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Structural analysis of the capsular polysaccharide of Escherichia coli K34 and studies of the glycanase… Kuma-Mintah, Agyeman 1985

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STRUCTURAL ANALYSIS OF THE CAPSULAR POLYSACCHARIDE OF ESCHERICHIA COLI K34 AND STUDIES OF THE GLYCANASE ACTIVITY OF SPECIFIC BACTERIOPHAGE ENZYMES BY AGYEMAN KUMA-MINTAH .Sc. (Hons.), U n i v e r s i t y of Science and Technology, Ghana, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER, 1985 Agyeman Kuma-Mintah. 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date i g l t f , b^cewgiPiZ, i f gr DE-6(3/81) i i ABSTRACT To further the understanding of the chemical basis f o r s e r o l o g i c a l d i f f e r e n t i a t i o n , 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 capsular polysaccha-r i d e s , produced by the various Escherichia c o l i (E. c o l i ) s t r a i n s has been embarked upon i n t h i s and other l a b o r a t o r i e s . The s t r u c t u r a l e l u c i d a t i o n of E. c o l i K34 capsular polysaccharide and r e l a t e d studies are reported i n t h i s communication. N.m.r. spectroscopy and sugar analysis data on E. c o l i K34 capsular polysaccharide indicated that i t consists of a pentasaccharide repeating u n i t with glucose, galactose and glucuronic a c i d as i t s sugar components. The nature of the anomeric linkages was established by ^H-n.m.r. spectroscopy and confirmed by the r e s u l t s of oxidation of the f u l l y acetylated polysaccharide with chromic a c i d . Methylation, periodate oxidation - Smith hydrolysis and uronic a c i d degradation studies on the capsular polysaccharide and on the carboxyl reduced polymer, of E. c o l i K34 show the structure to co n s i s t of a repeating u n i t , - G l c A p ^ D - Galpi-^D-Galpi-0 3 0 0 0 1 D-Galp 4 a 1 D-Glcp i i i A l l the sugar residues i n t h i s capsular polysaccharide were assigned the D-configuration from c i r c u l a r dichroism measurements. The D configura-t i o n of the a-glucose was confirmed by treatment of E. c o l i K34 bacteriophage generated oligosaccharide with a-D-glucosidase. From the r e s u l t s of the cross-reaction between E. c o l i K34 and other group 09 E. c o l i s t r a i n s (K28, K31, K32 and K33), we suggest that the immunodominant sugar of E. c o l i K34 capsular polysaccharide may ei t h e r be 1 — > 2 li n k e d glucuronic acid or the terminal glucose. The occurrence of 1 — > 2 li n k e d glucuronic a c i d i n E. c o l i K34 polysaccharide i s the f i r s t to be reported i n b a c t e r i a l polysaccharide. In t h i s study E. c o l i K34 and K31 capsular polysaccharides were depolymerized using t h e i r corresponding bacteriophages. Characteriza-t i o n of the bacteriophage generated oligosaccharides revealed that the E. c o l i K34 and K31 bacteriophage-borne glycanases are galactosidase and glucosidase r e s p e c t i v e l y . iv TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF APPENDICES v i i LIST OF TABLES v i i i LIST OF FIGURES ix LIST OF SCHEMES X ACKNOWLEDGEMENTS x i I. INTRODUCTION 1 II. METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES 11 11.1 Isolation and purification 12 11.2 Sugar analysis 14 11.2.1 Total hydrolysis 14 11.2.2 Characterization and quantitation of sugars 16 11.2.3 Determinationof the configuration (D or L) of sugars 17 11.3 Position of linkage 18 11.3.1 Methylation analysis 18 11.3.2 Characterization and quantitation of methylated sugars 22 V 11.4 Sugar sequence 23 11.4.1 Periodate oxidation and Smith hydrolysis 23 11.4.2 Uronic acid degradation 26 11.4.3 Bacteriophage degradation 29 11.5 Determination of anomeric resonance 30 11.5.1 Nuclear magnetic resonance spectroscopy 30 11.5.1.1 ^H-n.m.r. spectroscopy 30 11.5.1.2 l^C-n.m.r. spectroscopy 35 11.5.2 Chromium trioxide oxidation 37 11.5.3 Other techniques 38 III. .RESULTS AND DISCUSSION 40 111.1 Composition and n.m.r. studies 41 111.2 Chromium trioxide oxidation 44 111.3 Methylation analysis 44 111.4 Periodate oxidation - Smith hydrolysis 46 111.5 Selective Smith degradation 49 111.6 Determination of the configuration (D or L) of the sugar 50 111.7 Isolation of bacteriophages ((f>31 and <£34) and cross-reactions 51 111.8 Depolymerization with E. c o l i K31 bacteriophage (c£31) 54 111.9 Depolymerization with E. c o l i K34 bacteriophage (<£34) 56 IV. EXPERIMENTAL IV.1 General methods 61 62 v i IV.2 I s o l a t i o n and p u r i f i c a t i o n of E. c o l i K34 capsular polysaccharide 66 IV.3 Sugar analysis and composition 67 IV.4 Chromium t r i o x i d e oxidation 69 IV.5 Methylation analysis 69 IV.6 Uronic a c i d degradation 71 IV.7 Carbodiimide reduction of K34 polysaccharide 71 IV.8 Periodate oxidation and Smith hydrolysis of carboxyl reduced K34 polysaccharide 72 IV.9 S e l e c t i v e Smith degradation 74 IV.10 Determination of the configuration (D or L) of the sugars 75 IV.11 S e r o l o g i c a l cross-reactions 75 IV.12 Bacteriophage depolymerization of E. c o l i K31 capsular polysaccharide 76 IV.13 Bacteriophage depolymerization of E. c o l i K34 capsular polysaccharide 77 V. BIBLIOGRAPHY 80 V l l LIST OF APPENDICES Appendix Page I The known structures of the Escherichia c o l i K antigens 87 II Mass Spectra 96 III -^H and 13C-n.m.r. spectra 103 v i i i LIST OF TABLES Table Page 1.1 Familia Enterobacteriaceae II.1 Three regions of a carbohydrate n.m.r. spectrum 33 111.1 Sugar analysis of K34 polysaccharide and derived products 42 111.2 N.m.r. data for E. c o l i K34 capsular polysaccharide and derived products 43 111.3 Methylation analysis of K34 polysaccharide and derived products 45 111.4 Configuration of sugar residues of E. c o l i K34 capsular polysaccharide 51 III. 5 Propagation of bacteriophage #31 '54 111.6 Determination of the reducing end of E. c o l i K31 oligosaccharide isolated after bacteriophage degradation of E. c o l i K31 polysaccharide 55 111.7 Propagation of bacteriophage #34 56 111.8 Proton n.m.r. data (400 MHz) for the oligo-saccharide generated in bacteriophage depolymerization of E. c o l i K34 capsular polysaccharide 58 111.9 Determination of the reducing end of E. c o l i K34 oligosaccharide isolated after bacteriophage degradation of E. c o l i K34 polysaccharide 59 ix LIST OF FIGURES Figure Page 1.1 Schematic representation of the Gram-positive and Gram-negative c e l l wall of bacteria 4 1.2 Basic morphological types of bacteriophages with the types of nucleic acid 7 1.3 The mechanics of inf e c t i o n by bacteriophage 9 1.4 The structures of capsular polysaccharides of K l e b s i e l l a K58 and Escherichia c o l i K33 10 11.1 Mass spectra of (a) 1,3,4,5-tetra-O-acetyl 2,6-dimethylgalactitol (b) 1,2,5,6-tetra-0-3,4-dimethylglucitol 24 11.2 Structure of K l e b s i e l l a K44 bacteriophage oligosaccharide 30 11.3 Relationship between dihedral angle (#) and coupling constants for a- and /?- hexoses 34 111.1 Calibration graph of absorbance versus I O 4 V I O 3 " 47 111.2 Periodate consumption by K34 polysaccharide with respect to time 47 111.3 Separation of the depolymerization products of E. c o l i K34 by gel-permeation chromatography (Bio-Gel P-2) 57 X LIST OF SCHEMES Scheme Page II.1 Methylation analysis of E. c o l i K34 polysaccharide 20 II.2 Fragmentation patterns of a l d i t o l d e r i v a t i v e s 21 II.3 Selective Smith degradation of E. c o l i K34 polysaccharide 27 x i ACKNOWLEDGEMENTS I would l i k e to express my sincere gratitude to Professor G.G.S. Dutton f o r h i s guidance and i n t e r e s t throughout the course of this t h e s i s . I am thankful to my colleagues i n the laboratory f o r t h e i r encouragement and h e l p f u l discussions. My g r a t e f u l thanks to Dr. S.O. Chan and the s t a f f of the n.m.r. services and Dr. G. Eigendorf and the s t a f f of the mass spectrometry services f or t h e i r assistance. My s p e c i a l thanks to Rani Theeparajah for typing t h i s t h e s i s . x i i DEDICATED TO MY GRANDPARENTS - 1 -CHAPTER I INTRODUCTION 2 I. INTRODUCTION Carbohydrates are polyhydroxyaldehydes or ketones, u s u a l l y i n the acetal or hemiacetal forms or substances which may be hydrolyzed by d i l u t e a c i d to these compounds. Carbohydrate-containing macromolecules are of widespread occurrence i n most l i v i n g organisms^. Polysaccharides are carbohydrate polymers but may not be confined s o l e l y to O-glycosidi-c a l l y l i n k e d carbohydrates. Polysaccharides are of great commercial^ and b i o l o g i c a l ^ importance. The organism Escherichia c o l i belongs to the family Enterobacteriaceae whose normal habitat i s the i n t e s t i n a l t r a c t of man and animals^. E. c o l i f i r s t i s o l a t e d from faeces by Escherich i n 1885, i s often found i n human urinary t r a c t i n f e c t i o n s and i s associated with severe i n f a n t i l e diarrhea^. Enterobacteriaceae are Gram-negative b a c t e r i a and c l a s s i f i c a t i o n of the family (Enterobacteriaceae) by Edwards and Ewing^ (shown i n Table 1.1) has been updated by Kauffmann^. An i n t e r e s t i n Escherichia c o l i i n recent years from both human and v e t e r i n a r y medicine has been followed by an i n t e r e s t i n the surface structure of these b a c t e r i a because of t h e i r s p e c i a l r o l e i n pathophysiological process, t h e i r usefulness i n epidemiological studies and t h e i r importance for the normal immunological status of the host. Escherichia c o l i are Gram-negative b a c t e r i a which produce e x t r a c e l l u l a r polysaccharides. S i m p l i f i e d pictures of the b a c t e r i a l c e l l f o r a Gram-negative and Gram-positive b a c t e r i a are shown i n Fig. 1.1. These polysaccharides, together with the f l a g e l l a H antigen 3 TABLE 1.1 T r i b e s Genera A. E s c h e r i c h e a e 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 . S a l m o n e l l a i v . C i t r o b a c t e r B. K l e b s i e l l e a e i . K l e b s i e l l a i i . E n t e r o b a c t e r i i i . H a f n i a i v . S e r r a t i a C. P r o t e a e i . P r o t e u s i i . M o r g a n e l l a 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 c o n s t i t u t e t h e p r i n c i p a l immunogens and a n t i g e n s o f the E. c o l i b a c t e r i a . 0, H and K a n t i g e n s a r e l o c a t e d on the c e l l s u r f a c e o f E.  c o l i b a c t e r i a ( F i g . 1.1) and c o u l d s t i m u l a t e p r o d u c t i o n o f a n t i b o d i e s A -Copula' Polyiotchorid* .Xapfjlo' Prottm M t n o r Of C t l l Copult r\jptidoglyeon With TtKhoK Acid Polymers with iviOkis fntmbron* proteins, tnzrmts one Ctll Woll Cytoplasmic Membrane The envelope of the Gram-positive c e l l wall The envelope of the Gram-negative c e l l wall Fig. 1.1 Schematic representation of the Gram-positive and the Gram-negative c e l l wall of bacteria. From T.J. Mackie and J.E. McCartney, "Medical Microbiology", Vol. 1: "Microbial Infections", 13th edn., Churchill Livingstone, Edinburgh, 1978. - 5 -(immunoglobulins produced by immune system which i n t e r a c t with antigens). As surface components of the ba c t e r i a , these antigens are implicated i n the complex host versus micro-organism i n t e r a c t i o n and are not only responsible f o r the stimulation of the human immune system against the invading b a c t e r i a but also f o r the vir u l e n c e of the encapsulated b a c t e r i a . 0 and K antigens were shown to be carbohydrate i n nature by Toenniessen^>^ i n 1914. The 0 antigen i s the 0 - s p e c i f i c polysaccharide of the c e l l wall lipopolysaccharide. I t i s a thermostable surface antigen (the b a c t e r i a keep t h e i r immunogenic, agglutinating and agglutinin-binding capacity a f t e r b o i l i n g . ) The structure and known properties of b a c t e r i a l lipopolysaccharides have been reported-^ >11>12 The H antigen are heat l a b i l e proteins contained i n the f l a g e l l a of motile E. c o l i b a c t e r i a . Most E. c o l i s t r a i n s have a unique K antigen. The K antigens are capsular and envelope antigens and a l l are polysaccharides except f o r two that are proteins (K88 and K99). These polysaccharides are made up of oligosaccharide repeating u n i t s . For a cl e a r understanding of the chemical basis of the antigenic s p e c i f i c i t y of the E. c o l i capsular antigens, t h e i r structures need to be elucidated. The K antigens consist of three groups (A, B and L). By ele c t r o p h o r e t i c means two groups of K antigens can be d i f f e r e n t i a t e d : those with high e l e c t r o p h o r e t i c m o b i l i t y (L antigen) and those with very low ele c t r o p h o r e t i c m o b i l i t y (A and B antigens). Inspection by electron microscopy revealed that the a c i d i c polysaccharides with low molecular weight (high electrophoretic mobility) form t h i n patchy capsules while - 6 -t h o s e w i t h h i g h m o l e c u l a r w e i g h t ( l o w 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 c o p i o u s c a p s u l e s . I t has been shown t h a t E. c o l i s t r a i n s w i t h 0 and K a n t i g e n s e x h i b i t i n g t he same i m m u n o e l e c t r o p h o r e t i c p a t t e r n c o u l d cause t h e same disease-'--'. B a c t e r i a a r e r e c o g n i z e d by the immune system o f a h o s t t h r o u g h a n t i b o d y - a n t i g e n i n t e r a c t i o n s . The a n t i b o d i e s p r o d u c e d by t h e immune sy s t e m o f man o r a n i m a l s a g a i n s t a p a r t i c u l a r E. c o l i s t r a i n may be dependent on the a n t i g e n i c s p e c i f i c i t y o f the K a n t i g e n o f the s t r a i n i n q u e s t i o n . I t i s known t h a t o n l y a r e l a t i v e l y s m a l l p o r t i o n o f a p o l y s a c c h a r i d e i s t h e major s i t e o f a n t i b o d y s p e c i f i c i t y and t h i s p a r t i s termed t h e d e t e r m i n a n t g r o u p i e . A d e t e r m i n a n t group may c o n s i s t o f s e v e r a l m o n o s a c c h a r i d e r e s i d u e s , one o f w h i c h c o n t r i b u t e s most t o t h e s p e c i f i c i t y . T h i s m o n o s a c c h a r i d e r e s i d u e i s termed the immunodominant s u g a r . C e r t a i n n o n - c a r b o h y d r a t e groups such as p y r u v a t e and 0 - a c e t y l 1 9 may f u n c t i o n as a n t i g e n i c determinants- 1- . S t r u c t u r e e l u c i d a t i o n o f the a n t i g e n ( p o l y s a c c h a r i d e ) i s n e c e s s a r y f o r the i d e n t i f i c a t i o n o f the a n t i g e n i c d e t e r m i n a n t . B a c t e r i o p h a g e s (40 a r e v i r u s e s t h a t i n f e c t b a c t e r i a and m u l t i p l y w i t h i n them. B a c t e r i o p h a g e s a r e c l a s s i f i e d i n t o m o r p h o l o g i c a l g r o u p s ^ ^ (see F i g . 1.2). Most a r e s p e c i f i c r e g a r d i n g the s p e c i e s o f b a c t e r i a t h e y w i l l i n f e c t a l t h o u g h some have a b r o a d o r l e s s r e s t r i c t e d h o s t r a n g e . T h i s s p e c i f i c i t y depends on the p r e s e n c e o f a s p e c i f i c r e c e p t o r s i t e on t h e c e l l s u r f a c e . P r o t e a s e can be us e d t o de m o n s t r a t e the b a c t e r i o p h a g e r e c e p t o r s i t e f o r E. c o l i s t r a i n s - * - - ^ . The v i r a l i n f e c t i o n o f a h o s t by b a c t e r i o p h a g e s i s u s u a l l y c h a r a c t e r i z e d by f o u r phases. These a r e : ( i ) a d s o r p t i o n o f t h e phage p a r t i c l e onto the s u s c e p t i b l e 7 2-DNA 2-DNA 2-DNA 1-DNA 1-RNA 1-DNA Fig. 1.2 Basic morphological types of bacteriophages with the type nucleic acid (from ref. 100). - 8 -host, ( i i ) i n j e c t i o n of the v i r a l DNA (or RNA) into the host, ( i i i ) r e p l i c a t i o n of the phage nuc l e i c a c i d and phage p r o t e i n at the expense of the metabolic process of the host, (iv) phage maturation and release which r e s u l t s i n the l y s i s of the host c e l l (see F i g . 1.3). Bacterio-phage -borne glycanases-'-O-'- may be hydrolases- 1^ or lyases-*-^, 104 These enzymes (glycanases), which occur i n bacteriophages, are generally s p e c i f i c f o r one or a few substrates. K i n e t i c studies on these bacteriophage-borne enzymes revealed that the a c t i v i t y i s i n h i b i t e d by products and high substrate concentrations^-^. Bacteriophage-borne enzymes (glycanases) are capable of depolymerizing polysaccharides into oligosaccharide repeating units without the removal of the possibly immunologically s i g n i f i c a n t O-acetyl and pyruvic a c e t a l groups-^»20 Bacteriophage-depolymerization and r e l a t e d studies are an important t o o l i n s t r u c t u r a l studies on carbohydrate polymers. Dutton and coworkers are c u r r e n t l y i n v e s t i g a t i n g the p o s s i b i l i t y of e l u c i d a t i n g the structure of E. c o l i capsular polysaccharides using bacteriophage degradation, 2D-n.m.r. spectroscopy and F.A.B. mass spectrometry. Stirm and Rieger-Hug employing seventy-four 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 , tested the host range of f i f t y - f i v e K l e b s i e l l a bacteriophages^. These 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 cross-reacting with none). Recently Beynon reported a study of the cross-absorption of c e r t a i n E.  c o l i bacteriophages with a number of E. c o l i s t r a i n s ^ - ^ . Antigens cross react when t h e i r immunodominant groups are s i m i l a r i n nature. Heidelberger has used cross-reactions extensively i n the p r e d i c t i o n of 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 - 9 -Fig. 1.3 The Mechanics of Infection by Bacteriophage A. Free phage. B. Phage attaches to c e l l wall with fibres, base plate in close contact with outer layers of c e l l wall. C. Sheath contracts and central core is pushed through the c e l l wall and DNA transfer begins. D. Transfer of DNA completed. Phage head is now empty and early events of phage growth cycle begins. (From T.J. Mackie and J.E. McCartney, "Medical Microbiology", Vol. 1, "Microbial Infections", 13th edn., Churchill Livingstone, Edinburgh, 1978). - 10 -c h e m i c a l l y ^ ~ . Dutton et a l . observed that E. c o l i K33 cross reacted with K l e b s i e l l a K58 since t h e i r capsular polysaccharides are i d e n t i c a l i n structure ( Fig. 1.4)21. I t i s c l e a r that b a c t e r i a l antigens of the type discussed above play a r o l e i n pathogenic processes. The understanding of these roles c a l l s f o r chemical analysis of b a c t e r i a l antigens, which has been embarked upon. In t h i s study, the structure of Escherichia c o l i K34 polysaccharide (K antigen) was elucidated and the nature of the antigenic determinant was deduced. In the course of t h i s thesis two E.  c o l i capsular polysaccharides (K31 and K34) were depolymerized with t h e i r respective bacteriophage (#31 and #34) and the degradation products characterized. —>3( -a-D-Glcp.- (1—>4) -/3-D-GlcpA- (1—>) -a-L-Fucp- (l-> 3 2 \ / C / \ Me COOH OAc a-D-Galp_ Klebsiella K58 >3(-a-D-Glcp.- (1—>4) -0-D-GlcpA-(1—>) -o-L-Fucp- (l-> 3 2 \ / C / \ Me COOH +0Ac Q-D-Galp. E. c o l i K33 Fig. 1.4 The structures of capsular polysaccharides of Klebsiella K58 and Escherichia c o l i K33. - 11 -CHAPTER II METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES 12 II . METHODOLOGY OF STRUCTURAL ANALYSIS OF POLYSACCHARIDES B a c t e r i a l p o l y s a c c h a r i d e s have complex and immensely d i v e r s i f i e d s t r u c t u r a l p a t t e r n s ^ . 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 have such c o m p l e x i t y and t h e i r s t r u c t u r a l e l u c i d a t i o n n e c e s s i t a t e s t h e use o f d i f f e r e n t c h e m i c a l methods as w e l l as methods i n v o l v i n g t h e use o f enzymes. The methodology o f s t r u c t u r a l s t u d i e s o f p o l y s a c c h a r i d e s i n c l u d e s ( i ) 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 o f t h e su g a r c o n s t i t u e n t s ; ( i i ) a n a l y s i s f o r n o n - c a r b o h y d r a t e s u b s t i t u e n t groups ( O - a c e t y l , N-a c e t y l , p h osphate e t c . ) ; ( i i i ) d e t e r m i n a t i o n o f the l i n k a g e c o n f i g u r a t i o n ; ( i v ) d e t e r m i n a t i o n o f the p o s i t i o n o f l i n k a g e ; (v) d e t e r m i n a t i o n o f the su g a r sequence. Some known a n a l y t i c a l methods employed f o r t h e s e g o a l s a r e d e s c r i b e d i n t h e f o l l o w i n g s e c t i o n s . II.1 ISOLATION AND PURIFICATION 2 3" 2 7 A major t a s k i n p o l y s a c c h a r i d e c h e m i s t r y i s o b t a i n i n g t h e m a t e r i a l under i n v e s t i g a t i o n i n a pure form. I n t h i s s t u d y t h e p o l y s a c c h a r i d e o f i n t e r e s t i s the K a n t i g e n . The i s o l a t i o n and p u r i f i c a t i o n p r o c e s s 13 -involves three stages: ( i ) b a c t e r i a growth and harvest of crude polysaccharide components; ( i i ) the i s o l a t i o n of the polysaccharide such that i t i s free from low molecular weight material and other high molecular weight material, and ( i i i ) i s o l a t i o n of a s i n g l e , monodispersed, polysaccharide species. The p u r i t y of the polysaccharide, the absence of heterogeneity, rather than the presence of homogeneity can be demonstrated by many independent techniques such as nuclear magnetic resonance spectroscopy, o p t i c a l r o t a t i o n measurements, electrophoresis^>24 and gel-permeation chromatography'1 . Escherichia c o l i b a c t e r i a serotype K31 and K34 were received as stab cultures from Dr. Ida Orskov (Copenhagen). These b a c t e r i a l culture were streaked on Mueller-Hinton agar plates at 37°C u n t i l large, i n d i v i d u a l capsular colonies were obtained. E. c o l i K34 was grown by i n o c u l a t i n g Mueller-Hinton broth medium with s i n g l e colonies and incubating the r e s u l t ant l i q u i d culture at 37°C on Mueller-Hinton agar f o r four days. The lawn of capsular b a c t e r i a was harvested by scraping from the agar surface. The K34 b a c t e r i a were k i l l e d by adding phenol s o l u t i o n and s t i r r i n g the mixture for f i v e hours. The phenol i n t h i s mixture (polysaccharide component plus b a c t e r i a debris) was dialyzed out. The polysaccharide components were separated from the high molecular components (e.g. b a c t e r i a l 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 . I s o l a t i o n from aqueous s o l u t i o n by the a d d i t i o n of a water-miscible - 14 -9 ft solvent^-" (e.g. ethanol, acetone) r e s u l t s i n the p r e c i p i t a t i o n of neutral and a c i d i c polysaccharides. The resultant s t r i n g y p r e c i p i t a t e was dissol v e d i n water and treated with C e t a v l o n 2 7 ( c e t y l t r i m e t h y l -ammonium bromide) solu t i o n , which s e l e c t i v e l y p r e c i p i t a t e d the a c i d i c polysaccharide. The neutral polysaccharide present, remained i n s o l u t i o n and the p r e c i p i t a t e ( a c i d i c polysaccharide) was separated from the supernatant by ce n t r i f u g a t i o n . D i s s o l u t i o n of the p r e c i p i t a t e i n 4M sodium chlo r i d e , p r e c i p i t a t i o n with ethanol and c e n t r i f u g a t i o n were c a r r i e d out twice. The f i n a l p r e c i p i t a t e was dissolved i n water, dia l y z e d f o r two days and freeze d r i e d to y i e l d the p u r i f i e d E. c o l i K34 capsular polysaccharide. I s o l a t i o n and p u r i f i c a t i o n of E, c o l i K31 was c a r r i e d out as described for E. c o l i K34. Chromatographic techniques l i k e paper chromatography, gel-permeation and ion-exchange chromatography may be used to enhance the p u r i t y and homogeneity of capsular polysaccharides. Traces of low molecular weight contaminants i n both E. c o l i K34 and E. c o l i K31 capsular polysaccharides were removed by gel-permeation chromatography (Bio-Gel P2). II.2 SUGAR ANALYSIS II.2.1 Total h y d r o l y s i s 2 8 " 3 3 The i n i t i a l step i n the s t r u c t u r a l study of a polysaccharide i s the quantitative a c i d hydrolysis of the polysaccharide into i n d i v i d u a l 9 p monosaccharides with minimum degradation. D u t t o n z o reviewed the - 15 advantages and disadvantages i n the use of d i f f e r e n t acids. Sulphuric acid, hydrochloric acid, formic a c i d and t r i f l u o r o a c e t i c a c i d are the most commonly used acids. T r i f l u o r o a c e t i c a c i d i s e a s i l y removed under diminished pressure and thus i s being used i n c r e a s i n g l y instead of the mineral acids. The conditions of hydrolysis must be c a r e f u l l y chosen and c o n t r o l l e d . In a t t a i n i n g the correct hydrolysis condition the hydrolyzate may be monitored by paper chromatography or h.p.l.c. The quantitative hydrolysis of E. c o l i K34 polysaccharide into i t s i n d i v i d u a l monosaccharides with minimum degradation was attained using 2M t r i f l u o r o a c e t i c a c i d for 20 hours. The h y d r o l y t i c rates of g l y c o s i d i c linkages vary greatly. Uronosyl linkages i n a c i d i c polysaccharides are more r e s i s t a n t to a c i d hydrolysis because of the presence of electron ' acceptor carboxyl groups which s t a b i l i z e s the uronosyl linkages through the h e t e r o c y c l i c oxygen. One of the means of overcoming the resistance of the uronosyl linkage to acid h y d r o l y s i s , i s the reduction of a l l carboxyl f u n c t i o n a l i t i e s i n the a c i d i c polysaccharide by d e r i v a t i z a t i o n into carbodiimide 2^ d e r i v a t i v e s followed by sodium borohydride reduction. An a l t e r n a t i v e method of reduction developed i n our l a b o r a t o r y 3 ^ to overcome the resistance of uronosyl linkages to a c i d h y d r o l y s i s , involves methanolysis of the g l y c o s y l linkages with the simultaneous e s t e r i f i c a t i o n of the carboxylic acid, which i s then reduced to the corresponding alcohol. The r e s u l t i n g mixture of neutral glycosides i s hydrolyzed with 2M t r i f l u o r o a c e t i c acid, to ensure complete release of sugar residues. Methanolic hydrogen chloride has the advantage of being less destructive to deoxy sugars and s i a l i c a c i d s 3 2 , 3 3 . Karunaratne has discussed the h y d r o l y t i c conditions - 16 -Of) f o r the h y d r o l y s i s of polysaccharides containing amino sugars and would therefore not be duplicated i n t h i s t h e s i s . I I . 2. 2 Characterization and q u a n t i f i c a t i o n of sugars^ 0'- 5 4~ 4- 5 The sugars released upon a c i d hydrolysis can be analyzed q u a l i t a t i v e l y using paper chromatography^'^, high performance l i q u i d chromatography^, paper electrophoresis-^, and t h i n layer chromato-graphy-^. Colorimetric-^ •^® analysis can be used i n the c l a s s i f i c a t i o n of sugars into broad classes (hexoses, pentoses, uronic acids, deoxy or amino sugars and s i a l i c acid) but has l i m i t e d applications for i n d i v i d u a l c h r a c t e r i z a t i o n . High performance l i q u i d chromatography (h.p.l.c.) can also be used for quantitative analysis of underivatized sugars. H.p.l.c. however has a l i m i t e d number of stationary phases compared to g a s - l i q u i d chromatography. The monosaccharides, released upon a c i d h y d r o l y s i s of polysaccharide, can be converted to v o l a t i l e d e r i v a t i v e s and analyzed using g a s - l i q u i d chromatography ( g . l . c ) . G.l.c. o f f e r s reproducible q u a n t i f i c a t i o n and c h a r a c t e r i z a t i o n of sugar residues of polysaccha-r i d e s . An extensive review of t h i s technique has been reported by Dutton^• . Sugars can be analyzed as t h e i r v o l a t i l e t r i m e t h y l s i l y l (TMS) d e r i v a t i v e s but the existence of anomeric forms of sugars at e q u i l i b r i u m y i e l d s a complicated chromatograph of multiple peaks. This problem was solved by converting the a c y c l i c sugar a l d i t o l s into the v o l a t i l e acetates, t r i f l u o r o a c e t a t e s or t r i m e t h y l s i l y l ethers. A l d i t o l - 17 -t r i f l u o r o acetates show p a r t i a l d e - e s t e r i f i c a t i o n on the column and the TMS d e r i v a t i v e s of the a l d i t o l s give poor r e s o l u t i o n ^ 2 on g . l . c . A l d i t o l acetates have good r e s o l u t i o n and short r e t e n t i o n times^ 3 and therefore, were used throughout t h i s i n v e s t i g a t i o n . Sugars separated by g . l . c . are u s u a l l y confirmed by g.l.c.-mass spectrometry. 44-47 II.2.3 Determination of the configuration (D or L) of sugars In general, chromatographic separation methods and spectroscopic analyses do not d i s t i n g u i s h between enantiomers. However enantiomers can be separated by g . l . c . using a c h i r a l column or converting the enantiomers into diastereomers using c h i r a l reagents (for example, (-)-2-butanol, (+)-2-octanol, or (+)-1-phenylethanethiol) and separation on a non-chiral p h a s e ^ • ^  • ^  . The D and L configuration of sugars can be determined by the i s o l a t i o n of the d i f f e r e n t monosaccharides and measurement of t h e i r o p t i c a l r o t a t i o n [ a ] n . S p e c i f i c oxidases (e.g. D-glucose and D-galactose oxidases) and enzymes can be used for the determination of D and L configuration of sugars. The method employed i n determining the D and L configuration of sugar i n t h i s i n v e s t i g a t i o n was by c i r c u l a r d i c h r o i s n A 7 measurement at 213 nm on a l d i t o l acetates, acetylated a l d o n o n i t r i l e or p a r t i a l l y methylated a l d i t o l acetates, where the acetoxy group acts as a chromophore. -. 18 -II. 3 POSITION OF LINKAGE II.3.1 Methylation a n a l y s i s 4 8 " 5 5 This technique involves the complete e t h e r i f i c a t i o n of the free hydroxyl groups of the sugar residues i n o l i g o - and poly- saccha-r i d e s 4 8 ' 4 ^ which acts as a l a b e l i n d i s t i n g u i s h i n g the o r i g i n a l unlinked p o s i t i o n from the l i n k e d p o s i t i o n . Methylation analysis 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 carbohydrates as a means to e s t a b l i s h ( i ) linkage p o s i t i o n s ; ( i i ) number and types of sugar per repeating unit; ( i i i ) i d e n t i t y of terminal u n i t ( s ) , branching u n i t ( s ) ; and (iv) the p o s i t i o n of base-stable substituents (e.g. pyruvate). In the early days, methyl ethers were formed by repeated r e a c t i o n with dimethyl s u l f a t e and sodium hydroxide 5^. Treatment of polysaccharide with s i l v e r oxide i n b o i l i n g methyliodide gives a f u l l y methylated polysaccharide according to Purdie and I r v i n e 5 ^ . Purdie's S 9 method was considerably improved by KuhnJZ- who used N,N-dimethyl-formamide as a solvent i n conjunction with methyl iodide and s i l v e r oxide. A more convenient method for methylating polysaccharides was devised by Hakomori 5^. This involves the treatment of a polysaccharide with 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 5 4 . Most undermethylations, be i t the Hakomori method or not, are due to incomplete d i s s o l u t i o n of the sample. The s o l u b i l i t y of a polysaccharide i n the appropriate organic solvent may be enhanced by c a r e f u l d e - i o n i z a t i o n of the polysaccharide, (for example, using - 19 -Amberlite IR-120 (H +) r e s i n ) . In cases where the Hakomori methylation gives an "undermethylated" product, complete methylation can be achieved by using the Purdie method. A second Hakomori methylation i s never conducted on a methylated a c i d i c o l i g o - or poly- saccharide as t h i s w i l l r e s u l t i n /3-elimination (see Section II.4.2). Methyl trifluoromethane i s a milder base and e f f e c t s methylation without cleavage of acyl groups i n the presence of 2 , 6 - d i t e r t i a r y b u t y l p y r i d i n e and trimethylphosphate as solvent-'-'. Polysaccharides containing uronic acids may be reduced with l i t h i u m aluminum hydride a f t e r the permethylation step. The methylated material i s u s u a l l y p u r i f i e d by d i a l y s i s , extrac-t i o n and gel-permeation chromatography (Sephadex LH 20). The complete-ness of methylation can be v e r i f i e d by i . r . spectroscopy (absence of hydroxyl absorption at 3600 cm"-'-) or by analysis of the methoxyl content. The methylated polymer i s usually hydrolyzed using 2M t r i f l u o r o a c e t i c a c i d at 95°C for about 18 h. The p a r t i a l l y methylated monosaccharides released on hydrolysis are reduced to t h e i r a l d i t o l s and acetylated to give v o l a t i l e p a r t i a l l y methylated a l d i t o l acetate de r i v a t i v e s f or g . l . c . and g.l.c.-m.s. analyses (see Scheme I I . 1). Uronic acids may be i d e n t i f i e d by comparison of methylation analysis r e s u l t s of the a c i d i c polysaccharide with those of the methylated-reduced polysaccharide. 20 f <rQ}l K34 polysaccharide r O * e _ Ox . I - OH <5 . 0 * 1. 3,4-OMe .-Glucose 2. 2,6-_OMe2-Galactose 3. 2,3,6-OMe3-Galactose 4. 2,4,6-OMe3-Galactose 5. 2,3,4,6-OMe^-Glucose 1) M s I H ( 11) -E_0/s>rTldll.t OAC OAc A c O • OHe A c O • — O A c - O A C - O K f - O A c -Ont 3Ac * — O n e - O A c "OMe - O A c " O H * -OUt - OAc -0 !_ Scheme II.1 Methylation analysis of E. c o l i K3A polysaccharide 21 Primary fragmentation: QJ t 2 117 161 HCCMe + MeOCH + HCCMe I HCQAc CH2CMe 205 161 45 Secondary fragmentation: © 0 HC=CMe HC=0Me I I HCCMe -AcOH CCMe I 9 II HCOAc CH2CMe m/z 205 CH CH 2aie lVz 145 CH_0Ac I 2 HCCMe H&gfe m/z 161 -AcOH CH. I 2 CCMe B>CMe rtVz 101 C H _ — > II© H C V Q / H Me ir/z 71 HC-OMe -AcOH HCOAc CH2CMe rr/z 161 -CH2CO © HC=CMe I c=o I CH 3 rr/z 87 Scheme II.2 Fragmentation patterns of a l d i t o l derivatives - 22 -I I . 3 . 2 C h a r a c t e r i z a t i o n and q u a n t i t a t i o n of methylated s u g a r s 2 8 > 4 1 > 5 6 > 5 7 P a r t i a l l y methylated monosaccharides released on t o t a l h y d r o l y s i s of the permethylated o l i g o - or poly- saccharide can be characterized using paper chromatography^. The methylated sugars are detected with _>-anisidine hydrochloride s p r a y ^ ^ followed by heating at liO°C for 5 min. These sugars are then t e n t a t i v e l y i d e n t i f i e d according to t h e i r r e l a t i v e m o b i l i t i e s (Rf values) and the d i f f e r e n t colours formed. Ga s - l i q u i d chromatography (g.l.c.) i s the most widely used technique f o r quantitative and q u a l i t a t i v e analysis of methylated sugars. A review of the applications of g. l . c . to carbohydrate analysis has been published by D u t t o n 2 8 - 4 ! . The methylated sugars are analyzed as t h e i r p a r t i a l l y methylated a l d i t o l acetates during t h i s work. The i d e n t i f i c a t i o n and quantitation of these p a r t i a l l y methylated a l d i t o l acetates are made by consideration of the r e l a t i v e r e t e n t i o n times and co-chromatography with authentic samples. For unambiguous i d e n t i f i c a t i o n of p a r t i a l l y methylated a l d i t o l acetates, g . l . c . r e s u l t s should be confirmed using g.l.c.-m.s. Studies done on the fragmentation of p a r t i a l l y methylated a l d i t o l acetates have been r e p o r t e d 5 7 . The primary fragments are formed as a r e s u l t of f i s s i o n of the C-C bond i n the a l d i t o l chain and t h i s cleavage follows the p r e f e r e n t i a l order shown below. 23 -CHOMe CHOMe CHOAc > CHOMe CHOAc CHOAc The i n t e n s i t y of the primary ion decreases with increasing molecular weight. Secondary ions can be obtained by loss of a c e t i c a c i d (M/z 60), ketene (m/z 42), methanol (m/z 32), methyl acetate (m/z 74), methoxy-methyl acetate (m/z 104) or acetoxymethyl acetate (m/z 132). Figure II.1 i l l u s t r a t e s the difference i n the mass spectra of 1,3,4,5-tetra-0-acetyl-2,6-di-0-methyl-D-galactitol and 1,2,5,6-tetra-0-acetyl-3,4-di-O-methyl-D-glucitol. II.4 SUGAR SEQUENCE The e l u c i d a t i o n of the sequence of sugars i n a polysaccharide involves the i s o l a t i o n and ch a r a c t e r i z a t i o n of oligosaccharide fragments. Lindberg and coworkers 5 8 have reviewed the various techniques employed i n the s p e c i f i c degradation of polysaccharide. The degradation techniques employed i n t h i s i n v e s t i g a t i o n are discussed i n t h i s section. II.4.1 Periodate oxidation and Smith H y d r o l y s i s 5 9 " 6 4 Oxidative cleavage of the C-C bond of v i c i n a l d i o l s by sodium 24 »f»° ei 7f t f Bf 4f 3f i f ' 41 61 III Iff r r r I 'i i ' • i-1 i i i i i | i i1 | r i i i i i i i i | i i i i i i i i i | ' i f f 25f 3tf 3Sf '»» (a) i«f *f i f ii te ** 4f If If If f l | I "I 6f i f f _Lii ?33 ,261 I I 1 "111 '| I' V I ( I I I i I I' I I 1 I I I t I I I I I I I I H 2ff 2Bf I I | I I I I I I I I I | I I I I I I I I I | I f f I t f 4ff (b) Figure I I . 1 Mass spectra of (a) 1 , 3 , 4 , 5 - t e t r a - 0 - a c e t y l - 2 , 6 -d i m e t h y l g a l a c t i t o l . (b) 1 , 2 , 5 , 6 - t e t r a - 0 - a c e t y l - 3 , 4 -d i m e t h y l g l u c i t o l 25 -metaperiodate i s of importance i n the s t r u c t u r a l determination of polysaccharides-^ a n d i t s uses are two f o l d . F i r s t , as an a n a l y t i c a l technique using small amounts of material and secondly, as a preparative technique namely Smith degradation^. Oxidations are usually c a r r i e d out i n aqueous media with the water soluble metaperiodate ion. Over-oxidation may be prevented by performing the r e a c t i o n i n the dark at about 4°C 60_ x;he periodate consumption can be monitored spectro-photometrically^O a n d the r e s u l t s i l l u s t r a t e the number of periodate s e n s i t i v e sugars per repeating u n i t i n a polysaccharide. Except for terminal side-chains and 1-6 l i n k e d sugars, one mole of periodate i s consumed for every oxidizable sugar i n a repeating u n i t ^ . The "polyaldehyde" produced, on periodate oxidation of a polysaccharide, i s u s u a l l y reduced with sodium borohydride into the p o l y o l . Cis g l y c o l s are observed to oxidize f a s t e r than trans and some trans g l y c o l are r e s i s t a n t to oxidation i f f i x e d i n an unfavourable conformation"''. Ebisu e t . a l . s e l e c t i v e l y oxidized the terminal /3-D-galactopyranosyl residues i n the Pneumococcus S-14 polysaccharide leaving the 1 — > 4 linked /3-D-glucose units i n the main chain i n t a c t ^ 3 . In a s i m i l a r exercise, the s e l e c t i v e oxidation of the terminal D-glucose, and 1 — > 4 l i n k e d galactose over 1 — > 2 l i n k e d glucuronic acid i n E. c o l i K34 was achieved. For a n a l y t i c a l purpose, methylation analysis or sugar analysis i s mostly performed on small quantities of the p o l y o l . Smith degradation i s an important modification of periodate oxidation devised by Smith and co-workers . I t gives valuable information on the sequencing of sugar residues i n a polysaccharide. - 26 -S m i t h d e g r a d a t i o n i n v o l v e s p e r i o d a t e o x i d a t i o n f o l l o w e d by m i l d a c i d h y d r o l y s i s on l a r g e q u a n t i t i e s o f the r e s u l t a n t p o l y o l . T h i s m i l d a c i d h y d r o l y s i s on t h e p o l y o l r e s u l t s i n t h e c l e a v a g e o f t h e a c e t a l l i n k a g e s l e a v i n g the g l y c o s i d i c l i n k a g e s i n t a c t . S m i th d e g r a d a t i o n y i e l d s o l i g o -o r p o l y - s a c c h a r i d e s and t h e s e o l i g o - o r p o l y - s a c c h a r i d e s may be c h a r a c t e r i z e d by s u g a r a n a l y s i s o r m e t h y l a t i o n a n a l y s i s . S e l e c t i v e S m i t h d e g r a d a t i o n can be a c h i e v e d by p e r f o r m i n g m i l d a c i d h y d r o l y s i s on a p o l y o l o b t a i n e d from s e l e c t i v e p e r i o d a t e o x i d a t i o n (see Scheme I I . 3 ) . I I . 4 . 2 U r o n i c a c i d d e g r a d a t i o n ( ^ - e l i m i n a t i o n ) 5 8 • 6 5 • 6 6 > 6 7 - 6 8 Base c a t a l y z e d d e g r a d a t i o n c a n be employed t o g e n e r a t e d e f i n e d o l i g o s a c c h a r i d e f r a g m e n t s from an a c i d i c p o l y s a c c h a r i d e 5 8 ' 6 5 ' 6 6 . The c a r b o x y l i c f u n c t i o n a l i t y o f the u r o n i c a c i d i n an a c i d i c p o l y s a c c h a r i d e i s e s t e r i f i e d on m e t h y l a t i o n . The e s t e r i f i e d u r o n i c a c i d r e s i d u e i s a s t r o n g e l e c t r o n - w i t h d r a w i n g group and t h u s enhances the a c i d i t y o f the r i n g p r o t o n a t C-5. When th e m e t h y l a t e d p o l y s a c c h a r i d e i s t r e a t e d w i t h base (sodium m e t h y l - s u l f i n y l m e t h a n i d e ) , the p r o t o n a t C-5 i s removed f o l l o w e d by t h e j3-elimination o f the 4 - 0 - s u b s t i t u e n t w i t h the f o r m a t i o n o f h e x - 4 - e n o - p y r a n o s i d u r o n a t e r e s i d u e s '. The main s t e p s o f t h i s d e g r a d a t i o n a r e o u t l i n e d as f o l l o w s : - 27 ,— OH 1. NaI0 4 C0.02M 3 hrs) OH 3. NaBH^ REDUCTION hrs CHjOH CM2OH CHjOH Scheme II.3 Selective Smith degradation of E. c o l i K34 polysaccharide - 28 A s p i n a l l and R o s e l l have shown i n t h e i r experiments that, under conditions normally used for base degradations, complete loss of uronic a c i d residues occurs and that the a c i d hydrolysis i s unnecessary 6 8. The free hydroxyl group exposed a f t e r /.-elimination, can be l a b e l l e d by a l k y l a t i o n with methyl iodide, e t h y l iodide or trideuteromethyl iodide. The r e s u l t a n t a l k y l a t e d oligosaccharide i s analyzed by g.l.c.-m.s. of the p a r t i a l l y methylated a l d i t o l acetates. In the structure e l u c i d a t i o n of E. c o l i K34, the s i t e of attachment of the uronic a c i d u n i t was determined by comparing g.l.c.-m.s. r e s u l t s of the 6-elimination and that of the methylation analysis. - 29 -II. 4 . 3 Bacteriophage degradation Bacter iophages ((}>) are v i r u s e s , i n f e c t i n g t h e i r h o s t s and m u l t i p l y i n g w i t h i n them. Bacter iophages c a r r y h o s t s u r f a c e carbohydrate degrading enzymes which i n c l u d e p o l y s a c c h a r i d e d e a c e t y l a s e s 6 9 , g l y c a n a s e s 7 ^ and l y a s e s 7 ! > 7 2 _ For most b a c t e r i a l p o l y s a c c h a r i d e s there e x i s t s an i n d i v i d u a l s p e c i f i c phage c o n t a i n i n g an endoglycanase 7 -^ . Each enzyme i s capable of h y d r o l y z i n g a p a r t i c u l a r c a p s u l a r p o l y s a c c h a r i d e i n t o o l i g o s a c c h a r i d e r e p e a t i n g u n i t s w i t h i t s a c i d or base l a b i l e non-carbohydrate s u b s t i t u e n t s i n t a c t ^ 9 . Bacter iophage d e g r a d a t i o n coupled w i t h c h a r a c t e r i z a t i o n techniques ( m e t h y l a t i o n a n a l y s i s , F . A . B . - M . S . e t c . ) may be used i n the sequencing of sugar r e s i d u e s i n a p o l y s a c c h a r i d e . Bacter iophages are u s u a l l y i s o l a t e d from sewage and may be propagated on b a c t e r i a a c c o r d i n g to the s tandard procedures of A d a m s 7 4 . To depolymer ize 1 g o f a b a c t e r i a l c a p s u l a r p o l y s a c c h a r i d e , 1 0 ^ o f the c o r r e s p o n d i n g bac ter iophage may be r e q u i r e d 7 5 . The s t r u c t u r e of an o l i g o s a c c h a r i d e s i n g l e r e p e a t i n g u n i t o b t a i n e d when K l e b s i e l l a K44 was degraded by i t s c o r r e s p o n d i n g bac ter iophage i s shown i n F i g . I I . 2 . N . m . r . and m e t h y l a t i o n analyses s t u d i e s on t h i s o l i g o s a c c h a r i d e r e v e a l e d t h a t g l u c u r o n i c a c i d and glucose were the n o n - r e d u c i n g end and r e d u c i n g end r e s p e c t i v e l y 7 6 . Thus g l u c u r o n i c a c i d i s l i n k e d to one of the g lucoses i n the main c h a i n of K l e b s i e l l a K44 c a p s u l a r p o l y s a c c h a -r i d e . R e c e n t l y F . A . B . - m a s s spectrometry s t u d i e s on K44 bac ter iophage o l i g o s a c c h a r i d e conf i rmed the sequence of the sugar r e s i d u e s i n the g i v e n s t r u c t u r e 7 7 (see F i g u r e I I . 2 ) . 30 -In t h i s study E, c o l i K31 and K34 p o l y s a c c h a r i d e s were depolymer ized u s i n g t h e i r cor responding b a c t e r i o p h a g e s . ^ >G 1 c A ^ R h a i - ^ R h a ^LJ - G 1 c i - ^ G 1 c — > /3 a a p 16 a I OR R=H or Ac F i g u r e I I .2 S t r u c t u r e of K l e b s i e l l a K44 bacter iophage o l i g o s a c c h a r i d e I I . 5 DETERMINATION OF ANOMERIC LINKAGE I I . 5 . 1 N u c l e a r magnetic resonance spectroscopy I I . 5 . 1 . 1 N . m . r . spec troscopy P r o t o n magnetic resonance spectroscopy i s w i d e l y used i n c o n f i g u r a t i o n a l c o n f o r m a t i o n a l and s t r u c t u r a l a n a l y s i s o f carbohydrates and t h e i r d e r i v a t i v e s . ^ H - n . m . r . spec troscopy was f i r s t a p p l i e d to s t r u c t u r a l problems i n carbohydrates by Lemieux and c o - w o r k e r s 7 9 . The OA i n t r o d u c t i o n of magnets based on superconduct ing s o l e n o i d s and F o u r i e r - t r a n s f o r m t e c h n i q u e 8 ^ have enhanced the r e s o l u t i o n and s e n s i t i v i t y of ^ H - n . m . r . s p e c t r o s c o p y . The use o f a h i g h - f i e l d spectrometer i s e s s e n t i a l f o r ^ H - n . m . r . spec troscopy o f p o l y s a c c h a r i d e s - 31 -due to the high v i s c o s i t y of polysaccharide samples. ^H-n.m.r. spectra of polysaccharides, run at ambient temperature, are characterized by si g n a l broadening which i s l a r g e l y due to short spin-spin r e l a x a t i o n times of the polymer protons. 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 spectra can be achieved by using elevated temperature at 60°-90° i n order to reduce v i s c o s i t y of the sample. Sharp signals i n the spectra obtained f o r b a c t e r i a l polysaccharides serve as proof of the regular repeating units present within them. Two dimensional homo- and hetero- nuclear n.m.r. has been employed i n the enhancement of r e s o l u t i o n as well as obtaining coupling information and assigning chemical s h i f t v a l u e s 8 3 . I n t e r p r e t a t i o n of a ^H-n.m.r. spectrum requires measurement of c e r t a i n parameters. ( i ) Chemical s h i f t The chemical s h i f t s of protons may depend on the following f a c t o r s , (a) s u b s t i t u t i o n , o r i e n t a t i o n of the molecule, 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 neighbouring and dis t a n t groups, and (b) the nature of the solvent can induce protons to resonate at d i f f e r e n t f i e l d strengths. Thus, equatorial ring-hydrogen atoms have lower chemical s h i f t s than t h e i r a x i a l counterparts. The n.m.r. spectrum of an ol i g o - or poly- saccharide i s made up of three main regions namely: ( i ) the anomeric region (5 4.5-5.5), ( i i ) the r i n g proton region (5 3.0-4.5), and (c) the high f i e l d region (S 1.15-2.5), where signals for CH3 of 6-deoxy sugars, pyruvate, 0-acetyl, N-acetyl etc. can be observed 32 -(see Table I I . 1). The anomeric region can be a r b i t r a r i l y divided i n two, that i s signals appearing u p f i e l d of S 5.0 are assigned to Q-linkages and those downfield are assigned to /3-linkages. Furthermore the linkage configuration (a and /!) can be determined from the combined measurements of the chemical s h i f t and coupling constant (see l a t e r ) . The number of sugar residues per repeating unit can be deduced from the number of anomeric signals and t h e i r corresponding i n t e g r a l s . The rin g proton region i s quite complex and assignments are d i f f i c u l t . H a l l 8 4 has proposed a s o l u t i o n to th i s problem. ( i i ) Coupling constants Nuclear spin-spin coupling constants over three bonds are designated by J J and are expressed as Hertz (Hz). In a f i r s t order spectrum, the magnitude of the coupling constants can be measured d i r e c t l y from the spectrum. Coupling constants can also be predicted using the Karplus equation and these values are u s u a l l y i n good agreement with observed values (see Figure II.3). The K a r p l u s 8 5 equation gives an approximate r e l a t i o n s h i p between the three-bond v i c i n a l coupling constant (^J) and the dihedral angle (#) between the protons 8.5 cos 2# - 0.28 0 ° ^ 9 0 ° 9.5 cos 2# - 0.28 90°£tel80° 33 Table II.1 Three regions of a carbohydrate spectrum Region Anomeric 4.5 - 5.5 >5.0 ( J 1 2 1-3 Hz) <5.0 ( J 1 > 2 7-9 Hz) 93 - 110 <101 >101 Ring H-2 Man H-5 GlcA 2°C 2°C (linked) 1°C 3.0 - 4.5 4.0 - 4.5 60 - 85 75 ± 5 80 - 85 60 - 65 High f i e l d N-acetate 0-acetate CH3 (6-deoxyhexose) CH3 (pyruvic a c i d ketal) a x i a l CH3 e q u a t o r i a l CH3 1.0 - 2.5 -2.03 -2.15 -1.33 -1.4 - -1.6 15 - 30 -21 -21 -17 -18 -26 £ C of C - 0 group (acetate, pyruvate and uronic acid) appear -170 p.p.m. 34 Figure II.3 Relationship between dihedral angle (^ ) and coupling constants for a - and ^-D-hexoses - 35 -The values are maximum when the dihedral angle (#) i s 0° or 180°, and minimum when i t i s 90°. In carbohydrate chemistry, the determination of values has been used to e s t a b l i s h configuration as well as conformational preferences for pyranose, furanose and a c y c l i c sugars. ( i i i ) Relative i n t e n s i t i e s of the signals The r e l a t i v e i n t e n s i t i e s of absorption signals f or 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 0 0 . The number of anomeric linkages, r e l a t i v e amounts of 6-deoxy sugars, 0-acetyl, N-acetyl and 1-carboxyethylidene substituents can be determined by computing the i n t e g r a l of t h e i r corresponding s i g n a l s . For o l i g o - or poly- saccharides, t h i s value permits a rapid quantitative analysis of the r a t i o of a to ^ linkages. II.5.1.2 i J C n.m.r. spectroscopy The advent of Fourier-transform n.m.r. spectroscopy and other Q "7 modern techniques ' have enhanced the s e n s i t i v i t y of the natural abundance of •LJC-n.m.r. ^C-n.m.r. spectroscopy which i s rapid and non-destructive has great p o t e n t i a l i n the study of polysaccharides of b i o l o g i c a l o r i g i n where only small amounts of material are a v a i l a b l e for an a l y s i s . In the study of complex molecules such as polysaccharides, the amount of information obtainable from lH-n.m.r. spectra i s usually - 36 -l i m i t e d compared to that revealed by -^C-n.m. r . 8 8 . 2-D N.m.r. spectroscopy has been employed i n s t r u c t u r a l studies of complex carbohydrates 8^•^0. The ^C-n m r spectrum of a polysaccharide l i k e the p.m.r. spectrum, can be categorized into three regions (see Table II.1). In C-n.m.r. the main parameter used f o r assignment i s chemical s h i f t . However, i f the spectra have a good s i g n a l to noise r a t i o (s/n), comparison of i n t e g r a l s of carbon atoms carrying the same number of hydrogen atoms often y i e l d s 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 ^ . An a r b i t r a r y d i v i s i o n of the anomeric region at 101 p.p.m. i s accepted but contrary to the p.m.r. the a-anomeric carbon appear u p f i e l d of the /9-anomeric carbons due to sh i e l d i n g e f f e c t s . and ^ H s h i f t s are af f e c t e d i n v e r s e l y ^ 2 because an increased s h i e l d i n g of a ^ 3C nucleus i s accompanied by a decrease i n the s h i e l d i n g of the attached proton. Signals f o r anomeric carbons of free sugars (reducing end) appear u p f i e l d , i n the region 93-97 p.p.m. 1 ^  Signals f o r CO group (acetate, pyruvate and uronic acid) and C=C group appear around 170 p.p.m. and 140-150 p.p.m. re s p e c t i v e l y . Signals due to C-2 to C-6 of sugar residues appear i n the r i n g carbon region. Carbon atoms of primary alcohols have c h a r a c t e r i s t i c signals between 60-65 p.p.m. which can be d i f f e r e n t i a t e d as lin k e d or non-linked by t h e i r chemical s h i f t s (non-linked, 60-62 p.p.m. when linked, they are s h i f t e d 7-10 p.p.m. downfield). The signals due to the carbons of secondary alcohols appear at 75 — 5 p.p.m., but on O-glycosylation or O-alkylation, the carbon(s) involved i s s u f f i c i e n t l y deshielded (by 7-11 p.p.m.) as to produce a s i g n a l well separated from - 37 -other r i n g carbons (80 — 5 p.p.m.). This deshielding phenomenon may r e s u l t i n an or-effect that i s the u p f i e l d s h i f t of the signa l ( s ) of the carbon atom(s) involved. However, carbons immediately adjacent to that carbon w i l l be s l i g h t l y shielded (1-2 p.p.m.) and t h i s i s the /3-effect. These a and /3 e f f e c t s must be taken into consideration during the -1 o no assignment of -LJC signals of ol i g o - and poly- saccharides^ . 1 3 •LJC Signals for 6-deoxysugars (-17 p.p.m.), acetate (-21 p.p.m.) and pyruvate appear i n the high f i e l d region. The stereochemistry of the a c e t a l carbon i n pyruvates can be d i f f e r e n t i a t e d by the chemical s h i f t of t h e i r methyl group 9 4. A x i a l methyl groups resonate at -18 p.p.m. and equatorial groups at ~26 p.p.m. II. 5 .2 Chromium t r i o x i d e oxidation The anomeric nature of sugar residues of poly- or o l i g o -saccharides can be determined by studying the reaction of t h e i r f u l l y acetylated d e r i v a t i v e s with chromium t r i o x i d e i n a c e t i c a c i d 9 5 . A f u l l y acetylated aldopyranoside, i n which the aglycon occupies an equatorial p o s i t i o n (g-linked) i s r e a d i l y oxidized by chromium t r i o x i d e while the anomer with an a x i a l aglycon (a-linked) i s quite r e s i s t a n t to oxida-t i o n ^ . However, s u b s t i t u t i o n i n ol i g o - and poly- saccharides may a l t e r the conformational equilibrium of a-fucosyl and a-rhamnosyl residues OA thus making them susceptible to oxidation- 3 . 38 -where, R = a l k y l group or sugar residue The oxidized product i s converted to a l d i t o l acetates or p a r t i a l l y methylated a l d i t o l acetates and analyzed by g.l.c.-m.s. In t h i s study chromium t r i o x i d e oxidation was used to determine the anomeric configuration of the sugar residues i n Escherichia c o l i K34 capsular polysaccharide. Sugar analysis performed on the oxidized product showed only glucose thus proving that i t was a-linked. I I . 5 . 3 Other techniques O p t i c a l r o t a t i o n can be used to confirm the anomeric configuration of sugar residues i n o l i g o - and polysaccharides. For example, i n t h i s i n v e s t i g a t i o n the anomeric configuration assigned to the sugar residues of Escherichia c o l i K34 capsular polysaccharide, from n.m.r. and chromium t r i o x i d e oxidation data, was confirmed by o p t i c a l r o t a t i o n . From, Hudson's Isorotaton Rules^ 7 one can p r e d i c t s p e c i f i c rotations of o l i g o - or poly- saccharides using the molecular r o t a t i o n values of the model methyl glycosides. Normally, the o p t i c a l r o t a t i o n i s measured at 39 the sodium D-line (589 nm) and the observed s p e c i f i c r o t a t i o n should agree with the value predicted by Hudson's Iso r a t i o n Rules. Enzymes, such as exoglycosidases, are s p e c i f i c f o r the sugar unit undergoing hydr o l y s i s and for i t s anomeric configuration. Thus, the c l a s s i c a l technique of enzymic h y d r o l y s i s 9 8 can be used to investigate g l y c o s i d i c linkage configurations. Recent observations i n our laboratory have shown that, enzymic hydrolysis on oligosaccharide gives more s a t i s f a c t o r y r e s u l t s . During t h i s study, E. c o l i K34 bacteriophage oligosaccharide was incubated (buffer pH «= 6.5, 37°C) with a-D-glucosidase f o r two days and the hydrolyzate was analyzed by paper chromatography. 40 -CHAPTER III RESULTS AND DISCUSSION 41 I I I . RESULTS AND DISCUSSION I I I . l Composi t ion and n . m . r . s t u d i e s E. c o l i K34 was grown on M u e l l e r H i n t o n agar and the a c i d i c p o l y s a c c h a r i d e p u r i f i e d as d e s c r i b e d l a t e r (see e x p e r i m e n t a l ) . The product monodispersed by g e l - p e r m e a t i o n , weighed 940 mg. T h i s p u r i f i e d p o l y s a c c h a r i d e has [a]n +24.2 which compares v e r y w e l l w i t h c a l c u l a t e d v a l u e o f 25.7 mg u s i n g Hudson's Rule o f I s o r o t a t i o n ^ 7 . Paper chromato-graphy of a n ' a c i d h y d r o l y z a t e of the p o l y s a c c h a r i d e showed ga lac tose glucose and g l u c u r o n i c a c i d . H y d r o l y s i s o f the p o l y s a c c h a r i d e and a n a l y s i s of the h y d r o l y z a t e as a l d i t o l ace ta tes by g . l . c . i n d i c a t e d glucose and g a l a c t o s e i n molar p r o p o r t i o n s of 1:2.6. M e t h a n o l y s i s and r e d u c t i o n o f the p o l y s a c c h a r i d e f o l l o w e d by h y d r o l y s i s , r e d u c t i o n , a c e t y l a t i o n and g . l . c . a n a l y s i s gave glucose and g a l a c t o s e i n molar p r o p o r t i o n s of 2:2.7 (see Table I I I . l ) . These r e s u l t s suggested t h a t E.  c o l i K34 c a p s u l a r p o l y s a c c h a r i d e c o n s i s t s of a pentasacchar ide r e p e a t i n g u n i t c o n t a i n i n g g a l a c t o s e , g l u c o s e , and g l u c u r o n i c a c i d r e s i d u e s i n the r a t i o s o f 3:1:1. The ^H and ^ C - n . m . r . s p e c t r a of the K34 p o l y s a c c h a r i d e i n d i c a t e d the presence o f f i v e sugar r e s i d u e s per r e p e a t i n g u n i t , c o r r e s p o n d i n g to one a and f o u r ^ - l i n k a g e s . The n . m . r . s p e c t r a showed the absence of deoxy s u g a r s , ace ta tes and p y r u v i c a c i d - k e t a l s (see Table I I I . 2). More p r e c i s e assignment of the s i g n a l s i n the n . m . r . s p e c t r a was a c h i e v e d a f t e r sugar a n a l y s i s o f chromium t r i o x i d e o x i d i z e d Table III . l - 42 -Sugar analysis of K34 polysaccharide and derived products Sugars^ (as alditol acetates) Galactose Glucose Glyceraldehyde Mole I II III 2.6 2.7 1.8 1 2 1.1 ratio  IV V VI 1.8 2.0 0.03 0.6 1 1.0 Using DB-17 column programmed for 180°C for 2 min, 5°C/min to 220°C I, original acid polysaccharide; II, carboxyl reduced polysaccha-ride; III, oligosaccharide obtained from Smith degradation of carboxyl reduced polysaccharide (P2); IV, oligosaccharide obtained from selective Smith degradation of acidic polysaccharide; V, Product obtained from selective Smith degradation of acidic polysaccharide; VI, product obtained from chromium trioxide oxidation of acidic polysaccharide - 43 Table III.2 Compound N.m.r. data for E. c o l l K34 capsular polysaccharide and derived products (see Appendix III) ^H-n.m.r. data 13 C-n.m.r. data a Integral Assignment— P-P m.d Assignment (H) K34 capsular 5.17 s 1 Q - G I C 98 71 Q-Glc polysaccharide 4.71 b 1 /9-GlcA 102 90 /S-GlcA 4.57 b 3 P-Qal 103 44 /9-Gal 104 44 0-Gal 179 45 CO of Q-GlcA Periodate 5.17 s 1 o-Glc oxidized K34 polysaccharide 4.71 b 1 /S-GlcA 4.57 8 3.1 0-Gal CHoi Gali-^Gal-O- 4.53 8 2 Gal CH20H ^ i c A ^ G a l l - ^ a l — i 4.71 4.57 GlcA Gal £ Chemical shift relative to internal acetone; 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 - singlet £ For example, /9-Gal - proton on C-l of ^ -linked-D-Gal residue & Chemical shift in p.p.m. downfield from Me^Si, relative to internal acetone; 31.07 p.p.m. downfield from D.S.S. — As for —, but for anomeric nuclei £ Oligosaccharide (P2) obtained from Smith degradation of carbodiimide reduced polysaccharide £ Polysaccharide obtained from selective Smith degradation of acidic polysaccharide - 44 polysaccharide and n.m.r. studies of products obtained from Smith degradation (see l a t e r and Table III.2). 111.2 Chromium t r i o x i d e Oxidation The anomeric nature of the sugar residues, i n K34 capsular polysaccharide was determined by chromium t r i o x i d e oxidation of the f u l l y acetylated polysaccharide, followed by sugar a n a l y s i s . G.l.c. r e s u l t s indicated that only glucose survived and other sugar residues were degraded (Table I I I . 2 ) . N.m.r. studies (Table III.2) showed that only one sugar residue i n K34 polysaccharide i s a-linked and t h i s sugar residue was deduced to be glucose from t h i s g . l . c . r e s u l t . Thus, the glucuronic a c i d and galactose residues are /3-linked i n the polysaccha-r i d e chain. 111.3 Methylation analysis On methylation, h y d r o l y s i s , reduction, and a c e t y l a t i o n the o r i g i n a l capsular polysaccharide gave the a l d i t o l acetates shown i n Table III.3 (column I ) . When the uronic a c i d i n the methylated polymer was reduced before h y d r o l y s i s , reduction, and a c e t y l a t i o n , the a l d i t o l acetates i n Table III.3 (column II) were obtained. These a l d i t o l acetates were analyzed by g . l . c . and g.l.c.-m.s. These r e s u l t s confirmed that the polysaccharide under i n v e s t i g a t i o n consists of a - 45 -Table III.3 Methylation analysis of K34 polysaccharide and derived products Methylated sugar^- Mole %^ (as a l d i t o l acetate) 6- l£ II 0- III 0- IV°- V°-2,3,4,6-Glc 28.4 18.7 23.3 2,4,6-Gal 19.8 21.9 50.6 58.3 47.5 2,3,6-Gal 22.2 21.9 26.1 40.9 2,6-Gal 29.6 19.4 12.6 3,4-Glc 18.1 2,3,4,6-Gal 41.7 2,3,4,6-Glc - l,5-di-0-acetyl-2,3,4,6-tetramethylglucitol etc. Values are corrected by use of the e f f e c t i v e , carbon-response factors given by Albersheim et a l . ^ 7 . Using DB-17 column programmed for 180°C for 1 min, 2°C/min to 250°C Retention times of these p a r t i a l l y methylated sugar was confirmed by t h e i r authentic standard samples. I, o r i g i n a l a c i d polysaccharide; I I , reduction of uronic ester; I I I , product from /^-elimination and remethylation; IV, product from Smith degradation; V, product from s e l e c t i v e Smith degradation 46 pentasaccharide repeating unit. Methylation analysis r e s u l t s also indicated that the polysaccharide has ( i ) a galactosyl residue linked at C - l , C-3 and C-4 as i t s branched point, and ( i i ) a glucose as i t s terminal non-reducing residue. The l o c a t i o n of the glucuronic acid i n the polysaccharide repeating unit, was determined by subjecting the permethylated polysaccharide to base-catalyzed uronic a c i d degradation ( ^ - e l i m i n a t i o n ) 6 7 > 6 8 . The product was d i r e c t l y a l k y l a t e d with methyl iodide and the i s o l a t e d residue was hydrolyzed, reduced, and acetylated. The g.l.c.-m.s. analysis of the resultant a l d i t o l acetates (Table I I I , column III) showed 2,4,6-tri-O-methylgalactose, 2,3,6-tri-0-methyl-galactose and 2 , 3 ,4, 6.-tetra-O-methylglucose i n the r a t i o 2:1:1. I t was deduced from these r e s u l t s that the glucuronic a c i d was linked to branched galactose at C-4. III.4 Periodate oxidation - Smith hydrolysis Carbodiimide-reduced K34 polysaccharide was oxidized with sodium periodate (122 h) and reduced with sodium borohydride. The consumption of periodate was monitored during the course of the rea c t i o n (see Figs. I I I . l and II I . 2 ) . About 4 moles of periodate per repeating u n i t were consumed and t h i s agreed with the t h e o r e t i c a l value. The pol y o l recovered a f t e r d i a l y s i s , was analyzed by n.m.r. spectroscopy (see Table I I I . 2 ) . The n.m.r. spectrum obtained was better than that of the parent polysaccharide due to the high s o l u b i l i t y of t h i s p o l y o l i n plus the low v i s c o s i t y of t h i s sample. Figure III.2 Periodate consumption by K34 polysaccharide with respect to time - 48 -This p o l y o l was subjected to Smith h y d r o l y s i s , during which the a c e t a l linkages of the modified sugar residues should be s e l e c t i v e l y hydrolyzed. The oligosaccharide ( P 2 ) i s o l a t e d by paper chromatography of the hydrolyzate, was also analysed by n.m.r. spectroscopy. The spectrum e x h i b i t s s i g n a l at 8 = 4.53, which integrate to two anomeric protons (Table I I I . 2). Sugar analysis conducted on the derived o l i g o -saccharide (P 2) indicated the presence of galactose and glyceraldehyde i n molar proportions of 1.8:1 (Table I I I . l ) . Methylation analysis of the derived oligosaccharide (P 2) (Table III.3, column IV) showed 2,4,6-tri-O-methyl-galactose and 2,3,4,6-tetra-O-methylgalactose, an i n d i c a t i o n that the two sugar residues (1,3,4 l i n k e d galactose and 1,3 l i n k e d galactose) r e s i s t a n t to periodate oxidation are l i n k e d to each other. Sugar analysis and methylation r e s u l t s therefore suggest that the o l i g o - saccharide (P 2) derived from Smith degradation has the structure below: CHO GalL-iGal-L-O— CH2OH According to the sugar analysis r e s u l t , (Table I I I . l ) , the o l i g o -saccharide obtained from Smith degradation of the native polysaccharide also has the structure shown above. The terminal glycerolaldehyde i s a fragment from the oxidized glucuronic a c i d residue. Thus, i t was speculated that the main chain of E. c o l i K34 capsular polysaccharide has the following structure: - 49 -- ^ G l c A p ^ G a l p i - ^ a l p — P 13 P This structure was confirmed by s e l e c t i v e Smith degradation of the native polysaccharide. III. 5 S e l e c t i v e Smith degradation The rate of periodate oxidation v a r i e s according to the configuration of the g l y c o l s . Sugars having trans g l y c o l s are oxidized more slowly or not at a l l i f f i x e d i n an unfavourable conformation 0 2. Observations i n our laboratory have shown that the uronic a c i d residue, having trans g l y c o l s , i s oxidized very slowly by sodium periodate. t h i s observation might be explained by the repulsion between the periodate ion and carboxylic anion. Thus s e l e c t i v e oxidation of 1 — > 4 l i n k e d galactopyranosyl and terminal glucopyranosyl residues over 1 — > 2 l i n k e d glucuronic a c i d was embarked upon i n t h i s study (see Scheme I I . 3 ) . The native polysaccharide was treated with 0.02 M sodium periodate f o r three hours followed by sodium borohydride reduction. The r e s u l t a n t p o l y o l which was p u r i f i e d by gel permeation chromatography (Sephadex LH-20) was subjected to mild a c i d h y d r o l y s i s (0.5 M TFA) for 48 hours. T o t a l sugar analysis of the non-dialyzable material y i e l d e d galactose and glucose i n molar proportions of 2.0:0.6 (Table I I I . l , column V). Methylation analysis indicated that the galactose residues i n 50 -the s e l e c t i v e Smith degradation product were 1 — > 4 l i n k e d and 1 — > 3 li n k e d galactopyranosides (Table III.3, column V). The ^H-n.m.r. spectrum of the degraded product showed signals for anomeric protons at 8 4.71 (J\2 ~ 8 **z) and 8 4.57 (J\2 ~8 anc* t n e s e signals integrate up to three anomeric protons. This degraded product therefore consists of a t r i s a c c h a r i d e repeating u n i t . Comparison of t h i s spectrum with the n.m.r. spectrum of the native polysaccharide indicates that t h i s n.m.r. spectrum has no s i g n a l at 8 5.17 (for the a-linked terminal glucopyranoside) and also the s i g n a l at 5 4.57 integrated up to two anomeric protons as compared to three i n the native polysaccharide. The o v e r a l l n.m.r. studies therefore suggest that the signals at 8 5.17, 8 4.71 and 8 4.57 can be assigned to a-glucose, /9-glucuronic a c i d and ^-galactoses r e s p e c t i v e l y . III.6 Determination of the configuration (D or L) of the sugar The configuration (D or L) of the sugar residues, contained i n E.  c o l i K34 capsular polysaccharide, was determined conveniently by c i r c u l a r dichroism measurements at 213 nm on t h e i r p a r t i a l l y methylated a l d i t o l acetates, where the acetoxy group acts as a chromophore. Glucose, glucuronic a c i d and galactoses were shown to be of the D c o n f i g u r a t i o n by the c i r c u l a r dichroism curves of the corresponding a l d i t o l acetates (Table III.4). These configurations were attained by comparing the c i r c u l a r dichroism measurements of these a l d i t o l acetates with those of the authentic standards. - 51 -Table III.4 Configuration of sugar residues of E. coli K34 capsular polysaccharide Sugar A l d i t o l C o n f i g u r a t i o n -2-GlcA^- 3 , 4 - D - O - m e t h y l g l u c i t o l D - ^ G a l i - 2 , 6 - D i - 0 2 m e t h y l g a l a c t i t o l D 3 -J±Gal±- 2 , 3 , 6 - t r i - O - m e t h y l g a l a c t i t o l D — 3 - G a l l - 2 , 4 , 6 - t r i - 0 - m e t h y l g a l a c t i t o l D -J-Glc 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l g l u c i t o l D The D c o n f i g u r a t i o n o f t h e t e r m i n a l g l u c o s e was c o n f i r m e d by t r e a t m e n t o f b a c t e r i o p h a g e o l i g o s a c c h a r i d e w i t h a - D - g l u c o s i d a s e . Paper c h r o m a t o g r a p h i c a n a l y s i s o f t h e h y d r o l y z a t e f r o m t h i s enzymic r e a c t i o n i n d i c a t e d t h a t g l u c o s e has been h y d r o l y z e d b y t h e a - D - g l u c o s i d a s e . III. 7 Isolation of bacteriophages (<^ 31 and ^34) and cross-reactions E. c o l i K31 and K34 b a c t e r i o p h a g e s (<£31 and <£34) o r i g i n a l l y i s o l a t e d f r o m Vancouver sewage were p r o p a g a t e d on t h e i r h o s t s t r a i n s . No p l a q u e s were formed when E. c o l i K34 b a c t e r i o p h a g e was s p o t t e d on a b a c t e r i a l lawn o f E. c o l i K31 o r v i c e v e r s a . Thus E. c o l i K34 does n o t 52 -s e r o l o g i c a l l y cross-react with E. c o l i K31. Hence the immunodeterminant group of E. c o l i K34 must d i f f e r from that of E. c o l i K31. In fact, t h i s i n v e s t i g a t i o n and previous studies have revealed that the structure of E. c o l i K34 capsular polysaccharide (K antigen) d i f f e r s from that of E, c o l i K31 (Appendix I ) . E. c o l i K34 bacteriophage d i d not give cross-absorption with the following E, c o l i s t r a i n s , K28, K32 and K33. -• Gali-^Glci-^-GlcrAi-^-Rhai^Rhal-B K Antigen of E. c o l i K31 1 1 _3_G i c 1_4_G 1 c A!_4 F u c!_ 4 a B a B 2 o r 3 1 I Gal 0 A c K Antigen of E. c o l i K28 OAc t 2 Q 3 ^ G l c L J l R h a i - ^ G a l i -B 1 GlcA K Antigen of E. c o l i K32 - 53 -^ I g l c ^ G l c A ^ F u c ^ a 3 2 8 3 V pyr 1 Gal + OAc K Antigen of E. c o l i K33 ^ I g i c A ^ G a l ^ G a l i -B 3 B 1 Gal 4 a 1 Glc K Antigen of E. c o l i K34 The immunodominant sugars of branched polysaccharides are usually located i n the side c h a i n - ^ 9 . E. c o l i K34 capsular polysaccharide has galactose and glucose i n i t s side chain. However E. c o l i s t r a i n s K33 and K28, which do not cross-react with E. c o l i K34, have galactose i n the side chain of t h e i r capsular polysaccharide. From cross-reaction r e s u l t s , we are l e d to suggest that the immunodominant sugar of E. c o l i K34 capsular polysaccharide may e i t h e r be the 1 — > 2 l i n k e d glucuronic a c i d or the terminal glucose. The occurrence of 1,2 l i n k e d glucuronic a c i d i n E. c o l i K34 capsular polysaccharide i s the f i r s t 1 — > 2 linked glucuronic a c i d to be reported i n b a c t e r i a l polysaccharides. - 54 -III.8 Depolymerization with E. c o l i K31 bacteriophage (<£31) Propagation of bacteriophage was continued on an increasing scale i n Mueller Hinton broth. The r e s u l t s of phage assays from tube l y s i s and f l a s k l y s i s are as tabulated i n Table I I I . 5. A f t e r d i a l y s i s f o r 2 1 9 days, the concentration of the bacteriophage s o l u t i o n was 1.8 x 10 L^ plaque-forming u n i t s . This bacteriophage s o l u t i o n was added to a s o l u t i o n of p u r i f i e d K31 capsular polysaccharide and the re a c t i o n mixture incubated at 37°C for a t o t a l of 48 h. A f t e r 24 h of incubation time, the re a c t i o n mixture appeared s i g n i f i c a n t l y l e s s viscous, an i n d i c a t i o n that depolymerization has occurred. The depolymerized product formed was separated from the polysaccharide-phage mixture by d i a l y s i s and then p u r i f i e d by ion exchange chromatography (Amberlite IR 120 (H +) r e s i n ) . Table III. 5 Propagation of Bacteriophage <j>31 II III T i t r e (p.f.u./mL)£ 4.95 x 1 0 1 0 5.5 x 1 0 1 0 Volume (mL) 30 100 180 To t a l (p.f-u.) -1.5 x 10 5.5 x 1 0 1 2 1.8 x 1 0 1 2 a b I, test-tube l y s i s ; I I , small f l a s k l y s i s ; I I I , a f t e r d i a l y z i n g p.f.u. = plaque forming u n i t . 55 The mixture of oligosaccharides obtained, a f t e r depolymerization of E.  c o l i K31 polysaccharide with phage #31, was then separated into pure components by gel permeation chromatography (85 mg, 47.2% y i e l d ) . The reducing end of the oligosaccharide was determined by Morrison's method^ 0. The r e s u l t s showed the presence of galactono-n i t r i l e , rhamnonitrile and g l u c i t o l i n d i c a t i n g that the glucosyl linkage was cleaved by bacteriophage-borne glycanase (see Table III.6). Thus, the type of enzyme that occurs i n E. c o l i K31 bacteriophage has a glucosidase a c t i v i t y . Table I I I . 6 Determination of the reducing end of E . c o l i K31 o l i g o -saccharide i s o l a t e d a f t e r bacteriophage #31 degradation of E . c o l i K31 polysaccharide Peracetylated T a d e r i v a t i v e of Column (DB 17) Rhamnonitrile 3.91 G a l a c t o n o n i t r i l e 7.33 G l u c i t o l 9.84 Column Temperature (180°C for 2 min, 5°C/min, to 220°C) Retention times confirmed by authentic standard samples - 56 -I I I . 9 Depolymerization with E . c o l i K34 bacteriophage (#34) E. c o l i K34 bacteriophage (#34) was also propagated on an increas -i n g sca le i n M u e l l e r Hinton b r o t h . The r e s u l t s of phage assays from tube l y s i s and f l a s k l y s i s are a lso tabulated i n Table I I I . 7 . The concentra t ion of the bacteriophage s o l u t i o n was 7.2 x 10^ 2 plaque-forming u n i t s a f t e r d i a l y s i n g f o r two days. Bacteriophage degradation of E . c o l i K34 capsular polysaccharide and i s o l a t i o n of the r e s u l t a n t o l i g o s a c c h a r i d e was conducted as p r e v i o u s l y d e s c r i b e d . Paper chromato-graphy i n d i c a t e d that f r a c t i o n I (see Figure I I I . 3 ) was a double repeat ing u n i t o l i g o s a c c h a r i d e (85.7 mg, 34% y i e l d ) . Resul ts f o r ^ H - n . m . r . spectroscopy s tudies on f r a c t i o n I , before and a f t e r sodium borohydride r e d u c t i o n , are shown i n Table I I I . 8 . Table I I I . 7 Propagation of bacteriophage #34 1= II I I I T i t r e ( p . f . u . / m L ) ^ 4.95 x 1 0 1 0 1.1 x 1 0 1 0 0.9 x 1 0 1 1 Volume (mL) 30 100 80 T o t a l ( p . f . u . ) 1.48 x 1 0 1 2 1.1 x 1 0 1 2 7.2 x 1 0 1 2 - I , t e s t - t u b e l y s i s ; I I , small f l a s k l y s i s ; I I I , a f t e r d i a l y z i n g — p . f . u . - plaque forming u n i t . eluted volume minus (mL) void volume F i g . I I I .3 Separation of the depolymerizat ion products of E . c o l l K34 by gel -permeation chromatography ( B i o - G e l P-2) 58 Table III.8 Proton n.m.r. data (400 MHz) f o r the oligosaccharide generated i n bacteriophage depolymerization of the E. c o l i K34 capsular polysaccharide Fract i o n I (p.p.m.)S 5.17 Integral (H) 1.0 Fr a c t i o n I (R)^ Integral (p.p.m.) (H) 5.16 1.0 4.97 0 .8 4.97 0.3 4.82 1.2 4.82 1.2 4.65 0.4 4.53 2.2 4.53 2.1 - Chemical s h i f t r e l a t i v e to i n t e r n a l acetone, 2.23 downfield from sodium 4,4-dimethyl-4-silapentane-l-sulfonate (DSS) Fracti o n I a f t e r reduction with sodium borohydride F r a c t i o n III had a low carbohydrate content as was judged from i t s ^H-n.m.r. spectrum. The r e s u l t s f o r the reducing end determination on * 1 0 ft f r a c t i o n I by Morrison's method^ 0 are shown i n Table I I I . 9. These r e s u l t s i l l u s t r a t e d that the E. c o l i K34 bacteriophage-borne enzyme has a ^-galactosidase a c t i v i t y . 59 Table III. 9 Determination of the reducing end of E. c o l i K34 oligosaccharide i s o l a t e d a f t e r bacteriophage degradation of E. c o l i K34 polysaccharide Peracetylated de r i v a t i v e of X— Column-(DB 17) Mole % Gl u c o n o n i t r i l e 7.73 G a l a c t o n o n i t r i l e 7.88 74 G a l a c t i t o l 10.04 19 £ Column Temperature, (180°C for 2 min, 5°C/min, to 220°C) b Retention times confirmed by authentic standard samples CONCLUSION This study revealed that E. c o l i K34 d i d not cross-react with other group 09 s t r a i n s l i k e K28, K31, K32, and K33. Studies conducted i n t h i s i n v e s t i g a t i o n revealed that K antigen of E. c o l i K34 has the structure below: -^2-D - GlcApi-^-D - Galpi—2-D - G a l p i -P 3 P P P 1 D-Galp 4 1 D-Glcp - 60 -A comparison of t h i s structure to the K antigen of the above mentioned E. c o l i s t r a i n s (see Appendix I ) , showed that E. c o l i K34 K antigen i s s i g n i f i c a n t l y d i f f e r e n t . The f a c t that E. c o l i K34 has a unique K antigen j u s t i f i e s i t s s e r o l o g i c a l d i f f e r e n t i a t i o n . E. c o l i K34 capsular polysaccharide i s novel i n that, t h i s i s the f i r s t time 1 —> 2 l i n k e d glucuronic a c i d has been reported i n b a c t e r i a l polysaccharides. CHAPTER I V EXPERIMENTAL - 62 -IV. EXPERIMENTAL IV.1 General methods Solutions were concentrated on a rotary evaporator with bath temperatures not exceeding 40°C. Frozen solutions were obtained using a dry ice-acetone mixture and l y o p h i l i z e d on a Unitrap II freeze-dryer. O p t i c a l r o t a t i o n measurements were conducted on aqueous solutions at 20°i3° using a Perkin-Elmer model 141 polarimeter with 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, with a quartz c e l l of 0.3 mL capacity and a path length of 0.1 cm. C d . samples were prepared by d i s s o l v i n g the appropriate a l d i t o l acetate i n spectroscopic grade a c e t o n i t r i l e . The c d . spectra were recorded i n the range 210-240 run. The i n f r a r e d ( i . r . ) spectra of methylated d e r i v a t i v e s were recorded on a Perkin Elmer model 457 spectrophotometer. The solvent used f o r sample preparation was spectroscopic grade carbon t e t r a c h l o r i d e . A n a l y t i c a l paper chromatography was performed by the descending method using Whatman No. 1 paper and the following systems: (1) 18:3:1:4 et h y l acetate-acetic acid-formic acid-water; (2) 8:2:1 ethyl acetate-pyridine-water; and, (3) upper phase of 4:1:5 1-butanol-ethanol-water. Preparative paper chromatography was c a r r i e d out using Whatman No. 3 paper and solvent system 1. Chromatograms were e i t h e r developed with 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 100°C f o r 10 min a f t e r being sprayed with p-anisidine hydrochloride-*-^ i n aqueous 1-butanol. - 63 -Sugars and oligosaccharide were detected by these methods. A Bio-Gel P-2 (400 mesh) column (2.5 x 100 cm) was used for preparative gel-permeation chromatography. The v o i d volume of the column and the e f f i c i e n c y of packing were determined using blue dextran (0.2%). The concentration of the samples applied to the column ranged from 40-100 mg/mL. Eluant was d i s t i l l e d water at a flow rate of approximately 6 mL/h. Fractions were c o l l e c t e d , freeze-dried, weighed and the e l u t i o n p r o f i l e was obtained. Sephadex LH-20 was used to p u r i f y large molecular weight carbohydrate material that i s soluble i n organic solvent, e.g. permethylated o l i g o - and poly- saccharides. A Hewlett-Packard 5890 instrument equipped with a dual flame-i o n i z a t i o n detector was used f o r a n a l y t i c a l g . l . c . separations. A Hewlett-Packard 3392A integrator was used to quantify the peak areas. Open tubular ( c a p i l l a r y ) columns were used with a helium carrier-gas flow rate of 48 mL/min. The columns used were: (A) fused s i l i c a c a p i l l a r y column (DB-17-15N); (B) fused s i l i c a c a p i l l a r y column (DB-225-15N). Preparative g . l . c . was c a r r i e d out with F & M model 720 dual column instrument f i t t e d with thermal conductivity detectors. S t a i n l e s s - s t e e l columns (1.8 m x 6.3 mm) were used with carrier-gas helium flow-rate of 60 mL/min. This column was packed with 3% of SP-2340 on Supelcoport (100-200 mesh) and programmed from 175°C to 240°C at l°/min. G.l.c.-m.s. analyses were performed with a V.C. Micromass 12 instrument equipped with a Watson-Biemann separator. Spectra were recorded at 70 eV with an i o n i z a t i o n current of 100 A and an ion source at 200°. The columns used for the separation were (A) and (B). l 3C-n.m.r. and lH-n.m.r. spectra were recorded on a Bruker WH-400 - 64 -instrument. Acetone was used as an internal standard for both lH-n.m.r. (2.23 p.p.m.) and 1 3 C-n.m.r. (31.07 p.p.m.) spectroscopy. ^H-n.m.r. spectra were recorded at elevated temperature and chemical shift values are given relative to that of external sodium-4,4-dimethyl-4-sila-pentanesulfonate (taken as zero). ^H-n.m.r. samples were prepared by dissolving in D2O and lyophilized three times from D2O solutions. These samples were dissolved in D2O and submitted in 5 mm diameter n.m.r. tubes. ""--X-n.m.r. spectra were recorded at ambient temperature. Samples were dissolved in the minimum of D2O and submitted in n.m.r. tubes of diameter sizes 5 mm or 10 mm. In our laboratory, we generally proceed as follows for the isolation of E. coli bacteriophages. Large samples of sewage are mixed with concentrated Mueller Hinton broth and then with an actively growing culture of the bacteria for which a virus shall be Isolated. This mixture is then incubated (37°C) overnight. The bacteria in this mixture are killed by adding chloroform and shaking vigorously. The crude bacteriophage solution is separated from the bacterial debris by centrifugation. The bacterial lawn needed for bacteriophage assay is prepared as follows. A Mueller Hinton agar plate was dried, upside down at 37°C in an incubator for 2 hours. Actively growing bacterial culture (3 mL) was pipetted on to the agar surface and after 20 min at 37°C, excess liquid was drained off. The plate was then incubated at 37°C for 1 hour to produce the bacterial lawn. An assay for determining bacteriophage concentration is described as follows. 0.3 mL portion of bacteriophage suspension was diluted - 65 -t e n - f o l d by adding 2.7 ml of s t e r i l e broth. 0.3 mL p o r t i o n of the r e s u l t a n t s o l u t i o n was further d i l u t e d t e n - f o l d ( i n a s i m i l a r manner) and the process repeated on subsequent solutions u n t i l a d i l u t i o n range of 10" 1 - l O - 1 0 was obtained. One small drop of bacteriophage suspension was spotted on the b a c t e r i a l lawn by means of a s t e r i l e p i p e t t e drawn to a f i n e t i p . A f t e r overnight incubation at 37°C, the number of plaques observed for the highest d i l u t i o n are counted. The counts of plaque-forming units (p.f.u.) per mL of undiluted phage suspension were c a l c u l a t e d based on the volume of the bacteriophage s o l u t i o n applied, the number of plaques and the d i l u t i o n that gave those plaques. The methods employed i n b u i l d i n g up the concentration of bacteriophage to a l e v e l s u f f i c i e n t f o r degrading the polysaccharide i n question are tube l y s i s 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 culture of E. c o l i was obtained by successive r e p l a t i n g on agar pl a t e s . A colony of t h i s a c t i v e l y growing b a c t e r i a was picked up into s t e r i l e broth (5 mL) and incubated at 37°C u n t i l the b a c t e r i a l culture became t u r b i d (4 hours). S t e r i l e broth ( 5 x 5 mL) i n culture test-tubes was then inoculated with the b a c t e r i a l culture (0.5 mL) and incubated at 37°C. A f t e r 30 minutes of incubation, bacteriophage suspension was added to the test-tubes consecutively at 30 minutes i n t e r v a l . Continued incubation r e s u l t s 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 the l a s t tube had cleared - 66 -(about 5 hours after the f i r s t addition of bacteriophage) a few drops of chloroform were added to each tube to prevent bacterial growth. The last two tubes were combined and bacteriophage separated from the bacterial debris by centrifugation. (b) Flask lysis This technique is quite similar to the tube lysis except that large volumes of bacteriophage are produced. 48 ml aliquots of sterile Mueller Hinton broth, in six Erlenmeyer flasks (125 ml), were each inoculated with 1 ml of actively growing culture. At 30 minute intervals, bacteriophage suspension (1 mL from the tube lysis or otherwise) was consecutively added to the flasks. The procedure was then continued as described for tube l y s i s . IV.2 Isolation and purification of E. c o l i K34 capsular polysaccharide The medium used for the growth of the bacteria was Mueller Hinton agar: beef extract (3.0 g), acid hydrolyzate of Casein (17.5 g), starch (1.5 g), and agar (12.0 g) per l i t e r of water. Sterilization of glassware and Mueller Hinton medium was done in a American Sterilizer model 57-CR for 15 minutes at 121° and 15-20 p.s.i. E. c o l i K34 culture was obtained from Dr. Ida Orskov (WHO International Escherichia Center, Copenhagen). Actively growing colonies of E. c o l i K34 were propagated by replating several times onto - 67 -P e t r i dishes (layered with s t e r i l e Mueller Hinton agar); a si n g l e colony being selected each time the b a c t e r i a were to be plated. Growth overnight of b a c t e r i a on P e t r i dishes at 37°C was s u f f i c i e n t . Broth (100 mL) was inoculated with E. c o l i K34 b a c t e r i a and incubated for 4 hours. A c t i v e l y growing E. c o l i K34 b a c t e r i a were poured onto a s t e r i l e , Mueller Hinton agar medium ( i n a metal tray 60 x 40 cm) and incubated f o r four days at 37°C. The K34 b a c t e r i a were scraped from the agar surface, d i l u t e d with 1% phenol s o l u t i o n and s t i r r e d at 4°C for 5 hours. The mixture of polysaccharide, b a c t e r i a l c e l l s and other debris was u l t r a c e n t r i f u g e d (for 4 hours at 15° on Beckmann L3-50 U l t r a -centrifuge using rotor 45 T i at 31000 r.p.m. or 80000 g) to separate the polysaccharide from the dead b a c t e r i a l c e l l s . The viscous honey-coloured supernatant was p r e c i p i t a t e d with ethanol. The resultant s t r i n g y p r e c i p i t a t e was dissolved i n water and treated with C e t a v l o n 2 7 (cetyltrimethylammonium bromide). The Cetavlon-polysaccharide complex was di s s o l v e d i n 4M NaCl solu t i o n , p r e c i p i t a t e d into ethanol, re-disso l v e d i n water and dialyzed against d i s t i l l e d water (two days). The polysaccharide was i s o l a t e d as a styrofoam-like material, by l y o p h i l i z a -t i o n . The i s o l a t e d polysaccharide was further p u r i f i e d by gel permeation chromatography using a Bio-Gel P2 column (100 cm x 2.5 cm). IV.3 Sugar analysis and composition Hydrolysis of a sample (20 mg) of K34 polysaccharide with 2M t r i f l u o r o a c e t i c a c i d (TFA) for 20 h at 95°C, removal of excess a c i d by - 68 -coevaporation with water, followed by paper chromatography (solvent (1)) showed glucose, galactose, glucuronic a c i d and an aldobiouronic acid. The sugars released were reduced (sodium borohydride i n water, for 4 hours) and the mixture was n e u t r a l i z e d with Amberlite IR-120 (H +) r e s i n . The r e a c t i o n mixture was f i l t e r e d , evaporated to dryness and c o d i s t i l l e d with portions of methanol (5 mL) i n order to remove the borate ion. The residue was treated with a c e t i c anhydride-pyridine (1:1) for 1 hour on a steam bath under anhydrous conditions. The resultant a l d i t o l acetates were analyzed by g . l . c . (column A, programmed from 180°C to 220°C at 5°C/min). The g . l . c . r e s u l t s are shown i n Table I I I . l , column 1. A sample of K34 polysaccharide (13 mg), d r i e d i n vacuo and under an i . r . lamp, was treated with methanolic hydrogen c h l o r i d e (3%) and refluxed overnight on a steam-bath under anhydrous conditions. The excess a c i d i n the r e a c t i o n mixture was n e u t r a l i z e d with lead carbonate. The r e s u l t a n t mixture was centrifuged to remove the lead chloride p r e c i p i t a t e . The supernatant was evaporated to dryness and the residue obtained was reduced with sodium borohydride i n anhydrous methanol. The r e a c t i o n mixture was n e u t r a l i z e d with Amberlite IR-120 (H +) r e s i n a f t e r 1 hour. The mixture was f i l t e r e d , the f i l t r a t e evaporated to dryness and c o d i s t i l l e d with three portions of methanol (5 mL) i n order to remove the borate ion. The residue was hydrolyzed with 2 M TFA on a steam-bath (20 hours) a f t e r which the TFA was removed by c o d i s t i l l a t i o n with water. The hydrolyzate was analyzed by paper chromatography. The sugars released were reduced with aqueous sodium borohydride s o l u t i o n for 2 hours at room temperature. The residue ( a l d i t o l ) was p u r i f i e d by cation-exchange chromatography and c o d i s t i l l a t i o n with methanol. The - 69 -a l d i t o l s were acetylated using a c e t i c anhydride-pyridine (1:1) at 95°C i n anhydrous conditions f o r 1 hour. The re s u l t a n t a l d i t o l acetates, d i s s o l v e d i n chloroform, were analyzed by g . l . c . using column A (see Table I I I . l , column I I ) . N.m.r. spectroscopy (^ H and ^C) was performed on the o r i g i n a l polysaccharide. The p r i n c i p a l signals for both ^H-n.m.r. and ^C-n.m.r, are recorded i n Table III.2 (see Appendix III f o r the reproduction of these n.m.r. spectra). IV.4 Chromium trioxide oxidation A sample (10 mg) of the polysaccharide was dissol v e d i n formamide (5 ml) and treated with a c e t i c anhydride (1 mL) and pyridine (1 mL) overnight at room temperature. The acetylated material (12 mg) was recovered by d i a l y s i s and freeze-drying. The acetylated polysaccharide dissolved i n a c e t i c a c i d was treated with chromium t r i o x i d e (100 mg) at 50°C for 2 hours. The material was recovered by p a r t i t i o n between chloroform and water. Sugar analysis was then performed on the recovered material. G.l.c. r e s u l t s are shown i n Table I I I . l , column VI. IV.5 Methylation analysis The capsular polysaccharide (30 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 - 70 -(H +) r e s i n , was dissolved i n anhydrous dimethyl sulfoxide (5 ml) and methylated 5 3 by treatment with 5 mL d i m e t h y l s u l f i n y l anion for 4 hours and then with 10 mL methyliodide for 1 hour. The methylated poly-saccharide was recovered by d i a l y s i s (M.W. cut o f f 13,500) against d i s t i l l e d water overnight. The methylated polysaccharide was p u r i f i e d by p a r t i t i o n between dichloromethane and water, as well as gel permeation chromatography (Sephadex LH 20). Drying i n vacuo and under i . r . lamp, followed by i . r . spectroscopic analysis i n d i c a t e d complete methylation (no absorptions at 3625 cm'*- and 3200-3500 cm"-*-). A p o r t i o n (15 mg) of t h i s product was hydrolyzed with 2 M TFA at 95°C for 20 hours. The excess a c i d was removed by c o d i s t i l l a t i o n with water and the hydrolyzate analyzed by paper chromatography (solvent (3) developed with p-anisidine hydrochloride). The hydrolyzate was converted to a l d i t o l acetates by sodium borohydride reduction followed by a c e t y l a t i o n with 1:1 a c e t i c anhydride-pyridine. These a l d i t o l acetates were analyzed by g . l . c . and g.l.c.-m.s. using columns A and B. G.l.c. and g.l.c.-m.s. r e s u l t s are as shown i n Table I I I . 3, column I, (see mass-spectra of the a l d i t o l acetates i n Appendix I I I ) . A p o r t i o n of the methylated polysaccharide (15 mg) was reduced with l i t h i u m aluminum hydride i n r e f l u x i n g oxolane overnight. The excess l i t h i u m aluminum hydride was decomposed by adding ethanol. The p r e c i p i t a t e formed was dissolved i n 10% hydrochloric a c i d and the product recovered by chloroform e x t r a c t i o n (3x). The residue was dried and analyzed by i n f r a - r e d spectroscopy. The methylated and carboxyl-reduced polysaccharide was hydrolyzed with 2 M TFA on a steam bath for 20 hours. Reduction of the hydrolyzate with sodium borohydride, - 71 -followed by a c e t y l a t i o n (with a c e t i c anhydride-pyridine), g . l . c . and g.l.c.-m.s. analyses (column A programmed from 180°C to 250°C at 2°C/min) gave the data i n Table III.3, column I I . IV.6 Uronic a c i d d e g r a d a t i o n 6 6 A sample (20 mg) of methylated K34 polysaccharide was d r i e d and then with a trace of p-toluenesulfonic acid, was disso l v e d i n 19:1 dimethylsulfoxide-2,3-dimethoxypropane (12 mL) and the f l a s k was sealed under nitrogen. Di 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 18 hours at room temperature. Methyl iodide (3 mL) was added to the cooled r e a c t i o n mixture and s t i r r i n g continued for an hour. The methylated, degraded product was i s o l a t e d by p a r t i t i o n between chloroform and water. The product was then p u r i f i e d by gel permeation chromatography (Sephadex LH-20). The degraded product was hydrolyzed with 2M TFA for 8 hours at 95°C and the p a r t i a l l y methylated a l d i t o l acetates were prepared as described e a r l i e r . G.l.c. analysis and g.l.c.-m.s. were conducted using column A programmed from 180°C to 250°C for 2°C/min. G.l.c. and g.l.c.-m.s. r e s u l t s are as shown i n Table I I I . 3, column I I I . IV. 7 Carbodiimide reduction of K34 polysaccharide 2^ A p o r t i o n (120 mg) of K34 polysaccharide (H + form) was dissolved 72 -i n water (30 mL). l-Cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMC, 423 mg) was added to the polysaccharide s o l u t i o n . As the r e a c t i o n proceeded (with consumption of hydrogen ions), the pH was maintained at 4.75 by adding hydrochloric a c i d (0.1M) dropwise. When consumption of hydrogen ions ceased, approximately 2 hours l a t e r , an aqueous s o l u t i o n of sodium borohydride (3M) was added dropwise. Foaming was c o n t r o l l e d by constant s t i r r i n g . Approximately 100 ml of sodium borohydride s o l u t i o n was added over a period of 2 hours. Throughout the base addition, the pH of the r e a c t i o n s o l u t i o n was maintained at about pH 7 by t i t r a t i n g with hydrochloric a c i d s o l u t i o n . A f t e r concentrating, the polysaccharide was dialyzed against d i s t i l l e d water f o r two days. The l y o p h i l i z e d product was then weighed ( y i e l d = 124 mg). IV.8 Periodate oxidation and Smith h y d r o l y s i s - ^ of carboxyl reduced K34 polysaccharide The consumption of periodate was monitored using spectrophotometer model 240 at 223 M. The c a l i b r a t i o n curve for monitoring periodate consumption was obtained as follows. ( i ) IO^'/IC^" solutions containing the following mmoles of NaI04 were prepared; 0.30, 0.225, 0.15 and 0.075. ( i i ) The absorbance of these solutions was measured by means of a spectrophotometer. ( i i i ) A p l o t of absorbance versus concentration was obtained (Figure I I I . l ) . The carboxyl reduced polysaccharide (120 mg) was d i s s o l v e d i n 30 73 -mL water and 20 mL 0.06M sodium metaperiodate was added. The reaction was conducted at room temperature and i n the dark. Aliquots (0.1 M) of the r e a c t i o n s o l u t i o n were withdrawn p e r i o d i c a l l y , d i l u t e d 250 times and analyzed on the spectrophotometer. The periodate consumption reached a plateau a f t e r about 122 hours (see Figure I I I . 2). The number of millimoles consumed was obtained from the c a l i b r a t i o n curve knowing the change i n absorbance a f t e r 122 hours. Approximately 4 moles 10^" were consumed per repeating u n i t . The excess periodate was destroyed by adding ethylene g l y c o l (1 mL) and the product was dia l y z e d overnight against d i s t i l l e d water. Sodium borohydride (0.5 g) was added and the s o l u t i o n was l e f t overnight. The s o l u t i o n was deionized with Amberlite IR 120 [H +] r e s i n and the product c o d i s t i l l e d with several portions of methanol. The derived polyalcohol was dissolved i n 10 mL 0.5 M TFA and s t i r r e d at room temperature for 48 h. The product was dialyzed against 2 L of d i s t i l l e d water. The dialy z a t e was concentrated and freeze-dried. Two compounds were i s o l a t e d by preparative paper chromatography on the freeze-dried product, using solvent B. The slowing moving compound P2 was analyzed by ^H-n.m.r. spectroscopy. Sugar analysis was performed on P2 as previously described (Table I I I . l , column I I I ) . P2 was methylated according to Hakomori's procedure-3-'. The methylated product was analyzed as p a r t i a l l y methylated a l d i t o l acetates by g . l . c . and g.l.c.-m.s. (column A programmed from 180°C to 250°C at 2°C/min). Smith degradation was also conducted on K34 polysaccharide (Na + form). - 74 -IV.9 Selective Smith degradation A solution of K34 polysaccharide (20 mg) in water was mixed with 0.02 M NalO^ (20 mL) and kept in the dark for 3 hours at room temperature. Ethylene glycol (0.2 mL) was added, the mixture was stirred for 1 hour and the polyaldehyde formed was dialyzed overnight. This polyaldehyde was reduced to the polyol by adding sodium borohydride to a concentrated solution of the non-dialyzable residue. Smith hydrolysis was effected by treating the product with 0.5 M TFA and s t i r r i n g for 24 hours at room temperature. The product was dialyzed against 1 L of d i s t i l l e d water. The substance that remained in the dialyzing sac was freeze dried and analyzed by ^H-n.m.r. spectroscopy (Table III.2) . This degraded product was methylated according to Hakomori's procedure 5 3. The methylated product was analyzed as partially methylated a l d i t o l acetates by g.l.c. and g.l.c.-m.s. (column A programmed from 180°C to 250°C at 2°C/min). Methylation analysis data are given in Table III.3. The degraded product, dried in vacuo and under i . r . lamp, was treated with methanolic hydrogen chloride (3%) and refluxed overnight on a steam bath under anhydrous conditions. The excess acid was removed by treatment of the reaction mixture with lead carbonate, followed by centrifugation. The supernatant was concentrated and the residue was converted to a l d i t o l acetates. These al d i t o l acetates were analyzed by g.l.c. and g.l.c.-m.s. (column programmed from 180°C to 220°C at 5°C/min). Sugar analysis data are shown in Table I I I . l . - 75 IV.10 Determination of the c o n f i g u r a t i o n (D or L) of the sugars The product from the methylat ion a n a l y s i s was separated into i n d i v i d u a l p a r t i a l l y methylated a l d i t o l acetates u s i n g preparat ive g . l . c . (column SP 2340 programmed from 175°C to 240°C at l ° C / m i n ) . The p a r t i a l l y methylated a l d i t o l acetates i s o l a t e d by the above mentioned technique are shown i n Table I I I . 4 . Each of these a l d i t o l acetates was d i s s o l v e d separate ly i n a c e t o n i t r i l e and t h e i r c d . spectra recorded. Comparison of the c d . spectra of these a l d i t o l acetates with that of t h e i r authentic standards showed that a l l the sugar residues have the D c o n f i g u r a t i o n . K34 bacteriophage o l igosacchar ide (3 mg) was d i s s o l v e d i n 1 mL of sodium acetate b u f f e r (pH - 7.0) and a s o l u t i o n of a - D - g l u c o s i d a s e (0.5 mg i n 1 mL b u f f e r ) was added. The mixture was incubated f o r 2 days at 3 7 ° C , then the r e a c t i o n was terminated by adding a t race 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 chromatograph. IV.11 S e r o l o g i c a l c r o s s - r e a c t i o n s E . c o l i K34 bacteriophage and E . c o l i K31 bacteriophage were i s o l a t e d from Vancouver Sewage. The b a c t e r i a l lawn f o r the E. c o l i s t r a i n s K28, K31, K32, K33 and K34 were made as p r e v i o u s l y d e s c r i b e d . By means of a s t e r i l e f i n e l y drawn p i p e t t e , E . c o l i K34 bacteriophage was spotted on the b a c t e r i a l lawns f o r the E. c o l i s t r a i n s K28, K31, K33 - 76 -and K34. E. coli K31 bacteriophage was also spotted on E. coli K34 bacterial lawn. After overnight incubation of these plates (containing bacterial lawn spotted with bacteriophages) the plates were inspected for plaques formed. IV.12 Bacteriophage depolymerization of E. coli K31 capsular polysaccharide E. coli K31 bacteriophage (#31) isolated from Vancouver sewage was propagated on E. coli K31 bacteria according to standard methods of Adam74. #31 was propagated using tube lysis and flask lysis (see Table III. 5). #31 solution (5.5 x 1012) was dialyzed (cut off 3500) against buffer pH - 7 (ammonium acetate and ammonium carbonate buffer) for 2 days. After concentrating by evaporation under reduced pressure, the concentration of #31 was 1.8 x lO^ 2 p . f . u . Stirm 7 5 had shown that lO^ 3 bacteriophages are required to degrade 1 g of the corresponding bacterial capsular polysaccharide. This concentrated #31 solution was added to E. coli K31 capsular polysaccharide (180 mg; prepared by Dr. E. Altman according to the procedure described in IV.2) solution. The depolymerization was conducted in an incubator at 37°C for 48 hours. A Molisch test^l^ a n c \ paper chromatography of the crude reaction mixture indicated the presence of an oligosaccharide. The reaction mixture was concentrated and dialyzed (M.W. cut off 3500) against disti l led water (3x). The dialyzate collected each time was combined and concentrated. The crude depolymerized product was subjected to Amberlite IR 120 (H+) c a t i o n i c exchange treatment and f r e e z e - d r i e d . A concentrated s o l u t i o n of the p u r i f i e d depolymerized product was placed on a column of B i o - G e l P2 (400 mesh) and e l u t e d at 6.8 m l / h . F rac t ions (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 . IV.13 Bacteriophage depolymerizat ion of E . c o l i K34 capsular polysaccharide E. c o l i K34 bacteriophage (#34) was a lso i s o l a t e d from Vancouver sewage and propagated on E. c o l i K34 b a c t e r i a according to the standard methods of Adam 7 ^. #34 was propagated us ing tube l y s i s and f l a s k l y s i s (Table I I I . 7 ) . #34 s o l u t i o n (1.1 x 1 0 1 3 ) was d i a l y z e d (M.W. cut o f f 3500) against b u f f e r pH - 7 (ammonium acetate and ammonium carbonate b u f f e r ) f o r 3 days. A f t e r concentrat ing by evaporat ion under reduced pressure , the concentra t ion of #34 was 7.2 x 10^ 2 p . f . u . The concen-t r a t e d #34 s o l u t i o n was added to E. c o l i K34 capsular polysacchar ide (250 mg, prepared according to the procedure descr ibed i n IV.2) s o l u t i o n . The depolymerizat ion was conducted i n an incubator at 37°C f o r 48 hours . Paper chromatography of the r e a c t i o n mixture i n d i c a t e d the presence of an o l i g o s a c c h a r i d e . The r e s u l t a n t degraded product was p u r i f i e d as p r e v i o u s l y d e s c r i b e d . A concentrated s o l u t i o n of the p u r i f i e d depolymerized product was placed on a column of B i o - G e l P2 (400 mesh) and e l u t e d at 6.6 mL/h. F rac t ions (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 . I I I . 3 . - 78 -N . m . r . study A s o l u t i o n of E. c o l i K34 bacteriophage generated o l i g o s a c c h a r i d e ( F r a c t i o n I) was reduced with sodium borohydride (45 min) and cation-exchanged with Amberli te IR 120 (H + ) r e s i n u n t i l the pH was a c i d i c . The eluant obtained was concentrated to dryness and the borate formed was removed by c o d i s t i l l a t i o n with methanol (3x). The reduced o l i g o s a c c h a r i d e (23 mg) was exchanged three times wi th D2O and submitted f o r ^ H - n . m . r . spectroscopy. Bacteriophage (#34) o l i g o s a c c h a r i d e (20 mg) was a lso exchanged three times with D2O and submitted f o r l H - n . m . r . spectroscopy. N . m . r . data f o r these o l i g o s a c c h a r i d e s are as shown i n Table I I I . 8 . Determination of the reducing e n d i U 0 NaBH^ (30 mg) was added to a s o l u t i o n of o l i g o s a c c h a r i d e ( 1 0 mg i n 5 ml of H 2 O ) . A f t e r s t i r r i n g f o r 5 hours the excess of sodium borohydride was removed by cation-exchange chromatography (IR 1 2 0 (H + ) ) and coevaporation with methanol. The reduced m a t e r i a l was hydrolyzed with 2M t r i f l u o r o a c e t i c a c i d on a steam bath f o r 2 0 hours and the excess a c i d removed by coevaporation with water. A hydroxylamine H C 1 s tock s o l u t i o n was prepared by d i s s o l v i n g 0 . 5 g of hydroxylamine H C 1 i n 2 0 ml of p y r i d i n e . 0 . 5 ml of h y d r o x y l -amine-HC1 s tock s o l u t i o n was added to the hydrolyzate and the r e a c t i o n mixture was heated on a steam bath f o r 15 minutes. A c e t i c anhydride (0.5 mL) was added to the cool reaction mixture and the resultant r e a c t i o n mixture was refluxed on a steam bath for 45 minutes. The mixture of peracetylated a l d o n o n i t r i l e s and peracetylated 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. The product was analyzed by g. l . c . and g.l.c.-m.s. using DB-17 c a p i l l a r y column programmed from 180°C to 220°C at 5°C/min. - 80 -BIBLIOGRAPHY 81 -BIBLIOGRAPHY 1. G.O. A s p i n a l l , i n G.O. A s p i n a l l ( E d . ) , "The P o l y s a c c h a r i d e s " , V o l . 1, Academic Press , New York, 1982, p . 1. 2. P . A . Sandford and J . 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Lepke and W.C Raschke, J . B a c t e r i o l . , 117. 1974, 461-467. 110. Z. Dische, Methods i n Carbohydr. Chem., Vol. 1, pp. 478. 111. V. Smirnyagin, C T . Bishop and F.P. Copper, Can. J . Chem., 43, 1965, 3109. 112. R. Morona, J . Tommassen and U. Henning, Eur. J . Biochem., 150 (1), 1985, 161-169. - ti / APPENDICES APPENDIX I THE KNOWN STRUCTURES OF THE ESCHERICHIA COLI K ANTIGENS (as of August 1, 1985) - 88 -E. COLI K ANTIGENS NANA5AC 2 — a E. c o l l KI ^ _ P _ 4 Gal L-2 Gly U21...,.? _ 5 G a l f 1_2 G l y H31} E. c o l i K2 A GlcA GlcNAc 1 -P E. c o l i K5 _2 R i b f 1_2 R i b f 1_7 2— ^ P P a -3. R i b f i - I KDO 2— 2 0 /9 P 1 R i b f E. c o l i 6a E. c o l l K6 3- ManNAcA -1—^ Glc - 1— OAc E. c o l i K7 and K56 - 89 -Rha I - 2 - Rha i—5—KDO 2— a ct 7/8 P I OAc E. c o l i K12 and K82 - i Rib f l~l KDO 2— P U P I OAc E. c o l i K13 —& GalNAc i - 5 - KDO 2_ tt 8 B I OAc E. c o l i K14 GlcNAc -^5- KDO 2_ E. c o l i K15 A Rib f L_Z KDO 2— 5, OAc E. c o l l K20 90 R i b f i _ Z KDO 2_ B B E. c o l i K23 A Glc LA GlcA F u c i . 3 O Q C Q 1 Gal E. c o l i K27 Glc LA GlcA LA Fuc -A— 3 Q a 1 Gal /3 o 2 or 3 I OAc E. c o l i K28 Man L-l Glc A-3- GlcA i - 3 - Gal a 1 Glc J--2- Man 4 6 b \ / pyr E . c o l i K29 M a n i _ 3 G a l 1_ 3 Q B a GlcA L-l Gal 0 E. c o l i K30 91 Gal i - 2 - Glc i - 3 - GlcA i - ^ Rha i - 2 - Rha 2-P E. coll K31 OAc 2 Glc 1-4 Rha i - 3 - Gal P 1 GlcA E. coli K32 _3 Glc GlcA i - 4 - Fuc ° 3 2 P 3 V pyr Gal OAc E. coli K33 -•—2- GlcA Gal I- 3- Gal 1-3 /* 1 Gal 4 a 1 Glc E. coli K34 - 92 _3 G a i 1_3 G a l A 1_3 F u c 1_ a a a E. c o l l K42 0 3 1 1 A Gal A 0 - P - 0 -OH 2 + OAc + OPr Fru E. c o l i K52 GlcA Uh. GlcA LA M a n i _ 3 M a n 1_3 G l c N A c 1_ M a n 1_3 M a n 1_3 G l c N A c 1_ 1 2 Rha Rha E . c o l l K85 ->-A GlcA LA FucNAc i - 3 - GlcNAc I- 6- Gal L 4 1 Glc 2-OAc E. c o l l K87 -2 NANA5AC "L-l NANA5Ac L. E. c o l i K92 93 — R i b f i - 2 - r i b i t o l 0 — r b 0 I P — OH E. c o l i K100 - 94 -References KI 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 . B a c t . , 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. R e s . , 105. 1982, 45-56. K7-K56 F . - P . T s u i , R . A . Boykins and W. Egan, Carbohydr. R e s . , 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 . Orskov and F. Orskov, 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 . Orskov and F. Orskov, I n f e c t . Immun., 39, 1983, 623-629. K27 K. Jann, B. Jann, K . F . Schneider, F . Orskov and I . Orskov, Eur . J . Biochem., 5, 1968, 456-465. K28 E . Altman and G . G . S . dutton, Carbohydr. R e s . , i n p r e s s . K29 Y . - M . Choy, F . Fehmel, N. Frank and S. S t i r m , J . V i r o l . , 16, 1975, 581-590. K30 A . K . Chakraborty, H. F r i e b o l i n and S. S t 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 . Orskov, F . Orskov, B. Jann and K. Jann, B a c t e r i o l . R e v . , 41 (1977) 667-710). K32 E . Altman, unpublished r e s u l t s . - 95 -K33 B . A . Lewis, unpublished r e s u l t s . K34 G . G . S . Dutton and A. Kuma-Mintah, 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. S t 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, Int . Symp. Carbohydr. Chem., 12th 1984 A b s t r a c t s , p . 367. K85 K. Jann, B. Jann, F . Orskov and I . Orskov, 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 press . - 96 -APPENDIX II MASS SPECTRA SPECTRUM NO. 1 HEXITOL HEXAACETATE MASS SPECTRUM 100 90 80 70 60 50 40 30 20 10 0 43 115 in 05 73 rt L U rr 145 r t 187 217 259 289 rVrr i i i i i r i i i i r i i i i i i i i TI i I i i i i | i i i f i 400 50 100 150 200 250 300 350 I I | I I I I I I I I I | 450 500 SPECTRUM NO. 2 1,2,5,6-TETRA-0-ACETYL-3,4-DI-O-METHYLGLUCITOL MASS SPECTRUM 100 90 80 70 60 50 40 30 20 10 0 43 71 .87 l"'l '| I "| I T T " I 50 129 I.. ..i.l 13 U51 r"r vi i-i-i '|11 i i f i i 150 189 233 261 i r r r 350 100 I | I I l " l I I I I I | I I 200 250 300 "TT 400 SPECTRUM NO. 3 1 , 3 , 4 , 5 - T E T R A - 0 - A C E T Y L - 2 , 6 - D I - M E T H Y L G A 1 A C T I T O L MASS SPECTRUM 100 90» 80 70 60 50 40 30 20 10 0 ' 43 1 58 74 87 117 Ii. ..lilll.i, ...Li 50 r r j r 100 143 t V i ' f e 150 185 T T - 'i i y "i r 200 i VO 305 231 I I"I T" I "I I I I I T 250 300 I I I I I I I | I 350 I I I I I I I | 400 SPECTRUM NO. 4 1,3,5-TRI-0-ACETYL-2,4,6-TRI-METHYLGALACTITOL MASS SPECTRUM 90 80 70 60 50 4Vl 30 '•' 20 10 0 43 117 58 17,1 T T 50 r r i 100 161 I T T | I 150 ilk o o 233 'i i | i i I I i • 200 T T 250 I 2 7 7  I I I I I | I I 300 I I I I | I 350 I I I I 400 SPECTRUM NO. 5 1,4,5-TRI-0-ACETYL-2,3,6-TRI-METHYLGALACTITOL MASS SPECTRUM 43 117 233 ,142 161 I I I I I I I | I I I 350 50 "i r'"|'"i "i' 100 T T I I I I I I I 150 T T T T 200 I I I | I I 250 I I I I' I | 300 400 SPECTRUM NO. 6 1, 5-DI-0-ACETYL-2,3,4,6-TETRA-O-METHYLGLUCITOL MASS SPECTRUM 100 90 80 70 60 50 40 30 20 10 0 43 71 4 i 0 i 87 161 129 205 1 1 " l " | ' ' I "I i "I I I I I J "I" I I' I I I i I I j I I 'I I I I I I I ( . , 150 200 250 300 I T 50 100 l l l l l I I | I I I I I 350 I I I 400 - 103 -APPENDIX III 1H AND 13C-N.M.R. SPECTRA K34 Polysaccharide ^-N.m.r. 400 MHz, 95° Spectrum No. 1 T 4 r 5 T 4 K34 Polysaccharide 13C-N.m.r. 100.6 MHz, 90° Spectrum No. 3 a c e t o n e 31.071 I i i K34 Polysaccharide (Selective Smith degraded) ^-N.m.r. 400 MHz, 95° HOD Spectrum No. 4 

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