Advances in Carbohydrate Chemistry and Biochemistry, Volume 48

Advances in Carbohydrate Chemistry and Biochemistry Volume 48

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Advances in Carbohydrate Chemistry and Biochemistry Editors R. STUART TIPSON DEREK HORTON Board of Advisors LAURENS ANDERSON J. GRANT BUCHANAN GUY G. S. DUTTON STEPHEN J. ANGYAL HANSH . BAER BENGTLINDBERG CLINTONE. BALLOU HANSPAULSEN JOHN S. BRIMACOMBE NATHANSHARON ROY L. WHISTLER

Volume 48

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

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Copyright 0 I990 by Academic Press. Inc. All Right5 Reserved. No p;in of this publicotion may be reproduced or transmitted in any form o r by any means, electronic or mech;rnicul, including photocopy. recording. o r any ~iifc)rniiitiotisttrrcigc and retrieval system, without permission i n writing from the puhlisher.

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

vii

Hamao Umezawa, 1914- 1986

TSUTOMU TSUCHIYA, KENJIMAEDA,A N D DEREKHORTON Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Chemistry of Carba-Sugars (Pseudo-Sugars)and Their Derivatives

TETSUO S U A MAI N D SEIICHIRO OGAWA

1. 11. 111. IV. V.

VI. VII. VIII. IX.

x.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of Racemic Carba-sugars ................ Synthesis of Enantiomeric Carba-sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6a-Carba-~-fructopyranoses ............................... Synthesis of Racemic Amin ............................... Synthesis of Enantiomeric Amino Carba-sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of Mono- and Dicarba-disaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . Synthesis of Biologically Active Carba-oligosaccharides . . . . . . . . . . . . . . . . . . . . Biological Effects of Carba-sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion ...................................

22 26 36 49 52 64 67 74 86 89

Chemistry and Developments of Fluorinated Carbohydrates

TSUTOMU TSUCHIYA I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 11. Basic Synthesis of Fluorinated Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 111. Synthesis and Biological Activities of Compounds Containing Fluorinated Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

Components of Bacterial Polysaccharides

BENGTLINDBERG 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 11. Aldoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 111. Glyculoses.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . 288

V

vi

CONTENTS

IV . AminoSugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Acidic Sugars VI . Ether and Acetal Substituents .........................................

289

................................ ............... X . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

311

299

317

Glycoside Hydrolases: Mechanistic Information from Studies with Reversible and Irreversible Inhibitors

GONTERLEGLER Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reversible Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Irreversible Inhibitors ..................... . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

319 327 362 378

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

385

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

413

I. I1. 111. IV .

PREFACE Analogs of the cyclic sugars in which the ring-oxygen atom is replaced by a methylene group were studied in 1966 by McCasland, who named them “pseudo-sugars.’’With the recognition that such compounds have a variety of interesting properties, especially as biochemical probes, and with the development of effective methods for their controlled synthesis and structural characterization, there has been much recent interest in this class of compounds, here surveyed by Suami (Tokyo) and Ogawa (Yokohama), who have themselves contributed a major proportion of the recent literature on these compounds. The “pseudo-sugar” terminology is unfortunately vague and not amenable to indexing. Neither the rational names based on the cyclitol terminology nor the fully systematic Geneva names are readily comprehended in reference to conventional carbohydrate nomenclature. The standard IUPAC “carba” prefix for replacement by carbon of a hetero atom in a compound having a recognized trivial name provides a rational solution to the problem of assigning explicit yet recognizable names to these cornpounds, and the “carba-sugar” names employed here should provide a superior compromise. The element fluorine is an atypical halogen, and likewise the fluorinated sugars are quite different in many respects from other halogenated sugars; their chemical synthesis frequently requires specialized methods. The development of effective new synthetic reagents, coupled with an extraordinary interest in the role of fluorinated sugars in biological processes, has led to an almost explosive growth of activity in this field. Although the subject was treated relatively recently, by Penglis in Volume 38, and the n.m.r. properties of fluorinated sugars were discussed by Czuk and Glanzer in Volume 46, the growth of the field has been so fruitful that the chapter here presented by Tsuchiya (Kawasaki) was obliged to occupy far more space than a normal Advances article; it was considered better to retain the subject material integrated into one large article than to fragment it into several shorter chapters. As early as Volume 2 of this series, the bacterial polysaccharides were surveyed in independent articles by Stacey and by Evans and Hibbert. There was little indication then of the profusion of structural types of sugar compounds and linkage patterns later to be found in these polysaccharides. Earlier analytical methods for separating and characterizing these components were very tedious. In the article presented here, Lindberg (Stockholm) provides a comprehensive yet compact overview of the current state of this greatly expanded field; the identification of close to 100 component sugar structures has largely been made possible through advances in structural analytical methodology pioneered in his own laboratory. The hydrolysis of glycosides by acid and by enzymes is one of the most important reactions encountered in the carbohydrate field. The mechanisms vii

viii

PREFACE

of acid hydrolysis of glycosideswas surveyed by BeMiller in Volume 22, and in this volume, Legler (Koln) provides an authoritative account of the glycoside hydrolases from the viewpoint of their mechanisms of action as probed by studies with various types of substrate analogs that inhibit these enzymes; the 1967 Phillips mechanism for lysozyme action remains of broad validity for the glycosidases in general. The prominent role played by Japanese investigators in carbohydrate science is underscored by the two substantial chapters by Japanese authorsin the current volume. This volume also pays tribute to one of the greatest Japanese carbohydrate scientists, Hamao Umezawa, in the obituary article contributed by Tsuchiya, Maeda, and Horton. Hamao Umezawa dedicated his entire, extraordinarily productive career to the development of antibiotics; his innovative contributions are exemplified by his chapter in Volume 30 of this series on the biochemical mechanism of inactivation of aminoglycoside antibiotics. With the completion of this volume, one of us, R. Stuart Tipson, terminates his function as Senior Editor and D. Horton continues as Editor. Tipson was a contributor to the founding volume in 1945 and has been a member of the editorial team since Volume 8 in 1954.

Kensington, Maryland Columbus, Ohio September 1990

R. STUARTTIPSON DEREK HORTON

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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL. 48

HAMAO UMEZAWA 1914-1986

Hamao Umezawa was born on October I , 19 14, the second son in a family of seven children, in Obama City, Fukui Prefecture, Japan. The city, which faces Obama Bay and has beautiful beaches connecting it to the Sea ofJapan, was a castle town ofthe Sakai family during the feudal age and is located just 50 kilometers north of Kyoto. The men of the Umezawa family had been physicians for generations. One of them (Ryoun Umezawa, 1839- 1922), who lived toward the end of the Tokugawa feudal age, was encouraged to become a surgeon after learning from an American missionary (a medical doctor) named Duane B. Simmons (1 834- 1889). Hamao’s paternal grandfather Ryozo was born in 1859 in Kurihashi, Saitama Prefecture, and became an oculist there. He was a country samurai and a skilled doctor, and was elected the 5th director of the Medical Association of the county. He died in 1930 at Kurihashi. Hamao’s father Junichi was born the eldest son in 1884 at Kurihashi. He graduated from the Medical Department ofthe University of Tokyo in 1910 with excellent records, and continued studies on biochemistry at the university. He became a physician (internal medicine), and in 19 13 was appointed the Director of the Obama Hospital. He mamed Taka Sat0 in 1908. In 1909, the first son, Sumio, was born (now Professor Emeritus of Keio University and current Director of the Institute of Bioorganic Chemistry, his speciality is organic chemistry, especially carbohydrates) and the second (Hamao) in 1914; their only daughter, Haruko, died in 1921 when she was only nine years old. Other sons of Junichi are Kuniomi (born in 1916, former ViceMinister of the Science and Technology Agency), Tsutomu (born in 1918, former Chief Director of the Health Center of the Metropolitan Police Board), Minoru (born in 1922, former Professor at the Centre de Recherches NuclCaires, Strasbourg), and Hiroomi (born in 1924, Professor of Theoretical Physics, University ofAlberta). In 19 19Junichi Umezawa returned, with his family, to Tokyo to study biochemistry again, under Professor Kakiuchi of the University of Tokyo. He completed his work for the Ph. D. degree in 1923 with a study of membrane osmosis (JotlmafOfBiochemistry, 1923). The brilliance of J. Umezawa was inherited by his sons. Hamao could write katakana and hiragana and do arithmetic when he was four years old, and it was easy for him to follow his lessons, which started in 1921 in

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TSUTOMU TSUCHIYA, KENJl MAEDA, AND DEREK HORTON

Hisamatsu Primary School in Tokyo. He soon mastered the Roman alphabet and the Hepburnian system. His excellent memory and quick understanding, and the ability to find a principle hidden in a phenomenon, seemed to emerge from his childhood, and these abilities remained throughout his life. In May 1923, the family moved to Sapporo, Hokkaido, when J. Umezawa was appointed to be the Chief Director of the Sapporo Hospital of the National Railways Corporation, and Hamao entered Kitakujo Primary School in Sapporo. After finishing primary school in only five years, he entered the middle school section of Musashi High School in Tokyo in 1926, and then the High School in 1930, finishing in 1933. He heartily enjoyed his school work, and was lucky to be educated by many good teachers, most notably by Prof. Bun-ichi Tamamushi (who died in 1982), the teacher of chemistry and physical chemistry at Musashi High School, who had studied colloid chemistry under Prof. H. Freundlich of the Kaiser-Wilhelm Institut in Berlin. Even after entering the University ofTokyo in 1933, he continued experiments in Tamamushi’s laboratory after school, and he learned the scientific method very early in his career. H. Umezawa’s clear and precise approach, and his ability to note unusual facets of detail in his researches (where sometimes truth was buried), were already evident at this time. He later related to others his deep gratitude to Prof. Tamamushi. He sometimes visited his father’s home in Sapporo. He loved the atmosphere of that city; the town streets lined with pleasant trees, the campus of Hokkaido University, and the Sapporo Botanical Garden, full ofdeep-green leaves in summer, the scene resembling, he thought later, that of Europe in summer. In 1933, he entered the Medical Department of the University of Tokyo. He felt the teaching system of the university to be incongruous: the professor always came 10 minutes late to give his lecture; after finishing the lecture, the assistants wiped the blackboard like servants, and the students were not expected to put questions to the professor. These customs were quite different from those of his high school, where teachers and students were much closer. The most dreaded course for him was anatomy. Autopsy gave him a severe loss of appetite, and he almost decided to move to another department. However, after two months, he gradually became accustomed to this work, and finally became comfortable. His excellent record won him a scholarship from the university in his last year. The greatest event in his university days was the discovery by Dr. Domagk ( 1932), ofthe sulfonamide drugs that had a major impact in the medical field in Japan, which had been very conservative. The War and Penicillin. After graduation in March, 1937, Hamao Umezawa became a Subassistant, and took a course in bacteriology under Prof. Matsujiro Takenouchi. His primary interest changed from biochemistry to bacteriology, despite his deep appreciation of the former; this came, in part, from his unconscious desire to pursue a more practical life relating to prob-

OBITUARY - H A M A 0 UMEZAWA

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lems of society. In July, he obtained his medical license. In those days, in Japan, there were signs of the impending World War, such as the Manchurian ( 1 93 l), Shanghai ( 1 932), and February 26th (1936) Incidents, and, in 1937, the Japan -China War broke out. In 1938, a national mobilization law was brought in, and Japan became completely under the control of the military. In 1937, cholera prevailed in the south of China, and H. Umezawa was sent to the Shimonoseki Harbor Quarantine Station in the Yamaguchi Prefecture in September to examine the feces of soldiers returning from the area. He examined a thousand samples a day by microscope for six months, and this resulted in his left eye becoming smaller than the right (he had big eyes), but this work made him skilled in the handling of bacteria. In April, 1938, he was drafted into the army, and sent to the Narashino Army Hospital. Chiba Prefecture. He became head ofthe Inspection Room and his work became well known in the military. In December, 194 I , the U.S. - Japan War broke out. In April, 1942, paratyphoid B was prevalent in the Takada Regiment of Niigata Prefecture, and the Inspection Room was instructed to move, with all of its facilities, to suppress the epidemic. The army doctor of the Regiment declared that the epidemic would take a half to one year to be suppressed. H. Umezawa, twenty-seven years old, had a thorough discussion with the talented doctor, and they came to the conclusion that, if no germ camer was found during a continuous 10-day inspection, the epidemic could be considered to be terminated. This simple criterion and rather hard everyday work by the members ofthe Room for the 2500 soldiers (the patients were isolated according to the degree of their sickness) brought the epidemic to an end in only twenty-five days. For this service, the director of the Narashino Army Hospital was commended, and H. Umezawa was rewarded with cancellation of the draft and allowed to return, in April, 1943, to his university. Such a decision during war-time by the military was quite rare. He became an Assistant in June, 1943. In the Fall of 1943, he was invited by Katsuhiko Inagaki, a Major, to join as a member of the Research Division of the Military Medical School. This Division had instructions to conduct investigations by able scientists on feeding, hygiene, and medicine to further the war effort. Such mobilization of scientists was commonly camed out at the time, but Inagalu always criticized the lack of evident success. He considered that the failure was attributable to the clumsiness of those in charge of policy, with no defined themes that would prove attractive for scientists. He, therefore, had to establish promptly the theme of investigation. Inagaki was a year senior to Umezawa in the Medical Department of the University of Tokyo. On Dec. 21, when K. Inagaki called on the Ministry of Education to announce the formation of the Division, the officer, Willy Nagai (a son of Nagayoshi Nagai, a famous chemist in Japan whose wife was German),

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handed him a couple ofjournals that had just amved from Germany aboard a Japanese submarine after a perilous journey through the guard of the Allied Forces. Inagaki remarked on an article written by Dr. M. Kiese, in Klinische Wochenschrjfi(August, 1949, about a new, clinically effective drug, penicillin. Inagaki took deep interest in this article, and showed it to the members of the Division: H. Umezawa, K. Sat0 (Nezu Chemical Institute, colloid chemistry), T. Torii (the Medical Department ofthe University ofTokyo, internal medicine), and M. Masuyama (the Central Meteorological Observatory, statistics). They jumped at the theme, and a decision was made “to try to produce this unbelievable drug, penicillin, as fast as possible.” As for the war situation, after General Isoroku Yamamoto, the commander in chief of all Japan Fleets, had been killed in action (April, 1943), the portents of defeat could not be concealed, even from the silent majority. H. Umezawa undertook the translation of the article. He read the article with enthusiasm and excitement because he was eager, after graduation, for such recent foreign reports. H. Umezawa completed the translation on the 5th ofJanuary 1944, and K. Inagaki, after deliberation, issued the translated article with his opinion to the Medical Bureau ofthe Department of War on the 18th ofJanuary, but obtained no response. However, when Asahi Shinbun on the 27th of January carried an article from their correspondent Imai in Buenos Aires on Winston Churchill, who had been cured of pneumonia by use of penicillin (this was later found to be only partially true), on the same day, T. Okada, Major General of the Bureau, at once issued a command to the Military Medical School to complete the basic investigation on penicillin production by August ofthe year with the budget of 150thousand yen (now estimated to approximately 200 million yen). The decision much delighted Inagaki, and the Penicillin Committee was immediately organized. He and Umezawa together called on prominent scholars to join the Committee, and the 1 st Committee, consisting of 20 persons, met on the 1st of February to investigate the production of penicillin. Among the members of the Committee was Hamao’s brother, Sumio Umezawa (Keio Univ.). The article translated by H. Umezawa, with other related papers, was widely distributed to many universities and institutes, and this, with the establishment of the Penicillin Committee, opened the door for antibiotics in Japan. Hamao’s premonition that microbes would be a mysterious box, full of hitherto unknown and valuable compounds, was very exciting. He was dissatisfied with the Japanese medical world at the time, which laid emphasis on the diagnosis and elucidation of diseases, but not on effective cure of patients having, for example, tuberculosis. The Penicillin Committee became the nucleus of research and production of penicillin, and the efforts of many laboratories, including those of Morinaga Confectionery, Banyu Pharmaceutical, and the Yamagata Union of Food Companies, brought penicillin into practical use in Japan, especially in the military, by the end of 1944, and many hopeless septicemia patients were

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dramatically cured. K. Inagalu, still only thirty-three years old, was always an excellent organizer in the production of penicillin, and H. Umezawa was continuously one of the chief leaders in the actual production. He, along with S. Umezawa (Assistant Prof. of Fujiwara Institute ofTechnology at that time) succeeded in isolating penicillin of high quality for the first time, from the fermentation broth of the Y 176 strain obtained from Prof. Yabuta, and demonstrated its excellent effectiveness in vivo (with T. Takeuchi; J. Penicillin, 1947). During this period, H. Umezawa acquired skills in managingand organizing many investigators; he later said that leaders must be patient, and also that any organization, even a government one, has the endeavors of one person behind it. In June 1944, H. Umezawa became an Associate Professor of the Institute of Infectious Diseases (IID; present name, the Institute for Medical Science), a part of the University of Tokyo. In the autumn, H. Umezawa engaged Tomio Takeuchi (now the director of both the Institute of Microbial Chemistry and the Microbial Chemistry Research Foundation) as his assistant, and Takeuchi, a lover ofexperimental research with an acute scientific sense, became the indispensable collaborator of H. Umezawa throughout his subsequent research life. In the midst of those busy days, Hamao Umezawa mamed Mieko Ishizaki on the 2nd of December 1944. She was a natural, pretty, and lovely girl of nineteen years at the time. Fortunately, the two were able to celebrate the wedding at the Imperial Hotel in Tokyo, because, on that cold day, there was no air raid by the American Air Force. Mieko’s father, Seiichi Ishizaki, was a successful wholesale merchant dealing in woolen yarn in downtown Tokyo from a shop named “Mitsubaya.” Her mother, Hana, was a daughter of Shukichi Takano, the owner of a big clockmaking establishment. Mieko was born on January 1, 1925, the last child in a family of four children. She was a trueborn Tokyoite, very often enjoyed Kabuki theatrical performances, and took lessons in classical Japanese dance from childhood. She wore the Kimono well, as befitted a daughter born to a traditional merchant. She graduated from the Japanese literature course at the University ofthe Sacred Heart in Tokyo in 1944. At their wedding reception, held on the night of their marriage, all of the food served to the guests had to be brought from outside by themselves. The next morning, Hamao went to his laboratory at eight o’clock as usual. They experienced a big air-raid by the American Force in the Tokyo area, and during the day were not able to determine the safety of each other because of the interruption of communications by this unusual wedding “present” from the U.S.A. H. Umezawa obtained the degree of Ph. D. (Medical Science) in April 1945. A.fier the War.The War ended on the 15th ofAugust in 1945. H. Umezawa considered the defeat to be a natural result. In that September, he was instructed by the last director of the Military Medical School to explain the Japanese state of penicillin production to the General Headquarters (GHQ)

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of the Occupation Forces of the Allies. He met a Lieutenant Colonel French of the GHQ, and they went together on tours of inspection of penicillin production. Dr. French later became the Head of the Department of Pathology of the University of Minnesota. In December, 1946, one half of the IID was transferred, by orders ofthe GHQ, to the Ministry of Public Welfare, and was named the National Institute of Health (NIH) of Japan. H. Umezawa became the first director ofthe Department ofAntibiotics ofthe NIH in 1947 ( I947 - 1978). This decision firmed his commitment to antibiotics. In October, 1946, Prof. J. W. Foster of the University of Texas visited Japan, by request of the GHQ, to assist in penicillin production in Japan. He met H. Umezawa and other important persons, and presented a strain suitable for tank fermentation of penicillin, which contributed to the practical production. Thus, H. Umezawa was busy until 1948 in establishing the commercial production of penicillin; the Meiji Seika Co. cooperated with him (Mr. Takeshi Nakagawa, later the President, and Dr. Harutaro Yasuda, later the Managing Director, were the principal persons involved), and became the largest penicillin-producing company in Japan in those days. Overlapping with the work on penicillin, H. Umezawa also worked until 1950 to increase the production of streptomycin. This antibiotic, discovered by Waksman in 1943, was difficult to purify, but proved effective against tuberculosis, the most feared disease in Japan in those days. Tuberculosis was called the national disease in Japan, and once established, the patient had, in many cases, to give up his future and was alienated from his neighbors. The purification problem was resolved by the use of a cation-exchange resin from Mitsui Chemicals Co. (in cooperation with H. Umezawa); Rohm & Haas Co. and Merck & Co. lnc. concurrently discovered the usefulness of this resin. After streptomycin, major discoveriesof useful antibiotics continued: chloramphenicol by Parke, Davis & Co. in 1945, chlortetracycline (aureomycin) by B. M. Duggar in 1947, and oxytetracycline (terramycin) by Chas. Pfizer Co. in 1948. On his first tour abroad, H. Umezawa spent October 1950 to February 1951 in the U.S.A. to learn more about the advances in antibiotics and biochemistry. He was impressed to see many large and fine buildings, even in Anchorage, Alaska, big salaries for important persons as compared with his own, and strong competition in research, and he was surprised to find every American he met very sympathetic. However, in contrast to Japan, he found that American research on antibiotics tended to be concentrated more in pharmaceutical companies than in university laboratories. Japan still emphasized a national demand for effective medicines, especially against tuberculosis. Discovery c f Kanamycin, and Establishment of'IMC. Chloramphenicol, chlor- and oxy-tetracyclines, and pyridomycin (H. Umezawa, 1967) were active, in in vitro experimcnts, against strains of tuberculosis, but these drugs, in contrast to streptomycin, were clinically inactive. H. Umezawa

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thought that this difference might be due to solubility differences in water, and therefore, targeted his research to find water-soluble, basic substances from Strqtomyces. As the rates of growth of the Mycobacterium tubercuh i s strains of clinical origin were very low and the organisms dangerous to handle, he brought M. smegmatis ATCC 607 back from the U S A . This was similar in character to the strains of clinical origin, but had a high growthrate and low risk, thus facilitating research work. Most of the compounds found in this system, however, demonstrated a delayed renal toxicity. After much difficult work on this line, which some persons criticized as a waste of time, he and his staff encountered in 1955 the first promising substance. It was isolated from the fermentation broth of a streptomycete, obtained from a soil sample from Nagano Prefecture, by using a screening system developed by himself and Y. Okami (now vice-director of IMC). The compound was crystallized in the form of a monosulfate monohydrate by one of the writers (K. Maeda; now vice-director of IMC) and named kanamycin. Crystallinity is rarely encountered in aminoglycoside antibiotics, and this feature facilitated the purification of kanamycin, and the X-ray crystal analysis carried out later. The efficacy of kanamycin against tuberculosis in mice was demonstrated by Dr. Ken Yanagisawa (then Director of the Department of Tuberculosis of NIH, Japan). Production on the 1-kg scale was established by the Meiji Seika Co., and the first clinical effectiveness was shown by Prof. Tokuji Ichikawa ofthe University ofTokyo. He was an authority on urology, the specialty most suitable for examining the drugs. The chemical structure of kanamycin, except for its absolute configuration, was determined in I959 by Prof. Sumio Umezawa of Keio University. In May 1958, a research conference on kanamycin was held by the Japan Medical Association, and in July, at the New York Academy of Sciences. During those days, Hamao Umezawa was anxious, because his work was being discussed by many Americans. This was the first time that Japanese work had been noted by the New York Academy. Kanamycin showed activities against resistant staphylococci, and resistant Gram-negative bacteria, including Shigella dysentery, and resistant strains of tuberculosis. Kanamycin was commercialized in 1958, and its clinical effectiveness attracted much public attention. As a result of his work on kanamycin, Hamao Umezawa was presented with many prizes: the Asahi Prize (from the Asahi Shinbun, 1959), Prize from the Minister of the Ministry of Science in Japan (1959), Commandeur de 1’Ordre de la Santk Publique (from France, 1960), and the Japan Academy Prize ( 1 962). The Order of Culture (Bunka-Kunsho), the most honorable prize for scholars in Japan, was conferred by the Emperor of Japan in 1962, when Hamao was forty-eight years old; the Umezawas’ two sons, Kazuo (now vice-director of the Institute of Microbial Chemistry) and Yoji (now chief researcher) were then sixteen and twelve years old, respectively. H. Umezawa wanted to have his own institute in order to develop his ideas

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TSUTOMU TSUCHIYA, KENJl MAEDA. AND DEREK HORTON

freely. Ryogo Hashimoto, then Minister of the Ministry of Health and Welfare, suggested that, as a reward for his efforts on kanamycin, there be established a foundation to receive the royalties from kanamycin. Thus, the Microbial Chemistry Research Foundation (MCRF) was founded in 1958, and H. Umezawa became its Director. In May 1962, the Institute of Microbial Chemistry (IMC) was built on a little hill near Meguro, Tokyo, and it was enlarged in 1966. The first key members of staff were Drs. Tomio Takeuchi (biology and anticancer antibiotics), Kenji Maeda (chemistry), and Yoshiro Okami (microbiology). Even before construction was complete, all members of IMC helped back up H. Umezawa in getting the new institute started. They knew they had to succeed, as otherwise the Institute would fail. Each person, regardless of his specialty, was engaged in the isolation of microbial products through fermentation and extraction. Mrs. (Dr.) Masa Hamada(nCe Kuroya; now Head of the Department of Microbiology Sect. I of IMC), having a trained sense and excellent memory for micro-organisms, examined all micro-organisms taken from soil samples. On the basis of morphological characteristics of the strain and the antibacterial spectrum of the crude product from the first fermentation, she selected promising-looking strains. These strains were then distributed to the hopeful members through the “Strain Meeting” that was spontaneously formed by all members of the Institute. Later, this unity gradually became diversified; for example Dr. Hiroshi Naganawa focused mainly on instrumental chemistry, and became the specialist in n.m.r. spectroscopy and mass spectrometry. During the search, M. Hamada and Y. Okami recognized a previously unknown, uncommon strain that produces a substance active against Pyriciiluriu oryzae, the causative strain of rice-blast disease. The culture broth of this strain did not inhibit the growth of P.oryzae when tested in ordinary culture media (pH 7), a factor that hindered the rapid investigation of this principle. H. Umezawa and T. Takeuchi thought that, because the component was active in the rice plant, the addition of rice-plant juice to the medium might change the situation, and this proved to be correct. At the same time, the juice was found to be acidic (pH - 3,and the active component inhibited the growth of P. oryzae best at this pH. The compound, named kasugamycin (1965), showed very low toxicity and has since then been widely used in rice fields in Japan, replacing the hazardous phenylmercuric compounds previously used. The pseudodisaccharide structure of kasugamycin was determined by Dr. Yasuji Suhara. For this contribution, H. Umezawa received a prize from the Ministry of Agriculture of Japan ( 1975). A pseudodisaccharide similar to kasugamycin was later found ( 1975); this was named minosaminomycin, a peptide antibiotic active against mycobacteria.

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9

Another useful substance that brought financial support to the Institute was josamycin, discovered in 1964; Dr. Takashi Osono of Yamanouchi Pharmaceutical Co. cooperated in this work. This macrolide antibiotic showed strong activity against Gram-positive bacteria and mycoplasmas, and was highly regarded as the macrolide of first choice because of its high affinity for the lungs, its lack of irritation to the stomach, and its low tendency to develop resistance. This work exemplifies well the type of mutual stimulation of conjoint research between members of the Institute and the collaborating drug companies, a style of research only possible under the strong leadership of H. Umezawa. Rcseurche.7 on Anticancer Agents. As early as 1949, H. Umezawa and T. Takeuchi looked for compounds active against viruses. They worked for two years in vain, but during that time they found that microbes often produce substances that inhibit the growth of the cells, whether or not they carried viruses. H. Umezawa also came to realize that most viral infections in patients were already at a peak when found, and it was difficult to effect cures with drugs. These concerns led his efforts to switch in 1951 from viruses to cancer, in cooperation with Drs. Tadashi Yamamoto (later, Director of the Institute for Medical Science), T. Takeuchi, Kazuo Nitta (now Director of the Chiba Cancer Center Institute), and Seizaburo Yamaoka (later, Vicepresident of Sumitomo Chemical Co. Ltd.). This pioneering work was rewarded in 1954 by the discovery of sarkomycin, a low-toxicity anticancer agent. Sarkomycin was the first antitumor substance clinically used in Japan: it was commercialized by Meiji Seika Co. and Banyu Pharmaceutical Co., but was discontinued because of its instability. In 1959, H. Umezawa and coworkers found a promising low-toxicity antitumor substance, phleomycin. Unfortunately, it was found to exhibit a delayed, irreversible renal toxicity in dogs. In 1963, however, a similar substance, having lower renal toxicity and higher acid-stability, was found. It was named bleomycin. Prof. Ichikawa of the First Tokyo National Hospital demonstrated the high therapeutic efficacy of bleomycin on squamous-cell carcinoma. Later, bleomycin was found highly effective against Hodgkin's disease. The selective effects on these diseases proved (1972) to be attributable to a high concentration of bleomycin and low content of a bleomycin-inactivating enzyme (bleomycin hydrolase) in the affected tissues. The complex chemical structure of bleomycin was largely elucidated in 1972 in cooperation with Dr. Tomohisa Takita (currently head of a chemistry section at IMC), and Drs. Yasuhiko Muraoka and Akio Fujii of Nippon Kayaku Co., and was further revised in 1978. Bleomycin is composed of five subunits, including a disaccharide, and its structural elucidation relied heavily on 100-MHz 'H- and 25-MHz 13C-n.m.r. spectroscopy with the established instruments of that time.

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TSUTOMU TSUCHIYA, KENJI MAEDA, AND DEREK HORTON

Cooperative work with Prof. Nobuo Tanaka (Professor Emeritus of the University of Tokyo) in 1969 showed the mechanism of action of bleomycin to involve DNA strand-scission. The difficult total synthesis of bleomycin was accomplished (1981) in cooperation with Takita and others, including Hamao’s son, Yoji Umezawa. H. Umezawa was very satisfied with the success of this total synthesis, and his sustained enthusiasm for improved bleomycins led to peplomycin ( 1 978;used clinically since 198 1)and libromycin ( 1985). After the discovery of sarkomycin, H. Umezawa’s research method was adopted by many groups in the world, and many antitumor antibiotics were found in microbes: mitomycin A, B (1 956),and C ( 1957)by Drs. Toju Hata (Professor Emeritus of Kitasato University) and Shigetoshi Wakaki (later, Vice-president of Kyowa Hakko Co. Ltd.), and doxorubicin (adriamycin, 1960)by Dr. F. Arcamone are significant examples. From 1971, H. Umezawa and T. Takeuchi also searched for anthracycline antibiotics having low cardiotoxicity, and found the aclacinomycins (1975)and baumycins ( 1977). The low cardiotoxicity of these compounds was confirmed by many groups, including Prof. G. Mathe of the Institut de Cancerologie et d’ Immunogenetique, and Dr. Shinichi Hirano of Sanraku-Ocean Co. As all of these compounds have, at OH-4 of the daunosamine or rhodosamine component, a sugar substitute or its equivalent to which the low toxicity ofthese antibiotics might be attributed, 4’-O-tetrahydropyranyladriamycin ( 1979)was synthesized, and its properties supported this assumption. A promising fluorinecontaining analog, 7-O-(2,6-dideoxy-2-fluoro-a-~-talopyranosyl)adriamycinone, was prepared in 1986 in cooperation with T. Tsuchiya, T. Takeuchi, and S. Umezawa. Another type of promising antitumor substance, spergualin, was discovered ( 198 1 ) by a special screening method. T. Takeuchi had often observed that microbes produce bioactive substances admixed with toxic compounds, but such mixtures were liable to be abandoned as useless by conventional screening. Thus, in order to detect the existence of low-toxicity antitumor substances in culture filtrates, it was necessary to perform simultaneous and comparative activity-measurements of the metabolite mixture against tumor and normal cells. On the basis of this new screening method, spergualin, showing strong activities against a variety of leukemia and adriamycinresistant leukemia cells, was discovered. Synthetic 15-deoxyspergualinlater proved to have higher activity, and is now in clinical use. Among some 70 new antitumor compounds discovered by H. Umezawa and coworkers, formycin ( 1966) constituted another important discovery. Produced in conjunction with coformycin, the latter inhibits the activity of adenosine deaminase ( 1967) and enhances the antitumor activity of formycin.

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Elucidation c?fthc. Resistance Mechanism of Aminoglycoside Antibiotics, and the Synthesis of Dibekacin. From the middle of the 1960’s, kanamycinresistant strains began to appear in hospital patients. In 1959, Prof. Kunitaro Ochiai of Eastern Citizen Hospital (Nagoya) and then Prof. Tomoichiro Akiba of the University of Tokyo independently discovered that the resistance of Bacilliis d-vsenntcryagainst the drug could be transferred to a different kind of bacterium, Escherichia coli, by mixing the cultures, and vice versa. This discovery attracted the attention of bacteriologistsworldwide, and soon the mechanism of resistance in bacteria was clarified as involving R-factors that carry genes conferring resistance to drugs, and that these R-factors could be transferred to other bacteria by direct contact or through bacteriophages. Thus, chloramphenicol was inactivated by acetylation caused by an enzyme expressed from the R-factor. However, in the case of aminoglycoside antibiotics, it was still thought that the bacteria became resistant through intensification of the surface bamer against the drug, although that seemed true only for the resistant bacteria formed in vitro by contact with a high concentration of the drug. However, H. Umezawa carefully examined kanamycin-resistant bacteria of clinical origin and found (Science, 1967) that inactivating enzymes exist inside the membrane and catalyze the phosphorylation of kanamycin at OH-3’ by ATP, to give the inactive kanamycin 3’-phosphate. Another discovery was the 6’-N-acetylation of kanamycin by acetylcoenzyme A under catalysis by another enzyme. He also found (1968) that streptomycin was inactivated by adenylylation at OH-3”. By many subsequent studies, it was concluded that these three modes of inactivation are of general occurrence in the aminoglycoside antibiotics. It is very interesting that bacteria cooperatively resist human artifacts. These studies were made mainly in cooperation with Dr. Shinichi Kondo (now Head of Department of Chemistry Sect. I1 of IMC) and also Dr. Morimasa Yagisawa (now Executive Director of the Japan Antibiotic Research Association). H. Umezawa received great admiration when he presented these results at the 5th International Congress of Chemotherapy in Vienna. He later related that 1967 was one of the brightest years of his life-after the discovery of kanamycin. The next problem for H. Umezawa was to use his findings to design new kanamycin derivatives effective against resistant bacteria. The synthetic work was undertaken in cooperation with his brother, Prof. Sumio Umezawa of Keio University, and one of the writers (T. Tsuchiya). The first useful derivatives active against resistant bacteria, namely, 3’,4’-dideoxykanamycin B (dibekacin)and 3’-deoxykanamycinA, were prepared in 1971. These were also found active against Pseudomonas known to have intrinsic resistance. These results supported the truth of H. Umezawa’s theory. In the synthesis of dibekacin, the Tipson-Cohen method for introducing unsaturation, developed by one of the writers (D. Horton, 1966) for pyranoside

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rings and on which Tsuchiya had studied with him, proved very useful. The production of dibekacin, in the first regular commercialization (1975) of a derivative of an aminoglycoside antibiotic in the world, was performed by the Meiji Seika Co. Takeshi Nakagawa; its president, Dr. Harutaro Yasuda (Senior Vice-president) and Dr. Shunzo Fukatsu (now Director of the Pharmaceutical Development Laboratories) were prime motivators. This task was really difficult, and the research meetings lasted intermittently for years under the baton of H. Umezawa until the production yield of dibekacin (from kanamycin B) attained 6090.H. Umezawa was greatly delighted by this success, because he was always anxious about the financial base of MCRF, and dibekacin brought substantial revenues to the Foundation. The success of dibekacin prompted worldwide attention to the removal of selected OH groups in aminoglycoside antibiotics susceptible to modification by resistant bacteria, and the chemical deoxygenation procedure of D. H. R. Barton was found particularly useful. Considering the rapid progress in chemistry, H. Umezawa felt the need to establish a new institute devoted to the chemistry of antibiotics. In early 197 1, he gave T. Tsuchiya the responsibility for constructing an institute near Keio University, even though H. Umezawa knew that Tsuchiya had no experience other than organic chemistry. Despite Tsuchiya’s misgivings, H. Umezawa taught him the basic knowledge needed for construction of the institute, as well as the difficultiesto be expected. He started out to search for suitable land, and met several difficulties, including trouble with one of the landowners, opposition by nearby inhabitants, a one-year bureaucratic delay in issuing the construction permit by the Kawasaki City Office, temporary suspension of construction by the discovery of ruins, and the steep rise in price caused by the so-called “Oil Shock.” Once H. Umezawa attended, with Dr. Kageaki Aibara (head ofa Department ofthe National Institutes of Health of Japan), a meeting with local inhabitants who opposed the construction as they feared that the work of the Institute in dealing with bacteria would be dangerous for the nearby inhabitants. H. Umezawa quietly answered even impolite questions from the audience in the manner of a physician treating his patients, and this welcome attitude allayed most of the suspicions. H. Umezawa’s strong will enabled Tsuchiya (now Vice-Director ofIBC) to overcome the problems, and the Institute of Bioorganic Chemistry became established in November, 1974. Further studies on the mechanism of resistance of aminoglycoside antibiotics focused on resistance genes existing in antibiotic-producer strains (mainly by Drs. Y. Okami and Kunimoto Hotta), and gradually clarified the relationship between the antibiotic-producing and -regulating mechanism. During this search, indolizomycin (1984) was discovered by cell fusion of two kinds of strains.

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Enzyme Inhibitors Having Low Molecular Weight. In 1965, after discussions with Prof. Toshiharu Nagatsu (biochemist), then of Aichi-Gakuin University, and T. Takeuchi, H. Umezawa decided to search for low-molecular-weight (1.m.w.)enzyme inhibitors from microbes. Their small molecular size would avoid problems arising from their behaving as antigens in the human body. This was the first such attempt in the world. H. Umezawa had long been thinking that microbes would be the source of all kinds of useful materials that might be found by altering the screening methods, and this was a good chance to explore his idea in a field other than antibacterial and anticancer antibiotics. Furthermore, there appeared to be a decline in the number of new antibiotics being discovered by classical methods. The first efforts were targeted toward inhibitors for norepinephrine biosynthesis, in the hope of finding compounds effective in alleviating high blood pressure in humans. The first such substance discovered was aquayamycin (1968), followed by fusaric acid ( 1969), oudenone ( 1970), dopastin ( 1972), and then many others. The research program was then expanded to inhibitors for such proteases (endo- and exo-peptidases) as plasmin, trypsin, papain, cathepsin B and D, chymotrypsin, pepsin, and renin. This work was conducted in cooperation with Drs. Takaaki Aoyagi (Head of the Department of Enzymology of IMC) and T. Takeuchi. Many promising substances were found, including leupeptin (1 969), pepstatin ( I970), antipain (1 972; identical with the compound found by the chemical screening method employing the Sakaguchi reagent for the guanidino group), and phosphoramidon ( 1973). The last is a thermolysin inhibitor and is identical with the substance found ( I 972) by the Ehrlich color-screening methods on culture filtrates of microbes that had been conducted with Prof. S. Umezawa and Drs. T. Tsuchiya and Kuniaki Tatsuta (now a Professor at Keio University). The compound contains a phosphoramide group, tryptophan, and L-rhamnose in the molecule. Chemical screening methods also disclosed dienomycin ( 1970), arglecin ( 197 1), spydrofuran ( 197 1 ), argvalin ( 1972), KD 16-U 1 ( 1974), and KF77-AG6 ( 1974), in addition to those compounds already noted. Pepstatin was found effective in treating gastric ulcer, and many of these protease inhibitors have been widely used for the identification and purification of the corresponding enzymes. For example, renin was purified for the first time by pepstatin affinity chromatography. H. Umezawa hoped to obtain immunostimulants from microbes, because, in cancer patients, the immune response is lowered. In 1972, H. Umezawa, T. Takeuchi, and M. Ishizuka (now Vice-Director of the Institute for Chemotherapy, a branch of IMC) found that the administration in mice of a small dose of diketocoriolin B, an oxidation derivative of the antitumor antibiotic coriolin B (1971), increases the number of mouse-spleen cells

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producing antibody to sheep red-blood cells that had been administered in advance. H. Umezawa and Dr. Takehiko Kunimoto also found that diketocoriolin B inhibited (Na+- K+)-ATPase, a cell membrane-bound enzyme. These results led to the idea that binding to cell-surface enzymes may promote the cell division of lymphocytes producing antibodies, consequently enhancing the immune response. H. Umezawa and coworkers found that aminopeptidases, alkaline phosphatases, and esterases exist on cell surfaces. Accordingly, they searched for compounds with which to inhibit these surface enzymes. Among the inhibitors thus found, immunostimulating compounds were further explored. By this procedure, bestatin ( I976), amastatin (1978), forphencine (1978), esterastin (1978), and ebelactone (1980) were discovered. Bestatin was commercialized in 1987, a year after H. Umezawa died. These studies on low-molecular-weight enzyme inhibitors, begun by H. Umezawa, are under continued development. Bioactivc Products from Marine Micro-orgunisrns. As it had been presumed that the strain-specific production of antibiotics is to some extent a response to environmental conditions for the strains, it was meaningful to extend the research for new sources to marine micro-organisms. Although most of the actinomycetes taken from a shallow-sea mud were found to be similar, or identical, to those of terrestrial origin, some were different; from one of them was discovered aplasrnornycin ( I 976) which inhibits plasmodium propagation and malarial disease in mice. It has a unique structure containing boron in the center of the molecule. Istamycin ( 1979), a fortimicin type of aminoglycoside antibiotic, was also found in marine actinomycetes that preferably grow in media containing sea water. Bisucaberin ( 1987), a cyclic compound (containing two hydroxamic acid moieties) which sensitizes tumor cells to cytolysis by murine peritoneal macrophages, was isolated from Alteromonus haloplant is obtained from deep-sea mud offshore from Akita Prefecture. It was recognized that, in sea water, there are various viscous constituents, such as agar or other biopolymers, as exemplified by the difficulty experienced in the filtration of water from shallow seas through a membrane filter. This observation suggested that the sea water might contain various degrading enzymes for such polymers. Following up on this lead, a new C Y - D - ~ ~ U canase ( 1980) having potential activity for hydrolyzing the insoluble D-glucan on the teeth was found. A polysaccharide named marinactan (1983) exhibiting complete tumor-regression was also isolated from a Flavohacteritim found by screening for strains producing polysaccharides. These studies were mainly performed by Dr. Y. Okami. Personal lije. H. Umezawa was always busy on week days, exchangng views with the staff of IMC and offering advice, and in meeting many visitors. He often enjoyed having dinner with foreign visitors, including one

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of the writers (D. Horton), in elegant restaurants. He appreciated good food and fine wines, and liked to criticize the policies of the top leaders in Japan and other countries during the meal. He had a unique ability to attract people. He appreciated persons of true ability and steadfastly treated them well. He despised persons who expressed opinions based on uncertain “facts,” and disliked commonplace men. At home, he could prepare papers even while watching television, or during the noisy play of his sons or the children of his relatives. In the early days of his marriage, he sometimes invited the young students to his home, where his wife, Mieko, would be busy finding food for them (right after the War, especially, food was very scarce). He was very kind to both his research group and Mieko’s friends. He also showed great devotion to his parents, especially his mother. On Sundays in his middle to late years, he very often went to his Institute to spend some quiet time reading the literature without disturbance. On such mornings, H. Umezawa and Mieko left home together in his car and, half way to the Institute, he dropped Mieko off at her church to attend the morning service and other activities in the church; H. Umezawa never complained, although he was then himself a Buddhist. Mieko Umezawa was baptized in her fortysixth year, and they had a tacit understanding to respect each other’s beliefs. She participated, for some 10years from 1973, in the volunteer activity “Life Line,” which gives advice by telephone to persons desperately seeking help; this was sometimes the last cry of these individuals before they went to their death. She often listened for one to two hours to a single person. In such miserable cases, she listened with patience, summarized what the person said, and offered her ideas for resolving the problem. She came to realize that such persons often had nothing to rely upon; she suggested, however, that every individual must live his or her own life to the end and must be aware of the grace ofGod. Although the activity itselfhas no direct relation to religion, it was motivated by her thought that Christian ideals must be realized in real life. The orderly life of the Umezawas was suddenly split, in August 1983, by Hamao’s falling victim to a cerebral infarction. Mieko nursed him day and night in the hospital for five months. Luckily he recovered, although his right hand would no longer move well. He soon began rehabilitation therapy, and continued it even in his Institute with the help ofDr. Takeshi Hara, and Miss Kyoko Kinjo, the secretary. He even visited the U.S.A. with Mrs. Umezawa in April, 1986, to receive the Smissman Award of the American Chemical Society for 1983. This was, for the Umezawas, an arduous tour which made Mieko remember the trip of I98 1, when they visited England in June, the first abroad for her, for H. Umezawa to receive the Doctor of Science, Honoris cuusu, from the University of Oxford. At that time, all things were bright, and she enjoyed the classic atmosphere of England free from daily

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household chores. In July, 1986, H. Umezawa developed pneumonia; his hospital room again changed to a research office, with visits from his staff, but this time, his disease gradually became worse. He sometimes had severe pain in his chest and difficulty in breathing, and he became a little imtable. He installed an electrocardiograph himself and checked his own cardiogram. He wanted to live longer; his mind was still full of ideas to be realized. On December 14, he suddenly told Mieko of his desire to be baptized. She was surprised, and wanted to ask “why?’, but could not do so from fear of hearing the next words. Soon a minister was called and H. Umezawa was baptized according to his own wishes. She felt quite happy and regarded the whole matter as the work ofGod. When he received a glass for the Sacrament in his bed, he said “this is just plain grape juice, not wine.” Throughout his hospital life, T. Takeuchi attended him and carefully observed his condition. Toward the end of December, Drs. K. Maeda, T. Hara, and Miss Kinjo also attended Umezawa every day, assisting Mrs. Umezawa, and sons Kazuo and Yoji came to understand Hamao’s life better than ever before. He died on Christmas Day, 1986. Mrs. Umezawa stated that these times in the hospital with her husband were just like their honeymoon days, wherein she found some of her husband’s hidden characteristics for the first time. Prof. J. C. Sheehan of MIT, one of Hamao’s best friends, said, during a symposium dedicated to H. Umezawa (Nov. 25, 1987), that H. Umezawa was a giant, and it is impossible to discuss the growth of antibiotics in the past four decades without him. A man of moderate stature, Hamao Umezawa was, until the time of his sickness, of robust appearance and engaging personality. He spoke English rapidly and with ease, with a characteristic inflection that demanded acute concentration by the listener. All of Hamao Umezawa’s work was closely connected with carbohydrates. The principal compounds that he developed, namely, kanamycin, dibekacin (with the elucidation of the resistance mechanism), kasugamycin, formycin, bleomycin, and anthracyclines, are all glycosides.The 1.m.w. enzyme-inhibitors, exemplified by bestatin, are mostly oligopeptides, and are the only exceptions. H. Umezawa established, in addition to the two institutes already described, Episome Institute ( 1 968) at Maebashi-shi, Gunma Prefecture (now directed by Prof. Susumu Mitsuhashi of Gunma University), and the Institute for Chemotherapy ( 1984) at Numazu-shi, Shizuoka Prefecture. Stages in H. Umezawa’s professional history not already described included the following: Professor of the University of Tokyo (1954- 1975), directing research in the 6th ( 1954- 1965)and the 12th Department ( 19651975) of the Institute of Applied Microbiology, and in the Cancer Biology Department of the Institute for Medical Sciences ( 1972- 1975); Professor

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Emeritus of the University of Tokyo ( 1977- 1986). He worked as a member of various Japanese Government Councils, such as the Central Pharmaceutical Affairs Council (an advisory committee of the Minister of Health and Welfare), the Science Council (an advisory committee of the Minister of Education, Science, and Culture), the Council for Science and Technology (an advisory committee of the Prime Minister), and others. With these services, he contributed in Japan not only to development of basic research but also to the improvement of administration for health and welfare, the advancement of science and education, the progress of technology concerning the life sciences, and other aspects. He held responsibilities in many associations: the International Society of Chemotherapy (Vice-president, 1963- 1967,and 1971 - 1975;Member of the Executive Committee, 1969I97 I), International Congress of Chemotherapy [President (6th ICC), 1967- 1969; Honorary President ( 14th ICC), I983 - 19851, Japan Association of Chemotherapy (Director, 1968- 1970), Japan Cancer Association (Chairman, I978), and Japan Antibiotic Research Association (Chairman, 1970- 1986). H. Umezawa received many awards not earlier mentioned: Ordre National de la Lkgion d’Honneur (Chevalier) (197 I), Fujiwara Prize from the Fujiwara Foundation (1 97 I), Princess Takamatsu Cancer Research Prize (l977), The Windsor C. Cutting Lectureship Award (Univ. of Hawaii, I977), Karl-August Forster Prize from Mainz Academy, Germany ( 1977), Paul-Ehrlich and Ludwig-Darmstaedter-Preis(Haupt Prize), Frankfurt-am-Main, Germany ( 1980)(H. Umezawa was especially delighted with the prize, as he respected P. Ehrlich and read his biography with much interest), Griffuel Prize, L’Association pour la Developpement de la Recherche sur le Cancer, France ( 198I), and the International Prize of Chemotherapy (1984). His last award was the First Order of the Sacred Treasure (Kun Itto, Zuihosho) conferred by the Emperor of Japan (Nov. 1986). H. Umezawa received several honorary doctorates: from the University of Santiago de Compostella, Spain ( 1977),the Royal Karolinska Institute, Sweden (1978), the University of Oxford (1981), and the University of Paris-Sud ( 1984). His academy memberships included: Member of Japan Academy ( I 969-), Mitglied der Deutsche Akademie der Naturforscher Leopordina (1973-), Member of the American Academy of Arts and Sciences, Boston, Massachusetts ( 1978-), Foreign Member of the Royal Swedish Academy, Sweden (1978-), and Full Member of the Pontifical Academy of Sciences, the Vatican ( 1983-). H. Umezawa held honorary memberships in the Japan Association of Medical Science ( 1963),the Brazil Cancer Society ( I969), the Poland Medical Association ( I970), the Pharmaceutical Society of Japan ( I974), the International Society of Chemotherapy ( 1977),the Japan Bacteriology Society ( 198I), and the Infectious Diseases Society of America ( I 984).

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ACKNOWLEDGMENTS The writers are grateful to Mrs. Mieko Umezawa, and Drs. S. Umezawa, K. lnagaki, T. Takeuchi, and M. Hamada for furnishing information. We obtained some of the information from the article “Biography on Research” written by H. Umezawa (“Shokim,” Nos. I -4, 1980, Bungei Shunju Co., in Japanese) and “Hekiso, the Japan Penicillin Story,” by Fusako Tsunoda ( 1978, Shincho-sha Co., in Japanese). We also express deep thanks to Mrs. Haruko Tsuchiya for help in preparing the manuscript.

TSUTOMU TSUCHIYA KENIIMAEDA DEREKHORTON

APPENDIX

H. Umezawa published some 1200 papers covering many new antibiotics

(- 100 for antibacterial, -70 for antitumor, and > 50 for enzyme inhibitors).

Here are listed only the most representative reports. “Studies on the penicillin. Relation between the growth inhibitory effect in vitroand the protective activity in vivo,” H. Umezawa and T. Takeuchi, J. Penicillin, 1 (1947) 14- 18; spoken at the Penicillin Committee in Jan., 1945.

“Sarkomycin, an anti-tumor substance produced by streptomyces,” H. Umezawa, T. Takeuchi, K. Nitta, T. Yamamoto, and S. Yamaoka, J. Antibiot., A6 (1953) 101. “Biological studies on kanamycin,” T. Takeuchi, T. Hikiji, K. Nitta, Y. Yamazaki, S. Abe, H. Takayama, and H. Umezawa, J. Antihiot., A 10( 1 957) 107- 114.

“Production and isolation of a new antibiotic, kanamycin,” H. Umezawa, M. Ueda, K. Maeda, K. Yagishita, S. Kondo, Y . Okami, R. Utahara, Y. Osato, K. Nitta, and T. Takeuchi, J. Antibiot., A 10 (1957) 18 1 - 188. “Kanamycin: its discovery,” H. Umezawa, Ann. N. Y. Acud. Sci., 76 (1958) 20-26. “A new antibiotic, formycin,” M. Hori, E. Ito, T. Takita, G. Koyama, T. Takeuchi, and H. Umezawa, J. Antibiot., A 17 ( 1964) 96 -99.

“Mode of inhibition ofcoformycin on adenosine deaminase,” T. Sawa, Y. Fukagawa, I. Homma, T. Takeuchi, and H. Umezawa, J. Antibiot., A20 (1967) 227-231.

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“A new antibiotic, kasugamycin,” H. Umezawa, Y. Okami, T. Hashimoto, Y. Suhara, M. Hamada, and T. Takeuchi, J. Antibiot., A18 (1965) 101- 103.

“Total synthesis of kasugamycin,” Y. Suhara, F. Sasaki, G . Koyama, K. Maeda, H. Umezawa, and M. Ohno, J. Am. Chem. Soc., 94 (1972) 6501 6507. “A new antibiotic, josamycin, I. Isolation and physico-chemical charac-

teristics,” T. Osono, Y. Oka, S. Watanabe, Y. Numazaki, K. Moriyama, H. Ishida, K. Suzaki, Y. Okami, and H. Umezawa, J. Antibiot., A20 (1967) 174- 180.

“New antibiotics, bleomycin A and B,” H. Umezawa, K. Maeda, T. Takeuchi, and Y. Okami, J. Antibiot., A19 (1966) 200-209. “Clinical study of a new antitumor antibiotic, bleomycin,” T. Ichikawa, K. Matsumoto, and H. Umezawa, Int. Congr. Chemother. 5th, Vienna, (1967) 507-516.

“Studies on the mechanism of antitumor effect of bleomycin on squamous cell carcinoma,” H. Umezawa, T. Takeuchi, S. Hori, T. Sawa, M. Ishizuka, T. Ichikawa, and T. Komai, J. Antibiot., 25 (1972) 409-420. “Chemistry of bleomycin. XIX. Revised structures of bleomycin and phleomycin,” T. Takita, Y. Muraoka, T. Nakatani, A. Fuji, Y. Umezawa, H. Naganawa, and H. Umezawa, J. Antibiot., 31 (1978) 801 -804. “Total synthesis of bleomycin A2,” T. Takita, Y. Umezawa, S. Saito, H. Morishima, H. Naganawa, H. Umezawa, T. Tsuchiya, T. Miyake, S. Kageyama, S. Umezawa, Y. Muraoka, M. Suzuki, M. Otsuka, M. Narita, S. Kobayashi, and M. Ohno, Tetrahedron Lett., 23 (1982) 521 -524. “New antitumor antibiotics, aclacinomycins A and B,” T. Oki, Y. Matsuzawa, A. Yoshimoto, K. Numata, I. Kitamura, S. Hori, A. Takamatsu, H. Umezawa, M. Ishizuka, H. Naganawa, H. Suda, M. Hamada, and T. Takeuchi, J. Antibiot., 28 (1975) 830-834. “Tetrahydropyranyl derivatives of daunomycin and adriamycin,” H. Umezawa, Y. Takahashi, M. Kinoshita, H. Naganawa, T. Masuda, M. Ishizuka, K. Tatsuta, and T. Takeuchi, J. Antibiot., 32 (1979) 1082- 1084. “Syntheses and antitumor activities of 7-0-(2,6-dideoxy-2-fluoro-a-~-talopyranosy1)-daunomycinone and -adriamycinone,” T. Tsuchiya, Y. Takagi, K. Ok, S. Umezawa, T. Takeuchi, N. Wako, and H. Umezawa, J. Antibiot., 39 (1986) 731 -733. “A new antitumor antibiotic, spergualin: isolation and antitumor activity,” T. Takeuchi, H. Iinuma, S. Kunimoto, T. Masuda, M. Ishizuka, M. Takeuchi, M. Hamada, H. Naganawa, S. Kondo, and H. Umezawa, J. Antibiot., 34 (198 1) 1619- 1621. “Phosphorylative inactivation of aminoglycosidic antibiotics by Escherichia coli carrying R factor,” H. Umezawa, M. Okanishi, S. Kondo, K.

20

TSUTOMU TSUCHIYA, KENJl MAEDA, AND DEREK HORTON

Hamana, R. Utahara, K. Maeda, and S . Mitsuhashi, Science, 157 (1967) 1559-1561. “3’,4’-Dideoxykanamycin B active against kanamycin-resistant Escherichia coli and Pseudomonas aeruginosa,” H. Umezawa, S. Umezawa, T. Tsuchiya, and Y . Okazaki, J . Antibiot., 24 (1971) 485-487. “Biochemical mechanism of resistance to aminoglycosidic antibiotics,” H. Umezawa, Adv. Carbohydr. Chern. Biochern., 30 (1974) 183-225. “Pepstatin, a new pepsin inhibitor produced by actinomycetes,” H. Umezawa, T. Aoyagi, H. Morishima, M. Matsuzaki, M. Hamada, and T. Takeuchi, J. Antibiot., 23 (1970) 259-262. “Mechanism of action of diketocoriolin B,” T. Kunimoto, M. Hori, and H. Umezawa, Biochirn. Biophys. Acta, 298 (1973) 5 13-525. “Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes,” H. Umezawa, T. Aoyagi, H. Suda, M. Hamada, and T. Takeuchi, J. Antibiot., 29 (1976) 97-99. “Low-molecular-weight enzyme inhibitors of microbial origin,” H. Umezawa, Annu. Rev. Microbiol., 36 (1982) 75-99. “Studies on marine micro-organisms, V. A new antibiotic, aplasmomycin, produced by a streptomycete isolated from shallow sea mud,” Y. Okami, T. Okazaki, T. Kitahara, and H. Umezawa, J. Antibiot., 29 (1 976) 1019- 1025. “Studies on antibiotics and enzyme inhibitors,” H. Umezawa, Rev. Infect. Dis., 9 (1987). “Institute of Microbial Chemistry, 1962- 1987” (including a list of all publications by H. Umezawa, 1943- 1987), (edited by Tomio Takeuchi). Microbial Chemistry Research Foundation, Business Center for Academic Societies Japan, Tokyo, 1987.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 48

CHEMISTRY OF CARBA-SUGARS (PSEUDO-SUGARS) AND THEIR DERIVATIVES

BY TETSUO SWAMI* Drpartmrnt qf Chemistry, Meisei University, Hino. Tokyo 191, Japan

AND

SEIICHIRO OGAWA~

Deparfment qf Applied Chemistry, Faculty of Science and Technology, Keio University, tliyashi, Yokohama 223, Japan

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 II. Synthesis of Racemic Carba-su 26 26 1. McCasland’s Three 28 2. From mycrlnositol 3. From the Diels-Al 29 Ill. Synthesis of Enantio . . . . . . . . . . . . . 36 I . Resolution of the Diels- Alder Adduct. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2. From Optically Active Natural Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3. From Truesugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 IV. 6a-Carba-~-fructopyranoses. . . . 49 I . Synthesis of Racemic 6a-Carb 50 2. Synthesis of Enantiomeric 6a51 V. Synthesis of Racemic Amino Carba-sugars. . . . . . . . . . . . . . . . . . 52 I . Carba-glycosylamines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2. 2-Amino-2-deoxycarba-hexosesand Relatives 59 V I . Synthesis of Enantiomeric Amino Carba-sugars. . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 I . Enantiomeric Carba-glycosylamines. . . . . . . . . . . . . . . . . . . . . . . 64 2. Enantiomeric 2-Amino-2-deoxycarba-hexoses and Other Amin Carba-sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

* Sections I-IV, IX, and X .

t Sections V - X . 21

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form r e ~ ~ e d .

22

TETSUO SUAMl AND SEllCHlRO OGAWA

VlI. Synthesis of Mono- and Dicarba-disaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . Synthesis of Carba-maltose. Carba-isomaltose, Carba-cellobiose, and the Like 2. Carba-trehaloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Carba-trehalosamine and Related Compounds. . . . . . . . . . . VIII. Synthesis of Biologically Active Carba-oligosaccharides . . . . . . . . . . . . . . . . . . 1. Antibiotic Validamycins and Related Compounds. . . . . . . . . . . . . . . 2. Carba-oligosaccharidic Alpha Amylase Inhibitors and Relat IX. Biological Effects of Carba-sugars ........................... X. Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61 68 69 12 14 74 81 86 89

I. INTRODUCTION The term “pseudo-sugar” is the name that has been used for a class of compounds wherein the ring-oxygen atom of a cyclic monosaccharide is replaced by a methylene group. The term, which is vague, was first proposed by the American Professor G. E. McCasland and coworkers’ when they synthesized the first such compound, which they called “pseudo-a-DL-talopyranose.” In the present article, the definitive prefix “carba,” preceded, where considered necessary, by the appropriate locant (“4a” for an aldofuranose, or “5a” for an aldopyranose), will be employed instead of “pseudo,” thus making the names amenable to indexing. The most reactive functional group in a true sugar, that is, an aldehyde or a ketone group, does not exist in the carba-sugars; and, accordingly, carba-sugars do not exhibit any characteristic reaction of a reducing sugar, such as reduction of heavy-metal salts in alkaline solution, mutarotation, and formation of osazones or hydrazones. Historically, the name pseudo-oligosaccharides had been used to designate oligosaccharides containing nontypical “sugars,” such as cyclitols or aminocyclitols, and also those containing carba-sugars or amino carbasugars (see Scheme I , p. 59). There are two forms of carba-sugar: carba-pyranoses and -furanoses. The former, especially the carba-hexopyranoses, have been extensively studied during the past two decades, ever since their derivatives were found in Nature as components of important antibiotics. However, very little is known about carba-furanoses, except for 4a-carba-P-~-arabinofuranose~.~ ( I ) G. E. McCasland, S. Furuta, and L. J. Durham, J. Org. Chem., 3 1 (1966) 1 5 16 - I52 1.

(la) The numbering used in this article for the carba-sugars is analogous to that for true sugars. The names employed do not replace the formal, systematic terms assigned by standard IUPAC rules and the cyclitol rules, but are convenient trivial names for use when the homomorphic relationships to true sugars are to be emphasized. ( 2 ) K. Tadano, H. Maeda, M. Hoshino, Y.Iimura, and T. Suami, Chem. Lett.. (1986) 1081 1084. (3) K. Tadano, H. Maeda, M. Hoshino, Y. limura, and T. Suami, J . Org. Chem., 52 (1987) 1946- 1956.

CARBA-SUGARS AND THEIR DERIVATIVES

23

40 HOH2C

4a-Carba-p-~-arabinofuranose 1

OH

HO

4'a-Carba-ansteromycin

QH2,

,

HO

OH

4a-Carba-p-D-ribofuranosylamlne

3

2

(l),and the 4'a-carba-P-~-ribofuranosyl moiety1aof the antibiotic anstero-

m y ~ i n (2) ~ -and ~ its racemic modification7-" (3). This article, therefore, focuses on the chemistry of carba-hexopyranoses and their derivatives, in which accomplishments are most evident. The first recognized carba-sugar, 5a-carba-a-~~-talopyranose (4), was synthesized by McCasland and coworkers,' and they prepared two more carba-sugars, 5a-carba-P-~~-gulopyranose~~ ( 5 ) and Sa-carba-a-~~-galactopyranose13 (6). They suggestedlZthat carba-sugars may possess biological effects, owing to their structurally close resemblance to sugars, and hope was expressed that, in some cases, a carba-sugar might be accepted by enzymes or biological systems in place of a true sugar and thus might serve to inhibit

HO

HO

OH 4

6 DL 70

(4) T. Kusaka, H. Yamamoto, M. Shibata, M. Muroi, T. Kishi, and K. Mizuno, J. Antibiot., 21 (1968) 255-263. (,5 _ ) T. Kishi, M. Muroi, T. Kusaka, M. Nishikawa, K. Kamiya, and K. Mizuno, Chem. Pharm. Bi~ll.,20 (1972) 940-946. (6) K. Tadano, M. Hoshino, S. Ogawa, and T. Suami, Tetrahedron Lett., 28 (1987) 2741 2144. (7) Y. F. Shealy and J. D. Clayton, J. Am. Chem. Soc., 88 (1966) 3885-3887. (8) Y. F. Shealy and J. D. Clayton, J. Am. Chem. Soc., 91 (1969) 3075-3083. (9) A. K. Saksena, Tetrahedron Lett., 21 (1980) 133-136. (10) R. C. Cermak and R. Vince, Tetrahedron Lett.. 22 (1981) 2331 -2332. ( 1 I ) H. Paulsen and U. Maass, Chem. Eer., 114 (1981) 346-358. (12) G. E. McCasland, S. Furuta, and L. J. Durham, J. Org. Chem.. 33 (1968) 2835-2841. (13) G. E. McCasland, S. Furuta, and L. J. Durham, J. Org. Chem., 33 (1968) 2841 -2844.

TETSUO SUAMl AND SEllCHlRO OGAWA

24

growth of malignant or pathogenic cells. In fact, 5a-carba-cu-~-galactopyranose (7) was dis~overed’~ in a fermentation broth of Streptomyces sp. MA-4145 in 1973, seven years after this suggestion. The carba-sugar 7 exhibits inhibitory activity against Klebsiella pneumonia, MB- 1264. Prior to the discovery of 7, carba-trisaccharidic antibiotics, validamycins, had been dis~overed’~ in 1970. Validamycins are obtained from a fermentation beer of Streptomyces hygroscopicus var. limoneus, and validamycin A (8)is the most active component, which exhibits strong inhibitory activity against the sheath blight of rice plants and “damping off’ of cucumber seedlings caused by an infection of Pellicularia sasakii and Rhizoctonia sol~ni.’~ Validamycins have been widely used in Japan as farming antibiotics. The carba-oligosaccharidic antibiotics acarbose16 (9), adiposin” (lo),

HOH-Q

o ! ! , , . CH20H

8 R

R‘

Validamycin A

H

11

B

OH

H

E

H

a-D-glucopyranosyl

(14) T. W. Miller, B. H. Arison, and G. Albers-Schonberg, Biotech. Eioeng., 15 (1973) 10751080. (15) T. Iwasa, H. Yarnarnoto, and M. Shibata, J . Antibiot., 32 (1970) 595-602. (16) D. D. Schmidt, W. Frornmer, B. Junge, W. Muller, W. Wingender, E. Truscheit, and D. Schafter, Naturwissenschafren, 64 (1977) 535 -536. ( 1 7) H. Seto, K. Furihata, N. Otake, S. Namiki, K. Kangouri, H. Ham, K. Mizokami, and S. Kimura, Meet. Jpn. Antibiot. Res. Assoc.. 223rd. Tokyo, Japan, March 198 1 .

CARBA-SUGARS A N D THEIR DERIVATIVES

25

CHzOH

I

HO

N H

q

CHZOH

CH20H

$i=O

Acarbose

a%&

HO= '

9

CHzOH I

H H

N H

CH2OH

O-

CH2OH

q 0-

Adiposin (TAI-2) 10

trestatins'* (1 l), S-AI,'9.20and oligostatins21.22 have been discovered in fermentation broths as enzyme inhibitors. In the 5a-carba-aldohexopyranose family, there are thirty-two stereoisomers theoretically possible, and all sixteen of the predicted racemic forms have been prepared, as well as fifteen of the enantiomers. (18) K. Yokose, S. Ogawa, Y . Suzulu, and P. Buchschacher,Symp. Natl. Org. Compds., 23rd,

Nagoya, Japan, Oct. 1980. (19) S. Murao and K. Ohyama, Agric. Biol. Chem.. 39 (1975) 2271 -2773. (20) S. Murao and K. Ohyama, Agric. Biol. Chem.. 43 (1979) 679-681. (21) J. Itoh, S. Omoto, T. Shomura, H. @no, K. Iwamatsu, and S. Inouye, J. Anfibiot., 34 (1981) 1424-1428. (22) S. Omoto, J. Itoh, H. Ogino, K. Iwamatsu, N. Nishizawa, and S. Inouye, J. Antibiot., 34 (1981) 1429-1433.

26

TETSUO SUAMl AND SEllCHIRO OGAWA

Among the carba-sugar derivatives, the most important and attractive members are amino carba-sugars, particularly 5a-carba-a-~-glucopyranosylamine ( ~ a l i d a m i n e )and ~ ~ its unsaturated derivative (~alienamine).~~ These amino carba-sugars have been synthesized successfully, as well as validamycin antibiotics and some of the aforedescribed enzyme inhibitors. In the present article, the preparation of carba-sugars, amino carba-sugars, and biologically active carba-oligosaccharides will be described. 11. SYNTHESIS OF RACEMIC CARBA-SUGARS The first three carba-sugars were synthesized by McCasland and coworkers. Two other carba-sugars were prepared from myo-inositol, and the remaining eleven carba-sugars have been synthesized from the Diels- Alder adduct of furan and acrylic acid. Conformational assignments of the carbasugars were established with the aid of 'H-n.m.r. spectroscopy. 1. McCasland's Three Carba-sugars

The first carba-sugar, 4, was synthesized from the keto acid 13, which was obtained by a two-step reaction from the Diels- Alder adduct (12) of 2-acetoxyfuran and maleic anhydride. Sodium borohydride reduction of 13, es(23) S. Horii, T. Iwasa, E. Mizuta, and Y . Kameda, J . Anfihiol., 24 (1971) 59-63. (24) Y. Kameda and S. Horii, J. C%em.Soc.. Ctiem. Commun., (1972) 746-747.

CARBA-SUGARS AND THEIR DERIVATIVES

14

13

12

21

RO

OR 15 R = A c 4 R=H

terification of the product, and acetylation, gave compound 14. Lithium aluminum hydride reduction of 14, and acetylation of the alcohol, gave 5a-carba-a-~~-talopyranose pentaacetate (15), which was converted into 4 by hydrolysis.' Sa-Carba-P-~~-gulopyranose ( 5 ) was prepared as follows. The Diels Alder cycloaddition of trans, trans- 1,4-diacetoxy-1,3-butadiene and ally1 acetate provided ( 1,2,5/0)-2,5-diacetoxy-1-(acetoxymethyl)-3-cyclohexene (16). Hydroxylation of 16 with osmium tetraoxide and hydrogen peroxide, followed by acetylation, gave Sa-carba-P-~~-gulopyranose pentaacetate (17). Hydrolysis of 17 affordedf25.

bAC

16

I?

5

Epimerization of 4 at C-2 provided Sa-carba-a-~~-galactopyranose (6). When the pentaacetate 15 was heated in acetic acid containing sulfuric acid, epimerization occurred at C-2 through an intermediary cyclic acetoxonium ion (18), with anchimeric assistanceofthe vicinal, axial acetoxyl group. After acetylation, 5a-carba-a-~~-galactopyranose pentaacetate (19) was obtained in a yield of 14%; it was converted into 6 by hydr01ysis.l~The antimicrobial activity of the racemate 6 was found to be about half that of the natural antibiotic 7 in the same assay system, indicating that the L-antipode is probably inactive.I4

TETSUO SUAMI AND SEIICHIRO W A W A

28

@

Ro CH2OR

CH20Ac

-

AcO

R

a

OAc

OR CH3

15

19 R = A c

18

6 R=H

2. From myo-Inositol Carba-P-DL-galactopyranose pentaacetate (27) and carba-0-DL-altropyranose pentaacetate (28) were synthe~ized~~ from myo-inositol (20), which was converted into DL- 1,2-anhydro-5,6-0-cyclohexylidene-chiro-inositol (21) by successive 0-cyclohexylidenation, tosylation, and epoxidation.26 Lithium aluminum hydride hydrogenation of 21 gavez7 DL- 1,2-0-cyclohexylidene-5-deoxy-chiro-inositol(22). 0-Isopropylidenation of 22 and Pfitzner- Moffatt oxidation of the product afforded the 0-isopropylidene inosose-1 derivative 23. Reaction of 23 with diazomethane provided the spiro epoxide 24 in 82Y0yield. Nucleophilic opening of the oxirane ring of 24 with hydriodic acid, followed by acetylation of the product, gave compound 25, which was converted into the exocyclic alkene 26 by an elimination reaction with zinc powder in glacial acetic acid. Hydroboration - oxidation reactions converted 26 into 27 and 28, after conventional acetylation. HO

HO

OH

20

OH

--C&pLOH--d~ 21

23

OH

OH

OH

22

24

25

T. Suarni, S . Ogawa, T. Ishibashi, and I. Kasahara, Bull. Chem. Soc. Jpn., 49 (1976) 1388-1390. T. Suami, S. Ogawa, S. Oki, and K. Ohashi, Bull. Chem. Soc. Jpn., 45 (1972) 2597-2602. T. Suarni, S. Ogawa, T. Ueda, and H. Uchino, Bull. Chem. Soc. Jpn.. 45 (1972) 32263227.

CARBA-SUGARS AND THEIR DERIVATIVES

A c O Y

29

OAc

OAC

26

27

28

3. From the Diels - Alder Adduct of Furan and Acrylic Acid The Diels- Alder cycloaddition of furan and acrylic acid, in the presence of hydroquinone as a polymerization inhibitor, provided endo-7-oxabicycl0[2.2.1]hept-5-ene-2-carboxylica ~ i d ~ *(29) , * ~in a yieldw of 45%. Compound 29 was found to be the most accessibleand important starting-material for the synthesis of various racemic carba-sugars, as well as their enantiomers. Hydroxylation of 29 with hydrogen peroxide and formic acid resulted in formation of the tricyclic lactone 30, with simultaneous lactonization of an initially formed glycol. Lithium aluminum hydride reduction of 30, and acetylation, gave the triacetate 31. Opening of the 1,4-cyclic ether linkage of 31 with I2 : 7 :0.7 acetic acid-acetic anhydride-sulfuric acid p r ~ v i d e d ~ ~ . ~ ' carba-a-DL-galactopyranosepentaacetate (19) and carba-P-DL-ghcopyranose pentaacetate (32). Hydrolysis of 19 and 32 gave3' the respective carbasugars 6 and 33.

29

30

0

CH2OR

RO CH20Ac

AcO

31

19 R = H 6 R=H

32 R = AC 33 R = H

(28) M. P. Kunstman, D. S. Tarbell, and R. L. Autrey, J. Am. Chem. Soc., 84 (1962) 41 154125. (29) W. L. Nelson and D. R. Allen, J. Heferucycl. Chem., 9 (1972) 561 -568. (30) T. Suami, S. Ogawa, K. Nakamoto, and 1. Kasahara, Curbohydr. Res.,58(1977) 240-244. (3 1 ) S. Ogawa, M. Ara, T. Kondoh, M. Saitoh, R. Masuda, T. Toyokuni, and T. Suami, Bull. Chern. Sucfpn., 53(1980) 1121-1126.

TETSUO SUAMI AND SEIICHIRO OGAWA

30

Starting from 29, four novel carba-sugars, of the a d o , a-manno, P-altro, and /?-mannoconfigurations, have been prepared. Hypobromous acid addition to 29 proceeded stereospecifically, and the tricyclic bromolactone 34 was obtained as a single product in 9 1Yo yield. Lithium aluminum hydride reduction and subsequent acetylation converted3234 into the bromo diacetate 35. Opening of the cyclic ether linkage of 35 with acetic acid-acetic anhydride - sulfuric acid gave 2-bromo-2-deoxy-carba-a-~~-galactopyranose tetraacetate (36) as the major and 2-bromo-2-deoxy-carba-~-~~-glucopyranose tetraacetate (37) as the minor product.

I

CH2OAc

34

I

35

38 R = A C

36

42 R = H

37

39 R = A c 43 R = H

40 R = A c 44 R = H

CH2OR

O R-“

41 R = A c 45 R = H

(32) S. Ogawa, 1. Kasahara, and T. Suami, Bull. Chem. SOC.Jpn., 52 (1979) 118- 123.

31

CARBA-SUGARS AND THEIR DERIVATIVES

Substitution reactions of 36 with nucleophilic acetate ions, followed by acetylation, furnished two carba-sugar pentaacetates of the C Y - D L - ~ (38; ~O 3 I Yo yield) and a - ~ ~ - g u l u cconfigurations to (19; 10%).The reactions seemed to involve formation of an intermediary 2,3-cyclic acetoxonium ion.3' Analogous reactions of37 provided3Itwo other carba-sugar pentaacetates, of the ( Y - D L - ~ U ~(39; ~ U 29% yield) and P - D L - u h o form (40; 27%). On the other hand, if substitutions of 37 were conducted with benzoate ions, instead of acetate ions, direct S N reactions ~ occurred predominantly, and carba-P-DL-mannopyranose pentaacetate (41) was ~ b t a i n e d . ~ ' 0-Deacetylation of 38, 39, 40, and 41 provided3' the corresponding carba-sugars 42, 43, 44, and 45. When 35 was heated in acetic acid containing hydrogen bromide, the tribromide 46 was obtained3*as a single product in 74% yield. Debromination of46 with zinc dust in acetic acid furnished the cyclohexene derivative 47, which was converted into compound 48 by osmium tetraoxide hydroxylation and a~etylation.~~ The substitution reaction of 48 with acetate ions provided carba-a-DL-glucopyranose pentaacetate (49), which gave the carba-sugar 50 on hydroly~is.~~ As an alternative reaction process, nucleophilic substitution reactions of ( I ,3/2,4,6)-4-bromo-6-(bromomethy1)-1,2,3-cyclohexanetrio1 triacetate (51) with benzoate ions furnished 49 in poor yield after exchange of the protective groups.35 A facile synthesis of 49 was described as follows. When the bromolactone 34 reacted with hydrogen bromide in acetic acid, regioselective cleavage of

35

-

Aco%Br

-

AcO

A c 0 ~ " ' " ' -

AcoAm CH$r

__

Br

46

49 R = A c

47

48

51 R = A c

50 R = H

(33) S. Ogawa, T. Toyokuni, T. Kondoh, Y. Hattori, S. Iwasaki, M. Suetsugu, and T. Suami, Bull. Clirm. SOC.Jpn.. 54 (1981) 2739-2746. (34) S. Ogawa, Y. Tsukiboshi, Y. Iwasawa, and T. Suami, Carbohydr. Res.. 136 (1985) 77-89. (35) S. Ogawa, N. Chida, and T. Suami, Chcm. Lett., (1980) 1559- 1562.

TETSUO SUAMl AND SEIICHIRO OGAWA

32

the 1,4-cyclic ether occurred, and ( 1,3,5/2,4)-2,3-diacetoxy-4,5-dibromocyclohexane- 1 -carboxylic acid (52) was obtained. Debromination of 52 with zinc dust in acetic acid gave the cyclohexene derivative 53, which was converted into the ester 54 with diazomethane. cis-Hydroxylation of 54 with osmium tetraoxide and hydrogen peroxide, followed by acetylation, gave compound 55. Lithium aluminum hydride reduction of 55 provided 49 in an overall yield of 16%, after conventional a ~ e t y l a t i o n . ~ ~

34

-

A

c

o

~

Ac 0

B

r

-

-AcIc*

C02Me

AC0* Ac 0

-

Br 52

53

54

C02Me

AcosiA Ac 0

49

OAc

55

Epimerization of 50 at C-3 furnished carba-a-DL-allopyranose(60). Stepwise, 0-isopropylidenation of 50 with 2,2-dimethoxypropane afforded compound 56. Ruthenium tetraoxide oxidation of 56 gave the %OX0 derivative 57, and catalytic hydrogenation over Raney nickel converted 57 into the 3-epimer 58 exclusively. Hydrolysis of 58, and acetylation, provided the pentaacetate 59, which was converted into 60 on hydroly~is.~~ The pentaacetate 59 was also obtained3’by a route from 29 to 61 in a better MC?,C,-OCH,

CMe,

56

CMe,

57

CMe,

58

59 R = A c

60 R = H

(36) S. Ogawa, Y. Yato, K. Nakamura. M. Takata, and T. Takagaki, Curbohydr. Res., 148 (1986) 249-255.

(37) S. Ogawa, M. Uemura, and T. Fujita, Curhohydr. Res.. 177 (1988) 213-221.

CARBA-SUGARS AND THEIR DERIVATIVES

33

yield (3 1 Yo). Esterification of 29, followed by cis-hydroxylation with potassium permanganate,j* acetylation, lithium aluminum hydride reduction, and acetylation, gave compound 62. Acetolysis of 62 provided 59 as the sole product, almost quantitatively. There are two possible reaction sites, C- 1 and C-4, in the reaction, and two carba-sugars, a-gulu and a-ah, should be obtained, but, during the course of the reaction with prolonged heating, the former carba-sugar is transformed into the latter by epimerization by way of 3,4-cyclic acetoxonium ions.37

4

-

29

-

-

Q J;A ;

59

CH2OAc

C02Me 61

62

Carba-P-DL-allopyranose (68) was synthesized from carba-P-DL-glucopyranose (33) by epimerization analogous to that employed in the aforedescribed reaction.340-Isopropylidenation of 33 provided two positional isomers, 63 and 64. Analogous reactions34converted 63 into 68 by way ofthe intermediary compounds 65,66, and 67. When carba-a-DL-galactopyranose (6) reacts with a, a-dimethoxytoluene, there are obtained two stereoisomers, 69 and 70, whose structures Me&-OC

33

-

-

Me&-OC

\

O

a

o O,

+* /

CMe,

63

O '

\ /O

OH

CMe,

64

-\omo

J

\ o w Me&-OC o

Me&-OC

o\ CMe, /

0 65

HO

-RO%

O\ 66

/ CMe,

OR RO

Ro

67 R = A c 68 R = H

(38) E. S. Gould, Mechanism and Sfructirrein Organic Chemistry. Holt, Rinehart and Winston, New York, 1959, p. 538.

TETSUO SUAMI AND SEllCHIRO OGAWA

34

were, by means of ‘H-n.m.r. spectroscopy, tentatively established to be as shown. Ruthenium tetraoxide oxidation of69 gave the 3-0x0 compound 71. Sodium borohydride reduction converted 71 into the two compounds, 69 and 72, in the ratio of - 1 :2. 0-Debenzylidenation of 72 and acetylation of the product gave carba-a-DL-gulopyranose pentaacetate (73), which provided the carba-sugar 74 on h y d r ~ l y s i s . ~ ~

+

/

69 Ptl,

I“

70

C

I‘

0 0

73 R = A c 71

74 R = H

Carba-P-DL-idopyranose (77) was prepared from (readily accessible) ( 1,2,4/3)-5-( hydroxymethyl)-5-cyclohexene- 1,2,3,4-tetr01~~ (75) by hydro-

genation over a platinum catalyst, acetylation of the product, and O-deacetylati~n.~~

75

77 R = H

The last then-unknown carba-sugar, carba-P-DL-talopyranose pentaacetate (83), was synthesized from” 36 as follows. Epoxidation of 36 with sodium methoxide gave, at the early stage of the reaction, the 2,3-epoxide (39) T. Toyokuni, Y. A h , S. Ogawa. and T. Suarni, 8~11. Chem. SOC.Jpn., 56 ( 1983) 505 - 5 1 3 .

35

CARBA-SUGARS AND THEIR DERIVATIVES

(78), which underwent migration of the epoxide ring, and, finally, the 1,6anhydro compound 80 was obtained, after acetylation. Sequential 0-deacetylation, 0-isopropylidenation, and acetylation transformed 80 into compound 81. 0-Deacetylation of 81, and ruthenium tetraoxide oxidation of the product, gave the 2-0x0 compound 82, which was converted into 83 by hydrogenation over a platinum catalyst and acetylation.@

OAc

80

79

78

H*C-

OAc

0

OAc

81

82

83

~xxo-7-Oxabicyclo[2.2.l]hept-5-ene-2-carboxylica ~ i d ~ ~(84) , ~was ~ - ~ ~ . ~ ~ isolated as a minor component from the mother liquor of 29. Compound 84 is an epimer of 29 at C-2, and three known carba-sugar pentaacetates have been prepared from 84 as follows. Sequential esterification, lithium aluminum hydride reduction, and acetylation converted 84 into compound 86. cis-Hydroxylation of 86 with

84

85

86

87 R = H = AC

88 R

(40)S. Ogawa, N. Kobayashi, K. Nakamura, M. Saitoh, and T. Suami, Curbohydr. Res., I53 (1986) 25-31. (41 ) R. J. Ouellette, K. Liptak, and G. E. Booth, J. Org. Chem.. 32 (1967) 2394-2397. (42) R. J. Ouellette, A. Roseblum, and G. Booth, J. Org. Chem., 33 (1968) 4302-4303.

36

TETSUO SUAMI A N D SEIICHIRO OGAWA

potassium ~ermanganate,~~ followed by acetylation, gave the triacetate 88, which was transformed into the three carba-sugar pentaacetates 41, 15, and 19 by acetoly~is.~' The following reaction mechanism is deduced for the acetolysis reaction. By cleavage of the 1,4-cyclic ether linkage with acetate ions, compounds having the /3-manno and an a-tulo configurations are formed in the early stage of the reaction, and the latter compound is subjected to epimerization to give the a-gulucto modification. The same epimerization had been described by McCasland and coworked3 under similar reaction conditions. Now, all sixteen of the predicted racemic carba-sugars have been synthesized, and their physical constants are listed in Table I. 111. SYNTHESIS OF ENANTIOMERIC CARBA-SUGARS Up to the present, fifteen enantiomers have been synthesized among the thirty-two carba-sugars theoretically predicted. Four of them have been prepared from the antipodes of the Diels- Alder adduct 29, two were obtained from quebrachitol, and a chiral synthesis, starting from true sugars, provided the thirteen enantiomers. 1. Resolution of the Diels-Alder Adduct

As 29 had been recognized as the most accessible starting-material for the synthesis of racemic carba-sugars, its resolution was successfully achieved with optically active a-methylbenzylamine as chiral reagent. Reaction of 29 with (+)-a-methylbenzylamine gave a mixture of two diastereoisomeric salts: [(+)-amine, (-)-29; and (+)-amhe, (+)-291, which were well separated, and the former salt was converted into (-)-29, [a]:2 -1 1 1.go (ethanol). Analogously, (+)-29, [a]i2 + I 10.7" (ethanol), was obtained.43 The absolute configuration of (-)-29 was established by X-ray crystal structure analysis of the bromolactone (89),which was prepared from (-)-29 by bromolactonization with hypobromous It was found that (-)-29 belongs to the D series of carba-sugars, and hence, (+)-29 corresponds to the L

Starting from (-)-29, carba-a-D-galactopyranose ( 7 ) and carba-P-D-glucopyranose (94)have been synthesized by a reaction analogous to that employed in the preparation of the r a ~ e m a t e s . ~ . ~ ~ (43) S . Ogawa, Y. Iwasawa, and T. Suami, Chem. Lett., (1984) 355-356. (44) S. Ogawa, Y. Iwasawa, T. Nose, T. Suami, S. Ohba, M. Ito, and Y. Saito, J. Chem. SOC., Perkin Trans. I . (1985) 903-906.

TABLE I Physical Constants of DL-Carba-SUgarS Free compound M.p. ("Cy

Pentaacetate Configuration M.p. ("C)" a-allo pallo

a-aliro p-aliro a-galacio

p-galacio a-glue0 pg1uco a-gufo pgu1u

~-id0 D-idu a-manno

&manno a-ialo pialo

120- 121 122-123 110-11 I 115-116 106-107 147-148. 137-138 142- 143 123-124

-

185-186

-

143.5- 144.5 173- I74* 167- 168

-

110-111

146- I47

-

106- 107 111-112 109- I10 132- 133' 106- I07 syrup 99.5- loo 123-125 125- 125.5 109- I lo* 110.5-112 117-119

cY-D-&'fl

p-D-g/IlCO

a-L-gIUco p-L-glUco a-L-idu @-id0 a-o-mannn b-o-manno a-L-mano &manno

135-136 syrup 84-85 syrup 143-144 144 syrup syrup SYNP 115-116 SYNP 114- I16 SYNP 111-112 86-87 115-1 17 80-81 I I9

84-86 syrup

34 41 34 25 31 13 31 41 25 33.34.35 44 30.3 I 34 12 31 34 31 31 41 I 41 37

syrup

SYNP 139- 140 syrup 154.5-156 SYWP syrup 198- I99

-

160-162.

-

Specific rotation (degrees) Solventb pL-allo a-o-allro a-L-aliro /%~-alrro a-o-galacto

References

+ +

[a]b8 3.7 [a1:4 14.4 - 13.7 [a];'+ 7 [a]:" 43.2

+ [a]:"+ 35.2 [a]:*+ 36.7 [a]:'+ 57 [a]:"+ 63.0 [a];0+ 13.8 [a]:"+ 8.9 [a]:'t 4.4 [a]:'- 56 [crib9 - 7.4 [a]:"- 36. I [a]:"+ 14.4 1 4 ~+ 9 27.8 [a]$ 2.9 [a]:' - 38.5 [a],- 1.1

+

Specific rotation (degrees) Solvent*

C C

C C C C C

C C C C

syrup syrup 161.5-162.5

[a]:' - 43.6 [a];' - 49.5

149+ 66.3

161 syrup SYWP SYNP syrup syrup

[a]z3+61.5f4 [ a p 47.9 [a]? 30 [a]:' 67 [a]i0 70.0 [a]:0 13.0 [a]go 10.9

syrup

[a];'- 67

+ + + + + +

m m W

w

m m m W W

W

C

C

m

C

C C C C C

C

127-128 syrup 217

[a];"- 45.7 8.5

+ [a]:o+ 11.9

W W

m

56 3 5435 49 43 14 46 48,49 45 57 43 57 54,55 45 3 57 57 56 46 3 2.3

'M.p.s were usually determined In a capdlary tube in a liquid bath. and those marked with an asterisk were measured on a hot-plate.

bc = chloroform; m = methanol: w =water.

TETSUO SUAMl AND SEllCHIRO OGAWA

38

4(-)-29

(+)- 29

Hydroxylactonization of (-)-29 with hydrogen peroxide and formic acid gave the tricyclic compound 90, [a]:*+47.9” (ethanol). Reduction of 90 with lithium aluminum hydride, followed by acetylation, provided the triacetate 91, which was converted into the pentaacetates 92 and 93 by acetolysis. 0-Deacetylation of 92 and 93 gave 7 and 94, respectively.4 The physical constants of all enantiomeric carba-sugars are listed in Table 1.

-

H

O

R

0-c

COZH

-

AcO&

CH20Ac

AcO

“0

90

(-)- 29

+

RO R Oq

92 R = A c 7 R=H

R O=“ R o

91

RO

93 R = A c 94 R = H

A facile synthesis of carba-a-D-ghcopyranose (99) and its L antipode45 (104) was accomplished by means of resolution36 of the starting compound DL-( 1,3,5/2,4)-2,3-diacetoxy-4,5-dibromocyclohexane1-carboxylic (45) S. Ogawa, K. Nakamura, and T. Takagaki, Bidl. Chem. Soc. Jpn., 59 (1986) 2956-2958.

39

CARBA-SUGARS AND THEIR DERIVATIVES

acid (52). Resolution of 52 with (+)- and (-)-a-methylbenzylamine provided (-)-52, [a];'-5.1 ' (ethanol), and (+)-52, [a];'+5.2" (ethanol). Analogously, for p r e p a r a t i ~ of n ~racemic ~ carba-a-glucopyranose 49 from 52, esterification of (-)-52 furnished the ester 95, which was transformed into compound 96 by debromination with zinc dust and acetic acid. Stereoselective hydroxylation of 96 with osmium tetraoxide and hydrogen peroxide, followed by acetylation, gave compound 97. Lithium aluminum hydride reduction of 97, and acetylation of the product, gave pentaacetate 98, which was converted into 99 by hydrolysi~.~~

" A cOO

B Br

h

r

-

(-1- 52

96

95

Acow - "-%A

CIiZOR

COZh1C

RO

AcO

OAc

OR

98 R = AC

97

99 R = H

Similarly,(+)-52 wasconverted into 104 by way of 100,101,102, and 103, which are the corresponding L antipodes45of 95,96,97, and 98.

(+)-

52

102

I00

103 R = A c 104 R = H

101

TETSUO SUAMI A N D SEIICHIRO OGAWA

40

2. From Optically Active Natural Products

Carba-a-D-galactopyranose(7) has also been synthesized from quebrachitol, by Paulsen and coworkers,* as well as carba-P-D-mannopyranose (I 14). Quebrachitol was converted into 1L-chiro-inositol (105). Exhaustive 0isopropylidenation of 105 with 2,2-dimethoxypropane, selective removal of the 3,4-0-protective group, and preferential 3-0-benzylation gave compound 106. Oxidation of 106 with dimethyl sulfoxide-oxalyl chloride provided the inosose 107. Wittig reaction of 107 with methyl(tripheny1)phosphonium bromide and butyllithium, and subsequent hydroboration and oxidation furnished compound 108. A series of reactions, namely, protection of the primary hydroxyl group, 0-debenzylation, formation ofS-methyl dithiocarbonate, deoxygenation with tributyltin hydride, and removal of the protective groups, converted* 108 into 7.

105

106

107

108

Halogenation of 106 with triphenylphosphine, iodine, and imidazole provided the iodo derivative 109. On treatment with lithium aluminum hydride, 109 was converted into two endocyclic alkenes, 110 and di-0-isopropylidenecyclohexanetetrol, in the ratio of 2 : 1. Oxidation of 110 with dimethyl sulfoxide- oxalyl chloride afforded the enone 111. 1,CAddition of ethyl 2-lithio-1,3-dithiane-2-carboxylateprovided compound 112. Reduction of 112 with lithium aluminum hydride, and shortening of the sidechain, gave compound 113, which was converted into I14by d e p r ~ t e c t i o n . ~ ~ (46) H. Paulsen, W. von Deyn, and W. Roben, Justus Liebigs Ann. Chem., (1984) 433-449.

CARBA-SUGARS AND THEIR DERIVATIVES

109

112

1 lo

113

41

111

114

3. From True Sugars From L-arabinose, carba-sugars of the a-D-gluco and P-L-aftro types were p r e ~ a r e d ~from ~ % ~D-arabinose, *; carba-a-~-mannopyranose~; from D-ribose, that of the P-L-mannu rnodificati~n~~~; from D-xylose, those of the P-L-gfuco and a-D-alfru forms3; and, from D - ~ ~ U C those O S ~ ,of the P-~-affu,4~ a-~~ltro,’~~’* a - ~ - g l 1 4 ~P0- ,D ~-~~ ~ U C a-~-ido,~~ U , ~ ’ , ~ ~P-~-ido,~~ and a-~-rnannd’~ types. a. From L-Arabinose.- L-Arabinose diethyl dithioaceta15’ (115) was converted into compound I16 by successive 0-tritylation, 0-benzylation, 0-detritylation, and 0-tosylation. The parent aldehyde (117) was regenerated from 116 with mercury(I1) chloride and calcium carbonate. Substitution of 117 with sodium iodide gave the iodo compound 118. Cyclization of I18 with dimethyl malonate and sodium hydride, followed by acetylation, provided the desired cyclohexane derivative 119 and a secondary pyranose derivative in the ratio of 1.3: 1. (47) T. Suami, K. Tadano, Y. Kameda, and Y.limura, Chem. Lefl., (1984) 1919- 1922. (48) K. Tadano, Y. Kameda, Y. Iimura, and T. Suami, J. Carbohydr. Chem.. 6 (1987) 23 1244. (49) K. Tadano, C. Fukabori, M. Miyazaki, H. Kimura, and T. Suami, Bull. Chem. SOC.Jpn., 60(1987) 2189-2196. (50) T. Suami, K. Tadano, Y. Ueno, and C. Fukabori, Chem. Lett., (1985) 1557- 1560. (51) K. Tadano, Y. Ueno, C. Fukabori, Y. Hotta, and T. Suami, Bull. Chem. SOC.Jpn., 60 (1987) 1727- 1739. (52) H. Paulsen and W. von Deyn, Jusius Liebigs Ann. Chem., ( 1987) 125 - 13 1. (53) E. Fischer, Ber.. 27 (1894) 673-679.

TETSUO SUAMI AND SEllCHlRO OCAWA

42

The cyclization involves a nucleophilic attack of the malonic ester carbanion on the carbonyl carbon atom of the aldehyde, and the substituted malonic ester carbanion attacks the electron-deficient carbon atom bearing the iodine atom, or in the reverse order, to give 119. The hydroxyl group generated in the first step of the reaction attacks the carbon atom, giving the pyranose product. Thermal decarboxylation of 119 provided the cyclohexene derivative 120, which gave compound 121 by lithium aluminum hydride reduction. Hydroboration - oxidation of 121, followed by acetylation, gave carba-sugar derivatives (122 and 123) in equal yields. HO

OH

HO

SEt

-

I BnO En = PhCH,

A

' SEt

En0

Ts = O,SC,H,Me-p

BnO

Brio

BnO

\

En0

Q -

B nO 08 n

CHZOH

qH,OR'

\

Q OBn

08 n

120

119

118

121

C0,Me

CO,Me

OBn

BnO

117

116

115

RO

122 R = Bn. R' = AC

98 R = R' = AC 99 R = R ' = H

+ 123 R = En. R ' = Ac 124 R = R ' = A C 125 R = R ' = H

0-Debenzylation of 122 and 123, followed by acetylation, afforded the pentaacetates 98 and 124, which were respectively converted into carba-aD-glucopyranose (99) and carba-P-L-altropyranose (125) on hydroly~is.~'.~~

b. From D-Arabinose.-The C-I, C-3 diepimer (133) of 121 was prepared from D-arabinoseby the modified method of the synthesis described in

CARBA-SUGARS A N D THEIR DERIVATIVES

43

Section 111, 3,a. From 133, carba-a-L-mannopyranose pentaacetate (135) was produced as follows. 5-0-Tntyl-~-arabinosediethyl d i t h i ~ a c e t a (I l ~26) ~ was converted into compound I27 by sequential 0-benzylation, 0-detritylation, 0-silylation with lert-butylchlorodiphenylsilane(Me,CPh,SiCI), and regeneration of the parent aldehyde group. Knoevenagel reaction of 127 with dimethyl malonate and pyridine (Doebner modification) provided compound 128, which gave compound 129 by catalytic hydrogenation and subsequent O-desilylation. The crucial cyclization of 129 was accomplished by oxidation with pyridinium chlorochromate (PCC) and acetylation, providing two cyclohexane derivatives (130 and 131) in the ratio of 10: 1. Thermal decarboxylation of 130 resulted in formation of the cyclohexene derivative 132, with concomitant elimination. Reduction of the ester group with diisobutylaluminum hydride converted 132 into 133. Hydroboration-oxidation of 133 gave the carba-sugar derivative 134 as a single product.

BnO R

o

a

QBn CO,Me C

0

2

M

BnO R

126

=

Me,CPh, SI

128

127

A c O C0,Me OBn

f

BnO

W

O

OBn

BnO

129

130

131

R'O

CH2OH

-

BnO -0.

BnO QOBn 132

(54) N. W. Bristow and

133

B

@ORCH,OR'

134 R = Bn, R ' = AC 135 R = R ' = Ac

B. J. Lythgoe, J. Chem. Soc., (1949) 2306-2309.

n

e

-

TETSUO SUAMI AND SEIICHIRO OGAWA

44

The hydroboration reaction occurs stereospecifically, and the boron attacks from the less-hindered side of the molecule by normal cis addition, owing to the presence of the bulky benzyloxy group in the axial position. 0-Debenzylation of 134 and acetylation gave3 135.

c. From D-ribose. -D-Ribose diethyl dithioacetaP (136) was transformed into the C-1 epimer (137) of 133 by an I 1-step reaction analogous to that described in (b). Hydroboration-oxidation of 137, followed by acetylation, gave compound 138 as the sole product. The hydroboration reaction proceeds stereospecifically,as observed in the case of (b). Compound 138 was converted into carba-P-L-mannopyranosepentaacetate (139) in the usual way.*s3 CH2OH

137

136

138 R = Bn. R' = AC 139 R = R' = Ac

d. From D-xy1ose.-Just as described in (b), the C-2 epimer (141) of 137 was prepared from D-xylose diethyl dithioacetaP by an 1 I-step reaction. Hydroboration -oxidation of 141 gave a mixture ofcompounds 142 and 143 in the ratio of 6 : 1 after acetylation. Compounds 142 and 143 were respectively converted into carba-P-L-glucopyranose pentaacetate (144) and carba-a-D-altropyranosepentaacetate (145) by Odebenzylation and acetylati~n.~

H OHO A A yOIHS E t

HO 140

SEt

-

6

-

BnO

OBn 141

CH,OR'

O

B RO

CH,OR'

OR

142 R = Bn. R = AC 144 R = R = A c

+

R

I

RO

O

H OR

143 R = Bn. R = Ac 145 R = R = AC

(55) G . W. Kenner, H. J. Rodda, and A. R. Todd,J . Chem. Soc., (1949) 1613- 1620. (56) L. Hough and T. J. Taylor, J . Chem. Soc.. (1955) 1213-1218.

CARBA-SUGARS AND THEIR DERIVATIVES

45

e. From D-glucose. -A mixture (146) of geometric isomers was prepared from D-glucose by a reported pro~edure.~’ Catalytic hydrogenation of 146 and oxidation with PCC gave the 3-C-propanoyl derivative 147. Selective hydrolysis of the 5,6-O-isopropylidene group, followed by periodic acid oxidation, provided the aldehyde 148. Cyclization of 148 with 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) and a subsequent elimination reaction with acetic anhydride and pyridine furnished compound 149.50,5’ Compound 149 was found to be an important key compound for the following synthesis of carba-sugars of the a-L-altro, P-D-gluco, P-L-allo, and a-D-manno modifications. /O% M ~ C \OCH

I

Q-

+HC 3

\

y C H 2

C

0-CMe,

O-CMe2 C

I1 0 146

cQ-

/CH2

H3C\

II

0 147

148

149

Epoxidation of 149 with hydrogen peroxide gave the epoxide 150 as a major component, which, by sodium borohydride reduction, was converted into compounds 151 and 152 in the ratio of 5 : 1. Opening of the oxirane rings of 151 and 152 by hydroxide anions provided the same compound, namely, 153. The reaction from 152 to 153 proceeds as a trans-diaxial opening of the oxirane ring. The reaction from 151 involves migration of the oxirane ring, with participation of the vicinal hydroxyl group, and the newly formed epoxide is opened in the same manner, to give 153. 0-Benzylation of 153, followed by hydrolysis and reduction, furnished compound 154. Periodic acid oxidation of 154 gave the aldehyde 155. (57) J. M. J. Tronchet and B. Gentile, Curbohydr. Res.. 44 (1975) 23-35.

TETSUO SUAMI A N D SEIlCHlRO OGAWA

46

Reduction of 155 and acetylation gave the carba-sugar derivative 156, which was converted into carba-a-L-altropyranose pentaacetate (157). The corresponding free carba-sugar 158 was ~ b t a i n e d ~from ~ . ~ 157 ' by hydrolysis.

p-:i

H

H CO-CMe,

0 149

H

CO--Me2

-

--;I

151

0 150

152

I

BnO

CH,OR

BnO 155

154

156 R=Bn.R' AC 157 R R1=AC 158 fl=fl'=H

Carba-sugarsof the a-L-altro and P-D-ghC0 modifications were preparedS' from 149 by way of 155. 0-Mesylation of 155 with an excess of mesyl chloride and pyridine resulted in formation of the cyclohexenealdehyde 159, accompanied by /?-elimination. Reduction of 159 with sodium borohydride gave the cyclohexenemethanol 160, which is the antipode OF 141. Analogously to the reaction 141 142 143, hydroboration - oxidation of 160 and acetylation provided carba-sugar derivatives of the a-L-altro (156) and P-D-~~uco (161) modification in the ratio of 1 :6, which were convertible into 158 and 93, respectively, in the usual way.5' Starting from 149, novel carba-sugar pentaacetates of the /?-L-allo (168) and ~ - D - M ~ W Z( IO71) configuration have been synthesized. Reduction of 149 with diisobutylaluminum hydride (DIBAL-H) and acetylation gave a mixture of acetates 162 and 163. Hydroxylation of the mixture with osmium tetraoxide and hydrogen peroxide provided compounds 164 and 165 in the ratio of 9 : I . Hydrolysis of 164 gave compound 166, which was transformed into 168 by a reaction analogous to that employed in the p r e p a r a t i ~ of n ~157 ~ from 153.

-

+

&

CARBA-SUGARS AND THEIR DERIVATIVES

155

------

BnO

-

OBn

&H

OBn

BnO

159

47

160

CH,OR'

+

156

R'o=&OR RO

OR

161 R = Bn, R' = AC 93 R = R ' = A c

149

AcO 162

163

HO OAc 164

OBn 166

167

168

TETSUO SUAMl AND SEIICHIRO OGAWA

48

From 167, compound 171 was obtained49by way of intermediary compound 170, the antipode of 133, by a reaction analogous to that used in the preparation of 93 from 155. C, HO

169

CH2OH

170

AcO

OAc 171

The reaction of the aldehyde 174, prepared from D-glucose diethyl dithioacetal by way of compounds 172 and 173, with lithium dimethyl methylphosphonate gave the adduct 175. Conversion of 175 into compound 176, followed by oxidation with dimethyl sulfoxide- oxalyl chloride, provided diketone 177. Cyclization of 177 with ethyldiisopropylamine gave the enone 178, which furnished compounds 179 and 180 on sodium borohydride reduction. 0-Desilylation, catalytic hydrogenation, 0-debenzylation, and acetylation converted52179 into the pentaacetate 93 and Sa-carba-a-~-idopyranose pentaacetate (181).

172 R=H 173 R=Bn

R=Me3CPh2Si

176

174

175

R=Me,CPh,Si 177

R=Me,CPh,Si 178

CARBA-SUGARS AND THEIR DERIVATIVES

49

CH,OR

un

OBn R = Me,CPh,Si

181 R = A c 182 R = H

179

B E n 0n

O

e

OH R = Me,CPh,Si

98

,

+

R o OR

R o R

CHzOR 183 R = Ac 184 R = H

180

Similarly, 180 was transformed into 5a-carba-cu-~-glucopyranosepentaacetate (98) and Sa-carba-P-~-idopyranosepentaacetate (183). Deacetylation of 181 and 183 by usual hydrolysis gaves2the corresponding carba-sugars 182 and 184. The hitherto-unknown carba-sugars having the a-(182) andP-L-ido (184) configuration were synthesized from ~-glucose,as well as those having the known a- (99) and P-D-gluco (94) c~nfiguration~~.

Iv.

6a-CARBA-P-FRUCTOPYRANOSES

D-Fructose is the sweetest sugar known in naturally occumng carbohydrates, and its intense sweetness is produced only by P-~-fructopyranose.~~The sweetness-elicitingunits, AH (a proton donor) and B (a proton acceptor) components were respectively assignedj9to the anomeric OH group on C-2 and the OH group on C- 1, and the CH, group of C-6 was ascribed61to a third (hydrophobic) component (X). The 6a-carba-P-~-fructopyranosemolecule may have this tripartite arrangement (AH, B, and X) in almost the same emplacement as has the P-D-fructopyranose molecule. (58) M. G . Lindley and G. G. Birch, J. Sci., FoudAgric., 26 (1975) 117- 124. (59) R. S. Shallenberger, PureAppl. Chem., 50 (1978) 1409- 1420 C.-K. Lee, Adv. Carbohydr. Chem. Biuchem., 45 (1987) 199-351. (60) 0.R. Martin, S. K. Tommola, and W. A. Szarek, Can. J . Chem.. 60 ( 1 982) 1857 - 1862. (61) L. B. Kier, J . Pharm. Sci., 61 (1972) 1394- 1397.

TETSUO SUAMI A N D SEIICHRO OGAWA

50

As it has been revealed that replacement of the ring-oxygen atom in a pyranoid sugar by a CH, group is not detrimental to its sweetness,626acarba-P-D-fructopyranose may have the same intense sweetness as D-fructose. To substantiate this prediction, the following two reaction routes have been successfullydeveloped for 6a-carba-P-~~-fmctopyranose, as well as for the enantiomers. 1. Synthesis of Racemic 6a-Carba-P-fructopyranose

a. From the Bromide 47.-D~ehydrobromination~~of 47 with DBU gave the diene 185, which was transformed into the spiro epoxide 186 by preferential epoxidation of the exocyclic C=C bond with m-chloroperoxybenzoic acid (mCPBA).Opening of the oxirane ring of 186 with acetate ions gave the tetraacetate 187, after acetylation with acetic anhydride-4-(dimethylamino)pyridine (DMAP). Sequential deacetylation, epoxidation with mCPBA, and conventional acetylation, converted 187 into the epoxide (188). Formation of the epoxide 188 proceeds stereospecifically by virtue of the cis-directing effect of the vicinal OH Reductive opening of the oxirane ring of 188 with lithium aluminum hydride, and acetylation with acetic anhydride - DMAP, provided the pen-

47

-

wcH2 __

W

C

OAc

Ac 0

Acb

185

187

H

OAc

188

186

189 R = A c 190 R = H

(62) T. Suami, S . Ogawa, and T. Toyokuni, Chrm. Ldf.,(1983) 61 1-612. (63) H. B. Henbest and R. A. L. Wilson, J. Clrem. Soc.. (1957) 1958- 1965.

,

51

CARBA-SUGARS AND THEIR DERIVATIVES

taacetate (189). Hydrolysis of 189 gave 6a-carba-P-~~-fructopyranose (190) as a s y r ~ p . ~ ~ ? ~ ~ b. A Facile Synthesis.-This was developed as follows. Hydrolysiss3of 47 with hydrochloric acid gave the cyclohexenediol 191. Stereospecific epoxidation of 191 with mCPBA, induced by the aforementioned cis-directing effect of the neighboring OH group, furnished compound 192 on acetylation of the product. Dehydrobromination of 192 with silver fluoride gave the exocyclic alkene 193. Reductive opening of the oxirane ring of 193 with lithium aluminum hydride, and acetylation, provided compound 194. Epoxidation of 194 with mCPBA gave the epoxide 195. Opening of the oxirane ring with acetate ions, followed by acetylation, gave the tetraacetate 196, or, by exhaustive acetylation with acetic anhydride- DMAP, the pentaacetate 189. Compounds 196 and 189 were readily transformed into 190 by hydrolysis.64-66

194

191

192

AC 0

RO

195

193

196 R = AC 190 R = H

2. Synthesis of Enantiomeric 6a-Carba-/?-Fructopyoses

Compound (-)-29 (Ref. 43) was converted into (1R)-( 1,3/2)-2,3-diacetoxy- I -(bromomethyl)cyclohex-4-ene(197) by reactions analogous to those used32.33 in the preparation of 47. Furthermore, reaction analogous to that (64) T . Suami, S. Ogawa, M. Takata, K. Yasuda, A. Suga, K. Takei, and Y. Uematsu, Chem. LcII..(1985) 719-722. (65) T. Suami, S. Ogawa, M. Takata, K. Yasuda, K. Takei, and A. Suga, Bull. Chem. SOC. Jpn., 59 (1986) 819-821. (66) T. Suami, S. Ogawa, Y. Uematsu, and A. Suga, Bull. Chem. SOC.Jpn., 59 (1986) 1261 1262.

TETSUO SUAMI AND SEllCHRO OGAWA

52

employed in the preparation ofthe racemate 196 in method (b) transformed 197 into 6a-carba-P-~-fructopyranosetetraacetate (198), m.p. 11 1- 112", [a]:'-50" (chloroform), and the free carba-sugar (199), a syrup, [a]2,2 -57" (methan~l).~~ Similarly, (+)-29 gave 6a-carba-P-~-fructopyranosetetraacetate (200), m.p. 113.5- 114.5", [a]i4 +46" (chloroform), and the respective free carbasugar (201), a syrup, [a]i3 +57" ( m e t h a n ~ l ) . ~ ~

197

198

R - Ac

200

R = AC

199

R=H

201

R

~

H

The racemate 190, the D antipode 199, and the L form (201) were almost equally as sweet as D-fructose, but 199 was f o ~ n d ~to~be , ~slightly * sweeter than 201. V. SYNTHESIS OF RACEMIC AMINOCARBA-SUGARS 1. Carba-glycosylamines

Carba-glycosylamines [2,3,4-trihydroxy-5-(hydroxymethyl)- I -cyclohexylamine] and related compounds are well known to exist as components of the antibiotic validamycin complex" and carba-oligosaccharidic alpha amylase inhibitor^.^^ Microbial degradationz3 of validamycin A (8)with a cell suspension of Pseudornonus dentrificuns afforded validamine (202) and valienaminez4 (203). Hydrogenolysis of validamycin B, followed by acid hydrolysis, yielded hydroxyvalidamine (204). Valiolamine (205) was isolated and shown70to be a component of validamycin G.70Biosynthesis of these carba-glycosylamines was extensively ~tudied,~' and the intramolecular aldol addition of the (67) S. Ogawa, Y. Uematsu, S. Yoshida, N. Sasaki, and T. Suarni, J. Curhohydr. Chem., 6 (1987) 471-478. (68) T. Suami, Pure Appl. Chem., 59 (1987) 1509- 1520. (69) E. Truscheit, W. Frommer, B. Junge, L. Miiller, D. D. Schmidt, and W. Wingender, Angew. Chem., In!. Ed. Engl., 20(1981) 744-761. (70) S. Horii, H. Fukase, Y. Kameda, N. Asano, T. Yarnaguchi, and K. Matsui, J. Aniibioi.. 39 (1986) I491 - 1494. (71) T. Toyokuni, W. -Z. Jin, and K. L. Rinehart, Jr., J . Am. Chem. Soc., 109 (1987) 34813482.

CARBA-SUGARS AND THEIR DERIVATIVES

53

2,6-diketose derived from sedoheptulose 7-phosphate was presumed to be involved in the formation of the key intermediate, the branched-chain inosose derivatives 203, 204, and 205. Compounds 202,203, and 205 possess detectable inhibitory activity against some hydrolases. Chemical modificat i ~ ofn 205, ~ ~the most potent among them, provided some inhibitors strong enough to be applicable to clinical treatment of diabetes. The first synthesis30of 202 was carried out by starting from the endo-adduct 29 of furan and acrylic acid. Treatment of the tosylate (206) obtained from 64 with sodium azide in DMF resulted in inversion ofthe configuration by direct S N displacement, ~ to give the azide 207, which was converted, by hydrogenation in the presence of Raney acid hydrolysis, and acetylation, into the penta-N,O-acetyl derivative of 202. The dibromide derived from 46 was converted into the epoxide (208) by treatment with sodium methoxide, followed by acetylation, and the bromogroup wasdisplaced with azide ion. The azide was converted into the N-acetyl derivative (209),which was treated with sodium acetate to give the validamine derivative selectively by way of neighboring-group as~istance.~~ Furthermore, bromide 210 was shown to be a most versatile intermediate, ~ o n v e r t i b l einto ~ ~ the azide 21 1. I'll , 0 l 1

Q

Oll t i I I 2

Ill1

202

o(, N112

I10

~

NilZ

110

Oil

203

,

I10

OH

1111

204

205

i

206

207

208

209

(72) S. Horii, H. Fukase, T. Matsuo, Y. Kameda, N. Asano, and K. Matsui, J. Med. Chem., 29 (1986) 1038-1046. (73) S. Nishimura, Bid/. Chem. SOC.Jpn., 32 (1959) 61 -64. (74) S. Ogawa, K. Nakamoto, M. Takahara, Y. Tanno, N. Chida, and T. Suami, Bull. Chem. Snc. Jpn.. 52 (1979) 1174- 1176.

1

TETSUO SUAMI AND SEIICHRO OGAWA

54

C1I20Ac

C02Me

I

OAC

I

OAC

21 0

dAC

212

211

The bromo methyl ester (212) formed by treatment of 30 with hydrogen bromide in acetic acid could also serve as a precursor to ~ a l i d a r n i n e . ~ ~ The 2-deoxy derivative (215) of 202 was prepared32 from the diacetate 213, obtained by debromination of 34, following a similar sequence starting from the dibromide 214. The 1-epivalidamine derivative 217 was prepared76by successive azidolysis, acetolysis, and hydrogenation of the dianhydride 216, derived from 51. CI12UAc

I

213

215

214

A1

0 UAL

216

217

(75) S. Ogawa, M. Suzuki, and T. Tonegawa, Bull. Chem. SOC. Jpn., 61 (1988) 1824- 1826. (76) S. Ogawa, M. Oya, T. Toyokuni, N. Chida, and T. Suami, E d / . Chem. SOC.Jpn., 56 (1983) 1441- 1445.

CARBA-SUGARS AND THEIR DERIVATIVES

55

m-Valienamine (203) was initially s y n t h e s i ~ e dfrom ~ ~ . ~the ~ crystalline conjugate diene79(218,45%)derived by treatment of 51 with DBU in toluene. Treatment of 218 with bromine in chloroform gave rise to a mixure of isomeric 1,4-dibromides(219a,b, go%), the primary bromo group of which was selectively replaced with the acetoxyl group, to give the ally1 bromides (220a,b). Azidolysis afforded the azides (221 and 222), separable by chromatography on silica gel, and selective reduction of the azido function with hydrogen sulfide in aqueous pyridinesOgave the penta-N,O-acetyl derivative of 203 and its 1 -epimer (223), respectively. In order to improve the selectivity of this sequence, 218 was first oxidized to the spiro epoxides. The major

218

219 X = Br 220 X = O A c

UAL

224

221

222 X = N, 223 X = NHAC

OAc

225

I

('Ill

226

Oil

227

(77) S. Ogawa, T. Toyokuni. and T. Suami, Chrm. Left., (1980) 713-716. (78) T. Toyokuni, S. Ogawa, and T. Suami, Bull. Chem. SOC.Jpn., 56 (1983) 1161 - 1 170. (79) S. Ogawa, T. Toyokuni, M. Omata, N. Chida, and T. Suami, Bull. Chem. SOC.Jpn., 53 (1980) 455-457. (80) T. Adachi, Y. Yamada. and 1. Inoue, Synthesis. (1977) 45-46.

TETSUO SUAMI A N D SEIICHRO OGAWA

56

isomer (224) was treated with hydrochloric acid to give the chloride (225), treatment of which with azide ion gave only 221 in 65% yield by the S N ~ ‘ mechanism (apofacial). Likewise, starting from 219, the 7-amino-7-deoxy (226) and the 1,7-diamino-1,7-dideoxy (227) analogs were readily pre~ared.~~,’~ Treatment of 51 with an excess of sodium benzoate in DMF resulted in substitution and elimination, to yield the cyclohexene derivatives1 (228, 36%). The yield was low, but 228 was later shown to be a useful compound for synthesisof carba-oligosaccharides.U-Deacylation of 228 and successive benzylidenation and acetylation gave the alkene 229, which was oxidized with a peroxy acid to give a single epoxide (230) in 60%yield. Treatment of 230 with sodium azide and ammonium chloride in aqueous 2-methoxyethanol gave the azide (231,5590)as the major product; this was converted into a hydroxyvalidamine derivative in the usual manner. On the other hand, an elimination reaction of the methanesulfonate of 231 with DBU in toluene gave the protected precursor (232, 8790) of 203.

Oh I

OAc

228

OAr

229

231

230

232

Acid hydrolysis of the epoxide (233) obtained from 46 gave, after acetylation, the acetate (234), which, on treatment with DBU, yielded the diene (235), useful for preparation of the azides (237 and 238), the precursors of valienamine analogs.**Treatment of 46 with DBU in toluene resulted in (81) S. Ogawa, N. Chida, and T. Suami, J. Org. Chcm..48 (1983) 1203- 1207. (82) S. Ogawa. T. Hattori, T. Toyokuni, and T. Suami, Bull. Chem. Soc. Jpn.. 56 (1983)

2077-2081.

CARBA-SUGARS AND THEIR DERIVATIVES

57

elimination of hydrogen bromide and substitution at the allylic carbon atom with an acetate ion to produce the diene (236), which was convertible into the a ~ i d (239). e~~

AcO

AcO

234

233

235

CH..

OAc AcO

236 CH,OAc

CII,OAc

AcO

AcO

237

238

CH,OAc

239

Reaction of 47 with NBS in carbon tetrachloride afforded the tribromide (240, 10090).After replacement of the primary bromo group with benzoyloxyl, the product (241,47%) was debrominated with zinc dust in ethanol to give the d i e ~ (242,6490). ~ e ~ ~ Epoxidation of 242 produced the isomeric compounds 243 and 244, which were transformed into the azides (245 and 246), convertible into valienamine isomers.84 (83) S. Ogawa, T. Toyokuni, M. Ara, M. Suetsugu, and T. Suami, Chem. Leff..(1980) 379382; Bull. Chem. SOC.Jpn., 56 (1983) 1710-1714. (84) S. Ogawa, M. Suetsugu, T. Toyokuni, and T. Suami, Nippon Kuguku Kuishi, (1982) 172 1 - 1726.

58

TETSUO SUAMl AND SEllCHRO OGAWA

240

241

X = Ur

242

X

=

243

244

OUz

ilA<

245

246

DL-Valiolamine (205) was synthesized from the exo-alkene (247)85,86 derived from 51 with silver fluoride in pyridine. Compound 247 was treated with a peroxy acid, to give a single spiro epoxide (248, 89%) which was cleaved by way of anchimeric reaction in the presence of acetate ion to give, after acetylation, the tetraacetate 249. The bromo group was directly displaced with azide ion, the product was hydrogenated, and the amine acetylated, to gve the penta-N,O-acetyl derivative (250,50%). On the other hand,

(I\,

247

249


0,\8

251

1 I '\

o,\c

lIA1

248

252

(85) S. Ogawa and Y . Shibata. Chan. Leii., (1985) I58 I - 1582. (86) S. Ogawa and Y. Shibata, Curbohydr. Res, 148 (1986) 257-263

250

59

CARBA-SUGARS AND THEIR DERIVATIVES

oxidation of 247 with osmium tetraoxide in tert-butanol afforded, after acetylation, the isomeric pentaacetate (251,6 1Yo). the bromo group ofwhich was preferentially replaced by an azido function, with retention of configuration, by way of neighboring assistance of the acetoxymethyl group. Reduction of the azide afforded the isomeP (252,45%) of 205. 2. 2-Amino-2-deoxycarba-hexosesand Relatives

Although aminodeoxycarba-hexosespossessing the amino group at C-2, C-3, C-4, or C-7 have not so far been discovered in Nature, they may constitute an important class of sugar analogs that are expected to be substrates or inhibitors of many kinds of enzymes. Up to the present, eighteen amino carba-sugars (ten 2-amino-2-deoxy-; five 3-amino-3-deoxy-; one 4-amino4-deoxy-; two 7-amino-7-deoxycarba-hexopyranoses) as racemic modifications, and four enantiomers have been synthesized (see Table 11). 2-Amino-2-deoxycarba-hexoses having the a d o (253) and a-gulucto (254) configurations were first synthesized3' by azidolysis of the bromide (36) in aqueous 2-methoxyethanol, followed by hydrogenation and acetylation. The reaction proceeded by way of diaxial opening of the cyclic acetoxonium ion formed by neighboring-groupparticipation, affording 253 as the major product. Similarly, from the bromide (37), a-DL-carba-mannopyranosylamine (255)and 3-amino-3-deoxy-~-~~-carba-altropyranose (256) were obtained as the penta-N,O-acetylderivatives. When the azidolysis of 37 was

w

HO CIiZOH oli

Hb

m-3p+

110

Oil

IlU

011

011

gulose

ldose

glucose

w3

HO CH201I

Oh

llow mannose

110

011

galactose

113 011

talose

altrose

5a-Carba-~d-hexopyranoses

SCHEME I.

Cli20tl

oil%i)l

Hil

allose

TETSUO SUAMI AND SEIICHRO OGAWA

60

TABLE I1 Physical Properties of Amino Carba-hexopyranose Penta-N, 0-Acetates Configuration

Substituents

Melting point ( " C )

References

a-allo /I-allo p-altro a-galacfo p-galacto a-gluco

2-amino-2-deoxy2-amino-2deoxy3-amino-3-deoxy2-amino-2deoxy2-amino-2-deoxy2-amino-2-deoxy3-amino-3-deoxy4-amino-4-deox y7-amino-7-deox y2-amino-2-deoxy3-amino-3-deoxy7-amino-7deoxy2-amino-2-deoxy2-amino-2-deoxy3-amino-3-deox y2-amino-2-deoxy2-amino-2deoxy3-amino-3-deoxy-

197- 198 122- 123.5 syrup 183-185 205 -205.5 134-136 144.5- 146 syrup 150-151 140- 141 146.5- 148 210-211 181.5- 182.5 148- I50 159- I60 SYWP 177- I79 154- 156

87 87 31 31,87 87 94 88 89 33 87 88 33 87 87 31 75 31 88

p-gluco a-gulo p-gulo a-ido a-manno p-manno

camed out in DMF, the sN2 reaction mainly occurred,87to yield, after the usual treatment, 2-amino-2-deoxy-~-~~-carba-mannopyranose (257, 5 1%). Cyclization of dialdehyde and nitromethane was utilized8' in carba-sugar chemistry, using the readily accessible 4,7-O-isopropylidene-a-~~-carba-galactose (258) and 4,7-O-benzylidene-P-~~-carba-glucose (259). The dialdehyde 260 generated by periodate oxidation of 258 reacted in the presence of sodium methoxide to give a 9 : 2 : 1 mixture of the nitrodiols having the P-galacto, a-galacfo,and P-gulo configurations in 86%combined yield. They were cleanly separated by chromatography on a column of silica gel. Hydrogenation in the presence of Raney nickel in methanol and acetic anhydride, hydrolysis, and acetylation afforded the respective penta-N,O-acetyl aminodeoxy carba-sugars (261, 262, and 263). Likewise, starting from the dialdehyde (264) derived from 259, two tetra-O-acetyl-2-acetamido-2-deoxycarba-hexoses having the P-gluco (265, 42%) and p-allo (266, 19%) configurations were obtained. The di-0-methylsulfonyl derivatives (267 and 268) gave, on treatment with sodium acetate followed by acetylation, a-DLcarba-gulosamine (269) and a-DL-carba-allosamine derivatives (270), respectively, with inversion of configuration. (87) S. Ogawaand M. Onhara, Carbohydr. Res., 177 (1988) 199-212.

CARBA-SUGARS AND THEIR DERIVATIVES

61

NHAc

NIIAc

253

254

C1I20Ac

CH20Ac

I

CH OAc 12

I

~ H A C

255

256

257

bll

ill3

258

259

Similarly, 1-0-acetyl-a-DL-carba-glucopyranose (271) was subjected to the dialdehyde- nitromethane condensation.88The reaction proceeded selectively, to give, after conventional treatment, penta-N,O-acetyl-3-amino3-deoxy-a-~~-carba-glucopyranose (273). The N-acetyl derivative (272) of 202 also afforded, exclusively, the diamino derivative (274) having the Qg/zico configuration. In contrast, under similar conditions, the P-gluco isomer (275) yielded the 3-amino-3-deoxycarba-hexosederivatives having the P-gluco (276) and /?-manno (277) configurations, and 273 formed by epimerization. Sharpless reaction of 47 occurred almost regioselectively to give the 2(278, 60%), together the 1-p-toluenesulfonamido derivatives (279, ( 8 8 ) S. Owaga and M. Orihara, Carbohydr. Rex, 189 (1989) 323-330.

TETSUO SUAMI AND SEIICHRO OGAWA

62

i*l,(p(;l +

QAc

Q,,' l,llLUAc

)il

AcO

CllO

ArO

NllAc

264 265

267

268

271 X =OAC 272 X = NHAC

(IAc

273 X=OAc 274 X = NHAc

NllAi

266

269

I

dll

ACO

270

CARBA-SUGARS AND THEIR DERIVATIVES

63

5%). Compound 278 was convertible into 2-amino-2-deoxy-(280) and 2,7diamino-2,7-dideoxy-a-~~-carba-glucopyranose (281) derivatives in satisfactory yields. The 4-0- (282) and 4,7-di-O-methanesulfonates(283) of a-DL-carbagalactopyranose obtained from 258 produced,89after azidolysis in DMF, hydrogenolysis, and acetylation, penta-N,O-acetyl-4-amino-(284) and 4,7diamino-4,7-dideoxy-a-~~-carba-glucopyranose (285).

278

279

Ts = SO,C,H,M,-p

CH20R

I

0.k

Bn

=

284

X = OAc

285

X

NHAc

Ph Ch,

(89) S. Ogawa, T. Taki, and A. Isaka, 191 (1989) 154-162.

280

S

=

OAc

281

X

=

NHAc

64

TETSUO SUAMI AND SEIICHRO OGAWA

VI. SYNTHESIS OF ENANTIOMERIC AMINOCARBA-SUGARS 1. Enantiomeric Carba-glycosylamines

(+)-Validamit~e~~ (202) and (+)+alienamhew (203) have been synthesized as the penta-N,O-acetates from the optically active d i b r ~ m i d e(51), ~~ following essentially the procedure described for the preparation of their racemic modifications. Paulsen and his coworkersg1first synthesized (+)-203 from L-quebrachitol (286) by a 2 I-step reaction as follows. The di-0-isopropylidene derivative was oxidized to the ketone (287),and then epoxidized with dimethyl sulfoxonium-methylide to give 288, which was subjected to benzoylation, mesylation, and demethylation, followed by benzylation, to afford 289. Introduction of unsaturation was accomplished by epoxidation of 289 with sodium methoxide to 290 and 291, and deoxygenation to 292. The azido group was introduced with azobis(dicarboxy1ate) to give 293, which was hydrogenolyzed, followed by deprotection to afford 203. They also prepared9*valienamine derivative (+)-227 from the ditosylate (294) derived from 286. The spiro epoxide (295) obtained from 294 was converted into 296 by treatment with selenoxobenzothiazole- tri-

tl0

AoO

ACO

290

291

292

293

(90) S. Ogawa, Y. Shibata, T. Nose, and T. Suami, Bull. Chem. Soc. Jpn.. 58 (1985) 33873388. (91) H. Paulsen and F. R. Heiker, Angew. Chem.. I n f . Ed. EngI., 19 (1980) 904-905; Justits Lirhigs Ann. Chem.. (1981) 2180-2203. (92) H. Paulsen and F. R. Heiker, Carhohydr. Rex. 102 (1982) 83-98.

CARBA-SUGARS AND THEIR DERIVATIVES

65

fluoroacetic acid, and mesylation. Reaction of 296 with sodium azide proceeded in the S N ~ fashion, ' to give the azide (297), which afforded the valienamine isomer. Treatment of 298 with sodium azide in DMFgave, by a [ 3.31-sigmatropic rearrangement (299 + 300), the diazido compound (301), hydrogenolysis of which yielded 227. Horii and F ~ k a s first e ~ succeeded ~ in synthesis of (+)-valiolamine (205)by stereoselectivechemical conversion of (+)-203 through the key intermediate (302) derived from the N-benzyloxycarbonyl derivative of 203, establishing its structure.

294

295

296

Kuzuhara and his coworkers94synthesized (+)-205 by hydroxylation of the alkene 304, which was prepared from the chiral azidocyclohexenederivative (303) derived from D-glucose, in which a novel rearrangement of the C - C double bond accompanying reduction of the azido group with lithium aluminum hydride was observed. (93) S. Horii, H. Fukase, and Y. Kameda, Curhohydr. Rex, 140 (1985) 185-200. (94) M. Hayashida, N. Sakairi, and H. Kuzuhara, J. Curbohydr. Chem., 7 (1988) 83-94.

TETSUO SUAMI AND SEllCHRO OGAWA

66

2. Enantiomeric 2-Amino-2-deoxycarba-hexosesand Other Amino Carba-sugars

Sharpless reaction of the enantiomeric alkenes (47) produced the precursors for 2-amino-2-deoxy-a-~-and -~-carba-glucopyranoses.~~ These cornpounds were converted into the penta-N,O-acetates by the following sequence. Replacement of the bromo group with an acetate ion, treatment with sodium in liquid ammonia, and peracetylation. Barton and his coworkers96succeeded9’ in the synthesis of two enantiomeric 2-amino-2-deoxycarba-hexosesstarting from the key substituted cyclohexanone 307, readily available from the D-glucosamine derivative (305) by way of the alkene (306). Treatment of 307 with Wittig reagents, using methoxy-methylenetriphenylphosphorane and methylenetriphenylphosphorane gave the vinyl ether (308,6Ooh)and the exocyclic alkene (309,67%), respectively. Compound 309 reacted with mercury(I1)acetate to give mainly the carba-D-glucosamine derivative (310),which was hydrolyzed, hydrogen-

1111

305

309

306

307

310

311

,I il

308

(95)S. Ogawa, N.Sasaki, T. Nose, T. Takagaki, and T. Suami, Symp. Eftro-Curhohydrutrs, Grenoble, France, Sept. 1985, Abstr. No. B-4-110. (96)D. H.R.Barton, S. D. Gero, S. Augy, and B.Quiclet-Sire.J. Chem.Soc.. Chem. Commun., (1986) 1399-1401. (97) G . Vass, P. Krausz, B.Quiclet-Sire, J.-M. Delaumeny,J. Cleophax, and S. D. Gero, C. H. Acad. Sci.. Srr. II, 301 (1985) 1345-1346.

CARBA-SUGARS AND THEIR DERIVATIVES

61

ated, and then acetylated to give the penta-N,O-acetate. While, hydroboration of 309 afforded the carba-L-idosamine derivative (31 I ) as the sole product in 60% yield, which was similarly converted into the penta-N,O-acetate. VII. SYNTHESIS OF MONO-AND DICARBA-DISACCHARIDES

Carba-disaccharidesare carbocyclic analogs of true disaccharides in which one or both of the hexose or pentose residues is (are) replaced with a carbasugar. Therefore, for instance, three types of carba-maltose are to be considered: type A is composed of two carba-D-glucopyranose units bonded by an ether linkage, and types B and C consist of one true and one carba-D-ghcopyranose unit, the first two (A and B) being ethers and the last (C), a glycoside. It may be possible to prepare a carba-maltose of biological interest by replacing ether and glucoside bonds with an imino and N-D-glucosyl or a thio ether and thio-D-glucoside bond, respectively. Carba-disaccharides of types A and B are chemically very stable and may be of interest in elucidating the roles of the hydroxyl groups of sugar residues, based on spatial geometry in biological systems. Carba-sugars of type C may perhaps be substrate analogs or inhibitors of certain enzymes, and be useful for elucidation of the mechanisms of their action.

011

OH

A

Maltose

B

C

TETSUO SUAMl AND SEIICHRO OGAWA

68

1. Synthesis of Carba-maltose, Carba-isomaltose, Carba-cellobiose, and the Like a-Carba-maltose and carba-isomaltose of type B were first synthesized by Paulsen and coworkers.98The 6-trifluoromethanesulfonate (312) of methyl 2,3,4-tri-0-benzyl-~-ghcopyranose was displaced with the protected CY-Dcarba-glucopyranose 313 in the presence of sodium hydride in tetrahydrofuran at room temperature to yield, after deprotection, the monocarba-isomaltose derivative 314 in 8690yield. Likewise, coupling of312 with the allyl alcohol 315 gave compound 316, which was hydrogenated to give the deoxy

CHIOI

f

I

312 TI

=

011

F,CCH,SO, 314

OBn

31 5

OB"

316

OAc

317

derivative 317. On the other hand, similar condensation of 313 with the 4-triflate (318) of 1,6-anhydro-2,3-di-O-benzyI-~-~-galactose in DMF gave the protected monocarba-disaccharide 319, which was converted into 4-0methylmonocarba-maltose (320, 7590)in the usual way. The branchedchain monocarba-disaccharide 322 was prepared by reaction of 318 with 321. The condensate (324), obtained by reaction with the isomeric allyl alcohol 323, was hydrogenated and deprotected, to afford monocarba-cellobiose (325). (98) H. Paulsen and W. von Deyn, Justus Liehigs Ann. Chem.,(1987) 141 - 152.

CARBA-SUGARS AND THEIR DERIVATIVES

69

Carba-disaccharides of type C were prepared by glycosylation of the appropriately protected carba-sugarswith glycosyl halides. The di-0-isopropylidene derivatives (327 and 329) were glycosylated with 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide (326a) in benzene in the presence of mercury(11) cyanide, giving the condensates, from which the respective monocarba-disa~charides~~ (328 and 330) were obtained.Iw The protected a-DL-carba-glucopyranose 332, derived from the benzylidene derivative 331, was glycosylated with 1,6-d~-O-acetyl-2,3,4-t~-0-~nzyl-a-~-glucose (333) or penta-0-acetyl-a-D-glucose (334), to give,1ooafter deprotection, monocarba-maltose (335) or monocarba-cellobiose (336). 2. Carba-trehaloses The carba-trehalosesare carbocyclic analogs of trehalose, in which one or both of the D-glucopyranose units is (are) replaced with D-carba-glucopyranose, and may be expected to act as a substrate analog or an inhibitor of (99) S. Ogawa, Y. Shibata, K. Miyazawa, T. Toyokuni, T. Iida, and T. Suami, Curbohydr. Rc>s..163 (1987) 53-62. (100) S. Ogawa and 1. Sugawa, unpublished results.

TETSUO SUAMI AND SEIICHRO OGAWA

70

(

ll.,llll

Cllz0ll

I

I -

326a X = Br

I

328

Ill/

326b X = CI I 1121111 I

Oil

329

I

330

Ollll

till

332

331

333

334

CII 201I

I

011 335

336

a,a-trehalose hydrolase (trehalase). All four of the diastereoisomeric pairs, theoretically possible, a,a, a$, p,a, and p,P, of carba-trehalose (type C), composed of D-glucopyranose and D- or L-carba-glucopyranosehave been synthesized.'O' (101) S. Ogawa, S. Yokoi, N. Kimura,Y. Shibata, and N.Chida, Carbohydr. Res., 181 (1988)

57-66.

CARBA-SUGARS AND THEIR DERIVATIVES

71

Four carba-glucopyranosyla-D-glucopyranosideswere obtained by coupling 2,3,4,7-tetra-O-benzyl-a(337) and -P-DL-carba-glucopyranose(338) with 333 in the presence of trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by separation of the diastereoisomers on a column of silica gel. On the other hand, coupling with 334 under similar conditions afforded four carba-glucopyranosyl P-D-glucopyranosides.Deprotection by hydrogenolysis in the presence of palladium-on-carbon, followed by acetylation, gave eight carba-trehalose octaacetates, 0-deacetylation of which afforded the free carba-disaccharides (339, 340, 341, and 342) and their diastereoisomers.Their absolute configurationswere deduced on the basis of ('11 >OBn

Q

Q

HnO

BnO

O h

OBn

337

338

(:I I201I

011 I

I

011

C11*O11 I

011

339

till

340

fq

110

011

OH

341

342

TETSUO SUAMI AND SEIICHRO OGAWA

72

optical rotations, and confirmed by calculation by HMP methods.lo2Compound 329 the absolute configuration of which corresponds to that of true trehalose has been shown'03to be a substrate (3390)of the trehalase obtained from cockchafer.

3. Carba-trehalosamine and Related Compounds Elucidation of the biological properties of carba-sugars may prove of help in understanding the roles that sugars play in biological systems. Aminoglycoside antibiotics composed partly of carba-sugars, instead of true sugars, have been synthesized. Trehalosamine,lM 2-amino-2-deoxy-a-~-glucopyranosyl a-~-glucopyranoside (343), is an antibiotic produced by Streptomyces lavendulae. Four related compounds, the 3-amino-3-deoxy and 4-amino-4-deoxy isomers,lo4 2-amino-2-deoxy-a-~-glucopyranosyl a-D-mannopyranoside (344),Io5and 3-amino-3-deoxy-a-~-~ucopyranosyl 3-amino-3-deoxy-~-~-glucopyranosidelMhave been shown to possess antibacterial activity.

NU2

NH2

343

344

Coupling of 237 with the protected 2-amino-2-deoxy-a-~-glucopyranosyl bromide (348) in dichloromethane in the presence of silver triflate afforded a diastereoisomeric mixture. The isomer related to the natural one was deprotected with sodium in liquid ammonia, to give the carba-trehalosamine (102) K. Bock, A. Bringnole, and B. W. Sigurskjold, J. Chem. Soc., Perkin Trans. 2. (1986) 1711-1713. (103) K. Bock, J. Defaye, H. Driguez, and E. Bar-Guilloux, Eur. J. Biochem., 131 (1983) 595-600. (104) F. Arcamone and F. Bizioli, Gazz. Chim. Ital., 87 (1957) 896-902; L. A. Dolak, T. M. Castle, and A. L. Laborde, J. Anlibiol., 33 (1980) 690-694; H. Naganawa, N. Usui, T. Takita. M. Hamada, K. Maeda, and H. Umezawa, ibid., 27 (1974) 145- 146. (105) M. Uramoto, N. Otake, and H. Yonezawa, J. Antibiot., 20 (1967) 236-237. (106) T. Tsuno, C. Ikeda, K. Numata, K. Tomita, M. Konishi, and H. Kawaguchi, J. Anlibiot.. 39(1986) 1001-1003.

CARBA-SUGARS AND THEIR DERIVATIVES

73

(345),which shows -25% of the antiviral activity of 2-trehalosamine against Klt.hsiella pne~rnoniae.'~'This result showed that, in biologically active oligosaccharides, partial replacement of a true sugar residue with a carbasugar unit does not always result in complete loss of its original activity. Similarly, the mannopyranosyl analog (346) was synthesized1O8by condensation of di-0-isopropylidene-a-DL-carba-mannopyranose (349) with 348. The other isomer (347) was obtained by reaction of 278 with tetra-0benzoyl-a-D-mannopyranosyl bromide (350).The four carba-glucopyranosyl D-mannopyranosides,including 346 and 347, show no antiviral activity.

Nil2

Nil2

346

345

(-&ao1, c112011

110

0

NII,

0

L

347

350

(107) S. Ogawa and Y . Shibata, Curbohydr. Res., 176 (1987) 309-315. (108) S. Ogawa and Y . Shibata, Curbohydr. Res., 170 (1987) 116- 123.

TETSUO SUAMl AND SEllCHRO OGAWA

14

VIII. SYNTHESIS OF BIOLOGICALLY ACTIVECARBA-OLIGOSACCHARIDES 1. Antibiotic Validamycins and Related Compounds

Seven validamycins and three validoxylamines have so far been isolated70Jmfrom the validamycin complex produced by Streptomyces hygroscopiczrs sp. limonew. Validamycins A, C, D, E, and F are D-glycopyranosides possessing validoxylamine A as their common aglycon, whereas validamycins B and G70have validoxylamines B and G, composed of hydroxyvalidamine (204) and valiolamine (205), respectively,instead of valid-

I

R4

1111

R3

R4

R5

I?

H

I1

I1

H

H

H

011

I1

H

H

I1

II

I1

0H

II

H

Validamycin A

H

11

II

H

II

p-Glcp

B

H

II

011

1%

H

p-Glcp

C

rr-Glcp

I1

I1

H

H

p-Glcp

D

I1

11

II

I1

u-Glcp

H

E

II

I1

II

H

H

p-Ma1

F

I1

a-Glcp

I1

H

II

B-Glcp

G

II

H

H

011

I1

p-Glcp

R'

R2

Validoxylamine A

I1

B

H

G

Synthetic studies of validamycins were initially devoted to establishing the structure of validamycin A. The structure in which a P-D-glucopyranosyl group is attached to 0-3 of the validamine residue was initially proposed on the basis of degradative studies. However, the original structure was re-

(109) T. Iwasa, Y. Kameda, M.Asai,S. Horii,andK. Mizuno,J. Anfihiof.,24( 1971) 119- 120: S. Horii, Y. Kameda, and K. Kawahara, ibid.. 25 (1972) 48-50.

CARBA-SUGARS AND THEIR DERIVATIVES

75

vised"oJ1l by the unequivocal synthesis of 3- (352)II2and 4-0-p-~-glucopyranosylvalidamine (355),'I I followed by comparison with the compound derived from validamycin A. The DL-validamine derivative 351,protected with the 4,7-0-isopropylidene and the N,2-0-cyclic carbamate functions, was p-D-glucosylated, to give p-D-glucopyranosylvalidamine (352),which was different from an authentic sample. Therefore, the dibromide 51 was 0-deacetylated, and the trio1 was treated with 2,2-dimethoxypropane in the presence of p-toluenesulfonic acid to give the 1,2-(353)and 2,3-O-isopropylidene derivatives. The bromo group of 353 was successively replaced with a benzoate ion and azide ion, to afford the protected precursor (354)of DL-validamhe. Compound 354 was p-D-glucosylated,and the resulting diastereoisomeric condensates were separated, to give carba-disaccharide 355,which was identical to the product obtained by hydrogenolysis of validamycin A.

352

CII Oil I ?

011

353

354

355

( I 10) S. Ogawa, N. Chida, and T. Suami, Chem. Lett., (1980) 139- 142; T. Suami, S. Ogawa, and N. Chida, J. Antihiot.. 33 (1980) 98-99. ( 1 1 I ) S. Ogawa, Y. Shibata, N. Chida, and T. Suami, Chem. Lett., ( 1980) I35 - 138; Bull. Chern. SOC.Jpn., 56 (1983) 494-498. ( 1 12) S. Ogawa, N. Chida, H. Ito, and T. Suami, Bull. Chem. SOC.Jpn., 56 (1983) 499-504.

76

TETSUO SUAMI AND SEIICHRO OGAWA

Construction of a carba-disaccharide structure bonded by an imino linkage may be a crucial step in the total synthesis of validamycins. The first attempt to fulfil this end involved coupling of a substituted cyclohexylamine with cyclohexenyl bromide (220). Di-0-isopropylidene-DL-validamine (358),readily accessible from 207, was used in this reaction. Coupling of 220 with 358 in DMF proceeded very slowly (room temperature, 20 days), to give a 40% yield of a diastereoisomeric mixture of the condensates which was separable by chromatography. The reaction involves formation of an acetoxonium ion through assistance of the acetoxyl group, followed by a rearside attack of the amine at the allylic position. Thus, the reaction occurs stereospecifically to give,113after deprotection and acetylation, compound (360).

OA,

(IAt

357

356

'

2

358

359

Next, condensation of di-0-isopropylidenevalienamine(359) with cyclohexene epoxide or cyclohexadiene epoxide was carried out in 2-propanol. Racemic validoxylamine B was synthesized as the per-N,O-acetyl derivative (356) by coupling 230 with 359 in DMF-2-propanol for 4 days at 50", ( 1 13) S. Ogawa, T. Toyokuni, and T. Suami, Chem. Leu., (1981) 947-950; T. Toyokuni, S. Ogawa, and T. Suami, Bull. Chem. Suc. Jpn., 56 (1983) 2999-3004.

CARBA-SUGARS AND THEIR DERIVATIVES

77

followed by deprotection and acetylation.114In this case, the epoxide group was cleaved diaxially, to generate a hydroxyl group having the desired configuration, but, in order to apply this reaction for a synthesisof validoxylamine A, conditions for removal of the hydroxyl group by deoxygenation or dehydration had to be envisaged. First, condensation of the epoxide 230 with the amine 358 was conducted, and dehydration of the condensate (361)was attempted by chlorination with sulfonyl or sulfuryl chloride in pyridine, followed by elimination with Cll O A i

C1l2OAc

L

I

(jAC

361

OA1

362

OAC

363

OAC

364

base.'15 The reaction proceeded readily, affording a mixture of products which was separated on a column of silica gel, to give the alkene (362), the chloride (363),and the aziridine (364).Compound 362 was deprotected, and ( 1 14) S. Ogawa, T. Toyokuni, Y.Iwasawa, Y. Abe, and T. Suami, Chern. Letf.,(1982) 279282. ( I 15) S. Ogawa, T. Ogawa, Y. Iwasawa, T. Toyokuni, N. Chida, and T. Suami, J. Org. Chem., 49 (1984) 2594-2599.

78

TETSUO SUAMI AND SEIICHRO OGAWA

the product characterized as the octaacetate (357), which was identical to an authentic sample of the octaacetate of validoxylamine A. On the other hand, the alkene obtained by dehydrochlorination of 363 with DBU differed from 362, indicating that the reaction with sulfuryl chloride proceeds in a diastereoselectivemanner. Mechanistically, the intermediate chlorosulfonyl esters would give rise to the alkene by dechlorosulfonation or the chloride by diaxial cleavage of the aziridine with chloride ion, the reaction course being diastereoselective. l6 Two validamycin A i ~ o m e r s ~were ~ ~ J synthesized ~* by coupling of the protected derivative of 355 and the epoxides 243 and 244. Synthesis of validamycin A has been a c ~ o r n p l i s h e dby ~ ~starting ~ from (+)-validoxylamine A derived from antibiotics. 0-Benzylidenation of it with I .3 mol. equiv. ofa,a-dimethoxytoluene in DMF in the presence of 1.1 mol. equiv. of p-toluenesulfonic acid afforded mainly the monobenzylidene derivative and a trace of the dibenzylidene acetal, which were separable as the peracetates (366 and 365), in 42 and 5% yield respectively. 0-Deacylation of 366, followed by benzylation, gave the hexabenzyl ether, which was treated with aqueous acetic acid to give the diol367. The primary hydroxyl group thereof was protected by selective acetylation, and the resulting validoxylamine A derivative 368 was treated with 2,3,4,6-tetra-0-acetyl-a-~-glucopyranosyl chloride (326b)in dichloromethane in the presence of trimethylsilyl trifluoromethanesulfonate and I , I ,3,3-tetramethylurea to afford a 74% yield of the P-D-glucoside (369). Deprotection of 369 with sodium in liquid ammonia, followed by acetylation, gave the undecaacetate, identical to that of validamycin A. Total synthesis of (+)-validamycins A and B starting from a common synthetic intermediate was elaborated by the following sequence. Tetra-0benzyl-(+)-valienamine (370),derived from 21 1, and the di-0-benzyl derivative (371) of the epoxide were coupled in 2-propanol to produce the protected dicarba compound (374), the structure of which was confirmed by conversion into (+)-validoxylamine B nonaacetate.'l9 Concurrently, compound 372 was glycosylated and the product oxidized with a peroxy acid, to afford a mixture of products from which the desired epoxide (373) was obtained in 70% yield. Coupling of 370 with 373 in 2-propanol at 120" afforded two carba-trisaccharides, and the major product (47%) was deprotected and characterized as the dodecaacetate of validamycin B.'19The pro15s1

( I 16) s. Ogawa, T. Ogawa, N. Chida, T. Toyokuni, and T. Suarni. Cliem. Lett., (1982) 749752. ( I 17) S. Ogawa, M. Inoue, Y. Iwasawa. T. Toyokuni, and T. Suarni, Chm. Lett., (1983) 1085- 1088. ( I 18) S. Ogawa and J. Urneda, unpublished results. ( I 19) S. Ogawa and Y. Miyarnoto, J. C%Pm.Sir..Cliem. Cornmiin.,(1987) 1843- 1844.

CARBA-SUGARS AND THEIR DERIVATIVES

366

19

OAC

Ol3lI

367 368

R1

=

R1

= H,

369

K1

=

Rz

=

I1

Rz = Ac p-(;lc , R z

= Ac

tected derivative of validoxylamine B was totally benzylated, and then the benzylidene group was removed. The OH-7 group was protected, and the resulting 4-hydroxy compound was D-glucosylated, followed by deprotection, to give validamycin B dode~aacetate."~ Removal of the hydroxyl group by way of the (methy1thio)thiocarbonyl derivative 375 failed. Compound 374 was transformed into the aziridine 377 with sulfuryl diimidazole in DMF in 89% yield; treatment of377 witha-toluenethiol afforded the sulfone (376) exclusively,in 9 1% yield, by diaxial attack of the reagent. Desulfurization was successfully achieved with inactivated Raney nickel, giving the same protected validoxylamine A as was previously obtained"' in the synthesis of validamycin A, thus constituting its total synthesis.120J21

(120) S. Ogawaand Y. Miyamoto. Chem. Leu., (1988) 889-89O;J. Chem. SOC.,Perkin Trans. I , (1988) 2675-2680. (121) Y. MiyamotoandS. Ogawa,J. Chem. Soc., Perkin Trans. I , (1989) 1013-1018.

80

TETSUO SUAMI AND SEIICHRO OGAWA

OBI,

370

OBI,

371

Q

110

OUn

372

I

O h

374

K

375

R = CSMc

=

011 II

S

377

OUll

CARBA-SUGARS AND THEIR DERIVATIVES

81

Reaction of 368 with hepta-0-acetyl-a-maltosyl, -cellobiosyl, and -lactosyl bromide in 1,2-dichIoroethanein the presence of silver triflate gave the respective carba-tetrasaccharides, including12' validamycin D. Glycosylation of the 7-hydroxy compound, prepared from 367 by tritylation, benzylation, and detritylation with acid, afforded the protected validamycin D in good yield.'22

2. Carba-oligosaccharidic Alpha Amylase Inhibitors and Related Compounds Several mono-carba-oligosaccharidic alpha amylase inhibitor^,^^ such as acarboseI6and its homologs, amylostatins,'* trestatins, oligostatins,2'adiposins, and so on, have been isolated from cultures of micro-organisms, and considerable interest in the biochemistry and chemistry of this class of inhibitors has been stimulated. The characteristic core-structure for inhibitory action is composed of a trihydroxy(hydroxymethy1)cyclohexenemoiety and a 4-amino-4,6-dideoxy-~-glucopyranose moiety, bonded by way of an imino linkage at the allylic position. A similar structural unit has been found in the antibiotic validamycins.

(&Q [oqoQ

I I ,OH 110

OH

011 Acarbose: Amyloslatin XG: Adiposin 2 : Adiposin 1 :

011

I1

OH

R = H, n = 1 R = H, n = 0 R = OH, n = 1 R = OH, n = 0

H,OH

01I

OH

OH

Oligostatin

(122) Y. Miyamoto and S. Ogawa, J. Chem. SOC.. Perkin Trans. 1, in preparation.

TETSUO SUAMI A N D SEIICHRO OGAWA

82

In a total synthesisofinhibitors ofthis kind, the following methods have so far been employed successfully:(a)reaction of a cyclohexenyl halide with an amine, (6) coupling ofan amine with an epoxide, and (c)condensation ofan amine with ketone and reduction of the resulting Schiff base (reductive alkylation of an amino sugar). The first synthesis, by method a, of amylostatin (XG) was reported by Kuzuhara and S a k a i ~ i .The ' ~ ~ synthon for the cyclohexene moiety was the benzylated ally1 bromide 382, derived from D-glucose by the sequence 378 + 382 of the Femer reaction. The coupling reaction of 382 using an excess of 4-amino- I ',6'-anhydro-4,6-dideoxymaltose tetrabenzyl ether (383), and sodium iodide in DMF for 3 days produced a mixture of the epimeric monocarba-trisaccharide derivatives, separation of which gave the protected derivatives in 15% yield.

OUIl

01!11

378

379

OHn

011:1

380

381

An alternative synthesis,124by method 6, was conducted by coupling 357 and 1,6-anhydro-4-0-(3,4-anhydro-6-deoxy-a-~-galactopyranosyl)-aD-glucopyranose (392a) in 2-propanol at 120"; this yielded a diastereoisomeric mixture, from which, after the usual treatment, amylostatin (XG) was isolated in 20% yield.125 Similarly, anhydro derivatives of monosaccharides and disaccharides readily react with 359 to give the respective carba-di- and -tri-saccharide. (123) N. Sakairi and H. Kuzuhara, Terruhedron Leu., (1982) 5327-5330. (124) S. Ogawa, H. Sugizalu, Y. Iwasawa. and T. Suami, Curbohvdr. Res., 140 (1985) 325331. (125) S. Ogawa, Y. Iwasawa, T. Toyokuni, and T. Suami, Cheni. Lett., (1983) 337-340.

CARBA-SUGARS AND THEIR DERIVATIVES

83

Thus, coupling of the epoxide 384 with 359 afforded126the core structure (386) of adiposins and the positional isomer (387). Adiposin- 1 was synthesized125p126 by use of 1,6-anhydr0-4-0-(3,4-anhydro-a-~-galactopyranosyl)P-D-glucopyranose (391a),prepared by the sequence 388a -,391a. In this method, removal ofthe 1,6-anhydro and the acetal rings is facile, even under mild conditions of acetolysis, giving, after deacylation, the free bases quantitatively. C,HZOH

OH

011

385a X = O H 385b X = H

384

(Ill

011

386

387

389a, b R = Bz 390a, b R = Ms

391a, b X = OH 392a, b X = H

(126) S. 0gawa.Y. Iwasawa,T.Toyokuni,andT.Suami, Curbohydr. Res.. 141 (1985)29-40.

TETSUO SUAMl AND SEllCHRO OGAWA

84

Likewise, complete synthesis of acarbose was conveniently accomplished by use of the anhydro derivative 392b prepared from maltotriose. Condensation of 392b with optically active 359 afforded two products,127one of which was, after deprotection, shown to be identical to an authentic sample of acarbose. When di-0-isopropylidenevalidamine (358) was used as the amine synthon, the saturated analogs of the inhibitors (393a and 394a) were obtained. 1 2 5 ~ 1 2 6 Modification of the cyclohexenyl moiety has been carried out by use of the cyclohexadiene epoxides 243 and 244, which were coupled with methyl 4-amino-4-deoxy- and -4,6-dideoxy-a-~-glucopyranoside (385a and 385b) to give *28 the isomers (395 and 396). b

~

~

(

~

o

o

o

H

110

011

OH

OIi

n

01I

393a,b X = H 394a,b X = O H

a n=O b n=l C ti K

QQoM I 0

CllZOll

011

dll

395 HO

OH

396

(127) S. Ogawa and Y. Shibata, J. Chem. Soc., Chem. Commun.. (1988) 605-606. (128) S. Ogawa, K. Yasuda, T. Takagaki, Y.Iwasawa, and T. Suami, Curbohydr. Rex. 141 (1985) 329-334.

CARBA-SUGARS AND THEIR DERIVATIVES

85

The core structure (397)of oligostatin, which possesses a tetrahydroxy(hydroxymethy1)cyclohexylmoiety, was readily accessible by coupling of the cyclohexene epoxide with 385b. Thus, 230 reacted with 385b, to give mainly the protected compound 397 by diaxial opening of the e p ~ x i d e ,along ' ~ ~ with 398 as the minor product. Alternatively, reaction of the protected hydroxyvalidamine 399 with 1,6 : 3,4-dianhydro-2-0-benzyl-~-~-galactose (400) afforded the monocarba-disaccharide (401) which was converted into the 4-0-methyl derivative'30(402).

(Ill

397

398

In order to develop potent D-glucosidase inhibitors, a synthesis of carbadisaccharides containing valiolamine (205)was attempted by Horii and his coworkers7*utilizing method c. Coupling 205 with the 4-ketose 403, using NaBH3CNand hydrochloric acid, was effective in DMF, giving, after deprotection, the epimeric carba-disaccharides (404 and 405). The saturated ( 129) S. Ogawa, Y. Iwasawa, T. Toyokuni, and T. Suami, Chem. Left.,( 1 982) I72 1 - 1732;

Carbohydr. Res.. 144 (1985) 155-162. ( I 30) H. Paulsen and W. Roben, Justus Liebigs Ann. Chem., (1985) 974-994.

86

TETSUO SUAMl AND SEIICHRO OGAWA

derivative (393b) of acarbose was prepared by a similar pr~cedure.'~' On the other hand, reaction of 205 with the carba-ketose 408 (derived from 407 by way of 406) gave the dicarba-disaccharides 409 and 410, of type A, having an imino bond.

408

011

OII

409 X = O H 410 X = NH,

Ix. BIOLOGICAL EFFECTS OF CARBA-SUGARS Besides sweetness, a carba-sugar may have biological activity, owing to its structurally close resemblance to a true sugar. As mentioned earlier, a-D-carba-galactopyranose (1)has been found in a fermentation broth of Streptomyces sp. MA-4145, as an antibiotic. The potency of the antibiotic was rather low. A concentration of 125 pg/mL is required in order to produce a standard inhibition zone of 25-mm diameter against Klebsiellu pneumoniue MB- 1264, using I 3-mm assay discs in a discplate assay. A sample of the synthetic a-~~-carba-galactopyranose~ (17) was

-

(131) H. Kuzuhara, M. Hayashilda, and N. Sakairi, Int. Symp. Chem. Nat. Prod., 15th, (IUPAC), The Hague, The Netherlands, Aug. 1986.

CARBA-SUGARS AND THEIR DERIVATIVES

87

about half as potent as the natural product (1) in the same assay system, indicating that the L enantiomer is inactive. An inhibition of D-glucose-stimulated release of insulin has been studied by using a-DL-carba-glucopyranose(54) as a glucokinase inhibitor. That is, 54 and P-DL-carba-glucopyranose (37) were used as synthetic analogs of D-glucose anomers in order to study the mechanism of D-glucose-stimulated release of insulin by the pancreatic islets. It was found that the carba-sugar was neither phosphorylated by liver glucokinase, nor stimulated release of insulin from the islets. Incubation of islets with 54 resulted in an accumulation of the carba-sugar, probably the D enantiomer, in the islets. Compound 54 inhibited both D-glucose-stimulated release of insulin (44% inhibition at 20 mM) and islet glucokinase activity (36% inhibition at 20 mM), but 37 did not show any activity.13* These results strongly suggested that the inhibition of D-glucose-stimulated release of insulin by 54 is due to the inhibition of islet glucokinase by the carba-sugar, providing additional evidence for the essential role of islet glucokinase in relation to D-glucose-stimulated insulin.52 The biological activity of simple amino carba-sugars and complex substances containing them has been investigated, stimulated by the unusual properties of antibiotic validamycins and carba-oligosaccharidic D-glucosidase inhibitors. The activity of validamycins against Pellicularia sasakii (sheath-blight disease of the rice plant) was thoroughly examined133;the relative activities are shown in Table 111. A considerable difference in activity would be due to their fine structures; however, a structure-activity relationship of this kind of carba-trisaccharide has not been fully established. TABLE Ill Activity of Validarnycins and Validoxylamines against Pellicularia sasakii Compound Validamycin A B C D E F Validoxylarnine A B

MIC @g/rnL)

0.0 I 0.5 10 100 0.013

0.013 10

>I00

( 1 32) 1. Miwa, H. Hara, J. Okuda, T. Suami, and S. Ogawa, Biochem. Int., 1 1 (1985) 809-8 16.

(133) S. Horii, Y. Kameda, and K. Kawahara, J. Antibiot., 25 (1972) 48-54.

88

TETSUO SUAMI AND SEIICHRO OGAWA

TABLEIV Inhibitory Activity ( M ) of Validamycin and Validoxylamine against Trehalase Compound Validamycin A B C D E

F G Validoxylamine A

B G

Trehalase inhibitory activity IC, (M)

Dendroid test method MIC W m L )

7.2 x 10-5 3.5 x 10-5

0.0 1 0.50

1.5

x 10-5

5.2 X lod 1.4 x 10-7 1.6 x 10-5 7.4 x 10-6

10

25 0.0 I 0.0 1 0.50 I .oo 50 2.50

Concerning the mechanism of inhibition of sucrase by acarbose, it was assumed that the unsaturated cyclitol unit (the valienamine part) of acarbose most likely interacts with the D-glucopyranosyl-binding center of sucrase, the axially attached nitrogen atom taking the place of the glycosyidic oxygen atom in the substrate molecule.69This consideration suggested that carbaoligosaccharides of type A bonded by an imino linkage, might be strong inhibitors of the hydrolasesof the corresponding normal oligosaccharides.In fact, the carba-disaccharide validoxylamine A and its derivatives show 134 strong inhibitory activity against the trehalase obtained from Rhizoctiu soluni (see Table IV). Because naturally occurring carba-glycosylaminesare readily obtained by degradation of certain antibiotics, or, more effectively, by isolation from the fermentation broth, systematic biological assays were carried o ~ t . Al~ ~ J ~ ~ though the compounds had no activity against sheath-blight disease, they were found to possess inhibitory activity against some enzymes, and valiolamine was found to be considerably more active than the others (see Table V). Therefore, extensive studies on chemical modification of valiolamine led to the discovery7*that its N-[2-hydroxy- 1-(hydroxymethyl)ethyl] derivative possesses strong a-D-glucosidase inhibitory activity against porcine intestinal maltase and sucrase. Carba-trehalosamineIMwas found to show 25% of the activity of 2-trehalosamine, which showed that, in biologically active oligosaccharides, re(134) N. Asano, T. Yamaguchi, Y. Kameda, and K. Matsui, J. Antibiot.. 40 (1987) 526-532. (135) Y. Kameda, N. Asano, T. Yamaguchi, and K. Matsui, J. Antibiot.. 40 (1987) 563-568.

CARBA-SUGARS AND THEIR DERIVATIVES

89

TABLE V Inhibitory Activity of Some Carba-glycosylamines against Three Enzymes IC, Value ( M ) against Enzyme Enzyme Compound

Sucrase

Validamine Deoxyvalidamine H ydroxyvalidamine Valienamine Valiolamine Epivaliolamine Validamycin A Validamycin G Validoxylamine A Validoxylamine G

1.5 X 2.8 x 10-4 4.2 - 10-4 5.3 x 10-5 4.9 x 10-8 5.0 x 10-5 >i.o x 10-3 1.1

x

10-4

> L O x 10-3 8.8 x

Maltase

lsomaltase

x 10-4 x 10-3 8.3 x 10-3 3.4 x 10-4 2.2 x 104 2.7 X lo4 1.0 x 10-3 >LO x 10-3 >LO x 10-3 >LO x 10-3 >LO x 10-3 , >i.o x 10-3 >i.o x 10-3 1.0 x lo-' 1.7 x 10-3 1.1 1.0

placement of the pyranoid-ring oxygen atom of one sugar residue with a methylene group may not result in complete loss of activity. Among the eight possible diastereoisomers of carba-trehalose of type C, only that isomer having the same configuration as the natural trehalose was found to be a substrate of certain maltases or treha1a~es.I~~

X. CONCLUSION All sixteen of the racemic carba-sugars predicted are known, as well as fifteen of the enantiomers. The most accessible starting-material for the synthesis of racemic carba-sugars is the Diels-Alder adduct of furan and acrylic acid, namely, endo-7-oxabicyclo[2.2.1]hept-5-ene-2-carboxylic acid (29). Furthermore, adduct 29 is readily resolved into the antipodes, (-)-29 and (+)-29, by use of optically active a-methylbenzylamine as the resolution agent. The antipodes were used for the synthesis of enantiomeric carbasugars by reactions analogous to those adopted in the preparation of the racemates. Among the known enantiomers of carba-sugars, thirteen have been synthesized by a chiral synthesis starting from a true sugar; the four modifica( I 36) K. Bock, J. F. B. Guzman, J. 0. Duus, S. Ogawa, and S. Yokoi, Curbohydr. Res., (1990) in press.

90

TETSUO SUAMl AND SEIICHRO OCiAWA

tions were prepared by the resolution of starting materials, and the two enantiomers were made from a naturally occumng cyclitol, quebrachitol. The seventeen unknown enantiomeric carba-sugars remaining will have to be obtained by one of the aforementioned methods in order to elaborate a complete set of the thirty-two enantiomers. Among other carba-sugar derivatives, the most important compounds are amino carba-sugars having an amino group at C- 1, They are known as validamine, valiolamine, hydroxyvalidamine, and valienamine, and are found in validamycin antibiotics as unique components; they have been synthesized in DL forms and also in optically active forms. Amino carba-sugars having an amino group other than at C- 1 have never been discovered in Nature, but the eighteen compounds have been prepared in DL forms, and four enantiomers were synthesized during the course of the studies. Such carba-disaccharidesas validoxylamines A and B were synthesized as intermediary compounds in the total synthesis of validamycin antibiotics. Carba-disaccharides related to maltose, isomaltose, cellobiose, the trehaloses, and trehalosamine modifications have also been synthesized. The total synthesisof the carba-trisaccharidic antibiotics known as validamycins A, B, and D was accomplished by using a carba-sugar as a key building-block, and carba-oligosaccharideshaving alpha amylase inhibitory activity, such as acarbose, adiposin, amylostatin, and oligostatin, have been synthesized. The chemistry of carba-sugars is a newly opened area ofchemistry, and the biological effects of these compounds, have not been well studied, except for (a) the equisweetness of D-carba-glucose, D-carba-galactose, and D-carbafructose with the respective true sugars, (b) the antibiotic activity of (Y-Dcarba-galactose,and (c) inhibition of a D-glucose-stimulated insulin release by D-carba-glucose. The increasing number of unusual carba-sugars and their presumably forthcoming derivatives will make the chemistry of carba-sugars an important new field of chemistry.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL. 48

CHEMISTRY AND DEVELOPMENTS OF FLUORINATED CARBOHYDRATES BY TSUTOMU TSUCHIYA Itisrititrc oJ'Bioorganic Chctnistrv, 1614, Ida, Nakahara-ku, Kawasaki, 21 1. Japan 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Basic Synthesis of Fluorin I . Glycosyl Fluorides and

2. Displacement of Sulfon

91 94 94 121 142 155 169 181 186 186 205 223 234

Fluorine is a female and diflerentfrom the other halogens I. INTRODUCTION Fluorine, a light element having the electronic structure lS'292P5, is located at the end of the first row of the Periodic Table. Fluorine is the most electronegativeelement known. The electronegativity,' x, of fluorine is 3.90, a value significantly higher than those for all other elements [0,3.50; C1, 3.15; N, 3.05; Br, 2.85; I, 2.65; C, 2.60; and H, 2.20; x i s a measure of the relative attracting power for the valence electrons of a covalent bond, and is proportional to the effectivenuclear charge and inversely proportional to the

( I ) J . A. Dean(Ed.), Lange's IIandbookofChemistry, 13thedn., McGraw-Hill BookCo.,New York, 1985.

91

Copyright 0 1990 by Academic Press,Inc. All rights of reproduction in any form rfferved.

92

TSUTOMU TSUCHIYA

covalent radius r, the effective distance from the center of a nucleus to the outer valence shell ofthat atom in a covalent bond]. The electronegativity of fluorine as alternatively expressed by a, (Ref. 2) is 0.52 (for OH, 0.43; NO,, 0.40; NH,, 0.33; C1, 0.28; CH,, 0.17; and H, 0.00). The atomic size of fluorine is slightly smaller than that of oxygen, as indicated by r 64 pm (0, 66 pm for the a bond) and the van der Waals radius ( J ) 135 pm (0,140 pm) the latter radius being the internuclear distance of closest approach of an atom to another, without bonding; the values of r and J for some other important elements are 37 ( r )and 120 pm (J) for H, N: 70, 154; C, 77,185; C1,99,181;Br, l14,195;andI, 133and215.Thecarbon-fluorinebond-energy” is extremely high (485 kJ/mol, calculated from the bond energy of CF,) in comparison to other bond energies (C-C1, 330; C-Br, 275; C-I, 220; C-H, 410; C-C, 350; and C-0,370 kJ/mol; all are the mean values obtained from many C - X compounds). The high C - F bond energy may be attributed to the high overlap of a hybridized s$ orbital of the carbon atom and the p orbital of the fluorine atom. The lower bond (C- X) energies of the other halogens (X = C1, Br, and I) may be attributed to the decreased overlap of carbon and halogen atoms, which, in turn, is a reflection of the broad p orbitals of the X atoms (C1 has the electronic structure of 1$2s22p63$3$). The characteristic features of fluorine, just described, namely, the high electron density on the fluorine atom polarizing the C-F bond, the relatively small atomic size, and the strong C-F bond-energy, with its lack of active hydrogen, make fluorine a unique element not directly comparable with the other halogens, and this influences the chemical reactions involving fluorine, and the characteristics of fluorine-containing sugars. As a reflection of these properties, fluoride ion is a hard base3bwith low nucleophilicity, and displacement reactions using fluoride ion do not proceed smoothly, even when similar reactions for other halide ions proceed readily (see Section 142). Another aspect important from the biological viewpoint is hydrogen bonding between4 C - F. - - * HO (or HN). However, evidence for hydrogen bonding is not necessarily clear, although it is very often anticipated (see Section 111,2). In this article, a treatment of n.m.r. studies on fluoro sugars is omitted, because fundamental studies in this field had already been de~cribed,~ and (2) S. Maniott, W. F. Reynolds, R. W. Tafi, and R. D. Topsorn, J. Org. Chem., 49 (1984) 959 -965; related references are cited therein. (3) J . March, Advanced Orgunic Chrmislry, 3rd edn., Wiley, New York, 1985, (a) p. 23. (b) pp. 21 8-236. (4) P. Murray-Rust, W. C. Stallings, C. T. Monti, R. K. Preston, and J. P. Glusker, J. Am. Chem. SOC.,105 (1983) 3206-3214.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

93

several excellent reviews are at hand.5-8 Therefore, n.m.r. data are only added when necessary in the description of compounds. Here we only show some fundamental studies on vicinal 19F-H (Refs. 9 and 10)and 19F-13C couplings," and on the conformation of fluorinated cyclohexanone~,~~J~ and and 1,2-di-substituted (including F and other halogens) c y c l o h e ~ a n e s . ~In~ - an ~ ~ n.rn.r. study on 3,4,6-tri-O-acety1-2-deoxy-2fluoro-p-D-glucopyranosyl fluoride, the I9F- 'H splittings were obtainedI9 separately from the homonuclear 'H - IH couplings by applying a skew-projection method to the 2D J-resolved spectrum. Excellent reviews on fluorinated sugars (and related fields) have been p u b l i ~ h e d . ~In~every - ~ ~ Section in the present article, work reported in 1978- I988 is emphasized, although important earlier studies are also included, and this article is fundamentally a succession to that by Penglis5in this Series.

(5) A. A. E. Penglis, Fluorinated Carbohydrates, Adv. Carbohydr.Chem. Biuchern.,38 (I98 1) I95 -285. (6) J. W. Emsley, L. Phillips, and V. Wray, Fluorine Coupling Constants, Prog. NMR Spec[rosc., 10 (1976) 83-756. (7) J. T. Gerig, B i d Magn. Reson., l(1978) 139-203. (8) R. Csuk and B. I. Glanzer, N.M.R. Spectroscopy of Fluorinated Monosaccharides, Adv. C'arbohj~dr.C'hem. Biuchern., 46 ( I 988) 73- 177. (9) K. L. Williamson, Y.-F. Li Hsu, F. H. Hall, S. Swager, and M. S. Coulter, J . Am. Chem. SOC.,90 (1968) 6717-6722. ( 10) T. C. Wong, V. Rutar, J.-S. Wang, M. S. Feather, and P. KovaE, J. Org. Chem., 49 (1984) 4358-4363. ( I I ) V. Wray, J. Am. Chem. Suc.. 103 (1981) 2503-2507. (12) J. Cantacuzene, R. Jantzen, and D. Ricard, Tetrahedron. 28 (1972) 717-734. (13) R. J. Abraham and L. Griffiths, Tetrahedron, 37 (1981) 575-583. (14) 0. A. Subbotin and N. M. Sergeyer, J. Am. Chem. Suc., 97 (1975) 1080- 1084. ( I 5) N. S. Zefirov, L. G. Gurvich, A. S. Shashkov, M. Z. Krimer, and E. A. Vorobeva, Tetrahedron, 32(1976) 1211-1219. (16) N. S. Zefirov, Tcfrahedron.33 (1977) 3193-3202. ( 17) N. S. Zefirov, V. V. Samoshin, 0.A. Subbotin, V. 1. Baranenkov, and S. Wolfe, Terrahedron. 34 (1978) 2953-2959. (18) L. Dosen-Micovic and N. L. Allinger, Telrahedrun,34 (1978) 3385-3393. (19) L. D. Hall and S. Sukumar, J. Am. Chem. Suc., 101 (1979) 3120-3121. (20) P. J. Card. J . Carbohydr. Chem., 4 (1985) 451 -487. (21) R. E. Banks and J. C. Tatlow, J. Niturine Chem., 33 (1986) 227-346. (22) J. Mann. Chem. Suc. Rev., 16 (1987) 381 -436. 43 (1987) 3123-3197. (23) J. T. Welch. 7i~[rahedron. (24) N. F. Taylor (Ed.), Fluorinated Carbohydrates.Chemical and Biochemical Aspects, ACS S p p . Ser. 374. Am. Chem. SOC.,Washington, D.C., 1988.

TSUTOMU TSUCHIYA

94

11. BASICSYNTHESIS OF FLUORINATED CARBOHYDRATES 1. Clycosyl Fluorides and Glycoside Formation

Because of the high C-F bond energy, glycosyl fluorides are stable25in comparison to the other glycosyl halides, and this character has attracted much attention. They have been prepared in many different ways. One of them, rather classical, is through addition of the elements of HF (for example, HF in benzene26),BrF, or IF to per-0-acylated glycals.*’ Glycosyl fluorides may be prepared by displacement of per-0-acyl or suitably protected 2-0-acyl I-halides (C1 or Br) with fluoride [AgF (Refs. 28 and 29), KHF, (Ref. 30) or AgBF, (Ref. 3 l)]. /?-D-Glucopyranosyl[18F]fl~o-

1 R=OAc 2 R=A

3 R=OAc 4 R=A

7

5 R=OAc 6 R=A

A= 1@-ditxnzoy1acicnin-9-y1

NH2

I

F. Micheel and A. Klemer, Adv. Carbohydr. Chem., 16 (1961) 85- 103. For example, see K. Bock and C. Pedersen, Acta Chem. Scarid., 25 (1971) 2757-2764. L. D. Hall and J . F. Manville, Can. J. Chcm., 47 (1969) 361 -377. For example, see L. D. Hall, J . F. Manville, and N. S. Bhacca, Can. J . Chem.. 47 (1969) 1-17. (29) F. Kong, J. Du, and H. Shang, Curbohydr. Res., 162 (1987) 217-225. (30) Ger. Pat. 3626028 Al(1987); Chem. Absrr., 107 (1987) 176,407e (31) K. Igarashi, T. Honma, and J . Irisawa, Carbohydr. Res., 11 (1969) 577-578; 13 (1970) 49-55. (25) (26) (27) (28)

DEVELOPMENTS O F FLUORINATED CARBOHYDRATES

95

ride,3z capable of being used as a tumor-localizing radiopharmaceutical compound, was prepared from tetra-0-acetyl-a-D-glucopyranosyl bromide by the AgF method,z8using Ag18F (for 15 min); also, l-O-acetyl-2,3,5-triO-benzoyl-4-fluoro-a-~-lyxofuranose (5), a mixture of 5 and 1 -0-acetyl2,3,5-tri-O-benzoyl-4-fluoro-~-~-ribofuranose (7), and 1,N6-dibenzoyl-9(2,3,5-tri-0-benzoyl-4-fluoro-a-~-lyxofuranosy1)adenine(6; a compound structurally related to n u ~ l e o c i d i nlo), ~ ~each of them being a glycosyl fluoride in nature, were prepared), from the 4-bromo-~-ribofuranosylderivative 3 and the adenosin-4’-yl bromide (4)by treatment with AgF, AgBF,, and AgF, respectively. Compounds 3 (and its 4-epimer) and 4 were prepared from the 0-benzoyl-P-D-ribofuranosyl compounds, 1 and 2, respectively, by photobromination. Similarly prepared were 1-0-acetyl-2,3,5,6-tetra~-benzoyl-4-fluoro-~-~-glucofuranose (8) and 1-0-acetyl-2,3,5,6-tetraO-benzoyl-4-fluoro-~-~-galactofuranose (9). Voznij and coworkers pre~ a r e d ~ ~per-0-acetyl-p-D-xylopyranosyl -~’ (llp), -a-L-arabinopyranosyl (1 2), -p-D-glucopyranosyl(13p),-a-D-mannopyranosyl(14a), -P-D-galactopyranosyl (15p),-a-L-rhamnopyranosyl (16), -P-cellobiosyl, -P-cellotriosyl, and -p-cellotetraosyl fluorides, and methyl (2,3,4-tri-0-acetyl-P-~-glucopyranosyl fluoride)uronate (17) in good yields, respectively, from the corresponding bromides by treatment with 2,4,6-trimethylpyridiniumfluoride in the presence of HgBr, in nitromethane. Glycosyl fluorides have also been preparedz5by treatment of per-0-acyl or partially 0-acylated sugars with hydrogen fluoride [liquid H F (for example, see Refs. 38 and 39) or HF in acetic acidm or dichloromethane], as exemplified by 2,3,4-tri-0-benzyl-a-~-xylopyranosyl(18), a-D-glucopyranosyl(19), tetra-O-pivaloyI-a-D-glucopyranosyl(20), and 2,3,5-tri-O-acetyl-~-xylofuranosyl fluorides (21) (see Table I). Frequently, HF treatmentsp4l leads to I ,2-acyloxonium ions and unexpected 1-fluorides. By the HF procedure, 2,3-di-0-benzoyl-4,6-di-O-methyl-aand -p-D-glucopyranosyl fluorides39 (32) A. E. Liemire and M. F. Reed, J. Label. Comp. Radiophurm., 15 (1978) 105- 109. (33) 1. D. Jenkins, J. P. H. Verheyden, and J. G. Moffatt, J. Am. Chem. Soc.. 93 (1971) 4323-4324. (34) R. J. Femer and S. R. Haines, J. Chem. Soc., Perkin Trans. 1, (1984) 1675- 1681. (35) Ya. V. Voznij, I. S. Kalicheva and A. A. Galoyan, Bioorg. Khim., 7 (1981) 406-409. (36) Ya. V. Voznij, I. S. Kalicheva, and A. A. Galoyan, Bioorg. Khim.,13 (1987) 1655- 1658. (37) Ya. V. Voznij, I. S. Kalicheva, and A. A. Galoyan, Bioorg. Khim., 13 (1987) 1659- 1664. (38) E. M. Bessel, A. B. Foster, J. H. Westwood, L. D. Hall, and R. N. Johnson, Curbohydr. Res., 19 (1971) 39-48. (39) (a) K. Bock and C. Pedersen, Acta Chem. Scand.. Ser. B, 30 (1976) 727-732; (b) C. Pedersen and S. Refn, ibid., 32 (1978) 687-689. (40) P. W. Kent and S. D. Dimitrijevich, J. Fluorine Chem., 10 (1977) 455-478. (41) H. Paulsen, Adv. Carbohjidr. Chem. Biochem., 26 (1975) 127- 195.

96

TSUTOMU TSUCHIYA

(major; they underwent rearrangement to the corresponding D-mannopyranosyl fluorides on longer treatment), 2,3-di-U-benzoyl-4,6-di-O-methyla-D-mannopyranosyl fluoride,393-deoxy-3-fluoro-a-~-~ucopyranosyl fluoride4* (see Section 11,3, Table 11) and hepta-U-acetyl-a-maltosyl f l ~ o r i d e(obtained ~ ~ , ~ quantitativelp) were prepared. polysaccharide~~~ to Hydrogen fluoride has also been used45to oligo- and mono-saccharides, as well as to release the sugar portion from glycopr~teins.~~~ The strongly acidic character of HF renders polysaccharides soluble in liquid HF through hydrogen bonding between the saccharides and HF, with cleavage of the intermolecular hydrogen bonds in the polysaccharide matrix. Following dissolution, some (or all) of the glycoside bonds are randomly or differentially cleaved, to give partially degraded glycosyl fluorides (in dilute solution, the monomeric fluorides): the latter are often converted by loss of the 1-fluorine atom into the corresponding oxocarbonium ions, and subsequently react with existing chemical species as well as such added substances as D-glUCitOl,45to give dimers, reversion oligosaccharides, and other coupled products. In these reactions, the concentration of the substrates in liquid HF, the temperature, and the water content are the most important factors45determining the nature of the final products. Sometimes the same end product@)are obtained starting either from a polysaccharide or its monosaccharide component, suggesting that most of the reaction intermediates are in equilibrium, thus finally giving the thermodynamically most-stable product(s). Because the degree of polymerization of the reversed products sometimes depends on the final concentration, isolation of the products present in the final solution should be carried out carefully, not by evaporation of the HF, but by addition of ether to precipitate the products instantly, or by rapid neutralization with CaCO,. For product analysis, ',C-n.m.r. spectroscopy has proved especially useful. The low-boiling hydrogen fluoride (b.p. 19.5") is, however, very hazardous, and should be handled carefully in special apparatus.49 (42) G . H. Klemm, R. J. Kaufman, and R. S. Sidhu, Tetrahedron Lett., 23 (1982) 2927 -2930. (43) D. S. Genghof, C. F. Brewer, and E. J. Hehre, Curbohydr. Res., 61 (1978) 291 -299. (44) K. Bock and H. Pedersen, Actu Chem. Scund.. Ser. B, 42 (1988) 75-85. (45) R. Franz, W. Fritsche-Lang, H.-M. Deger, R. Erckel, and M. Schlingmann, J.Appl. Polym. Sci.. 33 (1 987) 129 1 - 1306; a brief historical survey of the use of HF is given. (46) A. J. Mort and W. D. Bauer, J . B i d . Chem.. 257 (1982) 1870- 1875. (47) Yu. A. Knirel, E. V. Vinogradov, A. S. Shashkov, B. A. Dmitriev, and N. K. Kochetkov, Curbohydr. Res., 112 (1983) c4-c6. (48) Yu. A. Knirel, E. V. Vinogradov, and A. J. Mort, Adv. Curbohydr. Chem. Biochem., 47 (1989) 167-202. (48a) A. J. Mort and D. T. A. Lamport, Anal. Biochem.. 82 (1977) 289-309. (49) A. J . Mort, Curbohydr. Res.. 122 (1983) 315-321.

DEVELOPMENTS OF EUORINATED CARBOHYDRATES

97

Treatmentsoofcellulose with liquid HF for a short (20%solution, - lo", 5 min) or a long period (- 5 ",40 min) mainly gave partially degraded, waterinsoluble and water-soluble oligomers, respectively. At higher temperatures (- lo%,20", 45 min), a-D-glucopyranosyl fluoride (22) was the main product (neutralization with CaCO, gave5' 1,6-anhydro-~-~-glucopyranose in addition to 22); concentration of this solution gave a complex mixture consisting mainly of highly branched a-linked oligomers. Amylose also readily (- O", a few min) gave5' partially degraded a-(1 +4)-~-glucopyranosyl oligomers; longer reaction (- 10-20", 1 h) gave the same a-linked oligomers as those obtained from cellulose. In the case of D - ~ ~ u c othes use ~,~~~~~ of a large volume of liquid HF led to 22, and concentration of it gave the same a-linked oligomeric mixture (there is some controversy on this point45) as obtained from cellulose or amylose.

Pol y saccharide

Oligomer

I

- g) CHPH

Reversion products

HO

OH

22

Treatments3of inulin or D-fructose with liquid HF (neat or diluted with liquid SO,) gave a mixture of six di-D-fructose dianhydrides, including 23, 25, 27, and P-D-fructofuranosyl P-D-fructopyranose 2,l' :3,2'-dianhydride

(SO) J. Defaye, A. Gadelle, and C. Pedersen. Curbohydr. Rex, 110 (1982) 217-227. (51) H. Hardt and D. T. A. Lamport, Phytochemisfry, 21 (1982) 2301 -2303. (52) Ger. Pat. 3,432,565 Al (1986); Cliern.Abstr., 106 (1986) 53681. (53) J. Defaye, A. Gadelle, and C. Pedersen, Curbohydr. Rex. 136 (1985) 53-65.

TSUTOMU TSUCHIYA

98

(29). This reaction was explained on the basis ofthe formation 0fp-D-fructofuranosyl fluoride (31), followed by the oxocarbonium ion 33. Similar treatrnenP4 of L-sorbose also gave, similarly, six L-sorbose anhydrides, involving 24, 26, 28, and 30, possibly all through 32 and 34. The yields of 27 and 28 0 1I

HO

011

0 1I

23,24

25,26 0 1I I

23,25,27,29,31,33 : K'=K4= 11. K2=011,R'=CII$)I1 24,26,28,30,32,34 : it'= 01 I, u2=KLI I, u4=CI I$)II

HO 27.28 0 1I

hlI 31,32

01I 33.34

could be raised55by using protected materials: treatment of peracetylated inulin in I : I liquid HF- SOzgave mainly the hexaacetate of 27, and similar treatment of 2,3 :4,6-di-0-isopropylidene-a-~-sorbofuranose (- 20", 10 min) gave mainly 28. TreatmenP of an extracellular polysaccharide of Rhizobiumjaponicum (an important factor for nitrogen-fixing symbiosis between bacteria and soybeans) with liquid HF (- 40°, 30 min) gave mono- and oligo-saccharides involving 0-p-D-glucopyranosyl-( 1 3)-O-(4-O-acetyl-a-~-galactopyranosyluronic acid)-(I +3)-~-mannose and its I-fluoride. Chitin, a p-( 1 +4)-linked polysaccharide consisting mainly of 2-acetamido-2-deoxy-~-glucopyranoseunits, is soluble in liquid HF, and is grad-

-

(54) J. Defaye, A. Gadelle. and C . Pedersen, Carbohydr. Rex, 152 (1986) 89-98. ( 5 5 ) J. Defaye, A. Gadelle, and C. Pedersen, Carbohydr. Rex, 174 (1988) 323-329. (56) A. J. Mort, J.-P. Utille,G. Tom,andA. S. Perlin, Curhohydr. Rex. 121 (1983)221-232.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

99

ually degradeds7 into chitooligosaccharides more smo0thly4~than by the method using conventional mineral acids, giving finally 2-acetamido-2deoxy-D-glucopyranose(20",24 h). When 2-acetamido-2-deoxy-~-glucose or -D-galactose was treated similarlySS(HF, 0+20"), with subsequent addition of methanol, the corresponding methyl P-D-glycopyranosideswere obtained stereospecifically. Slow evaporation, however, gave mixtures of /?-D-( 1 +6)-linked di- to hexa-saccharides containing 2-acetamido-2-deoxyglucosyl and -galactosyl residues, respectively. Reactions of monosaccharides (for example, D-ribose, D-xylose, D-mannose) with cyclohexenes9and tetracyanoethylene"' in liquid HF have been studied. The preparation of glycosyl fluorides is described next. Aiming to have a convenient glycosyl donor convertible into 1,2-cis-furanosides,Mukaiyama and coworkers6' prepared 2,3,5-tri-O-benzyl-P-~-ribofuranosyl fluoride (368) by treatment of a protected D-ribofuranose (35) with 2-fluoro- 1-methylpyridinium tosylate62(FMPTs);the total yield was raised by anomerizing the simultaneously produced a- I -fluoride (36cy, JI,F 66, J2,F 24 Hz) to 368 (J,,F63.5 Hz, J2,F very sma1P3)by treatment with BF3.0Et,.

-

B ' l w i o H @-l +

OBn

Brio

N I ' -OTs

Me

BnO

FMPIs

35

36a

OBn 36

36P

(57) C. Bosso, J . Defaye, A. Domard, A. Gadelle, and C. Pedersen, Curbohydr.Res.. 156 (1986) 57-68. (58) J. Defaye, A. Gadelle, and C. Pedersen, Curbohydr. Res., 186 (1989) 177- 188. (59) F. Micheel, A. Klemer, M. Kohla, H. Peschke, and R. Mattes., Justits Liebigs Ann. Chem.. (1985) 383-395. (60) M. Kohla, A. Klemer, R. Mattes, and M. Leimkiihler, Justus Liebigs Ann. Chem.,(1986) 787 - 798. (61) T. Mukaiyama, Y. Hashimoto, and S. Shoda, Chem. Leu., (1983) 935-938. (62) T. Mukaiyama, Angew. Chem., Int. Ed. Engl., 18 (1979) 707-721. (63) G. H. Posner and S. R. Haines, Tetrahedron Lett., 26 (1985) 1823- 1826.

TSUTOMU TSUCHIYA

I00

Nicolaou and ~ o w o r k e r sreported ~ ~ , ~ ~ a new method for preparing glycosyl fluorides (38) from phenyl thioglycosides (37) by treatment with diethylaminosulfur trifluoride (DAST)- N-bromosuccinimide (NBS), or HF- pyridine- NBS, the phenylthio group of the thioglycosides being initially activated66by NBS. Thus prepared were compounds 39, 40, and 42 (see Table I) and 3,4-0-carbonyl-2,6-dideoxy-3-C-methyl-~-~~bo-hexopyranosyl, 2-azido-2,6-dideoxy-3,4-O-isopropylidene-~-altropyranosyl, -0

37

-0

38

5-0-acetyl-6-deoxy-2,3-0-isopropylidene-a-~-mannofuranosyl, 3,4,6-tri0-acetyl-2-deoxy-a-~-urubino-hexopyranosyl, and 4-0-benzoyl-2,6-dideoxy-3-0-methyl-a-~-ribo-hexopyranosylfluorides. Similarly, (6s)fluoro derivative@ (involving 145; see later) of 2,3,4-tn-O-acetyl- and -benzyl-1,6-anhydro-~-glucopyranose were prepared from the corresponding (6R)-phenylthio precursors. In 1984, Szarek and coworkers67and Noyori and coworkers68found concurrently that glycosyl fluorides may be readily prepared by treatment of 0-protected free sugars or 0-protected- 1-0-acyl sugars with pyridinium poly(hydrogen fluoride)(3 : 7 pyridine- hydrogen fluoride), a reagent milder than the hydrogen fluoride introduced by Olah and coworker^.^^ The reactions were camed out without solvent (HF content varied) or with solvent (dichloromethane, acetone, or benzene, with or without addition of pyridine or collidine). By this p r ~ c e d u r e , ~1la, ~ . ~13a, ~ . ~14a, ~ 15a, 36a, 36p, 2,3,5tri-0-benzyl-a- (41a)and -p-D-arabinofuranosy1(41/?),2,3,5-tri-O-benzoylD-ribofuranosyl(43), 2,3,5-t~-O-benzoyl-~-arabinofuranosyl(44), 2,3 : 5,6di-0-isopropylidene-a-D-mannofuranosyl (45a), 3,5-di-O-acetyl-2-deoxyD-erythro-pentofuranosyl (46), 2,3,4,6-tetra-O-benzyl-a-~-glucopyranosyl (64) K. C. Nicolaou, R. E. Dolle, D. P. Papahatjis,and J . L. Randall, J. Am. Chem. Soc., 106 (1984) 4189-4192. (65) R. E. Dolle and K. C. Nicolaou, J. Am. Chem. Soc., 107 (1985) 1691 - 1694. (66) K. C. Nicolaou, S. P. Seitz, and D. P. Papahatjis, J. Am. Chem. Soc., 105 (1983) 24302434. (67) W. A. Szarek, G. Grynkiewicz, B. Doboszewski, and G. W. Hay, Chem. Leu.. (1984) I75 I - 1754. (68) M. Hayashi, S. Hashimoto, and R. Noyori, Chem. Left.,(1984) 1747- 1750. (69) G. A. Olah, J . T. Welch, Y. D. Vankar, M. Nojima, 1. Kerekes, and J . A. Olah, J. Org. Chern.. 44 (1979) 3872-3881. (70) R. Noyori and M. Hayashi, Chem. Letl.. (1987) 57-60.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

101

(47a),2,3,4,6-tetra-O-benzyl-a-~-mannopyranosyl (48a),2,3,4,6-tetra-Obenzyl-a-D-galactopyranosyl(49a),and 3,4,6-tri-O-acetyl-2-deoxy-a-~-arahino-hexopyranosyl fluorides (50a)were prepared in mostly good isolated yields (see Table I). A characteristic feature of this procedure is that the thermodynamically favored a-D-glycopyranosyl fluorides are generally obtained. Ishido and coworkers prepared71-73 several D-ribofuranosyl fluorides by treatment of 1-free sugars with N,N-diethyl-1,1,2,3,3,3-hexafluoropropyl (CF,CHFCF2NEt2,Ishikawa reagent). Thus, 36 and 2,3,5-tri-Omethyl-, 2,3-di-O-benzyl-5-0-methyl-, 5-0-benzyl-2,3-di-O-methyl-aand -P-D-ribofuranosyl fluorides were prepared. Rosenbrook and coworker^'^ prepared glycosyl fluorides (41 and 47) by treatment of protected 1-free sugars with neat DAST (Et2NSF3)(see Table I). Posner and H a i n e ~ and, ~~ later, Ogawa and coworker^^^.^^ used the DAST reagent diluted with oxolane or dichloromethane to prepare 51, 52, 53,54,and 55 (see Table I). The solvent used77in the fluorination of 2,3,5-tn-O-benzyl-~-ribofuranose influenced the anomeric ratio @/a= 2.0-9.9). However when 2,3,4-tri-Obenzyl-D-glucopyranose (56)was treated with DAST in 1,2-dimethoxyethane at 70", the 3.6-anhydro derivative 59 was mainly produced,80 accompanied by the 1,6-difluoro-a-(57,trace) and -j?-~-glucoses 58(minor). In this reaction, 59 was presumed to be produced through intermediate 60, formed after rapid 1-fluorination of 56.The yields of 57 and 58 were raised when a base was added, because of increase of fluoride ion content. A unique preparation of 2,3 : 5,6-di-O-isopropylidene-a-~-mannofuranosyl fluoride (45)utilizing the Mitsunobu reaction8' [diethyl azodicarboxylate (DEAD)- triphenylphosphine in the presence of Et,O+BK in this case] has been reported82(see Table I). (71) Y. Araki, K. Watanabe, F.-H. Kuan, K. Itoh, N. Kobayashi, and Y. Ishido, Curbohydr. RL's..127 (1984) ~ 5 - C 9 . (72) F.-H. Kuan, N. Kobayashi, K. Watanabe, K. Itoh, Y. Araki, and Y. Ishido, Nippon Kugukii Kuishi, (1985) 2040-2047. (73) Y. Araki, N. Kobayashi, Y. Ishido. and J. Nagasawa, Curbohydr.Res.. 171 (1987) 125139. (74) A. Takaoka, H. Iwakiri, and N. Ishikawa, Bull. Chem. SOC.Jpn., 52 (1979) 3377-3380. (75) A. Takaoka. H. Iwakiri, N. Fujiwara, and N. Ishikawa, Nippon Kugukii Kuishi, (1985) 2161 -2168. (76) W. Rosenbrook, Jr., D. A. Riley, and P. A. Lartey, TetrahedronLett.. 26 (1985) 3-4. (77) G. H. Posner and S. R. Haines. Tetrahedron Let!.. 26 (1985) 5-8. (78) Y. Nakahara and T. Ogawa, Tetrahedron Lett., 28 (1987) 2731 -2734. (79) K. K. Sadozai. T. Nukada, Y. Ito, Y. Nakahara, T. Ogawa, and A. Kobata, Curbohydr. R C S ,157(1986) 101-123. (80) P. Kovaf, H. J . C. Yeh, and G. L. Jung, J . Curbohydr. Chem., 6 (1987) 423-439. (81) 0. Mitsunobu, Synthesis, (1981) 1-28. (82) H. Kunz and W. Sager, Helv. Chim. Actu. 68 (1985) 283-287.

TSUTOMU TSUCHIYA

102

0Un

OR11

56

57 K'= H, K'= 58 R'= F, R'=

I;

5')

ti

F

60

Treatmente3of the 3,4-trans-di-O-acylglycals61,64, and 67 with pyridinium poly(hydrogen fluoride)'j9gave, through the Femer rearrangement, the relatively unstable 2,3-unsaturated fluorides 62, 65 and 68, in some cases accompanied by minor amounts of the corresponding 2-deoxy-1-fluoro sugars63 and 66. However, 3,4-cis-di-O-acylglycals gave no 2,3-unsaturated fluorides. Treatmentg4 of glycals 61 with nitronium tetrafluoroborate (N0,BF4) gave the 2-nitroglycals 70 through the unstable 2-deoxy-2-nitroglycosyl fluorides 69 (some of them were isolated). The nitroglycals may be utilized to prepare 2-amino-2-deoxyglycosides. Reactionss5of 3-chloro-4-enouronate 71 with AgF in acetonitrile (room temp., 12 h) gave the crystalline 3-eno-5-fluorouronate 72 under the S N ~ ' type of allylic rearrangement; the product is unstable and is readily converted into 73 in saturated methanolic ammonia. Glycosylations utilizing the aforedescribed glycosyl fluorides are described next. In 1981, Mukaiyama and coworkerss6attempted to prepare I ,2-cis-glycosidesby utilizing the relatively stable (as compared with other glycosyl halides) 2,3,4,6-tetra-0-benzyl-~-~-glucopyranosyl fluoride (478) (83) S. J. F. Macdonald and T. C. McKenzie, Tetrahedron Le//.,29 (1988) 1363- 1366. (84) C. W. Holzapfel, C. F. Marais, and M. S. van Dyk,Synrh. Cornrnun., 18 (1988) 97- 1 14. (85) J. Kiss, P. C. Wyss, G . Flesch, W. Arnold, K. Noack, and P. Schonholzer, J. Curbohydr. Chern.. 4(1985) 347-361. (86) T. Mukaiyama, Y. Murai, and S. Shoda, Chern. Lett.. (1981) 431 -432.

DEVELOPMENTS OF FJSJORINATED CARBOHYDRATES ROCH, I

103

ROCH,

I

61

62

R=AcorBz

Atop -

AcoFo>F

+

63

‘ “ V F

AcO

AcO

64

65

66

A c o c o > F AcO

67

68

6),Fi -Rk2

ROCH,

ROCH,

ROCH,

RO

NO2

NO2

61 R = Ac, Bn, MeOlC

70

69

Bz or Me

60-Q-Q OBn

OBn

NHCOLBn

N1 ICO2Bn 71

COJNII2

<‘OzMC

72

OBI)

N ti (‘0:I3 TI 73

reacting in S N fashion ~ with alcohols. The best conditions found used 47Bin the presence of SnC1,- AgCIO, in ether with a variety ofalcohols (methanol, cyclohexanol, tut-butanol, cholesterol, cholestanol, and methyl 2,3,4- and 2,3,6-tn-0-benzyl-a-~-glucopyranosides) and afforded mainly the 1,2-cisglycosides 74, the ratio (Y (=cis) :p being 12 : 1 to 4 : 1. For the formation of

TSUTOMU TSUCHIYA

104

1,2-cis-glycosides,however, the species of solvent used was another important factor, as described later.87In the condensation involving some other protected furanosyl fluorides, however, the use of trityl perchlorate (TrCIO,), instead of AgClO,, was found successful6'in affording 1,2-cis-furanosides. Reactions of 2,3,5-tri-O-benzyl-~-~-ribofuranosyl fluoride (36) with several alcohols in the presence of SnC1,-TrC10,- molecular sieves in ether afforded the a-Danomers preponderantly (a: p = 8 1 : 19 to 88 : 12). A similar condensation of 2,3,5-tri-O-benzyl-a-~-arabinofuranosylfluoride ( 7 5 ~with ) cholestanol also gave a good result (a: p = 1 :4).

47P

74

The Mukaiyama condensation methods6'Ss6 using SnCl, - AgClO, or SnCI, - TrClO, have been widely used for the synthesis of a variety of glycosides, as follows: rhynchosporosides, toxic principles for plants produced by Rhynchosporium secalis, using 0-(2,3,4,6-tetra-0-acetyl-p-~-glucopyranosy1)-(1 -4)-(2,3,6-tri-0-acety1-~-~-glucopyranosyI fluoride) or the related fluorides of the higher congenerss8 77, those prepared by the Nicolaou method from 76; total s y n t h e s i ~ ~of~ cyclomaltohexaose -~' (a-cyclodextrin) and cyclomaltooctaose (y-cyclodextrin), using 0-(4-0-acetyl-2,3,6-tri-Obenzyl-a-D-glucopyran0syl)-( 1-4)-(2,3,6-tn-0-benzyl-p-~-glucopyranosyl fluoride) and the higher congeners 78 (each of them being prepared from the corresponding a-glycosyl chloride by the AgF methodzs), the coupling reactions (involving the intramolecular cyclization) being performed in the presence of SnCl, - AgSO,CF,; several L-rhamnopyranosides involving methyl 2-0- and 4-0-a-~-rhamnopyranosyl-~-~-glucopyranosides, using 2,3,4-tri-0-benzyl-6-deoxy-~-mannopyranosyl fluoride9, (79, see Table I); a heptasaccharide h a ~ t e n , using ~~ 0-(2,3,4,6-tetra-O-acetyl-p-~(87) (88) (89) (90) (91) (92)

S. Hashimoto, M. Hayashi, and R. Noyori, Tetrahedron Lcff.,25 (1984) 1379- 1382. K. C. Nicolaou, J. L. Randall, and G. T. Furst, J. Am. Chem. Soc., 107 (1985) 5556-5558. T. Ogawa and Y. Takahashi, Curbahydr. Rex, 138 (1985) c5-c9. Y. Takahashi and T. Ogawa, Curbohydr. Rex, 164 (1987) 277-296. Y. Takahashi and T. Ogawa, Curbohydr. Res., 169 (1987) 127- 149. S. Kamiya, S . Esaki, and R. Ito, Agric. Biol. Chem., 50 (1986) 1321 - 1322.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

76 77

R ' = SPh. R ' = f:,

105

R'=Ac or Bn R 2 = A c or Bn

OBn 78

( n =4,6)

R= H or Ac

galactopyranosy1)-(1-4)- 0-(2 -acetamido- 3,6-di-0-acetyl- 2 -deoxy -P- Dglucopyranosy1)-(1+2)-0-[(2,3,4,6-tetra-~-acetyl-P-~-galactopyranosyl)( 1 -4) - 0-(2 -acetamido- 3.6 -di - 0-acetyl- 2 -deoxy -P-~-glucopyranosyl)( I +4)]-3,6-di-U-acetyl-a- and -P-D-mannopyranosyl fluorides (prepared by the DAST method); glycolipid M 1-XGL-1 ,93 using peracetylated lactosyl (a,P)-fluoride and per-0-acetylated 0-(2-acetamido-2-deoxy-/?-~-glucopyranosy1)-( 1 +3')- 0-[(2-acetamido-2 -deoxy-P-~-galactopyranosy1)( 1 +4')]-lactosyl @-fluoride; glycosphingolipid~~~ such as triosyl-P-(1 1)2-N-tetracosanyl-(2S,3R,4E)-sphingenineand its protected analog, using 0(2-acetamido-3,4,6-t~-0-acetyl-2-deoxy-~-~-glucopyranosyl)-( 14 3 ) - 0 (2,4,6-tri - 0-acetyl -p- D -galactopyranosyl)-( 1-'4)-2,3,6- tri- 0-acetyl- Dglucopyranosyl fluoride (84) and 0-(2-acetamido-3,4,6-tri-O-acetyl-2deoxy-p-D-glucopyranosyl)-( 1 +4)-0-( 3-0-acetyl-2,6-di-0-benzoyl-P-~galactopyranosy1)-(1 4)-2,3,6-t~-O-benzoyl-~-glucopyranosyl fluoride (each a : p = - 1 : 3; prepared by the DAST method), and sphingosine derivatives; and several a!-(1 +4)-linked galacto-oligosaccharides78involving 82 (from 80) and 83 (from 81), using 53, 54, and 55 (see Table I). Methyl 2,3,6-tri-0-benzoyl-P-~-galactopyranoside (85) and its tri-0-benzyl analog (86) were condensed with 2,3,4-tri-O-benzyl-~-fucopyranosyl(87), 2,4,6tri-O-benzyl-3-deoxy-~-xylc-hexopyranosyl(88),and 2,3,6-tri-O-benzy1-4deoxy-o-xybhexopyranosyl fluorides (89; prepared from the corresponding protected 1-free sugars by treatment with DAST) to give95 the

-

-

(93) Y. Ito, M. Sugimoto, S. Sato. and T. Ogawa, Tetrahedron Lett., 27 (1986) 4753-4756. (94) Y. Ito. S. Sato, M . Mori, and T. Ogawa, J. Curbohydr. Chem., 7 (1988) 359-376. (95) J. Kihlberg, T. Frejd, K. Jansson, S. Kitzing, and G . Magnusson, Curbohydr. Res., 185 (1989) 171-190.

TSUTOMU TSUCHIYA

I06

CH20K2 I

-uF

\ ORn

ORn

ORn

ACO

F OAC

NHAc

+

corresponding 4-0-a- and -/3-D-glycosylgalactosides [85 87- P-D(3 1%); 86 87- a-D(5690) and P-D(10%);86 88- (Y-D (7 190)and P-D(1 5%); and 86 89- a-D(50%)and p-D(27%)]. The Mukaiyama condensation method was also utilized in the synthesis of several antibiotics and related bioactive substances. These synthesesinclude: a p r a m y ~ i nusing , ~ ~ 4-azido-2,3,6-t~-0-benzyl-4-deoxy-~-~-~ucopyranosyl fluoride (prepared from the corresponding a-D-glucopyranosyl chloride by the AgF method**);avermectin BLa,using protected sugar derivatives of oleandrosyl fluoride (a-L-Ole-F) and 0-(a-L-Ole)-( 1 -4)-a-~-Ole-F (both

+

+

+

(96) K. Tatsuta, K. Akimoto, H. Takahashi, T. Hamatsu, M. Annaka, and M. Kinoshita, TetrahedronLeu.. 24 (1983) 4846-4870; Bull. Chem. SOC.Jpn., 57 (1984) 529-538.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

107

prepared by the DAST - NBS method); C-2”a- and C-2”/3-fluoroavermectin9’ Bla; and a u r o d ~ xand ~ ~e f r o t o m y ~ i ncomponents ~~, of the antibiotic elfamycin complex, using protected 2,4-di-O-methyl-a-~-rhamnopyranosyl fluoride65and 0-(2,4-di-O-rnethyl-a-~-rhamnopyranosyl)-( 1 +4)-6-deoxy3-0-methyl-a-~-allopyranosylfluoride.9s Noyori and coworkerss7found that tetrafluorosilane or trimethylsilyl triflate catalyzes the condensation of appropriately protected glycopyranosyl fluorides with trimethylsilyl ethers or alcohols. The strong affinity of silicon for fluorine was considered to be the driving force for this reaction. In the case of SiF,, attack of a nucleophile on the glycosyl cation-SiFy ion-pair intermediate was a n t i ~ i p a t e d .Thus, ~ ~ condensation of 2,3,4,6-tetra-Obenzyl-a- and -P-D-glucopyranosyl fluorides (47a and 478) with methyl 2,3,4-tri-0-benzyl-6-O-(trimethylsilyl)or 2,3,6-tri-O-benzyl-4-O-(trimethylsily1)-a-D-glucopyranosidegaves7the 6-0- and 4-O-~-glucosylderivatives, respectively; the resultant anomeric configuration was largely influenced by the solvent used; in ether and acetonitrile a- (1,2-cis)and p-D anomers ( I ,2,-trans)preponderate, respectively, irrespectiveofthe anomeric configuration of the starting fluorides. This result indicates that the anomeric configuration of the starting 1-fluoride is not the sole factor in determining the anomeric configuration of the glycosides produced. The Noyori procedures7was applied to a total synthesis99of baiyunoside, a sweet principle, using 2,3,4-tri-0-benzyl-~-xylopyranosyl fluorides2(18; see Table I), and a synthesis ofglycotriosyl ceramide.lWA model experiment for the synthesis,99using 18, showed a solvent dependence for the a : P ratio of the products. In this case, the use of acetonitrile, oxolane, or ether gave the a anomer ( 1,2-cis),and the use of toluene or hexane gave the p anomer ( 1,2trans), preponderantly. Glycosylation utilizing glycosyl fluorides in the presence of BF, OEt, was reported concurrently by I ~ h i d o ,Voznij,lol ~’ Nico1aou,lo2and their respective coworkers, and Kunz and Sagar.s2 Ishido and coworkers7’ prepared 1 1 (a,a and P,P)-disaccharides under BF, catalysis by condensation of 2,3,5-tri-O-benzyl-~-~-ribofuranosyl fluoride (36p) and 2,3,5-tri-O-benzylD-ribofuranose, its 1-0-P-(trimethylsilyl), or 1-0-P-acetyl derivatives.

-

(97) C. Bliard, F. C. Escribano, G. Lukacs, A. Olesker, and P. Sarda, J. Chem. Soc., Chem. CO/V/722ln.,(1987) 368-370. (98) R. E. Dolle and K. C. Nicolaou, J . Am. Chem. Soc., 107 (1985) 1695- 1698. (99) H. Yamada and M. Nishizawa, Tetrahedron Lett., 28 (1987) 4315-4318. (100) S. Nunomura and T. Ogawa. Tetrahedron Lett.. 29 (1988) 5681 -5684. (101) Ya. V. Voznij, 1. S. Kalicheva,andA.A.Galoyan,Bioorg.Khim., lO(1984) 1256-1259. ( 1 02) K. C. Nicolaou, A. Chucholowski, R. E. Dolle, and J. L. Randall, J. Chem. Soc., Chem. Comtnlln.. (1984) 1155- 1156.

108

TSUTOMU TSUCHIYA

Voznij and C O W O ~ ~ ~prepared ~ S ~ a~ number ~ , ~ of ~ 1,2-trans-glycosides ~ J ~ by treatment of several 1,2-trans-glycosyl fluorides (90) with alkyl and aryl alcohols involving 11 - 16, and by treatment of 2,3,4,6-tetra-O-acetylp-D-galactofuranosyl fluoride with protected 0-(trimethylsilyl) sugars or 4-methyl-7-trimethylsilyloxycoumarin in the presence of BF, - OEt, in benzene. Similar condensation of per-0-acetyl-B-cellobiosyl,-B-cellotriosyl, and -p-cellotetraosyl fluorides, and 17 with the aforegoing coumarin or related compounds was also r e p ~ r t e d . ~ ~Generally, . ~ ' ~ ' ~ ~I ,2-trans aryl glycosides (92)are consideredlo' to be formed through the l ,3-dioxolan-2-ylium intermediate 91. Glycosylation of ",A"-bis(trimethy1silyl)uracil with 13p in the presence of BF, OEt, gave the corresponding I -B-D-glucopyranosyluracil derivatives.IM

w

YI

92

Nicolaou and coworkerslo2also found that, in the presence of BF, catalyst (in some instances, in the presence of MgBr,-OEt,, SnCl,, or Me,Al), some per-0-acetyl- and per-0-benzyl-D-glucopyranosylfluorides (93),upon condensation with several organic acids, 2,3,4,6-tetra-0-benzyl-a-~-glucopyranose, phenyl 2,3,4-tri-0-benzyl-I-thio-~-~-glucopyranoside, several thiols, tert-butyl peroxide, trimethylsilyl azide, several organic amines, and (PhCH,O),P(O)OSnBu, gave, respectively, the D-glucosyl esters, a D-glucosyl-( 1 l)-D-glucoside, a 6-0-~-glucosylD-glucoside, the 1-thio-D-glucosides, a D - ~ ~ U C O Speroxide, Y~ a D-glucosyl azide, the D-glucosylamines, and a D-glucosyl phosphate (represented by 94). Kunz and Sage?, also demonstrated the effectiveness of this procedure. Under BF, catalysis, reactions (in CH,Cl,) of l8,20a, and 4% with alcohols [including benzyl alcohol, cholesterol, their silyl ethers, and N-(benzyloxy-

-

(103) Ya. V. Voznij, A. A. Galoyan, and 0. S . Chizhov, Eioorg Khim., 1 I (1985) 276-278. (104) Ya. V. Voznij, I. S. Kalicheva, and A. A. Galoyan, Bioorg. Khim., 12 (1986) 521 -526. (105) A. M. Yegorov, A. N. Markaryan, Ya. V. Voznij, T. V. Cherednikova, M. V. Demcheva, and 1. V. Berezin. A n d . Leff.,21 (1988) 193-209. (106) Ya. V. Voznij, L. N. Koikov, and A. A. Galoyan, Eioorg. Khim., I I (1985) 534-535.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

R

Y3

R=Ac. Bn

109

R

Y4

X = OCOR', OC-, SR', O R ' ,N,, NHR', NR',, OP(O)-(OBn)Z R'= alkyl or x y l

carbony1)serineally1 ester] gave the corresponding glycosides, mostly in high yields (reactions with the silyl ethers were faster than those with the corresponding alcohols).Noteworthy was the fact that 18 and 45agave mainly the a-D-glycosides, whereas 20a gave the P-D-glycoside (an excess of BF, was also needed) on account of participation by the pivaloyl group. Condensation reactions utilizing glycosyl fluorideswere also carried out in the presence of titanium tetrafluoride catalyst. Thus, 138, 478, 47a, and methyl (2.3,4-tri-0-acetyl-~-~-galactopyranosyl fluoride)uronate (95) were condensedlo7with several alcohols, including 1,2 : 3,4-di-O-isopropylidenea-D-galactopyranose, 1,6 :2,3-dianhydro-P-~-mannopyranose, 1,6-anhydro-2-azido-4-O-benzyl-~-~-glucopyranose (96), and some of their trimethylsilyl ethers (experiments with SnF, or CF,SO,SiMe, as the catalyst were also reported). In these reactions, as would be expected, the 2-0-acetyl fluorides 13pand 95 gave only P-D-glucosides(involving97), and the 2-0-benzyl fluorides 478 and 47a, an anomeric mixture, the a:Pratio of the latter reactions being influenced by the solvent used [acetonitrile (rich in P anomer) and ether (rich in a anomer)]. The structure of the titanium tetrafluoride- ligand complex as the reaction intermediate was discussed.'07

(107) M. Kreuzer and J. Thiem, Curbohydr. Rex, 149 (1986) 347-361.

TSUTOMU TSUCHIYA

I10

Suzuki and coworkerslo8prepared glycosides of D-mycinose by condensation of 4-O-acetyl-~-~-mycinosyl fluoride (98, prepared by the Nicolaou m e t h ~ d ~with , ~ several ~) alcohols (benzyl, cyclohexyl, and tert-butyl alcohol) in the presence of a metallocene compound, Cp2MCI,-AgC10, (Cp: cyclopentadienyl, M: Ti, Zr, and Hf), the catalysts being chosen based on the soft - hard concept: hard acids prefer to bond to hard bases, and soft acids prefer to bond to soft b a ~ e s . ~ ~glycosyl A s chlorides and bromides are suitably activated by Hg2+and Ag+ catalysts, glycosyl fluorides (Fis a stronger hard base than CI- or Br-) may be more effectively activated by IVa (Si, Sn) and IVb group metals (Ti, Zr, Hf)cations than by Hg2+and Ag+,because cations of the former group are stronger hard acids than those of the latter. The reactions proceeded smoothly during a short period at low temperature (-20" -room temp.) and, when benzene was used as the solvent, the p-Dglycosides were obtained preponderantly (p: a = > 16 : < 1). In similar condensations using the 1-fluoro-D-desosamine derivative 99 (Ref. 109; prepared by the Nicolaou method using DAST- NBS- HF-pyridine), the use of Cp,HfCI, - AgCIO, or SnCI, - AgC10, (Ref. 86) was also found useful. The new procedure was applied"O in the synthesis of mycinamicins (macrolide antibiotics) from mycinolide IV, and also of several aryl glycosides of phenols. I I

98

OCO, Me 99

T. Matsumoto, H. Maeta, K. Suzuki, and G. Tsuchihashi, Tetrahedron Lett., 29 (1988) 3561 - 3510. K. Suzulu, H. Maeta, T. Matsumoto, and G. Tsuchihashi, Tiltrahedron Lett., 29 (1988) 3511-3514.

T. Matsumoto, H. Maeta, K. Suzuki, and G . Tsuchihashi, Tetrahedron Lett., 29 (1988) 3575-3578. T. Matsumoto, M. Katsuki, and K. Suzuki, Chem. Lett., (1989) 431-440.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

111

Voznij and coworkers112prepared 1,2-frans aryl glycopyranosides from the corresponding 1,2-frunsglycopyranosyl fluorides llp, 12,13p,lSp,and 16 by treatment with several sodium phenolates in an alcoholic medium. Similarly 1 ,?-trans aryl glycofuranosides (103)were obtained1l3from such glycofuranosyl fluorides (100) as tetra-O-acetyl-/?-D-gluco-1L4 and -galactofuranosyl fluorides,'I5 and tri-0-benzoyl-a-L-arabinofuranosyl (the enantiomer 44 was known1l6)and -P-D-nbofuranosyI fluoride^"^ (43),possibly through 1,2-anhydro intermediates (102)after deacetylation (to give 101). Also, when 3,4,6-tri-0-benzyl-~-glucopyranosyl fluoride, 2-0-acetyl-3,4,6tn-0-benzyl-P-D-galactopyranosyl fluoride29(1048;see Table I) and its p-

100

101

102

I03

bromobenzyl analog were treated with base (NaH or KOCMe, in oxolane), the corresponding 1,2-anhydro-(~-~-gluco-~~~ and -galacto-pyranose were produced, which are potential intermediate^"^ for the synthesis of/?-(I 4 2 ) linked D - ~ ~ U C Oand D-galacto-pyranan. Further application of the foregoing glycosyl fluoride methods to prepare biologically important principles is now described. 3-Deoxy-~-manno-2-0~tulopyranosonic acid'20 (Kdo) is a characteristic sugar component of cellsurface lipopolysaccharides (LPS) and capsular polysaccharides of Gramnegative bacteria, and is bound, a-ketosidically, to the 6-hydroxyl group of the nonreducing portion of the bis(D-glucosamine) moiety of lipid A. The

Ya. V. Voznij, I. S. Kalicheva, and A. A. Galoyan, Bioorg. Khim., 8 ( 1982) 1388 - 1392. Ya. V. Voznij, I. S. Kalicheva, and A. A. Galoyan, Biourg, Khim.. 1 1 (1985) 970-972. K. Bock and C. Pedersen, Actu Chem. Scund., 26 (1972) 2360-2366. K. Bock, C. Pedersen, and L. Wiebe, Actu Chem. Scund., 27 (1973) 3586-3590. A. K. Bhattacharya, R. K. Ness, and H. G. Fletcher, Jr., J. Org. Chem., 28 (1963) 428-435. (1 17) C . Pedersen and H. G. fletcher, Jr., J. Am. Chem. Soc.. 82 (1960) 941 -945. ( I 18) P. F. Sharkey, R. Eby, and C. Schuerch, Curbohydr. Res., 96 (1981) 223-229. ( I 19) C. Schuerch, Adv. Curbohydr. Chem. Biochem., 39 (1981) 157-212. (120) F. M. Unger, Adv. Curbohydr.C h m . Biochem., 38 (1981) 323-388. ( 1 12) ( I 13) ( 1 14) ( I 15) ( 1 16)

112

TSUTOMU TSUCHIYA

Kdo glycosides have usually been prepared by utilizinglzO-lzz 2-chloro and 2-bromo derivatives of Kdo. Rosenbrook and coworkers76first prepared the 2-fluoro derivative 112 (7390,a :P = 2 : 1) of Kdo and its 3-hydroxyl analog 113 (P-F, 60%) from 4,5,7,8-tetra-O-acetyl-Kdo methyl ester (111) and 4,5,7,8-tetra-0-aCetyl-a-D-g~~Ce~~D-gU~UC~~OCtUlOSOniC acid methyl ester, respectively, by applying76the DAST method. The 2-P-fluoride 107 (33%) was prepared1z3from 4,5: 7,8-di-O-isopropylidene-Kdo methyl ester (105, R = Me; the isopropylidene group was chosen so as to be removed selectively, in a later step, without affecting the acetyl groups of the products) by treatment with 2-fluoro- 1 -methylpyridinium tosylate61.62 (FMPTs); in this fluorination, if DAST was used, only a 2,3-unsaturated product was obtained. Treatment of the a-2-0-acetyl derivative 106 (R = Me and Bn) with the Olah reagent gave the a-fluorides 108 (58% for R = Me) with retention of configuration. Condensation of 108 (R = Bn) with the D-glucosamine derivative 109 by the BF, gave the disa~charide'~~ 110 (60%) of the desired a-anomeric configuration, with a trace (2%)of the P anomer. Compound 108 (R = Bn) was also used in the s y n t h e s i ~oftri~ ~ ~and J ~ ~tetra-saccharide part structures of LPS containing Kdo and 1-dephosphono lipid A. Similar condensationtz6of 115 (a:P= - 1 : 1, 639'0; prepared from 114 by use of DAST) with benzyl 2-deoxy-4-O-(diphenoxyphosphinyl)-3-0-tetradecanoyl- 2 - [(3R)-3-(tetradecanoyloxy)tetradecanamido]-P-~-glucopyranoside (120) gave 121 (27%; the p anomer: 4I Yo); whereas Koenigs- Knorr condensation of the 2-bromide 116 with 120 gave 121 in better yield (67%). Similar results were obtained126in the condensation of 118 (a: p = - I : 1, 60%; prepared from 117 by use of DAST) or the bromide 119 with 120 to give 122 (65% from 119, and a very poor yield from 118) indicating that, in these reactions, high C- F bond energy turned out to be inadequate to give the desired condensation product. Deblocking of 122 gave 123, a lipid A analog containing Kdo.

( I 2 I ) H. Paulsen, M. Stiem, and F. M. Unger, Tetrahedron Lett.. 27 (1986) 1 I35 - I 138. (122) H. Paulsen, Y. Hayauchi, and F. M. Unger, Justus Liebigs Ann. Chem.. (1984) 1270-

1287, 1288- 1297. ( I 23) M. Imoto, N. Kusunose, Y. Matsuura, S. Kusumoto, and T. Shiba, Tetrahedron Lett.. 28 ( 1987) 6277 - 6280. (124) M. Imoto, N. Kusunose, S. Kusumoto, and T. Shiba, Tetrahedron Lett., 29 (1988) 2227-2230. ( I 25) S. Kusumoto, N. Kusunose, T. Kamikawa. and T. Shiba, Tetrahedron Lett.. 29 (1988) 6325-6326. ( I 26) M. Kiso, M. Fujita, E. Hayashi, A. Hasegawa, and F. M. Unger, J.Carhohydr. Chem., 6 (1987) 691 -696; M. Kiso, M. Fujita, M. Tanahashi, Y. Fujishima, Y. Ogawa, A. Hasegawa, and F. M. Unger, Carhohydr. Re.% 177 (1988) 5 1-67.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

OH

KJ 0

OH

in5

106

c

R= Me or Bn

107

108

CH20H 108 (R=Bn)

t

TrocO H

O

S OBn Troc 109

R'OC\

1

R'WH ~

4

111 112 113 114 115 116 117 118 119

R'

R2

R'

X

Me Me

H

Ac Ac Ac

OH

H

Me Me Me Mc

OH H H

Bn Bn

H H H

Bn

H

CICHEO CICHfZO

ClCHEO CICHSO CICHKO CICHEO

F F OH F Br OH

F Br

\0

121 122 123

R'= OBn(P). R2= Ph, R3= Me, R1= OBn(P)).R2= Ph. R3= Bn, R'= OH. RfR3=R4= H

R4=ClCHs0

R4=CICH2C0

113

1 I4

TSUTOMU TSUCHIYA

N-Acetylneuraminic acid (5-acetamido-3,5-dideoxy-~-glycero-~-galucto-2-nonulopyranosonic acid) is one of the principal constituents of sialic acids,I2' which terminate the oligosaccharide chains of glycoproteins and glycolipids and have a variety of biological functions. In order to prepare neuraminic acid glycosides (especially natural ones having the 2-a configuration), the comparatively unstable peracetylated 2-chloro and 2-bromo neuraminic acid methyl esters128have usually been used, resulting in products manifesting low stereoselectivity in low yields. The instability may be mainly ascribed to the presence of the carboxyl group at C-I, inducing elimination reactions involving the anomeric center. Sharma and Eby129 prepared the stable 2-fluoro analogs 127 and 130 [127: 15%, JH-3cq,F 4.8, JH-3ax,F 24 HZ; 130: 50%, J H - ~ ~ 8.6, , F JH-3m.F 10 Hz] by displacement of the 2-chloro derivative of 124 with fluorine (AgF in MeCN). F l ~ o r i n a t i o n ' ~ ~ - ' ~ ~ of 124, and the peracetylneuraminic acid allyl esters 125 and 126 with the Olah reagent gave the desired @-fluoride127 (80%), 128 (JH-3m,F 33.8 Hz; the allyl group may be removed rather readily in a later step), and 128 with retention, retention, and inversion of configuration at C-2, respectively. C o n d e n s a t i ~ n ' ~of ~ J 128 ~ l with 1,2 :3,4-di-O-isopropylidene-a-~-galactopyranose under BF, catalysis, however, gave the desired a glycoside 131 as a minor product (131 : 132 = 1 : 5; total yield, 44%), whereas the 2-chloro analog 129, in the presence of Ag2C03-Drierite, gave mainly the a product (131 :132 = 6 : 1). Condensation of 128 with eicosan-1-01or 2-(octadecy1)eicosan- 1-01 also gave mainly the corresponding @-glyco~ides'~* (a:@ = 1 :2 1 :3). Deallylation of the products was readily achieved by using (PPh,),Pd(O)- morpholine. Similar attempted c o n d e n s a t i ~ nof ' ~the ~ 3-hy-

124 K'= OAc, K2= C(bMc 125 K'= O A c , K2= CO~C'HzC'H-C'H~ 126 K'= C02CH2('H=CHz3 K2= O A c

127 128 129

X=F, K=Mc X= F, K = Ally1 X= CI. K= Ally1

130

(127) R. Schauer, Adv. Curhuhydr. Chem. Biochem.. 40 (1982) 131-234. (128) C. Shimizu, K. Ikeda, and K. Achiwa, Chern. Pharm. Bull.. 36 (1988) 1772- 1778. ( 129) M. N. Sharma and R. Eby, Curhohydr. Re.s., I27 ( 1984) 20 I - 2 10. (130) H. Kunz and H. Waldmann, J. Chem. Soc., Chem. Cummun.. (1985) 638-640. (131) H. Kunz, H. Waldmann, and U. Klinkhammer, Helv. Chim. Acfu. 71 (1988) 18681874. (1 32) K. Okamoto, T. Kondo, and T. Goto, Tetrahedron. 43 (1 987) 59 19- 5928.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

1 I5

132

131

Gal = 1,2:3,4-di.0-isopropylidene-a-~-galactopyranose

droxyl (jl)analog of 127 with methyl 2,3,4-tri-O-benzyl-a-~-glucopyranoside showed no reaction. Compound 130, was however, utilized, through its 2-P-S-acetyl intermediate, in the synthesis of 5’-4(5-acetamido-3, 5-dideoxy-~-g~ycer~~-~-ga~act~~-nonu~opyranosy~onic acid)-5’-thiocytidine. 133 Ito and O g a ~ asucceeded I~~ in obtaining a-(2+6)-linked N-acetylneuraminic acid glycosides by utilizing the known neighboring-group participation of a /I-phenylselenyl group at C-3. Thus, the 2-fluoride 134 (a:/I = 20: 1; prepared from 133 by the DAST method) was treated with 1,2: 3,4-di-O-isopropylidene-a-~-galactopyranose in the presence of SnC1,-AgS0,CF3 to give the a glycoside 136 with an elimination product 138, the latter being recycled to give 133. The phenylselenyl group was then

QMc BI10 ” AcHN

OR11

-

CO ~MC

- ~ 0 e 6 1 G a l

CO~MC

+

-$ 138

133 134

135

X = O H , Y=ScPh X=F, Y=ScPh X= F, CI, or Rr, Y= SPh

136 Y=ScPh 137 Y=SPh

Gal = 1 , 2 : 3 , 4 - d i ~ ~ - i s o p r o p y l i d c n c - a - ~ ~ - ~ ~ l ~ ~ t o p y r ~ o s c

removed by a known method to give the 3-deoxy compound. The a-phenylselenyl isomer of 134, on the other hand, gave the undesired P-glycoside. Similarly, by using 134, N-acetylneuraminic acid derivatives a-(2-6)(133) 0. Kanie, J. Nakamura, M. Kiso, and A. Hasegawa, J. Carbohydr. Chem., 6 (1987) 105- 115. (134) Y. Ito and T. Ogawa,Tetrahedron Lett., 28 (1987) 6221 -6224.

TSUTOMU TSUCHIYA

116

linked to D-glucose and a-(2-3')- and a-(2+4')-linked to lactose were prepared. The use of 2-phenylthio analogs 135 (X = F, CI, Br) has been found')' more effective in obtaining the a glycosides (through 137). A ( 1 +3')-coupled compound between methyl (3R)-3-[(3'R)-3'-hydroxydecanoyloxy]decanoate and 2-0-a-~-rhamnopyranosyl-a-~-rhamnopyranose, a rhamnolipid from Pseudomonas aeruginosa, expected to have various biological activities, was prepared'36 by double couplings using 3,4-di-O-benzyl-2-0-chloroacetyl-c~-~-rhamnopyranosyl fluoride (by the BF,. OEt, method). Glycosides may also be prepared by enzyme-catalyzed condensation reactions utilizing a glycosyl fluoride. Thus 6-0-cu-maltosylcyclodextrins were prepared enzymically from a-maltosyl fluoride (obtained from the corresponding heptaacetate4) by Zemplh deacetylation) and cyclodextrins.137-139 C-Glycosyl derivatives may be prepared by utilizing glycosyl fluorides. Ishido and coworkers r e ~ o r t e d ~ 'that - ~ )the reaction of 2,3,5-tri-O-benzyl-c(36a) or -P-D-ribofuranosyl fluoride (36p) with isopropenyl trimethylsilyl ether under BF, catalysis (0.1 -0.05 mol. equiv. for the fluoride, in ether or acetonitrile) gave a mixture of 4,7-anhydro-5,6,8-trii-0-benzyl1,3-dideoxyD-altro- (139, major) and -~-allo-2-octulose(140); 139 was stated to be isomerized to 140 (should be vice versa) under Lewis acid catalysis. Similar

36 139 R'= 11, RZ=CIIZCOMe 140 K ' = C H ~ C O MR~ ,~ 11 =

reactions7)of 418 with (2)-1-ethyl-I-propenyl trimethylsilyl ether or 1 -isopropyl-2-methyl-1-propenyl trimethylsilyl ether gave, respectively, the expected ketone and no reaction product. Nicolaou and coworkersWJMalso (135) Y. Ito and T. Ogawa, Tetrahedron Lett.. 29 (1988) 3987-3990. ( 136) P. Westerduin, P. E. de Haan, M. J. Dees, and J. H. van Boom, Curbohydr. Rex. I80 (1988) 195-205. ( I 37) S. Kitahata, Y. Yoshimura, and S. Okada, Carbohydr. Rex. 159 ( 1 987) 303 - 3 13. (138) Y. Yoshimura, S. Kitahata, and S. Okada, Curbohydr. Res., 168 (1987) 285-294. (139) Y. Yoshimura, S. Kitahata, and S. Okada, Agric. B i d . Chem., 52 (1988) 1655- 1659. (140) K. C. Nicolaou, R. E. Dolle, A. Chucholowski, and J. L. Randall, J. Chem. Soc., Chem. Commiin., (1984) 1153- 1154.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

1 I7

prepared C-glycosyl compounds by using similar procedures, and enlarged the scope. Treatment of 2,3,4,6-tetra-0-benzyl-~-glucopyranosyl (47) and per-~-benzy~-~-~-~-D-~ucopyranosy~-a,~-D-~ucopyranosy~ fluorides with allyl-, cyanomethyl-, and cyano-trimethylsilane (Me,SiCH,CH=CH,, Me,SiCH,CN, and Me,SiCN) or vinyl silyl ethers [PhC(OSiMe,)=CH, and kH,(CH,),CH =dOSiMe,] under BF, catalysis gave the corresponding C-glycosyl derivatives 141, with the a-Danomers preponderant [aglycons of the C-glycosyl compounds being CH,CH=CH,, CH,CN,- CN,

-

CH,COPh, and CH(CH,),CO]. Compound 142 was also preparedi4' from both 36pand 36a. This suggeststhat the reaction proceeds under SN 1 conditions by way of the D-ribofuranosyl cation. Compound 142, in contrast to

&>

BnOCH2

47

--+

K

BnO

OBn

-

K= CHZ-CH=CHl, CHZCN, CN, CHICOPh, or

CH(CH2),C0

141

BnO

OBn

I42

139 and 140, was not i~omerized.~~ In another e ~ p e r i r n e n t ,47 ' ~ underwent radical coupling to CH,=CHCN [in the presence of MgBr,*OEt,, Bu,SnH, and 2,2'-azobis(isobutanonitrile)] to give the 1-C-(2-cyanoethyl)derivative. Araki and coworker^'^' studied in detail the mechanism of reaction ofglycosyl fluorides 36p, 47p, and 47a with Me,SiCN, and concluded that unstable glycosyl isocyanides 143 are formed first, and then isomerized to the cyanides 144. Nicolaou and coworkersIa prepared C-glycosyl compounds by treatment of the D-glucosyl fluorides involving 47 with Me,AI, Me,AlCN, MgBr, - OEt,, or AlH,, without catalyst, to give the C-Me, -CN, and -Br, and (141) Y. Araki, N. Kobayashi, K. Watanabe, and Y. Ishido, J. Curbohydr. Chem.. 4 (1985) 565-585.

TSUTOMU TSUCHIYA

1 IS

Bit0

ORn 36p

143

I44

-H (cyclic ether) compounds, with the a-Danomers preponderating. Posner and H a i n e ~also ~ ~employed the strong affinity of aluminum for fluoride ion14*to prepare C-glycosyl compounds. Thus, treatment of glycosyl fluorides 36a,36p,45a,45p,47p,and 75a with trialkyl-(or alkylary1)-aluminums [Et3AI, Ph,AlEtBu',Al(CH=CHC,H,,), and Et2AI(C=Cc6Hl3)] gave the corresponding C-glycosyl derivatives (aglycons being Et, Et, CH=CHC6H,3r and C=CC,H,,) with the a-Danomers preponderating. The use of Bu',AIH converted 45a into the oxolane. In the reaction of 145 with Et,AI, compound 146 was obtained.63This procedure was applied143to the reaction between glycosyl fluorides 36,47,and 48 (see Table I) and aryldiethylaluminum (aryl: furan-2-yl and 1-methylpyrrol-2-yl), and the corresponding aryl C-glycosyl compounds were obtained with retention of configuration at C- 1.

R= alkyl or aryl, R'= alkyl F

Et

0

0

0Bn

OBn

145

146

(142) G . H. Posner, J. W. Ellis, and J. Ponton,J. FluorineChm., 19 (1981) 191-198. (143) S. J. F. Macdonald, W. B. Huizinga, and T. C. McKenzie, J. Org. Chem., 53 (1988) 337 1-3373.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

119

C-Arylglycosyl derivatives (such as 149)of sugars have been preparedI4 by treatment of glycosyl fluorides (such as 147) with phenols under Lewis acid promotion (BF, - OEt,, SnCl,, or Cp2HfC1,- AgClO,). The reaction proceeds, as shown, through 0-glycosyl intermediates (such as 148)which are formed rapidly (-78", 10 min), and are gradually transformed into the C-glycosyl derivatives.

Some physical and chemical properties of glycosyl fluorides are now described. 2,3,4-Tri-0-benzoyl-P-~-xylopyranosyl fluoride and bromide are in the 'C, c ~ n f o r m a t i o n(all ' ~ ~substituents axial) both in solution and in the crystalline state, whereas the corresponding chloride crystallizes in a twist form (it also adopts the 'C, conformation in solution). The conformations of 2-deoxy-2-fluoro-~-~-mannopyranosyl fluoride in the solid state and in solution (D,O) were determined'46to be both ,C1, by X-ray crystallography and n.m.r. spectroscopy. 2,3,4,6-Tetra-0-acetyl-j?-~-gluco(13P) and related [ D-XY~O(I lp), L-arabino- (12a),and D-galacto-Ipyranosyl fluorides (I5p)a n ~ m e r i z ein ' ~the ~ presence of AgBF, and a trace of BF3.0Et2. It is presumed that the mechanism is that AgBF, furnishes a Lewis acid (BF,) in a solvent (the reaction rate is highly dependent on the species of the solvent), allowing equilibration as shown (see also the description on 91). Glycosyl fluorides generally resist hydrolysis (or solvolysis) under basic conditions. For example, 150,151 (see Section II,3 and Table 11), and the deprotected product (153)from 151 were unreactiveI4*to sodium methoxide in refluxing methanol (overnight), only 152 giving the correspondT. Matsumoto, M. Katsuki, and K. Suzuki, Tetrahedron Lett., 29 (1988) 6935-6938. K. Vangehr, P. Luger, and H. Paulsen, Chem. Ber., 113 (1980) 2609-2615. S. G. Withers, 1. P. Street, and S. J. Rettig. Can. J. Chem., 64 (1986) 232-236. Ya. V. Voznij, L. N. Koikov, and A. G. Galoyan, Carbohydr. Rex, I32 ( 1984) 339 - 34 I . (148) P. Kova?, H. J. C. Yeh, G. L. Jung, and C. P. J. Glaudemans, J. Carbohydr. Chem., 5 (1986) 497-512. (144) (145) (146) ( 147)

TSUTOMU TSUCHIYA

120

y2

-0

2 OAC - F

OAC

ing methyl a-D-ghcoside 154. Phase-transfer-catalyzed b e n ~ y l a t i o n ' ~ ~ (PhCH,CI, aq. NaOH, and Bu4NBr) of Ilp, 13p, lSp, and 20a gave the corresponding per-0-benzylated fluorides without anomerization. However, a-~-glucopyranosyl,~~ a-D-galactopyranosyl, a-Iactosyl, and 2-acetamido-2-deoxy-a-~-glucopyranosyl fluorides were alkylated150(with benzyl, allyl, and butyl bromides, and methyl and octyl iodides) in the presence of a basic reagent (Ag,O or KOH in DMF) in high yields in most cases. Aryl glycosides112J13 and 1,2-anhydro compounds118were also readily obtained. Glycosyl fluorides are generally cleaved under acidic conditions. For example, 150 and 151 each gave the same anomeric mixture148(155) on treatment with methanolic hydrogen chloride.

/

I loci 12

OCH2 I

I

OMe 150 It'=1;. RL= H 151 R ~ = H , R ~ = F

OMe

152 R ' = F, R ~ = R ~I I= 153 R1=R'= I I , RZ= f: 154 R'=R'= 1 1 , R ~ =OM^ 155 R1.Rz= H,OMe, R'= Bn

(149) V. S. Abramov, Ya. V. Voznij, and A. A. Galoyan, Zh. Obshch. Khim., 55 (1985) 1885- 1886. (150) J. Thiem and M. Wiesner, Synthesis, (1988) 124- 127.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

121

Chemical-ionization mass-spectral studies for typical per-0-acetylglycopyranosyl and -furanosyl fluorides have been reported. i 5 i ~ i 5 2 Several enzyme-catalyzed reactions of glycosyl fluorides have also been described. The a-glucan phosphorylase-catalyzed D-glucosyl transfer from a-D-glucopyranosyl fluoride to oligosaccharides(by way of the enzyme-Dglucosyl fluoride - primer complex) has been studied.'53 Mechanism-based inactivation of several P-D-ghcosidases by 2-deoxy-2-fluoro-P-~-gluco(I 56), -P-D-manno- (157), and -P-D-galacto-pyranosyl fluorides (158) was studied;ls4 the inactivating activities against the enzymes were found to decrease in the order of the compounds mentioned. Also, based on the 19F-n.m.r.spectra ofp-D-glucosidase-substrate (156 and 157) complexes [d ( 19F)of the complexes, formed after liberation of the 1-fluorine of the glycosyl fluorides as F- (6 12 l ) were 197.3 and 201, respectively], the glycosylenzyme structure, which was slowly hydrolyzed but had basically the same character as that of a complex formed in the usual, faster enzymic reaction, was c ~ n c l u d e to d ~have ~ ~ a~covalent ~ ~ ~ bond character (at C-1) with the a-D-anomeric configuration. This conclusion accorded with the generally accepted double-displacement reaction mechanism, including an oxocarbonium ion intermediate for the enzymic reaction. Cellobioside hydrolasecatalyzed hydrolysis of P-D-cellobiosyl fluoride has been reported. 156 2. Displacement of Sulfonyloxy Groups by Fluorine

Considerable interest has been focused on the efficient and rapid synthesis of 2-deoxy-2-[i8F]fluoro-~-glucose, a popular imaging agent for positronemission tomography (see Section II1,l). However, introduction of a fluorine atom at C-2 by nucleophilic displacement is generally not easyis7JSson account of the weak nucleophilic character of the fluoride ion. One possible

( I 5 I ) V. I. Kadentsev, 1. A. Trushkina. 0.S. Chizhov, and Ya. V. Voznij, Izvesf.Akud. Nuirk SSSR. Ser. Khim.. (1987) 2580-2584. ( I 52) V. 1. Kadentsev, I. A. Trushkina, 0.S. Chizhov, and Ya. V. Voznij, Izvest. Akud. Nuirk SSSR, Ser. Khim.. (1987) 2708-2711. ( I53) D. Palm, G. Blumenauer, H. W. Klein, and M. Blanc-Muesser, Biochem. Biophys. Res. Commzm.. 11 1 (1983) 530-536; related references are cited therein. ( I 54) S . G . Withers, K. Rupitz, and I. P. Street, J. Biol. Chem.. 263 (1988) 7929-7932. (155) S. G. Withers and 1. P. Street, J . Am. Chem. SOC.,110 (1988) 8551-8553. ( 1 56) J. K. C. Knowles, P. Lentovaara, M. Murray, and M. L. Sinnott, J. Chem. Soc.. Chem Commim., (1988) 1401- 1402. (157) A. C. Richardson, Carhohydr. Rex, 10 (1969) 395-402. (158) M. Miljkovii., M. GligorijeviC, and D. GliSin, J. Org. Chem.. 39 (1974) 3223-3226.

TSUTOMU TSUCHIYA

122

TABLE I Protected Glycosyl Fluorides

Compound

On C-1 of starting material

R

Reagent'

ff

:p

Yield (Yo)

References

2,3,4-Tri-O-R-o-xylopyranosyl fluoride OAc HF-Py 19:1 Br HF-TMP P OAc HF-CHIC12 Bn (18) ff 2,3,4-Tri-O-acetyI-~-arabinopyranosylfluoride (12) Br HF-TMP 2,3,4,6-Tetra-O-R-~-glucopyranosyI fluoride Ac (13) Br (a) W F , P HF-TMP Br P HF-Py OH ff 1:3 OAc (P) HF- Py 19:1 OAc (P) conc. HF-Py OH HF ff H (19) Piv (20) OPiv ( p ) HF (neat) ff Bn (47) CI (4 AgF P OH HF- Py a OH HF-Py 97:3 HF-Py OAc(a) 97: 3 1 :4 OH DAST OH DAST I :7.7 OH DAST I :45 Bz (51) OH DAST 2:21 4-0-Acetyl-2.3-di-O-R-6-0-( terf, Jtyldiphen) .lilyl)-D-~UCOpyranOSylfluoride SPh (a) DAST-NBS 5:1 SiBu'Me, (39) SPh (a) HF-Py-NBS 5:I Bn (40) SPh DAST- NBS - 1:l Methyl (2,3,4-tri-O-acetyl-~-o-glucopyranosyl flu0ride)uronate (I 7) Br(a) HF-TMP 2,3,4,6-Tetra-O-R-~-mannopyranosyI fluoride OH HF-Py ff Ac (14) Br HF-TMP P Bn (48) OAc HF-Py 19:I 2,4-Di-0-acetyl-3,6-di-O-benzyl-~-rnannopyranosyl fluoride (52) OH DAST-oxolane 4:1 6-Deoxy-2,3,4-tn-O-R-~-rnannopyranosyl fluoride Br HF-TMP ff (L) Ac (16) Bn (79) OH FMPTs 312 2,3:5,6-Di-O-isopropylidene-~-rnannopyranosyl fluoride (45) OH HF-Py ff OH Mitsunobu ff OH DAST 6.6:1 Ac ( 1 1 )

92 86 I00

68 35 81

62

35

10

82 68 89 91 99 92 92

30,I07 35 67 68 68 52 81 150 61 68 68 76 77 79 77

82 74 90

64 64 64

49

36

69 66 84

67 35 68

-

92

63

35 91

31 54 87

67 81 77

67 53 85 72 91 74

I

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

123

TABLE I (continued) On c - 1 of

Compound

R'

starting material

ReagenC

2-0-R'-3,4,6-Tri-0-R2-~-galactopyranosy~ fluoride R l = R 2 = A c(15) OH HF-Py Br HF-TMP OAc HF-Py R I = R Z = B n(49) R' = Ac, R2 = Bn (or pBrBn) ( 104) C1 (a) AgF-MeCN 2,3-Di-0-benzyl-4-0-R1-6-O-R2-~-galactopyfanosy1 fluoride OH R' = COCH2CI,R2 = Ac (53) DAST - oxolane R1 = Bn, R2 = Ac (54) OH DAST-oxolane OH DAST R1R2= CMe, (55) 2,3 : 5,6-Di-O-isopropylidene-~-gulofuranosyl fluoride (42) SPh ( P ) DAST-NBS HF-Py-NBS SPh ( P ) 3,4,6-Tri-0-acetyl-2-deoxy-~-arabIno-hexopyranosyl fluoride (SO) OAc HF-Py 2,3,5-Tri-~-acety~-~-xy~opyranosy~ fluoride (2 I ) 1.2-0-iso- HF (- 78") ProPYlidene 2,3,5-Tri-O-R-~-ribofuranosyl fluoride Bn (36) OH FMPTs OH HF-Py OH HF-Py OAc HF-Py OH Ishikawa OH DAST Bz (43) OH HF-Py OH DAST OH DAST 2.3,5-Tri-O-R-~-arabinofuranosyl fluoride Bn (41) OH HF-Py OH DAST Bz (44) OAC HF-Py 2.3,5-Tri-O-benzyl-~-arabinofuranosyl fluoride (75) OH DAST 3,5-Di-O-acetyl-2-deoxy-~-ery~~~r~pentofufanosy~ fluoride (46) OAC HF-Py

a:p

Yield (Yo)

References

19: 1

62 72 80

67 35 68

2:7

- 83

29

73

a

P

1.3 9:ll 1.3: 1

89

78 78 78

P P

91 80

64 64

19: I

82

68

1.4: 1

84

39b

79 68 89 88 90 79 99

62 67 68 68 72 77 67 77 61

58 78 -

67 76 70

10.5: 1

95

77

-

-

70

P -1:I 13:7 13:7 1:3 I :9.9 1 : 1.4

aP-P 37: 13 8:1 -

Abbreviations: TMP. 2,4,6-trimethylpyridinium fluoride: FMPTs, 2-nuoro-I-methylpyridinium tosylate; Ishikawa, Ishikawa reagent (Ref. 74): Mitsunobu, through Mitsunobu reaction (Ref. 80); NBS. N-bromosuccinimide; Piv, pivaloyl; and Py. pyridine.

I24

TSUTOMU TSUCHIYA

method for resolving the problem involves introduction of the strongly electron-withdrawing trifluoromethylsulfonyl (triflyl) g r o ~ p at ~ C-2 ~ ~ J ~ (acetolysis of CF,SO,Et is 30,000 times faster than that of ethyl tosylate161)with the combined use of nuked fluoride [tetraalkylammonium fluoride,162-164 tetrabutylammonium b i f l ~ o r i d e ' (Bu,N+HF;), ~~ crown ether- metal fluoride, and the like]. Levy and coworkers166treated methyl 4,6-0-benzylidene-3-0-methyl-2-0-triflyl-~-~-mannopyranoside (159; the corresponding (Y anomer being considered ineffective1s8)with fluoride ion [CsF in N,N-dimethylformamide (DMF), 1307 and obtained the 2-deoxy2-fluoro-~-~-glucoside derivative 160 in 42% yield. Removal of the 3-0methyl and other protecting groups [with BBr, in CH2C12,at room temperature (r.t.)] afforded1662-deoxy-2-fluoro-~-ghcose(2DFG, 909/0);however, as to this ready removal of the 3-0-methyl group, there was controversy (see later).

6

R~OCH~

R~O

159 161 163 164 165 166 168 170

R'= Me (POMc), R2=Me, R1= Bn @OBn), R'= Bn (a-OBn), R'= Me (a-OMe), R'= Me (a-OMe), R'= Me GOMe), R'= Me (POMe), R1= Me (POMe),

R2= R3= R4= R2= R3= R4= R2= Ac, R2= Me, R2= Bn, R2= Ac, R2= Rn,

R3R4= CHPh Bn Rn Rk4= CHPh R3R4= CHF'h R3R4= CHPh R3R4= CHPh R3= R4= Ac

-

-

?OR'

F 160 162

167 169 171

(159) R. W. Binkley and M. G. Ambrose, J. Carbohydr. Chem., 3 (1984) 1-49, (160) M. G. Ambrose and R. W. Binkley, J. Org Chem., 48 (1983) 674-677. (161) A. Streitwieser, Jr., C. L. Wilkins, and E. Kiehlmann, J. Am. Chem. Soc., 90 (1968) 1598- 1601. (162) L. A. Carpino and A. C. Sau, J. Chem. SOC.,Chem. Commun.,(1979) 5 14-515; related references are cited therein. (163) R.K.SharmaandJ.L.Fry,J. Org. Chem.,48(1983)2112-2114. (164) D. P. Cox, J. Terpinski, and W. Lawrynowicz, J. Org. Chem., 49 (1984) 3216-3219. (165) P. Bosch, F. Camps, E. Chamorro, V. Gasol, and A. Guerrero, TetrahedronLeu., 28 (1987) 4733-4736. (166) S . Levy, E. Livni, D. Elmaleh, and W. Curatolo, J. C h m Soc., Chem. Cornmun.,(1982) 972-973.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

172

173

174 175 176

125

R1=H, R2=OBn, R3=R4=Bn R'=OMe, R2=H, R3R4=CHPh R1=OMe, R2= H, R3= R4= Ac

Dessinges and coworkers'67prepared benzyl 3,4,6-tri-O-benzyl-2-0-triflyl-P-D-mannopyranoside (161) in three steps from the 1,2-0-dibutylstannylene complex 172, and treated it with fluoride ion [tetrabutylammonium fluoride (Bu,NF) at 60", or CsF at 130", both in DMF], to afford the 2deoxy-2-fluoro-~-~-glucopyranoside162 (45 - 509/0;see also, Section 11,3). Catalytic debenzylation gave 2DFG quantitatively. Haradahira and colleagues'68studied in detail the displacement reactions for several 2-0-triflylD-mannopyranosides (159,164,165,166,168,and 170), varying the fluorinating reagents [ KF, CsF, Bu,NF, and tetraethylammonium fluoride (Et,NF)] and solvents [MeCN, oxolane (THF), and DMF], and changing the reaction conditions (temperature and time). Although the (Y-Dcompounds (164 and 165) gave rise only to elimination products involving 173 (a on benzyl 3-0-benzyl-4,6-0-benzylidene-2-0-t~flyl-(~-~-mannopyranoside also gave the corresponding 2,3-unsaturated derivative, such as 174, exclusively), the p-D compounds (159, 166, 168, and 170) gave the corresponding 2-fluoro derivatives [159 160 (63-64%), 166 + 167 (4257%), 168 + 169 (21%), and 170 .--,171 (17-36%, mainly 176); the best yield for 166 + 167 being 57% accompanied by the enol ether 175 (21%; with Et4NF in MeCN, 50", 20 min)]. Although 159 gave 160 in good yield, removal of the 3-0-Me group from 160 was difficult, in contrast to the result of Levy and coworkers.'66The reactivity of the 2-triflate (178) of 177 was +

(167) A. Dessinges, A. Olesker, G. Lukacs, and T. T. Thang, Curbohydr. Rex, 126 (1984)

~6-~8. (168) T. Haradahira. M. Maeda, Y. Kai, H. Omae, and M.Kojima, Chem. Phurm. Bull., 33 (1985) 165-172. (169) W. Karpiesiuk, A. Banaszek, and A. Zamojski, Curbohydr. Rex, 186 (1989) 156- 162.

TSUTOMU TSUCHIYA

126

examined170with several reagents: Bu,NF on silica gel'71(MeCN, r.t., 8 h) gave the 2-deoxy-2-fluoro derivative 179 (40%) with the 2,3-unsaturated compound 180 (27%); the use of spray-dried KF (Ref. 172)-dibenzo-18crown-6 [MeCN, reflux (refl.), 1 h] gave 179 (48Yo) and 180 (37%), but, without the crown ether, the starting material was recovered; the use of CsF

I177 R = H 178 R=T~-I

180

I79

(DMF, 130", 30 min) gave 179 ( 1 1%) and 180 (2 1%); however, treatment of 177 with diethylaminosulfur trifluoride (DAST; see Section II,3) in pyridine-benzene (3 : 50,60", 3 h) gave 179 (78%) and 180 (6Yo). To avoid the elimination generally accompanying the foregoing fluorination, 1,6-anhydro derivative 181 was treated173with tetraalkylammonium fluoride (MeCN, THF, or Me,CO), whereupon the 1,6-anhydr0-2-fluoro derivative 182 was obtained in high yield (80-90%). Successive cleavage of the 1,6-anhydro ring and removal of the benzyl groups of 182 was performed by heating in 50%aqueous methanesulfonic acid ( I 20°, 30 min), to give 2DFG

t I82

ikahashi. d. Endo, S. Jmezawa, and H. Umezawa, Carbohydr. Chern.. 4 (1985) 587-61 1. (171) J. H. Clark, J. C'hern.SOC., Chem. Comrnun..(1978) 789-791. (172) N. Ishikawa, T. Kitazume, T. Yamazaki, Y. Mochida, and T. Tatsuno, Chem. Left., ( 170) T. Tsuchiya,

I

(1981) 761-764. ( 1 73) T. Haradahira, M. Maeda, Y. Kai, and M. Kojima, J. Chem. SOC.,Chem. Comrnun..

(1985) 364-365.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

127

(64%). 4-0-Benzoyl-6-deoxy-3-0-methyl-2-0-triflyl-a(183) and -p-~-glucopyranosyl fluorides (184) on treatment with Bu,NF or CsF, 2-deoxy-2-fluoro-~-mannoderivatives, 185 (75%) and 186 (55%), respectively.

183

R'=F,

184

R'=H.

K2= ti R~=F

185 1%

Some triflates were f l ~ o r i n a t e d ' ~in~a. ~short ~ ~ period of time at low temperature by using tris(dimethy1amino)sulfonium difluorotrimethylsiliate'^^ [(Me,N),S+F,SiMe;; (TASF)]. This reagent, an effective fluoride-ion donor, is a hygroscopic solid that is freely soluble in the common organic solvents, and scrupulously anhydrous conditions are essential for effecting fluorination. Using this reagent, the 2-triflates 159, 161, 163 (a-D-mannoside), and 166 (in CH,Cl, or CH,Cl,-MeCN) gave 160 (64%;0-20", < 10 rnin), 162 ( 1 1%;0-20", - 5 rnin), 174(-779owith no fluorineproduct; 23", 50 min), and 167 (45%; 40",10 min), respectively. In those cases where the yields of fluorinated products were low, unsaturated compounds were formed instead. prepared 2DFG by treatment of the 2,3-cyclic sulfate 187 with tetramethylammonium fluoride [Me,NF in refluxing (refl.) MeCN, 10 min]. The 2-fluoro-3-sulfate 189 was the major product, and it was converted into 2DFG (70%based on 187) on treatment with B(O,CCF,), (Min CF,CO,H, r.t., 5 min; the glycosidic bond was the most resistant). As the glycosidic bond of 189 was not cleaved readily (on account of the strongly electron-withdrawing fluorine atom at C-2), the 1-propenyl aglycon was used instead. Thus, glycoside 188 aff~rded,''~ through 190, 2DFG (85Yo) under milder reaction conditions (2 M HC1, refl., 10 min).

-

(174) W. A. Szarek, G. W. Hay, and B. Doboszewski, J. Chem. Soc., Chem. Comrnun.,(1985) 663-664. (175) B. Doboszewski, G. W. Hay, and W. A. Szarek, Can. J. Chem., 65 (1987) 412-419. (176) W. J. Middleton, U. S. Pat., 3,940,402 (1976); Org. Synth., 64 (1985) 221 -225. (177) T. J. Tewson, J. Label. Comp. Radiopharm., 19 (1982) 1629. (178) T. J. Tewson, J. Org Chem.. 48 (1983) 3507-3510. (179) T. J . Tewson and M. Soderlind, J. Carbohydr. Chem., 4 (1985) 529-543.

TSUTOMU TSUCHIYA

128

I: in7

R= ~e

188

K= CH=CIIMe

I89 I90

2-Deoxy-2-fluoro-~-mannose(2DFM) was also prepared by the S Nreac~ tion. TreatmentI8Oof benzyl(l91) or ally1 3,4,6-tri-O-benzyI-2-0-triflyl-PD-glucopyranoside (193) with Bu,NF (THF, 50") gave the 2-deoxy-2fluoro-D-manno derivatives, 192 (77%) and 194 (49%), respectively. Catalytic debenzylation or deallylation (PdCI,- NaOAc-aq. AcOH) of 192 or 194 gave the free sugar. Suitably protected methyl P-D-glucopyranoside 2-triflates (195, 197, 199, and 201) were treated181with tetraalkylammonium fluorides (Me,NF, Et4NF, or Bu,NF in refl. MeCN) to give 196 (32%), 198 (63-8190 with the 0-deacetylated isomer), 200 (81%), and 202 (687790).In the case of 201, the use of CsF (DMF or AcNH,) gave little or no 202, emphasizing the importance of selection of the appropriate fluorinating reagent. The 2-triflates 205 and 206, having an &-D-glucosidic bond, were treated similarly, but, as e ~ p e c t e d , ~ ' ~ 206 J ' ~gave the 2-deoxy-2-fluoro derivative 208 in only low (3090)yield (Bu,NF, refl. MeCN, 2.5 h), and 205 gave no fluorinated product; however, treatment of 207 with CsF (DMF, 120", 7 h) gave the 2-deoxy-2-fluoroderivative 209 in 6OYo yield'82(see also, 333 in Table 111). Cleavage of the glycosidic bond of 196, 198, 202, and 208 required,'*' as did the 2-deoxy-2-fluoro-~-gfucoisomers, relatively strongly acidic conditions(5 MHCI, refl. 30 min, or 30-5090 MeSO,H, refl. 20 min) to give 2-deoxy-2-fluoro-~-mannose(2DFM) [8 1 - 88%; 6790 for 208; 200 gave 2-deoxy-2-fluoro-3-0-methyl-~-mannopyranose (93?40)].Treatment181 of 200 or 208 with BBr, (CH,CI,, r.t., 50 min) did cleave theglycosidic bond, but the fluorine atoms were simultaneously replaced with bromine, to give the 2-bromo-2-deoxy derivative 210, respectively, in good yields. TreatrnentI7' of the 2-triflates 199 and 203 with TASF (CH,CI,) gave 200 (6590, -40",60 min) and 204 (2390,-40",5 h), respectively.

( I 80) T. Ogawa and Y. Takahashi, J. Curbohydr. Chem., 2 (1983) 46 1-467. ( 18 I ) T. Haradahira, M. Maeda, H. Omae, Y. Yano, and M. Kojima, Chem. Phurm. Bull.. 32

(1984) 4758-4766. ( 1 82) R. Faghih, F. C. Escnbano, S. Castillon, J. Garcia, G . Lukacs, A. Olesker, and T. T. Thang, J. Org. Chem., 5 I (1986) 4558-4564.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

129

OTtl 191 193 195 197 199 201 203

205 206 207

R’= R2= R’= RJ= Bn R’= CH2CH=CH,. R‘= R’= R4= Bn R’= Me, R’= R’= R4= Ac R’= Me, R’= Ac, R’R4= CHPh R ’ = M e , R’=Me, R’R‘=CHPh R’= Me, R‘= Bn. R3R4= CHPh R1= CH2CCIj, R?= Bn, R k J = CHPh

R’= Me, K?= OBz R’= Me, R’=OBn R’= Bn, R’=Nj

208 209

192 194 196 198 200 202 204

R’= Me, R’= OBn R’= Bn, R?=N?

210

The difference in reactivity between fluoride and the other halide ions was studied183in the displacement reactions of the 2-triflates (211 and 214) of 1,3,4,6-tetra-O-acetyl-~-~-glucoand -manno-pyranose. Treatment of 21 1 and 214 with tetrabutylammonium halides (halogen X = C1, Br, or I; refl. benzene) gave, respectively, the corresponding 2-halo-manno 212 and - g l z i c ~derivatives ~ 215 in high yields (the 2-iodo-~-mannoderivative from 211 was unstable and underwent gradual conversion into tri-0-acetyl-D-glucal), but, 211 and 214, on treatment with Bu,NF under the same conditions, gave, respectively, 5-(acetoxymethyl)-2-formylfuran(213) or a mixture of four products [216 (23%), 217, 218, and 2191. These differences in the reactions with F- and X- may be explained on the basis of the decreased (183) R. W. Binkley, M. G. Ambrose, and D. G. Hehemann, J. Carbohydr. Chem., 6 (1987) 203 -2 19.

I30

TSUTOMU TSUCHIYA

212

211

213

nucleophilicity and increased basicity of the fluoride ion. The formation of 213 and 218 (derived from 217) was explained183as involving initial binding of fluoride ion to the relatively unstable acetate groups ( I-C-OCOMe of 211 or 3-C-OCOMe of 217 F -C-OC(F)O-Me MeCOF -C-O-), with subsequent consecutive eliminations. Acid-catalyzed epimerization (>3 MHCI, 1 lo", 30 min) between 2DFG and 2DFM occurred, and was by means of I9F-n.m.r.spectros-

+

2DFG

+

-

+

2DFM

copy [19F6:-32.53,-32.38,-37.85,and-56.26in D,OfromC,F,(external reference) for a- and /3-2DFG, and a-and /3-2DFM, respectively]. This interconversion has also been observed in in vivo experiments: after injection of 2DFG into mice, formation of 2DFM and the 6-phosphates was ob( 184) T. Haradahira, M. Maeda, Y. Kanazawa, Y. Momozono, and M. Kojima, Chem. Pharm.

BUN.. 34 (1986) 1407-1410.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

131

served,lE5along with other metabolitesIg6in the tissues and urine, and by inJection18'of 2DFM, formation of 2DFG and the 6-phosphates [19F,Sfor aand P-2DFG 6-phosphate, and a- and P-2DFM 6-phosphate (in D,O): - 32.4, - 32.2, - 37.6, and -56.0 (from external C6F6),respectively]. TreatmentIEEof suitably protected 2-triflates 220 and 222 of methyl p-Dtalopyranoside with Et,NF (MeCN, 50", 30- 50 min) gave, respectively, the 2-fluoro-P-~-galactopyranoside221 (50%)and the unsaturated product 223 (74%).The mechanism was discussed.lE8Acid-catalyzed hydrolysis of 221 (5 TrOCH,

HOCH,

I

I

F

220

22 1

F 20FGal

Bn0

222

223

M HCl, refl. 30 min) gave 2-deoxy-2-fluoro-~-galactose(2DFGal,86%).The crystal structure of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-fluoro-~-~-galactopyranose has been reported.'89 Treatment of the 2-triflate (224) of methyl 3-azido-4,6-0-benzylidene3-deoxy-a-~-idopyranoside with Bu,NF (DMF, r.t.) readily gavelw the ( I 85) Y. Kanazawa, Y. Momozono, M. Ishikawa, T. Yamada, H. Yamane, T. Haradahira, M. Maeda, and M. Kojima, Lifi Sci., 39 (1986) 737-742. (186) T. Nakada, 1. L. Kwee, and C. B. Conboy, J. Neurochem.. 46 (1986) 198-201. ( I 87) Y. Kanazawa, Y. Momozono, H. Yamane, T. Haradahira, M. Maeda, and M. Kojima, Chem. Phurm. Bitll., 35 (1987) 895-897. (188) T. Haradahira, M. Maeda, Y. Yano, and M. Kojima, Chem. Phurm. Bull., 32 (1984) 33 17-33 19. (189) T. Srikrishnan andS. H. An,J. Carbohydr. Chem., 7 (1988) 571-581. (190) L. H. B. Baptistella, A. J. Marsaioli, P. M. Imamura, S. Castillon, A. Olesker, and G. Lukacs, Curhoh.vdr. Rex, 152 (1986) 310-315.

TSUTOMU TSUCHIYA

I32

2-fluoro-a-~-gulopyranoside225, despite the a-D-anomeric configuration of 224. This result was explained by a ,C, 'C, equilibrium, as shown. The structurally related, but conformationally rigid, a-D-altropyranoside 226 did not react." The situation seems analogous in the p-Dseries; thus, treatment of 227 gave the corresponding 2-deoxy-2-fluoro-~-~-gulopyranoside (CsF, 70", 5 h; 7 lYo), whereas 228 remained unchanged under the conditions.Is2

*

225

224

226

227

228

In the case of L-fucopyranosides 229 and 230, treatment with Et,N - 3HF (with Et,N, in MeCN, r.t., 16 h) gave a mixture of unstable, ring-contracted sugars,'91 231 and 232 (- 1 : 3 from 229; almost 1OOYo of 231 from 230) without the occurrence of any usual displacement or elimination [231: 2J1,F 63.7, 3J2,F 8, 'JF.C-2 30 Hz (indicating F-ring 0 being anti); 232: 2Jl,,63.4, 3J2,F25, 2JF,c-2 2 1.4 Hz]. A similar result was obtained from 233 (to give 234). Preparation of 2-fluorofuranoses is also important in relation to the synthesis of biologically active 2'4uor0 derivatives of nucleosides (see Section 111,4). Su and coworkers'92 prepared the 2-triflates 236 and 239 through acid-catalyzed methanolysis of 3,5-di-O-benzyl-1,2-O-isopropylidene-a-~ribofuranose [to give 235 (major) and 2381 and subsequent triflylation. On treatment with fluoride ion, the p anomer 236 afforded exclusively the furan derivative 237, whereas the a anomer 239 gave the 2-fluoro compound 240 (191) H. H. Baer, F. H. Mateo, and L. Siemsen, Curhohydr. Rex. 187 (1989)67-92.

(192)T.-L.Su,R.S.Klein,andJ.J.Fox,J.Org.Chem.,46(1981)1790-1792.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

Ic;l-.

133

MeO,

OMe

CI 1,

O

\ /O

229 230

233

R ' = O M c , K'= H K ' = 11, R'=OMe

23 I

232

234

in good yield (62%; Bu,NF in THF, - 10"; use ofLiF was unsuccessful).The lack of substitution reaction of 236 was attributed to hindrance by the 1-methoxyl group. Effective syntheses of the Q anomers 238 and 241 were acc~mplishedl~~ by treatment of a 1,2-complex with a stannane (formed by reaction of 3,5-di-O-benzyl-~-ribose and dibutyltin oxide) with Me1 or benzyl bromide to give 238 (49%) or 241 (83%); 241 was converted into 243 in good yield through 242. In the case of the 2-sulfonates (244) of 1 ,3,5-tri-O-benzoyl-~-~-ribofuranose, fluorination by Bu,NF was generally unsuccessful; only the triflate gave the corresponding 2-deoxy-2-fluoro-~-arabinofuranose245 (in only 20% yield).194However, fluorination of the imidazolylsulfonate 246 with KHF, 50% aq. HF (2,3-butanediol, 160", 1 h) gave 245 in 63% yield,195possibly by way of the 2-(fluorosulfonate)intermediate 247 (a group that was introduced as a leaving group by Hanessian and Vat~Ye'~~). Syntheses of 3-deoxy-3-fluoro sugars are described next. A rapid synthesis aimed at 3-deoxy-3-[1*F]fluoro-~-glucose (I8F-3DFG) was de~eloped.'~' (193) T.-L. Su. R. S. Klein. and J. J. Fox, J . Org. Chem., 47 (1982) 1506-1509. ( 194) D. F. Smee, M. Chernow, M. Krafi, P. M. Okamoto, and E. J. Prisbe, Nucleus. Nucleot., 7 (1988) 155-165. ( 195) C. H. Tann, P. R. Brodfuehrer, S. P. Brundidge, C. Sapino, Jr., and H. G. Howell, J. Org. C'ltcm., 50 (1985) 3644-3647. (196) S. Hanessian and J.-M. Vattle, Tetrahedron Lett.. 22 (1981) 3579-3582. (197) T. J. Tewson and M. J. Welch, J. Org. Chem., 43 (1978) 1090- 1092.

I34

TSUTOMU TSUCHIYA

U

HnO

OK

235 236

R=H R=Ttl

23H 239 241 242 244 246 247

R'= Me, R2= H, R3= Bn R'= Me, R2= Tfl, R'= Bn R'= R3= Bn, R2= H R'= R3= Rn. R2= Tfl R'=R'=Bz, R2=Ms, Tfl, or Ts R'= Rs= Bz, R2= SO2N2C.1133 R'= R3= Bz, R2= S02F

BnO

231

-

240

243 245

Treatment of 1,2 :5,6-di-O-isopropylidene-a-~-allofuranose 3-t1iflate'~~ (248) with CsF (refl. DMF, 25 min) gave 249 (7 1%), which, on deprotection (BC1, - CH2C1,, and then with water), afforded 3DFG. The crystal structure of 249 has been reported.199Compound 249 was also readily prepared from 248 by treatment*''' with Et,N. 3HF (MeCN, 80", 60 h; 65%) or with174J75 TASF (CH2C12,0-20", < 10 min; 66Yo), but 250 gave the 3,4-unsaturated compound 380 (see Section II,3 and Table 111). 3-Deoxy-3-fluoro-P-~-galactopyranose @-3DFGal) was obtained20'*202similarly, by treatment of L. D. Hall and D. C. Miller, Curbohydr. Rex, 47 (1976) 299-305. M. Argentini, R. Weinreich, R. Oberti, and L. Ungaretti, J. Fluorine Chem., 32 (1986) 239-254. D. Picq and D. Anker, Carbohydr. Rex. 166 (1987) 309-313. J. S. Brimacornbe, A. B. Foster, R. Hems, J. H. Westwood, and L. D. Hall, Can.J. Chem., 48 ( 1970) 3946 - 3952. P. KovaE and C. P. J. Glaudemans, Curbohydr. Res., 123 (1983) 326-331.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

Me C

135

HOCH, I

'OCH

>

-w

F ) . , O H R2

1;

0-CMe,

0 CMe,

249

0-CMc,

2.50

OH

3DFG R'=H. R2=OH ~ D F G ~R'=OH. I R ~ H=

F 25 1

1,2 : 5,6-di-O-isopropylidene-a-~-gulofuranose 3-sulfonates with fluoride ion (Bu,NF or resin-F-) accompanied by 380 (see Section 11,3). Methyl 2,3-dideoxy-3-fluoro-~-erythvo-pentofuranos~de (251) was prepared203.204 (Bu,NF, THF) from a protected 3-O-triflyl-~-threoprecursor. An improved synthesisZoS of 4-deoxy-4-fluoro-~-glucose(4DFG) was reported; treatment of methyl 2,3,6-tri-O-benzyl-4-O-mesyl-a-~-galactopyranoside (255) with Bu,NF (refl. MeCN, 3 d) gave the 4-deoxy-4-fluoro-~glucoside (256,73%). Later, the 4-triflate analog of 255 was reported to give 256 (67%) on treatment17swith TASF (CH,C12, 0-20", 10 min). Treatment20(jof methyl 2-benzamido-3-O-benzyl-2-deoxy-4-O-mesyl-6-0-trityla-D-galactopyranoside(257) with Bu,NF (refl. MeCN, 18 h) gave 258 (67%), but, with KF (a slightly less reactive nucleophilic reagent) in boiling ethanediol, gave no fluorinated product. Again, methyl 4-O-mesyl-2,3-di-0methyl-6-O-trityl-a-~-galactopyranoside (252) failed to react with CsF in ethanediol, whereas the detrityl derivatives (253 and 259) of 252 and 257, respectively, did reactzMp207with CsF or JW, to give 254 and 260, both in moderate yields. When the 4,6-dimesylate 261 was treated with KF (refl.

(203) G. W. J . Fleet and J. C. Son, Tetrahedron Letf..28 (1987) 3615-3618. (204) G. W. J . Fleet, J. C. Son, and A. E. Derome, Tetrahedron, 44 (1988) 625-636. (205) D. P. Lopes and N. F. Taylor, Carbohydr. Res., 73 (1979) 125- 134. (206) L. Hough, A. A. E. Penglis. and A. C. Richardson, Can. J. Chem.. 59 (1981) 396-405. (207) A. B. Foster, R. Hems, and J. H. Westwood, Carbohydr. Res., 15 (1970) 41 -49.

TSUTOMU TSUCHIYA

136 CH~R~

" O K )

R3CH,

- &)

OMe

OMe

R'

R1

252 253 255 257 259 261

R' OMe OMe OBn NHBz NHBz NHBz

R2 Me Me Bn Bn Bn Bn

FF)oM

R3 OTr OH OBn

OTr OH OMS

NHBz 263

254 256 258 260 262 R3=F

ethanediol, - 30 min) or Bu,NF (refl. MeCN, 24 h), a mixture of4,6-difluoride 262 and unsaturated product 263 was obtained (65%,262:263 = 3 :2 for the former case). RefluxingZ0*of methyl 2,3-di-0-benzyl-4-0-(p-bromophenylsulfonyl)-60-trityl-P-D-ghcopyranoside (264) with Bu,NF in acetonitnle gave the 4deoxy-4-fluoro-~-~-galactoside derivative 265 in low yield (30%),but, with an anion-exchange resin (dehydrated Amberlyst A-26, F form, refl. benzene, 3 -4 d), gave 265 in 77Y0yield.~~ Similar treatmentzI0[anion-exchange resin (F- form), refl. benzene] of the 6-0-benzoyl derivative 266 gave 267 in moderate yield, and this was converted into methyl 4-deoxy-4-fluoro-P-~galactopyranoside. Analogous reaction2I1(Bu,NF, DMF, 120°,48 h) of the 6-O-(tert-butyldimethylsilyl) derivative 268 gave 269 in good yield (72%) because of removal of the silyl group during the reaction. The 4,6-difluorogalactoside 276 was prepared2Mfrom 274 in two ways using Bu,NF (refl. MeCN, 4-5 d, 77%) or KF (refl. ethanediol, -30 min, 62%). Similarly, benzyl 2-acetamido-3,6-di-~-benzyl-2,4-dideoxy-4-fluoro-a-~-galactopyranoside2I2 (59%) and methyl 2,6-di-0-acetyl-3-(tert-butoxycarbonylamino)-3,4-dideoxy-4-fluoro-~-~-galactopyranos~de~~~ (70%) were pre(208) A. Maradufu and A. S. Perlin, Curbohydr. Res., 32 (1974) 26 I - 277. (209) P. KovaE and C. P. J. Glaudemanq J. Curbohydr. Chem.. 2 (1 983) 3 13 - 327. (210) Y. Ittah and C. P. J. Glaudemans, Curbohydr. Res.. 95 (1981) 189- 194. (21 1) J. E. Nam Shin, A. Maradufu, J. Marion, and A. S. Perlin, Curbohydr. Rrs., 84 (1980) 328-335. (212) R. L. Thomas, S. A. Abbas, and K. L. Math, Curbohydr. Res., 184 (1988) 77-85. (213) R. Albert, K. Dax, A. E. Stiitz, and H. Weidmann, J. Curbohydr. C'hem.. 1 (1982-83) 289-299.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

R'

264 266 268 270 272 274 275

K' OMe OMe OMe OMe OMe 1-1 H

R? H I1 tl ti H OMe OMe

137

R'

R"

R1

RS

ORn OBn ORn OBn OBn NHBz NHBz

SOlC,HjBrp M\ M\ Tfl Ttl M\ M\

OTr OBZ OSiMQBu' OBn OTfl

OMS F

265 261 26Y Rs=O H 271 273 R S = F 276 R S = F

pared from the corresponding 4-0-mesyl (or -triflyl)-D-glucopyranosides (Bu,NF, MeCN). The 4-deoxy-4-fluoro (271, 77%) and 4,6-dideoxy-4,6difluoro-D-galactosides 273 (39%) were readily prepared175from the 4-triflate 270 and 4,6-ditriflate 272, respectively, with TASF (CH,Cl,, 40", 20- 30 min). Compound 271 is quite resistant95to acid-catalyzedhydrolysis, and gave, under moderately strong conditions [AcOH-M aq. HCl (7 :2), 1 lo", 4 h], 2,3,6-t~-0-benzyl-4-deoxy-4-fluoro-~-galactopyranose (55%). The 4-triflates 277,279, and 281 of benzyl2,3-anhydro-a-~-and -P-L-~bopyranosides and -a-D-lyxopyranoside gave,*14s215 readily, on reaction with Bu4NF [C6H6(24 h) or MeCN (5-8 h), r.t.1, the respective 2,3-anhydro-4deoxy-4-fluoroderivatives 278,280, and 282 in good yields. The conformation ( O H s ) of the starting compounds remained the same after fluorination. Methyl 2,3-anhydro-4-deoxy-4-fluoro-a-~-lyxopyranoside (284, 86%) was prepared200from the 4-triflate 283 by treatment with Et,N. 3HF (CH,Cl,Et,N, 40°, 5 h). 5-Deoxy-~-fluoro-~-ghcose and -L-idose were synthesized216from 1,2O-isopropylidene-a-~-glucofuranurono-6,3-lactone (285). Treatment of 1,2-0-isopropylidene-~-0-trifly~-a-~-~uco(286; prepared from 285) and -~-~-ido-furanurono-6,3-lactones (289; prepared from 288), with Bu,NF

-

(214) F. Latif. A. Malik, and W. Voelter, Z. Nuturforsch., TeilE, 40(1985) 317-318. (215) F. Latif, A. Malik, and W. Voelter, Justus Liebigs Ann. Chem., (1987) 617-620. (216) R. Albert, K. Dax, S. Seidl, H. Sterk, and A. E. Stiitz, J. Curbohydr. Chem., 4 (1985) 5 13-520.

I38

TSUTOMU TSUCHIYA

277 278

283

284

R ' = I I,

K'=F.

R2= O'l'll R~=H

R ' = H . R2=OTtl R1= F. R 2 = H

(MeCN, r.t., 24 h) gave, respectively, a mixture of fluorinated derivatives 287 (67%) and 290 (6%),and 290 (549/0) and 287 (1 1Yo). In each case, the minor products were formed by C-5 epimerization of the starting compounds prior to the displacement, followed by fluorination. Reduction (NaBH,) of 287 and 290, followed by hydrolysis, gave an anomeric mixture of 5-deoxy-5fluoro-L-idofuranose (from 287) and 5-deoxy-5-fluoro-~-glucofuranose

291 285 2x6 2x7 2nx 289 2Yo

RLOH. R'=

[mi,

R'= tl, K'= H, R ' = ti, R'=F.

K?= t i K?= I I K2= F R'= OtI

RL= OTtl K2=11

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

139

(from 290) in high yields. In the n.m.r. spectra of 287,290, and some other products, long-range couplings were observed [JF,H-, 1.2 (287) and JF,H-2 3.5 Hz (29O)J. ( 1S,2R,3R,4S)- I -Azido-4-benzyloxy-3-(benzyloxymethyl)-2fluorocyclopentane (291), a synthon for carbocyclic nucleosides, was prepared2" from the 2-tnflate epimer of 291 with Bu,NF. Similarly, (a)- ( 1p,2a,3a,4/3,5p)- 2,3- (dimethylmethylenedioxy)- 5-fluoro- 4- [(phenylmethoxy)methyl]-1-cyclopentyl azide (using TASF) and (k)-( la,2a,3/3,4a,5a)-2-(p-anisyldiphenylrnethoxy)-4-fluoro-3[(phenylmethoxy)methyl]-6oxabicyclo[3.1.O]hexane (using Bu,NF) have been prepared.218 Displacement reactions of terminal sulfonic esters with fluorine may be expected to proceed smoothly, although, in contrast to the other halogens, this is not necessarily so. Treatment38J60of methyl 6-O-tosyl-a-~-glucopyranoside (KF in boiling ethanediol) or 1,2,3,4-tetra-O-acetyl-6-O-triflyl-pD-glucopyranose (Bu,NF) with fluoride ion gave only poor yields of the corresponding 6-deoxy-6-fluoro derivatives. In contrast,2Mtreatment of the 4,6-dimesylate 274 with Bu,NF (refl. MeCN, 1 h) gave the 6-fluoro derivative 275 in high yield (87%); demesylation (NaOH-MeOH) or inversion at C-4 (PhCO,Li, in hexamethylphosphoric triamide) afforded crystalline 2amino-2,6-dideoxy-6-fluoro-a-~-glucopyranose hydrochloride (292) or methyl 2-acetamido-2,6-dideoxy-6-fluoro-au-~-galactopyranoside (293,

I

NHR'

2Y2 293

R ' = H W J , R'= H, R'=OH R ' = Ac, K2=OH, K'= ti

-

30% based on 275), respectively. Treatment2I9of methyl 2,3,4-tri-O-benzyl-6-O-tosyl-a-~-glucopyranoside with KF in poly(ethy1ene glycol) 400 (mol. weight, 380-420; 70°, 44 h), gave the 6-fluoride in 63% yield without elimination. (217) K. Biggadike, A. D. Borthwick, D. Evans, A. M. Exall, B. E. Kirk, S. M. Roberts, L. Stephenson. and P. Youds, J. C h m . Soc., Perkin Trans. 1, (1988) 549-554. (218) G. V. B. Madhavan, D. P. C. McGee, R. M. Rydzewski, R. Boehme, J. C. Martin, and E. J. Prisbe, J. Med. Chem., 31 (1988) 1798- 1804. (219) D. Badone, G. Jommi, R. Pagliarin, and P. Tavecchia, Synthesis, (1987) 920-921.

TSUTOMU TSUCHIYA

I40

Direct conversion of the 6-sulfonates of D-galactopyranosides into their 6-deoxy-6-fluoro derivatives is usually hindered, because of the polar fieldeffect exerted by the lone electron pairs of the axial 0-4 (Ref. 158). Thus, the 6-0-mesylgalactoside 294 gave220the 3,6-anhydro derivative 297 (23%) as the only isolated product on reaction with CsF in boiling ethanediol. How-

NHAc 294 295 2%

R'= R2= Bn, R3= OMS R'R2= CMq, R3= OMS R ' R ~ = C M Q , R"=F

NHAc

298

291

299

300

OBz

301

0-CMq

R=OTs R=OTfl R=F

OBz

OBz

302

303

R= 2,3,4,6-tena-0-benzyIaD-galactopyranosyl

ever, when 2,3-d~-O-benzoyl-4-O-glycosyl-6-O-t~flyl-~-~-galactopyranoside (301)was treated22'with TASF, the 5,6-unsaturated compound302 was the major product (79%), along with the minor (16%) 6-deoxy-6-fluoro derivative 303. This reactivity difference between 294 and 301 may be ascribed principally to the substituent at C-3; the nucleophilicity of 0 - 3 bearing an acyl group should be weaker than that bearing a benzyl group. When, however, the 6-0-mesyl compound 295, having a 3,4-O-isopropylidene group, was treated similarly,220the 6-deoxy-6-fluoroderivative 296 was

(220) M. Sharma, G.G . Potti. 0.D. Simmons, and W. Korytnyk, Curbohydr.Rex, 162 (1987) 41-51. (221)J. Kihlberg, T. Frejd, K. Jansson, and G. Magnusson, Curhohydr. Res.. 176 (1988) 281-294.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

141

obtained in 67% yield. Also,2221,2 : 3,4-di-O-isopropylidene-6-O-tosyl-a-~galactopyranose (298) gave the 6-deoxy-6-fluoro derivative 300 in acceptable yield. Szarek and coworker^^^^,'^^ reported that the 6-triflate 299 gave 300 readily by reaction with TASF. Compound 300 was also preparedzm from 299 by treatment with Et,N - 3HF(MeCN-Et3N, 50", 6 d; 8790). These results suggest that the compounds having a 3,4-O-isopropylidene group adopt a conformation (more precisely, the orientations ofthe 0-4 lone pairs) that does not hinder the approach of the fluoride ion at C-6. Treatment of 6-0-tosylglycal304 with Bu,NF (DMF, 90°, 2 h) gave the corresponding 6-fluoride (305) in high yield.223

304

305

2,6-Dideoxy-6-fluoro compounds 306, 307, 308, and 309 were also formed224-227 from the corresponding 6-bromides by treatment with A@, with the object of preparing the 5-enopyranosides.

R'O 306 307 308 30Y

K'=Bz, R2=Me K'=Mc. R'=Bz K'= Bz, R'= Bn R'= Ac, R'= 4~-ocetyl-2.3,6-trideoxya-Leryrhro- hex-2-enopyranosyl

(222) D. R. Christman, Z. Orhanovic, W. W. Shreeve, and A. P. Wolf, J. Label. Comp. Radiopharm., 13 (1977) 555-559. (223) K. Bischofberger, R. H. Hall, A. Jordaan, and G . R. Woolard, S. Afr. J. Chem.. 33 (1980) 92-94. (224) J . Yoshimura,T. Yasumori,T. Kondo,andK.Sato, Carbohydr. Res., 106(1982)cl -c3. (225) J . Yoshimura, T. Yasumori, T. Kondo, and K. Sato, Bull. Chem. SOC.Jpn.. 57 (1984) 2535-2537. (226) T. Yasumori, K. Sato, H. Hashimoto, and J. Yoshimura, Bull. Chem. SOC.Jpn., 57 (1984) 2538-2542. (227) J . Thiem, H.-W. Kluge, and J. Schwentner, Chem. Ber., I13 (1980) 3497-3504.

TSUTOMU TSUCHIYA

I42

Two branched-chain sugars, methyl 3-azido-4,6-0-benzylidene-2,3,dideoxy-3-C-(fluoromethyl)-a-~-urub~~~hexopyranos~de and methyl 2azido-4,6-O-benzylidene-2,3-dideoxy-2-C-(fluoromethyl)-~-~-r~bohexopyranoside have been prepared228through the usual displacement reactions. Treatment of 1,2 :3,5-di-O-methylidene-6-O-tosyl-a-~-glucofuranose (310) with KF (in refl. ethylene glycol, 3 min) gave229a mixture of 6-deoxy-6fluoro (311, ~OYO), an alkene (312, l8Y0), and 6-0-(2-hydroxyethyl) derivatives ( 12%). H2C 0 1 ' s

I

r;Q

~

H2C F /Xv,I

+

y(Hy) CII2

II

H$2

0-CII,

310

31 I

312

6-Deoxy-6-fluoro-~-ascorbic (314) was prepared from methyl 2,3-O-isopropylidene-6-O-tosyl-a-~-gulosonate (313) by reaction with KF followed by isomerization of the product (with H+ cation-exchange resin).

3. Introduction of Fluorine by the DAST Reagent

Dialkylaminosulfur t r i f l ~ o r i d e s , ~exemplified ~ ' . ~ ~ ~ by diethylaminosulfur t r i f l ~ o r i d e(Et2NSF3, ~~~ DAST), were introduced into carbohydrate chemistry as fluorinating agents by Sharma and K ~ r y t n y k and , ~ ~they ~ can be T. T. Thang, M. A. Laborde, A. Olesker, andG. Lukacs, J. Chem. SOC.. Chem. Commun.. (1988) I58 I - 1582. H. C. Srivastava and V. K. Srivastava, Curbohydr. Rex. 60 (1978) 210-218. J. Kiss and W. Arnold, Experientia, 36 (1980) 1138- 1139. L. N. Markovskii, V. G. Pashinnik, and A. V. Kirsanov, Synthesis. (1973) 787-789. W. J. Middleton, J. Org. Chem., 40 (1975) 574-578. W. J. Middleton and E. M. Bingharn, Org. Synth., Col. Vol., 6 (1988) 440-441. M. Sharma and W. Korytnyk, Tetrahedron Lett., (1977) 573-576.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

143

handled without hazard (the similarly reactive sulfur tetrafluoride is much more hazardous). The DAST reacts with a hydroxyl group (C-OH), giving an unstable and intensely electron-withdrawing fragment, C-OSF2NEt2,with liberation of HF; approach of a fluoride ion (derived from the HF) then forms a C- F bond, with inversion of configuration. However, at positions where the S N reaction ~ is hindered, the reaction sometimes stops at the COSF2NEt2stage and the hydroxyl group is recovered after conventional isolation. When the approaching fluoride ion is somewhat hindered, and an electron-rich or a weakly bonded group is present near the COSF2NEt2 group, fluorination sometimes occurs by mechanisms other than the simple S N reaction, ~ and leads to migration of the neighboring group(& retention of configuration, anhydride formation, or elimination; in some cases, no fluorinated compound is obtained. These alternative reactions are described later. These rather unusual reactions are also observed in the displacement reactions of sulfonic esters by fluoride ion, but are less frequent. A characteristic property of the unstable fragment COSF2NEt2originates from the fact that the OSF2NEt2portion is sometimes cleaved rapidly, with only slight induction by the neighboring group(s), thus initiating the unusual reactions just noted. Fluorine- 18-labeled DAST has been ~ r e p a r e d . KovtiE ~ . ~ ~ ~and coworkers236stated that dimethylaminosulfur trifluoride is a slightly better reagent than DAST. N,N-Diethyl-l , l,2,3,3,3-hexafluoropropylamine74~75 reacts similarly, and has also been used for the fluorination of hydroxyl groups. In relation to preparation of the short-lived 2-deo~y-2-[’~F]fluoro-~-glucose (I8F-2DFG; see Section 111, I), a rapid and high-yielding synthesis of 2DFG was needed. TreatmentI6’ of benzyl 3,4,6-tri-0-benzyl-P-~-mannopyranoside (315) with DAST(CH2C12,40°, 5 min)gave the 2DFG derivative 162 (~OYO),the yield being higher and time being superior to those (45 -50%, 30 min) required for the displacement reaction of the 2-tnflate 161 by fluoride ion (Bu,NF or CsF; see Section 11,2). Treatment237of 1,3,4,6-tetra-O-

315 316

R=Rn R=Ac

162 317

318

(235) M. G. Straatmann and M. J. Welch, J . Nucl. Med.. 18 (1977) 151-158. (236) P. KovaE, V. Sklenii, and C. P.J. Glaudemans, Carbohydr. Res., 175 (1988) 201 -213. (237) P. KovaE, Curhohydr. Res., 153 (1986) 168-170.

TSUTOMU TSUCHIYA

I44

acetyl-P-D-mannopyranose (316), which was obtained from D-mannose in a one-vessel reaction, with DAST (diglyme, 100- 1 lo", 7 min) gave the 2deoxy-2-fluoro-~-~-gluco derivative 317 in 77% yield. TreatmentI'O of 177 (see Section II,2) with DAST gave 179 (78%).Similarly, the 4-O-benzoyl-2fluoro-L-oleandrosideanalog 318 (8090)was prepared238from benzyl 4-0benzoyl-6-deoxy-3-O-methyl-~-~-mannopyranoside. Treatment239of 1,2unprotected 3,4,6-tri-O-benzyl-~-mannose(319) with DAST [CH2Cl,, room temperature (r.t.), 24 h] gave 3,4,6-tri-O-benzyl-2-deoxy-2-fluoro-~D-glucopyranosyl fluoride (322, 3690) and 3,4,6-tri-O-benzyl-a-~-mannopyranosyl fluoride (321, 18%); in this reaction, 321 was not further fluorinated. Evidently, 322 is formed from the intermediate 320 by further

1;

319

320

32 I

322

fluorination, but the fluorination of 321 is inhibited by the electronegative a-fluorine atom, which hinders the same-side approach of fluoride ion. A similar result was obtained9' in the DAST treatment of 4-O-benzoyl-6deoxy-3-O-methyl-~-mannopyranose (323), when two fluorinated L-oleandrose analogs, 324 (62%) and 325 (34%), were formed.

OH

01I

323

324

325

(238) C. Bliard, P. Herczegh, A. Olesker, and G. Lukacs, J . Carhohydr. Chem., in press. (239) 1. P. Street and S. G. Withers, Can. J. Chem.. 64 (1986) 1400- 1403.

DEVELOPMENTS O F FLUORINATED CARBOHYDRATES

145

Lukacs and coworkersza observed the formation of three unexpected compounds on treatment of benzyl 3-azido-4,6-O-benzylidene-3-deoxy-aD-altropyranoside (326) with DAST [refluxing (refl.) benzene]; these were 3-azido-2-fluoro-a-~-altro-(327, 40%)and 2-azido-3-fluoro-a-~-gluco-pyranosides (328, 40%),and 3-azido-2-O-benzyl-~-~-allopyranosyl fluoride (329, 15%).Nicolaou and coworkers24'also observed similar reactions involving aglycon migration when a series of a-D-manno- (represented by 330), P-D-~~ucoand P-galacto-pyranosides (represented by 332) were treated with DAST (CH2Cl,, 0-45",56-93% yields of products). The l-fluorides 329, 331, and 333 can be utilized for glycosylation reactions (see Section II,1). Street and Withers,z39Kovai: and coworkers,148and Lukacs and c o ~ o r k e r s also ~ ~ Jobserved ~~ similar reactions; thus, benzyl 3-azido4,6-O-benzylidene-3-deoxy-a-~-glucopyranoside (334) gave, on treatment with DAST, the 2-deoxy-2-fluoro-a-~-mannoderivative 335 and a mixture of a- and P- 1-fluorides (337 and 336) with 1-benzyloxy-group migration. Also, methyl 3-0-benzyl-4,6-O-benzylidene-a-~-mannopyranoside (338) and the related D-manno type of compounds (339 and 340) having a free 2-hydroxyl group gave a similar mixture of 1-fluorides (see Table 11). The mechanism of these reactions for 338,339, and 340 involving a 1,2-shiftmay

326

327

330

328

329

33 I

X= OMe, OAC,SPh, N3. or a proiecied glycosyloxy group R=SiM%CMq. or Rn (240) S. Castillon, A. Dessinges, R. Faghih, G. Lukacs, A. Olesker, and T. T. Thang, J. Org. Chern..50 (1985) 4913-4917. (241) K. C. Nicolaou, T. Ladduwahetty, J. L. Randall, and A. Chucholowski, J.Am. Chem. SOC.,108 (1986) 2466-2467.

TSUTOMU TSUCHIYA

I46

333

332 X= SPh,N3,OBn, or a protected glycosyloxy group R=SiMqCMq. Me.or 3.4-0 -isopropylidene group

be explained as follows: the S-F bond of the intermediate I is cleaved, with concurrent migration of OR to give a 1,2-trans 1-fluoride, I1 (route a), or participation of the axial 1-OR group with concerted removal of the axial

OH

%OR

UK

OR

IV

V

2-sulfoxo group, gives the 1,2-alkoxonium ion inte~-mediate'~*.~~~ 111 (route b), which is then transformed into I1 by the action of fluoride ion, or into another intermediate (IV) of glycosyloxonium s t r u c t ~ r ewhich , ~ ~ is~then ~~~~ transformed into I1 and V ( I ,2-cis 1-fluoride)according to the direction of approach of the reacting fluoride ion. These reactions require, therefore, the 1,2-trans relationship of the substituents in the starting material. In the case of 326, the 2-sulfoxo group in VI is removed by participation of either the 3-azido or 1-benzyloxygroup, to give the azidonium or oxonium interme-

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

I47

F

F

/1

/1

VI

328

327

329

diate (VII or VIII), which then is converted by fluoride ion into 327 (from VII and VIII), 328 (from VII), or 329 (from VIII). In the reactions'82of334 and benzyl3-azido-4,6-O-benzylidene-3-deoxy-~-~-idopyranoside (341), unexpected cis-shifts of an OBn group were observed (see Table 11). These shifts were explained on the basis of participation of an azido group at C-3, as shown.

Fluorination with retention of configuration (such as in 327) has often been attributed to steric crowding on the opposite side of the hydroxyl group, hindering the back side of the fluoride ion for S N attack. ~ However, a possible SNi mechanism by loss of an OSF2NEt2fragment to give a carbonium cation, followed by front-side attack of fluoride ion, should be

148

TSUTOMU TSUCHIYA

carefully evaluated from the alternative viewpoint of neighboring-group participation, as just described. In the case of DL-1-0-benzoyl3,4,5,6-tetra-O-benzyl-2-(bromomethyl)-myo-inositol (342) or the 2-acetoxymethyl analog 343 (see Table 11), b r o m o n i ~ mor~ ~1,3-dioxolan-2~ y l i ~ m ion~intermediates, ~ ~ respectively, are considered to be formed after loss of the OSFzNEt, fragment. Fluorinations with retention of configuration under clear SNi conditions are although they are outside the scope of carbohydrate chemistry. Formation of fluorohydrin 345 from the protected (+)-aminotriol344, and related unusual reactions (see Table II), were explained by a hydride shift as ~ h o ~ n Other . ~ ~ unusual ~ , fluor~ ~ ~ * ~ ~ ~ inations by DAST on pyranoside and furanoside structures are described in Table 11. NHDNP

344 ( raccrnatc )

345 (raccmatc)

TIPS= -(W)zSiOSi(*

DNP= dinitrophcnyl

S. S. Yang, T. R. Beattie, and T. Y. Shen, Synfh. Cornmiin.. 16 (1986) I3 I - 138. S. S. Yangand T. R. Beattie, J. Org. Chem., 46 (1981) 1718- 1720. T. G. C. Bird, P. M. Fredericks, E. R. H. Jones, andG. D. Meakins, J . Chem. Soc., Chem. Cornmiin.,(1979) 65-66. K. Boulton and B. E. Cross, J. Chem. SOC.,Perkin Trans. 1, (1981) 427-432. K. Bannai, T. Tom, T. Oba, T. Tanaka, N. Okamura, K. Watanabe, A. Hazato, and S. Kurozumi, Tefrahedron,39 ( 1983) 3807 - 38 19. K. Biggadike, A. D. Borthwick, D. Evans, A. M. Exall, B. E. Kirk, S. M. Roberts. L. Stephenson, P. Youds, A. M. Z. Slawin, and D. J. Williams, J. Chem. SOC..Chem. Cornmiin.,(1987) 25 1-254. K. Biggadike, A. D. Borthwick, A. M. Exall, B. E. Kirk, and R. A. Ward, J. Chem. Suc.. Chem. Comrniin.. (1988) 898-900.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

I49

Benzyl 2-acetamido-6-O-benzyl-2,4-dideoxy-4-fluoro-~-~-glucopyranoand the corresponding free sugar250were prepared from benzyl2-acetamido-3-O-allyl-6-O-benzyl- (or -3,6-di-0-benzyl)-2-deoxy-a-~-galactopyranoside by treatment with DAST (in diglyme). A number of 6-deoxy-6-fluoroaldohexoseshave been prepareds~79,234~251 in mostly good yields by treatment with DAST of the 6-hydroxyl precursors (see also, Table 11). S ~ m a w a r d h a n atreated ~ ~ ~ non-protected methyl a - ~ glucopyranoside (346) with DAST in the absence of solvent, and obtained methyl 4,6-dideoxy-4,6-difluoro-a-~-galactopyranoside (350)in good yield. In a similar procedure,253methyl P-D-gluco- (352) and a-D-manno-pyranosides (369) respectively gave methyl 3,6-dideoxy-3,6-difluoro-~-~-allopyranoside (358) and 4,6-dideoxy-4,6-difluoro-a-~-talopyranoside (370). Card254,255 examined in detail the behavior of various non-protected (and some protected) D - ~ ~ U C Oand D-manno-pyranosides with DAST in dichloromethane (see Table III), and obtained the following results: (1) at short reaction times, a- and P-D-glucopyranosides346,348,352,354,356, and an a-D-mannopyranoside 373 give, selectively, the corresponding 6-deoxy-6fluoro derivatives 347, 349, 353, 355, 357, and 374 (2) at longer reaction times, a-D-glucopyranosides346,348,361, and the a-D-mannopyranoside 373 give 4,6-dideoxy-4,6-difluoro- (350 and 351) and 4-deoxy-4-fluoro-aD-galactopyranosides 362 and the 4,6-dideoxy-4,6-difluoro-a-~-talopyranoside 375, whereas P-D-glucopyranosides352,354,356,363,365, and 367 give 3,6-dideoxy-3,6-difluoro-(358,359, and 360) and 3-deoxy-3-fluoro-aD-allopyranosides364,366, and 368 more readily than for the reaction of the a-~-glucoisomers; (3) a-D-mannopyranosides 369, 376, and 378 give smoothly 4,6-dideoxy-4,6-difluoro-(370) and 4-deoxy-4-fluoro-talopyranosides 377 and 379; this is noteworthy, in that displacement reactions at C-4 of mannopyranosides are generally hindered by the axial 0-2. It was suggested that the reaction was facilitated by the formation of the intermediate IX, which undergoes attack by fluorine at (2-4, to give the 4-deoxy-4-fluoro

(249) R. L. Thomas, S. A. Abbas, C. F. Piskorz, and K. L. Matta, Curbohydr. Rex. 175 (1988) 158- 162. (250) R. L. Thomas, S. A. Abbas, and K. L. Matta, Curhohydr. Res.. 175 ( 1 988) 153- 157. (251) S. G. Withers, D. J. MacLennan. and I. P. Street, Curbohydr. Rex, 154 (1986) 127- 144. (252) C. W. Somawardhana. Curhohydr. Res.. 94 (1981) c14-cI5. (253) C. W. Somawardhana and E. G. Brunngraber, Curhohydr. R t x , 121 (1983) 51-60. (254) P. J. Card, J. Org. Chem.. 48 (1983) 393-395. (255) P. J. Card and G. S. Reddy, J. Org. C/zem.,48 (1983) 4734-4743.

TSUTOMU TSUCHIYA

I50

F

I

/

F-SNEt, \

IX

derivative. This was supported by the failure of DAST to give the 4-deoxy-4fluoro derivative for the 2-0-methylmannoside 371, only giving the 6deoxy-6-fluoromannoside(372). Fluorination of other related compounds is listed in Table 111. Methyl 6-deoxy-6-fluoro-a-~-glucopyranoside was conveniently prepared20(35%) by treatment of the (dichloromethane-soluble) per-0-(trimethylsily1)-a-D-glucopyranosidewith DAST, the silyl group at 0-6 being removed during the reaction. The tetraisopropyloxadisilyl group was reported to be stable217s247 to the DAST reagent. When methyl 2,3-di-0-benzyl-4-0-[2,3,4,6-tetra-O-benzyl-a-~-galactopyranosyl (=Gal)]-P-~-galactopyranoside (381) was treated with DAST (CH,CI,), the 3,6-anhydro derivative 382 (73%) was obtained221as the only product isolated. Similar treatment221of the 2,3-dibenzoate analog 383 gave the 6-0-methylgalactobiosyl fluorides 384a (9.5%) and 384p ( 5 lYo), the reaction possibly being initiated by 1-6 methoxyl group migration. This undesired reaction-pathway appears to be character is ti^'^^ for the nucleophilic displacement at C-6 of galactopyranosides, as already described in Section 142. Such a similarity for the DAST and S Nreactions ~ is also seen in the reaction with 1,2 : 3,4-di-O-isopropylidene-a-~-galactopyranose (see 300, in Section 11,2). Thus, treatment256of 1,2 : 3,4-di-O-isopropylidene-aL-galactopyranose with DAST gave the 6-deoxyd-fluoro derivative in good yield (73%), and this was then transformed into 6-deoxy-6-fluoro-~-galactopyranose (6-fluoro-~-fucose). 2,3,4-Trideoxy-2,3,4-trifluoro-~-galactoand -gluco-pyranosyl derivatives 391 and 392 have been prepared257from 1,6-anhydro-4-O-benzy1-2deoxy-2-fluoro-~-~-~ucopyranose (385) by use of DAST, through several intermediates, as shown [385+386,70%; 387-388,72%; 389+390,90%; refl. toluene (for 385) or dichloromethane (for 387 and 389), 24 h]. (256) J. R. Sufrin, R. J. Bernacki, M.J. Morin, and W. Korytnyk, J. Med. Chem.,23 (1980) 143- 149. (257) P. Sarda. F. C. Escribano, R. J . Alves, A. Olesker,andG. Lukacs,J. Curbohydr. Chem.,in

press.

CH@H I

OR

381 383

OBn

R=Bn R=B/

3x2

D

-!

3 OBz

Gal= 2,3,4.6-tclra-0-bcn/yl-

1

GaloF>F McOCH,

~ - D - ~ . I !ropyranosyl A

I

3x4

OBz

I

I;

385

386

F 388

R = Bn

387 R= H

Hry-67- t:/’.. H2C-

HZC-

AcOCH2

1

R2

E‘ 3x9

F 3!m

F 391 R L F , R ~ = H 392 R ‘ = H , R 2 = F

TSUTOMU TSUCHIYA

152

(-)- 1 L-1-Deoxy- I -fluoro-myo-inositol (393) and (+)- 1 D- 1-deoxy- 1fluoro-myo-inositol(394)have been preparedz5*from myo-inositol through multi-step reactions involving DAST treatment.

le

I10 ti0

OH

393

H & 0 4 7 [~

F

394

Difluoro compounds have been preparedzs9 by treatment of carbonyl compounds with DAST. These are methyl 5-deoxy-5,5-difluoro-2,3,0-isopropylidene-P-D-ribofuranoside, 6-deoxy-6,6-difluoro-1,2 :3,4-di0-isopropylidene-a-D-galactopyranose, methyl 2-deoxy-2,2-difluoro3,4-0-isopropylidene-P-~-erythro-pentopyranoside, and methyl 4-deoxy4,4-difluoro-2,3-0-isopropylidene-~-~-erythr~-pentopyranoside.As 6deoxy-6-fluoro-~-and -L-galactoses have been reportedz56to be inhibitors of ~-[~H]galactose and ~-[~H]fucose incorporation, respectively, into human tumor cells, 6-deoxy-6,6-difluoro-~-galactose~~ (396) was prepared by treatment of 1,2 : 3,4-di-0-isopropylidene-a-~-gulacto-hexodialdo1,5-

395

396

pyranose (395) with DAST. The compound, however, had almost no cytotoxic activity. (~)-4-Amino-3,3-difluoro-2-hydroxycyclopentylmethanol (398), a precursor for synthesis of the carbocyclic analogz47of 1-(2-deoxy2,2-difluoro-~-~-erythro-pentofuranosyl)-5-methylurac~l, was preparedz47 by treatment of the 0x0 compound 397 with DAST. Likewise, an anomeric (258) A. P. Kozikowski. Y. Xia, and J. M. Rusnak. J. Chem. Soc.. Chem. Cornmiin.,(1988) 1301 - 1303. (259) R. A. Sharma, 1. Kavai, Y.L. Fu, and M. Bobek, Tetrahedron Leu., (1977) 3433-3436. (260) H. H. Lee, P. G. Hodgson, R. J. Bernacki, W. Korytnyk, and M. Sharma, Carhohydr. Res..176 (1988) 59-72.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

I53

mixture of methyl 2-deoxy-2,2-d~fluoro-3,4-0-~sopropyl~dene-~-erythr pentopyranosides (399, 78%) was prepared261.262 by treatment of methyl 3,4-O-~sopropyl~dene-~-~-erythro-pentopyranos~d-2-u~ose with DAST. This compound and the 3,4-di-O-acetyl analog 400 are unstable, and, in the presence of strong acids (HCI or HBr in CH,Cl,, 0"-v.t.), give the 2-deoxy2-fluoro-2-halo derivatives, 401 and 402, respectively262(see also, Section 111.4).

397 (raccrnatc)

ROC

P

O

M

398 (raccmatc)

c

F

RO

399 400

cp

RO

RO R 3 R=CM% R=Ac

OMe

F

401 R . R = C M q , X= CI 402 R = A c , X = B r

When 1,2-O-isopropylidene- (285) or -benzylidene-a-D-glucofuranurono-6,3-lactones or I ,2-O-isopropylidene-~-~-idofuranurono-6,3-lactone (288) were treated263with DAST, 5-deoxy-5-fluoro-6,3-lactones 287 and 290 and 3,6-anhydro-6,6-difluorofuranoses 403 and 404 were formed in good total yield [287 :403 = 13 : 58 (7 1 Yo); 290 :404 = 2 : 3 (75%)]. As these products did not undergo further fluorination, an OH-5-assisted mechanism was proposed. On treatment with sulfur tetrafluoride in HF (-78", overnight) and subsequent glycosylation with methanol, an anomeric mixture of racemic (261) S.-H. An and M. Bobek, TefruhedronLnt., 27 (1986) 3219-3222. (262) M. Bobek, S.-H. An, D. Skrincosky, E. DeClercq, and R. J. Bernacki,J.Mrd. Chem.,32 (1989) 799-807. (263) R. Albert, K. Dax, U. Katzenbeisser, H. Sterk, and A. E. Stiitz, J. Curbohydr. Chem.. 4 (1985) 521 -528.

TSUTOMU TSUCHIYA

154

0-CMe, 285

287

403

290

404

OiMe, 288

c

t NEt, I

CH, 405 (racemate)

406 (racemate)

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

155

methyl 3-acetamido-2,3,6-trideoxy-arabino-hexopyranosides (405)gave, by fluorinative dehydroxylation, racemic methyl 3-acetamido-2,3,5,6-tetra-

deoxy-5-fluoro-ribo-hexopyranosides264 (406,63%). 4. Epoxide and Epimine Cleavage by Fluoride Ion Introduction of fluorine by oxirane-ring opening is described first. The 1,2-oxirane 408,prepared from nitroalkene 407 and hydrogen peroxide, was treated with KHF, (ethylene glycol, 20 min, - 112") to give274,275 2deoxy-2-fluoro-3,4: 5,6-di-O-isopropylidene-aldehydo-~-glucose (409)and the D-manno analog 410 (formed by epimerization of 409)with a minor hemiacetal 411 (formed from 408 and the solvent). Deacetalation of the CHN02

It I

,CHN02 O\

Ct(

q o/

I I

4

CMc,

1

-7" + 408

CHO

I

HCF

I

CHO

+

HOHC

I FCH vvcn

409

410

1

+

I

rhlh

-

"C\()/Ct12 411

AcOCH,

HCO, H,CO

/CMC2

C

Y

O

A

C

AcO F

216

J. T. Welch, B.-M. Svahn, S. Eswaraknshnan, J. P. Hutchinson, and J. Zubieta, CurbohJdr.Rcs., 132(1984)221-231. D. J. Baillargeon and G. S. Reddy, Curbohydr.Res., I54 ( 1 986) 275 - 279. C. Jiang. J. D. Moyer, and D. C. Baker, J. Curbohydr. Chem., 6 (1987) 319-355. A. Hasegawa, M. Goto, and M. Kiso, J. Curbohydr. Chem., 4 (1985) 627-638. K. Biggadike, A. D. Barthwick,A. M. Exall, B. E. Kirk,S. M. Roberts,P.Youds,A. M.Z. Slawin, and D. J. Williams, J. ChcJm.Soc., Chem. Commun., (1987) 255-256. S. G. Withers, M. D. Percival, and 1. P. Street, Curbohydr.Rex. 187 (1989) 43-66. T. C. Wong, R. R. Townsend, and Y. C. Lee, Curbohydr. Res., 170 (1987) 27-46. M. Sharma and W. Korytnyk, Curbohydr. Res., 83 (1980) 163- 169. J. Kihlberg, T. Frejd, K. Jansson, A. Sundin, and G. Magnusson, Curbohydr. Res., 176 ( 1988) 27 1 - 286. S. S. Yang, T. R. Beattie, and T. Y. Shen, TefruhedronLetf., 23 (1982) 5517-5520. W. A. Szarek, G. W. Hay, and M. M. Perlmutter, J. Chem. Soc., Chem. Commun.. ( 1982) 1253- 1254. W. A. Szarek, G. W. Hay, B. Doboszewski, and M. M. Perlmutter, Curbohydr.Res., 155 (1986) 107- 118.

TSUTOMU TSUCHIYA

156

TABLEI1 Fluorinated Products Obtained' by Treatment with DAST with Migration, or Retention of Configuration

Material

Reaction conditions

Product

B, reflux, 2 h

Yieldb (%)

References

16

182

44' I OBn

1

OH

334

335

336

I ORn

331

D, 100-110", 30 min

60

I48

16

338 338

D, Py, r.t., 3 d

- 20

I48

BnOCH,

Y OMe

339

A, reflux, 1.5 h J

23 24

239

80 (total yield)

91

B, reflux, I h

85

I82

A, r.t., 5 h

75

182

OMC F

B, reflux OH

MoO

340

N,

341

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

I57

TABLEI1 (continued) Material

Product

Reaction conditions

Yieldb (TO)

References

A, 25", 20 h

62

265

B, reflux., 30 min

78

182

31 8.8

266

T, 65", 4 h

14.5 35

266

T, 65", 30 min

83

242

A, r.t., 18 h

50

243

(continued)

TSUTOMU TSUCHIYA

I58

TABLEI1 (confinued) Material

Reaction conditions

Product

T, 60",2 h

Yieldb

Po)

References

50 R=Me

267

79

R=Bn

A

2:3

268

- 24

247 217

-25

247 217

B"0

, O C p HO H D N P

TIPS

,

O

C

p

P

A, 0" A, 0"

TIPS

F

0 ' 344 (rac.)

34s

VNHDNP A, - 30"

345

TIPS

0'

OH (rac.)

and TIPS

A, - 78" -+ r.t.

0'

- 18 - 74

247

61

217

- 50

248

-5

~~

Abbreviations: Cy, cyclohexylidene; DNP,2,4dinitrophenyl; Py, pyridine; TIPS, -(Pr'),SiOSi(Pr')2-; rac., racemate; r.t., room temperature; A, reaction in dichloromethane; B, reaction in benzene; D, reaction in diglyrne;T, reaction in toluene. Yields are described in the order of products, drawn from the left to the right. The sum of the last two compounds. dlnserted as a reference reaction.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

159

TABLE 111 Fluorinated Products Obtained" by Treatment with DAST with Normal Displacement Material

Reaction conditions

Product

Yield

(%I

References ~

[Q

HOQOMe

OMe

OH

A, r.t. 18 h

19

255

52

255

70

254

58

255

60

252

38

255

60

255

29

255

55

255

32 51

253 255

70

255

78

255

A, r.t., 1 h

56

269

neat, r.t., 18 h A, r.t., 72 h

42 23

253 255

OH

;G/lMe

A, r.t., 18 h

OH

HOCH,

110 (QR

011

OH

346 R= MK 348 R = Ph

I ('I I

FQOR OH

rcti,

HOCti,

347 (C346) A, r.t., I h 349 ( ~ 3 4 8 ) A, r.t., 2 h 350 (-346) neat, r.t., 18 h 351 ( ~ 3 4 8 ) A, r.t., 5 d 353 R = Me A, r.t., 15 min

fQ

110

011

352 R = M e

(Q

110

OH

FCH,

354 R = P b

355 R = Ph A, r.t., 25 min 357 R = C6H,NO,p A, r.t., 35 min 358 ( ~ 3 5 2 ) neat, r.t., 18 h A, r.t., 18 h 359 (+354) A, r.t., 18 h 360 ( ~ 3 5 6 ) A, r.t., 18 h

How OH

F

HOCH,

FCH, L

O

F

TTH;

F

TrOCH,

OH

OH

36 1

362

(continued)

TSUTOMU TSUCHIYA

160

TABLE 111 (continued) Material

OH

363 R = 0 7 r 365 R= OPiv 367 R = V,

Product

I

OII

Reaction conditions

Yield

(YO)

References

50

255

364 ( ~ 3 6 3 ) A, r.t., 18 h 366 ( ~ 3 6 5 ) A, r.t., 18 h 368 ( ~ 3 6 7 ) A. rd., 3 h

35

255

28

255

A, r.t., 18 h

45

255

71

255

A, r.t., I8 h

68

255

neat, r.t., 5 d +Ac,O, Py, MP

48

42

A, MP, r.t., 18 h

16

270

A, MP, r.t., 28 h

66

269

neat, r.t., 18 h A, r.t., 2 h

72 80

253 254

OH

R=F A, r.t., 18 h

R=N, t"

H,

OH

OH AiCKII,

NHAc HOCH,

OAL

HOCH,

369

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

161

TABLE111 (conlinued)

Material

Product

Reaction conditions

Yield (o/.)

References

IiocII~

371 R = Mc

A, r.t., I h

60

255

374 R' = H, R 2 = OH A, r.t., 15 min 375 R ' = F. R 2 = H A, r.t., 24 h

14

255

23

255

D, 40", 3 h

62

27 I

40

255

56

255

A, MP, r.t., 20 h

31

269

A, r.t., 18 h A, MP, r.t., 24 h

41 56

254 25 1

A, r.t.. 45 min

15

209

372

Ii(K't 1 2

OMK 373

377 (+376) A, r.t., 2 h 379 (+378) A, r.t., I h

OH

OH

(continued)

TSUTOMU TSUCHIYA

I62

TABLE I11 (continued) Material

Reaction conditions

Product

A, r.t., 2 d-reflux, 5h R 1= OH, R2= H

I

MOMOCH

,

CII

I

0

Q 0

0-CMc,

(To)

References 272

R 1= H,RZ= F

54 12

A, 0-25" T. 70-80"

86

273 273

A, r.t., 1.5 h

70

255

62

I97

A, Py,r.t.

61

197

A. r.t., I h

-

269

A,

HOCH

Yield

Py,r.t., 30 min

0

, A, MP, r.t., > 20 h R = CI

MOMQ)

R=F I

1

0-CMe2

49 63

269 269

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

163

TABLE 111 (continued) Material

I

Reaction conditions

Yield

(W

References

HCO,

HCO, H 2C’

Product

CMe,

I

H2C’

CMez

Abbreviations: All, allyl; MP, dimethylaminopyridine or 2,4,6-trimethylpyridine: MOM, methoxymethyl; Piv, pivaloyl: Tr. trityl: other abbreviations are the same. bObtained accompanied with the 3,4-0-(methyl orthoacety1)a-o-galactoside:the Odeacetyl derivative was also reported. Inserted as a reference reaction. A mixture of two diastereoisomers.‘On hydrolysis (R = F), 3,6dideoxy-3,6difluoro-~-glucopyranose is formed. Methanolysis gives methyl 3-deoxy-3-fluoro-c~-~-galactopyranoside.

mixture of 409 and 410, followed by acetylation, gave 1,3,4,6-tetra-O-acetyl-2-deoxy-2-fluoro-~-glucopyranose (216; ab mixture) and the corresponding D - ~ ~ W analog. K J Treatment276 of benzyl 2-acetamido-3,4-anhydro-2-deoxy-6-O-trityla-D-allopyranoside (412) with tetrabutylammonium fluoride [Bu,NF; in refluxing (refl.) MeCN, 24 h] gave, under diaxial ring-opening, benzyl 2acetamido-2,4-dideoxy-4-fluoro-6-O-t~tyl-a-~-gulopyranos~de (413), in CH,OTr I

CtiLOTr

agreement with the Furst - Plattner rule, the axial fluorine being confirmed by the gem coupling constant**(JF,H4 54 Hz) in the n.m.r. spectrum. 1,6Anhydro sugars having an epoxy ring are cleaved similarly. Thus, 1,6 :2,3dianhydro-4-O-benzyl-P-~-allopyranose (414) and 1,6 : 3,4-dianhydro-P-~(276) M. Sharma and W. Korytnyk, Curbohydr. Res., 79 (1980) 39-51.

TSUTOMU TSUCHIYA

164

altropyranose (415) afforded, by reaction with KHF, (refl. ethylene glycol, 4- 5 h), the 1,6-anhydr0-3-deoxy-3-fluoroderivatives 416 (Ref. 277) and 417 (Ref. 278) having an axial 3-fluorine, respectively. 1,6-Anhydro-ring cleavage (as. 5% CF,CO,H, 130- 140") of 417 gave crystalline 3-deoxy-3fluoro-D-mannose [4JF,H-, 5.1 (a anomer) and 2.1 Hz (p anomer) in D,O]. Reduction (by NaBH,) gave 3-deoxy-3-fluoro-~-mannitol.Similar treatment279.280 of the 2,3-anhydro-~-gulopyranoside 418 with KHF, (ethylene

414

glycol, 1 80°, 3 h) gave the 2-deoxy-2-fluoro-~-idoderivative 419 (44%), which, on oxidation - reduction (at C-3), gave the 2-deoxy-2-fluoro-~-talopyranoside 420. When, however, methyl 2,3-anhydro-4,6-di-O-methyl-a-

3H 418

419

F

420

D-allopyranoside (421) was treated similarly (KHF,, ethylene glycol, 160",4 h), the 3-fluoro-~-g/:luco derivative 422 (42%) was obtained281mainly, contrary to the Furst - Plattner rule, together with the ~-deoxy-~-fluoro-~-a~Z~o derivative 423 ( 17%). (277) T. B. Grindley,G.J . Reimer,J . Kralovec, R. G. Brown, and M. Anderson, Can.J.Chem.. 65(1987) 1065-1071. (278) M. Ctm9, J . Doleialova, J . Macova, J. Pacak, T. Tmka, and M. BudESinsky, Collect. Czech. Chem. Commun., 48 (1983) 2693-2700. (279) T. Tsuchiya, Y. Takagi, K. D. Ok, S. Umezawa, T. Takeuchi, N. Wako, and H. Umezawa, J. Anfihiof.. 39 (1986) 731 -733. (280) K. D. Ok, Y. Takagi, T. Tsuchiya, S. Umezawa, and H. Umezawa, Carbohydr. Res.. 169 (1987)69-81. (281) T. Tsuchiya, Inf. Chem. Congr. Puc~$cBasin Soc.. Honolulu, Hawaii, U.S.A., Dec. 17 (1984).

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

42 I

165

423

422

In the case of the cyclopentane oxirane 424, fluorination268[KHF,, MeO(CH,),OH] proceeded to give preferably one fluoroalcohol, 426 (6 190) over the isomeric one, 425 (i%o), possibly by the influence of the benzyloxymethyl group. Similarly2E2(KHF,, ethylene glycol, 160"),another oxirane, 427, was converted into 428 (30%). 2-C-(Fluoromethyl)-myo-inositol(429)

RnO

BnO

BnO 424

425

427

428

426

429

has been preparedzE3through ring-opening of the 2-oxiranyl precursors (KHF,, DMF, 160"). In an epoxy-ketone system such as 4~,5P-epoxy-6~-fluoroandrostane3,17-dione (430),fluorination (by the Olah reagent69)occurred as in an S N ~ type of mechanism, giving 2~,6P-difl~oroandro~t-4-ene-3,17-dione~~~ (431). This procedure may be applicable to sugars. (282) A. D. Borthwick, S. Butt, K. Biggadike, A. M. Exall, S. M. Roberts, P. M. Youds, B. E. Kirk, B. R. Booth, J. M. Cameron, S. W. Cox, C. L. P. Marr, and M. D. Shill, J. Chem. Soc.. Chetn. Cornmiin.. (1988) 656-658. (283) S. S. Yang, J . M. Min, and T. R. Beattie, Synrh. Commun., 18 (1988) 899-905. (284) J. Mann and B. Pietmk, J. Chem. Sot., Perkin Trans. 1. (1983) 2681 -2685.

TSUTOMU TSUCHIYA

166

9

430

Introduction of fluorine by epimine ring-opening is described next. Treatmentzssof methyl 2-benzamido-4,6-0-benzyl~dene-2-deoxy-3-~-tosyl-~-~glucopyranoside (432) with Bu,NF (in hexamethylphosphoric triamide, 8 5 " , 6 h) gave, through the N-benzoylepimine 435 (formed within 5 min), 3-benzamido-2,3-dideoxy-2-fluoro-cy-~-altropyranos~de (437, 35%) by trans-diaxial ring-opening. The use of oxolane as the solvent improvedzs6the yield of 437 (6 1Yo). Similar treatment2s7of the N-benzoylepimine 436 (prepared from 433) gave the 2-deoxy-2-fluoro-~-altroside438 (38Yo), together with the 3-deoxy-3-fluoro-~-glucoside439 (7%) and the N-debenzoylepimine (40%)of 436. Similar treatment288of the 3-0-mesyl derivative (434) under strictly dry conditions (in MeCN) gave a better yield of437 (63%).The

432 R'= Me, R2=Ts 433 R'= Bn, R2= Ts 434 R'= Mc, R2= Ms

435 R'= Me R'=Bn

436

437 R'= Me R'= Bn

439

438

(285) L. Hough, A. A. E. Penglis, and A. C. Richardson, Carbohvdr. Rex, 83 (1980) 142- 145. (286) M. K. Gurjar, V. J. Patil, J. S. Yadav, and A. V. Rama Rao, Carbohydr.Rex, 135 ( I 984) 174- 177. (287) L. H. B. Baptistella, A. J. Marsaioli, J. D. S. Filho, G . G. Oliveira, A. B. Oliveira, A. Dessinges. S. Castillon, A. Olesker, T. T. Thang, and G . Lukacs, Carbohydr. Res.. 140 (1985) 5 1-59. (288) H. H. Baer and A. Jaworska-Sobiesiak,Curbohydr. Res.. 140 (1985) 201 -214.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

167

n.m.r. data (JH,F) for 437,439, and the related compounds were given.Z85.Z88 Treatment289~z90 of some 0-mesyl derivatives of methyl (dially1amino)deoxyglycopyranosides440,442,444,446, and 448 with Et,N * 3HF com-

c/!-- (-TMe qOMe

OMS

F 441

440

R = CH2CH= CH,

4""' UMe--

RP

NH2

443

442

RO MsO 444

446

MsOFH~

NR2

RP 448

449

(289) D. Picq, D. Anker,C. Rousset,andA. Laurent, Telrahedron Letf.,24(1983)5619-5622. (290) D. Picq and D. Anker, J. Carbohydr. Chern.. 4 (1985) 113- 123.

TSUTOMU TSUCHIYA

168

plex gave the corresponding deoxyfluoro derivatives 441,443,445,447, and 449 (Ref. 200) in good yields after N-deallylation through the N,N-diallylaziridinium intermediates. Epimine-ring opening of several C-methyl 7-azabicyclo[4.1.O]heptanes (450,451,452, and 453) by the Olah reagent was studied,29’in that the results were explained292based on dynamic conformational analysis, utilizing the torsion-angle notation293(as illustrated by B); for example, 450 led preferentially to 454 (70%; 455: 30%) through a transition state of least energy (A) [in this case, this state equals the “pre-chair” state (instead of the “pre-twist” state)] for the “primary final product”293(C) of lowest energy. Me

450 R = H 451 R = M c

452 R = H 453 R=Mc

454

A

455

B

c

(

= 554)

(291) Y. Girault, M. Decouzon. M. Rouillard, and M. Azzaro, J. Fluorine Chem., 22 (1983) 253 -262. (292) Y. Girault, S. Genbaldi, M. Rouillard, and M. Azzaro, Tetrahedron,43 (1 987) 2485 2492. (293) E. Toromanoff, Telrahedron, 36 (1980) 2809-2931.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

I69

In the case of the 2-N,3-O-ditosyl-a-~-glucopyranoside 456, treatment with KHF, (DMF, 150") gave,281in the early stages, the 2,3-dideoxy-2fluoro-3-tosylamino-a-~-altropyranoside 458 (JF-2,H-I 9, JF-~,H-~ 45 Hz) with a minor proportion of 2,3-dideoxy-3-fluoro-2-tosylamino-a-~-glucopyranoside 459, (&-3,H-2= JF-3.H-4 12, JF-3,H-3 53 Hz) and N-tosylepimine 457, but, gradually, the thermodynamically stable 459 (apparent retention of configuration) became the major product. The mechanism suggested was confirmed experimentally. It is worth mentioning that compound 458, having a strong C-F bond, is equilibrated with the N-tosylepimine457; no such equilibrium is observed in the corresponding N-acyl corn pound^.^^^^^^^ The N-tosyl group of 459 was removed with sodium in liquid ammonia.281

456

457

459

5. Addition to Glycals

Introduction of a fluorine atom at C-2 by addition to glycals is effected by electrophilic reagents, Q6--Fd+ (Q is a strongly electron-withdrawing group). As C-2 of a glycal is more electronegative than C-1 because of the participation of -O+=C( l)-C(2), fluorination by Q-F always occurs at C-2.Reagents in this category are trifluorofluoroxymethane (CF,OF; another name is trifluoromethyl hypofluorite; the difference between this and (294) G. Alvernhe, S. Lacombe, and A. Laurent, Telruhedron Lett., 21 (1980) 289-292

TSUTOMU TSUCHIYA

I70

CF30CI has been discussed295),fluorine (Fz,diluted with inert gases), xenon difluoride (XeF,), acetyl hypofluorite (MeCO,F), and related compounds. As basic aspects of reactions using some of these reagents have been described by PenglisS in this Series, developments after 1978 will mainly be described. In 1969, Adamson, Foster, and reported the synthesis of 2deoxy-2-fluorosugars by addition ofCF,OF(in CFCl,, -80') to 3,4,6-tri-0acetyl- 1,5-anhydr0-2-deoxy-~-arabino-hex1-enitol (61; 3,4,6-tri-O-acetylD-glucal). The reagentz98~z99 fluorinates 61 e l e c t r ~ p h i l i c a l l at y ~C-2, ~ ~to~ ~ ~ ~ ~ ~ ~ afford cis-addition products: trifluoromethyl 3,4,6-tri-O-acetyl-2-deoxy-2-

461

463

K=OCF< R=F

483 484 485 486

I<'=OCF,, I<'=H, I<'=F, I<'=€],

R'=Il Kz=OCF3 K2= H RL=F

fluoro-a-D-gluco-(461,26%)and -P-D-manno-pyranosides(460, - 6%), and 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-cu-~-gluco(463, 34%) and -P-Dmanno-pyranosyl fluorides (462, - 8%) (see Table IV). Formation of 462 and 463 was explainedzg7by the reaction sequence, 0-C( 1)=C(2) C(2)F

F-OCF3+6+0=C( 1)-C(2)-F

OCFj"+O-C+( 1)l)F-C(2)F OCF,.

+ -OCF3 (-OCF3+OCFz + F-)-O-C(

' 1 .

+

Their structures were established by n.m.r. s t ~ d i e s [JF-Z,H-3 ~ ~ ~-~25 ~ ~ ' ~ ~ ~ ~

(460), 1 1.5 (461), 2 1.4 (462), and 12.0 Hz (463); JF-I,H-z 7.6 (462), and 23.8

(295) K. K. John and D. D. DesMarteau, J . Org. Chem., 48 (1983) 242-250. (296) J . Adamson, A. B. Foster, L. D. Hall, and R. H. Hesse, J. Chem. Soc., Chem. Comtnun., (1969) 309-310. (297) J. Adamson. A. B. Foster, L. D. Hall, R. N. Johnson, and R. H. Hesse, Curbohydr. Res., 15 (1970) 351-359. (298) L. B. Marantz, J. Org. Chem., 30 (1965) 4380-4381. (299) J. K. Ruff, A. R. Pitochelli, and M. Lustig, J. A m . Chern. SOC.,88 (1966) 4531 -4532. (300) D. H. R. Barton, L. S. Godinho, R. H. Hesse, and M. M. Pechet, J. Chem. Soc.. Chem. Cotnmun., (1968) 804-806. (301) E. L. Albano, R. L. Tolman, and R. K. Robins, Curbohydr. Rrs.. 19 (1971) 63-70. (302) L. D. Hall, R. N. Johnson, J . Adamson, and A. B. Foster, Can. J . Chem.. 49 (1971) 118- 123.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

171

Hz (463)l. Acid-catalyzed hydrolysis297(1 -5 M boiling aq. HCl) gave 2deoxy-2-fluoro-~-glucose(2DFG) and -D-mannose (2DFh4) in good yields. Similar treatment303 (CFCl,, - 80") of 3,4,6-tri-O-acetyl-1,5-anhydro-2deoxy-D-lymhex- 1-enitol (465, 3,4,6-tri-0-acetyl-~-galactal)gave 2deoxy-2-fluoro-a-~-galacto-(467 and 469) and -/?-D-talo-pyranosylderivatives (466 and 468; the product ratio being - 15: 1; see Table IV). L i k e ~ i s e , ~(CFCl,, ~ ~ , '~ 60 ~ ~to~-~70") 3,4-di-O-acetyl-1,5-anhydro-2,6dideoxy-L-lyxo-hex-1-enitol (473, di-0-acetyl-L-fucal), 3,4-di-O-acetyl-1,5anhydro-2,6-dideoxy-~-arabino-hex1-enitol (477, di-0-acetyl-L-rhamnal) and 4-O-benzoyl-6-deoxy-2-fluoro-3-O-methyl-~-glucal(482), the last compound being prepared from 4-0-benzoyl-2,6-dideoxy-2-fluoro-3-0-methyla-L-glucopyranosylbromide by the usual procedure, gave the corresponding 2-deoxy-2-fluoro cis-addition products (474 and 475, 478-481, and 483 486; 483-486 being the 2,2-difluoro compounds; see Table IV); in the case of 473, the quasiaxial OAc group at C-4 conformation) may additionally hinder303.306 the approach of the reagent from the a side, although the major product-determining factor remains the orientation at C-3 (that is, trans-addition with respect to the C-3 substituent). Some Jc,F-2 values in the I3C-n.m.r.s p e ~ t r a ~ ~ ~ o fand 4 8 a2 mixture , of483 and485 are: = 40.2 (482), - 30 and - 40 Hz (483 and 485); Jc-2,F-2 = 240 (482), - 245 and - 260 Hz (483 and 485); Jc-3,F-2 = 2 1.7 (482), - 19 and - 19 Hz (483 and 485); JC4,F-z = 8 Hz (483 and 485); and Jc-5,F-2 = 9 Hz (482). Applications of the procedure are described next. The 2,2-difluorodaunosamine derivative 495 (a four-compound mixture; JF-~=,H-~ 23 - 24, JF-2al,H-3 6 Hz) was prepared307 from 4-0-benzoyl-2,3,6-trideoxy-2-fluoro-3-(trifluoroacetamido)-a-~-galactopyranosyl bromide2"0(493) through dehydrobromination (to give the 2-fluoroglycal494; JF,H-I 5 , JF,c-I

-

493

494

495

R= OCF, or F (303) J . Adamson and D. M. Marcus, Carbohydr.Res., 22 (1972) 257-264. (304) C. G. Butchard and P. W. Kent, Tetrahedron, 35 (1979) 2551-2554. (305) C. G. Butchard and P. W. Kent, Tetrahedron, 35 (1979) 2439-2443. (306) R. U. Lemieux and B. Fraser-Reid, Can. J. Chem., 43 (1965) 1460- 1475. (307) A. Dessinges, F. C. Escribano, G. Lukacs, A. Olesker,and T. T. Thang,J. Org. Chem., 52 (1987) 1633- 1644.

I72

\o

IP

480(22)

481 (12)

305

C

485d (42) (58)

486d (9.5) (29)

238 238

B'

488 (-

489 (- 40)'

316

B'

491 (- 5)'

492 (- 35)'

316

A

B70R A

478(31)

479(11)

483d (16)

(5)

F

Me0

c'a

HOCH, I HO

490

a Isolated yield. A, CF,OF in CC1,F at - 60 to - 80' with or without CaO, B, F2 in Ar (or N2-CCIlF) at - 78'; B', F2 in N2-H20 at room temperature; C, XeF2-BF,. OEt2 in ether, benzene, toluene, dichloromethane, or a mixture of some of them (0"to room temperature). Yield of the deprotected compound after hydrolysis. 2.2-Difluoro compound, Estimated from the final 2deoxy-2-fluoroglycose.

I74

TSUTOMU TSUCHIYA

-

'CN

,

R 497 R=OCF3 498 R = F

496

500 X=OCFj 501 X = F

499

502

R= NHCqEt

40, JF,c-2240, JF,c-320, and JF,c-55 Hz) followed by addition of CF,OF. Treatment223of glycals 496 and 499 with CF,OF (CH,Cl,, 0') gave the TIO CH 2

EQC

J2? 0-CMe,

NHCHO

503

504

2-deoxy-2-fluoro derivatives 497 (2490)and 498 (3690), and 500 YO), 501 (1 OYo), and 502 (2890), respectively. Addition of CF,OF to enamines had been reported,300and the procedure was applied to a synthesis of showdomycin analogs308(503-504). Formation of perfluoroalkyl hypofluorites (which are more stable than AcOF) by the reaction of NaO(O)CCF, and F,, and the reaction with 61 (to give 2DFG), have been reported.309 (308) J. C. A. Boeyens, A. J. Brink, and A. Jordan, S. Ajk. J. Chem., 3 1 ( 1978) 7 - 13. (309) G. K. Mulholland and R. E. Ehrenkaufer, J. Org. Chem., 51 (1986) 1482- 1489.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

I75

Addition of F, to glycals had been considered to be a rather drastic reaction that gives no simple products, but several measures, such asdilution ofthe F2 with an inert gas, lowering the reaction temperature, and selection ofsuitable solvents moderate the reaction, and F, has become one of the most frequently used fluorinating reagents for glycals. TreatmenPo of 3,4,6-tri-0acetyl-D-glucal (61, CFCl,, -78") with F2 (diluted with argon) gave two 1,2-difluorocis-addition p r o d ~ c t s ,462 ~ ~and ~ . ~463 ~ ~(26 and 4090 isolated yield; see Table IV). The method is thus superior to that employing CF30F (Ref. 297), in that it produces fewer isomeric products. This procedure is conveniently used to prepare the short-lived 2-deo~y-2-[~~F]fluoro-~-glucose311(l8F-2DFG; see Section II1,l). Tri-0-acetyl-D-galactal (465) and 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-~-rib~-hexI -enit01 (471) were treated similarly,to give3', 2,4,6-tri-0-acetyl-2-deoxy-2-fluoro-a-~-galactopyranosyl(469) and 3,4,6-t~-~-acetyl-2-deoxy-2-fluoro-~-~-altropyranosyl fluorides (472), respectively (see Table IV). Fluorination of unprotected D-glucal(487) and D-galactal(490) in water with F, was ~ t u d i e d , ~ 'and ~-~'~ found to give the 1,2-difluorocis-addition products, 488 and 489, and 491 and 492, respectively (see Table IV; see also, Section II1,l). Fluorinations of glycals with F, and AcOF in water and other solvents have been compared.,18 were the first to apply XeF, (Refs. 32 1 Korytnyk and 323) in the synthesis of 1,2-difluoridesfrom glycals. This reagent, although expensive, is solid and can be conveniently used in the laboratory. Treat-

(310) T. Ido, C.-N. Wan, J. S. Fowler, and A. P. Wolf, J. Org. Chem., 42 (1977) 2341 -2342. (31 I ) T. Ido, C.-N. Wan, V. Casella, J. S. Fowler, A. P. Wolf, M. Reivich, and D. E. Kuhl, J. Label. Comp. Radiopharm., 14 (1978) 175- 183. (312) M. Tada, T. Matsuzawa, H. Ohrui, H. Fukuda, T. Ido, T. Takahashi, M. Shinohara, and K. Komatsu, Heterocycles, 22 (1984) 565-568. (313) G. T. Bida, N. Satyamurthy, H. C. Padgett, and J. R. Bamo, J. Label. Comp. Radiopharm., 21 (1984) 1196. (314) G. T. Bida, N. Satyamurthy, and J. R. Barrio, J. Nucl. Med., 25 (1984) 1327- 1334. (315) M. Diksic and D. Jolly, J. Carbokydr. Chem.. 4 (1985) 265-271. (316) M. Diksic and D. Jolly, Carbohydr. Rex, 153 (1986) 17-24. (317) P. D. Raddoand M. Diksic, Carbohydr. Rex, 153 (1986) 141-145. (318) N. Satyamurthy, G. T. Bida, H. C. Padgett, and J. R. Bamo, J. Carbohydr. Chem., 4 (1985) 489-512. (319) W. Korytnyk and S. Valentekovic-Horvath, Tetrahedron Lett., 21 (1980) 1493- 1496. (320) W. Korytnyk, S. Valentekovic-Horvath, and C. R. Petrie 111, Tetrahedron, 38 (1982) 2547 -2550. (321) R. Filler, Isr. J. Chem., 17 (1978) 71 -79; related references are cited therein. (322) A. Gregortit and M. Zupan, J. Org Chem.. (1979) 1255- 1258. (323) A. Gregortit and M. Zupan, J. Org. Chern.,44 (1979) 4120-4122.

TSUTOMU TSUCHIYA

176

ment of tri-0-acetyl-D-glucal (61) and -galactal (465), and di-0-acetyl-Lfucal(473) with XeF, in the presence of BF, * OEt, as the catalyst (30 min overnight, at room temp.) gave 1,2-cis-and 1,2-trans-difluoroproducts(462, 463, and 464 from 61; 468,469, and 470 from 465; and 475 and 476 from 473; see Table IV), the addition pattern being different from that of CF,OF or F,. For reactions with XeF,, both ionic and free-radical mechanisms have

BF3 + F-, lhcn F- attacks ai C-1

A : BF4-

from the a or p side

been proposed,323but, in the foregoing case, the ionic mechanism shown here3I9accounts for the formation of the trans-addition products. Zemplkn deacetytation of 463,469, and 475 gave the corresponding free 1,2-difluoro sugars, 505, 506 and 2,6-dideoxy-2-fluoro-c~-~-galactopyranosyl fluoride (507) in crystalline form. 2-Deoxy-2-fluoro-~-fucose,~~~ has been prepared from 507 by acid-catalyzed hydrolysis. Xenon difluoride is characteristic in reacting with polarized double bonds (glycals and enol acetates324)in the presence of acid or Lewis acid catalyst,

F 505

RLH. R~=OH

OH

507

506 R'= 011, Rz= I I

(324) B. Zajc and M. Zupan, J . Org Chern.. 47 (1982) 573-575.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

177

but not with non-polarized double bonds. Thus, benzyl2-acetamido-6-0acetyl-2,3,4-trideoxy-a-~-erythro-hex-3-enopyranoside and tri-O-acetyl-5thio-D-glucal showed no reaction320with XeF,. Some n.m.r. properties of the I ,2-difluoro sugars are as follows32o:F-1 resonates at 50-75 p.p.m. downfield in comparison with F-2; JF-lax,F-2q - 18 to -2 1 and J F - l e q , ~or - ~zeq - 13 to - 16 Hz; JF-I,H-I = JF-~,H-~ 48-54, 3 J ~ (tram-) , ~ 23-25, except for 3 J ~ , ~ . ~ - 14, 3JF,H (crseuche) - 14, except for 3JF,H-lan 3-59 and 3JF,H (trans-gauche) 8, except for 3JF,H.1 < 1 Hz. These results accorded well with those reported.5,302,325 The conformation of 2-deoxy-2-fluoro-~-~-mannopyranosylfluoride (157) was determined146by X-ray crystallography, as well as by IH- and 19F-n.m.r. spectroscopy; in D,O, the H - H homonuclear 2D J-resolved spectrum demonstrated the usefulness for discrimination between the J F , H and J H , H couplings; some Jvalues are: JF-I,H-I 47.4, JF-I.H-2 2.8, JF-2,H-I 17.8, JF-2,H-2 5 1.8, JF-2.H-3 30.0, and JF-I.F-2 12.7 Hz. These values are slightly different from those reported for the unstrained 4C1conformation, as a result of weak intramolecular hydrogen-bonding [F(2)-0H(3)] and interaction (in the crystal) between F( 1)-CH(2). Xenon [ 18F]difluoridewas ~ynthesized’,~by isotopic exchange between XeF2 and HISF, Si18F4,or As18F, and I8F-2DFG was prepared327.328 by the action of this reagent on tri-0-acetyl-D-glucal (61). In this reaction, the combination of ethyl ether (as the solvent) and BF, (as the Lewis acid catalyst) was found to give the best r e s ~ l t . ~ ~ ~ . ~ ~ ~ Bromofluorination of double bonds using polymer-supported hydrogen fluoride- N-bromosu~cinimide~~~ (NBS) or Bu4NF- NBS (Ref. 330) has been reported. Reaction of AcOF with glycals has been developed. Rozen and cow o r k e r ~observed ~ ~ ~ that elemental fluorine, which is practically insoluble in, and non-reactive with, acetic acid, gives rise to the active species, AcOF,

-

(325) L. Phillipsand V. Wray, J. Chem. Soc., B, (1971) 1618-1624. (326) G. Schrobilgen, G. Firnau, R. Chirakal, and E. S. Garnett, J. Chem. Soc., Chem. Commtm., (1981) 198-199. (327) S . Sood, G. Firnau, and E. S. Garnett, Int. J. Appl. Radial. Isot.. 34 (1983) 743-745. (328) C.-Y. Shiue, K.-C. To, and A. P. Wolf, J. Label. Comp. Radiopharm., 20 (1983) 157162. (329) A. GregorEii. and M. Zupan, J. Fluorine Chem., 24 (1984) 291 -302. (330) M. Maeda, M. Abe, and M. Kojima, J. Fluorine Chem.. 34 (1987) 337-346. (331) S. Rozen, 0. Lerman, and M.Kol, J. Chem. Soc., Chem. Commun., (1981) 443-444.

TSUTOMU TSUCHIYA

I78

when a salt such as NaF, NaOAc, or NaO(0)CCF3is present. Acetyl hypofluorite332[m.p. -96 f 1 ' (Ref. 333)] F,

+ AcOH + NaX

-

AcOF

+ HX + NaF (X=F,

AcO, CF3C0,)

is a strongly electrophilic reagent, and reacts331rapidly with double bonds, mainly in the syn mode, to give vicinal acetoxy fluorides (a similar reagent, CF3C02F, has also been reported334).This methodology was soon applied by Adam and coworker^^^^.^^^ to glycals and vinyl ethers. Treatment of tri-0-acetyl-D-glucal (61), -galactal (465), 3,4-di-0acetyl- 1,5-anhydro-2-deoxy-~-erythro-pent1-enitol (511, 3,4-di-O-acetylD-arabinal), 6-deoxy- 1,2 : 3,4-di-O-isopropylidene-~-~-arabino-hex-5-eno1,5-pyranose (516), 3,5-0-benzylidene-6-deoxy1,2-O-isopropylidene-a-~xylo-hex-5-eno-1,Cfuranose (527), and 5-deoxy- 1,2-O-isopropylidene-3-0tosyl-P-~-threo-pent-4-eno-1,bfuranose gave, respectively, the 1-0-acetyl2-deoxy-2-fluoro derivatives 508, 510, and 513, and 5(R)-5-C-acetoxy6 - deoxy - 6 - fluoro- 1,2 : 3,4 -di - 0-isopropylidene-P- L-arabino-hex0 - 1 3 pyranos-5-ulose (517), 5(S)-5-0-acetyl-3,5-O-benzylidene-6-deoxy-6fluoro-1,2-O-isopropylidene-a-~-xy~o-hexo1,4-furanos-5-ulose (528), and 4- 0-acetyl- 5-deoxy-5- fluoro- 1,2- 0-isopropylidene- 3 - 0-tosyl -P- L-threopento- 1,4-furanos-4-uloseas the major products (see also, Table V). This method has been extensively utilized in the synthesis of 18F-2DFG (see Section II1,l). However, there have been questions concerning the purity of the compound synthesized (see Section II1,l). To clarify the problem, Shiue and coworker^^^^,^^^ and Satyamurthy and coworkers318examined the behavior of tri-0-acetyl-D-glucal [also of tri-O-(2-methylpropyl)-~-glucal and tri-0benzoyl-~-glucal]and D-glucal toward F, and AcOF with variation of the reaction solvents. It was thus found that no combination of starting materials, reagents, and solvents (H,O, MeOH, AcOH, DMF, MeCN, Me,CO, CFCl,, CCI,, cyclohexane, and hexane) gave the desired 2DFG exclusively; the best combination was tri-O-acetyl-D-glucal - AcOF- solvent-of-low-

(332) D. M. Jewett, J. F. Potocki, and R. E. Ehrenkaufer, Synth. Commun., 14 (1984) 45-51. (333) E. H. Appleman, M. H. Mendelsohn, and H. Kim, J. Am. Chem. SOL-.,107 (1985) 65 15-65 18. (334) S. Rozen and 0. Lerman, J. Org. Chem.. 45 (1980) 672-678. (335) M. J. Adam, J. Chem. Soc., Chem. Commun., (1982) 730-731. (336) M. J. Adam, B. D. Pate, J.-R. Nesser, and L. D. Hall, Carbohydr. Rex, 124 (1983) 2 15 -224. (337) C.-Y. Shiue, J. S. Fowler, A. P. Wolf, D. Alexoff, and R. R. MacGregor, J . Label. Comp. Radiopharm., 22 ( 1985) 503 - 508. (338) C.-Y. Shiue and A. P. Wolf, J. Fluorine Chem., 31 (1986) 255-263.

TABLE V Reaction Products and the Yields from Glycals with MeC0,F

Product and yield (YO)

Material AcOCH

I

conditions"

References

F

,

Q

Ad)

AcOCH, I

P

508 (78)

61

(49)

509(20)

510 (84) (74)

465

336 340

513 (96)

51 1

512 (9)

336 340

(55)

336 340

515 (54)

340

519 (47)

340 340

Ad)

0

514 (31)

67

Ad)

516

R'O

518 R = H 520 R = B n

C C

521 (61)

522 R ' = H 524 H ' = B n R2= CH,OMe

C

523 (30)

C

525 (51) 526(12)R2=H

340 340 340

(continued)

TSUTOMU TSUCHIYA

180

TABLE V (continued) Product and yield (Yo)

Material

Reaction conditions"

A, Fz in He-AcOH-NaOAc-CCI,F at gas-CHzCIz- hexane at room temperature.

- 78"; B,

& TAc References

F

AcOF gas-AcOH at room temperature; C, AcOF

polarity (CCI,, CFCl,, hexane, or cyclohexane), which gave the product ratio of 2DFG : 2DFM = 96 :4-93 :7. Any combination of tri-0-acetyl-D-glucal (or D-glucal)- F, gave the ratio of 2DFG : 2DFM = 50 :50- 70 :30. It was also found that any reaction of D-glucal with AcOF in a polar solvent (H,O, AcOH, Me,CO, or MeCN) gave 2DFM-rich products (2DFG :2DFM = 45 : 5 5 - 12 :88). The collective results were explained3'* on the basis of the difference in stability of the transient, tight ion-pairs 529 and 530, generated by the initial reaction of the reagents, to the double bond from the a- and /?-sides(the stability being governed by the a n ~ m e r i and c ~ ~gauche ~ effects") and the species of the substituent at C-3, the polarity ofthe solvent used, and so on. In later experiment^,^^ when 61, 67, and 511 were treated with -0

+

(339) W. A. Szarek and D. Horton (Eds.), Anotnrric ,?fledcl. Origin and ConsequcwccJs..4CS Syrnp. Ser. 87. Am. Chem. SOC.,Washington, D.C., 1979. (340) K. Dax and B. 1. Glanzer, Carboliydr. Res., 162 (1987) 13-22.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

181

gaseous AcOF, a mixture ofcis-addition products 508 and 509,514 and 515, and 512 and 513 was respectively produced. Reactions of some furanoid glycals having free 3-hydroxyl(518 and 522) and 3-0-benzyl groups (520 and 524) with AcOF gave different products3"$ in the former and the latter groups of compounds, respectively, addition reaction occurred mainly from the same (to give 519 and 523, respectively) and from the opposite sides [to give 521 and 525 (with 526), respectively] of the substituents at C-3 (see Table V). 6. Miscellaneous

Several 2-deoxy-2-fluoro and 2-deoxy-2,2-difluoro sugars were synthesized by coupling of suitably protected simple aldehydo sugars (or their N-benzylimine derivatives) with monofluorohalo or difluorohalo acetates (or their equivalents). Reaction of 2,3-0-isopropylidene-~-glyceraldehyde (531) and lithio ethyl fluoroacetate (533) gave a mixture of condensation products 534 - 537, which were deprotected, and the products reduced with diisobutylaluminum hydride (DIBAL) to give a mixture of 2-deoxy2-fluoro-~-pentopyranoses~~~ (538-541). Condensation (Reformatskii reaction) of 531 and (bromodifluoromethyl) phenylacetylene gave the difluoroacetylene 544, which was converted into 1,3,4-tri-O-acetyl-2-

531 532

K=II K= M c

533

I

534 I

HO 538

539

540

5-41

(341) J. T. Welch and S. Eswarakrishnan, J. Chem. Soc., Chem. Commun.. (1985) 186- 188.

I82

TSUTOMU TSUCHIYA

deoxy-2,2-difluoro-a-~-erythro-pentopyranose~~~ (548). Compound 548 and l,3,4-tri-0-acetyl-2,6-dideoxy-2,2-difluoro-~-lyxo-hexopyranose (549) were also prepared343by condensation of 531 or 532 with the ketene silyl XCF&&R 542 543

545

X= Hr. K= h X= I . R = M c

546

K= H

547

R= Mc

54X K = H 549 K = M c

acetal545, derived from difluoroiodoacetate 543 (to give 546 and 547), and subsequent work-up involving reduction with DIBAL. Similarly, 2-deoxy2,2-difluoro-~-eryfhro-pentofuranose~~ was prepared through condensation (Reformatskii reaction) of 531 and 542. Likewise, 2,2-difluoro amino sugar derivatives 551 were prepared345through condensation (in the presence of zinc) of the N-benzylimine derivative of 532 with difluorohaloacetate (to give difluoro-P-lactams 550) and subsequent work-up involving reduction (DIBAL) and acetylation.

IN\\

€3I1

0 F

550

55 I

(342) Y. Hanzawa, K. Inazawa, A. Kon, H. Ac.1, and Y. Kobayashi, Tetrahedron Lett., 28 (1987) 659-662. (343) 0. Kitagawa, T. Taguchi, and Y. Kobayashi, Tetrahedron Lcti., 29 (1988) 1803- 1806. (344) L. W. Hertel, J. S. Kroin, J. W. Misner, and J. M. Tustin, J. Org. Chem.. 53 (1988) 2406- 2409. (345) T. Taguchi, 0.Kitagawa, Y. Suda, S. Ohkawa, A. Hashimoto, Y. Iitaka, and Y. Kobayashi, Tetrahedron Lett., 29 (1988) 529 1-5294.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

183

3-Deoxy-3-fluoro-~-glucose(25%) and -mannose (40%) were prepared3& from 2-deoxy-2-fluoro-~-arabinose'~~ by a chain-extension reaction (cyanohydrin synthesis).Likewise, 4-deoxy-4-fluoro-~-glucose~~~ (27%) and -mannose (45%) were prepared from 3-deoxy-3-fluoro-~-arabinose."~ 4-Deoxy-4-fluoro-~-fructose(552) was prepared348(59%)by fermentation of 3-deoxy-3-fluoro-~-mannitolwith Gluconobucter oxyduns.The structure of 552 (p-D form) was confirmed by the n.m.r. spectrum, which resembles that of 4-deoxy-4-fluoro-a-~-sorbopyranose~~~ (553);552 was identical with one of the products349obtained from the oxirane-ring opening of 3,4-anhydro- 1,2-0-isopropylidene-~-~-tagatopyranose with KHF,.

HO

553

552

Sugars having a difluoromethylene group were prepared350by reaction of an aldehyde group in sugars with difluoromethylene tris(dimethy1amin0)phosphorane; some of these were 554-559. Similarly, (Z)- (560)and (E)-gem-bromofluoroalkene 561 were prepared260from 1,2 :3,4-di-O-isopropylidene-a-D-gulucto-hexodialdo1,5-pyranose (395) by treatment with Ph3P= CFBr. HC=CF2

I

R'

OCH

YQ

R4

HC 0,

0-CMe,

554 555 556

R' OBn H H

R2 R' R4 H CH=CF2 H OMC CH=CF, t1 OMc H CH=CF2

I

/CMc2

H2CO

557

0 Me&-0

558

(346) J. R. Rasmussen, S. R. Tafuri, and S. T. Smale, Carbohydr. Res., I16 (1983) 21 -29. (347) J. A. Wright and N. F. Taylor, Curbohydr. Res., 3 (1967) 333-339. (348) M. BudEinski, M. tern$, and J. Doleialova, M. Kulhinek, J. PacBk, and M. Tadra, Collect. Czech. Chem. Commun., 49 ( 1 984) 267 -274. (349) G. V. Rao, L.Que, Jr., L. D. Hall,andT.P.Fondy,Curbohydr. Res., 40(1975)311-321. (350) J. M. J. Tronchet, A. Bonenfant, and F. Barbalat-Rey, Curbohydr. Res., 67 (1978) 564-573.

TSUTOMU TSUCHIYA

I84

R' I

R2-C

\

FK =ytl

CH

0-CMC,

0-CMC,

559

560 561

R'= F, R2= Br R'= Br, R 2 = F

Preparation of 0-(trifluoromethy1)ated sugars is generally difficult, in contrast to 0-methylation. Trainor succeeded in preparing351several O-trifluoromethyl sugars (563, 565, and 566) by treatment of the triflates 562, 299, and 248 with tris(dimethy1amino)sulfonium trifluoromethoxide, respectively, with the corresponding fluoro derivatives 564, 119, and 249 [CH,CI,, room temp., 2.5 h for 562; MeCN, reflux, 45 min for 299; and MeCN, reflux, 5 h for 2481. These ethers are stable in aqueous trifluoroacetic acid (room temp.), or during catalytic reduction, and are more volatile than the corresponding methyl ethers. Some trifluoromethyl glucosideswere also prepared351from the corresponding protected glycosyl bromides as shown (567 + 568 13p).

+

ORn 562

OBn 563 ( 7 6 % )

(351) G . L. Trainor. J. CurDohydr. Ctiem..4 (1985) 545-563.

OBn 564 ( 8 % )

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

-

OCH,

Mc2C(

Qy

I OCH

TflO

OCH,

OCH,

I O

Mc,C(

Mc,C< q +

C

O

I

C

-

AcOQr

249 (75%)

OAc

67

AcOCH~

AcOCH,

+

AcOQF3

AcO

OAc

567

7

0-CMe,

566 (16%)

AcOCH,

q

0-CMe,

0-CMe, 248

185

OAc 13B (37%)

568 (14%)

3-C-(Trifluoromethyl)-gulo- (570) and -talo-furanosides 571 were prepared352from 3-trifluoromethyl-A2-butenolide (569) through a sequence of reactions. RnOCHL

RIIOCH~

I

569

I

570 (racemate)

571 (racemate)

MOM= inethoxymethyl

The branched-chain sugar fluoromethylphosphonate (573) was prepared3s3 by treatment of I ,2 :5,6-di-O-isopropylidene-a-~-ribo-hexofuranos-3-ulose (572) with lithio(fluoromethy1)diisopropylphosphonate (oxolane, - 78" room temp.) to give a diastereoisomeric mixture having the D - d o configuration.

-

(352) K. Kawada, 0.Kitagawa, T. Taguchi, Y. Hanzawa, Y. Kobayashi, and Y. Iitaka, Chem. Pharm. Bull., 33 (1985) 4216-4222. (353) C. M. Blackburn and M. J. Parratt, J. Chem. Soc.. Chem. Cornmun.. (1983) 886-888.

TSUTOMU TSUCHIYA

186

0-CMc,

512

O-CMc2

573

111. SYNTHESIS A N D BIOLOGICAL ACTIVITIES OF COMPOUNDS

CONTAINING FLUORINATED CARBOHYDRATES 1. Isotopically Modified, Fluorinated Carbohydrates

a. Uses for Fluorine-18-labeled Deoxyfluoro Sugars.-Deoxyfluoro sugars labeled with fluorine- 18 have important applications as imaging agents. The basis354for these applications is described briefly. As the consumption of D-glucose in tissues is intimately correlated with their levels of metabolic activity, quantitative measurement of D-glucose uptake in brain, heart, or other organs in man and animals affords valuable information on the functions and activities ofthese organs in normal, stimulated, and pathological states. However, the use of such labeled D - ~ ~ U C Oderivatives S~ as [ 14C]and [3H]-glucosefor this purpose, coupled with a suitable detector, is not necessarily convenient, because D-glucose is rapidly metabolized to carbon dioxide. On the other hand, 2-deoxy-~-aruhino-hexose[“2-deoxy-~-glucose” (2DG)], and therefore the 14C-labeledanalog, 14C-2DG,administered to a mammalian system is metabolized, after transport from the blood to the tissues, to the 6-phosphate by hexokinase in the tissues,355and trapped there for a time without further metabolism because 2DG 6-phosphate is not356a substrate for either phosphohexose isomerase or D-glucose 6-phosphate dehydrogenase as it lacks an OH-2 group. This feature makes it convenient to evaluate the initial D-glucose uptake in the tissues by use of 2DG if it is assumed that the rate of initial uptake of 2DG (and of 14C-2DG)is proportional to that of D-glucose (and this has been confirmed). Sokoloff and coworkers357designed a mathematical model for the consumption of D-glu(354) (355) (356) (357)

A. P. Wolf, Semiti. Nucl. Med., I I (1981) 2- 12.

Related references are cited in Refs. 357 and 372. A. Sols and R. K. Crane, J. B i d . Chem.. 210 (1954) 581 -595. L. Sokoloff,M. Reivich, C. Kennedy, M. H . DesRosiers, C. S. Patlak, K. D. Pettigrew,0. Sakurada,and M. Shinohara, J. Neurochem., 28 (1977) 897-916.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

187

cose in brain, using 14C-2DG,under a series of reasonable assumptions, and proposed an operational equation for the net rate of cerebral D-glucose uptake (Riin Eq. 30 in Ref. 357); in practice, the Ri value is a function of a few determinable variables, such as concentrations of 14C-2DGand ~ - g l u cose in arterial plasma, the rate constant (kl*) for the transport of 14C-2DG from plasma to the tissue precursor pool, the rate constant (k2*)for transport back from tissue to plasma, the rate constant (k3*)for phosphorylation of I4C-2DG in the tissue, and the so-called "lumped constant" 357-360 (LC): ,IVmar*.K,,,/4. V,,.Km*.360"An extended R,value [Eq. 23 in Ref. 361 that takes into consideration the role of phosphatase (14C-2DG 6-phosphate + I4C-2DG; the rate constant being expressed as k4*)J was proposed361.362 late~-.~~~~ (358) M. Reivich, D. Kuhl, A. Wolf, J. Greenberg, M. Phelps, T. Ido, V. Casella, J. Fowler, E. Hoffman, A. Alavi, P. Som, and L. Sokoloff, Cir. Rex. 44 (1979) 127- 137. (359) H. Matsuda, H. Nakai, S. Jovkar, M. Diksic, A. C. Evans, E. Meyer, C. Redies, and Y.L. Yamamoto, J. Nucl. Med., 28 (1987) 471 -480. (360) R. A. Brooks, J. Hatazawa, G. Di Chiro, S. M. Larson, and D. S. Fishbein, J. Cerebr. Blood Flow Metab., 7 (1987) 427-432. (360a) The 1 value is the ratio ofthe distribution volume of 2DG (and IT-2DC) in the tissue to that of D-glucose;4 is the ratio ofthe difference between the rates ofphosphorylation and K,* and VmU*,and K,,, dephosphorylation Of D-glucoseto its rate of pho~phorylation~~'; and Vmarare the kinetic constants of the Michaelis-Menten equation [v = V,, [S]/ (IS] K,,,)] for the hexokinase-catalyzed phosphorylation reactions of 2DG and ~ - g l u P (product)]. This constant cose, respectively [S (substrate) E (enzyme) ES+E (LC) accounts for the ratio of the arteriovenous extraction fraction (by transport and phosphorylation) of 2DC to that of glucose ( L C = I ) under steady-state condit i ~ n s . " " JThis ~ ~ concept can be directly applied to the caseof2DFG by employing the LC (-0.5) for 2DFG. (361) M. E. Phelps, S.-C. Huang, E. J. Hoffman, C. Selin, L. Sokoloff, and D. E. Kuhl, Ann. Neurol.. 6 (1979) 371-388. (362) S.-C. Huang, M. E. Phelps, E. J. Hoffman, K. Sideris, C. J. Selin, and D. E. Kuhl, A m . J. Ph,~iol..238 (1980) ~ 6 9 - ~ 8 2 . (362a) These rate constants are more precisely determined363(especially for the uptake of IsF-2DFG in brain) by taking into consideration the other factors involving regional cerebral blood volume (rCBV). The importance of the first few minute's data on tissues has also been stressed. Thus, several alternative operational equations e m p h a ~ i z i n gthe ~~ ~ ~ ' a quick computing algorithm for calcuimportance of K B V have a ~ p e a r e d . ~ " .Also, lating the rate constant (k,*,kZ*,k3*)in the I8F-2DFG model has been developed.36s (363) A. C. Evans, M. Diksic, Y. L. Yamamoto, A. Kato, A. Dagher, C. Redies, and A. Hakim, J. Cerebr. Blood Flow Metab., 6 ( 1986) 724- 738; related references on the rate constants are cited therein. (364) A. A. Lammertsma, D. J. Brooks, R. S. J. Frackowiak, R. P. Beaney, S. Herold, J. D. Heather, A. J. Palmer, and T. Jones, J. Cerebr. Blood Flow Metab., 7 ( 1 987) 16 I - 172; related references on the operational equations are cited therein. (365) H. Sasaki, I. Kanno, M. Murakami, F. Shishido, and K. Uemura, J. Cerebr. Blood Flow Metab., 6 (1986) 447-454.

+

+

+

+

188

TSUTOMU TSUCHIYA

This principle is useful, coupled with autoradiography, for evaluating the initial regional D-glucose consumption (uptake) by use of 14C-2DG or 3H-2DGfor the frozen sliced tissues of sacrificed It cannot be applied, however, for living human beings, because external detection (for the brain, for example, detection must be from outside the skull) of the emissions of radionuclides by /? decay (for example, 14Cand 3H;these are the radionuclides most commonly used in drug distribution studies) is impossible, because of the short range of electron travel. In consequence, compounds labeled with nuclides of positron emitters (“C, 13N, 150,or leF) producing penetrating prays are to be desired. Thus, [ I-11C]-2DGwas ~ r e p a r e d , ~but ~ ~ the -~~ half-life ’ of “C is too short for it to be used conveniently (llC:T,,, = 20.4 min, 13N:10.0 min, I5O:123 s, and I8F:109.8 min). Therefore, 2DG analogs having a longer half-life, such as 2-deoxy-2[ 18F]fluoro-~-ghcose ( 18F-2DFG),were considered as candidates, although they contain the “foreign atom, fluorine,” an undesirable inclusion from the biochemical viewpoint. Coe372and Bessell and coworkers373studied the metabolic fates of 2deoxy-2-fluoro-~-glucose(2DFG) and related compounds by using yeast hexokinase (as a model for mammalian hexokinase), and determined373the kinetic constants ( K , and V-) of the Michaelis-Menten equation; ~ - g l u cose: 0.17 (K, in mM), 1.OO (relative Vmarvalue, D-glucose taken as 1); 2DG: 0.59 f0.1 1, 0.85; 2DFG: 0.19 f0.03, 0.50; 2-deoxy-2-fluoro-~-mannose (2DFM): 0.4 1 f 0.05, 0.85; 2-deoxy-2,2-difluoro-~-urubinu-hexose (2,2DFG):0.13 +0.02,0.53; 3-deoxy-3-fluoro-~-glucose(3DFG): 70 f30, 0.10; and 4-deoxy-4-fluoro-~-glucose(4DFG): 84 f 30,O. 10. These deoxyfluoro sugars are thus classified into three groups: (1) good substrates for hexokinase: 2DFG, 2,2DFG, 2DFM, and 2DG; (2) poor substrates: 3DFG and 4DFG; and (3) non-substrates: a-andp-D-glucopyranosyl fluorides, and

(366) K. Takahashi, M. Murakami, E. Hagami, F. Shishido, S. Miura, H. Sasaki, I. Kanno, and K. Uemura, Kukuiguku, 21 (1984) 1389- 1393. (367) J . L. Olds, K. A. Frey, R. L. Ehrenkaufer and B. W. Agranoff, Bruin Rex, 361 (1985) 217-224. (368) M. Murakami, K. Takahashi, E. Hagami, Y. Kondo, H. Iida, S. Miura, H. Sasaki, 1. Kanno, and K. Uemura, Kukuiguku, 23 (1986) 843-847. (369) J. S. Fowler, R. E. Lade, R. R. MacGregor, C. Shiue, C.-N. Wan, and A. P. Wolf,J. Label. Comp. Radiophurm., 16 (1979) 7-9. (370) C.-Y. Shiue, R. R. MacGregor,R. E. Lade,C.-N. Wan,andA. P. Wolf, Curbohydr. Res., 74 (1979) 323-326. (37 I ) R. R. MacGregor, J. S. Fowler, A. P. Wolf, C.-Y. Shiue, R. E. Lade, and C.-N. Wan, J . N d Med., 22 (1981) 800-803. (372) E. L. Coe, Biochim. Biophys. Actu, 264 (1972) 319-327. (373) E. M. Bessell, A. B. Foster, and J. H. Westwood, Biochem. J., 128 (1972) 199-204.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

I89

6-deoxy-6-fluoro-~-glucose(as it lacks an OH-6 group). It was concluded that the 2-deoxy compounds in group (1) behave similarly, to give the corresponding 6-phosphates. The poor substrate character of the 2DFG 6-phosphate thus produced for further metabolism, as with 2DG 6-phosphate described before, was also demonstrated.374375 It was concluded, therefore, that 18F-2DFG(Ref. 376) could effectively be used as a tool for the evaluation of initial D-glucose utilization in tissues. Fluorine-I 8 is not known naturally (natural fluorine is all I9F),but it may be prepared in a variety of artificial nuclear reactions. It has a short half-life (T,!*109.8 min), and decays377to stable oxygen-18 by emitting a positron /I+ which is annihilated when it encounters an electron /Iin the surrounding matter, to produce two 0.5 1 1-MeV photons (prays). Thus, administration of I8F-labeled2DFG or its equivalent, followed by measurement ofthe pray concentration in tissues with a tomographic scanner, situated around the living organs, and simultaneous computer-aided data-processing based on several assumptions involving an appropriate kinetic model (for example, the R j value 357 or a corrected gives transformed topological images358 of the regional D-glucose uptake (in other words, the initial rate of D-glucose consumption) of the tissues (brain, heart, and so on) at selected times after administration. This procedure is called positron-computed tomography358.378.379 (PCT). The term “emission-computed tomography” 380 (ECT) is also used. “Positron emission tomography” 381-384 (PET)is more generally used, but sometimes with less emphasis on the computer processing. At present, the most important drawback of the PET method is the limited spatial resolution.

(374) E. M. Bessell and P. Thomas, Biochem. J., 131 (1973) 77-82. (375) E. M. Bessell and P. Thomas, Biochem. J., 131 (1973) 83-89. (376) J. S. Fowler and A. P. Wolf, Appl. Radiat. Isot., 37 (1986) 663-668; a historical perspective of I8F-2DFG with related references. (377) B. M. Coursey, D. D. Hoppes, M. P. Unterweger, A. G . Malonda, R. A. Margolin, R. M. Kessler, and R. Manning, Int. J. Appl. Radial. Isot.. 34 (1983) 1181 - 1189. (378) M. E. Phelps, Semin. Nucl. Med., I 1 (1981) 32-49. (379) M. E. Phelps, J. C. Mazziotta, and S.-C. Huang, J. Cerebr. BloodFlow Metab.. 2 (1982) 113-162. (380) M. E. Phelps, E. J. Hoffman, S.-C. Huang, and D. E. Kuhl, J. Nucl. Med., 19 (1978) 635-647. (381) T. Greitz, D. H. Ingvar, and L. Widen (Eds.), The Metabolism ofthe Human Bruin Studied with Positron Emi.rsion Tomography, Raven Press, New York, 1985. (382) M. E. Phelps and J. C. Mazziotta, Science, 228 (1985) 799-809; references on PET on brain reported before 1984 are cited therein. (383) R. Dagani, Chem. Eng News, Aug. 15 (1988) 26-29. (384) N. C. Andreasen, Science, 239 (1988) I38 1 - 1388.

TSUTOMU TSUCHIYA

190

b. Preparation of Deoxy-18F-FluoroCarbohydrates.-In the synthesis of lsF-labeled sugars, fluorine-I8 reagents must first be prepared. Molecular [lsF]fluorine(lSF2)and [lsF]fluoride ( I 8 F ) are the two major reagents initially produced by the nuclear reactions. Anhydrous 18F2(Refs. 385 - 389) has been prepared by the accerelated deuteron bombardment of neon gas in a target chamber, the nuclear reaction being ,0Ne(d,a)18F(a:He nucleus), and the short-lived fluorine- I8 thus created is, without delay, allowed to react with glycals or utilized to prepare another 18Freagent. The deuteron beam is produced by different types of cyclotron (medical-, small-, baby-cyclotrons, and the like; Wolf and Jones3goclassified them into four levels, based on the maximum beam energy). The yield and purity of 18F2produced is significantly influenced by the quality of the materials of the target chamber (nickel is usually used) as well as by the con tarn in ant^^^^^^^^ in the target gas, which is usually a mixture of neon and a trace of fluorine (19F,). The added 19F2operates as a s c a ~ e n g e rof~ I8F ~ ~adsorbed . ~ ~ ~ on, or reacted with, the walls of the producing system's target-chamber (for example: I9F2 Ni18F, -+ I8F2 Ni19F2)and as a carrier of the I8F2gas; if 19F2is not added, the yield ofthe volatile IsF2is very Small proportions (>0.1Yo) of N,, CO,, or CF, in the target gas, neon, result in the production of N18F3 and P F , at the expense of 18F2.High yields of I8F2gas were reported388when the levels of N,, CO,, and CF, in the target-gas mixture were less than 0.0 lYo, and the carrier F, was - 0.1%.Factors influencing the yield of I8F2have been examined in detai1.39'.392 ['8F]Fluorine thus formed in the target chamber is purged into a solution containing a substrate or a precursor for another reagent. Introduction3" of I8F2 into 3,4,6-tri-O-acetyl-~-glucal (61) in CC13F (Freon 1 I ) at -78" in a manner used for the non-labeled compound310 (so-called cold synthesis) gave a 4 : 1 mixture of 3,4,6-tri-O-acetyl-2-deoxy2 4 18F]fluoro-a-D-gluco-(574) and P-D-manno-pyranosyl fluorides (575),

+

+

(385) T . Nozaki. M. Iwamoto, and T. Ido, Int. J. Appl. Radiat. Isot.. 25 (1974) 393-399. (386) R. M. Lambrecht, R. Neirinckx, and A. P. Wolf, Int. J. Appl. Radiat. hot., 29 (1978) I75 - 183; references before I976 for the production of anhydrous l*F, are cited therein. (387) V. Casella, T. Ido, A. P. Wolf, J. S. Fowler, R. R. MacGregor, and T. J. Ruth, J. Nucl. Med.. 21 (1980) 750-757. (388) G. T. Bida, R. L. Ehrenkaufer, A. P. Wolf, J. S. Fowler, R. R. MacGregor, andT. J. Ruth, J. Nucl. Med.. 21 (1980) 758-762. (389) G. Blessing, H. H. Coenen, K. Franken, and S. M. Qaim, Appl. Radial. Isot., 37 (1986) 1135- 1 139. (390) A. P. Wolf and W. B. Jones, Radiochim. Acta, 34 (1983) 1-7. (39 I ) W. J. Shaughnessy,S. J. Gatley, R.D. Hichwa, L. M. Lieberman, and R. J. Nickles, Int. J . Appl. Radiat. Isot., 32 (1981) 23-29. (392) M. Diksic and Y. Toda, Can. J. Chem., 61 (1983) 661 -664.

191

DEVELOPMENTS O F FLUORINATED CARBOHYDRATES

which were separated (preparative g.1.c.); 574 and 575 were then hydrolyzed (as. HCl, 130") to give I8F-2DFG3" (8% radiochemical yield) and I8F-2DFM.Many subsequent report^^^^-^ were basically developments of this proced~re,~" simplifying, semi-automating,"O' or automating it, and

6) - 6) &-$T

AcO CH 2

AcOCH2

I

+

AcO

AcOYH2

I

+

"F2

AcO

AcO

IXF

61

574

HOCH2

575

HOCH2

&?OH

&%OH

HO

HO

'XF ''F-2DFG

''F-2DFM

(393) M. Diksic and D. Jolly, Appl. Radial. Isot., 37 (1986) 1159- 1161. (394) D. L. Alexoff, J. A. G. Russell, C.-Y. Shiue, A. P. Wolf, J. S. Fowler, and R. R. Madiregor, Appl. Radiat. Isot.. 37 (1986) 1045- 1061. (395) J. R. Barrio, N. S. MacDonald, G. D. Robinson, Jr., A. Najafi, J. S. Cook, and D. E. Kuhl, J. Nucl. Med., 22 (1981) 372-375; key problems on the synthesis of I8F-2DFG are discussed and related references are cited therein. (396) J. S. Fowler, R. R. MacGregor, A. P. Wolf, A. A. Farrell, K. I. Karlstrom, and T. J. Ruth, J. N d . Med., 22 (1981) 376-380. (397) T. Irie, T. Ido, K. Fukushi, R. Iwata, M. Uoji, K. Tamate, T. Yamasaki, and Y. Kashida, Radioisotopes, 3 I ( 1982) 13- 15. (398) R. Iwata, T. Ido, T. Takahashi, and M. Monma, Int. J. Appl. Radial. Isot., 35 (1984) 445-454. (399) C.-Y. Shiue, C. D. Amett, and A. P. Wolf, Eur. J. Nucl. Med., 9 (1984) 77-80. (400) G . Mestelan, C. Crouzel, C. Cepeda, and J. C. Baron, Eur. J. Nucl. Med., 7 (1982) 379- 386. (401) G. D. Robinson, Jr., N. S. MacDonald, M. P. Easton, and J. S. Cook, J. Nut/. Med., 19 (1978) 701 -702.

TSUTOMU TSUCHIYA

192

employing remote and shielded operational systems to protect the operators from exposure to irradiation (in such steps as solvent transfer, chromatography, and evaporation). Minimizing the loss of radioactive fluorine distributed in the synthetic line, and lessening the total time required for the synthesis (less than 1 hour is desirable) are also factors to be considered. 2-Deoxy-2-[18F]fluoro-~-galactose (18F-2DFGal;a diagnostic liver-imaging agent), -maltrose, and -L-glucose were prepared3'2.a2-404by treatment of 465 (see Section 11,5), 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-~-ribo-hex1-enitol (471), and tri-0-acetyl-L-glucal, respectively, with 18F2or Ac0I8F (prepared from 18F2as described later), followed by acid-catalyzed hydrolysis. Preparation of I8F-2DFG from unprotected D-glucal ( 1,5-anhydro-2deoxy-D-urubino-hex-1-enitol) and 18F2would be advantageous in comparison with that through 61, in terms of decreasing the overall synthetic time and reaction steps by avoiding the use of protecting groups. Bida and cow o r k e r ~ ~ introduced ' ~ , ~ ' ~ 18F2into an aqueous solution of D-glucal(487) at room temperature, and, after acid-catalyzed hydrolysis of the resulting 1,2difluorides, the desired 18F-2DFGand I8F-2DFM were obtained in the ratio of 2 : 1. In this reaction, the 1,2-difluoro products were considered to be formed by addition of a fluorine molecule to D-glucal. However, considering the rapid reaction of fluorine with water to give hypofluorous acida5 (F2 H,O -,HF HOF), the real addition mechanism to give the 1,2-difluoride remains c o n t r o ~ e r s i a l . Diksic ~ ' ~ ~ ~and ~ ~ coworker^^^^-^^^ independently studied the reaction of D-glucal(487), tri-0-acetyl-D-glucal (61),and D-galactal (490) with F, (see Table IV in Section II,5) or I8F2in water, to give I8F-2DFG, I8F-2DFM, 18F-2DFGal,and 2-deoxy-2-[L8F]fluoro-~-talose, respectively. Preparation of 18F-2DFG through the reaction of tri-0-acetyl-D-glucal (61)and Xe18F2has been r e p ~ r t e d(see ~ ~Section ~ . ~ ~11,5). ~ In 1985,aquestion was raised concerning the purity of the synthetic I8F-2DFGthus far reported. Thus, van Rijn and coworkersm clarified, by t.1.c. (NaH,PO,-impregnated silica plates) and h.p.1.c. analysis, that the addition reactions of AcOF to

+

+

(402) M. Tada, T. Matsuzawa, K. Yamaguchi, Y. Abe, H. Fukuda, M. Itoh, H. Sugiyama, T. Ido, and T. Takahashi, Carhohydr. Rex, 161 (1987) 314-317. (403) T. Takahashi, T. Ido, M. Shinohara, R.Iwata, H. Fukuda, T. Matsuzawa, M. Tada, and H. Orui, J. Label. Comp. Radiopharm., 2 1 ( 1984) 12 15 - 12 17. (404) F. Oberdorfer, B.-C. Traving, W. Maier-Borst, and W. E. Hull, J. Label. Comp. Radiophnrm., 25 (1988) 465-481. (405) E. H. Appelman and R. C. Thompson, J. Am. Chem. Soc., 106 (1984) 4167-4172. (406) C. J. S. van Rijn, J. D. M. Herscheid, G . W. M. Visser, and A. Hoekstra, Int. J. Appl. Radial. I s o f . . 36 (1985) 1 I I - 115.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

193

tri-0-acetyl-D-glucaI(61) and D-glucal are not fully stereoselective,but give mixtures of 2DFG and 2DFM (2DFG :2DFM = 83 : 14-92 : 56). Shiue and coworkers337obtained similar results. This problem has been clarified further by detailed studiesN7(see also Refs. 3 18, 337, and 338). 18F-2DFGand I8F-2DFM were successfully separatedN8 (as confirmed by the IH- and 19F-n.m.r.data) by h.p.1.c. (developed with water) with lead(I1)-loaded cation-exchangeresin (Aminex HPX-87P), the separation being sensitive to the temperature and the flow rate. A rapid, enzymic method for determining the purity of I8F-2DFG preparations using hexokinase (to give 18F-2DFG6phosphate) coupled with use of a column of an anion-exchange resin (Dowex-I) has been devised.N9 Methods so far reported for preparing 18F-2DFGby electrophilic reaction were Synti-esis of 2-deoxy-2-['8F]fluoro sugars using I8F-labeled acetyl hypofluorite (AcO18F)is described next. The reagent was ~ r e p a r e d ~ lfrom l-~~~ 18F2according to the procedure for the cold synthesis (see Section 11,5). Introduction of 18F2at room temperature into acetic acid containing a small proportion of ammonium hydroxide (or sodium acetate) gave Ac018F (chemical and radiochemical yields were 80 and 40%, respectively). Subsequent reaction of Ac018F with tri-0-acetyl-D-glucal [reaction period: 2 - 5 min (Ref. 408)] gave the peracetates 576 and 577; 576 was converted into I8F-2DFG [20 - 3 1% radiochemical yield; reaction period 60 - 70 min after

AcOH

"F?

+

MKCO~Nlt,

M~co~'% + NH$'~F

576

577

(407) K. Ishiwata, T. Ido, H. Nakanishi, and R. Iwata, Appl. Radiat. Isot., 38 (1987) 463-466. (408) F. Oberdorfer, W. E. Hull, B. C. Traving, and W. Maier-Borst, Appl. Radiat. Isot., 37 (1986) 695-701. (409) S. J. Gatley, S. G. Brown, and C. M. Thompson, J. Nucl. Med., 29 (1988) 1443- 1447. (410) H. H. Coenen, V. W. Pike, G. Stocklin, and R. Wagner, Appl. Radiat. Isot., 38 (1987) 605-610. (41 1) C.-Y. Shiue, P. A. Salvadori, A. P. Wolf, J. S. Fowler, and R. R. MacGregor, J. Nucl. Med.. 23 (1982) 899-903. (412) J. S. Fowler, C.-Y. Shiue, A. P. Wolf, P. A. Salvadori, and R. R. MacGregor, J. Label. Cornp. Radiophurm., 19 (1982) 1634- 1636. (413) M. Diksic and D. Jolly, Int. J. Appl. Radiat. Isot.. 34 (1983) 893-896.

I94

TSUTOMU TSUCHIYA

the end of bombardment (EOB)]. The product was reported to be free from 2-deoxy-2-[18F]fluoro-~-mannose(I8F-2DFM);the purity was confirmed (also, see the later description) by comparison (g.1.c.) with authentic 18F-2DFM.When, in this reaction, I8F2was introduced into an acetic acid solution of 61,the resultant I8F-2DFG derivative was contaminated with two 1,2-cis difluoro To shorten the synthetic period and eliminate the complications accombY panied by solution transfer, gaseous Ac018F was first prepared386.414s415 introducing 18F2(diluted with an inert gas) into a column containing KOAc * (HOAc),,, (the particle size and dryness significantly influenced the yield of Ac018F),and the gas liberated was then introduced into a solution of 61 in Freon 1 1314.414 (CC13F), or of D-glucal in water.415Automatic syst e m ~ involving ~ ~ ~ a . modular ~ ~ ~autosynthesi~er~~~ for this reaction were developed. Palmer showed4I7that I8F-2DFG prepared from 61 and l8F2could be separated, by liquid chromatography on a cation-exchange resin (AminexA5, Rb+ form), from [18F]fluorideand less-polar impurities, but not from I8F-2DFM. 2-Deoxy-2-[18F]fluoro-~-galactose (18F-2DFGal) was prepared4I8 from 3,4,6-tn-0-aCetyl-D-galaCtal(465), and l8F2in the general manner described for the cold synthesis.312The 1-phosphate of 18F-2DFGalwas prepared.419 The phosphate and UDP-I8F-2DFGal were prepared4I9 enzymically in a manner analogous to that reported for 2-deoxygalactose. In contrast to the preparation of anhydrous 18F2so far described, [I8F]fluoride may be prepared comparatively easily.420-424 Nuclear reactions425fre(414) D. M. Jewett, J . F. Potocki, and R. E. Ehrenkaufer, J. Flzrorine Cliern., 24 (1984) 477-484. (415) R. E. Ehrenkaufer, J. F. Potocki, and D. M. Jewett, J . Nucl. Med., 25 (1984) 333-337. (416) M. J . Adam, T. J. Ruth, S. Jivan, and B. D. Pate, Inf. J. Appl. Radial. Isot., 35 (1984) 985-986. (4 17) A. J. Palmer, J . Label. Comp. Rudiopharm.. 18 ( I98 I ) 264- 266. (418) H. Fukuda, T. Matsuzawa, M. Tada, T. Takahashi, K. Ishiwata, K. Yamada, Y. Abe, S. Yoshioka, T. Sato, and T. Ido, Eur. J. Nucl. Med., 1 I (1986) 444-448. (4 19) K. Ishiwata, T. Ido, Y. Imahori, K. Yamaguchi, H. Fukuda, M. Tada, and T. Matsuzawa, Nucl. Med. Biol., 15 (1988) 27 I -276. (420) P. V. Harper, N. Lembares, and H. Krizek, J. Nucl. Med., 12 (1971) 362-363. (421) F. Helm, 0. Krauss, and W. Maier-Borst, Radiochem. Radioanal. Lett., 15 (1973) 225-230. (422) G. Firnau, G. Nahmias, and S. Garnett, Inf. J. Appl. Radial. Isot., 24 (1973) 182- 184. (423) B. E. Gnade, G. P. Schwaiger, C. L. Liotta, and R. W. Fink, Int. J. Appl. Radial. Isot., 32 (1981) 91 -95. (424) S. J. Gatley, R. D. Hichwa, W. J. Shaughnessy, and R. J. Nickles, Int. J. Appl. Radiat. I.W., 32 (1981) 21 1 -214. (425) J. Fitschen, R. Beckmann, U. Holm, and H. Neuert, Inf.J . rlppl. Radial. Isot., 28 (1977) 781 -784.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

195

quently used for this purpose have been: 6Li(n,a)3H followed by 160(3H,n)'8F [Ref. 426; target material being Li,CO, (Refs. 421 and 422), LiOH-H,O, or enriched 6LiOH-H,0 (Refs. 423 and 427), and 6LiF-160 (Ref. 428)], 160(4He,pn)'8F,160(3He,p)'8F (Refs. 425,429, and 430; target materials: oxygen or water), '80(p,n)18F(Refs. 429 and 431 -437; target materials: or H2I80),2oNe(3He,cup)'8F, and 2oNe(d,cu)'8F(described also in the preparation of '*F,). When a water target385,425~429.430~432.434*437-448 [H2160 or H 2 l 8 0 (enriched or non-enriched)] was employed in the 160(3He,p)18F or 180(p,n)18Freaction, the irradiated water in the target chamber was, at the EOB, pumped out from the chamber, and, after addition of a carrier (KF, CsF, KHF,, and the like), the solution was evaporated (426) H. V. Ruiz, Appl Radiut. /so/., 39 (19XX) 31 -39. (427) S. J. Gatley and W. J. Shaughnessy, Int. J . .4ppl. Rudia/. Isot., 33 (1982) 1325- 1330. (428) B. W. Wessels, W. R. Yusof, and D. Ercegovic, J . Radioanal. Nrrcl. Chern., 92 (1985) 27-35. (429) M. Vogt, R. Weinreich. E. J. Knust, and H.-J. Machulla, Appl. Radial. Isot.. 37 (1986) 873 - 875. (430) E. J. Knust and H.-J. Machulla. I w / . J. App/. Radiut. Isot.. 34 (1983) 1627- 1628. (431) T. J. Ruth and A. P. Wolf. Rudiodrini. Ac/a. 26 (1979) 21 -24. (432) M. R. Kilbourn. J. 7'. Hood, and M. J. Welch. In/.J. App/. Radial. Isot.. 35 (1984) 599-602. (433) C.-Y. Shiuc. J. S. Fowler, A. P. Wolf, M. Watanabe, and C. D. Amett, J . Nircl. A f d 26 (1985) 181-186. (434) A. Luxcn, N. Satyamurthy, G. T. Bida, and J. R. Barrio, Appl. Radiar. Isof..37 (1986) 409 - 4 13. (435) R. J. Nickles, R. D. Hichwa, M. E. Daube. G. D. Hutchins, and D. D. Congdon. Int. J . Appl. Rudial. Isot.. 34 (1983) 625-629. (436) R. J. Nickles, M. E. Daube, and T. J. Ruth, In/.J. ,4pp/. Radiat. Isot.. 35( 1984) 1 17- 122. (437) 0.7'.Dejesus, J. A. Martin, N. J. Yasillo, S. J. Gatley, and M. D. Cooper,Appl. Radrar. / s o t , 37 (1986) 397-401. (438) P. A. Beeley, W. A. Szarek, G. W. Hay, and M. M. Perlmutter, Can. J . Chern..62 (1984) 2709-271 I . (439) E. J. Knust, H.-J. Machulla, and W. Roden. App/. Radiut. Isol., 37 (1986) 853-856. (440) B. W. Wieland and A. P.Wolf, J. N r d Med.. 24 (1983) ~ 1 2 2 . (441) 1. HuszQr and R. Weinreich, .I. Radioanal. N U ( / . Cliem. Le/l.. 93 (1985) 349-354. (442) M. S. Bemdge and 7'.J. 'l'cwson, J. Label. Comp. Radiopharm., 23 ( 1986) I 177- 1 178. (443) M. R. Kilbourn. P. A. Jerabek. and M. J. Welch, Int. J. App/. Rndiat. Isot.. 36 (1985) 327- 328. (444) M. Vogt. 1. Huszar, M. Argentini, H. Oehninger, and R. Weinreich, Appl. Radial. I s o t . , 37 (1986) 448-449. (445) J. Keinonen, A. Fontell, and A.-L. Kairento, A p p / . Radial. Isot., 37 (1986) 631 -632. (446) R. Iwata. T. Ido, F. Brady, T. Takahashi, and A. Ujiie, Appl. Radiat. Isot.. 38 (1987) 979-984. (447) H. J. Tochon-Danguy, D. Townsend, M. Wensveen, P. Frey, A. Christin, A. Geissbuhler, A. Donath, H. Ravn. and R. Deltenre. J. Hrrorine CIwm., 41 (1988) 33-43. (448) 0. S o h . J. Bergrnan, M. Haaparanta, and A. Reissell, A& Radial. Iso/., 39 (1988) 1065- 1071.

196

TSUTOMU TSUCHIYA

and the residue dried (mostly by microwave heating) to give anhydrous 18F salts (K18F, C P F , KHL8F,, and so on) in a short reaction-period. The yield, purity, and ease of production of 18F- were compared385for several nuclear reactions involving 2oNe(d,a)1SF, 20Ne(3He,ap)1sF, 2oNe(3He,an)18Ne -, 18F,and 160(3He,p)18F. Camer-free H18Fand [ lsF]fluorides were also prepared.449These were NaI8F (Refs. 386 and 423), Agl8F, K18F (Refs. 429 and 434), lBF-Bu4NF(Refs. 434 and 450; they can also be prepared through reaction of trimethylsilyl [ 18F]fluorideand tetrabutylammonium hydroxide451),l8F-Et4NF(Refs. 424, 429. 452, and 453), W 8 F (Refs. 454 and 455; H18Fwas initially prepared from the ,ONe(d,a)I8Freaction, using a target of Ne containing 15% of H,, and the HI8Fproduced was allowed to react with CsOH), resin-CH,NMe3+18F-, and K18F comp l e ~ e d ~with * ~ 18-crown-6 [the nuclear reaction being 6Li(n,a)3H160(3H,n)18F and the 3H accompanied was removed by an alumina column, total time, 20- 30 min]. Effective extraction procedures of [18F]fluoride from the [ 180]watertarget were r e p ~ r t e d ,using ~ ~ ~a -column ~ ~ ~ of anion-exchange resin or of lipophilic K+ (or NH4+) cryptand (Kryptofix 222-D), followed by solvent extraction. Production of fluorine- 18 by high-energy proton bombardment against a molten aluminum target was the nuclear reaction being 27Al(p,2a2n)18Ne(1.67s) ISF. The I8Ne produced in the target can be continuously swept away from the irradiation zone with a stream of He gas; the carrier-free 18F2can then be converted into the electrophilic or nucleophilic 18F-reagents.

-

(449) A. J. Palmer, J. C. Clark, and R. W. Goulding, Inf. J . Appl. Radiaf. Isot.. 28 (1977) 53-65; reports on the preparation of I8F- before 1975 are cited therein. (450) M. Maeda, T. Fukumura, and M. Kojima, Appl. Radial. Isot., 38 (1987) 307-310. (451) R. Chirakal, G. Firnau, and E. S. Garnett, Appl. Radiat. Isot.. 39 (1988) 1099- 1101. (452) S. J. Gatley and W. J. Shaughnessy, Int. J. Appl. Radial. Isof.. 31 (1980) 339-341. (453) T. J. Tews0n.J. N u d MLd. 24(1983) 718-721. (454) T. J. Tewson, M. J. Welch, and M. E. Raichle, J. Nircl. Med., 19 (1978) 1339- 1345; reports on biochemical studies on 3DFG before 1977 are cited therein. (455) T. J . Tewson and M. J . Welch, J . Nucl. Med.. 21 (1980) 559-564. (456) H. H. Coenen, M. Colosimo. M. Schiiller, and G . Stocklin, J . Nucl. Med.. 26 (1985) ~ 3 7 ; H. H. Coenen, B. Klatte, A. Knochel, M. Schiiller, and G. Stikklin, J. Label. Comp. Radiopharm., 23 (1986) 455-466. (457) D. Block, B. Klatte, A. Knochel. R. Beckmann, and U. Holm, J. Label. Comp. Radiopharm.. 23 (1986) 467-477. (458) D. J. Schlyer, M. Bastos, and A. P. Wolf, J. Nucl. Med.. 28 (1987) 764. (459) D. M. Jewett, S. A. Toorongian, G. K. Mulholland, G. L. Watkins, and M. R. Kilbourn, Appl. Radiat. Isot.. 39 ( I 988) 1 109 - 1 1 1 I . (460) M. C. Lagunas-Solar, 0. F. Carvacho, and R. R. Cima, Appl. Radiat. Isot., 39 (1988) 4 I -47.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

197

Another method for the production of [ lEF]fluoridefrom fluorocarbons by the I9F(y,n)lEFreaction has been reported.46118F-DAST has been pre~ared.~.~~~ Preparation of I8F-2DFG by way of the S N reaction ~ with lEF-has attracted attention for the following reasons: (I) 1 8 F (with or without carrier) is obtained more readily than IEF2,(2) all of the expensive radioisotopes of the lEF-radiolabel can be utilized in principle (especially when an excess of the substrate is used), whereas the reaction of glycal with I8F2utilizes only half of the radioisotope, and (3) the fluorinated products are separable comparatively readily from the accompanying by-products; whereas fluorine addition to glycals usually occurs from both the a and /? sides, to give products difficultly separable. However, the S N reaction ~ at C-2 generally proceeds with difficulty, and takes a longer time to complete. One method for resolving the problem would be to utilize the triflate group (see Section 11,2). Thus, 18F-2DFGwas prepared462according to the cold synthesis166. treatment of l,3,4,6-tetra-O-acetyl-2-O-(tr~fluoromethylsulfonyl)-/?-~mannopyranose (578) with CsH18F2gave 1,3,4,6-tetra-O-acetyI-2-deoxy-2[ 18F]fluoro-/?-~-glucopyranose (579) in 30% yield [in N,N-dimethylformamide (DMF) or hexamethylphosphoric triamide (HMPA), 130°,25 min]. Robotic synthesis of 18F-2DFG using 578 and IEF-Bu,NF has been reported.463Carrier-free IEF-2DFGwas also prepared.464Thus, treatment of 578 with amino(po1yether)-supported, carrier-free [18F]fluoride [KL8FKryptofix 222; prepared from carrier-free HI8F (Ref. 386)and a polyether] gave 579, which, on acid-catalyzed hydrolysis, gave carrier-free 18F-2DFG (- 44%, total time 45 - 50 min). A computer-controlled synthesis of this product has been developed.465lEF-2DFG was also prepared429,437,453 bY AcOCI 1: I

I

IXF 578

579

(461) G. A. Brinkman and A. Wyand, Appl. Radial. Isot., 39 (1988) 1141- 1144. (462) S. Levy, D. R. Elmaleh, and E. Livni. J. Nttcl. Med., 23 (1982) 918-922. (463) J. W. Brodack, C. S. Dence, M. R. Kilbourn, and M. J. Welch, Appl. Radial. ISOI., 39 (1988) 699-703. (464) K. Hamacher. H. H. Coenen, and G. Stocklin, J. Niicl. Med.. 27 (1986) 235-238. (465) H. C. Padgett. D. G. Schmidt, A. Luxen, G. T. Bida, N. Satyamurthy, and J. R. Bamo, ilppl. Rudiut. Isot.. 40 (1989) 433-445.

198

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treatment of P-D-mannopyranoside 2,3-cyclic s ~ l f a t e ' (187) ~~,~ with ~ ~carrier-free lSF-Et4NF(Refs. 424 and 453) according to the method earlier described.178Detailed studies on this procedure have been reported.466The use467of purified 18F-Et4NFgave a better yield and quality of 18F-2DFG. However, a question was posed468as to the regiospecificityof this reaction (at C-2), based on the h.p.1.c. and g.1.c. analysis. Camer-free 18F-2DFGwas also preparedM9by treatment of methyl 3-0-benzyl-4,6-O-benzylidene-2-O-triflyl-P-D-mannopyranoside (166)or 1,6-anhydro-3,4-di-O-benzyl-2-O-triiflyl-P-D-mannopyranose (181)with fluoride, followed by acid-catalyzed hydrolysis (6 M HCl, 115", 20 min); among the reaction conditions tested, combination of 166 and K18F-Kryptofix 222 in acetonitrile in a poly(methylpentene) vessel gave the best yield and purity. Preparative methods for I8F-2DFG by the S N reaction ~ were compared.417I8F-2DFG was also prepared438through oxirane-ring opening of 407 (see Section II,4) with KH18F,. 2-Deoxy-2-[18F]fluoro-3-O-methyl-~-glucopyranose was prepared by the reaction of methyl 4,6-O-benzylidene-3-O-methyl-2-O-t~flyl-~-~-mannopyranoside (159)with CsH18F,, and the possibility of its use as an imaging agent was examined.470 2-Deoxy-2-[18F]fluoro-~-mannose(18F-2DFM)was prepared434through treatment of 201 (see Section 142) with l8F-Bu4NF(MeCN, 75", 30 min) or K18F crown ether, as reported for the cold synthesis.181I8F-2DFM was also prepared471from methyl 4,6-di-0-acetyl-3-0-benzoyl-2-O-triflyl-a-~-glucopyranoside with amino(po1yether)-supported, carrier-free [ 18F]fluoride. 2-Deoxy-2-[18F]fluoro-~-galactosecan be prepared through* an addition reaction to tri-O-acetyl-D-galactal(465),but better S Nreac~ tion (K18F- Kryptofix 222 in MeCN) of methyl 3,4-O-isopropylidene-2-0triflyl-6-O-trityl-P-~-talopyranoside (220; see Section 11,2), according to the cold synthesis.

(466) J. Z. Ginos, R. French, and R. Reamer, J. Label. Comp. Radiopharm., 24 (1987) 805815. (467) L. G. Hutchins, A. L. Bosch, M. S. Rosenthal, R. J. Nickles, and S. J. Gatley, Int. J. Appl. Radial. Isot., 36 (1985) 375-378. (468) M. M. Vora, T. F. Boothe, R. D. Finn, P. J. Kothari, A. M. Emran, S. T. Carroll, and A. J. Gilson, J. Label. Cornp. Radiopharm., 22 (1985) 953-960. (469) T. Haradahira, M. Maeda, and M. Kojima, J. Label. Comp. Radiopharm., 25 (1988) 497 - 507. (470) S. Levy, E. Livni, D. R. Elmaleh, D. A. Varnum, and G. L. Brownell. Int. J.Appl. Radial. Isof.,34 (1983) 1560- 1562. (47 I ) K. Hamacher, H. H. Coenen, and G. Stocklin, J.Label. Comp. Radiopharm., 23 (1986) 1095. (472) T. Haradahira, M. Maeda, Y. h i , and M. Kojima, J. Label. Comp. Radiopharm.. 25 (1988) 721 -729.

DEVELOPMENTS O F FLUORINATED CARBOHYDRATES

I99

3 - D e o x y - 3 - [ ~ 8 F ] f l ~ ~ r ~ -(I8F-3DFG) ~ - g l ~ ~ ~ ~ ewas prepared429g439.452.essentially according to the procedure for the cold synthesis'97 (see Section 11,2),namely, treatment of 248 with Cs'*F(in DMF or HMPA), tetraalkylammonium ['8F]fluoride (DMF, HMPA, or MeCN) or K18F (in acetamide). 6-Deoxy-6-[~8F]fluoro-cu-~-galactopyranos was likewise prepared222according to the cold synthesis.222 454,467,473-476

c. Biological Application of the Deoxy-'*F-Fluoro-Sugars With and Without Coupling with Positron Emission Tomography.-Numerous biological studies have been performed by using the I8F-2DFG- PET techn i q ~ eamong ~ ~ ~ them, , work on the brain382being the most prominent. The biodistribution of 18F-2DFG in mice was s t ~ d i e d . In ~ ~the~ brain . ~ ~ ~and heart, I8F-2DFG was, after injection, rapidly converted into 18F-2DFG6phosphate and trapped there for more than 2 hours, a convenient feature for application of PET. In contrast, in the lungs, liver, and kidneys, rapid clearance of radioactivity was observed. The I8Fthat cleared from the organs was excreted in the urine, mostly in the form of unchanged I8F-2DFG. Utilization of D-glucose (using I8F-2DFG)in gray and white matter in the brain was measured,358and the former was shown to be the higher, especially in the visual cortex ( 10.3 mg/ 100 g/min). Time- radiation-activity-change after intravenous (i. v.) injection of 18F-2DFG,in human bladder and brain, was The transport rates and the "lumped constants" (see a) of I8F-2DFG from plasma to a variety of human cerebral tissue portions were d e t e r m i r ~ e d . ~Similarly, ' ~ , ~ ~ ~ the transport and metabolism by hexokinase, in brain, of [ l-"C]-2DG and 18F-2DGwere Factors by which to (473) T. J. Tewson, M. J. Welch, and M. E. Raichle, J. Label. Comp. Radiopharm. (Symp. Abstr.), 16 (1979) 10-1 1. (474) S . J. Gatley and W. J. Shaughnessy, J. Labd Comp. Radiopharm., 18 (1981) 24-25. (475) M. M. Goodman, D. R. Elmaleh, K. J. Kearfott, R. H. Ackerman, B. Hoop, Jr., G. L. Brownell, N. M. Alpert, and H. W. Strauss, J. Nucl. Med., 22 (1981) 138- 144. (476) E. J. Knust, H.-J. Machulla, and K. Dutschka, Radiochem. Radioanal. Lett., 55 (1982) 21 -28. (477) Recent studies are to be found in J. Nucl. Mrd., 29 (1988) No. 5 (Proc. Annu. Meet. Soc.

Nucl. Med ... 3Sth). (478) B. M. Gallagher, J. S. Fowler, N. 1. Gutterson, R. R. Madregor, C.-N. Wan, and A. P. Wo1f.J. Nucl.Med., 19(1978) 1154-1161. (479) B. M. Gallagher, A. Ansari, H. Atkins, V. Casella, D. R. Christman, J. S. Fowler, T. Ido, R. R. MacGregor, P. Som, C. N. Wan, A. P. Wolf, D. E. Kuhl, and M. Reivich, J. Nucl. hied.. 18 (1977) 990-996. (480) S . C. Jones, A. Alavi, D. Christman, I. Montanez, A. P. Wolf, and M. Reivich, J. Nucl. Med., 23 (1982)613-617. (48 I ) M. Reivich, A. Alavi, A. Wolf, J. H. Greenberg, J. Fowler, D. Christman, R. Madregor, S. C. Jones, J. London, C. Shiue, and Y. Yonekura, J. Cerebr. Blood Flow Metab., 2 (1982) 307-319.

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increase the accuracy of PET using I8F-2DFGin the measurement of cerebral metabolic activity have been searched for.482.483 Receptor-site mapping of functional activities in brain was carried out by measuring the regional I8F-2DFGuptake in response to given stimuli against v i s ~ a 1 , ~audi~ ~ . ~ ~ ~ - ~ ~ tory,379,484485488 and tactile3799484.485 systems in resting,489 norma1,379.484.488 s t i m ~ l a t e d ,and ~ ~pathological379 ~.~~~ states. The combined use of short-lived [13N]ammoniaand long-lived I8F-2DFG(double-tracer method) was studied.490Also, the combined use of 18F-2DFG(short-lived) and a 14C-compound (long-lived)was undertaken491for the simultaneous measurement of local cerebral blood flow and local cerebral D-glucose utilization. The cerebral metabolic relationship between selected brain regions in healthy adults was (using 18F-2DFG)in order to examine the activity correlation among the regions. The IsF-2DFG- and I3NH, PCT methods were utilized to examine the local metabolism in human agmg,493.494 as well as in such pathologic states as chronic s~hizophrenia,~~’ e p i l e p ~ y ,stroke,494,498 ~~~,~~~ (482) B. Honvitz and S. 1. Rapoport, J. N i d . Med., 29 (1988) 392-399. (483) J. L. Tyler, S. C. Strother, R. J. Zatorre, B. Alivisatos, K. J. Worsley, M. Diksic, andY. L. Yamamoto, J. Niicl. Med., 29 (1988) 631 -642. (484) A. Alavi, M. Reivich, J. Greenberg, P. Hand, A. Rosenquist, W. Rintelmann, D. Christman, J. Fowler, A. Goldman, R. MacGregor, and A. Wolf, Semin. Nucl. Med., I 1 ( I98 1) 24-31. (485) J. H. Greenberg, M. Reivich, A. Alavi, P. Hand, A. Rosenquist, W. Rintelmann, A. Stein, R. Tusa, R. Dann, D. Christman, J. Fowler, B. MacGregor, and A. Wolf, Science, 2 I2 (1981) 678-680. (486) M. E. Phelps, D. E. Kuhl, and J. C. Mazziotta, Science, 21 1 (1981) 1445- 1448. (487) M. E. Phelps, J. C. Mazziotta, D. E. Kuhl, M. Nuwer, J. Packwood, J. Metter, and J. Engel, Jr., Neurologv. 31 (1981) 517-529. (488) J. C. Mazziotta, M. E. Phelps, R. E. Carson, and D. E. Kuhl, Neirrology, 32 (1982) 921 -937. (489) J. C. Mazziotta, M. E. Phelps, J. Miller, and D. E. Kuhl, Neurology, 31 (1981) 503-5 16. (490) S.-C. Huang, R. E. Carson, E. J. Hoffman, D. E. Kuhl,and M. E. Phelps,J. Nucl. Med.. 23 (1982) 816-822. (491) K. Sako, A. Kato, M. Diksic, and L. Y. Yamamoto, Stroke, 15 (1984) 896-900. (492) E. J. Metter, W. H. Riege, D. E. Kuhl, and M. E. Phelps, J. Cerebr. BloodFlow Metab.. 4 (1984) 1-7. (493) D. E. Kuhl, E. J. Metter, W. H. Riege, and M. E. Phelps, J . Cerebr. Blood Flow Metab.,2 (1982) 163-171. (494) M. Kushner, M. Tobin, A. Alavi, J. Chawluk, M. Rosen, F. Fazekas, J. Alavi, and M. Reivich, J. Nucl. Med.. 28 ( 1987) 1667- 1670. (495) T. Farkas, A. P. Wolf, J. Jaeger, J. D. Brodie, D. R. Christman, and J. S. Fowler, Arch Gen. P S J J C 41 ~ . ,(1984) 293-300. (496) D. E. Kuhl, J. Engel, Jr., M. E. Phelps, and C. Selin, Ann. Neiirol., 8 (1980) 348-360. (497) J. Engel, Jr., W. J. Brown, D. E. Kuhl, M. E. Phelps, J. C. Mazziotta, and P. H. Crandall, Ann. Nntrd.. 12 (1982) 518-528. (498) D. E. Kuhl. M. E. Phelps. A. P. Kowell, E. J. Metter, C. Selin. and J. Winter. Ann. Neiirol.. 8 (1980) 47-60.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

20I

embolic infarcti~n,"~~ and A l z h e i m e r ' ~ , ~park ~ ~ins~on'^,^^^^^^ ~ ~ . ~ ~ ~and Huntington's diseases502in humans. Human cerebellar D-glucose consumption in normal (and normal aging) and pathological states (for both ipsilatera1 and contralateral hemispheres in stroke, glioma, and Alzheimer's disease), using the I8F-2DFG- PET method, was studied.494The '*F2DFG-PET method was combined with the l5OZ(T,,2 123 s) inhalation method to study400 the difference in the metabolic rates of oxygen and D-glucose and the regional cerebral blood-flow between a normal man and a patient with ischemic stroke. The PET method was compared with that using n.m.r. imagng503 The 18F-2DFG-PET method was also utilized in examining the metabolism of D-glucose in the heart. Time-distribution patterns of uptake of 18F-2DFGin myocardial tissues in healthy animals and man were studied479,504 as a standard measure for D-glucose consumption in myocardial diseases. A kinetic model for regional myocardial D-glucose utilization, which is basically the same as that for brain, was propo~ed,~~-~@' but, at the same time, difficulty in evaluating the myocardial metabolism was indi~ a t e dTo . ~characterize ~~ the behavior of 2DFG as a D-glucose analog in myocardium, ~-[2-~H]glucose and I8F-2DFG were simultaneously ap-

(499)D. E. Kuhl, M. E. Phelps, E. J. Hoffman, G . D. Robinson, Jr., and N. S. MacDonald,Acta Neurol. Scand.. 56 (Suppl 64)(1977)192- 193. (500) 1. Nakano and A. Hirano, Ann. Ncw-ol.. 15 (1984)415-418. (501) J. B. Chawluk. A. Alavi, R. Dann, H. I. Hurtig, S. Bais, M. J. Kushner, R. A. Zimmerman. and M. Reivich. J. Nircl Med., 28 (1987)43 1-437. (502) D. E. Kuhl. E. J. Mettcr, W. H. Riege, and C. H. Markham, Ann. Neurol., I5 (1984) Sl19-Sl25. (503)A. Alavi and M. Reivich, Nertrol. Neurobiol.. 21 (1986)355-379; Chem. Abstr., 106 (1987)238 I/: (504)M.E. Phelps, E. J. Hoffman, C. Selin, S.-C. Huang, G.Robinson, N. MacDonald, H. Schelbert, and D. E. Kuhl, J . Nucl. Med.. 19 (1978)13 I I - I3 19. (505) M. E. Phelps, H. R. Schelbert, E. J. Hoffman, S.-C. Huang, and D. E. Kuhl, Prog. Nucl. M d 6 (1980)183-209. (506)0 .Ratib, M.E. Phelps, S.-C. Huang, E. Henze, C. E. Selin, and H. R. Schelbert, J. Nucl. h i d . . 23 (1982)577-586. (507)J. Krivokapich, S.-C. Huang, M. E. Phelps, J. R. Barrio, C. R. Watanabe,C. E. Selin,and K. I. Shine, Am. J. Physiol., 12 (1982)~884-~895. (508) R. C. Marshall, S.-C. Huang, W. W. Nash, and M. E. Phelps, J. Nucl. Med., 24 (1983) 1060- 1064. (509)E. Henze,S.-C. Huang,O. Ratib, E. Hoffman, M. E. Phelps,andH. R. Schelbert,J. Nucl. Med., 24 (1983)987-996.

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plied5I0(in rabbits). The PET method was applied511*512 to cases of human myocardial ischemia and infarction. Several cardiac nuclear medicines involving 18F-2DFGwere compared.513 The 18F-2DFG-PET method may also be utilized for tumor d e t e ~ t i o n . ~ ~ ~Rapid , ~ ' ~ -uptake518 ~'' of I8F-2DFG was observed in a variety of transplanted and spontaneous tumors in animals, the uptake ratio in tumor-to-normal tissues being in the range of 2.10-9.15. Distribution patterns of 18F-2DFG in various organs of rats carrying implanted Rous sarcoma were reported.519In rats, the activity-ratio of hexokinase/D-glucose 6-phosphatase was high520( 14.0) in the tumor tissues, and the injected (i. v.) 18F-2DFGwas trapped there (after 45 min) in the form of the 6-phosphate, suggesting that the PET method can also be applied to tumor diagnosis. In liver metastases from colon carcinoma in human patients, I8F-2DFG was ac~urnulated~~' in the tumor (the tumor to normal-liver ratio was 3.3 -4.7 at 50 min after injection), with the central area of the tumor showing less 18F-activity.A gamma camera was used522for imaging of tumor-bearing rats after i. v. injection of l8F-2DFG. After irradiation therapy, 18F-2DFGwas administered, demonstrating it to be a good marker with which to estimate

(510) S.-C. Huang. B. A. Williams, J. R. Bamo, J. Krivokapich, C. Nissenson, E. J. Hoffman, and M. E. Phelps, FEBS Lett., 216 (1987) 128- 132. (5 I 1) R. C. Marshall, J. H. Tillisch, M. E. Phelps, S.-C. Huang, R. Carson, E. Henze, and H. R. Schelbert, Circulation, 6 7 (1983) 766-778. (512) C. M. De Landsheere, Dev. Cardiovasc. Med., 55 (1987) 241-263; Chem. Absrr., 107 (1987) 20,078n. (513) A. Storch-Becker, K. P. Kaiser, and L. E. Feinendegen, Eur. J. Nucl. Med.. 13 (1988) 648-652. (514) G. D. Chiro, R. L. DeLaPaz, R. A. Brooks, L. Sokoloff, P. L. Kornblith, B. H. Smith, N. J. Patronas, C. V. Kufta, R. M. Kessler, G. S. Johnston, R. G. Manning, and A. P. Wolf, Neurology, 32 (1982) 1323- 1329. (515) R. P. Beaney, Semin. Nucl. Med., 14 (1984) 324-341. ( 5 16) Y. Abe, T. Matsuzawa, H. Fukuda, S. Endo, K. Yamada, T. Sato, M. Ito, K. Kubota, J. Hatazawa, S. Yoshioka, T. Fujiwara, and T. Ido, Kakuigaku, 22 (1985) 389-391. (517) H. Joensuu, A. Ahonen, and P. J. Klemi, Eur. J. Nucl. Med., 13 (1988) 502-506. (518) P. Som, H. L. Atkins, D. Bandoypadhyay, J. S. Fowler, R. R. MacGregor, K. Matsui, Z. H. Oster, D. F. Sacker, C. Y. Shiue, H. Turner, C.-N. Wan, A. P. Wolf, andS. V. Zabinski, J. Nucl. Med., 21 (1980) 670-675. (519) R. Paul, A. Iouru, K.-0. Soderstrom, J. Tuominen, R. Johansson, M. Haaparanta, D. Roeda, and 0. Solin, LijiSci., 4 0 (1987) 1609-1616. (520) E.-M. Suolinna, M. Haaparanta, R. Paul, P. Harkanen, 0. Solin, and H. SipilB, Nucl. Med. B i d , 13 (1986) 577-581. (521) Y. Yonekura, R. S. Benua,A. B. Brill, P. Som, S. D. J. Yeh, N. E. Kemeny, J. S. Fowler, R. R. MacGregor, R. Stamm, D. R. Christman, and A. P. Wolf, J. Nucl. Med.. 23 (1982) 1133-1137. (522) P. Paul and S. Parviainen, Res. Exp. Med., 186 (1986) 249-258.

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203

the state of tumors in mice523and humans.5242-Deoxy-2-[18F]fluoro-~-glucose and gallium-67 citrate, another imaging agent for tumors, were compared for detection of lymphoma.525 The finding that 18F-2DFGis toxic for mammalian cells when it is applied 5 hours after synthesis, but relatively non-toxic after 2 hours, was reported,526and the expected cause for that was discussed. Utilization of 18F-2DFMinstead of 18F-2DFGfor the PET method was examined.527A similar or indistinguishable level of uptake of both compounds in brain (human and rats) and heart (rats) was shown, as well as of renal excretion (rats). Also, almost the same distribution of both compounds in rat and rabbit tumors. was Biological studies on compounds similar to, but other than, 18F-2DFG and 18F-2DFM,have also been reported. Autoradiography in rats injected with I8F-2DFGal s h o ~ e d ~significant ~ . ~ ~uptake ~ * ~and ~ retention ~ of the compound only in the liver, as compared with the other tissues. It was suggested530 that 18F-2DFGalis trapped in the liver initially in the form ofthe I-phosphate, and then as the UDP d e r i ~ a t i v e ~(after ' ~ . ~1 ~ h). Organ distribution and toxicity of 18F-2DFGalwas r e p ~ r t e d . ~ ' ~ . ' ~ ~ d. Biological Studies of Other Deoxy-'8F-Fluoro Carbohydrates Not Described in c. -2-Deoxy-2-['8F]fluoro-~-glucopyranosyl ['8F]fluoridegave a tissue distribution pattern399(in mice) similar to that for 18F-2DFG,and was metabolized to 18F-2DFG and [ 18F]fluoride.2-Deoxy-2-[18F]fluoro-~-altrose and 2-deoxy-2-[18F]fluoro-~-glucose were rapidly cleared from all organs in rats.4032-Deoxy-2-['8F]fl~~r~-~-fu~~~e was prepared532through (523) Y. Abe, T. Matsuzawa, T. Fujiwara, H. Fukuda, M. Itoh, K. Yarnada, K. Yarnaguchi, T. Sato, and T. Ido, Eur. J. Nucl. Med., 12 (1986) 325-328. (524) H. Minn, R. Paul, and A. Ahonen, J. Nitcl. Med., 29 (1988) 1521- 1525. (525) R. Paul, J. Nucl. Med., 28 (1987) 288-292. (526) A. 1. Kassis, S . J. Adelstein, A. P. Wolf, J. G. Fowler and C.-Y. Shiue, J. Nucl. Med., 24 (1983) 1055- 1059. (527) G. D. Robinson, Jr., M. E. Phelps, and S.-C. Huang, J. Nucl. Med., 20 (1979) 672. (528) H. Fukuda, T. Matsuzawa, Y. Abe, S . Endo, K. Yamada, K. Kubota, J. Hatazawa, T. Sato. M. Ito, T. Takahashi, R. Iwata, and T. Ido, Eur. J. Nucl. Med., 7 (1982) 294-297. (529) H. Fukuda, K. Yamaguchi, T. Matsuzawa, M. Ito, Y. Abe, T. Fujiwara, T. Yamaguchi, H. Miyazawa, H. Kawai, H. Matsui, K. Ishiwata, and T. Ido, Kakuigaku. 24 (1987) 87 I -874. (530) K. Ishiwata, Y. Imahori, T. Ido, M. Tada, andT. Matsuzawa, Kukuigaku, 22 (1985) 1 142 (abstract). (531) H. Fukuda, K. Yamaguchi. T. Matsuzawa, Y. Abe, K. Yarnada, S. Yoshi0ka.T. Sato, M. Tada, Y. Ogata, T. Takahashi, and T. Ido, Kukuiguku, 24 (1987) 165- 169. (532) Y. Imahori, T. Ido, K. Ishiwata, T. Takahashi, Y. Yanai, Y. Miura, and R. Iwata,J. Nucl. Mrd.. 27 (1986) 983 (abstract).

204

TSUTOMU TSUCHIYA

treatment of 3,4-di-O-acetyl-~-fucal(473; see Section II,5) with gaseous Ac018F. In a biodistribution (in rats), this compound showed very low radioactivity in brain, possibly because of the blood- brain barrier. However, in the glioma cells of the brain, high accumulation was observed, suggesting rapid turnover of L-fucosyl glycoprotein synthesis in the tumor cells. Biologcal s t u d i e P on 18F-3DFG,involving the kinetic for PET, have also been reported. From a tissue-fraction e x t r a c t i o n - ~ t u d f ~of~ . ~ ~ ~ l8F-3DFG in monkeys, it was suggested that the compound crosses the blood - brain bamer by a facilitated diffusion mechanism in a manner analogous to that of D-glucose. Rapid and high accumulation and slow release of 18F-3DFGin brain and myocardium (mice and dogs, after i.v. injection) were r e p ~ r t e d ,but ~ ~other ~ . ~workers ~ ~ ~ b s e r v e dno ~~ such ~ . high ~ ~ accumu~ lation in the organs (in rats, mice, and dogs); I8F-2DFGshowed much higher accumulation in various organs (especially in the brain, heart, and tumors) but 18F-3DFGwas suggested to be useful for comparative measurement of D-glucose transport in tissues.536 e. Synthesis and Biological Application of Labeled Deoxfluoro Carbohydrates with Nuclides Other than l*F.-2-Deoxy-2-fluoro-~-[1-14C]glucose was prepared3" from 3,4,6-tri-u-acetyl-~-[1-14C]glucalby treatment with F2 or CF,OF. 3-Deoxy-3-fluoro-~-[1-'4C]mannose,346.537 4-deoxy-4fluoro-D-[ 1-14C]glucose,4-deoxy-4-fluoro-~-[1-14C]mannose,and 4-deoxy4-flUOrO-D-[ l-13C]mannosewere also ~ r e p a r e d . '4-Deoxy-4-fluoro-~-[6~ 3H]glucose was prepared538from 4-deoxy-4-fluoro-c~-~-glucopyranoside~~~ through oxidation - reduction (NaB3H4)reactions at C-6. The high purity of the compound was confirmed by reoxidation to give a product of only slight radioactivity. 14C-Labeled 1'-deoxy- 1 '-fluorosucrose was prepared539according to the cold synthesis (see Section 1142). (533) J. R. Halama, S. J. Gatley, T. R. DeCrado, D. R. Bernstein, C. K. Ng, and J. E. Holden, Am. J . Physiol., 247 (1984) H754-H759; references for biological studies on 3-deoxy-3fluoro-o-glucose are cited therein. (534) K. Vyska, H. M. Mehdorn, H.-J. Machulla, and E. J. Knust, Neurol. Rex. 7 (1985) 63-67. (535) E. J. Knust and H.-J. Machulla, Radiochem. Radioanal. Lett., 59 (1983) 7- 14. (536) K. J. Kearfott, D. R. Elmaleh, M. Goodman, J. A. Correia, N. M. Alpert, R. H. Ackerman, G. L. Brownell, and W. H. Strauss, Int. J. Nucl. Med. Bid.. 1 I (1984) 15-22. (537) T. J. Crier and J. R. Rasmussen, Biochem. J., 209 (1983) 677-685; related references on the metabolism of deoxyfluoro sugars are cited therein. (538) J. Samuel and N. F. Taylor, Carhohydr. Res., 133 (1984) 168-172. (539) P. J. Card and W. D. Hitz, J. Am. Chem. Soc., 106 (1984) 5348-5350.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

205

2. Biologically Active Saccharides In this Section, fluorine-containing mono-, di-, and oligo-saccharides are described. These compounds have been prepared with various intentions, but especially to clarify the binding mechanism between target enzymes and the parent substrates. Sometimes, useful inhibitors for the enzymes have been discovered. An advantage expected to be obtained by replacement of a hydroxyl group of saccharides by a fluorine atom may be the removal of the active hydrogen atom of the OH group with the least disturbance of the surroundings of the parent saccharides in terms of atomic size (0and F) and electronegativity.In this Section, studies on the metabolism of deoxyfluoro sugars intimately correlated to imaging agents are not described (for that, see Section II1,I). Several 1 -phosphates of deoxyfluoro sugars were prepared, and their acidcatalyzed hydrolysis was studied. 2-Deoxy-2-fluoro- (580), 3-deoxy-3fluoro- (582), 4-deoxy-4-fluoro- (583), and 6-deoxy-6-fluoro-a-~-glucopyranosyl phosphates (584) were prepared251by treatment of the corresponding per-0-acetylated P-D-glucopyranoseswith phosphoric acid [the p anomer (581) of 580 was prepared by a different method]. The first and second ionization constants (pKa, and pKa,) of these compounds were determined potentiometrically, as well as by the 19F-n.m.r.chemical shifts at a series of pH values, and then the rate constants of hydrolysis for neutral (B) and monoanion (C) were decided. The first-order rate-constants (k) for 580- 584 and a-D-glucopyranosyl phosphate (in MHC104, 25 ") were 0.068, 0.175, 0.480, 0.270, 1.12, and 4.10 (all as k x 105/s),respectively. The rate

- 4('

0

_f

F

A

+ 0- P- 0 1 I

0- P- OH

:),I

oti

H

t

II 0- P- OH I

0-

-'9 t-

0 II

0- P - 0I

0-

C

Ib

constants were found to be dependent on temperature [for 580: 1.17 (45") and 120 (82")] and hydrogen-ion concentration [for 580 at 80": 0.068 (pH 6.2), 0.199 (4.10), 0.726 (2.10), and 6.250 (l.OO)]. These results were ex~ l a i n e dbased ~ ~ ' on a (consensus) mechanism involving the three species:

TSUTOMU TSUCHIYA

206

conjugated acid (A; present principally at pH < l), neutral (B; pH 2 - 5), and monoanion (C; pH > 5 ) , which, respectively, are cleaved by C - 0 (principally C - 0, accompanied by 0 - P), C - 0 [through the oxocarbonium ion (D; route a); D has a 4H3conformation, in which the C-5 -0-5 -C-1 -C-2 atoms are coplanar], and 0 - P bond fissions (route b). In these hydrolyses, the position of the fluorine atom (especially at C-2) exerts a large influence on the transition state (0)The . study may be useful for discriminating between enzymes: enzymes engaged in route a (such as glycogen phosphorylase) cannot catalyze the hydrolysis of 580, because of destabilization of D by the presence of F-2, and the enzymes engaged in route b (such as phosphoglucomutase) can catalyze the hydrolysis. Absence of the latter kind of enzyme from brain makes the positron emission tomography (PET) method successful (see Section II1,l). a-1Phosphates of 2,6-dideoxy-2,6-difluoro-, 3,6-dideoxy-3,6-difluoro-, and 4,6-dideoxy-4,6-difluoro-~-glucopyranose,3,6-dideoxy-6-fluoro-~-ribohexopyranose, and 4,6-dideoxy-6-fluoro-~-xyfu-hexopyranose have also been prepared269from the corresponding precursors (see Table 111in Section II,3) by the same procedure, and their acid-catalyzed hydrolysis rates (with those for 580,582,583, and 584) were measured, and, in consequence, it was concluded that there is an electronic additive effect (caused by deoxygenation and deoxyfluorination) for the hydrolysis. The interaction (the transition-state structure) between rabbit muscle glycogen phosphorylase b and substrates ( 1-phosphates) was Michaelis- Menten kinetic parameters (K,,,and VmU)for the enzymic phosphorolysis of580,582,583, and 584 (in the presence ofAMPand glycogen),as well as the l9F-n.m.r.spectra ofthe enzyme - substrate mixtures, were measured, and an oxocarbonium ion-like transition state and existence of hydrogen b ~ n d i n g ' (involving ~' F- HO), which lowers the energy, were proposed. The incorporation and metabolism of 2-deoxy-2-fluoro-~-[~H]glucose and in yeast and chick-embryo cells has been 2-Deoxy-2-fluoro-~-glucose,2-deoxy-2-fluoro-~-mannose,and their GDP and UDP derivatives were found to interfere with protein (involving that of

mannose nose

(540) I. P. Street, K. Rupitz, and S. G. Withers, Biochemistry, 28 ( I 989) 158 1 - 1587. (541) I. P. Street, C. R. Amstrong, and S. G. Withers, Biochernistty, 25 (1986) 6021 -6027. (542) M. F. G. Schmidt, P. Biely, Z. Kritk9, and R. T. Schwarz, Eur. J. Biochem., 87 (1978) 55-68.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

207

enveloped viruses, such as influenza virus) g l y c o s y l a t i ~ nby ~ ~inhibiting ~-~~~ the incorporation of D-glucose or D-mannose into lipid-linked oligosaccharides. Similar effects of 4-deoxy-4-fluoro-~-mannoseand its GDP derivative were reported.550Metabolism of 2-deoxy-2-fluoro-~-glucosein rat brain was studiedls6 (see also, Section 11,2), without killing the animals, using I9F-n.m.r. spectroscopy (after i.v. injection, four 19F-resonancepeaks were observed). 2-Deoxy-2-fluoro-~-galactose (2DFGal) is metabolized551in mice to 2-deoxy-2-fluoro-~-glucose(2DFG) through UDP-2DFGa1419.530 (see Section III,1 ,c) and UDP-2DFG (this interconversion is catalyzed by UDP-D-galactose epimerase), the transformation being proved by the shiftvalues of the 19F-n.m.r. spectra of the excised organs of mice (see also, Section 11,2). 2-Deoxy-2-fluoro-~-fucose(see Section II,5) competes with L-fucose in glycoprotein b i o s y n t h e s i ~ . ~ ~ ~ Glucosidase inhibitors will be useful in therapy for diabetes. 2,4-Dinitrophenyl 2-deoxy-2-fluoro-~-~-glucopyranoside(585), a mechanism-based inhibitor553having a reactive glycosyl bond, inhibitP4 the Alcaligenes faec a h P-D-glucosidase by forming a 2-deoxy-2-fluoro-~-glucosyl -enzyme intermediate, which is reactivated by addition of 585 itself or other D-glucosides. The inactivation of several glycosidases by 2-deoxy-2-fluoro-a- and -P-D-glycosyl fluorides (of gluco, manno, and galacto types) was studiedls4 (see Section II, I). To assist in clarifying the three-dimensional structure of the glycogen phosphorylase- D-glucose complex, the crystal structure of 2-deoxy-2-fluoro-~-~-mannopyranosyl fluoride (see 157 in Section II,5) was determined.146 (543) M. F. G. Schmidt, R. T. Schwarz, and H. Ludwig, J. Virol.. 18 (1976) 819-823. (544) R. T. Schwarz, M. F. G. Schmidt, and R. Datema, Biochem. Soc. Trans., 7 (1979) 322-326. (545) R. Datema and R. T. Schwarz, Biochern. J.. 184 (1979) 113- 123. (546) R.Datema,R.T.Schwarz,andA. W.Jankowski, Eur.J. Biochem.. 109(1980)331-341;

related references are cited therein. (547) R. Datema. R. T. Schwarz, and J. Winkler, Eur. J. Biochem., 110 (1980) 355-361. (548) R. Datema and R. T. Schwarz, Biosci. Rep., 4 (1984) 213-221. (549) W. McDowell, R. Datema, P. A. Romero, and R. T. Schwarz, Biochemistry,24 (1985) 8 145-8 152. (550) W. McDowell, T. J. Crier, J. R. Rasmussen, and R. T. Schwarz, Biochem. J.,248 (1987) 523 - 53 1: related references are cited therein. (551) Y. Kanazawa, S. Kuribayashi, M. Kojima, and T. Haradahira, Chem. Pharm. Bull., 36 (1988) 4213-4216. (552) D. J. Winterbourne, C. G. Butchard, and P. W. Kent, Biochem. Biophys. Res. Commun.. 87 (1979) 989-992. (553) S. G. Withers, 1. P. Street, and M. D. Percival, Ref. 24., pp. 59-77. (554) S. G. Withers, 1. P. Street, P. Bird, and D. H. Dolphin, J. Am. Chem. Soc., 109 (1987) 7530- 7531.

208

TSUTOMU TSUCHIYA

In order to obtain P-D-glucosidase-resistant, bioactive substances, several D-glucosides containing the 2-deoxy-2-fluoro-~-~-glucosyl group were prepared.555Treatment of crystalline 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-a-~glucopyranosyl bromide (586) with phenol in quinoline in the presence of silver oxide gave the phenyl P-D-glucoside (40%). ZemplCn deacetylation gave phenyl 2-deoxy-2-fluoro-~-~-ghcopyranoside~~~ (587). Stability towards acid-catalyzed hydrolysis (0.75 M aq. H,SO,, 80') of 587 and

6)-- &>

AcO CH 2

I10CH2

0 OPh

Phenol

I10

AcO

F

f;

5n7

5x6

phenyl P-D-glucopyranoside was compared555:the latter was hydrolyzed about 15 times faster than 587. Condensation of 586 with estrone (quinoline- Ag,O; 45%) or 1,2 :3,4-di-O-isopropylidene-a-~-galactopyranose [Hg(CN),- 1 : 1 benzene-MeCN; 68%] gave the corresponding P-Dglycosides. As part of a study on cell-surface glycans, 2,3-bis(tetradecyloxy)propyl 2-deoxy-2-fluoro-a- and -P-D-mannopyranosides were preparedIs0 by condensation of 3,4,6-tn-0-benzyl-2-deoxy-2-fluoro-c~-~-mannopyranosyl chloride with 2,3-bis(tetradecyloxy)propanol. 3-Deoxy-3-fluoro-~-glucose'~~ (see Section 11,2), a weak substrate373for yeast hexokinase, is phosphorylated e n z y m i ~ a l l yto~give ~ ~ ~the~ ~ 6-phos~ phate 588, which is transformed into 2-deoxy-2-fluoro-~-arabinose5-phosphate (589) by lead tetraacetate oxidation.

OH

HO

(555) J. Pacak. Z.Kollnerova, and M. tern$, Collect. Czech. Chem. Comrnun.,44 (1979) 933-941. (556) D.G. Drueckhammer and C.-H. Wong, J . Org. Chem., 50 (1985) 5912-5913.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

209

3-Deoxy-3-fluoro-~-glucoseis non-toxic for rats, but shows weak toxicityS5’ (LD50 4.8 mg/g) for two locust species (when injected) having greater water retention than rats. A search was made for the toxic mechanism. The 3-deoxy-3-fluoro-~-glucoseis metabolized, in the locusts, to 3-deoxy-3fluoro-D-glucitol by aldose reductase, and the latter to 3-deoxy-3-fluoro-~fructose by D-glucitol dehydrogenase. Another metabolic fate of 3-deoxy3-fluoro-~-glucose(inhibition of glycolysis and fluoride ion release) using the 3-3H analog was reported.558Incubation559of the fat body or flightmuscle extracts with the 3-3H analog gave rise to formation of fluorinated glycogen and trehalose, and accumulation of ~-[~H]fructose. 3-Deoxy-3fluoro-D-glucose was actively transported5@’ into vesicles prepared from D-glucose-grown cells of Pseudomonas putida retaining D-glucose oxidase and D-gluconate dehydrogenase. The active-transport mechanism of (Y-Davubino-2-hexulopyranosonic acid (“2-keto-~-gluconate”)into the same vesicles was also studied56’; the transport is competitively inhibited by “3-deoxy-3-fluoro-2-keto-”or “4-deoxy-4-fluoro-2-keto-~-gluconate”, which were respectively prepared enzymically from 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-~-glucose.Uptake of 3-deoxy-3-fluoro-~-glucose(using the 3-3H analog) by synaptosomes in rat-brain cortex was reported.562 4 - D e o x y - 4 - f l u o r o - ~ - g ~ u c o s eunlike , ~ ~ ~ ~ 3-deoxy-3-fluoro-~-glucose, ~~~ was neither transported into563nor oxidized by D-glucose-grown cells of Ps. putidu, but extensive release of fluoride ion occurred. 4-Deoxy-4-fluoro-~[ 1 -14C]mannose and 3-deoxy-3-fluoro-~-[ I -14C]mannose (see Section III,1 ,e) were slightly, and much more slightly, transported537into yeast cells, respectively, than was 2-deoxy-~-[1-14C]glucose,but they were metabolized to the corresponding GDP-deoxyfluoro-D-[ 1-‘4C]mannoses, deoxyfluoroD-[ 1 - 1 4 C ] m a n n ~1,6-diphosphates, ~e and deoxyfluoro-D-[l-’4C]mannose6phosphates. Methyl 4-deoxy-4-fluoro-~-~-galactopyranoside (590) is a substrate for D-galactose oxidase, but neither 590 nor its a anomer is a substrate2” for /?-D- (from E. coh) or a-D-galactosidase (from Asp. filmigutus), suggesting that the axial OH-4 is important for binding to these D-galactosidases. Compound 590, however, functions as an inhibitor for /?-D-galactosidase.For immunoglobulin A 5539 (Fab’),590 gave2I0ligand-in-

(557) A. Romaschin. N. F. Taylor, D. A. Smith, and D. Lopes, Can. J. Biochem., 55 (1977) 369-375. (558) A. Romaschin and N. F. Taylor, Can. J. Biochem., 59 (1981) 262-268. (559) M. Agbanyo and N. F. Taylor, Biosci. Rep., 6 (1986) 309-316. (560) A. Al-Jobore, G. Moses, and N. F. Taylor, Can. J. Biochem.. 58 (1980) 1397- 1404. (561) F. Agbanyo and N. F. Taylor, Biochem. J., 228 (1985) 257-262. (562) D. M. Halton, N. F. Taylor, and D. P. Lopes, J. Neurosci. Res.. 5 (1980) 241 -252. (563) T. D’Amore and N. F. Taylor, FEBS Len., 143 (1982) 247-251.

210

TSUTOMU TSUCHIYA

duced changes in the tryptophanyl fluorescence, but methyl 2-deoxy-2fluoro-P-D-glucosidedid not, suggestingthat OH-2 is involved as a hydrogen donor in hydrogen bonding to the protein. Ally1 2-acetamido-2,4-dideoxy4-fluoro-a-~-galactopyranoside(see Table 111 in Section II,3) has been utilized in receptor-binding studies270in rat hepatocytes. As 2-amino-2-deoxy-~-mannose is tumorstatic and 2-acetamido-2deoxy-D-mannose 6-phosphate is an obligatory intermediate in the biosynthetic pathway to sialic acid, displacement of the essential OH-6 with a fluorine atom should be interesting from the biological viewpoint. 2Acetamido- 1,3,4-tri-0-acetyl-2,6-dideoxy-6-fluoro-~-mannopyranose (see Table 111 in Section I1,3)and its 0-and N,O-deacetyl derivatives were prepared271;the first compound showed weak anticancer activity. 5-N-Acetyl-9-deoxy-9-fluoroneuraminic acid (591) was prepared5w.565 by treatment of a protected 6-hydroxyl precursor with N,N-diethylaminosulfur trifluoride (DAST) or through condensation of 2-acetamido-2,6-dideoxy-6fluoro-~-mannopyranose~~' with potassium di(terf-butyl) oxaloacetate. Compound 591 is a substrate for cytidine monophosphate (CMP)-sialicacid synthetase, giving rise to CMP-5-N-acetyl-9-deoxy-9-fluoroneuraminic acid, which is cytotoxic against tumor cells. 5-N-Acetyl-3-fluoroneuraminic acids 592 - 594 were prepared566through fluorine (or acetyl hypofluorite) addition (in AcOH) to methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-2,3,5-tndeoxy-~-g~yc~r~~~-galact~non-2-enopyrano~te. Compound 592 was found to be a potent neuraminidase inhibitor.

As insects and certain other organisms utilize a,a-trehalose (a-~-glucopyranosyl a-D-glucopyranoside) as their storage carbohydrate, and rely on trehalase to release D-glucose, deoxyfluoro derivatives of trehalose may be (564) M. Sharmaand W. Korytnyk, J. Curbohydr. Chern., 1 (1982-83) 31 1-315. (565) M. Sharma, C. R. Petrie, IIIrd, and W. Korytnyk, Curbohydr. Res., 175 (1988) 25-34. (566) T. Nakajima, H. Hori, H. Ohrui, H. Meguro, and T. Ido, Agric. Biol. Chern.. 52 (1988) 1209- I2 IS.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

21 1

expected to show some inhibitory activity towards these organisms. 6Deoxy-6-fluoro-a,a-trehalose (595) was p r e ~ a r e d from ~ ~ ~2,3,2',3',4',6'J~~ hexa-0-benzyltrehalose 6 - t o ~ y l a t eor ~ ~2,3,2',3'-tetra-O-benzy1-4',6'-0~ benzylidenetrehalose 4,6-dirne~ylate~~~ (protection of the hydroxyl groups by acetyl groups is unsuitable) by treatment with tetrabutylammonium fluoride (Bu,NF) in MeCN, followed by deprotection. In the metabolism567 catalyzed by cockchafer trehalase, compound 595 showed a K,,, value (0.645 m M )almost equal to that of trehalose (0.6 17 mM), but a smaller V,, value (0.075: that of trehalose being taken as 1). Compound 595 also showed a competitive, reversible inhibition568for the trehalase taken from the flight muscle of the greenbottle fly. Similarly, 6-deo~y-6-fluoro-~~~ (596),4-deoxy4 - f l ~ o r o (597), - ~ ~ ~ 4,6-dideoxy-4,6-difl~oro-~~~ (598), 4-acetamido-4,6-dideoxy-6-fluor0-~~~ (599),and 4-amino-4,6-dideoxy-6-fluoro-a-~-galactopyranosyl a-~-glucopyranosides~~~ (600) were prepared by treatment of the corresponding sulfonate precursors with Bu,NF. Compounds 597 and 599

Ho I

OH 595 596 597 SYH 599 600

R'= tl, R'=OtI,

RLF,

RZ=OH, R7=F RL= H, R 3 = F R'= H, R3=0t1 R~=H

R'=R'=F, R'=NHAC, R ~ = H . R L F R'=NHZ. RZ=H, R 7 = F

(567) J. Defaye, H. Dnguez, B. Hennssat, J. Gelas, and E. Bar-Guilloux, Curbohydr. Res., 63 (1978) 41-49. (568) A. F. Hadfield, L. Hough, and A. C. Richardson, Curbohydr. Rex, 63 (1978) 51 -60. (569) A. F. Hadfield, L. Hough, and A. C. Richardson, Curbohydr. Rex, 7 1 (1979) 95 - 102. (570) A. F. Hadfield, L. Hough, and A. C. Richardson, Curbohydr. Res., 8 0 (1980) 123- 130.

TSUTOMU TSUCHIYA

212

had only weak biological activity. Although it is generally a ~ ~ e p t e d ~ ~ ~ , ~ ~ that, in 6-deoxy-6-fluoro-gluco- and -galacto-pyranosides, antiperiplanar (JF-6,H-s = 22 - 29 Hz) and gauche (- 15 Hz) arrangements between F-6 and H-5 preponderate, respectively, the large J value of 600 (30 Hz; compare 599: - 15 Hz) indicates that 600 adopts the former arrangement570because of attractive dipolar interaction between F-6 and C-NH3+-4. To examine the role ofthe hydroxyl groups in a mucin type ofoligosaccharide which could act as an acceptor for glycosyltransferases, partially fluorinated low-molecular-weight oligosaccharides(601-603) were synthesized573 through DAST treatment of the corresponding protected 6-hydroxyl precursors. The 4-deoxy-4-fluoro analog 604 was prepared249by glycosylation R’ CHlOH

R’

AcNH

ot

HO ORn

OH

NHAc

of benzyl 2-acetamido-6-O-benzyl-2,4-dideoxy-4-fluoro-a-~-glucopyranowith 2,3,4,6-tetra-O-acetyl-a-~-galactopyranosyl bromide, or by fluorination at C-4 of a disaccharide precursor with DAST. To study the transition state (oxocarbonium ion) ofthe action of lysozyme [p-(1 -+4)-~-galactosidase]on the substrate, it was intended to prepares74 ~-~-(~-deoxy-~-fluoro-~-D-ga~actopyranosy~)-~-~ucose (2’-deoxy-2’-fluorolactose). However, condensation of the 2-deoxy-2-fluoro-a-~-glycosylbromide 605 or 606 with 2,3-di-O-acetyl-1,6-anhydro-P-~-glucopyranose (607) (Ag2C03- AgS0,CF3- ether) gave the corresponding a-glycosides (608 and 609) exclusively. The a-glycoside formation (the structure of 609 was confirmed by X-ray crystallography) was attributed574to the use of a non-polar solvent (ether), the presence of the 3- and 6-OAc groups, and, especially,the 2-fluorine atom, each of which may facilitate reactive P-bromide formation by the action of bromide ion liberated during the reaction, thus facilitating the formation of the a-glycosides. Compound 609 was converted into 2‘deoxy-2’-fluoromaltose (610). (571) (572) (573) (574)

0. S. Chizhov and N. K. Kochetkov, Adv. Carbohydr. Chem., 21 (1966) 39-93. L. Evelyn and L. D. Hall, Chem. Ind. (London). (1968) 183- 184. R. L. Thomas, S. A. Abbas, and K. L. Matta, Curbohydr.Rex, 165 (1987) c 1 4 - c l 6 . J. G. Shelling, D. Dolphin, P. Win, R. E. Cobbledick. and F. W. B. Einstein, Carbohydr.

Rex, 132(1984) 241-259.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

-

CHZOAc

OAC )R{ K?

Br

+

OAc

HO

OAc

OAc

F

607

R'= OAc. R2= H R'=H, R2= OAc

H2C-

R'R t O L o ( Y

OAc

I ;

605 606

CH@Ac

213

OAc

608 R'=OAc, Rz= H 609 R ' = H , R~=OA~ CH20H

HOCH, I

I

OH

F

As part of a study on cell-surface glycans, 4-0-(2-deoxy-2-fluoro-a-~mannopyranosy1)-D-glucopyranose (612) was prepared. CondensationIso of 6-0-acetyl- 1,2,3-tri-0-benzyl-P-~-glucopyranose with the protected 2deoxy-2-fluoro-a-~-mannopyranosy~ chloride 611 gave the corresponding 4 - 0 4 ~ and ~ - P-D-mannopyranosy1)-D-glucopyranoside derivatives (alp = HWH2 I

HOCH2 I

ORn 61 I

OH 612

8.72,79%). Deprotection of the a-~-rnannoanomer gave 612. The 6-deoxy6-fluoro and 6'-deoxy-6'-fluoro analogs of methyl 4-O-a-~-glucopyranosylP-D-glucopyranoside (methyl P-maltoside) were prepared" through DAST treatment of suitably protected 6- and 6'-free methyl P-maltosides, respectively, and they were examined as the substrate for amyloglucosidase; although the former binds tightly to the enzyme, the latter is neithera substrate nor an inhibitor for the enzyme, indicating the importance of OH-6 in the nonreducing end. As part of a study on the epitope of globoside, methyl 6-deoxy-6-fluoro-4~-a-~-galactopyranosy~-~-D-galactopyranoside~~~ (613), and methyl 4-0(4-deoxy-4-fluoro-(614) and -6-deoxy-6-fluoro-a-~-galactopyranosyl)-~-~-

TSUTOMU TSUCHIYA

214

galactopyranoside (615) were prepared.95 Condensation of methyl 2,3-di-0-benzoyl-6-deoxy-6-fluoro-~-~-galactopyranos~de (see Table I11 in Section II,3) with 2,3,4,6-tetra-0-benzyl-a-~-galactopyranosyl chloride (AgSO,CF, - toluene), followed by deprotection, gave 613. The minimumenergy conformations of613 -615 were calculated272by computer (SUGAR R’ CH,R’

+&!H 0

613 614 615

R’ F

R’

OH F OH

01-1

OH

R’ OH 011

F

and Allinger’s M M 2 - 8 2 programs): the torsional angles [+H (H- l’-C-l’-O4-C-4)andqH(H-4-C-4-0-4-C-1’)] oftheglycosidelinkageand theangle for the C-5 -C-6 bond (0-5 -C-5 -C-6 -F-6) in the most-stable conformation of 613 were - 44”,- 13 ’, and 178 respectively. Various strains of oral streptococci produce D-glucosyltransferaseswhich utilize sucrose as a D - ~ ~ U C O Sdonor Y~ in the production of soluble and insoluble D-glucans. Consequently, it may be expected that some deoxyfluoro derivatives of sucrose function as competitive inhibitors for the dextransucrases of tooth bacteria, thus preventing decay, or at least may be used as active-site probes for the enzymes. Another aim ofthese researches is to find non-metabolizable sweeteners. 3-Deoxy-3-fluorosucroses7s(616), prepared through DAST treatment of 3’,4’,6’-tris(0-tert-butyldimethylsilyl)-I ’,2 :4,6-di-O-isopropylidenedo-sucrose, is digested576,in the presence of a glucosyltransferase (GTF-I; an insoluble-D-glucan-producingenzyme) and maltose (as an acceptor), to give 6- and 3-0-(3-deoxy-3-fluoro-a-~-ghcopyranosyl)maltose, indicating that 616 is able to donate a D-glucosyl residue to maltose. However, 616 is O ,

(575) T. P. Binder and J . F. Robyt, Curhohydr. Res., 147 (1986) 149- 154. (576) T. P. Binder and J. F. Robyt, Curbohydr. Res., 154 (1986) 229-238.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

215

not a substrate of the enzyme, and it inhibits the activity only weakly. Through a study involving other data, the importance of the 3-hydroxyl group of sucrose in binding with the enzyme was indicated. 4-Deoxy-4-fluoro-g~lacto-sucrose~~~ (617; 4-deoxy-4-fluoro-a-~-galactopyranosyl D-fmctofuranoside), 2,3,1’,3’,4’,6’-hexa-O-benzy1-4,6-dideoxy4,6-difl~orosucrose~~~ (618), 2,3, I ’,3’,4’,6’-hexa-O-benzyl-6-deoxy-6fluoro-g~lucto-sucrose~~~ (619), and 2,3,1’,3’,4’,6’-hexa-O-benzy1-4,6dideoxy-4,6-difluoro-g~fucto-sucrose~~~ (620) were prepared by displacement of the sulfonyloxy groups of the corresponding precursors with fluoride (Bu,NF in MeCN). 6 FH,R~

1

6’

616 617 618

619 620 621 622

0

R1

R2

Rj

R4

K’

Rh

II

F OH OBn OBn OBn OH OtI

H F H OH F [I

OH H F H H OH OH

OH OH F F F F F

OH OH OBn OBn OBn OH F

H Bn

Bn Bn H

H

t1

(577) L. Hough, A. K. M. S. Kabir, and A. C. Richardson, Curbohydr. Res., 131 (1984) 335-340. (578) L. Hough, A. K. M. S. Kabir, and A. C. Richardson, Curbohydr. Res., 125 (1984) 247-252.

216

TSUTOMU TSUCHIYA

6-Deoxy-6-fluorosucrose (621) was prepared through the displacement reaction578 of 2,3,1',3',4',6'-hepta-0-benzyl-4,6-di-0-mesylsucrose with Bu,NF (in MeCN) or from5792,3,4,1',3',4',6'-hepta-0-benzoylsucrose by treatment with DAST. 6-Deoxy-6-fluorosucrose (621) is an effective competitive inhibitor579for Leuconostoc mesenteroides B-5 12F dextransucrase and Streptococcus mutans 67 15 D-glucosyltransferases(GTF-S and -I), and is bound to the sucrose-binding-sitesapproximately ten times more strongly than sucrose, suggesting that OH-6 of sucrose is not required for binding to the enzymes. Compound 621 was also shownSBo to be a strong competitive inhibitor ( K j -0.5; sucrose: K, 200) for the amylosucrase of Neisseria perflava, an enzyme postulated to cause dental caries, but not a substrate for the enzyme. A comparative study using 6-deoxysucrose ( K j 6) and 621 suggested that F-6 is a hydrogen acceptor that enhances binding to the enzyme. 6,6'-Dideoxy-6,6'-difluorosucrose (622) was preparedS8' through treatment of 2,3,4,3',4'-penta-0-benzoylsucrose with DAST (OH-1' is inert). This compound seems582not to inhibit D-glucan formation caused by GTF-S, but it affects GTF-I. 1 '-Deoxy- 1 '-fluorosucrose (625) was prepared539by utilizing a sucrose synthetase. The starting 1-deoxy-1-fluoroD-fructose (624) was prepared from the readily available 2,3:4,5-di-0isopropylidene-1-0-triflyl-D-fructose(623) by treatment with tris(dimethy1-

-

-

':5

-

CMc2

O\/?

F,CSO,OH,

0

FCH,

+

it02H$'CH

HO

Me,?C-O

623 (579) (580) (581) (582)

sucrosc synthcllisc

624

625

S. H. Eklund and J. F. Robyt, Curbohydr. Res.. 177 (1988) 253-258. B. Y . Tao, P. J . Redly, and J. F. Robyt, Curhohydr. Rcs., 181 ( 1988) I63 - 174. J. N. Zikopoulos, S. H. Eklund, and J . F. Robyt, Curbohydr. Rex, 104(1982)245-251. T . P. Binder and J. F. Robyt, Curbohydr. Res.. 140 (1985) 9-20.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

217

amino)sulfonium diflu~rotrimethylsilicate*~~ (TASF see also Section II,2) in 80%yield. Incubation of 624 plus UDP-D-glucose with a sucrose synthetase preparation (from barley seeds)gave 625 in good yield. Compound 625 is not hydrolyzed by sucrose invertase, but is transported into plant tissues by a sucrose-carrier protein; this finding might be useful for studying sucrose transport in plants. A ‘‘C-labeled analog of 625 was also prepared.5396’Deoxy-6’-fluorosucrose (631) and 4’-deoxy-4’-fluorosucrose(630) were prepared583through similar chemoenzymic methods. Incubation of 6-deoxy-6fluoro-D-glucoseand UDP-D-glucose with D-glucose isomerase (to give 629) and sucrose synthetase preparations afforded 631 (739/0) in one vessel. In the case of 630, as the starting 4-deoxy-4-fluoro-~-glucoseis not a substrate for the isomerase, it was initially converted into the 6-phosphate by another enzyme and then transformed further: incubation of 4-deoxy-4-fluoro-~glucose with (a)ATP and hexokinase (to give the 6-phosphate), (b)phosphoglucose isomerase (to give 4-deoxy-4-fluoro-~-fructose 6-phosphate 627), (c) ~-fructose-6-phosphatekinase to afford the 1,6-diphosphate 628, in one vessel, and then ( d ) alkaline phosphatase, to give 4-deoxy-4-fluoro-~fructose 626. Incubation of 626 and UDP-glucose with sucrose synthetase HOCH,

I

I

R’ R‘

R‘

R‘

626

H

F

011

627

H

F

opq2-

628

PO’‘

F

OPQL-

629

H

OH

F

R‘

RZ

630

F

OH

631

OH F

(583) P. J. Card, W. D. Hitz, and K. G. Ripp, J. Am. Chem. Soc., 108 (1986) 158- 161.

TSUTOMU TSUCHIYA

218

then gave 630. Compound 625 and 631 have more hydrophobic character than sucrose, and this may be one of the reasons they bind strongly to sucrose-carrier protein. As part of a study on the substrate for glycosidases and glycosyltransferases, benzyl 2-acetamido-6-0-(2-acetamido-2,4-dideoxy-4-fluoro-~-~glucopyranosyl)-2-deoxy-a-~-galactopyranoside (634) was prepared.250 Condensation of 2-methyl-(3,6-di-O-acetyl- 1,2,4-trideoxy-4-fluoro-a-~glucopyrano)-[2,1-d]-2-oxazoline (632) with benzyi 2-acetamido-2-deoxya-D-galactoside 633 (TsOH - 1,2-dichloroethane) gave the 6-0-glycosyl product (6 IYo), which was converted into 634. HOCH2

-\

NHAc

Me

632

633

634

To investigate the mode of binding of saccharides (asligands) to monoclonal anti-( 1 +6)-P-~-gaiactan-~pecificimmunoglobulin A (IgA; an antibody), KovaE, Glaudemans and coworkers prepared specifically fluorinated mono-, di-, tri-, and tetra-saccharides having the ( 1 -6)-P-~-galactopyranosyl structure, and measured the binding constants against the IgA. Condensation584.585 of methyl 2,3,4-tri-O-acetyl-P-~-galactopyranoside or methyl 0-(2,3,4-tri-0-acetyl-/.-~-galactopyranosyl)-( 1 6)-2,3,4-tri-0-acetyl-P-~galactopyranoside with 2,4,6-tri-0-acetyl-3-deoxy-3-fluoro-a-~-galactopyranosyl bromide [Hg(CN),- HgBr, - Drierite - benzene] gave in good yields the ( 1 -.6)-P-~-galactosides (after removal of the accompanying a-Danomers by chromatography), which, on deacetylation, gave the desired D-galactosyl di- and tri-saccharides (635 and 636) having a 3-deoxy-3-

-

635 n = 1 636 n = 2

(584) P. KovaE and C. P. J. Glaudemans, Curbohydr. Rex, 123 (1983) c29-c30. (585) P. KovaT, H. J. C. Yeh, and C. P. J. Glaudemans, Curbohydr. Res., 140 (1985) 277-288.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

219

fluoro-p-D-galactopyranosyl group. Similar condensationss6of methyl 2,3di-O-benzyl-4-deoxy-4-fluoro-~-~-galactopyranoside with 2,3,4,6-tetra-Oacetyl-a-D-galactopyranosyl bromide gave the corresponding p-( 1 +6)-~-galactoside (93%; a anomer, 2.6%), which, by deprotection, gave the D-galactosyl disaccharide 637 having a 4-deoxy-4-fluoro-~-galactoside residue. Condensation587 of methyl 2,3-di-O-acetyl-3-deoxy-3-

637

fluoro-p-D-galactopyranoside with the foregoing bromide or 2,3,4-tri-Oacetyl-6-O-(bromoacetyl)-a-~-galactopyranosyl bromide under AgSO,CF, catalysis (in the presence of 2,4,6-trimethylpyridine in 1 : 1 toluenenitromethane) gave anomerically pure p-( 1-6)-disaccharides [when Hg(CN),-HgBr, was used as the catalysts, an anomeric mixture was obtained, making separation difficult]. Deprotection then gave the D-galactosyl disaccharide 638 having a 3-deoxy-3-fluoro-~-galactoside residue. In a similar fashion, involving de(bromoacety1)ation (with thiourea) of the condensation intermediates and subsequent glycosylation of the free hydroxyl group liberated, higher saccharides 639 and 640 were prepared. Similar fluorine-containing D-galactosyl saccharides, 641 (Ref. 588) and 642 (Ref. 589) were prepared similarly.Utilizing these deoxyfluoro D-galactosides, the

638 n = l 639 n = 2 640 n = 3

(586) P. KovaE and C. P. J. Glaudemans, J. Curbohydr. Chem.. 3 (1984) 349-358. (587) P. KovaE and C. P. J. Glaudemans, Curbohydr. Res., 140 (1985) 289-298. (588) P. KovaE, C. P. J. Glaudemans, W. Guo, and T. C. Wong, Curbohydr.Res., 140 (1985) 299-31 1. (589) P. Kovaf and C. P. J. Glaudemans, J. Curbohydr. Chem., 4 (1985) 613-626.

TSUTOMU TSUCHIYA

220

641 642

K’=OH R’=OH, R2= F K’=F,

binding modes of /I-(1 -6)-~-galactopyranan segments to IgA J539 (Fab’) were in~estigated.’~~-’~’ It is known that IgA 5539 can bind to four sequential D-galactosyl residues of a polysaccharide chain through the binding subsites of IgA named A , B, C, and D. The binding affinities of mono-, di-, tri-, and tetra$-( 1-*6)-~-galactopyranosides and their fluorinated analogs as just described to the antibody (IgA) were measured by the ligand (that is, the ga1actoside)-inducedfluorescence change of the tryptophan residues of the protein (IgA). It was clearly shown that replacement of OH-2 or OH-3 of the D-galactosides (but not of OH-4 or -6) by a fluorine atom causes cessation of binding to subsite A, the most sensitiveand important site ofthe four, by lack of hydrogen bonding (both OH-2 and -3 are required to bind to subsite A as hydrogen donor). Through these ingenious studies, it was con~ 1 ~ d e d ~ that ~ ~ (a) . ’the~ sequence , ~ ~ ~ of. the ~ ~four ~ subsites of the IgA is C-A-B-D,(b)the Cdirects to the light-chain of the protein (IgA), and (c) the relative binding affinities (as defined by the binding constants) of the four subsites to the individual galactosyl residues decrease in the order of A > B > C > D.Similar studies of the binding mode of a-(l+6)-linked D-glucopyranans to monoclonal antidextran IgA W3 129, by utilizing methyl a-D-ghcopyranoside (G),methyl 2-deoxy-2-fluoro- (2F), 3-deoxy3-flUOrO- (3F), 4-deoxy-4-fluoro (4F),and 6-deoxy-6-fluoro-a-~-glucopyranoside (6F)(effectivesyntheses of 2F,3F,and 4F from the corresponding existing deoxyfluoro sugars have been reporteds94),and a number of oligosaccharides (involving 644,646,650,651,654,655,and 657) were de~cribed.’~’Among the four ligand-binding subsites (A, B, C, and D)of the protein, A has the highest affinity ( A > > >B >C >D)and binds only to the D - ~ ~ U C O Sgroup Y ~ at the nonreducing end through hydrogen bonding with the (590) C. P. J. Glaudemans, P. KovaE, and K. Rasmussen, Biochemistry, 23 (1984) 6732 -6736; related references are cited therein. (591) C. P. J. Glaudemans and P. KoviE, in Ref. 24, pp. 78- 108. (592) C. P. J. Glaudemans and P. KovBE, Mol. Imrnunol., 22 (1985) 651 -653. (593) C. P. J. Glaudemans, Mol. Irnrnunol., 24 (1987) 371 -377. (594) P. KovBE, H. J . C. Yeh, and C. P. J. Glaudemans, Carbohydr. Res., 169 (1987) 23-34. (595) C. P. J. Glaudemans, P. KoviE, and A. S. Rao, Carbohydr. Res.. 190 (1989) 267-277.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

22 1

6- and 4-hydroxyl groups (hydrogen donors) of the residue [binding constants (Ka) for G, 2F, 3F, 4F, 6F, 644,646,650,651,654,655,and 657 were 1.8, 1.8,8.7,0,0, 16,0.05,67,0.26, 180,0.46,and 190(x 103),respectively]. More effective binding of 3F than that of natural G (see Ka) suggests the possibility that A has a binding portion donating a hydrogen atom to form a hydrogen bond with the substituent at C-3 ofthe residue (more effectivewith F-3 than with OH-3), or has a positive charge (to attract the F-3); enhancement of binding by increasing the acidity of OH-2 or -4 of the residue caused by the inductive effect of F-3 is an alternative explanation. To study the binding mode of the P-D-galactopyranosyl residue of the Lewis b human blood-group determinant to its monoclonal antibody or to a lectin, methyl 2-acetamido-2-deoxy-3-0-(6-deoxy-6-fluoro-2-0-a-~-fucopyranosyl-~-~-galactopyranosyl)-4-0-a-~-fucopyranosyl-~-~-glucopyranoside was synthesized.596 As a probe to use in investigating the binding mode of antidextran monoclonal antibody known to bind only to the nonreducing end of a-(1-6)linked D-glucopyranans, specifically fluorinated isomalto-oligosaccharides 646 and 651 were prepared.236Condensation of methyl 2,3,4-tri-O-benzyla-D-glucopyranoside with 2,3,4-tri-O-benzyl-6-deoxy-6-fluoro-~-~-glucopyranosyl fluoride79(58; see Section 11, 1) according to MukaiyamaS6gave a mixture of 645 (62%) and 647 (16%).Direct fluorination of the benzylated isomaltose derivative 643 with DAST also gave 645 (82%).Similar condensation of 643 with 61 gave a mixture of649 (68%) and 652 (16%). Debenzylation of 645,647,649, and 652 gave, respectively, 646,648,651, and 653. Similarly, methyl 0-(6-deoxy-6-fluoro-~-~-glucopyranosyl)-( 1 +2)-a-~glucopyranoside (methyl 6’-deoxy-6’-fluoro-a-sophoroside),methyl 046deoxy-6-fluoro-~-~-glucopyranosyl)-( 1 3)-a-D-glucopyranoside (methyl 6’-deoxy-6’-fluoro-a-laminarabioside), methyl 0-(6-deoxy-6-fluoro-a-~glucopyranosy1)-(1 2)-a-~-glucopyranoside (methyl 6’-deoxy-6’-fluoroa-kojibioside), and methyl 0-(6-deoxy-6-fluoro-a-~-glucopyranosyl)( I 3)-a-~-glucopyranoside(methyl 6’-deoxy-6’-fluoro-a-nigeroside) were prepared.597Compound 656 was also prepared.598 Mono-6-deoxy-6-fluorocyclomaltoheptaose(prepared from the mono-0tosyl precursor) was examined599for the substrate of porcine-pancreatic alpha amylase, which has five binding subsites for its substrate (one D-glucosyl residue per subsite); much less formation of 6-deoxy-6-fluoro-40-a-D-glucosyl-D-glucose than of 4-0-(6-deoxy-6-fluoro-a-~-glucosyl)-aD-glucose indicated that the enzymic hydrolysis of 6-deoxy-6-fluoro-~-glucosyl-(1 +4)-~-glucose(the reaction occurs at subsite 3) is restricted. +

+

-+

(596) (597) (598) (599)

R. U. Lemieux, R. Cromer, and U. Spohr, Can. J. Chrm., 66 (1988) 3083-3098. P. KovaC and C. P. J. Glaudemans, J. Curbohydr. Chem., 7 (1988) 317-335. P. KovaE and L. Lerner, Curbohydr. Rrs., 184 (1988) 87- 112. P. J. Braun. D. French, and J. F. Robyt, Curbohydr.Res., 143 (1985) 107- 116.

222

TSUTOMU TSUCHIYA

OR

OCH2

I

I

OR' d

n

643 644 645 646 641 648 649 650 651 652 653 654 655

a-D

0 0 0

656

F D

657

a-D

a-D a-D

a-D PD P D a-D

a-D

a-D F D &D

a-D a-D

0 0 0 1 1 I 1 I 2 2

2 3

R' Bn H Bn FI Bn €1

Bn H H Bn ti

H H Bn H

R?

OH H F F F F F H F F F H

I: F H

Several simple deoxyfluoro sugars and glycosyl fluorides were utilizeds4' as probes for hydrogen bonding in the glycogen phosphorylase - D-glucose complex. Deoxyfluorostarchesm were prepared from corn starch by treatment of the tosylated (mostly at 0-6 of the ~-glucosylresidues)starch precursors with KF in ethylene glycol ( 190", 15 min) or by treatment of starch with hydrofluoric acid (Me,SO, -30",4 h room temp., 10 min).

-

(600) H. C. Snvastava and V. K. Snvastava, Sraerke, 31 (1979) 265-267.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

223

3. Antimicrobial and Anticancer Fluoro Sugars Fluorination (by replacement of H, OH, or C=O) of aminoglycosides and anticancer antibiotics has been undertaken in order to obtain derivatives of enhanced activity and decreased toxicity. Because fluorine is the most electron-withdrawing atom ofall, replacement of any atom, or ofa common group, with fluorine gives rise to an electronic change in the molecule and induces, as the result, a stereochemical change as well (through bonds or through space). An amino or hydroxyl group located near to the fluorine atom may respectively change its basicity (decreased) and acidity (increased). As the C-F bond is polarized, the fluorine atom may respectively attract or repel electron-deficient or electron-rich atoms or groups situated nearby. Also, replacement of a hydroxyl group by a fluorine atom may cause conformational changes in the molecule through the loss of a hydrogenbond donor. These properties may give the fluoro or deoxyfluoro compounds changes in activity in comparison to the parent compounds. Treatment60'@'2 of sporaricin B (N-deglycylsporaricin A) derivatives 658 -660 with diethylaminosulfur trifluoride (DAST), followed by attachment of the glycyl group, gave, respectively, 3-epi-3-fluoro- (662), 3-fluoro(663), and 3,3-difluoro-3-de(methoxy)sporaricin A (664). Displacement of the Me0-3 group of sporaricin A (661) with a fluorine atom would be expected to decrease601the basicity of the NH,-1 group by virtue of the strongly electron-withdrawing property of the 3-fluorine atom, and, in consequence, to lower the toxicity of sporaricin A, because, generally, the basi-

I1 0

NHZ 658 659 (i6o

R ' = H , R2=OH R'= OH, R2= H R ' R ~ =o Z= CQCHZPh

(i6i 662

663 664

665

R'= H, R ~ =OMC, R3= OH R1= F, R2= H, R3=OH R'= H, R2= F, R3=OH R'=R~=F, R ~ OH = R'= H, R2= OMc, R3= F

(60 I ) T. Tsuchiya, T. Toni, and S. Umezawa, and H. Umezawa, J.Antibiot., 35 ( 1982) 1245 1247. (602) T. Tsuchiya, T. Toni, Y. Suzuki, and S. Umezawa, Curbohydr. Res., 116 (1983) 277287.

224

TSUTOMU TSUCHIYA

city and number of amino groups in amino sugar antibiotics are believed to be the origin of their toxicity (663 was prepared, and showed really enhanced antibiotic activity and lower toxicity60l in comparison to 661). Treatmentm3 of tetrakis(Nbenzyloxycarbony1)sporaricinB with DAST gave the 5-deoxy5-fluorosporaricin B derivative (31%) with retention of configuration, along with tris(N-benzyloxycarbonyl)-4-N,5-0-carbonylsporaricin B (39%); deprotection of the former with subsequent glycine coupling gave 5-deoxy-5fluorosporaricin A 665, which had no activity. Simple analogs of an aminoglycoside antibiotic, 2,6-dideoxy-4-0- (671) and -5-0-(2,3-dideoxy-2-fluoro-cu-~-ribo-hexopyranosyl)streptam~ne (672) were preparedm by coupling of tri-O-acetyl-2-fluoro-~-glucal~~ (666) with cyclitol derivatives 668 or 667 (through 669 and 670) as shown. Preparation of several fluorinated analogs of kanamycin A (673) were reported. 6”-Deoxy-6”-fluorokanamycinA (674) was preparedm through

i--

OTs 668 ( = R 2 H )

671

670

672

(603) T. Toni, T. Tsuchiya, and S. Umezawa, Carbohydr. Res., I I6 ( 1983) 289 - 294. (604) G. Vass, A. Rolland, J. Clkophax, D. Mercier, B. Quiclet,and S. D. Gero, J . Antibiot.. 32 (1979) 610-612. (605) J. Adamson, A. B. Foster, and J. H. Westwood, Carbohydr. Rcs., 18 (1971) 345-347. (606) R. Albert, K. Dax, and A. E. Stiitz, T&-uhLdronLeft., 24 (1983) 1763- 1766.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

225

the displacement reaction (76%) of 2’,3‘,4’,2”,4”-penta-O-acetyl-l,3,6’,3”tetrakis(N-terz-butoxycarbonyl)-6”-O-triflylkanamycin A with tetrabutylammonium fluoride (Bu,NF) in acetonitrile. Similarly, 4”-deoxy-4”-epi4”-fluoroamikacin and 6’’-deoxy-6’’-fluoroamikacin (amikacinm7 is 1-N-[(S)-4-amino-2-(hydroxybutanoyl]kanamycinA) were prepared,m8but both compounds were less active than amikacin. Compound 674 and 6”deoxy-6”-fluoroamikacinwere also prepared,609in low yields, through direct treatment of per(Nbenzyloxycarbony1) derivatives of kanamycin A and amikacin with a limited use of DAST. 5-Deoxy-5-epi-5-fluorokanamycin A (675), 5,6”-dideoxy-5-epi-5,6”-difluorokanamycin A (676), and 5-deoxy5,4”-diepi-5-fluorokanamycinA (677) were respectively prepared606p610 from tetrakis(N- tert - butoxycarbonyl) - 2’,3’,4’,2”,4”,6” - hexa - 0-acetyl -, -2’,3’,4’,2”,4”-penta-O-acetyl-, and -2’,3’,4’,2”,6”-penta-O-acetyl-kanamyh’

OH

3’

OH

673 674 675 676 677 678 679 680 681 682

R‘ OH OH H H H OH OH OH OH OH

R2 H H F F F H H H H H

R3 H H H H OH F H F F OH

R4 OH OH OH OH H H F H H H

R5 OH F OH F OH OH OH F H F

(607) H. Kawaguchi, T. Naito, S. Nakagawa, and K. Fujisawa, J.Anfibiof.,25 (1972) 695 -708. (608) R. Albert, K. Dax, A. E. Stiitz, and J. Hildebrandt, J. Anfibiof.,38 (1985) 275-278. (609) Y. Takagi, T. Tsuchiya, and S. Umezawa, Nippon Kuguku Kuishi, (1985) 2001-2009. (610) S. Afr. Pat. 78/6385 (1978); Chern. Absfr., 90 (1978) 104,301y.

226

TSUTOMU TSUCHIYA

cin A by the DAST treatment, the 4”-epimerization of the last compound being performed by the P-side attack of the 3”-N-(tert-butoxycarbonyl) group on the C-4’’ sulfoxo group formed as the reaction intermediate. 4”Deoxy-4“-epi-4”-fluorokanamycin A (678, 72%) was prepared213by treatment of 2’,3’~4’,2”,6”-penta-~-acetyl-tetrakis(N-~er~-butoxycarbon~l)-4”0-triflylkanamycin A with Bu,NF (MeCN, 20”, 4 h). 4”-Deoxy4”-fluorokanamycin A (679) was prepared6I1 by the double-inversion method at C-4” (for the first inversion step, sodium nitrite was used6’, on the 4”-O-triflyl derivative). Similarly, 4”,6”-dideoxy-4”-epi-4”,6”-difluorokanamycin A (680), 4”,6”-deoxy-4”-epi-4”-fluorokanamycin A (681), and 6”deoxy-4”-epi-6”-fluorokanamycin A (682) were respectively prepared6I3 from tetrakis(N-tert-butoxycarbonyl)-2’,3’,4’,2’’-tetra-O-acety1-4”,6’’-di-~triflyl-, -6”-O-(p-bromophenylsuIfonyl)-4”-O-triflyl-, and -6”-O-(p-bromophenylsulfonyl)-4”-epi-kanamycinA by treatment with Bu,NF in the second reaction, fluorination at C-4’’ was performed selectively in preference to the 6”-O-sulfonyl group. These compounds (674-682) ~ h o ~ e d ~ antibacterial ~ ~ , ~ activity ~ , ~comparable , ~ ~ ~ to that , ~of~kanamy~ cin A (673). Kanamycins are inactivated by enzymes of resistant bacteria capable of phosphorylating or adenylylating the 3’-, 4‘-, or 2”-hydroxyl groups, among which the 3’-hydroxyl group is the most frequently m ~ d i f i e d . ~ ’Removal ~.~’~ of those hydroxyl groups that can be modified is the major solution for recovery of activity, as exemplified by 3’-deoxykanamycin A and 3’,4’-dideoxykanamycin B (dibekacin). Replacement of the hydroxyl group to be modified by resistant bacteria with a fluorine atom has been considered to give a similar effect, in terms of biological activity, to deoxygenation. Thus, 3’-deoxy-3’-fluorokanamycin A (684) was prepared616through coupling of 6-azido-2,4-di-O-benzyl-3,6-dideoxy-3-fluoro-~-~-~ucopyranosyl bromide (683)with a pseudodisaccharide component of kanamycin. The same compound 684 was also prepared281.617 from the protected 3’-O-sulfonylkanamycin A derivative 689, after conversion into the 2‘,3‘-anhydro derivative 690 (with NaOMe in MeOH), by treatment with KHF, in ethylene glycol [to give the 3’-deoxy-3’-fluoro (691) and 2’-deoxy-2’-fluoro derivatives 692; see also, 421 in Section II,4]. 3’-Deoxy-3’-fluorokanamycinB (61 I ) R. Albert, K. Dax, and A. E. Stutz, Curbohydr. Res., 132 (1984) 162-167. (612) R. Albert, K. Dax, R. W. Link, and A. E. Stutz, Carbohydr.Res., 118 (1983) cS-c6. (613) R. Albert, K. Dax, and A. E. Stutz, J. Curbohydr. Chem.. 3 (1984) 267-278. (614) S. Umezawa, Adv. Curbohydr. Chem. Biochem.. 30 (1974) I I I - 182. (615) H. Umezawa, Adv. Carbohydr. Chem. Biochem., 30 (1974) 183-225. (616) T. Tsuchiya, Y. Takahashi, Y. Kobayashi, S. Umezawa, and H. Umezawa, J. Antihiol., 38 (1985) 1287- 1290. (6 17) T. Tsuchiya, E. Umemura, and S. Umezawa, Carbohydr. Rex. in press.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

227

(685; Ref. 6 16) was prepared from the per-N-tosyl-3’-O-sulfonylkanamycin B derivative 686, after conversion into the 2’,3’-N-tosylepimine 687 (with NaOMe in MeOH), by treatment with KHF, in N,N-dimethylformamide (DMF; 150”, 2 h, giving the 3’-deoxy-3’-fluoroderivative 688 in 60%yield),

ORn

R

683

6M 685

R=OH R=NH2

ChrnHNCH2

ChmHNL;H2

Ts 688

681

1

Cbm= CYMe

685 OR2

686 689

R1=N14Ts. R2=Ac R’=O11, R2= I I

t

690

691 ( 7 1 % )

692 (16%)

TSUTOMU TSUCHIYA

228

the fundamental study of this reaction being described281previously (see 456 in Section 11,4). Compounds 684 and 685 showed enhanced activity616 in comparison to that of 3’-deoxykanamycin A and dibekacin, respectively, against most of the resistant bacteria. 2’,3’-Dideoxy-2’-fluorokanamycinA (Ref. 170), a kanamycin derivative in which the OH-2’ or NH2-2‘ of kanamycin A and B, respectively, is replaced by a fluorine atom, was prepared by condensation of 6-azido-4-0benzoyl-2,3,6-tndeoxy-2-fluoro-cw-~-ribo-hexopyranosyl bromide, derived from 179 (see Section 11,2), with the pseudodisaccharide component of kanamycin. The compound showed mediocre antibacterial activity; however, on attachment of an (S)-4-amino-2-hydroxybutanoyl residue, as in amika~in,~ to’ the NH2-I group, the derivative showed considerably enhanced activity.618 5-Deoxy-5-epi-5-fluorosisomicin (695) was prepared6I9by mutasynthesis using 2,5-dideoxy-5-epi-5-fluorostreptamine (693). Compound 695 and 5deoxy-5-fluorosisomicin (696) were respectively synthesized from protected derivatives 697 and its 5-epimer by treatment with DAST. These compounds (695 and 696) showed antibacterial activity similar to that of sisomi-

F

OH

693

694

695 696

(sisomicin) R’=OH, RZ=H R’=H, R’= F R’=F, R2=H

(618) Jpn. Kokai, 63/208598 A2 (1988); C h m . Absfr., 110 (1988) 95,713m. (619) P. J. L. Daniels, D. F. Rane, S. W. McCombie, R. T. Testa, J. J. Wright, and T. L. Nagabhushan, in K. L. Rinehart, Jr., and T. Suami (Eds.), Aminocyclifol Anfihiofics, ACSSymp. Ser. 125, Am. Chem. SOC.,Washington,D.C., 1980, pp. 371 -392.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

229

NHZ

HO OH

697 Z= CQCHzPh

698 699

R=H R=OH

cin (694). 5-Deoxy-5-fluoro-dibekacin(698) and -tobramycin (699) were prepared620from the protected 5-epi- hydroxyl precursors, and found to show enhanced activity compared to that of dibekacin (3’,4’-dideoxykanamycin B) and tobramycin (3’-deoxykanamycinB), respectively. The basicity of the NH,-3 group (as indicated by the pKa values) of 698, 699 and the related compounds were shown to decrease621in comparison to those of parent compounds having the OH-5 group (from a study on the I3C-shift values). Lividomycin B (700) is inactivated by a group of resistant bacteria (producing 3’-phosphotransferase I) by conversion into the 5”-phosphate. 5”Deoxylividomycin B (701), however, showed significantly decreased activity. To examine the role of OH-5”, that is, would the OH play a hydrogen donor or acceptor role in its antibacterial action, 5”-deoxy-5”-fluorolividomycin B (704) was prepared622from the protected derivative 702 through treatment with DAST [to give the S’-deoxy-5”-fluoro derivative 703 (28%) and the 2‘-N, 5”-O-carbonyl compound 705 (61Yo)I. The synthetic 704 showed only slightly improved activity compared with that of 701, suggesting that OH-5 of 700 may act as a hydrogen donor. (620) T. Tsuchiya, T. Shitara, Y. Kobayashi, S. Umezawa, and H. Umezawa, Annu. Meel. Cliern. Soc. Jpn., Tokyo, Apr. 1, 1988 (Abstr. p. I 126); details in Carbohydr. Res., in press. (621) T. Tsuchiya. T. Miyake, Y. Takahashi, and S. Umezawa, to be published. (622) T. Toni, T. Tsuchiya, S. Umezawa, and H. Umezawa, Bull. Chem. SOC.Jpn., 56 (1983) 1522- 1526.

230

TSUTOMU TSUCHIYA NHZ

700 701 702

703 704

R’=R2= H, R3= OH R’=R2=R3= H R ’ = E COfZH2Ph, R2= Ac, R3= OH R’= Z, R2= Ac, R3= F R’=R2= H. R3=F

705

5 - 0 - (4 - Deoxy - 4 - fluoro -p- D - mycaminosy1)tylonolide(709) was prepared623in low yield from the suitably protected 4’-0-(benzylsulfonyl) derivative 706 by treatment with KHFzin ethylene glycol [ 120°, 1 h, to give the 4’-deoxy-4’-fluoro derivative 707 (20%) with retention of configuration, along with several by-products involving solvent-incorporated derivat i v e ( ~ ) ~In~ this ~ ] . reaction, when DMF (120”, 3 h) was used as the solvent, the undesired 4’-(dimethylamino)-3’-fluoroderivative 708 was the major compound formed (56%). As 708 was partially converted into 707 in the same treatment, a reaction mechanism similar to that already described (see 457 in Section II,4) involving an aziridinium ion intermediate (711) was anticipated. Compound 709 showed only slightly improved a ~ t i v i t y com6~~ pared with that of the parent compound, 5-O-~-~-mycaminosyltylonolide (710). (623) S. Kageyama, T. Onoda, T. Tsuchiya, S. Umezawa, and H. Umezawa, Curbohydr. Res.. 169 (1987) 241 -246. (624) L. Evelyn and L. D. Hall., Curbohydr. Rev., 47 (1976) 285-297.

23 1

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES 0.

yc

H~CIIO

,.*..

0

I."..,

()

1.1,.

Jy-

I l0Cl I,

Ac(K'llz

7M

NMc2

71I

0

706 707

+

()

709 710

H'= NMq, K2=K'= II. K4=OSwILJ'h H'= NMch K2=K'= II, K4= I; H'=R4= 11, H2= I:, K'= NMq

R=I: R=011

Anticancer agents fluorinated at the sugar portion are described next. In this field, only daunorubicin and doxorubicin analogs have been reported. In order to ~ t r e n g t h e n ' ~ the ~ ,glycosidic ~~~ bond of these anticancer antibiotics (the weak glycosidic bond of these antibiotics may be one of the causes of their toxicity) introduction of a fluorine atom at C-2' was undertaken. Starting from a 3-amino-2,3-dideoxy-2-fluoro-~-altroside 437,438, or 712 (Refs. 285 -288 and 290; see also Section 11,4), the 3-amino-2,3,6-trideoxy-2fluoro-L-galactoderivatives714 were prepared through the established route shown. The 4-en0 analog 713 was also reported.286Condensation240.626 of the XCI 12

d

Ph C H

0

R'O

NHR~

I or H X = Br o r I

R?= H I

437 R'= Me. R'= BI

438 R'= Bn, R?= B/ 712 R'= Mc. R2= CF'CO

R")

NHR*

oR4 713 R'= Mc, R2= B/

R4= OMc, ORn, or OH R5= BI. CF3C0,o r H R"= BI, CF,CO, or H

NHR' 714

( 6 2 5 ) M. cerny, V. Pnkrylovi, and J. Pacik, Collect. Czech. Chem. Commun., 37 (1972) 2978-2984. (626) H. H. Baer and L. Siemsen, Can. J. Chem., 66 (1988) 187- 190.

TSUTOMU TSUCHIYA

232

1-bromide493 (see Section II,5) or the 4-0-acetyl analog with daunomycinone [AgSO,CF, - CH,CI, (Ref. 240) or Hg(CN), - H@r2 - toluene626]gave the daunorubicin analog 715 (549/0),and then 716 and 2’-P-fluorodaunorubicin 717. Compound 717 was slightly less active626(L 12 10 murine leukemia) and had a higher active-dose range240(P 388 leukemia cells) than daunorubicin, but was fairly less toxic.

0

715 716 717 71%

R 1 = NHCOCF?. R2= B/, o r Ac R’= NHCOCF?, R2= H R’= N H , R2= ti R ’ = OH, k2= H

A 2-a-fluoro compound, methyl 3-acetamido-4-0-benzoyl-2,3,6-tndeoxy-2-fluoro-~-~-mannopyranoside (719) was preparedlWby utilizing 225 (see Section 142)through a route similar to that already described involving the 5-en0 derivative. The similar L-talo analog 720 was prepared627from 419 (see Section II,4) by a sequence of reactions involving displacement of the 3-0-triflyl derivative with an azido group (to give a derivative of the L-talo configuration), and then 720 was coupled to daunomycinone according to a reported method,62Eto give, after deprotection, 7-0-(3-amino-2,3,6-trideoxy-2-fluoro-a-~-talopyranosyl)daunomycinone 726 (2’-a-fluorodaunorubicin). Through the usual hydroxylation at C- 14 of the daunorubicin derivative, 2’-a-fluorodoxorubicin 727 was prepared. The 3’-hydroxyl compound, 7-0-(2,6-dideoxy-2-fluoro-~u-~-talopyranosyl)daunomycinone (722), its 14-hydroxyl analog 723, and the 14-0-substituted derivatives 724 (627) Y. Takagi, H A . Park, T. Tsuchiya, S. Umezawa, T. Takeuchi, K. Komuro, and C. Nosaka, J. Anrihior.. 42 ( 1 989) I3 15 - 1317. (628) Y. Kimura, M. Suzuki, T. Matsumoto, R. Abe, and S . Terashima, BUN.Chem. Soc. Jpn., 59 (1986)423-431.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

233

and 725 were prepared279.280.629 by similar condensation of 721, prepared from 420 (see Section 11,4), with daunomycinone. Compounds 722, 723, and 725 (especially 723 and 725) showed a broad active-dose range (6.25 -

c"p

OMe AcHN

AcO

FICCONI1

I; 71Y

F

720

AcO AcO

F 72 I

COCH?R I OH Me0

0

OH

0

722 72.3 724 725 726 727

K 1 = I I , RZ=O1-l R ' = O H , R>=OH K'= OSO{Na. R'= OH R ' = OCO(CH,),CO,H (n=2-6), R'= OH

R L H ,KLNH, R1=O1l, R Z = N H 2

100 pg, per mouse per day, intraperitoneally) against several tumor-bearing mice in vivo with low toxicity; however, the 2P-fluoro analog 718 showed629 almost no antitumor activity. These characteristics basically supported the suggestion by Horton and coworker^^^^-^^^ on the configuration at C-2' and

(629) T. Tsuchiya, Y . Takagi, S. Umezawa, T. Takeuchi, K. Komuro, C. Nosaka, H. Umezawa, S. Fukatsu, and T. Yoneta, J. Antihiof.,41 (1988) 988-991. (630) D. Horton, W. Priebe, and 0. Varela, Curbohydr. Rex, 144 (1985) 305-315. (63 I ) E.-F. Fuchs, D. Horton, and W. Weckerle, Curbohydr. Rex, 57 (1977) c36-c39. (632) D. Horton and W. R. Turner, Curbohydr. Res., 77 (1979) c 8 - c l l .

234

TSUTOMU TSUCHIYA

the substituent at C-3’ (NH, or OH) in terms of activity. The synthesis and activity of 4’-deoxy-4’-fluorodaunorubicinhave been reported.633 4. Nucleosides

Numerous 5-fluorouracil derivatives have been prepared as potential anticancer agents. In this section, however, only nucleosides containing fluoro sugars in their molecules are described. Nucleotides and polynucleotides containing fluoro sugars are excluded, owing to their vastly expanded areas, except for such compounds as are intimately correlated to the nucleosides described. Reports published before 1977 are omitted, except for those presumed to be important and fundamental. Fluoro- or fluorodeoxy-nucleosides synthesized are very often endowed with antiviral or anticancer activities, or both, and a number of analogs have been prepared. The order of description follows, if at all possible, from the compounds’ having ribo, xyfo, and urubino structures for the sugar portions. In this field, excellent rev i e w ~have ~ ~been ~ , published. ~ ~ ~ a. Synthesis and Physicochemical Properties of 2’-Fluoropyrimidine Nucleosides. -(a) Synthesis. -Fox and coworkers 2’deoxy-2’-fluorouridine (728), 2’-deoxy-2’,5-difluorouridine (729), and 2’a-fluorothymidine (732) from the corresponding 2,2’-anhydro- 1P-D-arabinofuranosylpynrnidines 733 - 735 with anhydrous liquid HF in 1,4-dioxane. 5-Bromo-2’-deoxy-2’-fluorouridine (730) and 2’-deoxy-2’fluoro-5-iodouridine (731) were also reported.639The mass ~ p e c t r u m ~ ~off j ~ ’ 728 and the I3C-n.m.r.spectrum642of the 3’,5’-di-O-acetyl derivative (with

(633) Ger. Pat. 3,428,945 Al (1985): Chem. Abstr., 102 (1985) 221,1481: (634) D. E. Bergstrom and D. J. Swartling: Fluorine Substituted Analogs of Nucleic Acid Components, in J. F. Liebman, A. Greenberg, and W. R. Dolbier, Jr., (Eds.), FluorineContaining Moleculm, VCH Publishers, New York, 1988, pp. 259-308. (635) P. Herdewijn. A. Van Aerschot, and L. Kerremans, Niicleos. Niicleor., 8 (1989) 65-96. (636) J. F. Codington, I. L. Doerr. D. Van Praag, A. Bendich, and J. J. Fox, J.Am. Ctiem. Soc., 83 (1961) 5030-5031. (637) J. F. Codington, I. L. Doerr, and J. J. Fox, J. Org. Chem., 29 (1964) 558-564. (638) J. F. Codington, I. L. Doerr, and J. J. Fox, J. Org. Chem., 29 (1964) 564-569. (639) K. A. Watanabe, T.-L. Su, R. S. Klein, C. K. Chu, A. Matsuda, M. W. Chun, C. Lopez, and J. J. Fox, J. Med. Chem., 26 ( 1983) I52 - 156. (640) M. Blandinand K. Jankowski, Bull. Acad. Pol. Sci.,Ser. Sci. Chim., 27 (1979) 563-570: Chern. Abstr.. 93 (1980) 186,7111: (641) M. Blandin and K. Jankowski. Eur. J. Mass Spectrom. Biochem.. Med. Environ. Res., I (1980) 129-133: Chon. Abstr., 95 (1981) 62,5701: (642) M. Blandin and K. Jankowski, Rev. Latinoam. Quim., 14 (1983) 45-47; Chem. Abstr.. 100 (1984) 68,641~.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

R

0

HO 728 729 730 731 732

F X=H X=F X= Br X=I X=Me

235

HO

733 734 735 736

R

X

0

H F Me H

0 0 NH

or without addition of a shift reagent) have been reported. The remarkable stability ofthese compounds (728,729, and 732) against acid and alkali (due to the presence of F-2’) was shown in comparison with other halogen congeners and uridine. 2’-Deoxy-2’-fluorocytidine (737) was prepareda3 from 728 through644 successive thiation (by P,S,, selective at C-4), S-methylation, and replacement of the S-Me group with an amino group (liquid NH,), or by4’ heating the hydrofluoride salt of 2,2’-anhydro-1-P-D-arabinofuranosylcytosine (736) in N,N-dimethylformamide (DMF). Compound 737 was later prepared646(40%) by treatment of 736 with KF-crown ether in DMF under strictly anhydrous conditions. In boiling water, compound 737 readily gave back 736 at a rate much higher than that of 728 to give 733, suggesting a difference in chemical reactivity between uridine and cytidine derivatives. The 2’-fluoro atom of 737 exerts643a slight base-weakening effect in comparison to 2’-deoxycytidine: the apparent pKa values (determined spectrophotometrically) are 3.9 and 4.3 for 737 and 2’-deoxycytidine, respectively. 2‘-Deoxy-2‘-fluoro-5-iodocytidine (738) was prepared639by direct iodination of an acetyl derivative of 737.

(643) 1. L. Doerr and J. J. Fox,J. Org. Chem., 32 (1967) 1462- 1471. (644) I. Wempen and J. J. Fox, Nucl. Acid Chem., I (1978) 299-302. (645) D. H. Shannahoff and R. A. Sanchez, J. Org Chem., 38 (1973) 593-598. (646) R. Mengel and W. Guschlbauer, Angew. Chem.,90 (1978) 557-558; Angew. Chem.,Int. Ed. Engl.. 17 (1978) 525.

TSUTOMU TSUCHIYA

236

0

NH, I

04

4

HOCH,

yJ F

"OCQ

737 738 739

X=H, X= 1, X=H.

R 1 = H , RZ=OH R'=H, R2=0t1 R'=OH, R*=H

740

1-(2-Deoxy-2-fluoro-~-~-xylofuranosy~)cytosine (739) was prepared647in good yield by condensation (in MeCN) of 3,5-di-O-benzoyl-2-deoxy-2fluoro-D-xylofuranosyl bromide with bis(trimethylsily1)cytosine. Because synthesis of 1-( 2-deoxy-2-fluoro-~-~-arabinofuranosyl)cytos~ne (744, FAC), an elementary arabino type of nucleoside having a growth-inhibitory effect against L 12 10 leukemia in mice,"' through direct introduction of a fluorine atom in the 2'-"up" (arabino) position was difficult, compound 744 was prepared648 by condensation of trimethylsilylated N4-acetylcytosinewith 3-0-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-~-arabinofuranosyl bromide (742), which had been prepared by periodate oxidation of 6-O-benzoyl-3-deoxy-3-fluoro-~-glucofuranose (741). Similar condensa-

74 I

712 743

K= Ac R=Bz

(647) J. A. Wright, D. P. Wilson, and J. J. Fox, J. Med. Chern., 13 (1970) 269-272. (648) U. Reichman. K. A. Watanabe, and J. J. Fox, Curbohvdr. Rex, 42 (1975) 233-240

DEVELOPMENTS O F FLUORINATED CARBOHYDRATES

237

tion649.650 of 742 with 5-halo-trimethylsilylated cytosinesor uracils (halogen: F, CI, Br, and I) gave, after deprotection, the corresponding 5-halo analogs [745,746,747,748 (FIAC), 754 (FFAU), 755,756, and 757 (FIAU)]; similarly, 753 (FAU) was also prepared. Direct fluorination (at C-5) of pyrimidine bases of existing nucleosides trifluoromethyl hypofluorite (CF,OF) or elemental F2 was also reported. A radiolabeled imaging agent, [2-I4C]-FAU,was prepared652by condensation of 742 and 2,4-bis(O-tnmethylsilyl)[2-'4C]uracil,and, after deacylation, the product was converted into [2-14C]-FFAUby treatment with fluorine in acetic acid. [1251]-FIAC and [1231]-,[lZ5I]-,[13'I]-FIAU (Ref. 653), and the 82Branalog of the last compound were also from 744 or 753. N.m.r. spectroscopy (in D,O) of 746-748 showed that these compounds are in an approximately equal mixture of N and S conformers,656respectively (see later description). Many 5-alkyl-, 5-alkenyl-, and 5-alkynyl-1-(2-deoxy-2-fluoro-P-~-arabinofuranosy1)pyrimidines(750 - 752 and 765 - 767) were prepared650.657-661 bY condensation of 742 (or the equivalent, such as 743) with C-5-substituted pyrimidine derivatives, or by attachmenP7 of a C-fragment to C-5 of the existing nucleosides through their 5-iOdO or 5-chloromercuri derivatives,660 or by further conversion of the attached C-5 substituent into another one.661

(649) K. A. Watanabe, U. Reichman, K. Hirota, C. Lopez,and J. J. Fox, J. h k d . Chem., 22 (1979) 2 1-24. (650) M. W. Chun, Arch. Phurm. Res., 6 (1983) 79-81. (65 I ) M. J. Robins, M. MacCoss, S. R. Naik, and G. Ramani, J. Am. Chem. Soc., 98 (1976) 738 1-7390. (652) H. K. Misra, L. 1. Wiebe, and E. E. Knaus, J. Label. Comp. Radiopharm., 24 (1987) 1107- I 116. (653) D. R. Tovell, H. P. Yacyshyn, H. K. Misra, E. E. Knaus, L. 1. Wiebe, J. Samuel, M. J. Gill, and D. L. J. Tyrrell, J. Med. Virol.,22 (1987) 183- 188. (654) H. K. Misra, E. E. Knaus, L. I. Wiebe, and D. L. Tyrrell, Appl. Radiat. Isot.. 37 (1986) 901 -905. (655) M. E. Perlman, P. S. Conti, B. Schmall, and K. A. Watanabe, Inf.J. Nucl. Med. Biol.. 1 1 (1984) 215-218. (656) R. L. Lipnick and J. D. Fissekis, Biochim. Biophys. Acta, 608 (1980) 96- 102. (657) M. E. Perlman, K. A. Watanabe, R. F. Schinazi, and J. J. Fox, J. Med. Chem.. 28 (1985) 74 1 - 748. (658) T.-L. Su, K. A. Watanabe, R. F. Schinazi, and J. J. Fox, J. Med. Chem.. 29 (1986) 15 I - 154. (659) K. A. Watanabe, T.-L. Su, U. Reichman, N. Greenberg, C. Lopez, and J. J. Fox, J. Med. Chetrl.,27 (1984) 91-94. (660) D. E. Bergstrom and J. L. Ruth, J. Carbohydr.Nucleos, Nucleot., 4 (1977) 257-269. (661) J. Matulic-Adamic, K. Takahashi, T.-C. Chou, H. Gadler, R. W. Price, A. R. V. Reddy, T. 1. Kalman, and K. A. Watanabe, J. Med. Chem., 31 (1988) 1642- 1647.

238

TSUTOMU TSUCHIYA

HO

744 745 746 747 748 749 750 751

752

X = H (FAC) X=F X=C1 X=Rr X = I (FIAC) X = M c (FMAC) X=Et (FEAC) X= CH=CH,, ( W H = C H R (R= Br, I), or (.EXH=CHCQR (R= EL,Na) X=C
HO

HO

753 754 755 756 757 758 759 760 761 762 763 764 765 766 767

X=H X=F

(FAU) (FFAU)

x= c1 X= Rr X=l (FIAU) X=Mc (FMAU) X= CHzOH X= CH2Rr X=CHO X=CH,F X=CHF, X=CFj X = E t (FEAU) X= CHzCHzF X= C H ~ C H ~ MCHMq, C, CH2CH=CH,, CHMeCH2Mc, CH+ZHMe2,CH=CH,, or (E)CH=CHR (R= CI, Br, I)

Among the simplest compounds of this series are I -(2-deoxy-2-fluoro-j?-~arabinof~ranosy1)thymine~~~ (758,FMAU) and 1 -(2-deoxy-2-fluoro-j?-~arabinofuranosy1)-5-methyl~ytosine~~~ (749,FMAC). Later FIAU (757)and FMAU (758)were prepared'95 in high yields (>95%; the ratio of j?/aanomen being 7/ 1 and 4/ 1, respectively) by condensation using 743 and 2,4bis(O-trimethylsilyl)-5-iodouracil(769),and 743 and 2,4-bis(O-trimethylsi1yl)thymine (770),respectively. Likewise, 753,757,758,and 765 (this was described later) were prepared662in high yields using pyrimidine derivatives (662) H. G. Howell, P. R. Brodfuehrer, S. P. Brundidge, D. A. Benigni, and C. Sapino, Jr., J. Org. Chem., 53 (1988) 8 5 - 8 8 .

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

239

768 -771 and 743. In these reactions, the a-1-bromideof 743 was very stable [no anomerization was observed in refluxing MeCN (during 18 h) as well as in non-polar solvents] and the condensation proceeded essentially under S N conditions ~ to give mainly the P-nucleosides LpIa:40-60 (in CCI,), 15- 30 (CHCI,), 10- 15 (CH,CI,), and 2-4 (MeCN)] without formation of intermediary oxocarbonium or 1,5-benzoxonium ions,662,663 which would give the a anomers in high proportion. OSiMeT

I

76n 769 770 771 772

X=H X=l X = M ~ X=Et X=CFj

HNSiMel I

773

The 5-mOnO- and 5-di-(fluoromethyl) analogs 762 and 763 of 758 (FMAU) were prepared66’from 3’,S’-bis(O-tert-butyldiphenylsilyl)-Fh4AU by a series of reactions involving photobromination [Me-S CH,Br (760)], hydrolysis (to give 759 after deprotection), and fluorination with diethylaminosulfur trifluoride (DAST) to give 762 after deprotection), or, after oxidation of the alcohol with MnO, (to give the 5-formyl nucleoside 761 after deprotection), with DAST (to give 763 after deprotection). The trifluoromethyl analog 764 was prepared66’by copper-catalyzed trifluoromethylation of 757 (FIAU), or by’94condensation using the 5-trifluoromethylpyrimidine derivative 772. I -(2-Deoxy-2,2-d~fluoro-~-~-erythro-pentofuranosyl)cytos~ne (775) was prepared344by condensation of trimethylsilylated cytosine (773) with the 2-deoxy-2,2-difluoropentose774 that had been prepared from 2-deoxy-2,2difluoro-D-erythro-pentofuranose (see Section 11,6). In a similar manner, the 2’-deoxy-2’,2’-difluoro analogs 776 and 777 were prepared. Different kinds of nucleosides (778) were prepared261.262 by condensation of compound 400 (see Section II,3) with trimethylsilylated uracils under Lewis acid catalysis, and removal of one fluorine atom at C-2 of the sugars. Uridine S’-(2-acetam~do-2,4-d~deoxy-4-fluoro-c~-~-galactopyranosyl di-

-

(663) C. P. Fei and T. H. Chan, Tetrahedron Len., 28 (1987) 849-852.

TSUTOMU TSUCHlYA

240

R

110

KO

;

I:

774 R= SiMc$u‘

775 776 777

R= NH,, X = I 1 R=NH2, X = M r R= 0 1 1 , X= H

0

AcO

I;

778 X= f I, F, of Me 779 R’= F, K2= H, R’=OH 780 R’= H , R2= OH, R’= F

phosphate) (779) and uridine 5’-(2-acetamido-2,6-dideoxy-6-fluoro-a-~glucopyranosyl diphosphate) (780) were

(b) Conformational analysis. -Knowledge as to the conformation of nucleosides is important in pursuing the relationship between structure and biological activity. Cushley and coworkers664studied the conformation of 2’-deoxy-2’-fluoroundine (728) in solution (D,O, and D 2 0- Me,SO-d,), and, based on the spin couplings (including JH-I,,H:2, 2.0, JH-It,F 18.6, and JH-3S.F 20.2 Hz in D20),concluded that the furanose nng adopts the envelope form 3E with C-3’ endo, which differs greatly from the conformation of (664) R.J. Cushley, J. F. Codington, and J . J. Fox, Can. J. Chewi., 46 (1968) I I3 I - 1140.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

24 1

2’-deoxyuridine [&-I‘,H-2’a - JH-Ir,H-2,% - 6.3 Hz (Ref. 665)]. Furanosyl conformations of a number of nucleosides were based on their JH,H coupling constants. From comparison of the shift-valuesof some typical nucleosides and their 3’-phosphates, exocyclic achiral hydrogen atoms of 5’-CHaHbOHwere di~criminated~’~ [Hbof the nucleosides is more shielded (0.09-0.12 ppm) than Ha, but only by 0.05-0.06 p.p.m. in the 3’-phosphates; 728: JHAt,H-ya 2.0, JH4#,He5%4.0 Hz (Ref. 664)], and the populations of the three rotamers around the C-4’-C-5’ bond were determined (gauchegauche, gauche-trans, and trans-gauche = 73 : 15 : 12 for 728). Guschlbauer and JankowskF studied the conformation of uridine, 2’-deoxy-, 2’-amino2’-deoxy-, 2’-azido-2’-deoxy-, and 2’-chloro-2’-deoxy-uridine, in addition to that of 728, in D20 and Me,SO-d6, and, based on ‘H- and I3C-n.m.r. analysis (precise data for the I3C and 2D spectra of 728 were reported late+”), the dihedral angles (4) relating to the D-ribosyl portion were estimated by utilizing the proposed equation JH,Hwobs) = ( 10cos24(R) 1.2~os4(~))[ 1 - 0.07 * (eR- &I, where eR is the electronegativity of the 2’-substituent calculated from the equation eR= 0.034.8, 0.84 (8, is the I3Cchemical shift of C-2’ of the compounds); the eRvalues thus determined (R=H:2.2, NH2:2.87, N,:3.16, OH:3.44,C1:2.95,andF:3.9) werein good agreement with those reported.664These results indicated that 728 resembles uridine more than it does 2’-deoxyuridine. To describe the conformational change of a furanose ring precisely, a concept of pseudorotation as designated by “phase angle” (P; variable between 0 and 360”)was intr~duced.~’~ According to this, a furanose ring is theoretically (pseudo)rotated through all possible twist (T)and envelope (E) forms starting from 3T2(P= through 3E(P= lSo), 3T4(P= 36”), E4, OT4, OE(P=90”), -, 2 T , ( P = 144”), 2 E ( P = 162”), 2T3(P= 180”), -, Eo(P= 270”), -, ending at 3 T 2 ( P =360” = Oo); the conformations between roughly P = 0 - 36’ and 144- 180”,which, respectively, may be expressed as type N(C-3 endo) and Sforms (C-2 endo),are usually in a region of

+

OO),

(665) W. Guschlbauer and K . Jankowski, Nucl. Acids Res., 8 (1980) 1421- 1433. (666) E. Westhof, H. Plach, 1. Cuno, and H.-D. Liidemann, Nucl. Acids Rex, 4 (1977) 939953. (667) H. Plach, E. Westhof, H.-D. Liidemann, and R. Mengel, Eur. J. Biochem.. 80 (1977) 295 - 304. (668) 1. Ekiel, M. Remin, E. Darzynkiewicz, and D. Shugar, Biochim. Eiuphys. Acfu, 562 (1979) 177-191. (669) G. Klimke, I. Cuno, H.-D. Liidemann, R. Mengel., and M. J . Robins, Z. NufurJursch., Teil C, 34 (1979) 1075- 1084. (670) M. Remin and D. Shugar, Biochem. Biuphys. Res. Cumrnun., 48 (1972) 636-642. (671) K. Jankowski and W. Guschlbauer, Specfrusc.Inf. J., 3 (1984) 473-478. (672) C. Altona and M. Sundaralingam,J. Am. Chem. Soc., 94 (1972) 8205-8212.

TSUTOMU TSUCHIYA

242

minimum energy, and there is an energy barrier between the two forms. It was concluded66sthat nucleosides are in equilibrium (on the n.m.r. timescale) between the N and S forms [in D,O, some 2’-substituted analogs of uridine are in the N form in the proportion (%) of 40 (2’-deoxyuridine), 25 (2’-NH,), 58 (2’-N3), 58 (2’-OH), 5 1 (2’-CI), and 87 (2’-F); thus, the 2’fluoro compound 728 has a remarkably high N form population]. Similar research673on the conformation of 728, its phosphates674[ 3’-phosphate, its methyl ester, and 2’-deoxy-2’-fluorouridylyl(3’- 5’)adenosine; the fraction of a stacked conformation of the last compound was 18% at 32”],compound 737 (Ref. 675),some other nucleosides, and the polymers ofthem, indicated, based on the n.m.r. spectra, the significant effect of 2’-fluorine (N-form population preponderates in most cases). A h initio molecular orbital calculations (using the Gaussian 80 computer program) on the barrier to pseudorotation (for the furanose ring) of two model compounds, 2-deoxy-~-D-g~ycero-tetrofuranosylamine (781) and 2deoxy-2-fluoro-~-~-erythrofuranosylamine (782) were reported.676 Al-

C-3-endd-2-exo P= 0’ in the Y state

C-2-endd-3+xo P= 1x0” in the S state

though, in 781, the average energy barrier for the N - S interconversion (when expressed by the pseudorotational cycle phase672P, it corresponds roughly to 0” 180”and 18Oo-36O0) is 1 1.3 kJ/mol and the energy difference (AEN.J is only 1.3 kJ/mol (the S state is the more stable), in 782, the energy barriers for the conversion of N - S and S - N are 20.0 and 8.0 kJ/ mol, respectively (thus the AEN-s value is 12 kJ/mol). Furthermore, it was clarified that the rotational energy level in any position is highly influenced

-

(673) J. B. Chiasson, K. Jankowski, and W. Brostow, J. Bioelecf.,2 (1983) 93- 110; related references on n.m.r. and conformation are cited therein. (674) I. V. Antonov, S. M. Dudkin, M. Ya. Karpeiskii, and G. I. Yakovlev, Bioorg. Khim., 2 (1976) 1209-1220; Chern. Ahstr., 85 (1976) 172,922~. (675) M. Blandin, T.-D. Son, J. C. Catlin, and W. Guschlbauer, Biochim. Biophys. A d a , 361 (1974) 249-256. (676) B. Lesyng, C. Marck, and W. Guschlbauer, Inr. J. Quantum Chem., 28 (1985) 517-523.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

243

by the orientation of the hydrogen atom of OH-3; in both the N (gauchedown for the OH bond) and S (gauche-outside) and any other states, the hydrogen atom of OH-3 always comes to occupy the shortest distance from the 2-fluoro atom (this does not necessarily mean hydrogen-bond formation); this situation is achieved by movement of the OH-3 analog synchronous with the pseudorotation of the furanose ring. The n.m.r. and X-ray crystal structure677of 3’,5’-di-O-acetyl-2’deoxy-2’-fluorouridine have been reported (the two sets of data on conformation agreed well). The X-ray crystal analysis678of 728 showed that the sugar ring has the O T 4 conformation ( P = 70.8”), with close proximity of F-2’ and OH-3’ (222 pm), that being different from the conformation of 728 in solution, and the base- plane orientation against the sugar ring, as defined by the torsional angle (jym) of C-6-N-1 -C-4-ring-O, is +54.4” (that is, anti: N- 1 -C-2 bond and the line connecting C- 1’ and the center of the sugar ring, are on opposite sides when viewed from the N- 1 -C- 1’ glycosyl bond). As may be seen with this compound, the conformations of a compound in crystal form and in solution often differ, but are complementary to each other. The crystal structure678of 737 [sugar ring puckering is C-3’ endo with P = 22.1” (close to 3E); base orientation against the sugar is anti km= 28.9”)] is significantly different from that of 728. For 748 (FIAC), the torsional angle xm of the crystal was estimated679to be 19” (base-sugar orientation being anti), a substantially smaller value than that reported for nine “usual” P-D-arabino nucleosides (+24.1 to +36.6”), and this may be ascribed to repulsive interaction between the 2’“‘up” fluoro group and H-6. With respect to the furanose ring, it was concluded that it has the C-3’ endo-C-2’ exo (3T2) conformation, in accordance with functioning of the “gauche effect”.” The results of ab initio molecular orbital calculations (the Gaussian 80 program) for 758 (FMAU) suggested680that the role of the 2/-fluoro atom in the antiviral activity would be raising of the syn-anti rotational bamer around the sugar-base bond to 52.8 kl/mol (for thymidine: 13.0 k.J/mol), thus “locking” the orientation into its anti structure [C-2’ endo conformation (C-2’ being above the sugar plane) of the sugar ring was also suggested];the locked state may contribute to ready attack of DNApolymerase on FMAU-triphosphate. A I9F-n.m.r. study on the conforma-

-+

(677) D. Suck, W. Saenger, P. Main, G. Germain, and J.-P. Declercq, Biochim. Biophys. Acta. 361 (1974) 257-265. (678) C. Marck, B. Lesyng, and W. Saenger, J. Mol. Strrrd.. 82 (1982) 77-94. (679) G. 1. Birnbaum, M.Cygler,K.A. Watanabe,andJ. J.Fox, J. Am. Chem. Soc.. 104( 1982) 7626- 7630. (680) A. M. Sapse and G. Snyder, Cancer Invest.,3 (1985) 115- 121.

244

TSUTOMU TSUCHIYA

tion of DNA-dinucleotides having a fluorine atom at C-3’681or (2-5’ (or both) has been reported.682

b. Biological Activities of 2’-Fluoropyrimidine Nucleosides. -Much effort has been devoted to preparing natural nucleoside analogs having effective antiviral (mostly a n t i h e r p e t i ~and ~~~ antitumor ) activities with low cytotoxicity for the host cells. Through these studies, the structure-activity relationship has gradually been clarified. Some basic aspects in this field will first be described. In pyrimidine nucleosides (the elementary pyrimidine bases are cytosine, uracil, and thymine), chemical modifications of the base portions have mainly been performed at C-5, the most chemically reactive position, and this frequently gives compounds of high biological activity. In cytidine-type nucleosides, however, the 4-amino group is readily deaminated enzymically (by deaminases) to afford uridine-type nucleosides; this biological transformation sometimes gives products of decreased biological activity or devoid thereof. Addition of a deaminase inhibitor sometimes makes the nucleosides remain active. Alteration of the natural P-D-ribosyl and 2-deoxy-~-~-eryfhrc1-pentofuranosyl groups to fluorine-containing p-Dribo-, -xylo-, and -arabino-furanosides is frequently successful in giving nucleosides of high biological activity. Nucleoside analogs having fluorinecontaining unnatural 5-membered carbocyclic residues or glycopyranosides are mostly lower in activity. Generally, however, most of the nucleosides thus synthesized do not exhibit biological activity as such (although some do), or as the 5’-mono- and 5’-di-phosphates, but they do exhibit it after being phosphorylated to the 5’-triphosphates enzymically, by several kinds of kinases of cellular, viral, and transformed cellular origins (these enzymes catalyze conversion of the nucleosides, nucleoside monophosphates, and nucleoside diphosphates into the corresponding mono-, di-, and tri-phosphates, respectively). Herpes simplex virus (HSV) type- 1 -encoded thymidine kinase and thymidylate (= thymidine 5’-phosphate) kinase have high multifunctional activities to phosphorylate thymidine, 2’-deoxyuridine, and other thymidine analogs to their monophosphates, and these phosphates to their dipho~phates,6~~ respectively. The diphosphates are further phosphorylated, inside the cells, by kinases of host-cell origin, and the triphosphates inhibit viral replication or tumor-cell division by inhibiting the DNA poly(681) A.Rosentha1, D.Cech,V. P. Veiko,andZ.A.Shabarova,Z.Chem., 23(1983) 178-179. (682) A. Rosenthal, D. Cech, A. Joecks, E. M. Ivanova, and A. V. Lebedev, Z. Cliem., 25 (1985) 26-27. (683) M. F. Jones, Chem. Br., (1988) 1122- 1126. (684) M.S. Chen, J. Walker, and W. H. Prusoff, J. Bid.Chem., 254 (1979) 10,747- 10,753; related references on virus-encoded enzymes are cited therein.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

245

merase of the transformed cells or (and) by terminating the chain growth of the DNA by incorporation into it. The value of synthetic nucleosides may be decided by the degree of inhibition of DNA polymerase or by the degree of incorporation into the DNA of transformed cells, relative to those of normal cells. Generally, however, inhibitors of any other enzyme correlating to the proliferation of (transformed) cells could also be useful drugs; for example, compounds that inhibit ribonucleotide reductase or thymidylate synthetase which respectively catalyze the reduction of ribonucleotides to the corresponding 2‘-deoxynucleotides and 5-methylation of 2’-deoxyuridine 5’monophosphate. Introduction of a fluorine atom at C-2’ will give a variety of effects on such activities. One of them is to strengthen the glycosyl bond, leading the nucleosides so prepared to resist enzymic hydrolysisby phosphorylases, which catalyze the splitting of nucleosides into their bases and glycosyl phosphates. Because the fluorine atom is strongly electron-withdrawing, and its atomic size is between that of H and OH, nucleic acid analogs (or polynucleotide analogs) containing 2-deoxy-2-fluoropentofuranose, if formed, resemble RNA from the electrochemical viewpoint (although F cannot be a hydrogen donor), and lie in between DNA and RNA from the stereochemical viewpoint. Nucleosides having a 2-deoxy-2-fluoro-~-~-~bofuranosyl group will be described first. 2’-Deoxy-2’-fluorocytidine(737) inhibits685the growth of various human lymphoblastic cell lines, but is rapidly deaminated (catalyzed by cytidine deaminase), to give the inactive 2’-deoxy-2’-fluorouridine (728). The susceptibility of P-D-nbo-, -xylo-, and -arabino-furanosylcytosine nucleosides to cytidine deaminase was omp pa red.^^.^^^*^^^ Generally, the 5fluoro and 5-methyl compounds are respectively deaminated more rapidly and more slowly than the parent compounds, and P-D-xylofuranosyl compounds are not deaminated. It was suggested that the 3‘“‘down” hydroxyl group (in the D-rib0 and D-arabino, but not the D-XYIO sugar portions) is a binding site for mammalian d e a m i n a ~ e s . ~Compound ~ ~ . ~ ~ ’ 737 is, to some extent, phosphorylated, by (probably) deoxycytidine kinase to the triphosphate level, and incorporated into DNA (proved by utilizing the 3H analog). The specificity of the 5’-triphosphatesof 728 and 737 as substrates for DNA polymerase I1 and I11 (from Escherichia coli) has been examined.6882’Deoxy-2’-fluorouridine 5’-diphosphate was shown689to be a useful probe (685) L. W. Brox, G. A. LePage, S. S. Hendler, and D. H. Shannahoff, Cancer Res., 34 (1974) 1838 - 1842. (686) G. W. Camiener, Biochem. Pharmacol.. 16 ( 1967) I69 I - 1702. (687) W. Kreis, K. A. Watanabe, and J. J. Fox, Helv. Chim. Acfa, 61 (1978) 1011 - 1016. (688) W. B. Helfman, S. S. Hendler, D. H. Shannahoff, and D. W. Smith, Biochemistry, 17 (1978) 1607-1611. (689) M. A. Ator, J . Stubbe, and T. Spector, J. Biol. Chem., 261 (1986) 3595-3599.

246

TSUTOMU TSUCHIYA

with which to study the mechanism of action of HSV-1-encoded ribonucleotide reductase, which is different from that of the host cell. The inactivation mechanism of the diphosphate for the enzyme was demonstrated to be initiated from the cleavage of H-3‘ (of the diphosphate) from the enzymephosphate complex. A similar study on the interaction between ribonucleoside triphosphate reductase (from Lactobacillus leichmannii) and the 5’-triphosphates of 728 and 737 has been reported.‘jWNo evidence was observed that 728 or 737 is cleaved, by phosphorylase, to the corresponding base and sugar. Biodistribution of 728 (using the 2J4C and 6-3H analogs) in tumor models has been The cytotoxic activities of the 2’,2’-difluoro analog (775) of 737 against Chinese hamster ovary and tumor cells, in comparison with those of I-P-Darabinofuran~sylcytosine~~~ (ara-C, a drug for leukemia), have been studied693;775 is transported the faster through membrane into cells, more effectively phosphorylated by the deoxycytidine kinase (to the 5’-mOnOphosphate) and, after conversion into the 5’-triphosphate, more highly accumulated in the cells, with longer duration time, than is ara-C, but nevertheless 775 is incorporated into the DNA to a lesser extent than is ara-C. These characteristics of 775 were discussed. I -(2-Deoxy-2-fluoro-~-~-arab~nofuranosyl)-5-~odoc~os~ne (748, FIAC), a typical antiviral agent, showed significant of the replications of Herpes simplex virus (HSV) type-I and -2, Varicella zoster virus (VZV), cytomegalovirus (CMV), and Epstein - Barr virus (EBV), and had weak cytotoxicity. On the other hand, 744 (FAC)and the 5-flUO1-0(745) and 5-chloro (746) analogs showeda9 moderate to appreciable cytotoxicity. The 5-bromo (747) and 5-methyl (749, FMAC) analogs showed639excellent activities against HSV-1 and -2. Thus, the balance between antiviral activity and cytotoxicity of synthetic nucleosides is significantly influenced by a slight change in the structure. The reason for the relative lacka9 of cytotoxicity of FIAC (and other related n u c l e ~ s i d e s against ~ ~ ~ ) host cells has been (690) G. Hams, G. W. Ashley, M. J. Robins, R. L. Tolman, and J. Stubbe, Biochemislry, 26 (1987) 1895-1902. (691) D. N. Abrams, Y. W. Lee, J. R. Mercer, E. E. Knaus, and L. I. Wiebe, Br. J. Radio/.,59 (1986) 263-270; Chem. Absfr., 105 (1986) I lox. (692) R. L. Tolman and R. K. Robins, J. Med. Chem.. 14 (1971) 1 1 12. (693) V. Heinemann, L. W. Hertel, G. B. Grindey, and W. Plunkett, Cancer Res.. 48 (1988) 4024 -403 1 ; related references on biological activity of 775 are cited therein. (694) E. De Clercq, J . Descamps, G. Verhelst, R. T. Walker, A. S. Jones, P. F.Torrence. and D. Shugar, J. lnjecf. Dis., 141 (1980) 563-574. (695) C. Lopez, K. A. Watanabe, and J. J. Fox, Anfimicrob. Agenfs Chemother., 17 (1980) 803 - 806. (696) Y.-C. Cheng, G.Dutschman. J. J. Fox, K. A. Watanabe, and H. Machida, Anrirnicrob. Agents Chemother., 20 (1 98 I ) 420-423.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

247

attributed695to FIAC’s being a better substrate for virus-induced thymidine kinase (TK; TK is a typical allosteric enzyme that converts thymidine, 2’-deoxyuridine, and 5-substituted 2’-deoxynucleosidesinto their 5’-mOnOphosphates) than the substrate for the normal, uninfected cellular deoxycytidine or thymidine kinase649*694*696*697 (however, in human CMV, this general chart should be altered698-700; see the next paragraph). The FIAC 5’-monophosphate thus formed is then converted into the 5’-di- and 5’-triphosphates, mainly by the cellular kinases for nucleoside mono- and diphosphates (virus-induced thymidylate kinase has only slight affinities for FIAC-, FIAU- and FMAU-mon~phosphates~~~), and the t r i p h o ~ p h a t e ~ ~ ~ ~ ~ ~ ~ thus formed inhibits virus-induced DNA polymerase more effectively than it does human DNA p o l y m e r a ~ e . This ~ ~ ~selective-inhibition -~~~ mechanism of FIAC (748) is genera1699p702*706 for other active nucleoside analogs. However, it was reported701that FIAC-triphosphate exhibits the activity only after it has been deaminated enzymically to FIAU-triphosphate. A basic, gene-level study on the origin of the antiviral activity of nucleoside analogs has been reported,707utilizing 757 (FIAU); according to this study, transfected cells are killed by producing FIAU phosphates that are caused by expression of the gene fragment for HSV- 1-TK (non-transfected cells are liable). Also, thymus weight-decreasewas observed in transgenic mice (nontransgenic mice were unaffected). Inhibitory activities of several of these nucleoside triphosphates against708ribonucleotide reductase (prepared from uninfected, and HSV- 1- and -2-infected, HeLa cells) and DNA p r i m a ~ e ~ ~ (from leukemia cells) were studied. (697) W. Kreis, L. Damin, J. Colacino, and C. Lopez, Biochem. Pharmacol.,31 (1982) 767773. (698) J. M. Colacino and C. Lopez, Anrirnicrob. Agenfs Chemother.,24 (1983) 505-508. (699) E.-C. Mar, J.-F. Chiou, Y.-C. Cheng, and E.-S. Huang, J. Virol.,56 (1985) 846-851. (700) J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother.,28 (1985) 252-258. (701 ) M. S. Chen, L. A. Amico, and D. J. Speelman, Antimicrob.Agents Chemother.,26( 1984) 778-780. (702) J. L. Ruth and Y.-C. Cheng, Mol. Pharmacol.. 20 (1981) 415-422. (703) H. S. Allaudeen, J. Descamps, R. K. Sehgal, and J. J. Fox, J. Biol. Chem.,257 (1982) 11,879- 11,882. (704) M. M. Mansuri, I. Ghazzouli, M. S. Chen, H. G. Howell, P. R. Brodfuehrer, D. A. Benigni, and J. C. Martin, J. Med. Chem.. 30 (1987) 867-871. (705) 0. Hantz, H. S. Allaudeen, T. Ooka, E. De Clercq., and C. Trepo, Antiv. Res., 4 (1984) 187-199. (706) S. Suzuki, H. K. Misra, L. 1. Wiebe, E. E. Knaus, and D. L. J. Tyrrell, Mol. Pharmacol.,3 1 (1987) 301-306. (707) E. Borrelli, R. Heyman, M. Hsi, and R. M. Evans, Proc.Natl. Acad. Sci. U.S.A.,85 (1988) 7572-7576. (708) K. Nakayama, J. L. Ruth, and Y.-C. Cheng, J. Virol., 43 (1982) 325-327. (709) W. B. Parker and Y.-C. Cheng, Mol. Pharrnacol.. 31 (1987) 146-151.

248

TSUTOMU TSUCHIYA

FIAC- (also FMAU-, FAU-, and FAC-)resistant variants of HSV-I and -2 are ~ n a b l e ~ to ' ~ phosphorylate ~~" these nucleosides because of decreased activity of the thymidine kinase (TK) and, therefore, are 6,000-fold less pathogenic7" (for the HSV-I variant) than the parent virus. This decreased TK activity (production) seems to be one of the characteristic features of resistant viruses. 5-Halo-4-0x0 nucleosides [754-756 and 757 (FIAU)] also showeda9 good antiviral activity, but with cytotoxicity. Compound 757 is active against seal herpes virus.712The 5-methyl analog 758 (FMAU) is639*657,713 more active against HSV- I and -2, VZV, CMV, and EBV,7'4but also showed cytotoxicity70"to normal cells without m u t a g e n i ~ i t y It . ~ showed ~ activity715,716 against mouse leukemia P 8 15 and L 1210 cells made resistant to 1-~-D-arabinofuranosykytosine(am-C). Compound 758 (FMAU) is superi0r717to 748 (FIAC), 9-@-~-arabinofuranosyladenine (mu-A), and 9-(2-hydroxyethoxymethy1)guanine (acyclovir, the first effective antiviral agent7'8,7'9)in mouse encephalitis caused by HSV-2, when measured by both its ED50 and its therapeutic index (IDSO/ED5,,).In mouse encephalitis caused by HSV-I, FMAU was accumulated720(measured by [2-14C]-FMAUcoupled with autoradiography for sectioned brain samples) in the infected regions (such as retina, suprachiasmatic nuclei, pineal gland, and superior cervical ganglion): however, similar accumulation in some uninfected regions was also recognized. Compounds 748 (FIAC), 749 (WAC), 757 (FIAU), and 758 (FMAU) were s ~ o w ~to be ~ potent ~ ~ *anti-human ~ ~ ' cyto(710) R. F. Schinazi, J. J. Fox, K. A. Watanabe, and A. J. Nahmias, Antimicmh. Agents Chemother., 29 (1986) 77-84. (71 I ) J. Colacino, E. Brownridge, N. Greenberg, and C. Lopez, Antimicrob. Agents Chemother., 29 (1986) 877-882. (712) A. D. M. E. Osterhaus, J. Groen, and E. De Clercq, Antiv. Rex, 7 (1987) 221 -226. (713) K. W. Pankiewicz, B. Nawrot, H. Gadler, R. W. Price, and K. A. Watanabe, J. Med. Chem.. 30(1987) 2314-2316. (714) J.-C. Lin, M. C. Smith, Y.-C. Cheng, and J. S. Pagano, Science. 221 (1983) 578-579. (7 15) J. H. Burchenal, T.-C. Chou, L. Lokys, R. S. Smith, K. A. Watanabe, T.-L. Su,and J. J. Fox, Cancer Re.q., 42 (1982) 2598-2600. (716) T.-C. Chou, J. H. Burchenal, F. A. Schmid, T. J. Braun,T.-L. Su, K. A. Watanabe, J. J. Fox,and F. S. Philips, Cancer Res., 42 ( 1 982) 3957 - 3963. (717) R. F. Schinazi, J. Peters, M. K. Sokol, and A. J. Nahmias, Antimicrob. Agents Chemother., 24 (1983) 95- 103. (7 18) G. B. Elion, P. A. Furman, J. A. Fyfe, P. de Miranda, L. Beauchamp, and H. J. Schaeffer, Proc. Natl. Acad. Sci.U.S.A.,74 (1977) 5716-5720. (719) G. B. Elion, Cancer Res.. 45 (1985) 2943-2950. (720) Y. Saito, R. Rubenstein, R. W. Price, J. J. Fox, and K. A. Watanabe, Ann. Neurol.. 15 (1984) 548-558. (721) E X . Mar, P. C. Patel, Y.-C. Cheng, J. J. Fox, K. A. Watanabe, and E.-S. Huang,J. Gen. Virol..65 (1984) 47-53.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

249

megalovirus (HCMV) agents having large therapeutic indexes, the mode of action being explained as impairment of viral DNA synthesis. HCMV, in contrast to most of the other herpes viruses, does not code for a virus-specific thymidine k i n a ~ ebut , ~ the ~ ~kinase activity is cell-specific, and, therefore, the activities of these nucleosides for HCMV must be explained otherwise. Suzuki and coworkers reported706that the effectiveness of these nucleosides [that examined was 754 (FFAU)] may be exerted by enhanced activity of cellular thymidine kinase, giving the 5’-monophosphate in HCMV-infected cells, in comparison to non-infected cells. It was r e p ~ r t e dthat ~ ~FIAC-, ~.~~ FFAU-, and other related nucleoside triphosphates, once produced, are efficiently incorporated into HCMV-DNA by virus-specific DNA polymerase. Activities of 748 (FIAC), 758 (FMAU), and related compounds against several viruses were compared,698.710*717.723-727 and the combined effects of 748 (FIAC)and 758 (FMAU) other antiviral drugs, or cyclophosphamide (for FMAU), an immunosuppressive agent, were examined. The 2’-chloro and 2’-bromo congeners639of either 748 (FIAC) or 758 (FMAU) are more cytotoxic than FIAC and FMAU, suggesting that these chloro and bromo nucleosides, in contrast to the 2’-fluoro compounds, are comparatively better substrates for deoxycytidine kinase of human lymphocytes than the substrates for viral-specificthymidine kinase. The disposition of the 2’-fluoro group may also be important from the biological viewpoint. It should be noted that the structural difference between RNA and DNA is at the 2’-position. The rib0 type of (738) of FIAC is lo3 times less effective in suppression of HSV replication than is FIAC. Thus Fox, and Watanabe and coworkers concluded that the 2’“‘up” fluorine disposition and the species of the substituent at C-5 are the two important factors influencing antiviral activity. Nevertheless, the mechanism of action of 2’deoxy-2’-fluorocytidine (737) on certain herpes viruses, including HSV- 1 (722) J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother., 24 (1983) 505-508. (723) J.-C. Lin, M. C. Smith, and J. S. Pagano, Antimicrob. Agents Chemother., 27 (1985) 971 -973. (724) H. Machida, Antimicrob. Agents Chemother., 29 (1986) 524-526. (725) A. Larsson and B. Oberg, Antimicrob. Agents Chemother., 19 (1981) 927-929. (726) M. D. Trousdale, A. B. Nesburn, T.-L. Su, C. Lopez, K. A. Watanabe, and J. J. Fox, Antimicrob. Agents Chemother., 23 (1983) 808-813. (727) H. Machida, A. Kuninaka, and H. Yoshino, Antimicrob. Agents Chemother., 21 (1982) 358-36 I . (728) R. F. Schinazi, T.-C. Chou, R. T. Scott, X. Yao, and A. J. Nahmias, Anrimicrob.Agents Chemother., 30 (1986) 491 -498. (729) Z. Zhi-Ming, M. L. Landry, D. R. Mayo, and G. D. Hsiung, Acta Pharmacol. Sinica, 8 (1987) 158-163.

250

TSUTOMU TSUCHIYA

and -2, was i n v e ~ t i g a t e dAlthough .~~ 737 is less effective than acyclovir, it is phosphorylated by the viral thymidine kinase, and incorporated into the DNA of infected cells. In the case of the moderately cytotoxic 1 -(2-deoxy-2-fluoro-p-~-arabinofuranosyl)-5-fluorouracil~9(754, FFAU), it was that the compound is converted into the corresponding nucleotide (5’-monophosphate) by a host nucleoside kinase (from S-49 mouse lymphoma cells) and then reversibly binds to the thymidylate ~ y n t h e t a s eand, ~ ~ ~in the presence of ~(+)-5,1 O-methyIene-5,6,7,8-tetrahydrofolate, forms a tight-binding covalent ~ o r n p l e x thus , ~ ~inhibiting ~ , ~ ~ ~ the incorporation of 2’-deoxyuridine into DNA (the tetrahydrofolate derivative is an essential cofactor for converting 2’-deoxyuridine monophosphate into thymidine monophosphate by donating a one-carbon unit at (2-5, in the presence of thymidylate synthetase). The stability of 754 to mammalian pyrimidine phosphorylase (to split the N-C bond between the sugar and the base) was also demonstrated, and this character was a s ~ r i b e d ~to~ the ’ . ~presence ~~ of the 2’-fluoro group, which strengthens the N-C bond. As the substituent at C-5 of 758 (FMAU) becomes larger than methyl, the antiviral activity becomes weakened, because of decreased conversion7@’ of the enzymically formed 5’-monophosphates into the 5’-diphosphates by thymidylate kinases. The 5-ethyl analog, 1-(2-deoxy-2-fluoro-P-~-arabinofuranosyl)-5-ethyl~racil~~~~~~~ (765, FEAU), however, exhibits only slightly lower antiviral activities (for HSV- 1 and -2, and encephalitis in mice inoculated with HSV-2) and has high therapeutic indexes657(ID5,/EDw’s for HSV-1 and -2 were > 769 and > 220). This weak toxicity was substantiated7@’.735 by the very weak affinity of 765 toward cellular thymidine kinases. All other 5-alkyl analogs (767; alkyl being propyl, isopropyl, allyl, 1and 2-methylpropyl) had significantly less antiviral Some 5-(2haloethyl) congeners, that is, 5-(2-fluoroethyl)-2’-deoxyuridine,and 5-(2-

(730) F.Wohlrab, A. T.Jamieson, J. Hay, R. Mengel, and W. Guschlbauer,Biochim. Biophys. ACtU, 824 (1985) 233-242. (73 1) J. A. Coderre,D. V. Santi, A. Matsuda, K. A. Watanabe, and J. J. Fox,J. Med. Chem.,26 (1983) 1149- 1152. (732) P. V. Danenberg, Biochim. Biophys. Acta. 473 (1977) 73-92. (733) R. A. Byrd, W. H. Dawson, P. D. Ellis, and R. B. Dunlap, J. Am. C h m . SOC..100 (1978) 7478 -7486. (734) J. D. Stoeckler, C. A. Bell, R. E. Parks, Jr., C. K. Chu, J. J. Fox,and M. Ikehara, Biochem. Pharmucol., 31 (1982) 1723-1728. (735) T.-C.Chou, X.-B. Kong, M. P. Fanucchi, Y.-C.Cheng, K.Takahashi, K. A. Watanabe, and J. J. Fox, Antimicroh. Agents Chemother.. 31 (1987) 1355- 1358.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

25 1

chloroethyl) and 5-(2-fluoroethyl)analogs (766) of 758 (FMAU) were prepared.736All of them showed low c y t o t o x i ~ i t y ~but ~ ~ .also ~ ~ ’low antiviral activity in comparison to those of FMAU. The 5-alkenyl analogs659(in 767) [alkene being vinyl and (E)-CH=CHR (R = C1, Br, and I)] were also less active than FIAC against HSV- 1, but much less cytotoxic against human lymphocytes than is FIAC. The 5-ethyl (750) and 5-alkenyl [751, alkene being vinyl, (E)-CH=CHR (R = Br and I), and (E)-CH=CHC02R (R = Et and Na)] analogP7 were also less active (and inactive for the last two); the iodovinyl analog, however, had high therapeutic indexes. A S-acetylene compound, 1-(2-deoxy-2-fluoro-~-~-arabinofuranosyl)-5-ethynylcyto~ i n e (752) ~ ~ showed ~ p ~ weak ~ ~ antiviral and moderate antitumor activities (L 12 10 and B 16 melanoma cell lines in culture). A compound that structurally resembles 758, namely, 5-(2-deoxy-2-fluoro-~-~-arabinofuranosyl)1 -rnethylura~il~~~ (784) was prepared from the 4,S-anhydro derivative 783 (Ref. 739) by treatment with tris(dimethy1amino)sulfonium difluorotrirnethylsili~ate’~~ (TASF; see also, Section 11,2), but 784 had only weak antiviral activity. 0

783

0

7M

Removal of the 5’-hydroxyl group of 748 (FIAC) or other related nucleosides, or substitution by another functional group, is interesting from the (736) H. Gnengl, E. Wanek, W. Schwarz, W. Streicher, B. Rosenwirth, and E. De Clercq, J. Mud. Chem., 30 (1987) 1199- 1204. (737) B. Rosenwirth, W. Streicher, E. DeClercq, E. Wanek, W. Schwarz,and H. Griengl,Anfiv. R ~ s .7. (1987) 27 1-287. (738) R. A. Sharma, 1. Kavai, R. G. Hughes, Jr., and M. Bobek, J. Med. Chem., 27 (1984) 410-412. (739) K. W. Pankiewicz, J.-H. Kim, and K. A. Watanabe, J. Org. Chem., 50 (1985) 33193322.

TSUTOMU TSUCHIYA

252

viewpoint of phosphate formation. The 5’-deoxy (785- 789), 5’-amino (790-792), and 5’-thiol compounds (793 and 794) were prepared7@from the 5’-tosylates by displacement reactions (with I-, N3-, and AcS-) followed by reduction or hydrolysis. Except for 792 (which was slightly active), they exhibited no activity against HSV- 1 and -2, with only weak cytotoxicity, suggesting the importance of the 5’-hydroxyl group for biological activity. However, 5‘-deoxy-5-fl~orouridine,~~’ in spite of its lack of a 5‘-hydroxyl group, exhibited antitumor activity. The reason was (see subsection c). K‘ I

HO 785 786 787 788 789 7’M 791 792 7Y3 794

X = H. X= H, X= I,

R’=

“12,

K’=011.

R’= NH2, R’=OH. X = Me, R’= OH, K’= ”12, X= I, x= 1, R’=OH, x= M ~ ,R’=OH, X= I, R’= Nlll, X= Me, R ’ = O H ,

x= 1.

R2= H RL= t1 R2= 11 K2= I 1 R’= H R2= N1 12 RL= NEIL R2= Nllz R2= SH R ~ SH =

Replacement of the ring-oxygen atom of the sugar moiety of nucleosides by a methylene group would be expected to strengthen the N-C bond. Thus, carbocyclic analogs (795-801) of 2’-fluoro and 2’,2’-difluoro nucleosides were synthesized247by utilizing (4-amino-3-fluoro-2-hydroxycyclopenty1)methanols and (4-amino-3,3-difluoro-2-hydroxycyclopentyl)(740) K. Harada, J. Matulic-Adamic, R. W. Price, R. F. Schinazi, K. A. Watanabe, and J. J. Fox, J. M L ~Chern., . 30 (1987) 226-229. (741) H. Hiebabecky and J. Beranek, Nucl. Acids Res.. 5 (1978) 1029- 1039. (742) A. F. Cook, M. J. Holman, M. J. Kramer, and P. W. Trown, J. Med. Chem.. 22 (1979) 1330-1335.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

253

methanol (for 801; on the syntheses of these compounds, see Section 11,3). Most of the carbocyclic analogs prepared were, however, biologically inactive. Another variation is the introduction of a fluoromethylene group instead of the ring-oxygen atom. Thus, compounds 802 -805 having fluorinecontaining, carbocyclic rings were prepared.268Among them, 803 was active against HSV- 1 .

HO

795 796 797 798 799 800 801

K’

x= I,

R’=OH, X = Me, R ’ = O t i , X= tl, R’=NHZ, X = H , R’=NHl, x= ti, R’=OH. X = Me, R ’ = O H , X- Me. R ’ = O H ,

R ~ = F , R?=H R’= F, R’= H R2= F, R7= H R2=H, R3=F R ~ H= , R ~ =F R2= ti, R’= F R2= b, hi= F

HO X 802 H 80.31 W H 8 0 5 1

R‘

R2

H H F F

F F H H

254

TSUTOMU TSUCHIYA

Distribution of the synthetic nucleosides into animal organs, as well as tumor and virus-infected cells, and the metabolic fate of the nucleosides have been actively studied by utilizing suitably radiolabeled (3H, I4C, 18F, W l , 82Br,1231, IZsI,and lS1I)nucleosides. Gati and studied the transport of [2-14C]thymidineinto human erythrocytes in the presence of extracellular 2’-deoxy-2’-halouridines. The influx of [2-14C]thymidine into human erythrocytes, and the release from them, were greatly inhibited and accelerated, respectively, by 2’-deoxy-2’-fluorouridine(728) or 2’-deoxy-2‘fluorocytidine (737). [6JH]-, [2-14C]-, and [2’-’8F]-2’-Deoxy-2’-fluorouridines were prepared744from [6-3H]-, [2-14C]-,745 and nonlabeled 2,2’-anhydr0-2’-epiuridines~~~ (733), respectively, by treatment with anhydrous HF (for the former two) or HI8F, and tumor uptake of [6-3H]- and [2-I4C]-2’deoxy-2‘-fluorouridineswas 5-Chloro- and 5-fluoro-2’-deoxy-2‘fluor0[2-~~C]uridines~~~ were also prepared, and the tissue distribution of them in mice bearing Lewis lung tumor was These compounds, especially the 5-flUOrO compound, accumulate in the organs of high mitosis (tumor, spleen, intestine, and bone), and are excreted in the urine unmetabolized, indicating resistance to enzymic phosphorolytic cleavage of them to the bases and sugar 1-phosphates, possibly by the influence of the 2’-fluorine atom in their molecules. [2-I4C]-FIACwas synthesized from [2-14C]cytosinein the general manner used for unlabeled 748 (FIAC), and its metabolic fate in mice was The compound (after i.v. injection) was deaminated by cytosine nucleoside deaminase and appeared as [2-14C]-FIAUin plasma, as confirmed by experiments on rats having a very low level of the deaminase, and by treatment with tetrahydrouridine, a nucleoside deaminase inhibitor. This was further confirmed by the use of purified human deoxycytidine d e a m i n a ~ eIt. ~was ~~

(743) W. P. Gati, E. E. Knaus, and L. 1. Wiebe, Mol. Pharmaml., 23 (1983) 146- 152. (744) D. N. Abrams, J . R. Mercer, E. E. Knaus, and L. I. Wiebe, Int. J. Appl. Radiat. Isot.. 36 (1985) 233-238. (745) J. Giziewicz, L. J. Gati, E. E. Knaus, J. R. Mercer, R. J. Flanagan, and L. 1. Wiebe, Inr. J. Appl. Radiat. Isor., 36 (1985) 227-231. (746) D. N. Abrams, Y. W. Lee, J. R. Mercer, E. E. Knaus, and L. I. Wiebe, Br. J. Radiol., 59 ( 1986) 263 - 270. (747) J. R . Mercer, E. E. Knaus, and L. 1. Wiebe, J. Med. Chern.. 30 (1987) 670-675. (748) T.-C.Chou,A. Feinberg, A. J. Grant, P. Vidal, U . Reichman, K. A. Watanabe, J . J . Fox, and F. S. Philips, CancerRes.. 41 (1981) 3336-3342. (749) Y. Cheng, R. Tan, J. L. Ruth, and G. Dutschman, Biochem. Pharmacol., 32 (1983) 726-729.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

255

further ~ l a r i f i e d ~ that ~ ~ 748 . ~ ~(FIAC) ~ , ~ is~converted ~ - ~ ~ ~into 757 (FIAU) and other metabolites, including 744 (FAC), 753 (FAU; both are de-iodinated products), 749 (FMAC), 758 (FMAU, a deaminated-de-iodinated meth~lated~’~ product), and the D-glucosiduronic acids of some of them, but the speed of formation of these products and their distribution in organs differed, depending on the animals tested; for example, 748 (FIAC) was poorly deaminated in dog^,^^^,^^^ and 744 (FAC) was a good substrate for mouse kidney d e a m i n a ~ e(both ~ ~ ~FIAC and FAC have a 3’-“down” hydroxyl g r o ~ p ~ ~In~contrast, v ~ ~ ~ 750 ) . (FEAC) was not d e a m i ~ ~ a t eThe d.~~~ degraded nucleosides were also found in DNA.756Analysis of the purified DNA, obtained from the small intestine of mice that had been treated with [ 2J4C]-FIAC, showed, after enzymic digestion, including treatment with DNAse, the presence of radiolabeled FAC, FMAU, and FIAU. [2-14C]-FIAC was incorporated more into DNA fractions than into RNA fractions of highly proliferating organs (intestine, spleen, and thymus) in the form of 757 (FIAU), 758 (FMAU), and 744 (FAC). In this case, it was suggested that deamination of 748 (FIAC) occurs after conversion into the nucleotide. Through the incorporation of FIAC (using [2-14C]-FIAC)into the DNA of Vero cells, it was shown that FIAC behaves metabolically like thymidine, 2’-deoxyuridine, or 2’-deoxy-5-iodouridine in HSV- 1-infected cells, but like 2’-deoxycytidine in non-infected cells; this suggests that the combined use of FIAC and 2’-deoxycytidinewould lessen the cytotoxicity of FIAC. 1-( 2-Deoxy-2-fluoro-~-~-arabinofuranosyl)-5-iodo[ 2-14C]uracil([2-14C]FIAU) was prepared758by condensation of 742 with 5-iOdO 2,4-bis(O-tri-

(750) A. Feinberg, J. Chromalogr.,210 (1981) 527-530. (75 I ) A. Feinberg, B. Leyland-Jones, M. P. Fanucchi, C. Hancock, J. J. Fox, K. A. Watanabe,

P. M. Vidal, L. Williams, C. W. Young, and F. S. Philips, Antimicrob. Agenls Chemoiher., 27 (1985) 733-738. (752) F. S. Philips. A. Feinberg, T.-C. Chou, P. M. Vidal, T.-L. Su, K. A. Watanabe, and J. J. Fox, CancerRes.. 43 (1983) 3619-3627. (753) X.-B. Kong, A. C. Scheck. R. W. Price, P. M. Vidal, M. P. Fanucchi, K. A. Watanabe, J. J. Fox, and T.-C. Chou, Anliv. R e x . 10 (1988) 153-166. (754) S. L. Commerford and D. D. Joel, Biochem. Biophys. Res. Commun., 86 (1979) 112118. (755) M. P. Fanucchi, K. A. Watanabe, J. J. Fox, and T.-C. Chou, Biochem. Pharmacol., 35 (1986) 1199-1201. (756) A. J. Grant, A. Feinberg, T.-C. Chou, K. A. Watanabe, J. J. Fox, and F. S. Philips, Biochern. Pharmacol.. 31 (1982) 1103-1 108. (757) T.-C. Chou, C. Lopez, J. M. Colacino, A. Feinberg, K. A. Watanabe, J. J. Fox, and F. S. Philips, Mol. Pharmacol., 26 (1984) 587-593. (758) J. E. Swigor and K. A. Pittman, J . Labd. Comp. Radiopharm., 22 (1985) 931 -937.

256

TSUTOMU TSUCHIYA

methylsilyl)[2-’4C]uracil, which had been prepared by iodination of [2J4C]uracil. Compound 758 (FMAU; by use of [2-14C]-FMAU661) administered in mice, rats, and dogs, was, in contrast to 748 (FIAC), mostly recovered unchanged,752with a slight proportion of practically inactive Shydroxymethyl d e r i ~ a t i v e ~759. ’ ~ . Incorporation ~~~ levels of [2-14C]-FMAU and [2-14C]-FEAUinto HSV- 1 infected and uninfected, or mock-infected, Vero cells were omp pa red.^^^,^^^ Although FMAU was incorporated into the DNA’s of both HSV-I-infected and mock-infected Vero cells, FEAU was incorporated only into the DNA of infected cells, supporting the low toxicity of the latter compound. Selective uptake of [2-I4C]-FMAUin the regions infected by HIV- 1 in the brain of rat encephalitis was s h o ~ n . ~ ~ ~ , ~ ~ c. Synthesis and Biological Activities of 3‘-Deoxy-3’-fluoro-, 4‘-Deoxy4’-flUOrO-, 5’-Deoxy-5’-fluoro-, and 6’-Deoxy-6’-fluoropyrimidineNucleosides.-The title nucleosides were prepared by ( a )the coupling method, (b) conversion of existing nucleoside precursors by S N displacement, ~ or (c) oxirane-ring and anhydro-ring opening (formed between 0-2 and a sugar ring position). 2’,3’-Dideoxy-3’-fluoro nucleosides are interesting from the biological viewpoint because of their lack of the 3’-hydroxyl group essential for DNA synthesis. 2’,3’-Dideoxy-3’,5-difluorouridine(811) was prepared76’from 2’-deoxy-5-fluorouridine (806),by way of the 2,3’-anhydro derivative 807, by fluorination (HF- AIF, in 1,4-dioxane),and the structure was confirmed by the n.m.r. spectrum (J2‘(up),F,JZ,(dorm,,F, Jy,F,and J4t,p were 40.5, 21.5, 53.5, and 28.5 Hz,respectively). Although 806 is a potent cytotoxic (antitumor) agent in cell-culture systems, it is cleaved, in vivo, into the pyrimidine base and 2-deoxy-~-erythro-pentofuranose1-phosphate by thymidine phosphorylase. In contrast, the 3’-flUOrO analog 811 is not cleaved by phosphorylase (from Lewis lung carcinoma), but was only weakly cytotoxic (against L 12 10 mouse leukemia cells). The parent compound, 806, was reported to be intracellularly converted by thymidine kinase into the 5‘phosphate, and the latter gives, in the presence of ~(+)-5,lO-methylene5,6,7,8-tetrahydrofolate,a tight-binding, covalent complex with thymidylate ~ y n t h e t a s e(a ~ ~target ~ enzyme in cancer c h e r n o t h e r a ~ y ~ thus ~~), inhibiting DNA synthesis (see also b). The 5’-ph0sphate’~’of 811,however, gave the corresponding complex only weakly. (759) A. Feinberg, P. M. Vidal, J. J. Fox, K. A. Watanabe, M. W. Chun, F. H. Field, A. Bencsath, B. Chait, and F. S. Philips, Drug Metab. Dispos., 12 (1984) 784-786. (760) Y. Saito, R. W. Price, D. A. Rottenberg, J. J. Fox, T.-L. Su, K. A. Watanabe, and F. S. Philips, Science, 2 17 ( 1 982) I I5 I - I 153. (76 I ) S. Ajmera, A. R. Bapat, K. Danenberg, and P. V. Danenberg, J. Mcd. Chem., 27 (1984) 11-14. (762) T. Haertle, F. Wohlrab, and W. Gushlbauer, Eur. J. Biochem., 102 (1979) 223-230.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES 0

0

F

110

807 808

X=F X=Mc

RHv F

810

nil 812 813 814 815 816 817

X=H, X=F, X=F, X= Mc, X= Mc, X= Mc, X=Mc, X=Et.

257

no9

@

HOCH,

F

R= OH

R= OH R= OP(O)(OH)? R= OH R= H

R= F R= I R= OH

3’-Deoxy-3’-fluorothymidine(813), a selective inhibitor763of DNA syn~ moderate ~ ~ - ~ ~ yields from 3’-O-mesyl- or 3’,5’-dithesis, was ~ r e p a r e d in 0-mesyl-thymidine, through 2,3‘-anhydro- I -(2deoxy-P-~-threu-pentofuranosy1)thymine (808), by treatment with hydrogen fluoride (0.1%HF in or 10%HF in DMF‘), l , C d i ~ x a n e - A l F ~1% , ~ HF ~ ~ .in~ DMF-A1F3,765 ~ (763) G. Etzold, R. Nintsche, G . Kowollik, and P. Langen, Tetrahedron, 27 (1971) 24632472. (764) G . Kowollik,G. Etzold, M. Janta-Lipinski,K. Gaertner,and P. Langen, J. Prukt. Chem., 3 I5 ( I 973) 895 -900. (765) A. Joecks, H. Kbppel, K. D. Schleinitz, and D. Cech, J. Prukt. Chem., 325 (1983) 88 I - 892. (766) M. Janta-Lipinski and P. Langen, Z. Chem., 23 (1983) 335.

258

TSUTOMU TSUCHIYA

or by treatment767of 1-( 5-O-trityl-2-deoxy-~-~-threo-pentofuranosyl)thymine with DAST. In similar ways, 2’,3’-dideoxy-3’-fluorocytidine(809, prepared from the corresponding uridine analog 810 or through DAST treatment767),810, and the analogs, 815,816, 817,767and 1-(2,3-dideoxy-3fluoro-~-~-erythro-pent-4-enofuranosyl)thymine (from 816) were prepared.764Compound 813 was also prepared by the condensation method.204 The carbocyclic analog 818 of 813 has also been prepared.768 3’-Deoxy-3’-fluorothymidine (813), and other nucleosides having a 2,3dideoxy-3-fluoro-~-~-erythro-pentofuranosyl group, are phosphorylated by cellular kinases (involving human T-cell kinases) to the triphosphate leve1769.770 (although the V,,,, value of the Michaelis- Menten equation for phosphorylation of the 5’-monophosphate of 813 is 25-fold less769than that for natural thymidine 5’-monophosphate in Ehrlich ascites carcinoma cells), and the triphosphates are incorporated into DNA, with removal of pyrophosphate, under catalysis of such enzymes as calf-thymus DNA polymerase, rat-liver polymerase, Escherichia coli DNA polymerase I, and bacteriophage T4 DNA polymerase, although the degree thereof is very small, and, in consequence, they terminate or inhibit,767-770-779 by lack of the OH-3’ group, further DNA chain-growth. A characteristic feature780of813 is that it (767) P. Herdewijn, J. Balzarini, E. De Clercq, R. Pauwels, M. Baba, S . Broder, and H. Vanderhaeghe, J. Med. Chem., 30 (1987) 1270- 1278. (768) H. Baumbartner, M. Bodenteich, and H. Griengl, Tetrahedron Left..29 (1988) 57455746. (769) P. Langen, G. Kowollik, G. Etzold, H. Venner, and H. Reinert. Actu Biol. Med. Germ.. 29 (1972) 483-494. (770) C . Schroeder and J. Jantschak, Z. Allg. Mikrohiol., 20 (1980) 657-662. (771) Z.G. Chidgeavadze, A. V. Scamrov, R. S. Beabealashvilli, E. I. Kvasyuk,G. V. Zaitseva, I. A. Mikhailopulo, G. Kowollik, and P. Langen, FEBS Lett., 183 (1985) 275-278. (772) E. Matthes, C. Lehmann, B. Drescher, W. Biittner, and P. Langen, Biomed. Biochim. Ada. 44 (1985) ~63-K73. (773) A. M. Atrazhev and M. K. Kukhanova, Bioorg. Khim., 1 1 (1985) 1627- 1635. (774) R. S. Beabealashvilli, A. V. Scamrov, T. V. Kutateladze, A. M. Mazo, A. A. Krayevsky, and M. K. Kukhanova, Biochim. Biophjx Acfa. 868 (1986) 136- 144. (775) Z. G. Chidgeavadze, R. S. Beabealashvilli, A. A. Krayevsky, and M. K. Kukhanova, Biochim. Biophys. .4cfa, 868 (1986) 145- 152. (776) Y. Cheng, G. E. Dutschman, K. F. Bastow, M. G. Sarngadharan, and R. Y. C. Ting, J. Biol. Chem.. 262 (1987) 2187-2189. (777) E. Matthes, C . Lehmann, D. Scholz, M. Janta-Lipinski, K. Gaertner, H. A. Rosenthal, and P. Langen, Biochem. Biophys. Res. Commun., 148 (1987) 78-85. (778) P. Herdewijn, J. Balzarini, M. Baba, R. Pauwels, A. Van Aerschot, G. Janssen, and E. De Clercq, J. Med. Chem.. 31 (1988) 2040-2048. (779) J. Balzarini, M. Baba, R. Pauwels, P. Herdewijn, andE. DeClercq, Biochem. Phnrmacol., 37 (1988) 2847-2856. (780) P. Langen, H. Graetz, M. V. Janta-Lipinski, and H. Weiss, Eur. J. Cancer, 14 (1978) 349 - 354.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

259

stops cell proliferation in the S-phase of the cell cycle (the period used to prepare DNA) rather than kills the cells, thus making accumulation of cells in the 5’-phase a desirable condition to make S-specific agents highly effective. These triphosphates also inhibit RNA-directed polymerase [reverse transcriptase (RT)] of avian myeloblastosis virus, and human immunodeficiency virus (HIV)-associatedRT [HIV is the cause ofthe acquired immune deficiency syndrome (AIDS)]. 3’-Deoxy-3’-fluorothymidine(813) inhibits HIV-RT more effectively than does 3’-azido-3’-deoxythymidine (AZT, an established AIDS drug), but the t o ~ i ~ i t y of ~ the ~ ~former, , ~ ~ as ~ expressed , ~ ~ * from the ratio of cytotoxic dose to effective dose (CD,o/ED,o) or the ratio of phosphorylation by HIV-RT to that by human cellular kinase, is smaller than that of AZT; however, in this respect there still remains contr~versy.~~’ Nucleoside analogs having an “up” 3’-hydroxyl group all seem inactive.767 3’-Deoxy-3’-fluorouridine (820) was prepared782according to H0lj1,’~~ through the reaction of 5-O-benzoyl-3-deoxy-3-fluoro-~-arabinose with cyanamide - methanolic ammonia, followed by methyl propiolate (to give a 2,2’-anhydrouridine derivative) in low overall yield. Compound 820 was prepared782in a yield of 3 1Yo by treatment of 2,3’-anhydro- I-P-D-xylofuranosyluracil(819) with hydrogen fluoride (0.19’0 HF in 1,4-dioxane-A1F3).In this reaction, 2’-deoxy-2’-fluorouridine (728) was simultaneously formed (the product ratio of 820:728 being 2: 3), possibly by way of the route shown. Compound 820 seems not to inhibit RNA synthesis.782 0

0

IOCII,

.;il 0

+

(78 I ) E. Matthes, C. Lehmann, D. Scholz, H. A. Rosenthal, and P. Langen, Biochem. Eiophys. Res. (bmmicn., 153 (1988) 825-831; related references on the toxicity of813 are cited

therein. (782) G. Kowollik, K. Gaertner. and P. Langen, J. Carhohydr. Nucleos. Nucleot., 2 (1975) I91 - 195. (783) A. Holp, TewahcJdronLeu., (1971) 189-192.

TSUTOMU TSUCHIYA

260

1 -(3-Deoxy-3-fluoro-~-~-xylofuranosyl)cytosine (821)was preparedu7 by condensation of 2,5-di-O-benzoyl-3deoxy-3-fluoro-~-xylofuranosyl bromide with bis(trimethylsily1)cytosine(773).It was proposed that 1 -( 3deoxy3-fluoro-~-~-arabinofuranosyl)uracil (823),preparedw from 1-(2,3-anhydro-P-D-lyxofuranosyl)uraciI (822) with anhydrous HF (- 8% in 1,4-dioxane; 1 16”,4 1 h, 3% yield), has a twist conformation of the furanose ring ( O T , ; acetone-d,-D,O; JH-*,+13.5, JH4,s- 27 Hz).Reexamination7” of the reaction under similar conditions gave 823 ( 13%)with 820 (1 1%) and uracil (6790). Conversion of 823 into 820 under the same conditions was verified experimentally, but the mechanism was not clarified. Kowollik and L a ~ ~ g e nobtained ’~~ 3’deoxy-3’-fluoro-j.l-~-arabinofuranosyl (825, 25%) and 2’-deoxy-2’-fluoro-~-~-xylofuranosy~ 826 ( 1 1 %) derivatives by treatment ofthe 5’-0-benzoyl derivative (824)of822with 10%hydrogen fluoride in 1,4-dioxane( 140”,2 d). Debenzoylation of825 and 826 respectively gave 823 and 1-(2-deoxy-2-fluoro-~-~-xylofuranosyl)uracil (740)in high yields. 1 -(3-Deoxy-3-fluoro-~-~-arabinofuranosyl)cytosine (827)was also prepared from 824.Gati and studied the influence of 823 on the influx and release of thymidine in mouse erythrocytes. 3’-Deoxy-3’,3’difluorothymidine [828,1-(2,3-dideoxy-3,3-difluor~~-~-glyce~~~ntofuranosyl)thymine] was prepared787from the corresponding 3’-0xo precursor by treatment with DAST. A nucleoside having a branched-chain sugar, 1 -[ 3-deoxy-3-fluoro-3-C-(h ydroxymethy1)-~~-xylofuranosyl]uracil, has been synthesized.788

OH n2 I

n22

(784) H. K. Misra, W. P. Gati, E. E. Knaus, and L. I. Wiebe, J . Heferocycl. Chern., 21 (1984) 773-775. (785) G . Kowollik and P. Langen, Z. Chern., 15 (1975) 147- 148. (786) W. P. Gati, H. K. Misra, E. E. Knaus, and L. I, Wiebe. Binchem. Pharmacol.. 33 (1984) 3325-3331. (787) D. Bergstrom, E. Romo, and P. Shum, Nucleos. Nucleot.. 6 (1987) 53-63. (788) A. J. Brink, 0. G . De Villiers, and A. Jordaan, Carbohydr. Res., 54 (1977) 285-291.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES uracil

uracil

26 I

ur:icil

n24

F

n2n

5’-Deo~y-5-fluorouridine~~’ has antitumor activity in spite of its lack of a 5‘-hydroxyl group to be phosphorylated (2‘,5‘-dideoxy-5-fluorouridine is inactive789).This result was e ~ p l a i n e das~ due ~ ~ to . ~the ~ circumstance that uridine phosphorylase or thymidine pho~phorylase,~~’ each being an enzyme abundant in tumor tissues but not in normal tissues, cleaves the glycosyl bond, to give 5-fluorouracil, which is then activated in vivo by conversion into 2’-deoxy-5-fluorouridine 5’-phosphate, and the latter inhibits the thymidylate synthetase. Among the analogs of 5’-deoxy-5’-fluorouridineprethe 5‘-0-mesyl, 5‘-0-tosyl, and 5’-bromo-5’-deoxy compounds exhibited activity against L 12 10 mouse leukemia more than did the parent compound, but the 5’-deoxy-5’-fluoro analog (5’-deoxy-5,5’-difluorouridine, 841) had less activity. To obtain a better substrate for the phosphorylase by weakening the glycosidic bond, the acid-labile analog 832, having a

(789) H. Hrebabecky and J. Beranek, Collect. Czech. Chern. Cornmiin.,43 (1978) 3268-3278. (790) H. Ishitsuka, M. Miwa, K. Takemoto, K. Fukuoka, A. Itoga, and H. B. Maruyama, Gann, 71 (1980) 112-123. (791) S. Sugata, A. Kono, Y. Hara, Y. Karube, and Y. Matsushima, Chern. Pharm. Bull., 34 (1986) 1219-1222. (792) S. Ajmera and P. V. Danenberg, J. Med. Chern., 25 (1982) 999- 1002.

262

TSUTOMU TSUCHIYA

fluorine atom at C-4', was prepared793according to the method of Moffatt and ~ o w o r k e r s .Treatment ~ ~ ~ , ~ ~ of ~ the 4'-eno compound 829 with iodine monofluoride, followed by reduction, gave the 4'-flUOrO derivative 831. Treatment of 829 with pyridinium poly(hydrogen fluoride) (the Olah reagent)69in oxolane at 0" and - 50" respectively gave the 4'-flUOrO derivatives, 833 and 831, in high yields. Because 831 could be converted into 833 at 0" by the Olah reagent, 833 is the thermodynamically more-stable compound. A n effective route to 832 consists in treating the 4'-eno compound 830 with the Olah reagent at - 50",followed by hydrogenolysis in the presence of the Pearlman catalyst. Compound 832 had the activity expected.

829 830

RW= C

M ~ ~

R'= R'= CI1JPh

831 R'R2=CMe2, It'= Me, R4= F 832 R'=K2= H , R3= Me, U 4 = F 833 R ' R ~ = C M C R?= ~ , F, RL~e

5'-Deoxy-5'-fluorothymidine (838) was prepared by Langen and Kowol1ik796,797from the 5'-0-tosyl precursor by treatment with fluoride. Compound 838 cannot be phosphorylated enzymically owing to the lack of OH-5', but it inhibits797the growth of carcinoma cells. This was explained as follows: the thymidine 5'-monophosphate (thymidylate) kinase in carcinoma cells, catalyzing the transformation of thymidine 5'-monophosphate into the diphosphate, is inhibited by 838, thus preventing the synthesis of

(793) S. Ajmera. A. R. Bapat, E. Stephanian, and P. V. Danenberg, J.Med. Chem., 31 (1988) 1094- 1098. (794) I. D. Jenkins. J. P. H. Verheyden, and J. G. Moffatt, J. Am. Chem. Soc., 98 (1976) 3346 - 3357. (795) G. R.0wen.J. P.H. Verheyden,andJ.G.Moffatt,J.Urg. Chem., 41 (1976)3010-3017. (796) P. Langen and G. Kowollik, Acta Bid. Med. Germ., 20 (1968) 417-419. (797) P. Langen and G. Kowollik, Eur. J. Biochern.. 6 (1968) 344-351.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

263

DNA. This concept, supported by other data, suggests that 838 may be a close analog of thymidine 5’-monopho~phate’~~ in terms of being a substrate for the enzyme, in spite of the large difference between a phosphate group and a fluoro atom. Inhibition of 5’-deoxy-5‘-halo analogs of 838 for the thymidylate kinase was compared798;the activity decreases in the order of 838, 5’-chloro-5’-deoxy-, 5’-bromo-5’-deoxy-, 5‘-deoxy-, and 5‘-deoxy-5‘iodothymidine. As regards inhibition of the DNA synthesis of intact carcinoma cells in vitro, 838 was also proved to be the most effective among them.798The glycosidic bond of 838 is only slowly cleaved by thymidine phosphorylase (from horse liver), and therefore the activity of838 lasts for a long time. 3’,5’-Dideoxy-3’-fluorothymidine(814), 3‘,5‘-dideoxy-3‘,5’0

NH?

0

FCHl

HO

834 835 836 837 838 839

HO

X=H X=F X=Br X=I X=Me X=CF?CF3

840 84 1 842 843 844 845 846 847 848 849 850 85 1

HO

R’

X

R’

R’

H F Br I H I CFzCFl OH see text H Br CHlOH

H H H H H H H H H OH OH H

OH OH OH OH F F OH OH OH H H OH

R2

R’

852 853 854

R’

H OH OH H H H

(798) P. Langen, G. Kowollik, M. Schiitt, and G. Etzold, Acta Biol. Med. Germ., 23 (1969) K 1 9 - K22.

264

TSUTOMU TSUCHIYA

difluorothymidine (815), and 3’-chloro-3‘,5’-dideoxy-5’-fluorothymidine were also prepared799by known methods; among them, 815 showed the highest inhibition for both DNA synthesis and thymidylate kinase (both from Ehrlich ascites carcinoma cells). 2’,5’-Dideoxy-5’-fluorouridine(834), 2’,5’-dideoxy-5,5 ’-difluorouridine (835), and 2’,5‘-dideoxy-5’-fluorocytidine (854) were prepared,s”’’ and 2’,5’-dideoxy-5’-fluoro-5-iodouridine (837), from 834 by iodination. The 2’,5’-dideoxy-5’-fluorouridine(834) and 5-bromo-2’,5’-dideoxy-5’-fluorouridine (836) were prepareds0’from the 2’hydroxyl precursors, 840 and 842, respectively, by a known method involving displacement (retention of configuration) of the OH-2’ group by C1 (with 2-acetoxyisobutanoylchloride) followed by dechlorination. The influence of the substituent at C-5 of 838 analogs for thymidylate kinase inhibition was compared.798This time also, the best one was 838, followed by 837 and 842 (834,835, and 840 were inactive). Compound 837 is a dead-end inhibitor of HSV- 1-encoded enzyme having thymidine and thymidylate kinase activities.6s4The preparation and activity of 5’-variants (other than fluorine) of 838 have also been reported.802 5’-Deoxy-5’-fluorourid~neBo3 (840), its 5-bromo (842) and 5-iodo analogs 843, 2’,5’-dideoxy-2’,5’-difluorouridine(844), its 5-iodo analog 845, 5’deoxy-5’-fluorocytidines03 (852), 1-(5-deoxy-5-fluoro-~-~-arabinofuranosy1)uracil (849), its 5-bromo analog 850, and 1-( 5-deoxy-5-fluoro-P-~-arabinofuranosy1)cytosine (853) were prepared8@’-8” by common methods. Compound 840, 5-fluorouridine, its mono-, di-, and tri-phosphates, and their analogs were separatedso7by reversed-phase ion-pair h.p.1.c. 5’-Deoxy5’-fluoro-5-(hydroxymethyl)uridines08 (851) and 5’-deoxy-5’-fluoro-orotidineBwwere also prepared. Compounds 840, 842, 843, 845, and 850 inhibit798,8M thymidine and uridine kinase activities, but, excepting 850, the degree of inhibition is lesssw than that of 837.800In the case of 2’-deoxy-5-

(799) P. Langen. G. Etzold, and G. Kowollik, Acta Biol. Med. Germ., 28 (1972) ~ 5 - ~ 1 0 . (800) G. Kowollik, K. Gaertner, G. Etzold, and P. Langen, Carbohydr. Res., 12 (1970) 301 31 I . (801) G. Herrmann, R. Staske, and D. Cech, Z. Cllem.. 18 (1978) 258-259. (802) J. J . Baker, P. Mellish, C. Riddle, A. R. Somerville, and J. R. Tittensor,J. Med. Chem., 17 (1974) 764-766. (803) H. M. Kissman and M. J . Weiss, J. Am. Chem. Soc.. 80 (1958) 5559-5564. (804) M. Schutt,G. Kowollik, G. Etzold,andP. Langen,J. Prakt. Chem., 314( 1972)251-265. (805) G . Herrmann, D. Cech, G. Kowollik, and P. Langen, Z. Chem., 19 (1979) 376-377. (806) J . D. Moyer, J. M. Karle, N. Malinowski, V. E. Marquez, M. A. Salam, L. Malspeis, and R. L. Cysyk, Mol. Pharmacol.. 28 (1985) 454-460. (807) J. L . 4 . Au, M. G . Wientjes, C. M. Luccioni, and Y. M. Rustum, J. Chromatogr., 228 (1982) 245-256. (808) G. Henmann, D. Cech. G. Kowollik, and P. Langen, Z . Chem., 19 (1979) 422-423. (809) G. Henmann, D. Cech, G. Kowollik, and P. Langen, Z. Chem., 2 0 (1980) 20.

DEVELOPMENTS OF F’LUORINATED CARBOHYDRATES

265

fluorouridine (806), it is intracellularlyphosphorylated by thymidine kinase, and the resulting 5’-monophosphate forms a tight-binding complex (ternary complex) with thymidylate synthetase and a tetrahydrofolate derivative and, in consequence, cell mitosis is strongly delayed.810.s11 The mode of phosphorolysis of 5’-deoxynucleoside analogs involving 840 in murine (mainly operated by uridine phosphorylase)and human tumors (mainly operated by thymidine phosphorylase) was found to and it was suggested that use of the mouse as a model system will, in some cases, be inadequate for finding new drugs for humans. Mitotic delay of Physarurn by 840 was discussed813in connection with the S-phase (refer to the similar description for 813) in the cell cycle. 5-(Perfluoroalkyl)-5’-deoxy-5’-fluoro- and 5-(perfluoroalkyl)-2’,5’-dideoxy-5’-fluoro-uridines were prepared8l4from 840 and 834, respectively, using perfluoroalkyl - copper complexes. Among them, 5’-deoxy-5’-fluoro(846) and 2’,5’-dideoxy-5’-fluoro-5-(perfluoroethyl)uridine (839) were particularly effective against Ehrlich ascites carcinoma. 5-Hydroxyl(847) and 5-amino or 5-alkylamino (5-NHMe, -NHBu, -NHCH2Ph, -morpholino, -piperidino, and -pyrrolo) analogs (848) of 840 were prepared.805The a anomer of 5’-deoxy-5’-fluorouridine (840) was also synthesi~ed.~’~ Much simpler fluorine-containing nucleosides, 855 and 856, were preparedsIa by the coupling method. 3’-Amino-3’,5’-dideoxy-5’-fluorothymi-

855 X = H, F, or Me

856 X= H, F, or Me

A. Lockshin and P. V. Danenberg, Biochemistry, 19 (1980) 4244-4251. P. Grobner and P. Loidl, FEBS Lett., 140 (1982) 41 -44. M. Miwa, A. Cook, and H. Ishitsuka, Chem. Pharm. Bull., 34 (1986) 4225-4232. S. A. Kauffman and R. M. Shymko, J. CellSci.. 53 (1982) 143- 154; Chem. Ahstr., 96 ( 1982) I 39.5 1 2 ~ . (814) D. Cech, G . Henmann, R. Staske, P. Langen, and B. Preussel, J. Prukt. Chem., 321 (1979) 488-494. (815) D. Cech, H.-H. Koitzsch, J. Konig, and T. Morsel, Z. Chem., 21 (1981) 449-450. (8 16) L. Kaulina, L. M. Yagupol’skii, N. V. Kondratenko, E. P. Vechirko, A. Berzina, E. Silina, M. Lidaks, and R. A. Zhuk, Khim. Geterolsikl. Soedin., (1982) 256-259; Chem. Abstr., 96 (1 982) 2 18, I68q.

(810) (81 I ) (812) (813)

266

TSUTOMU TSUCHIYA

dine and 3’-azido-3’,5’-dideoxy-5f-fluorothymidineE17 derived from AZT had no inhibitory activities against thymidine and deoxycytidine kinases. As a tool for a hybridization study of DNA, nucleosides having a difluoromethylphosphonate group were prepared. By treatment of thymidine with HCF,P(O)CI,, the 3’- and 5’-mOnO-, and 3’,5’-bis-(difluoromethylphosphonate), and 3’,5’-bis(thymidylyl) difluoromethylphosphonate (857) were Compound 857 was also synthesizedEI8through two successive couplings using difluoromethyl 0,O-bis( I -benzotriazolyl)phosphonate and 5’-0- and 3’-O-protected thymidines. IH-, I3C-, 19F-,and 3’P-n.m.r.spectra of 857 have been recorded and measured. The synthesis undertaken was based on the assumption that the CF, group may function as an isopolar and isosteric substitute for oxygen. The synthesis of dinucleotides containing fluorine in the sugar portion, such as 5’-deoxy-5’-fluorothymidyl-(3’+ 0

0

857

5 ‘)- 3‘-deoxy-3‘-fluorot hy midine, 5 ’-deoxy-5’-fluorothymid yl-( 3 ’+5 7-3 ’deoxy-3’-azidothymidine, 2’-deoxycytidyl-(3’+ 5’)-3’-deoxy-3’-fluorothymidine, and a higher nucleotide, has been reported.819 Nucleosides containing deoxyfluoroglycopyranosyl residues were also prepared. 1-(6-Deoxy-6-fluoro-~-~-glucoand -galacto-pyranosy1)thymine (858 and 860) were obtainedE20from I -p-~-gluco-and -galacto-pyranosylthymine by the usual displacement reaction, or by the condensation method. (817) T . 4 . Lin, Y.-S. Gao, and W. R. Mancini, J. Mrd. Chrm., 26 (1983) 1691-1696. (818) D. E. Bergstrom and P. W. Shum, J. Org. Chem.. 53 (1988) 3953-3958. (8 19) A. Rosenthal, D. Cech, V. P. Veiko, Z. A. Shabarova, M. Janta-Lipinski, and P. Langen. J. Prakr. Chrrn.. 324 (1982) 793-802. (820) G. Etzold, M.Janta-Lipinski, and P. Langen, J. Prukl. Cht.m., 3 18 (1976) 79-86.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

267

Removalsz' of the 2'-sulfonyloxy group of 859 in a basic medium, followed by reaction with metal halides (LiBr and NaI) or hydrogen halides (HCl1,4-dioxane,HBr-acetone, or 0. I % HF in 1,4-dioxane-A1F3) gave, by way of the 2,2'-anhydro intermediate 861, the 2'-halo derivatives 862 -865. The 2'-deoxy analog 866 and 1-(6-deoxy-6-fluoro-~-~-mannopyranosyl)thymine were also prepared from 864 (R2 = H) and 861 (R2 = H), respectively. 1-(4-Deoxy-4-fluoro-~-~-glucopyranosyl)thymine was obtained822by the condensation method. A different kind of nucleoside, 5-(5-deoxy-5-fluoro2,3-O-isopropylidene-a-~-ribofuranosyl)1,3-dimethyIuracil has also been prepared.823 0 .Mc

Mc

0

Mc

__*

R'

OR' 858

859 860

R'= R2= H (gluco) R ' = M S o r TS,R ~ H = or R'= R2= H (galack))

86 I AC (giuco)

862 863 864 865

x66

R'= F,

R2= H

R'=Cl, R2=H R'= Rr, R ~ H = OAC~ R'= I, R 2 = H o r Ac R'= R2= H

d. Synthesis and Biological Activities of C'-Fluoropurine Nucleosides. Purine nucleosides containing fluoro sugars are here described. The syntheses were generally performed either by condensation of fluoro sugar derivatives with purine bases or by introduction of fluorine into the sugar portions of existing, protected purine nucleosides through replacement of sulfonyloxy groups with fluorine, treatment of hydroxyl compounds with DAST, or oxirane-ring opening.

(821) M. Janta-Lipinski, G. Etzold, and P. Langen, J. Prukt. Chem., 320 (1978) 157- 165. (822) M. Janta-Lipinski, G. Etzold, and P. Langen, Z. Chem., 19 (1979) 106. (823) J.-H. Kim,G.-H. Jeon. and K. A. Watanabe, J. Org. Chem., 53 (1988) 5046-5050.

268

TSUTOMU TSUCHIYA

2’-Deoxy-2’-fluoroadenosine(867) was prepared by RanganathangZ4 by treatment of 9-[3,5-bis(O-tetrahydropyranyl)-2-O-(trifluoromethylsulfonyl)-P-~-arabinofuranosyl]adeninewith Bu,NF in oxolane (JH-,ts; JH-Z,,F and JH-3,,F are 16.5, 52, and 17 Hz,respectively). Similarly, 867 and 2’deoxy-2’-fluoroguanosine (868) were prepared.825-828 An n.m.r. study of 867 and 868 in solution indicated that the N conformer populations (see subsection a) of the sugar ring of both compounds are extremely high828-832 [679/0 (Ref. 829) for 867 in MezSO-d6-D20] as compared with those of the other 2‘-substituted compounds (the population decreases in the order F > N, > CI > Br > I). A crystallographic study of 867 indicated833that the sugar ring has a C-3’endo puckering, that is, the ,E conformation.

Ho?

HO

F

R’=H, R’=Ntl? 868 R ’ = N H ~ , R ~ = O H 869 R ’ = H , R~=OH 867

R. Ranganathan, Tetrahedron Lett., (1977) 1291 - 1294. M. lkehara and H. Miki, Chem. Pharm. Bull., 26 (1978) 2449-2453. M. Ikehara, A. Hasegawa, and J. Imura, J. Carbohydr. Nucleos. Nucleot.. 7 (1980) 131 - 140. M. Ikehara and J. Imura, Chem. Pharm. Bull., 29 (1981) 1034- 1038. M. Ikehara and J. Imura, Chem. Pharm. Bull., 29 (1981) 3281 -3285. S . Uesugi, H. Miki, M. Ikehara, H. Iwahashi, and Y . Kyogoku, Tetrahedron Lerr.,(1979) 4073-4076. S. Uesugi, H. Miki, and M. Ikehara, Chem. Pharm. Bull., 29 (1981) 2199-2204. S . Uesugi, T. Kaneyasu, J. Matsugi, and M. Ikehara, Nucleos. Nucleot.. 2 (1983) 373385. M. Ikehara, Helerocycles, 21 (1984) 75-90. K. Morishita, T. Hakoshima, T. Fujiwara, K. Tomita, T. Kaneyasu, S. Uesugi, and M. Ikehara, Acta Crysrallogr., Sect. C, 40 (1984) 434-436.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

269

The 5’-phosphate and 3’,5’-cyclic phosphate (870) of 867 were prepareda3*.a34 (adenosine 3’,5’-cyclic phosphate is a mediator for the action of many hormones) and the conformations were estimated from inspection of the n.m.r. spectra. As an extension of this study, three dinucleoside monophosphates (871,872,and 873) were s y n t h e ~ i z e d ~ ~their ~ - ~physicochemi~’; cal data indicated that each of the three compounds has a conformation similar to that of the corresponding adenosine dimer 874 but with a greater extent of base- base overlap (stacking of bases) than 874, with the 3E conformer preponderating. The analog 875 of873 having a guaninyl moiety was likewise p r e ~ a r e d , and ~ ~ ~a -similar ~ ~ conformation was indicated. The interaction between 875 and ribonuclease T,, which cleaves RNA chains specifically at the 3’-guanylic acid residue, was investigateda4’by using ‘H-n.m.r. spectroscopy: in the complex with the enzyme, the 3’-guanylic acid residue of 875 takes the syn disposition in which the N-9 - C-4 bond and a line connecting C- 1’ and the center of the sugar ring are on the same side, viewed from the N-9 - C- 1’ glycosyl bond, as proved by the nuclear Overhauser effect (n.0.e.) between H-8 and H- 1’. 2’-Deoxy-2’-fluoroinosine (869) monohydrate, prepared from 867 by treatment with nitrous acid, was founde4,to be, in crystals, a mixture of two different stereoisomers (syn and anti orientation of the base- sugar.) 9-(2,3-Dideoxy-2-fluoro-~-~-erythro-pentofuranosyl)adenine (877) was prepareda43J44from the 5’-0-protected precursors (876) by treatment with DAST (CH,CI,; 82% yield) or with Bu4NFfor the corresponding 2’-triflate. The corresponding threo isomer (879) was obtained by deoxygenation at (834) S. Uesugi, J. Matsugi, T. Kaneyasu, and M. Ikehara, Heterocycles, 17 (1982) 285-288. (835) S. Uesugi, Y. Takatsuka, M. Ikehara, D. M. Cheng, L. S. Kan, and P. 0. P. Ts’o, Biochemistry, 20 ( I98 1) 3056- 3062. (836) S. Uesugi, Y. Takatsuka, A. Ohta, and M. Ikehara, Nippon Kagaku Kaishi, (1981) 85 1 -859. (837) D. M. Cheng, L.4. Kan, P. 0. P. Ts’o, S. Uesugi, Y. Takatsuka, and M. Ikehara, Biopolymers, 22 (1983) 1427- 1444. (838) M. Ikehara and J. Imura, Chem. Pharm. Bull.. 29 (1981) 2408-2412. (839) S. Uesugi, T. Kaneyasu, and M. Ikehara, Biochemistry, 21 (1982) 5870-5877. (840) S. Uesugi, T. Kaneyasu, J. Imura, M. Ikehara, D. M. Cheng, L A . Kan, and P. 0.P. Ts’o, Biopolymers, 22 (1 983) I 189- 1202. (84 1) Y. Shibata, 1. Shimada, M. Ikehara, T. Miyazawa, and F. Inagalu, FEBSLett., 235 (1988) 237-240. (842) T. Hakoshima, H. Omori, K. Tomita, H. Miki, and M. Ikehara, Nucl. Acids Res., 9 (1981) 711-729. (843) P. Herdewijn, R. Pauwels, M. Baba, J. Balzarini, and E. De Clercq, J. Med. Chem., 30 (1987) 21 31 -21 37. (844) V. E. Marquez, C. K.-H. Tseng, J. A. Kelley, H. Mitsuya, S. Broder, J. S. Roth, and J. S. Driscoll, Biochem. Pharmacol., 36 (1987) 2719-2722.

270

TSUTOMU TSUCHIYA NI 12

I

870

871

872

873 874

875

R’= adenin-Y -yl. R’= R’= I: R’= ndenin-’S-yl. R’= F, K’= OH K’= adenin-Y-yl, R’= OH, R’= F K’=adenin-y-yi, R’= I$= 011 R’= g u a n i ~ ~ - - R y ~~, OH, = R ~ F=

C-3’ of 883 (see later). It should be noted843that, although 877 was obtained in good yield (76%)from 876, compound 879 was formed in only 10%yield by DAST treatment of the corresponding precursor, 878. Similar DAST treatment of 6-N,5’-O-dibenzoyl-2’-deoxyadenosine gave no fluorine compound, possibly because of formation of the 3’,5’-acyloxonium intermediate. The n.m.r. spectra843of these compounds were diagnostically useful for the determination of structure values for 877 and 879 were 36.6 and 17.1 Hz, respectively). Both 877 and 879 were stable in acidic medium. They showed a protecting effect,844for ATH8 cells, against the cytopathic action caused by HIV, but they were found less active843than 3’-azido-3’deoxythymidine (AZT) for inhibition of replication of HIV-infected MT-4 cells. C o n d e n s a t i ~ n ~of~ ~1,3-di-O-acetyl-5-O-benzyl-2-deoxy-2-fluoro-~.~~~ a r a b i n o f u r a n ~ s (880) e ~ ~ ~or 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-~-arabinofuranosyl bromide (881) with 2,6-dichloropurine (fusion procedure for the former) gave, respectively, an anomeric mixture of 9-glycosylpurine

(845) J. A. Wright, N. F. Taylor, and J. J. Fox, J. Org. Chem., 34 (1969) 2632-2636. (846) J. A. Montgomery, A. T. Shortnacy, D. A. Carson, and J. A. Secrist, 111, J. Med. Chem., 29 (1986) 2389-2392.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

876 877 878 879

R.'

R'

R2

OH

H

H (OMe),-;?Tr F H OH Tr

F

H

H

21 1

H

derivatives [in the 'H-n.m.r. spectra, the H-8 signals of the a and p anomers were singlets and doublets, respectively, in both cases; thepanomer obtained from 881 showed a n.0.e. between H-8 and H-5', indicating the antiorientation (compare with the case of 875)]. Selective displacementus of the chlorine atom at C-6 of the condensation products (from 880) with an amino group (NH, - MeOH), or dual displacements8* of the products (from 881) with azido groups (at C-2 and -6), followed by catalytic reduction and

HO

R' 880 881

882

OAC

R2

HO

R'

AC CHZPh Rr Ac COPh Rr COPh COPh

883 884 885

R'= H, R2= NH2 R'= R2=NH2 R ' = H , R2=SMe R'= R2= H

887 R ' = H , R 2 = 0 888 R ' = N H , R 2 = 0 889 R'=NH2, R2=S 890 R'= H, R2= S

272

TSUTOMU TSUCHIYA

deprotection, gave, respectively, 9-(2-deoxy-2-fluoro-a- and -P-D-arabinofuranosy1)adenine [thea anomer of883: J1S.F 16.3,J5t,F1 Hz;883: J1t.F 14.7, J8,F 2 Hz (H-8 giving a doublet), each845in Me,SO-d,] and 9-(2-deoxy2-fluoro-a- and -~-~-arabinofuranosyl)-9H-purine-2,6-diamine~~ (884, JH-8.F 2.3 and JC-8.F 3.7 Hz). Enzymic deamination (at C-6) of 884 gave 9-(2-deoxy-2-fluoro-~-~-arab~nofuranosyl)guan~ne~~ (888). Compound 888 was also preparedZB2 through coupling of 882 and 2-amino-6-chloro-2N,9-bis(trimethylsilyl)purine.Compound 888 is stableBMto hydrolysis catalyzed by purine nucleoside phosphorylase. It showed cytotoxic activity after phosphorylation by cellular deoxycytidine kinase. 9-(2-Deoxy-2-fluoro-PD-arabinofuranosy1)hypoxanthine(887) was reported847to have anti-leishmania1 (Leishmania tropica) activity. Similarly, 9-(2-deoxy-2-fluoro-p-~arabinofuranosy1)purine (886) and the sulfur-containing analogs, 885,889, and 890, were prepared.848 The carbocyclic analog 891 of 888 was preparedZBZ by way of coupling of 2-amino-4,6-dichloropyrimidine with a racemic fluor~aminodiol,~~~ or by coupling of a protected 0-tosyl analog of 428 (see Section II,4) with 2amino-6-chloropurine. Optically active 891 was then obtainedZB2 through elegant enzymic phosphorylation - dephosphorylation reactions of racemic 891 [the first phosphorylation being catalyzed by thymidine kinase from HSV- 1-infected Vero cells, and the second dephosphorylation by 5‘-nucleotidase (EC 3.1.3.5) from Crotalus atrox venom]. The carbocyclic 891 was lo3-fold more active than 888 against HSV-1 and -2 in vitro, with low toxicity.282The unnatural type of enantiomer of 891 was far less active. Another carbocyclic nucleoside (897)was prepared248starting from aristeromycin (892).Treatment of the 3’,5’-O-disiloxanediyl derivative of 892 with DAST gave the 2’-deoxy-2’-fluoro derivative in poor yield, but the 3’-0,5’0,6-N-tribenzoyl derivative 894 gave the desired derivative 896 in 55% yield, with a N-3 -C-2’-linked by-product (- 1OYo). Treatment248of the 2‘-0-triflyl derivative 895 with Bu4NF in oxolane gave 896 (-25%) with a different by-product (- 25%) having a 1’,2’-double bond. Deprotection of 896 gave 897, which was 10 times more active against HSV-1 and -2 than the corresponding 2’-hydroxyl (arabino type) analog 893. 3’-Fluor0-2’,3‘-dideoxyadenosine~~~ (898), 3’-fluoro-2’,3’-dideoxyguano ~ i n e(899) ~ ~ ~ and 9-(2,3-d~deoxy-3-fluoro-~-~-erythro-pentofuranosyl)2,6-diaminop~rine~~~ (900) were synthesized by way of DAST treatment of

(847) P. Rainey, P. A. Nolan, L. B. Townsend, R. K. Robins, J. J . Fox, J. A. Secrist, 111, and D. V. Santi, Pharm. Rex, (1985) 217-220. (848) C. K. Chu, J. Matulic-Adamic, J.-T.Huang, T.-C. Chou, J . H. Burchenal, J. J . Fox, and K. A. Watanabe, Chem. Pharm. Bull.. 37 (1989) 336-339.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

R' 89 I

892 893 894 8Y5 896

897

R'

11 OH OH tl H OH I4 OSQCFl F H r: 11

213

R3

H

H COPh COPh COPh

H

the corresponding 9-(5-0-protected 2-deoxy-P-~-threo-pentofuranosyl)purine derivatives;their anti-HIV activities were ~ o r n p a r e dwith ~ ~ those ~ . ~ ~for ~ the corresponding 3'-azido congeners, as well as AZT. 1 -(2,3-Dideoxy-3fluoro-p-D-erythru-pentofuranosy1)benzimidazole(901) was prepareds50 through transglycosylation between the 5'-0-acetylated derivative of 813 and benzimidazole. A deoxyfluoro analog (903)of 9-[( 1,3-dihydroxy-2-pro-

F 898 899

900 901

R = adeiiin-9-yl B = guntiiii-9-yl B = 2, h-dinminopurin-9-yl B = henzimidazol- I -yl

(849) J. Balzarini, M. Baba, R. Pauwels, P. Herdewijn, S . G . Wood, M. J. Robins, and E. De. Clercq, Mol. Pharrnacol.. 33 (1988) 243-249. (850) N . B. Dyatkina, L. A. Alexandrova, M. Janta-Lipinski, and P. Langen, Z . Chern., 25 (1985) 180.

274

TSUTOMU TSUCHIYA

R 902 903 904

R=OH, X = O R=F, X=O R= OH, X=CH2

acyclovir

poxy)methyl]guanine (902), the latter being an effective antiviral analog of a c y ~ l o v i r , ~was ' ~ .prepared,85'*852 ~~~ but 903 was found inactive, suggesting the importance of the hydroxyl group of 902. (f)-9-[(2-Fluoro-3-hydroxyand 3-fluoro-2-hydroxy-propoxy)methyl]guanineswere prepared,853and utilized as tools for study of the enzymic phosphorylation mechanism of 9-[(2,3-dihydroxypropoxy)methyl]guanine,an antiherpetic agent. Also, 9-[2- (Ref. 854) and 3-fluoro-4-hydroxy-3-(hydroxymethyl)butyl]guanine855were prepared, but they were both less active than the highly active parent nucleoside, 9-[4-hydroxy-3-(hydroxymethyl)butyl]guanine (904). Condensations56 of 2,5-di-O-benzoyl-3-deoxy-3-fluoro-~-xylofuranosyl bromide856(905) with 6-benzamidopurine or chloromercuri-6-benzamido-

J. C. Martin, D. P. C. McGee, G. A. Jeffrey, D. W. Hobbs, D. F. Smee. T. R. Matthews, and J. P. H. Verheyden, J. Med. Chem., 29 (1986) 1384- 1389. W. Streicher, G. Werner, and B. Rosenwirth, Chem. Scr., 26 (1986) 179- 183. J. D. Karkas, W. T. Ashton, L. F. Canning, R. Liou, J. Germershausen, R. Bostedor. B. Arison, A. K. Field, and R. L. Tolman, J. Med. Chem., 29 (1986) 842-848. M. R. Harnden, A. Parkin, and P. G. Wyatt, J. Chem. Soc.. Perkin Trans. 1. (1988) 2757 - 2765. S. Bailey and M. R. Harnden, J. Chem. Soc.. Perkin Trans. 1. (1988) 2761-2775. J. A. Wright and N. F. Taylor, Carbohydr. Rex. 6 (1968) 347-354.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

275

purine, followed by deprotection, gave 9-( 3-deoxy-3-fluoro-~-~-xylofuranosy1)adenine (906). Similar treatment of 2,5-di-O-benzoyl-3-deoxy3-fluoro-~-arabinofuranosylbromide856(907) with purine derivatives gave, however, mainly the a-D derivative 909. 9-(3-Deoxy-3-fluoro-P-~-

OBz

OH

Yo5

YO6

F

yo7

Yo8 YOY

I ’-!3 ilnonier 1 ’*anomer

arabinofuranosy1)adenine (908) has been prepared8s7(41%) by oxirane-ring opening of 9-(2,3-anhydro-~-~-lyxofuranosyl)adenine with KHF, (refluxing ethylene glycol, 55 min). Compound 906 was also obtainedss8by oxiranering opening (Et,NF- MeCN) of a protected 9-(2,3-anhydro-P-~-nbofuranosy1)adenine. The furanoside conformation of 908 was studiedBs9by (857) K. Miyai, R. K. Robins, and R. L. Tolrnan, J. Med. Chem., 15 (1972) 1092- 1093. (858) M. J . Robins, Y. Fouron, and R. Mengel, J. Org. Chem.. 39 (1974) 1564- 1570. (859) G. Klirnke, 1. Cuno, H.-D. Ludemann, R. Mengel, and M. J. Robins, Z . Nuturjorsch. Ted C, 35 (1980) 853-864.

276

TSUTOMU TSUCHIYA

‘H-n.m.r. spectroscopy. The data (Jlt,zt4.3, J2,,3, J3,,4,-2.5 Hz at -60” in ND,; confirmed by computer simulation) were found quite different from those of the 3’-chloro and 3’-bromo congeners (Jl,,z,-6.3, Jy,3.J3r,4,8 . 5 9.4 Hz) and indicated that the Sconformer preponderates (75%) in the sugar portion, in contrast to the aforementioned congeners (N conformer 9098%;see also, subsection a). In compound 906, the Nconformer preponderates (91%; J,r,z.= Jz.,y = 0, J3,,4,2.4 Hz at -60” in ND3).860 The susceptibilitiesof some of these fluorinated purine nucleosides to the action of enzymes are now described. In contrast to the inertness of the 2’-deoxy-2’-fluoro- and 3’-deoxy-3’-fluorocytidine analogs 739, 744, and 821 towards cytidine deaminase, the adenosine compounds 867,883, and 906 are readily d e a m i ~ ~ a t e d ~by~ the ’ . ~adenosine ,~ deaminase in erythrocytes and calf intestine, but the resulting (deaminated) inosine compounds (from 867 and 883), as well as 888, are highly r e s i ~ t a n t ~ to ” , cleavage ~~~ by purine nucleoside phosphorylase (to give hypoxanthine base for the first two). The reason was Both 867 and 883 can form the 5’-triphosphates, without deamination, in human erythrocytes or munne sarcoma cells in the presence of 2’-deo~ycoformycin,~~’ an adenosine deaminase inhibitor, but X

HO

910 911 912 913 914

k

X=H, R=OH X=NH2, R = O H x=cI, R = O H X=SH, R = O H X=NHz, R= H

YIS (racemate) R’= F, R ~ = H 916

(racemate) R ’ = H , it2= t:

(860) G. Klimke, 1. Cuno, H. -D. Liidemann, R. Mengel, and M. J. Robins, Z. Nuturjorsrh. Tril C, 35 (1980) 865-877. (861) J. C. Hanvey, E. S . Hawkins, D. C. Baker, and R. J. Suhadolnik, Biochemistry. 27 (1988) 5790- 5795; related references on 2’deoxycoformycin are cited therein.

DEVELOPMENTS OF FLUORINATED CARBOHYDRATES

277

only 867 is incorporateda62as the triphosphate into 2’-deoxycoformycintreated T and B cell-enriched lymphocytes. Compound 888, having no amino group at C-6, was also suggestedaMas being initially phosphorylated by deoxycytidine kinase in cells, and, after metabolism to the 5’-triphosphate, it inhibits DNA (but not RNA and protein) synthesis. Study of 5’deoxy-5’-halo(including F)inosines as purine nucleoside phosphorylase inhibitors has been reported.863 5’-Deoxy-5’-fluoroadenosine(911) and the analogs 910, 912, 913 were preparedao3by coupling of 5-deoxy-5-fluoro-~-ribofuranoseand 6-chloropurine. 2’,5’-Dideoxy-5’-fluoroadenosine (914) was preparedsm through a displacement reaction of the corresponding 5’-O-tosyl precursor with fluoride (Bu,NF in DMF). The carbocyclic nucleosides 915 and 916 have been prepared2I8and their antiviral activities evaluated. 5’-S-Alkyl- and 5’-S-aryl-5’-deoxy-5’-fluoro-5’-thioadenosines were prepared864by treatment of protected 5’-S-alkyl- and -aryl-thioadenosine sulfoxides with DAST - SbCl,, followed by deprotection. ACKNOWLEDGMENTS The author expresses his deep thanks to Dr. R. Stuart Tipson, and Prof. Derek Horton ofThe Ohio State University, for reading the manuscript and giving valuable advice, and to Prof. S. Umezawa of this Institute for supporting this work. He is also grateful to Mr. Yoshihiko Kobayashi, to Miss Yoko Matsuura, and to the staff(Ya. T., T. U., T. M., T. S.,Yo. T., Y. K., L. G., Y. S., and R. K.) of this Institute for the documentation research and computer-aided drawing of chemical formulas, for assistance in preparing the manuscript, and for assistance in examination of the manuscript. respectively.

(862) F. W. Burgess, J. D. Stoeckler, and R. E. Parks, Jr., Biochern. Pharmacol., 34 (1985) 3353- 3360. (863) J. D. Stoeckler, C. Cambor, V. Kuhns, S.-H. Chu, and R. E. Parks, Jr., Biochern. Pharmacol., 31 (1982) 163-171. (864) M. J. Robins and S. F. Wnuk, Tefrahedron Lett.. 29 (1988) 5729-5732.

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ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 48

COMPONENTS OF BACTERIAL POLYSACCHARIDES

BY BENGTLINDBERG Department of Organic Chemistry, Arrheniiis Laboratory, Universityof Stockholm, S-106 91. Stockholm, Sweden 1. Introduction. . . . . . . . . 11. Aldoses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

2. Hexoses.. . . . . . . . . 3. Heptoses.. . . . . . . . . . . . .

281 281

4. Branched-chain Sug 111. Glyculoses ...................................................

IV. Amino Sugars . . . . . . . 1. Monoamino Sugars ............................. 2. DiaminoSugars.. ................................................. V. AcidicSugars ......................................... 1 . Glycuronic Acids ................................... 2. Glyculosonic Acids. . .

290 292

1. Non-sugar Aglycons . . . . . . . . . . . . . . . . . . . 299 300 2. Methyl Ethers .....................................................

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

4. Acetals.. . . . . . . VII. Acyl Groups. . . . . .

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

304

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

313 317

X. Conclusions.. .......................................................

I. INTRODUCTION In two articles published in this Series in 1946, the chemistry of bacterial polysaccharideswas discussed.'#*All the sugar and non-sugar components of such polysaccharides that were known at that time had previously been isolated from plant or animal polysaccharides. It was thus not known that ( I ) M. Stacey, Adv. Carbohydr. Chern.. 2 (1946) 161-201. (2) T. H. Evans and H. Hibbert, Adv. Carbohydr. Chem., 2 (1946) 203-233.

219

Copyright 0 1990 by Academic Press, Inc.

AU rights of reproduction in any form reserved.

280

BENGT LINDBERG

the bacterial polysacchandes contain a number of components not present in other natural polysaccharides. Shortly afterwards, Westphal, Liideritz, and their coworker^;^ using the newly developed method of paper chromatography, found a new class of sugars in lipopolysaccharides(LPS) from Gram-negative bacteria, and identified them as 3,6-dideoxyhexoses.This work is summarized in Ref. 4.These discoveries initiated more-systematic investigations of hydrolyzates from bacterial polysaccharides,and a number of new monosaccharides were completely or partially identified. This development has been summarized by Ashwell and H i ~ k m a n . ~ It was with the introduction of Fourier-transform nuclear magnetic resonance spectroscopy (n.m.r.) that good IH- and I3C-n.m.r.spectra of polysaccharides could be obtained. Such spectra often revealed the presence ofsugar and non-sugar components that had been overlooked in previous studies, because, for example, they were decomposed during hydrolysis with acid. The number of such components that had been identified was thereby increased considerably. In a review article published in 1983, some 70 monosaccharide components and some 30 non-sugar components of bacterial polysaccharides were tabulated.6 These figures do not include methyl and 1-carboxyethy1ethers of sugars, fatty acids and hydroxy-fatty acids in LPS, or amino acids in peptidoglycans. Both these figures have now been increased considerably, and new classes of sugars, as well as new types of substituents, have been detected. In an article’ in this Series, on the biosynthesis of bacterial polysaccharides, the different monosaccharide components were also discussed. The aim of the present article is to account for all of the known components of bacterial polysaccharides, except the amino acids in peptidoglycans, that had been reported in the literature before 1989. For less-common sugars and for sugars in unusual ring-forms or anomeric configurations, their modes of linkage in the polysaccharides will also be briefly discussed. References will therefore not always be given to the first identification of a component, but often to publications in which this component is part of a defined polysaccharide structure. For the occurrence of the more-common components, no references will be given, but the reader is referred to previous summaries, for example, those in Ref. 6. (3) 0. Westphal, 0. Liideritz, I. Fromme, and N. Joseph,Angeew. Chern., 65 (1953) 555-557. (4) 0. Westphal and 0. Liideritz, Angew. Chern., 72 (1 960) 88 I - 89 I . (5) V. Ashwell and J . Hickman, in G. Weinbaum. S. Kadis, and S. J. Ajl (Eds.), Microbial Tarins, Vol. IV, Bacterial Endoroxins. Academic Press, New York, I97 I , pp. 235-266. (6) L. Kenne and B. Lindberg, in G. 0.Aspinall (Ed.), The Polysaccharides, Vol. 11, Academic Press, New York, 1983, pp. 287-363. (7) V. N. Shibaev, Adv. Carbohydr. Chern. Biochern.. 44 (1986) 277-339.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

28 1

11. ALDOSES 1. Pentoses

Five pentoses, namely, D-ribose, D- and L-arabinose, and D- and L-xylose, have been found in hydrolyzates of bacterial polysaccharides. D-Riboseis the most common of these, and is a component of different LPS, capsular polysaccharides, and teichoic acid type of polymers. In all these polymers, it occurs as the p-furanosyl group or residue. D-Arabinose occurs in arabinogalactans and arabinomannans elaborated by Mycobacterium species. When this had been determined, for example, for some arabinomannans,8 it was found to be furanosidic and a-linked. The arabinogalactan from Mycobacterium however, contains both a- and &linked D-arabinofuranosyl residues. It also occurs9 in the a-form in the LPS from Pseudomonas maltophila strain NCIB 9204. LArabinose is a component of the LPS from the purple, sulfur bacterium Chromaliurn vinosum.'O D-Xylose, which is one of the most abundant sugars in plant polysaccharides, is a rare component of bacterial polysaccharides. It is found in the LPS of Type 1 Neisseria gonorrhoeae strain" GC 6 . L-Xylose and its 3-methyl ether are components of the LPS of Pseudomonas maltophila strain NCTC 10257, and are P-pyranosidic.'* The D- and L-sugars, and different methyl ethers of these, have also been found in the LPS of some photosynthetic bacteria.I3

2. Hexoses Six of the 18 aldohexoses, namely, D-glucose, D- and L-mannose, D-galactose, D-allose, and L-altrose have been found in bacterial polysaccharides. D-Glucose is the most common sugar in Nature, and has always been found as the a- or ,!-pyranoside. The finding of /?-D-glucofuranosylresidues in the 0-antigen polysaccharide frornI4Erwinia arnylovora T was therefore unexpected, and should be confirmed. (8) A. Misaki, 1. Azuma, and Y. Yamamura, J. Blochem. (Tokyo),82 (1977) 1759- 1770. (8a) M. Daffe, P. J. Brennan, and M. McNeil, J. Biol. Chem., 265 (1990) 6734-6743. (9) S. G. Wilkinson, L. Galbraith, and W. J. Anderton, Curbohydr. Res., I 12 (1983) 24 I -252. (10) R. E. Hurlbert, J . Weckesser, H. Mayer, and I. Fromme, Eur. J. Biochem., 68 (1976) 365-37 1. ( I I ) M. B. Perry, V. Daoust, B. B. Diena, F. E. Ashton, and R. Wallace, Can. J. Biochem., 53 (1975) 623-629. [ 12) D. J. Neal and S. G. Wilkinson, Curbohydr. Res., 69 (1979) 19 1-201. (13) J. Weckesser, G. Drews, and H. Meyer, Annu. Rev. Microbiol.. 33 (1979) 215-239. (14) T. C. Ray, A. R. W. Smith, R. Wait, and R. C. Hignett, Eur. J. Biochem., 170 (1987) 357-361.

282

BENGT LINDBERG

D-Mannose is common, but L-mannose has only been found in a small group of extracellular polysaccharides of related structures, one of which is elab~rated'~ by Alcaligenes ATCC 3 1555. In these polysaccharides, it is a-linked and partially replaces an a-L-rhamnopyranosyl residue in the pentasaccharide repeating-unit. It seems possible that these sugar residues are scrambled, but the other possibility, that there are two populations of polysaccharides, has not yet been excluded. D-Galactose is common, and occurs both as a-and P-pyranosides and as P-furanosides. In natural furanosides, the aglycon is generally in the position trans to OH-2, as in P-D-galactofuranose(1). There is, however, one report of a-D-galactofuranosylresidues in a bacterial polysaccharide, the teichoic acid type of capsular antigen from E. coli K2, but this assignment is tentative only.16L-Galactose, which occurs in some plant and animal polysaccharides, has not been found in bacterial polysaccharides. D-Allose and L-altrose are components of the extracellular polysaccharides elaborated by Pseudomonas viscogena'' and Butyrovibrio fibrisolvens, respectively. 2-Deoxyhexoses have not been found in bacterial polysaccharides, but there is one example of a 4-deoxyhexose, namely, 4-deoxy-~-arabino-hexose. This sugar is a component of some 0-antigens from Citrobacter, for

I

CH,OH

OH

1

HO

2

( I 5) J.-E. Jansson, B. Lindberg, G .Widmalm, and P. A. Sandford, Curbohydr. Res.. I39 (1985) 2 17-223. (16) K. Jann, B. Jann, M. A. Schmidt, and W. F. Vann, J. Bacteriol., 143 (1980) I 108- 1 1 15. (17) A. Misaki, Y. Tsumaraya, M. Kakuta, H. Takemoto, and T. Igarashi, Curbohydr. Rex. 75 (1979) ~ 8 - c l O . (18) R. J. Stack, FEMSMicrobiol. Left.,48 (1987) 83-87.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

283

example, that19from Citrobacter 0 36, which is a homopolysaccharide composed of ( 1 +2)-linked P-pyranosyl residues (2). 6-Deoxy-~-mannose(L-rhamnose) is common, but D-rhamnose has only been found in some 0-antigens, as in that20of Pseudomonas cepacia IMV 3 I8 1, which is a homopolysaccharide containing both a- and P-pyranosyl residues. 6-Deoxy-~-galactose(L-fucose) is common, and has only been found as the a- or P-pyranoside. The rare D-fucose has, however, been found both as a-pyranoside, in the LPS from2' Pseudomonas cepacia serotypes B and El and as a-furanoside, in the cell-wall antigen22from Eubacterium saburreum L 452 and the 0-antigens from different strains of Psuedomonas syringae.23.24 The a-furanoside, as in 3, has a cis relationship between the aglycon and OH-2. The corresponding p form has not yet been found. 6-Deoxy-~and -L-taloseare components of the extracellular polysaccharidesfrom some strains of ButyrivibrioJibrisolvenP and of the LPS from some strains of E. coli,26respectively. The 0-antigen from Yersinia enterocolitica serotype 2 is a homopolysaccharide composed of 6-deoxy-P-~-altropyranosylresidues2' In the LPS from

I

OH

3

(19) E. Romanowska, A. Romanowska, C. Lugowski, and E. Katzenellenbogen, Eur. J. Biochem., 121 (1981) 119-123. (20) Yu. A. Knirel, A. S. Shaskhov, B. A. Dmitriev, N. K. Kochetkov, N. V. Kasyanchuk,and I. Ya. Zakharova, Bioorg. Khim., 6 (1980) 185 I - 1859. (21) Yu. A. Knirel, A. S. Shashkov, M. A. Soldatkina, N. A. Paramonov,andI. Ya. Zakharova,

Bioorg. Khim., 14 (1988) 1208-1213. (22) J. Hoffman, B. Lindberg, T. Hofstad, and H. Lygre, Carbohydr. Rex, 58 (1977) 439-442. (23) S. F. Osman, W. F. Fett, and K. B. Hicks, Carbohydr. Rex. 176 (1988) 205-210. (24) Yu. A. Knire1,G. M.Zdorovenko, A.S. Shashkov, N. Y.Gubanova, L. M. Yakovleva,and R. 1. Gvozdyak, Bioorg. Khim., 14 (1988) 92-91. (25) R. J. Stack, Int. Carbohydr. Symp., X I V th, Stockholm (1988), Abstract A 109. (26) F. Oerskov, 1. Oerskov, B. J a m , K. J a m , E. Miiller-Seitz, and 0. Westphal, Acfa Palhol. Microbiol. Scand., 71 (1967) 339-358. (27) J. Hoffman, B. Lindberg, and R. R. Brubaker, Carbohydr. Res., 78 (1980) 212-214.

BENGT LINDBERG

284

Y. pseudotuberculosis VB, however, the same sugar is a-furanosidic.28The LPS from other species of Yersinia contain 6-deoxy-~-gulose,which is apyranosidic in the LPS fromz9Y. enterocolitica serovar 0 :6,3 1. The 3,6-dideoxyhexoses were first observed3in the Salmonella LPS and have also been found in other bacteria belonging to the Enterobacteriaceae. The five natural sugars of this class are 3,6-dideoxy-~-ribu-hexose (paratose, 4), 3,6-dideoxy-~-arabino-hexose (tyvelose, 5), 3,6-dideoxy-~-arabino-hexose (ascarylose, 6), 3,6-dideoxy-~-xylo-hexose (abequose, 7), and 3,6-dideoxy-L-xylo-hexose(colitose,8).The use ofdifferent trivial names for the D and L forms is unfortunate, but has historical reasons. All five sugars are found in different types of Yersinia pseudotuberculosis. They are occasionally found outside the Enterobacteriaceae. Thus, tyvelose is a component of the cell-wall antigen from 30 Eubacterium saburreum strain L32, and ascarylose occurs3' in the 0-antigen of Vibrio cholerea 0-3. These sugars are generally found as terminal a-pyranosyl groups. There are only two exceptions; paratose occurs as terminal P-furanosyl groups (9) in the LPS from32

OH

4

OH

5

OH

(28) N. I. Kochagina, R. P. Gorshkova, and Yu. S. Ovodov, Bioorg. Khim.,8 (1982) 16661669. (29) N. Kalmykova, R. P. Gorshkova, V. V. Isakov, and Yu. S. Ovodov, Bioorg. Khim.,14 (1988) 652-657. (30) J. Hoffmann, B. Lindberg, T. Hofstad, and N. Skaug, Carbohvdr. Res.. 66 (1978) 67-70. (3 1) B. Lindberg, unpublished results. (32) V. V. Isakov, R. P. Gorshkova, S. V. Tomshich, Yu. S. Ovodov, and A. S. Shashkov, Bioorg. Khim., 7 (1981) 559-562.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

285

HO

h 7

OH

OH

OH

CH3

HO

8

I

I

OH

9

Yersinia pseudotuberculosistype IB, and as terminal /3-pyranosyl groups in the LPS from type 111 of the same species.33

3. Heptoses Three heptoses, namely, L-gfycero-D-manno-heptose, D-glycero-Dmanno-heptose, and D-gfyCero-D-gUfUcto-heptOSe have been found in bacterial polysaccharides. The first of these is a component of the common core in a number of LPS. D-gi.vcero-D-manno-Heptose, which is a precursor of Lgfycero-D-manno-heptose in the biosynthesis,’ is occasionally found in the common core of LPS. Both sugars have also been found in 0-antigen polysaccharides, L-glycero-D-manno-heptose in that fromMPseudomonas cepa(33) R. P. Gorshkova, N. A. Komandrova, A. I. Kalinovsky, and Yu. S. Ovodov, Eur. J. Biochrm.. 107(1980) 131-135. (34) Yu. A. Knirel. N. V. Tanatar, M. A. Soldatkina, A. S. Shashkov, and I. Ya. Zakharova, Bioorg. Kliim., 14 ( 1988) 77 - 8 I.

286

BENGT LINDBERG

cia IMV 61312, and D-glycero-D-manno-heptosein t h a P from Vibrio cholerae 0 2 1. L-glycero-D-manno-Heptopyranose, having an axial hydroxyl group on C- I in the most stable conformation (10) could be described as L-glycero-aD-manno-heptopyranose, according to the Anglo-American Rules,36or as p-L-glycero-D-manno-heptopyranose according to the IUPAC - IUB Recommendation~.~’ The forthcoming new IUPAC - IUB Recommendations will probably follow the Anglo-American Rules in this respect, and they will be followed in the present article. The anomeric configuration of this sugar, when it occurs in the core of LPS from Enterobacteriaceae, is L-glyceru-a-Dmanno, but there are numerous examples of incorrect or ambiguous naming in the literature. It is therefore advisable, in this and similar examples, to state explicitly, or indicate in a formula, which is the actual configuration. D-gfycero-D-galacto-Heptose has been found in different bacterial poly-

OH

10

saccharides. A cell-wall polysaccharide from Eubacterium saburreum L44 is a homop~lysaccharide~~ in which this sugar is /?-pyranosidic.Another strain, T27, of the same organism also contains a-furanosidic groups ofthis sugar.39 On reduction, L-glycero-D-manno-heptoseand D-glycero-D-gafactu-heptose give the same heptitol, and a sugar analysis based upon g.1.c. of the alditol acetates obviously does not distinguish between these two sugars. Two 6-deoxyheptoses, namely 6-deoxy-~-manno-heptoseand 6-deoxyD-aho-heptose, are components of bacterial polysaccharides. The former occurs as a-pyranosyl residues in the LPS from some strains of Yersinia pseudotuberculosis,40and the latter as terminal a-furanosyl groups (11) in

A. A. Ansari, L. Kenne, B. Lindberg, B. Gustafsson, and T. Holme, Curbohydr. Res., 150 (1986) 213-219. J . Org. Chem., 28 (1963) 281-291. Eur. J. Biochem.. 21 (1971) 455-477. J. Hoffman, B. Lindberg, S. Svensson, and T. Hofstad, Curbohydr. Rex. 35 ( I 974) 49- 53. W. Kondo, F. Nakazawa, M. Sato, and T. Ito, Curbohydr. Rcs., 117 (1983) 125-131. K. Samuelsson, B. Lindberg, and R. R. Brubaker, J. Bucteriol., 117 (1974) 1010- 1016.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

281

CH20H

I

CHZ

I HOCH

P HO

11

the cell-wall polysa~charides~' from Eubacterium saburreum, strain L49 and some other strains of this species.

4. Branched-chain Sugars Before 1983, branched-chain sugars had not been found in bacterial polysaccharides,but there are now five examples belongingto this class. The LPS from4*Coxielfa hurneti phase I contains both 6-deoxy-3-C-methyl-~-gulose (L-virenose)as pyranoside (12) and 3-C-(hydroxymethyl)-~-lyxose as furanoside (13). Another 6-deoxy-3-C-methylhexose,having the manno configuration, is a component of the Nitrobacter hamburgiensis O-antigen.43

HO

12

0

HO

OH

13

(41) J. Hoffman, B. Lindberg, J. Lonngren, and T. Hofstad, Carbohydr. Res., 47 (1976) 261 267. (42) S. Schramek, J. Radziejewska-Lebrecht, and H. Mayer, Eur. J. Biochem., 148 (1985) 455 -46 1. (43) H. Mayer, E. Bock, and J. Weckesser, FEMS Microbiol. Lett., 17 (1983) 93-96.

288

BENGT LINDBERG

Two 3,6-dideoxy-4-C-(I -hydroxyethyl)hexosesare components of O-antigens from Yersiniu species. One, yersiniose A, was first isolated from the Y. pseudot uberculosis VI serovar LPS.44The other, yersiniose B, was obtained from the Y. enterocoliticu 0 :4,32 LPS.45They both have the D-xyloconfiguration, but differ in the configuration of the side chain. Yersiniose A is 3,6-dideoxy-4-C- [ 1-(5')-hydroxyethyll-~-xylo-hexose, and yersiniose B the corresponding I -(R)-hydroxyethylisomer.46Two pairs of diastereoisomeric pyranose derivatives may be formed from either yersiniose A or B, and it is not clear if 14 and 15, which are the ( R )and (5')isomers of 3,6-dideoxy-4-C( 1-hydroxyethyl)-D-xylo-pyranose,represent the actual ring forms in the polysaccharides or only the favored ring forms of the free sugars in solution.

111. GLYCULOSES

In addition to the sugars discussed in this Section, a number of glyculosonic acids will be discussed in Section V. D-threo-Pentulose ("D-ribulose") is a component of some LPS from Pseudomunus and Yersinia species; for example, that4' from Y. enterocoliticu 0 :5,27, and occurs asp-D-furanosidic terminal groups (16). Two extracellular D-fmctans, (2 +6)-linked p-D-fructofuranan or levan' and the less common corresponding (2+ I)-linked polysaccharide,4*of the inulin type, are elaborated by different bacteria. These polysaccharides are formed from sucrose by the action ofsucrose fructosyltransferases.Terminal p-D-fructofuranosyl groups are present in some bacterial heteropolysacchar(44) R. P. Gorshkova, V. A. Zubkov, V. V. Isakov, and Yu. S. Ovodov, Carbohydr. Res., 126 (1984) 308-312. (45) R. P. Gorshkova, V. A. Zubkov, V. V. Isakov, and Yu. S. Ovodov, Bioorg. Khim., 13 (1987) 1146-1147. (46) V. A. Zubkov, A. F. Sviridov, R. P. Gorshkova, A. S. Shashkov, and Yu. S. Ovodov, Bioorg. Khim.. 15 (1989) 192-198. (47) M. B. Perry and L. L. MacLean, Biochem. Cell Biol.. 65 (1987) 1-7. (48) K.-G. Rose11 and D. Birkhed, Acla Chem. Scand., Ser. B. 28 (1974) 589.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

289

HO

16

ides, as in the capsular polysaccharide from49Haemophilus influenzae type e. A D-fructan from Zymomonas motilis contains both a- and @-linked D-fructofuranosylresidues.50The 0-antigen from Yersinia intermedia strain 680 is a (2- 1)-linked D-fructan having alternating a-and P-furanosidic residue^.^' D-Fructose generally occurs as @-furanosylresidues in Nature, and these seem to be the only known exceptions. The t ype-specific capsular polysaccharide from52Streptococcuspneumoniae type 5 contains 2-acetamido-2,6-dideoxy-~-~-xylo-hexopyranosyl-4ulose residues (17). Sugar nucleotides of hexos-4-uloses are important intermediates in the transformation of sugars during the biosynthesis,’ but this is the only known example of such a sugar as a polysaccharide component.

NHAc

17

IV. AMINOSUGARS

In this Section, aminodeoxyaldoses will be discussed. Sugars containing both amino and carboxyl groups will be dealt with in Section V. Most amino (49) P. Branefors-Helander, L. Kenne, B. Lindberg, K. Peterson, and P. Unger, Curbohydr. Res.. 88 (1981) 77-84. (50) K. D. Barrow, J. G. Collins, P. L. Rogers, and G. M. Smith, Eur. J. Eiochem., 145 (1984) 173- 179. (51) R. P. Gorshkova, S. V. Koval’chuk, V. V. Isakov, G. M. Frolova, and Yu. S. Ovodov, Bioorg. Khim., 13 (1987) 818-824. (52) P.-E. Jansson, B. Lindberg, and U. Lindquist, Curbohydr. Res., 140 (1985) 101- 110.

290

BENGT LINDBERG

sugars are N-acylated, and the different acyl groups will be discussed in Section VI. The aminodeoxyaldoses, when present in bacterial polysaccharides, are known only as pyranosides.

.

..

O-P-OI OH

18

1. Monoamino Sugars A monoaminopentose, 4-amino-4-deoxy-~-arabinos,is known as a component of some Gram-negative bacteria. It is linked, as the /I-pyranosyl phosphate (18), to a 2-amino-2-deoxy-~-D-glucopyranosyl residue in the lipid A part of the LPS.53 A considerable number of monoaminohexoses are known that have the amino group in the 2-, 3-, or 4-position. 6-Amino-6-deoxyhexoseshave not, however, been found in bacterial polysaccharides. 2-Amino-2-deoxy-~-glucose (D-glucosamine)and 2-amino-2-deoxy-~-galactose(D-galactosamine) are common, and 2-amino-2-deoxy-~-mannose(D-mannosamine)has been found in some LPS and extracellular polysaccharides. D-Glucosamine, having a free amino group, is sometimes found; for example, in the core region 0P4the Bordetella pertussis LPS. 2-Acetamido-2-deoxy-~-glucose a component of the LPS from Pseudomonas cepacia serogroup 0 1. D-G~UCOSamine, N-acylated by an (R)-3-hydroxyfatty acid, is present in the lipid A moiety of LPS. The 2-amino group in hexosamines is, however, generally acetylated. Amino groups in other positions are also most often acetylated, but acylation with other acids is not uncommon. Six 2-amino-2,6-dideoxyhexosesare known as components of bacterial polysaccharides, namely, those having the D- and L-ghco, L-manno, D- and L-galacto, and ~ - t a l oconfigurations. 2-Amino-2,6-dideoxy-~-glucose(Dquinovosamine) occurs in some LPS; for example, that from Pseudomonas

(53) M. Batley, N. H. Packer, and J. W. Redmond, Biochemistry, 21 (1982) 6580-6586. (54) M. Moreau, R. Chaby, and L. Szabo, J. Bacferiol.. 159 (1984) 61 1-617. (54a) A. D. Cox and S. G. Wilkinson, Curbohydr. Res., 195 (1990) 295-301.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

29 1

aeruginosa serotypeSS0 6 and, together with the L-isomer, in the capsular polysaccharide elaborared bys6Bacteroidesfragilis NCTC 9343.2-Amino2,6-dideoxy-~-mannose(L-rhamnosamine) is rare, but has been found in some polysaccharides; for example, the LPS froms7 E. coli 0 3 . 2-Amino2,6-dideoxy-~-galactose(L-fucosamine)is rather common. D-Fucosamine is less common, but is present in some LPS from Pseudomonas species, sometimes together with L-fucosamine, as in the LPS fromS8P. aeruginosa serotype 07.2-Amino-2,6-dideoxy-~-talose(“pneumosamine”) is a component of the Streptococcus pneumoniae type 5 capsular polysac~haride.~~ The name “pneumosamine”, and some similarly constructed names for natural amino sugars are not very suitable as the trivial name for the parent sugar, as “pneumose” for 6-deoxy-~-talose,is not used. Three 3-amino-3,6-dideoxyhexoses, having the D- and L-gluco and D-galacto configurations, have been found. The two D-sugars are not very common, but occur in some 0-antigens; for example, those from E. coli 01 14 (Ref. 60) and E. coli 0 2 (Ref. 6 1), respectively. The D-galuctoisomer has also been found in the cell-wall polysaccharide from6*Eubacterium saburreum strain L 13.3-Amino-3,6-dideoxy-~-glucose has been found in the core part63 of the Aerornonas hydrophila chemotype I11 LPS. Three 4-amino-4,6-dideoxyhexoses, having the D-gluco, D-manno, and D-galactoconfigurations, are known, and all three are components of O-antigens. Thus, 4-amino-4,6-dideoxy-~-glucose (“viosamine”) is present in the O - a n t i g e ~of~ ~E.~ coli 0 7 . 4-Amino-4,6-dideoxy-~-galactose(“thomosamine”) occurs in the E. coli 010 0-antigen6’ and also in the enterobacterial (55) B. A. Dmitriev, N. A. Kocharova, Yu. A. Knirel, A. S. Shashkov,N. K. Kochetkov, E. S . Stanislavsky, and G. M. Mashilova, Eur. J. Biochem.. 125 (1982) 229-237. (56) D. L. Kasper, A. Weintraub, A. A. Lindberg, and J. Lonngren, J. Bucteriol., 153 (1983) 991 -997. (57) B. Jann and K. Jann, Eur. J. Biochem., 5 (1967) 173- 177. (58) B.A. Dmitriev, Yu. A. Knirel, N.A. Kocharova,N. K. Kochetkov, E.S. Stanislavsky,and G. M. Mashilova, Eur. J. Biochem., 106 (1980) 643-651. (59) S. A. Barker, J. S. Brimacombe, M. J. How, M. Stacey, and J. M. Williams, Nature, (1 96 1) 303 - 304. (60) B. A. Dmitriev, V. L.’vov, N. V. Tochtamysheva, A. S . Shashkov, N. K. Kochetkov, B. Jann, and K. Jann, Eur. J. Biochem., 134 (1983) 517-521. (61) P.-E. Jansson, H. Lennholm, B. Lindberg, U. Lindquist, and S. B. Svenson, Curbohydr. Re.?., 161 (1987) 273-279. (62) P.-E. Jansson, B. Lindberg, M. Spellman, T. Hofstad, and N. Skaug, Curbohydr. Res., 137 (1983) 197-203. (63) J . H. Banoub and D. H. Shaw, Curbohydr. Res.. 98 (1981) 93- 103. (64) V. L. L‘vov, A. S. Shashkov, B. A. Dmitriev, N. K. Kochetkov, B. Jann, and K. Jann, Carbohydr. Res., 126 (1984) 249-259. (65) L. Kenne, B. Lindberg, C. Lugowski, and S. B. Svenson, Curbohydr. Rex, 151 (1986) 349-358.

292

BENGT LINDBERG

common antigen.664-Amino-4,6-dideoxy-~-mannose(“perosamine”) has been found in different 0-antigens, as in those from Vibrio ~ h o l e r a eYer,~~ sinia enterocolitica 0 9 (Ref. 68), and Brucella abortus (Ref. 68). A 2-amino-2-deoxyheptose of unknown configuration is a minor component of the LPS from the photosynthetic procaryote Anacystis n i d u l a n ~ . ~ ~ 2. Diamino Sugars

The lipid A from some species, belonging to Rhodopseudomonas, Pseudomonas, and some other groups of bacteria, contains 2,3-diamino-2,3-dideoxy-D-glucose instead of the usual disaccharide composed of D-glucosamine residues connected by a /?-(1 -6) linkage.70 2,4-Diamino-2,4,6-t~deoxy-~-glucose (“bacillosamine”) was first found as a component of cell walls from Bacillus lichen of or mi^.^^ It is also present in some 0-antigens from Pseudomonas a e r ~ g i n o s a2,4-Diamino-2,4,6.~~ trideoxy-D-galactose is a component of the Shigella sonnei O-antige~~,’~ the Streptococcus pneumoniae type 1 capsular p~lysaccharide,~~ and the S. pnsumoniae C - s ~ b s t a n c ewhich , ~ ~ is a cell-wall teichoic acid. V. ACIDIC SUGARS

The acidic sugars discussed in this Section are glycuronic acids and glyculosonic acids. Bacterial polysaccharides may also become acidic by substitution of sugar residues, for example by etherification with lactic acid, acetalation with pyruvic acid, or phosphorylation, and these possibilities will be discussed in the following Sections. A sugar that does not fall into any of

(66) A. Dell, J. Oates, C. Lugowski, E. Romanowska, L. Kenne, and B. Lindberg, Curbohydr R ~ s . 133 . (1984) 95-104. (67) L. Kenne, B. Lindberg, P. Unger, B. Gustafsson, and T. Holme, Curbohydr. Res., 100 ( 1982) 34 1 - 349. (68) M. Caroff, D. R. Bundle, and M. B. Perry, Eur. J. Biochem , 139 (1984) 195-200. (69) G. Weise, G. Drews, B. J a m , and K. Jann, Arch. Microbiol., 71 (1970) 89-98. (70) H. Mayer and J. Weckesser, in E. T. Rietschel (Ed). Handbook qf Endofoxin. Vol. I , Chemistry of Endotoxin, Elsevier, Amsterdam; 1984, pp. 22 1-241. (71) U. Zehavi and N. Sharon, J. Biol. Chem., 248 (1973) 433-438. (72) Yu. A. Knirel, E. V. Vinogradov, A. S. Shashkov, S. G. Wilkinson. Y. Tahara, B. A.

Dmitriev, N. K. Kochetkov, E. S. Stanislavsky, and G. M. Mashilova, Eur. J. Biochem., 155 (1986) 659-669. (73) L. Kenne, B. Lindberg, K. Peterson, E. Katzenellenbogen, and E. Romanowska, Curhohydr. Res., 78 (1980) 119- 126. (74) B. Lindberg, B. Lindqvist, J. Lonngren, and D. A. Powell, Curbohydr. Res.. 78 (1980) I 1 I - I 17. (75) H. J. Jennings, C. Lugowski, and N. M. Young, Biochemistry, 19 (1980) 4712-4719.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

293

these categories is a 2-amino-2,6-dideoxyhexose-6-sulfonicacid76 of unknown configuration, isolated from the cell walls of a Halococcus species. 1. Glycuronic Acids

The capsular polysaccharide from Rhizobium meliloti IF0 13336 contains terminal a-D-ribofuranosyluronic groups77 (19). With this obvious exception, all known glycuronic acids in bacterial polysaccharides are pyranosidic.

OH

HO 19

D-Glucuronic acid and D-galacturonic acid are common components of extracellular polysaccharides, and have also been found in different LPS. D-Mannuronic acid and its C-5 epimer, L-guluronic acid, are the components of bacterial alginic acid, elaborated by strains of Pseudornonas aerug i n o s ~ .The ~ * C-5 epimers of D-glucuronicand D-galacturonicacid, namely, L-iduronic acid and L-altruronic acid, are components of a dermatan sulfate-like polysaccharide from79Clostridium perfingens Hobbs 10 and of an extracellular polysaccharide from Aeromonas viridans var. h~mari,'~" respectively. All known hexosaminuronic acids have an amino group in the 2-position. Seven 2-amino-2-deoxyhexuronicacids are known, namely, those with the D - ~ ~ U C OD-manno, , D- and L-galacto, L-talo, L-altro, and L-gulo configurations. None of these is very common, and some have been found in one polysaccharide only. (76) R. Reistad, Curbohydr. Res., 54 (1977) 308-310. (77) A. Anemura, M. Hisamatsu, S. K. Ghai, and T. Harada, Carbohydr. Rex, 91 (1981) 59-65. (78) A. Linker and R. S. Jones, J. B d Chem., 241 (1966) 3845-3851. (79) L. Lee and R. Cherniak, Carbohydr. Res., 33 (1974) 387-390. (79a) K. Hermansson, L. Kenne, B. Lindberg, B. Ane, R. G. Brown, and J. E. Stewart,

Carbohydr. Res., in press.

294

BENGT LINDBERG

2-Amino-2-deoxy-~-glucuronicacid was first found in the cell-wall antigen fromSoStaphylococcus aureus Smith strain 05068, in which it occurs as P-D-pyranosyl residues. 2-Amino-2-deoxy-~-mannuronicacid is a component of some cell-wall polysaccharides; for example that from Micrococcus lysodeiticus,slof some capsular polysaccharides, such as that froms2Streptococcus pneumoniae type 12F, and of the enterobacterial common antigen.66 It has always been found as P-D-pyranosyl residues. The Vi-antigen is a homopolysaccharide, composed of 2-amino-2-deoxy-~-ga~acturonicacid residues.s3The same acid is a component of some other capsular polysaccharides and of some 0-antigens; for example, thats4 from Psuedomonas aeruginosa immunotype 1. It is a-pyranosidic in all known examples. 2-Amino2-deoxy-~-galacturonicacid, as a-L-pyranosyl residues, is a componentss of the Pseudomonas aeruginosa 0 2 0-antigen. 2-Amino-2-deoxy-~-taluronic acid is a component of pseudomurein in Methanobacterium,s6 a genus belonging to the taxon Archaeobacteria, which seems to be a departure from the main stem of bacteria. 2-Amino-2-deoxy-~-altruronicacid, as a-L-pyranosyl residues, has only been found in the 0-antigen from Shigella s ~ n n e i . ’ ~ 2-Amino-2-deoxy-~,~~ guluronic acid, as a-L-pyranosyl residues, has also only been found once, in the capsular polysaccharidess from Neisseria meningitides Group I, and occurs together with the D-mannoisomer. The presence ofthe two 5-epimers may indicate that epimerization takes place on the polymer level, as has been demonstrated for alginic acid. A group of 2,3-diamino-2,3-dideoxyhexuronic acids has been found in bacterial polysac~harides,~~ mainly in different 0-antigens from Pseudomonas aeruginosa. The D-gulo isomer (20) was first found in the 0-antigen fromw P. aeruginosa 06. The m man no and ~-guIoisomers (21 and 22) are (80) S. Hanessian and T. H. Haskell, J. Biol. Chem.. 239 (1964) 2758-2764. (81) Nasir-ud-Din and R. W. Jeanloz, Curbohydr. Res., 47 (1976) 245-260. (82) K. Leontein, B. Lindberg, and J. Ldnngren, Can. J. Chem., 59 (1981) 2081 -2085. (83) K. Heyns and G. Kiessling, Curbohydr. Res.. 3 (1967) 340-353. (84) Yu. A. Knirel, A. S . Shashkov, B. A. Dmitriev, and N. K. Kochetkov, Curbohydr. Res.. 133 (1984) ~ 1 2 - C I 4 . (85) Yu. A. Knirel, E. V. Vinogradov, A. S . Shashkov, B. A. Dmitriev, N. K. Kochetkov, E. S . Stanislavsky, and G. M. Mashilova, Eur. J. Biochem., 125 (1982) 221 -227. (86) H. Kdnig, 0. Kandler, M. Jensen, and E. T. Rietschel, Z. Physiol. Chem., 364 (1983) 627-636. (87) T. Kontrohr, Curbohydr. Rex. 58 (1977) 498-500. (88) F. Michon, J. R. Brisson, R. Roy, F. E. Ashton, and H. J. Jennings, Biochemistry, 24 (1985) 5592-5598. (89) Yu. A. Knirel and N. K. Kochetkov, FEMSMicrobiol. Rev.. 46 (1987) 381 -385. (90) B. A. Dmitriev, N. A. Kocharova, Yu. A. Knirel, A. S . Shashkov, N. K. Kochetkov, E. S. Stanislavsky, and G. M. Mashilova, Etir. J. Biochem.. 125 (1982) 220-237.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

295

components ofantigens from the serogroup 0 3 train.^' The two former are P-linked and the latter a-linked, and consequently, they all have the same absolute configuration at C- 1.

21

HO

22

2. Glyculosonic Acids Several glyculosonic acids have been identified as components of bacterial polysaccharides. D-fyxo-Hexulosonicacid, as a-D-pyranosyl residues (23), is a component of the extracellular polysaccharide from a Rhodococcus spec i e ~The . ~ LPS ~ from Acinetobacter cafcoaceticus NCTC 10305 contain^^^.^^ D-gfycero-D-talo-octulosonic acid (24). It is isosteric with 3-deoxy-~manno-octulosonic acid (25), which is a constituent of bacterial LPS and links the polysaccharide part to the lipid A region. It seems possible that D-gfycero-D-tafo-octulosonic acid replaces 3-deoxy-~-manno-octulosonic acid in the A . calcoaceticus LPS.

(91) Yu. A. Knirel, N. A. Paramonov, E. V. Vinogradov, A. S.Shashkov, B.A. Dmitriev, N. K. Kochetkov, E. V. Kholodkova, and E. S. Stanislavsky, Eur. J. Biochem., 167 (1987) 549-561. (92) E. V. Vinograqov, Yu. A. Knirel, A. S . Shashkov, S. E. Gorin, T. F. Vustina, V. S. Soyfer, S . E. Esipov, L. V. Lisak, and N. K. Kochetkov, Bioorg. Khim., 14 (1988) 1214- 1223. (93) K. Kawahara, H. Brade, E. T. Rietschel, and U. a h r i n g e r , Eur. J. Biochern.. 163 (1987) 489-495. (94) U. Zlhringer, K. Kawahara, P. Kosma, H. Paulsen, C. Krogrnann, V. Sinnwell, and E. T. Rietschel, Int. Curbohydr. Symp., XIV th, Stockholm ( 1 988), Late Abstract A 13 1.

BENGT LINDBERG

296

OH

23

I

24

OH

I tix

Ho OH

CH,OtI

25

I

OH

A number of 3-deoxyglyculosonicacids have been identified. These substances are acid-labile and are decomposed on hydrolysis with acid under normal conditions, and have therefore often escaped detection in the past. The simplest member of this class, 3-deoxy-~-glycero-pentulosonicacid (26), occurs as terminal groups in95the capsular polysaccharide from Klebsiefla K38.Pyranosidic 3-deoxy-~-threo-hexulosonicacid is a component96 of the Vibrio parahaemolyticiis 0 7 and 0 1 2 LPS. The same acid, as p-pyranosyl groups, is also present in the extracellular polysaccharide from Azo-

tohacter vinel~ndii.~'

26

(95) B. Lindberg, K. Samuelsson, and W. Nimmich, Carbohydr. Res., 30 (1973) 63-70. (96) S. Kondo, U. Zahringer, E. T. Rietschel, and K. Hisatsune, Carbohydr. Res.. 188 (1989) 99 - 104. (97) F. Ferreira, L. Kenne, B. Lindberg, and W. Nimmich, Carbohydr. Rex. in press.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

297

As just mentioned, 3-deoxy-~-manno-octulosonicacid is a component of almost all LPS from Gram-negative bacteria. The two abbreviations concurrently used for this sugar, KDO and dOcla, are not in accord with general practice in carbohydrate chemistry. In the forthcoming IUPAC-IUB Recommendations Kdo, which agrees better with the common three-letter abbreviations used for sugars, will probably be recommended. There has been considerable uncertainty about the anomeric configuration of Kdo in LPS, but it is now established that it is a-linked and pyranosidic,9*as in 25. In some LPS, however, as in that from Aerornonas sulrnonicidu, it is furanosid i ~Kdo . ~ is~also a component of several extracellular polysaccharides. It may be a-pyranosidic, as in that'" from E. coli LP 1092,j?-pyranosidic, as in E. coli K13, K20, and K23 (Ref. loo), or furanosidic, as in E. coli K95 (Ref. 10 1). 3-DeOXy-D-gl~Cer~~D-gUlUCf~nOnUlOSOniC acid, as a-pyranosyl groups, is present in the Klebsiellu K4 capsular polysaccharide.102It has the same stereochemistry as neuraminic acid. Three 3-deoxynonulosonic acids containing amino groups are known. The most abundant of these is ~-amino-~,~-deoxy-~-g~ycero-~-ga~ucfononulosonic acid (neuraminic acid, 27), which occurs in different extracellular polysaccharides. Some of these, like colominic acid103from E. coli K 1, are homopolysaccharides. Neuraminic acid is generally N-acetylated and, as in the animal glycoconjugates, has only been found in the a-pyranosyl form (27). It also occurs in some LPS, for example those from some Rhodobacter species.Io4

OH

OH

27

(98) H. Brade, U. Zahringer, E. T. Rietschel, R. Christian, G. Schulz, and F. M. Unger, Curhohydr. Rex. 134 (1984) 157- 166. (99) D. H. Shaw, M. J. Squires, E. E. Ishiguro, andT. J. Trust, Eur. J. Biochem., 161 (1986) 309-3 13. (100) A. Neszmtlyi, P. Kosma, R. Christian, G. Schulz, and F. M. Unger, Curbohydr.Rex, I39 (1985) 13-22. (101) T. Dengler. B. Jann, and K. Jann, Curbohydr. Rex, 142 (1985) 269-276. (102) Yu. A. Knirel, V. A. Mamontova, N. A. Kocharova, A. S. Shashkov, T. F. Solovtva, and N. K. Kochetkov, Bioorg. Khim., 14 (1988) 1583- 1585. (103) E. J. McGuire and S. B. Binkley, Biochemistry, 3 (1964) 247-251. (104) J. H. Krauss, G. Reuter. J. Schauer, J. Weckesser, and H. Mayer, Arch. Microbiol., 150 (1988) 584-589.

298

BENGT LINDBERG

Two isomeric 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acids have been found in LPS from Pseudumonas and Shigeffaspeciesa9One of these, 5,7-diamino-3,5,7,9-tetradeoxy-~-gfycer~~-mann~non~o~~c acid ("pseudaminic acid") occurs both asp-pyranosyl residues (28), as in the P. aeruginusa 0 10 s e r o g r ~ u pLPS, * ~ ~and as a-pyranosyl residues, as in the LPS from serogroup 0 5 of the same species.IMIt is also a constituent of the Vibrio choferae 0 2 0-antigen. lo' The D-glyceru-L-gafuctoisomer has only been found once, in the LPS from108P. aeruginusa 0 13, and occurs in the D-gfyceru-cY-L-gafactu-pyranosylform (29).

H3:w OH

CO;!H

H2 N

OH NH2

28

29

I

Oh

A 3-deoxyheptulosaricacid has been found in the LPS from10gAcinetobacter calcoaceticus NCTC 10305. Another acid of this class, 3-deoxy-~lyxu-heptulosaric acidl10(30),is a component ofa plant polysaccharide.One 4-deoxyhexulosonic acid, of unknown configuration, is known and is a component of the E. cufi K3 capsular po1ysaccharide.l" Yu. A. Knirel, E. V. Vinogradov, A. S. Shashkov, B. A. Dmitriev,N. K. Kochetkov, E. S. Stanislavsky, and G. M. Mashilova, Eur. J. Biochem.. 157 (1986) 129- 138. Yu. A. Knirel, N. A. Kocharova, A. S. Shashkov, B. A. Dmitriev,N. K. Kochetkov,E. S. Stanislavsky, and G. M. Mashilova, Eur. J. Biochem.. 163 (1987) 639-652. L. Kenne, B. Lindberg, E. Schweda, B. Gustafsson, and T. Holme, Curbohydr. Res.. I80 (1988) 285-294. Yu. A. Knirel, E. V. Vinogradov,A. S.Shashkov. B.A. Dmitriev,N. K. Kochetkov,E. S. Stanislavsky, and G. M. Mashilova, Eur. J. Biochem., 163 (1987) 627-637. H. Brade and E. T. Rietschel, Eur. J. Biochem., 153 (1985) 249-254. T. T. Stevenson, A. G. Darvill, and P. Albersheirn, Curbohydr. RPS.,179 (1988) 269288. T. Dengler, K. Himmelspach, B. Jann, and K. J a m , Curbohydr. Rex, 178 (1988) 191 201.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

299

tc

30

VI. ETHERAND ACETALSUBSTITUENTS In this Section, ether and acetal substituents will be discussed. In some polysaccharides, the terminal “reducing” sugar is glycosidically linked to a non-sugar aglycon, and this will be discussed in a special part. 1. Non-sugar Aglycons

It is often difficult to find and determine the terminals in a polysaccharide, and, for most bacterial polysaccharides, this has not been done. For some, however, it is known that the terminal “reducing” sugar is glycosidically linked to a non-sugar aglycon. The simplest example is an 0-methylated mannan from Mycobacterium smegmatis,”* which is terminated by a methyl a-D-mannopyranoside residue. In some LPS from mycobacteria, for example that from M. phlei,’I3the polysaccharide chain is terminated by a 2-O-c~-~-glucopyranosy1-~-glyceric acid residue (31). In other polysaccharides the “reducing” terminal is linked to 0-1 ofa 2,3-di-O-acylglyceroI(32). One example is a cell-wall LPS from Micrococcus CH,OH I

a - D - Glcp - OCIl I

a - D - Manp -OCH, I

CO,H

CHOCOR

31

CH,OCOR

I

32 ( 1 12) L. S. Hams and G. R. Gray, J. Biol. Chem., 252 (1977) 2470-2477. ( I 13) W. L. Smith and C. E. Ballou. J. Biol. Chem., 248 (1973) 71 18-7125. ( 1 14) D. A. Powell, M. Duckworth, and J. Baddiley, Biochem. J.. 151 (1975) 387-397.

300

BENGT LINDBERG

In the LPS from Termoplasma acidophilum, a mannan chain is terminated by an a-D-glucopyranosyl residue glycosidically linked to a macrocyclic aglycon consisting of two glycerol residues connected by two saturated, Ca isoprenoid diols115(33). This species belongs to the taxon Arachaeobacteria, which in several respects departs from other bacteria. --f

3) - a - D - GlcpOCH,

CH,

I

CH,

I

I

MCO(C117C112CHCH,),(CH,CHCl-I,('H,),OCl-II I

I

H,CO( CH,CH,CHCH,),( CH,CHCHZCH2),0CH I

I

CH,

CH,

I

CH,OH

33

2. Methyl Ethers 0-Methylated sugars are common in Nature, and the methyl groups originate from methionine. As the ether group is resistant to hydrolysis with acid, they are obtained as such in a sugar analysis, and are therefore often considered as component sugars. Some methylated sugars even have trivial names, which seems to be rather unnecessary. In this article, however, they are treated as derivatives of their parent sugars. Whereas methyl ethers of sugars are common in bacterial polysaccharides, there seems to be only one example of an N-methylated sugar, namely, a 3,6-dideoxy-3-(methy1amino)hexose of unknown configuration, which is a component of a Rhizobium LPS.1'6 Several methylated sugars have been identified in hydrolyzates of LPS, cell-wall polysaccharides, and extracellular polysaccharides. A considerable number of these have been found in the LPS from photosynthetic prokaryotes.I3Two polysaccharides from Mycobacterium species, a glucan' l 3 and a mannan l 2 are remarkable in that they contain high percentages of methylated sugars. Glycolipids from Mycobacterium speciesare also rich in methylated sugars, some ofwhich have not been found elsewhere, but this is beyond the scope of the present article.

( 1 15) K. J. Mayberry-Carson, T. A. Langworthy, W. R. Mayberry, and P. F. Smith, Biochim.

Biophys. Acra, 360 (1974) 217-229. ( 1 16) E. M. Hrabak. M. R. Urbano, and F. B. Dazzo, J. Bacteriol., 148 (1981) 697-71 I .

COMPONENTS OF BACTERIAL POLYSACCHARIDES

30 I

The methylated sugars may occur in stoichiometric proportions, and each repeating unit contains the methylated sugar residue. This is the situation in the extracellular polysaccharide elaborated by some strains of Rhizobium japonicum,'I7 which contains terminal 4-0-methyl-~-~-glucopyranosyluronic acid groups. The methylated sugar can also partially replace its parent sugar, as do 3-O-methyl-~-xyloseand L-xylose in the LPS from Pseudomonas maltophilia. '* Another example is the extracellular polysaccharide from Rhizobium strain CB744, in which 70% of the terminal P-D-galactopyranosyl groups are methylated in the 4-p0sition.~'*It is not known whether the two sugars are scrambled in the polysaccharide or if there are two different populations, one with, and the other without, the 0-methyl groups. A third possibility is that the methylated sugar only occurs in the terminal repeating unit, as in'19the LPS from Klebsiellu 0-10, which is terminated by a 3-0-methyl-a-~-rhamnopyranosylgroup. It seems possible that the presence of such a group may stop the elongation of the polysaccharide chain. For most polysaccharidescontaining methylated sugars, however, the structures are unknown or only partially known. The different methylated sugars known as components of bacterial polysaccharides are summarized in Table I. When possible, references to publications in which the methylated sugar is part of a known structure are preferred to references in which the component has merely been identified. References to sugars of undetermined configuration or absolute configuration have been omitted when there is reason to assume that they are identical to better characterized compounds from other sources.

( 1 17) W. F. Dudman, Curbohydr. Res., 66 (1978) 9-23. ( I 18) R. Beyer, L. D. Melton, and L. D. Kennedy, Carbohydr. Res., 122 ( 1 983) 155 - 163. ( 1 19) H. Bjorndal, G. Lindberg, and W. Nimmich, Actu Chem. Scund., 24 (1970) 3414-3415. ( I 20) G. Rosenfelder, 0. Liideritz, and 0. Westphal, Eur. J. Biochem., 44 (1974) 41 1-420. (121) L. D. Kennedy, Carbohydr. Rex. 61 (1978) 217-221. ( 1 22) L. D. Kennedy and R. W. Bailey, Curhohydr. Res., 49 (1976) 45 1-454. (123) P.-E. Jansson, J. Lonngren, G. Widmalm, K. Leontein, K. Slettengren, S. B. Svenson, G. Wrangsell, A. Dell, and P. R. Tiller, Curbohydr. Res., 145 (1985) 59-66. ( 124) A. J. Mort, J.-P. Utille, G. Tom, and A. S. Perlin, Carbohydr.Res., 12 1 ( 1983) 22 I -232. (125) L. D. Kennedy, Curbohydr. Res., 87 (1980) 156-160. ( 126) R. Russa and Z. Lorkiewicz, FEMS Microbiol. Lett., 6 ( 1 979) 7 I -74. ( I26a) S. M. Panasenko, B. Jann, and K. Jam, J. Bucteriol., 17 1 ( 1 989) 1835 - 1890. (127) J. H. Banoub, F. Michon, and F. Cooper, Biochem. Cell. Biol., 63 (1985) 1265- 1267. (128) J . A. Coria, S. Cavaignac, and R. A. Ugalde, J. Bid. Chem., 262 (1987) 10,601 - 10,607. (129) K. Miyano, M. Ishibashi, N. Kunita, Y. Takeda, and T. Miwatani, FEMS Microbiol. Lett., 20 (1983) 225-228.

BENGT LINDBERG

302

TABLEI Methylated Sugars in Bacterial Polysaccharides Parent sugar Arabinose D-Ribose D-XYlOSe

OMe in position

4 6 3 4 6 2 2 3 4 6 2,3 2 2 3 2 3 2,3 3 3 6 3,6 6 6

LPS, Photosynth. LPS, Photosynth. LPS, Myxococcus LPS, Photosynth. LPS, Pseudomonas LPS, Photosynth. LPS, Photosynth. LPS, Mycobacterium EPS, Rhizobium EPS, Rhizobium LPS, Mycobacterium LPS, E. coli LPS, Photosynth. LPS, Photosynth. LPS, Photosynth. LPS, Photosynth. LPS, Photosynth. EPS, Rhizobium EPS, Rhizobium LPS, Photosynth. LPS, Photosynth. LPS, Photosynth. LPS, Klebsiella LPS, Photosynth. EPS, Rhizobium LPS, Photosynth. LPS, Photosynth. LPS, Rhizobium LPS, Photosynth. LPS, Rhizobium LPS, Photosynth. LPS, Mycococcus

4 4 2 2

LPS, Vibrio EPS, Rhizobium EPS, Rhizobium LPS, Vibrio

2,3,4 3 3 4

L-X ylose

Xylose D-GIllCOse

D-Mannose Mannose galactose

D-Rhamnose r-Rhamnose L-FUCOS 6-Deoxy-~-talose Heptose ~-Glucosamine Galactosamine 3-Amino-3,6dideoxyL-glucose D-Glucuronic acid D-Galacturonic acid Ribitol

Source

3 4 2 3

References 13 13 120 13 12 13 13 1 I3 121 122 1 I3 123 13 13 13 13

13 122,124 121 13 13 13 1 I9 13 125 13

13 126 13 126 13 126a 127 1 I7

128 129

a Abbreviations:LPS, lipopolysaccharide;EPS,extracellularpolysaccharide;Photosynth., photosynthetic prokaryote.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

303

3. 1-Carboxyethyl Ethers Some sugar residues in bacterial polysaccharidesare etherified with lactic acid. The biosynthesis of these involves 0-alkylation, by reaction with enolpyruvate phosphate, to an enol ether (34) of pyruvic acid, followed by reduction to the (R) or (S)form of the lactic acid ether (35). The enol ether may also react in a different manner, giving a cyclic acetal(36) of pyruvic acid.

c-Oli

/ \ 34

\

35

/

36

The first known 1-carboxyethylether of a sugar was1302-amino-3-0-[(R)1-carboxyethy1]-2-deoxy-~-ghcoseor muramic acid (37).It is a component

of the polysaccharide moiety ofthe peptidoglycan in the bacterial cell-wall. It is partially replaced by the manno isomer, 2-amino-3-0-[(R)- I -carboxyethyl]-2-deoxy-~-mannose,in the peptidoglycan from Micrococcus lyso-

deikticus. 131

37

(130) R. E. Strange and L. H. Kent, Biochem. J.. 71 (1959) 333-339. ( 13 1 ) 0.Hoshino, U. Zehavi, P. Sinay and R. W. Jeanloz, J. B i d . Chern..247 ( 1972) 38 1 - 390.

304

BENGT LINDBERG

[(I?)- 1 -Carboxyethyl]- glucose occurs in the 0-antigen fromi32Shigella dysenteriae type 3 and also in the extracellular polysaccharide from133Klebsiella type 66. Another extracellular polysaccharide, from Aerococcus viridans var. homari, contains 4-0-[(S)- 1-carboxyethyl]-~-glucose.~~~~~~ The reduction of the intermediate enol ether (34), in the biosynthesis, may thus give rise to either isomer. 4-0-[(S)- 1-Carboxyethyl]- mann nose is a component of some extracellular polysaccharides from Mycobacterium species; for example, that from M. lacticolum. i 3 5 4-0-( 1 -Carboxyethyl)-D-galactose, of unknown absolute configuration of the lactic acid moiety, is a constituent of extracellular polysaccharides from different strains of Buturivibriofibrisolen^.^^^^ Some of these polysaccharides also seem to contain other sugar derivatives of this class. 3-0-[(S)- 1-Carboxyethyll-~-rhamnosehas been isolatedi36from the 0-antigen of Shigella dysenteriae type 5. 4-0-[(S)-1-Carboxyethyll-~-glucuronicacid is a component of the extracellular polysaccharide fromi3' Klebsiella K37. 4. Acetals

Cyclic acetals of pyruvic acid are common in extracellular polysaccharides (compare, for example, Ref. 6). They have also been found in some LPS, namely, those fromi3*Shigella dysenteriae type 6 and E. coli 0-149 (Ref. 139), and in the teichoic acid from Brevibacterium iodinum.I4 The biosynthesis of these acetals has already been discussed. Pyruvic acid is most often linked to the 4- and 6-positions of a hexopyranosyl residue. The absolute configuration at the acetal carbon atom may be

(132) B. A. Dmitriev, V. L. L'vov, and N. K. Kochetkov, Carhohydr. Res.. 56 (1977) 207-209. ( 1 33) P.-E. Jansson, B. Lindberg, J. Lonngren, C. Ortega, and W. Nimmich, Carbohydr. Res., 132 ( 1984) 297 - 305. ( 134) L. Kenne. B. Lindberg, B. Lindqvist, J. Lijnngren, B. Arie, R. B. Brown, and J. E. Stewart, Carbohydr. Rex. 51 (1976) 287-290. (135) N. K. Kochetkov, A. F. Sviridov, K. A. Arifkhodzhaev, 0. S. Chizhov, and A. S. Shashkov, Carbohydr. Rex, 71 (1979) 193-203. (1354 R. J. Stack, T. M. Stein, and R. D. Plattner, Biochem. J. 256 (1988) 769-773. (136) B. A. Dmitriev, L. V. Backinowsky, Yu. A. Knirel, N. K. Kochetkov, and I. L. Hofman, Eur. J . Biochem., 78 (1977) 381-387. (137) B. Lindberg, B. Lindqvist, J. Lonngren, and W. Nimmich, Carhohjdr. Res., 58 (1977) 443-45 I . ( I 38) B. A. Dmitriev, Yu. A. Knirel, E. V. Vinogradov, N. K. Kochetkov, and 1. L. Hofman. Bioorg. Kliitn., 4 (1978) 40-46. (139) A. Adeyeye, P.-E. Jansson. B. Lindberg, S. Abaas, and S. B. Svenson, Carbohydr. Rex, 176 (1988) 231 -236. (140) W. J. Anderton and S. G. Wilkinson, Biochem. J.. 226 (1985) 587-599.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

305

determined by 13C-n.m.r. When the methyl group is equatorial, as in the D-glucopyranosyl(38) or D-galactopyranosyl(39) residue, the carbon signal of this group appears at 6 26-27, and, when it is axial, at 6 17- 18. The methyl group is equatorial in all known natural examples, which means that the configuration at the acetalic carbon atom is (5') for the D-glucopyranosyl, D-mannopyranosyl, and 2-acetamido-2-deoxy-~-glucopyranosyl derivatives, and ( R ) for the D-galactopyranosyl derivatives. The cyclization of the intermediate enol ether (34) thus seems to follow a stereospecific course. 702H

HOPC

38

OH

HO

39

OH

The pyruvic acid may also be linked to vicinal positions. When linked to 0 - 3 and 0-4 of a D-galactopyranosyl residue (40), the dioxolane ring becomes cis-fused. In the limited number of known examples, the absolute configuration at the acetalic carbon atom is141(9,as in 40. There are some examples of trans-fused dioxolane rings, and these are more sensitive to hydrolysis with acid than the others. Thus, pyruvic acid is acetalically linked to 0 - 3 and 0-4 of an a-L-rhamnopyranosyl residue in the Klebsiellu type 72 capsular poly~accharide,'~~ to 0-2 and 0 - 3 of an a-D-galactopyranosyl residue in the Streptococcuspneumoniae type 4 capsular poly~accharide,~~~ and to 0 - 2 and 0 - 3 of a P-D-glucopyranosyluronic acid residue in the Klebsiellu K 1 capsular polysaccharide.144In the extracellular polysaccharide from (141) P. J . Garegg, P.-E. Jansson, B. Lindberg, F. Lindh, J. Lonngren, I. Kvarnstrom, and W. Nimmich, Curbohydr. Res., 78 ( 1 980) I27 - 132. (142) Y. M. Choy and G . G. S. Dutton, Can. J. Chem., 52 (1974) 684-687. (143) P.-E. Jansson. B. Lindberg, and U. Lindquist, Curbohydr. Res., 95 (1981) 73-80. (144) C. Erbing, L. Kenne, B. Lindberg, J. Lonngren, and I. W. Sutherland, Curbohydr. Rex, 50(1976) 115-120.

306

BENGT LINDBERG

40

Klebsiella K12, pyruvic acid is acetalically linked to 0-5 and 0-6 of a p-Dgalactofuranosyl residue.I4’ Pyruvic acid is further acetalically linked to 0-4 and 0-5 of a D-mannitol residue in an unusual type of teichoic acid from Brevibacteriurn iodinurn.L40The absolute configuration at the acetalic carbon atom is ( S ) in the s.pneurnoniae type 4 p o l y s a c ~ h a r i d ebut , ~ ~it~has ~ not yet been determined for the other polymers. In a group of polysaccharides,the M-antigens, elaborated by Enterobacteriaceae species when grown under special conditions, namely, low temperature and high salt concentration, the carbohydrate backbone is the same, but the group acetalically linked to a terminal P-D-galactopyranosyl group differs.146It can be pyruvic acid linked to 0 - 3 and 0-4 or to 0-4 and 0-6, formaldehyde linked to 0 - 4 and 0-6, or acetaldehyde linked to 0 - 3 and 0-4. These are the only observations ofacetals of formaldehyde and acetaldehyde in bacterial polysaccharides, and, as the work was done before the introduction of Fourier-transform n.m.r. spectroscopy, these polymers should be reinvestigated. VII. ACYLGROUPS Acyl groups are common in bacterial polysaccharides. The parent acids are fatty acids, hydroxy acids, and amino acids. The simplest acid, formic acid, has only been found as the amide. The occurrence of 0-formyl groups had been reported, but proved to be incorrect.14’N-Formyl groups have been found in different polysaccharides;for example, in the 0-specific side-chains of the L P P from Yersinia enterocolitica 0:9, which are composed of 4,6dideoxy-4-formamido-~-mannopyranosyl residues. The formyl group can assume two main conformation^,^^^ s-cis (41) and s-trans (42), which are ( 145) M. Beurret, J.-P. Joseleau, M. Vignon, G. G . S. Dutton, and A. V. Savage, Curbohydr.

Rcs., 189 (1989) 247-260. (l45a) C. Jones, Curbohydr. Res.. 198 (1990) 353-357. ( 146) P. J. Garegg, B. Lindberg, T. Onn, and 1. W. Sutherland, Acfa Chem. Scund., 25 (197 1 ) 2 103-2 108. ( 147) A. Dell, G. G. S. Dutton, P.-E. Jansson, B. Lindberg, U. Lindquist,and I. W. Sutherland, Curbohydr. Rex, 122 (1983) 340-343. ( 148) L. Kenne, P. Unger, and T. Wehler, J. Chem. Soc.. Perkin Trans. I , ( 1988) I 183 - 1 186.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

307

present in the ratio of 7 :3.The equilibrium between these, at room temperature, is slow on the n.m.r. time-scale, which complicates the 'H- and 13C-n.m.r.spectra. This effect is not observed for other amides, in which the s-cis form preponderates.

H

42

41

0-Acetyl and N-acetyl groups are very common. 0-Acetyl and other 0-acyl groups on a sugar residue may migrate, unless they are prevented from this by other substituents. If 0-acyl groups are found in two positions in a sugar residue, 0-acylation may, therefore, neverthelesshave occurred in a unique position during the biosynthesis. In the 0-antigen from Yersinia ruckeri, the amino group of a 4-amino-4,6-dideoxyhexoseis diacetylated.149 This seems to be the only example of a sugar containing a diacetamido group in a bacterial polysaccharide. N-Acetimidoyl groups have been found in several LPS from Pseudomonas species. It is generally N-3 of a 2,3-diamino-2,3-dideoxyhexuronic acid residue, as in 43, that cames this group,89which was originally mistaken for an imidazoline grouping. It has also been found linked to 2-amino-2,6-di-

r .,43

deoxy-L-galactose, in the LPS from P. aeruginosalo8and to N-7 of 5,7-diamino-3,5,7,9-tetradeoxy-~-glycero-~-manno-nonulosonic acid, in the LPS fromLo7Vibrio cholerae 0-2. This group is basic, and the first and the last residue just discussed therefore occur as zwitterions. (149) J. H. Banoub, N. A. Nakhlar, andT. R. Patel, Inf.Curbohydr. Symp.. XIVth, Stockholm, (1988), Abstract A 94.

308

BENGT LINDBERG

A lipopolysaccharidefrom Mycobacterium phlei is a D-glucan containing

ester-linked acetic, propanoic, isobutanoic, octanoic, and succinic acid.l13 LPS from M. lepraeand M. tuberculosisare arabinogalactans, esterified with lactic, succinic, palmitic, and 10-methyloctadecanoic acid. ISo Esters of propanoic acid have also been found in other polysaccharides, for example the extracellular polysaccharide from151E. coli K52 and the 0-antigen from Vibrio anguillarum. Half-esters of succinic acid occur in some extracellular polysaccharides from Rhizobium, Alcaligenes, and Agrobacterium spec i e ~ .As ' ~discussed ~ later, capsular polysaccharides may be linked through a phosphoric diester to 0-1 of a 2,3-di-O-a~ylglycerol.~~~~~~~ The acid in this moiety is often hexadecanoic acid. A number of amide- and ester-linked fatty acids and (R)-3-hydroxy acids are components of the lipid A part in the LPS from Gram-negative bacteria. The acids have been tabulatedIs6 and the chemistry of lipid A summaThe most common acids in lipid A from Enterobacteriaceae are the saturated I2 : 0, 14 :0, and 16 :0, and the (R)-3-hydroxy-14 :0, The last is linked to N-2 and 0-3 of the 2-amino-2-deoxy-~-glucopyranosylresidues, and the others are ester-linked to the hydroxy acid, as in the lipid A (44) of Salmonella minnesota. Other linear and branched fatty acids, unsaturated acids, (59-2- and (R)-3-hydroxyacids, and 3-oxotetradecanoic acid are components of lipid A from certain different species. In the lipid A from Rhizobium trifolii, 2,7-dihydroxyoctanoic acid is linked as amide to a 2-amino-2deoxy-D-glucopyranosyl residue.157a In the peptidoglycan from some mycobacteria, the muramic acid is N-glycolylated, not N-a~ety1ated.l~~ As already mentioned, ester-linked lactic acid S. W. Hunter, H. Gaylord, and P. J. Brennan, J. Biol. Chem., 26 1 ( 1986) 12,345- I2,35 I . (151) P. Hofmann, B. Jann, and K. J a m , Eur. J. Biochcm., 147 (1985) 601 -609. (152) J. H. Banoub, F. Michon, and H. J. Hodder, Biochem. Cell. Biol.. 65 (1987) 119- 126. (153) T. Harada, A. Anemura, P.-E. Jansson, and B. Lindberg, Carhohvdr. Rex. 77 (1979) 285-288. (154) E. C. Gotschlich, B. A. Fraser, 0. Nishimura, J. B. Robbins, and T.-Y. Liu, J. Biol. Chem., 256 (1981) 8915-8921. ( 1 5 5 ) M. A. Schmidt, B. Jam. and K. J a m , FEMS Microbiol. Lett.. 14 (1982) 69-74. ( 156) S. G. Wilkinson, in 1. Sutherland (Ed.), Surface Carbohydrates of the Prokaryotic Cell, Academic Press, London, 1977, pp. 97- 175. ( 157) E. T. Rietxhel, H.-W. Wollenweber, H. Brade, U. Zahringer, B. Lindner, U. Seydel, H. Bradaczek, G. Barnickel, H. Labischinski, and P. Giesbrecht, in E. T. Tietxhel (Ed), Handbook QfEndotoxin, Vol. I, Chemistry oj'Endotoxin. Elsevier, Amsterdam, 1984, pp. I87 -220. (157a) R. I. Hollingsworth and D. A. Lill-Elghanian, J. Biol. Chem., 264 (1989) 14,03914,042. (158) 1. Azuma, D. W. Thomas, A. Adam, J.-M. Ghuysen, R. Bonaly, J. F. Petit, and E. Lederer, Biochim. Biophys. Acta, 208 (1970) 444-45 I . ( 150)

COMPONENTS OF BACTERIAL POLYSACCHARIDES

309

c

NH,

is6

HG

Kdo-

HO

?

-.!

t

- OCti,Cti,rIH, 0

44 ( A dotted line indicates partial substitution \

occurs in some arabinogalactans from r n y c ~ b a c t e r i a An . ~ ~extracellular ~ polysaccharide from Pseudomonas eloderu, gellan gum, contains ester groups of L-glyceric acid.ls9 In the cell-wall antigen of Eubacterium suburreuin strain L 13,3-amino-3,6-dideoxy-~-galactose is N-acylated with ~ - g l y ceric acid.160Each residue is glycosidically linked to 0 - 2 in the glyceroyl moiety of the next residue (see 45). The polymer contains alternating glycoside and amide linkages, and thus differs from a conventional polysaccharide. An extracellular polysaccharide from Rhizobium trifolii contains esterlinked (R)-3-hydroxybutanoic acid,161 which was at first assumed to be ether-linked. The same acid is linked as an amide to a 4-amino-4,6-dideoxy-

( 159) M.-S. Kuo, A. J . Mort, and A. Dell, Carhohvdr. Res., I56 ( 1 986) I73 - 187.

(160) P.-E. Jansson. B. Lindberg, M. Spellman, T. Hofstad, and N. Skaug, Curbohydr. Rex, 137 (1985) 197-203. (161) R. 1. Hollingsworth, F. B. Dazzo, and A. J. Mort, J. Bucteriol., 169 (1987) 3369-3371.

310

BENGT LINDBERG HO

CH~OH

45

D-glucopyranosyl residue in the LPS from'62E. coli 010. It is also linked to N-7 of a 5,7-diamino-3,5,7,9-tetradeoxy-~-glycerononulosonic acid residue in the 0-specific side-chain~'~~ of the Pseudomonas aeruginosa 0 10 LPS. In the latter, the next sugar is linked to 0 - 3 of the hydroxybutanoyl moiety (46), and this is a second example of a natural polymer having alternating glycoside and amide linkages. (S)-3-Hydroxybutanoic acid is linked to N-4 of 2,4-diamino-2,4,6-t~deoxy-~-glucose in the LPS72from P. aeruginosa 0 1. A 3,5-dihydroxyhexanoic acid of unknown configuration is linked3' to the same position of this sugar in the LPS from Vibrio cholerae 0 3 . The V. cholerae 01 0-antigen is a homopolysaccharide composed of 4-amino-4,6-dideoxy-~-mannose residues N-acylated with (S)-2,4-dihydroxybutanoic (see 47). There are some examples of ester- and amide-linked amino acids. N-Acetylglycine is linked to N-4 of the 4-amino-4,6-dideoxy-~-glucopyranosyl residue in the 0-specific side-chains of the LPS from163Shigella dysenteriae type 7. Ester- linked D-alanine is common in the teichoic acids.IH L-Alanine is linked as an amide to D-galactosamine in the core part of a LPS from Pseudornonas aeruginosa.165 In the cell-wall antigen from a strain of Staphylococcus uureus, the amino group of a 2-amino-2-deoxy-~-glucosyluronic residue is acylated with N-acetyl-L-alanine.sON-Acetyl-L-serine is linked to N-3 of a 3-amino-3,6-dideoxy-~-glucopyranosyl residue (48) in the O-specific side-chains of the E.coli 01 14 LPS.'% A new amino acid, namely, a (162) L. Kenne, B. Lindberg, C. Lugowski, and S. B. Svenson, Carbohydr. Res., 151 (1986) 349-358. (163) Yu. A. Knirel, V. V. Dashunin. A. S. Shashkov, N.K. Kochetkov, B. A. Dmitriev, and I. L. Hofman, Carbohydr. Res.. 179 (1988) 51 -60. (164) M. Duckworth, in 1. Sutherland (Ed.), Surface Carbohydrates of the Prokaryotic Cell, Academic Press, London, 1977, pp. 177-208. (165) D. T . Drewry, K. C. Symes, G. W. Gray, and S. G. Wilkinson, Biochem. J., 149 (1975) 93- 106. (166) V. L. L'vov, N. V. Tochtamysheva, A. S. Shashkov, B. A. Dmitriev, and K. Capec, Carhohydr. Rex, 112 (1983) 233-239.

COMPONENTS OF BACTERIAL FQLYSACCHARIDES

31 1

C02H

I

I

46

I

HOCH

I CHZ

I

CH2OH

AcHN

47

CH I CH2OH

48

3-hydroxy-2,3-dimethyl-5-oxoproline, is linked to N-3 of the same sugar in the 0-antigen from Pseudomonasfluorescens. 167 VIII. AMIDES

In several polysaccharides containing glycuronic acid residues, the carboxyl groups of these are linked to the amino group of amino compounds, forming amides. In the simplest examples, these are primary amides, such as the 2-formamido- and 2-acetamido-2-deoxy-~-galacturonamide (49) residues in 0-specific polysaccharides from different strains of Pseudomonas (167) G . A. Naberezhnykh, V. A. Khomenko, V. V. Isakov, Y. N. El'kin, T. F. Solov'eva, and Yu. S. Ovodov, Bioorg. Khim., 13 (1987) 1428- 1429.

312

BENGT LINDBERG

aeruginosa. 16* In the 0-antigen from Shigellu boydii type 8,2-amino- 1,3-dihydroxypropane is linked as an amide (50) to a D-galacturonic acid residue.169All the other known linkages ofthis type are to the amino group of an amino acid.

49 HO

,NHCH(CH,OH),

(0-c,

0 50

I

In the cell-wall antigen from Staphylococcus aureiis M, taurine is linked as an amide (51) to a 2-acetamido-2-deoxy-~-galactosyluronic residue.170 LThreonine and L-glutamic acid are linked as amides to D-glucuronic acid residues in the LPS from'71Rhodopseudomonas sphaeroides ATCC 17023 and in the capsular polysac~haride~~~ from Klebsiellu K82, respectively. In the capsular polysacchande from E. coli K54,L-serine and L-threonine, in the ratio 1 : 9, are linked to the carboxyl group of a D-glucuronic acid residue.173In the capsular polysacchande from Haemophilus influenzae type d, ( 1 68) E. V. Vinogradov, Yu.A. Knirel, A. S. Shashkov, and N. K. Kochetkov, Curbohydr. Rex,

170 (1987) cl -c4. (169) V. L. L'vov, N. V. Tochtarnysheva, A. S. Shashkov, B. A. Drnitriev, and N. K. Kochetkov, Bioorg. Khim., 9 (1983) 60-73. (170) S. V. K. N. Murthy, M. A. Melly, T. M. Hams, C . G. Hellerqvist, and J. H. Hash, Curbohydr. Res., 117 (1983) 113-123. (171) P. V. Salirnath, R. N. Tharanathan, J. Weckesser, and H. Mayer, Eur. J. Biochem., 144 ( 1984) 227 - 232. (172) P.-E. Jansson, B. Lindberg, G. Widrnalrn, G. G. S. Dutton, A. V. S. Lim, and I. W. Sutherland, Carbohydr.Rex, 175 (1988) 103- 109. (1 73) P. Hofrnann, B. Jann, and K. Jann, Curbohydr. Res., I39 (1 985) 26 I - 27 I .

COMPONENTS OF BACTERIAL POLYSACCHARIDES HO

I ozc'

313

PJHCH2CH2S03 H

three amino acids, namely, L-alanine, L-serine, and L-threonine, in the ratios of 2 : 2 : 1, are linked to the carboxyl group of a 2-acetamido-2-deoxy-~mannuronic acid residue.'74 The LPS from Pruteus species contain amino acids linked as amides to acidic sugars. Thus, L-lysine is linked by way of N-6 to a D-galacturonic acid residue (52) in the LPS from P. h a ~ s e r i ,but ' ~ ~by way of N-2 to a D - ~ ~ U C U ronic acid residue in the LPS P. mirabilis 027. The latter LPS also contains L-alanine, linked to the carboxyl group of a D-galacturonic acid residue. 10

NH(CH;?),CHNH2COzH

52

IX. PHOSPHORIC ESTERS Several natural polysaccharides are esterified with sulfuric or phosphoric acid. Sulfated bacterial polysaccharides are not, however, very common. One example is a polysaccharide from an Arthrobacter specie^,'^' which is most probably linked to the proteoglycan and contains sulfated D-galactopyranosyl residues. An extracellular polysaccharide from a Phormidium spe-

( 174) P. Branefors-Helander, L. Kenne, B. Lindberg, K. Petersson, and P. Unger, Curbohydr.

RLJS.,97 (1981) 285-291. (175) E. V. Vinogradov. A. S. Shashkov, Yu. A. Knirel, N. K. Kochetkov, E. V. Kholodkova, and E. S. Stanislavski, Bioorg.Khim., 13 (1987) 660-669. (176) E. V. Vinogradov, D. Pietrasik, A. S. Shashkov, Yu. A. Knirel, and N. K. Kochetkov, Bioorg Khim.. 14 (1988) 1282- 1286. (177) K. Inoue, H. Korenaga, and S. Kadoya, J. Biochem. (Tokyo). 92 (1982) 1775- 1784.

BENGT LINDBERG

314

cies is also ~u1fated.l'~ The P-D-mannopyranosyl residues in the disaccharide repeating unit of the extracellular polysaccharide from the marine Pseudornonas No. 32 are sulfated in the 6-po~ition.l~~" Many bacterial polysaccharidescontain phosphoric ester groups. There is a limited number of examples of monoesters. More common are phosphoric diesters, connecting an amino alcohol or an alditol to the polysaccharide chain. Another possibility is that oligosaccharide or oligosaccharide-alditol repeating units are connected to a polymer by phosphoric diester linkages. In addition to the intracellular teichoic acids, several bacteria, for example, different types of Streptococcus pneumoniae, elaborate extracellular polymers of this type. These polymers are generally discussed in connection with the bacterial polysaccharides. Phosphate and pyrophosphate are linked157to the lipid A region of LPS (see 44). A 4-amino-4-deoxy-P-~-arabinopyranosyl group may be linked to the ester-linked phosphoryl group, and ethanolamine to the glycosidic pyrophosphate. Phosphate and ethanolamine phosphate are also linked to the heptose and Kdo regions of the common core in LPS.179Some extracellular polysaccharides, for example, that180from Streptococcus pneumoniae type 17F,are substituted by phosphate, but substitution by phosphoric esters is more common. A number of pneumococcal polysaccharides are thus substituted by choline phosphate,181a group that is immunologically significant. In the Proteus mirabilis 0 3 antigen, N-(2-hydroxyethyl)-~-alanine is linked,

HO

0 II p -OCH,CH,NHCH(CH,)CO,H

/ O 53

(178) Y. Bar-Or and M. Shilo, Appl. Environ. Microbiol., 53 (1987) 2226-2230. (178a) S. Tandavanitj and K. Okutani, Nippon Suisan Gnkkaishi, 55 (1989) 1845-1849; Chem. Abstr., 112 (1990) 51,867b. C. Galanos, 0. Luderitz, E. T. Rietschel, and 0. Westphal, in T. W. Goodwin (Ed.), International Review of Biochemistry, Vol. 14: Biochemistry of Lipids, I f , University Park Press, Baltimore, 1977, pp. 239-335. M. B. Perry, D. R. Bundle, V. Dauost, and D. J. Carlo, Mul. Immunol., 19 (1982) 235-246. U. B. S. Soerensen, R. Agger, J. Bennedsen, and J. Henrichsen,Infecf.Immun.. 43 ( 1 984) 876-878.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

315

through a phosphoric diester linkage, to 0-6 of an a-D-galactopyranosyl residue (53)in the tetrasaccharide repeating unit.18* In some polysaccharides, the “reducing” terminal is linked, through a phosphoric diester linkage, to 0-1 ofa 2,3-di-U-acylglycerol. This structural feature has been demonstrated for some capsular polysaccharides from E. coli and Neisseria s p e c i e ~ , but ~~~ is Jprobably ~~ more common than that. Non-covalent linkage between the lipid part and the cell membrane may explain why extracellular polysaccharides often occur as capsules, and the high (apparent) molecular weight observed for these polysaccharidesmay be due to micelle formation in aqueous solution. Several polysaccharides contain glycerol phosphate substituents. In the cell-wall polysaccharide from Bacillus cereus AHU 1356, sn-glycerol 1phosphate is linked to 0-2 an a-L-rhamnopyranosyl residue (54). When glycerol phosphate derives from CDP-glycerol, an sn-glycerol3-phosphate is expected,Ig4but this glycerol phosphate residue derives from phosphatidyl-glycerol. Different extracellular polysaccharides, such as that from185Slreptococcuspneurnoniae type 1 1A, contain a glycerol 1-phosphate substituent, but the absolute configuration of the glycerol moiety has not been determined. There are also some examples of substitution by glycerol 2-phosphate, as in the capsular polysaccharide from’86S. pneurnoniae type 23F. Ribitol I-phosphate is a substituent in the capsular polysaccharide from18’ S. pneurnoniae type I IF. The absolute configuration of the ribitol phosphate moiety has not been determined, but should be D-nbitol5-phosphate (55) if it is derived from CDP ribit01.l~~ Several of the intracellular teichoic acids are polymers of glycerol phosphate or ribitol phosphate.’” An unusual teichoic acid, composed of Dmannitol phosphate, and with pyruvic acid linked as an acetal to 0-4 and 0-5, has been isolated from Brevibacteriurn iodinurn.lw In different polysaccharides of the teichoic acid type, monosaccharides or oligosaccharides are connected by phosphoric diester linkages. Two examples are the capsular antigens from Neisseriu meningitides*8gtype A (56)and Huernophilus influenzae type clg9(57), respectively. Glycerol phosphate (182) E. V. Vinogradov, W. Kaca, A. S. Shashkov, D. Krajewska-Piestrasik, A. Rozalski, Yu. A. Knirel, and N. K. Kochetkov, Eur. J. Biochm., 188 (1990) 645 -65 I . (183) N. Kojima, Y. Araki, and E. Ito, Eur. J. Biochem., 148 (1985) 479-484. (184) A. R. Archibald and J. Baddiley, Adv. Curbohydr. Chem., 21 (1966) 323-375. ( 185) D. A. Kennedy, J. G. Buchanan, and J. Baddiley, Biochem. J., I 15 (1 969) 37 -45. (186) J. C. Richards and M. B. Perry, Biochem. Cell Biol.. 66 (1988) 758-771. ( I 87) J. C. Richards, M. B. Perry, and P. J. Kniskern, Biochem. Cell Biol., 63 (1985) 953-968. ( 188) D. R. Bundle, I. C. P. Smith, and H. J. Jennings, J. Bid. Chem.,249 (1 974) 2275 -228 1. ( 1 89) P. Branefors-Helander, B. Classon, L. Kenne, and B. Lindberg, Curbohydr. Res., 76 (1979) 197-202.

BENGT LINDBERG

316

p‘\ I O

54

H2C-

\ ’ O

OH

I

HOCH

I

CHpOH

HOCH

I

HOCH I

HO~H

I

CHpOH

55

may, however, also be part of the chain, as in the capsular polysaccharide from190N. meningitidis type Z (see 58). There are many examples of ribitol phosphate as part of the chain, as in the Streptococcuspneumoniaetype19134 (59)and the H. influenzue type a192(60)capsular polysaccharides. In several of these examples the absolute configuration of the ribitol phosphate moiety has been determined, and has always been D-ribitol Sphosphate, in agreement with the presumed biosynthetic route. There are solitary examples of other alditol phosphates as components of this class of polymers. Arabinitol 1-phosphate is part of the S. pneumoniue type 17Fcapsular p01ysaccharide.l~~ D-Glucitol6-phosphate is a component of the group-specific polysaccharide from group B Streptococcus,194 which has a most unusual, ramified structure. In a polysaccharide from Nocurdiu

(190) H. J. Jennings, K.-G. Rosell, and C. P. Kenny, Can. J. Chem.. 57 (1979) 2902-2907. (191) G. J. F. Chittenden, W. K. Roberts, J. G. Buchanan, and J. Baddiley, Biochem. J.. 109 (1968) 597-602. (192) P. Branefors-Helander, C. Erbing, L. Kenne, and B. Lindberg, Curbohydr. Res., 56 (1977) 117-122. (193) M. B. Perry, Personal communication. (194) F. Michon, J.-R. Brisson, A. Dell, D. L. Kasper, and H . J. Jennings, Biochemisfr,v,27 (1988) 5341-5351.

COMPONENTS OF BACTERIAL POLYSACCHARIDES

317

0 II 4 6) - Q - D - MmpNAC -(I - 0 - P - 0 I

OH

56 0 I1

+ 4) -p - D-GlcpNAc - ( 1 + 3)-a - D -Galp-(1 - 0 - P - O I OH

57

+ 3) - a

- D -GalpNAc

-(I

0

+ 1)-glycerol

II - 3 - yl - ( 3 - 0 - P - 0 I

OH

58 + 3) -p

-D

-Gay

- (1

+ 3)-a- D - G a l p - ( 1 + 2)- p-

D-

Gay -(1

--f

3 ) - a - D - Galp - (1 +

0 I1

3) - D-ribitol-(5 -0 - P-OI

OH

59 0 II

+ 4)- p - D-

Glcp -(+4) - D -ribit01 - ( 5 - O - P - 0 -

OH

60

asteroides, the arabinogalactan chain is linked to the peptidoglycan by a 6-deoxy-~-altritolphosphate residue.Ig5

X. CONCLUSIONS An intriguing question which is, of course, as yet impossible to answer, is why the bacteria synthesize so many and so diverse polysaccharide components. A common speculation is that this gives them an advantage in their protection against the bacteriophages. The latter have to develop specific (195) A. Voiland and G. Michel, Can.J. Microbiol., 31 (1985) 1011-1018.

318

BENGT LINDBERG

enzymes which catalyze the hydrolysis of the capsular or cell-wall polysaccharides surrounding the bacteria, or both, before they can invade them. A diversity of structures obviously limits the number of different bacteriophages capable of attacking each type of bacterium, and the bacteriophages are generally type-specific. Some bacterial types may also be converted by phages, resulting in a modification of their polysaccharides. For most of the sugar components, little or nothing is known about their biosynthesis. Nucleoside hexosyl-4-ulose diphosphates are, however, almost certainly key intermediates in the biosynthesis of several of these sugars, as discussed in Ref. 7. The biosynthesis of the 6-deoxyheptoses is probably analogous to that of the 6-deoxyhexoses, and proceeds by way of nucleoside heptosyl-4-ulose diphosphates. Epimerization at C-5of hexuronic acids is a reaction that proceeds both on the polymer and on the sugar nucleotide level. In addition to the three pairs of parent acids, namely, the D-ghcof L-ido-, D-mannofL-gulo-, and D-galuctu/L-ulfro-hexuronic acids, the 2-amino-2-deoxy acids belonging to the last two and the 2,3-diamino-2,3-dideoxy acids belonging to the middle pair have been found. The biosynthesis of Kdo and neuraminic acid is known to involve enolpyruvate phosphate and D-arabinose or 2-acetamido-2-deoxy-~-mannose, respectively. Nothing is known about the biosynthesis of all the other glyculosonic acids. One interesting problem is, for example, whether the two 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acids are synthesized analogously to neuraminic acid, from a three- and a six-carbon fragment, by modification of neuraminic acid on the sugar nucleotide level, or by a third, less obvious route. The number of known sugar components of bacterial polysaccharides at the end of 1988 was approximately 85.The figure refers to the parent sugars, independent of eventual substitution by 0-methyl, 0-(1-carboxyethyl), or other groups. The number of non-sugar components was also considerable, and the different combinations of a sugar and one or several non-sugar components present in bacterial polysaccharides was, of course, very much larger. It may safely be asumed that these figures will increase rapidly, the reason for this being twofold. Firstly, the methods for detecting and identifying new components have been greatly improved, particularly by the introduction of new, 2-D n.m.r.-spectral techniques. Secondly, only a limited number of all the bacterial families and tribes have until now been investigated for their cell-wall or extracellular polysaccharides, or both. The best studied families are Enterobacteriaceae, Pseudomonadaceae, and Streptococcaceae, but here also, there are considerable gaps.

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY. VOL. 48

GLYCOSIDE HYDROLASES: MECHANISTIC INFORMATION FROM STUDIES WITH REVERSIBLE AND IRREVERSIBLE INHIBITORS BY G ~ ~ N T LEGLER ER Institul fur Biochemie, Universitat Koln, 0-5000 Koln I , Federal Republic of Germany 1. Introduction. . . . . . . . . . . .......................................... ....................... I . Non-enzymic Hydrolysis of Glycosides.

....................... 2. Catalytic Efficiency of Glycoside Hydro1 3. Mechanistic Information from X-Ray Crystallography.....................

319

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

325 327

2. Basic Sugar Analogs ...... ....................................... 3. Pseudosubstrates ..............................................

333

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

362

I . Conduritol Epoxides (1, 2. Sugar-related Aziridines

1. Interpretation of Inhibition Results by Mechanistic Features. . . . . . . . . . . . . . . . 378 383 2. Generalizations and Exceptions. .......................................

I. INTRODUCTION Because of its importance to carbohydrate technology, biochemistry, and physical organic chemistry, the hydrolytic cleavage of glycosides has been extensively studied with respect to both acid and enzymic catalysis. Reviews on the acid-catalyzed hydrolysis have been presented by BeMiller,* Capon,z

(1) .I. N. BeMiller, Adv. Carbohydr. Chem.. 22 (1967) 25- 108;Adv. Curbohydr. Chern. Bio-

chem., 25 (1970) 544. (2) B. Capon, Chem. Rev., 69 (1969) 407-498. 319

Copyright 0 1990 by Academic Rey. Inc. All rights of reprodunion in any form rrurvcd.

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GUNTER LEGLER

and S i n n ~ t tGlycosidases .~ have been reviewed by Lalegerie and coworkers4 and, in greater detail, by S i n n ~ t t . ~ Hen-egg lysozyme is still the only enzyme for which detailed mechanistic information is available from X-ray structure analysis with respect to activesite structure and functional groups involved in catalysk6 Data from X-ray crystallography have also been published' for alpha amylase from Aspergillus wentii (Taka-amylase A), where enzyme - substrate interactions have been inferred from an enzyme - maltose complex and from model building. Interestingly,both enzymes are catenases or endo-glycanases,cleaving inner bonds of polysaccharide chains. For all other glycosidases, especially the vast number of exo-hydrolases, we depend on the interpretation of kinetic and inhibition studies with reversible and irreversible inhibitors. In this article are discussed the results ofthose studies which have become available over the past 15 years and which permit some generalizations on the catalytic mechanism of glycoside hydrolases from widely differing sources and with different sugar and aglycon specificities. It will be seen that, with few exceptions, the data support a mechanism almost identical to that proposed by Phillips and his group for lysozyme.6 1. Non-enzymic Hydrolysis of Glycosides

Hydrolysis of glycosides occurs by cleavage of the bond between the anomeric carbon atom and the glycosidic oxygen atom, except for glycosides of teriary alcohols or of aglycons of comparable carbenium ion stability.*The reaction is thus a nucleophilic substitution at C-1 (or C-2 in the case of 2-ketosides). Direct displacement of the aglycon by hydroxide or a water molecule in an S Nreaction ~ is strongly hindered, however, because it would require an inversion at the anomeric carbon atom. This would result in an intermediate of the sugar having an unfavorable skew or boat conformation, making the barrier of the activation energy prohibitively high, even with a proton-assisted departure of the aglycon as a neutral alcohol molecule. Nevertheless, there are enzymes that apparently catalyze glycoside hydrolysis by (3) M. L. Sinnott, in M. 1. Page (Ed.), The C'/2emistryofEnzymeilction, (New Comprehensive Biochemistry, Vol. 6). Elsevier, Amsterdam, 1984, pp. 389-43 I. (4) P. Lalegerie, G. Legler, and J. M. Yon, Biochimie, 64 (1982) 977- 1000. (5) M. L. Sinnott, in M. 1. Page and A. Williams (Eds.), Enzyme Mechanisms. The Royal Society of Chemistry, London, 1987, pp. 259-297. (6) C. C. F. Blake, L. N. Johnson, G. A. Mair, A. T. C. North, D. C. Phillips, and V. R. Sarma, Proc. R. Soc. London. Ser. B, 167 (1967) 378- 385. (7) Y. Matsuura, M. Kusunoki, W. Harada, and M. Kakudo, J. Biochem. (Tokyo). 95 (1984) 697 - 102. (8) T. E. Timell, Can. J. Chem., 42 (1964) 1456- 1472.

32 I

GLYCOSIDE HYDROLASES

a direct displacement reaction (see Section IV,2; Generalizations and Exceptions). A n SN1-like reaction, on the other hand, is much more favorable, because the glycosyl cation intermediate is stabilized by charge distribution between C- 1 and the ring-oxygen atom. The unfavorable formation of an ion pair on bond cleavage can be avoided by protonation of the glycosidic oxygen atom and thus the requirement for acid catalysis (see Scheme 1).

H,O'

kHZ0

SCHEME I . -Acid-catalyzed Hydrolysis of P-Glycoside (Hydroxyl substituents omitted).

Because of the low basicity of the glycosidic oxygen atom (pK, - 10 for aldehydes and pK, - 3.5 for ethers9), the equilibrium concentration of the protonated substrate is extremely small, and the rates of hydrolysis will strongly depend on the electronic properties of the substituents on the pyranose ring. For example, D-glucopyranosides are hydrolyzed at < lo-' times, and 2-deoxy-~-arabino-hexopyranosidesat < l 0-3 times, the rate of tetrahydropyranyl ethers.' Replacement of the hydroxyl group at C-2 by chlorine lowers the rate of acid-catalyzed hydrolysis more than 30-fold.1° As will be shown (see Section 11,3; Pseudosubtrates) the rate of enzyme-catalyzed hydrolysis is affected in a completely different way by the replacement of the 2-hydroxyl group by a hydrogen or a halogen atom. Effects of aglycon structure on hydrolysis rate, on the other hand, are much smaller. Decreased basicity caused by electron-withdrawing substituents is largely compensated for by a better leaving-group propensity and, with enzyme-catalyzed reactions, by specific aglycon interactions. As enzymes work best near neutral pH, where only general acid catalysis is possible, the problem of general vs. specific acid catalysis deserves special ( 9 ) J. March, Advanced Organic Chemistry, 3rd edn., Wiley, New York, 1985, p. 220. (10) E. Buncel and P. R. Bradley, ChnJ. Chem.. 45 (1967) 515-520.

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discussion. Because of the low basicity of glycosides, it is very difficult to detect general acid catalysis, and many studies (reviewed by Fife”) have been made in attempts to find it in non-enzymic systems. These investigations have shown that general acid catalysis is possible only when the acidic group is incorporated in the aglycon in a position that facilitates direct intramolecular proton-transfer to the glycosidic oxygen atom. Base-catalyzed hydrolysis is observed only with glycosides of phenols and enols. The former are cleaved by an intramolecular attack of the deprotonated 2-hydroxyl groups, to give the 1,2-epoxide,which then undergoes further reactions (such as formation of the 1,6-anhydro sugar, hydrolysis, and isomerization).12As phenols and enols are of comparable acidity, it is likely that enol glyc~sides’~ are also hydrolyzed by way of a 1,2-epoxide intermediate when treated with a strong base. A similar participation of the 2-hydroxyl group has also been discussed for the hydrolysis of certain aryl D - ~ u cosides by the P-D-glucosidase from a1rn0nds.l~ A different type of glycoside cleavage induced by strong bases is an elimination of the sugar, a reaction which is initiated by deprotonation of an activated -CHgroup in the position p to the glycosidic bond. Examples are ranunculin (the P-D-glucoside of 4,5-dihydroxy-2-hexenoicacid lactone),I4oligosaccharides having a free reducing-end group (“peeling reaction”),I5 glycosidic links in glycoproteins,16and polysaccharides having alduronic acids as building blocks.” For the latter group, there is also an enzymic counterpart for this type of degradation.l8 Hydrolysis of glycosides without acid or base catalysis is extremely slow, even at high temperatures. Thus, very few data are available for making an estimate of the true catalytic efficiency of D-glycosidases. In order to compare uncatalyzed and enzyme-catalyzed reactions directly, the hydrolysis of several P-D-glucosidesbetween pH 3 and 8 and from 80 to 1 10”(see Table I) has been rneas~red.’~ Only D-glucosides of phenols having pK, =s8 undergo spontaneous hydrolysis. The rate constants for phenyl, 2-(hydroxy( I I ) T. H. Fife, Ace. Chem. Res., 5 (1972) 264-272. (12) C. E. Ballou, Adv. Carbohydr. Chem., 9 (1954) 59-95. (13) C. E. BaIlou and K. P. Link, J. Am. Chem. Soc., 72 (1950) 3147-3152. (14) R. Hill and R. van Heyningen, Biochem. J., 49 (1951) 332-335. (15) R. L. Whistler and J. N. BeMiller,Adv. Carbohydr. Chem., 13 (1958) 289-329. (16) A. Neuberger, A. Gottschalk, R. D. Marshall, and R. G . Spiro, in A. Gottschalk (Ed.), Glycoproteins.2nd edn., Elsevier, Amsterdam, 1972, pp. 450-490. (17) J. Kiss, Adv. Carbohydr. Chem. Biochem., 29 (1974) 229-303. (18) G. Legler, M. L. Sinnott, and S. G . Withers, J. Chem. Soc.. Perkin Trans. 2, (1980) 1376- 1383. ( 19) G . Legler, Striiktur des aktiven Zentrurns glykosidspaltender Enzyme, Forschungsber. Nordrhein-Westfalen, Nr. 2846, Westdeutscher Verlag, Opladen, FRG, 1979.

GLYCOSIDE HYDROLASES

323

TABLE I Rate Constants and Activation Parameters for the Non-enzymic Hydroly~is'~ of &D-Glucopyranosides' at pH 5

AH*

As*

Aglycon

lo6 . k(100") (s-')

lo1* k(25")@-I)

(kJ . mol-I)

4-Nitrophenol 4-Methylumbelliferone D-GIUCOX

0.64

52

118

-32

0.28 0.22

4.7 13

135 123

- 3 -1 1

a

(kJ mol-I)

Extrapolated from data obtained at higher temperatures.

methyl)phenyl, and methyl P-D-glucoside at 105" were c0.02. s-'. At higher temperatures, decomposition reactions prevented accurate measurements. The cleavage of cellobiose is probably due to a degradation from the reducing end, as already mentioned; methyl P-cellobioside was not hydrolyzed. 2. Catalytic Efficiency of Glycoside Hydrolases

The catalytic efficiency of an enzyme is usually expressed by the ratio of its or turnover number (moles ofsubstrate reacted per mol of rate constant (LJ enzyme per unit time) relative to the rate constant of the uncatalyzed reaction. In our case, this amounts to a comparison of the susceptibility of a glycoside molecule towards hydrolysis when it is bound at the active site of the enzyme with that of the glycoside in solution. The data listed in Table I1 TABLE I1 Catalytic Constants and Activation Parameters for the Enzymic Hydrolysis of &~-Glucopyranosides~~

k, K, Aglycon

P-D-Glucosidase A, from Asp. nvntii, pH 4.0, 25" P-D-Ghcosidase A from bitter almonds, pH 5.0, 25"

(s-')

AH*

(mM)

(kJ . mol-I)

4-nitrophenol 4-methylumbelliferone

210 350

0.62 0.57

68.5

D-dUCOX

335

78

0.16 3.3

n.d. 49"

320

1.4

29.5b 5 7" 31 . 9

4-nitrophenol 4-methylumbelliferone D-@UCOSe

8

95

70

n.d.

As* (J mol-'

*

K-')

+ 146 + I42 n.d. + 73 + I05 n.d.

From 5" to26". * From26" to45" Thetwodifferent valuesforAH*arecaused byachangein therate-limitingstepat 26'.

324

GUNTER LEGLER

for a P-glucosidase from Aspergillus wentiP and from bitter almonds19 compared with those in Table I give acceleration factors for aryl P-glucosides from 10l2to > The acceleration factors are even larger, by more than an order of magnitude, for alkyl glucosides and for glucosides of non-acidic phenols, as their uncatalyzed hydrolysis was too slow to be measured. The activation parameters given in Tables I and I1 show that both enzymes accelerate the reaction by lowering the activation energy and by making the entropy of activation positive. The acceleration factors then calculated are by no means exceptional; a perusal of published data on other glycosidases reveals that most of them have turnover numbers of comparable magnitude for “good” or specific substrates, and it may be expected from the results of the acid-catalyzed hydrolysis’ that other glycosides behave similarly to the P-glucosides studied in Refs. 18 and 19. As only a tiny fraction of the substrate is normally present as enzymesubstrate complex, it might be argued that the foregoing comparison of rate constants results in an overestimation of the catalytic efficiency of enzymes. Another mode of expression could be based on a comparison of the enzymecatalyzed rate with that of the acid-catalyzed rate of hydrolysis. An equivalent to Ltwould be the rate constant for the decomposition of the protonated glycoside (slow step of Scheme 1). As there are no data on the protonation equilibrium, this constant cannot be deduced from measured rates of hydrolysis. However, the latter are proportional to the concentration of the acid (more precisely to aH+).We can, therefore, compare enzyme- and acid-catalyzed reactions under conditions where both obey the same rate law, that is, where the rate is of first order with respect to catalyst and substrate concentrations. For the enzymic reaction, this holds for substrate concentration [S] << Michaelis constant K,,, (where the rate constant approaches LJY,with decreasing concentrations of substrate). Extrapolation of the data compiled by BeMiller’ to 25 gives second-order rate-constants for the acid-catalyzed hydrolysis of alkyl and aryl glycosides around 10-8 M-1.s-1 . Reference to the values for LJK,,, in Table 11 shows that glycosidases are able to catalyze the hydrolysis of their substrates up to lOI4 times more efficiently than is the hydronium ion. A l O13-foldincrease in reaction rate requires that the free energy of activation be lowered by 75 kJ/mol. In order to “understand” how this is achieved, we should know as many details as possible about enzyme-substrate interactions and reaction pathway. Numerous weak, but synergistic, non-covalent bonds of active-site functional groups with sugar and aglycon provide a specificity which, in many cases, gives a better than 104-folddiscrimination between closely related isomers, for example, between D-glucosides and D-galactosides. At the same time, the intrinsically weak bonds ensure that enzyme - substrate and enzyme - product complexes form and dissociate

GLYCOSIDE HYDROLASES

325

rapidly. The following features have to be considered as means for lowering the free energy of activationz0:conversion of a bimolecular reaction between catalyst and substrate into a unimolecular one within the enzyme-substrate complex, precise orientation of catalytic groups with respect to the bonds to be broken, multifunctional (synergistic) catalysis by two or more of such groups, formation of reactive intermediates (chemical catalysis), favorable changes of the local dielectric constant by the exclusion of solvent water, and alterations of the ground-state and transition-state energes relative to that of the free, fully solvated molecule. Information on the extent to which the various factors contribute to catalysis by an enzyme can be obtained from studies on the influence of substrate structure on reaction kinetics and by the effects of reversible and irreversible inhibitors. The latter permit the identification of functional groups of the active site in the amino acid sequence and, from considerations of inactivation chemistry, their possible role in catalysis. The interpretation of results with reversible inhibitors is based on the assumption that the active sites of enzymes have evolved to a high complementarity to the transition state in a particular reaction-pathway rather than to the structure of the substrate.21This would stabilize the transition state and destabilize the ground state relative to the conditions of the unbound, fully solvated substrate, thus causing a rate acceleration corresponding to the decrease in the free energy of activation. Interactions of competitive, substrate-related inhibitors which are much stronger than with the substrate or part of the substrate itself are, therefore, considered to reflect features of the active site important for catalysis. The idea is that the energy needed to change the structure of the substrate in the ground to that of the transition state will partly show up as additional binding energy with compounds that resemble the transition state. Differences in the free energies of binding calculated from binding constants of the inhibitor and a reference compound may thus be used, with reservations, to estimate roughly the contribution of a particular structural feature to the rate enhancement by the enzyme.

3. Mechanistic Information from X-Ray Crystallography From crystal-structure analysis of hen-egg lysozyme and of its complex with the competitive inhibitor tn-N-acetylchitotriose, the following conclusions were drawn6:the active site consists of a cleft containing six sub-sites,A to F, of which each could accommodate ap-( 1 4)-linked N-acetylglucosa-

-

(20) M. I . Page in Ref. 3, pp. 1-54. (21) L. Pauling, Nature (London). 161 (1948) 707-710.

326

GUNTER LEGLER

0

-

I

ASP-52

FIG. I.-Hypothetical Transition State for the Cleavage of the Glycosidic Bond of a (GlcNAc), Chito-oligosaccharide Chain at Sub-Site D of the Substrate Binding Cleft of Lysozyme (from Ref. 65, with Permission).

mine unit of a chitopolysaccharide chain. Tri-N-acetylchitotriose occupied sub-sites A to C. Between sub-sites D and E were the only amino acids which could possibly participate in catalysis, namely, Asp-52 and Glu-35. In addition, an N-acetylglucosamineresidue could be accommodated at sub-site D if it was distorted towards a conformation having a planar arrangement of 0 - 5 (the ring-oxygen atom), C- 1, and C-2. Phillips therefore proposed that Glu-35 would act as a general, acid catalyst by protonating the glycosyl oxygen atom, and the carboxylate of Asp-52 would stabilize the glycosyl cation that would remain after departure of the aglycon (see Fig. 1). Formation of the glycosyl-cation intermediate would be aided by the electrostatic stabilization provided by asparate-52 and distortion of the normal chair conformation of the N-acetylglucosamine unit at sub-site D towards the planar geometry of the oxocarbenium ion. Later studies, reviewed by Sinnott? revealed that distortion of a monosaccharide unit bound at sub-site D does not occur, and that non-covalent interactions with the hydroxymethyl group of this unit play a decisive role. Support for this proposal came from kinetic studies with the 3,4-dinitrophenyl glycoside of tetra-N-acetylchitotetraose and the corresponding glycoside having the aglycon linked to a terminal N-acetylxylosamine residue. The xylose-containing substrate was not hydrolyzed by lysozyme. Crystal-structure analysis’ ofTaka amylase A gave similar results, in that it showed that it had an extended cleft which could accommodate six, or possibly seven, a-(1 44)-linked glucose units and two oppositely placed acidic amino acids (Asp-206 and Glu-230) which could interact with the bound substrate similarly to Asp-52 and Glu-35 in lysozyme.

GLYCOSIDE HYDROLASES

327

11. REVERSIBLE INHIBITORS

1. Aldonolactones and 5-Amino-5-deoxylactams

The strong inhibition of glycosidases by aldonolactones was first mentioned in 1940 by Japanese workers who studied /?-D-glucosidases from Aspergillits (Taka-diastase)22and almonds.23These studies were extended ten years later to P-D-glucosiduronase by L , e v ~ yand ~ ~to other glycosidases by Conchie and L e ~ v yThese . ~ ~ authors showed that the aldonic acids themselves are non-inhibitory, and that I ,5-lactones are better inhibitors than their 1,4-isomers (see Ref. 26 for a review). That the aldonolactones might exert their powerful inhibition by virtue of their structural similarity with a glycosyl oxocarbonium ion intermediate or a related transition state was first pointed out by L e a b a ~ kBoth . ~ ~ the lactone and the oxocarbonium ion have a trigonal, planar configuration at C- I, and adopt a half-chair conformation which is in marked contrast with the tetrahedral C-1 configuration and 'C., conformation of aldopyranoside substrates and aldoses (see Scheme 2).

1% AldoPyrdnOSide

Pryanosyl oxocarbenium don

Aldono-l,5-laclone

SCHEME 2. -p-Aldopyranoside tone.

and Glycopranosyl Oxocarbeniurn Ion vs. Aldono- 1,Mac-

(22) S. Ezaki, J. Biochem. (Tokyo),32 (1940) 104- 1 1 1. (23) K. Horikoshi, J. Biochem. (Tokyo), 35 (1942) 39-42. (24) G. A. Levvy, Biochem. J., 52 (1952) 464-412. (25) J. Conchie and G. A. Levvy, Biochem. J., 65 (1957) 389-395. (26) G. A. Levvyand S. M. Snaith,Adv. Enzymol., 36 (1972) 151-181. (27) D. H. Leaback, Biochem. Biophys. Res. Commun., 32 (1968) 1025- 1030.

328

GUNTER LEGLER

In addition to these geometrical factors, there have to be considered electrostatic interactions arising from the large dipole moment of the lactone which would enhance lactone binding if there is a negatively charged group in close proximity to C- I of the bound inhibitor. A new, unorthodox concept to explain the strong inhibition by aldonolactones was advanced by Sinnott and Suchard,28who proposed the reversible addition of an active-site nucleophile X (possibly a carboxylate) to C- 1.

FIG.2.-Possible Formation of an Enzyme-linked Acylal (-X- = Carboxylate) from an Aldono-1,5-lactone at the Active Site of a /P.3ycosidase (after Sinnot and SouchardZ8).

Evidence for this mode of lactone binding consisted in considerations of isotope effects, rate dependence on aglycon acidity, and microscopic reversibility which led to a mechanism for P-D-galactosidasefrom Escherichia coli which includes a galactosyl - enzyme intermediate consisting of a covalent galactosyl -enzyme in equilibrium with an ion pair consisting of oxocarbonium ion and X-. An experimental verification of the Sinnott-Souchard hypothesisz8could be made by a comparison of inhibition constants of aldonolactones and the corresponding lactams (see Table IV) The latter are much less susceptible to nucleophilic additions, and should inhibit less powerfully if a covalent complex is formed as shown in Fig. 2. Studies with P-D-glucosidases from Aspcrgillus wentii18 and almonds29have shown that this is not the case. A quantitative interpretation of aldonolactone inhibition in terms of an adaptation of the active site to a transition state approaching a planar, glycosyl oxocarbonium ion is made difficult for several reasons. Due to the interconversion between the 1,4- and 1,54actones, and their hydrolysis to the aldonic acids, their use is limited to kinetic studies with incubation times of 10 min or less. This was not realized by most investigators prior to 1970. In many cases, only the 1,4-lactonecan be isolated; its (partial) conversion into

(28) M. L. Sinnott and 1. J . L. Souchard, Biochm. J.. 133 (1973) 89-98. (29) M. P. Dale, H. E. Ensley, K. Kern. K. A. R. Sastry, and L. D. Byes, Biochemistry, 24 (1985) 3530-3539.

GLYCOSIDE HYDROLASES

329

the 1,5-lactone is then inferred from the increase in inhibitory power after incubation at slightly acid pH. In these cases, only approximate values for the inhibition constant K, can be obtained from kinetic data unless the relative amounts of the two lactones and the free acid are determined by 3C-n.m.r. spectros~opy.~~ Another point is that many investigators did not give K, values for the corresponding aldose; instead, a comparison of relative affinities was made with the Michaelis constant K, of the substrate. In many cases, this may be misleading, in that the contribution of the lactone structure to the binding energy is greatly underestimated. While K,,, may be similar to K, ofthe aldose in some cases (for example, P-D-ghcosidase A, from Asp. wentii”), it may be more than 1000 times smaller in others (for example, cytosolic p-D-glucosidase from calf liver,’). A 1000-fold difference may seem exceptional, but values for K, (aldose)/K, (aryl glycoside) from 20 to 100 are quite common. In these cases, enzyme - substrate interactions with the aglycon site not available to the aldose or lactone make a large contribution to the binding energy. There is a general agreement that the aldono- 1,5-lactonesare better inhibitors for glycosidases than are the 1,4-i~omers,~~ but few accurate data are available on differences. ff -D-Mannosidasefrom rat epididymis was found to be inhibited by ~-mannono-1,5-and -1,4-lactones, with K, 0.071 and 32 mM, re~pectively.~~ The latter value is in the range expected for inhibition by appropriate polyols, and the question is, if the 1 ,4-isomers show any specific binding by virtue of their lactone structure. In cases where the 1,4-lactone shows appreciable inhibition, for example, 2-acetamido-2-deoxy-~-galactonolactone with P-D-glucosaminidase from pig e p i d i d y m i ~or~ ~D-galactono- 1,4-lactonewith P-D-galactosidase from Escherkhia c~li,~O it may well be that the inhibition observed is caused by a small proportion of the 1 3 isomer rapidly formed in solution. A strong inhibition by 1,4-lactones having the D-gahcto configuration is difficult to rationalize, because in this case, the 1,5- to 1,4-isomerization produces an isomer having an inverted sidechain configuration, a structural alteration which probably cannot be accommodated by the glycon binding-site of the enzyme. The question of a correlation of anomeric specificity of glycosidases with their susceptibility to aldonolactone inhibition was addressed by Reese and coworkers34in a comparative study with six fungal a-D-ghcosidases and

’

(30) R. E. Huber and R. L. Brockbank, Biochemistry, 26 (1987) 1526- 1531. (31) G . Legler and E. Bieberich, Arch. Eiochem. Eiophys.. 260 (1988) 437-442. (32) G. A. Levvy, A. J . Hay, and J. Conchie, Eiochem. J., 91 (1964) 378-384. (33) J. Conchie, A. J. Hay, 1. Strachan, and G. A. Levvy, Eiochem. J., 102 (1967) 929-941. (34) E. T. Reese, F. W. Pamsh, and M. Ettlinger, Carbohydr. Res.. 18 (1971) 381-388.

330

GUNTER LEGLER

exo-a-glucanases and five p-D-glucosidases. It was found that the a-specific enzymes were inhibited by D-gluconolactone at least 100-fold less potently than the p-specific ones. This extended earlier observations with ( Y - D - ~ ~ U C O siduronase and N-acetyl-a-D-glucosaminidaseZ6 that aldonolactones are poor inhibitors for a-D-glycosidases. However, this rule is not without exceptions, as shown by the data for a-~-mannosidases.’~.~~ No exceptions seem to be known for P-D-glycosidases which are inhibited by aldono- 1 3 lactones several hundred- to many thousand-fold better than by the corresponding aldoses. A representative list for different enzymes from widely differing organisms is given in Table 111. No reliable data are available for galactosidases, because the concentration of galactono- 1,5-lactone in the assay is difficult to estirnate.’O The effect of lactone ring-size on the inhibition was studied, for N-acetylp-D-glucosaminidase from bovine epididymis, with lactones and lactone derivatives unable to undergo ring-isomenzation, by Pokorny and coworkers.“ From a comparison of K, values for 2-acetamido-2-deoxy-~-glucono-1,5-lactone (0.45 ph4) with the 1,4-lactone (4.5p M ) and of K, for the methylp-furanoside with that for the pyranose (4 mM), it was concluded that the 1,44actone has an - 10-fold lower inhibitory potency than the 1J-lactone. The weak inhibition by the 5,6-O-isopropylidene derivative of the 1,4-lactone (1,500 ph4) in comparison with K, for the 4,6-O-isopropylidene1,5-lactone ( 1.5 ,uM) is probably due to steric effects of the different acetal structures. The problems regarding ring size and stability that are encountered with aldonolactones disappear when the ring-oxygen atom is replaced by an NH group. The resulting 5-amino-5-deoxyglyconolactams constitute a new group of inhibitors, closely related to the lactones, which can be obtained by

(35) A. G. Day and S. G. Withers, Biochem. Cell. Biol., 64 (1986) 914-922. (36) L. B. Daniels, P. J. Coyle, Y.-B. Chiao, R. H. Glew, and R. S. Labow, J. Biol. Chem.. 256 (1981) 13,004-13,013. (37) A. Cogoli and G . Semenza, J. Biol.Chem., 250 (1975) 7802-7809. (38) T. Sukeno, A. Tarentino, T. Plummer, Jr., and F. Maley, Bitxhemisfry, I 1 (1972) 14931500. (39) Y.-T. Li, J. Biol. Chem., 242 (1967) 5474-5480. (40) W. Einhoff and H. Riidiger, Biol. Chem. Hoppe-Seyler, 369 (1988) 165- 169. (41) C.-C. Wang and 0. Touster, J. Biol. Chem., 247 (1972) 2650-2656. (42) S.-C. Li and Y.-T. Li, J. Biol. Chem.. 245 (1970) 5153-5160. (42a) K.Sandhoffand W. Wbsle, Hoppe-Seyler’sZ. Physiol. Chem.. 352(1971) 1119-1133. (42b) E. W a r , J. E. Cabezas, and P. Calvo, Biochirnie. 66 ( 1984) 29 I - 304. (43) I. I. Secemski and G. E. Lienhard, J. Biol. Chem., 249 (1974) 2932-2937. (44) N. Pokorny, E. Zissis, H. G. Fletcher, Jr., and N. Pravdk, Curbohydr. Res.. 37 (1974) 32 I - 329.

GLYCOSIDE HYDROLASES

33 1

TABLE Ill Inhibition of Glycosidases by Aldono-1.5-lactones and Aldohexoses Expressed by the Dissociation Constant K,of the Enzyme- Inhibitor Complex K, (Lactone)

(W

Enzyme (Source)

P-D-Glucosidase A lcaligenes faecalis Aspergillus wentii Sweet almonds, A B Human liver, cytosolic a-D-Ghcosidase Limpet Rabbit, intestinal sucrase P-D-Mannosidase (Hen oviduct) a-D-Mannosidase jack bean Rat, epididymis b-D-Glucosiduronase (Rat liver) N-Acet yl-P-D-glucosaminidase jack bean pig, epididymis human liver slug, Arion rufus Lysozyme (hen egg)

0.00 17 0.0095 0.20 0.036 0.0 I5 26 10

K, (Hexose) (W 6.4 2.8 189 80 55"

5' 19

& (Hexose) K, (Lactone)

References

3,700 290 950 2,200 3,700

35 18 29 19 31,36

-

32 37

1.9

0.0 I7

2.2

130

3

0.12 0.07 I

22 12d

180 170

39,40 32

> 3,300

41

15,000

42 33 42a 42b 43

0.000 I7 0.0008 0.0005 0.0001 0.005 0.0000838

>0.56' 12 21 5 3.6 0.01~

>4,000

50,000 700 I20

a K, with bovine enzyme which is very similar to the human Inhibitor concentration for 50% inhibition. K,,, of substrate (phenyl a-Dmannopyranoside). K, of substrate (4-nitrophenyl a-Dmannopyranoside). K,,, of substrate (4-nitrophenyl Po-glucosiduronic acid). 'K, of substrate (4-nitrophenyl 2-acetamido-2deoxy-P-o-glucoside).8 Inhibitors were chitotetraono- I ,S-lactone and chitotetraose, respectively.

oxidation of 5-amino-5-deoxyhexopyranoseswith iodine.45Like their oxygen analogs, they have a trigonal, planar configuration at C-1; the dipole moment of the carbonyl group is expected to be even larger than that of the lactone carbonyl, due to the larger contribution of the dipolar resonance structure. Differences in their interaction with glycosidases may arise if substrates and lactones are bound with the ring-oxygen atom in a closely fitting cleft of the active site. The NH group may then cause steric repulsion, or a hydrogen-bond donor for the ring-oxygen atom may fail to interact (45) S. Inouye, T. Tsuruoka, T. Ito, and T. Niida, Tetrahedron, 23 (1968) 2125-2144.

332

GUNTER LEGLER

properly with the NH group because of its amide resonance. The strong inhibition observed with the lactams tested so far (see Table IV) shows that these effects are of minor importance. In cases where the inhibition constants for lactams and lactones can be compared (a-and p-D-glucosidases, and N-acetyl-P-D-glucosaminidases), they have the same order of magnitude. This also holds for P-D-galactosidases if inhibition by 5-amino-5-deoxy-~galactonolactam and by D-galactose is compared. The weak inhibition of a-specific enzymes by lactams extends and confirms the rule of Reese and coworkers34cited already about the inhibition by aldonolactones, of glycosidases ofdifferent anomeric specificity. The reason for this may be seen in the greater structural similarity of lactones and lactams (orientation of the C=O dipole) with the orientation of the anomeric oxygen atom ofp-glycosides than of a-glycosides. If Pauling's hypothesis2' is followed about an evolution of enzyme-active sites towards complementarity to the transition TABLE IV Inhibition of Glycosidases by 5-Amino-5-deoxyaldonolactamsand Aldohexoses, as Expressed by the Dissociation Constant K, of the Enzyme - Inhibitor Complex Enzyme (Source) P-D-Ghcosidase Aspergillus wenlii Sweet almonds, B cu-D-Ghcosidase Rabbit intestinal sucrase P-D-Galactosidase Escherichiu coli Aspergillus "enfii b-Galactocerebrosidase Pig brain Cr-D-Galactosidase Escherichiu coli Coffee beans (I-D-Ghosiduronase Bovine liver

K, (Lsctam)

K, (Hexose)

h (Hexose)

(mM)

(mM)

K, (Lactam)

0.036 0.037 23

2.8 190 19

0.07 0.0037

34 I .3

0.0025

26

4 0.4

50 0.18

0.000039

78 5.100 0.8

References

18 29 46

480 350

47 47

I0,000

48

12

47 47

0.45

49

(46) G. Hanozet, H. P. Pircher, P. Vanni, B.Oesch, andG. Semenza,J. B i d . Chem.,256 (1981) 3703-371 I . (47) G. Legler and F. Kastenholz, unpublished data. (48) G. Legler, F. Kastenholz, and S. Pohl, unpublished data. (49) T. Niwa, T. Tsuruoka, S . Inouye, Y. Naito, T. Koeda, and T. Niida, J. Biorhem. (Tokyo). 72 (1972)207-211.

GLYCOSIDE HYDROLASES

333

state, this would indicate a considerable resemblance of the transition-state structure with that of the substrate. The results of inhibition studies with aldonolactones and 5-amino-5deoxyaldonolactams may be summarized as follows: P-D-glycosidases are inhibited by 1,5-lactonesand the lactams some 100- to > 10,000-foldbetter than by the parent aldoses, with K, values from 200 p M to
a. G1ycosylamines.-The first report on sugar analogs bearing a basic group which are strong inhibitors for glycosidases was by Lai and A x e l r ~ d . ~ ~ These authors found that glycosylamines derived from D-glucose, D-galactose, and D-mannose are bound by a- and P-glycoside hydrolases of correct glycon specificity with K, values ranging from 0.002 to 0.22 mM, that is, up to 1000-fold better than the corresponding aldoses. Extension of these studies by LeglerS1and Legler and coworker^'^^^^ showed that N-substituents increase the inhibitory potency of D - ~ ~ U C Oand D-galacto-sylamine, provided that they do not impair the basicity of the amino group. This is illustrated by the data listed in Table V for P-D-ghcosidases from almondsS1 and Asp. wentii.Is Similar results were obtained with P-D-galactosidase from E. c0l1.~*It was found that the amino group on C-1 gives an enhancement of the inhibition, over that by the aldose, ranging from 220-fold to several thousandfold. N-Alkylation permits additional interactions with the aglycon binding-site of the enzymes. In several cases, this site appears to be quite hydrophobic (for example, P-D-glucosidase from almonds,51and P-D-galac-

(50) H.-Y. L. Lai and B. Axelrod, Biochem. Biophys. Res. Cornmim., 54 (1973) 463-468. (51) G. Legler, Biochim. Biophja. Acta, 524 (1978) 94- 101.

GUNTER LEGLER

334

TABLE V Inhibition of BD-Ghcosidase A, from Aspergillus w e n f i P and /3-D-Glucosidase B (Ref. 51) from Bitter Almonds by Basic and Non-Basic Derivatives of D-Glucose Asp. wentii

Bitter almonds

Inhibitor

I(I (mM)

I(I (mM)

P-D-GlUCOSe

2.8

P-D-Glucosylamine P-D-Glucosylmethylamine 2-Amino-2deoxy-~-glucose N-(Bromoacetyl)-fi-D-~ucosylamine N-Benzyl-P-D-glucosylamine

0.00 16 0.14 18 0.26

N-P-D-Glucosyl-ptoluidine Benzyl I-thio-P-D-glucopyranoside N-P-D-Glucosylpipendine N-P-D-Glucosylpyndinium ion P-D-GhCOSylbenZeIIe I -P-D-Glucosylimidazole

0.09 I 0.3 96 5.9

450

2.0

-

0

60 0.00026 54 1.46

0.0035 40b 49 500

’

* -, not determined. For &D-@UCOSidaSe from sweet almonds.

tosidase from E. even though the natural substrates of these enzymes are probably the disaccharides amygdalin and lactose, respectively. N-Arylation or N-acylation, on the other hand, greatly lowers the inhibitory potency, as shown by the K, values for N-p-D-glucosyl-ptoluidineand N-(bromoacetyl)-~-D-glucosylamine. As this type of substitution lowers or abolishes the ability of the amino group to take up a proton, it may be postulated that the strong inhibition by glycosylamines is caused by the formation of an ion pair consisting of the protonated inhibitor and a negatively charged group of the active site in close proximity to the anomeric carbon atom. The weak inhibition by I -P-D-glucosylimidazole,which is of similar basicity to the N-alkyl-p-D-glucosylamines,is probably caused by the delocalization of the positive charge of its protonated form, which makes electrostatic interactions with a negatively charged group less effective, all the more so as the center of the positive charge is at a slightly greater distance from C- 1. The necessity of a direct attachment of the basic group, and thus of the positive charge, to the anomeric carbon atom is also demonstrated by the weak inhibition shown by 2-amino-2-deoxy-~-glucoseand -galactose54and p-D(52) G. Legler and M. Herrchen, Curbohydr. Rex, 116 (1983) 95- 103. (53) C. K. De Bruyneand M. Yde, Curbohydr. Rex, 56 (1977) 153-164. (54) R. E. Huber and M. T. Gaunt, Can. J. Biochem., 60 (1982) 608-612.

335

GLYCOSIDE HYDROLASES

glucosylmethylamine. Similar results were obtained with C-P-D-~~UCOSYImethylamine and bovine l y s ~ s o m aand l ~ ~cytosolic P-~-glucosidase~’. This strict requirement for a correct position of the amino group indicates a greatly restricted mobility of the bound inhibitor, the groups responsible for the strong inhibition by glycosylamines, or both types. The close alignment of the cationic group of the protonated inhibitor with an anionic group of the active site does not seem possible if the amino group is removed from the anomeric carbon atom by as little as a single bond. A detailed interpretation of inhibition data with glycosylamines is complicated by their susceptibility to spontaneous hydrolysis (half-life of Nalkylgalacto~ylamines,~~ 20 min at 25 o and pH 7) and by a rapid interconversion between the a and P a n o m e r ~The . ~ ~latter probably does not cause serious errors with unsubstituted glycosylamines. In cases where the anomeric specificity has been studied with hexoses (for example, P-D-glucosidase from almonds29and P-D-galactosidase from Asp. wentii”) both anomen were found to be bound with similar affinityand comparable relations with glycosylamines may be expected. Binding of their N-substituted derivatives may, however, depend strongly on the (generally unknown) anomeric composition, as interactions with the aglycon binding-site are involved. Another point to be considered is the dependence of the inhibitory strength on the pH, which will affect the ionization states both of inhitors and functional groups at the active site. This aspect will be discussed in the next Section.

-

b. Cyclic Sugar Analogs Having an Imino Group in the Ring. -In 1970, it was reported that nojirimycin, identified as 5-amino-5-deoxy-~-glucopyranose (l),is a powerful inhibitor of P-~-glucosidases.~~ It had been discovered by virtue of its antibiotic activity in culture filtrates of certain strains of CH,OH

CH,OH

H

HO

1

HO

2

( 5 5 ) H. Liedtke, Thesis, University of Cologne (1987). (56) G. P. Ellis and J. Honeyman, Adv. Curbnhydr. Chern., 10 (1955) 95- 168. (57) G. Coenen, Thesis (Diplom Biology), University of Cologne, (1980). (58) T. Niwa, S. Inouye, T. Tsuruoka, Y. Koaze, and T. Niida, Agric. B i d . Chem., 34 (1970) 966-972.

336

GUNTER LEGLER

S~reptomyces;~~ its I -deoxy derivative (2), a strong inhibitor Of a-D-glUCOSidases, is produced by certain strains of B a c i l l ~ sThe . ~ chemistry of this class of sugar derivatives has been discussed by Paulsen and Todt.61Subsequent studies with 5-amino-5-deoxyhexopyranosesand I ,5-dideoxy-1,5-iminohexitols related to D-mannose,62~ - g a l a c t o s eand , ~ ~2-acetamido-2-deoxy-~glucosea showed that the strong inhibition of glycosidases by these compounds is a general phenomenon probably based on common mechanistic features. In contrast with glycosylamines, the sugar analogs with nitrogen in the ring have much greater stability in aqueous solution. No spontaneous decomposition reactions are known for the 1,5-iminohexitols. The half-life at 25 and pH 5 of the D-manno analog of nojirimycin is 62 2 100 h and, for the 2-acetamido-2-deoxy analogMit is 50 h. The large inhibitory potential of these basic sugar derivatives, and their stability, have made them valuable tools not only for mechanistic studies but also for the investigation of problems in cell biology where glycosidases are involved, for example, in glycoprotein r e s e a r ~ h . ~ ~ - ~ ' Results of inhibition studies with nojirimycin and its analogs published up to 1988, and additional data from the author's laboratory, are summarized in Table VI. It should be noted that the inhibition constants are given in/M instead of mM (as in Tables I1 - V). Data for glycosylaminesare included, in order to facilitate an estimation of the effects caused by the different positions of the basic group in the two types of basic sugar derivative. Not included are the data of Reese and coworkers34and of Grover and Cushley'j8 on nojirimycin, because these authors were apparently unaware of the slow and only partial dissociation of the nojirimycin hydrogensulfite adducP9 which had been used instead of free nojirimycin. The data in Table VI demonstrate that, for most enzymes, 1,5-iminoalditols have an inhibitory potency similar to or even larger than that with

(59) S. Inouye, T. Tsuruoka, and T. Niida, J . Antibiof., 19 (1966) 288-291. (60) D. D. Schmidt, W. Frommer, L. Miiller, and E. Truscheit, Nururwissenschufren,66 (1 979) 584-585. (61) H. Paulsen and K. Todt, Adv. Carbohydr. Chem.. 23 (1968) 116-232. (62) G. Legler and E. Jiilich, Curhhydr. Res., 128 (1984) 61 -72. (63) G. Legler and S. Pohl, Curbohydr. Res., 155 (1986) 119- 129. (64) E. Kappes and G. Legler, J . Curbohydr. Chem., 8 (1989) 371 -388. (65) G. Legler. Pure Appl. Cllem., 59 (1987) 1457- 1464. (66) U. Fuhrmann, E. Bause, and H. Plough, Biochim. Biophys. .4ctu, 825 (1985) 95- 110. (67) A. Elbein, Annu. Rev Biochem., 56 (1987) 497-534. (68) A. K. Grover and R. J. Cushley, Biochim. Biophys. Acta. 482 (1977) 109- 124. (69) G. Legler and W. Becher, Curbohydr. Res., 101 (1982) 326-329. (70) G. Legler, R. Riitter, and E. Jiilich, unpublished results. (7 I ) U. Petzold, Thesis, University of Cologne, (1984).

TABLE VI Inhibition of a- and /l-Glycosidases by the Respective 5-Amino-S-deoxyhexopyranoses,1,5Dideoxy-1,S-iminohexitols, and Glycosylamines

Enzyme and source a-u-Glucosidases Yeast Rice Rabbit intestine (sucrase) &D-Glucosidases Aspergillirs uvntii Sweet almonds Calf liver (cytosol) Calf spleen (lysosomes) He1i.u pomu fin a-D-Galactosidases Coffee beans Eschcrichia coli &D-Galactosidases Escherichiu coli Aspergillus went ii Pig brain (galactocerebrosidase) a-u-Mannosidases Jack beans Sweet almonds Calf liver I ysosomes endoplasmic reticulum &u-Mannosidase Aspergillits wentii Goat liver isoenzyme A isoenzyme B a-L-Fucosidase Bovine epididymis PD-Glucosiduronase Human liver N-Acety l-/l-D-glucosaminidases Aspergillits niger Jack beans Helix pontutia Bovine kidney Human liver, isoenzyme A N- Acetylneuraminidase Vihrio cholerae a

5-Amino-5-deoxv1.5-Dideoxvhexopyranose 1 ,S-iminohexitol 6.3 0.015 0.13" 0.36" 0.89" 4.5 1.1" 0.0007" 0.17" 0.045"

0.0 I 1"

Glycosylamine

12.6 0.01" 0.032" 2.7" 47 210

I80 60

0.00 16" 0.24 12.5 0.16"

32 -b

-

References 46 46 45

1.7 310 240 65 -

18,70 29,5 1,7 1 31 55,72 73

7.5 80

63

7 13.4

63 63

63

I .5

125

-

74

I .2" 21

68 I10

-

62 62

4.4

83 7

-

62 75

4,600

-

62

> 10,000

-

-

76 76

-

77

-

78

1,400 12 34 43 -

64 64 64 64 64

-

79

-

7.7

-

-

300 0.0048

-

80

I .2 0.00 12" 0.5" 0.003" -

I ,OOo 0.14 80 0.6 1.o

-

12Q

Slow approach to the inhibition equilibrium. -, not determined. Inhibitor: HO

338

GUNTER LEGLER

glycosylamines, and that 5-amino-5-deoxyaldopyranosesare even better inhibitors. It remains to be established the extent to which the poor inhibition of P-D-mannosidase from Asp. wentii and the N-acetylglucosaminidase from Asp. niger by the respective 1,5-iminohexitolscan be traced to specific features of their active sites. Irrespective of individual variations, it can be stated that sugar derivatives having an imino group in the ring inhibit glycosidases from several hundredfold to more than ten thousandfold better than their oxygen analogs. This means that the electrostatic interactions of protonated inhibitor and negatively charged group of the active site discussed for glycosylamines is not impaired by the different position of the positive center in the N-cyclic inhibitors. The formation of an ion pair consisting of the protonated inhibitor and an anionic group at the active site can take place in two ways: (i) the enzyme binds the neutral form of the inhibitor, which then gets protonated, probably by the same group which protonates the glycosidic oxygen atom during substrate hydrolysis; (ii) the enzyme binds the inhibitor cation, which then forms an ion pair with a carboxylate of the active site. Both modes show a similar increase of inhibitory strength with pH, because, with increasing pH, an increasing proportion ofthe inhibitor will be present in the unprotonated form and ionization of an active-site carboxylic group will increase the proportion of the carboxylate responsible for the tight binding of cationic glycosyl derivatives. As there is a considerable overlap of pK, values for protonated inhibitors (pK, 5.3 to 6.1 forglu~osylamines,~~ pK, 5.1 to 5.6 for 5-amino-5-deoxyhexopyranoses,5*~6z~63 and pK, 6.3 to 7.2 for 1,5-dideoxy1,5-iminohexitols60~6z~63) and for carboxylic groups (pK, 4 to 6), it is not possible to discriminate between the two modes by pH studies with basic inhibitors alone. A straightforward discrimination between the two pathways is possible with enzymes that are inhibited by basic glycosyl derivatives as well as by permanently cationic ones. This is illustrated by P-D-glucosidase A, from Asp. wentii, where K, values for P-D-glucosylpyridinium ion, P-D-glucosyla(72) K. Osiecki-Newman, D. Fabgro, G. Legler, R. J. Desnick, and G . A. Grabowski, Biochim. B / ~ p h yA~c .~ u915 , (1987) 87- 100. (73) R. Donsimoni, G . Legler. R. Bourbouze, and P. Lalkgerie, Enzyme, 39 (1988) 78-89. (74) S. Pohl, Thesis, University of Cologne, (1988). (75) J. Schweden, G . Legler, and E. Bause, Eur. J. Biochem., 157 (1986) 563-570. (76) K. T. Cavanagh, R. A. Fisher, G. Legler, M. Herrchen, M. Z. Jones, E. Julich, R. P. Sewell-Alger,M. L. Sinnott, and F. E. Wikinson, Enzyme, 34 (1985) 75-82. (77) G . W. Fleet, A. N . Shaw, S. V. Evans, and L. E. Fellows, J. Chem. Soc., Chem. Comrnun., (1985) 841-842. (78) I. Cenci diBello, P. Dorling, L. Fellows, and B. Winchester, FEBS Leu., 176 ( 1 984) 6 1 ~ 6 4 . (79) F. Bamberger, A. Vasella, and R. Schauer, Helv. Chim. Actir. 71 (1988) 429-445.

GLYCOSIDE HYDROLASES

339

Y-

Y-

Q

Q

8t

t5 4

7

3

6 4

5

6

7

PH

FIG.3. -pH-Dependence ofthe Inhibition ofp-D-Glucosidase A, from Aspergillus wentii by P-D-GlucosylpyridiniumIon (-*-- , left ordinate) and the Protonated Form of D-Glucosylamine (-*-@-*, right, calc. with pK, 5.6) and Nojirimycin (----A--, right, calc. with pK, 5.3) (from Ref. 65, with Permission).

mine, and nojirimycin have the same pH dependence, provided that the concentration of the last two is based on the concentration of the protonated form.ls The most plausible explanation in this case is that the enzyme binds the inhibitor cation and the pH-dependence reflects the ionization of a carboxylic group having pK, 5.6 which provides strong binding only when it is present as carboxylate. Binding of the basic form of the inhibitor and subsequent protonation by an acidic group of the active site probably take place with /I-D-glucosidase from almondsS1and P-D-galactosidase from E. coli,52 because here the /I-glycosylpyridinium ions are bound no better than their uncharged analogs, the P-glycosylbenzenes. In cases where such isosteric pairs of cationic and neutral inhibitors as glycosylpyridinium ion and glycosylbenzene are not available, a permanently cationic derivative can be prepared by N-permethylation of a basic sugar analog. Interpretation of inhibition results presents no problems if the quaternary ammonium ion is bound with affinity similar to that of the parent compound. This was observed with an a-D-glucosidase of the endoplasmic reticulum involved in glycoprotein biosynthesis (glucosidase I) which is inhibited by 1-deoxy-N,N-dimethylnojirimycin, with K, 0.4 pM, whereas the monomethyl derivative inhibited with K, 0.07 p M and l-deoxynojirimycinsOwith K, 1pM. Similar results were obtained with lysosomal /I-D-glucosidasefrom calf spleenSSand human placenta,s1which were inhib(80) J . Schweden, C . Borgmann,G. Legler, and E. Bause, Arch. Biochem. Biophys., 248 (1986) 335-340. (8 I ) G. A. Grabowski, personal communication.

340

GUNTER LEGLER

ited by 1-deoxy-N,N-dimethylnojirimycin about 4-fold better than by the unmethylated compound. These results also showed that enzyme - substrate interactions cannot be very close to the ring-oxygen atom of the substrate; if they were, N-alkylation would have weakened the inhibition by steric interference, as it dida2with glucosidase 11, another a-D-glucosidase involved in glycoprotein biosynthesis and with a-D-galactosidase from coffee beans.63 With this type of enzyme, decisive evidence regarding the protonation state of the bound inhibitor can only be obtained from detailed studies on the pH-dependence of substrate K, and the K, values for neutral and basic inhibitors, as exemplified by the work of Dale and coworkers on almond /?-gluc~sidase.~~ A still enigmatic feature of glycosidase inhibition by sugar analogs with a nitrogen atom in the ring is its slow onset in many, but not all, cases, where K, is in the micromolar range or below. This was first reported for p-D-glucosidase from almonds and nojirimycin by Grover and Cushley,'ja and, for intestinal sucrase- isomaltase and nojirimycin, 1-deoxynojirimycin, and acarbose, by Hanozet and coworkers."j The approach to the steady-state inhibition took place on the time-scale of minutes. Studies with other enzymes (see Table VI) showed that this phenomenon is fairly widespread and is also observed with certain indolizine alkaloids (see Section 11,2,c). In all cases, the enzyme- inhibitor complex is formed at a rate that is of first order with respect to the inhibitor concentration, with rate constants in the range45,72*73 of lo3to 104M-' * s-* and thus three to four orders of magnitude below those of the reactions controlled by the rate of diffusion. As the association rate showed no saturation effects with inhibitor concentrations up to 20 K,, any loose, pre-steady-state complex must have a dissociation constant K, at least 50-fold larger than the steady-state K,. Inhibition constants for intestinal sucrase measured after 2 and 10 s with nojirimycin, 1-deoxynojirimycin,and acarbose were found to be 134-, 59-, and 106-fold larger than those measured45for 15 min. Inhibitors of this type were classified as slow, and slow, tight-binding inhibitors by Morrison and W a l ~ h . ~ ~ Several possible models can be discussed for the molecular basis of slow inhibition,a3but experimental evidence in support of one or the other is still lacking for glycosidases. A reversible chemical reaction at the active site, for example, formation of the cyclic imine 3 or a diffusion-controlled association with a trace of 3 in equilibrium with the 5-amino-5-deoxypyranose 1 can be precluded, because slow inhibition is also observed with ldeoxynojirimycin and its analogs and with acarbose (see Section II,2,d) and indoli-

(82) H. Hettkamp, G . W e r , and E. Bause, Eur. J. Biochem.. 142 (1984) 85-90. (83) J . F. Momson and C. T. Walsh, Adv. Enzymol.. 61 (1988) 201 -301.

GLYCOSIDE HYDROLASES

34 I

Hop OH

3

zine alkaloids. Another possibility could be the association of the inhibitor with a high-affinity conformer of the enzyme present in low, equilibrium concentration. Complex-formation would then shift the conformational equilibrium towards the high-affinity state. In this case, however, the rate constant should be independent of the inhibitor concentration, because the high-affinity conformer would always be saturated with the inhibitor. A third possibility is the formation of a loose complex, having a dissociation constant Ei, which then undergoes a slow conformational change to form the tight complex. Reversibility of the inhibition requires that the dissociation of the tight enzyme-inhibitor complex must also be a slow process. Experiments carried out to obtain experimental support for a slow, inhibitor-induced, con formational change were conducted with P-D-glucosidase from sweet almond^.'^ This enzyme had been shown to undergo, on binding D-glucono- I ,5-lactone, a conformational change which could be monitored with the fluorescent probe 1,8-anilinonaphthalenesulfonate(ANS).49The association of the enzyme with nojirimycin caused a change in the fluorescence of enzyme-bound ANS almost identical to the effect caused by ~ - g l u cono- 1,5-lactone. However, the dissociation constant for the nojirimycin complex determined fluorimetrically (4.4p k i ) differed strongly from K, (0.85 ptM), and the rate constant for the change of ANS fluorescence caused by nojirimycin binding was about 5-fold larger than the constant calculated from inhibition experiments. It may well be that the enzyme can adopt more than two conformations, which differ with respect to nojirimycin affinity and fluorescence ofbound ANS. Ifthe transition from a state ofintermediate affinity to the high-affinity state observed by virtue of the inhibition of substrate hydrolysis is without additional effect on ANS-fluorescence, it will go unnoticed by fluorescence measurements. c. Indolizine Alkaloids and Polyhydroxypyrrolidines. -Another group of specific, tight-binding, glycosidase inhibitors which have in common a basic, endocyclic nitrogen atom and a hydroxylation pattern of spatial similarity to that of specific hexoses is represented by certain plant alkaloids and synthetic pyrrolidine derivatives. Their inhibition constants (see Table VII)

GUNTER LEGLER

342

are of the same order of magnitude as those of 5-amino-5-deoxypyranoses and 1,5-iminohexitols,and, in some cases, a slow onset ofthe inhibition has also been observed. However, with some enzymes of identical glycon specificity and a similar response to the inhibitors of Section II,2,b, large differences in the inhibition by indolizine alkaloids have shown up which point to differences in active-site structure and mechanisms. Swainsonine (4), first isolated from the Australian plant Swainsonia canescens by Colgate and coworkers,84and later from spotted locoweed (Astrulagus lentiginosus) by Molyneux and James,85was characterized as a strong inhibitor of a-D-mannosidases by Dorling and coworkers.86Its inhibitory potency can be rationalized by the spatial arrangement of its hydroxyl group and its nitrogen atom: protonated swainsonine (4) greatly resembles the mannosyl cation (5),a putative intermediate in substrate hydrolysis, in its

HO

<9

4

CH,OH

5

hydroxylation pattern and position of the positive charge. A special feature of swainsonine is its ineffectivity against mannosidase I (no inhibition at 0.1 mM),which is in marked contrast to its strong inhibitionE7of mannosidase I1 [K, (app) -0.2 pw. Both a-D-mannosidases participate in N-glycoprotein biosynthesis and are located in the membranes of Gold vesicles. Later studiesEErevealed that the inhibition of a-mannosidases from jack beans and rat-liver lysosomes, and of Golgi mannosidase 11, is a slow process. Dissociation of the inhibitor from the jack-bean enzyme was immeasurably slow under native conditions, and required denaturation. Castanospermine (6), isolated from Castanospermum australe by Hohenschutz and coworkers89and characterized as a strong inhibitor for p - ~ (84) (85) (86) (87) (88)

S. M. Colgate, P. R. Dorling, and C. R. Huxtable, Aust. J. Chem.. 32 (1979) 2257-2264. R. J . Molyneuxand F. James, Science 216 (1982) 190-191. P. R. Dorling, C. R. Huxtable, and S. M. Colgate, Biochem. J., 191 (1980) 649-65 I . D. R. P. Tulsiani, T. M. Hams, and 0. Touster, J. Biol. Chem., 257 (1982) 7936-7939. D. R. P. Tulsiani, H. P. Broquist, and 0. Touster, Arch. Biochem. Biophys., 236 (1985)

421-434. (89) L. D. Hohenschutz. E. A. Bell, P. J. Jewess, D. P. Leworthy, R. J. Pyrce, E. Arnold, and J. Clardy, Phvtochetnistry. 20 (1981) 81 1-814.

GLYCOSIDE HYDROLASES

343

glucosidases by Saul and coworkers,g0can be considered to be a derivative of 1-deoxynojirimycin (2), with its hydroxymethyl group fixed in space by the ethylene bridge to the nitrogen atom which forms the pyrrolidine ring ofthe indolizine skeleton. The rigid, bicyclic structure of castanospermine and swainsonine may well be the cause of their strong inhibition of certain enzymes and weak inhibition of others (see Table MI). If the active site can adopt a conformation of high complementarity of its glycon part which happens to be identical to that of the indolizine alkaloids, their binding will be favored over that of the more flexible 1,5-iminoalditols:the entropic part of the free energy of binding will be much less negative for the rigid inhibitors. If, on the other hand, the glycon site corresponds to a conformation which is different from that of the rigid indolizine alkaloids, they will be bound much less tightly than the more flexible 1,5-iminoalditols, as exemplified by the strong inhibition of a-D-glucosidase from yeast by l-deoxynojirimycin and the absence of inhibition by ca~tanospermine.~~~'

OH 6

The importance of a basic nitrogen atom for strong inhibition by the indolizine type of inhibitor is demonstrated by the finding93that K, for inhibition by castanospermine N-oxide ofthep-D-glucosidase from almonds is 500-fold larger than K, of castanospermine itself (760puMvs. 1.5 pMat pH 6.0). The pH-dependence of the inhibition also indicated that unprotonated castanospermine is a better inhibitor than the protonated form. However, as essential carboxyl groups of the enzyme ionize in the same range of pH as castanospermine (pK, 6.09), it was not possible to estimate the inhibitory potency of protonated castanospermine. (90) R. Saul, J. P. Chambers, R. J . Molyneux, and A. D. Elbein, Arch. Eiochem. Biophys.. 22 1 (1983) 593-597. (91) L. Hosie, P. J. Marshall, and M. L. Sinnott, J . Chem. Soc., Perkin Trans. 2, (1984) 1121 - 1124. (92) B. R. Ellmers. B. L. Rhinehart, and K. M. Robinson, Eiochcm. Pharmacol.. 36 (1987) 2381 -2385. (93) R. Saul, R. J . Molyneux,and A. D. Elbein,Arch. Biochem. Eiophys., 230( 1984)668-675.

GUNTER LEGLER

344

TABLEV11 Inhibition of DMannosidases and Mlucosidases by Swainsonine and Castanospermine, Respectively Enzyme Swainsonine a-D-Mannosidase jack beans almonds rat liver, lysosomes Mannosidase I A/B rat liver, Golgi ves. I1 P-D-Mannosidase Aspergillus wentii Castanospermine a-~-Gl~co~idase Yeast Rice Rat liver, lysosomes Sucrase, rat intestine P-D-GluCOSidW almonds Aspergillus wentii human placenta, lysosomes

K,( p M )

0.001"

> 1,000

0.07"

> 1,000 0.1"

References

88 70 88 87 88

> 2,000

70

> 1,500

90,9 I 70 92 94

0.0 15" 0.1" 0.00055

1.5" 0.9" 7

70,93 70 81

Slow approach to the inhibition equilibrium.

A case similar to the slow, practically irreversible inhibition of jack bean a-D-mannosidase by swainsonine8*is represented by the interaction of castanospermine with isomaltase and rat-intestinal suc~ase.9~ Whereas the association constants for the formation of the enzyme-inhibitor complex were similar to those of other slow-binding glycosidase inhibitors (6.5 lo3and 0.3 lo3M- s- for sucrase and isomaltase, respectively), the dissociation constant of the enzyme-inhibitor complex was extremely low (3.6 s-l for sucrase) or could not be measured at all (isomaltase), resulting in a virtually irreversible inhibition. Danzin and Ehrhard94discussed the strong binding of castanospermine in terms of the similarity of the protonated inhibitor to a D-glucosyl oxocarbenium ion transition-state, but were unable to give an explanation for the extremely slow dissociation of the enzymeinhibitor complex. Polyhydroxypyrrolidines7 to 12 represent a group of compounds having only a marginal resemblance to monosaccharides, but which are, neverthe-

'

'

(94) C. Danzin and A. Erhard, Arch. Biochem. Biophys., 257 (1987) 472-475.

-

-

GLYCOSIDE HYDROLASES

345

less, potent inhibitors for many glycosidases. One, namely 8, has been isolated from plants95and synthesized by Fleet and Smith96;others have been prepared by Fleet and coworkers (7 and 9, in Ref. 97 and 10 in Ref. 98) and by Ganem and coworkers (11 in Ref. 99 and 12 in Ref. 100). As with the indolizine alkaloids discussed in the preceding Section, their high affinity for glycosidases probably results from a spatial arrangement of the hydroxyl groups resembling that ofthe glycon moiety ofthe substrates, combined with the ability to become protonated to form a cation which has its positive charge in a position that permits strong interactions with a carboxylate group of the active site. Inhibition constants in the micromolar range (see Table VIII) show that this feature can be realized even with compounds having only a cyclic, five-membered structure. In fact, compounds 7 to 11 inhibit yeast a-D-ghcosidase and jack bean a-D-mannosidase much better than the corresponding six-membered, 1,5-iminohexitols (see Table VI). That cation formation by proton transfer to the pyrrolidine nitrogen atom is required for strong inhibition was demonstrated by the conversion of 9 C H L OH

CH,OH

CH,OH

I

I

CH, O H 7

9

8

L I

10

11

12

(95) A. Welter, G. Dardenne, M. Marlier, and J. Casimir, Phytochemistry, 25 (1976) 747749. (96) G. W. J. Fleet and P. W. Smith, Tetrahedron Letf., (1976) 1469- 1471. (97) G. W. J. Fleet, S. J. Nicholas, P. W. Smith, S. V. Evans, and L. E. Fellows, Tetrahedron Lett., (1985) 3127-3130. (98) G. W. J. Fleet, P. W. Smith, S. V. Evans, and L. E. Fellows, J. Chem. Soc., Chem. Comrnun.,(1984) 1240- 1241. (99) M. J. Eis, C. J. Rule, B. A. Wurzburg, and B. Ganem, Tetrahedron Lett., (1985) 53975398. (100) C. J. Rule, B. A. Wurzburg, and B. Ganem, Tetrahedron Lett., (1985) 5379-5380.

346

GUNTER LEGLER

TABLE VIIl Inhibition of D-Glycosidases by Glycon-related Polyhydroxypyrrolidines, Expressed by the Inhibition Constants K,inpM (Adapted from Ref. 97) Inhibitor Enzyme

7

8

9

cu-D-Glucosidase, yeast P-D-Glucosidase, almonds a-D-Mannosidase jack beans rat liver, lysosomal a-D-Galactosidase Coffee beans P-D-Galactosidase,Asp. nfger P-D-Glucosiduronase

0.18 200

3.3 7.8

n. i. 350

100 -

n. i. * -

-

n. i. n. i.

n. i. n. i. -

0.2 140 -

-

14

10

11

500 450

-a 1000

12 -

-

IOd

-

-

400 160 -

n. I.

-

n. i.

90'

0.5

0.5'

-

-, not determined. n. i . , no inhibition up to 0.33 mM inhibitor. Ref. 99. Ref. 101. Ref.

100.

into the N-(4-methoxyphenyl) derivative,101thereby lowering the pK, by about four units. Rat-liver lysosomal a-D-mannosidase required 8 mM racemic N-(4-methoxyphenyl)-7 for 50% inhibition, whereas swainsonine (pK, 7.4), which can also be regarded as a N-substituted derivative of 9, inhibitP this enzyme with K, 0.07 p M . A detailed interpretation of the inhibition constants summarized in Table VlIl in terms ofstructure and orientation ofthe bound inhibitor at the active site is still a problem. In order to facilitate the discussion, formulas 7 to 12 were drawn in an orientation in which the position of the nitrogen atom would coincide with that of the ring-oxygen atom ofa bound glycoside, if the pyrrolidine alkyl substituent takes the position of the substituent on C-5 of the substrate. Discrimination between D-glycosidases and D-galactosidases by 7 and 8 on the one hand and by 9 on the other can be rationalized by an interaction of their hydroxyl groups with complementary groups at the active site responsible for binding to the hydroxyl groups on C-3 and C-4 of the substrate. The small difference between the &-values for 7 and 9 with almond P-D-glucosidase reflects the poor discrimination by this enzyme between inhibitors with D-gluco- and D - g d U C f O configuration (e.g.,glycosylamineslo3and 1,Sdideoxy-1,5-imino-~-hexitoIs~~). However, an explana-

-

(101) P. DeShong, D. R. Sidler. D. A. Kell, and N. N. Aronson, Jr., TefruhedronLerf..(1985) 3747-3148. (102) G. Palamarczyk, M. Mitchell, P. W. Smith, G. W. Fleet, and A. D. Elbein, Arch. Biochem. Biophys., 243 (1985) 35-45. (103) D. E. Walker and B. Axelrod, Arch. Biochem. Biophys., 187 (1978) 102- 107.

GLYCOSIDE HYDROLASES

34 7

tion of the strong inhibition of the two a-D-mannosidases by 9 and more so by 10 and 11 on this basis meets with considerable difficulties: neighboring cis-hydroxyl groups on the substrate are on C-2 and C-3, so that these inhibitors must be bound in a different orientation to provide strong interaction. Interestingly, a-specific enzymes are inhibited up to 1000-fold better than P-specific ones when enzymes of the same glycon specificity are compared. d. Aminocyclito1s.-Only a few of the many naturally occurring aminocyclitolshave been found to inhibit glycosidehydrolases. They are characterized by a hydroxymethyl substituent in the 1,3-positionrelative to the amino group, and a hydroxylation pattern corresponding to that of D-glucose (13 to 16); they were isolated from culture filtrates of Streptomyces hygroscopicus. 104-106 Except for a strong inhibition of intestinal sucrase - isomaltase (reviewed by Truscheit and coworkers’06),they are much less potent inhibitors for a-and P-D-glucosidases than are D-glucosylamine and 1-deoxynojirimycin (see Fig. 4 and Tables V and VI). Replacement of the ring-oxygen or nitrogen atom by a -CH,or =CH- group thus appears to be detrimental to an efficient interaction with a- andP-D-glucosidases.This could be explained by an adaptation of the active site favoring a half-chair conformation of the D-glucosyl residue. The inhibitors having an oxygen or nitrogen

oH2 HOCH,

HOCH,

HO

HO

OH

13 Valiolamine u-r-Glucosidase

HOCH,

H°CH,

HQH~

OH

OH

OH

H0QH2

OH

14

15

Valienearnine

Validamine

16

Hydroxyvalidamine

190

18

580

360

810

880

1500

740

(Yeast) !$o-Glucosidase (Almonds)

[Numbers give K, @M).]

(104) S. Hori, T. Iwasa, E. Mizuka, and Y. Kameda, J. Antibiof., 24 (1971) 59-63. (105) Y. Kameda, N. Asawa, M. Yoshikawa, M. Takeuchi, T. Yamaguchi, and K. Matsu, J. Antihiof., 27 (1984) 1301-1307. (106) E. Truscheit, 1. Hillebrand. B. Junge, L. Miiller, W. Puls, and D. Schmidt, Prog. Clin. Biochrrn.Med.. 7 (1988) 17-99; E. Truscheit, W. Frommer, B. Junge, L. Miiller, and D. D. Schmidt, Angew. Chrtn.,93( 1981)738-755;Angew. Chern.. Int. Ed. Engl., 20(1981) 744-76 I .

348

GUNTER LEGLER

atom in the ring might better adapt to this geometry than the more rigid cyclohexane or cyclohexene system. Incorporation of valienamine (14) into an oligosaccharide chain consisting of a+( 1 + 4)-linked D-glucosyl residues gives a group of pseudo-oligosaccharides (17) which are effective inhibitors for various amylases and for intestinal sucrase-isomaltase (see Ref. 106 for reviews). They are produced by various species of Actinomycetales and Streptomyces. Endo- and exo-glucanases such as alpha and beta amylase of microbial, plant, and mammalian origin, and glucoamylase, are inhibited at concentrations in the micromolar range. This is in marked contrast with the lack ofinhibition (K, > 10mM) by valienamine and its analogs.106Additional interactions with sugar-binding groups adjacent to the cleavage site appear to be essential for effective binding to glucanases. The only enzymes acting on saccharides of low molecular weight which are susceptible to the type 17 inhibitors appear to be intestinal sucrase and isomaltase, which are inhibited by acarbose (17, m = 0, n = 2) with K, = 0.47pMfor sucrase after a slow approach to the inhibition equilibri~rn.~’ OH

I

I

HO

Oh ’H

0

H

$H HO n

0-

rn

17

3. Pseudosubstrates The term “pseudosubstrate” as used in this article will comprise sugar-related compounds that are chemically transformed by glycosidases, often forming long-lived intermediates and thereby acting as reversible inhibitors. Even in cases ofweak inhibition, where the intermediate is too short-lived for chemical or physical characterization, the type of reaction catalyzed by the

GLYCOSIDE HYDROLASES

349

enzyme gives insight into details of the catalytic mechanism with normal substrates. A clear-cut separation of pseudosubstrates from normal substrates or reversible inhibitors on the one hand and from irreversible, activesite-directed inhibitors on the other is not always possible. For example, glycosylpyridinium salts are discussed as competitive inhibitors in Section II,2,a. Their hydrolytic cleavage is extremely slow, but they are transformed into moderate or even good substrates by the introduction of electron-withdrawing substituents in the pyridine or isoquinoline ring.lo7As acid catalysis cannot occur with these permanently cationic, glycosyl derivatives, it was possible to estimate catalytic forces and acceleration factors not related to acid catalysis by a detailed study of the correlation of aglycon basicity with enzymic and non-enzymic hydrolysis rates of /I-~-galactosyl-~~~ and p-Dglucosyl-pyridinium salts.'*An interesting outcome of these studies was the finding that the contribution of acid catalysis to the hydrolysis of normal glycosides was much less with p-D-galactosidase from Escherichia coli (pH optimum from pH 6 to 8, pK, 8.5 for the acidic group required for catalysis108.1w) than with P-D-glucosidase A, from Aspergillus wentii (plateau of maximal activity below pH 3.5, pK, 5.4 for the essential acidic group'1o). This is understandable, as, even with an active site shielded from the aqueous environment (see Section 11,2,b), water would compete effectively for the catalytic proton if the acidity of the proton-donating group was too high. A transition to irreversible inhibitors is seen in pseudosubstrates forming enzyme-bound intermediates which are cleaved very slowly, for example, the 2-deoxy-2-fluoro-~-glycosidesto be discussed in Section II,3,b.

-

a. D-Glycals (1,5-Anhydro-2-deoxy-~-hex-l-enitols and 2.6-Anhydro-2deoxy-D-heptenitols). -The first examples of strong glycal inhibition were described by Lee,'" who found that p-D-galactosidasesof bacterial, fungal, and plant origin are inhibited specifically by D-galactal(18), with K, values from 18 to 90 pM, that is, several hundredfold better than by D-galactose. The original explanation for the strong inhibition was based on its planar geometry at C- 1, which would result in a higher affinity for a binding site adapted to a half-chair conformation of the pyranoid ring, as proposed for lysozyme.6A detailed study of the inhibition of D-galactosidase from Escherichia coli by D-galactal, by Wentworth and Wolfenden,ll* revealed, how(107) M. L. Sinnott and S. G. Withers, Biochem. J.. 143 (1974) 751 -762. ( 108) J. P. Tenu, 0. M. Viratelle, J. Gamier, and J. Yon, Eur. J. Biochem.. 20 ( 1971 ) 363 - 370. (109) S. G. Withers, M. Jullien, M. L. Sinnott, 0.M. Viratelle, andJ. M. Yon, Eur. J. Biochem., 87 (1978) 249-256. ( 1 10) G. Legler, Hoppe-Se.vlerS Z. Physiol. Chem., 348 (1967) 1359-1366. ( 1 1 1 ) Y. C. Lee, Biochem. Biophys. Res. Commun., 35 (1969) 161- 167. ( I 12) D. F. Wentworth and R. Wolfenden, Biochemisrry, 13 ( 1974) 47 15-4720.

GUNTER LEGLER

350

ever, that D-galactal itself is only a weak inhibitor (K, 2 10 mM from presteady-state rates). Its strong inhibitory potency (K, 14pA4) was interpreted as the result of the slow formation of a 2-deoxy-~-hexosyl-enzymeintermediate which is slowly hydrolyzed, to regenerate the free enzyme and “2deoxy-D-galactose” (2-deoxy-~-lyxo-hexose).The slow hydration of D-galactal to 2-deoxy-~-/yxo-hexoseby P-D-galactosidase from E. coli had previously been reported by Lehmann and Schroter,113who had also shown that the enzyme binds 2-deoxy-~-lyxo-hexosylresidues which could not only be transferred to water but also to glycerol, to give I -deoxyglycerol-1-yl 2-deoxy-P-~-/yxo-hexopyranoside.

18

19

Studies with other glyco~idases~~.~’-’~~-~~’ showed, however, that effective inhibition by glycals is not a general phenomenon, and that inhibition does not correlate well with hydration to 2-deoxy-~-hexoses.Based on kinetic considerations, the interaction of glycosidases with D-glycals (A) can be described by the following scheme: E

+A

K: E .

4“

. . .A

ko,

E-A

7 E + 2-deoxyhexose khydr

“*O

With K, as dissociation constant of a loose, rapidly formed, non-covalent presteady-state complex E. * ‘A, the inhibition constant K, for the steady state is given by

y= 1

K: + kn/(koIT + khydr)

( I 13) J. Lehmann and E. Schroter, Curbohydr. Res., 23 (1972) 359-368. ( 1 14) E. J . Hehre, D. S. Genghof. H. Sternlicht, and C. F. Brewer, Biochemistry. 16 (1977) 1780- 1787. ( I 15) G. Legler, K.-R. Roeser, and H.-K. Illig, Eur. J. Biochem., 101 (1979) 85-92. ( 1 16) J . Lehmann and B. Zieger, Curbohydr. RKS.,58 (1977) 73-78. ( 1 17) T. Kanda, C. F. Brewer, G .Okada, and E. J. Hehre, Biochemistry,25 (1986) I 159- I 165.

GLYCOSIDE HYDROLASES

35 I

If, as it is usually done, the interaction of enzyme with glycal is studied in the presence of substrate S having Michaelis constant &,the observed rate constant bppr for the approach to the steady-state inhibition has to be corrected for the competition of substrate for the free enzyme, in order to calculate the rate constants kn,k,,, and k,, from the experimental data.

When the accessible concentration range of glycal [A] << K, and kO6 << khy, as for p-D-galactosidase from E. coli,112 this reduces to kPpr (1 [S]/ K,,J = (kon/K) * [A] 4-k,,,. The rate constant k,, for the addition of water has to be measured separately, by the appearance of the 2-deoxy-~hexose. With p-D-galactosidase from E. coli112and p-D-glucosidase from Asp. wentii115it has been found that k,, << khy,. Depending on the magnitude of k,,, b6,and k,,, different patterns of glycal inhibition are possible. A slow approach to the steady state requires that k,, and khy, be small. For strong inhibition, k6must also be small [k,, > (k,, khy,)], for example, as withp-D-galactosidase from E. cok and are large, kn/ p-D-glucosidase from Asp. wentii. When both bnand krr k,, >> 1, and k,, >> khy,, there is a rapid approach to the steady state, strong inhibition, and slow formation of 2-deoxy-~-hexoseas with the p-Dgalactosidase from Asp. ~entii.~'When all three constants are large and k,, = khy,, there is no detectable lag-phase in the onset of the inhibition, and, depending on k,,,,/(k,, khy,), moderate to weak inhibition combined with rapid hydration of D-glycal (for example, a-D-glucosidase from Candidu tropicalis'14or the hydration of cellobial by the exo-cellulase from Zrpex

+

+

lacteirs' 17).

The results of inhibition studies with D-glycals are summarized in Table IX. Also included are data for sialidases (N-acetylneuraminidase) with the D-glycal equivalent N-acetyl-2,3-dehydroneuraminicacid (N-acetyl-2deoxyneur-2-enaminic acid) (19), introduced by Meindl and T ~ p p y . "A~ comparison of the K a n d K, values with those for the corresponding hexoses shows that, with the exception of the p-D-galactosidase from Asp. wentii, D-glycals attain their inhibitory potency only after a slow reaction at the active site, combined with a slow hydrolysis of the intermediate. Sialidases appear to be different: the 2,3-unsaturated glycon analog is generally a good inhibitor but, in spite of good inhibition, there are no reports about a slow approach to the steady state or the hydration of the double bond with forma(1 18) E. J. Hehre, S. Gtahata, and C. F. Brewer, J. Eiol. Chem., 261 (1986) 2147-2 153. (1 19) P. Meindl and H. Tuppy, Hoppe-Seyler's Z . Physiol. Chem.. 350 (1969) 1088- 1094.

352

GUNTER LEGLER TABLE1X Interaction of Glycosidases with D-Glycals and Related Compounds (See Text for Definition of Kinetic Constants) Enzyme (source)

cy-D-Ghcosidases (D-glucal) Saccharomyces cerevisiae Candida tropicalis P-D-Glucosidases (D-ducal) Aspergillus wentii Almonds P-D-Galactosidases (D-Galactal) Escherichia coli Aspergillus wentii Cellulases (Cellobial) Aspergillus niger (endo) Irpex lacteus (exo) Beta amylase (maltal) Sweet potatoes Sialidases (N-acetyl-2,3dideoxy-2,3dehydroneuramink acid) Vibrio cholerae Influenza virus A Arthrobacter sialophilus 4-epi 4-OXO a

K;

K(

(mM)

(mM)

14" 5

0.19 10

a 10 0.013 -

0.0 14 -

kh,

(min-')

References

no reaction - I7Ob

I I3 1 I4

0.04 1 I .6

I I5 19

0.28 o.oO01

I12 57

- 1.4b

31"

- 16b

I17 I I7

9 5"

- 46

1 I8

0.01 0.0053 0.0016 0.048 0.06 1

no reaction no reaction no reaction no reaction no reaction

I I9 I20 121 I22 122

Michaelis constant K,, for ~ g l y c ahydration. l Approximate value calculated from specificactivity

with M,50,000.

tion of N-acetylneuraminic acid. The data for sialidasesalso show the extent of structural variation tolerated by these enzymes. The type of intermediate that is formed in the slow inhibition with ~ - g l y cals was identified, with the aid ofthep-D-glucosidase A, from Asp. wentii, as an ester of 2-deoxy-~-arubino-hexosewith an aspartic acid side-chain. Is The same aspartoyl residue had already been shown, by labeling with conduritol B e p ~ x i d e ' *(see ~ Section III,l), to be essential for p-D-glucoside hydrolysis. In addition, this aspartate was found to form a glycosyl-enzyme (120) P. Meindl, G. Bodo, J. Lindner, and H. Tuppy, Z. Nuturforsch., Teil B, 26 (1971) 792-796. (121) V. Kumar, J. Kessler, M. E. Scott, B. H. Patwardhan, S. W. Tanenbaum, and M. Flashner, Carbohydr. Res., 94 (1981) 124- 128. (122) V. Kumar, S. W. Tanenbaum, and M. Flashner, Carbohydr. Res., 103 (1982) 281 -285. (123) E. Bause and G. Legler, Hoppe-Seyler's 2. Physiol. Chem.. 355 (1974) 438-442.

GLYCOSIDE HYDROLASES

353

SCHEME3. -Formation of 2-Deoxy-cu-~-aruhino-hexosylEnzyme from ~-Glucalat the Active Site of a P-D-Glucosidase.

intermediate during the hydrolysis of 2-deoxy-~-arabinoihexosidesby this enzyme.'24The good agreement between btfor D-glucal hydration and kt for 2-deoxy-~-urubino-hexosidehydrolysi~'~~ can be taken as evidence that degl ycosylation of the intermediate is rate-limiting for both types of reaction. A difference of six orders of magnitude between btfor 2-deoxy-~-urabinohexosideIz4and D-glucoside hydrolysis'* shows the importance of the 2-hydroxyl group for interactions, at the active site of this enzyme, which are required for efficient catalysis. The stereochemistry of proton transfer to C-2 of D-glucal was investigated by Hehre and coworker^"^ with a-D-glucosidase from Cundidu tropicalis and P-D-ghcosidase from almonds, and by Lehmann and Zieger1I6with P-D-galactosidase from E. coli. Experiments camed out in D,O as solvent (with acidic protons of the enzymes exchanged for deuterium) and n.m.r. spectroscopyofthe resulting 2-deoxy-~-hexosesrevealed that, with all three enzymes, the protonation of C-2 occurs from the side opposite to the glycosidic oxygen atom of the respective substrates, if it is assumed that D-glucal is bound in the same orientation as the glycon moiety of the substrate (see Scheme 3). It was also shown that the attack ofwater results in the release of 2-deoxy-~-uruhino-hexosehaving the same anomeric configuration as that of the respective substrates. The same stereospecificity of glycal hydration was found with cellobial ~-~-glucopyranosyl-( 1 4)- 1 3 anhydro-2-deoxy-~-urubino-hex1-enitol] acted upon by an endo-cellulase from Aspergillus niger and an exo-cellulase from the wood-rotting fungus Irpex lucteus. 'I7 Although direct evidence is available only for P-D-glucosidase from almonds,125it can be assumed that the other enzymes, too, are "retaining" glycosidases, that is, the glycon part of the substrate is released with the same anomeric configuration as the glycosidic bond which is

-

(124) K.-R. Roeser and G. Legler, Biochirn. Biophys. Acfu, 657 (1981) 321-333. (125) D. E. Eveleigh and A. S. Perlin, Curbohydr. Rex, 10 (1969) 87-95.

354

GUNTER LEGLER

cleaved. Taking account of the stereochemistry of the reactions of glycosidases with glycon-related epoxides (see Section III,2) and the formation of a 2-deoxy-~-urubino-hexosy~ aspartic ester with P-D-glucosidase A, from Asp. wentii, the results of Hehre and coworkers can best be rationalized with a cyclic, six-membered, transition state in which the active-siteaspartate (or its equivalent) acts115as both an acid and a nucleophile (see Scheme 3). Special comments are appropriate on the stereochemistry of the hydration of maltal by sweet potato beta amylase,’ls an “inverting” a-glycosidase which degrades amylose chains from the nonreducing end, with release of p-maltose. In contrast with the double-displacement mechanism of the “retaining” glycosidases,the hydrolysis with inversion of the anomeric configuration is probably effected by a general, base-assisted, direct attack of a water molecule on the anomeric carbon atom, aided by proton transfer from an imidazole cation of the active site.126-128 Product analysis revealed that the proton donated to C-2 of the glucal moiety of maltal had come from “above” (see Scheme 3), that is, again from a group that is presumed to act as a base during substrate hydrolysis. This unexpected stereochemistry of the D-glycal hydration probably results from a combination of two factors: on chemical grounds, the protonation has to occur on C-2, because, on C-1 (with subsequent formation of 1,5-anhydro-~-hexitol),it would produce a highly unstable intermediate, with its (partial) positive charge localized on C-2, whereas protonation on C-2 results in the formation of a relatively stable 2-deoxy-~-glycosylcation with its charge delocalized over C- 1 and the ring-oxygen atom. However, the distance between C-2 and the acidic group responsible for proton-transfer to the glycosidic oxygen atom of the substrates appears to be too large for efficient proton-transfer. This can be taken as additional evidence for the restricted, spatial flexibility of the catalytic groups relative to the bound substrates or inhibitors, as discussed in Section II,2,a. The stereochemistry of the enzymic hydration of D-glycals demonstrates, on the other hand, a catalytic flexibilityofglycosidasesthat had not been envisaged before, and which is supported by their reactions with glycosyl fluorides (see Section 11,3,b). An extension of the concept of pseudosubstrates with planar, sp2-geometry of C- 1 of glycosyl derivatives was provided by the introduction of 2,6-anhydro- 1-deoxy-D-gufucto-hept- 1-enitol(20) by Brockhaus and Lehmann. 128 The authors showed that 20 is hydrated by P-D-galactosidase from Escherichiu coli, to 1-deoxy-D-galucto-heptulose(21) and that glycerol can be glycosylated to 1-deoxyglycerol-1-yl 1-deoxy-P-D-gu/ucto-heptuloside. The latter reaction resembles the formation of 1-deoxy-glycerol-1-yl2-deoxy-P(126) J. A. Thorna, J . Theor. Biol., 19 (1968) 297-310. (127) D. French, MTPInt. Rev.Sci., Ser. 1, Biochem.. 5 (1975) 267-335. (128) M. Brockhaus and J . Lehrnann, Curbohydr. Res.. 53 (1977) 21-31.

GLYCOSIDE HYDROLASES

355

20

D-lyxo-hexosidefrom D-galactal,' l 3 a reaction which defines the stereospecificity of glycosyl transfer to the acceptor oxygen atom. The kinetics of hydration of 20 by thep-D-galactosidasefrom E. coli were characterized by a Michaelis constant K,,, 60 mMand a turnover number k,, - 50 s-I (corresponding approximately to K', and khydrin the glycal-hydration scheme). Even though the individual rate-constants for the formation ofan enzyme - l -deoxyketosylintermediate and its hydrolysiscould not be determined, it is informative to compare the foregoing data with the corresponding ones for the hydration of D-galactal by the same enzyme. lI2As the methyl group of the protonated heptenitol will stabilize an oxocarbenium ion-like transition state, the glycosylation step (k,)will be favored compared with the D-galactal reaction. It is, therefore, safe to assume that, with heptenitol hydration, the deglycosylation step is rate-limiting, as with D-galactal hydration, that is, khydr= btfor both substrates. Comparison of the two rate-constants gives kt(heptenitol)/bt(D-galactal) = 1. I - lo4.As deglycosylation is likely to be hindered by the methyl group of the 1-deoxyD-heptulosyl residue rather than to be accelerated, it must be concluded that the 104-folddifference in the deglycosylation rate reflects favorable interactions (with the enzyme) of the hydroxyl group on C-3 on the heptenitol which are not possible with the 2-deoxy-~-lyxo-hexosylintermediate from D-galactal. Similar conclusions can also be drawn from a comparison of hydrolysis rates of glycosides with 2-deoxyglycosides (see Section 11,3,c). The Michaelis constant K,,, -60 mM for heptenitol hydration may be taken as a measure for enzyme substrate affinity, provided that deglycosylation is not rate-limiting. If it is, the true dissociation constant will be even larger. A comparison of the foregoing K, with K,-values for D-galactose (34 mM) and for 5-amino-5-deoxy-~-galactono-1$lactam (70 pM, see Table IV) and an expected value in the micromolar range for D-galactono-I ,5-lactone reveals that the sp2-geometry of C- I is not sufficient by itself to cause strong inhibition. Unless the two methylene hydrogen atoms of 20 interfere on steric grounds with strong binding, it must be concluded that the binding energy for aldonolactone and lactam inhibition (see Table IV) comes mainly from polar interaction of the large, carbonyl dipole with the active site.

-

GUNTER LEGLER

356

Studies by Hehre and coworkers in collaboration with Lehmann’s g r o ~ p ’with ~ ~2,6-anhydro, ~ ~ 1-deoxy-D-gluco-hept-I -enitol (22) and a-Dglucosidases from the yeast Cundida tropicalis and rice, P-D-glucosidase from almonds, and an exo-( 1 -+ 4)-a-glucanase from Arthrobacter globijbrrni.7 have extended the findings of Brockhaus and Lehmann12*to other enzymes. All four enzymes were found to effectthe stereospecific hydration of 22 to 1-deoxy-D-gluco-heptulose(23). The anomeric configuration of 23 before alp equilibration corresponded to the anomeric specificity of the three glucosidases, that is, the a-D-glucosidases formed a-,and the P-D-~~ucosidase formedp-, D-heptulose. In addition to the hydration product 23,the a-D-glucosidases and the exo-( I 4)a-glucanase formed l-deoxy-Dghco-heptulosides by transfer of the I -deoxyheptulosyl residue to hydroxyl groups ofthe heptenitol22 and/or the heptulose 23 present in the incubation mixtures. Glycosylation was 99% at the 7-hydroxyl group of the acceptor with the Cundida and the Arthrobacterenzyme, whereas the rice enzyme was more specific for the hydroxyl group on C-5 (89% at C-5, 1 I % at C-7). The anomeric configuration of the heptulosides formed with the a-D-glucosidases and the exo-( 1 6)-a-glucanase from Arthrobacter globiformis was identified as a.

-

-

-

CH,OH

22

23

24

The formation of the a-heptuloside by the Arthrobacter enzyme, an “inverting” glycosidase, is of special interest in conjunction with the formation of 1-deoxy-P-D-gluco-heptulose in the heptenitol hydration reaction. Whereas the stereochemistry of the hydration corresponds to that of substrate hydrolysis (that is, formation Of j?-D-glucose),the glycosylation reaction points to an active site that permits entry of the acceptor molecule only at the aglycon site, but not at the water site, and in which the acidic group proximal to the glycosidic oxygen atom of the substrate functions (after deprotonation) as base in the glycosylation reaction (see Scheme 4). This model implies a functional inversion of the role of the catalytic groups which is similar, but opposite, to that proposed for proton transfer in the D-glycal hydration. ( 129) E. J. Hehre, C. F. Brewer, T. Uchiyama, P. Schlesselmann, and J. Lehmann, Biochemistry. 19 (1980) 3557-3564. ( 130) P. Schlesselmann, H. Fritz, J. Lehmann, T. Uchiyama, C. F. Brewer, and E. J. Hehre, Biochemisfry, 24 (1982) 6606-6614.

GLYCOSIDE HYDROLASES

357

+o

+f*o

c H3 OO ,.\

0Y’ .-.o

CH3

0,:.;0 C

I

The stereochemistry of proton transfer to an exocyclic double bond was studied with the prochiral octenitol24 with the “retaining” a-D-glucosidases from Asp. niger and rice and the “inverting” trehalase from Trichoderma reesei.I 3 l The results with the “retaining” enzymes showed that proton transfer had occurred from “above” (re face), and addition of hydroxide from “below,” the ring (see Scheme 4). The ionization state of the functional acid and base were assumed to be opposite to that postulated for glycoside hydrolysis. Proton transfer is thus different from that found with hydration of D-glucal, both with respect to direction and proton-donating group. The “inverting” trehalase, on the other hand, catalyzed the formation of the /3 anomer of the octulose by cis hydroxylation. b. Glycosyl Fluorides.- In contrast with other glycosyl halides, both aand p-glycosyl fluorides are sufficiently resistant to spontaneous hydrolysis to permit their use in enzymic studies under the normal conditions of pH and temperature. Their non-enzymic hydrolysis is catalyzed both by acids and hydroxide ion,132the former probably being of importance for their susceptibility to enzymic hydrolysis. A comparison of the rate constants for the acid-catalyzed hydrolysis of a- and p-D-glucosyl fluoride with those for p-nitrophenyl a-andp-D-glucoside’showsthat, at 25 theglucosyl fluorides are cleaved - l O5 as rapidly. The enzymic hydrolysis of a-and p-D-glucosyl, a-D-galactosyl, and a-D-mannosyl fluoride by the corresponding glycosiO,

( 1 3 I ) W. Weiser, J. Lehmann, S. Chiba, H. Matzui, C. F. Brewer, and E. J. Hehre, Biochemis.

try, 27 (1988) 2294-2300. ( 132) J. E. G. Barnett, Curbohydr. Rex, 9 ( I 969) 2 I -3 I .

358

GUNTER LEGLER

dases was studied by Barnett and coworkers.133The I&, values of the fluorides were similar to or lower than, and the V, values similar to or larger than, the corresponding constants for the enzymic hydrolysis of 2- and 4-nitrophenyl glycosides. Glycosyl fluorides are thus seen to behave as normal substrates for glycosidases,where the low hydrogen acceptor potential of the fluorine substituent is more than compensated for by its better leavinggroup propensity. Their importance for mechanistic studies with glycosidases derives from the ability of their a andpanomers to react in stereocomplementary ways with “inverting” glycosidases. Hehre and coworkers134showed that beta amylase from sweet potatoes, an “inverting,” a-specific exo-( 1 4)-glucanase, catalyzes the hydrolysis of p-maltosyl fluoride with complex kinetics which indicated the participation of two substrate molecules in the release of fluoride ion. Furthermore, the reaction was strongly accelerated by the addition of methyl p-maltoside. Hydrolysis of a-maltosyl fluoride, on the other hand, obeyed MichaelisMenten kinetics. The main product with both a-and p-maltosyl fluoride was p-maltose. The results with p-maltosyl fluoride were interpreted by the assumption of a glycosylation reaction preceding hydrolysis by which a maltotetraoside is formed by the replacement of fluoride ion by a second substrate molecule or added methyl p-maltoside (see Scheme 5). The ability of “inverting” a-glucanases to cleave p-glycosyl fluorides by the intermediate formation of an a-glycosidic oligosaccharide appears to be a general feature of enzymes, of this type. Glucoamylase from Rhizopus niveiis and glucodextranase from Arthrobacter globiformis, which release p-D-glucose from the respective ( 1 4)- and ( 1 6)-a-~-glucans,were shown to catalyze the hydrolysis ofp-D-glucosyl fluoride similarly to the one observed with beta amylase and /I-maltosyl fluoride.135Here, too, the reaction was accelerated by added D-glucosides, and hydrolysis of a-D-glucosyl fluoride proceeded with Michaelis- Menten kinetics. Interestingly, all enzymes hydrolyzed the a-glycosyl fluoride more than tenfold better than thep anomer. Evidence for the intermediate formation of a-D-glucosyl transfer product was obtained by the isolation of methyl a-maltoside and methyl a-isomaltoside from digests of p-~-glucosylfluoride and methyl a-~-glucoside with either glucoamylase or glucodextranase. The orientation of the acceptor glucoside with respect to p-~-glucosylfluoride bound at the active site was not rigid, but corresponded, nevertheless, to the substrate specificity of the enzymes: the ratio of maltoside : isomaltoside was 2 : 1 with the glucoamylase, and 1 : 25 with the glucodextranase.

-

+

-

(133) J. E. G. Barnett, W. T. S. Jarvis. and K. A. Munday, Biochem. J.. 105 (1967) 669-672. (134) E. J . Hehre, C. F. Brewer, and D. S. Genghof, J. Biol. Chem., 254 (1979) 5942-5950. ( I 35) S. Kitahata, C. F. Brewer, D. S. Genghof, T. Sawai, and E. J. Hehre, J. Biol. Chem., 256 (1981) 6017-6020.

GLYCOSIDE HYDROLASES

F H 4

C

359

40

R'

0-R2

H

o, ,o

/L

SCHEME5 . -Hydrolysis of a-and /.-Maltosyl Fluoride by Beta Amylase (R1= ~-DGIUCOsyl, R2 = H or a-Maltosyl).

Other "inverting" glucosidases which conform to the pattern of direct hydrolysis of glycosyl fluorides having the correct anomeric configuration, and transglycosylationwith inversion if the anomeric configuration is opposite to that of the natural substrates are trehalase from rabbit renal cortex and from the yeast Candida t r ~ p i c a l i s ,and ' ~ ~P-D-xylosidase from Bacillus pumilis.13' c. 2-Deoxy- and 2-Deoxy-2-fluoro-glycosides. -Even though both of these groups of compounds are derived from normal substrates by only a minor modification of the glycon moiety, they are discussed together with pseudosubstrates, because their reactions with glycosidases show, in many cases, unusual kinetic features. 2-Deoxyglycosides are, in spite of the 2 2000-fold greater sensitivity against acid hydrolysis, only poor substrates for glycosidases,as shown by the ( 136) E. J. Hehre, T. Sawai, C. F. Brewer, M. Nakano, and T. Kanda, Biochemistry, 2 I (1982)

3090- 3097. ( I 37) T. Kasumi, Y. Tsumuraya, C. F. Brewer, H. Kersters-Hilderson, M. Claeyssens,and E. J. Hehre, Ahstr. Pup. Am. Chem. Soc. Meef.. 190 (1985) CARB 30.

GUNTER LEGLER

360

TABLE X Hydrolysis of 4-Methylumbelliferyl j3-D-Glycosides by j3-D-Clucosidase A from Almonds1= and by j3-DGIucosidase A, from Aspergillus w e n t i P

Almonds

Glycon

(mM)

(min-I)

1.7 0.9

26.400 245

D-XylOSe

0.7 0.57 2.9

12 21,600 34

2deoxy-~-arabinohexose

1

D-~~UCOS~

D-XYIOSe

2-deoxy-~-arabinohexose Asp. wentii

k,

K,

Enzyme source

glucose

.o

0.09

data in Table X. Whereas the replacement of the hydroxyl group on C-2 by hydrogen has only moderate effects on binding, as expressed by the Michaelis constant &, it is profoundly detrimental to the rate constant of product formation kt.For a meaningful interpretation of the results, it is necessary to take account of the fact that “retaining” glycosidases require at least two distinct chemical steps for substrate hydrolysis: (z] a first bondbreaking reaction (rate constant k,) leading to the release ofthe aglycon, and formation of a glycosyl-enzyme intermediate, (ii) cleavage of the glycosylenzyme by a water molecule, with release of the sugar with the same anomeric configuration as in the substrate, and regeneration of free enzyme (rate constant k,). If rate constants for any conformational changes are included with the kinetic constants for the chemical steps, the steady-state can be represented by the following scheme: H,O

E + S ek, E * . k-,

*

*S-E‘-P2-E+P2. A k -2

k -1

Under practically all conditions, the back reaction can be neglected. The observable constants &and ktare then given by

With P-D-glucosidase A, from Asp. wentii, the conversion of /h-glucosides into the 2-deoxy derivatives had a much larger effect on k, than on k,, so that deglycosylation became rate-limiting.138As a consequence of the (138) G. Legler, Act4 Microbiol. Acad. Sci.Hung., 22 (1975) 403-409.

GLYCOSIDE HYDROLASES

36 1

large decrease of k,, the steady-state rate was reached on the time-scale of minutes instead of fractions of a second. Under the conditions of k, > k,, comparisons of K,,, -values for P-D-glucoside and 2-deoxy-~-~-urubino-hexoside are meaningless, because the Michaelis constant &, no longer a measure of enzyme-substrate affinity, is lower than the dissociation constant K, = k l/kl. An estimate of the effect of deoxygenation at C-2 on the enzyme-glycon affinity was obtained from a comparison of the inhibition constants for P-D-glucosyl- and 2-deoxy-~-~-arubino-hexosyl-py~d~n~um ion, which showed a I 10-fold decrease in affinity for the 2-deoxy derivative. The deglycosylation rate, on the other hand, was decreased 2 106-fold. The greatly differing effects of deoxygenation at C-2 on enzyme-substrate affinity and catalysis can be rationalized on the grounds that the interaction energy with the hydroxyl group at C-2 not only shows up as binding energy expressed by K,,, or K,but is “used” to a large extent to lower the free energy of activation. It may well be that interactions with the 2-hydroxyl group are required in the sense of Koshland’s “induced-fit’’ theory139for an optimal orientation of the catalytic groups with respect to the bond to be cleaved. That these interactions are more important for k, than for k, is understandable, because the whole substrate molecule interacts with the enzyme in the first step, whereas, for the second step, only interactions with the glycon moiety are available. If k, > k,, the glycosyl-enzyme intermediate will accumulate, and may be trapped by the rapid denaturation of the enzyme in the presence of (saturating) amounts of substrate. With P-glucoside A, from Asp. wentii and 4-nitrophenyl[ 14C]-2-deoxy-~-~-urubino-hexopyranoside, it was possible to identify the intermediate as a glycosyl ester (acylal) of 2-deoxy-~-urubinohexose bound to the same aspartate residue“ that had previously been labeled with the active-site-directed inhibitor conduritol B ep~xide’,~ and with ~-glucal.ll~ This constituted an important proof that the carboxylate reacting with the epoxide is directly involved in catalysis. Replacement of the 2-hydroxyl group ofa glycosyl derivative by a fluorine atom has a large decelerating effect in all cleavage reactions that proceed by way of a glycosyl-oxocarbenium ion intermediate, because the destabilization of the transition state by the strongly electron-withdrawing fluorine atom leads to a large increase of the free energy of activation. Glycosidases that have a glycosyl ion or a glycosyl ion-like transition state on their reaction pathway should be similarly impaired in their catalytic capabilities. This was indeed confirmed by Withers and coworkers141for a number of a- and (139) D. E. Koshland, Jr., Prcx. Null. Acud. Sci. CJ. S. A , 44(1958) 98-101. (140) K.-R. Roeser and G. Legler, Biochim. Biophys. Acfu,657 (198 I ) 32 1-333. (141) S. G. Withers, K. Rupitz, and 1. P. Street, J. Biol. Chem., 263 (1988) 7929-7932.

362

GUNTER LEGLER

P-specific glycosidases. Based on their reactivity with the respective 2-deoxy2-fluoro-a- and -P-D-~~YcosY~ fluorides, the enzymes were found to fall into three groups. Group (a) was completely inactivated, with simple, first-order kinetics. The inactivation rate-constant, k,(obs.), could be described by the rapid formation of a non-covalent complex, having dissociation constant K,, which then gives the covalent enzyme- pseudosubstrate complex with a rate constant k. This gives k,(obs.) = k, [I]/(& [I]) where [I] = concentration of glycosyl fluoride. This group comprised a P-D-glucosidase from Alcaligenes faecalis and P-D-galactosidasesfrom E. coli, Asp. oryzae, and Asp. niger. The K, values for the 2-deoxy-2-fluoro-~-glycosyl fluorides were lower, or in the range of the K, values for the corresponding hexoses, indicating that noncovalent binding is not impaired by the two fluorine substituents. Group (6) comprised P-D-glucosidase from almonds, a-D-glucosidase from yeast, P-D-galactosidase from bovine liver, and a-D-galactosidasefrom Asp. niger, and was characterized by complex inactivation kinetics that did not, except for almond P-D-glucosidase,lead to complete loss of activity. A possible reason for an incomplete loss of activity could be a slow hydrolysis of the 2-deoxy-2-fluoro-~-glycosyl-enzyme intermediate, as discussed for the reaction of D-galactal with P-D-galactosidasefrom E. coli and of D-glucal with P-D-glucosidase from Asp. wentii. Group (c), a-D-mannosidase from jack beans and from almonds, and a-D-galactosidase from coffee beans, showed no inactivation. The results with these enzymes can possibly be explained by the formation of a (weak) non-covalent complex in which glycosylation is too slow to cause inactivation within the time period of measurements, or, less likely, rapid hydrolysis of the glycosyl-enzyme intermediate.

+

111. IRREVERSIBLE INHIBITORS

Information relevant to the mechanism of an enzyme-catalyzed reaction can, in general, only be obtained from irreversible inhibitors which react specifically at the active site and thereby inactivate the enzyme. As activesite-directed inhibition is treated in detail in Ref. 142; general aspects will be discussed here only briefly. In order to be suitable as an active-site-directed inhibitor, a compound must fulfil the following requirements. ( i )High non-covalent affinity for the active site, in order to avoid unspecific labeling by permitting the reaction to be camed out at low inhibitor (142) W. B. Jakoby and M. Wilchek (Eds.), Affrnity Labeling, Methods Enzymol.. Vol. 46 (1977).

GLYCOSIDE HYDROLASES

363

concentrations. (ii) Stability of the enzyme-inhibitor bond against denaturation, proteolysis, and sequential peptide degradation, in order to permit identification of that amino acid in the peptide sequence which has reacted with the inhibitor. (iii) Sufficient stability against spontaneous decomposition under the reaction conditions. According to Baker,143it is convenient to distinguish two kinds of activesite-directed inhibitors or affinity labeling reagents, namely, endo and exo. The former are inhibitors that react with functional amino acids directly participating in the catalytic mechanism; the others react with amino acids outside the catalytic center. With glycosidases, this would be that part of the active site which interacts with the aglycon or which is responsible for binding the sugar moiety ofthe substrate. As the glycon and aglycon binding-sites are unlikely to have any catalytic properties, exu-inhibitors must have a functional group of sufficient intrinsic reactivity. Suitable groups are bromoacetyl (reacting with the side chains ofcysteine, methionine, or histidine), isothiocyanato (N-terminal amino groups, and lysine), aryldiazonium ion (tyrosine and histidine), and groups which, on acidolytic or photolytic decomposition, give rise to carbenes or nitrenes, such as diazocarbonyl compounds, triazenes, diazirines, and azido compounds. Whereas the reaction with the first three groups depends on the fortuitous presence ofa susceptible amino acid residue within reach of the reagent, the carbenes and nitrenes have a much higher reactivity, which permits a reaction, at least with carbenes, even with C - H bonds. Depending on the non-covalent affinity and intrinsic reactivity, unspecific labeling may become important. Modification of a glycon-related compound with any of these groups will, because of the great sugar specificity of most glycosidases,strongly affect the non-covalent affinity, if this is done in the glycon moiety at positions different from C- 1. Introduction of such a group at C- 1, however, might provide the possibility of labeling an amino acid which is directly involved in catalysis, provided that it fulfils the requirements of chemical reactivity called for by the reactive group ofthe inhibitor and that it is within reach ofthat group. As will be shown later, the latter aspect presents a severe restriction, because of the limited flexibility of these amino acid side-chains. On the other hand, e m labeling reagents intended for exploring the aglycon site are subject only to the chemical restriction ofcompatibility ofthe reagent and the amino acid to be labeled. As glycon affinity of glycosidases is generally low for monosaccharides (see Tables I11 and IV), problems with unspecific labeling may arise with glyconderived inhibitors of high intrinsic reactivity which do not have additional features to provide enhanced affinity, for example, a suitably positioned (143) B. R. Baker, J . Pharm. Sci., 53 (1964) 347-353.

GUNTER LEGLER

364

basic group. These problems can be overcome with inhibitors which are of low intrinsic reactivity but which are specifically activated at the active site. As this usually occurs by functional groups which participate directly in catalysis, that is, substrate hydrolysis, they have been called ktinhibitors,'43 suicide inactivators,Iu or mechanism-based inhibitors. The inactivation is normally a first-order process, provided that the inhibitor is in large excess over the enzyme and is not depleted by spontaneous or enzyme-catalyzed side-reactions. The observed rate-constant for loss of activity in the presence of inhibitor at concentration [I] follows MichaelisMenten kinetics and is given by k,(obs) = k,(max) - [I]/(K, [I]), where K, is the dissociation constant of an initially formed, non-covalent, enzyme inhibitor complex which is converted into the covalent reaction product with the rate constant k,(max). For rapidly reacting inhibitors, it may not be possible to work at inhibitor concentrations near K,. In this case, only the second-order rate-constant k,(max)/K, can be obtained from the experiment. Evidence for a reaction of the inhibitor at the active site can be obtained from protection experiments with substrate [S] or a reversible, competitive inhibitor [I(rev)].In the presence of these compounds, the inactivation rate K,(obs) should be diminished by an increase of K, by the factor ( 1 [S]/K,,,) or ( 1 [I(rev)]/K,(rev)). From the dependence of k,(obs) on the inhibitor concentration [I] in the presence of a protecting agent, it may sometimes be possible to determine K, for inhibitors that react too rapidly in the accessible range of concentration.'&

+

+

+

I . Conduritol Epoxides (1,2-Anhydroinositols) and Sugar-derived Epoxides14'

The reactivity of the epoxide group in a tetrahydroxycyclohexane system against acid-catalyzedand nucleophilic addition is greatly diminished by the electron-withdrawing effect of the neighboring hydroxyl groups. This inertness makes conduritol epoxides (25 and 27-31] ideal ktinhibitors, as they are resistant against spontaneous hydration and only react with proteins if they are bound in a position that permits protonation of the epoxide oxygen atom by an acidic group in close proximity. The specific reaction of a conduritol epoxide requires, in addition to the acidic group for the protonation of the oxirane, a nucleophile for the formation of the covalent bond. In all cases studied so far, this is the carboxylate

(144) R. R. Rando, Science, 185 (1974) 320-324. (145) R. H. Abeles and A. L. Maycock, Acc. Chem. Rex. 9 (1976) 313-319. (146) G. Legler, Hoppe-Seyler's Z. Physiol. Chem., 349 (1968) 767-774. ( 147) G. Legler, Glucosidases, Ref. 142, pp. 368 - 38 1.

GLYCOSIDE HYDROLASES

365

\

\

OH 25

27

26

29

OH

OH

H 0'

30

20

31

group of an aspartate or, in the case ofp-D-galactosidasefrom E. c ~ l i , of ' ~a~ glutamate residue. In principle, the pH-dependence of k,(obs) should be governed by the ionization state of both groups; that is, a plot of k(obs) vs. pH should have a maximum and inflection points determined by the pK, of the acid on the alkaline and by the pK, of the nucleophile on the acidic side. However, in many cases, only the inflection determined by the acid is observed, as for /?-~-glucosidase'~~ and p-D-galactosidase from Asp. ~ e n t i i ' ~ ~ and P-D-glucosidase from almonds,149where a sigmoidal pH-dependence having a plateau on the acidic side was observed. A possible explanation could be an optimal orientation of the nucleophile with respect to the inhibitor, and rate-limiting proton-transfer to the epoxide oxygen atom. The ionization state of the nucleophilic carboxylate would not affect k,(obs) if the protonated epoxide should immediately react with the carboxylate, even if present in very small proportions. In other cases, for example, p-D-glucosidase from Asp. uryzael5Oand intestinal sucrase- i~omaltase,'~' a sigmoidal pH-dependence of k,(obs) is found which appears to be determined by the ionization of the nucleophilic carboxylate. The wide scope of the application of conduritol epoxides for the activesite-directed inhibition is seen from the data given in Table XI. Only a few

(148) M. Herrchen and G. Legler, Eur. J. Biochem.. 138 (1984) 527-531. (149) G. Legler and S. N. Hasnain, Hoppe-Seyler's Z. Physiol. Chem., 35 I (1970) 25-31. ( 1 50) G. Legler and L. M. Omar Osama, Hoppe-Seyler's Z. Physiol. Chem., 349 (1968) 14881492. (151) A. Quaroni, E. Gershon, and G. Semenza, J. Biol. Chem.. 249 (1974) 6424-6433.

366

GUNTER LEGLER

TABLE XI Kinetic Constants for the Inactivation of Glycosidases by Conduritol Epoxides Corresponding to their Respective Glycon Specificities (see Text) Enzymes a-D-Glucosidases (Conduritol B epoxide, 25) Yeast (S. cerevisieae) Rice Monascirs rirber Asprrgilliis niger (Maltase) Rabbit, intestine (Sucrase) (Isomaltase) Calf liver, endoplasm. ret. (Glucosidase I and 11) &D-Glucosidases (Conduritol B epoxide, 25) Aspergillus wentii, A, Aspergillus oryzae Sweet almonds A B Helix pornatia Human placenta (lysosomal, purified) Calf spleen, lysosomal purified crude Calf liver (cytosolic) a-D-Galactosidases (Conduritol C trans-epoxide, 27) Coffee beans Mortierella vinacea Escherichia coli Human liver (lysosomal) &D-Galactosidases (Conduritol C cis-epoxide, 28) Escherichia coli

-Mg*+ Aspergillirs wentii a-D-Mannosidases (Conduritol F trans-epoxide, 30) Jack beans Almonds /h-Mannosidase (Conduritol F cis-epoxide, 31) Goat liver

- 25

> 10 -

> 50 > 10

-0.16 -

-

-

0.6 -

1.7 2.3

0.13 0.08

0.17

0.05 1

4

M.6 0.17

4. I

60

-

0.65 0.0023

1.2

-

0.14 -

-

0.23 0.16

0.052 -

-

-65" 10 3.2 <0.02

0.8 I 6.5 <0.02

I504 130 15 76" 31

155 46 156

154 151 151

82

146 150

149 I49 73

300

157

360 3,700

158 158

0.57

I59

20 <0.02 <0.0 1

71,160 161 162 163

0.09

1.4

226 <0.0 I I400

162 I62 162

-

-

0.8 <0.02

154 154

-

-

I .o

23

16

GLYCOSIDE HYDROLASES

367

TABLE XI (continued) Enzymes a-1.-Fucosidase (Conduritol C /runs-epoxide, 27) Human liver Cellulases (4',5'-Epoxypentyl /l-cellobioside, 35, m = 3) O.\-lporlis sp. Lysozyme (2',3'-epoxypropylA', N'-diacetyl-/l-chitobioside,36, n = 2) Hen egg

43

0.29

6.8

164

33

0.0 I5

0.46

165

- 6b

166

0.17

-0.001

a Complex inactivation kinetics caused by enzyme-catalyzed decomposition of epoxide; kinetic constants calculated from initial rates of inactivation. Approximate value calculated from half-life in the presence of 50 m M inhibitor.

p-specific glycosidases appear to be resistant against the corresponding epoxides, for example the highly aglycon-specific@-glucosidases from garbanzo plant (Cicera r i e f ~ m and ) ' ~ from ~ Alocusia ma~rorhiza,'~~ and thep-D-mannosidase from Asp. ~entii.'~~ With a-specific enzymes, the lack of suscepti(152) W. Hosel and W. Ban, Eur. J. Biochrm., 57 (1975) 607-616. (153) A. Nahrstedt, W. Hosel, and A. Walther, Phytochernistry, 18 (1979) 1137- 1141; Resistance of the /l-D-@ucosidasesof Refs. I5 1 and 152 against inactivation by conduritol B epoxide, W. Hosel, personal communication. (154) G. Legler and E. Jiilich, unpublished results. ( 1 55) R. Winterhalter, Thesis (Diplom Biology), University of Cologne (1976). (156) S.-J. Yang, S.-G. Ge. Y.-C. Zeng, and S.-Z. Zhang, Biochirn. Biophys. Acta, 828 (1985) 236 - 240. (I 57) G. A. Grabowski, K. Osiecki-Newman, T. Dinur, D. Fabbro, G. Legler, S . Gatt, and R. J. Desnick, J. Biol. Chem., 261 (1986) 8263-8269. (158) H. Liedtke and G. Legler, in R. Salvayre, L. Douste-Blazy, and S. Gatt (Eds.), Lipid Storage Disordm. Plenum Press, New York, 1988, pp. 353-358. (159) G. Legler and E. Bieberich, Arch. Biochern. Biophys., 260 (1988) 437-442. (I 60) U. Petzold, Thesis. University of Cologne (1984). (161) H. Suzuki, S.-C. Li, and Y.-T. Li, J. Bid. Chern.. 245 (1970) 781-786; G. Legler, Resistance against inactivation by conduritol C trans-epoxide, unpublished results. (162) G. Leglerand M. Herrchen, FEBS Lett., 135 (1981) 139-144. (163) D.Bishop, personal communication. (164) W. J. White, Jr., K. J. Schray. G. Legler, and J. A. Alhadeff, Biochirn.Biophys. Acta, 873 (1986) 198-203. (165) G. Legler and E. Bause, Curbohydr. Res., 28 (1973) 45-52. (166) E. E. Thomas, J. F. McKelvy, and N. Sharon, Nature, 222 (1969) 485-487.

368

G m T E R LEGLER

bility against epoxide inhibition seems to be more widespread [see Table XI, enzymes with k(max) <0.02 M-' - miK4], probably because the trans-diequatorial ring-opening required for a-glycosidasesis more difficult than the trans-diaxial reaction of the P-specific ones (see Scheme 6). Reactivity of the different epoxides, and identification of the products cleaved from enzymes inactivated with radioactive epoxides, permit the following conclusions. (i) Release of the label by treatment with hydroxylamine indicates an ester linkage with a carboxylate group at the active site identified as an aspartate residue in P-D-glucosidase A, from Asp. ~ e n t i i ,A' ~ ~ from bitter almonds,'67and the lysosomal enzyme from human placenta,'68 in rabbit intestinal sucrase and i~omaltase,'~~ whereas it was identified as an L-glutamate residue in P-D-galactosidase from E. ~0li.I~~ The denaturing conditions required for release are in marked contrast with the lability at pH 3 8 of peptide fragments obtained from the labeled enzymes. This can be taken as evidence for the strong shielding of the active site from the aqueous environment. (ii) In all cases, the reaction product was identified as inositol produced by trans-opening of the epoxide. The configuration of the new hydroxyl groups was trans-diaxial with P-specific enzymes and trans-diequatorial with a-specific ones (P-~-glucosidases,'~,~~~ a-~-glucosidases,~~~ sucraseisomaltase,'70P-~-galactosidase,'~~ and a-~-galactosidase~'). A model for the stereochemistry of proton transfer and nucleophilic attack is shown in Scheme 6. This stereochemistry also explains the failure of epoxides to react with glycosidases if the oxirane ring does not have the correct orientation with respect to the acidic and nucleophilic groups; for example, only conduritol C trans-epoxide (27)reacts with a-D-galactosidases, and only conduritol C cis-epoxide (28) with P-D-galactosidases. Similar results were obtained with P-~-mannosidase.~~ As the trans-diequatorial opening of the oxirane ring is energetically ~nfavorable,'~' it becomes understandable that a-specific glycosidases react slowly or not at all with the corresponding conduritol epoxides. (iii) Conduritol epoxides are chiral molecules, but are usually prepared and applied as racemic mixtures. The identification of (+)-chiru-inositol as a

(167) G . Legler and A. Harder, Eiochirn. Biophys. Acta. 524 (1978) 102- 108. ( I 68) T. Dinur, K. M. Osiecki, G . Legler, S. Gatt, R. J. Desnick, and G . A. Grabowski, Proc. Natl. Acud. Sci. USA, 83 ( 1986) 1660- 1664. (169) A. Quaroni and G . Semenza, J. Biol. Chern.. 251 (1976) 3250-3253. (170) H. Braun, G . Legler, J. Deshusses, and G . Semenza, Eiochirn. Biophys. Acta, 483 ( I 977) 135- 140. ( I 7 I ) A. Fiirst and A. Plattner,Abstr.,Pap. I n [ . Con/:PureAppl. Chern., 12th, New York, 195 I , p. 409.

369

GLYCOSIDE HYDROLASES

I

HA

7 o:.!.

A

OH

-

A'

OH

/c~o

-0 A-

&

SCHEME6. -Reaction of Conduritol Epoxide (Anhydro-inositol, Hydroxyl groupsomitted) with p- and a-Specific Glycosidase, Respectively.

reaction product of conduritol B epoxide (see Scheme 6 ) with P-glucosidase A, from Asp. ~ e n t i ishowed ' ~ ~ that the enzyme specifically selects that enan-

tiomer which corresponds to the glycon configuration of the substrate. This was confirmed by studies with 1-L-1,2-anhydro-myu-inositol(28) which was unreactive with both a- and P-~-glucosidases.'~'-'~~ The successful inactivation of both a-D-galactosidase162and a-~-fucosidasel~~ by conduritol C trans-epoxide (27) can be ascribed to the presence of both the D and L specific enantiomer of the epoxide in the preparation. Replacement of the cis-hydroxyl group adjacent to the oxirane ring in conduritol B epoxide (25) by bromine results in some cases in a decreased, and in other cases in an increased, reactivity with P-D-glucosidases.Whereas ki(max)/& for the bromo epoxide (29)was 24-fold lower with the enzyme from Asp. ~ e n t i i and l ~ ~-50-fold lower with the enzyme from Asp. oryit was 5.5-fold larger with /h-glucosidase B from sweet almonds,*49 7-fold larger with the enzyme from Heli~pomatia,'~and 40-fold larger with the l y s ~ s o m a l ~and ~ J ~130-fold * larger with the cytoplasmicP-D-glucosidase from calf.'59It had been proposed173that the different response to the re(172) D. Mercier, A. Olesker, S. D. Gero, and J. E. G. Barnett, Carbohydr. Res.. 18 (1971) 135- 140. (1 73) G. Legler, Mol. Cell. Biochem.. 2 ( 1 973) 3 I - 38.

GUNTER LEGLER

370

placement of the hydrophilic hydroxyl group by the hydrophobic bromo substituent would reflect the presence of a hydrophobic area in the glycon binding-side complementary to C-6 of the substrate, because enzymes having enhanced reactivity with the bromo epoxide hydrolyzed 6-deoxy-P-~glucosides better than P-D-glucosides. This proposal could not be generalized, however, because the lysosomal P-D-glucosidase from calf spleen showed a higher activity with D-glucosides than with 6-deoxy-~-glucosides.174 The principle of active-site-directed inactivation of glycosidases by glycon-related epoxides can be extended to compounds having an exocyclic oxirane ring, either directly attached to the six-membered ring (32) or at some distance (33,34).Studies with P-D-glucosidase from sweet almonds175 and intestinal sucrase- i s o m a l t a ~ erevealed ~ ~ ~ that, in spite of the higher intrinsic reactivity of these epoxides, this shift of the position of the epoxide function causes a 10- to 30-fold decrease of b(max)/y, an effect which probably reflects the limited flexibility ofthe catalytic groups involved in the epoxide reaction.

32

33

34

A noteworthy point in inhibition experiments with enzymes which depend on additional factors for their activity is seen with mammalian lysosoma1 P-D-glucosidase. This enzyme requires anionic lipids, or activating proteins, or both, for maximal activity, which largely remain associated with crude enzyme preparations. Removal of these activating factors during purification not only impairs the catalytic activity with natural and synthetic substrates,1S*but also causes an almost tenfold decrease in the inactivation rate-constant b(max)/K, (see Table XI). This can be taken as evidence that the purified enzyme differs strongly in its intrinsic catalytic properties from its state in the native environment. A related case is seen with P-D-galactosidase from E. coli, which requires magnesium ions for a~tivity.”~ Removal of Mg2+ did not impair binding of conduritol C trans-epoxide (it acted as

(174) M. Bremer. Thesis (Diplom, Chemistry), University of Cologne, 1986. (175) M. L. Shulman, S . D. Shiyan. and A. Ya. Khorlin, Biochrm. Biophys. Acta. 445 (1976) 169- 181. (176) M. L. Sinnott, S. G. Withers, and 0. M. Viratelle, Biochem. J., 175 (1978) 539-546.

GLYCOSIDE HYDROLASES

37 1

competitive inhibitor, with K, 0.16 mM), but decreased the inactivation rate by more than four orders of magnitude.16* Glycoside hydrolases, like lysozyme, or cellulases which act on polysaccharides, do not hydrolyze mono- and di-saccharides, but require oligosaccharides of a certain minimal chain-length for effective binding and catalysis. It is, therefore, not surprising that they do not react with conduritol epoxides. Epoxide inactivators can, however, be obtained by the attachment of an w-epoxyalkyl chain of appropriate length at the reducing end of oligosaccharides of appropriate structure. This approach was successfully applied to lysozyme'66with chito-oligosaccharides35 and to c e l l ~ l a s e with s ~ ~ cello~ oligosaccharides36. For the latter, tested with n = 1 to 4,maximal reactivity was found with the 4',5'-epoxypentyl derivative (n = 3). The pH-dependence of the inactivation rate indicated activation ofthe epoxide by an acidic group, thus giving evidence for an active site of the cellulases similar to the one of lysozyme.6

35

36

Despite a higher intrinsic reactivity, epoxides of type 35 and 36 show a lower inactivation rate k,(max), as seen in Table XI, than the conduritol epoxides. This is probably caused by the greater flexibility of the epoxyalkyl chain in the active-site cleft, and by non-productive binding in positions where the oxirane is not within reach ofthe catalytic groups ofthe active site. For epoxypropyl oligosaccharides,this would hold even when the inhibitor occupies the correct subsites. 2. Sugar-related Aziridines A new type of mechanism-based enzyme-inactivators, which are related to conduritol epoxides with respect to activation at the active site, was introduced by Tong and Ganem,177who prepared the aziridine 37 from the D - ~ U ~ U Canalog ~O of I-deoxynojirimycin. Compound 37 proved to be a PQ(177) M. K. Tongand B. Ganem,J. Am. Chem. Soc., llO(1988) 312-313.

GUNTER LEGLER

372

tent, specific inactivator of a-~-galactosidasefrom coffee beans, with K, 7.1 p M and k(max)/K, 2540 M-' min-'. The authors assumed that protonated 37 inactivates by esterification ofthat carboxylate group on theP face of the bound inhibitor which is presumed to stabilize a cationic intermediate during hydrolyis of a-~-galactosides(see Scheme 7).

-

HO

HO

37

H

0,

F

,o

&

H

o.c 40 I

&

SCHEME7. -Reaction of Aziridine 37 with a-D-Galactosidase.

Comparison of the kinetic data for 37 with those with the same enzyme and conduritol C trans-epoxide (27, see Table XI) reveals an estimated 50-fold higher reactivity of 37 if based on k(max)/K,. (This estimate takes account of the different temperatures employed, namely, 37" in Ref. 178 and 25 for the data in Table XI, re-e~aluation~' of the data given in 16* gave the values listed in Table XI.) A detailed inspection of the inhibition data shows that the advantage of the aziridine 37 over the oxirane 27 lies in its almost l 04-foldhigher non-covalent affinity, which overcompensates for its - 60-fold lower covalent reactivity. It is interesting that the moderate structural alteration involved in the conversion O f D-gUlUCtU- 1-deoxynojirimycin (K, 0.00 16 pM, see Table VI) into 37 causes a 4500-fold decrease in affinity. Replacement of the oxirane oxygen atom of conduritol B epoxide (25) by the NH group gives an aziridine which should be an active-site-directed inhibitor of glucosidases. This was tested by Caron and Withers,*77*who synthesized this imino analog of 25 ( 1,2-dideoxy-1,2-epimino-myo-inositol) and found it to inhibit covalently a-D-ghcosidase from yeast and a-~-glucosidase from Alcaligenesfueculis. The kinetic parameters for the inactivation reaction were K, 9.5 mM and k,(max) 0.39 min-' for the a-D-glucosiO

(177a) G . Caron and S. G . Withers, Biochem. Biophys. Rrs. Commrtn.. 163 (1989) 495-499. (178) M. L. Sinnott and P. J. Smith, Biochem. J.. 175 (1978) 525-538.

GLYCOSIDE HYDROLASES

373

dase and K, 3.0 mM and k(max) 0.077 min-' for the P-D-glucosidase. Comparison of these data with the corresponding ones for 25 (see Table XI) shows both K, and k(max) to be in the same range as the values for 25. The apparent lack of effect on the non-covalent affinity (K,) of the basic epimino group clearly requires further studies.

3. Glycosylmethyltriazenes Reaction of glycosylmethylamines with aryldiazonium salts gives a class of compounds which, by acid catalysis or unknown factors of enzymic catalysis, generate glycosylmethyldiazoniumions. These, in turn, lose nitrogen, to yield highly electrophilic carbenium ions, as illustrated for the p-Dgalactosyl derivative 38 (see Scheme 8). If these reactions take place at the active site of a glycosidase, they can be used to alkylate a nucleophile, provided that the glycosyl moiety gives sufficient non-covalent affinity to make the reaction specific. With aryl groups having electron-withdrawing substituents, the spontaneous decomposition is, above pH 6, sufficientlyslow to make this approach feasible. This was first shown by Sinnott and Smith"* with P-D-galactosidase from E. coli and 38. The amino acid alkylated by the triazene 38 was identified179as methionine502 (formerly Met-500, new numbering according to Ref. 180), which had

bH,, DNo'

HO

HO

NH N ,

=N

OH

SCHEME 8. -Formation of /?-D-Galactosylmethyl Carbenium Ion from /?-D-Galactosylmethyl-44 nitropheny1)triazene. (179) A. V. Fowler. I. Zabin, M. L. Sinnott, and P. J. Smith, J. Eiol. Chem., 253 (1978) 5283 - 5285. (180) A. Kalmins, K. Otto, U. Riither, and B. Miiller-Hill, EMEO J.,2 (1983) 593-597.

GUNTER LEGLER

374

earlier been labeled with N-(bromoacetyl)-p-D-galactosylamine181 (see next Section). The scope and limitations of active-site-directed inhibition by glycosylmethyltriazenes was explored by Sinnott and his group182with the following results (see Table XII): “retaining” p-glycosidaseswere, with the exception of p-D-glucosidase B from sweet almonds, inactivated with second-order rateconstants b(max)/K, from 340 to 23,000 M-’ - min-’. a-Glycosidases reacted much more slowly, or not at all. “Inverting” glycosidases, such as glucoamylase from Aspergillus or p-D-xylosidase from Bacillus pumilis were not inactivated. The rapid inactivation of human lysosomal P-D-galactosidaseis noteworthy, as the reaction had to be camed out at 2 pH units above its pH optimum (pH 4.0) in order to minimize the spontaneous decomposition ofthe inhibitor. Similar conditions had to be employed for p-D-xylosidase from Penicillium wortmannii (optimum at pH 3.5), which was studied at pH 7.0. The fact that p-D-galactosidase from Escherichia coli is inactivated more rapidly in the absence of Mg2+than in its presence can be taken as evidence that the activation of the triazene 38, that is, formation of p-D-galactosylmethyldiazonium ion, proceeds without acid catalysis, because Mg2+is required for the proton-assisted catalysis of p-D-galactoside hydrolysis by this enzyme.177Additional evidence for the absence of acid catalysis in the de-

TABLE XI1 Kinetic Constants for the Inactivation of Some Glycosidases by the CorrespondingClycosylmethyl(4-nitrophenyl)triazeneslB2

~~

P-D-Galactosidase,E co/i +0.05 M Mg2+ 50 mM EDTA P-D-Galactosidase,human liver, lysosomal P-D-Ghcosidase, almonds a-~-Galactosidase,coffee /h-Xylosidase, B. picmilis

~

+

-

0.07 0.48

0.024 0.58

0.018

0.41 -

100

-

- 225

340 1.200 23,000 3.4 0.95 2,250

(181) F. Naider, Z. Bohak, and J. Yariv, Biochemistry. 1 1 (1972) 3202-3207. (182) P. J. Marshall, M. L. Sinnott, P. J. Smith, and D. Widdows, J. Chem. Soc.,Perkin Trans., 1, (1981) 366-376.

GLYCOSIDE HYDROLASES

375

composition of the enzyme-bound triazene was obtained from studies with P-D-galactosidase from E. coli and p-D-xylosidase from P. wortrnannii with glycosylmethyltriazenes having aryl substituents of different leaving-group p r ~ p e n s i t y .Nevertheless, ’~~ it could be shown that the triazene decomposes more rapidly at the active site than in free solution (5-fold and 800-fold with P-D-galactosidase from E. coli in the presence and absence, re~pectively”~ of Mg2+). It remains to be established if glycosylmethyltriazenes should be classified with ktor mechanism-based inhibitors, as it is still an open question if, and then to what extent, the enzymic effects on triazenes are also important for the catalysis of substrate hydrolysis. 4. Miscellaneous Inhibitors

a. Electrophiles Having Reactive Halogen Substituents.-As discussed in the introduction to Section 111, reactive groups can be introduced into a glycose molecule at C-1 only. If other positions are used, the noncovalent interactions at the active site will be strongly impaired, and specificitywill be lost. The first successful active-site-directed inhibitor with a glycon derivative having a reactive halogen atom was reported by Naider and coworkers,I8l who specifically inactivated p-D-galactosidase from E. coli with N-( bromoacety1)-P-D-galactosylamine (39).Consideration of the amino acid sequence of the enzyme produced by mutant strains of E. coli indicated that 39 had reacted with methionine-502, an amino acid that does not participate in catalysis, as it could be replaced by norleucine without loss of activity. Compound 39 can, therefore, be regarded as an exo-labeling reagent. Met-502 must, however, be very close to the catalytic site, as its neighbor Tyr-503 was shown to be directly involved. Replacement of the latter by phenylalanine by using site-directed mutagenesis caused an - 1000-fold deof kat.

Hob CH,OH

HO

OH

H c,c. I 1

H,Br

11

0 39 ( 1 83) M. L. Sinnott, G. T. Tzotzos, and S. E. Marshall, J. Chem. Soc., Perkin

Trans. 2, (1982)

I665 - 1670. (184) M. Ring, D. E. Bader, and R. E. Huber, Biochem. Biophys. Res. Commun., 152 (1988) 1050- 1055.

GUNTER LEGLER

376

The positions of the reactive groups in 39 and in the carbenium ion formed from the triazene 38 differ by two single bonds, but both react with the same amino acid, Met-502. This indicates considerable flexibility of the methionine side-chain of the bound inhibitors. The latter appears more likely, because N-(bromoacetyl)-b-D-glucosylamine was found to be also an active-site-directedinhibitor, which probably185alkylates the same methionine as 39. The kinetic constants for the two inhibitors were: K, 1.1 W a n d b(max) 0.063 M-' min-' (D-gulucfo ) and K, 220 mM and k,(max) 7.0 M-I * min-I (D-glumderivative). The interpretation by the authors was that both inhibitors are bound at the D-glucose subsite of the enzyme (normally accommodating the D-glucose moiety of the natural substrate lactose) where they could alkylate Met-502, and that 39 was, in addition, non-productively bound at the D-galactosesubsite, thus giving low values for K, and k(max). A comparison of the shape and reactivity of the two inhibitors with lactose make this mode of binding unlikely. A more plausible model would be that both bromoacetyl derivatives bind to the D-galactose site, but the greater flexibility of the loosely bound D-gluco derivative permits a more facile reaction with Met-502. A different type of reactive bromo compound having a moderate resemblance to hexoses is represented by the bromoconduritols B (40) and F (41), named after the respective parent tetrahydroxycyclohexene.lsg Even though their hydroxylation pattern resemblesthat O f D-glucose, only a few examples of D-glucosidase inhibition have been reported. The first was a-D-glucosidase from yeast,186which is inhibited by bromoconduritol B (formerly called bromoconduritol A), having k(max)/K, 69,000 M-' * min-', by alkylation of a histidine residue at the active site.

qBr

HoQo

HoQBr

HO

I

OH 40

I

OH 41

42

The pHdependence of the inactivation rate indicated the participation of both a basic and an acidic group in the reaction with 40. The latter could be explained by the formation at the active site of the highly reactive epoxide 1,2-anhydroconduritoI F (42) which is subsequently activated by the acidic (185) 0. M. Viratelle, J. M. Yon, and J. Yariv, FEBS Lett., 79 (1977) 109- 112. (186) G . Legler and W. Lotz, Hoppe-Seyler's Z. Physiol. Chem., 354 (1973) 243-254.

GLYCOSIDE HYDROLASES

377

group. As participation of a basic group did not show up in the pH-profile of the inactivation rate with 42, it was assumed that the basic group seen from the pH-profile of the inactivation with 40 was involved in the formation of 42 from 40. From the effects of bromoconduritol B and F (formerly A and B, respectively) on the structure of asparagine-linked oligosaccharides of glycoproteins from chick-embryo cells infected with influenza virus, it had been deducedI8' that 40 or41 had inactivated glucosidase I, an a-D-glucosidase (of the endoplasmic reticulum) required for glycoprotein biosynthesis. This is probably a special case, however; glucosidase I from calf liver was notE2 susceptible to inhibition by 40 or 41. The only other examples of bromoconduritol inhibition reported so far are a cytosolic P-D-ghcosidase from calf liver159and the lysosomal P-D-~~ucosidase from calf spleen.55In spite of the 6500-fold difference in their reactivity with conduritol B epoxide (see Table XI), both enzymes are rapidly inactivated by bromoconduritol F, with b(max)/K, lo5M-' min-' for the cytosolic enzyme and b(max)/K, 3.2 lo5 for the crude and 3.9 * lo4 M-' - min-I for the purified lysosomal enzyme. It should be noted that purification of the lysosomal P-D-glucosidase had effects on the reactivity with bromoconduritol F similar to those it had on the reactivity with conduritol B epoxide (see Table XI). Interestingly, both the cytosolic and the lysosomal enzyme regained most of their activity on prolonged standing after they had been inactivated to the extent of 2 98% with bromoconduritol F. The rate of reactivation was larger at pH 6 than at pH 4.6. It was concluded that a labile ester-bond had been formed in the inactivation reaction. From the stereochemistry of the hydroxyl groups and the bromine substituent, it could have been with the carboxyl group presumed to act as acid catalyst in the activation of substrate or epoxide (see Scheme 6).

-

-

b. Glycosyl Isothiocyanates. -The isothiocyanato group, an electrophile which easily reacts with amino groups near neutral pH to form a thiourea derivative, is widely used for the covalent attachment of ligands to proteins.Is8Unspecific reactions with accessible amino groups have, therefore, to be taken into consideration when glycosyl isothiocyanates are employed as active-site-directed inhibitors. Shulman and coworker^"^ showed that P-D-glucosyl isothiocyanate (43) can be used for the specific inactivation of P-D-glucosidase B from sweet ( I 87) R. Datema, P. A. Romero, G. Legler, and R. T. Schwarz, Proc. Nutl. Acud. Sci. U.S.A.,79 (1982) 6787-6791. ( 1 88) R. G. Langdon, Organic Isothiocyunatrs as Afinify Labels, in Ref. 142, pp. 164- 168.

378

GUNTER LEGLER

43

almonds, with K, 13 mMand k(max)/Ki 1.3 M-’ - min-’ at pH 5 and 37” (recalculated from the experimental data in Ref. 171).At pH 6, the inactivation rate was threefold lower. No information is available with respect to the functional group that had reacted with 43. IV. CONCLUSIONS 1. Interpretation of Inhibition Results by Mechanistic Features

a. Carboxylate and Proton-donating Groups at the Active Site.-All “retaining” /?-D-glycosidasesand most “retaining” a-D-glycosidases are inhibited by basic glycon-relatedcompounds several hundred- to almost 105-fold better than by non-basic structural analogs. This strong inhibition requires that the basic nitrogen atom be directly joined to the anomeric carbon atom or, in the case of pyrrolidine and indolizine derivatives, can adopt a position which is equivalent to the ring-oxygen atom of the bound substrate. This close, positional requirement is an indication for the restricted flexibility of the bound ligand and the functional groups responsible for the enhancement of affinity. The latter can be taken as evidence for a carboxylate group (in close proximity to the anomeric carbon atom) which forms an ion pair with the protonated inhibitor. The large differences seen in the enhancement of affinity by a correctly placed, basic group [more than two orders of magnitude in K,(non-basic analog)/& when different enzymes and different types of inhibitor are compared] probably reflects small individual differences in active-site structure. In order to account for the inability of many enzymes to bind the protonated form of the basic inhibitors or permanently cationic ones better than uncharged analogs (for example, P-D-galactosidase from E. coli, and p-Dglucosidase from almonds), it was proposed5’that the enzyme could protonate the inhibitor at the active site by a cationic acid (for example, protonated histidine). If proton transfer cannot occur, the attractive forces due to the carboxylate would be canceled by the repulsion from the cationic acid. Experimental evidence for this proposal is, however, still lacking. In p-D-galactosidase from E. coli, a tyrosine is presumed to be responsible for the protonation of substrates.la4

GLYCOSIDE HYDROLASES

379

Supporting evidence for a carboxylate group in close proximity to the anomeric carbon atom of the bound substrate came from the inactivation experiments with conduritol epoxides. In addition, insight into the stereochemical relations between the carboxylate and the acid required for the activation of the epoxide and, presumably, of the substrate, came from the structures of the inositols cleaved with hydroxylamine from epoxide-labeled enzymes. In all a- and P-glycosidases,the orientation of the catalytic groups was found to be complementary to the anomeric configuration of the substrate, that is, the acidic group acted from the side of the glycosidic oxygen atom and the carboxylate from the opposite side. As reactions with oxiranes involve protonation from one side of the ring and nucleophilic attack from the other, it is understandable that no “inverting” glycosidase has been found to be inactivated by an active-site-directed epoxide. Whether these enzymes catalyze their hydrolysis has, however, not yet been tested. Nevertheless, “inverting” glycosidases probably also have a carboxylate group at the bond cleavage site, because glucoamylase is inhibited by 1-deoxynojirimycin at submicromolar concentrations.Io2 Direct participation of a carboxylate group in substrate hydrolysis, possibly as a nucleophile, was obtained from quenching experiments with a-Dglucosidase from Saccharomyces o v i f ~ r r n i and s ~ ~ p-glucosidase ~ A, from Asp. ~ ~ e n t iUse i . ~of~substrates ~ labeled in the glycon moiety permitted isolation ofthe glycosylated enzyme by rapid denaturation in the presence of an excess of substrate. The bond between the glycosyl residue and the enzyme was characterized by its chemical reactivity as an acylal; with the Aspergillus enzyme, the carboxyl group involved was found to be part of the same aspartate residue that had been labeled with conduritol B e p 0 ~ i d e . I ~ ~ As deglycosylation has to be rate-limiting for a successful application of this approach, glycosides having a highly reactive aglycon, or a slowly reacting sugar moiety, or both, are needed. Evidence for a glycosyl-enzyme intermediate of finite lifetime with “inverting” a-D-glycosidases,and details of its reaction, came from studies with 2,6-anhydro- 1 -deoxyhept-I - e n i t 0 1 s ’ ~ and ~ J ~glycosyl ~ f l ~ o r i d e s . l ~Anal~,l~~ ysis of hydration and hydrolysis products on the one hand, and of glycosylation products on the other, indicated an intermediate that could be approached by water from the p-face only of the ring, and by other glycosyl acceptors only from the a-face (see Schemes4 and 5). This can be considered a proof of the principle of microscopic reversibility of chemical reactions.

(189) H.-Y. Lai. L. G. Butler, and B. Axelrod, Biochem. Biophys. Rrs. Commim., 60 (1974) 635-640.

GUNTER LEGLER

380

b. Access of Solvent Water to the Active Site.-Electrostatic interactions are very weak in aqueous solution because of the strong hydration of ions and the large dielectric constant of water. If the strong inhibition of glycosidases by basic sugar analogs is caused by the formation ofan ion pair, it must, therefore, be assumed that access of solvent water to the active-site region of the enzyme-inhibitor complex is largely restricted. According to the Debye-Huckel theory of strong electrolytes, there is a large effect of ionic strength on ionic interactions in solution. Determination of K, for a basic inhibitor and K, for a neutral substrate (see Table XIII) has shown the absence of such effects. Any alterations of the kinetic constants with buffer concentration were seen to affect the interaction of the enzyme with the basic (or cationic) inhibitor (K,) and with the substrate (k) to about the same extent. This restricted access of solvent water is probably important for the close alignment of the substrate with respect to the catalytic groups which is required for effective catalysis. In addition, it was shown by W a r ~ h e l ~ ~ J ~ ~ TABLEXI11 Effects of Ionic Strength on the Inhibition of D-Clucosidases by 1-Deoxynojirimycin Buffer conc. (mM)

K,(PM)

a-D-Clucosidase' (yeast)'54 5 66 I10 50 300 280 /%D-Glucosidase" (Asp. 5 760 50 760 300 440 /%D-Glucosidase" (calf-spleen lysosomesys 5 2 Ih 50 45 300 67

K,(PM)

lOOO%/Y,

Phosphate pH 6.8 9.6 145 17.4 159 44.3 158 Acetate pH 4.0 1.8 2.36 2.3 3.02 2.2 5.O Citrate pH 4.6 96 4,600 260 5,700 400 6,000

a Substrate, 4-Methylumbelliferyl a-@-D-glucoside. Substrate, 4-nonylumbelliferyl Po-glucoside.

(190) A. Warshel, Proc. Nad. Acad. Sci. U.S.A.,15 (1978) 5250-5254. (191) A. Warshel, Acc. Chem. Res.. 14(1981) 284-290.

GLYCOSIDE HYDROLASES

38 1

that exclusion of water from the active site creates, for the transition state, a "supersolvent" which greatly lowers the free energy of activation. Conclusions were based on theoretical calculations for the heterolytic cleavage of a glycoside bond at the active site of lysozyme and water, respectively. The developing charges of the transition state are stabilized much better by the fixed dipoles of the enzyme than by the dipoles of fluctuating water molecules. c. Sequence Homologies around the Catalytic Amino Acids.-Evolution of glycosidases is expected to have produced binding sites adapted to the subtle structural differences of the sugars of their natural substrates, and to the more widely varying structural features of the aglycon moieties. As seen from the results of inhibition studies, most of them seem to effect catalysis by the same basic mechanism, and it may be asked to what extent this functional homology shows up in homologies of primary structure. Labeling experiments with conduritol epoxides have permitted determination of the amino acid sequence around the essential aspartate (glutamate) with the following results. Lysozyme (hen egg)6

-Asp-Met-Ser-Thr-Asp-Tyr-Gly-Ile-Leu-GlnP-D-Glucosidase A, (Asp. ~ e n t i i ) ' ~ ~

-Val-Met-Ser-Asp-Trp-Ala-Ala-His-His-Ala-Gly-ValP-D-Glucosidase A (bitter almonds)167

Ile-Thr-Glx-Glx-Val-Phe-Gly-Asp-Ser-(Ala, Asx,, Glx, Pro)-Lys P-D-Glucosidase (human placenta, lysosomal)168 -Val-Ala-Ser-Gln-Lys-Asn-Asp-Leu-Asp-Ala-Val-Alaa-D-Glucosidase (sucrase, rabbit small intestine)'69 -1le-Asp-Met-Asn-Glu-Pro- Asn-

a-D-Glucosidase (isomaltase, rabbit small i n t e ~ t i n e ) ' ~ ~ -G1y-GIy-Gln-Ile-Asp-MetP-D-Galactosidase (Escherichia c ~ f i ) ' ~ ~ -Ser-Leu-G1y- Asn-Glu-Ser-G1y-His-GIy-AlaNo sequence homologies can be detected. This is, perhaps, not surprising. The X-ray structure analysis of lysozyme by Phillips6 has shown that the polypeptide chain is folded in a way which puts none of the amino acids in sequential vicinity of the catalytic Asp-52 and Glu-37 that are near to the bound substrate. Comparable folding patterns can probably be realized with widely differing arrangements of amino acids, and thus the apparent lack of homologies.

382

GUNTER LEGLER

d. Distortion of Substrate and Catalysis.-From the X-ray analysis of the lysozyme-chitotriose complex, it had been deduced that an N-acetylglucosamine residue could not be accommodated at the presumed cleavage site (sub-site D) unless its 4C, conformation was distorted towards a half-chair conformation resembling that of the presumed ionic transition-state. This, and the strong inhibition of lysozyme and most other glycosidases by hexono-1,5-lactones of appropriate structure (see Table 11), led to the proposal that substrate distortion towards the transition state constitutes a major contribution to catalysis. A later X-ray studyIg2 of another lysozymetrisaccharide complex, where the inhibitor occupied sub-sites B to D, revealed, however, that an N-acetylmuramic acid residue can be accommodated at sub-site D without distortion of the chair conformation, with only minor structural readjustments of the polypeptide chain, and calculations of the energies involved in these readjustments showed that the enzyme adjusts its conformation to the requirements of the ligand, rather than forcing a drastic conformational change upon the latter. 193 According to W a r ~ h e l , ' ~ .the ' ~ ' energy of the transition state is mainly lowered by electrostatic interactions of the substrate with the combined dipole and charge field of the catalytic site. Strong binding of hexono- 1 3 lactones and lactams would then reflect the interactions of these polar structures with the local field. 1,6Lactones would inhibit much less tightly, because of their different steric requirements. The relatively weak inhibition of a-D-glycosidases by 1,5-lactones can be explained by an adaptation of their active-site field to the axial dipole of the substrate, which differs strongly from the dipole of the lactones. In addition, hydrogen bonds with the acidic group involved in proton transfer to the glycosidic oxygen atom might form with a lactone bound to a P-D-glycosidase, which is not possible with an a-D-glycosidase. e. Slow, Tight-binding Inhibitors and the Transition State.-A number of basic sugar analogs having k-values below M (see Tables VI and VII) show a slow approach to the inhibition equilibrium, that is, the rate of association is considerably below the diffusion-controlled limit. The phenomenon of slow inhibition in the absence of covalent interaction with the enzyme was discussed by S c h l o s ~in' ~terms ~ of a high affinity of the enzyme for the transition state, and a somewhat lower affinity for reaction interme-

(192) J. A. Kelly. A. R. Sielecki, B. D. Sykes, M. N. G . James, and D. C. Phillips, Nature, 282 (1979) 875-877. (193) A. Warshel and M. Levitt, J . Mol. Biol., 103 (1976) 227-231. (194) J. V. Schloss. A K . Chon. Res.. 21 (1988) 348-353.

GLYCOSIDE HYDROLASES

383

diates more stable than the transition state. Release of these (metastable) intermediates caused by a lower binding-energy should be prevented. The thermodynamic dissociation constant K, is related to the standard free-energy of binding, AGO, and the kinetic constants for association k,and dissociation k,, by K, = b s / k , = exp (-AG”/RT), that is, slow dissociation and a low value for AGO require a low rate-constant k,. Slow binding of structural analogs is thus seen to be a consequence ofthe thermodynamics of keeping reaction intermediates bound at the active site. Application of this argument to the slow inhibition ofglycosidases by basic sugar analogs would point to the occurrence of a glycosyl-oxocarbenium ion on the reaction pathway. Partial formation of such an intermediate has been inferred from studies on secondary deuterium-isotope effects with lysozyme,195 p-D-galactosidase from E. ~ o l i , ~ O ’and p-D-glucosidasefrom Asp. wentii. Is Studies with P-D-galactosidase (from E. coli) and substrates where deglycosylationis rate-limiting2*have revealed similar isotope-effects, indicating that, in this step, also, a change in hybridization from sp’ to sp2is on the reaction pathway. In cases where no isotope-effect can be detected, for example, p-D-glucosidase from almonds,195a step different from the bond-breaking steps, such as a conformational change, could be rate-limiting. 2. Generalizations and Exceptions

Published data on the reversible inhibition by basic sugar analogs, and active site-directed inactivation by conduritol epoxides, show that the majority of “retaining” a- and p-glycosidases have an active site that features a proton-donating group close to the glycosidic oxygen atom, and a carboxylate group that can stabilize the positive (partial) charge developing on the anomeric carbon atom and that may possibly form a covalent glycosylenzyme intermediate that is subsequently hydrolyzed. There are, however, a number of cases that reportedly do not correspond to the inhibition pattern discussed in the preceding Sections. It may be asked whether these exceptions represent enzymes acting by different mechanisms, or if there are other causes for their apparent failure to be strongly inhibited by basic glycon analogs and be covalently inactivated by epoxides of appropriate structure. In many cases, inhibition studies were not carried out to obtain information on the reaction mechanism, but for other purposes. Thus, only inhibitors were tested that were considered suitable for the particular project, for example, studies on the biological function of the enzyme where glycosylamines and aldonolactones are unsuitable. Inhibition by 1 3 - and 1,4-dide(195) F. W. Dahlquist, T. Rand-Meir, and M. A. Raftery, Biochemistry. 8 (1969) 4214-4221.

384

GUNTER LEGLER

oxyiminohexitols may be weak, because structural readjustments required for optimal interaction with the endocyclic nitrogen atom are difficult, or the enzyme requires specific interactions with the aglycon for optimal orientation (induced fit) of the catalytic acid, or the carboxylate group, or both. They may fail to be strongly inhibited by the indolizine type of inhibitors because steric interactions or conformational constraints prevent strong binding. A failure to be inhibited by epoxides of appropriate structure may have one of the following causes: (i) specific aglycon interactions are required for protonation of and reaction with the epoxide, (ii) a-D-glycosidases react too slowly because of the energetic barrier of the tramdiequatorial ring-opening. Effective protonation of the epoxide is not possible, due to a limited flexibility of the active-site acid (for example, with the cytosolic p-D-glucosidase from calf liverIs9),the positive charge on the anomeric carbon atom is not stabilized by a carboxylate group but by the negative end of an a-helix of the polypeptide chain, which may give a ~tabilization'~~ of 50 kJ/mol, and the enzyme acts with inversion rather than with retention of the anomeric configuration. Careful and extensive studies are obviously required before a new reaction mechanism can be postulated.

-

(196) W. G. Hol, P.T. van Duijnen, and H. J. C. Berendsen, Noture. 273 (1978) 443-444.

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

A

Alivisatos, B., 200 Allaudeen, H. S., 247 Allen, D. R., 29 Allinger, N. L., 93 Alpert, N. M., 199,204(475) Altona, C., 241, 242(672) Alvernhe, G., 169 Alves, R. J., 150 Ambrose,M.G., 124, 129, 130(183), 139(160) Amico, L. A., 247 An, S. H., 131 An, S.-H., 153, 239(261), 239(262) Anderson, M., 164 Anderton, W. J., 281,304, 306( 140) Andreasen, N. C., 189 Anemura, A., 293, 308 Anker, D., 134, 141(200), 167 Annaka, M., 106 Ansari, A. A,, 286 Ansari, A., 199,201(479) Antonov, 1. V., 242 Aoki, H., 182 Appelman, E. H., 192 Appleman, E. H., 178 Ara, M., 29, 34(31), 56,60(31) Araki, Y., 101, 107(71), 112(71), I16(71, 72, 73), 117, 123(72) Arcamone, F., 72 Argentini, M., 134, 195 Ark, B., 293, 304 Arifkhodzhaev, K. A,, 304 Arison, B. H., 24,27( 14), 37( 14) Arison, B., 274 Armstrong, C. R., 206, 221(541) Arnett, C. D., 191, 195, 203(399) Arnold, E., 342 Arnold, W., 102, 142 Aronson, N. N., Jr., 346 Asai, M., 74 Asano, N., 52, 53, 74(70), 88

Abaas, S., 304 Abbas, S. A., 136. 149, 212, 218(250), 240(2 12) Abe. M., 177 Abe. R., 232 Abe, Y., 34, 77, 192, 194, 202, 203 Abeles, R. H., 364 Abraham, R. J., 93 Abramov, V. S., 120 Abrams, D. N., 246, 254 Achiwa, K., I 14 Ackerman, R. H., 199, 204(475) Adachi, T., 55 Adam, A,, 308 Adam, M. J., 178, 179(336), 194 Adamson, J., 170, 171, 172(297), 175(296, 297), 177(302), 224 Adelstein, S. J., 203 Adeyeye, A,, 304 Agbanyo, F., 209 Agbanyo, M., 209 Agranof, B. W.. 188 Ahonen, A., 202,203 Ajl, S. J., 280 Ajmera, S.. 256, 26 I , 262 Akimoto, K., 106 Al-Jobore, A., 209 Alavi, A., 187, 189(358), 199, 200, 201, 202(494) Alavi, J.. 200. 201(494) Albano, E. L., 170 Albers-Schonberg, G., 24, 27( 14), 37( 14) Albersheim, P., 298 Albert, R., 136, 137, 153,224,225.226. 226(2 13) Alexandrova, L. A,, 273 Alexoff, D. L., 191 Alexoff, D., 178, 193(337), 194(394) Alhadeff, J. A., 367, 369( 164) 385

386

AUTHOR INDEX

Asawa, N., 347 Ashley, G. W.. 246 Ashton, F. E., 28 I , 294 Ashton, W. T., 274 Ashwell, V., 280 Aspinall, G.O., 280 Atkins, H. L., 202 Atkins, H., 199, 201(479) Ator, M. A,, 245 Atrazhev, A. M., 258 Au, J. L.-S., 264 Augy. S., 66 Autrey, R. L., 29 Axelrod. B., 333, 346, 379 Azuma, I., 308 Azzaro, M., 168

B Baba, M., 258, 259(167, 778, 779), 269, 270(843), 272(778), 273 Backinowsky, L. V., 304 Baddiley, J., 299, 316 Bader, D. E., 375, 378(184) Badone. D.. 139 Baer, H. H., 132, 166, 167(288), 231, 232(626) Bailey, R. W., 30 I , 302( 122) Bailey, S., 274 Baillargeon. D. J., 155, 157(265) Bais, S., 201 Baker, B. R., 364 Baker, D. C., 155, 157(266), 276 Baker, J. J., 264 Ballou, C. E., 299, 300( 1 13). 302( 1 I3), 322 Balzarini, J., 258, 259(767, 778, 779). 269, 270(843), 272(767, 778), 273 Bamberger, F., 338 Banaszek, A., 125 Bandoypadhyay, D., 202 Banks, R. E., 93 Bannai, K., 148 Banoub, J. H.. 301, 302(127), 307, 308 Bapat, A. R., 256,262 Baptistella, L. H. B., 131, 132( 190). 166, 232( 190) Bar-Guilloux, E., 72, 2 I I Baranenkov, V. I., 93, 243( 17)

Barbalat-Rey, F., 183 Barker, S. A,, 29 I Barnett, J. E. G., 357, 358, 369 Barnickel, G., 308 Baron, J. C., 191, 201(400) Bamo, J. R., 175, 178(318), 191, 192(313, 314, 317, 318), 194(314), 195, 198(434), 201, 202 Barrow, K. D., 289 Barthwick, A. D., 155, 158(217, 247, 268), 165(268), 253(268) Barton, D. H. R., 66, 170 Ban, W.. 361 Bastos, M., 196 Bastow, K. F., 258 Batley, M., 290 Bauer, W. D., 96 Baumbartner, H., 258 Bause, E., 336, 338, 339, 340, 352. 361(123), 366(82), 367, 368( 123), 371(165), 377(82), 381(123) Beabealashvilli, R. S., 258 Beaney, R. P., 187, 202 Beattie, T. R., 148, 155, 157(242, 243), 162(273), 165 Beauchamp, L., 248,274(7 18) Becher, W., 336 Beckmann, R., 194, 195(425), 196 Beeley, P. A.. 195, 198(438) Bell, C. A., 250, 276(734) Bell, E. A.. 342 BeMiller, J. N., 319, 321(1), 322, 324(1), 327( I), 358( I ) Beneath, A., 256 Benigni, D. A,, 238, 239(662), 247, 248(704), 250(704) Benua, R. S., 202 Beranek, J., 252, 261 Berendsen, H. J. C., 384 Berezin, I. V., 108 Bergman, J., 195 Bergstrom. D. E., 234, 237, 266 Bergstrom, D., 260, 266(787) Bernacki, R. J., 150. 152, 153, 183(260), 239(262) Bernstein, D. R., 204, 208(533) Bemdge. M. S., 195 Berzina, A,, 265 Bessel, E. M., 95, 139(38)

AUTHOR INDEX Bessell, E. M., 188, 189, 208(373) Beuberger, A,, 322 Beurret, M., 306 Beyer, R., 30 I Bhacca, N. S.. 94. 95(28), 104(28), 106(28). 163(28) Bhattacharya, A. K., I I I Bida, G. T., 175, 178(318), 190, I92(3 13, 314, 318), 194(314), 195. 197, 198(434) Bieberich, E.. 329, 331(31), 335(31), 337(31). 367, 377(159), 384(159) Biely, P., 206, 207(542) Biggadike. K.. 139. 148, 152(247), 155, 158(217, 247, 248, 268), 165, 252(247), 253(268), 272(248, 282) Binder. T. P.. 214, 216 Bingham, E. M., 142 Binkley, R. W., 124, 129, 130(183), 139(160) Binkley, S . B., 297 Birch, G. G., 49 Bird, P.. 207 Bird, T. G. C., 148 Birkhed, D., 288 Birnbaum, G. I.. 243 Bischofberger, K., 14 I Bishop, D., 367 Biziori. F., 72 Bjorndal, H.. 301, 302( 119) Blackburn, C. M., 185 Blake, C. C. F., 320, 325(6), 326(6), 371(6), 381(6) Blanc-Muesser, M., 12 1 Blandin, M., 234, 242, 243(675) Blessing, G., 190 Bliard, C., 107, 127(97), 144, 145(97), 156(97). 171(238), 173(238) Block, D., 196 Blumenauer. G., 12 I Bobek, M., 152, 153,239(261,262), 251 Bock, E.. 287 Bock, K., 72. 89, 94, 95. 96, 1 I 1 Bodenteich, M., 258 Bodo. G.. 352 Boehme, R., I39,277(2 18) Boeyens. J. C. A,, 174 Bohak. Z., 374. 375(181) Bonaly, R., 308 Bonenfant, A,, I83 Booth, B. R., 165, 272(282)

387

Booth, G. E., 35, 37(41) Booth, G., 35 Boothe, T. F., 198 Borgmann, C., 339 Borrelli, E., 247 Borthwick, A. D., 139, 148, 152(247), 158(217, 247, 248), 165, 252(247). 272(248, 282) Bosch, A. L., 198, 199(467) Bosch, P., 124 Bosso, C., 99 Bostedor. R., 274 Boulton, K., 148 Bourbouze, R., 338, 341(73), 369(73) Bradaczek, H., 308 Brade, H., 295, 297, 298, 308 Bradley, P.R., 32 I Brady, F., 195 Branefors-Helander, P., 289. 3 13. 3 I6 Braun, H., 368 Braun, P. J., 22 I Bremer, M., 370 Brennan, P. J.. 28 I , 308, 309( 150) Brewer, C. F., 96, 116(43), 350, 35 I , 352(114, 117, 118), 353(114, 117), 354(118), 356, 357, 358, 359, 379( 129, 130, 134, 135) Brill. A. B., 202 Brimacombe, J. S., 134, 29 I Bringnole, A., 72 Brink, A. J., 174,260 Brinkman, G. A., 197 Brisson, J. R., 294 Brisson, J.-R., 316 Bristow, N. W., 43 Brockbank, R. L., 329 Brockhaus, M., 354, 356( 128) Brodack, J. W., 197 Broder, S., 258,259(767), 269, 270(844) Brodfuchrer, P. R., 133, 238, 239(662), 247, 248(704), 250(704) Brodie, J. D., 200 Brooks, D. J., 187 Brooks, R. A., 187,202 Broquist, H. P., 342, 344(88), 346(88) Brostow, W., 242 Brown, R. B., 304 Brown, R. G., 164, 293, 304(79a) Brown, S . G., I93

388

AUTHOR INDEX

Brown, W. J., 200 Brownell, G. L., 198, 199,204 Brownridge, E., 248, 249(7 I I ) Brox, L. W., 245 Brubaker, R. R., 283,286 Brundidge, S. P., 133, 238, 239(662) Brunngraber, E. G., 149, 158(253), 160(253) Buchanan, J. G., 316 Buchschacher, P., 25, 81( 18) BudESinsky, M., 164, 183 Buncel, E., 321 Bundle, D. R., 292 Burchenal, J. H., 248, 272 Burgess, F. W., 277 Butchard,C.G., 171,172(304), 173(305),207 Butler, L. G., 379 Butt, S., 165,272(282) Buttner, W., 258 Byers. L. D., 328, 331(29), 332(29). 335(29), 337(29), 340(29) Byrd, R. A., 250 C

Cabezas, J. E., 330, 331(42b) Calvo, P., 330, 331(42b) Cambor, C., 277 Cameron, J . M., 165, 272(282) Camiener, G. W., 245,255(686) Camps. F., 124 Canning, L. F., 274 Cantaeuzene, J., 93 Capec, K., 310 Capon, B., 3 19 Card, P. J., 93, 149, 150(20), 158(254, 255), 160(254,255), 161(254, 255), 162(255), 204, 216(539), 217 Caroff, M., 292 Caron, G., 372 Carpino, L. A., 124 Carroll, S. T., 198 Carson, D. A., 270, 27 1(846), 272(846), 277(846) Carson, R. E., 200 Carson, R., 202 Carvacho, 0. F., 196 Casella, V., 187, 189(358), 190, 191(31 I), 199,201(479),204(311) Casimir, J., 345 Castillon. S., 128, 131, 132(182, 190), 145,

147(182), 156(182), 157(182), 166, I7 1(240), 232( 190, 240) Castle, T. M., 72 Catlin, J. C., 242,243(675) Cavaignac, S., 301, 302(128) Cavanagh, K. T., 338, 366(76), 368(76) Cech, D., 244,257,264,265,266 Cenci deBello, I., 338 Cepeda, C., I9 I , 20 I(400) Cereny, M., 23 1 Cermak, R. C., 23 Ctmy, M., 164, 183, 208,231(555) Chaby, R., 290 Chait, B., 256 Chambers, J. P., 343, 344(90, 93) Chamorro, E., 124 Chan, T. H., 239 Chawluk, J. B., 201 Chawluk, J., 200, 201(494), 202(494) Chen, M. S., 244, 247, 248(704), 250(704), 264(684) Cheng, D. M., 269 Cheng, Y., 254,258 Cheng, Y.-C., 246,247, 248, 249(699), 250, 256(735) Cherednikova, T. V., 108 Cherniak, R., 293 Chernow, M., 133,239(194) Chiao, Y.-B., 330, 331(36) Chiasson, J. B., 242 Chiba, S., 357 Chida, N., 3 I , 37(35),53,54,55,70,75,77,78 Chigeavadze, Z. G., 258 Chiou, J.-F., 247,249(699) Chirakal, R., 177, 196 Chiro, G. D., 202 Chittenden, G. J. F., 3 16 Chizhov, 0. S., 108, 121, 212, 304 Chou, T.-C., 237, 239(661), 248, 249, 250, 254, 255, 256(66 I , 735,752, 753), 272 Choy, Y. M., 305 Christian, R., 297 Christin, A., 195 Christman, D. R., 141, 199, 200,201(479), 202 Christman, D., 200 Chu, C. K., 234,235(639), 238(639), 246(639), 248(639), 249(639), 250,272, 276(734) Chu, S.-H., 277

AUTHOR INDEX Chucholowski, A., 107, 108(102), 116, 117(140), 145 Chun, M. W., 234,235(639), 237, 238(639), 246(639), 248(639), 249(639), 256 Cima, R. R., 196 Claeyssens, M., 359 Clardy, J., 342 Clark, J. C., 196 Clark, J. H., 126 Clayton, J. D., 23 Cleophax, J., 66, 224 Cobbledick, R. E., 2 12 Coderre, J. A., 250 Codington, J. F., 234, 240, 241(664), 248(637), 254(637), 260(664) Coe, E. L., 188 Coenen, G., 335, 350(57), 351(57) Coenen, H. H., 190, 193, 196, 197, 198 Cogoli, A., 330, 331(37) Colacino, J. M., 247, 249, 255 Colacino. J., 247, 248, 249(698, 71 1) Colgate, S. M., 342 Collins, J. G., 289 Colosimo, M., 196 Commertord, S. L., 255 Conboy, C. B., 131,207(186) Conchie, J., 327, 329, 331(32, 33) Congdon, D. D., 195 Conti, P. S., 237 Cook, A. F., 252,261(742) Cook, A., 265 Cook, J. S., 191 Cooper, F., 301, 302( 127) Cooper, M. D., 195, 197(437) Coria, J. A,, 301, 302(128) Coulter, M. S., 93 Coursey, B. M., 189 Cox, A. D., 290 Cox, D. P., 124 Cox, S. W., 165,272(282) Coyle, P. J., 330, 331(36) Crane, R. K., 186 Cromer, R., 22 I Cross, B. E., 148 Crouzel, C., 191,201(400) Csuk, R., 93 Cuno. I., 241, 275, 276 Curatolo, W., 124, 125(166) Cushley, R. J., 240, 241(664), 260(664), 336, 340(68)

389

Cygler, M., 243 Cysyk, R. L., 264

D D’Amore, T., 209 Dafaye, J., 89 Dafe, M., 28 1 Dagani, R., 189 Dagher, A,, I87 Dahlquist, F. W., 383 Dale, M. P., 328, 331(29), 332(29), 335(29), 337(29), 340(29) Damin, L., 247 Danenberg, P. V., 250,256,261,262,265 Daniels, L. B., 330, 331(36) Daniels, P. J. L., 228 Dann, R.,201 Danzin, C., 344 Daoust, V., 281 Dardenne, G., 345 Darvill, A. G., 298 Darzynkjewicz, E., 241 Dashunin, V. V., 310 Datema, R., 207, 377 Daube, M. E., 195 Dawson, W. H., 250 Dax, K., 136, 137, 153, 180, 181(340), 224, 225,226 Day, A. G., 330, 331(35) Dazzo, F. B., 300, 309 De Bruyne, C. K., 334 De Clercq, E., 153, 239(262), 246, 247, 248, 251, 258,259(767, 778, 779), 269, 270(843), 272(778), 273 de Haan, P. E., 116 De Landsheere, C. M., 202 De Miranda, P., 248, 274(7 18) De Villiers, 0. G., 260 Dean, J. A., 91 Declercq, J.-P., 243 Decouzon, M., 168 Dees, M. J., 116 Defaye, J., 72, 97,98, 99, 21 I Deger, H.-M., 96,97(45), 99(45) DeGrado, T. R., 204, 208(533) Dejesus, 0. T., 195, 197(437) DeLaPaz, R. L., 202

390

AUTHOR INDEX

Delaumeny, J.-M., 66 Dell, A,, 292, 295(66), 301, 302(123), 306, 309, 316 Deltenre, R., 195 Demcheva, M. V., 108 Dence, C. S., 197 Denenberg, K., 256 Dengler, T., 297, 298 Derome, A. E., 135, 258(204) Des Rosiers, M. H., 186, 187(357), 189(357) Descamps, J., 246, 247 DeShong, P., 346 Deshusses, J., 368 DesMarteau, D. D., 170 Desnick, R. J., 338, 367, 368, 381(168) Dessinges, A., 125, 143(164), 145, 166, 171, 232(240) Di Chiro, G., 187 Diena, B. B., 28 1 Diksic, M., 175, 187, 190, 191, 192(315, 316, 317), 193, 194(393), 199(359), 200 Dimitrijevich, S. D., 95 Dinur. T., 367, 368, 381(168) Dmitriev, B. A,, 96, 29 I , 294, 295, 298. 304, 307( 108), 310,312 Doboszewski, B., 100, 122(67), 123(67), 127, 128(175), 134(174, 175), 135(175), 137(175), 141(174, 175), 155 Doerr, I. L., 234, 235, 248(637), 254(637) Dolak, L. A., 72 Doleialova, J., 164, 183 Dolle, R. E., 100, 107, 108(64, 65, 102), 116, 117(140), 122(64), 123(64) Dolphin, D. H., 207 Dolphin, D., 212 Domard, A., 99 Donath, A., 195 Donsimoni, R., 338, 341(73), 369(73) Dorling, P. R., 342 Dorling, P., 338 Dosen-Miovic, L., 93 Douste-Blazy, L., 367 Drescher, B., 258 Drewry, D. T., 3 10 Drews, G., 28 I , 292, 302( 13) Driguez, H., 72, 21 1 Driscoll, J. S., 269, 270(844) Drueckhammer, D. G., 208 Du, J., 94, I I1(29), 123(29) Duckworth, M., 299, 310

Dudkin, S. M., 242 Dudman, W. F., 30 I , 302( I 17) Dunlap, R. B., 250 Durham, L. J., 22, 23, 27(1, 12. 13), 36(13), 37(1, 12, 13) Dutschka, K., 199 Dutschman, G. E., 258 Dutschman, G., 246, 247(696), 254 Dutton, G. G.S., 305, 306, 3 12 Dyatkina, N. B., 273

E Easton, M. P., 191 Eby, R., I I I , 114. 120(118) Ehrenkaufer, R. E., 174, 178, 194 Ehrenkaufer, R. L., 188, 190 Einhof, W., 330, 331(40) Eis, M. J., 345 Ekiel, IO., 24 1 Eklund, S. H., 216 El’kin, Y. N., 31 I Elbein, A. D., 343, 344(90), 346, 379(102) Elbein, A,, 336 Elion, G. B., 248, 274(7 18, 7 19) Ellis, G. P., 335 Ellis, J. W., 118 Ellis, P. D., 250 Ellmers, B. R., 343, 344(92) Elmalch, D. R., 197, 198, 199, 204 Elmalch, D., 124, 125(166) Emran, A. M., 198 Emsley, J. W., 93 Endo, M.. 126, 144(170) Endo, S., 202, 203 Engel, J., Jr., 200 Ensley, H. E., 328, 33 l(29), 332(29), 335(29), 337(29), 340(29) Erbing, C., 305, 3 16 Erckel, R., 96,97(45), 99(45) Erecgovic, D., I95 Erhard, A., 344 Esaki, S., 104, 122(92) Escribano, E. C., 150, 156(182) Escribano, F. C., 107, 128, 127(97), 132(182), 144(97), 145(97, 182), 147(182), 156(97), 157(182), 171 Esipov, S. E., 295 Eswarakrishnan, S., 155, 181

A U T H O R INDEX

Ettlinger. M., 329, 332(34). 336(34), 350(34) Etzold, G.. 257, 258(764), 263, 264, 266, 267, 277(800) Evans, A. C., 187, 199(359),252(247) Evans, D., 139, 148, 152(247), 158(217, 247, 248) Evans, R. M., 247 Evans, S. V.. 338, 345 Evans, T. H., 279 Eveleigh, D. E., 353 Evelyn, L., 2 12, 230 Exall. A. M., 139, 148, 152(247), 155, I58(2 17, 247, 248, 268), 165(268), 253(268), 252(247), 272(248, 282) Ezaki, S., 327

F Fabbro. D., 367 Fabgro, D., 338 Fanucchi, M. P., 250, 255, 256(735), 256(753) Farkas, T., 200 Farrell, A. A,, I9 I Fazekas, F., 200, 201(494), 202(494) Fei, C. P., 239 Feinberg, A., 254, 255, 256 Fellows, L. E., 338, 345 Fellows, L.. 338 Fett, W. F., 283 Field, A. K., 274 Field, F. H.. 256 Fife. T. H., 322 Fink, R. W.. 194 Finn, R. D., 198 Firnau, G., 194, 195(422), 196 Fischer, E., 41, 51(5) Fisher, R. A., 338, 366(76), 368(76) Fissekis, J. D., 237 Fitschen. J., 194, 195(425) Flanagan, R. J., 254 Flashner, M., 352 Fleet, G. W. J., 345 Fleet, G. W., 338, 346, 379(102) Fletcher, H. G.. Jr., 330, 333(44) Fontell. A., 195 Foster, A. B., 188, 208(373), 224 Fouron. Y., 275 Fowler, A. V., 374, 375(179)

39 1

Fowler, J. G., 203 Fowler, J. S., 188, 189, 190, 191, 194(394, 41 I), 195, 199, 200,201(479), 202 Fowler, J., 199, 200 Fox, J . J., 234, 235, 236, 237, 238(639), 240, 241(664), 243, 245, 246, 247, 248, 249, 250,251(657), 252, 254, 255, 256, 260(647, 664). 270, 271(845), 272, 276(647, 734) Frackowiak, R. S. J., 187 Franken, K., 190 Fraser, B. A,, 308 French, D., 221, 354 French, R., 198 Frey, K. A,, 188 Frey, P., 195 Fritz, H., 356, 379(130) Frolova, G. M., 289 Fromme, I., 28 I Frommer, W., 24, 52, 81(69), 88(69), 336, 338(60), 347, 348( 106) Fuhrmann, U., 336 Fuhs, E.-F., 233 Fujisawa, K., 225, 228(607) Fujita, T., 33, 36(37), 37(37) Fujiwara, T., 202, 203, 268 Fukabori, C., 41,45(50, 51), 46(49, 50, 51), 48(49) Fukase, H., 52, 53, 65, 74(70), 88(72), 192, 194, 202, 203, 207(419) Fukumura, T., 196 Fukuoka, K., 261 Furihata, K., 24 Furman, P. A,, 248, 274(7 18) Furst, A., 368, 369( 171) Furuta, S., 22, 23, 27(1, 12, 13), 36(13), 37(1, 12, 13) Fyfe, J. A., 248, 274(718)

G Gadelle, A,, 97, 98, 99 Gadler. H., 237, 239(661), 248, 251(713), 256(661) Gaertner, K., 257, 258, 259, 264, 277(800) Gallagher, B. M., 199, 201(479) Gallbraith. L., 281 Galoyan, A. A., 95, 107, 108, 1 1 I , 120, 122(35, 36), 123(35)

392

AUTHOR INDEX

Galoyan, A. G., I 19 Ganem, B., 345, 371, 375(177) Gao, Y.-S., 266 Garcia, J., 128, 132(182), 145(182), 147(182), 156(182), 157(182) Garegg, P. J., 305, 306 Garnett, E. S., 177, 192(327), 196 Garnett, S., 194. 195(422) Gamier, J., 349 Gasol, V., 124 Gati, W. P., 254, 260 Gatley, S. J., 190, 193, 194, 195, 196, 197(437), 198, 199, 204,208(533) Gatt, S., 367, 368, 381(168) Gaunt, M. T., 334 Gaylord, H., 308, 309( 150) Ge, S.-G., 367 Geissbuhler, A., 195 Gelas, J., 21 1 Genghof, D. S., 96, I16(43), 350, 351(114), 352( 114), 353( 114), 358, 379( 134), 379(135) Gentile, B., 45 Geribaldi, S., 168 Gerig, J. T., 93 Germain, G., 243 Germenhausen, J., 274 Gero, S. D., 66, 224, 369 Gershon, E., 365, 366(151), 370(151) Ghai, S. K., 293 Ghazzouli, I., 247,248(704), 250(704) Ghuysen, J.-M., 308 Giesbrecht, P., 308 Gill, M. J., 237 Gilson, A. J., 198 Ginos. J. Z., 198 Girault, Y., 168 Giziewicz, J., 254 Glanzer, B. I., 93, 180, 181(340) Glaudemans, C. P. J., 119, 120( 148), 134, 136, 143, 145(148), 146(148), 156(148), 161(209), 163(202),209(210), 218, 219, 220,221 Clew, R. H., 330, 331(36) GligorijeviC, M., I2 1, 124(158), 128(158) GliSin, D., 12 I , I24( I58), I28( 158) Glusker, J. P., 92 Gnade, B. E., 194 Godinho, L. S., 170 Goldman, D., 200

Goodman, M.M., 199, 204(475) Goodman, M.,204 Gorin, S. E., 295 Gorshkova, R. P., 284, 285,288, 289 Goto, M., 155, 158(267), 158(267) Goto, T., 1 14 Gotschlich, E. C., 308 Gottschalk, A., 322 Could, E. S., 33, 36(38) Goulding, R. W., 196 Grabowski, G. A., 338, 339, 344(81), 367, 368, 381(168) Graetz, H., 258 Grant, A. J., 254, 255 Gray, G. R., 299 Gray, G. W., 310 Greenberg, J. H., 199 Greenberg, J., 187, 189(358), 199(358),200 Greenberg, N., 237,248,249(71 l), 250(659), 25 l(659) GregorEiE, A., 175, 176(323), 177 Greitz, T., 189 Griengl, H., 25 I , 258 Crier, T. J., 204, 207, 209(537) Griffiths, L., 93 Grindey, G. B., 246 Grindley, T. B., 164 Grobner, P., 265 Groen, J., 248 Grover, A. K., 336, 340(68) Grynkiewicz, G., 100, 122(67), 123(67) Gubanova, N. Y., 283 Guerrero, A., 124 Guo, W., 219 Gujar, M. K., 166, 231(286) Gurvich, L. G., 93 Guschlbauer, W., 235, 241, 242,243(675), 250 Gushlbauer, W., 256 Gustafsson, B., 286, 292,298 Gutterson, N. I., 199 Gvozdyak, R. I., 283

H Haaparanta, M., 195, 202 Hadfield, A. F., 21 I , 212(570) Haertle. T.. 256 Hagami, E.', 188

AUTHOR INDEX Haines, S. R., 95, 99, 100(63), 101, I17(63), 118(63), 122(77), 123(77) Hakim, A., 187 Hakoshima, T., 268,269 Halama, J. R., 204, 208(533) Hall, F. H., 93 Hall, L. D., 93,94,95, 104(28), 106(28), 134, 139(38), 163(28), 170, 171(297), 172(297), 175(296,297), 177(302), 178, 179(336), 183, 212, 230 Hall, R. H., 141 Halton, D. M., 209 Hamacher, K., 197, 198 Hamada, M., 72 Hamatsu, T., 106 Hancock, C., 255 Hand, P., 200 Hanessian, S., 133, 294, 310(80) Hanozet, G., 332, 337(46), 340(46) Hantz, O., 247 Hanzawa, Y., 182, 185 Hara, H., 24, 87 Hara, Y., 261 Harada, K., 252 Harada, T., 293,308 Harada, W., 320, 326(7) Haradahira, T., 125, 126, 128, 130, 131, 198,207 Harder, A,, 368, 38 1( 167) Hardt, H., 97 Harkonen, P., 202 Harnden, M. R., 274 Harper, P. V., 194 Hams, G., 246 Hams, L. S., 299 Hams, T. M., 312, 342,344(87) Hasegawa, A., 112, 115, 155, 158(267), 268 Hash, J. H., 3 12 Hashimoto, A,. I82 Hashimoto, H., 141 Hashimoto, S.. 104, 104(61), 107(87), 122(68), 123(68) Hashimoto, Y., 99, I12(61), 123(61) Haskell, T. H., 294, 310(80) Hasnain, S. N., 365, 366( 149), 368( 149), 369( 149) Hatazawa, J., 187, 202, 203 Hattori, Y., 31, 37(33), 50(33), 60(33) Hawkins, E. S., 276 Hay, A. J., 329, 331(32, 33)

393

Hay, G. W., 100, 122(67), 123(67), 127, 128(179, 134(174, 173, 135(173, 137(175), 141(174, 175), 155, 195, 198(438) Hay, J., 250 Hayashi, E., I12 Hayashi, M., 100, 104, 107(70, 87), 122(68), 123(68, 70) Hayashida, M., 65 Hayashilda, M., 86 Hazato, A,, 148 Heather, J. D., 187 Hehemann, D. G., 129, 130( 183) Hehre, E. J., 96, 116(43), 350, 35 I , 352( 114. 117, 118),353(114, 117),354(118), 356, 357, 359, 379( 129, 130, 134, 135) Heiker, F. R., 64 Heinemann, V., 246 Helfman, W. B., 245 Hellerqvist, C. G., 3 12 Helus, F., 194, 195(421) Hems, R., 134, 135 Henbest, H. B., 50 Hendler, S. S., 245 Henrissat, B., 2 I I Henvey, J. C., 276 Henze, E., 201, 202 Herdewijn, P., 234, 258,259(767, 778, 779), 269, 270(843), 272(635, 767, 778), 273 Herezegh, P., 144, 171(238), 173(238) Hermansson, K., 293, 304(79a) Herold, S., 187 Herrchen, M., 334, 338, 365, 366(76), 367, 368(76, 148), 369(162), 371( 162). 372(162), 381(148) H e m a n n , G., 264,265 Hersheid, J. D. M., 192 Hertel. L. W., 182, 239(344), 246 Hesse, R. H., 170, 171(297), 172(297), 175(296, 297) Hettkamp, H., 340, 366(82), 377(82) Hey, H. J. C., 101 Heyman, R., 247 Heyns, K., 294 Hibbert, H., 279 Hichwa, R. D., 190, 194, 204(391) Hickman, J., 280 Hicks, K. B., 283 Hiedtke. H.. 367 Hignett,' R. C., 28 I

AUTHOR INDEX

394

Hildebrandt, J., 225 Hill, R., 322 Hillebrand, I., 347,348(106) Himmelspach, K., 298 Hirano, A., 201 Hirota, K., 237,246(649), 247(649),

248(649), 250(649) Hisamatsu, M., 293 Hitz, W. D., 204,216(539), 217 Hobbs, D.W., 274 Hodder, H. J., 308 Hodgson, P.G., 152,183(260) Hoekstra, A., 192 Hoffman, E. J., 187,189,199(362),200,

201,202

Howell, H. G., 133,238,239(662),247,

248(704), 25O( 704) Hrabak, E. M., 300 Hiebabeckq, H., 252,26I Hsi, M., 247 Hsiung, G. D., 249 Huang, E.-S., 247,248,249(699) Huang, J.-T., 272 Huang, S.-C., 187,189,199(362), 200(379),

201,202,203 Huber, R.E., 329,334,375,378( 184) Hughes, R. G., Jr., 25 I Huizinga, W. B., I18 Hull, W. E., 192,193,198(404) Hunter, S . W., 308,309(150) Hurlbert, R. E., 281 Hurtig, H. I., 201 Huszar, I., 195 Hutchins, G. D., 195 Hutchins, L. G., 198,I99(467) Hutchinson, J. P.,155 Huxtable. C. R., 342

Hoffman, E., 187,189(358), 199(358) Hoffman, J., 283,286,287 Hoffmann, J., 284 Hofman, I. L., 304,3 10 Hofmann, P., 308,3 I2 Hofstad, T., 284,286,287,309 Hohenschutz, L. D., 342 Hol, W. G., 384 Holden, J. E., 204,208(533) Hollingsworth, R. I.. 308,309 I Holm, U.,194,195(425), 196 Holman, M.J., 252,261(742) Ido, T., 175. 187,189(358), 190,191,192, Holme, T., 286,292,298 194,195,199,201(479),202,203, Holy. A., 259 204(31 I , 532). 207(419,530).210 Homma, T., 94 Igarashi, K., 94 Honeyman, J., 335 Igarashi, T., 282 Hood, J. T., 195 Iida, H., 188 Hoop, B.,Jr., 199,204(475) Iida. T., 68 Hoppes. D. D., 189 limura, Y., 22,37(2,3), 41,42(47,48), Hori, S., 347 44(2,3) Horii, S., 26,52,53,65,74,87,88(72) litaka, Y.. 182,185 Horikoshi, K., 327 Ikeda, C.. 72,88( 106) Horton, D., 180,233 Ikeda, K., 114 Horwitz, B.,200 Ikehara, M., 250,268,269,276(734) Hiisel, W., 367 Illig, H.-K., 350,352(1 IS), 354( 115) Hoshino, M., 22,23,37(2,3), 41(2,3). 44(2, Imahori, Y ., 194,203,204(532), 207(4 19) Imamura, P.M.,13 I , 132( l9O),232(190) 3) Hoshino, 0..303 Imoto, M.,I12 Hosie, L., 343,344(9 1) Imura, J., 268,269 Hostad, T., 283 Inagaki, F., 269 Hotta, Y., 41,45(51),46(51) Inazawa, K.. 182 Hough, L., 44,135,I36(206), 139(206), Ingvar, D. H., 189 166,167(285), 169(285), 21 1 , 212(570), Inoue, I., 55 215,216(578) Inoue, K., 3 I3 How, M. J., 29I Inoue, M., 78,79(117)

AUTHOR INDEX Inouye. S., 25, 81(21), 331, 332, 335, 336, 337(45), 338(58), 348(45) Irie, T., 191 Irisawa, J., 94 Isaka, A,. 63 Isakov, V. V., 284, 288, 289, 31 I Ishibashi, M., 30 I , 302( 129) Ishibashi, T., 28, 37(25) Ishido, Y., 101, 107(71), 1 l2(7l), I l6(7l, 72, 73), 117, 123(72) Ishiguro, E. E., 297 Ishikawa, M., I3 1 Ishikawa, N., 101, 126, 143(74, 75) Ishitsuka, H., 26 I , 265 Ishiwata. K., 193, 194, 203, 204(532), 207(419, 530) Ito, H., 75 Ito, M., 36, 37(44), 38(44), 202. 203 Ito, R., 104, 105, 122(92) Ito. T.. 286, 331, 337(45) Ito, Y., 101, 104(79), 115, 116, 122(79), 149(79), 221(79) Itoh. J., 25, 81(21) Itoh, K., 101, 107(71), I 12(71),I 16(71,72), 123(72) Itoh, M.: 192, 203 Ittah, Y., 136, 209(210) Ivanova, E. M., 244 Iwahashi, H., 268 Iwakiri, H., 101, 143(74, 75) Iwamatsu, K., 25. 81(21) Iwamoto, M., 190, 195(385) Iwasa, T., 24. 26, 52(15, 23). 74, 347 Iwasaki, S., 31, 37(33), 50(33), 60(33) Iwasawa, Y., 31, 33(34), 34(34), 36, 37(34, 43,44), 38(44), 5 I (43), 63(43), 77, 78, 79( I 17), 82, 83, 84, 85 Iwata, R., 192. 193, 195, 203, 203. 204(532) Izuma. I., 28 1

J Jacoby, W. B., 363 Jaeger, J., 200 James, F., 342 James. M. N. G., 382 Jamieson, A. T., 250 Jankowski, A. W., 207 Jankowski, K., 234,241,242

395

Jann, B., 282, 283, 291, 292, 297, 298, 301, 302( 126a), 308, 312 Jann, K., 282, 283, 291, 292, 297. 298, 301, 302( I26a), 308, 3 12 Janssen. G., 258, 259(778), 272(778), 273(778) Jansson, J.-E., 282 Jansson, K., 105, 137(95), 140, 150(221), 155, 162(272), 213(272), 214(95, 272) Jansson, P.-E., 289, 301, 302(123), 304, 305, 306, 308,309,3 I2 Janta-Lipinski, M. V., 258 Janta-Lipinski, M., 257, 258, 266, 267, 273 Jantzen, R.,93 Jarvis, W. T. S., 358 Jaworska-Sobiesiak, A., 166, 167(288) Jeanloz, R. W., 294, 303 Jeffrey, G. A., 274 Jenkins, I. D., 95, 262 Jennings, H. J., 292, 294, 316 Jensen, M., 294 Jeon, G.-H., 267 Jerabek. P. A,, 195 Jewess, P. J., 342 Jewett, D. M., 178, 194, 196 Jiang, C.. 155, 157(266) Jin, W.-Z., 52 Jivan, S., 194 Joecks, A., 244,257 Joel, D. D., 255 Joensuu, H., 202 Johansson, R., 202 Johnson, L. N., 320. 325(6), 326(6), 371(6), 381(6) Johnson, R. N., 95, 139(38), 170, 171(297), 172(297, 303), 175(297), 177(302) Johnston, G. S., 202 John. K. K., 170 Jolly, D., 175, 191, 192(315, 316), 193, 194(393) Jommi, G., I39 Jones, A. S., 246, 247(694) Jones, C., 306 Jones, E. R. H., 148 Jones, M. F., 244 Jones, M. Z., 338, 366(76), 368(76) Jones, R. S., 293 Jones, S. C., 199 Jones, T., 187 Jones, W. B., 190

AUTHOR INDEX

396

Jordaan, A., 141, 174,260 Joseleau, J.-P., 306 Jovkar, S., 187, 199(359) Jiilich, E., 336, 337(62), 338, 344(70), 366(76), 367, 368(76) Jullien, M., 349 Jung, G . L., 101, 119, 120(148), 145(148), 146(148), l56( 148) Junge, B., 24, 52, 81(69), 88(69), 347, 348( 106)

K Kabir, A. K. M. S., 215, 216(578) Kadentsev, V. I., 121 Kadis, S., 280 Kageyama, S., 230 Kai, Y., 125, 126, 198 Kairento, A.-L., 195 Kaiser, K. P., 202 Kakudo, M., 320, 326(7) Kakuta, M., 282 Kalicheva, I. S., 95, 107, 108, 1 I I , 120(112, 113), 122(35, 36), 123(35) Kalinovsky, A. I., 285 Kalman, T. I., 237,239(661), 256(661) Kalmins, A,, 374 Kalmykova, N., 284 Kameda, Y., 26, 41, 42(47,48), 52, 53, 65, 74, 87, 88, 347 Kamikawa, T., 112 Kamiya, K., 23 Kamiya, S., 104, 122(92) Kan, L. S., 269 Kan, L.-S., 269 Kanayasu, T., 269 Kanazawa, Y., 130, 13I , 207 Kanda, T., 350,351( 117). 352( I17), 353( 1 17), 359 Kandler, O., 294 Kaneyasu, T., 268, 269 Kangouri, K., 24 Kanic, O., 115 Kanno, I., 187, 188 Kappes, E., 336,337(64) Karkas, J. D., 274 Karle, J. M., 264 Karlstrom, K. I., 191 Karpeiskii, M. Ya., 242

Karpiesiuk, W., 125 Karube, Y., 26 I Kasahara, I., 28, 29, 30, 31(32), 36(30), 37(25, 30), 53(30, 32), 54(32) Kasper, D. L., 3 I6 Kassis, A. I., 203 Kastenholz, F., 332 Kasumi, T., 359 Kato, A., 187 Katsuki, M., 110, 119, 213(144) Katzenbeisser, LJ.,I53 Katzenellenbogen, E., 283, 292 Kauffman, S. A., 265 Kaufman, R. J., 96, 160(42) Kaulina, L., 265 Kavai, I., 152, 25 1 Kawada, K., 185 Kawaguchi, H., 72, 88( l06), 88( l06), 225, 228(607) Kawahara, K., 74, 87, 295 Kawai, H., 203 Kearfott, K. J., 199, 204 Keene, L., 280 Keinonen, J., 195 Kell, D. A., 346 Kelley, J. A,, 269, 270(844) Kelly, J. A,, 382 Kemeny, N. E., 202 Kenne, I., 305 Kenne, L., 286, 289, 291, 292, 293, 295(66), 298, 304(79a), 306, 3 10, 3 13, 3 16 Kennedy, C., 186, 187(357), 189(357) Kennedy, L. D., 301, 302(121, 122, 125) Kenner, G. W., 44 Kenny, C. P., 3 16 Kent, L. H., 303 Kent,P. W., 95, 171, 172(304), 173(305),207 Kentovaara, P., 12 1 Kerekes, I., 100, 102(69), 165(69), 262(69) Kern, K., 328, 331(29), 332(29), 335(29), 337(29), 340(29) Kerremans, L.. 234, 272(635) Kersters-Hilderson, H., 359 Kessler, J., 352 Kessler, R. M., 189, 202 Kholodkova, E. V., 295. 3 I3 Khomenko, V. A., 3 I I Khorlin, A. Ya., 370, 377(175) Kiehlmann, E., 124 Kier, L. B., 49

AUTHOR INDEX Kiessling, G., 294 Kihlberg, J., 105, 137(95), 140, 150(221), 155, 162(272), 213(272), 214(95,272) Kilbourn, M. R., 195, 196, 197 Kim, H.. 178 Kim, J.-H., 25 I , 267 KImura, H., 41,46(49), 48(49) Kimura. N., 70 Kimura, S., 24 Kimura, Y., 232 Kinoshita, M., 106 Kirk, B. E., 139, 148, 152(247), 155, I58(2 17, 247, 248, 268), 165,252(247), 253(268), 272(248) Kirsanov, A. V., 142 Kishi, T., 23 Kiso, M., 112, I IS, 155, 158(267) KISS, J., 102, 142, 322 kssman, H. M., 264 182, 185 Kitagawa, 0.. Kitahata. S., 116, 351, 352(118), 354(118), 358, 379( 135) Kitazume, T., 126 Kitzing, S., 105, 137(95), 214(95) Kiuru, A., 202 Klatte, B., 196 Klein, H. W., 121 Klein, R. S., 132, 133, 234, 235(639), 238(639), 246(639), 248(639), 249(639) Klemer, A,, 94, 95(25), 99 Klemi, P. J., 202 Klemm, G. H., 96, 160(42) Klimke, G., 241,275,276 Klinkhammer. U., I14 Klug, H.-W., 141 Knaus, E. E., 237, 246, 247, 249(706), 254, 260 Knirel, Yu. A,, 96, 283, 285, 291,292, 294, 295, 298, 304, 307(89, 108), 310, 312, 313 Knockel, A,, 196 Knowles, J. K. C., I2 I Knust, E. J . , 195, 197(429), 199,204 Koaze, Y., 335, 338(58) Kobata, A,, 101, 104(79), 122(79), 149(79), 22 I(79) Kobayashi, N., 35, 101, 107(71), 112(71), I16(71, 72, 73), 117, 123(72) Kobayashi, Y., 182, 185, 226, 227(616), 228(6 I6), 229

397

Kochagina, N. I., 284 Kocharova, N. A., 291,294,297,298 Kochetkov, N. K., 96, 2 12, 29 I , 294, 295, 297,298, 304, 307(89, 108), 310, 312, 313 Kodaya, S., 3 13 Koeda, T., 332 Koharova, N. A., 29 I Kohla, M., 99 Koikov, L. N., 108, 119 Koitzsch, H.-H., 265 Kojima, M., 125, 126, 128, 130, 131, 177, 196, 198,207 Kol, M., 177, 178(318), 180(318) Koldatkina, N. A., 283 Kollnerova, Z., 208, 231(555) Komandrova, N. A,, 285 Komatsu, K., 175, 192(312), 194(312) Komuro, K., 232,233 Kon, A,, 182 Kondo, T., 114, 141 Kondo, W., 286 Kondo, Y., 188 Kondoh, T., 29, 31, 34(31, 34), 36(31), 37(31, 33). 50(33), 60(31, 33) Kondratenko, N. V., 265 Kong, F., 94, 11 1(29), 123(29) Kong, X.-B., 250, 255, 256(735, 753) Konig, H., 294 Konig, J., 265 Konishi, M., 72, 88( 106) Kono, A., 261 Kontrohr, T., 294 Koppel, H., 257 Korenaga, H., 3 13 Kornblith, P. L., 202 Korytnyk, W., 140, 142, 149(234), 150, 152, 155, 161(271), 163, 175, 176(319, 320), 177(320), 183(260), 210 Koshland, D. E., Jr., 361 Kosma, P., 295, 297 Kothari, P. J., 198 KovaE,P.,93, 101, 119, 120(148), 134, 136, 143, 145(148), 146(148), 156(148), 161(209), 163(202), 218,219, 220, 221 Koval’chuk, S. V., 289 Kowell, A. P., 200 Kowollik, G., 257, 258, 259, 260, 263, 264, 277(800) Kozikowski, A. P., 152

398

AUTHOR INDEX

Kraft, M., 133,239(194) Kralovec, J., 164 Kramer, M. J., 252, 261(742) Kratk9, Z., 206, 207(542) Krauss, J. H., 297 Krauss, O., 194, 195(421) Krausz, P., 66 Krayevsky, A. A., 258 Kreis, W.. 245, 247, 255(687) Kreuzer, M., 109, 122(107) Krimer, M. Z., 93 Krivokapich, J., 20 I , 202 Krizek, H., 194 Krogmann, C., 295 Kroin, J. S., 182, 239(344) Kuan, F.-H., 101, 107(71), 112(71), 116(71, 72), 123(72) Kubota. K., 202, 203 Kufta, C. V., 202 Kuhl, D. E., 175, 187, 189, 191, 199, 200, 201, 204(311) Kuhl, D., 187, 189(358), 190(31I), 191(311) Kuhns, V., 277 Kukhanova, M. K., 258 Kulhanck, M.. I83 Kumar, V., 352 Kuninaka, A,, 249 Kunita, N., 30 I , 302( 129) Kunstman, M. P., 29 Kunz, H., 102, 107(82), 108(82), I14 KUO,M.-S., 309 Kuribayashi, S., 207 Kurozumi, S., 148 Kusaka, T., 23 Kushner, M. J., 201, 202(494) Kushner, M., 200, 201(494), 202(494) Kusumoto, S.. I I2 Kusunoki, M., 320, 326(7) Kusunose, N., 112 Kuzuhara, H., 65, 82, 86 Kvarnstrom, I., 305 Kwee. 1. L., 13I , 207( 186) Kyogoku, Y., 268

L L'vov, V. L., 291, 310, 312 Labischinski, H., 308 Laborde, A. L., 72

Laborde, M. A,, 142 Labow, R. S., 330, 331(36) Lacombe, S., 169 Ladduwahetty, T., 145 Lade, R. E., 188 Lagunas-Solar, M. C., I96 Lai, H.-Y. L., 333 Lai, H.-Y., 379 LalCgerie, P., 320, 338, 341(73), 369(73) Lambrecht, R. M., 190 Lammertsma, A. A,, I87 Lamport, D. T. A,, 96,97 Langdon, R. G., 377 Langen, P., 257, 258, 259, 260, 263, 264. 265, 266, 267, 273, 277(800) Langworthy, T. A,, 300 Larson, S. M., 187 Larsson, A., 249 Lartey, P. A., 101, I12(76), 122(76), 123(76) Latif, F., 137 Laundry, M. L., 249 Laurent, A,, 167, 169 Lawrynowicz. W., 124 Leaback, D. H., 327 Lebedev, A. V., 244 Lederer, E., 308 Lee, H. H., 152, 183(260) Lee, L., 293 Lee, Y.C., 155, 160(270), 210(270), 349 Lee, Y. W., 246, 254 Legler, G., 320. 322, 324(18, 19), 328(18). 329, 331(18. 19, 31), 332, 333, 334, 335(31), 336, 337(18, 31, 51, 62,63, 64), 338, 339, 340, 341(73), 344(70), 346(63), 349, 350, 352, 353, 354(115), 360, 361, 364, 365, 366(76, 82, 146, 149, 150), 367, 368, 369, 371(162, 165), 372(162), 376. 377, 378(51). 379(138), 381(123, 148, 167, 168), 383(18), 384(159) Lehmann, C., 258,259 Lehmann, J., 350, 352(113), 354, 356, 357, 379( 129, 130) Leimkuhler, M., 99 Lemares, N., 194 Lemieux, R. U., 171, 221 Lemire, A. E., 95 Lengen. P., 258 Leontein, K., 301, 302(123) LePage. G. A,, 245

AUTHOR INDEX Lerman, O., 177, 178, 180(318) Lemer, L., 22 I Lesyng, B., 242, 243 Levitt, M., 382 Levvy, G. A., 327, 329, 330(26), 331(32, 33), 332(18), 333(18), 337(18), 339(18) Levy, S., 124, 125(166), 197, 198 Leworthy, D. P., 342 Leyland-Jones, B., 255 Li Hsu, Y.-F., 93 Li, S.-C., 330, 33 1(42), 367 Li, Y.-T.. 330, 331(39, 42), 367 Lidaks, M., 265 Lieberman, L. M., 190,204(391) Liedtke, H., 335, 337(55), 377(55) Lienhard. G. E., 330, 331(43) Lilli-Elghanian, D. A,, 308 Lim, A. V. S., 312 Lin. J.-C., 248, 249 Lin, T.-S.. 266 Lindberg, B., 280, 282. 283, 284, 286, 287, 289, 291, 292, 293, 294, 295(66), 296, 298, 304, 305, 306, 308, 309, 3 10, 3 12, 313, 316 Lindberg, G., 30 1, 302( I 19) Lindh, F., 305 Lindley, M. G., 49 Lindner, B., 308 Lindner, J., 352 Lindquist. U.,289, 305, 306 Lindqvist, B., 292, 304 Link, K. P., 322 Link, R. W., 226 Linker, A,, 293 Liotta, C. L., 194 Liou, R., 274 Lipnick, R. L., 237 Liptak, K., 35, 37(41) Lisak, L. V., 295 Liu, T.-Y., 308 Livni, E., 124, 125(166), 197, I98 Lockshin. A,, 265 Loentein, K., 294 Loidl, P., 265 Lokys, L., 248 London, J., 199 Lonngren, J., 287, 292, 301, 302(123), 304, 305 Lopes, D. P., 135, 183(205), 204(205), 209 Lopes, D., 209

399

Lopez, C., 234, 235(639), 237, 238(639), 246,247,248,249,250(649,659),

25 1(659), 255 Lorkiewicz, Z., 301, 302( 126) Lotz, W., 376 Luccioni, C. M., 264 Liidemann, H.-D., 241,275,276 Liideritz, O., 280, 301, 302(120) Luger, P., 119 Lugowski, C., 283,291,292,295(66), 310 Lukacs, G., 107, 125, 127(97), 128. 131, 132(182, 190) 142, 143(164), 144, 145, 147(182), 150, 156(97, 182), 157(I82), 166, 171, 173(238), 231(240), 232(190, 240) Lustig, M., I70 Luxen, A., 195, 197, 198(434) Lygre, H., 283 Lythgoe, B. J., 43

M Maass, U., 23 McCasland, G. E., 22, 23,27( I , 12, 13), 36(13), 37(1, 12, 13) McCombie, S. W., 228 MacDonald, N. S., 191,201 MacDonald, N., 201 Macdonald, S. J. F., 102, 118 McDowell, W., 207 McGee, D. P. C., 139, 274,277(218) MacGregor, B., 200 MacGregor,R.R., 178, 188, 190, 191, 193, 194(394,41 I), 199, 201(479), 202 MacGregor, R., 199 McGuire, E. J., 297 Machida, H., 246, 247(696), 249 Machulla, H.-J., 195, 197(429), 199, 204 McKelvy, J. F., 367, 371(166) McKenzie, T. C., 102, I18 MacLean, L. L., 288 MacLennan, D. J., 149, 161(251) McNeil, M., 28 I MaCoss, M., 237 MacovP, J., 164 Madhavan, G. V. B., 139, 277(2 18) Maeda, H., 22, 37(2, 3), 41(2, 3). 44(2, 3) Maeda, K., 72 Maeda,M., 125, 126, 128, 130, 131, 177, 196, 198

400

AUTHOR INDEX

Maeta, H., 110 Magnusson, G., 105, 137(95), 140, 150(221), 155, 162(272), 213(272), 2 14(95, 272) Maier-Borst, W., 192, 193, 194, 195(421), 198(404) Main, P., 243 Mair, G. A., 320, 325(6), 326(6), 371(6), 381(6) Maley, F., 330 Malik, A., 137 Malinowski, N., 264 Malonda, A. G., 189 Malspeis, L., 264 Mamontova, V. A,, 297 Mancini, W. R., 266 Mann, J., 93, 165 Manning, R. G., 202 Manning, R., 189 Mansuri, M. M., 247, 248(704), 250(704) Manville, J. F., 94,95(28), 104(28), 106(28), 163(28) Mar, E X . , 247,248, 249(699) Maradufu, A., 136, 209(2 I I ) Marais, C. F., 102 Marantz, L. B., 170 March, J., 92, 32 1 Marck, C., 242,243 Marcus, D. M., 171, 172(303) Margolin, R. A., 189 Marion, J., 136, 209(211) Markaryan, A. N., 108 Markham, C. H., 201 Markovskii, L. N., 142 Marlier, M., 345 Marquez, V. E., 264, 269,270(844) Marr, C. L. P., 165,272(282) Mamott, S., 92 Marsaioli, A. J., 131, 132(190), I66,232( 190) Marshall, P. J., 343, 344(91), 374 Marshall, R. C., 20 I , 202 Marshall, R. D., 322 Marshall, S. E., 375 Martin, J. A., 195, 197(437) Martin, J. C., 139,247, 248(704), 250(704), 274,277(2 18) Martin, 0. R., 49 Mashilova, G. M., 291,294, 298, 307(108), 3 10(105) Masuda,R., 29,34(31),36(31),37(31),60(31)

Mateo, F. H., 132 Matsu, K., 347 Matsuda, A., 234, 235(639), 238(639), 246(639), 248(639), 249(639), 250 Matsuda, H., 187, 199(359) Matsugi, J., 268, 269 Matsui, H., 203 Matsui, K., 52, 53, 74(70), 88, 202 Matsumoto, T., 110, 119,213(144), 232 Matsuo, T., 53, 88(72) Matsushima, Y ., 26 1 Matsuura, Y., 112, 320, 326(7) Matsuzawa, T., 175, 192, 194,202,203, 207(419, 530) Matta, K. L., 136, 149, 212, 218(250), 240(2 12) Mattes, R., 99 Matthes, E., 258, 259 Matthews, T. R., 274 Matulic-Adamic, J., 237, 239(66 I), 252, 256(661), 272 Matzui, H., 357 Maybeny, W. R., 300 Maybeny-Carson, K. J., 300 Maycock, A. L., 364 Mayer, H., 287, 292, 297, 3 12 Mayo, D. R., 249 Mazo, A. M., 258 Mazziotta, J. C., 189, I99(382), 200 Meakins, G . D., 148 Meguro, H., 210 Mehdorn, H. M., 204 Meindl, P., 35 1, 352, 352( 1 19) Mellish, P., 264 Melly, M. A,, 3 12 Melton, L. D., 30 I Mendelsohn, M. H., 178 Mengel, R., 235, 241, 250, 275, 276 Mercer, J. R., 246,254 Mercier, D., 224, 369 Mestelan, G., 191, 201(400) Metter, E. J., 200, 201 Metter, J., 200 Meyer, E., 187, 199(359) Meyer, H., 28 1, 302( 13) Micheel, F., 94, 95(25), 99 Michel, G., 317 Michon, F., 294, 301, 302( 127), 308, 316 Middleton, W. J., 127, 142, 2 17(176), 251(176)

A U T H O R INDEX

Miki, H., 268, 269 Miljkovit, M., 121, 124(158), 128(158) Miller, D. C., I34 Miller, T., 24, 27(14), 37(14) Min, J. M., 165 Minn, H., 203 Misaki, A,, 281,282 Misner, J. W., 182, 239(344) Misra, H. K., 237, 247, 249(706), 260 Mitchell, M., 346, 379(102) Mitsunobu, O., 101, 122(81) Mitsuya, H., 269, 270(844) Miura, S., I88 Miura, Y., 203, 204(532) Miwa, I., 87 Miwa, M., 261,265 Miwatani, T., 301, 302(129) Miyai, K., 275 Miyake, T., 229 Miyamoto, Y., 78, 79, 8 1 Miyano, K., 301, 302(129) Miyazaki, M., 41,46(49), 48(49) Miyazawa, H., 203 Miyazawa, K.. 68 Miyazawa, T., 269 Mizokami, K., 24 Mizuka, E., 347 Mizuno, K., 23, 74 Mizuta, E., 26, 52(23) Mochida, Y., 126 Moffatt, J. G., 95, 262 Molyneux, R. J., 342, 343, 344(90, 93) Momozono, Y., 130, I3 1 Monna, M., 191 Montanez, I., 199 Montgomery, J. A., 270, 271(846), 272(846), 277(846) Monti, C. T., 92 Moreau, M., 290 Mori, M., 105 Morin, M. J., 150, I52(256) Morishita, K., 268 Morrison, J. F., 340 Morsel, T., 265 Mort, A. J., 96,98, 301, 302(124), 309 Moses, G., 209 Moyer, J. D., 155, 157(266), 264 Mukaiyama, T., 99, 103, 104(61, 86), 112(61, 62), 123(61,62), 221(86) Mulholland, G. K., 174, 196

40 I

Miiller, L., 52, 81(69), 88(69), 336, 338(60), 347, 348( 106) Miiller, W., 24 Miiller-Hill, B., 374 Miiller-Seitz, E., 283 Munday, K. A., 358 Murai, Y., 103, 104(86), 221(86) Murakami, M., 187, 188 Murao, S., 25 Muroi, M., 23 Murray, M., 121 Mumy-RuSt, P., 92 Murthy, S . V. K. N., 312

N Naberezhnykh, G. A., 3 1 1 Nagabhushan, T. L., 228 Naganawa, H., 72 Nagasawa, J., 101, 116(73) Nahmias, A. J., 248,249,255(710), 256(710) Nahmias, G., 194, 195(422) Nahrstedt, A., 367 Naider, F., 374, 375(181) Naik, S. R., 237 Naito, T., 225, 228(607) Najafi, A., 191 Nakada, T., I3 I , 207( 186) Nakagawa, S., 225, 228(607) Nakahara, Y., 101, 104(79), 105(78), 123(78), 149(79), 221(79) Nakai, H., 187, 199(359) Nakamoto, K., 29, 53 Nakamura, J., 1 I5 Nakamura, K., 32, 35, 36(30), 37(30), 38, 39(45), 53(30) Nakanishi, H., 193 Nakano, I., 201 Nakano, M., 359 Nakayama, K., 247 Nakazawa, F., 286 Nakhlar, N. A., 307 Nam Shin, J. E., 136,209(2 1 I ) Namiki, S., 24 Nash, W. W., 201 Nasir-ud-Din, 294 Nawrot, B., 248, 251(713) Neal, D. J., 281, 301(12), 302(12) Neirinckx, R., 190

402

AUTHOR INDEX

Nelson, W. L., 29 Nesburn, A. B., 249 Ness, R. K., 1 I I Nesser, J.-R., 178, 179(336) Neszmklyi, A,, 297 Neuert, H., 194, 195(425) Ng, C. K., 204, 208(533) Nicholas, S. J., 345 Nickles, R. J., 190, 194, 195, 198, 199(467), 204(391) Nicolaou, K. C., 104, 107, 108(64, 65, 102), 116, 117(140), 122(64), 123(64), 145 Niida, T., 331, 332, 335, 336, 337(45), 338(58), 348(45) Nimmich, W., 296, 301, 302( I 19), 304, 305 Nintsche, R., 257 Niolaou, K. C., 100 Nishikawa, M., 23 Nishimura, O., 308 Nishimura, S., 53 Nishizawa, M., 107 Nishizawa, N., 25 Nissenson, C., 202 Niwa, T., 332, 335, 338(58) Nojima, M., 100, 102(69), 165(69), 262(69) Nolan, P. A., 272 North, A. T. C., 320, 325(6), 326(6), 371(6), 381(6) Nosaka, C., 232,233 Nose, T., 36, 37(44), 38(44), 64, 66 Noyori, R., 100, 104, 107(70, 87), 122(68), 123(68, 70) Nozaki, T., 190, 195(385) Nukada, T., 101, 104(79), 122(79), 149(79), 22 l(79) Numata, K., 72,88(106) Nunomura, S., 107 Nuwer, M., 200

0 Oates, J., 292, 295(66) Oba, T., 148 Oberdorfer, F., 192, 193, 198(404) Oberg, B., 249 Oberti, R., 134 Oehninger, H., 195 Oerskov, F., 283 Oerskov, I., 283

Oesch, B., 332, 337(46), 340(46) Ogata, Y., 203 Ogawa, S., 23, 25, 28, 29, 30, 31, 32. 33, 34, 35, 36, 37(25, 30, 31, 33, 34, 35, 37,43, 44), 38, 39(45), 50, 5 I , 52, 53, 54, 5 5 , 56, 58, 59(86), 60,63, 64,66,68, 70, 73,75,76,77,78,79,81,82, 83.84, 85, 87,89 Ogawa,T., 101, 104, 105, 107, 115, 116, 122(79,91), 123(78), 128, 149(79), 209, 208(180), 213(180), 221(79) Ogawa, Y ., I I2 Ogino, H., 25, 81(21) Ohashi, K., 28 Ohba, S., 36, 37(44), 38(44) Ohkawa, S., I82 Ohrui, H., 175, 192(312), 194(312) Ohta, A., 269 Ohyama, K., 25 Ok, K. D., 164, 233(279, 280) Okada,G., 350,351(117), 352(117), 353(117) Okada, S., I16 Okamoto, K., I 14 Okamoto, P. M., 133, 239(194) Okamura, N., 148 Oki, S., 28 Okuda, J., 87 Okuni, T., 56 Olah, G. A., 100, 102(69), 165(69), 262(69) Olah, J. A., 100, 102(69), 165(69), 262(69) Olds, J. L., 188 Olesker, A,, 107, 125, 127(97), 131, 132(190), 142, 143(164), 144, 145, 150, I56(97), 166, I7 I , 173(238), 23 I (240), 232( 190, 240) Olesker, R., 128, 132( 182). l45( I82), 147(182), 156(182), 157(182) Oliveira, A. B., 166 Oliveira, G. G., 166 Olseker, A., 369 Omae, H., 125, 128, 198(181) Omar Osama, L. M., 365, 366( 150) Omata, M., 5 5 Omori, H., 269 Omoto, S., 25, 81(21) Onn, T., 306 Onoda, T., 230 Ooka, T., 247 Orhanovic, Z., 141, 199(222) Orihara, M., 60

AUTHOR INDEX

Orui, H., 192, 203(403) Osieclu, K. M., 368, 381(168) Osiecki-Newman, K., 338, 367 Osman, S. F., 283 Oster, Z. H., 202 Osterhaus, A. D. M. E., 248 Otake, N., 24.72 Otto, K., 374 Ouellette, R. J., 35, 37(41) Ovodov, Yu. S., 284, 285, 288,289, 3 1 1 Owen, G. R., 262 Oya, M., 54

P Pacak, J., 164, 183, 208, 231 Packer, N. H., 290 Packwood, J., 200 Padgett, H. C., 175, 178(318), 192(313), I92(3 I8), I97 Pagano, J. S., 248, 249 Page, M. I., 320, 325, 331(3), 349(3) Pagliarin, R., 139 Palamarczyk, G., 346, 379(102) Palm, D., 121 Palmer, A. J., 187, 194, 196, l98(4 17) Panasenko, S. M., 301, 302( 126a) Pankiewicz, K. W., 248, 25 I Papahatjis, D. P., 100, 108(64), 116(64), 122(64), 123(64) Paramonov, N. A., 283,295 Park, H.-L., 232 Parker, W. B., 247 Parkin, A., 274 Parks, R. E., Jr., 250,276(734), 277 Parratt, M. J., 185 Panish, F. W., 329,332(34), 336(34), 350(34) Parviainen, S., 202 Pashinnik, V. G., 142 Pate, B. D., 178, 179(336), 194 Patel, P. C., 248 Patel, T. R., 307 Patil, V. J., 166, 231(286) Patlak, C. S., 186, 187(357), 189(357) Patronas, N. J., 202 Patwardhan, B. H., 352 Paul, P., 202 Paul, R., 202, 203 Pauling, L., 325, 332(21), 333(21)

403

Paulsen, H., 23,40,41,48(52), 49(52), 64, 68, 83, 87(52), 95, 112, 119, 295, 336 Pauwels, R., 258, 259(767, 778, 779), 269, 270(843), 272(778), 273 Pechet, M. M., 170 Pedersen, C., 94,95,96(39), 97,98, 99, 1 I I , 123(39b) Pedersen, H., 96 Penglis, A. A. E., 93,95(5), 135, 136(206), 139(206), 143(5), 149(5), 166, 167(285), 169(285), 170(5), 177(5), 197(5) Percival, M. D., 155, 159(269), 160(269), 161(269), 162(269), 206(269), 207 Perlin, A . S., 98, 136, 209(21 I), 301, 302( 124), 353 Perlman, M. E., 237, 250(657), 251(657) Perlmutter, M. M., 155, 195, 198(438) Perry, M. B., 28 I, 288,292,3 16 Pexhke, H., 99 Peters, J., 248 Peterson, K., 289, 292, 3 13 Petit, J. F., 308 Petrie,C. R.,III, 175,176(320), 177(320),210 Pettigrew, K. D., 186, 187(357), 189(357) Petzold, U., 336, 366(7 I), 367, 368(7 I), 372(7 I ) Phelps, M. E., 189, 199(382), 200, 201, 202, 203 Phelps, M. F., 187, 199(362) Phelps, M., 187, 189(358), 199(358) Philips, F. S., 248, 254, 255, 256 Phillips, D. C., 320, 325(6), 326(6), 371(6), 38 1(6), 382 Phillips, L., 93, 177, 2 12(325) Picq, D., 134, 141(200), 167 Pietrasik, D., 3 I3 Pietrzak, B., 165 Pike, V. W., 193 Pircher, H. P., 332, 337(46), 340(46) Piskorz, C. F., 149,212(249) Pitohelli, A. R., I70 Pittman, K. A., 255 Plach, H., 241 Plattner, A., 368, 369( 171) Plattner, R. D., 304 Plough, H., 336 Plummer, T., Jr., 330 Plunkett, W., 246 Pohl, S., 332, 336, 337(63), 338, 346(63)

404

AUTHOR INDEX

Pokorny, N., 330, 333(44) Ponton, J., 118 Posner, G. H., 99, 100(63), 101, 117(63), 118, 122(77), 123(77) Potocki, J. F., 178, 194 Potti, G. G., 140 Powell, D. A., 292, 299 Pravdii., N., 330, 333(44) Preston, R. K., 92 Preussel, B., 265 Price, R. W., 237,239(661), 248, 252, 251(713), 255,256 Priebe, W., 233 Pnirylova, V., 23 I Prisbe, E. J., 133, 139, 239(194), 277(218) Prusoff, W. H., 244, 264(684) Puls, W., 347, 348( 106) Pyrce, R. J., 342

Q Qaim, S. M., 190 Quaroni, A., 365,366(151), 368,370(151), 381( 169) Que, L., Jr., 183 Quiclet, B., 224 Quiclet-Sire, B., 66

R Raddo, P. D., 175 Radziejewska-Lebrecht,J., 287 Raftery, M. A., 383 Raichle, M. E., 196, 199, 204(454, 473) Rainey, P., 272 Rama Rao, A. V., 166,231(286) Ramani, G., 237 Rand-Meir, T., 383 Randall, J. L., 100, 104, 107, 108(64, 102), 116, I l7( l40), 122(64), 123(64), 145 Rando, R. R., 364 Rane, D. F., 228 Ranganathan, R., 268 Rao, A. S., 220 Rao, G . V., 183 Rapoport, S. I., 200 Rasmussen, J. R., 183, 204, 207, 209(346, 537) Rasmussen, K., 220

Ratib, O., 201 Ravn, H., 195 Ray, T. C., 28 I Reamer, R., 198 Reddy, A. R. V.,237,239(661),256(661) Reddy, G. S., 149, 158(255), 160(255), I6 1(255), I62(255) Redies, C., 187, 199(359) Redmond, J. W., 290 Reed, M. F., 95 Reedy, G. S., 155, I57(265) Reese, E. T., 329,332(34), 336(34), 350(34) Refn, S., 95, 123(39b) Reichman, U., 236, 237, 246(649), 247(649), 248(649), 250(649, 659), 251(659), 254, 255(748) Reilly, P. J., 2 I6 Reimer, G. J., 164 Reinendegen, L. E., 202 Reissell, A,, 195 Reistad, R., 293 Reitshel, E. T., 292 Reivich, M., 175, 186, 187, 189(357, 358), 190(3l I), 191(3l I), 199,200,201. 202(494), 204(311) Remin, M., 241 Rettig, S. J., 119, 207( 146) Reuter, G., 297 Reynolds, W. F., 92 Rhinehart, B. L., 343, 344(92) Ricard, D., 93 Richardson, A.C., 121, 128(157), 135, 136(206), 139(206), 150( 157), 166, 167(285), 169(285), 21 I , 212(570), 215, 2 16(578) Riddle, C., 264 Riege, W. H., 201 Rietschel, E. T., 294, 295, 297, 298, 308 Riley, D. A,, 101, I12(76), 122(76), 123(76) Rinehart, K. L., Jr., 52,228 Ring, M.. 375, 378(184) Rintelmann, W., 200 Ripp, K. G., 2 17 Robbins, J. B., 308 Roben, W., 40,83 Roberts, S. M., 139, 148, 152(247), 155, 158(217, 247, 268), 165, 252(247), 253(268), 272(282) Roberts, W. K., 316 Robins, M. J., 237, 241,246,273, 276, 277

AUTHOR INDEX Robins, R. K., 170, 246, 272, 275 Robinson, G. D., Jr., 191, 201, 203 Robinson, G., 201 Robinson, K. M., 343, 344(92) Robyt, J. F., 214,216,221 Rodda, H. J., 44 Roden, W., 195, 199(439) Roeda, D., 202 Roeser, K.-R., 350, 352(115), 353, 354( I I5),36 I Rogers, P. L., 289 Rolland, A., 224 Romanowska, A., 283 Romanowska, E., 283,292,295(66) Romaschin, A., 209 Romero, P. A,, 207, 377 Romo, E., 260, 266(787) Roseblum, A., 35 Resell, K.-G., 288, 3 16 Rosen, M., 200, 201(494), 202(494) Rosenbrook, W., Jr., 101, I12(76), 122(76), 123(76) Rosenfelder, G., 30 I , 302( 120) Rosenquist. A., 200 Rosenthal, A., 244, 266 Rosenthal, H. A., 258,259 Rosenthal, M. S., 198, 199(467) Rosenwirth, B., 25 I , 274 Roth, J. S., 269, 270(844) Rottenberg, D. A., 256 Rouillard, M., 168 Rousset, C., 167 Roy, R., 294 Rozen, S., 177, 178, 180(318) Rubenstein, R., 248, 256(720) Riidiger, H., 330, 331(40) Ruff, J. K., 170 Ruiz, H. V., 195 Rule, C. J., 345 Rupitz, K., I2 I , 206, 362 Rusnak, J. M., 152 Russa, R., 30 I , 302( 126) Russell, J. A. G., 191, 194(394) Rustum, Y. M., 264 Rutar, V., 93 Ruth, J. L., 237, 247, 254 Ruth, T. J., 190, 194, 195 Riither, U, 374 Riitter, R., 336, 344(70) Rydzewsla, R. M., 139,277(218)

405 S

Sacker, D. F., 202 Sadozai, K. K., 101, 104(79), 122(79), 149(79), 221(79) Saenger, W., 243 Sager, W., 102, 107(82), 108(82) Saito, Y.,36, 37(44), 38(44), 248,256 Saitoh, M., 29, 34(31), 35, 36(31), 37(31), 60(31) Sakairi, N., 65,82,86 Saksena, A. K., 23 Sakurada, O., 186, 187(357), 189(357) Salam, M. A., 264 Salimath, P. V., 312 Salvadori, P. A., 193, 194(411) Salvayre, R., 367 Samoshin, V. V., 93,243(17) Samuel, J., 204, 237 Samuelsson, K., 286, 296 Sanchez, R. A., 235 Sandhoff, K., 330, 331(42a) Sanford, P. A., 282 Santi, D. V., 250 Santi, V., 272 Sapino, C., Jr., 133,238, 239(662) Sapse, A. M., 243 Sarda, P., 107, 127(97), 144(97), 145(97), 150, 156(97) Sarma, V. R., 320, 325(6), 326(6), 371(6), 381(6) Sarngadharan, M. G., 258 Sasaki, H., 187, 188 Sasaki, N., 52, 66 Sastry, K. A. R., 328, 331(29), 332(29), 335(29), 337(29), 340(29) Sato, K., 141 Sato, M., 286 Sato, S., 105 Sato, T., 194, 202,203 Satyamurthy, N., 175, 178(318), 192(313, 314, 318), 194(314), 195, 198(434) Sau, A. C., 124 Saul, R., 343, 344(90,93) Savage, A. V., 306 Sawai, T., 358,359,379( 135) Schaeffer, H. J., 248, 274(718) Schater, D., 24 Schauer, J., 297 Schauer, R., 114,338

406

AUTHOR INDEX

Scheck, A. C., 255, 256(753) Schelbert, H. R., 202 Schelbert, H., 201 Schelert, H. R., 201 Schgal, R. K., 247 Schinazi, R. F., 237,246(658), 248, 249, 250(657,658), 251(657), 252, 255(710), 256(710) Schleinitz, K. D., 257 Schlesselmann, P., 356, 379( 129), 379(130) Schlingmann, M., 96, 97(45), 99(45) Schloss, J. V., 382 Schlyer, D. J., 196 Schmall, B., 237 Schmidt, D. D., 24, 52, 81(69), 88(69), 336, 338(60) Schmidt, D. G., 197 Schmidt, D., 347, 348(106) Schmidt, M. A., 282, 308 Schmidt, M. F. G., 206,207 Scholz, D., 258, 259 Schonholzer, P., 102 Schramek, S., 287 Schray, K. J., 367, 369(164) Schrobilgen, G., 177 Schroter, E., 350, 352( 1 13), 354( 1 13) Schuerch, C., 1 I I , I20( I 18) Schulz, G., 297 Schiitt, M., 263.264 Schwaiger, G. P., I94 Schwarz, R. T., 206,207, 377 Schwarz, W., 25 1 Schweda, E., 298 Schweden, J., 338, 339 Schwentner, J., 141 Scott, M. E., 352 Scott, R. T., 249 Secemski, I. I., 330, 331(43) Secnst, J. A., III,270,271(846), 272,277(846) Seidl, S., 137 Selin, C. E., 201 Selin, C., 187, 189(361), 199(362),200,201 Semenza, G., 330,331(37), 332, 337(46), 340(46), 365, 366(15 I), 368, 370( I5 I), 38 I( 169) Sergeyer, N. M., 93 Seto, H., 24 Sewell-Alger, R. P., 338, 366(76), 368(76) Seydel, U., 308 Shabarova, 2.A,, 244,266

Shallenberger, R. S., 49 Shang, H., 94, 11 1(29), 123(29) Shannahoff, D. H., 235,245 Sharkey, P. F., I I 1, 120(I 18) Sharma, M. N., 114 Sharma, M., 140, 142, 149(234), 152, 155, 161(271), 163, 183(260), 210 Sharma, R. A., 152,25 I Sharma, R. K., 124 Sharon, N., 292, 367, 371(166) Shashkov, A. S., 93, 96, 283, 284, 285, 288, 29 I , 292. 294, 295, 297,298, 304, 307(108), 310, 312, 313 Shashkov, M. A., 283 Shaughnessy, W. J., 190, 194, 195, 196, 199,204(391) Shaw, A. N., 338 Shaw, D. H., 297 Shealy, Y. F., 23 Shelling, J. G., 2 12 Shen, T. Y., 148, 155, 157(242), 162(273) Shiba, T., 112 Shibaev, V. N., 280, 285(7), 289(7), 318(7) Shibata, M., 23, 24, 52(15) Shibata, Y., 58, 59(86), 64, 68, 70, 73, 75, 84,269 Shill, M. D., 165, 272(282) Shimada, I., 269 Shimizu, C., I14 Shine, K. I., 201 Shinohara, M.,175, 186, 187(357), 189(357), 192, 194(312),203(403) Shishido, F., 187, 188 Shitara, T., 229 Shiue, C. Y., 202, 203(399) Shiue, C., 188, 199 Shiue, C.-Y., 177, 178, 191, 192(328), 193, 194(394, 41 I), 195, 203 Shiyan, S. D., 370, 377(175) Shoda, S., 99, 103, 104(86), 112(61), I23(6 I), 22 l(86) Shomura, T., 25, 81(21) Shortnacy, A. T., 270, 271(846), 272(846), 277(846) Shreeve, W. W., 141, 199(222) Shugar, D., 24 I , 246, 247(694) Shuller, M., 196 Shulman, M. L., 370, 377( 175) Shulz, G., 297 Shum. P. W., 266

AUTHOR INDEX Shum, P., 260, 266(787) Shymko, R. M., 265 Sideris, K., 187, 199(362) Sidhu, R. S., 96, 160(42) Sidler, D. R., 346 Sielecki, A. R., 382 Siemsen, L., 132, 23 1,232(626) Sigurskjold, B. W., 72 Silina, E., 265 Simmons, 0. D., 140 Sinay, P., 303 Sinnott, M. I., 374, 375(179) Sinnott, M. L., 12 1, 320, 322, 324( 18). 328, 329(18), 331(3, 18). 332(18), 333(18), 337( IS), 338, 339( IS), 343, 344(91), 349, 353( 18). 366(76), 368(76), 370, 372, 374, 375, 383(18,28, 107) Sinnwell, V., 295 Sipila, H., 202 Skaug, N., 284, 309 Sklenar, V., 143, 221(236) Skrincosky, D., 153, 239(262) Slawin, A. M. Z., 148, 152(247), 155, I58(247, 268), 165(268), 252(247), 253(268) Slettengren, K., 30 I , 302( 123) Smale, S. T., 183, 204(346), 209(346) Smee, D. F., 133, 239( l94), 274 Smith, A. R. W., 28 1 Smith, B. H., 202 Smith, D. A,, 209 Smith, D. W., 245 Smith, G. M., 289 Smith, M. C., 248, 249 Smith, P. F., 300 Smith, P. J., 372, 374, 375(179) Smith, P. W., 345, 346, 379( 102) Smith, R. S., 248 Smith, W. L., 299, 300( 1 13), 302( 1 13) Smitriev, B. A., 3 I0 Snaith, S. M., 327, 329(26), 330(26) Snyder, G., 243 Soderlind, M., 127 Soderstrom, K.-O., 202 Sokol, M. K., 248 SokoloK L., 186, 187, 189(357, 358, 361), 199(358), 202 Soldatkina, M. A,, 285 Solin, O., 195, 202 Solov’eva, T. F., 3 I I

407

SolovCva, T. F., 297 Sols, A., 186 Som, P., 187, 189(358), 199,201(479), 202 Somawardhana, C. W., 149, 158(252, 253), 160(253) Somerville, A. R., 264 Son, J. C., 135, 258(204) Son, T.-D., 242, 243(675) S o d , S., 177, 192(327) Souchard, I. J. L., 328, 383(28) Soyer, V. S., 295 Speclman, D. J., 247 Spector, T., 245 Spellman, M., 309 Spiro, R. G., 322 Spohr, U., 22 I Squires, M. J., 297 Srikrishnan, T., I3 1 Srivastava, H. C., 142,222 Srivastava, V. K., 142,222 Stacey, M., 279,291 Stack, R. J., 282,283, 304 Stallings, W. C., 92 Stamm, R., 202 Stanaislavsky, E. S., 29 I , 294, 295, 298, 307(108), 310(105) Stanislavslu, E. S., 3 13 Staske, R., 264,265 Stein, T. M., 304 Stephanian, E., 262 Stephenson, L., 139, 148, 152(247), 158(217, 247), 252(247) Sterk, H., 137, 153 Sternlicht, H., 350, 351( 114). 352( I14), 353( 114) Stevenson, T. T., 298 Stewart, J. E., 293, 304 Stiem, M., 112 Stocklin, G., 193, 196, 197, 198 Stoeckler, J. D., 250, 276(734), 277 Storch-Becker, A,, 202 Straatmann, M. G., 143, 197(235) Strachan, I., 329, 331(33) Strange, R. E., 303 Strauss, H. W., 199, 204 Street, I. P., 119, 121, 144, 145(239), 146(239), 149, 155, 156(239), 159(269), 160(269), 161(251, 269), 162(269), 177(146), 206, 207, 221(541), 362 Streicher, W., 251, 274

408

AUTHOR INDEX

Streitwieser, A,, Jr., 124 Strother, S. C., 200 Stubbe, J., 245,246 Stiitz, A. E., 136, 137, 153,224,225,226 SU,T.-L., 132, 133, 234,235(639), 237, 238(639), 246(639,658), 248, 249, 250(658), 255, 256 Suami, T., 22, 23, 28, 29, 30, 31, 33(34), 34, 35, 36, 37(2, 3, 25, 30, 31, 33, 34, 35, 43,44), 38(44), 41,42(47,48), 44(2, 3), 45(50, 51),46(49, 50, 51), 48(49), 50, 51, 52, 53, 54, 55, 56, 60(33), 63(43), 64, 66, 68, 75, 76, 77, 78, 79( I I7), 82, 83, 84, 85, 87, 228 Subbotin, 0.A,, 93,243( 17) Suck, D., 243 Suda, Y., 182 Suetsugu, M.,31, 37(33), 50(33). 56, 60(33) Suga, A., 5 I Sugata, S., 26 1 Sugawa, I., 68 Sugimoto, M., 105 Sugiyama, H., 192 Sugizaki, H., 82 Suhadolnik, R. J., 276 Sukeno, T., 330 Sukumar, S., 93 Sundaralingam, M., 241, 242(672) Sundin, A., 155, 162(272), 213(272), 2 14(272) Suolinna, E.-M., 202 Surin, J. R., 150, 152(256) Sutherland, 1. W., 305, 306, 312 Sutherland, I., 308 Suzuki, H., 367 Suzuki, K., 110, 119,213(144) Suzuki, M., 54,60(75), 232 Suzuki, S., 247, 249(706) Suzuki, Y., 25, S l ( l 8 ) Svahn, B.-M., 155 Svenson, S. B., 291, 301,302( 123), 304,3 10 Svensson, S., 286 Sviridov, A. F., 288, 304 Swager, S., 93 Swartling, D. J., 234 Swigor, J. E., 255 Sykes, B. D., 382 Symes, K. C., 3 10 Szabo, L., 290

Szarek, W. A., 49, 100, 122(67), 123(67), 127, 128(175), 134(174, 175), 135(175), 137(175), 141(174, 175), 155, 180, 195, 198(438)

T Tada, M., 175, 192, 194, 203,207(419), 207(530) Tadano, K., 22, 23, 37(2, 3), 41, 42(47, 48), 44(2, 3), 45(50, 51), 46(49, 50, 51). 48(49) Tadra, M., 183 Taft, R. W., 92 Tafuri, S. R., 183, 204(346), 209(346) Taguchi, T., 182, 185 Takagaki, T., 32, 38, 39(45), 66, 84 Takagi, Y., 164,225,226(609), 232,233 Takahara, M., 53 Takahashi, H., 106 Takahashi, K., 188, 237, 239(661), 250, 256(661, 735) Takahashi, T., 175, 191, 192, 194, 195,203, 204(532) Takahashi, Y., 104, 122(91), 126, 128, 143(74), l44( 170), 208( 180), 2 I3( I80), 226,227(616), 228(616), 229 Takaoka, A., 101, 143(74,75) Takashi, T., 203 Takata, M., 32, 38(36), 5 1 Takatsuka, Y., 269 Takeda, Y ., 30 I , 302( 129) Takei, K., 5 I Takemoto, H., 282 Takemoto, K., 26 1 Takeuchi, M., 347 Takeuchi, T., 164,232, 233 Taki. T., 63 Takita, T., 72 Tan, R., 254 Tanahashi, M.. I I2 Tanaka, T., 148 Tanatar, N. V., 285 Tanenbaum, S. W., 352 Tann, C. H., 133, 238(195) Tanno, Y.,53 Tao, B. Y., 216 Tarbell, D. S., 29

AUTHOR INDEX Tarentino, A., 330 Tatlow, J. C., 93 Tatsuno, T., 126 Tatsuta, K., 106 Tavecchia, P., 139 Taylor, N. F., 93, 135, 183, 204, 209, 270, 271(845), 272(845), 274 Taylor, T. J., 44 Tenu, J. P., 349 Terashima, S., 232 Terpinski, J., 124 Testa, R. T., 228 Tewson, T. J., 127, 133, 162(197), 195, 196, 197(453), 198(177, 178), 199, 204(454. 473), 208( 197) Thang, T. T., 125, 128, 132(182), 142, 143(164), 145, 147(182), 156(182), 157(182), 166, 171,231(240), 232(240) Tharanathan, R. N., 3 12 Thiem, J., 109, 120, 122(107), 141 Thoma, J. A., 354 Thomas, D. W., 308 Thomas, E. E., 367, 371( 166) Thomas, P., 189 Thomas, R. L., 136, 149,212, 218(250), 240(2 12) Thompson, C. M., 193 Thompson, R. C., 192 Tietxhel, E. T., 308 Tiller, P. R., 301, 302( 123) Tillixh, J. H., 202 Timell, T. E., 320 Ting, R. Y. C., 258 Tittensor. J. R., 264 TO, K.-C., 177, 192(328) Tobin, M., 200, 201(494), 202(494) Tochon-Danguy, H. J., 195 Tochtamysheva, N. V., 29 I , 3 10.3 I2 Toda, Y., 190 Todd, A. R., 44 Todt, K., 336 Tolman, R. L., 170, 246, 274, 275 Tomita, K., 72,88(106), 268,269 Tommola, S. K., 49 Tomshich, S. V., 284 Tonegawa, T., 54,60(75) Tong, M. K., 37 I , 3 7 3 177) Toorongian, S. A., 196

409

Topsom, R. D., 92 Toni, T., 223,229 Toromanof, E., 168 Torrence, P. F., 246, 247(694) Tom, G., 98, 301, 302(124) Tom, T., 148 Touster, O., 330, 331(41), 342, 344(87, 88), 346(88) Tovell, D. R., 237 Townsend, D., 195 Townsend, L. B., 212 Townsend, R. R., 155, 160(270), 2 lO(270) Toyokuni, T., 29, 31, 34, 36(31), 37(31), 50, 52, 54, 55,60(31, 33), 68, 76, 77,78, 79( I17), 82, 83, 84( 125), 85 Trainor, G. L., 184 Traving, B. C., 193, 198(404) Traving, B.-C., 192 Trepo, C., 247 Trnka, T., 164 Tronchet, J. M. J., 45, 183 Trousdale, M. D., 249 Trown, P. W., 252, 26 l(742) Truscheit, E., 24, 52, 81(69), 88(69), 336, 338(60), 347, 348( 106) Trushkina, I. A., 121 Trust, T. J., 297 Ts’o, P. 0. P., 269 269,270(844) Tseng, C. K.-H., Tsuchihashi, G., I10 Tsuchiya, T., 126, 144(170), 164, 169(281), 223, 225, 226,227(616), 228(218, 616), 229,230,232,233 Tsukiboshi, Y., 31, 33(34), 34(34), 37(34) Tsumaraya, Y., 282, 359 Tsuno, T., 72, 88( 106) Tsuruoka, T., 331, 332, 335, 336, 337(45), 338(58), 348(45) Tulsiani, D. R. P., 342, 344(87, 88), 346(88) Tuominen, J., 202 Tuppy, H., 35 I, 352,352( 1 19) Turner, H., 202 Turner, W. R., 233 Tustin, J. M., 182, 239(344) Tyler, J. L., 200 Tyrell, D. L. J., 237, 247, 249(706) Tzotzos, G. T., 375

410

AUTHOR INDEX

U Uchino, H., 28 Uchiyama, T., 356, 379(129, 130) Ueda, T., 28 Uematsu, Y., 51, 52 Uemura, K., 187, 188 Uemura, M., 33, 36(37), 37(37) Ueno, Y., 41,45(50, 5 I), 46(50, 5 I ) Uesugi, S., 268 Ugalde. R. A., 301, 302(128) Ujiie, A., 195 Umeda, J.. 78 Umezawa, H., 72, 126, 144(170), 164, 223, 226. 227(616), 228(616), 229,230, 233 Umezawa, S., 126, 144(170), 164, 223, 225, 226,227(616), 228(616), 229,230, 232, 233 Ungaretti, L., 134 Unger, F. M., I I I , 112,297 Unger, P., 289, 292, 306, 3 13 Unterweger, M. P., 189 Uramoto, M., 72 Urbano, M. R., 300 Usesugi, S., 269 Usui, N., 72 Utille. J.-P., 98, 301, 302(124) V Valentekovic-Horvath, S., 175, 176(319, 320), 177(320) Van Aerschot, A,, 234,258,259(778), 272(635, 778), 273(778) van Boom, J. H., I16 van Duijnen, P. T., 384 van Dyk, M. S., 102 van Heyningen, R., 322 van Rijn, C. J. S., 192 Vanderhaeghe, H., 258, 259(767) Vangehr, K., I 19 Vankar, Y.D., 100,102(69), 165(69),262(69) Vann, W. F., 282 Vanni, P., 332, 337(46), 340(46) Varela, O., 233 Vasella, A., 338 Vass, G., 66, 224 Vattle, J.-M., 133 Vechirko, E. P., 265 Veiko, V. P., 244, 266 Verhelst, G., 246, 247(694)

Verheyden, J. P. H., 95, 262, 274 Vidal, P. M., 255, 256 Vidal, P., 254 Vignon, M., 306 Villar, E., 330, 331(42b) Vince, R., 23 Vinogradov, E. V., 96, 292, 294, 295, 298, 304, 307(108), 310(105), 312, 313 Viratelle, 0. M., 349, 370, 376 Visser, G. W. M., 192 Voelter, W., 137 Vogt, M., 195, 197(429), 199(429) Voiland, A., 317 von Deyn, W., 40,41, 48(52), 49(52), 68, 87(52) Vora, M. M., 198 Vorobeva. E. A., 93 Voznij, Ya. V., 95, 107, 108, I 1 I , 119, 120, 121, 122(35, 36) Vustina, T. F., 295 Vyska, K., 204 W

Wagner, R., 193 Wait, R., 281 Wako, N., 164, 233(279) Waldmann, H., 114 Walker, D. E., 346 Walker, J., 244 Walker, R. T., 246, 247(694), 264(684) Wallace, R., 281 Walsh, C. T., 340 Walther, A., 367 Wan, C. N., 199 Wan,C.-N., 175, 188, 190(310, 311), 191(31I), 199,201(479), 202,204(311) Wanek, E., 25 I Wang, C.-C., 330, 331(41) Wang, J.-S., 93 Ward, R. A., 148, 158(248). 272(248) Warshel, A., 380, 382 Wassle, W., 330, 33 l(42a) Watanabe, C. R., 201 Watanabe, K. A., 234,235(639), 236,237, 238(639), 239(661), 243, 245, 246, 247(649, 695, 696), 248, 250, 251, 252, 254,255, 256.267, 272 Watanabe, K., 101, 107(71), Il2(71), 116(71, 72), 117, 123(72), 148

41 1

AUTHOR INDEX Watanabe, M., 195 Watkins. G., L., 196 Weckesser, J., 281, 287, 292,297, 302(13), 312 Weekerle, W., 233 Wehler, T., 306 Weidmann, H., 136, 226(2 13) Weinbaum, G., 280 Weinreich, R., 134, 195, 197(429), 199(429) Weise, G., 292 Weiser, W., 357 Weiss, H., 258 Weiss, M. J., 264 Welch, J. T., 93, 100, 102(69), 155, 165(69), 181, 262(69) Welch, M. J., 133, 143, 162(197), 195, 195, 196, 197, 199, 204(454,473), 208( 197) Welter, A., 345 Wensveen, M., 195 Wentworth, D. F., 349, 351(112), 352(112), 354( I 12) Werner, G., 274 Wessels, B. W., 195 Westerduin, P., I16 Westhof, E., 241 Westphal, O., 280,283, 301, 302(120) Westwood. J. H.. 95, 134, 135, 139(38), 188, 208(373), 224 Whistler, R. L., 322 White, W. J., Jr., 367, 369(164) Widdows, D., 374 Widen, L., 189 Widmalm, G., 282, 301, 302( 123), 3 12 Wiebe, L. I., 237, 246, 247. 249(706), 254, 260 Wiebe, L., 11 I Wieland, B. W., 195 Wientjes, M. G., 264 Wiesner, M., 120 Wikinson, F. E., 338. 366(76), 368(76) Wilchek, M., 363 Wilkins, C. L., 124 Wilkinson, S. G., 281, 290, 292, 301( 12), 302( I2), 304, 306( I40), 308, 3 I0 Williams, A., 320 Williams, B. A,, 202 Williams, D. J., 148, 152(247), 155, 158(247, 268). 165(268), 252(247), 253(268)

Williams, J. M., 29 I Williams, L., 255 Williamson, K. L., 93 Wilson, D. P., 236, 245(647), 260(647), 276(647) Wilson, R. A., 50 Winchester, B., 338 Wingender, W., 24, 52, 81(69), 88(69) Winkler, J., 207 Winter, J., 200 Winterbourne, D. J., 207 Winterhalter, R., 367, 368( 155) Win, P., 2 I2 Withers, S. G., 119, 121, 144, 145(239), 146(239), 149, 155, 156(239), 159(269), 160(269), 161(251, 269), 162(269), 177(146), 206, 207, 221(541), 322, 324( 18), 328( 18), 329( 18), 330, 33 1( 18, 3 9 , 332( I 8), 333( l8), 337( IS), 339( I8), 349, 383(18, 107), 362, 370, 372 Wnuk, S. F., 277 Wohlrab, F., 250, 256 Wolf, A. P., 141, 175, 177, 186, 188, 189, 190, 191, 192(328), 193, 194(394,41I), 195, 196, 199, 200, 201(479), 202, 203, 204(311) Wolf, A,, 187, 189(358), 200 Wolfe, S., 93 Wolfenden, R., 349, 351(112), 352(112), 354( 1 12) Wollenweber, H.-W., 308 Wong, C.-H., 208 Wong,T.C.,93,155,160(270),210(270),219 Wood, S. G., 273 Woolard, G. R., 141 Worsley, K. J., 200 Wrangsell, G., 30 I , 302( 123) Wray, V., 93, 177, 212(325) Wright, J. A., 183, 236, 245(647), 260(647), 270, 271(845), 272(845), 274, 276(647) Wright, J. J., 228 Wurzburg, B. A,, 345 Wyand, A., 197 Wyatt, P. G., 274 wyss, P. c., 102

X Xia, Y., 152

412

AUTHOR INDEX

Y Yacyshyn, H. P., 237 Yadav, J. S., 166,231(286) Yagupol’skii, L. M., 265 Yakovlev, G. I., 242 Yakovleva, L. M., 283 Yamada, H., 107 Yamada, K., 194,202,203 Yamada, T., I3 1 Yamada, Y., 55 Yamaguchi, K., 192, 194,203,207(419,530) Yamaguchi, T., 52,74(70), 88,203, 347 Yamamoto, H., 23, 24 Yamamoto, Y. L., 187, 199(359), 200 Yamamura, Y., 28 I Yamane, H., 131 Yamazaki, T., 126 Yanai, Y., 203,204(532) Yang, S. S., 148, 155, 157(242, 243), 162(273), 165 Yang, S.-J., 367 Yano, Y., 128, 131, 198(181) Yao, X., 249 Yanv, J., 374, 3 7 3 181), 376 Yasillo, N. J., 195, 197(437) Yasuda, K., 5 1, 84 Yasumon, T., 141 Yato, Y., 32, 38(36) Yde, M., 334 Yegorov, A. M., 108 Yeh, H. J. C., 119, 120(148), 145(148), 146(148), 156(148), 218,220 Yeh, S. D. J., 202 Yokoi, S., 70 Yokose, K., 25, 81(18) Yon, J. M., 320, 376 Yon, J., 349 Yonekura, Y., 199,202

Yonezawa, H., 72 Yoshida, S., 52 Yoshikawa, M., 347 Yoshimura, J., 141 Yoshimura, Y., 116 Yoshino, H., 249 Yoshioka, S., 194. 202, 203 Youds, P. M., 165, 272(282) Youds, P., 139, 148, 152(247), 155, 158(217, 247. 268), 165(268), 252(247), 253(268) Young, C. W., 255 Young, N. M., 292 YusofF, W. R., 195 Z Zabin, I., 374, 375(179) Zabinski, S. V., 202 Zahnnger, U., 295, 297, 308 Zajc, B., 176 Zakharova, I. Ya., 283, 285 Zamojski, A., 125 Zatorre, R. J., 200 Zdorovenko, G. M., 283 Zefirov. N. S., 93, 243( 17) Zehavi, U., 292,303 Zeng, Y.-C., 367 Zhang, S.-Z., 367 Zhi-Ming, Z., 249 Zhuk, R. A,, 265 Zieger, B., 350 Zikopoulos, J. N., 216 Zimmerman, R. A., 201 Zissis, E., 330, 333(44) Zubieta, J., 155 Zubkov, V. A., 288 Zupan, M., 175, 176, 177

SUBJECT INDEX

A

Acarbose. 24-25, 348 inhibition of sucrase by, 88 synthesis, 84 4-Acetamido-4,6-dideoxy-6-~uoro-cu-~galactopyranosy I-D-glucopyranosides, 21 I 2- Acetamido- 1,3,4-tri-O-acetyl-2,6-dideoxy6- tluoro-D-mannopyranose, 2 10 N-Acetimidoyl groups, bacterial polysaccharides, 307 I -O-Acetyl-a-DL-carba-glucopyranose, 61 -62 N-Acetyl-2,3-dehydro-neuraminicacid, 35 1 5-N-Acetyl-9-deoxy-9-tluoroneuram~nic acid, 2 I0 5-N-Acetyl-3-fluoroneuraminic acid, 2 I0 N-Acetylneuraminic acid, 1 I4 a-(2 6)-linked N-Acetylneuraminic acid glycosides, synthesis, I 15 Acidic sugars, 292 - 293 glyculosonic acids, 295-298 glycuronic acids, 293 -295 Aclacinomycins, 10 Acrylic acid, Diels- Alder cycloaddition, racemic carba-sugars, synthesis, 29- 36 Activation parameters, glycoside hydrolysis, 323-324 Acyl groups, bacterial polysaccharides, 306-31 I Adiposin. 24-25 Adiposin-I, synthesis, 85 Aglycons, non-sugar, 299- 300 Alditol phosphates, bacterial polysaccharides, 316-317 Aldono- I ,5-lactones, 328 - 330 Aldonolactones, reversible inhibitors of glycosidases, 327-333 P-Aldopyranoside, 327 Aldoses branched-chain sugars, 287 - 288 heptoses, 285 -287 hexoses, 281 -285 pentoses, 28 I D-AIIox, 282

-

413

L-Altrose, 282 Amides, bacterial polysaccharides, 3 I I -3 I3 Amino acids catalytic, sequence homologies around, 38 1 ester- and amide-linked, 310-31 1 Amino carba-hexopyranose, physical p r o p erties, 60 Amino carba-sugars, synthesis, 66 -67 Aminocyclitols, glycoside hydrolase inhibition, 347 - 348 5-Amino-5-deoxyaldonolactams,glycosidase inhibition, 330-332 3-Amino-3deoxy-/l-~~-carba-altropyranose, synthesis, 59, 6 1 2-Amino-2-deoxy-a-~~-carba-glycopyranose, 63 2-Amino-2deoxycarba-hexoses, synthesis, 59-63 enantiomeric, 66-67 2-Amino-2-deoxy-~-~r-carba-mannopyranose, synthesis, 60-61 2-Amino-2-deoxy-c~-~-glucopyranosyl bromide, 72-73 2-Amino-2deoxy-a-~-glucopyranosyl a-D-glucopyranoside, 72 2-Amino-2-deoxy-c~-~-~ucopyranosyl a-D-mannopyranoside, 72 5-Amino-5-deoxylactams, reversible inhibitors of glycosidases, 327-333

4-Am~no-4,6-dideoxy-6-fluoro-a-~-galactopyranosyl-a-D-@ucopyranosides, 21 1-212 2-Am~no-2,6-dideoxy-6-tluoro-cu-~-glucopyranose hydrochloride, I39 Amino-dideoxyhexoses, 290- 292

(+)-4-Amino-3,3-difluoro-2-hydroxycyclopentylmethanol, 152- I53 Aminoglycoside antibiotics, resistance mechanism, I 1 Amino sugars, 290-292 7-04 3-Amino-2,3.6-tndeoxy-2-fluoro-a-~talopyranosy1)daunom ycinone, 232-233 Amylostatin (XG), synthesis, 82 2.6-Anhydro- I -deoxy-D-galactuhept- 1 enitol, 354-355

SUBJECT INDEX

414

2,6-Anhydro- ldeoxy-~-g/uc@heptI-enitol, 356 3.6-Anhydro-6,6-difluorofuranoses, 1 53 2,3-Anhydro-~-gulopyranoside, 164 4,7-Anhydro-5,6,8-tri-O-benzylI ,3dideoxyD-2-OCtUlOse, synthesis, I 16 Antibiotics, synthesis, Mukaiyama condensation method, 106- 107 Anticancer agents fluoro sugars, 222-23 I research, Japan, 9- 10 Antimicrobial agents, fluoro sugars, 222 - 23 I D-Arabinose, enantiomeric Carba-sugars from, 42-44 L-Arabinose, enantiomeric carba-sugars from, 4 1 -42 I ,2-rrans Aryl glycofuranosides, synthesis,

non-sugar aglycons, 299 - 300 pentoses, 28 I phosphoric esters, 3 I3 - 3 I7 reducing terminal linkage, 3 I5 sulfated, 3 I 3 - 3 14 teichoic acids, 3 15 - 3 16 Baiyunoside, synthesis, 107 Baumycins, 10

3-Benzamido-2,3-dideoxy-2-fluoro-cr-~altropyranoside, 166 4-O-Benzoyl-6-deoxy-3-O-methyl-2-O-triflyl-L-glucopyranosyl fluorides, I27 DL- 1 -0-Benzoyl-3,4,5,6-tetra-O-benzyl-2-

(bromomethy1)-rnyuinositol, 148

Benzyl2-acetamido-6-0-(2-acetamido-2,4dideoxy-4-fluoro-~-~-glucopyranosyl)-2deoxy-a-D-galactopyranoside, 2 I8 Ill Benzyl 2-acetamido-6-O-benzyl-2,4-dideoxyI ,2-frans Aryl glycopyranosides, synthesis, 4-fluoro-~u-~-glucopyranoside, 149 Ill Benzyl 2-acetamido-2,4-dideoxy-4-fluoro-6( 1S,2R,3R,4S)- I -Azido-4-benzyloxy-3-(ben0-trityl-cu-D-gulopyranoside, 163 zyloxymethyl)-2-fluorocyclopentane, 4,7-O-Benzylidene-P-~~-carba-galactose. 139

3-Azido-2-fluoro-cu-~-altro-pyranosides, 145 2-Azido-3-fluoro-cxu-~-gluco-pyranosides, I45 Aziridines, sugar-related, glucosidase inhibition, 371 -373

B Bacterial polysaccharides acetyl-subst it uted, 304 - 306 acyl groups, 306 - 3 I 1 alditol phosphates, 3 I6 - 3 I7 amides, 3 I 1 - 3 I3 biosynthesis, 3 18 branched-chain sugars, 287 -288 I -carboxyethy1 ethers, 303 - 304 diarnino sugars, 292 ether and acetal substituents, 299-306 glycerol phosphate substituents, 3 15 glyculosonic acids, 295 - 298 glycuronic acids, 293 - 295 heptoses, 285-287 hexoses, 281 -285 methyl ethers, 300-302 monoamino sugars, 290-292

synthesis, 60-61 Benzyl 3,4,6-tri-0-benzyl-2-O-triflyl-P-~glucopyranoside, 124- 125 Beta amylase, catalysis of P-maltosyl fluoride hydrolysis, 358- 359 Bioactive products, from marine microorganisms, 14 3’5’,-Bis(thymidylyl) difluoromethylphosphonate, synthesis and biological activity, 266 Bleomycin, research, Japan, 9- 10 Branched-chain sugars, 287-288 N-( Brornoacety~)-~-~-galactosylamine, 375-376 ( I ,3/2,4,6)-4-Bromo-6-(bromomethyl)I ,2,3cyclohexanetriol triacetate, 3 I Bromoconduritols, 376- 377 2-Brorno-2-deoxy-carba-c*.-~r-galactopyranose tetraacetate, 30

C C-2, deoxygenation effects on enzymesubstrate affinity, 361 Carba-cr-DL-allopyranose,32 Carba-P-~~-allopyranose, 33

SUBJECT INDEX Carbaa-D-akropyranose pentaacetate, synthesis, 44 Carba-a-DL-altropyranose pentaacetate, synthesis, 28-29 Carba-cu-L-altropyranosepentaacetate, synthesis, 46 4a-Carba-P-~-arabinofuranose,22 - 23 4a-Carba-aristeromycin, 23 Carba-cellobiose, synthesis, 68 - 70 6a-Carba-P-~~-fructopyranose, synthesis, 50-51 Carba-furanoses, 22 Carba-a-D-galactopyranose, 24,86 - 87 synthesis, 36, 40 Carba-a-~~-galactopyranose, 23, 33 synthesis, 27-28 5a-Carba-c~-~-glucopyranose pentaacetate, synthesis, 49 Carba-cu-DL-galactopyranose pentaacetate, 29 synthesis, 27 - 28 Carba-P-DL-galactopyranose pentaacetate, synthesis, 28-29 Carba-a-D-glucopyranose, synthesis, 38 Carba-/3-D-glucopyranose,synthesis, 36 Carba-/3-DL-glucopyranose,33, 87 Carba-j?-L.-glucopyranosepentaacetate, synthesis, 44 Carba-glucopyranosyl a-D-glUCOpyI‘anOsides, synthesis, 7 1 Carba-glycosylamines enantiomeric, synthesis, 64-65 inhibitory activity, 88-89 synthesis, 52-59 5a-Carba-/3-~~-gulopyranose, 23 synthesis, 27 Carba-a-~~-gulopyranose pentaacetate, 27, 34 Carba-/3-DL-idopyranose,34 Sa-Carba-a-~-idopyranosepentaacetate, synthesis, 48-49 Carba-isomaltose, synthesis, 68- 70 Carba-maltose, synthesis, 68-70 Carba-a-DL-mannopyranose pentaacetate, 31 Carba-/3-L-mannopyranose pentaacetate, synthesis. 44 a-DL-Carba-mannopyranosylamine, synthesis, 59, 6 I

415

Carba-oligosaccharides, biologcall y active, 74-86.279-318 alpha amylase inhibitors, 81 -86 validamycins, 74- 78 Carba-pyranoses, 22 4a-Carba-P-~-nbofuranosylamine, 23 Carba-sugars, 2 I -90, see also Enantiomeric carba-sugars; Racemic carba-sugars 6a-carba-~fructopyranoses,49 52 amino, synthesis, 66-67 biological effects, 86-89 mono- and dicarba-disaccharides, 67 - 73 physical constants, 37 5a-Carba-a-~~-talopyranose, 23 5a-Carba-cu-D~-talopyranosepentaacetate, 27 Carba-P-DL-talopyranose pentaacetate, 34-35 Carba-trehalosamine, 88 89 synthesis, 72-73 Carba-trehaloses, synthesis, 69- 70 I-Carboxyethyl ethers, 303 -304 Carboxylate groups, at active site, 379 Castanospermine, P-D-glucosidase inhibition, 342 - 344 Catalysis, substrate distortion, glucosidase inhibition, 382 Catalytic efficiency, glycoside hydrolases, 323-325 Cellulose, treatment with HF, 97 C-F bond energy, 92 Chitin, treatment with HF, 98-99 3-Chloro-4-enouronate, reactions with AgF, 102-103 Conduritol epoxides glycoside hydrolase inhibition, 364-37 I kinetic constants, 366 reaction with glycosidases, 368 - 369 DL- 1,2-O-Cyclohexylidene-5-deoxy-c~~~~ inositol, 28

-

-

D DAST reagent, fluorine introduction, see Fluorine, introduction by DAST reagent 5-Deoxy-5,4”diepi-5-fluorokanamycin A, 225

4 I6

SUBJECT INDEX

6-Deoxy-6,6-difluoro-~-galactose, I52

2-Deoxy-2-fluoro-3,4:5,6-di-O-isopropyli2-Deoxy-2,2-difluoro-~-erythro-pentofurandene-aldehydo-D-glucose, 155, 163 ose, 182 2-Deoxy-2-fluoro-~-~-erythrofuranosy~I -( 2-Deoxy-2,2-difluoro-P-~-erythro-pentoamine, 242-243 furanosyl)cytosine, synthesis, 239 - 240 4-Deoxy-4-fluoro-~-fructose,183 2-Deoxy-2-fluoro-~-fucose, labeled, 2-Deoxy-2,2-difluorosugars,synthesis, I8 1 - I82 synthesis, 203-204 I -Deoxy-N,Ndimethylnojirimycin, 3-Deoxy-3-fluoro-~-D-ga~actopyranose, glucosidase inhibition, 339- 340 synthesis, 134- I35 5-Deoxy-5-epi-5-fluorokanamycin A, 225 4-Deoxy-4-fluoro-~u-~-galactopyranosides, 2’-Deoxy-2’-fluoroadenosine, synthesis, 149 268 - 269 Deoxy-fluoro-a-D-galactopyranosyl-D5‘-Deoxy-5‘-fluoroadenosine, synthesis, 277 glucopyranosides, 2 1 I 6-Deoxy-6-fluoroaldohexoses, synthesis, I49 2-Deoxy-2-fluoro-~-ga~actose, 13 I 2-Deoxy-2-fluoro-~-altroside,I 66 labeled, I92 - 194, I98 2-Deoxy-2-fluoro-~-arabinofuranose, metabolism, 207 133-134 4-Deoxy-4-fluoro-~-ga~actosides, 137 I -( 2-Deoxy-2-fluoro-~-~-arabinofuranosyl~ 4-Deoxy-4-fluoro-galacto-sucrose, 2 I 5 cytosine, synthesis, 236 I -( 6-Deoxy-6-fluoro-~-~-glucoand I -(2-Deoxy-2-fluoro-~-~-arabinofuranosyl)- -galacto-pyranosy1)thymine.synthesis 5-ethyluraci1, biological activity, 250 and biological activity, 266- 267 138- I 39 1 -(2-Deoxy-2-fluoro-&~-arabinofuranosy~)- 5-Deoxy-5-fluoro-~-glucofuranose, 5-fluorouracil, biological activity, 250 2-Deoxy-2-fluoro-~-glucopyranosyl fluoride, I -(2-Deoxy-2-fluoro-P~-arabinofuranosy~)labeled, 203 5-iodocytosine, biological activity, Deoxyfluoro-cu-D-glucopyranosyl phos246 - 248.254- 255 phates, 205 - 206 I7 I , I75 I -(2-Deoxy-2-fluoro-P-~-arabinofuranosyl)- 2-Deoxy-2-fluoro-~-~ucose, labeled, PET technique, 199-202 5-iodouraci1, labeled, biological activity, labeled, synthesis, 121, 124, 143- 144, 255-256 192- 194, 197-198 1-(2-Deoxy-2-fluoro-~-~-arabinofuranosyl)5-methylcytosine metabolic fate, 188- I89 biological activity, 248 - 249, 255 metabolism, 207 synthesis, 238 3 - ~ O X y - 3 - f l U O r O - D - ~ U C O 183 ~, metabolism, 209 I -(2-Deoxy-2-fluoro-&~-arabinofuranosy~)thymine labeled, 199 biological activity, 248-249, 255 4-Deoxy-4-fluoro-~-~ucose, synthesis, 135-136 synthesis, 238 2-Deoxy-2-fluoro-~-araabinose Sphosphate, 5-Deoxy-5-fluoro-~-glucose, synthesis, 137-138 208 6-Deoxy-6-fluoro-~-aorbicacid, I42 3-Deoxy-3-fluoro-~-glucoside,166 2-Deoxy-2-fluoroglycosides,glucosidase Deoxyfluoro carbohydrates, labeled inhibition, 359-362 biological studies, 203 -204 2-Deoxy-2-fluoro-~~-gulopyranoside. 132 synthesis and biological applications, 204 5-Deoxy-5-fluoro-~-idofuranose, 138- I39 Deoxyfluorodeoxy carbohydrates, labeled, 5-Deoxy-5-fluoro-~-idose,synthesis, synthesis, 190- I99 2’-Deoxy-2’-fluorocytidine 137-138 biological activity, 245 -246, 254 (-)- IL-I-Deoxy-I-fluoro-myo-inositol, I52 3’-Deoxy-3’-fluorokanamycinA, 226 synthesis, 235-236 5-Deoxy-5-fluorodibekacin,229 4”-Deoxy-4”-fluorokanamycinA, 226

SUBJECT INDEX

417

cytosine, synthesis and biological 6”-Deoxy-6”-fluorokanarnycinA, 224 - 226 activity, 260 3’-Deoxy-3’-fluorokanarnycinB, 226 -228 5-Deoxy-5-fluoro-6,3-lactones, I 53 2-Deoxyglycosides, glucosidase inhibition, 5”-Deoxy-5”-fluorolividornycinB, 229 -23C 359-362 2’-Deoxy-2’-fluorornaltose,2 I2 - 2 I3 3-Deoxyglyculosonic acids, 296 3-Deoxy-3-fluoro-~-mannito~,I64 1-Deoxy-D-g/uco-hept- 1-enitol, reaction 2-Deoxy-2-fluoro-&~-rnannopyranosyl with a-D-glucosidases, 356-357 fluoride, conformation, 177 6-Deoxyheptoses, 286-287 4-O-( 2-Deoxy-2-fluoro~-~-mannopyrano- 3-Deoxyheptulosanc acid, 298 1-Deoxy-D-ga/aclo-heptulose,354 - 355 syl)-D-glucopyranose, 2 13 I -Deoxy-D-g/ucc+heptulose,356 2-Deoxy-2-fluoro- D-rnannose, 17 I labeled 2-Deoxy-~-arabino-hexose, labeled, PET technique, 203 186- 188 synthesis, I92 - 194, I98 2-Deoxyhexoses, 282-283 2-Deoxy-~-arabino-hexoside,hydrolysis, 353 synthesis, 128- 129 3-Deoxy-3-fluoro-~-rnannose,164, I83 2-Deoxy-c~-~-arab~nuhexosy~, formation, 2-Deoxy-2-fluoro-3-O-rnethyl-~-glucopyran- 353-354 Deoxylividornycin B, 229 -230 ose, labeled, 198 5-0-(4-Deoxy-4-fluoro-~-~-rnycarn~nosyl)1-Deoxynojirirnycin, glucosidase inhibition, tylonolide, 230-23 1 380 3-Deoxynonulosonic acids, 297 2-Deoxy-2-fluoro-~-pentopyranoses, I8 1 3-Deoxy-~-rnanno-2-octulopyranosonic 5-Deoxy-5-fluorosisomicin,228 -229 acid, 111-112 4-Deoxy-4-fluoro-cu-~-sorbopyranose, I8 3 3-Deoxy-~-rnanno-octulosonic acid, 297 Deoxyfluorostarches, 222 2-Deoxy-&~-g~~vcero-tetrofuranosy~arnine, Deoxyfluorosucroses, 2 I4 - 2 I7 242 -243 Deoxyfluoro sugars, fluorine- 1 8-labeled ( I ,3,5/2,4)-2,3-Diacetoxy-4,5-dibrornocyclobiological application, 199-203 hexane-I-carboxylic acid, 32 UWS, 186- I89 3.4- Trans-Di-0-acylglycals, treatment with 2-Deoxy-2-fluoro sugars pyridiniurn poly(hydrogen fluoride), labeled, synthesis, I93 - 194 102- I03 synthesis, 170. 172-173, 181-182 3-Deoxy-3-fluoro sugars, synthesis, I33 - I34 2,7-Diamin0-2,7-dideoxy-cr-~~-carba-gluco2-Deoxy-2-fluoro-~-talopyranoside, 164 pyranose, 63 4.7 - Diarnino-4,7-dideoxy-crcu-~~-carba2-Deoxy-2-fluoro-~-talose,labeled, 192 3’-Deoxy-3”-fluorothymidine,synthesis and glucopyranose, 63 Diarnino sugars, 292 biological activity, 257-259 5,7-Diarnino-3,5,7,9-tetradeoxynonulosonic 5’-Deoxy-5-fluorothymidine,synthesis and acids, 298 biological activity, 262 - 264 I ,6:3,4-Dianhydro-&~-altropyranose, 5-Deoxy-5-fluorotobrarnycin, 229 6-Deoxy-6-tluoro-a,au-trehalose,2 1 I 163- I64 2’-Deoxy-2’-fluorouridine I ,6:2,3-Dianhydro-4-0-benzyl-&~-allopyrbiological activity, 254 anose, 163 conformation, 240- 243 Dibekacin, synthesis, I I - 12 Dicarbadisaccharides, synthesis, 67 - 73 synthesis, 234-235 5’-Deoxy-5’-fluorouridine,synthesis and 2,6-Dideoxy-0-( 2,3-dideoxy-2-fluoro-cu-~ribo-hexopyranosy1)streptarnine.224 biological activity, 26 I - 262, 264-265 9-(3-Deoxy-3-fluoro-~-~-xy~ofuranosy~)- 4,6-Dideoxy-4,6-difluoro-a-~-galactopyranadenosine, synthesis, 275-276 osyl-D-glucopyranosides, 2 I 1 1-( 3-Deoxy-3-fluoro-~-~-xy~ofuranosy~)-4,6-Dideoxy-4,6-difluoro-~-galactosides, 137

418

SUBJECT INDEX

6,6’-Dideoxy-6,6’-difluorosucrose, 2 I6 Enantiomeric carba-sugars, synthesis 4,6-Dideoxy-4,6-difluoro-cu-~-talopyrano- a-L-altro and P-D-glUC0 modifications, side, 149 46-47 2’,3’-Dideoxy-3’,5’difluorouridine, amino carba-sugars, synthesis, 64-67 synthesis and biological activity, 256 from L-arabinose, 4 1 -42 4”,6”-Dideoxy-4”-epi-4”,6”diIluorokanafrom D-arabinose, 42 - 44 mycin A, 226 Diels- Alder adduct, resolution, 36, 38- 39 5,6”-Dideoxy-5-epi-5,6”difluorokanamycin from D - ~ ~ U C 45-49 OS~, A, 225 from optically active natural products, 2,6-Dideoxy-2-fluoro~-~-galactopyranosyl 40-41 fluoride, 176 from D-ribose, 44 2’,3’-Dideoxy-2’-fluorokanamycin A, 228 from true sugars, 4 1 -49 9-(2,3-Dideoxy-2-fluoro-/3-~-erythro-pento- from D - X Y ~ O S ~44 , furanosyl)adenine, synthesis, 269 - 270 Enzyme inhibitors, low molecular weight, 7-0-(2,6-Dideoxy-2-fluoro-a-~-talopyranoresearch, Japan, I 3 - I4 syl)daunomycinone, 232 - 233 3-Epi-3-fluoro-3de(methoxy)sporaricinA, 2,3-D~deoxy-2-fluoro-3-tosylamino-cu-~223 altropyranoside, 169 Epimine, cleavage by fluoride ion, 155, 3,6-Dideoxyhexoses, 284- 285 163-169 Diels- Alder adduct. resolution, 36, 38-39 Epoxide, cleavage by fluoride ion, 155, Diels- Alder cycloaddition, furan and 163-169 acrylic acid, racemic carba-sugars, synthesis, 29-36 2P,6&Difluoroandrost-4-ene-3,17-dione, F 165 3,3-Difluoro-3-de(methoxy)sporaricinA, 223 Fluorinated carbohydrates, 9 1 -93, 121, Di-0-isopropylidene-a-m-carba-mannopyr124- 142, 142- 155, see also Fluorine anose, 73 addition to glycals, 169- I8 I Di-0-isopropylidenecyclohexaneterol, 40 from DAST treatment 2,3:5,6-Di-O-isopropylidene-cu-~-mannofurwith migration or configuration anosyl fluoride, synthesis, I0 I - 102 retention, 156- I58 Di-0-isopropylidene-DL-validamine, 76 with normal displacement, 159- 163 Di-0-isopropylidenevalinamine,76 epoxide and epimine cleavage by fluoride 4,7-Di-methanesulfonates, 63 ion, 155, 163-169 2,4-Dinitrophenyl 2-deoxy-2-fluoro-/3-~glycals with MeCO,F, 179- 180 glucopyranoside, 207 purity of compounds synthesized, 178 Dissociation constant. 330-33 I Fluorine, 9 I -92 introduction by DAST reagent, 142- 155 COSF,NEt, group, 143 retention of configuration, 147- 148 E I ,2-shift, 145- 146 sulfonyloxy group displacement, Electrophiles, with reactive halogen acid-catalyzed epimerization, 130 substituents, glucosidase inhibition, 131 375-377 Fluorine- 18, 189 Enantiomeric 2-amino-Z-deoxycarbaproduction, 196- 197 hexoses, synthesis, 66-67 3-Fluoro-3-de(methoxy)sporaricinA, 223 Enantiomenc 6a-carba-j?-fructopyrnoses, 2-Fluorofuranoses, synthesis, 132- I33 synthesis, 5 1 - 52 2-Fluoro-~-D-galactopyranoside,I 3 I Enan tiomeric carba-glycosylamines, 2-Fluoro-a-~-gulopyranoside, 13 1 - I32 synthesis, 64 -65

SUBJECT INDEX Fluorohydrin, synthesis, 148 2-C-(Fluoromethyl)-rnyo-inositol,165 Fluoromethylphosphonate, synthesis, I85 I86 C'Fluoropurine nucleosides. synthesis and biological activity, 267-277 2'-Fluoropyrimidine nucleosides biological activities, 244-256 conformational analysis, 240-244 synthesis, 234-240 Fluoro sugars, antimicrobial and anticancer, 222-234 Free energy of activation, glycoside hydrolases, 324- 325 D-Fructose, treatment with HF. 97-98 1-Fucopyranosides, 132- 133 L-FUCOX,283 Furan, Diels- Alder cycloaddition, racemic carba-sugars, synthesis, 29- 36

G D-GahCtal, 349 - 350 D-Galactose, 282 D-Galactosyl di- and tri-saccharides, 2 18-220 p-D-Galactosylmethyl carbenium ion, formation, 373-374 p-~-Galactosylmethyl-4-(nitropheny1)triazene, 373- 374 p-D-Glucopyranosides enzymic hydrolysis, 323 - 324 non-enzymic hydrolysis, 322- 323 P-D-Glucopyranosylvalidamine,75 D-GIUCOX, 281 -285 enantiomeric carba-sugars, synthesis, 45-49 regional consumption evaluation, 186-189 P-D-Glucosidases, inhibition, 207, 333-334 by castanospermine, 342 - 344 pH-dependence, 338-339 D-Glucosides, containing 2-deoxy-2flUOI'O-~-D-@UCOSyl POUP. 208 I -~-D-Glucosylirnidazole, inhibition by, 334-335 P-D-GIucos~Iisothiocyanate. 377- 378 Glycals, addition to, fluorinated carbohydrates, I69 - I8 I

419

D-Glycals, 349- 357 hydration stereochemistry, 354 inhibition patterns, 35 I interaction with glycosidases, 350- 352 intermediates formed in slow inhibition, 352-353 kinetics of hydration, 355 proton transfer to C-2, 353 Glycopranosyl oxocarbenium ion, 327 Glycoside hydrolases, 3 19- 384 active-sitedirected inactivation, 370 anomeric specificity, 329 - 330 catalytic efficiency, 323 - 325 inhibition access of solvent water to active site, 380-381 carboxylate and proton-donating groups at active site, 378-379 generalizations and exceptions, 383-384 ionic strength effects, 380 sequence homologies around, 38 I slow, tight-binding inhibitors and transition state, 382 - 383 substrate distortion and catalysis, 382 irreversible inhibitors, 362-364 conduritol epoxides, 364-37 1 electrophiles with reactive halogen substituents, 375-377 glycosyl isothiocyanates, 377- 378 glycosylmethyltriazenes, 373 - 375 sugar-related aziridines, 371 -373 mechanistic information from X-ray crystallography, 325-326 reversible inhibitors aldonolactones and 5-amino-5-deoxylactams. 327 - 333 aminocyclitols, 347- 348 cyclic sugar analogs having imino POUP, 335 - 34 I 2-deoxy- and 2-deoxy-2-fluoro-glycosides, 359-362 D-glycals, 349 - 357 glycosylarnines, 333- 335 glycosyl fluorides, 357-359 indolizine alkaloids, 341 344 polyhydroxypyrrolidines, 344 - 347 pseudosubstrates, 348-349 slow onset, 340

-

420

SUBJECT INDEX

G1ycosides low basicity, 32 I - 322 non-enzymic hydrolysis, 320- 323 1,2-trans-Glycosides, synthesis, 108 Glycosidic bond, cleavage, 128, 326 Glycosylamines, glycosidase inhibition, 333-335 Glycosylation, glycosyl fluorides, 103- 104, I07 - I08 Glycosyl fluorides cleavage, 358-359 glycoside hydrolase inhibition, 357-359 synthesis, 94- 12 I affinity of silicon, 107 C-arylglycosyl derivatives, I I9 BF, catalyst, 108 cellulose treatment with HF, 97 chitin treatment with HF, 98-99 condensation reactions, 109- I 10 enzyme-catalyzed reactions, I2 I D-fmCtOse treatment with HF, 97-98 glycosylations, 103- 104, 107- 108 C-glycosyl compounds, I I6 - 1 I8 inulin treatment with HF. 97-98 Mukaiyama condensation methods, 104- I07 Noyon procedure, 107 from 0-protected free sugars or 1-0-acyl sugars, 100- 101 per-0-acyl displacement, 94-95 from phenyl thioglycosides, 100 physical and chemical properties, I 19121 polysaccharide treatment with HF, 96 protected, 122- 123 D-ribofuranosyl fluorides, 10 1 treatment with HF, 95-96 treatment with pyridinium poly(hydr0gen fluoride), 102 Glycosyl isothiocyanates, glucosidase inhibition, 377-378 Glycosylmethyltriazenes, glucosidase inhibition, 373-375 Glycotriosyl ceramide, synthesis, 107 GI~CUIOXS, 288-289 Glyculosonic acids, 295 - 298 Glycuronic acids, 293 -295

H 5-Halo-4-0x0 nucleosides, biological activity, 248 Heptenitol, hydration, 355 Heptoses, 285-287 a-Heptuloside, formation, 356 Hexoses, 28 I -285 Hydrogen fluoride glycosyl fluoride synthesis, 95-96 polysaccharide degradation, 96 Hydrolysis 2-deoxy-~-aruhincrhexoside,353 enzymic, glucosides, 323 - 324 a-and P-maltosyl fluoride, 358 - 359 4-methylumbelliferyl P-D-glucosides, 359360 non-enzymic, glycosides, 320- 323 participation of carboxylate group, 379 Hydroxyvalidamine, 74 synthesis, 52-53 I I ,5-lminoalditols, glucosidase inhibition, 336, 338 Imino group, cyclic sugar analogs with, 335-341 Immunostimulants, 13- 14 lndolizine alkaloids, glucosidase inhibition, 341 -344 Inhibition constants, polyhydroxypyrrolidines, 345 - 346 I L-chirdnositol, 40 myo-Inositol, racemic carba-sugars from, 28-29 Institute of Bioorganic Chemistry, establishment, 12 Institute of Microbial Chemistry, establishment, 8 Inulin, treatment with HF, 97-98 Isomalto-oligosaccharides, fluorinated, 22 1 4,7-O-Isopropylidene-a-~~-carba-galactose, synthesis, 60 - 6 I

J Josamycin, 9

SUBJECT INDEX

42 I

K

pylidene-D-eryrhr@pn topyranosides, I53 Kanamycin, discovery, 6-7 4-deoxy-4-fluoro-P-~-ga~actopyranoMethyl Kanamycin A, fluorinated analogs, 224-228 side, 209 - 2 10, 136 Kasugamycin, 8 Methyl 4-~-(deoxy-fluoro-cu-~-ga~actopyC,inhibitors, 364 ranosyl)-P-D-galactopyranoside, 213-214 Methyl 6-deoxy-6-fluoro-4-U~-~-galactoL pyranosyl-P-D-galactopyranoside, 213-214 Lipid A Methyl 3,6-dideoxy-3.6difluoro-P-~components, 308 allopyranoside, 149 phosphate and pyrophosphate. 3 14- 3 15 Methyl 4,6-dideoxy-4,6-difluoro-cu-~Lividomycin B, 229 - 230 galactopyranoside, I49 Methyl 2,3dideoxy-3-fluoro-~-er.~~~ru pentofuranoside, 135 M Methyl ethers, 300-302 4-Methylumbelliferyl P-D-glucosides. 6-U-a-Maltosylcyclodextrins, synthesis, I 16 a-and P-Maltosyl fluoride, hydrolysis, hydrolysis, 359-360 358-359 Michaelis constant, D-glycal hydration, 355 D-Mannose, 282 Monoaminohexoses, 290 Monocarba-disaccharides, synthesis, 67 - 73 a-D-Mannosidase, inhibition by swainsonMono-6-deoxy-6-fluorocyclomaltoheptose, ine, 342. 344 Marine micro-organisms, bioactive products 22 I from, 14 Mukaiyarna condensation methods, glycosyl fluoride synthesis, 104- 107 Mechanism-based inhibitors, 364 Muramic acid, 303 4-Methanesulfonates, 63 Methyl 3-acetamido-4-U-benzoyl-2,3,6-tri- mycobacteria, 308-3 10 deoxy-2-tluoro-~~-mannopyranoside, Mycinamicins, synthesis, 1 10 Mycobacteria 232-233 muramic acid, 308-310 Methyl 2-acetamido-2,6-dideoxy-6-fluoro-cuD-galactopyranoside, I39 polysaccharides, 308 Methyl 3-acetamido-2,3,5,6-tetradeoxy-5fluoro-rihuhexopyranosides, I55 Methyl 2,3-anhydro-4-deoxy-4-fluoro-cu-~N lyxopyranoside, 137 Methylated sugars, 300-302 Neuraminic acid glycosides, synthesis, I 14 C-Methyl 7-azabicyclo[4. I .O]heptanes, Nojirimycin, glucosidase inhibition, epimine-ring opening, I68 335-336 Methyl 2-azido-4,6-0-benzylidene-2,3Non-sugar aglycons, 299 300

dideoxy-2-C-(fluoromethyl)-P-~-ribo-

-

hexopyranoside, 142 Methyl 3-azido-4.6-0-benzylidene-2,3dideoxy-3-C-(fluoromethyl)-cu-~-ara0 hinuhexopyranoside, 142 Methyl 2-benzamido-4.6-U-benzylidene-2- Octenitol. 356-357 deoxy-3-U-tosyl-cu-~-glucopyranoside, Oligosaccharides, partially fluorinated 166 low-molecular-weight, 2 12 Methyl 2-deoxy-2,2-difluoro-3,4-U-isopro- Oligostatin, 8 1, 83

422

SUBJECT INDEX

Optically active natural products, enantiomeric carba-sugars, synthesis, 40-4 1 e.uo-7-Oxabicyclo[2.2. I Ihept-5-ene-2carboxylic acid, 35

P

McCasland’s three carba-sugars, 26 -27 from myo-inositol, 28-29 L-Rhamnose, 283 Ribitol phosphate, bacterial polysaccharides, 316 D-Ribofuranosyl fluorides, synthesis, 10 1 D-Ribose, enantiomeric carba-sugars, synthesis, 44

Paratyphoid B epidemic, Japan, 3 Pellictilaria sasakii, validamycin and validoxylamine activity against, 87 S Penicillin production, Japan, 4 -6 Saccharides, biologically active, 205 -222 Penta-N,O-acetates, 59 -60 Penta-N,~-acety~-3-arnino-3-deoxy-c~-~~-metabolism, 206 Sialidases, interaction with glycosidases, carba-glucopyranose, 6 I -62 35 1-352 Penta-N,O-acetylamino-deoxycarba-sugars, 60,62 Solvent water, access to active site, 380-381 Penta-N,O-acetyl-4-arnino-4,7-dideoxy-a- Spergualin, 10 Streptomycin production, Japan, 6 DL-carba-glucopyranose, 63 Sucrase, inhibition by acarbose, 88 Pentoses, 28 I Sugars Peracetylneuraminic acid ally1 esters, I 14 branched-chain, 287 -288 PET technique, labeled 2-deoxy-2-fluoro-~cyclic analogs with imino group, 335 - 34 I glucose, 199-202 diamino, 292 Phenyl 2-deoxy-2-fluoro-~-~-glucopyranowith difluoromethylene group, synthesis, side, 208 183 Phosphoric esters, bacterial polysaccharides, enantiomeric carba-sugars from, 41 -49 313-317 Pol yhydrox ypyrrolidines, glucosidase methylated, 300-302 monoamino, 290-292 inhibition, 344-347 Polysaccharides, see also Bacterial polysacSuicide inactivators, 364 Sulfonyloxy group, displacement by charides degradation, 96 fluorine, 130- I3 1 Swainsonine, a-mmannosidase inhibition, Proton-donating groups, at active site, 378 Pseudo-oligosaccharides,348 342,344 Pseudosubstrates, 348 - 349 D-glycals, 349-357 glycosyl fluorides, 357- 359 T Pseudo-sugars, see Carba-sugars Pyruvic acid, cyclic acetals, 304- 306 Teichoic acids, bacterial polysaccharides, 315-316 Tetra-O-acetyl-2-acetamido-2-deoxy-carbahexoses, synthesis, 60.62 R 1,3,4,5-Tetra-O-acetyl-2-deoxy-2-fluoro-~Racemic amino carba-sugars, synthesis glucopyranose, 163 2-amino-2-deoxycarba-hexoses and relaTetra-0-benzoyl-a-D-mannopyranosyl tives, 59-63 bromide, 73 carba-glycosylamines, 52 - 59 Ti F, catalyst, glycosyl fluorides, synthesis, Racemic carba-sugars, synthesis I09 from Diels- Alder adduct of furan and Transition state, slow, tight-binding acrylic acid, 29- 36 inhibitors. 382-383

SUBJECT INDEX

423

Trehalase, validamycin and validoxylamine Validamycin A, 24 activity against, 88 microbial degradation, 52 Trehalosamine. 72 synthesis, 78-79 Trestatins. 25-26 Validamycin B, synthesis, 78 - 80 I ,3,4-Tri-0-acetyl-2-deoxy-2,2-difluoro-a-~Validam ycins ervthrtrpentopyranose, I8 I - I82 activity against 3,4.6-Tri-0-acetyl-2-deoxy-2-fluoro-~-~Pelliciilaria sasakii, 87 altropyranosyl fluorides. I75 trehalase, 88 2,4,6-Tri-0-acetyl-2-deoxy-2-fluoro-a-~- synthesis, 74-81 galactopyranosyl, I75 Validoxylamine B, synthesis, 76-77 3.4.6-Tri-0-acetyl-2-deoxy-2-['8F]luoro-aValidoxylamines, activity against D-ghco- and /h-manno-pyranosyl Pellicularia sasakii, 87 fluorides. 190- 192 trehalase, 88 I ,3,4-Tri-0-acetyl-2,6-dideoxy-2,2-difluoroValienamines, 348 L-lwo-hexopyranose, 182 synthesis, 52-53, 55, 6 4 2.3.5 -Tri-O-benzyl-P-D-ribofuranosyl Valiolamines, 52-53, 74, 83 fluoride, synthesis, 99 synthesis, 58-59, 65 0-(Trifluoromethy1)ated sugars, synthesis, 184- I85 2-O-T~flyl-~-mannopyranosides, 125 X

U Umezawa, Hamao, obituary, I - 17

Xenon difluoride, 176- 177 X-Ray crystallography, gl ycoside hydrolases, 325-326 ~-Xylose,enantiomenc carba-sugars, synthesis, 44

V Validamines, 74 synthesis, 52-53, 6 4

Y Yersiniose A and B, 288

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