Fluorinated Carbohydrates. Chemical and Biochemical Aspects 9780841214927, 9780841212268, 0-8412-1492-1

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Fluorinate Carbohydrate

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

ACSSYMPOSIUMSERIES374

Fluorinated Carbohydrates Chemical and Biochemical Aspects N.F.Taylor, EDITOR University of Windsor

Developed from a symposium sponsored by the Division of Carbohydrate Chemistry at the 194th Meeting of the American Chemical Society, New Orleans, Louisiana August 30-September 4, 1987

American Chemical Society, Washington, DC 1988 In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Library of Congress Cataloging-in-Publication Data Fluorinated carbohydrates: chemical and biochemical aspects N.F. Taylor, editor p.

cm.(ACS Symposium Series: 374).

Developed from a symposium sponsored by the Division of Carbohydrate Chemistry at the l94th Meeting of the American Chemical Sociefy. Ne August 20-September 4,1987 Includes bibliographies and ISBN 0-8412-1492-1 1. Fluorocarbohydrates—Physiological effectCongresses. 2. Fluorocarbohydrates—Therapeutic use— Testing—Congresses. 3. Fluorocarbohydrates— Synthesis—Congresses. I. Taylor, N . F . II. American Chemical Society. Division of Carbohydrate Chemistry. III. Series. QP702.F58F58 1988 547.7'.81-dcl9 88-19332 CIP

Copyright © 1988 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprograpnic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc.. 27 Congress Street, Salem, M A 01970, for copying beyond (hat permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, tor creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected oy law. PRINTED IN THE UNITED STATES OF AMERICA

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

ACS Symposium Series M . Joan Comstock, Series Editor 1988 ACS Books Advisory Board Paul S. Anderson

Vincent D. McGinniss

Merck Sharp & Dohme Research Laboratories

Battelle Columbus Laboratories

Harvey W. Blanch University of California—Berkeley

Malcolm H. Chisholm Indiana University

University of Iowa

James C. Randall Exxon Chemical Company

Alan Elzerman

E. Reichmanis

Clemson University

AT&T Bell Laboratories

John W. Finley Nabisco Brands, Inc.

Natalie Foster Lehigh University

C. M. Roland U.S. Naval Research Laboratory

W. D. Shults Oak Ridge National Laboratory

Marye Anne Fox The University of Texas—Austin

Roland F. Hirsch

Geoffrey K. Smith Rohm & Haas Co.

U.S. Department of Energy

Douglas B. Walters

G. Wayne Ivie

National Institute of Environmental Health

USDA, Agricultural Research Service

Michael R. Ladisch

Wendy A. Warr

Purdue University

Imperial Chemical Industries

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Foreword The ACS SYMPOSIUM SERIES was founded in 1974 to provide a

medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of th Editor with th assistanc f th Serie Advisory Board and symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Preface

THE DIVERSE CHEMICAL, BIOLOGICAL, AND INDUSTRIAL APPLICATIONS of the numerous synthetic carbon—fluorine compounds are almost legendary. In 1944, Marais identifiedfluoroacetatein the leaves of the South African shrub Dichapetalum biologists continue to b and properties of the C - F bond. A Ciba Foundation symposium was first published in 1972 to honor the contribution made by Sir Rudolph Peters to the biochemical aspects of the subject. This book was followed in 1976 by an ACS Symposium Series publication, Biochemistry Involving Carbon-Fluorine Bonds. The natural occurrence of the C - F bond in carbohydrates is restricted to nucleocidin, an antibiotic in which the hydrogen at C-4 of this ribofuranoside is replaced by fluorine. In contrast, during the last 10 years, new synthetic methods for the introduction of both F and F into carbohydrates have led to an explosive growth in the number and variety offluorinatedsugars. Therefore, compounds that were relatively rare have become readily accessible for chemical and biochemical research. The topics reported in this book reflect recent studies in the synthesis, reactivity, and some biochemical aspects of fluorinated carbohydrates. An appropriate combination of chemical and enzymatic methods now provides a versatile approach for the stereospecific introduction of fluorine into monosaccharides, disaccharides, oligosaccharides, and in the future, polysaccharides. A recurrent theme in biochemical studies is based on the rationale that F and F may replace the hydroxyl group of carbohydrates with minimal perturbance of molecular structure and conformation. The changes in biochemical reactivity, combined with the specific hydrogen-bonding capacity of the C - F bond, permit the use of deoxyfluorinated sugars as unique cellular and enzymatic probes. Studies illustrate carbohydrate metabolism, enzymology, antigen-antibody specificity, carbohydrate transport, and the design of new antiviral and antitumor agents. The inhibition and modification of viral-envelope glycoprotein synthesis by fluorinated 1 9

1 9

1 8

1 8

ix In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

sugars promises to be a new approach to the interference with virus-retrovirus-host cell membrane interactions. The research reported in this volume raises more questions than it answers. For this reason, the future of fluorinated carbohydrates remains assured and many new avenues of research await further interdisciplinary investigation. I hope the chemical and biochemical scope of this book will continue to stimulate those already active in the field and attract new researchers into this exciting and fascinating area of carbohydrates. Acknowledgments I am deeply indebted to all contributors; without their patience and cooperation this book would t hav bee possible I als wish t thank Robin Giroux and the staf and assistance when it was most needed. Finally, my thanks to Donna Dee and Maeve Doyle for their expert secretarial and administrative assistance. Additional financial support for the organization of the symposium was kindly provided by E. I. du Pont de Nemours and Company.

N. F. TAYLOR Department of Chemistry and Biochemistry University of Windsor Ontario N9B 3P4, Canada January 1988

x In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 1

Retrospect

and Prospect

P. W. Kent Nuffield Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford University, Oxford, 0X2 6HE, United Kingdom In the past quarter of a century interest has grown apace in these modified sugars, both i biological potentialitie only touch on a few of the developments which have brought us to our present state of knowledge. For the most part, attention is devoted to monosaccharides containing a single F atom though undoubtedly others in which -CF and -CF have been introduced are both interesting and potentially important. It was evident to early workers in the field that such F-sugars should be capable of existence if only on the grounds of the similarity of the F-atom to the OH group in size, electronegativity and ability to participate in H-bonded structures. Interest was further fuelled by the discovery of the high toxicity of monofluoroacetate and by the bizarre fact that this compound occurred in Nature in many plants. An extensive series of reviews of fluorinated biological analogues including F-carbohydrates was published in 1972 in the form of a CIBA Symposium volume (1) and an ACS Symposium in 1976 (2). A substantial, useful source book cataloguing properties of carbohydrate derivatives, including some fluorosugars has recently been published by Collins (3). 3

2

F o r b r e v i t y , F-sugar d e n o t e s a d e o x y f l u o r o c a r b o h y d r a t e , e g . 6Fg l u c o s e d e n o t e s 6 - d e o x y - 6 - f l u o r o - D - g l u c o s e . T h i s system i s used throughout t h i s chapter. RETROSPECT L i k e many o t h e r a r e a s o f o r g a n i c c h e m i s t r y , f l u o r o c a r b o h y d r a t e s had s m a l l b e g i n n i n g s . The f i r s t tenuous t e s t s seem t o have been c a r r i e d o u t b y M o i s s a n (£) h i m s e l f , t h e d i s c o v e r e r o f e l e m e n t a l fluorine. I n h i s monumental monograph ' F l u o r e t s e s Composes* p u b l i s h e d 1900, he wrote o f t h e r e a c t i o n between F and g l u c o s e : 2

"Rien a f r o i d : c h a u f f e legerment, i l e s t attaque p a r l e f l u o r avec d e p o t de c h a r b o n . Lorsque l a temperature s ' e l e v e p a r s u i t e de l a r e a c t i o n , l a d e s t r u c t i o n d e v i e n t

0097H5156/88/0374-0001$06.00/0 ° 1988 American Chemical Society

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FLUORINATED CARBOHYDRATES: C H E M I C A L AND BIOCHEMICAL ASPECTS

r a p i d e e t complete avec l e f l u o r e u s e de fluorhydrique."

carbone e t d ' a c i d e

S u c r o s e and m a n n i t o l f a r e d no b e t t e r . In the p e r i o d t o 1939, the l i m i t e d means o f s y n t h e s i s l e d t o a slow a c c u m u l a t i o n o f knowledge about f l u o r o - o r g a n i c compounds and t h e i r r e a c t i v i t y [a number o f r e v i e w s o f t h i s e a r l i e r work were p u b l i s h e d (5)]. R e s e a r c h i n P o l a n d , B e l g i u m and Germany, i n p a r t i c u l a r , opened t h e way t o f u r t h e r i n v e s t i g a t i o n s . In t h e c a r b o h y d r a t e f i e l d , t h e most i n t e r e s t i n g f i n d i n g s were o f t h e s y n t h e s i s and use o f g l y c o s y l f l u o r i d e s w i t h F a t C l , which c o u l d be e a s i l y o b t a i n e d by exchange r e a c t i o n s w i t h m e t a l l i c f l u o r i d e s o r by t h e a c t i o n o f l i q u i d HF on f u l l y a c e t y l a t e d m o n o s a c c h a r i d e s . Results i n t h i s f i e l d have been r e v i e w e d by M i c h e e l and Klemer ( 6 ) . These d e r i v a t i v e s p r o v i d e d a u s e f u l p r o t e c t i n g group f o r C l , s t a b l e i n a l k a l i n e c o n d i t i o n s b u t r e a d i l y removable by d i l u t e a c i d s . A c u r i o u s f i n d i n g mad g l u c o s e from wood r e v e a l e anhydrous HF t o g i v e g l u c o s y l f l u o r i d e i n h i g h y i e l d . I f traces of m o i s t u r e were p r e s e n t , t h i s p r o d u c t was n o t o b t a i n e d and i n s t e a d a n o n - l i n e a r g l u c a n , ' c e l l a n ' , r e s u l t e d ( 7 ) . T h i s was a random s t r u c t u r e , a h i g h l y b r a n c h e d p o l y s a c c h a r i d e i n which s e v e r a l t y p e s o f i n t e r g l y c o s i d i c l i n k a g e s were p r e s e n t (8) . G l u c o s e i t s e l f c o u l d be i n d u c e d t o p o l y m e r i s e i n a v a r i e t y o f d e h y d r a t i n g acidic c o n d i t i o n s (eg. r e f e r e n c e 9) t o g i v e a s y n t h e t i c p o l y s a c c h a r i d e w i t h p o t e n t i a l as a b l o o d plasma e x t e n d e r . D u r i n g the p e r i o d o f W o r l d War I I , much r e s e a r c h was u n d e r t a k e n , r e s u l t s o f which were not p u b l i s h e d u n t i l 1947 and a f t e r , owing t o q u e s t i o n s o f n a t i o n a l s e c u r i t y . In p a r t i c u l a r s t u d i e s were made o f fluorophosphidates [reviewed by Saunders (10)] and o f d e r i v a t i v e s o f f l u o r o a c e t i c a c i d and i t s h i g h e r homologues [reviewed by P a t t i s o n

uin. The d i s c o v e r y i n 1943 by M a r a i s (12) t h a t p o t a s s i u m f l u o r o a c e t a t e was the t o x i c m a t e r i a l p r e s e n t i n a number o f South A f r i c a n p l a n t s a t t r a c t e d n o t a b l e a t t e n t i o n , though t h e r e a s o n s f o r the t o x i c i t y were n o t t h e n known. F u r t h e r e x p l o r a t i o n showed the presence o f the s i m i l a r l y t o x i c c o - f l u o r o o l e i c a c i d i n other p l a n t s . At t h a t time, the techniques f o r q u a n t i t a t i v e m i c r o a n a l y s i s o f o r g a n i c f l u o r i d e s were q u i t e i n a d e q u a t e u n t i l an o u t s t a n d i n g complexometric method was d e v i s e d i n B e l c h e r ' s l a b o r a t o r y ( 1 3 ) . It appeared from e x t e n s i v e s u r v e y s t h a t f l u o r o f a t t y a c i d s i n f a c t were w i d e s p r e a d i n t r a c e q u a n t i t i e s i n many s p e c i e s o f g r e e n p l a n t s . The b i o c h e m i c a l mechanism o f the t o x i c i t y o f f l u o r o a c e t a t e i n mammals was f i r s t e l u c i d a t e d by t h e n o t a b l e r e s e a r c h e s o f t h e l a t e S i r Rudolph P e t e r s , who showed t h a t i n l i v e r and k i d n e y , the compound was m e t a b o l i c a l l y t r a n s f o r m e d i n t o an even more t o x i c f l u o r o c i t r i c a c i d i s o m e r , a p o w e r f u l i n h i b i t o r o f the t r i c a r b o x y l i c a c i d c y c l e . These r e s u l t s t o g e t h e r w i t h t h o s e o f o t h e r s , L i e b e c q , Bergmann, B u f f a , G a l , t o name b u t a few, e s t a b l i s h e d beyond doubt t h i s new b i o c h e m i c a l t w i s t i n which a p o t e n t i a l t o x i c a g e n t c o u l d be t r a n s formed by an e x i s t i n g enzyme pathway i n t o a n o t h e r o f g r e a t e r t o x i c i t y c r e a t i n g a p h y s i o l o g i c a l l y c r i t i c a l s i t u a t i o n , a phenomenon termed " L e t h a l S y n t h e s i s " by P e t e r s . W i t h t h i s i n mind, i t was even more c h a l l e n g i n g t o p u r s u e Fc a r b o h y d r a t e s t o see whether t h e s e would be m e t a b o l i s e d t o y i e l d

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Retrospect and Prospect

3

f l u o r o a c e t a t e a s t h e e n d - p r o d u c t . Two p r i n c i p a l approaches were followed f o r t h e i r chemical synthesis; t o t a l s y n t h e s e s and f l u o r i n a t i o n o f e x i s t i n g m o n o s a c c h a r i d e d e r i v a t i v e s . The s y s t e m a t i c s t u d y o f F - s u g a r s was begun i n O x f o r d i n 1951 on t h e s e l i n e s . Total

Syntheses

In t h i s s t r a t e g y , a s i m p l e s t a r t i n g compound, e g . e t h y l f l u o r o a c e t a t e w i t h t h e F-atom a l r e a d y p o s i t i o n e d , e q u i v a l e n t i n c u r r e n t i d e a s t o a s y n t h o n , was c o n v e r t e d b y C l a i s e n c o n d e n s a t i o n and s e l e c t i v e r e d u c t i o n i n t o a number o f s i m p l e F - a n a l o g u e s o f biochemical i n t e r e s t (Figure 1). By t h i s r o u t e , 2 F - g l y c e r o l and 2 F - g l y c e r i c a c i d were o b t a i n e d and l i k e l F - g l y c e r o l were found t o be m e t a b o l i z e d t o f l u o r o a c e t a t e and t h u s t o be h i g h l y t o x i c . 2FM a l a t e (2-hydroxy-3-fluoro(±)-butandioic a c i d ) on t h e o t h e r hand was r e l a t i v e l y n o n - t o x i c d e s p i t e i t s c l o s e s t r u c t u r a l r e l a t i o n s h i p t o m a l i c a c i d , a TCA c y c l be a u s e f u l i n d u c e r o f 2 F - E r y t h r i t o l (a c o m p e t i t i v e growth i n h i b i t o r o f the Brucella group) was a c r y s t a l l i n e compound, isomorphous w i t h e r y t h r i t o l i t s e l f , whose s t r u c t u r e h a d t o be e s t a b l i s h e d b y X - r a y d i f f r a c t i o n analysis. 2 F - R i b i t o l l i k e w i s e was o b t a i n e d b y a s i m i l a r s y n t h e t i c route. T o t a l s y n t h e s i s (14) however i s a s e l f - c o m p l i c a t i n g r o u t e g e n e r a t i n g m u l t i p l e asymmetric c e n t r e s which p r o v i d e s t h e i n v e s t i gator with a strong incentive t o explore a l t e r n a t i v e s t r a t e g i e s . M o d i f i c a t i o n o f Monosaccharide

Structures

Exchange R e a c t i o n s . I n v e s t i g a t i o n s h a d a l r e a d y been u n d e r t a k e n i n H e l f e r i c h ' s l a b o r a t o r y which i n 1941 (JL_5) l e d t o t h e s y n t h e s i s o f 6 F - g l u c o s e b y exchange o f a s u l p h o n y l o x y e s t e r b y KF i n m e t h a n o l . The sugar and i t s d e r i v a t i v e s were o b t a i n e d i n good y i e l d and appeared t o be s t a b l e . T h e r e was some f e a r a t t h e time t h a t l o s s o f HF b y e l i m i n a t i o n might r e a d i l y o c c u r a s a major s i d e - r e a c t i o n . In the 1950's f u r t h e r e x p l o r a t i o n o f t h i s r o u t e , m o d i f y i n g s o l v e n t , b l o c k i n g groups and c o n d i t i o n s , l e d t o 6 F - g a l a c t o s e and a number o f m o n o s a c c h a r i d e s F - s u b s t i t u t e d i n the e x t r a c y c l i c p o s i t i o n . U n l i k e t h e even-numbered 0)-f l u o r o f a t t y a c i d s , none o f t h e s e 0)s u b s t i t u t e d s u g a r s were m e t a b o l i s e d t o f l u o r o a c e t a t e and e x h i b i t e d l i t t l e o r no t o x i c i t y . Attempts t o s y n t h e s i s e s e c o n d a r y 2-, 3- o r 4 - f l u o r o s u g a r s met with l i t t l e success. T h i s was f o r a number o f r e a s o n s such as competing e l i m i n a t i o n r e a c t i o n s , t h e p o o r n u c l e o p h i l i c i t y o f F ~ , t h e s o l v e n t dependence o f t h e r e a c t i o n , t h e a v a i l a b i l i t y o f s u i t a b l y s t a b l e b l o c k e d s u g a r s and t h e need t o s e c u r e t h e a p p r o p r i a t e a x i a l conformation o f t h e d e p a r t i n g e s t e r group. These d i f f i c u l t i e s were p r o g r e s s i v e l y overcome b y c a r e f u l s y s t e m a t i c s t u d i e s o f t h e r e a c t i o n c o n d i t i o n s and by t h e i n t r o d u c t i o n o f p o w e r f u l new f l u o r i n a t i n g a g e n t s . Amongst t h e s e t e t r a b u t y l ammonium f l u o r i d e (BuifNF) was an o u t s t a n d i n g d i s c o v e r y (16) , as a n o n - m e t a l l i c i o n i c f l u o r i d e . By i n g e n i o u s u s e o f t h i s r e a g e n t , under r i g o r o u s l y anhydrous c o n d i t i o n s , F o s t e r and h i s group (17) i n 1967 a c h i e v e d a s y n t h e s i s o f 3 F - g l u c o s e ( F i g u r e 2 ) . The method

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

FLUORINATED CARBOHYDRATES: C H E M I C A L AND BIOCHEMICAL ASPECTS

F i g u r e 1. T o t a l s y n t h e s i s o f t h r e e - , f o u r - and f i v e - c a r b o n f l u o r o s u g a r s ( a l l asymmetric p r o d u c t s a r e r a c e m i c m i x t u r e s )

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5

i n v o l v e d the o x i d a t i o n o f a g l u c o s e d e r i v a t i v e w i t h i t s e q u a t o r i a l OH a t C-3 t o an oxo group which on BHi»"" r e d u c t i o n gave t h e a x i a l OH a t C-3 f a v o u r a b l e f o r F~" exchange o f t h e C H S 0 e s t e r . This i m p o r t a n t advance l e d t o f u r t h e r s e c o n d a r y F - s u g a r s , e g . 4 F - g l u c o s e , e s p e c i a l l y a s 0-methyl and 0 - b e n z y l groups came t o be u s e d f o r p r o t e c t i n g OH c e n t r e s (JJ^JJ)) . F u r t h e r advances were made a s a r e s u l t o f t h e s i g n a l d i s c o v e r y by M i d d l e t o n ( 2 0 ) , o f p o w e r f u l new f l u o r i n a t i n g a g e n t s , d i a l k y l a m i n o sulphur t r i f l u o r i d e (Et NSF o t h e r w i s e known as DAST) and b i s ( d i a l k y l a m i n o ) s u l p h u r d i f l u o r i d e [ ( E t N ) S F ] as e a s i l y h a n d a b l e r e a g e n t s f o r r e p l a c i n g OH groups b y F d i r e c t l y under m i l d conditions. The c o n d i t i o n s o f t h e exchange were such t h a t even 0a c e t a t e s p r o v i d e d adequate p r o t e c t i o n o f t h e s t a r t i n g s u g a r . Sharma and K o r y t n y k (21) t h u s o b t a i n e d 6 F - g l u c o s e b y t h i s method from 1 , 2 , 3 , 4 - t e t r a - O - a c e t y l - g l u c o s e i n good y i e l d and w i t h o u t s i d e reactions. T h i s method p r o v e d t e r t i a r y a l c o h o l groups attainable. The approach h a s been f u r t h e r extended b y r e s e a r c h e s i n S z a r e k ' s l a b o r a t o r y (22) which showed t h a t t r i s ( t r i m e t h y l a m i n o ) s u l p h o n i u m d i f l u o r o t r i m e t h y l s i l i c a t e (TSAF) (23) was a n o t h e r most e f f e c t i v e f l u o r i n a t i n g agent f o r OH compounds. The i n t r o d u c t i o n o f improved l e a v i n g groups such as C F S 0 ~ ( t r i f l a t e ) l e d t o t h e s y n t h e s i s o f l F - f r u c t o s e (24) and a range o f o t h e r secondary F-monosaccharides. These r e a c t i o n s p r o c e e d r a p i d l y and unambiguously under m i l d anhydrous c o n d i t i o n s . This reaction o f f e r s c o n s i d e r a b l e p r o m i s e f o r a f a s t means o f i n t r o d u c i n g F ~ i n t o b i o l o g i c a l a n a l o g u e s f o r p o s i t r o n e m i s s i o n t r a n s a x i a l tomography (PETT). A number o f r e v i e w s o f exchange r e a c t i o n s and t h e i r p r o d u c t s have been p u b l i s h e d (25-27). The l i s t o f e f f i c i e n t f l u o r i n a t i n g a g e n t s c o n t i n u e s t o grow. 3

2

2

3

2

2

2

3

1

3

8

Epoxide S c i s s i o n . The s u c c e s s o f p a r a l l e l i n v e s t i g a t i o n s i n t h e s t e r o i d f i e l d i n o b t a i n i n g f l u o r o d e r i v a t i v e s by c l e a v a g e o f e p o x i d e s by HF and s i m i l a r r e a g e n t s even when s e c o n d a r y and t e r t i a r y c e n t r e s were i n v o l v e d gave c o n s i d e r a b l e encouragement t o a p p l y i n g t h e method t o sugar e p o x i d e s ( 2 8 ) . The f i r s t p r a c t i c a b l e r e s u l t s were o b t a i n e d b y T a y l o r and h i s coworkers (29) and b y Bergmann's group ( 3 0 ) . These i n v e s t i g a t i o n s l e d t o 3 F - x y l o s e i n good y i e l d b y r e a c t i o n o f a 5 - 0 - b e n z y l - 2 : 3 - a n h y d r o r i b o s i d e w i t h HF o r KHF i n ethandiol. T h i s was a s i g n i f i c a n t b r e a k t h r o u g h and t h i s r o u t e f i r s t l e d Pacak and coworkers (31) t o t h e i m p o r t a n t probe compound, 2F-glucose. 2

A d d i t i o n Reactions. A f u r t h e r important s y n t h e t i c route l a y i n h a l o g e n and a c t i v a t e d F donor a d d i t i o n t o 1:2 g l y c o s e e n s . R e s u l t s d e s c r i b i n g B r and o t h e r h a l o g e n a d d i t i o n s were a l r e a d y a v a i l a b l e i n t h e e x t e n s i v e and d a u n t i n g p a p e r b y F i s c h e r , Bergmann and S c h o t t e (32) p u b l i s h e d i n 1920. A d d i t i o n s u s i n g i n t e r h a l o g e n s , 'BrF' and IF (formed in situ) w i t h t r i a c e t y l g l u c a l showed t h a t t h e p r e d o m i nant p r o d u c t s were t h e gluoo and manno e p i m e r s o f 2-deoxy-2-bromo (or i o d o ) - g l y c o s y l f l u o r i d e s . The r e a c t i o n was c h a r a c t e r i s t i c a l l y a 2

1

1

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

t r a n s a d d i t i o n w i t h F t a k i n g up t h e a x i a l C - l p o s i t i o n and bromine (or i o d i n e ) a t C-2. The r o u t e was i n e f f e c t i v e f o r f l u o r i n a t i o n a t other positions. The mechanism o f t h i s t y p e o f r e a c t i o n i n a c c o u n t i n g f o r t h e m u l t i p l e i s o m e r i c p r o d u c t s was s u b s t a n t i a l l y e l u c i d a t e d b y t h e e l e g a n t work o f Lemieux, F r a z e r - R e i d and c o l l e a g u e s (33.) and by p i o n e e r development o f H and F-NMR t e c h n i q u e s e s p e c i a l l y b y L.D. H a l l (3£) and by R.A. Dwek (35) . S i g n a l s u c c e s s was o b t a i n e d by t h e u s e o f f l u o r o - o x y t r i f l u o r o methane (CF OF) i n t r o d u c e d b y B a r t o n (36) i n 1969. Carbohydrate a p p l i c a t i o n s were u n d e r t a k e n i n l a b o r a t o r i e s i n London and O x f o r d . I t was shown (_37) t h a t C F 0 F r e a c t e d r e a d i l y w i t h t r i a c e t y l g l u c a l a t -78° g i v i n g c h i e f l y t h e c o r r e s p o n d i n g 2 F - g l u c o s y l a - f l u o r i d e (34%) and i t s C F 0 a - g l y c o s i d e (26%) . I n a d d i t i o n t h e c o r r e s p o n d i n g 3-manno- compounds were p r e s e n t as minor p r o d u c t s ( F i g u r e 3 ) . T h i s was a s c r i b e d t o t h e d i s m u t a t i o n : 1

19

3

3

3

CF 0F ^ 3

A t t h e same t i m e , comparable p r o d u c t s r e s u l t e d i n t h e r e a c t i o n s w i t h t r i a c e t y l g a l a c t a l , d i a c e t y l x y l a l and d i a c e t y l a r a b i n a l (38-40). C o n f o r m a t i o n a l e f f e c t s a r i s i n g from t h e c o n f i g u r a t i o n o f t h e s u b s t i t u e n t a t C-4 p l a y e d a s i g n i f i c a n t r o l e i n c o n t r o l l i n g t h e s t e r e o c h e m i s t r y o f a d d i t i o n p r o d u c t s (41) such as t h o s e a r i s i n g from d i a c e t y l r h a m n a l (42) , d i a c e t y l f u c a l (_43) and h e p t a c e t y l - l a c t a l (44) . P r o g r e s s i n t h e f i e l d was g r e a t l y a i d e d by r e s u l t s from W o l f ' s l a b o r a t o r y (_45,46) i n 1977 on t h e d i r e c t f l u o r i n a t i o n o f t r i a c e t y l g l u c a l w i t h F . C o n d i t i o n s were found i n which a r a p i d and c o n t r o l l e d r e a c t i o n took p l a c e g i v i n g t h e 2 F - g l u c o s y l a - f l u o r i d e (40% y i e l d ) and t h e 2F-mannosyl 3 - f l u o r i d e (26%) d e r i v a t i v e s . These r e s u l t s p r o v e d t o be o f utmost v a l u e f o r t h e s y n t h e s i s o f t h e F - l a b e l l e d compounds f o r p o s i t r o n e m i s s i o n tomography. The speed w i t h which t h e a d d i t i o n and subsequent s e p a r a t i o n s c o u l d be a c h i e v e d e x p e r i m e n t a l l y e n a b l e d t h e 2 - F - g l u c o s e t o be o b t a i n e d w i t h a r a d i o a c t i v e p u r i t y o f o v e r 98% and w i t h h i g h s p e c i f i c a c t i v i t y , e g . 8750 mCi/mmol. The l a b e l l e d sugar has come i n t o e x t e n s i v e prominence f o r t h e mapping in vivo o f r e g i o n s o f e l e v a t e d g l u c o s e r e q u i r e m e n t s a s i n d i c a t i v e o f t i s s u e a b n o r m a l i t i e s . The b i o c h e m i c a l c o m p a r a b i l i t y o f 2 F - g l u c o s e , i t s a p p a r e n t n o n - t o x i c i t y and t h e s h o r t h a l f - l i f e o f t h e i s o t o p e ( F , t ^ 110 mins) o f f e r s d i s t i n c t i v e s u i t a b i l i t y f o r n o n - i n v a s i v e d i a g n o s t i c u s e b y PETT. 2

1 8

1 8

1 8

Some B i o l o g i c a l

Effects

Though i n a n i m a l s F-monosaccharides a r e r a r e l y i f e v e r m e t a b o l i s e d t o F - a c e t a t e , n e v e r t h e l e s s t h e y e n t e r i n t o a number o f b i o c h e m i c a l pathways i n c l u d i n g o x i d a t i o n , p r i m a r y p h o s p h o r y l a t i o n b y k i n a s e s and c o n v e r s i o n i n t o n u c l e o t i d e F-sugar p h o s p h a t e s , thence t o make t h e i r appearance i n a number o f g l y c o p r o t e i n s and g l y c o l i p i d s . Schmidt, Schwarz and c o l l e a g u e s (£7,48) f u r t h e r showed t h a t 2Fmannose and 2 F - g l u c o s e i n h i b i t e d t h e b i o s y n t h e s e s o f i n f e c t i v e S e m l i k i v i r u s and f o w l p l a g u e v i r u s ( i n f l u e n z a v i r u s A) i n c h i c k embryo c e l l s and t h a t o f p s e u d o r a b i e s v i r u s i n r a b b i t k i d n e y c e l l s . T h i s was a t t r i b u t e d t o f a i l u r e t o complete t h e v i r a l e n v e l o p e (49)

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

KENT

Retrospect and Prospect

1. BH " 2. p-MeC H S0 CI 4

6

4

2

- pyridine

/ 3 - deoxy - 3 - fluoro -1,2 - 5,6 - O diisopropylidene - a - D - glucofuranose F i g u r e 2.

F i g u r e 3.

Synthesis

S0 C H Me(p) 2

6

4

o f 3-deoxy-3-fluoro-D-glucose

A d d i t i o n o f CF OF t o 3 , 4 , 6 - t r i - O - a c e t y l - D - g l u c a l 3

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

i n a manner a n a l o g o u s t o 2-deoxyglucose (2DG) which underwent c o n v e r s i o n v i a t h e GDP i n t e r m e d i a t e t o g i v e a c h a i n - t e r m i n a t i n g l i p i d 2DG(GlcNAc) PP D o l i c h o l . The i n h i b i t i o n was r e l i e v e d by a d d i t i o n o f mannose. The b l o c k e d l i p i d was i n c a p a b l e o f mannosylat i o n r e q u i r e d f o r f o r m a t i o n o f N - l i n k e d o l i g o s a c c h a r i d e components i n appropriate glycoproteins. These 2F-sugars caused e x t e n s i v e o l i g o s a c c h a r i d e c h a i n s h o r t e n i n g i n g l y c o l i p i d s o f i n f l u e n z a A i n f e c t e d c h i c k c e l l s and i n y e a s t ( 5 0 ) . The a l t e r a t i o n i n membrane m o l e c u l a r a r c h i t e c t u r e consequent on t h e s e m o d i f i c a t i o n s p o i n t s t o s u b s t a n t i a l p r o s p e c t s f o r r e d u c i n g v i r a l i n f e c t i v i t y , as Blough and G i u n t o l i (5j_) found i n t h e t r e a t m e n t o f human g e n i t a l h e r p e s w i t h 2DG. The f i e l d was r e v i e w e d i n 1982 by Schwarz and Datema (_52) . Amongst the g l y c o s a m i n o g l y c a n s , N-fluoro[ H]acetyl-D-glucosamine and N - f l u o r o a c e t y l - D - [ 1 - ' * C ] glucosamine was found (53) t o be i n c o r p o r a t e d i n t o h y a l u r o n a t e by mammalian c e l l s v i a t h e UDP i n t e r m e d i a t e s ( 5 4 ) . Th acetylglucosaminyl residues l - C - r i n g l a b e l l e d , appeared t o be more r e s i s t a n t t o h y a l u r o n i d a s e d e g r a d a t i o n t h a n t h e f l u o r i n e - f r e e c o u n t e r p a r t from t h e same t i s s u e , s u g g e s t i n g t h a t c a r b o h y d r a t e complexes, b i o s y n t h e t i c a l l y m o d i f i e d i n t h i s f a s h i o n , may have extended h a l f - l i v e s in vivo. 2F-Glucose and o t h e r F-monosaccharides can promote r e d u c t i o n i n c e l l growth o f a number o f tumour c e l l l i n e s i n c u l t u r e (55,56) b u t appear t o have l i t t l e e f f e c t on tumours in vivo. I n murine lymphoma c e l l s , 2 F - g l u c o s e was an e f f e c t i v e growth i n h i b i t o r , w h i l e N - t r i f l u o r o a c e t y l - D - g l u c o s a m i n e d i m i n i s h e d the i n c o r p o r a t i o n o f glucosamine i n t o c e l l components. The u n d e r l y i n g mechanisms t o a c c o u n t f o r growth i n h i b i t i o n a r e c o n s i d e r e d t o be due i n p a r t t o m e t a b o l i c imbalance between i n t r a c e l l u l a r p o o l s o f c r i t i c a l b i o c h e m i c a l i n t e r m e d i a t e s , as w e l l as by t h e c e l l s u r f a c e m o d i f i c a tions. D i s t u r b a n c e o f g l y c o p r o t e i n s t r u c t u r e has a l s o been a c h i e v e d by d e p l e t i o n o f g l y c o s y l a t i o n t h r o u g h m e t a b o l i c i n c o r p o r a t i o n o f t h r e o $ - f l u o r o a s p a r a g i n e i n t o the p o l y p e p t i d e sequence t h u s d e n y i n g N - g l y c o s y l a t i o n [reviewed by E l b e i n (J57) ] . 2

3

1

1 I f

A f u r t h e r i m p o r t a n t mechanism a f f e c t i n g mammalian c e l l metabol i s m was found by B a r n e t t (58) i n sugar t r a n s p o r t phenomena. I n the mammalian i n t e s t i n a l mucosa and i n the r e d c e l l , 3 F - g l u c o s e , f o r example, was a s p e c i f i c c o m p e t i t i v e i n h i b i t o r o f the t r a n s p o r t o f the p a r e n t s u g a r . C e l l s grown i n t h e p r e s e n c e o f s u b - l i m i t i n g c o n c e n t r a t i o n s o f t h e 3F-sugar e x p e r i e n c e d g l u c o s e d e p r i v a t i o n and c a r b o h y d r a t e s t a r v a t i o n w i t h consequent r e s t r i c t i o n i n c e l l growth. I n t e r e s t i n g l y , a - D - g l u c o p y r a n o s y l f l u o r i d e p r o v e d t o be a s u b s t r a t e f o r i n t e s t i n a l a - g l u c o s i d a s e , a s u b s t r a t e w i t h the s m a l l e s t aglycone p o s s i b l e . In non-mammalian systems, F - s u g a r s have o t h e r b i o c h e m i c a l properties. F o r example, 3 F - g l u c o s e i s o x i d i z e d i n Pseudomonas putida t o F - a l d o n i c a c i d s (59), whereas 4 F - g l u c o s e i s m e t a b o l i z e d o n l y a f t e r c l e a v a g e o f the C-F bond by an o u t e r membrane p r o t e i n (60). The end p r o d u c t o f t h i s r e a c t i o n has been i d e n t i f i e d as 4,5d i h y d r o x y p e n t a n o i c a c i d ( 6 1 ) . In Locusta migratoria, T a y l o r has shown a f u r t h e r u n u s u a l m e t a b o l i c d e g r a d a t i o n o f 3 F - g l u c o s e w i t h l i b e r a t i o n o f F~. The i n c o r p o r a t i o n o f 3 F - g l u c o s e i n t o t r e h a l o s e and i n t o m o d i f i e d g l y c o g e n was a l s o shown t o o c c u r ( 6 2 ) .

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.

Retrospect and Prospect

KENT

9

PROSPECT The ease with which F-monosaccharides can now be synthesized by r e l a t i v e l y straightforward chemical or chemoenzymatic (63) methods makes t h i s class o f sugar a t t r a c t i v e i n many areas of research. To the synthetic organic chemist, these compounds o f f e r a range of s t a r t i n g materials, with one or more fluorine atoms stereochemically s p e c i f i e d , suitable for the chiron methods of assembly (64) of more complex molecules. Design of retro-synthetic routes could enable precise fluoro-analogues of hormones, surfaceactive l i p i d s and vitamins f o r example to be synthesized with a view to seeking agents of enhanced b i o l o g i c a l a c t i v i t y and of longer retention time in vivo. I t w i l l be r e c a l l e d that 9-a-fluorjocortisol i s a well known instance amongst the steroid hormones of such enhancement. There i s also substantial promise i n semi-synthetic approaches where, with ever-improvin molecular s i t e s i n existin important molecular modifications can be envisaged. The p o s s i b i l i t i e s which may be opened by introducing even a single F-atom at a c r u c i a l p o s i t i o n i n , say, the p e n i c i l l i n s , t e t r a c y c l i n s or cephalosporins are immense e s p e c i a l l y as the questions of microbial resistance become the more pressing. One notes the existence of a t least one naturally occurring a n t i b i o t i c , nucleocidin (65) containing an F-atom attached to C4 of a ribofuranosyl residue. Beginnings have been made using F-sugar derivatives as a n t i - v i r a l agents and the p o s s i b i l i t i e s of influencing in vivo t h e i r growth and i n f e c t i v i t y s p e c i f i c a l l y and with minimal disturbance to the host hold out great promise (66). A further aspect of these p o s s i b i l i t i e s i s i n r e l a t i o n to the powerful new non-invasive imaging techniques which NMR and PETT (67) o f f e r and no doubt even more powerful techniques w i l l follow i n the future. The NMR d i s t i n c t i v e chemical s h i f t s a r i s i n g from F suggest that methods are r e a l i s a b l e for locating F-containing pharmacological agents in vivo and possibly for t h e i r pharmacodynamic investigation. In vivo probes as well as those in vitro can be envisaged as leading to new understanding of membrane binding and retention of s e l e c t i v e l y transported molecules. The central enigma of the biochemical means by which the C-F bond i s synthesized s t i l l remains unsolved even i n outline. Though t h i s i s assumed to be a phenomenon r e s t r i c t e d to plants, t h i s may not be wholly the case. Not only i s the o r i g i n of F-acetate ripe for reinvestigation but also that of v o l a t i l e plant products such as FCH C0CH and FCH CH 0CH CH . Whatever the pathway of F-acetate formation, i t can be presumed to be an unusual one and may be of substantial i n t e r e s t to the inorganic chemist. I t i s interesting to speculate whether a f l u o r o - s i l i c o n intermediate i s involved which participates i n an exchange with an activated phosphate ester. The question i s only marginally relevant to F-sugars at the present time however. To the biochemist and physiologist, fluoro-containing b i o l o g i c a l analogues are of great potential i n t e r e s t . The development of routes for the b i o l o g i c a l incorporation of F-sugars into strategic biopolymers i s p a r t i c u l a r l y so. For example, i t i s of 1 9

2

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2

2

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In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: C H E M I C A L AND BIOCHEMICAL ASPECTS

i n t e r e s t t o e x p l o r e i n much g r e a t e r d e t a i l t h e p o s s i b l e r o l e o f 4 F - g l u c o s e i n g l y c o g e n s y n t h e s i s and g l y c o g e n o l y s i s , where even p a r t i a l i n c o r p o r a t i o n o f t h e F - s u g a r can be e n v i s a g e d a s a c h a i n t e r m i n a t i n g step i n outer-branch chains o f the molecule. In g l y c o g e n s t o r a g e d i s e a s e s , where o v e r - p r o d u c t i o n and d e p o s i t i o n o f t h e p o l y s a c c h a r i d e o c c u r s , t h e approach c o u l d be a v a l u a b l e one. And a range o f o t h e r s t o r a g e d i s e a s e s a w a i t i n v e s t i g a t i o n . The more complex c h e m i c a l s t r u c t u r e s and b i o l o g i c a l r e c o g n i t i o n s i t e s amongst t h e g l y c o p r o t e i n s i n some c a s e s m e d i a t e d t h r o u g h c e l l surface oligosaccharides, presents legion p o s s i b i l i t i e s f o r s i m i l a r m e t a b o l i c i n t e r v e n t i o n by i n t r o d u c t i o n o f F - s u g a r s o r d e o x y s u g a r s . M i l d and s e l e c t i v e f l u o r i n a t i o n methods may come h e r e t o have importance i n s e m i - s y n t h e t i c m o d i f i c a t i o n s o f p r e - e x i s t i n g p r o t e i n s , g l y c o p r o t e i n s and p o l y p e p t i d e s . Again the p o s s i b i l i t y o f i n c r e a s i n g in vivo t h e r e t e n t i o n time i n c i r c u l a t i o n , p o i n t s t o ways o f r e d u c i n g t h e dose l e v e l s o f a d m i n i s t e r e d t h e r a p e u t i c a g e n t s and p e r h a p s o f t a r g e t i n g the The p r o g r e s s i n t h has been i m p r e s s i v e b u t many more e x c i t i n g developments y e t a w a i t u s . Acknowledgments I thank D r . G . E . Newman and M r s . Jean Sfrrith f o r t h e i r h e l p preparing t h i s chapter.

in

Literature Cited 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11.

CIBA Symposium, Carbon-Fluorine Compounds: Chemistry, Biochemistry and Biological Activities; Associated Scientific Publishers: Amsterdam, 1972. Biochemistry Involving Carbon Fluorine Bonds: F i l l e r , R. (ed.); ACS Symposium Series 28, American Chemical Society, Washington, D.C., 1976, 1-214. Collins, P.M. Carbohydrates; Chapman Hall: London, U.K., 1987. Moissan, H. Le Fluor et ses Composés; G. Steinheil: Paris, 1900; p.244. (i) Lovelace, A.M.; Rausch, D.A.; Postelnek, W. Aliphatic Fluorine Compounds; Reinhold Publishing Corp.: New York, 1958. (ii) Hudlický, M. Chemistry of Organic Fluorine Compounds; Pergamon Press: Oxford, U.K., 1961. (iii) New Methods of Preparative Organic Chemistry; Interscience Publications Inc.: New York, 1948 (English translation of German researches published in 1944) Bockeműller, W., pp.229-45 and Weichert, K . , pp.315-62. Micheel, F . ; Klemer, A. Adv. Carbohydr. Chem. 1961, 16, 85-103. Fredenhagen, K.; Cadenbach, G. Angew. Chem. 1933, 46, 113-117. Helferich, B.; Peters, O. Annalen 1932, 494, 101-106, and references therein. Pacsu, E . ; Mora, P.T. J . Am. Chem. Soc. 1950, 72, 1045. Saunders, B.C. Some Aspects of the Chemistry and Toxic Action of Organic Compounds containing Phosphorus and Fluorine; Cambridge University Press: Cambridge, U.K., 1957. Pattison, F.L.M. Toxic Aliphatic Fluorine Compounds; Elsevier Publishing Co.: Amsterdam, 1959.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.

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Retrospect and Prospect

11

12. Marais, J.S.C. Onderstepoort J . Vet. Sci. Anim. Ind. 1943, 18, 203-206. 13. Belcher, R.; Leonard, M.A.; West, T.S. J . Chem. Soc., 1959, 3577-79. 14. Zamojski, A.; Banaszek, A . ; Grynkiewicz, G. Adv. Carbohydr. Chem. Biochem. 1982, 40, 1-20. 15. Helferich, B.; Gnűchtel, A. Chem. Ber. 1941, 74, 1035-39. 16. Henbest, H.B.; Jackson, W.R. J . Chem. Soc. 1962, 954-59. 17. Foster, A.B.; Hems, R.; Webber, J.M. Carbohydr. Res. 1967, 5, 292-301. 18. Foster, A.B.; Hems, R.; Westwood, J.M. Carbohydr. Res. 1970, 15, 41-49. 19. Lopes, D.P.; Taylor, N.F. Carbohydr. Res. 1979, 73, 125-134. 20. Middleton, W.J. J . Org. Chem. 1975, 40, 547-78. 21. Sharma, M.; Korytnyk, W. Tetrahedron Letters 1977, 6, 573-76. 22. Szarek, W.A.; Hay, G.W.; Doboszewski, B. Chem. Comm. 1985, 663-64. 23. Middleton, W.J. U.S 24. Card, P . J . ; Hitz, W.D. J . Am. Chem. Soc. 1984, 106, 5348-50. 25. Barnett, J.E.G. Adv. Carbohydr. Chem. Biochem. 1967, 22, 177-227. 26. Penglis, A.A.E. Adv. Carbohydr. Chem. Biochem. 1981, 38, 195-285. 27. Kent, P.W. in CIBA Symposium, Carbon-Fluorine Compounds; Chemistry, Biochemistry and Biological Activities; Associated Scientific Publishers: Amsterdam, 1972, pp.168-208. 28. Taylor, N.F.; Kent, P.W. Adv. Fluorine Chem. 1963, 4, 113-141. 29. Taylor, N.F.; Childs, R . F . ; Brunt, R.V. Chem. & Ind. 1964, 928-929. 30. Cohen, S.; Levy, D.; Bergmann, E.D. Chem. & Ind. 1964, 1802-3. 31. Pacák, J.; Tocík, Z . ; Cerný, M. Chem. Comm. 1969, 77. 32. Fischer, E . ; Bergmann, M.; Schotte, H. Chem. Ber. 1920, 53B, 509-47. 33. Lemieux, R.U.; Frazer-Reid, B. Can. J. Chem. 1965, 43, 1460-75. 34. Hall, L.D.; Manville, J.F.; Bhacca, N.S. Can. J. Chem. 1969, 47, 1-18. 35. Dwek, R.A. in CIBA Symposium, Carbon-Fluorine Compounds: Chemistry, Biochemistry and Biological Activities; Associated Scientific Publishers: Amsterdam, 1972, pp.239-71. 36. Barton, D.R.H.; Danko, L.J.; Ganguly, A.K.; Hesse, R.H.; Tarzia, G . ; Pecket, M.M. Chem. Comm. 1969, 227. 37. Adamson, J.; Foster, A.B.; Hall, L . D . ; Hesse, R.H. Chem. Comm. 1969, 309-310. 38. Dwek, R.A.; Kent, P.W.; Kirby, P.T.; Harrison, A.S. Tetrahedron Letters 1970, 2987-90. 39. Butchard, G.C.; Kent, P.W. Tetrahedron 1971, 27, 3457-63. 40. Adamson, I . ; Marcus, D.M. Carbohydr. Res. 1970, 13, 314-16. 41. Evelyn, L.; Hall, L.D. Carbohydr. Res. 1976, 47, 285-97. 42. Butchard, G.C.; Kent, P.W. Tetrahedron 1975, 35, 2439-43. 43. Butchard, G.C.; Kent, P.W. Tetrahedron 1975, 35, 2551-54. 44. Kent, P.W.; Dimitrijevich, S.D. J . Fluorine Chem. 1977, 10, 455-78. 45. Ido, T . ; Wan, C-N.; Fowler, J . S . ; Wolf, A.P. J . Org. Chem. 1977, 42, 2431-32.

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46. Ido, T . ; Wan, C-N.; Casella, V . ; Fowler, J . S . ; Wolf, A.P.; Reivich, M.; Kuhl, D.E. J . Labelled Compounds & Radiopharmaceuticals 1978, 19, 173-183. 47. Schmidt, M.G.; Schwarz, R.T.; Ludwig, H. J . Virol. 1976, 18, 819-23. 48. Schmidt, M.G.; Biely, P.; Kratz, Z . ; Schwarz, R.T. Eur. J . Biochem. 1978, 87, 55-68. 49. Schwarz, R.T.; Schmidt, M.G.; Datema, R. Biochem. Soc. Trans. 1979, 7, 322-26. 50. Datema, R.; Schwarz, R.T. Biochem. J. 1979, 184, 113-123. 51. Blough, H.A.; Giuntoli, R.L. J . Am. Med. Soc. Ass. 1979, 241, 2798-2801. 52. Schwarz, R.T.; Datema, R. Adv. Carbohydr. Chem. Biochem. 1982, 40, 287-379. 53. Winterbourne, D . J . ; Barnby, R.; Mian, N . ; Kent, P.W. Biochem. J. 1979, 182, 707-16. 54. Schulz, A.M.; Mora 55. Bernacki, R . J . ; Sharma Korytnyk, W. J . Supermol. Structure 1977, 7, 235-50. 56. Paul, B.; Korytnyk, W. in Cell Surface Carbohydrate Chemistry; Academic Press: New York, 1978; pp.311-35. 57. Elbein, A.D. Ann. Rev. Biochem. 1987, 56, 497-534. 58. Barnett, J.E.G. in CIBA Symposium, Carbon-Fluorine Compounds: Chemistry, Biochemistry and Biological Activities; Associated Scientific Publishers: Amsterdam, 1972, pp.95-109. 59. Taylor, N.F.; Hill, L . ; Eisenthal, R. Can. J. Biochem. 1975, 53, 57-64. 60. D'Amore, T . ; Taylor, N.F. FEBS Lett. 1982, 143, 247-51. 61. Sbrissa, D.; McIntosh, J.M.; Taylor, N.F. Proc. Can Fed. Biol. Soc. Abstr. 1987, 30, 141. 62. Agbanyo, M.; Taylor, N.F. Bioscience Reports 1986, 6, 309-16. 63. Drueckhammer, D.G.; Wong, C-H. J . Org. Chem. 1985, 50, 5912-13. 64. Henessian, S. Total Synthesis of Natural Products: The 'Chiron' Approach; Pergamon Press: Oxford, U.K., 1983. 65. Morton, G.O.; Lancaster, J.E.; Van Lear, G . E . ; Fulmor, W.; Meyer, W.E. J . Am. Chem. Soc. 1969, 91, 1535-37. 66. Leyland-Jones, B.; Donnelly, H . ; Groshen, S.; Myskowski, P.; Fox, J . J . J . Infectious D i s . 1987, 154, 430-36. 67. Dagani, R. Chem. & Eng. News 1981, 9 Nov., 30-37. RECEIVED January 8, 1988

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 2

Preparation and Reactions of Glycosyl Fluorides 1

Jared L. Randall and K. C. Nicolaou Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104 In recent years studied i n t e n s i v e l y glycosylating agents. In t h i s paper, the preparation and reactions of glycosyl fluorides will be reviewed with emphasis on the use of glycosyl fluorides prepared from thioglycosides. Applications of t h i s technology to the synthesis of carbohydrate - containing natural products will be presented. A d d i t i o n a l l y , the novel 1,2-migration reaction induced by diethylaminosulfur t r i f l u o r i d e (DAST) will be discussed.

Although f l u o r i n a t e d carbohydrates have been a s u b j e c t o f i n t e r e s t f o r decades (1), t h e recent r e a l i z a t i o n »that g l y c o s y l f l u o r i d e s can be s y n t h e t i c a l l y u s e f u l has sparked renewed i n v e s t i g a t i o n i n t h i s a r e a . The modern advances i n the p r e p a r a t i o n and r e a c t i o n s o f g l y c o ­ s y l f l u o r i d e s have been surveyed r e c e n t l y i n an e x t e n s i v e review (2.); t h e r e f o r e , t h i s l e c t u r e w i l l focus on the use o f g l y c o s y l f l u o r i d e s i n n a t u r a l product s y n t h e s i s and t h e s t u d i e s c a r r i e d out i n o u r laboratories. Preparation

of Glycosyl

Fluorides

The review by Card (2.) c o n c i s e l y p r e s e n t s t h e v a r i e t y o f r e p o r t e d methods f o r the p r e p a r a t i o n o f g l y c o s y l f l u o r i d e s from a number o f p r e c u r s o r s . Although they have been prepared from g l y c o s y l c h l o r i d e s , a c e t a t e s , and p i v a l o a t e s and g l y c o s e s , we have found t h i o g l y c o s i d e s t o be more v e r s a t i l e p r e c u r s o r s t o g l y c o s y l f l u o r i d e s . The employment of t h i o g l y c o s i d e s a l l o w s t h e s e l e c t i v e a c t i v a t i o n o f t h e anomeric c e n t e r e a r l y i n the s y n t h e t i c scheme, o b v i a t i n g the s e l e c t i v e deprot e c t i o n o r a c t i v a t i o n o f the anomeric c e n t e r d i r e c t l y b e f o r e f l u o r i ­ nation. The N i c o l a o u group's i n t e r e s t i n t h e development o f m i l d methods f o r the formation o f 0 - g l y c o s i d i c l i n k a g e s t o complex n a t u r a l Current address: The Procter and Gamble Company, Miami Valley Laboratories, P.O. 398707, Cinncinnati, O H 45239-8707

•

0097-6156/88/0374-0013$06.00A) 1988 American Chemical Society

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Box

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FLUORINATED CARBOHYDRATES: C H E M I C A L AND

BIOCHEMICAL ASPECTS

products l e d t o i n v e s t i g a t i o n s of g l y c o s y l f l u o r i d e s as g l y c o s y l donors. The key d i s c o v e r y t h a t l e d t o these a p p l i c a t i o n s was the r e a l i z a t i o n t h a t phenyl t h i o g l y c o s i d e s can be e f f i c i e n t l y converted t o g l y c o s y l f l u o r i d e s (Figure 1) Q) . Treatment of phenyl t h i o g l y c o ­ sides (1) w i t h d i e t h y l a m i n o s u l f u r trifluoride (DAST) or hydrogen f l u o r i d e - p y r i d i n e complex (HF-pyr), and N-bromosuccinimide (NBS) in dichloromethane at -15-0°C p r o v i d e s g l y c o s y l f l u o r i d e s (3) i n good t o e x c e l l e n t y i e l d s . A d d i t i o n a l l y , i t was found t h a t many p r o t e c t i n g groups, which cannot w i t h s t a n d the more d r a s t i c c o n d i t i o n s commonly used f o r g l y c o s y l donor formation, t o l e r a t e these c o n d i t i o n s ; indeed, even s i l y l e t h e r s are s t a b l e t o the r e a c t i o n c o n d i t i o n s when DAST i s u t i l i z e d as the f l u o r i n a t i n g agent. I t i s reasonable t o assume t h a t t h i s f l u o r i n a t i o n r e a c t i o n proceeds v i a an i n t e r m e d i a t e bromosulfonium i o n (2, F i g u r e 1) which i s a t t a c k e d by a f l u o r i d e i o n . The f l u o r i d e i o n donor a b i l i t y of HFp y r i d i n e complex i s w e l l known; however i n t h i s case the mechanism by which DAST i s able t forward. We propose two may proceed (Figure 2) . Mechanism A i n v o l v e s the s u c c i n i m i d e ion which i s formed as a byproduct i n the b r o m i n a t i o n r e a c t i o n . The s u c c i n i m i d e i o n may a t t a c k the s u l f u r atom i n the DAST reagent and d i s p l a c e a f l u o r i d e i o n which, i n t u r n , opens the bromo-sulphonium i o n (4). An a l t e r n a t i v e mechanism (B) i n v o l v e s the lone p a i r of e l e c t r o n s i n the DAST reagent which d i s p l a c e s the f l u o r i d e i o n which then a t t a c k s the bromosulphonium s p e c i e s (5) . The second mechanism seems more p l a u s i b l e i n view of the f a c t t h a t t h i s r e a c t i o n , i n most cases, proceeds t o completion v e r y q u i c k l y . R e a c t i o n s of G l y c o s y l

Fluorides

The use of g l y c o s y l f l u o r i d e s as g l y c o s y l donors i s advantageous f o r the s y n t h e t i c chemist because, they are r e a d i l y prepared, s t a b l e t o column chromatography, and t o l e r a n t of storage f o r extended p e r i o d s of time. U n t i l r e c e n t l y , g l y c o s y l f l u o r i d e s were c o n s i d e r e d t o be too s t a b l e f o r use as g l y c o s y l donors (1) . However, the advent of the Mukaiyama c o n d i t i o n s f o r the formation of the O - g l y c o s i d i c linkage generated i n t e n s i v e study of the r e a c t i o n s of g l y c o s y l f l u o r i d e s ilrl) • A f t e r s u r v e y i n g many a c t i v a t i n g agents, Mukaiyama and cowork­ e r s found t h a t u t i l i z i n g a combination of s i l v e r p e r c h l o r a t e and stannous c h l o r i d e as promoters c o n v e r t e d p - g l y c o s y l f l u o r i d e s t o 0g l y c o s i d e s i n h i g h y i e l d s and w i t h good a - g l y c o s i d e selectivities. We have found the Mukaiyama r e a c t i o n t o be e x t r e m e l y u s e f u l and have extended the a p p l i c a t i o n of t h i s method t o the s e l e c t i v e synthe­ s i s of p - g l y c o s i d e s as w e l l as a - g l y c o s i d e s . By t a k i n g advantage of s o l v e n t e f f e c t s and p r o t e c t i n g groups at the 2 - p o s i t i o n , remarkable s t e r e o s e l e c t i v i t y i n the formation of O - g l y c o s i d i c l i n k a g e s has been obtained (£) . Once the optimum c o n d i t i o n s were a s c e r t a i n e d , the anomeric c o n f i g u r a t i o n of the g l y c o s y l f l u o r i d e was not found t o have any s i g n i f i c a n t e f f e c t on the s t e r e o s e l e c t i v i t y of the g l y c o s i d a t i o n r e a c t i o n Q ) . This phenomenon has a l s o been r e p o r t e d by Ogawa e£ a l . (£,1) . We concur w i t h these i n v e s t i g a t o r s ' p r o p o s i t i o n t h a t the high s t e r e o s e l e c t i v i t y of these r e a c t i o n s i s due t o the formation of i n t i m a t e i o n p a i r s , as p r e v i o u s l y proposed by Lemieux (£) and Paulsen (2) f o r o t h e r g l y c o s y l donors.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

2.

R A N D A L L & NICOLAOU

Preparation and Reactions of Glycosyl Fluorides 15

When used i n c o n j u n c t i o n w i t h t h e m i l d p r e p a r a t i o n o f g l y c o s y l f l u o r i d e s from t h i o g l y c o s i d e s , t h e Mukaiyama c o n d i t i o n s p r o v i d e t h e key t o a m i l d , s e l e c t i v e method f o r t h e s y n t h e s i s o f t h e O - g l y c o s i d i c l i n k a g e . We have found t h i s method t o be p r a c t i c a b l e f o r t h e forma­ t i o n o f g l y c o s i d e bonds t o s e n s i t i v e , complex a g l y c o n e s and f o r t h e r e p e t i t i v e , b l o c k - t y p e s y n t h e s e s o f o l i g o s a c c h a r i d e s . The u s e o f t h i o g l y c o s i d e s as p r e c u r s o r s t o g l y c o s y l f l u o r i d e s i s k e e n l y s u i t e d f o r t h e s e a p p l i c a t i o n s because t h e t h i o g l y c o s i d e f u n c t i o n a l i t y c a n : 1) be used t o mask t h e anomeric c e n t e r e a r l y i n t h e s y n t h e t i c scheme; 2) e x i s t i n t h e g l y c o s y l a c c e p t o r w i t h o u t i n t e r f e r i n g w i t h t h e g l y c o s i d a t i o n r e a c t i o n ; and 3) when d e s i r e d , be c o n v e r t e d t o t h e g l y c o s y l f l u o r i d e under m i l d c o n d i t i o n s . Formation o f O - G l y c o s i d i c Linkages t o S e n s i t i v e Aalvcones. The f i r s t a p p l i c a t i o n o f t h i s t e c h n o l o g y t o n a t u r a l p r o d u c t s s y n t h e s i s was t h e p a r t i a l s y n t h e s i s o f avermecti B Q ) .Th o l e a n d r o s derivativ (6 F i g u r e 3) was p r e p a r e d f l u o r i d e (7) i n 80% y i e l t h e s i l y l e t h e r p r o t e c t i n g group o f t h e t h i o g l y c o s i d e (6) a f f o r d e d t h e h y d r o x y l component (8) . C o u p l i n g o f these two components, u t i l i z i n g t h e Mukaiyama c o n d i t i o n s , s e l e c t i v e l y p r o v i d e d t h e al i n k e d d i s a c c h a r i d e (9) . The d i s a c c h a r i d e was t h e n c o n v e r t e d t o g l y c o s y l f l u o r i d e (10) (a:P r a t i o = 5:1) i n 85% y i e l d by t r e a t m e n t w i t h NBS and DAST. C o u p l i n g o f t h e d i s a c c h a r i d e g l y c o s y l donor t o t h e a v e r m e c t i n a g l y c o n e d e r i v a t i v e proceeded w i t h complete a-anomeric s e l e c t i v i t y i n 62% y i e l d . D e s i l y l a t i o n o f t h e r e s u l t a n t p r o d u c t p r o ­ v i d e d avermectin B . T h i s s y n t h e s i s demonstrates t h e s y n t h e t i c u t i l i t y o f t h e method f o r t h e f o r m a t i o n o f O - g l y c o s i d i c l i n k a g e s t o complex, s e n s i t i v e a g l y c o n e s . Our method was a l s o employed i n t h e t o t a l s y n t h e s i s o f e f r o t o m y c i n (jJl) . The d e s i r e d c a r b o h y d r a t e u n i t s (11 and 12, F i g u r e 4) were e f f i c i e n t l y p r e p a r e d from D - a l l o s e and L-rhamnose, r e s p e c t i v e l y . Con­ v e r s i o n o f t h e rhamnose-derived t h i o g l y c o s i d e (12) t o t h e c o r r e s p o n d ­ i n g g l y c o s y l f l u o r i d e (13) was a c c o m p l i s h e d i n 88% y i e l d w i t h t h e NBS/DAST c o n d i t i o n s . By e m p l o y i n g t h e Mukaiyama c o n d i t i o n s , t h i s g l y c o s y l f l u o r i d e was e f f i c i e n t l y c o u p l e d w i t h g l y c o s y l a c c e p t o r (11) i n 75% y i e l d t o p r o v i d e d i s a c c h a r i d e (14) w i t h t o t a l a-anomeric s e l e c t i v i t y . B u t , when t h e t h i o g l y c o s i d e o b t a i n e d (14) was c o n v e r t e d to t h e g l y c o s y l f l u o r i d e (15) w i t h t h e NBS/DAST c o n d i t i o n s and c o u p l e d w i t h g o l d i n o l a c t o n e ( 1 8 ) , t h e u n d e s i r e d a-anomeric p r o d u c t was o b t a i n e d . N e v e r t h e l e s s , p r o t e c t i n g group i n t e r c h a n g e o f t h e s i l y l p r o t e c t i n g groups t o n e i g h b o r i n g - g r o u p - a c t i v e a c e t y l groups p r o v i d e d d i s a c c h a r i d e (16). A f t e r conversion o f t h i s d i s a c c h a r i d e t o g l y c o s y l f l u o r i d e (17), c o u p l i n g t o goldinolactone allowed t h e p r e p a r a t i o n o f t h e d e s i r e d p - g l y c o s i d e ( 1 9 ) , e x c l u s i v e l y , i n 86% y i e l d . T h i s i n t e r ­ m e d i a t e was t h e n f u r t h e r e l a b o r a t e d t o p r o v i d e t h e f i r s t total synthesis of efrotomycin. la

Synthesis o f Oligosaccharides. To demonstrate t h e e f f e c t i v e n e s s o f t h i s t e c h n o l o g y f o r t h e r e p e t i t i v e and b l o c k - t y p e s y n t h e s i s o f o l i g o ­ s a c c h a r i d e s , we t a r g e t e d t h e l i n e a r h e x a s a c c h a r i d e (28, F i g u r e 5) c o n s i s t i n g o f s i x g l u c o s e u n i t s l i n k e d i n an a(l-*6) f a s h i o n Q ) . From t h e r e a d i l y p r e p a r e d phenyl t h i o g l y c o s i d e (20), both t h e g l y c o s y l f l u o r i d e (22, NBS/DAST, 90%, a:p r a t i o = 1 : 1 ) and t h e g l y c o -

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

16

FLUORINATED CARBOHYDRATES: C H E M I C A L AND BIOCHEMICAL ASPECTS

NBS

-SPh

HF-pyr. L_

Figure

1.

2

Formation o f G l y c o s y l F l u o r i d e s from T h i o g l y c o s i d e s .

F -S—NEt 2

F i g u r e 2.

3

2

The Mechanism

o f t h e DAST R e a c t i o n .

MeO

RO Me

a(85/o) | _ Y

a(80/o)|__

7

p _ si BuMe , Y = F 2

2

1) Aglycone-Bis(silyl ether), c (62%) 2) b (89%)

b(100%)

t

t

p _ Si BuMe , Y = F

1 0

8 R = H, Y = SPh Avermectin B

1 a

n

a) NBS, DAST, CH CI , -15°C; b) Bu NF, THF, 0°C; c) AgCI0 , SnCI , 4A MS, Et 0, -15-*20°C. 2

4

Figure

3.

P a r t i a l Synthesis

2

2

4

2

o f A v e r m e c t i n B . ( A d a p t e d from R e f . 3.) la

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Preparation and Reactions of Glycosyl Fluorides

R A N D A L L & NICOLAOU

a) NBS, DAST, CH CI , -15°C; b) AgCI0 , SnCI , 4A MS, Et 0, -15— 0°C; c) i) Bu NF, THF, 0-25°C, ii) Ac 0, DMAP, CH CI , 25°C. 2

2

4

2

2

n

4

F i g u r e 4.

2

2

2

Attachment o f t h e C a r b o h y d r a t e P o r t i o n o f E f r o t o m y c i n . ( A d a p t e d f r o m R e f . 10.)

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

F L U O R I N A T E D CARBOHYDRATES: C H E M I C A L AND B I O C H E M I C A L ASPECTS

'BuPh SiO 2

AcO SPh

PhCH 0 2

20

a (90%)/

OCH Ph

(98%)

2

>v

HO

AcO SPh

PhCH 0

_in n = 0, R=OSi«BuPh , X=F . 25 I

a ( 8 8 % )

n = 2, R=OSi'BuPh , X=F . 27 J22,c(66%)

a ( 8 9 % )

2

,

R=Si BuPh X=SPh(|5) 23

PhCHzOOs^-^-A^x OCH Ph 2

b ( 9 7 / o )

• R=H, X=SPh (P)

2>

2 4

1 | c (70%)

2

R=Si90% yield. S i m i l a r s e l e c t i v i t y was o b s e r v e d i n t h e h y d r o l y s i s o f t h e o t h e r a c y l d e r i v a t i v e s o f a - and pm e t h y l D - h e x o p y r a n o s i d e s i n c l u d i n g g a l a c t o s e , mannose, N a c e t y l g l u c o s a m i n e and N - a c e t y l m a n n o s a m i n e . (22) T h e s e 6OH s u g a r d e r i v a t i v e s c a n be u s e d f o r s y n t h e s i s o f 6-F h e x o s e s by r e a c t i o n w i t h DAST. Table I indicates r e l a t i v e and s p e c i f i c a c t i v i t i e s o f C a n d i d a l i p a s e c a t a l y z e d h y d r o l y s i s of d i f f e r e n t sugar e s t e r s at C-6 position. A l l t h e p r o d u c t s were i s o l a t e d by s i l i c a g e l

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

3.

WONGETAL.

Cmpd

ChemoenzymaticSynthesisof Fluorosugars

M

N

O

P

Q

R

X

K

m

(mM)

Vmax (Umg" )

0.17

300

1

a

OH

b

F

0.19

15 0

c

H

F

0.41

260

d

F

F

0.13

1 60

H

H

OH

OH

H

0

•

35

e f 9

F

h

H

84

30

-

3

F

F i g u r e 5. H e x o k i n a s e - c a t a l y z e d p h o s p h o r y l a t i o n o f f l u o r i n a t e d h e x o s e s c o u p l e d w i t h a n ATP r e g e n e r a t i o n system u s i n g phosphoenol pyruvate as p h o s p h o r y l a t i n g agent and pyruvate k i n a s e as c a t a l y s t .

column chromatography e l u t e d w i t h CHC1 : MeOH (10:1) a n d t h e s t r u c t u r e s p r o v e n b y m i c r o a n a l y s i s a n d C-NMR a n a l y s i s u s i n g t h e d e u t e r i u m i n d u c e d s h i f t s (DIS) t e c h n i q u e (24.) • T h e a d d i t i o n o f d i m e t h y l f o r m a m i d e (10%) t o t h e Candida l i p a s e - c a t a l y z e d h y d r o l y s i s o f g l u c o s e p e n t a a c e t a t e changes t h e r e g i o s e l e c t i v i t y . In reaction with glucose pentaacetate, 1,2,3-tri-O-acetyl-a-D-glucose and 1 , 2 , 3 , 4 - t e t r a - O - a c e t y l - ^ - D - g l u c o s e were i s o l a t e d i n 73% a n d 27% y i e l d r e s p e c t i v e l y . T h e s t r u c t u r e s o f t h e compounds a r e shown i n T a b l e I I I . A l l s h i f t s a r e i n ppm f r o m TMS. T h e DIS numbers a r e i n p a r e n t h e s e s . Negative values indicate upfield s h i f t s . B o t h compounds w e r e i d e n t i f i e d b y u s i n g t h e DIS t e c h n i q u e s ( T a b l e I I ) . T h e y show e x p e c t e d u p f i e l d s h i f t o f t h e d e a c y l a t e d c a r b o n . When t h e p e r a c e t y l a t e d s u g a r s were h y d r o l y z e d b y p o r c i n e p a n c r e a s l i p a s e i n t h e p r e s e n c e o r a b s e n c e o f 10% DMF, t h e same r e g i o s e l e c t i v i t y was o b s e r v e d , a l l b e i n g hydrolyzed a t C - l p o s i t i o n p r e f e r e n t i a l l y (Table I I I ) . The p r o d u c t s were i s o l a t e d b y e x t r a c t i o n w i t h e t h y l 3

13

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

36

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

F i g u r e 6. Preparation of 2-deoxy-2-fluoro-arabinose5 - p h o s p h a t e ( u p p e r scheme) and 2 - d e o x y - 2 - D - r i b o s e - 5 p h o s p h a t e ( l o w e r scheme). U p p e r scheme: a) 1, p y r i d i u m d i c h r o m a t e . 2, NaBH /70% a q . e t h a n o l , 92% yield, b) D A S T / C H C l / p y r i d i n e , 88 % y i e l d . c) Dowex 50 ( H + ) / H 0 92% Y i e l d . d) ATP/hexokinase/phosphoenol pyruvate/pyruvate kinase, 90% y i e l d , e) P b ( 0 A c ) / H , 64% y i e l d . Lower scheme: a) P h C C l / p y r i d i n e 70 % y i e l d , b) D A S T / C H C 1 , 50% yield. c) 1.80% AcOH/Dowex 50 ( H ) ; 2. A T P / h e x o k i n a s e s y s t e m a s above, 90% y i e l d , d) P b ( O A c ) / H , 60 % y i e l d . 4

2

2

2

/

+

4

3

/

2

2

+

+

4

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

3.

37

ChemoenzymaticSynthesisofFluorosugars

WONGETAL.

Table I. Lipase-Catalyzed Selective Hydrolysis of D i f f e r e n t S u g a r E s t e r s a t C-6 p o s i t i o n Specific Activity

Substrate

Me a - D - g l u c o p y r a n o s i d e , t e t r a p e n t a n o a t e Me a - D - g l u c o p y r a n o s i d e , t e t r a o c t a n o a t e Me B - D - g l u c o p y r a n o s i d e , t e t r a o c t a n o a t e Me a - D - g a l a c t o p y r a n o s i d e , t e t r a p e n t a n o a t e Me a-D-mannopyranoside, t e t r a p e n t a n o a t e aD-glucose, pentaacetate B-D-glucose, p e n t a a c e t a t e Me N - A c e t y l g l u c o s a m i n e , t r i p e n t a n o a t e Me N-acetylmanosamine, t r i p e n t a n o a t e

0.,020 0,.153 0..044 0..017 0..015 0..00 0.,04b

4

1

>

6

F

( t r a n s ) * 6',F ( t r a n s ) ? H4,F ( c i s ) 6.F ( c i s ) " 4 - 8 Hz, H-4, H-6), 4.10 11H, H, J l 2 " 2 3 >1 H z , Anal. Calc'd for C H F 0 : C, 36.01;rf,5.04;'F, 1 8 . 9 9 F o u n d : C, 3 5 - 9 4 ; H, 5.08; F, 19.01. J

=

2

1

6H

z

J

=

J

J

6

1 0

2

5

Biological Results. I n c o r p o r a t i o n o f t h e s e a n a l o g u e s o f myo­ i n o s i t o l (1) ( s e e F i g u r e 2) i n t o p h o s p h o l i p i d w o u l d b e e x p e c t e d t o be a p r e r e q u i s i t e f o r a c t i v i t y a s a n i n h i b i t o r o f e i t h e r t h e P I

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

52

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

0 I

0 1 OBzl

(OH

0 J

)

+

0 I OBzl

(oBzl

)

OBzl

OH 19

^°

)

B z l

)

f

Ho\

OH

20

21

22

F i g u r e 5. I m p r o v e d s y n t h e s i s o f 5 - d e o x y - 5 - f l u o r o - m y o - i n o s i t o l (4). R e a g e n t s : a. J D - T S O H , H 0 C H C H 0 H ; b. P h C H B r - n Bu NHS0 /NaOH; c. ( C F , S 0 ) 0 - P y r i d i n e ; C s 0 C C H C H - DMF; d. NaOH - MeOH; e. D i e t h y l a m i n o s u l f u r t r i f l u o r i d e (DAST) - DMAP; f. H - P d ( 0 H ) / C , HOAc. 2

4

4

2

2

2

2

2

2

2

3

2

HO

BzIO n — ( d

R»m\^

B z l 0 \ _ Y

|/

H

°

I

OH , OH

-

1

OBzl

18

23

24

6

F i g u r e 6. S y n t h e s i s o f 5 - d e o x y - 5 , 5 - d i f l u o r o - m y o - i n o s i t o l (6). R e a g e n t s : a. CrO* - P y r i d i n e - A c 0 ; b. D i e t h y l a m i n o s u l f u r t r i f l u o r i d e (DAST); C. H - Pd(0H) /C, HOAc. 2

2

2

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

4.

MOYERETAL.

53

myoinositols as Biological Probes

k i n a s e o r p h o s p h o l i p a s e C, a s t h e s u b s t r a t e s f o r b o t h e n z y m e s a r e p h o s p h o l i p i d s c o n t a i n i n g m y o - i n o s i t o l and not f r e e m y o - i n o s i t o l i t s e l f . P r e v i o u s s t u d i e s o f P I s y n t h e t a s e had s u g g e s t e d t h a t t h e r e q u i r e m e n t s f o r the i n o s i t o l s u b s t r a t e were v e r y s t r i n g e n t ( 3 2 , 33)* Our r e s u l t s ( T a b l e i ) r e v e a l e d t h a t e i t h e r r e p l a c e m e n t , o r replacement w i t h i n v e r s i o n , o f the hydroxyl a t the 2 o r 4 ( 6 ) p o s i t i o n w i t h f l u o r i n e l e d t o l o s s o f a l l d e t e c t a b l e a b i l i t y o f the a n a l o g u e t o be i n c o r p o r a t e d i n t o p h o s p h o l i p i d a s a s s a y e d b y t h e s t o i c h i o m e t r i c f o r m a t i o n o f CMP f r o m C D P - d i g l y c e r i d e . [it i s n o t e w o r t h y t h a t , s i n c e 3 i s a m i x t u r e o f 4 - and 6 - i s o m e r s , i n t e r p r e t a t i o n o f t h e r e s u l t s f o r 3 i s n o t s t r a i g h t f o r w a r d a s one i s o m e r c o u l d be a n i n h i b i t o r and the o t h e r a s u b s t r a t e . R e s u l t s o f a separate experiment ( 2 3 ) using [^H]myo-inositol, however, i n d i c a t e d t h a t t h e r a c e m i c m i x t u r e was n o t a n i n h i b i t o r o f P I synthetase.] Table I .

Fluorinate as s u b s t r a t e brain£

Analogue (no.)

Substrate A c t i v i t y CMP F o r m a t i o n (% o f m y o - i n o s i t o l )

myo-inositol ( l )

100

1-deoxy-1-fluoro-scyllo-inositol ( 2 )

2 - f l u o r o , which i s the i n v e r s e of the o r d e r o b s e r v e d f o r the a c i d - c a t a l y s e d h y d r o l y s i s of s e r i e s o f a l k y l (40) and a r y l (41) deoxy p - D - g l u c o p y r a n o s i d e s ( i . e . 2-deoxy > 4-deoxy > 3-deoxy > 6 - d e o x y ) . T h i s inverse r e l a t i o n s h i p suggests a common, p r o b a b l y e l e c t r o n i c , o r i g i n and we p r o v i d e two p o s s i b l e

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

r a t i o n a l e s as f o l l o w s . C l e a r l y i n d u c t i v e e f f e c t s w i l l be i m p o r t a n t at the 2 - p o s i t i o n , but these would be expected to be a t t e n u a t e d r a p i d l y w i t h d i s t a n c e from the anomeric c e n t r e . They m i g h t , however, be s u f f i c i e n t to at l e a s t p a r t i a l l y e x p l a i n the observed o r d e r of r a t e c o n s t a n t s i f one assumes t h a t the m a j o r i t y of the p o s i t i v e charge i n the oxocarbonium i o n r e s i d e s on the r i n g oxygen. In t h a t case the 4 - s u b s t i t u e n t i s c l o s e r to the charge than i s the 3 - p o s i t i o n so i t s e f f e c t s h o u l d be g r e a t e r . However, the 6 - p o s i t i o n i s a l s o j u s t as c l o s e , yet the 6 - f l u o r o d e r i v a t i v e h y d r o l y s e s more r a p i d l y than the 3 - f l u o r o compound. Another p o s s i b l e e x p l a n a t i o n r e l a t e s t o the r e l a t i v e o r i e n t a t i o n of d i p o l e s a s s o c i a t e d w i t h C-OH and C - F bonds i n the ground s t a t e and i n the h a l f c h a i r t r a n s i t i o n s t a t e , s i n c e i n c r e a s e d a l i g n m e n t of d i p o l e s a t the t r a n s i t i o n s t a t e would be expected to r e s u l t i n r a t e d e c r e a s e s . The importance of such d i p o l a r i n t e r a c t i o n s has been amply i l l u s t r a t e d by the anomeric e f f e c t . As d e t a i l e d elsewhere ( 3 9 ) , t h e r e i s an o v e r a l l d e c r e a s e i n d i p o l a r a l i g n m e n t f o r the 3 - f l u o r which s h o u l d i n c r e a s e i t the 2 - and 4 - p o s i t i o n s r e s u l t s , r e s p e c t i v e l y , i n an i n c r e a s e i n a l i g n m e n t and i n no net change. The r e s u l t of t h i s would be t o d e c r e a s e the r a t e of the 2 - f l u o r o r e l a t i v e l y and to l e a v e the 4 - f l u o r o slower than the 3 - f l u o r o . T h i s c o u l d be the cause of the o r d e r i n v e r s i o n and the c o n v e r s e would c l e a r l y f o l l o w f o r the deoxyglucosides. In r e a l i t y both e f f e c t s p r o b a b l y c o n t r i b u t e . determined f o r the The a c t i v a t i o n 2 - f l u o r o analogue (AH = 113.5 k J m o l " ; *5 ) compared w i t h those (Air = 116.6 k J m o l " ; A S * = 14.9 eu) f o r the p a r e n t sugar phosphate ( 3 3 ) . S u r p r i s i n g l y , the major d i f f e r e n c e i s i n the a c t i v a t i o n e n t r o p y which s u g g e s t s a more S 2 - l i k e t r a n s i t i o n s t a t e i n v o l v i n g c o n s i d e r a b l e p r e a s s o c i a t i o n of the water to the g l y c o s i d i c c a r b o n atom. This is quite consistent with current suggestions ( 3 8 , 4 2 ) t h a t a l l s o l v o l y s e s at sugar anomeric carbons proceed t h r o u g h a p r e - a s s o c i a t i o n mechanism and a l s o w i t h the r e c e n t o b s e r v a t i o n s (43) of i n d u c t i v e enhancement of p r e - a s s o c i a t i v e p a r t i c i p a t i o n i n other displacement r e a c t i o n s . Clearly, therefore, electronic effects can be v e r y i m p o r t a n t , but the magnitude of the e l e c t r o n i c e f f e c t s o b s e r v e d i n any case may not be p r e d i c t a b l e s i n c e the t r a n s i t i o n s t a t e s t r u c t u r e can v a r y c o n s i d e r a b l y . T h i s w i l l be p a r t i c u l a r l y t r u e , and h a r d to e v a l u a t e , at an enzyme a c t i v e s i t e . 1

A S

=

e u

a

n

d

1

5

N

Enzyme S t u d i e s ;

Glycogen Phosphorylase.

We have now s t u d i e d f l u o r i n a t e d sugars as s u b s t r a t e s of a number o f g l y c o s y l t r a n s f e r a s e s ; our most complete s t u d y to date b e i n g w i t h r a b b i t muscle g l y c o g e n p h o s p h o r y l a s e . T h i s enzyme, which c a t a l y s e s the r e v e r s i b l e p h o s p h o r o l y s i s of g l y c o g e n , p r o d u c i n g g l u c o s e - 1 phosphate i s thought to o p e r a t e v i a the e l e m e n t a r y mechanism shown i n Scheme 1 (R « p h o s p h a t e , R'OH = g l y c o g e n ) . As can be s e e n , b i n d i n g of g l u c o s e - l - p h o s p h a t e i s f o l l o w e d by the a c i d - c a t a l y s e d c l e a v a g e of the anomeric l i n k a g e l i b e r a t i n g phosphate and p r o d u c i n g a c a r b o x y l a t e - s t a b i l i s e d g l u c o s y l oxocarbonium i o n i n t e r m e d i a t e , o r more l i k e l y a c o v a l e n t g l u c o s y l enzyme. D e g l u c o s y l a t i o n of the enzyme, and consequent g l u c o s y l t r a n s f e r , w i l l o c c u r upon a t t a c k of the 4 - h y d r o x y l at the n o n - r e d u c i n g t e r m i n u s of g l y c o g e n . Thus both

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the g l u c o s y l a t i o n and the d e g l u c o s y l a t i o n of the enzyme are c o n s i d e r e d to p r o c e e d v i a oxocarbonium i o n - l i k e t r a n s i t i o n s t a t e s . I n l i g h t of t h i s proposed mechanism i t i s of i n t e r e s t to a n t i c i p a t e what would be the e f f e c t s on t u r n o v e r , of s u b s t i t u t i n g v a r i o u s sugar h y d r o x y l s by hydrogen and by f l u o r i n e . Two major e f f e c t s come to mind, namely e l e c t r o n i c e f f e c t s and b i n d i n g e f f e c t s . The p r e v i o u s study of the a c i d - c a t a l y s e d h y d r o l y s i s of the s e r i e s of d e o x y f l u o r o - and d e o x y - a - D - g l u c o s e - l - p h o s p h a t e analogues has amply demonstrated the importance of e l e c t r o n i c e f f e c t s from such s u b s t i t u e n t s on r e a c t i o n s p r o c e e d i n g v i a c a t i o n i c t r a n s i t i o n s t a t e s . As was seen i n Scheme 1, t h i s enzymic r e a c t i o n i s a l s o thought to p r o c e e d v i a oxocarbonium i o n - l i k e t r a n s i t i o n s t a t e s . On the b a s i s of electronic effects alone, therefore, the deoxy s u b s t r a t e s would be expected to t u r n over more r a p i d l y than the normal s u b s t r a t e and the d e o x y f l u o r o s u b s t r a t e s more s l o w l y . B i n d i n g e f f e c t s are somewhat more complex As has been d i s cussed many times p r e v i o u s l by s p e c i f i c a l l y b i n d i n g t h e r e b y s t a b i l i s i n g i t and l o w e r i n g the o v e r a l l a c t i v a t i o n f r e e energy. Thus b i n d i n g energy i s used i n a f a i r l y d i r e c t way to e f f e c t catalysis. Some v e r y e l e g a n t s t u d i e s i n v o l v i n g p r o t e i n mutagenesis have been performed to measure the s t r e n g t h s of i n d i v i d u a l hydrogen bonds i n v o l v e d i n s t a b i l i s i n g the r e a c t i o n t r a n s i t i o n s t a t e f o r the enzyme t y r o s y l t-RNA s y n t h e t a s e ( 1 3 , 4 6 ) . These s t u d i e s s u g g e s t e d t h a t t h i s enzyme takes advantage of the g e o m e t r i c a l changes i n the s u b s t r a t e s t r u c t u r e on a p p r o a c h i n g the t r a n s i t i o n s t a t e , to s e l e c t i v e l y s t a b i l i z e the t r a n s i t i o n s t a t e , t h e r e b y f a c i l i t a t i n g the reaction. A s i m i l a r s i t u a t i o n may o b t a i n w i t h g l y c o g e n p h o s p h o r y l a s e , s i n c e the t r a n s i t i o n s t a t e oxocarbonium i o n s p e c i e s w i l l have a h a l f - c h a i r c o n f o r m a t i o n , d i s t i n c t l y d i f f e r e n t from the ground-state chair conformation. Thus i n d i v i d u a l hydrogen bonds may be v e r y i m p o r t a n t i n promoting such a t r a n s i t i o n . Any s u b s t i t u t i o n i n the sugar r i n g which e l i m i n a t e s hydrogen bonds i n v o l v e d i n t r a n s i t i o n s t a t e b i n d i n g w i l l t h e r e f o r e r e s u l t i n lower t u r n o v e r rates. On t h i s b a s i s a l o n e , t h e r e f o r e , the deoxy s u b s t r a t e s s h o u l d be the s l o w e s t , s i n c e no hydrogen bonding i s p o s s i b l e to the hydrogen substituent. The d e o x y f l u o r o s u b s t r a t e s may be as poor as the deoxy, b u t , on a v e r a g e , s h o u l d be s l i g h t l y b e t t e r due to t h e i r c a p a c i t y f o r a c c e p t i n g hydrogen bonds. These two f a c t o r s , b i n d i n g , and i n t r i n s i c e l e c t r o n i c e f f e c t s can be e x p e c t e d , t h e r e f o r e , to combine and to a f f e c t r a t e s of t u r n o v e r . U n f o r t u n a t e l y , however, i t i s not a s i m p l e t a s k to d i s s e c t t h e s e two e f f e c t s w i t h any p r e c i s i o n . Even though the i n t r i n s i c e l e c t r o n i c e f f e c t s a r e known f o r a model r e a c t i o n , the a c i d - c a t a l y s e d h y d r o l y s i s of the same compounds, t h e s e cannot be a p p l i e d d i r e c t l y to the e n z y m a t i c r e a c t i o n s i n c e the a b s o l u t e magnitude of such e l e c t r o n i c e f f e c t s depends upon the p r e c i s e t r a n s i t i o n s t a t e s t r u c t u r e . It i s v e r y u n l i k e l y t h a t these t r a n s i t i o n s t a t e s t r u c t u r e s f o r the enzymec a t a l y z e d and a c i d - c a t a l y z e d r e a c t i o n s w i l l be i d e n t i c a l . T a b l e V g i v e s the M i c h a e l i s - M e n t e n p a r a m e t e r s , V and Km, measured f o r t u r n o v e r of t h e s e s u b s t r a t e analogues by r a b b i t muscle phosphorylase b. V x/* used i n t h i s a n a l y s i s s i n c e they r e p r e s e n t the s e c o n d o r d e r r a t e f o r r e a c t i o n of f r e e enzyme and f r e e s u b s t r a t e which can be r e l a t e d to o v e r a l l a c t i v a t i o n f r e e e n e r g i e s . m

m

v

a

l

u

e

s

a

r

a

x

e

Ja3i

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Table V .

Kinetic

Parameters f o r G l y c o g e n P h o s p h o r y l a s e b

MUSCLE PHOSPHORYLASE b COMPOUND Vm/Km x 10"

AAG?

4

G1P

187500

-

2-FLUORO G1P

1.08

7.3

3-FLUORO G1P

2.2

6.8

4-FLUORO G1P

37

5.1

6-FLUORO G1P

0.26

8.1

2-DEOXY G1P

n.d.

n.d.

3-DEOXY G1P

5.8

6.3

4-DEOXY G1P

400

3.7

6-DEOXY G1P

11.1

5.9

AAG^ r e p r e s e n t s the d i f f e r e n c e i n a c t i v a t i o n f r e e e n e r g i e s of c a t a l y s e d r e a c t i o n of g l u c o s e - l - p h o s p h a t e and the r e s p e c t i v e analogue. T h i s i s c a l c u l a t e d from E q u a t i o n 3 where (y^x^^i v a l u e f o r g l u c o s e - l - p h o s p h a t e and ( / )2 analogue. V

K m

i

s

t

n

a

t

f

o

r

t

n

e

m a x

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enzymeIs

the

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C l e a r l y they a r e a l l v e r y slow s u b s t r a t e s which r e a c t at r a t e s some 10 t o 10 times slower than the normal s u b s t r a t e . D e s p i t e the a f o r e m e n t i o n e d d i f f i c u l t i e s i n d i s s e c t i n g the e l e c t r o n i c and b i n d i n g f a c t o r s s e v e r a l i m p o r t a n t q u e s t i o n s can be a d d r e s s e d as f o l l o w s : (a) does e n z y m a t i c c a t a l y s i s p r o c e e d v i a an oxocarbonium i o n t r a n s i t i o n state? I t was proposed e a r l i e r t h a t , on the b a s i s of b i n d i n g f a c t o r s o n l y , the deoxy sugars s h o u l d g e n e r a l l y be worse s u b s t r a t e s than the d e o x y f l u o r o s u g a r s . However, the c o n v e r s e i s c l e a r l y seen to be t r u e i n comparing V / K m values. T h i s can o n l y be e x p l a i n e d i f the i n t r i n s i c e l e c t r o n i c e f f e c t s a r e i m p o r t a n t and the t r a n s i t i o n s t a t e has s u b s t a n t i a l oxonium i o n c h a r a c t e r . These d a t a t h e r e f o r e p r o v i d e s u b s t a n t i a l s u p p o r t f o r the mechanism proposed, (b) How s t r o n g a r e the hydrogen bonds at the t r a n s i t i o n s t a t e ? A minimum e s t i m a t e of such hydrogen bond s t r e n g t h s can be o b t a i n e d by c o n s i d e r i n g V ^ ^ K m v a l u e s f o r the d e o x y - s u g a r s e r i e s . Since i n t r i n s i c e l e c t r o n i effect w i l l tend t d thei t u r n o v e r , then any r a t e p o o r e r b i n d i n g at: the t r a n s i t i o f r e e e n e r g y , AAG* can be r e a d i l y c a l c u l a t e d from E q u a t i o n 3. These v a l u e s are l i s t e d i n T a b l e V and a r e minimum e s t i m a t e s of the s t r e n g t h s of hydrogen bonds to the m a x

M G * = RT i n W * » > 2 (

(3)

< W * « > i c o r r e s p o n d i n g h y d r o x y l a t the t r a n s i t i o n s t a t e . The v a l u e s o b t a i n e d a r e c o n s i d e r a b l y l a r g e r than those measured i n the study of g l u c o s e b i n d i n g to the T - s t a t e enzyme. This is consistent with expectations r e g a r d i n g i n c r e a s e d hydrogen bond s t r e n g t h s at the t r a n s i t i o n s t a t e . F u r t h e r , i t i s c l e a r t h a t the hydrogen bonds at the 3- and 6p o s i t i o n s a r e the s t r o n g e s t , as was observed f o r g l u c o s e b i n d i n g to T - s t a t e enzymes. T h i s s u g g e s t s a common g l u c o s e s u b - s i t e w h i c h remains i n t a c t d u r i n g the T*R t r a n s i t i o n . A Mechanism-Based I n h i b i t o r f o r a g - G l u c o s i d a s e . Glucosidasec a t a l y s e d h y d r o l y s e s are g e n e r a l l y c o n s i d e r e d to proceed v i a a "lysozyme type" of mechanism s i m i l a r to t h a t d i s c u s s e d p r e v i o u s l y f o r g l y c o g e n p h o s p h o r y l a s e and i l l u s t r a t e d i n Scheme 2 f o r a " r e t a i n i n g " glucosidase. Thus g l u c o s i d e h y d r o l y s i s proceeds through some form of g l u c o s y l - e n z y m e i n t e r m e d i a t e and v i a a t r a n s i t i o n s t a t e w i t h s u b s t a n t i a l oxocarbonium i o n c h a r a c t e r . On the b a s i s of our p r e v i o u s s t u d i e s we reasoned t h a t s u b s t i t u t i o n of the 2 - h y d r o x y l by an e l e c t r o n e g a t i v e f l u o r i n e s h o u l d d e s t a b i l i s e the t r a n s i t i o n s t a t e s f o r g l u c o s y l a t i o n (k^) and d e g l u c o s y l a t i o n (k2) of the enzyme, thus s l o w i n g down both these s t e p s . However, f u r t h e r i n c o r p o r a t i o n of a r e a c t i v e l e a v i n g group as the a g l y c o n e i n t o such d e a c t i v a t e d s u b s t r a t e s might i n c r e a s e the g l u c o s y l a t i o n r a t e s u f f i c i e n t l y ( w i t h o u t a f f e c t i n g the d e g l u c o s y l a t i o n r a t e ) to p e r m i t t r a p p i n g o f the 2 - d e o x y - 2 - f l u o r o - D - g l u c o s y l enzyme i n t e r m e d i a t e . I f the d e g l u c o s y l a t i o n r a t e i s s u f f i c i e n t l y slow t h i s might p e r m i t i s o l a t i o n of an i n a c t i v a t e d enzyme. Thus, 2 , 4 - d i n i t r o p h e n y l 2-deoxy-2-fluoro-0-D-glucopyranoside s y n t h e s i z e d by d e p r o t e c t i o n of i t s t r i - O - a c e t a t e , which had been

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Scheme 2. G e n e r a l mechanism f o r 3 - g l u c o s i d a s e s r e a c t i o n w i t h r e t e n t i o n of c o n f i g u r a t i o n .

catalysing

p r e p a r e d by t r e a t m e n t of 2 , 3 , 6 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 - f l u o r o - D g l u c o p y r a n o s e w i t h f l u o r o d i n i t r o b e n z e n e , i n the presence of base (l,4-diazabicyclo[2.2.2]octane) (47). I n c u b a t i o n of A l c a l i g e n e s f a e c a l i s p - g l u c o s i d a s e (48) w i t h t h i s compound r e s u l t e d i n a r a p i d , t i m e - d e p e n d e n t l o s s of enzyme a c t i v i t y . T h i s f o l l o w e d pseudof i r s t - o r d e r k i n e t i c s as shown i n F i g u r e 2, p e r m i t t i n g e s t i m a t i o n of a d i s s o c a t i o n c o n s t a n t (K^) of 0.05 mM and an i n a c t i v a t i o n r a t e c o n s t a n t (k^) of 25 minute . The c o m p e t i t i v e i n h i b i t o r i s o p r o p y l t h i o 3 - D - g l u c o p y r a n o s i d e p r o v i d e d p r o t e c t i o n a g a i n s t i n a c t i v a t i o n as shown i n F i g u r e 2, p r o v i d i n g e v i d e n c e t h a t i n a c t i v a t i o n i s due to r e a c t i o n at the a c t i v e s i t e .

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F i g u r e 2. I n a c t i v a t i o n of A . f a e c a l i s g - g l u c o s i d a s e w i t h 2 , 4 dinitrophenyl 2-deoxy-2-fluoro 3-D-glucopyranoside. a) I n a c t i v a t i o n at the f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s ( O 0.5 uM; • = 1.0 uH; • = 2.0uM; • = 3.0 uM; A = 4 . 0 uM; = 5.0 uM). b) P r o t e c t i o n a g a i n s t i n a c t i v a t i o n g i v e n by i s o p r o p y l 3-D-thioglucopyranoside. VQ i s the i n i t i a l enzyme a c t i v i t y , V i s the o b s e r v e d enzyme a c t i v i t y at the times i n d i c a t e d . Enzyme a c t i v i t y was measured by s p e c t r o p h o t o m e t r y a s s a y of p - n i t r o p h e n o l r e l e a s e from p - n i t r o p h e n y l - p - D - g l u c o p y r a n o s i d e i n 50 mM sodium phosphate b u f f e r , pH 6 . 8 . A

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T h i s t h e r e f o r e r e p r e s e n t s a n o v e l type of mechanism-based i n h i b i t o r of g l y c o s i d a s e s which s h o u l d be f a i r l y g e n e r a l l y a p p l i c a b l e to t h i s c l a s s of enzymes, p r o v i d e d t h a t the d e g l y c o s y l a t i o n r a t e f o r the analogue i s s u f f i c i e n t l y slow r e l a t i v e to the g l y c o s y l a t i o n r a t e . E x p e r i m e n t s are c u r r e n t l y planned to i d e n t i f y the a c t i v e s i t e n u c l e o p h i l e i n t h i s enzyme u s i n g such an i n h i b i t o r and to c o n f i r m i t s mode of a c t i o n by p u r i f y i n g the i n a c t i v e d e o x y f l u o r o g l u c o s y l enzyme and s t u d y i n g i t by F NMR. The g e n e r a l a p p l i c a b i l i t y of t h i s a p p r o a c h i s a l s o b e i n g t e s t e d w i t h a v a r i e t y of g l y c o s i d a s e s by use of the a p p r o p r i a t e 2 - d e o x y - 2 - f l u o r o - g l y c o s i d e i n each c a s e . 1

9

Conclusions S u i t a b l e a p p l i c a t i o n of deoxy- and d e o x y f l u o r o - analogues of e n z y m a t i c s u b s t r a t e s has p r o v i d e d v a l u a b l e i n s i g h t i n t o the mechanisms of e n z y m e - c a t a l y s e d g l u c o s y l t r a n s f e r and i n t o the r o l e of hydrogen bonding i n s u b s t r a t p e r m i t t e d d e s i g n of a n o v e inhibitors. A n o t h e r a p p e a l i n g a p p l i c a t i o n of such compounds i s t h e i r use i n F nmr s t u d i e s of e n z y m e - s u b s t r a t e complexes. We have r e c e n t l y i n i t i a t e d such s t u d i e s on the enzyme phosphoglucomutase. Acknowledgments T h i s work was s u p p o r t e d by g r a n t s from the N a t u r a l S c i e n c e s and E n g i n e e r i n g R e s e a r c h C o u n c i l of Canada and from the B r i t i s h Columbia H e a l t h Care R e s e a r c h F o u n d a t i o n .

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Walsh, C. Adv. Enzymol. 1983, 55, 197. Quicho, F.A.; Vyas, N.K. Nature 1984, 310, 381. Barnett, J.E.G. In Ciba Fdn. Symp. Elsevier & Associated Scientific Press, Amsterdam, 1972; p95. Sheppard, W.A.; Shantz, C.M. In Organic Fluorine Chemistry; W.A. Benjamin: New York, 1964; p 40. Curtiss, L.A.; Frurip, D.J.; Blander, M. J . Am. Chem. Soc. 1978, 100, 79. Murray-Rust, P.; Stallings, W.G.; Monti, C.T.; Preston, R.K.; Glusker, J.P. J . Am. Chem. Soc. 1983, 105, 3206. Karipides, A.; Miller, C. J . Am. Chem Soc. 1984, 106, 1494. Withers, S.G.; Street, I.P.; Rettig, S.J. Can. J. Chem. 1986, 64, 232. Klotz, I.M.; Franzen, J.S. J. Am. Chem. Soc. 1962, 84, 3461. Jencks, W.P. In Catalysis in Chemistry and Enzymology, McGraw Hill: New York, 1969. Fersht, A.R. Trends Biochem. Sci. (pers. Ed.) 1984, 9, 145. Hines, J . J . Am. Chem. Soc. 1972, 94, 5766. Fersht, A.R.; Shi, J.P.; Knill-Jones, J.; Lowe, D.M.; Wilkinson, A.J.; Blow, D.M.; Brick, P.; Cortes, P.; Waye, M.M.Y.; Winter, G. Nature 1985, 314, 235. Hehre, E . J . ; Brewer, C.F.; Genghof, D.S. J. Biol. Chem. 1979, 254, 5942. Kasumi, T.; Tsumuraya, Y.; Brewer, C.F.; Kersters-Hilderson, H.; Claeyssens, M.; Hehre, E.J. Biochemistry 1987, 26, 3010.

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WITHERS ET AL.

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.

Enzyme Specificity and Mechanism

11

Treder, W.; Thiem, J.; Schlingmann, M. Tetrahedron Lett. 1986, 27, 5605. Barnett, J.E.G. Biochem. J. 1971, 123, 607. Palm, D.; Blumenauer, G.; Klein, H.W.; Blanc-Muesser, M. Biochem. Biophys. Res. Commun. 1983, 111, 530. Bessell, E.M.; Foster, A.B.; Westwood, J.H. Biochem. J. 1972, 128, 199. Thomas, P.; Bessell, E.M.; Westwood, J.H. Biochem J. 1974, 139, 661. Bessell, E.M.; Thomas, P. Biochem. J. 1973, 131, 77. Drueckhammer, D.G.; Wong, C.-H. J . Org. Chem. 1985, 50, 5913. Bessell, E.M.; Thomas, P. Biochem. J. 1973, 131, 83. Maradufu, A.; Perlin, A.S. Carbohydr. Res. 1974, 32, 93. Taylor, N.F.; Romaschin, A.; Smith, D. In Biochemistry Involving Carbon-Fluorine Bonds, ACS Symposium Series 1976, 28, 99. Wright, J.A.; Taylor Soc., Chem. Commun Taylor, N.F.; Hill, L . ; Eisenthal, R. Can. J. Biochem. 1975, 53, 57. Squire, S.; Taylor, N.F. Proc. Can. Fed. Biol. Soc. Abstr. 1985, 28, 226. Penglis, A. Adv. Carb. Chem. Biochem. 1981, 38, 195. Street, I.P.; Armstrong, C.R.; Withers, S.G. Biochemistry 1986, 15, 6021. Sprang, S.R.; Fletterick, R.J. J. Mol. Biol. 1979, 131, 523. Sprang, S.R.; Goldsmith, E . J . ; Fletterick, R.J.; Withers, S.G.; Madsen, N.B. Biochemistry 1982, 21, 5364. Bunton, C.A.; Llewellyn, D.R.; Oldham, K.G.; Vernon, CA. J . Chem. Soc. C 1958, 3574. Bunton, C.A.; Humeres, E. J. Org. Chem. 1969, 34, 572. O'Connor, J.V.; Barker, R. Carbohydr. Res. 1979, 73, 227. Capon, B. Chem. Rev. 1969, 69, 407. BeMiller, J.N. Adv. Carbohydr. Chem. 1967, 22, 25. Sinnott, M.L. In The Chemistry of Enzyme Action, Elsevier: New York, 1984; p 389. Withers, S.G.; MacLennan, D.J.; Street, I.P. Carbohydr. Res. 1986, 154, 127. Overend, W.G.; Rees, C.W.; Sequeira, J.S. J. Chem. Soc. 1962, 3429. Mega, T.; Matsushima, Y. J. Biochem. (Tokyo) 1983, 94, 1637. Jencks, W.P. Chem. Soc. Rev. 1981, 10, 345. Lambert, J.B.; Larson, E.G. J. Am. Chem. Soc. 1985, 107, 7546. Pauling, L. Chem. Eng. News 1946, 24, 1375. Fersht, A.R. In Enzyme Structure and Mechanism (2nd Edn.) W.H. Freeman 1985. Fersht, A.R.; Leatherbarrow, R.J.; Wells, T.N.C. Trends Biochem. Sci. (Pers. Ed.) 1986, 9, 145. Withers, S.G.; Street, I.P.; Bird, P.; Dolphin, D.H. J. Am. Chem. Soc. 1987, 109, 7530. Day, A.G.; Withers, S.G. Biochem. Cell Biol. 1986, 64, 914.

RECEIVED March 14, 1988

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 6

Deoxyfluoro Carbohydrates as Probes of Binding Sites of Monoclonal Antisaccharide Antibodies Cornells P. J. Glaudemans and Pavol Kováč National Institutes of Health, Bethesda, MD 20892 The mode of bindin to a group o monoclona antibodies is examined. The combining area in these antibodies has four subsites, each capable of binding to a single galactosyl residue in a sequence of four in the antigen. One subsite has the highest affinity for its residue amongst the four, and that subsite binds through H-bonding to the hydroxyl groups at positions 2 and 3 of the galactosyl residue i t associates with. Thus, by using galacto oligosaccharides bearing a 3-deoxyfluoro group at defined positions, and thereby prohibiting the binding of that (substituted) galactosyl residue to the subsite with the highest affinity, the order of the affinities of the four subsites could be measured. Correlation of the binding data with the amino acid sequences of the antibodies studied has led to a proposal for the arrangement of the bound antigen on the surface of the immunoglobulins. The synthetic routes to a variety of the ligands used is discussed in detail. Immunoglobulins - or antibodies - are a class of proteins secreted by plasma cells. The clonal selection theory, now generally accepted, proposes that precommitted B-lymphocytes (whose progeny is the plasma cell) have the specificity of the immunoglobulin they produce, and which occurs on their c e l l surface in small amounts, encoded in their genome (1). An immunogen, upon entering the body selects lymphocytes that carry immunoglobulin of the appropriate specificity and then combines with that immunoglobulin, thus stimulating the proliferation of these cells. By a process not entirely understood, B-lymphocytes, now stimulated by immunogen-contact, differentiate through a lymphoblast into a plasma cell which produces large amounts of immunoglobulin (2). The combination of immunogen with surface immunoglobulin appears to be the driving force of this This chapter not subject to U.S. copyright Published 1988 American Chemical Society

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d i f f e r e n t i a t i o n , s i n c e B-lymphocytes can a l s o be t r i g g e r e d to differentiate by c r o s s linking their surface proteins with pokeweed mitogen ( 3 ) . S i n c e even a simple antigen u s u a l l y has many s t r u c t u r a l d e t e r m i n a n t s , it can combine w i t h the s u r f a c e immunoglobulins o f a l a r g e number o f d i f f e r e n t immunocompetent cells. A l t h o u g h each of t h e s e c e l l s produces a s i n g l e , u n i q u e immunoglobulin, the o v e r a l l response would thus still yield a heterogeneous pool of immunoglobulins. It is t r u e t h a t some immune r e s p o n s e s e x p r e s s t h e m s e l v e s m o n o c l o n a l l y ( o r nearly so), and t h u s produce a s i n g l e molecular species of antibody ( 4 , 5 ) , but t h e predominant s o u r c e of monoclonal a n t i b o d i e s i s through immunoglobulin-producing neoplasms, i.e. proliferative plasma cells or plasma cytomas (6-8). Originally, spontaneous, or randomly induced plasma cytomas were t h e major s o u r c e of m o n o c l o n a l a n t i b o d i e s . T h u s , t h e m o n o c l o n a l immunoglobulins ( 6 , 7 ) were s c r e e n e d for thei specificitie (9-12) l e d d i r e c t l y t o an e a r l response, as well a a n t i g e n - a n t i b o d y i n t e r a c t i o n . In 1965 S a c h , H o r i b a t a , Lennox and Cohn began in vitro c u l t u r i n g of murine plasma cytomas (see r e f e r e n c e 12 f o r a d e s c r i p t i o n o f t h e s e e v e n t s ) and t h i s led to the discovery by K o h l e r and M i l s t e i n (13) t h a t plasma cytomas can be f u s e d with normal plasma cells to yield a hybridoma p r o d u c i n g m o n o c l o n a l a n t i b o d y o f p r e d e t e r m i n e d s p e c i f i c i t y . These hybridomas a r e p a t h o l o g i c a l l y p r o l i f e r a t i v e , are transplantable, and form t h e b a s i s f o r o b t a i n i n g l a r g e amounts o f immunoglobulins of d e s i r e d s p e c i f i c i t y . As t h e number o f m o n o c l o n a l a n t i b o d i e s increased (12,14,15) they were used f o r a v a r i e t y of s t u d i e s i n c l u d i n g t h e e l u c i d a t i o n o f the g e n e t i c s o f t h e immune r e s p o n s e . This a r t i c l e w i l l b r i n g up t o d a t e c e r t a i n a s p e c t s o f a p r e v i o u s r e v i e w on t h i s s u b j e c t (16) as w e l l as p r e s e n t a new method t o elucidate the location of binding s i t e s and s u b - s i t e s in monoclonal a n t i b o d i e s . GENERAL FEATURES OF IMMUNOGLOBULINS S e v e r a l immunoglobulin fragments have been i n v e s t i g a t e d by X - r a y diffraction of their crystals (17-22). These studies have r e v e a l e d t h a t c o r r e s p o n d i n g immunoglobulin domains g e n e r a l l y have very s i m i l a r s t r u c t u r e s . An e x c e l l e n t r e v i e w has been p u b l i s h e d (22) . In summary: A n t i b o d i e s a r e made up o f two i d e n t i c a l heavy (H) chains of molecular weight ca. 50,000 D a l t o n s , and two i d e n t i c a l l i g h t ( L ) c h a i n s o f m o l e c u l a r weight c a . 25,000 D a l t o n s (23) . These four chains form a number o f g l o b u l a r domains, each domain c o n t a i n i n g a p p r o x i m a t e l y 100 amino a c i d s . The N - t e r m i n a l domains o f o f each H , L p a i r t o g e t h e r h a r b o r one combining s i t e a t t h e s o l v e n t exposed end o f t h e molecule. Thus, each f o u r c h a i n unit H 2 L 2 p o s e s s e s two combining s i t e s , and t h e y a r e i d e n t i c a l . The N - t e r m i n a l domains o f both the H and the L c h a i n v a r y from immunoglobulin to immunoglobulin, while for a given c l a s s of immunoglobulins the remainder of the H and L c h a i n s are i d e n t i c a l . The v a r i a b i l i t y o f t h e f i r s t N - t e r m i n a l domain o f b o t h t h e H and L c h a i n e x p r e s s e s i t s e l f as a unique s p e c i f i c i t y , and this v a r i a b i l i t y resides particularly in c e r t a i n areas w i t h i n t h e domain c a l l e d h y p e r v a r i a b l e r e g i o n s ( 2 4 ) . These a r e loops of

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS about h a l f a dozen o r so amino a c i d r e s i d u e s , and t h e r e a r e t h r e e such regions in both the H and t h e L chain. When the immunoglobulin ct-carbon backbone is observed i t i s noted that t h e s e h y p e r v a r i a b l e (hv) l o o p s , although separated i n sequence, occur near one a n o t h e r i n space, and can t o g e t h e r make up t h e combining site (24). Diversity of immunoglobulins a r i s e s by s e v e r a l ways (25), mostly a t the g e n e t i c l e v e l ( 2 6 - 2 8 ) , and i t would be i n a p p r o p r i a t e t o discuss this here. Suffice i t t o say that enormous diversity can be generated, but it must be remembered t h a t v a r i a t i o n s i n amino a c i d sequence do not always l e a d t o changes i n b i n d i n g s p e c i f i c i t y . Cohn o r i g i n a l l y d e s c r i b e d a myeloma immunoglobulin A (9) which p r e c i p i t a t e d w i t h the species-specific cell wall-teichoic acid occuring in Streptococcus Pneumoniae. The haptenic d e t e r m i n a n t t u r n e d out t o be p h o s p h o r y l c h o l i n e a n d , since then, innumerable myeloma and hybridom monoclonal a n t i b o d i e with this specificity hav p h o s p h o r y l c h o l i n e IgA whose s t r u c t u r e has been solved by X - r a y d i f f r a c t i o n studies (17,19,22,32,33). Thus, detailed knowledge of the threed i m e n s i o n a l s t r u c t u r e o f i t s combining s i t e , a n d t h e i n t e r a c t i o n of the s i t e with the l i g a n d , has become the f o c a l p o i n t o f our knowledge about t h e s u b j e c t . In o b s e r v i n g the c o m b i n i n g s i t e o f Mc603 it can be seen that the cavity is defined by the surrounding surfaces formed by the h y p e r v a r i a b l e (hv) l o o p s on the face of the I g A . The f i r s t hv r e g i o n o f t h e L c h a i n ( t w ^ - l ) i s u n u s u a l l y l o n g , and p r e v e n t s hv^-2 from r e a c h i n g the c a v i t y . In a d d i t i o n , b o t h hvjj-2 and hvjj-3 a r e l o n g e r t h a n u s u a l , and t h e o v e r a l l e f f e c t i s the c r e a t i o n of a deep cavity. This i s not a general feature for immunoglobulins, and i n the a n t i - g a l a c t a n monoclonal antibodies these hv r e g i o n s are s h o r t e r , thereby c r e a t i n g a shallower combining area (33). ANTI-GALACTAN MONOCLONAL ANTIBODIES One o f the l a r g e s t groups o f m o n o c l o n a l murine immunoglobulins w i t h a n t i c a r b o h y d r a t e s p e c i f i c i t y i s t h e group o f a n t i - g a l a c t a n s . In addition to the myeloma immunoglobulins (14,16), a large number o f anti-galactan hybridoma immunoglobulins have been e l i c i t e d (34). One o f t h e major a r e a s t h a t our l a b o r a t o r y has worked on i n t h e p a s t y e a r s a r e t h e s e m o n o c l o n a l immunoglobulins with specificity for P( l - > 6 ) - D - g a l a c t o p y r a n a n (7,35-37). C o n s i d e r a b l e p r o g r e s s has been made i n t h e e l u c i d a t i o n o f d e t a i l s of the interaction of that antigen w i t h a n t i b o d y , but t h e s e d e t a i l s a r e f a r from complete. T h i s i s p a r t l y due t o the f a c t that although X-ray diffraction studies of one of these a n t i b o d i e s (J539 F a b ) has been r e p o r t e d (33) no c r y s t a l s from these antibodies have been obtained with the antigenic determinant occupying the complementarity determining region ( 3 8 ) . We have shown t h a t one o f t h e members o f t h i s g r o u p , IgA J539, binds along the length of the c h a i n ( i n t e r c a t e n a r i l y ) to i t s l i n e a r (i( l - > 6 ) - D - g a l a c t o p y r a n a n a n t i g e n ( 3 9 ) , and t h e a n t i b o d y makes c o n t a c t w i t h f o u r sequential galactosyl residues (40,41). The a r e a s of importance t o b i n d i n g i n t h e antigenic determinant have been mapped f o r two immunoglobulins i n t h i s group (J539 and 1

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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X24), and i t has been shown t h a t t h e a n t i b o d y ' s h i g h e s t b i n d i n g subsite interacts with the C1-C2-C3 face of the g a l a c t o s y l residue it binds (40-42). This laboratory proposed t h a t the 3 ( l - » 6 ) - D - l i n k e d g a l a c t o b i o s e l i g a n d , which b i n d s t o the a n t i b o d y , i s i n a c o n f o r m a t i o n h a v i n g i t s r i n g oxygens on o p p o s i t e s i d e s o f the d i s a c c h a r i d e . In that conformation the g a l a c t o b i o s e would bind with o n l y one s i d e to the a n t i b o d y ( 4 1 , see F i g . 1). In addition, derivatives of methyl 3-D-galactopyranoside carrying

Fig. I. The postulated conformation of 6-0-/?-D-galactopyranosyl-D-galactose . (The heavy bonds indicate the area of strongest binding to the immunoglobulin A J539 (Fab').]

bulky s u b s t i t u e n t s on C6 bound as d i d the u n s u b s t i t u t e d s u g a r , i n d i c a t i n g t h a t the h i g h e s t binding s u b s i t e was not s p a c i a l l y r e s t r i c t e d , and p r o b a b l y was on the s u r f a c e o f the p r o t e i n ( 4 2 ) . The amino a c i d sequences o f the v a r i a b l e p a r t o f b o t h the H and the L c h a i n o f immunoglobulins o f t h i s group have been r e p o r t e d ( 2 8 , 3 4 , 4 3 ) . In addition, it has now been shown t h a t t h i s e n t i r e group o f a n t i b o d i e s i s d e r i v e d by s o m a t i c m u t a t i o n o p e r a t i n g on two genes V y G a l 39.1 and VjjGal 55.1 ( 4 4 ) . D a t a o b t a i n e d from a f f i n i t y l a b e l i n g may n o t always be u s e f u l , s i n c e the a r e a around the s i t e of protein-attachment would be e q u a l i n r a d i u s t o the l e n g t h o f t h e l i g a n d , i t would q u i c k l y exceed t h e known a r e a - s i z e for the combining site of antibodies. We a r e nevertheless pursuing that approach (45-48), and t h e use of extremely reactive, indiscriminate (photo)affinity labels, such as diazirino glycosides of (3-galactose is showing great promise (48). Most o f our present insight results from our work on c h e m i c a l l y a l t e r e d l i g a n d probes (40-42) c u l m i n a t i n g i n t h e use of g a l a c t o s y l ( o l i g o ) s a c c h a r i d e s b e a r i n g f l u o r i n e i n the p l a c e o f hydroxyl groups at defined positions (49) to probe the p o s s i b i l i t y o f h y d r o g e n - b o n d i n g ( 5 0 ) . T h i s has l e d t o the mapping of s u b s i t e s i n the combining a r e a of a n t i g a l a c t a n IgA J539 (3537). Thus, if hydrogen-bonding i n d e e d t a k e s p l a c e , and i f t h e protein were t h e hydrogen donor i n bonding w i t h any oxygen atoms in the sugar molecule, replacement of the hydroxyl groups involved by f l u o r i n e could increase the affinity for the hydrogen-bonding due to the higher electronegativity o f the f l u o r i n e . I f , on the other hand, hydrogen bonding should occur t h r o u g h hydrogen donation by a sugar h y d r o x y l group t o the protein, synthetic carbohydrate-ligands bearing deoxyfluoro groups at the required position would p r o b a b l y show g r e a t l y d i m i n i s h e d b i n d i n g ( o r none a t a l l ) . S h o u l d hydrogen bonding n o t

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS be mediated by any o f t h e h y d r o x y l groups s u b s t i t u t e d by f l u o r i n e in the D-galactoside, the affinities would p r o b a b l y n o t be greatly affected. I t had been mentioned above t h a t we had shown t h a t t h e a n t i b o d y combining a r e a can accommodate f o u r s e q u e n t i a l galactopyranosyl residues, and t h a t the antibody binds to interchain 3( l - > 6 ) - l i n k e d tetrasaccharide segments of galactopyranans (39). It is to be expected that these four a n t i b o d y s u b s i t e s each have their own, differing a f f i n i t y for "their" galactosyl residue, since the protein in general including the combining area has an a n i s o t r o p i c nature. Examination of the data presented i n Table I permits an assignment of the relative strength of interaction o f each antibody subsite w i t h i t s g a l a c t o s y l r e s i d u e , and a l s o p e r m i t s a c o n c l u s i o n about the d i r e c t i o n of the p o l y s a c c h a r i d e c h a i n when i t is bound t o t h e a n t i b o d y combining a r e a . C o n s i d e r i n g o n l y t h e spacial exclusion f o r each atom e proposed a c o n f o r m a t i o n f o r 3(l-»6)-D-galactooligosaccharide ring oxygen of eac s i d e s of the p o l y s a c c h a r i d e c h a i n (41). Since then we have made e n e r g y - m i n i m i z a t i o n (HSEA, 51) c a l c u l a t i o n s (we a r e g r a t e f u l t o D r . K l a u s Bock f o r a s s i s t i n g u s ) f o r the t e t r a s a c c h a r i d e O-fS-Dg a l a c t o p y r a n o s y l - ( 1^6 ) - O - 3 - D - g a l a c t o p y r a n o s y l - ( l - > 6 )-O-p-Dgalactopyranosyl-(l-»6)-3-D-galactopyranose (see Fig. 2). This h y p o t h e t i c a l c o n f o r m a t i o n , which may o r may n o t be t h e one t h e

Figure 2. Intersaccharidic bond angles f o r p-(1-*6)-linked p-D-galactopyranotetraose as calculated by the IISEA method (51).

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

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Monoclonal Antisaccharide Antibodies

o l i g o s a c c h a r i d e assumes when actually bound t o the a n t i b o d y , i s used i n t h i s c h a p t e r t o i l l u s t r a t e the b i n d i n g mode o f s a c c h a r i d e l i g a n d s t o t h e m o n o c l o n a l a n t i b o d i e s i n our f o l l o w i n g d i s c u s s i o n . A tetrasaccharide segment in this c o n f o r m a t i o n has a l i n e a r d i m e n s i o n o f c a . 17 A. The s u b s i t e w i t h t h e h i g h e s t a f f i n i t y for a galactosyl residue is likely to be n e a r a s o l v e n t exposed t r y p t o p h a n y l r e s i d u e , s i n c e even t h e s i m p l e s t l i g a n d m e t h y l (3D-galactopyranoside (5 i n Table I) shows ligand-induced antibody tryptophanyl fluorescence change (41; for a detailed description of using l i g a n d - i n d u c e d changes of the antibody t r y p t o p h a n y l f l u o r e s c e n c e t o measure a f f i n i t y c o n s t a n t s , see r e f . 5 2 ) . On the b a s i s o f an e x a m i n a t i o n o f t h e c o o r d i n a t e s o f IgA J539 Fab obtained by X - r a y diffraction studies at 2.7 A resolution (33), it can be seen that the two t r y p t o p h a n y l r e s i d u e s i n the complementarity determining regions (CDRs, see r e f . 38) o f IgA J 5 3 9 namely t r y p t o p h a n (TRP) 33H and TRP 91L ( 2 8 , 3 8 , 5 3 ) appear s o l v e n exposed) and a r e at immunoglobulin combining a r e a . These two TRP residues are separated by c a . 9-10 A. The o n l y o t h e r t r y p t o p h a n y l r e s i d u e s near t h e general combining a r e a , TRP 36H and TRP 3 5 L , are not i n t h e CDR and a r e b u r i e d where they would not be e x p e c t e d t o be perturbable by l i g a n d s bound on t h e p r o t e i n s u r f a c e . The f i r s t antibody subsite t o a t t r a c t a l l o r p a r t o f t h e l i g a n d appears t o be near TRP 33H, since a heterologous H / L - c h a i n recombinant immunoglobulin we p r e p a r e d (54), derived from t h e two a n t i g a l a c t a n I g A ' s S10 and J 5 3 9 , showed fluorescence characteristics upon b i n d i n g m e t h y l 0 - D - g a l a c t o p y r a n o s i d e s i m i l a r t o t h o s e o f t h e H - c h a i n d o n o r - p r o t e i n . B o t h immunoglobulins S10 and J539 have t h e invariant TRP 33H r e s i d u e (53, J. Pumphry and S. R u d i k o f f , u n p u b l i s h e d r e s u l t s ) . We l a b e l that subsite as A and w i l l show evidence that - when f o u r s u b s i t e s a r e l a b e l e d D, B, A and C as shown i n F i g . 3-5 a c r o s s t h e c o m b i n i n g a r e a w i t h t h e H - c h a i n on the right the polysaccharide binds with i t s r e d u c i n g end p r o j e c t e d towards t h e L c h a i n and t h a t t h e order of a f f i n i t i e s for the subsites is A > B > C > D. Two modes o f b i n d i n g f o r t h e p o l y s a c c h a r i d e c h a i n a r e a priori p o s s i b l e (as shown i n F i g u r e s 3 and 4). We have discussed above that the s i d e of the f i r s t g a l a c t o s y l r e s i d u e to bind (to s u b s i t e A) is the face of the p y r a n o s y l r i n g b e a r i n g t h e 2- and 3-0H groups ( 4 1 , 4 2 , 5 5 ) . The C-6 ( t h e h y d r o x y m e t h y l group) p r o j e c t s away from t h e p r o t e i n toward the solvent as i n d i c a t e d in Figures 3 - 5 . We have a l s o r e p o r t e d t h a t r e p l a c e m e n t o f e i t h e r t h e 2o r 3-0H group i n m e t h y l (3-Dgalactopyranoside by f l u o r i n e appears to lead to non-binding ( 4 9 , 3 5 , see T a b l e I ) , w h i c h i s highly indicative of there being r e q u i r e d H-bonding i n s u b s i t e A. It appears from t h e d a t a (see below) t h a t H - b o n d i n g o c c u r s o n l y i n s u b s i t e A , a t l e a s t f o r the 3-0H group o f any g a l a c t o s y l moiety i n an o l i g o s a c c h a r i d e . F l u o r i n e i t s e l f does not i n t e r f e r e due t o s i z e or charge per se when s u b s t i t u t i n g f o r an OH group ( 5 6 ) . T h u s , from t h e s e d a t a t h e p o l y s a c c h a r i d e can be p l a c e d i n the combining a r e a as shown i n either F i g . 3 o r F i g . 4. We w i l l show t h a t e v i d e n c e s u p p o r t s t h e b i n d i n g mode shown i n F i g . 3 and not that of F i g . 4. Table I lists the maximum changes in the protein tryptophanyl 1

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83

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS L

CHAIN D

TRP B

A TRP

H CHAIN

reducing end of the chain

3: Schematic representation of binding mode of a 0(1-* 6)-D-galactopyranan segment to IgA J539, with the reducing end of the polysaccharid of the IgA is calle the protein molecule, looking down along the face of the antibody combining area. The 3-OH groups of each galactosyl residue are boldface. Protein subsites are labeled C, A, B, and D.

FIGURE

L

F I G U R E 4: Schematic representation of the binding mode of a 0(l-*6)-r>galactopyranan segment to IgA J539, with the reducing end of the polysaccharide oriented toward the H chain. For other particulars, see legend to Figure 3. f l u o r e s c e n c e (52) i n d u c e d by a number o f our s y n t h e t i c l i g a n d s ( a d i s c u s s i o n of t h e i r p r e p a r a t i o n w i l l c o n s t i t u t e the l a s t p a r t of t h i s a r t i c l e ) . I t can be seen that for t h e m o n o s a c c h a r i d e s 3-6 this value i s c a . 20% ( o f t h e t o t a l t r y p t o p h a n y l f l u o r e s c e n c e o f the antibody). When t h e ligand increases its length to approximately twice the size, i.e. in going from the monosaccharide methyl p-D-galactopyranoside to 6-0-(}-Dgalactopyranosyl-D-galactose (Ga^), the value goes up s u b s t a n t i a l l y ( t o 33 %), and even more so for the m e t h y l 0g l y c o s i d e of G a l 2 (7, 36 %) o r f o r t h e c o r r e s p o n d i n g m e t h y l - G a l 3 (10, A3 %). G a l has a l e n g t h o f c a . 9 A. The X - r a y d i f f r a c t i o n studies of J539 (33) show t h e two TRP r e s i d u e s 33H and 91L t o be c a . 9 . 5 A a p a r t . T h e r e f o r e , t h e above d i s a c c h a r i d e G a l with i t s n o n - r e d u c i n g r e s i d u e i n s u b s i t e A (and so i n t e r f a c i n g t h e p r o t e i n 2

2

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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GLAUDEMANS & K O V A C

85

MonoclonalAntisaccharideAntibodies L-chain

H-chain TUP A

•jjfB

CH3O^C H , O H ^ O H ^

C

cHjo

H

5

7

3 0 ^ P ^ o

vT tb°^-

C H j O - ^ ^ O - ^ -

10

8

CH 0 3

Fig. 5. Schematic representation of the binding of the galactosyl derivatives studied to subsites D, B, A and C . w i t h i t s 2- and 3-OH g r o u p s ) , would r e a c h towards TRP 91L w i t h the second galactose unit and p e r t u r b that tryptophanyl s f l u o r s c e n c e only i f i n t h e mode shown i n F i g . 3 and n o t as i n F i g . 4. In the l a t t e r mode t h e second g a l a c t o s e r e s i d u e would p r o j e c t away from t h e TRP a t 91L and would o r i e n t more towards 1

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TABLE I

L OING CONSTANTS (Ka) AND MAXIMAL FLUORESCENCE CHANGE lAFmax) FOR IgA J539 FAB' WITH GALACTOSE DERIVATIVES

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

GLAUDEMANS & KOVAC

Monoclonal Antisaccharide Antibodies

the H - c h a i n . T h u s , t h e i n c r e a s e d maximal f l u o r e s c e n c e o f t h e d i and higher 3(1*6)-linked galactooligosaccharides supports the b i n d i n g mode shown i n F i g . 3, and we c o n c l u d e t h a t t h e r e d u c i n g end o f the p o l y s a c c h a r i d e a n t i g e n p r o j e c t s towards t h e L chain. Turning to T a b l e I we can i n t e r p r e t t h e b i n d i n g d a t a as f o l l o w s : Methyl galactoside 5 binds at subsite A (see Fig. 5 for a schematic representation). The m e t h y l g a l a c t o b i o s i d e 7 shows a large increase in the maximally induced p r o t e i n f l u o r e s c e n c e , indicating that it is now p e r t u r b i n g b o t h solvent exposed tryptophanyl residues. We t h e r e f o r e place that bioside 7 in s u b s i t e s A and B ( r e a c h i n g towards t h e TRP a t 9 1 L ) . R e p l a c i n g t h e 3'-OH i n 7 w i t h f l u o r i n e to o b t a i n 9, b r i n g s about a 5-6 t i m e s reduction in binding a f f i n i t y (Table I ) . I t i s impossible to accomodate t h e t e r m i n a l r e s i d u e bearing the 3 -deoxy-3 -fluoro group o f 9 i n s u b s i t e A ( s u b s i t e A r e q u i r e s p r o t e i n c o n t a c t w i t h 2- and 3-0H g r o u p s ) . T h e r e f o r e t h e m e t h y l d e o x y - f l u o r o bioside 9 s h i f t s to s u b s i t e C an methyl g a l a c t o s i d e moiet to subsite A in order for 9 to u t i l i z e that h i g h e s t b i n d i n g s u b s i t e . As a r e s u l t , t h e 3 - d e o x y - 3 - f l u o r o g a l a c t o s y l group o f 9 shifts to s u b s i t e C (we w i l l p r e s e n t d a t a l a t e r t o show t h a t i n d e e d s u b s i t e C must be p l a c e d n e x t t o s u b s i t e A and not n e x t t o subsite B). If that is correct, the methyl deoxyfluoro galactobioside 9 - unlike the methyl galactobioside 7 - should cause a maximum f l u o r e s c e n c e change in the protein like a m o n o s a c c h a r i d e , namely c a . 20% ( i t does not come any n e a r e r t h e TRP 91L t h a n does 5, and thus o n l y perturbs the TRP 33H n e a r subsite A ) . Table I shows t h a t this is s o . The e v i d e n c e above s u g g e s t s an o r d e r o f a f f i n i t y f o r t h e s e t h r e e s u b s i t e s as A > B > C. To v e r i f y this we measured the affinity of the methyl g a l a c t o t r i o s i d e 10. I f the above i s c o r r e c t i t must f o l l o w t h a t t r i o s i d e 10 s h o u l d a s s o c i a t e w i t h s u b s i t e s A , B and C ( w i t h an i n c r e a s e d a f f i n i t y compared t o t h a t o f 7 ) , and t h a t t h e 3"-deoxy3 - f l u o r o d e r i v a t i v e o f 10, namely 11, s h o u l d b i n d w i t h t h e same affinity as 10. T h i s , because t h e t e r m i n a l 3 - d e o x y - 3 " - f l u o r o g a l a c t o s y l group o f 11 has i t s f l u o r i n e - b e a r i n g g a l a c t o s y l group bound t o s u b s i t e C without i n t e r f e r e n c e (see F i g . 5 ) . A l s o , i f t h e t r i s a c c h a r i d e s 10 and 11 b i n d i n t h e same s u b s i t e s A , B and C, they s h o u l d show t h e same l i g a n d - i n d u c e d maximal f l u o r e s c e n c e change. The v a l u e s i n T a b l e I show t h i s to be so. Next, the d e o x y - f l u o r o g a l a c t o b i o s i d e 8 was measured. Compound 9 was f o r c e d t o s h i f t t o s u b s i t e s A and C (see F i g . 5) i n order to optimize b i n d i n g t h e r e b y c h a n g i n g t h e maximal f l u o r e s c e n c e change from c a . 40% t o 20%. A l t e r n a t i v e l y , saccharide 8 should be capable of b i n d i n g t o s u b s i t e s A and B w i t h t h e C 2 - C 3 f a c e o f t h e t e r m i n a l g a l a c t o s y l group hydrogen-bonded i n s u b s i t e A o f t h e a n t i b o d y and t h e 3 - d e o x y - 3 - f l u o r o g a l a c t o s y l group l o c a t e d i n s u b s i t e B . Hence i t s K and A F s h o u l d be t h e same as t h a t f o r 7, and T a b l e I bears this out. Similarly, t r i s a c c h a r i d e s 12 and 13 s h o u l d be a b l e t o b i n d t o s u b s i t e s B A C , as do 10 and 11 a n d , a g a i n , T a b l e I shows t h e i r K s and maximal changes i n f l u o r e s c e n c e t o be e s s e n t i a l l y t h e same. I n o r d e r t o l o c a t e s u b s i t e D a t e i t h e r the L or t h e H c h a i n s i d e o f t h e B A C sequence t h e t r i s a c c h a r i d e 14 1

1

1

1

1 1

M

f

a

m

a

!

x

f

a

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

was p r e p a r e d ( 3 7 ) . L e t us a priori place s u b s i t e D near the L c h a i n . T h i s i s reasonable, s i n c e i f p l a c e d near the H c h a i n (next t o s u b s i t e C) i t would appear to p r o j e c t too f a r away from the general antibody combining a r e a . The a f f i n i t y of 14 for m o n o c l o n a l IgA J539 F a b was found t o be 4.2 x 10 L / M . Since s u b s i t e A r e q u i r e s i n t e r a c t i o n o f a g a l a c t o s y l 2- and 3-OH group w i t h t h e s u r f a c e o f t h e p r o t e i n , 14 w i l l be i n c a p a b l e of having its internal D-galactosyl residue engage t h a t s u b s i t e , and t h i s t r i s a c c h a r i d e can t h e r e f o r e n o t b i n d t o t h e B A C sequence (see F i g . 5 ) . Assuming t h e p o s i t i o n i n g o f s u b s i t e D t o be c o r r e c t , t h e t r i s a c c h a r i d e 14 c o u l d b i n d e i t h e r t o s u b s i t e A C o r t o A B D, as indicated in Fig. 5. Methyl 6 - 0 - (3* - d e o x y - 3 - f l u o r o - ( 3 - D g a l a c t o p y r a n o s y l ) - p - D - g a l a c t o p y r a n o s i d e (9) b i n d s t o s u b s i t e s A C with a K of 0.8 x 1 0 L / M . T h u s , 14, w i t h a K some f i v e t i m e s l a r g e r must i n v o l v e a d d i t i o n a l b i n d i n g c o n t a c t s , i . e . subsites A B D. T r i s a c c h a r i d e 14 would a l s o have t h e p o s s i b i l i t y t o b i n d t o three s u b s i t e s i f s u b s i t 1

4

1

4

a

a

C. In that case the A F around 20%, s i n c e i t would t h e n o n l y p e r t u r b TRP 33H. However, 14 shows a £s max ° ^ 40.6% and thus appears t o engage TRP 91L. Hence, s u b s i t e D must be on t h e L c h a i n s i d e o f B. T h i s a l s o confirms the D B A C arrangement o f s u b s i t e s , r a t h e r t h a n C B A D, which c o u l d have l i g a n d 9 binding to subsites C B i n that s e q u e n c e . Such a mode would lower t h e A F observed, which i s a s s o c i a t e d with the p o s s i b l e p e r t u r b a t i o n of one TRP r e s i d u e , here residue 91L. However, if that were the c a s e , l i g a n d 14 s h o u l d have t h e same K as does l i g a n d 10, and n o t some t e n t i m e s F

m

a

x

a

l e s s , as o b s e r v e d . (The f a c t t h a t l i g a n d 11 shows t h e same K and AF as l i g a n d 10 a l s o shows t h a t t h e s u b s i t e sequence D B A C is correct and n o t t h e sequence C B A D). The above o b s e r v a t i o n s on the l o c a t i o n o f s u b s i t e D a l s o agree w i t h t h e f i n d i n g t h a t t h e K found f o r 14 ( 4 . 2 x 1 0 L / M ) i s o n l y s l i g t h l y h i g h e r than t h e previously reported K for l i g a n d 8 (3.6 x 10 ). This reflects only a small additional b i n d i n g due t o s u b s i t e D. We have shown that the maximally b i n d i n g methyl fl-glycoside of p(l->6)-linked Dg a l a c t o t e t r a o s e (15) b i n d s (39) t o IgA J539 F a b with a K of 0.59 x 10" L / M , w h i l e t h e c o r r e s p o n d i n g t r i o s i d e 10 b i n d s with a K of 0.48 x 1 0 L / M , r e v e a l i n g the s m a l l a d d i t i o n a l b i n d i n g of the f o u r t h subsite. Lastly, note (Table I) that 4 shows a significant increase in b i n d i n g compared t o 5. T h i s does n o t appear t o be due t o H-bond a c c e p t a n c e by t h e 6 - p o s i t i o n , since compound 6, l a c k i n g the 6-0H g r o u p , shows an i d e n t i c a l i n c r e a s e i n b i n d i n g . We have no e x p l a n a t i o n f o r t h i s o b s e r v a t i o n . a

m a x

4

a

4

a

1

a

6

a

Having thus e s t a b l i s h e d t h e l i k e l y arrangement o f g a l a c t o s y l binding subsites in IgA J 5 3 9 , we t u r n our a t t e n t i o n t o o t h e r members of the 3( l - » 6 ) - D - g a l a c t o p y r a n a n b i n d i n g monoclonal antibodies. Potter et al. (43) and o u r l a b o r a t o r y ( 1 4 , 5 7 ) have over the years i d e n t i f i e d many i n d i v i d u a l myeloma and hybridoma monoclonal a n t i b o d i e s with t h i s s p e c i f i c i t y . I t i s important to see i f a l l t h e s e a n t i - g a l a c t a n i m m u n o g l o b u l i n s , whose amino a c i d sequence we know, show similar or dissimilar subsite a r r a n g e m e n t s . T h u s , i t may be possible to propose or dismiss c e r t a i n amino a c i d r e s i d u e s r e s p o n s i b l e f o r bonding t o t h e l i g a n d by comparing t h e sequences o c c u r i n g i n t h e h y p e r v a r i a b l e r e g i o n s .

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GLAUDEMANS & KOVAC

89

In Table II are listed the constants of a s s o c i a t i o n ( K ) of compounds 2, 5, 7-11, 15 and 16 w i t h seven m o n o c l o n a l a n t i p(l-»6)-D-galactopyranans, as well as the maximal f l u o r e s c e n c e changes o f t h e s e p r o t e i n s on b i n d i n g t h e l i g a n d i n q u e s t i o n ( 5 8 ) . These seven antibodies a l l belong to t h e same f a m i l y (44) and t h e i r amino a c i d sequence i s known ( 2 8 , 3 4 , 4 3 ) . From t h e K v a l u e s of 5 , 7, 10, 15, and 16 w i t h IgA ( F a b ) J539 i t can be deduced t h a t the f o u r s u b s i t e s C, A, B and D have vastly different a f f i n i t i e s for "their i n d i v i d u a l g a l a c t o s y l r e s i d u e , namely 1 0 ( A ) , 47 ( B ) , 10 (C) and 1.3 ( D ) , and thus only subsite A has a high affinity. It is interesting that that highest binding s u b s i t e i s l o c a t e d internally i n the sequence o f subsites (59). It can a l s o be learned from T a b l e I I t h a t a l l a n t i - g a l a c t a n a n t i b o d i e s show t h e same K p a t t e r n : m e t h y l 3 - d e o x y - 3 - f l u o r o - p - D g a l a c t o p y r a n o s i d e (2) shows no n o t i c a b l e b i n d i n g , w h i l e m e t h y l p D-galactopyranoside (5) show relatively th strongest bindin (as —AG/sugar residue galactopyranosyl-( l - » 6 ) - p - D - g a l a c t o p y r a n o s i d corresponding tri(10), t e t r a - (15) and p e n t a s a c h h a r i d e ( 1 6 ) . T h a t i s t o s a y , f o r l i g a n d s 5 , 7, 10, 15 and 16 t h e A G ( A G =R T l n K ) of binding are such t h a t t h e - &G f o r 5 i s t h e h i g h e s t per sugar residue. Therefore, all these antibodies p o s e s s one galactosyl-binding subsite having an a f f i n i t y f o r " i t s " s u g a r r e s i d u e w h i c h f a r exceeds the a f f i n i t i e s which the other three s u b s i t e s have for t h e i r g a l a c t o s y l residues. Methyl 0-3-deoxy-3f l u o r o - p - D - g a l a c t o p y r a n o s y l - ( l - » 6 ) - p - D - g a l a c t o p y r a n o s i d e (9) b i n d s substantially less than the nonfluorinated disaccharide 7, whereas t h e i s o m e r i c d i s a c c h a r i d e 8 b i n d s t o t h e p r o t e i n s s t u d i e d w i t h s i m i l a r K ' s as does 7. As we p o i n t e d out above, t h i s i s due t o the f a c t t h a t 7 binds t o the two s u b s i t e s w i t h the h i g h e s t a f f i n i t i e s (A and B) whereas 9 must s h i f t t o t h e s u b s i t e s A and C i n o r d e r t o be a b l e to use the two r e q u i r e d hydrogen-bonding h y d r o x y l groups demanded by s u b s i t e A . A g a i n , i n so d o i n g l i g a n d 9 s h o u l d t h e n o n l y p e r t u r b t h e TRP a t p o s i t i o n 33H, and i t can be seen i n T a b l e I I that i t s maximal f l u o r e s c e n c e change i s l e s s than t h a t for 7 in all cases except for IgA X44 ( f o r an e x p l a n a t i o n see b e l o w ) . L i g a n d 8 can a g a i n b i n d t o s u b s i t e s A and B , and thus w i l l show e s s e n t i a l l y t h e same K and ^max as does 7. T h i s pattern holds for a l l the a n t i b o d i e s s t u d i e d here ( f o r IgA X44 t h e F stays the same, see below). A l l this is consistent with the same s u b s i t e arrangement f o r a l l o f t h e s e a n t i b o d i e s . In a l l cases, except f o r hybridoma HyG 1 ( l a c k o f antibody supply), t h i s was moreover c o n f i r m e d by c o m p a r i s o n o f t h e K and F values for the trioside 10 w i t h those f o r methyl 0-3-deoxy-3-fluoro-p-D-galactopyranosyl-(l-*6)-0-p-Da

a

1

1 1

3

a

a

a

a

a

m

a

x

m

a

x

galactopyranosyl-(l-»6)-p-D-galactopyranoside (11), which should be, and a r e , very similar. T h e r e f o r e , t h e way i n which t h e s e l i g a n d s c o n t a c t t h e a n t i b o d i e s i n v o l v e d appears t o be the same i n a l l cases. Table III l i s t s the known ( 2 8 , 3 4 , 4 3 ) amino a c i d sequences for the v a r i a b l e p a r t s of the H and L c h a i n s of t h e seven antibodies studied. Kabat et al. (38) have t a b u l a t e d t h e known amino a c i d sequences o f i m m u n o g l o b u l i n s . In t h e r e t h e sequences

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988. »1

0

S

9 .6xlO (-9)

7.0xl0 (+23)

3

4. l x l O 1 (-11)

8

11

3.7xl0 (+45)

4

3. 3 x l 0 1[-7)

16

0

4.1xl0 (+42)

5

3

4

5

5.9xl0 (+42)

S

2xlO 1-9)

S

5

4

5.9xlO (+41)

4.8xl0 (+43)

4.7xl0 (+41)

l.OxlO ( + 21)

3

5

5

4

3

J539

3. 2 x l 0 (-9)

15

5%

1. 9 x l 0 (-8)

7 lxlO -11)

0. 4 x l 0 (-9)

HO CH|OH

X:-44

5

10

-

3

1.5xl0 (+42) 0 0 0

5

5

3 3

3.0xl0 (+24) 4.0xl0 ( + 15)

0

-

2.2xl0 (+9)

4

3

1.6xl0 ( + 19)

4

3.1xl0 (+46) 4

l.SxlO ( + 31)

S

S

1.8xlO (+20)

4

3.1xl0 ( + 18)

S

-

1.6xl0 (+21)

S

3.2xl0 (+43)

S

4

2.1xlO (+41)

3.1xl0 (+41)

3

1.3xl0 ( + 10)

HyG-1

3.9xl0 (+42)

-

S

3.2xlO ( + 32)

S

2.6xlO ( + 31)

4

1.3xl0 ( + 30)

0.7xl0 ( + 26)

3

HyG-10

2.1xl0 ( + 31) 5

3

3

0.4xl0 ( + 15)

T601

3.0xl0 ( + 38)

6.3xl0 (+20)

4

1.7xl0 ( + 38)

5

6.2xl0 ( + 40)

5

5

4

5.7xl0 (+40)

3.2xl0 (+41)

2.1xl0 (+35)

O.SxlO (+21)

X-24

Immunoglobulin

4

2.6xl0 (+10)

0

l.OxlO (+9)

(-2)

S

S

2.0xlO (+10)

2.1xlO (+12)

5

4

l.OxlO (+9)

2.0xl0 (+9)

(-3)

S-10

5

in parentheses) for monoclonal immunoglobulins with, galactose derivatives

t

11. Binding constants, K , and percentage maximalfluorescencechange ( A / ^

Compound

TABLE

I

8 =

w

1

s

0

1

as

0 2

o

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

T-601

X-44

HyG 1 HyG 10 S-10 J-539

T-601 HyG-10 hyG-1 S-10*«

X-24

J-539

X-44

III*

Ht

TL

FDV,—A—T-

—GH—LS—i -QGVH— GG ' —,'QKLF-LG -

-LG

H3

EIVLTQSPAITAJ^WKVTITCSAdSSVSYMITOQQK^^

-W

- SG-

L-chain V-region sequences of murine B A L B / c galactan-binding monoclonal immunoglobulins

H2

H-chain V-rcgion sequences of murine B A L B / c galactan-binding monoclonal immunoglobulins

EVlUU£SGGGLVQPGGSLKX£CJUtSGrDFSRYt^

T A B L E

92

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS of some 51 monoclonal immunoglobulins o f v a r i e d s p e c i f i c i t i e s (anti-phosphorylcholine, -2,1-fructan, 2,6-fructan, -dextran, -pN-acetyl-glucosaminyl, -galactan) and h a v i n g H c h a i n s b e l o n g i n g t o s u b - g r o u p I I I have been l i s t e d . Some twenty o t h e r s have been p a r t l y sequenced t o r e s i d u e 36. I t i s i n t e r e s t i n g t h a t - w i t h t h e e x c e p t i o n o f IgG MOPC 173 h a v i n g no known s p e c i f i c i t y o n l y the a n t i - c a r b o h y d r a t e immunoglobulins have a TRP a t p o s i t i o n 33H (and we have found t h a t MOPC 173 i s " r e l a t e d " t o a n t i - 2 , 6 - f r u c t a n , and thus t o an a n t i - c a r b o h y d r a t e , see r e f . 6 0 ) . Of t h e s e sequenced a n t i b o d i e s , o n l y t h e a n t i - f r u c t a n s and t h e a n t i - g a l a c t a n s show an a s p a r a g i n e (ASN) a t p o s i t i o n 58H i n a d d i t i o n t o t h e TRP a t 33H. Of some 85 m o n o c l o n a l murine kappa L c h a i n s w h i c h have been completely sequenced, only t h e a n t i - g a l a c t a n s (and t h r e e o t h e r immunoglobulins o f as y e t unknown s p e c i f i c i t y ) have a TRP a t p o s i t i o n 91L and a p r o l i n e (PRO) a t p o s i t i o n 94L. I t can be seen from t h e X - r a y d a t a (33) t h a t ASN 58H TRP 33H and PRO 94L a r e clustered together wit around 7 A and t h e d i s t a n c t h e TRP 33H r i n g system from 9-13 A and 7-11 A r e s p e c t i v e l y (see s t e r e o F i g . 6 ) . The s u b s i t e w i t h the h i g h e s t g a l a c t o s y l - b i n d i n g a f f i n i t y appears n e a r the H c h a i n , and i t i s q u i t e p o s s i b l e t h a t t h e above t h r e e residues make up t h i s subsite. In a d d i t i o n , certain differing amino a c i d s i n the second h y p e r v a r i a b l e r e g i o n of t h e heavy c h a i n ( i n Table III referred to as H2) may be eliminated as likely candidates f o r d i r e c t hydrogen-bonding to t h e ( p o l y ) s a c c h a r i d e : h i s t i d i n e 52H ( t h i s confirms our e a r l i e r work, see r e f . 61) i n J 5 3 9 , a s p a r t i c a c i d 53H i n X44, J 5 3 9 , T601, HyG10 and H y G l , s e r i n e 55H i n a l l (J539 has a glycine at that

Fig. 6. The complementarity determining regions (CDRs) of IgA J539 according to Suh et at. ( 33). Alternating CDRs are drawn lightly and boldly. The TRP (33H), ASN (58H) and PRO (94L) in the right lower quadrant are clearly visible.

p o s i t i o n ) and t h r e o n i n e 60H i n a l l but HyG 10, which has a l y s i n e there. A l s o , from T a b l e I I I i t can be seen that the the t h i r d hypervariable region i n these a n t i b o d i e s show much v a r i a t i o n i n s e q u e n c e . S i n c e b i n d i n g b e h a v i o u r i s so s i m i l a r , i t appears t h a t the c o n t r i b u t i o n to b i n d i n g by t h i s e p i t o p e can be i g n o r e d . L a s t l y , i t can be seen i n T a b l e I I t h a t XAA has an u n u s u a l l i g a n d

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

GLAUDEMANS & KOVAC

Monoclonal Antisaccharide Antibodies

i n d u c e d f l u o r e s c e n c e b e h a v i o u r , i n t h a t the change i s essentially i n v a r i a n t w i t h t h e s i z e o f the ligand in c o n t r a s t to the other antibodies. Visual examination of t h e c r y s t a l s t r u c t u r e o f J539 ( F a b ) (33) shows t h a t t h e TRP 9 1 L , a l t h o u g h t u r n e d towards the protein, is s i t u a t e d as p a r t of the w a l l of the f r o n t a l c a v i t y , and thus appears p e r t u r b a b l e . I t can be seen t h a t IgA X44 i s t h e only protein in this group which has an a d d i t i o n a l TRP a t p o s i t i o n 96L i n s t e a d o f an i s o l e u c i n e ( I L E U ) . Examination of the hvL~3 loop of J539 in the X-ray structure (33) shows t h a t p o s i t i o n 96L i s r e l a t i v e l y c l o s e t o p o s i t i o n 91L. X44 may have a three-dimensional structure which i s r e l a t i v e l y c l o s e t o t h a t o f J 5 3 9 . I f s o , may i t be p o s s i b l e t h a t i n X44 t h e s e two t r y p t o p h a n residues are stacked so that t h e energy can t r a n s f e r between them? I f so t h e TRP 91L i n X44 may n o t be e a s i l y p e r t u r b a b l e by ligands. 1

PREPARATION OF LIGANDS Our p r e p a r a t i o n of the many m e t h y l 3 ~ g l y c o s i d e s o f p - ( l + 6 ) - D g a l a c t o - o l i g o s a c c h a r i d e s - some o f w h i c h c a r r i e d one o r more deoxyfluoro groups was instrumental in the e l u c i d a t i o n of b i n d i n g modes d e s c r i b e d above. The l a t t e r were c o n c e r n e d more w i t h the b i n d i n g c o n t r i b u t i o n o f t h e immunoglobulin than w i t h the c o n t r i b u t i o n o f t h e ligand. This laboratory had shown e a r l i e r (40-42,49,55,59) the a r e a s o f importance t o b i n d i n g o c c u r r i n g on the l i g a n d . S y n t h e t i c procedures associated with that e a r l i e r work w i l l n o t be reviewed h e r e . We h e r e d e s c r i b e o u r a p p r o a c h t o the syntheses of d e r i v a t i v e s of 1) deoxyfluoro d e r i v a t i v e s of methyl 3~D-galactopyranoside, as w e l l as 2) m e t h y l 3 - g l y c o s i d e s of (l+6)-3-D-galacto-oligosaccharides and t h e i r specifically f l u o r i n a t e d analogs. In our initial s t u d i e s (40, 41) r e d u c i n g o l i g o s a c c h a r i d e s had been u s e d . However, m e t h y l 3 " g l y c o s i d e s more c l o s e l y i m i t a t e the s i t u a t i o n i n the polymeric a n t i g e n , (l->6)-3"D-galactopyranan (39), and a l l our oligoand deoxyfluoro-oligosaccharides, p r e p a r e d s i n c e , were m e t h y l 3 ~ g l y c o s i d e s . Monodeoxyfluoro d e r i v a t i v e s of methyl 3 D - g a l a c t o p y r a n o s i d e were s y n t h e s i z e d by t h e i n t r o d u c t i o n of fluorine at s p e c i f i c positions. Certain h y d r o x y l groups i n a sugar d e r i v a t i v e ( n o t n e c e s s a r i l y h a v i n g a D-galacto configuration) were b l o c k e d t o a r r i v e at a p r e c u r s o r s u i t a b l e f o r a C-OH -» C - F c o n v e r s i o n . The only exception to direct C-OH -> C - F t r a n s f o r m a t i o n was t h e i n t r o d u c t i o n of f l u o r i n e via D - g a l a c t a l , u s i n g xenon d i f l u o r i d e , l e a d i n g (49) t o 1 (Scheme 1 ) . To e f f e c t o t h e r f l u o r i n a t i o n s , we have used a number o f reagents ranging from a common s o u r c e of f l u o r i d e i o n s u c h as cesium f l u o r i d e (62) t o one such as polymer-bound f l u o r i d e ( A - 2 6 F, ion exchange r e s i n ) . Using the l a t t e r r e a g e n t , we have been a b l e t o improve ( 4 9 , 6 2 , 6 3 ) the y i e l d (64) o f methyl 4-deoxy-4fluoro-3-D-galactopyranoside ( 3 ) , obtained a f t e r d e b l o c k i n g of its 2,3-di-0-benzyl-6-0-trityl derivative (Scheme 2). The most versatile fluorinating reagents, now available for the replacement of a h y d r o x y l group for fluorine, seem t o be diethylaminosulfur trifluoride (DAST) and i t s methyl analog _

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

93

94

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Scheme 1

CH OTr

F CH OTr

2

2

OBn

OBn

HO

HO

„

I CHjF

HO

4

Scheme 3 ( m e t h y l DAST, 6 5 ) . We used DAST t o p r e p a r e (Scheme 3) m e t h y l 6deoxy-6-fluoro-p-D-galactopyranoside (4, 6 3 ) , as w e l l as i t s 3fluoro analog (2, 62). DAST was especially useful in the preparation of the latter d e r i v a t i v e (Scheme 4 ) . F l u o r i n a t i o n with i n v e r s i o n of c o n f i g u r a t i o n of position 3 of the D-gulose d e r i v a t i v e 17, p r e p a r e d from D - g l u c o s e , as shown below, c o u l d be markedly improved i n comparison to fluorinations mediated by tetraethylammonium bromide o r by an i o n exchange r e s i n i n t h e f l u o r i d e form. A f t e r i t had become a p p a r e n t t h a t hydrogen bonding i n v o l v i n g h y d r o x y l groups at p o s i t i o n s 2 (ref. 49) and 3 ( r e f . 62) i n D g a l a c t o s e p l a y an i m p o r t a n t r o l e i n b i n d i n g of the c a r b o h y d r a t e to a n t i g a l a c t a n m o n o c l o n a l i m m u n o g l o b u l i n s , our n e x t t a s k was t o obtain deoxyfluoro ligands closely related to t h e (l->6)-fl-Dgalactopyranan antigen. From the studies involving monodeoxyfluoro d e r i v a t i v e s o f methyl 3 - D - g a l a c t o p y r a n o s i d e , we knew t h a t replacement of either the 2-OH o r 3-OH group by f l u o r i n e causes cessation of binding to the highest binding antibody subsite A (35,49). Thus, if the modified oligos a c c h a r i d e s would c o n t a i n a 2- o r 3-deoxyfluoro-D-galactose at a certain position ( o r p o s i t i o n s ) i n the o l i g o s a c c h a r i d e sequence, i t would f o r c e that g a l a c t o s y l m o i e t y not t o b i n d t o the s u b s i t e A. C l e a r l y , only the use o f 3 - d e o x y - 3 - f l u o r o - D - g a l a c t o p y r a n o s y l h a l i d e d e r i v a t i v e s would a l l o w us t o use a 2 - O - a c y l group as a blocking moiety. This, in t u r n , through i t s anchimeric t r a n s d i r e c t i n g a c t i o n , would s t e r e o s e l e c t i v e l y r e s u l t i n t h e f o r m a t i o n of the desired {S-galactosidic linkage. The u s e f u l starting

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

GLAUDEMANS & KOVAC

Monoclonal Antisaccharide Antibodies

90% m a t e r i a l f o r the s y n t h e s i s o f such a halide appeared to be a 2,4,6-tri-0-acyl-3-deoxy-3-fluoro-aor -p-D-galactopyranose. A c e t y l a t i o n of 3-deoxy-3-fluoro-D-galactose with a c e t i c anhydride in the presence of sodium a c e t a t e a t an e l e v a t e d t e m p e r a t u r e , c o n d i t i o n s under which ( 3 - 1 - O - a c e t y l d e r i v a t i v e s a r e o f t e n formed in high y i e l d s , had been previously d e s c r i b e d . However, t h e reported low yield (35%, 66) of the only p y r a n o s y l isomer isolated, crystalline (3-1-O-acetate, was due to extensive formation of acetates having the furanose structure. Clearly, a p r e r e q u i s i t e o f an efficient c o n v e r s i o n t o a h a l i d e such as 19 i s finding reaction conditions for the acetylation of 3-deoxy-3fluoro-D-galactose whereby the combined y i e l d o f 18a and 18fl would be h i g h . T h i s we r e a l i z e d (Scheme 5) when a c e t y l a t i o n o f 3deoxy-3-fluoro-D-galactose with a c e t i c a n h y d r i d e i n p y r i d i n e was conducted i n i t i a l l y at subzero temperature. That the formation of acetyl derivatives having furanose s t r u c t u r e was m i n i m a l can be deduced from t h e o b s e r v e d h i g h y i e l d o f c r y s t a l l i n e 19. C o n s t r u c t i o n of methyl ^-glycosides of (l->6)-3-D-galactooligosaccharides followed the s t r a t e g y developed e a r l i e r i n t h i s laboratory (67). Accordingly, a selectively removable

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

95

FLUORINATED CARBOHYDRATES: C H E M I C A L AND BIOCHEMICAL ASPECTS

(haloacetyl) group was used for the temporary p r o t e c t i o n o f position 6 in the D-galactosyl halides employed for the construction of these oligosaccharides. Such a h a l i d e has t o h a v e , i n a d d i t i o n , a group a t p o s i t i o n 2 capable of anchimeric assistance in r e p l a c i n g t h e l e a v i n g group a t C - l . O r i g i n a l l y , we used 2,3,4-tri-0-acetyl-6-0-(chloroacetyl)-a-D-galactopyranosyl bromide (20, ref. 67). In this way, t h e m e t h y l 3 - g l y c o s i d e s

Br Scheme 5

18a,3

** m.p. 101 102° lo| -«-25l

o

0

of (l->6)-(S-linked D-galactotriose (10) and the related, fluorinated trisaccharide 11 were s y n t h e s i z e d ( 6 8 ) . However, we observed that noticeable migration of acetyl groups o c c u r r e d during de(chloroacetyl)ation of intermediates with thiourea, H y d r o x y l groups protected with the bromoacetyl group can be regenerated under much m i l d e r c o n d i t i o n s than those p r o t e c t e d with the chloroacetyl groups. Therefore, to construct (69)

internal units of the fluorinated o l i g o s a c c h a r i d e s 12 and 21 2 , 3 , 4 - t r i - O - a c e t y l - 6 - O - ( b r o m o a c e t y l ) - a - D - g a l a c t o p y r a n o s y l bromide (22, 69) was used as t h e g l y c o s y l donor (Scheme 6 ) . A c e t y l group migration during de(haloacetyl)ation could be m i n i m i z e d i n t h i s way. However, formation of by-products, mainly O-acetyl derivatives of the employed nucleophiles (70), during

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

GLAUDEMANS & KOVAC

MonoclonalAntisaccharideAntibodies

I

ϋ

u w

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97

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS g l y c o s y l a t i o n s promoted by s i l v e r t r i f l u o r o m e t h a n e s u l f o n a t e c o u l d s t i l l n o t be e n t i r e l y overcome when O - a c e t y l a t e d r e a c t a n t s were used. T h e r e f o r e , i n subsequent syntheses of methyl 3~glycosides of (1+6)-0-D-galacto-oligosaccharides, and t h e i r specifically fluorinated analogs, benzoylated intermediates were u s e d . The strategy of using the b r o m o a c e t y l group f o r the temporary protection of position 6 i n otherwise fully benzoylated d e r i v a t i v e s of D - g a l a c t o s e r e q u i r e d the p r e p a r a t i o n of a l a r g e number o f v a r i o u s l y s u b s t i t u t e d i n t e r m e d i a t e s . F o r example ( F i g . 7 ) , the s p e c i f i c a l l y f l u o r i n a t e d t r i s a c c h a r i d e 13 c o n t a i n s 3d e o x y - 3 - f l u o r o - D - g a l a c t o s e i n both e n d - u n i t s , and i t s s y n t h e s i s requires two different derivatives of 3-deoxy-3-fluoro-Dgalactose as intermediates. In a d d i t i o n , t o form t h e i n t e r n a l u n i t o f the t r i s a c c h a r i d e , a complex g l y c o s y l donor d e r i v e d from D-galactose is required. Such a s y n t h o n , 2 , 3 , 4 - t r i - 0 - b e n z o y l - 6 O - b r o m o a c e t y l - a - D - g a l a c t o p y r a n o s y l bromide (23 Scheme 7) was prepared (71) from D-galactos d e r i v a t i v e s 24 o r 25 6-Tri-0-benzoyl-3-deoxy-3-fluoro-a-D-galacto-pyranosyl bromide (26), used to construct the glycosyl end-group of the oligosaccharide (Fig. 7), was p r e p a r e d (71) from 3 - d e o x y - 3 f l u o r o - D - g a l a c t o s e as shown i n Scheme 8. Referring to F i g . 7, methyl 2,4-di-0-benzoyl-3-deoxy-3-fluoro-0-D-galactopyranoside (shown a t t h e bottom r i g h t o f F i g . 7) was condensed with a f u l l y b l o c k e d g a l a c t o s y l h a l i d e (23) b e a r i n g a group a t 0-6 which c o u l d be removed s e l e c t i v e l y . Subsequent removal o f that group, followed by c o n d e n s a t i o n of the product with the deoxyfluoro HO I CH OH 2

OMe

23

Br

Figure 7. Specifically fluorinated trisaccharide 13 containing 3-deoxy-3-fluoro-D-galactose in both end-units.

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g a l a c t o s y l h a l i d e (26), followed Dy complete d e b l o c k i n g , then y i e l d e d 13. The s y n t h e s i s o f t h e c l o s e l y r e l a t e d t r i s a c c h a r i d e 14 required different intermediates. Synthesis of its fully benzoylated precursor i s shown i n Scheme 9. The key i n t e r m e d i a t e used t o form the internal 3-deoxy-3-fluoro-3-D-galactopyranosyl r e s i d u e of the o l i g o s a c c h a r i d e , 2 , 4 - d i - O - b e n z o y l - 6 - 0 - b r o m o a c e t y l 3 - d e o x y - 3 - f l u o r o - a - D - g a l a c t o p y r a n o s y l c h l o r i d e ( 2 7 ) , was o b t a i n e d (37) from the c o r r e s p o n d i n g m e t h y l 3 ~ g l y c o s i d e by c l e a v a g e (72) w i t h d i c h l o r o m e t h y l m e t h y l e t h e r (DCMME)/ZnCl2 r e a g e n t . G l y c o s y l bromide 23 proved to be an e x c e l l e n t g l y c o s y l d o n o r , but i t s l a r g e - s c a l e p r e p a r a t i o n , r e q u i r e d f o r the s t e p w i s e c o n s t r u c t i o n of higher (l+6)-linked 3-D-galacto-oligosaccharides or t h e i r methyl g l y c o s i d e s , i s somewhat t e d i o u s (Scheme 7 ) . I t requires isolation of all intermediates by chromatography, i n v o l v i n g , inter alia, the separation of four isomeric t e t r a - O benzoyl-D-galactoses, t

Scheme 7

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100

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS HO

BrO |CH 0B«

B2O

?CH,0H

l

C H

2° « B

OH

2

OBx

OBd OBz 74%

24%

BzO

„

26 Schem

OBzl Br

8

f u r a n o s e isomers forme g benzoylatio 6-O-trityl-D galactose. Therefore, a more e f f i c i e n t source of a suitable g l y c o s y l a t i n g reagent r e l a t e d to 23 was s o u g h t . To p r e v e n t t h e formation of 5-membered ring forms during synthetic m a n i p u l a t i o n s , we changed t h e s y n t h e t i c s t r a t e g y and d e c i d e d t o use as a key intermediate a compound i n which t h e p y r a n o s e s t r u c t u r e would be l o c k e d b e f o r e f u r t h e r t r a n s f o r m a t i o n s would be attempted. These r e q u i r e m e n t s a r e met i n an e x c e l l e n t way by methyl 2 , 3 , 4 - t r i - O - b e n z o y l - p - D - g a l a c t o p y r a n o s i d e (28), a r e a d i l y o b t a i n a b l e c r y s t a l l i n e compound ( 7 3 ) , w h i c h we used as a pivotal intermediate to synthesize methyl 3-glycosides of (l+6)-p-Dg a l a c t o - o l i g o s a c c h a r i d e s by a s t e p w i s e and a b l o c k w i s e a p p r o a c h ( 7 4 ) . Compound 28 was used f i r s t l y , i n a way s i m i l a r t o t h a t i n t h e s y n t h e s i s o f 14 ( c . f . Scheme 9 ) , as the n u c l e o p h i l e i n the syntheses of the said o l i g o s a c c h a r i d e s , up t o and i n c l u d i n g a h e x a s a c c h a r i d e . S e c o n d l y , i t was t h e key i n t e r m e d i a t e i n t h e preparation of 2,3,4-tri-0-benzoyl-6-0-(bromoacetyl)-a-Dg a l a c t o p y r a n o s y l c h l o r i d e ( 2 9 ) . T h u s , (Scheme 10) compound 28 was ( b r o m o a c e t y l ) a t e d and t h e r e s u l t i n g 6-0-(bromoacetyl) d e r i v a t i v e was c l e a v e d w i t h DCMME/ZnCl t o y i e l d (84%) t h e g l y c o s y l c h l o r i d e 29. C r y s t a l l i n e g l y c o s y l chloride 29 is sufficiently reactive under the conditions of g l y c o s y l a t i o n promoted by s i l v e r t r i f l u o r o m e t h a n e s u l f o n a t e , t o y i e l d t h e d e s i r e d d i s a c c h a r i d e 30 w i t h high stereoselectivity. The s t e p w i s e a p p r o a c h was a p p l i e d t o t h e s y n t h e s i s o f lower o l i g o s a c c h a r i d e s i n t h i s s e r i e s , as shown i n Scheme 11. 2

To obtain the higher members of methyl p - g l y c o s i d e s of (1+6)-3-D-galacto-oligosaccharides a more e f f i c i e n t , blockwise s y n t h e t i c a p p r o a c h was a p p l i e d . I t r e q u i r e d a complex g l y c o s y l donor d e r i v e d from an o l i g o s a c c h a r i d e , b e a r i n g a t p o s i t i o n 6 of its glycosyl end-group a s e l e c t i v e l y removable b l o c k i n g g r o u p . D e r i v a t i v e s 24 and 25, i s o l a t e d i n c o n n e c t i o n w i t h the s y n t h e s i s of 23 (Scheme 7 ) , were u s e d as i n t e r m e d i a t e s i n t h e p r e p a r a t i o n of s u c h a g l y c o s y l donor ( 3 1 ) . As shown i n Scheme 12, 24 and 25 were each detritylated and then condensed with the

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FLUORINATED CARBOHYDRATES: C H E M I C A L AND BIOCHEMICAL ASPECTS

Scheme

10

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( b r o m o a c e t y l ) a t e d g l y c o s y l h a l i d e 29, t o a f f o r d the c o r r e s p o n d i n g d i s a c c h a r i d e s . Each o f t h e l a t t e r c o u l d be c l e a v e d s u b s e q u e n t l y w i t h DCMME/ZnCl t o y i e l d the same g l y c o s y l chloride (31), a derivative of (1+6)-p-linked galactobiose. Its use in the b l o c k w i s e s y n t h e s i s o f the t a r g e t o l i g o s a c c h a r i d e s i s e x e m p l i f i e d in Scheme 13, showing the pathway leading to the fully s u b s t i t u t e d p r e c u r s o r o f t h e p e n t a s a c c h a r i d e 16. F i n a l l y , from t h e H NMR s p e c t r a o f our d e o x y f l u o r o l i g a n d s , i t has become c l e a r t h a t t h e r e i s no e v i d e n c e f o r any c o n f o r m a t i o n a l change due to t h e i n t r o d u c t i o n o f fluorine into these molecules ( c f . r e f . 37 and papers c i t e d t h e r e i n ) . 2

1

CONCLUSION This review shows t h e application of the combination of bromoacetyl and b e n z o y l groups, as temporary and permanent blocking groups in oligosaccharide s y n t h e s i s . I n t h i s way, we have been a b l e t o p r e p a r e s y s t e m a t i c a l l y , and w i t h a h i g h degree of s t e r e o s e l e c i v i t y , specifically fluorinated ligands for a n t i g a l a c t a n m o n o c l o n a l i m m u n o g l o b u l i n s . N e x t , our work has shown t h e extraordinary usefulness of these deoxyfluoro (oligo)saccharides in u n r a v e l i n g details of the binding of these monoclonal antibodies to t h e i r carbohydrate antigens.

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32. 33. 34. 35. 36. 37. 38.

39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58.

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Satow, Y . ; Cohen, G. H . ; Padlan, E. A . ; Davies, D. R. J. Mol. Biol. 1986, 190, 593. Suh, S. W.; Bhat, T. N.; Navia, M. A.; Cohen, G. H . ; Rao, D. N.; Rudikoff, S.; Davies, D. R. Proteins: Structure, Function and Genetics 1986, 1, 74. Pawlita, M.; Potter, M.; Rudikoff, S. J . Immunol. 1982, 129, 615. Glaudemans, C. P. J.; Kováč, P.; Rasmussen, K. Biochem. 1984, 23, 6732. Glaudemans, C. P. J.; Kováč, P. Mol. Immunol. 1985, 22, 651. Kováč, P.; Glaudemans, C. P. J . J. Carbohydr. Chem. 1986, 4, 613. Kabat, E. A.; Wu, T. T . ; Bilofsky, H . ; Reid-Miller, M.; Perry, M.; Gottesman, K. Sequences of Proteins of Immunological Interest. U.S Dept of H.H.S. P.H.S. N.I.H., Bethesda Glaudemans, C. P Molec. Immunol. 1986, 23, 655. Jolley, M. E.; Rudikoff, S.; Potter, M.; Glaudemans, C. P. J . Biochem. 1973, 12, 3039. Jolley, M. E . ; Glaudemans, C. P. J.; Rudikoff, S.; Potter, M. Biochem. 1974, 13, 3179. Ekborg, G.; Ittah, Y . ; Glaudemans, C. P. J . Molec. Immunol. 1983, 20, 235. Rudikoff, S.; Pawlita, M.; Pumphry, J.; Mushinsky, E. B.; Potter, M. J. Exp. Med. 1983, 158, 1385. Hartman, A. B.; Rudikoff, S. EMBO J. 1984, 3, 3023. Ekborg, G.; Vranešić, B.; Bhattacharjee, A. K.; Kováč, P.; Glaudemans, C. P. J . Carbohydr. Res. 1985, 143, 203. Falent-Kwast, E.M.; Kováč, P.; Bax, A.; Glaudemans, C. P. J . Carbohydr. Res. 1986, 145, 332. Nashed, E. M.; Glaudemans, C. P. J . Carbohydr. Res. 1986, 158, 125. Jung, G. L . ; Holme, K. R.; Glaudemans, C. P. J.; Lehmann, J.; Lay, H. Carbohydr. Res. ms. submitted. Ittah, Y . ; Glaudemans, C. P. J . Carbohydr. Res. 1981, 95, 189. Goldstein, I. J.; Hayes, C. E. Adv. Carbohydr. Chem.Biochem. 1978, 35, 127, and references therein. Lemieux, R. U.; Bock, K. Arch. Biochem. Biophys. 1983, 221, 125. Jolley, M. E.; Glaudemans, C. P. J . Carbohydr. Res. 1974, 33, 377. Rao, D. N.; Rudikoff, S.; Krutzsch, H . ; Potter, M. Proc. Nat. Acad. Sci. U.S.A. 1979, 76, 2890. Manjula, B. N.; Mushinski, E. B.; Glaudemans, C. P. J . J. Immunol. 1979, 119, 867. Glaudemans, C. P. J.; Zissis, E . ; Jolley, M. E. Carbohydr. Res. 1975, 40, 129. Murray-Rust, P.; Stallings, W. C.; Monti, C. T . ; Preston, R. K.; Glusker, J . P. J. Amer. Chem. Soc. 1983, 105, 3206. Manjula, B. N.; Glaudemans, C. P. J.; Mushinsky, E. B.; Potter, M. Carbohydr. Res. 1975, 40, 37. Glaudemans, C. P. J . Mol. Immunol. 1987, 24, 371.

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Glaudemans, C. P. J . Mol. Immunol. 1986, 23, 917. Enghofer, E . ; Glaudemans, C. P. J.; Bosma, M. J . Molec. Immunol. 1979, 16, 1103. Gettings, P.; Boyd, J.; Glaudemans, C. P. J.; Potter, M.; Dwek, R. A. Biochem. 1981, 20, 7463. Kováč, P.; Glaudemans, C. P. J . Carbohydr. Res. 1983, 123, 326. Kováč, P.; Glaudemans, C. P. J . J . Carbohydr. Chem. 1983, 2, 313. Maradufu, A.; Perlin, A. S. Carbohydr. Res. 1974, 32, 261. Kováč, P.; Sklenar, V.; Glaudemans, C. P. J . Carbohydr. Res. ms. submitted. Brimacombe, J . S.; Foster, A. B.; Hems, R.; Westwood, J . H.; Hall, L. D. Can. J. Chem. 1970, 48, 3946. Bhattacharjee, A. K.; Zissis, E . ; Glaudemans, C. P.J. Carbohydr. Res. 1981, 89, 249. Kováč, P.; Yeh Res. 1985, 140, 277 Kováč, P.; Glaudemans, C. P. J . Carbohydr. Res. 1985, 140, 289. Kováč, P.; Glaudemans, C. P. J . Carbohydr. Res. 1985, 140, 313. Kováč, P.; Glaudemans, C. P. J.; Guo, W.; Wong, T. Carbohydr. Res. 1985, 140, 294. Gross, H . ; Farkas, I . ; Bognár, R. Z. Chem. 1978, 18, 201. Szabó, P.; Szabó, L. J. Chem. Soc. 1960, 3762. Kováč, P. Carbohydr. Res. 1986, 153, 237.

RECEIVED March 14, 1988

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Chapter 7

Metabolic and Enzymatic Studies with Deoxyfluoro Carbohydrates 1

2

N. F. Taylor, D. Sbrissa, S. T. Squire , T. D'Amore , and J. M. McIntosh Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada The biochemica of fluorine int exemplified by a review of transport, metabolic and enzymatic studies with deoxyfluorosugars. Recent biochemical results with 3-deoxy-3fluoro-D-glucose (3FG) and 4-deoxy-4-fluoro-D-glucose (4FG) i l l u s t r a t e the reactivity of the C-F bond. Thus unlike 3FG, when 4FG is incubated with Pseudomonas putida extensive fluoride ion release occurs due to the presence of an outer membrane protein. Using D-[6-3H]-4FG and D-[U14C]-4FG a de-fluorinated metabolite is isolated and identified by C and H NMR analysis to be 4,5-dihydroxypentanoic acid. Evidence is also presented for the incorporation of radiolabel into a peptidylglycan fraction of the c e l l envelope. The s p e c i f i c i t y and kinetics of pyranose-2-oxidase (E.C. 1.1.3.10) towards glucose, 4FG and 3FG is examined. Whereas 4FG is a substrate and competitive inhibitor, 3FG is not. The synthesis of 4-deoxy-4-fluoro-D-fructose via the immobilized enzyme is also reported. 13

1

The rationale for the introduction of fluorine into carbohydrates has been extensively cited (1). Initially, the idea was based on the close similarity in size and electronegativity between fluorine and a hydroxyl group when attached to carbon (2). Support for this contention was provided by a comparative X-ray crystallographic Current address: Laboratory of Intermediary Metabolism, Institut de Recherches Clinique de Montreal, Montreal, Quebec H2W 1R7, Canada Current address: Labatts Brewing Company, Ltd., 150 Simcoe Streeet, London, Ontario N6A 4M3, Canada

2

0097-6156/88/0374-0109$08.25/0 • 1988 American Chemical Society

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

a n a l y s i s of e r y t h r i t o l and 2 - d e o x y - 2 - f l u o r o - D L - e r y t h r i t o l which revealed an almost identical crystal lattice structure f o r the two compounds (3). These early observations suggested t h a t f l u o r i n e might r e p l a c e a hydroxyl group i n carbohydrates with minimal p e r t u r b a t i o n of molecular s t r u c t u r e and conformation, whilst still c o n f e r r i n g s u b t l e d i f f e r e n c e s i n chemical and biochemical reactivity (4). Subsequently, a physico-chemical comparison of bond length, Van der Waals r a d i u s and e l e c t r o n e g a t i v i t y r e v e a l e d a c l o s e s i m i l a r i t y between the c a r b o n - f l u o r i n e and carbon-oxygen bond (5). An important aspect of the biochemical rationale, emphasized by Barnett (5), i s i n the f a c t t h a t the hydroxyl group i n a carbohydrate may act as a donor or acceptor of hydrogen bonding, w h i l s t a deoxyfluoro s u b s t i t u e n t may act only as a hydrogen bond acceptor Thus the replacement of the hydroxyl group by f l u o r i n about the d i r e c t i o n b i n d i n g to a v a r i e t y of p r o t e i n s . There are now many i n vitro and i n v i v o biochemical s t u d i e s t h a t support this idea (e.g. enzyme s p e c i f i c i t y and mechanism, t r a n s p o r t p r o t e i n s , antibody-antigen s p e c i f i c i t y and metabolism). Furthermore, the presence of f l u o r i n e i n the substrate permits a study of s u b s t r a t e - p r o t e i n i n t e r a c t i o n s by using F nmr spectroscopy (6). These ideas and the wider i n t e r e s t i n the biochemistry of the c a r b o n - f l u o r i n e bond (2/8)/ especially with regard to mechanism of f l u o r o a c e t a t e p o i s o n i n g (£,i0) and the concept of " l e t h a l s y n t h e s i s " developed by R.A. Peters ( i l ) , undoubtedly stimulated the growth and i n t e r e s t i n the s y n t h e s i s , r e a c t i v i t y and biochemistry of f l u o r o c a r b o h y d r a t e s . After a brief review of fluorocarbohydrate biochemistry i n the areas of t r a n s p o r t , enzymes and metabolism, a t t e n t i o n w i l l be confined to our recent metabolic and enzymatic s t u d i e s with 3-deoxy-3-fluoroand 4-deoxy-4-fluoro-D-glucose. 1 9

Transport Studies Such s t u d i e s i n c l u d e : 3-deoxy-3-fluoro-D-glucose and glucose t r a n s p o r t mutants i n E s c h e r i c h i a c o l i (12); 2deoxy-2-fluoro-D-glucose and the i s o l a t i o n , k i n e t i c s and c h a r a c t e r i z a t i o n of hexose t r a n s p o r t mutants i n L6 r a t myoblasts (13,14); comparative binding studies with deoxyfluoro and deoxyhexoses to determine the structural requirements and k i n e t i c s of glucose t r a n s p o r t i n the human e r y t h r o c y t e (15,16), the hamster i n t e s t i n e (12) and r a t b r a i n synaptosomes (18); the use of 3-deoxy-3-fluoroand 4-deoxy-4-fluoro-D-glucose i n the k i n e t i c s and c h a r a c t e r i z a t i o n of the D-glucose, D-gluconate and 2-oxoD-gluconate t r a n s p o r t c a r r i e r systems i n the cytoplasmic membrane of Pseudomonas p u t i d a (1£,2J)). Recently (21), deoxyfluoro-sucroses have been used to study the s p e c i f i c i t y of the sucrose c a r r i e r p r o t e i n i n p l a n t s .

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Studies

A number o f enzyme specificity studies have been u n d e r t a k e n b a s e d on k i n e t i c m e a s u r e m e n t s . I d e a l l y , a s i n the case o f t r a n s p o r t s t u d i e s , t h e methodology i s based on a comparison o f t h e Km a n d / o r the competitive Ki values of the appropriate s u b s t i t u t e d deoxyfluoro and d e o x y a n a l o g u e w i t h t h a t o f t h e n a t u r a l s u b s t r a t e . Such a comparison of k i n e t i c data allows assignment of the direction and s t e r e o s p e c i f i c i t y o f hydrogen bonding of the s u b s t r a t e a t t h e c a t a l y t i c s i t e o f t h e enzyme. This approach has b e e n u s e d t o o b t a i n i n f o r m a t i o n about t h e specificity o f g l y c e r o l k i n a s e from Candida mycoderma (22.), yeast hexokinase (22), glucosephosphate isomerase ( 2 4 ) / g a l a c t o k i n a s e (2^3) a n d U D P G - p y r o p h o s p h o r y l a s e ( 2 6 ) . The glucansucrases of Leuconostoc mesenteroides and S t r e p t o c o c c u s mutan for the synthesis o fluorosucrose competitively i n h i b i t s the glucansucrases of t h e above organisms (Eklund, S.H.; Robyt, J.F. Carbohydr. Res. i n p r e s s ) . G l y c o s y l fluorides are e x c e l l e n t s u b s t r a t e s f o r g l y c o s i d a s e s a n d t h e y have been used t o a s c e r t a i n w h e t h e r enzyme a c t i o n o c c u r s with or without i n v e r s i o n of c o n f i g u r a t i o n a t t h e anomeric c e n t r e of t h e s u b s t r a t e s ( 2 8 - 3 0 ) . The enzymic h y d r o l y s i s of g l y c o s y l f l u o r i d e s i s n o t c o n f i n e d t o g l u c o s i d a s e s . Thus, sucrose phosphorylase w i l l utilize o 95%) fluoride ion release (Scheme l b ) . Fractionation of the cell envelope (76) i n d i c a t e d t h a t t h i s C-F b o n d cleavage in 4FG i s c o n f i n e d t o a p e p t i d y l g l y c a n a s s o c i a t e d p r o t e i n o f t h e o r g a n i s m . No C-F b o n d c l e a v a g e i s o b s e r v e d w i t h c e l l free extracts or the c y t o p l a s m i c membrane fraction. Instead, 4FG i s o x i d i z e d , t o t h e e x t e n t o f 2 g atoms of oxygen/mol, by t h e c y t o p l a s m i c membrane bound enzymes glucose o x i d a s e (GOX) and g l u c o n a t e d e h y d r o g e n a s e (GDH) (41/50)• The p r o d u c t s o f o x i d a t i o n a r e 4-deoxy-4-fluoroD-gluconate (4FGA) and 2-keto-4-deoxy-4-fluoro-Dg l u c o n a t e (2K4FGA) r e s p e c t i v e l y (Scheme l c ) . Scheme 1 (a) w h o l e c e l l s :

3FG

(b) w h o l e c e l l s :

4FG

(c) c e l l - f r e e

G

Q

X

extracts:

>

3FGA

G

D

H

>

2K3FGA

> F" + d e f l u o r i n a t e d 4FG

G

Q

X

> 4FGA

G

D

H

product(s)

> 2K4FGA

U s i n g c h l o r o a m p h e n i c o l t r e a t e d g l u c o s e o r s u c c i n a t e grown cells, i t was a l s o d e m o n s t r a t e d t h a t t h e defluorination was due to the presence of an inducible/respressible protein. The fluoride release from 4FG by chloroamphenicol t r e a t e d g l u c o s e grown c e l l s , displayed s a t u r a t i o n k i n e t i c s (Km, 3.6 m; Vmax, 1 nmol fluoride/mg protein/min) and c o u l d be p r e v e n t e d i n t h e presence of glucose, gluconate and 2-ketogluconate. Further, the complete p r o t e c t i o n o f f l u o r i d e r e l e a s e was a c h i e v e d in

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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the presence o f N-ethyl maleimide which suggested t h e involvement o f a p r o t e i n -SH g r o u p i n t h e C-F bond cleavage. These r e s u l t s are c o n s i d e r e d t o be c o n s i s t e n t with a n i n i t i a l e q u i l i b r i u m b i n d i n g o f 4FG ( b y p o s s i b l e f l u o r i n e - h y d r o g e n b o n d i n g ) t o a n -SH c o n t a i n i n g protein, f o l l o w e d by a slow f l u o r i d e e l i m i n a t i o n o r d i s p l a c e m e n t . In o r d e r t o i n v e s t i g a t e t h e pathway a n d mechanism o f fluoride release from 4FG f u r t h e r , we initially synthesized 4-deoxy-4-fluoro-D-[6- H]glucose (D-[6- H ] 4FG) ( 7 2 ) . I n c u b a t i o n o f g l u c o s e grown w h o l e c e l l s o f P. putida w i t h t h e r a d i o l a b e l e d 4FG ( s p e c i f i c a c t i v i t y 7.2 mCi/mmol), however, gave a n u n e x p e c t e d r e s u l t . T h u s , a 24 h i n c u b a t i o n o f t h e o r g a n i s m w i t h 1 mM D-[6- H]-4FG gave 100% f l u o r i d e r e l e a s e a s w e l l a s a n u n e x p e c t e d extensive release of radiolabel from the substrate. After centrifugation, approximatel 70% f th total r a d i o a c t i v i t y was f o u n the remainder i n th radioactivity remained c e l l envelope associated after extensive dialysis o f the c e l l pellet. Borate anione x c h a n g e column a n a l y s i s (28) o f t h e s u p e r n a t a n t f r a c t i o n emonstrated t h a t t h e l o s s o f r a d i o a c t i v i t y was d u e t o H9O (Figure 3, Peak A) a n d t h a t a s i g n i f i c a n t amount (20%) o f a t r i t i a t e d u n i d e n t i f i e d component (Figure 3, Peak B) was produced. Peak B was shown, by chromatographic and F o u r i e r t r a n s f o r m F NMR analysis, t o be a n o n - f l u o r i n a t e d a c i d i c c a r b o h y d r a t e . To overcome t h e e x t e n s i v e t r i t i u m l o s s encountered with D-[6- H1-4FG, we have now s y n t h e s i z e d 4-deoxy-4f l u o r o - D - [ U - C ] - g l u c o s e (D-[U- C]-4FG) by the a c t i o n o f diethylaminosulphur t r i f l u o r i d e on methyl 2,3,6-tri-ObenzoylCX - D - [ U - C ] g a l a c t o p y r a n o s i d e (Sbrissa and Taylor, unpublished r e s u l t s ) . Table I summarizes t h e distribution of r a d i o a c t i v i t y i n various fractions obtained f r o m a 24 h i n c u b a t i o n o f g l u c o s e grown whole c e l l s o f P. p u t i d a (600 mg p r o t e i n ) w i t h 1 mM D-[U- C]4FG (specific a c t i v i t y : 10,600 dpm/umol) i n 0.1 M, pH 7.1, p o t a s s i u m phosphate b u f f e r , a t 30°C. Fluoride i o n measurements i n t h e s u p e r n a t a n t f r a c t i o n s i n d i c a t e d a 95 + 5% r e l e a s e o f F~ i n two i n c u b a t i o n s . A d d i t i o n a l l y , two independent smaller scale incubations, designed t o trap any l i b e r a t e d c a r b o n d i o x i d e , showed t h a t a s much a s 4.8 ± 0.2% o f t h e r a d i o l a b e l was l o s t a s C 0 . When t h e supernatant f r a c t i o n , w h i c h a c c o u n t e d f o r more t h a n 50% o f t h e i n i t i a l r a d i o a c t i v i t y ( T a b l e I ) , was s u b m i t t e d t o borate anion-exchange column chromatography and e l u t e d with a linear ammonium tetraborate gradient, the radiolabeled m a t e r i a l was r e s o l v e d i n t o a s i n g l e poorly retained "minor peak" a n d a s t r o n g l y retained "major peak" metabolite. Internal carbohydrate standards were d e t e c t e d a t 420 nm b y t h e o r c i n o l c o l o u r i m e t r i c p r o c e d u r e (21)/ (Figure 4). A similar control chromatographic analysis o f a sample o b t a i n e d f r o m a 24 h i n c u b a t i o n o f 1.0 mM D - [ U - C ] - 4 F G u n d e r i d e n t i c a l c o n d i t i o n s , i n t h e 1

1 4

9

1 4

1 4

1 4

2

1 4

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Water

0.25 M Ammonium

tetraborate

Eluate mL F i g u r e 3. Dowex AB 1-X8 b o r a t e r e s i n m i c r o - c o l u m n e s t i m a t i o n o f t r i t i a t e d w a t e r r e l e a s e d f r o m 1 mM D-(6-^H)-4FG a f t e r 2b h i n c u b a t i o n w i t h P. p u t i d a . Column a p p l i c a t i o n s : • 25yM

10

20

Figure 11. Pyranose-2-oxidase; plots # Glucose; Km, 3.2 mM; Vmax, 126 micromol/min/mg p r o t e i n . •4-Deoxy-^-fluoro-D-glucose; Km, 5'5 mM; Vmax, 10^ micromol/min/mg p r o t e i n . Each point represents the mean o f three determinations. Data p l o t t e d by the method of l e a s t squares. S, s u b s t r a t e c o n c e n t r a t i o n (mM); V, micromol hydrogen peroxide/ min/mg p r o t e i n .

0-5-

o

10 2 S" xio mM 1

2

Figure 12. Pyranose-2-oxidase. Competitive i n h i b i t i o n of glucose o x i d a t i o n by ^-deoxy-4-fluoro-D-glucose. • Glucose; • glucose i n the presence o f 15 mM i n h i b i t o r . Data obtained as i n Figure 11. V, micromol/min/mg p r o t e i n ; S, glucose c o n c e n t r a t i o n (mM).

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

TAYLOR ET AL.

Metabolic and Enzymatic Studies

o

10 W S~ x 10 mM 1

J

F i g u r e 13- P y r a n o s e - 2 - o x i d a s e . The e f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n s of 3-deoxy-3-fluoro-Dg l u c o s e on t h e a c t i v i t y o f t h e enzyme t o w a r d glucos • g l u c o s e o n l y ; i n t h e p r e s e n c e o f : H 5 mM, A 10 mM • 15 mM 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e . D a t a o b t a i n e d a s i n F i g u r e 11. V , micromol/min/mg p r o t e i n ; 3, g l u c o s e c o n e n t r a t i c n (mM).

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

4-Deoxy-4-fluoro-D-glucitol (11)

H

F i g u r e 14. The chemoenzymatic s y n t h e s i s of 4 - d e o x y - 4 - f l u o r o D - f r u c t o s e and some mass spectrum peaks o f the p e r a c e t y l a t e d derivative. (a) I m m o b i l i z e d p y r a n o s e - 2 - o x i d a s e ; (c) sodium borohydride reduction. The o b s e r v e d m/e 129, d e r i v e d from (10), i s d i a g n o s t i c for a 2-ketopyranose.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Metabolic and Enzymatic Studies

The structure of this compound was s u p p o r t e d by a comparative e l e c t r o n i m p a c t mass s p e c t r o m e t r i c analysis of the derivatized glucose, glucosone a n d 4-deoxy-4fluoroglucosone (8) ( T a b l e I I ) . A l l t h e sugars were rendered volatile by c o n v e r s i o n to the peracetylated hydrazones o r osazones by e s t a b l i s h e d procedures (96). The t r i - O - a c e t y l - d i p h e n y l h y d r a z o n e o f ( 8 ) showed i n t e n s e

8 2 The common p e a k s a t 223 a r e d i a g n o s t i c ! o r t f i e glucosone structure ( 9 2 , 9 2 ) . The 40 mass u n i t difference between p e a k s 472 a n d 512, d e r i v e d f r o m t h e h y d r a z o n e s o f ( 8 ) a n d glucosone, i s due t o t h e p r e s e n c e o f f l u o r i n e (m/e, 19) instead o f a c e t o x y (m/e, 59) r e s p e c t i v e l y (Table I I ) . corresponding Similarly, t h e mas osazones were foun respective structures. to catalytic After isolation, ( 8 ) was s u b j e c t e d the reaction hydrogenation (Pd/C). HPLC analysis of component (9) mixture showed t h e f o r m a t i o n o f a new i s o l a t e d as a syrup which f a i l e d t o c r y s t a l l i z e . Although not crystalline, thetetra-O-acetyl derivatives o f (9) gave the correct elemental analysis and mass spectrometric fragmentation pattern f o r the formation of 4 - d e o x y - 4 - f l u o r o - t e t r a - O - a c e t y l - ap - D - f r u c t o p y r a n o s e the (10) ( a - i s o m e r shown F i g u r e 1 4 ) . T h i s i s b a s e d on and fragmentation of peracetylated fluoro-hexoses pentoses p r e v i o u s l y d i s c u s s e d (98)• Table

I I . Comparison o f m a j o r mass s p e c t r u m peaks from diphenylhydrazone O-acetylated d e r i v a t i v e s of g l u c o s e ( a ) , g l u c o s o n e ( b ) a n d 4-deoxy-4fluoro-glucosone (c) Diphenylhydrazones

(a) Penta-Oacetate m/e 556

145

Fragment

(b) Tetra-Oacetate m/e

(c) Tri-Oacetate m/e

512 223

472 223

Hydrazone (mol.ion) OC-CH=NN(Ph) diagnostic f o r glucosone

145

145

CH CO-8-COCH OCCH

2

3

3

3

103

103

103

43

43

43

CH3CO-O-COCH3

CH CO

+

3

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Thus (10) w i l l d i f f e r f r o m 4FG b y an interchange of s u b s t i t u e n t s a t C l a n d C5. As a r e s u l t , t h e p e r a c e t y l a t e d pyranose f o r m o f (9) w i l l b e more h i g h l y s u b s t i t u t e d at the ketal carbon than t h e corresponding form o f 4FG. C o n s e q u e n t l y , a s o b s e r v e d ( F i g u r e 1 4 ) , (10) w i l l be s p l i t a t C6 a n d C3 l e a d i n g t o m/e f r a g m e n t s 129, 84 a n d 55. The presence o f t h e f l u o r i n e i n (10) a t C4 a l s o makes t h e cleavage between C4-C5 a n d C4-C3 unfavourable (98) resulting, i n t h i s c a s e , w i t h a mass u n i t o f 174. The s t r u c t u r e o f 4 - d e o x y - 4 - f l u o r o - D - f r u c t o s e (9) was further confirmed b y s o d i u m b o r o h y d r i d e r e d u c t i o n t o an alditol w h i c h was shown t o be i d e n t i c a l ( o p t i c a l r o t a t i o n , IR a n d F NMR) t o 4 - d e o x y - 4 - f l u o r o - D - g l u c i t o l ( 1 1 ) , o b t a i n e d b y a s i m i l a r r e d u c t i o n o f 4FG. i y

C o n c l u s i o n and F u t u r e P e r s p e c t i v e s 1 4

Our studies with g l u c o s e (4FG) d e m o n s t r a t e t h a t t h i s s u g a r i s m e t a b o l i z e d in whole c e l l s o f P. p u t i d a w i t h c l e a v a g e o f t h e C-F bond. The i s o m e r i c 3-deoxy-3-fluoro-D-glucose (3FG), however, i s m e t a b o l i z e d w i t h r e t e n t i o n o f t h e C-F bond. This s t e r e o s p e c i f i c d e f l u o r i n a t i o n o f 4FG a p p e a r s t o be i n i t i a t e d b y an i n d u c i b l e / r e p r e s s i b l e p r o t e i n associated w i t h t h e o u t e r membrane w h i c h i s i n v o l v e d w i t h t h e u p t a k e of glucose, g l u c o n a t e and 2-ketogluconate. Subsequent i n t r a c e l l u l a r metabolism o f t h e d e f l u o r i n a t e d sugar, v i a d e c a r b o x y l a t i o n o f an a s y e t unknown m e t a b o l i t e , l e a d s t o the formation o f a 5-carbon sugar, i d e n t i f i e d a s 2,3dideoxyribonic a c i d . In a d d i t i o n t o t h e above e v e n t s , a s i g n i f i c a n t amount o f r a d i o l a b e l i s i n c o r p o r a t e d i n t o t h e peptidylglycan component o f t h e c e l l w a l l . The C-F bond is c o n s i d e r e d t o be t h e s t r o n g e s t s i n g l e bond (450-485 kJ) formed by carbon ( 9 9 ) . C l e a r l y , in a biological m i l i e u , t h i s s i t u a t i o n i s not a p p l i c a b l e . This i s f u r t h e r i l l u s t r a t e d w i t h t h e enzyme p y r a n o s e - 2 - o x i d a s e , isolated f r o m P o l y p o r u s o b t u s u s . T h u s , 4FG i s a s u b s t r a t e w h e r e a s interaction o f t h e enzyme w i t h t h e i s o m e r i c 3FG results in C-F bond c l e a v a g e a n d f l u o r i d e i o n i s r e l e a s e d . The mechanism of t h i s defluorination and t h e apparent allosteric stimulation of the c a t a l y t i c a c t i v i t y of the enzyme, r e m a i n s t o be d e t e r m i n e d . Immediate future s t u d i e s w i t h P. p u t i d a are: the i s o l a t i o n and f u n c t i o n o f t h e o u t e r membrane p r o t e i n and t o e s t a b l i s h t h e mechanism o f f l u o r i d e r e l e a s e f r o m 4FG; e l u c i d a t e t h e pathway i n t e r m e d i a t e s f o r t h e m e t a b o l i s m o f 4FG t o 2 , 3 - d i d e o x y r i b o n i c a c i d i n whole c e l l s by high resolution w i d e b o r e NMR s p e c t r o m e t r y (100); ascertain the l o c a t i o n and n a t u r e o f t h e r a d i o l a b e l incorporated into the peptidylglycan component. Whether the incorporation leads t o a modified peptidylglycan (cell wall) i s a question of p a r t i c u l a r interest i n r e l a t i o n t o t h e e f f e c t s on c e l l g r o w t h . P r e l i m i n a r y e x p e r i m e n t s show that 4FG i n h i b i t s t h e growth o f P. putida a n d P.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Metabolic and Enzymatic Studies

133

a e r u g i n o s a . I n t h e l o n g e r t e r m , w h e t h e r t h i s f o r m o f 4FG metabolism i s confined t o t h e Pseudomonads or i s a p p l i c a b l e t o o t h e r g r a m - n e g a t i v e b a c t e r i a , r e m a i n s t o be determined. I n g e n e r a l , when t h e C-F b o n d i s r e t a i n e d , t h e e a r l y biochemical rationale for the synthesis of deoxyfluorinated sugars h a s now b e e n i l l u s t r a t e d by numerous examples. O u r own s t u d i e s indicate that deoxyfluoro s u g a r s may s e r v e a s n o v e l membrane, enzyme and metabolic probes. A d d i t i o n a l l y , as a r e s u l t o f demonstrated incorporation and/or "lethal synthesis", f l u o r i n a t e d s u g a r s have c o n s i d e r a b l e p o t e n t i a l t o augment the a n t i b i o t i c and a n t i v i r a l a r s e n a l . Acknowledgments One o f us (NFT Engineering Researc c o n t i n u e d s u p p o r t , w i t h o u t w h i c h much o f t h i s work would not have been possible. The awards of an NSERC Scholarship (TD), an O n t a r i o Graduate S c h o l a r s h i p (DS) and a U n i v e r s i t y o f Windsor Scholarship (STS) a r e g r a t e f u l l y acknowledged.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Penglis, A . A . E . Adv. Carbohydr. Chem. Biochem. 1981, 38, 195-285. Taylor, N . F . ; Kent, P.W. J. Chem. Soc. 1958, 168, 872-875. Bekoe, A . ; Powell, H.M. Proc. R. Soc. Ser. A. 1959, 250, 301-315. Kent, P.W. Chem. Ind. (London) 1969, 1128-1132. Barnett, J . E . G . In Ciba Fdn. Symp. Carbon Fluorine Compounds: Elsevier & Associated Scientific Publishers, Amsterdam, 1972; 95-115. Dwek, R.A. In Ciba Fdn. Symp. Carbon Fluorine Compounds; Elsevier & Associated Scientific Publishers, Amsterdam, 1972; 239-279. Ciba Fdn. Symp.: Carbon-Fluorine Compounds; Elsevier & Associated Scientific Publishers, Amsterdam, 1972, 1-417. Biochemistry Involving Carbon-Fluorine Bonds: Filler, R., E d . ; ACS Symposium Series 28, American Chemical Society, Washington, D . C . , 1976, 1-214. Peters, R . A . ; Wakelin, R.W.; Buffa, P . ; Thomas, L . C . Proc. R. Soc. Ser. B 1953, 140, 497-506. Kirsten, E.; Sharma, M . L . ; Kun, E . Molec. Pharmacol. 1978, 14, 172-184. Peters, R.A. In Biochemical Lesions and Lethal Synthesis; Alexander, P. and Bacq, Z . M . , Eds. Pergamon Press, Oxford, 1963, 88-130. Kornberg, H . L . ; Smith, J . FEBS Lett. 1972, 20 270272.

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13. D'Amore, T.; Duronio, V.; Cheung, M.O.; Lo, T.C.Y. J. Cell. Physiol. 1986, 126, 29-36. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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D'Amore, T . ; Lo, T.C.Y. J . Cell Physiol. 1986, 127, 95-105. Barnett, J . E . G . ; Holman, J . D . ; Chalkley, R . A . ; Biochem. J . 1975, Biochem. J. 145, 417-4229. Lopes, D . P . ; Taylor, N.F. Carbohydr. Res. 1979, 73, 124-134. Barnett, J . E . G . ; Holman, J . D . ; Munday, K.A. Biochem. J . 1973, 131, 24-221. Halton, D.M.; Taylor, N . F . ; Lopes, D. J. Neurosci. Res. 1980, 5, 241-252. Al-Jobore, A . ; Moses, G . C . ; Taylor, N.F. Can, J. Biochem. 1980, 58, 1397-1404. Agbanyo, F . R . ; Taylor N.F Biochem J. 1983 223 257-262. Schmalstig, J.G. 85, 407-12. Eisenthal, R.; Harrison, R.; Lloyd, W . J . ; Taylor, N.F. Biochem. J . 1972, 130, 199-205. Bessell, E . M . ; Foster, A . B . ; Westwood, J . H . Biochem. J. 1972 128, 199-204. Bessell, E . M . ; Thomas, P. Biochem. J. 1972, 131, 7782. Thomas, P . ; Bessell, E . M . ; Westwood, J . H . Biochem. J. 1974, 139, 661-664. Wright, J . A . ; Taylor, N . F . ; Brunt, R . V . ; Brownsey, R.W.; Chem. Commun. 1972, 691-692. Robyt, J . F . In Encyclopedia of Polymer Science and Engineering: Mark, H . F . , Bikales, N.M. and Overberger, C . G . , Eds.; Wiley, New York, 1986, 2nd Edition; V o l . 4, pp. 752-767. Barnett, J . E . G . Biochem. J. 1971, 123, 607-611. Hehre, E.J.; Brewer, C . F . ; Ganghof, D.S. J. B i o l . Chem. 1979, 254, 5942-5950 Kasumi, T . ; Tsumuraya, Y . ; Brewer, C . F . ; KerstersHilderson, H . ; Claeyssens, M . ; Hehre, E . J . Biochemistry 1987, 26, 3010- . Gold, A . M . ; Osber, M.P. Biochem. Biophys. Res. Commun. 1971, 42, 469-474. Treder, W.; Thiem, J.; Schlingmann, M. Tetrahedron Lett. 1986, 27, 5605-5608. Sadozi, K . K . ; Nukada, T . ; Ito, Y . ; Nakahara, Y . ; Ogawa, T . ; Kobata, A. Carbohvdr. Res. 1986, 157, 101-123 Butchard, C . G . ; Dwek, R . A . ; Kent, P.W.; Williams, R . J . P . ; Xavier, A.V. Eur. J. Biochem. 1972, 27, 548553. Hoffman, R . A . ; Forsen, S.W. Progress in NMR Spectroscopy: Emsley, J.W.; Feeny, J. and Sutcliffe, Eds. Pergamon Press, Oxford, 1963, 1, 15204.

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Metabolic and Enzymatic Studies

Street, I . P . ; Armstrong, C . R . ; Withers, S.G. Biochemistry 1986, 25, 6021-6027. Withers, S . G . ; MacLennan, D . J . ; Street, I.P. Carbohydr. Res. 1986, 154, 127-144. Taylor, N . F . ; Hill, L . ; Eisenthal, R. Can. J. Biochem. 1975, 53, 57-64. Drueckhammer, D . G . ; Wong, C-H. J. Org. Chem. 1985, 50, 5912-5913. Squire, S . T . ; Taylor, N.F. Proc. Can. Fed. B i o l . Soc. Abstr. 1985, 28, 226. Brunt, R . V . ; Taylor, N.F. Biochem. J. 1967, 105, 41c. Brunt, R . V . ; Taylor, N.F. Biochem. J. 1969, 114, 445-447. Woodward, B . ; Taylor, N . F . ; Brunt, R.V. Biochem. Pharmacol. 1971 20 1071-1077 Heredia, C . F . Biophys. Acta 1964 Miles, R . J . ; P i r t , S . J . ; J. Gen. Microbiol. 1973, 76, 305-318. Taylor, N . F . ; Louie, L.; Can. J. Biochem. 1977, 55, 911-915. Peterkofsky, A . ; Trends Biochem. S c i . 1977 2, 12-14. White, F . H . ; Taylor, N.F. FEBS Lett. 1970, 11, 268272. Midgley, M . ; Dawes, E . A . ; Biochem. J. 1973 132, 141154. Roberts, B . K . ; Midgley, M . ; Dawes, E . A . J. Gen. Microbiol. 1973, 78, 319-329. Taylor, N . F . ; Hill, L . ; Eisenthal, R. Can. J. Biochem. 1975, 53, 57-64. Entner, N . ; Doudoroff, M. J. B i o l . Chem. 1952, 196, 853-862. D'Amore, T . ; Taylor, N.F. FEBS Lett. 1982, 143, 247251. Romaschin, A . ; Taylor, N . F . ; Smith, D . A . ; Lopes, D. Can. J. Biochem. 1977, 55, 369-375. Romaschin, A . ; Taylor, N . F . ; Can. J. Biochem. 1981, 59, 262-268. L a i , C . Y . ; Horecker, B . L . i n Essays i n Biochemistry; Campbell, P.M. and Dicknes, F . eds. Academic Press, London, U.K. 1972, 8, p. 149. Rose, J . A . ; Adv. Enzymol. Relat. Areas Mol. B i o l . 1975, 43, 491. Agbanyo, M . ; Taylor, N.F. Bioscience. Rep. 1986, 6, 309-316. Defay, J.; Driguez, H . ; Henrissat, B. Carbohydr. Res. 1975, 63, 41-49. Wang, T . ; Himoe, A. J. B i o l . Chem. 1974, 249, 38953902. Grier, T.J.; Rasmussen, J . R . Biochem. J. 1983 209, 677-685.

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136

62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86.

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Schwarz, R . T . ; Schmidt, M . F . G . ; Datema, R. Biochem. Soc. Trans. 1979, 7, 322-326. Schwarz, R . T . ; Datema, R. Trends. Biochem. S c i . 1980, 5, 65-67. Hubbard, S . C . ; Robbins, P.W. J. B i o l . Chem. 1979, 254, 4568-4576. Datema, R.; Schwarz, R.T. Biochem. J. 1979, 184, 113-123. Datema, R.; Schwarz, R . T . ; Jankowski, A.W. Eur. J. Biochem. 1980, 109, 331-341. Datema, R.; Schwarz, R . T . ; Winkler, J . Eur. Biochem. 1980, 110, 355-361. McDowell, W.; Datema, R.; Romero, P . A . ; Schwarz, R.T. Biochemistry. 1985, 24, 8145-8152. Kornfeld, R.; Kornfeld, S. Ann. Rev. Biochem. 1985, 54, 631-664. Grier, T.J.; Rasmusssen 127, 100-104. Grier, T.J.; Rasmussen, J . R . J. B i o l . Chem. 1984 259, 1027-1030. Rottenberg, D . A . ; Cooper, A . J . L . Trends Biochem. Sci. 1981, 6, 120-122. Ter-Pogossian, M.M., Raichle, M . E . ; Sobel, B . E . S c i . Amer. 1980 243, 171-181. Greenberg, G . H . ; Reivich, M . ; A l a v i , A . ; Hand, P . ; Rosequist, A . ; Rintelmann, W.; Stein, A . ; Tusa, R.; Dann, R.; Christman, D . ; Fowler, J.; MacGregor, B . ; Wolf, A. Science 1981, 212, 678-680. Gallagher, B . M . ; Fowler, J . S . ; Gutterson, N . I . ; MacGregory, R.R.; Wan, C . N . ; Wolf, A . P . J. Nucl. Med. 1978, 19, 1154-1161. Mizuno, T . ; Kageyama, J. Biochem. 1978, 84 179-191. Samuel, J.; Taylor, N.F. Carbohydr. Res. 1984, 133, 168-172. Clark, M.G. Int. J. Biochem. 1978, 9, 17-18. F l o r i d i , A. J. Chromatog. 1971, 59, 61-70. Dangyan, M . T . ; Arakelyan, S.V. Nauch. Trudy Esevan. Gosudarst. Univ. Ser. Khim., 1954, 44, 35-39; Chem. Abstr. 1959, 53, 21648i. Janssen, F.W.; Ruelius, H.W. Biochim. Biophys. Acta 1968, 167, 501-510. Ruelius, H.W.; Kerrwin, R.M.; Janssen, F.W. Biochim. Biophys. Acta 1968, 167, 493-500. Janssen, F.W.; Ruelius, H.W. In Methods in Enzymology: Wood, W.A. E d . ; Academic: New York, 1975, 41, 170-173.' Geigert, J.; Neidelman, S . L . ; Hirano, D.S. Carbohydr. Res. 1983, 113, 159-162. Geigert, J.; Neidelman, S . L . ; Hirano, D . S . ; Wolf, B.; Pauschar, B.M. Carbohydr. Res. 1983, 113, 163165. Yozo, M . ; Toru, N. Agric. B i o l . Chem. 1984, 48, 2463-2470.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

7. TAYLOR ETAL.

Metabolic and Enzymatic Studies

87.

137

Lineweaver, H . ; Burk, D. J. Amer. Chem. Soc. 1934, 56 568-666. 88. Eisenthal, R.; Cornish-Bowden, A. Biochem. J. 1974 139, 715-720. 89. Hanes, C.S. Biochem. J. 1932, 26, 1406-1421. 90. Hofstee, B . H . J . J. B i o l . Chem. 1952, 199, 357-364. 91. Krupka, R.M. Biochem. 1966, 5 1988-1998. 92. Volo, J.; Sedmera, P . ; Musilek, V. F o l i a Microbiol. 1978, 23, 292-298. 93. Barnett, J . E . G . ; Atkins, G.R.S. Carbohydr. Res. 1972, 25, 511-515. 94. Pacak, J.; Halaskova, J.; Stepan, V . ; Cerny, M. Collect. Czech. Chem. Comm. 1972, 37, 3646-3651. 95. Tipson, R.; Brady, R . F . ; West, B . F . Carbohydr. Res. 1971, 16, 383-393. 96. Rao, G . V . ; Que L.; H a l l L . D . ; Foudy R.P Carbohydr. Res 97. L u i , F - N . E . ; Wolf Geigert S . L . ; Chin, J . D . ; Hirano, D.S. Carbohydr. Res. 1983, 113, 151-157. 98. Chizov, O . S . ; Kadentsev, V . I . ; Zolotarev, B . M . ; Foster, A . B . ; Jarman, M . ; Westwood, J . H . Org. Mass Spectrum 1971, 5 437-445. 99. Sharpe, A . G . i n Ciba Fdn. Symp. Carbon Fluorine Compounds; Elsevier Associated Scientific Publishers, Amsterdam, 1972; 33-54. 100. Campbell-Burk, S . L . ; Shulman, R.G. Ann. Rev. Microbiol. 1987, 47, 595-616. RECEIVED January 15, 1988

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 8

Sucrose Transport in Plants Using Monofluorinated Sucroses and Glucosides William D. Hitz Central Research and Development Department, E. I. du Pont de Nemours and Company, Experimental Station, Wilmington, DE 19898 T h e s u c r o s e carrie developing s o y b e a s u c r o s e derivitives which are singly fluorinated at C-1', C-4', a n d C-6'. S o m e a -glucosides are also recognized and transported and may be used a s sucrose analogs in studying carrier-substrate interaction. Phenyl a -D-thioglucopyranosides fluorinated singly at positions C - 3 , C-4, a n d C-6 along with the deoxy-glucosides at these positions indicate that recognition by the plant carrier requires interaction at these three hydroxyls along with hydrophobic interaction with the β -face of the g l u c o s e moiety a n d the a -face of the fructose moiety in sucrose. 1'-Deoxy-1'-fluorosucrose is also recognized a n d transported by the carrier proteins in the vascular tissues of leaves a n d is thus moved in long distance transport in a manner identical to s u c r o s e . At limiting substrate c o n c e n t r a t i o n s , 1'-deoxy-1'-fluorosucrose is metabolized by s u c r o s e synthase at a rate 3.6 times slower than s u c r o s e . Hydrolysis by invertase however occurs at a rate 4200 times slower than sucrose. In vivo rates of metabolism for the 1'-fluoro derivitive a n d for s u c r o s e showed that s u c r o s e is metabolized by both e n z y m e s acting in parallel, a n d that the relative contribution of the individual e n z y m e s varies with tissue development.

In many plant s p e c i e s , including virtually all major field crops and many vegetable crops, s u c r o s e is the carbohydrate utilized 0

0097-6156/88/0374-0138$06.00/0 1988 American Chemical Society

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8.

139

Sucrose Transport in Plants

HITZ

for long distance transport a n d for soluble, short term

storage.

T h e carbon which makes up the bulk of the dry weight harvested for economic yield is p r o c e s s e d through the e n z y m e s a n d other proteins in the physical a n d metabolic pathways of s u c r o s e metabolism.

S i n c e much of the increased yield potential

achieved in breeding improved varieties of crop plants h a s c o m e from a more efficient transfer of carbon fixed in photosynthesis to harvestable portions of the plant (1), there is continuing interest in detailing the early steps of s u c r o s e

metabolism in

order to test direct methods of improving yields through or c h e m i c a l

modification

of controlling

steps

genetic

in metabolism.

Two plant e n z y m e s are capable of catalyzing the breakage of the glucose to fructose bond in sucrose hydrolysis

either

Invertase catalyzes

in th

extra-cellular s p a c e , a n d s u c r o s e synthase c a t a l y z e s the transfer of the glucose residue to Uridine diphosphate Since

extra-cellular

membrane

invertase

is present

transport of s u c r o s e

h a s two possibilities

Uptake of the intact molecule occurs a s d o e s followed by uptake of the hexoses produced. fluorinated

carbohydrates

a s alternate

(2.).

in s o m e tissues, the also.

hydrolysis W e have used

substrates

for these

e n z y m e s a n d transport proteins in order to determine both how substrate interaction occurs a n d to determine which of the alternative elements

pathways

is functioning

of substrate

interaction

in vivo . with

T h e important

carrier

determined from the interaction of fluorinated

proteins sucroses and

s u c r o s e analogs w a s used to guide the synthesis of affinity probes

for protein

identification

a n d the fluorinated

sucroses

themselves have been used to determine the relative flux of s u c r o s e through paralell enzymic pathways in vivo. Substrate

Recognition

bv S u c r o s e

Transporters

T r a n s m e m b r a n e movement of sucrose is accomplished by transport proteins in several, but not all tissue types. T h e most obvious example of a specific tissue type is the phloem. T h e s u c r o s e concentration in this tissue c a n approach 0.8 M in contrast to m M concentration in the surrounding tissues a n d probably s u b m M concentration in the extra cellular s p a c e s surrounding the phloem (2). Transport into the cells of the phloem is difficult to study a s they are an integral part of the leaf or stem structure, a n d may comprise only 5 to 10% of the total leaf mass. Another example of s u c r o s e transport is the accumulation of s u c r o s e a n d other nutrients by cells of

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

140

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

developing embryos or embryo storage tissue.

Large

accumulations are not obvious in these tissues, but the physiological evidence suggests that s u c r o s e uptake occurs, a n d that in the c a s e of the developing s o y b e a n cotyledon, it has many of the s a m e characteristics a s phloem uptake

(4*5).

S i n c e the soybean cotyledon is quite homogeneous a s to tissue type (S.)

a n d c a n be readily manipulated to remove cell

walls in tissue slices to yield protoplasts (7), we have these protoplasts a s a model system to study transport of s u c r o s e in general.

used

membrane

T h e concentration kinetics of

s u c r o s e influx suggest that at least two p r o c e s s e s are operating in parallel. O n e of these is saturable with respect to s u c r o s e concentration

of comparatively

low capacity a n d

inhibited by any of severa specificity of this system c a n be studied by using substrates sucrose

a s apparent

inhibitors of the influx of

alternate radiolabled

present at a concentration well below the concentration

required for half-maximal monosaccharides

uptake

(K ). m

Studies in which

a n d natural disaccharides other than

sucrose

were tested a s substrates show the carrier to be quite for s u c r o s e (8.).

singly substituted s u c r o s e s , the

specific

Accordingly, we c h o s e to make a series of using fluorine a s a substitute for

hydroxyl, to systematically

probe the substrate

binding

site

in the s u c r o s e carrier protein in these cells. Fructosvl-Substituted S u c r o s e s . T h e deoxy-fluoro-derivatives of sucrose at C - 1 \ C-4' a n d C-6', along with deoxy a n d deoxyazido-derivatives at C-6' a n d C - V respectively were prepared by the sucrose synthase catalyzed coupling of U D P - g l u c o s e and the substituted fructose (9.10). T h o s e structures a n d their binding constants relative to s u c r o s e (estimated from their respective Kj's) are given in Figure 1a. Substitutions at C - V and C-6' which were more hydrophobic than the hydroxyl they replaced, bound about two fold more tightly than sucrose. T h e amino substitution at C - V (formed by catalytic reduction of the V-azido) bound about one-half a s well a s the native structure. W e reason that this binding pattern could occur if the three dimensional structure of s u c r o s e a s s o c i a t e s with the binding site of the protein such that the relatively hydrophobic surface of s u c r o s e formed by the portions of the two monosaccharide rings which are s h a d e d in Figure 2 a interact with a similarly shaped, hydrophobic site on the protein. T h e hydrophobic substitutions at C - V and C-6' act to increase the size of this

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8. HITZ

Sucrose Transport in Plants

141

Figure 1a and 1b. T h e relative binding constants for 6 substituted s u c r o s e s (1a) and 10 phenyl

RELATI

1

40

O

a X

20 it )

n 1 10

fit 20

a

$

S

A

-

A

-

•

D

1 40

30 TIME (min)

50

i 60

i 70

Figure 6. Translocation of C - s u c r o s e ( x,d) a n d C-1'deoxy-V-fluorosucrose (A, O) into young s o y b e a n leaves. Counts per minute ( C P M ) , measured by Geiger-Mueller counting, were corrected for background a n d expressed a s per cent of C P M at 66 min. C - s u g a r s were supplied to an exporting leaf at time zero by application of a buffered solution containing the sugar to an a b r a d e d surface. (Reproduced with permission from ref. 16. Copyright 1987 T h e A m e r i c a n Society of Plant Physiologists.) 1 4

1 4

1 4

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8.

HITZ

Sucrose Transport in Plants 40

151

A - 4% FLL

30 20

O

10 40

B - 7% FLL

30 20 10 CO

z> -J

o z

50 -

C - 11% FLL

40

id 30 m

3 g o < cc

20 10

50

8

D - 17% FLL O

40

OQ

30 20 10 40 •

E - 40% FLL

30 o

20

„ o o o ° o oS^b o o „^ o

10 • 20

40

60 80 TIME (min.)

100

120

Figure 7. The time course of percent incorporation into ethanol insolubles of phloem translocated sucrose (o) and 1 '-deoxy-1 'fluorosucrose (•) in soybeanfirsttrifoliolate leaves of various ages (%FLL is the percent offinalleaf length of a comparable leaf at maturity). Radiolabled sucrose and/or 1'-deoxy-1'-fluorosucrose were supplied as described in Figure 3 and importing leaves were either removedfromsampling at time points or parts of the leaf were removed at time points and extracted as described in the text. (Reproduced with permissionfromref. 16. Copyright 1987 The American Society of Plant Physiologists.)

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

152

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS Quantitative

determination

of the relative

contributions

of s u c r o s e synthase and invertase to the breakdown of phloemsupplied s u c r o s e c a n b e determined from the total

metabolism

rate for e a c h sugar a n d the discrimination factor for the two sugars a s substrates for invertase and sucrose synthase.

The

total rate of breakdown of either sucrose or V - F S is the s u m of the fluxes through the two e n z y m e s so that: R and

= Is + S S

s

Rf

s

(3)

S

= Ifs + S S f

(4)

s

where R is the total rat the contribution of invertas contribution of s u c r o s e synthase. At the tracer concentrations arriving in the importing leaf, the ratio of s u c r o s e to V - F S metabolism by s u c r o s e synthase is 3.6 times the ratio of substrate concentration, a n d the s a m e ratio for invertase is 4200 times the ratio of substrate concentration. Thus:

SS

S

= 3.6 S S f s

[

1

4

C

-

S

U

C

r

°

S

e

l

[14 c - V - F S ]

and

l

s

= 4200 l f

[

1 4

(5)

C-sucrose]

s

[14c- V-FS]

(6)

S i n c e the two substrates are transported identically, a n d supplied at the s a m e concentration to the mature leaves, their initial concentrations in their respective importing l e a v e s will be very similar a n d the substrate ratios in equations 5 a n d 6 will be essentially o n e . S i n c e s u c r o s e is always metabolized faster than V - F S , the ratio will b e c o m e less than o n e with time and the apparent relative metabolism rates for the two sugars will c h a n g e a s c a n be s e e n in figure 7. During the initial, linear period of label incorporation into insolubles however the ratio can be a s s u m e d to be o n e . Assuming this simplification, dividing equation 3 by equation 4 and substituting for S S f s and Ifs from equations 5 and 6 respectively, then solving for I s / S S s , gives

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8. HITZ

153

Sucrose Transport in Plants

,

s

/

S

S

s

m

1-(Rs/Rfs)/3.6 (Rs/Rfs)4200-1

T h e ratios Rs/Rfs from the data of figure 7 and the corresponding contributions of invertase and s u c r o s e

synthase

calculated from those ratios are given in T a b l e I for leaves of different T a b l e I.

developmental

ages.

T h e relative contribution of invertase and

synthase to s u c r o s e metabolis Leaf A g e

i

developin

sucrose

soybea

Rate of Sugar Metabolism

(% F L L i a

_Rs_.

leave

% of Total. Metabolism

b _R /Rfs_ _ I / S S

_Rfs_

S

S

C S

_. Js_

0.25

0.074

3.4

0

0

100

7.6

0.64

0.095

6.7

0.86

46

54

1.5

0.19

7.9

1.20

54

46 58 57

17.0

1.0

0.16

6.2

0.72

42

40.0

0.37

0.059

6.5

0.75

43

L e a f age as determined by the % of length attained by a

comparable leaf b

S

4.0 11.0

a

_SS _

at full development.

R a t e of sucrose or 1'-FS

metabolism in % of total radiolabel in

the leaf converted to insolubles per minute. c

C a l c u l a t e d using equation 7.

T h e rate of carbon import and s u c r o s e metabolism increased very rapidly during early development. Most of the increased rate of sucrose metabolism could be accounted for by a sharp rise in the flux through invertase. A s the leaf b e c a m e photosynthetically competent, the rate of utilization of c a r b o n imported from mature leaves declined, however about one-half of s u c r o s e

metabolism

remained through

invertase.

In leaves at early stages of development, extractable invertase activity (measured at its p H optima) c a n e x c e e d extractable s u c r o s e synthase activity by 10 to 20 fold (15L), yet compartmentalization of e n z y m e and substrate may make the relative flux through these parallel paths quite different from

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

154

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

the proportionate activities.

T h e method described above is o n e

of the few that c a n discern in vivo flux in this way.

As a

general method it could be applied to any system for which differentially

metabolizable

substrates c a n be produced.

In the

c a s e of 1'-FS used in this way, a problem arises d u e to the extreme discrimination against 1'-FS by invertase.

A very

small change in the ratio Rs/Rfs corresponds to a large change in the flux through invertase, sensitive to errors

making the calculation

very

in the ratio.

Conclusions a n d Future Studies T h e tenative identificatio f carrie protei many a v e n u e s of researc s u c r o s e binding proteins in other tissues using the s u c r o s e photoprobe a n d utilizing antibody c r o s s reactivity. Ultimately the function of the 62 kD membrane protein from cotyledons needs to be proven either by the biochemical methods of reconstitution into artificial m e m b r a n e v e s i c l e s , by c o m p a r i s o n of the implied protein structure (obtained from the structural g e n e sequence) with other, known transporters, or by genetically manipulating transport. T h e use of fluorinated s u c r o s e s a s tracers of s u c r o s e metabolism in order to differentiate between glycolysis started by invertase a n d glycolysis started by s u c r o s e synthase should be quite useful in determining the in vivo flux of these paths and the large differences in phosphate metabolism which a c c o m p a n y the two routes of carbon metabolism.

Acknowledgments T h e active collaborations of Dr. Peter C a r d in the synthesis of fluorinated carbohydrates, Mr. Kevin Ripp in protein purification and preparation of antibodies, Dr. Vincent Francheschi in immunohistochemistry a n d Dr. Judy Schmalstig in the m e t a b o l i s m of 1 '-deoxy-1 '-flurosucrose are greatfully a c k n o w l e d g e d .

Literature 1.

Cited

Gifford, R. M.; Thorne, J . ; Hitz, W.; Giaquinta, R. 1984, 225, 801-8.

Science

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8. IIITZ 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Sucrose Transport in Plants

155

Avigad, G. In Plant Carbohydrates I. Encyclopedia of Plant Physiology. New Series: Springer-Verlag: Berlin, 1982; vol. 13A; pp. 217-347. Giaquinta, R. T. In The Biochemistry of Plants: Academic: New York, 1980; vol. 3; pp. 271-320. Lichtner, F. T.; Spanswick, R. Plant Physiol. 1981, 68, 693-8. Thome, J. H. Plant Physiol. 1982, 70,953-8. Thome, J. H. Plant Physiol. 1981, 67, 1016-25. Lin, W.; Schmitt, M.; Hitz, W.; Giaquinta, R. Plant Physiol. 1984, 75, 936-40. Schmitt, M. R.; Hitz, W.; Lin, W.; Giaquinta, R. Plant Physiol. 1984, 75, 941-6 Card, P. J.; Hitz Card, P. J.; Hitz, W. J. Am. Chem. Soc. 1986, 108, 158-61. Hitz, W. D.; Card, P.; Ripp, K. J. Biol. Chem. 1986, 261, 11986-91. Mathlouthi, M.; Cholli, A.; Koenig, J. Carbo. Res. 1986, 147, 1-9. Hindsgual, O.; Norberg, T.; Lependu, J.; Lemeiux, R. Carbo. Res. 1982, 109, 109-142. Guthrie, R. D.; Jenkins, I.; Yamasaki, R. Aust. J. Chem. 1982, 35, 1003-8. Giaquinta, R. T. Plant Physiol. 1978, 61, 380-5. Schmalstig, J. G.; Hitz, W. Plant Physiol. 1987, 85, 40712.

RECEIVED January 15, 1988

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 9

Preparation of Fluorine-18 Deoxyfluorohexoses for Metabolism and Transport Studies 1

2

2

2

S. John Gatley , James E. Holden , Timothy R. DeGrado , Chin K. Ng , James R. Halama , and Robert A. Koeppe 2

2

1The University of Chicago, Franklin McLean Institute, Box 433, 5841 South Maryland Avenue, Chicago, IL 60637 Medical Physics Department Avenue

2

The preparation of deoxyfluorohexoses labeled with the positron emitter Fluorine-18, and their use in studies of hexose transport and phosphorylation with isolated perfused working rat hearts is described. Compartmental analysis methods were applied to time courses of F-18, measured with scintillation probes, in hearts given 2-deoxy-2-fluoro-D-glucose (2FDG). Rate constants determined from model fits were used to calculate 2FDG distribution volumes and phosphorylation rates for hearts operated without insulin at two work loads and several perfusate glucose concentrations. Estimates of intracellular glucose contents were made from the F-18 data and independent measures of glucose utilization. The results confirm the saturability of glucose transport with increasing perfusate glucose and showed enhanced glucose content at higher workloads. The growing interest in sugars labeled with Fluorine-18 over the last dozen years has been fuelled by the development of positron emission tomography (PET) as a medical imaging modality (JL) . The concentration of a radionuclide which decays with emission of a positron can be measured accurately within the human body, by coincidence detection of the pair of 511keV gamma rays which are created when each positron and an atomic electron are mutually annihilated. Commercial tomographs are now available with a spatial resolution of about 5mm, and with suitable radiotracers the rates of metabolic and physiological processes can be estimated in the brains and other organs of patients and experimental subjects. PET is distinguished from routine, clinical nuclear medical imaging by its superior quantitation and spatial isolation. In addition, an accident of nature has decreed that the longest lived externally detectable isotopes of the most 0097-6156/88/0374-0156$06.00/0 • 1988 American Chemical Society

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

9, GATLEY ET AL.

157

Fluorine-18 Deoxyfluorohexoses

i m p o r t a n t b i o l o g i c a l e l e m e n t s , oxygen, n i t r o g e n , a n d c a r b o n , a r e a l l positron emitters. Thus, i m a g i n g b a s e d on p o s i t r o n a n n i h i l a t i o n r a d i a t i o n i s most a p p r o p r i a t e f o r j j i v i v o s t u d i e s w i t h a m a j o r i t y o f m e t a b o l i c a l l y a c t i v e compounds. T h e r e a r e no i s o t o p e s o f h y d r o g e n w h i c h emit gamma r a y s o r p o s i t r o n s , b u t f l u o r i n e c a n o f t e n s u b s t i t u t e f o r h y d r o g e n (or h y d r o x y l g r o u p s ) w i t h r e t e n t i o n o f b i o l o g i c a l a c t i v i t y , a n d F-18 i s a r e a d i l y a v a i l a b l e n u c l i d e w i t h an e x t r e m e l y c o n v e n i e n t h a l f - l i f e (110 m i n u t e s ) f o r i n v i v o medical use. Short a s t h i s h a l f - l i f e may a p p e a r t o w o r k e r s accustomed t o c o n v e n t i o n a l biomedical t r a c e r s with half-lives r a n g i n g f r o m weeks t o m i l l e n i a , r e c e n t d e v e l o p m e n t s have made i t p o s s i b l e t o p r e p a r e many F-18 compounds i n h i g h y i e l d s b a s e d on the i s o t o p e (2.) • The o t h e r i m p o r t a n t p o s i t r o n e m i t t e r s , C - l l , N-13, a n d 0-15, i n f a c t have c o n s i d e r a b l y s h o r t e r h a l f - l i v e s o f 20, 10, a n d 2 m i n u t e s r e s p e c t i v e l y (3.) . F-18 h a s two o t h e r advantages apart from i t s h a l f - l i f e One i s t h a t i t i s a v a i l a b l e i n moderate a c t i v i t i e s cyclotrons (or other c h a r g e d - p a r t i c l N-13, and C - l l a r e only a v a i l a b l e from c y c l o t r o n s . The s e c o n d advantage i s t h a t t h e r a n g e o f t h e p o s i t r o n i n t i s s u e (about 2mm) i s s h o r t e r than f o r t h e other n u c l i d e s , so t h a t h i g h e r r e s o l u t i o n images a r e i n p r i n c i p l e a v a i l a b l e , a l t h o u g h t h e s t a t e - o f - t h e - a r t i n tomograph d e s i g n does n o t y e t make t h i s i m p o r t a n t . The most important organic PET radiotracer i s 2-deoxy-2-fluoro-D-glucose (IL) , w h i c h i s a b b r e v i a t e d t o 2FDG b y workers i n t h i s f i e l d , p r o b a b l y because t h e t r a c e r k i n e t i c t h e o r y underlying i t s use i s t h a t developed by S o k o l o f f ' s group f o r a n i m a l a u t o r a d i o g r a p h i c s t u d i e s w i t h C-14 2-deoxy-D-glucose, which i s commonly t e r m e d 2DG (£.) . Thus, t h e a b b r e v i a t i o n 2FDG c a r r i e s w i t h i t t h e i m p l i c a t i o n t h a t i t s d i s t r i b u t i o n c a n be i n t e r p r e t e d i n t h e same way a s t h a t o f 2DG. The r a t i o n a l e o f t h e 2 ( F ) D G method i s t h e i s o l a t i o n o f t h e h e x o k i n a s e r e a c t i o n w h i c h o c c u r s b e c a u s e t h e r e s u l t i n g 2 (F)DG-6-phosphate i s s t r u c t u r a l l y i n c a p a b l e of t a k i n g p a r t i n t h e subsequent hexose-6-phosphate isomerase r e a c t i o n o f t h e normal g l y c o l y t i c pathway (2)• The p h o s p h o r y l a t e d tracer i s said t o be m e t a b o l i c a l l y trapped (JL) , since d e p h o s p h o r y l a t i o n i s v e r y slow i n most t i s s u e s , such as b r a i n (JL) , w h i l e d i r e c t d i f f u s i o n o u t o f t h e c e l l o r m e t a b o l i s m t o more d i f f u s i b l e s p e c i e s do n o t o c c u r t o s i g n i f i c a n t e x t e n t s . The amount o f 2(F)DG-6-P a c c u m u l a t e d c a n be d e t e r m i n e d f r o m t h e t o t a l r a d i o a c t i v i t y i n e a c h b r a i n r e g i o n , by a p p l y i n g a t r a c e r k i n e t i c model (£_, 1£L) Local rates of glucose metabolism a r e then e s t i m a t e d f r o m t h e known p r o p o r t i o n a l i t y b e t w e e n g l u c o s e a n d 2(F)DG p h o s p h o r y l a t i o n r a t e s (11). Extensive d i s c u s s i o n of these a s p e c t s o f t h e 2(F)DG method c a n be f o u n d i n t h e c i t e d r e f e r e n c e s . Preparation

o f F-18

Fluorosugars

The p r e p a r a t i o n o f r e a c t i v e F-18 h a s r e c e n t l y been r e v i e w e d (4) . L a b e l e d m o l e c u l a r f l u o r i n e was f i r s t p r e p a r e d f r o m t h e N e ( d , alpha) F r e a c t i o n by b o m b a r d i n g Ne-20 c o n t a i n i n g a t r a c e o f F w i t h a d e u t e r o n beam i n an a l l - n i c k e l c y c l o t r o n t a r g e t (12.) . A p p r o x i m a t e l y one t h i r d o f t h e r a d i o i s o t o p e i s e x c h a n g e d i n t o t h e d e s i r e d c h e m i c a l form, t h e r e s t b e i n g e i t h e r bound t o t h e n i c k e l 2 0

1 8

2

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

s u r f a c e o r i n u n r e a c t i v e F-18 g a s e s . L a t e r t e c h n i c a l developments a l l o w e d t h e use o f a p r o t o n beam v i a t h e 0(p,n) F nuclear reaction (13) . T e c h n i q u e s were a l s o d e v e l o p e d f o r conveniently c o n v e r t i n g F-18 F i n t o t h e more s e l e c t i v e f l u o r i n a t i n g a g e n t s , C H C 0 F and X e F (14-16) . A p a r a l l e l d e v e l o p m e n t a l p r o c e s s has o c c u r r e d w i t h p r o d u c t i o n o f F-18 f l u o r i d e i o n and w i t h r e a c t i o n conditions f o r displacement reactions. T h e r e a r e many n u c l e a r r e a c t i o n s which l e a d t o F-18, b u t t h e most c o n v e n i e n t o f t h e s e p r o d u c e t h e n u c l i d e as aqueous s o l u t i o n s o f f l u o r i d e . These t e n d e d t o be a v o i d e d a d e c a d e ago, i n part because i t was recognized that f l u o r i d e takes a long time to dry. In f a c t , y i e l d s o b t a i n e d from n u c l e o p h i l i c s u b s t i t u t i o n r e a c t i o n s of F-18 f l u o r i d e a t t h a t t i m e t e n d e d t o be low (17). However, t h i s was p r o b a b l y due t o t h e use o f p o o r l y r e a c t i v e s t a r t i n g m a t e r i a l s , i n h i b i t o r y c o n t a m i n a n t s o f F-18 s o l u t i o n s and s u b - o p t i m a l r e a c t i o n c o n d i t i o n s , as much as t o t h e p r e s e n c e o f w a t e r Conditions for successful nucleophili fluorosugars involve a c e t o n i t r i l e w h i c h has the u s e f u l p r o p e r t y of forming a low b o i l i n g a z e o t r o p e w i t h water (UL) ; a s u p p o r t i n g s a l t , which consists of a large easily dehydrated cation like t e t r a e t h y l a m m o n i u m and a b a s i c a n i o n s u c h as h y d r o x i d e , w h i c h p r o b a b l y p u l l s water from f l u o r i d e i o n s (1JL) , and a r e a c t i v e l e a v i n g g r o u p s u c h as t r i f l a t e o r c y c l i c s u l f a t e . In p r a c t i c e , 5-50 m i c r o m o l e o f s u p p o r t i n g s a l t i s added t o t h e aqueous s o l u t i o n o f F-18, w h i c h i s t h e n e v a p o r a t e d t o d r y n e s s in v a c u o o r i n a stream of nitrogen. Starting material is then added, in s t o i c h i o m e t r i c q u a n t i t y w i t h t h e s u p p o r t i n g s a l t , w i t h l-10mL o f acetonitrile. I n c o r p o r a t i o n r e a c t i o n s are g e n e r a l l y complete i n a few m i n u t e s a t r e f l u x t e m p e r a t u r e (2H) . Higher y i e l d s are o f t e n obtained with potassium plus Kryptofix 2,2,2 rather than a tetraalkylammonium cation (2JL.) . T h i s may be because the a m i n o p o l y e t h e r can a l s o e n c r y p t t r a c e s o f c a t i o n s , d e r i v e d f r o m t h e c y c l o t r o n t a r g e t , which b i n d f l u o r i d e t i g h t l y . A less basic c a t i o n t h a n h y d r o x i d e , such as c a r b o n a t e , may a l s o i n c r e a s e y i e l d s when a b a s e s e n s i t i v e p r o t e c t i v e group, l i k e a c e t a t e , i s p r e s e n t i n the s t a r t i n g m a t e r i a l . The use o f p o t a s s i u m / K r y p t o f i x i s not n e c e s s a r y i f t h e F-18 i s p u r i f i e d v i a f l u o r o t r i m e t h y l s i l a n e (22.) • T h i s i s e a s i l y g e n e r a t e d by r e a c t i o n o f a c r u d e F-18 fluoride s o l u t i o n w i t h c h l o r o t r i m e t h y l s i l a n e and can be swept out o f t h e r e a c t i o n v e s s e l i n a stream of a i r . A f t e r s c r u b b i n g t h e gas can be c r y o t r a p p e d f o r subsequent h y d r o l y s i s , o r d i r e c t l y c o n v e r t e d t o f l u o r i d e by b u b b l i n g t h e gas s t r e a m t h r o u g h aqueous a c e t o n i t r i l e containing tetraethylammonium hydroxide. This procedure takes only a b o u t f i v e m i n u t e s and is virtually quantitative. It s e p a r a t e s the F-18 f r o m t r a c e s o f e l e m e n t s which o t h e r w i s e t i e up added f l u o r i d e w i t h e l e c t r o s t a t i c or c o v a l e n t bonds u n d e r the c o n d i t i o n s used f o r the s u b s t i t u t i o n r e a c t i o n . 1 8

1 8

2

3

2

2

Routes t o 2FDG The preparation e l e c t r o p h i l i c and

of 2FDG has n u c l e o p h i l i c F-18

been approached i n t e r m e d i a t e s (22.)

.

from The

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

both first

9. GATLEY ET AL.

159

Fluorine-18 Deoxyfluorohexoses

reported r o u t e employed a d d i t i o n o f F-18 l a b e l e d molecular fluorine (0.1-1% F d i l u t e d w i t h Ne) a c r o s s t h e 1,2 d o u b l e bond i n 3 , 4 , 6 - t r i a c e t y l g l u c a l (24.). A d d i t i o n o c c u r s p r e d o m i n a n t l y , b u t not e x c l u s i v e l y , t o g i v e t h e g l u c o s e d e r i v a t i v e ; about one q u a r t e r of t h e F adds t o g i v e t h e 2 - d e o x y - 2 - f l u o r o m a n n o s y l f l u o r i d e . Greater stereospecificity i s achieved using F-18 a c e t y l h y p o f l u o r i t e w i t h a b o u t 5% o f t h e mannose compound b e i n g produced under o p t i m a l c o n d i t i o n s (25,26). In contrast, d i r e c t F-18 f l u o r i n a t i o n o f g l u c a l i n water g i v e s a h i g h e r p r o p o r t i o n o f t h e mannose compound (27-29). Xenon d i f l u o r i d e l a b e l e d w i t h F-18 has a l s o been u s e d t o f l u o r i n a t e t r i a c e t y l g l u c a l (3Hf31) • While o p t i m i z a t i o n o f f l u o r i n a t i n g agent and r e a c t i o n c o n d i t i o n s g r e a t l y i m p r o v e d t h e y i e l d o f 2FDG, t h e s e e l e c t r o p h i l i c s y n t h e s e s were i n t r i n s i c a l l y l e s s e f f i c i e n t than d e s i r e d because t h e F and C H C 0 F a r e t h e m s e l v e s p r o d u c e d i n p o o r y i e l d (22-2A) . I t was e a r l y recognized that a nucleophili displacement reactio involvin F-18 f l u o r i d e a n i o n woul greater y i e l d , but s e v e r a m a t e r i a l s and r e a c t i o n c o n d i t i o n s were d e v e l o p e d . 2

2

2

3

2

Tewson's u s e o f l - b e t a - O - m e t h y l - 4 , 6 - b e n z y l i d e n e - D mannose 2,3-cyclic sulfate provided the f i r s t practicable nucleophilic s y n t h e s i s o f 2FDG (2h. 2JL) . The c o r r e s p o n d i n g 1 - a l p h a - O - m e t h y l compound f a i l e d t o g i v e good i n c o r p o r a t i o n y i e l d s , but the 2 - f l u o r o - b e t a - O - m e t h y l p r o d u c t was d i f f i c u l t t o d e p r o t e c t . Boron t r i s (trifluoroacetate) i n trifluoroacetic a c i d gave adequate yields o f 2FDG, a n d HPLC (aminopropyl column with 70% a c e t o n i t r i l e ) showed a s m a l l i m p u r i t y peak w h i c h was a s c r i b e d t o r e s i d u a l beta-methyl 2FDG. Subsequent e x p e r i e n c e demonstrated t h a t t h e s i t u a t i o n was more c o m p l i c a t e d . HPLC a n a l y s e s i n r e a c t i o n m i x t u r e s r e v e a l e d two peaks o f r o u g h l y e q u a l s i z e and a t l e a s t t h r e e s m a l l i m p u r i t y peaks a f t e r d e p r o t e c t i o n and d e - s a l t i n g (12.) . F u t h e r m o r e , i n o u r hands t h e y i e l d o f 2FDG i n t h e d e p r o t e c t i o n s t e p was a l w a y s c l o s e t o 50%, a n d most o f t h e r e m a i n i n g F-18 b e h a v e d l i k e f l u o r i d e . I t thus appears t h a t o n l y h a l f t h e o r g a n i c l a b e l i n r e a c t i o n m i x t u r e s i s p r o t e c t e d 2FDG. The other half i s largely d e c o m p o s e d t o HF b y b o r o n tris (trifluoroacetate). Whether t h i s f r a c t i o n i s t h e p r o d u c t o f n u c l e o p h i l i c a t t a c k a t C-3, o r o f a r e a r r a n g e m e n t r e a c t i o n o f t h e p r o t e c t e d 2FDG i s n o t a t p r e s e n t c l e a r . The r e l a t e d b e t a - O - v i n y l s t a r t i n g m a t e r i a l (38) d e p r o t e c t s e a s i l y w i t h 2N-HC1, b u t a l s o gives a f i n a l product with s e v e r a l percent o f l a b e l e d i m p u r i t i e s . A s t a r t i n g m a t e r i a l w i t h a t r i f l a t e g r o u p on C-2 o f a p r o t e c t e d mannose ( 4 , 6 - b e n z y l i d e n e - 2 - t r i f l y l s u l f o n y l - 3 - O - m e t h y l beta-O-methyl-D-mannose) was f i r s t r e p o r t e d by E l m a l e h e t a l ( 3 9 ) . Good i n c o r p o r a t i o n was a c h i e v e d , b u t t h e 3-O-methyl g r o u p p r o v e d i m p o s s i b l e t o remove e f f i c i e n t l y u n d e r t h e t i m e c o n s t r a i n t s o f w o r k i n g w i t h F-18. S y n t h e s i s o f t h e analogous 3 - 0 - b e n z y l compound was l a t e r r e p o r t e d (AO.) • The c u r r e n t s t a r t i n g m a t e r i a l o f c h o i c e f o r n u c l e o p h i l i c 2FDG production i s 1,3,4,6-tetra-acetyl-2-0t r i f l y l - D mannose ( H ) . T h i s i n c o r p o r a t e s F-18 r a p i d l y i n h i g h yield f r o m p r e p a r a t i o n s o f r e a c t i v e F-18 f l u o r i d e , and the t e t r a a c e t y l 2FDG i s c l e a n l y d e p r o t e c t e d by 1N-HC1. Net y i e l d s o f a r o u n d 50% a r e o b t a i n e d i n 45 m i n u t e s s t a r t i n g w i t h an aqueous r

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s o l u t i o n o f F-18 f l u o r i d e . F-18 2FDG has a l s o been p r o d u c e d i n low y i e l d by e p o x i d e c l e a v a g e

with

labeled

6-di-O-isopropylidene-l-C-nitro-D O t h e r F-18

KHF

mannitol

2

i n 1,2-anhydro-3,4:5, (AZ).

Sugars.

Since a wide range o f F-18 r e a c t i v e i n t e r m e d i a t e s i s now a v a i l a b l e , any p u b l i s h e d s y n t h e s i s o f a f l u o r o c a r b o h y d r a t e c a n i n p r i n c i p l e be a d a p t e d t o u s e t h e r a d i o i s o t o p e . The s y n t h e s i s must be c o m p l e t e d w i t h i n a t most an h o u r o r two, w h i c h n e c e s s i t a t e s c o n s i d e r a b l e a t t e n t i o n t o t h e r a t e o f each s t e p which occurs a f t e r incorporation of the isotope. T h i s i n c o r p o r a t i o n s t e p s h o u l d be as l a t e i n t h e s y n t h e t i c scheme as p o s s i b l e . C h r i s t m a n e t a l . (A2L) i n v e s t i g a t e d t h e i n f l u e n c e o f s e v e r a l r e a c t i o n p a r a m e t e r s on t h e s y n t h e s i s o f 6-deoxy 6 - f l u o r o - D - g a l a c t o s e . They u s e d a l a b e l e d tetraalkylammonium f l u o r i d a variety of leavin 1,2-3,4-diisopropylidenegalactose similar s y n t h e s i s o f F-18 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e (4A-4JL) (3FDG) f r o m 1 , 2 - 5 , 6 - d i i s o p r o p y l i d e n e a l l o s e e m p l o y e d t h e more r e a c t i v e t r i f l a t e group i n s t e a d o f t h e t o s y l group o r i g i n a l l y used for unlabeled 3FDG b y F o s t e r e t a l (Al) . Incorporation of f l u o r i d e f r o m t e t r a e t h y l a m m o n i u m f l u o r i d e was f o u n d t o be c o m p l e t e i n a few m i n u t e s i n r e f l u x i n g a c e t o n i t r i l e (2JI) , whereas 48 h o u r s (Al) was a l l o w e d f o r r e a c t i o n w i t h t h e t o s y l a t e . P a p e r s f e a t u r i n g F-18 3FDG h a v e a p p e a r e d i n the nuclear medicine (4 8) and physiology (4.2.) l i t e r a t u r e . I t i s a good s u b s t r a t e f o r g l u c o s e t r a n s p o r t , b u t a p o o r s u b s t r a t e f o r h e x o k i n a s e (hSL) a n d has been u s e d t o s t u d y l o c a l r a t e s o f g l u c o s e t r a n s p o r t i n PET e x p e r i m e n t s (hi.) . T h i s i s i n c o n t r a s t t o s t u d i e s w i t h 2FDG w h i c h g i v e l o c a l rates of glucose phosphorylation. As m e n t i o n e d a b o v e , t h e e l e c t r o p h i l i c s y n t h e s e s o f 2FDG y i e l d 3,4,6-triacetyl-2-deoxy-2f l u o r o - D - m a n n o s y l f l u o r i d e o r t h e c o r r e s p o n d i n g 1 - a c e t y l compound as a s i d e p r o d u c t . T h i s c a n be s e p a r a t e d and d e p r o t e c t e d t o g i v e 2-deoxy-2-fluoro-D-mannose (2FDM) (52.) though y i e l d s a r e p o o r and it is difficult to avoid contamination with 2FDG. The corresponding p r o b l e m o f 2FDG p r e p a r a t i o n s c o n t a i n i n g v a r i a b l e amounts o f 2FDM h a s c a u s e d c o n s i d e r a b l e anxiety i n t h e PET community. A l t h o u g h 2FDM, l i k e 2FDG, i s a good s u b s t r a t e f o r b o t h t r a n s p o r t and p h o s p h o r y l a t i o n , t h e r e l a t i v e r a t e s o f t h e e p i m e r s for t h e two p r o c e s s e s a r e almost certainly different, and c o n t a m i n a t i o n w i t h 2FDM w i l l , t h e r e f o r e , c a u s e e r r o n e o u s v a l u e s o f g l u c o s e m e t a b o l i c r a t e t o be c a l c u l a t e d i n 2FDG s t u d i e s . The two sugars are hard t o separate chromatographically, so t h a t t h e s e v e r i t y o f t h e problem i n each batch o f f l u o r o s u g a r i s not easy to determine. J u s t as t h e n u c l e o p h i l i c r o u t e s t o 2FDG do n o t p r o d u c e 2FDM as a c o n t a m i n a n t , a n u c l e o p h i l i c pathway w h i c h i s r e p o r t e d t o g i v e p u r e 2FDM i s now a v a i l a b l e . The s t a r t i n g m a t e r i a l is beta-0-methyl-4,6-benzylidene-3-0-benzyl-2-0triflyl-Dg l u c o p y r a n o s e (hi) . I n c o r p o r a t i o n o f F-18 p r o c e e d s r e a d i l y , b u t the product requires quite vigorous conditions (5N-HC1 u n d e r r e f l u x f o r 30 m i n u t e s ) f o r d e p r o t e c t i o n ( 5 4 ) . F i g u r e 1 shows t h e s t r u c t u r e s o f s e v e r a l o f t h e s t a r t i n g materials u s e d i n s y n t h e s e s o f f l u o r o s u g a r s f r o m F-18 f l u o r i d e .

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

9.

GATLEYETAL.

Fluorine-18 Deoxyfluorohexoses

161

Included i s 2 , 3 , 6 - t r i b e n z o y l - 4 - t r i f l y l alpha-methyl-D-galactoside which we a r e e x a m i n i n g as a p r e c u r s o r o f F-18 4-deoxy-4fluoro-D-glucose: n u c l e o p h i l i c s u b s t i t u t i o n has o c c u r r e d r e a d i l y t o g i v e a s i n g l e p r o d u c t i n most t e s t r e a c t i o n s , b u t p a r t i a l d e p r o t e c t i o n h a s been seen i n some. We a r e p r e s e n t l y optimizing c o n d i t i o n s f o r c o n t r o l l e d removal o f t h e p r o t e c t i v e groups from the i n c o r p o r a t i o n product. Some t u m o r s c o n t a i n l a r g e q u a n t i t i e s o f L - f u c o s e . This encouraged t h e development o f 2 - d e o x y - 2 - f l u o r o - L - f u c o s e as a potential imaging agent f o r detecting and m o n i t o r i n g such m a l i g n a n c i e s (IL5.) • © route i n v o l v e d a d d i t i o n o f e l e c t r o p h i l i c F-18 t o d i a c e t y l f u c a l i n g l a c i a l a c e t i c a c i d o r F r e o n 11. T

n

R a p i d P u r f i c a t i o n and Q u a l i t y

Control

T h e r e a r e s e v e r a l s i g n i f i c a n t p r a c t i c a l d i f f e r e n c e s between t h e preparation o f compound h a l f - l i v e s , and t r a d i t i o n a the need f o r r a d i a t i o n p r o t e c t i o n ; t h e s m a l l s c a l e ; t h e need f o r g r e a t s p e e d ; t h a t y i e l d s a r e b a s e d on t h e i n o r g a n i c r a d i o n u c l i d e rather than t h e organic starting material; some s i m p l e , y e t p o w e r f u l methods s u c h a s r e c r y s t a l l i z a t i o n a n d m e l t i n g point determination c a n n o t be a p p l i e d . C l e a r l y , a f r e s h b a t c h o f any F-18 s u g a r has t o be p r e p a r e d on a t l e a s t a d a i l y b a s i s , a n d t h e term " q u a l i t y c o n t r o l " r e f l e c t s t h i s . B a t c h e s o f F - 1 8 s u g a r s a r e t y p i c a l l y p r e p a r e d u s i n g a few milligrams of organic precursor, while the s t a r t i n g r a d i o a c t i v i t y o f F - 1 8 may be a n y t h i n g up t o 1 C u r i e . E l e c t r o p h i l i c F-18 f l u o r i n a t i o n s a r e g e n e r a l l y performed with tens o f micromoles o f c a r r i e r f l u o r i n e . In c o n t r a s t , f o r n u c l e o p h i l i c F-18 f l u o r i n a t i o n s t h e mass o f f l u o r i n e i s g e n e r a l l y t i n y , c o n s i s t i n g o f t h e F - 1 8 itself (about 0.6 nanomole p e r C u r i e ) p l u s a n y n o n - r a d i o a c t i v e F-19 present i n r e a g e n t s , which i s u s u a l l y o f t h e o r d e r o f 1 nanomole. These a r e r e f e r r e d t o as "no c a r r i e r added" c o n d i t i o n s . The l a b e l e d p r o d u c t t h e n h a s a v e r y h i g h s p e c i f i c r a d i o a c t i v i t y , w h i c h c i r c u m v e n t s any p o s s i b l e t o x i c o r p h a r m a c o l o g i c e f f e c t s d u r i n g human u s e . The o v e r a l l s c a l e o f b o t h e l e c t r o p h i l i c a n d nucleophilic reactions i s small enough t o a l l o w a n a l y t i c a l methodologies t o be u s e d p r e p a r a t i v e l y during work-up a n d purification. F o r example, a c i d s u s e d i n d e p r o t e c t i o n c a n be removed by p a s s a g e t h r o u g h s m a l l columns o f i o n - r e t a r d a t i o n r e s i n . T r a c e s o f F - 1 8 f l u o r i d e a r e removed w i t h a l u m i n a c o l u m n s , a n d u n h y d r o l y z e d i n i t i a l l a b e l e d p r o d u c t s , i f n e u t r a l , a r e r e t a i n e d on o c t a d e c y l s i l a n e bonded s i l i c a g e l . O b j e c t i o n a b l e c o l o r may o f t e n be removed w i t h a c t i v a t e d c h a r c o a l , a n d s t e r i l i z a t i o n achieved w i t h a 0.2 m i c r o n M i l l i p o r e f i l t e r . Labeled r e a c t i o n products not h e l d up i n t h i s c a s c a d e o f a b s o r b e n t s a n d f i l t e r s w o u l d i n c l u d e a l k y l g l y c o s i d e s and n e u t r a l rearrangement p r o d u c t s . Most o f t h e s t a r t i n g materials f o r f l u o r i d e based synthesis o f deoxyfluoro sugars y i e l d t h e c o r r e s p o n d i n g parent sugar, o r i g i n a t i n g from n u c l e o p h i l i c a t t a c k o f h y d r o x i d e i o n o r w a t e r , a s t h e dominant unlabeled impurity. B e c a u s e o f t h e s m a l l amount o f s t a r t i n g material, the concentration of unlabeled material i s not o b j e c t i o n a b l e f o r PET work. F o r example, g l u c o s e i n human b l o o d

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plasma i s p r e s e n t a t about 5 micromole/mL i n about 5L o f f l u i d ; t h u s v a s c u l a r i n j e c t i o n o f 5ml o f 2FDG p r e p a r a t i o n containing 10-20 m i c r o m o l e o f g l u c o s e does n o t p e r t u r b g l u c o s e h o m e o s t a s i s . F o r work w i t h i s o l a t e d o r g a n s (see b e l o w ) , however, t h e i n j e c t a t e must be f r e e o f g l u c o s e and o t h e r u n l a b e l e d i m p u r i t i e s . The method o f c h o i c e f o r p r e p a r a t i v e s e p a r a t i o n o f d e s i r e d F-18 f l u o r o s u g a r from e i t h e r l a b e l e d o r u n l a b e l e d i m p u r i t i e s i s h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC), a n d t h i s i s a l s o the most common o p t i o n for quality control. Two popular s t a t i o n a r y phases a r e aminopropyl d e r i v a t i z e d s i l i c a and c e r t a i n c a t i o n exchange r e s i n s . The f o r m e r i s c h e a p e r a n d e a s i e r t o use, but t h e l a t t e r has s u p e r i o r r e s o l u t i o n f o r many s u g a r s a n d has t h e a d v a n t a g e f o r p r e p a r a t i v e u s e o f u s i n g w a t e r as m o b i l e p h a s e r a t h e r t h a n aqueous a c e t o n i t r i l e . F l u o r i d e i o n a n d some o t h e r compounds have v e r y l o n g r e t e n t i o n t i m e s i n b o t h t y p e s o f HPLC column, a n d i t i s e a s y t o o v e r e s t i m a t e t h e r a d i o c h e m i c a l p u r i t i e s of f l u o r o s u g a r s by f a i l u r peaks. T h i n l a y e r chromatograph HPLC b u t t h e a d v a n t a g e t h a t l a b e l e d s p o t s c l o s e t o t h e o r i g i n a r e as e a s i l y d e t e c t e d on a u t o r a d i o g r a p h s as compounds w i t h l a r g e RF values. A n o t h e r a n a l y t i c a l s t r a t e g y f o r some F-18 f l u o r o s u g a r s i s t o compare chromatograms b e f o r e a n d a f t e r u s i n g h e x o k i n a s e p l u s ATP/Mg t o g e n e r a t e t h e 6-phosphates . F i g u r e 2 shows a 2 +

chromatogram o f a p r e p a r a t i o n o f 2FDG made f r o m t h e b e t a - m e t h y l c y c l i c s u l f a t e s t a r t i n g m a t e r i a l . In t h i s case t h e r e i s a s m a l l l a b e l e d i m p u r i t y c o - e l u t i n g w i t h 2FDG on t h e amino column, w h i c h i s d e m o n s t r a t e d by t h e r e s i d u a l peak l e f t a f t e r t r e a t m e n t w i t h h e x o k i n a s e . T h i s i l l u s t r a t e s a p o t e n t i a l p r o b l e m i n r e l y i n g on r e t e n t i o n t i m e a l o n e , o r i n a s i n g l e system, f o r q u a l i t y c o n t r o l . The 2FDG-6-phosphate i s r e t a i n e d on t h e column i n d e f i n i t e l y under t h e s e c o n d i t i o n s . The s u g a r p h o s p h a t e s c a n a l s o be e a s i l y t r a p p e d w i t h a n i o n exchange r e s i n s a n d t h e p h o s p h o r y l a t a b l e f r a c t i o n o f a p r e p a r a t i o n determined. A p p l i c a t i o n o f F-18

Fluorosugars

S y n t h e s e s o f s e v e r a l F-18 f l u o r o s u g a r s have now been d e v e l o p e d t o t h e p o i n t a t w h i c h t h e y c a n e a s i l y be p r e p a r e d on a s m a l l s c a l e where r a d i a t i o n e x p o s u r e i s n o t a s e r i o u s i s s u e (say, up t o 1 mCi) i n any l a b o r a t o r y s i t u a t e d w i t h i n s e v e r a l h o u r s o f a s u i t a b l e n u c l e a r r e a c t o r (JLZ) o r a c c e l e r a t o r . M o l e c u l a r and b i o l o g i c a l s c i e n t i s t s can, t h e r e f o r e , c o n s i d e r working with t h i s i s o t o p e . One a d v a n t a g e i s t h a t F-18 i s e a s i l y detected and counted quantitatively, i n any k i n d o f sample, w i t h o u t the tedious p r e p a r a t i o n needed f o r C-14 o r t r i t i u m ; t h e t i m e s a v e d more t h a n compensates f o r t h e n e e d t o p r e p a r e t h e F-18 t r a c e r e v e r y d a y . Another i s the extremely high s p e c i f i c r a d i o a c t i v i t y o b t a i n a b l e , which a l l o w s a t r a c e r s t a t e ( i . e . , no p e r t u r b a t i o n o f t h e s y s t e m u n d e r s t u d y ) t o be m a i n t a i n e d , and w h i c h p e r m i t s s l o w p r o c e s s e s and binding sites present i n low concentrations t o be investigated.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

GATLEY ET AL.

Fluorine-18 Deoxyfluorohexoses

2FDM

4FD

F i g u r e 1. S t r u c t u r e s o f p r e c u r s o r s f o r n u c l e o p h i l i c r o u t e s to deoxyfluorohexoses.

2 MIN

CONTROL

A

+ HEXOKINASE

F i g u r e 2. H i g h performance liquid chromatogram of a preparation of 2-deoxy-2-fluoro-D-glucose. Stationary phase, a m i n o p r o p y l banded s i l i c a ; m o b i l e phase, 70% aqueous a c e t o n i t r i l e a t 1.5mL/min. The l a r g e peak i n t h e u p p e r p a n e l i s 2FDG.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Animal Experiments D a t a f r o m e x p e r i m e n t s {22,12.) i n which i n j e c t e d i n t o r a t s a r e shown i n T a b l e I .

three

F-18 s u g a r s

were

TABLE I . T i s s u e - t o - b l o o d r a t i o s o f D-2FDG, D-3FDG and L-3FDG i n r a t s a t 60 min Organ

D-2FDG

D-3FDG

L-3FDG

Brain Heart Kidney Liver Muscle Urine

6.22±1.2 12.9±5.6 4.38±4.3 1.28±0.09 0.71+0.17

0.82+0.25 1.17±0.47

0.13+0.12 0.69±0.17 3.59±1.67

1.43±0.63 0.90±0.31 0.53+0.29

0.80±0.23 0.47+0.23 50-60

The d i s t r i b u t i o n o f t h e u n n a t u r a l L-3FDG, w h i c h we e x p e c t t o be n e i t h e r a s u b s t r a t e f o r t r a n s p o r t systems, n o r t o be m e t a b o l i z e d , i s consistent with i t s being l a r g e l y confined t o the e x t r a c e l l u l a r f l u i d , a n d i t s r a p i d e l i m i n a t i o n v i a t h e k i d n e y s . The b r a i n , w i t h i t s t i g h t c a p i l l a r y s t r u c t u r e , p a r t i c u l a r l y e x c l u d e s L-3FDG. A s t r i k i n g l y d i f f e r e n t p i c t u r e i s g i v e n by D-3FDG, w i t h much l e s s F-18 i n u r i n e a n d more F-18 i n b r a i n a n d h e a r t . T h i s b e h a v i o r confirms t h a t D-3FDG i s s p e c i f i c a l l y t r a n s p o r t e d i n s e v e r a l t i s s u e s , and t h e r e l a t i v e a c c u m u l a t i o n seen i n h e a r t h i n t s a t a s p e c i f i c t r a p p i n g mechanism. Much g r e a t e r t i s s u e - t o - b l o o d r a t i o s i n h e a r t and b r a i n were seen i n a n i m a l s g i v e n D-2FDG, which i s n o t only transported into these organs, but i s trapped as t h e 6-phosphate t o a g r e a t e r d e g r e e t h a n D-3FDG. However, D-2FDG i s n o t , u n l i k e D-3FDG, a s u b s t r a t e f o r t h e e n e r g y - l i n k e d c a r r i e r i n t h e k i d n e y t u b u l e s , a n d so i s e l i m i n a t e d f a s t e r . T h i s i s an a d v a n t a g e i n whole body i m a g i n g s t u d i e s , b e c a u s e i t c a u s e s t h e "background" due t o F-18 i n b l o o d t o d e c r e a s e r a p i d l y . Isolated Perfused

Hearts

D i f f e r e n c e s i n t h e b e h a v i o r o f D-2FDG, D-3FDG and L-3FDG were seen more c l e a r l y when t h e y were a d m i n i s t e r e d t o i s o l a t e d p e r f u s e d r a t hearts (Ail) ( F i g u r e 3) . B r i e f l y , 10 m i c r o l i t e r s o f t r a c e r i n p e r f u s i o n medium was p r e s e n t e d i n a n e a r l y i n s t a n t a n e o u s (

+k )

(5)

43

When the f l u x through hexokinase i s low r e l a t i v e t o the c e l l u l a r transport step, e . g . at high perfusate glucose concentration, or i n the presence of i n s u l i n , and k > k , the equations approximate t o V = k / k . There i s good reason t o believe that k / k , the volume of d i s t r i b u t i o n i n the absence of metabolism, has the same value f o r a l l sugars sharing the same c a r r i e r (iL2.) . I t f o l l o w s that when c e l l t r a n s p o r t i s r a p i d compared with phosphorylation 2 3

3 2

3 2

4 3

2 3

2 3

LC (limiting) = k * / k 4 3

(6)

4 3

A large number of experiments has given the value of 0.56 as the l i m i t i n g lower bound f o r LC f o r 2FDG i n the r a t heart (£A) , i n good agreement with k * / k c a l c u l a t e d from the hexokinase k i n e t i c parameters (50). 4 3

4 3

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

170

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Table I I I : P r e l i m i n a r y estimates of heart c e l l u l a r glucose Pre/After l o a d (mmHg)

15/140 15/140 15/140 15/140 15/140 5/70

Perfusate g l u c o s e (mM)

2 5 10 15 30 5

MRglc (mmol/min/g)

9.7 11.6+1.0 12.1+1.7 11.4±1.6 12.2±2.5 4 . 6±0 . 6

Cellular g l u c o s e content (micromol/heart)

1.95 2.10±.36 1.71+.40 1.59±.14 1.59±.27 1.18±.31

content Lumped constant

0.52 1.19+.39 1.70+.51 1.59±.20 2.39±.40 0.72±.09

Table I I I gives estimates of the glucose content f o r the same groups of hearts as Table I I . Our data show that c e l l u l a r glucose content r i s e s with perfusate glucose, but less than l i n e a r l y . In other words, t r a n s p o r t i s s a t u r a b l e . Under low workload conditions, where the rate of glucose u t i l i z a t i o n was about h a l f that at high workloads, glucose content was decreased. This suggests modulation of the transport step by metabolic needs. Table I I I also gives values f o r the lumped constant, which tends to decrease with higher perfusate glucose. Preliminary s t u d i e s have been done i n h e a r t s t r e a t e d with m e t a b o l i c i n h i b i t o r s , i n hearts perfused with long chain f a t t y acids i n addition to glucose, and i n hearts t r e a t e d with i n s u l i n . In the l a t t e r s i t u a t i o n there i s a large decrease i n lumped constant and a r i s e i n estimated c e l l u l a r glucose content. I n s u l i n i s well known t o s t i m u l a t e glucose t r a n s p o r t i n heart, probably by i n c r e a s i n g the number of a c t i v e c a r r i e r s (£5.) , but i t also has s e v e r a l other e f f e c t s . The f a l l i n lumped constant t o 0.56 r e f l e c t s the change i n r e l a t i v e c o n t r o l strengths (iL£) of transport and phosphorylation i n the presence and absence of i n s u l i n . With the hormone, the c o n t r o l strength of transport i s very small, and the lumped constant r e f l e c t s almost purely the r e l a t i v e a c t i v i t i e s of 2FDG and glucose with hexokinase. The analog i s phosphorylated more slowly than the mother substance. The value of the lumped constant under these conditions i s more s i m i l a r to that obtained i n normal r a t b r a i n (e.g. 10), where there i s a l s o a l a r g e excess of t r a n s p o r t c a p a b i l i t y over hexokinase a c t i v i t y . Furthermore, i s o l a t e d yeast hexokinase has an a c t i v i t y r a t i o (Vmax/Km) f o r 2FDG h a l f that of glucose (id) . When the control strength of transport i s high, i n hearts perfused without i n s u l i n , the lumped constant r e f l e c t s p r i m a r i l y the r e l a t i v e a c t i v i t i e s of 2FDG and glucose f o r the c a r r i e r . The analog i s the better substrate f o r transport, and thus the lumped constant r i s e s .

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

9. GATLEY ET AL. PET

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171

Studies

The e x t e n s i v e use o f 2FDG i n human p o s i t r o n e m i s s i o n t o m o g r a p h i c studies is assured. Its vast potential in clinical and e x p e r i m e n t a l work has d r i v e n t h e development o f i t s s y n t h e s i s t o h i g h y i e l d and p u r i t y , and w i l l a s s u r e i t s a v a i l a b i l i t y f o r b o t h PET and non-PET a p p l i c a t i o n s i n t h e f u t u r e . I t i s beyond t h e scope of t h i s c h a p t e r t o d i s c u s s PET s t u d i e s o f l o c a l v a l u e s o f t h e r a t e of g l u c o s e u t i l i z a t i o n g e n e r a t e d w i t h 2FDG. However, i t may be w o r t h p o i n t i n g out t h a t s m a l l volumes i n t h e q u a n t i t a t i v e images g e n e r a t e d by PET s c a n n e r s c a n be r e g a r d e d somewhat like an i s o l a t e d p e r f u s e d organ; the i s o l a t i o n i s tomographic, r a t h e r than p h y s i c a l . F i g u r e 7 shows t o m o g r a p h i c d a t a f r o m a human b r a i n g r a y m a t t e r r e g i o n o b t a i n e d w i t h 3FDG (JL1) . Two computer g e n e r a t e d f i t s a r e shown. The two p a r a m e t e r s r e f e r r e d t o i n t h e l o w e r p a n e l a r e r a t e c o n s t a n t s f o r t r a n s f e r o f 3FDG between c a p i l l a r y b l o o d and t h e p o o l o f f r e e 3FDG takes into account rat d e p h o s p h o r y l a t i o n , analogous t o k * and k * i n F i g u r e 4. The b e s t 4 3

3 4

estimates of the transport rate constant are obtained when c o r r e c t i o n s f o r t h e slow p h o s p h o r y l a t i o n o f 3FDG a r e made. T h u s , t h e t r a n s p o r t o f 3FDG t h r o u g h t h e b l o o d b r a i n b a r r i e r c a n be studied i n small regions of the human b r a i n . Corresponding e s t i m a t e s made w i t h 2FDG a r e more u n c e r t a i n b e c a u s e t h e c o r r e c t i o n f o r p h o s p h o r y l a t i o n i s much g r e a t e r . Conclusions We have shown t h a t i n d i v i d u a l r a t e c o n s t a n t s i n r e s i d u e curves obtained with i s o l a t e d perfused rat hearts c a n be measured w i t h much g r e a t e r p r e c i s i o n t h a n t h e c o r r e s p o n d i n g p a r a m e t e r s i n P E T . A single s t u d y w i t h 2FDG y i e l d s good v a l u e s of c e l l membrane extraction, dephosphorylation rate constant and fluorosugar d i s t r i b u t i o n volume i n a d d i t i o n t o t h e f l u o r o s u g a r p h o s p h o r y l a t i o n r a t e . From a s e r i e s o f 2FDG s t u d i e s and an i n d e p e n d e n t measure o f the r a t e of glucose u t i l i z a t i o n , the g l u c o s e content of the hearts can be c a l c u l a t e d under a range o f c o n d i t i o n s . A f t e r s p e n d i n g s i x y e a r s d e v e l o p i n g our a p p r o a c h , we now p l a n to extend it to s t u d i e s of hearts under abnormal m e t a b o l i c c o n d i t i o n s , s u c h as i s c h e m i a and h y p o x i a . We w i l l a l s o be a b l e t o examine t h e e f f e c t s o f hormones and o f p h a r m a c o l o g i c a l l y a c t i v e compounds i n a n o v e l way. We a l s o e x p e c t t h e use o f F - 1 8 l a b e l e d fluorosugars in isolated perfused organs to continue its i n t e r a c t i v e r e l a t i o n s h i p w i t h P E T . On t h e one h a n d , new t r a c e r s and a d m i n i s t r a t i o n p r o t o c o l s can be e x p l o r e d much more q u i c k l y and c h e a p l y t h a n i n i m a g i n g s t u d i e s . On t h e o t h e r , i t may be p o s s i b l e t o a t t a c k q u e s t i o n s g e n e r a t e d by p a t i e n t s t u d i e s , s u c h as t h o s e r e l a t i n g t o changes i n the v a l u e of the lumped c o n s t a n t in pathological conditions.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

F i g u r e 7. T i m e c o u r s e o f F-18 i n human brain after i n t r a v e n o u s i n j e c t i o n o f 3FDG. Upper p a n e l , f i t a l l o w i n g f o r m e t a b o l i s m t o a t a p p e d s p e c i e s such as 3FDG-6-phosphate; lower p a n e l , f i t assuming no m e t a b o l i s m .

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Fluorine-18 Deoxyfluorohexoses

9. GATLEY ETAL.

173

Acknowledgments T h i s work was f u n d e d b y t h e N a t i o n a l G r a n t s HL29046, HL27970 and HL36534.

Institutes

o f Health

under

Literature Cited

1.

Phelps M.E.; Mazziota J.; Schelbert H. Positron Emission Tomography and Quantitative Autoradiography: Raven Press: New York, 1986. 2. Palmer A.J.; Taylor D.M. (Eds): Radiopharmaceuticals Labeled with Halogen Isotopes. Published as Int. J . Appl.Radiat. Isotopes 37, 645-921, 1986. 3. Lederer C.M.; Shirley V.M. (Eds) Table of Isotopes. (7th edition); Wiley: New York, 1978. 4. Nickles, R.J.; Gatley, S.J.; Votaw, J.R.; Kornguth, M.L. Int. J. Appl. Radiat. Isotopes 1986 37 649-661 5. Reivich M.; Kuhl D. T.; Casella V.; Hoffma Circulation Res. 1979, 44, 127-137. 6. Sokoloff L. Thr Harvey Lectures. Series 79, 1985, pp 77-143. 7. Sols A.; Crane R.A. J . Biol. Chem 1954, 210, 581-595. 8. Gallagher B.M.; Ansari A.; Atkins H.; Casella V.; Christman D.R.; Fowler J.S.; Ido T.; MacGregor R.R.; Som P.; Wan C-N.; Wolf A.P.; Kuhl D.E.; Reivich M. J . Nucl. Med. 1977, 18, 990-996. 9. Gjedde A.; Wienhard K.; Heiss W-D.; Kloster G.; Diemer N.H.; Herhoiz K. Pawlik G. J. Cereb. Blood Flow Metab. 1985, 5, 163-178. 10. Sokoloff, L . ; Reivich, M.; Kennedy, C.; DesRosiers, M.; Patlak, C.S.; Pettigrew, K.D.; Sakarada, O.; Shinohara, M. J. Neurochem. 1977, 28, 897-916. 11. Reivich M.; Alavi A.; Wolf A.; Fowler J.; Russell J.; Arnett D.; MacGregor R.R.; Shiue C-Y.; Atkins H.; Anand A.; Dann R.; Greenberg J.H. J . Cereb .Blood Flow Metab. 1985, 5, 179-192. 12. Fowler J.S.; Finn R.D.; Lambrecht R.M.; Wolf A.P. J . Nucl. Med. 1973, 14, 63-64. 13. Nickles R.J.; Daube M.E. Int. J . Appl. Radiat. Isot. 1984, 35, 117-122. 14. Bida G.T.; Satyamurthy N.; Barrio J.R. J . Nucl. Med. 1984, 25, 1327-1334. 15. Schrobilgen G.; Firnau G.; Chirakal R.; Garnett E.S. J . Chem. Soc. Chem. Commun. 1981, 198-199. 16. Chirakal R.; Firnau G.; Schrobilgen G.J.; McKay J.; Garnett E. S. Int. J . Appl. Radiat. Isot. 1984, 35, 401-404. 17. Palmer A . J . ; Clark J.C.; Goulding R.W. Int.J.Appl.Radiat. Isot. 1977, 28, 53-65. 18. DeKleijn J.P. J . Fluorine Chem. 1977, 10, 341-350. 19. Landini, D., Maia, A.; and Podda, G. J . Org. Chem. 1982, 47, 2264-2268. 20. Gatley S.J.; Shaughnessy W.J. J . Labelled. Compounds 1981, 18, 24-25. 21. Gatley S.J.; Kornguth M.L.; De Grado T.R.; Holden J.E. J . Nucl. Med, 1987, 28, 635. 22. Rosenthal, M.S.; Bosch, A.L.; Nickles, R.J.; Gatley, S.J. Int. J . Appl. Radiat. Isotopes 1985, 36, 318. ;

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Fowler, J . S . ; Wolf, A.P. Int. J . Appl. Radiat. Isotopes 1986, 37, 663-668. 24. Ido T.; Wan C-N.; Fowler J . S . ; Wolf A.P. J . Org. Chem. 1977, 42, 2341-2342. 25. Shiue C-Y.; Salvadori P.A.; Wolf A.P.; Fowler J . S . ; MacGregor R.R. J . Nucl. Med. 1982, 23, 899-903. 26. Adam M.J. J . Chem. Soc. Chem. Commun. 1982, 730-731. 27. Jewett D.M.; Potocki J.F.; Ehrenkaufer R.E. J . Fluorine Chem. 1984, 24, 477-484. 28. Ehrenkaufer R.E.; Potocki J.F.; Jewett D.M. J . Nucl. Med. 1984, 25, 333-337. 29. Shiue C-Y.; Fowler J . S . ; Wolf A.P.; Alexoff D.; MacGregor R.R. J . Labelled Compd. Radiopharm. 1985, 22, 503. 30. Shiue C-Y.; To K . C . ; Wolf A.P. J . Labelled Compd. Radiopharm 1983, 20, 157. 31. Sood S.; Firnau G.; Garnett ES Int J Appl Radiat Isot 1983, 34, 743-745 32. Shaughnessy W.J.; Gatle Nickles R.J. Int. J . Appl. Radiat. Isotopes 1981, 32, 23-29. 33. Casella V . ; Ido T . ; Wolf A.P.; Fowler J . S . ; MacGregor R.R.; Ruth T . J . J . Nucl. Med. 1980, 21, 750-757. 34. Bida G.T.; Ehrenkaufer R.L.; Wolf A.P.; Fowler J . S . ; MacGregor R.R.; Ruth T . J . J . Nucl. Med. 1980, 21, 758-762. 35. Tewson T . J . J . Nucl. Med. 1983, 24, 718-721. 36. Tewson T . J . J . Org. Chem. 1983, 48, 3507. 37. Hutchins L . G . ; Bosch A . L . ; Rosenthal M.S.; Nickles R . J . ; Gatley S.J. Int. J. Appl. Radiat. Isot 1985, 36, 375-378. 38. Tewson T.J.; Soderlind M. J . Nucl. Med. 1985, 26, 129. 39. Levy S.; Livine E.; Elmaleh D.; Curatolo W. J . Chem. Soc. Chem. Commun. 1982, 972-973. 40. Haradahira, T . ; Maeda, M.; Omae, H . ; Kojima, M. J. Label. Compd. Radiopharm. 1984, 21, 1218-1219. 41. Hamacher, K.; Coenen, H.H.; Stocklin, G. J. Nucl. Med. 1986, 27, 235-238. 42. Beeley P.A.; Szarek W.A.; Hay G.W.; Perlmutter M.M. Can. J . Chem 1984, 62, 2709-2711. 43. Christman D.R.; Ohranovic Z . ; Shreave W.W. J . Labeled Compd. Radiopharm. 1977, 13, 555-559. 44. Tewson T.J.; Welch M.J. J . Org. Chem. 1978, 43, 1090-1099. 45. Tewson T.J.; Welch M.J.; Raichle M.E. J . Nucl. Med. 1978, 19, 1339-1345. 46. Shaughnessy W.J.; Gatley S.J. Int. J . Appl. Radiat. Isot. 1980, 31, 339-341. 47. Foster, A.B.; Hems, R.; Webber, J.M. Carbohyd. Res. 1967, 5, 292-301. 48. Goodman, M.M.; Elmaleh, D.R.; Kearfoot, K . J . ; Ackerman, R.H.; Hoop, B.; Brownell, G . L . ; Alpert, N.M.; Strauss, H.W. J. Nucl. Med. 1981, 22, 138-144. 49. Halama J.R.; Gatley S . J . ; DeGrado T.R.; Bernstein D.R.; Ng C.K.; Holden J . E . Am. J. Physiol. 1984, 247, H756-H759. 50. Bessell E.M.; Foster A . B . ; Westwood J . H . Biochem. J . 1972, 128, 199-204. 51. Koeppe R.A. Ph.D. Thesis. University of Wisconsin, 1985. 52. Ido, T . ; Wan, C-N.; Casella, V . ; Fowler, J . S . ; Wolf, A . P . ; Reivich, M.; Kuhl, D.E. J. Label. Comp. Radiopharm. 1978, 14, 175-18. In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Haradahira, T.; Maeda, M.; Omae, H.; Yano, Y.; Kojima, M. Chem. Pharm, Bull. 1984, 32, 4758-4766. 54. Luxen, A.; Satyamurthy, N.; Bida, G.T.; Barrio, J.R. Int. J . Appl. Radiat. Isotopes 1986, 37, 409-414. 55. Imahori, Y.; Ido, T.; Ishiwata, K.; Takahashi, T.; Kawashima, K.; Yanai, K.; Miura, Y.; Mouma, M.; Watanabe, S.; and Ujiie, A. Annual Report of the Cyclotron and Radioisotope Center, Tohoku University, 1984, pp 119-130. 56. Gatley S.J.; Martin J.A.; Cooper M.D. J . Nucl. Med. 1987, 28, 624. 57. Gatley, S.J.; Shaughnessy, W.J. Int. J . Appl. Radiat. Isotopes 1982, 33, 1325-1330. 58. Nickles, R.J.; Hutchins, G.D.; Daube, M.E. J . Nucl. Med. 1982, 23, p60. 59. Taegtmeyer, H.; Hems, R.; Krebs, H.A. Biochem. J . 1980, 186, 701-711. 60. Chu, S.C.; Berman 699-702. 61. Halama J.R. Ph.D. Thesis. University of Wisconsin, 1983. 62. Krivokapich, J.; Huang, S.C.; Phelps, M.E.; Barrio, J.R.; Watanabe, C.R.; Selin, E.E.; Shine, K.I. Am. J . Physiol. 1982, 243, H884-H896. 63. Nakada, T.; Kwee, I.L.; Conboy, C.B. J . Neurochem. 1986, 46, 198. 64. Ng, C.K.; Holden, J.E.; DeGrado, T.R.; Kornguth, M.L.; Raffel, D.M.; Gatley, S.J. J . Nucl. Med. 1986, 27, 966. 65. Cheung, J.Y.; Conover, C.; Regen, D.M.; Whitfield, C.F.; Morgan, H.E. Am. J . Physiol. 1978, 234, E70-E78. 66. Kacser, H.; Burns, J.A. Biochem. Soc. Trans. 1979, 7, 1149-1160. RECEIVED February 5, 1988

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 10

Antiviral Activities of 2'-Fluorinated Arabinosyl—Pyrimidine Nucleosides 1

1

1

2

3

J. J. Fox , K. A. Watanabe , T. C. Chou , R. F. Schinazi , K. F. Soike , I. Fourel , G. Hantz , and C. Trepo 4

4

4

1Memorial Sloan-Kettering Cancer Center, New York, NY 10021 Emory University-Veterans Administration Medical Center, 2

3

Delta Regional Primat Hepatitis Virus Unit, Institut National de la Santéet de la Recherche Médicale, U 271, 69003 Lyon, France

4

Comparative biochemical and antiviral studies are described for the 2'-fluoro-substituted arabinosyl-pyrimidine nucleosides 1-(2'-deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-methyluracil [FMAU] and 1-(2'deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-ethyluracil [FEAU]. Biochemical studies indicated that FEAU should be a selective antiherpesvirus agent that is less toxic than FMAU. FEAU was evaluated against simian varicella virus infection in African green monkeys and, like FMAU, was highly effective in preventing rash and reducing viremia without apparent toxicity at doses of 30, 10, or 3 mg/kg/day x 10 administered intravenously. Oral administration of FEAU in those monkeys at 10, 3, and 1 mg/kg/day x 10 was equally effective. FEAU and FMAU were evaluated against woodchuck hepatitis virus (WHV) in chronically-infected woodchucks (an animal model of choice for evaluation of potential antihepatitis B virus agents in humans). FEAU inhibits WHV replication at 2.0 and 0.2 mg/kg/day x 10 in a l l animals tested. The inhibitory effect was immediate, nontoxic and long-lasting. Preliminary studies indicated that FEAU is also effective against WHV when given by the oral route. FMAU also produced immediate inhibitory effects against WHV replication at doses of 2.0 and 0.2 mg/kg/day x 5: however, unacceptable toxicities were observed with FMAU at these dosages. FEAU appears to be the most promising of the nucleoside analogs tested thus far against Hepadnaviridae and may be an effective agent clinically against Hepatitis B virus. 0097-6156/88/0374-0176$06.00/0 • 1988 American Chemical Society

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Arabinosyl—Pyrimidine Nucleosides

177

Since our o r i g i n a l report (1.) on the synthesis and anti-herpes v i r u s a c t i v i t i e s of several 5-substituted pyrimidine nucleosides bearing the l-(2'-deoxy-2'-fluoro-0-D-arabinofuranosyl) moiety, structurea c t i v i t y studies have indicated that the 2'-fluoro substituent i n the "up" (arabino) configuration conferred more potent a n t i v i r a l a c t i v i t y than did a 2'-OH, hydrogen, or other halogens (2). Moreover, where studied (3.,4), the 2'-fluoro nucleosides were resistant to catabolic cleavage by nucleoside phosphorylases, presumably a r e s u l t of the increased metabolic s t a b i l i t y of the Ng l y c o s y l linkage imposed by this electronegative 2'-substituent. Of the several 2'-fluoro-5-substituted-arabinosyl pyrimidine nucleosides synthesized (1), l-(2'-deoxy-2'-fluoro-0-D-arabinofuranosy 1 )-5-iodocytosine [FIAC] has demonstrated c l i n i c a l e f f i c a c y against Herpesv i r u s infections i n Phase 1 (5.) and against V a r i c e l l a zoster v i r u s i n Phase 2 (6) c l i n i c a l t r i a l s i n immunocompromised cancer patients. The corresponding 5-methyluracil analog l-(2'-deoxy-2'-fluoro-p-Darabinofuranosyl)-5-methyluraci in mice infected with Herpe without t o x i c i t y at e f f e c t i v e dose l e v e l s . FMAU was a l s o found to be active i n v i t r o and i n v i v o against P-815 and L-1210 leukemia c e l l l i n e s resistant to the antileukemic agent, 1-0-D-arabinofuranosylcytosine [Ara-C]. A Phase 1 t r i a l of FMAU i n patients with advanced cancer showed that drug-induced central nervous system (CNS) dysfunction was the dose-limiting t o x i c i t y (J).

FIAC

FMAU

With FMAU as a lead compound, the syntheses of other 5 - a l k y l substituted 2'-fluoro-ara-uracils were undertaken (8,£) including 1(2 '-deoxy-2'-f 1 uoro-0-D-arab inof urano sy 1 )-5-e thy 1 urac i 1 [FEAU]. As shown i n Table I, though FEAU was approximately one log order less potent than FMAU against HSV-1 and HSV-2 infected Vero c e l l s i n culture, the former exhibited f a r less host c e l l t o x i c i t y , r e s u l t i n g in an extremely favorable therapeutic index (9,10). A comparison of the a n t i v i r a l a c t i v i t y of FMAU, FEAU, and 5ethyl-2'-deoxyuridine (EDU) i n mice inoculated intracerebral l y with HSV-2 i s given i n Table II (10). FEAU showed a c t i v i t y at 50 mg/kg/day f o r 4 days and was h i g h l y e f f e c t i v e i n reducing m o r t a l i t y in these mice at doses of 100 - 200 mg/kg/day. At these dose l e v e l s , no t o x i c i t y was observed. In normal (uninfected) mice, FEAU

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Table I. Comparative Anti-HSV A c t i v i t y of FMAU and FEAU i n Plaque Reduction Assays i n Vero C e l l s

HSV-KF)*

HSV-2 (G)*

^90 (uM)

ID*

E D

^50

90 (jiM)

(

»

# ft

l M )

Therapeutic Index I D

/ E D

50 90 HSV-1 HSV-2

FMAU

0.010

0.042

0.023

0.09

2.8

67

FEAU

0.024

0.26

0.24

0.91

>200

>769

31 >220

•Correlation c o e f f i c i e n t >. 0.86. #Cytotoxic effect measured i n r a p i d l y d i v i d i n g c e l l s . ID^Q = concentration required to i n h i b i t c e l l growth by 50%. **E HSV r e p l i c a t i o n by the

Table I I .

Treatment

A n t i v i r a l Effects of FEAU, FMAU and EDU i n Mice Inoculated Intracerebrally with HSV-2 (Strain G) Dose* mg/kg/day

MDD**

-

% Survivors

%Wt Gain on Day 7 14 21 30

16 17

6.7

100 7

-3 -22

9 -14

13 8

10 50 100 200

9.4 11.8 11.0^ (14)

8 25 72 93

-7 3 2 0

-13 -10 7 1

+

31 16 13 9

36 25 13 13

FMAU

0.5

9.4

67

1

4

5

7

EDU

800 1000

7.6 8.6

13 33

-10 3

-6 13

Neg Control (no virus) PBSA (virus control)

FEAU

#

+

+

+

+

+

2 6 34 43

•Given 5 hr after intracerebral inoculation. Dose schedule, twice a day for 4 days. "Median day of death c a l c u l a t e d on day 21. #Numbers i n parentheses indicate death of a single animal. §As compared to day 1. Based on weight of a single surviving animal only. APhosphate buffer s a l i n e . +

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was nontoxic at doses of 800 mg/kg/day given twice d a i l y for four days. I t i s a l s o noteworthy that EDU, which d i f f e r s s t r u c t u r a l l y from FEAU only by the absence of the 2'-fluoro substituent, i s much less e f f e c t i v e than FEAU i n this HSV-2 mouse encephalitis model. This finding i s consistent with our previous observation (1) attesting to the importance of the 2'-fluoro substituent f o r the anti-HSV a c t i v i t y exhibited by these arabinofuranosyl-pyrimidine nucleosides. It was concluded p r e v i o u s l y (11) that FMAU i s a most potent and s e l e c t i v e a n t i v i r a l compound f o r the treatment of mouse encephalitis caused by HSV-2 and therefore deserved consideration as a p o t e n t i a l agent i n human t r i a l s f o r the treatment of HSV encephalitis i n neonates and adults at low dose l e v e l s . The preliminary data described i n Tables I and II f o r FEAU suggested (10) that this compound may a l s o be worthy of s i m i l a r consideration. Based upon these e a r l i e r findings (10) d preliminar biochemical t (12), further comparativ undertaken with FMAU an against Simian v a r i c e l l a v i r u s i n the African green monkey and against h e p a t i t i s v i r u s i n the woodchuck animal model. In these studies (13) an a l t e r n a t i v e synthesis of FEAU i s a l s o described. COMPARATIVE BIOCHEMICAL STUDIES Biochemical studies at Memorial Sloan-Kettering Cancer Center (13) on the r e l a t i v e effects of FEAU and FMAU on the growth of mammalian c e l l s i s shown i n Table III. Note that against the human

Table I I I . Comparison of E f f e c t s of FEAU and FMAU i n Mammalian C e l l s * ED (in |iM) f o r Inhibiting C e l l growth i n :

FEAU

FMAU

HL-60 C e l l s Vero C e l l s

2060 >200

15.4 2.8

ED (jiM) f o r i n h i b i t i n g thymidine incorporation into DNA

FEAU

FMAU

700 630 3,700

14.0 28.0 8.9

5Q

5Q

P-815 C e l l s L-1210 Cells Rat Bone Marrow C e l l s •See reference 13 f o r experimental

FEAU/ 'FMAU 133 >71

FEAU/ 'FMAU 50 22 415

procedures

promyelocytic leukemia c e l l l i n e (HL-60) as w e l l as against Vero c e l l s (derived from the African green monkey), FEAU i s f a r less growth-inhibitory than FMAU, which compares favorably to the i n v i t r o study given i n Table I. S i m i l a r l y , against rodent c e l l l i n e s (P-815, L-1210 and rat bone marrow c e l l s ) , FEAU i s s u b s t a n t i a l l y l e s s inhibitory of thymidine incorporation than FMAU (Table III).

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

C e l l u l a r k i n e t i c constants (K^) were a l s o determined (13) f o r the i n h i b i t i o n of natural precursor incorporation into the DNA of L1210 c e l l s by FEAU and FMAU. Using t r i t i u m - l a b e l led natural precursors as substrates (thymidine, 2'-deoxyuridine and 2'deoxycytidine), i t was found that the FEAU/FMAU K^ ratios were 61, 111, and 8.1, r e s p e c t i v e l y . These r e s u l t s a l s o indicated that FEAU is a weaker i n h i b i t o r of natural nucleoside anabolism than FMAU i n these mammalian c e l l s .

14

14

Table IV. Incorporation of [2- C] FEAU or [2- C]FMAU Into DNA of Mammalian Cells Incorporation at 10 uM ( i n p mole/10 c e l l s / h r ) 6

C e l l Line

FEAU

FMAU

L-1210 Vero HSV-1 Infected Vero C e l l s

ND* 0.48

1.3 3.4

•Not detectable.

Also not detectable at 100 jiM.

14

Studies on the incorporation of 2 - C - l a b e l led FEAU and FMAU into the DNA of mammalian c e l l s showed very s i g n i f i c a n t differences (Table IV). There was no detectable incorporation of FEAU radioa c t i v i t y into the DNA of either L-1210 or Vero e e l 1 l i n e s , but substantial amounts of FMAU r a d i o a c t i v i t y were incorporated into the DNA of both c e l l l i n e s . When HSV-1 infected Vero c e l l s were exposed to C - l a b e l l e d FEAU and FMAU, both nucleosides were incorporated into the DNA of these v i r u s - i n f e c t e d c e l l s . Under these conditions, FMAU incorporation into HSV-1 infected Vero c e l l s was 7-fold greater than that observed for FEAU. This difference in incorporation may be due to the greater a f f i n i t y of FMAU for viral-encoded DNA polymerase and i s comparable to the magnitude of the difference of t h e i r a n t i he rpe t i c potencies i n v i t r o (13). It i s g e n e r a l l y accepted that the s e l e c t i v e antiherpes a c t i v i t y of 2'-fluoro-arabinosylpyrimidine nucleosides i s associated i n large measure with their a b i l i t y to be recognized as good substrates f o r HSV-specified thymidine kinase (TK), but not by the host TK (14*11). As shown in Table V, FMAU i s a good substrate ( r e l a t i v e to thymidine) for cytosol and mitochondrial TK's derived from the HL-60 human c e l l l i n e , as w e l l as f o r HSV-1 and HSV-2 derived TK's. By contrast, FEAU is a very poor substrate for the host HL-60 derived cytosol TK and an excellent substrate for HSV-1 and HSV-2 derived TK's (13). These data are consistent with the recent report by Mansuri et a l . , (16) who showed that FEAU has a very low a f f i n i t y for Vero c e l l TK (compared to thymidine) but a high a f f i n i t y toward HSV-1 and HSV-2 encoded TK's. Their data a l s o indicate that, in uninfected Vero c e l l s , FEAU would be phosphorylated at a very low rate i n the presence of thymidine. Their report (16) a l s o describes a modified chemical synthesis of FEAU.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Arabinosyl-Pyrimidine Nucleosides

Table V. Percentage Rates of Phosphorylation of FEAU and FMAU (Relative to Thymidine*) by Various Thymidine Kinases** Enzyme Source

% Rates of Phosphorylation of Various Thymidine Kinases FEAU

FMAU

HL-60 C e l l s Cytosol TK Mitochondrial TK

0 - 2.1 N/A#

81.7 219.0

HSV-1 (Strain KOS) TK HSV-2 (Strain 333) TK

82.7 203.2

42.0 146.6

•Thymidine at 400 jiM **Measured by procedure of Cheng et a l . , (14). #Not assayed

These biochemical and a n t i v i r a l screening studies (10,12-16) suggest that FEAU should be more s e l e c t i v e i n i t s a n t i v i r a l a c t i v i t y and thus offer less host t o x i c i t y . In v i v o experiments i n mice (10,13) show that both FMAU and FEAU are r e l a t i v e l y nontoxic. However, FMAU i s very neurotoxic i n dogs ( l e t h a l dose 2.5 mg/kg/day, i.v. x 5), while preliminary studies on FEAU i n dogs at 50 mg/kg/day x 10 show no encephalopathy ( l e t h a l dose 100 mg/kg/day x 10). As mentioned previously, FMAU exhibited dosel i m i t i n g CNS t o x i c i t y i n patients with advanced cancer (1) at an intravenous dose of 0.8 mg/kg/day x 5. The t o x i c i t y of FEAU i n humans i s not known. COMPARATIVE STUDIES IN MONKEYS AGAINST SIMIAN VARICELLA VIRUS. Studies at the Delta Regional Primate Research Center compared the a c t i v i t i e s of FEAU and FMAU i n African green monkeys infected with simian v a r i c e l l a v i r u s (SVV). As shown i n Table VI, the three untreated controls exhibited marked viremia and died by day 11. Monkeys treated with FEAU at three dose l e v e l s (intravenous route) showed no apparent t o x i c i t y even at the higher dose of 30 mg/kg/day x 10. Hematology tests and serum chemistries f o r a l l treated monkeys were normal and viremia ( r e l a t i v e to the controls) was minimal even at the low dose of 3 mg/kg/day. While the control monkeys developed severe rash, none of the FEAU-treated monkeys developed rash at these drug l e v e l s . Further studies showed that a lower dose of 1 mg/kg/day prevented development of rash but did not reduce viremia i n two out of three monkeys. These data suggest that the minimal e f f e c t i v e dose i n this system for FEAU i s ~1 mg/kg/day. Concurrent studies with FMAU showed i t to be ~40-fold more potent against SVV with a minimal e f f e c t i v e dose of ~0.04 mg/kg/day x 10. FEAU was a l s o h i g h l y e f f e c t i v e i n the treatment of Simian v a r i c e l l a v i r u s by the o r a l route. Oral administration at dose l e v e l s of 10, 3, or 1 mg/kg/day x 10 prevented rash (Table VII) and reduced viremia s i g n i f i c a n t l y . Even at the 1 mg dose, rash was almost e n t i r e l y prevented (two v e s i c l e s appeared on day 9, then promptly disappeared). Doses at 10 mg/kg/day x 10 by the o r a l route

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Table VI. Evaluation of FEAU in Treatment of Simian V a r i c e l l a Virus Infection in the African Green Monkey: Effect on Viremia Viremia** - Mean PFU on Days p . i . Treatment Group*

Monkey Number

3

5

7

9

G029 GO 30 G031

1 3 1

140 163 99

>400 >400 >400

Dead >400 Dead

Dead

FEAU 30 mg/kg/day

G023 G024

1 2

0 0

0 0

0 0

0 0

FEAU 10 mg/kg/day

G025 G026

1 0

14 1

5 1

0 0

0 0

FEAU 3 mg/kg/day

G027 GO 2 8

0

1

1

0

0

Control H0

-

2

11

•Treatment was administered by i.v. i n j e c t i o n twice d a i l y beginning 48 hours after virus inoculation and continuing for ten days ••Viremia was determined by culture of lymphocytes c o l l e c t e d from 3 mL of heparinized blood on indicated days post-infection (p.i.). The mean plaque-forming units expressed i s the average number of plaques present in two f l a s k s of Vero c e l l co-cultures inoculated with each lymphocyte suspension.

were without any observed t o x i c i t y . Thus, FEAU i s shown to be a highly e f f e c t i v e and s e l e c t i v e a n t i v i r a l in the treatment of SVV infection by both the intravenous and o r a l routes. In view of: a) the in v i t r o a c t i v i t y (15,18) of FIAC and FMAU against v a r i c e l l a zoster v i r u s (VZV, a member of the human herpesv i r u s group), b) the reported e f f i c a c y of FIAC for the treatment of v a r i c e l l a zoster v i r u s in Phase 2 t r i a l s in immunosuppressed patients (6), and c) the in v i v o a c t i v i t i e s of FMAU and FEAU against simian v a r i c e l l a v i r u s described herein and elsewhere ( 18), one may expect that FEAU w i l l also exhibit s i g n i f i c a n t s e l e c t i v e a c t i v i t y against VZV in humans. COMPARATIVE ANTI-HEPATITIS VIRUS STUDIES IN WOODCHUCKS. Hepatitis B v i r u s (HBV), a member of the Hepadna viruses, causes acute and chronic h e p a t i t i s in humans. It i s estimated that ~200 m i l l i o n people are c a r r i e r s of this v i r u s . HBV may be the primary causative agent of h e p a t o c e l l u l a r carcinoma (19). Hepadna viruses have also been discovered in other animals such as the woodchuck (Marmota Monax). The close s t r u c t u r a l and c l i n i c a l pathological s i m i l a r i t i e s , including nucleic acid homology (20), noted between woodchuck h e p a t i t i s v i r u s (WHV) and HBV suggest that the woodchuck may represent a promising model for studying persistent h e p a t i t i s v i r u s infections as w e l l as t h e i r r e l a t i o n s h i p to the development of l i v e r cancer (21). Like HBV, the woodchuck h e p a t i t i s v i r u s

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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elaborates a very s i m i l a r DNA polymerase for i t s r e p l i c a t i o n and integration. Potential anti-HBV agents can be detected by their i n h i b i t i o n of endogenous WHV or HBV DNA polymerase obtained from sera prepared from chronic-carrier woodchucks and from an immunosuppressed patient p o s i t i v e for the hepatitis B surface antigen. Such studies were undertaken at the Hepatitis Virus Unit, INSERM, Lyon, France, to measure the inhibitory effects of certain nucleoside triphosphates on these endogenous v i r a l DNA polymerases. In a series of assays (23,24* « Trepo, personal communication), the r e l a t i v e s e n s i t i v i t i e s of HBV and WHV DNA polymerases to several nucleoside triphosphates were determined (Table VIII). Of the six nucleoside triphosphates examined, FMAU was the most e f f i c i e n t i n h i b i t o r of both HBV and WHV DNA polymerases, followed c l o s e l y by FIAC. Moreover, the potencies (ID Q» ) of each of these six triphosphates against HBV or WHV DNA polymerases, though not i d e n t i c a l , were rather close More important the orders of potency as i n h i b i t o r s of these r e s u l t s attest furthe WHV and point to the v a l i d i t y of the woodchuck as an animal model of choice for the i n v i v o evaluation of p o t e n t i a l a n t i h e p a t i t i s B v i r u s agents (24,). Studies were then undertaken to evaluate FMAU and FEAU i n this animal model using woodchucks n a t u r a l l y c h r o n i c a l l y - i n f e c t e d with woodchuck h e p a t i t i s v i r u s (25). [It was assumed that these nucleosides would be anabolized to their nucleoside triphosphates within the animal.] WHV r e p l i c a t i o n was measured p e r i o d i c a l l y by the endogenous DNA polymerase a c t i v i t y and by the detection of WHV DNA by the dot-blot hybridization assay. The v i r a l DNA polymerase a c t i v i t i e s of untreated woodchucks are given i n Figure 1. Three d i f f e r e n t dosages of FMAU and FEAU were investigated. FMAU at 2.0 mg/kg/day x 5, (given i n t r a p e r i t o n e a l l y beginning at day 0), produced marked i n h i b i t i o n of WHV r e p l i c a t i o n as shown by the complete suppression of WHV DNA polymerase a c t i v i t y (Figure 2). However, at this dose, severe CNS t o x i c i t y was observed, which was e v e n t u a l l y l e t h a l . FEAU administered i.p. at 2.0 mg/kg/day x 10 a l s o produced almost immediate suppression of WHV r e p l i c a t i o n and did not exhibit any toxic effects. At 0.2 mg/kg/day, FMAU (given x 5) and FEAU (given x 10) were e q u a l l y e f f e c t i v e i n suppressing v i r a l r e p l i c a t i o n (Figure 3). At this dose, FMAU exhibited a less severe and delayed t o x i c i t y , whereas FEAU was again nontoxic. Even at doses of 0.04 mg/kg/day x 10, FEAU exerted a somewhat diminished anti-WHV effect. A preliminary study with one woodchuck given FEAU by o r a l administration at a dose of 0.2 mg/kg/day x 10 gave s i g n i f i c a n t suppression of WHV r e p l i c a t i o n , again without any observed t o x i c i t y (Figure 4). After cessation of drug administration, the i n h i b i t o r y a c t i v i t y of FEAU at 0.2 or 2.0 mg doses remained s i g n i f i c a n t over a six-week period while returning slowly to pretreatment l e v e l s . FEAU i n h i b i t i o n of WHV r e p l i c a t i o n was almost immediate and was markedly more sustained than i s the case with other a n t i v i r a l s such as 6deoxyacyclovir [9-(2-hydroxyethoxymethyl)-2-aminopurine], DHPG [9(l,3-dihydroxy-2-propoxymethyl)guanine], or Ara-AMP [9-(0-Da

n

d c

5

s

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Table VII. Evaluation of Oral Dosage of FEAU i n the Treatment of Simian V a r i c e l l a Virus Infection i n the African Green Monkey: Effect on Rash* Rash _ jSeverity on Days p . i . Treatment Group*

Monkey Number

6

Control PBS**

F644 G668 G604

—

FEAU 10 mg/kg/day

G249 G267 G264

FEAU 3 mg/kg/day

G665

FEAU 1 mg/kg/day

7

8

9

10

11

12

14

+

3+

4+

-

-

+

±

3+

Dead 1+ 4+

±

+

4+ 2+ 4+

2+

+

+

-

-

-

-

-

G257 G268 G269 G270 G274

+

•Treatment was administered by stomach tube twice d a i l y beginning 48 hours after v i r u s inoculation and continuing f o r ten days. ••Phosphate buffer saline. #Severity of rash was graded on a scale of + to 4+. A + rash was scored when several v e s i c l e s were observed while a 4+ rash indicated the widespread d i s t r i b u t i o n of rash over the body surface. Table VIII. Comparative Inhibitory A c t i v i t i e s of Nucleoside Triphosphate Analogs on DNA Polymerases of Human Hepatitis Virus (HBV) and Woodchuck Hepatitis Virus (WHV) ID Inhibitors" FMAU-TP FIAC-TP BVDU-TP Ara T-TP ACV-TP Ara C-TP

5 0

HBV DNA Polymerase 0.025 0.05 0.25 0.30 0.90 1.10

+

(uM)* 1

WHV DNA Polymerase" " 0.05 0.10 0.30 0.40 0.70 1.20

'ID50 = Concentration of i n h i b i t o r giving 50% i n h i b i t i o n of DNA polymerase a c t i v i t y . ••These inhibitors are the nucleoside 5'triphosphate d e r i v a t i v e s of: FMAU, l-(2'-deoxy-2'-fluoro-0-Darabinofuranosy 1 )-5-methyluraci 1; FIAC, l-(2'-deoxy-2'-fluoro-p-Darabinofuranosyl)-5-iodocytosine; BVDU, E-5(2-bromovinyl)-2 deoxyuridine; Ara-T, 1-0-D-arabinofuranosylthymine; ACV, 9-(2hydroxyethoxymethy 1)guanine; Ara-A, 9-0-D-arabinofuranosy 1 adenine. DNA polymerase was assayed as described by Kaplan et a l . (22), as modified by Hantz et a l . (23) r

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FOXETAL.

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Figure 1. WHV DNA Polymerase A c t i v i t y in Untreated C h r o n i c a l l y Infected Woodchucks (Controls). Each symbol (o—0, T—T, or •) represents i n d i v i d u a l woodchucks, with points on the l i n e indicating the day when blood was taken for polymerase assay. DNA polymerase a c t i v i t y was measured as described by Kaplan et a l . (22), as m o d i f i e d by Hantz et a l . (23.).

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

FMAU

-10

0

10

20

DAYS

Figure 2. Comparison of Inhibition of WHV R e p l i c a t i o n in C h r o n i c a l l y infected Woodchucks by Treatment with FMAU and FEAU at Dosage and Schedule Indicated. The length of the bar gives the drug treatment time period in days indicated on the abscissa. Each of the symbols (A—A, • — o r A—A) refers to i n d i v i d u a l woodchucks, with points on the l i n e representing the days when bloods were drawn for polymerase assay. WHV DNA polymerase a c t i v i t y was measured according to Kaplan et a l . (22), as modified by Hantz et a l . (23.).

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

10. FOX ET AL.

Arabinosyl-PyrimidineNucleosides

F i g u r e 3. I n h i b i t i o n o f WHV R e p l i c a t i o n i n C h r o n i c a l l y I n f e c t e d Woodchucks by Treatment w i t h FMAU and FEAU at Lower Dosages. (See F i g u r e 2 f o r l e g e n d ) .

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Figure 4. I n h i b i t i o n of WHV Replication i n C h r o n i c a l l y Infected Woodchucks Treated with FEAU by the Oral Route. FEAU was administered by stomach tube. (See Figures 1 and 2 for legend.)

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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arabinofuranosyl)adenine 5'-phosphate. These l a t t e r drugs a l s o diminished WHV DNA polymerase l e v e l s , but they soon rebounded to pretreatment l e v e l s after cessation of drug administration (C, Trepo, personal communication). In contrast to previous r e s u l t s obtained with FMAU and FIAC (where l e t h a l t o x i c i t y was demonstrated), only an ~10% weight loss was observed following FEAU treatment (24). The observed e f f i c a c y of FEAU against woodchuck h e p a t i t i s v i r u s r e p l i c a t i o n at these low doses was rather surprising. On the basis of the i n v i t r o studies on Herpes simplex viruses (9,10), the i n v i v o studies on the herpes encephalitis model in mice (10), and the simian v a r i c e l l a v i r u s studies i n the African green monkey reported herein, one would have expected that FEAU would be much less potent than FMAU. The data suggest that FEAU may be an e f f e c t i v e agent c l i n i c a l l y against h e p a t i t i s B v i r u s . On the basis of i t s potency and s e l e c t i v i t y , i t appears to be the most promising of the nucleoside analogs tested thus far. Further evaluation of FEAU i s i n progress.

ACKNOWLEDGMENTS These studies were supported i n part by National Cancer Institute grants CA-08748, 18601 and 18856 (JJF, KAW, TCC), by CA-44094 and The Veterans Administration (RFS), by the National Institute of A l l e r g y and Infectious Diseases grant NO-A1-62521 (KFS), and by the I n s t i t u t National de l a Sante' et de Recherche Medicale (IF, OH, CT).

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9.

Watanabe, K. A.; Reichman, U.; Hirota, K.; Lopez, C.; Fox, J. J. J. Med. Chem. 1979, 22 21-24. Fox, J. J.; Lopez, C.; Watanabe, K. A., in Medicinal Chemistry Advances; de las Heras, F. G.; Vega, S., Eds.; Pergamon Press: Oxford, 1981; pp 27-40. Chou, T. C.; Feinberg, A.; Grant, A. J.; Vidal, P.; Reichman, U.; Watanabe, K. A.; Fox, J. J. Cancer Res. 1981, 41, 3336-3342. Coderre, J. A.; Santi, D. V.; Matsuda, A.; Watanabe, K. A.; Fox, J. J. J. Med. Chem. 1983, 26, 1149-1152. Young, C. W.; Schneider, R.; Leyland-Jones, B.; Armstrong, D.; Tan, C.; Lopez, C.; Watanabe, K. A.; Fox, J. J.; Philips, F. S. Cancer Res. 1983, 43, 5006-5009. Leyland-Jones, B.; Donnelly, H.; Groshen, S.; Myskowski, P.; Donner, A. L.; Fanucchi, M.; Fox, J. J. J. Infectious Dis. 1986, 154, 430-436. Fanucchi, M. P.; Leyland-Jones, B.; Young, C. W.; Burchenal, J. H.; Watanabe, K. A.; Fox, J. J . Cancer Treatment Rep. 1985, 69, 55-59. Watanabe, K. A.; Su, T-L.; Reichman, U.; Greenberg, N.; Lopez, C.; Fox, J. J. J. Med. Chem. 1984, 27, 91-94. Perlman, M. E.; Watanabe, K. A.; Schinazi, R. F.; Fox, J. J. J. Med. Chem. 1985, 28, 741-748.

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10. Fox, J. J.; Watanabe, K. A.; Schinazi, R. F.; Lopez, C. In Herpes Viruses and Virus Chemotherapy; Kano, R.; Nakajima, A., Eds.; Excerpta Medica: Amsterdam, 1985; pp 53-56. Based upon proceedings of International Symposium on Pharmacological and Clinical Approaches to Herpes Viruses and Virus chemotherapy presented in Oiso, Japan, September, 1984. 11. Schinazi, R. F.; Peters, J.; Sokol, M. K.; Nahmias, A. J. Antimicrobial Agents Chemother. 1983, 24, 95-103. 12. Chou, T-C.; Kong, X-B.; Potter, V. P.; Schmid, F. A.; Fox, J . J . ; Watanabe, K. A.; Fanucchi, M. Proc. Amer. Assoc. Cancer Res., 1985, 26, 333. 13. Chou, T-C.; Kong, X-B.; Fanucchi, M. P.; Cheng, Y-C.; Takahashi, K.; Watanabe, K. A.; Fox, J. J. Antimicrobial Agents Chemother. 1987, 31, 1355-1358. 14. Cheng, Y-C.; Dutschman, G.; Fox, J. J.; Watanabe, K. A.; Machida, H. Antimicrobial Agents Chemother 1981 20, 420-423 15. Lopez, C.; Watanabe Chemother. 1980, 17 16. Mansuri, M. M.; Ghazzouli, I.; Chen, M. S.; Howel, H. G.; Brodfuehrer, P. R.; Benigni, D. A.; Martin, J. C. J. Med. Chem. 1987, 30, 867-871. 17. Machida, H.; Kuninaka, A.; Yoshino, H. Antimicrobial Agents Chemother. 1982, 21, 358-361. 18. Soike, K. F.; Cantrell, C.; Gerone, P. J. Ibid., 1986, 29, 2025. 19. Blumberg, B. S.; London, W. T. In Clinical Management of Gastrointestinal Cancer: Decosse, J. J.; Sherlock, P., Eds.; Martinus Nijhoff Publishers: Boston, 1984; pp. 71-91. 20. Galibert, F.; Chen, T. V.; Mandart, E. Proc. Natl. Acad. Sci. U.S.A., 1981, 78, 5315-5319. 21. Blumberg, B. S. Hum. Pathol. 1981, 12, 1107-1113. 22. Kaplan, P. M.; Greenman, R. I.; Gerin, J. L.; Purcell, R. H.; Robinson, W. S. J . Virol. 1973, 12, 995-1005. 23. Hantz, O.; Ooka, T.; Vitvitski, L.; Pichoud, C.; Trepo, C. Antimicrobial Agents Chemother. 1984, 25, 242-246. 24. Hantz, O.; Allaudeen, H. S.; Ooka, T.; De Clercq, E.; Trepo, C. Antiviral Res. 1984, 4, 187-199. 25. Fourel, I.; Hantz, O.; Watanabe, K. A.; Fox, J. J.; Trepo, C. Hepatology 1987, 7, 1122. RECEIVED January 15, 1988

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 11

Fluorinated Analogs of Cell-Surface Carbohydrates as Potential Chemotherapeutic Agents Moheswar Sharma, Ralph J. Bernacki, and Walter Korytnyk Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute 666 Elm Street Buffalo NY 14263 The rationale for designing fluorinated analogs of cell surface carbohydrates as anticancer agents is described. Using diethylaminosulfur trifluoride, a general and a convenient method for synthesis of 6-deoxy-6-fluoro-hexosamines and 9-deoxy-9-fluoro-N-acetylneuraminic acid has been developed. This re-agent has also been used to introduce geminal fluorine both in the 6-position and 4-position (6,6-difluoro-D-galactose and 4,4-difluoro-N-acetyl-D-glucosamine). Another method for incorporation of fluorine is the addition of fluorine to the double bond in the acetylated glycals using XeF2 in the presence of BF3. This process is shown to be a convenient route to synthesize 1,2-dideoxy-1,2-difluoro sugars and 2-deoxy-2,3-di-fluoro-N-acetyl-D-neuraminic acid. Conventional methods of fluorination are used to synthesize 6-deoxy-6-fluoro-D-galactosamine and 4-deoxy-4-fluoro-D-galactosamine. The effects of these analogs on the growth of tumor cells (in vitro) and inhibition of macromolecular biosynthesis, as well as their therapeutic activity on syngenic mice with ascites L1210 leukemia (in vivo) are reported. As p a r t o f our program on a n t i t u m o r plasma-membrane m o d i f i e r s and i n h i b i t o r s , we have s y n t h e s i z e d s e v e r a l c a r b o h y d r a t e anal o g s , s u b s t i t u t i n g a f l u o r i n e f o r a hydroxyl group. These a n a l o g s were d e s i g n e d t o a c t as c a r b o h y d r a t e c h a i n t e r m i n a t o r s o f c e l l s u r f a c e g l y c o c o n j u g a t e s o r as i n h i b i t o r s o f v a r i o u s enzymes involved i n g l y c o p r o t e i n metabolism. A l t e r a t i o n o f the c e l l s u r f a c e c a r b o h y d r a t e s c o u l d be a c h i e v e d e i t h e r by i n h i b i t i o n o f N O T E : This chapter is dedicated to the late Walter Korytnyk.

0097-6156/88/0374-0191$06.00/0 • 1988 American Chemical Society

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t h e i r b i o s y n t h e s i s o r by i n c o r p o r a t i o n o f m o d i f i e d derivatives. T h i s c o u l d r e s u l t i n s i g n i f i c a n t changes i n the membrane s t r u c t u r e . The c h e m i c a l s y n t h e s i s o f t h e f l u o r i n a t e d d e r i v a t i v e s o f c e l l surface carbohydrates, namely galactose, L-fucose, galactosamine, g l u c o s a m i n e , mannosamine and M - a c e t y l - D - n e u r a m i n i c a c i d as w e l l as t h e i r b i o l o g i c a l e f f e c t s w i l l be reviewed i n t h e c o n t e x t o f ongoing research in t h i s area. There has been considerable interest to pursue approaches d i r e c t e d towards the m o d i f i c a t i o n and i n h i b i t i o n o f c e l l - s u r f a c e glycoprotein. Such m o d i f i c a t i o n may change the immunogenicity, t u m o r o g e n i c i t y and/or m e t a s t a t i c p o t e n t i a l o f cancer c e l l s . The p r i m a r y t a r g e t o f such m o d i f i c a t i o n a r e membrane s i a l i c a c i d , amino s u g a r s , and the o t h e r c e l l - s u r f a c e n e u t r a l s u g a r s . In a d d i t i o n , some c a r b o h y d r a t e a n a l o g s a r e e x p e c t e d t o modify the n u c l e o t i d e pool s i z e s and hence have p o t e n t i a l use i n c o m b i n a t i o n chemotherapy with n u c l e o s i d e a n a l o g s Plasma-membrane components a r e i n v o l v e d in c e l l growth, d i v i s i o n and a n t i g e n i c i t y . Fxtensiv occur i n these c e l l - s u r f a c e p r o p e r t i e s f o l l o w i n g oncogenic t r a n s f o r m a t i o n by v i r u s e s o r c a r c i n o g e n s . One o f the major components o f t h e c e l l s u r f a c e found t o be a l t e r e d f o l l o w i n g t r a n s f o r mation are the glycoconjugates. The g l y c o c o n j u g a t e s comprise glycoproteins and g l y c o l i p i d s t h a t a r e a t t a c h e d by t h e i r hydrophobic region to the l i p i d b i l a y e r . The c a r b o h y d r a t e c h a i n s o f these m o l e c u l e s a r e found on the o u t e r s u r f a c e o f t h e plasma membrane and c o n s i s t of fa) amino sugars, M-acetyl-P-glucosamine (GlcMAc), N-acetyl-D-galactosamine (GalMAc) and N - a c e t y l - D - n e u r a m i n i c a c i d "(MAMA); (b) n e u t r a l s u g a r s l i k e L - f u c o s e , D-mannose and D-galactose. The attachment t o membrane p r o t e i n o c c u r s e i t h e r as GlcNAc-asparagine l i n k e d N - g l y c o s i d i c a l l y o r as G a l N A c - s e r i n e ( o r t h r e o n i n e ) which a r e l i n k e d £ - g l y c o s i d i c a l l y . L-Fucose and s i a l i c a c i d s g e n e r a l l y o c c u r as t h e t e r m i n a l sugar (4). T h i s c o m p l e x i t y c o n f e r s a good deal o f i n f o r m a t i o n a l p o t e n t i a l t o t h e s u r f a c e c a r bohydrate p r e s e n t i n the v a r i o u s branched o l i g o s a c c h a r i d e structures (5). The r a t i o n a l e t o pursue s t u d i e s on the s y n t h e s i s and b i o c h e m i s t r y o f f l u o r o s u g a r s i s based on the o b s e r v a t i o n s t h a t : I. Biochemical d i f f e r e n c e s e x i s t i n the c e l l s u r f a c e complex carbohyd r a t e s o f tumor c e l l s . II. Sialic a c i d s may mask cell-surface a n t i g e n ; i t s removal enhances c e l l u l a r i m m u n o g e n i c i t y . I I I . Carboh y d r a t e s a r e i n v o l v e d i n the s o c i a l b e h a v i o r o f c e l l s such as c e l l to c e l l communications r e s u l t i n g i n c o n t a c t i n h i b i t i o n . I V . Changes in c e l l s u r f a c e c a r b o h y d r a t e s a r e found t o e f f e c t t h e uptake o f drugs and n u t r i e n t s . V . D i f f e r e n c e s a r e found t o d e v e l o p i n the m e t a b o l i s m o f membrane sugar f o l l o w i n g o n c o g e n i c t r a n s f o r m a t i o n . V I . Some membrane sugar and t h e i r a n a l o g s a r e c y t o t o x i c t o tumor cells (£). V I I . Uptake and a c c u m u l a t i o n o f some membrane sugars results in the lowering of specific intracellular nucleotide pools. V I I I . Some membrane sugars and t h e i r a n a l o g s have been shown t o i n h i b i t e n v e l o p e DNA and RMA v i r u s p r o l i f e r a t i o n . IX. Membrane s i g n a l t r a n s d u c t i o n i s a l t e r e d by oncogene p r o d u c t s following t r a n s f o r m a t i o n . The membrane sugar a n a l o g s under development i n t h i s program, may a c t on tumor o r v i r a l l y i n f e c t e d c e l l s i n t h e f o l l o w i n g manner:

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SHARMAET AL.

Fluorinated Analogs ofCell-Surface Carbohydrates

(a) In c o m p e t i t i o n as an a n t i m e t a b o l i t e w i t h the n a t u r a l sugar subs t r a t e l e a d i n g t o the i n h i b i t i o n o f membrane g l y c o c o n j u g a t e form a t i o n , (b) V i a i n c o r p o r a t i o n o f s t r u c t u r a l l y s i m i l a r a n a l o g s i n t o g l y c o n j u g a t e l e a d i n g t o c h a i n t e r m i n a t i o n , (c) In c o n j u g a t i o n with s p e c i f i c n u c l e o t i d e s l e a d i n g t o an a l t e r a t i o n i n n u c l e o t i d e pool s i z e s ( p o t e n t i a l use i n c o m b i n a t i o n chemotherapy), (d) Through i n t r o d u c t i o n o f changes i n c e l l s u r f a c e a n t i g e n i c i t y and immunogenicity. (e) By m o d i f i c a t i o n o f the s o c i a l b e h a v i o r o f c e l l s l e a d i n g to d e c r e a s e d tumor growth and m e t a s t a s i s , (f) A n t i v i r a l a c t i v i t y . S y n t h e s i s o f F l u o r i n a t e d Membrane Sugar A n a l o g s F u l l y a c e t y l a t e d membrane sugars and t h e i r a n a l o g s a r e found to be more c y t o t o x i c than t h e i r corresponding unacetylated derivatives. These d i f f e r e n c e s may have been the r e s u l t o f an i n c r e a s e i n the a g e n t s c e l l u l a r p e r m e a b i l i t y and p a s s i v e u p t a k e , s i n c e it has been found t h a t f u l l y a c e t y l a t e d glucosamine (e-PAGlcNAc) has a h i g h e r o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t ( 0 . 4 2 ) than the p a r e n t compound, N - a c e t y l - D - g l u c o s a m i n t a k e , PA-GTcMAc was O - d e a c e t y l a t e d t o form UDP-GlcNAc, r e s u l t i n g i n a s i g n i f i c a n t l o w e r i n g o f c e l l u l a r UTP and PTP p o o l s ( 6 ) . A general and c o n v e n i e n t method f o r the s y n t h e s i s o f 6 - d e o x y - 6 f l u o r o h e x o s e s has been d e v e l o p e d u s i n g the f l u o r i n a t i n g agent N_,Ndiethylaminosulfur trifluoride (DAST) (7h This reagent permits the use o f the a c e t y l as the p r o t e c t i n g g r o u p ; however, the r e a c t i o n a l s o runs smoothly when the p r o t e c t i n g groups a r e b e n z y l o r methyl e t h e r s as w e l l ( 8 ) . DAST a l s o d i s p e n s e s w i t h the n e c e s s i t y o f i n t r o d u c i n g a l e a v i n g group as the h y d r o x y l group i s being directly r e p l a c e d with f l u o r i n e . The f o l l o w i n g 6-deoxy-6-fluoro hexopyranoses have been prepared using this reagent from the 6-hydroxy-acetylated hexoses: D-glucose, N-acetyl-D-glucosamine, M - a c e t y l - D - g a l a c t o s a m i n e and M-acetyl-D-mannosamine. In t h e p r e p a r a t i o n o f 6 - d e o x y - 6 - f l u o r o - D - m a n n o s a m i n e from 6 - d e o x y - 6 - f l u o r o - D glucosamine by base e p i m e r i z a t i o n o f the M - a c e t y l - d e r i v a t i v e , the s e p a r a t i o n was v e r y d i f f i c u l t for large-scale preparations, even though the e p i m e r i c m i x t u r e c o n t a i n e d about 20% o f the D-mannoepimer. We r e s o r t e d , t h e r e f o r e , t o the use o f N-acetyl-D-mannosamine ( 9 J . The usual p r o c e d u r e f o r the p r e p a r a t i o n o T the 6 - h y d r o x y t e t r a a c e t y l - d e r i v a t i v e c o u l d not be a p p l i e d w i t h N-acetyl-D-mannosamine as t h e i n i t i a l t r i t y l a t i o n s t e p i n v o l v e d p a r t i a l epimerizat i o n to N-acetyl-P-glucosamine i n the p r e s e n c e o f p y r i d i n e . The s t a r t i n g m a t e r i a l was 2 - a c e t a m i d o - l , 3 - d i - 0 - a c e t y l - 2 - d e o x y - 4 , 6 - C i s o p r o p y l idene-D-manno-pyranose (1) (10F. The a c e t a l group was opened up w i t h aqueous a c e t i c a c i d (60%T t o g i v e (2) ( F i g u r e 1) and the p r i m a r y h y d r o x y l group was t r i t y l a t e d i n the usual way t o g i v e ( 3 ) . The h y d r o x y l group i n the 4 - p o s i t i o n was a c e t y l a t e d and the t r i t y l group was removed t o y i e l d (4) which was then f l u o r i n a t e d w i t h DAST i n anhydrous diglyme t o g i v e ( 5 ) . Anhydrous diglyme was a l s o used as the s o l v e n t i n the p r e p a r a t i o n o f 6 - d e o x y - 6 - f l u o r o - D - g a l a c t o s e (11) and 6 - d e o x y - 6 , 6 d i f l u o r o - D - g a l a c t o s e (8) ( L e e , H . H . ; Hodgson, P . G . ; B e r n a c k i , R . J . ; K o r y t n y k , W.; Sharma, M. Carbohydr. Res., in press) (34) from 1,2:3,4-di-0-isopropyl idene-a-D-galactose and 1,2:3,4-dl-Cpsopropylidene-a-D-oalactohexo-dialdo-1,5-pyranose (6) respectively, by t r e a t m e n t w i t h DAST. D e b l o c k i n g o f the i n t e r m e d i a t e d i f l u o r o d e r i -

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v a t i v e (7) t o g i v e t h e t a r g e t compound (8) was a c c o m p l i s h e d by c o n ventional a c i d h y d r o l y s i s (Figure 2 ) . S i m i l a r l y , the preparation o f the 6 - d e o x y - 6 - f l u o r o - L - g a l a c t o s e o r 6 - f l u o r o - L - f u c o s e , from 1 , 2 : 3,4-di-O-isopropylidene-L-galactopyranose (12) has been reported (13). F l u o r i n a t i o n o f N - a c e t y l - D - g a l a c t o s a m i n e by t r e a t m e n t o f 1 , 3 , 4 t r i - O - a c e t y l - N - a c e t y l - D - g a l a c t o s a m i n e w i t h DAST gave low y i e l d s o f the H e s i red p r o d u c t . An a l t e r n a t i v e p r o c e d u r e was adopted t o p r e pare 6 - d e o x y - 6 - f l u o r o - N - a c e t y l - D - g a l a c t o s a m i n e (11) ( 1 4 ) . Treatment o f benzyl 2-acetamldo-2-deoxy-3,4-0-isopropylidene3"-0-mesyl-aD - g a l a c t o p y r a n o s i d e (9) ( F i g u r e 3) witFT cesium f l u o r i d e Th b o i l i n g ethanediol gave (10) whicfT was d e b l o c k e d by c a t a l y t i c hydrogenol y s i s t o (11) and then a c e t y l a t e d t o ( 1 2 ) . —

F l u o r i n a t i o n o f the anomeric carbon atom a l o n g w i t h the a d j a c e n t c a r b o n atom, o c c u r s by t h e a c t i o n o f f l u o r i n e on a c e t y l a t e d g l y c a l s (15). A n o t h e r method o f f l u o r i n a t i o n o f a c e t y l a t e d g l y c a l s i s t h e use o f t r i f l u o r o m e t h y s u l t s i n a s i d e produc xenon d i f l u o r i d e ( 1 8 ) , a r e a g e n t t h a t can be handled w i t h o u t spec i a l care or apparatus. The c o n v e n i e n c e o f t h i s r e a g e n t a l l o w s t h e preparation of F - l a b e l l e d compounds and hence i s o f potential use i n p o s i t r o n emmission tomography. Boron trifluoride-etherate was c a r e f u l l y used as a m i l d a c i d c a t a l y s t t o e f f e c t t h e f l u o r i n a t i o n and b e n z e n e - e t h e r m i x t u r e as a s o l v e n t t o a v o i d d i m e r i c and polymeric products. Thus, 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-a-Dglucopyranosyl f l u o r i d e (14), 3 , 4 , 6 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 - f l u o r o - e D - g l u c o p y r a n o s y l f l u o r i d e (15) and 3 , 4 , 6 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 f l u o r o - e - D - m a n n o p y r a n o s y l f l u o r i d e (16) were a l l o b t a i n e d by f l u o r i nation o f 3 , 4 , 6 - t r i - O - a c e t y l - D - g l u c a l (13) w i t h xenon difluoride ( F i g u r e 4) through both c i s and t r a n s a d d i t i o n o f f l u o r i n e ( 1 9 ) . The same mode o f r e a c t i o n was o b s e r v e d w i t h t r i - 0 - a c e t y l g a l a c t a T T 1 8

The composition of products resulting from cis and trans a d d i t i o n o f f l u o r i n e was found t o be the same when t h e r e a c t i o n was c a r r i e d o u t i n an oxygen o r n i t r o g e n atmosphere t o a v o i d any f r e e r a d i c a l r e a c t i o n mechanism. This r e a c t i o n with 3 , 4 - d i - 0 - a c e t y l L - f u c a l (17) (20) gave a l m o s t e x c l u s i v e l y t h e s i n g l e a d d i t i o n p r o duct 3,4-di-(Tacetyl-l,2-dideoxy-1,2-difluoro-L-fucose (18) ( 1 9 ) , which on a c i c T h y d r o l y s i s gave 2 - d e o x y - L - f u c o s e (19) ( F i g u r e T ) . From o u r e x p e r i m e n t a l data i t appears t h a t the r e a c t i o n proceeds as shown i n F i g u r e 6 . The r e a c t i o n o f u n s a t u r a t e d s i a l i c a c i d w i t h xenon d i f l u o r i d e was a l s o s t u d i e d i n t h e s i m i l a r f a s h i o n ( F i g u r e 7 ) . Methyl 4 , 7 , 8 , 9 t e t r a - 0 - a c e t y l - 2 , 3 - d e h y d r o - 2 - d e o x y - N - a c e t y l n e u r a m i n a t e (20)(21) r e a c t e d with xenon d i f l u o r i d e i n methylene c h l o r i d e i n t h e p r e s e n c e of boron t r i f l u o r i d e e t h e r a t e i n an oxygen atmosphere t o g i v e t h e 2,3-difluoro-derivative (21). In t h e absence o f oxygen the r e a c t i o n was v e r y s l u g g i s h and c o n s i d e r a b l e d e c o m p o s i t i o n o c c u r r e d . The a c e t y l a t e d 2 , 3 - d i f l u o r o - m e t h y l e s t e r (21) was d e a c e t y l a t e d t o the c o r r e s p o n d i n g d i f l u o r o a c i d (22) a t pH 7 w i t h o u t l o s s o f f l u o r ine. The l a t t e r compound on a c i d h y d r o l y s i s gave t h e 3 - f l u o r o - N a c e t y l n e u r a m i n i c a c i d (23) w i t h the h y d r o l y t i c removal o f anomeric fluorine. T h i s p r o c e s s s i g n i f i c a n t l y improved t h e y i e l d o f 3 f l u o r o - N - a c e t y l n e u r a m i n i c a c i d o v e r the p r e v i o u s l y r e p o r t e d p r o c e -

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

11. SHARMAETAL.

Fluorinated Analogs ofCell-Surface Carbohydrates

(I)

(2)

,(3)

(5)

(4)

F i g u r e 1. S y n t h e s i s o f t e t r a - 0 - a c e t y l - 6 - d e o x y - 6 - f l u o r o - D mannosamine (5). (a A c 0 / P y . ( c ) Pd/c-AcOH ?

F i g u r e 3 . Scheme f o r s y n t h e s i s o f N - a c e t y l - 3 , 4 - d i - 0 - a c e t y 1 - 6 deoxv-6-fluoro-Dgalactosamine (11). (a) CsF/ethyTene-alycol (200 C ) , (b) Pd/C i n AcOH, [ H ] , ( c ) A c 0 / P y . 2

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

F i g u r e 5. Synthesis of 2-deoxy-?-fluoro-L-fucose (19). (a) X e F , B F - e t h e r a t e , (b) 0 . 5 M HC1. 2

3

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SHARMA ET AL.

Fluorinated Analogs of Cell-Surface Carbohydrates

Figure 6. Possible mechanism of addition of f l u o r i n e to glycals with XeF . (a) E l e c t r o p h i l i c addition of F from XeF -BF3 complex, (b) addition of F on carboxonium i o n , (c) anomerization of pyranoxylfluoride with BF3. 2

2

Figure 7. Synthesis of 3-deoxy-3-fluoro-NANA (23). (a) XeF , BF -etherate, (b) NaOMe-MEOH, (c) 0.5 N HC1. 2

3

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dure (22). The F-NKR data were used t o d e t e r m i n e the conf i g u r a t i o n o f t h e f l u o r i n e atom o f methyl 4 , 7 , 8 , 9 - t e t r a - 0 - a c e t y l - 2 deoxy-2,3-difluoro-N-acetylneuraminic acid (21). The magnitude o f the J p _ 2 , F - 3 (I -5" Hz) indicates an a x i a l - e q u i t o r i a l arrangement. The c o u p l i n g c o n s t a n t J p _ H-3 d * Hz) i n d i c a t e s a d i a x i a l o r i e n t a t i o n o f the anomeric' f l u o r i n e and t h e H-3. These c o u p l i n g c o n s t a n t s and the absence o f any long range c o u p l i n g b e tween f l u o r i n e and hydrogen s u g g e s t t h e r i n g c o n f o r m a t i o n t o be C 5 (D) w i t h F-2 a x i a l and F-3 e q u i t o r i a l . 19

9

9

5

2

2

The 9 - p o s i t i o n o f N - a c e t y l n e u r a m i n i c a c i d (NANA) was s u b s t i t u t e d f o l l o w i n g two r o u t e s : Figure 8a, 2-Acetamido-2,6-dideoxy-6fluoro-D-glucopyranose (24) (8^,23) was condensed with potassium di-tert-butyloxaloacetate (Sharma, M.; Korytnyk, W., Carbohydr. R e s . , i n p r e s s ) and s u b s e q u e n t l y h y d r o l y z e d to 9 - d e o x y - 9 - f l u o r o NANA. Figure 8b, Methyl 2-0-methyl-4,7,8-tri-0-benzyl-N-acetylneuraminate (30) was o b t a i n e d by b e n z y l a t i o n o f meThyl 2 - 0 - m e t h y l 9-0-trityl-N-acetylneuraminat Treatment o7 the d e t r i t y l a t e hydrogenolysis and hydrolysis, gave the 9-deoxy-9-fluoro-NANA (27). The d e t r i t y l a t e d i n t e r m e d i a t e p r o d u c t (30) was o x i d i z e d a t the 9 - p o s i t i o n t o t h e c o r r e s p o n d i n g 9 - a l d e h y d e d e r i v a t i v e (31) and then c o n v e r t e d to the 9 - d e o x y - 9 - d i f l u o r o - N - a c e t y l n e u r a m i n i c acid derivative (32). The C MMR spectrum o f 9-deoxy-9-fluoro-NANA (27) showed t h a t t h e i n t r o d u c t i o n o f f l u o r i n e a t the 9 - p o s i t i o n d e s h i e l d e d C-9 from 663.5 p . p . m . t o 8 1 . 4 (as a d o u b l e t Jrj_p 164.9 Hz). The l - F NMR spectrum o f 9 - d e o x y - 9 - f l u o r o - N A N A gave a c l e a r sextet (-235.4 p . p . m . , J _ g , 4 6 . 8 Hz; J H-8> -° ) ind i c a t i n g t h e absence o f a n y ' a n o m e r i c i s o m e r . 1 3

2

F

j

H

F

6

H z

>

S u b s t i t u t i o n o f the secondary h y d r o x y l group o f c a r b o h y d r a t e s with f l u o r i n e o c c a s s i o n a l l y g i v e s r i s e t o t h e problem o f i n v e r s i o n , both i n a c o n v e n t i o n a l d i s p l a c e m e n t r e a c t i o n o r r e a c t i o n w i t h DAST. However, t h e use o f t h e s e methods i n t h e f l u o r i n a t i o n r e a c t i o n a r e w o r k a b l e , i n such c a s e s , with a p r e c e e d i n g s t e p o f i n v e r s i o n when n e c e s s a r y , t o o b t a i n the r e q u i r e d e p i m e r i c f l u o r i n a t e d p r o d u c t ( 2 4 , 25). We were i n t e r e s t e d i n i n t r o d u c i n g f l u o r i n e i n the C-4 p o s i t i o n o f 2 - a c e t a m i d o - 2 - d e o x y - D - g l u c o p y r a n o s e and 2 - a c e t a m i d o - 2 deoxy-D-galactopyranose s i n c e these molecules occur i n the core r e g i o n o f the g l y c o p r o t e i n s . Our f i r s t attempt was t h e opening o f the 3 , 4 - e p o x i d e w i t h the f l u o r i d e n u c l e o p h i l e . Benzyl 2 - a c e t a m i d o 3,4-anhydro-2-deoxy-6-0-trityl-a-D-glucopyranoside (33) was t r e a t e d w i t h tetrabutylammonium f l u o r i d e i n a c e t o n i t r i l e t o g i v e the c o r r e s p o n d i n g D-gulosamine d e r i v a t i v e (26) ( F i g u r e 9 ) . Inversion of the h y d r o x y l group o f (34) a t C-3 co~UTd not be a c h i e v e d t o o b t a i n the D - g a l a c t o s a m i n e d e r i v a t i v e . However, 2 - a c e t a m i d o - l , 3 , 6 - t r i - 0 acetyl-2,4-dideoxy-4-fluoro-D-gulose (35) was i s o l a t e d and showed e n c o u r a g i n g i n h i b i t o r y a c t i v i t y on t h e growth o f L1210 leukemia cells. 2-Acetamido-2,4-dideoxy-4-fluoro-D-galactopyranose (38) and 2-acetamido-2,4,6-trideoxy-4,6-difluoro-D-galactopyranose (43) were s y n t h e s i z e d from b e n z y l 2-acetamido-3,4-di-0-benzyl-2-deoxy-4O - m e s y l - a - D - g l u c o p y r a n o s i d e (36) and b e n z y l 2 - a c e t a m i d o - 3 - 0 - b e n z y l 2-deoxy-4,6-di-P-mesyl-a-D-glucopyranoside (41) (Figure 10), res p e c t i v e l y , by d i s p l a c e m e n t o f the methanesulphonyloxy groups with tetrabutylammonium f l u o r i d e . These f l u o r o sugar d e r i v a t i v e s were

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

FluorinatedAnalogs of Cell-Surface Carbohydrates

SHARMA ET AL.

F i g u r e 8. T o p , s y n t h e s i s o f 9 - d e o x y - 9 - f l u o r o - N A N A from 6-deoxy6 - f l u o r o - N - a c e t y l - D - g l u c o s a m i n e ( 2 7 ) . (a) Potassium d i - t e r t b u t y l - o x a l o a c e t a t e - M E O H ( e p i m e r i z a t i o n ) , (b) c o n d e n s a t i o n and h y d r o l y s i s . Bottom, s y n t h e s i s o f 9 - d e o x y - 9 - f l u o r o - N A N A from NANA ( 2 7 ) . (a) Benzyl b r o m i d e - B a ( 0 H ) - D M F , (b) AcOH-H 0 (70%), 90 ° C , (c) DAST-CHoClo, NaOH-HoO, 0.05 N HC1, (d) Py-CrOo-CHoCl (e) D A S T - C H C 1 . 2

2

2

2

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200

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

0-\-^0CH Ph 2

NHAc

\

/nr^PK

HO HNAc

(33)

(34)

F i g u r e 9. S y n t h e s i s fluoro-D-gulosamine [ H ] , Py/AcOH.

\

/ OAc HNAc (35)

of N-acetyl-1,3,6-tri-0-acetyl 4-deoxy-4( 3 D " , ( a ) BU4NF-CH3CN, (b) Pd/C-AcOH

HNAC

HNAc

(39)

(38)

HNAc (44)

HNAc (40)

HNAc (45)

F i g u r e 1 0 . Scheme f o r s y n t h e s i s o f ^ - a c e t y l - 4 - d e o x y - 4 - f l u o r o and N-acetyl-4,6-dideoxy-4,6-difluoro-D-galactosamine (39) and R 5 ) . (a) BU4NF-CH3CN, (b) [ H ] , Pd/c-AcOH, (c) Pt/C-H 0-0 , (d) Ac 0/Py, (e) BU4NF-CH3CM, (f) [H], Pd/C-AcOH, ( g ) P t / C - H 0 - 0 , (h) A c 0 - P y . 2

2

2

2

2

2

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SHARMAETAL.

Fluorinated Analogs ofCell-Surface Carbohydrates

a l s o o b t a i n e d by t r e a t i n g b e n z y l 2 - a c e t a m i d o - 3 , 6 - d i - 0 - b e n z y l - 2 d e o x y - a - D - g l u c o p y r a n o s i d e and b e n z y l 2-acetamido-3-(P>enzyl-a-Dglucopyranoside (27-29) w i t h DAST i n methylene c T i l o r i d e , but in much l o w e r y i e l d . Tfie" i n t e r m e d i a t e s were d e p r o t e c t e d by hydrogenol y s i s t o g i v e the f l u o r i n a t e d sugars (38) and ( 4 3 ) . The a l d o n o l a c tones (40) and (44) d e r i v e d from t h e s e s u g a r s by o x i d a t i o n w i t h m o l e c u l a r oxygen i n the p r e s e n c e o f p l a t i n u m on a c t i v a t e d c h a r c o a l were found t o be s t r o n g i n h i b i t o r s ( K i 27.2 uM and 41.4 nM, r e s p e c t i v e l y ) of hexosaminidases ( 2 5 , 3 2 ) . The a c e t y l a t e d d e r i v a t i v e s o f t h e s e s u g a r s (39) and (45) were shown t o be very good a n t i t u m o r agents in t i s s u e c u l t u r e (Table I ) . In the F NMR spectrum of the 4,6-dideoxy-4,6-difluoro-galactosamine derivative (42), it appeared from the v i c i n a l c o u p l i n g c o n s t a n t s ( J p . ^ H-5 ) t h a t t h e f a v o r e d rotamer about the C - 5 - C - 6 has F-6 a ' n t i - p e r i p l a n a r to C - 4 - C - 5 . 1 9

1

2

H

z

The b i o l o g i c a l activit f thes fluor suggested th p r e p a r a t i o n o f the 4 - d e o x y - 4 , 4 - d i f l u o r M-acetyl-P-glucosamine d e r i v a t i v e s "5enzyl-2-deoxy-a-D-xylo-4-uloside (46) ( F i g u r e 11) was t r e a t e d with DAST to g i v e the 4 - d e o x y - 4 , 4 - d i f l u o r o - D - g l u c o s a m i n e d e r i v a t i v e (47) i n v e r y good y i e l d . A f t e r d e p r o t e c t i o n and a c e t y l a t i o n t h e d i f l u o r o compound (48) showed v e r y good i n h i b i t o r y a c t i v i t y o f c e l l growth (L1210) i n v i t r o ( T a b l e I ) . 2 - A c e t a m i d o - l , 3 , 6 - t r i - 0 - a c e t y l 2,4-dideoxy-4-fTuoro-P-glucosamine (52) was s y n t h e s i z e d by double i n v e r s i o n a t the 4 - p o s i t i o n f o l l o w i n g the scheme as shown i n F i g u r e 12. Compound (49) was o b t a i n e d from (36) by t r e a t m e n t w i t h l i t h i u m benzoate i n hot N , M - d i m e t h y l f o r m a m i d e , f o l l o w e d by d e b e n z o y l a t i o n with Amberlyst A-26-(0H) r e s i n i n methanol. Treatment o f (49) w i t h DAST and subsequent h y d r o g e n o l y s i s gave 2 - a c e t a m i d o - 2 , 4 - d i d e o x y - 4 f l u o r o - D - g l u c o s a m i n e (51) which was a c e t y l a t e d t o ( 5 2 ) . B i o l o g i c a l A c t i v i t y o f F l u o r i n a t e d Carbohydrates In view o f the p h y s i c o - c h e m i c a l p r o p e r t i e s o f the CF b o n d , which by s i z e f a l l s between the CH and C-OH b o n d , i t s i n t r o d u c t i o n i n t o carbohydrates should r e s u l t in b i o l o g i c a l l y analogous molecules. Numerous b i o c h e m i c a l a p p l i c a t i o n s o f f l u o r o s u g a r s have appeared i n the e a r l i e r literature (30,31), and t h e s e studies prompted the development o f s e v e r a l f l u o r o s u g a r s w i t h i n t h i s program. A number o f t h e s e sugars have been t e s t e d f o r t h e i r e f f e c t on tumor c e l l growth iji v i t r o , t h e i r e f f e c t s on m a c r o m o l e c u l a r b i o s y n t h e s i s and t h e i r antitumor a c t i v i t y in mice. These p r e l i m i n a r y s t u d i e s demons t r a t e t h a t the f l u o r o s u g a r s as a group a r e b i o l o g i c a l l y active. Their addition at concentrations ranging from 10~ M to 10~ M ( T a b l e I) to L1210 leukemia c u l t u r e s r e s u l t e d i n c e l l growth i n hibition. G e n e r a l l y , the f u l l y a c e t y l a t e d compounds h a v i n g g r e a t e r lipophilicity were more c y t o t o x i c to c e l l s . The nonacetylated a g e n t s , 6 - F - D - g a l a c t o s e and 9-F-MANA demonstrated c e l l growth i n h i b i t i o n at higher concentration, 1.0 and 0 . 8 mM, respectively. These two agents in addition demonstrated an a b i l i t y to alter c e l l u l a r s i a l i c acid metabolism. Thus, 6-F-D-galactose treatment resulted in d e c r e a s e d L1210 leukemia cell ectosialyltransferase a c t i v i t y (34) w h i l e 9-F-NANA was found t o a c t as a s u b s t r a t e f o r CMP-MAMA synthetase (Petrie, C.R.; Sharma, M.; Simmons, O.D.; Korytnyk, W., unpublished results 1987) and thus serves as a p r e c u r s o r f o r membrane i n c o r p o r a t i o n . 3

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202

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

The f l u o r i n a t e d n e u t r a l s u g a r s a l s o mimicked t h e i r p a r e n t sugars by competing w i t h them f o r i n c o r p o r a t i o n i n t o m a c r o m o l e c u l a r components ( T a b l e I I ) . 6 - F - P - G a l a c t o s e and 6 - F - L - f u c o s e specifically d e c r e a s e d t h e i n c o r p o r a t i o n o f t h e i r c o r r e s p o n d i n g p a r e n t sugar t o 39 and 9%, r e s p e c t i v e l y , i n murine P288 lymphoma c e l l s grown i n tissue culture. L i t t l e s p e c i f i c e f f e c t was noted on leukemia c e l l i n c o r p o r a t i o n o f glcNAc o r l e u c i n e ( l e u ) w i t h t h e s e two a g e n t s , w h i l e f u l l y a c e t y l a t e d sugars reduced t h e i n c o r p o r a t i o n o f a l l p r e c u r s o r s , n o n s e l e c t i v e l y (Table I I ) . The a d m i n i s t r a t i o n o f f l u o r o sugars t o mice w i t h L1210 leukemia r e s u l t e d , i n a l l c a s e s , with small i n c r e a s e s i n l i f e span (% I L S ) , r a n g i n g from 11 t o 68% ( T a b l e III). The most a c t i v e a g e n t , 4 - F - g a l N A c - t r i - 0 - a c e t a t e or f l u g a l (68% ILS) i s c u r r e n t l y b e i n g e v a l u a t e d i n mice w i t h v a r i o u s o t h e r tumors t o more f u l l y a s s e s s i t s a n t i t u m o r p o t e n t i a l .

Table

I.

Fluoro

F f f e c t s o f F l u o r o - S u g a r A n a l o g s on Murine L1210 Leukemia C e l l Growth in v i t r o I C f ) 9 M* LeuK,emia L1210 5

Sugar

6-F-G1cNAc-tri-O-acetate 6-F-ManNAc-tri-U-acetate 6-F-GalNAc-tri-U-acetate 6-F-Gul NAc-tri-TJ-acetate 6-F-4-ene-lyxNAc-di-0-acetate 6-F-4-ene-lyx-tri-0-acetate 6 - F - G a l (34) 6-F-Mann-tetra-0-acetate 6-F-Glc-tetra-O-acetate 4-F-GalNAc-tri-O-acetate (flugal) 4,4-Di-F-GlcNAc-tri-O-acetate 4,6-Di-F-GalMAc-di-0-acetate 4-F-G1cNAc-Tri-O-acetate 9-F-MAMA 9-F-MAMA

5.0 1.0 1.7 6.0 1.0 3.2 1.0 5.5 5.0 3.0 3.4 2.0 3.0 3.6 8.0

X X X X X X X X X X X X X X X

10-5 10-4 10

1

10-5 io-; 10-6 10-3 10-5 lO10-5 10-5 10-5 10-5 lO-* l O " (TA3) 5

4

*The c o n c e n t r a t i o n o f f l u o r o s u g a r which i n h i b i t e d murine L1210 l e u kemia c e l l growth i n c u l t u r e by 50% o v e r a two-day p e r i o d ( 3 3 ) . C u l t u r e s were i n o c u l a t e d w i t h 5 x 1 0 c e l l s / m L i n RPMI 1640 medium c o n t a i n i n g 10% f e t a l b o v i n e serum. Sugar a n a l o g s were added and growth a l l o w e d f o r 24 h . C e l l number was measured and % c o n trol (no sugar a n a l o g added) growth was c a l c u l a t e d . The IC50 (50% growth i n h i b i t o r y c o n c e n t r a t i o n ) was d e t e r m i n e d from the dose response c u r v e . A l l a s s a y s were performed i n d u p l i c a t e on a t l e a s t two s e p a r a t e o c c a s s i o n s . TA3:Mouse mammary adenocarcinoma. 4

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

SHARMA ET AL.

Fluorinated Analogs of Cell-Surface Carbohydrates

(46)

(47)

F i g u r e 11. Synthesi 4 , 4 - d i f l u o r o glucosamine T48). Pd/C-ACOH, Ac^O-Py.

(48)

(a) DAST-CH2CT2, (b)

[H],

NHAc

(52)

(51)

F i g u r e 12. Synthesis of N-acetyl-l,3,6-tri-0-acetyl-4-deoxy4-fluoro-D-glucosamine fB2). (a) DAST-CH2CI2, (b) [H], Pd/C-AcOH, ( c ) A c 0 - P y . 2

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

204 Table

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS I I . E f f e c t s of Fluoro-Sugars P288 Lymphoma C e l l s

Fluoro-Sugars,

Cone.

6 - F - G a l (14) 6 - F - L - F u c o s e (14) 6-F-Glc-B-tri-flT

~T0=^ 10-

on M a c r o m o l e c u l a r B i o s y n t h e s i s

P288 Lymphoma Incorporation, and Leu % Control**

[M]

L-Fuc* 9

3

lO-

acetate, 6-F-ManNAc-tri-0acetate, 4-F-GalNAc-tri-Oacetate, 6-F-Mann

lO-

Gal* 39

3

3

GlcNac* 89 88

in

o f [ H ] Sugars 3

Leu* 84

-

46

51

-

55

71

IPlO"

**P288 lymphoma c e l l s (10 cells/mL) were i n o c u l a t e d i n medium RPMI 1640 c o n t a i n i n g 10% FBS (fetal bovine serum) with the i n d i c a t e d [ H ] r a d i o l a b e l e d p r e c u r s o r * (1 n C i / m L ) . A f t e r 5 h the amount o f i n c o r p o r a t e d r a d i o a c t i v i t y was d e t e r m i n e d and compared with c o n t r o l . A l l a s s a y s were performed i n d u p l i c a t e on t h r e e s e p a r a t e o c c a s s i o n s and the r e s u l t s l i s t e d a r e means. 5

3

Table

III. Effects vivo *

of Fluoro-Sugars

Fluoro-Sugar

on L1210 Leukemia i n Mice

Tumor

6-F-GlcNAc-tri-P-acetate 6-F-Mann-tri-0-acetate 4,6-Pi-F-GalNAc-tri-0-acetate 4-F-G1cNAc-tri-O-acetate 6-F-Mann 6-F-Mann-tetra-O-acetate 6-F Gal 4-F-GalNAc-tri-O-acetate ( F l u g a l ) (30) ~ 4-F-GulNAc^Tri-0-acetate 9-F-NANA 6-F-G1c-tetra-O-acetate 1,2-Pi-F-Glc-tri-O-acetate

Dose(mg/kg)

in

%ILS

L1210 L1210 L1210 L1210 L1210 L1210 L1210

6.25 25.0 10.0 100.0 200.0 200.0 25.0

12.2 11.5 11.4 33.0 17.8 27.4 32.0

L1210 L1210 L1210 L1210 L1210

50.0 50.0 50.0 6.25 400.0

68.0 12.2 19.0 21.0 35.0

l a t e d i . p . w i t h ID* L1210 leukemia c e l l s on day z e r o . Starting on day one mice were g i v e n v a r i o u s dosages (5 mice/dosage l e v e l ) o f the t e s t agent i . p . , once p e r day through day 5 . L i f e span was checked d a i l y . The % measure i n l i f e s p a n (% ILS) was c a l c u l a t e d as compared t o t h e c o n t r o l g r o u p , c o n s i s t i n g o f 8-10 a n i m a l s . The optimum % ILS i s l i s t e d . 5

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

11. SHARMA ETAL.

Fluorinated Analogs ofCell-Surface Carbohydrates

C o n c l u s i o n s and F u t u r e S t u d i e s T h i s program has attempted t o demonstrate t h a t , by i n t e r f e r i n g o r m o d i f y i n g t h e b i o s y n t h e s i s o f components o f tumor c e l l surface glycoconjugates, development o f chemotherapeutic agents may r e sult. In p u r s u i n g t h i s avenue several analogs of cell-surface carbohydrates were s y n t h e s i z e d and t e s t e d both i_n v i t r o and i j i vivo as a n t i t u m o r a g e n t s . C o m b i n a t i o n chemotherapy w i t h nucleos i d e a n a l o g s a l s o may improve t h e t h e r a p e u t i c r e s p o n s e as noted with P-glucosamine and p e n t a - O - a c e t y l - B - D - g l u c o s a m i n e (B-PAG1CNAC) in combination with a z a r i b i n e (330. I t appears from t h e p r e liminary experimental data i n our l a b o r a t o r y and o t h e r s that f l u o r i n a t e d s u g a r a n a l o g s a r e membrane-active a g e n t s and a r e worth f u r t h e r s y n t h e t i c and b i o c h e m i c a l investigation. Acknowledgments We would l i k e t o thank P r . E . M i h i c h f o r h i s a c t i v e e n c o u r a g e ment t h r o u g h o u t t h e program W would a l s lik t thank D r B Paul and M r . N . J . A n g e l i n gram. T h i s s t u d y wa from t h e N a t i o n a l Cancer I n s t i t u t e .

LITERATURE CITED 1. Nicolson, G.L. Biochem. Biophys. Acta. 1976, 458, 1-72. 2. Nicolson, G.L.; Poste, G; New England J. Med. 1976, 295 197203;253-258. 3. Poste, G, Weiss, L. "Fundamental Aspects of Metastasis", Weiss, L . , Ed.; North Holland Publishing Co., Amsterdam 1976, p. 25. 4. Hughes, R.C. "Membrane Glycoproteins". Butterworth, LondonBoston, 1976. 5. Winzler, R.J. In "Membrane Mediated Information". Kent, P.W., Ed., Medical and Technical Publishing Co. Ltd., 1973, Vol. 1, p. 3-19. 6. Bernacki, R.J.; Sharma, M.; Porter, N.K.; Rustum, Y.; Paul, B.; Korytnyk, W. J . Supramol. Structure, 1978, 7, 235-250. 7. Middleton, W.J., J. Org. Chem., 1975, 40, 574-578. 8. Sharma, M.; Korytnyk, W. Tetrahedron Lett. 1977, 573-576. 9. Sharma, M.; Korytnyk, W. Carbohydr. Res. 1980, 83, 163-169. 10. Hasegawa, A.; Fletcher, H.G. Carbohydrates. 1977, 29, 209-222. 11. Taylor, M.F.; Kent, P.W. J . Chem. Soc. 1958, 872-875. 12. Fush, H.L.; Isbell, H.S. Methods Carbohydr. Chem. 1962, 1, 127-130. 13. Sufrin, J.R.; Bernacki, R.J.; Morin, M.J.; Korytnyk, W. J . Med. Chem 1980, 23, 143-149. 14. Sharma, M.; Potti, P.G.; Simmons, O.; Korytnyk, W. Carbohydr. Res. 1987, 162, 41-52. 15. Ido, T.; Wan, C.N.; Fowler, J.S.; Wolf, A.P. J . Org. Chem. 1977, 42, 2341-2342. 16. Adamson, J.; Mercus, D.M. Carbohydr. Res. 1970, 13, 314-316. 17. Butchard, G.C.; Kent, P.W. Tetrahedron 1979, 35, 2551-2554. 18. Greigorcic, A.; Zupan, M. J . Org. Chem. 1979, 44, 4120-4122. 19. Korytnyk, W.; Valentekovic-Horvath; S, Petrie, C.R. Tetrahedron. 1982, 38, 2547-2550. 20. Isetin, B.; Reichstein, T. Helv. Chim. Acta. 1944, 27, 1146-1149.

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

205

206

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

21. Meindle, P.; Tuppy, H., Monatsh. Chem. 1969, 100, 1295-1306. 22. Gantt, R.; Millner, S.; Binkley, S.B. Biochemistry. 1964, 3, 1952-1960. 23. Shulman, M.L.; Khorlin, A. Ya. Carbohydr. Res. 1973, 27, 141-147. 24. Hough, L . ; Penglis, A.E.; Richardson, A.C. Can. J. Chem. 1981, 59, 396-404. 25. Sharma, M.; Korytnyk, W. XIIIth. International Carbohydr. Symposium. 1986, 93. 26. Sharma, M.; Korytnyk, W. Carbohydr. Res. 1980, 79, 39-51. 27. Korytnyk, W.; Sharma, M.; Angelino, N.; Bernacki, R.J. XIIth International Carbohydr. Symposium. 1982, IV 40. 28. Garegg, P.J., Hultberg, H. Carbohydr Res. 1981, 93, C10-C11. 29. Jaquinet, J.C.; Petil, J.M.; Sinay, P. Carbohydr. Res. 1974, 38, 305-311. 30. Burnett, J.E.G. Cib Fdn Symp Elsevie d Associated Scientific Publishers 31. Taylor, N.F. Cib Symp Scientific Publishers, Amsterdam, 1972, 215-238. 32. Niedbala, M.J.; Madiyalakin, R.; Matta, K., Crickard, K.; Sharma, M; and Bernacki, R.J. Cancer Res. 1987, 47, 4634-4641. 33. Bernacki, R.J.; Porter, C.W.; Korytnyk, W. and Mihich, E. Adv. Enzyme Regulat. 1977, 16, 217-237. 34. Morin, M.J.; Porter, C.W.; Petrie III, C.R.; Korytnyk, W. and Bernacki, R.J. Biochem. Pharmacol. 1973, 32, 553-561. 35. Sharma, R.A.; Kavai, I.; Fu, Y.L. and Bobek, M. Tetrahedron Lett. 1977, 39, 3433-3436. RECEIVED

January 18, 1988

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Author Index Baker, David C , 43 Bernacki, Ralph J., 191 Chou, T. C , 176 D'Amore, T., 109 DeGrado, Timothy R., 156 Dreckhammer, D. G., 29 Fourel, I., 176 Fox, J. J., 176 Gatley, S. John, 156 Glaudemans, Cornelis P. J., 78 Halama, James R. Hantz, G., 176 Hitz, William D., Holden, James E., 156 Jiang, Cong, 43 Kent, P. W., 1 Koeppe, Robert A., 156 Korytnyk, Walter, 191 Kovac, Pavol, 78 Malinowski, Nancy, 43

Mcintosh, J. M , 109 Mover, James D., 43 Ng, Chin K., 156 Nicolaou, K. C , 13 Percival, Michael D., 59 Randall, Jared L., 13 Reizes, Ofer, 43 Sbrissa, D., 109 Schinazi, R. F., 176 Sharma, Moheswar, 191

, , Sweers, H. M., 29 Taylor, N. F., 109 Trepo, C , 176 Watanabe, JL A., 176 Withers, Stephen G., 59 Wong, C.-R, 29

Affiliation Index Delta Regional Primate Research Center, 176 E. I. du Pont de Nemours and Company, 138 Emory University-Veterans Administration Medical Center, 176 Institut National de la Sante et de la Recherche Me'dicale, 176 Memorial Sloan-Kettering Cancer Center, 176 National Cancer Institute, 43 National Institutes of Health, 78

Oxford University, 1 Roswell Park Memorial Institute, 191 Texas A & M University, 29 University of Alabama, 43 University of British Columbia, 59 University of Chicago, 156 University of Pennsylvania, 13 University of Windsor, 109 University of Wisconsin, 156

Subject Index A

N-Acetyl-4-deoxy-4-fluoro-D-galactosamine, synthesis, 198,200^,201 N-Acetyl-3,4-di-0-acetyl-6-deoxy-6fluoro-D-galactosamine synthesis, 194,195/

Acetylated glycals, fluorination mechanism, 194,197/,198

207

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Author Index Baker, David C , 43 Bernacki, Ralph J., 191 Chou, T. C , 176 D'Amore, T., 109 DeGrado, Timothy R., 156 Dreckhammer, D. G., 29 Fourel, I., 176 Fox, J. J., 176 Gatley, S. John, 156 Glaudemans, Cornelis P. J., 78 Halama, James R. Hantz, G., 176 Hitz, William D., Holden, James E., 156 Jiang, Cong, 43 Kent, P. W., 1 Koeppe, Robert A., 156 Korytnyk, Walter, 191 Kovac, Pavol, 78 Malinowski, Nancy, 43

Mcintosh, J. M , 109 Mover, James D., 43 Ng, Chin K., 156 Nicolaou, K. C , 13 Percival, Michael D., 59 Randall, Jared L., 13 Reizes, Ofer, 43 Sbrissa, D., 109 Schinazi, R. F., 176 Sharma, Moheswar, 191

, , Sweers, H. M., 29 Taylor, N. F., 109 Trepo, C , 176 Watanabe, JL A., 176 Withers, Stephen G., 59 Wong, C.-R, 29

Affiliation Index Delta Regional Primate Research Center, 176 E. I. du Pont de Nemours and Company, 138 Emory University-Veterans Administration Medical Center, 176 Institut National de la Sante et de la Recherche Me'dicale, 176 Memorial Sloan-Kettering Cancer Center, 176 National Cancer Institute, 43 National Institutes of Health, 78

Oxford University, 1 Roswell Park Memorial Institute, 191 Texas A & M University, 29 University of Alabama, 43 University of British Columbia, 59 University of Chicago, 156 University of Pennsylvania, 13 University of Windsor, 109 University of Wisconsin, 156

Subject Index A

N-Acetyl-4-deoxy-4-fluoro-D-galactosamine, synthesis, 198,200^,201 N-Acetyl-3,4-di-0-acetyl-6-deoxy-6fluoro-D-galactosamine synthesis, 194,195/

Acetylated glycals, fluorination mechanism, 194,197/,198

207

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

208

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

N-Acetyl-4,6-dideoxy-4,6-difluoro-Dgalactosamine synthesis, 198,200^,201 N-Acetylneuraminic acid, substitution of 9-position, 198,199/* N- Acetyl-1,3,6-tri-0-acetyl-4-deoxy4,4-difluoroglucosamine synthesis, 201,203/ N- Acetyl-1,3,6-tri-0-acetyl-4-deoxy4-fluoro-D-glucosamine synthesis, 201,203/ N- Acetyl-l,3,6-tri-0-acetyl-4-deoxy-4fluorosamine synthesis, 198,200/* Acid-catalyzed hydrolysis data of sugar phosphate effect of fluorine substitution, 69-70 rate constants, 69/ Acylated sugars, lipase-catalyzed deprotection, 33-39 Addition reactions, synthesis of fluorocarbohydrates, 5,6,7/ Aldolase-isomerase-catalyzed reactions, syntheses of hexoketoses, 30-34 Antigalactan monoclonal antibodies antigen-antibody interaction, 80 binding constants and maximal fluorescence change, 82,83/,89,90/ binding sites, 80-81 complementarity-determining regions, 92f,93 effect of fluorine substitution on hydrogen bonding, 81—82 H-chain V-region sequences, 89,91/,92 schematic of binding mode, 84,85,86f,87 Antiphosphorylcholine IgA, structure, 80 Avermectin B , partial synthesis, 15,16/ l a

B Biological effects of fluoromonosaccharides, examples, 6,8 C Carbohydrates, advantages of fluorine substitution, 109-110 Cellan, description, 2 Cell-surface glycoprotein modification, 192 Clonal selection theory, 78-79 D 6'-Deoxy-6'-(4-azido-2-hydroxybenzamido)sucrose inhibition of sucrose influx, 144 structure, 144

6-Deoxy-6,6-difluoro-D-galactose synthesis, 193-194,195/ 5- Deoxy-5,5-difluoro-m)>0-inositol, synthesis, 51,52/* l-(2'-Deoxy-2'-fluoro-0-D-arabinofuranosyl)5-ethyluracil antiviral activity, 177,178/,179 cellular kinetic constants, 180 effects on mammalian cells, 179* effects on viremia, 181,182/ incorporation into DNA of mammalian cells, 180r rate of phosphorylation by thymidine kinases, 177 structures, 177 treatment of woodchuck hepatitis virus, 184,186-188/",189

activity, 177,178 structure, 177 1- (2'-Deoxy-2'-fluoro-)9-D-arabinofuranosyl)5-methyluracil antiviral activity, 177,178/ cellular kinetic constants, 180 effects on mammalian cells, 177 effects on viremia, 181,182/ incorporation into DNA of mammalian cells, 180/ rate of phosphorylation by thymidine kinases, 177 structure, 177 treatment of woodchuck hepatitis virus, 184,186^,187/ 4-Deoxy-4-fluoro-D-arabino-hexos-2-ulose catalytic hydrogenation, 131-132 structure, 127,13Qf,131 2- Deoxy-2-fluoro-D-arabinose5-phosphate chemoenzymatic synthesis, 112,113/ preparation, 36 Deoxyfluorocarbohydrates, total synthesis, 3,4/ Deoxyfluorocyclitols functions, 45,47 structures, 45,46/* syntheses, 47-52 use as substrates for phosphatidylinositol synthetase, 45 4-Deoxy-4-fluoro-D-fructose, chemoenzymatic synthesis, 127,13Q/;i31 2- Deoxy-2-fluoro-L-fucose, synthesis, 161,194,196/* 3- Deoxy-3-fluoro-D-galactose acetylation, 95—96 6- Deoxy-6-fluoro-D-galactose synthesis, 160

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX

209

2- Deoxy-2-fluoro-D-glucose behavior isolated in perfused hearts, 164-170 calculation of glucose content, 169,170/ calculation of lumped constant, 169,170/ distribution behavior in tissue, 164/ distribution volumes and transported and phosphorylation fractions, 167/ high-performance liquid chromatography, 162,163/ inhibition of glycoprotein synthesis, 116-117 preparation, 158-160 use as PET radiotracer, 157 3- Deoxy-3-fluoro-D-glucose behavior isolated in perfused hearts, 164-170 distribution behavior in tissue, 164/ high-performance liquid chromatography, 167,168/" locust toxicity, 114,115/,116 metabolism, 112-116,132 oxidation, 118 PET data, 171,172/" synthesis, 160 4- Deoxy-4-fluoro-D-glucose chromatographic analysis of sodium dodecyl sulfate solubilized cell envelope, 121,124,125/ chromatographic analysis of supernatants, 119,121,122/" fluoride release, 118-119 Fourier transform NMR spectrum of metabolite, 121,123/ growth inhibition, 132-133 locust toxicity, 114,116 mechanism of fluoride release, 132 metabolism, 112-116,132 pathway for the loss of H 0,124 proton-coupled C - N M R spectrum of metabolite, 121,122/" proton-decoupled C - N M R spectrum of metabolite, 121,123/ radioactivity distribution, 119,121/ tritiated water release, 119,120/ 2- Deoxy-2-fluoro-L-glucose behavior isolated in perfused hearts, 164-170 distribution behavior in tissue, 164/ 3- Deoxy-3-fluoro-D-glucose-6-phosphate, chemoenzymatic synthesis, 112,113/ 6-Deoxy-6-fluorohexopyranoses, synthesis, 193 Deoxyfluorohexoses, structures of precursors for nucleophilic synthesis, 160-161,163/ 3

2

13

13

6-Deoxy-6-fluorohexoses synthesis, 193 2-Deoxy-2-fluoro-£-D-mannopyranosyl fluoride, stereoview of packing, 60,61/62 2-Deoxy-2-fluoro-D-mannose inhibition of glycoprotein synthesis, 116-117 synthesis, 160 4(6)-Deoxy-4(6)-fluoro-myo-inositol, synthesis, 47,48/ S-Deoxy-S-fluoro-myo-inositol improved synthesis, 49,51,52/ incorporation into leukemia cells, 54,56/" radiolabeling, 51 substrate for phosphatidylinositol synthetase, 54,55/ synthesis, 47,49,5Qf

-Deoxy- -fluoro-scy/fo-inosito synthesis, 47 1'-Deoxy-1 '-fluorosucrose rate of metabolism, 149 relative contributions of sucrose synthase and invertase to sucrose breakdown, 152,153/,154 [ C]-1 '-Deoxy-l'-fluorosucrose time course of label incorporation, 149,151/ translocation by invertase and sucrose synthase, 149,15Q/* r-Deoxy-r-fluorosucrose hydrolysis concentration kinetics by invertase, 146,147/ concentration kinetics by sucrose synthase, 146,148/" Deoxyfluoro sugars inhibition of glycoprotein biosynthesis, 116-117 reasons for study, 59 2-Deoxyglycosides, synthesis, 23-27 2-Deoxy-2-D-ribose-5-phosphate, preparation, 33,36/" Deoxy sugars, 60 (Diethylamino)sulfur trifluoride fluorination, 47 preparation of glycosyl fluorides, 14,16/ use as fluorinating agent, 193 Diphenylhydrazone O-acetylated derivatives of glucose, glucosone, and 4-deoxy-4-fluoroglucosone, comparison of major mass spectrum peaks, 131/ 14

Efrotomycin, total synthesis, 15,17/ Electrophilic synthesis, 2-deoxy2-fluoro-D-glucose, 158-159

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

210

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Embryo storage tissue, transmembrane movement of sucrose, 140 Enzyme studies of fluorocarbohydrates, 111-112 Epoxide scission, synthesis of fluorocarbohydrates, 5 5-Ethyl-2'-deoxyuridine, antiviral activity, 177,178/,179 Exchange reactions, synthesis of fluorocarbohydrates, 3,5,7/

18

F-fluoro sugars application, 162 behavior in isolated perfused hearts, 164-170 distribution behavior in tissues preparation, 157-158 quality control, 163/ rapid purification, 161-162 Fluorinated analogues of cell-surface carbohydrates rationale for study, 192-193 use as anticancer agents, 191-192 2'-Fluorinated arabinosylpyrimidine nucleosides structure-activity studies, 177 treatment of woodchuck hepatitis virus, 184,186f-18^,189 Fluorinated carbohydrates, biological activity, 201-202,204/ Fluorinated disaccharides, synthesis, 39,4Qf Fluorinated hexoses, hexokinase-catalyzed phosphorylation, 32-33,35/ Fluorinated membrane sugar analogues, synthesis, 193-203 Fluorinated oligosaccharides, synthesis, 39,4Qf Fluorinated sugars, use as pharmaceuticals or pharmacological probes, 29-30 Fluoroacetate, biochemical mechanism of toxicity, 2 Fluorocarbohydrates advantages of fluorine substitution, 110 discovery, 1-2 enzyme studies, 111-112 metabolic studies, 112-118 synthesis, 2-10 transport studies, 110 use in positron emission transaxial tomography, 117-118 2-Fluoroerythritol synthesis, 3 1-Fluorofructose synthesis, 5

3-Fluoroglucose, synthesis from tetrabutylammonium fluoride, 3,5,7/ 6-Fluoroglucose synthesis, 3 2-Fluoroglyceric acid synthesis, 3 1- Fluoroglycerol synthesis, 3 2- Fluoroglycerol synthesis, 3 2-Fluoromaleate synthesis, 3 Fluoromonosaccharides incorporation into hyaluronate, 8 oligosaccharide chain shortening, 8 reduction of cell number in tumors in vitro, 8 role in glycogen synthesis, 9 sugar transport inhibition, 6 use as in vivo probes, 9 use as starting materials, 9 use in molecular modifications, 9

Fluorosugar(s) definition, 1 effects on leukemia in mice, 202,204/ effects on macromolecular biosynthesis in lymphoma cells, 204 Fluorosugar analogues, effects on leukemia cell growth, 201,202/ 3- Fluoroxylose synthesis, 5 F - N M R spectroscopy, use in enzyme studies offluorocarbohydrates,111-112 Fructose-1-diphosphate aldolase inhibition constant, 3234/" preparation of fluorinated sugars, 30^1/ reaction mechanisms, 3031/32 reaction rate constant, 32,33/ Fructosyl-substituted sucroses relative binding constants, 140,141/ space-filling model, 140,142^,143 Functional groups hydrogen-bonding capabilities, 60 size comparison, 59,60/ 19

G ^-D-Galacto ligands, preparation, 93-113 6-0-0-D-Galactopyranosyl-D-galactose, postulated conformation, 81/ £-D-Galactopyranotetraose, intersaccharidic bond angles, 82f,84 [2- F]Glucose synthesis, 6 Glucose content, calculation, 169,170/ 0-Glucosidase, mechanism-based inhibitor, 73-75,76/" a-Glucosyl transferases, mechanism, 66,68-69 Glycoconjugates, description, 192 18

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX

211

Glycogen phosphorylase binding effects of hydrogen and fluorine substitution, 71 electronic effects of hydrogen and fluorine substitution, 71 mechanism, 70-71 Michaelis-Menten parameters, 71,72/,73 Glycogen phosphorylase-glucose complex, hydrogen bonding, 64,65/,66,67/ C-Glycosides, preparation from glycosyl fluorides, 21,2^ N-Glycosides, preparation from glycosyl fluorides, 21,22/ 0-Glycosides, preparation from glycosyl fluorides, 21,2^f ^-Glycosides, preparationfromglycosyl fluorides, 21,22/" O-Glycosidic linkages, formatio sensitive aglycons, 15,1^,17 Glycosyl fluorides advantages as glycosyl donors, 14 applications as glycosyl donors, 27 enzymatic hydrolysis, 111 formation of O-glycosidic linkages to sensitive aglycons, 15 Mukaiyama conditions, 14-15 natural product synthesis, 21,22/" other reactions, 21,22/" preparation, 13-14,16/" reactions, 14-22 synthesis of oligosaccharides, 15 synthesis of rhynchosporosides, 19,20/;21 use as substrates, 63 Glycosyl-substituted thiophenyl glycopyranosides relative binding constants, 141/,143 space-filling model, 142/",143

Immunoglobulin(s) clonal selection theory, 78-79 definition, 78 structures, 79-80 Immunoglobulin-producing neoplasms, definition, 79 myo-inositol discovery, 43-44 structure, 43-44 synthesis of analogues, 43-44 myo-inositol analogues, See Deoxyfluorocyclitols Invertase breakage of glucose tofructosebond in sucrose, 139

, relative contribution to breakdown of sucrose, 152,153/,154 Isolated perfused hearts, behavior of Ffluorosugars,164-170 1 8

Lipase-catalyzed deprotection of acylated sugars application, 33—34 " C shifts of deuterium-induced shift experiments, 35,37/,39/ relative and specific activities, 34-35,37/ selective hydrolysis at C - l position, 35^8/^9 Lumped constant, calculation, 169,170/ ^-Lymphocytes, differentiation, 79

H Hepatitis B virus description, 182-183 inhibitory activities of nucleoside triphosphate analogues, 183,184/ Hexokinase-catalyzed reactions, synthesis of fluorinated hexoses, 32-33,37/,37/ Hexopyranose-binding enzymes, specificity studies, 63 Hydrogen bond(s) bond energy, 62-63 detection, 62 Hydrogen bonding in glycogen phosphorylase-glucose complex hydrogen-bond strengths, 66 inhibition constants, 64,65/ interactions, 66,67/

M Metabolic studies of fluorocarbohydrates, 112-118 Methyl 3-deoxy-3-fluoro-j0-Dgalactopyranoside synthesis, 94-95 Methyl 4-deoxy-4-fluoro-/?-Dgalactopyranoside synthesis, 93—94 Methyl 6-deoxy-6-fluoro-/?-Dgalactopyranoside synthesis, 94 Methyl /9-glycosides of (l-6)-/0-D-galactooligosaccharides, synthesis of higher members, 100,104-106 Methyl 2,3,4-tri-O-benzoyl-^-Dgalactopyranoside synthesis, 100,102-103 1,2-Migration reaction mechanism, 21,23,24/

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

212

FLUORINATED CARBOHYDRATES: CHEMICAL AND BIOCHEMICAL ASPECTS

Monodeoxyfluoro derivatives of methyl /7-D-galactopyranoside synthesis, 93-94 Monosaccharide structure modification addition reactions, 5-6,7/ epoxide scission, 5 exchange reactions, 3,5,7/ Mukaiyama reaction, synthesis of glycosides, 14 Myeloma immunoglobulin A, description, 80

N Natural products, synthesis by glycosyl fluorides, 21,22/" Nucleophilic displacement reactions, preparation of F fluorosugars Nucleophilic synthesis of 2-deoxy-2-fiuoro-D-glucose, 159-160

Pyranose 2-oxidase—Continued Lineweaver-Burk plots, 124,127,128/

Rhynchosporosides, synthesis, 19,2Q/",21

Substrate recognition by sucrose transporters fructosyl-substituted sucroses, 140,141/,142/*,143 glycosyl-substituted thiophenyl glycopyranosides 143,145

1 8

O a(l-4)-Ofigogalactosides, syntheses, 21,22/* Oligosaccharides, synthesis, 15,18/",19

P (2-Phenylthio)-a-glucosides, formation mechanism, 25,2^,27 (2-Phenylthio)-£-glucosides, formation mechanism, 25,26/",27 Phloem, transmembrane movement of sucrose, 139 Phosphatidylinositol synthetase, use of deoxyfluorocyclitols as substrates, 51,53-59 Phosphoinositides pathway, 45,46/" role in action of hormones and growth factors, 44-45 Phospholipase C, role in biological processes, 45 Positron emission tomography (PET) advantages of using fluorine as emitter, 157 description, 156-157 time course for 3-deoxy-3-fluoro-Dglucose, 156,171,172/* Positron emission transaxial tomography, description, 117-118 Potassiumfluoroacetate,toxicity, 2 Pyranose 2-oxidase competitive inhibition of glucose oxidation, 127,128/" description, 124 effect of sugar concentration on activity, 127,125!/'

importance in plants, 138-139 rate of metabolism, 149 relative contributions of sucrose synthase and invertase to breakdown, 152,153f,154 [ C]Sucrose time course of label incorporation, 149 translocation by invertase and sucrose synthase, 149,15Qf Sucrose hydrolysis concentration kinetics by sucrose synthase, 146,148/" ratio of kinetic constants, 146,149,15Q/" Sucrose synthase breakage of glucose-to-fructose bond in sucrose, 139 determination of in vivo enzyme activity, 145 relative activities, 149-154 relative contribution to breakdown of sucrose, 152 Sucrose transporters, substrate recognition, 139—145 Synthesis of 2-deoxyglycosides, mechanism, 23,24/,25 14

T Tetra-0-acetyl-6-deoxy-6-fluoro-Dmannosamine synthesis, 193,195/ Tetrabutylammoniumfluoride,synthesis of 3-fluoroglucose, 3,5,7/ Thioglycosides, preparation of glycosyl fluorides, 13 Transport studies of fluorocarbohydrates, 110

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX

213

2,3,4-Tri-0-acetyl-6-0-(bromoacetyl)-a-Dgalactopyranosyl bromide, use as glycosyl donor, 96-98 2,3,4-Tri-0-acetyl-6-0-(chloroacetyl)-a-Dgalactopyranosyl bromide, structure, 96 3,4,6-Tri-O-acetyl-D-glucal fluorination, 194,196f 2,3,4-Tri-0-benzoyl-6-0-(bromoacetyl)-a-Dgalactopyranosyl bromide synthesis, 98-99,101 2,3,4-Tri-0-benzoyl-6-0-(bromoacetyl)-a-Dgalactopyranosyl chloride synthesis, 100,102-103

2,4,6-Tri-0-benzoyl-3-deoxy-3-fluoro-a-Dgalactopyranosyl bromide synthesis, 98,100

W Woodchuck hepatitis virus D N A polymerase activity, 184,185/ treatment with 2'-fluorinated arabinosylpyramidine nucleosides, 184,186f-188f,189

Production by Barbara J. Libengood Indexing by Deborah H. Steiner and Linda R Ross Jacket design by Carla L. Clemens Elements typeset by Hot Type Ltd, Washington, DC Printed and bound by Maple Press, York, PA

In Fluorinated Carbohydrates; TAYLOR, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.