucofuranoside was obtained, again demonstrating that pathway C had been followed. It may also be pointed out that the conversion of the monothioacetal into the l-thioaldofuranoside is improbable, as it involves the preferential displacement of an alkoxy group, instead of an ethylthio group; the former is a leaving group that leaves with difficulty, and the latter leaves wit,h ease, being acidic in nature. (75) L. Hough and M. I. Taha, J . Cheni. Soc., 2042 (1956). (76) J. Fried and L). E. Walr, J . Am. Chem. Soc., 71, 140 (1949). (77) M.L. Wolfrom, D. I. Weisblat, and A. R. Hanze, J . Am. Chew. Sor., 66, 2065 (1944).

JOHN W. GHEEN

118

Pathway C' has been demonstrated for both the ethyl and benayl 1-thio-a-wglucofuranosides;these compounds were uonverted,'* in ethanol

PR HtSEt H OH I

HT:H (29)

\ B

CHOH H t O H

(24)

at 70°, into 8 nonreducing sirup, presumably ethyl D-glucofuranoside,which W&B not investigated further. A mechanism is here proposed which is speculative in nature, but which affords a possible explanation of the reactivity of the acyclic monothioacetal (23) and the lack of reactivity of the 1-thioaldofuranoside (26). It

119


in b w d on the participation of the bulky, mercuric chloride molecule in the tranuition state for the reaction of the monothioacetal (30),the dithioacetal (29), or the 1-thioaldofuranoside (31) with a nucleophile (which may be an alcohol, water, or the C-4 hydroxyl group of the aldohexose or aldopentose concerned). This nucleophilic displacement of the ethylthio (or other ttlkylthio) groups from the dithioacetal or from subsequent products is aided by the mercuric chloride molecule. Shafizadeh' has suggested that a unimolecular (SNJ reaction is operative. In this type of reaction, the rate-determining step would be the formation of a carbonium ion, with the removal of the ethylthio group; subsequent attack on this ion by the nucleophile would be rapid. In an SN2 reaction, the removal of the ethylthio group and the attack by the nucleophile would be simultaneous. The SN1 reaction seems more probable here. (1) For the transition states (29) and (30) of the acyclic thioacetals, RO H \ / Et:---C---HOR HAOH HgC1, I

Etp---y---HOR Et\ /H 1'

&Cl*

\

HCOH

1

(30)

(29)

there is free rotation of the bond between C-1 and C-2, and the possibility of any steric interference by the C-2 hydroxyl group with the bulky mercuric chloride molecule is readily alleviated by rotation around this bond. So, the displacement of the ethylthio group, to form a carbonium ion, is readily accomplished, and steps A , B, or D should be fairly rapid. A similar possibility may be proposed for steps E and F. (2) With the cyclic 1-thioaldofuranoside, there is restricted rotation about the carbon-carbon bonds. The inclusion of the mercuric chloride molecule in the transition-state complex (31) of this cis compound would

Q \SEt

(31)

be severely hindered by tlic acljttaciit C-2 hydroxyl group. Reactions of the (68-1,2)-l-thioaldofuranoside with nucleophiles would then be relatively slow. The frequent isolation of 1-t,hioaldofuranosidesfrom reaction systcnis coiitaitiing either alcohols or watcr shows that rettctions C' and F'

120

JOHN W. QlZElN

do not occur readily. (The rapid, acid hydrolysis of l-thioddofuranosidea may be explained by the much smaller steric requirements of the proton; no such crowding will occur.) (3) The favored formation of (ch-1 ,2)-l-thioaldofuranosides and of tram-1 ,%aldofuranosides is more difficult to explain. One possibility is 811 orientation of the intermediate carbonium ions (32) and (33). There may

be more repulsion between the alkoxy group at C-1 and the adjacent hydroxyl group at C-2 than there is between the ethylthio group and the hydroxyl group. There seems at present to be no really adequate explanation here; in,each caae, the attacking nucleophile is the C-4 hydroxyl group (in order to effect ring closure). (4) There is also no adequate explanation of the fact that path A or E is observed with mme sugars (*galactose, n-mannoae, D or Irarabinoee, wxylose, and D-lyxose) and path D with others (wglucoae, n-glucuronic acid, and wribose). The C-2 hydroxyl group haa been ahown to have an effect in the c&ge of D-glucose. The l-thio-cr-D-glucofurmoside is readily formed from the dithioacetal, but 2-deoxy-wurubino-hexose dithioacetal gives only free sugar and unconverted dithioacetal under the same conditions. In methanol, the methyl 2-deoxy-D-urubino-hexofuranosidesare readily formed from the dithioaaetal, in contrast to the behavior of the D - ~ ~ U C O dithioacetal. S~ It is not yet known whether pathway D-c' or pathway A-C is being obaerved for the Zdeoxy-n-arubino-hexose system. Work by Capon and Thacker4*l on the formation of aldofuranoaides from the corresponding acyclic dialkyl metals in aqueous acid haa suggested that the C-4 hydroxyl group of the n-glucose derivative is much more nucleophilic than that of the wgalactose derivative, and that both of these groups are able to compete successfully with the water present aa a solvent; thus, ring closure to the furanoside occurs in preference to formation of a free sugar. This concept might be extended to the dialkyl dithioacetals. For wglucose dialkyl dithioacetds, the C-4 hydroxyl group successfully displaces a thioalkyl group at C-1, in competition with the solvent (either an alcohol or water), and the resulting product is a l-thio-Dglucofuranoside. With the D-galactose derivative, the C-4 hydroxyl group is, presumably, weaker niicleophile than the solvent, and cannot compete sucaendully; the


121

product is a mixed monothioacetal which is subsequently convert,ed into the D-gaJactofuranoside.

V. GENERAL PREPARATIVE METHODS 1. Preparation from Furanose Esters and Halides

The furanose acetates and bensoates may be used to advantage in the preparation both of alkyl and aryl aldofuranosides. The first synthesis of a wgalactofuranoside was achieved by Schlubach and Mei~enheimer~~; treatment of 2 ,3 ,5 ,6-tetra-O-acetyl-wgalactofurmosewith ethyl iodide and silver oxide gave ethyl 8-D-galactofuranoside. Application of this reaction may also be made to the l-thiofuranosides. Reaction of penta-0benzoyl-@-D-glucofuranosewith 2-methyl-2-propanethiol and zinc chloride gave the tertbutyl l-thio-a- and -fl-D-glucofuranosides in 48 and 25% yields, respectively.7e The aryl aldofuranosides have generally been prepared by fusing the furanose acetate with the appropriate phenol in the presence of ptoluenesulfonic acid. Tsou and Seligmanso prepared phenyl and Znaphthyl 8-D-ghcofuranosiduronolactonein this way, and Ishidate and MatsuP obtained the p-nitrophenyl P-D analog. The phenyl p-sfuranosides of wxylose, carabinose, D-glucose, and wgalactose were prepared similarly by Lindterg and coworkers.82~89 p-Chlorophenyl 8-D-ribofuranoside was the product from fusion of either the a- or 8-wribofuranose tetraacetate with p-chlorophenol and p-toluenesulfonic acid, but use of zinc chloride gave the a-wribofuranoside in~tead.~' Fletchsr and coworkers have utilized the poly-0-acylaldofuranosyl halides. Treatment of crude tri-0-bensoyl-wribofuranosyl bromide with sodium phenoxide in 1,2-dimethoxyethane, gave phenyl 0-wribofuranosidee6; use of sodium methoxide gave methyl /3-D-ribofuranoside.88 (78) H.H. Schluhach and X. Meisenheimer, Ber., 67, 429 (1934). (70) H. B. Wood, Jr., H. W. Diehl, and H. G. Fletcher, Jr., J . Org. Chem., 29, 461 (1964). (80)K . 4 . Teou and A. M. Seligman, J . Am. Chem. Soc., 74, 5606 (1952); 76, 1042 (1953). (81) M. Ishidata and M. Mateui, Yakugaku Zaeahi, 82, 662 (1962); Chem. Abstracta, 68, 4639 (1963). (82) H. Borjeeon, P. Jerkeman, and B. Lindberg, A c h Chem.Scad., I T , 1705 (1963). (83) P.Jerkemsn and B. Lindberg, Ada Chem. Scud., 17, 1709 (1963). (84) T. Shimidate, Nippon Kagaku Zaaahi, 88, 214 (1962); Chem. Abafracia, 69, 6498 ( 1963). (85) E. Vie and H. G. Fletcher, Jr., J . Am. Chem. SOC.,79, 1182 (1957). (86) C. Pederaen and H. G. Fletcher, Jr., J . Am. Chem. SOC.,84, 941 (1950).

122

JOHN W. GREEN

Methyl a-xdyxofuranoside was preparedw from tri-O-benroyl-wlyxofuranosyl bromide. PedersetP treatcd tetra-0-benzoyl-cr-mlyxopyranose with hydrofluorio wid and obtained tri-0-benroyl-cu-Plyxofuranosyl fluoride; reaction of the residual sirup, obtained from the mot,her liquor, with methanol and sodium hydrosido gave niet hyl cu-mlysofurlinc~idc. Although the neighboring-group c?!€ect8 gave the knm-1 ,2-glycwide in thc above examples, Barker and Fletcher“ found that 2,3,fi-tri-O-benzylPribofuranosyl bromide and methanol, with silver carbonate, gives mainly methyl a-mribofuranoside. Perline0 converted 5 ,&di-O-acetyl-a-wmannofuranosyl bromide, with sodium hydroxide in methanol, into methyl a-Dmannofuranoside in high yield; treatment of the bromide with methanol and silver oxide gave, instead, a sirup tentatively identified aa the 5,6diacetate 2 ,3-carbonate of methyl Pmmannofuranoside. 2. Glycofuranosylamines The subject of glycosylamineshas already been reviewed in thia Series,W and the nomenclature of these compounds, formerly called N-glycosides, amidea, anilides, etc., was discussed. The poly-0-acylglycofuraosyl halides have been valuable for use in oondensation resations with purines and pyrimidines, in order to prepare the naturally occurring nucleosides and various isomers containing sugar residues other than the Pribofuranosyl and 2-deoxy-mqthro-pentofuranosyl residues normally found. This subject, also, has been thoroughly reviewed in this Series.B1-04Such condensations are generally carried out either with the silvergsor the chloromercuri saltwof the nitrogenous base, and the furanoid structure of the products has been thoroughly established. The “trans” rule of Tipsonw has been used to explain the configuration of the products. Various epimerizations and other transformations of the sugar residues have been performed, primarily with the aid of the 2,3anhydro derivatives; the chemistry of methyl 2 ,3-anhydro-~-lyxofurano(87) A. K. Bhlrttaoharya, R, K. Ness, and H. G. Fletcher, Jr., J . Otg. Chem., 48, 428 (1983). (88) C. Pedersen, Acfa Chem. Scad., 18,BO (1904). (89)A. 8.Perlin, Can. J . Chem., 49, 1306 (1904). (90)a. P. Ellie and J. Honeynan, Advon. CarbohydrofeChem.,10,95 (1965). (91) R. 8. Tipaon, Advan. CarbohydrafeChem. 1, 193 (l&). (92) cf. R.Barker, Advan. Carbhydmb Chsm.,11,286 (1950). (98) J. J. Fox and I. Wempen, Advan. Carbohydrah Chetn., 14, 283 (1969). (94)J. A. Montgomery and H. J. Thomes, Advan. Corbohyhfe Chem.,17,301 (1962). (96) E.Fiecher and B. Helferiob, Ber., 47,210 (1914). (90)J. Davoll and B. A. Lowy, J . Am. C h . &c., 78, 1060 (1961). (97)R. 8. Tipeon, J . Ei01. C h . ,180, 66 (1939).


123

deg’ h a been developed as a preliminary to such reactions with the nucleosidcs. These transformations have been described in detail by Montgomery and The 2 ,2‘-anhydro derivatives of the nucleosides have also been used; in this type of derivative, the anhydro ring may be broken without inversion at the C-2 hydroxyl group of the sugar. The lability of the 2deoxy sugars originally necessitated use of methods involving the introduction of the methylene group at C-2 of the nucleoside, instead of starting with the difficultly available di-O-acyl-2-deoxy-Deylthro-pentofuranosyl halides. This problem was finally solved by use of the less reactive chlorides. Zorbach and Payneggaprepared a 2 ,6-dideoxyD-ribo-hexopyranosyl chloride 3,4-di-p-nitrobenzoate by treatment of the 1,3 ,Ptri-pnitrobenzoate with hydrogen chloride in dichloromethane. The first 2-deoxy-ddofuranosyl chloride was made by Fox and coworkersegbby treating the 3,B-di-ptoluoyl ester of methyl 2-deoxy-~erythro-pentofuranoside with hydrogen chloride in acetic acid : condensation with monomercuri-thymine and subsequent deacylation gave 60yo of thymidine and 4% of the a-Danomer. Ness and Fletcher’oo synthesized 2-deoxyadenosine from chloromercuri-6-benzamidopurine and 2-deoxy3,5di-O-p-nitrobenzoyl-D-erythro-pentofuranosylchloride : the latter compound was prepared by the method of Zorbach and P a ~ n e . ~No ~ ’steric control (“trans” rule) is, of course, observed with 2-deoxy-werythro-pentose or other 2-deoxy sugars. A new route, from l-thio-waldohexofuranosides, was developed by Wolfrom and coworkers; ethyl l-thio-a-D-glucofuranosidewas converted by chlorine into the chloride,lol and this wm condensed with the chloromercuri derivative of a 2 ,6-diacetamidopurine to give, on partial deacetylaD-Galactofuranosyl anation, a 2-acetamido-9-~-~-glucofuranosyladenine.~~ logs were also prepared. In 1946, Berger and LeeIo2heated &ribose with aniline in ethanol, and obtained a crystalline product to which they ascribed an a-D-furanoid structure. This N-phenyl-a-D-ribofuranosylamineshowed mutarotation in water, and was hydrolyzed by water, by aqueous acid, and by alkali. It was distinguished from the pyranoid isomer by differences in optical rotation and mutarotation. The furanoid structure was allegedly established (98) B. R. Baker, R. E. Schaub, and J. H. Williams, J . Am. Chem. Soc., 77, 7 (1955). (99) J. A. Montgomery and H. J. Thomas, Ref. 94, pp. 313-326 and 331-335. (!)Ha) W. W. Zorbach and T. A . Payne, Jr., J . A m . Chem. Soc., 80, 5564 (1958). (Wb) M. Hoffer, R. Duwchinsky, J. J. Fox, and N. Yung, J . Am. Chem. SOC.,81, 4112

(1959). (100) R. K. Neas and H. G. Fletcher, Jr., J . Am. Chem. Soc., 82, 3434 (1960). (101) M. L.Wolfrom and W. Groebke, J . Org. C h . , 28,2986 (1963); F. Weygand and H. Ziemann, Ann., 667, 179 (1962). (102) L. brger and J. Lee,J . Org. C h . ,11, 75 (1946).

1%

JOHN W. QRElN

by the preparation of a trityl derivative; periodate oxidation and methylation techaiques could not be applied,because of occurrence of extensive decomposition under the conditions used. Todd end coworkers reported108 that N-phenyl-D-ribopyranoaylamine is isomerieed by boiling ethanol to give the furanoid isomer. The rate of hydrolysia of the furanoaylmine was studied by Stacey and coworkera.m' Other N-arylglycosylamines have been prepared. Kuhn and Kirschenlohr'" isolated N-benzyl- and N-phenyl-carabinosylamine.Berezovskii and coworkerP prepared the N-(3 ,P.xylyl) derivatives of D-xylosylamine and D-arabinosylamine, and, from the reaction of 3 ,4 ,5-trimethylaniline with each of the aldopentoaes, obtained products whose respective configurations were cu-Darabinofuranosyl, cu-D-ribofuranosyl, &~-lyxofuranoayl, and @-Dxylofuranosyl.All of these products showed mutarotation in solution. Hockett and Chandler107 prepared a N-D-glucofuranosylacetamide (N-acetyl-Pglucofuranosylamine)as a product from the Wohl degradationIo8 of hexa-O-acetyl-D-gZuco-D-gubheptononitrile. This compound, having an amide grouping, is more stable t h w the arylglycosylamines discussed previously, and its rate of mutarotation is lower. A N-D-ribofuranosylurea was prepared by Benn and JoneslOg; the structure of this compound was inferred from its optical rotation and its relative mobility on a paper chromatogram. Considerable over-oxidation waa encountered in periodate oxidation of both of these compounds. Baddiley and coworkers1mprepared a D-ribofuranosylamine by condensing sodium azide with 2 ,3 ,6-tri-O-benzoyl-~-ribofuranosylchloride in acetonitrile and catalytically reducing the resulting glycosyl azide. The unstable amine waa converted, by reaction with (benzy1oxy)carbonylglycyl ethyl carbonate and subsequent debenzoylation and hydrogenolysis, into the anomers of N-glycyl-D-ribofuranosylamine.Each anomer wm oxidized (103) G. A. Howard, G. W. Kenner, B. Lythgoe, and A. R. Todd, J. Chem. Soc,, 856 ( 1946). (104) K. Butler, S. Lalsnd, W. G. Overend, and M. Stacey, J. Chem. Soc., 1433 (1960). (106) R. Kuhn and W. Kbachenlohr, Ann., 800, 116 (1956). (106) V. M. Berezovskii and V. A. Kurdyukovs, Do&. AM. Navk SSSR, 76, 839 (1961); C h m . Akhack, 46, 8464 (1961); V. M. Berezovakii, E. P. Rodionova, and L. I. Strel'chunas, Zh. Okhch. Khim., 44, 628 (1964) ; Chem, Abubacb, 48, 10696 (1964); V. M. Berezovslrii and E. P. Rodionova, Zh. Obshch. Khim, 26, 746 (1966); Chcm. Absfrack, 60, 146S7 (1966). (107) R. C.Hockett and L. B. Chandler, J. Am. Cham. SOC.,66, 067 (1944). (108) V. Deulofeu, Aduan. Carbohydrate Chcm.. 4, 110 (1949); V. Deulofeu and J. 0. Def~rrfbri,Analse Asoc. @im. Arg., 88, 241 (1960); Cham. Abelracfs, 46, 5110 (1961); J . Org. Chcm.,17, 1087 (1962). (109) M. H. Benn and A. 13. Jonep, J. Chem. Soc., 3887 (1960). (110) J. Baddiley, J. G. Buohanan, R. Hodgee, and J. F. Prenaott, J. Chem. SOC.,4769 (1967).

THE GLYCOFURAXOSIDES

129

successfully with periodate to establish its structure. The same diddeliydc was obtained from the j3-~anonier and from iV-glycyl-j3+glucopyranosylamine. The 2 ,3,5-tri-O-ben~l-D-ribosylamilie mentioned above was condensed by Shaw and Warrenerll' with a substituted acrylamide, to give uridine. Ellis and Honeyman" have raised strong doubts as to the validity of the structural determinations that have been applied to glycosylamines; these doubts are based on the fact that such compounds are readily isomerized, as evidenced by their mutarotation. The formation of a trityl ether cannot be considered valid evidence for the presence of a primary hydroxyl group in the compound tritylated, and periodate oxidation, which is often excessive, may lead to faulty conclusions. 3. Miscellaneous Preparations

A general route to alkyl D-glucofuranosides was initiated by ReeveP; this consisted in the reduction of methyl 2,5-di-O-methyl-~~-wglucofuranusidurono-6, 3-lactone with lithium aluminum hydride to give methyl 2,5-di-O-methyl-~~-~-glucofuranoside. The procedure was developed into a general method by D. D. Phillips,22with the preparation of methyl CY- and Bwglucofuranoside, and ethyl p-D-glucofuranoside; the reducing agent he employed was sodium borohydride.1128A significant method for the reduction of an acylated aldono-l,4-lactone to the acylated furanose employsll28 This procedure has been used especially bis( 1 ,Zdimethylpropyl)borane.112b in nucleoside synthesis with hexoses.1120 As a modification of the Fischer method, alcohols other than methanol have been used. Purves and Hudson*1aprepared benzyl a-wfructofuranoside by alcoholysis of methyl a-D-fructofuranoside in benzyl alcohol containing hydrogen chloride; the product, isolated aa the tetraacetate, waa unaffected by invertase. Treatment of %ribose with benzyl alcohol-hydrogen chloride until the reducing power had practically disappeared gave benzyl a similar procedure reported1148 gives 49% of product. &~-ribofuranoside~~'; An anomeric mixture of isopropyl wglucofuranosides was obtained1l6 (111)G.Shaw and R. N. Warrener, J . Chem. SOC.,2294 (1958). (112) R. E.ItBevea, J . Am. Chem. Soc., 78, 934 (1954). (1124 P. Kohn, R. H. Semaritano, and L. M. Lerner, J . Am. Chem. Soc., 88, 1457 (1964). (112b) G. Zweifel, K. Nagase, and H. C . Brown, J . A m . Chem. SOC.,84, 190 (1962). (112~) P. Kohn, R. H. Samaritano, and L. M. Lerner, J . Am. Chem. SOC.,87, 5475 (1965);J . Org. Chem., 31, 1603 (1966). (113) C.B. Purves and C. S. Hudson, J . Am. Chem. SOC.,60, 49 (1937). (114)R. K.Netu and H. G. Fletcher, Jr., J . A m Chem. SOC.,76, 3289 (1953). (114s)K. Heyns and J. h n z , C h .Ber., W, 348 (1961). (116)J. Kiss and H. Spiegelberg, Helu. Chim. Acta, 47, 398 (1964).

126

JOHN W. GREEN

from 3 ,5,6-tri-0-beneoyl-1 ,2-O-isopropylidene-a-~-glucofuranose in acidic isopropyl alcohol. The first preparation of crystalline methyl a-wmannofuranoside wt18 made by Haworth and Porter,%' by treating D-mmnofuranose 2 ,3 :5,6&carbonate with diasomethane, a neutral alkylating agent. Use of an alkalime reagent reverses the anomer distribution obtained with other reagents. Walker, Gee, and McCready116applied the Kuhnreagents (silver oxide and methyl iodide in N,Ndimethylformamide) to aeverd sugars, and analyzed the completely methylated glycosides by gas chromatography. D-Xylose, wmannose, and wglucose gave only traces of furanosides, and carabinose gave about 14% of an unresolved mixtwe. DGalactose gave Soyoof a- and 10% of Bfuranoside, and wgdacturonic acid gave 93 and 7%, respectively. Thus, a preponderance of the cis-l ,2-glycoside is'obtained in each instance. D-Fructose was converted into a mixture of 19% of a- and Slyo of 8-furanoside, a decided shift from the ratio observed in acidic reagents. The preponderance of furanoside had been noted by Haworth and coworkers'" in the methylation of wgalactose with dimethyl sulfate and alkali. A biochemical approach has been utiliaed in the preparation of &Dfructofuranosides. Invert-catalyzed transglycosylation118 of sucrose in methanol gives methyl Bwfructofuranoside; use of ethanol gives ethyl fl-D-fructofuranoside.ll@ Bacon and Edt$mqlm treated an tiqueous solution of glycerol with sucrose and invertase in order to prepare an 0-D-fructosylglycerol. Presumably, all of these compounds are fructofuranosides. Purves and HudsonMJ1*used the hydrolyzing power of invertaae to prepare methyl and benzyl a-D-fructofuranoside; wfructose and the &D anomer were fermented from the reaction mixture by the enzyme, thus allowing the ready isolation of the invertaae-resistant a-Danomer. This biochemical technique has been extended to the preparation of many di- and oligo-saccharides. The a-D-fructofuranoeyl derivatives of 2-deoxyD-@rabino-hexose(8-linked)lg1 and of wxyloseL** have been prepared. (118) H.C;. Walker, Jr., M. Gee, and R. M.McCready, J . Org. Chcm., 27,2100 (1982). (117) W. N. Haworth, D. A. Ruell, and G . C. Weatgarth, J . Chem. SOC., 126, 2488

(10%); J. Pryde, J . c h .SOC.,118, 1808 (1023). (118) T.Miwa, Koeo Kagoku Shiumpoaiumu, 8, 57 (1053);Chem.Abakaefs,47, 10676 (1983);K.Iahiaawa and T. Miwe, ibid., 9, 40 (1964);C h . Abuhrrcle,48, 7083 (1064). (119) A. I. Oparin and M. S. Bradinakaya, DoM. A M . Nauk SSSR,89, 631 (1953); Chem. Abatracb, 47,8113 (1863);M.8. Bardineksya, Tr. Komissii And. Khim., Akad. Nauk SSSR,Inel Gmkhim. iAnal. Khim., 6, 486 (lo&); Chem. Abahrrcb, 50, 13122 (1088).. (120) J. 5. D.Baaon and J. Edelman, Arch. Biochem., 18, 467 (1960). (121) G.A. Barber, J . Am. Chem. Soc.,81,3722 (1960). (122) a.Avigad, D.S. Feinpld, and 8.Herrtrin, Bdoclrim. Biophgu. Acb,PO, 129 (1068); Chom. AbaWcb, 50,11387 (1953).

THE GLYCOFURAKOSIDES

127

deveral oligosaccharides containing wfructofuranose residues have been as well as an a-D-glucopyranosyl ~-galactofuranoside.~** Special examples of stable glycofuranosides will be discussed in Section VI,4, but mention may be made here of two distinctive glycofuranosides. in Ballou and Fischera refluxed di-0-isopropylidene-D-~~~~~o-hexodialdo methanolic hydrogen chloride, and obtained a 20% yield of a compound containing two furanoside rings, namely, the dimethyl a ,d i u r a n o s i d e of =manno-hexodialdose. It waa identified by methylation, by its strong dextrorotation, and by its rate of hydrolysis by 0.01 N hydrochloric acid. Finan and Warren126 conducted a Koenigs-Knorr condensation of benzyl 3,5,6-tri-O-benzyl-a-~-glucofuranosidewith tetra-0-acetyl-a-Dglucopyranosyl bromide, and obtained a derivative in which the reducing residue of aophorose was present aa its benzyl furanoside.

VI. STRUCTURE OF GLYCOFURANOSIDES The uniqueness of the aldofuranosides, aa compared to the aldopyranosides, waa recognized initially through the difference in rates of hydrolysis by acid. This difference WBB confirmed by the use of methylation techniques; the latter have, to a great extent, been supplanted by the periodate oxidation of Jackson and HudsonlZ6and by oxidation with lead tetraacetate. Hudson's rules of isorotation were also employed to a great extent. The present discussion will deal mostly with hydrolysis and oxidation, although isorotation will receive consideration.

1. Application of Isomtation to Furanoid Struetures One of the primary functions of the rules of isorotation of Hudsonlfl was the demonstration that two glycosides can be a- and /3-anomers; this waa applied by Haworth and Portera to ethyl a- and &D-glucofuranosides; the difference in rotation of these two compounds is 184', whereaa that for the ethyl D-glucopyranosides is 183.7'. Similarly, establishment of a furanoid ring in a compound of undetermined structure waa applied by Green and PacsuS2to ethyl 1-thio-a-.p glucofuranoside; the observed specific rotation of this compound in aqueous (123)

(124) (126) (126) (127)

J. 8. D. Bacon and D. J. Bell, J . Chem. Soc., 2628 (1963); 8. A. Barker and T. R. Camngton, ibid., 2126 (1964); D.Grose, P. H. Blanohard, and D. J. Bell, ibid.,

1727 (1964); P. J. Allen and J. 8. D. Bacon, B i d e m . J., 88, 200 (1966); 8.A. Barker, E. J. Bourne, and 0. Theander, J . C h . Soc., 2064 (1967); G. Avigad, J . Bio2. Chem., 329, 121 (1967). E. J. Bourne, J. Hartiigan, and H. Weigel, J . C h .Soc., 1088 (1961). P.A. Finan and C. D. Warren, J . C h . Soc., 6229 (1963). E. L. Jackson and C. 8. Hudmn, J . Am. Chem. Soc., 69, 994 (1937). C. 8.Hudmn, J . Am. Chem. SOC.,91, 66 (1909).

128

JOHN W. GRBEN

solution was +120.7' and the theoretical value, calculated from data for Haworth's ethyl D-glucofuranosides,l and for the known ethyl 1-thi0-fl-Dglucopyranoside, prepared by Schneider and SeppJo1wm +120.7". Pacsu and coworkers60*Malso found good agreement between the calculated and oberved optical rotations for alkyl D-galactofuranosidea, barabinofuranosides, and wmannofuranosides, A final example is the work of Wolfrom and Shafizadehl*aon the structure of sucrose, for which Hudson had given an (Y-D configuration for the Dglucopyranosyl moiety, based on the results of invertasecatalyzed hydrolysis. These workers,l*ausing data from the methyl hfructofuranosides, demonstrated that this hydrolysis is not accompanied by a Walden inversion and that the original work of Hudson waa correct. 2. Acid Hydrolysis of Glycofuranosides The relatively rapid hydrolysis of glycofuranosides by acids was the first property used for differentiating these compounds from the more normally encountered, more stable glycopyranosides. In Tables VIII and IX are assembled data from the literature; the rate constants are given in set.-' and common logarithms.120The conditions of acid ooncentration and temperature varied; in the data r e p ~ r t e d , ' ~the * ~conditions ~ were the same, and so the rate constants can be compared. Heidt and P u r v e ~used ~ ~a term, k* = k/[H], to eliminate differencesin the concentration of acid used. The data in Table VIII are presented in the order of the conformational stability observed fop methyl glycosidation (see Table I on p. 99 and Table I1 on p. 106).In each of the three cmes where data for both anomers are available, the trans-1,2 anomer is the more stable, and a lower rateconstant is observed. This is the same pattern as that observed in Table 11. However the order of conformational stability given earlier is not observed here. The arabinofuranosides show the maximum stability, and the wgalactofuranosides and cfucofuranoeides, having similar conformations, show a similar stability, However, the furanosidea of ~-lyxoee,wmannose, and brhamnose show an unexpectedly high stability which is almost aa great as those of the furanosides of the first-mentioned sugars. These data lead to the conclusion that the conformational stability for tranaition complexes (128)M. L. Wolfrom and F. Sh&adeh, J . olg. C h . ,21,88 (1956). (129) The dimemions wed for rate constants in the literature vary, being given in eeconds, in minutes, and even in h o w ; normally, common (decimal) logarithms 81x9 used, although ocoaaionally natural 10a;srithms rn employed. Since common logarithma have been uaed in other articles in thin €jerk,the practice ie being

continued.

(130) L. J. Heidt and C. B. Purves, J . Am.

C h .Soc., 68, 1385 (1044).

THE GLYCOFUHASOSIDES

129

TABLE1111 Hydrolysis0 of Some Methyl A l d o f u r a n ~ s i d e a ~ ~ ~ * Methyl furanoeide of

Anomer

106 k (am.-')

a

1.5 7.7

B

B

49

B

54 92

a

-

11.3 46 0.89 7.5

B a

B

2.4 34

a

3.6

a

0

1.1 3.5

The reaction conditions were: 1.0 N hydrochloric acid at 20".

encountered in the hydrolysis mechanism is not necessarily similar to that encountered in the mechanism of methyl glycosidation.la1 In Table IX are presented data for the hydrolysis of aldofuranosides and of fructofuranosides, together with those for some pyranosides as reference compounds. The fructosides have been explored in great detail by Purves and coworkers2sJ1aJa2-4; the difference in rate of acid hydrolysis of the D-fructofuranosides and Dfructopyranosides is much less than the differences observed for the two kinds of aldosides. The activation energies foynd by Purvesl**are very similar for furanoid and pyranoid derivatives, and it WM concluded that the small difference in these energies (or in the rate constants) could not be used to distinguish between the two types of ring structure. (131) B. Capon and W. G. Overend, Aduan. Carbohydrate C b . ,16, ll(eee especially p. 34) (1960). (132) C. B. Purves, J . Am. Chem. Soc., 66, 1969 (1934).

JOHN W. GREEN

130

TAB- IX Acid Hydrolyda of S o m e Alkyl Glycofuranoridea and Glycopyranosidea Glycoride

Methyl 2-deoxy-u ,&wrabinOhexofuranomde Benzyl a-D-fructofurenoside Benzyl a-D-fruotopyranoeide Methyl a-D-fructofuranoeide Methyl a-wfructopyranoaide Methyl a-wgltwofuranoiide Ethyl crwglucofuranoside B anomer Sucrose Methyl a-D-mannofuranoside Ethyl 8-D-gelaotofuranoside u anomer Methyl a-warabinofuranoside Methyl 2 ,6di-O-methyl-w gluooaiduronolactone Benzyl 1-thio-a-D-glucofuranoeide Ethyl 1-thio-a-D-glucofuranoside Methyl a-D-arabinopyranoside Methyl 8-p.glucopyranoeide Methyl a-D-mannopynmoside Methyl a-wgalactopyranoside Benzyl a-D-fructofuranoeide Methyl a-D-fructofuranoaide Sucroae Methyl &D-fructopyranoside Ethyl pD-gshctofuranoside Ethyl 8-D-galactopyranoside Ethyl 8-wglucopyranoaide

Normality Temp., of Acid

OC.

10% (-.-I)

References

0.006

16

1700

60

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0 -01 0 -01 0.01 0.01 0.01

40

150 20

66-60 65-80 65-60 96-100 95-100 96-100 98 88 100 100

134 134 134 134 E3 23

0.01

100

0.01 0.01 0.01 0.01 0.01 0.26 0.26 0.26 0.33 0.33

100 100 96-100 95-100 95-100 20 20.2 20 20 20 19.8

2

.o

2 .o 2 .o 2 .o

40

50 50

300 100 128 380 207

890 830 260 133 133 113 100

23 137 137 137 50

m

27 138

10.6

64

9 .a

62 27 137 137 137 113 132 113 133 133 136 136 136 136

10.7 6 1.7 3-8 1%

58

80

7 21.6 16 .O 26.6 2880

60 60

12.6

m

(133) C. B. Purvea and C. 8.Hudson, J . Am. Chem.Soc., 68, 1170 (1937). (134) L. J. Heidt and C. B. Purvee, J. Am. Chsm. doc., 60, 1206 (1938). (135) W. 0.Overend, C. W. Rees, und J. S. Sequeira, J . C h . Soc., 3429 (1062). (136) F. A. Img, J. 0.Pritchard, and F. E. Stufford, J. Am. Chem. Soc., 78, 2362 ( 1867). (137) W.N. Haworth and E. L. Riret, J. Clmr. Soc., 2616 (1930). (138) R.E. Reeves, J . Am. Chem. Soc., 62,1616 (1940).

131


An entropy value for the acidic hydrolysis of ethyl BD-galactofuranoside by Overend and coworkers."6 The negative value (- 7.1 e.u.) waa interpreted aa being diagnostic of an A-2 mechanism, in contrast to the positive values (+13.7 e.u., mean value) and A-1 mechanism found for a large number of pyranosides. The proposed intermediate is shown in formula, (34). It waa considered that the planar, furanoid ring is capable of

WBR reported

accommodating a crowded transition-state. Entropy values have now been reported for several methyl aldof~ranosides*~* (see Table X); these are also negative, and thus support the A-2 mechanism. The rapid hydrolysis of ethyl BD-galactofuranoside, in comparison with the slower hydrolyses of the pyranosides, was attributedlss to the much lower activation energy (22.7 kcal.) compared with an average value of 31.6 kcal. found for the pyranosides. It is interesting that sucrose, having a fructofuranose residue, is hydrolyzed by an A-1 mechanism; a positive entropy value (+9.7 e.u.) has been reported.la6 TABLEX Entropies of Activation for Acid Hydrolysis of Methyl Aldoaides Methyl Aldoside

AS for the furanoside,a e. u.

a-D-Gluco B-D-G~uco a-D-Gslacto

&D-Galacto a-D-Xy lo P-D-XylO 4 Detern~ined'~* in 1 M HClO, at 25". ~niiied'gb in 1 M HClOd st 25".

-11.1

-9 .oE -9.4 -8.7 -8.3 -8.8

AS for the pyranoride,b e. U .

+14.8 +16.5 4-17.7 +13.3 +15.7 -1-17.5

* Determined'Jb in 2 N

HCI at 60".0 Deter-

132

JOHN

W. GREEN

3. Oxidation of Glycol Groups Two reagents have been used in determining the number and configuration of the glycol groups in glycofuranosides. The action of p e r i ~ d a t e ' ~ ~ is generally nonstereospecific, and the use of this oxidant i R confined mostly to stoichiometry. Lead tetraacetate" is more stereospecific in its nctioii on cis- and trans-glycol groups, and rate studies are generally made of its action. The consumption of one mole of oxidant per mole, with no formation of formic acid, is used to detect a furanoid structure in aldopentofuranosides. The consumption of two moles of oxidant per mole, with the formation of one mole of formaldehyde and no formic acid, is used in the detection of aldohexofuranosides. Overoxidation sometimes occurs. leaves only one Periodate oxidation of the ~-aldotetrofuranosides~~ optically active center, at the original C-1, and both &D anomers, (35) and (36),give a product (37)of [ a ] ~ -116 to -119O, whereas the CT-D

anomers give a product of [ a ]113 ~ to 120'. Similar data have been obtained for the D-aldopentofuranosidda6:here, there is also an asymmetric center left at the original C-4, as shown in (39).In both instmces, only one mole of oxidant is consumed per mole, and no formaldehyde or formic acid is formed. In the case of the D-aldopentofuranosides, for example, (38), the dialds hyde (39) can be equated with the same dialdehyde formed from an aldohexopyranoside, for example, (W),and hence the anomeric centers of

(139) J. M. Bobbitt, Advan. Carbohydrale ClLent., 11, 1 (1956). (140) A. 8. Perlin, Advan. Carbohydmk Chem., 14, 1 (1959).


133

the furanoside and the pyranodde are related. This has been demonstrated with the methyl D- and tarabinofuranosides,l% methyl a-wxylofurano~ide,"~ met hy 1 8-wrihof~ranoside~~'~ and N-glycyl-bwribofuranosylamine."o For such comparisons, it is not necessary that the products be isolated.141. Phenyl 8-D-ribofuranoside was equated with phenyl 8-wglucopyranosides6; with an excess of sodium metaperiodate, 0.313 M solutions of each gave constant, observed rotations of - 1.68" (after 3 minutes for the D-ribofuranosidr and after 6 hours for the D-ghcopyranoside). Similar results were obtained for benzyl 8-D-ribofuranoside and benzyl P-Dglucopyranoside.110 The aldotetro- and aldopento-furanosides each have a vic-diol grouping at C-2, C-3, and the rate of oxidation of these 2, 3diols by lead tetraacetate is determined by the cis or trans configuration, and, to a smaller extent, by the cis or trans relationship of the aglycon group to the adjacent C-2 hydroxyl group (that is, the amount of C-1, C-2 nonbonded interaction). The rate of oxidation of methyl CY- and 8-werythrofuranosides is very high compared with that of the D-threofuranosides, and is complete in 45 seconds at 0" (0.01 M sol~tion).'~ The rate of oxidation of very dilute solutions (0.OOOl M ) may be measuredi4zby spectral determination of Pbw at 280-300 mp; the rate for the CY-Danomer, having a cis-l,2 relationship of the aglycon and C-2 hydroxyl groups, is 3.5 times that of the @-D anomer. The slower oxidation of the methyl D-threofuranosides was determined at normal concentrations&; the rate for the 8-Danomer, having a C-1, C-2 interaction, is six times that of the a-Danomer. For the aldohexofuranosides, there are two sets of glycol groups, namely, a 2 ,3-glycol in the ring, and a 5,6-glycol outside the ring. It has been established that, with lead tetraacetate, the rate of oxidation is in the order cis8 ,3-glycol > 5 ,6-glycol > trans-2, 3-glycol. Thus, one mole of methyl a-wmannofuranoside (41) reacts very rapidly with one mole of oxidant14a;the second mole is consumed very slowly, and formaldehyde is formed only in small amount. This action is attributed to the fast oxidation of the cis-2,3-glycol; the subsequent, slow reaction of the 5,6-glycol of the dialdehyde (42) is attributed to the formation of hemicetals between these hydroxyl groups and the aldehyde groups. A similar type of reaction was observed with methyl 8-wgulof~ranoside.~~~ (141) E. Berner and 0. Kjoelberg, Ackr Chem. Scad., 14, 909 (1960). ( 1 4 1 ~ )However, we T. L. Hullar and F. Smith [J. 078. Chem., 31, 1657 (1966)] for rulwquent horohydride reduction, and cmvomion of tha rcsult,ing alcohols into the triH-p-nitrot)enzoateR. (142) A. S. Perlin and 8.Suriiki, Can. J . C h m . , 40, 1226 (1962). (143) R. C. Hookett, M. H. Nickernon, and W. H. Reeder, 111, J . Am. Che7-n. Soc., 66, 471 (1!)44). (144) H. Q. Fletcher, Jr., H. W. Diehl, and R. K. Nem, J . Am. Chem. Soc., 76, 3029

(1964).

JOHN W. QREEW

134

In contrast, ethyl 8-D-galactofuranoside (43) is oxidized at a lower but steadier rate1'*; more than two moles of oxidant are consumed per mole, and one mole of formaldehyde is formed. The 5,6-glycol is attacked first, to give (44), and then the truns-2,3-glycol is attacked, to afford the trialdehyde (45). Compound (44) cannot give a hemiacetal. The consumption

HOH& (49)

(44)

(45)

of more than two moles of oxidant per mole is attributed to the oxidation of the malonaldehyde grouping in (45) ; such compounds have been shown to be unstable to oxidants for glycols. Although periodate is less specific in its attack, use of only one mole of the oxidant per mole of an aldohexofuranoside having a 2,3-truns-glycol grouping gives one mole of formaldehyde, with cleavage of the C-6 to C-6 bond."' Subsequent reduction of the oxidation product with sodium borohydride gives an Jdopentofuranoside aa a product, and this procedure has been utilized to co-relate aldohexofuranosides with aldopentofuranosides.14* Methyl a- and &wgalactofuranoside were converted into the corresponding L-arabinofuranosides, and the methyl a-and &~-glucofuranosides into the Pxylofuranosides. Identity of products was shown by optical rotation, hydrolysis to the free pentose, or further oxidation of the pontofuranoside and isolation of glycolaldehyde as the (2,ainitrophenyl)hydraaone benzoate. Similar, controlled oxidation and subsequent reduction converted phenyl &.D-glucofuranosideinto the 8-D-xylofuranoside, and phenyl@-wgalactofuranosideinto the a-L-arabinofuranoside. ** Mitra. and Per1inlMemphasized the rapid oxidation of furanosides by lead tetraacetate. In 150 minutes, 0.88 mole of this oxidant w a consumed ~ by one mole of methyl 8-o-fructofuranoside, whereaa only 0.21 mole of (145) 0. Kjoelberg, Acfa C h .Soand., 14, 1118 (1960). (146) A. K.Mitra and A. 9. Perlin, Can. J . C h . ,87, 2047 (1959).

135

THE GLYCOFURAXOSIDES

perirnlatc was consumed. A reversal of the relative speeds for the two oxidants was uhown with methyl a-wglucopyranoside, and a corresponding selective oxidation was shown for the two sugar residues in sucrose. 4. Ring Stability

The stability of the furanoid ring may be altered in three ways. First, the polar nature of the groups at C-2 and C-3 can be altered. Secondly, another five-membered ring can be introduced into the molecule. Finally, the oxygen atom in the ring or attached to the aglycon group can be replaced by a different atom. Any or all of these transformations may raise or lower the stability of the aldofuranoside. The C-2 hydroxyl group has a very strong stabilizing effect' on the group at C-1. Replacement of this group by a more polar group increases this effect ; for example, methyl 2-O-p-tolylsulfonyl-cr-~-ribofuranoside~~~ is very stable, being unchanged by 0.33 N sulfuric acid during eight hours at 70". Conversely replacement of this hy droxyl group by a hydrogen atom results in less stable derivatives; for example, methyl 2-deoxy-~-erythropentofuranoside is completely hydrolyzedm by 0.005 N hydrochloric acid during three minutes at 100". The rate of hydrolysis of methyl 2-deoxya,/?-warabino-hexofuranosidedOin 0.005 N acid at 15" (see Table IX, p. 130) was found to be fifteen times that for methyl a+arabinofuranosiden under much more drastic conditions. Replacing the hydroxyl groups at both C-2 and C-3 by hydrogen atoms creates an even greater instability; Stacey and coworkers104were unable to prepare ethyl 2 ,3-dideoxy-werythrohexofuranoside, because of its great lability. Replacement of the C-3 hydroxyl group by a hydrogen atom, with retention of the stabilizing hydroxyl group at C-2, removes nonbonded interactions. 3-Deoxy-~-lyxo-hexose forms only furanosides with acidic methanol, at room temperature, under reflux, or1(*at 100". The removal of interactions, at C-2, C-3, and a t C-3, C-4, is far more effective than the loss of the C-3 hydroxyl group as a stabilising influence on the relatively distant group a t C-1. For the Z-deoxy derivatives, the loss of the hydroxyl group affects the stability to a greater degree than the removal of nonbonded interactions. The introduction of a second, five-membered ring haa already been discussed in reference to the ready formation of furanosides from Dgluaurono-(i,3-1a(!to1ic.~~~ The ratc (;onstant,given in Table VIII for methyl 2 , R-tli-O-iucl,liyl-cu-D-glucofuraiioxidurorio-O, 3-lactone is much smaller than thst for cthyl C3-D-Rlucofurrttiouidc. The effect is also shown in the ready (147) I). M. Hrowii, (1. I). Fenner, I). I. McCrath, and A. R. Todd, J . Chem. Soc., 1448 (1954).

(148) (149)

P. Weygand and H. Wols, Chem. Ber., 86, 466 (1952). L. N. Owen, S. Peat, and W. J. G . Jones, J . Chem. Soc.,

339 (1941).

130

JOHN W. (IItMEN

cowereion of tho mathyl pyranosidoe of 3, &anhydro-~-gluco~c~~ and of 3, B-anhydro-~-mannosc'" into tho more stable furanosides, by acidic in 0.1 methanol. Methyl 3, S-anhydro-2-deo~y-warabino-hexopyranoside~~~ N sulfuric acid givos a 70% yield of the furanoside; this is a marked incrciisc in stability over that of the 2-deoxy-warabino-hoxofuraiimide. Nicholas and coworkers16* converted methyl 3,0 :3', cj'-dianhydro-flcellobioside in acidic methanol into methyl 3, &anhydro-a-D-glucofuranoside; some fl-D anomer wa8 also obtained. This stabilizing effect of another ring has also been shown in thermal reactions. Bishop, Cooper, and Murray16*injected methyl 3,6-anhydro-a-~mannopyranoside ihto a gas chromatograph, operatigg at a column temperature of 225', and found 75% conversion into the furanoside. In some instances, another ring may introduce an * instability factor. Methyl and ethyl 2,5-anhydro-tarabinofuranosideshave been prepared,lM but are very unstable: they decompose in aqueous solution. The steric requirements of 3,Banhydro-=galactose are even more adverse : treatment166 of a"dialky1 dithiqacetal of this sugar under conditions designed to give furanosides (with methanol, mercuric chloride, and mercuric oxide) gave only the methyl pyranoside. Stabilising rings may also be created by forming 0-isopropylidene acetals. Condensation of methyl wribopyranoside with acetone wa8 shown by Levene and Stiller'w to give equal amounts of the 2,3-O-isopropylidene furanoside and the 3,4-O-isopropylidene pyranoside ; Barker and Spoors,167 investigating this reaction further, concluded that the isomerization occurs after the condensation with acetone. Several 4- and Sthioaldoses have now been prepared. Both 4-thio-~and +ribose form Cthiofuranosides in hot acidic methano1.168 In contrast, bthio-carabinose and 5thio-L-idose form 5-thiopyranoside~.~~~-~~~ In thew (150) W. N. Haworth, L. N. Owen, and F. S,mith, J . C h . doc., 88 (1941). (151) A, B. Foster, W. G. Overend, M. Stacey, and G. Vaughan, J . C h . sbc., 3367 ( 1964). (152) F. H. Newth, S. D. Nicholas, F. Smith, and L. F. Wiggins, J . Chem. Soc., 2550 (1949). (153) C. T. Bishop, F. P. Cooper, and R. K. Murray, Can. J . Chsm., 41, 2246 (1963). (154) M. Cifonelli, J. A. Cifonelli, R. Montgomery, and F. Smith, J . Am. Chem.,Soc., 77, 121 (1966). (165) H. Zinner, K.-H. Btark, E. Michalzik, and H. Kristen, Chsm. Ber., 06, 1391 (1962). (186) P. A. Ihvene and E. T. Stiller, J . Biol. Chsm., 100,421 (1934). (157) G.It. Barker and J. W. Sporrs, J . Chsm. Soc., 1192 (1956). (168) E. J. Reist, 1).E. Gueffroy, and L. Goodman,J . Am. C h .doc.,86,6658 (1964);

R. L. Whistler, W. E. Diak, T. R. Ingle, R. M. Rowell, and B. Urbas, J . Org. Chem., 20, 3723 (1964). (150) R.1,. WhiRtler, M. S.Feather, and D. L. Itigles, J . Am. Chem.Sor., 84, 122 (1962); It. L. Wliilitler and R. M. Rowell, J . Org. Chem., 20, 1250 (1964). (180) L. Goodman and J. E. Christensen, J . fhg. Chem., 20, 1787 (1964).

137

THE OLYCOFUHANOSIDES

three examples, the ring contains the hetero atom. In the formation of the ring of a glycoside, there is a nucleophilic attack by the C 1 or C-5 hydroxyl group on the protonated C-1; the nucleophilicit,y of these two groups is, apparently, very similar. The -8H group is a stronger nucleophile than the -OH group,1a1and so, nucleophilic attack by this group predominates, so that the sulfur atom becomes included in the ring. The lower rates of hydrolysis (see Table IX) found for l-thio-wglucofuranosidea are attributable to the lower basicity of the sulfur atom,161and thus, less protonation of the 1-thio-wglucoside occurs in acid solution, in comparison to the wglucofuranosides.

VII. GENERAL 1. Action of Alkali

Jansen and LindberglB2have determined the effect on various aldosides of 10% sodium hydroxide at 170'; the rate constants are given in Table XI. The aldofuranosides are much less stable in alkali than are the corresponding aldopyranosides, and the trans-1 ,Zanomers are less resistant than the cis anomers. The lower stability of the trans-glycosides has been attributed to the formation of a 1,2-anhydrideJ with elimination of the methoxyl group. TABLBI XI Alkaline Hydrolysis' of Methyl Aldonides'" lWk (sec.-I)

Methyl glycoside

Furanoside

Pyranoside

8.9

2.8 0.78 0.33

a-L-Arabino j3 anomer a-D-xylO j3 anomer u-D-GahCtO j3 anomer o-D-Gluco j3 anomer a-D-manno j3 anomer 0

-

2.2 above 28 2.2 7.8

-

above 28 8.3

-

1.60

0.28 1.6

0.28 0.70

0.78 03 0

With 10% sodium hydroxide at 170".

The high rate-constants found for the D-glucofyanosides and ~-xylofuranosides were unexpected. No correlation is observable of the relative stability of the various aldofuranosides with the order shown in acidic (161) D. Horton and D. H. Hutson, Ref. 6, see especially p. 124. (182) J. Jansen and B. Lindberg, Acfa Chem. Seam#., 14, 2061 (1960).

138

JOHN W. OHPEN

methanolysis or acidic hydrolysis. There ie, however, a correlation of the greater alkali stability of the cis arromers arid the high yields of cis anomers of methylated aldofuranmides formed in the Kuhir mcthylatioii reaction."* Also, a sorrelation can be made with the absorption on btuia, ioii-exchunge rosins; Bnddiley aiid coworkcrd4 foutrd that, ftiriwolcidcs, uxpwitilly tlw j3 anoniers, are more slowly eluted t,han tho pyrunoddee from such resins. The aryl aldofuranosides show a greater lability to alkali than the alkyl aldofuranosides. Phenyl 0-~xylofuranosideis very labile to alkali.82Treatwith dilute alkali ment of Znaphthyl ~~-glucofuranosidurono-6,3-lactone opens the lactone ring, and rapid hydrolysis occurs.BoIshidate and Matsuial is most noted that pnitrophenyl ~-~glucofuranosidurono-6,3-lactone sensitive to alkali, the amide next, and then the pyranoid derivatives; toward acids, the relative sensitivities of the lactone and the amide are reversed. 2. Formation of Furanose Polymers

Many of the methyl glycofuranosidea form polymers when distilled under reduced pressure. Bott, Hirst, and Smithleanoted such polymerizathey tion during the distillation of methyl tri-0-methyl-D-lyxofuranoside; obtained a high-boiling fraction, which changed to a solid melting at 77O, believed to be a dipentose polymethyl ether. This behavior has also been 2-deoxynoted with the fnethyl furanosides of Zdeoxy-~-arabino-hexose,~6~ D-lyxo-hexose,le6 and 2-deoxy-D-erythro-pentose.l~Il~The hard, glassy products obtained are believed to consist of 5 to 7 residues] linked through C-1 and the primary hydroxyl groups. In heating the polymer of 2-deoxy-~erythro-pentose with acidic methanol, the methyl furanoside wm re-formed. The benzyl furanosides behave similarly to the methyl furanosides. Peat and coworkerslg investigated the reversion products formed by the action of acid on &glucose and found, by periodate oxidation] that tho mixed anhydrides contain 18 to 47% of material having a furanoid structure. Some 1,6-anhydro-&~-glucofuranose was isolated. Other sugars apparently give only pyranoside reversion products. Dutton and Unrau168studied synthetic polymers obtained by the technique of heating the sugar with phosphorous acid.l" These products (163) H. G . Bott, E. L. Hirst, and J. A. B. Smith, J . Cham. Soc., 668 (1930). (164) I. W. Hughes, W. 0.Overend, and M. Stacey, J . Chem. Soc., 2846 (1949). (165) M. Stacey and W. G. Overend, British Pat. 884,689 (1953); Chem. Absfracfs, 47, 6166 (1963). (166) W.G. Overend, F. Shefiaadeh, and M. Stacey, J. Chem. Soc., 994 (1951). (167) S. Pest, W. J. Whelan, T. E. Edwards, and 0. Owen, J . Chem. Soc., 586 (1968). (168) G. G. S. Dutton and A. M. Unrau, Can. J . Chen., 40, 2105 (1962); 41, 2439 (1963); 481 924 (1966). (169) P. T. More, J. W. Wood,P. Msury, and B. G. Young, J. Am. Chsm. Soc., 80, 693 (1968).

THE OLYCOFUIlANOSIDES

139

were all highly hranc:hwl. Mothylation whowed that, in a xylan, 27% of the ititernal rc~idiiw,uric1 :$tif& of thc tn!rrninal rcsitlues, arc furtinoid. For a gluean, 201% of the tortnintti residucx arid wme of the internal re&lueu were in the furanose form. An arabinan also contained a large proportion of furanoid residues. 3. Natural Occurrence of Furanose Residues

Four sugars quite frequently occur as furanose residues in Nature. These sugars are wfructose, carabinose, D-ribose, and Zdeoxy-~-erythro-pentose D-Galactose, L-fucose, and Dpsicose have also besn found naturally in the furanose form. The /3-D-fructofuranose residue is present in sucrose17o;it also occurs in the levans and in inulin, as (2 + 1) and (2 + 6)-linked p01ymers.l~'The a-Larabinofuranose residue occurs in arabinan, generally combined with Dgalactopyranose residues17*:the backbone of such a polymer is (1 5)linked, with (1 + 3)-linked side-chains. This sugar residue also occurs as a side chain in hemicelluloses, often linked to D-arabinopyranose residues.178 The p-D-ribofuranosyl and 2-deoxy-~-~-e~i!h~o-pentofuranosy~ groups have already been discussed as the sugar residues in the n u c l e o ~ i d e s . ~Oc~-~~ casionally, an a-wribofuranosyl residue is found, as in a-riba~ole.~~' A galactan of short chain-length, containing &wgalactofuranose residues, has been r e ~ 0 r t e d . lL-Fucofuranose ~~ has been found as a residue in a polysa~charidp.'~~ An antibiotic substance, 6-amino-9-D-psicofuranosylpurine has also been in~estigated.'~~ The isolation of oligosaccharides after graded, acid hydrolysis of these polysaccharides is often difficult. Aspinall and Nicolson17B oxidized the primary hydroxyl group of the arabinofuranose residue in a European larch galactan, and obtained a (6-D-galactose L-arabinofuranosid)uronic acid. This strengthening of the furanoside linkage was also applied to an arabin~xylan.'~~ (170) I. Levi and C. B. Purves, Advan. Carbohydrak Chem., 4, 1 (1949). (171)E.J. McDonald, hoban. Carbohydrale Chem., 2, 253 (1946). (172)A. E. Goodban and H. S. Owena, J . Polymer Sci., 2S, 825 (1957). (173) G. 0.b i n a l l , Aduan. Carbohydrate Chem., 14,429 (1959). (174) N.G.Brink, F. W. Holly, C. H. Shunk, E. W. Peel,J. J. Cahill, and K. Folkere, J . Am. Chem. SOC.,72, 1866 (1960). (175)P: W. Clutterbuck, W. N. Haworth, H. Raietrick, G. Smith, and M. Stacey, Biochem. J., 28, 94 (1934);W.N. Haworth, H. Raiatrick, and M. Stacey, ibid., 31,640 (1937);P.A. J. Oorin and J. F. T. Spencer, Can. J. Chem., 31,499 (1959). (176)G. 0.Aepinall, R. S. P. Jamieson, and J. F.Wilkinson, J. Chem. SOC.,3483 (1956). (177)W. Schroeder and H. Hoeksema, J . Am. Chem. SOC.,81, 1767 (1958). (178)G. 0.Aspinall and A. Nicolson, J. Chem. ~ o c . ,2SO3 (1960). (179) G. 0.Aspinall and I. M. Cairncroaa, J . Chem.Soc., 3877, 3998 (1960).

140

JOHN W. GREEN

The above sugar residues have furanoid rings of high conformational stability, and their natural occurrence may be attributed to this factor. The various polymers also possess free primary hydroxyl groups, which may confer tz certain amount of water solubility upon them. These primary hykboxyl groups’would not be available in an aldoyentopyrauoid structure.

VIII. TABLES Tables XI1 to XV list the melting point and specific optical rotation of some methyl glycofuranosides, phenyl aldofuranosides, alkyl glycofur. anosides, and ethyl 1-thioaldofuranosidea. Tasm XI1 Melting Point and Specific Optical Rotation of Methyl Glyoofuranosides Methyl glycofuranoclide of

a-D-EqthroSe fl anomer a-D-Threw fl anomer a-tArabinose anomer a-D- Arebinom 4 anomer U-D-LYXOS~

M. p., ‘C. Simp sirup sirup Simp 520

58 66-7. 66-7 93-4 SiNP

4 anomer

79-80 84 46 91-2 69 63 sirup 100-1 118-19 46-7 80.6-1

[ a ] ~ ,degrees (water)

+133 148 +97 193 -128 +118 +123 -119 1366 +131 +146.90 +146

-

-

+

-50

-62.40

+182

-89.5 +lo4 -112 +ll8 1366 -77 108

-

+113

-112.6 +93.05 +91.64

@ anomer 0

-50.4

Hygroscopic. * Benzene aa solvent. * Methanol 88 solvent.

(180) J. 8.D. Becon and D.J. Bell, J . C h . Soc., 81181 (1957).

References

40 40 40 40 31 31 27 31 51 87 33 15 48,41 33 31 31 29 29 23 23 23 144 24 54 26 26 180

-

141


TABLE XI11 Melting h i n t and S p i f i c Optical Rutotion of Some Yhenyl Aldofuranogides Phenyl furanoside of

&L-Arabinose &n-Ribose &D-XyloSe 8-D-Galactose ~~-D-GIucoM fl-~-G~ucofuranurono-6,3lactone, diacetate 0

M. p., OC. 63-6 106-7 114-16 82-3 79-80 188-9

[ a ] ~ degreea , (water)

- 159 -99 - 128 - 148

-142 +74.50

References

82 85 82 83 83 80

In chloroform.

TABLE XIV Melting Point and Specific Optical Rotation of Some Miscellaneous Alkyl Glycofuranosides Furanoside

Ethyl a-tarabinoBenzyl BD-riboEthyl a-D-galactofl anomer Propyl fl-wgalactoBenzyl &n-gslactoEthyl CU-D-~~UCOB anomer Ethyl a-n-mannoPropyl Cr-D-rnannoIsopropyl a-n-mannoMethyl a-n-gluco~idurono-6,3lactone fl anomer Benryl WD-fructoMethyl a-L-rhamnoEthyl a-L-rhamnofl anomer Methyl a-L-fUcofl anomer Methyl bdeoxy-u-D-xyloMethyl 2,3-anhydro-a-n-lyxofl anomer

M. p.,

OC.

48-9 95-6 134-40 85-6 89-90 80-1 82-3 61-3 90 98 82-4 148-9 138-9 89 62 56.5-7.5 21-4 127-8 sirup 83-5 80-2 74-5

[ a ] ~ degrees , (water)

References

-116 -60.5 +92 - 102

50 114 53 52 50 50 2 22 54 54 54 35

-1w -96

+lo6 -76 105 +96 +96.7 149

+

+

-57 45.7 -98.6 -98 105 -108 +113 149.I4 67 - 102

+

149 113 48 56 56 47 34 181 98 98

In chloroform.

(181) K.J. Ryan, H. Araoumanian, E. M. Acton, and L. Goodman, J . Am. Chem. Soc., 88,2497(1964).

JOHN W. Q R B W

142

TABLE XV Melting Point and Specltia Optical Rotation of Some Ethyl 1-Thioaldofuranwider Ethyl 1-thiofuranoeideof

M. p., "C.

[ah,degreca

ReCeroncer

(water) ~

a-~-Ar~bin~ee

fl anomer a-D-Ribose a-D-Galactose a-~-Gluc~ fl anomer a-D-Glucofursnuronat, sodium salt ZAcetamida-Zdeoxy-&Irarabinme ZAcetamido-2deoxy-a-~-xylow ZAcetamido-Zdeoxy-cu-D-gahctow fl anomer 2.Acet.amido-2-deoxy-a-o-glucose fl anomer, triacetste

* In methanol. b In chloroform.

02-3 49-60 71-2 sirup

153 8iNP

210-2 127-9 157-8 81-3 108-10 1 19-20 174.80

+24@ -la@ +176 +124 +121 -104 +110

05 65 06 69 64 64 67

+172 +222

72 73 71 71 08 68

+1M)

- 1350 +170 -4!2b

DEOXY SUGARS BY STEPHEN HANESSIAN Research Labomtwiee, Parke, Davis & Company, Ann Arbor, Michigan I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 11. Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 111. Monodeoxy Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 1. ZDeoxy Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 2. 3-Deoxy Sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 3. 4DeoxySugars.. ................................................. 166 . . . . . . . . . . . . . . . . 167 4. 5-Deoxy Sugars.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Terminaldeoxy Sugars.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 IV. Dideoxy Sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 1. 2,BDideoxyhexoses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 2. 3,6Dideoxyhexoses. ..................... ...................... 187 ...................... 190 3. 4,6Dideoxyhexoses. ..................... 4. 5,6Dideoxyhexoses... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 5. Other Dideoxy ....... - Sugars. V. Trideoxyhexoses.... VI. Chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 1. Paper Chromatography... . . 2. Thin-layer Chro 3. Ionophoresie.. . . . . . . . . . . . . . . . . . . . . . . . . . 200 4. Gas-Liquid Chromatography... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 . . . . . . . . . . . . 201 VII. Nuclear Magnetic Resonance Spectroscopy VIII. Msss Spectrometry.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

I. INTRODUCTION The deoxy sugars, long known as components of natural products, are an important class of carbohydrates. Unlike other classes, many deoxy sugars confer unique biological properties on the natural substances of which they are a part. The commonly recognized members that have received attention from the biological standpoint have been the terminal-deoxy hexoses and the dideoxy-hexoses occurring as components of cardiac glycosides and as antigenic determinants in bacterial polysaccharides. Several unusual deoxy sugars have been isolated from other natural products in recent years. The advent of modern physical methods and the adaptation of new synthetic reactions to carbohydrates have greatly facilitated the elucidation of the structure of these uncommon deoxy sugars. Reviews on this subject have 143

144

STEPHEN HANEBBIAN

been very selective arid few in number. The deoxy sugars of the cardiac glycosides were reviewed by Elderfield’ in the first Volume of this Series and, more recently, by Reichstein and W e k a Other reviews on these compouiidtl have been published elsewhere.“’ The chemistry of the 2-dcoxy sugarv was discussed by Overend and Stticoys in Voluiiic 8 of this Scriw, and that of 2-deoxy-~-erylhro-petitotle was reviewed by the same uuthors’ in 1955. The chemistry and biology of the 3 ,O-dideoxyhexoses have been outlined in two article^.^^^ In recent years, significant advances have been made in our knowledge of the biosynthesis and metabolism of deoxy sugars.lo The objective of the present article is to discuss the natural occyrreme, synthesis, and chemical and physical properties of the deoxy sugars, and to outline chromatographic and physicochemical methods for their separation, characterization, and identification. Although the term deoxy sugar is often used loosely, the present review will be restricted to the openchain deoxyaldoses and deoxyketoses. Biochemical aspects of deoxy sugars, such as their biosynthesis, metabolism, and other biological transformations which might involve them, will not be discussed. Despite these obvious but unavoidable shortcomings, it is hoped that, with the emphasis on the chemistry of the deoxy sugars, the article will nevertheless be useful to the reader.

11. NOMENCLATURE The nomenclature adopted in this review will conform with the rules of carbohydrate nomenclature established cooperatively by the British and American committees.108The deoxy sugars have been subjected to a great deal bf misleading naming; the main difficulties have been occasioned by the unwarranted practice of assigning a name on the basis of source m a terial or preparation rather than on the structure of the sugar itself. The complexity of the nomenclature in this class of sugars has been discussed previously.@Although the present rules provide a simple, consistent system R. C. Elderfield, Advan. Carbohydrate Chem., 1, 147 (1945). T. hiohstein and E. Weiss, Advan. Carbohydrate C h . ,17,65 (1962). H. Heuaser, Forlschr. Chem. Org. Nalursbfe, 7,87 (1950) T. Reichstein, Angew. Chem., 08, 412 (1951). C. Tamm, Forbchr. Chem. Org. Naturstofle, 18, 137 (1956). (a) W. G. Overend and M. StSfiRY, Advan. Carbohydrate Cheni., 8 , 45 (1053). (7) W. G. Overend and M. Stscey, in “The Nucleic Acids,” E. Chargaff and J. N . Davidson, eds., Academic Press Ino., New York, N.Y., Vol. 1, 1955, p. 9. (8) 0. Weetphd and 0.Lfideritz, Angew. Cham.,72,881 (1960). (9) 0. Ltideritz, Bull. SOC.Chim. Biol., 41, 1355 (1960). (10) L.Claser, Phyeiol. Rev., 43,215 (1963). (108) J . Org. Cham., 28,281 (1963). (1) (2) (3) (4) (5)

PEOXY GUGAHS

145

for naming deoxy sugars, the efforts made to unify the nonienrlature systein of this class are impaired by those scientists who, presumably for reasons of supposed simplicity, continue to use unsatisfactory names. The term deoxy indicates the replacement of a hydroxyl group by a hydrogen atom. In naming a deoxy sugar, the prefix “deoxy” appears before the name of the sugar, preceded by the appropriate numeral indicating its position. The configuration of the sugar is denoted by the group prefix (such as eryulro and ribo) indicating the number of asymmetric centers, which should be consecutive but need not be contiguous. The following are some examples of correct nomenclature; incorrect names are given in parentheses: methyl 2-deoxy-a-~-arabino-htxopyranoside(methyl 2-deoxy-cu-~-glucoside); 3deoxy-~-ribo-hexose (3deoxy-~-glucose); 4deoxy-D-xyb-hexose (4deoxy-~-glucose); methyl 2,3-dideoxy-ar-~-erythrohexopyranoside (methyl 2,3-dideoxy-a-~-glucoside). The accepted trivial names rhamnose and fucose will be used in this Chapter. 111. MONODEOXY SUGARS

1. 2-Deoxy Sugars a. General Considerations.-Since this subject has been dealt with extensively in previous reviewsJ2-’ emphasis will be laid on the newer developments in the synthesis and characterization of Zdeoxy sugars. A Zdeoxy-3-O-methylpentose (corchsularose) was isolated, as the crystalline phenylhydrazone, from corchsularinJ1la bitter principle from the alcoholic extract of jute seeds. According to this investigation, the structure of the pentose derivative was adequately corroborated by the isolation of methoxysuccinic acid after permanganate oxidation. It seems remarkable, however, that a 2-deoxypentose derivative should withstand the conditions of hydrolysis (concentrated hydrochloric acid) which were employed to liberate it. Among the capbohydrate analogs which have been tested as glycolytic inhibitors, only 2-deoxy-~-ribo-hexose and 2-deoxy-~-xylo-hexose have shown significant inhibition in the glycolysis of human leucocytes, human leukemic cells, and a number of animal tumors.1aThis class of deoxy sugars and their derivatives should, therefore, be selected candidates as potential antimetabolites for cancer chemotherapy. Two new nucleosides, 9- (ZdeoxyD*rabino-hexopgranosyl) adenine and its 2,3-unsaturated analog, were found1*to be highly effective against AK leukemia 120. (11) M. A. Khalique and M. D. Ahmed, J . Org. Chem., lS, 1523 (1954). (12) J. Lmzlo, B. Landau, K. Wright, and D. Burk, J . Natt. Cancer Inst., 21,475 (1958). (13) J. J. K. Novak and F. germ, Colleclwn Czech. Chem. Commun., 27,904 (1962).

146

STEPHEN HANESSIAN

Considerable progress has been made in the past few years in obtaining crystalline esters, such aa the benzoates,+l6 acetates,17 and pnitrobenzoates,*30 of Zdeoxy sugars (and, in particular, of 2-deoxy-~-erythropentose) , The availability of these important intermediates as pure, crystalline substalices potentiates their utilization as precursors of the corresponding glycosyl halides and, thence, of various glycosides. The utility of crystalline p-chlorobenzoyl and ptoluoyl esters of Zdeoxy-D-erythro-pentofuranoeyl chloride for ready coupling with mercuripyrimidines was shown by Fox and coworkers.?1I n connection with the etudy of acid-catalyzed anomerizations, BonneP obtained pure 2-deoxy-a-~-ara~nohexopyranose tetraacetate and the corresponding B-D anomer by fractional recrystallization. Acetylation with hot acetic anhydride and sodium acetate led to a product containing 65% of the CY-Danomer. Although 2deoxy-~-erz~thropentose was recognized by Levene as being a component of one kind of nucleic acid over 35 years ago: only in the past few years has the role of this sugar and its phosphate esters been the target of many investigators. The phosphate esters, which are important intermediates in biological transformations, have been synthesized by strictly chemical means. Prepared by well established phosphorylation methods, the 5-phosphates have been reported from several laboratories.u-s Comparison of the physical properties of the momeric 2-deoxy-~-erythro-pentosylphosphates" with those of the natural ester showed that the natural anomer has the (Y-D configuration. Although the characterization of bugars and their derivatives as (substituted) hydrazones requires relatively mild conditions, an unexpected reaction was observedn when 3,5-di-O-benzoyl-2-deoxy-~-eryerythro-pentose (1) (14) H. Zinner end H. Nims, C h n . Ber., 01,1657 (1958). (16) H.Zinner, H.Nims, and E. Wittenburg, Clum. Bet-., 98,340 (1960). (16) C. Pedersen, H. W. Diehl, and H. G . Fletcher, Jr., J . Am. C h . SOC.,82, 3425 (1960). (17) H. Venner and H. Zinner, Chem. Ber., 98, 137 (1960). (18) W. W. Zorbach and T. A. Payne, J . Am. C b m . Soc., 80,5564 (1958). (19) R. K. New and H. G . Fletcher, Jr., J . Am. Clum. Sm.,82,3434 (1960). (20) W. W. Zorbach and G . Pietsch, Ann., 666,28 (1962); W. W. Zorbach and W. Birchler, Ann., 890, 118 (1963); K. V. Bhat and W. W. Zorbach, Carbohydrate Ree., 1, 93 (1986). (21) M. Hoffer, R. Dumhinsky, J. J. Fnx, and N. Yung, J . Am. C h n . Soc., 81,4112 (1959). (22) W. A. Bnnnw, J . Org. Chem., 26, (308(1961). (28) R. Allerton, W. G . Overend, and M. Stacey, Chem. I d . (London), 963 (1962). (24) T. Ukita and K. Nagasawe, Chem. Pharm. Bull. (Tokyo), 7,655 (1959). (25) D. L. MacDonald and H. G . Fletcher, Jr., J . Am. Chem. SOC.,81,3719 (1959). (26) D. L. hfacDonlrld and H. Q. Fletcher, Jr., J . Am. Chem. SOC.,84,1212 (1962). (27) M. G. Blair, D. Lipkin, J. C. Sowden, and D. R. Strobach, J . Org. Chem., 26, 1079 (1960).

DEOXY SUGARS

147

ww treated with l-benzyl-l-phenylhydrazine.An unsaturated hydrazone (2) waa formed by an a,~eliminationof the benzoyloxy group from G 3 ,CaK

OH

HC=N-N I HC ‘CH*CaH, HL! I HCOH

Bz0H9cP I

Bz

CH,OIjz

(1)

(2)

and a hydrogen atom from C-2. The product was reduced to the corresponding 2,Q-dideoxy derivative, debenzoylated, and finally degraded to succinic acid. Another example of based-catalyzed elimination in the 2-deoxy sugar series is that of 1-[2-deoxy-3-0-( methylsulfonyl)-5-O-trityl-p-~-erythropentofuranosyl]uracil and 2’ ,3-anhydro-l- (2deoxy-5-O-trityl-p-~-threopentofuranosyl) uracil, which, when treated with potassium tertbutoxide in methyl sulfoxide, give a 70% yield of a 2,3-unsaturated nucleoside.a8 A stepwise, chemical degradation reported20 for the 2-deoxy sugars permits each of the carbon atoms to be isolated, eventually, as barium carbonate, and their radioactivity, if any, to be determined. Yields as high as 90% (or even higher) of the available carbon may be obtained. In this method, the aldose is converted into the alditol, which is oxidized with periodate, to give three readily differentiatable products (namely, formaldehyde, formic acid, and 3-hydroxypropionaldehyde) which are, in turn, further degraded.

b. Syntheeis.-Several novel approaches to the synthesis of 2-deoxy sugars have been reported. However, some of the standard methods (such as the glycal method, the reductive cleavage of epoxides, and the FischerSowden method) are still in use. These general methods have been reviewed in detail.‘*sOThe glycals and their reactions have been reviewed by Helferich.81 Several of the methods included herein are described in detail and in revised form in the Methods in Carbohydrate Chemistry series.82 (28)

J. P.Horwitz, J. Chua,I. L.Klundt, M. A. DaRooge, and M. Noel, J . Am. Chem.

Soc., 88, 1896 (1964). (29)A. M.Unrau and D.T.Canvin, Can. J . Chem., 41,607 (1963). (30) J. C.Sowden, Aduun. Curbohydrute Chem., 6,291 (1951); see also, W. W. Zorbach and A. P. Ollapally, J . Org. Chem., 29, 1790 (1964). (31) €3. Helferich, Aduun. Curbohydrute Chem., 7, 209 (1952). (32) Methods Carbohydrate Chem., Vols. 1and 2 (1963).

148

STEPHEN HANEBBIAN

(i)The Glycal Method.-This method, which is considered to be the most direct route to Zdeoxy sugars, was first reported by Emil Fischer.aa It involves the conversion of an acetylated glycosyl halide into an acetylated glycal by the action of zinc dust in acetic aaid. The acctylated deoxy sugar is then obtained by mild treatment of the glycal with aqueous acid at low temperatures. This method has been the subject of extensive iuvestigation, particularly as regards improving yields and minimizing sideproducts. Iselin and Reichsteinw have obtained improved yields of the acetylated glycals by omitting the isolation of the acetylated aldosyl bromides. Although universal, the glycal method suffers from the disadvantage of affording low yields in certain cases and failing in others. An extreme caae was the failure of the glycal method in the synthesis of 2-deoxy-3-O-methyl-~-ribo-hexose.~ Prinssd attributed the variable yields to steric effects associated with the substituenta and with the configuration of the original aldose. According to the mechanism proposed" for the formation of glycals, two possible by-products could be the anhydroalditol (3) and the aldose peracetate (4), as shown in the Rcheme below:

AcOQ@

AAcoqc@ AcOQOR

0 0

AcO

R

-

(4)

OH, OAc

Ac

bAc

A detailed study of the mechanism of glycal formation and subsequent transformations in the light of modern concepts of nonclassical carbonium iona might prove uReful. Many years ago, use was made of glycals for the (33) (34) (35) (30) (37)

E. Fischer, Ber., 47, 196 (1914). B. Iselin and T. Reichstein, Helu. Chim. .Acb, 27, 1200 (1944). C. A. Grob and D. A. Prins, Helu. Chim. Acb, 28, 840 (1946). D.,A. Prins, Helv. Chim. Acla, 99, 1 (1946). D. A. Prim and R. W. Jeanlos, Ann. Rev. B i o c h . , 17,67 (1948).

DEOXY SUGARS

149

general synthesis of 2deoxy sugars.' 3,4,6-Tri-O-acetyl-~-g1ucal, on treatment with bromine or chlorine, afforded the respective 3,4,G-tri-O4cetyl2deoxy-2-halo-hexopynosyl halides, which were then converted into methyl 2-deoxy-~-arabino-hexopyranoside. An extension of this method was reported by Vargha and Kuszmann,* who treated di-0-acetyl-narabinal with chlorine in carbon tetrachloride, and obtained a mixture of 3,4-di-O-acetyl-2-chloro-2-deoxypentopyranosyl chlorides. This mixture was converted into a mixture of anomeric methyl glycosides which, on treatment with hydrogen and Raney nickel, afforded crystalline methyl 2-deoxy-a-D-erythro-pentopyranoside. Mild hydrolysis with aqueous beneoic acid the? gave a 70% yield of crystalline 2-deoxy-D-erythra-pentose (2530% from D-arabhose) . The methoxymercuration of glycals has been another potential synthesis investigated by two g r ~ u p s ~ it~ ~constitutes ~"; of 2-deoxy sugars. Thus, Schwarz and cow0rkem4~have obtained good from the 2-acetoxyyields of methyl 2deoxy-j3-~-arabino-hexopyranoside mercuri derivatives. A synthesis of 2-deoxy nucleosides involves the acidcatalyzed condensation of acetylated glycals with purines.'* This method is based OD.previous experiences with the condensation of 2,3-dihydropyran with certain purines."

(ii) The Fischer-Sowden Method.-One of the earliest synthetic applications of carbohydrate nitro-olefins was their successful conversion into 2-deoxy sugars. H.0. L. Fischer and Sowden4' showed that condensation of an aldose with nitromethane yields a mixture of epimeric C-nitro alcohols which can be acetylated and the acetates converted into an acetylated nitro-olefin by treatment with sodium bicarbonate in a nonpolar solvent such as benzene. The olefinic derivatives can be selectively hydrogenated to 1,2dideoxy-1-nitroalditolswhich, in the form of their aei salts, undergo the Nef reactions0to give the corresponding 2-deoxy sugars. Crystalline 2-deoxy-~-arabino-hexosewas obtained by this method, and purified through its benzylphenylhydrazone.44 Essentially similar results have been obtained by Stacey and coworkers,&who were able to isolate the crystalline sugar directly. The synthesis of 2-deoxy-D-erythro-pentose was also reported by this method.q Good yields are obtained at all stages (38) E. Fischer, M. Bergmann, and H. Schotte, Ber., 69,509 (1920). (39) L. Vargha and J. Kuszmann, Chem. Ber., 96,411 (1963). (40)G. R. IngliR, J . C. P. Rchwarz, and L. McLaren, J . Chem. Soc., 1014 (1962). (41).'1 Mnnolopoulos, M. Mdnick, and N. N. Lichtin, J . Am. Chem. SOC.,84, 2203 (1 9G2). (42) W. A. 13owles and R. K. Robins, J . Am. Chem. Soc., 86, 1253 (1964). (43) R. K. Rohina, E. F.Godefroi, E. C. Taylor, L. R. Lewis, and A. Jackson, J . Am. Che?tl. SOC.,88,2574 (1961).

L.Fischer and J. C. Sowdon, J . Am. Chem. Soc., 69, 1048(1947). (45) W.U. Overend, M. Stacey, and J. Stanek, J . C b m . Soc., 2841 (1949). (46) J. C: Sowden, J . Am. Chon.Soc., 71, 1897 (1949); 72,808 (1950). (44) H. 0

150

STEPHEN IIANESSIAN

in the synthesis of the 2-deoxypentose, and Sowdenaohas pointed out that,, for preparative purposes, isolation of intermediates may be omitted. Although thie practice would be satisfactory up to the acetylated nitro-olefin stage, particular care should be taken in the selective hydrogenation of the olefinic double bond, since this select ivity apparobtly 3optmdx markcdly on the solvent employed.a The Nef reaction," which gives rim to thr\ acid-labile 2deoxy sugars, requires a strongly acid medium, and should be conducted at low temperature. After considering the several methods that were available for the synthesis of 2-deoxy-~-e~ythro-pentose, Murray and Butler'? chose the FischerSowden method for the preparation of 2-deoxy-D-erythro-pentose-l-"C. The source of the D-erythrose was 4,6-0bensylidene-D-glucitol. In pilot experiments employing an excess of radioactive nitromethane without isolation of the intermediates, yields of 2-deoxy-n-erylhro-pentose-1-14C of about 30% (based on starting material) were obtained." (iii) From Anhydro Sugars.-The cleavage of 2 ,3-anhydroaldose derivatives with various reagents constitutes another method for the introduction of a deoxy function at C-2. An epoxide ring may open in two possible ways when attacked by a nucleophile. The stereoselectivity of ring opening, discussed later, depends on conformational factors. Selection of a suitable uucleophile capable of being reduced at a later stage of the synthesis would provide a convenient way of introducing a deoxy function into the molecule; this forms the basis of a method developed some twenty years ago by Reichstein and coworkers" for the preparation of 2deoxy sugars. Treatment of methyl 2 ,3-anhydro-4,6-0-benzylidenoa-D-allopyranoside in refluxing methanol with sodium thiomethoxide afforded, after chromatobenzylidene-2-S-methyl-2-thio-ar-~graphic purification, methyl 4,6-0altmside. Hydrogenation of the latter converted it into methyl 2-deoxya-D-ribo-hexopyranoside. The position of the deoxy function was proved'g by methylation of the product of ring opening, followed by reduction, hydrolysis, oxidation, and isolation of L-(-) -methoxysuccinic acid arising from C-1 to C-4 of the original hexose. The generality of the opening of the 2,&snhydro ring by thioalkoxide ions as a route to deoxy sugars is dependent on conformational factors3.*~w~61 The anhydro sugar derivatives comnionly used are fixed as fused, cis- or tranalinked, 4 ,&O-benzylidene acetals. The mode of epoxide ring opening follows the Fiirst-Plattner rule,*6 and results in the formation of the 2,3diaxial derivative. Of the four (47) (48) (49) (60) (61) (62)

D. H. Murray and G. C. Butler, Can J . Chem., 87, 1776 (1969). R. W.Jesnloz, D. A. Prins,and T. Reichstein, Ezperientia, 1, 336 (1946). R. W.Jeanloz, D. A. Prins, and T. Reichetein, Helu. Chim. Ada, 29, 371 (1946). W. G. Overend and G. Vaughan, Chent. Znd. (London), 996 (1966). F. H. Newth, Quart. Rev. (London), 18,30 (1969). A. mr8t and P.A. Plettner, Helu. Chim. Ada, 82, 276 (1949).

DEOXY SUGARS

151

methyl 2,3-auhydrd, ~ ~ ~ ~ y l i ~ e I i ~ psiblc, ~ h e ody s o ~ ~ ~ the Meoxy sugars possessing the ribo and xylo configurations*can be obtained by ring opening with a thioalkoxide. The derivatives available for synthmis are thus methyl 2 ,3-anhydro-4 ,6-O-ben~ylidene-ar-Plo~ide~~~~ and methyl 2,3-snhydro-4,6-0-benxylidene-a-~-guloside.~ The stereochemical predictions regarding the ring opening of sugar epoxides cannot be made with certainty for molecules which lack a stabilizing, fused ring. It had been maintained that the generalized rules of epoxide opening were not applicable to 2 ,3-anhydropentosides, and that attempts to convert these into 2-deoxypentosides afforded, instead, the oorregDoqding 3-deoxypentosides. However, Casini and Goodmans6 have now described the first example of a predominant attack by a nucleophile at C-2 3f an anhydropentoside. Treatment of methyl 2,3-anhydro-b-~lyxofuranoside with sodium a-toluenethioxide afforded a mixture of the correeponding 2- and 3-benxylthio derivatives, which could be separated as their p-nitrobenzoates. Reductive desulfurixation and hydrolysis afforded 2-deoxy-~-threo-pentoseas a sirup that was characterized as the crystalline benzylphenylhydrazone. Successful conversion of a 2,3-anhydropentoside into a 2-deoxypentoside by an indirect procedure has been reported by Baker and coworkers.67Ring cleavage of methyl 2,3-anhydro-j3-~-ribofuranoside with sodium ethanethioxide was found to proceed as expected. to give the 3-S-ethyl-3-thio-~-xylosederivative (5). On treatment of (5) with ptoluenesulfonyl chloride, a mixture of 2-chloro-3- S-ethyl-3-thio- and 3-chloro-2-S-ethyl-2-thio-pentoseswas formed, instead of the expected 2-p-toluenesulfonate. Acetolysis of the mixture gave methyl 3,5-di-0acetyl-2-S-ethyl-2-thio-~-~-arabinofuranoside (7) by way of an epi0 Desulfurization sulfonium intermediate (6) having the ~ 4 y i - configuration. with h n e y nickel, followed by acid hydrolysis, afforded 2-deoxy-~-eryfhropentose.

whcrc Ts is p-tolyltwlfonyl. (53) G. J. Robertson and C. F. Griffith, J . Chem. Soc., 1193 (1935). (54) N.K.Richtmyer and C. S. Hudson, J . Am. Chem. Soe., 63, 1730 (1941). (55) E. Sorkin and T. Reichstein, Helv. Chitn. A d a , 28, 1, 662, 9.20 (1045). (56)G.Cllsini and L. Goodman, J . Am. Chcm. Soc., 86, 1.127 (1964). (57) C.D.Anderson, L. Goodman, and B. R. Baker, J . Am Chem SOC.,81,898(1959).

152

STEPHEN HANBSBIAN

In 1948, Prink introduced a new niethod for the reduction of sugar epoxides to deoxy sugars by employing lithium aluminum hydride. It was subsequently found that the generalizations drawn for the ring opening of the anhydrohexosides are applicable to opening by hydride ion. This niethod is coiisidered to be nmre praaticd and low lrttmriouw tlran tlw thioxide met,hod. Schniid and I<arn?ld@have ijhown that tlic rwction of crtrbobydrate ptoluenesulfoiiates with lithium duminum hydride depends on the poeition of the ester function.o0Primary ptoluenesulfonates, except for 1-0-p-tolylsulfonylketoses, are invariably desulfonyloxylated to the deoxy compounds. Secondary ptoluenesulfonates undergo, even under prolonged treatment, simple desulfonylation to regenerate the secondary alcohol. -CHt4SO*R

-CH,

+

I -CHOH

I

-CH--OSOsR

+ RSOaH

4

+ WOsH

In a study of the cleavage of sulfonic esters of carbohydrates with lithium aluminum hydride, Allerton and Overendo' reported that treatment of with this methyl 3,4-O-isopropylidene-2-0-p-tolylsulfonyl-~-~ar~binoside hydride in dry ether afforded, after complete acid hydrolysis, only Garabinose, indicating mere ester cleavage. With methyl 2-0-ptolylsulfonyl-13-carabinoside, however, the preponderant product was methyl 2-dcoxy-8-1.-erylhro-pentoside and methyl 8-carabinoside, with a small proportion of the 3-deoxy derivative. Since, from paat experience, hydrogenolysis of epoxideswas known to give the 3-deofrypeqtosides,the formation of a ccnsiderable proportion of the 2-deoxy derivative by the ring opening of an intermediate, 2,3-anhydro derivative was ruled out; instead, a direct, reductive cleavage of the secondary p-tolylsulfonyloxy group at C-2 was assumed to have taken place. Allowing for minor steric effects, the corresponding 3,4-0-isopropylidene acetal would be expected to undergo the =me type of cleavage also, to give a 2deoxy sugar derivative. Another reductive method involves the use of Rrtney nickel; the direction of ring opening is, however, such that the products are invariably 3-deoxy sugar derivatives. Epoxide rings in sugars have been opened by the action of halogen acids. Both possible products of ring scission were observed in the reaction of methyl 2,8-anhydro-Q, fJ-O-benzylidene-a-~-alloside6~~~~ with hydrobromic (66) D. A. Prins, J . Am. Chem. Soc., 70,3965 (1948). (69) H. Bchmid and P. Karrer, Helu. Chim. Acta, ST, 1371 (1949). (80)R. 8.Tipson, Advan. Carbohydrate Chern., 8 , 107 (1963). (61) R. Ailerton and W. G. Overend, J . Chem. Soc., 3020 (1954).

DEOXY SUGARS

153

acid.“ ‘l’hc preponderant product was the 3-bromo derivative, so this method has no practical value for the synthesis of 2deoxy sugars. (iv) By Degradation of Higher Sugars.-Under this general title are included methods utilizing oxidative glycol cleavage, Ruff degradation, and elimination reactions. In preliminary experiments, Gorin and Jonese3partially oxidized 3-deoxyD-m’bo-hexose at C-1 to C-2 with periodate, and obtained a-deoxy-~erythro-pentose (as the “anilide”64) in 29% yield. Rembaraa6treated one mole of 3-deoxy-~-arabino-hexosewith one mole of lead tetraacetate in benzena, and obtained 2-deoxy-~-erythro-pentoseas the crystalline “anilide” in 60% yicld. The same selective cleavage between C-1 and C-2 of 3-deoxywxylo-hexose was effected& to give 2-deoxy-~-threo-pentose.Szab6 and coworkersB6obtained 2-deoxy-~-threo-pentose5-phosphate (as the crystalline barium salt) by the selective cleavage of 3-deoxy-~-xylo-hexose 6-phosphat’ewith sodium periodate. The first application of the Ruffs7degradation in the deoxy sugar series was repcrted by Kdiani and Naegeli,sEwho converted a 3deoxyhexonic acid into a crystalline 2-deoxypentose which had constants similar to those reported years later by Levene and Moriss for 2-deoxy-~-erythro-pentosc. Sowden70 found that a combination of Nef’s alkaline isomeri~ation~’ of aldoses (to the corresponding3-deoxyaldonolactones) with the modification of Ruff’s degradation readily afforded 2-deoxypentoses, The same sequence was reported independently by Richards.7a Further refinements of this general method have resulted in a simplified pro~edure’~ for obtaining large quantities of 2-deoxy-~-erythro-pentose.The method involves the treatment of D-glucose with calcium hydroxide, and submitting the resulting metasaccharinic acids (without isolation) to degradation in the presence of hydrogen peroxide and ferric acetate. The crude pentose is isolated as the “anilide” in 6 4 % overall yield, and is regenerated from the latter in high yield. Although the yields are low, the procedures involved are simple, (62) F. H. Newth, W. G. Overend, and L. F. Wiggins, J . Chern. Soc., 10 (1947). (63) P. A. J. Gorin and J. K. N. Jones, Nature, 172, 105 (1953). (64) R. E. Deriaz, W. G. Overend, M. Stscey, E. G. Teece, and L. F. Wiggins, J . Chem. Soc., 1’379 (1949). (65) G. Itembars, Chem. Ber., 96, 1565 (1962). (66) K. Antonakis, A. Dowgiallo, and 1,. SsaM, Bull. Soc. Chint. France, 1355 (1962). (67) 0. Ruff, Ber., 94, 1362 (1901). (68) H. Kiliani and H. Neegeli, Ber., 36,3628 (1902). (69) P. A. Levene, L. A. Mikeska, and T. Mori, J . Biol. Chem., 86, 785 (1930). (70) J. C. Sowden, J . Am. Chem. Soc., 76,3541 (1954). (71) J. U. Nef, Ann., 376, 1 (1910). (72) G. N. Richards, Chem. Znd. (London), 1035 (1953). (73) H. W. Diehl and H. G. Fletcher, Jr., Biochem. Prepn., 8,49 (1961).

154

STEPHEN HANESSIAN

axid tho starting material is cheap. V e n i ~ eobtaiiied r~~ 3deoxypeutonic acids from tarabinose and D-xylose, respectively, by reaction in aqueous alkali at 100' under nitrogen. Degradation of the resulting aldooic acid salts gave crystalline 2deoxy-Pglyccru-tetrose (10-117$ froni tho ponbosu) , 111 the sa111ereport,, Ve~iiicr~~ claimed the isolutioti of 8 (yuitrophotyL)o~rrr,u~', 1n.p. Q ~ Y - % ~ O O (50' ing.) froin the reactioii of 2-deoxy-u-erUlhro-peiito~ (1 g.) with an excess of (pnitropheny1)hydrasiiie in acetic acid. It is not unlikely that a slight contamination (less than 0.5%) by the starting 2-deoxypentose could have been responsible for the production of the osalione. The Ruff degradation has been applied76 to substituted, deoxy sugar derivatives to give correspondingly substituted 2-deoxypentoses. Thus, calcium 6-O-benzyl-~-gluconatewas subjected to degradation in the presence of barium acetate, ferric sulfate, and hydrogen peroxide to give sirupy 5-0-beneyl-2-deoxy-~-eryth~o-pentose. Similarly, 3 ,5-0-benzylidene-2-deoxy-o-ery#m-pentose was obtained from the appropriate metasrlccharinic acid. That the general conditions of degradation were suitable in the presence of a phosphate eater function was demonstrated by Szab6 and coworker^.^^ Reaction of 1,2-0-isopropylidene-3-0- (methylsulfonyl) -Dglucose with diphenylphosphorochloridate afforded the 6- (diphenylphosphate) , which apparently decomposed instantaneously in the presence of water. This phenomenon, which was attributed to transesterification with the GE,hydroxyl group to give a cyclic phosphate, was not observed when the C-5 hydroxyl group was substituted. Hydrolysis was, therefore, effected in dry benzene containing a cation-exchange resin. The resulting 3-0(methylsulfonyl)-D-glucose 6-phosphate (barium salt) was converted into metasaccharinic acid &phosphate with barium hydroxide, and this was degraded to 2-deoxy-~-erythro-pentosebphosphate.2a-'"6Crystalline 2-deoxy-D-threo-pentose has been obtained in 35-41 % yield from 3-deoxy-Dxylo-hexose by way of the oqrresponding calcium hexonate." D-Glucose substituted a t C-3 with various groups has been converted into 2-deoxy-~-erythro-pentoseby Revera1 investigators, SmithTsreported that, ( 8 ) is treated with 2 when onc mole of 3-0-(methy~su~fony~)-~-glucoee moles of sodium hydroxide a t room temperature, a rapid reaction occurs, to give the 2deoxypentose, D-arabinal, and formate (the last two being detected chromatographically). By partition chromatography on cellulose, (74) H. Venner, Nalumissenschafh, u),278 (19511); Chem. Ber., 90, 121 (1957). (75) J. Kenner and G. N. Richards, Brit, Pat. 768,250 (1057); Chum. Abstracts, 61, 16529 ;1957). (76) S. Lewak, R. Derache, und L. Szab6, Compt. Rend., 248, 1837 (1959). (77) H. Zinner, G. Wulf, and R. Heinatz, Chem. Ber., 97,3538 (1031). (78) D. C. C. Smith, Chem. Ind. (London), W (1955).

155

DEOXY SUGARS

a .5.5yGyield of the dcoq-pexitose (baed on the starting wgliicose d~rim-

tive) wm obtained. A mechanism may be formulated as follows: O Y O \ HC=CHOH

I

OH

H c=o I CH,

OH I

HCOH (!!&OH

Later, employing 1 ,4 ,6-tri-0-acetyl-3-0- (methylsulfonyl) +glucose, Smith79 reported the isolation of the enolate intermediate ( 9 ) as a sirup. A re-inveetigation of this reaction by Hardegger and coworkers,80who cited analogies in the steroid field, revealed that the alkaline degradation could be conducted in the pH range 8-9 at 45" for 2 hr., optimal yields of 4749% of the deoxypentose being produced. Furthermore, examination of the mother liquors provided no indication of the presence of D-arabinal, as originally suggested by Smith.T8 Since the yields of the required intermediates are very good, 100-200 g. of 2-deoxy-n-erythro-perltose could be prepared in one o p e r a t i ~ nAccording . ~ ~ ~ ~ ~to these authors,aothe yield of the deoxypentose was only 20-26% when 3-O-p-tolylsulfonyl-r>-glucosewas used. Kenner and Richardss1 studied the effect of the 3-substituent on the behavior toward alkali, and found, in contrast to the results of Hardegger and cowr)i'kers,80that, the 3-p-toluenesulfonate is an excellent source of 2-deoxy-~-er!ithro-pei~~,ose. Treatment of this ester with dilute lime-water (0.04N ) a t 25" resulted in a rapid reaction, with formation of the crystalline deoxypentose in 76% yield.s1 The action of limewater was founds1to be (7!)) D. C. C. Smith, J . Chem. Soc., 2690 (1957). (80)E. fisrdagger, M. Schellenbnum, R. Wyler, and A. Ziilst, Helv. Chim. Ada, 40, 1815 (1957). (81) J. Kenner and G . N. Richards, J . Chem. Soc., 3019 (1957).

156

STEPHEN HANESSIAN

more rapid than that of 2 N sodium hydroxide, showing the importance of the cation in this reaction. The preference for the ptolylsulfonyl, rather than the methylsulfonyl, derivative was based on the lower yield (65%) obtained from the latter.s1 A simplified method for the preparat,ioii of 2deory-o-erylhro-pentosefrom ( 8 ) was described by Rcroiido uitd Riirdcrknecht.*? The use of sodiuni carbonate for the dcgiadatioii wtts fouid to give particularly high yields (4740%) of the crystalline 2-deoxypentosc. A specific anion effect during the degradation was observed by these authors.s2The order of effectiveness of anions was CO, > HP04 > PO4 > SO, > Cl. Although the reactions discussed above provide an elegant synthesis for 2-deoxypentoses, several aspects are still riot settled. The variable effects of the 3-sulfonic esters and the apparent mtionic and anionic effects await explanation. Weygand and Woh88degraded 3-deoxy-~-xylo-hexoseoxime in the presence of 1-9uoro-2, .l-dinitrobemene, and obtained 2-deoxy-~-threo-pentose. The MacDonald-Fischefi degradation was used by Hough and T a y l o i for obt'aining 2-deoxy-~-erylhro-pentosefrom 3deoxy-D-ribo-hexose diethyl dithioacetal. (v) Other Methods.-F€ougha outlined a method for the synthesis of 2-deoxy-~-gylhro-pentosewhich involves the reaction of 2 ,3-0-isopropylidene-D-glyceraldehydewith allylmagnesium bromide. The resulting 5 ,6-0-isoyropylidene-l-hexene-~-erythro-4 ,5,6-triol, obtained in excellent yield, was hydroxylated to a mixture of products. Periodate oxidation of the hexitol derivatives, followed by hydrolysis, afforded the 2-deoxypentose. As essentially one deoxypentose was obtained, it appears that the Grignard synthesis is asymmetric in this case, since the product consists largely of the erylhro isomer. This attractive synthesis, which could be applied to a variety of deoxy sugars using different Grignard reagents, has not yet been exploited. The reactions of carbohydrate derivatives with certain organometallic reagents (Grignard and Friedel-Crafts processes) have been reviewed in this Series and elsewhere.syThe attempted application of a Chugaev reaction to sugar xanthates was reported by Wolfrom and Foster." It was found, (82) E. Recondo and H. Rinderknecht, Helv. Chim. Acta, 48, 1663 (1980). (83) F.Weygand and H. Wolr, Chem. Ber., 86,256 (1952). (84) D. L. MaoDQnald 8nd H. 0. L. Fischer, J . Am. Chem.Soc., 74, 2087 (1952). (&) L. Hough and T. J. Taylor, Chem. Znd. (London), 875 (1864) (86) L. HoLgh, Chem. Id.(London), 406 (1961). (87)(a) W. A. Bonner, Aduan. Carbohydrate Chem., 6, 261 (1961); (b) S. Haneseian, Ph.11. Dissertation, The Ohio Stab University (1960); Diseertdion Abslr., 31, 3268 (1961). I (88)M. L. Wolfrom and A. B. Foster. J . Am. C h .Soc.. 78. 1399 (1966).

DEOXY SUGARS

157

however, that a thermal rearraugerueut o c c d , Luted of the rypected elimination. A low yield of 2deoxy-D-erythro-piitose could be obtaiiicd from the rearranged 2-5- (methylthiocarbonate) . Another relatively unexplored reaction that might lead to a potential synthesis of 2-deoxy sugars is the selective acetylation of sugar dithioacetals and the subsequent reaction with a mercaptan. Thus, if 3,4,5,6-tetra-0-benzoyl-~-g1ucose diethyl dithioacetal is treated with ethanethiol and hydrochloric acid, a 2-S-ethyl-2-thio derivative result~P;this has been converted, in a number of steps, into 2-deoxy-~-urubino-hexosein good yield.w Similarly, 3,4,!j-tri0-bewmyl-wxylose diethyl dithioacetaP has been converted into a 2-S? ethyld-thio derivative,s2 but no attempt at subsequent conversion into a 2-deoxy-~-threo-pentosewas reported. To provide proof for the position of the cewly introduced ethylthio function, the product was reductively desulfurized to 1,2-dideoxy-u-threo-pentitol.The configuration at C-2 in these two ethylthio derivatives is still unknown. A synthesis of 2deoxym-erythro-pentose from 1-methoxy-1-buten-3-yne was described by Weygrtnd and Leube.ea 2. 3-Deoxy Sugars a. General Considerations.-To date, only one naturally occurring 3-deoxy sugar (namely, 3-deoxy-~-erythro-pentose) has been isolated. There has, however, been considerable controversy concerning the identity of this sugar, as will be outlined. The antibiotic substance cordycepin, an adenine nucleoside, was reportede4to contain a branched-chain sugar (cordycepose) having the molecular formula CsHlOO~. Cordycepose forms a crystalline (pnitrophenyl) osazone, indicating that there is no deoxy function at C-2. A crystalline phenylhydrazide from cordyceponic lactone allegedly did not conform with any of the theoretically possible, open-chain, 3-deoxypentonic phenylhydrazides. The riucleoside did not react with periodate, and theyefore a 3-deoxy structure was possible. Based on the above observations, a 3 deoxy-3-C- (hydroxymethyl)aldotetrose structure was assigned to cordy~epose.~' A supposed synthesis of Dbcordycepose was reported by Raphael and Roxburgh,gsstarting with diethyl 2-( diethoxyethyl)malonate. The (p-nitrophenyl) osasone from the synthetic productg6 seemed to be identical with an authentic sample prepared from the natural sugar.94 Following the publication of this supposedly overwhelming evidence in (89) P. Brig], H. Miihlschlegel, and R. Schinle, Ber., 64, 2921 (1931). (90)B.R. Bolligcr and M. D. Schmid, Helv. Chim. Acfa, 54, 1671 (1951). (91) M. L. Wolfrom and W. von Bebenburg, J . Am. Chem. Soc., 81, 6705 (1950). (92) M. L. Wolfrom and W. von Bebenburg, J . rlm. Chem. Soc., 82,2817 (1960). (93) F. Weygand and H. Leube, Chem. Ber., 89, 1914 (1958). (94) H. R. Bentley, K. G. Cunningham, and F. G. Spring, J . Chem. Soc., 2301 (1951). (95) R. A. Raphael and C. M. Roxburgh, J . Chem. SOC.,3405 (1955).

158

STEPHlN HANEBSIAN

favor of the branched-chain structure proposetl for aordycepose, a (%deoxypentosyl) adeninea obtaiiied from cultures of .4apergillus nidulans (Eidam) Wint. was found to be identical with cordycepiiiw and with a synthetic with respect to sample of 9-(3-depxy-~-er~lhro-peritofurariosyl)adenine~ nuclear magnetic remiance, infrared, a i d othor physioal data. Tlra rvrorukrl melting points for the (piiitrophrny1)oeasoticlJ froin 3-dwxy-I)- lttiti -L-erylhro-pentose, namely, m.p.b71m253-255.5O andm 254-250' (decomp.) , respectively are fairly close to that [2W0(decomp.)] reportedM for the osazone obtained from the natural product which partly explains the reasons for the ambiguity. The corrected structure of cordycepin has been corroborated- by mass-spectral studies on natural snd syntheticw cordycepin. Chemical evidence in favor of the 3-deoxy-wsrythro-pentose structure was obtained from degradative studies on W-labeled cordycopose.gg* Cordycepin has also been isolated from a new strain of Cordycepa militaria, and has been identified by infrared Many reactions of 3-deoxyhexoses involve their degradation by various means to Zdeoxypentoses, as discussed in the previous Section. The synthesis of 3-deoxy-~-m'bo-hexose6-phosphate for testing as a potential antimetaholite in cancer chemotherapy was reported by Dahlgard and Kaufmann.lmThe anomeric 3-deoxy-~-xylo-hexopynosylphosphates have been obtained101 as the crystalline barium salts from the corresponding acetylated glycosyl bromides; it was observedlol that the @-D anomer is hydrolyzed .more rapidly than the CY-Danomer by 0.02 N hydrochloric acid at 15'. A synthesis of 3-deoxy-~-ergthro-pentosc? 5-phosphate from Sdeoxywribo-hexose was described by the S ~ a b b sAlkaline . ~ ~ ~ treatment of pentose 3, &(hydrogen phosphates) produaes a mixture of 3-deoxy-threo- and erythro-pentonic acid

b. Syntheei8.-By far the moat commonly used method for the synthesis of 3-deoxy sugars involves the opening of anhydro rings by nucleophiles (98) E. A. Knckzka, N . R. Trenner, B.Arieon, R. W. Walker, and K. Folkers, Biochem. Biophye. Res. comm?4n.,14, 468 (1084). (97) W. W. Lee, A. Benitaa, C. D. Anderson, L. Goodman, and B. R. Baker, J . Am. Chem. Soc., 88, 1906 (1981). (98) P. W. Kent, M. Btaceg, and L. F. Wiggins, J . Chsm Soc., 1232 (1959). (99) 9. Mukherjee and A. R. Todd, J . Chem. Soc., 989 (1949). (99a) R. J. Suhadolnik and J. G. Cory, Biochim. Bwphys. Acta, 81, 861 (1964). (99b) S. Frederiksen and H.Malling, Biochim. Biophyu. Ada, 86, 189 (1965). (990) 8. Hanmdan, D. C. Ddongh, and J. A. McCloskey, Biochim. Bwphys. Acta. 117, 480 (1sSe). (100) M. Dahlgmrd and E. Kaufmann, J . Org. Chsm., 46,781 (1960). (101) K. Antonakis, Compf.find., 468,3611 (1964). (102) P. Sra'o6 and L. Saab6, J . C h . Soc., 2944 (1986). (102a) W. Jachymcayk, L. Menager, and L. Szab6, Tetrahsdrm, 41, 2049 (1985).

DEOXY SUGARS

199

aapable of being subsequently reduced, by hydride ion or by hydrogenolysis, to the deoxy function. Several other new methods are available, but have not yet been extensively tested. (i) From Anhydro Sugars.-Provided that the anhydro ring has the requisite stereochemistry (as discussed in the previous Section), such nucleophiles as methoxide or thiomethoxide ions attack a 2,3-anhydro ring predomimntly at C-3, in a stereoselective manner. Reaction of methyl with sodium thiometh2,3-anhydro-4 ,6-O-benzylidene-fl-~-talopyranoside oxide in methanol afforded the corresponding 3- S-methyl-3-thio derivative in quantitative yieldlo*; reductive desulfurization of this gave methyl 3-deoxy-4 ,6-O-benzylidene-~-lyxo-hexopyranoside.In the -me year, Mukherjee and Toddw reported the synthesis of methyl 3-deoxy-cergthropentoside by a similar procedure, A number of such applications, using different thioalkoxides, have since been reported. Of particular interest is the ring opening of 2,3-anhydropentofuranosidederivatives which leads to a predominant attack at C-3. The synthesis of 3deoxy-~-erythro-pentose from methyl 2, 3-anhydro-/3-D-ribofuranosideJby way of the 3-S-ethyl-3thio derivative, is a typical example.b7 An exception to the generalized behavior in the anhydro-ring opening of furanosides is found in the reaction of methyl 2,3-anhydro-fl-D-lyxofuranoside with sodium a-toluenethioxide.MSurprisingly, predominant attack occurs a t C-2, resulting in a 3:2 ratio of the 2-S-benzyl-2-thio- and 3-S-benzyl-3-thio-pentosides(isolated as the crystalline pnitrobenzoates) . A nucleophile which has some advantages from the practical standpoint is the thiocyanate ion. The resulting ring-cleavage products are amenable to*reduction in the presence of Raney nickel, or other suitable agents, to the corresponding deoxy sugar derivatives. An added asset associated with the use of thiocyanate ion is its good nucleophilicityand the facile characterization of the reaction products by virtue of the familiar infrared bands for SCN a t 2135-2170 cm.-'. The reductive cleavage of epoxides is one of the earliest and most universal routes to 3-deoxy sugars. All past experience has shown that the catalytic hydrogenation of substituted 2,3-anhydrohexosides affords, irrespective of the stereochemistry of the epoxide ring, a 3-deoxyhexoside. Frequently, a benzylidene protecting group is removed during this reactionsb or is hydrogenated to the 4,6-0-cyclohexylidene acetal. 3-Deoxy-~-arabinoand 3-deoxy-Plyxo-hexosela have been hexose,lo4:3-deoxy-D-x:ylo-hexose,1a syuthesizsd by this procedure. Crystalline :3-deoxy-D-ribo-hexose10'Jwas (103) M. Gut, 1).A. Prim, and T.Reichstein, Helu. Chim.Aciu, 90,743 (1947). (104) H . R. Bolliger and D. A. Prins, Helv. Chim.Ada, 29, 1061 (1946). (106) H. Kuber and T. Reichstein, Helv. Chim. Ada, 81, 1645 (1948). (106) J. W. Prntt and N. K. Richtmyer, J . Am. Chem. Soc., 79, 2597 (1957).

160

STEPHEN HANESSIAN

obtained by the catalytic hydrogenation of methyl 2,3-anhydro-4 ,GObenzylidene-a-D-alloside,followed by acetolysis and deacetylation. Induced acid treatment converted the 3-deoxyhexose into its 1 ,&anhydride to the extent of lo%,compared to 20% for the 8-deoxy-tarabitw auulog. The fuilurt) of soma of the related, iwnieric, dcwxy s u g m to ctptdliw i w y ho trttributablc to the presence of variable proportions of their 1,O-anhydridcs. Indeed, Pratt and Richtmyer'Q suggested that the C-3 substituent plays an important role in controlling the point of equilibrium. A useful, practical method for separating the resulting methyl 3-deoxyhexosides from other by-products consists in re-benzylidenation of the crude, reaction product and isolstion of the crystalline methyl 4 ,6-0-benzylidene-3-deoxyhexosides. In a re-exa~ination~~' of the hydrogenolysisabof methyl 2 ,3-anhydro-4,6-0benzylidene-u-D-allosidein the presence of h n e y nickel, it was found that the 2-deoxy- and 2,3-dideoxy-hcxosides are formed (in addition to the previously reporteda 3-deoxy derivative). Ionophoresis in borate buffer proved useful in establishing the heterogeneous nature of the reaction.lOl Since the introduction of lithium aluminum hydride for the cleavage of sugar epoxides," its use in this route to 3-deoxy sugars has become the method of choice. Thus, treatment of methyl 2,3-anhydro-4 ,6-0-benzylidene-u-D-mannopyranoside with lithium aluminum hydride affords methyl 4,6-0benzylidene-3deoxy-u-D-arabinehexopyranosideas the sole product in good yieldw; this product was also prepared by this multi-step procedure some years later.'m Hydrolysis with dilute acid, however, afforded sirupy 1,&anhydm-3-deoxy-/3-~-urubin~hexopyranose. Equilibration studieslm in dilute acid gave a mixture whose optical rotation properties indicated that it consisted of a 7:3 mixture of 3-deoxy-~-arab&no-hexose with its 1,6 anhydride; this permitted the calculation of the optical rotation of 3-deoxyD-arabino-hexose as being [&ID47 f lo.It became evident that the same deoxy sugar obtained as a sirup by Bolliger and PriqP by acid treatment contained a considerable proportion of the levorotatory 1,6anhydride. RembarzlQ was successful in obtaining crystalline 3deoxy-&~-arabinohexose in 8 total yield of 61% by acid hydrolysis of the corresponding glyooside; the 1,6-anhydride was found in the mother liquors. It is interesting to note that the initial value- of [ ~ J D 46.1' agreers remarkably well with that calculated from the equilibration &udiea.1m Two anomeric forms of 3-deoxy-D-ribo-hexose have been obtained in crystalline condition by Anetm; the high rate of mutarotation of the u-D anomer suggested that it has a furanose ring. (107) F. J. Hedgley, a. Overend, and R.A. C. Rennie, J . Chem. rsoC., 4701 (1903).

+

v.

(108) 0.Reknbers, Chem. Ber., 98,622 (1980). (108) E.F. L.J. Anat, C h . Ind. (London), 345 (1960).

DEOXY YIlGAHS

161

The preparatiouof M e o x y sugars may also bc d r i c w x i by Ity,yrtnyyc~alysis of the 3 ,hpoxides. This type of ring cleavage is not so storcosclcctivc as that of the 2,3-epoxides, and the mixture of 3- arid 4-dcoxy sugar derivatives resulting has to be separated. Methyl 3,4-anhydro-cu-~-galactopyranoside,l10on treatment with Raiiey nickel and hydrogen, affords a mixtuie of the 3- and 4-deoxy glycosides, from which methyl 4,6-0-benzylidene-3-deoxy-u-~-xylo-hexopyranoside may be obtained by rebenzylidenation of the crude mixture.111 Treatment of the @-D-glycoside1l2(10) with lithium aluminum hydride gave a 73y0yield of crystalline methyl 3-deoxy@-D-xylo-hexoside (12) and a 5y0 yield of the corresponding 4-deoxy derivative (1l).1laReduction with Rmey nickel, on the other hand, re-

sulted in the formation of only 10% of the 3-deoxy and 30% of the 4-deoxy derivative.118The ratio of products was found to depend on the age of the catalyst. These authors11sreported the isolation of 3-deoxy-~-xy20-hexose as an oil, and its conversion, in theoretical yield, into the corresponding (2 ,Pdiitrophenyl) hydralrone and hexitol derivative. It follows that this sugar, which is related to D-galactose and wgulose, apparently has litt.le tendency toward 1,6-anhydride formation during acid hydrolysis. V i and Karrer114have found that treatment of methyl 4 ,&O-benzylidene-2,3- 0-p-tolylsulfonyl-cu-Dglucoside( 13) with lithium aluminum hy.(15) in dride provides methyl 4,6-0-benzylidene-3-deoxy-a-~-ribo-hexoside good yield. Since hydrogenolysis is possible at both C-2 and C-3 of the two possible 2,3-anhydro dcrivatives, only the a110 isomer (14) could give the 3-deoxy derivative by further reaction with a reagent. Indeed, none of the alternative 2-deoxy derivative was found. This transformation is, un(110) J. C. Buchanan, J . Chem. Soc., 2511 (1968). (111) E. J. Hedgley, 0. Memz, W.G. Overend, end R. A. C. Rennie, Chem. Ind. (London), 938 (1960). (112) A. hlitller, M. Mbricz, and G . Verner, Ber., 72,745 (1939). (113) M. Dshlgard, B. H. Chastain, and RuJen Lee Han, J . 078. Chem., 27, 929 (1962). (114) E. Vis and P. Karrer, Helu. Chim. Acta, 97,378 (1964).

102

8TEPHEN HANEIIBIAN

doubtetily, an isolated example, and it must depend on the orientation of substituents. That this is 80 was shown by Overend and coworkers*" by

OTe (19)

\P

PC"

phcHQMe- I%

PhCyQOMe

OH

0

(14)

(16)

applying the eame treatment to the corresponding 8-D-glycoside; the expected product was isolated in only 38% yield. Changing the solvent from tetrahydrofuran to pdioxane gave even lower yields. The by-product in these reactions was found to be methyl 4,6-0-benzylidene-2,3-dideoxy0-D-erythro-hexoside, presumably resultingl0l from C-0 fission at C-2 and c-3. Anhydro sugars have been treated with hydrogen halides to give 2- and 3-halo derivatives which are amenable to reduction and provide 3deoxy sugars,wJu Some years ago, Newth and coworkers116 observed that methyl 2 ,3-anhyd*o-Q,6-0-benz;ylidene-a-~-alloside reacts with methylmagnesium iodide to give, as the sole product, methyl 4,6-0-beneylidene-3-deoxy-3iodo-a-D-glucoside in 80% yield. Richards11Thas found the same behavior with the u-manno isomer when methyl- or phenyl-magnesium iodides are used. Treatment of the epoxide with ethylmagnesium iodide, however, produces, instead of thc expected 3-iodo derivative, methyl 4 ,6-0-benzyliden~~-cleoxy-a-n-~~f~hcxoRide dircctly (in 53% yield) , presumably as tho result of rductioii by hyclrogcii trniiflfcr. Tho rcactioii of sugar epoxides with orgniiomstallic rcageiitH has not, yet becii followed up. (115) R. Allerton Rnd W. (1. Ovt*rend,J . Chen. Soc., 1480 (1951). (110) F. H.Newth, G.N. Richards, and L. F. WigninR, J . Chem. Soc., 2350 (1950). (117) Q. N. Ric*hnrda,J . (Ihenr. Soc., 4511 (1964).

163

DEOXY SUGARS

(ii) From Vinylic Ethers.-The basecatalyzed elinlitlation of ccrtain tosyloxy functions in carbohydrates was demonstrated as early as 1922 by Freudenberg and Brauns.l18 From the reaction of 1,2:5,6-di-O-isopropylidene-3-O-p-tolylsulfonyl-~glucofuranose ( 16) with anhydrous hydrazine, they obtained, in addition to the 3-hydrazino derivative, 3-deoxy-1 ,2 :5 ,6di-0-~propylidene-~-erythro-hex-3-enofurar10se( 17). Hydrogenation of

H

c=o I

HCOH I

yH*

HOCH I HCOH I CH,OH (19)

(18)

(17) was originally assumed to have given a 3-deoxy-~-m'bo-hexosederivative. The product has been shown by Weygand and W O ~ to Z be ~~ 3deoxy-l,2 :5,6-di-O-isopropylidene-~-xyZo-hexofuranose( 18). The hydrogenation step therefore prqceeds in a stereospecific manner. These authorssa also obtained the same intermediates in 67% yield by effecting the elimination in xylene in t8hepresence of sodium carbonate. Catalytic reduction and hydrolysis of the product gave 3-deoxy-D-xylo-hexose (19). A modification" involves heating ( 16) with sodium carbonate at 210" under vacuum, and affords a 70-75% yield of the unsaturated intermediatc.

(iii) From Sugar XanthateH.-This method is currently claimed10'to be the mont, Ratisfactory for the preparation of 3-deoxy-D-ribo-hexose. (118) I -

I;cG

(27)

- -CH,

&

(28)

C&OH &-Ia 0

QOH OH

(2Q)

with a known aample.laeAfter Wolfrom and Whiteley's preliminary communication,laa Overend and coworkers"' disclosed another synthesis for (28) ; this irivolved the catalytic hydrogeiiolysis of 5,6-anhydro-l,2-0(186) P. Itegna, J . Am. Chem. Soc., 68, 246 (1947). (187)(a) M. L. Wolfrom and T. E. Whitely, Abstracts Papers Am. Chem. Soc. Meeting, 187, 20 (1960). (b) M. L. Wolfrom, K. Mstsuda, F. Komitsky, Jr., and T. E. Whikly, J . Org. Chem., 28, 3661 (1903). (138) H.C. Brown and B. C. Subba Rao, J . Am. Chem. Soc., 78, 2682 (1966);H.C. Brown, J. K.Murray, L.J. Murray, J. A. hover, and G. Zweifel, ibid., 82, 4233

OW).

(139)(a) H. Ohle and E. Dickhtiuser, Ber., 68, 2693 (1925);(b) J. K. N. Jones and J. L. Thompnon, can. J . Chem., 86, 955 (1967);(c) L. D. Hall, T,. Hough, nntl li, A. Pritchard, J . C h .Soc., 1637 (1961).

DEOXY SUGARS

169

isopropylidene-a-D-glucof uranose in the presence of Rantly niclir.1 in nwt 11anol a t 100' and 110 atmospheres of hydrogen. The liydrogetiolysis of thc terminal anhydro ring apparently depends on the nature of the catalyst and on the medium.'" In the presence of alkali, a terminal deoxy sugar is produced exclusively, but, in acidic media, the product also contains 1 ,2-O-isopropylidene-6-O-methyl-a-~-glucofumnose. The product of reduction of 6-O-benzoyl-1,2- O-isopropylid~ne-5-~-p-to~y~s~fony~-a-~-g~ucofuranose with a hydride, previously assumed,141without proof, to be a 6-deoxy derivative, has been shown"* to be (28). The hydroboration reactionl37J" has been extended in the carbohydrate series by Goodman and coworker~.~4~ Hydroboration of the 3-methanesulfonate of (27) affords 5-deoxy-1 ,2-O-isopropylidene-3-O-(methylsulfonyl)-a-D-zylo-hexose; this is de-estersed by methanolic potassium hydroxide to give (28), and the latter may be hydrolyzed to the free sugar (29) . Methyl 2 6-di-O-benzoyl-,5-deoxy-~-ribo-hexopyranoside was obtained'd2 (by an inversion) by the action of sodium benzoate in N , N dimethylformamide on methyl 2 6-di-O-benzoyl-5-deoxy-3-O-(methylsulfonyl)-a-u-zylo-hexopyranoside,and this was converted into U~OXY-Dribo-hexose (33).The above sequence of reactions illustrates the use of a neighboring benzoate group in aiding the solvolytic displacement reactions of secondary sulfonates in cyclic carbohydrate derivatives. Compound (28) was formed in low yield (9%) by the Raney nickel desulfurization of 3 ,6-di-O-acetyl-5- S-acetyl-1 ,2-0-isopropylidene-5-thio-~-idofuranose;an accompanying product, namely, 5 ,6-dideoxy-1 ,2-O-isopropylidene-~-q&ohexose, was formed in 57% yield. A Chugaev elimination reaction was successfully performed on the 5-S-methylxanthate (30)of methyl 2 ,3-0-isopropylidene-6-deoxy-~-~-allofuranoside.~~~ The product was shown, by nuclear magnetic resonance studies, to be the allylic olefin (31), not the vinylic olefin (34).It should be noted that the related elimination reaction in cyclic derivatives gives rearranged products." The rather unexpected selectivity obtained during the pyrolysis was explained by these authors1* on the basis of steric hindrance in the transition state required for the formation of (34).Hydroboration of (31), and acid hydrolysis of the product after fractionation, afforded (33).The presence of u-deoxyhexose side-products (40%) in the hydroboration reaction mixture prompted these workers to use a bulkier borane, namely, cis- (3-methyl-2-butyl) borane, in an effort to increase the stereoselectivity of the reaction; the yield of (32) was unaffected. )

)

(140) E. J. Hedgley, Private communication. (141) E. J. Reisq R. R. Spencer, and B. R. Baker, J . 0t.g. Cham., 23, 1757 (1958). (142) K. J. Ryan, H. Arzoumanian, E. M. Acton, and L. Goodman, J . Am. C h m . SOC., 86,2503 (1964).

170

STIPHEN HANESSIAN

uocH'-

ROCH

o

CqOH

&Q HO

OH

This reaction should be extended to other types of olefinic carbohydrates containing different substituents and ring sizes, in order to ascertain whether coordination of the reagent with one or more of the several oxygen functions in the molecule could possibly account for the variable selectivity. Another synthesis of (28), and thence of (29) in 25% overall yield from n-glucose, utilizes a @-eliminationreaction of 3-O-acetyl-l , 2-0-isopropylidene-BO-ptoly~s~fony~-6-~-trity~-a-~-g~ucofuranose by the action of sodium metk.oxide.1" An interesting variation in the processing of the interuranose mediate A-deoxy-1 ,2-0- isopropylidene-&O-trityl-~-x~b-hex-5-enof (35) m-as independently reported by Bucharian and Oakes.1"" Acid-catalyzed detritylation of (35) afforded a dialdose derivative (36)as the difuranose, with concomitant production of a deoxy function at Cb, as in the glycal series. Borohydride reduction then produced the knownlp] (28). Montgomery and Hewson'* have described yet another synthesis for this class of deoxy sugars. Treatment of methyl 2,3-0-isopropylidene-50-(p-nitrophenylsulfanyl)-8-D-ribofuranoside with sodium cyanide in N ,Ndimethylforniamide afforded the corresponding 5-cyano-5deoxy derivative (143) R,E. Gramera, T. R. Ingle, and R. L. Whistler, J . Ore. Chem., 29, 2074 (1984). (144) J. G.Buchanan and E. M. Oakes, Tetrubdron Letters, 2013 (1964); Carbohydrate Ree., 1, 242 (1966). (146)J. A. Montgomery and K. Hewson, J . 0t.o.Chem., 29,3436 (1964); J . Med. C h m . , 9, 234 (1966)

-

DEOXY BUGARS

171

in modemtc yield. Rduction and dizotization then gave methyl hleoxy2,3-O-isopropylidone~-~~~~hexofurtLnoside as the sole product which, on acid hydrolysis, afforded (33) as an oil.

‘ko>

S ci

CHOTr II

0-c-CH, I CHa (55)

Xi@

- ‘“QY

-

08)

O-C-CH, I CH,

(36)

5. Terminal-deoxy Sugars a. General Considerations.-The w-deoxyhexoses are the most widespread class of deoxy sugar derivatives in the plant and microbial world. The large volume of historical background and literature concerning their occurrence has been adequately documented in a textbook by S t a ~ S k l ~ ~ and, in part, in several reviews.1-6 There are many reports in the literature in which 6-deoxyhexoses isolated from natural substances have been “characterized” by chromatographic techniques only. In many other instances, however, they have been isolated in crystalline condition. A selection of the more recent reports of their occurrence will be stressed in this brief outline. To date, no derivative of 6deoxyidose has been found in natural products. A reasonable explanation for this, on the basis of the conformational instability147inherent in the idopyranoss structure, has been proposed.* One of the 6deoxy sugars earliest isolated, 6-deoxy-~-mannose(crhamnose) , was obtained by Rigaudla from the plant glycoside quercetin. Although sugars are seldom found in the free form in Nature, a paper14g report+$ the occurrence of free brhamnose in the roots of Datisca cannabinae. LRhamnose is an abundant member of bacterial polysaccharides obtained from Gram-negative bacteria.I6O It has also been isolatedl5l from mycoside (146) J. Stanlik, M. cernf, J. Kocourek, and J. Pa&, “The Monosaccharides,” Academic Prws Inc., New York, N. Y., 1963, p. 403. (147) R. I!: Reeves, Advan. Carbohydrate Chem., 6, 108 (1951). (148) L. Rigaud, Ann., 90,283 (1854). (149) J. A. Mikhailova, L. N . Efremova, and A. A. Pryanishnikov, Tr. Vses. Nuuchn.Issled. Inst. Khim. Reaktivov, as, 65 (1959); Chem. Absiracts, 06,2493 (1961). (150) M. R. J. Salton, Biochim. Biophys. Acta, 46, 364 (1960). (151) A. P. MacLennan, Bwchern. J., 82, 394 (1962).

172

STEPHEN HANESSIAN

C, a mixture of glycolipides produced by strains of Mycobaclerium avium. D-Rhamnose, rarely found in Nature, has been isolated from a capsular polysaccharide of Gram-negative bacteria.ua McKinnell and Percivall63 have isolated crystalline Irrhamnose from the hydrolyzate of a watersoluble polysaccharide from the green seaweed E’nleromorpha cmnpressa. The authors speculated that the majority of sulfate ester groups in algal polysaccharides are probably carried by the brhamnose residues. Another report described the isolation and identification of L-rhamnose from rabbit skin.u4 Although neutral sugars frequently occur in glycoproteins and blood-group substances, Lrhamnose had not been previously reported as a constituent of mammalian tissue. Another common 6-deoxyhexose1 6-deoxy-Lgalactose (cfucoue) was isolated from an bfucose-containing polysaccharidelfi in 1912. D-Fucose, on the other hand, was obtained by the hydrolysis of convolvulin,’66and characterized as the crystalline 2-benzyl-2-phenylhydrazorle; this sugar is a constituent of numerous polysaccharides and plant glycoside~.~J46 The immunological significance of D- and bfucose and their methyl ethers in polysaccharides of the blood-group substances has been discussed in detai1.u’ Several antibiotic substancesu8 have been shown to have a fucose or a fucose methyl ether as a constituent. Galmarini and Deulofeulb~ isolated crystalline 4-0-methyl-~-fucose (D-curacose) from the antibiotic curamycin produced in the cultures of Streptmnycee mra-coi; this constitutes the first example of the isolation of such 8 D-fucose derivative from natural sources. Another antibiotic substance, chartreusin,lWwas also shown to contain wfucose. Finally, crystalline 2,4di-0-methyl-~-fucose (labilose) has been isolated from labilomycin,161 an antimycobacterial antibiotic substance. Methanolysis of the antibiotic material afforded a mixture of glycosides which, on hydrolysis by aqueous acid, gave crystalline labilose. Demethylation with hydrobromic acid gave D-fucose, thus proving the (182) A. Markovitz, J . Biol. Chem., 287, 1707 (1902). (163) J. P.Mal Ke; and I* > BB > OAce > Cle. The pronounced effect of the anions strongly suggests” an important role for anions in the formation of complexes, Because electrophoretic studies in both water and alcohol (see Section II,8, p. 234) offer evidence that free univalent anions do not complex significantly, if at all, with carbohydrates in solution, the effect of the anion on the optical rotation may well stem from an interaction between the carbohydrate and undiseociated salt molecules. Ramaiah and Vishnu66 offered a different explanation for the anionic effect. They suggested that this difference between the anions is due to the difference in their ability to alter the refractive index of water. However, this hypothesis fails to explain why, for example, lead nitratew has no effect on the optical rotation of sucrose. The refractivity of an aqueous solution of lead nitrate is greater than that of an equimolar solution of sodium br0mide.a Salts of alkalineearth metals do not generally differ greatly from salts of alkali metals in their effect upon the specific rotation of carbohydrates. The variation in the rotation of sucmse,(~~6@ D-gZpm-D-gdo-heptose:6 Cr-D-gulose,’& and methyl D-gulopyranosides,” as a function of calcium chloride concentration in aqueous solution, has been mathematically expressed in empirical equations. Aqueous mixtures of sucrose with magneeium sulfate, calcium acetate, barium chloride, and lead nitrate, respectively, have been studied polarimetrically.’O Lead nitrate differs from the other salts in having no apparent effect upon the rotation of sucrose. However, this salt does affect the rotation of Dfructose. The equilibrium rotation of ~guloeeAl. and of D-gZycero-D-gulo-heptosedb v d e s considerably with the concentration of calcium chloride (see Fig. 4). On the other hand, that of pglucose is not greatly influenced. Frush and Isbell” have adequately shown that the influence of calcium chloride is predominantly caused by an adduct formation that leads to a marked shift in the equilibrium between the a- and p-D-pyranose modifications. The higher the concentration of calcium chloride, the greater is the proportion of the u-D modification. The presence of ethanol in the system affects the position of the equilibrium rotation in such a way aa to suggest (68) “International Critical Tablee,” MoGraw-Hi11 Book Co.,Inc., New York, 1930, (69)

VOl. VII. D. 63 ff * If,. F. Ja&son and C. Id. Gillis, Bur. Sbndarda Sci. Pamra. No. 376. 126 (1920)

ALKALI AND ALKALINE-EARTH METAL COMPLEXES

231

+ 10 0

8 2 -10 9

- 20 0

2

4

6

CaClp, g/IOO ml of

8

10

solution

FIG.4.-Effect of Calcium Chloride on the Equilibrium Rotation of ~glycerct~-guloI3eptose.Q (Conaentration of sugar ia 4 g./lOO ml. of solution.)

an increme in the proportion of the CX-Danomer. The mutarotation coefficients for pure D-gulose and wgtycero-wgulo-heptose in aqueous solution are in accord with those of the corresponding calcium chloride adducts. Interpretation of the rotational data on reducing sugars in salt solution must necessarily take into account the possibility that changes in specific rotation of the sugar can be caused, at least partially, by carbohydrat-lt interactions that have no effect upon the proportion of the CY-(D or L) modification. Solutions containing both a salt and a reducing sugar contain two classes of substance: (1) free, uncomplexed sugar consisting of CY and fl (D or L) anomera in equilibrium, and (2) complex4 sugar. The latter class is possibly composed both of charged and uncharged species, formed by the interaction of the sugar with free cations and with undissociated molecules of the salt, respectively. The optical rotation of the solution is, thus, equal to the s u m of the rotations of the different complex species plus the rotation of the free anomers of the sugar. The possibility that both the pyranose and the furanose structures contribute to the overall rotation introduces additional complications. Polarimetric studies have shown that dilution of a solution of a sugar and a salt leads to a lowering in the percentage of sugar in the complex form. For example,@.in a 0.03 M solutionof cu-D-gulose*CaCl2.H2O in water, the percentage of Dgulose in the complex form, estimated from rotatiQnal measurements, was reported to be 0.9%, whereas, at a concentration of 0.34 M ,the percentage indicated waR 15%. 8. Electrophoresis

Electrophoretic migration of carbohydrates in solutions of alkali metal salts or alkaline-earth metal salts demonstrates the ability of carbohydrates

232

J. A. BENDLEMAN, JR.

to unite with free ions; however, it cannot detect the union of carbohydrate molecules with undissociated molecules of salt. The rate of migration is a function of many variables, such as stability of the complex, the stoichiometry, the concentration of salt, the cationic radius, the favored coordination geometry of the cation, and the size, configuration, and confiorniation of the polyhydroxy compound. Obviously, the electrophoretic data done would not permit the determination of the relative complexing abilities of carbohydrates; however, they do permit qualitative information to be obtained concerning the relative complexing abilities of alkali and alkalineearth metal ions (see Section 11,6,p. 227). a. Aqueous Mdia.--Mill@ has used cellulose-paper electrophoresis to provide evidence for the existence, in dilute aqueous mlution, of complexes of polyhydroxy compounds with cations of alkali metals and alkalineearth metals. The relative effectiveness of the different cations in promoting migration toward the cathode has already been given in Section 11,6 (see p. 227). No evidence was found for complexing between the carbohydrate and the anions acetate, nitrate, and perchlorate; however, the sulfate ion appeared to have some complexing ability. Of all the polyhydroxy compounds studied in aqueous solution,"* cisinositol exhibits the greatest mobility. The epi- and allo-inositol and cis-quercitol also show considerable movement, but to a lower degree; other cyclitols are less mobile. Reducing sugars and alditols generally show very little or no movement in the presence of Mg*@and alkali metal ions; all move in the presence of Caa, Sr@, and Baa, but the rates are only moderate. Table I11 gives the relative mobilities of several polyhydroxy compounds in aqueous solutions of various metal ions.

Turn I11 Relative Mobilitles. of Polyhydmxy Compound8 in Aquaour S O ~ U ~ ~ofO X I B Metallic Ionra Compound

Bag

Md'

Na (s

K '

&Inoaitol 6pi-Inodtol GIditol Allitol ~-Talom

82 26

20 2

13

10 3

6 3

5

1 1

1s

0

1 1 2

1 2

1

4 Cstionio movements are given as peroentagsa of the anionio movement (about 10 cm.)of p-tnitropenienmlfonic acid on the mme strip, with 2,3,Btri-O-methyl-~-glucose MI the marker for Bero migration. The eleotrolyte was a 0.1 M solution of the metal acetate in 0.2 A4 Boetio acid. Electrophoreds wae performed for 1 hr. at a potential gradient of about 20 v./om. on Whatman No.4 paper under a uniform preseure of 0.4 atm., with oooling by tep water. Compounds were applied as 0.1 M aqueous solutione.

ALKALI AND ALKALINBEARTH METAL COMPLEXES

233

OH I

FIG.6.4-Inoaitol.

Mills6*attributed the outstanding complexing power of cis-inositol to the presence of three axial hydroxyl groups in a chair conformation (see Fig. 5 ) ; these groups are suitably oriented for close approach to a cation. Arrangements of three hydroxyl groups that are close enough together to be associated with a cation can be discerned in certain of the conformations of the other cyclitols that show catiohc migration. Charley and Saltmane studied the migration of radioactive Cate in the presence and absence of lactose in aqueous solution at pH 7.0. The solutions were buffered with sodium hydrogen carbonate. The inability of calcium to migrate in the presence of lactose indicated that Cat” had reacted with the sugar to form a soluble, uncharged complex.

b. Alcoholic Media.-Studies of the interaction of polyhydroxy compounds with alkali metal salts in alcoholic solutiona6have shown that electrophoretic migration is much faster in methanolic and ethanolic solution than in aqueous solution. This observation indicates that the stability of a complex is much greater in alcoholic than in aqueous systems. Even 2,3,4 ,6-tetra-0-methyl-D-glucose migrates at a small, but measurable, rate. Glass-fiber paper, instead of cellulose paper, was employed in the studies, in order to eliminate the possibility of errors that could arise from the formation of complexes between cellulose and metal ions. The effectiveness with which the solvent promotes migration decreases in the order: methanol > ethanol >> water. The extremely low stability of complexes in water can be explained by the relatively great tendency of metal ions to associate with water molecules.70The difference between the rate in ethanol and that in methanol can be attributed, at least partly, to the fact that salts we more highly dissociated into free ions in methanoP; a higher concentration of free cations would permit a higher concentration of positively charged carbohydrate species. Relative rates of carbohydrate migration are spread over a wider range of values in methanol than in ethanol: this can be attributed to a lower E. Martell and M. Calvin, “Chemistry of the Metal Chelate Compounds,” PrenticA3aI1, New York, 1962, p. 239. (71) N. A. I z d o v , E. I. Vd,and N. N. Salstnikov. Uch.Zap. Kb’kouak. Qos. Univ. Tr. Khim. Fak. i Nauchn.-Isaled. Znut. Khim., 71, No. 14, 29 (1956).

(70) A.

J. A. RENDLEMAN, JR.

234

stability of complexes in methuriolic media. Stability of chelates is known to bo greater in solvents of lower than in those of high The relative rates in an alcoholic medium are virtually independent of the nature of the anion (see Table IV) ; this constitutes a strong indication that free anions do not complex with carbohydrates to a n y significaiit extent. On the other hand, anions do play an important role in determining absolute TABLIC IV Rater of Eleatmphoreticr MigratJona of ~ - X y l ~ rand e D - G I U O Oin ~ Methanolic %duthnr of Varloua Electmly te#

Salt

M Temp., Migration "C.' rate,

Maib

mm./hr.

NaI NaBr NaCl NsOAo

Licl

NI4C1 CaCl, M s 1 ~ Ha0 6

0.03 0.16 0.16 0.03 0.16

0.30 0.30 0.16 0.16 0.16

38

-

-

0.61

38 34

7.2 3.8 6.7 4.7 0.1 3.0 14.0

0.57 0.68 0.66

0.8

0.2

34 38 43 37 39

44

4.0

-

0.60

0.06 0.67 0.56

Temp., Migration "CSb rate, mm./hr.

M~lb

-

-

-

47 38

10.1 8.2

0.64

-

0.66

34

6.6 4.3

-

-

0.64 0.40

39 44

10.2 1.7

0.40

38 -

-

-

0.41

a Zone electrophoresis WM on glass-fiber paper at a potentiel gradient of 16.7 v./in.; referenae (nonmigrating) oompouad waa ohrysene, whioh ie visible under ultraviolet lieht when dry, but not when wet; oompounds were applied aa alooholio solutions (0 .O& 0.08 M,solubility permitting); all carbohydrates migrated towerd the oathode; Mart. rate of migration relative to that of ~-ribose.'Maximum temperature to whioh the system rose.

-

rates of migration. In promoting migration, metal halides are more effective than the corresponding acetates. In methanol, magnesium acetate causes no migration, whereas magnesium chloride (hexahydrate) effects a memurable movement that is roughly comparable to that of ammonium chloride and lithium chloride. This differencein the ability of different salts (poseessing a common cation) to promote migration is possibly due to a difference in the degree of ionic dissociation, and not to complexing between the carbohydrate and the free anion. (72) A. Brtlndstr6m, Arkiu Kemi, 7, 81 (1964).

235

ALKALI APSD ALKALINE-EARTH METAL COMPLEXES

Table V contains a list of the absolute rates and relative rates of niigration for various carbohydrates in methanolic solutions of potassium acet'ate and sodium acetate. Table I V shows the effect of different salts on the absolute rates and relatlve rates of wxylose and wglucose in methanol. The ability TABLEV Rate8 of Electrophoretic Migrationa of Polyhydroxy Compounds in 0.3 M Solution8 of Potaadum Acetate and Sodium Acetate in Methanol= KOAc Compound

Temp.? Migration "C. rate, mm./hr.

D-Xylose

D-Lyxose D-Arabinose a-D-Glucose fructose Sucrose Maltose Ibffinose Meleaitoee 1,6-Anhydro-p-D-g1uc0pyranose Methyl a-D-glucopyranoside B momer Methyl a - ~ msnnopy ranoside D-Glucitol Erythritol Q

See Table IV, footnote a.

42 48 42 48

48 48 42 42 42 42

7.3 10 .a 4.6 6 .O 7.4 9.1 4.8 10.2 4.1 4.8

NaOAc MRib

1.oo 1.oo

0.60

37 38 38

-

-

6 .O

.oo

38 38 38 a7

0.58 0.73 0.90 0.64 1 0 -68 0.64

-

-

42 42 42

4.6 4.4 6.7

0.63 0.62 0.94

42 42

6.2 4 .O

0.69

-

-

Temp.? Migration "C. rate, mm./hr.

0.66

9.4 9.3 4.7

7.8 4.3 7.9

1.oo 1.oo

0.60

-

0.65 0.83

0.46 0.84

-

-

-

37 37 37

4.3 3.4 12.3

0.36 1.31

-

-

-

-

-

-

-

-

MRib

0.4

* Maximum temperatwe to which the system rose.

of a cation to promote migration in methanolic salt solution decreases in the order: Ca2e > Na" > Ke > NH4@> Lie, There is no clear correlation between the electrophoretic data available and the geometry of polyhydroxy compounds. The unusually high, relative rate of migration of 1,6anhydro-~-wglucopyrrtnosemay be due to the presence of the two axially oriented cis hydroxyl groups on G 2 and C-4 of a boat conformation. Such an orientation in a rigid molecule would be

236

J. A. RBINDLDMAN,

JR.

uxpoc:tcd to givo u uo,omplex of stability higher than average, aimilar to that of cia-inoeitol in aqueous media, Electrophoresis in nonaqueous media may be an effective means of separating polyhydroxy compounds from mixtures which would otherwise be difficult to resolve. Subsequent separation of salt from carbohydrate could then be agcon@ished by means of ion-exchange techniquee.

9. Structure of the Complex The true structure of a carbohydrate-salt adduct can be determined only from detailed x-ray diffraction studies, a few of which have been made. Such studies enabled Beevers and Cochrad2 to determine the complete structure of suprose-NaBr.2 HSO. Each Na@ion was found to have sixfold coordination, with almost regular octahedral symmetry. The Bre ion also has sixfold coordination, but the coordination group has no regular shape. Each Na@ion is close to one Bre ion, two water molecules, and three carbohydrate hydroxyl groups. There are only two direct intermolecular bonds between hydroxyl groups themselves; the remaining hydroxyl groups are linked through Na@and Bre ions and the water molecules. The hydroxyl groups of both the D-glucose sjld the D-fructose moiety participate in the bonding with oation and anion. The separation between Na@and B e in the complex (2.94 A.) is actually maller than that in pure, crystalline sodium bromide (2.98 A,). Senti and WitnauerSO made similar x-ray studies of potassium salt adducts of amylose. Adducts having a 2:l ratio of D-glucose residue to salt (iodide, bromide, formate, acetate, and bicarbonate) were found to have tetragonal lattices with fourfold screw symmetry; 1 : l adducts (acetate and propionate) were orthorhombic. The structure of the tetragonal adducts is determined, not by amylose-amylose contacts, but lsrgely by amylose-cation contacts. Amylose-anion contacts appear to be of minor importance, for the unit-cell dimensions are relatively insensitive to the size of the anion in an isomorphous series of potassium salt adducts. The elements of symmetry in these complexes indicate that the D-glucose residues in the amylose chain, or, at least, those chains in the cry-stalline portions (of the complex) that are responsible for the discrete diffraction patterns, are equivalent. The conformation of the &glucose residues was not determined. By means of three-dimensional, x-ray diffraction data, Hybl, Rundle, and W i l l i w solved the crystal and molecular structure of the potassium acetate adduct of cyclohexsamylose, a Schardinger dextrin. Cyclohexaamylose is a macro-ring consisting of six D-glucopyranose rings connected by a - ~(-1-4) -glucosidic linkages. Because there are six D-glucopyranose

ALKALI AND ALKALINHbIMRTH METAL COMPLEXEB

237

residuw in each turn of the helical structure of amylose, cyclohexaamylose is an ideal model compound on which to base a study of both the conformation of the D-glucopyranose rwiduea and the geometry of the CY-D( 1 4 ) -gluoosidic linkage in amylose. The analysis of the complex 2 cyclohexaamylose*3.08KOAc-19.4 HtO showed a translational stacking of the cyclohexaamylose molecules, to yield a cylindrical, carbohydrate canal structure. The a-n-glucose residues are all in the pyranose form, and this i s in,the C1 (D) cgnfonnation (la283e4e5e). Each molecule of cyclohexaamylose has six pocket positions along its surface that are occupied by water molecules and potaasium ions. The potassium ions are in distorted octahedral environments, outside the carbohydrate channels. Two of the three acetate ions in each unit cell are at highly anisotropic, disordered sites inside the cyclohexaamylose macro-rings. The third acetate ion has not yet been accounted for. There are intermolecular hydrogen bonds between the hydroxyl groups at C-2 and C 3 of each pair of contiguous P-glucose residues.

111. COMPLEXES FROM THE INTERACTION OF CAF~BOHYDRATES WITHMETALBASES The interaction of strong bases with polyhydroxy compounds, a1though extensively studied, has not yet been fully clarified. The available evidence indicates that the removal of a proton by a basic anion, to give an alcoholate (reaction 1 ), and the formation of an adduct (reaction 2 ) can both occur. In alcoholic media, reaction 1 has been definitely shown to occur. However, in aqueous media, differentiation between reactions 1 and S has not yet been possible. ROH + MB ROM + HB (11 ROH

+ MB

ROH*MB

0)

where M = a metal ion; and B = OH*, CNe, or an alkoxide ion. Alcoholates of polyhydroxy compounds will be included in the category of complexes, because of the probability that most, if not all, of them are stabilized by inner chelation of the metal ion with neighboring hydroxyl groups, similar to that illustrated in Fig. 6, Adducts, a h , should be stabilized by chelation.

FIQ.&-Chelate Structure of

the Sodium Alcohblste of a 1 ,ZDiol.

238

J. A. RENDLEMAN, JR.

There is considerable supporting evidence for the existence of undissociated inner chelates in aqueous solution. Potentiometric pH meaaurementsn and nuclear magnetic resonance studies74.76 of tiqueous solutions of a- and phydroxy carboxylic acid salta indicate an appreciable association between the alkali metal cation and the organic anion; alkaliie-earth metal ions associate even more strongly than do alkali metal ions. The stability of the complex increases a,s the radius of the cation decreases." All alkali metal cations form complexes with malate ion. The observation that the tetramethylammonium ion is much less strongly bound than an alkali metal ion is understandable, in view of the fact that quaternary ammonium ions are known to resist being solvated, even by water. The constants of formation of various metal kojates, including that of calcium, have been determined by potentiometric titration?' Possibly, the &membered, chelate ring of the metal kojate (see Fig. 7) contributes significantly to ita stability. Chsistepenn has suggested that chelation between alkali metals and

FIQ. 7.--Chehte Structure of Calcium Kojate.

pyridoxal plays a role in biologic4 transport; he studied both the alkali metals and the alkaline-earth metals. The formation of complexes of wgluconate ion with alkaline-earth metal ions has been studied." Supporting the concept of the existence of alkali metal hydroxide adducts is the isolation of highly crystalline, 1:1 molar adducts of potassium hydroxide with certain tertiary acetylenic carbinole and glycols.~~ However, these complexes cannot be strictly compared to alkali metal hydroxidecarbohydrate adducts, because of the probable involvement of the r shell of the carbon-carbon triple bond. Weizmann,@on the other hand, has reported the formation of potassium hydroxide complexes of acetala and of (73) L. E. Erickmn and J. A. Denbo, J . Phyu. C b m . , 67, 707 (1983). (74) 0. Jsrdetriky and J. E. We&, J . Am. Chem. Soc., 84, 318 (1880). (76) L. E. Erickson and R. A. Alberty, J . Phy8. Chem., 66, 1702 (1062). (76) B. E. Bryant and W. C. Fernelius, J . Am. Chem. Soc., 76, 6361 (1964). (77) H. N. ChristenMn, dlcimcc,ll,1087 (1066). (78) R. K. Canand A. Kibrick, J . Am. Chem. Soc., 60, 2314 (1038). (70) R. J. Tedeschi, M. F. Wilson,J. Scanlon, M. Pawlak, and V. Cunicella, J . &g. C h . ,48, 2480 (1963). (80)C. Weimmann, British Pat. 682,191 (1948); C h .AMroatb, 41, a630 (1047).


239

dialkyl ethers of glycols; this indicates that alkoxyl groups call act 89 electron donors, in the same capacity as hydroxyl and keto groups. However, the electrondonating ability of an alkoxyl group would undoubtedly be much less than that of a hydroxyl or keto group. Both the pH and the conductivity of an aqueous solution of sodium hydroxide are markedly decreased by the presence of polyhydroxy compounds. By assuming that the reactions are due entirely to the removal of one or more hydroxylic protons, several inveatigabrs have calculated ionization constants for a number of simple carbohydrates.a1-’J4A tabulation of most of these constants (as p&) is availab1e.w Hirsch and Schlage showed that the reactions are reversible, and that the extraordinarily high “acidity” of reducing sugars is not due to enolization of the aldehydo form. An aldehydo or keto group is not essential for high reactivity toward alkali metal hydroxide, as shown by a comparison of selected p& (18’) values for D-glucose (12.43), sucrose (-12.6) , and ~-glucitol (13.57). The lesults of calculations in which these values were used indicate that, for aqueous solutions that are 0.16 M in carbohydrate and 0.16 M in sodium hydroxide, the extents to which D - ~ ~ U C O Ssucrose, ~, and wglucitol react to give alcoholate and water (reaction 1 ) are, roughly, 70%, So%, and 20010, respectively. These percentages must, of course, be viewed with some skepticism, since their calculation was based on the assumption that the contribution of reaction 8 is negligible. Apparent ionization constants wheat starchJWand alginate.” have also been determined for In Fig. 8, tha number of hydroxide ions consumed per molecule of carbohydrate, in an aqueous solution that is 0.25 M in sodium hydroxide, has been plotted against the initial concentration of carbohydrste. The ease with which a single molecule of disaccharide can effect the disappearance of more than one hydroxide ion is readily apparent. Starch and dextrin are unable to consume more than 0.5 hydroxide ion per wglucose residue. Should the &values for oligosaccharidea be expressed as molecules of sodium hydroxide consumed per monosaccharide residue, it would at once be apparent that the difference between oligosaccharideand plysaccharide is not very large. Furthermore, on this basis, pure *glucose is more reactive than any of the oligosaccharides studied. Smoleiiski and Porejko8astudied the pH of aqueous solutions containing (81)L. Michaelie and P. Ilona, Biocham. Z., 4@,232 (1913). (82)J. Thamsen, Ada C b m . S m d . 6, 270 (1952).

(83)P. Hirech and R. Sohlags, 1.Phyeilc. Chern. (Leipzig), A, 141, 387 (1929). (84)P.Souchay and R. &had, BuU. Soc. Chim. Fronts, 819 (19M)). (85) B.Capon and W. G. Overend, Advan. Ca&oh&ate Chsm., 16,32 (1960). (80) 8.M. Neale, J . Teztile Inst., 20, 373T (1929). (87)8.P.Sari0 and R. K. Schofield, Proc. Roy. Soc. (London), Ssd.A, 186, 431 (1948). (88)K.Smolehki and 8. Porejko, Roczniki Chem., 16,281 (1936).

J. A. RIJNDLEMAN, JR.

240

8FIO.8.--Relation between Consumption of Sodium Hydroxide and Conoentration of Carbohydrrh in Aqueous Solution." (Concentration of polymcaharidea h a x p r e g e e d in mole of p.plucose &due per liter of solution. Initial conaentration of sodium hydroxide is 0.26 M.e Imole of sodium hydroxide oonsumed psr mole of carbohydrate. Carbohydratea: D-glucoee 0,D-fruotOrw 0, BUDIOBB X, leotoee 0,meltose A, staroh @, and dextrin A.)

both calcium hydroxide and sucro~e.Their reaults were similar to those obtained with sodium hydroxide and ~umom,in that sucrose behaves a~ a weak acid. Measurementsof the pH of alcoholic solutions containing alksli metal hydroxide and carbohydrate have not yet been reported. A pH meter is capable of giving reproducible e.m.f. values in alcohol or alcoholwater solvents,m but the pH numbers read from the inetrument ere subject to no simple, clear interpretation in term of chemical equilibrium. There isJ at this time, no universal scale of acidity for mlventa differing in w a t e ~ alcohol ratio. Studies of the heats of reaction of sucrose with sodium hydroxide, and of sucrose with barium hydtoxide, in aqueous solution have shown that neither reaction ie as simple aa that of an wid-base neutralismtion; the experimental reeults indicated the possible involvement of hydrogen bonding.88. After devoting considerable thought to the problem of the chemical structure of alkali cellulose, B l e a M I and Lositsksyaw have suggested (89) R.Gi. Bab, M.Pesbo, and R. A. Robiaeon, J . Phya. Chenr., 67, 1838 (1983). (SQa) E.Calvet, H.Thibon, and P. Leydet, Bull. Soc. Chim. Fmncs, 2187 (1%). (90)8. V, BleehinskiI and 8. F. Lositateya, Tr. I&. Khim. A W . Not& Kim. 88R. I , 73 (1961).

ALJCALI AND ALKALINE-EARTH METAL COMPLEXES

241

that alkali cellulose exists as a mixture of metal hydroxide adduct and metal alcoholate, the adduct preponderating. They hypothesized that the ratio of adduct to alcoholate vanes with’thesize of the metal cation; the stronger the ion-dipole bonding between a cation and a carbohydrate hydroxyl group, the greater should be the proportion of the adduct. However, there is no unequivocal evidence to confirm their hypothesis. The same attractive force between cation and hydroxyl group that would stabilize an adduct might just as readily stabilize an alcoholate. The metal cation of a carbohydrate alcoholate is, very probably, chelated to neighboring hydroxyl groups. 1. Reactions in Aqueous Media

In homogeneous, aqueous solution, alkali metal hydroxides react with carbohydrates to produce negatively charged carbohydrate species. Although the general feeling among chemists is that these species are free alcoholate anions, the possibility that they are composed, at least partially, of carbohydratehydroxide ion adducts cannot be dismissed. There is electrophoretic evidence that the S0,S ion is capable of weak bonding with polyhydroxy compounds.” If this is true, perhaps the hydroxide ion and certain other oxyanions are likewise capable of weak bonding. Detailed x-ray studies of crystalline lithium hydroxide monohydratee’ and of sodium hydroxide tetrahydratee*have shown that hydrogen bonds can form between water molecules and hydroxide ions. Because contact of a metal ion with a hydroxide ion can increase the dipole moment of a hydroxide ion, and enhance its ability to form hydrogen bonds with hydroxyl groups,g* it is conceivable that undissociated, metal hydroxide molecules should form hydrogen bonds with carbohydrate hydmxyl groups more readily than can free hydroxide ions. In either situation, bonding would be expected to be stronger, the greater the partial positive character of the hydrogen atom on the hydroxyl group of the carbohydrate. Evidence for the existence of OHe(ROH)s, where ROH is an alkyl alcohol, in solutions of alcohols in benzene and in nitrobenzene, has been obtained by Agarwal and Diamond.” Their studies, which involved the use of various quaternary ammonium hydroxides, indicated that the hydroxide ion is capable of associating with three alcohol molecules, regardless of whether it IR in the free form or nmciated with a cation. (91) R. Pepinsky, 2.Krist., la, 119 (1940). (92) G. Beurskena and G. A. Jeffrey, J . Chem. Phge., 41, 924 (1904). (Q3) A. F. Wells,“Structural Inorganic Chemistry,” University Press, Oxford, 1947, p.

360.

(94) B. R. Agarwal and R. M. Diamond, J . Phys. Chem., 67, 2785 (1963).

242

J. A. RBNDLEMAN, JR.

Makolking6studied the rate of exchange of l80between labeled water and alkali cellulose, and between labeled water and the trisodium alcoholate of cellulose, and concluded that merceriration proceeds by reaction 8 ; that is, alkali cellulose is an adduct. He b d his conchpion on the fact that theN is no measurable exchange between water and the alooholate, whereas there is measurable exchange between alkali cellulose and water. His conclusion, which is based upon difference of rate, is, however, not necessarily valid. The trisodium alcoholate may not possess a structure that is as accessible to water aa is that of alkali cellulose. The complex 2 sucrose-NaOH, prepared in aqueous alcoholic media, loses the elements of water at 110"under vacuum, to give the corresponding alcoho1ate.m Treatment of the alcoholate with glacial acetic acid permits B U C ~ Y to ) ~ be ~ recovered in 90% yield. Ahali metal complexes may be analyred for their metal content by simple acidimetric titration. Analysis for adduct (hydroxide) content is more involved, and entails the aseumption that there can be no water of hydration attached to an alcoholate anion. The methodm involves: first, dissolving the complex in anhydrous methanol, and then, treating the resulting solution with an appropriate anhydrous acid, such as tartaric acid. The acid servea to convert any hydroxide ion into water (reaction S),

which can then be quantitatively determined by titration with the Karl Fischer reagent. The amount of water thereby measured is assumed to equal the amount of hydroxide originally preaent in the adduct. By similar means, it has been shown@@. that, when alkali cellulose (prepared in an aqueous sodium hydroxide medium) is dried under vacuum at 6 5 O , the reaulting material consists of both alcoholate and hydroxide adduct. There is the poseibility that complexesieolated from aqueous (or aqueous alcoholic) media are not hydroxide adducts, but, instead, hydrated alcoholatea. The above adaptation of the Karl Fisoher analysis does not, unfortunately, distinguish between a hydroxide ion and a water molecule. Such a distinction could posaibly be made by detailed x-ray analysis; however, neither alcoholates nor adducts have, as yet, been obtained in a form suitable for such a study. Tablo VI contains a comprehensive list of known alkali metal hydroxide ndducts. Complexm prepared by the interaction of carbohydrates or naotiitcn of ctirhohydmtewwith nlkdi motd hydroxide in anh!/drrgusalcoholic (95) I. A. Mskolkin, Zh. Ob8hch. Khim. 12, 306 (1042). (96)J. A. Nendleinn, Jr., J . Org. C h . ,81, 1845 (1966). (!IOU) E.(ifligerand H.Noh, Helu. Chim. A&, 40, 660 (1987).

TABLE VI Adducta of Carbohydrates with Alkali Metal Hydroxides Carbohydrate &and)

Amylose

MOH

CsOH LiOH KOH

Molar r ~ t i o , ~ ligand-MOH

References

Solvent of solvation, molecules/cation

Solvent medium

3: 1 3:l 1:l 3:l

-

H&EtOH H&EtOH H&-EtOH H&EtOH

97 97

-

s

U

k

1:l

-

H&EtOH

96

cellobiose

KOH

1:l 1:2

-

H&EtOH HaEtOH

99 100

Celluld

CsOH

3: 1 1:l 2: 1 2: 1 3:2 4:3 3: 1 1:l 1:l 1:l 2: 1 2:l 3:l

-

Hto H&EtOH HeEtOH HZO

RbOH

NaOH

-

3 HZO HZO

-

3 HtO

f:2

-

4:3

-

HaEtOH H&EtOH HZO H@-EtOH H+EtOH H&EtOH H&EtOH

El20

HZO-EtOH HeEtOH HeEtOH

*

z

97

NaOH

-

c (

98

~Arabiiose

KOH

Tr

101 9% 102

102 101 102 102 101 103

103 104 104 101 103 104 104

*

ET ss

i ! b c1

0

5 Fi

L

h3

8

Tmuc VI (Continued) Adducts of Gubohyhtca w i t h Alkali Metal H y M d e s Carbohydrate

(ligand)

MOH

Molar mttio,ligand:MOH

Solvent of &ation,

Solvent medium

Refere-

molec&/~tion

D-GlUcoSe

Starch

SUCroSe

NaOH

1.S:l 1:l 1:1*2 2.0:l

KOH

(2-3) KOH

KOH NaOH

1:3 1:l

HJO-EtOH H&-EtQH

NaOH

1:1

H&-EtOH

KOH NaOH

1:1 1:l

H&-EtOH H&WH

106 106

NaOH

1:l 1:2

H&EtOH H&-EtOH

107 107

NaOH

1:1 1 :2 2:1

Hto HZO

Hso

108, 109 108,109

2:1 1:l 1:2 1:3

H&EtOH HeEtOH H&EtOH H&EtQH

96 110,111 110 110

LiOH KOH

CsOH KOH

no &O

-

0.2 &o 0.1 H a , 0.08 EtOH

H&-EtQH H&WH H&EtOH H&WH

96 99 96 96

H&EtOH

108,109

Q

-r

NaOH

1:l 1.7:l 1:1.8 1:2.5 1:5.1

0.3 HzO

-

no HtO 0.22 HsO, 0.04EtOH

HeEtOH HeEtOH HeEtOH H&EtOH HtO-EtOH

111 96 96 96

96

r

The Jigand in a polysaccharide adduct is the D-glucose residue. In other adducts, it is the entire carbohydrate molecule. b For adducts of cellulose with lithium hydroxide, 6ee the discussion in Section III, la (p. 250).

r

6

(9i)F.R. Senti and L. P. Witnauer, J. Am. Chern. Soc., 70, 1438 (1948). (98)W. J. Heddle and E. G. V. Percival, J. Chin. Soc., 1690 (1938). (99)E. G.V.Percival, J. C h .Soc., 1160 (1934). (100) E.G.V. Percival and G. G. Richie, J. Chem. Soc., 1765 (1936). (101) E. Heuser and R. Bartunek, CeUu2oseehemie, 6,19 (1925). (102) K. G.Ashar, J . Cltim. Phys., 48, 583 (1951). (103) I. Sakurada and S. Okamura, Kouoid-Z., 81, 199 (1937). (104) G.Champtier and J. Nhl, BuU. Soc. Chim. FMW, 930 (1949). (105) A. Herzfeld, Ann., aa0,206 (1883). (106) A. Bau, Z.Vet. Deoct. Zzccker-lnd., M,481 (1904). (107)K.Beythien and B. Tollens, Ann., 266, 195 (1889). (108) G.Champtier and 0.Yovanovitch, J. Chim. Phys., 48,587 (1951J. (109) 0. Yovanovitch, Compt. Rend..,292, 1833 (1951). (110)E.G.V. Percival, J. Chem. Soc., 648 (1935). (111) E. Soubeiran, Ann., 43, 223 (1842).

E

-.

U

>

E

E 3r

J. A. RENDLEMAN, Jll.

246

TABLEm1 Complexea of Carbohydrates with Alkaline-earth Metal Hydroxides Carbohydrate (ligand) Hydroxide

drabhose p.F~ctose D-G~UCOW

Lactose Maltose

Manninotrioso RaffiIlOee

Be Sr Ca BE Ca Ca Ba Ca Sr Blb Ba Ca

Stachyose

Sr Be

Sucrose

Ba

Sr

C€+

Sr Q

,P-Trehalose

D-XJ’hi?

CR

BE Sr

Molar ratio, ligandiaation

2:1 2:1 1:l 2:1 1.1

1:l 1:1 1:l 1:1

1:l 1:l 1:2 1:l 1:3 1:2 2:3 1 :2 1:6

1:1 1:l 3:l 1:1 1:l 1:2 1:3 1:l 1:2 2:3 2:1 2: 1

Solvent

References

medium HeEtOH HgO-EtOH HlO HZO-EtOH HQ-EtOH HsO Ha-EtOH Ha-EtOH Ha-EtOK HaO-EtOH H&EtOH HiO-EtOH HsO-EtOH HlO HiO-EtOH HaEtOH Hi0 HlO HiO-EtOH

HlO HlO Ha0 HgO-EtOH H&-EtOE HlO Hi0 HqO HsO-EtOH H&EtOH H&EtOH

(112) E. Poligot, Compt. Rand.,90, 163 (1880). (113) H.Wintw, Ann., 144, 296 (1888). (114) H. Will, Arch. Phrni., 816, 812 (1887). (116) C.Tanret, Bull. Soc. Chim. Frame, 87, 947 (1802). (118) L.Lindet, J . Fabr. S w e , 81, 19 (1880). (117) G. Tanret, Compt. Rend.,166, 1620 (1912). (118)P. Horsin-DOon, Bull. doc. Chim. Frunce, 17, 166 (1872). (119) I. Schukow, 2. Ver. Dad. ZuckerZnd., 50,818 (1800).

4

4 69,112,113 114 59 69 105 59, 105 105 115 107 107 116 107 107 115 117 117 59 19 19 69 69 59, 118 69 19,59 19,69 119 4 4


247

rneclia*'WJ'O are not listed, b e c a w of the probability that they are, preponderantly ,alcoholates. With the exception of magnesium hydroxide, alkaline-earth metal hydroxides are similar to alkali metal hydroxides in that both types are strongly basic and highly dissociated in aqueous solution. Interaction of an alkaline-earth metal hydroxide or oxide with a carbohydrate results in an increased solubility of the hydroxide or oxide, apparently through the formation of either an alcoholate, a carbohydratemetal hydroxide adduct, or a carbohydrate-metal oxide adduct. Mackenaie and Quin"' have suggested that, in the compounds of reducing sugars with calcium hydroxide, the calcium is united with the hydroxyl group at C-1, possibly in the form 0

/ \

-CH

Ca

or

\ / 0

\

HC-O-Ca-OH,

/

and that, in the compounds of nonreducing sugars, the calcium is present simply aa calcium hydroxide bound to the carbohydrate in a manner similar to that of a salt in a salt-carbohydrate adduct. Because there has been little experimental work with either alkaline-earth metal hydroxides or oxides since that of Mackenzie and Quin:Q any attempt to draw conclusions concerning the composition and structure of the complexes must await further experimentation. For convenience, therefore, all complexes formed from hydroxides or oxides will be called adducts of alkaline-earth metal hydroxides, and they are listed aa such in Table VII. Water of hydration is omitted from Table VII for several reasons. (1) Many investigators did not consider water of hydration in determining the formula for a complex. (2) In all instances where water was determined, it was determined indirectly. The difference between the weight of a complex before and after the complex had been subjected to dehydrating conditions (for example, under vacuum at 100') was often assumed to equal the weight of water of hydration. (3) Dehydration of a complex could involve either the removal of water of hydration or the removal of water formed by chemical reaction, or both. And (4) , there is considerable question concerning the chemical structure of the complex. There has been no reported isolation of a magnesium hydroxide complex. Attempts by BenedikP to prepare such a complex of sucrose were unsuccessful. A barium hydroxide adduct of amylose was prepared by Senti and WitnaueP7 by means of an exchange of barium hydroxide for potassium hydroxide in a potassium hydroxide-amylose adduct; however, the exact (120) R.Benedikt, Ber., 6, 413 (1873).

248

J. A. RENDLEMAN, JH.

composition of the adduct was not reported. Dextran, also, reacts with hydroxides of calcium, strontium, and barium to give complexes; these complexes have pravidg a method of fractionating dextran for molecular size,'21 the process being based upon the fact that fractional acidification of an aqueous suspension of the complex results first in the dissolution of the component having the highest molecular weight. Dialysis studies'" of d u l o s e and dextran in aqueous solutions of barium hydroxide, sodium hydroxide, and "cadoxene" (cadmium hydroxide in ethylenediamine) showed no difference in complexing ability between the two polysaccharides. Furthermore, in solutions having equal base normality, Ba2@,Nae, qnd Cd(e$hylenedismine)P had loughly equal complexing abilities. a. Stoichiometry of Alkali-Metal Hydroxide Reactions.-The combining ratio for alkali metal hydroxide adducts is variable, as with that for alkali metal salt adducts. The ratio is dependent on the concentration both of hydroxide ion and carbohydrate, and on the size both of alkali metal ion and carbohydrate. At low concentrations (’*18The wall comprises 10-40~~ of the dry weight of the intact organism, and may vary in thickneas from 100 A. to a,s much a8 800 A. Examination of bacteria by electron microsoopy clearly reveals the prwnce of such walls, which can usually be differentiated from such surfaoe appendages as flagella, from the capsular and other extracellular substances which coat many bacteria, and from the underlying protoplasm with its surrounding membrane. The production of capsular material is subject to genetic and environmental control, and it is often possible to remove capsules from encapsulated organisms without affecting the morphological integrity and viability of the cell.l+*1 Dissolution of the wall of certain organism may be effected with muramidase (formerly known aa lysozyme)e.B; this results in the production of fragile, spherical protoplasts which are readily ruptured by osmotic pressure. The shape and integrity of the cell is thus principally due to the cell wall. The component common to all bacterial cell-walls, and primarily responsible for their strength and rigidity, is a glycosaminopeptide (mucgpeptide) composed of 2-acetamido-2deoxy-~-glucose,Nacetylmuramic acid18~’8~24~26 [Zacetamido-%deoxy- (3-O-lactoy1-~-glucose)l, and four or five amino acids (typically, alanine, glutamic acid, glycine, and either lysine or 2 ,6diaminoheptanedioic acid), some of which have the D configuration. The other components usually found in cell walls are proteins (in small proportion), polysaccharides, and teichoic acids. Complete removal of these soluble components can often be achieved without disintegration of the wall, and their function is, therefore, presumably not structural.I6 The protoplast membrane differs markedly in properties and composition from the wall, and is characterized by a high content of lipid and by the virtual absencflv” of the characteristic components of wall glycosamino(17) I. C. Gunsalus and R. Y. Stanier, “The Bacteria,” Academic Press Inc., New York, N.Y.,1960, Vol. 1.

(18) M. R. J. Salton, “The Bacterial Cell Wall,” Elsevier Publishing Co., Amsterdam, 1984. (19) 0. T. Avery and R. Dubos, J . EzpU. Med., 64, 73 (1931). (20) M.Tod, Med. J . Osaka Unw.,6,726 (1955). (21) M. H. Adams and H. R. Park, Vitology, 2,719 (1956). (22) C. Weibull, Ann. Rev. MicrobioZ., 12, 1 (1968). (23) M. R. J. Salton, Bacterial. h., 21,82 (1967). (24) H. J. Rogers, in “Menibranes and Surfaces of Cells,” Biochem. SOC. Symp. (Cnmbridge, Engl.), 24, 55 (1962). (26) H. J. Rogers, in “Function and Structure in Micrc-organisms,” Symp. SOC.Gen. Mirrobiol., 16, 186 (1965). (26) C. Wiebull and L. Bergstrom, Biochim. Biophys. Ada, 80,340 (1968). (27) G. D. Shockman, J. J. Kolb, B. Bakay, M. J. Conover, and G. Toenniea, J . B p e f d l . , 86, 168 (1963).

A. R. ARCHIBALD AND J. BADDILEY

326

peptide. The teichoic acids formerly described w “intracellular” appear to be closely sssociated with the membrane,’ as are at least some of the enzymes concerned with the biosynthd of both “intracellular” and wall teichoic w i d s . % ~ ~ * ~ ~ 111. DISCOVERY OF THE TEICHOIC ACIDS The application of electron microscopy to the study of fractionation of disrupted bacteria, and the subsequent isolation of homogeneous preparations of wall fractions,a have led to an increasing interest in their chemistry. This interest has been further stimulated by the recognition of the metabolic importance of the wall, and the discovery that the lethal action of such antibiotic substances as the penicillins is principally due to inhibition of wall synthesis (compare Ref. 33). The discovery of teichoic acid aa a major component of the walls of several bacteria was not a direct consequence of thia interest in cell w d s , but followed from the discovery of two new nucleotides, first detected in extracts of LactobadUue arabinoeueJ- but now known to be widely distributed. By degradative studies and by chemical synthesis these were shown to be Pl-cytidine b(P-D-glycerol l-pyrophosphate) (1)*“ and Pl-cytidine Ci-(P-cribitol l-pyrophosphate) (2).@,@*&The con!iguration@b of the glycerol phosphate residue in Phytidine 5-(P%-glycerol l-pyre phosphate) is that of the naturally occurring wglycerol l-phosphate (“cglycerol3-phosphate”),and it seemed likely that this nucleotide would (28) L. Gleser and M. Burger, J . BW. C h . , 989,3187 (1964). (29) M. Burger, Biochim. Biophga. A&, 71,496 (1963). (30) S. G. Nathenson and J. L. Stmminger, J . Biol. C h . , 487, ~63839(1962). (31) S. G. Netheneon and J. L. Strominger, J . Bid. C k . ,oS8, 3161 (1903). (32) M. R. J. Salton and R. W. Home, Biyhim. Biophys. Ada, 7,177 (1951). (33) E.P.Abraham, Endeauour, 18,212 (1969). (34) J. Baddiley end A. P. Mathiaa, J . Chbm. &., 2723 (1964). (36) J. Baddiley, J. 0.Buchanan, B, Cam, A. P. Mathias, and A. R. Sanderson, Biochem. J., 64,699 (1956). (36) J. Baddiley, h. G . Buchanen, A. P. Math-, end A. R. Sanderaon, J . C h . Soc., 4188 (1966). (37) J. Baddiley, J. G. Buchanan, and B. CWM, J . C h . Isloc., 1869 (1967). (38) J. Baddiley, J. G. Buchanan, and A. R. Senderaon, J . C h .Soc., 3107 (1968). (39) J. Baddiley, J. G. Buohanan, B. Cam, and A. P. Ma&, J . C h . Soc., 4683 (1966). (40) J. Baddiley, J. G . Buchanan, and C. P. Fawcett, J . C h . Soc., 2192 (1969). (404 6ee 6.F. Neufeld and W. 2.Hamid, Aduan. CarbolLyh Chum., 18,309 (1983).

(40b) In the original publications on teichoio wide, the numbering of dditob doee not conform to the Rules. The names and formulaa used in the present article are in conformity with the Rules of Cwbohydrate Nomenclature, J . Org. Chem., 98, 281 (1963).

THE TEICHOIC ACIDS

327

be involved in the biosynthesis of known glycerol phosphate derivatives.

No ribitol phosphate was, however, known in Nature, and it was considered that (2) might function either in transformations or in transfer processes leading to the formation of polymeric substances containing ribitol or ribitol phosphate residues. Thus, the cytidine derivatives would be analogous to nucleotides containing either 2-acet.amido-2-deoxy-~-glucoseor N-acetylmuramic acid peptides, which are recognized intermediates in the biosynthesis of the glycosaminopeptide component of bacterial walls.

The correctness of this reasoning has been established by the isolation of polymers cantaining residues of ribitol phosphate or glycerol phosp h a t e t h e teichoic acids-and by the direct demonstration of the participation of the nucleotides in their biosynthesis in cell-free systems. The existence of the polymers was demonstrated by addition of ethanol to trichloroacetic acid extracts of whole cells of Lactobacillus arabinow, whereupon a polymeric material was precipitated which contained derivatives both of glycerol phosphate and of ribitol phosphate." Examination of the isolated walls of Lactobacillus arabinosus and of Bacillus subtilis showed that these contain substantial proportions of phosphorus, which is present as a ribitol phosphate polymer'$; D-glucose and alanine are also compoThe alanine has the D configuration, and is nents of the new sub~tances.~~ attached to the polyribitol plioflphnte through unusually labile, ester linkagc~Similar cmmpound~wcre dctected in the walls of other bacteria, (41) J. Birddilcy, J. C.Buahanan, and G. R. Greenberg, Biochem. J., 66, 5 1 (1957). ~ (42) J. h d d i l ( y , J. (;. Ruchnntm, and B. Cam, Biochim. Biophys A c b , 27,220 (1958). (43) J. J. AriiraLroiig, J. Bnddiley, J. G. BuoBnnnn, and B. Cars, Nature, 181, 1092 (1968).

328

A. R. ARCHIBALD AND J. BADDILUY

and such compounds were called teichoic acids.U Most teichoic acids conform to the general structure (3), Jthough variations on this structure have been noted. The glycerol phosphate polymere isolated from whole cells also contain sugar residues and walanine residues, and are related in structure to the polymere in the cell walls. Although the “intracellular” polymers are now believed to be associated with the cell membrane, they, too, are cdled teichoic acids.

p~

sugar I

HO-P-0-alditol-0 I1 I 0 D-alanine

fs. 87

P-0-aalditol-0 0 D-alanhe

j-~

sugar I P-0-alditol I .o D-alanine

(3)

It is apparent from the literature that teichoic acids had been observed on earlier oacaaions, although their nature had not been elucidated. Thus, in 1935, Julianelle and WieghmP isolated a carbohydrate antigen from staphylocooci which they called plysaccharide A; this has now been shown to be identical in serological properties with the teichoic acid extracted from the walls of SlCsphy2ococcus aureu8.18Similarly, in 1951, Mitchell and Moyle47e@detected a phosphate in the walls and intracellular fractions of Staphylococcus aureus which appeared to be mainly a derivative of glycerol phosphate: it now seems likely that this was a mixture of glycerol teichoic acid and ribitol teichoic acid. McCarty’D haa independently isolated an antigenic material from several bacteria, and shown that it is a polymer of glycerol phoephate. No sugar or D-alanine residues were present, but it is likely that the conditions used during extraction would remove D danine residues, and might remove sugar residues if only small proportions were present originally. IV. THIIHYDROIAXIIS OF EWERS OF PHOSPHORIC ACID The elucidation of the detailed structure of the teichoic acids has been greatly facilitated by advances in the chemistry of phosphate eatersadvanceg which have been stimulated by work on nucleic acids and phospholipids. The development of greatest significance to studies on the (44)J. J. Armstrong, J. Baddiley, J. Q. Buohanan, B. Cam, and Q. R. Oreenberg, J . Chcm. SOC.,4344 (1958). (45) L. A. Jdianelle and C. W. Wieghard, J . EzpU. Med., 6 2 , l l (1935).

(46)G . Hauksnes, D. C. Ellwood,J. Baddiley, and P. Oediig, Biochim. Biophvu. Ada, MI, 425 (1961). (47) P. Mitcheil and J. Moyle, J . Urn. Microbiot., 6,986 (1961). (48) P. Mitchell and J. Moyle, J . Urn. Miwobiol., 6,981 (1951). (49) M. McCarty, J . Exptl. Med., 109,381 (lQ59).

THE TEICHOIC ACIDS

329

structure of naturally occurring esters of phosphoric acid has been the elucidation of the manner in which they undergo hydrolytic cleavage (compare Refs. 50 and 51). Information obtained from the study of model compounds has been applied with great succeas to structural work on naturally occurring phosphates, and a brief summary of the relevant aspects is given here. Simple phosphomonoesters are stable to alkali, but are readily hydrolyzed at pH 4, when the species undergoing hydrolysis is the mono-anion. In more midic solution, the rate decreases, but, at pH values below about 0.5, the rate again increases due to hydrolysis of the protonated phosphate group. In molecules having a hydroxyl group adjacent to the phosphate group, the pattern of hydrolysis is similar; but at low pH values the phosphate grmp can migrate reversibly to the neighboring hydroxyl group, so that, for example, on heating D-glycerol l-phosphate in acid solution, a racemic mixture of 1- and %phosphates is formed." Simple phosphodiesters (such as dimethyl phosphate) are stable under alkaline conditions and a t pH 4, but are hydrolyzed a t low pH values. The presence of a hydroxyl group adjacent to the phosphate group markedly increases the eaae of hydrolyais of phosphodiesters, both in alkali and in acid, the hydroxyl group participating, in each case to effect hydrolysis through an intermediate, five-membered, cyclic phosphate which is readily hydrolyzed to a mixture of isomeric monoesters." Thus, Brown and Todd and their collaborators (see Ref. 50) found that acid- or alkali-catalyzed hydrolysis of alkyl esters of 2- and 3-monoribonucleotides follows the path outlined in Fig. 1. Examination of the products of incomplete hydrolysis of cytidine 3-(benzyl hydrogen phosphate) (4, R = CHGHb) has shown significant difference between the two pathways. Under alkaline conditions, no migration of the alkyl phosphate group in unhydrolyzed material occurs, whereas, in acid, significant migration, (4)+(8), is found. These results suggest that the step (4)+(5) in alkali involves an unsymmetrical transitionstate which may be represented aa (11) and, in acid, involves the formap tion of protonated species derived from intermediates of types (9) and (101, which can then undergo P-O fission to give either ( 6 ) or (7). Triesters of phosphoric acid are readily hydrolyzed under mildly acidic or alkaline conditions. (50) C. A. Vernon, C h . Soc. (London) Spec. Publ., 8,17 (1957). (51) D. M. Brown, Advan. Org. Chem., 8, 75 (y962). (52) P. W. C.Barnard, C. A. Burton, D. R. Llewellyn,K. G . Oldham, B. L. Silver, and C. A. Vernon, Chem. Znd. (London), 760 (1955). (53) D. Y. Brown and A. R. Todd, J . C h . Soc., 52 (1952). (54) D. M. Brown, D. I. Magrath, A. M. Niehn, and A. R. Todd, Nature, 177, 1124 (1956).

330

A.

n. ARCHIBALD

(a)

(8 )

FIQ.1-Participation Phosphodieetsr.

AND J. BADDILEY

(11)

(10)

of an Adjaaent Hydroxyl Group in the Hydrolysis of a

During structural studies on (2), it waa found that inorganic phosphate is readily produced when the nucelotide is heated at 100' in N hydrochloric acid. Synthetic L-ribitol 1-phosphate behaves similarly, whereas glycerol phosphates and wmannitol 1-phosphatebehave normally, and do not give signifiaant amounts of inorganic phosphate under these conditions. A study of the hydrolysis of L-ribitol 1-phosphate (wribitol bphwphate)n*M showed that, in addition to the expected racemisation due to the reversible migration of the phosphate group, large proportions both of inorganic phosphate and 1,$-anhydroribit01 (13) are produced. The mechanism suggested includes protonation of the ester oxygen atom followed by the electronic displacements shown in Fig. 2; the hydroxyl group at C 4 of the D-ribitol ester (12) is, presumably, in a sterically favorable position for the intramolecular,

HOGC

+ HSPO, HO

OH (12)

(13)

FIO. 2.-Formation of Anhydroribitol from Ribitol &phosphate. (66) J. Baddiley, J. G. Buchanan, and B. Car=, J . C h m . Soc., 4058 (1957).

331

THE TEICHOIC ACID8

nucloophilia whtitutiori. liibitol is converted into 1,Panhydroribitol in the presence of dilute acid, presumably by a mechanism which is similar but in which a primary hydroxyl group is protonated; however, this conversion occcrs more slowly than for ribitol phosphate. Traces of an optically active anhydroribitol and its phosphates are proNo anhydroduced when some teichoic acids are hydrolyzed with alkaJi.w*67 ribitol is formed by similar treatment of ribitol, its 1-, 2-, or 3-phosphates, or ribitol 1,9-diphosphate." However, small proportions of anhydroribitol and its phosphates are produced by the action of alkali on a synthetic poly(ribito1 phosphate) prepared by the action of diphenyl phosphorochloridate oil 3,4-O-isopropylideneribitol1-phosphate and 2-pho~phate.~ This observation suggests that 1,Canhydroribitol (13) or its derivatives (IS) are produced by fission of a phosphodiester, for example (14), in the manner indicated in Fig. 3, and that this reaction occurs together

ROH,C

/0 0

vG OH

HO

(14)

00

I H,~O-POR~ II 0

-

+ R'OPOS& OH

HO (15)

FIQ.3.-Formation of Anhydroribitol from a Ribitol Phosphodiester.

with the normal, cyclic phosphate sequence. Hydrolysis of certain phosphodiesters by C-0 fission, resulting from nucleophilic attack by a vicinal hydroxyl group, has been described by Brown and UsherJS0and the intermediate formation of a 3-membered oxide ring haa been demonstrated. However, in the case of a ribitol 1,5-di(phosphodiester), in which the linkages are between positions 1 and 5 of adjacent ribitol residues, it is likely that hydroxyl groups at C-4 of the ribitol are suitably oriented for direct attack on C-1, thereby eliminating phosphate and yielding 1,4anhydroribitol. Supporting this conclusion is the observation6O that a &membered epoxide is probably not an intermediate in the formation of 1,4anhydroerythritol when 1-0-p-tolylsulfonyl-DerythritoI is treated with alkali. (66)J. J. Armstrong, J. Baddiley, and J. G. Buchanan, Biochsm. J., 80, 264 (1961). (67) A. R. Amhibald, J. Baddiley, and J. G. Buchanan, Bioehem. J., 81,124 (1961). (58) D. A. Applegarth, J. (3. Buchanan, and J. Beddiley, J . Chem. Soc., 1213 (1966). (69) D. M. Brown and D. A. Usher, Proc. Chem. Soc., 309 (1963). (80)F. C. Hartman and R. Barker, J . Org. Cham., Y, 1004 (1983).

332


The proportioq of 1,4-anhydroribitol formed by treatment of teichoic acids and synthetic poly(ribito1 phosphate) with alkali is small, and the major hydrolytic pathway involves the cyclic phosphate sequence. No 1,4anhydroribitol glycosides have been observed in the alkaline hydrolyzates of teichoic acids; possibly, tho prwnco of a glycosyl substituent makos the reaction stericttlly l a s favorablethan when such substituerits are absent.

V. MEMBRANE TEICHOIC ACIDS As the compounds described hitherto as “intracellular” teichoic acids are now known to occur, at least in those cases studied so far, in the region between the wall and membrane, and are probably attached to the membrane, the term “intracellular” is correct but misleading. It is now proposed to call these compounds membrane teichoic acids. All of those so far examined are polymers of glycerol phosphate in which glycerol residues are joined together through phosphodiester groups at the 1-and 3-positions. The %-positionsof the glycerol residua bear glycosyl or D-alanyl substituents, aa shown in structure (16). Membrane teichoic acids have been prepared by extraction of whole cells with trichloroacetic acida1 or by similar extraction of the cell contents (which contain fragments of membranes) obtained by the removal of walls from suspensions of disrupted bacteria.62-66 In addition to the teichoic acid, such extracts contain large proportions of polysaccharides and polynucleotides; small proportions of ribitol teichoic acid are frequently preaent in acid extracts of cell contents of organisms which have a ribitol teichoic acid in the wall. The significance of the presence of some (ribitol) wall teichoic acid in the cell contents fraction is at present uncertain, but its presence may be a consequence of release during the disintegration of the cells. The particulate fraction obtained by high-speed centrifugation of the cell contents (twp. 366) contains most of the membrane teichoic acid, and extracts of this fraction contain very little of the wall teichoic acid. HO

-

-Lo-,-% II A

CH,- o ~ ~ c l - w - ~ , c ~ - o f ~ o - Hf C &WE a

(16)

where R ~-0lYlinsor glycoryl

(61) (62) (63) (64) (66)

M. V. Kelemen and J. Baddiley, Biochsm. J., 80,246 (1961). P. Critchley, A. R. Archibald, and J. Baddiley, Biochar. J., 85,420 (1962). A. J. Wicken and J. Baddiley, Biochern. J., 87,64 (1883). A. J. Wicken, 8. D. Elliott, and J. Baddiley, J . h. Microbhl., 81,231 (1963). U. L. RRjBhandsry and J. Baddiley, Biochar. J., 87,429 (1965).

THE TEICHOIC ACIDS

333

Essentially pure saniples of teichoic acid have been obtained from the several types of extract by procedures involving fractional precipitation, gel filtration, and ion-exchange chromatography. Such preparations contain equal proportions of glycerol and phosphate, a somewhat smaller proportion of D-ulanine, and a variable proportion of sugar. The alanine from several teichoic acids has been isolated as its hydrochloride and shown by the positive action of Damino acid oxidase to have the D configuration. The alanine residues are attached to the rest of the chain through ester linkageswhich are readily hydrolyzed by ammonia or dilute hydroxylamine, to give alanine and its amide or hydroxamate. Kinetic studies of the reaction with neutral hydroxylamine have shown that the alanine ester groups in the membrane teichoic acids from L. c u a e i S 1 and L. arabinosuSa2exhibit the characteristic, high reactivity of those in the ribitol teichoic acids.66 These ester groups are much more labile than alanine methyl ester toward hydroxylamine. The remarkable lability of the alanine ester residues is characteriRtic of most of the teichoic acids studied, and is comparable to that of the amino acid ester residues in the aminoacyl nucleic acids which are concerned in protein biosynthesis.B7The high reactivity of the nucleic acid derivatives was originally attributed to the presence of a hydroxyl group on the carbon atom adjacent to that carrying the ester linkage.*M The lability of the alanine in the ribitol teichoic acids could also be explained on this basis, but the comparable lability in the glycerol teichoic acids clearly could not be, since in this caae there is no adjacent hydroxyl group. It appears from this conclusion that a neighboring phosphate group might also confer high reactivity on the ester linkages. Accordingly, ~~-2-(alanyloxy) ethanol and its dihydrogen phosphate were prepared and their rates of reaction with neutral hydroxylamine were compared with that of Dkalanine methyl ester." Both of the synthetic model compounds were hydrolyzed at a rate which was similar to that for the teichoic acids and much greater than those for simple, amino acid esters. Activation of the ester linkage may therefore be effected either by vicinal hydroxyl or phosphate groups. Either or both of such groups may be responsible for the lability of the alanine in the ribitol teichoic acids, whereas in the glycerol polymers, the phosphate ester groups are sufficient to cause the observed reactivity. It is of interest that the alanine ester groups in the membrane teichoic (68)J. Baddiley and F. C. Neuhaus, Biochem. J., IS, 579 (1960). (67) P. Berg and E. J. Ofengand, Proc. Natl. Acad. Sn'. U.S., 44, 78 (1958). (68) T. Wieland, J. Merz, and G . Pfieiderer, C h a . Ber., 98, 1816 (1960). (09) H. G. Zachau, Chem. Ber., 98,1822 (1960). (70) H. G. Zachau and W. Ksrau, C h m . Bet., 98,1830 (1960). (71) 2.A. Shabarova, N. A. Hughes, and J. Baddiley, Bioehem. J., 88,216 (1962).

334


acids of group D streptococci are appreciably more stable toward alkali than are those of the other teichoic acids examined.a The alanine in the streptococcal compounds is attached to D-glucose reaidues, and c o w quently its stability may be ascribed to the absence either of phosphate or hydroxyl groups in sterically suitable proximity. Membrane teichoic acids have now been detected in a large number of bacteria, including almost all of the Gram-positive organisms examined. Neverthelesa, the proportion present is sometimes small, and separation of the acids from other macromoleculq cell-components, such as nucleic acids, peptides, and polysaccharides, is difEcult. Consequently, few have been obtaiaed in an amount and of a purity adequate for detailed chemical study. Even from the limited studies so far made, it is clear that structural details differ considerably from case to case, and it is convenient to classify these teichoic acids according to the organisms from which they have been isolated. 1. Lactobacillus arablnosus 17-5s" The glycerol teichoic acid obtained by fractionation of cold dilute trichloroacetic acid extracts of the "cell contents" fraction of L.aTabinosua contains 24% of a ribitol teichoic acid, presumably that which is present in the wall of this organism. Ion-exchange chromatography of the mixture, after removal of the alanine ester groups with dilute ammonia, gave a pure preparation of glycerol teichoic acid; it contained glycerol phosphate and glucose in the molar ratio of 1:0.11. Acid hydrolysis gave glycerol (characterized as its tribemoate) , D-glucose (characterbed with &glucose oxidase), glycerol mono- and di-phosphates, and a trace of inorganic phosphate. The formation of these products indicates that the polymer is composed of a chain of glycerol phosphate residues in which glycerol residues are attached to each other through a phosphate group. Acid hydrolysis can occur at either side of the phosphodieater linkages, and will thus give glycerol and its diphosphlltesin addition to the monophosphates. This is illubtrated for an unsubstituted polymer (17) in Fig. 4. The presence of phosphodiester groups was confirmed by potentiometric titration. On treatment with hot alkali, most of the phosphodiester groups were

~.o--f.~-o-i"i-Y-fc~~o{E"

.*....-

-0-EI&

{

-c&-o-

1

O l g C ~ ~

monopbo8phater

I

glycerol

(17)

-......

i

I

I

p,

I

glgcerol dipborph.tr.

FIQ.4.-Hydrolysis of a 1,%LinkedPolymer of Glycerol Phoaphab by Acid.

335

THE TEICHOIC ACIDS

H&H

1

HO,

,o-CH

CH20ap*0 \

A

I C&O
+" It was biologicdly active. Two major, metabolio conjugates were detected, the second apparently being the aspartate. A second glucose ester, possibly the 6-esterJ was not split by the enzyme, and there was also evidence for the presence (SO) C. A. Carlier and C. Van Horne, Nature, 901, 677 (1964). (81) K.J. Scott, J. Daly, and H. H. Smith, Plant PhysioZ., 89, 709 (1964). (82) H. D. Kllmbt, Pkanta, 66, 618 (1981); 67, 339, 391 (1961). (83) M. H. Zenk, Nature, lftl, 493 (1961). (84)M. H. Zenk, Plan&, 66,688 (1902).

PLANT-GHOWTH SUBSTASCES

391

of the &wglucoside of 2-hydroxyindole-3-aceticacid, which was biologically inactive.*W Animals excrete the indole auxin as the l-ter of BDglucuronic acid.” Zenk has suggestedeethat the natural growth-regulator indole3-acetic acid forms stable compounds with =glucose or Gaspartic acid, competitively, depending on the plant species. The glucose conjugate was believed to be the more primitive, as it appears in all plants, including bacteria and fungi. Aspartate competition occurs in higher plants only: 33 of 38 monocotyledonous and 72 of 75 dicotyledonous species form the aspartate, compared to 25 and 48 which form the glucoae ester, respectively. The glucose ester is labile to ammonium ion, ethanol, or amino acids, but Zenk has presented evidence that the glucoside is not an intermediate in aspartate formation. The glucoside is formed rapidly, reaching a maximum in 4-8 hours after the addition of label@ auxin, followed by slow hydrolysis, whereas the formation of aspartate occurs very slowly, but continually, after 6-8 hours. Free, unbound auxin remained at a more-or-less constant, low level of 0.04 pM per g. in St. John’s wort (Hyperium hircinum) leaf. Since there are plants which form only the aspartate or the glucoside, “it is unlikely that either conjugate is an essential part of the growth induction mechanism. We therefore assume that both conjugates are true detoxication products.”8oAlthough the auxin was added at 5 X 1W6M , and was therefore present in considerable excess in the external medium, i t seems curious that the natural, planegrowth regulator requires a “detoxication mechanism,” unless this conjugate formation is a means of regulating the amount of the free, more bioactive, acid. 1-0-(Indole-3-propionyl)-B.~-glucosewas found , by similar means, in incubation cultures of BaciUus megatheriumsO and 1-0-(indolp3-acetyl) arabinose was, from the results of paper-chromatographic separation, believed present in hydrolyzed, immature corn kernels?’ The presence of carbohydrate esters in plants is not unusual; there is good evidence for 1-estem of glucose and gentiobiose, even of esters of phenolic acids having free hydroxyl groups, such as caffeic, ferulic, and pcoumaric acid.@* >Ascorbic acid, considered to be a growth regulator (although whether (86) H.I). Klilmbt, Nalu~un’s8en8chafkn,46, 649 (1959). (86) H.I). Kliirgbt, I:lanb, 66, 309 (1961). (87) H.1). KlfiniI)t, in “Rrguhteim Nutiirrls de In CroiRRltnce Vegetale,” J. P. Nitsch, rd., C o i i l ~ Nirt ~ ion:rl tlr IHi t d i c w h c Sririitifiqur, PILI-~R, 1964, p. 235. (88)J. 13. Jtppwoii, Hiurhsnr. J . , 69, 82 (1958). (89) M. H. Zenk, in “Iiegulateura Naturds de la Croisaance Vegetale,” J. P. Nitsch, ed., Centre National de la Recherche Scientifique, Paris, 1964, p. 241. (90)J. Tabone and D. Tabone, Cmipl. Rend., 297, 943 (1953). (91) E. M. Shsntz arid F. C. Steward, Plunf Physiol., S2, Suppl. viii (1957). (92) I,. Birkofer, C. Kaiser, and H. Kosmol, Nalum*ssmcha&n, 47, 409 (1960).

392

H. W. HILTON

it is inhibiting or promoting depends on the source of information), forms, in Brassica species, an inactive compound with indol&acetic acid. The Czech literature on this compound, known aa aacorbigen, has been r e viewed by Bentley.”~~‘ The goitrogenic “bramica factors” from various species of cabbrrgo (Bramica) seem to inhibit uptake of iodine by the thymid. The inhibition has been attributed to SCNe, which appears as one of the hydrolysis products of mustard oil glycosidea, such m glucobrassicin (3).

(3 1

5( 8-p.Glucopyranosyl)-3-indolylthioacetylhydroximyl0-sulfate Compound (3) was obtained as a crystalline salt, in yields of up to 3% (based on the dry weight of cabbage). The N-methylindole analog, neoglucobrassicin, from the same species, has similir properties!6-@‘ 1-Naphthaleneacetic acid-I4C formed an emulsin-hydrolyzed D-glucose 1-ester in wheat, together with a Pglucoside of 8-hydroxy-1-naphthaleneacetic acid.82-m** Neither waa crystallired. 2-Naphthyloxyacetic acid formed both the 6- and 8-hydroxylated glucosides. The naphthalene auxins show considerably more stability and are converted less rapidly into the COIG jugate form. 111. PLANT-GROWTH SUBSTANCES USEDAS HERBICIDE~S

1. (2,4=Dichforophenoxy)acetioAcid and Related Compounds (2 ,.Q-Dichlorophenoxy)acetic acid, together with the 2-methyl4chloro and the 2,4, &trichloro analogs, 2 4 2,4,5-trichlorophenoxy)propionic acid , and many other related compounds, constitute one of the major classes of herbicides, selective primarily to dicotyledonous plants. Monocotyledons, (93)J. A. Bentley, Ann. Rsu. Pkrnt Phu&ol., 8, 47 (1968). (94) J. A. Bentley, in “Enoyclopedia of Plant Physiology,” W.Ruhland, ed., SpringerVerlsg, Berlin, 1961, Vol. 14, p. f309. (96) R. Gmelin and A. I. Virtannen, Ada Chem. Soond.,14, 607 (1960); 18,1378 (1962). (96) R. Omelin and A. I. Virtannen, Ann. Amd. 815.F m n h , 8er. A . IZ, lW,3 (1961). (97) A. I. V i n n e n , Pkyf0ch3mh3CryJ4, 207 (1966). (98) M. H. Zenk, Plank, MI, 76 (1962).

PLANTGROWTH SUBSTANCES

393

\KY‘:\UW of poor. absorption by the leaf and poor trsllsiocation within the plant. Abnornlal stem-twisting, leaf fusion and curling (epinasty), and rooting at nodes and leaf sxils are characteristic of these auxinic herbicides. The principal differences from the auxins previously discussed are the persistence and the high degree of phytotoxic effect of the phenoxy compounds. The various compounds are similar in their responses to the biological-growth tests; for each compound, the herbicidal properties appear at a critical concentration for the individual species and stage of growth. In common with the natural (and less phytotoxic) auxins, (2,4dichlorophenoxy) acetic acid in low concentrations decreases the reserve carbohydrate, temporarily increases the reducing substances, and increases the content of protein and amino acid, especially of the roots and stems. Although the exhaustive synthesis of nucleic acids and protein was suggested as a mechanism for herbicidal activity,a the depletion of reserves is not a sin,ple starvation process. For example, (2,4-dichlorophenoxy)acetic acid at a toxic concentration of lo00 mg./l. depletes sucrose only in the leaves and roots of kidney bean in 6 days, without altering the reducing substances, starch, acid-hydrolyzable polysaccharides, crude fiber, ash, ether extxact, uhsaponifiable materials, and fatty acids; and yet the reducing and nonreducing carbohydrates are depleted, and starch, crude fiber, and hydrolyzed polysaccharides are diminished in the stems.”Jm Soluble carbohydrate in buckwheat stems and leaves treated with 50, 100, 500, hnd lo00 mg. per liter increased the first day and decreased to 48% of that in the controls in 8 days. Starch in the stems declined immediately, increased above the controls in 12 hours, and then was depleted in 8 days at the highest concentrations. Root-starch levels fell contin~ously.~~ Starch formation wag prevented in tomato leaves, although sucrose increased continuously with low levels of (2-methyl-4-chlorophenoxy ) acetic acid.lO’ Many of the same conclusions may be drawn from results with synthetic auxins as from indole-3-acetic acid: water intake increased, there was a lessening of the downward translocation of photosynthate with temporary increases of soluble carbohydrates in the leaves, and alterations appeared in the metabolic rate and direction and in enzyme activity. However, the direct B i t e of action is not known, and many of the effects are puzzling. &Fructose oligosaccharides in artichoke and chicory storage-tissue were diminished by 70y0 (calculated on the content of dry matter) in 6 days

apwiaUy the grasses, an! not very nmvptible, ptwtly

(99) H. M. Sell, R. W. Leucke, B. M. Taylor, and C. L. Hamner, Phnt Phyehl., 24, 295 (I 949). (100) L. E. Weller, R. W. Leucke, C. L. Hamner, and H. M. Sell, Phnd Phyewl., 25, 289 (1950). (101) A. Rhociees, J . Ezptl. Botany, 2, 129 (1952).

394

H. W. HILTON

with M (2,4-dit:hlorophcnoxy):~cctiaacid, and oiily by 25% with the less toxic ( 3 , bdichlorophenoxy) acetic acid." Increases in hemicellulose, cellulom, sucrose, and total rarhohydrate, and decreases in starch arid of ( 2 , &dic*hlor.ophmrcducing subrstaticw were notd :It nd)lctohttlIcvc~l~ cwd xoybssii"M Iccwcw. Tho diniiiiut8ioiii i i tho oxy)ncetic*wid iii c*otloii*o" ratmeof downward transport of photosyiithete is put icwltrrly soveie with the herbicidal auxins, arid there is interference with photmynthwis.lMThe ripening of detached bananas by (2,4dichlorophenoxy)acetic acid and similar compounds by the more rapid conversion of starch into soluble carbohydrate could only have come about through a direct or indirect effect on the enzyme systems.IwThe ripening of fruit by means of chemicals is discuseed in Section VII (see p. 429). Uptake of carbon-l'C dioxide by tomato leaves was greatly increased with (2 ,.l-dichlorophenoxy)acetic acid, although the pattern of utilization was altered from leaves to fruiLIO"This contrasts with the assumption that auxin diminishes the rate of photosynthesis.lm The incorporation of carbon from sucrosd4C into ylheat and pea-stem segment cellulose and hemicellulose was "greatly enhanced,"'" and yet the comprehensive studies by Stevens and coworkerslw and Bourke and c0workers7~with ~-glucos&~C on pea-root tissue did not confirm this work. The two latter studies, with gluco~e-l-~~C, glu~ose-&~~C, g1ucoseJ4C6, acetate-l-14C,and acetate-PC, and a group of eight chlorinated phonoxyacetic and phenoxypropionic acids at lG-*M, showed that there wm no correlation of phytotoxioity with metabolic changes. All of the compounds inhibited uptake of acetate and increased respiration (as the ratio of carbon dioxide to residue), but did not alter the.distribution of radioactivity in respiratory WOt (from C-1 or C-6 of gluhose, or C-1 or C-2 of acetate), or in the ethanol-soluble or in ethanol-insoluble fractions. Glucose uptake was inhibited by all of the compounds, with little effect on the C-6/C-1ratio at lo-" M.At lo-&M, the C-6/C-1 ratio increased; this wm interpreted as indicating a stimulation of glycolysis. At lo-* M, the C-6/C-1 ratio was lowered, probably by inhibition of glycolysis. Increased radioactivity wm found in the ethanol(102) R. L. Wain, P. P. Rutherford, E. W. Weaton, and C. M. Griffiths, Nature, 208, 504 (1904). (103) D. R. Ergle and A. A. Dunlap, T e r n Agr. Ezpt. 8tu.Bull., 718, 18 pp. (1949). (104) D. E. Wolf, G. Vermillion, A. W a k e , and G. H. Ahlgren, Botan. Um., 112, 188 (1960). (105) A. J. Loustalot and T. J. Muaik, Botan. Uar., 111, 60 (1963). (106) 8.R.Freiberg, Botan. Urn., 117, 113 (1965) (107) N. I. Yakushkina, Fiziol. R a t . , 8, 111 (1982). (108) F. Wightmah and A. C. Noiah, Proe. Iniern. Botan. Congr., 9th Montreal, 1869, p. 430. (100) V L. Stevens, J. S.Butte, and 9. C. Fang, Plan4 P h y h l . , S7, 215 (1962).

PLAXT-QBOWTH SUBSTAHCES

395

dwreafed incopration w w irotd in the itdublc, ''cell-wall" fraction. The apparent alteration, by auxin, of the carbohydrate reserves, especially cjterch, in plants has had two consequences in essentially opposite directionn. First, there have been attempts to correlate the effectiveness of herbicide treatments with the natural variation in the root reserves of various species of weed. Although there has been some evidence of succm with Canadian thistle (Cirsium arvense)', in which the root reserves (fructan) are normally lowest, and most affected by herbicide, just before flowering,'1° the results have in general been disappointing. It is more likely that effective translocation of the foliar-applied herbicide throughoat the plant and into the root system depends on the downward movement of food reserves from the leaves, which in turn is related to the synthetic activity of the plant as a whole. This possibility agrees with a number of observations that treatment with a herbicide is most effective during active, vegetgtive growth.lllJ1*There have been few attempts to determine how much of the applied herbicide enters the phloem system of the plant, or the degree of translocation to various organs of the plant in relation to the effects observed. It also seems unfortunate that some of the published work has considered only the variations in the soluble carbohydrates m a function of the treatment.111,11a,114 The total volume of the root or the root weight will also determine whether recovery will take place.116 Second, attempts have been made to increase the storage reserves both of root crops and stem crops, or to decrease the reducing carbohydrate without altering the reserve of starch or sucrose (see also, Section VI). The accumulation of soluble carbohydrates in the leaves hais been attributed to poor translocation in the stem without immediate cessation of photosynthesis,llBwhich may or may not influence the reserve. Potato plants sprayed with (2,4-dichlorophenoxy) acetic acid at low concentrations, a t eight ounces per acre in August, showed no change in tuber sucrose in October, but had an increased proportion of reducing carbohydrate, whereas a July treatment decreased the reducing carbohydrate and increased the sucrose.117Increased starch and decreased reducing sub-

?dJhltdf2 extract, aid

B. Granstdim, Kgl. Liaabnclce-hg8kOl. Ann., 11, 281 (1954). H. P. Cords and A. A. Bandiei, Weeds, 12, 299 (1964). A. F. W i w and H. E. Rea, Weeds, 10, 58 (1962). H. M.LeBaron, Univ.Mimfilma 6!2-149,213 pp.Dis~erlationAb8tr.~ 12,2542 (1982). C. G. McWhohr, Weed, 9, 563 (1961). D. L. Linscott and M. K. McCarty, Weeds, 10, 298 (1962) L. G. Ganyushkina, uch. z a p . Karehk. Ped. Inat., 6, 135 (1958); Chem. Absh.acte, 68, 20307 (1959). (117) M. G. Payne and J. L. Fults, Am. Po@to J., 81, 144 (1955). (110) (111) (112) (113) (114) (115) (116)

H. W. HILTON

890

cwe*18;in anothcr, there were no differences”*; third showcd 3274 less of tliv rc!ducing substances, but no difference in suc’rosc or nitrogen in riincb i w w s . I w Variety, treatment, climate, and thc agc of thc trctttd plnnts huvc d l vwicd, and t hc cttrbohydrntnc.diffcrewes observed lruvc iwt been 1ttr.p)ailid liavc vwicd with thc oltbpxtd tinic? dtor treutnieiit. Phosphorus, in sll fornis rxcvpt in conibinnt ion in iwlcic acids, wns diminished in several plnrit species treated69 with aP. The particularly large diminution in organic phosphates, coupled with large decreases in incorporation of phosphorus into $,he high-energy adenosine di- and tri-phosphates suggests a major dislocation of the phosphorylase systems. Examination of isolated phosphorylation enzymes, however, shows no consistent alteration of activity. (2,4-Dichlorophengxy)acetic acid-I’C is metabolized by plant tissue to inactive, wt?,ter-soluble substances, some of which regenerate the parent acid on hydrolysis. The rate of metabolism differs with different species, but is usually low in comparison with the indole or naphthalene auxins. One of the earlier studies described a single major metabolite in corn, wheat, peas, and tomatoes,lZ1the identity of which is still uncertain. In another study of differenceR between species which were susceptible and those which were resistant to the herbicide, the resistant red currant metabolized 50% of the carboxyl-labeled and 20% of the methylene-labeled (2,4dichlorophenoxy)acetic acid to “COP in seven days, whereas the susceptible black currant alteredln only 2%. Similar effects were found with apple varieties. Strawberry and lilac decarboxylated the herbicide readily; however, 16 other species, of various susceptibilities to herbicide, were unable to decarboxylate the acid to any significant extent. In both varieties of currant, 5 to 10% of the herbicide was converted into watersoluble derivatives of the parent acid, and 10 to 30% was bound in the leaf tissue in an unextractable form. Two essentially different views of the metabolism of (2 ,4-dichlorophenoxy)acetic acid have been presented. The firstlP8Ja4was based on a study with bean stems, which converted 42% of (2,4dichlorophenoxy)acetic acidJ4C into acidic, water-soluble substances. Two ether-soluble metabolites were formed, and the same two metabolites could be recovered, (118) M. (i.Psync, J. L. Fults, R. J. Hay, and C. H. Livingston, Am. Potato J., 80, stuticw was indic:cttd in OIIC

aid

8

(119) (120) (121) (122) (123) (124)

46 (1!)53). TI. It. l’rrtcrpon, ZX8sertufion Abatr., 18, 147 (1953); 47, 7149 (1953). D. J. Wort, World Rev. Pest Conirol, 1, 6 (1962). 8.C. Fang and J. S.Butts, Plant Physiol., 19, 56 (1954). 5. C. Luckwill and C. P. Lloyd-Jonee, Ann. Appl. B i d , 48, 613, 626 (1980). M. K. Bach, Plant Physiol., 88, 668 (1961). M. K ‘Bach and J. Fellig, in “Plant Growth Regulation,” R. M. Klein, ed., Iowa State Univ. Press, Amee, Iowa, 1981, p. 273.

PLANT-GROWTH SUBBTAXCES

397

after hydmlysis of the water-soluble fmction, ‘‘mi~tainiihatvdwith sugars arid arrrino at-ids.” Spit her was tlir origiirrl wid or 2,~ ~ ~ Hydrogellation, and rc:tc*tion wit11 hydriorlic- wid, p;:t\lr? only pmdwts having intact ring~,bclievcd to t-ontttin Icngthcncd sidc-chains; this is

similar to formation of fatty acid by the addition of tcctyl groups. This view does not agree with earlier observations that the phenoxyalkanoic acids having an even number of carbon atoms in the side chain are converted by &oxidation into the phenoxyacetic acid. Neither does it agree with the conclusion derived from other studies, in which the major metabolites were believed to be either the glucose eater or a reaction product Mineral acids, takadiastase, or emulsin with aspartic acid, or both.126J28 regeperated the parent acid. Radioactive, chromatographic artefacts were found when leaves were dried before extraction with ethanol. 2 ,4-Dichloroanisole, but not 2 ,4-dichlorophenol, appeared to be a likely minor product in the latter work and in that of Crosby, who investigated two major, water-so!uble metabolites.127He believed one to be the glucose ester described by Klambt,B7and the other to be a structurally altered molecule, probably aJm present as a glucose derivative. Kliimbt had suggested the slow formation of (2 ,4dichloro-3-hydroxyphenoxy)acetic acid, but a more definitive study’m with oat tissue failed to reveal its presence. Phenoxyacetic acids, such as the 2-chloro and 2,&dichloro derivatives, with the C-4 unsubstituted, formed the 4-hydroxyl derivative and accumulated as the 4-/3-~-glucoside.The 4-substituted acids, such as (Pchlorophenoxy)acetic acid arid (2,4dichlorophenoxy)acetic acid, were not hydroxylated to any appreciable extent, but formed the glucose ester at equimolar ratios. The infrared epectrum of the acetylated ester isolated was identical with that of synthetic 2 ,3,4,6-tetra-O-acetyl-l-O- (2 ,Michlorophenoxy) acetyl-&r+ glucopyranose. (2,GDichlorophenoxy)acetic acid formed both the glucoside of the Phydroxy derivative and the glucose ester of the parent acid, acetic acid was exceptional in that it formed but (2,4,6-trichlorophenoxy) a glucoside of the 3-hydroxyl derivative. All derivatives could be hydrolyzed with acid or with &glucosidase. Other herbicidal phenoxyacetic and phenoxypropionic acids have some properties Rimilar to those of (2,4dichlorophenoxy) acetic acid, but often have quite different species selectivity. Some of the differences can be explained on thc basis of molecular stability, persistence, or mobility in the toxic form, as well as on the basis of differences in solubility and in absorption through leaves or roots. (126) (120) (127) (128)

R. W. Holley, F. P. Boyle, and D. B. Hand, Arch. Biochem., 87, 143 (1950). E. G. Jaworski and J. S.Butts, Arch. Biochem. Biophys., 88, 207 (1952). D. G. Croeby, J . Agr. Food Chem., 10, 3 (1964). E. W. Thomas, B. C. Laughman, and R. G. Powell, N u b e , 204,286 (1964).

~

398

H. W. HILTON

Of the thrcc monochlorophenoxyacotic acidR, only the 4-chloro derivative is very active and is used to retard abscission of fruit. A study of the effects of the isomers on the carbohydrates of the bean plant showed that the alterations of the carbohydrates were not related to the growth-inhibiting mechanism.’” Reducing substances, starch, mid s u c m decserwed with dl treatments, and the 2-chloro compound had the least effect; polyssccharides other than starch were not much altered. It is of interest that Aspergillus niger detoxified the (2-chlorophenoxy)acetic and (4-chlorophenoxy)acetic acid8 by hydroxylation of the ring, with remctval of the chlorine atom.’” 2. 1,2-Dihydro-3,6-.pyridazinedione

Maleic hydrazide (4) is not active in the Avena auxin test, and is inhibitory to growth, especially of grasses, at all measurable concentrations. 0

One of its curious manifestations is the inhibition of terminal growth without major inhibition of photosynthesis. It has been used to inhibit the sprouting of stored potatoes and to repress the growth of tobacco suckers. Starch content and “quality” of potatoes, and the sucrose in sugarbeets, treated with mrtleic hydrazide, did not change appreciably with storage.1s1Ja2 no effe~t,’**J*~ The content of reducing substances varied :dimin~tion,~~7J~’ or an being noted, depending on the elapsed time of treatment and the iength of storage. The sucrose content either remained unchanged or increased.lg7Plants sprayed with solutions of maleic hydrazide have shown moderate to large incremes in the amount of sucrose in the shoots

H.M. Sell, C. L, Hamner, T. L. Rebsfock, and L. E. Weller, Mich. State Uniu. Agt. E x P ~Sla. . Q’WTt. Bull., 40,306 (1957). (130) J. K.Fadkner and D. Woodcock, J . C k m . Soc., 5397 (1961). (131) D. R. Pateraon, S. H. Wittwer, L. E. Weller, and H. M. Sell, Plant Physiol., 27, (129)

135 (1952). (132) 8.H. Wittwer and C. M. Hansen, PTW. Am. SOC.Sugar Beet Techmlogiets, 6, 90 (1Qb;o). (133) M. E. Highlands, J. J. Licciardello, and C. E. Cunningham, Am. Pdoto J., 29, 226 (1952). (134) E. J. Kennedy and 0. Smith, Proc. Am. Soc. Hwt. Sd.,61, 395 (1953).

PLAATSROWTH SUBSTASCGS

399

c ~ r n , l and ~ J ~of surmse auld shrc.h itr r.I)tt8ir,1* wheprt,'a and bean.'" In t h e latter work, only the frnctan rwervc in barley innremed. Tho gluvoxe content was cithcr iinttltered or w w lower than that of thc aoiit,roln i n thtrxc irivcnt,ilr;utioiin. Three di ffcretit workom have dcwribcd oornlmted niuiiily of (IU(:MSB -from barlcyllm c o r ~ i , and ~~J~ leaf exudi&+q wheat.*a Synthesis or metabolism of starch does tiot appear to have been altered,140at least in tomato or bean plants. The apparent reason for the altered distribution of carbohydrate is that the sucrose or starch photorjynthate accumulates in the leaves, while transport and utilization are hindered. Sugarbeets and sugarcane (see Section VI) treated with foliar sprays of maleic hydrazide have not shown significant increases in reserve sucrose, although minor increases have been found for short, initial periods after treatment.14'-143 N o changes in oxidative phosphorylation, acetate uptake or metabolism, or pathways of carbohydrate metabolism were evident with this compound. The &D-glucoside of maleic hydrazide has been postulated as being one of two metabolites in wheat leaves and in apple, willow, tobacco, and other plants. These are formed144when the plants are supplied with maleic14Chydrazide or with inactive maleic hydrazide and glucoseJ4C. As rather large proportions of the parent compound are needed in order to produce plant responses, it is possible that the glucosidation is a detoxifying mechanism, as the authors suggest, although the activity level of the glucoside has not been determined, and other, alternative mechanisms of erratic performance, such as poor absorption by the leaf and poor translocation, seem equally likely.

and letves cf

3. 3-Amino-s-triazole

The most obvious effect of 3-amino-8-triazole (amitrole) (5) in plants a bleached appearance and diminished photosynthesis, although an effect on the synthesis of protein is probably involved as a more fundamental mechanism of herbicidal activity

is the lack of chloroplwta, resulting in

.

A. W. Naylor and E. A. Davis, Boban. Uaz., 112, 112 (1950). A. W. Naylor, Arch. Bdochem. Bzophys., 98, 340 (1951). W. J. McIlrath, Am. J . Bobany, 37, 816 (1950). D. J. Samborski and M. Shsw, Can. J . Botany, 36, 457 (1957). 'H. B. Currier, B. E. Day, and A. 5. Crafts, Botan. Uaz., 112, 272 (1951). V. A. Greulaoh, Botan. Gaz., 114, 480 (1953). M. Stout, PTOC. Am. Soc. Sugar Beet Technologiste, 8, 95 (1950). D. Ride, D. 8. Mikkelson, and R. S. Bsskett, Proc. Am. Soc. Sugar Beet T e c h t w b ~ t a7, , 88 (1952). (143) F. H. Peto, w.G.Smith, and F. R. Law,PTOC. Am. Soc. Sugar Beet !/'&-fa,

(135) (136) (137) (138) (139) (140) (141) (142)

7, 101 (1962). (144) G . H N. Towers, A. Hutchinson, and W. H. Andreae, Nature, 181, 1536 (1958).

400

H. W. HILTON

Amitrole had a drastic effect on the fixation of WOr by illuminated chloroplasts of ChZoreZZu pyrenoidosa, lowering the incorporation into sucrose by 95% at a concentration of 500 mg. per liter; it did pot affect phosphorylated compounds.146Carbohydrates hydrolynable by acids were higher (510j0) in treated, chlorotic corn leavea than in controls (32%), and this resdt was interpreted aa due to increased metabolism of proteins and fats. The respiratory quotient of 0.8 to 0.88, compared to controls near 1.O, supported this interpretation.146

I

H

Thera is considerable evidence that amitrole does not remain in the plant system as the free amine. For a perit$ of about 12 hours, amitrole does not move readily out of a leaf to which it is applied, although it is eventually quite mobile throughout the leaves, stems, and roots. The lag period has been interpreted as being connected with the rate of formation of one or more bioactive, translocation substances. Sorting out the various metabolic fragments has been fairly unrewarding and has been complicated by the presence of many minor metabolites. Most of the evidence seems to point to two major metabolites: one formed fairly rapidly and translocated easily in the plant; the second formed more slowly, possibly consisting of a transformation product of the first into a more stable form, and translocated with more difficulty. The evidence for transformation of the first compound into the second, as opposed to competitive formation at different rates, is not very clear. Disagreement exists aa to whether the metabolites are compounds formed with glucose, or with glycine or serine; each theory has proponents, and some scientists favor competitive ,or intermedigte formation of both metabolites. From all the evidence, the triazole ring appears to remain intact, even when incorporated into protein. Amitiwle-6-W formed 13 labeled, water-soluble metabolitea, with 50 to 90% transformation, in a variety of resistant and susceptible plant species. The principal compound wm formed with glycine or Gserine; it was ninhydrin-positive, and extremely resistant to hydrolysis (16 hours with 6 N hydrochloric acid in an autoclave sufficed to regenerate amitrole, but (146) a. Gu&in-Dumartrait, C m p t . Rend., 969,1837 (1961). (146) C.G.MoWhorter and W.II. Porter, Physid. Plontcmrm, 18,444 (1880).

PLANT-GROWTH SUBSTANCES

401

not the amino acid)>*--leTwo major metabolites were found in Canadian thistle, but only one in soybeanl". RogerS'Qproposed that a glucose adduct was the more labile compound in thistle; Herrett and Lincklm claimed that this v~aaformed exclusively if D-glucose was fed, with amitrole, to the plants; apparently, it is formed by reaction of amitrole with ~-glucosyl phosphate.-lS1Carter and Naylor149 could not incorporate ~-glucose--l~Ce into their major compound, whereas glycine-l4C entered readily. The Dg1ucow--l4C6 in the presence of amitrole increased the labeling of citric and malic acids, with no change in sucrose or phosphorylated compounds. Later, Naylorla demonstrated some increase in phosphorylated compounds and D-fructose, with inhibition of sucrose incorporation; he believed that the glucose adduct was questionable as a limiting factor in metabolism. Re-examination of three metabolites in Canadian thistle1" showed two to be inactive; a third was more bioactive than the parent compound, although it was different from the synthetic D-glucoside. Translocation of the herbicide required light, which may have brought about reaction to the active, transport form. Neither the synthetic Pglucoside nor reaction mixtures of amitrole with D-mannose, carabinose, D-xylose, or =ribose seemed to be involved in the two major metabolites in cotton.'M Massini166Jm first synthesized 3- (3-amino-s-triazolyl-l-)-Galanine, and identified it as being identical with one plant metabolite in tomato, presumably formed by the reaction of amitrole with serine. He investigated only two m e tabolites, which occurred in the diazotizable fractions: the alanine compound, and a second substance which may be the corresponding glycine compound. The substituted alanine was optically active, [ah -43' (water) , crystalline, and biologically inactive; it was translocated poorly. It appeared to be the most stable metabolite in tomato, increasing in quantity with time. Its similarity to histidine has been noted as a r e w n for its inccrporation into protein.U' Dospite the rather preponderant odds in favor of amino acid reaction, (147) (148) (149) (150) (151) (182) (163) (164) (156) (156) (157)

B. J. Rogers, Weeds, 6, 5 (1967). M. C. Carter and A. W. Naylor, Bdan. olaz., 122, 138 (1960). M. C . Carter and A. W. Naylor, PhysioZ. Plankarum, 14, 20, 62 (1961). R. A. Herrett and A. J. Linck, Physiol. Plankarum, 14,767 (1961). R. B. Shimsbukuro and A. J. Linck, Phyeiol. Planlarum, 17, 100 (1964). A. W. Neylor, J . dgr. Food C h . , 12, 21 (1964). R. A. Hemtt and W. P. Begley, J . Agr. Food Chem., 12, 17 (1964). C. 8.Miller and W. C. Hall, J . Agr. Food Chem., 9, 210 (1961). P. Msseini, Biochim. Biophys. A&, 86, 648 (1969). P. Messini, Acta Bolan. Need., 12, 64 (1963). A. K. William, 5. T. Cox, and R. G. Eagon, Bioehem. Biophy.9. Ree. Commun., 18, 260 (1966).

402

€1. W. HIWON

amitrole does react with carbohydrates to form stable products, but this reaction has not been unequivocally demonstrated in intact, plant systems. However, amitrole and D-glucoselhlQ and D-glucosyl phosphate161J62were condensed in rritro to form crystalline compound, m.p. 215” (dec.) , believed to be 3-(Boglucopyrsnosylamino)-s-tria~le(6) , on the basis of

OH (6)

its spectral properties in the ultraviolet and infrared, its molecular weight, and its slow phosphorylation with yeast hexokinase. It is biologically inactive, nonreducing, ninhydrin-negative, and not diazotized,which would mean that it would not have been investigated by Massini. A compound (presumably the same) referred to aa “triazole glucosazone” waa biologically inactive to wheat roots.Ioa 4. Chlorinated Aliphatic Acids

Two aliphatic acids possess, for grasses, many of the growth-distortion and toxicity effects associated with the synthetic auxins on dicotyledonous plants. Trichloroacetic acid and 2,2-dichloropropionic acid (dalapon), as although the sodium salts, have been called grass “hormones” or “auxinsJJJ Wilkinson’64 could find no growth stimulation at low concentrations, and described dalapon as an “antiauxin” from its interference with indole-3acetic acid effects. The herbicidal properties of trichloroacetate do not depend on ito proteindenaturing ability, and those of 2,2dichloropropionic acid involve, at least indirectly, the synthesis of pantothenic acid. Treatment of various plants with the compounds, at least up to herbicidal levels, has riot shown much alteration in the proportion of soluble carbohydrate, but ha8 shown rather wide variation in the distribution among the forms of the carbohydrates. flugarbeet seedlings increased in (168) A. C. Gentile and J. F. Frcdrick, Phyeiol. Plantarum, 19,862 (1969). (169) J. I?’. Fredrick m d A. C. Gentile, Arch. Biochem. Biophys., 86, 30 (1960). (100) J. F. Fredrick and A. C. Gentile, Arch. Biochem. Biophys., BP, 366 (1961). (161) J. F. Fredrick and A. C. Gentile, Physiol. Planbrum, 18, 761 (1960). (162) J. F. Fredrick and A. C. Gentile, Phytun (Buenos Aires), 16, 1 (1960). (163) E. E. Bchweiaer and B. J. Rogers, weeds, 12, 7 (1984). (164) I%. E. Wilkinson, Weeds, 10, 276 (1962).


403

reducing substances, imparting freeze resistance to treated plants.1a Sucrose increased in Johnson grass at the expense of reducing substances,114and trichioroacetate lowered sucrose to a quarter of that in controls in stems and shoots of wheat, and increased starch and other root polyaaccharides.la Organic acids remained relatively unchanged in dalapon-treated wheatelm Acid-soluble and acid-insoluble phosphates in ChZoreUa vulgaris increased 2 to 4 fold, depending on the concentration of trichloroacetate, with the soluble phosphorus most affected. However, neither respiration nor the carbohydrate-metabolizing enzymes were particularly sensitive. Little change occurred in the hydrolyzable polysaccharides.l@ Dalapon is hydrolyzed in plants to pyruvic acid.169

5. Carbamates and Carbanilates Several herbicides for monocotyledonous plants are carbamates, as are numerous insecticides and fungicides. Two of the principal herbicides, represeatative of the class, are isopropyl 3-chlorocarbanilate (CIPC) (7) , and S-ehhyl N ,Ndipropylthiocarbamate (EPTC) ( 8 ) .Although the herbi-

0)

(8)

cide activity appears to be associated with interference of cell mitosis, increases occurred in reducing carbohydrates and sucrose in corn and soybeans at herbicidal rates of application of isopropyl 3-chlorocarbanilateJ resulting in an overall increase of 90% in soluble carbohydrate in leaves and ~tems.l7~ The typical, dark blue-green color of the young plants, especially of the cotyledons, suggested a greater content of chlorophyll, but no increase in photosynthesis was S-Ethyl N ,Ndipropylthiocarbamate only moderately inhibited uptake of 14C02 at 1 0 - 2 M in red kidney-bean. No alteration appeared in the distribution of ~ucrose,amino (165) S. R. Miller and W. G. corn^, Cun. J . Botany, 96, 5 (1957). (168) T.L.Rebstock, C. L.Hamer, R. W.Luecke, and H. M. Sell, Plant Phpiol., 28, 437 (1953). (167) C. Oyolu and R. C. Huffaker, Crop Csci., 4, 95 (1964). (168) K. A. H m l l , Physiol. P&darum, 14, 140 (1961). (169) J. K. Lessure, J . A@. Food Chem., 12, 40 (1964). (170) J. A. M ede and A. 0. Kuhn, Wee&, 4 4 3 (1956); 6,68 (1958). (171) F. M Ashton, Wee&, 11, 295 (1963).

404

H. W. HILTON

acids, or organic acids; respiration of bean embryos was unchanged. Phosphorylation was more pronounced, but was not enough to be the cause of the altered growth. Inhibition of fl-amylrtse activity by a series of ringsubstituted carbanilates,with CFa, Br, and C1 substituents, was associated with electmnegativity and solubility, a strong argument for a physical adsorption on enzymes or other actively changing surfaces.ln Diminished carbohydrate reserves resulted in correspondingly greater reducing substances in another study.*TaAmylase, phosphatase, and invertase activity increased with isopropyl carbanilate (IPC) , but reducing carbohydrate did not increase proportionately. Sucrose decreased in proportion to invertase activity.lT4Zinc N ,Ndimethyldithiocarbamate, a fungicide-herbicide, at two to eight pounds per acre, increased glutamic acid about 100% but did not affect yield of sucrose in sugarbeets.lTSVarious carbamates inhibited the Hill reaction (see Section 111,6, p. 405), but not efficiently (as compared with the substituted ureas). No really satisfactory hypothesis explaining the various observed eff ects has yet been offered. The l-thio-r3-P.glucopyranosideof N ,Ndimethyldithiocarbamic acid was isolated in crystalline form, by countercurrent extraction, from potatoes treated with the sodium salt of the fungicide. No data are given in the reference,"a but the compound was said to have been compared with an authentic sample prepared from tetra-0-acetyl-D-glucopyranosyl bromide. The fl-D configuration was inferred from the synthesis, and the compound was a weak fungicide. Another carbanilate herbicide, 4-chloro-2-butynyl 3-chlorocarbanilate (bwban) , formed a water-soluble metabolic product in various plants. The metabolite could not be identified as any simple reaction-product, and it was tentatively proposed that the original herbicide might be sssociated with a plant component such as a flrtvonoid. 3-Chloroaniline could be distilled out after hydrolysis, and several hydrolytic fractions contained carbohydrates but no nitrogen.In 6. Substituted Ureas A group of substituted ureas used as herbicides has a profound inhibiting effect on the photosynthetic mechanism in plants. The relatively advanced (172) D. E. Moreland and J. C. Davis,Proc. sbuthmn Weed Cmf., 9, 150 (1956). (173) V. H. Freed, J . Agr. Food Chem., 1, 47 (1953). (174) C. Tomisirwn arid H. Koikc, Nogyo Gijuku KenkyLsho Hokoku, Byon' Konchu, 4, 26 (1954). (175) J. L.Fults, M. G . Payne, J. 0.Grmkill, L. R. Hao, and A. C. Walker, Botan. Gaz., 113, 207 (1951). (176) J. Ksslander, K. Kaars-Sijpesteijn, and G.J. M. van der Kerk, Biochim. Biophva. Acla, 62, 396 (1961). (177) J. R. Riden and T.R. Hopkins, J . Agr. Food Chem., 10, 455 (1962).

PLANT-OROWTH SUBSTANCES

405

state of photosynthetic research has made possible the study of the inhibition in some detail. The most active compound, which severely inhibits or kills seedlings by root uptake of 1 X 10-7 M solutions, is 3-(3,4dichloropheny1)-1,1-dimethylurea (diuron) (9). A dozen or so other am0

II

c

1

N- C-N, pA1

,cH, CH,

c1 (9)

logs are herbicidal, with 3- (pchloropheny1)-1,1-dimethylurea (monuron) being the most common. Their activity apparently lies in their ability to inhibit photosynthetic phosphcrylation; probably, the photoreduction of adenine nicotinamide dinucleotido or its phosphate is blocked, leading to the lack of an oxygen receptor from the photolysis of water. The effect is meaaured aa an inhibition of the Hill reaction, in which the photoreduction of ferricyanide or pbeneoquinone added to iIluminated suspensionsof chlorophyll is measured with and without the chemical.l*lB1 Diuron inhibition of the Hill reaction was found to be 2500 times that of phenylurethan; if it is not too severe, the inhibition can be partly reversed by ~-gIucose,sucrose, or flavine mononucleotide.182Ja The last compound is a catalyst in the reaction which involves production and reutilization of oxygen from water. There is also apparent interference with the hexokimsecatalyzed photophosphorylation of D-glucose with adenosine triphosphate to give 0-glucose 6-phosphateJ demonstrated1” with *,P,Herbicidal members of many groups of compounds-the ureas, carbanilates, s-triazines, acylamides, and uracils-aJl have essentially the same mode of action, with differences of degree and species selectivity. The mechanism for oxidation of water to oxygen appears to be primarily affected in each case. The urea herbicides do not inhibit seed-germination to any extent, or the growth of nonphotosynthetic tissue, nor do they interfere with darkfixation reactions. Monuron-treated bean-leaves which were fed 14C02in the light, however, decreased in sucrose content (as percent of ethanol-soluble materials) from 70 to 2% in 72 hours.186Most of the carbon-14 label (178) The Hill reaction has been reviewed by I(. A. Clendenning, Ann. Rey. P2cmt Phlpwl., 8, 137 (1957). (179)A. It. Conkc, North Cmtral Weed Cbntrol Cmf., Ree. Rept., 12, 181 (1955). (180) J. 8.C. W e w h and R. van der Veen, Biochim. Biophyu. A h , 19, 548 (1956). (181) B.Exer, Weed &8. 1, 233 (1961). (182)M.J. Geoghegan, New Phytolopiat, 66, 71 (1957). (183) W.A. Gentner and J. L. Hilton, Weeds, 8, 413 (1960). (184) N. E.Good, Plan4 PhfpiOl., 36, 788 (1961). (185) B’. M.Ashton, F. G . Uribe, and G . Zweig, Wee& 91676 (l9Gl).

406

H. W. HILTON

appeared in aspartic acid (58.60j0). Although neither the total percentage of amino acids nor that of organic acids changed much, the distribution ratios changed considerably. All carbohydrates decreased, as measured by dry weight, as "water-insoluble" carbohydrates presumed to be starch, and as soluble substances.188 Amylase activit,y decreased ; within the series of chlorosubstituted ureas, decreased activity varied directly with water solubility, suggesting an adsorptive effect on the cnxyme surface. The enzyme effects were not, considered to be major causes of growth inhibiti~n."~ An inac.tive metabolite formed from monuron in bean plants has noC yet been idcntificd.I87 7. SubBtituted n-Triazines

Members of an extensive group of sym-triazine herbicidos, usually having one or two secondary amine substituents, block the Hill reaction and inhibit photosynthesis in a manner quite similar to that of the urea herbicides. The most widely used, 2-chloro-4-(ethylamino) -6-(isopropy1amino)s-triazine (atraxine), (lo), is one of several hundred herbicidal analogs 'N

R

N

(10)

differing in the three substituents attached to the ring-carbon atoms. An interesting difference in species selcctivity involves the ability of certain plants (and soil) to render the molecule biologically inactive by hydrolytic removal of one or more of the attached groups. Ring fission may also occur. The photosynthetic block precedes sucrose synthesis, and sucrose partially revwscs the inhibition caused by the lack of fixation of carbon dioxide. D-Fructose disappears first, after treatment, followed by sucrose, and then r~-glucose.lssIt is likely that other metabolic systems are involved as well, since the amino acid distributions were not identical to the I4CO2 dark-fixation products.1se Uptake of s u ~ r o s e - ~iricreased ~C some acids (as(186) W. H. Minshall, Can.J. Botany, 88, 201 (1960). (187) S. C. Fang, V. H. Freed, R. B. Johnson, and D. R. Coffee, J . A p . Food Chem., 8, 400 (1955). (188) S. M. Mashtakov and R. A. Prochornik, Dokl. Akad. Nauk Bellmask. SSR, 6,1367 (1962). (18'3) P.M Ashton, G . Zwcig, mid C;. W. Mason, Weeda, 8, 448 (1960).


407

partic and glutamio) at the expense of others (seine, alsnine, glyceric acid) in the light, but not in the dark. Serine-I4Cdid not form any sucrose; and an increase in glyceric acid suggested a block in the glycolytic scheme wherein the acid required energy from adenosine triphosphate for conversion into glyceric acid 3-phosphate. Acetate-2-14Cor -8-l*C uptake and utilization were decreased by simazine, the bis(ethy1amino) analog.1oo Starch production in leaves cemed after one to six days, depending on the species; su~rose-~~C could be substituted m a source of substrate for starch.'@JCorn (maize) tolerates many of the triazine herbicides, but its isolated chloroplasts do not. Treatment did not influence catalase activity or respiration,Ig1 but stopped oxygen output immediately.102 Simazine and atrazine have little effect on corn, sorghum, sugarcane, and some other grasses, apparently because of the ability of the plant to metabolize the parent molecule. Corn and wheat gave a cyclic hydroxamate, namely, the 2-~-glucosideof 2,4dihydroxy-7-methoxy-l,Pbenzoxazin-3-one ( l l ) , m.p. 168-70",aglycoii m.p. 1587" (dec.), believed to

have replaced the chlorine atom catalytically by a hydroxyl group.lg&l" The campound is not known in other tolerant plants, and it is likely that enzymic metabolism is also involved. 8. Substituted Benzoic Acids

There is little published information of interest to carbohydrate chemists concerning the benzoic acid herbicides. The predominant member, 2,3,6trichlorobenzoic acid, inhibited uptake and utilizationlWof acetate-W. It shows some auxin-like properties, whereas 2 , 3 ,5triiodobenzoic acid appears to block the transport of natural and synthetic auxins, and has often been termed an "antiauxin." The triiodo compound inhibited oxygen (86% at (190) (191) (192) (193) (194) (195) (106)

A. Gast, Ezperidia, 14, 134 (1958). B.Exer, Ezperidia, 14, 136 (1958). W. Roth, Ea~perientiu,14, 137 (1958). R. H. Hamilton and D. E. Moreland, Science, 181,373 (1962). W. Roth and E. Knusli, Ezperientia, 11, 312 (1961). R. H. Hamilton, J . Agr. Food Chem., 12, 14 (1984). 0. WulilrooB iirid A. I. Virtannen, Actu Chem. Sand., 18, 1908 (1959).

408

H. W. HILTON

2 X 1 P M) and phosphorus uptake:o

and the uptake and utilization of acetate.1w Benmic wid, the three monohydroxybenmic acids, and the three monochlorobenzoia acidH decreased carbohydratm in barley leaves, d retarded respiration, although none are particularly phytotoxic. Added sucrose increased the output of carbon dioxide, but did not increw the carbohydrate content in the plants. Plant sucrose was depletd.’@’J@ Many other herbicides and compounds having growth-alteration properties are known, and new ones appear a n n d y from commercial screening. It has been quite evident so far that no correlation has been demonstrated between chemical structure or physical properties and biological activity. The empirical search continues for new structures having biological propertieg, arid bmic research studies continue with newer instrumental techniques for investigating the mechanisms of activity.

IV. GLYCOSIDES AND OTHERCARBOHYDRATE DERIVATIVES AS ~ T - G R O W T HSUBSTANCES 1. Natural Glycosides

In ddition to the naturally occurring indole and gibberellin growthregulators, mrtny “growth factors,” accessory factors, and inhibitory substances have boon either isolated or at least suggested to cover unexplained results. One area of lively academic and practical interest involves the natural inhibitors of growth and of germination of seeds and axillasy buds. Many plantdrgan and seed extracts are inhibitory in a gross sense to other plants, and many seeds will not germinate until some inhibitory fraction has been extracted. Three proposed mechanisms for the activity of the inhibitors are: (1) antagonism, competitive or otherwise, with essential auxins, vit,amins, and so forth; (2) direct inhibition of some growth or development process at sites other than those of direct, hormone activity; and (3) indirect inhibition of growth, affecting enzymes, nutrient uptake, or utilization. There is evidence for all three. This discussion will not cover the direct enzyme and metabolic inhibitora or poisons, which have been so comprehensively reviewed by Webb.1Bg The characterization of many of the growth inhibitors is not definitive, and it would be extremely premature at this point to try to assess the physiological or chemical importance of those that are known. However, (197) M. I. Naguib, Ccn. J . Bdcmy, 41, 939 (1W). (198) M. I. Naguib, Plonla, 04, 20 (1006). (109) J. 1,. Webb, “Enzyme and Metabolic Inhibitors,” Academic Press Ino., New York,

N. Y.,and London, 1083.

PWNT-GROWTH SUBSTANCES

409

so many belong to the flavonoid and relatcd groups, or arc dcrivtitivos of coumarin, that it is believed worthwhile to review them. Most of them exist in the plant as glycosides of phenolic or enolic groups, or as carbohydrate esters. Glycoside formation has generally been considered to be a detoxification, the “more or less universal reaction (together with destructive breakdown) of all organisms to the presence of foreign compounds,” and anthocyanin pigments have been considered to be growthinactive end-products. It is likely, however, that the glycosides and the aglycons should either be treated as individual entities, or that the glycoside formation should be considered to be a regulatory mechanism for controlling the amount of active substance. It seems strange that the plant should treat those compounds which it produces as “foreign substances” requiring detoxification, unless glycoside formation provides some such regulatory mechanism. The statement of Gibbsm is applicable: “Many of the substances, such as the alkaloids, the cyanogenetic glycosides, the phenolics, and so on, that seem to be so useful as taxonomic guides have no obvious usefulness to the plant.. . When we know more of them we may think diffelently.” So far, relatively few glycosides are known to have growth-alteration properties. The earlier work (to about 1958) has been reviewed by Hembergml and Mayer and P~ljakoff-Mayber.~~ Many of the simple phenolic acids are inhibitory, including coumarin, trans- (but not cis-) cinnamic acid, salicylic acid, and 0- and p-coumaric acids, among others. Precursors of the various coumarin derivatives appear to be substituted cinnamic acids. o-Coumaric acid (truns-o-hydroxycinnamic acid) , as the &wglucoside in sweet-clover leaves, formed a n equilibrium mixture containing 89% of the Cis isomer in normal sunlight, with subsequent ring closure to coumarin.*m6 It has been suggested that the glycoside is formed first, and is involved in the Cis-trana isomerization and in the biosynthesis of the coumarin derivatives which are predominantly present as g1ycosides.m Precursors of the flavones and flavanones are probably hydroxylated benzoic acids. Light decreased the auxin response of etiolated peas without affecting the auxin level; if light activates or produces an inhibitor which interacts

.

(200) R. D. Gibbs, in “Chemical Plant Taxonomy,” T. Swain, ed., Academic P m Inc., New York, N. Y., and London, 1963, p. 80. (201) T. Hemberg, in “Encyolopedia of Plant Physiology,” W. Ruhland, ed., SpringerVerlag, Berlin, 1961, Vol. 14, p. 1162. (202) A. M. Mayer and A. Poljakoff-Mayber, in “Plant Growth Ragulation,” R. M. mein, ed., Iowa State Univ. h, Am-, Iowa, 1961, p. 735. (203) 8. A. Brown, G. H. N. Towers, and D. Wright, Cun. J . Biochem., 88, 143 (1960). (204) T. Komge and E. E. Conn, J . BWZ. Chtnn., U,2133 (1969). (206) F. A. Haskins, L. 0. William, and H. J. Gors, P & d Phyeiol., 89, 777 (1964). (208) D. J. Austin and M. B. Meyera, Phyfoehemishy, 4, 256 (1965).

H. W. HILTON

410

with the auxin, it might be poasible to explain the resu1ts.m Galstoil and his group believed that they had characterized the natural inhibiting substances in peas aa being derivatives of the two flavonols: quercetin and kaempferol (12), which exist as mixtures of the 3-O-"tri-r+glucosyl"

GR-

HO

-

R H in kaampferol derivatives

R = OH in quercetin derivatives (la)

pcoumarate (shown),and the 3-O-"tri-~-glucoside~~ forms.208 The structure of the "tri-&glucose" portion is not known. Other derivatives of quercetin had earlier been implicated as inhibitors of pollen germinationm : rutin [quercetin 3- (0-0-/3+rhamnopyranosyl) -Dglucopyranoside], and quercetrin (3-O-8-crhannopyranosylquercetin) Naringenin, (13), isolated m a glycoside from peach buds, inhibited

.

I,7,4'-TrihydroxYfhWne (13)

seed and peach-bud g e r m i n a t i o ~ i ? possibly ~ ~ * ~ ~ by interaction with gibberellins. Since naringenin is structurally similar to kaempferol, it may be t r d o r m e d into the latter as the active form. Other related compounds, such as the flavone apigenin 'I-~-~ucoside, had similar growth-activity. Ten other flavanones, flavones, and isoflavones were found to stimulate (207) A. W. Galston and M. E. Hand, Am. J . Bdany, 86, 86 (1949). (208)M. Furuya and A. W. Galston, Ph@ochmiu@, 4,285 (1966). (209)F. Moewua, Aqpsw. Chm., 63,486 (1960). (210) C.H. Henderehott and D. R. Walker) Bn'ancs, 180, 798 (1959). (211) C.H. Hendersbott and D. R. Wfilker, Proc. Am. Boc. Hort. Bci., 74, 121 (1959). (212) I. D.J. Phillips, Nature, 1011 240 (1961). (213) I. D.J. Phillips, J . Ezpdl. Botany, 18, 213 (1962). (214) C. F.Eagles and P. F. Wareing, PhysME. Plontarum, 17, 697 (1864).

PLANT-QROWTH SUBSTANCES

411

wheat rookgrowth at 1od to l(r M ,but were inhibitory at higher levels. Stenlid216 used the aglycons in the study, but noted that they existed mainly aa glycosides. He suggested that they affected enzymic oxidation of phenols and L-ascorbic acid, The compounds reversed the mobinhibiting properties of indole-%acetic acid, (2,4dichlorophenoxy) acetic acid, D-mannose, D-galactose, and 2-deoxy-~-arabino-hexoseto some extent. Quercetin was not too effective as an antagonist for (2,4dichlorophenoxy)acetic acid (4 X 10-7 M) ,indole-3-acetic acid (4 X lo-*M ),D-mannose (7 X 1 P M ), or 2deoxy-~-urubino-hexose(3 X M), and it failed to show auxinic activity. The uptakes of phosphate, chloride, and nitrate were inhibited, which led to the conclusion that quercetin was growth specific, since n-glucose can reverse D-mannose inhibition, but does not depress ion uptake. No complete parallel existed between the effects of the externally applied substances and the endogenous growth mechanism.216 Work with the kaempferol- and quercetin-“triglucose” esters of p hydroxycinnamic acid has established their structures by spectral data, carbon-hydrogen analysis, and chromatography; the esters examined were not crystdliie. The 1:3 molar ratio of aglycon to n-glucose, and the attachment of the cinnamic acid to the D-glucose chain were determined.*17e218 The present view is that, in etiolated peas, and possibly in all green plants, kaempferol exists as a mixture of the “triglucoside” and “triglucosyl’’ p-coumarate forms, with only traces of the quercetin derivatives, whereas the latter (again as the same kind of mixture) are predominant in green plants in the light.m The kaempferol-3-“triglucosyl” p-coumarate waa found only in the leaves, kaempferol-3-“triglucoside” occurred in leaves and stems; quercetin-3-“triglucosyl~’pcoumarate occurred throughout the plants (except in the roots), and the triglucoside wm abaent from roots and stems. Each organ appeared to have its own pattern of flavonoid synthesis, which could be modified by light. Far-red and red light altered leaf-growth production of kaempferol-3-“triglucosyl” p-coumarate. These effects of light were most likely indirect, ultimately changing the flavonoid synthesis.21eKaempferol “hexaglucoside” has been reported in pea seedlings treated with red light.= The physiological effects of the kaempferol and quercetin derivatives are uncertain: they inhibited indole-3-acetic acid oxidase in vitro; they may induce dormancy, uncouple oxidative phosphorylation, stimulate plant (215) (216) (217) (218) (219) (220)

G. Stenlid, Physiol, Phnfamm, 14,659 (1961); 16,698 (1962). G. Stenlid, Physiot. Phntumrn, 10, 807, 924 (1957); 12, 199, 218 (1959). F. E. Mumford, D. H. Smith, and J. E. Castle, Plutzt Phyeiol., 86, 752 (1961). M.Fwuya, A. W. Galston, and B. B. Stowe, Nuture, 198, 456 (1962). M. Furuya and R. G . Thomas, Plant PhysioZ., 89, 634 (1964) F. E.Mumford, D. H. Smith, and P. G. Heytler, B i o c h . J., 91, 517 (1964).

412

H.

W. HILTON

growlh, or iiihibit flowering. 15sglcx aiid WareingZi4proposed that they be called growthdormancy regulators, to distinguish them from the synthetic growth-inhihitors. FIeterN of chismio acids are more common than glyconides of phenols. Tho moiiocstmv of D-g1uco8el rutiiiosc, or goritiobiow with tho four naturally occurriiig cinnniuic acid8 (cinnamic, ferulic, caffeic, and sinltyic: mid) have been recognized as iiaturttl products--some, as complex products containing more than one cirinamoyl Both kaempferol and quercetin exist as 3-glucosides1rhamnosides, galactosides, arabinosides, gentiobiosides, and more complex forms in numerous p1ants.m Kaempferol 3-~-glucoside (astraglin) and kaempferol 3-~glucosylpcoumarate (tiliroside) increased indole-3-acetic acid activity by inhibiting the oxida~e.**S*~ An auxin (1-naphthaleneacetic acid), 2,3 ,&triiodobenzoic acid, and a gibberellin interfered with formation of anthocyanin pigment in Impatiens balsamina. The first compound increased, and the second and third decreased, the anthocyanin formation. This interaction shifted the genetically controlled pattern of quantitative distribution of anthocyanin toward the base of the stem segments.n6 A series of 36 plants showed only two that form no glucosides when sucrose is injected into the leaves. In the two exceptions, the sucrose was converted into nonsugars. Phosphorylation uncouplers (2,4dinitrophenol, iodoacetate, and the like) did not affect Dglucoside formation.m Plant-growth factors producing positive responses of rapid, controlled cell-growth and celldivision have been found in many places; undoubtedly, some are mixtures of vitamins, carbohydrates and amino acids with the natural growth-regulators. None have proved to be more interesting, more useful, or more difficult to characterize than those from coconut milk or from the corresponding, immature, liquid endosperm of various plants, such as horae-chestnut ( Aeeeulw woerlilzenais) and corn (Zea mays). Fractionstion of the active substances, reviewed by Steward and Shantz,n7 has so far produced only 1,3diphenylurea, a possible indole-3-acetic acid eater of arp,binose, and another substance, 20 mg. of which waa saidm to (221) J. J. Corner, J. B. Hsrborne, 8.G. Humphriea, and W. D. Ollie, Phytochmbfty, 1, 73 (1902). (222) See, for example, J. B. Hsrborne, Arch. Ba&m. Btbphys., 96, 171 (1962). (223) J. B. Harborne, Phybockmbtty, 1, 161 (1964). (224) J. P. Niteoh and R. Parip, BuU. Soc. Botan. Frame, 109, 241 (1902). (228) A. W. Arnold and L.8. Albert, Plant Phyeiol., 19, 307 (1964). (226) D.I. Lisitayn, Uglcvody a Uglcvodnyi Obmsn v Z h i v o t m i Rastilsl'nom Organ&mukh, M M a y KW.,Moeeow, 1868, (Publ. 1969); C h m . Abstraete, 66, 7665 (1961). (227) F. C. Steward and E. M. Shantz, Ann. Rev. Plant Phyeiol., 10, 379 (1959). (228) E. M.Shantz and F. 0.Steward, Plant Physiol., 80, xxxv (1866).

PWNT-GROWTH SUBSTANCES

413

equal the activity of coconut milk at 10%. It w a proposed that the product might be the mono-wglucoside of either a leucoanthocyaninm (14) or a ledcocyanidinm ’(15). No other report of its biological activity has been published.

Leucoanthocyanin hm been found in buckwheat, and is formed both in light and darkness, whereas the anthocyanin pigments are formed independently and only in the light.281 The aglycon had little activity. It is not clear whether these compounds are responsible for the major activity of the endosperm source or for only a d part of it. The constituents of the endosperm are diEcult to isolate and purify. The phenolic constituents should be investigated in greater detail. The 5-&~-glucopyranosideof 2 ,5dihydroxybenzoic acid (gentisic acid) waa identified as an accumulation product in borondeficient sunflower.2n.2ss A number of glycosylamine derivatives are growth factors for fungi and bacteria, notably derivatives of riboflavine, coenzyme A, vitamin Bu, myo-inositol, N-glucosylglycine, Zacetamido-Zdeoxy-0-(D-galactosyl)-Dglucose, and others. These will not be discumd here’ePr,2as 2. Synthetic Glycosides, Other Carbohydrates, and Carbohydrate Derivgtives Glycosides are formed by higher planta from a variety of externally applied substances, regardless of whether or not the substance is toxic or growth active. The herbicidal chemicals have been discussed in other Sections, and this Section will deal with glycoside formation of some other compounds, and with some carbohydrates and derivatives that have shown growth activity. Nearly all plants, excepting the lower plants, algae, and fungi, form (229) G. M. Robirison and R. Robinson, Biochcm. J., 27, 206 (1933). (230) T. Swain, Chem.In&. (London), 1144 (1954). (231) J. R. Troyer, Phyfochniehy,8, 638 (1964). (232) R. Watmabe, W. Chorney, J. Skok, and 8.Wender, Phyiochemiu6q 8,391 (1964). (233) -4. Zaue and 8.H. Wender, C h m . I d . (London), 1836 (1964). (234) Reviewed by N. Fries, in “Encyclopedia of Plant Physiology,” W. Ruhland, ed., Springer-Verlag, Berlin, 1961. Vol. 14, D. 332. (235) W.8.McNutt, C h n . Org. Naturs&$e, 9, 401 (1952).

414

H. W. HIL'TON

mono-&D-glucosides with either quinol (hydroquinone) or resorcinol as ail externd phenol. This phenol glycosylation reaction hss been reviewed.2aa Phloroglucinol-14Cand D-glucose formed the corresponding j3+glucoside, phlorin, in many Those species which seemed to have no glycosylatioq mechanism converted the D-glucose into sucmse or, occasionally, into p-fructose or D-glucitol. MilleP-w has studied the glycosides produced (in proportions aa great aa 13% of the dry weight) when chloral hydrate, 2,2 ,2-trichloroethanol, ethylene chlorohydrin, and o-chlorophenol were added to plants. He originally investigated these compounds as tuberdormancy regulators, and found them to have a low degree of phytotoxicity, except at high concentrations. Seven species formed the gentiobioside instead of the D-glucoside, although gentiqbiose was not known to be present in the plants. The derivatives were compared with authentic compounds. A third, unauthenticated carbohydrate material may have been present aa a primeveroside. The carbohydrate moiety present depended on the aglycon applied. The glycosides did not move readily, were not found in sprouts, and, therefore, were considered to be detoxication products. Compounds isolated and synthesized are listed in Table I. Apart from the utiliestion of the common mono- and di-saccharides as metabolic substrates for growth and energy, there is little information on the growth effects of externally applied carbohydrates. Some, like D-mannose, *galactose, and Zdeoxy-D-urabino-hexose, are inhibitory to root growth,*lapossibly because they provide a high osmotic concentration in solution, from which they are only indifferently utilized, without being actively phytotoxic. J. B. Pridhsm, Phyfochsmistry, 8, 493 (1864). A. Hutahinson, C. Roy, and 0.H. N.Towers, Nature, 181, 841 (1968). L. P. Miller, Confrib. Boyce Thompson Znet., 0, 426 (1938). L. P. Miller, Csontrib. Boyw Thumpson Znef., 10, 139 (1939). L. P. Miller, Codrib. Boyce Thornpuson Znet., 11, 28 (1939). L. P. Miller, Am. J . Botany, 96, 14a (1939). L. P. Miller, Conkib. Boyw Thompon Zneb., 11, 271 (1940). L. P. Miller, Seisnce, 91, 42 (1940). L. P. Miller, Codrib, Boy@Thumpuson Znet., 11, 387 (1941). L. P. Miller, Codrib. Bww Thumpuson Znet., 19, 16 (1041). L. P. Miller, Cdrdb. Boyce Thompm Znet., 19, 26 (1941). L. P. Miller, C d r i b . Boyce Thompson Znat.,14, 29 (1941). L.P. Miller, Contnb. Boycs T h p s o n Zteet., 19, 163 (1941). L. P. Miller, Codrib. Boyw T h p o n Inst., 19, 167 (1941). (250)L. P. Miller, J . Am. C h .SOL, 68,3342 (1941). (251) L. P. Miller, Con&&. Boyes Thampsson Znet., 18,369 (1942). (262) L. P. Miller, Contrib. Boyce Thpson Zmt., 19, 466 (1943). (263) L. P. Miller, Confrdb. Bqce Thompson Znuf., 18, 186 (1943). (254) L. P. Miller, Contra%.Boyce Thompson Znef., 18, 113 (1967).

(236) (237) (238) (239) (240) (241) (242) (243) (244) (246) (240) (247) (248) (249)

TABLE I P k n t Glycosidea from Chloral Hydrate, Ethylene Chlorohydrin, and 0-Chlorophenol

Free

Glyeoside

Peracetate

presence

Refennees

in plant@

glycoside

8-D-Glucopyranoside 2,2,2trichlorOethyl

2-chlomethyl whlorophenylb

153

-

-40.5

172

-65.3

205 (dec.)

-41.2

+ +

249,251-253 238,239, slci

+-

245,247,

-49.5

+

250 242-244,Ws

-47.2 -39.9

+-

250,252 252,254

145 118 150

-29 -13.4 -44.6

184 168 208

-28.5 -m.2

170 176

240

8-Gentiobioeide 2 ,2 ,2-trichloroethyl ZChlmOethyP o-chloropheny1

-

-

amorph.

&Primevenmide 2,2,2trichlor0ethyl~ ZChloroethyl* a

*

+

-

Key: means present; - means absent. Synthetic. Glycoside structure uncertain, but may be a primeveroside.

9 s

H. W. HIYMN

416

The cyclitol, myo-inositol, is a growth promoter for lower plants, and the group of cyclibls is widely distributed in higher plants. Their physiological function has not yet been fuUy established, but they are known to be involved in the storage of phosphate, as phytin, and in the biosynthesis of aromatic rings from carbohydrates. Spruce tissue-cultures were maintained with myo-inositol or sequoyitol (a monomethyl ether) at 50 to 100 mg. per liter. (+)-Pinitol, Anositol, and ecyllo-inositol had slight activity, whereas quebrachitol, and L-, epi-, mwo-, and neo-inositol were all inactive.n-manno-Heptulose inhibited phosphorylation of glucose by inhibiting hexokinase?" and the aldono-1,&lactones inhibited carbohydraaes having the same configuration." R. Brown and coworkersm~360 observed that seeds of Striga, known as witchweed, a semi-parasitic plant of gnaws and corn, germinated only in the presence of root exudates from the living host. They isolated the sirupy exudate, [a33 20' (water), resemblinga pentose, but did not specifically identify the active substance. A study of many carbohydrates, mostly pentoses, showed that only wthreo-pentulose, -32' (water), had the required activity; cthreo-pentulose, [a& 35" (water) ,was not active. Two groups of sucrose derivatives of herbicidal acids have been reported. The herbicidal properties of the sucrose estera of (2 ,P-dichlorophenoxy)acetic acid and other analogs differed somewhat from the salts of the free herbicide acids. This could be accounted for as being due to differences in solubility and penetration, since it is unlikely, by analogy to the fatty acid esters, that the sucrose esters would remain intact in the plant.m The second set of sucrose esters were water-soluble sirups, having surfactant properties, prepared from reaction products of hydroxyethyl .ethers of sucro~eor diglycidyl ethers of poly(oxyethy1ene glycol) with (2,4,5-trichlorophenoxy) acetic acid or other herbicidd acids.Ml

+

+

[aso

V. GIBBERELLINS AND KININS Gibberellic acid and related compounds, referred to generically as gibberellins, form another class of naturally occurring, plant-growth regulators (265) C. Steinhart, L.Anderson, and F. Skoog, P h i Phgeiol., 81, 60 (1962). (266) H. G . Coore and P. J. Randle, Biochcm. J., Q1,bB(1964).

(267) G. A. Lewy, A. J. Hay, and J. Conchie, Biochsnr. J., 89, 102~) 103~(1964);Ql, 378 (l9M). (258) R.Brown and M. Edwarda, Ann. Botany (London), 8, 131 (1944). (269) R.Brown, A.W. J o h n , E. Robinson, and A. R. Todd, Proc. Roy. 8oc. (London), SW. B, i a ~ 1, (1949). (260) H.Domaneka and 1;. Eckstein, Romiki Nauk RolniugcA Ser. A, 88, 69 (1963); &gar Id.Abetr., 48, 671 (1964). (261) A. W.Andenson, U.S.Pat. 2,927,919(DowChemical Go.), imed March 8, 1960; Chem. Abelractu, 66, 14143 (1980).

PUNT-OROWTH SUBSTANCE8

417

or hormones. They produce cell, leaf, and stem extension, especially of dwarf mutants, induce flowering in longday plants during shorbday growth, and break dormancy in seeds and buds. Gibberellic acid salts are URed commercidly to induce flower-induction, to enlarge fruitrsize (e+ pecially in grapes), and to accelerate barley-mlting. The chemical structure

a

Gibberellic Acid (m. p. 232-235’ (dec.), [ u ] z +Q2.0’) (16)

according to Cross and co~orkers,2d8-*~~ namely (16) , as revised, has been derived from that of terpene precursors. The most obvious external effect of gibberellic acid on flowering plants is the rapid increase in shoot length. The total (green) weight of the plant may, however, increase, remain unchanged, or decrease, depending on the genotype, on the amount of applied chemical, and on the growth environment. The gibberellins are not particularly phytotoxic, even at excessive dosages, producing only minor rookinhibition and leaf chlorosis. The chemistry and growth responses have been extensively r e ~ e ~ e d . ~ l ~ ~ The effect of gibberellic acid on amylase in barley was first noted by Hayashi.*” The grains softened, releasing soluble carbohydrates into the medium, and increasing the quantity of extractable amylase without affecting the enzyme activity. The symptoms were analogous to those of the breaking of dormancy just prior to germination; maltose was formed from the starch reserve in the endosperm. If the embryo portion was cut away and discarded, gibberellic acid could replace the natural substances (from the embryo) which diffused into the endosperm under conditions (202) B. E. Crow, J. F. Grove, J. F. MscMillan, and T. P. C. Mulholland, Chem. I d . (London), 954 (1950).

(203)

B. E. Cross, J. F. Grove, P. McCloakey, and T. P. C. Mulholland, C h . Id.

(London), 1345 (1959). (204) G. Stork and H. Newman, J. Am. Chem. Soc., 81, 5618 (1959). (265) F. H. Stodola, “Source Book on Gibberellins (1928-1967),” Agr. Res. Service, U. S. Dept. of Agr., Peoria,Illinois, 1958. (266) B. B. Stowe and T. Yamaki, Ann. Reu. Plant Phyaiol., 8, 181 (1957). (267) T. Hayashi, Bull. Agt. Chem. Sw. Japan, 18, 531 (1940).

418

H. W. HILTON

favorable to germinstion. Gibberellic acid concentrations of from 2 X 10-4 pg to 2 X 104 bg promoted hydrolysis of starch; maltose accounted for 15%, n-glucose for So%, and D-fructose, sucroee~,and, probably, maltotriose and maltotetraose, made up the remainder of the reducing substances.268 With sterile conditions, oxygen uptake remained constant, and gibberellic acid activity WM due entirely to induced production of enzymes capable of hydrolyzing starch. The enzyme resembled amylase, rather than Bamylase, as measured by the shift in the wavelength maximum of the iodine reaction and by the heat stability of the enzyme from potato starch. At the optimum concentration of gibberellic acid (5.8 X 1od M ) at 30°, barley endosperms lost 50% of their dry weight in three days, and this loss was attributed to hydrolysis of starch, protein, and part of the cell-wall constituents. Eighty percent of the soluble portion could be accounted for as D-~IUCOSBor protein nitrogen. Iodoacetate and other enzyme poisons inhibited starch conversion by reacting with thiol groups of the enzyme,28Q and oxygen,270 Case, and n1K@ promoted enzyme activity. The effects suggested participation by several enzymes, although a-amylase is currently considered to be the major product.*s4 Evidence of cell-wall breakdown rests on the identification,in treated malt after seven days, of small amounts of arabinose and xylose which are not present in untreated malt.n6 The carbohydrates, separated chromatographically on carbon by gradient elution, inoreased (in the treated malt) in maltotriose (188%) ,maltose (33%) , isomaltose (13%) , D-glucose (820/,), wfructose (13%) , sucrose (16%), “glucodifructose” (5773, and unidentified di-, tri-, and tetrtcaaccharides. The stimulating effect on ketoses could not be entirely explained by a-amylase production. It is now supposed that gibberellic acid stimulates tho hormonal production of a-amylase (and, probably, of &amylase and others) in the aleurone layer surrounding the endosperm starch-reserve. Gibberellic acid released 400 mg. equivalents of wglucose per g. in 90 hours in wheat grains, with a maximum hydrolytic activity after 60 hours. The effect on pamylase was thought to be a release (to the extent of 85%) from the protein-bound, insoluble form.fl6 (268) L. C;. Paleg, Plant Phyeiol., 86, 293, 902 (1960); 86, 829 (1961). (269) J. E.Varner, Plant Physid., 89, 413 (1964). (270) D.E.Brigge, J . Iml. Brewing, 68, I3 (1963). (271) (272) (273) (274) (276) (276)

J. Yomo and H. Iinumlt, Agr. I3ioZ. Chem (Tokyo), 27, 70 (1963). L. (4. Paleg, D.H. B. Sparrow, snd A. Jenninw, Plant Physiol., 37, 679 (1962). L. G. Paleg, B. G. Coombe, and M. 9. Butt-, Phnf Phyttiol., 87, 798 (1962). L. G. Pdeg and B. Hyde, Plant Phy.iol., SB, 673 (1864). B. Drewa, H. Specht, and H. J. Pieper, Branntweinwirtschuft, 109, 377 (1963). E. V. Rowsell and L. J. Goad, Biochem. J . , 90, 1 1 ~ 1, 2 (1964). ~

PIJANT-QROWTH SUBSTANCE8

419

One paper (not seen by the present author in the original) suggested that gibberellic acid increased the growth in starch-containing plants (such aa taro, water chestnut, canna, and rice) by increasing amylase activity to make the reserve polysaccharide available. Plants lacking reserve starch (garlic, onion, and narcissus) did not respond.” Whether this increased hydrolysis would l e d to decreased (fresh or dry) weights fromthe loss of reserve, or to increases by a shift to other plysaccharides or proteins, fats, and 80 forth, is not apparent from the ab8tract.m Perhaps, favorable growth-conditions, temperature, day length, and other variables would influence the ultimate yield more than the available carbohydrate. Stem elongation induced by gibberellic acid is generally accompanied by a reduction in stem diameter, and by reduced and altered leaf shape and size. It seems unlikely that increases in dry weight occur with any regularity, except perhaps in the dwarfed genotypes. Effects on the starch content from applications to starch-storage crops have been variable. Soluble carbohydrates in other plants (such as sugarbeetsnaBnoand cabbagem) have been claimed to be increased, those in corn were increased”’ or decreaaed,m and they decreased in other p1ants.m In cotton, gibberellic acid so reduced the reserve polysaccharide that the cell-wall and protoplasm substances must have been metabolized to provide energy for celldivision activity. The reduction in dry weight accompanying elongation must have resulted in “diluted” or thinned cell-walls. Inhibition of cell division with indole-3-acetic acid had the opposite effect, of increasing starch and dry weight.” Reduction in starch and increased soluble carbohydrate coincided with a reduced uptake (40%) of mPin corn, in which duration of the effect depended on the concentration.% Photosynthetic activity, measured aa uptake of carbon dioxide, depended on the leaf area. Gibberellic acid did not influence photosynthesis per unit (277) S. Lo and H. Wang, Shih Yen S h g Wu Hmeh Poo, 8, (3-4), 576 (1963); C h . Abekoete, 60, 16433 (1964). (278) G. A. Evtuehenko, Akad. Nauk SSSR, Inst. Fizwl. Rast., 110 (1963); C h m . Abatr&, 60, 16067 (1964). (279) N. I. Yakushkina and E. K. Artemova, Akud. Nauk SflSR, Inst. Fiziol. f i s t . , 121 (1963); C h . Abstracfs, 80, 16067 (1964). (280) E. Ye. Ermolaeva, N. A. Koslova, and A. F. Bel’denkova, A&&. Nu& SSSR, Imt. Fizkd. h l . , 149 (1963); Chem.Ab8fr&s, 60, 16066 (1964). (281) I. L. Zakhar’yants and A. 8.Ioneaova, A M . Nauk SSSR, Inst. Fitiol. Raet., 161 (1963); C h m . Ab8trmt8, 60, 16066 (1964). (282) S. Iatatkov, lev. Inat. po Biol.MetodiiPopov, Bulgar. A W . Nauk., 18, 121 (1983). (283) L. A. Lebedenko, Acta Botan. A d . Sci. Hung., 9 (1-2), 86 (1963). (284) L. 8.Dure and W. A. Jensen, Botan. Om.,118,264 (1956). (286) Yu. P. Starchenkov, Vim. 8il18’kog~dar.Nuuki, 8 (7), 36 (1960); Chem. Ahtracte, 66, 1826 (1961).

420

H. W. HIWON

of leaf area, although gibberellic acid indirectly affected photosynthesis by altering the leaf area: treated rice did not change in leaf area, but decreased in its content of starch, whereas tomato increaaed its leaf area, but showed little change in the ratio of starch to reducing oarb0hydrate.m Gibberellic acid and (2 ,Uichlorophenoxy)acetic’ acid increased the glycoside content of Ormithogalurn umbeZEatum, as meeeured by a biological assay of extracts. The active principle, strophanthidin, is one of the cardiac g1ycosides.m There is little reference in the literature to this type of work, but the hydrolytic effects of gibberellic mid on starch may shift the equilibrium toward phenyl glycoside formation in some plant species. Various gibberellins exist as glycosides in the plant system. The 0-w glucoside of gibberellic acid waa prepared, M a noncrystalline substance, in cucumber leaves in vitro, as establiied by Bwglucosidase hydrolysis and by chromatography.mo”o A presumed P-glucoside has also been isolated from sugarcane as a gummy substance sohble in water.m Another compound, isolated originally from autoclaved or aged deoxyribonucleic acid, and identified as 6-(2-furfurylamino)purine (kinetin), (17) has a powerful initiating effect on cell division. The adenine moiety is

necessary; other replacements (such as phenyl, benzyl, and amyl) give products that are known generically as kinins or kinetinoids. Not one has been conclusively demonstrated in plants, but they are of considerable interest, as their activity resembles the action of red At an optimum concentration of lo-‘ M , kinetin acted similarly to gibberellic (286) T.Hayaahi, in “Plant Growth Regulation,” R. M. Klein, ed.,Iowa State Univ. Press, Amea, Iowa, 1901, p. 679. (287) P. Soresuchart, J. A. Smith, and G. R. Patarson, Can. Pharm. J., Sci Sect., 96, 496 (1962); C h .Abatmctu, 68, 4979 (1863). (288) G. Sembdner,G. Sohneider,J. Weiland, and EL.Sahreiber, Ezperientia, 10,89 (1964). (289) Y. Murakami, Botan. Mag. (Tokyo),74, 424 (1981). (280) B. H. Moat and A. Hughes, Ann. Rapt., Tat6 and Lyk Centml Agr. Rss. Sia., Trinidad, Weat Idkf,lW, p. 279. (291) H. Buratdim, in “Encyclopedia of Plant Phyaiology,”W. Ruhland, ed., Springer-

Verlag, Berlin, 1961, Vol. 14, p. 1166. (292) J. A. Zwar, M. I. Bruce, W. Bottomley, and N. P. Kefford, in “Regulateurs Nature18 de la Croiasanoe Vegetale,” J. P. Nitach, ad., Centre National de la

Recherche Scientifique, Paris, 1964, p. 123.

PWNTGROWTE SUBSTANCES

421

acid at 1W6M in stimulating amylase activity or production in wheat, without affecting phosphorylation. The 42% increased elongation of stem segments corresponded to that produced by D-glucose at 0.1 M ,suggesting that liberation of D-glucose from starch brought about growth as an indirect effect.2g8There is a conflicting view that kinetin stimulates the formation of polysaccharides in pea epicotyl at the expense of mono- and di-saccharides. The polysaccharide formation proceeded first in cellulose and othor cell-wall constituents, an8 in starch; considerable phosphorylase stimulation occurred in this case.294

VI. THEEFFECTS OF PUNT-GROWTH SUBSTANCES ON SUQARCANE Sugarcane is an intensively cultivated, perennial-grass crop grown throughout the world in a belt lying between about 30"N.and 30%. latitude. Its ability to store sucrose in the stalk over long periods of time (2 years or more), without a definite maturity or death, makes it one of the most efficient converters of the energy of sunlight into food. The rate of growth is not uniform throughout the plant's life, because of competition for nutrient and sunlight; and practical considerations make necessary the harvesting and processing of the crop at somewhat less than ultimate yield. Excess nitrogen and reducing carbohydrates are brought to a minimum at harvesting, because they are detrimental to processing, especially because of color formation due to heat, or interfelrencewith sucrose crystallization. Green leaves and apical stem tissue are undesirable, as they contribute a considerable amount of reducing carbohydrate, colored pigments, amino acids, and other organic substances detrimental to factory recovery of sucrose. Finally, the water content of the stalk should be lowered before harvesting, both for greater sucrose concentration in the stalk juice and for lowered D-glucose and D-fructose contents. The many aspects of sugarcane culture have been discussed by Burr and coworkers,@6and the physiological aspects of production of sucrose in the leaves, and its transport and storage in the stalk, have been detailed.2w-2ge (293) D. Boothby and 8.T. C. Wright,Ndute, 198, 389 (1962). (294) W. Maciejeweke-Potapcaykowa and H. Lukasiak, Ada Soc. Botan. Polon., 28, 96 (19S9). (296) G. 0. Burr, C. E. Hartt, H. W. Brodie, T. Tanimoto, H. P. Kortschak, D. Takahashi, F. M. Ashtan, and R. E. Coleman, Ann. Rev. Plant Physdol., 8, 275 (1957). (!!96) c. E. Hartt, Huwnaiian P&a&r8' &cord, 41, 33 (1937); 4 , 8 9 (1940); 47,113,166, ,223 (1943); 48, 31 (1944). (297) C. E.'Hartt.,H. P. Kortschak, A. J. ForbeB, and G . 0. Burr, Phn6 Physiol., 88, 306 (1963); (298) H. P. Kortschak, C. E. Hartt, and Q. 0. Burr, Plant Physiol., 40, 209 (1966). (299) K. T. Glasriou, Plant Phyuiol., 86, 896 (1960).

422

H. W. HIWON

Sucrose storage is an active process in which the stored sucrose in the stem cells is in equilibrium with small proportions of *glucose and D-fructose. Growth-promoting mechanisms (chemical or otherwise) are more apt to stimulate hydrolysis of sucrose, a d utilization of the monosaccharides in respiration and formation of new tissue, than to promote storage. In spite of this somewhat obvious conclusion, numerous attempts have been and are being d e to increase sucrose production both in sugarbeet and sugarcane by the use 6f growth-promoting, chemical treatments, Three areas have received the m?st attention: control of flowering in sugarcane to increase vegetative growth, desiccation of green leaves at harvest to lower the water content and the extraneous leaf trash, and control either of sucrose production or recovery by the use of chemical treatments, usually applied just prior to the harvesting period. The use of chemicttls for weed and insect control in sugarcane is common, but there is little information that shows that sucrose or yield is affected, unless injury is Revere and prolonged. Effects of externdlly applied chemicals on the living, intact plant are usually reversible, at least up to the point of extreme phytotoxicity. Chemical effects on (a) sucrose production, (b) equilibrium of sucrose with reducing carbohydrates, and (c) conversion into new tissue or respiration energy are alao likely to be reversible with time. The direction and amount of an effect will depend, as in other plants, on concentrations of the chemical, the stage of plant growth, the variety of plant, and environmental factors. The rate of growth determines the ultimate crop yield, but the rate of sucrose production may or may not follow the total-yield curve. This suggests that chemical treatment may aim either for increased total yield or for increased sucrose recoverable from the same yield. The latter might be accomplished by decreasing water, nitrogen, or reducing carbohydrates, ILS well as by increasing the percentage of sucrose per unit of dry matter. It seems obvious that there would be an osmotic limit to the sucrose concentration in the cell sap, and that more energy would be required to accumulatesucrose against an increasing concentration gradient. Sugarcane flower initiation is dependent on day length, temperature, age, moisture, and variety, and can be prevented by chemical applications at, or very close to, the date of floral initiation. The effective chemicals have been of two types: (a) photosynthetic inhibitors, such as 3- (p-chloropheny1)1, ldimethylurea (monuron), or (b) led-burning, contact chemicals. The very effective bipyridylium herbicides: 6,7dihydrodipyrido[l ,2-a:2’ ,1’clpyrazidinium dibromide (diquat) and 1 ,l’dimethyl-4,4’-bipyridinium bis(methy1 sulfate) (paraquat) combine the two properties, although the

PIA?N“+ROWI”T BUBSTANCES

423

mechanism for flower prevention is unknown. The herbicidal effect is temporary, and newly emerging leaves show no burn or chlorosis. Vegetative growth continues, resulting in higher ultimate yield than in flowering plants. The bipyridylium compounds have also been dsed experimentally to desiccate sugarcane leaves, and are more effective for this purpose than substituted phenols, oils, and chlorates. The yield of treated cane remained more or less constant for several weeks, and then increased again as new leaves formed. The yield of sucrose decreased as a result of treatment, partly because of the temporary reduction of photosynthesis, and partly as a result of increased stalk-moisture.800 Sugarcane can be artificially “ripened” near the harvesting period to increase tlucrose at the expense of water, nitrogen, and reducing substances. The choice of the term is unfortunate, for a plant which has no physiological ripening processes comparable to those of semnal plants. The st&moisture level decreases with age, from a maximum of 80-8501, to a minimum of about 70% in “ripened” cane. Sucrose formation is almost a mirror image of moisture content, varying from less than 10 to more than 45% of the dry weight. The older view has been that all factors affecting maturity or “ripening” do so by affecting the water content: increased water content results in decreased sucrose content.w1A more recent view is that nitrogen is 12180 intimately involved, and that “ripening” is essentially a reversible reaction which can be altered by water, nitrogen, growth substances, enzymes, decreased respiration, and photosynthetic a1teration.m Ideally, during “ripening,” photosynthesisshould continue while respiration decreases; however, photosynthesis is more sensitive to alteration than is respiration, and respiratory inhibition is likely to affect photosynthesis. Auxins stimulate both proceEses at low concentrations, but repress both at higher levels, although nothing is yet known as to the critical amounts present at the active site. Inhibitors of growth are likely to be more promising than stimulants for sugarcane “ripening.” Burr and coworkersm*observed that many factors have been found to decrease the formation of sucrose in sugarcane, but, up to that time (1956), the only factors which were known consistently to increase the formation Were a r i s i i temperature, improved aeration, and the addition of malic acid. (300) Reviewed by L. G. Davidson, Proc. Intern. Soc. Sugadane Techmlogiekl, 11, 319 (1962) (Publ. 1963). (301) S. V. Parthaeartithy and M. V. Rams Rao, Proc. Bien. Conj. Sugarcane Res. Workers Indkn Union, 1, Pt. 11, P-2, 5 (1951). (302) R. A. Yates and J. F. Bates, Proc. Brit. West Indies Sugar Technologists, 174 (1957).

424

H. W. HILTON

The auxins indole-&acetic acid and indole3-butyric acid, injected into sugarcane at the leaf base, stimulated adventitious rootdevelopment.aOa The same auxins plus l-naphthaleneacetic acid and 2-(o-chlorophenoxy) propionic acid, as soaking treatments for cuttings, lowered the reducing substances and increased the sucrose, perhaps by inhibition of sucrose phosphorylase. High concentrationsof the stme auxins reversed the carbohydrate effects." Invertase activation or synthesis by l-naphthaleneacetic acid, proposed by Sacher and Glaaaiou,806is reminiscent of the effect of gibberellic acid on a-amylase in starch-storage plants. However, indole-3acetic acid had the opposite effect on amylase in cotton.ou (2,4-Dichlorophenoxy)acetic acid and, to a lesser extent, the 2,4,5trichloro and other analogs have been tested and used on sugarcane more widely than any other chemicals. They are among the most common herbicides for dicotyledonous weeds in sugarcane, as they have very little effect on grasses. Beauchamp" in Cuba claimed to have found increases in sucrose averaging 0.5% on cane weight, from sprays of 1.25 lb. per acre applied to the soil 48 days prior to sampling. Five Ib. per acre decreased the yield of total carbohydrate by about the same proportion.amFoliar sprays or dusts, at even lower rates of application, all gave increases in sucrose after 20 and 40 days (over the untreated checks). Expanded testing, however, produced variable results; of 18 field tests, seven showed sucrose improvement, five showed decreases, and six were either not significant or inconclusive. Drought-stressed and mature sugarcane ftiiled to improve.m Beauchamp blamed over-mature cane and excessive applications of (2,Cdichlorophenoxy)acetic acid (about 1.5 lb. per acre) for the poor results in Puerto Rico reported by Loustalot and coworkers.MaBrazilian work in general confirmed the Cuban results, with increased sucrose, purities, and juice solids content up to ten days after foliar application of (2,4dichlorophenoxy)acetic acid salts and powders at low rates (of about 10 to 30 g. per acre). The discussion described a need for many precise experiments to provide rigorous, statistical analysis, but there is no published evidence of further testing either in Cuba or Indian work has tended to support the claims of increased sugar production, approximating 0.5% of sucrose on cane tonnage, from applications of (2,4dichlorophenoxy)acetic acid of from 0.025 lb. per acre to 3 lb. or more per acre, on different varieties, autumn- and spring-planted cane, (Ms) Anon., Prm. Htawaiian S W T Plantcre' A88OC., Rspf., 68, 30 (1938) (Publ. 1939). (304) W. C. Hall and M. A. Kahn,Bohn. Gtu., 116, 274 (1966). (306) J. A. Sacher and K. T. Glanziou, Biochdm. Biophp. Rss. Commun., 8, 280 (1962). (306) 9. E. Beauchamp, PTOC. A8m. Tec. A-T. Cubu, 48, 69 (1949); 24, 147 (1950). ($07) C. E.Bsauohamp, Sugar J., 13 (51, 57 (Oat. 19aO); 13 (61,20 (Nov. 1950). (308) A. J. Loustalot,, H. J. Cruzado, and T. J. Muzik, S U ~J., T 13 (5), 78 (Oct. 1950). (309) Anon., B t a ~ i Acucarciro, l 87, 328 (1951).

PLANT-OROWTH SUBSTANCES

425

irrigated and nori-irrigated, and from two to about 30 days after treatment.a’* *’) I t is difficult to arrive at a definite conclusion from this work. The major criticism which has been made of this and similar testing is that at least 15 samples are required in order to provide statistical significance at 0.5 70 sucrose based on cane yield. Variations in the yield from field blocks are commonly as high as lo%, and often more. Some of the demonstrated “increases” in the percentage of sucrose in the sugarcane have been accompanied by overall decreases in sugarcane yield per upit area. Other experiments have shown no effects on yield. In Jamaica,a no effect wm found from (2,4-dichlorophenoxy)acetic acid, but positive responses were obtained from low rates of application (less than 0.009 lb. per acre) of (2,4,5-trichlorophenoxy)acetic acid asd 2- (2,4,5trichlorophenoxy) propionic acid. Louisiana sugarcane failed to respond to the low rates of (2,Cdichloro- or (2,4,5-trichloro-phenoxy)acetic acid, but (2,4dichlorophenoxy) acetic acid at one and three lb. per acre increased the sugar yield significantly at eight days, although not at 25 or 39 days.S16 No significant differences in sucrose a t rates of 0.25, 0.5, 1, 2, 10, 20, and 30 lb. per acre were reported from Puerto Rico,117and from experiments in Australiaal*nl and in Hawaii,a22 where sucrose decreased at higher rates. Maleic hydrazide has been applied to sugarcane in numerous tests, because it was thought to slow terminal-growth without affecting photosynthetiis. Only one positive report has been madeaz8of 0.86% sucrose (310)S. C. Varma, Proc. B i a . Cmcf. Sugarcane Res. Deuel. Workers Indian Union, 2, Pt. 11, 626 (1954). (311) P.S. Mathur, Proc. Bien. Conf. Sugarcam Res. Devel. Workers Indian Union, 2, Pt. XI, 637 (1954). (312) A. 8. Chacravarti, D. P. Srivasteva, and K. L. Khanna, Sugar J . , 18 (6), 23 (Nov. 1955). (313)A. S. Chacravarti, D. P. Srivastava, and K. L. Khanna, Current Sci. (India), fu, 316 (1955);26, 302 (1956).Chm. Abstracts, 60, 17299 (1956). (314) A. S.Chacravarti, D. P. Srivastava, and K. L.Khanna, Proc. Inlcn. Soc. SugarCane T e c h n o ~ b9,, (1956),Val. 1, 355 (Publ. 1957). (316) A. 9. Chacravarti, D. P. Srivastava, R. D. Sahi, and K. L. Khanna, Proc. Zndiun Acad. Sci., Sect. B, 46, 9 (1957). (316) R. E.Coleman and L.P. Hebert, Sugar Bull., 86, 389 (1957). (317) M. A. Lugo-Upez and R. Grant, J . Agr. Uniu. P w ! o Rico, 86, 187 (1952). (318) H.C. Haskew, Cane &mers’ Quart. BuU., 17,52 (1953). (319) H. C. Haskew, Sugar J., 17 (l), 34 (1964). (320) L.G.Vallance, Ann. Rept. Bur. Sugar Exp. 8b.,Brdsbane, 66, 21 (1955). (321) Anon., AuakalQanSugar J., 47.37 (1956). (322) Anon., Hawaiian Sugar Planbe’ Assoc. Ezp. Sta. C m m . Rcpt., 67 (1956). (323) M.8.Subba Rm, M. H. Haque, R. B. Prasad, and K. L. Khanna, Current Sn’. (India), 26, 116 (1967).

426

H. W. HIXA'ON

increase per unit weight of cane on four varieties, using 100 mg. per liter spray solutions. No significant differences have been found in Puerto Rico:Z4 Louisiana?"JJamaica,8o2Hawaii,aB and Australia,8*6although in the ~ found at 49 days in young cane, laat country, small gains in R U O ~ Rwero but not in niore mature cane. l'ctmporrwy uxuewcs of BUOIUIJOiir the leaves and upper portions of the treated stalk call be accounted for aa being the result of depressed growth or poor trtmnslocation. Varietal, seamnal, and age effects cannot be ruled out, but it is obvious that little information of general applicability has been gained from this type of experiment. Such effects as have been noted appeared during younger, more vigorous growth of cane, not at the more mature, harvatabie age when the increases are most desirable. AlexandeP has tried to assess the effect of indole-3-acetic acid, (2 ,4 dichlorophenoxy)acetic acid , and maleic hydrazide on the soluble carbohydrates and enzyme systems in sugarcane leaves. All of the chemical treatments increased sucrose, total reducing value, wfructose, and D-glucose in leaves (compared with the controls), with a maximum at nine days after applying about two g. per plant. The indole auxin increased sucrose most, followed by the phenoxy compound and the hydrazide; D-glucose increased in the reverse order. Poor translocation from the leaves may have caused the temporary increase in leaf photmynthate. Alterations in the enzyme activities 811 a result of the chemical treatment are difficult to interpret, partly aince so little is known about their relative importance, and partly because the activity in the controls varied by as much tm 100% from one sampling period to the next. Many other compounds have been included in studies on sucrose response. Moat of these have been herbicides or ensyme poisons. None of the common herbicides had any positive effect on sucrose at rates up to that causing severe foliar injury. Earlier reports of response from 2-(2,4,5trichlorophenoxy) propionic acid and 2 ,2dichloropropionic acid could not be substantiated in British Guiana and &ueensland.Pn Some compounds, such as 3- (pchlorophenyl) -1,ldimethylurea (monuron), (2 ,Cdichlorophenoxy) acetic acid in soil, ethylenediaminetetrucetic acid, and leaf desiccants decreased aucroae and juice solids content.8s Field trials with several chemicals in Trinidad showed enhanced sucrose at 14 to 28 days before harvest resulting from the application of 8 and 12 lb. (per acre) of (324) M. A. Lugo-Lbpez, 0. Samuels, and R. Grant, J . Agr. Univ. Puerto Rico, 57, 44 (1983). (326) J. C. Skinner, Tech. Cmmun., Bur. Sugar Exp. Sh.,Briebaw, No. 1 (1960). (320) A. G. Alexander, J . Agr. Univ. P w d o R h , 49, 1 (19sa). (327) R.A. Yatee, Tropical A@. (London), 41, 2% (1964). (328) H. Evans and J. F. Batee, Proc. Intern. Soc. Sugat4aw Technobgiata, 11, (1962) 298 (1883).

PLANT-QROWTH SUBSTANCES

427

2,3,6trichlorobenmic acid or its mixture with (2-methyl-4-chlorophenoxy) acetic acid. One application by air, at four lb. of the mixture per acre, significantly increased sucrose at the 0.01 significant level at 28 days, but not at 9, 14, or 20 days. Lawrie believed that the trichlorobenmic acid was the main cause of the improved quality.aa No further report was made in 1963 or 1964, but this is the only report of positive response which k not been tested and reported elsewhere. Enzyme poisons have been studied mainly in attempts to understand the normal physiological processes. As would be expected, synthesis and accumulation of sucrose have often been drastically lowered. In particular, the inhibition of the formation of D-fructose diphosphate and the conversion of D-glucose into =fructose inhibited the formation of sucrose.290~a90 3-(3,CDichlorophenyl)-1 ,1-dimethylurea (diuron) treatment of a plant crop of sugarcane has been proposed as the cause of severely reduced crop yields and altered enzyme activities in the ratoon crop 25 months after the treatment, but without determining the presence of diuron residue. The sucrose level in cane was not significantly alter+, although the enzyme activities were considered in relation to their effect on sucrose synthesis. In particular, the amylase activity decreased while maltase increased."' The conclusion that, in sugarcane leaves, starch constitutes a primary source of &glucose for sucrose synthesis is not supported by the results of radiocarbon work~Ja2Jsa in which only a trawglycosylase could be demonstrated to synthesize sucrose by transfer of D - ~ ~ U C OtSo~ D-fructose from uridine 5-(~-glucosylpyrophosphate) The glucose is formed in sugarcane by direct photosynthesis. Gibberellic acid applied to stem cuttings before planting, to the leaves, or to the apex of growing plants affects elongation markedly, produ~in@*~*

.

(329) I. D.Lawrie, Ann. Rept. T a b and Lyla Central Agr. a s . Sta., Trinidad, Wed Indim, 96 (1961-2). (330) R. L. Bieleski, Ausbalian J . Biol. A%., 18, 221 (1960). (331) A. G.Alexander and J. G. Ibtifiez, J . Agr. Univ. Puarb Rko, 48,284 (1964). (332) C. E.Cardini, L. F. Leloir, and J. Chiriboga, J . Bid. Chem.,284, 149 (1966). (333) R.B.Frydman and W. Z. Hamid, Nature, 199, 382 (1963). (334)C. E.Chardon, Ann. Meeting Aseoc. Sugar TcchnicMIM, Puerto R h , 1966. (336) Anon.,HawaiMn Sugar Planted Aseoc.Ezp.Sfa. Comm.R@., 11 (1956);19 (1967). (336) R.E.Coleman, Sugar Bull., 86, 24 (1967). (337),R. E. Coleman, Sugar J., 20 (ll), 23 (April 1968). (338) R.E.Coleman, E. H. Todd,I. E. Stokes, and 0. H. Coleman, Proc. Infern. Soc. Sugar-Cane T e c h m ~ b10, , 688 (1969,h b l . 1980). (339) R.E.Coleman, E. H. Todd, I. E. Stokes, and 0. H. Coleman, Sugar J., 2S (3), 11 (Aug. 1980). (340) H. Chang and R. Lm, Rept. Taiwan Sugar Expt. Sta. (Taiwan), 28, 121 (1962), having an English summary and tables. (341) H.P.Varma and S. A. Ali, Indian S u p , 12, 636 (1983). (342)T. A. Bull, Awrtrdian J . Agr. Res., 16, 77 (1984).

TABLE II The ~

e t rof , G;bbenllr' c

Treatment

Cuttings soaked, or spray Apex, weekly treatdnents Spray, 3 treatments Cuttings soaked, leaf w y , soil drench a

Key:

Rderences

J3ffectsnon

Length

Apex, 4 treatme& at %week btervah

Acid on Sugarcane

+

+ (early1; +

nf4

(-1

+ (early);nf4 (finsl) + (early); (final) 119

Fnehwt.

Sucrose or

Per plant

quality increase

-

+

-

341

-

11s

336,337

-

+

+

9

342

2:

119

ns

119

335,340

L

119

ns

338,339

Girth

+, increase over control; -, decrease over control; ns, no aignificant difference.

T:

a

z

PIAVI"TR0WTH SUBSTANCES

429

increases in length of 30 to 100%. Girth and overall platit weight measuremcritn have hccn variable, as Ttiblc I1 shows. Variety, age, temperature, arid timing of applications produced differences. Multiple treatments at thrcc- to four-week iiitervals were needed in order to maintain significant differences for proloiiged periods. Treatments reduced tillering (suckering) and germination of buds, but did not affect flowering. Repeated sprayings produced length increases of up to 30 inches (over controls), but did not affect final yield, juice solids content, sucrose, or purity. Higher concentrations (loo0 mg. per liter) lowered the yields. Gibberellic acid responses appeared to be greater where growth conditions were least favorable and normal growth was suppressed.

VII. ABSCISSION AND RIPENING Defoliation and fruit- and flowerdrop are responses to auxin changes. A number of synthetic compounds, such rn l-naphthaleneacetic acid or its amide, l-naphthyl N-methylcarbamate, (2,4 ,Btrichlorophenoxy)acetic acid, and other phenoxyacetic and phenoxypropionic acids, are used for promoting or inhibiting fruit- and flower-set or -drop, for regulating fruit size and maturity, and for defoliating such plants as cotton or potatoes.'8-a"6 The auxins generally retard abscission, probably by stimulating growthprocesses rather than by a direct effect?4aThe carbamate compound promotes fruit drop, and higher concentrations of the auxins have a similar result. At optimal concentrations, the synthetic auxins apparently make up for a deficit of natural auxin, which is, perhaps, one of the effects of aging, leading to a breakdown of the cell-wall membranes, possibly by polygalacturonase or pectic methylesterase. From the carbohydrate point of view, the main point is that the cell wall (including the cellulose) finally dissolves, probably by enzymic hydrolysis; whether this 'occurs as the result of an auxin gradient across the petiole has been argued."'JU Nitrogen, sulfur, magnesium, and zinc deficiencies, and 'presence of oxygen, ethylene, and alanine stimulated abscission; carbohydrate applied distally (on the leaf side) delayed it. 2,3 ,bTriiodobenzoic acid produced effects opposite to those of auxin addition. Gibberellic acid promoted abscission at all concentrations, possibly by acceleration of protein hydrolysis and polysaccharide hydrolysis; kinetin (343) P. C Marth, W. V. Audia, and J. W. Mitchell, J . Agr. Food C h . ,7, 122 (1959). (344) F. Addicott, in "Encyclopedia of Plant Physiology," W. Ruhland, ed., SpringerVerlag, Berlin, 1901, Vol. 14, p. 829. (346) A. C. Leopold, Ann. Rev. Plant Phyeiol., B, 281 (1958). (346) W. P. Jacobs, M. P. Kaushik, and P. G. Rochmis, Am. J . Botany, 51,893 (1984). (347) B. Rubenstein and A. C. Leopold, PZud Phyeiol., 88, 262 (1963). (348) S. K. Chatterjee and A. C. Leopold, Plunf Phyeiol., 88, 268 (1963).

430

H. W. HILTON

had little effect, except to delay abscissionw at relatively high mncentrations of M. A relationship was found between the awin present and ethylene evolution, and it has been suggested that aoceleration of abscission may be due to ethylene.am The concentration of ethylene increaaes in fruit during ripening, and it is considered to be the natural ripening hormone.”ldM It is formed from G 5 and G6 of D-glucose, presumably through a triose, and BurgF-W believes it to be present throughout the life of the plant, but to be triggered to greater production by some regulatory mechdsm. The conversion of starch to soluble carbohydrate, associated with ripening of many fruits, is probably a result of the overall aging processes rather than a direct effect of the ethylene. Yang and How proposed that phosphorylase, not amylase, hydrolyzes starch to Dglucosyl phosphate, which then forms sucrose. The soluble pectin fraction increases. The concentration gradient across the fruit skin wm related by Bur@a2 to the rate of ethylene production by a factor of 2 ppm/pl./kg./hr., and the response was related to the log of the internal content of ethylene. Application of ethylene-“(7 to plants resulted in only a 2.4% conversion into soluble carbohydrates, 11% into ether-soluble materials, S.9yo into phytol, 31.7% into cellulose and lignin, and 9.6% into soluble protein and non-protein material, mainly ph0sphates.m Treatment of detached fruit (such ae apples, bananas, peaches, figs, and pears) with synthetic auxins, especially (2 ,4,5-trichlorophenoxy)acetic acid, speeded up ripening, aa indicated by color, taste, softness, and starch breakdown.” Other fruits have been similarly ripened,’oBe-861 and the treatments are effective both on climacteric and non-climacteric fruit. (349) (360) (361) (362) (363) (364) (366) (368) (357)

(368) (889) (360) (381) (382)

8.K. Chatterjee and A. C. Leopold, P h d P h W . , 80, 334 (1984). F. B. Abelea and B. Rubenetain, Phnt Phw&Z., 80, 903 (1984). 9. Y. Burg, Ann. Rev. Plant Phyaiol., 18, 286 (1982). 8.P. Burg and E. A. Burg, Phnt Phybiol., 87, 179 (1982). S. P. Burg and E.A. Burg, Science,l#, 1190 (1986). S.P. Burg, in “Ri3gulaburs Naturela de la Crohanae Vegetale,” J. P.Nitsch, ed., Centre National de la Reohemhe hientifique, Paria, 1984, p. 719. 8.Yang and H. Ho,J . Chiwe Chsm. Soc. (Taiwan), 6,71 (1968). W. C. Hall, C. 8. Miller, and F. A. H e m , in “Plant Growth Regulation,” R. M. Klein, ed., Iowa State Univ. Preea, Am-, Iowa, 1981 p. 761. J. W. Mitahell and P. C. Msrth, B&n. Ow., 106, 199 (1944). J. R. Blske and C. D. Steveneon, Qumdund J . A p . Sci., 18, 87 (1969). W. 8.Kemp and D. W. W h n , New Zsakrnd J . Ap., 01, 106,607 (1956). P. C. Marth and J. W. Mitahell, Bobn. Qaa., 110,614 (1949). P. 0.Marth, W. V. Audia, and J. W. Mitahell, B&n. Ght., 118, 108 (1958). W.B. Data and P. B. Mathur, Food Sci. (Mysore), 9, 248 (1960).

CHEMICAL SYNTHESIS OF POLYSACCHARIDES* BYI. J. GOLDSTEIN AND T. L. HULLAR Department of Biologiml Chemistr& Univweily of Mi&gan, Ann Arbor, Michigan, a d Departmen4 of M e d i c i d Chmiatw, S h l of P h i a m , State University of New York at Bug&, Buflalo, New York

Dedicated to the late Professor Fred Smith, who taught ua our carbohydrate chemistry. I. Introduction. ........................................... ............. 431 1. s c o p e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .433 ....... 11. Condensation Polymerization.. . . . . . . . . . . . . . .. .. .. . . . . . . . . . . .. .. . 434 1. General.. . . . . . . . .. . . . . ...... . . ... .. . . , .. . . .. .. . . .. . 434 2. Polymerization in a Solvent. , . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 441 3. Polymerization in the Solid State.... . . . . . . . . . . . . .. .. . . . . . . . .. . . . . . 461 111. Addition Polymerisation.. . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . , . . . . . . 477 1. General ..... ....... .. . . .. ........ .,.... .. .. . .. .... . . .. .. .. .. ..... 477 2. Anhydro sugam.. ...* . . .. . . . . . . . . . . . .. . . . . . . . . * .. . . . .. ., * . 478 3. other............................................................. 491 IV. Methods of Study. ... . . .... . . . . . , . .. . . . . . . . . .. . . .. . . . .. . .. .. . . . 491 1. Ieole~on.......................................................... 492 2. F ~ d i o ~ t i ...................................................... on 492 3. Meeaurement of Homogeneity. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 494 4. Structural Analysis. . . . . . . . . . . . .. .... ... . . . . . . . . . . . . . .. . . . 494 6. Use of Eneymea ,... . . . . . . . . . ... . . . . ... .. . . . . . . . . . . . . . . . . . . . 502 6. Use of Immunochemistry., . . . . . . . .. . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . 504 V. Applications of Synthetic Polysaccharidea.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

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

.. ..

..

.... .. . . .. . .. . ... . . . I

.

.

.

.

I. INTRODUCTION Over the pmt century, organic chemists attempting the chemical syntheRia of specific polysaccharides have invariably obtained polymers having indefinite composition. However, a polysaccharide has now been synthesized which apparently possesses properties similar to that of naturally occurring cellulose.1 Synthesis has also been achieved1*of an apparently linear polysaccharide containing only (1 -+ 6)-linked a-D-glucopyranosyl

* The preparation of ‘thisreview waa supported, in pert, by a grant (GM 12992201) from the National h t i t u t e a of Health, U.S. Public Health SeMce. (1) E. Hueemenn and 0. J. M. MWer, MakrmZ. C h . , 91, 212 (1986);privsta communication from E. Husemann. (la)E.R. Ruckel and C. Schuerch, J. Am. C h m . h., 88,2606 (1968). 431

432

I. J. QOLDSTEIN AND T. L. HULLAR

residues. If confirmed, these would represent the first successful, stereospecific syntheses of carbohydrate polymers. This subject has been of continuing interest for several reasons. First, the present concepts of the chemical constitution of such important biopolymers tw cellulose, amylose, and chitin can be confirmed by their adequate chemicd synthesis. Second, synthetic polysaccharides of defined structure can be used to study the action pattern of enzymes, the induction and reaction of antibodies, and the effect of structure on biological activity in the interaction of proteins, nucleic acids, and lipides with polyhydroxylic macromolecules. Third,it is anticipated that synthetic polysaccharides of known structure and molecular size will provide ideal systems for the correlation of chemical and physical properties with chemical constitution and macromolecular conformation. Fihally, synthetic polysaccharides and their derivatives should furnish a large variety of potentially useful materials whose properties can be widely varied; these substances may find new applications in biology, medicine, and industry. For some purposes, it is sufficient to obtain polymers (of high molecular weight) whose structural features are highly varied and generally undetermined. Considerable success has been achieved in synthe&i this type of polymer. For other purposes, however, it is highly desirable to obtain polymers which possess a defined structure. Chemical synthesis of such a structure requires that there be rigorous control of the position and configuration of the glycosidic bond being formed, the ring size of the incorporated sugar residues, the extent of branching (if desired) , and the molecular size and distribution of the polymers. Adequate control of these several variables requires that suitably protected sugars enter into a mild, facile method of polymerization which insures stereospecific synthesis of the glycosidic bond and formation of the desired ring size. Only very limited efforts toward this more demanding objective have thus far been made. In the biological synthesis of carbohydrate polymers, there is complete control of the factors noted above. Enzymes (transglycosyhes) effect the serial transfer of glycosyl groups, in a highly stereospecific manner, to the ends of a growing polymer chain, all transfers being under strict physiological conditions of pH and temperature? It is the duplication of these syntheses, md,’furthermow,the preparation of new, highly ordered carbohydrate polymers that will occupy the carbohydrate-polymer chemist in the foreseeable future. Because the chemical synthesis of defined polysaccharides is in a crucial stage of its development, and because these synthetic polymers and their derivatives show definite signs of having application in the biological (2) P. Bernfeld, in “Biogeneeis of Natural Compounde,” P. Bernfeld, ed., Pergamon P m ,The Maomillan Company, New York, N. Y.,1963,p. 233.

CHEMICAL SYNTHESIS OF POLYSACCHARIDES

433

sciences, it was considered appropriate to review the subject at this time and to assem the prospects for future progrm. Thia topic will be divided into condensation and addition polymerizations, aa defined in the subsequent discussion. Polysaccharides will arbitrarily be defined as polymers of ten or more eimple augar residues united by glycosidic bonds. Polymers containing less than ten such units will be considered to be oligosaccharides. In keeping with current usage, synthetic polysaccharides will be called “poIyglycoBe8,” for example, polyglucose, to d i a t i i h them from the naturally occurring polysaccharides.

1. scope Only syntheses which involve the formation of new glycosidic linkages will be considered in this article. This restriction excludes many interesting examples of copolymerization in which only one of the monomers is a carbohydrate (or carbohydrate derivative),Ithe polymerization of carbohydrate derivatives which contain a polymerizable group‘ (such aa acrylate) , and the polymerization of sugar 1actoncsP Many of these topics have already been discussed in reviews.6-8Ale0 outside the scope of this article is the chemical modification of naturally occurring polysaccharides~; thus, we have excluded the industrially important process of dextrinization,“Jexcept as it may pertain to acid condensation processes. The radiation-catalyzed polymerization and modification of carbohydrate poly(3) W, for example, J. M. van Bemmelen, J. Pmkt. C h . ,[l] 69, 84 (1856);M. L. Wolfrom, M. 8. Toy, and A. Chaney, J. Am. Chem. h c . , 80, 6328 (1958);M. L. Wolfrom, J. 0. WehrmWer, E. P.Swan, and A. Chaney, J. Org. Chum., 9, 1556 (1958);W. N.Haworth, R. L. Heath, and L. F. Wiggins, J. Chem. Soc., 155 (1944). (4) See, for example, P. L. Nichole and E. Yanovsky, J. Am. Chum. SOC.,66, 1626 (1944);R. H.Treadway and E. Yanovsky, ibid., 67, 1038 (1945);W. N. Haworth, H. Gregory, and L. F. Wiggins, J. C h . Soc., 488 (1946);T. P. Bird, W. A. P. Black, E. T. Dewar, and D. Rutherford, C h .I d . (London), 1331 (1960);8. Kimum apd M, Imqoto, Maktomd. Chum., 60, 155 (1961);R. L. Whistler, H.P. Pwer, and H. J. Roberta, J. Org. C h . ,26,1683 (1961); R.S. Theobald, J . C h . Soc., 6359,5365(1961);W.T.Bird, W. A. P.Blaak, J. A. Colquhoun, E. T. Dewar, and D. Rutherford, C b m . I d . (London), 1073 (1965). (6) H.D. K. Drew and W. N. Haworth, J . Chem. Soc., 776 (1927). (6)Yu. L. Pogoeov and Z. A. Rogovin, Rw8. Chum. Reu. (English Transl.), SO, 645 (1961). ’ (7) J. Klar, ChumiAw ZQ., 87, 731 (1963). (8)J. K.N.Jonerr, Pure A w l . Chum., 4,469 (1962). (9) B. J. Binee and W. J. Whelan, Chum. Id.(London), 997 (1960);B. J. Bines, Z. H. Gunja, and W. J. Whelan, ibid., 1358 (1960);R. L. Whistler and S. Hiram, J . Org. Chum.,26,4800 (1961);E. Husemano and M. Reinhardt, M a k r m l . Chum., 57,109,129(1962);M.L.Wolfrom, M. I. Taha, and D. Horton, J. Org. Chum., 28, 3563 (1963). (10)See, for example, “The Chemistry and Industry of Staroh,” R. W.Kerr, ed., Academic Press Inc., New York, N. Y., 2nd Edition, 1950,pp. 345,351;J. R.Kate, Rsc. Trw. Chim., MI, 565 (1934);B.Brimhall, Znd. Eng. Chum.,86,72 (1944).

434

I. J. GOLDIJTEIN AND T. L. HULLAR

mere is under active investigation, and has been surveyed in reviews.11 Three reviewe of the chemical synthesis of polysaccharideshave appeared.“* The historical aspects of the chemical synthesia of polysaccharideawill be considered in some detail. However, the main emphasis is on progress made during the paat 26 years. Selected examples of the synthesis of higher oliosaccharides are also included, since the methods used and the results obtained bear on the synthesis of polysaccharides. Discussion of modern analytical tools and techniques available for studying the nature of the synthetic products is included and, where possible, application of these techniques to the study of synthetic polysaccharides is described. 11.

CONDENBATION POLYMERIZATION

1. General Condensation polymerization has been defined by Floryl2 as a “polymerilitition process which proceeds by a reaction between pairs of functional groups with the formation of a type of interunit functional group not present in the monomer.” In this review, condemalion polynerization of carbohydrates is defined as a polymerization of eugm or their derivatives to give a polymer of sugar residuea joined together by glycoddic bonds and a by-product composed of the conjugate acid of the substituent originally present on the anomeric carbon atom.’* Thie definition may be illustrated by equation 1 , where HO is an (the) alcoholic hydroxyl group of a sugar moiety, X is the substituent on the anomeric carbon atom, G is the remainder of the sugar moiety, and G-4-G is the newly formed glycosidic 12 HO+X

HW-(O--G-),+-X

+ ( n - 1) HX

(1)

linkage. In the majority of the condensation polymerizatiom of sugars or sugar derivatives, X haa been a hydroxyl group; lesa frequently, it haa been a halogen atom or a carboxylic acid group. Before discussing the various methods used to achieve condensation polymerization of sugars and sugar derivatives, several factors will be (11) [Joe, for example, J. B. Snell, J . Pol-

Sci., Pt. A, 8, 2591 (19615);F. A. Bovey, “The Effects of Ionizing Radiation on Natural and Synthetic High Polymers,” Intemcience Publiuhers, Ino., New York, N. Y., 1958; a. 0. Phillip, Aduan. Ca&ohydv& C h . ,16, 13 (1881). (12) P. J. Flory, “Principles of Polymer Chemiwtry,” Cornell Univereity Prees, Ithaca, N. Y., 10153, p. 39. (13) For oonvenience, “polycondenaation” will be used as a synonym for oondeneation polymerbation, and “polycondeneate” will be ueed aa a eynonym for the product of a condewtion polymerbation.

CHEMICAL 8YNTHESI8 OF POLYSACCHARIDES

435

considered which iduence the course of the polycondenaation and t.he structures of the products. First, condensation polymerization of sugars and their derivatives is an equilibrium process (see, for example, equation W) , a nearly stoichiometric a C&O6

S

+ (a - 1) HsO

C~l,O,(C,H,,O,),,C3llOI

(8)

proportion of a by-product (for example, water) being liberated. Zechmeister14 suggested that the polycondensation of sugars is a reversible process; this was confirmed by Frahm,16 who determined the reducing values of solutions of D-glucose and cellulose in 40.8% hydrochloric acid, and found that both solutions reached the same equilibrium. To support further the idea of revenibility, Frahm" found that a 57% solution of D-glucose in,40.8% hydrochloric acid gave a reducing value 35y0 that of wglucose. Dilution of this solution to 15% of D-glucose in 40.8% hydrochloric acid gave, at equilibrium, the same reducing value (68% that of D-glucose) a8 wa8 obtained when a solution of 15% of D-glucose in 40.8% hydrochloric acid was allowed to reach equilibrium. This increase in reducing value upon addition of water corresponds to a decreaae in molecular size, and is the expected result if equations 1 and W are governed by the law of maas action. The position of equilibriumwas examined further by Silin and Srtpegina,l'J They found that both R 20% solution of wglucose and its polymeric equivalent, an 18% solution of starch, in 0.5 N hydrochloric acid, gave at equilibrium a reducing value 91.8% that of D-~~UCOSELFrom a solution of D - ~ ~ U C O in S ~ 75% sulfuric acid, a mixture of oligomers OF' p,, 5 waa obtained.11) Efficient removal of the by-product, HX, of a polycondenaation will shift the equilibrium of the polymerbation so as to provide high yields of material of high molecular weight. The efforts directed to this goal will be discussed in Section II,2 and 3 (see pp. 441 and 461). Second, the positions of the linkages between sugar residues of a polymer from a polycondensation are dependent on the number and reactivity of the hydroxyl groups which can enter into glycoside formation, (14) L. zechmeieter, 2.Physik. C h . ,108,316 (1922). (15)H.Frahm, Ber., 74, 622 (1941). (16) P. M. Silin and E. A. Sapegina, Tr. Voronashsk. Khirn.-Teknol. Inst., 8-4, 79 (1939);Clrtnr. Abstrads, 86, 8338 (1941). (17)Abbdationn employed throughout are: D.8., average degree of substitution; number average molecular weight; aW, weight average molecular weight; and p,, average degree of polymerbation. (18) P. N. Odincova and A. I. Preobraehenskii, Latvdjoa P8R Zinalnu A M . V C S l i S , lB66,No. 2,73;Chem. Ahlmds, 60, 15107 (1966).

1

a,,

-'

436

I. J. QOLDBTEIN AN9 T.

?J.HULLAR

An uneubstituted hexose, such m D-glucopyranw ( l ) , conbins four alcoholic hydroxyl groups and one hemiacetal hydroxyl group. Such a polyfunctionallg monomer can polymeriae to give a multibranched polymer, such 88 (2). A trisubstituted hexose, such as 2,3,&tri-O-(N-phenylcontains only one alcoholic hydroxyl carbamoyl)-D-glucopyranose (3)

OH

-

OH (11

OH

OH

(2) (after Erlander and Frencha1)

group and one hemiacetal hydroxyl group. This bifunctional monomer can give rise only to a linear polymer (4). However, even with a monomer such as (3), the synthesis of polymer (4) ia not assured, since it must be assumed that, a t lesst at the outeet, no (141) (trehaloee type) linkages are formed and that no isomeriaation or removal of the protecting groups occurred during the reaction. The actual position of the linkagea in a branched polymer, such as (2), will depend on the relative reactivities of the hydroxyl groups. These reactivities are known to be different both in monomeric and polymeric carbohydrates.a* In a studya of the condenaation polymerization of un(lQ)W . 11. Carothers, Tram. Faraday ~ o c . ,82, 39 (1936); C h . Rev., 8, 353 (1931); Ilef. 12, pp. 31-32. (20) E. Huuemann and G . J. M. Mllller, Makromol. CAem., 4@,238 (1961). (21) 8.Erlander and I). French, J . Polyiner Sci., SO, 7 (1956). (22) For a genersl review of the relative wactivitiea of the hydroxyl groups of carbohydrates, aee J. M. Sugihara, Aduan. Carbohydrate C h . ,8, 1 (1953). (23) H. Frahm, Ann., 666, 187 (1944).

CHEMICAL SYNTHElsIS OF POhYSACCHARIDES

437

OR 0

II (S), R = -CNHC,H,

I

(4), R =

-8CNHC,H,

substituted and partially methylated D-glucose in 40.8% hydrochloric acid, the reactivity of the C-6 hydroxyl group was found to be approximately four times that of the C-2, C-3, or C-4 hydroxyl groups. The predominance of ( 1+6) -1inkagea in polyglycoses haa been repeatedly observed. The lower reactivity of the C-3hydroxyl group, relative to the C-2 and C-4 hydroxyl ~ the reactivity of the several hygroups, has been n ~ t e d . * ~In. *addition, droxyl groups may ohange during the course of polycondenaation. Methylation studiee of polysaccharidea under alkaline conditions ahowedl6 that alkylation of one hydroxyl group altered the reactivity of the adjacent hydroxyl group. Such altered reactivity may also prevail in polycondensations of free augara. For example, the reactivity of the G 3 hydmxyl group of a reeidue such as (5) may be different from that of the C-3 hydroxyl group of a rssidue such as (6).The relative reactivities of these hydmxyl groups wiIl depend on their location on the sugar residues,* the substituents on the adjacent carbon atoms, and their location within the polymeric matrix. Thus, any statistical treatment211wof the polyconden(24) A. Bhattacherya and C. Schuerch, J . Org. Chem., 36, 3101 (1961). (25) G. G. 8.Dutton and A. M. Unrau, Can. J . Chem., 41, 2439 (1963). (26) I. Croon, S u m k PaPpe?’.sttid.,88, 247 (1960). (27) P.J. Flory, J . Am. Chsm. h c . , 74,2718 (1952); Ref. 12, pp. 102-103, Chaptar IX.

438


sation of polyfunctional sugar monomers must allow for the differing reactivityll of the hydroxyl groups instead of considering them to be of equal reactivity,* even though the differenoes in reaativity may be constant throughout the polymerimtion.

0

HO

0

HO

I

OH

0(5 1

(6)

Third, the ring size of the constituent glycosyl residues of a polyglycose will depend on the condition5 used for the polymerization. Thus,mixtures of 5- and 6membered ring-forms are formed, and transformed into one anotherJo)under conditions of acid-catalyzed, glycoside formation. It is, therefore, not surprising that both furanosyl and pyranosyl residues have been found in the acid-catalyzed, vacuum polycondensation of ~glucoses (see Section IIJ3,d).Even a protected sugar [such as (3), which possesses a C-4 hydmxyl group] a n exist and be polymerized aa a mixture of ring forms (equation S), the pyranoid form probably preponderating. Acyclic RO&C

Q;

HO

OR

sugar residues can also be incorporated.*'*" This isomerization of ring forms can often be avoided by utilizing monomers whose ring size is fixed

(28) C. T.Bishop and F.P. Cooper, Can. J . Chem.,40,224 (1982); 41, 2743 (1963). (29) G. G. S. Dutton and A. M.Unrau, Can. J . Chm., 40, 1198 (1982).

CHEMICAL SYNTHE8IS OF WLYSACCWARIDES

439

by the p m n c e of a suitable substituent at C-1. Thus, formation of the glycoaidic bond by direct displacement (equation 4) of the C-1 substituent or by effecting displacement a t C-1 through neighboring-group participation,’OJ1followed by attack with hydroxyl (equation 6 ) resulta in retention of the ring form present in the monomer, since, in neither caae (equations 4 and 6), would the expected transition state allow interconversion of ring forms.*2

mr;

I

1

Fourth, the configuration of the glycosidic bonds in the polyglycose will depend upon the polymerization conditions. In an acid-catalyzed polycondensation of an unsubstituted sugar, the intermediate, a stabilized carbonium ion (7), reacts with a hydroxyl group to give the thermodynamically controlled mixtun9 of anomeric glycosides (equation 6 ) .

A more stereoselective formation of the glycosidic bond, assuming nonequilibrium conditions for the polycondensation, involves the use of a large counter-ion (Ae).a4 The intermediate carbonium ion (7) would be stabilized preferentially from the least hindered side of the ring tw in (8) and, comequently, would furnish one glycoside anomer such as (9) (see Section (30) B. R. Baker,J. P. Joseph, R. E. Schrtub, and J. H. Williams, J. Org. C h . ,19,1786 ( 1954). (31) For a recent, general review of this important mbject, see B. Capon, @art. Rev. (London), 18, 45 (1964). (32) Compare with C. G. Swain, J. Am. Chem. Boc., 14, 4578 (1950); C. G. Swain and J. F.Brown, ibid., 74,2534 (1952). (33) R. U. Lemieux, in “Molecular ftearrangements,” P. de Mayo, ed., Interscience Publishers, New York, N. Y.,1964, Vol. 2, p. 735. (34) J. Kope and C. Schuerch, Proc. Cellulose COY$.,6th, Syacuse, (1965); J . Polymer rsn’., Pt. C., 11, 119 (1966).


440

1142,b). Stabilization of the carbonium ion can also be achieved by neighboring-group participation.a1 Thie waa probably the principal factor operative in the condensation of (3) to form a largely, possibly completely, B-D-lhked polymer' (equation 7). Incidentally, if the conversion of (3) into (3a) occurred by protonation of the C-1hydroxyl group followed by

OH -

ROH$C

n

H

n

OR

[.@) OR

n

neighboring-group displacement of water by the N-phenylcarbamoyl group. (as seems likely), the pyranoid form would be retained, as in (10). Defined stereochomistry a t the anomeric carbon atom is also possible of achievement by means of displacement reactions at C-1 (see equations 4 arid 6 ) . Only oligomere containing &D-( l-S)-linkages were obtained when

441


2,3,4-tri-O-acetyl-a-~glucopyranmyl bromide (11) was subjected to selfcondensation under Koenigs-Knorr conditions, giving (12).'O

QBr-:H0W

HOH$

RO

OR

(ll), R = Ac

OR

(12),

R = AC

In all successful syntheses of polysaccharides, it is essential that the equilibrium be so shifted as to afford good yields of material of high molecular weight. The extent to which the other factors must be controlled will depend upon the type of polymer deaired. 2. Polymerization in a Solvent a. Aqueous Solution.-Polycondeneation of wglucoee was probably' obtained first by Berthelot" by heating D-glUCOSe with sirupy phosphoric acid. M u s c ~ I u found s ~ ~ ~that ~ treatment of wglucose (20g.) with concen[.ID 131 trated sulfuric acid gave a white, hygroscopic product (10g.) ( to 134") possessing only slight reducing power. Only 0.54% of this product dialyzed through parchment paper in 24 hr., being similar in this respect to the so-called ydextrin which Musculus obtained by the action of diastase on starch. The material was hydrolyzed to wglucose by dilute acid, but misted fermentation by yeast. Similar products were obtained by the action of gaseous hydrogen chloridea and of heat (170°)'* on D-glucose. It was found" that treatment of cellulose, starch, or =glucose with concentrated sulfuric acid a t 30-35' gave similar products having optical rotations of +llO" to +138". Chemical evidence for the condensation of Dglucose units under strongly acidic conditions was obtained when FischeP isolated

+

(36) G. ZemplOn and A. Gerece, Ber., 64,1646 (1931). (36) S. Haq and W.J. Whelan, J . Chm. SOC.,4643 (1966); W.J. Whelan and S. Haq, Chem. I d . (London), 800 (1966). (37) M. Berthelot, Ann. chim. et phyu., M, 74 (1868). (38) M. Muaculw, Bull. SOC.Chim. (France), [2] 18, 66 (1872). and A. Meyer, Bull. SOC.Chim. (France), [2] 96,368 (1881); Ber., 14, (39) M.MUECU~UE 850 (1881): (40)A. Gautier, Bull. SOC.Chim. (France), [2] 22, 145 (1874); Ref. 7, footnote 36. (41) See Ref. 7, footnote 67. (42) M. H.H6nig and H. 8. Sahubert, Monatsh., 7,466 (1886). (43) E.Fischer, Ber., 09,3687 (1890); 18,3024 (1896).

442

I. J. QOLDBTEIN AND T. L. HULlrhR

a disaccharide44 (as its osazone), together with dextrinous material, dter D-glucose was treated with concentrated hydrochloric acid. This result was confirmed by Szheibler and Mittelmeier?‘ who employed hot, dilute sulfuric actid to obtain tho same disac!aharido from ~gluc!ostt. Crimaux and I~fevrc‘’ obtaiiiod UII cllaohol-pwoipilablc iiiutcrial wlieii a solution of n-gluco& in five per cent hydrochloric acid waa evaporated under diminished pressure on a waterbath. The properties of this material were simiIar to those of the dextrins obtained from the hydrolysis of starch. WohP extendcd these observations by studying the effect of dilute acid on D-fructose, D-glucose, sucrose, and starch. He observed that the hydrolysis of polysaccharides is not a firstcorder reaction, since the monosaccharide units condensed in acid to form polymeric materials. Moel~yn-Hughes~~ drew similar conclusions, and obtained optical rotatory evidence for the self-condensatim of D-glucose in N hydrochloric acid. WohP wea the first to refer to the formation of higher saccharides by the action of acids on monosaccharides as “reversion.” This process, the acidcatalyzed formation of glycosidic bonds between sugar residues, constitutes the most rudimentary form of condensation polymerization. Reversion of oligosaccharides has also been observed.@Jl The relationehips between the hydrolysis of polysaccharides and the reversion of mono- and oligo-saccharides is illustrated for amylose in Fig. 1. Since these early studies, the reversion of carbohydrates has proved to be of considerable importance in the hydrolysis of starch:*@ in the elucidation of polysaccharide structure,w*61*- and in the synthesis of oligonamed iaomaltoee by Fischer.u Later studies showed46 that the dieanbride waa more likely to have been gentiobiose, not iaomaltose. (46) H. Berlin, J . Am. C h . Soc., 48, 1107, 2627 (1926); G. H. Coleman, M. A. Buchanan, and P. T. Paul, ibid., 67,1119 (1936). For a summary of the hietory of the structural elucidation of hmaltoee, we M. L. Wolfrom, L. W. Georgee., and I. L. Miller, iW.,71, 126 (1940). (48) C. Scheiblor and H. Mittelmeier, Ber., 44, 301 (1891). (47) E. ctrimaux and T. Lefevre, Cmnpt. Rend., 108, 146 (1886). (48)A. Wohl, Ber., UI, 2084 (1890). (49) E. A. Mdwyn-Hughee, Trans. Fcrroday Soc., 46, 603 (1928). (60)D. J. Manners, G. A. Mercer, and J. J. M. %we, J . Chem. Soc., 2150 (1866). (61) K. Taufol, H. Iwainsky, and H. Rutloff,J . Pmkt. Chum., 4, 89 (1956). (62) W. R. Fetzer, E. K. Croaby, C. E. Engel, and L. C. Kirst, Ind. Eng. Chem., 46, (44) The diaaccbaride was

1076 (1963).

(63) G, Graefe, Lsbrke, 4, 27 (1960). (64) A. Thornpeon, K. Anno, M. L. Wolfrom, and M. Inahme, J . Am. C h m . SOC.,76, 1309 (T954). (66)A. Thompson, M. L. Wolfrom, and E. J. Quinn, J . Am. Chcm. SOC.,76,3003 (1953). (66) 8.Peat, W.J. Whelm, T. E. Edwards, and 0. Owen, J . Chem. Soc., 586 (1968).

CHEMICAL 8YNTHESI8 OF POLYBACCHARIDES

Hydrolysis

Reversion

Amylose

Polysac,$Kuides

Maltodextrlns

FIG.1.-Relationship

-

443

it

Higher Ollgoraccharides

Between Hydrolysis and Reveraion of Some Carbohydrates.62

saccharides.” The extensive literature that haa arisen because of this interest puts a complete discussion of reversion beyond the scope of this article.68 Consequently, only a few selected, synthetic applications of the reversion reaction i ~ i lbe l considered. As mer;tioned above, a disaccharide, probably gentiobiose, was obtained by FiecheP and Scheibler and Mittelmeiefl when D-glucose was treated with concentrated hydrochloric acid or dilute sulfuric acid. An oligosaccharide fraction, characterized as a tetramer, was obtained by Myrback and eovorkerso2when D-glucose was treated with 3740% hydrochloric acid. Methylation analysis suggests that the fraction had a branched structure. By using chromatographic methods, some of the disaccharidea obtained by treatlnent OP D- and mwabinose,‘.86 wxyloseJw ~galactose,” and D-mannose&with concentrated acids have been characterized (see Table I). Virtually all of the possible disaccharides of D-glucose have been isolated from the reversion products of D-~~UCOR?*** In addition to lower oligosaccharides, condensation products having high molecular weight were also obtained by early workers. Thus, by subjecting (67) J. Stan&, M. Cerng, J. Kocourek, and J. PacBk, “The Monosaccharidea,” Academic Prm Inc., New York, N. Y., 1963, pp. 110-111. (58) For reviqwa of reversion, aee Refs. 8, M)-67,6981; and K. MUer and K. Taufel, 2.?~bmem.UnteraZlch.-F~~ch., 100, 437 (1966). (59) J. C. Sowden and A. S. Sprigga, J . Am. C h . SOC.,18,2603 (1966). (60)P.S. O’Colla, E. E. Lee, and D. McGrath, J . C h . &c., 2730 (1962). (61) H. 13. Schlubach and E. Lilhra, Ann., 647, 73 (1941). (62) K. Myrbbck, M. Hammarstrand, and H. Gelinder, Atkiu Kemi, 1, 235 (1949). (63) F. A. H. Rice, J . Am. Chem. Soc., 78, 6167 (1956). (04) L. Hough and J. B. Pridham, C h . Id.(London), 1178 (1967). (65)J. K. N. Jones and W. H. Nicholson, J . C h .Soc., 27 (1958). (06) D. H. Ball and J. K. N. Jones, J . Chsm.rsoC., 33 (1968). (67) C. N. Turton, A. Beddington, 8.Dixon, and E. Pacau, J . Am. C h . Soc., 77,2506 (1955).

444

1. J. QOLDllTlilIN AND

T. L. HULLAR

TABLE I Preparation of Dinaooharidesby Reversion of Monosaccharidee

Monosamharide

Disamharides identified

References

~-Arabinosea

&D-Arap(l 4 3)-D-Ara

63

i.-Arsbinoeeb

p t A r a p ( l + 3)-cAra p t A r a p ( 1 4)-~Araa &x.-Arap-( 1 + l)-StArapc

64,66

D-Xyloseb

D-Galactose“

67

a-~-Manp-(1 + @&Man & ~ - M a n p1( -+ @+Man i3+Msnp-( 1 -+ 3)-~-Man & ~ - M a n p1 ( 4)-~-Mana

D-Mmnd

-

65

a I n 20 N sulfuric acid. * In 6 N hydrochloric acid. 0 Tentatively identified. d In 11.7 N hydrochloric acid.

D-glucose to the action of fuming hydrochloric acid, Ost@obtained a preparation which had [ a ] ~ 124’ and a reducing value 11.3% of that of maltose. The so-called “isomaltoee” of Georg and Pictetegwaa prepared in the same way. This preparation waa later stated by Zempldn and Bruckner’o to be a mixture of a disaccharide and higher saccharides; the higher saccharides were subsequently considered61 to be similar to the polyglucose prepared by the action of hydrogen chloride gas on p.glucose.el Reversion of D-glucose in 75% sulfuric acid at 20” gave polymeric material which, after fractionation, showed i?, ranging from 2 to 18 and1*averaging 5. In 75% sulfuric acid, reversion waa slower than in 41% hydrochloric acid; bowever, products of higher polymeriaation were obtained in the sulfuric acid system.71

+

(68) H. Oat, Chemiker Ztg., 19, 1506 (1895);2. A n g ~ C. h . ,17, 1663 (1904); H. Oat and T. Broedkorb, Chcmiksr Ztg., 86, 11% (1911). (69) A. aeorg and A. Pictet, H&. CAim. Ado, 9,612 (1926). (70) 0. Zemplbn and Z.BrucLner, Ber., 64, 1862 (1831). (71) P. N. Odinoova and A. I. Preobrashenskii, Ldvijccr P8R Zinatnu Akad. V d 8 ,

1966, No. 2 (Whole No. 91), 43; Chem. Abtrads, 49, 16393 (1968); ibid., 1966, No. 11,M; C h . A W o d s , 60,14329 (1966).

CHEMICAL BYNTHESIS OF POLYBACCHARIDES

445

A three-step procedure7*has been employed by Merck and Company to obtain a plyglucose suitable for use as a plasma volume-expander. A 50% D-glupose solution (w/v) containing 1 mole yo of phosphoric acid (based on D-glucose) was hcatctl for a period of approximately 20 hours at 150160"/5-10 mm. pressure. The partially water-soluble, crude polymer was then degraded in an unspecified manner, to give a yellow powder which was fractiomted by precipitation from aqueous alcohol. The polyglucose contained 0.244% of phosphorus which appeared to be in the bound form. The polymers were undoubtedly of high molecular weight, and this, perhaps, accounted in some measure for their limited solubility in water. As pointed out in Section II,l, polycondensation of sugars in aqueous acid is a reversible process made inefficient by the presence of water. Polycondensation can be conducted more efficientlyin anhydrous solvents, with the removal of the evolved water. Alternatively, it may be carried out by using derivatives which possess a t C-1 nonhydroxylic substituenta that, on displacement, may be conveniently removed from the reaction system. These methods will be discussed in turn.

b. Non-aqueous Solution-Some polycondensations conducted in partially aqueous media fit more conveniently into this Section and will be discussed here. A short review of acid-catalyzed synthesis of oligo- and poly-saccharides has appeared.7a Liquid hydrogen fluoride was found to convert filter paper into a pulpy m.ma.74 Larger proportions of anhydrous hydrogen fluoride dissolved the filter paper completely within a few seconds76to give D-glucopyranosyl fluoride.76 The polymeric products derived from treatment of cellulose with hydrogen fluoride were studied by Helferich and Btittger." In the presence of anhydrous hydrogen fluoride a t 30", cellulose is transformed into a polymeric product, called celIan.n This substance is water-soluble, strongly dextrorotatory ( [.ID 143'), completely converted i n k D-glucose on refluxing with dilute mineral acid, and only faintly reducing. Molecular-weight determinations (by cryoscopy) on cellan acetate ( [ a ] ~ 128') and on the regenerated polymer indicated it to have a p , of 14. When treated with hydrogen fluoride, D-glucose was converted, in 90% yield, into 9 polymeric material," similar to cellan in its solubility and

+

+

(72) A. J. Zambito, Merck and Co.,Inc., Private oommunication. (73) S. Suauki, Tobku Yakka Daigaku Kiyo, 6, 1 (1958); Chem. Abstracts, 63, 10053 (1969;. (74) J. Gore, J . Chem. h e . , 22, 396 (1809). (76) K. Fredenhagen and G.Cadenbach, 2.Anorg. Allgem. C h . ,178, 289 (1929). (76) K. Fredenhagen and G.Cadenbach, Angeur. C h . ,46, 113 (1933). (77) B. Helfeiich and A. BBttger, Ann., 476, 150 (1929).

446

I. J. GOLDSTEIN AND T. L. HULLAR

optical rotatory properties. However, the molecular weight of the acetate and regenerated polymer showed the P a to be approximately 7. The action of anhydrous hydrogen fluoride on cellobiose gave a polymer similar to that obtained from D - ~ ~ U C O S ~ . In an amlogous fashion, starch in hydrogen fluoride at -20' gave a polymer ([.ID 145O), called amylan," which was similar to cellan. However, amylan differa from cellan in the higher rotation ([a]D 142') of its acetyl derivative and in ita precipitability from aqueous soIution by ethanol. Under similar treatment with hydrogen fluoride, maltose gave a polymer, apparently identical with amylan. The similarity in properties of these several preparations suggeets a similarity in structure. The high, positive rotation is indicative of a preponderanc2of a-D-glucosidic linkages, and the isolation of a large proportion of 2,3,4,6-tetra-O-methy1-~-glucose from the hydrolytic fragments of methylated cellan" showed the polymem to be highly branched. The formation of polymera of Iow molecular weight derived from P-glucose and cellobiose may be due to the greater proportion of water released during condensation, preventing formation of higher polymers. Alternatively, the higher P, of cellan may be due to ita being derived largely through traneglucoeidati~n.~~~~~ Russian workeram found that cellulose and glucose furnish ensantially identical products on treatment with 93% hydrogen fluoride. Analysis of the fractions showed that 7% of the product had a P, of approximately 10 (by iodine and copper numbers) and [a]~ 84'; 20% had a P n of approximately 5, [a]~ 141'; and the remainder had a P n of 2, [a]D 152'. An a-D-(l-b6)-gluaosidic linkage was considered responsible for the high, positive rotation of this "biose" fraction. Polycondensrttion of D-glucose (10-20% concentration) in 45%870 hydrogen fluoride at 10-30" furnished a product consisting of 3-5 reaiduea at equilibrium.** Early studies by mu soul us^ indicated that concentrated sulfuric acid effecta polymerizatiou of ~glucose.Nabmuram found that optimal conditions for this polycondmtion involve hating of D-glucose (20 g.) with concentrated sulfuric acid (1 ml.) at 95' for 10-20 min. Carbonization was conspicuous when 4 ml. of sulfuric acid was used. Under these optimal

+

+

+

+

+

(78) B. Helferioh, A. Stllrker, end 0. Peters, Ann., 489, 183 (1930). (79) B. Helferih and 0. Petem, Ann., 44, 101 (1832). (80)Z. A. Rogovin and Yu. L. Pogoeov, Nauchn. Dokl. VywM Shhbly, Khim. i Khim. Tekhn~l.,19691 NO.2, 868; C h . Abstm~ls,6 8 8 22912 (1959); V. I. ShWkOV md M. G. Smirnova, Zh. Prikl. Khim., 87, 976 (1954); C h . Absfrade, 49, 10869 (1956); J . Appl. C h . USSR. (Englieh Trmsl.), 17, 911 (1954). (81) Yu. L. Pogoeov and Z. A. Rogovin, Uabskak. Khim. Zh., 1980, No. 3, 68; C h . Akfracls,66, 24100 (1981). (82) T. Ne,kamura,Kogyo Kagah Zasshi, 68, 1789 (1960).


447

coudition8, polymers of P, 9-18 (by iodine methods) were obtained in 20-3OOJoyield following fractionation, by alcohol, of the dialyzed reaction products. (The Pn,based on reducing value, before fractionation was approximately 4.) The polymers gave wglucom on acid hydrolysis, but were unaffected by tt crude, d i v a r y amylase preparation. Methylation analysis of a polyglucose fraction having P n 17 suggested that ( 1 4 ) linkages preponderate, with (l+b)-linkages the next most common. The presence of n few branch points at both C-4 and C-6 was also shown, but a relatively low proportion of nonreducing, terminal glycosyl p u p s were indicated. This rather linear structure containing a sizable proportion of (1+4)-bonds is in contrast to the highly branched structures containing (14)-linkages obtained in the presence of other acid catalysts (such as thionyl chloride or hydrogen chloride). Polycondensation of D-glucose also followed when a mixture of D-glucose (20g.) and water (2 ml.) was heated at 95' with phosphorus trichloride (13), phosphorus pentachloride ( 14), or phosphorus pentsoxide (15) .s2 The addition of water waa essential, since these three compounds apparently failed to catalyze polycondeiisation under anhydrous conditions. Under optimum conditions, polymers of P n 11-13 were obtained in 10-15% yield after 20-30 min. reaction times, when (13) and (14) were used for catalysis. With (ls), polymers of P , 8-11 were obtained in 10-30% yield, but such polycondensations required 1-2 hr., instead of a few minutes (as for 13 and 14). In no case did polymerization appear to be as efficient as when sulfuric acid waa used. Kentm capitalized on the waction between thionyl chloride and water (to form sulfur dioxide and hydrogen chloride) for removing the water of condenaation and generating a gaseous by-product to aid in driving the polymerization to completion. In support of this role of thionyl chloride is the observation that polymerization did not proceed under strictly anhydrous conditions. A small proportion of water facilitated the polymerization, probably by permitting initial formation of hydrogen chloride. The optimum conditions for this type of polymerkationWinvoIved heating a solution of D-glucose monohydrate (18 g.) in thionyl chloride (3 ml.) a t 100' for 45-60 minutes. In this manner, a polymer preparation of P, 12 was obtained in 75% yield. The P. could be increased to approximately 30, if the first polymer preparation was re-treated with thionyl chloride under similar conditions. The optical rotation ( [ a ] ~ 100') suggested that a-Sglucosidic linkages were present. Limited analysis, by periodate oxidation, of the structure of a polyglucose of Pn20 prspared by this thionyl chloride procedure showed 58% of the

+

(83)P.W.Kent, Bwchm. J., 66, 361 (1956).

448


residues to be either (l+6)-linked or terminal.a4Graded hydrolysis, with acid, of the polymer permitted isolation of isomaltose, thereby demonstrating the presence of a--( 1-+6)-glucosidic linkages. Infrared analysis (see Section IV,4,b) showed the presence of a-w(1+6)-linkagesJ and also suggested the presence of a few WD-( 1-*3)-1inkagea. The thionyl chloride method has also been used@for the condensation polymeriza+ionof mgalactoee, &mannose, and lactose, and for the copolymerizaticn of mixtures of &glucose and D-galactose, wglucose and crhamThe p,, ranged from 4.5 nose, and D-glucose and ~-glucurono-6,3-lactone. (for the product from wglucose and wglucuronolactone) to 12 (for the product from D-wnnose, and from lactose). No structural studies, or efforts to repolymerize thew products to material of higher molecular weight, have been reported. Polyphosphoric acid este196 has been employed for activating carboxyl groups for peptide synthesis," and hrts also been used to activate the hemiacetal hydroxyl group of sugars for glycoside synthesis.a Schramm and coworkers reporteda that a solution of polyphosphoric acid ester and wglucose k~f o m m i d e furnished, after dialysis and deionization, a phosphate-free polymeric material of molecular weight approximately 50,OOO (in 10-20(r0 overall yield). By similar procedures, wribose and D-fructose were found to give polymers having molecular weighte of approximateIy

40,Ooo.

+

The optical rotation ( [ a ] ~ 16") of the polyglucose, together with periodate-oxidation data (one mole of periodate consumed per mole equivalent of sugar residue) was taken as evidence for &n-( 14)-glucosidic linkages (see, however, Section IV,4,c). The high viscosity, [ q ] = 100, of a polyglucose containing aome phosphate ester groups, prepared by using N,N-dimethylformamide as the solvent,@ was stated to indicate linear, unbranched molecules. These data prompted the interpretationa that the polyglucosa contained &D- ( 1 4 )-glycosidic linkages almost exclusively. Similar arkuments permitted the conclusion that the polyribose was chiefly a-w(1-+5)-Iinked.a The high degree of stereoselectivity claimed is surprising. In the system used, no directive effects causing exclusive reaction of the (3-4hydroxyl group of D-glucose are apparent. Indeed, in an independent study using this (84) E. Loudon, R. 8. Theobald, and 0. D. Twigg, Chsm. Id.(London), 1060 (1955). (86) W. Pollmann and G . Schramm, Bwchim. Biophgu. A&, 80, 1 (1964). (86) G.Schramm and H. Wiaamann, C h . Ber., 81, 1073 (1968). (87) G.Schramm, H. Gr8tsch, and W. Pollmann, Angew. C h . , 78, 619 (1961). (88)G.Schramm, H. GrWch, and W. Pollmann, Angew. C h . ,74,53 (1962); Angm. Chem. Inlem. Ed. Engl., 1, 1 (1962). (89) Formamide ia eaidu to retard phwphorylation by the polyphoRphoric acid ester.

CHEMICAL SYNTHESIS OF POLYSACCWRIDES

449

method of polycondensation of wglucose, it was concluded that the syntht+ sis proceeds in a nonspecific mnner, to give branched polyglucosee.w This result is to be expected, since, under conditions of acid-catalyzed polycondensation, the C-2, C-3, and G 4 hydroxyl groups are of approximately equal reactivity, but are less reactive than the C-6 hydroxyl Furthermore, the selective formation of the 8-wglucoeidic linkage usually requires neighboring-group participation by a substituent at C-2.” No such group is evident in this system. Consequently, it seems that a mixture of CY-D and p-D anomeric configurations was, most probably, obtained, the proportions of each being dependent on the position of the thermodynamic equilibrium in the acidic medium employed. A (1+5)-linkage for the polyribose is expected to be a preponderant linkage, based on the relative reactivities of primary and secondary hydroxyl groupsp2and on the tendencyQ1of &ribose to adopt the furanoae form. However, the anomeric configuration of the polyribose is, most ~ since a mixture of D-ribose and probably, a mixture of O-D and p - linkages, adenine, under the influence of polyphosphoric acid esters,= gave, among other products, approximately equal proportions of adenosine and its CY-Disomer.g2 Even though this method of polycondensation probably gives polymers containing several different types of glycosidic linkage, the mildness of the method and the high molecular weights of the polyglycoses obtained suggest that the method itself, or modifications of it, when applied to appropriately substituted sugars, may well constitute a convenient synthesis of structurally defined polyglycoses of high molecular weight. Methyl sulfoxide has been used, chiefly by Micheel and his coworkers, as an effective solvent for the acid-catalyzed polycondensation of s ~ g a r s . ~ In * - view ~ ~ ~of the good yields of material of high molecular weight, the methods developed offered considerable promise. Unfortunately, it was subsequently that, in acidic media, methyl sulfoxide undergoes a (90)E.Husemann, Private communication of unpublished reaulta. (91) See,for example, S. Haneasian and T. H. Hmkell, J . Org. Chem., 28, 2604 (I=),

but compare with Ref. 28. (92) J. A. Carbon, Chem. I d . (London), 529 (1963). (93)F. Micheel and W. Greaser, C h . Ber., B l , 1214 (1958). (94) F. Micheel, W. Neilinger, and F. Zerhueen, Tetrahedron Letters, 1205 (1963). (95) F. Micheel, A. Bbckmann, and W. Mecketroth, M a k r m l . Chem., 48, 1 (1961); F. Micheel and A. Btickmann, Angew. Chem., 72, 2096 (1960). (96) F. Micheel and D. Mempel, Makromol. Chem., 48, 24 (1961). (97) F. Micheel and A. Btickmann, M a k r m l . Chem.,61, 97 (1962). (98) F. Micheel and H. Alfes, Makroml. Chem., 48, 33 (1961). (99) F. Micheel and R. Puchta, Malcroml. Chem., 48, 17 (1961). (100) F. Micheel and A. Bockmann, M a k r m l . Chem., 61, 102 (1982). (101) E.Hueemann and J. Klar, Methods Carbohydrate Chem., 6, 180 (1965).

I. J. QOLDBTIPIN AND T. L HULLAR

450

slow disproportioiiation to form methanethiol and formaldehyde. In an analogous reaction, thionyl chloride is known to react with an excem of methyl sulfoxide, to give, among other products, formaldehyde and hydrogen chloride.lm In the presence of acid, formaldehyde is known to undergo polymerization*oato poly (oxymethylene) and to form methylene acetalP with alcohols. Analysis of the polyglycoees for oxymethylene groups (derived from formaldehyde) showed that the polyglycosea contained between 0.5% and 15% of formaldehyde. The presence of polp (oxymethy!ene) as a contaminating polymer was excluded on the basis of infrared spectral analysis and solubility properties. The presence of cyclic, methylene acetals was considered unlikely, since amylose or starch under the reaction conditions employed for polycondensation did not contain cyclic acehl groupings. In order to study the manner in which oxymethylene unita are bound to sugar residua, methyl 2,4 ,6tri-O-methyl-/3-~glucsoside( 16) and 2,3,4,6-

OR

-

(le), R -CH,

(102) F.G. Bordwell and B. M. Pitt, J . Am. C h .rsoC., 77,672 (1966). (103) J. F. Walker, “Formaldehyde,” Reinhold Book Division, New York, N. Y., 3rd Edition, 1864, p. 160. (104) Ref. 103, pp. 264-276; T. G. Bonner, Methode Carbohydrde Chent., 2, 433 (1963).

CHEMICAL SYNTHEBIB OF POLYBACCHhRIDES

451

tetra-O-methyl-~glucoae (17)-neither of which can undergo polymerization-were treated with methyl sulfoxide and acid under the conditions uaed for the polymerizations of sugars. From the reaction of (16), a aeries of compounds, (18), was obtained, in which two residues of (16) were united by oxymethylene units of various chain lengths. In a similar manner, compound (17) gave a mixture of homologous, oxymethylene compounds, (19), of which the first member, n = 1, was chamcterized, and higher members, n = 2, 3, , were identified chromatographically. Compound (17) also furnished the octa-O-methyl-a,cr- and a,Btrehalose~(20) and (21), respectively. It is poasible that oxymethylene units can also form bridges between nonanorneric and anomeric hydroxyl groups, tw in (22). The observation that hydrolysis by 0.1 N aqueous acid causes ti large decrease in molecular weight of the synthetic polymers constitutes further support for the presence of cxymethylene groups between sugar residues.

I t is clear, &herefore,that polymers synthesized by polycondensation of sugars in acidic methyl sulfoxide are, actually, copolymers of sugars and formaldehyde in which some of the glycosyl residues are joined by glycoeidic bonds, and others are linked by “bridged’ of oxymethylene units. Nonetheleas, because polycondensation of sugars can be effected in methyl sulfoxide, and because the methods developed for the polycondenaation may be the extensive studies reported by applicable to other solvent Micheel and his coworkers will be discussed. The general methodg6developed by Micheel and coworkers consists of allowing a 20% solution of sugar in methyl sulfoxide to undergo condensation in the presence of an acid catalyst for two to eight days, usually at 40’. The acid cahlysts used were (a) hydrogen chloride,06(b) hydrogen chloride together with small proportions of hydrogen bromide:& (c) a mixture of hydrogen chloride, hydrogen bromide, and phosphorus pentaoxide,” and (d) thionyl chloride.06 The weight ratio of catalyst to monomer was 0.3-0.5 to 1.0. For polymerizations catalyzed by hydrogen chloride and (106) Other solventa, such aa dimethyl sulfone, tetramethylene sulfone, or acetic acid, have been stated*4 to give polyglycoaea containing no oxymethylene ~ O U P R .

452

I. J. OOLDBTEIN AND T. L, HULLAR

hydrogen bromide, the water of condensation was removed by azeotropic distiIlation with benrene at 14 Tom at 40".Thionyl chloride and phosphorus pentaoxide served as internal, water scavengers, as well as catalysts. Methyl sulfoxide waa removed by rapid distillation, and the polymer was precipitated by the addition of methanol. Methanol-insoluble polymer was diaIyzed to remove the material of low molecular weight. The sugars used in these polycondensations, and some of the oharacteristics of the methanolinsoluble, dialyzed polymers are summarized in Table 11. Effective polymerization occurred only when the water of condensation was removed by one of the methods mentioned above. The maximal yield of material of high molecular weight waa obtained after a reaction time of 5-12 days (see Table 11, Nos. 2 and 3). The molecular weight and yield of the products depended on the concentration of monomer in solution (see Table 11, Nos. 4 and 5 ) and on the catalyst used. For the neutral sugars, hydrogen chloride furnished preparations of the highest molecular weight in the highest yield; the addition of hydrogen bromide resulted in a 50% diminution in the molecular weight and in a 20% diminution in the yield.06 For 2-acet~mid6-2-deoxy-~-glucose , however, the hydrogen chloridehydrogen bromide system proved to be a catalyst superior to hydrogen chloride alone. From wglticose and from 2-acetamido-2-deoxy-~-glucose, thionyl chloride and hydrogen chloride gave similar yields of polymers, but those obtained by using thionyl chloride were of lower molecular weight. Poly (2-acetamido2-deoxy-D-glucose) prepared by means of thionyl chloride was also reported to contain more furanosyl residues than the polymer prepared by using hydrogen chloride. Phosphorus pentaoxide in combination with hydrogen chloride and hydrogen bromide proved to be an effective catalyst for the polymerization of 2 ,3 ,6 - t r ~ - ~ - m e t h y ~ - ~ - ~ uHowever, c o s e . ~ this polymer may have contained a considerable proportion of oxymethylene bridges, since it has been observeds" that polymers prepared in the presence of phosphoric acid are richer in oxymethylene groups than those prepared by using hydrogen chloride-hydrogen bromide. Calculations based on the formaldehyde content of the polyglycoses indicated that, on the average, blocks of four glycosyl residues are distributed randomly. In view of the ease with which formaldehyde undergoes polymerization, it is more probable that blocks of oxymethylene residues are interposed betwecn blocks of glycosyl residua. Complete hydrolysis of the polyglycoses gave the starting monomer. Hydrolysb with dilute acid gave an initial rise in optical rotation which was attributed to the hydrolysis of furanosidic bonds. The positive rotations suggest the presence of CY-Daa well as of &D-glycosidic linkages. The isolation

TABLE I1 Acid-catalyzed Polycondensationof Sugars in Methyl Sulfoxide

No.

-

Monomer

1 2 3 4 5 6 7 8 9 10

c-(=lucose D-Glucose D-Glucose D-Ghd D-Glumseb D-Glucosec D-GdaCtoSe D-Mannose D-xylQSe 2-Acetsmido-Zdeoxy-D-

11

2-Acetsmid~zdeox~-~ glum& 2,3,6T~i-@rne%hyl-~-

glUCOSe

12

glUCOSe*

13

D-Glucose (6.67 g.) plus Dglucuronic acid (3.33 g.)

14 15 16 17

Maltose

18

Cellobioae

Lsctod

Lactoset Polymaltose (10 g.) plus wglucose (25 9.)

Catalyst (g./10 g. of monomer/!%ml. Time of methyl sulfoxide) (days)

Temper- Yield,o ture ("C.) (%)

40 40

Mae.

References

+97

11 ,900 6,200

-1

3,200

96

+I24

5,600

97

23

+75

12,000

98

2

40 40 40 40

49 36 44 33

+86 +67 +76 +79

%ooo

36,300 10,800 6,300

99 99 99

?i

40

60

+90

32,100

4 5 12

6.60 g. SOCI:' 0.73 g. HCl 0.10 g. HBr 1.50 g. Pa' 4.66 g. HCI 0.133 g. HBr 4.80 g. HCl 4.25 g. HC1 5.1 g. HCl 5.7 g. HCl 0 . 2 g. HBr 4.0 g. HCl 1.0 g. HBr

6

2

30

6

45

31

4

40

5.5 5 5 5 4.5

5 5 6 7 4 6

watep)

95 95 95 95 95 95 95 95 95 96

4.M g. HCI 3.08 g. HC1 0.10 g. HBr 3.18 g. RCl 0.08 g. HBr 6.60 g. SOCls' 4.40 g. HCl 3.60 g. HCl 3.20 g. HCl 3.50 g. HCl 0.60 g. HBr

3

Cab

(degrees, in

40 40 40 20 40 40 40 40

46 40 48 47 46 57 45 53 19 16

4-90 +89 +89 +95 +94 +88

+79

+I03 +96

24,000 12,400

13,800 7,300 7,300 12,500 24, loo

24,400

-3

X

p:

6

0

G m

4

$ c) ! ? I

m

2!

8

*5

zE

U

99 99

For methanol-insoluble, nondialysable-mterial. * Used 20 g. of wglucose/50 ml. of methyl sulfoxide. Used 10 g. of Dgiuoose in 20 d In 20 ml. of methyl sulfoxide. Used 6 g. of monomer in 10 ml. of methyl sulfoxide, with the catalyst mixture given. f Used 13.3 g. of lactose/50 ml. of methyl sulfoxide. a

ml. of methyl sulfoxide.

W

454


of high proportions of 2 ,3,4 ,6-tetra-0-methylhexoses and of 2 ,3 ,4-tri-Omethyl-D-xyloae from the hydrolygateo of the methylsted products indicated highly branched po1ymers.06A high degree of branching is also suggested by the difficulty06lB6of methylation, relative to acetylation. Periodattwxidation data reveal that, for D-glucoseat lertst, the hydrogen chloride-hydrogen bromide catalyst system gives a polymer having a considerably greater proportion of terminal or ( l 4 ) - l i n k e d residues than does that produced by hydrogen chloride alone. The periodate-oxidation &fa further show that, for polymers derived from D-glucose and wgahctose (using hydrogen chloride as catalyst) , a considerable proportion (30-4070) of the glycosyl residues are substituted on the C-2 and C-4, or on the C-3, hydroxyl groups, as shown by their resistance to oxidation; some are substituted on the (2-2 or C-4 hydroxyl groups, as indicated by periodateconsumption data,whereas others are unsubstituted at the hydroxyl groups on C-2, C.3, and (2-4, aa shown by the formation of formic acid. Radioisotope-incorporation studiesn indicate that the polymers are products of an equilibrium process (see Section I1,1, p. 435). In summary, the acid-catalyzed condensation polymerization of sugars in methyl sulfoxide results in the formation of copolymers of the sugars with formaldehyde. The glycosyl residues probably occur in blocks, instead of being evenly separated by methylene bridges. The polymers are highly branched, and the glycosyl residues appear to be substituted in a random fashion. Altholigh the use of methyl sulfoxide as a solvent for the polycondensation of sugars did not, in them studies, lead to the synthesis of homopolymers, it did provide the basis%for an elegant synthesis of an apparently linear, @ - ~ ( 1 4 ) - l i n k e dpolymer of D-glucose (see equation ‘7).lm To direct polyglycoside formation between the C-1 and C-4 hydroxyl groups, thereby preveqting synthesis of branched polymers, the hydroxyl groups at C-2, C-3, and C-6 were protected by a substituent which was relatively stclble to acid but could be removed by alkali. The N-phenylcarbamoyl group fuIiiLs these requirements.lw To prepare the desired intermediate, celluloee was carbanilatedl*wJO’J@ with phenyl isocyanate, the cellulose derivative was depolymerized in Zmethoxyethanol to the D-glucopyranoside corresponding to 2 ,3,&tri-0- (N-phenylcarbamoyl) -D-glucopyranose (3), which was then hydrolyzed to afford (3). It was found, by study1 of reaction conditions, that polycondensation of (3) to form polymeric products is most satisfactory when a solution of (3) (1 g.) and phosphorus pentaoxide (1 g.) in methyl sulfoxide (1 ml.) and (106) (107)

E.Husamnn and G. J. M. MUer, Angm. Cham.,IS, 377 (1963). H. 0. Eouveng, Ada C h .Smnd., 16, 87 (1961); W.M. Hearon, Methoda Carbo-

h y d w C h . , I,239 (1963). (108) J. N. BeMiller, Methods Carbohydrate Chem., 5 , 400 (1906); 4, 301 (1964).


455

chloroform (9 ml.) is stirred for 2-14 daysma at 30".Under these conditions, the methyl sulfoxide did not disproportionate to formaldehyde; increase in the proportion of methyl sulfoxide gave traces of formaldehyde. A higher proportion of phosphorus pentaoxide (2g./lO ml.) gave a product containing 0.08% of phosphorus. Phosphorus penlaoxide in methyl sulfoxide is also known to oxidize secondary hydroxyl groups of carbohydrate In the absence of methyl sulfoxide, polymerization in derivatives. chloroform gave products of1low (4-8)P,. Under the optimal conditions, polymers were obtained in 7540% yield, and showed [ q ] of 22-78 and Bwaa high tw 335,000 (by light-scattering) , corresponding to a P, of 640. Protecting groups were removed by treatment of the polymer with sodium methoxide in pdioxane, followed by tetraethylammonium hydroxide. Although nitrogen-free polymers were obtained by this procedure, the polymers had suffered a 16-20'3, decrease in p,,. The resulting polymers were insoluble in water, sodium hydroxide, N ,N-dimethylformamide, and methyl sulfoxide, but were soluble in tetraethylammonium hydroxide and in Schweizer reagent. Total hydrolysis of the polymers gave D-glucose only. Water-soluble derivatkes (ethyl or carboxymethyl ethers) of the polymers were unaffected by a-amylase, but were partially hydrolyzed by a cellulase preparation from Acetobacter xylinum. The optical rotations of several preparations of this polyglucose and of cellulose ( P a 1150) in tetraethylammonium hydroxide were all O", thereby strongly suggesting that the polyglucoses are @-wlinked."'Q On the ba& of the data, it was concluded' that the synthetic polyglucoses obtained by this method are linear, Bn-( 1 4 )-linked gIucans which possess properties similar to those of cellulose. It is especially significant that this polymerization proceeded with apparent stereospecificity, to form only the @-D anomeric linbge. The mild conditions under which the polycondensation occurs probably contribute to this selectivity of reaction. (lo&) A second report of conducting polycondenaationa in methyl sulfoxide and phoaphorua penwxide haa appeared [K.Onodera, 8.Himno, and N.Kaahimura, J . Am. Chem. Soc., 87, 4651 (1966)], but experimental reeults are not yet available. (109) This view is further supported by the following observations. Acid-catalyzed aolvolysia of 2,3,6tri-O-(N-phenylcarbemoyl)celluloae by Zmethoxyethanol allows isolation of a ~-glucoeide, Zmethoxyethyl 2,3,6tri-O-(N-phenylcarbamoyl!-D-glucopyranoside, which shows [PI# - 88" (in pdioxane).' The struo turally AimilrLr a-n-glncmide, methyl 2,3,4,~~t,ra~-(N-phenylcarbamoyl)-cr-Dglitcopyranoaido, lrliows [a]# 73" (iii acelone).'IO The diaparity of these two rotstions in solvent8 of aimilar properties suggeeb that the compound of [a]# -88' ie the &D anomer. Since the glucoside formation and the polycondensation are both carried out under conditions that are undoubtedly equilibrium conditions, the glycoaide formation to afford a polymeric derivative may be expected to p r o d analogously to the glycoside formation to give a &-linked monomeric derivative. Hence, the polymer ahould also have the &slinkage.

+


456

c. Methods Based on Glycoeide Syntheees.-Condensation polymerization of eugam which possess at C-1 such ncmhydroxylic groups as a halogen atom or an acyloxy group hm been studied. The simplest examplea of such condensetion reactions are the KoenigsKnorr synthews of glycoside8.111 In principle, any of the modifications of this general reaction are available for purposes of polycondensation. Thus, Helferich and Gootz,11*synthesized gentiotetraoae in a stepwise manner by condensing 2,3,4,2’, 3‘ ,4‘, 6’-hepta-O-acetyl-a-~-gentiobiosylbromide (23)118with 1,2,3,Ctetra-O-acetyl-ar-mglucopyranose(24)114 in the presence of silver carbonate, to give the protected trisaccharide (25a). Bromination of (23aj at C-1 (by hydrogen bromide in acetic acid) to give (25b), Ac

-

%

Ac

Ac

A

c

O

Ac*QAC

AC r

Ac

AQ--jQ-Q-j$&

Ac

OAC

C

AcO

OAc

C

(26)

(110) (111)

M.L. Wolfmm and D.E.Pletaher, J . Am. Chcm. Csoc., 63, 1161 (1940). W.L.Ev&1111,D. D. Rsynolde, and E. A. Tdey, Adoon. Carbohydrate C h . ,6,

n

(1951). (112) B. Hnlierich and R. GOO~II, Bsr., 04, 109 (1931). (113) Q. ZempIBn, Bsr., 67, 702 (1924). (114) B. Helferiah and W.Klein, Ann., 460,219 (1926).

457

CHEMICAL BYNTHEBIB OF POLYBACCHARIDEB

followed by condensation of (25b) with (24), gave the blocked tetraectccharide (26) in a 5% overall yield from (23). It is clear that the stepwke synthesis of polyglycoses by such a procedure is laborious and gives low yields. As an alternative approach to the synthesis of polyglycoaes, a monomer possessing both a free hydroxyl group and a 1-bromo substituent should, n HO-M-Br

+

(--O-M-),,

+

HBr

under suitable conditions, self-condense to give a polymer. To this end, Whelan and Haqw treated 2,3,4-tri-O-acetyl-a-D-glucopyranosylbromide (11)a with silver oxide and, after deacetylation and carbon-column chromatography, isolated a series of gentiodextrins (27). The di-, tri-, tetra-, and penta-saccharides [(27), n = 0, 1, 2,and 3,respectively] were characterized &B their crystalline acetatee, and the hexasaccharide [(27),

HOH&

kO\

I

OH

on

n = 41 by its 1"JleM~and1" R M values. Reasonable yields of oligosaccharides wer8 obtained: dimer, 14%; trimer, 22%; tetmmer, 5%; pentamer, 2.3%; and hexamer, 1%. 1 ,6-Anhydro-&~-glucopyranose derived from intramolecular condensation constituted only 25% of the condensation products. The low yield of the higher oligosaccharides, together with the isolation of wglucom, are believed due to reaction of the water (released when the liberated hydrogen bromids reacts with the silver oxide) with the unreacted 1-bromide. The stereospecific formation of the 8-D anomeric configuration is due to the (116) K. Freudenberg and G. Blomqviet, Ber., 68, 2070 (1936). (116) W. J. Wheld, J. M. Bailey, and P. J. P. Roberta, J . Chem. Soc., 1293 (1963); B. Lindberg and J. McPheraon, Adcr C h .Scud., 8,986 (1954). (117) D. French and G. M. Wild, J . Am. Chem. Soc., 76, 2612 (1963).

458

I. J. OOLDSTEIN AND T. L. HULLAR

influence of tho neighborbig C-2 hydroxyl group.w If m a n s are found to remove the water of reaction (whioh results in consumption of monomer) , a method of polycondensation brteed upon the Koeniga-Knorr reaction may be of consiclerphle promise for tho stercospccific syntheees of other polyglycoses. Condensation polymerbation of u-D-mannopyranoeyl fluoride (28) haa been attempted.ll* Reaction of (28) with sodium methoxide gave methyl a-D-mannopyranoside (30) in 75% yield. Like other derivatives posseasing a fluoro group tram to the C-2 substituent (hydroxylllg or ptolylsulfonamidom), the stereospecifio formation of (30) undoubtedly proceeds by way of a 1,2-epoxide intermediate (29) which then undergoes facile ringopening at C-1, to give (30). In support of this mechanism is the obser-

vation that Z-O-methyl-a-P~Mopyranosylfluoridell* (31) reacts with (32), sodium methoxide to give methyl 2-O-methyl-&.~mannopyranoside the product expected by direct S32 displacement of the fluorine atom in the absence of participation by the C-2 methoxyl group.1g1-126 In an effort to extend theae etereospecific reactions to the formation of polymers, (28) waa treated with 50% sodium hydroxide. (See Section 111,2,c,p. 400 for the addition polyrnerizatiorP of an analogous compound, (118) F. Michoel and D. Borrmann, C h . Ber., B3, 1143 (1960). (119) F. Micheel and A. Klemer, Chem. Bsr., 85,187 (1962); F. Micheel, A. Klemer, and 0. b u m , tW.,88, 476 (1965). (120) F. Micheel and H. WuW, Chsm. Bar., 89,1621 (1966); F. Micheel and E. Michaelis, W.,91, 188 (1968). (121) C. M. McCloskey and G. H. Coleman, J . Org. Chem.,10, 184 (1946). (122) M. P. nardolph and 0. H. Coleman, J . Org. Chem.,16, 169 (1960). (123) R. U. Lemieux and 0. Brice,Can. J . Cham., SO, 296 (19Sb). (124) A. Dyfvennan and B. Wndberg, Ada chsnr. Scad., 4,878 (1958). (1248) R. C. Gesman and D. C. Johneon, J . Or#. Chum., 81, 1830 (1968). (126) F. Mi-I end A. Khmer, Char. Bss., Bl, 104,683 (1968). (126) However, the methoxyl group ie known" to lresiet in the dieplacement, by silver aoetab, of a traw, vicinal bromide atom attaohed to s cyclohmme ring. (127) 8. Winntein and R. B. Hendemon, J . Am. Char. Soc., 66, 2196 (1943). (128) 8. Hsq md W. J. Whelan, Nature, 198, 1222 (1966).

459

CHEMICAL SYNTHESIS OF POLYBACCHARIDEB

HOSy:

HOHa?

Brigl’s anhydride, 3,4,6-tri-O-acetyl-l , 2-anhydro-~-glucopyrnose.)Two dieaccbarides.[(34) and (35)] of the trehalose type were isolated in 5.3% yield each, and a trisaccharide (36) was isola.ted in 8.5’% yield. Higher

&C----d

t

Q

HO

HOH,C

0

HO (34)

(35)

oligomccharides were isolated in 7.8% yield, but were not obtained in the pure state. The disaccharides (34) and (35) arose by stereospecificreaction of the 1,%anhydro intermediate (29) with the anomers of wmannose (33), the latter wising from the reaction of (29) with the water or hydroxide ion of the medium. The trisaccharide (36) reaulted from reaotion of the disaccharide (34) with (29). It is clear that the reaction conditions used in this study do not allow formation of polyglycoses from (28). The facility of the 1,2kpoxide formation and of the subsequent ringspening do suggest, however, that different reaction conditions should permit ready synthesis of higher oligo-

460


wtccharides, aid, poesibly, of polywtcciuirides. lt'or example, reaction of a suitable 3,4di-O-substituted wmannosyl fluoride with an appropriate base should furnish oligomers and polymers aomposed largely of a+( 146)linked wmannopyranosyl residues. The polymerbation of several uilaubetitutud aldosyl fluorides's under mildly acidic conditions has been briefly described.1w Treatment of the aldosyl fluorides with pyridinium hydrochloride or hydrofluoride under diminished pressure gave good yields (see Table 111) of nondialymble, polymeric material. TABU I11 PolyglyocMee from Aldopyranmyl Fluoriddm

Polyaaccharide Fluoride

PD-GIUCORY~ 8-p-GluWsyl

arD-Xyloeyl fl-D-Arabinosyl

Yield, %

72 67 81 66

[ o h m Odegreea ,

+66

+68 +32

- 121

No structural studies of these products have been reported. However, from the greater reactivity of the C-6 hydroxyl groupmunder conditions of acid-catalyaed condensation, it may be expected that (1-+6)-linked wglucosy1 residues preponderate. The positive, optical rotations of the polymers from wglucose and D-xylose indicate the presence of some a-D-glycosidic linkages; the levorotation of the poly(D-arabhose) suggests it to be mainly FD-linked. These anomeric configurations, together with the observation that the a- and pwglucopyranosyl fluorides furnish polymers having similar rotations, suggest thst the polymerbation proceeds by way of a carbonium ion intermediate, to give a thermodynamically controlled mixture of a-3 and P-D linkages. The high yields of polymer obtained suggest that further development of this procedure would be desirable, particularly if partially substituted aldosyl fluorides were used. Acid-catalyzed condensation polymerhation of p-D-glucopyranosyl met+ itoate (37) has also been explored. This method takes advantage of the acid-catalyaed, stereospecific displacement of the mesitoyl group of &Dgluaopyranoeyl mesitoat@ by alcohols to give, with methanol,18amethyl (laS)For B review of glyaoayl fluorides, nw F. Mioheel and A. Klemer, Adrxm. Carbohydrats C h . ,16, 86 (1981). (180) F. Miaheel and 0.Hallerman, Telralledron h & a , 19 (1962). (131)F. Miaheel and Q. Baum,Chsm. Ber., 88,2020 (1966);H.B. Wood, Jr., and H. G. FIetaher, Jr., J . Am. C h m . &oc., 78, !207, 2849 (1966). (132)B. Helfeich and D. V. Haahelika,Chem. Ber., 90,2084 (1957).

461


(u-i,-14Iucol,yranoside (38). Thus, self-condensation of (37) in pdioxane, using ptoluenssulfonic acid aa the catalyst, gave, in 88% yield, a identified by molecular-weight studies as being a tetrasaccharide. The optical rotation ([aID 92') indimtcd the prescncc of a-D-linkages; methylation analysis gave 2 ,Y,4,&tetra-0-methyl-%glucose and unidentified di-0- and tri-0-methyl-mglucos, indicating the materiil to be branched. This method may also have synthetic utility, particularly if reaction should be confined to specific hydroxyl groups. In a newly developed, elegant method of a-D-glucoside synthesis,'u the ezo isomer of a 3,4,6-tri-O-acetyl-a-D-glucopyranose1,2-alkyl orthoacetatel" (39) undergoes acid-catalyzed rearrangement, to give the corresponding alkyl2 ,3 ,4, 6-tetra-O-acetyl-a-~-glucopyranoside (40). It r e m b

+

H@ AcO

-Ac

OR OAc

to be seen whether this facile reaction can be extended to the synthesis of oligo- and poly-saccharides possessing glycosidic linkages a.8 to the C-2 hydroxyl groups. In a related reaction,'". 3,4 ,Btri-0-acetyl-cu-Pglucopyranose 1,%(ethyl orthoacetate) reacts in nitromethane containing cholesterol, mercuric bromide, and a trace of ptoluenesulfonic acid to give the transglucoside, cholesteryl 2 ,3 ,4 ,6-tetra-O-acetyl-~-~-glucopyranoside. This method has been applied to the synthesis of polysaccharides. Thus, barabinofuranose 1 $ 2,&orthobenzoate has been polymerized in nitromethane containing mercuric bromide, to furnish'sb a poly(carabinose) containing about 70y0of (1 + 51-linkages. 3. Polymerization in the Solid State

Some of the investigations discussed here do not conform strictly to this (133) B. Helferich, H.G . Germscheid, W. Pid,and W. Oat, Chem.Ber., 91,1354 (1962). (134)R.U. Lemieux, Abetrada Papers Am. Chem.Soc. Mectillg, 148,lO D (1964);R.U. Lemieux and A. R. Morgan, Can. J . Chem.,To be published. (135) R. U. Lemieux and A. R. Morgan, Can. J . C h . , 48,2199 (1906). (135s) P!. K.Kochetkov, A. J. Khorlii, and A. F. Bochkov, Tetrahedron Leusrs, 289 (1884). (135b) N. Ihyl 4-0-benroyl-2,3-0reversion of, to disaccharides, 444 carbonyl-6-deoxy-a-n-, 295 Monoses, configuration of, 8 -, methyl 4-O-benzoyl-2,3-O-carbonyl-6- Monuron deoxy-pD-, 296 effect on sugarcane, 422, 426 -, methyl 44-benzoyl-2,3-O-carbonyl- as herbicide, 405 6-O-p-tolylsulfonyl-~-~,295 Mycinose, 173, 179 Mannosyl bromide, 4-0-benzoyl-2 ,Scarbonyl-B-deoxy-WD-, 296 N -, 2,3,4,&tetra-O-acetyl-a-I, 291 -, 2,3,4tri-O-benzoyl-&deoxy-a-u-,290 1-Naphthaleneacetamide, effect on abscission and ripening, 429 Ma.. spectra 1-Naphthaleneacetic acid of acetals, 74, 70 effect on abscission and ripening, 429 of aldom and ketoses, 66 on cell-wall polyeaccharides, 381 of amino sugars, 67, 93 on sugarcane, 424 ~urw of anhydro sugars, 93 Naringenin, as plant-growth substance, of carbohydrate derivatives, 46 410 of deoxy sugars, 204 Nef reaction, synthesis of 2aeoxy sugars of dimcchaddes, 69 by, 150 effect of substituents on, 54 Neoglucobrassicin, 392 of nucleosides, 90 Neriifolin, 286 of oligosaccharides, 93 Neutramycin, 173 principles of interpretation of, 43, 45 Nomenclature stereochemistry and, 79, 92 of deoxy sugars, 1 4 of stereoisomers, 59 of tartaric acid, 34 of thioacetals, 93, 205, 206 Nuclear magnetic resonance, 191, 195 Mass spectrnmeterx, 40 of chromoses, 185 Mam Rpectromeiry conformation of glyoofiiranosides and, of carbohydrate derivatives, 39-93 97 wrope aiid limitiitioiis of, 43 of deoxy Rugars, 202, 203 Melibione, 313 of polywarcharidex, 5131 Meri*trptalr,of aldosen, I!, Nuc.leoAlefi, 28 Mewsptrrris, react ioir with nldmes, 19 mmr hpertra of, 90 Mcwciric- cyaiiide, in cwdenolido q n pyrimidine, rorltaining 2,3-dideoxy suthesis, 278 gar, 194

550

SUBJECT INDEX, VOLUME

Nucleotidea, 24 Numbering, of carbon atom in sugars by Fischer, 10

-, -,

21

zdeOXy-DLc@h-, 157 Zdeoxy-D-threa-, 151, 153 -, Sde~~y-wryUltb, 157, 159 5-phosphateJ 158 0 -, 3-deoxy-catyihro-, 158 -, 4-deoxy-cerylhro-, 166 Oligosaccharides, 27 -, 2,3-dideoxy-cglycei-o-, 194 complexes with alkali metd hydroxides, -, 2, Mideoxy-n-ctythro-, 194 254 Pentase-l-le, 2deoxy-~-ery&ro-,150 with metal nalts, 221 Pentasidq methyl 3deoxy-Lerythro-, 159 dofinition of, 4d3 Pentulose, wihreo-, as plant-growth subfuranoid, 126 stance, 416 mass spectra of, 46, 93 -, 1-amino-1 ,&anhydro-ldeoxy-D-threo-, Ryntheais of, 457 ma88 spectrum of, 91 Olioee, acetyl-, 184, 186 -, Saeoxy-D-threo-, 175 Olivomose, 184 Periplogenin, 3-@-glucopyranosyl-, 282 Olivomycin, 184, 186 Perseitol, 17 Olivomycose, 184 Phosphatm (estcm), hydrolysis of, 328 Olivose, 184 Phosphorus pentachloride, polycondensaOptical rotation tion of Dglucoee in aqueous, 447 effect of carbohydrate-complex ealt Phosphorus pentaoxide, &a catalyst for formation on, 213 polycondensation of sugars, 447, of complexing on, of carbohydrates, 452,454 228 Phosphorus trichloride, polycondensation of metal hydroxides on, of carboof mglucose in aqueous, 447 hydrates, 253 Physical properties, of herbicides, biologof glycofuranosides, 140, 141 ical activity and, 408 of sucrose, effect of salts on, 225 Pinitol, as plant-growth substance, 416 of teichoic acids, 344 Plant-growth substances of 1-thioaldofuranosides, 142 effect on carbohydrate systems, 377-430 Organic compounds, m w spectrometry on sugarcane, 421 and structure of, 39 flavonoids, 409 Orthoacetates, of sugars, 20 glycosidea and other carbohydrate deOsaaones, diecovery of, 10 rivatives as, 408, 413 OsoneR, 11 aa herbicides, 392 Oxidation, of glycol groups in glycofuranoPlant physiology, carbohydrate chemistry sides, 132 and, 378 Oxymethylene groups, in polyglycoses, 450 Pollen germination, inhibitors Of, 410 Polyarabinose, 474 P Polygalactoglucose, 483 Paraquat, effect on sugarcane, 422 Polygalactose, 483 Paratose, 188, 189 in immunochemistry, 506 Pentitol, 1,2dideoxy-~-threo-,157 Polyglucose, 462, 463, 465, 467, 469, 471, Pentofuranoside, methyl 2,Mideoxy-o-D481 erythro-, 194 fractionation of, 505 -, methyl 3,5-dideoxy-o-n-erythro-,194 glycosidia linkages in, 448 Pentofuranosyl chloride, !2deoxy-~in immunochemistry, 504 wthro-, esters, 146 Polyglycoses, 433 Pentose, 2deoxy-~-e@hro-, 149, 151, 153, from aldopyranosyl fluorides, 460 156 nuclear magnetic raqoriance smctrosesters, 146 copy of, 501

SUBJECT IMDEX, VOLUME

21

551

preparation of, 491 Potassium chloride, complex with sucrose, 223 sulfates, as heparin substitutes, 508 x-ray diffraction of, 501 Potassium iodide, complex with s u m e , Polymaltose, 463, 475 215, 223 in immunochemistry, 506 Propionic acid, Z(o-chlorophenoxy)-, efPolymannose, 466,483 fect on sugarcane, 424 -, 2,2dichloroPolymerization effect on sugarcane, 426 addition, 477 as plant-growth substance, 402 catalysis by boric acid, 466 -, 2-(2,4,5-trichlorophenoxy)by hydrogen chloride, 462 by ion-exchange resins, 464 effect on sugarcane, 425 as herbicide, 392 catalysts for, of sugars, 447, 452 Peicofuranine, 174 condensation, of carbohydrates, 434 Purine, 4(2,3-dideoxy-19-Dglycer~pentoeffect of pressure on, 467 epoxide, 491 furanosy1)-, 194 Pyran, 2, Wiacetoxytetrahydro-, m&89 of glycofuranosides, 138 spectra of cis- and trans-, 62 in methyl sulfoxide, 449, 453 Pyranose ring in solid state, 461 thermal, 476 fragmentation of, 47 Polymers, furanose, 138 maea spectrum of, 61 Polyphosphoric acid esters, in glycoside stability of, 136 3,6-Pyrazinedione, 1,2dihydro-, see Masynthesis, 448 Polyrhamnose, 474 leic hydraside Purines, N-glycosyl derivatives, 23 Polyribose, glycosidic linkages in, 448 Polysaccharides, 27 adducts with alkali metal hydroxides, Q 254 branching, determination of degree of, Quebrachitol, as plant-growth substance, 496 416 complexes with alkali-metal and alka- Quercetin, 3-O-&crhamnopyranosyl-, as line-earth-metal salts, 221, 224 plant-growth substance, 410 cell-wall, effect of indole-3-acetic acid -, 3-(&O-~~rhamnopyranosyl-~-glucoand of 1-naphthaleneacetic acid pyranosy1)-, &B plantrgmwth subon, 381 stance, 410 characteriration of, 473 Quercetin derivatives, as plant-growth configurrrtion of anomeric linkages, 496 substances, 410 fraotioriation of, 493 Quercetrin, as plant-growth substance, glycosidic linkages in, 498 410 homogeneity of, 494 isolation of, 402 R mass spectra of, 46 structural analysis of, 494 Itaffinose, 30 structure of, enzymes in analysis of, 502 Resins immiirioc*hemixtryin study of, 504 catalysis of polymerixat,ion by ionexsynthe& of, 431-512 change, 4G4 w s of synthetic, 507 ion-exchange, aa catalysts iri glycohiranPolyxylose, 463, 474 oside formation, 104 Potassium acetate, complex with sucrose, ReBpiration, in plants, effeat of aiixiiw 0 1 1 , 223 387 Potamium broinide, romplex with xucrose, 'Iteversion, of saccharide#, 442 215 Rhamnofuranoside, ethyl a(arid &L-, 112

552


Rhamnose configuration of, 17 D-

occmnca in Nature, 172 synthesis of, 177, 289 I-

1 ,2-(methyl orthoamtab), 20 occurrence in Nature, 171 relation to galactose, 16 Rhodinw, 195 Rhodomycin, 195 Ribitol. U)-&D-glucopyranosyl-n(andL)degradation of, 356 Ftibofuranoside, benrsyl &D-, 125 -, pchlorophenyl PD-, 121 -, ethyl l-thio-a-D-, 115, 116 -, isopropyl 1-thio-arD-, 115 -, methyl WD,122 conformation of, 98 -, methyl &D-, 121 conformation of, 98 -, methyl 2,3-anhydro-a(and a)-~-,conformation of, 99 -, methyl l-thio-crD-, 115 -, phenyl &D-, 121 -, propyl 1-thio-a-m, 115 Ribopyranoside, methyl tri-0-methyl-B-D-, m&88 spectrum of, 60 Ribose, D-, 17 -, 5db00xy-~-, 176,177, 179 Rings furanoid, stability of, 135 pyranoid, stability of, 136 Ripening, 429 Ruff degradation, of higher sugars to deoxy sugars, 153, 177 Rutin, BB plantgrowth substance, 410 I

S

Salicylaldehydc, alkali metal chelates, 265 Salicylic acid BB herbicide, 408 as plant-growth substance, 409 Sambunigrin, 23 Bequoyitol, as plant-growth substance, 416 Silicon compounds, trimethylsiiyl ethers of oligoaaccharidea, mass Rpectra and, 93 Simazine, BX herbiaide, 407

21

Sodium bromide, complex with sucrose, Structure of, 236 Sodium carbonate, complex with sucrose, 213 Sodium chloride, system with D-glucoee, 211 Sodium ethoxide, complex with wglucose, 258 Sodium iodide camplex with aucrose, 215 systems with sucrose, 211 Sodium methoxide, complex with D glucorw, 258 Solvation, of carbohydrate-metal salt complexw, 226 Sorbose, DL-, 12, 13 Stability of carbohydrabmetal salt complexes, 227 of furanoid and pyranoid rings, 135 Staphylomcua a u m Copenhagen, teichoic acid from, 360 H,teichoic acid from, 359 teichoic acid from, 342 8 t a p h ~ ~ c o c wIrrdis, u teichoic acid from, 342,347,350 Starch effect of auxins on, 386 polymerination in hydrogen fluoride, 446 Stereochemistry biochemistry and, 34 mass spectrometry and, 79, 92 Stereoieomsre, mars spectra of, 59 Stoichiometry of cerbohydrahlkali metal dcoholate formation, 259 of carbohydrate-alkali metal hydroxide complexes, 248 of carbohydrate-alkaline-earth metal complexes, 251 of carbohydratemetal salt complexes, 222 StreproooeCi, teichoic acid from group D, 344 Streptolydigin, 186 Strontium oxide, complex with sucrose, 213 Strophanthidin, 274 -, 3rr-carabinopymnosyl-, 281, 285 -, 3-(6-deoxy-&D-gulosyl)-, 286

kUl3JECT INDEX, VOLUME

,

21

553

fhibntituenb :!-~-(~-t~~!~JXy-~-lrlIlJt?lll~JpyrtLn~lHyl)-,

determination by nuss spectrometry, 44 nee Corrvalltitoxiri 3-(2,6-dideoxy-&~-n'bo-hexopyraneffect on ma^^ spectra, 54 0syl)-, 308 Sulfuric acid, polymerization of Dglucose -, 3-j3-~-glucopyranosy1-, 281, 288, 312 in, 446 -, o-j3-D-glucosyl-o-~-D-glucosyl-&cyT ~ ~ ~ r o s y293 l-, -, 3-o-(a-D-lyXOSyl)-, 286 Talose, ~ - ~ B ~ x Y - D -173 , -, 3-~-~-mannopyranosyl-,292 -, 6-deo~y-1.~~ 173, 179, 181 -, 3a-~-rhamnopyranosyl-,296 Tartaric acid, configuration of, 32 -, 3-&~-rhamnopyranosyl-,296, 313 nomenclature of, 34 -, 3-fi-~-xylopyranosyl-,281 Teichoic acids, 323-375 Strophanthidin glycosides from actincmycetes, 363 cardiotonic activities of, 314 from Bacillus subtilie, 350, 354 biosynthesis of, 372 properties of, 313, 319 degradation of, 340 Strophanthidol, 3,19-di-O-(a-brhamnopydiscovery of, 326 ranosy1)-, 294 -, 3-&D-glucopyranosyl-, 288 function of, 371 -, 1W-a-~rhamnopyranosyls294 glycerol, 334, 346 Ic-Strophanthin-fi, 293 hydrolysis of, 331, 345, 346,352 from Laclobacillus aralvinosus, 334,361 k-strophanthoside, 292 from Laclobacillus buchneri, 350 Structure from Laclobacillus casei, 341 of carbohydrates, mass spectrometry location in relation to cell structure, 365 and, 39 (Jf herbicides, biological activity and, 408 membrane, 332 Sucrose, 29 ribitol, 354 alkali action on, 358 complexos with alkali metals, 223 from Staphylococcus arabinosus 17-5, 363 with sodium bromide, structure of, 236 from Stuphylococcwr aureua, 343 with sodium carbonate, 213 with sodium iodide, 215 from Staphylococcus aureus Copenhagen, esters, herbicidal properties of, 416 360 hydrolysis of, 131 from Staphylococcus aurewr H, 359 optical rotation of, effect of salts on, 225 from Stuphylococcwr laclie, 342, 347, 350 from Sikeptowcei, 344 in solution of metal salts, 229, 230 structure of, 128 wall, 346 system with barium oxide, 213 Tetrose, 2-deoxy-~-glycero-, 154 with sodium iodide, 211 Theophylline, N-glucosyl-, 24 with strontium oxide, 213 Thevetose, cardenolides containing, 286 Sugarcane, effect of plant-growth sub- Thioacetala stances on, 421 aldofuranosides and thioaldofuranoSugars sides from, mechanism of, 117 acetohalogens, 20 glycofuranoside preparation from di-, amino, 17 112, 113 mass spectra of, 67, 93 mass spectra of, 85, 93 anhydro, see Anhydro sugars of deoxy sugars, 205, 206 deoxy, see Deoxy sugars Thioaldofuranosides, 114 epoxide, deoxy sugam from, 152, 159 ethyl, melting points and specific optical polycondensation in methyl sulfoxide, rotations of, 142 449, 453 mechanism of formation of, 117 uneaturated, synthesis of, 194 Thiols, reaction with aldoseu, 19

-,

554


Thioiiyl chloritlo, polymcristltion of augam by, 447 34 Threaric acid, L,-(+)-, Tilironide, aa plant-growth aubatanm, 412 Trelialow, 29 cl-Tritlrilie, 8diloro-4,6bib.(uthylaiiliiio)-, aa herbicide, 407 -, 2-chloro-4(ethylamino)-B(isopropylamino)-, ae herbicide, 408 e-Triaaole, 3-amino-, aa plantgrowth substance, 399 -, 3-(&D-glucopJrranosylamino)-, 402 Trideoxyhexoses, 195 Trideuteriomethyl group, in identification of methylated monosaccharides by II~&BBspectra, 71 Trieaccharides, 28 Turanoae, 30 Tylosin, 173 Tyvelom, 187,189, 190

U

Urea, %@-chlorophenyl)-l ,l-dimethyleffect on sugarcane, 422,426 aa herbicide, 406 -, 343,1Michlorophenyl)-l,l-dimethyleffect on sugarcane, 427 aa herbicide, 405 -, D - ~ u c o s ~24 ~-, -, glucosylthio-, 24 Uearigenin, 274

21

V Vecciniiti, 24, 26 Vallarose, 173 Vieconity, eKec:h of cerbohydratecomplex salt formation on, 213 Volemitol, 17

W Walden inversion, 20 Wohl degradation, 5-deoxy pentosea by, 177

X Xanthatea, deoxy sugara from, 163 X-ray diffraction conformationof glycofuranosidesand, 97 of polyglycOses, 501 Xylitol, 1-acetamidotri-0-acetyl-1 ,5-anhydro-ldeoxy-, maas spectrum of, 91 Xylofuranoae, 1,2 :4 ,5di-o-iaopropylidencm-, maw spectrum of, 78 Xylofuranoaide, ethyl Zacetamido-2deoxy-1-thio-on-, 116 -, msthyl a(and P)-D-, conformation of, 98

Xylopyranoside, methyl tri-Gmethyl-pD-, maaa spectrum of, 60 Xylose D-

-,

electrophoretic migration of, 234 polymerization by hydrogen chloride, 463 Saeoxy-~-,synthesis of, 177

CUMULATIVE AUTHOR INDEX FOR VOLS. 1-21 A

ADAMS,MILDRED.See Caldwell, Mary L. ANDERSON,ERNEST, and SANDS,LILA,A Discussion of Methods of Value in Research on Plant Polyuronides, 1, 329-344

ANDER~ON, LAURENS. See Angyal, S. J. ANET, E. F. L. J., 3-Deoxyglycosuloses (BDeoxyglycosones) and the Degradation of Carbohydrates, 19, 181-218 ANQYAL,S. J., and ANDERSON, LAWNS, The Cyclitols, 14, 135-212 ARCHIBALD,A. R., and BADDILEY,J., The Teichoic Acids, 21, 323-375 ASPINALL, G. O., The Methyl Ethers of Hexuronic Acids, 9, 131-148 ASPINALL, G. O., The Methyl Ethers of n-Mannose, 8, 217-230 ASPINALL,G. O., Structural Chemistry of the Hemicelluloses, 14, 429-4458

B

BEBLIK, ANDREW, Kojic Acid, 11, 145-183 BELL, D. J., The Methyl Ethers of DGalactose, 6, 11-25 BEMILLER,J. N. See Whistler, Roy L. See Zorbach, BHAT,K. VENKATRAMANA. W. Werner. BINKLEY,W. W., Column Chromatography of Sugars and Their Derivatives, 10, 55-94 BINKLEY,W. W., and WOLFROM, M. L., Composition of Cane Juice and Cane Final Molasses, 8,291-314 BIRCH, GORDONG., Trehaloses, 18, 201-225

BISHOP,C. T., Gas-liquid Chromatography of Carbohydrate Derivativea, 19, 95-147

BLAIR, MARYGRACE,The ZHydroxyglycals, 9, 97-129 BOBBITT,J. M., Periodate Oxidation of Carbohydrates, 11, 1 4 1 BOESEKEN, J., The Use of Boric Acid for the Determination of the Configuration of Carbohydrates, 4, 189-210 BONNER, T. G., Applications of Trifluoroacetic Anhydride in Carbohydrate Chemistry, 16, 59-84 BONNER, WILLIAM A., Friede1-Craft.a and Grignard Processes in the Carbohydrate Series, 6, 251-289 BOURNE,E. J., and PEAT,STANLEY, The Methyl Ethers of DGlucose, 6,

BADDILEY, J. See Archibald, A. R. BAILEY, R. W., and PRIDHAM, J. B., Oligosaccharides, 17, 121-167 BALLOU,CLINTONE., Alkali-sensitive Glycosides, 9, 59-95 BANKS,W., and GREENWOOD, C. T., Physical Properties of Solutions of Polysaccharides, 18, 357-398 BARKER,G. R., Nucleic Acids, 11,285-333 145-190 BARKER, S. A., and BOURNE,E. J., E. J. See also, Barker, S. A. Aoetals and Ketals of the Tetritols, BOURNE, BOUVENQ,H. O., and LINDBERG,B., Pentitols and Hexitols, 7, 137-207 Methods in Structural Polyeaccharide BAR~TT,ELLIOTT, P., Trends in the Chemistry, 16, 53-89 Development of Granular AdsorbBRAY, H. G., n-Glucuronic Acid in ents for Sugar Refining, 6, 205-230 Metabolism, 8, 251-275 BARRY,C. P., and HONEYMAN, JOHN, Fructose and its Derivatives, 7, 53-98 BRAY, H. G., and STACEY,M., Blood Group Polysaccharides, 4,37-55 BAYNE,S., and F E W S ~J. R ,A., The BRIMACOMBE], J. 8.See How,M. J. Osones, 11, 43-96 5515

5%

CUMULATIVE AUTHOR INDEX FOR VOLS.

C

CAESAR,GEORCIE V., Starch Nitrate, 18, 331-345

1-21

~IIMLER, R. J., 1 ,6-Anhydrohexofura-

noses, A New Clam of Hexosana, 7, 37-52

M. See IIitmid, W. Z. CALDWELL, MARYL., and ADAMB,MIL- I)OUDOROFI, I ~ R A CP. HS , ee Mehta, N. C. DRED, Action of Certain Alpha I ~ T C H E RJAMEB , D., Clieniid,ry of the Aniylwtw, 6, 2 2 ! ! 8 8 Amino Sugars Derived from AntiCANTOR,SIDNEY M., [Obituary of] John biotic Subshncas, 18, 259-308 C. Sowden, 20, 1-10 CANTOR,SIDNEYM. See also, Miller, Robert Ellsworth. E CAPON,B., and OVEREND, W. G., Constitution and PhysicochemicalProper- ELDERFIELD,ROBERTC., The Carboties of Carbohydratea, 16, 11-51 hydrate Components of the Cardiac CARR, C. JELLEFF, and KRANTZ,JOHN Glycosidea, 1, 147-173 C., JR., Metabolism of the Sugar EL KHADEM, HASBAN, Chemistry of OsaAlcohols and Their Derivativw, 1, zones, 20, 139-181 175-192 E L KHADEM HASSAN,Chemistry of OsoCHIZHOV, 0.9.See Kochetkov, N. K. thaoles, 18, 99-121 CLAMP,JOHN R., HOIJQH,L., HICKSON, ELLIS,G. P., The Maillard Reaction, 14, JOHN L., and WHISTLER,ROY L., 63-134 Lactose, 16, 159-206 ELLIS, 0. P., and HONEYMAN, JOHN, COMPTON, JACK, The Molecular ConstituGlycosylaminea, 10, 95-168 tion of Cellulose, 8, 185-228 EVANS,TAYLORH., and HIBBERT, CONCHIE,J., LEVVY,G. A., and MARSH, HAROLD,Bacterial Polysaccharides, C. A., Methyl and Phenyl Glyco2, 203-233 sides of the Common Sugars, 12, EVANS,W. L., REYNOLDS, D. D., and 157-187 TALLEY, E. A., The Synthesis of COURTOIS,JEANEMILE, [Obituary of] Oligosaccharides, 6, 27-81 Emile Bourquelot, 18, 1-8 CRUM,JAMES D., The Four-carbon SacF charinic Acids, 18, 169-188

D

FERRIER, R. J., Unsaturated Sugars, 20, 67-137 I ~ V I E BD. , A. L., Polysaccharides of J. A. See Bayne, 8. Gram-negative Bacteria, 16, 271-340 FEWSTER, HEWITTG., JR., The ChemDEAN,G . H., mid GOTTFRIED, J. B., The FLETCHER, istry and Configuration of the CycliCommercial Production of Cryetalline tole, 3, 45-77 Dextrose, 6, 127-143 HEWITT G., JR., and RICHTDIo BELD~ER, A. N., Cyclic Amtala of the FLETCHER, Aldonun and AldoRides, 20, 219-302 MYER, NELSON K., Applications in the Carbohydrate Field of Reductive DEITZ,VICTORR. See Liggett, R.W. Deaulfurization by Raney Nickel, 6, DEWEL,1%.See Mehta, N. C. 1-28 DEUEL,HARRY J., JR.,and MOREHOUSE, MARGARET G., The Interrelation of FLETCHER, HEWITT G., JR. See alao, Carbonydrate and Fat Metabolism, Jeanloe, Roger W. FORDYCE, CHARLESR., Cellulose Eaters I, 119-160 of Organic Acids, 1, 309-327 DEULOFRW,VENANCIO,The Acylated Nitrilea of Aldonic Acids and Their FOSTER, A. B., Zone Electrophoresis of Degradation, 4, 119-151 Carbohydrates, 12, 81-115

CUMULATIVE AUTHOR IYDEX FOR VOLS.

FOL~TER, A. I%, arid ~ ~ O R T I)., O H Asperts , of i tic Chemistry of t,lrc Amino Siigarli, 14, 2l:%-2Xl

I ~ + T E R ,A. J3., arid I ~ ~ J I W AA.R J., I ) ,‘l%e Ctioniihlry c i f I lcparin, 10, :$:$5-;168 FOSTER, A. 13., a i d STACEY,M., The Chemitllry of the %Amino Sugars (2-Amino-2-deoxy-sugars),7, 247-288 FOSTER, A. B., and WEBBER,J. M., Chitin, 16, 371-393 Fox, J. J., and WEMPEN,I., Pyrimidine Nucleosides, 14, 283-380 FRENCH, DEXTER,The Raffinose Family of Oligosaccharides, 9, 149-184 FRENCH,DEXTER,The Schardinger Dextrins, 12, 189-260 FREUDENBERG, KARL, Emil Fischer and his Contribution to Carbohydrate Chemistry, 21, 1-38 G

GARCfA, GONZALEZ,F., Reactions of Monosaccharides with beta-Ketonic Esters and Related Subst.ances, 11, 97-143 GARL‘fA

Advances in Carbohydrate Chemistry, Volume 21

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