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The carbonate catalyzed anomerization of protected 2, 4-dinitrophenyl glucopyranosides : a mechanistic… Berven, Leise Ann 1987

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THE CARBONATE CATALYZED ANOMERIZATION OF PROTECTED 2,4-DINITROPHENYL GLUCOPYRANOSIDES: A MECHANISTIC STUDY by LEISE ANN BERVEN B . S c , U n i v e r s i t y o f Minnesota, M i n n e a p o l i s , M i n n e s o t a , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department o f Chem 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 to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA May 1987 © L e i s e Ann Berven, 1987 4 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 or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of CHEMISTRY The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date JULY 20, 1987  DE-6(3/81) ABSTRACT The mechanism o f the c a r b o n a t e c a t a l y z e d c o n v e r s i o n o f 2 , 4 - d i n i t r o -p h e n y l 2 ,3 , 4 , 6 - t e t r a - 0 - a c e t y l - / S - D - g l u c o p y r a n o s i d e i n DMSO t o an e q u i -l i b r a t e d m i x t u r e o f the a- and ^ - g l y c o s i d e s has been i n v e s t i g a t e d u s i n g a v a r i e t y o f t e c h n i q u e s . P s e u d o - f i r s t - o r d e r r a t e c o n s t a n t s (k) measured f o r the a n o m e r i z a t i o n o f the p a r e n t s u b s t r a t e and the 1 - d e u t e r i o sub-s t r a t e i n d i c a t e d a secondary d e u t e r i u m i s o t o p e e f f e c t o f ^ / i c D = 1.09 ± 0.06. P s e u d o - f i r s t - o r d e r r a t e c o n s t a n t s measured f o r s e v e r a l deoxy and d e o x y f l u o r o d e r i v a t i v e s o f the p a r e n t sugar showed t h a t the d e o x y f l u o r o s u g a r s r e a c t a t l e a s t as f a s t as the p a r e n t sugar whereas the deoxy s u g a r s r e a c t e d more s l o w l y . I n a d d i t i o n t o t h e 2 , 4 - d i n i t r o -p h e n y l g l u c o s i d e , 2 , 6 - d i n i t r o p h e n y l g l u c o s i d e a l s o was found to anomerize, y e t attempts to exchange the 2 , 4 - d i n i t r o p h e n o l a t e groups o f the g l u c o s i d e w i t h added 2 , 6 - d i n i t r o p h e n o l a t e a n i o n and v i c e v e r s a were u n s u c c e s s f u l . Exchange o f the p r o t o n a t the anomeric c a r b o n w i t h a d e u t e r o n a l s o does not o c c u r when the a n o m e r i z a t i o n i s p e r f o r m e d i n the p r e s e n c e o f a d e u t e r o n s o u r c e (and v i c e v e r s a ) . Exchange o f the g l u c o s y l r e s i d u e was o b s e r v e d , however, when the 1 - d e u t e r i o s u b s t r a t e was anomerized i n the p r e s e n c e o f n o n - d e u t e r a t e d 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l -D - g l u c o p y r a n o s e . ^H-n.m.r. o f the 2 , 4 - d i n i t r o p h e n y l a - g l u c o s i d e i s o l a t e d from t h i s r e a c t i o n i n d i c a t e d t h a t the a - g l u c o s i d e p o s s e s s e d o n l y 50% o f the d e u t e r i u m l a b e l , a t the anomeric c e n t e r . These r e s u l t s a l o n g w i t h the o b s e r v a t i o n o f a Meisenheimer i n t e r m e d i a t e i n d i c a t e t h a t i i i -the a n o m e r i z a t i o n p r o c e e d s v i a n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n and as such i s n o v e l mechanism f o r g l y c o s i d e a n o m e r i z a t i o n . - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i L IST OF FIGURES i x ABBREVIATIONS x i i i ACKNOWLEDGEMENTS x i v INTRODUCTION 1 1. The Anomeric E f f e c t 3 2. M u t a r o t a t i o n o f D-Glucose 7 3. A c i d C a t a l y z e d A n o m e r i z a t i o n 9 3.1 U n p r o t e c t e d G l u c o p y r a n o s i d e s 10 3.2 A c e t y l a t e d G l y c o p y r a n o s i d e s 12 3.3 P e n t a - O - a c e t y l G l y c o p y r a n o s e s 13 4. H a l i d e Ion C a t a l y z e d A n o m e r i z a t i o n 16 5. Base C a t a l y z e d R e a c t i o n s o f G l y c o p y r a n o s i d e s . . . . 20 5.1 H y d r o l y s i s 20 5.2 A n o m e r i z a t i o n 22 - V -RESULTS 26 1. S y n t h e s i s 26 1.1 2 , 4 - D i n i t r o p h e n y l $ - D - g l u c o p y r a n o s i d e s . . . . 26 1.2 Deuterium L a b e l l e d 2 , 4 - D i n i t r o p h e n y l ^ - D - g l u c o p y r a n o s i d e s 26 1.3 Deoxy and D e o x y f l u o r o 2 , 4 - D i n i t r o -p h e n y l /9-D-glucopyranosides 28 1.4 P e r a c e t y l a t e d S u b s t i t u t e d P h e n y l G l u c o p y r a n o s i d e s 31 2. M e c h a n i s t i c I n v e s t i g a t i o n s 31 2.1 G e n e r a l A n o m e r i z a t i o n Procedure 31 2.2 A n o m e r i z a t i o n o f S u b s t i t u t e d P h e n y l G l u c o p y r a n o s i d e s 36 2.3 Exchange R e a c t i o n s 39 2.4 K i n e t i c I s o t o p e E f f e c t s 49 2.4.1 Survey o f S o l v e n t s 50 2.4.2 Survey o f C a t a l y s t s 50 2.4.3 Measurement o f Rates 54 2.5 Remote S u b s t i t u e n t E f f e c t s 61 DISCUSSION 67 MATERIALS AND METHODS 80 1. S y n t h e s i s 80 1.1 G e n e r a l P r o c e d u r e s and M a t e r i a l s 80 1.2 G e n e r a l P r e p a r a t i o n o f 2 , 4 - D i n i t r o -p h e n y l /3-D- g l u c o p y r a n o s i d e s 84 v i -1.3 Deuterium Labelled Glucopyranosides 86 1.3.1 2,4-Dinitrophenyl 2,3,4,6-tetra-0-acetyl-/?-D-[1-^H]-glucopyranoside 86 1.3.2 2,4-Dinitrophenyl 2,3,4,6-tetra-O-benzyl-/3-D-[1-^H]-glucopyranoside 87 1.4 Deoxy and Deoxyfluoro Glucopyranosides . . . 88 1.4.1 2,4-Dinitrophenyl 2,3,6-tri-0-acetyl-4-deoxy-4-fluoro-/?-D-glucopyranoside 88 1.4.2 2,4-Dinitrophenyl 2,3,6-tri-O-acetyl-4-deoxy-j8-D-glucopyranoside 90 1.4.3 2,4-Dinitrophenyl 2, 3 , 6 - t r i - 0 - a c e t y l -6-deoxy-/?-D-glucopyranoside 92 1.5 Peracetylated Substituted Phenyl Glucopyranosides 93 2. Anomerization Reactions 97 2.1 Preparative Scale Anomerization 97 2.1.1 2,4-Dinitrophenyl 2,3,4,6-tetra-O-acetyl-/J-D-glucopyranosides 97 2.1.2 Peracetylated Substituted Phenyl Glucopyranosides 98 2.1.3 Small Scale Reactions: V a r i a t i o n of Solvents and Catalysts 99 2.2 Exchange Reactions 100 2.2.1 Exchange of H or D at Anomeric Carbon . . . . 100 2.2.2 Exchange of Phenolate 101 2.2.3 Exchange v i a a Nucleophilic Attack Mechanism. . 102 3. K i n e t i c s 104 3.1 Polarimetric Studies 104 3.2 -^H-N.m.r. Studies 105 - v i i -APPENDIX 1: KINETIC ISOTOPE EFFECTS 109 APPENDIX 2: KINETIC DATA 120 BIBLIOGRAPHY 124 - v i i i -L I S T OF TABLES T a b l e Page 1 S u b s t i t u t e d p h e n y l g l u c o p y r a n o s i d e s and o b t a i n e d y i e l d s 32 2 A n o m e r i z a t i o n o f s u b s t i t u t e d p h e n y l g l u c o p y r a n o s i d e s 38 3 Survey o f s o l v e n t s f o r a n o m e r i z a t i o n o f /3-2,4-DNPG w i t h K 2 C 0 3 51 4 Survey o f c a t a l y s t s f o r a n o m e r i z a t i o n o f 0-2,4-DNPG 53 5 Sample k i n e t i c d a t a f o r KIE measurements 58 6 Summary o f r e s u l t s : KIE c a l c u l a t e d from r a t e o f d i s a p p e a r a n c e o f /J-2.4-DNPG (29) and (32) : 60 7 Remote s u b s t i t u e n t e f f e c t on a n o m e r i z a t i o n : r a t e s o f d i s a p p e a r a n c e f o r /9-2,4-DNPG s u b s t r a t e s 63 8 Chemical s h i f t s (5) f o r H(6)-DNP group and H ( l ) o f the 2,4-DNPG d e r i v a t i v e s i n DMS0-d 6 107 9 Remote s u b s t i t u e n t e f f e c t s : 2 , 4 - d i n i t r o p h e n y l 2, 3 , 6 - t r i - 0 - a c e t y l - 4 - d e o x y - 4 - f luoro-j8-D-g l u c o p y r a n o s i d e (40) 120 10 Remote s u b s t i t u e n t e f f e c t s : 2 , 4 - d i n i t r o p h e n y l 2,3 ,6- t r i - 0 - a c e t y l - 4 - d e o x y - / 3 - D - g l u c o p y r a n o s i d e (44) 121 11 Remote s u b s t i t u e n t e f f e c t s : 2 , 4 - d i n i t r o p h e n y l 2,3,4, - t r i - 0 - a c e t y l - 6 - d e o x y - y 9 - D - g l u c o p y r a n o s i d e (49) 121 12 Remote s u b s t i t u e n t e f f e c t s : 2 , 4 - d i n i t r o p h e n y l 2,3,4,6-tetra-0-acetyl-£-D-glucopyranoside (29) . . 122 13 Remote s u b s t i t u e n t e f f e c t s : 2 , 4 - d i n i t r o p h e n 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 - i 9 - D -g l u c o p y r a n o s i d e (39) 122 14 Remote s u b s t i t u e n t e f f e c t s : 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 , 6 - t r i - 0 - a c e t y l - y 9 - D - g l u c o p y r a n o s i d e (29) . . . 123 i x -LIST OF FIGURES F i g u r e Page 1 The s t r u c t u r e o f g l y c o s i d e s 1 2 The anomers o f D - g l u c o p y r a n o s i d e 2 3 The anomeric e f f e c t : e l e c t r o s t a t i c i n t e r a c t i o n s 4 4 The anomeric e f f e c t : m o l e c u l a r o r b i t a l i n t e r a c t i o n s 5 5 The r e v e r s e anomeric e f f e c t 6 6 The p r o t o n a t i o n o f 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l -a - D - g l u c o s y l i m i d a z o l e 7 7 The mechanism f o r m u t a r o t a t i o n o f D-glucopyranose 8 8 The m u t a r o t a t i o n o f D-glucopyranose v i a n u c l e o p h i l i c a t t a c k by water 8 9 Mechanisms f o r a c i d c a t a l y z e d a n o m e r i z a t i o n o f methyl D - g l u c o p y r a n o s i d e 11 10 The s t a b i l i z a t i o n o f the oxocarbonium i o n t r a n s i t i o n s t a t e 12 11 Lewis a c i d c a t a l y z e d r e a c t i o n o f g l y c o s i d e s 13 12 The mechanism f o r a c i d c a t a l y z e d anomeriza-t i o n o f 1,2,3,4,6-penta-O-acetyl-D-g l u c o p y r a n o s e 14 13 The mechanism f o r a c i d c a t a l y z e d anomeriza-t i o n o f 1, 2,3,4,6-penta-O-acetyl-D-mannopyranose 15 14 The K o e n i g s - K n o r r g l y c o s i d a t i o n 17 15 The h a l i d e i o n c a t a l y z e d g l y c o s i d a t i o n 18 16 The mechanism o f h a l i d e exchange 19 - X -17 The base c a t a l y z e d h y d r o l y s i s o f 4 - n i t r o -p h e n y l a - D - g l u c o p y r a n o s i d e 21 18 The h y d r o l y s i s o f 4 - n i t r o p h e n y l y9-D-g a l a c t o p y r a n o s i d e 22 19 The h y d r o l y s i s o f 4 - n i t r o p h e n y l a-D-mannopyranoside 22 20 The p r e p a r a t i o n and a n o m e r i z a t i o n o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - 0 -a c e t y l - ^ - D - g l u c o p y r a n o s i d e 24 21 S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2,3,4,6-t e t r a - O - a c e t y l - 0 - D - [ 1 - 2 H ] - g l u c o -p y r a n o s i d e 27 22 S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2,3,4,6-tetra-0-benzyl-/9-D- [1- 2H] - g l u c o -p y r a n o s i d e 28 23 S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2,3,6-t r i - 0 - a c e t y l - 4 - d e o x y - 4 - f luoro-/8-D-g l u c o p y r a n o s i d e 29 24 S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 6 - t r i -0-acetyl-4-deoxy-/3-D-glucopyranoside 30 25 S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 - t r i -0-acetyl-6-deoxy-/9-D-glucopyranoside 30 26 S y n t h e s i s o f s u b s t i t u t e d p h e n y l /3-D-glucopyranosides 31 27 ^-N.m.r. s p e c t r a o f 0-2,4-DNPG and Q-2.4-DNPG (29) i n CDC1 3 34 28 ^-N.m.r. s p e c t r a o f /9-2,4-DNPG and a-2,4-DNPG (29) i n DMS0-d 6 35 29 Absorbance v e r s u s time f o r g e n e r a t i o n o f 2 , 4 - d i n i t r o p h e n o l a t e d u r i n g the a n o m e r i z a t i o n o f (29) 37 30 P o s s i b l e s i t e s o f bond c l e a v a g e 40 31 1-H-N.m.r. spectrum o f H/D exchange experiment . . 41 32 ^-H-N.m.r. spectrum o f D/H exchange experiment . . 42 - x i -33 •LH-N.m.r. s p e c t r a o f p h e n o l a t e exchange experiments 44 34 A l t e r n a t i v e r o u t e t o C ( l ) - 0 ( 1 ) bond c l e a v a g e 45 35 ^H-N.m.r. f o r exchange v i a 0 ( l ) - a r y l bond c l e a v a g e (experiment 1) 47 36 Exchange experiment f o r 0 ( l ) - a r y l bond c l e a v a g e 48 37 1-H-N.m.r. f o r exchange v i a 0 ( l ) - a r y l bond c l e a v a g e (experiment 2) 48 38 ^H-N.m.r. s p e c t r a f o r KIE measurements . . . . 56 39 ^H-N.m.r. s p e c t r a f o r KIE measurements . . . . 57 40 KIE measured f o r a n o m e r i z a t i o n o f /9-2,4-DNPG 59 41 U . v . / v i s i b l e absorbance s p e c t r a f o r a n o m e r i z a t i o n r e a c t i o n o f (29) 62 42 R e a c t i o n r a t e s measured f o r remote s u b s t i t u e n t e f f e c t s 65 43 Mechanism ( i ) . Base c a t a l y z e d p r o t o n a b s t r a c t i o n 67 44 Mechanism ( i i ) . P h e n o l a t e a b s t r a c t i o n and f o r m a t i o n o f an oxocarbonium i o n i n t e r m e d i a t e 69 45 Mechanism ( i i i ) . P r o t o n a b s t r a c t i o n a t C(2) and f o r m a t i o n o f a g l u c a l i n t e r m e d i a t e 71 46 Mechanism ( i v ) . A r o m a t i c n u c l e o p h i l i c s u b s t i t u t i o n 72 47 Meisenheimer i n t e r m e d i a t e formed by the r e a c t i o n o f 1 - s u b s t i t u t e d 2 , 4 - d i n i t r o -benzenes and methoxide i n DMSO 73 48 F o r m a t i o n o f the 2 , 4 - d i n i t r o p h e n o l a t e a n i o n 77 - x i i 49 P y r i d i n e c a t a l y z e d a n o m e r i z a t i o n o f 1,2,3,4,6-penta-O-acetyl-£-D-g l u c o p y r a n o s e 79 50 The r e a c t i o n c o o r d i n a t e diagram f o r a p r i m a r y i s o t o p e e f f e c t I l l 51 Bending v i b r a t i o n a l f r e q u e n c i e s o f sp^ and sp^ h y b r i d i z e d c a r b o n 112 52 The r e a c t i o n c o o r d i n a t e diagram f o r a a - s e c o n d a r y i s o t o p e e f f e c t 113 53 H y p e r c o n j u g a t i o n i n an oxocarbonium i o n . . . . 115 54 The i s o t o p e e f f e c t s measured f o r the a c i d c a t a l y z e d h y d r o l y s i s o f methyl a- and ^ - g l u c o p y r a n o s i d e 117 55 The a c i d c a t a l y z e d h y d r o l y s i s o f m ethyl a- and / J - g l u c o p y r a n o s i d e s 117 - x i i i -ABBREVIATIONS DMSO = d i m e t h y l s u l p h o x i d e Ac = a c e t y l Me = methyl DNP - d i n i t r o p h e n y l FDNB = l - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e DABCO - l , 4 - d i a z a b i c y c l o [ 2 . 2 . 2 ] - o c t a n e DMF = N,N-dimethylformamide DNPG - d i n i t r o p h e n y l g l u c o p y r a n o s i d e Bn - b e n z y l THF = t e t r a h y d r o f u r a n t . l . c . = t h i n l a y e r chromatography n.m.r. = n u c l e a r magnetic resonance u . v . / v i s — u l t r a v i o l e t / v i s i b l e t-BuOH = t - b u t a n o l o r 2-methyl-2-propanol KIE — k i n e t i c i s o t o p e e f f e c t MeCN = a c e t o n i t r i l e d i m s y l = d i m e t h y l s u l f i n y l m.p. = m e l t i n g p o i n t b.p. = b o i l i n g p o i n t ppm = p a r t s p e r m i l l i o n s — s i n g l e t d = d o u b l e t t — t r i p l e t m — m u l t i p l e t Bz — b e n z o y l c - c o n c e n t r a t i o n i n mg/ml eu = e n t r o p y u n i t s M = molar mmol — m i l l i m o l e s - x i v -ACKNOWLEDGEMENTS I would l i k e t o thank P r o f e s s o r s S.G. W i t h e r s and D.H. D o l p h i n f o r the s u p p o r t and encouragement they p r o v i d e d t hroughout t h e i r s u p e r v i s i o n o f the p r o j e c t and the p r e p a r a t i o n o f t h i s t h e s i s . I a l s o would l i k e t o thank the r e s e a r c h group o f S.G. W i t h e r s f o r t h e i r s u g g e s t i o n s and h e l p f u l d i s c u s s i o n s . F i n a n c i a l s u p p o r t from the Department o f C h e m i s t r y i n the form o f a T e a c h i n g A s s i s t a n t s h i p (1984-87) i s g r a t e f u l l y acknowledged. I a l s o e x t e n d s p e c i a l thanks t o the t e c h n i c a l s t a f f o f the department whose h e l p has been g r e a t l y a p p r e c i a t e d . F i n a l l y , I w i s h t o ext e n d my g r a t i t u d e t o the s t u d e n t s , s t a f f , and F a c u l t y f o r t h e i r a d v i c e , s u p p o r t , and most o f a l l , f o r t h e i r f r i e n d -s h i p . - 1 -INTRODUCTION G l y c o s i d e s a r e c y c l i c a c e t a l s which a r e d e r i v e d from the h e m i a c e t a l form o f a sugar and an a l c o h o l o r p h e n o l ( F i g u r e 1 ) . Each g l y c o s i d e has A hemiacetal An acetal F i g u r e 1: The s t r u c t u r e o f g l y c o s i d e s (R" = a l k y l o r a r y l ) two p o s s i b l e isomers c a l l e d anomers which d i f f e r o n l y i n the c o n f i g u r a -t i o n a t C ( l ) ( t h e anomeric carbon) . F o r D - g l y c o p y r a n o s i d e s i n the c o n f o r m a t i on, the a-anomer (2) has an a x i a l s u b s t i t u e n t a t C ( l ) whereas the y(3-anomer (1) has an e q u a t o r i a l s u b s t i t u e n t a t C ( l ) . I n t e r c o n v e r s i o n o f the two isomers i s termed a n o m e r i z a t i o n ( F i g u r e 2 ) . T h i s i n t r o d u c -- 2 -t i o n o u t l i n e s the thermodynamic and k i n e t i c a s p e c t s o f a n o m e r i z a t i o n f o r a v a r i e t y o f g l y c o s i d e s and sim p l e s u g a r s . F i g u r e 2: The anomers o f D - g l u c o p y r a n o s i d e (R = H, a l k y l o r a r y l ) The e l u c i d a t i o n o f r e a c t i o n mechanisms a t g l y c o s i d i c c e n t e r s has a t t r a c t e d the i n t e r e s t o f many i n v e s t i g a t o r s . One r e a s o n f o r t h i s i n t e r e s t c o n c e r n s the f u n c t i o n o f g l y c o s i d e s i n p l a n t s and an i m a l s . Many c a r b o h y d r a t e - c o n t a i n i n g b i o p o l y m e r s and n a t u r a l l y o c c u r r i n g mono-s a c c h a r i d e s a r e g l y c o s i d e s and i t i s the g l y c o s i d i c l i n k a g e which i s p r e f e r e n t i a l l y c l e a v e d i n the breakdown o f such compounds. One example i s the enzymic breakdown o f g l y c o s i d e s o r o l i g o s a c c h a r i d e s by g l y c o s i -dases which h y d r o l y z e the g l y c o s i d i c l i n k a g e t o produce a sugar r e s i d u e and an a l c o h o l ( the a g l y c o n e ) i n the case o f a s i m p l e g l y c o s i d e , or two or more sugar r e s i d u e s i n the case o f an o l i g o s a c c h a r i d e . S i n c e these t y p e s o f b i o c h e m i c a l t r a n s f o r m a t i o n s can o f t e n be r e l a t e d to the an a l o -gous c h e m i c a l t r a n s f o r m a t i o n s , the mechanisms f o r r e a c t i o n s such as g l y c o s i d e h y d r o l y s i s , a n o m e r i z a t i o n , and g l y c o s y l t r a n s f e r are o f - 3 -i n t e r e s t . A second r e a s o n f o r s t u d y i n g a n o m e r i z a t i o n i s t h a t the p r o c e s s o f t e n o c c u r s d u r i n g the p r e p a r a t i o n s and r e a c t i o n s o f s i m p l e sugars and g l y c o s i d e s , thus p r o d u c i n g low y i e l d s and u n d e s i r e d b y - p r o d u c t s . There-f o r e , m e c h a n i s t i c s t u d i e s can l e a d to the development o f improved s y n t h e t i c methods. 1. The Anomeric E f f e c t The anomeric e f f e c t i s d e f i n e d as the tendency 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 a t the anomeric c e n t e r t o adopt the a x i a l p o s i t i o n i n p yranose r i n g s . T h i s p r e f e r e n c e f o r the a x i a l p o s i t i o n c o n t r a s t s p r e d i c t i o n s b a s e d s o l e l y on s t e r i c i n t e r a c t i o n s which f a v o r the equato-r i a l o r i e n t a t i o n . Two t h e o r i e s have been p r o p o s e d to e x p l a i n the anomeric e f f e c t . One s i m p l e r a t i o n a l i z a t i o n p o s t u l a t e s e l e c t r o s t a t i c i n t e r a c t i o n s e x i s t -i n g between the anomeric s u b s t i t u e n t and the l o n e p a i r e l e c t r o n s on the a c e t a l oxygen i n the pyranose r i n g . 1 ' 2 F o r the /8-anomer i n the ^C^ c o n f o r m a t i o n , the d i p o l e a s s o c i a t e d w i t h the C ( l ) s u b s t i t u e n t e x a c t l y e c l i p s e s t h e d i p o l e from the l o n e p a i r e l e c t r o n s on the oxygen ( F i g u r e 3 ) . D e s t a b i l i z a t i o n o f the /3-anomer i s due t o r e p u l s i o n between the e c l i p s i n g d i p o l e s . In the case o f the a-anomer, t h i s u n f a v o r a b l e d i p o l e - d i p o l e i n t e r a c t i o n does not o c c u r as shown by the Newman p r o j e c -t i o n s i n F i g u r e 3. A second e x p l a n a t i o n o f the anomeric e f f e c t p r oposes the s t a b i l i z a -- 4 -t i o n o f the a-anomer by e l e c t r o n d e l o c a l i z a t i o n through the m o l e c u l a r o r b i t a l s on the r i n g oxygen t o the C ( l ) s u b s t i t u e n t . - ^ I n the case of F i g u r e 3: The anomeric e f f e c t : e l e c t r o s t a t i c i n t e r a c t i o n s the a-anomer, the e l e c t r o n p a i r on the r i n g oxygen i s o r i e n t e d a n t i p e r i -p l a n a r t o the a x i a l C-X bond. S t a b i l i z a t i o n r e s u l t s from p a r t i a l e l e c t r o n t r a n s f e r from t h i s l o n e p a i r t o the a n t i b o n d i n g a* o r b i t a l o f the bond t o 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 a t the anomeric c e n t e r ( F i g u r e 4 ) . T h i s e x p l a n a t i o n can a l s o be i l l u s t r a t e d by the i d e a o f a "double bond - no bond resonance" s t r u c t u r e i n which the e l e c t r o n i c d e l o c a l i z a t i o n i s due to the o v e r l a p o f an e l e c t r o n p a i r p - o r b i t a l on the oxygen w i t h the a n t i b o n d i n g a o r b i t a l o f the C-X bond ( F i g u r e 4 ) . - 5 -O v e r l a p o f t h e se o r b i t a l s i s p o s s i b l e o n l y i n the a x i a l o r i e n t a t i o n s i n c e i n the e q u a t o r i a l o r i e n t a t i o n the o r b i t a l s a r e o r t h o g o n a l . F i g u r e 4: The anomeric e f f e c t : m o l e c u l a r o r b i t a l i n t e r a c t i o n s . F i g u r e a t the r i g h t shows the "double bond - no bond r e s o n a n c e " s t r u c t u r e E v i d e n c e f o r the anomeric e f f e c t a r i s i n g from these i n t e r a c t i o n s i s w e l l e s t a b l i s h e d . F o r example, the e x t e n t o f the p r e f e r e n c e f o r the a x i a l p o s i t i o n d e c r e a s e s i n s o l v e n t s o f i n c r e a s i n g p o l a r i t y . ^ S o l v e n t s o f r e l a t i v e l y h i g h p o l a r i t y were found to d e c r e a s e the anomeric e f f e c t s i n c e t h e se s o l v e n t s w i l l s t a b i l i z e the n e t d i p o l e p r e s e n t i n the /fl-anomer. A l s o , the anomeric e f f e c t i s m a g n i f i e d w i t h i n c r e a s i n g e l e c t r o n e g a t i v i t y o f the anomeric s u b s t i t u e n t X. T h i s i s p a r t i c u l a r l y t r u e f o r g l y c o s y l h a l i d e s : an e q u i l i b r i u m m i x t u r e o f 2,3,4,6-tetra-O-a c e t y l - / J - D - g l u c o p y r a n o s y l c h l o r i d e i n d r y a c e t o n i t r i l e c o n t a i n s 93-95% o f the a-anomer i n the p r e s e n c e o f added c h l o r i d e ion.-* F i n a l l y , groups which r e v e r s e the d i p o l e o f the bond a t t a c h i n g them to the anomeric - 6 -c a r b o n show a " r e v e r s e anomeric e f f e c t " . Lemieux s t u d i e d the anomeric e f f e c t f o r N - ( 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - a - D - g l u c o p y r a n o s y l ) - 4 - m e t h y l p y r i d i n i u m bromide (3) and found t h a t the p o s i t i v e l y c h a r g e d p y r i d i n i u m group p r e f e r s t o adopt the e q u a t o r i a l o r i e n t a t i o n shown i n F i g u r e 5. ^ F i g u r e 5: The r e v e r s e anomeric e f f e c t I t was a l s o shown t h a t t h i s n o r m a l l y u n f a v o r e d c o n f o r m a t i o n was n o t s i m p l y a r e s u l t o f the s t e r i c b u l k o f the p y r i d i n i u m group by comparison w i t h the i m i d a z o l e d e r i v a t i v e (4) ( F i g u r e 6 ) . Compound (4) p r e f e r s the c h a i r c o n f o r m a t i o n b u t upon p r o t o n a t i o n , f l i p s t o the boat c o n f o r m a t i o n . T h e r e f o r e , the p r e f e r e n c e f o r the e q u a t o r i a l o r i e n t a t i o n i n (3) and (4) may be due to s t a b i l i z a t i o n o f the p o s i t i v e charge by an e c l i p s i n g i n t e r a c t i o n w i t h the c o n c e n t r a t i o n o f n e g a t i v e charge on the r i n g oxygen. T h i s o b s e r v a t i o n o f a r e v e r s e anomeric e f f e c t i s one major f l a w i n the "double bond - no bond resonance" argument f o r the anomeric e f f e c t s i n c e t h i s t h e o r y would p r e d i c t t h a t the a x i a l o r i e n t a t i o n i n ( 3 ) - 7 -would s t i l l be p r e f e r r e d d e s p i t e the p o s i t i v e l y c h a r g e d p y r i d i n i u m s u b s t i t u e n t . H F i g u r e 6: The p r o t o n a t i o n o f 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - a - g l u c o s y l i m i d a z o l e 2. M u t a r o t a t i o n o f D-Glucose When /3-D-glucopyranose (1, R = H) i s d i s s o l v e d i n water, a change i n o p t i c a l r o t a t i o n i s o b s e r v e d as i t anomerizes to the a-form (2, R = H). T h i s phenomenon i s termed m u t a r o t a t i o n . The r e a c t i o n i s w e l l c h a r a c t e r i z e d f o r most r e d u c i n g sugars such as D -glucose and the mecha-nism i s c o n s i d e r e d t o i n v o l v e a c i d / b a s e c a t a l y s i s w i t h the f o r m a t i o n o f an a l d e h y d o - s u g a r i n t e r m e d i a t e as shown i n F i g u r e 7. The b e s t e v i d e n c e f o r the a c y c l i c i n t e r m e d i a t e i s the o b s e r v a t i o n t h a t [ 1 - - ^ 0 ] - D - g l u c o s e undergoes oxygen exchange w i t h water 30 times more s l o w l y than the m u t a r o t a t i o n r e a c t i o n i t s e l f . ^ T h i s r e s u l t e x c l u d e s a p o s s i b l e a l t e r n a t i v e mechanism i n which the h y d r o x y l group a t C ( l ) undergoes 8 -n u c l e o p h i l i c d i s p l a c e m e n t by water and oxygen exchange would be as f a s t as m u t a r o t a t i o n ( F i g u r e 8 ) . The mechanism g i v e n i n F i g u r e 7 i n v o l v e s . O H • ° H O ^ - l ^ V - H O H H H O ^ - C ^ A ® H ^ p - H H O V - ^ J J Q ^ O H O H H 0 ^ V 4 ^ ° \ O H + H 2 0 / 0 H £ O H Figure 7: The mechanism f o r mutarotat ion of D- glucopyranose F igure 8: ^ m u t a r o t a t i o n of D-glucopyranose v i a n u c l e o p h i l i c a t tack - 9 -b o t h removal and a d d i t i o n o f a p r o t o n . Indeed i t has been determined t h a t the r e a c t i o n i s c a t a l y z e d by the pre s e n c e o f b o t h a c i d and base. C o n s e q u e n t l y , the m u t a r o t a t i o n o f D-glucose p r o c e e d s v e r y s l o w l y i n a p r o t i c s o l v e n t s b u t i s a c c e l e r a t e d by the a d d i t i o n o f water which then s e r v e s as a s p e c i f i c a c i d / b a s e c a t a l y s t . In a d d i t i o n , i t was found t h a t 2 ,3,4,6-tetra-O-methyl-D-glucopyranose m u t a r o t a t e s i n a m i x t u r e o f p y r i d i n e and c r e s o l where the p y r i d i n e i s thought t o a c t as a base and the c r e s o l as an a c i d . However, v i r t u a l l y no m u t a r o t a t i o n was o b s e r v e d i n e i t h e r p y r i d i n e o r c r e s o l a l o n e s i n c e the p r o t o n donor and a c c e p t o r a r e n o t p r e s e n t s i m u l t a n e o u s l y i n the system.** Furthermore, 2-hydroxy-p y r i d i n e was found t o be a v e r y e f f e c t i v e b i f u n c t i o n a l c a t a l y s t . A l t h o u g h 2 - h y d r o x y p y r i d i n e i s a weaker a c i d or base than e i t h e r p y r i d i n e o r c r e s o l , i t c a t a l y z e s the r e a c t i o n even f a s t e r than a m i x t u r e o f these two c a t a l y s t s . ^ 3. A c i d C a t a l y z e d M u t a r o t a t i o n A n o m e r i z a t i o n o f g l y c o s i d e s and o t h e r s i m p l e c a r b o h y d r a t e d e r i v a -t i v e s i s g e n e r a l l y e i t h e r a c i d o r base c a t a l y z e d . Examples o f a c i d c a t a l y z e d r e a c t i o n s are f a r more p r e v a l e n t and t h e r e f o r e , more i s known about the mechanisms o f t h e i r r e a c t i o n s . A c i d c a t a l y z e d r e a c t i o n s are t h e r e f o r e d i s c u s s e d i n t h i s i n t r o d u c t i o n a t g r e a t e r l e n g t h t h a n base c a t a l y z e d r e a c t i o n s . 10 -3.1 U n p r o t e c t e d G l u c o p y r a n o s i d e s The f i n a l s t e p i n the F i s c h e r s y n t h e s i s o f g l y c o s i d e s i s the e q u i l i b r a t i o n o f a- and 0- f u r a n o s i d e s and p y r a n o s i d e s . j n systems where p y r a n o s i d e f o r m a t i o n i s f a v o r e d , an e q u i l i b r i u m m i x t u r e o f methyl-D - g l u c o p y r a n o s i d e s c o n t a i n s 73% o f the a-anomer and 27% o f the /3-anomer.H The mechanism o f t h i s f i n a l e q u i l i b r a t i o n s t e p has been of much i n t e r e s t . Capon has p r oposed t h r e e p o s s i b l e mechanisms f o r the e q u i l i b r a t i o n o f m e t h y l a-and /3-D-glucopyranosides ( 5 ) . ^ The f i r s t s u g g e s t s a c y c l i c oxocarbonium i o n i n t e r m e d i a t e ( ( 6 ) , mechanism i ) , the second s u g g e s t s an a c y c l i c i n t e r m e d i a t e ( ( 7 ) , mechanism i i ) , and the t h i r d , an a c e t a l i n t e r m e d i a t e ( ( 8 ) , mechanism i i i ) ( F i g u r e 9 ) . By p e r f o r m i n g the a c i d c a t a l y z e d a n o m e r i z a t i o n o f (5) i n CD3OD, i t was found t h a t the methoxyl group c o m p l e t e l y exchanges w i t h the s o l v e n t , t h e r e b y e x c l u d i n g mechanism ( i i ) . Mechanism ( i i i ) was e x c l u d e d by s y n t h e s i z i n g the d i m e t h y l a c e t a l (8) s e p a r a t e l y and s u b s e q u e n t l y s u b j e c t i n g i t to the a n o m e r i z a t i o n c o n d i t i o n s . Here i t was found t h a t the methyl f u r a n o s i d e s were formed, as opposed to p y r a n o s i d e s , and any r i n g e x p a n s i o n to form the pyrano-s i d e s shown i n F i g u r e 9 went s l o w l y compared to the r a t e o f anomeriza-t i o n f o r (5-/3) o r ( 5 - a ) . In a d d i t i o n , an e a r l i e r p u b l i c a t i o n by Capon e t a l . ^ r e p o r t e d e n t r o p i e s o f a c t i v a t i o n o f +5.7 ± 1.0 eu f o r (5-/3) and +7.7 ± 1.0 eu f o r (5-a) a t 35°C, s u g g e s t i n g a u n i m o l e c u l a r mechanism f o r the r e a c t i o n . R e c e n t l y , J e n s e n i n v e s t i g a t e d the e f f e c t s o f methanol a c t i n g as a s o l v e n t as opposed to i t a c t i n g as a r e a g e n t i n t h i s anomer-i z a t i o n . X J By measuring the e f f e c t o f d e c r e a s i n g methanol c o n c e n t r a t i o n 11 -F i g u r e 9: Mechanisms f o r a c i d c a t a l y z e d a n o m e r i z a t i o n o f m e t h y l D-g l u c o p y r a n o s i d e i n CH3OH/DMSO s o l u t i o n s on the r a t e o f a n o m e r i z a t i o n , i t was dete r m i n e d t h a t the r e a c t i o n i s z e r o o r d e r i n methanol as i s n e c e s s a r y f o r mecha-nism ( i ) . However, i t s t i l l r e q u i r e s methanol o r a s i m i l a r n u c l e o p h i l i c s p e c i e s f o r the t r a n s f o r m a t i o n t o o c c u r . J e n s e n u s e d t h i s e v i d e n c e to propose an " i n t e r m e d i a t e " o f the type shown i n F i g u r e 10 where the oxocarbonium i o n formed i s not a f r e e s p e c i e s b u t r e q u i r e s i n t e r a c t i o n w i t h s o l v e n t m o l e c u l e s i n the s o l v e n t cage f o r s t a b i l i z a t i o n . I t was - 12 -a l s o s u g g e s t e d t h a t the f o u r h y d r o x y l groups on the p y r a n o s i d e r i n g might i n t e r a c t w i t h the s o l v e n t cage by d e r e a l i z a t i o n o f charge to p r o v i d e f u r t h e r t r a n s i t i o n s t a t e s t a b i l i z a t i o n . F i g u r e 10: The s t a b i l i z a t i o n o f the oxocarbonium i o n t r a n s i t i o n s t a t e ( r e a c t i o n p e rformed i n CD3OD). 3.2. A c e t y l a t e d G l y c o p y r a n o s i d e s A n o m e r i z a t i o n o f a c e t y l a t e d g l y c o s i d e s has been found to occur u s i n g s t r o n g Lewis a c i d c a t a l y s t s such as s t a n n i c c h l o r i d e , - ^ t i t a n i u m t e t r a c h l o r i d e , l - * and b o r o n t r i f l u o r i d e . ^ E x a m i n a t i o n o f the r e a c t i o n o f v a r i o u s a c e t y l a t e d g l u c o s i d e s w i t h b o r o n t r i f l u o r i d e showed t h a t the r a t e o f a n o m e r i z a t i o n depends on the n a t u r e o f the a g l y c o n : the r e a c t i -v i t y o f d i f f e r e n t g l u c o s i d e s d e c r e a s e s f o l l o w i n g the o r d e r i s o p r o p y l > e t h y l > methyl = a l l y l = b e n z y l . T h e r e f o r e , e l e c t r o n - d o n a t i n g s u b s t i -t u e n t s seem to a c c e l e r a t e the r e a c t i o n . These r e s u l t s can be e x p l a i n e d - 13 -i n terms o f a mechanism i n which the a c i d c a t a l y s t c o o r d i n a t e s w i t h the r i n g oxygen atom, f a c i l i t a t i n g the f o r m a t i o n o f an open c h a i n i n t e r m e d i -a t e s i m i l a r t o ( 8 ) . ^ F u r t h e r e v i d e n c e f o r the open c h a i n i n t e r m e d i a t e was shown by the r e a c t i o n o f methyl t r i - O - a c e t y l - ^ - D - a r a b i n o p y r a n o s i d e (9) w i t h e i t h e r 8% z i n c c h l o r i d e or 0.16% s u l f u r i c a c i d i n a 7:3 a c e t i c a n h y d r i d e - a c e t i c a c i d m i x t u r e . T h e p r o d u c t s i s o l a t e d from t h i s r e a c t i o n were b o t h anomeric forms o f the O - a c e t y l a t e d m e t h y l h e m i a c e t a l (10) i n good and a p p r o x i m a t e l y e q u a l y i e l d s ( F i g u r e 11). Figure 11: Lewis a c i d catalyzed reaction of glycosides 3.3 Penta-O-acetyl Glycopyranoses E q u i l i b r a t i o n o f 1,2,3,4,6-penta-O-acetyl-D-glucopyranose (11) i n a c e t i c a n h y d r i d e w i t h s u l f u r i c a c i d produces a m i x t u r e c o n t a i n i n g 87% o f the a-anomer and 13% o f the /3-anomer.^ A s w i t h the a n o m e r i z a t i o n of - 14 -methyl g l u c o p y r a n o s i d e s , the mechanism i s c o n s i d e r e d t o i n v o l v e unimo-l e c u l a r d i s s o c i a t i o n o f the a c e t a t e a t C ( l ) f o r m i n g an oxocarbonium i o n i n t e r m e d i a t e (12) ( F i g u r e 12). I n v e s t i g a t i o n s by Lemieux e t a l . , ^ 0 - 2 1 and Bonner'^ s u p p o r t t h i s mechanism by comparing the r a t e s o f anomeriza-t i o n w i t h the r a t e s o f a c e t a t e exchange u s i n g compounds l a b e l l e d w i t h •^C a t the C ( l ) a c e t y l group. For the a-anomer (11-a) the r a t e o f a c e t a t e exchange was almost the same as the r a t e o f a n o m e r i z a t i o n s u g g e s t i n g t h a t t h e s e two p r o c e s s e s o c c u r v i a the same i n t e r m e d i a t e (1:1 a c e t i c a n h y d r i d e - a c e t i c a c i d w i t h 0.50 M s u l f u r i c a c i d a t 25°C, k e x c ^ = 0.703 x l O ^ s e c " 1 , k a n o m = 0.68 x 1 0 ' 4 s e c " 1 ) . The /3-peracetate (11-/9), however, was found to exchange a c e t a t e a p p r o x i m a t e l y 15 times f a s t e r t h a n i t anomerized (same c o n d i t i o n s , k e x c Y l = 72.2 x 1 0 " 4 s e c " 1 , k a n o m = 4.93 x 1 0 " 4 sec"-'-). T h i s d i f f e r e n c e i n r a t e s f o r (11-/3) was e x p l a i n e d OAc AcO A c 0 X - ^ b ^ ° A C (11-/8) A c O \ ^ T > A / O A c AcO © - H O A c OAc (12) C H 3 OAC .OAc H O A c (11-«) OAc ©OAc H OAC A c O / V ^ ° \ I ®J (13) O - C F i g u r e 12: The mechanism f o r a c i d c a t a l y z e d a n o m e r i z a t i o n o f 1,2,3,4,6-penta-O-acetyl-D-glucopyranose 15 i n terms o f a n c h i m e r i c a s s i s t a n c e o f the a c e t a t e group a t C(2) to g i v e the acetoxonium i o n (13) shown i n F i g u r e 12. T h i s i n t e r m e d i a t e can be a t t a c k e d o n l y on the /3-face t o r e f o r m (11-/9). T h e r e f o r e , many exchanges o f the C ( l ) a c e t a t e group o c c u r b e f o r e the oxocarbonium i o n i s a t t a c k e d by s o l v e n t on the a - f a c e t o form ( 1 1 - a ) . A n c h i m e r i c a s s i s t a n c e o f t h i s type r e q u i r e s t h a t the groups a t C ( l ) and C(2) have a t r a n s r e l a t i o n -s h i p . Indeed, s i m i l a r r e s u l t s were o b t a i n e d w i t h 1,2,3,4,6-penta-O-acetyl-D-mannopyranose (14). In t h i s c a s e , (14-a) exchanged f a s t e r than i t anomerized s i n c e i t now has a t r a n s d i s p o s i t i o n w i t h the a x i a l a c e t y l group a t C(2) and can form the acetoxonium i o n r e a d i l y (1:1 a c e t i c a c i d - a c e t i c a n h y d r i d e , 0.50 M s u l f u r i c a c i d a t 25°C, k e x c h (14-a) = 3.15 x 1 0 ' 4 s e c " 1 , k a n o m (14-a) = 0.33 x 1 0 " 4 s e c " 1 , k e x c h " (14-0) = 5.5 x 1 0 " 4 s e c - 1 , k a n o m (14-0) = 5.23 x 1 0 " 4 s e c - 1 ) ( F i g u r e 13). F i g u r e 13: The mechanism f o r a c i d c a t a l y z e d a n o m e r i z a t i o n o f 1,2,3,4,6-penta-O-acetyl-D-mannopyranose 16 F u r t h e r e v i d e n c e f o r the above mechanism i s g i v e n by s u c c e s s i v e r e p l a c e m e n t o f the hydrogen atoms on the C(2) a c e t o x y group by c h l o r i n e atoms which causes a d e c r e a s e i n the r a t e s o f b o t h a n o m e r i z a t i o n and exchange.20 T h i s c o n s t i t u t e s e v i d e n c e f o r a carbonium i o n i n t e r m e d i a t e or t r a n s i t i o n s t a t e s i n c e the g r e a t e r e l e c t r o n - w i t h d r a w i n g power o f c h l o r i n e r e l a t i v e t o hydrogen w i l l d e s t a b i l i z e the p o s i t i v e charge a t the anomeric c a r b o n and t h e r e b y slow the r a t e o f the a n o m e r i z a t i o n . Furthermore, the r a t e o f the exchange r e a c t i o n i s r e t a r d e d s i n c e the e l e c t r o n - w i t h d r a w i n g c h l o r i n e atoms d e c r e a s e the n u c l e o p h i l i c i t y o f the c a r b o n y l oxygen on the C(2) a c e t a t e group. 4. H a l i d e Ion C a t a l y z e d A n o m e r i z a t i o n K o e n i g s - K n o r r g l y c o s i d a t i o n i s a method o f p r e p a r i n g 1 , 2 - t r a n s - a r y l g l y c o p y r a n o s i d e s from g l y c o s y l h a l i d e s . 2 ^ For example, t r e a t m e n t o f 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - a - D - g l u c o p y r a n o s y l bromide (16) w i t h 2 - n i t r o -p h e n o l i n the p r e s e n c e o f an e l e c t r o p h i l i c c a t a l y s t (Ag2CC>3) y i e l d s 2 - n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - / 3 - D - g l u c o p y r a n o s i d e (17). The r e a c t i o n o c c u r s v i a u n i m o l e c u l a r d i s s o c i a t i o n o f the bromide i o n f o r m i n g an oxocarbonium i o n which i s t h e n s u b j e c t t o n u c l e o p h i l i c a t t a c k . The s t e r e o s p e c i f i c i t y f o r the f o r m a t i o n o f the /3-anomer i s thought to be due to p a r t i c i p a t i o n by the a c e t y l group a t C(2) f o r m i n g an acetoxonium i o n i n t e r m e d i a t e (18) which can o n l y be s u b s t i t u t e d on the /3-face ( F i g u r e 14). Because o f t h i s s t e r e o s e l e c t i v i t y and the r e l a t i v e s t a b i l i t y o f Q - g l y c o s y l h a l i d e s , t h i s p r o c e d u r e p r o v i d e s a good method f o r p r e p a r i n g a r y l / 3-glucopyranosides. 17 © ,OAc -Br j^. AcO' AcO Br (16) ACQ' F i g u r e 14: The K o e n i g s - K n o r r g l y c o s i d a t i o n S t u d i e s by Lemieux e t a l . 2 4 showed t h a t the above r e a c t i o n can be m o d i f i e d w i t h the a d d i t i o n o f h a l i d e i o n to produce the a - g l y c o s i d e i n e x c e s s . T h i s was termed " h a l i d e i o n c a t a l y z e d g l y c o s i d a t i o n " . In the i n i t i a l s t u d i e s o f the r e a c t i o n between (16-a) and p y r i d i n e , 2 - ^ b o t h anomers o f the p y r i d i n i u m bromide (19) were o b t a i n e d i n n e a r l y , e q u a l amounts. Under the same r e a c t i o n c o n d i t i o n s b u t w i t h a d d i t i o n of tetra-n-butylammonium p e r c h l o r a t e , a 10% e x c ess o f the a-anomer (19-a) was o b t a i n e d . With a d d i t i o n o f tetra-n-butylammonium bromide, the amount o f the a-anomer (19-a) was i n c r e a s e d to >90%. These r e s u l t s were used to p ropose the mechanism shown i n F i g u r e 15. The g l y c o s y l h a l i d e (16-a) i n i t i a l l y undergoes n u c l e o p h i l i c a t t a c k by p y r i d i n e to produce the 0 - p y r i d i n i u m bromide (19-0). The bromide i o n l i b e r a t e d c a t a l y z e s the a n o m e r i z a t i o n o f the a - g l y c o s y l bromide ( 1 6 - a ) . The 0 - g l y c o s y l - 18 -F i g u r e 15: The h a l i d e i o n c a t a l y z e d g l y c o s i d a t i o n . The a - p y r i d i n i u m bromide has been found t o p r e f e r t h e boat c o n f o r m a t i o n shown due t o the r e v e r s e anomeric e f f e c t and the s t e r i c b u l k o f the p y r i d i n i u m group^ bromide (16-/3) formed undergoes n u c l e o p h i l i c a t t a c k f a s t e r t h a n the more s t a b l e a-anomer and hence the a-anomer (19-Q) i s o b t a i n e d . Furthermore, the e f f e c t o f added h a l i d e i o n i s so s t r o n g t h a t the p a r t i c i p a t i o n o f the a c e t a t e a t C(2), which f a c i l i t a t e s ^ - g l y c o s i d e f o r m a t i o n ( F i g u r e 14), becomes i n s i g n i f i c a n t . I n f a c t , a - g l y c o s y l h a l i d e s w i t h non-p a r t i c i p a t i n g groups a t C(2) a r e commonly used i n h a l i d e i o n c a t a l y z e d g l y c o s i d a t i o n s t o form the a - g l y c o s i d e . ^ 6 Thus, r e a c t i o n o f 2,3,4,6-t e t r a - O - b e n z y l - a - D - g l u c o p y r a n o s y l c h l o r i d e w i t h methanol i n the pr e s e n c e o f tetraethylammonium c h l o r i d e y i e l d s 83% o f the methyl a - D - g l y c o s i d e . - 19 Measurement o f f i r s t o r d e r r a t e c o n s t a n t s f o r 2,3,4,6-tetra-O-a c e t y l - / 3 - D - g l u c o p y r a n o s y l c h l o r i d e (20-0) showed t h a t the r a t e i s p r o p o r t i o n a l t o the added c h l o r i d e c o n c e n t r a t i o n . ^ A l s o , the r a t e o f exchange o f the a-anomer (20-a) w i t h r a d i o a c t i v e c h l o r i d e was n e a r l y e q u a l t o the r a t e o f a n o m e r i z a t i o n ( k e x c ^ = 2.0 x 1 0 " 4 min"-*-, k anom = 2.3 ± 0.4 x 1 0 " 4 m i n ' 1 , [20] = 0.2 M, [Me 4NCl] = 0.2 M, 30 ° C ) . These r e s u l t s s u g g e s t an S N 2 - t y p e p r o c e s s . As was seen w i t h the a c i d c a t a l y z e d a n o m e r i z a t i o n o f p e n t a - O - a c e t y l - D - g l u c o p y r a n o s e (11), the r a t e o f exchange o f the 0-glucosyl c h l o r i d e (20-0) was about f o u r times g r e a t e r t h a n i t s r a t e o f a n o m e r i z a t i o n . T h i s o b s e r v a t i o n s u g g e s t s a n c h i m e r i c a s s i s t a n c e by the C(2) a c e t o x y group. The acetoxonium i o n thus p r o d u c e d r e a c t s w i t h the c h l o r i d e i o n to r e f o r m the 0-anomer (20-0) ( F i g u r e 16). F i g u r e 16: The mechanism o f h a l i d e exchange - 20 -5. Base C a t a l y z e d R e a c t i o n s o f G l y c o p y r a n o s i d e s 5.1 H y d r o l y s i s Base c a t a l y z e d r e a c t i o n s i n v o l v i n g the g l y c o s i d i c l i n k a g e are much l e s s common than a c i d c a t a l y z e d r e a c t i o n s . C o n s e q u e n t l y , v e r y l i t t l e i s known about the mechanism and k i n e t i c s o f base c a t a l y z e d t r a n s f o r m a -t i o n s . A l k a l i n e h y d r o l y s i s and d e g r a d a t i o n o f g l y c o s i d e s perhaps have been the most e x t e n s i v e l y s t u d i e d i n t h i s a r e a , y e t the mechanisms are s t i l l v e r y u n c l e a r . G e n e r a l l y , the g l y c o s i d i c l i n k a g e i s more s e n s i t i v e to b a s i c c o n d i t i o n s when the a g l y c o n i s e l e c t r o n - w i t h d r a w i n g . For example, the a l k a l i n e h y d r o l y s i s o f 4 - n i t r o p h e n y l a - D - g l u c o p y r a n o s i d e i s a p p r o x i m a t e l y 600,000 times f a s t e r than p h e n y l a - D - g l u c o p y r a n o s i d e ( [ g l y c o s i d e ] = 0.002 M, [NaOH] = 3.9 M, 7 0 ° C ) . 2 7 I t has a l s o been s u g g e s t e d t h a t i n some c a s e s a r y l - D - g l u c o p y r a n o s i d e s a r e c l e a v e d by a l k a l i w i t h a r y l - o x y g e n f i s s i o n . An u n u s u a l case o f t h i s r e a c t i o n i s the a l k a l i n e h y d r o l y s i s o f 4 - n i t r o p h e n y l a - D - g l u c o p y r a n o s i d e ( 2 1 ) . ^ The mechanism i s c o n s i d e r e d t o i n v o l v e 0(1) to 0(2) m i g r a t i o n o f the 4 - n i t r o p h e n y l group f o l l o w e d by 0(2) to 0(3) m i g r a t i o n o f the a r y l group to g i v e 3 - 0 - ( 4 - n i t r o p h e n y l ) - D - g l u c o p y r a n o s e . Subsequent h y d r o l y s i s l i b e r a t e s the 4 - n i t r o p h e n o x i d e a n i o n v i a a / 3 - e l i m i n a t i o n pathway and produces the sugar r e s i d u e as a m i x t u r e o f s a c c h a r i n i c a c i d s (23) ( F i g u r e 17). The mechanism i s s u p p o r t e d by the i s o l a t i o n and c h a r a c t e r -i z a t i o n o f the 2 - 0 - ( 4 - n i t r o p h e n y l ) - D - g l u c o p y r a n o s e (22). T h i s m i g r a t i o n p a t t e r n i s u n u s u a l s i n c e i t may i n v o l v e an i n t r a m o l e c u l a r , n u c l e o p h i l i c , a r o m a t i c s u b s t i t u t i o n r e a c t i o n and c l e a v a g e o f the a r y l - o x y g e n bond. - 21 -F i g u r e 17: The base c a t a l y z e d h y d r o l y s i s o f 4 - n i t r o p h e n y l a-D-gluco-p y r a n o s i d e O t h e r mechanisms f o r a l k a l i f i s s i o n o f g l y c o s i d e s a r e v a r i e d and o f t e n depend on the o r i e n t a t i o n o f h y d r o x y l groups around the pyranose r i n g . 2 9 ^ - G l y c o s i d e s w i t h the C(2) h y d r o x y l t r a n s to the a g l y c o n f r e q u e n t l y y i e l d the 1,6 anhydro compound v i a i n i t i a l f o r m a t i o n o f the 1,2-epoxide. Thus, 4 - n i t r o p h e n y l - / 3 - D - g a l a c t o p y r a n o s i d e (24) r e a c t s w i t h sodium methoxide i n methanol to y i e l d 76% o f 1 , 6 - a n h y d r o g a l a c t o s e (25) and 8% methyl 0 - D - g a l a c t o p y r a n o s i d e (26) ( F i g u r e 18). E v i d e n c e f o r p a r t i c i p a t i o n o f the C(2) h y d r o x y l and the i n i t i a l f o r m a t i o n o f the 1,2-epoxide was o b t a i n e d by p e r f o r m i n g the same r e a c t i o n w i t h 4 - n i t r o -p h e n y l -a-D-mannopyranoside ( 2 7 ) . H e r e , o n l y methyl a-D-manno-p y r a n o s i d e (28) was produced s i n c e the 1,2-epoxide i n i t i a l l y formed has the wrong c o n f i g u r a t i o n f o r r e a c t i o n w i t h the C(6) h y d r o x y l group ( F i g u r e 19). - 22 F i g u r e 18: The h y d r o l y s i s o f 4 - n i t r o p h e n y l /3-D-galactopyranoside A r = 4 - n i t r o p h e n y l v — H O ' x ^ - © < ; HO (27) OAr OAr -ArO^ H 0 ^ \ £ ^ < \ M e 0 H F i g u r e 19: (28) The h y d r o l y s i s o f 4 - n i t r o p h e n y l a-D-mannopyranoside A r = 4 - n i t r o p h e n y l OMe 5.2 A n o m e r i z a t i o n The p r e c e d e n c e f o r base c a t a l y z e d a n o m e r i z a t i o n r e a c t i o n s i s v e r y l i m i t e d . Up to 1937, the o n l y example o f base c a t a l y z e d a n o m e r i z a t i o n 23 -was the m u t a r o t a t i o n o f n o n - s u b s t i t u t e d r e d u c i n g sugars such as g l u c o s e . As e x p l a i n e d e a r l i e r , t h i s t r a n s f o r m a t i o n i s e f f e c t e d by b o t h a c i d and base c a t a l y s i s . Wolfrom and Husted were the f i r s t t o r e p o r t the a n o m e r i z a t i o n o f a f u l l y p r o t e c t e d sugar u s i n g a l k a l i i n s t e a d o f a c i d . 3 ! They c o n v e r t e d 1, 2 , 3 ,4, 6-penta-0-acetyl-/9-D-glucopyranose (11-/3) to the a-form u s i n g a s u s p e n s i o n o f the sugar and s o l i d sodium h y d r o x i d e i n anhydrous e t h e r . The method was n o t an improvement over a c i d c a t a l y z e d p r e p a r a t i o n s s i n c e i t was accompanied by a t l e a s t a s m a l l amount o f d e a c e t y l a t i o n . S t i l l , i t p r o v i d e d an u n u s u a l example o f the base c a t a l y z e d i n t e r c o n v e r s i o n o f anomeric s u g a r s . L i n d b e r g s u g g e s t e d t h a t the above r e a c t i o n i n v o l v e s heterogeneous op c a t a l y s i s . c He r e p o r t e d t h a t when a m i x t u r e o f s o l i d sodium h y d r o x i d e and D r i e r i t e was shaken i n e t h e r , f i l t e r e d , and the f i l t r a t e used as the r e a g e n t f o r the a n o m e r i z a t i o n o f (11-/3) , t h e r e was no t r a c e o f base i n the s o l u t i o n and the t r a n s f o r m a t i o n from 3 to a d i d n o t o c c u r . L i n d b e r g a l s o showed t h a t the a n o m e r i z a t i o n o f 2 , 4 - d i n i t r o p h e n y l 2,3,4,6-tetra-O-a c e t y l - / 3 - D - g l u c o p y r a n o s i d e (29-3) c o u l d be e f f e c t e d u s i n g A s c a r i t e i n anhydrous p y r i d i n e . A l t h o u g h the y i e l d o f the a-anomer (29-a) was o n l y 30%, t h i s r e a c t i o n was one o f the f i r s t methods found to t r a n s f o r m a p h e n y l /9-glycoside i n t o the a - g l y c o s i d e . I n t e r e s t i n g l y , a n o m e r i z a t i o n o f the ^ - g l u c o s i d e s o f p h e n o l , 2 - n i t r o p h e n o l and 4 - n i t r o p h e n o l by the same method were u n s u c c e s s f u l . I n 1980, van Boom e t a l . r e p o r t e d f u r t h e r s t u d i e s on the anomeri-z a t i o n o f ( 2 9 - / 8 ) . 3 3 They p r e p a r e d the 2 , 4 - d i n i t r o p h e n y l (DNP) /9-D-glucoside (29-8) from 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - D - g l u c o p y r a n o s e (30) 24 -and f l u o r o d i n i t r o b e n z e n e (FDNB) i n DMF u s i n g 1 , 4 - d i a z a b i c y c l o - [ 2 . 2 . 2 ] -octane (DABCO) as a base c a t a l y s t ( F i g u r e 20). E x c l u s i v e p r o d u c t i o n o f the /3-glucoside i s due to k i n e t i c c o n t r o l o f the g l y c o s i d a t i o n r e a c t i o n . Because o f the s t e r i c b u l k o f the d i n i t r o p h e n y l group, the r e a c t i o n o f FDNB w i t h the /9-anomer, which has a l e s s h i n d e r e d e q u a t o r i a l h y d r o x y l a t C ( l ) , p r o c e e d s f a s t e r t h a n the r e a c t i o n w i t h the a-anomer. Subsequent r e - e q u i l i b r a t i o n o f (30-a) and (30-6) d r i v e s the r e a c t i o n towards e x c l u s i v e p r o d u c t i o n o f the l e s s t h e r m o d y n a m i c a l l y s t a b l e /3-anomer (29-/9). Furthermore, no a n o m e r i z a t i o n o r rearrangement o f the DNP e t h e r group was obser v e d , and a h i g h y i e l d (82%) o f (29-/9) was o b t a i n e d . In comparison, p r e v i o u s r o u t e s t o the DNP-glycoside i n v o l v e d t r e a t m e n t o f t e t r a - O - a c e t y l g l u c o s y l h a l i d e s w i t h 2 , 4 - d i n i t r o p h e n o l i n the p r e s e n c e o f p o t a s s i u m c a r b o n a t e t o a f f o r d the p r o t e c t e d 1,2-trans g l y c o s i d e s i n r e l a t i v e l y low y i e l d s ( 8 - 5 0 % ) . 3 4 • 3 5 F i g u r e 20: The p r e p a r a t i o n and a n o m e r i z a t i o n o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - 6 - D - g l u c o p y r a n o s i d e - 25 -Treatment o f (29-B) w i t h s o l i d p o t a s s i u m c a r b o n a t e i n DMF a f f o r d e d an e q u i l i b r i u m m i x t u r e o f the a- and B- anomers (80:20 r e s p e c t i v e l y ) i n good y i e l d ( F i g u r e 20). A c l u e to the mechanism o f the a n o m e r i z a t i o n was o b t a i n e d when they r e a c t e d 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - 0 - b e n z y l -^ - D - g l u c o p y r a n o s i d e (31) under the same r e a c t i o n c o n d i t i o n s and found the r e a c t i o n t o p r o c e e d a p p r o x i m a t e l y 70 times more s l o w l y than the a c e t y l a t e d DNP sugar ( 2 9 ) . Hence i t was s u g g e s t e d t h a t the a c e t y l group a t C(2) may p r o v i d e some n e i g h b o r i n g group a s s i s t a n c e t o the r e a c t i o n . F u r t h e r s t u d i e s towards the mechanism o f t h i s a n o m e r i z a t i o n and i n v e s -t i g a t i o n i n t o the r o l e o f the p o t a s s i u m c a r b o n a t e a r e the s u b j e c t o f t h i s t h e s i s . - 26 -RESULTS 1. S y n t h e s i s 1.1 2 , 4 - D i n i t r o p h e n y l /J-D-glucopyranosides P e r - O - a c e t y l a t e d 2 , 4 - d i n i t r o p h e n y l /3-D-glucopyranosides were r o u t i n e l y p r e p a r e d u s i n g the method o f van Boom e t . a l . ^  i n which the a p p r o p r i a t e sugar was r e a c t e d w i t h FDNB i n DMF u s i n g DABCO as a base c a t a l y s t . A l l o f the /9-2,4-DNPG d e r i v a t i v e s were h i g h l y c r y s t a l l i n e and were r e c r y s t a l l i z e d from warm e t h a n o l . Y i e l d s f o r the d e r i v a t i z a -t i o n r e a c t i o n were g r e a t e r t h a n 80%. 1.2 Deuterium L a b e l l e d 2 , 4 - D i n i t r o p h e n y l 0 - D - g l u c o p y r a n o s i d e s The r o u t e f o r the p r e p a r a t i o n o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - 0 -a c e t y l - / 3 - D - [ 1 - 2 H ] - g l u c o p y r a n o s i d e (32) i s g i v e n i n F i g u r e 21.^6 Lewis a c i d c a t a l y z e d a c e t y l a t i o n D - g l u c o n o - 1 , 5 - l a c t o n e was pe r f o r m e d essen-t i a l l y a c c o r d i n g to N e l s o n - ^ u s i n g a c e t i c a n h y d r i d e and z i n c c h l o r i d e . Removal o f t r a c e s o f a c e t i c a n h y d r i d e was a c h i e v e d by s t i r r i n g the r e a c t i o n m i x t u r e i n s a t u r a t e d sodium b i c a r b o n a t e . Simple work-up and e v a p o r a t i o n o f the s o l v e n t produced the t e t r a - O - a c e t y l l a c t o n e (33) as a c o l o r l e s s gum i n 92% y i e l d . The l a c t o n e was red u c e d w i t h sodium boro-d e u t e r i d e , p r o d u c i n g a m i x t u r e o f the a- and / 9 - [ 1 - 2 H ] - t e t r a a c e t a t e s (34) 27 -F i g u r e 21: S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - / 3 -D - [ l - ^ H ] - g l u c o p y r a n o s i d e which were d e r i v a t i z e d t o the /3-2,4-DNPG compound ( 3 2 ) . The c r y s t a l -l i n e /3-2,4-DNPG d e r i v a t i v e was o b t a i n e d i n 6 6 % o v e r a l l y i e l d from the l a c t o n e . A l t e r n a t i v e methods o f deu t e r i u m s u b s t i t u t i o n r e p o r t e d i n the l i t e r a t u r e 3 ^ ' 3 ^ . 4 0 w h i c h were c o n s i d e r e d h e r e i n c l u d e d i r e c t r e d u c t i o n o f D - g l u c o n o - 1 , 5 - l a c t o n e i n s o l u t i o n w i t h sodium amalgam i n the pr e s e n c e o f p h o s p h o r i c a c i d - d 3 , f o l l o w e d by a c e t y l a t i o n , o r r e d u c t i o n o f the t e t r a h y d r o p y r a n y l d e r i v a t i v e o f D - g l u c o n o - 1 , 5 - l a c t o n e w i t h sodium b o r o d e u t e r i d e i n THF, a c i d - c a t a l y z e d h y d r o l y s i s i n D 2 O o f the p r o t e c t i n g groups, and a c e t y l a t i o n w i t h a c e t i c a n h y d r i d e and sodium a c e t a t e . Y i e l d s f o r b o t h o f these approaches, however, a r e r e p o r t e d t o be o n l y about 30%. The d e u t e r a t e d t e t r a - O - b e n z y l d e r i v a t i v e (35) was p r e p a r e d a c c o r d -i n g t o the scheme i n F i g u r e 22. The o x i d a t i o n o f 2 , 3 , 4 , 6 - t e t r a - 0 -28 OBn B n 0 ^ 1 ^ ° \ B n o X ^ o V (36) AC2O OBn OH D M S 0 B n O ^ X - - - - A N a B D 4 ^ * B n o V ^ o \ TD„° (37) ,OBn B n o V S o V D ™CF° B n 0 ^ ? o V (38) 0 H (35) ODNP F i g u r e 22: S y n t h e s i s o f B-2,4-dinitrophenyl 2 , 3 , 4 , 6 - t e t r a - 0 - b e n z y l -/3-D- [1- 2H] - g l u c o p y r a n o s e b e n z y l - D - g l u c o p y r a n o s e (36) was p e r f o r m e d by the method o f Kuzuhara and F l e t c h e r 4 1 and the l a c t o n e (37) was i s o l a t e d as a c o l o r l e s s syrup i n h i g h y i e l d . Subsequent r e d u c t i o n to (38) and d e r i v a t i z a t i o n a f f o r d e d the d e u t e r a t e d DNP d e r i v a t i v e (35) i n 62% o v e r a l l y i e l d . 1.3 Deoxy and D e o x y f l u o r o 2 , 4 - D i n i t r o p h e n y l /3-D-glucopyranosides The s y n t h e t i c r o u t e f o r the p r e p a r a t i o n o f the 4 - d e o x y - 4 - f l u o r o d e r i v a t i v e (40) i s g i v e n i n F i g u r e 23. Treatment o f 1,2,3,6-tetra-0-a c e t y l - 4 - d e o x y - 4 - f l u o r o - / 9 - D - g l u c o p y r a n o s e (41) w i t h 45% hydrobromic a c i d i n a c e t i c a c i d and a c e t i c a n h y d r i d e 4 2 a f f o r d e d the bromide (42) as a c o l o r l e s s s y r u p i n 85% y i e l d . H y d r o l y s i s o f the bromide (42) w i t h s i l v e r c a r b o n a t e and water i n a c e t o n e 4 3 y i e l d e d a brown syrup which c o n s i s t e d p r i m a r i l y o f the h y d r o l y s i s p r o d u c t (43) (-70%) and was not 29 -ODNP F i g u r e 23: S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 6 - t r i - 0 - a c e t y l - 4 - d e o x y 4 - f l u o r o - 0 - D - g l u c o p y r a n o s i d e p u r i f i e d . D e r i v a t i z a t i o n by the s t a n d a r d method gave the 4-deoxy-4-f l u o r o DNPG (40) as y e l l o w c r y s t a l s i n 34% o v e r a l l y i e l d . S y n t h e s i s o f the 4-deoxy DNPG d e r i v a t i v e (44) was a c h i e v e d from m e t h y l 2 , 3 , 6 - t r i - 0 - b e n z o y l - 4 - d e o x y - a - D - g l u c o p y r a n o s i d e (45) as shown i n F i g u r e 24. D e p r o t e c t i o n o f (45) w i t h sodium methoxide i n m e t h a n o l 4 4 f o l l o w e d by a c e t i c a n h y d r i d e / p y r i d i n e a c e t y l a t i o n a f f o r d e d (46) as a whit e s o l i d . F o r m a t i o n o f the g l y c o s y l c h l o r i d e (47) was a c h i e v e d by the method o f Kovac e t . a l . 4 ^ u s i n g z i n c c h l o r i d e and d i c h l o r o m e t h y l m e t h y l e t h e r t o o b t a i n a brown syrup which c o n s i s t e d o f o n l y one com-ponent by t . l . c . (presumably the c h l o r o s u g a r ) . Subsequent h y d r o l y s i s 4 - ^ to form (48) and d e r i v a t i z a t i o n a f f o r d e d the 4-deoxy DNPG (44) as y e l l o w c r y s t a l s i n 51% o v e r a l l y i e l d . S y n t h e s i s o f the 6-deoxy DNPG (49) was a c h i e v e d by the same r o u t e u s e d f o r the p r e p a r a t i o n o f the 4 - d e o x y - 4 - f l u o r o d e r i v a t i v e (40) ( F i g u r e 30 -F i g u r e 24: S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 6 - t r i - 0 - a c e t y l - 4 - d e o x y -/3-D-glucopyranose .ODNP (52) (49) F i g u r e 25: S y n t h e s i s o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 - t r i - O - a c e t y l - 6 - d e o x y -0 - D - g l u c o p y r a n o s i d e 25). The 6-deoxy DNPG (49) was o b t a i n e d as y e l l o w c r y s t a l s i n 83% o v e r a l l y i e l d . 31 1.4 P e r a c e t y l a t e d S u b s t i t u t e d P h e n y l G l u c o p y r a n o s i d e s The s u b s t i t u t e d p h e n y l /3-D-glucopyranosides l i s t e d i n T a b l e 1 were p r e p a r e d by c o n d e n s a t i o n o f the sodium s a l t o f the p h e n o l w i t h 2,3,4,6-t e t r a - O - a c e t y l - a - D - g l u c o p y r a n o s y l bromide (16) i n aqueous a c e t o n e , ^ The /3-D-glucopyranoside was formed e x c l u s i v e l y i n each c a s e . However, y i e l d s were g e n e r a l l y low due to the f o r m a t i o n o f 2 , 3 , 4 , 6 - t e t r a - 0 -a c e t y l - D - g l u c o p y r a n o s e by h y d r o l y s i s o f the bromide ( F i g u r e 26). F i g u r e 26: S y n t h e s i s o f s u b s t i t u t e d p h e n y l /3-D-glucopyranosides 2. M e c h a n i s t i c I n v e s t i g a t i o n s 2.1 G e n e r a l A n o m e r i z a t i o n Procedure The /3-2,4-DNPG d e r i v a t i v e s (29,31,32,35,39,40,44,49) as w e l l as the s u b s t i t u t e d p h e n y l g l u c o p y r a n o s i d e s (53-60) were anomerized by the method o f van Boom e t a l . ^ 3 u s i n g p o t a s s i u m c a r b o n a t e as a c a t a l y s t . Due t o the p o s s i b l e d e c o m p o s i t i o n o f DMF under b a s i c c o n d i t i o n s , 4 ^ however, DMSO was s u b s t i t u t e d as the s o l v e n t i n most e x p e r i m e n t s . In g e n e r a l , the r e a c t i o n m i x t u r e was s t i r r e d o v e r n i g h t o r l o n g e r to o b t a i n 32 -T a b l e 1: S u b s t i t u t e d p h e n y l g l u c o p y r a n o s i d e s and o b t a i n e d y i e l d s R % y i e l d R % y i e l d (53) 46 (57) -< f ) > 51 N02 N02 (54) NOo 35 NO, (58) ~\^_J > 3 3 N02 (55) F 51 CI (59) - ^ ^ ^ - N Q 2 58 CI (56) NO, N02 57 N O (60) 55 NOo - 33 an e q u i l i b r i u m m i x t u r e o f the anomers. S e p a r a t i o n o f the m i x t u r e o f a-and 0-anomers was a c h i e v e d by f r a c t i o n a l c r y s t a l l i z a t i o n i n warm e t h a n o l . The a n o m e r i z a t i o n r e a c t i o n s were r o u t i n e l y m o n i t o r e d by t . I . e . i n which the a- and 0-glucopyranosides were d e t e c t e d by u.v. l i g h t o r c h a r r i n g and the a- and 0-anomers t y p i c a l l y had R f v a l u e s which d i f f e r e d by about 0.16 ( s t a n d a r d t . l . c . c o n d i t i o n s , Rf(/3) = 0.36, R f ( a ) = 0.52). When a c c u r a t e d e t e r m i n a t i o n o f the amounts o f each anomer was n e c e s s a r y , the r e a c t i o n m i x t u r e was worked-up w i t h o u t s e p a r a t i o n o f the p r o d u c t s . The amount o f each anomer formed was d e t e r m i n e d by i n t e g r a t i o n o f the •'•H-n.m.r. spectrum o f the p r o d u c t m i x t u r e . When d i s s o l v e d i n C D C I 3 , the r a t i o o f a- and /3-anomers g e n e r a l l y was d e t e r m i n e d by i n t e g r a t i o n o f the resonance f o r H(6) o f the p h e n y l r i n g ( F i g u r e 27) i n which the resonance f o r the 0-anomer i s s h i f t e d about 0.10 ppm u p f i e l d from t h a t o f the a-anomer. I n DMSO-dg, resonances f o r the sugar r i n g p r o t o n s were more d i s t i n c t and the a/0 r a t i o c o u l d be de t e r m i n e d by i n t e g r a t i o n o f the anomeric resonance f o r each compound ( F i g u r e 28). I n i t i a l l y m i n i m a l c a r e was taken t o remove water from the r e a c t i o n m i x t u r e . I n g e n e r a l , s o l v e n t s were d i s t i l l e d and r e a c t a n t s were d r i e d o v e r n i g h t i n vacuo, b u t the r e a c t i o n i t s e l f was n o t p e r f o r m e d i n an anhydrous environment. L a t e r i t was d e t e r m i n e d t h a t the p r e s e n c e o f even a s m a l l amount o f water has a pronounced e f f e c t on the anomeriza-t i o n , p a r t i c u l a r l y on r a t e measurements. T h i s e f f e c t was o r i g i n a l l y b e l i e v e d t o be due to a h y d r o l y s i s s i d e r e a c t i o n s i n c e t . l . c , and n.m.r. and u . v . / v i s s p e c t r o s c o p y showed the p r e s e n c e o f the 2 , 4 - d i n i t r o p h e n o l a t e a n i o n a f t e r a n o m e r i z a t i o n o f ( 2 9 ) . I n o r d e r to - 34 -F i g u r e 27: H-N.m.r. spectrum o f /9-2,4-DNPG (29-/3) and a-2,4-DNPG (29-a) i n CDC1 3 35 -lH-N.m.r. spectrum o f /3-2,4-DNPG <29-£) and a-2,4-DNPG (29-a) In DMSO-Dg 36 determine the e f f e c t o f water on the a n o m e r i z a t i o n and on the g e n e r a t i o n o f the 2 , 4 - d i n i t r o p h e n o l a t e a n i o n , the r e a c t i o n was m o n i t o r e d by means of u . v . / v i s s p e c t r o p h o t o m e t r y a t the wavelength o f the 2 , 4 - d i n i t r o -p h e n o l a t e absorbance ( A m a x = 429 nm). These r e s u l t s a r e shown i n F i g u r e 29. As c a n be seen, the a d d i t i o n o f water r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n the r a t e o f g e n e r a t i o n o f d i n i t r o p h e n o l a t e , whereas scrupu-l o u s removal o f water r e s u l t e d i n a d r a m a t i c i n c r e a s e i n the r a t e . S imultaneous t . l . c . o f these r e a c t i o n m i x t u r e s showed t h a t a l t h o u g h a d d i t i o n o f water a p p a r e n t l y slows the r e a c t i o n , i t does not p r e v e n t a n o m e r i z a t i o n e n t i r e l y . These r e s u l t s suggest t h a t f o r m a t i o n o f the d i n i t r o p h e n o l a t e a n i o n i s n o t a consequence o f h y d r o l y s i s , b u t perhaps i s formed by some o t h e r s i d e r e a c t i o n . I n any case, water d r a m a t i c a l l y a f f e c t s the r a t e o f the r e a c t i o n and the r e a s o n f o r t h i s o b s e r v a t i o n i s n o t e n t i r e l y c l e a r . As a r e s u l t , g r e a t e r c a r e was n e c e s s a r y to e l i m i -n a t e water from the system, p a r t i c u l a r l y w i t h k i n e t i c measurements. 2.2 A n o m e r i z a t i o n o f S u b s t i t u t e d P h e n y l G l u c o p y r a n o s i d e s The p e r - O - a c e t y l a t e d a r y l g l u c o p y r a n o s i d e s i n T a b l e 2 were sub-m i t t e d t o the a n o m e r i z a t i o n c o n d i t i o n s ( p o t a s s i u m c a r b o n a t e , DMSO) to determine the e f f e c t o f s u b s t i t u t i o n o f the p h e n y l group on the r e a c -t i o n . O nly the 2 , 6 - d i n i t r o p h e n y l g l u c o p y r a n o s i d e (60) and the 2 , 6 - d i c h l o r o - 4 - n i t r o p h e n y l g l u c o p y r a n o s i d e (59) anomerized and the r a t e s o f a n o m e r i z a t i o n were c o n s i d e r a b l y s l o w e r than t h a t f o r the 2 , 4 - d i n i t r o -p h e n y l g l u c o p y r a n o s i d e (29) as d e t e r m i n e d by t . l . c . o f the r e a c t i o n - 37 -TIME (hours) Figure 29 : Absorbance vs time f o r generation of 2 , 4-dinitrophenolate ( A m a x - 429 nm) during the anomerization of (29) ; • water added, • no water added, no s p e c i a l drying precautions, • a l l reagents dried In vacuo - 38 -Table 2: Anomerization of substituted phenyl glucopyranosides. pK a values are those f o r the corresponding phenol and were determined i n water.48,49 Glucoside R pK a B -» a Glucoside R p K a B - a (53) NO, 9.98 no (58) 5 .15 no (54) CI 7.15 no (59) ~^Q^-N0 2 3 .15 yes CI (55) - ^ ^ ^ - F 5 .32 no NO, (60) —C( y > 3 .77 yes NOz ( 5 6 ) - f ^ ) - N 0 2 5 .36 no (29) ^ N O , N0 2 0 2 4 . 0 0 yes (57) - U )) 4 . 8 9 no N0 2 N0 2 - 39 -m i x t u r e and n.m.r. a n a l y s i s o f the p r o d u c t m i x t u r e . S i n c e 2 , 6 - d i c h l o r o -4 - n i t r o p h e n o l and 2 , 6 - d i n i t r o p h e n o l a r e the o n l y two a g l y c o n e s which have pKa's lower than t h a t o f 2 , 4 - d i n i t r o p h e n o l , t h e n the r e a c t i o n appears t o depend on the r e l a t i v e e l e c t r o n - w i t h d r a w i n g power o f the s u b s t i t u t e d p h e n y l group. T h i s e f f e c t may be combined w i t h i n h i b i t o r y s t e r i c f a c t o r s a s s o c i a t e d w i t h the e x t r a o r t h o s u b s t i t u t i o n i n the p h e n y l group which a r e not p r e s e n t w i t h 2,4-DNPG (29) and thus may e x p l a i n the r e l a t i v e l y slow r a t e s o f a n o m e r i z a t i o n f o r (59) and (60). I n a d d i t i o n , the s u l f u r analogue (61) was s u b m i t t e d t o the a n o m e r i z a t i o n c o n d i t i o n s and was found to be u n r e a c t i v e . 2.3 Exchange R e a c t i o n s The a n o m e r i z a t i o n o f /3-2,4-DNPG (29) may p r o c e e d v i a t h r e e p o s s i b l e mechanisms i n v o l v i n g e i t h e r ( i ) C ( l ) - H ( l ) bond c l e a v a g e ( p r o t o n a b s t r a c -t i o n ) , ( i i ) C ( l ) - 0 ( 1 ) bond c l e a v a g e ( p h e n o l a t e d e p a r t u r e ) , or ( i i i ) 0 ( l ) - a r y l bond c l e a v a g e ( n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n ) ( F i g u r e 30). ( D e t a i l s o f these mechanisms a r e c o n s i d e r e d i n the D i s c u s s i o n N02 (61) AO -OAc N 0 2 F i g u r e 30: P o s s i b l e s i t e s o f bond c l e a v a g e s e c t i o n ) . I n a l l t h r e e c a s e s , the i n t e r m e d i a t e s g e n e r a t e d by bond c l e a v a g e s h o u l d be exchangeable w i t h a p p r o p r i a t e e q u i v a l e n t s u b s t a n c e s added to the r e a c t i o n m i x t u r e . T h e r e f o r e a s e r i e s o f exchange r e a c t i o n s was p e r f o r m e d i n an attempt to determine which bond i s c l e a v e d d u r i n g a n o m e r i z a t i o n . 41 -P r o t o n a b s t r a c t i o n , case ( i ) , was t e s t e d i n two exper i m e n t s . The f i r s t i n v o l v e d r e a c t i n g /3-2,4-DNPG (29) under normal a n o m e r i z a t i o n c o n d i t i o n s , b u t w i t h the a d d i t i o n o f t-BuOD ( p r e p a r e d from t-BuOH and D 2 0 ) . Exchange between the d e u t e r o n and H ( l ) on /3-2,4-DNPG (29) s h o u l d o c c u r i f the r e a c t i o n proceeds v i a anomeric p r o t o n a b s t r a c t i o n . Deuter-a t e d t - b u t a n o l was chosen as a d e u t e r o n s o u r c e s i n c e t - b u t o x i d e i s a r e l a t i v e l y poor n u c l e o p h i l e and t h e r e f o r e , would be u n l i k e l y t o cause s i d e r e a c t i o n s . The r e a c t i o n was c a r r i e d out i n the p r e s e n c e o f a 100 f o l d mole excess o f t-BuOD and the m i x t u r e was s t i r r e d f o r 2 days, a f t e r which time a p p r o x i m a t e l y 50% c o n v e r s i o n t o the a-anomer had o c c u r r e d . ^H-N.m.r. a n a l y s i s i n v o l v i n g a comparison o f the i n t e g r a t i o n v a l u e s f o r the a-, /3- a r o m a t i c resonances w i t h those o f the a-,/3-anomeric r e s o n a n c e s showed t h a t no exchange had o c c u r r e d ( F i g u r e 31). T — r 6.0 7.5 7.0 6.5 6 0 PPM F i g u r e 31: iH-N.m.r. o f H/D exchange experiment (DMSO-dg) - 42 The c o n v e r s e H/D exchange experiment a l s o was performed. In t h i s experiment, d e u t e r i u m l a b e l l e d /3-2,4-DNPG (32) was r e a c t e d under the same a n o m e r i z a t i o n c o n d i t i o n s i n the p r e s e n c e o f normal t-BuOH (100 f o l d mole e x c e s s ) . •'-H-N.m.r. a n a l y s i s i n t h i s c ase would show H/D exchange v e r y c l e a r l y s i n c e the anomeric resonance absent i n the •'-H-n.m.r. spectrum o f [1- 2H]-2,4-DNPG would be r e a d i l y a p p a r e n t i f the de u t e r i u m atoms a r e exchanged f o r hydrogen. A g a i n , no exchange was o b s e r v e d ( F i g u r e 32) . -v r * * * * | *—v—»—»—|—>—•—v—t—j—v—t—v—r—|—u—IT——r—j—r—>—t—»—(•—»—•—r—•—r 8.0 7.0 6.0 F i g u r e 32: 1H-N.m.r. o f D/H exchange experiment (CDC1 3) The p o s s i b i l i t y o f p h e n o l a t e d e p a r t u r e and r e c o m b i n a t i o n , case ( i i ) , was i n v e s t i g a t e d by a t t e m p t i n g to exchange the 2,4-DNP group f o r - 43 -the 2,6-DNP group. ( R e c a l l t h a t o f a l l the g l y c o s i d e s s t u d i e d i n T a b l e 2, /8-2,6-DNPG (60) was one o f o n l y t h r e e g l y c o s i d e s found to anom e r i z e ) . I f the r e a c t i o n o c c u r s v i a c l e a v a g e o f the C ( l ) - 0 ( 1 ) bond, then 2 , 4 - d i n i t r o p h e n o l a t e i s ge n e r a t e d , and i f an o t h e r p h e n o l a t e o f s i m i l a r pKa such as 2 , 6 - d i n i t r o p h e n o l a t e i s p r e s e n t , t h e n a m i x t u r e o f a-,0-2,4-DNPG (29) and a-,6-2,6-DNPG (60) s h o u l d r e s u l t . ^-H-N.m.r. a n a l y s i s o f the p r o d u c t m i x t u r e would i n d i c a t e i f exchange had o c c u r r e d . A g a i n , two experim e n t s were performed. The f i r s t i n v o l v e d r e a c t i n g /?-2,4-DNPG (29) under the normal a n o m e r i z a t i o n c o n d i t i o n s i n the pr e s e n c e o f added p o t a s s i u m 2 , 6 - d i n i t r o p h e n o l a t e (one e q u i v a l e n t ) . The second i n v o l v e d r e a c t i n g 6-2,6-DNPG (60) under the same c o n d i t i o n s i n the p r e s e n c e o f added p o t a s s i u m 2 , 4 - d i n i t r o p h e n o l a t e (one e q u i v a l e n t ) . The r e a c t i o n s were s t i r r e d f o r t h r e e days a f t e r which time t . l . c . i n d i c a t e d t h a t 80% c o n v e r s i o n t o the a-anomer ( e i t h e r 2,6-DNPG o r 2,4-DNPG) h a d o c c u r r e d i n the f i r s t r e a c t i o n , and 50% c o n v e r s i o n i n the second r e a c t i o n . I n b o t h c a s e s , ^H-n.m.r. a n a l y s i s o f the p r o d u c t m i x t u r e i n d i c a t e d t h a t no exchange had o c c u r r e d ( F i g u r e 33). The r e a c t i o n o f the 0-2,6-DNPG (60) was r e p e a t e d w i t h 10 f o l d mole excess o f the 2 , 4 - d i n i t r o p h e n o l a t e . A g a i n ^H-n.m.r. i n d i c a t e d t h a t no exchange had o c c u r r e d . An a l t e r n a t i v e mechanism l e a d i n g t o the c l e a v a g e o f the C ( l ) - 0 ( 1 ) bond would i n v o l v e p r o t o n a b s t r a c t i o n a t C(2) and r e s u l t i n g e n e r a t i o n o f a p r o t e c t e d g l u c a l i n t e r m e d i a t e v i a e l i m i n a t i o n o f 2 , 4 - d i n i t r o -p h e n o l a t e ( F i g u r e 34). However, t h i s r o u t e s h o u l d a l s o l e a d t o exchange between 2,6-DNP and 2,4-DNP. Furthermore, p r o t o n a b s t r a c t i o n a t C(2) s h o u l d l e a d t o H/D exchange a t C(2) and perhaps some e p i m e r i z a t i o n t o - 44 -2,6-DNPG + 2,4-DNP® H(3),H(5) 2,6-DNPG 2,4-DNPG +2,6-DNP H(3),H(5) Figure 33: •"-H-N.m.r. spectra of attempted phenolate exchange experiments (CDCI3) - 45 -the marine-side d e r i v a t i v e . A g a i n , ^H-n.m.r. s p e c t r a o f the p r o d u c t m i x t u r e from the a n o m e r i z a t i o n o f /3-2,4-DNPG (29) i n the p r e s e n c e o f a d e u t e r o n s o u r c e showed no H/D exchange o r e p i m e r i z a t i o n a t C ( 2 ) . Furthermore, when 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - D - g l u c a l (62) was r e a c t e d w i t h 2 , 4 - d i n i t r o p h e n o l i n p o t a s s i u m carbonate/DMSO, no DNPG was formed, a l s o p r o v i d i n g e v i d e n c e a g a i n s t the g l u c a l (62) as an i n t e r m e d i a t e . .OAc A c O T ' ^ * ^ y A c 0 ^ ACO (29) ODNP OAc A c O ^ l ^ f v A c O V ^ J o ^ Q D N P F i g u r e 34: A l t e r n a t i v e r o u t e t o C ( l ) - 0 ( 1 ) bond c l e a v a g e C l e a v a g e o f the 0 ( 1 ) - a r y l bond o r case ( i i i ) i s p o s s i b l e i f the r e a c t i o n p r o c e e d s v i a n u c l e o p h i l i c s u b s t i t u t i o n on the a r o m a t i c r i n g , perhaps w i t h the c a r b o n a t e a n i o n a c t i n g as a n u c l e o p h i l e . The /3-glyco-s y l a n i o n which i s g e n e r a t e d c o u l d anomerize t o the a - g l y c o s y l a n i o n and then r e - a t t a c k the a r o m a t i c i n t e r m e d i a t e v i a n u c l e o p h i l i c s u b s t i t u t i o n . - 46 -To t e s t c ase ( i i i ) , /3-2,6-DNPG (60) was anomerized i n the p r e s e n c e o f l - f l u o r o - 2 , 4 - d i n i t r o b e n z e n e (FDNB) under s t a n d a r d c o n d i t i o n s . I f the r e a c t i o n i n v o l v e s 0 ( l ) - a r y l bond c l e a v a g e , exchange o f the a r y l s u b s t i -t u e n t s s h o u l d o c c u r v i a a t t a c k o f the i n t e r m e d i a t e g l y c o s y l a n i o n on the FDNB. Indeed, exchange was o b s e r v e d as shown by the ^-H-n.m.r. spectrum o f the p r o d u c t m i x t u r e ( F i g u r e 35). A l t h o u g h o n l y about 30% o f the /3-2,6-DNPG (60) had r e a c t e d as j u d g e d by b o t h t . l . c . and 1H-n.m.r. a f t e r s t i r r i n g f o r 8 days, the •'•H-n.m.r. spectrum ( C D C I 3 ) showed a t l e a s t a s m a l l amount o f the o-2,4-DNPG (29-a) and p o s s i b l y some o f the /3-2,4-DNPG (29-/3) had been formed. Furthermore, no a-2,6-DNPG (60-a) was formed as j u d g e d by the absence o f the H(6)-DNP resonance o f the a-2,6-DNPG a t 7.87 ppm. A l t h o u g h the a g l y c o n exchange o b s e r v e d h e r e had o c c u r r e d o n l y t o a s m a l l e x t e n t , i t s u p p o r t s a mechanism i n v o l v i n g 0 ( 1 ) - a r y l bond c l e a v a g e . A second exchange experiment was pe r f o r m e d t o p r o v i d e f u r t h e r e v i d e n c e f o r the n u c l e o p h i l i c s u b s t i t u t i o n mechanism. I n t h i s c a s e , 0-2,4-DNPG which was d e u t e r a t e d a t C ( l ) (32) was anomerized as u s u a l b u t i n the p r e s e n c e o f one e q u i v a l e n t o f 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - D - g l u c o -pyranose ( 3 0 ) . Under the b a s i c c o n d i t i o n s , the anomeric h y d r o x y l o f (30) w i l l be d e p r o t o n a t e d t o produce the same g l u c o s y l a n i o n as t h a t formed by n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n on (3 2 ) . T h e r e f o r e , the g l u c o s y l a n i o n i n t e r m e d i a t e w i l l c o n s i s t o f b o t h d e u t e r i u m l a b e l l e d and u n l a b e l l e d s p e c i e s . Subsequent a n o m e r i z a t i o n o f the a n i o n and recombi-n a t i o n w i t h the a r o m a t i c i n t e r m e d i a t e s h o u l d produce a-2,4-DNPG which has l o s t a t l e a s t some o f i t s d e u t e r i u m l a b e l ( F i g u r e 36). Indeed, the •'•H-n.ra.r. spectrum o f i s o l a t e d a-2,4-DNPG showed a resonance f o r the - 47 -REACTION MIXTURE FDNB F i g u r e 35: •LH-N.m.r. f o r exchange v i a 0 ( 1 ) -(experiment 1) (CDC1 3) - 48 -anomeric hydrogen and comparison between the i n t e g r a t e d a r e a o f t h i peak and t h a t f o r H(6) o f the a r o m a t i c group i n d i c a t e d t h a t t h a - 2 , 4 - D N P G was o n l y 50% d e u t e r a t e d ( F i g u r e 37). F i g u r e 36: Exchange experiment f o r 0 ( 1 ) - a r y l bond c l e a v a g e (experiment H(6)-DNP • 1 1 I ; 1 1 • I 1 1 1 1 I • ' 1 ' I ' • 1 • I 1 • 1 • I • • • ' ! • • i I I 1 1 1 1 I • : • 1 I • 1 • • I 9 6 7 6 5 * PPM F i g u r e 37: ^H-N.m.r. f o r exchange v i a 0 ( 1 ) - a r y l bond c l e a v a g e (experiment 2) (DMS0-d 6) - 49 -2.4 K i n e t i c Isotope E f f e c t s K i n e t i c isotope e f f e c t s (KIEs) were measured as another method to probe the s i t e of bond cleavage f o r the anomerization reaction. (See appendix 1 f o r an out l i n e of the theory behind KIEs.) Deuterium was substituted at the anomeric center f o r the per-O-acetylated DNPG (32) and the per-O-benzylated DNPG ( 3 5 ) . For. both of these substrates, the magnitude of the isotope e f f e c t should ind i c a t e which bond i s cleaved at the anomeric center. I f the re a c t i o n proceeds v i a C ( l ) - H ( l ) bond cleavage, then a primary KIE would be expected i n which ^ / k j ) = 2—7. On the other hand, i f the reac t i o n goes v i a C(l)-0(1) bond cleavage, then a secondary KIE would be expected i n which ^ / f c n ~ 1.1-1.2. The absence of an isotope e f f e c t or an isotope e f f e c t of less than 1.1 would suggest an a l t e r n a t i v e mechanism, i n which the bond being cleaved does not involve the anomeric center, or that some step i n the mechanism other than bond cleavage i s r a t e - l i m i t i n g . Anomerization by the method of van Boom 3 3 r e s u l t s i n a heterogenous mixture since potassium carbonate i s v i r t u a l l y i n s o l u b l e i n DMF. Reac-t i o n rates f o r t h i s system may be measured by removing aliquots from the rea c t i o n mixture at s p e c i f i e d times, performing a mini-work-up, and f i n a l l y analyzing f o r products by ^H-n.m.r. spectroscopy. This method does not ensure, however, that products are not l o s t during the work-up consequently a f f e c t i n g the measured rates. In order to monitor the react i o n d i r e c t l y and therefore obtain more accurate k i n e t i c data, i t was necessary to f i n d a homogeneous system. This i n v e s t i g a t i o n e n t a i l e d a thorough survey of c a t a l y s t s which e f f e c t the anomerization and 50 -s o l v e n t s i n which b o t h the sugar and the c a t a l y s t a r e s o l u b l e . 2.4.1 Survey o f S o l v e n t s The a n o m e r i z a t i o n o f the a c e t y l a t e d /9-2,4-DNPG (29) was pe r f o r m e d i n each o f the s o l v e n t s l i s t e d i n T a b l e 3. The sugar was s o l u b l e i n most r e l a t i v e l y p o l a r s o l v e n t s b u t p o t a s s i u m c a r b o n a t e was v i r t u a l l y i n s o l u b l e i n a l l o f the s o l v e n t s . However, i n c a s e s where a n o m e r i z a t i o n d i d o c c u r , i t was presumed t h a t the p o t a s s i u m c a r b o n a t e must be a t l e a s t s p a r i n g l y s o l u b l e i n o r d e r t o c a t a l y z e the r e a c t i o n . 2.4.2 Survey o f C a t a l y s t s I n o r d e r t o i n c r e a s e t h e s o l u b i l i t y o f the c a r b o n a t e c a t a l y s t , the p o t a s s i u m c a t i o n was r e p l a c e d by the tetraalkylammonium c a t i o n . T e t r a -m e t h y l , t e t r a e t h y l and tetrabutylammonium c a r b o n a t e were s y n t h e s i z e d by b u b b l i n g c a r b o n d i o x i d e t h r o u g h a s o l u t i o n o f the tetraalkylammonium h y d r o x i d e i n water. The s o l u t i o n was s a t u r a t e d w i t h c a r b o n d i o x i d e and the tetraalkylammonium c a r b o n a t e s a l t was p r e c i p i t a t e d . A l t h o u g h the tetramethylammonium c a r b o n a t e was the most h y g r o s c o p i c , i t was the e a s i e s t t o p u r i f y by r e c r y s t a l l i z a t i o n and so was u s e d most e x t e n s i v e l y f o r the a n o m e r i z a t i o n . A l l t h r e e c a r b o n a t e s , however, were found to be a t l e a s t somewhat s o l u b l e i n DMSO, DMF, THF, and MeCN. A n o m e r i z a t i o n w i t h the tetraalkylammonium s a l t s i n these s o l v e n t s p r o c e e d e d to the u s u a l p r o d u c t m i x t u r e . O t h e r c a t a l y s t s employed f o r the a n o m e r i z a t i o n as p a r t o f the 51 -T a b l e 3: I n v e s t i g a t i o n o f s o l v e n t s f o r a n o m e r i z a t i o n o f (29) w i t h K2CO3. "Dec" i n d i c a t e s d e c o m p o s i t i o n o f (29) S o l v e n t D i e l e c t r i c c o n s t a n t (e) 50 K 2 C 0 3 /3-2,4-DNPG B -» a DMSO DMF 49 36.7 s i . s o l . s o l u b l e yes s i . s o l . s o l u b l e yes P r o p y l e n e c a r b o n a t e T e t r a h y d r o f u r a n E t h y l a c e t a t e 65.1 7.32 6.02 s o l u b l e s o l u b l e yes yes s o l u b l e yes (dec) P y r i d i n e 12.3 N-methyl- 2 - p y r r o l i d i n o n e 32.0 C h l o r o f o r m 4.7 t - B u t a n o l 10.9 Toluene 2.38 D i e t h y l e t h e r 4.34 Methanol 32.6 1,4-Dioxane 2.21 Dic h l o r o m e t h a n e 8.9 Acetone 20.7 A c e t o n i t r i l e 36.2 s o l u b l e s o l u b l e s o l u b l e s o l u b l e s o l u b l e s o l u b l e s o l u b l e s o l u b l e s o l u b l e s o l u b l e s o l u b l e yes yes yes no no no dec yes (dec) no yes (dec) yes 52 -s e a r c h f o r a s o l u b l e system are l i s t e d i n T a b l e 4. The p K a v a l u e s l i s t e d are deter m i n e d i n w a t e r ^ and these v a l u e s are known t o be h i g h l y dependent on the s o l v e n t system i n which they a r e d e t e r -mined. T h e r e f o r e , the v a l u e s l i s t e d can o n l y be used to compare r e a c t i v i t i e s o f DNPG s u b s t r a t e s and b a s i c i t y o f the c a t a l y s t as a rough a p p r o x i m a t i o n . D i s s o c i a t i o n c o n s t a n t s have been i n v e s t i g a t e d i n DMSO s o l u t i o n s and f o r c h a r g e d a n i o n s , such as a l c o h o l s and c a r b o x y l i c a c i d s , the p K a has been p r e d i c t e d t o i n c r e a s e by as much as 7 p K a u n i t s r e l a t i v e to v a l u e s d e t e r m i n e d i n water.53,54 T h e r e f o r e , i t i s presumed t h a t the b a s i c i t y o f c a t a l y s t s such as c a r b o n a t e and phosphate, which are b o t h h i g h l y charged d e l o c a l i z e d a n i o n s , i s c o n s i d e r a b l y h i g h e r i n DMSO than i n water. As can be seen i n T a b l e 4, the r e a c t i o n does appear t o depend on the b a s i c i t y o f the c a t a l y s t used s i n c e weakly a c i d i c compounds such as S0^z and C I O 4 " and n e u t r a l bases such as 1 , 8 - b i s ( d i m e t h y l a m i n o ) -n a p h t h a l e n e do not c a t a l y z e the a n o m e r i z a t i o n . T h i s s u g g e s t s a base c a t a l y z e d type mechanism i n v o l v i n g p r o t o n a b s t r a c t i o n a t the anomeric c a r b o n (case ( i ) , i n F i g u r e 30). The ani o n s which c a t a l y z e the r e a c t i o n might be c o n s i d e r e d t o be r e l a t i v e l y n u c l e o p h i l i c , p a r t i c u l a r l y i n a p r o t i c s o l v e n t s such as DMSO. A l t e r n a t i v e l y , t h e se a n i o n s may be b a s i c enough t o d e p r o t o n a t e DMSO and form the d i m e t h y l s u l f i n y l a n i o n which a l s o c a t a l y z e s the r e a c t i o n . T h e r e f o r e , the r e s u l t s found i n T a b l e 4 may a l s o s u g g e s t a mechanism v i a n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n ( c a s e ( i i i ) i n F i g u r e 30). 53 -T a b l e 4: Survey o f c a t a l y s t s f o r a n o m e r i z a t i o n o f 0-2,4-DNPG (29). A l l were r e a c t i o n s performed i n DMSO exce p t where o t h e r w i s e n o t e d . C a t a l y s t s 51 p K a (H 20) Carbo n a t e s K2CO3, N a 2 C 0 3 , BaC03, CSCO3, ( M e 4 N ) 2 C 0 3 > ( E t 4 N ) 2 C 0 3 , ( B u 4 N ) 2 C 0 3 KHCO3 Phosphates ( t r i b a s i c ) N a 3 P 0 4 - H 2 0 ( M e 4 N ) 3 P 0 4 Phosphates ( d i b a s i c ) ( M e 4 N ) 2 H P 0 4 KC10 4 K 2 S 0 4 Amines t r i - n - b u t y l a m i n e 1 , 8 - b i s ( d i m e t h y l a m i n o ) n a p h t h a l e n e E t 4 N B r D i m e t h y l s u l f i n y l a n i o n Sodium h y d r i d e P o t a s s i u m t - b u t o x i d e n - B u t y l l i t h i u m i n hexanes 10.33 Yes 6.35 12.67 7.21 ~-8 1.92 -10 12.37 31.3 35 19 >40 Yes Yes No Yes No No No No No Yes Yes Yes No - 54 -2.4.3 Measurement o f Rates P o l a r i m e t r y i s the most common method used t o measure r a t e s o f a n o m e r i z a t i o n r e a c t i o n s . S i n c e a n o m e r i z a t i o n r e s u l t s i n an i n v e r s i o n o f s t e r e o c h e m i s t r y a t C ( l ) , the o p t i c a l r o t a t i o n o f the r e a c t i o n m i x t u r e changes d r a m a t i c a l l y as the c o n c e n t r a t i o n s o f the a-anomer i n c r e a s e and the ^-anomer d e c r e a s e . A d i s a d v a n t a g e o f t h i s method i s t h a t s i d e r e a c t i o n s cannot be r e a d i l y d e t e c t e d so the measured change i n o p t i c a l r o t a t i o n may be due i n p a r t t o the f o r m a t i o n o f s i d e p r o d u c t s . T h i s was fou n d t o be the problem i n the a n o m e r i z a t i o n o f /J-2.4-DNPG ( 2 9 ) . DMSO was u s e d as a s o l v e n t f o r t h i s s t u d y s i n c e i t i s r e l a t i v e l y i n e r t under b a s i c c o n d i t i o n s and s i n c e bis(tetramethylammonium) carbo -n a t e i s r e l a t i v e l y s o l u b l e i n DMSO. The r e a c t i o n was m o n i t o r e d s i m u l t a -n e o u s l y by p o l a r i m e t r y and t . l . c . A f t e r s e v e r a l h o u r s , the anomeriza-t i o n had p r o c e e d e d t o about 50% as j u d g e d by t . l . c . o f the p r o d u c t m i x t u r e b u t the o p t i c a l r o t a t i o n o f the s o l u t i o n i n d i c a t e d t h a t the r e a c t i o n had pr o c e e d e d t o o n l y a p p r o x i m a t e l y 10% o f the a-anomer. A t t h i s p o i n t the s o l u t i o n was dark brown, presumably due to the f o r m a t i o n o f 2 , 4 - d i n i t r o p h e n o l a t e as shown by comparison o f the u . v . / v i s i b l e s pectrum o f the r e a c t i o n m i x t u r e and t h a t o f a s t a n d a r d s o l u t i o n o f p o t a s s i u m 2 , 4 - d i n i t r o p h e n o l a t e . T h e r e f o r e , i t was c o n c l u d e d t h a t a s i d e r e a c t i o n i n v o l v i n g the f o r m a t i o n o f the d i n i t r o p h e n o l a t e i o n , perhaps due t o the p r e s e n c e o f water i n the m i x t u r e , was a f f e c t i n g the o p t i c a l r o t a t i o n measurement. S i n c e complete e l i m i n a t i o n o f water from the p o l a r i m e t r y c e l l i s d i f f i c u l t , p o l a r i m e t r y was found t o be an u n s u i t a b l e method t o measure k i n e t i c d a t a . Because o f the p o t e n t i a l problems w i t h s i d e r e a c t i o n s , •'-H-n.m.r. s p e c t r o s c o p y was found to be the most s u i t e d f o r k i n e t i c measurements f o r s e v e r a l r e a s o n s . F i r s t l y , the r e l a t i v e amounts o f a- and /3-anomers formed a r e e a s i l y d e t e r m i n e d by i n t e g r a t i o n o f e i t h e r anomeric r e s o -nances o r the resonances o f H(6) o f the DNP r i n g ( F i g u r e s 27 and 28). Seco n d l y , the r e a c t i o n can be performed i n DMSO-dg, i n which b o t h the sugar and the c a t a l y s t a r e s o l u b l e , so t h a t the r e a c t i o n can be moni-t o r e d d i r e c t l y i n the n.m.r. tube. A l s o , anhydrous r e a c t i o n m i x t u r e s can be m a i n t a i n e d i n s e a l e d n.m.r. tubes. F i n a l l y , n.m.r. s p e c t r o s c o p y a l s o a l l o w s easy d e t e c t i o n o f any s i d e r e a c t i o n s as w e l l as i d e n t i f i c a -t i o n and q u a n t i f i c a t i o n o f p r o d u c t s from these r e a c t i o n s . Sample •'-H-n.m.r. s p e c t r a taken over the c o u r s e o f the r e a c t i o n f o r a n o m e r i z a t i o n o f /3-2,4-DNPG (29) and [1- 2H]-2,4-DNPG (32) a r e shown i n F i g u r e s 38 and 39. I n t e g r a t i o n v a l u e s i n a l l s p e c t r a were s t a n d a r d i z e d to the sum o f the -OAc peak a t 5 2.0-2.4 (not shown) f o r which the i n t e g r a l d i d n o t change d u r i n g the r e a c t i o n . I n the s p e c t r a f o r [1- H]-DNPG, the s i g n a l a t S 6.3 was n o t due to the anomeric resonance o f a-2,4-DNPG but was the s i g n a l f o r H(6) o f the 2 , 4 - d i n i t r o - p h e n o l a t e a n i o n which was formed t o a s m a l l e x t e n t . T a b l e 5 r e p o r t s some t y p i c a l r e s u l t s f o r the a n o m e r i z a t i o n o f (29) and (32) . The d a t a p r o v i d e an example o f the raw i n t e g r a l v a l u e s o b t a i n e d and the c a l c u l a t e d amounts o f /3-anomer r e m a i n i n g a t the n o t e d i n t e r v a l s o f the r e a c t i o n . The r a t e s were dete r m i n e d by p l o t t i n g the i n t e g r a t i o n v a l u e f o r the /3-anomer v e r s u s time ( i . e . , the raw data) and by p l o t t i n g the c a l c u l a t e d l o g ( a t - a e ) v e r s u s t i m e ^ to o b t a i n a p s e u d o - f i r s t - o r d e r r a t e c o n s t a n t k (ae = amount o f /3-DNPG p r e s e n t a t - 56 -T= 0 Hours 0 10 T=1 Hour 0 0 a 'i 1 1 1 1 i 1 1 1 ' i ' ' i i i i « 0 7 5 7 0 f S a IJL 6 0 7 * 7 c « j ' e ' e ' p P M T = 2 Hours T=4 Hours Figure 38: l-H-N.m.r. f o r KIE measurements: non-deuterated substrate (29) (DMS0-d6) - 57 -T = 0 Hours 0 1 r™1—-,—1—'—r~<—"—<—•—i—i—i i i | — i — i — i — i — j — T - - T -5 C 7 5 7 0 6 5 6 0 P F M T = 0.7 Hour 0 a : 1 ' '— '—|—i— i— r— T — I— i— I — I — i— I— i—p— i— i—i— i— i— r -= & 7 5 7 0 6 5 6 0 P P M T = 2 Hours T = 4 Hours 0 a •i* ^ — r 1—<—•—•-!— 1—•— 1— 1—r~ 1— 1— 1— 1—i— 1— 1— 1— 1—;—>--E C 7 1 7 0 6 5 6 C P P M IF ' I ' • 1 1 I 1 1 1 1 I 1 1 1 i I I 1—r 7 5 7 0 6 5 6 0 P P M Figure 39: ^-N.m.r. f o r KIE measurements: deuterated substrate (32) (DMS0-d6) 58 -Table 5: K i n e t i c data f o r KIE measurement. Data f o r one t r i a l only are given. [(Me4N) 2C0 3] = 0.0182 M (DMS0-d6). Temperature = 25°. 1-H-0-D-DNPG (29), [DNPG] = 0.0121 M (DMSO-dg) I n t e g r a l A r e a f o r 0-DNPG H(6)-DNP f o r 0-anomer (mmol x 10^) l o g ( a t - ae) time (min) 5. .94 6. .78 -2, .27 6 4. .93 6. .08 -2 .33 20 4, .55 5. .57 -2, .38 40 3. .82 4. .78 -2, .48 60 3. .32 4. .13 -2, .57 80 2. .72 3. ,43 -2. .70 100 2. .58 3. .21 -2. .76 120 2. .29 2. .89 -2. .84 140 2. .03 2. .53 -2. .97 160 1. ,86 2. .35 -3, .05 180 1. ,63 2. .04 -3. .28 240 1. .40 1. .76 -3, .63 284 [l-2H]-0-D-DNPG (32), [DNPG] = 0.0127 M (DMS0-d6) I n t e g r a l A r e a f o r 0-DNPG H(6)-DNP f o r 0-anomer (mmol x 10^) l o g ( a t - a e ) time (min) 6. ,20 7. ,65 -2. .21 5 5. ,17 6. ,93 -2. .27 20 4. .60 6, .18 -2, .33 40 3. .95 5, .38 -2, .41 60 3. .55 4. .77 -2. .49 75 3. .03 4. .08 -2 .59 100 2. .60 3, .58 -2 .69 120 2, .65 3, .59 -2, .69 140 2. .36 3, .17 -2 .78 160 1. .80 2. .50 -3 .01 200 1. .68 2, .26 -3 .13 220 1, .45 1, .92 -3 .40 260 1 .40 1, .93 -3, .42 280 59 -e q u i l i b r i u m , a t = amount o f /3-2,4-DNPG p r e s e n t a t time t , F i g u r e 40). The KIE f o r a n o m e r i z a t i o n o f /3-2,4-DNPG from b o t h methods was c a l c u l a t e d t o be 1.09 ± 0.06. The r e s u l t s o b t a i n e d from f o u r r e p e a t e d determina-t i o n s a r e summarized i n T a b l e 6. The r e l a t i v e l y s m a l l i s o t o p e e f f e c t might be r e p r e s e n t a t i v e o f a s e c o n d a r y KIE but more i m p o r t a n t l y , i t i s too s m a l l to be a p r i m a r y KIE. T h i s s u g g e s t s t h a t the r e a c t i o n does not p r o c e e d v i a p r o t o n a b s t r a c t i o n a t C ( l ) . The p e r - O - b e n z y l a t e d DNPG (31) anomerized much more s l o w l y than (29) and the i s o t o p e e f f e c t c o u l d n o t be d e t e r m i n e d due to a l a r g e amount o f decompositon to the 2 , 4 - d i n i t r o p h e n o l a t e a n i o n which c o m p l i -c a t e d the •'•H-n.m.r. spectrum. The i s o t o p e e f f e c t f o r the p r o d u c t i o n o f t h e d i n i t r o p h e n o l a t e a n i o n , however, was measured t o be 1.09. T h i s i s i n a c c o r d a n c e w i t h the KIE f o r the a n o m e r i z a t i o n o f (29) and t h e r e f o r e , the a n i o n may be formed by a s i m i l a r mechanism. 0 100 200 300 TIME (minuttt) F i g u r e 40: KIE f o r a n o m e r i z a t i o n o f 0-2,4-DNPG 60 T a b l e 6: Summary o f r e s u l t s : P s e u d o - f i r s t - o r d e r r a t e c o n s t a n t s c a l c u l a t e d f o r the d i s a p p e a r a n c e o f /3-2,4-DNPG (29) and (32) kH 1-H-/3-2.4-DNPG (min" •*•) C o r r e l a t i o n C o e f f i c i e n t -4.81 x 1 0 " 3 -.9945 -4.96 x 10" - .9980 -4.87 x 1 0 " 3 -4.72 x 1 0 " 3 -.9895 -.9982 kD [1- 2H]-/3-2,4-DNPG C o r r e l a t i o n ( m i n " 1 ) C o e f f i c i e n t -4.47 x 1 0 " 3 -4.47 x 1 0 " 3 -4.65 x 1 0 ' 3 -4.15 x 1 0 " 3 .9917 ,9934 - .9982 - .9975 Average kH = -4.84 x 1 0 ° ± 1.27 x 10" Average kD = -4.44 x 1 0 " 3 ± 1.16 x 10" KIE = kH/kD = 1.09 ± 0.06 61 I n t e r e s t i n g l y , under these anhydrous c o n d i t i o n s , the r e a c t i o n m i x t u r e t a k e s on a deep v i o l e t c o l o r which e v e n t u a l l y becomes red-orange (1 hour) and t h e n v e r y dark brown (8-10 h o u r s ) . The dark brown c o l o r was a t t r i b u t e d to the 2 , 4 - d i n i t r o p h e n o l a t e a n i o n formed by a s i d e r e a c t i o n . The pr e s e n c e o f the a n i o n was appa r e n t i n the ^-H-n.m.r. spectrum (H(6) 5 6.3). Attempts t o i d e n t i f y the s p e c i e s c a u s i n g the deep p u r p l e c o l o r were u n s u c c e s s f u l by ^H-n.m.r. s p e c t r o s c o p y s i n c e the spectrum showed no d i s t i n c t anomalous peaks. The u . v . / v i s i b l e spectrum, however, showed an u n u s u a l charge t r a n s f e r a b s o r p t i o n a t 570 nm. (DNPG A m a x = 275 nm, po t a s s i u m 2 , 4 - d i n i t r o p h e n o l a t e -X m a x = 429 nm i n DMSO). Furthermore, a mix t u r e o f /3-2,4-DNPG i n DMSO and a s m a l l amount o f d i m e t h y l s u l f i n y l ( d i m s y l ) a n i o n , which may be p r e s e n t i n the anomeriza-t i o n m i x t u r e due to the h i g h b a s i c i t y o f c a r b o n a t e i n DMSO, showed the same p u r p l e c o l o r and an a b s o r p t i o n a t 574 nm. The r e a c t i o n o f d i m s y l a n i o n w i t h FDNB i n DMSO a l s o p r o d u c e d the p u r p l e c o l o r and an a b s o r p t i o n a t 548 nm ( a l o n g w i t h a b s o r p t i o n s a t 642 nm, 436 nm, and 370 nm) ( F i g u r e 4 1 ) . The p u r p l e c o l o r o f t h i s s o l u t i o n d i s a p p e a r e d w i t h i n 2-3 minutes p r o d u c i n g a brown s o l u t i o n whereas the p u r p l e c o l o r o f the DNPG s o l u t i o n p e r s i s t e d f o r s e v e r a l hours b e f o r e decomposing to the brown c o l o r . The p r o b a b l e o r i g i n o f t h i s u n u s u a l c o l o r a t i o n i s pr o p o s e d i n the D i s c u s s i o n s e c t i o n . 2.5 Remote S u b s t i t u e n t E f f e c t s S u b s t i t u t i o n 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 around the sugar r i n g i s known t o cause pronounced e f f e c t s on the r a t e s o f g l y c o s i d e h y d r o l y -- 62 -(B) F D N B + D I M S Y L A N I O N ( D M S O ) 400 500 600 700 600 WAVELENGTH (nm) F i g u r e A l : U . v . / v i s i b l e absorbance s p e c t r a f o r a n o m e r i z a t i o n r e a c t i o n (A) 2,4-DNPG w i t h d i m s y l a n i o n and (B) r e a c t i o n o f FDNB w i t h d i m s y l a n i o n s i s . ' F o r example, i t was r e c e n t l y shown t h a t s u b s t i t u t i o n o f f l u o r i n e f o r h y d r o x y l s a t the 2,3,4, o r 6 - p o s i t i o n o f a - D - g l u c o p y r a n o s y l phosphate lowered the r a t e s o f a c i d c a t a l y z e d h y d r o l y s i s . The e l e c -t r o n e g a t i v e f l u o r i n e d e s t a b i l i l z e s the e l e c t r o n d e f i c i e n t i n t e r m e d i a t e and slows the r e a c t i o n . I n t h i s s e r i e s o f d e o x y f l u o r o s u g a r s , the 2 - d e o x y - 2 - f l u o r o and the 4 - d e o x y - 4 - f l u o r o d e r i v a t i v e s showed the most d r a m a t i c e f f e c t . T h e r e f o r e , i t was b e l i e v e d t h a t s i m i l a r e f f e c t s might be o b s e r v e d w i t h the a n o m e r i z a t i o n o f d i n i t r o p h e n y l g l u c o p y r a n o s i d e s i f the r e a c t i o n p r o c e e d s v i a an oxocarbonium i o n formed by c l e a v a g e o f the C ( l ) - 0 ( 1 ) bond. A l t e r n a t i v e l y , the o p p o s i t e e f f e c t might be o b s e r v e d i f the r e a c t i o n i n v o l v e s a c a r b a n i o n i n t e r m e d i a t e formed by a mechanism i n v o l v i n g p r o t o n a b s t r a c t i o n a t the r e a c t i o n c e n t e r . I n t h i s c a s e, 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 such as f l u o r i n e w i l l s t a b i l i z e the a n i o n i c i n t e r m e d i a t e and t h e r e b y i n c r e a s e the r a t e o f a n o m e r i z a t i o n . To probe the e f f e c t o f s u b s t i t u e n t s around the sugar r i n g , pseudo-f i r s t - o r d e r r a t e c o n s t a n t s (k) f o r the 4 - d e o x y - 4 - f l u o r o , 4-deoxy, and 6-deoxy DNPG (40, 44, and 49, r e s p e c t i v e l y ) a l o n g w i t h the p a r e n t sugar (29), were measured as d e s c r i b e d p r e v i o u s l y . The r e s u l t s are summarized i n T a b l e 7 (experiment 1; p l o t shown i n F i g u r e 42). As can be seen, the 4 - d e o x y - 4 - f l u o r o DNPG (40) showed a s l i g h t r a t e enhancement over the r a t e o f a n o m e r i z a t i o n o f (29), whereas the 4-deoxy DNPG (44) anomerized an o r d e r o f magnitude slower than ( 29). The r a t e measured f o r the 6-deoxy DNPG (49) i s s i m i l a r t o the r a t e measured f o r (44) s i n c e t h i s a l s o showed a s l i g h t d e c r e a s e i n r a t e compared t o ( 2 9 ) . In a d d i t i o n , a s e p a r a t e experiment was performed i n which the r a t e o f a n o m e r i z a t i o n o f 2 - d e o x y - 2 - f l u o r o DNPG (39) was compared to t h a t o f - 64 T a b l e 7: Remote s u b s t i t u e n t e f f e c t s o f a n o m e r i z a t i o n : C a l c u l a t e d p s e u d o - f i r s t - o r d e r r a t e c o n s t a n t s f o r the d i s a p p e a r a n c e o f /3-2,4-DNPG d e r i v a t i v e s . Temperature = 25° Experiment 1: [ ( M e 4 N ) 2 C 0 3 ] - 0.0167 M S u b s t r a t e C o n c e n t r a t i o n (M) kxlO^ ( m i n " l ) C o r r e l a t i o n C o e f f i c i e n t 4 - D e o x y - 4 - f l u o r o (40) 0.0138 •3.82 9923 4-Deoxy (44) 0.0150 -0.456 .9952 6-Deoxy (49) DNPG (29) 0.0153 0.0136 •1.45 •3.63 9897 .9901 Experiment 2: [ ( M e A N ) 2 C 0 3 ] = 0.0182 M 3 S u b s t r a t e C o n c e n t r a t i o n k x l O C o r r e l a t i o n (M) ( m i n " 1 ) C o e f f i c i e n t 2 - D e o x y - 2 - f l u o r o (39) DNPG (29) 0.0121 0.0134 -3.56 -3.97 .9881 .9969 - 65 -2.CH T 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 — 0 100 200 300 TIME(minutes) Reaction rates measured f o r remote substituent e f f e c t s (A) experiment 1; (B) experiment 2. - 66 -(29) (experiment 2 ) . S i m i l a r t o (40), the 2 - d e o x y - 2 - f l u o r o DNPG r e a c t e d a t n e a r l y the same r a t e as the p a r e n t sugar (29) ( F i g u r e 4 2 ) . Mechanis-t i c i m p l i c a t i o n s o f these r e s u l t s w i l l be c o n s i d e r e d i n the D i s c u s s i o n s e c t i o n . - 67 -DISCUSSION Four mechanisms c a n be p r o p o s e d f o r the c a r b o n a t e c a t a l y z e d anomer-i z a t i o n o f p r o t e c t e d 2 , 4 - d i n i t r o p h e n y l /3-D-glucopyranosides. B r i e f l y , t h e s e are ( i ) p r o t o n a b s t r a c t i o n a t C ( l ) ; ( i i ) p h e n o l a t e a b s t r a c t i o n ; ( i i i ) p r o t o n a b s t r a c t i o n a t C ( 2 ) ; and ( i v ) n u c l e o p h i l i c a t t a c k on the a r o m a t i c r i n g . Mechanism ( i ) ( F i g u r e 43) was s u g g e s t e d by F e r r i e r - ^ as an u n u s u a l y e t v i a b l e pathway t o the a-anomer. The mechanism i n v o l v e s base c a t a l y z e d removal o f the anomeric p r o t o n and f o r m a t i o n o f a c a r b a n i o n i n t e r m e d i a t e ( 6 3 ) . A l t h o u g h the p K a o f the anomeric p r o t o n would be e x p e c t e d t o be h i g h (>20), the a c i d i t y o f H ( l ) may be i n c r e a s e d by the p r e s e n c e o f the s t r o n g l y e l e c t r o n - w i t h d r a w i n g d i n i t r o p h e n y l group which Figure 43: Mechanism ( i ) . Base catalyzed proton abstr a c t i o n - 68 -would f a c i l i t a t e t h i s mechanism. Furthermore, the c a r b o n a t e a n i o n i s p r o b a b l y a much s t r o n g e r base i n DMSO-^ than i n water ( p K a = 10.33) and may be s t r o n g enough t o remove the r e l a t i v e l y n o n - a c i d i c H ( l ) . The t o t a l l a c k o f H/D exchange a t C ( l ) when the a n o m e r i z a t i o n o f 0-2,4-DNPG (29) i s performed i n the p r e s e n c e o f a d e u t e r o n s o u r c e , or when the a n o m e r i z a t i o n o f [1- 2H] - 0-2,4-DNPG (32) i s p e r f o r m e d i n the p r e s e n c e o f a p r o t o n s o u r c e , i s s t r o n g e v i d e n c e a g a i n s t mechanism ( i ) . T h i s absence o f H/D exchange might be r a t i o n a l i z e d t h r o u g h the f o r m a t i o n o f an i n t i m a t e i o n p a i r i n t e r m e d i a t e i n which the p r o t o n i s removed then r e - a t t a c h e d w i t h o u t e v e r l e a v i n g the s o l v e n t cage s u r r o u n d i n g the r e a c -t i o n c e n t e r . T h i s e x p l a n a t i o n i s u n l i k e l y , however, s i n c e an i o n p a i r i n t e r m e d i a t e would be e x p e c t e d t o exchange t o a t l e a s t a s m a l l e x t e n t . The measurement o f a s m a l l i s o t o p e e f f e c t (^/^D = 1.09) i s a l s o e v i d e n c e a g a i n s t mechanism ( i ) . P r o t o n a b s t r a c t i o n a t C ( l ) would g i v e a p r i m a r y i s o t o p e e f f e c t i n the o r d e r o f ^/^D = 2-7 i f bond c l e a v a g e i s r a t e - d e t e r m i n i n g , as would be exp e c t e d . A l t h o u g h p r i m a r y i s o t o p e e f f e c t s c a n be s u p p r e s s e d by f a c t o r s such as p r o t o n t u n n e l i n g o r non-l i n e a r i t y i n the t r a n s i t i o n s t a t e , these f a c t o r s c a n be p r e c l u d e d i n l i g h t o f the absence o f H/D exchange. Mechanism ( i i ) ( F i g u r e 44) i n v o l v e s the removal o f the p h e n o l a t e a n i o n and f o r m a t i o n o f an oxocarbonium i o n i n t e r m e d i a t e (64) which may be f u r t h e r s t a b i l i z e d by the p a r t i c i p a t i o n o f the C(2) a c e t o x y group and the f o r m a t i o n o f an acetoxonium i o n (65) . T h i s mechanism i n i t i a l l y was c o n s i d e r e d t o be the most l i k e l y r o u t e f o r s e v e r a l r e a s o n s . E a r l y e xperiments i n t h i s s t u d y showed the r e l e a s e o f the d i n i t r o p h e n o l a t e a n i o n by the orange c o l o r , t . l . c , and the u . v . / v i s i b l e spectrum o f the - 69 -Figure 44: Mechanism ( i i ) . Phenolate abstraction and formation of an oxocarbonium ion intermediate re a c t i o n mixture. The measurement of a small isotope e f f e c t and lack of H/D exchange are i n accord with mechanism ( i i ) . F i n a l l y , van Boom et. a l . reported a 70 f o l d decrease i n the rate of anomerization f o r the non- p a r t i c i p a t i n g tetra-O-benzyl DNPG (31), suggesting formation of the acetoxonium ion which a s s i s t s phenolate departure. Our study confirmed t h i s r e s u l t . There i s strong evidence, however, which may be used to discount mechanism ( i i ) . F i r s t l y , only substrates with aglycons having pK a values lower than 4.00 (2,4-DNPG (29), 2,6-DNPG (60), and 2,6-dichloro-4-nitrophenyl glucopyranoside (59)) were found to anomer-iz e , although to a small extent, under these conditions. This dramatic influence of electron-withdrawing substituents on the phenyl r i n g i s s u r p r i s i n g since studies on the ac i d catalyzed hyd r o l y s i s of glycopyra-- 70 -n o s i d e s ( a l s o c o n s i d e r e d t o o c c u r v i a an oxocarbonium i o n mechanism) show o n l y a s m a l l dependence o f r a t e on the e l e c t r o n e g a t i v i t y o f the a g l y c o n . ^ S e c o n d l y , p h e n o l a t e exchange experiments i n which the 2,6-DNPG (60) was anomerized i n the p r e s e n c e o f added 2 , 4 - d i n i t r o -p h e n o l a t e i n d i c a t e d t h a t no exchange o f the p h e n o l a t e s u b s t i t u e n t s o c c u r r e d . S i m i l a r l y , no exchange was o b s e r v e d when 2,4-DNPG (29) was anomerized i n the p r e s e n c e o f added 2 , 6 - d i n i t r o p h e n o l a t e . T h i r d l y , the r a t e o f a n o m e r i z a t i o n measured f o r the 2 - d e o x y - 2 - f l u o r o DNPG (39) i s i n the same o r d e r as the r a t e o f normal DNPG (29 ) . I t i s u n l i k e l y , t h e r e -f o r e , t h a t the C ( 2 ) - a c e t o x y group p a r t i c i p a t e s i n t h i s mechanism as s u g g e s t e d e a r l i e r , assuming t h e r e i s no change i n mechanism f o r the two s u b s t r a t e s . Furthermore, s u b s t i t u e n t e f f e c t s a t C ( 2 ) , C(4) and C(6) p r o v i d e e v i d e n c e a g a i n s t the f o r m a t i o n o f an oxocarbonium i o n s i n c e the r e l a t i v e r a t e s o f the 4-deoxy and the 6-deoxy d e r i v a t i v e s (44 and 49, r e s p e c t i v e l y ) a r e lower than t h a t f o r the p a r e n t sugar (29) , b u t the 4 - d e o x y - 4 - f l u o r o d e r i v a t i v e (40), l i k e the 2 - d e o x y - 2 - f l u o r o d e r i v a t i v e ( 3 9 ) , anomerized as f a s t as (29) . I f an oxocarbonium i o n i s i n v o l v e d i n the mechanism, the e l e c t r o n e g a t i v e f l u o r i n e s u b s t i t u e n t s h o u l d d e s t a b i -l i z e the t r a n s i t i o n s t a t e l e a d i n g t o the i n t e r m e d i a t e , and slow the r e a c t i o n . F i n a l l y , i t i s d i f f i c u l t t o f i n d a r o l e f o r the ca r b o n a t e c a t a l y s t I n such a mechanism. Mechanism ( i i i ) ( F i g u r e 45) a l s o i n v o l v e s the removal o f the p h e n o l a t e a n i o n v i a p r o t o n a b s t r a c t i o n a t C(2) f o r m i n g a g l u c a l i n t e r m e d i a t e (62) o r an open c h a i n i n t e r m e d i a t e (66) . T h i s mechanism i s s u p p o r t e d by the s m a l l i s o t o p e e f f e c t which, i n t h i s c a s e, would be a f5-secondary i s o t o p e e f f e c t . A l s o , the r e l a t i v e r a t e s measured f o r the - 71 -Figure 45: Mechanism ( i i i ) . Proton abstraction at C(2) and formation of a g l u c a l intermediate 2-deoxy-2-fluoro (39) and the 4-deoxy-4-fluoro (40) d e r i v a t i v e s favor the mechanism since the electronegative f l u o r i n e substituents would s t a b i l i z e the carbanion intermediate. Against t h i s mechanism, however, i s the complete lack of exchange between phenolate substituents as explained for mechanism ( i i ) and which would also be expected i n t h i s case. In addition, there was no H/D exchange at C(2) when the anomerization of (29) was performed i n the presence of a deuteron source, nor was there epimerization to the D-mannoside d e r i v a t i v e . F i n a l l y , 2,3,4,6-tetra-O-acetyl-glucal (62), a p o s s i b l e intermediate i n t h i s mechanism, di d not react with potassium 2,4-dinitrophenolate to form the a- or /?-glucopyranoside. Therefore, - 72 -mechanism ( i i i ) can also be discounted as a reasonable pathway for anomerization. Mechanism ( i v ) (Figure 46) i s perhaps the most unusual pathway and i n i t i a l l y was not considered i n t h i s study. The mechanism proceeds v i a n u c l e o p h i l i c aromatic s u b s t i t u t i o n on the phenyl r i n g and formation of the g l y c o s y l oxyanion (68) which can anomerize v i a the open chain Figure 46: Mechanism ( I v ) . Aromatic n u c l e o p h i l i c s u b s t i t u t i o n . Nuc = nucleophile ( C 0 3 = or CH3SOCH2") - 73 -aldehyde form (69) of the sugar. The anomerization process i t s e l f i s mechanistically s i m i l a r , therefore, to the acid/base catalyzed mutarota-t i o n of D-glucose. In addition, t h i s mechanism involves the formation of a hi g h l y d e l o c a l i z e d charged intermediate (67), which i s known as a Meisenheimer intermediate. The occurrence of Meisenheimer intermediates i s common f o r n u c l e o p h i l i c reactions i n v o l v i n g highly a c t i v a t e d aromatic compounds.^ For example, n u c l e o p h i l i c aromatic s u b s t i t u t i o n reactions of 1-substituted 2,4-dinitrobenzenes i n DMSO are well characterized.^1 In a study of the reac t i o n of the 1-halo-substituted compounds with methoxide i n methanolic DMSO (Figure 47), the re a c t i o n rates followed the expected order: fluoro » chloro > bromo > iodo. In each case, the product was 2,4-dinitroanisole which was i s o l a t e d q u a n t i t a t i v e l y and characterized. A p a r t i c u l a r l y i n t e r e s t i n g observation i n t h i s i n v e s t i -gation was the co l o r change which occurred during the course of the OMe N02 N02 NO 2 N02 A N02 x x MeO N02 + N02 Me NOz NOj Figure 47: Meisenheimer intermediate (70) formed by the r e a c t i o n of 1-substituted 2,4-dinitrobenzenes and methoxide i n DMSO (X - F, CI, Br, I) 74 -r e a c t i o n . I n a s o l v e n t m i x t u r e c o n t a i n i n g >55 mol% DMSO, the m i x t u r e i n s t a n t l y became p u r p l e upon a d d i t i o n o f the s u b s t r a t e and the n gradu-a l l y became more r e d t o brown. The u . v . / v i s i b l e spectrum o f t h i s c o l o r e d s o l u t i o n showed an a b s o r p t i o n a t 295 run due t o the p r o d u c t , 2 , 4 - d i n i t r o a n i s o l e , b u t a l s o a l a r g e absorbance a t 500 nra. T h i s new absorbance was a t t r i b u t e d t o the f o r m a t i o n o f the Meisenheimer complex (70) shown i n F i g u r e 47. N u c l e o p h i l i c a t t a c k a t the 3- and the 5 - p o s i -t i o n s o f these h i g h l y a c t i v a t e d benzene r i n g s a l s o has been r e p o r t e d . ^ 2 The r e a c t i o n o f l - c h l o r o - 2 , 4 , 6 - t r i n i t r o b e n z e n e w i t h sodium h y d r o x i d e i n DMS0-dg/D20 s o l u t i o n s showed r a p i d hydrogen exchange o f the a r o m a t i c p r o t o n s by ^H-n.m.r. s p e c t r o s c o p y and i s o l a t e d s t a r t i n g m a t e r i a l a f t e r 30 seconds r e a c t i o n time showed f u l l exchange o f t h e s e p r o t o n s by mass s p e c t r o m e t r y . N u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n r e a c t i o n s a r e r a r e i n a r y l g l u c o p y r a n o s i d e c h e m i s t r y . I n f a c t , o n l y one example was f o u n d i n the l i t e r a t u r e which r e p o r t s a mechanism s i m i l a r t o the mechanism p r o p o s e d h e r e . T h i s i s the base c a t a l y z e d h y d r o l y s i s o f 4 - n i t r o p h e n y l a-D-gluco-p y r a n o s i d e (21) which was d e s c r i b e d p r e v i o u s l y I n the I n t r o d u c t i o n . 2 8 I n s h o r t , the h y d r o l y s i s i n v o l v e d base c a t a l y z e d 0(1) to 0(2) m i g r a t i o n o f the 4 - n i t r o p h e n y l group v i a an i n t r a m o l e c u l a r , n u c l e o p h i l i c , a r o m a t i c s u b s t i t u t i o n mechanism ( F i g u r e 17). The e v i d e n c e p r e s e n t e d h e r e sug-g e s t s a s i m i l a r mechanism (but w i t h no i n t r a m o l e c u l a r component) f o r the a n o m e r i z a t i o n o f p r o t e c t e d 2 , 4 - d i n i t r o p h e n y l g l u c o p y r a n o s i d e s . A l l o f the e v i d e n c e used i n s u p p o r t i n g o r o p p o s i n g the p r e v i o u s t h r e e mechanisms s u p p o r t s o r a t l e a s t does not d i s c o u n t mechanism ( i v ) . The absence o f H/D exchange a t C ( l ) , the absence o f p h e n o l a t e exchange, - 75 -and the measurement o f a s m a l l i s o t o p e e f f e c t a r e c o n s i s t e n t w i t h t h i s mechanism, b u t by no means prove i t . The marked i n f l u e n c e o f e l e c t r o n -w i t h d r a w i n g groups on the o r t h o - and p a r a - p o s i t i o n s o f the p h e n y l r i n g s u p p o r t s t h i s mechanism s i n c e s u b s t i t u t i o n a t these p o s i t i o n s has the l a r g e s t e f f e c t on the r e a c t i v i t y o f the a r o m a t i c group. The r e l a t i v e l y slow r e a c t i o n r a t e f o r the 2 , 6 - d i n i t r o p h e n y l and t h e 2 , 6 - d i c h l o r o - 4 -n i t r o p h e n y l g l u c o p y r a n o s i d e s (60 and 59, r e s p e c t i v e l y ) may be e x p l a i n e d by the i n c r e a s e i n s t e r i c b u l k a t the r e a c t i o n c e n t e r when the a r y l group i s s u b s t i t u t e d a t b o t h the 2- and the 6 - p o s i t i o n s . The u n r e a c t i -v i t y o f the 2 , 4 - d i n i t r o p h e n y l 1 - t h i o - g l u c o p y r a n o s i d e (61) i s c o n s i s t e n t w i t h the o b s e r v e d g e n e r a l t r e n d t h a t 1 - 0 - s u b s t i t u t e d compounds a r e more r e a c t i v e t o n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n t h a n a r e 1 - S - s u b s t i t u t e d compounds.^ The r e a c t i o n r a t e s measured f o r the 4 - d e o x y - 4 - f l u o r o (40) and the 2 - d e o x y - 2 - f l u o r o (39) d e r i v a t i v e s a r e c o n s i s t e n t w i t h t h i s mechanism s i n c e t h e e l e c t r o n e g a t i v e f l u o r i n e s u b s t i t u e n t s w i l l s t a b i l i z e the g l y c o s y l o x y a n i o n i n t e r m e d i a t e . Furthermore, the 4-deoxy (44) and the 6-deoxy (49) compounds showed the e x p e c t e d d e c r e a s e i n r a t e r e s u l t -i n g from the absence o f e l e c t r o n - w i t h d r a w i n g groups a t C(4) o r C ( 6 ) . The l a r g e d e c r e a s e i n r a t e o b s e r v e d f o r 2 , 3 , 4 , 6 - t e t r a - O - b e n z y l DNPG (31) may be due to the g r e a t e r b u l k o f the b e n z y l groups o r p o s s i b l y to s l o w e r a n o m e r i z a t i o n o f the t e t r a - O - b e n z y l g l y c o s y l o x y a n i o n . T h i s i s c o n s i s t e n t w i t h o t h e r f i n d i n g s from t h i s l a b o r a t o r y i n v o l v i n g the W i t t i g r e a c t i o n o f 2 , 3 , 4 , 6 - t e t r a - O - b e n z y l - D - g l u c o p y r a n o s e (36) w i t h v a r i o u s y l i d e s i n which (36) was found t o r e a c t v e r y s l o w l y and o n l y i n the p r e s e n c e o f v e r y s t r o n g base. The mechanism o f the W i t t i g r e a c t i o n i s s i m i l a r t o mechanism ( i v ) s i n c e i t a l s o p r o c e e d s v i a the open c h a i n form 76 -o f the sugar. I t i s p o s s i b l e , t h e r e f o r e , t h a t the slowness o f b o t h r e a c t i o n s may be due to the r e l a t i v e i n a b i l i t y o f the b e n z y l group to s t a b i l i z e the o x y a n i o n compared to the a c e t a t e group which i s c o n s i d e r -a b l y more e l e c t r o n - w i t h d r a w i n g . The most c o n c l u s i v e e v i d e n c e f o r mechanism ( i v ) comes from the exchange r e a c t i o n s where B-2,6-DNPG (60) was anomerized i n the pres e n c e o f FDNB, and where [1- 2H] - B - 2 ,4-DNPG (32) was anomerized i n the pr e s e n c e o f 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - D - g l u c o p y r a n o s e ( 3 0 ) . I n the f i r s t exper-iment, the 2,6-DNPG (60) r e a c t e d o n l y t o a s m a l l e x t e n t b u t the ^H-n.m.r. spectrum ( F i g u r e 35) o f the p r o d u c t m i x t u r e showed t h a t a- and B-2,4-DNPG (29) were formed, y e t no a-2,6-DNPG was formed. I n the second experiment, [1- 2H]-2,4-DNPG (32) l o s t 50% o f i t s d e u t e r i u m l a b e l upon a n o m e r i z a t i o n . T h i s second experiment c o n c l u s i v e l y demonstrates t h a t the o x y a n i o n o f 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - D - g l u c o p y r a n o s e must be an i n t e r m e d i a t e i n the r e a c t i o n mechanism. A d d i t i o n a l e v i d e n c e f o r mechanism ( i v ) a r e t h e c o l o r changes o b s e r v e d d u r i n g the a n o m e r i z a t i o n which a r e c h a r a c t e r i s t i c o f those found i n the f o r m a t i o n o f the p r e v i o u s l y d e s c r i b e d Meisenheimer com-p l e x e s . U . v . / v i s i b l e a b s o r p t i o n s p e c t r a o f the p u r p l e r e a c t i o n m i x t u r e r e s u l t i n g from m i x i n g B-2,4-DNPG (29) w i t h d i m s y l a n i o n o r w i t h bis(tetramethyl-ammonium) c a r b o n a t e i n DMSO showed an absorbance a t about 574 nm. As mentioned e a r l i e r , n u c l e o p h i l i c a r o m a t i c s u b s t i t u t i o n r e a c t i o n s o f 1 - s u b s t i t u t e d 2 , 4 - d i n i t r o b e n z e n e s a l s o e x h i b i t t h i s p u r p l e c o l o r and the u . v . / v i s i b l e spectrum showed an absorbance a t 500 nm.^i A l t h o u g h i t i s u n c l e a r whether the ca r b o n a t e o r the d i m s y l a n i o n a c t s as a n u c l e o p h i l e i n the a n o m e r i z a t i o n r e a c t i o n , i t seems more l i k e l y t h a t - 77 -i t i s the dimsyl anion since t h i s has been shown previously to be nu c l e o p h i l i c i n aromatic s u b s t i t u t i o n r e a c t i o n s . ^ 4 i n reactions where the anomerization was performed with the carbonate c a t a l y s t i n DMSO, the carbonate must be basic enough to deprotonate DMSO and form the dimsyl anion i n s i t u . Predictions of the b a s i c i t y of h i g h l y charged anions i n polar a p r o t i c solvents suggest that carbonate may be much more basic i n DMSO than i n water,-*3 and therefore i n s i t u formation of the dimsyl anion i s not unreasonable. The a l t e r n a t i v e , i n v o l v i n g the carbonate anion a c t i n g as the nucleophile, i s less l i k e l y but possible i n an aprotic solvent such as DMSO. The formation of the dinitrophenolate anion which i s apparent from the ^H-n.m.r. s p e c t r a l data of the anomerization r e a c t i o n mixture (Figure 39), can be r a t i o n a l i z e d by the act i o n of carbonate as a nucleophile (Figure 48). The intermediate (71) presumably would eliminate carbon dioxide quite r e a d i l y forming the Figure 48: Formation of the 2,4-dinitrophenolate anion during anomerization of (29) - 78 -2,4-dinitrophenolate anion. Although the re s u l t a n t protected glucose residue was not apparent by t . l . c . or ^H-n.m.r. spectra of the r e a c t i o n mixture, i t i s believed that the acetate groups on the glucosyl oxyanion (68) could e a s i l y rearrange and cause decomposition of the glucose residue to other sugars. Indeed, t h i s type of decomposition was observed with the previously described base catalyzed h y d r o l y s i s of 4-nitrophenyl a-D-glucopyranoside i n which saccharinic acids were the f i n a l carbohydrate products of the reaction. F i n a l l y , the mechanism proposed here f o r the anomerization of 0-2,4-DNPG (29) i s consistent with the ea r l y reports by Lindberg on the anomerization of 1,2,3,4,6-penta-O-acetyl - 0-D-glucopyranose (11) using a l k a l i i n pyridine.-^ 2 In t h i s case and i n accordance with mechanism ( i v ) , pyridine acts as a nucleophile and attacks the carbonyl group of the anomeric acetate, forming the highly reactive a c y l a t i n g reagent (72) shown i n Figure 49. Anomerization of the r e s u l t i n g glucosyl anion (68) and r e a c y l a t i o n affords the thermodynamically favored a-pentaacetate. Lindberg reported that the anomerization of the 0-pentaacetate (11) i s 6-7 times slower when the r e a c t i o n i s performed using a l k a l i i n ether-dioxane i n place of pyridine. This i s not s u r p r i s i n g since these conditions are more l i k e l y to cause deacetylation at the anomeric center v i a n u c l e o p h i l i c attack of hydroxide forming the glucosyl residue (68) and the acetate anion. Deacetylation and r e l a t i v e l y low y i e l d s were found i n Wolfrom and Hudsted's e a r l y study on anomerization of f u l l y a cetylated sugars under basic c o n d i t i o n s . ^ Further work towards proving mechanism (iv) should include synthe-s i z i n g the aromatic intermediates thought to be formed i n the reaction - 79 -Figure 49: Pyridine catalyzed anomerization of 1,2,3,4,6-penta-O-acetyl-/3-D-glucopyranose by a separate route, then c h a r a c t e r i z a t i o n of these intermediates (by u . v . / v i s i b l e and ^H-n.m.r. spectroscopy) and f i n a l l y , determining that they are present i n the anomerization mixture. I t should also be determined i f these intermediates react with 2,3,4,6-tetra-O-acetyl-glucopyranose (30) to form the expected equilibrium mixture of the a-and B- 2,4-DNPG. Measurements of rates of t h i s g l y c o s i d a t i o n reaction and comparison with measured rates of anomerization would be required to prove the k i n e t i c competence of these species as possible intermediates i n the anomerization. - 80 -MATERIALS AND METHODS 1. S y n t h e s i s 1.1 G e n e r a l P r o c e d u r e s and M a t e r i a l s M e l t i n g p o i n t s (m.p.) were d e t e r m i n e d w i t h a B r i s t o l i n e 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 . •^H-nuclear magnetic resonance (n.m.r.) s p e c t r a were r e c o r d e d w i t h a 300 MHz V a r i a n XL-300 i n s t r u m e n t . Chemical s h i f t s a r e g i v e n i n the d e l t a ( 5 ) s c a l e . Samples which were d i s s o l v e d i n C D C I 3 a r e r e f e r e n c e d to i n t e r n a l t e t r a m e t h y l s i l a n e (5 - 0.00). Samples which were d i s s o l v e d i n DMSO-dg a r e r e f e r e n c e d t o the r e s i d u a l p r o t o n r e s o n a n c e s from DMSO (5 = 2.49). S i g n a l m u l t i p l i c i t y , i n t e g r a t e d a r e a , c o u p l i n g c o n s t a n t s (where n e c e s s a r y ) , and resonance assignments a r e i n d i c a t e d i n paren-t h e s e s . 1 3 L C-n.m.r. s p e c t r a were r e c o r d e d on a V a r i a n XL-300 MHz i n s t r u m e n t . Samples d i s s o l v e d i n DMSO-dg were r e f e r e n c e d u s i n g the DMSO resonances as the i n t e r n a l r e f e r e n c e (39.5 ppm r e l a t i v e t o TMS). Che m i c a l s h i f t s a r e g i v e n i n ppm. ^F-n.m.r. s p e c t r a were r e c o r d e d a t 254 MHz on a Br u k e r model HX7-270 s p e c t r o m e t e r e q u i p p e d w i t h a N i c o l e t 1180 computer, a D i a b l o d i s k d r i v e and a 5 mm probe. -^F-Chemical s h i f t s were measured a g a i n s t e x t e r n a l t r i f l u o r o a c e t i c a c i d and a r e g i v e n i n ppm u p f i e l d from C F C I 3 . T r i f l u o r o a c e t i c a c i d r e s o n a t e s 76.53 ppm u p f i e l d from C F C I 3 . - 81 -I n f r a r e d s p e c t r a were r e c o r d e d on a N i c o l e t 5DX f o u r i e r t r a n s f o r m s p e c t r o p h o t o m e t e r u s i n g NaCl p l a t e s . C h e m i c a l - i o n i z a t i o n mass s p e c t r a were r e c o r d e d w i t h a KRATOS MS50 s p e c t r o m e t e r . U . v . / v i s i b l e absorbance s p e c t r a were r e c o r d e d on a Pye Unicam PU 8800 u . v . / v i s i b l e s p e c t r o p h o t o m e t e r u s i n g q u a r t z c e l l s . M i c r o - a n a l y s e s were performed by Mr. P. Borda, M i c r o - a n a l y t i c a l l a b o r a t o r y , U n i v e r s i t y o f B r i t i s h Columbia, Vancouver. S o l v e n t s and r e a g e n t s used were e i t h e r r e a g e n t grade, c e r t i f i e d or s p e c t r a l grade. Dry s o l v e n t s were p r e p a r e d as f o l l o w s . THF was r e f l u x e d o v e r c a l c i u m h y d r i d e , d i s t i l l e d , t h e n r e f l u x e d w i t h sodium benzophenone and d i s t i l l e d . DMF was s t i r r e d o v e r n i g h t w i t h p o t a s s i u m h y d r o x i d e t h e n d i s t i l l e d a t red u c e d p r e s s u r e from c a l c i u m o x i d e . DMSO was p r e d r i e d o v e r n i g h t w i t h sodium h y d r o x i d e p e l l e t s t h e n d i s t i l l e d a t re d u c e d p r e s s u r e from sodium h y d r o x i d e . D i s t i l l e d DMF and DMSO were s t o r e d o v e r 3A m o l e c u l a r s i e v e s . P y r i d i n e was p r e d r i e d o v e r n i g h t w i t h p o t a s s i u m h y d r o x i d e p e l l e t s f o l l o w e d by d i s t i l l a t i o n from b a r i u m o x i d e . Methanol was d i s t i l l e d from magnesium methoxide p r e p a r e d i n s i t u by the r e a c t i o n o f methanol w i t h magnesium t u r n i n g s i n the p r e s e n c e o f i o d i n e . Acetone was d r i e d o v e r p o t a s s i u m c a r b o n a t e . Anhydrous d i e t h y l e t h e r was use d as s u p p l i e d . DABCO, FDNB, D- g l u c o n o - 1 , 5 - l a c t o n e and 2 , 4 - d i n i t r o p h e n o l were o b t a i n e d from Sigma Chemical Co. Sodium b o r o d e u t e r i d e was o b t a i n e d from A l d r i c h C h e m i c a l s and was s t o r e d under n i t r o g e n . The f o l l o w i n g s u b s t i -t u t e d p h e n o l s were o b t a i n e d from F l u k a C h e m i c a l Co.: p e n t a f l u o r o p h e n o l , 3 , 4 - d i n i t r o p h e n o l , 2 , 3 - d i n i t r o p h e n o l , 2 , 5 - d i n i t r o p h e n o l , 2 , 6 - d i c h l o r o - 4 -- 82 -nitrophenol. 2,6-Dinitrophenol was obtained from J.T. Baker Chemical Co. A l l of the above chemicals were used as received without further p u r i f i c a t i o n . The following compounds and precursors were synthesized by other workers i n t h i s laboratory: 2,3,4,6-tetra-O-benzyl-D-glucopyranose (36); 1,2,3,6-tetra-O-acetyl-4-deoxy-4-fluoro-/9-D-glucopyranose (41); methyl 2,3,6-tri-0-benzoyl-4-deoxy-a-D-glucopyranoside (45); 1,2,3,4-tetra-O-acetyl-6 -deoxy-D-glucopyranose (50); 2,3,4,6-tetra-O-acetyl-D-glucal (62); 2,4-dini trophenyl 2,3,4,6-tetra-O-ace tyl-1-thio -B-D-glucopyrano-side (61); 2,4-dinitrophenyl 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-£-D-glucopyranoside (39). Dimethylsulfinyl anion was prepared from sodium hydride and DMSO and was ki n d l y provided by the laboratory of Dr. G.G.S. Dutton. Thin layer chromatography ( t . l . c . ) separations were performed using K i e s e l g e l 60 F-254 (Merck) a n a l y t i c a l p l a t e s . The spots were detected with u.v. l i g h t when possible, or by charring with 10% s u l f u r i c a c i d i n methanol. The solvent system was 1:1 (V/V) e t h y l acetate/petroleum ether unless otherwise stated and the Rf of the product In t h i s solvent system i s noted. Column chromatography was performed on Merck s i l i c a g el 60 (180-230 mesh). Solvents were evaporated i n vacuo at <50". Products were d r i e d i n vacuo and were generally stored i n a vacuum dessicator over phosphorous pentoxide. 2-methyl-2-propan(ol-d) (t-butanol) .t-Butanol was deuterated by d i s s o l v i n g t-butanol (3.9 g, 53 mmol) i n D 20 (5.0 ml, 250 mmol), mixing w e l l , and ext r a c t i n g out the deuterated t-butanol with pentane. The - 83 -organic ex t rac ts were d r i e d over calc ium oxide for 20 hours and a small amount o f deuterated t -butano l was i s o l a t e d by d i s t i l l a t i o n ; b . p . 81 -82° ( l i t . b . p . 8 2 ° ) ; ^H-n .m. r . : S (CDC1 3) 1.21 (s , 3 x CH3), no s i g n a l 5 2.40 due to -OH; IR: (neat) no -OH absorbance at 3600 c m " 1 . Bis(tetraalkylammonium)carbonates Te t ramethy l - , t e t r a e t h y l - , and tetrabutylammonium carbonate s a l t s were prepared from the t e t r a a l k y l -ammonium hydroxide and carbon d iox ide as descr ibed here f o r b i s ( t e t r a -methylammonium) carbonate. Carbon d iox ide was bubbled in to 25% t e t r a -methyl ammonium hydroxide i n water (25 ml) over a p e r i o d o f s e v e r a l hours dur ing which time a white p r e c i p i t a t e formed. The water was removed i n vacuo and a white s o l i d was obtained which was c r y s t a l l i z e d from warm a c e t o n i t r i l e ; m.p. > 3 0 0 ° , ^-H-n.m.r. 6 (DMS0-d 6) 3.10 (s , CH3). 1 3 C - n . m . r . : 6 (DMS0-d 6) 158.6 (s , C 0 3 " ) , 56.0 (q, CH3). A n a l . C a l c d . f o r C Q H 2 4 N 2 0 3 : C, 51.87; H, 11.61; N, 13.50. Found: C, 51.47; H, 11.29; N, 13.44. The other carbonate s a l t s were prepared i n a s i m i l a r manner but were not c h a r a c t e r i z e d . 2 . 3 . 4 . 6 - T e t r a - O - a c e t y l - q - D - g l u c o p v r a n o s v l bromide (16) a -D-g lucose (10 g , 56 mmol) was ace ty la ted us ing p y r i d i n e and a c e t i c anhydride by the method o f Wo1from and Thompson 6 5 to a f f o r d the a-pentaacetate (11) as a c r y s t a l l i n e s o l i d which was r e c r y s t a l l i z e d from 95% ethanol ; 16.5 g, 42.3 mmol, 76%; m.p. 110 -111° ( l i t . 6 5 m.p. 1 1 2 - 1 1 3 ° ) . The per -ace ta te (11) (10.0 g , 25.6 mmol) was brominated by the method of Haynes and Newth 4 2 us ing 45% HBr i n a c e t i c a c i d . 2 , 3 , 4 , 6 - t e t r a - 0 -a c e t y l - a - D - g l u c o p y r a n o s y l bromide was obtained as a c o l o r l e s s syrup and - 84 -was c r y s t a l l i z e d from d i e t h y l ether/petroleum ether. The pure (16) was stored at 0-5° i n the presence of potassium hydroxide p e l l e t s (8.47 g, 20.6 mmol, 79%) m.p. 89-90° ( l i t . 6 6 m.p. 88-89°). 2.3.4.6-Tetra-O-acetyl-fl-D-glucopyranose (30) — Compound (30) was prepared by hydrolysis of the bromide (16) (5.5 g, 3.0 mmol) using s i l v e r carbonate i n aqueous a c e t o n e 4 3 and was i s o l a t e d as a c r y s t a l l i n e s o l i d (3.07 g, 8.8 mmol, 68%); m.p. 131-134° ( l i t . 4 3 m.p. 132-134°). 1.2 General Preparation of 2,4-Dinitrophenyl ^-D-Glucopyranosides The method of van Boom 3 3 was employed f o r the syntheses of 2,4-dinitrophenyl glucopyranosides and i s described here f o r the pre-paration of 2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-^-D-glucopyranoside (29). 2,3,4,6-tetra-O-acetyl-0-D-glucopyranose (30) (1.25 g, 3.6 mmol) and DABCO (1.37 g, 12.2 mmol) were dissolved i n DMF and the s o l u t i o n s t i r r e d over 3A molecular sieves to remove traces of water. FDNB (0.78 g, 4.2 mmol) was added and the s o l u t i o n s t i r r e d f o r 2 hours at which time t . l . c . showed one u.v. act i v e , charring component (Rf - 0.36). The s o l u t i o n was concentrated i n vacuo to a syrup, then d i s s o l v e d i n chloro-form, washed with 10% aqueous sodium bicarbonate and water, respec-t i v e l y , and f i n a l l y the organic phase was dr i e d with magnesium sulphate. Removal of the solvent produced the product (29) as a yellow syrup which c r y s t a l l i z e d Immediately upon addition of 95% ethanol. Repeated recrys-t a l l i z a t i o n afforded the pure (29) as white needles (1.50 g, 2.9 mmol, - 85 -81%); m.p. 177-178° ( l i t . " m.p. 174-175°); iH-n.m.r. data: S (CDC13) 8.68 (d, 1H, H(3) of DNP), 8.40 (dd, 1H, H(5) of DNP), 7.49 (d, 1H, H(6) of DNP), 5.4-5.1 (m, 3H, H(2), H(3), H(4)), 4.25 (m, 2H, H(6), H(6')), 4.OA (m, 1H, H(5)) and 2.2-2.0 (4S, 12H, 4CH3C0); S (DMS0-d6) 8.81 (d, 1H, H(3) of DNP), 8.58 (dd, 1H, J 5 6 10 Hz, J53 3 Hz, H(5) of DNP), 7.66 (d, 1H, J 6 5 10 Hz, H(6) of DNP), 5.93 (d, 1H, J 1 2 9 H z -H ( l ) ) , 5.43 (dd, 1H, J 3 4 9 Hz, J 3 2 9 Hz, H(3)), 5.20-5.00 (m, 2H, H(2) and H(4)), 4.38 (m, 1H, H(5)), 4.23 (dd, 1H, J 6 6 - 12 Hz, J 6 5 - 6 Hz, H(6)), 4.13 (dd, 1H, J 6 6 12 Hz, J 6 > 5 3 Hz, H(6')), 2.05 (s, 3H CH3CO), 2.00 (s, 6H, 2 x CH3CO), 1.97 (s, 3H, CH3CO). D e r i v a t i z a t i o n of 2,3,4,6-tetra-O-benzyl-D-glucopyranose (0.81 g, 1.5 mmol) by the foregoing procedure produced 2,4-dinitrophenyl 2,3,4,6-tetra-O-benzyl-0-D-glucopyranoside (31) (1.70 g, 1.2 mmol, 81%); m.p. 99-100° ( l i t . 3 3 m.p. 99-100°); ^-n.m.r. data: S (CDCI3) 8.64 (d, 1H, J35 3 Hz, H(3) of DNP), 8.11 (dd, 1H, J 5 6 10 Hz, J53 3 Hz, H(5) of DNP), 7.27-7.20 (m, 21H, H(6) of DNP and aromatic H of benzyl groups), 5.15 (d, 1H, J 1 > 2 7 Hz, H (D ) , 5.04-4.46 (m, , 10H, methylene H), 3.87-3.40 (m, 6H, H(2), H(3), H(4), H(5), H(6) and H(6'); 6 (DMS0-d6) 8.80 (d, 1H, J35 3 Hz, H(3) of DNP), 8.42 (dd, 1H, J 5 6 10 Hz, J53 3 Hz, H(5) of DNP), 7.67 (d, 1H, H 6 5 10 Hz, H(6) of DNP), 7.60-7.02 (m, 20H, aromatic H of benzyl groups), 5.71 (d, 1H, J 1 2 6 Hz, H ( l ) ) , 4.95-4.33 (m, 10H, methylene), 3.96 (m, 1H, H(5)), 3.87-3.47 (m, 6H, H(2), H(3), H(4), H(5), H(6) andH(6'). - 86 -1.3 Deuterium Labe l l ed Glucopyranosides 1.3.1 2 ,4 -D in i t ropheny l 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - [ 1 - 2 H ] - / 9 - D - g l u c o pyranoside (32) 2 .3.4.6-Tetra-O-acetyl - f l -D-T1 - 2 H 1 -glucopyranose (34) — The procedure I T of N e l s o n J / was used fo r the a c e t y l a t i o n of D -g lucono-1 ,5 - l ac tone . A mixture o f D -g lucono-1 ,5 - lac tone (5.0 g , 28 mmol), and z i n c c h l o r i d e (2.5 g , 1.8 mmol) i n a c e t i c anhydride (25 ml) was s t i r r e d at room temperature f o r 1 h , poured in to a c o l d , saturated sodium bicarbonate s o l u t i o n (250 mL), s t i r r e d fo r 1 h , ex t rac ted three times wi th c h l o r o -form (100 mL) and the ex t rac ts combined, washed twice wi th c o l d water (100 mL), d r i e d wi th magnesium sulphate , and evaporated to a c o l o r l e s s syrup which c o n s i s t e d of one component by t . l . c . (Rf - 0.52, H2SO4 s p r a y ) . Reduct ion of the lactone (33) was performed by the method of Hosie and S innot t . 3 * * The syrup was d i s s o l v e d i n te t rahydrofuran (60 mL) and coo led to 0 ° . A c o l d s o l u t i o n of sodium borodeuter ide (0.44 g , 11 mmol) i n D 2 0 (2.1 mL) was added dropwise with s t i r r i n g , and the mixture s t i r r e d f o r 2 h , the base was n e u t r a l i z e d with Dowex 50-W (H +) i on -exchange r e s i n , the suspension f i l t e r e d , and the f i l t r a t e evaporated i n vacuo. The r e s u l t i n g syrup was washed with methanol to remove b o r i c a c i d . The product (34) was obtained as a c o l o r l e s s syrup which was a mixture of the anomers (8.02 g, 23 mmol, 82%), R f - 0.42 (H2SO4 spray) . - 87 -2.4- D ln i t rophenv l 2 . 3 . 4 . 6 - t e t r a - O - a c e t y l - f l - D -ri- 2Hl - g l u c o p y r a n o s i d e (32) — The t e t r a acetate (34) was d e r i v a t i z e d by the method descr ibed fo r (29). The deuterated 0-DNPG (32) was i s o l a t e d as white needles (9.62 g, 19 mmol, 66% from the l a c t o n e ) ; m.p. 176 -177° ( l i t . 3 3 m.p. 1 7 4 - 1 7 5 ° ) ; ^H-n.m.r . showed an i d e n t i c a l spectrum to (29) except peaks at 5 5 .1-5.4 in tegra ted f o r 3 (not 4) protons i n C D C I 3 and there was no resonance at 6 5.93 (H( l ) ) i n DMSO-d 6 i n d i c a t i n g l o s s of H ( l ) . I so top ic p u r i t y at the anomeric center was confirmed by mass spectrometry of 1 ,2 ,3 ,4 ,6 -penta -O-ace ty l - 0 -D -g lucopyranose which was prepared us ing a m o d i f i c a t i o n o f the foregoing p r o c e d u r e ; 3 6 m/z ( r e i . i n t e n s i t y ) 332 [5.51, [ 2H] (M+-0Ac) - C 1 4 H 1 8 D 0 9 ] and 331 [0.15, (M+-0Ac) - C 1 4 H 1 9 0 9 ] . 1.3.2 2 , 4 - D i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - O - b e n z y l - 0 - D - [ 1 - 2 H ] - g l u c o -pyranoside (35) 2 . 3 . 4 . 6 - T e t r a - O - b e n z v l - D - \ 1 - 2 H 1 -glucopvranose (38) Ox idat ion of 2 , 3 ,4 ,6 - t e t ra -O-benzy l -D -g lucopyranose (36) (1.23 g , 2.3 mmol) with a s o l u t i o n of a c e t i c anhydride (4.7 mL) and DMSO (7.0 m L ) 4 1 a f forded the benzy la ted lactone (37) as a c o l o r l e s s syrup; Rf - 0.74 (7:1 V/V benzene/ether , H2SO4 spray and u . v . ) ; IR (neat) 1757 c m " 1 ( O O , 1.5- l a c t o n e ) , no -OH absorp t ion . Reduction of the lactone by the method employed f o r reduct ion of (33) produced the product (38) as a white c r y s t a l l i n e s o l i d which was r e c r y s t a l l i z e d from warm e thano l ; (1.06 g, 2.0 mmol, 87% from (36)); m.p. 150 -151° ( l i t . 6 7 m.p. 151 -152° fo r nondeuterated (38)) . - 88 -2 . 4-Dinitrophenyl 2 . 3 . 4.6-tetra-O-benzvl-fl-D-f1 - 2 H 1-glucopyranoside (35) - Compound (38) was d e r i v a t i z e d a c c o r d i n g t o the g e n e r a l method t o a f f o r d the b e n z y l a t e d /3-DNPG (35) as a y e l l o w syrup which was c r y s t a l -l i z e d from 95% e t h a n o l ; (1.30 g, 1.8 mmol, 79% from ( 3 6 ) ) ; R f = 0.71 (98 :2 V/V c h l o r o f o r m / m e t h a n o l ) ; m.p. 99-100° ( l i t . 3 3 m.p. 99-100°); •'-H-n.m.r. d a t a : i d e n t i c a l spectrum t o (31) exc e p t t h e r e was no resonance f o r H ( l ) a t 5 5.15 i n CDC13 o r a t 5 5.71 i n DMS0-d6. 1.4 Deoxy and Deoxyfluoro Glucopyranosides 1.4.1 2,4-Dinitrophenyl 2,3,6-tri-0-acetyl-4-deoxy-4-fluoro-^-D-glucopyranoside (40) 2 . 3 .6-Tri - 0-acetyl - 4-deoxy - 4-fluoro-a-D-glucopyranosyl bromide (42) — The bromide (42) was p r e p a r e d e s s e n t i a l l y by the method o f Haynes and N e w t h 4 2 from 1 , 2 , 3,6-tetra - 0-acetyl - 4-deoxy - 4-fluoro - / 3-D-glucopyranose ( 4 1 ) . A s o l u t i o n o f (41) (0.100 g, 0.29 mmol) i n 45% HBr/HOAc (3.0 mL) and a c e t i c a n h y d r i d e (0.30 mL) was s t i r r e d f o r 2 h a t room temperature t h e n c o o l e d t o 0°, d i l u t e d w i t h d i c h l o r o m e t h a n e (30 mL), washed f i v e times w i t h i c e c o l d water, and the o r g a n i c phase d r i e d w i t h sodium s u l p h a t e . Removal o f the s o l v e n t produced the p r o d u c t as a c o l o r l e s s s y rup which was c r y s t a l l i z e d from d i e t h y l e t h e r / h e x a n e s ; Rf = 0.61 (H2SO4 s p r a y ) ; 90 mg, 0.24 mmol, 85%. - 89 -2.3.6-Tri-0-acetvl-4-deoxy-4-fluoro-D-glucopvranose (43) — Hydrolysis of the bromide (42) was performed according to the method of McCloskey and Coleman. 4 3 A s o l u t i o n of (42) (90 mg, 0.24 mmol) was di s s o l v e d i n acetone (0.20 mL) and cooled to 0°. Water (10 fiL) was added followed by add i t i o n of s i l v e r carbonate (0.10 g, 0.36 mmol) ( f r e s h l y prepared from sodium carbonate and s i l v e r n i t r a t e i n small portions. The s o l u t i o n was s t i r r e d f o r 20 h at room temperature i n the dark, warmed to 50°, and the s i l v e r s a l t s were f i l t e r e d and washed with two portions of warm acetone. The solvent was removed from the combined f i l t r a t e s to produce a brown syrup which was not p u r i f i e d ; 64 mg, 0.21 mmol. 2 . 4-Dinitrophenyl 2 . 3 . 6 -tri - 0 -acetyl - 4 -deoxv - 4-fluoro-fl-D-gluco-pyranoside (40) D e r i v a t i z a t i o n of the u n p u r i f i e d t r i - a c e t a t e (43) was performed by the general method to a f f o r d (40) as yellow needles (45 mg, 0.091 mmol, 34% from tetra-acetate (41)); R f - 0.74 (H2SO4 spray, u.v.); m.p. 171-173°; ^-n.m.r. data: S (CDCI3) 8.72 (d, IH, J35 3 Hz, H(3) of DNP), 8.44 (dd, IH, J 5 > 6 10 Hz, J 5 3 3 Hz, H(5) of DNP), 7.47 (d, IH, J 6 5 10 Hz, H(6) of DNP), 5.50-5.23 (m, 3H, H ( l ) , H(2), H(4)), 4.68 (dt, IH, J F 4 50, J 9 Hz, H(4)), 4.55 (dd, IH, J 6 6 12 Hz, H(6) or H(6')), 4.28 (dd, IH, J 6 6 , 12 Hz, H(6) or H(6')), 4.20 (m, IH, H(5)), 2.14 (s, 6H, 2 x CH3CO), 2.09 (s, 3H, CH3CO); 6 (DMS0-d6) 7.62 (d, IH, J 6 5 10 Hz, H(6) of DNP), 5.93 (d, IH, J 1 2 8 Hz, H ( l ) ; 1 9F-n.m.r. data: 6 (CDCI3) 199.8 (dd, J F 4 50.2 Hz, J F 3 15.3 Hz). Anal. Calcd. for C18 H19 F N2 ° 1 2 : c> 4 5 - 5 7 : H> 4.04; N, 5.93. Found: C, 45.50; H, 3.82; N, 6.01. - 90 -1.4.2 2,4-Dinitrophenyl 2,3,6-tri-O-acetyl-4-deoxy-0-D-gluco-pyranoside (44) Methyl 2.3.6-tri-0-acetvl-4-deoxv-a-D-glucopyranoside (46) — To a suspension of methyl 2,3,6-tri-0-benzoyl-4-deoxy-a-D-glucopyranoside (45) (0.20 g, 0.41 mmol) i n methanol (1.0 mL) was added 1 M sodium methoxide i n methanol (0.010 mL). 4 4 The mixture was s t i r r e d f o r 2 days at room temperature, n e u t r a l i z e d with Dowex 50W-X8 (H +) ion exchange r e s i n , and the suspension f i l t e r e d . The f i l t r a t e was concentrated i n vacuo and the concentrate was p u r i f i e d by column chromatography (90:10 V/V e t h y l acetate/methanol). The r e s u l t i n g syrup (72 mg, 88%) was immediately acetylated e s s e n t i a l l y by the method of Wolfrom and Thomp-s o n . 6 5 A s o l u t i o n of a c e t i c anhydride (0.37 mL, 3.9 mmol) and pyridine (0.47 mL) was cooled to 0° and added to the syrup. The mixture was s t i r r e d f o r 1 h at 0" then warmed to room temperature and s t i r r e d overnight. The reac t i o n was quenched by ad d i t i o n of methanol to the cooled mixture and s t i r r e d at 0° for 1 h. The s o l u t i o n was d i l u t e d with dichloromethane, washed three times with i c e c o l d water and the organic phase was d r i e d with sodium sulphate. Removal of the solvent i n vacuo afforded the product (46) as a c o l o r l e s s syrup which was c r y s t a l l i z e d from d i e t h y l ether (87 mg, 0.29 mmol, 71%); R f - 0.50 (H2SO4 spray). 2.3.6-tri-Q-acetyl-4-deoxv-q-D-glucopyranosvl chloride (47) — C h l o r i -nation of (46) was performed by the method of Kovac e t . a l . 4 5 The methyl glucopyranoside (46) was dissolv e d i n dichloromethyl methyl ether (0.40 mL) and a c a t a l y t i c amount of f r e s h l y fused zinc c h l o r i d e was added. - 91 -The s o l u t i o n was s t i r r e d at 65-70° f o r 1 hr at which time t . l . c . showed only one component, Rj - 0.59 (H2S04 spray). The s o l u t i o n was concentrated i n vacuo, d i l u t e d with chloroform, washed twice with 10 mL of c o l d saturated sodium bicarbonate s o l u t i o n . The organic phase was dr i e d with sodium sulphate and removal of the solvent produced a brown syrup (65 mg, 74% from (45)). 2.3.6-Tri-0-acetyl-4-deoxv-D-glucopvranose (48) The chloro sugar (47) was hydrolyzed by the method employed f o r (42) to produce (48) as a yellow syrup (82 mg). 2.4-Dinltrophenvl 2 . 3.6-tri - 0-acetvl-4-deoxv-fl-D-glucopyranoside (44) -Compound (48) was reacted with FDNB by the usual method to produce (44) as a yellow c r y s t a l l i n e s o l i d (96 mg, 0.21 mmol, 51% from the t r i - 0 -benzoate (45)); R f - 0.45 (H2S04 spray, u.v.); m.p. 180-181°; ^H-n.m.r. data: S (CDCI3) 8.71 (d, 1H, J35 3 Hz, H(3) of DNP), 8.42 (dd, 1H, J 5 6 10 Hz, J 5 3 3 Hz, H(5) of DNP), 7.48 (d, 1H, J 6 5 10 Hz, H(6) of DNP), 5.32-5.05 (m, 3H, H ( l ) , H(2), H(3)), 4.22 (d, 2H, J 6 6 7 Hz, H(6) and H(6'), 4.40 (m, 1H, H(5)); S (DMS0-d6) 7.60 (d, 1H, J 6 5 10 Hz, H(6) of DNP), 5.72 (d, 1H, J 1 > 2 8 Hz, H ( l ) ) . Anal. Calcd. f o r C l gH 2 0N 2O 1 2: C, 47.36; H, 4.42; N, 6.16. Found: C, 47.27; H, 4.40; N, 6.18. - 92 -1.4.3 2,4-Dinitrophenyl 2, 3,4-tri-0-acetyl-6-deoxy-/9-D-gluco -pyranoside (49) 2.3.4-Tri-0-acetyl-6-deoxv-q-D-glucopyranosvl bromide (51) — 1,2,3,4-tetra - 0-acetyl-6-deoxy-D-glucopyranose (50) (0.40 g, 1.2 mmol) was brominated by the method employed f o r the preparation of (42). A f t e r s t i r r i n g f o r 2 h at room temperature, t . l . c . showed one component, Rf -0.69 (H2SO4 spray)* ^ e Product was i s o l a t e d as a white s o l i d (420 mg, 1.06 mmol, 99%). 2.3.4-Tri-0-acetvl-6-deoxy-a-D-glucopyranose (52) — Hydrolysis of the bromide (51) was performed exactly as for (43), y i e l d i n g (52) as a c o l o r l e s s gum (306 mg, 1.05 mmol, 99% from bromide); R f - 0.42 (H2S04 spray). 2 , 4-Dinitrophenvl 2 .3.4-tri-0-acetyl-6-deoxv-fl-D-glucopyranoside (49) — Compound (52) was d e r i v a t i z e d by the general method to a f f o r d (49) as white needles (424 mg, 0.93 mmol, 77% from the tetra-acetate (50)); Rf -0.58 (H2S04 spray, u.v.); m.p. 198-199°; ( l i t . 3 5 m.p. 152-156°); iH-n.m.r. data: 6 (CDCI3) 8.70 (d, 1H, J35 3 Hz, H(3) of DNP), 8.43 (dd, 1H, J 5 6 10 Hz, J53 3 Hz, H(5) of DNP), 7.45 (d, 1H, J6_5 10 Hz, H(6) of DNP), 5.46-5.20 (m, 3H, H ( l ) , H(2), H(3)), 4.95 (dd, 1H, J 4 5 J 4 3 10 Hz, H(4)), 3.83 (m, 1H, H(5)), 2.12 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO), 2.05 (s, 3H, CH3C0), 1.35 (d, 3H, J 6 5 4 Hz, 3 xH(6)); 5 (DMS0-d6) 7.66 (d, 1H, J 6 5 10 Hz, J(6) of DNP), 5.86 (d, 1H, J 1 2 7 H z • H ( l ) ) . Anal. Calcd. f o r C 1 8H 2oN20 1 2: C, 47.36; H, 4.42; N, 6.16. Found: C, 47,34; H, 4.35; N. 6.10. 1.5 P e r a c e t y l a t e d S u b s t i t u t e d P h e n y l G l u c o p y r a n o s i d e s P r e p a r a t i o n s o f a c e t y l a t e d g l u c o p y r a n o s i d e s (53-60) were c a r r i e d out by the method o f S i n n o t t and V i r a t e l l e 4 6 as f o l l o w s . 2 , 3 , 4 , 6 - t e t r a -O - a c e t y l - a - D - g l u c o p y r a n o s y l bromide (16) (1.0 g, 2.4 mmol) was d i s s o l v e d i n a c etone (6.0 mL) and the s o l u t i o n mixed w i t h the a p p r o p r i a t e p h e n o l (4.0 mmol) d i s s o l v e d i n 1.0 M sodium h y d r o x i d e (4.0 mL). The m i x t u r e was s t i r r e d a t room temperature f o r 20 hours a t which time t . l . c . showed one u.v. a c t i v e , c h a r r i n g component and some p r o p o r t i o n o f a component c o r r e s p o n d i n g t o 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - D - g l u c o p y r a n o s e (Rf = 0.42, H 2 S 0 4 s p r a y ) . The acetone was removed i n vacuo and the m i x t u r e d i l u t e d w i t h water (20 mL). The s o l u t i o n was e x t r a c t e d 3 times w i t h d i c h l o r o -methane (25 mL) and the o r g a n i c phase was the n washed t w i c e w i t h 50 mL o f 2 M sodium h y d r o x i d e , washed once w i t h 50 mL o f water and d r i e d w i t h magnesium s u l p h a t e . E v a p o r a t i o n o f the s o l v e n t a f f o r d e d the g l u c o p y r a -n o s i d e as a y e l l o w syrup which c r y s t a l l i z e d i m m e d i a t e l y upon a d d i t i o n o f 95% e t h a n o l . The p r o d u c t s were r e c r y s t a l l i z e d from warm e t h a n o l . Compounds and c h a r a c t e r i z a t i o n d a t a a r e l i s t e d below. Ph e n y l 2 . 3 . 4 . 6 - t e t r a - 0 - a c e t v l - g - D - g l u c o p v r a n o s i d e (53) — (0.25 g, 0.60 mmol, 4 6 % ) , R f = 0.50 ( H 2 S 0 4 s p r a y , u . v . ) , m.p. 124-126° ( l i t . 6 8 m.p. 125-126°) ^-n.m.r. d a t a : 6 ( C D C I 3 ) 7.38-6.80 (m, 5H, a r o m a t i c ) , 5.35-4.93 (m, 4H, H ( l ) , H(2), H(3), H ( 4 ) ) , 4.25 (dd, IH, H 6 6 , , 12 Hz, - 94 -J 6 > 5 4 hz, H(6)), 4.10 (dd, IH, J 6 6 12 Hz, J 6 - 5 2 Hz, H(6')), 2.08 (s, 3H, CH3C0), 2.05 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 1.98 (s, 3H, CH3CO) . 4-Nitrophenvl 2.3.4.6-tetra-O-acetyl-fl-D-glucopyranoside (54) — (0.39 g, 0.83 mmol, 35%); R f 0.40 (H 2S0 4 spray, UV); m.p. 178-180° ( l i t . 6 8 m.p. 174-175°); ^-n.m.r. data: 5 (CDCI3) 8.15 (dd, 2H, J 3 2 and J 5 6 11 Hz, J 3 5 4 Hz, H(3) and H(5), aromatic), 5.35-5.15 (m, 4H, H ( l ) , H(2), H(3), H(4)), 4.30-4.15 (m, 2H, H(6) and H(6')) 3.88 (m, IH, H(5)), 2.08-1.95 (4S, 12H, 4 x OCOCH3). Pentafluorophenvl 2.3.4.6-tetra-O-acetyl-fl-D-glucopvranoside (55) — (0.64 g, 1.24 mmol, 51%); R f - 0.51 (H 2S0 4 spray, u.v.); m.p. 144-145°; ^-n.m.r. data: 6 (CDCI3) 5.34-5.12 (m, 3H, H(2), H(3), H(4)), 4.97 (d, IH, J 1 2 8 Hz, H ( l ) ) , 4.25-4.10 (2dd, J 6 > 5 4 Hz, J 6 , 5 2 Hz, J 6 6 . 12 Hz, H(6) and H(6')), 3.70 (m, IH, H(5)), 2.10 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.02 9s, 6H, 2 x CH3CO); 1 9F-n.m.r. data: 6 (CDCI3) 166.73 (t , IF, J F 4 i F 3 and J F 4 . F 5 21 Hz, F(4)), 163.30 (m, 2F, F(3) and F(5)). 147.32 (dd, 2F, J F 2 . F 3 and J F6-F5 * 9 H z> F(2) and F(6)), Anal. Calcd. f o r C 2 0 H 1 9 F 5 0 1 0 : C, 46.71; H, 3.72. Found: C, 45.90; H, 3.56. 3.4-Dinitrophenyl 2.3.4.6-tetra-0-acetyl-5-D-glucopyranoside (56) — (0.71 g, 1.38 mmol, 57%); R f - 0.63 (H 2S0 4 spray, u.v.) m.p. 171-172°; ^•H-n.m.r. data: 6 (CDCI3) 8.03 (d, IH, J 5 6 8 Hz, H(5) of DNP) 7.42 (d, IH, J 2 6 2 Hz, H(2) of DNP), 6 7.25 (IH, H(6) of DNP, hidden by CHCI3 is o t o p i c impurity peak) 5.42-5.22 (m, 4H, H ( l ) , H(2), H(3), H(4)), 4.22 - 95 -(d, 2H, J 6 5 and H 6 . 5 4 Hz, H(6) and H(6'), 3.97 (dt, 1H, J 4 11 Hz, J 5 6 and H 5 6 . 5 Hz H(5)), 2.08-1.98 (4S, 12H, 4 x CH3CO). Anal. C a l c d . for C 2oH22 N2°14 : c» 4 6 - 6 9 I H . 4.31; N, 5.47. Found: C, 46.49; H, 4.22; N, 5.52. 2.3 Dinitrophenyl 2.3.4.6-tetra-O-acetvl-fl-D-glucopyranoside (57) — (0.33 g, 0.64 mmol, 51%); R f - 0.35 (H 2S0 4 spray, u.v.); m.p. 191-192°; iH-n.m.r. data: S (CDCI3) 7.96 (dd, 1H, J 4 5 8 Hz, J 4 > 6 2 Hz, H(4) of DNP), 7.68 (dd, 1H, J 6 5 8 Hz, J 6 4 2 Hz, H(6) of DNP), 7.62 (dd, 1H, J 5 4 8 Hz, J 5 6 8 Hz, H(5) of DNP), 5.32-5.05 (m, 4H, H ( l ) , H(2), H(3), H(4)), 4.24 (d, 2H, J 6 5 and J 6» 5 4 Hz, H(6) and H(6'), 3.86 (dt, 1H, J 5 > 4 10 Hz, J 5 > 6 and J 5 6 , 4 Hz, H(5)), 2.14 (s, 3H, CH3CO), 2.09 (s, 3H, CH3CO), 2.05 (s, 3H, CH3CO), 2.03 (s, 3H, CH3CO). Anal. Calcd. for C 2 0H 2 2N 2O 1 4: C, 46.69; H, 4.31; N, 5.47. Found: C, 46.32; H, 4.51; N, 5.42. 2.5-Dinitrophenvl 2.3.4.6-tetra-Q-acetvl-fl-D-glucopvranoside (58) — (0.41 g, 0.80 mmol, 33%); R f - 0.60 (H 2S0 4 spray, u.v.); m.p. 145-147°; ^H-n.m.r. data: S (CDC13) 8.23 (d, 1H, J 6 4 2 Hz, H(6) of DNP), 8.06 (dd, 1H, J 4 3 9 Hz, J 4 6 2 Hz, H(4) of DNP), 7.89 (d, 1H, J 3 4 9 Hz, H(3) of DNP), 5.37-5.06 (m, 4H, H ( l ) , H(2), H(3), H(4)), 4.30 (dd, 1H, J 6 , > 6 12 Hz, J 6 , 5 3 Hz, H(6')), 4.19 (dd, 1H, J 6 > 6 , 12 Hz, J 6 > 5 6 Hz, H(6)), 4.02 (m, 1H, H(5)), 2.13 (s, 3H, CH3CO), 2.11 (s, 3H, CH3CO), 2.05 (s, 3H, CH 3C0), 2.02 (s, 3H, CH3CO). - 96 -2.6-Dichloro-4-nitrophenyl 2 . 3 . 4 .6-tetra-O-acetyl-0-D-glucopyranoside (59) (0.75 g, 1.4 mmol, 58%); R f - 0.60 (H 2S0 4 spray, u.v.); m.p. 181-182°; ^-n.m.r. data: S (CDC13) 8.20 (s, 2H, H(3) and H(4) of aromatic group), 5.46-5.13 (m, 4H, H ( l ) , H(2), H(3), H(4)), 4.18 (dd, 1H, J 6 6 11 Hz, H(6')), 4.07 (dd, 1H, J 6 . ( 6 , 11 Hz, J 6 ' , 5 , 2 Hz, H(6')), 3.65 (m, 1H, H(5), 2.09 (s, 3H, CH3CO), 2.04 (s, 3H, CH3CO), 2.02 (6H, 2 x CH3CO). Anal. Calcd. f o r C2oH2iCl2N08: C, 44.62; H, 3.93; N, 2.61. Found: C, 44.39; H, 4.06; N, 2.79. 2.6-Dinitrophenyl 2 . 3 . 4.6-tetra - 0 -acetyl-fl-D-glucopyranoside ( 6 0 ) — — (0.34 g, 0.66 mmol, 55%); Rf - 0.40 (H2S04 spray, u.v.); m.p. 190-191°; ^-n.m.r. data: S (CDCI3) 7.98 (d, 2H, J35 8 Hz, H(3) and H(5) of DNP), 7.47 ( t , 1H, J 4 5 and J 4 3 8 Hz, H(4) of DNP), 5.36-5.08 (m, 4H, H ( l ) , H(2), H(3), H(4)), 4.03 (d, 2H, J 6 > 5 and J 6 , 5 4 Hz, H(6) and H(6'), 3.62 (dt, 1H, H 5 4 10 Hz, J 5 6 and J 5 6 » 4 Hz, H(5)), 2.13 (s, 3H, CH3CO), 2.10 (s, 3H, CH3CO), 2.03 (s, 3H, CH3CO), 2.00 (s, 3H, CH3CO). Anal. Calcd. f o r C 2 0H 2 2N 2 0 1 4 : C, 46.69; H, 4.31; N, 5.47. Found: C, 46.45; H, 4.23; N, 5.29. - 97 -2. A n o m e r i z a t i o n R e a c t i o n s 2.1 P r e p a r a t i v e S c a l e A n o m e r i z a t i o n 2.1.1 2 , 4 - D i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - a - D - g l u c o p y r a n o s i d e (29-cO The a-anomer (29-a) was p r e p a r e d e s s e n t i a l l y by t h e method o f van Boom 3 3 as f o l l o w s . 2 , 4 - D i n i t r o p h e n y l 2,3,4,6-tetra-0-acetyl-/9-D-g l u c o p y r a n o s i d e (29-/9) (0.453 g, 0.88 mmol) was d i s s o l v e d i n DMF (1.7 mL) c o n t a i n i n g 3A m o l e c u l a r s i e v e s t o remove t r a c e s o f water. Anhydrous p o t a s s i u m c a r b o n a t e (0.320 g, 2.3 mmol) was added and the m i x t u r e was s t i r r e d f o r 20 h a t room temperature a f t e r which time t . l . c . (H2SO4 s p r a y , u.v.) i n d i c a t e d t h a t the m i x t u r e c o n t a i n e d the a- and B- anomers (-80:20) ( R f (a) = 0.52, R f (0) = 0.36, H 2 S 0 4 s p r a y and u . v . ) . The mi x t u r e was d i l u t e d w i t h c h l o r o f o r m , f i l t e r e d and c o n c e n t r a t e d i n vacuo to a r e d s y r u p . The syrup was r e d i s s o l v e d i n c h l o r o f o r m (50 mL), washed t w i c e w i t h 10% sodium b i c a r b o n a t e s o l u t i o n (50 mL), t h e n t w i c e w i t h water (50 mL). The o r g a n i c phase was d r i e d w i t h magnesium s u l p h a t e and e v a p o r a t e d i n vacuo t o a y e l l o w s y r u p . The syrup was d i s s o l v e d i n warm 95% e t h a n o l and the a-anomer f r a c t i o n a l l y c r y s t a l l i z e d out from the s o l u t i o n (211 mg, 0.41 mmol, 4 7 % ) . The c r y s t a l s were f i l t e r e d and the f i l t r a t e c o n c e n t r a t e d and c o o l e d t o produce c r y s t a l s which were a m i x t u r e o f a- and B- anomers (200 mg, 0.39 mmol, 4 4 % ) . T h i s m i x t u r e was no t p u r i f i e d f u r t h e r , b u t s e p a r a t i o n o f the anomers c o u l d be a c h i e v e d r e a d i l y by column chromatography as d e s c r i b e d by van Boom. a-2,4-DNPG - 98 -( 2 9 - a ) : m.p. 184-185° ( l i t . 3 3 m.p. 181-183°); ^-H-n.m.r. d a t a : 5 ( C D C 1 3 ) 8.76 (d, IH, J 3 5 4 Hz, H ( 3 ) o f DNP), 8 . 4 5 (dd, IH, J 5 6 9 Hz, J 5 3 4 Hz, H ( 5 ) o f DNP), 7 . 5 4 (d, IH, J 6 5 9 Hz, H ( 6 ) o f DNP), 6 . 0 2 (d, IH, J 1 2 3 Hz, H ( l ) ) , 5.67 (dd, IH, J 8 Hz, H(3) , a s s i g n e d by d e c o u p l e d -^H a t S 5.09, H ( 2 ) ) , 5.23 (dd, IH, J 8 Hz, H ( 4 ) ) , 5.09 (dd, J 2 > 3 8 Hz, J 1 2 4 Hz, H ( 2 ) ) , 4 . 2 4 - 4 . 1 0 (m, 3 H , H ( 6 ), H ( 6 ' ) , H ( 5 ) ) , 2.03 ( s , 3 H , C H 3 C O ) , 2 . 0 0 ( s , 3 H , C H 3 C O ) , 1.98 ( s , 6 H , 2 x CH 3 C 0 ); 5 (DMSO-dg) 8.82 (d, IH, J 3 5 4 Hz, H ( 3 ) o f DNP), 8.56 (dd, IH, J 5 _ 6 1 0 Hz, J 5 _ 3 3 Hz, H ( 5 ) o f DNP), 7 . 7 4 (d, IH, J 6 5 10 Hz, H ( 6 ) o f DNP), 6.32 (d, IH, J 1 > 2 4 Hz, H ( l ) , 5.48 (dd, IH, J Hz, H ( 3 ) ) , 5.25 - 5 . 1 2 (m, 2 H , H ( 2 ), H ( 4 ) ) , 4.19 (dd, J 6 | 6 , 12 Hz, J 6 , 5 4 Hz, H ( 6 ) ) , 4.09 (m, IH, H ( 5 ) ) , 3 . 9 7 (dd, J 6 6 , 12 Hz, J 6 ' , 5 2 Hz, H ( 6 ' ) ) , 2 . 0 2 ( s , 6 H , 2 x C H 3 C O ) , 2 . 0 0 ( s , 3 H , C H 3 C O ) , 1.96 ( s , 3 H , C H 3 C O ) . [1-2H]-B-2,4-DNPG (32) ( 4 4 7 mg, 0.87 mmmol) was anomerized by the f o r e g o i n g p r o c e d u r e t o produce the pure a-anomer (180 mg, 0 . 3 5 mmol, 40%) and a m i x t u r e o f anomers (225 mg, 0 . 4 4 mmol, 50%); Rf (a) = 0.56 (H2SO4 s p r a y , u . v . ) ; m.p. 183-185°; •'•H-n.m.r. d a t a : spectrum i d e n t i c a l to n o n - d e u t e r a t e d a-2,4-DNPG (29-a) ex c e p t no s i g n a l f o r H ( l ) a t 6" 6 . 0 2 i n C D C I 3 and S 6 . 3 3 i n D M S 0-d 6. 2.1.2 P e r a c e t y l a t e d S u b s t i t u t e d P h e n y l G l u c o p y r a n o s i d e s The p r e c e d i n g a n o m e r i z a t i o n method was m o d i f i e d s l i g h t l y t o d e t e r -mine the a/0 p r o d u c t c o m p o s i t i o n f o r the a r y l g l u c o p y r a n o s i d e s ( 5 3-60). Thus, the ^ - g l u c o p y r a n o s i d e (0.036 mmol) was s t i r r e d w i t h anhydrous - 99 -p o t a s s i u m c a r b o n a t e (0.10 mmol) i n DMSO (0.20 mL) c o n t a i n i n g 3A molecu-l a r s i e v e s . The m i x t u r e was s t i r r e d f o r 3-5 days a t room temperature a t which time the r e a c t i o n was worked-up as p r e v i o u s l y d e s c r i b e d e x c e p t no attempt was made t o s e p a r a t e the anomers. T . l . c . and n.m.r. a n a l y s e s o f the p r o d u c t m i x t u r e i n d i c a t e d when a n o m e r i z a t i o n had o c c u r r e d s i n c e the a-anomers have v e r y c h a r a c t e r i s t i c t . l . c . and n.m.r. p r o p e r t i e s . Compounds 53-58 showed no change by t . l . c . o r n.m.r. a n a l y s e s a f t e r b e i n g s u b m i t t e d to the above c o n d i t i o n s and t h e r e f o r e , a n o m e r i z a t i o n had no t o c c u r r e d . 2 . 6 - D i c h l o r o - 4 - n i t r o p h e n y l 2 . 3 . 4 . 6 - t e t r a - 0 - a c e t y l - a - D - g l u c o p y r a n o s i d e  (59-q) — R f = 0.70 (H2S04 s p r a y , u . v . ) ; ^-n.m.r. d a t a : S (CDC13) 7.88 (d, J 3 4 J 5 4 9 Hz, H(3) and H(5) o f DNP), 6.32 (d, J 1 2 3 H z > * * ( ! ) ) • 2 . 6 - D i n i t r o p h e n y l 2 . 3 . 4 . 6 - t e t r a - 0 - a c e t y l - a - D - g l u c o p y r a n o s i d e (60-a) — R f = 0.52 (H2S04 s p r a y , u . v . ) ; ^-n.m.r. d a t a : 8 (CDCI3) 7.87 (d, H(3) and H(5) a r o m a t i c ) . 2.1.3 S m a l l S c a l e R e a c t i o n s : V a r i a t i o n o f S o l v e n t s and C a t a l y s t s A t tempted a n o m e r i z a t i o n o f 2 , 4 - d i n i t r o p h e n y l 2 , 3 , 4 , 6 - t e t r a - 0 -a c e t y l - 0 - D - g l u c o p y r a n o s i d e (29) u s i n g v a r i o u s c a t a l y s t s and s o l v e n t were p e r f o r m e d u s i n g 10 mg (0.018 mmol) o f (29 ) , 0.05 mmol o f c a t a l y s t and 0.10 mL o f s o l v e n t . M i x t u r e s were s t i r r e d f o r 1-3 days and the e x t e n t o f a n o m e r i z a t i o n ( i f any) was de t e r m i n e d by t . l . c . o n l y (Rf(0) = 0.36, - 100 -R f ( a ) = 0.52, H 2 S O 4 s p r a y and u . v . ) . 2.2 Exchange R e a c t i o n s 2.2.1 Exchange o f H o r D a t Anomeric Carbon 2 . 4 - D i n i t r o p h e n y l g l u c o p v r a n o s i d e (29) w i t h t - b u t a n ( o l - d ) — A 0.12 mM s o l u t i o n o f (29) i n DMS0-d 6 (0.200 mL) was mixed w i t h a 0.18 mM s o l u t i o n o f ( M e 4 N ) 2 C 0 3 i n DMSO-dg (0.400 mL) and t - b u t a n ( o l - d ) (0.100 mL, 0.84 mmol) i n a n.m.r. tube and the tube was s e a l e d o f f u s i n g an oxygen t o r c h (see a l s o p r e p a r a t i o n o f samples f o r k i n e t i c s t u d i e s ) . A f t e r 2 days a t room temperature the ^H-n.m.r. spectrum showed t h a t a n o m e r i z a t i o n had o c c u r r e d t o 50% and the amount o f a-anomer p r e s e n t d e t e r m i n e d by i n t e g r a t i o n o f H ( l ) (5 6.32, DMSO-dg) was e q u a l t o the amount de t e r m i n e d by i n t e g r a t i o n o f H(6)-DNP (5 7.74, DMS0-d 6) (53% a-anomer by H ( l ) , 52% a-anomer by H(6) o f DNP). T h e r e f o r e , no H/D exchange had o c c u r r e d . 2 , 4 - D i n i t r o p h e n v l f 1 - 2 H 1 - g l u c o p v r a n o s i d e (32) w i t h t - b u t a n o l — 2,4-D i n i t r o p h e n y l 2,3,4,6-tetra-0-acetyl-£-D-[l- 2H]-glucopyranoside (32) (45 mg, 0.087 mmol) and (Me4N) 2C0 3 (31 mg, 0.23 mmol) was s t i r r e d i n DMSO (0.20 mL) c o n t a i n i n g t - b u t a n o l (0.70 g, 9.4 mmol) f o r 7 days. The mi x t u r e was worked-up a c c o r d i n g t o the p r o c e d u r e g i v e n f o r a n o m e r i z a t i o n o f s u b s t i t u t e d p h e n y l g l u c o p y r a n o s i d e s . ^H-n.m.r. o f the p r o d u c t mix-t u r e i n d i c a t e d t h a t a n o m e r i z a t i o n had o c c u r r e d t o 70% by the i n t e g r a t i o n o f the resonance o f H(6)-DNP a t 5 7.54 o f the a-anomer (CDC1 3) b u t t h e r e - 101 -was c l e a r l y no resonance a t S 6.02 p.p.m. ( C D C I 3 ) c o r r e s p o n d i n g to a p r o t o n a t the a r o m a t i c c e n t e r . The p r e c e d i n g H/D exchange experiments were r e p e a t e d u s i n g d i m s y l a n i o n as a c a t a l y s t where DMSO i s the o n l y p r o t o n o r d e u t e r o n s o u r c e . [1- 2H]-/3-D-2.4-DNPG (32) (20 mg, 0.039 mmol) was anomerized w i t h 0.064 M d i m s y l a n i o n i n DMSO (1.0 mL), and n o n - d e u t e r a t e d /3-2,4-DNPG (29) (20 mg, 0.039 mmol) was anomerized w i t h 0.064 M d i m s y l a n i o n i n DMSO-dg (1.0 mL). ^H-n.m.r. s p e c t r a f o r b o t h experiments showed t h a t a n o m e r i z a t i o n had o c c u r r e d w i t h no H/D exchange a t the anomeric c e n t e r o r w i t h any o t h e r sugar r i n g p r o t o n s . 2.2.2 Exchange of Phenolate 2 . 4 - D i n i t r o p h e n v l g l u c o p y r a n o s i d e (29) w i t h 2 . 6 - d i n i t r o p h e n o l a t e — A m i x t u r e c o n t a i n i n g 0-2,4-DNPG (29) (45 mg, 0.087 mmol), anhydrous p o t a s s i u m c a r b o n a t e (32 mg, 0.23 mmol), and p o t a s s i u m 2 , 6 - d i n i t r o -p h e n o l a t e (19 mg, 0.087 mmol) i n DMF (0.20 mL) was s t i r r e d a t room temperature f o r 3 days. T . l . c . o f the mi x t u r e a f t e r t h i s time showed the p r e s e n c e o f a p p r o x i m a t e l y 80% o f a component w i t h an Rf o f 0.56 ( H 2 S O 4 s p r a y , u.v.) c o r r e s p o n d i n g e i t h e r t o the or-2,4-DNPG o r the a-2,6-DNPG. the m i x t u r e was worked-up a c c o r d i n g t o the p r o c e d u r e f o r a n o m e r i z a t i o n o f a r y l g l u c o p y r a n o s i d e s . -'-H-n.m.r. ( C D C I 3 ) o f the p r o d u c t m i x t u r e showed o n l y the pr e s e n c e o f the 2 , 4 - d i n i t r o p h e n y l anomers. There was no resonance 5 7.87 c o r r e s p o n d i n g t o H(3) and H(5) o f the a r o m a t i c group f o r the a-2,6-DNPG (60 - a ) . 102 -2 .6 -D in i t ropheny l glucopvranoside (60) with 2 .4 -d in l t ropheno la te — A mixture conta in ing 0-2,6-DNPG (60) (20 mg, 0.039 mmol), anhydrous potassium carbonate (15 mg, 0.11 mmol) and potassium 2 , 4 - d i n i t r o -phenolate (9 mg, 0.041 mmol) i n DMF (0.20 mL) was s t i r r e d at room temperature f o r 3 days. T . l . c . of t h i s product mixture i n d i c a t e d that approximately 50% of the 0-2,6-DNPG (60-0) had reacted to form e i t h e r the a-2,6-DNPG (60-a) or the a-2,4-DNPG (29-a) . T-H-n.m.r. of the worked-up r e a c t i o n mixture showed the presence o f only a - and 0-2,6-DNPG and no s i g n a l s at p o s i t i o n s corresponding to the 2,4-DNPG anomers. 2 . 3 . 4 . 6 - T e t r a - O - a c e t v l g l u c a l (62) and 2 .4 -d in i t ropheno la te — The g l u c a l (10 mg, 0.030 mmol) was mixed wi th 0.60 mL of potassium 2 ,4 -d in i t ropheno la te (8 mg, 0.036 mmol) i n DMSO-dg. The ^H-n.m.r . spectrum of the sample a f t e r severa l days i n d i c a t e d the presence of only s t a r t i n g m a t e r i a l s . 2 .2 .3 Exchange v i a a N u c l e o p h i l i c At tack Mechanism 2 . 6 - D i n i t r o p h e n y l glucopvranoside (60) with FDNB — 0-2,6-DNPG (60) (50 mg, 0.097 mmol) was mixed with FDNB (165 mg, 0.99 mmol) and potas-sium carbonate (70 mg, 0.51 mmol) i n DMSO (0.50 mL) conta in ing 3A molecular s i e v e s . The mixture was s t i r r e d fo r 8 days at which time the r e a c t i o n was worked-up according to the procedure f o r anomerizat ion of a r y l g lucopyranosides . ^H-n.m.r . a n a l y s i s i n CDCI3 of the product mixture showed the presence of predominantly 0-2,6-DNPG (60-0) (-60% by - 103 -H(6)-aromatic a t 6 7.98) and a s m a l l amount o f a-2,4-DNPG (29) (-10% by H ( l ) a t 8 6.02), b u t no a-2,6-DNPG (60-a) no s i g n a l s f o r (H(3) and H(5) o f DNP a t 8 7.87). A l l o t h e r a r o m a t i c peaks c o u l d be a s s i g n e d t o FDNB, 0-2,6-DNPG (60) o r a-,0-2,4-DNPG (29). 2 . 4-Dinitrophenyl f l ^ H ] - g l u c o p y r a n o s i d e (32) w i t h 2.3.4.6-tetra-0- a c e t v l - D - g l u c o p v r a n o s e (30) — [1-2H]-0-D-2,4-DNPG (32) (40 mg, 0.078 mmol) and the t e t r a - a c e t a t e (30) (27 mg, 0.078 mmol) were s t i r r e d w i t h p o t a s s i u m c a r b o n a t e (57 mg, 0.41 mmol) i n DMSO (0.80 mL) c o n t a i n i n g 3A m o l e c u l a r s i e v e s f o r 2 days a t which time t . l . c . i n d i c a t e d t h a t the 0-2,4-DNPG had anomerized. The m i x t u r e was worked-up as u s u a l . ^H-n.m.r. a n a l y s i s o f the crude p r o d u c t m i x t u r e i n DMSO-dg showed the Q-and 0-DNPG anomers i n a p p r o x i m a t e l y e q u i l i b r i u m p r o p o r t i o n s (80:20 r e s p e c t i v e l y ) . The a r e a o f the peak a t 5 6.33 ( H ( l ) o f a-2,4-DNPG) i n t e g r a t e d t o o n l y about one h a l f the a r e a o f the peak a t 5 7.74 (H(6)-aromatic o f a-2,4-DNPG). The a-2,4-DNPG was f r a c t i o n a l l y c r y s t a l l i z e d from warm 95% e t h a n o l and ^-H-n.m.r. o f the i s o l a t e d pure a-anomer i n DMSO-dg c l e a r l y c o n f i r m s t h i s r e s u l t (26 mg, 0.051 mmol, 64%); m.p. 183-184° ( l i t . m.p. 181-183°); iH-n.m.r. d a t a : 5 (DMS0-d6) 8.82 (d, 1H, H(3) o f DNP), 8.56 (dd, 1H, H(5) o f DNP), 7.74 (d, 1H, H(6) o f DNP), 6.32 (d, 0.5H, H ( l ) ) , 5.48 (dd, 1H, H(3)), 5.25-4.12 (m, 2H, H(2) and H(4)), 4.19 (dd, 1H, H(6)), 4.09 (m, 1H, H(5)), 3.97 (dd, 1H, H(6')), 2.02 ( s , 6H, 2 x CH3C0), 2.00 ( s , 3H, CH3CO), 1.96 ( s , 3H, CH3CO). There was no o b s e r v e d l o s s o f the de u t e r i u m l a b e l when [l- 2 H ] - 0 - D -- 104 -2,4-DNPG (32) was anomerized w i t h p o t a s s i u m c a r b o n a t e and DMSO i n a s e p a r a t e , c o n t r o l experiment. 3. KINETICS 3.1 P o l a r i m e t r i c S t u d i e s O p t i c a l r o t a t i o n s were measured w i t h a P e r k i n Elmer 141 p o l a r i m e t e r u s i n g a sodium lamp. A 1-dm, 1.0 ml, w a t e r - j a c k e t e d c e l l was u s e d and temperature o f the s o l u t i o n was m a i n t a i n e d a t 25°. The o p t i c a l 25 r o t a t i o n s , [ a ] D , o f 0- and a-2,4-DNPG (29) measured i n DMSO were +12.3° (c 5.7) and +187.7° (c 9.3), r e s p e c t i v e l y . A f t e r s e v e r a l m o d i f i c a t i o n s , the f o l l o w i n g p r o c e d u r e was u s e d i n an attempt t o measure r a t e s o f a n o m e r i z a t i o n . Bis(tetramethylammonium) c a r b o n a t e (0.156 g, 0.702 mmol) was d i s s o l v e d i n DMSO (10 mL) by s t i r -r i n g o v e r n i g h t a t room temperature. T h i s s o l u t i o n (2.0 mL) was added to 0-2,4-DNPG (29) (0.335 g, 0.651 mmol) i n a 2.0 mL v o l u m e t r i c t e s t tube. The s o l u t i o n was mixed and t r a n s f e r r e d t o the p o l a r i m e t e r c e l l as q u i c k l y as p o s s i b l e t o p r e v e n t the s o l u t i o n from coming i n t o c o n t a c t w i t h the atmosphere and to measure the o p t i c a l r o t a t i o n as soon as p o s s i b l e a f t e r m i x i n g the r e a g e n t s . O p t i c a l r o t a t i o n r e a d i n g s were t a k e n a t 10, 20 o r 30 minute i n t e r v a l s . A f t e r 90 minutes, o p t i c a l r o t a t i o n i n d i c a t e d t h a t a n o m e r i z a t i o n had o c c u r r e d t o o n l y 5 t o 10% whereas t . l . c . o f the same r e a c t i o n m i x t u r e showed t h a t 30 t o 40% o f the a-anomer had formed. A f t e r 5 ho u r s , t h e r e was no l o n g e r any change 105 -i n o p t i c a l r o t a t i o n b u t t . l . c . o f the m i x t u r e i n d i c a t e d t h a t anomeriza-t i o n was n e a r l y complete. The r e a c t i o n m i x t u r e a t t h i s time was r e d t o brown i n c o l o r p o s s i b l y due t o the f o r m a t i o n o f 2 , 4 - d i n i t r o p h e n o l a t e . These e r r o n e o u s o p t i c a l r o t a t i o n measurements do n o t appear to be an a r t i f a c t o f the h i g h l y a b s o r b i n g background s i n c e a s o l u t i o n o f 0-2,4-DNPG (29) w i t h added p o t a s s i u m 2 , 4 - d i n i t r o p h e n o l a t e i n DMSO showed an e s s e n t i a l l y i d e n t i c a l o p t i c a l r o t a t i o n t o t h a t o f a s t a n d a r d 25 b-2,4-DNPG s o l u t i o n ( [ a ] D = +14.89°, c 14.2, DMSO). 3.2 ^-N.m.r. S t u d i e s The k i n e t i c s o l u t i o n s were p r e p a r e d i n s e a l e d n.m.r. tubes and the f o l l o w i n g p r e c a u t i o n s were t a k e n t o a v o i d c o n t a m i n a t i o n by water. The 0-2,4-DNPG (29) and bis(tetramethylammonium) c a r b o n a t e were weighed out i n approximate amounts i n t o s e p a r a t e pre-weighed v i a l s t h e n d r i e d i n vacuo. The v i a l s were s t o p p e r e d w i t h rubber s e p t a i n a g l o v e box then reweighed t o o b t a i n the a c c u r a t e amounts o f d r i e d s u b s t r a t e and c a t a -l y s t . The v i a l s were t r a n s f e r r e d a g a i n t o the g l o v e box and the appro-p r i a t e amount o f DMSO-dg ( s e a l e d under n i t r o g e n and us e d as r e c e i v e d ) was added. The bis(tetramethylammonium) c a r b o n a t e was d i s s o l v e d a f t e r s t i r r i n g o v e r n i g h t a t room temperature. N.m.r. tubes (5 mm) w i t h a 14/20 ground g l a s s j o i n t a t the top and were o v e n - d r i e d (>100°) b e f o r e use, t h e n c o o l e d w h i l e f l o w i n g anhydrous n i t r o g e n t h r o u g h the tube. The a p p r o p r i a t e amount o f the c a t a l y s t s o l u t i o n was added v i a m i c r o - s y r i n g e and the s o l u t i o n was f r o z e n w i t h l i q u i d n i t r o g e n , f o l l o w e d by a d d i t i o n - 106 -o f the a p p r o p r i a t e amount o f s u b s t r a t e s o l u t i o n v i a m i c r o - s y r i n g e and a g a i n f r e e z i n g w i t h l i q u i d n i t r o g e n . I n t h i s way the r e a g e n t s are f r o z e n i n l a y e r s t o p r e v e n t m i x i n g u n t i l the m i x t u r e i s warmed to room temperature. While f r o z e n , the n.m.r. tubes were e v a c u a t e d and s e a l e d u s i n g an oxygen t o r c h . R e a c t i o n s were i n i t i a t e d by warming the tube to room temperature as q u i c k l y as p o s s i b l e (over a p e r i o d o f about 3 minutes by r u b b i n g the o u t s i d e o f the tube) and m i x i n g t h o r o u g h l y . The •^H-n.m.r. s p e c t r a were immediately r e c o r d e d u s i n g the V a r i a n XL-300 s p e c t r o m e t e r a t a probe temperature o f 25°. P r o g r e s s o f the r e a c t i o n was f o l l o w e d by r e c o r d i n g s p e c t r a a t a p p r o p r i a t e i n t e r v a l s . R a tes o f a n o m e r i z a t i o n were o b t a i n e d by f o l l o w i n g the d i s a p p e a r a n c e o f the /J-anomer o r by f o l l o w i n g the appearance o f the a-anomer, the former method g i v i n g more a c c u r a t e r e s u l t s . The H(6) reson a n c e s o f the DNP group and the anomeric resonances were used t o determ i n e the amounts o f each anomer and the c h e m i c a l s h i f t s o f t h e s e r e s o n a n c e s f o r each s u b s t r a t e a r e summarized i n T a b l e 8. The H ( l ) s i g n a l and the H(6) s i g n a l o f the DNP group were i n t e g r a t e d u s i n g a c e t a t e r e s o n a n c e s a t S 2.2-2.0 as the i n t e r n a l r e f e r e n c e . F o r the d e u t e r i u m l a b e l l e d sub-s t r a t e , the r e a c t i o n c o u l d be f o l l o w e d o n l y by i n t e g r a t i o n o f the H(6) s i g n a l o f the DNP group. The r e l a t i v e i n t e g r a t i o n s were measured a t v a r i o u s t i m e s , t , and the amount o f r e m a i n i n g /9-anomer was denoted a t (mmol). The e q u i l i b r i u m amount o f the /9-anomer, a e (mmol), was d e t e r m i n e d a f t e r a r e a c t i o n time o f 8-12 h o u r s . R e a c t i o n r a t e p l o t s o f l o g ( a t - a e ) a g a i n s t t i m e 1 3 were l i n e a r f o r 2 h a l f l i v e s ( c o r r e l a t i o n c o e f f i c i e n t , r > 0.98). R e a c t i o n r a t e s - 107 -Table 8: Chemical s h i f t s (£) f o r H(6)-DNP group and H(l) of the 2,4-DNPG derivatives i n DMSO-dg. Peaks are referenced i n t e r n a l l y to DMSO-dg at S 2.49 r e l a t i v e to TMS Compound B-anomer a-anomer  H(6) of DNP H(l) H(6) of DNP H(l) (d,J 6 > 510Hz) ( d . J 1 2 8 H z ) ( d , J 6 5 1 0 H z ) ( d , J 1 2 3 H z ) (29) 7.64 5.93 7.74 6.33 (32) 7.67 5.71 7.72 6.33 (39) 7.73 6.57 7.65 6.09 (40) 7.62 5.93 7.74 6.32 (44) 7.60 5.72 7.70 6.26 (49) 7.66 5.86 7.72 6.27 - 108 -reported are the rates of disappearance of the ^-anomer and were c a l c u l a t e d us ing l i n e a r regress ion a n a l y s i s . E r r o r i n the measured i n t e g r a t i o n values i s ± 5%. K i n e t i c isotope e f f e c t s were c a l c u l a t e d from the r a t i o of r e a c t i o n rates f o r the i s o t o p i c a l l y s u b s t i t u t e d and unsubst i tu ted subs t ra tes , or Rate (H)/Rate (D). - 109 -APPENDIX 1 KINETIC ISOTOPE EFFECTS 5 8 1. D e f i n i t i o n of Deuterium Isotope E f f e c t s An isotope e f f e c t i s the observed difference i n rate of a reaction when a reactant i s i s o t o p i c a l l y substituted. Therefore, the k i n e t i c isotope e f f e c t (KIE) i s given by the r a t i o of rates f o r the i s o t o p i c a l l y unsubstituted and substituted substrates or ^ / j j ) f o r deuterium e f f e c t s . Since a change i n the rate of a rea c t i o n r e f l e c t s a change i n the energy of the t r a n s i t i o n state f o r that reaction, isotope e f f e c t s provide a method for probing the structure of the t r a n s i t i o n state and the forma-t i o n of intermediates during the reaction. Use of an isotope e f f e c t i n t h i s way i s based upon the f a c t that s u b s t i t u t i o n of an isotope i n the substrate molecule does not appreciably perturb the p o t e n t i a l energy surface of the reaction. That i s , the e l e c t r o n i c structure of the molecule i s e s s e n t i a l l y i n s e n s i t i v e to differences i n nuclear masses (the Born-Oppenheimer p r i n c i p l e ) . Therefore, the isotope e f f e c t may be treated only i n terms of v i b r a t i o n a l energies, making i n t e r p r e t a t i o n simpler. The following sections w i l l review types of KIEs and give a basic i n t r o d u c t i o n to the theory behind these e f f e c t s . The scope of t h i s discussion w i l l be l i m i t e d to deuterium isotope e f f e c t s . I t should be noted, however, that heavier atom KIEs, such as ^C/^C, i 4 N / 1 5 N , and 16 0/18 0 w h i c h h a v e D e en d i f f i c u l t to measure accurately are being used more commonly as methods to measure them become more preci s e . - 110 -2. Theory 2.1 P r i m a r y Isotope E f f e c t s Primary isotope e f f e c t s can be measured when there i s i s o t o p i c s u b s t i t u t i o n i n a bond which i s being cleaved i n a reaction. The di f f e r e n c e i n observed rates r e l a t e s to changes i n the v i b r a t i o n a l frequency of a bond upon i s o t o p i c s u b s t i t u t i o n . For a deuterium KIE, t h i s i s most e a s i l y explained by comparing the ground state energies of C-H and C-D bonds. I f the bonds are assumed to act as simple harmonic o s c i l l a t o r s , then the v i b r a t i o n a l frequency v of the bond i s given by v — — 7k//i where fi i s the reduced mass and k i s the force constant. I f 2w k i s constant for i s o t o p i c a l l y s ubstituted and unsubstituted molecules and frequency and therefore the energy (given by E - i hu where h -Plancks constant) depend only on the reduced mass. The C-D bond, having a higher reduced mass, w i l l have a lower ground state energy. As a r e s u l t , the C-D bond w i l l require a higher a c t i v a t i o n energy for bond cleavage than the C-H bond. This i s shown schematically In Figure 50. For the primary KIE i t i s considered that the i s o t o p i c a l l y substituted and unsubstituted molecules go through the same t r a n s i t i o n state or intermediate so that the energy difference between the C-H and C-D bonds disappears as the re a c t i o n approaches the t r a n s i t i o n state. The magni-tude of the isotope e f f e c t can be used to estimate the r e l a t i v e p o s i t i o n of the t r a n s i t i o n state. I f there i s complete bond cleavage at the t r a n s i t i o n state, a l a r g e r KIE would be expected than i f the t r a n s i t i o n state more nearly resembled the reactants where very l i t t l e bond - I l l -cleavage has occurred. The maximum KIE possible f o r deuterium i s 7.0 which corresponds to the t o t a l disappearance of the C-H or C-D s t r e t c h -ing v i b r a t i o n i n the t r a n s i t i o n state. In general, a deuterium KIE ranging from 2 to 7 i s a primary isotope e f f e c t . 2.2 Secondary Isotope E f f e c t s Secondary isotope e f f e c t s a r i s e from changes i n the bonding of the i s o t o p i c a l l y l a b e l l e d carbon, but the isotope i s substituted i n a bond which i s not being cleaved i n the reaction. Two types of secondary KIEs - 112 -e x i s t : a-secondary KIEs where the isotope i s substituted d i r e c t l y at the r e a c t i o n center; or secondary KIEs where the Isotope i s substi-tuted on an adjacent atom to the reaction center. a. a-Secondary KIEs a-Secondary KIEs may be observed when the rea c t i v e carbon undergoes a change i n h y b r i d i z a t i o n i n the rate c o n t r o l l i n g step of the reaction. A change from s p 3 to s p 2 h y b r i d i z a t i o n at the t r a n s i t i o n state produces a change i n the zero-point energies of the C-H and C-D bonds as a r e s u l t of changes i n the bonding v i b r a t i o n s i n reaching the t r a n s i t i o n state (Figure 51). Again, because of the lower reduced mass of the C-H bond \ - C - H bend 1350 cm -1 out • of-plane bend 800 cm"1 \ - C - D bend 1040 cm -1 out-of-plane bend 600 cm"1 Figure 51: The bending v i b r a t i o n a l frequencies of s p 3 and sp hybridized carbon - 113 -the v i b r a t i o n a l frequency of the C-H bond i s generally higher. In going from s p 3 to s p 2 h y b r i d i z a t i o n , there i s an o v e r a l l lowering of the v i b r a t i o n a l frequencies and the lowering i s greatest f o r the C-H bond (550 cm"1 f o r C-H versus 440 cm"1 for C-D). The rate of the re a c t i o n of the i s o t o p i c a l l y unsubstituted molecule w i l l be greater since the a c t i v a t i o n energy w i l l be lower. Therefore, the isotope e f f e c t of ^ / k n w i l l be greater than unity. The energy diagram for an a-secondary KIE i s shown i n Figure 52. ENERGY REACTION COORDINATE Figure 52: The r e a c t i o n coordinate diagram f o r an a - s e c o n d a r y isotope e f f e c t The converse i s true f o r a re a c t i o n going from sp^ to sp h y b r i d i z a t i o n i n the r a t e - c o n t r o l l i n g step. In t h i s case there i s an o v e r a l l increase i n the v i b r a t i o n a l frequencies of the C-H and C-D - 114 -bonds, and the I n c r e a s e i s g r e a t e r f o r the C-H bond. Thus, the r e a c t i o n i n v o l v i n g the C-H bond w i l l have a lower r a t e s i n c e i t w i l l r e q u i r e a h i g h e r a c t i v a t i o n energy. The i s o t o p e e f f e c t o r ^ /^D w i l l he l e s s than u n i t y and i s t h e r e f o r e termed an i n v e r s e i s o t o p e e f f e c t . I n b o t h c a s e s , the d i f f e r e n c e i n r a t e s o r the magnitude o f the i s o t o p e e f f e c t w i l l r e f l e c t the n a t u r e o f the t r a n s i t i o n s t a t e . Rate d i f f e r e n c e s f o r a- s e c o n d a r y KIEs a r e g e n e r a l l y i n the o r d e r o f 10-20% bu t may r e a c h a maximum o f 40%. S j j l - t y p e r e a c t i o n s which i n v o l v e the f o r m a t i o n o f a carbonium 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 i n v o l v e a t r a n s i t i o n s t a t e w i t h carbonium i o n c h a r a c t e r t y p i c a l l y g i v e an a-secon-d a r y KIE about ^ / k D = 1 . 2 . On the o t h e r hand, an S N2 type mechanism, where t h e r e i s no n e t change i n the h y b r i d i z a t i o n o f the r e a c t i o n c e n t e r d u r i n g the r e a c t i o n , would g i v e an i s o t o p e e f f e c t o f n e a r l y u n i t y . b. 6-Secondary KIEs Secondary d e u t e r i u m i s o t o p e e f f e c t s a l s o a r i s e from changes i n h y b r i d i z a t i o n and a r e g e n e r a l l y o b s e r v e d when the r e a c t i o n i n v o l v e s the f o r m a t i o n o f a carbonium i o n . When the a d j a c e n t c a r b o n i s deu t e r i u m s u b s t i t u t e d , the d i f f e r e n c e i n r a t e between the p r o t i o - and de u t e r o -s u b s t r a t e s i s due t o the r e l a t i v e a b i l i t y o f the C-H o r C-D bond to s t a b i l i z e the e l e c t r o n d e f i c i e n t r e a c t i o n c e n t e r through h y p e r c o n j u g a -t i o n . T h at i s , the d i f f e r e n c e i n r a t e depends on the a b i l i t y o f an a d j a c e n t C-H o r C-D a bond t o donate e l e c t r o n d e n s i t y to an e l e c t r o n d e f i c i e n t p - o r b i t a l on the r e a c t i v e c a rbon. F o r example, a c i d c a t a l y z e d - 115 -glycoside h y d r o l y s i s i s considered to occur v i a an oxocarbonium ion intermediate. Hyperconjugative s t a b i l i z a t i o n of t h i s intermediate or the t r a n s i t i o n state leading to i t i s represented by the structures shown i n Figure 53. The structure at the r i g h t shows the possible influence of resonance int e r a c t i o n s on hyperconjugation. Since the deuterium atom i s s l i g h t l y e l e c t r o n donating r e l a t i v e to hydrogen, a C-D bond i s stronger and the electrons on the deuterium atom are less a v a i l a b l e f o r hyperconjugation. Therefore, deuterium s u b s t i t u t i o n at C(2) of the structure In Figure 53 causes a decrease i n r e a c t i o n rate because i t s t r a n s i t i o n state w i l l be l e s s stable and higher i n energy. The hyperconjugative component of a secondary isotope e f f e c t i s , how-ever, geometry dependent and w i l l have no influence I f the C-H or C-D bonds are orthogonal to the e l e c t r o n d e f i c i e n t p - o r b i t a l on the reactive carbon. In t h i s case, i t i s possible that the C-D bond may show an inductive e f f e c t . Here the C-D bond, being s l i g h t l y more e l e c t r o n H H HO III HI HO Figure 53: Hyperconjugation i n an oxocarbonium ion - 116 -donating, w i l l s t a b i l i z e the charge at the reaction center r e l a t i v e to the C-H bond. The deuterated species then reacts f a s t e r and an inverse isotope e f f e c t w i l l r e s u l t . 0-Secondary KIEs are generally no greater than about 10% and u n t i l recently, with the development of more accurate methods of measurement, have been used very l i t t l e . 3. Use i n Studying Reaction Mechanisms A recent p u b l i c a t i o n by Sinnott and Bennet 6 9 exemplifies the power of isotope e f f e c t s as a method of studying r e a c t i o n mechanisms. Here a number of isotope e f f e c t s on the a c i d catalyzed h y d r o l y s i s of methyl cr-and /3-glucopyranosides (5) were measured. The p o s i t i o n s of s u b s t i t u t i o n f o r each isotope e f f e c t are shown i n Figure 54. Some of the r e s u l t s of t h i s study In consideration of the p r e v a i l i n g mechanism on the a c i d catalyzed h y d r o l y s i s (Figure 55) are summarized as follows. ( A l l KIEs measured i n 2.0 M HC10 4 at 80°C). ( i ) Leaving group ^ 0 e f f e c t s (primary KIE) were determined to be k 1 6 0 / k 1 8 0 - 1.026 f o r (5-a) and k 1 6 0 / k 1 8 0 - 1.024 f o r (5-/3). The magnitude of the KIEs were found to be very close to isotope e f f e c t s c a l c u l a t e d f o r the d i s s o c i a t i o n of 4-nitrophenyl B-gluco-pyranoside where 4-nitrophenol and a glucopyranosyl c a t i o n are generated. Thus, i t was concluded that there i s a high degree of glycosyl-oxygen bond cleavage i n the t r a n s i t i o n state leading to the oxocarbonium ion. - 117 -Figure 55: The a c i d catalyzed hydrolysis of methyl a- and 0-D-gl pyranosides - 118 -( i i ) a-Secondary deuterium e f f e c t s were determined to be ^ / ^ n - 1.137 for (5-a) and ^V^n - 1.089 for ( 5 - 0 ) . Allowing for the inductive e f f e c t of deuterium which decreases the KIE, the magnitude of these isotope e f f e c t s suggests that the t r a n s i t i o n state Is nearly s p 2 hybridized and C(l)-0(1) bond cleavage i s e s s e n t i a l l y complete. ( i i i ) Ring 1 8 0 e f f e c t s (secondary KIE) were found to be inverse f or both anomers ( k 1 6 0 / k 1 8 0 (5-a) - 0.996, k 1 6 Q / k 1 8 0 (5-/3) - 0.991). The inverse KIE r e f l e c t s the double bond character of the C(l)-0(5) bond i n the t r a n s i t i o n state. Since 1 8 0 i s s l i g h t l y more electron-donating than 1 6 0 , d e r e a l i z a t i o n by the oxygen lone p a i r i s greater f o r the i s o t o p i c a l l y substituted glycoside, thus s t a b i l i z -ing the t r a n s i t i o n state f o r t h i s substrate. The magnitude of these e f f e c t s was c o r r e l a t e d with the amount of o r b i t a l overlap between C ( l ) and the r i n g oxygen i n the t r a n s i t i o n state i n order to derive the dihedral angle about the C(l)-0(5) bond. (iv) 0-Secondary deuterium e f f e c t s were measured to be ^ / ^ n ~ 1-073 f o r the a-anomer and ^ / ^ n - 1.045 for the /9-anomer. As with the r i n g 1 8 0 e f f e c t s , the secondary 0 KIE was c o r r e l a t e d with o r b i t a l overlap of the C(2)-H(2) or C(2)-D(2) bond with the e l e c t r o n d e f i c i e n t p - o r b i t a l at C ( l ) and the dihedral angle between the p - o r b i t a l and the C(2)-H(2) or C(2)-D(2) bond was calculated. These r e s u l t s i n conjunction with r e s u l t s from the r i n g 1 8 0 e f f e c t s were used to propose probable conformations of the tran-s i t i o n state i n the reaction. - 119 -As seen with the above example, measurement of isotope e f f e c t s i s a powerful t o o l i n the mechanistic investigations of chemical reactions. I t should be noted that isotope e f f e c t s alone cannot give a complete pi c t u r e of a reaction mechanism. Therefore, other aspects such as products, stereochemistry, k i n e t i c s , and s a l t or solvent e f f e c t s should be considered. - 120 -APPENDIX 2 KINETIC DATA FOR ANOMERIZATION REACTIONS R e a c t i o n r a t e d a t a o b t a i n e d f o r the a n o m e r i z a t i o n r e a c t i o n s are g i v e n i n the f o l l o w i n g t a b l e s . A l l measurements were p e r f o r m e d by the method d e s c r i b e d i n M a t e r i a l s and Methods. C o n c e n t r a t i o n s o f s u b s t r a t e and c a t a l y s t a r e i n d i c a t e d . Table 9: Remote substituent e f f e c t s : 2,4-dinitrophenyl 2,3,6-tri-O-acetyl-4-deoxy-4-fluoro - 0-D-glucopyranoside (40) . [DNPG] = 0.0138 M. [(Me 4N) 2C0 3] = 0.0167 M. a e = 1.42 x 10" 3 mmol I n t e g r a l A r e a f o r 0-DNPG l o g ( a t - a e ) time (min) H ( i ) o f 0-anomer (mmol x 10" 3) 7.80 7.08 -2.25 37 7.75 5.77 -2.36 56 6.73 4.96 -2.45 93 5.50 4.09 -2.57 123 3.58 3.33 -2.72 153 4.08 3.06 -2.78 183 3.72 2.89 -2.84 213 - 121 -Table 10: Remote substituent e f f e c t s : 2,4-dinitrophenyl 2,3,6-tri-O-acetyl-4-deoxy-0-D-glucopyranoside (44). [DNPG] = 0.0150 M. [(Me 4N) 2C0 3] = 0.0167 M. ae = 2.08 x 10' 3 mmol Integral Area f o r H(l) of 0-anomer 0-DNPG (mmol x 10" 3) log ( o t - ae) time (min) 12. ,90 10, .4 -2. ,08 14 12. ,85 10. .4 -2. ,08 34 12. .50 10, .2 -2. .09 64 12. ,05 9, .89 -2. .11 94 11. ,65 9, .59 -2. .12 124 11. ,35 9 .30 -2. .14 154 10. ,32 8, .52 -2. .16 214 10. ,10 8 .44 -2. .20 319 9. .83 7, .80 -2. .24 369 Table 11: Remote substituent e f f e c t s : 2,4-dinitrophenyl 2,3,4-tri-0-acetyl-6-deoxy-£-D-glucopyranoside (49). [DNPG] = 0.0153 M. [(Me 4N) 2C0 3] = 0.0167 M. a e = 2.06 x 1 0 - 3 mmol Integral Area f o r H(l) of /9-anomer 0-DNPG (mmol x 10" 3) log ( a t - a e) time (min) 13. .72 10. .3 -2. .08 6 13. .60 10. ,1 -2. .09 30 13. .42 10. .0 -2. .10 62 12. .53 9. ,10 -2. .15 97 11. .68 8. .72 -2, .18 123 10. .82 8. .21 -2, .21 150 10, .05 7. .37 -2 .29 180 9, .60 7. .17 -2, .35 212 8. .70 6, .51 -2 .44 242 7, .55 5. .65 -2, .52 270 - 122 -Table 12: Remote substituent e f f e c t s : 2,4-dinitrophenyl 2,3,4,6-tetra-0-acetyl-/3-D-glucopyranoside (29). [DNPG] = 0.0136 M. [(Me 4N) 2C0 3] = 0.0167 M. a e = 1.76 x 1 0 - 3 mmol Integral Area f o r H(l) of /3-anomer 0-DNPG (mmol x 10" 3) log (Qft - a e) time (min) 12. .30 8. .78 -2. .15 5 12. .15 8. .42 -2. .18 30 10. .40 7, .28 -2. .26 60 8. .90 6, .22 -2. .35 90 7. .00 4. .95 -2. .50 120 6, .10 4, .32 -2. .59 150 5. .03 3. .59 -2. .74 180 4, .15 2, .95 -2. .92 210 3. .62 2. .57 -3. .09 240 3 .40 2, .42 -3. .18 270 3, .30 2, .38 -3. .21 355 Table 13: Remote substituent e f f e c t s : 2,4-dinitrophenyl 3,4,6-tri-0-acetyl-2-deoxy-2-fluoro-0-D-glucopyranoside (39). JTDNPG] = 0.0134 M. [(Me 4N) 2C0 3] = 0.0182 M. a e = 9.85 x 1 0 - 3 mmol Integral Area f o r H(l) of /3-anomer /3-DNPG (mmol x 10" 3) log (at - a e) time (min) 5. .10 6. .61 -2. .24 6 4, .00 5, .89 -2, .30 32 3. .20 4. .72 -2. .42 60 2. .50 3, .69 -2. .55 90 1. .90 3. .42 -2. .60 120 1. .45 2. .48 -2 .80 150 1. .20 2, .07 -2, .93 190 1. .12 1. .76 -3. .06 212 0. .80 1. .56 -3, .17 291 - 123 -Table 14: Remote substituent e f f e c t s : 2,4-dinitrophenyl 2,3,4,6-tetra-0-acetyl-/3-D-glucopyranoside. [DNPG] = 0.0121 M. 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