UBC Theses and Dissertations

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UBC Theses and Dissertations

The synthesis of several fluorinated disaccharides and the elucidation of their conformation using ¹H… Shelling, Judith Grace 1979

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SYNTHESIS OF SEVERAL FLUORINATED DISACCHARIDES AND THE ELUCIDATION OF THEIR CONFORMATION USING 1 H AND 1 9 F NMR by JUDITH GRACE SHELLING B . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f C h e m i s t r y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l , 1979 (c) Judith Grace Shelling, 1979 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date D E - 6 B P 7 5 - 5 1 1 E i i ABSTRACT The g o a l o f t h i s p r o j e c t was t o s y n t h e s i z e a f l u o r i n a t e d d i s a c c h a r i d e as a s u b s t r a t e o r i n h i b i t o r f o r a g l y c o s i d a s e enzyme. A s u b s t r a t e a n a l o g u e w i t h o n l y one s t r a t e g i c a l l y p o s i t i o n e d f l u o r i n e a t o m c o u l d be u s e d t o p r o b e s u c h a c o m p l e x i n t e r a c t i o n by u s i n g 1 9 F NMR t o m o n i t o r any c h a n g e s i n c h e m i c a l s h i f t , l i n e w i d t h , and p c o u p l i n g c o n s t a n t s w h i c h a r i s e when t h e a n a l o g u e b i n d s t o t h e enzyme. S u c h c h a n g e s w o u l d be more 19 e a s i l y o b s e r v e d by u s i n g F NMR t o m o n i t o r t h e p e r t u r b a t i o n s a r o u n d t h e s i n g l e F n u c l e u s t h a n by m o n i t o r i n g t h e "*"H NMR b e c a u s e t h e s p e c t r u m i n t h e l a t t e r c a s e i s much more c o m p l e x due t o t h e number o f p r o t o n s i n h e r e n t i n b o t h t h e n a t u r a l s u b s t r a t e and t h e enzyme. The compounds 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 - a - D -g l u c o p y r a n o s y l b r o m i d e [5] and 2 , 3 - d i - 0 - a c e t y l - l , 6 - a n h y d r o - 3 - D -g l u c o s e [2] were s y n t h e s i z e d as a p p r o p r i a t e s t a r t i n g m a t e r i a l s and t h e n c o u p l e d i n d i e t h y l e t h e r i n t h e p r e s e n c e o f Ag^jCO^j u s i n g AgSO^CF^ as a c a t a l y s t ; t h e s e a r e t h e s i m p l e c o n d i t i o n s o f t h e w e l l - k n o w n K o e n i g s - K n o r r r e a c t i o n . The c o u p l i n g o f a h a l o s u g a r b e a r i n g a n o n - p a r t i c i p a t i n g g r o u p a t C-2 w i t h an a l c o h o l i n l o w c o n c e n t r a t i o n h a s b e e n shown by many o t h e r w o r k e r s t o r e s u l t i n a m i x t u r e o f a- and 3 - l i n k e d p r o d u c t s , w i t h t h e f o r m e r b e i n g p r e d o m i n a n t . I n t h i s s t u d y , o n l y t h e a - l i n k e d d i s a c c h a r i d e 2 , 3 - d i - 0 - a c e t y l - l , 6 - a n h y d r o - 4 - 0 -( 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 - a - D - g l y c o p y r a n o s y l ) - B - D -g l u c o p y r a n o s e [12] was o b t a i n e d , t h e c o r r e s p o n d i n g g - l i n k e d i i i d i s a c c h a r i d e was n o t o b s e r v e d i n t h e r e a c t i o n m i x t u r e . T h i s f a c t s u g g e s t s t h a t some f a c t o r i n e i t h e r t h e r e a c t i o n c o n d i t i o n s e m p l o y e d o r i n t h e n a t u r e o f t h e two s u g a r s i n -v o l v e d i n t h e c o u p l i n g was r e s p o n s i b l e f o r t h e e x c l u s i v e p r o d u c t i o n o f o n l y one t y p e o f l i n k a g e . The c t - l i n k e d d i s a c c h a r i d e [12] was d e p r o t e c t e d t o g i v e t h e f r e e s u g a r 2 ' - d e o x y - 2 ' - f l u o r o - m a l t o s e ( 4 - 0 - ( 2 - d e o x y - 2 -f l u o r o - a - D - g l u c o p y r a n o s y l ) - D - g l u c o p y r a n o s e [ 1 4 ] ) w h i c h i s t h e C-2' f l u o r i n a t e d a n a l o g u e o f m a l t o s e . T h i s compound s h o u l d be a s u i t a b l e p r o b e f o r enzyme s y s t e m s s u c h as 19 M a l t a s e o r a - g l y c o s i d a s e . The p r o t o n d e c o u p l e d P NMR o f [14] d i s p l a y e d two d i s t i n c t r e s o n a n c e s s e p a r a t e d by 0 . 1 ppm w h i c h c o u l d be due t o t h e a and 3 a n o m e r i c f o r m s o f t h a t compound. The o p t i c a l r o t a t i o n s v a l u e s and t h e a/3 a n o m e r i c r a t i o s d e d u c e d f r o m t h e "'"H NMR s p e c t r a f o r m a l t o s e and [14] were c o m p a r a b l e , s u g g e s t i n g t h a t t h i s may i n d e e d be t h e c a s e . The s y n t h e s i s o f 4 - 0 - ( 2 - d e o x y - 2 - f l u o r o - a - D - g a l a c t o -p y r a n o s y l ) - D - g l u c o p y r a n o s e [17] was i n i t i a t e d w i t h t h e v i e w o f u s i n g s u c h a compound t o s t u d y an a - g a l a c t o s i d a s e s y s t e m ; t h e p a r t i a l s y n t h e s i s o f t h i s m o l e c u l e i s a l s o i n c l u d e d h e r e i n . The c o n f o r m a t i o n a l a s p e c t s o f a l l t h e s u g a r s s y n t h e s i z e d i n t h i s work were d e d u c e d f r o m "*"H NMR and t h e n c o n f i r m e d by 1 9 F NMR a n d c 6 p t i c a T c r ; o t a t i o n ( . . j . . a T h e o l 9 F NMR s p e c t r a o f t h e d i s a c c h a r i d e s were s i m p l e and were shown t o p r o v i d e s u f f i c i e n t i n f o r m a t i o n f o r t h e d e d u c t i o n o f t h e c o n f o r m a t i o n a l and i v c o n f i g u r a t i o n a l a s p e c t s o f t h e s e s u g a r s i n t h e r e g i o n o f i n t e r e s t . T h i s i n d i c a t e d t h a t c h a n g e s i n t h e r e g i o n a b o u t 19 t h e f l u o r i n e c o u l d be m o n i t o r e d u s i n g P NMR and t h a t t h e s e s u g a r s c o u l d t h u s p r o v e t o be v a l u a b l e p r o b e s f o r t h e e l u c i d a t i o n o f enzyme m e c h a n i s m s . V TABLE OF CONTENTS PAGE L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i Acknowledgements i x I I n t r o d u c t i o n 1 . I I E x p e r i m e n t a l 18 P r e p a r a t i o n o f the Anhydro Sugars [1] and [2] 19 P r e p a r a t i o n o f the F l u o r i n a t e d Sugars [ 4 ] , [51, [ 1 2 ] , [ 1 2 a ] , [ 1 3 ] , [ 1 4 ] , and [ 18] 21 P r e p a r a t i o n o f T r i f l u o r o m e t h y l h y p o f l u o r i t e [CF 3OF] 32 P r e p a r a t i o n o f t h e F l u o r i n a t e d Sugars [ 7 ] , [ 8 ] , and [ 16] 33 I I I C h e m i c a l S h i f t s and C o u p l i n g C o n s t a n t s 40 IV I n t e r p r e t a t i o n o f the NMR Data 65 V D i s c u s s i o n 83 VI B i b l i o g r a p h y 107 v i L I S T OF TABLES TABLE I . C h e m i c a l S h i f t ' A s s i g n m e n t s f o r t h e A n h y d r o S u g a r s [1] and [2] TABLE I I . C h e m i c a l S h i f t A s s i g n m e n t s f o r t h e A n h y d r o M o i e t y o f t h e D i s a c c h a r i d e s [12] and [15] TABLE I I I . C h e m i c a l S h i f t A s s i g n m e n t s f o r t h e 2 - D e o x y - 2 - F l u o r o M o n o s a c c h a r i d e [4] and t h e A n a l o g o u s B r o m i n a t e d D e r i v a t i v e [5] TABLE I V . C h e m i c a l S h i f t A s s i g n m e n t s f o r t h e 2 - D e o x y - 2 - F l u o r o M o n o s a c c h a r i d e [7] and t h e A n a l o g o u s B r o m i n a t e d D e r i v a t i v e [8] TABLE V, TABLE V I TABLE V I I TABLE V I I I C h e m i c a l S h i f t A s s i g n m e n t s f o r t h e D i s a c c h a r i d e s [ 1 2 ] , [ 1 3 ] , and [14] C h e m i c a l S h i f t A s s i g n m e n t s f o r t h e D i s a c c h a r i d e s [ 15 ] . and [ 16] C o u p l i n g C o n s t a n t s as D e t e r m i n e d f r o m t h e T-H NMR S p e c t r a f o r t h e M o n o s a c c h a r i d e s . [4] , '.[5], [71 > [ 8 ] , and [ 18] C o u p l i n g C o n s t a n t s as D e t e r m i n e d f r o m t h e -^H NMR S p e c t r a f o r t h e D i s a c c h a r i d e s [ 1 2 ] , [ 1 3 ] , [ 1 4 ] , [ 1 5 ], and [ 16] TABLE I X . C o u p l i n g C o n s t a n t s as D e t e r m i n e d f r o m t h e 1°-F NMR S p e c t r a f o r t h e M o n o s a c c h a r i d e s [ 4 ] , [5] , [7] , [ 8 ] , and [ 18] TABLE X. C o u p l i n g C o n s t a n t s as D e t e r m i n e d f r o m t h e x 9 F NMR S p e c t r a f o r t h e D i s a c c h a r i d e s [ 1 2 ] , [ 1 3 ] , [ 1 4 ] , [ 1 5 ], and [16] v i i LIST OF FIGURES AND ILLUSTRATIONS PAGE F i g jure 1. The N a t u r a l S u b s t r a t e o f Lysozyme Showing P o i n t s o f Attachment i n the B i n d i n g S i t e . 7 Fig Sure 2 . Proposed Induced Mechanism o f the H y d r o l y s i s by Lysozyme. 8 F i g sure 3- Proposed f l u o r o - a [14] . S y n t h e s i s o f 4 - 0 - ( 2 - d e o x y - 2 -- g l u c o p y r a n o s y l ) - D - g l u c o p y r a n o s e 14 Fig Sure 4. Proposed f l u o r o - a S y n t h e s i s o f 4 - 0 - ( 2 - d e o x y - 2 -- D - g a l a c t o p y r a n o s y l ) - D - g l u c o s e . 15 F i g Sure 5. P a r t i a l [12] . . 1H NMR S p e c t r a ( 1 0 0 MHz) o f 51 Fig Sure 6 . P a r t i a l [13] • 1 H NMR S p e c t r a ( 1 0 0 MHz) o f 52 Fig Sure 7 . 1 H NMR Spectrum ( 2 7 0 MHz) o f [ 1 4 ] . 53 F i g u r e 8 . 1 H NMR S p e c t r a i n D 2 0 at 270 MHz [A] Compound [14] [B] M a l t o s e 54 F i g u r e 9 • P a r t i a l [15] . 1 H NMR S p e c t r a ( 1 0 0 MHz) of 55 Fiji j u r e 1 0 . P a r t i a l [16] . 1 H NMR S p e c t r a ( 1 0 0 MHz) o f 56 F i g u r e 11. 1 9 F NMR Spectrum ( 9 4.1 MHz) o f [ 4 ] . 77 Fi£ gure 1 2 . 1 9 F NMR Spectrum ( 9 4.1 MHz) o f [ 7 ] . 78 F i ^ gure 13> 1 9 F NMR S p e c t r a o f [ 5 ] , [ 1 2 ] , and [131- 79 Fi£ gure 14. 1 9 F NMR Spectrum o f [ 1 4 ] . 80 F i | gure 1 5 . 1 9 F NMR S p e c t r a o f [ 8 ] , [ 1 5 ] , and [ 16] . 81 Fid Sure 1 6 . 1 9 F NMR S p e c t r a o f [ 18] i n C D C l ^ 82 F i , gure 17- C r y s t a l S t r u c t u r e o f [ 1 2 ] . 92 v i i i F i g u r e 1 8 . C r y s t a l S t r u c t u r e o f [ 1 2 ] . 93 F i g u r e 1 9 . 1 H NMR S p e c t r a o f S i d e P r o d u c t [ 1 2 a]-..in-.CDCl 3. 94 [A] N o r m a l S p e c t r u m [B] S p e c t r u m i n t h e P r e s e n c e o f D ? 0 i x ACKNOWLEDGEMENTS I w o u l d l i k e t o t h a n k D r . D.H. D o l p h i n f o r h i s s u p e r v i s i o n t h r o u g h o u t t h e s y n t h e s e s i n v o l v e d i n - t h i s s t u d y , as w e l l as f o r h i s h e l p i n w r i t i n g t h i s t h e s i s . I w o u l d l i k e t o t h a n k Ms. D. M i l l e r and Mr. S.O. Chan f o r r u n n i n g t h e NMR s p e c t r a c o n t a i n e d h e r e i n . A l s o t h a n k s t o D r . F.W.B. E i n s t e i n a n d Mr. R.E. C o b b l e d i c k f o r d o i n g t h e c r y s t a l s t r u c t u r e o f t h e f u l l y p r o t e c t e d d i s a c c h a r i d e [ 1 2 ] . S p e c i a l t h a n k s t o D r . M. R a d c l i f f f o r h i s p e r s o n a l com-m u n i c a t i o n s w i t h r e g a r d s t o t h i s work and t o h i m and a l l t h e o t h e r s who h e l p e d p r o o f r e a d t h i s t h e s i s ; t h e i r comments and s u g g e s t i o n s were much a p p r e c i a t e d . T h a n k s a l s o t o D r . L. Vegh and D r . P . W i r t z f o r p r o v i d i n g me w i t h two o f t h e s t a r t i n g m a t e r i a l s i n v o l v e d i n t h i s w o r k . S p e c i a l t h a n k s t o D r . P. Aubke f o r h i s a i d i n s y n t h e s i z i n g t r i -f l u o r o m e t h y l h y p o f l u o r i t e a n d t o t h e m a c h i n e s h o p f o r b u i l d i n g t h e a p p a r a t u s u s e d i n t h a t s y n t h e s i s . I INTRODUCTION 1 The e l u c i d a t i o n o f enzyme mechanisms has f o r some t i m e b e e n t h e f o c u s o f much r e s e a r c h due t o i t s r e l e v a n c e t o o u r b a s i c u n d e r s t a n d i n g o f b i o c h e m i c a l r e a c t i o n s . Of p r i m e i m p o r t a n c e among t h e s e i s t h e u s e o f ' N u c l e a r M a g n e t i c R e s o n a n c e (NMR) t e c h n i q u e s t o l o o k a t t h e d y n a m i c , as o p p o s e d t o t h e s t a t i c ( e . g . X - r a y d i f f r a c t i o n ) p i c t u r e o f m o l e c u l e s , e s p e c i a l l y f o r enzymes. Enzymes u n d e r g o a s e r i e s o f c o n f o r m a t i o n a l t r a n s i t i o n s on r e a c t i o n w i t h s p e c i f i c s u b s t r a t e m o l e c u l e s and t h e s e c h a n g e s c a n be f o l l o w e d by NMR; t h e r e i s l i t t l e d o u b t t h a t t h i s s o r t o f d y n a m i c i n f o r m a t i o n w i l l l e a d t o a new u n d e r s t a n d i n g o f enzyme c a t a l y s i s . 19 The u s e o f F as a d e t e c t i n g p r o b e i n l a r g e r m o l e c u l e s s u c h as enzymes has s e v e r a l a d v a n t a g e s . ^ Enzymes a r e l a r g e o r g a n i c m o l e c u l e s w h i c h h a v e many p r o t o n s a s s o c i a t e d w i t h them, t h u s t h e "'"H s p e c t r u m u s u a l l y i s t o o c o m p l e x t o be u s e d t o o b s e r v e t h e r e s o n a n c e s a s s o c i a t e d w i t h s p e c i f i c p o r t i o n s o f t h e m o l e c u l e , n o t t o m e n t i o n t h e s m a l l c h a n g e s i n t h e s h i f t s a n d / o r c o u p l i n g c o n s t a n t s w h i c h s h o u l d o c c u r d u r i n g i t s a c t i v e p r o c e s s e s a s a r e s u l t o f s l i g h t c h a n g e s i n t h e o v e r a l l c o n f o r m a t i o n o f t h e m o l e c u l e . Enzymes g e n -e r a l l y i n t e r a c t w i t h f a i r l y l a r g e o r g a n i c m o l e c u l e s , w h i c h a l s o c o n t a i n many p r o t o n s a n d t h u s t h e same l i m i t a t i o n s a r i s e f r o m l o o k i n g a t t h e s p e c t r u m ' o f t h e n a t u r a l s u b s t r a t e . 19 The u s e o f F NMR p r o v i d e s a much s i m p l e r s p e c t r u m as o n l y t h e r e s o n a n c e s o f t h e p r o b e i t s e l f a r e v i s i b l e , t h u s f a c i l i t a t i n g v i s u a l i z a t i o n a n d 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 , I t h a s a d i s t i n c t a d v a n t a g e o v e r o t h e r n u c l e i a s , a t a g i v e n m a g n e t i c f i e l d , t h e s e n s i t i v i t y o f d e t e c t i o n o f t h e 19 1 19 F n u c l e u s i s s e c o n d o n l y t o t h a t o f H, and F i s f o u n d i n 1 00% n a t u r a l a b u n d a n c e . A n o t h e r a d v a n t a g e i s t h a t t h e c o u p l i n g c o n s t a n t s a nd c h e m i c a l s h i f t s a s s o c i a t e d w i t h t h e f l u o r i n e m o i e t y a r e a t l e a s t an o r d e r o f m a g n i t u d e l a r g e r t h a n t h o s e o f t h e c o r r e s p o n d i n g p r o t o n a n a l o g u e s , t h u s s m a l l c h a n g e s i n t h e s e v a l u e s a r e more r e a d i l y o b s e r v e d . The s m a l l s i z e ( c a . t h a t o f a h y d r o x y l g r o u p ) a n d l o w r e a c t i v i t y o f t h e C-F bond (whose i n t r o d u c t i o n i n t o t h e m o l e c u l e s h o u l d t h u s c a u s e o n l y a s m a l l p e r t u r b a t i o n ) p r o v i d e t h e i n g r e d i e n t s o f a n e x c e l l e n t r e p o r t i n g p r o b e . One l a s t a d v a n t a g e i s t h e r e l a t i v e e a s e o f s e l e c t i v e f l u o r i n a t i o n c o m p a r e d w i t h o t h e r t e c h n i q u e s s u c h as g r o s s d e u t e r a t i o n , w h i c h i s o f t e n u s e d t o s i m p l i f y t h e i n t e r p r e t a t i o n o f "'"H NMR f o r more c o m p l e x m o l e c u l e s . By i n c o r p o r a t i n g t h e f l u o r i n e a t o m a t a s p e c i f i c s i t e on a s u b s t r a t e (S) m o l e c u l e , one e x p e c t s t h e c h e m i c a l s h i f t a n d l i n e w i d t h o f t h e r e s o n a n c e t o a l t e r when i t b i n d s t o t h e enzyme (E) s i n c e t h e e n v i r o n m e n t o f t h e f l u o r i n e • n u c l e u s w i l l c h a n g e ; i t i s t h e r e f o r e u n d e r s t a n d a b l e t h a t t h e i d e a o f u s i n g s u c h p r o b e s t o s t u d y t h e e f f e c t s o f 1 2 3 4 5 i n h i b i t o r s o r s u b s t r a t e s on a n enzyme i s n o t new. ' ' ' ' * f j 8 9 ' 3 ' As p r e v i o u s l y o u t l i n e d , t h e s p e c t r u m o f t h e s u b -s t r a t e o r i n h i b i t o r ( I ) bound t o t h e enzyme i s much s i m p l e r t h a n any p r o t o n s p e c t r u m due t o t h e l a r g e number o f p r o t o n s 3 i n b o t h the enzyme and the s u b s t r a t e , thus d i r e c t i n f o r m a t i o n about E-S or E-I complex can be o b t a i n e d i n a s i m p l e r manner. Where c h e m i c a l exchange between the bound and unbound s u b s t r a t e environments i s i m p o r t a n t , one s h o u l d t h e n observe the u s u a l b e h a v i o r f o r the case o f e i t h e r slow or f a s t exchange."'" I n the slow exchange l i m i t where the sub-s t r a t e i s s t r o n g l y bound t o t h e enzyme, p r i m a r i l y t h e resonance o f the bound s u b s t r a t e i t s e l f may be o b s e r v e d , as i l l u s t r a t e d by the examples o f the b i n d i n g o f N - f l u o r o -7 a c e t y l - D - g l u c o s a m i n e ( G l c - N A c F ) , N - t r i f l u o r o r a c e t y l - D -g glucosamine (G'lc-NTFAc), and 3 - m e t h y l - N - f l u o r o a c e t y l - D -q glucosamine (3-Me-Glc-NAcF), whose s t r u c t u r e s are i n d i c a t e d on_the f o l l o w i n g page. I f , however, t h e r e i s a s i g n i f i c a n t amount of f r e e l i g a n d , t h e n two s u b s t r a t e r e s o n a n c e s are o b s e r v e d which c o r r e s p o n d t o each environment. I n the case o f f a s t exchange, o n l y a s i n g l e l i n e i s o b s e r v e d which i s dependent on the p o s i t i o n o f the two resonances i n -d i c a t e d above. With any o f t h e s e t y p e s o f exchange r a t e s , the c h e m i c a l s h i f t s o f the s u b s t r a t e analogue can be ob-g s e r v e d and i n t e r p r e t e d . The i n t e r p r e t a t i o n o f c h e m i c a l s h i f t s from a knowledge o f the c r y s t a l s t r u c t u r e o f the E-I complex i s p o s s i b l e i f the mode of b i n d i n g o f the i n h i b i t o r i s known or assumed; the c r y s t a l s t r u c t u r e g i v e s the i n t e r a c t i o n o f the s u b s t r a t e w i t h s p e c i f i c groups on the e n z y m e . 1 ' 1 0 I f the f l u o r i n a t e d analogue b i n d s s i m i l a r l y , the b i n d i n g o f t h i s m o l e c u l e t o t h e enzyme s h o u l d cause a change i n t h e c h e m i c a l s h i f t o f 19 7 9' t h e F probe, as has been obse r v e d i n p r e v i o u s work. 3 4 5 The d e p e n d e n c e on pH o f s u c h s h i f t c h a n g e s h a s b e e n shown t o c o r r e l a t e w e l l w i t h t h e pK v a l u e s o f amino a c i d r e s i d u e s a t o w h i c h t h e l a b e l l e d p o r t i o n s o f t h e s u b s t r a t e a r e 8 9 a s s o c i a t e d . I n s u c h s t u d i e s . M i l l e t a n d R a f t e r y o b-s e r v e d c o n f o r m a t i o n a l • c h a n g e s i n t h e c o m p l e x e s o f l y s o z y m e w i t h i t s s u b s t r a t e a n a l o g u e s due t o i o n i z a t i o n o f a l l t h r e e 8 a c t i v e s i t e c a r b o x y l g r o u p s , Asp 5 2 , G l u 3 5 , and Asp 1 0 1 . I t i s p o s s i b l e t h a t s i m i l a r c o n f o r m a t i o n a l ; , c h a n g e s o c c u r i n t h e a c t u a l c a t a l y t i c p r o c e s s a n d a r e i m p o r t a n t i n l o w e r i n g t h e e n e r g y o f t h e t r a n s i t i o n s t a t e . B e c a u s e so much work on t h i s p a r t i c u l a r enzyme h a s b e e n d o n e , i t w o u l d be i d e a l t o make a p r o b e t o s t u d y i t s m e c h a n i s m , w h i c h i s t h o u g h t t o be f a i r l y w e l l e s t a b l i s h e d ( F i g u r e 2 ) . ' ' ' ' J Thus j u d g e m e n t on t h e r e l a t i v e c o r r e c t n e s s o f any o b s e r v e d c h a n g e s i n s h i f t o r c o u p l i n g c o n s t a n t s , c a n be e a s i l y v e r i f i e d by c o m p a r i s o n w i t h t h o s e e x p e c t e d on t h e b a s i s o f t h e p r o p o s e d m e c h a n i s m , a s h a s b e e n o u t l i n e d p r e v i o u s l y . A l s o , t h e c r y s t a l s t r u c t u r e o f t h e E - I c o m p l e x h a s b e e n r e p o r t e d so t h e mode o f b i n d i n g o f t h e s u b s t r a t e h a s b e e n d e t e r m i n e d a s shown i n F i g u r e 1 . L y s o z y m e i s a n enzyme w h i c h a t t a c k s b a c t e r i a by h y d r o l y z i n g c e l l w a l l p o l y s a c c h a r i d e s . One common c e l l w a l l f r a g m e n t h y d r o l y z e d by t h i s enzyme i s composed o f a l t e r n a t i n g 3 ( 1 - 4 ) l i n k e d u n i t s o f N - a c e t y l g l u c o s a m i n e (NAG) and N - a c e t y l m uramic a c i d (NAM) as shown i n F i g u r e 1. The p r o p o s e d i n t e r m e d i a t e I n t h i s p r o c e s s i s a c a r b o n i u m . i o n w h i c h i s i n a h a l f c h a i r c o n f o r m a t i o n as shown c l e a r l y '.on.the f o l l o w i n g 1 2 page where t h e C - 5 , r i n g 0 , C - l , C - 2 , and C - 3 atoms a l l (A) Chair conformation of the 2-acetamido-2-deoxy-D-glucopyranose ring. The number's indicate the position cf hydrogens. The dotted line marks the intersection between the C-2-C-1-H-1 plane and the H-2-C-2-N plane. The dihedral arigle 6 is the angle between the H-2-C-S-C-1 plane, and the C-2-C-1-H-1 plane. (B) Half-chair conformation of the 2-acetamido-2-deos:y-D-gluco-pyranose ring. With the C-S-O-C-l confined to a plane, the equatorial groups attached to C-l and C-Z rotate to nearly axial positions, thus eliminating the substrate-enzyme Alter-ation at C-6 and closing the dihedral angle 6 toward 0 . l i e i n a s i n g l e p l a n e . The k n o w l e d g e o f t h e c r y s t a l s t r u c t u r e a n d t h e m e c h a n i s m o f a c t i o n o f t h e enzyme a i d s i n i n t e r p r e t i n g t h e s p e c t r a on t h e b a s i s o f e x p e c t e d s h i f t and c o u p l i n g c o n s t a n t c h a n g e s f o r t h e p r o b e . P a t t a n d 12 c o w o r k e r s f o l l o w e d t h e f o r m a t i o n o f t h e p r o p o s e d h a l f c h a i r i n t e r m e d i a t e by m o n i t o r i n g t h e J„ u c o u p l i n g c o n -s t a n t . T h i s J v a l u e c h a n g e s due t o t h e d e c r e a s e i n t h e d i h e d r a l a n g l e 0 shown a b o v e as t h e i n t e r m e d i a t e i s f o r m e d . The m a j o r d i s a d v a n t a g e s i n s u c h a s t u d y a r i s e f r o m t h e f a c t t h a t t h i s c o u p l i n g i s v e r y s m a l l t o b e g i n w i t h , a l t h o u g h I t i n c r e a s e s when 0 d e c r e a s e s . I n a d d i t i o n , t h e s u b s t r a t e was c l e a v e d r a p i d l y t h u s m o n i t o r i n g t h e chan g e i n t h e c o u p l i n g c o n s t a n t was d i f f i c u l t . The u s e o f a s u b s t r a t e a n a l o g u e w i t h a C-2 f l u o r i n e s u b s t i t u e n t c o u l d o v e r c o m e t h e d i f f i c u l t i e s e n c o u n t e r e d a b o v e b e c a u s e t h e F .atom s h o u l d d e s t a b i l i z e t h e p r o p o s e c L c a r b o n i u m i o n i n t e r m e d i a t e and t h u s c l e a v a g e o f t h e s u b s t r a t e w o u l d A the darker edges of the rings exposed to tlie outside, the lighter ones buried at ere vice bottom.—OR = -0-CH(CH,)-C0OH. NAG NAM NAG .NAM FIGURE 1. A. The natural substrate of the enzyme showing points of attachment i n the binding s i t e . The l e t t e r s A,B,C,D,E, and F displayed w i t h i n r e f e r to the subsites on the enzyme to which each respective sugar binds. B. A schematic view of the sugars C,D,E, and F portrayed i n a more commonly accepted fashion to f a c i l i t a t e : an ap p r e c i a t i o n of the conformational aspects of each sugar. 8 o The mechanism of cleavage of the polysaccliaride bond in lysozyme. FIGURE 2. P r o p o s e d m e c h a n i s m o f t h e h y d r o l y s i s i n d u c e d by L y s o z y m e . 9 be h i n d e r e d . The m a j o r a d v a n t a g e , h o w e v e r , a r i s e s f r o m t h e f a c t t h a t t h e J T T „ c o u p l i n g c o n s t a n t s a r e much l a r g e r t h a n t h e c o r r e s p o n d i n g H c o u p l i n g c o n s t a n t s . T h u s , i d e a l l y , m o n i t o r i n g t h e c o u p l i n g J w „ s h o u l d g i v e a d e t e c t a b l e i n c r e a s e i n t h i s c o u p l i n g as t h e s u b s t r a t e a n a l o g u e i s f o r c e d i n t o t h e h a l f c h a i r c o n f o r m a t i o n . A s i m i l a r o b-s e r v a t i o n i s e x p e c t e d f o r t h e J„ „ c o u p l i n g c o n s t a n t . The m a j o r p r o b l e m t h a t e x i s t s f o r s t u d y i n g t h i s s y s t e m u s i n g s u c h a l a b e l l e d s u b s t r a t e i s t h a t t h e s y n t h e t i c r o u t e s r e q u i r e d t o c o n s t r u c t s u c h a p r o b e a r e v e r y d i f f i c u l t . I n s t e a d , i t was h o p e d t h a t a s i m p l e r p r o b e c o u l d be made w h i c h w o u l d be u s e d t o s t u d y a d i f f e r e n t g l y c o s i d a s e 1 5> 1 6> 1 7 (any enzyme w h i c h h y d r o l y s e s a g l y c o s i d i c l i n k a g e ) s y s t e m t o s e e i f t h e method w o u l d be f e a s i b l e f o r t h e s t u d y o f s u c h enzyme mechanisms i n g e n e r a l . The b a s i c a p p r o a c h w o u l d be t o s y n t h e s i z e t h e f l u o r i n a t e d s u b s t r a t e a n a l o g u e s d e p i c t e d i n F i g u r e 3 . S u c h d i s a c c h a r i d e s c o u l d f e a s i b l y be u s e d t o s t u d y enzyme s y s t e m s s u c h as t h o s e w h i c h a r e i n d i c a t e d b e l o w . S t r u c t u r e Enzyme A. M a l t a s e , a - g l u c o s i d a s e B. 3 - g l u c o s i d a s e C. a - g a l a c t o s i d a s e D. 3 — g a l a c t o s i d a s e I n t h i s w o r k , t h e s y n t h e s i s o f A ( F i g u r e 3) was a c h i e v e d . I n g e n e r a l , t h e c h o i c e o f t h e t y p e and p o s i t i o n o f t h e f l u o r i n e p r o b e was b a s e d on t h e f o l l o w i n g i d e a l c o n d i t i o n s . 10 B,OH C D A. 4 - 0 - C 2 - d e o x y - 2 - f l u o r o - a - D - g l u c o p y r a n o s y l ) -D - g l u c o p y r a n o s e [ 2 ' - d e o x y - 2 ' - f l u o r o - m a l t o s e ] B . 4 - 0 - ( 2 - d e o x y - 2 - f l u o r o t - a - D - g a l a c t o p y r a n o s y l ) -D - g l u c o p y r a n o s e [ 2 ' - d e o x y - 2 ' - f l u o r o - c e l l o b l o s e ] C. 4 - 0 - ( 2 - d e o x y - 2 - f l u o r o - a - D - g a l a c t o p y r a n o s y l ) - D -g l u c o p y r a n o s e [ A 2 ' - d e o x y - 2 ' f l u o r o - a - g a l a c t o s l d e ] D. 4 - 0 - ( 2 - d e o x y - 2 - f l u o r o - B - D - g a l a c t o p y r a n o s y l ) - D -g l u c o p y r a n o s e [ 2 ' - d e o x y - 2 ' - f l u o r o - l a c t o s e ] 11 I t d o e s n o t i n t e r f e r e w i t h t h e n a t u r a l b i n d i n g s i t e s b e t w e e n t h e enzyme and t h e s u b s t r a t e . I n t h e a b o v e c a s e , t h i s i s o n l y a n i d e a l a s s u m p t i o n as t h e c r y s t a l s t r u c t u r e s o f t h e s e enzymes h a v e n o t b e e n d o n e . I t i s n o t l a r g e e nough t o p e r t u r b t h e m o l e c u l e so t h a t i t i n t e r f e r e s w i t h t h e n a t u r a l b i n d i n g mode. I t i s c l o s e e nough t o t h e a c t u a l s i t e o f c l e a v a g e so t h a t c h e m i c a l s h i f t and c o u p l i n g c o n s t a n t c h a n g e s i n t h e f l u o r i n e s p e c t r u m a r e l i k e l y t o b o t h o c c u r and be c l e a r l y v i s i b l e . As i n d i c a t e d e a r l i e r , t h e C-2' p o s i t i o n i s e s p e c i a l l y a d v a n t a g e o u s as one w o u l d a l s o e x p e c t t o s e e a c h a n g e i n t h e c o u p l i n g c o n s t a n t s J ^ ' ^ , and J ^ ' p s h o u l d a h a l f c h a i r t r a n s i t i o n s t a t e be f o r m e d . The r e s u l t i n g f l u o r i n e s p e c t r u m s h o u l d be s i m p l e as t h e r e i s o n l y a s i n g l e f l u o r i n e n u c l e u s i n t h e p r o b e and t h u s t h e r e s h o u l d be no e x t e n s i v e c o u p l i n g w i t h o t h e r n u c l e i i n t h e m o l e c u l e . D e c o u p l i n g o f t h e p r o t o n s s h o u l d r e s u l t i n a s i n g l e p eak where c l a r i f -i c a t i o n o f s h i f t c h a n g e s i s n e c e s s a r y . The e l e c t r o n e g a t i v e F atom s h o u l d , a t t h i s p o s i t i o n , d e s t a b i l i z e t h e p r o p o s e d c a r b o n i u m i o n i n t e r m e d i a t e and t h e r e f o r e t h e s u b s t r a t e a n a l o g u e s h o u l d n o t be c l e a v e d as r a p i d l y as t h e n o r m a l s u b s t r a t e . O n l y u n d e r t h e s e c i r c u m s t a n c e s w o u l d one e x p e c t t o g e t a c hange w h i c h w o u l d be s l o w enough t o o b s e r v e . 19 The l i t e r a t u r e o f t h e F NMR o f m o n o s a c c h a r i d e s w i t h a C-2 f l u o r i n e s u b s t i t u e n t e n c o m p a s s e s s e v e r a l p a p e r s . 18,19,20,21,22,23,24 m u , , . „ ' ' ' ' ' ' The d e p e n d e n c i e s o f c o u p l i n g 1 2 c o n s t a n t s on t h e configuration o f v a r i o u s s u b s t i t u e n t s on t h e a n o m e r i c c a r b o n h a v e b e e n f a i r l y w e l l e s t a b l i s h e d i n t h e s e r e p o r t s , b o t h by 1 H and 1 9 F NMR. T h i s f a c i l i t a t e s b o t h t h e d e d u c t i o n o f t h e configuration- a t t h e a n o m e r i c c e n t r e f o r t h e d i s a c c h a r i d e s and mono-s a c c h a r i d e s s y n t h e s i z e d i n t h i s w o r k , as w e l l as s i m p l i f y i n g t h e i n t e r p r e t a t i o n o f any o b s e r v e d c h a n g e s i n t h e s h i f t s and c o u p l i n g c o n s t a n t s l i k e l y t o o c c u r when t h e s u b s t r a t e a n a l o g u e b i n d s t o t h e enzyme. The r a t e d e t e r m i n i n g s t e p i n t h i s k i n d o f a p p r o a c h i n v o l v e s t h e s y n t h e s i s o f t h e l a b e l l e d s u b s t r a t e , t h e g r e a t e s t c h a l l e n g e l y i n g i n t h e s y n t h e s i s o f t h e d i s a c c h a r i d e p r o b e s . The most common s y n t h e s i s o f s u c h d i s a c c h a r i d e m o l e c u l e s i n v o l v e s t h e u s e o f t h e c l a s s i c a l K o e n i g s - K n o r r r e a c t i o n as o u t l i n e d b e l o w . R=alkyl or a ry l group K o e n i g s a n d K n o r r p r e p a r e d v a r i o u s d e r i v a t i v e s o f compound I I f r o m t h e c o r r e s p o n d i n g b r o m i d e I w i t h s i l v e r c a r b o n a t e , s i l v e r n i t r a t e , o r p y r i d i n e as t h e a c i d a c c e p t o r , and o t h e r s h a v e done s i m i l a r r e a c t i o n s u n d e r t h e s e same 2 6 " t y p i c a l " c o n d i t i o n s . The r e a c t i o n as o u t l i n e d a bove p r o c e e d s w i t h n e t i n v e r s i o n o f c o n f i g u r a t i o n a t t h e a n o m e r i c c a r b o n atom u n d e r s u c h c o n d i t i o n s . I n g e n e r a l , t h e p r o c e d u r e i n v o l v e s t h e t r e a t m e n t o f a p e r - 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 w i t h a n a l c o h o l i n t h e p r e s e n c e o f a h e a v y - m e t a l s a l t o r o r g a n i c b a s e as t h e a c i d a c c e p t o r ; t h e l a t t e r e n h a n c e s t h e r a t e o f r e a c t i o n and a l s o p r e v e n t s s i d e r e a c t i o n s s u c h as 2 6 d e a c e t y l a t i o n . As m e n t i o n e d a b o v e , 1,2 t r a n s - g l y -c o p y r a n o s i d e s ( 3-anomer) a r e u s u a l l y o b t a i n e d by t h e r e a c t i o n o f t h e c o r r e s p o n d i n g 1,2 c i s - ( a - a n o m e r ) o r 1,2 t r a n s -g l u c o p y r a n o s y l b r o m i d e s w i t h an a l c o h o l i n t h e p r e s e n c e o f s i l v e r c a r b o n a t e o r s i l v e r o x i d e . 1,2 c i s - g l y c o s i d e s a r e , h o w e v e r , u s u a l l y t h e m a j o r p r o d u c t o b t a i n e d f r o m g l y c o p y r a n o s y l 2 6 h a l i d e s h a v i n g a n o n - p a r t i c i p a t i n g g r o u p a t C-2 w h i l e t h e m i n o r p r o d u c t i s t h e 1,2 t r a n s - g l y c o p y r a n o s i d e . I n t h i s w o r k , t h e l a t t e r r e s u l t was e x p e c t e d a s t h e C-2 s u b s t i t u e n t o f t h e h a l o s u g a r s [51 and [7] ( F i g u r e s 4 and 5 r e s p e c t i v e l y ) b o r e a f l u o r i n e a t o m w h i c h was n o n - p a r t i c i p a t i n g . The p r o p o s e d m e c h a n i s m o f t h e K o e n i g s - K n o r r r e a c t i o n i s shown 26 b e l o w where t h e e f f e c t o f p a r t i c i p a t i o n o f t h e C-2 s u b s t i t u e n t c a n be f u l l y a p p r e c i a t e d . I n t h e a b s e n c e o f a H 7C 0 A c 2 ° ^ pyr id ine Ac OAc [ 1 ] CHjOAc AcO CF 3OF C F C 1 , 2. 0 AcO 6AC CH2OAc AcO r OCF CH2OAc [3] H 2C 0 Aco ^ r OH CHjOAc y ~ ~ \ OCF, OAc [ 2 ] CH2OAc AcO CHjOAc [4] [51 CH2OAc [ 1 2 ] CH2OAc H2C -0 CH OAc OH F OAc [ 1 2 ] S.P.-[12a] CH2OAc NaOCH./CH.OH OAc CH2OH CH2OH ,0H [14] FIGURE 3- Synthesis of 4-0-(2-deoxy-2-fluoro-a-D-glucopyranosyl)-D-glucopyranose [Hi] [10] [ 1 7 ] FIGURE 4. Proposed syn t h e s i s o f 4-0-(2-deoxy-2-fluoro-a-D-galactopyranosyl)-D-glucose [17], showing the s y n t h e t i c route o f 1,2,3,6-tetra-0-acetyl-4-0-(3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-a-D-galactopyranosyl)-D-glucopyranose [16]. 1 6 p a r t i c i p a t i n g g r o u p a t C-2, t h e r e a c t i o n may p r o c e e d v i a an i n t i m a t e i o n - p a i r ( D ) , l e a d i n g t o t h e 1,2 t r a n s -g l y c o p y r a n p s i d e (A) and a s o l v e n t - s e p a r a t e d i o n o r a f r e e i o n ( E ) . The i o n (E) r e a c t s w i t h a l c o h o l t o g i v e t h e 1 5 2 - t r a n s - ( A ) a nd - c i s - g l y c o p y r a n o s i d e s ( I ) . S o m e t i m e s , i t ha s b e e n p o s t u l a t e d t h a t s o l v e n t p a r t i c i p a t i o n c a n o c c u r t o g i v e a n i o n (H) w h i c h i s m a i n l y o b t a i n e d f r o m (C) i n some c a s e s when a m e r c u r y ( I I ) s a l t i s u s e d a s t h e c a t a l y s t , b u t t h i s m e c h a n i s m i s s t i l l u n c l e a r . What i s e v i d e n t , h o w e v e r , i s t h a t one s h o u l d e x p e c t a m i x t u r e o f a - and 3 - l i n k e d p r o d u c t s I n t h e a b s e n c e o f a p a r t i c i p a t i n g g r o u p a t C-2. The r a t i o o f t h e s e p r o d u c t s h a s b e e n shown t o be a f u n c t i o n o f a l c o h o l c o n c e n t r a t i o n , t y p e o f s i l v e r s a l t u s e d , t h e 2 p o l a r i t y o f t h e s o l v e n t , and t h e p r e s e n c e o f h a l i d e i o n s . T h u s , u n d e r t h e c o n d i t i o n s e m p l o y e d i n a " t y p i c a l " K o e n i g s - K n o r r r e a c t i o n ( s i l v e r t r i f l a t e was a l s o i n c l u d e d i n o u r s y n t h e s i s t o a c t a s a c a t a l y s t ) , one w o u l d e x p e c t t h a t t h e s y n t h e s i s o f [12] and [ 1 5 ] , as o u t l i n e d i n F i g u r e s 4 and 5 r e s p e c t i v e l y , w o u l d y i e l d p r i m a r i l y t h e a - l i n k e d anomer. I n t h i s s t u d y , t h e f l u o r i n a t e d a n a l o g u e o f m a l t o s e [14] was s y n t h e s i z e d ( F i g u r e 3) and c h a r a c t e r i z e d . U n d e r t h e c o n d i t i o n s o f t h e K o e n i g s - K n o r r r e a c t i o n , o n l y t h e a - l i n k e d p r e c u r s o r i { 1 2 ] " was p r o d u c e d , none o f t h e e x p e c t e d g - l i n k e d d i s a c c h a r i d e was o b t a i n e d . P o s s i b l e r e a s o n s f o r 19 t h i s r e s u l t a r e o u t l i n e d i n t h e D i s c u s s i o n . The F NMR s p e c t r a o f t h e s u g a r s ( F i g u r e s 4 and 5) s y n t h e s i z e d v e r i f i e d t h e c o n f i g u r a t i o n s d e d u c e d f r o m "'"H NMR s p e c t r u m . 17 The p a r t i a l s y n t h e s i s o f a 2 ' - d e o x y - 2 ' - f l u o r o - a - D - g a l a c t o s i d e 19 ( F i g u r e 3 C ) was a l s o a t t e m p t e d and t h e F NMR s p e c t r a o f t h e s u g a r s ( F i g u r e 5) i n v o l v e d i n t h i s w ork a l s o c o n f i r m e d t h e "'"H NMR c o n f o r m a t i o n a l a s s i g n m e n t s . I n b o t h c a s e s , t h e f o c u s i s on t h e u s e o f t h e c o u p l i n g c o n s t a n t s J ^ ' p, J^.' p, J ' and J ' „ i n d e d u c i n g t h e c o n f o r m a t i o n o f t h e -> H T i l l d i s a c c h a r i d e s u b s t i t u e n t s a t , C 2 3 ' ^4 ' N o enzyme b i n d i n g s t u d i e s were a t t e m p t e d due t o t h e i n t r i c a t e and t i m e - c o n s u m i n g p r o c e d u r e s n e c e s s a r y t o i s o l a t e and p u r i f y t h e e n z y m e s . 1 8 I I EXPERIMENTAL Those p r o d u c t s w h i c h were o i l s w ere t a k e n t o d r y n e s s on a r o t a r y e v a p o r a t o r , c o e v a p o r a t e d w i t h b e n z e n e , and t h e n d r i e d u n d e r h i g h vacuum. A f t e r r e c r y s t a l l i z a t i o n , s o l i d s w ere d r i e d u n d e r h i g h vacuum o v e r ^2^5 ^ o r h o u r s . M e l t i n g p o i n t s were m e a s u r e d i n c a p i l l a r y t u b e s on a Thomas H o o v e r U n i m e l t C a p i l l a r y M e l t i n g P o i n t A p p a r a t u s and a r e u n c o r r e c t e d . T h i n l a y e r c h r o m a t o g r a p h y ( t i c ) was p e r f o r m e d on s i l i c a g e l GF ( M e r c k , 10 cm) and d e t e c t i o n o b t a i n e d by c h a r r i n g w i t h H^O^. E l e m e n t a l a n a l y s i s o f a l l e l e m e n t s e x c e p t f l u o r i n e were p e r f o r m e d by Mr. P. B o r d a o f t h i s d e p a r t m e n t ; f l u o r i n e a n a l y s i s was o b t a i n e d f r o m A n a l y t i s c h e L a b o r a t o r i e n , Germany. The NMR s p e c t r a were t a k e n by Mr. S.O. Chan and Ms. D. M i l l e r on t h e f o l l o w i n g i n s t r u m e n t s ; MHz I n s t r u m e n t 100 CW V a r i a n HA 100 100 FT V a r i a n XL 100 270 FT B r u k e r ( C o n s o l e ) O x f o r d I n s t r u m e n t s ( M a g n e t ) t h e r e s t o f t h e 270 MHz i n s t r u m e n t a t i o n was c o n s t r u c t e d a t U.B.C. The s p e c t r a w i l l be i n t e r p r e t e d a nd d i s c u s s e d i n S e c t i o n s I I I and I V . O p t i c a l r o t a t i o n s were m e a s u r e d on e i t h e r a P e r k i n - E l m e r l 4 l P o l a r i m e t e r o r on a P e r k i n - E l m e r 249 MC P o l a r i m e t e r . The h i g h r e s o l u t i o n mass s p e c t r a w e re 19 o b t a i n e d on a K r a t o s / A E I M S 50 ( d i r e c t p r o b e i n l e t a t 70 eV) and t h e l o w r e s o l u t i o n mass s p e c t r a were o b t a i n e d on a V a r i a n / M A T CHYB ( d i r e c t i n l e t p r o b e a t 70 e V ) ; t h e y were t a k e n by D r . G.K. E i g e n d o r f a n d Mr. G. Gunn. The c r y s t a l s t r u c t u r e s were o b t a i n e d by D r . F.W.B. E i n s t e i n a n d Mr. R.E. C o b b l e d i c k . P r e p a r a t i o n o f t h e A n h y d r o - S u g a r s [1] and [2] 27 1 , 6 - A n h y d r o - B - D - g l u c o s e [1] A 500 m l r o u n d - b o t t o m f l a s k c o n t a i n i n g Canada C o r n S t a r c h ( 1 9 4 . 5 g) was c o n n e c t e d ( v i a a 6 0 ° t u b u l a r g l a s s c o n n e c t o r ) t o a 1 l i t r e r o u n d - b o t t o m f l a s k ( i n a d r y i c e -a c e t o n e b a t h ) f i t t e d w i t h a c o n d e n s e r . The s t a r c h was d r i e d a t 150°C u n d e r h i g h vacuum ( 0 . 2 mm Hg) and t h e n h e a t e d c a r e f u l l y w i t h a l a r g e l u m i n o u s f l a m e . C o n d e n s a t i o n o f t h e r e s u l t i n g o i l i n b o t h t h e r e a c t i o n f l a s k and t h e g l a s s c o n n e c t o r was m i n i m i z e d by l o c a l h e a t i n g w i t h a s e c o n d b u n s e n b u r n e r . H e a t i n g was c o n t i n u e d u n t i l no f u r t h e r v a p o u r i z a t i o n was e v i d e n t and o n l y c h a r c o a l ( 3 7 - 5 g) r e m a i n e d i n t h e f l a s k . The c o n d e n s e d o i l was removed f r o m t h e 1 l i t r e f l a s k u s i n g a c e t o n e ( 2 * 200 ml) and e v a p o r a t e d t o d r y n e s s . The a d d i t i o n o f a minimum amount o f a c e t o n e t o d i s s o l v e t h e o i l , f o l l o w e d by s c r a t c h i n g w i t h a g l a s s r o d , Starch 20 gave 3 0 . 1 0 g o f c r u d e t a n - c o l o u r e d c r y s t a l s . T h e s e were d i s s o l v e d i n m e t h a n o l ( 5 0 ml) and c a . 2 g o f a 1 : 1 0 c e l i t e / c h a r c o a l m i x t u r e was a d d e d . The s o l u t i o n was h e a t e d t o t h e b o i l i n g p o i n t w i t h s t i r r i n g a n d t h e n f i l t e r e d h o t . The f i l t r a t e was r e d u c e d t o c a . 25 ml and a l l o w e d t o c r y s t a l l i z e a t 0°C t o g i v e 2 1 . 0 7 g o f p u r e w h i t e c r y s t a l s ( 1 5 . 5 $ ) . m.p. = 1 7 4 - 1 7 5°C ( l i t . 1 7 2 ° C ) 2 8 R f = 0 . 6 2 i n 4 : 1 CHClg/MeOH [ a ] 2 3 = - 6 8 . 5 ° ' ( c . 0 . 8 1 5 , H 2 0 ) ( l i t . [ a ] j p = - 6 6 . 2 ° , H A n a l y s i s ; C a l c . f o r : C, 4 4 . 4 5 ; H, 6 . 2 2 P o u n d : C, 4 4 . 3 0 ; H, 6 . 2 2 r 2 9 2 , 3 - D i - 0 - a c e t y l - l , 6 - a n h y d r o - g - D - g l u c o s e [2] in ( 2 ) 13 g ( 0 . 0 8 0 mmoles) o f [1] were d i s s o l v e d i n d r y p y r i d i n e (100 ml) w i t h s t i r r i n g . A c e t i c a n h y d r i d e (19 ml) was a d d e d and t h e s o l u t i o n was s t i r r e d f o r 10 h o u r s a t room t e m p e r a t u r e . The f l a s k was t h e n h e a t e d t o 50°C f o r one h o u r , c o e v a p o r a t e d t h r e e t i m e s w i t h t o l u e n e (25 ml e a c h ) and d r i e d t o g i v e 24.09 g o f a t a n - c o l o u r e d o i l . The o i l was d i s s o l v e d i n a minimum o f e t h e r and s e e d e d t o g i v e 5.01 g o f t h e f u l l y a c e t y l a t e d p r o d u c t ( 2 , 3 , 4 - t r i - O r - a c e . t y l 1 , 6 - a n h y d r o - g - D - g l u c o s e ) . The r e m a i n i n g o i l was s e p a r a t e d 21 on s i l i c a g e l (Woelm, A c t i v i t y 1.5, 120 g) by e l u t i o n w i t h 1:1 b e n z e n e / e t h y l a c e t a t e t o y i e l d 2.20 g o f t h e d e s i r e d p r o d u c t ( 1 1 . 1 5 $ ) as a n o i l . R f = 0 . 3 9 4 i n 1:1 b e n z e n e / e t h y l a c e t a t e H i g h r e s o l u t i o n mass s p e c t r a : C a l c . f o r c ^ H ^ O : 2 4 6 . 0 7 4 P o u n d : 2 4 6 . 0 7 4 [ a ] 2 3 ' 5 = - 4 9 . 3 ° ( c . 0 . 7 6 9 , CHC1 3) ( l i t . [ a ] 2 7 = - 4 5 ° c. 4 . 9 , CHC1 3) 2 9 P r e p a r a t i o n o f t h e F l u o r i n a t e d S u g a r s  [ 4 ] , [ 5 ] , [ 1 2 ] , [ 1 2 a ] , [ 1 3 1 , [14] and [ 18] 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 - a - D - g l u c o p y r a n o s y l f l u o r i d e [4] 2 5 . 0 2 g ( 0 . 0 9 3 mmoles) o f [3] ( A c c u r a t e C h e m i c a l s a nd S c i e n t i f i c C o r p o r a t i o n ) were d i s s o l v e d i n F r e o n 11 ( C F C 1 3 ) ( 6 1 0 ml) a t room t e m p e r a t u r e w i t h s t i r r i n g . The s o l u t i o n was f l u s h e d w i t h N 2 , c o o l e d i n an i c e - w a t e r b a t h , a nd f u r t h e r c o o l e d t o - 7 8 ° C i n a d r y i c e - a c e t o n e b a t h w i t h c o n s t a n t N 2 b u b b l i n g t o p r e v e n t i n l e t c l o g g i n g . C F 3 0 F ( p r e p a r e d a s o u t l i n e d on p 33) i n a g l a s s t r a p was p l a c e d i n a N„/ 22 d i c h l o r o e t h y l e n e Dewar f l a s k and t h e i n l e t o p e n e d i n t o t h e r e a c t i o n v e s s e l t o p e r m i t s l o w b u b b l i n g . A f t e r 5 h o u r s , a p p r o x i m a t e l y 38 g o f CF^OF ( t o t a l c o n t e n t s o f t h e t r a p ) were u s e d up -and t h e s o l u t i o n was s t i r r e d a t room t e m p e r a t u r e f o r a n a d d i t i o n a l 2 hours:. A r g o n was b u b b l e d t h r o u g h f o r 0.5 h o u r s a n d t h e n ¥1^ was b u b b l e d t h r o u g h o v e r n i g h t . The s o l u t i o n was l e f t t o come t o room t e m p e r a t u r e w i t h s t i r r i n g a n d t h e n m e t h y l e n e c h l o r i d e (500 m l ) and d e c o l o u r i z i n g c a r b o n (2 g) were a d d e d ; t h e c o n t e n t s were l e f t s t i r r i n g f o r 0.5 h o u r s . E v a p o r a t i o n t o d r y n e s s gave 32.15 g o f a t a n -c o l o u r e d o i l w h i c h was d i s s o l v e d i n a minimum o f m e t h y l e n e c h l o r i d e a n d s e p a r a t e d on s i l i c a g e l (Woelm, A c t i v i t y 2, 600 g) where e l u t i o n w i t h 1:1 e t h e r / l i g h t p e t r o l e u m e t h e r ( b . p . 3 0 ° - 6 0°C) gave t h e p r o d u c t as a n o i l . The o i l was c r y s t a l l i z e d f r o m d r y e t h a n o l - l i g h t p e t r o l e u m e t h e r t o g i v e 4.8 g o f w h i t e c r y s t a l s (17-8%). m.p. = 89-91°C ( l i t . 91-92°C) 2 0 R f = 0.395 i n 1:1 b e n z e n e / e t h y l a c e t a t e [ a ] 2 2 ' 5 = +132?:" ( c . 3-317, C H C 1 3 ) ( l i t . [a] 2 5 = + 1 3 8 0 ) 2 0 A n a l y s i s ; C a l c . f o r c 1 2 H i 6 ° 7 F 2 : C ' ^ 6 - i j 5 ; H, 5-20; F, 12.25 F o u n d : C, 46 .16; H, 5-18; F, 12.45 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 - a - D - g l u c o p y r a n o s y l  b r o m i d e [5] [A] (5] 23 1 .99 g (6.42 mmoles) o f [4] were d i s s o l v e d i n 30-32% HBr i n AcOH ( E a s t m a n - K o d a k Co., 25 m l ) . A c e t i c a n h y d r i d e (1 ml) was a d d e d and t h e s o l u t i o n was s t i r r e d f o r 2 h o u r s a t room t e m p e r a t u r e i n t h e a b s e n c e o f l i g h t . A r g o n was b u b b l e d t h r o u g h f o r 2 h o u r s t o p u r g e HBr and t h e s o l u t i o n was t a k e n t o d r y n e s s on a r o t a r y e v a p o r a t o r a t 30°C. C o e v a p o r a t i o n w i t h t o l u e n e (4 x 20 m l ) a t t h e same t e m p e r a t u r e a n d f u r t h e r d r y i n g u n d e r h i g h vacuum gave a t a n - c o l o u r e d o i l . T h i s p r o d u c t was d i s s o l v e d i n a minimum o f e t h e r , an e q u i v a l e n t v o l u m e o f l i g h t p e t r o l e u m e t h e r was a d d e d , a n d t h e s o l u t i o n was a l l o w e d t o c r y s t a l i z e a t 0°C. The r e s u l t i n g w h i t e c r y s t a l s were f i l t e r e d , washed w i t h l i g h t p e t r o l e u m e t h e r , and d r i e d t o g i v e 2 .17 g o f p u r e p r o d u c t ( 9 1.0$). m.p. = 75-76°C ( l i t . 79-80°C) 2 1 R f = 0 .473 i n 1:1 b e n z e n e / e t h y l a c e t a t e [ a ] 2 3 ' 5 = +209°: ( c . 1 . 0 8 , CHC1 3) ( l i t . [ a ] 2 5 = +229° 1-2%, CHC1 3) 2 1 A n a l y s i s ; C a l c . f o r C-^H-^O^FBr: C, 3 8 . 8 3 ; H, 4 . 3 5 ; F, 5-12; B r , 21 . 5 3 P o u n d : C, 38 .84; H, 4 .44 ; P, 5-26; B r , 21 . 8 0 2 , 3 - D i - O - a c e t y l - l , 6 - a n h y d r o - 4 - 0 - ( 3 , 4 , 6 - t r i - 0 - a c e t y l - 2 - d e o x y - 2 - f l u o r o - a - D - g l u c o p y r a n o s y l ) - g - D - g l u c o p y r a n o s e [12] CHjOAc CHjOAc 2, 2 [2] [5] [12) S . P . - [ 1 2 « ] F r — F 24 A. E x c e s s o f [2] To a 100 m l d r y * f l a s k w e r e a d d e d 292 mg (1 . 1 9 mmoles) o f d r y [ 2 ] , 92.0 mg AgSO^CF.^ (0 . 3 5 8 m m o l e s ) . 276 . 6 mg A g 2 C 0 3 3 0 (1.00 m m o l e s ) , 2 .79 g D r i e r i t e (CaSO^) (0.146 m o l e s ) [ a l l d r i e d two d a y s o v e r ^2®$ u n c ^ e r h i g h vacuum i n t h e a b s e n c e o f l i g h t ] , a n d a b s o l u t e e t h e r (50 m l ) . The s o l u t i o n was s t i r r e d f o r 8 h o u r s i n t h e a b s e n c e o f l i g h t a t room t e m p e r a t u r e i n a s t o p p e r e d f l a s k . 312.0 mg ( 0 . 8 4 2 mmoles) o f [5] were a d d e d i n f o u r e q u a l p o r t i o n s a t 2 h o u r i n t e r v a l s . The s o l u t i o n was s t i r r e d o v e r n i g h t u n d e r t h e c o n d i t i o n s o u t l i n e d a b o v e , f i l t e r e d t h r o u g h a f i n e s i n t e r e d g l a s s f u n n e l and t h e r e s i d u e was washed t h o r o u g h l y w i t h m e t h y l e n e c h l o r i d e . T i c u s i n g 1:1 b e n z e n e / e t h y l a c e t a t e showed t h r e e s p o t s as i n d i c a t e d b e l o w . The d r i e d 604 mg o f o i l was d i s s o l v e d i n a minimum o f m e t h y l e n e c h l o r i d e and s e p a r a t e d on s i l i c a g e l (Woelm, A c t i v i t y 1 . 5 5 30 g ) . E l u t i o n w i t h 1:1 b e n z e n e / e t h y l a c e t a t e gave t h e p r o d u c t s o u t l i n e d b e l o w . The d i s a c c h a r i d e was c r y s t a l l i z e d f r o m e t h e r R f PRODUCT mg 0.473 s i d e p r o d u c t = [12a] 162 . 9 0.407 d i s a c c h a r i d e = [12] 240.0 0 .229 R-OH = [2] 80.7 and r e c r y s t a l l i z e d f r o m d r y e t h a n o l t o g i v e 2 0 8 . 4 . mg o f p r o d u c t (46 . 2%) . F l a s k was h e a t e d and c o o l e d t o room t e m p e r a t u r e u n d e r a c o n s t a n t s t r e a m o f N~. 25 m.p. = 150-151°C R f = 0 . 4 0 7 i n 1:1 b e n z e n e / e t h y l a c e t a t e [ a ] 2 2 ' 1 = +49 .4 .° ( c . 0 . 7 7 7 , CHC1 3) [ a ] 2 5 = + 4 8 . 4 0 ° ( C H C 1 3 ) f o r 2 , 3 , 2 ' 3 ' 4 ' 6 ' - h e x a - 0 -31 a c e t y l - 1 , 6 - a n h y d r o m a l t o s e shown b e l o w . AcO A n a l y s i s ; C a L a . f o r C ^ H ^ O ^ F . i C, 49-25.; H:, 5.45.; F, 3-54 Pound : C, 4 9 . 0 1 ; H, 5 .43 ; F, 3-68 B. E x c e s s o f [51 To a 35 ml d r y r o u n d - b o t t o m f l a s k were a d d e d 62.6 mg (0.254 mmoles) o f [ 2 ] , 67-9 mg (0.256 mmoles) A g 2 C 0 3 3 ° , 18.7 mg (0.073 mmoles) A g SO^CF^ , 309-6 mg (0.162 mmoles) o f D r i e r l t e [ a l l d r i e d two d a y s o v e r Po0,_ u n d e r h i g h vacuum ^ 5 i n t h e a b s e n c e o f l i g h t ] a n d a b s o l u t e e t h e r (10 m l ) . The s o l u t i o n was s t i r r e d a t room t e m p e r a t u r e i n a s t o p p e r e d f l a s k i n t h e a b s e n c e o f l i g h t f o r 0.5 h o u r s . 92.7 mg o f [5] were a d d e d u n d e r t h e same c o n d i t i o n s a n d , a f t e r 20 m i n u t e s an a d d i t i o n a l 29-9 mg o f t h e same r e a c t a n t were a d d e d (0.330 mmoles t o t a l ) . The s o l u t i o n was s t i r r e d o v e r n i g h t , f i l t e r e d t h r o u g h a f i n e s i n t e r e d g l a s s f u n n e l , and washed w e l l w i t h m e t h y l e n e c h l o r i d e . T i c o f t h e f i l t r a t e showed t h r e e s p o t s , as i n d i c a t e d b e l o w . The 272.5 mg o f t h e d r i e d f i l t r a t e w e r e s e p a r a t e d on a s i l i c a g e l (Woelm, 26 A c t i v i t y 1.5, 100g) by e l u t i o n w i t h 7:4 b e n z e n e / e t h y l a c e t a t e ( a f t e r i t was d i s s o l v e d i n a minimum o f m e t h y l e n e c h l o r i d e ) t o g i v e t h e y i e l d s i n d i c a t e d b e l o w . R f PRODUCTS mg 0.507 s i d e p r o d u c t = [12a] 34.9 0.422 d i s a c c h a r i d e = [12] 82.8 0.273 R-OH = 2 The c r u d e o i l o f [12] was c r y s t a l l i z e d f r o m e t h e r R e c r y s t a l l i z a t i o n f r o m d r y e t h a n o l g ave 74 mg p r o d u c t (54.4$) w i t h t h e p r o p e r t i e s i n d i c a t e d i n s e c t i o n A. The s i d e p r o d u c t o b t a i n e d f r o m t h e r e a c t i o n m i x t u r e s A and B o f s y n t h e s i s o f [12] was p u r i f i e d on a s i l i c a g e l c o l u m n (Woelm, A c t i v i t y 1.5, 30g) w i t h 1:1 b e n z e n e / e t h y l a c e t a t e and t h e n d r i e d . The r e s u l t i n g o i l had t h e p r o p e r t i e s i n d i c a t e d b e l o w . R f = 0.539 i n 1:1 b e n z e n e / e t h y l a c e t a t e [ a ] 2 1 , 7 = +100° ( c . 0.777, CHC13) H i g h r e s o l u t i o n mass s p e c t r u m : C a l c . f o r C 1 2 H 2 7 ° 8 F : 308.090 P o u n d : 308.087 T i c o f t h e p u r i f i e d s i d e p r o d u c t showed t h e p a t t e r n s s u m m a r i z e d b e l o w . A t t e m p t s t o s e p a r a t e t h e two s p e c i e s o b s e r v e d u s i n g t h e s i l i c a g e l c o l u m n s i n d i c a t e d b e l o w were u n s u c c e s s f u l . The p r e s e n c e o f a t r a c e o f AcOH was n e c -e s s a r y i n a l l c a s e s t o a c h i e v e s e p a r a t i o n on t h e t i c p l a t e s . 27 (1) (2) (3) T i c s o l v e n t " 1:1:0.1 e t h e r / p e t r o l e u m e t h e r (b.p. 30-60°C)/AcOH as above but at 4°C R f c a . r e l a t i v e q u a n t i t y * 0.455 0.344 0.276 0 . 1 9 1 2 : 1 e t h e r / p e t r o l e u m e t h e r 0 . 6 1 2 (b.p. 30-60°C)/trace o f AcOH 0 . 5 1 0 2 1 2 1 2 1 E l u t i n g S o l v e n t (1) (2) (3) (no AcOH) Column A c t i v i t y 1 . 0 (Woelm, 40 g) A c t i v i t y 1 . 0 (Woelm, 50 g) A c t i v i t y 1 . 0 (Woelm, 100 g) Sample S i z e 300 mg 410.9 m 303-8 mt 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 - D - g l u c o s e 32 [ 1 2 a ] A c O C H j O A c [5] C H j O A c H 20 ether A c O ™ f OH S.P . - l l 2a] 46 . 5 mg ( 0 . 1 2 5 mmoles) o f [5] were added t o e t h y l e t h e r (5 ml) and water ( 1 0 m l ) . 3 6 . 1 mg ( 0 . 0 3 0 mmoles) 30 were added and the s o l u t i o n s t i r r e d o v e r n i g h t a t A g 2 C 0 3 room temperature i n the absence o f l i g h t . The r e s u l t i n g s o l u t i o n was f i l t e r e d and the f i l t r a t e e x t r a c t e d w i t h * Based on i n t e n s i t y o f spot a f t e r c h a r r i n g . ^ Si.ll.ca g e l columns were r u n at room temperature u n l e s s o t h e r w i s e i n d i c a t e d . 28 CHC1 2 (3 x 100 m i s ) . The C H 2 C 1 2 e x t r a c t s were washed w i t h w a t e r (100 m l ) , 2N K H C 0 3 (100 m l ) and w a t e r (100 m l ) , t h e n d r i e d o v e r MgSO^ and e v a p o r a t e d t o d r y n e s s t o g i v e 0.0653 g o f o i l . The o i l was d r i e d and l o a d e d on a s i l i c a g e l c o l u m n (Woelm, A c t i v i t y 1 . 5 , 30 g) i n a minimum o f m e t h y l e n e c h l o r i d e a n d e l u t e d w i t h 1:1 b e n z e n e / e t h y l a c e t a t e t o y i e l d 35.45 mg p r o d u c t (91.9$) as an o i l . R f = 0.546 i n 1:1 b e n z e n e / e t h y l a c e t a t e [a] 21.9 = + 99 . 80° ( c . 0 . 9 08 , C H C l q ) M e t h y l 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 - 3 - D - g l u c o p y r a n o s e 3 3 [18] [51 [18] A t w o - n e c k e d 25 m l r o u n d b o t t o m f l a s k was f l u s h e d w i t h N 2 and f l a m e d . 308.5 mg ( 1 . 6 l mmoles) D r i e r i t e , a b s o l u t e m e t h a n o l (1 ml) (0.025 m o l e s ) , a n d a b s o l u t e e t h e r (5 ml) 3 0 were a d d e d u n d e r a s t r e a m of N^. 77.0 mg A g 2 C 0 3 (0.312 m m o l e s ) , 18 mg AgSO^CF^ (0.071 mmoles) were added s i m i l a r l y a n d t h e s o l u t i o n was s t i r r e d a t room t e m p e r a t u r e f o r 30 m i n u t e s i n t h e a b s e n c e o f l i g h t . 0.100 g o f [5] (0.269 mmoles) i n a b s o l u t e e t h e r (5 ml) were added d r o p w i s e 29 o v e r a p e r i o d o f 4 h o u r s a n d p o s i t i v e p r e s s u r e o f ¥S^ was m a i n t a i n e d t h r o u g h o u t . The s o l u t i o n was s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e , f i l t e r e d , a nd t h e r e s i d u e was washed w e l l w i t h C l ^ C ^ . D r y i n g g ave 69.8 mg o f a w h i t e powder (79.5$) w h i c h was r e c r y s t a l l i z e d f r o m e t h e r / l i g h t p e t r o l e u m e t h e r t o g i v e O.O65 g o f w h i t e c r y s t a l s ( 7 4 . 3 % ) . m.p. = 126-126.5°C ( l i t . 130-132°C) 3 3 = O.689 i n 1:1 b e n z e n e / e t h y l a c e t a t e [ a ] 2 4 ' 0 = + 3 4 . 2 ° ( c , 1.21, CHC1 3) ( l i t . [ a ] p 5 = +33° c. 0 .58 , CHC1 3) 3 3 A n a l y s i s : C a l c . f o r C 1 3 H i g O g P : C, 48.44; H, 5 -94; F, 5-89 F o u n d : C, 48.36; H, 5 -89; F, 5-91 1 , 2 , 3 , 6 - T e t r a - 0 - a c e t y l - 4 - 0 - ( 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 - a - D - g l u c o p y r a n o s y l ) - a - D - g l u c o p y r a n o s e [13] 70 mg (0.130 mmoles) o f d r y [12] were s t i r r e d f o r 1 h o u r a t room t e m p e r a t u r e i n a n a c e t y l a t i n g s o l u t i o n (1.1 ml) composed o f A c 2 0 (45 m l ) , AcOH (15 m l ) , and H^SO^ (1 m l ) . F u s e d a n h y d r o u s NaOAc (300 mg) ( F i s h e r S c i e n t i f i c ) was a d d e d and t h e s o l u t i o n was s t i r r e d f o r a n o t h e r 10 m i n u t e s . I c e 30 (20 g) was a d d e d a n d t h e m i x t u r e was s t i r r e d f o r a n o t h e r 30 m i n u t e s . The r e s u l t i n g s o l u t i o n was e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e (3 x 100 ml) and t h e c o m b i n e d e x t r a c t s w ere washed s u c c e s s i v e l y w i t h w a t e r (100 m l ) , 2N KHCO^ (100 m l ) , w a t e r (100 m l ) , and t h e n d r i e d 0.5 h o u r s o v e r MgSO^. The s o l u t i o n was f i l t e r e d a n d t h e f i l t r a t e t a k e n t o d r y n e s s t o g i v e 135-1 mg o f an o i l . The a d d i t i o n o f a b s o l u t e e t h a n o l (2 m l ) f o l l o w e d by c o o l i n g t o 0°C gave w h i t e c r y s t a l s w h i c h were f i l t e r e d and washed w i t h p e t r o l e u m e t h e r ( b . p . 30-60°C) and t h e n d r i e d t o g i v e 50 mg p r o d u c t ( 6 0 $ ) , w h i c h was t h e oc-anomer. m.p. 1 4 8 . 5 - 1 4 9 - 5°C R f = 0.525 i n 1:1 b e n z e n e / e t h y l a c e t a t e (3-anomer h a d R f = 0.577) [ a ] 2 1 , 7 = +107° ( c . 0 .815 , CHC13) [ a ] 2 0 = +122.8°(CHC1 3) f o r 1 , 2 , 3 , 6 - t e t r a - 0 - a c e t y l - 4 - 0 ( 2 , 3 , 4 , 6 -t e t r a - O - a c e t y l - a - D - g l y c o p y r a n o s y l ) - a - D - g l u c o -34 p y r a n o s e as shown b e l o w . AcO OAc A n a l y s i s : C a l c . f o r C 2 6 H 3 5 0 1 7 F : C, 4 8 . 9 0 ; H, 5 - 5 3 ; P, 2.98 F o u n d : C, 4 8 . 6 5 ; H, 5 - 3 3 ; F, 3 . 1 4 31 4 - 0 - ( 2 - D e o x y - 2 - f l u o r o - a - D - g l u c o p y r a n o s y l ) - D - g l u c o p y r a n o s e [14] (.2 ' -Deoxy-2 ' - f l u o r o - m a l t o s e ) 34.4 mg (0.054 mmoles) o f d r y [13] were d i s s o l v e d i n a b s o l u t e m e t h a n o l (4.9 ml) u n d e r a s t r e a m o f . The s o l u t i o n was t h e n s y r i n g e d i n t o a f l a s k u n d e r p o s i t i v e p r e s s u r e o f N^. 1.0 N m e t h a n o l i c NaOCH^.(0.1 m l , f r e s h l y p r e p a r e d ) was t h e n a d d e d s i m i l a r l y a n d t h e s o l u t i o n s t i r r e d f o r 3 h o u r s a t room t e m p e r a t u r e . A s m a l l amount o f c h a r c o a l (500 mg) was a d d e d w i t h s t i r r i n g a n d a l s o A m b e r l i t e IR - 1 2 0 ( H , 300 mg) r e s i n t o remove N a + i o n s . When t h e m i x t u r e r e a c h e d n e u t r a l i t y ( a s i n d i c a t e d by pH p a p e r ) , i t was f i l t e r e d t h r o u g h a f i n e g l a s s f i l t e r f u n n e l and t h e r e s i d u e was w ashed w e l l w i t h m e t h a n o l . The f i l t r a t e was t a k e n t o d r y n e s s t o g i v e 18.42 mg p r o d u c t (99-5$) as a t r a n s p a r e n t g l a s s . R f = 0.342 i n 1:1 C H C l 3 / e t h a n o l [ a ] 2 3 ' 5 = +109° ( c . 0 .892, p y r i d i n e , 20 h r s ) . ?0 7 [ a ] ^ ' = +106° ( c . 1.12, p y r i d i n e ; 20 h r s ) f o r m a l t o s e * w h i c h i s shown b e l o w . (D+) M a l t o s e - N u t r i t i o n a l B i o c h e m i c a l s . - C o r p o r a t i o n 32 3 5 P r e p a r a t i o n o f T r i f l u o r o m e t h y l h y p o f l u o r i t e [CP^OF] CO, + P- / s £ C F o 0 F ( g ) 2 ( g ) 3 A s p e c i a l a p p a r a t u s was c o n s t r u c t e d f o r t h i s s y n t h e s i s a c c o r d i n g t o t h e d i r e c t i o n s g i v e n i n t h e r e f e r e n c e s t a t e d a b o v e and . i t i s shown on p 3 3 - The f u r n a c e was h e a t e d t o 350°C o v e r n i g h t a n d f l u s h e d w i t h N 2 f o r .1 h o u r b e f o r e t h e f l o w o f CO, v and F„, N was i n i t i a t e d ; t h e two g l a s s ( g ) 2 ( g ) t r a p s were c o o l e d w i t h l i q u i d 0^- The c a l i b r a t e d f l o w -m e t e r s were u s e d t o e n s u r e t h a t t h e gas f l o w s w e re a p p r o x i m a t e l y 5 l i t r e s / h o u r f o r F 2 and 2 l i t r e s / h o u r f o r CO. The f l o w o f t h e two g a s e s was i n i t i a t e d a t t h e same t i m e and p r o d u c t c o n d e n s a t i o n was o b s e r v e d one t o two m i n u t e s l a t e r , c o n s i s t i n g o f a y e l l o w l i q u i d a l o n g w i t h some w h i t e p r e c i p i t a t e , t h e l a t t e r b e i n g l i k e l y C OF 2 a n d / o r P^CO-OCF^. The r e a c t i o n was r u n f o r 3 .hours a f t e r w h i c h t h e t r a p s were s e a l e d o f f f r o m t h e r e s t o f t h e s y s t e m and t h e l i q u i d was pumped f r o m t h e f i r s t t r a p i n t o t h e s e c o n d t r a p , t h u s b e i n g f i l t e r e d t h r o u g h s i n t e r e d g l a s s a n d d r a w n up t h r o u g h t h e c a p i l l a r y c o n n e c t i n g t h e two t r a p s u n t i l a l l o f t h e p r o d u c t was c o n t a i n e d i n t h e s e c o n d t r a p . T h i s t r a p was t h e n r e m o v e d f r o m t h e a p p a r a t u s , a s o d a - l i m e t r a p was a t t a c h e d , a n d t h e F 2 ( y e l l o w c o n t a m i n a n t ) was pumped o f f u n t i l t h e l i q u i d became c o l o u r l e s s . S i n c e t h e 3 5 d e n s i t y o f t h e l i q u i d p r o d u c t was known t o be 1.9 g / c c J , t h e t o t a l y i e l d was e s t i m a t e d by c o m p a r i s o n w i t h a s i m i l a r v o l u m e o f w a t e r i n a t r a p o f t h e same d i m e n s i o n s . Re-p e a t i n g t h i s s y n t h e s i s u n d e r s i m i l a r c o n d i t i o n s gave a 33 NaF trap flowmeter Copper iurnace a . thermocouple/heating u n i t b'. g l a s s c a p i l l a r y J(. swage lock connections CO N 2 s i n t e r e d glass _> f i l t e r _1 b glass traps t o t a l o f 38 g o f p r o d u c t w h i c h was u s e d t o f l u o r i n a t e [3] as o u t l i n e d p r e v i o u s l y . The compound may now be p u r c h a s e d f r o m PCR. P r e p a r a t i o n o f t h e F l u o r i n a t e d S u g a r s  [7] , [ 8 ], and [16] 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 - q - D - g a l a c t o p y r a n o s y l 20 22 f l u o r i d e ^ u><^ [7] T r i - O - a c e t y l - g a l a c t a l [ 6 ], o b t a i n e d f r o m T e r o c h e m L a b o r a t o r i e s as a 1Q>% s o l u t i o n i n b e n z e n e , was d r i e d a t room t e m p e r a t u r e u n d e r h i g h vacuum t o g i v e an o i l . A 1 l i t r e r o u n d - b o t t o m f l a s k was f l a m e d and f l u s h e d w i t h N 2 f o r 2 h o u r s . 10.94 g (0.04 m o l e s ) o f t h e g a l a c t a l were d i s s o l v e d F F [7] 3.4 i n F r e o n 11 (400 m l ) and t h e f l a s k was c o o l e d w i t h an i c e -w a t e r b a t h w h i l e Ng was b u b b l e d t h r o u g h t h e s o l u t i o n . The f l a s k was f u r t h e r c o o l e d t o - 78°C i n a Dewar f l a s k c o n -t a i n i n g N g / d i c h l o r o e t h y l e n e a n d Ng was b u b b l e d t h r o u g h a t t h i s t e m p e r a t u r e f o r a f u r t h e r 2 h o u r s . An e x c e s s o f CF^OF (21.2 g, 4.91 m o l e s , PCR) was r a p i d l y b u b b l e d t h r o u g h t h e s o l u t i o n f o r 2 h o u r s a n d t h e n t h e s y s t e m was f l u s h e d a f u r t h e r 2 h o u r s w i t h Ng. The Dewar f l a s k was r e m o v e d , t h e s o l u t i o n l e f t t o warm t o room t e m p e r a t u r e w i t h c o n s t a n t Ng b u b b l i n g , and t h e n t a k e n t o d r y n e s s t o g i v e 14 . 5 8 g o f o i l . T h i s o i l was d i s s o l v e d i n a minimum o f c h l o r o f o r m ( c a . 5 ml) and s e p a r a t e d on d r y K i e s e l g e l (200 g, M a c h e r y , N a g e l , & Co.) by e l u t i o n w i t h 2:1 p e t r o l e u m e t h e r ( b . p . ' 3 0 -60°C)/ether t o g i v e 3-06 g o f [7] as an o i l ( 2 4 . 5 % ) w h i c h w o u l d n o t c r y s t a l l i z e d e s p i t e r e p e a t e d a t t e m p t s i n v a r i o u s s o l v e n t s . R f = 0 .576 i n 2:1 e t h e r / p e t r o l e u m e t h e r ( b . p . 30-60 C) [ a ] 2 2 = +123.0° ( c . 2 . 7 5 , CHC1 3) ? n ( l i t . [ a ] D = +136° c. 2 . 8 , CHCl- 3) H i g h R e s o l u t i o n Mass S p e c t r u m : C a l c . f o r c 1 2 H i 6 ° 7 F 2 : 310.087 F o u n d 310.087 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 - a - D - g a l a c t o p y r a n o s y l b r o m i d e [8] 35 A . Compound [11] was p r o v i d e d b y D r . P. W i r t z whose mode o f s y n t h e s i s i s i n d i c a t e d on t h e o v e r a l l f l o w s h e e t 20 20 shown a b o v e . Compound [10] was made by D r . L. V e g h . 40 .8 mg (0.117 mmo le s ) o f d r y [11] w e r e d i s s o l v e d i n 30-32% HB r i n CH^COgH ( E a s t m a n - K o d a k ) (1 m l ) a n d t h e s o l u t i o n was s t i r r e d a t r oom t e m p e r a t u r e f o r 2 h o u r s i n t h e a b s e n c e o f l i g h t . A r g o n was b u b b l e d t h r o u g h f o r an a d d i t i o n a l 2 h o u r s a n d t h e s o l u t i o n was c o e v a p o r a t e d w i t h 3 x 10 m l o f t o l u e n e a t 30°C t o g i v e 33-10 mg p r o d u c t a s an o i l ( 7 6 . 6 % ) . The o i l d i d n o t c r y s t a l l i z e d e s p i t e r e p e a t e d a t t e m p t s i n v a r i o u s s o l v e n t s . R f = 0.689 i n 2:1 b e n z e n e e t h y l a c e t a t e [ a ] 2 1 , 9 = + 64 . 2 ? " ( c : 2 .59 , C H C l ^ ) H i g h R e s o l u t i o n Mas s S p e c t r u m : C a l c . f o r C - ^ H ^ O ^ F B r : 371.013 F o u n d : 371.011 36 C a l c . f o r c 1 2 H i 6 ° 7 P : 2 9 1 - 0 8 8 F o u n d : 291.088 The h i g h r e s o l u t i o n mass s p e c t r a l d a t a were o b t a i n e d a f t e r p u r i f i c a t i o n o f t h e s a m p l e on a s i l i c a g e l c o l u m n (Woelm, A c t i v i t y 1.5, 30 g) t h r o u g h e l u t i o n i n t h e same s o l v e n t s y s t e m i n d i c a t e d f o r t h e t i c v a l u e . (7J [8] B. 200.2 mg (0 .645 mmoles) o f d r y [7] were d i s s o l v e d i n 30-32$ HBr i n C H ^ C O ^ (4 m l ) and a c e t i c a n h y d r i d e (20 d r o p s ) was a d d e d . The s o l u t i o n was s t i r r e d o v e r n i g h t a t room t e m p e r a t u r e i n t h e a b s e n c e o f l i g h t . A r g o n was b u b b l e d t h r o u g h t h e r e a c t i o n m i x t u r e f o r 2 h o u r s t o p u r g e HBr a n d t h e n t o l u e n e (5 m l ) was a d d e d a n d t h e s o l u t i o n t a k e n t o d r y n e s s a t 30°C. The r e s u l t i n g o i l was c o -e v a p o r a t e d w i t h t o l u e n e (3 x 10 ml) a t t h e same t e m p e r a t u r e , 1:1 m e t h y l e n e c h l o r i d e / b e n z e n e (10 ml) and d e c o l o u r i z i n g c a r b o n ( c a . 500 mg) were a d d e d , a n d t h e s o l u t i o n s t i r r e d 0.5 h o u r s . The m i x t u r e was f i l t e r e d and washed w e l l w i t h e t h a n o l a n d m e t h y l e n e c h l o r i d e t o g i v e a l i g h t y e l l o w f i l t r a t e . D r y i n g t h e f i l t r a t e gave 236.7 mg o f o i l (99.3$) w h i c h h a d t h e p r o p e r t i e s o u t l i n e d a b o v e i n s e c t i o n A. T h i s 37 p r o d u c t decomposed upon s t a n d i n g t o a compound w i t h R f 0.326 and t h u s was s t o r e d a t 0°C. 2 , 3 - D i - 0 - a c e t y 1 - 1 , 6 - a n h y d r 0 - 4 - 0 - ( 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 - q - D - g a l a c t o p y r a n o s y l ) - g - D - g l u c o p y r a n o s e [15] T h i s compound was p r o v i d e d by D r . P. W i r t z and was made u n d e r c o n d i t i o n s s i m i l a r t o t h o s e u s e d i n t h e s y n t h e s i s o f [12] where a n e x c e s s o f b r o m i n a t e d mono-s a c c h a r i d e was u s e d . The compound was c h a r a c t e r i z e d so t h a t an o p t i c a l r o t a t i o n measurement c o u l d be t a k e n w h i c h c o u l d be compa r e d w i t h t h a t o f t h e n o n - f l u o r i n a t e d a n a l o g u e i n d i c a t e d b e l o w . m.p. = 175-176°C R f = 0 . 43 i n 1:1 b e n z e n e / e t h y l a c e t a t e [ a ] 2 3 * 5 = +53.7° ( c . 0 . 9 4 6 , CHC1 3) [ a ] 2 3 = + 5 4 . 4 ° ( c . 1.1, CHC1 3) f o r 2 , 3 - d i - 0 - a c e t y l - l , 6 -a n h y d r o - 4 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 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 ) -g - D - g l u c o p y r a n o s e shown b e l o w . 38 A n a l y s i s : C a l c . f o r C 2 2 H 2 g O l 4 F : C 3 4 9 -25 , H, 5 -45 ; F, 3-5^ P o u n d : C, 4 9 -05 ; H, 5 -26; P, 3-36 1 , 2 , 3 , 6 - T e t r a - 0 - a c e t y l - 4 - 0 - ( 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 - g - D - g a l a c t o p y r a n o s y l ) - q - D - g l u c o p y r a n o s e [16] The same s y n t h e t i c r o u t e a s f o r [13] was u s e d . 30 mg (0.056 mmoles) o f d r y [15] gave 28 mg p r o d u c t (78.4%) w h i c h was t h e a anomer. m.p. = 158-159°C R f = 0.50 ( a ) i n 1:1 b e n z e n e e t h y l a c e t a t e [ a ] 2 3 ' 5 = +101° ( c . 0 .952 , CHC1 3) [ a ] 2 7 = +117° ( c . 1 . 1 4 , CHC1 3) f o r 1 , 2 , 3 3 6 - t e t r a - 0 -a c e t y l - 4 - 0 - ( 2 , 3 , 4 , 6 - t e t r a - 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 ) -31 a - D - g l u c o p y r a n o s e shown b e l o w . F 39 O A c A n a l y s i s : C a l c . f o r C 2 6 H 3 5 0 1 7 F : C, 4 8 .90 , H, 5 -53 , F, 2 .98 F o u n d : C, 4 8 . 71 , H, 5 -50, F, 2 .81 The s u g a r s s y n t h e s i z e d as o u t l i n e d i n t h i s s e c t i o n were 19 1 t h e n s t u d i e d u s i n g F and H NMR. The r e s u l t s o f t h i s w ork a r e d i s p l a y e d i n t h e f o l l o w i n g s e c t i o n ( I I I ) and i n t e r p r e t e d i n t h e s u b s e q u e n t s e c t i o n ( I V ) . 4-Q. -I I I CHEMICAL SHIFTS AND COUPLING CONSTANTS The i n i t i a l c h e m i c a l s h i f t a s s i g n m e n t s were made on t h e known m o n o s a c c h a r i d e s [ 1 ] , [ 2 ] , [ 4 ] , and [7] shown b e l o w t h r o u g h c o n s i d e r a t i o n o f e x p e c t e d s p l i t t i n g p a t t e r n s and by c o m p a r i s o n w i t h t h e l i t e r a t u r e v a l u e s f o r t h e s e c a r b o h y d r a t e s . T h i s method o f a n a l y s i s was u s e d i n o r d e r [1] •- [2] oac t o t e s t t h e v a l i d i t y o f t h i s p r o c e d u r e , w h i c h was a l s o e m p l o y e d t o i n t e r p r e t t h e NMR s p e c t r a o f t h e o t h e r s u g a r s . The b a s i c s t r u c t u r e o f t h e two m o n o s a c c h a r i d e u n i t s [4] and [7] v a r i e s l i t t l e t h r o u g h o u t t h e s y n t h e s i s , e x c e p t t h a t t h e C - l s u b s t i t u e n t i n t h e h a l o s u g a r s [5] and [8] 41 i s a B r atom, a n d f o r t h e d i s a c c h a r i d e s i t i s t h e o t h e r s u g a r m o i e t y . Thus l i t t l e c h a n g e i n t h e s h i f t p o s i t i o n o f t h e r e s p e c t i v e r i n g p r o t o n s ( e x c l u d i n g H^) was e x p e c t e d o r o b s e r v e d . The s p l i t t i n g p a t t e r n s were q u i t e c h a r a c -t e r i s t i c f o r e a c h p r o t o n a nd were e a s i l y r e c o g n i z a b l e i n a l l o f t h e compounds; thess< p r o t o n a s s i g n m e n t s were 19 v e r i f i e d by t h e F d e c o u p l i n g e x p e r i m e n t s as w i l l be o u t l i n e d s h o r t l y . The o n l y e x c e p t i o n t o t h i s p r o c e d u r e a r o s e i n t h e c a s e o f compound [14] ( t h e f l u o r i n a t e d a n a l o g u e o f m a l t o s e ) f o r w h i c h t h e a s s i g n m e n t s w e re made s o l e l y on t h e b a s i s o f e x p e c t e d s p l i t t i n g p a t t e r n s , t h e e f f e c t o f t h e a d d i t i o n o f D^O on t h e s p e c t r u m , a nd com-p a r i s o n w i t h t h e s p e c t r a o f t h e f u l l y a c e t y l a t e d a n a l o g u e [13]. T h i s was n e c e s s a r y s i n c e 100 MHz s p e c t r u m was 19 e x t r e m e l y c o m p l e x and t h u s F d e c o u p l i n g gave no d e f i n i t e i n f o r m a t i o n a b o u t t h i s s p e c i e s . The c o u p l i n g s were t a k e n f r o m a 270 MHz s p e c t r u m where t h e r e s o n a n c e s w e re much more c l e a r l y r e s o l v e d . A l l o f t h e "*"H s p e c t r a were r u n a t 100 o r 270 MHz ( a s i n d i c a t e d i n t h e T a b l e s ) a nd t h e 1 9 F s p e c t r a were r u n a t 94.1 MHz. T a b l e s I t o V I p r e s e n t t h e c h e m i c a l s h i f t a s s i g n m e n t s f o r t h e s u g a r s i n d i c a t e d i n e a c h t a b l e w h i l e T a b l e s V I I t o X p r e s e n t t h e c o u p l i n g c o n s t a n t s d e r i v e d f r o m t h e "*"H and 19 F s p e c t r a . I n t h e f o r m e r c a s e , t h e r e l a t i v e i n t e g r a l v a l u e s f o r t h e OAc r e s o n a n c e s a r e shown i n b r a c k e t s t o t h e l e f t o f t h e c h e m i c a l s h i f t a s s i g n m e n t . T a b l e I encompases t h e c h e m i c a l s h i f t a s s i g n m e n t s f o r t h e two a n h y d r o s u g a r s [1] and [2] w h i l e T a b l e I I p r e s e n t s TABLE I CHEMICAL SHIFT ASSIGNMENTS FOR THE ANHYDRO SUGARS [1] AND [ 2 j a [1] [2] COMPOUND OAc H± H 2 H 3 H^ H^ Hgexo Hg.endo 0H 2 QH'3 OH^ SOLVENT [1] 37? 4.83 6.80 6.62 6.65 5.63 6.48 6.11 5.3?. 5-12 5-20 DMSO-dg [1] — ' 4.82 6.82 6.60 6.66 5.68 6.47 6.12 5.28 5-10 5-21 [2] ('6)7.94 '4.66 6.51 6.38 6.09 5-36 5.64 5-50 [2] (6)7-93 4.61 6.48 6.22 5.94 5-24 5-48 5 -4 l 7.89 6.99 CDC1-a . The chemica l s h i f t s a re g i ven i n T and a re r e l a t i v e t o e x t e r n a l TMS. ment i s ± 0.05 x. A l l s pec t r a were run at 100 MHz b. Actual literature values from the reference cited. Maximum e r r o r i n measure-COMPOUND OAc ^ E? H 3 Hgexo Hg.endo SOLVENT [12] (9)8.00 4.56 6.43 6.24 6.02 5.20 5-44 5-40 CDC1 (6)7.03 [15] (6)8.02 4.56 6.58 6.25 6.04 5-24 5-46 5-50 (9)7-92 a. The chemical s h i f t s are given i n x and are r e l a t i v e t o e x t e r n a l TMS. Maximum e r r o r i n measure-ment i s ± 0.05 T • A l l spectra, were r u n at 100 MHz. -Cr LAJ-44 t h e d a t a f o r t h e c o r r e s p o n d i n g a n h y d r o m o i e t y i n t h e d i s a c c h a r i d e s [12] and [ 1 5 ] . I t was r e l a t i v e l y s i m p l e t o a s s i g n t h e c h e m i c a l s h i f t v a l u e s i n t h e l a t t e r two c a s e s as t h e y a r e q u i t e d i s t i n c t f r o m t h o s e o f t h e p r o t o n s a t t a c h e d t o t h e o t h e r h a l f o f t h e d i s a c c h a r i d e m o l e c u l e . F o r t h e o t h e r d i s a c c h a r i d e s [13] , [ 1 4 ] , a n d [16] , h o w e v e r , t h i s a n h y d r o - l i n k a g e i s l o s t a n d t h e r e s o n a n c e s s h i f t t o l o w e r f i e l d w h ere t h e y a r e o f t e n o b s c u r e d by t h e r e s o n a n c e s o f t h e o t h e r p r o t o n s i n t h e m o l e c u l e ; e x a c t a s s i g n m e n t s were t h u s t e d i o u s o r i m p o s s i b l e w i t h o u t t h e a i d o f p r o t o n d e c o u p l i n g e x p e r i m e n t s . T h i s was e x p e c t e d due t o t h e s i m i l a r i t y i n e n v i r o n m e n t s o f t h e r e s p e c t i v e p r o t o n s i n e a c h r i n g , as w i l l be o u t l i n e d s h o r t l y . A l t h o u g h t h e r e s o n a n c e s o f [2] a p p e a r t o s h i f t down-f i e l d i n C D C l ^ o v e r t h o s e m e a s u r e d i n DMSO-dg, t h e r e l a t i v e p o s i t i o n s o f t h e r e s o n a n c e s a r e m a i n t a i n e d . The s h i f t s o f t h e 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 ( T a b l e I I ) , h o w e v e r , a r e v e r y s i m i l a r t o t h o s e o f t h e a n a l a g o u s s u g a r [ 2 ] , as was e x p e c t e d due t o t h e r e t e n t i o n o f c o n f i g u r a t i o n i n t h e o v e r a l l s t r u c t u r e . The a b s e n c e o f t h e OH (C^.-OH) r e s o n a n c e f o r [2] i n DMSO-dg was s u r p r i s i n g as g e n e r a l l y d e p r o t e c t e d s u g a r s i n t h i s s o l v e n t h a v e v e r y d i s t i n c t h y d r o x y r e s -O Q o n o n a n c e s ' w h i c h a r e w e l l - r e s o l v e d due t o i n t e r a c t i o n o f t h e s o l v e n t . L i k e l y t h e p e a k was e i t h e r s u p e r i m p o s e d upon a n o t h e r o r t h e i n t e g r a t i o n was n o t s u f f i c i e n t l y s e n s i t i v e t o d e t e c t i t . I t was, h o w e v e r , c l e a r l y v i s i b l e i n t h e CDC1, s p e c t r u m so t h e o t h e r c h e m i c a l s h i f t v a l u e s 45 were i n c l u d e d i n t h i s T a b l e s i m p l y f o r t h e o b s e r v a t i o n o f t h e e f f e c t s o f DMSO-dg on t o t a l c o n f o r m a t i o n ; t h e s e a p p e a r t o be s l i g h t . I n T a b l e I I I , t h e c h e m i c a l s h i f t a s s i g n m e n t s f o r t h e compounds [4] a n d [5] a r e g i v e n . The r e p l a c e m e n t o f t h e f l u o r i n e atom a t t h e a n o m e r i c c a r b o n by. a r b r o m i n e t a t o m - r e s u l t s i n a d o w n f i e l d s h i f t o f t h e r e s o n a n c e . C o m p a r i s o n o f t h e c h e m i c a l s h i f t s o f [4] i n DMSO-dg w i t h t h o s e o f t h e r e f e r e n c e i n C D C l ^ i n d i c a t e s t h a t a ch a n g e i n s o l v e n t h a s o n l y a s l i g h t e f f e c t on t h e s e v a l u e s , a l t h o u g h t h e r e s o n a n c e i s o b s c u r e d by t h o s e o f Hg and Hg,, w h i c h were n o t c l e a r l y r e s o l v e d . S i n c e t h e r e l a t i v e p o s i t i o n s ^ Of a l l t h e c h e m i c a l s h i f t s a r e m a i n t a i n e d I n t h e t r a n s i t i o n f r o m [4] t o [5] and t h e o n l y l a r g e c h a n g e i n s h i f t i s f o r H^, one c a n c o n c l u d e t h a t t h e o v e r a l l s t r u c t u r e o f t h e m o l e c u l e has b e e n m a i n t a i n e d , e x c e p t f o r t h e s u b s t i t u e n t a t C - l . I n T a b l e I V , t h e c h e m i c a l s h i f t a s s i g n m e n t s f o r compounds [7] and [8] a r e shown and b a s i c a l l y t h e same c o n c l u s i o n s c a n be drawn f r o m t h i s d a t a as f o r t h o s e s t a t e d a b o v e r e g a r d i n g T a b l e I I I . The o n l y r e a l d i f f e r e n c e l i e s i n t h e H 2 r e s o n a n c e s . I n T a b l e I I I , t h e s e r e s o n a n c e s s h i f t t o a l a r g e r d e g r e e when one c o n v e r t s t h e C - l f l u o r i n e t o a b r o m i n e a n d t h e s h i f t i s u p f i e l d r a t h e r t h a n d o w n f i e l d ( a s s e e n f o r H^) a l t h o u g h i t i s n o t l a r g e e nough t o s i g n i f y a l a r g e c o n f o r m a t i o n a l c h a n g e . I n T a b l e I V , t h i s t y p e o f s h i f t i n i s n o t r e a l l y n o t i c e a b l e . T a b l e s V and V I c o n t a i n t h e c h e m i c a l s h i f t v a l u e s f o r t h o s e p r o t o n s i n t h e d i s a c c h a r i d e s w h i c h b e l o n g t o t h e s u g a r TABLE I I I CHEMICAL SHIFT ASSIGNMENTS FOR THE 2-DE0XY-2-FLU0R0 MONOSACCHARIDE [4] AND THE ANALOGOUS BROMINATED DERIVATIVE [5] & [4] [5] MHz COMPOUND OAc E1 H 2 H^ H^ H 5 H gexo Hg.endo SOLVENT 270 • [4] (6)7.90 4.21 5-23 4.84 5.11 6.05 —5.94— (3)7.87 DMSO-cV 100 [5] (9)8.02 OBSC 5.45 4.62 5-18 8.00 —-6: . .22 - to -5 .48— 100 [4J19- (4)8.03 4.22 5.28 4.70 4.96 (5)7.99 —6.06-to-5.68— CDC1 3 100 [5] (9)8.00 3.54 5-50 4.40 4.92 7-96 —6.02-to-5.62 a. The chemical s h i f t s are given i n T and are r e l a t i v e t o e x t e r n a l TMS, OBSC = resonance obscured by other peaks. Maximum e r r o r i n measurement i s ± 0.05 T ON TABLE IV CHEMICAL SHIFT ASSIGNMENTS FOR THE 2-DEOXY-2-FLUORO MONOSACCHARIDE [7] AND THE ANALOGOUS BROMTNATED DERIVATIVE.[8] a MHZ COMPOUND OAC ^ H 2 H 3 H^ Hgexo Hg.endo SOLVENT 270 [7] (6)8.04 3-92 5-12 4.97 4.58 5-55 —6.03-to - 5 . 94— DMSO-dg (3)7-92 100 [ 7 ] " 2 0 (6)8.02 4.21 5-28 4.64 4.50 5-64 _ 5 . 9 8 - t c ~ 5 . 9 i — CDC1 (3)7.92 6 100 [8] (6)8.00 3-44 5-30 4.58 4.53 5-56 —5.94-to -5 .87— " (3)7-91 a. The chemical s h i f t s are given i n x and are r e l a t i v e to e x t e r n a l TMS. Maximum e r r o r i n measure-ment i s ± 0.05 x TABLE V CHEMICAL SHIFT ASSIGNMENTS FOR THE DISACCHARIDES [12], [13], AND [14] a [12] [133 [14] MHZ COMPOUND OAc H ^ H~2, H ^ H^, H , H g, 6 , t SOLVENT E± 100 [12] (9)8.00 4.68 5-52 4.42 5.02 -5.91-to-5.59- CDCl, (6)7.93 5 100 [13] (21)7-96 4.69 5-47 4.49 4.96 t o 7.70 OBSC 3.68 270 [14] 5.02 5-90 5-44 5.70 -OBSC DMSO-cL The chemical s h i f t s are gi v e n i n x and are r e l a t i v e t o e x t e r n a l TMS. Maximum e r r o r i n measure-ment i s ± 0.05 x TABLE VT CHEMICAL SHIFT ASSIGNMENTS FOR THE DISACCHARIDES [15lyjAND [ l 6 ] a COMPOUND OAc [15] (6)8.02 4.63 5.28 4.57 4.53 5.55 5-9.6 CDC1 3 (.9)7.92 [16] (21)7-99 4.74 5-27 4.60 4.51 OBSC 11 3-73 The chemical s h i f t s are given i n x and are r e l a t i v e to e x t e r n a l TMS. Maximum e r r o r i n measure-ment i s ±"0.05 x. A l l s p e c t r a were r un a t 100 MHz. 50 m o i e t y c o r r e s p o n d i n g t o t h e a n a l o g o u s h a l o s u g a r f r o m w h i c h t h e y were d e r i v e d . S i n c e t h e m a j o r theme o f t h i s w ork was t o l o o k a t t h e f l u o r i n e NMR, t h e a n a l y s i s o f t h e s u g a r s p e c t r a f o r t h e s e l a t t e r compounds was c o n c e n t r a t e d on 19 t h e s u g a r m o i e t y c o n t a i n i n g t h e f l u o r i n e atom w i t h F d e c o u p l i n g e x p e r i m e n t s u s e d t o a i d i n t h e a s s i g n m e n t o f t h e r e s o n a n c e v a l u e s f o r t h e p r o t o n s i n t h i s r i n g ; t h e r e s u l t s o f t h e s e e x p e r i m e n t s a r e shown I n F i g u r e s 5 3 6, 9, and 10. I n f a c t , none o f t h e 1 H s h i f t a s s i g n m e n t s were v e r i f i e d by p r o t o n d e c o u p l i n g e x p e r i m e n t s as t h e f o c u s 19 r e m a i n e d on t h e u s e o f t h e F NMR i n t h e 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 , w i t h t h e "^"H NMR s e r v i n g as a c o n f i r m a t i o n o f t h e s e i n t e r p r e t a t i o n s . F o r [ 1 4 ] , t h e a s s i g n m e n t o f c h e m i c a l s h i f t s i n DMSO-dg was made on t h e b a s i s o f e x p e c t e d s p l i t -t i n g p a t t e r n s o n l y , as o u t l i n e d i n F i g u r e 1. T h i s was n e c e s s a r y as t h e r e s o l u t i o n o f t h e 100 MHz s p e c t r u m was n o t 19 good enough f o r F d e c o u p l i n g e x p e r i m e n t t o g i v e any i n t e r p r e t a b l e i n f o r m a t i o n . F i g u r e 8 d i s p l a y s t h e s p e c t r u m o f [14] and m a l t o s e . S h i f t s a r e r e l a t i v e t o DSS* and t h e r e -f o r e were n o t r e p o r t e d . The s p e c t r a show some s i m i l a r i t y ; ' t h e a/3 r a t i o f o r t h e a n o m e r i c p r o t o n was 2/3 i n b o t h c a s e s . The t r a n s i t i o n f r o m [12] t o [13] ( T a b l e V) o r [14] t o [15] ( T a b l e V I ) seems t o ha v e l i t t l e e f f e c t on t h e s h i f t v a l u e s f o r t h i s p o r t i o n o f t h e s u g a r . The r e s o n a n c e s H ,-i * 2 , 2 - d i m e t h y l - 2 - s i l a p e n t a n e - 5 - s u l f o n i c a c i d 1-1 FIGURE 5- P a r t i a l 1H NMR s p e c t r a (100 MHz) o f [12] A. Normal spectrum B. Spectrum w i t h simultaneous i r r a d i a t i o n a t the F resonance Frequency (56000 Hz) Spectra were recorded i n CDC1 0 w i t h e x t e r n a l TKS as the r e f e r e n c e . 52 FIGURE 6. P a r t i a l H NMR s p e c t r a (100 MHz) o f [13]. A. Normal spectrum B. Spectrum w i t h simultaneous I r r a d i a t i o n at the F resonance frequency (59501 Hz) Spectra were recorded .in CDC1-, with e x t e r n a l TM3 as the r e f e r e n c e . FIGURE 7. H NMR s p e c t r u m (270 MHz) o f [ 1 4 ] . S p e c t r u m was r e c o r d e d i n DMSO-dg w i t h e x t e r n a l TMS as t h e r e f e r e n c e , a and 3 r e f e r t o t h e a n o m e r i c H r e s o n a n c e s . V a l u e s i n d i c a t e d f o r c h e m i c a l s h i f t a r e i n ppm. oo 54 I F i g u r e 8. H NMR s p e c t r a i n DgO a t 270 MHZ. a and 3 r e f e r t o t h e a n o m e r i c p r o t o n . A. Compound [14] B. M a l t o s e 55 FIGURE 9- P a r t i a l NMR s p e c t r a (100 MHz) o f [15]. A. Normal spectrum B. Spectrum w i t h simultaneous i r r a d i a t i o n at the F resonance frequency (62501 Hz) S p e c t r a were recorded i n CDCl.^ w i t h e x t e r n a l IMS as a r e f e r e n c e . FIGURE 10. P a r t i a l 1H NMR s p e c t r a (100 MHz) o f [ 16 ] . A. Normal spectrum B. Spectrum w i t h simultaneous i r r a d i a t i o n a t the F resonance frequency (59501 Hz). Spectra were recorded i n CDC1, w i t h e x t e r n a l TMS as a r e f e r e n c e . 57 and Hg.. g, become o b s c u r e d when t h e a n h y d r o - l i n k a g e i s b r o k e n as t h e r e s o n a n c e s o f t h e p r o t o n s i n o t h e r r i n g s h i f t d o w n f i e l d . C o m p a r i s o n o f t h e e n v i r o n m e n t s o f any one p r o t o n p o s i t i o n b e t w e e n t h e two r i n g s i n [12] and [13] o r [14] a nd [15] i n F i g u r e 14 w i l l i n d i c a t e why t h i s d o w n f i e l d s h i f t i s o b s e r v e d . F o r e x a m p l e , compare t h e c o n f i g u r a t i o n o f w i t h H~3, i n [12] and [13]. I n [12] H^, i s a x i a l and i s e q u a t o r i a l , w h i l e i n [13], b o t h p r o t o n s a r e a x i a l . The same i s t r u e f o r H 1 , E^s and H,- when one compares them w i t h H^,, H 2,, , and H,_, r e s p e c t i v e l y b e t w e e n t h e two compounds. I t i s now e v i d e n t why t h e i n t e r p r e t a t i o n o f t h e "^"H s p e c t r a becomes so d i f f i c u l t a f t e r t h e l o s s o f t h e a n h y d r o - l i n k a g e due t o t h e s i m i l a r i t y o f t h e e n v i r o n m e n t s o f r e s p e c t i v e p r o t o n s b e t w e e n t h e two r i n g s . 58. F l u o r i n e d e c o u p l i n g e x p e r i m e n t s were t h e o n l y d e f i n i t e means o f a s s i g n i n g some o f t h e c h e m i c a l s h i f t v a l u e s shown f o r t h e d i s a c c h a r i d e s , a s i d e f r o m t h e a n o m e r i c p r o t o n (H^) w h i c h was e a s i l y v i s i b l e i n t h e p e r - O - a c e t y l a t e d s u g a r s as t h e r e s o n a n c e a t l o w e s t f i e l d . The d e c o u p l e d a n d n o r m a l s p e c t r u m o f t h e d i s a c c h a r i d e s [13] and [15] o u t l i n e d a r e g i v e n i n F i g u r e 9 and F i g u r e 10. L o o k i n g a t t h e s e s p e c t r a , 19 i t i s e a s y t o u n d e r s t a n d why t h e u s e o f F NMR i s d e s i r e a b l e due t o t h e c o m p l e x i t y o f t h e "*"H r e s u l t s . The c h e m i c a l s h i f t s b e t w e e n [13] t o [14] as o u t l i n e d i n T a b l e V a p p e a r t o a l t e r d r a s t i c a l l y b u t t h i s i s l i k e l y due t o t h e u s e o f DMS0-d ( as a s o l v e n t f o r t h e l a t t e r s u g a r ; h y d r o x y l a t e d m o l e c u l e s a r e u s u a l l y s t r o n g l y a s s o c i a t e d t o DMSO t h r o u g h h y d r o g e n b o n d i n g and t h e i r c o n f o r m a t i o n and r e a c t i v i t y c a n be O Q o n a f f e c t e d by t h e s o l v e n t - s o l u t e i n t e r a c t i o n . ' I n p a r t i c u l a r , t h i s h o l d s f o r f l e x i b l e m o l e c u l e s , w h i c h c a n a d a p t t h e i r s h a p e t o g i v e t h e s t r o n g e s t a s s o c i a t i o n w i t h t h e s o l v e n t , b a l a n c i n g t h e i n t r a m o l e c u l a r and i n t e r -o D m o l e c u l a r H - b o n d i n g e n e r g i e s . I n T a b l e s V I I and V I I I , t h e c o u p l i n g c o n s t a n t s as d e t e r m i n e d f r o m t h e "'"H NMR d e c o u p l i n g e x p e r i m e n t s a r e shown. I n T a b l e V I I one c a n v i e w t h e d i f f e r e n c e i n c o u p l i n g c o n s t a n t s b e t w e e n t h e m o n o s a c c h a r i d e s w h i c h a r e i n t h e a c o n f o r m a t i o n ( [4] , [ 5 ] , [7]> [ 8 ]) as o p p o s e d t o t h o s e w h i c h a r e i n t h e 3 c o n f o r m a t i o n ( [ 1 8 ] , [ X ] b ) . The c o u p l i n g c o n s t a n t s f o r t h e a s u g a r s a r e v e r y s i m i l a r , a g a i n s u g g e s t i n g t h a t no l a r g e c h a n g e i n t h e o v e r a l l c o n f i g u r a t i o n o f t h e COMPOUND HpH 2 H^ ,F-2.9 53.0 19 c 2.9 53.3 [5] n.o — [18] 7.5 — 6.1 51.7 [T] 2.75 53.3 2.7 52.5 L8J 4.0 — ^ 2 H 2,F 1 HgjFj 0.0 24.0 48.0 9-8 0.0 23.8 48.3 9-6 0.0 — 48.4 9.6 3-0 — 51-0 9.0 4.0 11.2 49 ..0 7.9 0.0 23.0 48.5 9-9 0.5 23.0 48.0 9-7 0.0 — 49.0 10.5 d H 3' F2 Hl)>F2 9-5 12.3 0.0 9.5 9.5 12.3 0.0 9.5 9.6 11.6 0.0 9.6 9-5' 14.8 0.0 9-5 8.9 15.0 0.0 9.0 3.4 11.5 3.5 1.25 3.2 11.0 3-5 1.0 3 . " 11.2 3.5 1.15 a. Coupling constants values are given in Hz. A l l spectra were recorded in CDC1-, at 100 MHz with TMS as the external reference. Maximum error in measurement is ± 0.5 Hz. b. Reference compound which was not synthesized in this study; values are taken from the reference indicated. Spectrum was recorded at 100 MHz in CDClv-CFCl,-TMS. c. Reference values given in the literature indicated. "The spectra reported in 19 was recorded under the same conditions as outlined in b. The spectrum reported in 20 refers to a solution in CDC1, with TMS as a reference. d. An average value derived from the splittings measured at H 0 and H.,. TABLE V I I I COUPLING CONSTANTS AS DETERMINED PTOM THE hi NMR SPECTRA FOR THE DISACCHARIDES [12]. [13], [14], [15], AND [ l 6 j a IS COMPOUND H 2,,P H 3,,F H V , F [12] 3-9 0.0 19-0 9.5 9.5 11.8 0.0 9.8 — [13] 3.8 0.0 19.5 9-6 : 9.5 11.5 0.0 9.5 3.8 [Iii] b 3-8 0.0 19.1 9.5 9.5 13.2 0.0 9-5 — [I'f] c 1.0 — 19.0 9-6 ^— — — — [15] 1.0 0.0 19-5 10.5 d 3.1 11.8 3.5 1.13d [16] 1.0 0.0 18.5 9.5 3-5 11.8 3.5 1.20 3-7 a. Coupling constant values are given in Hz. All spectra were recorded in CDC1, at 100 MHz unless otherwise stated. Maximum error in measurements is ± 0.5 Hz. b. Spectrum recorded in DMSO-dg at 270 MHz. c. Spectrum recorded in DjO at 270 fflz, d. An average value derived from the splittings measured at H 0 and H... 61 s u g a r o c c u r s w i t h r e s p e c t t o H^, H^, o r Hj- r e l a t i v e t o t h e r e s t o f t h e m o l e c u l e . The v a l u e s f o r [ 4 ] c and [ 7 ] C were i n c l u d e d t o show t h e s i m i l a r i t y b e t w e e n t h e c o u p l i n g c o n -19 20 s t a n t s m e a s u r e d i n t h i s work t o t h o s e o f o t h e r w o r k e r s . ' T h i s i s i m p o r t a n t as t h e a s s i g n m e n t o f c o n f o r m a t i o n was b a s e d on t h e s e s i m i l a r i t i e s ; [ X ] b was n o t s y n t h e s i z e d i n t h i s s t u d y b u t was i n c l u d e d f o r t h e same r e a s o n s . T a b l e V I I I p r e s e n t s t h e c o u p l i n g c o n s t a n t s d e t e r m i n e d f o r t h e d i s a c c h a r i d e s a n d t h e s e compare f a v o u r a b l y w i t h t h o s e o f t h e a n a l o g o u s m o n o s a c c h a r i d e s i n T a b l e V I I w h i c h a r e i n t h e a c o n f o r m a t i o n . The s i g n i f i c a n c e o f t h e m a g n i t u d e o f t h e s e c o u p l i n g s w i l l be d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n . The s p e c t r u m f o r [14] i n D 20 was p a r t i a l l y o b s c u r e d by H 2 0 p e a k s b u t , f o r t h e t h r e e c o u p l i n g s g i v e n i n t h e T a b l e , one c a n s e e t h a t t h e c h a n g e o f s o l v e n t s l i k e l y d o e s n o t a f f e c t t h e s e v a l u e s t o any g r e a t e x t e n t , as expected. T a b l e s I X and X c o n t a i n t h e c o u p l i n g c o n s t a n t s d e t -19 e r m i n e d f r o m t h e F NMR s p e c t r a and t h e y compare f a v o u r a b l y w i t h t h o s e d e t e r m i n e d f r o m t h e c o r r e s p o n d i n g """H NMR. A g a i n , t h e e x p e c t e d d i f f e r e n c e s i n c o u p l i n g c o n s t a n t s f o r t h e a - g a l a c t o s i d e s [15] and [16] a r i s e f r o m t h e p r e s e n c e o f a g a l a c t o s e 2 - d e o x y - 2 - f l u o r o m o i e t y r a t h e r t h a n t h e c o r r e s p o n d i n g g l u c o s e s u g a r p r e s e n t i n [12] , 1 1 3 ] } and [14]. B e c a u s e t h e c o u p l i n g c o n s t a n t s do n o t c h a n g e r a d i c a l l y a s t h e s u b s t i t u e n t s a r o u n d t h e s u g a r s c h a n g e , i t a p p e a r s t h a t t h e o v e r a l l c o n -f o r m a t i o n i s r e t a i n e d t h r o u g h o u t t h e s y n t h e s i s . The v a l u e s shown were a v e r a g e d as t h e s p e c t r a t h e m s e l v e s were n o t c o m p l e t e l y s y m m e t r i c a l . T h i s a s y m m e t r y a r i s e s f r o m TABLE IX COUPLING CONSTANTS AS DETERMINED FROM THE 1 9 F NMR SPECTRA FOR THE MONOSACCHARIDES [4] , [5], [7], [8], AND [ l 8 ] a COMPOUND F ppm F F F V * 2 F r H i F 1 H 2 ^ 1 F 2 H 2 ^ 3 P 2 H 4 SOLVENT Pl r2 150 .7 203.8 19-3 53.1 23.8 0.0 47.3 12.5 DMSO- dg [5] — 188.4 — — — 0.0 49.0 11.3 it [7] 151 .6 209.6 18.6 53.7 23.1 0.0 47-9 12.3 3.8 it [8] — 193-3 — — — 0.0 48.2 10.9 3.6 n [7] 152, .5 211.2 18.5 52.9 22.8 0.0 48.6 12.1 3.8 CDCl^ [8] — 195-3 — — — 0.0 48.6 9.9 3.4 3 n [18] — 195.8 — — — U.R. 51.8 14.8 II a. Coupling constant values are given i n Hz and ppm values are a l l u p f i e l d o f CFC1 A l l s n e r f r a were recorded a t 94.1 MHz. 3 ' t , p e L t i a ro TABLE X COUPLING CONSTANTS AS DETERMINED FROM THE 1 9 F SPECTRA FOR THE DISACCHARIDES [12], [13], [1*0, [15], AND [16]' COMPOUND ppm F,H l t F,Hp, F,H 3 ? [12] [13] [14] [15] [16] 198.c£ 198. r 200.2 199-5 198.2 198.4 206.1 205.5 0.0 0.0 0.0 0.0 0.0 48.9 48.4 49.2 47.5 48.6 12.0 11.6 13.1 11.8 11.5 [12] [13] [15] [16] 200.9 195.5 208.0 207-1 0.0 0.0 0.0 0.0 48.8 48.8 48.8 49.8 11.5 11.4 9.3 9-9 [14] 197.?* 197.3 197.0 197.1 - « — — « . F,H 4, SOLVENT DMSO-dg 2.4 2.3 2.8 2.4 t ! II CDClo i t ~> i t t i D 20 a. Coupling constant values are gi v e n i n Hz and ppm values are a l l u p f i e l d o f CFC1 were recorded a t 94.1 MHz. Maximum e r r o r i n ppm values i s ca. ± 1.0 Hz. b. ppm values measured from the s p e c t r a which were not H decoupled. 64 t h e f a c t t h a t t h i s i s an AXY s y s t e m where A ( F ) , X ( H ^ ) , and Y(H^) d i s p l a y l a r g e d i f f e r e n c e s i n c h e m i c a l s h i f t s ; t h e c o u p l i n g c o n s t a n t s a r e t h e r e f o r e s u b j e c t t o s e c o n d o r d e r e f f e c t s . T h i s l a c k o f symmetry was n o t , h o w e v e r , so g r e a t t h a t t h e s p e c t r a c o u l d n o t be j u s t i f i a b l y i n t e r p r e t e d as f i r s t o r d e r . SUMMARY I n g e n e r a l t h e n , a c h a n g e i n s o l v e n t seems t o a f f e c t o n l y t h e c h e m i c a l s h i f t s t o any a p p r e c i a b l e e x t e n t , t h e c o u p l i n g c o n s t a n t s a r e n o t a f f e c t e d a nd t h e r e l a t i v e p o s i t i o n s o f t h e c h e m i c a l s h i f t s a r e m a i n t a i n e d . I t a p p e a r s t h a t a l l o f t h e d i s a c c h a r i d e s s y n t h e s i z e d i n t h i s s t u d y a r e a - l i n k e d a n d t h i s d e d u c t i o n w i l l be o u t l i n e d i n g r e a t e r d e p t h i n t h e f o l l o w i n g s e c t i o n . 65 I V INTERPRETATION OF THE NMR DATA The c h e m i c a l s h i f t s a n d c o u p l i n g c o n s t a n t s o f c a r b o -h y d r a t e s and t h e i r d e r i v a t i v e s h ave g e n e r a l l y b e e n u s e d t o d e t e r m i n e c o n s t i t u t i o n a l , c o n f i g u r a t i o n a l , a nd c o n f o r m a t i o n a l 40 p r o p e r t i e s . As a g e n e r a l r u l e i n p y r a n o i d r i n g s , e q u a t o r i a l p r o t o n s r e s o n a t e a t l o w e r f i e l d t h a n a x i a l p r o t o n s ( i n t h e a b s e n c e o f e l e c t r o n e g a t i v e s u b s t i t u e n t s ) and t h i s was, i n f a c t , o b s e r v e d t o h o l d t r u e i n t h i s w o r k . One c l e a r e x a m p l e o f t h i s c a n be i l l u s t r a t e d by t h e i n f o r m a t i o n b e l o w w h i c h was t a k e n f r o m T a b l e I I I and T a b l e I V . I n c o n t r a s t , OAC [4] [71 H^ = 4.96 x H^ = 4.50 T u s u a l l y t h e p r o t o n s a s s o c i a t e d w i t h t h e m e t h y l g r o u p s o f a x i a l a c e t o x y g r o u p s g i v e r i s e t o s i g n a l s a t l o w e r f i e l d t h a n t h e c o r r e s p o n d i n g p r o t o n s i n e q u a t o r i a l a c e t o x y g r o u p s , a l t h o u g h t h i s d i f f e r e n t i a t i o n i s n o t r e g a r d e d as s u f f i c i e n t l y r e l i a b l e t o s e r v e as a b a s i s f o r m a k i n g c o n f i g u r a t i o n a l 40 a s s i g n m e n t s . " I n t h e p r e v i o u s t a b l e s , t h e o n l y c a s e s where b o t h t y p e s o f OAc g r o u p s w ere p r e s e n t a r e [12] ( T a b l e V) and 66 [15] ( T a b l e V I ) . The a s s u m p t i o n made abo v e a p p e a r s t o h o l d t r u e i n both cases. I t i s a l s o a f a i r l y g e n e r a l r u l e ( i n t h e a b s e n c e o f e l e c t r o n e g a t i v e s u b s t i t u e n t s ) t h a t a n o m e r i c p r o t o n s g i v e r i s e 41 t o s i g n a l s a t l o w e r f i e l d t h a n do t h e o t h e r r i n g p r o t o n s ; t h i s a r i s e s f r o m t h e f a c t t h a t t h e a n o m e r i c c a r b o n a t om i s b o n d e d t o two o x y g e n a t o m s , t h u s c a u s i n g , i t t o be somewhat more d e s h i e l d e d t h a n t h e o t h e r r i n g p r o t o n s . T h e s e r e s o n a n c e s were c l e a r l y d i s t i n g u i s h a b l e f r o m t h e o t h e r r e s o n a n c e s i n t h i s s t u d y , as i n d i c a t e d by t h e a n o m e r i c and , c h e m i c a l s h i f t v a l u e s i n T a b l e s I t o V I . 19 The c h e m i c a l s h i f t s o f t h e f l u o r i n e n u c l e i i n t h e F NMR s p e c t r a o f h e x o p y r a n o s y l f l u o r i d e d e r i v a t i v e s a r e a l s o known t o be s u b j e c t t o some v a r i a t i o n t h a t d e p e n d s upon t h e c o n f i g u r a t i o n a t t h e o t h e r c h i r a l c a r b o n atoms a r o u n d t h e 19 42 p y r a n o s i d e r i n g . 5 I f one c o m p a r e s t h e c h e m i c a l s h i f t v a l u e s o f t h e f l u o r i n e a t o m a t t a c h e d t o t h e m a l t o s e d e r -i v a t i v e s [ 1 2 ] , [13]> and [14] w i t h t h o s e o f t h e c o r r e s p o n d i n g a - g a l a c t o s i d e a n a l o g u e s [15] and [16] g i v e n i n T a b l e X, a s l i g h t s h i f t u p f i e l d i n t h e l a t t e r c a s e c a n be o b s e r v e d ; t h i s c o u l d be t h e r e s u l t o f t h e d i f f e r e n c e i n c o n f o r m a t i o n a t C-4. The m a g n i t u d e o f t h e c o u p l i n g c o n s t a n t b e t w e e n h y d r o g e n atoms on a d j a c e n t c a r b o n atoms o f t e n y i e l d s v a l u a b l e i n -f o r m a t i o n a b o u t t h e c o n f o r m a t i o n a l and c o n f i g u r a t i o n a l p r o p e r t i e s o f c a r b o h y d r a t e s . I n t h e s i m p l e s y s t e m - -C„ - H„, t h e p r i m a r y f a c t o r w h i c h i n f l u e n c e s t h e m a g n i t u d e 67 o f J„ „ i s t h e s i z e o f t h e t o r s i o n a l a n g l e <j> a s s o c i a t e d H, ,H Q 4 l w i t h t h e s e two p r o t o n s . T h i s a n g u l a r d e p e n d e n c e f i n d s q u a n t i t a t i v e e x p r e s s i o n i n t h e w e l l - k n o w n K a r p l u s r e l a t i o n -44 43 s h i p shown b e l o w . J J.„ „ = A cos2cf> - C f o r 0 < c|) < 9'0° J n „ = B cos 2cj> - C f o r 90 ^ <j> ^ 180° H l j H 2 where A, B, and C a r e c o n s t a n t s . The o r i g i n a l c a l c u l a t i o n f o r t h e " e t h a n i c " t y p e f r a g m e n t i n d i c a t e d e a r l i e r y i e l d e d v a l u e s 44 f o r A, B, and C o f 8 .5 , 9 -5 , and 0.3 Hz r e s p e c t i v e l y . S u b s t i t u t i o n o f t h e s e v a l u e s i n t o t h e e q u a t i o n s a b o v e a l l o w s one t o p r o d u c e t h e w e l l - k n o w n " K a r p l u s C u r v e " w h i c h i s shown 43 b e l o w . A plot of the Karplus r e l a t i o n s h i p between J„ „ and <J>. H1'H2 68 I n a c c o r d a n c e w i t h t h i s g r a p h , p r o t o n s w h i c h a r e a n t i - p e r i p l a n a r (tt = l 8 0 ° ) on a p y r a n o s e r i n g e x h i b i t a l a r g e v i c i n a l c o u p l i n g c o n s t a n t o f 7 - 10 Hz, w h e r e a s p r o t o n s w h i c h a r e s y n - c l i n a l (a) = 60°) u s u a l l y e x h i b i t a s m a l l v a l u e o f 1 - 4 Hz. I n a l l o f t h e s p e c t r a d e t e r m i n e d i n t h i s w o r k , t h e v a l u e s f o r e a c h t y p e o f c o u p l i n g f e l l w i t h i n t h i s r a n g e , some e x a m p l e s o f w h i c h a r e shown b e l o w ; t h e c o r r e s p o n d i n g s t r u c t u r e s a r e g i v e n on page 69-COMPOUND COUPLING CONFORMATION J (Hz) [ 5] H-j^H^ 60 °" e,a*'* 4 . 0 § H ^ H ^ ; H ^ j H ^ ; H ^ , H C J 180° a,a 9.5-9 .8 [ 7] H-j^  ,H 2 60° e ,a 2 . 7 5 * H ^ H ^ 180° a,a 9.9 •60° a,e 3 .4 H 4 JH r- 60° e ,a 1 . 2 4 * [12] 60° e ,a § 3 - 9 ^2? J H ^ I j H ^ , ,H|j, ;H|j, ,Hr- 180° Si 3 3. 9-5-9 .8 [18] H 1 ' H 2 180° a,a 7.5 H 2 , H 3 ; H ^ H ^ ; Hi|,Hrr_ 180° 3, j 8, 9.0-9 .5 ** e = e q u a t o r i a l , a = a x i a l , e,a -- t h e r e s p e c t i v e H o r i e n t -a t i o n i s e q u a t o r i a l , a x i a l The d a t a s e r v e t o p o i n t o u t some i n t e r e s t i n g f a c e t s a b o u t c o u p l i n g c o n s t a n t s I n c a r b o h y d r a t e s . I f one t a k e s a g a i n t h e s i m p l e " e t h a n i c " l i n k a g e H^ - C^ - C 2 - H 2 and t h e n adds an e l e c t r o n e g a t i v e s u b s t i t u e n t t o one o f t h e c a r b o n s t h e n t h e m a g n i t u d e o f t h e v i c i n a l c o u p l i n g c o n s t a n t f o r a 69 70 p a r t i c u l a r t o r s i o n a l a n g l e i s n o t as l a r g e as p r e d i c t e d by 45 46 47 t h e K a r p l u s r e l a t i o n s h i p . 5 ' The e l e c t r o n e g a t i v e s u b s t i t u e n t e x e r t s i t s maximum e f f e c t on t h e v i c i n a l c o u p l i n g c o n s t a n t when i t i s a n t i - p e r i p l a n a r t o one o f t h e two p r o t o n s 48 49 i n v o l v e d i n t h e c o u p l i n g . 5 F o r p y r a n o s e s w i t h an a x i a l p r o t o n on C - l and an e q u a t o r i a l p r o t o n on C - 2 , t h e c o u p l i n g c o n s t a n t i s u s u a l l y w i t h i n t h e r a n g e o f 1.0 t o 1.5 Hz; t h i s c a n be c o r r e l a t e d t o t h e r e s u l t i n d i c a t e d a bove (*) by l e t t i n g C - l = C - 5 and C - 2 = C - 4 i n t h e s i m p l e s y s t e m as shown b e l o w f o r [ 7 ] . I n t h e s c h e m a t i c v i e w b e l o w , one c a n s e e t h a t H^ and t h e r i n g o x y g e n a r e a n t i - p e r i p l a n a r . F o r p y r a n o s e s w i t h an e q u a t o r i a l p r o t o n on C - l and an a x i a l p r o t o n on C - 2 , t h e c o u p l i n g c o n s t a n t i s u s u a l l y w i t h i n t h e r a n g e o f 2 . 5 t o 3.5 Hz; t h i s c a n be a c c o u n t e d f o r i n t h e d a t a g i v e n a b o v e (*) where H^ and F^ a r e a n t i - p e r i p l a n a r . The c o r r e s p o n d i n g v a l u e s i n d i c a t e d (§) a r e somewhat l a r g e r as t h e y a r e a n t i -p e r i p l a n a r t o t h e r i n g o x y g e n i n t h e g l y c o s i d i c l i n k , r a t h e r t h a n t o t h e f l u o r i n e . A more a p p r o p r i a t e f o r m o f t h e K a r p l u s e q u a t i o n h a s b e e n d e d u c e d w h i c h t a k e s i n t o a c c o u n t t h e 5 0 p r e s e n c e o f an e l e c t r o n e g a t i v e s u b s t i t u e n t b u t i t . g i v e s 49 r e s u l t s so s i m i l a r n u m e r i c a l l y t h a t , i n t h i s s t u d y , i t s a p p l i c a t i o n was n o t r e a l l y j u s t i f i e d , e s p e c i a l l y i n v i e w o f t h e u n c e r t a i n t i e s a s s o c i a t e d w i t h e l e c t r o n e g a t i v e s u b s t i t u e n t s . * In case [5], one of the protons is antiperiplanar to a brcmine atom, rather than to the ring oxygen. 71 A l s o , t h e r e l a t i v e m a g n i t u d e s o f t h e c o u p l i n g c o n s t a n t s o b t a i n e d f r o m t h e s p e c t r a b e t w e e n t h e a - and t h e 3 - l i n k e d s u g a r s were c l e a r l y d i f f e r e n t i a t e d and t h u s t h e configurations o f t h e s p e c i e s c o u l d be e s t a b l i s h e d w i t h o u t t h e u s e o f t h i s m o d i f i c a t i o n o f t h e K a r p l u s e q u a t i o n ; t h e b a s i c f o r m u l a as o u t l i n e d p r e v i o u s l y was a d e q u a t e . A l t h o u g h t h e u s e o f t h e b a s i c f o r m u l a p r o v e d t o be s u f f i c i e n t , i t i s i m p o r t a n t t o r e a l i z e t h a t i t i s now g e n e r a l l y f e l t t h a t t h e a n g u l a r d e p e n d e n c e o f v i c i n a l c o u p l i n g c o n s t a n t s i s c o n s i d e r a b l y more 51 52 53 c o m p l e x t h a n f i r s t a p p r e c i a t e d . . ' J E v e n i n t h e a b s e n c e o f an e l e c t r o n e g a t i v e s u b s t i t u e n t o r an a d j a c e n t C-0 b o n d , t h e J„ c o u p l i n g c o n s t a n t s d i d v a r y ri -,.rl x' y i n t h i s e x p e r i m e n t . L o o k i n g a t many o f t h e v a l u e s r e f e r r i n g t o p r o t o n s w h i c h a r e e a , ; e , e ; o r a,e i n r e l a t i v e o r i e n t a t i o n t h e v a l u e s do d i f f e r , a l t h o u g h t h e d i f f e r e n c e i s s l i g h t . B e c a u s e a l l o f t h e s e e n t i t i e s h ave b e t w e e n them a d i h e d r a l a n g l e o f c a . 60°, one c a n n o t assume t h a t t h e c a r b o n atoms o f c a r b o h y d r a t e d e r i v a t i v e s h a v e p r e c i s e l y t e t r a h e d r a l c o n f o r m a t i o n s . T h i s a s s u m p t i o n i s s u p p o r t e d by t h e f a c t t h a t X - r a y 54 d i f f r a c t i o n s t u d i e s s u g g e s t t h a t t h e p r e c i s e h y b r i d i z a t i o n o f e a c h ccar b a n a t om o f t h e r i n g d e p e n d s on t h e o r i e n t a t i o n o f t h e s u b s t i t u e n t s a t t a c h e d t o t h a t c a r b o n and t h u s o v e r a l l s t e r o c . h e m i s t r y o f t h e i n d i v i d u a l c a r b o n atoms i n t h e p y r a n o s e r i n g i s a f a c t o r i n t h e f i n a l v i c i n a l c o u p l i n g c o n s t a n t s o b s e r v e d . The c o u p l i n g c o n s t a n t s s t i l l p r o v i d e t h e means o f a s s e s s i n g t h e c o n f o r m a t i o n o f t h e s u g a r s s y n t h e s i z e d i n t h i s w o r k . The s m a l l v a l u e s o f J u „ f o r [ 4 ] , [ 5 ] , [ 7 ] , and [8] H 1 ' H 2 72 i n T a b l e V I I i n d i c a t e t h a t t h e c o n f o r m a t i o n a t t h e a n o m e r i c c a r b o n i s a ( f o r t h e s u b s t i t u e n t ) . T h i s v a l u e i n d i c a t e s t h a t t h e d i h e d r a l a n g l e b e t w e e n t h e s e two p r o t o n s i s c l o s e r t o 60° ( 1 - 4 Hz) t h a n t o 1 8 0 ° ( 7 - 9 - 5 H z ) . 1 9 3 2 0 , 4 4 s l m l l a r l V j t h e v a l u e s g i v e n f o r [18] and [X] i n T a b l e V I I i n d i c a t e a 3 c o n f o r m a t i o n , as does t h e f a c t t h a t J u „ i s a l a r g e r v a l u e H 1 ' F 2 t h a n t h e c o r r e s p o n d i n g a c o n f o r m e r s . I n a d d i t i o n , t h e v a l u e s o f J„ _ d i f f e r s i g n i f i c a n t l y b e t w e e n t h e two c o n -f o r m a t i o n s , a l a r g e r v a l u e b e i n g e v i d e n t f o r t h e 3 anomer; some e x a m p l e s a r e l i s t e d b e l o w . COUPLING RANGE CONFORMATION H 1 , H 2 ' H i " H 2 » 2 ' 9 ~ 4 , 0 a H 1 S P 2 ; H l f , P 0.5 a H 1 , H 2 6.1- 7-5 3 H 1 , F 2 3-0- 4.0 3 H 3 , F 2 ; H 3,,F 11.6-12.3 a H 3 , F 2 14.8-15-0 3 T h i s l a t t e r d i f f e r e n c e i s ' i n i m p o r t a n t one as J„ „ i s H 1 ' F 2 a s m a l l v a l u e f o r e i t h e r c o n f o r m a t i o n . The l a r g e v a l u e o f J„ „ makes i t a l s o a l i k e l y c a n d i d a t e f o r m o n i t o r i n g c h a n g e H 3,,F i n c o u p l i n g c o n s t a n t s d u r i n g enzyme b i n d i n g . The v a l u e s o f t h e c o u p l i n g c o n s t a n t s J T T T T , „ , H3 > H 4 H 4 3 H 5 and J„ „ a r e much s m a l l e r i n t h e g a l a c t o s e and c o r -r e s p o n d i n g ( i . e . J„ J„ ) g a l a c t o s i d e d e r i v a t i v e s [ 7 ] , 3 ' ' "14' [ 8 ] , [ 1 5 1 , and [16] ( T a b l e s V I I and V I I I ) as e x p e c t e d due t o t h e c o n f o r m a t i o n o f t h e a c e t y l s u b s t i t u e n t a t C-4. F o r a l l o f t h e s u g a r s d e p i c t e d i n T a b l e s I t o X t h e s p l i t t i n g 73 o f t h e a n o m e r i c p r o t o n i s e s s e n t i a l l y e q u a l t o _ ' J w „ s i n c e 1 ' 2 t h e r e s o n a n c e s a nd H 2 h a v e s i g n i f i c a n t l y d i f f e r e n t c h e m i c a l 52 4 s h i f t s a n d and a r e w e a k l y c o u p l e d . The J c o u p l i n g o f 3-5 Hz b e t w e e n F 2 and H^ f o r t h e g a l a c t o s e d e r i v a t i v e s i s c o n s i s t e n t w i t h t h e .planar-VI.. re/lati©nship...inherent.; i n t h e 20 D - g a l a c t o p y r a n d i d c o n f i g u r a t i o n i n t h e C^ (D) c o n f o r m a t i o n . The s m a l l m a g n i t u d e o f l e s s t h a n 0.5 Hz f o r t h e J,-, „ i i 2 , H - L c o u p l i n g c o n s t a n t r e f l e c t s t h e h i g h t o t a l e l e c t r o n e g a t i v i t y o f t h e s u b s t i t u e n t s a t t a c h e d t o C - l and C-2 a n d , i n p a r t i c u l a r , t h e a n t i - p e r i p l a n a r r e l a t i o n s h i p b e t w e e n t h e F - C 2 , and C ^ - r i n g o x y g e n b o n d s . T h i s i s g e n e r a l l y s e e n i n s u g a r s w i t h t h e D - g l u c o c o n f i g u r a t i o n a t C - l , C-2, and C-3 and h a s b e e n A K 18,20 o b s e r v e d by o t h e r s . ' F o r t h e c a s e o f t h e F nF„ and F H c o u p l i n g c o n s t a n t s , 1 2 x y ^ to ' t h e y r e m a i n e d f a i r l y c o n s t a n t t h r o u g h o u t t h e s t u d y , i n d i c a t i n g a r e t e n t i o n o f c o n f i g u r a t i o n i n t h a t a r e a o f t h e s u g a r t h r o u g h o u t t h e s y n t h e s i s . The s i m i l a r i t y b e t w e e n t h e two s e t s o f c o u p l i n g s b e l o n g i n g t o t h e a c e t y l a t e d [13] and f r e e s u g a r [14] o f t h e m a l t o s e a n a l o g u e i m p l i e s t h a t t h e s u b s t i t u e n t s o f two s p e c i e s h a v e t h e same c o n f i g u r a t i o n a t any one c a r b o n a nd t h u s t h e r e p l a c e m e n t o f t h e OAc g r o u p s w i t h OH g r o u p s has no s i g -n i f i c a n t i n f l u e n c e on t h e m a g n i t u d e o f any a s s o c i a t e d c o u p l i n g ; 47 t h i s t y p e o f o b s e r v a t i o n h a s b e e n n o t e d p r e v i o u s l y . I n some i n s t a n c e s , t h e m a g n i t u d e o f l o n g - r a n g e s p i n - s p i n 4 5 c o u p l i n g s b e t w e e n p r o t o n s s e p a r a t e d by f o u r ( J ) o r f i v e ( J ) s i n g l e b onds may h e l p t o c o n f i r m a c o n f o r m a t i o n a l a s s i g n m e n t . The v a l u e s o f t h e s e c o u p l i n g s h o w e v e r , were t y p i c a l l y q u i t e 74 s m a l l ( J 1 2.0 Hz) and w e r e , f o r t h e most p a r t , b e y o n d t h e s c o p e o f t h i s w o r k ; t h e r e s o l u t i o n i n most c a s e s was n o t good enough t o c o m p l e t e l y v e r i f y s u c h a n a s s i g n m e n t . W i t h a l l o f t h e d a t a a c c u m u l a t e d , t h e s e t y p e s o f v a l u e s were n o t r e a l l y n e c e s s a r y t o a i d i n t h e f i n a l d e t e r m i n a t i o n o f t h e c o n f o r m a t i o n o f t h e s u g a r s s y n t h e s i z e d i n t h i s s t u d y . The a s s i g n m e n t o f an a o r 3 c o n f i g u r a t i o n c a n be s u b -s t a n t i a t e d s i m p l y on t h e b a s i s o f t h e c h e m i c a l s h i f t v a l u e 43 f o r H,. L o o k i n g a t t h e v a l u e s i n d i c a t e d b e l o w f o r t h e o-D-Glucopyr»nos» p-D-Glucoprranojt a c o n f o r m a t i o n , one w o u l d e x p e c t t h e s e s p e c i e s t o have a c h e m i c a l s h i f t o f c a . 5-32 ppm (4.68T), w h i l e f o r t h e 3 c o n f o r m a t i o n , a v a l u e o f 4.74 ppm (5.26T) i s a n t i c i p a t e d . The r a n g e f o r t h e d i s a c c h a r i d e s as g i v e n i n T a b l e s V and V I I was 4.63-5-02T, t h u s i n d i c a t i n g t h a t t h e H^, i s , i n f a c t , e q u a t o r i a l . The C^ -OH i n t h e scheme a b o v e c o r r e l a t e s w e l l w i t h t h o s e i n d i c a t e d i n F i g u r e 7 f o r t h e m a l t o s e d e r i v a t i v e ; t h e o b s e r v e d OH r e s o n a n c e s i n DMSO were a t 6.69 (3) and 6.34 ppm ( a ) . The s m a l l v a l u e o f J„ „ f o r t h e p e r - O - a c e t y l a t e d s u g a r s [13] and [16] i n T a b l e s V and V I I i n d i c a t e s t h a t t h e a c e t y l g r o u p a t t h e a n o m e r i c c a r b o n i s i n an a c o n f o r m a t i o n . The ''"H d e c o u p l i n g e x p e r i m e n t s s e r v e d t o v e r i f y t h e i n i t i a l c h e m i c a l s h i f t a s s i g n m e n t s f o r t h e p r o t o n s o u t l i n e d T a b l e s V I and V I I , as i n d i c a t e d i n t h e s p e c t r a a c c o m p a n y i n g 75 t h e s e T a b l e s . The c o u p l i n g c o n s t a n t s d e r i v e d from t h e s e 19 s p e c t r a were c o n f i r m e d by the F s p e c t r a which are g i v e n i n F i g u r e s 11 t o 16. The l a r g e s p l i t t i n g o f J„ , „ was f o l l o w e d Hp , r by the s p l i t t i n g o f t h i s d o u b l e t by the s m a l l e r c o u p l i n g , p i n t o a d o u b l e t o f d o u b l e t s * . The o n l y case where t h i s was not apparent was f o r [14] , as shown i n F i g u r e 14. Here 19 the i n t e r p r e t a t i o n o f the F spectrum i n d i c a t e d t h a t t h e r e were two f l u o r i n e resonances which were v e r y c l o s e t o g e t h e r ; the "*"H d e c o u p l e d spectrum v e r i f i e d t h i s i n t e r p r e t a t i o n as . shown i n F i g u r e 14. The resonances were e x t r e m e l y c l o s e t o g e t h e r , as i n d i c a t e d i n T a b l e X, thus s u g g e s t i n g t h a t t h e r e were two v e r y s i m i l a r s p e c i e s i n s o l u t i o n . T h i s anomaly w i l l be f u r t h e r e x p l o r e d i n the D i s c u s s i o n . The NMR o f [18] showed the same b a s i c s p l i t t i n g p a t t e r n as f o r the o t h e r s u g a r s , a l t h o u g h the measured c o u p l i n g s d i f f e r e d as o u t l i n e d i n Table I X ; i t s spectrum i s shown i n F i g u r e 16. 19 The F NMR s p e c t r a were a l l c h a r a c t e r i z e d by a l a c k o f symmetry i n the c o u p l i n g c o n s t a n t s between the d o u b l e t p a i r s as w e l l as a l a c k o f o v e r a l l symmetry s i n c e not a l l o f the f o u r resonances o f the d o u b l e t s have the same h e i g h t and w i d t h . T h i s a r i s e s from the f a c t t h a t t h i s i s an AX.Y system and, because the c o u p l i n g c o n s t a n t s here a r e l a r g e r t h a n t h o s e f o r the "*"H NMR s p e c t r a , the second o r d e r e f f e c t s ( o u t l i n e d p r e v i o u s l y ) a re much more a p p a r e n t . I n t e g r a t i o n o f some o f the s p e c t r a i n d i c a t e d t h a t the a r e a encompassed by each d o u b l e t o f the p a i r , as w e l l as f o r each resonance i n any one d o u b l e t , i s the same. * See F i g u r e 16 76 SUMMARY 19 I n g e n e r a l , t h e F NMR s p e c t r a s u b s t a n t i a t e d t h e c o u p l i n g c o n s t a n t s d e r i v e d f r o m t h e "*"H NMR s p e c t r a , a l t h o u g h s e c o n d o r d e r e f f e c t s r e q u i r e d s l i g h t a v e r a g i n g t o be e m p l o y e d . From t h e c o u p l i n g c o n s t a n t s o b s e r v e d , t h e o v e r a l l c o n f o r m a t i o n o f t h e s u g a r s was d e d u c e d ; t h e s e a s s i g n m e n t s w i l l be f u r t h e r s u b s t a n t i a t e d i n t h e D i s c u s s i o n . FIGURE 1 1 . ±yF NMR spectrum ( 9 4 . 1 MHz) o f [ 4 ] . A. resonances o f B. resonances o f F^ The spectrum was r u n i n DMSO-dg and resonances are u p f i e l d o f CFC1 s p e c t r u m was r u n i n DMSO-dg and r e s o n a n c e s a r e u p f i e l d o f CPC1 79 F i g u r e 13 . 1 9 F NMR s p e c t r a o f [ 5 ] , [ 1 2 ] , and [13] 80 FIGURE.14. F NMR s p e c t r a o f [ 1 4 ] , A. N o r m a l s p e c t r a . B. "'"H d e c o u p l e d s p e c t r a . 81 FIGURE 15. F NMR s p e c t r a o f [ 8 ] , [15] and [16] 8 3 V DISCUSSION The s y n t h e s i s o f 1 , 6 - a n h y d r o - B - D - g l u c o s e [ 1 ] shown b e l o w i s w e l l known, b o t h t h r o u g h t h e method e m p l o y e d i n t h i s ^ 6 ^ 7 ^ 8 3 7 5 9 s t u d y JJi'' 3 ^ 1 3 - ^ a n ( j t,y o t h e r r o u t e s . ~" > ^  B e c a u s e a c i d s a r e p r o d u c e d , i t i s l i k e l y t h a t h y d r o l y s i s t o 3 - D - g l u c o s e i s an e s s e n t i a l f a c t o r a n d t h a t t h e s u g a r i s t h e r e a f t e r c o n -" 57 v e r t e d i n t o t h e c o r r e s p o n d i n g a n h y d r i d e ; The i n d i v i d u a l p r o t o n r e s o n a n c e s o f t h i s c o u p o u n d , as o u t l i n e d i n T a b l e I , c o r r e s p o n d w e l l w i t h t h o s e g i v e n i n t h e r e f e r e n c e i n d i c a t e d ; t h i s a g r e e m e n t , a l o n g w i t h t h e o p t i c a l r o t a t i o n measurement and e l e m e n t a l a n a l y s i s , s e r v e d t o c o n f i r m t h e s t r u c t u r e a nd p u r i t y o f t h e compound o b t a i n e d . P a r t i a l a c e t y l a t i o n o f compound [ 1 ] i n p y r i d i n e a nd a c e t i c a n h y d r i d e gave t h e f o u r p r o d u c t s o u t l i n e d b e l o w a l o n g w i t h 84 some m o n o a c e t a t e t h e d e s i r e d compound [2] was s e p a r a t e d on s i l i c a g e l , w i t h the r e l a t i v e e l u t i o n o r d e r i n d i c a t e d above. T h i s compound i s a l s o w e l l known and has been s y n t h e s i z e d by s e v e r a l d i f f e r e n t r o u t e s . 2 9 > 6 0 i t i s w e l l known t h a t the C-4 hydroxy group i n the c o n f o r m a t i o n o f g l y c o p y r a n o s e , a l t h o u g h e q u a t o r i a l l y o r i e n t e d , e x h i b i t s r a t h e r low r e -a c t i v i t y . ^ T h i s d i f f e r e n t i a l r e a c t i v i t y o f the secondary h y d r o x y l groups i s not s o l e l y dependent on c o n f o r m a t i o n but i s a l s o s t e r i c a l l y h i n d e r e d by a d j a c e n t s u b s t i t u e n t s 29 e s p e c i a l l y by the 5 - a c e t y l o x y - m e t h y l group. Thus com-pound [2] shown below was chosen as a means o f o b t a i n i n g a s u i t a b l y s u b s t i t u t e d g l u c o s e d e r i v a t i v e i n which the f r e e C-4 h y d r o x y l group would have enhanced r e a c t i v i t y . 1,6-anhydro-3-D-glucopyranose [1] e x i s t s i n the IC c o n f o r m a t i o n and, a l t h o u g h a l l o f the h y d r o x y l groups a r e a x i a l l y o r i e n t e d , s t e r i c c o n s i d e r a t i o n s i n d i c a t e d t h a t t h o s e i n p o s i t i o n s 2 and 4 would r e a c t p r e f e r e n t i a l l y ( t h e C-3 h y d r o x y l i s the most h i n d e r e d one, owing t o the h e m i a c e t a l and anhydro r i n g s , and to the C-C l i n k a t C - 5 ) • 6 1 E s t e r i f i c a t i o n experiments 6 2> 6 3> 6 4 have shown h i g h y i e l d s o f the 2 , 4 - d i a c y l d e r i v a t i v e s t o sup p o r t t h i s a s s u m p t i o n . W i t h the a d d i t i o n o f s i l v e r t r i f l a t e as a c a t a l y s t , compound [2] proved t o be a s u i t a b l y sub-s t i t u t e d d e r i v a t i v e f o r our purposes f o r the f o l l o w i n g r e a s o n s : 85 (1) i t h a s a s i m p l e ''"H NMR s p e c t r u m , (2) i t i s s t a b l e u n d e r t h e c o n d i t i o n s u s e d i n t h e d i s a c c h a r i d e s y n t h e s i s , (3) i t i s e a s i l y p u r i f i e d , and (4) c o n v e r s i o n t o t h e f r e e s u g a r c a n be o b t a i n e d u n d e r m i l d c o n d i t i o n s . I t h a s , i n f a c t , b e e n u s e d p r e v i o u s l y i n t h e s y n t h e s i s o f l a c t o s e ( I I I ) i n h i g h y i e l d s v i a c o u p l i n g w i t h 2 , 3 , 4 , 6 - t e t r a -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 b r o m i d e ( I ) and s u b s e q u e n t fill -d e a c e t y l a t i o n o f t h e p r o d u c t as i n d i c a t e d b e l o w , and i n t h e s y n t h e s i s o f o t h e r d i s a c c h a r i d e s ( u s i n g p a r t i c i p a t i n g B ,OH g r o u p s on C -2 o f t h e h a l o s u g a r ) s u c h as 4 - 0 - ( 2 - a c e t a m i d o - 2 -29 d e o x y - 3 - D - g l u c o p y r a n o s y l ) - D - g l u c o p y r a n o s e ' shown b e l o w . R = c 6 H 5 C O [2] 4-0-(2-acetamido-2-deoxy-B -D-glucopyranosyl)-D-glucopyranose R = H The h i g h r e s o l u t i o n mass s p e c t r u m a n d c o m p a r i s o n o f t h e 86 s p e c i f i c o p t i c a l r o t a t i o n w i t h t h e l i t e r a t u r e v a l u e c o n f i r m e d t h e s t r u c t u r e ; t h e compound i s an o i l a n d , a l t h o u g h p r e -v i o u s l y s y n t h e s i z e d , h a s n e v e r b e e n c r y s t a l l i z e d . T r i f l u o r o m e t h y l h y p o f l u o r i t e [CF^OF] i s a l s o a known compound ' ' and i t s s y n t h e s i s n e c e s s i t a t e d t h e b u i l d i n g o f a s p e c i a l a p p a r a t u s ( o u t l i n e d p r e v i o u s l y ) as w e l l as e x t r e m e c a r e i n h a n d l i n g b o t h t h e r e a c t a n t s a n d t h e p r o d u c t s . The compound was n o t c o m m e r c i a l l y a v a i l a b l e a t t h e t i m e t h a t t h i s s t u d y was i n i t i a t e d b u t i s now a v a i l a b l e f r o m PCR. I n g e n e r a l , u n d e r m o d e r a t e c o n d i t i o n s , h y p o f l u o r i t e w i l l a dd 68 a c r o s s a C=C b o n d a nd t h e u s e o f t h i s r e a g e n t t o f l u o r i n a t e 19 21 b o t h D - g l u c a l - t r i a c e t a t e a n d [ 3 ] , and D - g a l a c t a l 20 2 2 t r i a c e t a t e [ 6 ] , ' h a s b e e n r e p o r t e d , as w e l l as i t s u s e i n t h e f l u o r i n a t i o n o f o t h e r u n s a t u r a t e d o r g a n i c c o m p o u n d s . ^ 9 ' ^ The r e a c t i o n i s a p p l i c a b l e t o c o m p l e x , s e n s i t i v e s u b s t r a t e s and p r o c e e d s w i t h o u t c o m p l i c a t i o n i n t h e p r e s e n c e o f a v a r i e t y o f f u n c t i o n a l g r o u p s ( e . g . k e t o - , a l k o x y - , and 69 c a e y l o x y - g r o u p s h a v e b e e n shown t o s u r v i v e u n d e r t h e c o n d i t i o n s e m p l o y e d i n t h i s e x p e r i m e n t ) . The n a t u r e o f t h e m i x t u r e o f i s o m e r s o b t a i n e d , as o u t l i n e d b e l o w f o r t h e s y n t h e s i s o f compound [ 4 ] , i s b e s t d e s c r i b e d as r e s u l t i n g f r o m n u c l e o p h i l i c a t t a c k o f t h e TT e l e c t r o n s o f t h e C=C bond C H 2 O A c CH20Ac CH20Ac CjH2 0 A c CHjOAc J—Ov J—0. ) ( X / — ° \ ° C F . / " ^ ° \ T. AcO . . . . 3 F 13] % y i e l d 14.3 17.8 5.82 7.24 87 u pon t h e f l u o r i n e t o a f f o r d a c a t i o n i c i n t e r m e d i a t e w h i c h i s s u s c e p t i b l e t o a t t a c k o f t h e "OCF^ m o i e t y as shown b e l o w , RV \ x _ Y R ^ V £ > ^ R ' c + - 0 - K K 69 The f o r m a t i o n o f t h e d i f l u o r o - a d d u c t s may be e x p l a i n e d by t h e 43 f o l l o w i n g e q u i l i b r i u m . J W i t h s u g a r s , t h i s a d d i t i o n o f t h e CF^O~ t COF 2 + F~ 20 21 71 F and OCP^ f u n c t i o n a l g r o u p s has a l w a y s b e e n c i s , ' '' and g e n e r a l l y t h e s e two f u n c t i o n a l i t i e s a r e a d d e d p r i m a r i l y i n s u c h a way a s t o g i v e t h e p r o d u c t s w i t h t h e D - g l u c o p y r a n o -configuration , as was o b s e r v e d i n t h i s s t u d y ( t h e y i e l d s a r e i n d i c a t e d i n t h e s y n t h e t i c scheme o u t l i n e d on t h e p r e v i o u s p a g e ) . T h i s f u l f i l l s t h e e x p e c t a t i o n t h a t t h e a x i a l l y o r i e n t e d p o r b i t a l o f t h e r i n g o x y g e n atom i s more f a v o u r a b l y d i s p o s e d f o r t h e p a r t i c i p a t i o n i n d e r e a l i z a t i o n o f d e -v e l o p i n g c h a r g e a t t h e a n o m e r i c c e n t r e t h a n i s t h e e q u a t o r i a l l y o r i e n t e d p o r b i t a l a n d a l s o t h e e x p e c t a t i o n t h a t a x i a l a t t a c k a t t h e a n o m e r i c c e n t r e i s more f a v o u r a b l e ( f o r r e a s o n s 72 o f t h e a n o m e r i c e f f e c t by a n e g a t i v e l y c h a r g e d i o n t h a n by a n e u t r a l m o l e c u l e . The a n o m e r i c e f f e c t d e s c r i b e s t h e i n t e r a c t i o n b e t w e e n an e l e c t r o n e g a t i v e s u b s t i t u e n t on t h e a n o m e r i c c a r b o n a tom and t h e r i n g o x y g e n atom, and i s 88 l a r g e r w i t h h a l o g e n o - t h a n w i t h o x y g e n - c o n t a i n i n g s u b -s t i t u e n t s . As a r e s u l t o f t h i s e l e c t r o n i c i n t e r a c t i o n , w h i c h i s o f t e n d i s c u s s e d i n t e r m s o f a c l a s s i c a l t y p e o f e l e c t r o s t a t i c i n t e r a c t i o n , e l e c t r o n e g a t i v e s u b s t i t u e n t s on t h e a n o m e r i c c a r b o n a tom p r e f e r t o assume a x i a l r a t h e r t h a n e q u a t o r i a l o r i e n t a t i o n s . As shown b e l o w , 4 3 t h e o r i e n t a t i o n (A) i n w h i c h t h e p o l a r b o nd i s s t a g g e r e d b e t w e e n t h e l o n e p a i r s i s d e s t a b i l i z e d w i t h r e s p e c t t o t h e o r i e n t a t i o n (B) i n w h i c h t h e p o l a r b o nd i s g a u c h e t o one l o n e p a i r and t r a n s t o t h e o t h e r . The e l e m e n t a l a n a l y s i s v e r i f i e d t h e p u r i t y o f [4] w h i l e t h e h i g h r e s o l u t i o n mass s p e c t r u m o f [7] c o n f i r m e d t h e p r e s e n c e o f t h a t s u g a r . The NMR c o u p l i n g 1 19 c o n s t a n t s , b o t h H and F as g i v e n i n T a b l e s V I I and I X c o n f i r m e d t h e a c o n f o r m a t i o n o f t h e s e s u g a r s . The b r o m i n a t e d s u g a r s [5] and [8] shown b e l o w were o b t a i n e d by r e p l a c e m e n t o f t h e F o r OAc m o i e t y a t C - l w i t h a b r o m i n e v i a a S^;2 mechanism- so t h a t r e t e n t i o n o f c o n f i g u r a t i o n a t C - l was o b t a i n e d ; t h e f i r s t - f o r m e d • 3 p r o d u c t i s u n s t a b l e 89 due t o t h e a n o m e r i c e f f e c t a n d t h u s was n o t o b s e r v e d . The s y n t h e s i s o f t h i s b r o m i n a t e d d e r i v a t i v e was n e c e s s a r y a s g l y c o s y l f l u o r i d e s do n o t r e a c t u n d e r t h e c o n d i t i o n s o f t h e K o e n i g s - K n o r r r e a c t i o n . Compound [5] has b e e n p r e v i o u s l y s y n t h e s i z e d i n o t h e r s t u d i e s , as g i v e n i n t h e e x p e r i m e n t a l s e c t i o n . The c o u p l i n g c o n s t a n t s g i v e n i n T a b l e s V I I and I X v e r i f y t h e a s s i g n e d a c o n f i g u r a t i o n ~ f o r b o t h [5] and [8] . The o p t i c a l r o t a t i o n o f [5] i s s l i g h t l y l o w e r t h a n t h e l i t e r a t u r e v a l u e b u t t h e e l e m e n t a l a n a l y s i s v e r i f i e d t h e p u r i t y o f t h i s compound. Compound [8] c o u l d n o t be c r y s t a l -l i z e d b u t t h e NMR and h i g h r e s o l u t i o n mass s p e c t r u m i n d i c a t e d t h a t t h i s compound was s y n t h e s i z e d by b o t h r o u t e s i n d i c a t e d . B o t h compounds were u n s t a b l e ( I . e . became d a r k b rown) a t room t e m p e r a t u r e and were k e p t a t 0°C. Once t h e b r o m i n a t e d m o n o s a c c h a r i d e w i t h a n o n - p a r -t i c i p a t i n g g r o u p a t C-2 was o b t a i n e d , t h e s y n t h e s i s o f t h e d i s a c c h a r i d e s was a t t e m p t e d . The i m p o r t a n t f e a t u r e s t h a t one has t o c o n t e n d w i t h i n a " t y p i c a l " K o e n i g s - K n o r r r e a c t i o n a r e : (1) t h e p r e s e n c e o f a n o n - p a r t i c i p a t i n g g r o u p a t C-2 o f t h e h a l o s u g a r , (2) t h e e f f e c t o f t h e C-3,0-4, and C-6 s u b s t i t u e n t s o f t h e h a l o s u g a r , . ( 3 ) t h e r e l a t i v e amount o f t h e h a l o s u g a r a n d t h e a l c o h o l ( i . e . w h i c h r e a c t a n t i s i n e x c e s s ) , (4) s o l v e n t p o l a r i t y , a n d (5 ) t h e t e m p e r a t u r e a t w h i c h t h e r e a c t i o n i s c a r r i e d o u t . The a d v a n t a g e o f h a v i n g a n o n - p a r t i c i p a t i n g g r o u p a t C-2 a r i s e s f r o m t h e f a c t t h a t c h a r g e d e r e a l i z a t i o n i s t h e b a s i c i n i t i a l d r i v i n g f o r c e f o r t h e r e l a t i v e l y h i g h r e a c t i v i t y 90 o f a - h a l o - e t h e r s . S u b s t i t u e n t s a t C-2, s u c h as a c e t o x y g r o u p s , c a n p r o v i d e a more e f f e c t i v e means o f c h a r g e de-72 l o c a l i z a t i o n a nd t h u s a l t e r t h e c o u r s e o f t h e s e r e a c t i o n s as o u t l i n e d p r e v i o u s l y . The p r o p o s e d m e c h a n i s m o f t h e s e r e a c t i o n s i s r e p e a t e d b e l o w . U n d e r b a s i c c o n d i t i o n s , t h e 1,2 i R H _ _ I . . . -a c e t o x o n i u m i o n r e a c t s w i t h an a l c o h o l t o g i v e a 1 , 2 - o r t h o -72 74 a c e t a t e d e r i v a t i v e i n s t e a d o f a g l u e o s i d e . ' U n d e r a c i d i c c o n d i t i o n s , t h e 1 , 2 - a c e t o x o n i u m i o n g i v e s r i s e t o a 72 3 - D - g l u c o p y r a n o s i d e . I n a " t y p i c a l " K o e n i g s - K n o r r r e a c t i o n , u s i n g a h a l o - s u g a r w i t h a n o n - p a r t i c i p a t i n g g r o u p a t C-2, a m i x t u r e o f a - and 3 - l i n k e d s u g a r s a r e o b t a i n e d as p r o d u c t s , w i t h t h e f o r m e r b e i n g p r e d o m i n a n t due t o t h e l a c k o f 2 6 a p a r t i c i p a t i n g g r o u p a t C-2. The p r e d o m i n a n c e o f one c o n -f i g u r a t i o n shows t h a t t h e s e r e a c t i o n s do n o t n e c e s s a r i l y p r o c e e d v i a a s i m p l e S ^ l m e c h a n i s m . The p r e d o m i n a n c e o f t h e a anomer a l s o s u g g e s t s t h a t t h e c a r b o n i u m i o n i n t e r m e d i a t e 91 must h a v e a p r e f e r r e d o v e r a l l c o n f i g u r a t i o n and t h i s i s l i k e l y t h e r e s u l t o f s t e r i c and d i p o l e e f f e c t s a r i s i n g f r o m t h e n a t u r e , s i z e , and p o s i t i o n o f t h e s u b s t i t u e n t g r o u p s . The i n t e r e s t i n g o b s e r v a t i o n i n t h i s work was t h a t ONLY t h e a - g l y c o s i d e [ 12] was o b t a i n e d . The a - c o n f o r m a t i o n o f t h e g l y c o s i d i c l i n k a g e was d e d u c e d f r o m t h e c o u p l i n g c o n -s t a n t s J„ „ ( T a b l e V I I I ) and <!„ „, J„ „ ( T a b l e X) and 1 ' ' 2 ' 1 1 5 3 ' ' I t was v e r i f i e d by t h e c r y s t a l s t r u c t u r e ( F i g u r e s 17 and 1 8 ) . The s i d e p r o d u c t o b t a i n e d , S.P. - [ 1 2 a ] was d e d u c e d t o have t h e f o l l o w i n g s t r u c t u r e f r o m t h e h i g h r e s o l u t i o n mass s p e c t r u m and f r o m c o m p a r i s o n o f t h e R^ and o p t i c a l r o t a t i o n v a l u e s o f t h i s s i d e p r o d u c t w i t h t h o s e o f . c o m p o u n d [12a] w h i c h was s y n t h e s i z e d d i r e c t l y f r o m t h e c o r r e s p o n d i n g h a l o -s u g a r [ 5 ]• The p r e s e n c e o f a C -OH g r o u p was v e r i f i e d by c o m p a r i n g t h e n o r m a l s p e c t r u m w i t h t h e s p e c t r u m i n t h e p r e s e n c e o f B^O as o u t l i n e d i n F i g u r e 1 9 . B o t h o f t h e s e compounds showed t h e same two s p o t s on t i c p l a t e s , as o u t l i n e d i n t h e e x p e r i m e n t a l s e c t i o n , b u t c o u l d n o t be s e p a r a t e d on t h e c o l u m n s i n d i c a t e d . T i c o f t h e compound as i t was e l u t e d o f f t h e c o l u m n s seemed t o i n d i c a t e t h a t s e p a r a t i o n had b e e n a c h i e v e d b u t , a f t e r a p e r i o d o f t i m e , t h e same f r a c t i o n w o u l d p r o d u c e a t i c I n d i c a t i v e o f t h e s t a r t i n g m i x t u r e . The o n l y p l a u s i b l e e x p l a n a t i o n o f s u c h a r e s u l t i s t h a t one i s l o o k i n g F i g u r e 17. C r y s t a l s t r u c t u r e o f [ 1 2 ] F i g u r e 18. C r y s t a l s t r u c t u r e o f [12] 94 ' ' • 1 ' 1 • 1 I ' • 1 1 1 ; 1 1 1 | 1 ' 1 1 ' 1 ' 1 ' | 1 1 1 ' 1 ' 1 I i | • ] i : i , i . , [ . i j j u J j J - i u - f j L x ^ x J L u ^ L i - j ' . . I i . ! | FIGURE 1 9 . H NMR s p e c t r a o f s i d e p r o d u c t [ 12a ] i n CDC1 ppm v a l u e s a r e r e l a t i v e t o e x t e r n a l TMS. [A] N o r m a l s p e c t r u m . [B] S p e c t r u m i n t h e p r e s e n c e o f D 9 0 . 95 a t t h e a and g a n o m e r i c f o r m s o f t h e compound, w h i c h c a n be a t l e a s t p a r t i a l l y s e p a r a t e d by t h e c o l u m n s b u t w h i c h , a f t e r a s h o r t p e r i o d o f t i m e i n s o l u t i o n , come t o e q u i l i b r i u m . S u c h an a s s u m p t i o n i s n o t t o t a l l y u n r e a s o n a b l e as s u g a r s u n d e r g o a n o m e r i z a t i o n t h r o u g h an i n t e r m e d i a t e w i t h c a r b o n i u m i o n c h a r a c t e r and t h i s i n t e r m e d i a t e c o u l d f e a s i b l y be d e s t a b i l i z e d by t h e Cp-P s u b s t i t u e n t . S u c h a s l o w a n o m e r i z a t i o n c o u l d o n l y be c o n c l u s i v e l y c o n f i r m e d by o b t a i n i n g t h e compound as a c r y s t a l a n d o b s e r v i n g m u t a r o t a t i o n i n s o l u t i o n . Un-f o r t u n a t e l y , t h i s compound c o u l d n o t be c r y s t a l l i z e d i n t h i s w o r k , b u t t h e p r e v i o u s l y d i s c u s s e d d a t a s t i l l i n d i c a t e t h a t t h e compound shown above was, i n f a c t , t h e s i d e p r o d u c t o f t h e s y n t h e s i s o f t h e d i s a c c h a r i d e [12] . T h i s compound c o u l d n o t be t h e 3 - l i n k e d d i s a c c h a r i d e due t o t h e l a c k o f r e s o n a n c e s i g n a l s b e t w e e n 3-0 and 4.0 ppm, w h i c h a r e t y p i c a l o f t h e 1 , 6 - a n h y d r o g l u c o s e a c e t a t e s , as i n d i c a t e d i n T a b l e s I and I I , a l o n g w i t h t h e f a c t t h a t t h e h i g h r e s o l u t i o n mass s p e c t r a l d a t a gave no m/e p e a k s i n d i c a t i v e o f s u c h a l a r g e m o l e c u l e . The s p e c i f i c s y n t h e s i s o f o n l y an a - l i n k e d d i s a c c h a r i d e h a s b e e n a c h i e v e d p r e v i o u s l y u s i n g t h e K n o e n i n g s - K n o r r r e a c t i o n . 7 5 F l o w e r s f o u n d t h a t no 3 - D - l i n k e d d i s a c c h a r i d e was d e -t e c t e d i n t h e r e a c t i o n m i x t u r e o f 2 - 0 - b e n z y l - 3 , 4 , 6 - t r i - 0 - p -CH2OR OCH2Ph A. R = H I R"= Br ; R"= O C C 5 H 4 N 0 2 lp> B . ? R = BrJR°= H ; R"= OCC 6 H 4 N0 2W If) • _ R = OCC 6 H,Nq /p; 96 n i t r o b e n z o y l - a ( o r 3 ) - D - g l u c o p y r a n o s y l b r o m i d e (A,B) w i t h 1 , 2 : 3 j 4 — d i — O — i s o p r o p y l i d e n e — o i — D — g a l a c t o s e (C) shown o n l t h e p r e c e d i n g p a g e , o r i n t h e c a s e o f t h e same h a l o - s u g a r ( s ) r e -a c t i n g w i t h 4 , 6 - 0 - e t h y l i d e n e - l , 2 - 0 - i s o p r o p y l i d i n e - a - D - g a l a c t o s e (D) i n d i c a t e d i n t h e scheme b e l o w . The a - l i n k e d d i s a c c h a r i d e was o b t a i n e d i n b o t h c a s e s , r e g a r d l e s s o f t h e a n o m e r i c c o n f i g u r a t i o n o f t h e s u g a r h a l i d e u s e d . I n t h e a b o v e c a s e s , a h a l o - s u g a r w i t h a n o n - p a r t i c i p a t i n g g r o u p a t C - 2 was e m p l o y e d b u t HgCN was u s e d i n p l a c e o f Ag^O^ and t h e f o r m e r i s known t o r e s u l t i n a h i g h y i e l d o f a - l i n k e d d i s a c c h a r i d e s e v e n w i t h s u g a r s 2 6 w h i c h do have a p a r t i c i p a t i n g g r o u p a t C - 2 . Thus t h e s e t y p e s o f r e a c t i o n s i n t h e p r e s e n c e o f m e r c u r i c s a l t s a r e n o t " t y p i c a l " K o e n i g s - K n o r r r e a c t i o n s and c a n n o t be d i r e c t l y c o m p a r e d t o t h o s e p r e s e n t e d i n t h i s s t u d y . To o b s e r v e t h e e f f e c t s o f t h e p a r t i c i p a t i o n o f OAc s u b s t i t u e n t s on t h e h a l o - s u g a r , one c a n l o o k a t t h e work o f D e j t e r - J u s z y n s k i and F l o w e r s who u s e d t h e t h r e e p o s s i b l e m o n o - O - a c e t y l - L - F u c o p y r a n o s y l b r o m i d e s shown b e l o w ( I ) and CH2OR A o r B + IP R = COC 6H 4NO z Cp) OR" [ I ] 97 c o n d e n s e d them w i t h b e n z y l - 2 - a c e t a m i d o - 0 - 3 , 4 - d i - 0 - a c e t y l - l , 2 -d e o x y - a - D - g l u c o p y r a n o s i d e ( I I ) . CH2'OH AcO OCH 2Ph NHAc ... t n L The 2-OAc d e r i v a t i v e o f t h e h a l o - s u g a r gave p r i m a r i l y t h e 3 p r o d u c t i n t h i s c o n d e n s a t i o n , as e x p e c t e d , w h i l e t h e 4-OAc and 3-OAc d e r i v a t i v e s h a d a m a r k e d a - d i r e c t i n g i n f l u e n c e . I n t h e R=CH2Ph c a s e o f t h e 4-OAc s u b s t i t u e n t , i t c o u l d be p o s s i b l e t o e v o k e p a r t i c i p a t i o n o f t h e a c y l g r o u p b u t t h e r e l a t i v e l y h i g h s t e r e o s e l e c t i v i t y shown by t h e 3-OAc s u b s t i t u e n t ( i n w h i c h i t i s n o t p o s s i b l e t o e v o k e p a r t i c i p a t i o n o f t h e a c y l g r o u p f r o m t h e 3 s i d e o f t h e m o l e c u l e t o d i r e c t t h e i n c o m i n g n u c l e o p h i l e a) shows t h a t t h e d i r e c t i n g a f f e c t o f t h e 3 - 0 - a c e t y l g r o u p r e q u i r e s a d i f f e r e n t e x p l a n a t i o n . P e r h a p s , as h a s b e e n 7 7 7 r\ p r e v i o u s l y s u g g e s t e d , 1 , 1 t h i s p r o c e s s o c c u r s t h r o u g h e l e c t r o n i c i n f l u e n c e o f t h e C -3 a c e t y l g r o u p , t h e m a g n i t u d e o f w h i c h i s d e p e n d e n t on i t s e l e c t r o n a t t r a c t i n g power. 98 A l t e r n a t i v e l y , t h e r e c o u l d be an i n t e r m e d i a t e f o r m a t i o n o f t h e more r e a c t i v e 3 h a l i d e f r o m t h e h a l i d e i o n f o r m e d d u r i n g 7 9 t h e c o u r s e o f t h e r e a c t i o n . The p r o p o r t i o n o f anomers f o r m e d w i l l t h e n be a f f e c t e d by t h e l o c a t i o n o f t h e s u b -s t i t u e n t s w h i c h h i n d e r t h e a p p r o a c h o f t h e n u c l e o p h i l e f r o m t h e a s i d e o f t h e m o l e c u l e , t o a t t a c k t h e 3 i o n p a i r t h u s f o r m e d . A c y l g r o u p s s u b s t i t u t e d a t d i f f e r e n t p o s i t i o n s on t h e r i n g c o u l d , p r e s u m a b l y , h a v e d i f f e r e n t e f f e c t s on t h e . f o r m a t i o n a n d r e a c t i v i t y o f t h e 3 i o n p a i r . I n t h i s w o r k , t h e C - 3 , C - 4 , and C-6 p o s i t i o n s on t h e h a l o - s u g a r a l l b o r e OAc g r o u p s . W h e t h e r o r n o t t h i s t y p e o f s u b s t i t u e n t i s s i g n i f i c a n t w i t h r e g a r d t o t h e s y n t h e s i s o f o n l y a n a - l i n k e d d i s a c c h a r i d e ( e . g . [12]) c o u l d be a n i m p o r t a n t f a c t o r . The p o s i t i o n o f t h e OAc g r o u p a t C -4 d i d n o t , h o w e v e r , a p p e a r t o i n f l u e n c e t h e t y p e o f p r o d u c t o b s e r v e d i n t h i s s t u d y as e v i d e n c e d by t h e r e s u l t s o f t h e s y n t h e s i s o f [12] i n w h i c h t h e h a l o - s u g a r o f a g l u c o s e d e r i v a t i v e was u s e d , a nd t h a t o f t h e s y n t h e s i s o f [15] i n w h i c h t h e h a l o s u g a r o f a g a l a c t o s e d e r i v a t i v e was e m p l o y e d . 8 0 The w ork o f K r o n z e r and S c h e u r c h i n c l u d e d t h e e f f e c t s o f u s i n g a n e x c e s s o f a l c o h o l c o m p a r e d w i t h u s i n g a n e x c e s s o f t h e h a l o - s u g a r i n a " t y p i c a l " K o e n i g s - K n o r r r e a c t i o n . I n c o n t r a s t t o r e a c t i o n w i t h a h i g h c o n c e n t r a t i o n o f a l c o h o l , r e a c t i o n s o f s e v e r a l d e r i v a t i v e s o f 2 , 3 > 4 - t r i - O - b e n z y l - a - D -g l u c o p y r a n o s y l b r o m i d e w i t h a l o w c o n c e n t r a t i o n o f m e t h a n o l ( i n t h e ^absence o f s i l v e r t r i f l a t e ) g a ve p r i m a r i l y t h e a - D - g l u c o s i d e s , r e g a r d l e s s o f t h e C -6 s u b s t i t u e n t . I n -t e r e s t i n g l y , t h e same b r o m i d e (shown n e x t p a g e ) gave a h i g h 99 p r o p o r t i o n o f g - D - g l u c o s i d e when a* l a r g e e x c e s s o f m e t h a n o l was u s e d i n e i t h e r t h e d i r e c t o r s i l v e r t r i f l a t e - a s s i s t e d m e t h a n o l y s i s . The r e l a t i v e amount o f a and 3 g l u c o s i d e s p r o d u c e d i n t h e p r e s e n c e o f s i l v e r t r i f l a t e d e p e n d e d on t h e t y p e o f C - s u b s t i t u e n t . One e x a m p l e o f t h i s r e a c t i o n w here t h e h a l o - s u g a r o u t l i n e d a b o v e has..a C -6 a c e t y l g r o u p was c i t e d and t h e d a t a i s g i v e n b e l o w . S o l v e n t [MeOH] M e t h a n o l / B r o m i d e R a t i o o f D - g l u c o s i d e s (M) ( m o l e s / m o l e ) ( a : g )  A c e t o n e [18] 390 7:13 I n a l o w c o n c e n t r a t i o n o f m e t h a n o l , t h e h a l o - s u g a r w i t h t h e C-6 OAc s u b s t i t u e n t gave e s s e n t i a l l y a 1:1 m i x t u r e o f a -and g - D - g l u c o s i d e s w i t h s i l v e r t r i f l a t e , w h i l e t h e same r e a c t i o n i n d i c h l o r o m e t h a n e gave o n l y t h e g - D - g l u c o s i d e . I n f a c t , t h e y f o u n d t h a t a - g l u c o s i d e s were g e n e r a l l y t h e predo.m-^ i n a n t - p r o d u c t i f t h e r e a c t i o n was r u n i n n o n - p o l a r s o l v e n t s s u c h as e t h e r ( a t l o w c o n c e n t r a t i o n s o f a l c o h o l ) . I n t h e c a s e o f s y n t h e s i s ' o f [18] ( c a r r i e d o u t i n t h i s w o r k ) o n l y t h e 3 - g l u c o s i d e was o b t a i n e d and t h i s r e a c t i o n was done i n 100 e t h e r I n t h e p r e s e n c e o f s i l v e r t r i f l a t e . The d a t a f o r t h i s r e a c t i o n i s shown b e l o w . S o l v e n t [MeOH] M e t h a n o l / B r o m i d e R a t i o o f D - g l u c o s i d e s (M) ( m o l e s / m o l e ) ( a : g )  E t h e r 4.17 92.9 0;100 I t seems t h a t t h e p o l a r i t y o f t h e s o l v e n t h a s l i t t l e e f f e c t u n d e r c o n d i t i o n s o f h i g h c o n c e n t r a t i o n s o f a l c o h o l s a s . t h e B - g l u c o s i d e [18] was t h e o n l y p r o d u c t . The o n l y d i s a c c h a r i d e s y n t h e s i z e d i n t h i s w ork h a d an a - g l y c o s i d e l i n k a g e ; t h e d a t a f o r s y n t h e s i s o f t h i s d i s a c c h a r i d e ( [ 1 2 ] ) i s g i v e n b e l o w . SOLVENT [2] [ 2 ] / B r o m i d e R a t i o o f D - g l u c o i d e s (M) ( m o l e s / m o l e ) ( a : g )  E t h e r 2.38 1 0 ~ 2 1 . 4 l 100: 0j E t h e r 2.54 1 0 - 2 0.77 100:Q A l t h o u g h t h e r e was..an e x c e s s o f t h e a l c o h o l [2] i n one s y n t h e s i s o f t h e d i s a c c h a r i d e , t h e e x c e s s may n o t h a v e b e e n g r e a t e nough t o a l l o w t h e p r o d u c t i o n o f s u c h a 3 - l i n k a g e . A r e a c t i o n u s i n g a s l a r g e a r a t i o o f a l c o h o l t o h a l o - s u g a r as t h a t e m p l o y e d i n t h e s y n t h e s i s o f [18] was n o t f e a s i b l e f o r t h e s y n t h e s i s o f [12] due t o t h e s m a l l amount o f t h e 8 0 a l c o h o l [2] a v a i l a b l e . S t i l l , K r o n z e r a n d S c h e u r c h a l w a y s f o u n d some o f t h e 3 - g l u c o s i d e , e v e n w i t h a s m a l l c o n -c e n t r a t i o n o f m e t h a n o l , w h i l e i n t h i s w ork o n l y t h e (De-l i n k e d d i s a c c h a r i d e - [12] was o b s e r v e d . The u s e o f t h e n o n -p o l a r s o l v e n t ( e t h e r ) as w e l l as t h e p r e s e n c e o f a C^-OAc, C^-OAc, and Cg-OAc s u b s t i t u e n t s may have b e e n i m p o r t a n t f a c t o r s i n t h i s r e s u l t , as w e l l as t h e u s e o f s m a l l c o n c e n t r a t i o n s 101 o f t h e a l c o h o l [2] ; i t i s o b v i o u s t h a t ; . a d e l i c a t e b a l a n c e o f c o n d i t i o n s i s n e c e s s a r y t o a c h i e v e s u c h a n a - l i n k e d d i s -a c c h a r i d e . I t s h o u l d be p o i n t e d o u t t h a t t h e t r i f l a t e - a s s i s t e d r e a c t i o n s i n t h e l i t e r a t u r e e x p l a i n e d a b o v e were a l l c a r r i e d o u t a t - 78°C and a t r i f l a t e e s t e r was t h e p o s t u l a t e d i n t e r -m e d i a t e . I n c o n t r a s t , t h e r e a c t i o n s i n t h i s s t u d y were done a t room t e m p e r a t u r e , t h e e f f e c t o f w h i c h i s d i s c u s s e d 81 b e l o w . S c h e u r c h a n d c o w o r k e r s s t u d i e d t h e r e a c t i o n o f g l y c o p y r a n o s y l t r i f l u o r o m e t h a n s u l p h o n a t e s w i t h a l c o h o l ; t h e y were f o u n d t o be e x t r e m e l y r e a c t i v e , g i v i n g h i g h y i e l d s o f a - g l u c o s i d e s a t l o w c o n c e n t r a t i o n s o f a l c o h o l a t - 78°C i n 30 m i n u t e s o r l e s s . M e t h y l - a - i s o m a l t o - o l i g o s a c c h a r i d e s w ere o b t a i n e d i n h i g h y i e l d s b u t i n c r e a s i n g t h e t e m p e r a t u r e r e s u l t e d i n t h e g l u c o p y r a n o s y l m e t h a n e s u l p h o n a t e s r e a c t i n g w i t h t h e s o l v e n t o r d e c o m p o s i n g t o g i v e a m i x t u r e o f u n -d e s i r e d p r o d u c t s . Thus t h i s i n f o r m a t i o n w a r n s a g a i n s t t h e p o s t u l a t i o n o f a t r i f l a t e e s t e r i n o u r r e a c t i o n , s i n c e i t was done a t room t e m p e r a t u r e , as w e l l as t h e f a c t t h a t AgSO^CF^ i s a s t r o n g a c i d a n d t h u s SO^CF^ w o u l d be e x p e c t e d t o be a ' j . r a t h e r p o o r n u c l e o p h i l e . I t i s , h o w e v e r , i n t e r e s t i n g t o n o t e t h a t s u c h an e s t e r w o u l d be an e x t r e m e l y r e a c t i v e a l k y l a t i n g e s t e r a n d ^ t h u s w o u l d be a d e s i r a b l e i n t e r m e d i a t e f o r t h e s u b s e q u e n t a d d i t i o n o f t h e w e a k l y n u c l e o p h i l i c 82 e q u a t o r i a l C^-OH o f g l u c o s e s h o u l d one d e s i r e t h e a - l i n k e d d i s a c c h a r i d e . a - D - g l u c o p y r a n o s i d e s , i n c l u d i n g s u c h d i s a c c h a r i d e s as 8 3 m a l t o s e h a v e b e e n p r e p a r e d s t a r t i n g f r o m t r i - O - a c e t y l - 1 , 2 -102 8 r3.nhyd.ro—a—D—glueopyra.nose, a l s o known as B r i g l ' s A n h y d r i d e , g o g r-a t e l e v a t e d t e m p e r a t u r e s . * M o d e r n c h e m i c a l t h e o r y s u g g e s t s , t h a t t h e r e a c t i v e i n t e r m e d i a t e r e q u i r e d f o r t h e o b s e r v e d s t e r e o s p e c i f i c a - g l u c o s i d a t i o n i n t h i s p r o c e s s i s t h e 3 , 4 , 6 - t r i - O - a c e t y l - D - g l u c o p y r a n o s y l c a t i o n shown b e l o w s i n c e t h e r e a c t i o n p r o c e e d s w i t h n e t r e t e n t i o n o f a n o m e r i c c o n f i g u r a t i o n . C e r t a i n l y t h e s t e r e o e l e c t r o n i c r e q u i r e m e n t s f o r t h e r e a c t i o n o f t h i s c a t i o n w i t h t h e a l c o h o l a r e e x p e c t e d t o l e a d p r e f e r e n t i a l l y t o t h e f o r m a t i o n o f t h e a - g l y c o s i d i c b o n d . S u c h may be t h e c a s e i n t h i s w o r k t o o , p a r t i c u l a r l y i n l i g h t o f t h e f a c t t h a t t h e a l c o h o l u s e d [2] i s s u c h a r i g i d , b u l k y g r o u p and t h u s c o u l d d e s t a b i l i z e t h e h a l f - c h a i r c o n -f o r m a t i o n w h i c h i s t h e s u g g e s t e d t r a n s i t i o n s t a t e i n t h e S ^ l m e c h a n i s m . I n s u c h a manner one c o u l d a r r i v e a t a c a r b o n i u m i o n i n t e r m e d i a t e w i t h a p r e f e r r e d c o n f o r m a t i o n . The C-2 f l u o r i n e a t o m w o u l d p l a y a l a r g e p a r t i n s u c h a r e s u l t due t o i t s l a r g e d i p o l e i n t e r a c t i n g w i t h t h e p o r b i t a l s o f t h e r i n g o x y g e n , as w e l l as t h e f a c t t h a t i t i s an e l e c t r o n w i t h d r a w i n g g r o u p a n d , as s u c h , w i l l d e s t a b i l i z e any c a r b o n i u m i o n i n t e r m e d i a t e . 103 I n l i g h t o f t h e i n f o r m a t i o n o u t l i n e d a b o v e , any d e f i n i t e c o n c l u s i o n s r e g a r d i n g t h e i n t e r m e d i a t e i n t h i s r e a c t i o n must a w a i t f u r t h e r i n f o r m a t i o n . R e t e n t i o n o f c o n f i g u r a t i o n i s n o t , on I t s own, c o n c l u s i v e e v i d e n c e a g a i n s t a b i m o l e c u l a r p r o c e s s i n w h i c h a g - l i n k e d d i s a c c h a r i d e w o u l d be f o r m e d , as t h e p o s s i b i l i t y o f i n v e r s i o n o f a f i r s t f o r m e d 3 - h a l o - s u g a r c a n n o t be e x c l u d e d . The c a r b o n i u m i o n f o r m e d i n t h e p r o c e s s c o u l d be s t a b i l i z e d by d e r e a l i z a t i o n o f t h e c h a r g e i n an o r b i t a l 2 f o r m e d by o v e r l a p o f a v a c a n t p o r b i t a l o f sp h y b r i d i z e d c a r b o n w i t h a p o r b i t a l o f t h e l a c t o l o x y g e n . T h i s s t a -b i l i z a t i o n , as e x p e c t e d , w i l l be m a x i m i z e d by a c o p l a n a r c o n f i g u r a t i o n o f t h e r i n g 0, C - l , C-2, and C-5 a s i n d i c a t e d p r e v i o u s l y . The i n f l u e n c e o f s t e r i c f a c t o r s on t h e u n i -m o l e c u l a r p r o c e s s w i l l a r i s e f r o m t h e manner i n w h i c h 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 t e d g l y c o s y l h a l i d e i n f l u e n c e s t h e e n e r g y o f t h e p a r t l y f o r m e d , p a r t l y p l a n a r c a r b o n i u m i o n , r e l a t i v e t o t h a t o f t h e i n i t i a l c h a i r f o r m o f t h e h a l i d e ; t h i s i n f l u e n c e t h u s may be r e g a r d e d as a s e c o n d a r y s t e r i c e f f e c t . P e r h a p s t h e f l u o r i n e d e s t a b i l i z e s t h e S ^ l t r a n s i t i o n s t a t e i n s u c h a manner t h a t f o r m a t i o n o f t h e 3 - l i n k e d d i s a c c h a r i d e i s u n f a v o u r a b l e . The e f f e c t o f s t e r i c f a c t o r s a n d t h e p o s s i b i l i t y o f p a r t i c i p a t i o n o f t h e OAc s u b s t i t u e n t s s h o u l d n o t be o v e r l o o k e d . T h r e e m e c h a n i s t i c r o u t e s seem p r o b a b l e , n a m e l y (1) i s o m e r i z a t i o n o f t h e b r o m i d e p r i o r t o s u b s t i t u t i o n , (2) a t r i f l a t e i n t e r m e d i a t e as i n d i c a t e d b e l o w where one h a s two s e q u e n t i a l S 2 r e a c t i o n s o c c u r r i n g , and 104 (3) an S j j l r e a c t i o n where t h e r e i s a p r e f e r r e d c o n -f o r m a t i o n f o r t h e c a r b o n i u m i o n i n t e r m e d i a t e s u c h t h a t 3 a t t a c k o f t h e a l c o h o l i s h i n d e r e d . The c r y s t a l s t r u c t u r e v e r i f i e d t h e a - g l y c o s i d i c l i n k a g e f o r t h e d i s a c c h a r i d e [12] and t h e o p t i c a l r o t a t i o n f o r a l l o f t h e 2 ' - d e o x y - 2 ' - f l u o r o - d i s a c c h a r i d e s was c o m p a t i b l e w i t h t h e c o r r e s p o n d i n g 2'-OAc d i s a c c h a r i d e s a s i n d i c a t e d i n t h e e x p e r i m e n t a l s e c t i o n ; t h i s i n d i c a t e d t h a t t h i s c o n f o r m a t i o n was m a i n t a i n e d i n a l l o f t h e d i s a c c h a r i d e s s y n t h e s i z e d ; t h i s a s s u m p t i o n was s u p p o r t e d by t h e c o u p l i n g c o n s t a n t d a t a f o r t h e s e s u g a r s . The m a j o r g o a l o f t h i s w ork was t o 19 s y n t h e s i z e 2 ' - d e o x y - 2 ' - f l u o r o - m a l t o s e [ 1 4 ] , and t h e F NMR 19 d a t a i n d i c a t e t h a t t h i s was a c h i e v e d . The p r e s e n c e o f two ^F r e s o n a n c e s w h i c h w e re e x t r e m e l y c l o s e t o g e t h e r ( e . g . 198.2 ppm; 198.4 ppm; DMSO-dg) i n d i c a t e d t h a t t h e r e were two s p e c i e s i n s o l u t i o n . I t seemed a t f i r s t u n l i k e l y t h a t t h e s e two r e s -o n a n c e s c o r r e s p o n d t o t h e two d i f f e r e n t a n o m e r i c f o r m s , a l t h o u g h 2 - d e o x y - 2 - f l u o r o - D - g l u c o s e has b e e n shown t o have two c l o s e l y s p a c e d f l u o r i n e r e s o n a n c e s c o r r e s p o n d i n g t o t h e 71 two a n o m e r i c f o r m s o f t h a t m o l e c u l e . The s t r u c t u r e s o f t h e l a t t e r and [14] a r e shown b e l o w . 105 H, 4' 2-deoxy-2-fluoro-D-glucose '4 H. l3 'H,OH HO The f i r s t p o s s i b i l i t y w h i c h a r o s e was t h a t d e a c e t y l a t i o n was n o t c o m p l e t e u n d e r t h e c o n d i t i o n s u s e d . T h i s seemed u n l i k e l y as t h e "*"H NMR s p e c t r a h a d no r e s o n a n c e s i n t h e r a n g e o f a c e t a t e 13 s h i f t s . The C s p e c t r a o f [12] a l s o h a d no a c e t a t e r e s -o n a n c e s so one must c o n c l u d e t h a t t h e s u g a r was f u l l y d e p r o t e c t e d . The s e c o n d p o s s i b i l i t y was t h a t somehow t h e s u g a r h a d b e e n d e s t r o y e d d u r i n g t h e d e p r o t e c t i o n e x p e r i m e n t s a n d t h u s 19 t h e e x t r a r e s o n a n c e o b s e r v e d i n t h e F NMR s p e c t r u m i s due t o some s o r t o f d e c o m p o s i t i o n p r o d u c t o r t o a s i d e p r o d u c t o f t h e r e a c t i o n i t s e l f . The c o n d i t i o n s e m p l o y e d i n t h e d e -p r o t e c t i o n e x p e r i m e n t a r e common ones and no s i d e p r o d u c t h a s b e e n o b s e r v e d by o t h e r w o r k e r s . T r e a t m e n t o f [14] i n a c e t i c a n h y d r i d e a n d p y r i d i n e f o l l o w e d by t i c o f t h e r e a c t i o n m i x t u r e gave two s p o t s w h i c h w e re i d e n t i c a l w i t h t h o s e o f t h e a and 3 a n o m e r i c f o r m s o f t h e p e r - O - a c e t y l a t e d compound [13] f r o m w h i c h t h e m a l t o s e a n a l o g u e [14] h a d o r i g i n a l l y b e e n 106 made (Figure 14); thus i t appears likely that the integrity of the rings was maintained during deprotection. The optical rotations for maltose and 21-deoxy-21-fluoro-maltose were similar in value, indicating apparent similarity in overall conformation of the two species. In addition, the a/g ratio of the anomeric protons (H^ )., as deduced from integration of the "4i NMR spectrum in (Figure 8), was 2/3 for both species. 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