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Structural studies on Klebsiella capsular polysaccharides Paulin, Marcel 1980

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STRUCTURAL STUD I ES ON K L E B S I E L L A CAPSULAR POLYSACCHARIDES by MARCEL PAULIN B . S c . , The U n i v e r s i t y o f M o n c t o n , 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f C h e m i s t r y ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l 1980 © . M a r c e l P a u l i n , 1980 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 o r an 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 t h e L i b r a r y s h a l l make 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 and 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 Head 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 Chemistry 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 A p r i l 29, 1980 i i TO MY WEST COAST PARENTS M r . & M r s . A l l e n C o o k e i i a ABSTRACT The g e n u s K I e b s i e l l a i s d i v i s i b l e i n t o a p p r o x i m a t e l y e i g h t y d i f f e r e n t s t r a i n s c h a r a c t e r i z e d on t h e b a s i s o f i m m u n o c h e m i c a l t e s t s . T h e s e G r a m - n e g a t i v e b a c t e r i a p r o d u c e l a r g e c a p s u l a r p o l y s a c c h a r i d e s w h i c h a r e t h e i r a n t i g e n i c d e t e r m i n a n t s . I n o r d e r t o u n d e r s t a n d t h e c h e m i c a l b a s i s o f s e r o l o g i c a l d i f f e r e n t i a t i o n , t h e s t r u c t u r a l i n v e s t i g a t i o n o f a l l 8 0 s t r a i n s i s t a k i n g p l a c e . U n t i l now a b o u t f i f t y s t r u c t u r e s h a v e b e e n e l u c i d a t e d . The c a p s u l a r a n t i g e n s i s o l a t e d f r o m K l e b s i e l l a s e r o t y p e s K53 a n d K74 a r e p r e s e n t e d h e r e a n d w e r e d e t e r m i n e d u s i n g many d i f f e r e n t t e c h n i q u e s . 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 , a n d u r o n i c a c i d d e g r a d a t i o n w e r e u s e d t o s t u d y t h e s e q u e n t i a l a r r a n g e m e n t o f t h e s u g a r c o n s t i t u e n t s i n t h e p o l y s a c c h a r i d e . 1 13 E x t e n s i v e u s e was made o f H - a n d 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 d e t e r m i n e a n o m e r i c l i n k a g e s w i t h i n i s o l a t e d o l i g o s a c c h a r i d e s a n d t h e n a t i v e p o l y s a c c h a r i d e . M e t h o d s s u c h a s g a s l i q u i d c h r o m a t o g r a p h y , g a s 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 , p a p e r c h r o m a t o g r a p h y , a n d p o l a r i m e t r y h a v e b e e n u s e d t o i s o l a t e a n d c h a r a c t e r i z e p r o d u c t s o b t a i n e d f r o m d i f f e r e n t d e g r a d a t i v e t e c h n i q u e s . The c a p s u l a r p o l y s a c c h a r i d e s f r o m K l e b s i e l l a s e r o t y p e s K53 a n d K74 a r e f o u n d t o c o n s i s t o f r e p e a t i n g u n i t s o f t h e f o l l o w i n g s t r u c t u r e s : ^ O - G l cpA ^ y O - M a n j D ^ 0 - M a n p _ ^ 0 - G a l p _ ^ y i - R h a £ 1 a 1 1 L - R h a £ K53 _ln m -^>-Galp_ 1^ 0-Manp_ 1^ 0-Manp_ 1 D-Glc£A 4, 1 D-Galp_ \/6 A CH 3 COOH i v TABLE OF CONTENTS Rage ABSTRACT . ' . i i TABLE OF CONTENTS i v L I S T OF TABLES v f L I S T OF FIGURES v i i ACKNOWLEDGEMENTS v i i i PREFACE i x I INTRODUCTION 2 I I METHODOLOGY OF STRUCTURAL A N A L Y S I S OF POLYSACCHARIDES 8 11.1. I s o l a t i o n a n d P u r i f i c a t i o n 8 11.2. 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 . . . . 9 11.2 . 1 . ^ H - n . m . r . S p e c t r o s c o p y 9 13 11.2 . 2 . C - n . m . r . S p e c t r o s c o p y 14 11.3. T o t a l S u g a r R a t i o 16 11.4. S t r u c t u r a l A n a l y s i s 19 1 1 . 4 . 1 . M e t h y l a t i o n A n a l y s i s . 19 11.4 . 2 . G . l . c . 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 . . . 22 11.4 .3. Mass S p e c t r o m e t r y 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 24 11.5 . S u g a r S e q u e n c e D e t e r m i n a t i o n by D e g r a d a t i o n M e t h o d s 26 11.5 . 1 . P a r t i a l H y d r o l y s i s 26 11.5 . 2 . U r o n i c A c i d D e g r a d a t i o n 30 V P a o e I I . 6 . D e t e r m i n a t i o n o f D - o r L - C o n f i g u r a t i o n o f Component S u g a r s . . . . . . 33 I I I STRUCTURAL INVESTIGATION OF K L E B S I E L L A SEROTYPE K53 CAPSULAR POLYSACCHARIDE 34 I I I . 1 . A b s t r a c t 35 1 1 1 . 2 . I n t r o d u c t i o n 35 111.3 . R e s u l t s a n d D i s c u s s i o n 36 111.4. E x p e r i m e n t a l 50 I V , STRUCTURAL INVESTIGATION OF K L E B S I E L L A SEROTYPE K74 CAPSULAR POLYSACCHARIDE . 58 I V . 1. A b s t r a c t 59 I V . 2 . I n t r o d u c t i o n 60 I V . 3 . R e s u l t s a n d D i s c u s s i o n 61 I V . 4 . E x p e r i m e n t a l 74 V BIBLIOGRAPHY 79 APPENDIX I : S t r u c t u r a l P a t t e r n s o f 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 84 APPENDIX I I . 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 ( K 1 - K 8 3 ) G r o u p e d A c c o r d i n g t o C h e m o t y p e 88 APPENDIX I I I . N . m . r . S p e c t r a 89 v i L I S T OF TABLES T a b l e Rage 111.1 N . m . r . D a t a o f K l e b s i e l l a K53 C a p s u l a r P o l y s a c c h a r i d e a n d D e r i v e d P o l y - a n d O l i g o s a c c h a r i d e s 38 1 1 1 . 2 M e t h y l a t i o n A n a l y s e s o f K53 C a p s u l a r P o l y s a c c h a r i d e a n d D e r i v e d P o l y - a n d O l i g o s a c c h a r i d e s 4 0 I V . 1 N . m . r . D a t a o f K l e b s i e l l a K74 C a p s u l a r P o l y s a c c h a r i d e a n d D e r i v e d O l i g o s a c c h a r i d e s . . . . 63 I V . 2 M e t h y l a t i o n A n a l y s e s o f N a t i v e , a n d D e g r a d e d K74 C a p s u l a r P o l y s a c c h a r i d e a n d D e r i v e d O l i g o s a c c h a r i d e s 66 v i i L I S T OF FIGURES F i g u r e P a g e I . 1 . 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 o f a b a c t e r i a l c e l l w a l l 3 I I . 1 V a r i a t i o n o f t h e d i h e d r a l a n g l e d ' r e l a t i v e t o t h e a n o m e r i c c a r b o n c o n f i g u r a t i o n o f D - g l u c o p y r a n o s e 11 11.2 ^ H - n . m . r . s p e c t r a o f K53 p o l y s a c c h a r i d e 13 13 11.3 C - n . m . r . s p e c t r u m o f K l e b s i e l l a K53 c a p s u l a r p o l y s a c c h a r i d e 17 11.4 M e t h y l a t i o n A n a l y s i s scheme f o r K53 p o l y s a c c h a r i d e . 21 11.5 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 K53 p o l y s a c c h a r i d e 23 1 1 . 6 Mass s p e c t r u m o f a 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 f r o m K53 c o m p a r e d t o t h e s p e c t r u m o f a s t a n d a r d d e r i v a t i v e . . 27 1 1 . 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 K74 p o l y s a c c h a r i d e 32 I I I . l G e l 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 c i d i c o l i g o m e r 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 K53 p o l y s a c c h a r i d e 42 I V . 1 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 K74 p o l y s a c c h a r i d e 67 I V . 2 G e l 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 c i d i c o l i g o m e r 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 K74 p o l y s a c c h a r i d e 69 A c k n o w l e d g e m e n t s 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 g r a t i t u d e t o P r o f e s s o r G . G . S . D u t t o n f o r h i s p a t i e n c e , e n c o u r a g e m e n t , a n d a d v i c e d u r i n g t h e r e s e a r c h a n d p r e p a r a t i o n o f t h i s t h e s i s . I g r a t e f u l l y a c k n o w l e d g e D r . A . M. S t e p h e n , D r . M. V i g n o n , D r . E. H. M e r r i f i e l d , A n g e l a S a v a g e , T i m F o l k m a n , a n d J o s e Di F a b i o f o r t h e i r h e l p f u l d i s c u s s i o n s . I s h o u l d a l s o l i k e t o t h a n k t h e o t h e r two members o f t h e l a b o r a t o r y , Don L e e k a n d E l e n o r a K a t s i n , f o r t h e i r i n t e r e s t . A s p e c i a l m e n t i o n g o e s t o t h e members o f t h e d e p a r t m e n t who h a v e h e l p e d i n d i r e c t l y d u r i n g t h e c o u r s e o f t h i s w o r k , v i z . t h e members o f t h e N . m . r . a n d M a s s - s p e c t r o m e t r y S e r v i c e s a n d D r . L. D. H a y w a r d a n d h i s t e c h n i c i a n R o b e r t N g . F i n a l l y , I w a n t t o t h a n k C e l i n e G u n a w a r d e n e f o r t y p i n g t h i s t h e s i s . i x P R E F A C E * I n 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 w o r k 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 c m o d i f i c a t 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 . N u m b e r i n g commences f r o m 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 t e 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 r o m D - g l u c o s e ( I ) o n l y •CHO 1 2 H 0 - ! h OH 3 4r~ OH OH 6 C H 2 0 H H0-CHO - O H '-OH - O H COOH CHO - O H L-OH HO HO CH-D - g l u c o s e D - g l u c u r o n i c a c i d L - r h a m n o s e ' ( I ) ( I I ) ( 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 - r h a m n o s e ( I I I ) i s p a r t o f a m e t h y l g r o u p a n d 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 - d e o x y - L - m a n n o s e . 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 r k e d 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 t o 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 i g u r a t i o n ) t h a t i s i m p l i e d b y 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 e a c h o f 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 - n u m b e r e d 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 , a n d t h e o t h e r s t o t h e L - s e r i e s . L - O H CH 2 0H D - s e r i e s CH 2 0H L - s e r i e s P h y s i c a l a n d 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 a t o m a t C - 5 on t h e a l d e h y d i c c a r b o n a t o m , 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 - l . T h i s r e s u l t s i n two a n o m e r s , r e p r e s e n t e d b e l o w H OH \/ C — OH HO I L - O H C H 2 0 H a - D - g l u c o s e ( I V ) CH 2 0H p - D - g l u c o s e ( V ) xi in the Tollens formulae. It should be noted that C-l is unique in having two attached oxygen atoms, formally making i t a hemiacetal carbon. Since the Tollens formulae have obvious limitations with their unequal bond lengths,Haworth-developed a perspective method of looking at the six-membered ring (VI and VII). This improvement recognizes that the ring oxygen atom lies behind the carbon chain and that bond lengths are approximately equal. Often in practise regular hexagons are used in Haworth projections, OH OH a-D-glucopyranose B-D-glucopyranose pyran (VI) (VII.) (VIII) which he related to such rings at the heterocyclic compound pyran (VIII) and named them pyranoses. Note that hydroxyl groups not involved in ring formation on the right in Fischer and Tollens formulae point down in the Haworth projections and those on the l e f t point up. Similarly, for aldopyranoses, the group on C-5 points up for D (IX) and down for the L enantiomer (X). It follows, then, that when sugar residues are attached there are two possible configurations, an a - or a 3-pyranoside, for each linkage. x i i Hi OH OH HO OH. a-D-rhamnopyranose (IX) a-L-rhamnopyranose (X) 1 The true conformation of pyranoid carbohydrates is related to the chair form of cyclohexane. X-ray diffraction analysis has shown that a hexose, such as a-D-glucose (XI), consists of a puckered, six-membered, oxygen-containing carbon ring, with hydroxyl substituents at C-l through C-4, and a hydroxymethyl group at C-5. All substituents on the ring, except for that at C-l, are equatorial. Two isomers (anomers) are possible in relation to the anomeric center (C-l), depending on whether a substituent is axial (a-anomer; XII) OH (XI) x i i i o r e q u a t o r i a l ( e - 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 , a n d 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 - , a n d 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 t 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 c a n 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 . OR H ( X I I ) . ( X I I I ) H a w o r t h p r o j e c t i o n s a r e m o s t u s e f u l a n d w i l l be u s e d i n t h i s t h e s i s , e v e n 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 . * R e p r o d u c e d w i t h t h e k i n d p e r m i s s i o n o f T . E . F o l k m a n f r o m h i s M . S c . t h e s i s e n t i t l e d " S t r u c t u r a l S t u d i e s on 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 " , 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 , A p r i l 1 9 7 9 . I . I n t r o d u c t i o n I I . M e t h o d o l o g y o f S t r u c t u r a l A n a l y s i s o f P o l y s a c c h a r i d e s -2-I. INTRODUCTION Occurrence and Nature of Klebsiella Bacteria The genus Klebsiella is composed of Gram-negative, nonmotile bacteria that conform to the definitions of the family Enterobacteriaceae and the tribe Klebsielleae. Approximately eighty strains of Klebsiella are known grouped in three species: K. pneumoniae, K. ozaenae, and 1 2 K. rhinoschleromatis. ' Many of these microorganisms are normally found in healthy carriers in the upper respiratory, intestinal, and genito-urinary tracts. Their pathogenicity to man is well known although some strains are not toxic. They are present in the lungs of patients with respiratory diseases and in the suppurative infections in other parts of the body. K.pneumoniae is the most important member of the group. It is the primary cause of pneumonia in 3 per cent of a l l bacterial pneumonias and has been isolated from 3 patients with pleurisy, appendicitis, c y s t i t i s , and pyelonephritis. This species is notable for i t s destructive action on the tissues, producing abscesses and cavities. The two other members of the group have been isolated from disease conditions of the upper respiratory track of man. K.rhinoschleromatis was isolated from the granulomatous nasal lesions of patients with rhinoschleroma, and K.ozaenae has been cultured from nasal secretions of individuals with ozena, a fetid , catarrhal condition of the nose. Other sources of Klebsiella included feces, pus, blood, abscesses, bones and joints. Klebsiella strains are resistant to pe n i c i l l i n in standard 2 • doses but may be sensitive to high concentrations. Sulfadiazine and -3-streptomycin have proved to be of value in therapy as well as chloramphenicol, tetracycline, neomycin, kanamycin, etc... It seems that proportion of resistant strains is increasing steadily, which may be due to mutations. Functions and Composition of Klebsiella Capsular polysaccharides Much attention has been given to the morphology, composition and structure of the bacterial cell wall.f The plasma membrane of the cell is surrounded by a cell wall composed of murein which in turn is enveloped by a lipophilic complex of lipopolysaccharide (LPS), phospholipid, and protein. The whole cellular structure of some organisms is surrounded by a capsule which is sometimes a protein but more often a polysaccharide. Finally, some bacteria have flagella which are proteins passing through the cell wall. In Figure 1.1 a diagrammatic representation of a bacterial cell wall is shown. flagella: H capsule: K complex-j_ Fl ipopolysaccharide:0 murein phospholipids _p_roteins plasma membrane Figure I. 1 Diagrammatic representation of a bacterial cell wall -4-According to the general structure of the bacterial cell wall just described many types of antigens have been found: - antigens H (German; Hauch = fog) corresponding to the flagella - protein - antigens 0 (German; ohne Hauch = without fog) also called somatic - 1ipopolysaccharide - antigens K (German; Kapsel) corresponding to the presence of a capsule - protein or polysaccharide It should be noted that as nonmotile bacteria, Klebsiella do not have flagella or H antigens. A common feature of Gram-negative organisms is the occurrence of polysaccharides on the cell surface. These are either lipopolysaccharides, capsular polysaccharides or both. It has been known for some time that Klebsiel!a produce large capsules and slime. 5 The polysaccharide nature of both was established by Emmerling, fi 7 " Pi Schardinger, Toenniessen and Kramar. Dudman and Wilkinson found that capsular and slime polysaccharides are identical in chemical composition.^ Thus, slime can be considered as excreted capsular polysaccharide. For non-capsulated (K~) Gram-- negative bacteria, serological classification is based on specific reactions of somatic 0 - antigens with respective 0 - antibodies. In the case of the genus Klebsiella most bacteria are heavily encapsulated (K +) and non - capsulated (K~) variants are d i f f i c u l t to obtain. This heavy capsule shields completely the 0 - antigen. Consequently, serological classification - 5 -o f K l e b s i e l l a s t r a i n s i s s o l e l y b a s e d on t h e i r c a p s u l a r K - a n t i g e n s . To d a t e , a p p r o x i m a t e l y 80 s t r a i n s h a v e b e e n c h a r a c t e r i z e d o n t h e b a s i s o f i m m u n o c h e m i c a l t e s t s . R e c e n t l y , 0 r s k o v a n d F i f e - A s b u r y ^ h a v e a d d e d K 8 2 , a n d d e l e t e d K 7 3 , 7 5 , 7 6 , 7 7 , a n d 7 8 . The e x a c t r o l e o f c a p s u l a r a n t i g e n s i s n o t w e l l u n d e r s t o o d . Among t h e v a r i o u s f u n c t i o n s i t h a s b e e n s u g g e s t e d t h a t 13 t h e y may a c t a s s t o r a g e m a t e r i a l i n c a s e o f n e e d , be a p r o t e c t i o n 14 15 1 a g a i n s t d e s i c c a t i o n , a g a i n s t p h a g o c y t o s i s o r a g a i n s t b a c t e r i o p h a g e s . The l a t t e r h y p o t h e s i s i s n o t a l w a y s t r u e . As i t i s shown by w o r k s t a k i n g p l a c e i n t h i s l a b o r a t o r y , 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 e x c e l l e n t s u b s t r a t e s o f p h a g e e n z y m e s . ^ P o l y s a c c h a r i d e s as a n t i g e n s i n d u c e , t h o u g h w e a k l y , t h e f o r m a t i o n o f a n t i b o d i e s i n man a n d a n i m a l s a n d r e a c t s e r o l o g i c a l l y w i t h t h e s e a n t i b o d i e s . I n o t h e r t e r m s t h e y p o s s e s s i m m u n o g e n i c i t y ( c a p a c i t y t o i n d u c e f o r m a t i o n o f a n t i b o d i e s i n mammals) and a n t i g e n i c i t y ( r e a c t i v i t y w i t h a n t i b o d i e s ) . H e i d e l b e r g e r ^ t aj_ 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 . The b a s i s o f t h e s e i m m u n o c h e m i c a l phenomena i s t h e r e c o g n i t i o n o f p a r t i a l s t r u c t u r e s o f t h e p o l y s a c c h a r i d e a n t i g e n s by a n t i b o d y m o l e c u l e s o r immune c e l l r e c e p t o r s . T h e s e p a r t i a l s t r u c t u r e s ( a n t i g e n i c d e t e r m i n a n t s ) may h a v e t h e s i z e o f o l i g o s a c c h a r i d e s o r m o n o s a c c h a r i d e 20 c o n s t i t u e n t s a n d may be o f a c a r b o h y d r a t e o r n o n - c a r b o h y d r a t e n a t u r e . F o r a q u a n t i t a t i v e c o n s i d e r a t i o n o f t h e a n t i g e n - a n t i b o d y r e a c t i o n k n o w l e d g e o f t h e s t r u c t u r e o f t h e p o l y s a c c h a r i d e a n t i g e n i s n e c e s s a r y . F o r t h i s r e a s o n s t r u c t u r a l i n v e s t i g a t i o n o f a l l K l e b s i e l l a s t r a i n s -6-h a s b e e n u n d e r t a k e n a n d w i l l s o o n be c o m p l e t e d . The i n v e s t i g a t i o n o f K53 a n d K74 i s r e p o r t e d i n t h i s t h e s i s . One o f t h e m o s t o u t s t a n d i n g f e a t u r e s o f 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 t h e i r o r d e r e d s t r u c t u r e c o m p o s e d o f o l i g o s a c c h a r i d e r e p e a t i n g u n i t s . T h i s i s d e m o n s t r a t e d i n t h e c a p s u l a r a n t i g e n s o f K l e b s i e l l a . N i m m i c h ^ ' ^ ' 2 ^ 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 a l l K - t y p e s . He f o u n d t h a t t h e m a j o r i t y c o n t a i n e d o n l y a c h a r g e d m o n o s a c c h a r i d e c o n s t i t u e n t e i t h e r D - g l u c u r o n i c a c i d o r D - g a l a c t u r o n i c a c i d a n d h e x o s e s l i k e D - m a n n o s e , D - g l u c o s e a n d D - g a l a c t o s e ; i n some s t r a i n s 6 - d e o x y h e x o s e s w e r e a l s o p r e s e n t s u c h a s L - r h a m n o s e a n d L - f u c o s e . I n a d d i t i o n , a c e t y l a n d f o r m y l g r o u p s , k e t a l - 1 i n k e d p y r u v a t e a n d , v e r y s e l d o m , k e t o a c i d w e r e f o u n d . T h e s e n o n - c a r b o h y d r a t e c o n s t i t u e n t s may f u n c t i o n a s d e t e r m i n a n t g r o u p s a n d may be t h e c a u s e o f c r o s s - r e a c t i v i t i e s o f some 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 . C a p s u l a r p o l y s a c c h a r i d e ; a n t i g e n s o f K l e b s i e l l a h a v e b e e n s t u d i e d i n many l a b o r a t o r i e s a n d d i f f e r e n t s t r u c t u r a l p a t t e r n s ( s e e A p p e n d i x I ) h a v e e m e r g e d f r o m t h e p r o p o s e d s t r u c t u r e s . Some a r e l i n e a r ( K 6 , K70) 2 2 ' 2 3 a n d many a r e b r a n c h e d ( K i l , K 2 8 ) 2 4 ' 2 5 s o much s o t h a t an i n t r i c a t e comb - l i k e s t r u c t u r e i s o b t a i n e d . The number o f 26 27 s u g a r s p e r 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 I , K63) ' t o s e v e n 28 (K-.41). M o s t o f t h e m o n o s a c c h a r i d e c o n s t i t u e n t s e x i s t i n t h e p y r a n o s e f o r m e v e n t h o u g h some f u r a n o s e f o r m s h a v e b e e n f o u n d i n a f e w 28 29 p o l y s a c c h a r i d e s ( K 1 2 , K 4 1 ) . ' A c c o r d i n g t o t h e i r c o m p o s i t i o n t h e d i f f e r e n t s t r a i n s o f K l e b s i e l ! a h a v e b e e n c l a s s i f i e d i n c h e m o t y p e s 18 ( s e e A p p e n d i x I I ) by H e i d e l b e r g e r a n d N i m m i c h . E v e n i f t h e y a r e o f - 7 -t h e same c h e m o t y p e , two p o l y s a c c h a r i d e s c a n h a v e d i f f e r e n t s t r u c t u r a l 30 31 p a t t e r n s as i l l u s t r a t e d by K31 a n d K 3 3 . T h e s e two p o l y s a c c h a r i d e s h a v e 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 a n d c o n t a i n D - g l u c u r o n i c a c i d , D - g a l a c t o s e , D - g l u c o s e , a n d D-mannose w i t h p y r u v a t e s u b s t i t u t i o n . I n K31 t h e g l u c u r o n i c a c i d i s t h e b r a n c h i n g s u g a r , w h i l e i n K33 a mannose r e s i d u e i s t h e b r a n c h i n g s u g a r a n d b e a r s two s i d e - c h a i n s . I n t h i s 32 l a b o r a t o r y an u n u s u a l s t r u c t u r e f o r K60 h a s b e e n f o u n d . The r e p e a t i n g u n i t o f K60 i s an h e p t a s a c c h a r i d e h a v i n g t h r e e s i d e - c h a i n s l i n k e d t o t h r e e d i f f e r e n t s u g a r r e s i d u e s . The d i v e r s i t y o f s t r u c t u r e s p r e s e n t e d by t h e d i f f e r e n t K - t y p e s t r a i n s p r o b a b l y j u s t i f i e s t h e i r s e r o l o g i c a l d i f f e r e n t i a t i o n . - 8 -I I . METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES To u n d e r s t a n d w h a t makes a p o l y s a c c h a r i d e i m m u n o g e n i c a n d w h a t i s t h e c h e m i c a l b a s i s o f i t s a n t i g e n i c i t y i t i s d e s i r a b l e t o know i t s s t r u c t u r e as p r e c i s e l y a s p o s s i b l e . S t r u c t u r a l a n a l y s i s o f t h e p o l y s a c c h a r i d e h a s t o c l a r i f y i t s s u g a r c o m p o s i t i o n , t h e s e q u e n c e o f s u g a r c o m p o n e n t s , a n d t h e n a t u r e o f t h e l i n k a g e s ( p o s i t i o n s o f s u b s t i t u t i o n a n d a n o m e r i c c o n f i g u r a t i o n s ) . I n t h e f o l l o w i n g t h e m o s t w i d e l y u s e d m e t h o d s a r e m e n t i o n e d . I I . 1. I s o l a t i o n a n d P u r i f i c a t i o n S t r a i n s o f K l e b s i e l l a s e r o t y p e s K53 a n d K74 w e r e o b t a i n e d a s a g a r s t a b c u l t u r e s f r o m D r . I . 0 r s k o v i n C o p e n h a g e n . U s i n g s t r o n g g r o w i n g c o l o n i e s t h e b a c t e r i a w e r e p l a t e d o u t t w i c e o n a g a r p l a t e s . E a c h s t r a i n was g r o w n i n a s u c r o s e - y e a s t e x t r a c t medium f o r 4 h o u r s a t 37° C a n d t h e n i n c u b a t e d f o r t h r e e d a y s on l a r g e t r a y s o f s u c r o s e - y e a s t e x t r a c t - a g a r . S l i m e was h a r v e s t e d , d i l u t e d w i t h a q u e o u s p h e n o l t o k i l l t h e b a c t e r i a a n d c e n t r i f u g e d . The v i s c o u s s u p e r n a t a n t c o n t a i n e d n e u t r a l a n d a c i d i c p o l y s a c c h a r i d e s . The s u p e r n a t a n t was f i r s t p r e c i p i t a t e d i n e t h a n o l . The p r e c i p i t a t e was d i s s o l v e d i n w a t e r a n d r e p r e c i p i t a t e d u s i n g 10 p e r c e n t h e x a d e c y l t r i m e t h y l ammonium b r o m i d e ( C e t a v l o n ) . A d d i t i o n o f an a q u e o u s s o l u t i o n o f c a t i o n i c d e t e r g e n t s t o s o l u t i o n s o f c r u d e p o l y s a c c h a r i d e s i n w a t e r r e s u l t s i n t h e p r e c i p i t a t i o n o f C e t a v l o n 33 s a l t o f t h e a c i d i c p o l y s a c c h a r i d e . N e u t r a l p o l y s a c c h a r i d e s a r e n o t p r e c i p i t a t e d by C e t a v l o n a n d c a n t h u s e a s i l y be s e p a r a t e d f r o m a c i d i c -9-o n e s . T h i s p r e c i p i t a t e was d i s s o l v e d i n 4M s o d i u m c h l o r i d e a n d p r e c i p i t a t e d a g a i n i n e t h a n o l . The p o l y s a c c h a r i d e was d i s s o l v e d i n w a t e r , d i a l y s e d f o r t h r e e d a y s a g a i n s t r u n n i n g t a p w a t e r a n d f r e e z e - d r i e d . I I . 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 I I .2.1. ^ H - n . m . r . s p e c t r o s c o p y N . m . r . s p e c t r o s c o p y h a s b e e n u s e d a l m o s t e x c l u s i v e l y f o r s i m p l e m o l e c u l e s b e c a u s e s p e c t r a o b t a i n e d f o r p o l y m e r s w e r e t o o c o m p l e x . L a t e l y , t h i s t e c h n i q u e h a s b e e n a p p l i e d s u c c e s s f u l l y i n t h e s t r u c t u r a l 34 35 a n a l y s i s o f 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 . ' B e c a u s e o f t h e i r r e g u l a r r e p e a t i n g u n i t s s p e c t r a o f t h e s e p o l y m e r s a r e v e r y c l o s e t o t h o s e o b t a i n e d f o r t h e i r o l i g o m e r s . S e v e r a l p r o b l e m s a r e e n c o u n t e r e d i n p e r f o r m i n g a p . m . r . e x p e r i m e n t on a p o l y s a c c h a r i d e . D e u t e r a t i o n o f a l l l a b i l e h y d r o g e n atoms by r e p e a t e d e x c h a n g e w i t h d e u t e r i u m o x i d e b e f o r e m e a s u r e m e n t o f t h e s p e c t r u m i s n e c e s s a r y a n d p r e v e n t s i n t e r f e r e n c e f r o m t h e l a r g e HOD r e s o n a n c e . T h i s e x c h a n g e i s p e r f o r m e d by d i s s o l v i n g t h e p o l y s a c c h a r i d e i n D2O f o l l o w e d by f r e e z e - d r y i n g . The s a m p l e i s t h e n d r i e d u n d e r vacuum f o r a few h o u r s a n d t h e p r o c e s s i s r e p e a t e d two o r t h r e e t i m e s . E v e n a f t e r c a r e f u l d e u t e r a t i o n , a HOD p e a k i s u s u a l l y o b s e r v e d . T h i s p e a k a p p e a r s i n t h e r e g i o n o f t h e s p e c t r u m a s s o c i a t e d w i t h t h e a n o m e r i c p r o t o n s (<5 4.5 - 5.5). I n s u c h c a s e s i t i s a d v a n t a g e o u s t o i n d u c e a c h a n g e i n t h e c h e m i c a l s h i f t o f t h e HOD p e a k . T h i s c a n be done by a l t e r i n g e i t h e r t h e pH ( a d d i t i o n o f t r i f l u o r o a c e t i c a c i d ) o r by r e c o r d i n g t h e s p e c t r u m a t h i g h t e m p e r a t u r e w h i c h h a s t h e e f f e c t t o s h i f t t h e HOD p e a k u p f i e l d . R e c o r d i n g t h e s p e c t r u m a t h i g h t e m p e r a t u r e h e l p s t o e l i m i n a t e a n o t h e r p r o b l e m r e l a t e d t o t h e v i s c o s i t y o f t h e s a m p l e . To r e c o r d a p . m . r . s p e c t r u m 1 - 2 p e r c e n t s o l u t i o n s o f t h e p o l y s a c c h a r i d e a r e n e c e s s a r y b u t t h e s e s o l u t i o n s a r e s o v i s c o u s t h a t t h e r e i s a l o s s o f r e s o l u t i o n when t h e ^ H - n . m . r . e x p e r i m e n t i s p e r f o r m e d . U s i n g h i g h t e m p e r a t u r e r e d u c e s t h e v i s c o s i t y a n d h o m o g e n i z e s t h e s a m p l e . L e s s v i s c o u s s a m p l e s c a n a l s o be o b t a i n e d by p e r f o r m i n g a m i l d a c i d h y d r o l y s i s on t h e n a t i v e p o l y s a c c h a r i d e . H y d r o l y s i s c o n d i t i o n s h a v e t o be s e l e c t e d s o t h a t a c i d l a b i l e g r o u p s , s u c h a s p y r u v a t e a n d a c e t a t e , a r e n o t c o m p l e t e l y r e m o v e d . T h i s t e c h n i q u e , i n some c a s e s , g r e a t l y i m p r o v e s t h e r e s o l u t i o n o f t h e s p e c t r u m . Nowadays a n o m e r i c l i n k a g e s a r e a s s i g n e d on t h e b a s i s o f n . r n . r . d a t a . I n ^ H - n . m . r . s p e c t r o s c o p y t h e a n o m e r i c r e g i o n c o v e r s a r a n g e b e t w e e n 6 4 . 5 t o 6 . 5 . 5 . I t i s w e l l a c c e p t e d t h a t s i g n a l s a p p e a r i n g d o w n f i e l d o f 6 5 . 0 a r e a s s i g n e d t o a - l i n k a g e s a n d t h o s e u p f i e l d t o 3 - l i n k a g e s . The b o r d e r l i n e a t 6 5 . 0 i s a r b i t r a r y b u t h a s b e e n f o u n d t o be v a l i d i n m o s t c a s e s . To a a n d g c o n f i g u r a t i o n s c o r r e s p o n d s p i n - s p i n c o u p l i n g c o n s t a n t s J b e t w e e n t h e p r o t o n s l i n k e d t o c a r b o n s CI a n d C2.. K a r p l u s h a s d e m o n s t r a t e d t h a t t h e c o u p l i n g c o n s t a n t i s a f f e c t e d by t h e d i h e d r a l a n g l e <|) f o r m e d by t h e two b o n d s i n v o l v e d i n t h e c o u p l i n g . As i l l u s t r a t e d i n F i g u r e I I . l , f o r t h e g a n o m e r o f D - g l u c o s e t h e HI a n d H2 p r o t o n s a r e t r a n s - d i a x i a l (<() = 1 8 0 ° ) , t h e c o u p l i n g c o n s t a n t i s m a x i m u m , J ] 2 = 7 " 9 0 n t n e o t n e r h a n d , f o r t h e a - a n o m e r p r o t o n s HJ a n d H2^  a r e i n a g a u c h e c o n f o r m a t i o n (<j)=60°) and t h e - n -c o u p l i n g c o n s t a n t i s s m a l l e r , 2 = 2 - 3 H z . However, f o r L-rhamnose t h e s i t u a t i o n i s r e v e r s e d w i t h t h e a - L - a n o m e r i c p r o t o n h a v i n g J-j 2 ~ 2 Hz and the g - L - a n o m e r i c p r o t o n 2 ~ 1 H z . F o r t h i s 6 - d e o x y h e x o s e , because o f an a x i a l OH a t C 2 , p r o t o n s HI and H2. a r e i n a gauche c o n f o r m a t i o n f o r both the B ( a , e ) and a ( e , e ) f o r m s . T h i s i s a n o t h e r f e a t u r e w h i c h a i d s i d e n t i f y i n g component s u g a r s though l e s s i m p o r t a n t than c h e m i c a l s h i f t s . OR K C o n f i g u r a t i o n a C o n f i g u r a t i o n B F i g u r e I I . 1 V a r i a t i o n o f t h e d i h e d r a l a n g l e $ r e l a t i v e to t h e anomeric carbon c o n f i g u r a t i o n o f D - g l u c o p y r a n o s e . -12-O t h e r i n f o r m a t i o n o b t a i n a b l e by ^ H - n . m . r . i n c l u d e t h e p r e s e n c e o r a b s e n c e o f p y r u v a t e a c e t a l s , 6 - d e o x y s u g a r s , a c e t a t e g r o u p s , a n d t h e number o f s u g a r c o n s t i t u e n t s p e r r e p e a t i n g u n i t . F i g u r e 1 1 . 2 shows t y p i c a l s p e c t r a o f K l e b s i e l l a K53 p o l y s a c c h a r i d e . S p e c t r u m A h a s b e e n r e c o r d e d on t h e n a t i v e p o l y s a c c h a r i d e w i t h o u t an i n t e r n a l s t a n d a r d . The s p e c t r u m e x h i b i t s a d o u b l e t a t fil.29 due t o t h e m e t h y l g r o u p s o f L - r h a m n o s e u n i t s . I n t h e a n o m e r i c r e g i o n (6 4 . 5 - ^ 5 . 5 ) t h e s i g n a l s a r e n o t v e r y w e l l r e s o l v e d . A b r o a d p e a k a p p e a r s u p f i e l d o f 6 5 . 0 p . p . m . a n d c a n be a s s i g n e d t o g - l i n k a g e s w h i l e t h e t h r e e l a r g e s i g n a l s d o w n f i e l d o f 5 5 . 0 p . p . m . c a n be a s s i g n e d t o a - l i n k a g e s . I n c o m p a r i n g t h e i n t e g r a t i o n o f t h e d i f f e r e n t p e a k s i t i s p o s s i b l e t o e s t i m a t e t h a t t h e m e t h y l p r o t o n s o f t h e r h a m n o s e u n i t s a n d t h e a n o m e r i c p r o t o n s a r e i n a 1 :1 r a t i o . A l s o , t h e r a t i o o f t h e a - p r o t o n s t o t h e 6 - p r o t o n s seems t o be 2 : 1 . A b s e n c e o f p e a k s a t 6 2 . 2 0 a n d 6 1 . 5 0 shows t h a t t h e p o l y s a c c h a r i d e d o e s n o t c o n t a i n a n y a c e t a t e g r o u p o r p y r u v a t e a c e t a l . S p e c t r u m B h a s b e e n o b t a i n e d a f t e r t h e p o l y s a c c h a r i d e u n d e r w e n t a m i l d h y d r o l y s i s . As c a n be s e e n t h i s t r e a t m e n t d i d n o t i m p r o v e v e r y much t h e s p e c t r u m e v e n i f t h e p r o d u c t was more s o l u b l e a n d t h e s o l u t i o n l e s s v i s c o u s . - 1 3 -1 i i i i W3 10 ZO 10 F i g u r e 1 1 . 2 ^ H - n . m . r . s p e c t r a o f K53 p o l y s a c c h a r i d e . A . N a t i v e p o l y s a c c h a r i d e , r e c o r d e d w i t h o u t i n t e r n a l s t a n d a r d . B . P a r t i a l l y d e p o l y m e r i z e d K 5 3 , 0 . 5 M T F A , 30 m i n a t 9 5 o c . - 1 4 -13 I I . 2 . 2 . C - n . m . r . S p e c t r o s c o p y One o f t h e m o s t i m p o r t a n t p h y s i c a l m e t h o d s b e i n g u s e d i n 13 o r g a n i c c h e m i s t r y i s w i t h o u t a n y d o u b t C - n . m . r . s p e c t r o s c o p y . The 13 m a i n d i f f i c u l t y i n C - n . m . r . i s t h e l o w n a t u r a l a b u n d a n c e o f t h e c a r b o n - 13 n u c l e u s (1.1%) w h i c h makes t h e t e c h n i q u e much l e s s s e n s i t i v e ( 1 . 6 % ) t h a n ^ H - n . m . r . ( 1 0 0 % ) . H o w e v e r , i m p r o v e m e n t s i n i n s t r u m e n t a t i o n 13 a n d t e c h n i q u e h a v e e n a b l e d C - n . m . r . s p e c t r a o f m o l e c u l e s o f c o n s i d e r -a b l e c o m p l e x i t y a n d h i g h m o l e c u l a r w e i g h t t o be d e t e r m i n e d . Many m e t h o d s h a v e b e e n d e v e l o p e d t o i n c r e a s e t h e s e n s i t i v i t y 13 o f C - n . m . r . O u t s t a n d i n g i n i m p o r t a n c e i s t h e u s e o f p u l s e F o u r i e r -t r a n s f o r m n . m . r . s p e c t r o s c o p y w h i c h i s r a p i d a n d g i v e s i m p r o v e d s i g n a l s . The m e t h o d c o n s i s t s e s s e n t i a l l y o f i r r a d i a t i n g t h e s a m p l e w i t h a s h o r t 13 r a d i o f r e q u e n c y p u l s e w h i c h e x c i t e s s i m u l t a n e o u s l y a l l t h e C n u c l e i . D e p e n d i n g on t h e p u l s e w i d t h t h i s i r r a d i a t i o n t i p s t h e v e c t o r o f m a g n e t i z a t i o n by a f i n i t e a n g l e ( 3 0 ° , 9 0 ° o r 1 8 0 ° ) . When t h e n u c l e i r e l a x t o t h e i r o r i g i n a l s t a t e t h e e n e r g y a b s o r b e d i s e m i t t e d ; t h i s i s c a l l e d a f r e e i n d u c t i o n d e c a y ( F . I . D . ) . A c c u m u l a t i o n o f t h e F . I . D . i n a c o m p u t e r a n d t h e i r F o u r i e r t r a n s f o r m a t i o n g i v e r i s e t o t h e 13 C s p e c t r u m o f t h e p r o d u c t . T h i s m e t h o d h a s a l s o t h e a d v a n t a g e t o i m p r o v e t h e s i g n a l t o n o i s e r a t i o . . T h i s r a t i o i m p r o v e s a s t h e s q u a r e r o o t o f t h e number o f t o t a l t r a n s i e n t s . B e c a u s e o f t h e i r l o w s o l u b i l i t y a n d h i g h v i s c o s i t y , 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 o l u t i o n s n e c e s s i t a t e a l a r g e number o f t r a n s i e n t s (> 1 0 0 , 0 0 0 ) t o o b t a i n a r e a s o n a b l e s i g n a l t o n o i s e r a t i o . ' . 13 A n o t h e r m a j o r b r e a k t h r o u g h i n C - n . m . r . s p e c t r o s c o p y was - 1 5 -t h e d i s c o v e r y o f p r o t o n b r o a d b a n d d e c o u p l i n g i n 1966 . S p i n d e c o u p l i n g o r n u c l e a r m a g n e t i c d o u b l e r e s o n a n c e i s a c h i e v e d by i r r a d i a t i n g an e n s e m b l e o f n u c l e i n o t o n l y w i t h a r a d i o - f r e q u e n c y a t r e s o n a n c e w i t h t h e n u c l e i t o be o b s e r v e d b u t a d d i t i o n a l l y w i t h a s e c o n d a l t e r n i n g f i e l d a t r e s o n a n c e w i t h t h e n u c l e i t o b e d e c o u p l e d . T h i s h a s t h e e f f e c t o f c o l l a p s i n g t h e s p i n m u l t i p l e t s i n t o s i n g l e t s m a k i n g t h e s p e c t r u m e a s i e r t o i n t e r p r e t a n d i n c r e a s i n g t h e s e n s i t i v i t y o f n . m . r . m e a s u r e m e n t s . A d d i t i o n a l s e n s i t i v i t y e n h a n c e m e n t i s p r o d u c e d a s a s i d e e f f e c t o f p r o t o n d e c o u p l i n g . T h i s phenomenon a r i s e s f r o m a n i n t r a m o l e c u l a r d i p o l e - d i p o l e r e l a x a t i o n m e c h a n i s m 38 a n d i s known as t h e n u c l e a r O v e r h a u s e r e f f e c t . 1 3 The C - n . m . r . s p e c t r u m o f a p o l y s a c c h a r i d e c o n t a i n s u s e f u l i n f o r m a t i o n , * r e l a t e d t o t h e f i n e s t r u c t u r e o f t h e m o l e c u l e . I n t h e l i g h t o f t h e b r o a d g e n e r a l i s a t i o n r e c o g n i z e d i n e a r l i e r s t u d i e s o f m o n o s a c c h a r i d e s , r e s o n a n c e s n e a r 9 1 - 1 0 5 p . p . m . c o u l d be a t t r i b u t a b l e t o a n o m e r i c c a r b o n a t o m s . C o n t r a r y t o 1 H - n . m . r . t h e a - a n o m e r i c c a r b o n s a p p e a r u p f i e l d o f t h e g - a n o m e r i c c a r b o n s due t o a s h i e l d i n g 13 e f f e c t . I t h a s b e e n f o u n d t h a t i n c r e a s e d s h i e l d i n g o f a C n u c l e u s i s a c c o m p a n i e d by a d e c r e a s e i n t h e s h i e l d i n g o f t h e a p p e n d e d p r o t o n , 13 1 39 i . e . , C a n d H s h i f t s a r e a f f e c t e d i n v e r s e l y . U s u a l l y c h e m i c a l s h i f t s a p p e a r i n g d o w n f i e l d o f 101 p . p . m . r e p r e s e n t 3 - l i n k e d s u g a r s a n d t h o s e u p f i e l d a - l i n k e d s u g a r s . T h i s r u l e i s n o t a l w a y s v a l i d a s i t h a s b e e n r e p o r t e d , 4 0 a n d o b s e r v e d i n t h e c o u r s e o f t h i s w o r k i n t h e i n v e s t i g a t i o n o f K 7 4 . C a u t i o n m u s t be e x e r c i s e d when a p p l y i n g - 1 6 -t h i s g e n e r a l r u l e e s p e c i a l l y when t h e m o l e c u l e c o n t a i n s r h a m n o s e o r mannose c o n s t i t u e n t s . I n a d d i t i o n t o i n f o r m a t i o n a b o u t a n o m e r i c l i n k a g e s , t h e p r e s e n c e o f d e o x y s u g a r s , p y r u v a t e a c e t a l and a c e t a t e 13 g r o u p may be d e t e c t e d f r o m a C - n . m . r . s p e c t r u m . 13 Q u i t e r e c e n t l y C - n . m . r . s p e c t r o s c o p y h a s b e e n a p p l i e d t o 41 42 4 3 - 4 c a r b o h y d r a t e s i n t h e s t u d y o f m o n o 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 , 4 6 - 4 9 a n d p o l y s a c c h a r i d e s . T h i s p h y s i c a l m e t h o d h a s b e e n u s e d s u c c e s s f u l l y f o r t h e s t r u c t u r a l a n a l y s . i s o f t h e p o l y s a c c h a r i d e s .K53 a n d K74 a n d t h e i r o l i g o s a c c h a r i d e s . The s p e c t r a w e r e r e c o r d e d a t 20 MHz a n d w e r e p r o t o n d e c o u p l e d . D e u t e r i u m o x i d e was u s e d a s s o l v e n t w i t h a c e t o n e a s i n t e r n a l s t a n d a r d g i v i n g a r e f e r e n c e p e a k a t 3 1 . 0 7 p . p . m . 13 F i g u r e 1 1 . 3 shows t h e C - n . m . r . p r o t o n d e c o u p l e d s p e c t r u m o f a s a m p l e o f K53 h a v i n g u n d e r g o n e a m i l d h y d r o l y s i s . A t a p p r o x i m a t e l y 100 p . p . m . s e v e n s i g n a l s a r i s i n g f r o m a n o m e r i c c a r b o n s a r e a p p a r e n t b u t two ( 1 0 2 . 2 3 a n d 1 0 3 . 4 4 p . p . m . ) h a v e b e e n a s s i g n e d 13 t o t h e same r e s i d u e . I n t h e r e g i o n u s u a l l y a s s o c i a t e d w i t h C n u c l e i b e a r i n g a p r i m a r y a l c o h o l ( 6 0 - 6 2 p . p . m . ) o n l y one s t r o n g p e a k i s o b s e r v e d . T h i s s i g n a l p r o b a b l y a r i s e s f r o m C 6 ' s o f t h e g a l a c t o s e a n d t h e two mannose m o . i e t i e s i n K 5 3 . U p f i e l d o f t h e a c e t o n e p e a k o n e 13 s i g n a l a p p e a r s a t 17 p . p . m . a t t r i b u t a b l e t o m e t h y l C n u c l e i f r o m r h a m n o s e r e s i d u e s . T h i s s p e c t r u m i s i n g o o d a g r e e m e n t w i t h t h e h e x a s a c c h a r i d e r e p e a t i n g u n i t o f K 5 3 . I I . 3 . T o t a l S u g a r R a t i o The n e x t s t e p i n t h e i n v e s t i g a t i o n o f a p o l y s a c c h a r i d e i s t o t a l . h y d r o l y s i s f o l l o w e d by a n a l y s i s o f t h e h y d r o l y s a t e q u a l i t a t i v e l y i—I—r —i i i 3 F i g u r e 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 K53 c a p s u l a r p o l y s a c c h a r i d e - 1 8 -a n d q u a n t i t a t i v e l y . A c i d s commonly u s e d f o r t h i s p u r p o s e a r e s u l f u r i c , h y d r o c h l o r i c , f o r m i c , a n d t r i f l u o r o a c e t i c . A d v a n t a g e s a n d d i s a d v a n t a g e s 50 51 o f t h e d i f f e r e n t m e t h o d s h a v e b e e n d i s c u s s e d e x t e n s i v e l y . ' T r i f l u o r o a c e t i c a c i d has t h e a d v a n t a g e o f b e i n g e a s i l y r e m o v e d u n d e r d i m i n i s h e d p r e s s u r e f o l l o w i n g t h e h y d r o l y s i s . T h i s a c i d w i l l n o r m a l l y h y d r o l y s e a n e u t r a l p o l y s a c c h a r i d e w i t h i n a f e w h o u r s w i t h a minimum o f d e g r a d a t i o n . 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 u r o n i c a c i d r e s i d u e s a r e more r e s i s t a n t t o a c i d h y d r o l y s i s . The p r e s e n c e o f e l e c t r o n a c c e p t o r , c a r b o x y l - g r o u p s s t a b i l i z e u r o n o s y l l i n k a g e s t h r o u g h t h e h e t e r o c y c l i c o x y g e n . To h y d r o l y s e a c i d i c 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 a m e t h o d h a s 52 b e e n d e v e l o p e d i n t h i s l a b o r a t o r y i n v o l v i n g t h e u s e o f m e t h a n o l y s i s . T r e a t m e n t w i t h m e t h a n o l i c h y d r o g e n c h l o r i d e c l e a v e s m o s t g l y c o s i d i c b o n d s b u t n o t a l l u r o n o s y l l i n k a g e s . A t t h e same t i m e t h e m e t h y l e s t e r - o f t h e u r o n i c a c i d i s f o r m e d a n d c a n be r e d u c e d u s i n g s o d i u m b o r o h y d r i d e i n a n h y d r o u s m e t h a n o l . R e d u c t i o n i s p e r f o r m e d i n m e t h a n o l s i n c e i n w a t e r t h e e s t e r w o u l d be s a p o n i f i e d . F o l l o w i n g t h e r e d u c t i o n t h e p r o d u c t i s h y d r o l y s e d w i t h 2M t r i f l u o r o a c e t i c a c i d . The m o n o s a c c h a r i d e s a r e t h e n r e d u c e d and a c e t y l a t e d t o t h e i r a l d i t o l p e r a c e t a t e s - f o r g a s -l i q u i d c h r o m a t o g r a p h y ; ( g . l . c . ) a n a l y s i s . The u s e o f 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 t h r o u g h o u t t h i s s t u d y i s b e c a u s e t h e y a r e much more v o l a t i l e t h a n t h e i r n a t i v e s u g a r c o u n t e r p a r t s . A l s o , t h e y g i v e r i s e t o o n l y o n e p e a k i n g . l . c . a n a l y s i s , m a k i n g q u a n t i t a t i o n e a s i e r . T h e o r e t i c a l l y , f o r a f r e e s u g a r f i v e f o r m s a r e p o s s i b l e (a- a n d g- p y r a n o s i d e s , a- a n d 3 --19-furanosides and the linear form) but in practice only four seem to be formed. Trimethylsilylation is fast enough to preserve the equilibrium between the different forms and produces a multiple peak chromatogram for each sugar residue. II. 4. Structural Analysis II. 4. 1. Methylation Analysis Methylation analysis is a facile method for determining the substitution of monosaccharide units constituting oligo - and polysaccharides. The method gives details of the structural units inr.the .polymer, the presence of branching and terminal residues, but no information on their sequence or the Anomeric nature of their linkages. The analysis relies upon the methoxyl substitution of a l l free hydroxyl groups in the native polysaccharide. This can be achieved by using several well known procedures, e.g., Purdie 53 54 55 56 methylation , Kuhn methylation , and Hak-omdri. methylation. ' The most widely used method is that described by Hakomori because i t involves milder conditions and gives good results. In this procedure the polysaccharide or oligosaccharide substrate, dissolved in anhydrous dimethyl sulfoxide, is f i r s t treated with sodium dimethylsulfinylmethanide (dimsyl sodium) and methyl iodide is subsequently added to effect methylation. Usually the Hakomori procedure gives complete etherification in one step but in this study a subsequent Purdie methylation was necessary for the native polysaccharide. It should be noted that Hakdmon" methylation - 2 0 -c a n n o t be r e p e a t e d on a p o l y s a c c h a r i d e c o n t a i n i n g u r o n i c a c i d b e c a u s e t h e m e t h y l e s t e r o f t h e u r o n i c a c i d may be d e g r a d e d by 3 - e l i m i n a t i o n 57 d u r i n g b a s e t r e a t m e n t ( s e e S e c t i o n 1 1 . 5 . 2 . ) . F o l l o w i n g m e t h y l a t i o n , t h e m a t e r i a l i s r e c o v e r e d by d i a l y s i s i n t h e c a s e o f a p o l y s a c c h a r i d e o r by e x t r a c t i o n w i t h c h l o r o f o r m f o r 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 p r o d u c t i s r e d u c e d w i t h l i t h i u m a l u m i n u m 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 a l u m i n u m h y d r o x i d e p r o d u c e d a f t e r t h e 1 i t h i u m a l u m i n u m h y d r i d e h a s b e e n d e s t r o y e d t e n d s t o a d s o r b m a t e r i a l s h a v i n g f r e e h y d r o x y l g r o u p s g i v i n g r i s e t o l o s s e s . T h i s p r o b l e m c a n be a l l e v i a t e d by e x t r a c t i n g no q u a n t i t a t i v e l y t h e p r o d u c t by t h e m e t h o d o f D u t t o n and S m i t h . The r e d u c e d p o l y m e r o r o l i g o m e r i s t h e n h y d r o l y s e d , r e d u c e d t o a l d i t o l s a n d a c e t y l a t e d . F i g u r e I I . 4 i l l u s t r a t e s t h e s e s t e p s i n a r e a c t i o n s c h e m e . The u n m e t h y l a t e d p o s i t i o n s o f t h e a l d i t o l r e p r e s e n t s i t e s o f l i n k a g e , e x c e p t i n t h e c a s e s o f a u r o n i c a c i d r e s i d u e a n d a r e s i d u e w i t h p y r u v a t e s u b s t i t u t i o n . F o r t h e s e r e s i d u e s , t h e r e d u c e d c a r b o x y l f u n c t i o n a n d t h e s i t e s o f p y r u v a t e s u b s t i t u t i o n w i l l be a c e t y l a t e d t o o . The d e r i v a t i v e s o f s u c h s u g a r s c a n 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 t h e same s e q u e n c e o f r e a c t i o n s on t h e c a r b o x y l r e d u c e d a n d / o r d e p y r u v y l a t e d . - p o l y s a c c h a r i d e . The r e s u l t s w i l l be t h e d i s a p p e a r a n c e o f c e r t a i n p e a k s a n d t h e a p p e a r a n c e o f new o n e s i n t h e g . l . c . t r a c i n g . - 2 1 -- D-G1CD_A Ig 2 - D-Man£ ~ - Manp_ ~ - D - G a l £ L-RhajD -J-COOCH 1 L - R h a £ CrLOCH-C H 3 0 ' 6CH 1) C H 3 S 0 C H 2 " Na / C H g l 2) C H 3 I / A g 2 0 C H 2 O C H 3 C H 2 O C H 3 ^ \ C H 3 ° / ° \ r x C H 3 0 / E n " ° -0 C H 3 G H 3 0 ^ * 0 L i A l H 4 i n T H F CH2OH C H 2 0 C H 3 C H 2 0 C H 3 C H 2 O C H 3 ) Ov C H 3 0 04 CrLO/j 0 3 / C H 3 C H 3 0 0 C H 3 0 C H 3 C H 3 0 0 1) a c i d h y d r o l y s i s 2) Na BH Z 3) A c 2 0 / p y r i d i n e 1 , 5 - d i - O - a c e t y l - 2 , 3 , 4 - t r i - 0 _ - m e t h y l - L - r h a m n i t o l 1 , 2 , 5 - t r i - 0 - a c e t y l - 3 , 4 - d i - C J - m e t h y l - L - r h a m n i t o l 1 , 2 , 5 - t r i - 0 - a c e t y l - 3 , 4 , 6 - t r i - O - m e t h y l - D - m a n n i t o ! 1 , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 , 6 - t r i - O - m e t h y l - D - g a l a c t i t o l 1 . 2 . 3 . 5 - t e t r a - O - a c e t y l - 4 , 6 - d i - O - m e t h y l - D - m a n n i t o l 1 . 4 . 5 . 6 - t e t r a - 0 - a c e t y l - 2 , 3 - d i - 0 - m e t h y l - D - g l u c i t o l F i g u r e I I . 4 M e t h y l a t i o n a n a l y s i s scheme f o r K53 p o l y s a c c h a r i d e - 2 2 -I I . 4 . 2 . G . l . c . 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 G a s - l i q u i d c h r o m a t o g r a p h y h a s b e e n m o d i f i e d r e p e a t e d l y a n d i s now a s i m p l e a n d r e l i a b l e m e t h o d f o r t h e s e p a r a t i o n a n d q u a n t i -t a t i o 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 g e n e r a l i t c a n be s t a t e d t h a t t h e t e c h n i q u e t o d a y h a s n e a r l y r e a c h e d p e r f e c t i o n . The 51 59 60 f i e l d h a s b e e n r e v i e w e d e x t e n s i v e l y . ' ' V a r i o u s l i q u i d p h a s e s a r e a v a i l a b l e f o r s e p a r a t i o n o f m i x t u r e 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 c . a c e t a t e s . B e s t s e p a r a t i o n s a r e o b t a i n e d on medium - p o l a r c o l u m n s s u c h a s : - E C N S S - M : a e t h y l e n e s u c c i n a t e - c y a n o e t h y l s i l i c o n e c o p o l y m e r - 0 V - 1 7 : a p h e n y l , m e t h y l s i l i c o n e p o l y m e r - 0 V - 2 2 5 : a p h e n y l , c y a n o p r o p y T , m e t h y l s i l i c o n e p o l y m e r - H I - E F F - 1 B : d i e t h y l e n e g l y c o l s u c c i n a t e D e p e n d i n g on t h e d i f f i c u l t i e s o f s e p a r a t i o n one m u s t u s e d i f f e r e n t c o l u m n s f o r o p t i m u m s e p a r a t i o n . Good s e p a r a t i o n o f t h e t h r e e d i - O - m e t h y l - L - r h a m n o s e i s o m e r s c a n be a c h i e v e d u s i n g H I - E F F - 1 B c o l u m n b u t 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l - D - g l u c o s e c a n n o t be s e p a r a t e d f r o m t h e 2 , 4 - d i - 0 - m e t h y l - L - r h a m n o s e . To s e p a r a t e t h e l a t t e r two compounds 48 c o l u m n 0 V - 1 7 c a n be u s e d . I n t h e i n v e s t i g a t i o n o f K l e b s i e l l a K53 a n d K74 c o l u m n ECNSS-M was u s e d a l m o s t e x c l u s i v e l y b e c a u s e i t g a v e g o o d s e p a r a t i o n s , ( s e e F i g u r e H . 5 . ) . P u b l i c a t i o n s by L i n d b e r g e t a j _ 6 0 a n d A l b e r s h e i m e t a l 6 1 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 n u m e r o u 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 . In t h e p r e s e n t w o r k i d e n t i f i c a t i o n o f v a r i o u s g . l . c . d e r i v a t i v e s was made by u s i n g t h e s e r e t e n t i o n t i m e s d a t a w h i c h was - 2 3 -2 , 3 , 4 - R h a C o l u m n : ECNSS - M P r o g r a m : 1 7 0 ° C i s o t h e r m a l C a r r i e r g a s : N 9 , 20 m L / m i n 3 , 4 - R h a 2 , 3 , 4 , 6 - G l c ( s t d ) 4 8 12 16 20 T i m e ( m i n . ) F i g u r e 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 K53 p o l y s a c c h a r i d e . - 2 4 -c o n f i r m e d 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 . 4 . 3 ) . Q u a n t i t a t i o n a s d e t e r m i n e d by p e a k i n t e g r a t i o n was c o r r e c t e d u s i n g m o l a r r e s p o n s e f a c t o r s ^ , t h o u g h L i n d b e r g e_t a l _ ^ s u g g e s t e d t h a t t h i s c o r r e c t i o n i s n o t n e c e s s a r y f o 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 . 1 1 . 4 . 3 . Mass S p e c t r o m e t r y 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 D a t a o b t a i n e d f r o m g a s - l i q u i d c h r o m a t o g r a p h y a n a l y s i s a r e i n f o r m a t i v e b u t do n o t a l l o w u n a m b i g u o u s i d e n t i f i c a t i o n o f t h e c o m p o n e n t s . H o w e v e r , c o m b i n e d g a s - 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 . l . c . - m . s . ) has become i n c r e a s i n g l y i m p o r t a n t t o c o n f i r m t h e i d e n t i f i c a t i o n o f t h e c o m p o n e n t s 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 . The f r a g m e n t a t i o n p a t h w a y s o f t h e 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 on e l e c t r o n i m p a c t h a v e b e e n s t u d i e d i n g r e a t d e t a i l by some S w e d i s h w o r k e r s ^ a n d c o n s i d e r a b l e d a t a a r e now a v a i l a b l e . ^ On e l e c t r o n i m p a c t 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 no m o l e c u l a r i o n i s f o u n d i n t h e s p e c t r a , a n d d e r i v a t i v e s h a v i n g t h e same s u b s t i t u t i o n , p a t t e r n s g i v e s i m i l a r mass s p e c t r a . F o r e x a m p l e , i t i s n o t p o s s i b l e t o d i f f e r e n t i a t e s t e r e o i s o m e r i c d e r i v a t i v e s o f g l u c o s e , g a l a c t o s e , a n d m a n n o s e . P r i m a r y f r a g m e n t s a r e f o r m e d by f i s s i o n b e t w e e n c a r b o n a t o m s i n t h e a l d i t o l c h a i n . F i s s i o n b e t w e e n two m e t h o x y l a t e d c a r b o n s i s more a b u n d a n t t h a n f i s s i o n b e t w e e n o n e m e t h o x y l a t e d a n d one a c e t o x y l a t e d c a r b o n w h i c h i n t u r n i s p r e f e r r e d t o f i s s i o n b e t w e e n two a c e t o x y l a t e d c a r b o n s , as shown b e l o w . - 2 5 -R R. R H - C - OCH 3 :> H - C - OCH Q i •J H - C - OAc H - C - OCH 0 i 3 H - C - OAc . H - C - OAc R, 2 P r i m a r y f r a g m e n t s g i v e r i s e t o s e c o n d a r y f r a g m e n t s . T h e s e a r e f o r m e d by l o s s o f o n e o r more o f t h e f o l l o w i n g : a c e t i c a c i d ( M . W . 6 0 ) , m e t h a n o l ( M . W . 3 2 ) , k e t e n e ( M . W . 4 2 ) , o r f o r m a l d e h y d e ( M . W . 3 0 ) . compounds f o r w h i c h s t a n d a r d s p e c t r a a r e n o t a v a i l a b l e . 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 t h e s i t e o f l i n k a g e on t h e s u g a r r e s i d u e b e a r i n g t h e u r o n i c a c i d c o m p o n e n t i s e t h y l a t e d a f t e r d e g r a d a t i o n . T h i s p r o d u c e an e t h y l a t e d 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 . I n t h e 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 K53 c a p s u l a r p o l y s a c c h a r i d e , 1 , 3 , 5 - t r i - ^ - a c e t y l - 2 - J ) - e t h y l - 4 , 6 - d i - j O - m e t h y l - D - m a n n i t o l was o b t a i n e d . By c o m p a r i n g t h e mass s p e c t r u m o f t h e 2 - 0 - e t h y l i s o m e r t o t h a t o f 1 , 3 , 5 - t r i - ^ - a c e t y l - 2 , 4 , 6 - t r i - _ 0 - m e t h y l - D - m a n n i t o l a c h a r a c t e r i s t i c s h i f t o f 14 mass u n i t s was o b s e r v e d . The s p e c t r u m o f t h e t r i - C ) - m e t h y l i s o m e r e x h i b i t s a s t r o n g p e a k f r o m t h e p r i m a r y f r a g m e n t m/e 1 1 7 , w h i l e i n t h e s p e c t r u m o f t h e 2 - 0 - e t h y l i s o m e r t h i s p e a k i s s h i f t e d t o m/e 1 3 1 . P r i m a r y f r a g m e n t a t i o n o f t h e s e t w o i s o m e r s a r e shown b e l o w a n d t h e i r mass s p e c t r a a r e p r e s e n t e d i n F i g u r e : I I . 6 The p r e v i o u s i n f o r m a t i o n i s u s e f u l t o i d e n t i f y l a b e l l e d - 2 6 -C H 0 - OAc I c. C H 2 - OAc C H 3 0 - CH AcO - CH 117 2 3 3 HC. • - OCH, HC • - OAc C H 2 - 0 C H 3 189 T61' 233 117 305 " 4 5 C H 3 C H 2 0 - CH AcO - CH HC - 0 C H 3 HC - OAc 131 233 C H 2 - OCH 3 203 161 247 117 319 ' 4 5 " 1 1 . 5 S u g a r S e q u e n c e D e t e r m i n a t i o n by D e g r a d a t i o n M e t h o d s I I . 5 . 1 . P a r t i a l H y d r o l y s i s I s o l a t i o n o f o l i g o s a c c h a r i d e s f r o m p a r t i a l h y d r o l y s i s o f a p o l y s a c c h a r i d e i s t h e k e y t o t h e d e t e r m i n a t i o n o f t h e s e q u e n t i a l a r r a n g e m e n t o f t h e c o n s t i t u e n t m o n o s a c c h a r i d e s i n t h a t p o l y m e r . T h e s e o l i g o s a c c h a r i d e s o r b l o c k u n i t s p e r m i t t h e r e c o n s t r u c t i o n o f t h e r e p e a t i n g u n i t i n an a d d i t i v e m a n n e r . T h e r e i s no s t a n d a r d m e t h o d o f p a r t i a l h y d r o l y s i s g i v i n g o p t i m u m y i e l d s . D e p e n d i n g on t h e t y p e o f m o n o s a c c h a r i d e r e s i d u e s , t h e a n o m e r i c c o n f i g u r a t i o n o f t h e g l y c o s i d i c l i n k a g e s , t h e p o s i t i o n o f l i n k a g e , e t c . , o n e h a s t o d e t e r m i n e t h e c o n d i t i o n s o f h y d r o l y s i s i n o r d e r t o g e t t h e maximum amount o f o l i g o s a c c h a r i d e s . Capon has r e v i e w e d t h e r a t e c o n s t a n t f o r t h e a c i d c a t a l y z e d h y d r o l y s i s o f a - 2 7 -100 _ ( a ) 1 , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 , . 6 - t r i - 0 - m e t h y l - D - m a n n i t o l 75 R e l . i n t . % 50 _J 25-^ I i i i r 11 i i i I' M I i i i i 11 i r' i i i i M 11 i i M I i u 50 100 150 200 250 IOO-II ( b ) 1 , 3 , 5 - t r i - 0 - a c e t y l - 2 - 0 - e t h y l - 4 , 6 - d i - O - m e t h y l - D - m a n n i t o l 7 5 J 50-J | i | I | I I I I I | ' I I- I I I l I I | i 50 100 150 I 1 I 1 I I f I I I I I | I | i | 200. 250 m/e F i g u r e 11.6 Mass s p e c t r u m o f a 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 f r o m K53(b) c o m p a r e d t o t h e s p e c t r u m o f a s t a n d a r d d e r i v a t i v e ( a ) . -28-large variety-of glycosides. He found that furanosides are more labile than pyranosides, deoxy sugars are more easily hydrolysed than hexoses, a - glycosidic bonds are cleaved more easily than 3 - glycosidic bonds and that uronic acids are resistant to hydrolysis. The resistance of the uronosyl bond is an advantage and permits the isolation of large proportions of aldobiouronic acid, and to a lesser extent aldotriouronic acid. Residues present in the side chain are hydrolysed more quickly than residues in the chain. This fact was used in the analysis of K53 where the single unit L-rhamnose side chain was removed to produce a linear polysaccharide. Pyruvate acetals spanning carbons four and six of an hexose are moderately stable as Tittle steric strain is involved. In some 64 cases, they have been found to survive during partial hydrolysis. During partial hydrolysis studies on K74 no oligomers retaining the pyruvate acetal were isolated but an interesting observation was made. In K74 the pyruvate acetal is linked to the terminal galactose of the side chain as 4,6-0-(l-carboxyethylidene-)-D-galactose, as shown below. A sample of the polysaccharide treated with 0.5M trifluoroacetic acid at 100°C for 1 hour was not completely depyruvylated. On the other hand, the acetal was completely removed by hydrolysing another sample with 0.01M trifluoroacetic.:acid at 100°C for 6 hours. This could possibly suggest that removal of the acetal is more dependent on the time of hydrolysis than on the acid strength. In order to improve yields, Galanos et a l have developed an apparatus whose basic principle is the continuous removal of -29,-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 t o p r e v e n t t o t a l h y d r o l y s i s t o monomers. A l t h o u g h t h i s a p p a r a t u s i s a v a i l a b l e i n t h i s l a b o r a t o r y i t was not used i n the c o u r s e o f t h i s work because o f the problems a s s o c i a t e d w i t h i t . The use o f t h i s a p p a r a t u s i s t e d i o u s , t i m e - c o n s u m i n g , r e q u i r e s l a r g e amounts o f s t a r t i n g m a t e r i a l , and does n o t guarantee good y i e l d s o f o l i g o m e r s . In the p r e s e n t s t u d y o n e - p o t p a r t i a l h y d r o l y s e s were performed on K53 and K74. B e f o r e p r o c e e d i n g on a l a r g e s c a l e , p a r t i a l h y d r o l y s e s m o n i t e r e d by paper chromatography were made on s m a l l samples t o determine the b e s t h y d r o l y s i s c o n d i t i o n s . T h i s b a t c h h y d r o l y s i s p r o c e d u r e was s u c c e s s f u l f o r both p o l y s a c c h a r i d e s . For K53, d i - , t r i - , t e t r a - , and p e n t a s a c c h a r i d e s were i s o l a t e d ; f o r K74, d i - , t r i - , and t e t r a s a c c h a r i d e s were c o l l e c t e d . 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 were p u r i f i e d by 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 q u e s . The h y d r o l y s a t e w a s . 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 f r a c t i o n s by i o n - e x c h a n g e - 3 0 -chromatography. The acidic oligomers were separated according to molecular size by gel f i l t r a t i o n chromatography and purified by descending paper chromatography. II. 5.2. Uronic Acid Degradation ( 3 - elimination) The 3-elimination reaction for uronic acids and their derivatives has been reviewed by Kiss. Recently, the technique has been applied with success to the structural investigation of acidic polysaccharides. The main steps of this degradation are outlined as follows: (I) OHC 0 COOCH C 0 0 C H 3 ,C = 0 (IV) (HI) - 3 1 -On m e t h y l a t i o n , t h e e s t e r i f i e d c a r b o x y l g r o u p i n u r o n i c a c i d r e s i d u e s becomes e l e c t r o n — w i t h d r a w i n g a n d i n c r e a s e s t h e a c i d i t y o f t h e r i n g p r o t o n a t C 5 . On s u b s e q u e n t t r e a t m e n t w i t h b a s e t h e p r o t o n a t C5 i s r e m o v e d a n d t h e s u b s t i t u e n t i n t h e 3 - p o s i t i o n (R^O") i s e l i m i n a t e d . The h e x - 4 - e n o - p y r a n o s y l u r o n i c r e s i d u e ( I I ) i s a c i d s e n s i t i v e . T r e a t m e n t u n d e r m i l d a c i d c o n d i t i o n s w i l l d e g r a d e t h i s u n s a t u r a t e d r e s i d u e w i t h o u t a f f e c t i n g i n t a c t g l y c o s i d i c b o n d s . A s p i n a l l a n d R o s e l l have r e p o r t e d t h a t t r e a t m e n t w i t h b a s e r e s u l t s i n c o m p l e t e l o s s o f CQ t h e u n s a t u r a t e d r e s i d u e ( I I ) a n d a c i d h y d r o l y s i s i s u n n e c e s s a r y . T h e y s u g g e s t e d t h a t a f t e r t r e a t m e n t w i t h b a s e , t h e p r o d u c t i s d i r e c t l y a l k y l a t e d w i t h t r i d e u t e r i o m e t h y l o r e t h y l i o d i d e t o l a b e l t h e s i t e ( s ) t o w h i c h u r o n i c a c i d s w e r e a t t a c h e d . The p r o c e d u r e a v o i d s i n t e r m e d i a t e i s o l a t i o n o f d e g r a d e d p o l y s a c c h a r i d e a n d p o s s i b l e l o s s o f a c i d l a b i l e g l y c o s y l s u b s t i t u e n t s . The d e g r a d a t i o n becomes more c o m p l e x , b u t a l s o more i n f o r m a t i v e , when t h e s u b s t i t u e n t on C4 o f t h e u r o n i c a c i d s i s a n o t h e r s u g a r . On t r e a t m e n t w i t h b a s e , a s u g a r r e s i d u e R^OH i s r e l e a s e d , a n d t h i s , h a v i n g a g o o d l e a v i n g g r o u p a t C 3 , w i l l f u r t h e r r e a c t by a s e c o n d 3 - e l i m i n a t i o n . I f t h i s l e a v i n g g r o u p a t C3 h a p p e n s t o be a n o t h e r s u g a r r e s i d u e , d e g r a d a t i o n w i l l c o n t i n u e g i v i n g r i s e t o t h e s o - c a l l e d p e e l i n g o f t h e p o l y s a c c h a r i d e . T h i s c a n be p r e v e n t e d by p r o t e c t i n g r e d u c i n g s u g a r s by a c e t y l a t i o n when e l i m i n a t i o n i s 69 p e r f o r m e d i n t h e p r e s e n c e o f a c e t i c a n h y d r i d e . The n a t u r e o f r e s i d u e s r e l e a s e d d u r i n g t h e d e g r a d a t i o n i s r e v e a l e d by a n a l y s i s o f t h e r e m a i n i n g s u g a r s , as 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 , by g a s - 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 . F i g u r e I I . 7 i l l u s t r a t e s - 3 2 -C H 2 0 C H 3 C H 2 O C H 3 C H 2 0 C H 3 CH 3 X C H 3 0 0 C - ^ ^ 0 , C H 2 0 C H 3 C H 3 0 0 C " 0 C H 2 0 C H 3 C H 2 0 C H 3 ^ C H 3 o / - ° \ ^ 0 3 K O C H ^ O C H 3 0 \ L i , , c „ 3 o 3) A c 2 0 / p y r i d i n e 1 , 2 , 5 - t r i - 0 - a c e t y l - 3 , 4 , 6 - t r i - 0 - m e t h y l - D - m a n n i t o l 1 , 3 , 5 - t r i - 0 - a c e t y l - 2 , 4 , 6 - t r i - 0 - m e t h y l - D - g a l a c t i t o l 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 o f K l e b s i e l l a K74 p o l y s a c c h a r i d e - 3 3 -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 K74 p o l y s a c c h a r i d e . I I . 6 . D e t e r m i n a t i o n o f D - o r ' L - C o n f i g u r a t i o n o f  Component S u g a r s The D o r L c o n f i g u r a t i o n o f i n d i v i d u a l s u g a r r e s i d u e s c a n d e t e r m i n e d by t h e s i g n o f t h e i r c i r c u l a r d i c h r o i s m c u r v e s m e a s u r e d on s u i t a b l e d e r i v a t i v e s . 7 0 T h e s e d e r i v a t i v e s w h i c h c a n be a l d i t o . 1 a c e t a t e s o 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 a r e c o l l e c t e d by p r e p a r a t i v e g a s - l i q u i d c h r o m a t o g r a p h y . M e a s u r e m e n t s a r e made i n a c e t o n i t r i l e a t 213 nm a n d s p e c t r a a r e c o m p a r e d w i t h t h o s e o b t a i n e d f r o m a u t h e n t i c s a m p l e s . S t r u c t u r a l I n v e s t i g a t i o n o f K l e b s i e l l a S e r o t y p e K53 C a p s u l a r P o l y s a c c h a r i d e - 3 5 -I I I . 1 A b s t r a c t The s t r u c t u r e o f t h e K l e b s i e l l a s e r o t y p e K53 c a p s u l a r p o l y s a c c h a r i d e h a s b e e n e l u c i d a t e d by 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 , a n d c h a r a c t e r i z a t i o n o f o l i g o s a c c h a r i d e s o b t a i n e d by p a r t i a l h y d r o l y s i s . 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 s u g a r r e s i d u e s 1 13 w e r e d e t e r m i n e d by H - a n d C - n . m . r . s p e c t r o s c o p y on t h e o r i g i n a l p o l y s a c c h a r i d e a n d o l i g o s a c c h a r i d e s . The p o l y m e r c o n s i s t s o f h e x 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 t h e f o l l o w i n g s t r u c t u r e . - D - G l c £ A ^ ~ D - M a n £ ~ - D-Manp ~ - D - G a l £ ^ ~ L - R h a £ ~ a 1 L - R h a p I I I . 2 I n t r o d u c t i o n N n m m i c h . ^ ' ^ h a s i n v e s t i g a 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 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 f r o m K l e b s i e l l a s e r o t y p e K53 a n d f o u n d t h a t i t c o n t a i n e d m a n n o s e , g a l a c t o s e , r h a m n o s e , a n d g l u c u r o n i c a c i d . Two o t h e r K l e b s i e l l a c a p s u l a r a n t i g e n s c o n t a i n t h e s e s u g a r s , n a m e l y K40 a n d K 8 0 , b u t t h e i r s t r u c t u r e s a r e n o t y e t k n o w n . I n an e f f o r t t o c o r r e l a t e c h e m i c a l 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 s t r u c t u r a l s t u d i e s o f K 5 3 . -se-l l i . 3. Results and Discussion Composition and n.m.r. Spectra Klebsiella K53 bacteria were grown on an agar medium, and the capsular polysaccharide was purified by one precipitation with Cetavlon. The product moved as one band during electrophoresis and had [a]D -12°, which compares well to the calculated value of [a]Q +2.3° using Hudson's rules of isorotation.^ The molecular weight of the polysaccharide was determined by gel chromatography to be 1.2x10 . Paper chromatography of an acid hydrolysate of the polysaccharide showed the presence of rhamnose, galactose, mannose, and 52 glucuronic acid. Methanolysis of K53 , reduction of the hydrolysate, and analysis of the derived alditol acetates by gas-liquid chromatography confirmed the presence of rhamnose, mannose, galactose, and glucose in the approximate molar ratio of 2.3:1.9:1:1 respectively. The H^-n.m.r. spectrum of the native polysaccharide (PI) was recorded in D2O at 90°C without acetone as internal standard (see Appendix I I I , spectrum No. 1). Chemical shift assignments were made relative to that of the methyl protons of the rhamnose residues. The spectrum exhibits a strong doublet at 61.28, fi 6 Hz, and four broad signals in the anomeric region at 64.55, 65.05, 65.23, and 65.29. The signal at 61.28 was assigned to methyl protons of the rhamnose sugars, and that upfield of 65.0 to p-linkages and those downfield to a-linkages. The integral ratio indicates that the repeating unit contains six monosaccharide constituents, two of which are rhamnose, and that two are B-linked and four a-linked. Absence of signals at 61.5 and 62.2 shows - 3 7 -t h a t t h e p o l y s a c c h a r i d e d o e s n o t c o n t a i n a n y p y r u v a t e k e t a l o r 34 35 C j - a c e t y l g r o u p . ' 13 The C - n . m . r . s p e c t r u m o f K53 ( s e e A p p e n d i x I I I , s p e c t r u m N o . 2 ) c o r r o b o r a t e s t h e r e s u l t s o b t a i n e d i n t h e ^ H - n . m . r . e x p e r i m e n t . S i x s i g n a l s a p p e a r i n t h e a n o m e r i c r e g i o n a t 9 6 . 1 4 , 9 7 . 3 1 , 1 0 0 . 4 4 , 1 0 1 . 2 8 , 1 0 3 . 3 5 , a n d 1 0 5 . 3 2 p . p . m . The s i g n a l a t 6 1 . 9 1 p . p . m . , a t t r i b u t a b l e t o C 6 ' s o f mannose a n d g a l a c t o s e , i n d i c a t e s t h a t t h e s e r e s i d u e s a r e 28 n o t l i n k e d t o o t h e r s u g a r r e s i d u e s a t p o s i t i o n 6 . The s i g n a l a t 1 7 . A 2 p . p . m . w a s a s s i g n e d t o C 6 ' s o f r h a m n o s e r e s i d u e s . 1 1 3 H - a n d C - n . m . r . s p e c t r a w e r e a l s o r e c o r d e d on a s a m p l e ( P l a ) o f K53 h a v i n g u n d e r g o n e a m i l d h y d r o l y s i s , 0 . 5 M t r i f l u o r o a c e t i c a c i d a t 9 5 ° C f o r 30 m i n . ( s e e A p p e n d i x I I I , S p e c t r a N o . 3 and N o . 4 ) . The t r e a t m e n t d i d n o t i m p r o v e v e r y much t h e ^ H - n . m . r . s p e c t r u m . The 13 C s p e c t r u m i s q u i t e s i m i l a r t o t h a t o f t h e n a t i v e p o l y s a c c h a r i d e e x c e p t t h a t s e v e n s i g n a l s a p p e a r i n t h e a n o m e r i c r e g i o n i n t e a d o f s i x . H o w e v e r , two s i g n a l s , 1 0 2 . 2 3 a n d 1 0 3 . 4 4 p . p . m . , w e r e a s s i g n e d t o t h e g l u c u r o n i c a c i d r e s i d u e . P r e c i s e a s s i g n m e n t o f t h e s i g n a l s was a c h i e v e d a f t e r s t u d y i n g 1 13 H - a n d C - n . m . r . s p e c t r a o f o l i g o 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 ; s e e T a b l e I I I . l . M e t h y l a t i o n A n a l y s i s 55 56 M e t h y l a t i o n 5 o f K53 p o l y s a c c h a r i d e a n d s u b s e q u e n t c o n v e r s i o n t o a l d i t o l a c e t a t e s a n d g . l . c . - m . s . a n a l y s i s 6 0 » 6 2 i n d i c a t e d t h a t K53 i s c o m p o s e d o f a h e x a s a c c h a r i d e r e p e a t i n g u n i t T A B L E I I I . I N . m . r . D a t a o f K l e b s i e l l a K 5 3 C a p s u l a r P o l y s a c c h a r i d e a n d D e r i v e d P o l y - a n d O l i g o s a c c h a r i d e s COMPOUND u l , 2 ( H z ) b H - n . m . r . d a t a I n t e g r a l ( H ) A s s i g n m e n t 1 3 C - n . m . r . d a t a p . p . m . 3 A s s i g n m e n t d G l c A 1 1 M a n - O H p A l 5 . 3 0 4 . 9 9 4 . 5 7 0 . 7 0 . 3 1 a - M a n - O H B - M a n - O H e - G l c A 1 0 2 . 4 9 9 2 . 9 1 6 1 . 4 0 8 - G l c A a , B - M a n - 0 H C 6 o f M a n G l c A 1 1 M a n — M a n - O H 6 a A 2 5 . 3 7 5 . 2 5 5 . 1 6 4 . 9 3 4 . 5 8 0 . 7 0 . 3 1 0 . 3 1 a - M a n - O H u n k n o w n o r i g i n a - M a n - M a n B - M a n - O H 8 - G l c A 1 0 2 . 3 6 1 0 0 . 8 4 9 3 . 4 0 6 1 . 8 4 ] 6 1 . 3 8 J 6 - G 1 c A a - M a n - M a n a , e - M a n - 0 H C 6 ' s o f M a n G l c A 1 1 M a n — M a n — G a l - O H p a a A 3 G l c A 1 1 M a n 1 1 M a n 1 1 G a l 1 - 1 R h a - O H A 4 [ ? G l c A 1 1 M a n 1 1 M a n l ^ G a l l^Rha i - l L_ P a a B a _ J n P 2 5 . 2 9 5 . 1 8 4 . 6 5 4 . 6 1 1 . 2 9 5 . 3 5 5 . 2 6 5 . 1 9 4 . 8 4 4 . 6 3 4 . 5 9 S S 0 1} 1 . 2 1 1 . 8 0 . 7 s i g n a l f o r H 6 ' s o f R h a ( 6 1 . 2 8 ) w a s n o t r e c o r d e d H n . m . r . s p e c t r u m w a s n o t r e c o r d e d a - M a n - G a l a - M a n - M a n B - G a l - O H B - G l c A u n k n o w n o r i g i n a - R h a -OH a - M a n - G a l a - M a n - M a n B - R h a-OH B - G a l B - G l c A 1 0 2 . 3 2 1 0 1 . 0 2 9 7 . 2 1 9 5 . 4 1 ] 9 5 . 1 2 J 9 3 . 1 2 6 1 . 7 1 1 0 5 . 4 4 1 0 2 . 3 1 1 0 1 . 1 1 9 5 . 5 3 9 3 . 9 1 9 3 . 6 8 6 1 . 7 9 1 7 . 6 3 1 0 5 . 2 7 1 0 2 . 2 0 1 0 1 . 1 2 1 0 0 . 4 3 9 5 . 5 0 6 1 . 7 2 1 7 . 2 8 B - G l c A a - M a n - M a n B - G a l - O H a - M a n - G a l a - G a l - O H C 6 ' s o f M a n . G a l B - G a l - R h a B - G l c A a - M a n - M a n a - M a n - G a l a - R h a - O H B - R h a - O H C 6 ' s o f M a n , G a l C 6 o f R h a - O H B - G a l - R h a B - G l c A a - M a n - M a n a - R h a - G l c A a - M a n - G a l C 6 ' s o f M a n , G a l C 6 o f R h a i CO CO I COMPOUND J 1 , 2 K (Hz)b H-n.m.r. data I n t e g r a l (H) A s s i g n m e n t c 13 C-n.m.r. da t a p.p.m.a Assignments! ^ G l cA Man — Man — Gal V ^ h a -0 3 a a 3 «_i n 1 Rha P l a 5.27 5.20 5.03 4.55 1.28 3.2 2 4 a-anomeric H 0-anomeric H H6's o f Rha 105.27 103.44' 102.23 101.16 100.37 96.77 95.51 61.73 17.42 0-Gal-Rha B-GlcA a-Man-Men a-Rha-GlcA a-Rha t e r m i n a l a-Man-Gal Cjj's o f Man, Gal C6's o f Rha - r e 4 1 2 1 2 1 3 1 2 1 - GlcA Man — Man — Gal Rha -B a a 6 cc 1 Rha 5.29 5.23 5.05 4.55 1.28 a-anomeric H B-anomeric H H6's o f Rha PI 105.32 103.35 101.28 100.44 97.31 96.14 61.91 17.42 g-Gal-Rha B-GlcA a-Man-Man a-Rha-GlcA a-Rha t e r m i n a l a-Man-Gal C6's o f Man, C6's o f Rha Gal i t o t o i a C h e m i c a l s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e ; 62.23 f o r ^ -n.m.r. and 31.07 p.p.m. f o r 1 3C-n.m.r. d o w n f i e l d from sodium 4 , 4 - d i m e t h y l - 4 - s i l a p e n t a n e - l - s u l f o n a t e (D.S.S.). b c b=broad; s = s i n g l e t . For example, a-Man-0H=protonon Cl o f a - l i n k e d Man r e s i d u e . d F o r example, e-Gal-Rha= 1 3C-1 o f 6-1 i n k e d n o n - r e d u c i n g Gal r e s i d u e . S p e c t r a r e c o r d e d w i t h o u t i n t e r n a l s t a n d a r d ; c h e m i c a l s h i f t s a s s i g n e d r e l a t i v e to the c h e m i c a l s h i f t s o f H6's and C6 o f rhamnose r e s i d u e , 61.28 f o r lH-n.m.r. and 17.42 p.p.m. f o r 1 3C-n.m.r. TABLE III.2 Methylation Analyses of K53 Capsular Polysaccharide and Derived Poly-and Oligosaccharides Methylated Sugars9 Tb Mole % c (as alditol acetates) Column (ECNSS-M) II III IV V VI VII VIII 2,3,4-Rha 0.44 18.6 19.5 30.1 3,4-Rha 0.89 19.0 18.4 18.8 16.2 2-Et-4,6-Man 1.82 20.5 3,4,6-Man 1.94 17.6 18.9 45.1 50.8 68.3 54.0 45.3 26.1 2,4,6-Gal 2.27 17.2 15.4 30.5 21.4 20.5 23.3 2,3,4-Glc 2.48 49.2 31.7 24.6 18.0 2,3,6-Glc 2.50 11.6 4,6-Man 3.31 16.0 14.1 2,3-Glc 5.32 11.6 2.0 5.6 3 2,3,4-Rha = 1,5-di-0-acetyl-2,3,4-tri-0-methyl-L-rhamnito.l, etc. b Retention time relative to that of 1,5-di-0-acetyl-2,3,4,6-tetra-0rmethyl-D-glucitol on an ECNSS-M column, isothermal at 170°C. C 61 Values are corrected by use of effective, carbon response factors given by Albersheim et a l . ^ I, methylated original polysaccharide PI; I I , methylated polysaccharide, reduced, remethylated; I I I , straight chain methylated polysaccharide P_2; IV, aldobiouronic acide Al_; V, aldotriouronic acid A2; IV, aldotetraouronic acid A3; VII, aldopentaouronic acid A4; VIII, ethylated product from uronic acid degradation. -41-(Table III. 2, column I). The presence of a dimethyl mannose residue is attributable to a branch point and that of a trimethyl rhamnose to the terminal sugar of a side-chain. Absence of any alditol methylated at the 5 position is an indication of the pyranosidic nature of the sugar constituents. The 2,3-di-()-methyl glucose is the only derivative which could have arisen from the glucuronic acid. This has been shown by the appearance in the g.l.c. tracing of a peak corresponding to the acetate derivative of 2,3,6-tri-O-methyl glucitol and the almost complete disappearance of the 2,3-di-0--methyl glucitol acetate peak, after the permethylated polysaccharide was reduced and remethylated (Table III.2, column II). A small amount of the dimethyl glucose residue in this second analysis was because of incomplete methylation. Partial Hydrolysis Partial acid hydrolysis of K53 polysaccharide was followed by separation of acidic and neutral fractions by ion-exchange chromatography. The acidic components were separated by gel f i l t r a t i o n chromatography. Figure III. 1 illustrates the chromatogram produced from the weight of fractions collected. Some fractions were pooled and purified by descending paper chromatography. Four oligomers were thus collected. i) Compound Al_, [ a ] D -38.9° (c_ 1.97, water), 18mg. Refer to Table III. 1 and Table III. 2, column IV, Appendix I I I , spectra No. 5 and No. 6. The H^-n.m.r. spectrum (D^O,;ambient temperature) shows anomeric .signals at 65 . 30 ( 0 . 7 H, J ] 2 2 Hz), 64.99 (0.3 H, singlet), and 4.57 (1 H, 2 7 Hz). 1 3 ' In the C-n.m.r. spectrum, two signals occur in the anomeric region -42-10 120 30 40 50 Fraction no. ure 111.1 Gel chromatography separation of acidic oligomers obtained from partial hydrolysis of K53 polysaccharide -43-at 102.49 and 92.91 p.p.m., and one in the region associated with 28 -CH^ OH groups at 61.40 p.p.m. These results are in good agreement with a disaccharide having a reducing hexose and a non-reducing 3-linked glycoside. Methylation, reduction, hydrolysis of Al_, and subsequent g.l.c.-m.s. analysis of i t s alditol acetates^ 0'^ gave 3,4,6-tri-fJ-methylmannose and 2,3,4-tri-O-methylglucose in a molar ratio of 1:1. Compound Al_. i s , therefore, established as being Glc£A ^j- Man£ A]_ i i ) Compound A2, [ a ] D -5.8° (c_1.17, water), 35mg. Refer to Table I I I . l and Table III.2, column V, Appendix I I I , spectra No. 7 and No.8. The H^-n.m.r. spectrum (D20, ambient temperature) exhibits anomeric signals at 65.37 (0.7 H, singlet), 65.25 (0.3 H, unknown origin), 65.16 (1 H, . singlet), 64.93 (0.3 H, singlet) and 64.58 (1 H, J ] 2 6 Hz). In the 13 C-n.m.r. spectrum, three anomeric signals occur at 102.36, 100.84 and 28 93.40 p.p.m., and two in the region associated with non-linked C6 at 61.84 and 61.38 p.p.m. Methylation of A2 and subsequent g.l.c. - m.s. CA CO analysis D U' D < 1 0 f i t s alditol acetates gave 3,4,6-tri-0-methylmannose; and 2,3,4-tri-0-methylglucose in a molar ratio of 2:1. The structure of A2 is thus established as Gl c£A ~ - Man£ — - Man£ A2 i i i ) Compound A3, [ a ] D +47.1° (£4.27, water), 91mg. Refer to Table I I I . l and Table III.2, column VI, Appendix I I I , spectra No. 9 and.No.. 10. A3 was shown by n.m.r. spectroscopy to consist of one reducing hexose and three non-reducing glycosyl units,two a-linked and one - 44 -3-linked. The 'H-n.m.r. spectrum shows four anomeric signals at .;65.29 (1 .2 H, singlet), 65.18 (1 H, singlet), two overlapping doublets corresponding to two protons at 64.65 (J-j 2 1 Hz) and 64.61 (J-j 2 1 Hz). One more signal of unknown origin occurs upfield of the acetone peak at 61.29 (0.7 H, singlet). This peak is characteristic of the chemical shift of methyl protons of a 6-deoxyhexose, such as 13 rhamnose. However no rhamnose was detected either in the C-n.m.r. spectrum or in the methylation analysis of the oligomer. 13 The C-n.m.r. spectrum presents some interesting aspects. In this spectrum only five signals were expected in the anomeric region but six appear at 102.32, 101 .02 , 9 7 . 2 1 , 9 5 . 4 1 , 9 5 . 1 2 , and 93.12 p.p.m. The chemical shifts at 97.21 and 93.12 p.p.m. were assigned respectively to the 3 and a forms of the reducing galactose and those at 95v41 and 95.12 p.p.m. to the a-mannopyranosyl residue linked to the C3 of the galactose. The i n i t i a l reaction to these results is that the sample is a mixture of oligosaccharides. This possibility was eliminated by checking the purity of the sample by paper chromatography, only one spot was detected, and by determining the number of reducing end in 72 the sample by the method of Morrison. This method measures the ratio of acetylated aldononitrile to acetylated alditol and gives the degree of polymerisation of the oligomer; only one reducing end 73 was found. Colson and King , have reported for some g-D-glycopyranosyl-L-rhamnose disaccharides that the anomeric signal of the glycopyranosyl residue, when linked to C2 of the rhamnose, is s p l i t by the passage from the equatorial (3) to axial (a) position of the OH linked to the -45-anomeric carbon of the reducing rhamnose. Such a phenomenon could explain the splitting in A3 of the Cl signal of the mannopyranosyl residue linked to the C3 of the reducing galactose. Similar observations were made for another tetrasaccharide isolated in the structural investigation of K74 and having the same terminal disaccharide. Methylation of A3 and subsequent reduction, hydrolysis and g.l.c. - m.s. analysis of i t s alditol acetates^'^ 2 gave 3,4,6-tri-0_-methylmannose, 2,4,6-tri-O-methylgalactose, and 2,3,4-tri-0-methylglucose in the approximate molar ratio of 2.3:0.9:1.0. Compound A3_ i s , there-fore, established as being Gl c£A Man£ ^  Man£ \^Gal£ A3 iv) Compound A4, [ a ] D + 28.5° {c 0.74, water), 29 mg. Refer to Table I I I . l and Table III.2, column VII, Appendix I I I , spectra No. 11 and No. 12. This oligosaccharide was shown by n.m.r. spectroscopy to contain one reducing 6-deoxyhexose and four non-reducing glycosyl constituents, two of which are a-linked and two B-linked. The ^ H-n.m.r. spectrum exhibits six peaks in the anomeric region at 65.35 (0.5H, singlet), 65.26(1H, singlet), 65.19 (IH, singlet), -64.84 (0.5 H, singlet) and two overlapping doublets corresponding to two protons at 64.63 (J-j ^  7 Hz) and 64.59 (J-j 2 1 Hz). The signal due to the methyl protons of the rhamnose residue does.not appear because the spectrum was not recorded upfield 13 of the acetone peak. In the C-n.m.r. spectrum, six anomeric signals occur at 105.44, 102.31, 101.11, 95.53, 93.91, and 93.68 p.p.m., and one 28 in the region associated with non-linked C6 at 61.79 p.p.m. Finally, the peak' -46-at 17.63 p.p.m. was assigned to the C6 of the rhamnose residue. Methylation of A4 and subsequent reduction, hydrolysis and g. 1.c-,m.s. analysis of its alditol acetates ' gave 3,4,6-tri-0-methylmannose, 2,4,6-tri-fJ-methylgalactose, 2,3,4-tri-fJ-methyglucose, and 3-4-di-CJ-methylrhamnose in the approximate molar ratio of 2.3:1.0:0.9:0.8. The structure of A4 is established as GlcpA ^ -y Man£ ^  Manp_ ^  Gal£ ^  Rha£ A4 Uronic Acid Degradation The permethylated polysaccharide K53 was subjected to a C O ~7A base-catalyzed uronic acid degradation. ' In a single operation, the polymer was treated with sodium methyl sulfinylmethanide in methyl sulfoxide and directly alkylated with ethyl iodide. Hydrolysis, reduction of the degraded product and subsequent g.l.c.-m.s. analysis fin fi? of i t s alditol acetates ' gave 2,3,4-tri-0-methylrhamnose, 2-0-ethyl-4T6-di-O-methylmannose, 3,4,6-tri-fJ-methylmannose, and 2,4,6-tri-0-methylgalactose (see Table III.2, column VIII). Loss of glucuronic acid residues was accompanied by further degradation of exposed reducing groups as witnessed by complete loss of the 3,4-di-0-methylrhamnose residue. Further degradation was halted at this point because of formation of base-stable 3,6-dideoxy-hex-2-enopyranose units as shown below. \ H,0H 4,6-Man — 3,4,6-Man — 2,4,6-Gal - 6 3 1 2,3,4-Rha -47-Degradation of the glucuronic acid and dimethylrhamnose units and the presence of 2-0-ethyl-4,6-dimethylmannose indicate that glucuronic acid is linked to C2 of the branching mannose and to 01_ of the in-the-chain rhamnose residue. These results also prove that the terminal non-reducing rhamnose is the only sugar in the side chain linked to C3 of the branching mannose. The mass spectrum of the 2-0-ethyl isomer exhibits primary fragments at m/e 45, 131, 161, and 247. By comparing this spectrum with that of 1,3,5-tri-fJ-acetyl-2,4,6-tri-fJ-methylmannitol a characteristic shift of 14 mass units is observed (see Figure II.6). Study of Partially Depolymerized K53 Polysaccharide A mild hydrolysis, 2M TFA at 95°C, 30 min., was performed on a sample of K53 capsular polysaccharide. Methylation of the non-55 56 dialyzable product, ' reduction, hydrolysis, and subsequent g.l.c.-m.s. analysis of its alditol acetates^ 0'^ gave the results shown in Table III.2, column III. Complete loss of 2,3,4-tri-O-methylrhamnose and absence of any other terminal non-reducing residue indicated that the depolymerized K53 was a straight chain polysaccharide, P2 [a]p +14.2° {c 0.84, water). The specific rotation is in good agreement with the calculated specific rotation, [a]^ +15.2°, using Hudson's rules of isorotation.^ The low proportion of 2,3-dimethylglucose was because of incomplete reduction of the glucuronic acid methyl ester. 1 3 In the C-n.m.r. spectrum of P2 (see Appendix I I I , spectrum No.13) five peaks appear in the anomeric range at 105.27, 102.20, 101.12, 100.43, and 95.50 p.p.m., and another one at 17.28 p.p.m. owing to the C6 of the -48-rhamnose residue. Comparing this spectrum to that of the native K53 polysaccharide Pl_, one can see that the peak at 97.31 p.p.m. disappeared and was thus assigned to the a-rhamnose of the side-chain. Removal of the side-chain moved the chemical shift of the glucuronic acid by about 1 p.p.m. upfield, from 103.35 to 102.20 p.p.m. Finally, on the basis of previous n.m.r. data the chemical shift appearing at 100.44 p.p.m. in the spectrum of the native K53 could be assigned to the a-rhamnose linked in the1 chain. D or L configuration of the component sugars The D or L configuration of the component sugars was determined by comparing the sign of the circular dichroism (cd.) curves measured on the corresponding alditol acetates of those obtained from authentic samples.7 0 Since galactitol is a meso-alditol the configuration of galactose was determined relative to the peracetyl derivative of 2,4,6-tri-0-methylgalactitol. These derivatives were isolated by preparative gas-liquid chromatography from samples obtained from total sugar ratio and methylation analyses of K53. Measurements were made in acetonitrile at 215-250 nm. Rhamnose was confirmed to be of the L configuration, and glucose, mannose, and galactose were of the D configuration. Measurements were also made on partially methylated alditol acetates of rhamnose, mannose, and glucose. It showed that 3,4-di-0-methyl-L-rhamnose and 2,3,4-tri-O-methyl-L-rhamnose have a negative c d . curve, while 3,4,6-tri-O-methyl-D-mannose, 4,6-di-O-methyl-D-mannose, and 2,3-di-O-methyl-D-glucose have a positive c d . curve. Proposed Structure -49-Data obtained in the present structural investigation demonstrate that the capsular polysaccharide from Klebsiella serotype K53 is composed of hexasaccharide repeating units having the following structure. ^O-GlcpA ^ 0-Man£ -^0-Man£ ^ O - G a ^ 1-^ L-Rha£ ^  1 L-Rha£ Of the Klebsiella capsular polysaccharide reported to date, 75 only K52 has the same structural pattern but is not of the same chemotype (see Appendices I and II). Capsular polysaccharides K53 and K52 cross-react heavily in 19 anti-K47 serum. Both antigens, as well as the capsular polysaccharide K47,^ contain a common disaccharide; i\e.' a-L-Rha£-(1^4)-g-D-Glc£A-. In K47, this disaccharide constitutes the lateral side-chain while in K52 and K53, i t is part of the backbone. This feature suggests that this dimer is an antigenic determinant. Cross-reactivity of K53 with anti-PnXXIII also suggests that;the non-reducing a-L-rhamnose lateral end group could be an immunodominant sugar because the capsular polysaccharide 19 PnSXXIII has the same side-chain. -50-III. 4. Experimental  General Methods Descending paper chromatography was carried out using Whatman No. 1 paper for analytical purposes and Whatman 3MM for preparative paper chromatography. The following solvent systems (v/v) were used: (A) ethyl acetate - acetic acid - formic acid - water (18:3:1:4); (B) ethyl acetate - pyridine..- water (8:2:1); (C) 1-butanol - acetic acid -water (2:1:1). Chromatograms were developed using alkaline silver nitrate reagent.^ Gel f i l t r a t i o n chromatography was conducted on a column (100 x 3 cm) of Bio-Gel P-2(100-200 mesh). The column was irrigated with water - pyridine - acetic acid (500:5:2) at a flow rate ^ 10mL/h. Fractions of 2.0 - 2.5 mL were collected with a Gibson FC-80K micro fractionator or a LKB Radi Rac 3403 B fraction collector. Fractions were freeze-dried, weighed in tared tubes and the results plotted on graph paper to produce a chromatogram. Analytical g.l.c. separations were performed using a Hewlett Packard model 571 OA gas chromatograph fitted with dual flame ionization detectors. A stainless steel column (1.8 m x 3 mm) of 5% ECNSS-M on Gas Chrom Q (100 - 120 mesh) was exclusively used, operated at 170°C isothermal, except as otherwise stated. Preparative g.l.c. was performed on an F & M model 720 gas chromatograph with dual thermal conductivity detectors. A column (1.8 m x 6.3 mm) of 5% Silar 10C on Gas Chrom Q (100-120;mesh) was used for preparative separations. An Infotronics CRS-100 electronic integrator was used to measure peak areas. -51-G.l.c. - m.s. was performed using a Micromass 12 instrument fitted with a Watson-Biemann separator. Spectra were recorded at 70 eV with an ionization current of 100 uA and an ion source temperature of 200°C. H^-n.m.r. spectra were recorded on.a Varian XL-100 spectrometer at approximately 90°C and, in some cases, at ambient temperature (for water peak suppression). Samples run in D20 were 13 exchanged and freeze-dried three or four times in 99.7% D^ O. C-n.m.r. spectra were recorded on a Varian CFT-20 instrument at ambient temperature in 50 per cent D20. In a l l cases acetone ( 2.23 for 1 13 H-n.m.r. and 31.07 p.p.m. for C-n.m.r. measured against aqueous sodium 4,4-dimethyl-4-silapentane-l-sul fonate, D.S.S.) was used as 13 internal standard. In addition, a C-n.m.r. spectrum of the native polysaccharide (Pl_), recorded at 95°C, was obtained by courtesy of • Dr. Michel Vignon, CERMAV/CNRS, Grenoble, France on a Cameca 250 MHz.; instrument. Circular dichroism measurements were made on a Jasco-J-20 automatic recording spectropolarimeter with a quartz cell of path length 0.01 cm. Optical rotations of previously dried samples were measured at room temperature on a Perkin-Elmer model 141 polarimeter using a 10 cm c e l l . Infrared spectra were recorded on a Perkin-Elmer 457 spectrophotometer. Solutions were concentrated under reduced pressure at temperatures not exceeding 40°C. -52-Isolation and Purification of Klebsiella K53 Polysaccharide A culture of Klebsiella K53 (1756/51) was obtained from Dr. Ida 0rskov, Copenhagen, and was grown on a 3% sucrose - yeast extract - agar medium composed of 5g NaCl, 2.5g ^ HPO^, 0.62g MgSO^ . 7H2O, 0.5g CaCOj, 75g sucrose, 5g Bacto yeast extract, and 37.5g agar in 2.5L of water. The cells were harvested after 3 days, IL of very viscous slime was collected. Slime was diluted to 3L with 1% aqueous phenol, and centrifuged in batches for 8-12h at 30,000 rvp.m. in a Beckman model L3-50 ultracentrifuge with rotor type 35. The clear, supernatant liquids were decanted, combined (^2L), and precipitated by pouring into 1OL of Solvent IK (ethanol-methanol, 95:5). Crude polysaccharide was dissolved in IL of water and precipitated with a 10% Cetavlon solution. The precipitate was isolated by centrifugation, redissolved in IL of 4M NaCl, and reprecipitated by pouring into Solvent IK. The purified polysaccharide was collected, dissolved in water, and dialyzed against running tap-water for three days. Freeze-drying of this solution yielded 13g of the sodium salt of the capsular polysaccharide, [ a ] Q -12° (£0.24, water). Purity of the polysaccharide was checked by electrophoresis using a 1% solution on a cellulose acetate strip (Sepraphore II I ; 15 x 2.5 cm) in Veronal buffer pH 8.6 (LKB-Produkter AB,Stockholm 12, Sweden) at 300V for 60 min and then development in alcian blue. Homogeneity was also confirmed by gel chromatography by courtesy of Dr. S.C. Churms, University of Cape Town South Africa, and the molecular weight of K53 polysaccharide was-determined to be 1.2x10.6 -53-Some spectroscopic analyses were performed on K53 polysaccharide that had been partially depolymerized to reduce viscosity. This was achieved by mild hydrolysis in 0.5M trifluoroacetic acid for 30 min at 95°C, and then concentration under reduced pressure and evaporation several times with water to eliminate excess of acid. The hydrolysate was dissolved in water, dialyzed overnight against running tap-water and freeze-dried. Analysis of Component Sugars Polysaccharide was hydrolysed overnight with 2M trifluoroacetic acid at 95°C. After evaporation, the hydrolysate was found, by paper chromatography in solvents A and B, to contain rhamnose, galactose, mannose and glucuronic acid. Methanolysis of 30mg of Klebsiella K53 capsular polysaccharide with 3% methanolic hydrochloric acid and subsequent treatment with sodium borohydride in anhydrous methanol depolymerized the substrate 52 and reduced uronic acid residues. Total hydrolysis, reduction of the free sugars to alditols with sodium borohydride, and acetylation with acetic anhydride-pyridine (1:1) at room temperature overnight yielded rhamnitol pentaacetate, and the hexaacetates of mannitol, galactitol, and glucitol in the g.l.c. tracing using a column of 5% ECNSS-M; programmed at 170°C for 8 min and then at 4°/min to 190°C. Methylation Analysis a) Native Polysaccharide Pl_ About 500 mg of dried K53 capsular polysaccharide was dissolved in 50 mL anhydrous dimethyl sulfoxide with ultrasonic agitation and methylated by treatment with lOmL dimethylsulfinyl anion for 6h, and -54-then 8mli methyl iodide for lh. After removal of excess reagents by dialysis against running tap-water for three days, the methylated polysaccharide was recovered by freeze-drying, yield 352mg. Subsequent 78 Purdie treatment with silver oxide and methyl iodide gave a permethylated product that showed no hydroxyl group absorption at 3600 cnf^ in the infrared spectrum, yield 373 mg. A sample of 55mg of this dried material was reduced with lithium aluminum hydride in refluxing tetrahydrofuran, 15mL, for 3h and then at room temperature overnight. The white precipitate of aluminum hydroxide was dissolved with 4% hydrochloric acid and the reduced product was extracted with chloroform (5xl0mL). The combined extracts were washed with water (3xl0ml_) and evaporated to dryness under reduced pressure. The reduced polysaccharide, 45mg, was separated into two equal fractions. Fraction one was hydrolysed with 2M trifluoroacetic acid overnight at 95°C, reduced with sodium borohydride, and acetylated with acetic anhydride:pyridine (1:1) at room temperature overnight. 78 The second fraction was remethylated by one Purdie treatment and converted to the corresponding alditol acetates as for fraction one. G.l.c. and g.l.c. - m.s. analyses of both mixtures of partially methylated alditol acetates allowed the assignments given in Table III.2, columns I and II. b) Partially Depolymerized K53 Polysaccharide, £2 A sample (215mg) of K53 polysaccharide was partially depolymerized with 2M trifluoroacetic acid at 95°C for 30 min. The hydrolysate was concentrated under reduced pressure, evaporated several times with water to eliminate excess of acid, and then dissolved in -55-water and dialyzed overnight against running tap-water. Freeze-drying of the solution yielded 165 mg of polymeric material, P2_, ,[a] D + 14.2° (c 0.82, water). Methylation of 16 mg of £2 by 55 56 Hakomori's procedure, ' derivatisation to the corresponding alditol acetates as for the original polysaccharide, and g.l.c. analysis gave the results listed in Table III.2, column III. Partial hydrolysis A batch hydrolysis was performed on 640 mg of K53 polysaccharide with 0.5M trifluoroacetic acid at 95°C for 5 hours. After removal of the acid by evaporation with several portions of water, paper chromatography (solvent A) of the hydrolysate showed the presence of several oligosaccharides. The total amount of material was applied to the top of a column (2x20cm) of Bio-Rad A61-X2 resin in the formate form. The column was f i r s t eluted with 700 mL of water, and then with 500 mL of 10% formic acid. Paper chromatography (solvent B) of the neutral fraction, 288 mg, showed that i t contained mainly monosaccharides and very l i t t l e oligosaccharide. The acidic fraction, 322 mg, was separated by gel f i l t r a t i o n chromatography on a column of Bio-Gel P-2, 100x3 cm. The column was irrigated with a buffer composed of water: pyridine: acetic acid (500:5:2). Fractions of 2.0 - 2.5 mL were collected, freeze-dried, and weighed in tared tubes. The results were plotted on graph paper to produce the chromatogram shown in Figure I I I . l , page 42. Fractions 20-26 were pooled as well as fractions 29-34, 35-40, 43-46, and purified by descending paper chromatography on Whatman 3 MM paper using -56-solvent C for three to six days. Four pure oligosaccharides were thus collected, i.e. Al^, A2, A3, and A4. Methylation analysis of these oligosaccharides was performed as follow. Dried samples of 7-10 mg were dissolved in 5 mL anhydrous dimethyl sulfoxide, treated with 3 mL dimethylsulfinyl anion for 6h 55 56 and 4 mL of methyl iodide for 1 hour. ' The mixtures were diluted with water, neutralized with 10% acetic acid, transferred to separatory funnels and extracted with chloroform (5 x 10 mL). The combined extracts were back extracted with water (3 x 10 mL) and evaporated to dryness under reduced pressure. Permethylated oligosaccharides were reduced with lithium aluminum hydride and converted to the corresponding alditol acetates as previously described for the methylation analysis of the original polysaccharide. G.l.c. analysis of the mixtures of partially methylated alditol acetates allowed the assignments shown in Table III.2, columns IV-VII; results confirmed by g.l.c. - m.s. analyses. Uronic Acid Degradation^'^ 4 A sample (100 mg) of dried, methylated polysaccharide and 1 mg of £ - toluenesulfonic acid were dissolved in 20 mL of a 19:1 (v/v) mixture of dimethyl sulfoxide—2,2 - dimethoxypropane in a flask sealed with a rubber cap. The flask was flushed with nitrogen and 10 mL of dimethylsulfinyl anion was added with a syringe and the mixture was stirred overnight at room temperature. The substrate was then directly re-alkylated by adding 7 mL of ethyl iodide with external cooling and the reaction mixture was stirred one more hour. The mixture -57-was neutralized with 10% acetic acid and extracted with chloroform (4 x 10 mL). The combined extracts were washed with water (3 x 10 mL) and evaporated to dryness. Hydrolysis of the degraded product with 2 M trifluoroacetic acid, reduction with sodium borohydride, acetylation with a 1:1 (v/v) mixture of acetic anhydride: pyridine overnight at room temperature, and g.l.c. analysis of the corresponding alditol acetates gave the results listed in Table 111.2, column VIII; results fin fi? confirmed by g.l.c. - m.s. analysis. ' -58-Structural Investigation of Klebsiella Serotype K74 Capsular Polysaccharide -59-IV. 1. Abstract By using the techniques of methylation analysis, partial hydrolysis, and uronic acid degradation, the structure of the capsular polysaccharide from Klebsiel!a serotype K74 has been investigated. The anomeric natures of glycosidic linkages were determined by 1 13 H-and C-n.m.r. spectroscopy on the original polysaccharide and on oligosaccharides obtained from partial hydrolysis. The polymer was shown to consist of pentasaccharide repeating units as shown below 1 2 D-Man£ 31 12 a D-Manp_ •  a n a D-Glc£A 4 D-Gal£ \ / C -60-IV. 2. Introduction In a qualitative analysis of the capsular polysaccharide from Klebsiella type K74, Nimmich^ reported the presence of glucuronic acid, galactose, mannose, and pyruvate acetal. Six other Klebsiella polysaccharides contain these sugars,^0 and structural investigation of two of these, type K20 7 9 and type K 2 1 h a v e been published. We now report the structural investigation of K74. - 6 1 -IV. 3 Results and Discussion Composition and n.m.r. spectra Isolation and purification of the polysaccharide, as previously described in Section 111.4, provided a homogeneous polymer as indicated by electrophoresis. The product had [a]^ + 66° (£0.21, water), which compares very well with the. calculated specific rotation (+65°) using Hudson's rules of isorotation^ , and a molecular weight of 4.7 x'.lO as determined by gel chromatography. 1 34 35 The H-n.m.r. spectrum ' of the original polysaccharide exhibits a sharp singlet at 6.1 .51, characteristic of the methyl protons of the pyruvate acetal. Absence of signal at 6 2.20 indicates that the polymer does not contain any acetate group. The anomeric region (64.5 - 5.5), though poorly resolved, shows one broad signal at 64.55 assigned to B-linkages and two broad signals at 65.26 and 6.5.37 attributable to o>-linkages. By integration the ratio of the a-anomeric protons to the g-anomeric ones seems to be 3:2. Comparison of the integrals also indicates that there is one pyruvate acetal per pentasaccharide repeating unit. This result has been confirmed by courtesy of Dr. S.C. Churms, University of Cape. Town, South Africa, who found that the polysaccharide contains 11.9% of pyruvic acid (calculated, 11.7%). However, Nimmich has 81 reported that K74 contains only 4% of pyruvate acetal. 1 13 The H-n.m.r. analysis was confirmed by C-n.m.r. spectroscopy of the polysaccharide. The spectrum shows five peaks in the anomeric region at 103.94, 103.26, 100.96, 100.50, and -62-96.16 p.p.m., and three in the region associated with -CHgOH groups at 62.19, 62.00, and 61.85 p.p.m. Another peak, characteristic of the methyl group of the pyruvate acetal, appears upfield of the acetone signal at 26.12 p.p.m. According to observations reported by Garegg et a l , the acetal carbon atom of 82 the pyruvate group is thus assigned to be of the R configuration. For n.m.r. data refer to Table IV.1 and Appendix I I I , spectra No.20 and No.21. Total hydrolysis of Klebsiella K74 capsular polysaccharide and subsequent g.l.c. analysis of the corresponding alditol acetates confirmed the presence of mannose, galactose, and glucose in the approximate molar ratio of 2:2:1. Glucose and mannose were confirmed to be of the D configuration by circular dichroism (cd.) measurements of their alditol acetates.'7 0 Galactose was also shown to be of the D configuration based on the c d . of the acetate derivatives of 2,3-di-O-methylgalactitol and 2,4,6-tri-0-methylgalactitol. The derivatives were isolated by preparative gas liquid chromato-graphy from samples prepared for the total sugar ratio and methylation analyses of K74. Measurements were made in acetonir•.ile• at 215-250 nm. Methylation Analysis Complete methylation of K74 polysaccharide in i t s sodium salt form, as well as its^free acid form, proved to be d i f f i c u l t owing to the low solubility of the polymer in dimethyl sulfoxide. 55 56 78 One Hakomori methylation ' and one Purdie treatment were necessary in order to obtain a product that showed no hydroxyl T a b l e I V . 1 N . m . r . d a t a o f K l e b s i e l l a K74 c a p s u l a r p o l y s a c c h a r i d e a n d d e r i v e d o l i g o s a c c h a r i d e s Compound 6 a J . , ^ H - n . m . r . d a t a 1 3 C - n . m . r . d a t a i 2 n—II.in. r. uow u - n . m . r . a a t a H ' \ b I n t e g r a l ( H ) A s s i g n m e n t ^ p . p . m . a A s s i g n m e n t d ( z) G l c A — Man-OH a B l G l c A I 3- Man ^ Man-OH B2 G l c A 1 1 Man ^ Man I 3- G a l - O H B3 i ? i ? G a l Man Man B 3 | 0 a 1 a G l c A 1 1 G a l V C H 3 COOH 5 . 3 4 5 . 1 8 4 . 9 4 5 . 3 7 5 . 3 4 5 . 1 7 5 . 0 8 4 . 9 4 5 . 3 4 5 . 2 9 5 . 1 7 5 . 0 7 4 . 6 4 5 . 3 7 5 . 2 6 4 . 5 5 1 . 5 1 3 1 a - G l c A 1 0 1 . 4 2 a - G l c A S 0 . 6 a-Man-OH 9 4 . 8 2 a-Man-OH S 0 . 4 B-Man-OH 9 4 . 3 4 B-Man-OH 6 1 . 7 3 C6 o f Man 2 . 1 a-Man-OH 1 0 2 . 8 5 a - M a n - M a n 3 J a - G l c A 1 0 1 . 3 7 a - G l c A 2 0 . 3 unknown o r i g i n 9 3 . 3 8 a , B - M a n - O H 2 1 . 0 a - M a n - M a n 6 1 . 9 4 C 6 ' s o f Man 1 0 . 3 B-Man-OH l\ 2 a - G l c A 1 0 3 . 0 7 a - M a n - M a n 4 J a - M a n - G a l 1 0 1 . 3 3 a - G l c A - O H S 0 . 3 a - G a l - O H 9 7 . 2 1 , B - G a l - O H 2 1 a - M a n - M a n 9 5 . 3 3 1 a - M a n - G a l 7 0 . 9 B - G a l - O H 9 5 . 0 8 J 9 3 . 0 9 a - G a l - O H 6 1 . 9 2 C 6 ' s o f M a n , G a l 3 a - a n o m e r i c H 1 0 3 . 9 4 1 B - G a l b J 1 0 3 . 2 6 J b 2 B - a n o m e r i c H 1 0 0 . 9 6 a - G l c A S 3 - C H 3 o f p u r u v a t e 1 0 0 . 5 0 B-Man-Man 9 6 . 1 6 , a - M a n - G a l 6 2 . 1 9 1 6 2 . 0 0 \ C 6 ' s M a n , G a l 6 1 . 8 5 J 2 6 . 1 2 - C H j o f p y r u v a t e K74 p o l y s a c c h a r i d e I I Chemical s h i f t r e l a t i v e t o i n t e r n a l a c e t o n e ; 62.23 f o r 'H-n.m.r. and 31.07 p.p.m. f o r uC-n.m.r. d o w n f i e l d from sodium 4 , 4 - d i m e t h y l - 4 -s i l a p e n t a n e - l - s u l f o n a t e (D.S.S.). b c b=broad, s = s i n g l e t . F o r example, a-GlcA = p r o t o n on Cl_ o f a - l i n k e d G l c A ; a-Man-Man = p r o t o n on CI o f the no n - r e d u c i n g a - l i n k e d Man As f o r c , but f o r a n o m e r i c , 1 3 C n u c l e i . e ^H-n.m.r. spectrum o f 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 c o r d e d w i t h o u t i n t e r n a l s t a n d a r d ; c h e m i c a l s h i f t s a s s i g n e d r e l a t i v e t o -CH, o f p y r u v a t e a c e t a l , 61.51. -65-absorption in the infrared spectrum. Reduction of the permethylated sample' with lithium aluminum hydride, total hydrolysis with 2M trifluoroacetic acid, and subsequent g.l.c. analysis of the corresponding alditol acetates gave the results listed in Table IV.2, column I. These results seem to corroborate those obtained from n.m.r. experiments despite the low molar percentages of the dimethylglucose and the dimethyl galactose units. The low abundance of these two compounds could be due to the fact that they have large retention times and show not well separated, broad peaks in the g.l.c. tracing. Consequently, integration of their peak areas is not very good. When the experiment is repeated using a programme the peaks of these two isomers are sharper and almost as large as the peaks of the other three partially methylated alditol acetates (see Figure IV.-.T). Integration is then better and the molar percentage ratio is improved (see Table IV.2, column I, values in parentheses). The s t i l l low proportion of 2*3-dimethylglucose was probably because of incomplete reduction of the glucuronic acid methyl esters. As demonstrated by further studies (see below), the presence of 4,6-di-0-methylmannose is due to a branch point and the 1-carboxyethylidene group is present as an acetal spanning 04 and 06 of the terminal 2,3-di-fJ-methyl galactose. Partial Hydrolysis Partial, acidic hydrolysis of the native K74 polysaccharide was followed by separation of the acidic and neutral fractions by ion-exchange chromatography. The neutral fraction was not studied TABLE IV. 2 Methylation Analysis of Native, and Degraded K74 Capsular Polysaccharide and Derived Oligosaccharides Methylated sugars Tb Mole % c (as alditol acetates) ECNSS - M I d II 11 I-l III-2 IV V 3,4,6 - Man 1.93 (1.98) e 23.3(21.2)e 16.8 41.5 23.6 58.6 2,4,6 - Man 2.08 51.5 49.2 39.8 32.5 2,4,6 - Gal 2.25 (2.34) 25.9(23.2) 20.2 35.0 2,3,4 - Glc 2.45 48.5 34.3 18.7 23.7 4,6 - Man 3.24 (3.34) 22.0(22.0) 6.4 2,3 - Glc 5.27 (4.53) 13.4(13.9) 2,3 - Gal 5.71 (4.72) 15.4(19.7) a 3,4,6 - Man = 1,2,5-tri-0-acetyl-3,4,6-tri-0-methyl-D-mannitol, etc. Retention time relative to 1,5-di-0-acetyl-2,3,4,6-tetra-0-methyl-D-glucitol on an ECNSS-M column operated at 17QOC, isothermal. Values corrected by use of effective, carbon response factors given by Albersheim et a l . I, methylated original polysaccharide; I I , aldobiouronic acid, Bl_; 111 -1 and III-2, aldotriouronic acid, B2; IV, aldotetraouronic acid,_B3j V, uronic acid degradation product. Numbers in parentheses refer to data obtained when temperature programming was used; 160°C for 13 min and then 2o/min to 190°C. -67-3,4,6-Man Column : ECNSS-M 4 8 12 16 20 Column: ECNSS-M Program: 160°C for 13 min then at 20/min to 19QOC Time (min.) Figure IV.T G.l.c. separation of a mixture of partially methylated alditol acetates obtained from KIebsiella K74 polysaccharide. -68-further, while the acidic one was separated by gel f i l t r a t i o n chromatography. The chromatogram produced from the weight of the fractions collected is shown in Figure IV.2. Some fractions were pooled and purified by descending paper chromatography. Three pure oligosaccharides were thus collected. i) Compound Bl_, [ a ] D + 80.7° [c 2.41, water), 52 mg. Refer to Table IV.1 and Table IV.2, column I I , and Appendix I I I , spectra No. 14 and No. 15. The "'rl-n.m.r. spectrum showsrsignals at 55;34 (1 -H, J-| 2 3 H z ) ' 5 5 - 1 8 (°-6 H, singlet), and 64.94 (0.4 H, singlet) in the anomeric region. In the 13 C-n.m.r. spectrum three signals occur in the anomeric region at 101.42, 94.82, and 94.34 p.p.m., and one in the region associated with non-linked C6 at 61.73 p.p.m. These data correspond to a compound having one reducing hexose and one a-linked, non-reducing glycosyl 55 56 residue. Methylation ' of Bl_ yielded a product that, on hydrolysis and conversion of the products into alditol acetates, gave components corresponding to 2,4,6-tri-fJ-methylmannose and 2,3,4-tri-0--methy!glucose in the molar ratio of 1:1; results confirmed by g.l.c.-m.s.^'^2 The structure of Bl_ is thus established as D-Glc£A ^  D-Man£ B l i i ) Compound B2,[a] D + 89.4° {c 1.61, water), 60 mg. Refer to Table IV.1 and Table IV.2, columns 111-1 and III,2, and Appendix I I I , spectra No. 16 and No.17. The H^-n.m.r. spectrum exhibits two overlapping doublets corresponding to 2.1 H at 65.37 (J-j 2 2 Hz) and 65.34 ( J 1 2 3 Hz), and three more peaks at 65.17 (0.3 H, J ] 2 2 Hz), -69--70-65.08 (1 H, J 1 2 2 Hz), and 6 4.94 (0.3 H, J-, 2 1 Hz). In the 13 C-n.m.r. spectrum three.,signals appear in the anomeric region at 102.85, 101.37, and 93.38 p.p.m., and one at 61.94 p.p.m. due 55 56 to non-linked C6_'s of the mannose residues. Methylation ' of B2 and subsequent g.l.c. - i i i .s. analysis of the corresponding alditol acetates ^°'^ 2 was performed twice. In the f i r s t case the analysis gave 3,4,6-tri-0-methylmannose, 2,4,6-tri-0-methylmannose, and 2,3,4-tri-0-methylglucose in the approximate molar ratio of 1:3:2. In the second analysis the molar ratio was approximately 1:1:0.5. These inconsistent results could possibly be attributable to some degradation during the methylation analysis and to incomplete reduction of the glucuronic acid methyl ester. However, the structure of B2_ is established as being D- 61 cpA — D- Ma np — D- Manp B2 i i i ) Compound B3, [a] + 130° (c 2.10, water), 72 mg. Refer to Table IV.1 and Table IV.2, column IV, and Appendix I I I , spectra No. 18 and No. 19. In the H^-n.m.r. spectrum two overlapping doublets corresponding to two protons occur at 65.34 (J-j ^ 3 Hz) and 65.29 (J-j ^ 4 Hz), and three additional signals at 65.17 (0.3 H, singlet),-65.07 (1 H, J-j 2 2 Hz), and 64.64 (0.9 H, J-j 2 7 Hz). The 1 3 C-n.m.r. spectrum exhibits six peaks in the anomeric region at 103.07, 101.33, 97.21, 95.33, 95.08, and 93.09 p.p.m., and one signal in the region associated with -^OH groups at 61.92 p.p.m. The spectrum presents 13 the same interesting feature observed in the C-n.m.r. spectrum of the tetraouronic acid isolated from K53 (see Section III.3, p 43 ); -71-i.e. splitting of the anomeric signal of the mannopyranosyl residue linked to C3 of the reducing galactose. Chemical shifts at 95.33 and 95.08 p.p.m. have been assigned to this mannose residue. As reported 73 for some 1 -* 2 linked disaccharides , such a splitting could possibly be due to the passage from the equatorial to axial position of the OH linked to Cl_ of the reducing galactose. The tetrasaccharide B3 was shown not to be a mixture of 72 oligosaccharides by the method of Morrison. The sample was reduced with sodium borohydride, hydrolysed, and converted to the corresponding alditol acetate and aldononitrile acetate. vG.l.c. analysis of the mixture gave mannononitrile pentaacetate and galactitol hexaacetate in the ratio of 2:1. GIucononitrile acetate did not appear in the g.l.c. tracing because the glucuronic acid was not reduced before conversion to the aldononitriles. These results confirmed that galactose is the reducing sugar in the tetrasaccharide. Knowing the structure of the aldobiouronic acid Bl_, i t is also possible to conclude that the two mannose residues are linked together. 55 56 Methylation of B3, ' followed by reduction, hydrolysis, and subsequent g.l.c. analysis of the derived alditol acetates gave 3,4,6-tri-fJ-methylmannose, 2,4,6-tri-0-methylmannose, 2,4,6-tri-0-methylgalactose, and 2,3,4-tri-0-methylglucose in a molar ratio of 1:1.5:1:1; results confirmed by g.l.c. - m.s.^0'^ Compound B3 is therefore established as D-GlcpA — D-Manp — D-Manp — D-Galp B3 -72-Uronic Acid Degradation In a single operation, the permethylated polysaccharide was subjected to a base-catalysed uronic acid degradation, using dimethylsulfi 68 74 anion, and directly alkylated with methyl iodide. ' Hydrolysis of the remethylated, degraded material and analysis of the products 60 62 by g.l.c. -m.s. ' of the alditol acetates gave the results listed in Table IV-.2, Column V.'The presence of some 4,6-di-(3-methylmannose is due to incomplete methylation of the degraded polymer. Loss of glucuronic acid residues was accompanied by further degradation of exposed, reducing groups as witnessed by the complete disappearance of 2,3-di-0-methylgalactose. These results indicate that the side-chain of K74 polysaccharide is a pseudo-aldobiouronic acid linked to C3 of the branching mannose and whose structure is established as From the structure of the tetrasaccharide B_3, i t has been established that the glucuronic acid is linked to a neutral trisaccharide in 1 3 12 1 3 the following sequence: GlcA Man Man Gal. Since the glucuronic acid is part of the side-chain, this neutral trimer constitutes the backbone of the polymer. Now connecting the side-chain to the backbone, the structure of the capsular polysaccharide D-GalE !-^D-Glc£A \--73-from Klebsiella serotype K74 is established as 1 2 1 2 D - Gal£ D - Man£ -^D-Man£ 1 D-Glc£A 41 1 D-Gal£ A I CH COOH Immunology The capsular antigen from Klebsiella K74 cross-reacts 19 weakly in antipneumococcal sera Pn I I , PnVIII, and PnXX . However, no useful information can be obtained from these reactions since there is no common structural feature between K74 and the pneumococcal capsular polysaccharides PnS I I , PnS VIII, and PnS XX. -74-IV. 4 Experimental General Methods Instrumentation used has been described previously (see Section III.4, page 50). For descending paper chromatography, the following solvent systems (v/v) were used: (A) ethyl acetate -acetic acid - formic acid - water (18:3:1:4); (B) ethyl acetate -pyridine - water (8:2:1); (G) freshly prepared 1-butandl-acetie a c i d -water (2:1:1). Analytical g.l.c. separations were performed using the following stainless steel columns (1.8 m x 3: mm): (A) 3% of SP - 2340 on Supelcoport (100-130 mesh); (B) 5% of ECNSS - M on Gas Chrom Q (100 - 120 mesh); (C) 3% of 0V - 225 on the same support. Preparative g.l.c. separations were performed using a column (D) (1.8 m x 6.3 mm) of 5% Silar 10C on Gas Chrom Q (100 - 120 mesh). 13 The C-n.m.r. spectrum of the native polysaccharide was obtained by courtesy of Dr. Michel Vignon, CERMAV/CNRS, Grenoble, France on a Bruker Spectrospin instrument. Isolation and Purification of Klebsiella K74 Capsular Polysaccharide A culture of Klebsiella serotype K74 (371), obtained by courtesy of Dr. Ida 0rskov, was grown as described previously for Klebsiella K53 (see Section III.4, page 52 ). The isolated polysaccharide (10 g), in the sodium salt form, had [a] D + 66? (c 0.21, water). Purity of the polysaccharide was checked by electrophoresis using a 1% solution on cellulose acetate strips (Sepraphore I I I ; 15 x 2.5 cm) in Veronal buffer pH 8.6 (LKB - Produkter AB, Stockholm 12, Sweden) -75-at 300 V for 60 min and then developed- , in alcian blue in citrate buffered ethanol. Homogeneity was confirmed by gel chromatography by courtesy of Dr. S.C. Churms, University of Cape Town, South Africa, and the molecular weight of K74 polysaccharide determined to be 4.7 x 106. Analysis of Sugar Constituents Sugar analysis was performed as described in Section 111.4, page 53. The alditol acetates of mannose, galactose, and glucose were identified by g.l.c. (column A; programmed at 195°C for 4 min and then 2°/min to 260°C) by comparison with authentic samples and found to be present in the approximate molar ratio of 2:2:1. Paper chromatography (solvents A and B) run on an hydrolysate of the polysaccharide confirmed the presence of mannose, galactose, glucuronic acid, and pyruvic acid. The D or L configuration of the constituent sugars was determined by measurement of the circular dichroism curve of the alditol acetates of mannose and glucose, and of the partially methylated alditol acetates of galactose. Samples were isolated by preparative g.l.c. (column D; 240°C isothermal). Comparison with authentic standards confirmed the D configuration of a l l the sugar constituents. Methylation Analysis A dried sample (127 mg) of K 74 polysaccharide was dissolved in 100 mL anhydrous dimethyl sulfoxide using ultrasonic agitation and methylated by treatment with 12 mL dimethyl sulfinyl anion overnight, and then 8 mL methyl iodide for 1% h. The reaction mixture -76-was dialyzed against running tap-water for three days and freeze-dried. The product was dissolved in chloroform, fi l t e r e d , and subsequent 78 Purdie treatment of the soluble fraction (153 mg) with methyl iodide and silver oxide gave a product that showed no hydroxyl absorption in the infrared spectrum. The permethylated sample was reduced with lithium aluminum hydride in refluxing tetrahydrofuran for 5 hours, and the reaction was continued at room temperature overnight. Excess of hydride was destroyed with water and the product was recovered by the method of Dutton and Smith.^ Hydrolysis of the reduced product with 2M trifluoroacetic acid at 95°C overnight, reduction with sodium borohydride, and g.l.x. - m.s.^'^2 analysis of the partially methylated alditol acetates gave the results listed in Table IV.2, column I. Partial Hydrolysis About 540 mg of K74 polysaccharide was partially hydrolysed with 2M trifluoroacetic acid at 95°C for 2.5 h. The solution was concentrated to dryness under reduced pressure and evaporated several times with water to eliminate excess of acid. The product was applied to a column (30 x 1.5 cm) of Bio - Rad AG 1 - X2 resin in the formate form. The neutral fraction was eluted with 600 mL of water and freeze - dried, yield 254 mg. The acidic fraction was eluted with 500 mL of 10% formic acid, evaporated to dryness under reduced pressure several times with water, and freeze -dried, yield 293 mg. Paper chromatography (solvent C) run on the -77-acidic fraction showed that i t contained a disaccharide and higher oligosaccharides. The acidic oligomers were separated by gel f i l t r a t i o n chromatography on a Bio - Gel P-2 column (100 x 3 cm) using a buffer composed of water: pyridine: acetic acid (500:5:2) for irrigation at a flow rate of 10 mL/h. Fractions of 2.0 - 2.5 mL were collected in tared tubes, freeze - dried, and weighed. The chromatogram produced by the weight of the fractions collected is shown in Figure IV.2. Fractions 41-47 were pooled as well as fractions 35-40 and 29-34, and purified by descending paper chromatography for three days using solvent C. Three pure oligosaccharides were thus collected, Bl_, B2, and B3. Methylation analysis of these oligosaccharides was performed as described for the oligomers isolated from K53 (see Section III.4, page 55 ). Results of the analyses are shown in Table IV.2, columns 11-IV. The purity of the tetrasaccharide B3 was checked by g.l.c. (column C; 210 C isothermal) according to the method of Morrison. Uronic Acid Degradation^' 7^ A dried sample (15 mg) of methylated K74 polysaccharide and 1 mg of £ - toluenesulfonic acid were dissolved in 10 mL of a 19:1 (v/v) mixture of dimethyl sulfoxide - 2,2-dimethoxypropane, and treated with 5 mL dimethylsulfinyl anion overnight. An excess of methyl iodide (7 mU) was added with external cooling, the mixture was stirred one hour, and dialyzed against running tap - water for -78-two days. The polymeric material was extracted with chloroform (5 x 10 mL) and the combined-extracts were evaporated to dryness under reduced pressure. Hydrolysis of the product with 2M trifluoroacetic acid, reduction with sodium borohydride, acetylation with a 1:1 (v/v) mixture of acetic anhydride.: pyridine overnight at room temperature, and g.l.c. analysis of the alditol acetates gave the results listed in Table IV.2, column V. -79-V BIBLIOGRAPHY 1. Edwards, P.R. and W.H. Ewing, "Identification of Enterobacteriaceae,1 Burgess Publishing Company, Minneapolis, 1972. 2. 0rskov, I., Bergey's Manual of Determinative Bacteriology, 8th ed., 321-324 (1974) 3. Morgan, H.R., "Bacterial and Mycotic Infections of Man (ed. Dubos and Hirsch), 610-648, J.B. Lippincott Company, Philadelphia, Montreal, 1965. 4. Ghuysen, J.M., J.L. Strominger and D.J. 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Choy, Y.M., and G.G.S. Dutton, Can. J. Chem., 5J_, 3015-3020 (1973). -83-80. Choy, Y.M. and G.G.S. Dutton, Can. J. Chem., 51, 198-207 (1973). 81. Nimmich, W., Z. Allg. Mikrobiol., 19 (5), 343-347 (1979). 82. Garegg, P.J., P.E. Jansson, B. Lindberg, F. Lindh, J. Lonngren, I. Kvarnstrom, and W. Nimmich, Carbohydr. Res., 78, 127-132, (1980). 83. Niemann, H., N. Frank, and S. Stirm, Carbohydr. Res., 59_, 165-177 (1977). -84-Appendix I Structural Patterns of Klebsiella Capsular Polysaccharides Key : X = Uronic Acid 0 = Neutral sugar; pyruvate and acetate omitted A. Uronic acid absent ^ I - 0 - 0 - 0 - 0 - - o - o - o - o - - 0 - 0 - 0 -K32, K72 I I 0 0 K56 K38 - 0 - 0 - —,0 - 0 -0 0 1 A [X] <X> K37 K22 (X) = 3-deoxy-L^-glycero-pentulosonic acid [X] = 4-0^  [(s)-l-carboxyethyl]-D-glucuronic acid (x) = 2R,3R-hex-4-enopyranosyluronic acid B. Uronic acid in chain a) linear - :X - 0 - 0 - - X - 0 - 0 - 0 - X - 0 - 0 - 0 - 0 KI, K5, K63 K4, K6 K9*, K44 - X - 0 - 0 - 0 - 0 - 0 -K70, K81 -85-b) branch point on uronic acid i) single unit side chain - X - 0 - 0 I 0 K11, K51, K57 - X - 0 - 0 - 0 I 0 K21, K24 i i ) two unit side chain - X - 0 - 0 I 0 ! 0 K31 c) branch not on uronic acid i i i ) plus branch points neutral sugars - X - 0 - 0 - 0 I I I 0 0 0 K60 X - 0 - 0 -I 0 K16, K54 X - 0 - 0 - 0 0 K7, K61, K62 X - 0 - 0 - 0 0 K52, K53 X - 0 - 0 - 0 I 0 K17 X - 0 - 0 I 0 K58 d) double branch not on uronic acid 0 - X - 0 - 0 - 0 -! 0 K 6 4 - 8 6 -C. U r o n i c a c i d i n s i d e c h a i n a ) s i n g l e u n i t s i d e c h a i n - 0 - 0 - 0 - - 0 - 0 - 0 - 0 I I X X K 2 , K 8 , K51 K 9 , K59 b) t w o s i n g l e u n i t s i d e c h a i n s f o r m i n g a d o u b l e b r a n c h 0 0 1 I - o - o - o - _ 6 - o - o - o I • I X X K 3 0 , K33 K27 c ) two u n i t s i d e c h a i n i ) u r o n i c a c i d t e r m i n a l - 0 - 0 -I 0 I X K 2 0 , K 2 3 , K 5 5 , K83 i i ) u r o n i c a c i d n o n - t e r m i n a l - 0 - 0 - - 0 - 0 - 0 - - 0 - 0 - 0 - 0 I I I X X X I I I 0 0 0 K 2 5 , K47 K 1 3 , K74 K I 2 , K 2 8 , K36 -87-Th re e unit side chain i ) uronic acid non-terminal - 0 - 0 - 0 - - 0 - 0 - 0 - 0 I I 0 X 1 I X 0 I I . 0 0 KI 8 K41 Note: K9 and K9* are supposed to be from the same strain of Klebsiella. This strain has been investigated in two laboratories but two different structures have been proposed. - 8 8 -APPENDIX II KLEBSIELLA CAPSULAR POLYSACCHARIDES (KI - K83) GROUPED ACCORDING TO CHEMOTYPE G l u c u r o n i c A c i d , G a l a c t o s e , G l u c o s e 8 P , l l p , 1 5 , 5 1 , 2 5 , 27 P G l u c u r o n i c A c i d , G a l a c t o s e , Mannose 20, 2 1 p , 2 9 p , 4 2 p , 4 3 , 6 6 , 7 4 p G l u c u r o n i c A c i d , G a l a c t o s e , Rhamnose 9 , 4 7 , 5 2 , 9 * , 8 1 , 83 G l u c u r o n i c A c i d , G l u c o s e , Mannose 2 , 4 , 5 P , 24 G l u c u r o n i c A c i d , G l u c o s e , Rhamnose 17, 4 4 , 71 G l u c u r o n i c A c i d , G l u c o s e , Fucose 1 , 54 G l u c u r o n i c A c i d , G a l a c t o s e , G l u c o s e , Mannose 1 0 , 2 8 , 3 9 , 5 0 , 5 9 , 6 1 , 6 2 , < 7 P , 1 3 p , 2 6 p , 3 0 p , 3 1 P , 3 3 p , 3 5 p , 4 6 p , 6 9 p , 60 G l u c u r o n i c A c i d , G a l a c t o s e , G l u c o s e , Fucose 1 6 , 5 8 p G l u c u r o n i c A c i d , G a l a c t o s e , G l u c o s e , Rhamnose 1 8 , 1 9 , 2 3 , 4 1 , 7 9 , 1 2 p , 3 6 p , 4 5 p , 5 5 p , 70 p G l u c u r o n i c A c i d , G a l a c t o s e , Mannose, Rhamnose 5 3 , 4 0 , 8 0 P G l u c u r o n i c A c i d , G l u c o s e , Mannose, Fucose 6 p G l u c u r o n i c A c i d , G l u c o s e , Mannose, Rhamnose 6 4 p , 6 5 p G l u c u r o n i c A c i d , G a l a c t o s e , G l u c o s e , Mannose, Fucose 6 8 p G l u c u r o n i c A c i d , G a l a c t o s e , G l u c o s e , Mannose, Rhamnose 1 4 p , 67 G a l a c t u r o n i c A c i d , G a l a c t o s e , Mannose 3 P , 4 9 , 57 G a l a c t u r o n i c A c i d , G l u c o s e , Rhamnose 3 4 , 48 G a l a c t u r o n i c A c i d , G a l a c t o s e , F u c o s e , Rhamnose 63 P y r u v i c A c i d , G l u c o s e , Rhamnose 72 P y r u v i c A c i d , G a l a c t o s e , Rhamnose 32 P y r u v i c A c i d , G a l a c t o s e , G l u c o s e , Rhamnose 56 Keto A c i d , G a l a c t o s e , G l u c o s e 2 2 , 3 7 , 38 K82 has been added b u t i t s q u a l i t a t i v e c o m p o s i t i o n i s not y e t known. P_ P y r u v i c a c i d p r e s e n t i n a d d i t i o n N o t e : K9 and K 9 * , see Appendix I , p . 8 7 . - 8 9 -Appendix III N.m.r. Spectra K53 polysaccharide (PI) Solvent D20 Temp. 95°C S.W. 12,500 Hz 100 Spectrum No. 2 K53 polysaccharide (Pla) Solvent D20 S . W . 8000 Hz A.T. 0.5 sec P.W. 15 usee P.D. 0 sec N.T. 446,200 105.27 \ 103.44 -102.23 I | I | I | I | I | I | I | T Spectrum NO. 4 I i I i 1 I | K53 Compound Al_ GlcA ^ Man-OH p Solvent D20 Temp, ambient Water peak suppression S.W. 1000 Hz HOD 4.57 I i i i i I ' ' i i l i i i i I i i i i i i i i I i i i i i i i i i i i i i i i i Spectrum NO. 5 acetone (2.23) i I .i i i i l i i i i i i i i I I I I I I I I I 1 I I I I I I I I I I I I I I • 1 1 1 1 I I I I I I I I ' I I I I I I I ' I I Spectrum No. 6 K53 Compound Al_ GlcA Man-OH S.W. 5000 Hz A.T. 0.8 sec P.W. 21 usee P.D. 0/sec N.T. 108,500 61.40 102.49 92.91 K53 Compound A2 GlcA 1/ Man — Man-OH — g a Solvent D20 S.W. 1000 Hz Temp, ambient Water peak suppression HOD 5.16 5. 371 I 4.58 n 4.93 Spectrum No. 7 acetone (2.23) i i I I I I I I I L _ L _ I I I I I I I l I l l I I ' I I K53 Compound A2 GlcA i ^ - Man — "Man-OH B ct S.W. 5000 Hz A.T. 0.8 sec P.W. 21 usee N.T. 101,200 102.36 100.84 93.40 Spectrum No. 8 acetone (31.07) K53 Compound A3 Gl cA IT2- Man — Man — Solvent D20 S.W. 1000 Hz Temp, ambient Water peak suppression i i i i I i i i i J i i i i i i i >i i i i i i i i i i i i i i . ' I I I I i i i i Spectrum No. 9 acetone (2.23) 1.29 (unknown origin) CO K53 Compound A4 1 2 1 ? 1 1 1 2 GlcA -4^ Man — Man — Gal Rha-3 a a 3 Solvent D20 Temp. 90°C 5 104.63^ ^4.59 rti Spectrum No. 11 acetone (2.23) 1.28 (not recorded) K53 Compound A4 GlcA Man — Man — Gal ~ - Rha-OH S.W. 5000 Hz A.T. 0.8 sec P.W. 18 usee P.D. 0 sec N.T. 278,300 | I | I | I | I | I | I | I | i | i I i I i | i Spectrum No. 12 acetone (31.07) 61.79 K53 straight chain polysaccharide (P2) ' I I I 1 I I I 1 I 1 I r Spectrum No. acetone (31.07) Spectrum No. 14 K74 Compound B]_ GlcA ^  Man-OH S .W. 5000 Hz A.T. 0.8 sec P.W. 18 usee P.D. 0 sec N.T. 110,200 42 94.82 94.34 Spectrum No. 15 61.73 acetone (31.07) K74 Compound E2 GlcA — Man — Man-OH a a Solvent D20 S . W . 1000 Hz Temp. 90°C l— l—i—I— I— I— l— l l i i _ l I i ; L_J i l i i i i i i I—L Spectrum No. 16 acetone (2.23) o en i Spectrum No. 17 K74 Compound B3 GlcA — Man — Man — Gal-a a a S.W. 5000 Hz A . T . 0 .8 sec P.W. 21 visec P.D. 0 sec N.T. 87,300 101.33 103.071 1 97.21 95.33 95.08 93.09 61.92 Spectrum No. 19 o OO I acetone (31.07) I | I 1 I 1 I | I | I K74 polysaccharide Solvent D20 Temp. 90°C Spectrum No. 20 1 . 5 1 CH^  pyruvate K74 polysaccharide Solvent D20 N.T. 53,152 103.26 103.94 i. i I i i 100.96 100.5 Spectrum No. 21 62.00 2.19 V 61.85 acetone (31.07) 26.12 CH3 pyruvate 

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