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UBC Theses and Dissertations

Structural studies on Klebsiella capsular polysaccharides Folkman, Timothy Edward 1979

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STRUCTURAL STUDIES ON K L E B S I E L L A CAPSULAR POLYSACCHARIDES BY TIMOTHY EDWARD FOLKMAN B . Sc .'( Hons . ) , U n i v e r s i t y of A l b e r t a , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t of C h e m i s t r y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRI T I S H COLUMBIA A p r i l , 1 9 7 9 © T i m o t h y Edward F o l k m a n , 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f d l \ e < m I . S ^ Y y The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 D a t e k ^ J ^ 4 . l 9 l C f - • - i i -ABSTRACT To d a t e , s t u d i e s have r e v e a l e d t h a t s e v e n t y - e i g h t s e r o l o g i -c a l l y d i s t i n c t s t r a i n s o f K l e b s i e l l a b a c t e r i a e x i s t . The a n t i -g e n i c c h a r a c t e r o f each s t r a i n i n an i m m u n o l o g i c a l r e a c t i o n i s c o n s i d e r e d t o be d e t e r m i n e d by t h e c a p s u l a r p o l y s a c c h a r i d e w h i c h s u r r o u n d s t h e b a c t e r i a l c e l l . S t r u c t u r e s o f t h e c a p s u l a r p o l y -s a c c h a r i d e s from w e l l o v e r h a l f t h e known K l e b s i e l l a s t r a i n s have been e s t a b l i s h e d ; a l l o f them u n i q u e . In a c o n t i n u i n g p r o g r a m t o e l u c i d a t e t h e c h e m i c a l s t r u c t u r e s o f t h e s e p o l y s a c -c h a r i d e s i n o r d e r to e x p l a i n i m m u n o l o g i c a l r e s p o n s e s , e v i d e n c e f o r t h e s t r u c t u r e o f t h e a n t i g e n s from K l e b s i e l l a s e r o t y p e s K17 and K44 i s p r e s e n t e d . M e t h y l a t i o n a n a l y s i s was u s e d t o e s t a b l i s h l i n k a g e p o s i t i o n s o f t h e m o n o s a c c h a r i d e u n i t s . U r o n i c a c i d d e g r a d a t i o n , p e r i o d a t e o x i d a t i o n - S m i t h d e g r a d a t i o n , and p a r t i a l h y d r o l y s i s were t h e t e c h n i q u e s e m p l o y e d t o g e n e r a t e and s t u d y o l i g o s a c c h a r i d e s from 1 13 t h e more c o m p l e x p o l y m e r . E x t e n s i v e u s e was made o f H and C n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y t o i n d i c a t e a n o m e r i c c o n f i g u r a t i o n s o f t h e o l i g o - and p o l y s a c c h a r i d e s a l i k e . M ethods suc h as gas l i q u i d c h r o m a t o g r a p h y , gas l i q u i d c h r o m a t o g r a p h y -mass s p e c t r o m e t r y , g e l f i l t r a t i o n c h r o m a t o g r a p h y , p a p e r c h r o m a t o -g r a p h y , and p o l a r i m e t r y have been u s e d t o i s o l a t e and i d e n t i f y t h e p r o d u c t s o b t a i n e d from t h e v a r i o u s d e g r a d a t i v e t e c h n i q u e s . A l t h o u g h t h e p o l y s a c c h a r i d e s o f K - t y p e s 17 and 44 a r e p a r t o f a s m a l l g r oup w i t h t h e same q u a l i t a t i v e c o m p o s i t i o n , t h e i r s t r u c t u r e s a r e f o u n d t o c o n s i s t o f t h e f o l l o w i n g d i s s i m i l a r r e p e a t i n g u n i t s : - i i i -K17 ^ - G l c p ^ L - R h a p ^ ^ - G l c A p ^ L - R h a p ^ -L-Rhap K44 ^0-Glcp^-^O-G l c p ^ - ^ D - G 1 c A p ^ - ^ - L - R h a p ^ - L - R h a p - — 3 a 3 a a The l a t t e r r e p r e s e n t s p e n t a s a c c h a r i d e r e p e a t i n g s a c c h a r i d e . t h e f i r s t u n i t i n a ex a m p l e o f K l e b s i e l l a a l i n e a r c a p s u l a r p o l y -- i v -TABLE OF CONTENTS Page ABSTRACT ; i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES , v i i ACKNOWLEDGEMENTS v i i i PREFACE i x I INTRODUCTION 2 I I METHODS USED IN STRUCTURAL ANALYSES OF POLYSACCHARIDES 5 I I . 1 I s o l a t i o n and P u r i f i c a t i o n . 5 .11.2 T o t a l H y d r o l y s i s and C h a r a c t e r i z a t i o n of Component S u g a r s 6 I I . 2 . 1 T o t a l s u g a r r a t i o 7 I I . 2 . 2 . N u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y (1H and 13c ) 8 11.3 A n a l y s i s of L i n k a g e P o s i t i o n f o r Component S u g a r s 14 11.3.1 M e t h y l a t i o n a n a l y s i s 16 11.3.2 G . l . c . a n a l y s i s o f m e t h y l a t e d s u g a r s 18 11.3.3 Mass s p e c t r o m e t r y o f m e t h y l a t e d s u g a r s 20 11.3.4 "*"H n.m.r. s p e c t r o s c o p y o f m e t h y l a t e d p o l y s a c c h a r i d e s and o l i g o s a c c h a r i d e s 24 11.4 S u g a r S equence D e t e r m i n a t i o n by U r o n i c A c i d D e g r a d a t i o n 24 - V -Pag_e 1 1 . 5 S u g a r Sequence D e t e r m i n a t i o n from P a r t i a l H y d r o l y s i s 26 1 1 . 6 P e r i o d a t e O x i d a t i o n 29 11.7 A n o m e r i c L i n k a g e D e t e r m i n a t i o n 34 11.8 D e t e r m i n a t i o n o f C o n f i g u r a t i o n of Compon en t S u g a r s 34 11.9 B i b l i o g r a p h y f o r S e c t i o n s I and I I 37 I I I STRUCTURAL INVESTIGATION OF K l e b s i e l l a SEROTYPE K17 CAPSULAR POLYSACCHARIDE 40 111.1 A b s t r a c t . . . . 41 111.2 I n t r o d u c t i o n 42 111.3 R e s u l t s and D i s c u s s i o n 43 111.4 E x p e r i m e n t a l 59 111.5 B i b l i o g r a p h y f o r S e c t i o n I I I 74 IV STRUCTURAL INVESTIGATION OF K l e b s i e l l a K44 CAPSULAR POLYSACCHARIDE 76 IV.1 A b s t r a c t 77 IV. 2 I n t r o d u c t i o n 78 IV. 3 R e s u l t s and D i s c u s s i o n 79 IV. 4 E x p e r i m e n t a l 93 IV . 5 B i b l i o g r a p h y f o r S e c t i o n IV 103 APPENDIX: N.M.R. SPECTRA 105 - v i -LIST OF TABLES T a b l e Page I I . 1 C a l c u l a t e d and measured v a l u e s f o r s p e c i f i c r o t a t i o n s o f K l e b s i e l l a K17 and K44 p o l y - and o l i g o s a c c h a r i d e s : 3 5 111.1 N.m.r. d a t a o f K l e b s i e l l a K17 c a p s u l a r p o l y -s a c c h a r i d e and d e r i v e d p o l y - and o l i g o s a c c h a r i d e s 44 111.2 M e t h y l a t i o n a n a l y s i s o f o r i g i n a l K17 c a p s u l a r p o l y s a c c h a r i d e 48 111.3 M e t h y l a t i o n a n a l y s e s o f K17 c a p s u l a r p o l y -s a c c h a r i d e and d e r i v e d p o l y - and o l i g o s a c c h a r i d e s 50 111.4 G . l . c . a n a l y s i s o f K17 p o l y s a c c h a r i d e a f t e r u r o n i c a c i d d e g r a d a t i o n 56 IV. 1 N.m.r. d a t a o f K l e b s i e l l a K44 c a p s u l a r p o l y -s a c c h a r i d e and d e r i v e d o l i g o s a c c h a r i d e s 80 IV.2 M e t h y l a t i o n a n a l y s i s o f o r i g i n a l and d e g r a d e d K44 c a p s u l a r p o l y s a c c h a r i d e 84 IV.3 M e t h y l a t i o n a n a l y s i s of a c i d i c o l i g o s a c c h a r i d e s i s o l a t e d a f t e r p a r t i a l , a c i d h y d r o l y s i s o f K44 c a p s u l a r p o l y s a c c h a r i d e ' 88 - v i i -L I S T OF F I G U R E S F i g u r e P a g e 1.1 D i a g r a m a t i c r e p r e s e n t a t i o n o f a b a c t e r i a l c e l l w i t h l i p o p o l y s a c c h a r i d e , c a p s u l e a n d s l i m e . . . . 2 1 1 . 1 P r o t o n n.m.r. s p e c t r a s h o w i n g t h e c h a n g e i n HOD s i g n a l l o c a t i o n w i t h t e m p e r a t u r e 10 11.2 "''H n . m . r . s p e c t r a o f K17 p o l y s a c c h a r i d e d e p i c t i n g s i g n a l s o t h e r t h a n a n o m e r i c o n e s i n t h e r e g i o n 64.0 t o 6.0 12 1 1 . 3 "^C n.m.r. s p e c t r u m o f K l e b s i e l l a K17 c a p s u l a r p o l y s a c c h a r i d e 15 1 1 . 4 M e t h y l a t i o n a n a l y s i s s c h e m e f o r K17 p o l y s a c c h a r i d e 17 1 1 . 5 G . l . c . s e p a r a t i o n o f a m i x t u r e o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s o b t a i n e d f r o m K l e b s i e l l a K17 p o l y s a c c h a r i d e 19 11.6 M a s s s p e c t r a o f u r o n i c a c i d d e g r a d a t i o n d e r i v a t i v e s f r o m K17 a n d K 4 4 23 11.7 U r o n i c a c i d d e g r a d a t i o n o f K l e b s i e l l a K 4 4 p o l y s a c c h a r i d e 27 11.8 P e r i o d a t e - S m i t h d e g r a d a t i o n o f K l e b s i e l l a K44 p o l y s a c c h a r i d e 31 1 1 . 9 S e l e c t i v e p e r i o d a t e - S m i t h d e g r a d a t i o n o f K17 p o l y s a c c h a r i d e 33 I V . 1 G e l f i l t r a t i o n c h r o m a t o g r a p h y s e p a r a t i o n o f a m i x t u r e o f s a c c h a r i d e s o b t a i n e d f r o m p a r t i a l h y d r o l y s i s o f K l e b s i e l l a K 4 4 p o l y s a c c h a r i d e . . . 90 -'/VI11 -ACKNOWLEDGEMENTS I s h o u l d l i k e t o e x p r e s s my s i n c e r e t h a n k s t o P r o f e s s o r G.G.S. D u t t o n whose g u i d a n c e and u n d e r s t a n d i n g b r o u g h t t h i s t h e s i s t o a s u c c e s s f u l c o n c l u s i o n . To name o n l y a few o f t h e many p e o p l e whose h e l p f u l d i s -c u s s i o n s and s e r v i c e s have a i d e d d u r i n g t h e c o u r s e o f t h i s work, I w i s h t o show my a p p r e c i a t i o n t o D r . M. V i g n o n , D r . A. Z a n l u n g o D r . A.M. S t e p h e n , and a l s o t o t h e o t h e r members o f t h e l a b o r a t o r A n g e l a S a v a g e , J o s e d i F a b i o , and M a r c e l P a u l i n . C r e d i t must a l s o go t o my w i f e L i n d a , f o r h e r c o n s t a n t p a t i e n c e and s u p p o r t . - i x -PREFACE In an e f f o r t t o f a m i l i a r i z e r e a d e r s who do n o t work i n t h e p a r t i c u l a r a r e a o f o r g a n i c c h e m i s t r y t o w h i c h t h i s t h e s i s r e f e r s , t h e f o l l o w i n g e x p l a n a t i o n o f t e r m s u s e d i s o f f e r e d . F i s c h e r p r o j e c t i o n f o r m u l a e a r e u s e d t o r e p r e s e n t t h e a c y c l i m o d i f i c a t i o n o f s u g a r s . Some e x a m p l e s a r e shown b e l o w . Numberin commences from t h e c a r b o n y l g r o u p a t t h e t o p o f t h e c h a i n ( I ) . N o te t h a t D - g l u c u r o n i c a c i d ( I I ) d i f f e r s f rom D - g l u c o s e ( I ) o n l y HO CHO 1—OH 3 OH OH CH 20H CHO — OH HOH OH —OH COOH CHO OH I—OH H0-HO—I CH. D - g l u c o s e ( I ) D - g l u c u r o n i c a c i d ( I D L-rhamnose ( I I I ) i n t h a t C-6 i s o x i d i z e d t o a c a r b o x y l i c a c i d g r o u p . The C-6 o f L-rhamnose ( I I I ) i s p a r t o f a m e t h y l group and i s r e f e r r e d t o a l s o by a n o t h e r common name, 6-deoxy-L-mannose. T h e r e a r e f o u r c h i r a l c e n t e r s i n t h e s e s i x - c a r b o n c h a i n s ( m a rked w i t h a s t e r i s k s i n s t r u c t u r e I I I ) m a k i n g i t i m p o r t a n t to a p p r e c i a t e t h e s p a t i a l a r r a n g e m e n t o f atoms ( c o n f o r m a t i o n ) t h a t i s i m p l i e d by t h e s e F i s c h e r r e p r e s e n t a t i o n s . To s i m p l i f y t h e n o m e n c l a t u r e o f a l l t h e p o s s i b l e i s o m e r s (16 f o r each o f - X -I , I I , I I I ) , a l l t h o s e h a v i n g t h e h y d r o x y l g r o u p a t t h e h i g h e s t -numbered c h i r a l c e n t e r (C-5) p r o j e c t i n g t o t h e r i g h t i n t h e F i s c h e r p r o j e c t i o n f o r m u l a e b e l o n g t o t h e D - s e r i e s , and t h e o t h e r s t o t h e L - s e r i e s . P h y s i c a l and c h e m i c a l e v i d e n c e i n d i c a t e s t h a t , i n f a c t , t h e s e s i x - c a r b o n p o l y h y d r o x y a l d e h y d e s e x i s t i n a c y c l i c f o r m . The r i n g c l o s u r e o c c u r s by n u c l e o p h i l i c a t t a c k o f t h e o x y g e n atom a t C - 5 on t h e a l d e h y d i c c a r b o n atom, g e n e r a t i n g a new c h i r a l ( a n o m e r i c ) c e n t e r a t C-1. T h i s r e s u l t s i n two anomers, r e p r e s e n t e d b e l o w \—0H HCH D - s e r i e s L - s e r i e s H OH HO H C h-OH h-OH HO — 0 HO 0 —OH hOH - g l u c o s e ( I V ) $-D - g l u c o s e (V) - x i -i n t h e T o l l e n s f o r m u l a e . I t s h o u l d be n o t e d t h a t C - l i s u n i q u e i n h a v i n g two a t t a c h e d oxygen atoms, f o r m a l l y m a k i n g i t a h e m i a -c e t a l c a r b o n . S i n c e t h e T o l l e n s f o r m u l a e have o b v i o u s l i m i t a t i o n s w i t h t h e i r u n e q u a l bond l e n g t h s , H aworth d e v e l o p e d a p e r s p e c t i v e method o f l o o k i n g a t t h e s i x - m e m b e r e d r i n g ( V I and V I I ) . T h i s i m p r o v e m e n t r e c o g n i z e s t h a t t h e r i n g oxygen atom l i e s b e h i n d t h e c a r b o n c h a i n and t h a t bond l e n g t h s a r e a p p r o x i m a t e l y e q u a l . O f t e n i n p r a c t i s e r e g u l a r h e x a g o n s a r e u s e d i n Ha w o r t h p r o j e c t i o n s , a-D-g l u copy r a n ose g - D - g l u c o p y r a n o s e p y r a n ( V I ) ( V I I ) ( V I I I ) w h i c h he r e l a t e d t o su c h r i n g s as t h e h e t e r o c y c l i c compound p y r a n ( V I I I ) and named them p y r a n o s e s . Note t h a t h y d r o x y l g r o u p s n o t i n v o l v e d i n r i n g f o r m a t i o n on t h e r i g h t i n F i s c h e r and T o l l e n s f o r m u l a e p o i n t down i n t h e Haworth p r o j e c t i o n s and t h o s e on t h e l e f t p o i n t up. S i m i l a r l y , f o r a l d o p y r a n o s e s , t h e g r o u p on C - 5 p o i n t s up f o r D ( I X ) and down f o r t h e L e n a n t i o m e r ( X ) , I t f o l l o w s , t h e n , t h a t when s u g a r r e s i d u e s a r e a t t a c h e d t h e r e a r e two p o s s i b l e c o n f i g u r a t i o n s , an a - o r a 3 - p y r a n o s i d e , f o r ea ch l i n k a g e . - X 1 1 -HO OH HO °xOH a - D - r h a m n o p y r a n o s e ( I X ) T h e t r u e c o n f o r m a t i o n o f p y r a n o i d c a r b o h y d r a t e s i s r e l a t e d t o t h e c h a i r f o r m o f c y c l o h e x a n e. X - r a y d i f f r a c t i o n a n a l y s i s h a s s h o w n t h a t a h e x o s e , s u c h a s a - D - g l u c o s e ( X I ) , c o n s i s t s o f a p u c k e r e d , s i x - m e m b e r e d , o x y g e n - c o n t a i n i n g c a r b o n r i n g , w i t h h y d r o x y l s u b s t i t u e n t s a t C - 1 t h r o u g h C - 4 , a n d a h y d r o xy m e t h y l g r o u p a t C - 5 . A l l s u b s t i t u e n t s on t h e r i n g , e x c e p t f o r t h a t a t C - 1 , a r e e q u a t o r i a l . Two i s o m e r s ( a n o m e r s ) a r e p o s s i b l e i n r e l a t i o n t o t h e a n o m e r i c c e n t e r ( C - 1 ) , d e p e n d i n g on w h e t h e r a s u b s t i t u e n t i s ( X I ) - X l l l -a x i a l ( a - a n o m e r ; X I I ) o r e q u a t o r i a l ( g - a n o m e r ; X I I I ) , w h e r e R = h y d r o g e n , f o r m o n o s a c c h a r i d e s , and R = a n o t h e r s u g a r r e s i d u e , f o r d i - , o l i g o - , and p o l y s a c c h a r i d e s . S i n c e H - l i s i n a d i f f e r e n c h e m i c a l e n v i r o n m e n t f o r t h e two a n o m e r s , n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r o s c o p y can e a s i l y d i s t i n g u i s h b e t w e e n them a n d , t h e r e b y , p r o v i d e s i n v a l u a b l e a s s i s t a n c e i n a s s i g n i n g a n o m e r i c c o n f i g u r a t i o n s . H a w o r t h p r o j e c t i o n s a r e most u s e f u l and w i l l be u s e d i n t h i s t h e s i s , even t h o u g h t h e y g i v e no i n d i c a t i o n o f t h r e e -d i m e n s i o n a l m o l e c u l a r s h a p e . T h e r e seems t o be l i t t l e j u s t i f i -c a t i o n f o r t h e u s e o f f o r m u l a e w h i c h d e p i c t s t a t e s o f m o l e c u l e s as w e l l a s s t r u c t u r e s , when t h e t r u e s t a t e s a r e o f t e n unknown o r v a r i a b l e . I N T R O D U C T I ON M E T H O D S U S E D I N S T R U C T U R A L A N A L Y S E S OF P O L Y S A C C H A R I D E S -2-I IN TR ODUCTION B a c t e r i a of t h e genus K l e b s i e l l a b e l o n g t o t h e f a m i l y E n t e r o b a c t e r i a c e a e ( g a s t r o i n t e s t i n a l f l o r a ) w h i c h i n c l u d e s o t h e r ""well known members su c h as t h e g e n e r a Salm on e l l a and E s c h e r i c h i a  co l i ( E . c o l i ) . The K l e b s i e l l a b a c t e r i a l c e l l has t h e n o t a b l e c h a r a c t e r i s t i c of p r o d u c i n g a c a p s u l a r p o l y s a c c h a r i d e . In c u l t u r e s of some K l e b s i e l l a s t r a i n s an e x c e s s of p o l y s a c c h a r i d e i s r e l e a s e d f r o m t h e c e l l f o r m i n g a " s l i m e " . Dudman and W i l k i n -son"'" have shown t h a t t h e s l i m e and p o l y s a c c h a r i d e a r e i d e n t i c a l i n c h e m i c a l c o m p o s i t i o n . In F i g u r e 1.1, a d i a g r a m m a t i c r e p r e -s e n t a t i o n of a b a c t e r i a l c e l l c o n t a i n i n g j u s t s u c h an " e x o p o l y -s a c c h a r i d e " i s shown. CELL WALL M E M B R A N E CYTOPLASM UPOPOLYSACCHARIDE C A P S U L E SLIME Figure 1.1 Diagrammatic representation of a b a c t e r i a l c e l l with lipopolysaccharide, capsule and slime. - 3 -The p o l y s a c c h a r i d e e n v e l o p e s t h e c e l l p r o v i d i n g t h e f i r s t s u r f a c e f o r e x t e r n a l c o n t a c t . K l e b s i e l l a a r e s e r o l o g i c a l l y t y p e d s o l e l y on t h e b a s i s o f c a p s u l a r a n t i g e n s and i t i s t h i s e n v e l o p e o f p o l y s a c c h a r i d e w h i c h i s t h o u g h t t o c a u s e i m m u n o -l o g i c a l r e s p o n s e s . S e v e n t y - e i g h t s e r o l o g i c a l l y d i s t i n c t s t r a i n s o f K l e b s i e l l a h a v e been e s t a b l i s h e d t o d a t e and d e s i g n a t e d K - t y p e s f r o m t h e German word " K a p s e l " . 0 r s k o v and 2 F i f e - A s b u r y h a v e r e c e n t l y amended t h e number o f known s t r a i n s by a d d i n g K82 t o , and d e l e t i n g K 7 3 , 7 5 , 7 6 , 7 7 , and 78 f r o m t h e s c h em e . P o l y s a c c h a r i d e s a r e g e n e r a l l y c o n s i d e r e d t o be m i l d l y o r n o n - a n t i g e n i c b u t a n t i b o d i e s h a v e been i s o l a t e d f r o m h o r s e s and r a b b i t s . H e i d e l b e r g e r e t a l ^ h a v e u s e d a n t i - K s e r a t o s t u d y c r o s s - r e a c t i o n s w i t h o t h e r b a c t e r i a l s p e c i e s . I m m u n o c h e m i c a l r e l a t i o n s h i p s can t h e r e b y be p r e d i c t e d i n m i c r o o r g a n i s m s b e -l o n g i n g t o w i d e l y d i s p a r a t e f a m i l i e s . U n f o r t u n a t e l y t h e s p e c i f i c a n t i g e n i c d e t e r m i n a n t c a n n o t be a s s i g n e d u n t i l t h e s t r u c t u r e s o f t h e s e c a p s u l a r p o l y s a c c h a r i d e s a r e k n o w n . Thus s t r u c t u r a l s t u d i e s o f a l l known K l eb s i e l l a s t r a i n s a r e u n d e r w a y . The i n v e s t i g a t i o n o f K17 and K44 i s p r e s e n t e d i n t h i s t h e s i s . I n c i d e n t a l l y , t h e p r e d i c t i o n o f H e i d e l b e r g e r e t a l ^ t h a t K17 c o n t a i n s e n d - g r o u p s o f L - r h a m n o s e , a s a r e s u l t o f c r o s s - r e a c t i -v i t y w i t h a n t i - K 1 9 and a n t i - K 4 7 , i s c o n f i r m e d by s t r u c t u r a l s t u d i e s i n t h i s t h e s i s . N i m m i c h ^ ' ^ ' ^ ' h a s r e p o r t e d t h e q u a l i t a t i v e c o m p o s i t i o n o f K - t y p e s 1 t o 81 a n d . h a s f o u n d them a l l t o be a c i d i c p o l y s a c c h a -r i d e s c o n t a i n i n g e i t h e r D - g l u c u r o n i c a c i d , D - g a l a c t u r o n i c a c i d , - 4 -p y r u v i c a c i d i n t h e f o r m o f 1 - c a r b o x y e t h y l i d e n e a c e t a l s , o r v e r y s e l d o m , a k e t o a c i d ( K 2 2 , 3 7 , 3 8 ) . In a d d i t i o n , D - g a l a c t o s e , D - g l u c o s e , D - m a n n o s e , L - r h a m n o s e and t o a l e s s e r e x t e n t , L - f u c o s e can a p p e a r . A l t h o u g h no s y s t e m a t i c p a t t e r n i s e v i d e n t f r o m t h e p o l y -s a c c h a r i d e s t r u c t u r e s o f K l eb s i e l l a K - t y p e s known t o d a t e , w h i c h number a p p r o x i m a t e l y 4 0 , some g e n e r a l p o i n t s h a v e e m e r g e d . The number o f s u g a r r e s i d u e s i n t h e r e p e a t i n g u n i t v a r i e s f r o m t h r e e ( K 1 , K 5 , K 6 3 ) t o s e v e n ( K 1 2 , K 4 1 , K 7 9 ) . O n l y o n e a c i d i c s u g a r ( p e r r e p e a t u n i t ) i s p r e s e n t , e x c e p t when i n c o m b i n a t i o n w i t h t h e p y r u v i c a c e t a l . S t r u c t u r e s a r e known t o e x i s t a s s t r a i g h t c h a i n p o l y m e r s o r a s b r a n c h e d c h a i n s w i t h o n e , two o r t h r e e u n i t s i d e c h a i n s . K l e b s i e l l a K - t y p e s can be g r o u p e d a c c o r d i n g t o t h e i r q u a l i t a t i v e c o n s t i t u e n t s b u t t h e i r s t r u c t u r e s can be w i d e l y d i v e r s e . F o r i n s t a n c e , K17 and K44 b o t h c o n t a i n D - g l u c u r o n i c a c i d , D - g l u c o s e and L - r h a m n o s e w h e r e a s K44 i s l i n e a r and K17 i s b r a n c h e d w i t h a s i n g l e u n i t s i d e c h a i n . O r i g i n a l l y t h o u g h t t o be t h e o n l y two c a p s u l a r p o l y s a c c h a r i d e s i n t h i s g r o u p , i t 7 8 h a s r e c e n t l y been r e p o r t e d t h a t K23 and K45 , a t f i r s t b e l i e v e d t o i n c l u d e D - g a l a c t o s e , can now be c o m b i n e d w i t h K17 and K 4 4 . M o s t i m p o r t a n t l y , t h e s e s t r u c t u r a l d i f f e r e n c e s e x p l a i n t h e i r s p e c i f i c s e r o l o g i c a l r e a c t i v i t y . - 5 -I I METHODS USED IN STRUCTURAL ANALYSIS OF POLYSACCHARIDES Techniques and methods used i n s t r u c t u r a l a n a l y s i s of 9 10 p o l y s a c c h a r i d e s are w e l l e s t a b l i s h e d . ' Many modern t e c h -n i q u e s are e x p l o i t e d i n order to g i v e a d d i t i o n a l i n f o r m a t i o n or to c o n f i r m i n t e r p r e t a t i o n s from c l a s s i c a l methods w h i l e making use of s m a l l e r q u a n t i t i e s and s h o r t e r a n a l y s i s t i m e s . N u c l e a r magnetic resonance, both proton ("''H) and carbon 13 (~^C), g a s - l i q u i d chromatography and mass s p e c t r o m e t r y were employed i n the p r e s e n t i n v e s t i g a t i o n . However, each K l e b s i e l l a K-type e x h i b i t s i t s own p h y s i c a l c h a r a c t e r i s t i c s such as quan-t i t y of p o l y s a c c h a r i d e produced, s o l u b i l i t y , degree of a c i d i t y and s u s c e p t i b i l i t y to h y d r o l y s i s . With t h i s i n mind, the f o l l o w i n g i s an o u t l i n e of the methods used i n t h i s s t u d y , w i t h emphasis on problems encountered w i t h K17 and K44. I I . 1 I s o l a t i o n and P u r i f i c a t i o n In September, 1977 b a c t e r i a l samples of K l e b s i e l l a K17 and K44 were r e c e i v e d from Dr. I . jSrskov i n the form of stab c u l t u r e s . These c u l t u r e s were s t r e a k e d out an agar p l a t e s and found to grow w e l l on i n c u b a t i o n at 37 °C. Healthy s i n g l e c o l o n i e s from the second p l a t i n g were used to i n o c u l a t e a beef e x t r a c t medium. The b a c t e r i a m u l t i p l i e d p r o f u s e l y w h i l e s h a k i n g i n a water bath at 37°C f o r 3-4 hours and were subse-q u e n t l y grown on l a r g e t r a y s of s u c r o s e - y e a s t e x t r a c t - a g a r medium f o r t h r e e days. A f t e r h a r v e s t i n g , the b a c t e r i a were d e s t r o y e d w i t h a 1% phenol s o l u t i o n and s e p a r a t e d from the p o l y s a c c h a r i d e - c o n t a i n i n g s l i m e by u 1 1 r a - c e n t r i f u g a t i o n . - 6 -The v i s c o u s s u p e r n a t a n t vi/as p u r i f i e d by t h r e e p r e c i p i t a t i o n s . F i r s t l y i n t o e t h a n o l and s e c o n d l y , a f t e r d i s s o l v i n g i n water, w i t h c e t y l t r i m e t h y l a m m o n i u m bromide (CETAVLON), a l a r g e qua-t e r n a r y ammonium c a t i o n which forms a water i n s o l u b l e s a l t w i t h the p o l y a n i o n of a c i d i c polysaccharides."'""'" F i n a l l y , a f t e r d i s s o l v i n g the CETAVLON-polysaccharide p r e c i p i t a t e i n 4 M sodium c h l o r i d e , the s o l u t i o n was again p r e c i p i t a t e d i n t o e t h a n o l to remove the l a r g e c a t i o n . The l a s t p r e c i p i t a t i o n i s r e q u i r e d to prevent r e f o r m a t i o n of i n s o l u b l e s a l t s as sodium c h l o r i d e i s removed d u r i n g f i n a l d i a l y s i s . L y o p h i l i z a t i o n of the d i a l y z e d m a t e r i a l completes the p u r i f i c a t i o n . Of the two p o l y s a c c h a r i d e s i s o l a t e d i n t h i s manner K17 produced about t w i c e as much as K44. I I . 2 T o t a l H y d r o l y s i s and C h a r a c t e r i z a t i o n of Component Sugars To begin s t r u c t u r a l a n a l y s i s of a p o l y s a c c h a r i d e one must know the q u a n t i t a t i v e c o m p o s i t i o n . T h i s i s somewhat s i m p l i f i e d i f the s t r u c t u r e i s composed of r e p e a t i n g u n i t s as i n K l e b s i e l l a K-types. T o t a l h y d r o l y s i s w i t h a c i d a f t e r f i r s t r e d u c i n g the c a r b o x y l i c a c i d f u n c t i o n l e a d s to a n a l y s i s by g a s - l i q u i d chroma-tography y i e l d i n g the d e s i r e d t o t a l sugar r a t i o . N u c l e a r magnetic resonance (n.m.r.) s p e c t r o s c o p y i s a l s o ; a ; u s e f u l a i d to t h i s end. Moreover, t h i s t e c h n i q u e c o n f i r m s the presence of r e p e a t i n g u n i t s by the r e l a t i v e l y s i m p l i f i e d s p e c t r a of a m o lecule of K l e b s i e l l a p o l y s a c c h a r i d e w i t h m o l e c u l a r weight a p p r o x i m a t i n g one m i l l i o n . E f f e c t i v e l y , n.m.r. "sees" a m o l e c u l a r weight of between 600 and 1400 depending on the number of sugars i n the r e p e a t i n g u n i t . - 7 -I I . 2 . 1 T o t a l sugar r a t i o A n e u t r a l p o l y s a c c h a r i d e w i l l n o r m a l l y be t o t a l l y hydro-l y z e d by a s t r o n g a c i d , commonly s u l f u r i c , h y d r o c h l o r i c or t r i f l u o r o a c e t i c a c i d , w i t h n e g l i g i b l e d e g r a d a t i o n of the monosaccharides formed. However, u r o n i c a c i d l i n k a g e s are c o n s i d e r a b l y more r e s i s t a n t and w i l l not h y d r o l y z e q u a n t i t a -t i v e l y under these c o n d i t i o n s . 12 By a method developed i n t h i s l a b o r a t o r y , m e t h a n o l y s i s i s used m a i n l y to e s t e r i f y the c a r b o x y l i c a c i d moiety. The methyl e s t e r i s then reduced w i t h sodium b o r o h y d r i d e i n anhydrous methanol, s i n c e the e s t e r would be s a p o n i f i e d i n aqueous media. Although m e t h a n o l y s i s c l e a v e s most g l y c o s i d i c bonds, the remainder, now made s u s c e p t i b l e , a l l o w the p o l y -s a c c h a r i d e to be t o t a l l y h y d r o l y z e d by 2 |1 t r i f ilu o roa ce t i c a c i d . The monosaccharides themselves are then reduced and a c e t y l a t e d to produce a l d i t o l p e r a c e t a t e s 1 ^ ' 1 ^ ' x ^ f o r g a s - l i q u i d c h romatographic ( g . l . c . ) a n a l y s i s . U r o n i c a c i d s , when p r e s e n t , appear as t h e i r reduced hexoses. D - g l u c u r o n i c a c i d , f o r example, would appear as D - g l u c i t o l h e x a c e t a t e a f t e r the above-mentioned s e r i e s of r e a c t i o n s . G . l . c . i s a w i d e l y accepted t o o l i n the c a r b o h y d r a t e f i e l d and has been reviewed extensively."*"^ T h i s method only r e q u i r e s sample s i z e s i n the microgram range and performs s e p a r a t i o n of c l o s e l y s i m i l a r compounds w i t h ease. A wide s e l e c t i o n of s t a t i o n a r y phases was employed i n t h i s s t u d y . I n c l u d e d are SP-2340, a h i g h l y p o l a r s i l i c o n e c o n t a i n i n g 7 5% c y a n o p r o p y l groups, and S i l a r IOC, having s i m i l a r c o m p o s i t i o n , which can - 8 -r e s o l v e the a l d i t o l a c e t a t e s of L-rrhamnose, D-mannose, D- g a l a c t o s e and D-glucose. The use of a l d i t o l a c e t a t e d e r i v a t i v e s throughout t h i s i n v e s t i g a t i o n i s because they g i v e r i s e t o only one peak, making q u a n t i t a t i o n e a s i e r . Other d e r i v a t i v e s t h a t p r e s e r v e the anomeric c e n t e r may r e s u l t i n two peaks r e p r e s e n t i n g the a- and 8- anomers, f o r i n s t a n c e , t r imet hy 1 s i l y 1 d e r i v a t i v e s . The f a c t t h a t s t a b l e , v o l a t i l e d e r i v a t i v e s are n e c e s s a r y f o r g . l . c . c o u l d be c o n s i d e r e d d i s a d v a n t a g e o u s and c o n t i n u e d r e s e a r c h i n t o h i g h performance l i q u i d chromatography ( h . p . l . c . ) as a complementary t e c h n i q u e i n c a r b o h y d r a t e c h e m i s t r y i s in ev i t a b l e . II . 2 . 2 N u c l e a r magnetic resonance s p e c t r o s c o p y ("'"H and ^ C ) Proton n.m.r. s p e c t r o s c o p y , an i n d i s p e n s a b l e t o o l f o r the o r g a n i c c h e m i s t , has been used p r o f i t a b l y i n the s t r u c t u r a l 17 18 a n a l y s i s of K l e b s i e l l a p o l y s a c c h a r i d e s . ' S i n c e b e i n g 19 13 i n t r o d u c e d i n 1969 the t e c h n i q u e of C n.m.r. has been a p p l i e d e x t e n s i v e l y t o mono- and h i g h e r - s a c c h a r i d e s as seen 20 i n a re c e n t r e v i e w . P o l y s a c c h a r i d e s are amenable t o s t r u c t u r a l a n a l y s i s by "^C n.m.r. s p e c t r o s c o p y ^ 3 but t h i s method has not been p r a c t i s e d u n t i l r e c e n t l y i n t h i s l a b o r a t o r y l a r g e l y due to the g r e a t success i n i n t e r p r e t i n g "''H n.m.r. s p e c t r a . In t h i s s tudy, n.m.r. s p e c t r a were o b t a i n e d a f t e r the p o l y s a c c h a r i d e underwent m i l d h y d r o l y s i s c o n d i t i o n s (0.4 M TFA, 20 min, 95°C) and then was d i s s o l v e d i n D^ O s o l v e n t . Kleb s i e l l a p o l y s a c c h a r i d e s are d i f f i c u l t to d i s s o l v e i n water, - 9 -even as the sodium s a l t form,and r e d u c i n g the polymer s i z e and v i s c o s i t y ever so s l i g h t l y sharpens the n.m.r. s p e c t r a remarkably. Sample s i z e s were i n the range 20 to 30 m i l l i g r a m s and 80 to 120 m i l l i g r a m s f o r ~*"H and "^C n.m.r. r e s p e c t i v e l y . With o l i g o s a c c h a r i d e s , however, very r e a s o n a b l e s p e c t r a were o b t a i n e d w i t h as s m a l l as 10 m i l l i g r a m s f o r both -^"H and "^C n.m.r. Acetone has been employed as a s t a n d a r d because of i t s s o l u b i l i t y and the f a c t t h a t i t s chemical s h i f t i s i n d e -pendent of temperature. A l a r g e HOD peak, always present due to i n c o m p l e t e ex-change w i t h D^O, p a r t i a l l y obscures the anomeric r e g i o n of the "'"H n.m.r. s p e c t r a , 64.5 to 5.5 r e l a t i v e to*aceto n e . S i n c e the i n t e r f e r i n g peak i s temperature dependent, advantage was taken to secure s p e c t r a at d i f f e r e n t temperatures thus o b s e r v i n g the HOD s i g n a l at s e p a r a t e areas of the anomeric r e g i o n and t h e r e -by p i e c i n g t o g e t h e r the whole r e g i o n . T h i s was ac c o m p l i s h e d u s i n g the 100 MHz and 270 MHz i n s t r u m e n t s whose ambient tem-p e r a t u r e s are 32°C and 23°C r e s p e c t i v e l y . However, as can be seen i n F i g u r e I I . 1 , s p e c t r a o b t a i n e d at h i g h temperature (95°C) can be i n t e r p r e t e d w i t h much more c o n f i d e n c e s i n c e the whole anomeric r e g i o n can be observed w i t h o u t i n t e r f e r e n c e . In "*"H n.m.r. s p e c t r o s c o p y the anomeric r e g i o n can n o m i n a l l y be d i v i d e d at 65.0. Meaning t h a t s i g n a l s d o w n f i e l d of 65.0 can be t e n t a t i v e l y a s s i g n e d to ^ - l i n k a g e s and those u p f i e l d to g - l i n k a g e s . K17 f o l l o w s t h i s g e n e r a l r u l e but e xh i b i ts an o th er i n t e r e s t i n g s i d e l i g h t (see F i g u r e I I . 2 ) . Normally emphasis i s p l a c e d on t h a t p o r t i o n of s p e c t r a between 64.5 to 65.5 but i f - 10 -80°C 90°C HOD 4. 25 6 HOD 4.126 F i g u r e I I . 1 Proton n.m.r. s p e c t r a showing the change i n HOD s i g n a l l o c a t i o n w i t h temperature i n the anomeric r e g i o n 64. 0 to 6.0. A. 270 MHz spectrum, U n i v e r s i t y of B.C. B. 100 MHz spectrum, U n i v e r s i t y of B.C. C. 250 MHz spectrum, C.N.R.S., Grenoble, France D. 220 MHz spectrum, U n i v e r s i t y of Toronto - 11 -one extends o b s e r v a t i o n up to 64.0 a d d i t i o n a l i n f o r m a t i o n Q may be gleaned from the r e c o r d i n g . F i r s t observed i n K45 , s i g n a l s at 64.5-4.0 may diagnose e i t h e r a hydrogen bonded to carbon-5( H-5) of u r o n i c a c i d s or o t h e r r i n g p r o t o n s of 21 2 2 n e u t r a l s u g a r s . I t has been shown ' t h a t H-5 of and 3 -gal a ctu ron i c a c i d s produces a "''H n.m.r. s i g n a l between 64.40-4.75 wit h the a-anomer always f u r t h e r d o w n f i e l d . By i n f e r e n c e then, H-5 of D - g l u c u r o n i c a c i d s would appear s l i g h t l y u p f i e l d s i n c e the average d e s h i e l d i n g e f f e c t of g a l a c t o p y r a n o s e 22 over g l u c o p y r a n o s e i s about 0.26 p.p.m. K17, which c o n t a i n s an a - D - g l u c u r o n i c a c i d component, g i v e s s i g n a l s at 64.60 and 4.51, one of which may be due to H-5 of the u r o n i c a c i d . Other evidence f o r t h i s f i r s t l y comes from the f a c t t h a t the t o t a l sugar r a t i o y i e l d s a rhamnose-glucose r a t i o of 3:2 i n d i c a t i n g f i v e sugar r e s i d u e s i n the r e p e a t i n g u n i t a c c o u n t i n g f o r only 5 of the 6 a n o m e r - l i k e s i g n a l s i n the "*"H n.m.r. spectrum. Secondly, the proton s i g n a l at 64.51 appears to be a t r i p l e t i n s t e a d of the normal d o u b l e t . I t i s a l s o noteworthy t h a t the s i g n a l at 64.60 shows a c o u p l i n g of 10 Hz ( g e n e r a l l y 7-QBz f o r 3-glucose) whereas the s i g n a l at 64.51 shows two c o u p l i n g s of a p p r o x i m a t e l y 7 H z . As can be seen i n F i g u r e I I . 2 , o t h e r s i g n a l s a l s o appear between 64.0-4.5 i n the spectrum of K17. P r o t o n s on carbon number two (C-2) and carbon number t h r e e (C-3) of L-rhamnose 2 5 and D-mannose produce s i g n a l s i n the range 63.80-3.9 5. The e f f e c t of bonding on C-2 and/or C-3 as i n a p o l y s a c c h a r i d e tends to p u l l these s i g n a l s d o w n f i e l d towards the anomeric r e g i o n - 12 -"*"H n.m.r. s p e c t r a of K17 p o l y s a c c h a r i d e d e p i c t i n g s i g n a l s o t h e r than anomeric ones i n the r e g i o n 64.0 to 6.0. Shown at arrows. A. At 80°C, showing s i g n a l at 64.51. ii. At 25°C, showing s i g n a l at 64.29. - 13 -perhaps as much as +0.4 p.p.m. I n d i c a t i o n s a r e , t h e r e f o r e , t h a t the n.m.r. s i g n a l at 64.29 f o r K17 may a r i s e from the H-2 proton of L-rhamnose, where an L-rhamnose u n i t o c c u r s as a b r a n c h i n g p o i n t l i n k e d at r i n g p o s i t i o n s 2 and 3. As s t a t e d b e f o r e , the assignment of ''"H n.m.r. s i g n a l s t o other than anomeric p r o t o n s may be a u s e f u l d i a g n o s t i c t o o l i n p o l y s a c c h a r i d e s t r u c t u r a l a n a l y s i s . The presence and r e l a t i v e p r o p o r t i o n of deoxy sugars are e v i d e n t i n the r e g i o n u p f i e l d from the acetone s t a n d a r d i . e . a p p r o x i m a t e l y 61.30. Another f e a t u r e which a i d s i n i d e n t i f y i n g component sugars i s t h e i r s p i n - s p i n c o u p l i n g cons-21 t a n t s e s p e c i a l l y i n the anomeric r e g i o n . ^ ^ O T 3-D-glucose and 3-D-galactose are of the order 7-9 Hz whereas f o r t h e i r a-anomers J , 9= 2-3 Hz. For L-rhamnose the s i t u a t i o n i s r e v e r s e d w i t h a - L - a n o m e r i c proton having J , 2 Hz and the B-L-an omer i c 1 J ^ proton a lower J , 9 ^ 1 Hz. However, chemical s h i f t i s more 1 > ^  important d i a g n o s t i c a l l y because th e s e v a l u e s are s m a l l and n o r m a l l y d i f f i c u l t to measure. "^C n.m.r. s p e c t r o s c o p y has been used throughout t h i s work f o r the p o l y s a c c h a r i d e s and f o r o l i g o s a c c h a r i d e s o b t a i n e d from d e g r a d a t i o n s t u d i e s as w e l l . Samples were run i n D^ O w i t h acetone as s t a n d a r d g i v i n g a r e f e r e n c e peak at 31.07 p.p.m. As a complement t o "*"H n.m.r., i n f o r m a t i o n on the number of monosaccharides i n the r e p e a t i n g u n i t of a K l e b s i e l l a p o l y s a c -c h a r i d e , the c o n f i g u r a t i o n at the anomeric c e n t e r s and the 13 presence of deoxy sugars can be o b t a i n e d from C n.m.r. - 14 -2 6 e x p e r i m e n t s . The d i f f e r e n c e b e i n g t h a t s p e c t r a were run a t ambient temperature s i n c e no i n t e r f e r e n c e from s o l v e n t re,S;On.an'ces i s expected as i n n.m.r. G e n e r a l l y , a d i v i d i n g l i n e around 101 p.p.m. may be drawn such t h a t "^C s i g n a l s a p p e a r i n g down-f i e l d r e p r e s e n t 3 - l i n k e d sugars and those u p f i e l d a - l i n k e d sugars. However, c a u t i o n must be e x e r c i s e d when a p p l y i n g t h i s g e n e r a l r u l e to a- and B-L-rhamnose l i n k a g e s , as seen i n the i n v e s t i -g a t i o n of K17. 27 13 Bork and coworkers have shown t h a t C-H c o u p l i n g c o n s t a n t s can be a p p l i e d to a s s i g n anomeric c o n f i g u r a t i o n . How-ever, l o s s of s i g n a l s t r e n g t h due to s p l i t t i n g and the a d d i t i o n a l number of peaks, a l r e a d y about 30 i n decoupled "^C s p e c t r a , p r e c l u d e the use of c o u p l i n g c o n s t a n t s f o r p o l y s a c c h a r i d e samples. T o t a l assignment of even anomeric carbon s i g n a l s has 2 8 proven d i f f i c u l t . Some r e f e r e n c e m a t e r i a l on methyl g l y c o s i d e s 29 and o t h e r K l e b s i e l l a p o l y s a c c h a r i d e s e x i s t but i n s u f f i c i e n t data are a v a i l a b l e to make a b s o l u t e assignments. A "^C n.m.r. s p e c t r a of K17 i s i l l u s t r a t e d i n F i g u r e I I . 3 , t y p i c a l of K l e b s i e l l a c a p s u l a r p o l y s a c c h a r i d e s . No problem i n d e t e c t i n g f i v e anomeric c e n t e r s e x i s t s w i t h t h i s s p e c t r a as i n "^H n.m.r. s p e c t r a d i s c u s s e d e a r l i e r . I I . 3 A n a l y s i s o f L i n k a g e P o s i t i o n f o r Component Sugars A f t e r d e t e r m i n i n g the number and types of sugar r e s i d u e s in a p o l y s a c c h a r i d e the next step i n v o l v e s a n a l y s i s of where t h e s e components are l i n k e d to form the p o l y m e r i c c h a i n . T h i s i s a c c o m p l i s h e d through m e t h y l a t i o n and subsequent c h a r a c t e r i z a t i o n Field; 20 MHz S.W.j 6 KHz N.T., 357,900 A.T.; 0.5 sec P.W.; 18 qsec P.D.; 0 sec 5 anomeric carbons C-6 of glucuronic acid | (-C00H) C-6 of /rhamnose (~CHg) acetone C-6 of ^glucose (-CH20H) 175 150 100 I ppm F i g u r e I I . 3 C n.m.r. s p e c t r u m of K l e b s i e l l a K17 c a p s u l a r p o l y s a c c h a r i d e . - 16 -of p r o d u c t s by g a s - l i q u i d chromatography and g a s - l i q u i d chromatography-mass s p e c t r o m e t r y (g.1.c.-m.s.) t e c h n i q u e s . I n f o r m a t i o n on whether b r a n c h i n g o c c u r s i n a p o l y s a c c h a r i d e i s i n c l u d e d i n m e t h y l a t i o n a n a l y s i s as w e l l as c o n f i r m a t i v e data on the number and types of sugar components p r e s e n t . I I . 3 . 1 M e t h y l a t i o n a n a l y s i s M e t h y l a t i o n of c a r b o h y d r a t e s i s a w e l l known p r o c e s s . In order to a c h i e v e complete m e t h y l a t i o n (judged by the absence of -OH a b s o r p t i o n at 3500cm ^ i n the i n f r a r e d spectrum) a s i n g l e Hakomori p r o c e d u r e ^ ' ^ was s u f f i c i e n t i n t h i s study of o l i g o -s a c c h a r i d e s ( o b t a i n e d from p a r t i a l h y d r o l y s i s , see s e c t i o n 32 I I . 5 ) , but f o r the n a t i v e p o l y s a c c h a r i d e a subsequent P u r d i e m e t h y l a t i o n was n e c e s s a r y . A second Hakomori m e t h y l a t i o n would have r e s u l t e d i n 3 - e l i m i n a t i o n s i d e r e a c t i o n s (see s e c t i o n I I . 4 ) F o l l o w i n g m e t h y l a t i o n , the m a t e r i a l was h y d r o l y z e d , reduced to a l d i t o l s and then a c e t y l a t e d . The unmethylated p o s i t i o n s of the a l d i t o l r e p r e s e n t s i t e s of l i n k age,, excep t i n the case of a u r o n i c a c i d r e s i d u e . Due to r e d u c t i o n of the c a r b o x y l f u n c t i o n p r i o r to h y d r o l y s i s and a l d i t o l a c e t a t e workup the C-6 p o s i t i o n as w e l l as l i n k a g e s i t e s would be a c e t y l a t e d . The d e r i v a t i v e of a u r o n i c a c i d moiety can e a s i l y be i d e n t i f i e d by r e p e a t i n g the same sequence of r e a c t i o n s on the c a r b o x y l -reduced p o l y s a c c h a r i d e . The r e s u l t w i l l be the d i s a p p e a r a n c e of a C-6 a c e t y l a t e d d e r i v a t i v e peak and the appearance of a C-6 m e t h y l a t e d d e r i v a t i v e peak i n the g . l . c . t r a c i n g . F i g u r e I I . 4 i l l u s t r a t e s these p o i n t s i n a r e a c t i o n scheme (page 17). - 1 7 -0 „ 3 a R h a ^ G I c A L ^ R h a 1 ^ -C H 2 O H OH OH 1. CH 3S0CH~Na 4 /CH 3 I W CH 30 O C H 3 C H ^ O C ^ N a B H ^ in THF / EtOH ( 1 : 1 ) CH^OH C H 3 0 O C H 3 C H - O A c C H - O A c I 2 l 2 H - C - O C H , H - C - O A c 3 0 - C - H H - C - O A c C H j . H - C - O A c A c O - C - H C'H2OCH3 C H 3 ( A ) (B) 1. A c i d h y d r o l y s i s 2. N a B H ^ 3. AC2O / P y r i d i n e C H 2 O A c H - C - O C H , I 3 C H J ) A c I 2 H ~ C ~ O C H 3 H - i - O A c C H 2 O A c H - C - O C H 3 H - C - O C H 3 H - C - O A c C H 3 0 - C - H . . . . O - C - H H - C - O A c C H 3 0 - 6 - H CH- jO - f J -H H - C - O A c A c O - C - H A c O - C - H C H 2 O A c ( C ) .AH. (D) C H -(E) F i g u r e II.4 M e t h y l a t i o n a n a l y s i s scheme f o r K17 p o l y s a c c h a r i d e . - 18 -An i n t e r e s t i n g o b s e r v a t i o n was made i n t h i s work d u r i n g the m e t h y l a t i o n a n a l y s i s of K17 p o l y - and o l i g o s a c c h a r i d e s . The m e t h y l a t e d c a r b o x y l groups of the p o l y s a c c h a r i d e were e a s i l y reduced w i t h sodium b o r o h y d r i d e i n anhydrous e t h a n o l - t e t r a h y d r o -furan (1:1) at room temperature. M e t h y l a t e d a c i d i c o l i g o s a c -c h a r i d e s , however, were not s u c c e s s f u l l y reduced i n t h i s manner but r e q u i r e d r e a c t i o n w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g t e t r a h y d r o f u r a n . Normally the f i r s t r e a c t i o n i s p r e f e r r e d because the l a t t e r r e s u l t s i n aluminum h y d r o x i d e being formed which tends to abosrb any m a t e r i a l which has f r e e h y d r o x y l groups p r e s e n t . T h e r e f o r e to q u a n t i t a t i v e l y r e c o v e r p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s from o l i g o s a c c h a r i d e s i t was nec e s s a r y at t i m e s to e x t r a c t the m a t e r i a l from aluminum h y d r o x i d e by the method of Dutton and Smith"^. K44 was then t r e a t e d i n the same way w i t h no problem. I I . 3 . 2 G ; l . c . a n a l y s i s of m e t h y l a t e d sugars R e v i e w s ^ ' ^  are a v a i l a b l e on the a p p l i c a t i o n of g . l . c . to the a n a l y s i s o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s . Systems of c h o i c e i n t h i s study were made to e x p l o i t the r e s o l v i n g c a p a b i l i t i e s of a c e r t a i n l i q u i d phase. One s t a t i o n a r y phase w i l l not ade q u a t e l y s e p a r a t e a l l p o s s i b l e c o m b i n a t i o n s o f methy-l a t e d s u g a r s , but by e x e r c i s i n g the r e s o l v i n g power of s e v e r a l l i q u i d s almost any m i x t u r e can be a n a l y z e d . M e t h y l a t i o n of K17 n a t i v e p o l y s a c c h a r i d e p r o v i d e d an e a s i l y s e p a r a t e d m i x t u r e of t r i - , d i - and mono-methyl ated L -r h a m n i t o l a c e t a t e s and t r i - and d i - m e t h y l ated D - g l u c i t o l a c e t a t e s (see F i g u r e 11. 5 ) . Columns c o n t a i n i n g OV-17, OS-138 - 19 -2 , 3 , 4 - R H A C o l u m n : 0 V - 1 7 ( 1 . 8 m x 3 mm) 3 % o n G a s C h r o m Q. P r o g r a m : 175° 8 m i n , 2°/min to 210°C. C a r r i e r g a s : N L . 2 0 m l / m i n . 2 , 4 - R H A I U-RHA 2,3,6-GLC II 2 , 3 - G L C l 175 ~ i — 175 ~185 i — 195 T E M P E R A T U R E (°C) F i g u r e I I . 5 ' G . l . c . s e p a r a t i o n of a m i x t u r e of p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s o b t a i n e d from Kleb s i e l l a K17 p o l y s a c c h a r i d e . - 20 -and ECNSS-M a l l gave good s e p a r a t i o n s , however 0V-17 was u s e d p r e d o m i n a n t l y b e c a u s e o f e x c e l l e n t s t a b i l i t y up t o tem-p e r a t u r e s o f 37 5 UC. A p a r t i c u l a r l y d i f f i c u l t s e p a r a t i o n between 2 , 3 , 4-1 r i - 0 - m e t h y l - 1, 5-di-0-a c e t y l - L - r h a m n i t o l and 2 , 4 - d i - 0 -m e t h y l - 3 - 0 - e t h y l - 1 , 5 - d i - 0 - a c e t y l - L - r h a m n i t o l a f t e r u r o n i c a c i d d e g r a d a t i o n e x p e r i m e n t s ( s e e s e c t i o n I I . 4 ) was p o s s i b l e o n l y by r e d u c i n g t h e i n i t i a l t e m p e r a t u r e on c o l u m n s o f 01/-17 and H I E F F - 1 B , w i t h t h e f o r m e r c h o s e n a g a i n f o r t h e above s t a t e d r e a s o n . I n t e r e s t i n g l y t h e e t h y l a t e d compound e l u t e s b e f o r e t h e o t h e r on t h e HIEFF-1B column and i n r e v e r s e o r d e r on 0U-17. F o r K44 t h e 01/-17 column c o u l d n o t r e s o l v e a l d i t o l a c e t a t e s o f 2,3,6- and 2 , 4 , 6 - t r i - O - m e t h y l - D - g l u c o s e . A medium p o l a r column of OV-225 gave good s e p a r a t i o n o f t h e s e compounds and i n r e t u r n gave o n l y a d e q u a t e s e p a r a t i o n o f d i - 0 - m e t h y l - L - r h a m n o s e i s o m e r s where OV-17 p l a y e d a b e t t e r r o l e . 3 5 3 6 P u b l i c a t i o n s by L i n d b e r g h et a l and A l b e r s h e i m et a l p r o v i d e r e l a t i v e r e t e n t i o n t i m e s f o r numerous p a r t i a l l y methy-l a t e d a l d i t o l a c e t a t e s . A l t h o u g h i d e n t i f i c a t i o n o f g . l . c . d e r i v a t i v e s i n t h e p r e s e n t s t u d y was p o s s i b l e from r e t e n t i o n t i m e d a t a a l o n e , c o n f i r m a t i o n was made by mass s p e c t r o m e t r y ( s e e s e c t i o n I I . 3 . 3 ) . Q u a n t i t a t i o n o f peak a r e a s was done a p p l y i n g r e s p o n s e f a c t o r s d e t e r m i n e d by A l b e r s h e i m e t a 1 . I I . 3 . 3 Mass s p e c t r o m e t r y o f m e t h y l a t e d s u g a r s Mass s p e c t r o m e t r y (m.s.) i s e m p l o y e d t o c o n f i r m t h e m e t h y l a t i o n p a t t e r n o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s i n d i c a t e d from g . l . c . r e c o r d i n g s and, i n p a r t i c u l a r , t h r o u g h - 2>1 -a p p l i c a t i o n of g.l.c.-m.s. t e c h n i q u e . C o n s i d e r a b l e data i s now a v a i l a b l e o n f r a g m e n t a t i o n of the s e c a r b o h y d r a t e d e r i v a t i v e s and t h i s l a b o r a t o r y a l s o keeps a supply of s t a n d a r d mass s p e c t r a on hand. 3 8 Swedish workers have made s y s t e m a t i c i n v e s t i g a t i o n s of f r a g m e n t a t i o n p a t t e r n s o f p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s . G e n e r a l i z a t i o n s made by them w i l l be i l l u s t r a t e d by examples i n t h i s s t u d y . U r o n i c a c i d d e g r a d a t i o n and subsequent e t h y l a t i o n p r o v i d e d di-O-methy1-0-ethy 1-L-rhamnose isomers from K17 and K44 m e t h y l a t e d p o l y s a c c h a r i d e s . F i r s t of a l l i t s h o u l d be mentioned t h a t mass s p e c t r o m e t r y w i l l not d i s t i n g u i s h between d i a s t e r e o m e r i c p a r t i a l l y methy-l a t e d a l d i t o l a c e t a t e s . T h e r e f o r e , these d e r i v a t i v e s of 2,3,4-t r i - 0 - m e t h y 1-L-rhamnose (6-deoxy-L-mannose) and 2 , 3 , 4 - t r i - O -m e t h y l - L - f u c o s e ( 6 - d e o x y - L - g a l a c t o s e ) g i v e e s s e n t i a l l y the same mass spectrum. E l e c t r o n impact m.s. does not y i e l d a m o l e c u l a r ion peak, r a t h e r , primary fragments occur by f i s s i o n between carbon atoms. Cleavage of carbon-carbon bonds a d j a c e n t to me t h o x y l a t e d carbons i s p r e f e r r e d to those a d j a c e n t to a c e t o x y l a t e d carbons, as shown below. — g — O C H 3 H C OCH, •l 3 - 22 -Secondary fragments then o r i g i n a t e from primary f r a g -ments by l o s s of one or more of the f o l l o w i n g : formaldehyde (M.W.30), methanol (M.W.32), ketene (M.W.42), or a c e t i c a c i d (M . W . 60 ) . When s t a n d a r d compounds are u n a v a i l a b l e , as may occur i n u r o n i c a c i d d e g r a d a t i o n s t u d i e s , mass s p e c t r a may be i n t e r p r e t e d u s i n g t h i s i n f o r m a t i o n . Thus, one would expect to observe s t r o n g peaks from primary fragments m/el31 and m/el75 of the 2 - 0 - e t h y l isomer. Although these fragments are a l s o p r e s e n t i n the mass spectrum of the 3 - 0 - e t h y l isomer, appea-rance of a d d i t i o n a l peaks at m/ e l l 7 and m/el89 s e r v e s to d i s t i n g u i s h these compounds. Primary f r a g m e n t a t i o n i s shown below. Mass s p e c t r a of these compounds i s demonstrated i n F i g u r e I I . 6 . H oC-0Ac HC-0CH oChU 131 hUC-OAc 175 HC-0CH„CH, 139 I 2 3 HC-OCH 175 175 131 ChUO-CH 3 i 131 AcO-CH AcO-CH CH 3 - 23 -(a) 3 - 0 - e t h y l - 2 ^ - d i - 0 - m e t h y l 4 5 - d i - 0 - a c e t y l - L - r h a m n i t o l 754' R E L . INT. 50H % 2 5 ^ 1 ^njtni I ijimjiXf I ' llll|llll|IUl|u muiiyn|«it^ inHim['i|H»"['i' ntfwyntniiimt""!" li|l'liyiii|iirt|B>t|*iii|«i»|iiii|iiii[iiiipiii|iiH|"iittiii|»i»|Hi<|iiii|iiiijnntwmii inuiinni[iiii|iiit| im[iJii|i] 11^ 50 100 1 5 0 2 0 0 2 5 0 100 - 1 7 5 ^ 5 0 H 2 5 -(b) 2 - 0 - e t h y l - 3 . 4 - d i - 0 - m e t h y l - 1 , 5 - d i - 0 - a c e t y l - L - r h a m n i t o l ii|i»|iii|ii^iiinuinn>oiiiipii 5 0 100 150 2 0 0 2 5 0 M / E F i g u r e I I . 6 Mass s p e c t r a of u r o n i c a c i d d e g r a d a t i o n d e r i v a t i v e s from K17(a) and K44(b). - 24 -I I . 3 . 4 "'"H n.m.r. s p e c t r o s c o p y of m e t h y l a t e d p o l y s a c c h a r i d e s and o l i g o s a c c h a r i d e s Some r e s e a r c h has been done on permethy1 ated and p a r t i a l l y m e t h y l a t e d monosaccharides and d i s a c c h a r i d e s ^ ' ^ and on perme-t h y l a t e d p o l y s a c c h a r i d e s of algaef"'"'^ however, g e n e r a l l y , i n f o r m a t i o n on me t h y l a t e d b a c t e r i a l p o l y s a c c h a r i d e s i s s c a r c e . P r e l i m i n a r y o b s e r v a t i o n s i n t h i s study i n d i c a t e a p r o m i s i n g f u t u r e f o r n.m.r. s p e c t r o s c o p y of m e t h y l a t e d p o l y - and o l i g o -s a c c h a r i d e s . Some advantages are t h a t by employing o r g a n i c s o l v e n t s , such as d e u t e r a t e d c h l o r o f o r m or a c e t o n i t r i l e , i n t e r -f e r e n c e of the k i n d from the l a r g e HOD peak e x p e r i e n c e d i n "'"H n.m.r. s p e c t r a o f n a t i v e p o l y s a c c h a r i d e s i s not a problem. Loss of homogeneity due to high v i s c o s i t y as i n water s o l u b l e n a t i v e p o l y s a c c h a r i d e s poses no b a r r i e r f o r m e t h y l a t e d m a t e r i a l . Q u a n t i t i e s used were i n the range of 25 mg f o r m e t h y l a t e d p o l y s a c c h a r i d e s and l e s s f o r me t h y l a t e d o l i g o s a c c h a r i d e s . One p o i n t to remember i s t h a t samples must be p u r i f i e d i n o r d e r to o b t a i n r e l i a b l e r e s u l t s . D i a l y s i s i n the case of met h y l a t e d p o l y s a c c h a r i d e s and c h l o r o f o r m e x t r a c t i o n f o r met h y l a t e d o l i g o s a c c h a r i d e s y i e l d e d l i g h t brown p r o d u c t s i n -stead of pure white samples. By p a s s i n g impure p r o d u c t s through a s m a l l s i l i c a g e l column u s i n g a s o l v e n t m i x t u r e , f o r i n s t a n c e c h l o r o f o r m - m e t h a n o l ( 9 5 : 5 ) , determined i n i t i a l l y from v a l u e s on a t h i n l a y e r chromatographic p l a t e , s h o u l d p r o v i d e s u f f i c i e n t pu r i f i c a t i o n . I I . 4 Sugar Sequence D e t e r m i n a t i o n by U r o n i c A c i d D e g r a d a t i o n S p e c i f i c d e g r a d a t i o n of p o l y s a c c h a r i d e s has been an area - 25 -43 44 of keen i n t e r e s t r e c e n t l y . ' F u n c t i o n a l l y - s e l e c t i v e methods, such as u r o n i c a c i d d e g r a d a t i o n employed i n t h i s study, p r o v i d e 45 s o l u t i o n s t o d i f f i c u l t s t r u c t u r a l problems. Some advantages a s s o c i a t e d w i t h these s p e c i f i c e l i m i n a t i o n s a r e , r e a c t i o n s are quick and easy t o perform, the u r o n i c a c i d moiety n o r m a l l y d i f f i c u l t t o degrade i s d e s t r o y e d , and s p e c i f i c c l e a v a g e s t h a t r e s u l t g i v e e a s i l y ^ i n t e r p r e t e d sequence i n f o r m a t i o n . The b a s i s f o r c l e a v a g e i s o u t l i n e d as below. COOCH COOCH-3 )~0R1 •+ R V 1 rs 3 2 3 OCH/O^COOCH £ = 0 R Z 0H ,R J 0H + Y J i H / 3 yH0 + R1QH ( I V ) ( I I I ) OR' M e t h y l a t e d p o l y s a c c h a r i d e s from K17 and K44 were t r e a t e d w i t h sodium m e t h y l s u l f i n y l m e t h a n i d e a c t i n g as base i n d i m e t h y l -45 s u l f o x i d e . Normally the L i n d b e r g method c a l l s f o r m i l d a c i d i c t reatment t o e f f e c t f i n a l d e g r a d a t i o n of the hex-4-eno-pyranosy1-u r o n i c r e s i d u e ( I I ) . However, A s p i n a l l et a l have r e p o r t e d ^ t h a t treatment w i t h the above base r e s u l t s i n complete l o s s of the h e x u r o n i c a c i d r e s i d u e ( I I ) and subsequent a c i d h y d r o l y s i s i s unnecessary. These workers a l s o submit t h a t i n a " s i n g l e 45 o p e r a t i o n " , m e t h y l a t e d p o l y s a c c h a r i d e i s t r e a t e d w i t h base - 26 -and t h e n , w i t h o u t i s o l a t i o n , a l k y l a t e d w i t h e t h y l i o d i d e to l a b e l the s i t e ( s ) t o which u r o n i c a c i d r e s i d u e s were a t t a c h e d . These m o d i f i c a t i o n s were a p p l i e d w i t h s u c c e s s i n the present i n v e st i g at ion . I t s h ould be mentioned t h a t f u r t h e r d e g r a d a t i o n can t a k e p l a c e i f the s u b s t i t u e n t on C-4 of the u r o n i c a c i d component i s another sugar. When the a c i d i c sugar i s e l i m i n a t e d the r e s u l t i n g r e d u c i n g end i s a l s o s e n s i t i v e t o the b a s i c environment and i n a c h a i n - t y p e r e a c t i o n e l i m i n a t e s the C-3 s u b s t i t u e n t which, i f i t happens t o be another sugar, c o n t i n u e s t o degrade the p o l y s a c c h a r i d e . Exposed r e d u c i n g sugars can be p r o t e c t e d from f u r t h e r d e g r a d a t i o n by a c e t y l a t i o n when the e l i m i n a t i o n i s performed i n the presence of a c e t i c a n h y d r i d e . ^ P r o d u c t s from both s i d e s of the bond c l e a v a g e can thus be i d e n t i f i e d . However, when a b a s e - s t a b l e u n i t , such as 3-deoxy-hex-2-enopyranose, ( r e s i d u e A on page 27) i s formed f u r t h e r r e -a c t i o n w i l l not o c c u r . These a c i d - l a b i l e u n i t s are d e s t r o y e d d u r i n g the a n a l y s i s of m e t h y l a t e d sugars and d i s a p p e a r a n c e of such a c o n s t i t u e n t p r o v i d e s e v i d e n c e f o r p l a c i n g i t i n sequence immediately a d j a c e n t t o the degraded hexuronate r e s i d u e . F i g u r e I I . 7 i l l u s t r a t e s t h e s e p o i n t s f o r the 8 - e l i m i n a t i o n r e a c t i o n of K44. I I . 5 Sugar Sequence D e t e r m i n a t i o n from P a r t i a l H y d r o l y s i s P a r t i a l or graded a c i d h y d r o l y s i s i s a well-known method f o r the d e t e r m i n a t i o n of sugar sequence and anomeric c o n f i g u r a -t i o n of p o l y s a c c h a r i d e s . O l i g o s a c c h a r i d e s , produced under c e r t a i n c o n d i t i o n s of h y d r o l y s i s ( a c i d c o n c e n t r a t i o n , temperature 27 ^ G l c ^ G t c L - ^ G l c A 1 — ^ R h a l — ^ R h a 1 — (X j9 cx c* CH 9OCH •o CH-0CH--o. OCH CH?OCH~ -o OCH OCH. CH 2 OCH 3 COOCH, CH.O/—0 OCH 3 CH 30 1. B A S E 2. Ethyl Iodide OCH 2 CH 3 C H J V T ~ ° 3/cH 3 CH_0/T~0 0 T H 3 OCK CH 30 0CH 2 CH 3 OCH. H + 2-0-ethyl-3.£-di-0-methyl-L-rhamnose 2,4-di-O-methyl-L-rhamnose 2A6-tri-0-methyl-D-glucose F i g u r e I I . 7 U r o n i c a c i d d e g r a d a t i o n poly s a c c h a r i d e . of K l e b s i e l l a K44 - 28 -and l e n g t h of h y d r o l y s i s ) , can be c h a r a c t e r i z e d by m e t h y l a t i o n and n.m.r. s t u d i e s i n orde r to e l u c i d a t e the more complex s t r u c t u r e of the t o t a l m o l e c u l e . T h i s i s e s p e c i a l l y so i n work w i t h K l e b s i e l l a where the r e p e a t i n g u n i t may be e s t a b -l i s h e d by i s o l a t i n g only two or t h r e e o l i g o s a c c h a r i d e s . H y d r o l y s i s of p o l y s a c c h a r i d e s i s a f f e c t e d by the type, r i n g s i z e and co n f o r m a t i o n of the monosaccharide r e s i d u e s , the anomeric c o n f i g u r a t i o n of the g l y c o s i d i c l i n k a g e s , the p o s i t i o n of l i n k a g e such as ( (1*2 ) , (1*3) , (1*4) and ( 1 * 6 ) ) , the presence of f u n c t i o n a l groups, and the i n t e n s i t y of i n t e r - and i n t r a -m o l e c u l a r i n t e r a c t i o n s . Rate c o n s t a n t s f o r the a c i d c a t a l y z e d 48 h y d r o l y s i s of a l a r g e v a r i e t y of g l y c o s i d e s have been rev i e w e d , a l t h o u g h data on p o l y s a c c h a r i d e s are mainly u n p u b l i s h e d and e m p i r i c a l . I n f e r e n c e s can be made, however, and are g e n e r a l l y a c c e p t e d For i n s t a n c e , the g l y c o s i d i c bond of u r o n i c a c i d i s e x t r e m e l y r e s i s t a n t to h y d r o l y s i s so t h a t i n the m a j o r i t y of K l e b s i e l l a s t r u c t u r a l s t u d i e s a l d o b i o u r o n i c a c i d and u s u a l l y a l d o t r i o u r o n i c a c i d have been i s o l a t e d . A g l y c o s i d i c l i n k a g e at a non-r e d u c i n g end i s h y d r o l y z e d more r a p i d l y than o t h e r s . T h i s f a c t i s made use of i n a n a l y s i s of K17 where the s i n g l e u n i t L-rhamnose s i d e chain was c l e a v e d by m i l d a c i d i c c o n d i t i o n s to produce the s t r a i g h t chain p o l y s a c c h a r i d e "backbone". The d i s a d v a n t a g e of p a r t i a l h y d r o l y s i s i s t h a t very poor y i e l d s of o l i g o s a c c h a r i d e s are r e a l i z e d . In an attempt to produc b e t t e r y i e l d s a m o d i f i e d a p p a r a t u s s i m i l a r to t h a t used by Galanos et a l " ^ was employed here. The p r i n c i p l e of c o n t i n u o u s - 29 -r e m o v a l o f o l i g o s a c c h a r i d e s from s o l u t i o n to p r e v e n t t o t a l h y d r o l y s i s to m o n o s a c c h a r i d e s i s the b a s i c t h e o r y . The c h a r -c o a l column i s removed i n our a p p a r a t u s s i n c e i t i s f e l t s u i -t a b l e m o n o s a c c h a r i d e se pa r a t i o n ; can be a c c o m p l i s h e d t h r o u g h s u b s e q u e n t g e l f i l t r a t i o n c h r o m a t o g r a p h y . In f a c t , when t h i s a p p a r a t u s was a p p l i e d to the p a r t i a l h y d r o l y s i s of K17, t w i c e the amount o f a c i d i c t e t r a s a c c h a r i d e (90 mg) was g e n e r a t e d as a l d o b i o u r o n i c a c i d (45 mg) from one gram of p o l y s a c c h a r i d e . In o r d e r to p u r i f y o l i g o s a c c h a r i d e s o b t a i n e d from p a r t i a l h y d r o l y s i s a number of s t a n d a r d c h r o m a t o g r a p h i c t e c h n i -ques were employed. The m a t e r i a l p r o d u c e d from t h e h y d r o l y s i s a p p a r a t u s was f i r s t s e p a r a t e d i n t o a c i d i c and n e u t r a l p o r t i o n s by i o n exchange c h r o m a t o g r a p h y , Dowex 1-X2 i n the c h l o r i d e or f o r m a t e form. These p o r t i o n s were then i n d i v i d u a l l y s e p a r a t e d a c c o r d i n g to m o l e c u l a r s i z e by g e l f i l t r a t i o n c h r o m a t o g r a p h y on a column o f B i o - G e l P-2. Two m i l l i l i t e r f r a c t i o n s c o l l e c t e d from the m o l e c u l a r s i e v e were, a f t e r 1 y o p h i l i z a t i o n , m o n i t o r e d by p a p e r c h r o m a t o g r a p h y . I I . 6 P e r i o d a t e O x i d a t i o n The term " c l a s s i c a l method" c o u l d w e l l be u s e d to d e s c r i b p e r i o d a t e o x i d a t i o n i n t h e a r e a of p o l y s a c c h a r i d e s t r u c t u r a l i n v e s t i g a t i o n s . " ^ Aqueous s o l u t i o n s of p e r i o d i c a c i d and i t s s a l t s ( g e n e r a l l y sodium) w i l l c l e a v e t h e bond between a 1,2-d i o l system as shown on page 30. T h i s r e a c t i o n i s w e l l s u i t e d to t h e w a t e r - s o l u b l e c a r b o h y d r a t e s which a r e p o l y h y d r o x y l i c by n a t u r e and whose c y c l i c 1 , 2 - d i o l s t r u c t u r e s would then be o x i d i z e d t o a s t r a i g h t c h a i n - 30 -+ CHOH CHOH HC = 0 + R R a, oj-d i a l dehy de . The p o l y a l d e h y d e p r o d u c t f r o m t h e c a r b o h y -d r a t e p o l y m e r i s r e d u c e d w i t h s odium b o r o h y d r i d e t o y i e l d t h e p o l y o l f o r ease o f h a n d l i n g . The u s e s o f p e r i o d a t e o x i d a t i o n a r e t w o f o l d . One i s an a n a l y t i c a l t e c h n i q u e u s i n g s m a l l amounts o f m a t e r i a l and d i l u t e p e r i o d a t e s o l u t i o n s t o m a i n t a i n s e l e c t i v e o x i d a t i o n . I n f o r m a t i o n on t h e number o f p e r i o d a t e s e n s i t i v e s u g a r s i n a p o l y s a c c h a r i d e can be d e t e r m i n e d (one mole o f p e r i o d a t e i s consumed p e r o x i d i -z a b l e s u g a r i n t h e r e p e a t i n g u n i t , e x c e p t f o r t h o s e w i t h 1-6 l i n k a g e s , and two m o l e s i n t h e c a s e o f a n o n - r e d u c i n g t e r m i n a l s u g a r and 1-6 1 i n ked , i i n - ch a i n s u g a r s ) T h i s y i e l d s c o n f i r m -a t o r y d a t a on l i n k a g e s i t e s t o t h a t f o u n d i n m e t h y l a t i o n a n a l y s i s . S e c o n d l y and more i m p o r t a n t i s a p r e p a r a t i v e o r s y n t h e t i c t e c h n i q u e done w i t h l a r g e r q u a n t i t i e s w i t h t h e aim o f i s o l a t i n g o l i g o s a c c h a r i d e f r a g m e n t s composed o f p e r i o d a t e 52 i n s e n s i t i v e s u g a r c o m p o n e n t s . S m i t h d e g r a d a t i o n i s em p l o y e d t o l i b e r a t e o l i g o m e r i c u n i t s . T h i s method t a k e s a d v a n t a g e o f t h e more a c i d l a b i l e a c e t a l l i n k a g e s r e s u l t i n g f r o m t h e o x i -d i z e d s u g a r r e s i d u e s compared t o t h e more s t a b l e i n t a c t - 31 -— G l c ~ ~ G lc 1—^ Glc A 1 — ? Rha 1—? Rha 1 — C H 2 O H C H 2 O H * = site of periodate attack H O ~ O H "polysaccharide" C H 2 O H C H 2 O H C O O H O H II || II II V II - * ^ 0 C H ? O H 0 0 C H 2 O H 0. 0 0 B H ; C O O H J W O H 0 "polyaldehyde" H O ' I H~C C H 0 HJC d OH 2 I I 2 2 I I HO OH " h~0. H O / ~ 0 HO / dT^s ^ C H 3 V - O 2| HO OH C H . H O Smith degradation O H 'polyalcohol' m ^ T ^ o — r t - 6H'OH H O O H O H ^ C H "oligomer' 2 x glycolaldehyde 1.2-propanediol C H 2 0 H glyceraldehyde D-erythronic acid F i g u r e I I . 8 P e r i o d a t e - S m i t h d e g r a d a t i o n of K l e b s i e l l a K44 p o l y s a c c h a r i d e . - 32 -g l y c o s i d i c l i n k a g e s o f t h e n o n o x i d i z e d o n e s . F i g u r e I I . 8 d e m o n s t r a t e s how t h i s s y n t h e t i c t e c h n i q u e was u s e d i n t h e p e r i o d a t e o x i d a t i o n o f K44. A n o t h e r i n t e r e s t i n g a p p r o a c h t o p e r i o d a t e o x i d a t i o n a c h i e v e d i n t h i s s t u d y i s t h e a p p l i c a t i o n o f s e l e c t i v e S m i t h d e g r a d a t i o n . As i n d i c a t e d i n a r e c e n t c o m m u n i c a t i o n f r o m P a i n t e r e t a l " ^ c e r t a i n s u g a r r e s i d u e s a r e more s u s c e p t i b l e t o p e r i o d a t e a t t a c k t h a n o t h e r s . T h i s can be shown q u a n t i t a -t i v e l y by c o m p a r i n g t h e s e c o n d - o r d e r r a t e c o n s t a n t s f o r p e r i o -d a t e o x i d a t i o n o f m e t h y l g l y c o p y r a n o s i d e s . F o r e x a m p l e , t h e r a t e c o n s t a n t f o r m e t h y l a - L - r h a m n o p y r a n o s i d e i s s e v e n t i m e s t h a t o f m e t h y l 3 - D - g l u c o p y r a n o s i d e . A l s o t h e f a c t t h a t a c i d i c p o l y s a c c h a r i d e s show an i n h i b i t i v e e f f e c t b e c a u s e o f e l e c t r o -s t a t i c r e p u l s i o n b e t w e e n t h e u r o n a t e a n i o n and t h e a t t a c k i n g 54 p e r i o d a t e i o n a d d s v e r s a t i l i t y t o p e r i o d a t e o x i d a t i o n s . F o r K17j s o d i u m p e r c h l d r a t e was u s e d t o s u p p r e s s t h i s r e p u l s i o n and p r e v e n t u n d e r - o x i d a t i o n i n t h e " t o t a l " o x i d a t i o n o f t h e p o l y m e r ( s e e e x p e r i m e n t a l f o r K17 page 6-8' ) . H o w e v e r , no s a l t was added i n t h e e x p e r i m e n t t o s e l e c t i v e l y o x i d i z e t h e r h a m n o s e s i d e - c h a i n o f K17 i n t h e p r e s e n c e o f o t h e r o x i d i z a b l e u n i t s . ( S e e F i g u r e I I . 9 , p a g e 3 3 ) . T h i s p a r t i c u l a r e x p e r i m e n t was a l s o a i d e d by t h e f a c t t h a t t h e t a r g e t f o r p e r i o d a t e a t t a c k was a more e a s i l y a c c e s s i b l e s i d e - c h a i n t e r m i n a l s u g a r r a t h e r t h a n an " i n -c h a i n " r e s i d u e . H o w e v e r , t h e s u c c e s s o f t h e s e l e c t i v e S m i t h d e g r a d a t i o n c o n c l u s i v e l y y i e l d e d t h e s i d e - c h a i n l i n k a g e s i t e on t h e b r a n c h i n g s u g a r a f t e r s u b s e q u e n t m e t h y l a t i o n a n a l y s i s , w h e r e s e v e r a l a t t e m p t s a t p a r t i a l h y d r o l y s i s had f a i l e d . - ^G lc 1 —^Rha 1-r^GlcA 1 - ^RhJ CK CX Rha CH 20H ~0 * most vulnerable site for periodate attack HO* OH 1. 0.027 M 10". 2 h 2. BH7 H 2 t C H 2 HO OH 3. 0.5 M trifluoroacetic acid, •17 h, R.T. 2,3,5-tri -O-methylglucose 3,4-di-O-methylrhamnose OH U. methylation 5. carboxyl reduction 6. hydrolysis 2.3- di-O-methylglucose 2.4- di-O-methylrhamnose gu r e I I . 9 S e l e c t i v e p e r i o d a t e -K17 p o l y s a c c h a r i d e . S m i t h d e g r a d a t i o n o f - 34 -11.7 Anomeric Linkage D e t e r m i n a t i o n The assignment of anomeric c o n f i g u r a t i o n to s p e c i f i c g l y c o s i d i c l i n k a g e s i n a p o l y s a c c h a r i d e i s acc o m p l i s h e d through the s i m p l e r n u c l e a r magnetic resonance s p e c t r a of t h e i r o l i -1 13 gomeric s u b u n i t s . The t e c h n i q u e of H and C n.m.r. has been d i s c u s s e d i n terms o f p o l y s a c c h a r i d e s i n S e c t i o n I I . 2 . 2 . In t h i s i n v e s t i g a t i o n "*"H n.m.r. and "^C n.m.r. s p e c t r a were o b t a i n e d f o r o l i g o s a c c h a r i d e s from p a r t i a l a c i d h y d r o l y s i s (Section I I . 5 ) and "*"H n.m.r. s p e c t r a were o b t a i n e d f o r methy-l a t e d o l i g o s a c c h a r i d e s from s p e c i f i c d e g r a d a t i o n ( S e c t i o n I I . 4 ) . The c l a s s i c a l t e c h n i q u e of o p t i c a l r o t a t i o n measurement has l o n g been c o n s i d e r e d the f r i e n d of c a r b o h y d r a t e c h e m i s t s . T h i s i n f o r m a t i o n w i l l g i v e i n d i c a t i o n s of the o v e r a l l anomeric c o n f i g u r a t i o n of a p o l y s a c c h a r i d e but no d e t a i l c o n c e r n i n g the i n d i v i d u a l sugar r e s i d u e s . A s e r i e s of o l i g o s a c c h a r i d e s would a i d i n t h i s r e g a r d but u n d e r s t a n d a b l y t h i s method cannot compete w i t h the wealth of i n f o r m a t i o n i n a n u c l e a r magnetic resonance experiment. (See Table I I . 1 , page 35). 11.8 D e t e r m i n a t i o n of C o n f i g u r a t i o n of Component Sugars The D and L c o n f i g u r a t i o n s of component sugars are d e t e r -mined sim p l y and r o u t i n e l y by c i r c u l a r d i c h r o i s m measurements. ^ Use i s made of d e r i v a t i v e s a l r e a d y a v a i l a b l e from sugar q u a n t i -t a t i o n s t u d i e s ( a l d i t o l a c e t a t e s ) or m e t h y l a t i o n s t u d i e s ( p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s ) . These v o l a t i l e g . l . c . d e r i v a t i v e s are c o n v e n i e n t l y c o l l e c t e d as they are s e p a r a t e d and e l u t e d from a s u i t a b l e column. ( S e c t i o n s I I . 3 . 2 and I I . 2 . 1 ) ; TABLE I I . 1 C a l c u l a t e d and M e a s u r e d V a l u e s f o r S p e c i f i c R o t a t i o n s o f K l e b s i e l l a K17 and K44 P o l y - and O l i g o s a c c h a r i d e s Compound O p t i c a l R o t a t i o n a t S p e c i f i c C o n e 3 W a v e l e n g t h (nm) K „ A " Na589 FTTgTTS FTg54~S PTgTTJ^ FT7gT63 j j c ^ m e a s . [ a ] D c a l c ? A K17 c a p s u l a r p o l y s a c c h a r i d e ( N a + ) 0.57 +0.17 +0.18 +0.20 +0.33 +0.48 + 30 + 15 +15 p o l y s a c c h a r i d e P I 6 1.81 +0.80 +0.83 +0.94 +1.55 +2.27 +444 + 34 +10 t e t r a s a c c h a r i d e 1 . 38 +0 . 52 +0.55 +0 . 62 +1.02 +1.54 + 38 + 14 +24 a l d o b i o u r o n i c a c i d 1.12 +1.24 +1.30 +1.46 +2.43 +3.69 +111 + 77 +34 d i s a c c h a r i d e 0.82 +0.03 +0.03 +3.7 -1Z5 +16 c a p s u l a r p o l y s a c c h a r i d e ( N a + ) 1 .08 +0 . 043 +0 . 0 48 +0. 0 58 +0 . 080 + 4.0 - 8 + 12 c a p s u l a r p o l y s a c c h a r i d e 1 .23 +0 . 10 +0. 11 +0 . 12 +0. 18 +0 . 21 +8 . 1 - 8 + 16 p e n t a s a c c h a r i d e 1 .95 + 0 . 48 +0 . 50 + 0 . 56 + 0 . 90 + 1. 22 + 25 + 7 + 18 f t e t r a s a c c h a r i d e 1 . 53 +0 . 62 + 0 . 65 +0 . 73 + 1. 19 + 1. 74 + 41 + 24 + 17 t r i s a c c h a r i d e ^ 1 . 44 -0 . 27 -0. 28 -0 . 31 -0 . 53 -0. 77 -19 - 31 + 12 a l d o b i o u r o n i c a c i d 0 . 46 -0 . 032 -0 . 032 -0 . 035 -0 . 0 62 -0. 11 -7.0 - 14 + 7 d i s a c c h a r i d e ^ 0 .87 -0 . 38 -0 . 40 -0. 74 -44 - 48 + 4 C o n c e n t r a t i o n i n g/100 ml w a t e r . M e a s u r e d s p e c i f i c r o t a t i o n ? d e g r e e s . c C a l c u l a t e d s p e c i f i c - d e r o t a t i o n , d e g r e e s . D i f f e r e n c e between measured and c a l c u l a t e d s p e c i f i c r o t a t i o n s . P a r t i a l l y d e p o l y m e r i z e d ( 0 . 4 M t r i f l u o r o a c e t i c a c i d , 20 m i n , 95°C). Not p u r e s a m p l e s , s e e e x p e r i m e n t a l page 9;8.( ^From p e r i o d a t e o x i d a t i o n ; c a l c u l a t e d v a l u e does n o t t a k e e r y t h r i t o l i n t o a c c o u n t . - 3 6 -Q u a n t i t i e s r e q u i r e d a r e o f t h e o r d e r o f 1 mg and t h e t e c h n i q u e i s n o n d e s t r u c t i v e . 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STRUCTURAL INVESTIGATION OF K l e b s i e l l a SEROTYPE K17 CAPSULAR POLYSACCHARIDE - 41 -I I I . 1 A b s t r a c t K l e b s i e l l a K17 c a p s u l a r p o l y s a c c h a r i d e has been i n v e s t i -g ated by the t e c h n i q u e s of m e t h y l a t i o n , p a r t i a l h y d r o l y s i s , p e r i o d a t e o x i d a t i o n , and u r o n i c a c i d d e g r a d a t i o n . In a d d i t i o n , the concept of s e l e c t i v e p e r i o d a t e o x i d a t i o n - S m i t h d e g r a d a t i o n was employed to remove a rhamnose s i d e c h a i n . The s t r u c t u r e was found to c o n s i s t of the p e n t a s a c c h a r i d e r e p e a t i n g u n i t shown below. Anomeric c o n f i g u r a t i o n s of the sugar r e s i d u e s were determined by "'"H and X^C n.m.r. s p e c t r o s c o p y on the p o l y s a c c h a r i d e and d e r i v e d p o l y - and o l i g o s a c c h a r i d e s o b t a i n e d through d e g r a d a t i v e p r o c e d u r e s . _ B-D-Glcp-(l~-2)- a-L-Rhap-( 1*4) - O-D-G 1 cAp-( 1*3) - 6-L-R hap-( 1* t 3 1 a-L-Rhap - 42 -111.2 In trodu c t i o n Of the a p p r o x i m a t e l y 80 s e r o l o g i c a l l y d i f f e r e n t 1 2 s t r a i n s of K l e b s i e l l a b a c t e r i a t h a t have been grouped ' a c c o r d i n g to the q u a l i t a t i v e c o m p o s i t i o n of t h e i r c a p s u l a r p o l y s a c c h a r i d e s , a s m a l l group of these s e r o t y p e s c o n s i s t s of the sugars D - g l u c u r o n i c a c i d , D-glucose, and L-rhamnose. At f i r s t t h o u g h t to i n c l u d e o n l y K17 and K44, r e c e n t f i n d i n g s have 3 4 shown t h a t K23 and K45 are p a r t of t h i s group a l s o . Each c a p s u l a r p o l y s a c c h a r i d e has a unique s t r u c t u r e , however, which determines i t s s e r o l o g i c a l s p e c i f i c i t y . We now r e p o r t on the s t r u c t u r e of the c a p s u l a r p o l y s a c c h a r i d e of type K17. The p r e d i c t i o n , ^ ' ^ from immunochemical r e l a t i o n s h i p s by c r o s s -r e a c t i o n s of K l e b s i e l l a i n a n t i p n e u m o c o c c a l , a n t i - S a l m o n e l l a , and a n t i - K l e b s i e l l a s e r a , t h a t K17 c a p s u l a r p o l y s a c c h a r i d e would have a n o n r e d u c i n g L-rhamnose end-group has been con f i r m e d by t h i s s t u d y . - 43 -111 . 3 R e s u l t s and D i s c u s s i o n  Composition and n.m.r. s p e c t r a K l e b s i e l l a K17 was grown on an agar medium; the cap-s u l a r p o l y s a c c h a r i d e was i s o l a t e d by u 1 t r a c e n t r i f u g a t i o n and p u r i f i e d by C e t a v l o n p r e c i p i t a t i o n . The p u r i f i e d m a t e r i a l moved as one band d u r i n g e l e c t r o p h o r e s i s and had C°G Q +30° . The m o l e c u l a r weight of the p o l y s a c c h a r i d e was determined by g e l chromatography to be 9.4 x 10"'. An a c i d h y d r o l y z a t e of the c a r b o x y l - r e d u c e d ^ p o l y s a c -c h a r i d e c o n t a i n e d rhamnose and g l u c o s e i n the r e l a t i v e pro-p o r t i o n s 3:2, a n a l y z e d as t h e i r a l d i t o l a c e t a t e s by g a s - l i q u i d chromatography ( g . l . c ) . Rhamnose was conf i r m e d to be of the L c o n f i g u r a t i o n and g l u c o s e of the D c o n f i g u r a t i o n by c i r c u l a r 8 d i c h r o i s m ( c d . ) measurement on t h e i r a l d i t o l a c e t a t e s . The "^C n.m.r. spectrum i n d i c a t e s t h a t the p o l y s a c c h a r i has f i v e anomeric carbon atoms w i t h s i g n a l s at 105.0, 103.2, 101.3, 100.8 and 96.4 p.p.m. The s i g n a l at 61.5 p.p.m., a t t r i b u t a b l e to C-6 of g l u c o s e , i n d i c a t e s t h a t the g l u c o s e i s not l i n k e d t o oth e r sugar r e s i d u e s at the 6 p o s i t i o n s i n c e l i n k a g e would r e s u l t i n a s i g n i f i c a n t d o w n f i e l d s h i f t of the 9 C-6 s i g n a l . The r e l a t i v e l y l a r g e s i g n a l at 17.4 p.p.m. can be a s s i g n e d to 0-6 of rhamnose (CH^) i n d i c a t i n g t h a t more than one rhamnose r e s i d u e i s p r e s e n t i n the r e p e a t i n g u n i t of K17. F i n a l l y , the s i g n a l at 175 p.p.m. was due to the c a r b o x y l group of g l u c u r o n i c a c i d . Assignment of the anomeric s i g n a l s was made a f t e r e x a m i n a t i o n of the X^C n.m.r. s p e c t r a of d e r i v e d p o l y - and o l i g o s a c c h a r i d e s (see Table I I I . l p a g e 4 4 ) . TABLE I I I . l N.m.r. data of K l e b s i e l l a K17 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d p o l y -and o l i g o s a c c h a r i d e s C ompoun d . 1 _C 6 (J-, 9 ) , i n t e g r a l , assignment p.p.m. assignment 1 > ^  G lc ^ - R h a - O H p GlcA^-^Rha-OH a Gl c^-^R h a^—-Gl cA-^—R h a-OH D Oo LX 5. 36 (1), 0.5, H-l a-Rha-OH 105. 1 C-1 B - G l c 4. 84 <T<1), 0.5, H-l B-Rha-OH 93. 7 C-1 aY6-Rha-4. 63 (7), 1, H-l 3-Glc 61. 4 C-6 G l c 1. 28 (J , , 6 ) , 3, H-6 Rha 17. 6 C-6 Rha 5. 17 ( b ) , 1.6, H-l a-GlcA and 96. 7 C-1 a-Rha-OH H-l a-Rha-OH 96. 3 C-1 B-Rha-OH 4. 87 ( b ) , 0.4, H-l B-Rha-OH 94. 4 C-1 a - G l c A 1. 33 ( J c .6), 3, H-6 Rha 5, 6 17 . 7 C-6 Rha 5. 17 ( b ) , 1.5, H-l a-GlcA and 105. 0 C-1 B - G l c H-l a-Rha-OH 100. 5 C-1 a-Rha 5. 11 (2), 1, H-l a-Rha 96. 6 C-1 a-Rha-OH 4. 87 (5), 0.5, H-l B-Rha-OH 96. 2 C-1 B-Rha-OH 4. 57 (7-8), 1, H-l B-Glc 94. 4 C-1 a-Gl cA 1. 31 ( J . , 6 ) , 6, H-6 Rha 61. 4 C-6 Glc 5, 6 17. 3 C-6 Rha TABLE I I I . l ( c o n t i n u e d ) Compound 1^ 13^, 6 a ( J ^ i n t e g r a l , assignment p.p.m.6 assignment 4 1 2 1 4 1 3 1 1 C H A V ° 1 C A H A V P l a 5.17 ( 3 ) , 1, H-l a-GlcA 104. 8 C-1 B-Gl c 5.11 ( s ) , 1, H-l a-Rha 101. 3 C-1 B-Rha 4.87 ( s ) , 1, H-l B-Rha 100 . 3 C-1 a-Rha 4. 56 (7 . 5 ) , 1, H--1 B-Glc 96. 1 C-1 a-GlcA 1.34 ( 6 ) , 3, H-6 Rha 61. 4 C-6 of G l c 1.27 ( 6 ) , . 3 , H-6 'Rha 17. 5 C-6 of Rha Un 4 1 2 1 4 1 3 1 -^G1 c^r^R h a^-^G 1 cA^^R h a ^ — • a a B a B 1 Rha c a p s u l a r p o l y s a c c h a r i d e 5.15 ( 3 - 4 ) , 1, H-l a-GlcA 5.04 ( d ) , 2, H-l a-Rha,a-Rha 4.85 ( s ) , 1, H-l B-Rha 4.60 ( 1 0 ) , 1, H-l B-Glc and 4.51 ( 7 ) e , 1, H-5 a-GlcA 1.30 ( 6 ) , 9, H-6 Rha 105.0 C-1 B-Glc 103.2 C-1 a - R h a ( t e r m i n a l ) 101. 3 C-1 B-Rha 100.8 "C-1 a-Rha 96.4 C-1 a-GdcA-. 61.5 C-6 G l c 17.4 C-6 Rha a C h e m i c a l s h i f t r e l a t i v e to i n t e r n a l acetone; 52.23 d o w n f i e l d from sodium 4, 4-dim et h y l - 4-s i l a p e n t a n e - 1 - s u 1 f o n ate (D.S.S.) ^ V a l u e s i n Hz; b, broad; s, s i n g l e t . C hemical s h i f t r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. d o w n f i e l d from D.S.S. ^A p a i r of u n r e s o l v e d s i n g l e t s . A t r i p l e t w i t h c o u p l i n g c o n s t a n t of unknown o r i g i n . - 46 -I n i t i a l l y the "'"H n.m.r. spectrum of K17 was d i f f i c u l t to i n t e r p r e t . The d i f f i c u l t y arose when s i x s i g n a l s c o u l d be d i s c e r n e d i n the anomeric r e g i o n (64.5-5.5). The "'"H n.m.r. spectrum from a 2 50 MHz i n s t r u m e n t shows t h i s c l e a r l y (see Appendix, spectrum no. 1 ) . Assignment of the anomeric s i g n a l s was a c h i e v e d by examination of "*"H n.m.r. s p e c t r a of p o l y - and o l i g o s a c c h a r i d e s d e r i v e d from the c a p s u l a r p o l y s a c c h a r i d e d u r i n g the s t r u c t u r a l i n v e s t i g a t i o n (see Table 111.1, page 44) S t u d i e s done i n t h i s laboratory"''^ have p o i n t e d out t h a t i t i s p o s s i b l e f o r the n.m.r. s i g n a l of the H-5 proton of g l u c u r o n i c a c i d , and e s p e c i a l l y of a - D - g l u c u r o n i c a c i d , to appear down-f i e l d a p p r o a c h i n g the anomeric r e g i o n ( 64.5). T h e r e f o r e , at pre s e n t the s i g n a l at 64.51 has been a s s i g n e d to H-5 of B-D-g l u c u r o n i c a c i d . I t i s n o t a b l e a l s o t h a t the s i g n a l at 64.60 has a r a t h e r l a r g e c o u p l i n g c o n s t a n t (10 Hz) f o r assignment to H-l of a 8-D-glucose component. The i n t e g r a l r a t i o of 5:9 f o r anomeric p r o t o n s t o rhamnose methyl p r o t o n s c o n f i r m s the presence of t h r e e rhamnose r e s i d u e s p-e r -r e p e a L i n g .urnfitt. The absence of p y r u v a t e and a c e t a t e i n K17 c a p s u l a r p o l y s a c c h a r i i s demonstrated by the "*"H n.m.r. spectrum. These t h r e e s e t s of data show K17 p o l y s a c c h a r i d e to be composed of t h r e e r e s i d u e s of L-rhamnose, one of D-glucose, one of D - g l u c u r o n i c a c i d , and to have t h r e e a- and two B - g l y c o s i d i c l i n k a g e s . M e t h y l a t i o n a n a l y s i s 11 12 M e t h y l a t i o n ' of the a c i d i c p o l y s a c c h a r i d e , subse-quent r e d u c t i o n w i t h sodium b o r o h y d r i d e , h y d r o l y s i s , and - 47 -d e r i v a t i z a t i o n as a l d i t o l a c e t a t e s i n d i c a t e d t h a t K17 i s composed of a p e n t a s a c c h a r i d e r e p e a t i n g u n i t (see Table I I I . 2 , page 48). The presence of a monomethyl rhamnose r e s i d u e i s a t t r i b u t a b l e t o a branch p o i n t and t h a t of a t r i m e t h y l rhamnose r e s i d u e t o the t e r m i n a l sugar of a s i d e c h a i n . The 2,3-di^O-methylg1ucose i s the only d e r i v a t i v e which c o u l d have a r i s e n from the D - g l u c u r o n i c a c i d component. The p y r a n o s i d i c n a t u r e of a l l the c o n s t i t u e n t rhamnose sugars i s demonstrated by the m e t h y l a t i o n r e s u l t s . P o s i t i o n s of m e t h y l a t i o n i n the d e r i v e d a l d i t o l a c e t a t e s were determined by mass s p e c t r o m e t r y ( m . s . ) ^ 3 and, i n t h i s i n v e s t i g a t i o n , i n d i v i d u a l s t e r e o i s o m e r s were r e a d i l y i d e n t i f i e d by t h e i r c h a r a c t e r i s t i c r e t e n t i o n t i m e s on g . l . c . The presence of 3, 4-di-O-methylrhamnose i n t h e i n i t i a l m e t h y l a t i o n a n a l y s i s can be e x p l a i n e d by a s m a l l amount of h y d r o l y s i s (~25?o) t a k i n g p l a c e upon prolonged exposure t o the a c i d i c ion-exchange r e s i n when c o n v e r t i n g the i s o l a t e d p o l y -s a c c h a r i d e t o the a c i d form. The r e l a t i v e l y l a b i l e rhamnose s i d e chain would thus be removed and subsequent m e t h y l a t i o n of the branch p o i n t rhamnose would occur at the l i n k a g e s i t e ( p o s i t i o n 3). The s i t e of s i d e chain l i n k a g e t o p o s i t i o n 3 of the branch p o i n t rhamnose i s supported by l a t e r p e r i o d a t e o x i d a t i o n a n a l y s i s . A second m e t h y l a t i o n a n a l y s i s showed t h a t no h y d r o l y s i s o c c u r r e d when the time of c o n t a c t w i t h the r e s i n was reduced, (see T a b l e I I I . 3 , column I, page 50). TABLE I I I . 2 M e t h y l a t i o n a n a l y s i s of o r i g i n a l K17 c a p s u l a r p o l y s a c c h a r i d e M e t h y l a t e d s u g a r s (as a l d i t o l a c e t a t e s ) T' Mole % Column B d Column C e Column D f Column D 9 Column B Column D f Column D 9 (OV-17) (OS-138) (ECNSS-M) (ECNSS-M) 2,3,4-Rha 0. 54 0 . 53 3,4-Rha 0.89 0.86 2,4-Rha 1.00 1.00 4-Rha 1.41 1.40 2, 3, 6 - G l c 1.77 2.02 2, 3 - G l c 2 . 42 3.58 a 2,3,4-Rha = 1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 - t r i -b R e t e n t i o n t i m e r e l a t i v e to 1 , 3 , 5 - t r i - O -c V a l u e s a r e c o r r e c t e d u s i n g t h e e f f e c t i v 29 et-al . Peak a r e a s f o r column C vi/ere n d Program; 175° f o r 8 min and then 2°/min e Program; 190°C i s o t h e r m a l , f Program: 170 UC i s o t h e r m a l , g Program; 180°and then 2°/min t o 2 2 0 U C . h P r o b a b l y due t o h y d r o l y s i s of rhamnose 0.46 0. 51 19. 3 20 .9 13 .9 0.91 0.91 5.1 h 3.7 4.4 1.00 1.00 22 . 5 2 5.6 23.8 1.71 1. 51 15. 8 h 17 .8 15.5 2.48 1.94 20 . 3 20 . 4 19 . 7 5. 40 3.14 17. 1 11.6 17. 6 0 - m e t h y 1 - L - r h a m n i t o l , e t c . a c e t y l - 2 - 4 - d i - 0 - m e t h y 1 - L - r h a mn i t o 1 e carbon r e s p o n s e f a c t o r s g i v a i b y A l b e r s h i e m ot a v a i l a b l e , t o 210°C. s i d e c h a i n by IR-120 r e s i n (see t e x t f o r d e t a i l ) - 49 -P a r t i a l h y d r o l y s i s P a r t i a l , a c i d i c h y d r o l y s i s of the n a t i v e p o l y s a c c h a r i d e was f o l l o w e d by s e p a r a t i o n of the a c i d i c and n e u t r a l f r a c t i o n s by ion-exchange chromatography. The n e u t r a l f r a c t i o n , a f t e r s e p a r a t i o n by g e l f i l t r a t i o n chromatography, c o n t a i n e d mono-s a c c h a r i d e s , m a i n l y rhamnose, and a d i s a c c h a r i d e (_1) . Chroma-t o g r a p h i c s e p a r a t i o n of the a c i d i c f r a c t i o n y i e l d e d two a c i d i c o l i g o m e r s (2_ and _3) . Compound 1^ , [of] n +3.7°, was shown by "'"H and ^Cn.m.r. sp e c t r o s c o p y (see Table I I I . l , page 44) to c o n t a i n one non-r e d u c i n g , 3 - a n o m e r i c s i g n a l and two anomeric s i g n a l s a t t r i b u -t a b l e to a r e d u c i n g 6-deoxyhexose (see Appendix, s p e c t r a no.'s 5,6). N.m.r. s p e c t r o s c o p i c s t u d i e s ' on t h i s compound have been r e p o r t e d by K i n g and Bishop ( ^ H ) , ^ and Colson and King ( 3 C ) . M e t h y l a t i o n , h y d r o l y s i s , and g.1.c.-m.s. 1 3 of the a l d i t o l a c e t a t e s gave 2,3,4,6-tetra-0-methy1glucose and 3,4-di-0-methylrhamnose (see Table I I I . 3 , column V, page 50). The s t r u c t u r e of 1 i s thus e s t a b l i s h e d as 3-D-*Glcp-(l*>2)-L-Rhap Q ) 1 13 Compound 2_, [[of] ^  +111°, was shown by H and C n.m.r, sp e c t r o s c o p y (see T a b l e I I I . l , page 44) to g i v e one anomeric s i g n a l due to the combined s i g n a l s of a n o n - r e d u c i n g and a r e d u c i n g a - g l y c o s i d i c l i n k a g e and another anomeric s i g n a l TABLE I I I . 3 M e t h y l a t i o n a n a l y s e s of K17 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d p o l y -and o l i g o s a c c h a r i d e s a T b Mole % M e t h y l a t e d sugar T . (as a l d i t o l a c e t a t e ) I I I I I I IV 2,3,4-Rha 0 . 48 2 1 . 6 ( 1 9 . 3 ) e 7.2 f 3,4-Rha 0 . 79 (5.1) 23.9 26. 3 49 .9 2,4-Rha 0.89 21.1(22.5) 27.9 27.9 48.2 2,3,4, 6-Glc 1.00 30.6 50 .1 4-Rha 1.25 21.8(15.8) 2, 3, 6-Glc 1 . 57 20.2(20.3) 24.2 2, 3, 4-Glc 1.61 51.8 2, 3-Glc 2.15 15.4(17.1) 16.8 15.2 a2,3,4-Rha = 1,5-di - 0 - a c e t y l - 2 , 3 , 4 - t r i - O - m e t h y l - L-rhamn i t o l , e t c . R^ eten t ion time r e l a t i v e to 1, 5 - d i - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0-m e t h y l - D - g l u c i t o l on an OV-17 column prog ramm ed at 17 5 0 f o r 8 min and then 2° /min to 210 UC. °Values are c o r r e c t e d by use of e f f e c t i v e carbon response f a c t o r s given by A l b e r s h e i m d T - A -I, a c i d i c c a p s u l a r p o l y s a c c h a r i d e , second m e t h y l a t i o n ana l y s i s ; I I , PI; I I I , a c i d i c t e t r a s a c c h a r i d e ;3| IV, a l d o b i o u r o n i c a c i d 2; and V, n e u t r a l d i s a c c h a r i d e 1. For comparison, numbers i n b r a c k e t s r e f e r t o the f i r s t m e t h y l a t i o n r e s u l t s , T a b l e I I I . 2 page 48(See t e x t f o r d e t a i l s ) . A s m a l l amount of 2 - 0 - a c e t y 1 - 1 , 3 , 4 - t r i - 0 - m e t h y 1 e r y t h r i t o l a l s o appears i n the chromatogram - 51 -a t t r i b u t a b l e to a r e d u c i n g 3 -6-deoxyhexose (see Appendix, s p e c t r a no.'s 7,8). M e t h y l a t i o n of 2_ and subsequent r e d u c t i o n w i t h l i t h i u m aluminum h y d r i d e , h y d r o l y s i s , and d e r i v a t i z a t i o n as a l d i t o l a c e t a t e s gave p r o d u c t s c o r r e s p o n d i n g to 2 , 3 , 4 - t r i -0 - m ethylglucose and 2,4-di-0-methy1-rhamnose, as a n a l y z e d by g.1.c.-m.s. X^ (see Table I I I . 3 , column IV, page 50). Compound 2_, the a l dobiou ron i c a c i d , i s t h u s e s t a b l i s h e d as having the s t r u ctu re a-D-GlcAp-(1>3)-L-Rhap (2) T h i s i s the f i r s t o c c u r r e n c e , t o date, of the above a l d o b i o u r o n i c a c i d i n a Kleb s i e l 1 a b a c t e r i a l p o l y s a c c h a r i d e . Compound 3^ , [of] ^  +38°, was shown by "''H and ''""'n.m.r. sp e c t r o s c o p y (see Table I I I . l , page 44) to produce f o u r s i g n a l s i n the anomeric r e g i o n c h a r a c t e r i s t i c of a n o n - r e d u c i n g a - g l y -c o s y l and a r e d u c i n g a - g l y c o s y l l i n k a g e ( u n r e s o l v e d ) , one non-r e d u c i n g a - g l y c o s y l bond, a g - g l y c o s y l bond due to a r e d u c i n g 6 - deo xy h exo se, and one B - g l y c o s y l bond a t t r i b u t a b l e to a non-r e d u c i n g g l u c o s e u n i t ( i m p l i e d by the l a r g e c o u p l i n g c o n s t a n t of the 3-D s i g n a l i n the "'"H n.m.r. spect r u m " ^ ) . See Appendix, s p e c t r a no.'s 9,10. M e t h y l a t i o n a n a l y s i s (as d e s c r i b e d f o r compound 2) y i e l d e d 2 , 3, 4, 6 - t e t r a - 0 - m e t h y l g l u c o s e , 3,4-di-0-methylrhamnose, 2 , 3 - d i - 0 - m e t h y l g l u c o s e , and 2,4-di-0-methylrham-nose (see Table I I I . 3 , column I I I , page 50) • Having e s t a b l i s h e d - 52 -the s t r u c t u r e s o f _1 and 1_ i t i s p o s s i b l e to a s s i g n the s t r u c -t u r e of 3 as B-D^ l c p - ( l * 2 ) - a - L - R h a p - ( l * - 4 ) - a - D - G l c A p - ( l * - 3 ) - L - R h a p (3) P e r i o d a t e o x i d a t i o n A complete p e r i o d a t e o x i d a t i o n of K17 p o l y s a c c h a r i d e was a c h i e v e d a f t e r two t r e a t m e n t s w i t h sodium periodate"*"^ and 18 sodium p e r c h l o r a t e . C a r b o x y l r e d u c t i o n and h y d r o l y s i s of the sodium b o r o h y d r i d e - r e d u c e d , p e r i o d a t e - o x i d i z e d polymer gave equimolar amounts of e r y t h r i t o l and rhamnose a n a l y z e d as t h e i r a l d i t o l a c e t a t e s by g . l . c . These r e s u l t s p r o v i d e complementary data to the m e t h y l a t i o n a n a l y s i s of the p o l y -s a c c h a r i d e . In o r d e r to e s t a b l i s h the l e n g t h of the s i d e chain and the r e s i d u e , and p o s i t i o n on t h a t r e s i d u e , to which the t e r m i n a l 19 rhamnose i s a t t a c h e d , s e l e c t i v e p e r i o d a t e o x i d a t i o n was per-formed. Knowing the s t r u c t u r e O f compound _3, two d i s t i n c t p o s s i b i l i t i e s f o r the s t r u c t u r e of the r e p e a t i n g u n i t o f K17 e x i — G l c^-^R h a^-^-Gl cA^-^R h a ^ 3 1 Rha a or -R h a^-^G 1 c A^-^R h a — a a 6 GI ? • i 1 Rha - 53 -The p e r i o d a t e s e n s i t i v e components of the p o l y s a c c h a r i d e , i . e . 1 , 4 - l i n k e d 3-D-glucopy ranose, g l u c u r o n i c a c i d , and t e r m i n a l rhamnose, p r o v i d e e x c e l l e n t o p p o r t u n i t y f o r p a r t i a l o x i d a t i o n to oc c u r . The low r e a c t i v i t i e s of 1 , 4 - l i n k e d 19 3-D-glu co py ran o se , due to s t e r i c h i n d r a n c e , and g l u c u r o n i c a c i d , due to e l e c t r o s t a t i c r e p u l s i o n between the ur o n a t e anion and the a t t a c k i n g p e r i o d a t e i o n } ^ are opposed by the high r e a c t i v i t i y of a t e r m i n a l a - L - rh amn opy ran ose . A s e l e c t i v e , p a r t i a l p e r i o d a t e o x i d a t i o n of the i n t a c t p o l y s a c c h a r i d e u s i n g 0.05 M_ sodium p e r i o d a t e proceeded r a p i d l y w i t h 2.6 moles of p e r i o d a t e b e i n g consumed per mole of r e p e a t i n g u n i t a f t e r 100 min ( t h e o r e t i c a l = 4 moles, f o r complete o x i d a -t i o n ) . An a n a l y s i s of the sugar components of the sodium b o r o h y d r i d e - r e d u c e d , p e r i o d a t e - o x i d i z e d product r e v e a l e d t h a t the p e r a c e t a t e s of e r y t h r i t o l , r h a m n i t o l and g l u c i t o l were p r e s e n t i n the r a t i o 1:3.2:1.9. These r e s u l t s show t h a t the p a r t i a l o x i d a t i o n proceeded beyond t h a t d e s i r e d , i . e . e r y t h -r i t o l came from the unwanted p e r i o d a t e o x i d a t i o n of g l u c o s e or g l u c u r o n i c a c i d . 20 Smith d e g r a d a t i o n of the reduced, o x i d i z e d product u s i n g 0.5 M_ t r i f l u o roa ce t i c a c i d f o r 17 h at room temperature, y i e l d e d a p o l y s a c c h a r i d e d e s i g n a t e d Pl_. The "^C n.m.r. spectrum of P_l_ i n d i c a t e d only f o u r anomeric s i g n a l s at 104.8, 101.2, 100.0 and 96.0 p.p.m. as w e l l as two s i g n a l s at 17.6 and 17.4 p.p.m. a t t r i b u t a b l e to the C-6 of two rhamnose u n i t s . How-ever, s i g n a l s at 63.4, 61.5, and 61.2 p.p.m. were a l s o observed c o n f i r m i n g the presence of e r y t h r i t o l i n the polymer (see Appendix, spectrum no. 4 ) . M e t h y l a t i o n a n a l y s i s of P_l y i e l d e d - 54 -the sugar d e r i v a t i v e s shown i n Table I I I . 3 , column I I , page 50 In p a r t i c u l a r , no 4-0-methy1rhamnose was generated and i n s t e a d 3,4-di-0-methy 1rhamnose appeared, e s t a b l i s h i n g t h a t the s i d e chain of K17 p o l y s a c c h a r i d e c o n s i s t s of one u n i t and t h a t the t e r m i n a l rhamnose i s a t t a c h e d to 0-3 of the branch p o i n t rhamnose. (See F i g u r e I I . 9 , page 33). Another s e l e c t i v e p e r i o d a t e o x i d a t i o n of the i n t a c t p o l y s a c c h a r i d e was done u s i n g only a t h r e e - f o l d excess of sodium p e r i o d a t e (0.014 M_) . The r e a c t i o n proceeded more s l o w l y consuming 1.1 moles of p e r i o d a t e per mole of r e p e a t i n g u n i t a f t e r 120 min. Smith d e g r a d a t i o n (same as f o r P_l) of the sodium b o r o h y d r i d e - r e d u c e d , o x i d i z e d product y i e l d e d a p o l y -s a c c h a r i d e ( P l a ) having an unbranched, s t r a i g h t c h a i n , f o u r -sugar r e p e a t i n g u n i t . No o v e r - o x i d a t i o n o c c u r r e d as judged by "'"H and n.m.r. s p e c t r o s c o p y (see Table I I I . l , page 48). The "*"H n.m.r. spectrum (see Appendix, spectrum no. 3) of P1 a, when compared w i t h t h a t of the o r i g i n a l p o l y s a c c h a r i d e , r e v e a l e d the absence of a s i g n a l c o r r e s p o n d i n g to the anomeric proton of an a - l i n k e d 6-deoxyhexose. Thus, the t e r m i n a l rhamnose r e s i d u e i s a - l i n k e d and t h e r e f o r e , by e l i m i n a t i o n , the rhamnose a t t a c h e d at 0-1 to the g l u c u r o n i c a c i d r e s i d u e i s B - l i n k e d . The i n c r e a s e i n o p t i c a l r o t a t i o n from +30° f o r the o r i g i n a l p o l y s a c c h a r i d e to +44° f o r P l a i s i n agreement w i t h these assignments. The r e s u l t s o b t a i n e d from m e t h y l a t i o n a n a l y s i s , p a r t i a l h y d r o l y s i s and p e r i o d a t e o x i d a t i o n s t u d i e s are s u f f i c i e n t to e s t a b l i s h the s t r u c t u r e of K17 p o l y s a c c h a r i d e . However, u r o n i c - 55 -a c i d d e g r a d a t i o n s t u d i e s are p r e s e n t e d as w e l l because they supply c o n f i r m a t i o n of the proposed s t r u c t u r e . U r o n i c a c i d d e g r a d a t i o n The p e r m e t h y l a t e d p o l y s a c c h a r i d e was s u b j e c t e d to a 21 b a s e - c a t a l y z e d u r o n i c a c i d d e g r a d a t i o n u s i n g dime t h y 1 s u f i n y 1 a n i o n . A f t e r n e u t r a l i z a t i o n , the degraded m a t e r i a l was i s o l a t e d and then r e - a l k y l a t e d u s i n g e t h y l i o d i d e and s i l v e r o x i d e . H y d r o l y s i s of the e t h y l a t e d , degraded m a t e r i a l and a n a l y s i s of the p r o d u c t s by g.1.c.-m.s.^^ of the a l d i t o l a c e t a t e s gave the r e s u l t s shown i n Table I I I . 4 , Columns I and I I , page 56.. Another u r o n i c a c i d d e g r a d a t i o n was performed on the permethy-l a t e d p o l y s a c c h a r i d e under the same c o n d i t i o n s as b e f o r e except t h a t a l k y l a t i o n w i t h e t h y l i o d i d e o c c u r r e d d i r e c t l y i n the r e a c t i o n m i x t u r e w i t h o u t p r i o r i s o l a t i o n of the 22 degraded m a t e r i a l . The r e s u l t was e s s e n t i a l l y the same except f o r a l a r g e r r e l a t i v e amount of the e t h y l a t e d compound i n d i c a t i n g a r e a c t i o n c l o s e r to c o m p l e t i o n (see Table I I I . 4 , column I I I page 56). The presence of 3-0-ethyl-2,4-di-O-methylrhamnose i n d i c a t e s t h a t i t was the r e s i d u e a t t a c h e d to 0-1 of the g l u c u r o n i c a c i d , c o n f i r m i n g the s t r u c t u r e of the a l d o b i o u r o n i c a c i d ( 2 ) . S i n c e not a l l the 2 , 4-d i-0-rn e t hy 1 rh amn o se component i s e t h y l a t e d , i t may be assumed t h a t a l l the enol e t h e r s formed d u r i n g the e l i m i n a t i o n r e a c t i o n are not c l e a v e d , 21 s u g g e s t i n g t h a t some m i l d a c i d t r e a t m e n t would have been a p p r o p r i a t e i n t h i s case. Loss of g l u c u r o n i c a c i d r e s i d u e i n v o l v e s cleavage of the main chain and was accompanied by some TABLE I I I . 4 G . l . c . a n a l y s i s of K17 p o l y s a c c h a r i d e a f t e r U r o n i c A c i d D e g r a d a t i o n M e t h y l a t e d sugars (as a l d i t o l a c e t a t e s ) T b Mole ? o C Column B (OU-17) C olu mn (HIEFF E 6 -IB) If I I I I I 2 , 3 , 4-R h a 0. 54 0. 42 16. 6 19. 3 17 . 6 3-Et-2,4-Rha 9 0. 57 0. 37 19. 9 19.0 22 . 5 2,4-Rha 1.00 1.00 17. 6 16.6 11. 7 4-Rha 1. 41 1.55 14. 1 11.9 16.8 2, 3, 6-Glc 1.78 1.94 31. 8 33.2 31. 4 a2,3,4-Rha = 1 , 5 - d i - 0 - a c e t y 1 - 2 , 3 , 4 - 1 r i - 0 - m e t h y 1 - L - r h a m n i t o l , e t c . ^ R e t e n t i o n time r e l a t i v e to 1 , 3 , 5 - 1 r i - 0 - a c e t y l - 2 , 4 - d i - 0 - m e t h y 1 - L - r h a m n i t o l . c 2 V a l u e s are c o r r e c t e d u s i n g e f f e c t i v e carbon response f a c t o r given by A l b e r s h e i m et a l ^Program: 175° f o r 8 min and then 2°/min to 210 °'C. eProgram: 1 6 0 u f o r 8 min then 2°/min to 200°C. I, u r o n i c a c i d d e g r a d a t i o n of p o l y s a c c h a r i d e , i s o l a t i o n of p r o d u c t s , and then e t h y l a t i o n , column B; I I , same, column E; I I I , u r o n i c a c i d d e g r a d a t i o n of p o l y -s a c c h a r i d e w i t h d i r e c t e t h y l a t i o n , column B (see t e x t f o r d e t a i l s , page72 ). g3-E-t-2, 4-Rha = 1, 5 - d i - 0 - a c e t y 1-3-0-ethyl-2 , 4 - d i - 0 - m e t h y l - L r rhamn i t o l . - 57 -f u r t h e r d e g r a d a t i o n of exposed r e d u c i n g - g r o u p s as seen by lower r e l a t i v e amounts of 4-0-methylrhamnose and 2 , 3 , 4 - t r i -O-methylrhamnose i n Table I I I . 4 . T h e r e f o r e , the b r a n c h i n g rhamnose r e s i d u e i s more l i k e l y a t t a c h e d to the o t h e r s i d e of the g l u c u r o n i c a c i d ( t h r o u g h 0-4) j u d g i n g by the quan-t i t a t i v e g . l . c . d a t a . The r e s u l t s r e p o r t e d here demonstrate t h a t the c a p s u l a r p o l y s a c c h a r i d e from K l e b s i e l l a s e r o t y p e K17 i s composed of p e n t a s a c c h a r i d e r e p e a t i n g u n i t s h a v i n g the f o l l o w i n g s t r u c t u r e . • 4 ) - B-D-Glcp-( 1+-2) - a- L-R h ap-( 1*4) - a-D - Gl cAp-( 1*3)- 6-L-R h ap-( 1*. t 3 1 a-L -R h ap Of the K l e b s i e l l a c a p s u l a r p o l y s a c c h a r i d e r e p o r t e d to 2 3 date, the s t r u c t u r e of K62 , h a v i n g a f i v e - s u g a r r e p e a t i n g u n i t w i t h a s i n g l e u n i t s i d e chain and g l u c u r o n i c a c i d i n the main c h a i n , i s the c l o s e s t analogue to K17. The presence of a g - l i n k e d L-rhamnose r e s i d u e has been r e p o r t e d i n only one o t h e r K l e b s i e l l a c a p s u l a r p o l y s a c c h a r i d e , 2 4 t h a t of s e r o t y p e K32. However, i n the p r e s e n t i n v e s t i g a t i o n , the absence of an i s o l a t e d o l i g o s a c c h a r i d e w i t h the g-L-rhamnose l i n k a g e i n t a c t and the u n u s u a l l y h i g h y i e l d of compound _3 l e a d s to the c o n c l u s i o n t h a t the g-L-rhamnose l i n k a g e i s more suscep-t i b l e to a c i d h y d r o l y s i s than the comparable main chain - 58 -ex-L-rhamn ose l i n k a g e . The "*"H n.m.r. spectrum of K17 p o l y s a c c h a r i d e i n c l u d e s s i x s i g n a l s i n the anomeric r e g i o n from a p e n t a s a c c h a r i d e r e p e a t i n g u n i t , i l l u s t r a t i n g t h a t c a u t i o n must be e x e r c i s e d when i n t e r p r e t i n g t h i s type of d a t a . In "^C n.m.r. s p e c t r o -scopy, as w e l l , a c l e a r d i v i d i n g l i n e f o r d i s t i n g u i s h i n g anomeric carbon atoms from a- and g-L-rhamnose r e s i d u e s does not appear to e x i s t . Assuming t h a t a change i n s u b s t i t u e n t s does not make a l a r g e d i f f e r e n c e i n che m i c a l s h i f t s , the s i g n a l at 103.2 p.p.m. has been a s s i g n e d to C-1 of an a-L-rhamnose component, s u b s t a n t i a l l y d o w n f i e l d from a nominal maximum of 101 p.p.m. f o r a - l i n k e d hexoses. The g-L-rhamnose anomeric s i g n a l at 101.3 p.p.m. appears between those of the two a-L-rhamnose s i g n a l s . Of cou r s e , "''H n.m.r. s p e c t r o s c o p y d i s t i n g u i s h e s the two anomers of L-rhamnose e a s i l y . - 59 -I I I . 4 E xperiroen t a l G e n e r a l methods Descending paper chromatography was c a r r i e d out u s i n g Whatman No. 1 paper and the f o l l o w i n g s o l v e n t systems ( v / v ) : (A) e t h y l a c e t a t e - a c e t i c a c i d - f o r m i c a c i d - water (18:3:1:4); (B) e t h y l a c e t a t e - p y r i d i n e - water ( 8 : 2 : 1 ) . ? 5 Chromatograms were developed u s i n g s i l v e r n i t r a t e . "~ Gel f i l t r a t i o n chromatography was conducted on columns (150 X 3 cm or 100 X 3 cm) of B i o - G e l P-2 (100-200 mesh). The columns were i r r i g a t e d w i t h water - p y r i d i n e - a c e t i c a c i d (1000:10:4) at a f l o w r a t e of -10 ml/h. F r a c t i o n s (2.0-2.5 ml) were c o l l e c t e d , f r e e z e - d r i e d , and chromatographed on paper or weighed i n t a r e d tubes and the r e s u l t s were p l o t t e d on graph paper to produce a chromatogram. A n a l y t i c a l g . l . c . s e p a r a t i o n s were performed u s i n g a Hewlett Packard model 5710A gas chromatograph f i t t e d w i t h d u a l flame i o n i z a t i o n d e t e c t o r s . S t a i n l e s s s t e e l columns (1.8 m X 3 mm) were used w i t h a c a r r i e r gas f l o w r a t e of 20 ml/min. Columns used were (A) 3?o of SP-2340 on S u p e l c o p o r t (100-130 mesh); (B) 3% of OV-17 on Gas Chrom Q (100-120); (C) 10% of OS-138 on the same s u p p o r t ; (D) 5% of ECNSS-M on the same s u p p o r t ; (E) 3% of HIEFF-1B on Chrom W(HP) (80-100 mesh). P r e p a r a t i v e g . l . c . was performed u s i n g an F and M model 7 20 i n s t r u m e n t w i t h d u a l thermal c o n d u c t i v i t y d e t e c t o r s . Column (F) (1.8 m X 6.3 mm) 5% of S i l a r 10C on Gas Chrom Q (100-120 mesh), analagous to column A, was used f o r p r e p a r a t i v e s e p a r a t i o n s . An I n f o t r o n i c s CRS-100 e l e c t r o n i c i n t e g r a t o r was used t o measure peak a r e a s . -60-G.l.c.-m.s. was pe r f orm ed ,.u s i n g a Micromass 12 i n s t - i : rument f i t t e d w i t h a Watson-Biemann s e p a r a t o r . S p e c t r a were re c o r d e d at 70 and 20 eV w i t h an i o n i z a t i o n c u r r e n t of 100 uA and an ion source temperature of 200 UC. "*"H 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-100 i n s t r u m e n t . Samples d i s s o l v e d i n D2O were exchanged and f r e e z d r i e d t h r e e or f o u r times u s i n g 99.1% D^ O. Acetone ( §2.23, measured a g a i n s t aqueous sodium 2 , 2 - d im e t h y 1 - 2-s i l a pen t an e-5-s u l f o n a t e (DSS)) was used as the i n t e r n a l s t a n d a r d . S p e c t r a were rec o r d e d at a p p r o x i m a t e l y 90°C. S p e c t r a of m e t h y l a t e d d e r i v a t i v e s were o b t a i n e d u s i n g CDCl^ as s o l v e n t w i t h TMS as i n t e r n a l s t a n d a r d on the U.B.C. 270 MHz i n s t r u m e n t at ambient t e m p e r a t u r e . X^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 CFT-20 i n s t r u m e n t and were o b t a i n e d at ambient temperature u s i n g 99.1% D^0 as s o l v e n t and acetone (31.07 p.p.m. from DSS) as i n t e r n a l s t a n d a r d . In a d d i t i o n , ''"H n.m.r. s p e c t r a of the p o l y s a c c h a r i d e and o l i g o s a c c h a r i d e s 2_ and 3. were o b t a i n e d c o u r t e s y of Dr. A. A. Grey, Canadian 220 MHz NMR Ce n t e r , Department of M e d i c a l G e n e t i c s , U n i v e r s i t y of Toronto on a V a r i a n HR-220 i n s t r u m e n t . "*"H and X^C n.m.r. s p e c t r a of the p o l y s a c c h a r i d e were o b t a i n e d c o u r t e s y of Dr. M. Vignon, CERMAV/CNRS, Gre n o b l e , France on a Cameca 2 50 MHz i n s t r u m e n t . C i r c u l a r d i c h r o i s m measurements were made u s i n g a Jasco J-20 a u t o m a t i c r e c o r d i n g s p e c t r o p o l a r i m e t e r w i t h a quart c e l l of path l e n g t h 0.01 cm. O p t i c a l r o t a t i o n s were measured at 23 - 2 UC on a P e r k i n - E l m e r model 241 MC p o l a r i m e t e r u s i n g - 61 -a 10 cm c e l l . I n f r a r e d s p e c t r a were r e c o r d e d u s i n g a P e r k i n -Elmer model 457 s p e c t r o p h o t o m e t e r . S o l u t i o n s were c o n c e n t r a t e d under reduced p r e s s u r e at bath temperatures not exceeding 40°C. P r e p a r a t i o n and p r o p e r t i e s of K l e b s i e l l a K17 c a p s u l a r p o l y s a c c h a r i d e A c u l t u r e of K l e b s i e l l a K17 (2005/49) was o b t a i n e d from Dr. I. JZlrskov, Copenhagen, and was grown on a 3% su c r o s e -yeast e x t r a c t - agar medium composed of NaCl (5 g ) , K^PO^ (2.5 g ) , MgS0 4.7 H 20 (0.62 g ) , CaSO^ (1.25 g ) , su c r o s e (75 g ) , Bacto yeast e x t r a c t (5 g ) , and agar (37.5 g) i n water (2.5 1). The c e l l s were h a r v e s t e d a f t e r 3 days, d i l u t e d to 2 1 w i t h water c o n t a i n i n g l ? o phenol, and c e n t r i f u g e d i n batches f o r 3 h at 34,000 r.p.m. i n a Beckman model L3-50 u 1 t r a c e n t r i f u g e w i t h r o t o r type 35. The c l e a r , s u p e r n a t a n t l i q u o r s were decanted, combined (^1300 ml), and p r e c i p i t a t e d by p o u r i n g i n t o 10 1 of s o l v e n t IK ( e t h a n o l - methanol, 95:5) Crude p o l y s a c c h a r i d e , i s o l a t e d by d e c a n t a t i o n and c e n t r i f u g a t i o n , was d i s s o l v e d i n water (800 ml) and then p r e c i p i t a t e d w i t h a 10% C e t a v l o n s o l u t i o n . The p r e c i p i t a t e was i s o l a t e d by c e n t r i f u g a t i o n , r e d i s s o l v e d i n 4 H NaCl (800 m l ) , and r e p r e c i p i t a t e d by p o u r i n g i n t o S o l v e n t IK ( 8 1 ) . The C e t a v l o n - p u r i f i e d p o l y s a c c h a r i d e was i s o l a t e d by c e n t r i f u g a t i o n , d i s s o l v e d i n water, and d i a l -yzed 2 days a g a i n s t r u n n i n g t a p - w a t e r . F r e e z e - d r y i n g of t h i s s o l u t i o n y i e l d e d 7.4 g of the sodium s a l t of the p o l y s a c c h a r i d e ( a p p r o x i m a t e l y 2.5 g per 2.5 1 of medium), [a] ^  +30 u(£ 0.57, w a t e r ) . P u r i t y of the p o l y s a c c h a r i d e was checked by e l e c t r o -p h o r e i s u s i n g a l ? o s o l u t i o n on c e l l u l o s e a c e t a t e s t r i p s - 62 -(Sepraphore I I I ; 15 x 2.5 cm) i n v e r o n a l b u f f e r pH8.6 (LKB-Produkter AB, Stockholm 12, Sweden) at 300 V f o r 90 min and then development i n e i t h e r a l c i a n b l u e i n c i t r a t e b u f f e r e d e t h a n o l (pH4) or p e r i o d a t e - S c h i f f r e a g e n t . Homogeneity was a l s o c o n f i r m e d by g e l chromatography c o u r t e s y of Dr. S. C. Churms, U n i v e r s i t y of Capetown, South A f r i c a , and the mole-c u l a r weight of K17 p o l y s a c c h a r i d e determined to be 9.4 x 10^. The f r e e - a c i d form of the p o l y s a c c h a r i d e was o b t a i n e d by p a s s i n g a s o l u t i o n of the sodium s a l t through a column of A m b e r l i t e 1R-120 (H ) r e s i n , f o l l o w e d by f r e e z e - d r y i n g . S p e c t r o s c o p i c a n a l y s e s were performed on K17 p o l y -s a c c h a r i d e and the d e r i v e d p o l y s a c c h a r i d e t h a t had been p a r t i a l l y d e p o l y m e r i z e d to reduce v i s c o s i t y . T h i s was a c h i e v e d by m i l d h y d r o l y s i s i n 0.4 M_ t r i f 1 u or oa ce t i c a c i d f o r 15 min at 95°C, and then e v a p o r a t i o n under reduced p r e s s u r e and washing w i t h water s e v e r a l times to e l i m i n a t e t r i f l u o r o a e e t i c a c i d . The n.m.r. spectrum of p a r t i a l l y d e p o l y m e r i z e d K17 p o l y s a c c h a r i d e i n D^0 at 90°C showed s i g n a l s r e l a t i n g to s i x pr o t o n s i n the anomeric r e g i o n , observed at 65.15 (1H, 2^~^ Hz), 5.04 (2H, J x 2 b r o a d ) , 4.85 (1H, s i n g l e t ) , 4.60 (1H, 2 10 Hz), 4.51 (1H, c o u p l i n g c o n s t a n t 7 Hz). Three s e t s of unr e s o -l v e d d o u b l e t s due to the CH^ of rhamnose appeared around 61.30 ( J , 6 Hz) and i n t e g r a t e d to n i n e p r o t o n s . (See Table I I I . l z>, 6 page 44, and Appendix, spectrum no. 1.) The n.m.r. spectrum of p a r t i a l l y d e p o l y m e r i z e d K17 p o l y s a c c h a r i d e c o n f i r m e d the presence of more than one rham-- 63 -nose r e s i d u e ( l a r g e s i g n a l f o r C-6 at 17.4 p.p.m.). One s i g n a l a t t r i b u t a b l e to C-6 of a hexose was observed at 61.5 p.p.m. In the anomeric r e g i o n , f i v e s i g n a l s c o u l d be d i s t i n -g u i s hed at 105.0, 103.2, 101.3, 100.8, and 96.4 p.p.m. (see Table I I I . l , page 44, and Appendix, spectrum no. 2 ) . A n a l y s i s of c o n s t i t u e n t sugars M e t h a n o l y s i s of a sample (30 mg) of K l e b s i e l l a K17 c a p s u l a r p o l y s a c c h a r i d e w i t h 3?o m e t h a n o l i c h y d r o c h l o r i c a c i d and subsequent treatment w i t h sodium b o r o h y d r i d e i n anhydrous methanol reduced the u r o n i c a c i d r e s i d u e s i n the p o l y s a c c h a r i d e . T o t a l h y d r o l y s i s w i t h 2 M_ t r i f l u oroa cet i c a c i d o v e r n i g h t at 95°C, r e d u c t i o n of the l i b e r a t e d monosaccharides to a l d i t o l s w i t h sodium b o r o h y d r i d e , 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 -p y r i d i n e (1:1) at room temperature o v e r n i g h t y i e l d e d L - r h a m n i t o l p e n t a a c e t a t e and D - g l u c i t o l h e x a a c e t a t e i n the r a t i o of 1.6:1.0 (column A; programmed at 1 9 5 u f o r 4 min and then at 2 °/min to 260°C). C i r c u l a r d i c h r o i s m of the r h a m n i t o l p e n t a a c e t a t e MiCN MeCN showed Ae 2 j ^ - l . l a n ^ *-he g l u c i t o l h e x a a c e t a t e As +0»32 a f t e r c o l l e c t i o n of the a l d i t o l a c e t a t e s by p r e p a r a t i v e g . l . c . (column F; programmed at 210°and then at 4 /'min t o 250°C). M e t h y l a t i o n a n a l y s i s of n a t i v e p o l y s a c c h a r i d e K17 p o l y s a c c h a r i d e (285 mg) i n the a c i d form was 11 12 meth y l a t e d ' i n d i m e t h y l s u l f o x i d e (20 ml) by treatment w i t h dim e t h y l s u 1 f i n y l anion (10 ml) f o r 4 h, and then methyl i o d i d e (4 ml) f o r 1 h. A f t e r removal of excess r e a g e n t s by d i a l y s i s , the m e t h y l a t e d p o l y s a c c h a r i d e was i s o l a t e d by f r e e z e - d r y i n g 7 - 64 -2 6 ( y i e l d 283 mg). Subsequent P u r d i e treatment w i t h s i l v e r o x i d e and methyl i o d i d e y i e l d e d a p e r m e t h y l a t e d p o l y s a c c h a -r i d e t h a t showed no h y d r o x y l group a b s o r p t i o n i n the i n f r a -red spectrum ( y i e l d 297 mg). A sample (35 mg) of t h i s m a t e r i a l was reduced o v e r n i g h t w i t h sodium b o r o h y d r i d e (100 mg) in t e t r a h y d r o f u r a n - e t h a n o l (1:1) at room tempera-t u r e . H y d r o l y s i s of the m e t h y l a t e d , reduced p o l y s a c c h a r i d e w i t h 2 lj_ t r i f l u o r o a c e t i c a c i d o v e r n i g h t at 95°C was f o l l o w e d by r e d u c t i o n of the h y d r o l y z a t e w i t h sodium b o r o h y d r i d e and then a c e t y l a t i o n of the a l d i t o l s . G . l . c . (column C; 190°C i s o t h e r m a l : column D; 170 uC i s o t h e r m a l : column D; programmed at 180 °C and then 2°/min to 220 UG) and g.l.c.-m.s. a n a l y s i s (column B) of m e t h y l a t e d a l d i t o l a c e t a t e s produced a l l o w e d the assignments given i n Table I I I . 2 , page 48. A second m e t h y l a t i o n of K17 p o l y s a c c h a r i d e was done on a sample (200 mg) which had been d e i o n i z e d by s t i r r i n g i n s o l u t i o n w i t h IR-120(Ht) r e s i n over 2 days, f i l t e r e d , d i a l y z e d , and f r e e z e - d r i e d ( y i e l d 163 mg). A f t e r one Hakomori treatment and one P u r d i e treatment the m e t h y l a t e d product showed no h y d r o x y l group a b s o r p t i o n i n the i n f r a r e d spectrum. R e d u c t i o n , h y d r o l y s i s , r e d u c t i o n , and a c e t y l a t i o n y i e l d e d a m i x t u r e of me t h y l a t e d a l d i t o l a c e t a t e s t h a t were i d e n t i f i e d by compara-t i v e g . l . c . and g.l.c.-m.s. (see Table I I I . 3 , column I, page 50). P a r t i a l , a c i d i c h y d r o l y s i s of p o l y s a c c h a r i d e A c i d i c p o l y s a c c h a r i d e (942 mg) was p a r t i a l l y hydro-l y z e d i n 0.1 II t r i f l u o r o a c e t i c a c i d at 95 °C f o r 3 days u s i n g - 65 -an a p p a r a t u s s i m i l a r to t h a t d e s c r i b e d by Galanos and co-27 workers. A f t e r each 24 h p e r i o d the d i a l y s i s s o l u t i o n was changed and the d i a l y z e d m a t e r i a l i s o l a t e d by removal of most of the water by e v a p o r a t i o n under reduced p r e s s u r e and then f r e e z e - d r y i n g the c o n c e n t r a t e d s o l u t i o n i n a t a r e d f l a s k . As judged by q u a n t i t y of d i a l y z a b l e m a t e r i a l r e c o v e r e d , 80% was o b t a i n e d a f t e r 24 h and v i r t u a l l y 100% a f t e r 48 h. No r e s i -dual p o l y s a c c h a r i d e remained i n the d i a l y s i s sac. Paper chromatography ( s o l v e n t A) of the d i a l y z e d , h y d r o l y z e d m a t e r i a l showed the presence of s e v e r a l o l i g o s a c c h a r i d e s , g l u c o s e and a l a r g e c o n c e n t r a t i o n of rhamnose. The t o t a l amount of d i a l y -z a b l e m a t e r i a l was n e u t r a l i z e d (0.1 M_ NaOH) and a p p l i e d to a column (30 X 1.5 cm) of Bio-Rad A G l - X 2 ( C l " ) r e s i n . The column was f i r s t e l u t e d w i t h water (1 1), y i e l d i n g n e u t r a l mono- and o l i g o s a c c h a r i d e s , and then w i t h 10% f o r m i c a c i d (100 m l ) , y i e l d i n g the a c i d i c o l i g o s a c c h a r i d e s (shown to be f r e e of n e u t r a l components by paper chromatography, s o l v e n t B ) . G e l chromatography ( B i o - G e l P-2; 100 X 3 cm) of the n e u t r a l components p r o v i d e d a n e u t r a l d i s a c c h a r i d e 1_ (9 mg).Compound 1_ had [ajp +3.7 u ( c 0.82, w a t e r ) . The 1H n.m.r. spectrum (0^0, 90 °C) showed a d o u b l e t ( J , ,6 Hz) at 61.28 c h a r a c t e r i s t i c of j 6 CH-j of rhamnose, and anomeric s i g n a l s at §4.63 (IH, J ^  ^1 Hz), 4.84(0.5H, J <1 Hz), and 5.36 (0.5H, J 1 H z ) . 1 4 See l , z l , z Table I I I . l , page 44 and Appendix, spectrum no. 5. The 1 3 C n.m.r. spectrum showed s i g n a l s at 17.6 p.p.m. from Cr6 of rhamnose, 61.4 p.p.m. from C-6 of g l u c o s e , and i n the anomeric r e g i o n , 93.7 and 105.1 p.p.m. In a d d i t i o n a s i g n a l - 66 -at 81.7 p.p.m. i s a t t r i b u t a b l e t o the l i n k a g e c a r b o n . ^ See Table I I I . 1 , page 44 and Appendix, spectrum no. 6. Hakomori m e t h y l a t i o n of 1_ (6 mg) was performed i n d i m e t h y l s u l f o x i d e (4 ml) u s i n g dim e t h y l s u 1 f i n y l anion (2 ml, 4 h) and methyl i o d i d e (1 ml, 1 h ) . The m i x t u r e was then t r a n s f e r r e d to a s e p a r a t o r y f u n n e l w i t h water (15 m l ) , n e u t r a l i z e d w i t h 10% a c e t i c a c i d (2 m l ) , and e x t r a c t e d w i t h c h l o r o f o r m (3 X 10 m l ) . The combined c h l o r o f o r m e x t r a c t s were back e x t r a c t e d w i t h H^ O (4 X 10 ml) and evaporated to dryness under reduced p r e s s u r e . Any r e s i d u a l s o l v e n t was removed by f r e e z e - d r y i n g w i t h water (15 ml) s e v e r a l t i m e s . H y d r o l y s i s of the m e t h y l a t e d d i s a c c h a r i d e w i t h t r i f l u o ro a ce t i c a c i d (2 M_, 16 h, 95°C), r e d u c t i o n and a c e t y l a t i o n y i e l d e d a l d i t o l a c e t a t e s of 3,4-di-0-methy1rhamnose and 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e i n the r a t i o 1:1 ( g . l . c : column B ). G.l.c.-m.s. confirmed the assignments of the above components (see Table I I I . 3 , column V , page50 f o r r e l a t i v e amounts and column d e t a i l s ) . Compound _1 i s t h e r e f o r e 2-0 - 3-D-glu copy ran o sy 1 - L - rhamn op y ranose Gel chromatography ( B i o - G e l ; 150 X 3 cm) of the a c i d i c components p r o v i d e d an a l d o b i o u r o n i c a c i d 2 (47 mg) and an a c i d i c t e t r a s a c c h a r i d e 3_ (92 mg) . The a l d o b i o u r o n i c a c i d 2 (R , 0.87, s o l v e n t A) had — g l c ' [ a ] D +111° (c 1.12,water). The 1H n.m.r. spectrum (D 20, 90 UC) showed anomeric s i g n a l s at 65.17 (1.6H, 2 b r o a d ) and 4.87 (0.4H, J , abroad), as w e l l as, two u n r e s o l v e d d o u b l e t s around 1 5 ^ 61.33 (3H, J r , 6 Hz) r e p r e s e n t i n g the CH^ of rhamnose (see j, 6 J - 67 -Table I I I . 1 , page 44 and Appendix, spectrum no. 8 ) . Hakomori m e t h y l a t i o n of 2 (24 mg) y i e l d e d p e r m e t h y l a t e d a l d o b i o u r o n i c a c i d which was then reduced w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g t e t r a h y d r o f u r a n . The n e u t r a l , m e t h y l a t e d d i s a c c h a r i d was h y d r o l y z e d , reduced, and a c e t y l a t e d y i e l d i n g e q uimolar amounts of 2,4-di-0-methy1-rhamnose and 2 , 3 , 4 - t r i - O - m e t h y l -g l u c o s e as t h e i r a l d i t o l a c e t a t e s (see Table I I I . 3 , column IV, page 50). I d e n t i f i c a t i o n «/as made by comparative g . l . c . and g.l.c.-m.s. a n a l y s i s (column B ) . Compound J5 ( R g l c 0 . 4 8 , s o l v e n t A) h a d [ a ] D +38° (c 1.38, w a t e r ) . The "''H n.m.r. spectrum (D2O, 90°C) showed s i g n a l s i n the anomeric r e g i o n at 65.17 (1.5H, b r o a d ) , 5.11 (1H, J „ 2 Hz 1 > ^ 4.87 (0.5H, s i n g l e t ) , and 4.57 (1H, J 7-8 Hz), as w e l l as, • 1 > ^ a group of d o u b l e t s around 61.31 (6H, 3^ ^ 6 Hz) from CH^ of rhamnose (see Table I I I . 1, page 44, and Appendix, spectrum no. 9 ) . The "^C n.m.r. spectrum showed s i g n a l s due to anomeric carbon atoms at 105.0, 100.5, 96.6, 96.2, and 94.4 p.p.m., due to C-6 o f g l u c o s e at 61.4 p.p.m., and due to C-6 rhamnose (two components) at 17.7 and 17.3 p.p.m.(see Table I I I . l , page 44 and Appendix, spectrum no. 10). Hakomori m e t h y l a t i o n of 2_ (35 mg) gave the p e r m e t h y l a t e d d e r i v a t i v e which was c o n v e r t e d to a l d i t o l a c e t a t e s (as f o r compound 2) y i e l d i n g f o u r compor- ::. ne n t s . G . l . c . and g.l.c.-m.s. (column B) i d e n t i f i e d t hese as being the a l d i t o l a c e t a t e s of 3,4-di-0-methylrhamnose,2,4-di-0-methylrhamnose, 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l g l u c o s e , and 2,3-di-0-methy1-glucose (see Ta b l e I I I . 3 , column I I I , page 50). The sm a l l amount of 2 , 3-d i-0-m eth y 1 g l u cose i s most l i k e l y due to - 68 -an un der-redu ced p r o duct of the methyl e s t e r a f t e r m e t h y l a t i o n of 2- Compound 3 i s , t h e r e f o r e , 3-D-Gl cp-(1-2 )-ct-L-Rhap-(1-4) - a-D-Gl cA p-(1-3 )-L-Rh ap , knowing what the s t r u c t u r e s of com-pounds 1_ and 2_ a r e . P e r i o d a t e o x i d a t i o n of p o l y s a c c h a r i d e 1 ^ C a p s u l a r p o l y s a c c h a r i d e (529 mg) i n the sodium s a l t form was d i s s o l v e d i n water (100 ml) and to t h i s was added a s o l u t i o n (100 ml) which was 0.1 M_ of sodium p e r i o d a t e and 0.4 18 H of sodium p e r c h l o r a t e . The s o l u t i o n was kept i n the dark at 4°C. A f t e r 120 h, e t h y l e n e g l y c o l (10 ml) was added and the s o l u t i o n was s t i r r e d 1 h b e f o r e d i a l y s i s f o r 3 days. F o l l o w i n g r e d u c t i o n w i t h sodium b o r o h y d r i d e (1 g) f o r 24 h, a d d i t i o n of 10% a c e t i c a c i d to d e s t r o y excess h y d r i d e (pH 6), and d i a l y s i s , the s o l u t i o n was f r e e z e - d r i e d y i e l d i n g 3 50 mg of p o l y m e r i c m a t e r i a l . To ensure complete o x i d a t i o n , the product from the f i r s t p e r i o d a t e o x i d a t i o n was d i s s o l v e d i n water (50 ml) and an equal amount of the s o l u t i o n of sodium p e r i o d a t e (0.1 FO and sodium p e r c h l o r a t e (0.4 M_) was added. The m i x t u r e was kept i n the dark at 4°C f o r 94 h. A d d i t i o n of e t h y l e n e g l y c o l (5 m l ) , d i a l y s i s , and f r e e z e - d r y i n g gave 324 mg of p o l y a l d e h y d e . F u r t h e r treatment w i t h sodium b o r o h y d r i d e (0.2 g ) , d i a l y s i s and f r e e z e - d r y i n g y i e l d e d 303 mg of p o l y o l which was c o n s i d e r e d to have undergone complete p e r i o d a t e o x i d a t i o n . Usual d e r i v a t i v e s were prepared w i t h a sample (15 mg) of the p o l y o l by m e t h a n o l y s i s w i t h 3% m e t h a n o l i c h y d r o c h l o r i c - 69 -a c i d , r e d u c t i o n w i t h sodium b o r o h y d r i d e i n anhydrous methanol, h y d r o l y s i s , r e d u c t i o n and a c e t y l a t i o n . The product was a n a l y z e d by comparative g . l . c . (column A; programmed at 150°for 4 min and then at 4 °/min to 260°C) w i t h a u t h e n t i c s t a n d a r d s . Only two compounds were p r e s e n t , i d e n t i f i e d as e r y t h r i t o l t e t r a -a c e t a t e and r h a m n i t o l p e n t a c c e t a t e i n the r a t i o 1:1. 19 S e l e c t i v e p e r i o d a t e o x i d a t i o n of p o l y s a c c h a r i d e A sample (525 mg) of p o l y s a c c h a r i d e i n the sodium s a l t form was d i s s o l v e d i n water (100 ml) and to i t was added a s o l u t i o n of 0.1 M_ sodium p e r i o d a t e (100 m l ) . The r e a c t i o n o was a l l o w e d to proceed at 4 C i n the dark and the p e r i o d a t e consumption was monitored by removing 1 ml a l i q u o t s which were 2 8 a n a l y z e d by the F l e u r y - L a n g e method. P e r i o d a t e consumption reached a l e v e l of 2.6 moles per r e p e a t i n g u n i t of K l e b s i e l l a K17 a f t e r only 1.7 h,whereupon the r e a c t i o n was t e r m i n a t e d w i t h e t h y l e n e g l y c o l (10 m l ) . F o l l o w i n g d i a l y s i s , r e d u c t i o n w i t h sodium b o r o h y d r i d e , d i a l y s i s , and f r e e z e - d r y i n g , 396 mg of p o l y m e r i c m a t e r i a l was i s o l a t e d . The i s o l a t e d m a t e r i a l (17 mg) was a n a l y z e d f o r sugar components i n the u s u a l manner: m e t h a n o l y s i s , r e d u c t i o n , h y d r o l y s i s , r e d u c t i o n , and a c e t y l a t i o n . G . l . c . data (column A) showed t h a t the p e r a c e t a t e s of e r y t h r i t o l , r h a m n i t o l , and g l u c i t o l were p r e s e n t i n the r a t i o n 1:3.2:1.9 r e s p e c t i v e l y . The v o l a t i l e a c e t a t e s of e t h y l e n e g l y c o l and 1 , 2 - p r o p a n e d i o l were l o s t under d i m i n i s h e d p r e s s u r e d u r i n g work-up. The p e r i o d a t e o x i d i z e d m a t e r i a l (120 mg) was s u b j e c t e d 20 to Smith d e g r a d a t i o n u s i n g 0.5 M_ t r i f l u o r o a c e t i c a c i d at room - 7o : -temperature f o r 17 h. F o l l o w i n g d i a l y s i s and f r e e z e - d r y i n g ( y i e l d 59 mg), the r e s u l t a n t polymer P_l gave s i g n a l s i n the 1 3 C n.m.r. spectrum at 104.8, 101.2, 100.0, and 96.0 p.p.m. in the anomeric r e g i o n . S i g n a l s were a l s o observed at 6 3 . 4 , 61.5, and 61.2 p.p.m. a t t r i b u t a b l e to carbons of a primary a l c o h o l group from C-6 of a hexose and C - l and C-4 of e r y t h r i t o l . F i n a l l y , two s i g n a l s at 17.6 and 17.4 p.p.m. were a s s i g n e d to CH-j of two 6-deoxy sugars (See Appendix, spectrum no. 4 ) . The o x i d i z e d and degraded p o l y s a c c h a r i d e Pl_ was then m e t h y l a t e d under Hakomori c o n d i t i o n s f o l l o w e d by a P u r d i e t r e a t m e n t (absence of h y d r o x y l a b s o r p t i o n i n the i n f r a r e d spectrum). T h i s m e t h y l a t e d m a t e r i a l was reduced o v e r n i g h t w i t h l i t h i u m aluminum h y d r i d e i n r e f l u x i n g t e t r a h y d r o f u r a n , hydro-l z e d , reduced, and a c e t y l a t e d . A n a l y s i s by g . l . c . and g.l.c.-m.s. r e v e a l e d t h a t the a l d i t o l a c e t a t e s of 3,4-di-0-methy1rhamnose, 2,4-di-0-methylrhamnose, 2 , 3 , 6 - t r i - 0 - m e t h y 1 g l u c o s e , and 2 , 3 - d i -0-methy1glucose were o b t a i n e d , as w e l l as, minor peaks c o r r e s -ponding to 1 , 3 , 4 - 1 r i - 0 - m e t h y 1 e r y t h r i t o l and 2 , 3 , 4- 1ri-0-methy1-rhamnose. (See Table I I I . 3 , column I I , page 50 f o r r e l a t i v e amounts and column d e t a i l s . ) A subsequent s e l e c t i v e p e r i o d a t e o x i d a t i o n was performed by d i s s o l v i n g K17 p o l y s a c c h a r i d e (289 mg, s a l t form) i n water (50 m l ) , and adding a t h r e e - f o l d excess of sodium p e r i o d a t e (0 . 0 2 8 M, 50 m l ) . R e a c t i o n c o n d i t i o n s and a n a l y s i s of p e r i o d a t e consumption were the same as d e s c r i b e d above. A f t e r 2 h the r e a c t i o n was stopped by adding e t h y l e n e g l y c o l , p e r i o d a t e consumption had reached 1.1 moles per r e p e a t i n g u n i t . I s o l a t i o n - 71 -of the p o l y o l as d e s c r i b e d above produced 238 mg of m a t e r i a l . The p o l y o l (90 mg) was then s u b j e c t e d to Smith d e g r a d a t i o n u s i n g 0. 5 M_ t r i f 1 uoroaceti c a c i d at room temperature f o r 17 h. A f t e r d i a l y s i s and f r e e z e - d r y i n g , the p o l y s a c c h a r i d e PI a was c o n s i d e r e d to be n e a r l y q u a n t i t a t i v e l y f r e e of t e r m i n a l rham-nose branch u n i t s , i . e . a s t r a i g h t chain polymer ( y i e l d 76 mg). T h i s p o l y s a c c h a r i d e PI a o b t a i n e d i n the above manner was taken to be i n a purer form than P^L from the i n i t i a l s e l e c t i v e p e r i o d a t e o x i d a t i o n experiment, i . e . v i r t u a l l y no o v e r - o x i d a t i o n took p l a c e , as judged by the n.m.r. s p e c t r a . The "*"H n.m.r. spectrum (D2O, 90 UC) of p a r t i a l l y d e p o l y m e r i z e d P l a (0.4 t r i -f l u o r o a c e t i c a c i d , 20 min, 95°C) c o n t a i n e d two o v e r l a p p i n g d o u b l e t s at 61-27 (3H, J , , 6 Hz) and 1.34 (3H, J , , 6 Hz) r e p r e s e n t i n g the CH^ of two rhamnose components, and anomeric s i g n a l s at 64.56 (1H, J 7.5 Hz), 4.87 (1H, s i n g l e t ) , 5.11 (1H, s i n g l e t ) , and 5.17 (1H, J , „ 3 Hz). See Table I I I . l , page 44, and Appendix, spectrum no. 3. The C n.m.r. spectrum of P l a c o n t a i n e d a l a r g e s i g n a l at 17.5 p.p.m. r e p r e s e n t i n g the C-6 of more than one rhamnose, a s i g n a l at 61.4 p.p.m. from C-6 of g l u c o s e , and f o u r s i g n a l s i n the anomeric r e g i o n at 96.1, 100.3, 101.3, and 104.8 p.p.m. (see Table I I I . l , page 44). P a r t i a l l y d e p o l y m e r i z e d P l a had £ofj ^  + 44° • (_c 1.81, a c i d d e g r a d a t i o n ; ' A sample of c a r e f u l l y d r i e d , m e t h y l a t e d p o l y s a c c h a r i d e (99 mg) and t o l u e n e - p - s u 1 f o n i c a c i d ( t r a c e ) were s e a l e d i n a 13 water) . B a s e - c a t a l y z e d deqradat ion of me t h y l a t e d p o l y s a c c h a r i d e ( U r o n i c - 72 -f l a s k w i t h a rubber cap. With the a i d of a s y r i n g e , d i m e t h y l -s u l f o x i d e (19 ml) and 2,2-dimethoxypropane (1 ml) were added. The m i x t u r e was s t i r r e d under n i t r o g e n f o r s e v e r a l hours. D i m e t h y l s u 1 f i n y 1 anion (10 ml) was then added by s y r i n g e and the r e a c t i o n was s t i r r e d f o r 18 h at room tem p e r a t u r e . F o l -l o w i n g the slow a d d i t i o n of 50% a c e t i c a c i d (~10 ml) to a d j u s t the pH t o 6, the s o l u t i o n was d i l u t e d w i t h water (50 ml) and e x t r a c t e d w i t h c h l o r o f d r m (3 :x 25 m l ) . The combined c h l o r o f o r m e x t r a c t s were back e x t r a c t e d w i t h water (4 X 25 ml) and evapor-ated to dryness under reduced, p r e s s u r e and f r e e z e - d r i e d . H a l f the degraded m a t e r i a l was e t h y l a t e d u s i n g e t h y l i o d i d e and s i l v e r o x i d e . H y d r o l y s i s of a p o r t i o n of the e t h y l a t e d m a t e r i a l and a n a l y s i s by g . l . c . and g.l.c.-m.s. as a l d i t o l a c e t a t e s gave peaks c o r r e s p o n d i n g to 2,3,4-tri-0-methy1rhamnose, 3 - 0 - e t h y l -2,4-di-0-methylrhamnose, 2,4-di-0-methy1rhamnose, 4-0-methyl-rhamnose, and 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e (see Table I I I . 4 , columns I and I I , page 56 f o r r e l a t i v e amounts and column d e t a i l s ) Another e t h y l a t i o n of the e t h y l a t e d m a t e r i a l and subsequent a n a l y s i s as a l d i t o l a c e t a t e s d i d not change the r e l a t i v e amounts of the components mentioned above. A second u r o n i c a c i d d e g r a d a t i o n was performed on m e t h y l a t e d K17 p o l y s a c c h a r i d e (36 mg) u s i n g the above procedure w i t h the e x c e p t i o n t h a t e t h y l a t i o n was done d i r e c t l y w i t h o u t 2 2 i s o l a t i o n of the degraded m a t e r i a l . That i s , a f t e r r e a c t i o n w i t h d i m e t h y l s u l f i n y l anion f o r 18 h, e t h y l i o d i d e (3 ml) was added dropwise d i r e c t l y i n t o the b a s i c s o l u t i o n u s i n g a s y r i n g e and e x t e r n a l c o o l i n g . A f t e r 1 h, 50?o a c e t i c a c i d (10 ml) was added s l o w l y to a d j u s t the pH to 6. E x t r a c t i o n , back e x t r a c t i o n , - 73 -and f r e e z e - d r y i n g y i e l d e d the e t h y l a t e d , degraded m a t e r i a l . H y d r o l y s i s , r e d u c t i o n , a c e t y l a t i o n , and g . l . c . and g.l.c.-m.s. a n a l y s e s showed the same components were present as i n the f i r s t u r o n i c a c i d d e g r a d a t i o n . However, a h i g h e r r a t i o of 3-0-ethyl-2,4-di-0-methylrhamnose to 2,4-di-0-methylrhamnose i s i n d i c a t e d even though not a l l the 2,4-di-0-methy1rhamnose had been ethy-l a t e d . (See Table I I I . 4 , column I I I , page56 f o r r e l a t i v e amounts and column d e t a i l s . ) - 74 -111. 5 B i b l i o g r a p h y f o r Sect-i ori'- J I I 1. W. Nimmich, A c t a B i o l . Med. Ger., 2_6, 397( 1971). 2. W. Nimmich, Z. M i c r o b i o l . Immunol., 154, 117(1968). 3. G.G.S. Dutton, K. L. Mackie, A. V . Savage, and M. D. Stephenson, Carbohyd. Res., 6j6, 125( 1978 ). 4. A. M. Zanlungo, u n p u b l i s h e d r e s u l t s from t h i s l a b o r a t o r y . 5. M. H e i d e l b e r g e r and W. Nimmich, Immunochemistry , 13, 67(1976). 6. M. H e i d e l b e r g e r , W. Nimmich, J . E r i k s e n , G.G.S. Dutton, S. S t i r m , and C. T. Fang, A c t a Path. M i c r o b i o l . Scand. S e c t . C, 8_3, 397( 1975). 7. M. T. Yang, Ph.D. t h e s i s , U n i v e r s i t y of B r i t i s h Columbia (1974). 8. G. M. B e b a u l t , J . M. B e r r y , Y. M. Choy, G.G.S. Dutton, N. F u n n e l l , L. D. Hayward, and A. M. Stephen, Can. J . Chem., 51, 324(1973). 9. J.-P. J o s e l e a u , M. LePeyre, M. Vignon, and G.G.S. Dutton, Carbohyd. Res., 67., 197 ( 1978 ). 10. A. Zanlungo, p e r s o n a l communication. 11. S. Hakomori, J . Biochem, (Tokyo), _55, 20 5( 1964). 12. H. E. Conrad, Methods Carbohyd. Chem., 6, 361(1972). 13. H. B j o r n d a l , C. G. H e l l e r q v i s t , B. L i n d b e r g , and S. Svensson, Angew. Chem. I n t . Ed. E n g l . , 9, 610(1970). 14. R.R. King and C. T. B i s h o p , Can. J . Chem., _52, 3913( 1974). 15. P. Colson and R. R. K i n g , Carbohyd. Res., _47, 1( 1976). 16. S. J . Angyal and V . A. P i c k l e s , A u s t . J . Chem., 2_5, 169 5( 1972). 17. G. W. Hay, B. A. Lewis, and F. Smith, Methods Carbohyd. Chem., 5, 357(1965). 18. J . E. S c o t t and R. J . H a r b i n s o n , H i s t o c h e m i e , 1_4, 215( 1968). 19. K. M. Aalmo, M. F. Ishak, and T. J . P a i n t e r , Carbohyd Res., 63, C3( 1978 ). 20. I . J . G o l d s t e i n , G. W. Hay, B. A. Lewis, and F. Smith, Methods Carbohyd. Chem., 5,361( 1965). - 75 -21. B. L i n d b e r g , J . Lonngren and J . L. Thompson, Carbohyd. Res., 2_8, 351( 1973). 22. G. 0. A s p i n a l l and K. -G . R o s e l l , Carbohyd. Res., 57_, C23( 1977). 23. G.G.S. Dutton and M. T. Yang, C a rbo h y d.. R es. , b9_, 179 ( 1977). 24. G. M. B e b a u l t , G.G.S. Du t t o n , N.A. F u n n e l l , and K. L. Mackie, Carbohyd. Res., 63, 183(1978). 25. W. E. T r e v e l y a n , D. P. P r o c t o r and J . S. H a r r i s o n , N ature, 166, 444(1950). 26. , E . L . H i r s t and E. P e r c i v a l , Methods Carbohyd Chem., _5> 287 ( 1965). 27. C. Galanos, K. Himmelspach, and 0. L u d e r i t z , European J . Biochem., 8_, 332( 1969 ). 28. R. D. G u t h r i e , Methods Carbohyd. Chem., 1_, 435( 1962). 29. D. P. Sweet, R. H. S h a p i r o , and P. A l b e r s h e i m , Carbohyd. Res., 40, 217 ( 1975). STRUCTURAL INVESTIGATION OF K l e b s i e l l a SEROTYPE K44 CAPSULAR POLYSACCHARIDE - 77 -IV . 1 A b s t r a c t The t e c h n i q u e s of m e t h y l a t i o n a n a l y s i s , u r o n i c a c i d d e g r a d a t i o n , p e r i o d a t e o x i d a t i o n , and p a r t i a l h y d r o l y s i s were used to i n v e s t i g a t e the s t r u c t u r e of K l eb s i e l l a s e r o t y p e K44 c a p s u l a r p o l y s a c c h a r i d e . N u c l e a r magnetic resonance s p e c t r o -scopy was used e x t e n s i v e l y to determine the anomeric n a t u r e of g l y c o s i d e l i n k a g e s i n the fragments i s o l a t e d from the v a r i o u s d e g r a d a t i v e p r o cedures used. The e x i s t e n c e of an 0 - a c e t y l group was e s t a b l i s h e d , but p r e s e n t o n l y i n the approximate r a t i o of one per f i f t e e n sugar r e s i d u e s . The s t r u c t u r e was shown to c o n s i s t of the f o l l o w i n g p e n t a s a c c h a r i d e r e p e a t i n g u n i t . +. 3) - 8-D-G l c p - ( 1*4) -ouD-Glcp-( 1*4) - B.-D-GlcAp-( 1*2) -o-L-Rhap-( l * 3 ) - a - L - R h a p - ( l -- 78 -IV . 2 In t ro du ct ion In a q u a l i t a t i v e a n a l y s i s of the c a p s u l a r p o l y s a c c h a r i d e 1 2 from Kleb s i e l 1 a K44, Nimmich ' r e p o r t e d the presence of g l u c u r o n i c a c i d , g l u c o s e , and rhamnose. H e i d e l b e r g e r and Nimmich 3 p r e d i c t e d t h a t two or t h r e e c o n s e c u t i v e rhamnose r e s i d u e s would be found i n K44, a c c o r d i n g to immunochemical r e l a t i o n s h i p s between the f a m i l i e s of pneumococci and K l e b s i e l l a b a c t e r i a . In an e f f o r t to c o r r e l a t e chemical s t r u c t u r e w i t h i m m u n o l o g i c a l s p e c i f i c i t y we now r e p o r t on the s t r u c t u r a l d e t e r m i n a t i o n of s e r o t y p e K44 c a p s u l a r p o l y s a c c h a r i d e . K-types 17 ( see S e c t i o n I I I , page 40) and 2 3 4 , whose c a p s u l a r p o l y s a c c h a r i d e s have the same q u a l i t a t i v e sugar c o m p o s i t i o n as K44, have been i n v e s t i g a t e d p r e v i o u s l y . - 79 -IV . 3 R e s u l t s and D i s c u s s i o n  Composition and n.m.r. s p e c t r a I s o l a t i o n and p u r i f i c a t i o n of the p o l y s a c c h a r i d e , as p r e v i o u s l y d e s c r i b e d ( S e c t i o n I I I . 4 ) , p r o v i d e d a homogeneous polymer as i n d i c a t e d by e l e c t r o p h o r e s i s . The product had Caj" Q  + 4.0 U(£ 1.08, water) and a m o l e c u l a r weight of 2.6 x lO"' as determined by g e l chromatography. The "''H n.m.r. spectrum"''^' i n d i c a t e d a sharp s i n g l e t at 62.17, c h a r a c t e r i s t i c of 0 - a c e t y l , and the absence o f p y r u v a t e . D e t e r m i n a t i o n of the a c e t y l : s u g a r r a t i o (1:1.5) was made by comparison of the i n t e g r a l s f o r the a c e t a t e s i g n a l and the rhamnose methyl group s i g n a l . In the anomeric r e g i o n ( 64.5-5.5), f i v e proton s i g n a l s were observed, and a s i x - p r o t o n d o u b l e t at 61.28, due to the methyl groups of two 6-deoxy sugars, was a l s o apparent (See Ta b l e IV.1, page 80 and appendix s p e c t r a no.'s 20,21). 1 3 C n.m.r. s p e c t r o s c o p i c i n f o r m a t i o n ^ was i n agree-ment wit h the "'"H n.m.r. dat a , and i n a d d i t i o n , showed the presence of two hexoses (two s i g n a l s were observed at 61.6 and 60.2 p.p.m., i n d i c a t i v e of the s i g n a l s from C-6 of two hexoses; see Table IV.1 and Appendix spectrum no. 22). n.m.r. and 1 3 C n.m.r. s p e c t r a i n d i c a t e the presence of two rhamnose and two hexose r e s i d u e s . From the q u a l i t a t i v e c o m p o s i t i o n of K44 c a p s u l a r p o l y s a c c h a r i d e , i t i s p o s s i b l e to deduce t h a t the re m a i n i n g sugar r e s i d u e i n the r e p e a t i n g u n i t i s t h a t of g l u c u r o n i c a c i d . M e t h a n o l y s i s of K l eb s i e l 1 a K44 p o l y s a c c h a r i d e , r e d u c t i o n of the product w i t h sodium b o r o h y d r i d e i n dry methanol, and TABLE IV.1 N.M.R. data of K l e b s i e l l a K44 c a p s u l a r p o l y s a c c h a r i d e and d e r i v e d o l i g o s a c c h a r i d e s 1 H 13 Compound L 6 a ( c o u p l i n g con s t a n t ) ^ , i h t e g r a l , assignment p.p.m.° assignment (Hz) 1 3 1 2 R h a G1 c—r—e r y t h r i t o l a 8 7 GlcA^-Rha-OH 5. 1 8 ( s ) , 1H, H - l a-Rha 103. 3 C-1 B-Glc 4. 60(8 ), 1H, H-l B - G l c 101. 9 C-1 a-Rha 1. 28(6) , 3H, H-6 Rha 63. ? C-6 G l c 62 . 0 C-1 e r y t h r i t o l 61. 6J [C-4 e r y t h r i t o l 17. 3 C-6 Rha 5. 34 ( b ) , 0.6H, H - l a-Rha-OH 105. 0 C-1 B-G IcA 4. 8 5 ( s ) , 0.4H, H - l B-Rha-OH 93. 8 C-1 a, B-Rha 4. 7 0 ( 6 ) , IH,-, H - l 3_GlcA 17. 6 C-6 Rha 1. 28(6) , 3H, H-6 Rha G 1 c A^Q-^-R h a - i - ^ R h a - 0 H E p a 3a 5. 32(s) , IH, H- 1 a-Rha 105. 1 C- 1 B - G l cA 5. 1 K b ) , 0 . 4H, H -1 a-Rha-OH 101. 6 C- 1 a-Rha 4. 8 8 ( s ) , 0.6H, H -1 B-Rha-0H 94. 8 C- 1 a-Rha-OH 4. 6 9 ( 7 ) , IH, H- 1 B -G 1 cA 94. 2 C- 1 B-Rha-OH 1. 2 9 ( 6 ) , 6H, H-6 Rha 17 . 5 C- 6 Rha TABLE IV . 1 ( c o n t i n u e d ) Gl c H 4a \l c A — -^Rha-0He 5. 42 ( 3 ) , IH, H- 1 a-G l c 105. 1 C- 1 3-GIcA 5. 30(b) , 0.6H, H -1 a-Rha-OH 10 3. 3 C- 1 3-Glc 4. Q 3 ( s ) , 0 . 4H , H -1 6-Rha-OH 99 . 3 C- 1 a-Glc 4. 69(8) , IH, H- 1 B-GlcA 93. 8 C- 1 a, 3-Rha 4. 53(7), 1H, H- 1 3-Glc 61. 41 6 a-Glc 1. 28(6) , 3H, H-6 Rha 60 . 2J L c - 6 '3-Glc 17 . 6 c - 6 Rha G l c 1 4, G l c- — G l c A — •^R h a — a ^flha-OH 5. 4 1 ( 3 ) , IH, H -1 a - G l c 10 5. 2 C-1 3-GlcA 5. 29(b) , IH, H -1 a-Rha 103. 2 C-1 3-Glc 5. 0 9 ( b ) , 0 . 5H, H- 1 a-Rha -OH 101. 7 C-1 a-Rha 4. 8 6 ( s ) , 0 . 5H, H- 1 3-Rha -OH 99 . 4 C-1 a - G } c 4. 69(8) , IH, H -1 8-GlcA 94. 8 C-1 a-Rha-0H 4. 53( 7 ) , IH, H -1 8-Glc 94. 2 C-1 3-Rha-OH 1. 2 7 ( 6 ) , 6H, H -6 Rha 61. 5) fC-6 a - G l c 60 . 2J [C-6 3-G l c 17 . 4 C-6 Rha TABLE IV.1 ( c o n t i n u e d ) 3 P 1 1 4 r i 1.4 r, ,1 2n,. 1 3 D . l —G1 c G1 c G1 c A =R h a R h a p a B a a K44 p o l y s a c c h a r i d e 5.41(4.5), IH, H - l a - G l c 5.28(~1), IH, H-l a,Rha 5.15(1.5), IH, H - l a-Rha 4.71(8), IH, H - l B-GlcA 4.54(8), IH, H-l B - G l c ' l . 2 8 ( 6 . 5 ) , 6H, H-6 Rha 105. 2 C -1 B-GIcA 103. 2 C -1 B-Glc 101. 8 c -1 a-Rha(2) 99 . 8 c -1 a - G l c 61. 6 -6 B-Glc 60 . 2 rip -6 a - G l c 17. 4 c -6 Rha CO ho I F o o t n o t e s : g Chemical s h i f t r e l a t i v e t o i n t e r n a l acetone; 6 2.23 d o w n f i e l d from D.S.S. ^ s , s i n g l e t ; b, broad, unable t o a s s i g n a c c u r a t e c o u p l i n g c o n s t a n t . For H-6 of Rha c o u p l i n g c o n s t a n t r e f e r s - t o J5,6. Chemical s h i f t r e l a t i v e t o i n t e r n a l acetone; 31.07 p.p.m. dovi/nfield from D.S.S. ^Two u n r e s o l v e d d o u b l e t s ; see Appendix, s p e c t r a no. 20,21. e O l i g o s a c c h a r i d e s 3 and 4 were not c o m p l e t e l y pure; minor n.m.r. s i g n a l s are o m i t t e d f o r c l a r i t y (see t e x t f o r d e t a i l s ) . - 83 -then h y d r o l y s i s of the c a r b o x y l - r e d u c e d m a t e r i a l , y i e l d e d a m i x t u r e of rhamnose and g l u c o s e i n the p r o p o r t i o n 45:55. I d e n t i f i c a t i o n was made by g . l . c . a n a l y s i s of t h e i r a l d i t o l a c e t a t e s . Rhamnose and g l u c o s e were shown to be of the L and D c o n f i g u r a t i o n r e s p e c t i v e l y by the c i r c u l a r d i c h r o i s m ^ curves of the c o r r e s p o n d i n g a l d i t o l a c e t a t e s . These r e s u l t s show t h a t the r e p e a t i n g u n i t of K44 p o l y s a c c h a r i d e i s composed of two r e s i d u e s of L-rhamnose, two of D-glucose, one of D - g l u c u r o n i c a c i d , and has t h r e e a- and two 3 - g l y c o s i d i c l i n k a g e s . F i n a l assignments of the anomeric l i n k a g e s were determined a f t e r examination of o l i g o s a c c h a r i d e s d e r i v e d from the o r i g i n a l p o l y s a c c h a r i d e . M e t h y l a t i o n a n a l y s i s 9 M e t h y l a t i o n a n a l y s i s of the a c i d i c p o l y s a c c h a r i d e confirmed t h a t K44 i s composed of a p e n t a s a c c h a r i d e r e p e a t i n g u n i t and i n d i c a t e d t h a t i t i s a l i n e a r p o l y s a c c h a r i d e (see Table IV.2, columns 1 and I I , page 8 4). Complete r e s o l u t i o n of a l l peaks from the g . l . c . a n a l y s i s of the m i x t u r e of p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s was accomplished on columns of OV-17 and OV-225. Lower amounts of the d i m e t h y l g l u c o s e r e s i d u e than expected were o b t a i n e d most', l i k e l y because of the i n c o m p l e t e c a r b o x y l r e d u c t i o n of the g l u c u -r o n o s y l methyl e s t e r . U r o n i c a c i d d e g r a d a t i o n A sample of m e t h y l a t e d K44 c a p s u l a r p o l y s a c c h a r i d e was TABLE IV.2 M e t h y l a t i o n a n a l y s i s of o r i g i n a l and degraded K44 c a p s u l a r p o l y s a c c h a r i d e M e t h y l a t e d s u g a r s 3 b c (as a l d i t o l a c e t a t e s ) M o l e °°  Column B d Column C e I f I I I I I IV 2,3,4-Rha 2-E t - 3 , 4-Rha g 3, 4.-Rha 2,4-Rha 2 , 4, 6-Glc 2,3,6-Glc 2,3-Glc 0 . 48 0 . 77 0 .90 1.56 1. 56 2.14 49 .8 0. 50 0.89 0. 95 1. 51 1.72 2.41 19 . 9 22 . 4 22.0 21.8 13.9 26:4 31.9 34.0 7.7 33. 5 3 2,3,4-Rha = 1, 5-di-0-acety1-2 , 3 , 4 - 1 r i - 0 - m e t h y 1 - L - r h a m n i t o l , e t c . R e t e n t i o n time r e l a t i v e to 1 , 5 - d i - 0 - a c e t y 1 - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y 1 - D - g l u c i t o l . c 2 4 V a l u e s . c o r r e c t e d by use of e f f e c t i v e carbon response f a c t o r s given by A l b e r s h e i m e t a l OV-17 column, programmed at 175° f o r 8 min and then 2 u/min to 210°C. e0V-225 column, programmed at 180°for 4 min then at 2 u/min t o 230°C. f I, o r i g i n a l p o l y s a c c h a r i d e , column B; I I , o r i g i n a l p o l y s a c c h a r i d e , column C; I I I , p o l y -s a c c h a r i d e a f t e r u r o n i c a c i d d e g r a d a t i o n , column C; IV, p o l y s a c c h a r i d e a f t e r p e r i o d a t e o x i d a t i o n - S m i t h d e g r a d a t i o n , compound jL,. column B. 9 l , 5 - d i - 0 - a c e t y l - 2 - 0 - e t h y l - 3 , 4 - d i - O - m e t h y l - L - r h a m n i t o l . ^Remainder of the t o t a l i s made up of 2 - 0 - a c e t y 1 - 1 , 3 , 4 - t r i - 0 - m e t h y l - D - e r y t h r i t o l , 16.7%. - 85 -s u b j e c t e d to a u r o n i c a c i d d e g r a d a t i o n 1 0 , and the product was d i r e c t l y a l k y l a t e d w i t h e t h y l i o d i d e 1 1 . H y d r o l y s i s of 12 the degraded, e t h y l a t e d m a t e r i a l and g.1.c.-m,s. . a n a l y s i s of the a l d i t o l a c e t a t e s y i e l d e d the compounds l i s t e d i n Table IV.2, column I I I , page 84 . Loss of g l u c u r o n i c a c i d r e s i d u e s i n v o l v e s cleavage of the l i n e a r chain and was accom-panied, by f u r t h e r d e g r a d a t i o n of exposed r e d u c i n g - g r o u p s as wit n e s s e d by almost complete l o s s of the 2 , 3, 6-1ri-0-methy1 -gl u c o s e r e s i d u e . S t i l l f u r t h e r d e g r a d a t i o n or " p e e l i n g " was h a l t e d at t h i s - p o i n t because of f o r m a t i o n of the b a s e - s t a b l e , 3-deoxy-hex-2-enopyranose u n i t (see S e c t i o n I I . 4 , page 24 and F i g u r e I I . 7, page 27). The appearance of 2 - 0 - e t h y l - 3 , 4-di-O-methylrhamnose, accompanied by the d i s a p p e a r a n c e of the 3,4-di-0-methy1rhamnose r e s i d u e , determines the sugar l i n k e d to 0-1 of g l u c u r o n i c a c i d . T h e r e f o r e , the sequence of t h r e e sugars of the p e n t a s a c c h a r i d e r e p e a t i n g u n i t of K44 p o l y -s a c c h a r i d e i s *4)-D-Glcp-(l-»-4)-D-GlcAp-(l*2)-L-Rhap-(l-»-. P e r i o d a t e o x i d a t i o n P e r i o d a t e o x i d a t i o n 1 " 5 of p a r t i a l l y d e p o l y m e r i z e d K44 p o l y s a c c h a r i d e (see F i g u r e I I . 8 , page 31) proceeded r a p i d l y w i t h 3.6 moles of p e r i o d a t e being consumed per mole of r e p e a t i n g u n i t a f t e r 24 h ( t h e o r e t i c a l = 3 m o l e s ) . Smith d e g r a d a t i o n 1 ^ of the sodium b o r o h y d r i d e - r e d u c e d , p e r i o d a t e - o x i d i z e d product f o l l o w e d by g e l chromatography y i e l d e d o l i g o m e r 1_, Cofjp -44°. *H and 1 3 C n.m.r. s p e c t r o s c o p y of 1 (see Table IV.1, page 80) i n d i c a t e d the presence of two non - r e d u c i n g anomeric s i g n a l s ; - 86 -one 3-hexose s i g n a l ( J ^ ^  8 Hz) and another s i g n a l ( s i n g l e t ) a t t r i b u t a b l e to an ot-L-rhamn ose r e s i d u e (see Appendix, s p e c t r a no.'s 11,12). In a d d i t i o n , the 1 3 C n.m.r. spectrum showed 15 carbon s i g n a l s ( t h e o r e t i c a l l y 16 s i g n a l s s hould appear but one l a r g e s i g n a l accounted f o r two carbon atoms), i n c l u d i n g t h r e e s i g n a l s at 61-63 p.p.m. ( i n d i c a t i v e of -CH^OH g r o u p s ) , s i g n i f y i n g ' ' . t h a t , b e s i d e s the two hexose s u g a r s , a f o u r carbon f ra gm en t (e r y.th r i t o l ) was pre s e n t on the r e d u c i n g t e r m i n u s . 12 M e t h y l a t i o n of j^, h y d r o l y s i s , and g.l.c.-m.s. of the a l d i t o l a c e t a t e s gave 2,3,4-tri-0-methy1rhamnose, 2 , 4 , 6 - t r i - O - m e t h y l -g l u c o s e , and 1 , 3 , 4 - t r i - 0 - m e t h y 1 e r y t h r i t o l (see Table IV.2, column IV, page 84). The s t r u c t u r e of compound 1 i s thus e s t a b l i s h e d as: a-L-Rhap-(l->-3) - 3-D-G l c p - ( 1*2) -D-ery t h r i t o l (j.) The r e s u l t s o b t a i n e d from u r o n i c a c i d d e g r a d a t i o n and p e r i o d a t e o x i d a t i o n are s u f f i c i e n t to e s t a b l i s h the sequence of the r e p e a t i n g u n i t of K44 p o l y s a c c h a r i d e as bei n g * 3 ) - L -Rhap-(1*3)-D-G 1 cp-(1*4)-D-G1 cp-(1*4)-D-G 1 cAp-(1*2 ) -L-R hap-( 1-^ . In order to a s s i g n the rem a i n i n g anomeric c o n f i g u r a t i o n s to g l y c o s i d i c l i n k a g e s , examination of the pro d u c t s from p a r t i a l h y d r o l y s i s was undert a k e n . P a r t i a l h y d r o l y s i s P a r t i a l , a c i d i c h y d r o l y s i s of K44 p o l y s a c c h a r i d e .and - 87 -s e p a r a t i o n of a c i d i c and n e u t r a l components by ion exchange chromatography y i e l d e d a m i x t u r e of a c i d i c o l i g o m e r s t h a t were s e p a r a t e d by g e l f i l t r a t i o n chromatography. A pure ol i g o m e r (_5) and u n r e s o l v e d components (>) were o b t a i n e d . Compound _5, Ca3 Q + 25°, was shown by "'"H and 1 3 C n.m.r. spe c t r o s c o p y (see Table IV.1, page 80 and Appendix, s p e c t r a no. 's 18,19) to generate two n on - redu c in g , ^-anomeric s i g n a l s , two n on - redu c i n g, a-anomeric s i g n a l s , and two s i g n a l s a t t r i -b u t a b l e t o a r e d u c i n g 6-deoxyhexose. M e t h y l a t i o n , r e d u c t i o n 12 w i t h l i t h i u m aluminum h y d r i d e , h y d r o l y s i s , and g.l.c.-m.s. a n a l y s i s of the a l d i t o l a c e t a t e s produced the compounds l i s t e d i n Table IV.3, column I, page 88. F r a c t i o n _3> H p "» i n i t i a l l y thought to be a pure a l do t r iou r on i c a c i d , c o n t a i n e d a c e r t a i n amount (~20?o) of another a c i d i c t r i m e r as w e l l . These two compounds had e s s e n t i a l l y i d e n t i c a l p r o p e r t i e s i n g e l f i l t r a t i o n chromatography and, hence, were i n s e p a r a b l e by t h i s method. The "'"H and 1 3 C n.m.r. s p e c t r a of the m i x t u r e i n d i c a t e d t h a t the major component (3a) c o n t a i n s one 8 - l i n k e d hexose, one a - l i n k e d rhamnose, and a re d u c i n g rhamnose u n i t (see Table I V . 1 ) . Minor s i g n a l s were a l s o observed i n the 1H (65.43, 0.2 H) and 1 3 C (C-1 at 99.6 and C-6 at 60.9 p.p.m.) n.m.r. s p e c t r a i n d i c a t i v e of an a-hexose (see Appendix, s p e c t r a no.'s 14.15). M e t h y l a t i o n a n a l y s i s (as d e s c r i b e d f o r compound _5) showed the r e s i d u e s and r e l a t i v e p r o p o r t i o n s of the two t r i s a c c h a r i d e s i n v o l v e d (see Table IV.3, column I I I ) . The s t r u c t u r e s of the components of 3 are d i s c u s s e d l a t e r . TABLE IV. 3 M e t h y l a t i o n a n a l y s i s of a c i d i c o l i g o s a c c h a r i d e s h y d r o l y s i s of K44 c a p s u l a r p o l y s a c c h a r i d e i s o l a t e d a f t e r p a r t i a l , a c i d M e t h y l a t e d s u g a r 3 T b Mole %° (as a l d i t o l a c e t a t e ) ^d ^ j 3, 4-Rha 2,4-Rha 2,3,4, 6-Glc 2,3,6-Glc 2, 3, 4-Glc 2, 3-Glc 0 .77 0.90 1.00 1 . 56 1.62 2 . 14 24.8 19 .9 22 . 6 20 . 4 12 .3 27 . 5 7 . 5 28 .0 18 . 1 18 .9 36.2 22.9 6.7 27.0 7.2 61.3 38 .7 g 3,4-Rha = 1 , 2 , 5 - t r i - 0 - a c e t y 1 - 3 , 4 - d i - 0 - m e t h y 1 - L - r h a m n i t o l , e t c . R e t e n t i o n time r e l a t i v e t o 1 , 5 - d i - 0 - a c e t y 1 - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y 1 - D - g l u c i t o l on an OV-17 column, programmed at 175° f o r 8 min and then 2 u/min t o 210°C. c 2 4 Valu e s c o r r e c t e d by use of e f f e c t i v e carbon response f a c t o r s given by A l b e r s h e i m et a l I, p e n t a s a c c h a r i d e , compound _5> U> t e t r a s a c c h a r i d e , component 4-; I I I , t r i s a cch ar i d e, component2; IV, a l d o b i o u r o n i c a c i d , compound 2_. - 89 -Another p a r t i a l h y d r o l y s i s of the p o l y s a c c h a r i d e was performed i n o r d e r to i s o l a t e the a l d o b i o u r o n i c a c i d . A f t e r s e p a r a t i o n of a c i d i c and n e u t r a l components, the a c i d i c com-ponents were s e p a r a t e d by g e l f i l t r a t i o n chromatography. The chromatogram produced from the weight of the f r a c t i o n s c o l l e c t e d i s shown in F i g u r e IV.1, page 80. In a d d i t i o n to components _3 and _5, a s m a l l amount of a l d o b i o u r o n i c a c i d 2 and another m i x t u r e , _4, of t e t r a s a c c h a r i d e s were o b t a i n e d . Compound 2, [>] - 7.0°, was shown by "*"H and 1 3 C n.m.r. spe c t r o s c o p y (see Table IV.1) to generate anomeric s i g n a l s a t t r i b u t a b l e to one 8-nexose and one r e d u c i n g rhamnose u n i t (see Appendix spectrum no. 13). M e t h y l a t i o n of 2, subsequent r e d u c t i o n w i t h l i t h i u m aluminum h y d r i d e , h y d r o l y s i s , and d e r i v a t i z a t i o n as a l d i t o l a c e t a t e s gave 3, 4 - d i - 0 - m e t h y l -rhamnose and 2 , 3 , 4 - t r i - O - m e t h y l g l u c o s e (see Table IV.3, column I V ) . Thus, the s t r u c t u r e of compound 2_ i s shown to be: !-D-GlcAp-(l+-2)-L-Rhap (2) The o c c u r r e n c e of t h i s a l d o b i o u r o n i c a c i d in b a c t e r i a l p o l y -s a c c h a r i d e s has been r e p o r t e d f o r K l eb s i e l l a K-types 361"* and 1 8 1 6 . The components 3. were i d e n t i c a l to those o b t a i n e d in the p r e v i o u s p a r t i a l h y d r o l y s i s experiment, as judged by and 1 3C n.m.r. s p e c t r o s c o p y . The f o l l o w i n g s t r u c t u r e s of 1 2 n 1 0 H 8-WT. (mg) 6j 4H penta-Column: Bio-Gel P-2 (100 x 3 cm). Flow rate: 10 ml/h. Collection: 4 fractions/h after void volume of 150 ml. 20 ' 7o FRACTION NO. loo" M3 O ilo 40 80 F i q u re IV.1 G e l f i l t r a t i o n chromatography s e p a r a t i o n of a m i x t u r e of s a c c h a r i d e s o b t a i n e d from p a r t i a l h y d r o l y s i s of Kleb s i e l l a K 44 p o l y s a c c h a r i d e (0.5 M t r i f l u o r o a c e t i c a c i d , l h , 9 5 U C ) . - 91 -the major component _3_a and minor component _3b are c o n s i s t e n t w i t h the m e t h y l a t i o n and n.m.r. s p e c t r o s c o p i c data o b t a i n e d , and wi t h the known s t r u c t u r e of compound 2. 3-D-GlcAp-(l*2)-a-L-Rhap-(l*3)-L-Rhap (3a) and a - D - G l c p - ( l * 4 ) - 3-D-GlcAp-( 1*2)-L-Rhap (3b) The a l d o t r i o u ron i c a c i d 3a. n a s been i s o l a t e d p r e v i o u s l y i n the s t r u c t u r a l i n v e s t i g a t i o n s of K l e b s i e l l a K36 1'' and K S l 1 ^ . F r a c t i o n 4-, [oQn +41 °, was again a m i x t u r e i n s e p a r a b l e by the g e l f i l t r a t i o n t e c h n i q u e . As f o r f r a c t i o n 3_, a major component 4-a i s d i s c e r n a b l e . The "'"H and 1 3 C n.m.r. s p e c t r a (see Table IV.1) of the m i x t u r e i n d i c a t e d t h a t the major component c o n t a i n s two 3 - l i n k e d hexoses and one a - l i n k e d hexose per r e d u c i n g rhamnose u n i t . S i g n a l s of a minor n a t u r e 1 x 13 were observed in H (°5.10, 0.2 H) and C (101.7 p.p.m.) n.m.r. s p e c t r a i n d i c a t i v e of an a - l i n k e d rhamnose (see Appendix, s p e c t r a no.'s 16,17). M e t h y l a t i o n a n a l y s i s as d e s c r i b e d b e f o r e y i e l d e d the r e s u l t s shown i n Table IV.3, column I I . From these data i t i s p o s s i b l e to propose s t r u c t u r e s f o r the major component _4a and the minor component j4b_, knowing the s t r u c t u r e s of compounds 2_, _3a, and _3b_, as f o l l o w s : 3 -D-G l c p - ( l * 4 ) - a - D - G l c p - ( 1*4) -3-D-GlcAp-( 1*2 )-L-Rhap (_4a) and - 92 -o - D - G l c p - ( l * 4 ) - 3-D-Glcp-( 1*2)- a -L-Rhap-(1*3 )-L-Rhap (4b) Component _5 was i d e n t i c a l to t h a t o b t a i n e d i n the p r e v i o u s p a r t i a l h y d r o l y s i s experiment, as judged by "'"H n.m.r. s p e c t r o s c o p y . Based on the m e t h y l a t i o n and n.m.r. s p e c t r o -s c o p i c data and knowledge of o l i g o s a c c h a r i d e s t r u c t u r e s d i s c u s s e d above, the s t r u c t u r e of compound _5 i s e s t a b l i s h e d as: 3-D-G l c p - ( l * 4 ) - a - D - G l c p - ( 1 * 4 ) - 3-D-G IcAp-(1*2 ) - a-L-R hap-(_5) (1*3)-L-Rhap The evidence p r e s e n t e d f o r K l e b s i e l l a K44 p o l y s a c c h a r i d e i s c o n s i s t e n t w i t h the f o l l o w i n g s t r u c t u r e . * 3 ) - 3 - D - G l c p - ( l * 4 ) - a - D - G l c p - ( 1 * 4 ) - 3-D-GlcAp-(1*2)-a-L-Rhap-(l*3)-°<-L-Rhap-(l* The e x i s t e n c e of an 0 - a c e t y l group on o t h e r than every r e p e a t i n g u n i t i s not uncommon and has been demonstrated i n p r e v i o u s s t r u c t u r a l i n v e s t i g a t i o n s of K l e b s i e l l a c a p s u l a r 18 19 p o l y s a c c h a r i d e s , those of K20 and K59 - 93 -IV . 4 E x p e r i m e n t a l G e n e r a l methods I n s t r u m e n t a t i o n used has been d e s c r i b e d p r e v i o u s l y (see S e c t i o n I I I . 4 , page 59). For descending paper chroma-tography, the f o l l o w i n g s o l v e n t systems (v/v) were used: (A) e t h y l a c e t a t e - a c e t i c a c i d - f o r m i c a c i d - water (18:3.1:4); (B) e t h y l a c e t a t e - p y r i d i n e - water ( 8 : 2 : 1 ) . A n a l y t i c a l g . l . c . s e p a r a t i o n s were performed u s i n g s t a i n l e s s s t e e l columns (1.8 m X 3 mm) w i t h a c a r r i e r gas f l o w r a t e of 20 ml/ min. Columns used were (A) 3% of SP-2340 on S u p e l c o p o r t (100-130 mesh); (B) 3% of 0V-17 on Gas Chrom Q (100-120 mesh); (C) 3% of OV-225 on SPT (100-120 mesh). P r e p a r a t i v e g . l . c . s e p a r a t i o n s were performed u s i n g a l a r g e r (1.8 m X 6.3 mm) column (D) 5% of S i l a r 10C on Gas Chrom Q (100-120 mesh). P r e p a r a t i o n and p r o p e r t i e s of K l e b s i e l l a K44 c a p s u l a r p o l y s a c c h a r i d e A c u l t u r e of K l e b s i e l l a K44 (7730), o b t a i n e d by c o u r t e s y of Dr. I. 0 r s k o v , was grown and i s o l a t e d as d e s c r i b e d p r e v i o u s l y f o r K l e b s i e l l a K17 (see S e c t i o n I I I . 4 , page 59). The i s o l a t e d p o l y s a c c h a r i d e , i n the sodium s a l t form, had [of] ^  +4.0° (£ 1.08, w a t e r ) . P u r i t y of the p o l y s a c c h a r i d e was checked by e l e c t r o -p h o r e s i s u s i n g a l?o s o l u t i o n on c e l l u l o s e a c e t a t e s t r i p s (Sepraphore I I I ; 15 X 2.5 cm) i n v e r o n a l b u f f e r pH 8.6 (LKB-P r o d u k t e r AB, Stockholm 12, Sweden) at 300 V f o r 90 min and then development i n e i t h e r a l c i a n b l u e i n c i t r a t e b u f f e r e d e t h a n o l (pH4) or p e r i o d a t e - S c h i f f reagent. Homogeneity was a l s o confirmed by g e l chromatography c o u r t e s y of Dr. S. C. Churms, - 94 -U n i v e r s i t y of CapeTown, South A f r i c a , and the m o l e c u l a r weight of K44 p o l y s a c c h a r i d e determined to be 2.6 X 1 0 5 . The ''"H n.m.r. spectrum of p a r t i a l l y d e p o l y m e r i z e d K44 p o l y s a c c h a r i d e (0.4 M t r i f l u o r o a c e t i c a c e t i c a c i d , 15 min, 95°C), i n D^ O at 90 u C, r e v e a l e d s i g n a l s c o r r e s p o n d i n g to f i v e anomeric p r o t o n s at 65.41 (IH, J 4.5 Hz), 5.28 (1H,J ~ 1 Hz), 5.15 (IH, J 1.5 Hz), 4.71 (IH, J 8 Hz), and 4.54 (IH, J 8 Hz). 1 , Z 1 , Z 1 , Z Two u n r e s o l v e d d o u b l e t s c e n t e r e d at £1.28 (6H, J , , 6.5 Hz) 5 » & were s i g n a l s a t t r i b u t a b l e to the methyl groups of two 6-deoxy-hexose r e s i d u e s . (see Table IV.1 and Appendix, s p e c t r a no.'s 20,21). A sharp s i n g l e t at <$2.17 i n t e g r a t e d to a p p r o x i m a t e l y one proton and was a s s i g n e d to the methyl group of an a c e t a t e . M i g r a t i o n of the a c e t a t e ' s s i g n a l to 61-91 upon a d d i t i o n of sodium h y d r o x i d e to the sample tube confirmed t h i s assignment. The 1 3 C n.m.r. spectrum of p a r t i a l l y d e p o l y m e r i z e d K44 p o l y s a c c h a r i d e showed f o u r s i g n a l s i n the anomeric r e g i o n at 105.2, 103.2, 101.8, and 99.8 p.p.m. with the s i g n a l at 101.8 p.p.m. being a p p r o x i m a t e l y t w i c e the h e i g h t of the other t h r e e s i g n a l s . S i g n a l s at 61.6 and 60.2 p.p.m. due to C-6 of two hexose u n i t s and at 17.4 p.p.m. due to C-6 of a 6-deoxyhexose u n i t a l s o appeared. No s i g n a l from the CH-j of a c e t a t e was d i s t i n g u i s h a b l e . See Table IV.1, page 80 and Appendix, spectrum no. 22. A n a l y s i s of sugar c o n s t i t u e n t s Sugar a n a l y s i s was performed as p r e v i o u s l y d e s c r i b e d (see S e c t i o n I I I . 4, page 59). The a l d i t o l a c e t a t e s of rhamnose and - 95 -gl u c o s e were i d e n t i f i e d by g . l . c . (column A; programmed at 195° f o r 4 min and then at 2 °/min to 260 °C) and found to be prese n t i n the r a t i o of 45:55 r e s p e c t i v e l y . P r e p a r a t i v e g . l . c , (column D; programmed at 210° and then at 4 u/min to 250 °C) to i s o l a t e the d e r i v a t i v e s and thenc.d. measurement showed MeCN MeCN ^ e 2 1 5 ^ o r ^he r h a m n i t o l p e n t a a c e t a t e andAe^^^ +0.30 f o r the g l u c i t o l h e x a a c e t a t e . M e t h y l a t i o n a n a l y s i s of n a t i v e p o l y s a c c h a r i d e A sample of i s o l a t e d K44 p o l y s a c c h a r i d e was passed through a column of A m b e r l i t e IR-120(H +) r e s i n and then methy-20 l a t e d by the llak om o r i procedure (see S e c t i o n I I I . 4 , page 59). M e t h y l a t i o n was i n c o m p l e t e and treatment w i t h P u r d i e ' s 21 reagents was r e q u i r e d t o o b t a i n a product t h a t showed n e g l i -g i b l e a b s o r p t i o n i n the h y d r o x y l group r e g i o n (~3500 cm-"'") of the i n f r a r e d spectrum. C a r b o x y l r e d u c t i o n of the methy-l a t e d p o l y s a c c h a r i d e w i t h sodium b o r o h y d r i d e i n t e t r a h y d r o -furan - e t h a n o l ( 1 : 1 ) , h y d r o l y s i s w i t h 2 M. t r i f l u o r o a c e t i c a c i d , and r e d u c t i o n w i t h sodium b o r o h y d r i d e was f o l l o w e d by a c e t y l a t i o n i n p y r i d i n e - a c e t i c anyhdride ( 1 : 1 ) . The m i x t u r e of p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s was a n a l y z e d by g . l . c . 12 m.s. . The a l d i t o l a c e t a t e s of 3,4-di-0-methylrhamnose, 2,4-di-0-methylrhamnose, 2 , 4 , 6 - t r i - 0 - m e t h y l g l u c o s e , 2 , 3 , 6 - t r i - 0 -m e t h y l g l u c o s e , and 2 , 3 - d i - 0 - m e t h y l g l u c o s e were i d e n t i f i e d (see Table IV.2, columns I and I I , page 8 4 f o r exact r a t i o s and column suu se d ) . U r o n i c a c i d d e g r a d a t i o n - 96 -10 , 11 M e t h y l a t e d K44 p o l y s a c c h a r i d e (37 mg) was d r i e d c a r e -f u l l y and then, a l o n g w i t h a t r a c e of to l u en e-p-su 1 f on i c a c i d , d i s s o l v e d i n a m i x t u r e (20 ml) of d i m e t h y l s u 1 f o x i d e and 2,2-dimethoxy-propane (19:1) under n i t r o g e n i n a s e a l e d f l a s k . The base, d i m e t h y 1 s u 1 f i n y 1 anion (10 m l ) , was added and a l l o w e d to r e a c t at room temp e r a t u r e . A f t e r 18 h, the degraded m a t e r i a l was a l k y l a t e d directly"'""'" w i t h e t h y l i o d i d e . F o l l o w i n g n e u t r a l i z a t i o n w i t h 5Q% a c e t i c a c i d and a d d i t i o n of water, the e t h y l a t e d , degraded product was i s o l a t e d by p a r t i t i o n between c h l o r o f o r m and the aqueous s o l u t i o n . Hydro-l y s i s of the i s o l a t e d p r o d uct u s i n g 2 M. t r i f l u o ra ce t i c a c i d and g.l.c.-m.s. a n a l y s i s of the a l d i t o l a c e t a t e d e r i v a t i v e s y i e l d e d peaks c o r r e s p o n d i n g to 2-0-ethy1-3,4-di-0-methy1-rhamnose, 2,A-di-0-methy 1rhamnose, 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e , and a minor peak due to 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e . (See Table IV. 2, column I I I , page :8'4 f o r exact r a t i o s and column used.) P e r i o d a t e o x i d a t i o n of K44 p o l y s a c c h a r i d e ' " K44 p o l y s a c c h a r i d e (300 mg) was d i s s o l v e d i n water (50 ml) and the s o l u t i o n was mixed w i t h 0.1 M. sodium p e r i o d a t e (50 m l ) . The r e a c t i o n was a l l o w e d to proceed at 4 UC i n the dark and the p e r i o d a t e consumption was f o l l o w e d by removing 22 1 ml a l i q u o t s which were a n a l y z e d by the F l e u r y - L a n g e method, *In o r d e r to conserve m a t e r i a l , p a r t i a l l y d e p o l y m e r i z e d K44 p o l y s a c c h a r i d e , r e c o v e r e d from an attempted p a r t i a l h y d r o l y s i s experiment, was used i n the p e r i o d a t e s t u d y . The sample of p o l y s a c c h a r i d e had undergone tr e a t m e n t w i t h 0.01 M_ t r i f l u o r o -a c e t i c a c i d , f o r 70 h a t 95°C and then f o r the remainder o f s i x days at room temperature, i n the Galanos appa r atu s 42 3J) N e g l i g i b l e amounts of o l i g o s a c c h a r i d e s were o b t a i n e d and the p o l y s a c c h a r i d e r e c o v e r e d from the d i a l y s i s was c o n s i d e r e d tco l-.b:e e s s e n t i a l l y unchanged. - 97 -P e r i o d a t e consumption was r a p i d and reached a l e v e l of 3.6 moles per r e p e a t i n g u n i t of K l e b s i e l l a K44 ( t h e o r t i c a l = 3 moles) a f t e r 24 h. F o l l o w i n g the a d d i t i o n of e t h y l e n e g l y c o l , d i a l y s i s , r e d u c t i o n w i t h sodium b o r o h y d r i d e , r e - d i a l y s i s , and f r e e z e - d r y i n g , the p o l y a l c o h o l ( p o l y o l ) was o b t a i n e d ( y i e l d 192 mg). 14 Smith d e g r a d a t i o n (0.5 IA_ t r i f l u o roa cet i c a c i d , 17 h, room temperature) of the p o l y o l (105 mg) y i e l d e d a m i x t u r e of pr o d u c t s which were s e p a r a t e d by g e l f i l t r a t i o n chromatography ( B i o - G e l P-2; 100 x 3 cm column). S e v e r a l f r a c t i o n s ( t o t a l weight 82 mg) were o b t a i n e d , but only one was found to be a pure compound, as judged by "''H n.m.r. s p e c t r o s c o p y . A c c o r d i n g to the chromatogram produced from the weight of the f r e e z e -d r i e d , c o l l e c t e d f r a c t i o n s , pure o l i g o s a c c h a r i d e 1_ (6 mg) was co n t a i n e d i n tube numbers 72-76 i n c l u s i v e . Compound J. (R l c 0.44, s o l v e n t B) had [of] D -44° (£0.87, w a t e r ) . In the "''H n.m.r. spectrum, s i g n a l s a t t r i b u t a b l e to anomeric p r o t o n s were observed at 65.18 (IH, s i n g l e t ) and 4.60 (IH, j \ „ 8 Hz), as w e l l as, 1.28 (3H,J. , 6 Hz) due to the 1 , Z 5,6 methyl group of rhamnose (see Table IV.1, page 80 and Appendix, spectrum no. 11). The 1 3 C n.m.r. spectrum showed s i g n a l s at 103.3 and 101.9 p.p.m. due to anomeric carbon atoms, at 63.2, 62.0, and 61.6 p.p.m. due to C-6 of g l u c o s e and C-1 and C-4 of e r y t h r i t o l , and at 17.3 p.p.m. from C-6 of rhamnose (see Table IV.1 and Appendix, spectrum no. 12). Hakomori m e t h y l a t i o n (see S e c t i o n I I I . 4 , page 59) of 1 f o l l o w e d by h y d r o l y s i s , r e d u c t i o n and a c e t y l a t i o n y i e l d e d the a l d i t o l a c e t a t e s 2,3,4-- 98 -t r i - ) - m e t h y l r h a m n o s e and 2 , 4 , 6 - t r i - O - m e t h y l g l u c o s e , as a n a l y s e d by g.l.c.-m.s. Only a minor amount of 2 - 0 - a c e t y l -1 , 3 , 4 - t r i - O - m e t h y l e r y t h r i t o l was present because of i t s v o l a -t i l i t y under reduced p r e s s u r e d u r i n g work-up. See Table IV.2, column IV, page 84 f o r r e l a t i v e amounts and column d e t a i l s . P a r t i a l , a c i d i c h y d r o l y s i s of K44 p o l y s a c c h a r i d e K44 p o l y s a c c h a r i d e (666 mg) was p a r t i a l l y h y d r o l y z e d i n 0.1 M_ t r i f l u o r a c e t i c a c i d at 9 5° C u s i n g an apparatus s i m i l a r 2 3 t o t h a t d e s c r i b e d by Galanos and coworkers . A f t e r 38 h, most of the m a t e r i a l had been d i a l y z e d , only a s m a l l amount (40 mg) of p o l y s a c c h a r i d e remained i n the d i a l y s i s s ac. A l l of the d i a l y z e d m a t e r i a l was then a p p l i e d on a column (30 X 1.5 cm) of Bio-Rad AG1-X2(C1 ) r e s i n i n as s m a l l as volume as p o s s i b l e (5-10 m l ) . N e u t r a l compounds were e l u t e d w i t h water (1600 ml) and f r e e z e - d r i e d ( Y i e l d 340 mg) . A c i d i c compounds were e l u t e d w i t h 10% f o r m i c a c i d (200 m l ) , evaporated to dryness under reduced p r e s s u r e s e v e r a l times w i t h water t o e l i m i n a t e f o r m i c a c i d , and then f r e e z e - d r i e d ( y i e l d 220 mg) . The a c i d i c f r a c t i o n , f r e e from n e u t r a l compounds as judged by paper chromatography ( s o l v e n t B), was s e p a r a t e d by g e l f i l t r a t i o n chromatography ( B i o - G e l P-2; 100 X 3 cm) u s i n g a s o l u t i o n of w a t e r - p y r i d i n e - a c e t i c a c i d (1000:10:4) f o r i r r i g a t i o n at a f l o w r a t e of 10 ml/h. No m a t e r i a l was present i n the v o i d volume (160 ml; blue d e x t r a n ) a f t e r c o l l e c t i o n and f r e e z e - d r y i n g . F r a c t i o n s (2-2.5 ml) were c o l l e c t e d i n t a r e d t u bes, f r e e z e - d r i e d , weighed, and the weights p l o t t e d t o produce a chromatogram. Every t h i r d tube was a n a l y z e d by paper chroma-- 99 -tography ( s o l v e n t A ) . A major component (j?, 32 mg) w i t h Rg^ c 0.14 ( s o l v e n t A) and C°Q n +25° (c_ 1.95, water) was o b t a i n e d . "'"H n.m.r. spectrum o f _5 showed s i g n a l s a t t r i b u t a b l e to anomeric p r o t o n s at 65.41 (IH, J 3 Hz), 5.29 (IH, b r o a d ) , 5.09 (0. 5H, b r o a d ) , 4.86 (0.5H, s i n g l e t ) , 4.69 ( I H , J 8 Hz), and 4.53 (IH, J 7 Hz). A doublet ( J ^ ^  6 Hz) at $1.27 (6H) was a l s o apparent (see Table IV.1, page 80 and Appendix, spectrum no. 18). In the 1 3 C n.m.r. spectrum, f o u r n o n - r e d u c i n g anomeric s i g n a l s at 105. 2, 103. 2, 101 .7, and 99.4 p.p.m. and two r e d u c i n g anomeric s i g n a l s at 94.8 and 94.2 p.p.m. were observed. Two s i g n a l s at 61.5 and 60.2 p.p.m. due to C-6 of two g l u c o s e u n i t s and a l a r g e one at 17.4 p.p.m. due to C-6 of two rhamnose u n i t s were a l s o e v i d e n t (see Table IV . 1 and Appendix, spectrum no. 19). A p o r t i o n (16 mg) of compound jj was m e t h y l a t e d under Hakomori c o n d i t i o n s . The perm e t h y l at ed product was then reduced w i t h l i t h i u m aluminum h y d r i d e i n r e l u x i n g t e t r a h y d r o -fu ran o v e r n i g h t , h y d r o l y z e d w i t h 2 M. t r i f l u o r a c e t i c a c i d , reduced w i t h sodium b o r o h y d r i d e and a c e t y l a t e d i n p y r i d i n e -a c e t i c a n h y dride ( 1 : 1 ) . G.l.c.-m.s. a n a l y s i s i n d i c a t e d the presence of 3,4-di-0-methylrhamnose, 2,4-di-0-methylrhamnose, 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l g l u c o s e , 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e , and 2, 3 - d i - 0 - m e t h y l g l u c o s e as t h e i r a l d i t o l a c e t a t e d e r i v a t i v e s . See. Table IV. 3, column I, page 88 f o r r e l a t i v e amounts and column used. A minor f r a c t i o n (.3; 15 mg), a m i x t u r e i n s e p a r a b l e by t h i s t e c h n i q u e , w i t h R q 1 c 0.77 ( s o l v e n t A) and [of] ^  -19° - 100 -(c 1.44, water) was a l s o o b t a i n e d . The major s i g n a l s i n the •*"H n.m.r. spectrum o c c u r r e d at 65 . 32 , 5. 11, 4 . 88, and 4.69 i n the anomeric r e g i o n and at 6i.29 due to CH^ of rhamnose (see Table I V . 1 ) . A s m a l l s i g n a l at 65.43 which i n t e g r a t e s t o <~0.2 proton i n d i c a t e s the presence of an a-D-glucose component a l s o (see Appendix, spectrum no. 14). In the 1 3 C n.m.r. spectrum, two n o n - r e d u c i n g anomeric s i g n a l s at 105.1 and 101.6 p.p.m. and two r e d u c i n g anomeric s i g n a l s at 94.8 and 94.2 p.p.m., as w e l l as, a s i g n a l at 17.5 p.p.m. from a rhamnose methyl group were observed (see Table I V . 1 ) . Other, l e s s i n t e n s e s i g n a l s were p r e s e n t at 99.6 (n on-redu c i n g a-D-g l u c o s e ) , 93.7 ( r e d u c i n g rhamnose), and 60.9 p.p.m. (C-6 of g l u c o s e ) . See Appendix, spectrum no. 15; Hakomori m e t h y l a t i o n of 3.> f o l l o w e d by ca rbo x y l - redu ct ion , h y d r o l y s i s and d e r i v a -t i z a t i o n were c a r r i e d out under the same c o n d i t i o n s as com-pound _5. G.l.c.-m.s. a n a l y s i s i d e n t i f i e d a m i x t u r e of a l d i t o l a c e t a t e s c o r r e s p o n d i n g t o 3,4-di-0-methylrhamnose, 2 , 4 - d i - 0 -methylrhamnose, 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c o s e , 2 , 3 , 4 - t r i - 0 -m e t h y l g l u c o s e , and 2 , 3 - d i - 0 - m e t h y l g l u c o s e i n the approximate r a t i o n 1.25:1:0.25:1:0.25 (see Table IV.3, column I I I , page 88, f o r exact r a t i o s and column d e t a i l s ) . Another p a r t i a l h y d r o l y s i s experiment was performed on K44 p o l y s a c c h a r i d e (240 mg) under the c o n d i t i o n s of 0.5 M. t r i f l u o r o a c e t i c a c i d at 9 5°C f o r 1 h. A f t e r removal of the a c i d by s e v e r a l s u c c e s s i v e e v a p o r a t i o n s w i t h water and n e u t r a -l i z a t i o n w i t h 0.1 \A sodium h y d r o x i d e , the h y d r o l y z e d m a t e r i a l was s e p a r a t e d i n t o n e u t r a l and a c i d i c f r a c t i o n s u s i n g AG1-X2(C1 ) - 1 0 1 -r e s i n . The a c i d i c c o mponents (200 mg), when s e p a r a t e d by g e l c h r o m a t o g r a p h y (same p r o c e d u r e as d e s c r i b e d p r e v i o u s l y ) , p r o v i d e d an a l d o b i o u r o n i c a c i d (2_; 7 mg), an a c i d i c t r i s a c -c h a r i d e m i x t u r e (3_; 17 mg), an a c i d i c t e t r a s a c c h a r i d e m i x t u r e (4-; 17 mg) , and an a c i d i c p e n t a m e r (_5; 39 mg). See F i g u r e I V . 1 , page 90. The a l d o b i o u r o n i c a c i d 2^^,16 J - ^ - J _ 7 . o u (g_ 0.46, w a t e r ) . The "*"H n.m.r. s p e c t r u m c o n t a i n e d s i g n a l s a t 65.34 (0.6H, b r o a d ) , 4.85 (0.4H, s i n g l e t ) , and 4.70 ( I H J 6 Hz) 1 ? ^  i n t h e a n o m e r i c r e g i o n , as w e l l a s , a t 6 1.28 (3H, 3^.6 Hz) due t o t h e m e t h y l g r o u p o f rhamnose ( s e e T a b l e I V . 1 and A p p e n d i x , s p e c t r u m no. 1 3 ) . In t h e 1 3 C n.m.r. s p e c t r u m , s i g n a l s o c c u r r e d i n t h e a n o m e r i c r e g i o n a t 105.0 (n on - redu c i n g) and 93.8 p.p.m. ( r e d u c i n g ) and a t 17.6 p.p.m. f o r C-6 o f rhamnose ( s e e T a b l e I V . 1 ) . H a k o m o r i m e t h y l a t i o n o f Z, f o l l o w e d by c a r b o x y 1 - r e d u c -t i o n , h y d r o l y s i s and d e r i v a t i z a t i o n i n t h e u s u a l manner p r o d u c e d t h e a l d i t o l a c e t a t e s o f 3 , 4 - d i - O - m e t h y l r h a m n o s e and 2 , 3 , 4 - 1 r i - 0 - m e t h y 1 g l u c o s e ( g . l . c . - m . s . ; T a b l e I V . 3 , column IV, page 88) . F r a c t i o n ~b_ was t h e same m i x t u r e as t h a t o b t a i n e d f r o m t h e f i r s t p a r t i a l h y d r o l y s i s , as j u d g e d by t h e i d e n t i c a l s p e c t r a o b t a i n e d f r o m "''H and 1 3 C n.m.r. s p e c t r o s c o p y . F r a c t i o n 4-, a t e t r a s a c c h a r i d e m i x t u r e w h i c h was n o t c o m p l e t e l y s e p a r a t e d by g e l c h r o m a t o g r a p h y , had []oTJ Q +41° ( c 1. 53,wate r ) . The "'"H n.m.r. s p e c t r u m c o n t a i n e d m a j o r a n o m e r i c s i g n a l s a t 65.42, 5.30, 4.83, 4.69, and 4.53 and two s e t s o f u n r e s o l v e d d o u b l e t s ( J R c 6 Hz) c e n t e r e d a t 61.28 ( s e e T a b l e - 102 -I V . 1 ) . An a d d i t i o n a l s i g n a l (~0.2H) i s apparent at 65.10 s u g g e s t i n g a minor a-L-rhamnose component as an i m p u r i t y (see Appendix, spectrum no. 16). Four major anomeric s i g n a l s are pr e s e n t i n the 1 3 C n.m.r. spectrum at 105.1, 103.3, 99.3, and 93.8 p.p.m., al o n g w i t h 61.4 (C-6 g l u c o s e ) , 60.2 (C-6 g l u c o s e ) and 17.6 p.p.m. (C-6 rhamnose) as l i s t e d i n Table IV.1. The s i g n a l at 101.7 p.p.m. c o n f i r m s the presence of an a-L-rhamnose, i n some form, as an i m p u r i t y (see Appendix, spectrum no. 1 7 ) . D e r i v a t i z a t i o n of 4- i n t o p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s in the u s u a l manner produced a m i x t u r e , i d e n t i f i e d by g.l.c.-m.s., c o r r e s p o n d i n g to 3,4-di-0-methy1rhamnose, 2,4-di-0-methylrham-nose, 2 , 3 , 4 , 6-1 e t ra-0-m e t hy 1 g l u c os e , 2 , 3 , 6-1 ri-0-m e t hy 1 g l u co se , and 2 , 3 - d i - 0 - m e t h y l g l u c o s e (see Table IV.3, column I I ) . Compound _5> the a c i d i c p e n t a s a c c h a r i d e , gave a "''H n.m.r. spectrum i d e n t i c a l to t h a t from the pentamer i s o l a t e d a f t e r the f i r s t p a r t i a l h y d r o l y s i s experiment. - 103 -IV . 5 B i b l i o g r a p h y f o r S e c t i o n IV 1. W. Nimmich, Z. M i c r o b i o l . Immunol., 154, 117(1968). 2. W. Nimmich, A c t a B i o l . Med. Ger., 2_6, 397( 1971). 3. M. H e i d e l b e r g e r and W. Nimmich, Immun o ch emi s t r y , 1_3, 67 (1976) . 4. G.G.S. D u t t o n , K. L. Mackie, A. V. Savage, and M. D. Stephenson, Carbohyd. Res., 6_6, 125( 1978 ). 5. Y. M. Choy, G.G.S. Dutton, A. M. Stephen, and M. T. Yang, A n a l . L e t t . , 5, 675(1972). 6. G. M. B e b a u l t , Y. M. Choy, G.G.S. D u t t o n , N. F u n n e l l , A. M. Stephen, and M. T. Yang, J . B a c t e r i o l . , 1_13, 1345( 1973). 7. J . M. B e r r y , G.G.S. D u t t o n , L. D. H a l l , and K. L. Mackie, Carbohyd. Res., b±, C8(1977). 8. G. M. B e b a u l t , J . M. B e r r y , Y. M. Choy, G.G.S. Dut t o n , N. Funnell, L. D. Hayward, and A. M. Stephen, Can J . Chem., 51, 324(1973). 9. H. B j o r n d a l , C.G. H e l l e r q v i s t , B. L i n d b e r g , and S. Svensson, Angew. Chem. I n t . Ed. E n g l . , 9, 610(1970). 10. B. L i n d b e r g , J . Lonngren, and J . L. Thompson, Carbohyd. Res, 2_8, 351( 1973). 11. G. 0. A s p i n a l l and K. -G . R o s e l l , Carbohyd. Res., _57, c23 (1977) . 12. J . Lonngren and S. Svensson, Adv. Carbohyd.Chem. Biochem., 29_, 41(1974). 13. G. W. Hay, B. A. Lewis, and F. Smith, Methods Carbohyd. Chem., 5, 357( 1965). 14. I . J . G o l d s t e i n , G. W. Hay, B. A. Lewis, and F. Smith, Methods Carbohyd. Chem., 5, 361(1965). 15. G.G.S. Dutton and K. L. Mackie, Carbohyd. Res., 5J5, 49 ( 1977) . 16. G.G.S. Dut t o n , K. L. Mackie, and M. T. Yang, Carbohyd.Res. , 251(1978). 17. M. C u r v a l l , B. L i n d b e r g , J . Lonngren, and W. Nimmich, Carbohyd Res., 42, 73(1975). - 104 -18. Y. M. Choy and G.G.S. Du t t o n , Can. J . Chem., _51, 3015(1973). 19. B. L i n d b e r g , J . Lonngren, U. Ruden, and W. Nimmich, Carbohyd Res., 42, 83(1975). 20. S. Hakomori, J . Biochem. (Tokyo), 55, 205(1964). 21. E. L. H i r s t and E. P e r c i v a l , Methods Carbohyd. Chem., _5, 287(1965). 22. R. D. G u t h r i e , Methods Carbohyd. Chem., _1, 435( 1962). 23. C. Galanos, 0. L i / d e r i t z , and K. Himmelspach, Eur. J . Biochem., 8, 332(1969). 24. D. P. Sweet, R. H. S h a p i r o , and P. A l b e r s h e i m , Carbohyd. Res., 40, 217(1975). APPENDIX: N.M.R. SPECTRA K17 p o l y s a c c h a r i d e S o l v e n t D^O Tern p • 8 0 °C S.W. 3000 Hz 3- 0, 4 4.35 65. 15 4. 60 4 6.0 5.0 Spectrum Mo. 1 1 . 30 K17 p o l y s a c c h a r i d e S.W. 8000 Hz A.T. 0.5 sec P.W. 18 ysec P.D. 0 sec N .T . 357, 900 K17 p o l y s a c c h a r i d e P l a S o l v e n t D^ O Temp. 9 0 °C S.W. 1000 Hz K17 n e u t r a l d i s a c c h a r i d e Glc-^-Rha-^OH S o l v e n t D ^ 0 Temp. 95°C S.W. 1000 Hz ••6 5. Spectrum No. 5 . 28 17 . 6 Spectrum No. 6 K17 a l d o b i o u r o n i c G l cA—-Rha~0H a S o l v e n t D^ O Temp. 85 UC S.W. 1000 Hz 6 5. 17 Spectrum No. 7 1.33 K17 a l d o b i o u r o n i c a c i d (47 mg/ml) GlcA - i -^Rha-OH et', S . W . 8 0 0 0 Hz Spectrum No. 8 K17 a c i d i c t e t r a s a c c h a r i d e G1 c-i-^-R h a-^-^G 1 cA^-^R h a~0 H 8 a a S o l v e n t D^O Temp. 35°C S.W. 1000 Hz 4. 52 4. 57 65.17? ! 5.11 4.87 Spectrum- No. 9 K17 a c i d i c t e t r a s a c c h a r i d e (9 2 mg/ml) G 1 c-i-^R h a - — G l cA^-^R h a~OH B a a S.W. 8000 Hz A.T. 0.507 sec P . W . 7 Usee P.D. 0 sec N.T. 160,300 105 Spectrum No. 10 a c e t,o n e 17 . 61. 4 i K44 Smith d e g r a d a t i o n p r o d u c t 1 3 1 2 R h a G1 c e r y t h r i t o l a 3 S o l v e n t D^ O Temp. 95°C S.W. 1000 Hz Spectrum No. 11 1. 28 s . K44 Smith d e g r a d a t i o n product 1 3 1 2 R h a——G1 c—|—e r y t h r i t o l 8000 Hz A . T. 0.5 sec P.W. 15 psec P.D. 0 sec N.T. 90,709 - 118 -i i K44 t r i s a c c h a r i d e (impure) S o l v e n t D 20 Temp. 9 5 °C S.W. 1000 Hz j .j i ! Spectrum No. 14 K44 a l d o t r i o u r o n i c a c i d (impure) Gl cA-^r^-R h a-—^ -R h a~0H S.W. 8 0 0 0 H z A.T. 0.5 sec P.W. 18 usee P.D. 0 sec N.T. 3 43,622 Spectrum No. 15 KA4 t e t r a s a c c h a r i d e (impure) S o l v e n t D^0 Temp. 9 5° C S.W. 1000 Hz Spectrum No. 16 K 4 4 t e t r a s a c c h a r i d e (impure) G 1 c^-A 1 c^-^G 1 C A ^ T ^ - R h a~0H s . W . 8 0 0 0 Hz A . T . 0 . 5 0 7 sec P .W. 2 0 ysec P .D . 0 sec N . f 7 1 4 2 , 0 0 0 i ! ; 1 0 1 . 7 Spectrum N o . 1 7 K44 p e n t a s a c c h a r i d e S o l v e n t D^ O Temp 95 °C S.W. 1000 Hz Spectrum No. 18 K44 p e n t a s a c c h a r i d e G l c — ^ G l c ^ c i c A 1 S , W . 8000 Hz A . T . 0.5 sec P .W . 18 ysec P.D. 0 sec N . T . 438 , 90 0.. Spectrum No. 19 K44 p o l y s a c c h a r i d e S o l v e n t D 20 Temp 9 0u C ~ S .W. 2 500 Hz Spectrum No. 20 K44 p o l y s a c c h a r i d e S o l v e n t D Temp. 90°C S.W. 500 Hz O f f s e t 720 Hz Spectrum No. 21 K4A p o l y s a c c h a r i d e S.W. 8000 Hz A.T. 0.5 sec P.W. 18 ysec P.D. 0 sec ISt.T . 220 , 300 : Spectrum No. 22 

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