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Capsular antigens of gram-negative bacteria Altman, Eleonora 1984

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CAPSULAR ANTIGENS OF GRAM-NEGATIVE BACTERIA by ELEONORA ALTMAN B . S c , The Hebrew U n i v e r s i t y of Jerusalem, I s r a e l , 1975 M.Sc., The Hebrew U n i v e r s i t y of Jerusalem, I s r a e l , 1977. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1984 © Eleonora Altman, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of CHiSTfl Y The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Sefit i i ABSTRACT K l e b s i e l l a , E s c h e r i c h i a c o l i , S a l m o n e l l a a n d S h i g e l l a a r e among t h e m o s t f r e q u e n t l y f o u n d p a t h o g e n i c E n t e r o b a c t e r i a . T h e c l a s s i f i c a t i o n o f S a l m o n e l l a a n d S h i g e l l a r e l a t e s m a i n l y t o t h e 0 a n t i g e n s w h i c h a r e l i p o p o l y s a c c h a r i d e s , w h e r e a s f o r K l e b s i e l l a a n d E . c o l i c a p s u l a r p o l y -s a c c h a r i d e s ( K a n t i g e n s ) p l a y a n i m p o r t a n t r o l e . A p p r o x i m a t e l y e i g h t y s e r o l o g i c a l l y d i f f e r e n t K l e b s i e l l a s t r a i n s a r e k n o w n o f w h i c h s e v e n t y s t r u c t u r e s h a v e b e e n d e t e r m i n e d . T h e s t r u c t u r e 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 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 K 5 0 i s p r e s e n t e d h e r e . I t i s u n i q u e among t h e K l e b s i e l l a K a n t i g e n s i n h a v i n g a 1 f i v e - p l u s - t w o ' r e p e a t i n g u n i t . - • 3 ) - p - D - G a l - ( l - > 3 ) - a - D - G l c - ( l - » 4 ) - a - D - G l c A - ( l - » 3 ) - a - D - M a n - ( l - > - 2 ) - a - D - M a n - ( 1 + 6 1 a - D - G l c 6 1 P - D - G a l K l e b s i e l l a K 50 The K a n t i g e n s o f E s c h e r i c h i a c o l i c a n be d i v i d e d i n t o t h r e e g r o u p s ( A , B , L ) o n t h e b a s i s o f t h e i r t h e r m o l a b i l i t y , a l l o f w h i c h c o m p r i s e a c i d i c p o l y s a c c h a r i d e s . The e x t r a c e l l u l a r A a n t i g e n s o f E .  c o l i b e a r a s t r o n g s i m i l a r i t y t o t h e K a n t i g e n s o f K l e b s i e l l a . i i i The present i n v e s t i g a t i o n describes the i s o l a t i o n and the s t r u c -t u r a l analyses of a c i d i c polysaccharides obtained from E s c h e r i c h i a c o l i 09:K28(A):H- (K28 antigen) and E s c h e r i c h i a c o l i 09:K32(A):H19 (K32 a n t i g e n ) . ->3)-ct-D-Glc-( l+4)-B-D-GlcA-( l*4)-a-L-Fuc-( 1* 4 2 and 3 • I 1 OAc B-D-Gal E s c h e r i c h i a c o l i K28 OAc I 2 -•3 ) - a-D-Glc- (1^4)-a-L-Rha- (l->3)-a-D-Gal-( 1 •* 3 • 1 B-D-GlcA E s c h e r i c h i a c o l i K32 S p e c i f i c bacteriophage-borne glucanases were u t i l i z e d to degrade the two E s c h e r i c h i a c o l i capsular polysaccharides. E. c o l i K32 p o l y -saccharide has been degraded using a p u r i f i e d <}>32 bacteriophage w i t h a-glucopyranosidase a c t i v i t y , while E. c o l i K28 polysaccharide has been degraded using crude s o l u t i o n s of the bacteriophages <t>28—1 and tj>28—2 (both with a-glucopyranosidase a c t i v i t y ) , and the r e s u l t s have been compared. i v TABLE OF CONTENTS Page ABSTRACT ' i i TABLE OF CONTENTS i v LIST OF APPENDICES i x LIST OF TABLES x LIST OF FIGURES x i i LIST OF SCHEMES x i v ACKNOWLEDGEMENTS xv PREFACE x v i 1 I . INTRODUCTION 1 I I . METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES . . . 22 11.1 I s o l a t i o n and p u r i f i c a t i o n 23 11.1.1 K l e b s i e l l a polysaccharides 25 11.1.2 E s c h e r i c h i a c o l i polysaccharides 26 11.2 Sugar a n a l y s i s 26 11.2.1 T o t a l h y d r o l y s i s and methanolysis . . . . 26 11.2.2 C h a r a c t e r i z a t i o n s and q u a n t i t a t i o n of sugars 28 11.2.3 Carboxyl reduction of a c i d i c polysaccharides 28 V II.2.4 Determination of the c o n f i g u r a t i o n of the sugars 30 I I . 3 P o s i t i o n of l i n k a g e 31 11.3.1 Met h y l a t i o n a n a l y s i s 31 11.3.2 G a s - l i q u i d chromatography ( g . l . c . ) . . . . 33 11.3.3 Mass-spectrometry (m.s.) 37 I I . 4 Sequencing of sugars 44 11.4.1 P a r t i a l h y d r o l y s i s 44 11.4.2 Periodate o x i d a t i o n and Smith degradation 46 11.4.3 Base-catalyzed degradation 53 I I . 5 Determination of l i n k a g e 55 11.5.1 O p t i c a l r o t a t i o n 55 11.5.2 Nuclear magnetic resonance spectroscopy 58 11.5.2.1 1H-n.m.r. spectroscopy 58 11.5.2.2 1 3C-n.m.r. spectroscopy . . . . 64 11.5.3 Other techniques 71 11.5.3.1 Enzymatic h y d r o l y s i s 71 11.5.3.2 Chromium t r i o x i d e o x i d a t i o n . . . 71 II.6 L o c ation of 0-acetyl group 73 v i I I I . GENERAL EXPERIMENTAL CONDITIONS 76 111.1 Paper chromatography 77 111.2 G a s - l i q u i d chromatography and g.I.e.-mass spectrometry 77 111.3 Gel-permeation chromatography 78 111.4 O p t i c a l r o t a t i o n and c i r c u l a r dichroism 79 111.5 Nuclear magnetic resonance 79 111.6 General c o n d i t i o n s 80 111.7 I s o l a t i o n and p u r i f i c a t i o n of the polysaccharides . . 80 111.7.1 K l e b s i e l l a polysaccharides 80 111.7.2 E s c h e r i c h i a c o l i polysaccharides . . . . 82 111.8 Bacteriophage propagation 83 111.8.1 Tube and f l a s k l y s i s 83 111.8.2 Large-scale propagation of the bacteriophage. 84 IV. STRUCTURAL INVESTIGATION OF K l e b s i e l l a SEROTYPE K50 CAPSULAR POLYSACCHARIDE 86 IV. 1 Abstract 87 IV. 2 I n t r o d u c t i o n 88 IV.3 Results and d i s c u s s i o n 88 IV. 4 Conclusion 100 IV. 5 Experimental 101 v i i V. STRUCTURAL INVESTIGATION OF E s c h e r i c h i a c o l i CAPSULAR POLYSACCHARIDES 109 V . l S t r u c t u r a l i n v e s t i g a t i o n of E s c h e r i c h i a c o l i 09:K28(A):H~ (K28 antigen) capsular polysaccharide 110 V . l . l A b s t r a c t 110 V.1.2 I n t r o d u c t i o n I l l V.1.3 Results and Dis c u s s i o n I l l V.1.4 Conclusion 123 V.1.5 Experimental 125 V.2 S t r u c t u r a l i n v e s t i g a t i o n of E s c h e r i c h i a c o l l 09:K32(A):H19 (K32 antigen) capsular polysaccharide 133 V.2.1 Abstract 133 V.2.2 I n t r o d u c t i o n 134 V.2.3 Results and d i s c u s s i o n 134 V.2.4 Conclusion 141 V.2.5 Experimental 143 v i i i VI. BACTERIOPHAGE DEGRADATION OF E s c h e r i c h i a c o l i CAPSULAR POLYSACCHARIDES SEROTYPES K28 and K32 151 VI. 1 I n t r o d u c t i o n 152 VI. 2 Results 161 VI. 3 D i s c u s s i o n 170 VI. 4 Experimental 174 VI I . BIBLIOGRAPHY 179 i x LIST OF APPENDICES Appendix Page I The known s t r u c t u r e s of the E s c h e r i c h i a c o l i 0 antigens 191 I I The known s t r u c t u r e s of the E s c h e r i c h i a c o l i K antigens 196 I I I LH and 1 3C-n.m.r. spectra 204 X LIST OF TABLES Table Page 1.1 K l e b s i e l l a capsular polysaccharides (K1-K83). Q u a l i t a t i v e a n a l y s i s and chemotype grouping . . . . 10 1.2 Schematic r e p r e s e n t a t i o n of the a g g l u t i n a t i o n r e s u l t s on which d e f i n i t i o n s of A, B, and L antigens are based 12 IV.1 N.m.r. data f o r K l e b s i e l l a K50 polysaccharide and derived o l i g o s a c c h a r i d e s 90 IV.2 Methylation a n a l y s i s of K l e b s i e l l a K50 polysaccharide and d e r i v a t i v e s 95 IV. 3 Analyses of a c i d i c o l i g o s a c c h a r i d e s from K l e b s i e l l a K50 polysaccharide . 98 V. l 1H-n.m.r. data f o r E s c h e r i c h i a c o l l K28 polysaccharide 113 V.2 l 3C-n.m.r. data f o r the n a t i v e and O-deacetylated E. c o l i K28 polysaccharide 116 V.3 Me t h y l a t i o n a n a l y s i s of E s c h e r i c h i a c o l i K28 polysaccharide and derived products 118 V.4 N.m.r. data f o r E s c h e r i c h i a c o l i K28 o l i g o -saccharides derived from p a r t i a l h y d r o l y s i s of the polysaccharide 120 V.5 A n a l y s i s of the ol i g o s a c c h a r i d e s from p a r t i a l h y d r o l y s i s of E s c h e r i c h i a c o l i K28 polysaccharide. . 121 x i V.6 N.m.r. data f o r E s c h e r i c h i a c o l i K32 n a t i v e and O-deacetylated polysaccharides 137 V. 7 M e t h y l a t i o n a n a l y s i s of E s c h e r i c h i a c o l i K32 polysaccharide and derived products 138 VI. 1 M e t h y l a t i o n a n a l y s i s and reducing end determina-t i o n of E. c o l i K28 o l i g o s a c c h a r i d e i s o l a t e d a f t e r bacteriophage <J>28—1 degradation of E. c o l i K28 polysaccharide 163 VI.2 Determination of the degree of poly m e r i z a t i o n and the reducing end of E. c o l i K28 o l i g o s a c c h a r i d e i s o l a t e d a f t e r bacteriophage <b28—1 degradation of E. c o l i K28 polysaccharide 164 VI.3 Proton assignments (400 MHz) f o r the o l i g o -saccharides and r e l a t e d compounds generated by bacteriophage depolymerization of the E. c o l i K32 capsular polysaccharide 169 VI.4 M e t h y l a t i o n a n a l y s i s of the reduced f r a c t i o n I I obtained a f t e r the separation of the depolymeriza-t i o n products of E. c o l i K32 polysaccharide . . . . 171 x i i LIST OF FIGURES Figure Page 1.1 Schematic r e p r e s e n t a t i o n of the Gram-positive and Gram-negative c e l l w a l l of b a c t e r i a 3 1.2 Electronmicrograph of a c r o s s - s e c t i o n of E. c o l i 09:K29 a f t e r contrast s t a i n i n g w i t h ruthenium red . . 5 1.3 Schematic diagram of the general s t r u c t u r e of b a c t e r i a l l i p o p o l y s a c c h a r i d e s (LPS) 13 1.4 Schematic r e p r e s e n t a t i o n of the s t r u c t u r e s of N-acetylneuraminic a c i d and 2-keto-3-deoxy-D-manno-2-octulosonic a c i d 15 1.5 a) E. c o l i K7 = K56 c r o s s - r e a c t s w i t h antiserum to S. pneumoniae type 3 and type 8. b) E. c o l i K30 c r o s s - r e a c t s w i t h antiserum to S. pneumoniae type 2 . 19 1.6 The s t r u c t u r e s of capsular polysaccharides of H. i n f l u e n z a e type b and E s c h e r i c h i a c o l i K100 . . . 20 11.1 Mass spectrum of a uronic a c i d degradation d e r i v a -t i v e from K l e b s i e l l a K50 polysaccharide (a) com-pared to the spectrum of a standard d e r i v a t i v e (b) . . 41 11.2 Common products formed on periodate o x i d a t i o n , followed by borohydride reduction and h y d r o l y s i s of t e r m i n a l and monosubstituted hexoses 48 11.3 Sequential periodate o x i d a t i o n and borohydride reduction of a l g i n a t e 51 11.4 R e l a t i o n s h i p between d i h e d r a l angle ( <)>) and coupling constants f o r a- and 6-D-hexoses 61 11.5 Schematic r e p r e s e n t a t i o n of d i f f e r e n t regions i n the ^H-n.m.r. spectrum of polysaccharides 63 11.6 The -^H-n.m.r. spectra of n a t i v e (top) and deacety-l a t e d (bottom) E. c o l i K28 capsular polysaccharides . 65 x i i i 11.7 The c h a r a c t e r i s t i c regions f o r resonances of carbon atoms belonging to d i f f e r e n t monosaccharide residues i n polysaccharides 68 11.8 The *3C-n.m.r. spectrum of deacetylated E. c o l i K28 capsular polysaccharide . . . 70 IV. 1 Gel-permeation chromatography of the product obtained a f t e r s e l e c t i v e , p a r t i a l h y d r o l y s i s of K l e b s i e l l a K50 polysaccharide 96 V. l P a r t i a l s t r u c t u r e of E. c o l l K28 polysaccharide . . . 117 VI.1 Schematic diagram demonstrating the s t r u c t u r e of T 2 bacteriophage 154 VI.2 Basic morphological types of bacteriophages w i t h the types of n u c l e i c a c i d 154 VI.3 The mechanics of i n f e c t i o n by bacteriophage . . . . 156 VI.4 Capsulated E. c o l l K29 exposed to a m.o.i. (the m u l t i p l i c i t y of i n f e c t i o n ) of 300 phage f o r 8 min at 37° 158 VI.5 Separation of the depolymerizatlon products of E. c o l i K32 by gel-permeation chromatography (Bio-Gel P-4) 167 VI.6 Molecular weight d i s t r i b u t i o n of f r a c t i o n I I . . . . 168 x i v LIST OF SCHEMES Scheme Page 11.1 Reduction of c a r b o x y l i c a c i d i n aqueous s o l u t i o n using carbodiimide reagent 29 11.2 M e t h y l a t i o n a n a l y s i s of K l e b s i e l l a K50 p o l y -saccharide 34 11.3 The mass spectra of the acetates (R = Ac ) , methyl ethers (R = Me), and t r i f l u o r o a c e t a t e s (R = COCF 3) of a l d i t o l s . Only primary fragments are shown . . . 39 11.4 The A- s e r i e s of fragments f o r the degradation of a di s a c c h a r i d e methyl g l y c o s i d e 42 11.5 Smith degradation of K l e b s i e l l a K50 capsular polysaccharide 52 11.6 Uronic a c i d degradation of K l e b l s i e l l a K50 polysaccharide 56 11.7 Location of O-acetyl s u b s t i t u e n t s according to the de Belder and Norrman procedure 74 XV ACKNOWLEDGEMENTS I would l i k e to express my sinc e r e g r a t i t u d e to Professor G.G.S. Dutton f o r h i s guidance, encouragement and i n t e r e s t throughout the course of t h i s work. I wish to thank my colleagues i n the l a b o r a t o r y f o r t h e i r support and h e l p f u l d i s c u s s i o n s and Dr. E.H. M e r r i f i e l d ( U n i v e r s i t y of Cape Town, South A f r i c a ) f o r h i s a s s i s t a n c e during the e a r l y stages of t h i s work. Thanks are a l s o due to Dr. S.C. Churms ( U n i v e r s i t y of Cape Town, South A f r i c a ) f o r gel-permeation measurements; Dr. S.O. Chan and the s t a f f of the n.m.r. s e r v i c e and Dr. G. Eigendorf and the s t a f f of the mass spectrometry s e r v i c e f o r t h e i r p a t i e n t a s s i s t a n c e . My s p e c i a l thanks to Dr. B. Lewis ( C o r n e l l U n i v e r s i t y ) f o r proof reading of t h i s t h e s i s . I should a l s o l i k e to thank Rani Theeparajah f o r typing t h i s t h e s i s . F i n a l l y , my g r a t e f u l thanks to my husband B o r i s f o r h i s encouragement and moral support. x v i DEDICATED TO THE MEMORY OF MY LATE FATHER ISRAEL KATSIN xv i i PREFACE The t o p i c of t h i s t h e s i s i s concerned w i t h the s t r u c t u r e e l u c i d a -t i o n of b a c t e r i a l polysaccharides. K l e b s i e l l a and E s c h e r i c h i a c o l i have many features i n common and our la b o r a t o r y has f o r s e v e r a l years stu d i e d the capsular antigens of K l e b s i e l l a . Now that the s t r u c t u r e s of almost a l l these 80 K s t r a i n s are known we are embarking on the examination of-the capsular polysaccharides of E s c h e r i c h i a c o l i . This t h e s i s there-f o r e , deals mainly w i t h s t r u c t u r e s of E s c h e r i c h i a c o l i K28 and K32 capsular polysaccharides. The s t r u c t u r e of K l e b s i e l l a K50 polysaccha-r i d e was studied f i r s t i n order to become acquainted w i t h the methodol-ogy of the carbohydrate research. In the I n t r o d u c t i o n I have attempted to give a concise account of E s c h e r i c h i a c o l i p olysaccharides, t h e i r b i o l o g i c a l importance, s e r o l o g i -c a l c l a s s i f i c a t i o n and s t r u c t u r a l d i v e r s i t y . The Methodology s e c t i o n deals w i t h the standard techniques of s t r u c t u r a l a n a l y s i s together w i t h more modern ones, such as the use of nuclear magnetic resonance spectroscopy. Examples from the s t r u c t u r a l i n v e s t i g a t i o n of K l e b s i e l l a and E s c h e r i c h i a c o l i capsular polysaccharides are chosen to best i l l u s t r a t e the methods used. Because of the i n c r e a s i n g importance of bacteriophage-associated glycanases f o r the s t r u c t u r a l a n a l y s i s of b a c t e r i a l surface carbo-hydrates, a chapter which deals w i t h the c l a s s i f i c a t i o n , s t r u c t u r e and a p p l i c a t i o n s of bacteriophages i s included. Appendix I contains the l i s t of known s t r u c t u r e s of E s c h e r i c h i a  c o l i 0 antigens. The l i s t of known s t r u c t u r e s of E s c h e r i c h i a c o l i K antigens along w i t h the l i t e r a t u r e references i s included i n Appendix I I . 1 CHAPTER I INTRODUCTION 2 I . INTRODUCTION Natural macromolecules c o n t a i n i n g carbohydrate u n i t s are of wide-spread occurrence i n a l l l i v i n g organisms and include (a) polysaccha-r i d e s as e x c l u s i v e l y carbohydrate polymers; (b) g l y c o p r o t e i n s , proteo-glycans, and peptidoglycans; (c) g l y c o l i p i d s and l i p o p o l y s a c c h a r i d e s ; (d) t e i c h o i c acids and r e l a t e d macromolecules co n t a i n i n g phosphodiester-l i n k e d o l i g o s a c c h a r i d e repeating u n i t s ; and (e) n u c l e i c a c i d s . 1 Commercially, i n t e r e s t i n polysaccharides has extended from s t a r c h and c e l l u l o s e i n food, pulp and paper i n d u s t r i e s to n a t u r a l gums and mucilages. The usefulness of most commercial polysaccharides i s based on t h e i r capacity to a l t e r the bas i c p r o p e r t i e s of water (e.g. t h i c k e n i n g and g e l l i n g ) . Polysaccharides a l s o play an important r o l e i n c o n t r o l l -i n g the texture of foods as w e l l as t h e i r f l a v o r , appearance and c o l o r . They perform as t h i c k e n i n g and s i z i n g agents i n i n d u s t r i a l a p p l i c a t i o n s (the t e x t i l e and paper i n d u s t r i e s ) and as d r i l l i n g f l u i d s i n o i l f i e l d a p p l i c a t i o n s (e.g. xanthan gum). 2 U n t i l r e c e n t l y , the b i o l o g i c a l f u n c t i o n s of polysaccharides were thought to be l i m i t e d to s e r v i n g as s t r u c t u r a l polymers and energy reserves. I t i s now w e l l e s t a b l i s h e d that complex carbohydrates play an important r o l e i n b i o l o g i c a l r e c o g n i t i o n as: receptors f o r phage and b a c t e r i o c i n s ; s p e c i f i c surface antigens; h i g h l y s p e c i f i c receptors i n eukaryotes f o r v i r u s e s , b a c t e r i a , hormones and t o x i n s ; and determinants of secreted g l y c o p r o t e i n s w i t h i n the c e l l s . C e r t a i n complex carbo-3 - Capsular Polysaccharide , Capsular Protein Capsule Interior Of Cell Peptidoglycan With Teichoic Acid Polymers Phospholipid bilayer with various membrane proteins, enzymes and permeases Cell Wall Cytoplasmic Membrane The envelope of the Gram-positive c e l l wall The envelope of the Gram-negative c e l l wall Fi g . 1.1: Schematic representation of the Gram-positive and the Gram-negative c e l l w all of bacteria. From T.J. Mackie and J.E. McCartney, "Medical Microbiology", Vol. 1: "Microbial Infections", 13th edn., C h u r c h i l l Livingstone, Edinburgh, 1978. 4 hydrates are chemical messengers and are e s p e c i a l l y important i n regu-l a t i n g growth, development, reproduction, and disease r e s i s t a n c e i n p l a n t s . 3 The m a j o r i t y of immunologically s i g n i f i c a n t polysaccharides are of m i c r o b i a l o r i g i n . A s i m p l i f i e d p i c t u r e of the b a c t e r i a l c e l l i s shown i n F i g . 1.1. Outside the plasma membrane i s the c e l l w a l l which can be of two general types: one that has an outer membrane over a peptidoglycan l a y e r (Gram-positive c e l l ) and one that l a c k s the outer membrane but has a d d i t i o n a l components w i t h i n the peptidoglycan l a y e r (Gram-negative c e l l ) . The main component i s l i p o p o l y s a c c h a r i d e , which c o n s t i t u t e s 10-15% of the dry c e l l w a l l and exerts both immunogenicity and f u l l e n d o t o x i c i t y . The s p e c i e s - s p e c i f i c somatic polysaccharide antigen i n the c e l l w a l l of Gram-negative b a c t e r i a i s c a l l e d the b a c t e r i a l 0 antigen. Many b a c t e r i a produce e x t r a c e l l u l a r polysaccharides (exopoly-s a c c h a r i d e s ) . They may e x i s t i n the form of a d i s c r e t e capsule surrounding the b a c t e r i a l c e l l or i n the form of a loose slime, unattached to the c e l l surface. 1* Capsules "mask" the c e l l w a l l 0 antigens and i n t e r f e r e w i t h t h e i r s e r o l o g i c a l d e t e c t i o n . 5 They can be recognized by the I n d i a ink s t a i n i n g technique or by e l e c t r o n microscopy (see F i g . 1.2). Capsules render b a c t e r i a r e s i s t a n t to phagocytosis and to the a c t i o n of the complement. This capsular m a t e r i a l i s al s o immunogenic and gives r i s e to s p e c i f i c a n t i - c a p s u l a r a n t i b o d i e s which react d i r e c t l y w i t h the encapsu-l a t e d b a c t e r i a . 5 They were f i r s t found i n e a r l y s e r o l o g i c a l studies of E. c o l i and the term K antigen (from the German word 5 F i g I . 2: Electronmlcrograph of a c r o s s - s e c t i o n of E. c o l i 09:K29 a f t e r c ontrast s t a i n i n g w i t h ruthenium red. (From r e f . 5 ) . 6 7 "Kapsel") was introduced i n 1945 by Kauffmann. Capsular polysaccharides play an important r o l e i n the immune response to b a c t e r i a l i n f e c t i o n due to t h e i r l o c a t i o n on the outer surface of the b a c t e r i a and c o n s t i t u t e the p r i n c i p a l antigens i n most of the pathogenic, Gram-negative and Gram-positive organisms. However, other antigens, such as p r o t e i n s and li p o p o l y s a c c h a r i d e s (0 a n t i g e n s ) , can also play a s i g n i f i c a n t r o l e i n the human immune-response to b a c t e r i a l i n f e c t i o n . 6 The m a j o r i t y of b a c t e r i a l polysaccharides are heteroglycans composed of o l i g o s a c c h a r i d e repeating u n i t s . This can be shown by mole-c u l a r weight d i s t r i b u t i o n s t u d i e s 7 and by nuclear magnetic resonance spectroscopy. 8 The e x t r a c e l l u l a r polysaccharides are g e n e r a l l y , but not always, a c i d i c . The a c i d i c component i s most often a uronic a c i d , a p y r u v i c a c i d a c e t a l , or a phosphoric d i e s t e r grouping. The capsule creates a simple, p h y s i c a l b a r r i e r p r o t e c t i n g the underlying surface of the b a c t e r i a and f o r any given pathogenic s t r a i n of b a c t e r i a , i t s v i r u l e n c e i s d i r e c t l y r e l a t e d to the amount of c a p s u l e . 6 The nomenclature of b a c t e r i a l polysaccharides i s complicated and i s based on the c l a s s i f i c a t i o n of the corresponding b a c t e r i a . Enterobacteriaceae i s a fa m i l y of Gram-negative, non-sporulating rods, e i t h e r m o t i l e or w i t h p e r i t r i c h o u s f l a g e l l a or non-motile. They grow on ordinary media and ferment glucose r a p i d l y w i t h or without gas production. The fa m i l y i s sub-divided i n t o t r i b e s , genera and s p e c i e s , which are the fundamental u n i t s of c l a s s i f i c a t i o n . 9 The system r e s u l t e d from comparative studies of the biochemical r e a c t i o n s given by 8 r e l a t i v e l y l a r ge numbers of c u l t u r e s of each of the genera. Kauffmann has proposed the f o l l o w i n g c l a s s i f i c a t i o n of E n t e r o b a c t e r i a c e a e : 9 F a m i l i a Enterobacteriaceae Tribes A. Eschericheae B. K l e b s i e l l e a e Genera i . E s c h e r i c h i a i i . S h i g e l l a i i i . Salmonella i v . C i t r o b a c t e r i . K l e b s i e l l a i i . Enterobacter i i i . Hafnia i v . S e r r a t i a C. Proteae i . Proteus i i . Morganella i i i . R e t t g e r e l l a i v . P r o v i d e n c i a Knowledge of the chemical s t r u c t u r e of b a c t e r i a l p o lysaccharides i s of great s i g n i f i c a n c e f o r understanding the molecular p r i n c i p l e s of 9 t h e i r b i o l o g i c a l a c t i v i t i e s . Most of the b a c t e r i a are pathogenic f o r man, form small groups of s e r o l o g i c a l l y typed species and are convenient f o r comparative immunochemical research. P u r i f i e d polysaccharides can be used as human vaccines (e.g. Streptococcus pneumoniae) and a con-s i d e r a b l e amount of research has been done i n t h i s f i e l d . 1 0 Klebsiella polysaccharides The genus K l e b s i e l l a i s composed of three species: K. pneumoniae, K. ozaenae and K. r h i n o s c h l e r o m a t i s . 1 1 K l e b s i e l l a c u l t u r e s were c l a s s i -f i e d by 0rskov on the basis of t h e i r K (capsular) and 0 (somatic) a n t i g e n s . 1 2 , 1 3 K. pneumoniae i s the most important member of the f a m i l y . I t i s found i n the r e s p i r a t o r y t r a c t of 5% of normal i n d i v i d u a l s and i s the primary cause of pneumonia i n 3% of a l l b a c t e r i a l pneumonias. 1 1* Nimmich has reported the q u a l i t a t i v e composition of the a p p r o x i -mately 80 d i f f e r e n t K l e b s i e l l a K s e r o t y p e s 1 5 ' 1 6 and has c l a s s i f i e d them i n t o chemotypes 1 7 (see Table 1.1). The s t r u c t u r e s of seventy K l e b s i e l l a capsular polysaccharides are known to date. Various s t r u c t u r a l patterns have emerged. These may be di v i d e d i n t o four types: 1) those l a c k i n g uronic a c i d ; 2) those where the uronic a c i d i s a component of the main chain; 3) those where the uronic a c i d i s i n a side chain; and 4) those with side chains of three u n i t s . A d e t a i l e d c o m p i l a t i o n of d i f f e r e n t K l e b s i e l l a s t r u c t u r e s was done by Di F a b i o 1 8 and i s not, t h e r e f o r e , repeated i n t h i s t h e s i s . I n 10 TABLE 1.1; Klebsiella capsular polysaccharides (K1-K83). Qualitative analysis and chemotype grouping GlcA Gal Glc GlcA Gal Man GlcA Gal Rha GlcA Glc Man GlcA Glc Rha GlcA Glc Fuc GlcA Gal Glc Man GlcA Gal Glc Fuc GlcA Gal Glc Rha GlcA Gal Man Rha GlcA Glc Man Fuc GlcA Glc Man Rha GlcA Gal Glc Man GlcA Gal Glc Man GalA Gal Man GalA Glc Rha GalA Gal Fuc Fuc Rha P P P 8 , 11 , 15, 25, 27 , 51 20, 2 1 P , 29 P, 42 P, 43, 66, 74 P 9, 9*, 47, 52, 81, 83 2, 4, 5 P, 24 17, 23, 44, 45, 71 1, 54 P P P P P P 7 , 10, 13 , 26 , 28, 30 , 31 , 33 , 35 P, 39, 46 P, 50, 59, 60, 61, 62, 69 ? 16, 58 P 12 P, 18, 19, 36 P, 41, 55 P, 70 P, 79 40, 53, 8 0 P 6 P P P 64 , 65 68 P 14 P, 67 3 , 49, 57 34, 48 63 PyrA Glc Rha 72 PyrA Gal Rha 32 PyrA Gal Glc Rha 56 KetoA Gal Glc 22 , 37, 38 GlcA g l u c u r o n i c a c i d GalA g a l a c t u r o n i c a c i d PyrA p y r u v i c a c i d KetoA rare uronic a c i d Glc glucose Gal galactose Man mannose Rha rhamnose Fuc fucose P pyruvic a c i d present i n a d d i t i o n 11 Appendix I and I I the l i s t of known s t r u c t u r e s of E. c o l i 0 and K antigens i s given. E s c h e r i c h i a c o l i polysaccharides The organism E s c h e r i c h i a c o l i was f i r s t i s o l a t e d from faeces by Esc h e r i c h i n 1885. I t belongs to the family Enterobacteriaceae whose normal h a b i t a t i s the i n t e s t i n a l t r a c t of man and a n i m a l s . 1 9 E. c o l i i s ofte n found i n human u r i n a r y t r a c t i n f e c t i o n s and i s associated w i t h severe i n f a n t i l e d i a r r h e a . 2 0 Within the spe c i e s , many d i f f e r e n t serotypes are recognized. The serotyping scheme i s based on the i d e n t i f i c a t i o n of surface 'K', somatic '0' and f l a g e l l a *H' antigens. The 0 antigens are thermostable somatic antigens, r e s i s t i n g heating at 100°, and are not destroyed by a l c o h o l . The term K antigen covers a group of e i t h e r envelope or capsular antigens which can be div i d e d i n t o three groups (A, B, L) (see Table 1.2). S t r a i n s which conta i n the thermolabile L or B antigens do not u s u a l l y possess morphological capsules, whereas s t r a i n s w i t h thermostable A antigen are ca p s u l a t e d . 9 The main t e s t s to demonstrate the presence of a K antigen are 0 i n a g g l u t i n a b i l i t y of the l i v i n g b a c t e r i a and t h e i r a g g l u t i n a t i o n w i t h K sera. The f i r s t a n t i g e n i c scheme, comprising 25 0 antigens, was es t a b l i s h e d by Kauffmann i n 1947. 2 1 Since then, many 0 antigens have been added and approximately 100 'K', 164 '0* and 56 'H' are c u r r e n t l y r e c o g n i z e d . 9 12 TABLE 1.2; Schematic presentation of the agglutination results on which definitions of A, B, and L antigens are based K Antigen 0 OK Serum type pre p a r a t i o n Serum Absorbed by c u l t u r e heated at 100° f o r 2 h Unab-sorbed L L i v e (or f o r m a l i n t r e a t e d _a + B o i l e d (100° f o r 1 h) + _c + B L i v e - - + B o i l e d + - + A L i v e - - + B o i l e d - - + a Negative or s i g n i f i c a n t l y lower than that of b o i l e d c u l t u r e b +, A g g l u t i n a t i o n c No a g g l u t i n a t i o n 13 0 antigens 0 Antigens c o n s i s t of three regions: l i p i d A, an o l i g o s a c c h a r i d e core, and the O - s p e c i f i c polysaccharide chain (see F i g . 1.3). i r O - s p e c i f i c polysaccharide Core o l i g o s a c c h a r i d e L i p i d A F i g . 1.3: Schematic diagram of the general s t r u c t u r e of b a c t e r i a l l i p o p o l y s a c c h a r i d e s (LPS). L i p i d A (region 1) i s buried i n the outer membrane of the b a c t e r i a l c e l l and i s responsible f o r the general endotoxic p r o p e r t i e s of l i p o p o l y s a c c h a r i d e . 2 2 I t c o n s i s t s of glucosamine, phosphate and f a t t y a c i d s . The s t r u c t u r e of l i p i d A has been described r e c e n t l y . 2 3 The core (region 2) i s l i n k e d to l i p i d A v i a a carbohydrate component that i s t y p i c a l f o r the LPS of Gram-negative b a c t e r i a , 2-keto-3-deoxy-D-manno-2-octulosonic a c i d (KDO). Five d i f f e r e n t core s t r u c t u r e s have been described so f a r . I t has been known f o r a long time that wild-type Enterobacteriaceae growing on agar may undergo a spontaneous S (smooth) — > R (rough) mutation which i s associated w i t h 14 the disappearance of the 0 antigen and l o s s of the p a t h o g e n i c i t y . A n a l y s i s of the R mutants revealed that they lack the O - s p e c i f i c chain and c o n s i s t only of the core bound to l i p i d A. 2 3 Region 3 i s the s p e c i f i c 0 polysaccharide of the LPS of b a c t e r i a l S forms. I t i s b u i l t up from repeating u n i t s of o l i g o s a c c h a r i d e s which may contain up to 6-7 sugars. For a long time a l l E. c o l l 0 antigens were thought to contain only n e u t r a l polysaccharide chains. More r e c e n t l y , LPS were i s o l a t e d that contain a c i d i c components such as g l y c e r o l phosphate, hexuronic a c i d s , and neuraminic a c i d . 2 2 The s t r u c t u r e s of the 0 antigens that have been published u n t i l now are given i n Appendix I . K antigens The K antigens are capsular or envelope antigens and can be detected by Immunoelectrophoresis. They are a l l polysaccharides except f o r two that are p r o t e i n s (K88 and K 9 9 ) . 2 2 . Since these capsular polysaccharides are not immunogenic f o r humans and animals, they w i l l have to be chemically modified to become immunogens. For such s t u d i e s knowledge of the polysaccharide s t r u c t u r e s i s extremely v a l u a b l e . 2 4 The d i f f i c u l t y i n s t r u c t u r a l i n v e s t i g a t i o n of E. c o l i capsular polysaccharides a r i s e s from the f a c t , that u n l i k e f o r K l e b s i e l l a p o l ysaccharides, the q u a l i t a t i v e composition of the most of the E. c o l l serotypes i s not known. This places the researcher i n a d i f f i c u l t p o s i t i o n , c o n s i d e r i n g the f a c t that E. c o l i polysaccharides are extremely diverse and contain uncommon and sometimes rare sugars. The 15 most prominent feature of these capsular polysaccharides i s the frequent occurrence of 2-keto-3-deoxy-D-manno-2-octulosonic a c i d (KDO) and al s o the occurrence of N-acetylneuraminic a c i d (NeuNAc or NANA) (see F i g . 1.4). N-Acetylmannosaminuronic a c i d (ManNAcA) was found i n the K7 a n t i g e n . 2 5 H N-acetylneuraminic a c i d 2-keto-3-deoxy-D-manno-2-(NeuNAc or NANA) o c t u l o s o n i c a c i d (KDO) F i g . 1.4: Schematic r e p r e s e n t a t i o n of the s t r u c t u r e s of N-acetylneuraminic a c i d and 2-keto-3-deoxy-D-manno-2-octulosonic a c i d . A number of K antigens occur e x c l u s i v e l y i n 0 groups 08, 09 and 0101. 2 2 The K antigens of these groups are of two types, one w i t h amino sugars and one without. Those devoid of amino sugars have high molecular weights (3 x 10 5 - 10 6 d a l t o n s ) . They are p h y s i c a l l y heterogeneous and become homogeneous a f t e r m i l d a l k a l i t r e a t m e n t . 2 6 These K antigens ( a l l A antigens and some B antigens) have a very low e l e c t r o p h o r e t i c m o b i l i t y , form t h i c k and copious capsules and bear a strong resemblance to the K antigens of K l e b s i e l l a . 2 7 I t i s noteworthy 16 that the 08 and 09 antigens are themselves r e l a t e d to K l e b s i e l l a 0 antigens. Thus the 09 antigen of E. c o l i i s i d e n t i c a l w i t h the K l e b s i e l l a 03 antigen, and the E. c o l i 08 antigen i s i d e n t i c a l w i t h the K l e b s i e l l a ' 05 a n t i g e n . 5 The K antigens of a group that contains amino sugars were thought to be e x t r a c e l l u l a r polysaccharides but they were found to be l i p o p o l y s a c c h a r i d e s . 5 E. c o l i w i t h these K antigens c o n t a i n two c e l l w a l l l i p o p o l y s a c c h a r i d e s : an a c i d i c one which i s termed a K antigen i n a d d i t i o n to a n e u t r a l one (the 08, 09 or 0101 a n t i g e n ) . The a c i d i c l i p o p o l y s a c c h a r i d e s are not capsular (K) antigens i n the true sense; they were c a l l e d t hermolabile B antigens i n the nomenclature of Kauffmann. 9 K antigens o c c u r r i n g i n 0 groups other than 08, 09 and 0101 are a l l a c i d i c polysaccharides w i t h rather low molecular weights (below 50,000 daltons) and high e l e c t r o p h o r e t i c m o b i l i t y . 5 ' 2 2 Kauffmann has c a l l e d them thermolabile L antigens, and they were found i n most E s c h e r i c h i a c o l i s t r a i n s i s o l a t e d from p a t h o l o g i c a l m a t e r i a l . 9 They contai n rare sugar c o n s t i t u e n t s , such as N-acetylneuraminic a c i d or N-acetylmannosaminuronic a c i d . The K l antigen, a l s o known as colominic a c i d , i s a poly-N-acetyl neuraminic a c i d . 2 8 K2 i s a t e i c h o i c a c i d - l i k e p o l y m e r . 2 4 Although p a r t i a l s t r u c t u r e s have been determined f o r s e v e r a l E.  c o l i p o l ysaccharides, r e l a t i v e l y few complete s t r u c t u r e s have been published. The s t r u c t u r e s of E. c o l i K42 and K l e b s i e l l a K63 are i d e n t i -c a l and c r o s s - r e a c t s e r o l o g i c a l l y . 2 9 The E. c o l l K 3 0 3 0 and K l e b s i e l l a K20 antigens are i d e n t i c a l , and E. c o l i K100 antigen i s s t r u c t u r a l l y 17 r e l a t e d to the Haemophilis i n f l u e n z a e type b capsular a n t i g e n . d l The s t r u c t u r e s of the E. c o l i K antigens that have been published u n t i l now are given i n Appendix I I . Immunology of polysaccharides The polysaccharides are true immunoantigens i n that they induce an immune response and the generation of s p e c i f i c a n t i b o d i e s . I t was shown that only a r e l a t i v e l y small p o r t i o n of a polysaccharide i s the major s i t e of antibody s p e c i f i c i t y and that part i s known as the d e t e r -minant group. 1* A determinant group may comprise s e v e r a l monosaccharide r e s i d u e s , one of which c o n t r i b u t e s most to the s p e c i f i c i t y ; that monosaccharide residue i s termed the immunodominant sugar. The c l a s s i c a l s tudies of K a b a t 3 2 on an isomaltose o l i g o s a c c h a r i d e s e r i e s have shown that the non-reducing t e r m i n a l residue c o n t r i b u t e d about 40% and the next two residues together about 60% to the t o t a l binding energy. These r e s u l t s i n d i c a t e that i n a l i n e a r polysaccharide the immunological s p e c i f i c i t y r e s i d e s p r i m a r i l y i n the t e r m i n a l sugar residue and extends along the polysaccharide chain. The s i t u a t i o n i s d i f f e r e n t f o r branched polysaccharides i n which the immunodominant sugars are those which are located on the side c h a i n s . 3 3 C e r t a i n noncarbohydrate groups may f u n c t i o n as a n t i g e n i c determinants. Jann and W e s t p h a l 2 7 have shown that 0-acetyl groups are e s s e n t i a l parts of the determinant regions i n some Salmonella polysaccharides. 18 Pyruvic a c i d , attached to a sugar as an a c e t a l was found to be the immunologic determinant i n the capsular polysaccharides of K l e b s i e l l a and Streptococcus pneumoniae. 3 4 Cross-reactions have been used e x t e n s i v e l y i n immunochemical a n a l y s i s and they can be used to e s t a b l i s h the s t r u c t u r e s of immuno-determinant groups. Heidelberger has used t h i s approach e x t e n s i v e l y and was able to p r e d i c t the presence of some s t r u c t u r a l features before they were v e r i f i e d c h e m i c a l l y . 3 5 - 3 8 Studies of c r o s s - r e a c t i o n s of more than 60 capsular, t y p e - s p e c i f i c polysaccharides of K l e b s i e l l a w i t h 26 s p e c i f i c types of antipneumococcal sera permitted the assignment of se v e r a l s t r u c t u r a l features such as non-reducing t e r m i n a l residues of D-glucuronic a c i d , L-rhamnose, and D-galacturonic a c i d and some linkages w i t h i n the polysaccharide chain. The molecular basis of some cr o s s - r e a c t i o n s i n which E. c o l i 0 and K antigens p a r t i c i p a t e has been e l u c i d a t e d 2 2 (see F i g . 1.5). Capsular polysaccharides are important v i r u l e n c e f a c t o r s i n many b a c t e r i a l i n f e c t i o n s i n c l u d i n g those caused by Streptococcus pneumoniae, N e i s s e r i a m e n i n g i t i d i s , Hemophilus i n f l u e n z a e , E s c h e r i c h i a c o l i , Salmonella typhosa, K l e b s i e l l a pneumoniae, and Staphylococcus aureus.^ The f i r s t capsular polysaccharide vaccine arose from the e a r l y work on Streptococcus pneumoniae. 3 9 I t i s important to r e a l i z e , however, that the immunity received on recovery from i n f e c t i o n by encapsulated b a c t e r i a d i f f e r s from that generated by immunization w i t h p u r i f i e d capsular polysaccharide vaccines. In young c h i l d r e n polysaccharide vaccines give r i s e to only IgM antibodies and there i s no memory response, whereas p r o t e i n vaccines 19 a) — ManNAcA Glc — P 6 8 OAc E. c o l i K7 = K56 — GlcA — Glc — 8 8 S. pneumoniae type 3 - GlcA — Glc — Glc — Gal — B 8 a c S. pneumoniae type 8 b) — Man — Gal — 3 j o B l l a Gal GlcA E. c o l i K30 Rha 1 3 a Rha 2I l l a G l c 1 a GlcA 1 3 Rha 1 4 Glc S. pneumoniae type 2 F i g . 1.5: a) E. c o l i K7 « K56 CROSS-REACTS WITH ANTISERUM TO S. pneumoniae TYPE 3 AND TYPE 8. b) E. c o l i K30 CROSS-REACTS WITH ANTISERUM TO S. pneumoniae TYPE 2 20 induce both IgM and IgG antibodies and an immunological memory.H There have been s e v e r a l attempts to overcome t h i s problem by conjugating p o l y -the development of the vaccine against pathogenic E. c o l i and the K l and K5 antigens i n p a r t i c u l a r . Both antigens belong to the most v i r u l e n t organisms encountered. 5 M e n i n g i t i s of small c h i l d r e n i s o f t e n caused by N. m e n i n g i t i d i s group B and a l s o by E. c o l i K l . The capsular antigens of both of these pathogens are i d e n t i c a l , both s t r u c t u r a l l y and s e r o l o g i c a l l y . A proposal has been made to use a c r o s s - r e a c t i n g E. c o l i K l polysaccharide as an a l t e r n a t i v e vaccine. 1* 1 E. c o l i 100 capsular polysaccharide c r o s s - r e a c t s w i t h H. i n f l u e n z a e type b polysaccharide (see F i g . 1.6). H. i n f l u e n z a type b may cause s e v e r a l diseases, of which m e n i n g i t i s i s the most frequent and more serious h e a l t h problem. Conjugates were prepared w i t h E. c o l i K100 capsular polysaccharide because of i t s s t r u c t u r a l relatedness to H. i n f l u e n z a e type b. F i r s t p o s i t i v e r e s u l t s have been obtained i n animal experiments. 1* 1 saccharides to a p r o t e i n c a r r i e r . 0 This approach has been used a l s o i n 3 — Rib 1 1 5 3 1 2 5 f P r i b i t o l Rib f P r i b i t o l Haemophilus i n f l u e n z a e type b E. c o l i K100 F i g . 1.6; The s t r u c t u r e s of capsular polysaccharides of H. i n f l u e n z a e type b and E s c h e r i c h i a c o l i K100. 21 A new p o t e n t i a l i n polysaccharide immunochemistry i s provided by homogeneous immunoglobulins that bind carbohydrate polymers. Here, the s p e c i f i c immunoglobulin-hapten i n t e r a c t i o n can be studied i n d e t a i l . 1 * 2 The o l i g o s a c c h a r i d e s obtained by bacteriophage degradation of b a c t e r i a l surface carbohydrates may be coupled to the p r o t e i n c a r r i e r s s e r v i n g as immunogens, r e p r e s e n t a t i v e of the corresponding b a c t e r i a l glycans. 1* 3 Bacteriophage-induced degradation of capsular polysaccha-r i d e s i s discussed i n Section VI of t h i s t h e s i s . Polysaccharides on the surface of a m i c r o b i a l c e l l are the s e r o -l o g i c a l determinants of that organism and therefore represent a h i g h l y s p e c i f i c means of i d e n t i f i c a t i o n . Their study by chemical and genetic approaches, as w e l l as the i n v e s t i g a t i o n of t h e i r b i o s y n t h e t i c pathways, are e s s e n t i a l f o r our understanding of b a c t e r i a l p a t h o g e n i c i t y . In the course of t h i s t h e s i s , the s t r u c t u r e s of K l e b s i e l l a K50 polysaccharide and two E s c h e r i c h i a c o l i p olysaccharides, serotype K28 and K32, were i n v e s t i g a t e d . Two E s c h e r i c h i a c o l i capsular polysaccha-r i d e s (K28 and K32) were degraded by t h e i r r e s p e c t i v e bacteriophages (<(>28-l, <))28-2 and <j>32) and the degradation products were c h a r a c t e r i z e d . 22 CHAPTER I I METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES 23 II. METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES An almost i n f i n i t e v a r i e t y of s t r u c t u r a l types i s p o s s i b l e among polysaccharides. Each sugar residue can e x i s t i n the pyranose or furanose form, each g l y c o s i d i c l inkage may have the oc- or B-configura-t i o n and the g l y c o s i d i c l inkage may inv o l v e s u b s t i t u t i o n of d i f f e r e n t hydroxyl groups i n an adjacent sugar residue. Chemical methods of determination of the s t r u c t u r e of polysaccha-r i d e s i n v o l v e : 1) q u a l i t a t i v e and q u a n t i t a t i v e e s t i m a t i o n of the sugar c o n s t i t u e n t s ; 2) a n a l y s i s f o r removable s u b s t l t u e n t s ( O - a c e t y l , N_-acetyl, phosphate, e t c . ) ; 3) determination of the linkage c o n f i g u r a -t i o n ; 4) determination of the p o s i t i o n of li n k a g e ; 5) determination of the sugar sequence. I t i s obvious that no one s t r u c t u r a l method can give answers to a l l these questions. In the f o l l o w i n g s e c t i o n an attempt i s made to describe each of the known s t r u c t u r a l methods together w i t h some of i t s l i m i t a t i o n s . II. 1 ISOLATION AND PURIFICATION1* One of the most important and d i f f i c u l t steps i n the i n v e s t i g a -t i o n of polysaccharides i s t h e i r p u r i f i c a t i o n and f r a c t i o n a t i o n i n t o homogeneous i n d i v i d u a l polysaccharides. 24 The simplest e x t r a c t i o n methods f o r polysaccharides are those using water alone at various temperatures. Some polysaccharides can be brought i n t o s o l u t i o n by the use of p o l a r nonaqueous s o l v e n t s , such as dimethyl s u l f o x i d e f o r s t a r c h and glycogen 1* 1* or N-methylmorpholine N-oxide f o r c e l l u l o s e . 1 + 5 D i l u t e a l k a l i has been used f o r polysaccharide e x t r a c t i o n s , but one has to be aware of p o s s i b l e base-catalyzed degrada-t i o n . E x t r a c t i o n of polysaccharides under a c i d i c c o n d i t i o n s i s normally avoided due to the p o s s i b l e cleavage of g l y c o s i d i c bonds. In order to obtain the carbohydrate p o r t i o n of g l y c o p r o t e i n s , extensive d i g e s t i o n w i t h protease of low s p e c i f i c i t y can be performed. 1 + 6 Mucopolysaccha-r i d e s , l i p o p o l y s a c c h a r i d e s and n u c l e i c acids can be p r e c i p i t a t e d from aqueous s o l u t i o n by adding l i q u i d phenol. 1* 7 The next step i n v o l v e s r e s o l u t i o n of a polysaccharide mixture i n t o i t s components. Methods f o r f r a c t i o n a t i o n of polysaccharides f a l l i n t o three broad c a t e g o r i e s : 1) those based on s e l e c t i v e p r e c i p i t a t i o n of polysaccharide themselves (by a d d i t i o n of a water-miscible solvent such as acetone or ethanol 1* 8) or of t h e i r s a l t s ( p r e c i p i t a t i o n of a c i d i c polysaccharides w i t h potassium c h l o r i d e , 1 * 9 c u p r i c acetate or s u l f a t e , 5 0 or w i t h c a t i o n i c detergents such as cetyltrimethylammonium bromide ( C e t a v l o n ) ) 5 * ; 2) those based on formation of complexes (use of F e h l i n g s o l u t i o n f o r mannans, 5 1 borate f o r galactomannans, 5 2 barium hydroxide f o r gluco- and galactomannans 5 3); 3) those based on chromatographic procedures ( g e l f i l t r a t i o n 5 1 * or molecular sie v e chromatography, 5 5 i o n - exchange chromatography, 5 6 a f f i n i t y chromatography 5 7 ) . 25 The p r a c t i c a l problem i s to e s t a b l i s h the p u r i t y of a polysaccha-r i d e or rather to demonstrate the absence of heterogeneity by as many independent c r i t e r i a as p o s s i b l e . This includes demonstration of constancy i n chemical composition (based on sugar r a t i o s ; spectroscopic examination by nuclear magnetic resonance spectroscopy) and p h y s i c a l p r o p e r t i e s ( o p t i c a l r o t a t i o n ; molecular weight determination by g e l permeation chromatography 5 8; e l e c t r o p h o r e s i s 5 9 ' 6 0 ) . II.1.1 Klebsiella polysaccharides 1 5' 6 1' 6 2 A c u l t u r e of K l e b s i e l l a K50, obtained from Dr; Ida 0rskov, was grown as p r e v i o u s l y d e s c r i b e d . 6 1 ' 6 2 B r i e f l y , b a c t e r i a l c u l t u r e was streaked on agar p l a t e s and grown at 37° u n t i l l a r g e , i n d i v i d u a l c o l o n i e s were obtained. B a c t e r i a were grown by i n o c u l a t i o n of beef-e x t r a c t medium and incu b a t i o n at 37° u n t i l a d e f i n i t e growth was observed ( u s u a l l y 3-5 h ) . The l i q u i d c u l t u r e thus obtained was incuba-ted on a tr a y of sucrose-yeast e x t r a c t agar f o r 3 days. The lawn of capsular b a c t e r i a produced was harvested and the b a c t e r i a were destroyed w i t h 1% phenol s o l u t i o n . The dead c e l l s were spun down by u l t r a - c e n t r i -f u g a t i o n and the polysaccharide was p r e c i p i t a t e d from the supernatant ' w i t h ethanol. The p r e c i p i t a t e was d i s s o l v e d i n water and tr e a t e d w i t h Cetavlon (cetyltrimethylammonium bromide) to p r e c i p i t a t e the a c i d i c polysaccharide. Further p u r i f i c a t i o n i n v o l v e d d i s s o l u t i o n i n 4M sodium c h l o r i d e , r e p r e c i p i t a t i o n i n t o ethanol, d i s s o l u t i o n i n water and d i a l y s i s . The d i a l y z e d s o l u t i o n was f r e e z e - d r i e d to y i e l d p u r i f i e d capsular polysaccharide. 26 II.1.2 Escherichia c o l i polysaccharides Cultures of E s c h e r i c h i a c o l i K28 and K32 from Dr. I . I&rskov (Copenhagen) were grown on Mueller-Hinton agar w i t h the a d d i t i o n of sodium c h l o r i d e (5% w/v). Each tray was layered with an a c t i v e l y growing l i q u i d c u l t u r e of E. c o l i b a c t e r i a , obtained by i n o c u l a t i o n of Mueller-Hinton broth w i t h a s i n g l e f r e s h colony of E. c o l i and f u r t h e r i n c u b a t i o n of the r e s u l t i n g s o l u t i o n f o r 5 h at 37°. The trays were l e f t i n the incubator f o r 5 d. The p u r i f i c a t i o n procedure was c a r r i e d out as p r e v i o u s l y described f o r K l e b s i e l l a p olysaccharides. II.2 SUGAR ANALYSIS II.2.1 Total hydrolysis and methanolysis 6 1* 6 3 - 6 7 Determination of the chemical composition of a polysaccharide i n v o l v e s an i n i t i a l a c i d h y d r o l y s i s i n t o c o n s t i t u e n t monosaccharides. A l l sugars are, to some extent, degraded by a c i d . Thus, the c o n d i t i o n s of h y d r o l y s i s must be c a r e f u l l y chosen and c o n t r o l l e d . D u t t o n 6 3 review-ed the advantages and disadvantages i n the use of d i f f e r e n t a c i d s . Hyd r o c h l o r i c a c i d i s commonly employed f o r g l y c o p r o t e i n s . 6 1 * T r i f l u o r o a c e t i c a c i d was f i r s t used by Albersheim and c o - w o r k e r s 6 5 f o r h y d r o l y s i s of plant c e l l - w a l l polysaccharides and i t has since been widely used f o r h y d r o l y s i s of other polysaccharides. I t i s v o l a t i l e and thus r e a d i l y removed. Aldose-containing polysaccharides can be completely hydrolyzed with minimum l o s s of sugar with 2M t r i f l u o r o a c e t i c 27 a c i d at 100°C f o r 6-8 h. However, other sugars, such as 2-deoxyaldoses, ketoses i n c l u d i n g s i a l i c a c i d s , and anhydro sugars are l a r g e l y destroyed under these c o n d i t i o n s . These sugars can be completely released under extremely mi l d c o n d i t i o n s , f o r example, s i a l i c a c i d w i t h 0.025-0.05M s u l f u r i c a c i d at 80° f o r 1 h . 6 6 S i a l i c acids may be s t a b i l i z e d a l s o by methanolysis, g i v i n g methyl g l y c o s i d e methyl e s t e r s . 6 7 Incomplete h y d r o l y s i s i s encountered w i t h uronic acids and 2-amlno-2-deoxyglycosidic l i n k a g e s . In the case of uronic a c i d s , that might be of an advantage, si n c e such r e s i s t a n c e to h y d r o l y s i s permits i s o l a t i o n of uronic a c i d - c o n t a i n i n g o l i g o s a c c h a r i d e s as p a r t i a l h y d r o l y s i s products. The reduction of a hexouronic a c i d to a hexose residue p r i o r to h y d r o l y s i s w i t h a water-soluble carbodiimide and sodium borohydride overcomes t h i s d i f f i c u l t y (see l a t e r ) . A technique, developed i n t h i s l a b o r a t o r y , i n v o l v e s use of methanolysis f o r uronic a c i d - c o n t a i n i n g p o l y s a c c h a r i d e s . 6 1 According to t h i s method, the polysaccharide i s f i r s t t r e a t e d w i t h methanolic hydrogen c h l o r i d e which, together w i t h the cleavage of g l y c o s i d i c bonds, causes an e s t e r i f i c a t i o n of u r o n i c a c i d residues. Treatment w i t h sodium borohydride i n anhydrous methanol reduces the uronic e s t e r s to the corresponding a l c o h o l s . The mixture of methyl glyc o s i d e s i s then hydrolyzed with 2M t r i f l u o r o a c e t i c a c i d (TFA) to give the n e u t r a l sugars which are converted i n t o a l d i t o l acetates and analyzed by g a s - l i q u i d chromatography ( g . l . c ) . 28 1 1 . 2 . 2 Characterization and quantitation of sugars 6 8" 7** The c h a r a c t e r i z a t i o n of the sugars formed on h y d r o l y s i s i s the s t a r t i n g point i n an i n v e s t i g a t i o n of the s t r u c t u r e of a polysaccharide. The conventional techniques i n v o l v e paper chromatography, 6 8' 6 9 t h i n l a y e r chromatography 7 0 and paper e l e c t r o p h o r e s i s . 7 1 Analyses of c e r t a i n broad cl a s s e s of sugars can be performed spectrophotometri-c a l l y . 7 2 ' 7 3 G a s - l i q u i d chromatographic methods i n v o l v e formation of s u i t a b l e v o l a t i l e d e r i v a t i v e s (see l a t e r ) . I n c r e a s i n g l y , high performance l i q u i d chromatography (HPLC) columns are being developed f o r the separation and q u a n t i t a t i v e a n a l y s i s of sugars. 7 l* 1 1 . 2 . 3 Carboxyl reduction of acidic polysaccharides 7 S"" 7 8 The r e s i s t a n c e of g l y c o s i d u r o n i c acids to complete h y r o l y s i s creates d i f f i c u l t i e s i n compositional and s t r u c t u r a l a n a l y s i s of polysaccharides. The best approach i s to reduce uronic acids to the corresponding hexose residues and then to carry out the i n v e s t i g a t i o n on the carboxyl-reduced p o l y s a c c h a r i d e . Taylor and C o n r a d 7 5 have developed a method i n which the a c i d i c polysaccharide i n aqueous s o l u t i o n i s treated w i t h a water-soluble carbodiimide to give O-acylisourea, which i s then reduced w i t h sodium borohydride. Both stages i n t h i s r e a c t i o n require c a r e f u l pH c o n t r o l (see Scheme I I . 1 ) . The a l t e r n a t i v e method in v o l v e s reduction of a permethylated polysaccharide, u s u a l l y w i t h l i t h i u m aluminum hydride i n t e t r a h y d r o f u r a n 29 RCOOH + RCOCTH IR" + H IHR" NR' E.D.C or C.M.C. RCH20H NoBH4 pH475 RCOl RCH + NHR' N0BH4 pH 5-7 NHR" + H + NHR1 E . D . C . = l - e t h y l - 3 - ( 3 - d i m e t h y l a m i n o p r o p y l ) c a r b o d i i m i d e C . M . C . = l - c y c l o h e x y l - 3 - ( 2 - m o r p h o l i n o e t h y l ) c a r b o d i i m i d e m e t h o - _ p _ - t o l u e n e s u l p h o n a t e Scheme I I . 1 : R e d u c t i o n o f c a r b o x y l i c a c i d i n a q u e o u s s o l u t i o n u s i n g c a r b o d i i m i d e r e a g e n t 30 or s i m i l a r s o l v e n t . ° This reagent, however, i s not s u i t a b l e f o r the r e d u c t i o n of a c i d groups i n unsubstituted polysaccharides because of t h e i r i n s o l u b i l i t y i n ether-type s o l v e n t s . In t h i s case methyl e s t e r s may be formed by treatment of the a c i d i c polysaccharide w i t h diazomethane and then reduction of the e s t e r groups using sodium borohydride i n aqueous s o l u t i o n . 7 7 II.2.4 Determination of the configuration (D or L) of s u g a r s 7 8 - 8 1 In general, chromatographic separation methods and s p e c t r o s c o p i c analyses do not d i s t i n g u i s h between enantiomers. Enantiomeric d i f f e r e n -t i a t i o n of sugars can be achieved on m i l l i g r a m q u a n t i t i e s by c i r c u l a r dichroism of a l d l t o l acetates or the p a r t i a l l y methylated a l d i t o l a c e t a t e s . 7 8 Recently, f o r even smaller amounts of m a t e r i a l and f o r mixtures of sugars, enantiomers have been d i s t i n g u i s h e d by conversion to e q u i -l i b r i u m mixtures of g l y c o s i d e s of c h i r a l a l c o h o l s (e.g., (+) or (-)-2-butanol or (+)- or (-)-2-octanol), followed by g . l . c . separation of v o l a t i l e d e r i v a t i v e s such as acetate or t r i m e t h y l s i l y l e t h e r s . 7 9 ' 8 0 . I n t h i s procedure, enantiomeric sugars form diastereomeric mixtures of d e r i v a t i v e s whose chromatographic separations provide a c h a r a c t e r i s t i c f i n g e r p r i n t . For a few sugars enzymatic assay can be used f o r enantiomeric c h a r a c t e r i z a t i o n . For example, the enzyme D-glucose oxidase i s used f o r the q u a n t i t a t i v e assay of D-glucose i n a m i x t u r e . 8 1 31 I I . 3 POSITION OF LINKAGE II.3.1 Methylation analysis'* 5>* 2~ 9 t* The technique of methylation a n a l y s i s 8 2 ' 8 3 i s r o u t i n e l y employed i n the s t r u c t u r a l c h a r a c t e r i z a t i o n of complex carbohyrates as a means to e s t a b l i s h the linkage p o s i t i o n s of the c o n s t i t u e n t monosaccharides. This method i s based on the a b i l i t y to separate and c h a r a c t e r i z e the p a r t i a l l y methylated monosaccharides generated v i a h y d r o l y s i s of the f u l l y methylated polysaccharide, which i s accomplished by combined gas-l i q u i d chromatography/mass spectrometry of t h e i r a l d i t o l acetate d e r i v a -t i v e s . The method, however, gives no information on the sequence or the anomeric nature of the l i n k a g e s i n the polysaccharide. The aim of methylation 8** i s to achieve e t h e r i f i c a t i o n of a l l free hydroxyl groups i n the polysaccharide. In the o r i g i n a l procedure used by Haworth 8 5 t h i s was achieved by repeated r e a c t i o n with dimethyl s u l f a t e and sodium hydroxide. The p a r t i a l l y methylated product obtained was then treated w i t h s i l v e r oxide i n b o i l i n g methyl i o d i d e , according to Purdie and I r v i n e , 8 6 to give f u l l y methylated polysaccharide. Purdie's method was considerably improved by Kuhn and co-workers 8 7 who used N^,N-dimethylformamide as a solvent i n conjunction w i t h methyl i o d i d e and s i l v e r oxide. The simplest and most convenient method f o r methylation of polysaccharides was developed by Hakomori. 8 8 This method was f i r s t a p p l i e d to capsular polysaccharides by Sandford and Conrad. 8 9 The polysaccharide i s t r e a t e d w i t h the strong base sodium m e t h y l s u l f i n y l methanide (dimsyl sodium) and methyl i o d i d e i s subsequently added to 32 e f f e c t methylation. The Hakomori procedure u s u a l l y gives complete methylation i n one step. I f t h i s i s not the case complete methylation can be achieved using Purdie's method, since a second Hakomori treatment would r e s u l t i n B - e l i m i n a t i o n i f the polysaccharide contains uronic a c i d . O-Acyl groups present i n many polysaccharides and glycoconjugates are completely cleaved under the strong a l k a l i n e c o n d i t i o n s , but pyruvic a c i d a c e t a l s are s t a b l e . S u b s t i t u t i o n of the carbohydrate residues by O-acyl groups can be determined using the Prehm 9 0 methylation procedure, where the polysaccharide i s d i s s o l v e d i n t r i m e t h y l phosphate and then methylated w i t h methyl tr i f l u o r o m e t h a n e s u l f o n a t e and 2 , 6 - d i - ( t e r t -b u t y l ) p y r i d i n e as proton scavenger. I f the substrate contains u r o n i c a c i d s these are transformed i n t o methyl e s t e r s . Most "undermethy-l a t i o n s " are due to incomplete d i s s o l u t i o n of a sample. This s i t u a t i o n may be improved by c a r e f u l d e - i o n i z a t i o n of a polysaccharide ( f o r example, using Amberlite IR-120 (H +) r e s i n ) . D e t a i l e d methylation procedures have been p u b l i s h e d . 8 2 ' 8 9 ' 9 * Recently, potassium methyl-s u l f i n y l methanide has been s u c c e s s f u l l y used i n Hakomori methyla-t i o n s . 9 2 Some polysaccharides, such as c e l l u l o s e , are i n s o l u b l e i n dimethyl s u l f o x i d e (DMSO) alone. Recently, N-methylmorpholine N-oxide (MMNO) has been shown to d i s s o l v e polysaccharides, so that the Hakomori methylation can be c a r r i e d out i n MMNO-DMSO mixtures. 1* 5 The methylated m a t e r i a l i s recovered by d i a l y s i s (polysaccharide) or by p a r t i t i o n between water and chloroform ( o l i g o s a c c h a r i d e ) . The completeness of methylation i s checked by i . r . spectroscopy (absence of hyro x y l absorption at 3600 cm - 1) or by a n a l y s i s of the methoxyl content. The subsequent h y d r o l y s i s i s performed w i t h 2M t r i f l u o r o a c e t i c a c i d on a 33 steam bath f o r 16 h. Polysaccharides c o n t a i n i n g uronic acids may be carboxyl-reduced before (carbodiimide reduction) or a f t e r ( l i t h i u m aluminum hydride) the permethylation step. Scheme I I . 2 i l l u s t r a t e s a t y p i c a l r e a c t i o n sequence. A recent report by Reinhold and co-workers 9 3 and Gray and co-workers 9 1* describes a new technique f o r determining the s t r u c t u r e of polysaccharides, t h a t , p o t e n t i a l l y , has s i g n i f i c a n t advantages over standard methylation a n a l y s i s . This method involv e s the i o n i c hydro-genation of a l l g l y c o s i d i c carbon-oxygen bonds i n the f u l l y methylated polysaccharide w i t h t r i e t h y l s i l a n e c a t a l y z e d by boron t r i f l u o r o e t h e r a t e . This reductive cleavage a f f o r d s a s e r i e s of p a r t i a l l y methylated a n h y d r o a l d i t o l s , which are subsequently a c e t y l a t e d In s i t u , and analyzed by g.l.c.-m.s. II.3.2 G a s - l i q u i d chromatography ( C U C . ) 6 3 ' 9 5 - 1 0 6 Gas chromatography i s a separation process i n which the components to be separated are v o l a t i l i z e d and d i s t r i b u t e d between a moving gas phase and a s t a t i o n a r y absorbent phase, which may be a s o l i d ( g a s - s o l i d chromatography), or a l i q u i d ( g a s - l i q u i d chromatography) adsorbed on an i n e r t support. The carbohydrate d e r i v a t i v e s used i n g a s - l i q u i d chromatography must be v o l a t i l e , yet s t a b l e at the operating temperature of the column and must not be adsorbed i r r e v e r s i b l y on the s t a t i o n a r y phase. G a s - l i q u i d chromatography of carbohydrates was f i r s t performed f o r methylated methyl g l y c o s i d e s . 9 5 The discovery of t r i m e t h y l s i l y l 34 KLEBSIELLA K50 POLYSACCHARIDE Dbose(CH3SCH2"No+) 2)CH3I Ch^OMe MeO }—0, MeO MeO OMe OMe ,OMe r~ (} C H2 2 — ( ,  f H2 0 H MeO ) 0 I J 0 ) — 0 OMe CH„OMe S~~OCH2 OMe MeO> OMe OMe 1. 2,4,6-OMe3-Galactose 2. 2,4-OMe2~Glucose 3. 2,3-OMe2-Glucose 4. 2,4,6-OMe3-Mannose OMe H 5. 3,4,6-OMe3-Mannose 6. 2,3,4-OMe3-Glucose 7. 2,3,4,6-0Me4-Galactose 0NOBH4 2)Ac20/Pyr g.l.c.-m.s. r—OAc |— OAc — OAc OAc OAc AcO-— OMe AcO— — OMe MeO-— OMe MeO— AcC— AcO-MeO-MeO-— OAc — OMe — OAc — OAc — OAc OMe OAc OMe OAc — OMe — OAc — OMe OMe OMe MeO — OAc |— OAc OMe — OMe MeO— OMe MeO— OAc OAc OAc OMe Scheme I I . 2 : M e t h y l a t i o n A n a l y s i s of K l e b s i e l l a K50 polys a c c h a r i d e 35 d e r i v a t i v e s by Sweeley and co-workers 6 i n 1963 has r e v o l u t i o n i z e d the a n a l y s i s of carbohydrates by g a s - l i q u i d chromatography. Extensive reviews of the a p p l i c a t i o n s of g . l . c . to carbohydrate a n a l y s i s have been published by D u t t o n . 6 3 ' 9 7 Although t r i m e t h y l s i l y l d e r i v a t i v e s are r e a d i l y formed and are v o l a t i l e , t h e i r obvious disadvantage i s a m u l t i p l i c i t y of peaks due to the isomeric forms present at e q u i l i b r i u m . Separations obtained by Jones and co- w o r k e r s 9 8 w i t h complex mixtures of a l d i t o l acetates are superior to those obtained f o r the corresponding mixtures of the t r i -m e t h y l s i l y l d e r i v a t i v e s . The carbohydrates present i n g l y c o p r o t e i n s were q u a n t i t a t i v e l y determined by g . l . c . of t h e i r a l d i t o l a c e t a t e s 9 9 and t h i s procedure i s widely used i n the a n a l y s i s of gly c o p r o t e i n s and ol i g o s a c c h a r i d e s . When the mixture i s not too complex, a n a l y s i s of the t r i m e t h y l s i l y l ethers i s p r e f e r r e d , since the r e a c t i o n r e q u i r e s only 5-15 m i n . 1 0 0 Jeanes and c o - w o r k e r s 1 0 1 i n v e s t i g a t e d s e v e r a l column packings, and found that an o r g a n o s i l i c o n e p o l y e s t e r (ECNSS-M) gives good separation of acetates of common a l d i t o l s . However, the maximum operating-temperature of t h i s column i s rather low (200°) and SP-2340 (75% cyanopropyl s i l i c o n e ) i s the s t a t i n a r y phase of choice f o r a n a l y s i s of a l d i t o l acetates and i t was used i n t h i s study. To determine the degree of po l y m e r i z a t i o n of an o l i g o s a c c h a r i d e and to i d e n t i f y the reducing sugar, p e r a c e t y l a t e d a l d o n o n i t r i l e s are u s e d . 1 0 2 M e t h y l a t i o n a n a l y s i s i s an important method i n s t r u c t u r a l p o l y -saccharide chemistry. G.l.c. o f f e r s the best method f o r the sep a r a t i o n and q u a n t i t a t i o n of the methylated sugars obtained on h y d r o l y s i s of a methylated polysaccharide or glycoconjugate. P a r t i a l l y methylated 36 a l d i t o l acetates have been used e x t e n s i v e l y i n the methylation a n a l y s i s . One advantage of using a l d i t o l acetates i s that each aldose d e r i v a t i v e w i l l give only one peak on g . l . c . Another advantage of p a r t i a l l y methylated a l d i t o l acetates i s that the q u a n t i t a t i o n can be performed without the use of response f a c t o r s 1 0 3 ( w i t h i n ±5%) except f o r the a n a l y s i s of N-acetamido sugars. The best separations of p a r t i a l l y methylated a l d i t o l acetates are obtained on medium-polar columns such as ECNSS-M or OV-225 (a s i l i c o n polymer c o n t a i n i n g methyl, phenyl and n i t r i l e groups). OV-225 i s u s u a l l y p r e f e r r e d f o r r o u t i n e work because of i t s thermal s t a b i l i t y . A manual on methylation a n a l y s i s , g i v i n g r e l a t i v e r e t e n t i o n times on g a s - l i q u i d chromatography of p a r t i a l l y methylated a l d i t o l acetates as w e l l as a c o l l e c t i o n of computer-drawn bar graph mass spectra of these d e r i v a t i v e s has been p u b l i s h e d . 9 1 Albersheim and co-workers have reported r e l a t i v e r e t e n t i o n times f o r numerous p a r t i a l l y e t h y l a t e d a l d i t o l acetates. They can be used to resol v e some of the polysaccharide components that are co-eluted as t h e i r p a r t i a l l y methylated a l d i t o l acetates. 1 0 1 + The i d e n t i f i c a t i o n of methylated sugars obtained on methylation a n a l y s i s by g . l . c . may be confirmed by g . l . c . on other columns and the s u b s t i t u t i o n p a t t e r n can be determined unambiguously by g.l.c.-mass spectrometry (see l a t e r ) . The a p p l i c a t i o n of HPLC (high performance l i q u i d chromatography) to carbohydrate a n a l y s i s was concentrated o r i g i n a l l y on the q u a n t i t a t i v e a n a l y s i s of sugars i n food and b e v e r a g e s . 1 0 5 Recently, Albersheim and co-workers have a p p l i e d HPLC to the separation of mixtures of small amounts of p e r a l k y l a t e d o l i g o s a c c h a r i d e a l d i t o l s . 1 0 6 Thus sequencing 37 complex carbohydrates by formation, separation and c h a r a c t e r i z a t i o n of p e r a l k y l a t e d o l i g o s a c c h a r i d e a l d i t o l s has developed i n t o a powerful method of s t r u c t u r a l a n a l y s i s . II.3.3 Mass s p e c t r o m e t r y 8 2 ' 1 0 7 - 1 1 7 Mass spectrometry (m.s.) has become an important and v e r s a t i l e t o o l i n the s t r u c t u r a l a n a l y s i s of carbohydrates. Mass spectrometry i s based on the i o n i z a t i o n of compounds when they are bombarded w i t h a beam of el e c t r o n s to form p o s i t i v e molecular ions which subsequently may break down to smaller fragments. The r e l a -t i v e peak i n t e n s i t y i s p r o p o r t i o n a l to the number of ions of the appro-p r i a t e m/z (mass-to-charge r a t i o ) value. The number of Ions depends on t h e i r s t a b i l i t y . 1 0 7 Carbohydrate d e r i v a t i v e s give weak or no molecular ions on e l e c t r o n impact ( e . i . ) mass spectrometry. Molecular weights may more r e a d i l y be determined by f i e l d i o n i z a t i o n ( f . i . ) , f i e l d desorption (f.d.) or chemical i o n i z a t i o n ( c . i . ) t e c h n i q u e s . 1 0 8 ' 1 0 9 Since u n d e r i v a t i z e d mono- and o l i g o s a c c h a r i d e s are thermally unstable and p r a c t i c a l y n o n - v o l a t i l e , they are converted Into more v o l a t i l e d e r i v a t i v e s such as methyl or t r i m e t h y l s i l y l e t h e r s , a c e t a t e s , t r i f l u o r o a c e t a t e s or a l k y l i d e n e d e r i v a t i v e s . The use of combined g.l.c.-mass spectrometry, i n which the components from the chromatographic column are Introduced d i r e c t l y Into the i o n i z a t i o n chamber of the mass spectrometer, has l e d to new methods 38 f o r the q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s of the mixtures of methy-l a t e d s u g a r s . 8 2 A l d i t o l d e r i v a t i v e s e x h i b i t the simplest patterns f o r fragmenta-t i o n of carbohydrate molecular ions. The mass spectra of the a c e t a t e s , methyl ethers, and t r i f l u o r o a c e t a t e s of a l d i t o l s d i s p l a y primary fragments corresponding to a l l p r i n c i p a l s c i s s i o n s of the compound (see Scheme I I . 3 ) . 1 0 7 A systematic i n v e s t i g a t i o n of mass spectra of p a r t i a l l y methyla-ted a l d i t o l acetates by Lindberg and co-workers has l e d to the f o l l o w i n g g e n e r a l i z a t i o n s . 8 2 1. D e r i v a t i v e s w i t h the same s u b s t i t u t i o n p a t t e r n give very s i m i l a r mass spec t r a , t y p i c a l of that s u b s t i t u t i o n p a t t e r n . 2. The base peak of the spectrum i s g e n e r a l l y m/z 43 (CH 3C — 0 ) . 3. F i s s i o n between a methoxylated and an acetoxylated carbon i s p r e f e r r e d over f i s s i o n between two acetoxylated carbons. 4. When the molecule contains two adjacent methoxylated carbons, f i s s i o n between them i s p r e f e r r e d over f i s s i o n between one of these and a neighboring acetoxylated carbon. 5. Secondary fragments are formed from the primary fragments by s i n g l e or consecutive l o s s of a c e t i c a c i d (m/z 60), ketene (m/z 42), methanol (m/z 32), or formaldehyde (m/z 30). This i n f o r m a t i o n was used to i d e n t i f y l a b e l l e d compounds f o r which standard spectra were not a v a i l a b l e . During the s t r u c t u r a l i n v e s t i g a t i o n on K l e b s i e l l a K50 capsular polysaccharide the p o s i t i o n of 39 ChfeOR HC=OR C 2 R = A c , m/ z 145 R=Me, m/z 8 9 R = C O C F 3 , m/z 2 5 3 RO=CH HCOR CH2OR R = A c , m/ z 3 6 2 R=Me, m/z 2 2 2 R = C O C F 3 , m/z 6 3 2 H 2C =0R Cl R = A c , m/z 73 R=Me, m/ z 45 R = C O C F 3 , m/z 127 HC=OR ROCH HCOR CH2OR C 3 R = A c , m / z 217 R=Me, m/ z 133 R = C O C F 3 , m/z 3 7 9 C. R = A c , m/ z 2 8 9 4 R=Me, m/ z 177 R = C O C F 3 , m/z 5 05 S cheme I I . 3 : The m a s s s p e c t r a o f t h e a c e t a t e s ( R = A c ) , m e t h y l e t h e r s ( R = M e ) , a n d t r i f l u o r o a c e t a t e s ( R = C 0 C F 3 ) o f a l d i t o l s . O n l y p r i m a r y f r a g m e n t s a r e s h o w n . 40 linkage between D-glucuronic a c i d and D-mannose was determined by u r o n i c a c i d degradation followed by l a b e l l i n g w i t h e t h y l i o d i d e . The e t h y l a -ted, p a r t i a l l y methylated a l d i t o l acetate ( l , 5 - d i - 0 - a c e t y l - 3 - 0 - e t h y l -2,4,6-tri-O—methylmannitol) was obtained and analyzed by means of g.l.c.-m.s. Several masses are s h i f t e d by 14 u n i t s as i l l u s t r a t e d i n Figure I I . 1 . On reduction some p a i r s of methylated sugars (e.g. a 3-0-methyl-and a 4-0-methyl hexose) give a l d i t o l s w i t h the same s u b s t i t u t i o n p a t t e r n . The l o s s of informa t i o n when sugars are reduced to a l d i t o l s can be r e a d i l y prevented i f the reduction i s performed w i t h sodium deuterioborate. This l a b e l l i n g technique, when used during reduction of the uronic a c i d residues to n e u t r a l sugars, permits one to d i s t i n g u i s h the n e u t r a l sugar residues obtained from other sugar residues present i n the same polysaccharide. G.l.c.-m.s. of p a r t i a l l y methylated a l d i t o l acetates i s now widely used i n conjunction w i t h the methylation a n a l y s i s of polysaccha-r i d e s and other m a t e r i a l s c o n t a i n i n g carbohydrates. For m a t e r i a l s having only one sugar of any one c l a s s (such as pentose, hexose, or 6-deoxyhexose), the i d e n t i f i c a t i o n of the components by m.s. i s unambiguous. 1 0 8 I f , however, the polysaccharide contains d i f f e r e n t sugars of the same c l a s s an i d e n t i f i c a t i o n of the d i f f e r e n t methylated sugars can be accomplished only i f the r e l a t i v e r e t e n t i o n times (T- values) of the relevant a l d i t o l acetates are s u f f i c i e n t l y d i f f e r e n t . A l d o n o n i t r i l e acetates are s u i t a b l e f o r a n a l y s i s of sugars by g . l . c . and give c h a r a c t e r i s t i c mass spectra that are easy to i n t e r p r e t . 41 a) 1,5-di-0-acetyl-3-0-ethyl-2,4,6-tri-0-methyl-D-mannitol Re l . I n t . % | ' " I ' " " T I 1 "—"J1 | i i r 50 "*T—r * I ' I — i — i — i — 1 — i — i — r — 100 ' 150 200 250 r-10 300 b) 1,5-di-0-acetyl-2,3,4,6-tetra-O-methyl-D-mannitol Re l . i n t . % 10 300 5 0 1 6 0 - • - i4o • ' • '200 ' ' ' '250 F i g . I I . 1 : Mass spectrum of a uronic a c i d degradation d e r i v a t i v e from K50 polysaccharide (a) compared to the spectrum of a standard d e r i v a t i v e (b). 42 P a r t i a l l y methylated a l d o n o n i t r i l e acetates have been i n v e s t i g a t e d by Kochetkov and c o - w o r k e r s . 1 1 0 The mass-spectrometric behavior of per-methylated d i s a c c h a r i d e s having (1 •*• 2 ) - , (1 -> 4) - , and (1 •* 6 ) - l i n k e d hexose residues has been i n v e s t i g a t e d by Kochetkov and c o - w o r k e r s . 1 1 1 I t was found that the fragmentation of both moieties of the d i s a c c h a r i d e f o l l o w s p r i n c i p l e s s i m i l a r to those f o r permethylated g l y c o s i d e s . The nomenclature f o r the d i f f e r e n t fragmentation s e r i e s of permethylated glyc o s i d e s was introduced by Chizhov and K o c h e t k o v 1 1 1 and modified l a t e r by Kovacik and c o - w o r k e r s . 1 1 2 ' 1 1 3 The nomenclature i s ex e m p l i f i e d f o r the degradation of a di s a c c h a r i d e methyl g l y c o s i d e (see Scheme I I . 4 ) . aA| baA, Scheme I I . 4 : The A- s e r i e s of fragments f o r the degradation of a dis a c c h a r i d e methyl g l y c o s i d e The A - s e r i e s of fragments serves to e s t a b l i s h the molecular weights of the di s a c c h a r i d e and i t s component sugar residues. The B-s e r i e s of fragments obtained by degradation of r i n g b can be used to e s t a b l i s h the nature of the linkage between two sugar r e s i d u e s . 1 0 8 4 3 The separation and s t r u c t u r a l a n a l y s i s of 21 permethylated t r i -saccharides by g a s - l i q u i d chromatography-mass spectrometry (g.l.c.-m.s.) was described by Karkkainen. 1 l l* The p o s i t i o n of the g l y c o s i d i c l i n k a g e next to the reducing end of s t r a i g h t - c h a i n t r i s a c c h a r i d e s can g e n e r a l l y be e s t a b l i s h e d by m.s., whereas d i f f e r e n t i a t i o n of (1 •> 6 ) - and (1 •*• 4 ) -linkages next to the non-reducing end may r e q u i r e previous knowledge of the stereochemistry of the sugar u n i t s . l l l + In recent years, m.s. procedures have found a wider a p p l i c a t i o n i n the s t r u c t u r e determination of o l i g o s a c c h a r i d e s and g l y c o l i p i d s hav-i n g high molecular weights. This has been made p o s s i b l e l a r g e l y as a r e s u l t of instrumental developments a l l o w i n g high s e n s i t i v i t y at high mass, and improvements i n sample handling. E l e c t r o n i o n i z a t i o n mass-spectrometry ( e . i . m.s.) of these high-mass substances s u f f e r s , however, from some l i m i t a t i o n s , namely, the l o s s i n s i g n a l i n t e n s i t y at high mass, which r e s u l t s i n the absence of the molecular i o n and high-mass sequence ions. The method can be improved by combining e . i . m.s. w i t h the r e s u l t s of f i e l d - d e s o r p t i o n ( f . d . ) s t u d i e s . However, the t e c h n i c a l d i f f i c u l t i e s a s s o c iated w i t h f.d. m.s. have l i m i t e d i t s use. The development of fast-atom-bombardment mass spectrometry (f.a.b.m.s.) has been of considerable importance f o r carbohydrate-structure e l u c i d a -t i o n . 1 1 5 F.a.b. spectra are r e l a t i v e l y easy to acquire, and e a r l y reports have shown that f.a.b.m.s. i s capable of o b t a i n i n g both molecu-l a r weights and fragment data from small q u a n t i t i e s of p o l a r , b i o l o g i c a l substances, i n c l u d i n g unmodified carbohydrates and g l y c o l i p i d s . 1 1 6 ' 1 1 7 44 I I . 4 SEQUENCING OF SUGARS II.4.1 P a r t i a l h y d r o l y s i s 1 1 8 " 1 2 5 P a r t i a l h y d r o l y s i s followed by c h a r a c t e r i z a t i o n of the product(s) i s often used i n s t r u c t u r a l carbohydrate chemistry. The method i s of p a r t i c u l a r value when a polymer contains a l i m i t e d number of a c i d - l a b i l e g l y c o s i d i c l i n k a g e s , which may be cleaved without s i g n i f i c a n t h y d r o l y s i s of other g l y c o s i d i c l i n k a g e s . I t i s a d v i s a b l e , t h e r e f o r e , to perform some p i l o t experiments i n order to determine optimal c o n d i t i o n s f o r the p a r t i a l a c i d h y d r o l y s i s . P a r t i a l h y d r o l y s i s may a l s o be combined w i t h methylation a n a l y s i s of the r e s u l t i n g o l i g o s a c c h a r i d e s . Many f a c t o r s seem to i n f l u e n c e the rate of h y d r o l y s i s , i n c l u d i n g the r i n g s i z e , c o n f i g u r a t i o n , conformation, and p o l a r i t y of the sugar as w e l l as the s i z e and p o l a r i t y of the a g l y c o n . 1 1 8 Hence, i t i s oft e n impossible to point to a s i n g l e f a c t o r that e x p l a i n s the d i f f e r e n c e s i n h y d r o l y s i s rates between two g l y c o s i d e s . 1 1 8 Capon has reviewed the rate constants f o r the a c i d - c a t a l y z e d h y d r o l y s i s of a large number of g l y c o s i d e s . 1 1 9 In general, furanosides are hydrolyzed more r e a d i l y than pyranosides, deoxyglycopyranosides are more a c i d l a b i l e than glycopyranosides, and aminosugars and uronic acids are r e l a t i v e l y r e s i s t a n t to h y d r o l y s i s . Graded h y d r o l y s i s of ur o n i c a c i d - c o n t a i n i n g polysaccharides leads to i s o l a t i o n of a c i d i c disaccharides ( a l d o b l o u r o n i c a c i d s ) and higher o l i g o s a c c h a r i d e s . The e f f e c t of the type of linkage on the rates of h y d r o l y s i s was studied f o r the D-glucose d i s a c c h a r i d e s . 1 1 9 In a l l cases, except f o r 45 the (1 6 ) - l i n k e d d i s a c c h a r i d e s , the a-D-linkage was more r e a d i l y hydrolyzed than the 6-D-linkage. For d i f f e r e n t types of lin k a g e s i n polysaccharides, the rates of h y d r o l y s i s seem to p a r a l l e l the rates of h y d r o l y s i s of d i s a c c h a r i d e s : (1 •*• 3 ) - l i n k a g e s are hydrolyzed f a s t e r than (1 -> 4 ) - and (1 -> 2 ) - l i n k a g e s w i t h (1 •> 6)-linkages being most r e s i s t a n t . The r a t e of h y d r o l y s i s depends on the l o c a t i o n of the linkage w i t h i n the polysaccharide chain. There i s a higher r a t e of h y d r o l y s i s of non-reducing t e r m i n a l and s i d e - c h a i n bonds, as compared wi t h the main i n - c h a i n b o n d s . 1 1 8 A number of a l t e r n a t i v e procedures may be used f o r the a c i d -catalyzed cleavage of g l y c o s i d i c l i n k a g e s , i n c l u d i n g a c e t o l y s i s , 1 2 0 methanolysis, and mercaptolysis. The l a t t e r two techniques are used mainly i n the s t r u c t u r a l a n a l y s i s of the s u l f a t e d polysaccharides from seaweeds. 1 2 1 The usefulness of a c e t o l y s i s i s that i t i s complementary to a c i d h y d r o l y s i s . In the former, (1 •*• 6)-linkages are the most s u s c e p t i b l e to a t t a c k ; i n the l a t t e r , they are the l e a s t s e a s i l y r u p t u r e d . 1 2 0 The mixture of mono, d i , and higher o l i g o s a c c h a r i d e s formed on p a r t i a l h y d r o l y s i s may be f r a c t i o n a t e d by a v a r i e t y of chromatographic procedures, i n c l u d i n g paper chromatography, gel-permeation chromatography, paper e l e c t r o p h o r e s i s , ion-exchange chromatography of a c i d i c o l i g o s a c c h a r i d e s , g a s - l i q u i d chromatography, and high performance l i q u i d chromatography. L i q u i d hydrogen f l u o r i d e has been s u c c e s s f u l l y used i n p a r t i a l h y d r o l y s i s of polysaccharides c o n t a i n i n g amino sugars. Mort and L a m p o r t 1 2 2 found that hydrogen f l u o r i d e could cleave sugars from 4 6 g l y c o p r o t e i n s , l e a v i n g the peptide moiety i n t a c t . They al s o observed a la r g e d i f f e r e n c e i n the rat e of cleavage of g l y c o s i d i c l i n k a g e s of amino sugars and n e u t r a l sugars i n hydrogen f l u o r i d e at 0°: the n e u t r a l sugar l i n k a g e s could be broken while l e a v i n g those of amino sugars i n t a c t . 1 2 2 Recently, M o r t 1 2 3 reported that i n hydrogen f l u o r i d e at subzero temperatures, d i f f e r e n t i a l cleavage of linkages of n e u t r a l and a c i d i c sugars can be obtained, and K n i r e l et a l . 1 2 1 t found d i f f e r e n t i a l cleavage of amino sugar linkag e s at 25°. The method i s of p a r t i c u l a r advantage f o r production of l a r g e r o l i g o s a c c h a r i d e s containing 0-acyl g r o u p s . 1 2 5 Another way of i s o l a t i n g o l i g o s a c c h a r i d e s c o n t a i n i n g a c i d - l a b i l e components, i s to use the bacteriophage-induced depolymerization of polysaccharides (see l a t e r ) . In the present study p a r t i a l h y d r o l y s i s was used i n the s t r u c t u r a l i n v e s t i g a t i o n s of K l e b s i e l l a K50 and E s c h e r i c h i a c o l i K28 polysaccharides. For K50, d i ~ , t r i - and t e t r a s a c c h a r i d e s were i s o l a t e d ; f o r E. c o l l K28, an al d o b i o u r o n i c a c i d and n e u t r a l d i s a c c h a r i d e were i s o l a t e d . II.4.2 Periodate o x i d a t i o n 1 2 6 " 1 3 8 G l y c o l - c l e a v i n g reagents, e s p e c i a l l y p e r i o d i c a c i d and i t s s a l t s , and lead t e t r a a c e t a t e have found widespread a p p l i c a t i o n s i n carbohydrate chemistry. Lead t e t r a a c e t a t e 1 2 6 had been l i t t l e used i n st u d i e s on polysaccharides, because they are i n s o l u b l e i n the solvents g e n e r a l l y used f o r such o x i d a t i o n s . 47 Treatment of g l y c o l groups w i t h p e r i o d i c a c i d and i t s s a l t s 1 2 ' r e s u l t s i n cleavage of carbon bonds and the formation of two aldehydic groups, one molecular p r o p o r t i o n of periodate being reduced. In g e n e r a l , open chain g l y c o l s are most r e a d i l y o x i d i z e d , followed by c y c l i c c i s -g l y c o l s ; c y c l i c t r a n s - g l y c o l s are more slowly o x i d i z e d , or not o x i d i z e d at a l l i f f i x e d i n an unfavorable conformation ( b i c y c l i c anhydrohexo-s e s ) . The r e a c t i v i t y may a l s o be a f f e c t e d by the s t e r i c e f f e c t s of neighboring g r o u p s . 1 2 8 Smith and c o - w o r k e r s 1 2 9 have studied the products obtained from polysaccharides a f t e r periodate o x i d a t i o n , borohydride reduction and h y d r o l y s i s . Hexose residues s u b s t i t u t e d i n the 4 - p o s i t i o n give e r y t h r i -t o l or t h r e i t o l together w i t h glycoaldehyde, while t e r m i n a l and 6-s u b s t i t u t e d residues give g l y c e r i t o l and glycoaldehyde, 2 - s u b s t i t u t e d residues give g l y c e r i t o l and g l y c e r o s e , and 3-substituted residues a f f o r d the i n t a c t hexose (see F i g . I I . 2 ) . Where three adjacent hydroxyl groups are present, a double cleavage of the carbon chain occurs w i t h formation of two aldehydic groups, the reduction of two molecular proportions of periodate and the l i b e r a t i o n of one molecular p r o p o r t i o n of formic a c i d . Periodate o x i d a t i o n s t u d i e s can be, t h e r e f o r e , used f o r l i n k a g e a n a l y s i s , determination of degree of p o l y m e r i z a t i o n and chain length of polysaccharides (by measuring the p r o p o r t i o n of formic a c i d or formalde-hyde released using the o x i d a t i o n ) , and degree of branching. Smith and c o - w o r k e r s 1 3 0 have given the general procedures used i n the o x i d a t i o n of polysaccharides. The r e a c t i o n i s normally c a r r i e d out i n the dark at 5°, e i t h e r i n d i s t i l l e d water or i n b u f f e r w i t h i n pH 4-5 to avoid a c i d 48 Terminal and mono-s u b s t i t u t e d hexoses Number of molecules of 10. 4 Products formed a f t e r o x i d a t i o n , NaBH. reduction 4 and h y d r o l y s i s CH„OH HO ( \ 0-H0 OH 2 ( :H0OH 2 ( OH + ( :H2OH 3H0 ^ 2OH CH 20-HO—( y—o HO' \)H 2 ( ( ]H2OH OH + ( :H2OH :HO :H2OH CH20H HO OH 1 ( :H2OH CH2OH OH — OH or + —OH HO— :H2OH CH2OH CHO CH2OH CHo0H HO / } 0-p \>H 0 CH„0H HO OH CH,OH HO — — ( \ 0-J \ . 1 ( ( M 20H CH0 —OH —OH or HO— :H2OH CH2OH 3H0 :H2OH F i g . I I . 2: Common products formed on periodate o x i d a t i o n , followed by borohydride re d u c t i o n and h y d r o l y s i s of t e r m i n a l and mono-s u b s t i t u t e d hexoses 49 h y d r o l y s i s and to minimize s i d e - r e a c t i o n s i n v o l v i n g n o n - s e l e c t i v e o x i d a -t i o n s . Because of the marked d i f f e r e n c e i n s t a b i l i t y between true a c e t a l s and g l y c o s i d e s , i t i s p o s s i b l e by m i l d a c i d h y d r o l y s i s to cleave the a c e t a l linkages i n p o l y a l c o h o l s r e s u l t i n g from periodate o x i d a t i o n and borohydride reduction of polysaccharides, and to leave any g l y c o s i d i c linkages i n t a c t . This important m o d i f i c a t i o n of the periodate o x i d a t i o n , devised by Smith and h i s c o - w o r k e r s , 1 3 1 gives valuable s t r u c t u r a l i n f o r m a t i o n on the f i n e s t r u c t u r e of the parent polysaccharide. Depending on the r e l a t i v e l o c a t i o n of the p e r i o d a t e -r e s i s t a n t sugar r e s i d u e s , the degradation may r e s u l t i n formation of g l y c o s i d e s of mono- or o l i g o - s a c c h a r i d e s , or degraded p o l y s a c c h a r i d e s , which can be subjected to repeated Smith degradation. A s e l e c t i v i t y i n the a c i d h y d r o l y s i s step i s sometimes d i f f i c u l t to achieve, since h y d r o l y s i s of normal g l y c o s i d i c bonds might occur together w i t h the removal of cleaved fragments. A m o d i f i c a t i o n of the Smith h y d r o l y s i s , introduced by Lindberg and c o - w o r k e r s , 1 3 2 i n v o l v e s methylation of the reduced o x i d i z e d polysaccharide ("polyalcohol") p r i o r to a c i d h y d r o l y s i s , which ensures complete removal of glycoaldehyde fragments from o x i d i z e d residues. Periodate o x i d a t i o n i s complicated by both under- and over-oxida-t i o n . P a i n t e r and L a r s e n 1 3 3 ' 1 3 , 4 have demonstrated that the o x i d a t i o n i s incomplete i n c e r t a i n cases due to the formation of i n t e r - r e s i d u e hemi-ac e t a l s between the aldehyde groups of o x i d i z e d hexuronic a c i d residues and the c l o s e s t hydroxyl groups of unoxidized residues. Those residues protected through the hemiacetal can be exposed to o x i d a t i o n by f i r s t 50 s u b j e c t i n g them to borohydride reduction (see F i g . I I . 3 ) . Incomplete o x i d a t i o n may occur due to e l e c t r o s t a t i c r e p u l s i o n s between the p e r i o -date ions and weakly a c i d i c groups (-C00H) of a c i d p o l y s a c c h a r i d e s . 1 3 5 This e f f e c t i s suppressed by adding a s a l t (0.2M sodium p e r c h l o r a t e ) . 1 3 5 O v e r - o x i d a t i o n 1 3 6 i s minimized i f the r e a c t i o n i s c a r r i e d out i n the dark, at low temperatures, without great excess of reagent and at pH 3.6-4.5, so that the formyl e s t e r i n i t i a l l y formed i s not hydrolyzed at a s i g n i f i c a n t r a t e . During the present study on the s t r u c t u r e of E s c h e r i c h i a c o l i K32 the periodate o x i d a t i o n conducted on the o r i g i n a l polysaccharide at room temperature and i n unbuffered sodium metaperiodate (NalO^) y i e l d e d a 46% o v e r - o x i d a t i o n of 3-linked oc-galactose, due to the p a r t i a l h y d r o l y s i s of the rhamnosyl bond (see experimental). When the o x i d a t i o n was performed i n buffered periodate s o l u t i o n (pH 4.5), 3-linked galactose was recovered q u a n t i t a t i v e l y . Recently, k i n e t i c s tudies c a r r i e d out by P a i n t e r and co-work-e r s 1 3 7 have shown that a f t e r a l i m i t e d period of o x i d a t i o n the 8-D-galactopyranosyl side groups i n a polysaccharide could be s e l e c t i v e l y removed by Smith degradation, l e a v i n g the 1,4-linked residues i n the main chain i n t a c t . Numerous p o s s i b i l i t i e s f o r s e l e c t i v e o x i d a t i o n can be demonstrated by comparison of second-order ra t e c o e f f i c i e n t s f o r periodate o x i d a t i o n of various methyl g l y c o s i d e s . 1 3 7 S e l e c t i v e o x i d a -t i o n of the t e r m i n a l a-rhamnosyl residue was f i r s t demonstrated i n the s t r u c t u r a l i n v e s t i g a t i o n of K l e b s i e l l a serotype K17 p o l y s a c c h a r i d e . 1 3 8 A t y p i c a l sequence of periodate o x i d a t i o n and Smith h y d r o l y s i s i s shown i n Scheme I I . 5 f o r K l e b s i e l l a K50 capsular polysaccharide. 0 * • 12 16 h An unoxidised doublet, with each unit protected by one oxidised neighbour. (See curve A) F i g . I I . 3 : Sequential periodate o x i d a t i o n (0.025 M NalO^, 20°) and borohydride r e d u c t i o n of al g i n a t e . At the po i n t s l a b e l l e d R, samples were reduced w i t h sodium borohydride and then o x i d i z e d f u r t h e r (curves B and C). From r e f . 133. 52 CH20H Scheme I I . 5 : Smith degradation of K l e b s i e l l a K50 capsular polysaccharide 53 II.4.3 Base-catalyzed degradation 1 3 9" 1** 6 In general, g l y c o s i d i c linkages are s t a b l e under a l k a l i n e c o n d i -t i o n s . However, base-catalyzed 8-eliminations can be i n i t i a t e d by s t r o n g l y electron-withdrawing f u n c t i o n a l groups w i t h consequent cleavage of g l y c o s i d i c l i n k a g e s . Groups that may act i n t h i s manner in c l u d e the carbonyl groups of reducing r e s i d u e s , a c t i v a t e d hexuronic a c i d d e r i v a -t i v e s ( e s t e r s ) , carbonyl groups introduced by o x i d a t i o n at s e l e c t e d s i t e s , and sulfone groups formed i n s t r u c t u r a l m o d i f i c a t i o n s . 1 3 9 Reducing sugars undergo a number of competing r e a c t i o n s when tre a t e d w i t h base. The r e a c t i o n i n v o l v e s base-catalyzed 8 - e l i m i n a t i o n (normally i n aqueous s o l u t i o n ) w i t h formation of 3-deoxyhex-2-eno-pyranoses. This e l i m i n a t i o n occurs most e a s i l y when there i s a 3-0-s u b s t i t u e n t to provide a good l e a v i n g group. The a l k a l i n e degradation of polysaccharides i s known as a " p e e l i n g " r e a c t i o n . Base-catalyzed degradations from hexuronic a c i d residues occur when the uronic a c i d i s e s t e r i f i e d and 4-0-substituted and r e s u l t i n the 8 - e l i m i n a t i o n of the 4-0-substituent w i t h the formation of hex-4-eno-pyranosiduronate r e s i d u e s . The most widely used r e a c t i o n sequence i s that developed by Llndberg and co-workers. 1 1 + 0 ' 1 **1 The main steps of t h i s degradation are o u t l i n e d as f o l l o w s : 54 The unsaturated product i s l a b i l e to acids and on m i l d h y d r o l y s i s w i t h a c i d releases the aglycon (R^OH) 1 1* 2 that f u r t h e r y i e l d s the furan w i t h the simultaneous release of the s u b s t i t u e n t s at 0-2 and 0-3. When R^OH i s a s i n g l e residue or chain of sugar r e s i d u e s , a second 8-elimina-t i o n r e a c t i o n occurs, and the next sugar may be released on the subse-quent mil d a c i d treatment. I f R^OH i s an aldose s u b s t i t u t e d at 0-3, f u r t h e r degradation of the polysaccharide w i l l occur during the t r e a t -ment w i t h a l k a l i . In an a l t e r n a t i v e procedure 1 1* 3 exposed reducing groups are s i m u l -taneously protected by a c e t y l a t i o n w i t h a c e t i c anhydride i f degradation i s performed using the organic base l,5-diazabicyclo[5.4.0]undec-5-ene (DBU). 55 L a t e r experiments have shown t h a t , under co n d i t i o n s normally used f o r base degradations, complete l o s s of ur o n i c a c i d residues occurs and that the a c i d h y d r o l y s i s i s unnecessary. 1 1 + l t The nature of residues released during the degradation i s r e v e a l -ed by f u r t h e r a l k y l a t i o n w i t h t r i d e u t e r i o m e t h y l i o d i d e or e t h y l i o d i d e , h y d r o l y s i s , and a n a l y s i s of the r e s u l t i n g sugars, as a l d i t o l a c e t a t e s , by g a s - l i q u i d chromatography - mass spectrometry (g.l.c.-m.s.). Comparison of t h i s a n a l y s i s w i t h the methylation a n a l y s i s of the o r i g i n a l polysaccharide reveals the s i t e of attachment of uronic a c i d u n i t s . The degradative sequence developed by Svensson and co-workers 1 1 + 5 i n v o l v e s s e l e c t i v e o x i d a t i o n of the exposed hydroxyl groups i n the methylated polysaccharide ( e i t h e r by base-catalyzed uronic a c i d degradation or by s e l e c t i v e h y d r o l y s i s of a c i d - l a b i l e g l y c o s i d i c linkages such as pyruvate), a l k a l i n e degradation and mild a c i d h y d r o l y s i s of enol g l y c o s i d i c bonds. S p e c i f i c o x i d a t i o n i s e f f e c t e d w i t h chlorine-DMSO. 1 4 6 Scheme II . 6 shows a t y p i c a l r e a c t i o n sequence f o r 8-el i m i n a t i o n of K l e b s i e l l a K50 polysaccharide. II.5 DETERMINATION OF LINKAGE II.5.1 O p t i c a l r o t a t i o n 1 * * 7 - 1 1 * 9 O p t i c a l r o t a t i o n can be used to determine the anomeric c o n f i g u r a -t i o n of sugar residues i n o l i g o - and polysaccharides. 56 OOMe CH20Me O-feOMe j)H+ 2)NoBH4 $Ac 20/Pyr 1 , 5 - d i - 0 - a c e t y l - 3 - 0 - e t h y l - 2 , 4 , 6 - t r i - O - m e t h y l m a n n i t o l 1 , 5 - d i - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l g a l a c t i t o l 1 , 2 , 5 - t r i - 0 _ - a c e t y l - 3 , 4 , 6 - t r i - Q - m e t h y l m a n n i t o l 1 . 3 . 5 - t r i - O ^ a c e t y l - 2 , 4 , 6 - t r i - O - m e t h y l g a l a c t i t o l 1 . 5 . 6 - t r i - 0 - a c e t y l - 2 , 3 , 4 - t r l - O - m e t h y l g l u c l t o l 1 , 3 , 5 , 6 - t e t r a - 0 - a c e t y l - 2 , 4 - d i - 0 - m e t h y l g l u c i t o l S cheme I I . 6 : U r o n i c a c i d d e g r a d a t i o n o f K l e b s i e l l a K 5 0 p o l y s a c c h a r i d e 57 Although o p t i c a l a c t i v i t y i s one of the p h y s i c a l p r o p e r t i e s of carbohydrates most often measured, i t probably i s more complex and l e s s understood than any other. The simplest approach invol v e s a p p l i c a t i o n of Van't Hoff's P r i n c i p l e of O p t i c a l S u p e r p o s i t i o n , 1 1 + 7 which proposes the a d d i t i v i t y of the r o t a t i o n a l c o n t r i b u t i o n s of d i f f e r e n t asymmetric centers i n a complex molecule. I t was found by Hudson 1 1* 8 to be a p p l i -cable to the determination of c o n f i g u r a t i o n at the anomeric center of the free sugars and g l y c o s i d e s . A p p l i c a t i o n of Hudson's I s o r o t a t i o n Rules gives i n f o r m a t i o n only on o v e r a l l molecular r o t a t i o n of p o l y - and o l i g o s a c c h a r i d e s . The c o n t r i b u t i o n s of O-acyl and pyruvate groups to the t o t a l molecular r o t a t i o n are neglected. The molecular r o t a t i o n (M) i s defined: where, [a] - s p e c i f i c r o t a t i o n M.W. - molecular weight Normally, the o p t i c a l r o t a t i o n i s measured at the D-line of sodium (589 nm). Using Hudson's I s o r o t a t i o n Rules one can p r e d i c t s p e c i f i c r o t a t i o n of o l i g o s a c c h a r i d e s and p o l y s a c c h a r i d e s 1 1 + 9 using the molecular r o t a t i o n values of model methyl g l y c o s i d e s . x M.W. 100 58 . . M i x 100 , . . . _ . f al = r r — where, IMi - sum of molecular r o t a t i o n M.W. values of model methyl glyc o s i d e s M.W. - molecular weight of polysaccharide. II.5.2 Nuclear magnetic resonance spectroscopy II.5.2.1 ^-n.m.r. sp e c t r o s c o p y 1 5 0 - 6 0 Proton magnetic resonance spectroscopy o f f e r s to carbohydrate chemists a method both f o r determining the c o n f i g u r a t i o n of unknown carbohydrates and f o r a s c e r t a i n i n g the conformations of known carbo-hydrates i n s o l u t i o n . 1 5 0 The fundamental work of Lemieux and co-workers introduced the s u c c e s s f u l a p p l i c a t i o n of ^-H-n.m.r. spectroscopy to s t r u c t u r a l problems i n the carbohydrate f i e l d . 1 5 1 An instrumental development of c o n s i d e r -able importance to p.m.r. spectroscopy of carbohydrates has been the i n t r o d u c t i o n of h i g h - r e s o l u t i o n magnets based on superconducting s o l e n o i d s . 1 5 2 The most s i g n i f i c a n t advance i n n.m.r. spectroscopy s i n c e 1964 has been the development of F o u r i e r - t r a n s f o r m techniques which a f f o r d a large enhancement i n s e n s i t i v i t y . 1 5 3 The use of a h i g h - f i e l d spectrometer i s e s p e c i a l l y b e n e f i c i a l f o r ^-n.m.r. spectroscopy of polysaccharides w i t h r e g u l a r repeating u n i t s . Numerous examples of t h i s 59 type of a p p l i c a t i o n are found i n st u d i e s on capsular polysaccharides of K l e b s i e l l a . These polysaccharides and a v a r i e t y of fragments prepared from them (by p a r t i a l or enzymatic h y d r o l y s i s , Smith degradation) were examined by *H- and 1 3C-n.m.r. spectroscopy i n combination w i t h chemical methods, and unique s t r u c t u r e s were determined. 1 5 t | H i g h - f i e l d ^-H-n.m.r. spectroscopy has been u t i l i z e d e x t e n s i v e l y i n determining the s t r u c t u r e s of g l y c o p r o t e i n s . 1 5 5 Two-dimensional (2-D) homo- and heteronuclear n.m.r. methods have been used as an a i d i n the assignment of the proton spectra of o l i g o s a c c h a r i d e moieties of g l y c o - c o n j u g a t e s . 1 5 6 A p p l i c a t i o n s of p.m.r. spectroscopy to problems of carbohydrate chemistry i n v o l v e measurement of four parameters. ( i ) R e l a t i v e i n t e n s i t i e s of the s i g n a l s The property t h a t , under proper operating c o n d i t i o n s , the r e l a -t i v e i n t e n s i t i e s of absorption s i g n a l s f o r d i f f e r e n t hydrogens are equal to the r e l a t i v e numbers of the hydrogens producing the s i g n a l s , has been p a r t i c u l a r l y important to a n a l y t i c a l carbohydrate c h e m i s t r y . 1 5 7 The number of anomeric l i n k a g e s , r e l a t i v e amounts of 6-deoxy sugars, O-acetyl, N-acetyl and 1-carboxyethylidene s u b s t i t u e n t s can be determin-ed. For monosaccharides i t also permits a r a p i d q u a n t i t a t i v e a n a l y s i s of the proportions of anomers, i n c l u d i n g furanose and pyranose forms. ( i i ) Coupling constants Nuclear s p i n - s p i n coupling constants are designated by J and are expressed as he r t z (Hz). When a f i r s t - o r d e r spectrum i s observed, the 60 magnitudes of the coupling constants may be determined d i r e c t l y from the spectrum. The r e l a t i o n s h i p between the v i c i n a l coupling constant (J) and the d i h e d r a l angle (<t>) between protons i s given approximately by the Karplus e q u a t i o n . 1 5 8 8.5 cos2<)>_0.28, 0° < <t> < 90° ^ ( H ^ H 2) = { 9.5 cos2<()_0-28, 90° < $ < 180° The values are maximum when the d i h e d r a l angle (<]>) i s 0° or 180°, and minimum when i t i s 90°. One of the most important consequences of the Karplus equation i s that the order of magnitude of d i a x i a l , a x i a l - e q u a t o r i a l and diequa-t o r i a l coupling constants (Jaa, Jae and Jee r e s p e c t i v e l y ) i n a c y c l o -hexane r i n g c h a i r system can be p r e d i c t e d and i s i n reasonable agreement w i t h the observed values. This i s shown i n F i g . I I . 4 . In carbohydrate chemistry, the determination of 3J(H,H) values has been used to e s t a b l i s h c o n f i g u r a t i o n as w e l l as conformational preferences f o r pyranose, furanose and a c y c l i c sugars. ( i i i ) Chemical s h i f t The chemical s h i f t s of protons are s t r o n g l y dependent upon s u b s t i t u t i o n a l , o r i e n t a t i o n a l and e l e c t r o n e g a t i v i t y e f f e c t s of neighbor-ing and d i s t a n t g r o u p s . 1 5 7 The e a r l y observations that the chemical s h i f t of a proton depends on i t s environment i n the molecule was demonstrated i n 1958 by Lemieux and c o - w o r k e r s . 1 5 1 Thus, e q u a t o r i a l 61 R 2 = H ,OH F i g . I I . 4 : R e l a t i o n s h i p b e t w e e n d i h e d r a l a n g l e (<)>) a n d c o u p l i n g c o n s t a n t s f o r a - a n d g - D - h e x o s e s 62 ring-hydrogen atoms had lower chemical s h i f t s than t h e i r a x i a l counterparts. The most important d i r e c t s h i e l d i n g e f f e c t i n carbohydrates i s that of the ring-oxygen atom, which causes the c h a r a c t e r i s t i c , l o w - f i e l d s h i f t of the anomeric hydrogen a t o m . 1 5 0 In the spectrum of an o l i g o s a c c h a r i d e or a p o l y s a c c h a r i d e , three main regions can be observed: a) the anomeric region (6 4.5-5.5), b) the r i n g proton region (6 3.0-4.5) and c) the high f i e l d r e g ion (6 1.15-2.5) where CH 3 groups of 6-deoxy sugars, pyruvates, 0 - a c e t y l , N - a c e t y l , e t c . can be observed (see F i g . I I . 5 ) . The c o n f i g u r a t i o n at the a c e t a l carbon atom of pyruvic a c i d a c e t a l s present i n some e x t r a c e l l u l a r b a c t e r i a l polysaccharides has been i n v e s t i g a t e d by Garegg and co-workers. The chemical s h i f t s 1 5 9 f o r the methyl groups of the pyruvic a c i d a c e t a l d i f f e r s i g n i f i c a n t l y depending upon whether these groups are a x i a l or e q u a t o r i a l . The r i n g protons are u s u a l l y d i f f i c u l t to assign except f o r cases where s p e c i f i c protons resonate at lower f i e l d (H-5 of g l u c u r o n i c a c i d and g a l a c t u r o n i c a c i d , H-5 of fucose, H-2 of mannose, e t c . ) . Two f a c t o r s dominate the a c q u i s i t i o n of high-res o l u t i o n xH-n .m.r. spectra of p o l y s a c c h a r i d e s , i n t e r f e r e n c e by exchangeable protons (0-H, N-H) and l i n e broadening of s i g n a l s . 1 5 4 The preparation of aqueous s o l u t i o n s of polysaccharides i n v o l v e s a p r i o r exchange treatment w i t h deuterium oxide ( p r e f e r a b l y 99.95 atom %) and the use of deuterium oxide as a s o l v e n t . Nevertheless a strong peak due to r e s i d u a l water (HOD s i g n a l ) i s o f t e n obtained. The chemical s h i f t of the HOD s i g n a l at room temperature (6 ~4.8 p.p.m.) i n t e r f e r e s w i t h the anomeric region and i t i s close to the H-l s i g n a l of B-glycopyranosyl residues. By r a i s i n g the ring protons N-Ac H-3a of KDO CH 3 of pyruvate CH 3 of u> 6(p.p.m. ) F i g . I I . 5 : Schematic representation of d i f f e r e n t regions i n the H-n.m.r. spectrum of polysaccharides 64 temperature one can d i s p l a c e the HOD s i g n a l u p f i e l d . There are a number of FT techniques f o r minimizing the i n t e r f e r e n c e by the HOD s i g n a l . 1 6 0 The problem of s i g n a l broadening i s l a r g e l y due to the f a c t that polymer protons have long r e l a x a t i o n times. A s u b s t a n t i a l enhancement i n the q u a l i t y of most polysaccharide *H spectra can be achieved by using elevated temperatures at 60°-90° or by performing a very m i l d h y d r o l y s i s i n order to reduce v i s c o s i t y of the sample (however, p o s s i b l e l o s s of l a b i l e groups should be considered). The *H s i g n a l s of many polysaccharides become appreciably sharper and, when a spectrum i s recorded at high f i e l d , there i s a corresponding enhancement i n s i g n a l s e p aration. Some b a c t e r i a l polysaccharides c o n t a i n O-acetyl groups which are i r r e g u l a r l y d i s t r i b u t e d along the polymeric c h a i n . This causes c e r t a i n anomeric s i g n a l s to be twinned. Such twinning disappears a f t e r O-de-a c e t y l a t i o n of the polysaccharide g i v i n g r i s e to a b e t t e r resolved spec-trum. This i s i l l u s t r a t e d i n F i g . I I . 6 which shows the ^H-n.m.r. spectra of E. c o l i K28 polysaccharide ( a f t e r a u t o h y d r o l y s i s , 95°, over-n i g h t ) and the same polysaccharide a f t e r O-deacetylation (0.01M NaOH, 23°, o v e r n i g h t ) . Both spectra were recorded w i t h acetone as an i n t e r n a l standard (6 2.23). II.5.2.2 Carbon-13 n.m.r. s p e c t r o s c o p y 1 5 9 ' 1 6 1 - 1 7 2 Carbon-13 nuclear magnetic resonance ( 1 3C-n.m.r.) has proved to be a powerful technique, y i e l d i n g i n f o r m a t i o n on composition, sequence and conformation of the polysaccharides. Using the F o u r i e r transform (FT) method, i t allows spectra of polysaccharides to be obtained using 65 F i g . I I . 6 : The H-n.m. r. sp e c t r a (400 MHz, 95° ) of n a t i v e (top) and deacetylated (bottom) E. c o l i K28 capsular polysaccharides 66 only t h e i r n a t u r a l abundance 1 3C atoms; i t complements ^-H-n.m.r. spectroscopy i n that i t gives b e t t e r s i g n a l separation owing to the wider range of chemical s h i f t s i n v o l v e d . 1 6 1 In many cases, i n p a r t i c u l a r when deal i n g w i t h complex molecules such as polysaccharides, the amount of informat i o n obtainable from 1 H_ n.m.r. spectra i s l i m i t e d compared to that revealed by 1 3C-n.m.r. 1 6 2 The technique i s r a p i d and nondestructive and can be used on r e l a t i v e l y small amounts of m a t e r i a l . Most of the studies to date have been concerned w i t h the proton-decoupled s p e c t r a . 1 6 1 However, a proton-coupled spectrum contains information about the 1 3C- 1H coupling constants that may be u s e f u l f o r the assignment of anomeric c o n f i g u r a t i o n . 1 6 3 In pyranosides C l - H l coupling 1 J [ 1 3 C H ( 1 ) ] i s l a r g e r when H-l i s e q u a t o r i a l (~170 Hz) than when H-l i s a x i a l (~160 Hz). A large number of hexopyranose d e r i v a t i v e s and t h e i r 1 J [ 1 3 C H ( 1 ) ] values have been examined by Bock and Pedersen. 1 6 1 * ' 1 6 5 The method of a n a l y s i s of 1 3C-n.m.r. spectra of polysaccharides i s to a large extent based on the comparison of the resonances Of the i n d i v i d u a l carbon atoms of the polysaccharide w i t h those of the p r e v i o u s l y assigned m o n o s a c c h a r i d e 1 6 2 ' 1 6 6 and o l i g o s a c c h a r i d e c o n s t i -t u e n t s . 1 6 7 ' 1 6 8 The chemical s h i f t s of the monosaccharides are s i m i l a r to those of the monosaccharide u n i t s w i t h i n the polysaccharide except f o r s u b s t i t u e n t e f f e c t s . These e f f e c t s produced by the attachment of any s u b s t i t u e n t to a sugar moiety cause an increase i n chemical s h i f t of the carbon d i r e c t l y i n v o l v e d i n the l i n k a g e ; t h i s i s u s u a l l y accompanied 67 by a decrease of smaller magnitude (sometimes an increase) i n the chemi-c a l s h i f t s of the neighboring (3-carbons. 1 6 1 The s e n s i t i v i t y of carbon-13 chemical s h i f t s towards changes i n s u b s t i t u t i o n permits l o c a t i o n of such s u b s t i t u e n t s as acetate, malonate, phosphate, or s u l f a t e groups. I n t r o d u c t i o n of an a c y l group onto oxygen causes a small (1.5-4 p.p.m.) downfield s h i f t of the oc-carbon atom. However, as Oj-acylation causes the s i g n a l of the (3-carbon atom to s h i f t u p f i e l d (1-5 p.p.m.), the cumulative e f f e c t of se v e r a l a c y l groups may be d i f f i c u l t to p r e d i c t . 1 6 2 An a l t e r a t i o n i n r i n g s i z e i s als o accom-panied by a change of chemical s h i f t s ; thus, furanoses have chemical s h i f t s downfield from those of the pyranoses. Sometimes, an immediate i d e n t i f i c a t i o n may be made when very l o w - f i e l d s i g n a l s at 6 = 107 p.p.m. or more are present, f o r example, f o r p-galactofuranoside and a-arabino-f u r a n o s i d e . 1 6 9 The s i g n a l s i n 1 3C-n.m.r. spectra of polysaccharides are known to d i s t r i b u t e i n groups each of which occupies s t r i c t l y d e f i n i t e regions. The t y p i c a l resonance regions u s e f u l f o r the f i r s t order a n a l y s i s of the 1 3C-n.m.r. spectrum are shown i n F i g . I I . 7 . 1 7 0 They i n c l u d e : a) carbonyl groups from u r o n i c a c i d , p y r u v i c a c i d , N- and 0-acetyl s u b s t i -tuents (175 ± 6 p.p.m.); b) anomeric carbons (100 ± 8 p.p.m.); c) secondary carbons (75 ± 5 p.p.m.); d) primary carbons (65 ± 5 p.p.m.); e) methyl groups from 0-acetyl and N-acetyl s u b s t i t u e n t s (20-28 p.p.m.), pyruvate (18-26 p.p.m.) and 6-deoxy sugars (~15-16 p.p.m.). The chemical s h i f t s f o r the R- and S-forms of the pyruvate d i f f e r s i g n i f i c a n t l y being ~18 p.p.m. f o r the a x i a l methyl groups and ~26 p.p.m. f o r the e q u a t o r i a l . 1 5 9 The anomeric c o n f i g u r a t i o n s of KDO AcO CO C 1 f I I T B a g C I HCOR HCOH HCN A c N "| CH2OH I I CCH , 00 I — 175 l r~"r_ 1 1 0 1 0 0 80 70 —r~ 60 r~ 5 0 -n r 4 0 —1~ 2 0 0 p . p . m . F i g . I I . 7 : The c h a r a c t e r i s t i c r e g i o n s f o r r e s o n a n c e s o f c a r b o n a t o m s b e l o n g i n g t o d i f f e r e n t m o n o s a c c h a r i d e r e s i d u e s i n p o l y s a c c h a r i d e s 69 residues can be e s t a b l i s h e d by 1 3C-n.m.r. spectroscopy. The C - l resonance i s s e n s i t i v e to anomeric c o n f i g u r a t i o n being 6 = 174.8 p.p.m. f o r the p-anomeric form and 6 = 176.5 p.p.m. f o r the a-anomer. 1 7 1 In the case of N-acetylneuraminic a c i d (NANA) the d i f f e r e n c e between chemi-c a l s h i f t s of C-l i n a- and B-forms i s sm a l l e r , being 6 = 175.9 p.p.m. f o r the 8-anomeric form and 6 = 174.6 p.p.m. fo r the oc-anomer. 1 7 2 Q u a n t i t a t i v e data cannot be s a t i s f a c t o r i l y obtained from i n t e g r a -ted 1 3C-n.m.r. sp e c t r a , because of s a t u r a t i o n phenomena and nuclear Overhauser e f f e c t s . However, i f spectra are measured under s u i t a b l e c o n d i t i o n s and i f i n t e g r a l s (or peak heights) of s i g n a l s from carbon atoms c a r r y i n g the same number of hydrogen atoms are compared, i t i s po s s i b l e to obta i n r a t h e r accurate information about the r e l a t i v e amounts of components i n a m i x t u r e . 1 6 2 An important l i m i t i n g f a c t o r i n the 1 3C-n.m.r. technique i s the s i g n a l - t o - n o i s e r a t i o obtained i n the spectra. A h i g h - f i e l d instrument, l a r g e sample tubes, and increased concentration of the sample r e s u l t i n a l a r g e r s i g n a l - t o - n o i s e r a t i o ( s / n ) . I f , however, a l i m i t e d amount of compound i s a v a i l a b l e , i t may be advantageous to use a smaller probe-i n s e r t . 1 6 2 In contrast to ^-H-n.m.r., p a r t i c l e s or undissolved m a t e r i a l make very l i t t l e d i f f e r e n c e to the q u a l i t y of the s p e c t r a . 1 6 1 F i g . II.8 shows the 1 3C-n.m.r. spectrum (proton decoupled) of the E. c o l i K28 deacetylated polysaccharide. Three s i g n a l s can be observed i n the anomeric region corresponding to four anomeric carbons (2 8 - l i n k -ed and 2 a - l l n k e d ) . The s i g n a l at 16.1 p.p.m. corresponds to the CH 3 group of fucose. acetone capsular polysaccharide 71 II.5.3 Other techniques 11.5.3.1 Enzymatic h y d r o l y s i s 1 3 9 * 1 7 3 A l l glycosidases are specific for the sugar unit undergoing hydrolysis and for i t s anomeric configuration. Exoglycosidases act on polysaccharide (or other glycoconjugate) substrates by the removal of non-reducing terminal units. These enzymes, however, can approach only to within a limited distance of other structural features such as other linkages or branch points. Thus, the oligosaccharides, generated by partial hydrolysis or bacteriophage degradation usually give more satis-factory results. Incubation with glycosidases i s usually performed in buffer at 37° at the appropriate pH for varying periods of time (some-times as long as 6-7 da y s ) . 1 7 3 In contrast, endo enzymes are not l i m i t -ed by action pattern and can cleave unbranched regions of both external and internal c h a i n s . 1 3 9 Highly specific endoglycanases are derived from bacteriophages (see Section VI). 11.5.3.2 Chromium trioxide o x i d a t i o n 1 7 4 * - 1 7 6 Angyal and James 1 7 < t showed that a fu l l y acetylated aldopyrano-side, in which the aglycon occupies an equatorial position in the most stable chair form (generally the B-anomer) is readily oxidized when treated with chromium trioxide in acetic acid. The anomer with an axial aglycon (generally the o-form) Is only slowly oxidized. 72 OR where, R = alkyl group or sugar residue This r e a c t i o n has been used to determine the anomeric c o n f i g u r a -t i o n of sugar residues i n o l i g o - and p o l y s a c c h a r i d e s . 1 7 5 The f u l l y a c e t y l a t e d m a t e r i a l and an i n t e r n a l standard are t r e a t e d w i t h chromium t r i o x i d e i n a c e t i c a c i d . Sugar a n a l y s i s of the o r i g i n a l m a t e r i a l and the o x i d i z e d product show which sugar residues have been o x i d i z e d . The method i s v a l i d f o r gluco-, g a l a c t o - , manno-, and x y l o - d e r i -v a t i v e s . However, s u b s t i t u t i o n i n o l i g o - and polysaccharides may a l t e r the conformational e q u i l i b r i u m of o-fucosyl and oc-rhamnosyl residues thus making them s u s c e p t i b l e to o x i d a t i o n . In some s i t u a t i o n s , i t may be p o s s i b l e to I s o l a t e oligomers a f t e r reduction of the o x i d i z e d product. This method was used i n s t u d i e s of the K l e b s i e l l a type 37 capsular p o l y s a c c h a r i d e . 1 7 6 In t h i s study chromium t r i o x i d e o x i d a t i o n was used to determine the anomeric c o n f i g u r a t i o n of g l u c u r o n i c a c i d i n E s c h e r i c h i a c o l i K32 capsular p o l y s a c c h a r i d e . Sugar a n a l y s i s performed on the o x i d i z e d product showed the disappearance of g l u c u r o n i c a c i d thus proving that i t was 8-1inked. 73 II.6 LOCATION OF O-ACETYL GROUPS161-177-180 Some polysaccharides w i t h l a b i l e O-acyl groups occur n a t u r a l l y . The removal of such s u b s t i t u e n t s may a l t e r p h y s i c a l and immunological p r o p e r t i e s of these polysaccharides. Since some of these acetate groups are extremely l a b i l e and can be e a s i l y removed, they create c e r t a i n d i f f i c u l t i e s i n ob t a i n i n g unambiguous evidence f o r t h e i r o r i g i n a l l o c a t i o n . The f i r s t and simplest method to use i s nuclear magnetic resonance spectroscopy ( I I . 5 . 2 ) . Proton nuclear magnetic resonance of the n a t i v e a c e t y l a t e d polysaccharide would show a s i n g l e t at 6 = 2.15-2.28 due to the presence of an O-acyl group. Presence of the acetate group can cause a d d i t i o n a l chemical s h i f t s of the neighboring p r o t o n s ; 1 7 7 however, the t o t a l cumulative e f f e c t may be d i f f i c u l t to p r e d i c t . 1 3C-n.m.r. spectroscopy i s more informative i n t h i s case, and comparison of the 1 3C-n.m.r. spectra of a c e t y l a t e d and deacetylated polysaccharides can of t e n provide very u s e f u l information on the 0_-acyl g r o u p 1 6 1 and sometimes unambiguous evidence f o r i t s l o c a t i o n ( f o r example, when present on C-6 of hexopyranose 2 5). S p e c t r o p h o t o m e t r y 1 7 8 and gas ch r o m a t o g r a p h i c 1 7 9 methods can be used f o r q u a n t i t a t i v e determination of the 0-acetyl groups. Chemical methods f o r l o c a t i n g these s u b s t i t u e n t s i n c l u d e periodate o x i d a t i o n (see Section I I . 4 . 2 ) , which i n some cases permits i d e n t i f i c a t i o n of sugar residues containing the acetate group. Prehm m e t h y l a t i o n 9 0 (see Section II.3.1.1) and the method of de Belder and 74 Norrman, l b U which i n v o l v e s the conversion of unsubstituted hydroxyl groups to methoxyethylacetals on r e a c t i o n w i t h methyl v i n y l ether (see Scheme II.7), followed by base-catalyzed de-O-acetylation and methylation. Scheme I I . 7 : Location of 0-acetyl s u b s t i t u e n t s according to the de Belder and Norrman procedure H y d r o l y s i s of the modified polysaccharide then gives sugar d e r i v a t i v e s l a b e l l e d w i t h O-methyl groups at the s i t e s o r i g i n a l l y occupied by 0-acetyl s u b s t i t u e n t s . This method gives the most s a t i s f a c t o r y results*, however, p o s s i b i l t y of "underprotection" should be considered i n the case of the pol y s a c c h a r i d e s . Oligosaccharides bearing 0-acetyl groups obtained by bacteriophage-induced degradation of polysaccharides (see 75 Section VI) or by p a r t i a l h y d r o l y s i s using hydrogen f l u o r i d e (see Section II.4.1) give more r e l i a b l e r e s u l t s . In t h i s study the p o s i t i o n of an CHacetyl group was loc a t e d us the methyl v i n y l ether procedure. The r e s u l t s obtained were f u r t h e r confirmed by -^H- and 1 3C-n.m.r. f i n d i n g s ( f o r d e t a i l e d procedure see Section IV). 76 CHAPTER I I I GENERAL EXPERIMENTAL CONDITIONS 77 III GENERAL EXPERIMENTAL CONDITIONS H I . l PAPER CHROMATOGRAPHY Paper chromatography was performed by the descending method using Whatman No. 1 paper and the f o l l o w i n g solvent systems: A) e t h y l a c e t a t e : a c e t i c a c i d : f o r m i c acid:water (18:3:1:4) B) e t h y l acetate:pyridine:water (8:2:1) C) 1-butanol:acetic acid:water (2:1:1) D) 1-butanol:ethanol:water (4:1:5, upper phase) Chromatograms were developed w i t h a l k a l i n e s i l v e r n i t r a t e or by heating at 110° f o r 5-10 min a f t e r spraying w i t h j>-anisidine hydro-c h l o r i d e i n aqueous 1-butanol. P r e p a r a t i v e paper chromatography was c a r r i e d out by the descend-ing method using Whatman 3MM paper and solvent C (unless otherwise s t a t e d ) . The relevant s t r i p s were cut out and elu t e d w i t h water f o r 6 h. The aqueous s o l u t i o n s were f i l t e r e d , concentrated and f r e e z e - d r i e d . III.2 GAS-LIQUID CHROMATOGRAPHY AND G.L.C.^IASS SPECTROMETRY A n a l y t i c a l g . l . c . separations were performed using a Hewlett-Packard 5700 instrument f i t t e d w i t h dual f l a m e - i o n i z a t i o n d e t e c t o r s . S t a i n l e s s - s t e e l columns (1.8 m x 3 mm) were used w i t h a c a r r i e r - g a s n i t r o g e n f l o w - r a t e of 20 mL/min. The f o l l o w i n g packing m a t e r i a l s and 78 programs were used (unless otherwise s t a t e d ) : (A) 3% of SP-2340 on Supelcoport (100-120 mesh), programmed from 195° f o r 4 min, and then at 2°/min to 260°; (B) 5% of ECNSS^l on Gas Chrom Q (100-120 mesh), isothermal at 170°, or programmed from 180° f o r 4 min, and then at 2°/min to 200°; (C) 3% of OV-225 on Gas Chrom Q (100-120 mesh), isothermal at 170°, or programmed from 180° f o r 4 min, and then at 2°/min to 230°. A l l q u a n t i t a t i v e data present are based on the average of at l e a s t three runs. P r e p a r a t i v e g . l . c . was c a r r i e d out with a F & M model 720 dual column instrument f i t t e d w i t h thermal c o n d u c t i v i t y d e t e c t o r s . S t a i n l e s s - s t e e l columns (1.8 m x 6.3 mm) were used w i t h c a r r i e r - g a s helium flow-rate of 60 mL/min. The f o l l o w i n g packing m a t e r i a l s and programs were used (unless otherwise s t a t e d ) : (D) 3% of SP-2340 on Supelcoport (100-120 mesh), programmed from 195° to 240° at 2°/min; (E) 3% of 0V-225 on Gas Chrom Q (100-120 mesh), programmed from 180° to 230° at 2°/min. G.l.c.-m.s. analyses were performed with a V.G. Micromass 12 instrument f i t t e d w i t h a Watson-Biemann separator. Spectra were r e c o r d -ed at 70 eV with an i o n i z a t i o n current of 100uA and an ion source at 200° (unless otherwise s t a t e d ) . I I I . 3 GEL-PERMEATION CHROMATOGRAPHY Preparative gel-permeation chromatography was performed using a column (2.5 x 100 cm) of Bio-Gel P-4 (400 mesh). The concentration of 79 the sample a p p l i e d to the column was 100 mg/mL. The column was i r r i g a t e d w i t h w a t e r : p y r i d i n e : a c e t i c a c i d (500:5:2) at a flow r a t e of 10.5 mL/hour. F r a c t i o n s of 2 mL were c o l l e c t e d , f r e e z e - d r i e d , weighed and an e l u t i o n p r o f i l e was obtained. III .4 OPTICAL ROTATION AND CIRCULAR DICHROISM O p t i c a l r o t a t i o n s were measured on aqueous s o l u t i o n s at 20°±3° on a Perkin-Elmer model 141 polarimeter w i t h a 1 dm c e l l (5 mL). C i r c u l a r dichroism spectra (c.d.) were recorded on a Jasco J-500A automatic recording spectropolarimeter w i t h a quartz c e l l of 0.3 mL capacity and a path length of 0.1 cm or 0.2 cm. Compounds were d i s s o l v e d i n a c e t o n i t r i l e ( s p e c t r o s c o p i c grade) and the spectra recorded i n the range of 190-260 nm. I I I . 5 NUCLEAR MAGNETIC RESONANCE Proton magnetic resonance spectra were recorded on a Var i a n XL-100, Bruker WP-80, Nic o l e t - O x f o r d H-270, or Bruker WH-400 instruments. Spectra were recorded at a temperature of 90°±5° and acetone was used as an i n t e r n a l standard. A l l values are given r e l a t i v e l y to that of i n t e r n a l sodium 4,4-dimethyl-4-silapentane-sul f o n a t e taken as 0. Samples (10-20 mg) were prepared by d i s s o l v i n g i n D 20 and fr e e z e - d r y i n g 2-3 times from D 20 s o l u t i o n s . Tubes of 5 mm i n diameter were used. 1 3C-n.m.r. spectra were recorded on a Bruker WP-80 or Bruker WH-400 spectrometer at ambient temperature. Samples (30-50 mg) were 80 d i s s o l v e d i n D 20 and acetone was used as an i n t e r n a l standard. Tubes of 5 or 10 mm i n diameter were used. 111.6 GENERAL CONDITIONS The I.R. spectra of methylated d e r i v a t i v e s were recorded on a Perkin-Elmer model 710B spectrophotometer i n carbon t e t r a c h l o r i d e ( spectroscopic grade). A l l s o l u t i o n s were concentrated on a r o t a r y evaporator i n vacuo at a bath temperature of 40°. Ion-exchange chromatography f o r separation of a c i d i c and n e u t r a l o l i g o s a c c h a r i d e s was performed on a column (2.0 x 28 cm) of Bio-Rad AG-1-X2 (formate form, 200-400 mesh). The n e u t r a l f r a c t i o n was el u t e d w i t h water and the a c i d i c w i t h 10% formic a c i d . Sephadex LH-20 was used f o r p u r i f i c a t i o n of permethylated o l i g o - and polysaccharides. D e - i o n i z a t i o n s were c a r r i e d out w i t h Amberlite IR-120(H +) r e s i n . 111.7 ISOLATION AND PURIFICATION OF THE POLYSACCHARIDES I I I . 7 . 1 K l e b s i e l l a polysaccharides The f o l l o w i n g media were used to grow the b a c t e r i a . 1. Beef-extract medium ("nutrient broth") 5 g of Bactopeptone 3 g Bacto beef e x t r a c t 2 g of NaCl 81 1 L of H 20 2. Sucrose-yeast extract-agar 75 g of sucrose 5 g of Bacto yeast e x t r a c t 37.5 g of agar 5 g of NaCl 2.5 g of KH2POl+ 0.625 g of MgSO^H^O 1.25 g of CaS0 1 + 2.5 L of H 20 A sample of K l e b s i e l l a b a c t e r i a serotype K50 was received as a stab c u l t u r e from Dr I. 0rskov (Copenhagen). The b a c t e r i a were streaked on agar p l a t e s and incubated at 37°. An i n d i v i d u a l colony was in o c u l a t e d i n beef-extract medium and b a c t e r i a were grown f o r 5 h at 37° wi t h continuous shaking. This l i q u i d c u l t u r e (50 mL) was incubated on a t r a y (86 cm x 46 cm) of sucrose-yeast extract-agar f o r 3 d. The lawn of capsular b a c t e r i a produced was harvested by scraping from the agar su r f a c e , and the b a c t e r i a were destroyed with 1% phenol s o l u t i o n . The polysaccharide was separated from the c e l l s by u l t r a c e n t r i f u g a t i o n (30,000 rpm). The viscous supernatant was p r e c i p i t a t e d i n t o ethanol (3 volumes). The p r e c i p i t a t e was d i s s o l v e d i n the minimum amount of water and r e p r e c i p i t a t e d w i t h a saturated (10%) s o l u t i o n of Cetavlon (cetyltrimethylammonium bromide). The p r e c i p i t a t e d a c i d i c polysaccha-r i d e was i s o l a t e d by c e n t r i f u g a t i o n . I t was d i s s o l v e d i n 4M NaCl, r e p r e c i p i t a t e d i n t o ethanol and the p r e c i p i t a t e was d i s s o l v e d i n water and d i a l y z e d against running tap water f o r 2 d. The d i a l y z e d s o l u t i o n 82 of polysaccharide was u l t r a c e n t r i f u g e d and the supernatant was f r e e z e -d r i e d . I I I . 7 . 2 E s c h e r i c h i a c o l i polysaccharides Stab c u l t u r e s of E s c h e r i c h i a c o l i b a c t e r i a K28 and K32 were obtained from Dr. I . 0rskov (Copenhagen). The f o l l o w i n g media were used to grow the b a c t e r i a . 1) Bacto Mueller Hinton Broth, dehydrated ( D i f c o ) 2) Mueller Hinton agar (BBL) w i t h a d d i t i o n of NaCl 0.5% (w/v). The streaked p l a t e s were incubated at 37° overnight. The b a c t e r i a were c u l t u r e d as described f o r the K l e b s i e l l a polysaccharides (see Section III.7.1) and were grown i n three small t r a y s (30 x 50 cm) using 1.5 L of Mueller Hinton agar and 5 g NaCl per t r a y . One t r a y was f i l l e d w i t h water and used as a humidity s o u r c e . 1 8 1 Each t r a y was layered w i t h an a c t i v e l y growing l i q u i d c u l t u r e of E. c o l i b a c t e r i a , obtained by i n o c u l a t i o n of 50 mL of Mueller Hinton broth w i t h a s i n g l e f r e s h colony of E. c o l i and f u r t h e r i n c u b a t i o n of the r e s u l t i n g s o l u t i o n f o r 5 h at 37°. The t r a y s were l e f t i n the i n c u -bator f o r 5 d and then the slime was c o l l e c t e d . The p u r i f i c a t i o n procedure was c a r r i e d out as described f o r the K l e b s i e l l a p olysaccharide. 83 I I I . 8 BACTERIOPHAGE PROPAGATION I I I . 8 . 1 Tube and f l a s k l y s i s The bacteriophages <J>28—1, <J>28-2, and <j>32 were i s o l a t e d from sewage (courtesy of Dr. S. Stirm, F r e i b u r g , Germany) and propagated on t h e i r host s t r a i n s i n n u t r i e n t broth by tube and f l a s k l y s i s . a) Tube l y s i s . An a c t i v e l y growing b a c t e r i a l c u l t u r e of E s c h e r i c h i a  c o l i was obtained by successive r e p l a t i n g s on agar p l a t e s . An a c t i v e l y growing b a c t e r i a l c u l t u r e (0.5 mL) was i n o c u l a t e d w i t h 6 x 5 mL of s t e r i l e n u t r i e n t broth and a f t e r 1 h of in c u b a t i o n 0.5 mL of a bacteriophage-containing s o l u t i o n was added to each t e s t tube at 30 min i n t e r v a l s . Continued i n c u b a t i o n r e s u l t e d i n gradual c l e a r i n g of the cloudy s o l u t i o n due to c e l l l y s i s . A f t e r 4-5 h of inc u b a t i o n a few drops of chloroform were added to each tube to prevent b a c t e r i a l growth and the contents of the f i r s t three and l a s t three t e s t tubes were combined. The b a c t e r i a l debris was then sedimented by c e n t r i f u g a t i o n and the phage s o l u t i o n s were analyzed by the "plaque assay technique". The technique was based on a s e r i e s of successive d i l u t i o n s of phage by t r a n s f e r r i n g 0.3 mL p o r t i o n s of phage to 2.7 mL portions of d i l u e n t ( n u t r i e n t b r o t h ) , thus making 10~ 2, 10~h, 10~ 6 and 10~ 8 d i l u t i o n s of o r i g i n a l s o l u t i o n . One drop from each d i l u t i o n was then placed on a b a c t e r i a l "lawn". (The "lawn" was prepared by i n o c u l a t i o n of 2 mL l i q u i d medium w i t h an a c t i v e l y growing colony and incu b a t i n g t h i s c u l t u r e f o r 4-5 h. An agar p l a t e , p r e v i o u s l y d r i e d i n the incubator at 84 37° was covered w i t h t h i s l i q u i d c u l t u r e , l e f t f o r 15 min and the excess of l i q u i d was removed. Incubation f o r 1 h gave a stab l e "lawn"). The p l a t e was then incubated at 37° overnight. At high phage concentrations (normally d i l u t i o n s 10 - t t, 10~ 5, and 1 0 - 6 ) i n d i v i d u a l phage p a r t i c l e s could not be d i s t i n g u i s h e d , but at s u i t a b l e d i l u t i o n s ( 1 0 - 7 and 10~ 8) i n d i v i d u a l plaques surrounded by halos (not always) could be e a s i l y counted. The t i t e r of the bacteriophage s o l u t i o n was then c a l c u l a t e d . number of plaques x d i l u t i o n Bacteriophage t i t e r drop s i z e ( i n plaque forming u n i t s (P.F.U.) per mL) b) F l a s k l y s i s . This technique was e s s e n t i a l l y the same as described f o r the tube l y s i s , except that l a r g e r amounts of bacteriophage could be produced. An overnight a c t i v e l y growing c u l t u r e (5 mL) was added to 6 x 50 mL of n u t r i e n t broth and a f t e r 1.5 h of incubation 5 mL of bacteriophage s o l u t i o n i s added to each f l a s k at 30 min i n t e r v a l s . The procedure was then continued as described f o r the tube l y s i s . A f l a s k l y s i s t y p i c a l l y y i e l d e d 300 mL of phage s o l u t i o n w i t h a t i t e r of 1 0 1 0 P.F.U./mL III.8,2 Large-scale propagation of the bacteriophage Bacteriophage <J>32 was propagated i n a 14 L fermentor j a r , fermentor model Microferm, New Brunswick S c i e n t i f i c . The f o l l o w i n g 85 co n d i t i o n s were used: a e r a t i o n , 5 L.p.m.; a g i t a t i o n , 500 r.p.m.; temperature, 37°. Nu t r i e n t broth (10 L) was autoclaved i n the fermentor j a r f o r 40 min at 121°. The s t e r i l e medium was placed i n a fermentor and a g i t a t e d f o r 15 min, then i t was in o c u l a t e d w i t h 450 mL of a c t i v e l y growing b a c t e r i a l c u l t u r e and s t i r r e d f o r 1 h. To t h i s s o l u t i o n a t o t a l of 9.6 x 1 0 1 0 P.F.U. i n 400 mL n u t r i e n t broth was added. A f t e r 2.5 h of incub a t i o n 400 mL of CHC1 3 was added to prevent b a c t e r i a l growth. The b a c t e r i a l debris was removed by continuous c e n t r i f u g a t i o n using a h i g h -speed Cepa-Schnell Model LE bench-scale c e n t r i f u g e cooled w i t h tap water and operated at 38,000 r.p.m., and with a flow rate of ~250 mL/min. 86 CHAPTER IV STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE OF K l e b s i e l l a SEROTYPE K50 87 IV STRUCTURAL INVESTIGATION OF THE CAPSULAR POLYSACCHARIDE OF Klebsiella SEROTYPE K50. IV.1 ABSTRACT The s t r u c t u r e of the capsular polysaccharide from K l e b s i e l l a K50 has been determined by using the techniques of methylation, periodate o x i d a t i o n and p a r t i a l h y d r o l y s i s and 8- e l i m i n a t i o n . N.m.r. s p e c t r o s -copy (*H and 1 3C) was used to e s t a b l i s h the nature of the anomeric l i n k -ages. The polysaccharide i s comprised of repeating u n i t s of the hepta-saccharide shown and i s the one of 19 capsular polysaccharides that are composed of D-glucuronic a c i d , D-galactose, D-glucose, and D-mannose residu e s . I t has the only known " f i v e - p l u s - t w o " repeating u n i t ; the st r u c t u r e of the polysaccharide from K l e b s i e l l a K50 i s , t h e r e f o r e , unique i n t h i s s e r i e s . -•3 ) - 8-D-Gal-(1 ->-3 ) - a-D-Glc- ( 1 -»4 ) - a-D-GlcA- (1 -»-3 ) - a-D-Man- (1 -»-2 ) - ct-D-Man- (1 6 t 1 a-D-Glc 6 + 1 8-D-Gal 88 IV.2 INTRODUCTION The capsular polysaccharide from K l e b s i e l l a serotype K50 i s composed of D-glucuronic a c i d , D-glucose, D-galactose, and D-mannose res i d u e s , and i s one of those, from almost twenty s t r a i n s , having the same q u a l i t a t i v e c o m p o s i t i o n . 1 5 I d e n t i t y of q u a l i t a t i v e a n a l y s i s , however, bears no r e l a t i o n s h i p to the s t r u c t u r a l p a t t e r n of the p o l y -saccharide, and examination of the capsular m a t e r i a l from K l e b s i e l l a K50 has shown i t to have a unique s t r u c t u r e i n t h i s s e r i e s . Several capsular polysaccharides based on a heptasaccharide repeating u n i t are known, but t h i s i s the f i r s t instance where such a u n i t i s of the "5 + 2" type. Furthermore, i n those capsular polysaccharides i n which the uron i c a c i d residue i s part of the main chain, only s i n g l e - u n i t l a t e r a l s u b s t i t u e n t s have p r e v i o u s l y been found. The polysaccharide of serotype K50 i s thus, on two counts, unique among the some s i x t y s t r u c t u r e s now known i n t h i s s e r i e s . The experimental evidence on which t h i s s t r u c t u r e i s based i s summarized next. IV.3 RESULTS AND DISCUSSION The i s o l a t i o n and p u r i f i c a t i o n of the polysaccharide were achieved as described i n Section I I I . 7 . 1 . 1 5 , 6 1 , 6 2 The a c i d i c p o l y -saccharide obtained a f t e r one p r e c i p i t a t i o n w i t h cetyltrimethylammonium bromide had t a ] D +102°, which compares w e l l w i t h the c a l c u l a t e d value of +96° using Hudson's Rules of I s o r o t a t i o n . 1 4 8 The polysaccharide was 89 shown to be homogeneous by gel-permeation chromatography with = 3.2 x 10 6 daltons. Paper chromatography of an a c i d hydrolyzate of the polysaccharide showed ga l a c t o s e , glucose, g l u c u r o n i c a c i d , mannose and an a l d o b i o u r o n i c a c i d . A c i d h y d r o l y s i s of carboxyl-reduced p o l y s a c c h a r i d e 7 5 and conversion to a l d i t o l acetates gave mannose, galactose, and glucose i n the molar r a t i o s of 2.0:2.4:3.0. Glucose was proved to be of the D-con-f i g u r a t i o n by c i r c u l a r dichroism (c.d.) measurements made on g l u c i t o l hexaacetate. Galactose and glucose were assigned the D c o n f i g u r a t i o n from c.d. measurements made on p a r t i a l l y methylated d e r i v a t i v e s . 7 9 The ^H-n.m.r. spectrum of p a r t i a l l y hydrolyzed polysaccharide was recorded i n D 20 at 90° w i t h acetone as i n t e r n a l standard (see Appendix I I I , Spectrum NO. 1). The spectrum e x h i b i t s 7 s i g n a l s i n the anomeric region: 6 5.46 (1H); 6 5.29 (1H); 6 5.24 (1H); 6 5.06 (1H); 6 5.01 (1H); 6 4.70 (^ 2 8 Hz, 1H) and 6 4.49 ( J x 2 8 Hz, 1H). From the values of the chemical s h i f t s and coupling constants, 5 a- and 2 6-anomeric linkages were assigned. No deoxy-sugar, 0_-acetyl or pyruvate could be detected. The 1 3C-n.m.r. spectrum confirmed these r e s u l t s (see Appendix I I I , Spectrum No. 2). Five s i g n a l s appear i n the anomeric region at 104.04; 103.97; 103.17; 101.30 and 99.16 p.p.m. The s i g n a l s at 104.04 and 99.16 p.p.m. each corresponded to two anomeric carbons. P r e c i s e assignment of the anomeric s i g n a l s was achieved a f t e r comparison of the *H- and 1 3C-n.m.r. spectra of the polysaccharide w i t h the corresponding spectra of o l i g o s a c c h a r i d e s obtained a f t e r p a r t i a l TABLE IV.1 N.M.R. DATA FOR Klebsiella K50 POLYSACCHARIDE AND THE DERIVED OLIGOSACCHARIDES Compound -^ H-N.m.r. data _-g "1,2 (Hz) Integral Assignment P.p.m proton 13C-N.m.r. data Assignment 1 3 GlcA-^-Man-OH Al 4.93 s 5.19 s 5.34 2.5 0.4 0.6 1.0 -Man-p-OH 3 -Man- -OH GlcA-1 3 1 2 GlcA-^-Man-^an-OH A2 4.93 5.08 2 2 0.3 0.7 2 —Man- -OH 3 -Man- -OH 103.4 101.93 3 -Mar GlcA—-a 5.32 2.5 2.0 GlcA-2 —Man-93.81 2 —Man-5.37 1 3 1 2 1 3 GlcA-^Ian-j-Man-^-Gal-OH A3 4.65 5.08 0.8 1 3 -Gal- -OH 3 -Man-103.1 101.34 3 —Man-GlcA-Compound* -^H-N.m.r. data 13C-N.m.r. data  J j 2 Integral Assignment0 P.p.m. Assignment (Hz) proton 3 3 5.19 - -Gal-j-OH 97.16 -Gal-p-OH 2 G l c A — — 95.41 -Man——Gal-o-OH OC u p 2 2 5.31 2 - M a n — 95.10 -Man-^-Gal-^-OH 3 93.04 -Gal——OH CHOH 1 3 1 2 I Gal-p-€lc-^-OCH 4.65 8 0.9 Gal—p- 104.11 Gal—p-CH20H 3 £H1 99.62 - G l c — 5.20 4 1 — G l c — — — 3 1 3 Ik 1 3 1 2 1 -Gal—s-Glc—_<;I CA——Han—r-Man—-T <x a a — a 1 Glc 6 1 Gal S50 4.49 4.70 3 - G a l 104.04 Gal-Gal-3 -Gal-103.97 -Man-Compound lH-N.m.r. data •V (Hz) Integral Assignment P.p.m. proton 13C-N.m.r. data a Assignment 5.01 5.06 5.24 5.29 5.46 2-Man-3-Glc-3-Mar 6-Glc - G l c / 103.17 101.30 99.16 6 -Glc 4 -GlcA-2 -Mar 3 -Glc 3 1 3 1 H 1 3 1 2 1 -Gal- c „ GlcA „ Man _ Man—— Glc Degraded K50 a a 4.51 5.02 5.27 3 -Gal-2 -Man-3 -Gal-3 —Man-Glc-103.83 3 -Gal-3 -Man-103.16 Glc-101.30 -GlcA- a t Compound H^-N.m. r . data 13C-N.m. r. data (Hz) Integral proton Assignment-0 P.p.m. • Assignment6 5.51 1 4 - G l c A — — a 99.16 98.80 2 -Man 3 - G l c — — a For the source of A l , A2, A3, and SHI, see t e x t . Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; 6 2.23 downfield from sodium 4,4-dimethyl-4-silapentahe-l-sulfonate (DSS). The numerical p r e f i x i n d i c a t e s the p o s i t i o n i n which the sugar i s s u b s t i t u t e d ; the a or B, the c o n f i g u r a t i o n of the g l y c o s i d i c bond, or the anomer i n the case of a (ter m i n a l ) reducing-sugar residue. Thus 3 - G a l — — r e f e r s to the anomeric proton of a 3-linked g a l a c t o s y l residue i n the a-anomeric c o n f i g u r a t i o n . The absence of a numerical p r e f i x i n d i c a t e s a (te r m i n a l ) nonreducing group. Chemical s h i f t i n p.p.m. downfield from Me^Si, r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. downfield from DSS. As i n c, but f o r 1 3C n u c l e i . See t e x t . 94 h y d r o l y s i s and of degraded polysaccharide a f t e r s e l e c t i v e h y d r o l y s i s (see Table IV.1). Methylation analysis M e t h y l a t i o n a n a l y s i s of the K50 polysaccharide, followed by h y d r o l y s i s , d e r i v a t i z a t i o n as a l d i t o l a cetates, and g.l.c.-m.s. analy-s i s , gave the values shown i n Table IV.2, column I . These r e s u l t s i n d i c a t e d that the polysaccharide c o n s i s t s of a heptasaccharide repeat-in g u n i t having a branch on glucose, w i t h galactose as the t e r m i n a l group. By reduction of the methylated polysaccharide (see column I I ) , the p r o p o r t i o n of 2,4,6-tri-O-methylmannose was increased and 2,3-di-0-methylglucose was formed, i n d i c a t i n g that g l u c u r o n i c a c i d i s s u b s t i t u t e d at 0_-4, and that i t i s l i n k e d to mannose. M e t h y l a t i o n - e t h y l a t i o n a n a l y s i s of the product obtained by base-catalyzed degradation of the uronic a c i d showed the presence of 3-0-ethyl-2,4,6-tri-0-methylmannose derived from e t h y l a t i o n at 0-3 of the mannose of the a l d o b i o u r o n i c a c i d (see column I I I ) . Selective partial hydrolysis Treatment of K50 polysaccharide w i t h very d i l u t e a c i d , and d i a l y -s i s of the products against d i s t i l l e d water, afforded a non-dialyzable polymeric m a t e r i a l and a d i a l y z a t e . The d i a l y z a t e contained g a l a c t o s e , as was shown by paper chromatography and g . l . c . (as a l d i t o l a c e t a t e ) . Examination of the non-dialyzable p o r t i o n by gel-permeation chromato-graphy showed the polymer to be e x t e n s i v e l y depolymerized (see F i g . IV.1), and t h i s was a t t r i b u t e d to the a c i d l a b i l i t y of the 3 - s u b s t i t u t e d 95 TABLE IV.2 METHYLATION ANALYSES OF Klebsiella K50 POLYSACCHARIDE AND DERIVATIVES Methylated sugars T a Mole % b (as a l d i t o l acetates) Column B C (ECNSS-M) I II III 2,3,4,6-Man6 0.87 ..- _ . . 16.5 2,3,4,6-Gal 1.00 19.8 14.5 23.5 3,4,6-Man 1.42 12.9 10.1 2,4,6-Man 1.49 8.7 18.4 25.6 2,4,6-Gal 1.59 15.8 14.5 8.9 2,3,4-Glc 1.66 26.7 15.2 20.9 2,4-Glc 2.52 16.0 11.1 4.4 2,3-Glc 2.59 - 15.5 -a b R e l a t i v e r e t e n t i o n time, r e f e r r e d to 2,3,4,6-Gal as 1.00. Values are corrected by use of the e f f e c t i v e , carbon-response f a c t o r s given by Albersheim et a l . 1 0 3 C Programmed f o r 4min at 160°, and then at 2°/min to 200°. ^ I , o r i g i n a l p o lysaccharide; I I , carboxyl-reduced polysaccharide; I I I , u r o n i c acid-degraded polysaccharide. 2,3,4,6-Man = 3-0-ethyl-2,4,6-tri-O-methylmannitol, e t c . 96 F i g . IV.1: Gel-permeation chromatography of the product obtained a f t e r s e l e c t i v e , p a r t i a l h y d r o l y s i s of K l e b s i e l l a K50 polysaccharide. (Bio-Gel P-300 column 85 x 1.5 cm, M NaCl e l u a n t , f l o w - r a t e 3 mL/h). Courtesy of Dr. S.C. Churms, Cape Town, South A f r i c a 97 8-D-galactopyranosyl u n i t , even under these mild c o n d i t i o n s . The *H-n.m.r. spectrum of t h i s m a t e r i a l lacked one s i g n a l (6 4.7) corresponding to a 6-linkage, but i n t e g r a t i o n of the s i g n a l s f o r anomeric protons was u n s a t i s f a c t o r y due to the depolymerization (see Table IV.1). Methyla-t i o n a n a l y s i s of the non-dialyzable m a t e r i a l showed that 2,3,4,6-tetra-O-methylglucose l a r g e l y replaced the corresponding galactose compound. P a r t i a l hydrolysis P a r t i a l h y d r o l y s i s of the n a t i v e polysaccharide w i t h a c i d was followed by separation of the a c i d i c and the n e u t r a l f r a c t i o n s by i o n -exchange chromatography. The n e u t r a l f r a c t i o n contained monosaccha-r i d e s . The a c i d i c f r a c t i o n contained three a c i d i c o l i g o s a c c h a r i d e s . On the basis of paper chromatography, t h e i r n.m.r.-spectral data (see Table IV.1), and methylation a n a l y s i s (see Table IV.3), the s t r u c t u r e s of these compounds were shown to be as f o l l o w s . M a-GlcA-( 1 ->3 ) -Man A2 ct-GlcA-(l+3)-a-Man-(l->2)-Man A3 a-GlcA-( l->-3)-a-Man-( l*2)-crt!an-( l+3)-Gal The a l d o t e t r a o u r o n i c a c i d (A3) obtained from p a r t i a l h y d r o l y s i s had p r e v i o u s l y been i s o l a t e d from other K l e b s i e l l a capsular polysaccha-r i d e s , namely K21, K26 and K74. 98 TABLE IV.3 ANALYSES OF ACIDIC OLIGOSACCHARIDES FROM Klebsiella K50 POLYSACCHARIDE Oligosaccharide Paper Sugar Methylation a b c chromatography analysis analysis (molar proportions) (molar proportions) A l GlcA Man -Man Glc (GlcA) < J > -A2 GlcA Man (1.5) 2,4,6-Man ( 0 . 7 ) d Man Glc (GlcA) (1) 3,4,6-Man (1.0) A3 GlcA Man (1.75) 2,4,6-Gal (1.0) Man Gal (1.2) 2,4,6-Man ( 0 . 7 ) d Gal Glc (GlcA) (1) 3,4,6-Man ( 0 . 8 ) d Solvents A and B. 15 As a l d i t o l c . acetates. As a l d i t o l a c e t a t e s , n e u t r a l sugars only. Ratios are low, due to incomplete h y d r o l y s i s of the g l u c o s y l u r o n i c l i n k a g e . 99 Periodate oxidation Periodate o x i d a t i o n of carboxyl-reduced p o l y s a c c h a r i d e 7 5 , f o l l o w -ed by methylation, Smith h y d r o l y s i s , r e m e t h y l a t i o n , 1 8 2 and h y d r o l y s i s , gave a mixture that was found, by g . l . c . a n a l y s i s of the a l d i t o l a c e t a t e s , to contai n d e r i v a t i v e s of 2,3,4,6-tetra-O-methylmannose, 2,3,4,6-tetra-O-methylgalactose, and 2,4,6-tri-O-methylglucose, i n d i c a t i n g that the galactose i n the main chain i s 3-linked to glucose, the branch p o i n t . The f o l l o w i n g s t r u c t u r e may, therefore be proposed; i n i t , the c o n f i g u r a t i o n of three linkages has yet to be determined. -•3 )-? -Gal-( 1 -»3 )-? - G l c - ( 1 +4 )-cc-GlcA-( 1+3 )-<x-Man-( 1 -»2 ) - a-Man-( 1 + 6 + 1 ?-Glc 6 + 1 B-Gal Smith degradation of the o r i g i n a l K50 polysaccharide gave an ol i g o s a c c h a r i d e , a n a l y s i s of which showed i t to be COOH I HCOH I Gal-(l-»3)-Glc-0CH I CH2OH and i t ' s '•H-n.m.r. spectrum i n d i c a t e d the presence of one a and one 8 lin k a g e . Because the two monosaccharides t h e r e i n e x h i b i t s i m i l a r c o u p l -100 ing-constants; no d e f i n i t i v e assignment could be made by such s p e c t r o s -copy. Incubation w i t h a 8-D-galactosidase caused cleavage (a-D-galacto-s i d a s e , negative) of galac t o s e , thus demonstrating that the i n - c h a i n galactose has the (3 c o n f i g u r a t i o n . S i x of the seven anomeric l i n k a g e s , i n c l u d i n g both of the 8 s i g n a l s , having been p o s i t i v e l y assigned, i t fo l l o w s that the side chain must be attached to the main chain by an a - g l y c o s i d i c bond. IV.4 CONCLUSION From the sum of these experiments, the complete s t r u c t u r e of the capsular polysaccharide from K l e b s i e l l a serotype K50 may be w r i t t e n -•3 ) - 8-D-Gal -(1 -»3 ) - cHD-Glc - (1 -»4 ) - a-D-Gl c A- (1 +3 ) - a-D-Man- (1>2)-ct-D-Man- ( 1 -> 6 t 1 a-D-Glc 6 + 1 0-D-Gal I t i s co n s i s t e n t w i t h the q u a l i t a t i v e a n a l y s i s o r i g i n a l l y r e p o r t -ed by Nimmich. 1 5 The s t r u c t u r e of the K50 polysaccharide i s unique among the K l e b s i e l l a K antigens i n having a "f i v e - p l u s - t w o " repeating u n i t . 101 IV.5 EXPERIMENTAL General methods The instrumentation used f o r n.m.r., g . l . c , g.l.c.-m.s. i n f r a r e d , c.d., and measurements of o p t i c a l r o t a t i o n has been described i n Section I I I . Paper chromatography, g a s - l i q u i d chromatography, and ion-exchange chromatography were performed as described i n Section I I I . Preparation and properties of K50 capsular polysaccharide A c u l t u r e of K l e b s i e l l a K50, obtained from Dr. Ida 0rskov (Copenhagen), was grown as described i n Section I I I . 7 . 1 . 1 5 ' 6 1 ' 6 2 The i s o l a t e d polysaccharide (3.4 g) had [ a ] 2 5 +102° (c 0.095, water). The average molecular weight was determined by g e l chromatography (courtesy of Dr. S.C. Churms, U n i v e r s i t y of Cape Town, South A f r i c a ) to be 3.2 x 10 6 daltons. N.m.r. spectroscopy ( *H and 1 3C) was performed on the o r i g i n a l K50 polysaccharide, but much be t t e r spectra were obtained a f t e r treatment of the polysaccharide w i t h 0.01M t r i f l u o r o a c e t i c a c i d during 2 h at 95°, i n order to lower the v i s c o s i t y . The p r i n c i p a l s i g n a l s and t h e i r assignments f o r the *H- and 1 3C- n.m.r. spectra are recorded i n Table IV.1. Hydrolysis of the polysaccharide H y d r o l y s i s of a sample (20 mg) of K50 polysaccharide w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) overnight at 95°, removal of the a c i d by repeated coevaporation w i t h water, followed by paper chromatography (solvents A and B, see Section I I I . l ) , showed the presence of D-mannose, 102 D-galactose, D-glucose, D-glucuronic a c i d and an aldo b i o u r o n i c a c i d . H y d r o l y s i s of carboxyl-reduced p o l y s a c c h a r i d e 7 5 w i t h 2M t r i f l u o r o a c e t i c a c i d overnight at 95°, followed by re d u c t i o n w i t h NaBH^ and a c e t y l a t i o n w i t h 1:1 a c e t i c anhydride-pyridine afforded the a l d i t o l a c e t a t e s 1 8 3 of mannose, galactose, and glucose which were i d e n t i f i e d by g . l . c . (column A, see Section I I I . 2 ) and found to be present i n the r a t i o s of 2.0:2.4:3.0. Circular dichroism measurements Glucose was proved to be of the D-configuration by c i r c u l a r d i c hroism (c.d.) measurements made on g l u c l t o l hexaacetate. Galactose and mannose were assigned D c o n f i g u r a t i o n from c.d. measurements made on p a r t i a l l y methylated d e r i v a t i v e s . 7 8 A l d i t o l acetates were separated p r e p a r a t i v e l y i n column D. P a r t i a l l y methylated a l d i t o l acetates were separated i n column E. For experimental d e t a i l s see Section I I I . 2 and MeCN Section I I I . 4 . The f o l l o w i n g values of &e2\3 were obtained f o r the acetates of: g l u c i t o l , +1.3; 2,3,4,6-tetra-0_-methylgalactltol, +0.7; and 2,4,6-tri-O-methylmannitol, +0.64. Methylation analysis The capsular polysaccharide (297 mg) i n the f r e e - a c i d form, obtained by passing the sodium s a l t through a column of Amberlite IR-120 (H +) r e s i n , was d i s s o l v e d i n dry dimethyl s u l f o x i d e (20 mL) and m e t h y l a t e d 8 8 by treatment w i t h 10 mL d i m e t h y l s u l f i n y l anion f o r 4 h, and then w i t h 15 mL methyl i o d i d e f o r 1 h. The product (316 mg), recovered 103 a f t e r d i a l y s i s against tap water, had been completely methylated (no hydroxyl absorption i n the i . r . spectrum). A sample (27.8 mg) of t h i s m a t e r i a l was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d , reduced w i t h sodium borohydride, a c e t y l a t e d w i t h 1:1 a c e t i c anhydride-pyridine, and analyzed as a l d i t o l acetates by g . l . c . and g.l.c.-m.s. i n columns B and C (see Table IV.2, column I ) . Carboxyl reduction of the f u l l y methylated polysaccharide (103 mg) w i t h L i A l H ^ i n anhydrous oxolane (10 mL) at room temperature overnight, h y d r o l y s i s of the product w i t h 2M t r i f l u o r o a c e t i c a c i d , followed by reduction of sugars w i t h sodium borohydride, and a c e t y l a t i o n of the a l d i t o l s w i t h 1:1 a c e t i c anhydride-pyridine gave a mixture of p a r t i a l l y methylated a l d i t o l acetates which was analyzed by g . l . c . and g.l.c.-m.s. i n columns B and C (see Table IV.2, column I I ) . Uronic a c i d degradation 1** 2 A sample (76 mg) of methylated K50 polysaccharide was d r i e d and, together w i t h a trace of _p_-toluenesulfonic a c i d , was d i s s o l v e d i n 19:1 dimethyl sulfoxide-2,3-dimethoxypropane (20 mL) under N 2 i n a f l a s k that was then sealed. D i m e t h y l s u l f i n y l anion (5 mL) was added and allowed to react f o r 16 h at room temperature, when e t h y l i o d i d e (7 mL) was added. The s o l u t i o n was s t i r r e d f o r 1 h, and the methylated, degraded product was i s o l a t e d by p a r t i t i o n between water and chloroform. H y d r o l y s i s and g.l.c.-m.s. a n a l y s i s of the a l d i t o l acetate d e r i v a t i v e s showed the presence of 3-0-ethyl-2,4,6-tri-0-methylmannose (see Table IV.2, column I I I ) . 104 Selective, partial h y d r o l y s i s i B H A s o l u t i o n of K50 polysaccharide (500 mg) i n 0.01M TFA (50 mL) was heated on a steam bath f o r 12 h. The a c i d was removed and the product was d i a l y z e d against d i s t i l l e d water (1 L ) , to a f f o r d a p o l y -meric m a t e r i a l (250 mg). Paper chromatography of the d i a l y z a b l e f r a c -t i o n showed galactose, confirmed by g . l . c . as g a l a c t i t o l hexaacetate. Me t h y l a t i o n a n a l y s i s of the polymeric m a t e r i a l and g . l . c . (column C) i n d i c a t e d the presence of 2,3,4,6-tetra-0-methylglucose i n s t e a d of the 2,3,4-tri-0-methylglucose found o r i g i n a l l y . Partial hydrolysis A s o l u t i o n of K50 polysaccharide (710 mg) i n 1M TFA (50 mL) was heated f o r 5 h on a steam bath. A f t e r removal of the a c i d by repeated coevaporation w i t h water, an a c i d i c and a n e u t r a l f r a c t i o n were separated on a column of Bio-Rad AG1-X2 (formate form) ion-exchange r e s i n . The a c i d i c f r a c t i o n (198 mg) was separated by pr e p a r a t i v e paper chromatography (solvent C), to give 51.5 mg of a pure a l d o b i o u r o n i c a c i d ( A l ) , 19.1 mg of a pure a l d o t r i o u r o n i c a c i d (A2), and 27.7 mg of a pure a l d o t e t r a o u r o n i c a c i d (A3). Paper chromatography of the n e u t r a l f r a c t i o n showed glucose, galactose and mannose. The analyses performed on each o l i g o s a c c h a r i d e were as f o l l o w s , (a) Paper chromatography. A c i d i c o l i g o s a c c h a r i d e s were tre a t e d w i t h 2M TFA overnight, and the hydrolyzates were examined by paper chromato-graphy (so l v e n t s A and B). (b) Sugar a n a l y s i s . The hydrolyzates were then reduced w i t h NaBH^, and the a l d i t o l s were a c e t y l a t e d w i t h 1:1 a c e t i c anhydride-pyridine. The a l d i t o l acetates obtained were analyzed 1 0 5 by g . l . c . i n column A. (c) M e t h y l a t i o n a n a l y s i s . A l l methylations were conducted by the method of Hakomori. 8 8 Dried samples of 5-6 mg were d i s s o l v e d i n 1 mL anhydrous dimethyl s u l f o x i d e , t r e a t e d w i t h 1 mL d i m e t h y l s u l f i n y l anion f o r 2 h and then w i t h 2 mL methyl i o d i d e f o r 1 h. The mixtures were d i l u t e d w i t h water and e x t r a c t e d w i t h chloroform (4 x 10 mL). The combined e x t r a c t s were back ex t r a c t e d w i t h water (3 x 10 mL) and evaporated to dryness under reduced pressure. The products were hydrolyzed w i t h 2M TFA, and analyzed (as a l d i t o l acetates) by g . l . c . and g.l.c.-m.s. i n columns B and C. The r e s u l t s obtained f o r each o l i g o s a c c h a r i d e are given i n Table IV.3, and the n.m.r. data, i n Table IV.1. Carbodiimide reduction of capsular polysaccharide 7 5 A sample of K50 polysaccharide ( N a + s a l t , 502 mg) was d i s s o l v e d i n 80 mL water. l-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p_-toluenesulfonate (CMC, 2.2 g) was added. As the r e a c t i o n proceeded, the pH was maintained at 4.75 by t i t r a t i o n w i t h 0.1 N HC1. The r e a c t i o n was allowed to proceed f o r at l e a s t two h. When the consumpton of h y d r o c h l o r i c a c i d ceased, an aqueous s o l u t i o n of 2M sodium borohydride (12 g/150 mL H 20) was added s l o w l y . The pH rose r a p i d l y to 7.0 and i t was maintained between 5-7 by t i t r a t i o n w i t h 4M HC1. A drop of 2-octanol (antifoaming agent) was added p e r i o d i c a l l y to c o n t r o l the amount of foam. The reduction was completed i n one h, and the r e a c t i o n mixture was s t i r r e d f o r an a d d i t i o n a l 30 min. The mixture was d i a l y z e d against tap water during 24 h, concentrated and f r e e z e - d r i e d . A second 1 0 6 treatment was c a r r i e d out. A t o t a l of 412.5 mg of the product was recovered a f t e r f r e e z e - d r y i n g . A sample of the reduced polysaccharide (10 mg) was hydrolyzed overnight w i t h 2M t r i f l u o r o a c e t i c a c i d on a steam bath and the sugars i n the hydrolyzate were converted i n t o a l d i t o l a c e t a t e s , g . l . c . a n a l y s i s of which showed mannitol, g a l a c t i t o l and g l u c i t o l hexaacetates i n a r a t i o 2.0:2.4:3.0, i n d i c a t i n g complete reduction of uronic a c i d . Periodate oxidation of carboxyl-reduced K50 polysaccharide Carbodiimide-reduced K50 polysaccharide (46.9 mg) i n water (20 mL) was mixed w i t h a s o l u t i o n (5 mL) of 0.1M NalO^ and 0.4M NaClO^, and kept i n the dark at 4°. The periodate consumption was followed on 1 mL a l i q u o t s by the Fleury-Lange method 1 8 5 and reached a plateau a f t e r 6 d (7.62 moles of periodate per mole of p o l y s a c c h a r i d e ) . Ethylene g l y c o l (10 mL) was added, the polyaldehyde was reduced to the p o l y a l c o h o l w i t h NaBH^, the base n e u t r a l i z e d w i t h 50% a c e t i c a c i d , and the s o l u t i o n was d i a l y z e d overnight, and f r e e z e - d r i e d to y i e l d the p o l y a l c o h o l (50.9 mg) which was methylated by Hakomori p r o c e d u r e . 8 8 One-third of the methyla-ted product was hydrolyzed w i t h 50% a c e t i c a c i d f o r 90 min, and then remethylated. The product was hydrolyzed, and the sugars were c h a r a c t e r i z e d as a l d i t o l acetates by g . l . c . i n column B. The remainder of the m a t e r i a l was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d overnight on a steam bath. Conversion of the p a r t i a l l y methylated sugars i n t o a l d i t o l acetates and g . l . c . thereof i n column B, showed the presence of 2,4,6-tri-O-methylmannose, 2,4,6-tri-O-methylgalactose, and 2,4-di-0-methylglucose i n equimolar p r o p o r t i o n s . 107 Smith degradation Polysaccharide (295 mg) i n water (100 mL) was mixed w i t h a s o l u -t i o n (28 mL) of 0.1M NaI0 4 and 0.4M NaC10 4, and kept i n the dark at 4°. A f t e r 95 h, ethylene g l y c o l (10 mL) was added. The s o l u t i o n was d i a l y z e d overnight, the polyaldehyde was reduced to the p o l y a l c o h o l w i t h NaBH^ (1 g ) , the base was n e u t r a l i z e d w i t h 50% a c e t i c a c i d , and the s o l u t i o n was d i a l y z e d , and f r e e z e - d r i e d , to y i e l d p o l y a l c o h o l (258 mg) which was subjected to Smith h y d r o l y s i s w i t h 0.5M t r i f l u o r o a c e t i c a c i d f o r 20 h at room temperature. Paper chromatography (solvent C) of the products showed g l y c e r o l and three o l i g o s a c c h a r i d e s . The chromatograms obtained i n d i c a t e d that the Smith h y d r o l y s i s was not complete, but 49.6 mg of a pure, a c i d i c oligomer (SH^) was i s o l a t e d , [ a ] ^ 5 +59° (c_ 0.08, water) and R„ t 0.55 (solvent C). N.m.r. data are given i n Table IV.1. Sugar a n a l y s i s of the product (by g . l . c , as the a l d i t o l acetates) gave galactose and glucose i n the r a t i o of 1:1. M e t h y l a t i o n by the Hakomori method 8 8 gave the p a r t i a l l y methylated a l d i t o l acetates corresponding to 2,3,4,6-tetra-0-methylgalactose and 2,4,6-tri-O-methylglucose. Enzymatic h y d r o l y s i s The a c i d i c product (SH^, 2.3 mg) was d i s s o l v e d i n 1 mL of b u f f e r , pH 7.3, and a s o l u t i o n of 8-D-galactosidase (1.1 mg, from E. c o l l , Sigma) i n 0.1 mL of the same buffer, was added. The mixture was incuba-ted f o r 3 d at 37°; then, the r e a c t i o n was terminated by a d d i t i o n of 50% a c e t i c a c i d . The product, i s o l a t e d by l y o p h i l i z a t i o n , was examined by paper chromatography (solvent B), which showed the presence of 108 galactose, the i d e n t i t y of which was confirmed by g . l . c . as g a l a c t i t o l hexaacetate. In a c o n t r o l experiment, compound SH^ and mellbiose were separately incubated w i t h a-D-galactosidase (pH 4). Galactose was released from melibiose (a-D-galactopyranosyl-(l^-6)-D-glucose), but not from SH,. 109 CHAPTER V STRUCTURAL INVESTIGATION OF Escherichia c o l i CAPSULAR POLYSACCHARIDES n o V.1 STRUCTURAL INVESTIGATION OF ESCHERICHIA COLI 09:K28(A):H~ (K28 ANTIGEN) CAPSULAR POLYSACCHARIDE. V . l . l ABSTRACT The s t r u c t u r e of the capsular polysaccharide from E s c h e r i c h i a  c o l i 09:K28(A):H~ (K28 antigen) has been determined by using the techniques of methylation, periodate o x i d a t i o n and p a r t i a l h y d r o l y s i s . N.m.r. spectroscopy (*H and 1 3C) was used to e s t a b l i s h the nature of the anomeric l i n k a g e s . Q u a n t i t a t i v e determination of 0-ac e t y l groups was done s p e c t r o p h o t o m e t r i c a l l y . The l o c a t i o n of the 0-acetyl group was determined using methyl v i n y l ether as a p r o t e c t i v e reagent. The polysaccharide i s comprised of repeating u n i t s of the t e t r a -saccharide shown (three-plus-one type) w i t h 70% of the f u c o s y l residues being 0-acetylated at e i t h e r 0-2 or 0-3. -•3 ) - a-D-Glc-(1 -»-4 ) - 8-D-Glc A- (1 -»-4 ) - a-L-Fuc-( 1 -> 4 2 or 3 • I 1 OAc 8-D-Gal I l l V.1.2 INTRODUCTION The K antigens of E. c o l i can be d i v i d e d i n t o three groups (A, B, L ) , a l l of which comprise a c i d i c polysaccharides. D i s t i n c t i v e features of A antigens are that they occur only together w i t h 08 and 09 antigens, that they form t h i c k capsules and that they are free of amino s u g a r s . 2 7 The e x t r a c e l l u l a r A antigens of E. c o l i bear a strong s i m i l a r i t y to the K antigens of K l e b s i e l l a . 2 7 The present i n v e s t i g a t i o n i s thus a l o g i c a l extension and describes the i s o l a t i o n and s t r u c t u r a l a n a l y s i s of an a c i d i c polysaccharide obtained from E. c o l i 09:K28(A):H~. V.1.3 RESULTS AND DISCUSSION Isolation and characterization A c u l t u r e of E s c h e r i c h i a c o l i K28 was grown on Muel l e r Hinton agar and the i s o l a t i o n and p u r i f i c a t i o n of the polysaccharide were achieved as described i n Section I I I . 7 . 2 . The p u r i f i e d product obtained a f t e r Cetavlon p r e c i p i t a t i o n had [ot] D -18.2° which compares w e l l w i t h the c a l c u l a t e d value of -19.3° using Hudson's Rules of I s o r o t a t i o n , 1 1 * 8 and was shown to be heterogeneous by gel-permeation chromatography, con t a i n i n g two components w i t h molecular weight 9 x 10 6 daltons (30% by weight) and 450,000 daltons (70% by weight); the average molecular weight M^was 3 x 10 6 daltons. The polysaccharide was found to be homogeneous a f t e r mild a l k a l i treatment (M = 350,000 d a l t o n s ) . This i s w a well-known phenomenon f o r E. c o l i K antigens associated w i t h 0 groups 112 08, 09 and 0101. Capsular (K) antigens belonging to t h i s group form very viscous aqueous s o l u t i o n s . Treatment with d i l u t e a l k a l i reduces the v i s c o s i t y d r a s t i c a l l y . 1 8 6 ' 1 8 7 These f i n d i n g s i n d i c a t e the presence of i n t e r - c h a i n e s t e r linkages between c a r b o x y l i c groups of hexuronic a c i d c o n s t i t u e n t s and hydroxyl groups of sugar r i n g s . 1 8 6 ' 1 8 7 The presence of acetate groups could a l s o c o n t r i b u t e to the formation of aggregates, since the removal of acetate y i e l d s a homogeneous polysaccharide. The K28 polysaccharide i s composed of D-glucose, D-galactose, D-glucuronic a c i d and L-fucose. I t does not contain D-galacturonic a c i d and D-mannose as was e a r l i e r t h o u g h t . 1 8 8 The presence of glucose, ga l a c t o s e , fucose, g l u c u r o n i c a c i d and an al d o b i o u r o n i c a c i d i n the a c i d hydrolyzate of the polysaccharide was observed by paper chromatography. Determination of the n e u t r a l sugars as the a l d i t o l acetates gave fucose, galactose and glucose i n the r a t i o s of 0.45:1:0.76. The carboxyl-reduc-ed p o l y s a c c h a r i d e 7 5 gave fucose, galactose and glucose i n the r a t i o s of 1:1.1:1.58, i n d i c a t i n g that the uronic a c i d i s glucuro n i c a c i d . JH-N.m.r. spectroscopy The ^-H-n.m.r. spectrum of the E. c o l i K28 polysaccharide (see Appendix I I I , spectrum No. ) i n d i c a t e d the repeating u n i t to be a te t r a s a c c h a r i d e and to conta i n 50% of 0-acetyl groups (see Table V . l ) . The spectrophotometric 0-acetyl determination showed that 70% of the polysaccharide was a c e t y l a t e d w i t h one a c e t y l group per repeat u n i t . The spectrum e x h i b i t s a s i g n a l at 6 = 1.3 which a r i s e s from the CH 3 group of L-fucose. Two s i n g l e t s at 6 2.15 and 6 2.18 are due to the 113 TABLE V.l H^-N.M.R. DATA FOR ESCHERICHIA COLI K28 POLYSACCHARIDE Polysaccharide H^-N.m.r. data J l , 2 (Hz) Integral proton Assignment Native 5.41 4.93 4.86 4.48 4.44 2.18 2.15 1.30 b b b b s s b 2.0 } 1.0 1.0 1.0 -1 3.0 a-Glc a-Fuc B-GlcA 8-Gal r i n g proton CH 3 of 0- a c e t y l CH 3 of a-Fuc Na t i v e , a f t e r a u t o h y d r o l y s i s (100°, overnight) 5.41 5.39 4.94 4.83 4.48 4.42 2.18 2.15 1.30 1.0 1.0 } >1 1.0 1.0 } 1.4 6.0 ( J 5 6 ) 3.0 s 8.0 8.0 8.0 8.0 s s or Glc a-Fuc 8-GlcA 8-Gal r i n g proton CH 3 of 0_-acetyl CH 3of a-Fuc 114 Native, a f t e r carbodiimide r e d u c t i o n Deacetylated 5.43 s 5.37 s 4.95 b 4.85 b 4.47 b 2.20 s 2.16 s 1.31 b 5.41 s 5.39 s 4.83 8.1 4.48 6.. 4.42 6. 1.30 b } 2.0 } 0.76 2.0 } 0.6 3.0 1.0 1.0 1.0 1.0 1.0 3.0 a-Glc a-Fuc 6-Glc B-Gal r i n g proton CH 3 of 0 - a c e t y l CH 3 of a-Fuc a-Glc a-Fuc 6-GlcA 8-Gal r i n g proton CH 3 of a-Fuc Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; 6 2.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (D.S.S.). ^ Key: b = broad, unable to assign accurate coupling constant; s = s i n g l e t . 115 presence of OCOCH3 groups. The presence of twin s i g n a l s can be a t t r i b u -ted to the l o c a t i o n of 0-acetyl groups on both 0-2 and 0-3 p o s i t i o n s of fucose. In the spectrum of deacetylated E. c o l i K28 polysaccharide those s i g n a l s were absent. In the anomeric region four s i g n a l s were detected. The s i g n a l at 6 4.48 represents B-linked galactose. Two s i g n a l s at 6 4.86 and 6 4.93 belong to the 8-glucuronic acd. The twinn-i n g i s a t t r i b u t e d to the presence of 0-ac e t y l groups on the adjacent fucose, s i n c e , a f t e r d e a c e t y l a t i o n , i t disappeared g i v i n g r i s e to a s i g n a l at 6 4.83. A broad s i g n a l at 6 5.41 represents the two anomeric protons of a-L-fucose and o-D-glucose. Gen e r a l l y , the *H-n.m.r. spectrum was not w e l l resolved due to the extreme v i s c o s i t y of the s o l u -t i o n . The q u a l i t y of the spectrum was improved a f t e r a u t o h y d r o l y s i s of the polysaccharide (100°, o v e r n i g h t ) . A good spectrum was als o obtained a f t e r d e a c e t y l a t i o n of the polysaccharide due to the decrease i n v i s c o s i t y . 13C-N.m.r. spectroscopy In the 1 3C-n.m.r. spectra of the n a t i v e and 0_-deacetylated K28 polysaccharides (see Appendix I I I , Spectra No. 12) the s i g n a l s a r i s i n g from CH3C0 (21.39 p.p.m.) and CH3C=0 (175.62 p.p.m.) were absent i n the spectrum of the deacetylated polysaccharide (see Table V.2). The s i g n a l s f o r anomeric carbons i n the deacetylated polysaccharide were only s l i g h t l y changed i n p o s i t i o n (see Table V.2). Carbons 2 and 3 of the f u c o s y l residue e x h i b i t e d downfield s h i f t s (A6 = 2.9 p.p.m. f o r C-2 and A6 = 2.67 p.p.m. f o r C-3) i n the n a t i v e polysaccharide due to the presence of acetate. Once the acetate was removed the s i g n a l s f o r C-2 116 TABLE V.2 13C-N.M.R. DATA FOR THE NATIVE AND O-DEACETYLATED E. COLI K28 POLYSACCHARIDE Native polysaccharide O-Deacetylated polysaccharide a b p.p.m. Assignment p.p.m. Assignment 102.79 } C-1 C-1 6-GlcA B-Gal 103.91 102.81 C-1 C-1 B-GlcA B-Gal 99.30 99.26 C-1 C-1 a-Fuc a-Glc 99.32 } C-1 C-1 a-Fuc a-Glc 76.94 C-3 B-GlcA 77.97 C-3 B-GlcA 76.89 C-4 a-Fuc 76.88 C-4 a-Fuc 75.91 C-5 B-Gal 75.93 C-5 B-Gal 74.34 C-3 a-Glc 74.40 C-3 a-Glc 73.74 C-4 B-GlcA 73.74 C-4 B-GlcA 73.46 73.33 C-2 C-4 B-GlcA B-Gal 73.43 { C-2 C-4 B-GlcA p-Gal 72.27 C-4 a-Fuc 72.21 C-4 a-Fuc 72.14 C-3 a-Fuc 69.47 C-3 a-Fuc 72.10 C-2 a-Fuc 69.19 C-2 a-Fuc 69.11 unassigned 67.57 unassigned 62.28 C-6 a-Glc, 8-Gal 62.26 C-6 a-Glc, p-Gal 21.39 CH 3 of acetate 16.02 15.97 CH 3 of a-Fuc 16.12 16.06 } CH 3 of a-Fuc a Chemical s h i f t i n p.p.m. downfield from Me^Si, r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. downfield from DSS. ^ The assignments were made by comparison w i t h the l i t e r a t u r e v alues, see r e f . 166. 117 and C-3 carbons of fucose s h i f t e d u p f i e l d and are i n agreement w i t h the l i t e r a t u r e v a l u e s . 1 6 6 These data, together w i t h ^-n.m.r. f i n d i n g s , suggest that CHacetyl groups are d i s t r i b u t e d between 0-2 and 0-3 of the oc-L-fucosyl residues. The presence of 0-acetyl on 0-3 of fucose can e x p l a i n the s p l i t t i n g of the anomeric s i g n a l assigned to B-D-glucuronic a c i d . A molecular model (Dreiding) shows that only i n t h i s case i s there a p o s s i b i l i t y of i n t e r m o l e c u l a r I n t e r a c t i o n between H-l of gluc u r o n i c a c i d and the carbonyl group of the acetate (see F i g . V . l ) . F i g . V . l : P a r t i a l s t r u c t u r e o f E . c o l i K 2 8 p o l y s a c c h a r i d e These conclusions were r e i n f o r c e d by the r e s u l t s of periodate o x i d a t i o n of the na t i v e and deacetylated polysaccharides and the p o s i t i o n s of the 0-acetyl groups were determined by e t h y l a t i o n (see l a t e r ) . M e t h y l a t i o n a n a l y s i s M e t h y l a t i o n of the K28 polysa c c h a r i d e , followed by h y d r o l y s i s , d e r i v a t i z a t i o n as a l d i t o l a c e t a t e s , and g.l.c.-m.s. a n a l y s i s , gave the values shown i n Table V.3, column I . These r e s u l t s i n d i c a t e d that the polysaccharide c o n s i s t s of a t e t r a s a c c h a r i d e repeating u n i t having a branch on glucose, w i t h galactose as the ter m i n a l group. By r e d u c t i o n 118 TABLE V.3 METHYLATION ANALYSIS OF Escherichia c o l i K28 POLYSACCHARIDE AND DERIVED PRODUCTS 3, Methylated sugar (as a l d i t o l acetate) Mol % b I C - • II III IV V 2,3,4-Fuc - - - 42 -' 2,3-Fuc 23 28 26 - 21 2,3,4,6-Glc - - - 20 -2,3,4,6-Gal 37 22 24 . - -2,4,6-Glc - - - 38 -2,3,6-Glc - - 18 - -2,6-Glc 40 26 31 - 79 2,3-Glc - 23 - - -a 2,3,4-Fuc=l,5-di-C^-acetyl-2,3,4-tri-0-methylfucitol, e t c . Values are cor r e c t e d by use of the e f f e c t i v e , carbon-response f a c t o r s given by Albersheim et a l . 1 0 3 using an OV-225 column, programmed from 180° f o r 4 min, and then at 2°/min to 230°. c I , o r i g i n a l polysaccharide; I I , compounds from L i A l H ^ r e d u c t i o n of methylated E. c o l i K28; I I I , carbodiimide-reduced polysaccharide; IV, product from Smith degradation of the o r i g i n a l polysaccharide; V, product from periodate o x i d a t i o n of deacetylated polysaccharide. 119 of the methylated polysaccharide (see Table V.3, column I I ) , the p r o p o r t i o n of 2,3-di-O-methylfucose was increased, and 2,3-di-O-methyl-glucose was formed, i n d i c a t i n g that g l u c u r o n i c a c i d i s l i n k e d through 0-4, and that i t i s j o i n e d to fucose. M e t h y l a t i o n a n a l y s i s of c a r b o d i -imide-reduced p o l y s a c c h a r i d e 7 5 showed the presence of 2 , 3 , 6 - t r i - 0 -methylglucose, derived from reduction of the carboxyl group of the g l u c u r o n i c a c i d (see Table V.3, column I I I ) . Partial hydrolysis P a r t i a l h y d r o l y s i s of the n a t i v e polysaccharide w i t h a c i d was followed by separation of the a c i d i c and the n e u t r a l f r a c t i o n s by i o n -exchange chromatography. The n e u t r a l f r a c t i o n contained monosaccharides and a d i s a c c h a r i d e ( N l ) . The a c i d i c f r a c t i o n contained an a l d o b i o u r o n i c a c i d ( A l ) . On the basis of t h e i r n.m.r. s p e c t r a l data (Table V.4) and t h e i r methylation a n a l y s i s (Table V.5), the s t r u c t u r e s of these compounds were shown to be as f o l l o w s : A l 8-GlcA-(1^4)-Fuc Nl B-Gal-(l->4)-Glc Periodate oxidation Smith degradation of the n a t i v e polysaccharide, followed by methylation and h y d r o l y s i s showed the presence of 2,3,4-tri-O-methyl-fucose, 2,3,4,6-tetra-0-methylglucose and 2,4,6-tri-O-methylglucose (see Table V.3, column I V ) . These r e s u l t s show that terminal galactose and the g l u c u r o n i c a c i d were completely o x i d i z e d thus i n d i c a t i n g the absence 120 TABLE V.4 N.M.R. DATA FOR Escherichia c o l l K28 OLIGOSACCHARIDES DERIVED FROM PARTIAL HYDROLYSIS OF THE POLYSACCHARIDE Compound *H-N.m.r. data "1,2 (Hz) Integral proton Assignment 13C-N.m.r. data c d P.p.m. Assignment GlcA-^Fuc-OH P A l 5.24 4.63 4.54 4.29 1.33 1.29 2.7 5.4 8.0 q 6.75 6.75 0.3 0.7 1.0 1.0 } 3.0 4-Fuc OH a 4-Fuc—r-OH 8 GlcA-B H-5 of Fuc CH 3 of 8-Fuc-OH CH 3 of a-Fuc-OH 103.89 G1CA-T-P 97.04 4-Fuc-s-OH P 93.11 4-Fuc- -0H 16.23 CH 3 of Fuc G a l ^ G l c - O H P Nl 5.23 4 0.4 4-Glc-4.67 7 0.6 4-Gle 4.45 7.5 1.0 Gal p 103.83 Gal-P 96.62 4-Glc- -OH 92.68 4-Glc- -0H Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; 6 2.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (D.S.S.). ^ The numerical p r e f i x i n d i c a t e s the p o s i t i o n i n which the sugar i s s u b s t i t u t e d ; the a or 8, the c o n f i g u r a t i o n of the g l y c o s i d i c bond, or the anomer i n the case of a (term i n a l ) reducing-sugar residue. Chemical s h i f t i n p.p.m. downfield from Me^Si, r e l a t i v e to i n t e r n a l acetone; 31.07 p.p.m. downfield from D.S.S. ^ As i n c, but f o r 1 3C n u c l e i . 121 TABLE V.5 ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF Escherichia c o l l K28 POLYSACCHARIDE Oligosaccharide Sugar As a l d i t o l Methylation As a l d i t o l analysis acetates analysis acetates (molar (molar proportions) proportions) A l Fuc 1.0 Glc(GlcA) 0.8 Nl Gal 1.0 2,3,4,6-Gal 0.95 Glc 0.9 2,3,6-Glc 1.00 122 of an 0-a c e t y l group on e i t h e r of them. The f u c o s y l linkage was hydrolyzed during the Smith degradation g i v i n g r i s e to 2,3,4,6-tetra-O-methylglucose, proving that fucose and glucose are engaged i n a 1+3 l i n k a g e . The pr o p o r t i o n of 2,3,4-tri-O-methylfucose found i n d i c a t e s that 70% of the fucose survived periodate o x i d a t i o n . This r e s u l t suggests that 70% of the f u c o s y l l i n k a g e s are O-acetylated at 0-2 or 0-3 and are thus protected against o x i d a t i v e degradation. Periodate o x i d a t i o n of deacetylated polysaccharide followed by methylation and h y d r o l y s i s showed that only 25% of the fucose had survived (see Table V.3, column V). The incomplete o x i d a t i o n of fucose could be explained on the basis of s t e r i c hindrance and p o s s i b l e hemiacetal f o r m a t i o n . 1 3 3 Quantitative determination of Q-acetyl groups The percentage of 0-a c e t y l groups present i n E. c o l i K28 polysaccharide was determined spectrophotomet r i c a l l y . 1 7 8 I t was found that 72-74% of the n a t i v e polysaccharide was O-acetylated. The acetate groups could be e a s i l y removed by mild a l k a l i treatment. These f i n d i n g s are i n a very good agreement w i t h the r e s u l t s obtained a f t e r periodate o x i d a t i o n of the a c e t y l a t e d and deacetylated polysaccharides. P a r t i a l removal of 0-acetyl groups during the high temperature n.m.r. can be explained on the basis of the l a b i l i t y of the 0-acetyl groups located on the f u c o s y l r e s i d u e s . 123 L o c a t i o n of P j-acetyl g r o u p s 1 8 0 O-Acetyl groups i n the polysaccharide were l o c a t e d 1 3 by r e a c t i o n w i t h methyl v i n y l ether and an a c i d i c c a t a l y s t , followed by e t h y l a t i o n a n a l y s i s of the product. I t was found that e i t h e r 0-2 or 0-3 of the a-L-fucosyl residues i s a c e t y l a t e d but no 2,3-di-O-ethylfucose was obtained. V.. 1.4 CONCLUSION From the sum of these experiments the complete s t r u c t u r e of the capsular polysaccharide from E s c h e r i c h i a c o l i K28 may be w r i t t e n . +3 ) - <x-D-Glc-( 1 ->4)- 8-D-GlcA-( 1+4)-a-L-Fuc~( 1 -> 4 2 or 3 1 OAc 8-D-Gal This s t r u c t u r e c l o s e l y resembles that of the capsular antigens from E_. c o l i K 2 7 1 8 6 ' 1 8 9 and i s of the same s t r u c t u r a l p a t t e r n as that of the capsular polysaccharide from K l e b s i e l l a K 5 4 . 8 9 , 1 9 0 124 +4) - o-Glc-(1+4)-a-GlcA-(1+3)-a-Fuc-(1+ 3 + 1 a-Gal E. c o l i K27 +3)-S-Glc-(l-»4)-a-GlcA-(l+3)-a-Fuc(l+ 4 2 • I 1 OAc 8-Glc K l e b s i e l l a K54 125 V . l . 5 EXPERIMENTAL General methods The instrumentation used f o r i n f r a r e d and n.m.r. spectroscopy, g . l . c , and g.l.c.-m.s., c.d., and measurements of o p t i c a l r o t a t i o n has been described i n Sectio n I I I . Spectrophotometric measurements were made by using a Perkin-Elmer 552A UV/VIS spectrophotometer. Paper chromatography, g a s - l i q u i d chromatography, and ion-exchange chromato-graphy were performed as described i n Section I I I . Preparation and properties of E. c o l i K28 capsular polysaccharide A c u l t u r e of E. c o l i K28 was obtained from Dr. Ida 0rskov (Copenhagen). The b a c t e r i a were f i r s t grown on s u c r o s e - r i c h medium as p r e v i o u s l y described f o r K l e b s i e l l a capsular polysaccharides. The r e s u l t s were not s a t i s f a c t o r y and the y i e l d of polysaccharide was very poor. In order to f i n d s u i t a b l e growth c o n d i t i o n s s i x d i f f e r e n t media were t r i e d : (1) t r y p t i c a s e soy agar (BBL); (2) L u r i a broth ( D i f c o ) ; (3) n u t r i e n t broth ( D i f c o ) ; (4) n u t r i e n t broth + yeast e x t r a c t ( D i f c o ) ; (5) beef heart i n f u s i o n ( D i f c o ) ; (6) Mueller Hinton agar (BBL). Sodium c h l o r i d e improves the growth of E. c o l i 1 9 1 and was used i n prepa r a t i o n of a l l s i x media (0.5% w/v). The streaked p l a t e s were incubated at 37° overnight. The best r e s u l t s were obtained on Mueller Hinton agar and i t was l a t e r used as a growth medium w i t h the a d d i t i o n of a small amount of NaCl (0.5% w/v). The p u r i f i c a t i o n procedure was c a r r i e d out as described i n Section I I I . 7 . 2 . Y i e l d : a c i d i c polysaccharide ~ 400 mg, n e u t r a l p o l y -126 saccharide ~ 100 mg (per batch). Three d i f f e r e n t batches of the polysaccharide were grown. The molecular weight of the polysaccharide was determined by g e l -permeation chromatography (courtesy of Dr. S.C. Churms, U n i v e r s i t y of Cape Town, South A f r i c a ) . The n a t i v e polysaccharide was shown to be heterogeneous, but became homogeneous a f t e r m i l d a l k a l i treatment (M = w 350,000 d a l t o n s ) . N.m.r. spectroscopy ( l H and 1 3C) was performed on the o r i g i n a l and d e a c e t y l a t e d K28 polysaccharide. The p r i n c i p a l s i g n a l s i n the *H- and 1 3C- n.m.r. spectra and t h e i r assignments are recorded i n Tables V . l and V.2 r e s p e c t i v e l y . Deacetylation of polysaccharide The polysaccharide was d i s s o l v e d i n 0.01M NaOH and s t i r r e d overnight at room temperature. The product was d i a l y z e d against tap water and f r e e z e - d r i e d . The completeness of d e a c e t y l a t i o n was checked by *H-n.m.r. spectroscopy. Hydrolysis of the polysaccharide H y d r o l y s i s of a sample (4 mg) of K28 polysaccharide w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) f o r 18 h at 95°, removal of the a c i d by successive evaporations w i t h water, followed by paper chromatography (s o l v e n t s 1 and 2) showed fucose, glucose, galactose, g l u c u r o n i c a c i d , and an a l d o b i o u r o n i c a c i d . Q u a n t i t a t i v e sugar a n a l y s i s of the carboxyl-reduced polysaccharide was performed, and the a l d i t o l acetates of fucose, glucose, and galactose were i d e n t i f i e d by g . l . c . (column A) 127 and found to be present i n the r a t i o s of 1:1.1:1.58. Preparative g . l . c . (column D), followed by measurements of the c i r c u l a r dichroism s p e c t r a , 7 8 showed the g l u c i t o l hexaacetate to be of the D c o n f i g u r a t i o n , and the f u c i t o l pentaacetate to be of the L c o n f i g u r a t i o n . Galactose was assigned the D c o n f i g u r a t i o n by the p o s i t i v e a c t i o n of D-galactose oxidase (Worthington Biochem. Co.) on the h y d r o l y s i s product of the p o l y s a c c h a r i d e . 1 9 2 Methylation analysis The capsular polysaccharide (60 mg) i n the f r e e - a c i d form, obtained by passing the sodium s a l t through a column of Amberlite IR-120 (H +) r e s i n , was d i s s o l v e d i n dry dimethyl s u l f o x i d e (6 mL) and methylated by the Hakomori p r o c e d u r e 8 8 . The product, recovered a f t e r d i a l y s i s against tap water, was not completely methylated (hydroxyl absorption i n the i . r . spectrum). I t was d i s s o l v e d i n chloroform and subjected to Purdie m e t h y l a t i o n 8 1 * w i t h methyl i o d i d e and s i l v e r oxide. This treatment y i e l d e d a f u l l y methylated polysaccharide (57 mg). A p o r t i o n of t h i s product (5 mg) was hydrolyzed 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 sugars were reduced w i t h sodium borohydride, the a l d i t o l s were ac e t y l a t e d w i t h 1:1 a c e t i c anhydride — p y r i d i n e , and analyzed by g . l . c . i n column C (see Table V.3, column I ) . Carboxyl reduction of the f u l l y methylated polysaccharide (12.1 mg) w i t h L i A l H ^ i n anhydrous oxolane at room temperature overnight, h y d r o l y s i s of the product w i t h 2M t r i f l u o r o a c e t i c a c i d , followed by reduction of sugars w i t h sodium-borohydride, and a c e t y l a t i o n of the a l d i t o l s w i t h 1:1 a c e t i c anhydride-p y r i d i n e gave a mixture of p a r t i a l l y methylated a l d i t o l acetates which 128 was analyzed by g . l . c . and g.l.c.-m.s. i n column C (see Table V.3, column I ) . The n e u t r a l polysaccharide obtained by carbodiimide r e d u c t i o n 7 5 was a l s o subjected to methylation a n a l y s i s . A sample (9.4 mg) of carbodiimide-reduced polysaccharide was d i s s o l v e d i n dry dimethyl s u l f o x i d e (1 mL) and methylated by treatment w i t h 1 mL d i m e t h y l s u l f i n y l anion f o r 4 h, and then 2 mL methyl i o d i d e f o r 1 h. The product recovered a f t e r d i a l y s i s against tap water, was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d overnight at 95°, converted i n t o p a r t i a l l y methyla-ted a l d i t o l acetates and analyzed, w i t h the r e s u l t s shown i n Table V.3, column I I I . Carbodiimide reduction of the capsular polysaccharide 7 5 A sample of K28 polysaccharide ( N a + s a l t , 36.2 mg) was d i s s o l v e d i n 20 mL water. l-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC, 0.4 g) was added. As the r e a c t i o n proceeded, the pH was maintained at 4.75 by t i t r a t i o n w i t h 0.1 N HC1. The r e a c t i o n was allowed to proceed f o r at l e a s t two h. When the consumption of h y d r o c h l o r i c a c i d ceased, an aqueous s o l u t i o n of 2M sodium borohydride (1 g/15 mL H 20) was added s l o w l y . The pH rose r a p i d l y to 7.0 and i t was maintained between 5-7 by t i t r a t i o n w i t h 4M HC1. A drop of 2-octanol (antlfoaming agent) was added p e r i o d i c a l l y to c o n t r o l the amount of foam. The reduction was completed i n one h. The mixture was d i a l y z e d against tap water during 24 h, concentrated and f r e e z e - d r i e d . A second treatment was c a r r i e d out. Y i e l d : 32.3 mg. A sample of the reduced polysaccharide (11.1 mg) was hydrolyzed overnight w i t h 2M t r i f l u o r o -129 a c e t i c a c i d at 95°, and the hydrolyzate was converted i n t o a l d i t o l acetates as before. G.l.c. a n a l y s i s was conducted i n column A. P a r t i a l hydrolysis The K28 polysaccharide (514 mg) was d i s s o l v e d i n 125 mL of 0.01M t r i f l u o r o a c e t i c a c i d (TFA) and the s o l u t i o n was heated f o r 32 h on a steam bath. A f t e r removal of the a c i d by successive evaporations w i t h water, an a c i d i c and a n e u t r a l f r a c t i o n were separated on a column of Bio-Rad AG1-X2 ion-exchange r e s i n . The a c i d i c f r a c t i o n was separated by prepa r a t i v e paper chromatography (solvent 3 ) , to give 45.7 mg of a pure al d o b i o u r o n i c a c i d ( A l ) . Paper chromatography of the n e u t r a l f r a c t i o n showed fucose, glucose, galactose and a n e u t r a l disaccharide (Nl^), which was i s o l a t e d by pr e p a r a t i v e paper chromatography (solvent 3) to give 21 mg of N l . P.m.r. s p e c t r a l data are recorded (Table V.4) f o r each o l i g o s a c c h a r i d e and analyses were performed as f o l l o w s : (a) Sugar a n a l y s i s . The a c i d i c o l i g o s a c c h a r i d e was tr e a t e d w i t h 3% HC1 i n anhy-drous methanol f o r 18 h on a steam bath. The methyl ester methyl g l y c o -side obtained was reduced w i t h sodium borohydride i n anhydrous methanol, followed by h y d r o l y s i s w i t h 2M TFA, reduction to the a l d i t o l s , and a c e t y l a t i o n w i t h 1:1 a c e t i c a n h y d r i d e — p y r i d i n e . The a l d i t o l acetates obtained were analyzed by g . l . c . i n column A. The n e u t r a l o l i g o s a c c h a -r i d e was hydrolyzed, and analyzed s i m i l a r l y , (b) Me t h y l a t i o n a n a l y s i s . Compound Nl was methylated by the method of Hakomori 8 8 and the r e s u l t s given i n Table V.5. 130 Periodate oxidation A s o l u t i o n of K28 polysaccharide (21.5 mg) i n water (10 mL) was mixed w i t h 0.03M NalO^ (10 mL) and s t i r r e d i n the dark at room tempera-ture (23°) f o r 6 d. A f t e r ethylene g l y c o l (2 mL) was added, the polyaldehyde was reduced to the p o l y a l c o h o l w i t h NaBH^, the base was n e u t r a l i z e d w i t h 50% AcOH, the s o l u t i o n was d i a l y z e d overnight, and f r e e z e - d r i e d to y i e l d the p o l y a l c o h o l (15 mg). A p o r t i o n (4.2 mg) was hydrolyzed wth 2M TFA overnight on a steam bath and converted i n t o a l d i t o l acetates. A n a l y s i s by g . l . c . i n column A showed f u c i t o l , g a l a c -t i t o l and g l u c i t o l i n the r a t i o s of 0.72:0.10:1. The remainder of the m a t e r i a l was tr e a t e d w i t h 0.5M TFA f o r 48 h at room temperature. The product (10.7 mg) was methylated by the Hakomori p r o c e d u r e , 8 8 hydrolyzed w i t h 2M TFA overnight on a steam bath and converted i n t o a l d i t o l a c etates. G.l.c. a n a l y s i s , conducted i n column B showed the presence of 2,3,4-tri-0-methylfucose, 2,3,4,6-tetra-0-methylglucose and 2 , 4 , 6 - t r i -0-methylglucose i n the r a t i o s of 0.70:0.35:0.65. These r e s u l t s i n d i c a t e that during the Smith degradation p a r t i a l h y d r o l y s i s of the f u c o s y l l inkage has occurred. Periodate o x i d a t i o n of deacetylated polysaccharide was performed s i m i l a r l y . A f r a c t i o n of the p o l y a l c o h o l product (1.6 mg) was analyzed f o r c o n s t i t u e n t sugars and the remainder of the m a t e r i a l was methylated by the Hakomori p r o c e d u r e . 8 8 Conversion of the p a r t i a l l y methylated sugars i n t o a l d i t o l a c e t a t e s , and g . l . c . thereof i n column B, showed the presence of 2,3-di-0-methylfucose and 2,6-di-0-methylglucose i n the r a t i o s 0.25:1.00. 131 Quantitative determination of 0,-acetyl groups 1 7 8 O-Acetyl groups were determined s p e c t r o p h o t o m e t r i c a l l y . The r e a c t i o n of 0-acetyl groups w i t h hydroxylamine In a l k a l i to form hydroxamic a c i d was employed. The hydroxamic a c i d was measured by the formation of a colored complex w i t h F e 3 + i n a c i d s o l u t i o n . To 1 mL of s o l u t i o n which contained 40 ug of the E. c o l i K28 polysaccharide i n 0.001M sodium acetate b u f f e r 2 mL of a f r e s h l y prepared 1:1 mixture of 2M hydroxylamine hydrochloride-3.5M NaOH was added. A f t e r 2 min at room temperature 1 mL of 12% h y d r o c h l o r i c a c i d was added followed by 1 mL of 0.37M FeCl 3»6H 20 i n 0.1N HC1. During the a d d i t i o n of each reagent the s o l u t i o n was s w i r l e d r a p i d l y . The absorbance at 540 nm of the r e s u l t i n g purple-brown s o l u t i o n was measured i n a Perkin-Elmer 552A UV/VIS spectrophotometer. A f t e r c o r r e c t i n g f o r n o n - s p e c i f i c c o l o r , the q u a n t i t y of 0-acetyl w a s c a l c u l a t e d from a standard curve prepared using 0.004M a c e t y l c h o l i n e c h l o r i d e i n 0.001M sodium acetate pH 4.5. Location of Q-acetyl groups 1 8 0 E. c o l i K28 polysaccharide (17.6 mg) and p_-TsOH (5 mg) were d r i e d overnight under vacuum and d i s s o l v e d i n dry dimethyl s u l f o x i d e (10 mL). Methyl v i n y l ether (3 mL) was added to the frozen s o l u t i o n , and the r e a c t i o n mixture was brought to room temperature and allowed to s t i r f o r 4 h. Then a second p o r t i o n of methyl v i n y l ether (3 mL) was introduced i n a s i m i l a r manner. The c l e a r , red s o l u t i o n obtained was placed on a Sephadex LH-20 column (58 cm x 1.5 cm) and e l u t e d w i t h acetone ( w i t h s l i g h t s u c t i o n ) . The product was c o l l e c t e d and concentrated. H a l f of the residue was used f o r e t h y l a t i o n according to the Hakomori 132 p r o c e d u r e . 8 8 The product, a dark red-orange o i l , was d i a l y z e d ( c u t o f f 3,500) overnight against tap water and ex t r a c t e d w i t h chloroform. The p o r t i o n of e t h y l a t e d , methyl v i n y l ether protected polysaccharide was hydrolyzed w i t h 2M TFA overnight at 95° and converted i n t o a l d i t o l a c e t a t e s . G.l.c. a n a l y s i s , conducted i n column B (210° isothermal) showed the presence of 2-0-ethy1fucose (16.9%), 3-0-ethylfucose (23.8%), fucose (12.7%), galactose (25.1%), and glucose (21.6%). These r e s u l t s were confirmed by g.l.c.-m.s., which was performed on a KRATOS MS80RFA instrument, using DB-225 c a p i l l a r y column (150° f o r 1 min, and then 10°/min to 210°). 133 V.2 STRUCTURAL INVESTIGATION OF E s c h e r i c h i a c o l l 09:K32(A):H19 (K32 ANTIGEN) CAPSULAR POLYSACCHARIDE V.2.1 ABSTRACT The s t r u c t u r e of the capsular polysaccharide from E s c h e r i c h i a  c o l i 09:K32(A):H19 (K32 antigen) has been determined by using the techniques of methylation, carboxyl reduction and Smith degradation. The nature of the anomeric linkages was e s t a b l i s h e d by using 1H- and 1 3C-n.m.r. spectroscopy, and was f u r t h e r confirmed by chromium t r i o x i d e o x i d a t i o n of the f u l l y a c e t y l a t e d polysaccharide. The l o c a t i o n of 0-acetyl groups was determined using methyl v i n y l ether as a p r o t e c t i v e reagent. The polymer was found to c o n s i s t of the t e t r a s a c c h a r i d e repeating u n i t shown (three-plus-one type) w i t h h a l f of the L-rhamnosyl residues being O-acetylated at 0-2. OAc I 2 ->3)-<x-D-Glc-( l+4)-oc-L-Rha-( l+3)-a-D-Gal-( l-> 3 1 B-D-GlcA 134 V.2.2 INTRODUCTION E s c h e r i c h i a c o l i serotype K32 i s of i n t e r e s t i n t h i s s e r i e s , being a s p e c i f i c host f o r three d i f f e r e n t E. c o l i capsular b a c t e r i o -phages <p28—1, <(>31 and <J>32.193 Because of the i n c r e a s i n g importance of s t u d i e s on the substrate s p e c i f i c i t y of the bacteriophage-borne enzymes, the complete s t r u c t u r a l a n a l y s i s of E. c o l i polysaccharide i s needed. Although the composition of the E. c o l i K32 capsule i s known, 2 2 i t s s t r u c t u r e i s not. In c o n t i n u a t i o n of our chemical examination of t h i s genus, we now report the primary s t r u c t u r e of E s c h e r i c h i a c o l l K32. V.2.3 RESULTS AND DISCUSSION A c u l t u r e of E s c h e r i c h i a c o l i K32 was grown on M u e l l e r Hinton agar, and the a c i d i c polysaccharide was p u r i f i e d by one p r e c i p i t a t i o n w i t h cetyltrimethylammonium bromide. The polysaccharide obtained had [<x]p +80.9° which compares w e l l w i t h the c a l c u l a t e d value of +71.5° using Hudson's Rules of I s o r o t a t i o n . i l + 8 I t was shown to be homogeneous by gel-permeation chromatography w i t h M^ =9 x 10 6 d a l t o n s . Treatment w i t h d i l u t e a l k a l i d i d not reduce the v i s c o s i t y of the polysaccharide s i g n i f i c a n t l y (M = 6 x 10 6 d a l t o n s ) . w Paper chromatography of an a c i d hydrolyzate of the polysaccharide showed ga l a c t o s e , glucose, g l u c u r o n i c a c i d , rhamnose and an a l d o b i o -u r o n i c a c i d . Determination of the n e u t r a l sugars as the a l d i t o l 135 acetates gave rhamnose, gala c t o s e , and glucose i n the r a t i o s of 0.7:1.0:1.5. The carboxyl-reduced polysaccharide gave rhamnose, galactose, and glucose i n the r a t i o s of 0.9:1.0:1.9, i n d i c a t i n g that the uronic a c i d i s gl u c u r o n i c a c i d . The glucose was proved to be of the D and rhamnose to be of the L c o n f i g u r a t i o n by c i r c u l a r dichroism measurements 7 8 made on the a l d i t o l acetates. Galactose was assigned the D c o n f i g u r a t i o n by the p o s i t i v e a c t i o n of D-galactose o x i d a s e 1 9 2 on the h y d r o l y s i s product of the polysaccharide. N.m.r. spectroscopy The 1H-n.m.r. spectrum of the E. c o l i K32 polysaccharide (see Appendix I I I , spectrum No. 2 0 ) i n d i c a t e d the repeating u n i t to be a te t r a s a c c h a r i d e and to contain 0.5 0-acetyl groups. The spectrum e x h i b i t s a s i g n a l at 6 = 1.34 which a r i s e s from the CH 3 group of L-rhamnose. The s i g n a l at 6 = 2.18 Is due to the presence of 0C0CH 3 groups. In the anomeric region s i x p r i n c i p a l s i g n a l s can be detected. The s i g n a l at 6 = 4.73 represents (3-linked g l u c u r o n i c a c i d which was f u r t h e r confirmed by chromic a c i d o x i d a t i o n of the f u l l y a c e t y l a t e d polysaccharide (see l a t e r ) . The s i g n a l at 6 = 5.52 belongs to the a-rhamnosyl residue bearing an 0-acetyl group, and i t s h i f t s to 6 = 5.25 on the d e a c e t y l a t i o n of the polysaccharide. This suggests that the 0-acetyl group i s loc a t e d on ()-2 of the a-rhamnosyl residue. The s i g n a l at 6 = 5.11 belongs probably to a-Glc and i t remains unchanged a f t e r O-deacetylation of the polysaccharide. The twinned s i g n a l s at 6 = 5.19 and 6 = 5.16 disappear on d e a c e t y l a t i o n g i v i n g r i s e to a s i n g l e t at 6 = 5.20. However, the d e f i n i t i v e assignment of a-Glc and a-Gal cannot be 136 made on the basis of the ^ -n.m.r. s p e c t r a l data (see Table V.6). In the 1 3C-n.m.r. spectra of the na t i v e and O-deacetylated E.  c o l i K32 polysaccharides (see Appendix I I I , Spectra No. 21 and 23) the s i g n a l a r i s i n g from CH^CO (21.18 p.p.m.) was absent i n the spectrum of the deacetylated polysaccharide. The p o s i t i o n s of the anomeric s i g n a l s were changed. Carbon 1 of the a-rhamnosyl residue e x h i b i t e d a d e f i n i t e downfield s h i f t due to the presence of the acetate i n the n a t i v e polysaccharide. However, the unambiguous assignment of the anomeric carbons could not be made (see Table V.6). ' Methylation analysis M e t h y l a t i o n a n a l y s i s of the E. c o l i K32 polysaccharide, followed by h y d r o l y s i s , d e r i v a t i z a t i o n as a l d i t o l a cetates, and g.l.c.-m.s. a n a l y s i s , gave the values shown i n Table V.7, column I . These r e s u l t s i n d i c a t e that the polysaccharide c o n s i s t s of a te t r a s a c c h a r i d e repeat-ing u n i t having a branch on rhamnose w i t h g l u c u r o n i c a c i d as the ter m i n a l group. By reduction of the methylated polysaccharide (see column I I ) , the pro p o r t i o n of 2-0-methylrhamnose was increased, and 2,3,4-tri-0_-methylglucose was formed, i n d i c a t i n g that g l u c u r o n i c a c i d i s t e r m i n a l , and that i t i s l i n k e d to rhamnose. Me t h y l a t i o n a n a l y s i s of carbodiimide-reduced p o l y s a c c h a r i d e 7 5 showed the presence of 2,3,4,6-tetra-O-methylglucose, derived from reduction of the c a r b o x y l i c group of the glucu r o n i c a c i d (see Table V.7, column I I I ) . 137 TABLE V.6 N.M.R. DATA FOR Escherichia c o l i K32 NATIVE AND O-DEACETYLATED POLYSACCHARIDES Compound -^N.m. r. . data 13C-N.m.r, . data . A* Integral (H) Assignment P.p.m.*1 Assignment native 5.52 5.25 1.0 0.8 a-Rha (with 0-Ac) a-Rha 173.85 COOH of B-GlcA 5.19 1 5.16 1.8 c a-Gal 102.98 } 8-GlcA a-Rha a-Gal 5.11 1.9 a-Glc C 95.97 a-Glc C 5.05 , 5.02 I 1.1 unknown origin 21.18 17.99 CH3 of 0-Ac CH3 of a-Rha 4.73 ( J 1 2=8Hz) 9 1.8 B-GlcA 2.18 3.0 CH3 of 0-Ac 1.34 6.1 CH3 of a-Rha 3-deacetylated 5.52 0.2 a-Rha (with 0-Ac) 102.97 B-GlcA 5.25 0.8 a-Rha 101.68 } a-Rha^ a-Gal C 5.20 0.9 a-Gal C 96.66 a-Glc C 5.11 1.0 a-Glc C 18.01 CH3 of a-Rha 4.73 (J, 2=8Hz) 0.9 B-GlcA 2ll8 0.5 CH3 of 0-Ac 1.34 3.2 CH3 of a-Rha 3 Chemical shift relative to internal acetone; 6 2.23 for ^ -H-n.m.r. and 31.07 p.p.m. for 13C-n.m.r. downfield from sodium 4,4-dimethyl-4-sila-pentane-l-sulfonate (D.S.S.). The assignment was made by comparison with literature values, see ref. 166. A definite assignment could not be made; the assignments of a-Glc and a-Gal are tentative. 138 TABLE V.7 METHYLATION ANALYSIS OF ESCHERICHIA COLI K32 POLYSACCHARIDE AND DERIVED PRODUCTS Methylated sugar (as a l d i t o l acetate) 2,3-Rha e 0.99 , ~. 42.7 2,3,4,6-Glc 1.00 - - 26.5 -2,3,4,6-Gal 1.24 - - - 17.5 2-Rha 1.55 32.5 16.4 26.5 10.3 2,4,6-Glc 1.96 53.6 38.7 23.5 29.3 2,4,6-Gal 2.26 13.6 25.7 23.5 -2,3,4-Glc 2.49 19.2 R e l a t i v e r e t e n t i o n time, r e f e r r e d to 2,3,4,6-Glc as 1.00. b Values corrected by use of the e f f e c t i v e , carbon-response f a c t o r s given by Albersheim et a l . 1 0 3 C Isothermal; 170°. d I , o r i g i n a l p o l y s a c c h a r i d e ; I I , reduction of uro n i c e s t e r ; I I I , carbodiimide-reduced polysaccharide; IV, product from Smith degradation. e 2,3-Rha = 1 , 4 , 5 - t r i - 0 - a c e t y l - 2 , 3 -di-O-methylrhamnitol, e t c . T a Mol % b Column B C I d II III IV (ECNSS-M) 139 Chromium t r i o x l d e o x i d a t i o n 1 7 a The anomeric nature of the glucuronic acid was confirmed by chromium trioxide oxidation of the fully acetylated polysaccharide, followed by sugar analysis. 8-Linked residues should be oxidized under these conditions, but the corresponding a-linked residues should be resistant. Determination, as a l d i t o l acetates, of the sugars obtained from the chromium trioxide oxidation gave rhamnose, galactose, and glucose in the molar ratios of 0.9:1.0:0.5 indicating that D-glucuronic acid i s B-linked. The following two structures are consistent with the results obtained so far. +3)-o-Glc-(l+3 or 4)-a-Rha-(l->-3)-a-Gal-(l+ 4 or 3 + 1 + 0-Ac B-GlcA +3)- a-Gal-(1+3 or 4)-a-Rha-(l+3)-a-Glc-(1+ 4 or 3 + 1 + 0-Ac B-GlcA B 140 Periodate oxidation Periodate o x i d a t i o n of the o r i g i n a l E. c o l i K32 polysaccharide followed by h y d r o l y s i s and g . l . c . a n a l y s i s of the a l d i t o l acetates gave rhamnose, galactose and glucose i n the molar r a t i o s of 1.0:0.5:1.0, i n d i c a t i n g that p a r t i a l h y d r o l y s i s of the rhamnosyl bond with f u r t h e r degradation of galactose had occurred. The o x i d a t i o n was then repeated on the carbodiimide-reduced p o l y s a c c h a r i d e 7 5 i n the presence of 0.1M sodium acetate b u f f e r pH 4.5. G.l.c. a n a l y s i s , as a l d i t o l a c e t a t e s , of the o x i d i z e d and borohydride reduced product gave rhamnose, gala c t o s e , and glucose i n the r a t i o s of 0.9:1.0:1.0. These r e s u l t s are c o n s i s t e n t w i t h the concept that only one te r m i n a l residue ( g l u c u r o n i c a c i d ) i s o x i d i z e d . Smith degradation Periodate o x i d a t i o n of the o r i g i n a l p olysaccharide, followed by methylation, Smith h y d r o l y s i s , remethylation, and h y d r o l y s i s , gave a mixture that was found, by g . l . c . a n a l y s i s of the a l d i t o l a c e t a t e s , t o co n t a i n d e r i v a t i v e s of 2,3-di-0-methylrhamnose, 2,3,4,6-tetra-O-methyl-galactose, 2-0-methylrhamnose, and 2,4,6-tri-0-methylglucose (see Table V.7, column IV). These r e s u l t s i n d i c a t e that the glucose i n the main chain i s 4-linked to rhamnose, the branch p o i n t . The rhamnosyl l i n k a g e was hydrolyzed during the Smith h y d r o l y s i s g i v i n g r i s e to 2,3,4,6-tetra-CHmethylgalactose, proving that the rhamnose i n the main chain i s l i n k e d to the galactose and not the glucose. The unusual i n s t a b i i t y of the 2,4,6-tri-O-methylgalactose was n o t i c e d throughout the s t r u c t u r a l 141 i n v e s t i g a t i o n of the E. c o l i K32 polysaccharide, e s p e c i a l l y i n the methylation analyses of the o r i g i n a l , a c i d i c polysaccharide. L o c a t i o n of the fl-acetyl group Since the complete b l o c k i n g of the polysaccharide w i t h methyl v i n y l ether proved d i f f i c u l t , the 0-acetyl l o c a t i o n procedure of de Belder and N orrman 1 8 0 was performed on the o l i g o s a c c h a r i d e , obtained a f t e r bacteriophage depolymerization of the E. c o l i K32 capsular p o l y -saccharide (see S e c t i o n V I ) . E t h y l a t i o n of the protected o l i g o s a c c h a -r i d e , followed by h y d r o l y s i s , conversion to the a l d i t o l a c e t a t e s , and g.l.c.-m.s. a n a l y s i s of the mixture of p a r t i a l l y e t h y l a t e d a l d i t o l acetates gave a r a t i o of 2-0-ethylrhamnose to rhamnose of 4.5:1 i n d i c a t -i n g that the O-acetyl group i s attached to 0-2 of rhamnose. V.2.4 CONCLUSION The sum of these experiments demonstrates that the s t r u c t u r e of the capsular polysaccharide from E s c h e r i c h i a c o l i 09:K32(A):H19 i s based on the t e t r a s a c c h a r i d e repeating u n i t shown w i t h h a l f of the L-rhamnosyl 142 OAc I 2 +3 ) - cc-D-Glc-( 1+4)-o-L-Rha-( 1+3)-cc-D-Gal-( 1+ 3 • 1 B-D-GlcA E. c o l l K32 residues being O-acetylated at 0-2. The s t r u c t u r e resembles that of K l e b s i e l l a K82; 1 9 t f i t has the same s t r u c t u r a l p a t t e r n ("three-plus-one" type) and a l a t e r a l p-D-glucosyluronic a c i d group as a t e r m i n a l u n i t . In common with K l e b s i e l l a K 5 5 , 1 9 5 a-L-rhamnose i s a branch point w i t h the CHacetyl group present on p o s i t i o n 0-2 of L-rhamnose. The s t r u c t u -r a l patterns of the two polysaccharides are, however, d i f f e r e n t . OAc I 2 +3)-8-Glc-( 1 +3)-a-Gal-( 1 +3)- 8-Gal-( 1 * ->3)-8-Glc-( l->4)-<r-Rha-( l-> 4 3 t + 1 1 8-GlcA a-Gal 3 t 1 oc-GlcA K l e b s i e l l a K82 K l e b s i e l l a K55 143 V.2.5 EXPERIMENTAL General methods The instrumentation used f o r n.m.r., g . l . c , i n f r a r e d , c.d., and measurements of o p t i c a l r o t a t i o n has been described i n Section I I I . G.l.c.-m.s. was performed w i t h the NERMAG R10-10 instrument. The c a p i l l a r y columns used were : (F) DB-225, programmed from 195° f o r 8 min, and then 4°/min to 220°, (G) SE-30, programmed from 70° f o r 1 min, and then 10°/min to 250°. Paper chromatography, g a s - l i q u i d chromato-graphy, and ion-exchange chromatography were performed as described i n Section I I I . Preparation and properties of E. c o l i K32 polysaccharide A c u l t u r e of E s c h e r i c h i a c o l i K32, obtained from Dr. I. 0rskov, (Copenhagen), was grown on Mu e l l e r Hinton agar as described i n Section I I I . 7 . 2 . Y i e l d : a c i d i c polysaccharide ~660 mg, n e u t r a l polysaccharide ~130 mg. The i s o l a t e d a c i d i c polysaccharide had [or.] 2 5 +80.9 (c 0.147, water), and a n a l y s i s by gel-permeation chromatography (courtesy of Dr. S.C. Churms, Cape Town, South A f r i c a ) showed i t to be homogeneous, w i t h an average molecular weight of 9 x 10 6 d a l t o n s . N.m.r. spectroscopy (*H and 1 3C) was performed on the o r i g i n a l and the deacetylated K32 polysaccharide. The p r i n c i p a l s i g n a l s i n the *H- and 1 3C-n.m.r. spectra and t h e i r assignments are recorded i n Table V.6. 144 Deacetylation of polysaccharide The polysaccharide was d i s s o l v e d i n 0.01M NaOH and s t i r r e d overnight at room temperature. The product was d i a l y z e d against tap water and f r e e z e - d r i e d . The d e a c e t y l a t i o n was almost complete (as judged from ^-H-n.m.r. spectrum) and 90% of the 0-acetyl groups were removed. A n a l y s i s of the deacetylated polysaccharide by gel-permeatlon chromatography showed i t to be homogeneous w i t h M w = 6 x 10 6 d a l t o n s . Hydrolysis of the polysaccharide H y d r o l y s i s of a sample (3 mg) of E. c o l i K32 polysaccharide w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) f o r 18 h at 95°, removal of the a c i d by successive evaporations w i t h water, followed by paper chromatography (sol v e n t s A and B), showed gala c t o s e , glucose, g l u c u r o n i c a c i d , and rhamnose. N e u t r a l sugars were q u a n t i t a t i v e l y determined by g . l . c . as t h e i r a l d i t o l acetates. The uronic a c i d was reduced by r e f l u x i n g a sample (6 mg) of K32 polysaccharide w i t h 3% HC1 i n methanol (4 mL) over-n i g h t , n e u t r a l i z i n g the HC1 w i t h PbC0 3, removing P b C l 2 , t r e a t i n g the d r i e d product w i t h NaBH^ (50 mg) i n anhydrous methanol (4 mL) and s t i r r -i n g overnight. The excess of NaBH^ was n e u t r a l i z e d w i t h Amberlite IR-120 (H +) r e s i n , and the b o r i c a c i d , as methyl borate, was removed by co-evaporation w i t h methanol. The sample was then hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d f o r 18 h at 95° and the a l d i t o l acetates were prepared and i d e n t i f i e d by g . l . c . (column A). Preparative g . l . c . (column D), followed by measurements of the c i r c u l a r d i chroism 145 s p e c t r a , showed the g l u c i t o l hexaacetate to be of the D c o n f i g u r a t i o n , and the rhamnitol pentaacetate to be of the L c o n f i g u r a t i o n . Methylation analysis The polysaccharide (29.8 mg), converted i n t o the f r e e a c i d form by passing the sodium s a l t through a column of Amberlite IR-120 (H +) r e s i n , was d i s s o l v e d i n dry dimethyl s u l f o x i d e (4 mL) and m e t h y l a t e d 8 8 by treatment w i t h 3 mL d i m e t h y l s u l f i n y l anion f o r 4 h, and then 6 mL of methyl i o d i d e f o r 1 h. The product, recovered a f t e r d i a l y s i s against tap water, was not completely methylated (hydroxyl absorption i n the i . r . spectrum). I t was d i s s o l v e d i n chloroform and subjected to Purdie methylation 8 1* w i t h methyl i o d i d e and s i l v e r oxide. This treatment y i e l d e d a f u l l y methylated polysaccharide (15.2 mg). A p o r t i o n of t h i s product (5 mg) was hydrolyzed 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 sugars were reduced w i t h sodium borohydride, and the a l d i t o l s were a c e t y l a t e d w i t h 1:1 a c e t i c a nhydride-pyridine, and analyzed by g . l . c . i n , column B (see Table V.7, column I ) . Carboxyl reduction of f u l l y methy-l a t e d polysaccharide (10.2 mg) w i t h L i A l H ^ i n anhydrous oxolane (5 mL) at room temperature overnight, h y d r o l y s i s of the product with 2M t r i f l u o r o a c e t i c a c i d , followed by reduction of sugars w i t h sodium boro-hydride, and a c e t y l a t i o n of the a l d i t o l s w i t h 1:1 a c e t i c anhydride-p y r i d i n e gave a mixture of p a r t i a l l y methylated a l d i t o l acetates which was analyzed by g . l . c . (column B) and g.l.c.-m.s. (column F and G). G.l.c. a n a l y s i s data f o r the p a r t i a l l y methylated a l d i t o l acetates are shown i n Table V.7, column I I . The n e u t r a l polysaccharide obtained by carbodiimide r e d u c t i o n 7 5 was a l s o subjected to methylation a n a l y s i s . A 146 sample (8.1 mg) of carbodiimide-reduced polysaccharide was d i s s o l v e d i n dry dimethyl s u l f o x i d e (1.5 mL) and methylated by the treatment w i t h 1.5 mL d i m e t h y l s u l f i n y l anion f o r 4 h, and then 3 mL methyl i o d i d e f o r 1 h. The product, recovered a f t e r d i a l y s i s against tap water, was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d overnight at 95°, reduced w i t h NaBH^ and the a l d i t o l s were a c e t y l a t e d w i t h 1:1 a c e t i c anhydride-pyridine. Subsequent a n a l y s i s by g . l . c . (column B) and g.l.c.-m.s. (column F) gave the r e s u l t s presented i n Table V.7, column I I I . Carbodiimide reduction of capsular polysaccharide 7 5 A sample of E. c o l i K32 polysaccharide (Na"!" s a l t , 50.15 mg) was d i s s o l v e d i n 30 mL water ( i n i t i a l pH 5.4). l - C y c l o h e x y l - 3 - ( 2 -morpholinoethyl)carbodiimide metho-p_-toluenesulfonate (CMC, 0.5 g) was added. As the r e a c t i o n proceeded, the pH was maintained at 4.75 by t i t r a t i o n w i t h 0.1N HC1. The r e a c t i o n was allowed to proceed f o r 2 h. An aqueous s o l u t i o n of 2M sodium borohydride (1.3 g/15 mL H 20) was added slow l y . The pH was maintained between 5-7 by t i t r a t i o n w i t h 4M HC1. The reduction was completed i n one h. The mixture was d i a l y z e d against tap water f o r two days, concentrated and f r e z e - d r i e d . Then a second treatment was c a r r i e d out s i m i l a r l y . A t o t a l of 47.2 mg of the product was recovered a f t e r f r e e z e - d r y i n g . A sample of the c a r b o d i i m i d e - r e d u c e d 7 5 polysaccharide (2.4 mg) was hydrolyzed overnight w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) on a steam bath and the hydrolyzate of sugars was converted i n t o a l d i t o l a c e t a t e s , g . l . c . a n a l y s i s of which (column A) showed rhamnitol pentaacetate, 147 g a l a c t i t o l hexaacetate and g l u c i t o l hexaacetate i n r a t i o s of 0.7:1.0:1.70, i n d i c a t i n g 70% re d u c t i o n . Chromium trioxide o x i d a t i o n 1 7 5 A sample (16.1 mg) of the polysaccharide was d i s s o l v e d i n formamide (5 mL), and t r e a t e d w i t h a c e t i c anhydride (1 mL) and p y r i d i n e (1 mL) overnight at room temperature. The a c e t y l a t e d m a t e r i a l (22.9 mg) was recovered by d i a l y s i s and f r e e z e - d r y i n g , and d i s s o l v e d i n a c e t i c a c i d (1 mL). The a c e t i c a c i d s o l u t i o n was t r e a t e d w i t h chromium t r i o x i d e (50 mg) at 50° f o r 1 h. The m a t e r i a l was recovered by p a r t i -t i o n between chloroform and water. The product was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d overnight, converted i n t o a l d i t o l acetates and analyzed by g . l . c . i n column A. Periodate oxidation A s o l u t i o n of K32 polysaccharide (42.5 mg) i n water (10 mL) was mixed w i t h 0.03M NalO^ (10 mL) and s t i r r e d i n the dark at room tempera-ture (23°). The r e a c t i o n was c a r r i e d out f o r 6 d. A f t e r ethylene g l y c o l (2 mL) was added to terminate the r e a c t i o n , the polyaldehyde was reduced to the p o l y a l c o h o l w i t h NaBH^, the base was n e u t r a l i z e d w i t h 50% a c e t i c a c i d , and the s o l u t i o n was d i a l y z e d overnight and f r e e z e - d r i e d to y i e l d e d the p o l y a l c o h o l (28.35 mg). A p o r t i o n (3.4 mg) was hydrolyzed w i t h 2M TFA overnight at 95° and converted i n t o a l d i t o l acetates. A n a l y s i s by g . l . c . i n column A showed rh a m n i t o l , g a l a c t i t o l and g l u c i t o l i n the r a t i o s of 1.0:0.5:1.0, i n d i c a t i n g that p a r t i a l degradation of the galactose had occurred. Periodate o x i d a t i o n was then repeated on the 148 carbodiimide-reduced polysaccharide' 5 3 using 0.1M sodium acetate b u f f e r and low temperature (4°) i n order to avoid p a r t i a l h y d r o l y s i s of the rhamnosyl bond and o v e r - o x i d a t i o n of the galactose. A sample of the carbodiimide-reduced polysaccharide (15.7 mg) was d i s s o l v e d i n 0.1M sodium acetate b u f f e r (pH 4.5), mixed w i t h 0.015M NalO^ (6 mL) and s t i r r e d i n the dark at 4°. A l i q u o t s (0.1 mL) were withdrawn p e r i o d i c a l l y , and d i l u t e d 250 times w i t h water. The a b s o r b a n c e s 1 8 5 at 223 nm of the r e s u l t i n g s o l u t i o n s were measured i n a Perkin-Elmer 552A UV/VIS spectrophotometer. The periodate consumption reached a plateau a f t e r ~10 days. Ethylene g l y c o l (2 mL) was added to decompose the excess of periodate, the polyaldehyde was reduced w i t h sodium borohydride, the base was n e u t r a l i z e d w i t h 50% a c e t i c a c i d , and the s o l u t i o n was d i a l y z e d and l y o p h i l i z e d to y i e l d the p o l y a l c o h o l . A p o r t i o n of t h i s p o l y a l c o h o l (7.2 mg) was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d overnight at 95° and converted i n t o a l d i t o l acetates. A n a l y s i s by g . l . c . i n column A showed rhamnitol, galactose, and glucose i n the r a t i o s of 0.9:1.0:1.0. Smith degradation A sample of the p o l y a l c o h o l (6.1 mg) obtained a f t e r periodate o x i d a t i o n of the K32 polysaccharide was d i s s o l v e d i n dry dimethyl s u l -foxide (3 mL) and methylated by treatment w i t h 2 mL of d i m e t h y l s u l f i n y l anion f o r 4 h, and then 4 mL of methyl i o d i d e f o r 1 h. The product was recovered by p a r t i t i o n between water and chloroform, and i t s i . r . spectrum showed complete methylation (no hydroxyl absorption i n the i . r . spectrum). I t was then subjected to the Smith h y d r o l y s i s by treatment 149 w i t h 50% a c e t i c a c i d f o r 90 min at 95°. A c e t i c a c i d was removed by co-evaporation with water and the dry residue was subjected to remethyla-t i o n by the Hakomori p r o c e d u r e . 8 8 The methylated product was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d (TFA) overnight at 95°, and the p a r t i a l l y methylated sugars were converted i n t o a l d i t o l acetates and analyzed by g . l . c . i n column B (see Table V.7, column IV) and g.l.c.-m.s. In column E. Location of the Q-acetyl g r o u p 1 8 0 A sample (5 mg) of the o l i g o s a c c h a r i d e obtained a f t e r b a c t e r i o -phage degradation of the E. c o l i K32 polysaccharide ( f r a c t i o n I I ) was d r i e d together w i t h a t r a c e of j v - t o l u e n e s u l f o n i c a c i d and then d i s s o l v e d i n dry dimethyl s u l f o x i d e (4 mL). Methyl v i n y l ether (3 mL) was added to a frozen s o l u t i o n , and the r e a c t i o n mixture was brought to 23° and allowed to s t i r f o r 4 h. Then a second p o r t i o n of methyl v i n y l ether (3 mL) was introduced i n a s i m i l a r manner. The c l e a r red s o l u t i o n was obtained. I t was placed on a Sephadex LH-20 column (16 cm x 2 cm) and e l u t e d w i t h acetone ( w i t h s l i g h t s u c t i o n ) . The product was concentrated and the residue was subjected to e t h y l a t i o n . I t was d i s s o l v e d i n dry dimethyl s u l f o x i d e (4 mL) and t r e a t e d w i t h 2 mL of d i m e t h y l s u l f i n y l anion f o r 4 h, and then w i t h 3 mL of e t h y l i o d i d e f o r 1 h. The dark-red product was e x t r a c t e d w i t h chloroform and p u r i f i e d on a Sephadex LH-20 column (14.5 cm x 2 cm) by e l u t i o n w i t h methanol. The product, a dark-red o i l , was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d overnight at 95° and converted i n t o a l d i t o l acetates. G.l.c. a n a l y s i s , conducted i n column A, programmed from 195° f o r 8 min, and then at 4°/min to 260°, 150 showed the presence of 2-0-ethylrhamnose (43%), rhamnose (9 . 6 % ) , galactose (16.4%), and glucose (30.8%). These r e s u l t s were confirmed by g.l.c.-m.s. (column F ) . CHAPTER VI BACTERIOPHAGE DEGRADATION OF Escherichia c o l l CAPSULAR POLYSACCHARIDES K28 and K32 152 VI. BACTERIOPHAGE DEGRADATION OF Escherichia c o l i CAPSULAR POLYSACCHARIDES K28 and K32. VI.1 INTRODUCTION L i k e many other organisms, b a c t e r i a are subject to i n f e c t i o n by a range of v i r u s e s or v i r u s - l i k e p a r t i c l e s which f a l l n a t u r a l l y i n t o two p h y s i o l o g i c a l l y separate groups, bacteriophages and b a c t e r i o c i n s . Bacteriophages (<)>) are true v i r u s e s , i n f e c t i n g t h e i r hosts and m u l t i p l y -i n g w i t h i n them. The members of the second group d i f f e r i n that they do not m u l t i p l y i n the c e l l a f t e r i n f e c t i n g i t , but only k i l l i t . B a c t e r i o c i n s may be defined as a n a t u r a l c l a s s of h i g h l y s p e c i f i c a n t i -b i o t i c s . The f i r s t account of the bacteriophage was published by Twort i n 1915. He demonstrated that the c u l t u r e s of b a c t e r i a l c e l l s could be i n f e c t e d w i t h and destroyed by f i l t e r a b l e agents that were subsequently termed bacteriophages. Two years l a t e r , d'Herelle independently i s o l a t e d a dysentery bacteriophage, c h a r a c t e r i z i n g i t as an u l t r a m i c r o s c o p i c p a r a s i t e of b a c t e r i a , and g i v i n g i t the name "bacteriophage", which means " b a c t e r i a - e a t e r " . Although bacteriophages (phages) were the l a s t major group of vi r u s e s to be recognized, today they are the best c h a r a c t e r i z e d and st u d i e d . The reasons f o r that l i e i n the f a c t that propagation and manipulation of phages has proven t e c h n i c a l l y much e a s i e r than equiva-l e n t s tudies on the other types of v i r u s e s . 153 Many d i f f e r e n t s t r a i n s of phages have been i s o l a t e d and c h a r a c t e r i z e d since they were f i r s t demonstrated i n 1915. Probably each b a c t e r i a l s t r a i n i s s u s c e p t i b l e to s e v e r a l d i f f e r e n t types of phage. Phages are r e l a t i v e l y easy to i s o l a t e from almost any b a c t e r i a l e n v i r o n -ment i n which a number of c l o s e l y r e l a t e d b a c t e r i a l s t r a i n s c o e x i s t (e.g., the g a s t r o i n t e s t i n a l t r a c t , sewage). I f a sample of sewage f i l t r a t e i s mixed w i t h a growing c u l t u r e of an e n t e r i c bacterium and spread on a p l a t e , the ensuing c u l t u r e w i l l show growth of the organism on the p l a t e i n t e r r u p t e d by small zones of c l e a r i n g , which are termed plaques. Each plaque represents the propagation of a s i n g l e phage p a r t i c l e i n the growing lawn of b a c t e r i a l c e l l s and i s analogous to an i s o l a t e d b a c t e r i a l colony. M o r p h o l o g i c a l l y phages are quite d i s t i n c t from other v i r u s types i n that they tend to be s t r u c t u r a l l y more complex. Bacteriophages have been i n t e n s e l y studied by many d i f f e r e n t techniques, but one of the most s i g n i f i c a n t c o n t r i b u t i o n s to our knowledge of these v i r u s e s has been made by e l e c t r o n microscopy. The morphological c l a s s i f i c a t i o n of the bacteriophages was introduced by B r a d l e y . 1 9 6 The phage head (see F i g . VI.1) contains the v i r u s n u c l e i c a c i d . In phage the n u c l e i c a c i d i s u s u a l l y i n the form of double-stranded DNA. The head has b a s i c a l l y an i c o s a h e d r a l s t r u c t u r e . I t i s composed of repeating i d e n t i c a l p r o t e i n chains and w i l l vary i n s i z e according to the s t r a i n , approximately 50 nm i n diameter. The phage t a i l i s more complex i n s t r u c t u r e than i s the head. A l a y e r of h e l i c a l l y arranged p r o t e i n molecules forms the inner tube of the t a i l . The tube i s encased by the c o n t r a c t i l e sheath, which extends from the c o l l a r to the end 154 Head F i g . VI.1: Schematic diagram demonstrating the s t r u c t u r e of T bacteriophage. 2 - D N A 2 - D N A 2 - D N A 1 - D N A 1 - K N A 1 - D N A F i g . VI.2t Basic morphological types of bacteriophages w i t h the types of n u c l e i c a c i d (from r e f . 196). 155 p l a t e . The sheath plays an important r o l e i n the i n f e c t i v e process by f o r c i n g the phage n u c l e i c a c i d through the hollow t a i l i n t o the host bacterium. The p l a t e contains small pins to which are attached s i x very long, f i n e t a i l f i b e r s by which phage p a r t i c l e s a t t a c h themselves to the c e l l w a l l s of s u s c e p t i b l e host c e l l s . 1 9 7 The b a c t e r i a l v i r u s e s e x h i b i t a great d i v e r s i t y of forms but i t i s p o s s i b l e to d i v i d e them i n t o s i x b a s i c morphological types (see F i g . VI.2). The f i r s t four groups (with t a i l s ) are unique to the b a c t e r i a l v i r u s e s . Groups E and F are d i f f e r e n t ; they resemble many p l a n t , animal, and i n s e c t v i r u s e s . 1 9 6 When a phage p a r t i c l e i n f e c t s a s u s c e p t i b l e host i t causes that c e l l to l y s e . The phases of the l y t i c c y c l e (see F i g . VI.3) in c l u d e the f o l l o w i n g : 1 9 7 ( i ) adsorption of the phage p a r t i c l e s to the s u s c e p t i b l e host ( i i ) i n j e c t i o n of v i r a l DNA (or RNA) i n t o the host ( i i i ) r e p l i c a t i o n of the phage n u c l e i c a c i d and synthesis of the phage p r o t e i n ( i v ) phage maturation and release Adsorption i s very host s p e c i f i c and depends on the presence i n the b a c t e r i a l c e l l w a l l of p r e c i s e receptor s i t e s . Adsorption of a phage to i t s receptor on the c e l l i s , i n most i n s t a n c e s , followed by pe n e t r a t i o n of the n u c l e i c a c i d through the cytoplasmic membrane. 1 9 8 Bacteriophages a c t i v e on exopolysaccharide-producing b a c t e r i a l s t r a i n s are g e n e r a l l y exopolysaccharide s p e c i f i c , 1 9 9 non-capsulate or 156 F i g . VI.3: The Mechanics of I n f e c t i o n by Bacteriophage A. Free phage. B. Phage attaches to c e l l w a l l w i t h f i b r e s , base p l a t e i n c l o s e contact w i t h outer l a y e r s of c e l l w a l l . C. Sheath c o n t r a c t s and c e n t r a l core i s pushed through the c e l l w a l l and DNA t r a n s f e r begins. D. Transfer of DNA completed. Phage head i s now empty and e a r l y events of phage growth c y c l e begin. (From T.J. Mackie and J.E. McCartney, "Medical M i c r o b i o l o g y " , V o l . 1, " M i c r o b i a l I n f e c t i o n s " , 13th edn., C h u r c h i l l L i v i n g s t o n e , Edinburgh, 1978). 157 non-slime producing mutants are r e s i s t a n t to the phages. Morphological examination of e x o p o l y s a c c h a r i d e - s p e c i f i c phages has revealed that most of them belong to group C, t h e i r base-plates are provided w i t h the spikes and no t a i l f i b r e s are s e e n . 1 9 8 Attack of bacteriophages on exopolysaccharide-producing b a c t e r i a i s often revealed by occurrence of halos around the c l e a r plaques i n the lawn of capsulated b a c t e r i a . 1 9 8 Within the halo the b a c t e r i a l lawn i s decapsulated. These halos are, at l e a s t p a r t l y , the r e s u l t of d i f f u s i o n of a phage-induced enzyme which hydrolyzes the capsule without k i l l i n g the b a c t e r i a . As shown by Bayer et a l . , 2 0 0 such phages can be v i s u a l i z e d under the e l e c t r o n microscope on t h e i r way from the outer surface of the b a c t e r i a l capsule to the c e l l w a l l underneath (see F i g . VI.4). The bacteriophage-associated enzymes may be c l a s s i f i e d according to the type of r e a c t i o n they c a t a l y z e , and according to the genus of the r e s p e c t i v e b a c t e r i a l host. Most v i r a l penetrases are hydrolases, but a few lyases have a l s o been found. The hydrolases are e i t h e r glycanases ( g l y c o s i d e hydrolases) or they are "deacetylases" which cleave o f f a c e t y l s u b s t i t u e n t s . 2 0 1 To date, v i r u s - a s s o c i a t e d enzymes of these types have mainly been studied using phages f o r b a c t e r i a belonging to the f a m i l y of Enterobacteriaceae, and w i t h i n the f a m i l y to the genera E s c h e r i c h i a c o l i , K l e b s i e l l a , Salmonella, S h i g e l l a and Proteus. In an extensive s t u d y , 2 0 2 55 d i f f e r e n t K l e b s i e l l a bacteriophages were tested f o r t h e i r enzymic a c t i o n on 74 d i f f e r e n t ( a c i d i c ) K l e b s i e l l a capsular polysaccharides (serotypes K1-K72, K74 and K80). The r e s u l t s may be summarized as f o l l o w s . 2 0 2 158 F i g . VI.4: Capsulated E. c o l i K29 exposed to a m.o.i. (the m u l t i p l i c i t y of i n f e c t i o n ) of 300 phage f o r 8 min at 37°. V i r u s p a r t i c l e s can be seen on the outer membrane surface. One v i r u s p a r t i c l e (upper l e f t corner) has apparently released most of i t s DNA. (From r e f . 200). 159 160 ( i ) The K l e b s i e l l a v i r u s - a s s o c i a t e d glycanases were found to be very s p e c i f i c , 33 c r o s s - r e a c t i n g with none, 18 w i t h one, two w i t h two, and one each w i t h 3 or 4 of the 73 heterologous polysaccharides; ( i i ) In most cases cleavage occurred on e i t h e r side of the sugar u n i t c a r r y i n g the negative charge, but reducing g l u c u r o n i c acids are not produced; ( i i i ) Most o f t e n , the reducing end sugar formed i s s u b s t i t u t e d at p o s i -t i o n 3; ( i v ) In the m a j o r i t y of cases, 6 - g l y c o s i d i c linkages are hydrolyzed; (v) In most polysaccharides which are acted upon by s e v e r a l phage enzymes, the same g l y c o s i d i c bonds are s p l i t by the d i f f e r e n t agents. The bacteriophage-associated glycanases allow the p r e p a r a t i v e i s o l a t i o n of o l i g o s a c c h a r i d e fragments. With some a c i d - l a b i l e components, bacteriophage degradation may be the only method f o r the i s o l a t i o n of repeating u n i t o l i g o m e r s . 2 0 3 Bacteriophage degradation may be used as a complement to other methods f o r the p a r t i a l degradation of b a c t e r i a l p olysaccharides. The l a r g e - s c a l e a c c e s s i b i l i t y of these repeating u n i t oligomers i s a l s o of advantage f o r analyses of nu c l e a r magnetic resonance spectroscopy. When coupled to s u i t a b l e p r o t e i n c a r r i e r s , b a c t e r i a l surface o l i g o s a c c h a r i d e s of two or more rep e a t i n g u n i t s may serve as immunogens, r e p r e s e n t a t i v e of the corresponding b a c t e r i a l g l y c a n s . 2 0 1 Although a number of h i g h l y s p e c i f i c "K bacteriophages" have been found f o r E s c h e r i c h i a c o l i capsular s t r a i n s , 1 9 3 the enzymic a c t i o n of 161 these phages has been studied to a much l e s s e r extent. The i n t e r a c t i o n between the capsulated E s c h e r i c h i a c o l i s t r a i n of serotype K29 and a capsule K 2 9 - s p e c i f i c bacteriophage has been s t u d i e d , using v i r u s adsorp-t i o n k i n e t i c s and immunological methods i n combination w i t h e l e c t r o n m i c r o s c o p y . 2 0 0 Recently, the capsule-degrading enzymic a c t i v i t y of two E. c o l i bacteriophages (<t>92 and <t>1.2) has been t e s t e d . 2 0 4 ' 2 0 5 The r e s u l t s of the degradation of two E s c h e r i c h i a c o l i capsular polysaccharides (K28 and K32) w i t h t h e i r r e s p e c t i v e bacteriophages ((j)28-l and (p28—2, and $32, r e s p e c t i v e l y ) are presented here. VI. RESULTS E s c h e r i c h i a c o l i bacteriophages were i s o l a t e d from sewage (courtesy of Dr. S. Stirm , F r e i b u r g , Germany). The bacteriophages have been c h a r a c t e r i z e d by Stirm and Freund-MOlbert and t h e i r morphology i s known. 1 9 3 Phage <p28—1 belongs to Bradley group A, and phages $28-2 and <b32 belong to Bradley group C. Most of the bacteriophages that are capable of i n f e c t i n g encapsulated Enterobacteriaceae belong to Bradley group C, 2 0 6 and bacteriophage-borne enzymatic a c t i v i t y seems to be a s s o c i a t e d w i t h spike s t r u c t u r e s . Depolymerization w i t h bacteriophages $28-1 and <t>28—2 The bacteriophages $28-1 and $28-2 were propagated on t h e i r host s t r a i n E s c h e r i c h i a c o l i K28 using n u t r i e n t broth as a medium. Propaga-t i o n was continued on an i n c r e a s i n g s c a l e u n t i l the crude l y s a t e s contained a t o t a l of ~ 1 0 1 3 plaque-forming u n i t s , an amount s u f f i c i e n t to 162 degrade one gram of the p o l y s a c c h a r i d e . Z [ ) 1 The depolymerization of E.  c o l i K28 capsular polysaccharide was conducted with the crude s o l u t i o n s of bacteriophages d>28—1 and <j>28-2,208 r e s p e c t i v e l y . I t was allowed to proceed f o r three days, chloroform being added to prevent b a c t e r i a l growth; the mixture was then concentrated, and the concentrate d i a l y z e d against d i s t i l l e d water. The process of concentration and d i a l y s i s was repeated s i x times, the d i a l y z a t e s were combined and concentrated. The concentrated d i a l y z a t e was t r e a t e d w i t h ion-exchange r e s i n Amberlite IR-120 (H +) three times and f r e e z e - d r i e d . The mixture of o l i g o s a c c h a r i d e s obtained a f t e r depolymerization of E. c o l i K28 polysaccharide w i t h phage <J>28—1 was then separated i n t o pure components by p r e p a r a t i v e paper chromatography. Only base l i n e carbohydrate m a t e r i a l was obtained. I t was i s o l a t e d and examined by gel-permeation chromatography (courtesy of Dr. S.C. Churms, Cape Town, South A f r i c a ) . The r e s u l t s showed that the i s o l a t e d m a t e r i a l was a mixture of high o l i g o s a c c h a r i d e s w i t h the average molecular weight M w = 2100 daltons. The degree of p o l y m e r i z a t i o n was determined by Morrison's m e t h o d 1 0 2 and by methylation a n a l y s i s on the mixture of corresponding o l i g o s a c c h a r i d e - a l d i t o l s (see Table VI.1 and Table VI.2). They i n d i c a t e d that the average length of the o l i g o s a c c h a r i d e i s 20 sugars or 5 repeating u n i t s and that the g l u c o s y l residue i s a reducing sugar. This i s i n agreement with ^-H-n.m.r. f i n d i n g s which show the presence of two s i g n a l s at 6 = 4.67 p.p.m. ( J , o = ^ **z^ a T U* a t ° = P«P«m« ( ^ 1 2 = 4 Hz) corresponding to the B-glucosyl and a-glucosyl residues 163 TABLE VI.1 METHYLATION ANALYSIS AND THE REDUCING END DETERMINATION OF E. c o l l K28 OLIGOSACCHARIDE ISOLATED AFTER BACTERIOPHAGE $28-1 DEGRADATION OF E. c o l i K28 POLYSACCHARIDE Methylated T b Mole %° a sugars Column Bb (as a l d i t o l ) (ECNSS-M) acetates) 1,2,5,6-Glc 0.8 15.3 f 2,3-Fuc 1.14 12.9 2,3,4,6-Gal 1.24 71.7 1,2,5,6-Glc = 3 , 4 - d i - 0 - a c e t y l - l , 2 , 5 , 6 - t e t r a - 0 - m e t h y l g l u c i t o l , e t c . R e l a t i v e r e t e n t i o n time r e f e r r e d to 2,3,4,6-Glc as 1.00. Values are corrected by use of the e f f e c t i v e , c a r b o n response f a c t o r s given by Albersheim et a l . 1 0 3 Ratios are low, due to incomplete h y d r o l y s i s of the g l u c o s y l u r o n i c linkage (2,3-Fuc) and high v o l a t i l i t y of the d e r i v a t i v e (1,2,5,6-Glc). 164 TABLE VI.2 DETERMINATION OF THE DEGREE OF POLYMERIZATION AND THE REDUCING END OF E. c o l i K28 OLIGOSACCHARIDE ISOLATED AFTER BACTERIOPHAGE <J»28-1 DEGRADATION OF E. c o l i K28 POLYSACCHARIDE P e r a c e t y l a t e d d e r i v a t i v e of Column C (OV-225) Mole % F u c o n o n i t r i l e 0.35 24.8 G l u c o n o n i t r i l e 1.00 39.9 G a l a c t o n o n i t r i l e 1.08 30.0 G l u c i t o l 1.20 5.2 a Isothermal at 230°. 165 r e s p e c t i v e l y . Both s i g n a l s disappeared a f t e r r eduction w i t h sodium borohydride due to the conversion of the reducing sugar i n t o the a l d i t o l . The molecular weight d i s t r i b u t i o n of the non-dialyzable p o r t i o n gave a mixture of high o l i g o s a c c h a r i d e s with the average molecular weight = 4500. These r e s u l t s i n d i c a t e that only p a r t i a l depolymerization had occurred. Depolymerization of E. c o l i K28 capsular polysaccharide w i t h <t>28-2 gave s i m i l a r r e s u l t s . Confirmation of the reducing end was obtained by Morrison's method, 1 0 2 whereby the o l i g o s a c c h a r i d e i s reduced to the a l d i t o l and a f t e r h y d r o l y s i s , the f r e e sugars are converted i n t o the p e r a c e t y l a t e d a l d o n o n i t r i l e s w i t h the reducing end being converted i n t o the perac e t y l a t e d a l d i t o l . The r e s u l t s showed the presence of g l u c i t o l , g l ' u c o n o n i t r i l e , g a l a c t o n o n i t r i l e and f u c o n o n i t r i l e i n d i c a t i n g that the g l u c o s y l l i n k a g e was cleaved by bacteriophage a c t i o n . Depolymerization with bacteriophage <p32 The bacteriophage 4>32 was propagated on i t s host s t r a i n E s c h e r i c h i a c o l i K32 using n u t r i e n t broth as a medium. When the phage concentration reached ~ 1 0 1 0 P.F.U./mL i t was f u r t h e r propagated i n a fermentor (see Section I I I . 8 . 2 ) . Bacteriophage <j>32 was p u r i f i e d by p r e c i p i t a t i o n with polyethylene g l y c o l 6000 (10% w / v ) . 2 1 3 The depolymerization was c a r r i e d out i n a v o l a t i l e b u f f e r 2 0 1 * f o r two days i n the presence of some chloroform to ensure s t e r i l i t y . The depolymerization mixture was d i a l y z e d against 166 d i s t i l l e d water overnight. The d i a l y s i s was repeated twice more. The d i a l y z a t e s were combined, concentrated and f r e e z e - d r i e d . The d i a l y z a b l e p o r t i o n was separated by gel-permeation chromatography on a column of Bio-Gel P-4.. The e l u t i o n p a t t e r n i s shown i n F i g . VI.5. Three f r a c t i o n s were obtained. The second f r a c t i o n was f u r t h e r examined by gel-permeation chromatography (courtesy of Dr. S.C. Churms, Cape Town, South A f r i c a ) f o r molecular weight d i s t r i b u t i o n . I t showed that the f r a c t i o n c o n s i s t e d mainly of an octasaccharide (M = 1600 daltons) (see F i g . VI.6). Analysis of the depolymerization products of E. c o l i K32  polysaccharide F r a c t i o n s I , I I and I I I were examined by iH-n.m.r. spectroscopy. F r a c t i o n I contained h i g h l y polymeric m a t e r i a l , and the spectrum of t h i s f r a c t i o n was very s i m i l a r to that of the n a t i v e a c e t y l a t e d p o l y -saccharide. The presence of an octasaccharide was not obvious from the 1H-n.m.r. spectrum of the second f r a c t i o n . However, a f t e r sodium boro-hydride reduction of that f r a c t i o n the ^-n.m.r. spectrum became b e t t e r r e s o l v e d , and was s i m i l a r to the spectrum of the deacetylated E. c o l i K32 polysaccharide. The comparison of two spectra d i d not permit the assignment of the reducing end (due to the p o s s i b l e overlapping w i t h other p r i n c i p a l s i g n a l s ) . F r a c t i o n I I I had a low carbohydrate content as was judged from i t s ^ -H-n.m.r. spectrum. I t revealed the presence of an octasaccharide w i t h a low acetate content (30% by n.m.r.). The 1H-n.m.r. data of a l l three f r a c t i o n s i s summarized i n Table VI.3. F i g . V I . 5 : S e p a r a t i o n o f t h e d e p o l y m e r i z a t i o n p r o d u c t s o f E . c o l i K 3 2 b y g e l - p e r m e c h r o m a t o g r a p h y ( B i o - G e l P - 4 ) 168 1600 8 0 1 © 70-E l u t e d volume (mL) Molecular weight distribution Mol. wt. % by wt. mol. % 4400 13 6 3300 11 7 2700 18 14 1600 58 73 Fig. VI.6: Molecular weight distribution of fraction II (Bio-Gel P-10 column 52 x 1.5 cm, M NaCl eluant, flow-rate 20mL/h). Courtesy of Dr. S.C Churms, Cape Town, South Africa. TABLE VI.3 PROTON ASSIGNMENTS (400 MHz) FOR THE OLIGOSACCHARIDES AND RELATED COMPOUNDS GENERATED IN BACTERIOPHAGE DEPOLYMERIZATION OF THE E. c o l i K32 CAPSULAR POLYSACCHARIDE. Fraction I- Fraction II Fraction II (R) C Fraction III A Integral A Integral A Integral A Integral Assignment^ (p.p.m.) (H) (p.p.m.) (H) (p.p.m.) (H) (p.p.m.) (H) 5.51 5.45 1.2 0.4 5.51 5.48 5.44 0.6 0.4 0.6 5.52 i 5.47 > 0.25 i a-Rha w i t h ' 0 - a c e t y l 5.24 1.0 5.24 1.0 5.23 1.0 5.25 1.0 a-Rha 5.18 5.14 } 5.3 5.18 5.15 } 5.2 5.17 5.14 } 3.0 5.19 | 5.16 2.0 } a-Gal 5.10 5.11 5.10 5.11 1.0 a-Glc 5.05 i 5.03 1.3 5.05 , 5.02 ' 1.5 5.07 0.4 5.05 0.5 unassigned 4.72(8Hz) 2.2 4.71(8Hz) 4.68(8Hz) } 2.0 4.66(8Hz) 1.0 4.72(8Hz) 2.0 j 6-GlcA 2.19 i 2.16 2.6 2.17(8Hz) 2.7 2.19(8Hz) 1.5 CH 3 of 0-ace t y l 1.35 5.0 1.35(6Hz) 1.33(6Hz) } 6.5 1.32 6.0 1.35(6Hz) 6.3 i CH 3 of ' a-Rha a For the source of Fr: I , I I and I I I i see t e x t . k Chemical s h i f t r e l a t i v e to i n t e r n a l acetone; 6 2.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (DSS). c d F r a c t i o n I I a f t e r reduction with sodium borohydride. A d e f i n i t e assignment could not be made, the assignments of a-Gal and a-Glc are t e n t a t i v e . 170 Meth y l a t i o n of the reduced f r a c t i o n I I , followed by h y d r o l y s i s , d e r i v a t i z a t i o n as a l d i t o l a cetates, and g.l.c.-m.s. a n a l y s i s gave values shown i n Table VI.4. The data demonstrate that the bacteriophage enzyme i s an <x-D-glucosidase and that the glucose i s present at the reducing end of the o l i g o s a c c h a r i d e which i s comprised of two repeating u n i t s . I t a l s o shows that the bacteriophage-borne enzyme does not have a "deacetylase" a c t i v i t y , since acetate-bearing o l i g o s a c c h a r i d e s were i s o l a t e d a f t e r the bacteriophage degradation of E. c o l i K32 polysaccharide. VI.3 DISCUSSION The main purpose of the bacteriophage work c a r r i e d out on E s c h e r i c h i a c o l i bacteriophages $28-1, cb28—2 and <j>32 was to o b t a i n o l i g o s a c c h a r i d e s representing subunits of the polysaccharides degraded, w i t h the l a b i l e 0-acetyl s u b s t i t u e n t s present as i n the o r i g i n a l p o l y -saccharide. These o l i g o s a c c h a r i d e s could be then studied by -^H-n.m. r . and 1 3C-n.m.r. spectroscopy i n order to l o c a t e the acetate p o s i t i o n . However, t h i s aim has not been achieved. The bacteriophage degradation of the polysaccharide was c a r r i e d out using two methods. According to the f i r s t method, the bacteriophage was propagated on an i n c r e a s i n g scale u n t i l a t o t a l of ~ 1 0 1 3 P.F.U. was obtained and a f t e r d i a l y s i s a concentrated crude s o l u t i o n of the bacteriophage was used d i r e c t l y f o r the depolymerization. This p r o c e d u r e 2 0 8 was developed i n our group and gave e x c e l l e n t r e s u l t s w i t h K l e b s i e l l a bacteriophages. However, use of t h i s method f o r b a c t e r i o -1.71 TABLE VI.4 METHYLATION ANALYSIS OF THE REDUCED FRACTION II OBTAINED AFTER THE SEPARATION OF THE DEPOLYMERIZATION PRODUCTS OF E. c o l i K32 POLYSACCHARIDE Methylated sugars (as a l d i t o l acetates) T b Column B b (ECNSS-M) Mole %° 1,2,4,5,6-Glc 0.35 4.9 d 2,4-Rha 1.01 11.9 2,3,4,6-Gal 1.55 28.6 2-Rha 1.96 35.5 2,4,6-Glc 2.26 19.1 1,2,4,5,6-Glc = 3-0-acetyl-l,2,4,5,6-penta-0-methylglucitol, e t c . R e l a t i v e r e t e n t i o n time r e f e r r e d to 2,3,4,6-Glc as 1.00. Values are correct e d by use of the e f f e c t i v e , carbon-response f a c t o r s given by Albersheim et a l . 1 0 3 Ratios of c e r t a i n sugars are low, due to incomplete h y d r o l y s i s of the gl u c o s y l u r o n i c linkage (2,4-Rha) and high v o l a t i l i t y of the d e r i v a t i v e (1,2,4,5,6-Glc). 172 phages $28-1 and $28-2 degradation of E. c o l i K28 polysaccharide d i d not give s a t i s f a c t o r y r e s u l t s . Only p a r t i a l depolymerization was achieved. Since these bacteriophages form very small "halos", i t i s p o s s i b l e that they have low enzymic a c t i v i t y , and do not produce o l i g o s a c c h a r i d e s corresponding to one or two repeating u n i t ( s ) of the polysaccharide. Bearing t h i s i n mind, the second method was used f o r b a c t e r i o -phage $32. I t was p u r i f i e d by p r e c i p i t a t i o n w i t h polyethylene g l y c o l 6000 p r i o r to the depolymerization. The degradation was done i n " v o l a t i l e " b u f f e r i n order to avoid p o s s i b l e l o s s of the a c t i v i t y during the depolymerization. The r e s u l t s obtained were more encouraging, and a small amount of the octasaccharide (two repeating u n i t s ) bearing an 0 - a c e t y l group was obtained. A l l three bacteriophages ($28-1, $28-2 and $32) e x h i b i t a-D-glucosidase a c t i v i t y . The bacteriophages $28-1 and $28-2 cleaved the same g l y c o s y l l i n k a g e , ->3)-oc-D-Glc-( 1->4)-8-D-G1CA-( l+4)-<x-L-Fuc-( 1-+ 4 1 $28-1, $28-2 8-D-Gal E. c o l i K28 although morphologically they belong to the d i f f e r e n t Bradley groups, 1- 9 3 $28-1 (group A) and $28-2 (group C), and d i f f e r e n t enzymic a c t i v i t y was expected. 173 In the case of bacteriophage <p32 oc-glucosidase a c t i v i t y was q u i t e unexpected, since the rhamnosyl linkage i s the most l a b i l e one (to a c i d ) . •>3 ) - a-D-Glc-(1 ->4) - a-L-Rha-(1 +3 ) - a-D-Gal-(1 -> 3 <J>32 1 B-D-GlcA E. c o l i K32 Here again the tendency of the bacteriophage to produce a l i n e a r s t r u c -ture can be n o t i c e d . Another i n t e r e s t i n g aspect of t h i s work i s the f a c t that bacteriophages $28-1 and $32 give cross-adsorption w i t h the b a c t e r i a l s t r a i n of E s c h e r i c h i a c o l i K32, but not the phage $28-2. 1 9 3 Comparison of the s t r u c t u r e s of E. c o l i K28 and E. c o l i K32 suggests c e r t a i n s i m i l a r i t y i n t h e i r s t r u c t u r a l p a t t e r n . Both s t r u c t u r e s are of a "three-plus-one" type, and t h e i r q u a l i t a t i v e composition i s s i m i l a r . However, despite these s i m i l a r i t i e s the two st r u c t u r e s are d i f f e r e n t . This suggests that the bacteriophage recog-n i z e s only a small p o r t i o n of the polysaccharide chain. The a c t i o n of the bacteriophage $28-1 on the b a c t e r i a l s t r a i n of E s c h e r i c h i a c o l i K32 has yet to be determined. 174 VI. 4 EXPERIMENTAL The bacteriophages d>28—1, $28-2 and $32 were received from Dr. S. Stirm ( F r e i b u r g , Germany) and propagated on t h e i r host s t r a i n s . For experimental d e t a i l s see Section I I I . 8 . Paper chromatography, gas-l i q u i d chromatography and gel-permeation chromatography were performed as described i n Section I I I . P r e p a r a t i v e paper chromatography was performed by the descending method using solvent C (see Section I I I . l ) . The Instrumentation used f o r n.m.r., g . l . c . and I n f r a r e d has been described i n Section I I I . G.l.c.-m.s. was performed on the NERMAG R10-10 instrument using c a p i l l a r y column of DB-225, programmed from 195° f o r 8 min, and then 4°/min to 220°. Incubation of E. c o l i K28 polysaccharide w i t h bacteriophages P u r i f i e d , capsular polysaccharide (100 mg) from E s c h e r i c h i a c o l i K28 was d i s s o l v e d i n 10 mL of d i s t i l l e d water. A phage $28-1 s o l u t i o n c o n t a i n i n g 2 x 1 0 1 3 P.F.U. was concentrated to a small volume p r i o r to the depolymerization, d i a l y z e d against tap water f o r three days and concentrated again to 100 mL. A polysaccharide s o l u t i o n was mixed w i t h a phage s o l u t i o n (100 mL) i n a n u t r i e n t broth, c o n t a i n i n g a t o t a l of 2.0 x 1 0 1 3 plaque-forming u n i t s (P.F.U.) and incubated f o r 48 h at 37°. A s i g n i f i c a n t drop i n the v i s c o s i t y of the o r i g i n a l polysaccharide s o l u -t i o n was n o t i c e d a f t e r approximately 3 h of i n c u b a t i o n . 175 Purification and separation of depolymerlzed material The r e s u l t i n g crude depolymerization mixture was d i a l y z e d a gainst 250 mL of d i s t i l l e d water. The procedure was repeated s i x times and the d i a l y s i s bag was changed a f t e r each time. The d i a l y z a t e s were combined, concentrated to a small volume on a r o t a r y evaporator and exchanged three times on Amberlite IR-120(H +) r e s i n . The r e s u l t i n g s o l u t i o n was f r e e z e - d r i e d . The degradation products were i s o l a t e d by preparative paper chromatography i n solvent C. Only b a s e l i n e carbohydrate m a t e r i a l was obtained (21.7 mg). The content of a d i a l y s i s bag was exchanged w i t h Amberlite IR-120(H +) ion-exchange r e s i n and f r e e z e - d r i e d . I t y i e l -ded 92.5 mg of the non-dialyzable m a t e r i a l . In a s i m i l a r way a degradation of E s c h e r i c h i a c o l i K28 using crude bacteriophage $28-2 suspension was done. The bacteriophage degra-dation was not complete and y i e l d e d only high oligomers as was judged by molecular weight d i s t r i b u t i o n of a d i a l y z a b l e f r a c t i o n (see Section VI.2). Purification of the bacteriophage $32 Bacteriophage $32 was propagated i n a fermentor (see S e c t i o n I I I . 8 . 2 ) to give 9 L of a c l e a r s o l u t i o n w i t h the t i t e r 1.6 x 1 0 1 0 P.F.U./mL. From t h i s p r e p a r a t i o n 2.5 L was p u r i f i e d by p r e c i p i t a t i o n w i t h polyethylene g l y c o l 6 0 0 0 2 0 9 i n the presence of NaCl (73.1 g, 0.5M). The s o l u t i o n was then placed i n a c o l d room and kept f o r 42 h at 4°. A very f i n e p r e c i p i t a t e of a bacteriophage appeared at the bottom of a beaker. I t was spun down i n a c e n t r i f u g e at 1400 r.p.m. The p r e c i p i t a t e was r e d i s s o l v e d i n 50 mL of 10 mM (pH 7.1) t r i s ( h y d r o x y -176 methyl)methylamine ( T r i s ) - hydrochloride b u f f e r which contained 10 mM NaCl. In order to remove polyethylene g l y c o l the milky and opalescent phage s o l u t i o n was d i a l y z e d against the same bu f f e r f i r s t at room temperature (23°) f o r 18 h and then at 4° f o r an a d d i t i o n a l 24 h. The T r i s - H C l b u f f e r was changed once during the d i a l y s i s . The phage s o l u t i o n was then spun at low speed 1200 r.p.m. and assayed. The bacteriophage t i t e r was 2.5 x 1 0 1 1 P.F.U./mL or a t o t a l of 1.25 x 1 0 1 3 P.F.U. ( f o r 50 mL of phage s o l u t i o n ) . The assay of the supernatant gave a t i t e r of 7.2 x 10 8 P.F.U./mL. This r e s u l t shows that the b a c t e r i o -phage p r e c i p i t a t i o n was not complete and that some a c t i v i t y was s t i l l present i n the supernatant. The phage was then d i a l y z e d against v o l a t i l e b u f f e r which contained 0.05M ammonium carbonate and 0.1M ammonium acetate and was adjusted to pH 7.2 w i t h 50% a c e t i c a c i d . 2 0 4 Depolymerization of the capsular polysaccharide from E. c o l i  K32 by bacteriophage 4>32 E. c o l i K32 polysaccharide (195 mg) was d i s s o l v e d i n 15 mL of a v o l a t i l e b u f f e r and to t h i s s o l u t i o n a t o t a l of 0.85 x 1 0 1 3 P.F.U. i n 50 mL of a v o l a t i l e b u f f e r was added. The mixture was kept f o r 48 h at 37°, chloroform being added to prevent b a c t e r i a l growth. The depolymerization mixture was t r a n s f e r r e d i n t o a d i a l y s i s bag and d i a l y z e d against d i s t i l l e d water overnight. The d i a l y s i s was repeated twice more, and the d i a l y z a t e s were combined and f r e e z e - d r i e d . The residue was r e d i s s o l v e d i n d i s t i l l e d water and f r e e z e - d r i e d again. This process was repeated s e v e r a l times u n t i l the weight of the sample 177 remained unchanged (114.2 mg). The contents of the d i a l y s i s bag was f r e e z e - d r i e d separately to y i e l d 260.5 mg. Separation of the depolymerized material by gel-permeation  chromatography The crude d i a l y z a b l e m a t e r i a l (114.2 mg) was placed on a column of Bio-Gel P-4 (400 mesh) and e l u t e d at 10.5 mL/h. F r a c t i o n s (2 mL each) were c o l l e c t e d and f r e e z e - d r i e d . The e l u t i o n p r o f i l e i s shown i n F i g . VI.1 (see Section VI.2). Three f r a c t i o n s of the degradation products were obtained: f r a c t i o n I - tubes 9-14 (17.4 mg); f r a c t i o n I I - tubes 15-21 (20 mg); f r a c t i o n I I I - tubes 22-34 (22.75 mg). Molecular weight d i s t r i b u t i o n of f r a c t i o n I I showed the presence of an octasaccharide, which represented a double repeating u n i t of E. c o l i K32 polysaccharide (see Section VI.2). Determination of the reducing end and the degree of  depolymerization A sample of o l i g o s a c c h a r i d e (10 mg) was d i s s o l v e d i n H 20 (5 mL) and to t h i s NaBH^ (15 mg) was added. A f t e r s t i r r i n g f o r 2 h, the excess of sodium borohydride was removed by treatment w i t h Amberlite IR-120(H +) ion-exchange r e s i n . The d r i e d , reduced o l i g o s a c c h a r i d e was r e f l u x e d i n 3% methanolic HC1 overnight. A f t e r n e u t r a l i z a t i o n w i t h Ag 2C0 3, and evaporation of the solvent a f t e r c e n t r i f u g a t i o n , the uronic e s t e r was reduced w i t h NaBH^ i n anhydrous methanol (5 mL). The reduced m a t e r i a l was hydrolyzed w i t h 2M t r i f l u o r o a c e t i c a c i d on a steam bath overnight and the excess a c i d was removed by co-evaporations w i t h water. A s o l u -178 t i o n (0.5 mL) of 5% hydroxylamine hydrochloride i n p y r i d i n e was then added and the r e a c t i o n mixture was heated on a steam bath f o r 15 min. A c e t i c anhydride (0.5 mL) was added to the cooled s o l u t i o n which was heated on a steam bath f o r 1 h. The mixture of pe r a c e t y l a t e d aldono-n i t r i l e s and p e r a c e t y l a t e d a l d i t o l acetates was i s o l a t e d by p a r t i t i o n between water and chloroform. G.l.c. a n a l y s i s was performed on column C i s o t h e r m a l l y at 230°. Methylation analysis and reducing end determination The o l i g o s a c c h a r i d e (5 mg) was reduced w i t h NaBH^ p r i o r to methy-l a t i o n by the Hakomori p r o c e d u r e . 8 8 The reduced m a t e r i a l was d i s s o l v e d i n dry dimethyl s u l f o x i d e (2 mL) and methylated by treatment w i t h 1 mL of d i m e t h y l s u l f i n y l anion f o r 2 h, and then 2 mL of methyl i o d i d e f o r 1 h. 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R.D. Guth r i e , Methods Carbohydr. Chem., ± (1962) 445-447. 186 K. Jann, B. Jann, and K.F. Schneider, Eur. J . Biochem., 5^  (1968) 456-465. 187 D. Hungerer, K. Jann, B. Jann, F. 0rskov, and I. 0rskov, Eur.  J . Biochem., 2_ (1967) 115-126. 188 B.B. Wiley and H.W. Scherp. Can. J . M i c r o b i o l . , A_ (1958) 505-516. 189 A.K. Chakraborty, Macromol. Chem., 183 (1982) 2881-2887. 190 P.A. Sandford, J.R. Bamburg, E.D. Epley, and T.J. K i n d t , Biochemistry, _5 (1966) 2808-2817. 191 R.E.W. Hancock, Department of M i c r o b i o l o g y , U n i v e r s i t y of B r i t i s h Columbia, personal communication. 192 Worthington Enzyme Manual, Worthington Biochemical C o r p o r a t i o n , Freehold, New Jersey, U.S.A. 07728, 1972, p. 21. 193 S. Stirm and E. Freund-MOlbert, J . V i r o l . , 8 (1971) 330-342. 194 G.G.S. Dutton and A.V.S. Lim, Carbohydr. Res., 123 (1983) 247-257. 190 195 G.M. Bebault and G.G.S. Dutton, Carbohydr. Res., 64 (1978) 199-213. 196 D.B. Bradley, B a c t e r i o l . Rev., 31 (1967) 230-314. 197 J . Levy, J.J.R. Campbell, T.H. Blackburn, "Introductory M i c r o -b i o l o g y " , John Wiley & sons, i n c . , New York, 1973. 198 A.A. Lindberg, i n I.W. Sutherland (Ed.), "Surface Carbohydrates of the P r o k a r i o t i c C e l l " , Academic Press, New York, 1977, pp. 289-356. 199 S. St i r m , J . V i r o l . , 2 (1968) 1107-1114. 200 M.E. Bayer, H. Thurow, and M.H. Bayer, V i r o l o g y , 94 (1979) 95-118. 201 H. Geyer, K. Himmelspach, B. Kwiatkowski, S. Schlecht, and S. Stirm, Pure & Appl. Chem., 55 (1983) 637-653. 202 D. Rieger-Hug and S. St i r m , V i r o l o g y , 113 (1981) 363-378. 203 G.G.S. Dutton, K.L. Mackie, A.V. Savage, D. Rieger-Hug, and S. Stirm, Carbohydr. Res., 84 (1980) 161-170. 204 B. Kwiatkowski, B. Boschek, H. T h i e l e , and S. St i r m , J . V i r o l . , 43 (1982) 697-704. 205 B. Kwiatkowski, B. Boschek, H. T h i e l e , and S. St i r m , J . V i r o l . , 45 (1982) 367-374. 206 D. Rieger, E. Freund-MOlbert, and S. Stirm, J . V i r o l . , 17 (1976) 859-864. 207 W. B e s s l e r , E. Freund-MOlbert, H. Kntffermann, C. Rudolph, H. Thurow, and S. Stirm, V i r o l o g y , 56 (1973) 134-151. 208 G.G.S. Dutton, J.L. Di Fabio, D.M. Leek, E.H. M e r r i f i e l d , J.R. Nunn, and A.M. Stephen, Carbohydr. Res., 9T_ (1981) 127-138. 209 K.R. Yamamoto, B.M. A l b e r t s , R. Benzinger, L. Lawthorne, and G. T r e i b e r , V i r o l o g y , 40 (1970) 734-744. 191 APPENDIX I THE KNOWN STRUCTURES OF THE Escherichia c o l l 0 Antigens (as of August 1, 1984) 192 APPENDIX I E. c o l l 0 antigens 3 1 4 1 3 1 4 1 06 — Man — — Man - r - GlcNAc GalNAc — . 8 8 a a 'I? Glc 3 1 3 , 1 2 lk 1 07 — GlcNac Qui 4Nac Man Gal — a p a f 3 a Rha Quip4NAc=4-acetamido-4,6-dideoxy-D-glucopyranose 3 1 2 1 2 1 08 — Man Man Man — a a a 3 1 3 1 2 1 2 1 2 1 09 — Man Man Man Man Man — a a a a a 2 Ik 1 6 1 3 1 018ac — Rha Gal Glc GlcNAc — a . 3 a a a • I . GlcNAc 193 020 — Gal 1 2 1 Rha 3 a i 3 1 3 1 3 1 025 — FucNAc GlcNAc - 5 - Glc — a B 6 P 1 Glc 055 Gal 1 3 GalNAc 1 6 GlcNAc 3 P 1 Gal 2 a Co l Col=3,6-dideoxy-L-xylopyranose 069 3 1 2 1 2 1 2 1 — GlcNAc - 5 - Rha Rha Gal — 8 a a a 194 075 3 1 3 1 k 1 - GlcNAc Gal Rha — a i+ -1 Man 086 — Gal GalNAc GalNAc I I Fuc Glc C o l 1 0111 GlcNAc 1 h Glc 6 C o l 1 i+ Gal 3 1 4 1 3 1 k 1 0114 — GlcNAc Qui 3N - 5 - R i b . - 5 - Gal — r a P I P f P P C=0 I AcHNCH CH20H } N- a c e t y l - L - s e r i n e where Qui 3N = 3-amino-3,6-dideoxy-B-D-glucopyranose 195 References 06 V. VSisa"nen-Rhen, J . E l o , E. VSisa'nen, A. Stftonen, I . 0rskov, F. 0rskov, S.B. Svenson, P.H. Ma*kela\ and T.K. Korhonen, I n f e c t . Immun., 43 (1984) 149. 07 V.L. L'vov, A.S. Shashkov, B„A. Dmitriev, N.K. Kochetkov, B. Jann, and K. Jann, Carbohydr. Res., 126 (1984) 249-259. 08 K. Reske and K. Jann, Eur. J . Biochem., 31_ (1972) 320-328. 09 P. Prehm, B. Jann, and K. Jann, Eur. J . Biochem., 67 (1976) 53-56. 018ac D.S. Gupta, B. Jann, and K. Jann, I n t . Symp. Carbohydr. Chem., 12th, 1984 A b s t r a c t , p. 373. 020 U.N. V a s i l i e u and I.Y. Zakharova, Bioorg. Chem., 2 (1976) 199-206. 025 L. Kenne and B. Lindberg, Carbohydr. Res., 122 (1983) 249-256. 055 B. Lindberg, F. Lindh, J . LOnngren, A.A. Lindberg, and S.B. Svensson, Carbohydr. Res., 97 (1981) 105-112. 069 C. E r b i n g , L. Kenne, B. Lindberg, G. Naumann, and W. Nimmich, Carbohydr. Res., 56 (1977) 371-376. 075 C. Erbi n g , L. Kenne, B. Lindberg, and S. Hammarstrtfm, Carbohydr. Res., 60 (1978) 400-403. 086 G.F. Springer, Ann. N.Y. Acad. S c i . , 169 (1970) 134-152. 0111 K. E k l i n d , P.J. Garegg, L. Kenne, A.A. Lindberg, and B. Lindberg, I n t . Symp. Carbohydr. Chem., 9th, 1978 A b s t r a c t , p. 493. 0114 B.A. Dmitriev, V.L. Lvov, N.V. Tochtamysheva, A.S. Shashkov, N.K. Kochetkov, B. Jann, and J . Jann, Eur. J . Biochem. ( i n p r e s s ) . 196 APPENDIX II THE KNOWN STRUCTURES OF THE Escherichia c o l l K Antigens (as of August 1, 1984) 197 APPENDIX II E. c o l l K antigens 8 2 — NANA5Ac — a E. c o l i Kl k 1 2 1(3) 5 12 1(3) x — P Gal Gly - — ( - P — Galc Gly ) a J 2n f a n E. c o l i K2 4 14 1 — GlcA — — GlcNAc — P a E. c o l i K5 2 12 17 2 3 17 2 — Rib. — 5 - Rib. - 5 - KDO or — Rib. —r- KDO — f 8 f P a — 2 | f P P Rib f E. c o l i 6a E. c o l i K6 198 — ManNAcA ^J1 Glc ±K P 6 J 8 OAc E. c o l i K7 and K56 Rha Rha KDO -a a 7 / 8 OAc E. c o l i K12 and K82 3 1 7 2 — R i b f ^j- KDO ^ OAc E. c o l i K13 — GalNAc KDO \ a 8 P OAc E. c o l i K14 — GlcNAc KDO a p E. c o l i K15 — R i b f KDO OAc E. c o l i K20 199 — R l b f KDO -2-p-E. c o l i K23 Gal E. c o l i K27 — Glc GlcA -^A FUC I -1 B 2 or 3 Gal OAc E. c o l i K28 8 — Man -i-3- Glc -M" GlcA -M" Gal a P k 1 a Glc -M- Man pyr E. c o l i K29 — V G a l ip. l l a GlcA -^p3- Gal E. c o l i K30 2 0 0 — Gal — Glc — GlcA Rha — Rha — E. c o l i K31 ?Ac - G l c V f a V Gal GlcA E. c o l i K32 - Glc GlcA ^ Fuc a 3 2 P 31 a V l l a + 0 A c Gal E. c o l i K33 — Gal Gal A - i - 3 - Fuc i -a a a E. c o l i K42 3 1 " — Gal - 0 - P - 0 — ^ 2 OH Fru + ° A c + ° P r E. c o l i K52 GlcA -2-^ t- GlcA -L-6- Man -L-3- Man -L-3- GlcNAc - — Man — Man -^ -3- GlcNAc — or 1 1 1 2 R h a Rha CO i l l K85 201 — GlcA - i r FucNAc GlcNAc -^ -6- Gal — l l 6 j Glc 2-OAc E. c o l i K87 — NANA5Ac NANA5Ac - 2-r a a E. c o l i K92 — R i b f ^ r i b i t o l — 0 E. c o l i K100 0 II T 1 -202 References K l E.J. McGuire and S.B. B i n k l e y , Biochemistry, 3_ (1964) 247-251. K2 K. Jann, B. Jann, and A.M. Schmidt,J. Bact., 143 (1980) 1108-1115. K5 W.F. Vann, M.A. Schmidt, B. Jann, and K. Jann, Eur. J . Biochem., 116 (1981) 359-364. K6 P. Messner and F.M. Unger, Biochem. Biophys. Res. Commun., 96, (1980) 1003-1010. K6a H.J. Jennings, K.-G. R o s e l l , and K.G. Johnson, Carbohydr. Res., 105 (1982) 45-56. K7=K56 F.-P. T s u i , R.A. Boykins, and W. Egan, Carbohydr. Res., 102 (1982) 263-271. K12=K82 M.A. Schmidt, B. Jann, and K. Jann, FEMS M i c r o b i o l . L e t t . , 14 (1982) 69-74. K13 W.F. Vann and K. Jann, I n f e c t . Immun., 25 (1979) 85-92. W.F. Vann, T. Soderstrom, W. Egan, F.-P. T s u i , R. Schneerson, I . 0rskov, and F. 0rskov, I n f e c t . Immun., 39 (1983) 623-929. K14 B. Jann, P. Hofmann, and K. Jann, (from K. Jann and B. Jann, Prog. A l l e r g y , 33 (1983) 53-79. K15 W. Vann, unpublished r e s u l t s . (From K. Jann and B. Jann, Prog. A l l e r g y , 33 (1983) 53-79). K20.K23 W.F. Vann, T. Soderstrom, W. Egan, F.-P. T s u i , R. Schneerson, I . 0rskov, and F. 0rskov, I n f e c t . Immun., 39 (1983) 623-629. K27 K. Jann, B. Jann, K.F. Schneider, F. 0rskov, and I . 0rskov, Eur. J . Biochem., 5_ (1968) 456-465. A.K. Chakraborty, Macromol. Chem., 183 (1982) 2881-2887. K28 E. Altman and G.G.S. Dutton, Carbohydr. Res., ( i n p r e s s ) . K29 Y.-M. Choy, F. Fehmel, N. Frank, and S. Stirm, J . V i r o l . , 16 (1975) 581-590. K30 A.K. Chakraborty, H. F r i e l b o l i n , and S. St i r m , J . B a c t e r i o l . , 141 (1980) 971-972. K31 K. Jann, unpublished r e s u l t s . (From I . 0rskov, F. 0rskov, B. Jann, and K. Jann, B a c t e r i o l . Rev., 41 (1977) 667-710. 203 K32 E. Altman, unpublished r e s u l t s . K33 B.A. Lewis, unpublished r e s u l t s . K42 H. Niemann, A.K. Chakraborty, H. F r i e b o l i n , and S. St i r m , J . B a c t e r i o l . , 133 (1978) 390-391. K52 P. Hofmann, B. Jann, and K. Jann, I n t . Symp. Carbohydr. Chem., 12th, 1984 A b s t r a c t s , p. 367. K85 K. Jann, B. Jann, F. 0rskov, and I . 0rskov, Biochem. Z., 346 (1966) 368-385. K87 L. Tarcsay, B. Jann, and K. Jann, Eur. J . Biochem., 23, (1971) 505-514. K92 W. Egan, T.-Y. L u i , D. Dorow, J.S. Cohen, J.D. Robbins, E.C. G o t s c h l i c h , and J.B. Robbins, Biochemistry, 16 (1977) 3687-3692. K100 W. Egan, F.P. T s u i , R. Schneerson, and J.B. Robbins, J . B i o l . Chem., ( i n p r e s s ) . 204 APPENDIX III XH AND 13C-n.m.r. SPECTRA Spectrum No. 1 j i 1 1 ' • 1 » ' ' T 4.0 3.0 K50 Polysaccharide C—n.m.r. 100.6 MHz, 90° 99.16 100 Spectrum No. 2 K50 Degraded Polysaccharide C-n.m.r. 20.1 MHz, ambient temp. 103.83 Spectrum No. 3 ho O To K50 Polysaccharide Compound A3 1 3 1 2 1 3 GlcA Man Man Gal^OH a a a H-n•m.r. 100 MHz, 90° 5.31 Spectrum No. 4 K50 Polysaccharide Compound Aj£ Gl c A ^ — ^ l a n ^ M a n ^ G a l ^ O H 13 C-n.m 20.1 MH;;, anbient :emp r; 103 i 101. 7 f !9 -\t _L. .95 ,93 41 i i o o/ J I L 100 K50 Polysaccharide Compound A2 1 3 1 2 GlcA Man ManMDH a a 1„ H-n.m. r. 100 MHz, 90° 5.37 5.32 -i—i—i—i—|—i—i—I—i—I—I—i—I—I | i i r 5.0 Spectrum No. 6 -I—i | I—I—i—I—[—i—i—I—I | i I i r 2.0 100 K50 Polysaccharide Compound GlcA^-Alan^OH „ H-n.m.r. 100 MHz, 90° ?7o JTo Spectrum No. 8 K50 Polysaccharide Compound SHI J r\/\f\j C0 2H CHOH 1 3 1 2 I Ga l — r - G l c OCH 8 a | CH OH 1 3 0 C—n.m.r. 400 MHz, ambient temp. 4.65 T " 5.0 Spectrum No. 9 i' 3.0 K50 Polysaccharide Compound SHI T V 104.11 CH0H Gal-Lr^GlcrL-^OCH 6 a | CH20H H-n.m.r. 100.6 MHz, ambienjt temp, 99.62 1— 100 Spectrum No. 10 acetone 31.07 E. c o l l K28 Polysaccharide 13_ C-n.m. r. 100.6 MHz, ambient temp. I'OO 90 8b 70 Spectrum No. 12 acetone 31.07 to I—1 16.02 ,15.97 AO 30 20 E. c o l i K28 Deacetylated Polysaccharide L—n. m. r • 100.6 MHz, ambient temp. Spectrum No. 15 acetone 31.07 0 K3 i — 1 -r— 1 " 1 1 — 1— 5.0 4.0 3.0 2.0 E. c o l i K28 Compound A^ 1- 4 GlcA——Fuc^OH ~^C—n. m. r. 20.1 MHz, 95 c iL03J8A -97 .100 04~ •t-i 3:. to i... •r~ 90 80 Spectrum No. 17 E. c o l i K28 Compound Nl G a l - V ^ l c % O H p H-n.m. r. 4.00 MHz, ambient temp. | . ; ! .: ! ! i . j • I i i . ; : . j I I- i : . I I 1 ! ' ' i 5.23 — i — 4.0 - i — 3.0 223 E. c o l i K32 Polysaccharid ^H-n.m.r. 400 MHz, 95° E. c o l l K32 Deacetylated Polysaccharide *H-n.m.r. 400 MHz, 95° 5.0 4 Spectrum No 1 . 22 .34 acetone 2.23 — i — 2.0 E. c o l i K32 Deacetylated P o l y s a c c h a r i d e C—n.m.r. 100.6 MHz, ambient temp. Spectrum No. 23 acetone 31.07 18.01 E. c o l l K28/bacteriophage <p28—1 H^-n.m.r. -400 MHz, 95° 5.0 r 4.0 E. c o l i K28/bacteriophage <j>28-l 575 ~ ' : ATO _ , Spectrum No. 25 acetone 2.23 2.0 r E. c o l i K32/bacteriophage $32 F r a c t i o n I 1„ H-n.m.r. 400 MHz, 95° E . c o l i K 3 2 / b a c t e r i o p h a g e <j>32 F r a c t i o n I I ^ H - n . m . r . 400 MHz, 95° S p e c t r u m N o . 27 1 . 1 2 5.0 4.0 2 . 0 

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