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Studies on bacterial capsular polysaccharides and on a plant gum Di Fabio, Jose Luis 1981

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STUDIES ON BACTERIAL CAPSULAR POLYSACCHARIDES AND ON A PLANT GUM by JOSE LUIS LDI FABIO B.Sc., Universidad de l a Republica, Uruguay, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1981 © Jose' Luis Di Fabio, 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f CH€HJS[«y  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e ^ 5 / P f S / / V a * DF-fi i?/7<n ABSTRACT The structure of the capsular polysaccharides from Klebsiella serotype K60 and K26 have been determined using the techniques of methylation.periodate oxidation,partial 1 13 hydrolysis, and B-elimination. H- and ^C-n.m.r. spectros-copy was used to establish the nature of the anomeric l i n -kages i n both polysaccharides and also in the oligosaccha-rides obtained by the different degradative techniques used. Specific hydrolases obtained from bacteriophages were uti l i z e d to degrade two Klebsiella polysaccharides.Larger quantities of oligosaccharide repeating units can be gen-erated i n this manner.Two bacteriophages, 060 and 046,the f i r s t one with B-glucosidase activity and the other with 8 -galactosidase activity,were used to degrade the cor-responding polysaccharides according to a new,simplified procedure. The purified gum exudate from Chorisia speciosa(palo borracho) was studied.The results from methylation anal-ysis, B -elimination and partial hydrolysis made possible a tentative assignment for an " average structure " of the gum polysaccharide. i i i D-G 1 l C £ K 6 0 —^D-Glcrr 1—^D-GlcpA^—^D-Galp^—2.D-Man£^— & 21 a 2 • " l l D-G1C2 1 D-G1C2 K 2 6 P k 6 D-Gal£ 1 [ D-Glcp I f 6 D-Glc£ • D - G a l j ^ — ^ D - G I C E A - -^D-Man£^—-D-ManjA iv TABLE OF CONTENTS P a g e ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x i LIST OF SCHEMES x i i i ACKNOWLEDGEMENTS xiv PREFACE XV I INTRODUCTION 1 II METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES 20 11.1 The structures of polysaccharides. . . . 21 11.2 Isolation and purification 22 11.2.1 Klebsiella polysaccharides . . . 23 11.2.2 Gum exudate of Chorisia speciosa 23 11.3 Sugar analysis Zk 11.3.1 Total hydrolysis Zk 11.3.2 Characterization and quantitation of the sugars . . . 25 11.3.3 Determination of the configuration of the sugars. . . 26 TL.h Position of linkage 28 II.k.1 Methylation analysis .28 II.k.1.1 Methylation procedures . 28 11.^.1.2 Characterization of the methylated sugars. . . . 29 V 11.5 Sequencing of sugars 36 11.5.1 P a r t i a l hydrolysis 36 11.5.2 Periodate oxidation and Smith hydrolysis 38 11.5.3 Degradation based on B-elimination. 41 11.5.3.1 Base catalyzed uronic acid degradation 43 11.5.3.2 Degradation preceded by oxidation 44 11.5.4 Separation of oligomers obtained from degradations ^7 11.6 Determination of the linkages 49 11.6.1 Optical r o t a t i o n 49 11.6.2 Nuclear magnetic resonance. . . . 51 11.6.2.1 "--H-n.m.r 51 11.6.2.2 1:3C-n.m.r 57 11.6.3 Other techniques . . . . . . . . 60 11.7 Various reactions on o l i g o - and poly-saccharides 61 II.7.1 Reduction 61 I I . 7.2 Oxidation 64 III GENERAL EXPERIMENTAL CONDITIONS 65 111.1 Paper chromatography 66 111.2 Gas-liquid chromatography and g.l.c.-m.s. spectroscopy 66 111.3 Gel-permeation chromatography 67 v i III.4 Optical r o t a t i o n and c i r c u l a r dichroism . 68 111.5 Nuclear magnetic resonance 68 111.6 General conditions 69 111.7 I s o l a t i o n and p u r i f i c a t i o n of the poly-saccharides 69 111.7.1 K l e b s i e l l a polysaccharides . . . . 69 111.7.2 Gum exudate of Chorisia speciosa . 70 111.8 Sugar analysis 71 111.9 Methylation analysis 72 111.10 Base catalyzed uronic acid degradation . 75 IV STRUCTURAL INVESTIGATIONS OF KLEBSIELLA CAPSULAR POLYSACCHARIDES 77 IV.1 S t r u c t u r a l i n v e s t i g a t i o n of K l e b s i e l l a serotype K60 capsular polysaccharide. . . 78 IV. 1.1 Abstract 78 IV. 1.2 Introduction 78 IV. 1.3 Results and discussion 79 IV. 1.4 Experimental 96 IV.2 S t r u c t u r a l i n v e s t i g a t i o n of K l e b s i e l l a serotype K26 capsular polysaccharide. . . 106 IV. 2.1 Abstract 106 IV. 2.2 Introduction 107 IV.2.3 Results and discussion 107 IV. 2.4 Experimental 121 IV.3 Bacteriophage degradation of K l e b s i e l l a polysaccharides K60 and K46 129 V I 1 IV. 3 . 1 Introduction 1 2 9 I V . 3 . 2 Results 1 3 2 I V . 3 . 3 Discussion. 1 5 2 IV. 3 . 4 Experimental 1 5 5 V STRUCTURAL STUDIES OF THE GUM EXUDATE OF CHORISIA SPECIOSA. 1 6 1 V . l Abstract 1 6 2 V . 2 Introduction 1 6 2 V . 3 Results and discussion 1 6 4 V . 4 Experimental 1 7 2 VI BIBLIOGRAPHY 1 7 7 • • • V l l l APPENDIX Page I S t r u c t u r a l patterns of K l e b s i e l l a capsular polysaccharides 1 9 0 II The structures of K l e b s i e l l a capsular poly-saccharides 1 9 5 III 1H- and ^C-n.m.r. spectra 2 1 2 IV Uses of peracetylated a l d o n o n i t r i l e s 2 ^ 5 Ix LIST OF TABLES Page TABLE 1.1 TABLE 1.2 TABLE 1.3 TABLE 1.1* K l e b s i e l l a capsular polysaccharides ( K l -K83).Qualitative analysis and chemo-type grouping. Aldobiouronic acids i n K l e b s i e l l a cap-sular polysaccharides Subsitution pattern of the sugar r e s i -dues i n the K l e b s i e l l a capsular poly-saccharides Percentage composition of a - and B -gl y c o s i d i c linkages i n K l e b s i e l l a cap-sular polysaccharides TABLE 1 . 5 Location of pyruvate i n K l e b s i e l l a capsular polysaccharides TABLE IV.1.1 N.m.r. data f o r K l e b s i e l l a K60 capsular polysaccharide and derived poly- and oligo-saccharides TABLE IV.1.2 Methylation analysis of K60 capsular polysaccharide and derived products TABLE IV.1.3 Analysis of the oligosaccharides from p a r t i a l hydrolysis of K60 polysaccharide TABLE IV.2.1 N.m.r. data f o r K l e b s i e l l a K26 capsular polysaccharide and derived ,poly- and oligo-saccharides TABLE IV.2.2 Methylation analysis of K26 capsular polysaccharide and derived products Analysis of the oligosaccharides from p a r t i a l hydrolysis of K26 polysaccharide 11 13 16 17 81 87 9^ 109 116 TABLE IV.2.3 TABLE IV.3.1 Propagation of bacteriophages 060 and ffo6 133 119 X TABLE IV.3 .2 Depolymerization of K l e b s i e l l a K60 and K46 capsular polysaccharides by bacteriophages 060 and 046 respectively TABLE I V . 3 . 3 a P.m.r. data f o r K l e b s i e l l a K60 capsular polysaccharide and the oligomers derived from bacteriophage degradation 3 b N.m.r. (^C) data f o r K l e b s i e l l a K60 capsular polysaccharide and the oligomers derived from bacteriophage degradation Determination of the degree of polymeriza-t i o n and the reducing end of K60 oligosac-charides (P^ and P 2) Methylation analysis of K60 oligosaccharides TABLE IV.3-TABLE I V . 3 - ^ TABLE IV.3 TABLE IV.3 (P.^  and P 2) from bacteriophage degradation TABLE IV. 3 -6a P.m.r data f o r K l e b s i e l l a K46 capsular polysaccharide and the oligomers derived from bacteriophage degradation 6b N.m.r. ("^O data f o r K l e b s i e l l a K46 capsular polysaccharide and the oligomers derived from bacteriophage degradation TABLE IV.3>7 Determination of the degree of polimeriza-t i o n and the reducing end of K46 oligosac-charides (P^ and Pg) 8 Methylation analysis of K46 oligosaccharides TABLE IV.3 TABLE V . l TABLE V . 2 TABLE V . 3 (P.^  and Pg) from bacteriophage degradation Methylation analysis of Chorisia speciosa gum exudate and derived products Analysis of the oligosaccharides from p a r t i a l hydrolysis of the gum exudate N.m.r. ("Hi) data of the a c i d i c oligosaccha-rides from p a r t i a l hydrolysis of the gum 1 3 4 1 3 8 140 143 146 148 1 5 0 1 5 1 1 6 5 1 6 7 1 7 2 x i LIST OF FIGURES ..Page Figure 1.1 Diagrammatic representation of the b a c t e r i a l c e l l envelope and d i f f e r e n t antigens 3 Figure 1.2 a ) K l e b s i e l l a K5 cross-reacts with anti-serum to Pneumococcus I I I ; b ) K l e b s i e l l a K26 cross-reacts with anti-serum to K l e b s i e l l a K l l 6 Figure II.1 M.s. of a) 1 , 2 , 5 - t r i - 0 - a c e t y l - 3 , 4 , 6 - t r i - 0 -methylgalactitol and b) l , 2 , 5 - t r i - 0 - a c e t y l -3 - 0 - e t h y l - 4 , 6 - d i - 0 - m e t h y l g a l a c t i t o l 3 3 Figure II.2 Common products formed on periodate oxidation of terminal and monosubstituted hexoses 40 Figure I I . 3 Relationship between dihedral angle(0) and coupling constant f o r a - and e -D-hexoses 5-^ Figure I I . 4 The """H-n.m.r. spectrum of K l e b s i e l l a K 6 0 capsular polysaccharide 5 6 Figure I I . 5 The ^C-n.m.r. spectrum of K l e b s i e l l a K 6 0 capsular polysaccharide 5 8 Figure IV.1 Separation of the a c i d i c oligosaccharides from p a r t i a l hydrolysis of P^ by gel-permea-t i o n chromatography 9 2 Figure IV.2 Separation of the a c i d i c oligosaccharides from p a r t i a l hydrolysis of K l e b s i e l l a K26 polysaccharide by gel-permeation chromato-graphy 1 1 5 Figure IV.3 Attack of a bacteriophage on an encapsulated bacteria 1 3 1 Figure IV.4 Separation of the depolymerization products of K60 by gel-permeation chromatography 136 Figure IV.5 Separation of the depolymerization products of K46 by gel-permeation chromatography 145 x i i Figure IV.6 Environment of the glycosidic linkage which undergoes enzymic hydrolysis (K60) and the one that does not (K60SH) Figure IV.? Growth curve and bacteriophage lysis of Klebsiella K60 bacteria Figure V.l One of the possible "average structure" for the gum of Chorisia.speciosa x i i i LIST OF SCHEMES Page Scheme II.1 Methylation analysis of Klebsiella K26 JO Scheme II.2 Smith degradation of Klebsiella K26 42 Scheme II.3 Uronic acid degradation of Klebsiella K26 polysaccharide 45 Scheme II.4 Degradation preceded by oxidation on the Klebsiella K26 polysaccharide 46 Scheme II.5 Reduction of a carboxylic acid i n aqueous solution using a carbodiimide reagent 63 x i v ACKNOWLEDGMENTS The d i r e c t i o n and i n t e r e s t of Prof. G.G.S. Dutton during t h i s study i s g r a t e f u l l y acknowledged. I wish to thank my collegues i n the laboratory for t h e i r support,specially Dr. A. Savage,and also the Professors who v i s i t e d the laboratory during t h i s time for t h e i r h e l p f u l discussions.Sincere thanks to Dr. E.H. M e r r i f i e l d for his assistance and proof-reading of t h i s t h e s is, and also Dr.S.C Churms (University of Cape Town) for the molecular weight determinations. My s p e c i a l thanks to my wif e , E t e l , f o r her encouragement and support i n thi s venture. I am g r a t e f u l to Mac M i l l a n Bloedel f o r the award of a Graduate Scholarship (1977-1978) and also the University of B r i t i s h Columbia for the award of a University Graduate F e l -lowship(1980-1981). XV PREFACE One of the most outstanding features of "bacterial poly-saccharides i s t h e i r ordered structures composed of oligo -saccharide repeating units.These surface carbohydrates can be extracted from bacteria and not only studied chemically, but also used as antigens.Such studies are very important i n the l i g h t of b a c t e r i a l i n f e c t i o n s and the production of protective vaccines.Oligosaccharides obtained from these polysaccharides can be coupled to c a r r i e r s and used for the induction of antibodies.A p r o l i f i c source of enzymes which depolymerize the capsular polysaccharides into t h e i r o l i g o -meric repeating units are the bacteriophages. The study on gum exudates has been car r i e d out f o r many years.The main goal i s to f i n d substitutes for the ones already known of commercial value, and f o r th e i r p o t e n t i a l use i n chemical taxonomy of plants. 1 I . INTRODUCTION 2 I. - INTRODUCTION The outermost surface of any organism i s of great im-portance i n the i n t e r r e l a t i o n between i t s e l f and i t s envi-ronment, the outermost mediator i s the f i r s t p o r t a l of entry and the l a s t b a r r i e r to excretion.In microorganisms i t must play an important r o l e i n the recognition of the c e l l by viruses,antibodies,etc. A b a c t e r i a l c e l l w all i s composed of several layers (see Figure 1.1), i ) the cytoplasmic membrane,ii) a peptido-glycan (murein) layer and i i i ) an outer membrane composed of lipopolysaccharides,proteins and polysaccharides. Many bacteria produce polysaccharides which are located outside the c e l l wall^". These polysaccharides may take the form of capsules or they may be an e x t r a c e l l u l a r slime un-attached to the b a c t e r i a l surface.Medical microbiologists were f i r s t interested i n these macromolecules as they play an important r o l e i n the pathogenicity of the bacteria(an-p tigens,toxins) .The i n t e r e s t increased with the i n d u s t r i a l need of new g e l l i n g and emulsifying agents (e.g. Xanthomonas  campestris^ produces an exopolysaccharide.xanthan gum,which i s used i n food industry). Although the function of these structures i s not well understood,they may be involved i n one or a l l of the follow-ing^ : i ) storage or reserve, i i ) v i r u l e n c e i p r o t e c t i o n against phagocitosis, i i i ) protection against predation, Capsular polysaccharide ( K antigen) Lipopolysaccha-: ride ( 0 antigen) Outer membrane Peptidoglycan layer Cytoplasmic membrane oooooooooooa Cytoplasm Figure 1.1 .Diagrammatic representation of the b a c t e r i a l c e l l envelope and the d i f f e r e n t antigens. iv) protection against desiccation, v) adhesion, vi) general barriers. The immunological defence system of higher organisms (e.g. mammals) is based on i t s capacity to recognise struc-tures as foreigners or as part of i t s e l f . I f the foreign structure has the property of inducing the formation of anti-bodies, i t i s called an antigen and the property immunogenici-ty.After an antibody is produced i t w i l l react specifically with the antigen that originated i t s production,this i s called antigenic specificity.However,there are small molecules.haptens, which,by themselves.cannot stimulate antibody synthesis but w i l l combine with the antibody once formed. Bacteria,when introduced into the blood stream of a mammal.will induce this immune response.Certain structures on the c e l l surface of the microorganism are antigens^,e.g. cap-sular material ( K antigens.protein or polysaccharide in natu-re) .lipopolysaccharides ( 0 antigens).flagella (H antigens,pro-tein in nature). Bacterial capsular polysaccharides have been recognised as antigens^''''for a long time and most of the pioneering work on quantitative immunochemistry was initiated by Heidelberger R—1 0 and coworkers ~ on pneumococcal capsular polysaccharides and their antigenic s p e c i f i c i t i e s . The purpose of the immunochemical analysis of polysaccha-rides is to find specific structures in them,which are the che-mical expression of the immunological character.These structu-res or antigenic determinants can be a single sugar linked in 5 a s p e c i f i c manner,an oligosaccharide or even non-carbohydra-te i n nature (e.g. polysaccharides with ketal-linked pyruvate). There i s some conclusive evidence that charged constituents on polysaccharides (uronic acids or other a c i d i c residues) are often part of the antigenic determinant or the immunodominant sugar.One polysaccharide can have'different antigenic determi-nants due to d i f f e r e n t sugar residues or d i f f e r e n t combina-tions of them.As the b a c t e r i a l polysaccharides are formed of oligosaccharide units that repeat,the antigenic determinants are expressed many times over. The same antigenic determinant may be present i n several polysaccharides.As a consequence,it i s recognised by i t s homo-logous antibody i n the d i f f e r e n t polysaccharides.no matter i n what organism they are produced.These polysaccharides that are immunologically related are said to cross-react s e r o l o g i c a l l y (see Figure I. 2 ) ^ ® .This immunological reaction has been used ingeniously by Heidelberger and coworkers"*"^"^ for the immune^ l o g i c a l determination of the structure of many unknown poly-saccharides . This approach i s based on the fact that the cross reaction of a polysaccharide of known chemical structure with antibodies to a polysaccharide of uncertain or unknown s t r u c -ture may y i e l d information as to one or more sugars contained i n the unknown and even as to the positions at which the su-gars are linked;conversely,cross-reactivity of a polysaccha-ride of known composition and linkage ,may be equally i n f o r -mative. The Gram-negative family Enterobacteriaceae i s of wide 6 a) Pn III K-5 _ 2 D-GlcjpA - — - D-Glcj? D-GlcpA - — - D-Glcp " 2 ? — 6AC D-Manjj b) K 11 K 26 D-Glcjp -—2. D-GlcjpA 6 4 D-Galp / 1 a ,'D-Galp ! —a D-Galp. ~ — - D-GICJDA -—2. D-Manp_ - — - D-Marffi — 1| D-Glcp_ 6 1| D-GlC£ 4| / 1 'D-Galp , Figure 1.2 a) K l e b s i e l l a K 5 cross-reacts with a n t i -serum to Pneumococcus I l l . b ) K l e b s i e l l a K26 cross-reacts with anti-serum to K l e b s i e l l a K 11. 7 i n t e r e s t because i t includes pathogens f o r man and other a n i -mals, e.g. Salmonella sp. and S h i g e l l a sp. are common cause of food poisoning.Yersinia pestis i s the cause of bubonic plague. By combination of morphological and biochemical c h a r a c t e r i s t i c s t h i s family has been divided i n 5 tribes"""'': Escherichieae , Klebsielleae.Proteceae.Yersineae and Erwinieae which are then subdivided i n several genuses. The genus K l e b s i e l l a ( t r i b e Klebsielleae ) i s composed of three species,K.pneumoniae,K.ozaenae and K.rhinoscleromatis. These microorganisms are normally found i n healthy c a r r i e r s i n the upper r e s p i r a t o r y , i n t e s t i n a l and genito-urinary t r a c t s . They may become pathogenic and have been shown to be responsi-ble for some respiratory diseases (3$ of b a c t e r i a l pneumonias). They can be isola t e d from feces,pus,blood.abscesses.bones and joints"*"^.Klebsiella cultures were c l a s s i f i e d s e r o l o g i c a l l y on 17 18 the basis of the i r K (capsular) and 0 (somatic) antigens '* As the number of 0 types i s small (11) compared with the K types ( 80) and as the determination i s d i f f i c u l t because of the heat stable K antigens.determination of the s e r o l o g i c a l type i s primarily based on the K determination.The capsule (K 19 antigen) has been demonstrated to be carbohydrate i n nature 7. Many immunological r e l a t i o n s h i p s (cross-reactions) have been 12 demonstrated between the K antigens of K l e b s i e l l a themsel-ves and with some K antigens of other bacteria ( Pneumococci) 13 14 J* .As t h i s c r o s s - r e a c t i v i t y i s based on the recognition of p a r t i a l structures of the antigen by the antibody molecules or immune c e l l receptors.knowledge of the structure of the 8 carbohydrate antigen i s fundamental.For t h i s reason a struc-t u r a l i n v e s t i g a t i o n of a l l K l e b s i e l l a capsular polysacchari-des has been undertaken. K l e b s i e l l a polysaccharides K l e b s i e l l a polysaccharides,as most of the b a c t e r i a l ex-20 t r a c e l l u l a r heteropolysaccharides ,are formed of oligosaccha-ride units composed of 3 to 7 sugar residues.which repeat r e -gu l a r l y to b u i l d up the high molecular weight polysaccharide ( 2*10-' - 2*10? daltons) .Qualitative analysis of the polysac-charides of the approximately 80 d i f f e r e n t K l e b s i e l l a K sero-21 22 types was done by Nimmich ' .which led to the grouping of the capsular polysaccharides into chemotypes2-^(see Table 1.1). This f i r s t d i v i s i o n i s not enough to explain t h e i r d i f f e r e n t immunological behaviour. The polysaccharides are a l l a c i d i c and t h i s i s due to the presence of uronic acidsjD-glucuronic acid i s the most common and D-galacturonic acid i s found i n a number of stra i n s . The capsular polysaccharides of K22, K37 and K38 have been shown to contain rare uronic acids.In polysaccharides devoid of uronic acids ( K32, K56 and K72 ) the a c i d i t y i s due to pyruvic acid that i s linked as an acetal to a sugar residue. Pyruvic acid also supplements the acid content.in other uronic acid containing K l e b s i e l l a polysaccharides.The neutral sugars found are the common hexoses, D-glucose, D-galactose and D-mannose, and the 6-deoxysugars,L-rhamnose and L-fucose.Non-carbohydrate substituents as a c e t y l or formyl may also be present i n addition to the monosaccharides constituents. 9 GlcA Gal Glc GlcA Gal Man GlcA Gal Rha GlcA Glc Man GlcA Glc Rha GlcA Glc Fuc GlcA Gal Glc Man GlcA Gal Glc Fuc GlcA Gal Glc Rha GlcA Gal Man Rha GlcA Glc Man Fuc GlcA Glc Man Rha GlcA Gal Glc Man Fuc GlcA Gal Glc Man Rha GalA Gal Man GalA Glc Rha GalA Gal Fuc PyrA Glc Rha PyrA Gal Rha PyrA Gal Glc Rha KetoA Gal Glc 8 p , l l p , 1 5 , 2 5 , 2 ? p , 5 1 2 0 , 2 1 P , 2 9 P , 4 2 P , 4 3 , 6 6 , 7 4 P 9 , 9 * . 4 7 , 5 2 , 8 1 , 8 3 2 , 4 , 5 P , 2 4 1 7 , 2 3 , 4 4 , 4 5 ,71 1,5^ 7 P , 1 0 , 1 3 P , 2 6 P , 2 8 , 3 0 P , 3 1 P , 3 3 P , 3 5 P , 3 9 , ^ 6 P , 5 0 , 5 9 . 6 0 , 6 l , 6 2 , 6 9 P 16 , 5 8 P 1 2 P , 1 8 , 1 9 , 3 6 P . 4 1 , 5 5 P , 7 0 P , 79 4 0 , 5 3 , 8 0 p 6 P 6 4 P , 6 5 P 6 8 p 1 4 P , 6 7 3 P , 4 9 , 5 7 3 4 ,48 63 72 32 56 2 2 , 3 7 . 3 8 GlcA glucuronic acid GalA galacturonic acid PyrA pyruvic acid KetoA rare uronic acid Glc glucose Gal galactose Man mannose Rha rhamnose Fuc fucose P pyruvic acid pre-sent i n addition TABLE I.1 K l e b s i e l l a capsular polysaccharides (KI-K83) Qualitative analysis and chemotype grouping. 1 0 The structures of f i f t y - f i v e K l e b s i e l l a polysaccharides are known to date and they may be c l a s s i f i e d according to the st r u c t u r a l pattern,as shown i n Appendix I. In spite of the great d i v e r s i t y of s t r u c t u r a l patterns,added to the possible multiple permutations of the d i f f e r e n t sugar residues i n the repeating unit,there are c e r t a i n combinations of sugars (aldobiouronic acid,see Table 1.2 ).substitutions(see Table 1.3) and anomeric linkages (see Table 1.4) that are preferred: i ) Aldobiouronic acids;D-Glucuronic acid and D-glucose are present together i n 5 0 strains,but of the 3 5 structures known that contain them,only i n two are they combined to form the aldobiouronic acid.D-Glucuronic acid and D-galactose form the aldobiouronic acids of 12 st r a i n s of known structure, a l -1 4 1 6 though GlcA Gal and GlcA Gal are very common i n plant 24 a B gums ,they are not as important among the K l e b s i e l l a polysac-charides. D-Glucuronic acid and D-mannose compose the aldobio-uronic acids of 16 strains and only two combinations are ob-1 2 1 3 served, GlcA Man and GlcA J Man>the f i r s t one i s also B 24 common i n plant gums .D-Glucuronic acid and L-rhamnose are found as the aldobiouronic acid i n 9 s t r a i n s and although a l l possible linkages have been determined ( 2 , 3 and 4 rhamnose ) ,8 of the 9 occur as B -glycosides.The aldobiouronic acids contain-ing D-glucuronic acid and L-fucose as well as those contain-ing D-galacturonic acid are also l i s t e d i n Table 1.2. P r i o r to the systematic i d e n t i f i c a t i o n of b a c t e r i a l poly-saccharides only a few aldobiouronic acids had been is o l a t e d . As a source of aldobiouronic acids.capsular polysaccharides 1 1 TABLE 1.2 Aldobiouronic acids i n K l e b s i e l l a capsular  polysaccharides. Aldobiouronic acid Klebsiella(K) Glucuronic acid and Glucose 1 4 GlcA Glc GlcA -"—- Glc 5 2 3 , 5 1 Glucuronic acid and Galactose GlcA -—2. G a l  l A 3 GlcA GlcA l P 4 Gal Gal 1 a | i GlcA - " — G a l GlcA 1 B 6 Gal 3 1 , 5 5 , 8 3 1 1 , 1 2 , 2 0 , 4 1 , 6 0 8 2 5 , 5 9 2 7 Glucuronic acid and Mannose GlcA - - - r - 2 - Man GlcA 1 _ H Man 7 , 2 8 , 5 3 , 6 1 , 6 2 2,4 , 13,21,24, 2 6 , 3 0 , 3 3 , 4 6 , 6 4 , 74 Glucuronic acid and Fucose GlcA 1 _ 1 Fuc 1 A 4 GlcA i - — Fuc 6 , 5 ^ 1 , 1 6 , 5 8 Glucuronic acid and Rhamnose GlcA GlcA GlcA GlcA 1 2 l a 3 1 B 4 Rha 2 Rha Rha Rha 9 * , 1 8 , 3 6 , 4 4 , 8 1 1 7 4 7 9 , 7 0 Galacturonic acid Gal A 1 2 Man 1 a 3 GalA i r — - Man GalA 1 1 1 Fuc a GalA - — - Rha 5 7 4 9 63 34,48 12 are,thus,of great importance. i i ) Substitution pattern; The conclusions that can be drawn from the s u b s t i t u t i o n pattern of the monosaccharide residues i n the K l e b s i e l l a capsular polysaccharides of known structure are the following: D-glucose appears mainly as an in-chain residue,linked through p o s i t i o n 3,k and 6 or as a terminal residue; D-galactose as an in-chain residue i s almost exclusively linked at p o s i t i o n 3 ( 30 s t r a i n s out of 3 D ; D-mannose does not have any p a r t i c u l a r characteristic.except that p o s i t i o n 6 i s not commonly substituted; L-rhamnose, l i k e D-mannose fdoes not have any p a r t i c u l a r c h a r a c t e r i s t i c ; D-glucuronic acid appears mainly as an in-chain residue (subs-t i t u t e d at p o s i t i o n k ) or as a terminal residue.No general conclusions can be drawn f o r L-fucose and D-galacturonic acid as they are not so common i n the capsular polysaccharides. i i i ) The d i s t r i b u t i o n of a - and B - g l y c o s i d i c linkages found i n K l e b s i e l l a capsular polysaccharides corresponds with the percentage composition of equilibrated aqueous sugar so-l u t i o n s . One would expect thermodynamic s t a b i l i t y to predomi-nate i n natural occurring substances.The 6 anomers of D-glu-cose ,D-galactose and D-glucuronic acid are more common,as the b u l k i e r aglycon groups tend to dispose themselves i n an equa-t o r i a l position.In the case of D-mannose and L-rhamnose,the a x i a l hydroxyl group at C-2 makes the anomeric e f f e c t increa-26 se i n significance,thus the a anomers are more stable . iv) Table 1.5 shows the preference for c e r t a i n pyruvyla-ted monosaccharide residues i n the K l e b s i e l l a capsular poly-13 TABLE 1.3 Substitution pattern of the sugar residues i n the K l e b s i e l l a capsular polysaccharides Substitution pattern  of the sugar residue a) terminal Glucose Galactose Mannose Rhamnose Fucose Glucuronic acid Galacturonic acid b) monosubstituted Glucose 2 3 4 6 K l e b s i e l l a (K) Galactose 2 3 18,25,27,28,38,41,46,5-+. 60(3) 7.16,52,58,61 24,57,62 17,3^7,53.56.64 2,8,9,20,23,27,30,33.51, 55,59,83 48,49 70 1,2,4(2),6,7,11,13,23,24, 2 8 , 3 1 , 3 ^ . ^ . ^ 8 , 5 5 . 5 8 , 5 9 , 60,61,64,72 2,5,13,17,22,25,26,30,33, 37,44,48,62 12,22,23.26,27,37,38,41(2) 51.61 28 8,9.9*.11.12,18,20(2),21, 26,27,31,32,36,38,41,46(2) 47.49(2),51.52,53.55,56(2) 57,62,63,70,74,81,83(2) 14 Mannose 2 7,21,24,26,28,31,53.57, 3 4,21,24,26,46,59.64 4 30,33 6 Rhamnose 2 9,9*(3),18 ,34(2),36,44, 48,52,53.70,81(2) 3 9,12,17,18,32;34,36,41 44,72(2),81(2) 4 32,52.70 Fucose 2 3 6,54,63 4 1,16 Glucuronic acid 2 4,25,63 3 7,28,53 4 5,6,9*,12,16,17,18,36,41, 44,47,52,54.61,64,70,74 Galacturonic acid 2 3 63 4 p c) d i s u b s t i t u t i o n ( branching or pyruvate ) Glucose 2,6 16,18 4,6 31 P ,36 P ,5^,64 P Galactose 2,3 12,41,52,56,60 3 , ^ 8 , 1 3 P , 2 2 , 2 5 , 3 0 P , 3 3 P . 3 7 . 51,59 4,6 1 1 P , 1 2 P , 2 1 P , 2 6 P , 7 4 P Mannose 2,3 7,20,28,49,53,60,61,62, 74 2,6 2,13 Rhamnose 2,3 17,23,36,48 3,^ 9,^7.55.83 1 5 Fucose 3tk 58 Glucuronic acid 2,4 24,26 3,4 11,21,31,46,60 Galacturonic acid 3,4 3^,57 p d) t r i s u b s t i t u t i o n ( double branching or pyruvate ) Glucose 3,^,6 7 P ,27 P,56 P Galactose 2,3,4 3 8 3,^,6 2 7 Mannose 2,3,4 64 3,^,6 5 P,6 P , 3 0 , 3 3,^6 P Rhamnose 2,3,4 1P,58P 16 TABLE 1.4 Percentage composition of a - and g-glycosidic linkages i n K l e b s i e l l a capsular polysaccharides. Sugar residue a - l i n k e d ( % ) B-linked(%) Glucose (65) 25 75 Galactose ( 5 * 0 4 8 52 Mannose (39) 74 26 Rhamnose ( 4 6 ) 95 5 Fucose ( 6) 100 0 Glucuronic acid (*5) 42 58 Galacturonic acid ( 5) 100 0 ( ) number of sugar residues considered from a l l K l e b s i e l l a capsular polysaccharides of known structure. 17 TABLE 1.5 Location of pyruvate i n K l e b s i e l l a capsular polysaccharides. Location of pyruvate K l e b s i e l l a (K) Glucose ^ 4 6 Glc — 31.36,64 8 — 3 Glc - r - 7,27,56 Galactose 4 / x6 Gal 11,21 a / x 6 Gal — 12,26,74 p 3 k Gal 13,30,33 Mannose Jt 4 6 — 3 Man --— 5,6,46 Rhamnose A 2 Rha 32,70,72 Glucuronic 4 GlcA 1,58 acid 3^2 B 18 saccharides. As a means of disease prevention the phenomenon of immu-niz a t i o n has been used by man f o r a long time.Immunization has been obtained, i n b a c t e r i a l induced diseases by i n j e c t i o n of the bacteria ( b a c t e r i a l vaccines) or the toxins produced by them.Protective immunization was also obtained with the bacte-r i a l polysaccharides 2"^' 2^ and with a r t i f i c i a l antigens 2^,where the haptenic oligosaccharide was coupled to c a r r i e r proteins, avoiding i n t h i s way the possible toxic e f f e c t s of bacteria. Due to the high s p e c i f i c i t y of the antigen-antibody reaction, p u r i f i c a t i o n of antibodies can be achieved by a f f i n i t y chrom-30 31 atography-^ * J ,where the haptenic oligosaccharide i s attached to a matrix,retaining i t s s p e c i f i c binding a c t i v i t y to the antibody. In either case,these haptenic oligosaccharides must be obtained i n large quantities.Several procedures can be used for this purposes a) Synthesis-^ 2"-^. When one considers that two glucoses can combine to form ten d i f f e r e n t disaccharides (considering only the pyranose form),the task i n synthesizing larger oligo-saccharides i s enormous.Specific protections,deprotections and glycosylation techniques must be used and the y i e l d s are generally low. b) P a r t i a l hydrolysis of the polysaccharide-^'-^?.As acid hydrolysis i s non-specific,the number of oligomers obtain-ed are large which implies tedious p u r i f i c a t i o n s and the yields 19 are low,Many polysaccharides have a c i d - l a b i l e substituents which i n some cases are.extremely important f o r the immunolo g i c a l a c t i v i t y . T h i s procedure generally y i e l d s oligomers which lack these substituents. c) Bacteriophage degradation of the polysaccharides-^ ' ^ 9 . Bacteriophages possess enzymes that s e l e c t i v e l y hydrolyze poly-saccharides . The y i e l d of oligomeric products i s usually high and mainly one or two repeating units are obtained(see Section IV.3)! In the course of t h i s Thesis, the structures of two K l e b s i e l l a polysaccharides,serotype K 60 and serotype K 2 6 , were investigated* two K l e b s i e l l a capsular polysaccharides ( K 6 0 and K 46) were degraded by t h e i r respective K l e b s i e l l a bacteriophages ( # 6 0 and 0 46 ) and the degradation products were i s o l a t e d , p u r i f i e d and characterized. 20 METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES 21 I I . - METHODOLOGY OF STRUCTURAL STUDIES ON POLYSACCHARIDES. II.1 The structures of polysaccharides. Polysaccharides can be c l a s s i f i e d i n the following way: a) Homopolysaccharides l i n e a r (based on one type of sugar) branched b) Heteropolysaccharides l i n e a r (based on two or more sugars) branched This c l a s s i f i c a t i o n could be extended depending on whether the polysaccharide contains regular repeating units or not.A s t r u c t u r a l study must characterize: a) the nature of the sugar residues and t h e i r proportions i n the polysaccharide, b) the p o s i t i o n of linkage,as the g l y c o s i d i c linkage may i n -volve s u b s t i t u t i o n of one of the several hydroxyl groups i n the adjacent sugar residue, c) the linkage configuration.as each g l y c o s i d i c linkage may have the a - or 8-configuration, d) the sequence of the sugar residues,as the various s t r u c t u -r a l units may be assembled i n many alte r n a t i v e sequences. With polysaccharides consisting of repeating units,the s t r u c t u r a l study w i l l lead to a d e f i n i t i v e structure,while on the other hand,an o v e r a l l sequence cannot be determined i n poly-saccharides lacking repeating units.The l a t t e r i n v e s t i g a t i o n can only provide information on an "average structure".sugar residues present,type of end groups.which sugars are branch 22 points.nature of some linkages.etc. The following discussion w i l l t r y to cover several tech-niques of s t r u c t u r a l e l u c i d a t i o n that are applicable to both types of polysaccharides. LQ-LLQ II.2 I s o l a t i o n and p u r i f i c a t i o n 7. The f i r s t major task i n polysaccharide chemistry i s to obtain the material to be studied i n a pure form.This involves a ) i s o l a t i o n of the carbohydrate material free from other sub-stances, and b ) i s o l a t i o n of a single polysaccharide species. The techniques used should be as mild as possible i n or-der to preserve i n t a c t the structure of the polysaccharide as i t exists i n nature.The f i r s t step consists of separating the polysaccharide from low molecular weight material (inorganic salts,amino acids,peptides,oligosaccharides,etc.) and other high molecular weight material (proteins,nucleic a c i d . l i g n i n ) . The i s o l a t i o n of a single polysaccharide species presents greater d i f f i c u l t i e s . S e v e r a l procedures have been used,such as f r a c t i o n a l p r e c i p i t a t i o n ^ . s e l e c t i v e p r e c i p i t a t i o n by com-+2 42 4? plex formation with Cu or CETAVLON ^,ion exchange chro-44 4*1 .46 matography ,gel chromatography ,electrophoresis ,etc. Indication of the presence of a single component by these and other c r i t e r i a of homogeneity ( [ a ] D . s u g a r a n a l y s i s ) p r o v i -des only evidence against heterogeneity and thus the impor -tance of specifying the c r i t e r i a used when dealing with new polysaccharides. The following i s o l a t i o n and p u r i f i c a t i o n procedures were usedr 2 3 hn 1 1 . 2 . 1 K l e b s i e l l a polysaccharides '. Each s t r a i n of K l e b s i e l l a was inoculated i n beef-extract medium and incubated at 3 7 ° u n t i l a d e f i n i t i v e growth was ob-served; t h i s l i q u i d culture was incubated i n a tray of sucrose-yeast extract-agar f o r three days.The lawn of capsular bacteria produced was harvested,diluted with aqueous phenol to k i l l the bacteria and ultracentrifuged.The polysaccharide was p r e c i p i -tated from the supernatant with ethanol.The p r e c i p i t a t e was dissolved i n water and treated with CETAVLON (c e t y l t r i m e t h y l -ammonium bromide) solution,which pre c i p i t a t e d the a c i d i c poly-saccharide. Further p u r i f i c a t i o n of the a c i d i c polysaccharide involved d i s s o l u t i o n i n 4M NaCl.reprecipitation into ethanol, d i s s o l u t i o n i n water and dialysis.The dialyzed s o l u t i o n yielded a f t e r freeze-drying the p u r i f i e d capsular polysaccharide. A c i d i c polysaccharides are normally easy to p u r i f y as they can be " s e l e c t i v e l y precipitated with CETAVLON,any neutral polysaccharides remaining i n solution.In case of heavy contam-ination with neutral polysaccharides t h i s p r e c i p i t a t i o n can be repeated. Further p u r i f i c a t i o n can be achieved by anion-exchange chromatography(e.g. diethylaminoethyl Sephadex,DEAE) or by g e l -permeation chromatography. LQ h.Q 11.2.2 Gum exudate of Chorisia speciosa ' . The gum co l l e c t e d from the trees of Chorisia speciosa as hard nodules was allowed to swell i n water.In order to help d i s s o l u t i o n i t was heated on a steam-bath for several hours , 24 the pH was kept at 7 i n order to prevent hydrolysis.The viscous s o l u t i o n obtained was p r e c i p i t a t e d with a c i d i f i e d ethanol.The pr e c i p i t a t e was dissolved i n water and a second p r e c i p i t a t i o n with ethanol was done.This p r e c i p i t a t e yielded upon d i s s o l u -t i o n and freeze -drying the gum polysaccharide. I I . 3 Sugar a n a l y s i s ^ 0 " 6 7 . I I . 3 . 1 Total hydrolysis. The f i r s t step i n the i n v e s t i g a t i o n of a polysaccharide i s the determination of the nature of the sugars released on hydro-l y s i s . As acid causes degradation of sugars,the conditions used (type of acid,concentration of acid,temperature and time)must be c a r e f u l l y chosen i n order to obtain a quantitative release of the sugars with a minimum of degradation.Dutton-^ reviewed the advantages and disadvantages i n the use of d i f f e r e n t acids. T r i f l u o r o a c e t i c acid (2M)-*1 has been found to be the best choice,, as i t has approximately the same hydrolytic strength as HCl(lM) and HgSO/j, (P.5M) ,does not s i g n i f i c a n t l y degrade sugars under the conditions normally used ( 6 - 8 h , 1 0 0 ° ) and because of i t s v o l a t i l i t y . i t i s e a s i l y removed. Although t r i f l u o r o a c e t i c acid (2M) w i l l break down comple-t e l y a neutral polysaccharide into i t s sugar constituents i n 6 to 8 hours at 1 0 0 ° , t h e s e conditions are not strong enough to ensure complete hydrolysis of glycuronosyl linkages.A consider-able amount of aldobiouronic acid thus survives the treatment. This w i l l cause discrepancies i n the sugar r a t i o of the hydro-lyzate and i n the polymer.However,incomplete hydrolysis provid-2 5 es a very useful i n d i c a t i o n as to the possible composition of the aldobiouronic acid. Several procedures have been devised to reduce the uronic acid residues to neutral sugars(see Section II.7.1).A technique t o has been developed i n t h i s laboratory^ which overcomes these d i f f i c u l t i e s . T h e polysaccharide i s treated with methanolic hy-drochloric acid.which cleaves most of the g l y c o s i d i c linkages forming the methyl glycosides and at the same time,the methyl esters of the uronic acids.Treatment with NaBH^ i n anhydrous methanol reduces the uronic esters to the corresponding a l c o -hols. The mixture of methyl glycosides i s then hydrolyzed with 2M t r i f l u o r o a c e t i c acid ( TFA ) to give the neutral sugars. By comparison of the r a t i o of neutral sugars only released upon hydrolysis of the a c i d i c polysaccharide and the r a t i o of neutral sugars and transformed uronic a c i d s , i t i s possible to i d e n t i f y the uronic acid as well as the molar proportions of the sugars present. II.3.2 Characterization and quantitation of the sugars. Through the years,the characterization and quantitation of the sugars released upon hydrolysis of polysaccharides has de-veloped from s p e c i f i c reactions and c r y s t a l l i z a t i o n of the de-r i v a t i v e s , through paper chromatography-^ -^ and thin-layer chro-matography-^, to more recent techniques of g a s - l i q u i d chromato-graphy and high pressure l i q u i d chromatography. Although the use of paper chromatography fo r quantitative purposes involves e l u t i o n of the d i f f e r e n t f r a c t i o n s and fur *» r ther quantitation (e.g.colorimetry) i t i s s t i l l a very useful 2 6 t o o l for q u a l i t a t i v e purposes. The development of gas-liquid chromatography (g.l.c.) i n -volved the conversion of the sugars to suitable v o l a t i l e deriva-t i v e s . An extensive review of t h i s technique and i t s applications has been done by Dutton^ 7'^^.The f i r s t v o l a t i l e derivatives used were the t r i m e t h y l s i l y l ethers-^,with the disadvantage that each sugar may give r i s e to four peaks ( a - and B-pyranosides and a -and 3-furanosides).The chromatograms were s i m p l i f i e d greatly upon reduction to the respective a l d i t o l s ^ 0 or conversion to the aldononitriles^""". I t must be kept i n mind that ketoses.such as fructose,give upon reduction two a l d i t o l s ( g l u c i t o l and mannitol) and i n t h i s case a more convenient derivative would be the aldo-nonitrile^ 2(oximes for ketoses). A wide s e l e c t i o n of stationary phases i s now available which give a good separation of the d i f f e r e n t v o l a t i l e d e r i v a t i -ves. During t h i s i n v e s t i g a t i o n the a l d i t o l acetates were used and good resolution,was obtained with the following stationary phases; SP - 2 3 4 0,a highly polar s i l i c o n e containing 7 5 $ of cyano-propyl groups,and ECNSS-M,a copolymer of ethylene g l y c o l s u c c i -nate polyester and a cyanoethyl s i l i c o n e . The l a t e s t advance i s the use of high pressure l i q u i d chro-matography (HPLC)^^'^ fo r quantitative analysis of sugars ,a technique which does not require d e r i v a t i z a t i o n . II.3-3 Determination of the configuration (D or L) of the sugars. The D or L configuration of the sugars was u n t i l recently determined by 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 ( l a ] D ) , or using s p e c i f i c 2 7 oxidases (e.g. D-glucose oxidase,D-galactose oxidase).The l a t t e r method i s not applicable to other commonly occuring sugars due to the absence of the corresponding oxidases. A more recent procedure i s the determination of the sign of t h e i r c i r c u l a r dichroism curves^measured on suitable d e r i -vatives. These derivatives.which can be the acetylated a l d i t o l s or the p a r t i a l l y methylated a l d i t o l acetates,can be e a s i l y i s o -lated by preparative gas - l i q u i d chromatography.A disadvantage of these derivatives i s that meso a l d i t o l s , s u c h as g a l a c t i t o l , are not o p t i c a l l y active.However,this can be overcome by using c h i r a l p a r t i a l l y methylated a l d i t o l acetates obtained i n , the methylation studies.Another p o s s i b i l i t y , w h i c h was developed i n the course of t h i s i n v e s t i g a t i o n ' s the use of the peracetyla-ted aldononitriles,which were synthesized and then iso l a t e d i n a pure form by preparative gas - l i q u i d chromatography (see Ap-pendix IV ). Recently,g;1 .c. has been used for the separation of enan-tiomers,using a c h i r a l stationary phase or converting the enan-tiomersinto diastereomers by c h i r a l reagents and separation on a non-chiral phase.Vliegenthart et a l . and Lindberg et a l . ' have used the second p r i n c i p l e to determine the absolute c o n f i -guration of the sugars»the f i r s t author used the trimethyl -s i l y l a t e d ( - ) - 2 - b u t y l glycosides and the l a t t e r the acetylated ( + ) - 2 - o c t y l glycosides,on c a p i l l a r y columns wall-coated with SE - 3 0 and S P - 1 0 0 0 , r e s p e c t i v e l y . 28 II.4 P o s i t i o n of linkage. After determining the number and type of sugar residues i n the polysaccharide,the next step involves methylation anal-y s i s to determine through which position(s) these components are linked to form the polymeric chain. II.4.1 Methylation analysis. This technique r e l i e s on the complete e t h e r i f i c a t i o n (methylation) of the free hydroxyl groups of the sugar r e s i -dues i n the polymer and the i d e n t i f i c a t i o n of the p a r t i a l l y methylated monosaccharides released a f t e r cleavage of a l l the gly c o s i d i c bonds i n the polysaccharide. II.4.1.1 Methylation procedures ~'->. Several procedures have been developed through the years each of which involves the treatment of the polysaccharide i n solutio n with a base and an alkylating(methylating) agent. Di f f e r e n t solvents ( HgO,dimethylformamide,dimethylsulfoxide), bases ( NaOH,Ag20,CH^S0CH2"Na+) and methylating agents (CH^I, CH2N2,(CH^JgSO^ ) have been used. The methods of methylation generally used are the Hakomori m e t h y l a t i o n ^ ' 7 0 and the Purdie-Irvine methylation 7 1.The f i r s t i s the more v e r s a t i l e and usually complete methylation i s a-chieved with one treatment.The polysaccharide i s dissolved i n dimethylsulfoxide (DMSO).the base i s sodium methyl s u l f i n y l -methanide (DMSO'Na*) and the a l k y l a t i n g agent i s methyl iodide (CH^I ).The main disadvantage of thi s procedure i s that i t i s not suitable for more than one treatment on a c i d i c polysaccha-ri d e s .because the methyl ester of the uronic acid can undergo 29 72 B -elimination' during the base treatment leading to degrada-t i o n of the polysaccharide.Purdie methylation i s used i n these cases to ensure complete methylation,a procedure which involves treatment of the polysaccharide with s i l v e r oxide i n r e f l u x i n g methyl iodide. Following the complete methylation of the polysaccharide , the product i s f u l l y hydrolyzed.Several procedures are used,but 2M TFA on a steam-bath f o r 16 hours ensures complete hydrolysis of a neutral methylated polysaccharide.The presence of uronic acids s t a b i l i z e s the uronosyl bonds which are not f u l l y hydro-lyzed under these conditions.Reduction of the methyl ester with LiAlH^ (see Section II.7.1) overcomes these d i f f i c u l t i e s . The reduced methylated polysaccharide can now be hydrolyzed com-p l e t e l y or remethylated p r i o r to the hydrolysis .Scheme II.1 i l -lustrates a t y p i c a l reaction sequence. The unmethylated positions of the sugars represent s i t e s of linkage,except i n the cases of uronic acid residues or r e s -idues with pyruvate attached.Uronic acids can be i d e n t i f i e d by comparison of the methylation r e s u l t s of;the a c i d i c polysaccha-r i d e with those of the reduced polysaccharide, or the reduced polysaccharide and the corresponding reduced remethylated pro-duct as shown i n the Scheme II.1.The p o s i t i o n of linkage of the pyruvate can be determined by comparison of the methyla-t i o n products of the o r i g i n a l and s e l e c t i v e l y depyruvylated polysaccharides as used for the K l e b s i e l l a K 26 polysaccharide. II.4". 1.2 Characterization of the methylated sugars. The techniques f o r separating and i d e n t i f y i n g the methyl-3 0 K26 POLYS ACCHARI DE Methylation l LiAlty / THF 2/»6-0Me3- Mannose 3,4,6-OMe3- Mannose 2,4.6-0Me3- Galactose Z3,4-OMe3- Glucose Z3.6-0Me3- Glucose 22-OMe2_ Galactose 3-0Me - Glucose 1. Remethylation 2. H + 2,A6-0Me3~ Mannose 3A,6-0Me3- Mannose 2A6-0Me3- Galactose 2.3,A-0Me3- Glucose 2,3.6-0Me3- Glucose 23-0Me2- Galactose 3,6-OMe2- Glucose Scheme I I . 1 M e t h y l a t i o n a n a l y s i s of K l e b s i e l l a K 26 3 1 ated sugars have improved considerably since the time they were separated by f r a c t i o n a l d i s t i l l a t i o n of t h e i r methyl glycosides. Adsorption chromatography,paper chromatography .paper e l e c t r o -phoresis ,etc. have been used extensively.Although many of these methods are seldom used at present ,paper chromato-graphy i s s t i l l a very useful t o o l i n the i d e n t i f i c a t i o n of methylated sugars.By developing the papers with _p_-anisidine 76 hydrochloride or other aromatic amines' and h e a t . i t i s po s s i -ble to do a primary i d e n t i f i c a t i o n of the methylated sugars ac-cording to the d i f f e r e n t colors formed as well as from t h e i r m o b i l i t i e s (shown by t h e i r R^ values). With the advent of g.I.e.,which provides a rapid and ac-curate q u a l i t a t i v e and quantitative a n a l y s i s . e f f o r t s have been made to render s u i t a b l e . v o l a t i l e derivatives of the methylated sugars.An extensive review of the f i e l d has been done by Dutton 5 ? . 5 8 < T n e derivatives of choice during the course of thi s i n -vesti g a t i o n were the p a r t i a l l y methylated a l d i t o l acetates, which can be e a s i l y obtained by reduction of the methylated sug-ars with NaBH^ followed by acet y l a t i o n with acetic anhydride-pyridine.Various l i q u i d phases (especially,medium polar ones) are available for the separation of mixtures of these deriv-atives and depending on the d i f f i c u l t i e s encountered on sepa-r a t i o n , several columns may need to be tried.Publications by Lindbergh and Albershein-7® provide r e l a t i v e retention times for numerous p a r t i a l l y methylated a l d i t o l acetates as well as molar response factors which enable correct quantitation of the components. 3 2 In the i n v e s t i g a t i o n of the K l e b s i e l l a K 2 6 capsular poly-saccharide, the peracetylated p a r t i a l l y methylated a l d o n o n i t r i l * . es were used to separate 2 , 3 , 4 - a n d 2 , 3 , 6 - t r i - 0 - m e t h y l g l u c o s e (although separation of the a l d i t o l acetates was achieved with a column of SP - 2 3 4 0 ).These derivatives were e a s i l y prepared by reaction of the methylated sugars with hydroxylamine hydrochlo-ride i n pyridine.followed by treatment with a c e t i c anhydride which dehydrates the oxime to the n i t r i l e and at the same time acetylates the free hydroxyl groups. The i d e n t i f i c a t i o n of an i n d i v i d u a l component can be a-chieved i n almost a l l cases by co-chromatography with authen-t i c standards,but by coupling the g . l . c . to a mass spectrome-ter (g. I.e.-m.s. ) .assignments can be made unambiguously. The fragmentation of the p a r t i a l l y methylated a l d i t o l acetates by 79 81 electron impact has been extensively studied y ~ .This tech -nique allows us to i d e n t i f y the s u b s t i t u t i o n pattern of the p a r t i a l l y methylated a l d i t o l acetates,but i t does not give the parent ion mass and cannot d i s t i n g u i s h between diastereomers, as mannose.glucose and galactose derivatives.The primary f r a g -ments are formed by the f i s s i o n of intercarbon bonds i n the a l -d i t o l chain.This cleavage follows a c e r t a i n preference : a bond between carbons that are methoxylated i s prefered over one methoxylated and the other acetoxylated,which i n turn i s prefered over two acetoxylated carbons: > > F i g u r e I I . 1 M.s. o f (a) 1 , 2 , 5 - t r i - 0 - a c e t y l - 3 » k , 6 - t r i - 0 -m e t h y l g a l a c t i t o l and (b) 1 , 2 , 5 - t r i - O - a c e t y l - 3 - 0 - e t h y l - 4 , 6 - d i - 0 - m e t h y l g a l a c t i t o l . 3k The primary fragments formed give r i s e to secondary f r a g -ments by loss of acetic acid (M.W. 60),ketene (M.W. 42),methan-o l (M.W. 3 2 ) or formaldehyde (M.W. 3 0 ) . This information was used to i d e n t i f y s p e c i f i c compounds for which standard spectra were not available.For example,when uronic acid degradation was performed on K l e b s i e l l a K 6 0 poly-saccharide, the p o s i t i o n where the uronic acid was attached was la b e l l e d with ethyl iodide.The e t h y l a t e d , p a r t i a l l y methylated a l d i t o l acetate thus obtained ( l , 2 , 5 - t r i - 0 - a c e t y l - 3 - 0 - e t h y l -4 , 6-di - 0-methyl g a l a c t i t o l ) was i d e n t i f i e d by means of i t s m.s. and comparison with the corresponding methylated deriva-t i v e ( l , 2 , 5 - t r i - 0 - a c e t y l - 3 t 4 , 6 - t r i - 0 - m e t h y l g a l a c t i t o l ) . S e v e r a l masses are s h i f t e d by 14 units as i l l u s t r a t e d i n Figure II.1. The fragmentation expected i s shown below: 35 Primary fragmentation CHgOCOCH^ CHOCH, CHOCOCH-I 3 CH £ 0 C H 3 CH ,0C0CHo I 2 3 CHOCOCH^ CH - 0 C H OCH~ + 2 3 (m/e 203) :H=OCH 2CH 3 ;HOCOCH 3 I H 2 0 C H 3 (m/e 219) CH-OCH, I 3 CHOCOCH^ CHgOCH^ (m/e 161) CHgOCOCH^ HOCOCH^ ; H O C H 2 C H 3 CH-OCH-+ 3 (m/e 2 4 7 ) Secondary fragmentation CH 2 0 C H 3 CH0C0CH"3 CH=0CH3 (m/e 161) -Ac OH CHOCH-II 3 H H=0CH3 (m/e 101) -Me OH CH, II z HOCOCH, H-OCH^ (m/e 129) -CH 2 C 0 CH 4 3 = 0 CH=0CH3 (m/e 8 7 ) H o 0C0CH^ CHOCOCH-2 3 -ArOH " H0C0CH 3 Z^im CH CH=0CH2CH3 CH=0CH2CH3 (m/e 203) (m/e 1 4 3 ) 3 6 II.5 Sequencing of sugars. The i s o l a t i o n and characterization of fragments of a poly-saccharide are the keys to the determination of the sequential arrangement of the constituent monosaccharides i n the polymer. By u t i l i z i n g d i f f e r e n t techniques i t i s possible to obtain these fragments and by determining the r e l a t i v e positions of one to the other,in an additive manner,is possible to bu i l d up the sequence i n the polysaccharide or i n the repeating unit. II.5.1 P a r t i a l h y d r o l y s i s 8 2 " 8 ^ . A considerable amount of data on rate constants and k i n e t i c parameters f o r the acid catalyzed 1 hydrolysis of glycosides of 82 83 monosaccharides has been presented i n several reviews ' J . Many factors seem to influence the rate of hydrolysis, such as r i n g size,configuration,conformation,polarity of the sugar , size and p o l a r i t y of the aglycon.Although data on polysaccha-rides are not so e a s i l y available.inferences can be made from the r e s u l t s on the monosaccharide glycosides,and may be sum-marized as follows: i ) furanosides are more l a b i l e than pyranosides, i i ) deoxysugars are more e a s i l y hydrolyzed than hexoses, i i i ) pentopyranosides are more acid l a b i l e than hexopyranosi -des, iv) a -glycosides are generally more l a b i l e than 3-glycosides, v) (1-6) linkages are more r e s i s t a n t to acid hydrolysis than (1-2),(1-3) and (1-4) linkages, v i ) residues present on side chains are often more e a s i l y hydrolyzed than when present i n the main chain, 37 v i i ) uronic acids are more r e s i s t a n t to hydrolysis, v i i i ) aminosugars are more acid r e s i s t a n t than common hexoses. In heteropolysaccharides we f i n d that some g l y c o s i d i c linkages are more r e s i s t a n t than others,so that, under c e r t a i n conditions of hydrolysis (acid concentration,temperature and time of hydrolysis) s e l e c t i v e cleavages w i l l occur i n the polysaccharide y i e l d i n g defined oligomeric units.For a c i d i c polysaccharides containing uronic acids .there s i s tance of the uronosyl bond leads to the accummulation of aldobiouronic acid and to a lesser extent the aldotriouronic and/or aldotetrao-uronic acid fragments.In polysaccharides with deoxysugars i t i s d i f f i c u l t to isolate,by p a r t i a l hydrolysis oligomers with deoxyhexosyl bonds.Usually a f t e r a p a r t i a l hydrolysis,the amount of monosaccharides i s high.By coupling'several separa-t i o n techniques i t i s possible to separate the oligomers from the monosaccharides as well as to i s o l a t e pure oligosacchari-des. The techniques most commonly used are paper chromatogra-phy ,gel-permeation chromatography,paper electrophoresis and gas - l i q u i d chromatography (see Section II .5.*0. Because acetals.acetates and formates that may be present i n the polysaccharides are more acid l a b i l e than g l y c o s i d i c bonds.it i s often possible to s e l e c t i v e l y remove these sub-sti t u e n t s without a f f e c t i n g the r e s t of the polysaccharide. P a r t i a l hydrolysis can also be c a r r i e d out on f u l l y methyl-ated polysaccharides. A complementary technique i s a c e t o l y s i s 8 - \ a s the r e l a t i v e rate of cleavage of the glycosides i n the two mechanisms i s 38 r e v e r s e d . D u r i n g a c e t o l y s i s , ( 1 - 6 ) l i n k a g e s a r e p r e f e r e n t i a l l y c l e a v e d , w h i l e t h e y a r e t h e m o s t s t a b l e d u r i n g a c i d h y d r o l y s i s . I I .5.2 P e r i o d a t e o x i d a t i o n a n d S m i t h h y d r o l y s i s ^ " ^ . 86 I t h a s b e e n k n o w n s i n c e 1928 t h a t p e r i o d i c a c i d a n d i t s s a l t s a r e c a p a b l e o f c l e a v i n g a , B - g l y c o l s q u a n t i t a t i v e l y t o g i v e t w o a l d e h y d e s : ?1 J l C H 0 H + I C v " C H 0 + H„0 + 10 ~ CHOH ^ CHO ^ J ^2 ^2 W h e n m o r e t h a n t w o h y d r o x y l g r o u p s a r e i n c o n t i g u o u s p o s i -t i o n s , f o r m i c a c i d i s r e l e a s e d , R , R , I 1 I 1 CHOH CHO I (j)H0H + 210^ +HC02H + H 20+ 2I0 3 CHOH CHO *2 R2 W h e n o n e o f t h e v i c i n a l h y d r o x y l g r o u p s i s a p r i m a r y a l c o -h o l , f o r m a l d e h y d e i s p r o d u c e d , CH 2 OH HCHQ CHOH + I OK CHO + H ?0 + 10 A R T h i s r e a c t i o n h a s f o u n d a w i d e r a n g e o f a p p l i c a t i o n s i n c a r b o h y d r a t e c h e m i s t r y . S i n c e t h e r e d u c t i o n o f p e r i o d a t e a n d t h e f o r m a t i o n o f f o r m i c a c i d a n d f o r m a l d e h y d e c a n a l l b e d e -t e r m i n e d a c c u r a t e l y o n t h e m i c r o s c a l e , p e r i o d a t e i s i n v a l u a b l e a s a n a n a l y t i c a l t e c h n i q u e , b u t i n a d d i t i o n . i t c a n b e u s e d a s a p r e p a r a t i v e p r o c e d u r e . 3 9 87 88 W h e n u s e d a s a n a n a l y t i c a l t e c h n i q u e , a k n o w n a m o u n t o f p o l y s a c c h a r i d e i s o x i d i z e d i n a s o l u t i o n o f p e r i o d a t e , t h e o x i d a t i o n b e i n g m o n i t o r e d b y m e a s u r i n g t h e c o n s u m p t i o n o f p e r i o d a t e a n d t h e r e l e a s e o f f o r m i c a c i d a n d / o r f o r m a l d e h y d e i f a n y i s f o r m e d . O v e r - , a n d u n d e r - o x i d a t i o n m a y h o w e v e r c o m -p l i c a t e r e s u l t s . O v e r o x i d a t i o n * ^ i s m i n i m i z e d b y w o r k i n g i n t h e d a r k a n d a t l o w t e m p e r a t u r e s w i t h b u f f e r e d s o l u t i o n s o f p e r i o d a t e . I n c o m p l e t e o x i d a t i o n 7 m a y o c c u r d u e t o e l e c t r o -s t a t i c r e p u l s i o n s b e t w e e n t h e p e r i o d a t e i o n s a n d t h e p o l y s a c -c h a r i d e i f a c i d i c ; f u r t h e r m o r e , s t e r i c h i n d r a n c e a f f e c t s b o t h t h e a c c e s i b i l i t y o f t h e v i c i n a l d i o l s t o t h e p e r i o d a t e i o n s i n c e r t a i n s u g a r r e s i d u e s , a n d t h e f o r m a t i o n o f i n t r a m o l e c u l a r 9 1 92 h e m i a c e t a l s 7 ' 7 t h r o u g h t h e d i a l d e h y d e s g e n e r a t e d b y o x i d a -t i o n a n d f r e e h y d r o x y l g r o u p s i n t h e p o l y s a c c h a r i d e . T h e f i r s t p r o b l e m i s o v e r c o m e b y a d d i t i o n o f s a l t s ( N a C l O ^ ) w h i c h i n h i b -i t t h e e l e c t r o s t a t i c r e p u l s i o n o f t h e s u b s t r a t e . T h e p r o t e c t e d r e s i d u e s ( w i t h t h e h e m i a c e t a l s ) c a n b e e x p o s e d t o o x i d a t i o n b y f i r s t s u b j e c t i n g t h e m t o b o r o h y d r i d e r e d u c t i o n . W h e n t h e o x i d a t i o n i s c o m p l e t e . a n a l y s i s o f t h e p r o d u c t s r e l e a s e d u p o n h y d r o l y s i s g i v e s v e r y u s e f u l i n f o r m a t i o n . T h e n u m b e r o f p e r i o -d a t e r e s i s t a n t s u g a r s i n t h e p o l y s a c c h a r i d e s h o u l d b e c o n s i s -t e n t w i t h t h e m e t h y l a t i o n d a t a . A s a n e x a m p l e . F i g u r e I I . 2 s h o w s t h e p o s s i b l e p r o d u c t s f o r m e d a n d m o l e s o f p e r i o d a t e c o n s u m e d u p o n o x i d a t i o n o f t e r m i n a l a n d m o n o s u b s t i t u t e d h e x o s e s . W h e n p e r i o d a t e o x i d a t i o n i s u s e d a s a f r a g m e n t a t i o n t e c h -n i q u e , t h e r e q u i s i t e i s t h a t s o m e o f t h e s u g a r 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 a r e n o t o x i d i z e d b y p e r i o d a t e a n d c a n b e s e -4 0 T e r m i n a l and mono-s u b s t i t u t e d hexoses P r o d u c t s formed a f t e r ox. and h y d r o l y s i s HO OH CHjOH HO—/ V-C— CH^H H O — / \ — 0 -CHnOH ' *- C H O h-OH + I C^OH CHUOH 1 CHO hOH • j CH^ DH CHJOH C H ^ D H —OH - O H - O H H O - * C H - O H CHJDH cr HjOH C H O IHJOH CHJOH UoH + CHO CHO h-OH HOH tHJDH CH-DH CH^H QL F i g u r e II.2 . Common p r o d u c t s formed on p e r i o d a t e o x i d a -t i o n o f t e r m i n a l and m o n o s u b s t i t u t e d h e x o s e s . 41 parated from the oxidized residues as mono-,oligo- or poly-saccharide derivatives a f t e r some chemical modifications.If, a f t e r oxidation with periodate,the aldehydes generated are reduced to alcohols with sodium borohydride,the polyalcohol (polyol) thus obtained i s constituted by g l y c o s i d i c linkages 0 3 olf as well as by a c y c l i c a c e t a l groupings.Smith degradation 7^ i s based on the d i f f e r e n t s t a b i l i t y towards acid of the gly-co s i d i c bond and an a c y c l i c acetal.The l a t t e r hydrolyzes much faster.The p o l y o l i s treated with mild acid and the products obtained,comprising; a) small fragments derived from oxidized, residues,or b) glycosides of mono- or oligo-saccharides (the aglycons being the small fragments mentioned before) and poly-saccharides derived from non-oxidized fragments are i s o l a t e d and p u r i f i e d by d i f f e r e n t separation techniques (see Section II.5.4) and characterized by conventional procedures.Based on the differences i n the rates of oxidation of v i c i n a l d i o l s ( c i s being oxidized f a s t e r than trans),the f a c t that termi-n a l residues are more accessible to periodate ions and that uronate anions tend to r e p e l l the periodate i o n s . i t i s pos-s i b l e to s e l e c t i v e l y oxidize c e r t a i n residues i n the polysac-c h a r i d e ^ , doing what may be c a l l e d a " s e l e c t i v e Smith degra-d a t i o n " 9 6 . Scheme II. 2 shows the Smith degradation of K l e b s i e l l a K 26 polysaccharide. II.5.3 Degradations based on g- e l i m i n a t i o n 7 ' . Several groups (alkoxyl,hydroxyl,etc.) i n the B- position to an electron withdrawing group,such as carbonyl,carboxylic kz 1. NalO^ 2. (CH2OH)2 3. NaBH4 Z>. 0.5MTFA r.t. 24h 5. NaBty Scheme 11,2 Smit h d e g r a d a t i o n of K l e b s i e l l a K 26 p o l y s a c c h a r i d e . *3 ester,amide or sulfone are eliminated on treatment with "base. The presence of a hydrogen atom i n the <*-position to these groups i s essential.These electron withdrawing groups can be introduced into the polysaccharides by e s t e r i f i c a t i o n of carboxylic acids,or by oxidation of alcohols to carbonyl com-pounds .Treatment with base w i l l y i e l d an unsaturated sugar residue which i s very acid l a b i l e and thus can be s e l e c t i v e -l y cleaved under mild acid conditions. Q O "i np I I . 5 - 3 * 1 Base catalyzed uronic acid degradation On methylation of an a c i d i c polysaccharide,the carboxylic acid (uronic acid) i s esterified,and on subsequent treatment with base,a 3 - e l i m i n a t i o n w i l l occur.eliminating the methoxyl or sugar residue attached to C-4 of the uronic acid residue and forming an hex-4-enopyranosyl uronate.lt has been observed that the base treatment w i l l cleave the g l y c o s y l uronic l i n k -a g e 1 0 0 , exposing the hydroxyl group to which the uronic acid residue was attached.This free hydroxyl group can be l a b e l l e d by a l k y l a t i o n with methyl iodide,ethyl iodide or t r i d e u t e r i o -methyl iodide. When the residue attached to C-4 of the uronic acid i s a sugar, a second B -elimination can take place because of the exposure of the reducing end to the base;if the aldose i s substituted at p o s i t i o n 3»the degradation can continue further. In order to avoid t h i s problem,the exposed reducing ends can be protected by using bases of low nucleophilicity,and by ac e t y l a t i o n with acetic a n h y d r i d e 1 0 1 . Comparison of the methylated sugars released upon hydro-l y s i s of the 3 -eliminated polysaccharide with the methylation analysis of the o r i g i n a l polysaccharide.reveals the environ-ment surrounding the uronic acid residues,i.e. the sugar r e -sidue to which the acid i s linked and the sugar residue at-tached to po s i t i o n C-4 of the acid ( i f any).An example of thi s procedure using K l e b s i e l l a K 26 i s shown i n Scheme II.3 • II.5 . 3 . 2 Degradation preceded by o x i d a t i o n 1 0 - ^ " 1 0 ^ . By using s e l e c t i v e procedures i t i s possible to obtain methylated polysaccharides with a li m i t e d number of free hydroxyl groups: s e l e c t i v e removal of acetal groups,from e l i -mination of uronic acids,by removal of acid l a b i l e sugar r e -sidues, etc. The free hydroxyl groups of the modified polysac-charides can then be s e l e c t i v e l y oxidized to carbonyl groups (see Section II.7.2) which can be the substrate for an alka-l i n e degradation. This procedure was used on the K 26 polysaccharide.The a c i d i c polysaccharide was reduced to the neutral polysaccha-ride by the carbodiimide procedure (see Section II.7.1) After methylation,the methylated reduced,acetal-linked pyru-v i c acid was s e l e c t i v e l y removed by hydrolysis.exposing free hydroxyl groups at C-4 and C-6 of the terminal galactose.The galactose was then oxidized (see Section II.7-2) to the d i -carbonyl derivative.degraded with base and removed by mild hydrolysis . The product was rernethylated,showing that the terminal galactose was linked to the C-4 of glucose (see Scheme II.4 ) . Scheme II.3 Uronic acid degradation of K l e b s i e l l a K 26 ^ polysaccharide. 46 Reduced K26 Fblysaccharide Methylation 1. Base 2. 50% Ch^COOH 3. Methylation 2NaBfy lAc^O/Py 1. 50V. CKjCOOH 2.0x.(DMSO/TFAA) 1.5 -OAc 2 - 2,3.446-OMe4-Glucitol 1.3,5-OAc3-2A5-OMe3- Mannitol 12,5-0^- 3.4.6 - 0Me3- Mannitol U5-OAc3-2A.6-OMe3-Galactitol t56-OAc3"-2,3.4-OMe3Glucitol 1.^5-OAcA-3.6-OMe2-Glucitol Scheme II.4 Degradation preceded by oxidation On the K l e b s i e l l a K 26 polysaccharide. 47 II.5.4 Separation of oligomers obtained from d e g r a d a t i o n s 1 0 6 " 1 0 9 . Normally,the degradation techniques described give mix-tures of oligomers.together with some unreacted polymeric ma-t e r i a l and contaminants from the side reactions.The desired oligomers must be separated and purified,the separation pro-cedures depending of the products.methylated or non-methyl-ated,basic or acidic,etc.The techniques normally used are pa-per chromatography,paper electrophoresis,gel-chromatography, gas- l i q u i d chromatography and more recently high pressure l i q u i d chromatography. Paper chromatography 1 0 6 , 1 0 7.Paper chromatography has been used extensively i n the f i e l d of carbohydrate chemistry.It has the a b i l i t y to separate components of complex mixtures accu-r a t e l y and simply.The p o s s i b i l i t y of using d i f f e r e n t solvent systems gives useful information on the nature of the carbo-hydrate material,e.g. a c i d i c oligomers do not move i n basic solvents.This procedure can be used on a preparative scale. Charged oligomers (acidic or basic) of high molecular weights move very slowly on paper,in which case paper e l e c t r o -phoresis can provide an alternative convenient method of sep-arating them.It i s faster and can also be carried out on a preparative scale. Gel chromatography 1 0^" 1 1 0.Gel chromatography i s a l i q u i d chromatographic method which separates molecules primarily according to differences i n molecular dimensions.Solutes are eluted i n the order of decreasing molecular sizes.This f i e l d 48 has been reviewed by Churms .Different types of gels have been produced,hydrophobic and hydrophilic gels which are used with organic and aqueous solvents respectively,with d i f f e r e n t exclusion l i m i t s , e t c . Gel chromatography has been used i n carbohydrate chemistry 109 for molecular-weight determination ,and for separation of products of p a r t i a l hydrolysis,periodate oxidation,etc.A-sep-aration based upon molecular size differences alone i s often not enough to obtain a completely pure product.This means i t must be used sequentially with other techniques such as ion-exchange chromatography,paper chromatography,For example, by p a r t i a l hydrolysis of the polysaccharide of K 6 0 a f t e r Smith degradation,mixtures of a c i d i c trisaccharides and tetrasac-charides were obtained by gel chromatography which had to be p u r i f i e d by paper chromatography. Gas-liquid chromatography 1 1 1" 1 1 6. The decrease i n vola-t i l i t y on going from mono- to d i - and higher oligo-sacchari-des i s the main disadvantage with using t h i s technique for such separations.Although suitable derivatives can be pro-duced , t r i m e t h y l s i l y l and permethyl e t h e r s , t r i f l u o r o a c e t y l , i t has not reached the point of excellence.Liquid phases for g . l . c . are available that are stable to high temperatures ( 2 5 0 - 3 0 0 ° ) . This allows separation of oligomers up to t e t r a -saccharides i n reasonable periods (30-40 minutes at 2 5 0 ° ) . The coupling of g.l.c to m.s. i s an important t o o l i n the s t r u c t u r a l studies of polysaccharides as shown i n several p u b l i c a t i o n s 1 1 5 ' 1 1 6 . 49 117-119 High pressure l i q u i d chromatography This technique has recently found increasing a p p l i c a t i o n i n carbohydrate chem-i s t r y . I t has the advantage over the other techniques of a high r e s o l u t i o n power as well as speed of separation.lt has been used i n the separation of methylated oligosaccharides and a re-cent publication by Albersheim 1 1 9shows the use of HPLC i n the sequencing of g l y c o s y l residues of complex carbohydrates. II.6 Determination of the linkages. The assignment of anomeric configuration to s p e c i f i c gly-c o s i d i c linkages i n a polysaccharide i s accomplished through the analysis of t h e i r oligomeric sub-units and derived poly-mers. The d i f f e r e n t techniques used may be c l a s s i f i e d as degra-dative (enzymic hydrolysis or chromium t r i o x i d e oxidation),or non-degradative ( o p t i c a l r o t a t i o n and nuclear magnetic r e -sonance ). II.6.1 Optical r o t a t i o n 1 2 0 " 1 2 3 . Optical r o t a t i o n has been used to d i s t i n g u i s h between enantiomorphs and as a c r i t e r i o n of purity and i d e n t i t y since i t can be as c h a r a c t e r i s t i c as a melting point i n the case of carbohydrates. The s p e c i f i c r o t a t i o n ( [ a ] ^ ) of a compound i s defined as: X [a){ =.«*100 1 x c where, a i s the observed r o t a t i o n 1 i s the length of the sample tube (dm) c i s the concentration of the s o l u t i o n (g/lOOmL) 5 0 t i s t h e t e m p e r a t u r e X i s t h e w a v e l e n g t h o f t h e p o l a r i z e d l i g h t T h e m o l e c u l a r r o t a t i o n ( M ) i s d e f i n e d a s £ _ [ a l x M . W . 100 N o r m a l l y , t h e w a v e l e n g t h u s e d i s t h e w e l l k n o w n s o d i u m D -l i n e . a l t h o u g h w h e n r o t a t i o n s a t t h e D - l i n e a r e v e r y s m a l l , t h e p r e s e n c e o f o p t i c a l a c t i v i t y c a n b e d e m o s t r a t e d a t s h o r t e r w a v e l e n g t h a n d a b e t t e r p h y s i c a l c o n s t a n t i s o b t a i n e d . S e v e r a l r u l e s h a v e b e e n p r o p o s e d t h a t s h o w t h e r e l a t i o n -s h i p b e t w e e n s t r u c t u r e a n d o p t i c a l a c t i v i t y , s u c h a s , V a n ' t 120 H o f f ' s R u l e o f O p t i c a l S u p e r p o s i t i o n . H u d s o n ' s R u l e o f I s o -121 r o t a t i o n , e t c . T h e y g i v e s a t i s f a c t o r y r e s u l t s w h e n c o m p a r i -s o n s a r e m a d e o n c l o s e l y r e l a t e d s u b s t a n c e s . H u d s o n ' s I s o r o t a t i o n R u l e s c a n b e a p p l i e d t o p r e d i c t s p e -122 c i f i c r o t a t i o n s o f o l i g o s a c c h a r i d e s a n d p o l y s a c c h a r i d e s b y c o n s i d e r a t i o n o f t h e i n d i v i d u a l g l y c o s i d i c l i n k a g e s i n v o l v e d a n d t h e m o l e c u l a r r o t a t i o n s o f m o d e l c o m p o u n d s ( e . g . m e t h y l g l y c o s i d e s ) NI = z M . w h e r e NI. i s t h e m o l e c u l a r r o t a t i o n o f t h e l l c o m p o n e n t s u g a r s a s i n t h e m e t h y l g l y c o s i -d e s . [ a ] - M x 100 M . W . T h e s e e q u a t i o n s a r e u s e f u l f o r c a l c u l a t i n g a n d d e t e r m i n -i n g t h e c o n f i g u r a t i o n o f s p e c i f i c l i n k a g e s f r o m t h e m e a s u r e d 5 1 o p t i c a l rotations. 124— 148 II.6.2 Nuclear magnetic resonance (n.m.r.) Current advances i n instrumentation (development of super-conducting solenoids.spectrometers with higher magnetic f i e l d s , etc.) have increased enormously the progress i n t h i s field.An extensive review with a l l the information available on carbo-hydrates i s a considerable task.The information that w i l l be given here r e f e r s only to the d i r e c t a p p l i c a t i o n of n.m.r. studies on s t r u c t u r a l analysis of oligosaccharides and poly-saccharides . The f a c t that the polysaccharides of K l e b s i e l l a , although of high molecular weight (~10 6),give interpretable spectra a r i s e s from t h e i r regular structures ->t .compared to the complex structures of the plant polysaccharides.where l i t t l e information can be obtained. 1 127—133 II.6.2.1 Proton magnetic resonance ( H-n.m.r.) ~ J J . The application of ^ H-n.m.r. to problems i n carbohydrate chemistry involves the measurement of several n.m.r. param-eters : i ) Chemical s h i f t . T h i s parameter depends upon the environ-ment of the proton i n the molecule,and i s strongly dependant on the s u b s t i t u t i o n a l , o r i e n t a t i o n a l and electronegativity e f f e c t s of the neighbouring groups.In the spectrum of an oligosaccharide or a polysaccharide,three main regions can be observed: a) the anomeric region ( 64.5-5.5)»b) the r i n g proton region ( 6 3-0-^.5) and c) the high f i e l d region,where s p e c i f i c groups can be observed such as methyl groups from 6-deoxy sugars.pyruvates and acetates.With some exceptions, 5 2 a general rule i s that a x i a l l y oriented protons resonate at higher f i e l d than the chemically s i m i l a r e q u a t o r i a l l y oriented protons.Several empirical rules have been proposed to calcu -> late chemical s h i f t s of protons. An a r b i t r a r y d i v i s i o n at <$ 5-0 was accepted which divides the anomeric region i n two,signals appearing downfield of 65.O are assigned to a-linkages(proton equatorially oriented) and signals appearing u p f i e l d are as-signed to 6-linkages(proton a x i a l l y oriented).When th i s i s applied to a polysaccharide spectrum.it i s possible to deter-mine the number of a - and B -linkages present,but no further assignment of p a r t i c u l a r g l y c o s i d i c bonds can be done,unless comparison with spectra of oligomers isolated.For the r i n g protons.assignments are more d i f f i c u l t to make,although i t has been observed that s p e c i f i c protons may resonate at lower f i e l d s ( 64.0-4.5)(H-2 of mannose residues,H -5 of glucuronic and galacturonic acid residues,etc).and can be as low as 1 p O 6 4.85 .In the high f i e l d region,particular groupings can be detected,including the methyl groups of L-rhamnose or L-129 fucose.acetate and pyruvate.Garegg and coworkers 7 have ob-served that the differences i n chemical s h i f t of stereoisome-r i c pairs of a c e t a l i c CH^- groups of pyruvate are great enough to be used f o r unequivocal determination of t h e i r stereochem-i s t r y . i i ) Coupling constant. This parameter.for v i c i n a l hydrogens, bears a r e l a t i o n s h i p to the tors i o n angle^3°(dihedral angle, 0 ) being a useful t o o l for the determination of configuration and/or conformation of carbohydrates.Karplus 1-^ 1 demonstrated 53 t h i s r e l a t i o n s h i p and gave an empirical equation which enables us to calculate coupling constants.The values are maximum when the dihedral angle(0) i s 0 or 180°,and minimum when i t i s 9 0 ° . 1 3 2 , A l t h o u g h the coupling constant depends on a va r i e t y of parameters besides the torsion angle (electronegativity of substituents,H(l)-C(l)-C(2) and C(l)-C(2)-H(2) bond angles,etc) i t i s very use f u l f o r the assignment of t r a n s - d i a x i a l protons ( 0=180°) and equa t o r i a l - a x i a l or equatorial-equatorial pro--tons ( gauche conformation ,0=60°) as a large coupling con-stant 7 - 9 Hz and a smaller coupling constant 1-3 Hz i s ob-tained respectively.This i s shown i n Figure II.3-i i i ) Relative i n t e n s i t y of the signals.Under proper operating conditions,the r e l a t i v e i n t e n s i t i e s of the d i f f e r e n t hydro-gens are equal to the r e l a t i v e amounts of hydrogen producing the signals.This allows a rapid quantitative analysis of the r a t i o of a - to B-linkages.number of 6-deoxy sugars.acetates, pyruvates.etc. present i n the spectrum.For oligosaccharides, the anomeric proton signals f o r the reducing end can be d i s -tinguished from signals of the other protons because i t d i s -plays the ef f e c t of mutarotation,showing two separate signals f o r the a - and B-anomers.Anomeric e q u i l i b r i a ( r a t i o a / B )can also be calculated. Several problems are encountered i n performing a proton n.m.r. experiment on a p o l y s a c c h a r i d e . F i r s t l y , a l l the protons from the free hydroxyl groups are interchanged by deuterium. This exchange i s done by succesive dissolutions of the sample i n 99.7% D o0 and freeze-drying.After heating the sample under 54 Figure I I . 3 Relationship between dihedral angle (0) and coupling constant for <* -and 8-D-hexoses. ( R=H,OH and R-,= H,etc. ) 55 vacuum fo r several hours.it i s dissolved i n 99-9% D^O.Despite the exchanges,a r e s i d u a l HOD peak i s always found which ap-pears i n the anomeric region of the spectrum.In order to e l i -minate t h i s interference several procedures have been devised, increase of the temperature,that s h i f t s the HOD peak to upper f i e l d s ( 6 4 . 1 - 4 . 2 ).addition of a c i d ( t r i f l u o r o a c e t i c acid)that s h i f t s the peak downfield, or a relaxation timeCl^) type ex-periment which eliminates the HOD peak1--^. Recording the spectra at higher temperatures ( 90-95°) not only eliminates the i n t e r -ference of HOD,but also helps to reduce problems associated with the v i s c o s i t y of the sample(loss of resolution).A less viscous sample can be prepared by performing a very mild hydro-l y s i s of the polysaccharide.The problem associated with t h i s procedure i s the p o s s i b i l i t y of loss of l a b i l e groups(acetate, pyruvate,etc.). Figure II.4 shows the """H n.m.r. spectrum of K l e b s i e l l a K60 polysaccharide.lt has been recorded with acetone as i n t e r -n a l standard ( 62.23).The spectrum exhibits 6 signals i n the anomeric region,4 i n the a - and 2 i n the B -region.From the integration data,one of the a-linkage signals corresponds to two protons.Further information can be obtained from the coup-l i n g constants,the polysaccharide i s known to contain 4 g l u -coses, 1 glucuronic a c i d , l galactose and 1 mannose per re -peating unit,and none of the B-linkages are associated with the mannosyl residue,as the coupling constant i n t h i s case should be 2-3 Hz.The conclusion i s that mannose must be a -linked. vip;iire II.4 The H-n.m.r. spectrum of K l e b s i e l l a K60 capsular polysaccharide. 57 13 \127 134-148 II.6.2.2 Carbon magnetic resonance ( ^C-n.m.r.) '' J . Many methods have been developed that increase the s e n s i -t i v i t y of natural abundance "^C-n.m.r.,including pulse-Fourier 134 transform n.m.r. spectroscopy.proton broad band decoupling J which e f f e c t s the collapsing of spin multiplets into singlets and as a side e f f e c t produces the nuclear Overhauser e f f e c t ^ v 13 that enhances the signals,etc. In recent years, -^C-n.m.r. has 136-141 found extensive use i n the study of carbohydrates J .The use of low molecular weight model compounds fo r analysis of spectra of polysaccharides i s generally valid.Assignment of ^ C spectra of polymeric carbohydrates i s generally done by c o r r e l a t i o n with the spectra of t h e i r oligomeric subunits as well as with monosaccharide glycosides,oligosaccharides and related p o l y s a c c h a r i d e s 1 * * 2 " 1 ^ . 13 The main parameter used for assignment of the spectra i s the chemical s h i f t . I n a spectrum of an oligosaccharide or a polysaccharide,four main regions can be observed:a) carbon-y l and carboxyl groups (around 170 ppm),b) anomeric carbons ( 93-HO ppm),c) the remainder of the r i n g carbons and the primary alcohols ( 6 0 - 8 5 ppm) and d) methyl groups of 6-deoxy sugars.acetates.pyruvates.The most useful region i s that of the anomeric carbons.As a general rule,these carbons are strongly deshielded (7-10 ppm) through glycoside formation and the C - l resonance of an a x i a l isomer i s shielded r e l a t i -ve to that of i t s equatorial isomer.With these generaliza-tions i n mind, g l y c o s i d i c linkages may be assigned by t h e i r p o s i t i o n r e l a t i v e to an a r b i t r a r y d i v i s i o n at 101 ppm,signals 58 Figure I I . 5 The^c-n.m.r. spectrum of K l e b s i e l l a K60 capsular polysaccharide. 5 9 appearing u p f i e l d represent a - l i n k e d sugars ( a x i a l anomers) and signals appearing downfield represent 3 - l i n k e d sugars (equatorial isomers) 1^.The anomeric carbons of free sugars (reducing end) appear u p f i e l d , i n the region 9 3 - 9 7 ppm. Very d i s t i n c t i v e are the signals due to the carbons of primary alcohol g r o u p s ( 6 0 - 6 5 ppm),which can be d i f f e r e n t i a -ted as linked or non-linked by t h e i r chemical s h i f t s (non-linked, 60 - 6 2 ppm;when linked,they are s h i f t e d 7-10 ppm down-f i e l d ) . Carbons involved i n g l y c o s i d i c bonding to the anomeric po s i t i o n of the adjacent residue are i n most-eases s u f f i c i e n t -l y deshielded as to produce signals well separated from the other r i n g carbons (~80 ppm). Methyl groups are d i s t i n c t l y separated and can be e a s i l y attributed to 6-deoxysugars ( ~ 1 7 ppm),acetate (~21 ppm)and pyruvate (~24 ppm).It has been shown that the stereochemistry of an a c e t a l i c linked pyruvate can be d i f f e r e n t i a t e d by the 148 chemical s h i f t of the methyl group 13 1 The coupling constants ( JC- H ) may be used to assign anomeric configuration,however,loss of si g n a l strength due to s p l i t t i n g and the ad d i t i o n a l number of peaks preclude the use of coupling constants for polysaccharide analysis. The ^ C spectra of oligosaccharides and polysaccharides were recorded with proton decoupling.Samples were dissolved i n DgO and acetone was used as i n t e r n a l standard ( 3 1 . 0 7 ppm). As the spectra had to be run at room temperature.difficulties with v i s c o s i t y were overcome by mild hydrolysis of the poly-6 0 saccharide. Figure II.5 shows the "^CJ-n.m.r. spectrum(proton decoup-led) of the K60 polysaccharide which had undergone mild hydro-l y s i s . Five signals can be observed i n the anomeric region cor-responding to seven carbons ( 4 6 -linked and 3 « -linked).In-tense signals are observed at 61.5 ppm corresponding to several free C-6 carbons. II.6.3 Other techniques. Among the degradative techniques used for determining the stereochemistry of the g l y c o s i d i c linkages only two w i l l be considered: a) Enzymic hydrolysis 1** - 9 , 1 5°.This technique was considered to be,together with the s p e c i f i c rotations,the c l a s s i c a l method of establishing the nature of the gl y c o s i d i c l i n k a g e s . l t i s based on the s u s c e p t i b i l i t y of c e r t a i n linkages to the hydro-l y t i c action of s p e c i f i c enzymes(glycosidases or glycohydro-lases).This procedure depends mainly on the a v a i l a b i l i t y of enzymes having the proper s p e c i f i c i t y f o r <* - and 8 - g l y c o s i -dic linkages.Pure enzymes having the proper s p e c i f i c i t y have, however,not always been a v a i l a b l e . l t should be emphasized that the rate of l i b e r a t i o n of a monosaccharide unit from d i f f e r -ent substrates by s p e c i f i c glycosidase varies.depending on the length and sequential arrangement of the oligosaccharide chain.The conclusion drawn i s that either a negative or a positi v e r e s u l t must be c a r e f u l l y examined. b) Chromium tri o x i d e o x i d a t i o n . l t has been observed that chro-mium tr i o x i d e i n ac e t i c acid r a p i d l y oxidizes peracetylated 6 1 hexopyranosides i n which the aglycone occupies an equatorial position;the peracetylated hexopyranosides with aglycons oc-cupying a x i a l positions are oxidized more slowly.These r e -s u l t s lead to a general method for d i s t i n g u i s h i n g a - and s _ pyranosidic linkages i n glycosides,oligosaccharides and poly-saccharides 1^"*"'" 1^. I I . 7 Various reactions on oligo-and poly-saccharides. II. 7 . 1 Reduction 1- 5**" 1^ Reduction of carboxyl groups i n a c i d i c carbohydrates can be carried out at d i f f e r e n t stages during a s t r u c t u r a l analy-s i s of a polysaccharide: i ) i n the t o t a l sugar r a t i o , i n order to obtain a neutral oligo-or polysaccharide so that i t can be e a s i l y hydrolyzed, i i ) during the methylation a n a l y s i s , f o r the i d e n t i f i c a t i o n of the s u b s t i t u t i o n pattern of the uronic acid, i i i ) p r i o r to periodate oxidation,in order to avoid e l e c t r o s -t a t i c repulsions between periodate ions and the polyanionic carbohydrate which leads to under-oxidation. Depending on the substrate (methylated or non-methylated, oligomeric or polymeric) several procedures can be followed. The reducing agents most commonly used are NaBH^ and LiAlH^ as well as some of t h e i r derivatives. NaBH;4. This reducing agent may be used i n aqueous solvents, and i t i s the reagent used f o r converting the free sugars to alditols.The disadvantage i s that i t does not reduce acids. This problem can"be.avoided by converting the acids into es-6 2 ters which can then he reduced with NaBH^ to the corresponding a l c o h o l s 1 5 6 . M e t h y l esters may be formed by treatment of the ac i d i c polysaccharide with methanolic hydrochloric acid or with diazomethane.With the f i r s t procedure.glycosidic linkages are cleaved at the same time.modifying the polysaccharide.This technique i s used extensively i n this laboratory f o r the t o t a l sugar ratio.The second procedure must be repeated i n order to ensure complete es t e r i f i c a t i o n . T h e use of NaBH^ to reduce es-ters has the disadvantage that hydrolysis of the sodium boro-hydride produces hydroxyl ions which can saponify the esters, thus decreasing the y i e l d of reduction.This d i f f i c u l t y can be overcome with the use of Ca(BU^)2* as the solutions of a l k a l i -ne borohydrides are nearly neutral 1 5 6.NaBH^ and Ca(BH^) 2 are soluble i n tetrahydrofuran and they are also used i n the r e -duction of permethylated a c i d i c oligosaccharides. Taylor and C o n r a d 1 5 7 have used a water soluble carbodiimi-de to activate the carboxylic acid which can then be reduced with NaBH^ according to the reaction mechanism outlined i n Scheme II.5 . LiAlH^.Although t h i s reducing agent i s powerful enough to r e -duce carboxylic acids i t s use i s limited to substrates soluble i n ether-type solvents.Permethylated a c i d i c o l i g o - and poly-saccharides are e a s i l y reduced with LiAlH^ i n oxolane.Reduc-t i o n of acylated polysaccharides which are soluble i n this type of solvent i s not suitable,as esters are reduced f a s t e r than the carboxylic acid with the subsequent i n s o l u b i l i z a t i o n of the polysaccharide.A modification involves the treatment 63 RCOOH R c o c r H+ R' I N I I C I I N I R" H + pH=A,75 NaBK pH-5-7 R" 0 *NH I I n RC-O-C NH R NH C=0 NH I R" 0 I I R-C-H R-CH 20H S c h e m e I I . 5 R e d u c t i o n o f a c a r b o x y l i c a c i d i n a q u e o u s s o l u t i o n u s i n g a c a r b o d i i m i d e r e a g e n t . 64 of the acylated (propionylated) polysaccharide with diazometh-ane to make the methyl ester of the carboxylic acids which are reduced f a s t e r than the propionic esters with LiBH^ D .This however requires several treatments i n order to achieve com-plete reduction.An alternative procedure involves the use of diborane.a reagent which reduces carboxylic acids i n prefer-ence to ester groups J 7 . I I . 7 . 2 Oxidation.. Two main types of oxidation may be used i n s t r u c t u r a l i n -vestigation of polysaccharides.The f i r s t involves the cleavage of the carbon-carbon bond of v i c i n a l d i o l s by reagents such as periodate or lead tetraacetate and has already been described (see Section I I . 5 . 2 ) . T h e second involves the oxidation of se-l e c t i v e hydroxyl groups to carbonyl or carboxyl groups. A s p i n a l l has used the oxidation of primary alcohols to carboxylic acids with 0^ and Pt as c a t a l y s t , i n order to obtain stable uronosyl bonds.As shown before, 3-elimination i s an important reaction i n s t r u c t u r a l investigations of polysac-charides and the substrates for these reactions are mainly carbonyl compounds.Several procedures have been developed through the years by which alcohols can be oxidized to carbo-nyIs.Oxidizing agents such as RuO^ have been used as well as the series based on dimethylsulfoxide,with Cl^^,P^,0^Z , acetic a c i d 1 ( ^ , t r i f l u o r o a c e t i c anhydride ( T F A A ) 1 ^ and carbo-diimide'*'^.During the course of thi s work,the DMSO/TFAA pro-cedure was used on the K 2 6 polysaccharide. 65 GENERAL EXPERIMENTAL CONDITIONS 66 I I I . - GENERAL EXPERIMENTAL CONDITIONS. 111.1 Paper chromatography. Paper chromatography was performed by the descending meth-od using Whatman No.l paper and the following solvent systems: A) ethyl acetate:acetic acid:formic acid:water (18:3slt4) B) ethyl acetate:pyridine:water ( 8:2:1 ) C) 1-butanol-acetic acid-water ( 2:1:1 ) D) 1-butanol-ethanol-water ( 4:1:5, upper phase ) Chromatograms were developed with alkaline s i l v e r n i t r a t e or by heating at 110° for 5-1° minutes a f t e r being sprayed with jj-anisidine hydrochloride i n aqueous 1-butanol. Preparative paper chromatography was ca r r i e d out by the descending method using Whatman 3MM paper and solvent(C)( un-less otherwise stated).The relevant s t r i p s were cut out and eluted with water for 6 hours.The aqueous solutions were f i l -tered ,concentrated and freeze-dried. 111.2 Gas-liquid chromatography and g.l.c.-mass spectrometry. A n a l y t i c a l g . l . c . separations were performed with a Hewlett-Packard 5700 instrument f i t t e d with dual flame-ioni-aation detectors.An Infotronics CRS-100 e l e c t r o n i c integrator was used to measure the ./peak areas. S t a i n l e s s - s t e e l columns ( 1.8mx3mm) were used with a carrier-gas flow-rate of 20mL per minute.The following packing materials were used: (a) 3% of SP-2340 on Supelcoport (100-120 mesh) (b) 5% of ECNSS-M on Gas Chrom Q (100-120 mesh) (c) 3% of 0V-225 on Gas Chrom Q (100-120 mesh) 67 (d) 5% of SP-1000 on Gas Chrom Q (100-120 mesh) (e) 5% of SE-52 on Chromosorb W ( 60- 80 mesh). The temperature programs used with each column r e s p e c t i -vely were (unless otherwise stated) t (a) 195° f o r 4 min.,2°/min , 260° f o r 32 min., (b) isothermal at 170° ,or 160° for 4 min.fZ°/min ,190° f o r 32 min., (c) isothermal at 170° ,or 180° for 4 min.,2°/min ,230° f o r 32 min., (d) isothermal at 220°, (e) isothermal at 170°. Preparative g . l . c . was carried out with a F&M model 720 dual column instrument f i t t e d with thermal conductivity de-tectors . S t a i n l e s s - s t e e l columns ( 1.8mx6.3mm) were used with the carrier-gas (helium) flow-rate of 60 mL/min.The following packing was used: (f) 5% of S i l a r 10 C on Gas Chrom Q (100-120 mesh). The temperature was programmed from 210° at 4 ° / min to 250°, and l e f t isothermal for 30 minutes. G^l.c.-m.s. analysis were performed with a V.G.Micromass 12 instrument f i t t e d 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 u A and an ion source temperature of 200°. I I I . 3 Gel-permeation chromatography. Preparative gel-permeation chromatography was performed using columns (2.5x 100cm) of Bio-Gel P-2 or Bio-Gel P-4 (both 400 mesh).The concentration of the samples applied to 68 the columns ranged from 40-100 mg/mL.Columns were i r r i g a t e d with water-pyridine-acetic acid ( 5 0 0 : 5 : 2 ) at a flow rate of 8 mL/hour.Fractions ( 2 - 3 mL) were collected,freeze-dried, weighed and a f t e r obtaining an e l u t i o n profile.chromatogra-phed on paper. 111.4 Optical r o t a t i o n and c i r c u l a r dichroism. Optical rotations were measured from aqueous solutions at 20 ± 3 ° on a Perkin-Elmer model 141 polarimeter with a 10 cm c e l l . C i r c u l a r dichroism spectra ( c d . ) were recorded on a Jasco J20 automatic recording spectropolarimeter with a quartz c e l l of 0 . 3 mL capacity and a path length of 0.1 cm.Compounds were dissolved i n high purity a c e t o n i t r i l e and the spectra record-ed i n the range 210-240 nm. 111.5 Nuclear magnetic resonance. Proton magnetic resonance spectra were recorded on Varian XL-100,Bruker WP-80,or Bruker WH-400 instruments.Spectra were recorded at temperatures of 9 0 ± 5 ° and acetone was used as an i n t e r n a l standard.All values are given r e l a t i v e to that of i n t e r n a l sodium -4,4-dimethyl-4-silapentanesulfonate taken as 0 .Samples were prepared by d i s s o l v i n g i n D^ O and freeze-dry-ing 2 - 3 times from DgO solutions. ^c-n.m.r. spectra were recorded on a Varian CFT-20 spec-trometer at ambient temperature.Samples were dissolved i n D o0 and acetone was used as i n t e r n a l standard. 6 9 1 1 1 . 6 General conditions. The I.R.spectra of methylated derivatives were recorded on a Perkin-Elmer model 4 5 7 spectrophotometer.The solvent used was carbon tetrachloride. A l l solutions were concentrated on a rotatory evaporator i n vacuo at a bath temperature of 4 0 ° . Ion-exchange chromatography for separation of a c i d i c and neutral oligomers was performed on a column ( 2.0x28cm) of Bio-Rad AG-1-X2 (formate form,200-400 mesh).The neutral f r a c -t i o n was eluted with water and the a c i d i c with 10% formic acid. De^-ionizations were carr i e d out with Amber l i t e IR-120(H +) res i n . 1 1 1 . 7 I s d l a t i o n and p u r i f i c a t i o n of the polysaccharides. III.7-1 K l e b s i e l l a polysaccharides. The following media were used to grow the bacteria: 1 . Beef-extract medium 5 g of Bactopeptone 3 g of Bacto beef extract 2 g of NaCl 1 L of H 20 2. Sucrose-yeast extract-agar 7 5 g of Sucrose 5 g of Bacto yeast extract 3 7 - 5 g of agar 5 g of NaCl 2 . 5 g of KH2P0^ 0 . 6 2 5 g of MgS0^.7H20 1 . 2 5 g of CaSO^ 2 . 5 L of H20 7 0 Samples of K l e b s i e l l a bacteria of serotypes K60 and K26 were received as stab cultures from Dr.I.0rskov(Copenhagen). They were streaked on agar plates at 37°.An i n d i v i d u a l co-lony was innoculated on beef-extract medium and bacteria were grown for 3 hours at 37° with continuous shaking.This l i q u i d culture was incubated on a tray of sucrose-yeast ex-tract-agar f o r three days.The lawn of capsular bacteria pro-duced was harvested by scraping the agar surface and the bac-t e r i a k i l l e d with 1% phenol solution.The polysaccharide was separated from the c e l l s by ultracentrifugation< ( 3 0 000 rpm). The viscous sol u t i o n of polysaccharide was precipitated with ethanol (3 volumes).The pr e c i p i t a t e was dissolved i n the minimum amount of water and reprecipitated with a saturated s o l u t i o n of C E T A V L 0 N (cetyltrimethylammonium bromide).Centri-fugation yielded the preci p i t a t e d a c i d i c polysaccharide.The Cetavlon-precipitated complex was dissolved i n kM NaCl,re-precipitated into ethanol (3 vol.) and the pr e c i p i t a t e d i a -lyzed against running tap water a f t e r d i s s o l u t i o n i n water. The dialyzed solution of polysaccharide was freeze-dried a f t e r a l l the s a l t had been removed. III.7 . 2 Gum exudate from Chorisis speciosa. The crude gum,which was obtained as l i g h t yellow nodules, was allowed to swell i n water f o r 2k hours.It was then heated on a steam-bath for 8 hours.The pH was kept constant around 7 i n order to avoid hydrolysis of the polysaccharide.After centrifugation,the s o l u t i o n was poured into a c i d i f i e d ethanol 71 ( 4 vols.).The pre c i p i t a t e d material was dissolved i n water and freeze-dried. I I I . 8 Sugar analysis. The i d e n t i f i c a t i o n and quantitation of the sugars present i n poly- and oligo-saccharides was done by g. l . c . of the d e r i -ved a l d i t o l acetates.Samples (2-10 mg) were hydrolyzed with 2M TFA on a steam-bath (time of hydrolysis:polysaccharides 8 hours and oligosaccharides 4 hours).The TFA was removed by co-d i s t i l l a t i o n with water.Part of the hydrolyzate was used f o r paper chromatography.while the r e s t was dissolved i n H"20(5mL) and reduced with NaBH^ (20 mg).After 3 hs.,the excess NaBH^ was decomposed with IR-120 (H +)resin,filtered,concentrated to dryness and c o d i s t i l l e d with three portions of methanol (5mL). The a l d i t o l s were acetylated with acetic anhydride-pyridine ( 1:1, 2mL) on a steam-bath for one hour and the excess r e -agents were removed by c o d i s t i l l a t i o n with ethanol and water. The a l d i t o l acetates were dissolved i n chloroform and i n j e c -ted into the g.l.c.The column used was (A) and the tempera-ture program as indicated before. Samples containing uronic acids were treated i n the f o l -lowing way before hydrolysis: Samples of oligosaccharides and polysaccharides(2-10 mg)dried i n vacuo were treated with Jfo HC1 i n methanol on a steam-bath for 8 hours.The acid was neutralized with PbCO^or Ag2C0^,cen-trifuged and concentrated to dryness.The residue was dissolved i n anhydrous methanol (5mL) and NaBH^ (20 mg) was added.The reaction was allowed to proceed at room temperature for 8 7 2 hours.The excess NaBH^ was decomposed with IR-120 (H +) re s i n , filtered,concentrated to dryness and c o d i s t i l l e d with three portions (5mL) of methanol.The samples thus obtained were treated as described before ( i . e . hydrolysis.reduction and acety l a t i o n ) . III.9 Methylation analysis. Methylation of polysaccharides and oligosaccharides was carried out by the Hakomori procedure ,and i n cases where complete methylation was not achieved with this method,a subsequent methylation by the Purdie-Irvine procedure was performed.Methylation was considered to be;complete when the product was devoid of hydroxyl absorption i n the infrared spectrum (3000-3200 cm" 1). Method A (for polysaccharides) A dried sample (100-300 mg) of the polysaccharide was d i s s o l -ved with s t i r r i n g and heating at 60° i n anhydrous dimethyl-sulfoxide (15-^5 mL) under Ng.After cooling to room tempera-ture, 2M sodium methylsulfinylmethanide (10-15 mL) was added to the solution.After 4 hours of s t i r r i n g , t h e s o l u t i o n was frozen and methyl iodide (5-1° mL) was added dropwise.It was then l e f t s t i r r i n g at room temperature f o r If hours before the excess methyl iodide was removed by rotatory evaporation and the reaction mixture was dialyzed overnight against run-ning tap water.The contents of the bag were freeze-dried to y i e l d the methylated polymer. I f methylation was incomplete,the p a r t i a l l y methylated polysaccharide was further methylated using the Purdie and 7 3 Irvine procedure as described below: The dried sample (100-200 mg) of p a r t i a l l y methylated poly-saccharide was dissolved i n dry methyl iodide ( 1 0 - 1 5 mL) and refluxed with s i l v e r (I) oxide ( 2 0 0 - 3 0 0 mg) for 1 - 3 days. The s i l v e r oxide and s i l v e r s a l t s were washed twice with: CHCl^ a f t e r centrifugation.The chloroform extracts were com-bined and the solvent was removed by rotatory evaporation. This procedure was repeated u n t i l no hydroxyl absorption was detected i n the i . r . spectrum. Method B ( for oligosaccharides) The methylation procedure i s as above,except for the solubi-. l i z a t i o n and recovery of the product.A sample of dried o l i g o -saccharide (1-10 mg) was dissolved i n anhydrous dimethylsul-foxide ( 1 - 3 mL).with s t i r r i n g under N 2. Sodium m e t h y l s u l f i n y l -methanide (2M, 1 - 3 mL) was added and the soluti o n s t i r r e d at room temperature for 2 hours before being frozen.Methyl iodide ( 1 - 3 mL) was added and was then allowed to s t i r at room temperature for 1 hour.The excess methyl iodide was r e -moved i n vacuo and the product was recovered by adding four volumes of water and extracting three times with one half volume of chloroform.The combined chloroform extracts were washed with water ( 3 times) and the solvent removed by r o -tatory evaporation.Residual DMS0 was removed i n vacuo by heating with an i . r . lamp. Hydrolysis of permethylated polysaccharides and ol i g o -saccharides was carr i e d out with 2M TFA on a steam-bath(time of hydrolysis, 16 hours for a polysaccharide,and 6 hours f o r 74 an oligosaccharide),the acid was then removed by c o d i s t i l l a -t i o n with water.Samples of the hydrolyzate were spotted on paper,and chromatographed i n solvent (D).The p a r t i a l l y meth-ylated sugars were i d e n t i f i e d by developing the papers with j)-anisidine hydrochloride i n aqueous 1-butanol,and heating for 5 - 1 0 minutes.The r e s t of the hydrolyzate was reduced with sodium borohydride (40 - 6 0 mg) i n water ( 5 - 1 ° mL).Residual sodium borohydride was decomposed with IR - 1 2 0 ( H + ) r e s i n , the s o l u t i o n was filtered,concentrated to dryness and codis-t i l l e d with three portions of methanol ( 5 mD.The r e s u l t i n g p a r t i a l l y methylated a l d i t o l s were acetylated with acetic anhydride-pyridine (Is 1 , 2 mL) on a steam-bath for 1 hour,fol-lowing which the excess reagents were removed by c o d i s t i l l a -t i o n with ethanol and water. Analysis of the p a r t i a l l y methylated a l d i t o l acetates was performed by g. l . c . using columns (b),(c) and (d). The i d e n t i t y of each component i n the mixture was confirmed by g. 1. c. -m. s. . The uronic methyl esters i n a c i d i c polysaccharides and oligosaccharides were converted into the corresponding sug-ars p r i o r to hydrolysis i n the following manners Method A (for polysaccharides) A sample of permethylated material ( 3 - 5 0 mg) was dissolved i n anhydrous oxolane ( 3 - 1 0 mL),LiAlH^ ( 3 ° - 5 0 mg) was added and the reaction was l e f t to s t i r at room temperature f o r 6 hours.Polysaccharides were refluxed for the same period. The excess L i A l H k was destroyed by dropwise addition of , 75 ethanol,the p r e c i p i t a t e formed was dissolved i n 4 volumes of 10% HCl.and the sol u t i o n was then extracted with CHCl^ ( 3 * 1 volume).The combined chloroform extracts were concentra-ted to dryness. I.r. spectroscopy of the sample dissolved i n CCl^ showed complete reduction when no carbonyl absorption was detected and hydroxyl absorption was observed. Method B ( for oligosaccharides) A dried sample (1 - 5 mg) of permethylated oligosaccharide was dissolved i n anhydrous oxolane ( 5 mL) fand treated with NaBH^ (20 mg) and CaCl 2 (20 mg),the Ca(BH^) 2 generated i n s i t u was the reducing agent used.The reaction was s t i r r e d during 8 hours at room temperature,and the solution was f i l t e r e d or centrifuged.Amberlite IR-120 (H +)resinwas added to decom-pose the excess borohydride,the solu t i o n was f i l t e r e d , c o n -centrated to dryness and c o d i s t i l l e d with three portions(5mL) of methanol.Reduction was checked by i . r . as above. III.10 Base catalyzed uronic acid degradation. Permethylated polysaccharide ( 3 0 - 5 0 mg) was c a r e f u l l y dried i n vacuo,dissolved i n dimethylsulfoxidei2,2-dimethoxy-propane ( 1 9 : 1 , 1 5 mL) with jD-toluenesulfonic acid (1-2 mg)and s t i r r e d under N 2 for 1 hour.Sodium methylsulfinylmethanide (2M) i n dimethylsulfoxide ( 1 5 mL) was added and the soluti o n was l e f t s t i r r i n g at room temperature overnight.After freez-ing the reaction mixture.methyl iodide (or ethyl iodide)(lOmL) was added and the so l u t i o n allowed to s t i r at room tempera-ture for l i hour.The excess methyl iodide was removed by rotatory evaporation,the s o l u t i o n was d i l u t e d with H ? 0 ( 4 vol.) 76 and then extracted with CHCl^ ( 3 x 1 5 mL).The product was hydrolyzed and the sugars released were analyzed as d e s c r i -bed previously for the methylation analysis. 77 STRUCTURAL INVESTIGATIONS OF KLEBSIELLA CAPSULAR POLYSACCHARIDES 78 IV.1 Structural investigation of Klebsiella serotype K 60  capsular polysaccharide. IV.1.1 Abstract. Non-linear capsular polysaccharides of Klebsiella bacteria usually have a single side chain per repeating unit, less commonly two side chains attached to the same sugar.The capsular polysaccharide from Klebsiella serotype K 60 is u-nique in having three side chains in the heptasaccharide re-peating unit shown.The structure,including the configuration of the glycosidic linkages,was established mainly by charac-terization of the oligosaccharides obtained by partial hydro-lysis of both,the original capsular polysaccharide,and the polymer resulting from the removal,by Smith degradation,of the side chains. D-Glc£ -—-- D-GlcpA -—2. D-Galjj - — 2 D-Manp - — B 4 f l l D-Glcp_ l l D-Glcp_ l l D-Glcp_ IV.1.2 Introduction. The capsular polysaccharides of Klebsiella that are com-posed of D-glucuronic acid,D-glucose,D-galactose and D-man-nose comprise the largest chemogroup in this genus with 20 strains represented,half of which incorporate pyruvic acid as an acetal into the polysaccharide.The capsular polysaccharide from Klebsiella serotype K 60 discussed here,represents a novel structural pattern in this series. 79 IV.1.3 Results and discussion. Composition and n.m.r. spectra.Klebsiella K 60 bacteria was grown on an agar medium and the capsular polysaccharide• was p u r i f i e d by one p r e c i p i t a t i o n with CETAVLON.The product moved as a single band on electrophoresis on c e l l u l o s e aceta-te and had[ a ] D +58°,which compares well with the calculated value of+4 7 ° using Hudson's Rule of Isorotation.The molecu-l a r weight of the polysaccharide was determined by gel chro-matography to be 8.1x 10^ daltons.The equivalent weight was of 1100 which corresponds to one uronic acid per seven sugar residues. Paper chromatography of the acid hydrolyzate of the polysaccharide showed the presence of mannose.glucose.galac-tose .glucuronic acid.glucuronolactone and an aldobiouronic acid.These r e s u l t s were i n contrast to the a n a l y t i c a l data of Nimmich where fucose and not galactose was detected.Acid hydrolysis of the carboxyl reduced polysaccharide and con-version to a l d i t o l acetates gave mannose.galactose and g l u -cose i n a r a t i o 1:1:5. The configuration of mannose and g l u -cose was determined to be D by measurements of the c i r c u l a r dichroism ( c d . ) of the a l d i t o l acetates;galactose was a s s i g -ned also a D configuration by c d . measurements of a methyl-ated derivative i s o l a t e d subsequently. The "'"H-n.m.r. spectrum of the native polysaccharide was recorded i n DgO at 90° with acetone as i n t e r n a l standard(see Appendix III,spectrum NaD.The spectrum exhibits s i x doublets i n the anomeric region: 6 5,47 ( J ^ 2 2Hz,2H)j 6 5.37 (J-j^ 2 2Hz, 80 1H)|«5.04 ( J 1 > 2 7Hz,lH); <5 4.85 ( J 1 > 2 7Hz,lH);6 4.70 ( J 1 ( 2 7Hz, IH) and <$ 4.54 ( J 1 2 7Hz,lH).From the values of the chemical shifts and coupling constants, 3 a -anomeric linkages and 4B -anomeric linkages were assigned for the heptasaccharide repeat-ing unit.No deoxy-sugar,0-acetyl or acetal-linked pyruvic acid could be detected.As mannose is present in the polysaccharide, i t was assigned an a-linkage,as the 6 -signals exhibit large coupling constants (7Hz) which do not correspond to B-linked mannose. The 1 3 c - , n # m < r > spectrum of K 60(see Appendix III,spec.No2) corroborates the results obtained by "'"H-n.m.r. spectroscopy. Six signals appear in the anomeric region at 99.56;100.60; 102.44;103.07;104.02 and 104.23 ppm.The signal at 99-56 cor-responded to two anomeric carbons.Several signals around 61.51 ppm are attributable to sugar residues that are not linked at position 6. Precise assignment of the anomeric signals was achieved 1 n after studying H- and -^ C-n.m.r. spectra of oligosaccharides and polysaccharides obtained by selective degradative techni-ques, see Table I V . 1.1 Methylation analysis. Methylation followed by carboxyl reduction,hydrolysis and conversion into a l d i t o l acetates gave the results shown inTablelV.1.2,column I,while further methylation after carboxyl reduction gave the data presented in column II.These results show that the polysaccharide con-sists of a heptasaccharide repeating unit,with three termi-nal glucose residues and one unit each of mannose.galactose TABLE IV.1.1 N.M.R. DATA FOR KLEBSIELLA K60 CAPSULAR POLYSACCHARIDE AND DERIVED POLY- AND OLIGO-SACCHARIDES H-n.m.r. Compound £l f2 (Hz) Integral  proton Assignment Spectrum No. GlcA 2—2 Gal-OH 5.30 4.74 4.60 2 8 7 0.4 1.0 0.6 -OH 3-Gal-G l c A " T 3-Gal OH GlcA-—^Gal-OH 6 2 1 Glc 5-38 4.70 4.66 8 8 3-^al-GlcA-G l c — -OH 3-Gal OH 2 B GlcA-—'hal-—^Man-OH B a 5.32 5.20 4. 94 4.78 2 2 2 7 1.0 0.5 0.5 1.0 3-Gal a 3-Man OH a 3-Man—OH GlcA 8 Glc-—^-LCA-I—^al-OH 5.28 4.80 2 7 0.4 1.0 3-Gal OH a G l c - — T A B L E I V . 1 . 1 ( c o n t . ) GlcA-—^Gal -—-Wn-—-<Jlc-0H B . a a A 5 Glc Man Glc-OH B a N l —-Glc-—-<UcA-—-<Jal-—-Man-B B a polysaccharide — - G l c - — - k l l c A - — - G a l - — - M a n -B 4 1 G 2 « 2 l c 1| G : 1.0 3-GlcA 0.6 3-Gal——OH B 1.0 1.4 1.6 1.0 2-Man-a 0.6 3-Glc- -OH a 1.0 Glc- 6 0.4 3-Glc- M B -OH 1.0 3-Man-a 1.0 3-Gal- a 1.0 3-Glc-"g~ 1.0 3-GlcA 2.0 3-Man-2 a Glc-a 1.0 3-Gal- a TABLE IV.1.1 (cont.) K60 capsular polysaccharide Compound  A l A 2 A3 5.04 4.85 4.70 4.54 7 7 7 7 1.0 1.0 1.0 1.0 3-Glc-G l c — G l c — 3-GlcA-4 ^C-n.m.r. Chemical s h i f t Assignment Spectrum 104.50 G l c A — 97.04 3-Gal—— OH 93.08 3-Gal OH a 104.21 GlcA-102.46 G l c — 92.87 3-Gal—OH 104.54 101.40 G l c A — 3 - G a l — TABLE IV.1.1 (cont.) 9^.82 94.30 104.35 103.52 97.04 93.00 104.51 101.51 101.35 96.76 93-04 102.54 99.85 96.80 93.07 104.19 103.34 101.42 3-Man—OH 3-Man OH 3-GlcA-G l c -3-Gal—OH 3-Gal OH a GlcA 8 3-Gal a 3-Man a 3 - G l c — O H 3-Glc OH a G l c — 2- Man--— 3- G l c — O H 3-Glc OH 3-GlcA-3-Glc-3-Gal- a 3-Man a TABLE IV.1.1 (cont.) K60 capsular polysaccharide 104.23 ^3-GlcA 4 B Glc 103.0? 3-GIC-I-102.44 G l c ~ 100.60 G l c — 3-Man— 99.56 i 2 a 3 - G a l — ? a {The numerical prefix indicates the position i n which the sugar i s substituted; the a or B to the configuration of the glycosidic bond or the anomer i n the case of a terminal reducing sugar.Thus, 3-Gal—-— refers to the anomeric proton of a 3 - l i n -ked galactosyl residue i n the a configuration.The absence of a numerical p r e f i x i n -dicates a terminal,non-reducing residue.( i n ^ C-n.m.r.spectra,the nuclei) The chemical s h i f t s are r e l a t i v e to i n t e r n a l acetones "hi-n.m.r at 6 2.23 »and ^C-n.m.r. at 31.0? p.p.m. 86 and glucuronic acid being the branching points. One may.therefore.write two possible patterns for the general structure of the K 60 polysaccharide: A) O O O 3Glc I I I Glc Glc Glc B) — O Glc Glc Glc 3 I Glc where represents galactose.mannose and glucuronic acid. In either c a s e . i t i s clear that only the three terminal g l u -cose residues are susceptible to periodate oxidation. Periodate oxidation.Periodate oxidation was carried out on the sodium s a l t of the s t a r t i n g material as well as on the carboxyl-reduced (carbodiimide procedure) polysaccharide.In both cases,the consumption of periodate reached a plateau (4 .85 and 4.95 moles of periodate per mol of repeating unit, respectively) i n 140 h.Reduction of the polyaldehyde and ox-id a t i o n of the polyalcohols thus obtained caused the consump-t i o n of a further 0.? and 0.9 moles of oxidant respectively. The t h e o r e t i c a l value i s 6 moles of periodate per mole of TABLE IV.1.2 METHYLATION ANALYSIS OF K60 CAPSULAR POLYSACCHARIDE AND DERIVED PRODUCTS. Methylated sugars - Relative retention times Mole % -(as a l d i t o l acetates) ECNSS-M 170° OV-225 170° SP-1000 220° I °- II III IV 2,3,4,6 - Glc 1.00 1.00 1.00 42 48 — 41 2,4,6 - Glc 1.85 1.83 1.68 16 16 28 25 2,4,6 - Man 1.91 - 1.82 — 30 — 2,4,6 Gal 2.12 - 1.91 — — 24 — 4,6 Man 3.13 2.96 2.60 13 13 — 18 4,6 Gal 3.50 3.26 2.82 14 12 — — 2,6 Glc 3.50 3.26 2.72 — 11 — — 2,4 Glc 4.75 - - — — 18 — 2 Glc 7.92 7.61 - 15 — — — 3?4,6 - Gal d- - - 0.92 — — — 16 - 2,3,4,6-Glc l , 5-di - 0-acetyl - 2 , 3 , 4 , 6-tetra - 0-methylglucitol,etc. - Values were corrected by use of ef f e c t i v e carbon-response factors given by Albersheim ejt a l . — I , o r i g i n a l capsular polysaccharide: II,remethylation a f t e r reduction of uronic ester 1 I I I , polymer P 1 : IV, product from B-elimination. - 3 - 0-ethyl- 4,6- d i -O-methylgalactose. 88 polysaccharide. The carboxyl-reduced product was subjected to a modified Smith degradation,that is,periodate oxidation,borohydride r e -duction, methylation, hydrolysis under mild conditions,borohyd-ride reduction and ethylation Hydrolysis of the product before the f i r s t borohydride reduction yielded mannose.galac-tose and glucose (as a l d i t o l acetates) i n the r a t i o of 1:1:2. After the f i r s t methylation,hydrolysis of part of the materi-a l and conversion into a l d i t o l acetates yielded 2 , 4 , 6 - t r i - 0 -methylglucose,4,6-di-0-methylmannose,2,6-di-0-methylglucose and 4 ,6-di -0-methylgalactose as expected.After the f i n a l step (ethylation).hydrolysis and conversion into a l d i t o l acetates yielded 2,4,6-tri-O-methylglucose and 3 other products that although they could not be separated and identified,correspond-ed to monoethyl-dimethylhexoses from glucose.galactose and mannose.These r e s u l t s showed that the s t r u c t u r a l pattern of the repeating unit must" be O 3Glc I Glc Glc Glc where represents mannose,galactose and glucuronic acid. Reduction of the product from periodate oxidation of the sodium s a l t of K 60 polysaccharide followed by mild hydroly-s i s (Smith degradation) yielded a polysaccharide ( P, ) com-89 posed of D-glucose,D-galactose,D-mannose and D-glucuronic 1 13 acid i n equimolar amounts.The H- and ^C-n.m.r. spectrum (see Appendix III,spec.6 and 7) of showed the presence of two a - and two B-anomeric linkages(see TablelV.1.1) and the methylation analysis(see TablelV.1.2,column III) demonstra-ted that P^ i s a l i n e a r polymer.lt follows from these data that of the three l a t e r a l glucose units,one i s a-and two are B-linked,and that the side chains are joined to 0-4 of the glucuronic acid and to 0-2 of both the D-mannose and the D-galactose units. Part of the methylated polysaccharide (P 1) was hydrolyzed p r i o r to carboxyl reduction and separated by ion-exchange chromatography into a c i d i c and neutral fractions.The a c i d i c f r a c t i o n was treated with 3% methanolic HC1 and the methyl esters formed were reduced with NaBH^ihydrolysis followed by conversion into a l d i t o l acetates showed on g.l.c. 2,4-di-0-methylglucose and 2,4,6-tri-0-methylgalactose.This indicated that the aldobiouronic acid i s GlcA — — 2 Gal . A p a r t i a l structure may now be elaborated: -1 Glc -—2. GlcA - — 2 G a l 4| 2" 1 Glc 1 Glc -2 Man 1 Glc a with the proviso that the sequence of the main chain i s un-known (r e l a t i v e p o s i t i o n of mannose and glucose) as are the anomeric configurations of almost a l l the linkages.These pro-9 0 blems may be resolved by i s o l a t i o n and characterization of suitable oligosaccharides. P a r t i a l hydrolysis.In effect,there are two polysacchari-des to be studied:the o r i g i n a l K 6 0 capsular polysaccharide which i s highly branched,and the l i n e a r polymer P^ obtained by periodate degradation. a) O r i g i n a l capsular polysaccharide.Partial hydrolysis of the st a r t i n g material gave two a c i d i c oligosaccharides (A^ and Ag) together with a neutral one (N^J.On the basis of t h e i r n.m.r. spectral data (see TablelV.1.1) and t h e i r methylation analyses (see TablelV.1.3)the structures of these compounds were shown to be: A, GlcA -—2. Gal ; A 0 GlcA -—2. Q a i •3 1 Glc N l Glc - — 2 Man -—2 G i c B a Comparison of the sp e c t r a l data of the o r i g i n a l polysac-charide and P 1 has already demonstrated that of the three l a t -e r a l glucose units one i s a - and two areB -linked.The l a t t e r two are shown d i r e c t l y , b y the structures of A 2 and N^,to be those linked to galactose and mannose{by elimination,the t h i r d glucose must be a -linked to 0 - 4 of the glucuronic ac-i d residue, whence i t follows that one may now write: 91 —2- 1—2 GlcA - — 2 Gal ^ — 2 Man - — ° a 4 a 3 2 B 2 1 1 1 Glc Glc Glc There are two anomeric linkages unassigned,those of the galactose and the in-chain glucose residue;according to the spectra of the o r i g i n a l polysaccharide,one i s a and the other e . b) Polysaccharide P^ . P a r t i a l hydrolysis of polysaccharide P 1 yielded several a c i d i c oligosaccharides.Separation by gel-per-meation chromatography (see Figure IV.1) and further p u r i f i c a -t i o n of each f r a c t i o n by paper chromatography afforded 2 pure aldotriouronic acids ( A^ and A^ ) and a mixture of two aldo-tetraouronic acids( A^ ) . Nuclear magnetic resonance data i n conjunction with ana-l y t i c a l and methylation data(see TablelV.1.3).showed these compounds to be: A~ GlcA - — 2 Gal -—2. M a n 3 B a Ai, Glc - — 2 GlcA - — 2 Gal A e i s a 3 to 1 mixture of GlcA - — 2 G a l - — 2 M a n 1—2. G l c 5 B a a and Man - — 2 G l c - — 2 GlcA - — 2 G a l a B B From these results,the structure of K 6 0 capsular poly-saccharide can unambiguously be assigned as: Figure IV.1 Separation of the a c i d i c oligosaccharides from p a r t i a l hydrolysis of P.^  by g e l -permeation chromatography ( Bio-Gel P-2) 9 3 _ 1 Glc 1 2 GlcA -2 Gal----- Man 4 * 2 A 2 1 a 1 6 1 Glc Glc Glc This structure was further confirmed by a base-catalyzed uronic acid degradation,where the uronic acid and the termi-nal glucose attached to i t s position 0-4 were degraded, and p o s i t i o n 0-3 of galactose was subsequently ethylated. Conclusion. The experiments described above lead to the conclusion that the structure of the capsular polysaccharide of K l e b s i e l l a serotype K 60 i s based on the repeating unit — 2 D-Glcp -—2. D-GlcpA - — 2 D-Galp - — 2 D-Manp. R J. i B 2 | A 2| A 1 D-Glc£ l l D-Glcp. l l D-Glcp The structure i s of a unique pattern i n t h i s series of b a c t e r i a l polysaccharide having three separate side chains per repeating unit.Based on the s p e c t r a l data of the o l i g o -saccharides i s o l a t e d . i t was then possible to assign the sig-nals i n the spectra of the o r i g i n a l polysaccharide (see Table IV.1.1). TABLE IV.1.3 ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF K60 POLYSACCHARIDE. Oligosaccharide [ a J D Sugar analysis (as (water) a l d i t o l acetates) A ± +12 Glc(GlcA) (1.0 Gal (1.0 A 2 ~ Glc (GlcA) (1.0 Glc (1.0 Gal (1.0 A^ + 49 Glc(GlcA) (1.0 Gal (1.0 Man (1.0 A^ +10 Glc(GlcA) (1.0 Glc (1.0 Gal (1.0 A^ +79-4 Glc(GlcA) (1.0 Glc (1.0 Gal (1.0 Man (1.0 Methylation analysis (as a l d i t o l acetates) 2,3.4 - Glc (1.0) 2,4,6 - Gal (1.0) 2,3,4,6- Glc (1.0) 2,3.4 - Glc (1.0) 1,4,5,6- Gal- — 2,3.4 - Glc (1.0) 2,4,6 - Gal (1.0) 2,4,6 - Man (0.9) 2,3,4,6- Glc (1.0) 2,4,6 - Gal (1.0) 2,4 Glc (0.9) 2,3,4,6- Man (0.3) 2,4,6 - Glc (1.2) 2,4,6 - Man (1.0) 2,4,6 - Gal (1.3) 2,3,4 - Glc (1.0) 2,4 Glc (0.3) TABLE IV.1.3 (cont.) +31 Glc Man (2.0) (1.0) 1.2,4,5.6 2,3,4,6 3.^.6 G l < £ ~ Glc (1.0) Man (1.0) a b — and — The oligosaccharides were reduced p r i o r to methylation. 9 6 IV.1.4 Experimental. General methods.The instrumentation used f o r n.m.r., g . l . c , g.I.e.-m-s..infrared,c.d.,and measurements of o p t i c a l r o t a t i o n has been described previously (see Section III).Paper chroma-tography, gas - l i q u i d chromatography,gel-permeation and ion-ex-change chromatography were performed as described i n Section III. Preparation and properties of K 6 0 capsular polysaccharide. A culture of K l e b s i e l l a K 6 0 ( 4 4 6 3 / 5 2 ) was obtained from Dr. I.jtfrskov,Copenhagen,and was grown by the procedure described i n Section I I I . 7 . 1 . I s o l a t i o n and p u r i f i c a t i o n of the polysac-charide was done as described i n Section I I I . 7 . 1 . ( y i e l d : 8g df polysaccharide: from 1 2 . 5 1 of medium). The product had[a] D + 5 8 ° ( c 0.33.water).The purity of the polysaccharide was checked by electrophoresis.using a 1% s o l u t i o n on ce l l u l o s e acetate s t r i p s (Sepraphore 1 1 1 , 1 5 x 2 . 5 cm) at 300V for 9 0 min. and then development i n either A l c i a n Blue i n citrate-buffered ethanol (pH 4),or periodate-Schiff reagent 1^?.The polysaccha-r i d e had a M i 810.000 daltons and was monodisperse according w to gel-permeation chromatography. The ^H-n.m.r. spectrum of K 6 0 polysaccharide i n D 20 at 9 0 ° , r e v e a l e d signals corresponding to 7 anomeric protons at 6 5 . ^ 7 ( 2 H . J i f 2 2Hz)| 6 5 . 3 7 - ( l H , J 1 2 2Hz)}6 5-04 ( 1 H , J 1 > 2 7Hz ) j 6 4 , 8 5 ( 1 H , J 1 ( 2 7 H z ) ; 6 4 . 7 0 (1H,J 1 2 7 H z ) and 6 4 . 5 ^ (IH, ^1 2 ? H z ) ( s e e T a ° l e IV.1 fo r assignments ).The "^C spectrum showed s i x signals i n the anomeric region at 104 . 2 3,104 . 0 2 , 1 0 3 . 0 7 , 1 0 2 . 4 4 , 1 0 0 . 6 0 and 9 9 . 5 6 p.p.m. with the s i g n a l at 9 7 9 9 * 5 6 p.p.m. being twice the height of any of the other f i v e signals.Several signals between 6 1 . 1 - 6 2 . 2 due to C - 6 of hexose units were also present (see TablelV. 1 . 1 f o r assign -ment). Hydrolysis of the polysaccharide.Hydrolysis of a sample (20 mg) of K 6 0 polysaccharide with 2M TFA overnight at 9 5 ° , removal of the acid by successive evaporations with water , followed by paper chromatography ( solvents (A) and (B)), showed D-mannose,D-galactose,D-glucose,D-glucuronolactone , D-glucuronic acid and an aldobiouronic acid.Sugar analysis was performed as previously described (see page 7 1 ).The a l d i -t o l acetates of mannose.galactose and glucose were i d e n t i f i e d by g.l.c. (column (a) .program (a) ) and found to be present i n a r a t i o of 1 : 1 : 5 . Preparative g . l . c . (column (f) ) f o l -lowed by measurements of the c d . spectra showed both the mannitol and g l u c i t o l hexaacetates to be of the D configura-tion. Methylation analysis.The capsular polysaccharide ( 3 0 0 mg) i n the free acid form (obtained by passing the sodium s a l t through a column of Amberlite IR-120 (H +) resin).was d i s s o l -ved i n 3 0 mL of anhydrous DMS0 and methylated by the Hakomori procedure (see page 7 2 ).Methylation was incomplete as shown by hydroxyl absorption i n the i . r . spectrum.A subsequent Pur-die (see page 7 3 ) treatment afforded complete methylation, ( y i e l d , 2 7 2 mg).Carboxyl-reduction of the f u l l y methylated polysaccharide with LiAlH^ i n anhydrous oxolane (see page 7k \ 98 hydrolysis (of a portion) with 2M TFA.and conversion into a l d i t o l acetates gave a mixture of p a r t i a l l y methylated a l d i -t o l acetates which was analyzed by g.l.c. on columns (b),(c) and (d) and by g.1.c.-m.s.(see TablelV.1.2.column I).The methylated and carboxyl-reduced polysaccharide ( 3 0 mg) was further methylated by the Hakomori method ;and a f t e r hydro-l y s i s .reduction and acetylation,analysis of the 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. on column (d) showed the replacement of 2-0-methylglucose by 2 ,6-di-0-methylglucose(see TablelV.1.2,column I I ) . P r e p a r a t i -ve g . l . c . on column (f) ( 2 1 5 ° isothermal)afforded a sample of 4 , 6-di-0-methylgalactitol acetate whose positive c d . curve indicated that galactose has the D-configuration. Carbodiimide reduction of capsular polysaccharide K 6 0 . A sample of K 6 0 polysaccharide ( Na +salt,1.02 g) was d i s s o l -ved i n 100 mL of water.l-Cyclohexyl - 3-(2-morpholinoethyl)-carbodiimide metho-p_-toluenesulfonate ( CMC,4.0 g) was added. This corresponds to ten times the equivalent of carboxylic acid i n the polysaccharide,based on one glucuronic acid r e -sidue per sequence.As the reaction proceded, the pH was main-tained at 4 . 7 5 by t i t r a t i n g when necessary with hydrogen chlo-r i d e (0. 10N) solution.When the consumption of HC1 ceased ( 6 . 7 0 mL).approximately two hours later,an aqueous so l u t i o n of sodium borohydride (2M) was added slowly.Bubble formation was minimized by a continuous flow of a i r blowing on the sur-face of the sdlution.A drop of 1-octanol was added p e r i o d i -c a l l y to control the amount of foam.At the same time the so-9 9 dium borohydride was added,the pH of the s o l u t i o n was main-tained at 6 . 5 with HC1 (4.OK).A t o t a l of 3 0 0 mL of NaBH^ so-l u t i o n was added over a period of two hours.The s o l u t i o n was concentrated and dialyzed against tap water during 48 h. and freeze-dried.A second treatment was carried out.The consump-t i o n of HC1(0.1 N )was 2.80 mL.A t o t a l of 780 mg of product was recovered a f t e r drying. A sample of the reduced polysaccharide (10 mg) was hydro-lyzed overnight with 2M TFA on a steam-bath and a f t e r conver-sion of the sugars into a l d i t o l acetates,g.1.c. showed man-n i t o l , g a l a c t i t o l and g l u c i t o l hexaacetates i n a r a t i o 1:1 : 5 , i n d i c a t i n g complete reduction of the uronic acids. Estimation of the equivalent weight of the polysacchari-de from the consumption of HC1 gives a value of 1100 ( theo-r e t i c a l , 1148 ). Periodate oxidation of carboxyl-reduced K 6 0 polysaccha-r i d e .A sample ( 6 5 mg) of the carboxyl-reduced K 6 0 polysac-charide was dissolved i n 1 5 mL of water.A s o l u t i o n of NalO^ (0.1 M,10 mL) was added.The reaction was allowed to proceed at 4° i n the dark.The periodate consumption was followed on 168 1 mL aliquots by the Fleury-Lange method and reached a plateau a f t e r 140 h. ( 4 . 9 5 moles of periodate per mole of polysaccharide).Ethylene g l y c o l ( 3 mL) was added,the p o l y a l -dehyde was dialyzed overnight.reduced with sodium borohydri-de (0.2 g).neutralized with 5 0 $ a c e t i c acid,dialyzed and freeze-dried to y i e l d the polyalcohol ( 4 5 mg).This material was further oxidized with periodate (20 mL of 0 . 0 5 M NalO^). 100 Periodate consumption was constant a f t e r ?2 h. (0.95 moles of periodate per mole of polysaccharide).Re-isolation gave the polyalcohol (35 mg). A sample of the polyalcohol (5 mg) was hydrolyzed with 2M TFA overnight on a steam-bath.Reduction and acetylation gave (on g.l.c.) mannitol,galactitol and g l u c i t o l hexaaceta-tes i n a r a t i o 1:1:2.The r e s t of the material (30 mg) was methylated by the Hakomori procedure.One t h i r d of the methyl-ated product was hydrolyzed with 2M TFA overnight on a steam-bath tconversion of the p a r t i a l l y methylated sugars into the p a r t i a l l y methylated a l d i t o l acetates and g.l.c. on column (d), showed the presence of 2 , 4 , 6-tri - 0-methylglucose , 4 , 6 -di-0-methylmannose,2,6-di-0-methylglucose and 4,6-di-0-methyl-galactose i n equimolar proportions.The r e s t of the methylated product was treated with $0% a c e t i c acid f o r one hour on a steam-bath.The acid was removed by several evaporations with water,the product was dissolved i n dioxane-ethanol(3«l»10 mL) and reduced with NaBH^ (50 mg).After elimination of the NaBH^ i n the usual manner,the material was dried and then ethylated by the Hakomori procedure.Hydrolysis,reduction and acetyla-t i o n gave a mixture of p a r t i a l l y methylated and p a r t i a l l y methylated ethylated a l d i t o l acetates which could not be se-parated by g. l . c . on any of the columns available.From the retention times i t was concluded that the mixture was com-posed of 3-or 4 - 0-ethyl-2 , 6-di - 0-methylglucose,2 , 4 , 6-tri - 0 -methylglucose and the 2-or 3 -0-ethyl -4,6-di -0-methyl deriva-t i v e s of both mannose and galactose. 101 Periodate oxidation of K 60 capsular polysaccharide.A solution of K 6 0 polysaccharide (1.0 g) i n water ( 1 5 0 mL) was mixed with 0.1M NalO^ and 0.4M NaClO^ ( 1 5 0 mL).The reaction was allowed to proceed at 4° i n the dark.The periodate con-sumption and release of formic acid were followed on 1 mL aliquots by the Fleury-Lange method and t i t r a t i o n against 0.001N NaOH,respectively.Periodate consumption and formic ac-i d production reached a plateau a f t e r 140 h. (4.8 moles of periodate and 1 . 7 moles of formic acid per mole of polysac-charide ) .Ethylene g l y c o l (10 mL) was added,the polyaldehyde was dialyzed overnight,reduced with NaBH^ (1 . 5 g) neutralized with 5 0 % acetic acid,dialyzed and freeze-dried to y i e l d the polyalcohol (680 mg).This material was further oxidized with periodate ( 200 mL of 0.05M NalO^ and 0.2M NaClO^ ).Periodate consumption was constant a f t e r 7 0 h. ( 0 . 7 moles of periodate per mole of polysaccharide).Re-isolation gave the polyalcohol ( 6 6 0 mg). This product was hydrolyzed (TFA, 0.5M ) at room temper-ature f o r 24 h.Paper chromatography i n solvent (A) showed the presence of one mobile compound id e n t i f i e d , b y comparison with a standard as glycerol,and a polymeric p r o d u c t j d i a l y s i s afforded 5 5 0 mg of polymeric material ( P 1 ) . T o t a l hydrolysis of P 1 and examination on paper (solvents (A) and (B)) showed D-mannose,D-galactose,D-glucose and D-glucuronic acidjan aldobiouronic acid was also present.Sugar analysis,as pre-viously described,gave (g.1.c..column (a)) mannitol,galacti-t o l and g l u c i t o l hexaacetates i n a r a t i o of It 1:2 ,where one 102 of the g l u c i t o l units i s derived from the glucuronic acid. Methylation of P-^  ( 2 5 mg) by the Hakomori procedure and one Purdie treatment afforded a f u l l y methylated polysaccharide with no hydroxyl absorption i n the infrared.The f u l l y methyl-ated material (10 mg) was reduced with LiAlH^,hydrolyzed with 2M TFA for 10 h,reduced with NaBH^ and acetylated.The p a r t i a l -l y methylated a l d i t o l acetates were analyzed by g.l. c . and g.l.c-m.s. on columns (b) and (d) with the r e s u l t s given i n TablelV. 1.2,column III.A portion (10 mg) of the methylated polysaccharide was hydrolyzed with 2M TFA f o r 5 h and sepa-rated into a c i d i c and neutral components on ion-exchange r e -s i n (Bio-Rad AGl-X2).The a c i d i c f r a c t i o n was refluxed i n 3% methanolic HC1,reduced with NaBH^ i n anhydrous methanol, hydrolyzed with 2M TFA f o r 3 h,reduced with NaBH^ and a c e t y l -ated.The r e s u l t s obtained by g. l . c . i n column (b) showed the presence of 2,4 , 6-tri-O-methylgalactose and 2,4-di-0-methyl-glucose i n a r a t i o of 1.0:1 .3. The ^H-n.m.r. spectrum of P^ showed signals signals at 6 5 . 3 3 (1H,J 1 2 2Hz)»6 5 . 2 7 ( 1 H , J 1 ( 2 2Hz);6 4.83 (1H,J 1 2 7Hz) and 6 4 . 7 7 (1H,J 1 2 7Hz) (see spectrum No.6).The ^ ^ g p e c t r u - i showed signals at 104 . 19 ( 1 C ) , 1 0 3 . 3 4 (IC), and 101,42 (2C)p.p.m i n the anomeric region,and four signals at 84.11 ,83.02,80 .13 and 7 6 . 6 0 p.p.m. due to C - 3 of the four hexoses.For assign -ments see Table IV.1.1 P a r t i a l hydrolysis of K 6 0 capsular polysaccharide.A portion of K 60 polysaccharide ( 5°0 mg) was dissolved i n TFA (0.5M,100 mL) and the sol u t i o n was refluxed for 3 h.The acid 1 0 3 was removed by evaporation,and a c i d i c and neutral components were separated on a column of Bio-Rad AGl-X2.The a c i d i c f r a c -t i o n ( 1 3 0 mg) was separated on Bio-Gel P -2 to give 2 5 mg of pure aldobiouronic acid(A 1) and 1 0 mg of pure aldotriouroriic acid ( A 2 ) . The aldobiouronic acid A 1 had R^.^0.28 (solvent (A)) and [ a ] D + 1 2 ° ( c 1 . 0 5 ,water).Sugar analysis showed g l u c i t o l (from uronic acid) and g a l a c t i t o l hexaacetates i n equimolar amounts. The aldotriouronic acid A 2 had R Q I C 0 ' 1 ^ (solvent (A)) and examination on paper following hydrolysis indicated (solvents (A) and (B)) glucose.galactose and an aldobiouronic acid.Acid A 2 was reduced with aqueous NaBH^,methylated,and the ester function was reduced with LiAlH^ i n anhydrous oxolane.Hydro-l y s i s and g . l . c . analysis ( column (b)) showed a component with R^ 0.8 together with peaks corresponding to 2 , 3 , 4 , 6 -tetra-O-methylglucose (R^ 1 . 0)and 2 , 3 , 4 - t r i - 0 - m e t h y l g l u c o s e (R_-t 1 . 9 ) .Mass spectrometry showed that the fas t e s t component was l , 4 , 5 , 6 - t e t r a - 0 - m e t h y l g a l a c t i t o l . The neutral f r a c t i o n was separated on Bio-Gel P - 2 to give i n a pure state,a trisaccharide ( N l t 3 0 mg) having E ( j i c 0 ' 3 2 (solvent (A)) and[ a] D + 3 1°(c 0 . 3 2 ,water).Analysis of gave (g.l.c.) mannitol and g l u c i t o l hexaacetates i n a r a t i o l t 2 . Methylation analysis of reduced N-^  gave (columns (c) and (d)) l , 2 , 4 , 5 , 6 - p e n t a - 0 - m e t h y l g l u c i t o l (R _ t 0 . 4 2 , ( d ) ) , 2 , 3 , 4 , 6 - t e t r a -O-methylglucose (R ^ . 1 . 0 0 ) and 3 , ^ , 6-tri - 0-methylraannose(R^.l.6 6 ) The n.m.r. data for A^,A2 and are presented i n Table IV. 1 . 1 104 P a r t i a l hydrolysis of polysaccharide P^ from periodate  oxidation. A sample of P^ (350 mg) was hydrolyzed with IM TFA (75 mL,lh) and the r e s u l t i n g oligosaccharides were separated into a c i d i c and neutral f r a c t i o n s on a column of Bio-Rad AG1-X2.The a c i d i c f r a c t i o n was separated by g e l chromatography on a column of Bio-Gel P-2. Three f r a c t i o n s were c o l l e c t e d (see Figure IV.1) corresponding to (l)the-aldobiouronic-acid(43 mg), (2)a mixture of two,trisaccharides ( 3 0 mg),and (3) a mixture of two tetrasaccharides ( 3 2 mg). Fraction 2 was p u r i f i e d by paper chromatography(solvent (C)) and two pure aldotriouronic acids were isolatedtA^ and A^) Oligosaccharide A^ (7 mg) had [ a ] D+49°(c 0.7,water) and -Wldobi 0 , 6 7 (solvent (C)).Proton n.m.r. showed signals at 6 5-32 ( l H , J 1 > 2 2 H z ) , 6 5.20 (0.5H,J ± 22Hz), 64.94 (0.5H,J ±^ 22Hz) and 6 4.78 (1H,J 1 2 7 H z ^ in d i c a t i n g onea-and one B-linkage and a reducing mannose (low coupling constant of the reducing signal).Oligosaccharide A^ (14 mg) had [ a] ^ +I0°(c 1.1,water) and Rgidoiji °.45 (solvent (C)).Sugar analysis of t h i s material, as previously described,showed on g. 1 . c . . g a l a c t i t o l and gl u -c i t o l hexaacetates i n a r a t i o 1»2.Methylation analysis gave 2,3,4,6-tetra-0-methylglucose,2,4,6-tri-O-methylgalactose and 2,4-di-0-methylglucose i n approximately equimolar amounts. Proton n.m.r. showed signals at 6 5.28 (0.4H,J 1 22HZ), <5 4.80 ( m t J 1 27Hz), 6 4.77 (1H,J 1 27Hz) and 64.64 ( 0 . 6 ^ ^ 27Hz). 13 From the -T spectrum of the mixture of aldotriouronic acids i t i s possible to assign signals to both oligosaccharides,by comparison to other oligosaccharide spectra (see spec.NoJLO 1 0 5 and TablelV. 1.1 f o r assignments ). Fraction 3 was p u r i f i e d by paper chromatography (solvent (C)) and a p a r t i a l l y p u r i f i e d tetrasaccharide was i s o l a t e d (A^,18 mg) having[a] D + 7 9°(c 1.7»water).Sugar analysis of t h i s material,as previously described,showed on g . l . c . mannit-o l , g a l a c t i t o l and g l u c i t o l hexaacetates i n a r a t i o Is 1:2. Methylation analysis of the r e d u c e d oligosaccharide gave on g.l.c. (column ( b ) ) , l , 2 , 4 , 5 . 6 - p e n t a - 0 - m e t h y l g l u c i t o l , l , 2 , 4 , 5 » 6 -penta-0-methylgalactitol,2 ,3.4,6-tetra-0-methylmannose,2,4 ,6 -tri-O-methylglucose,2 , 4 , 6-tri-0-methylmannose,2 , 4 , 6-tri-O-methylgalactose ,2 ,3 .4-tri-0-me thy lglucose and 2,4-di-0-methyl-glucose i n a r a t i o of 0 .3:1.0:0 .3:0 .3:1.0:1.0:1.0:0 .3 .show-ing that i t i s a mixture of two aldotetrauronic acids i n a r a t i o 3 * 1 -The "'"H-n.m.r. spectrum showed signals at <5 5 . 3 1 ( - H , J 1^ 22Hz), 6 5 . 2 5(1.4H,J 1 22Hz) and 6 4 . 7 7 ( 1 . 6 H , J l f 2 broad) and the 13C-n.m.r. spectrum at 104 . 51,104.28 , 103.48,101 . 51,101 . 35 , 9 6 , 7 6 , 9 4 . 7 and 93-04 p.p.m.From the r e l a t i v e i n t e n s i t i e s . a s -signments could be made f o r both oligosaccharides. Uronic acid degradation.Methylated and c a r e f u l l y dried K 6 0 polysaccharide was subjected to a uronic acid degradation as described before (see page75 ).The d i r e c t a l k y l a t i o n was ca r r i e d out with ethyl iodide instead of methyl iodide.Hydro-l y s i s of the product.reduction and a c e t y l a t i o n gave r e s u l t s shown i n TablelV. 1.2column I I . 106 IV.2 S t r u c t u r a l inves t i g a t i o n of K l e b s i e l l a serotype K 26  capsular polysaccharide. IV.2.1 Abstract. The structure of the capsular polysaccharide from K l e b s i e l l a K 26 has been determined using the techniques of methylation,periodate o x i d a t i o n , p a r t i a l hydrolysis and B -1 13 elimination. H- and ^C-n.m.r. spectroscopy was used to es-t a b l i s h the nature of the anomeric linkages and to i d e n t i f y oligosaccharides obtained by the d i f f e r e n t degradative tech-niques used. The polysaccharide i s shown to comprise the heptasaccha-ride repeating unit below. — 2 D-Galp_ 1 2 B D-GlcpA 41 * 1 _ 1 D-Manp. 1 2 D-Manp. - — a a a a 11 D-Gl 61 C £ II D-GL 41 1 D-i 1 0 ? I V . 2 . 2 Introduction. K l e b s i e l l a serotype K 2 6 i s one of 1 7 strains whose cap-sular polysaccharides are composed of D-glucuronic acid,D-gal actose,D-glucose and D-mannose.Eight of these polysaccharides have 1-carboxyethylidene substituents and i n t h i s subgroup the structures of K l e b s i e l l a K 7 l 6 9 , K 1 3 1 7 ° , K 3 0 1 7 1 , K 3 1 1 7 2 and K ^ 1 ^ are known. The structure of K l e b s i e l l a K26 polysaccha-r i d e , a member of t h i s subgroup,is presented here;the serotype K35 and K69 remain to be examined. This polymer i s shown to be based on a heptasaccharide repeating uni t (four plus three type) and,in t h i s respect,is s i m i l a r to the polysaccharide i s o l a t e d from K ^ l ^ ^ . I n the l a t t e r case.however,the D-glucuronic acid residue i s i n the side-chain and the branch point i s a unit of D-galactofuran-ose.The structure of the polysaccharide from K l e b s i e l l a K26 is,therefore,unique i n t h i s series. I V . 2 . 3 Results and discussion. Composition and n.m.r. spectra. The i s o l a t i o n and p u r i -f i c a t i o n of the polysaccharide was conducted as previously described.The p u r i f i e d product obtained a f t e r Cetavlon pre-c i p i t a t i o n had [ a ] D + 8 0 ° , w h i c h compares well to the calculated value of + 6 8 ° using Hudson's Rule of Isorotation.The mole-cular weight of the polysaccharide was determined by g e l chromatography to be 1 x 1 0 ? daltons jthe equivalent weight was 6 5 0 . Paper chromatography of an acid hydrolyzate of the poly-saccharide showed galactose,glucose.glucuronic acid,mannose 108 and an aldobiouronic acid.Determination of the sugars,as a l d i t o l acetates,of the carboxyl-reduced polysaccharide gave mannose.galactose and glucose i n a r a t i o of 1 . 0 j l . 0 t l . 5 • Glucose and mannose proved to be of the D-configuration by c i r c u l a r dichroism (c.d.)measurements of the a l d i t o l aceta-tes. Galactose and glucuronic acid were also assigned the D-configuration by c d . measurements of p a r t i a l l y methylated derivatives i s o l a t e d subsequently. The ^H-n.m.r. spectrum of the native polysaccharide was recorded i n D^O at 9 0 ° with acetone as i n t e r n a l standard.The spectrum exhibits the presence of seven anomeric protons cor-responding to four a - and three B - linkages;also one 1-carboxyethylidene acetal per repeating unit was detected.The "^ H-n.m.r. of the depyruvylated polysaccharide showed that both mannoses should be a -linked as the anomeric protons corresponding to the g -linkages exhibit large coupling con-stants (?Hz) which do not correspond to B - l i n k e d mannoses (see spectrum Nol3,and Table IV. 2.1). The ^C-n.m.r. spectrum confirmed the proton r e s u l t s and from the chemical s h i f t of the methyl group of the a c e t a l ( 2 5 - 7 p.p.m.) i t i s possible to assign the R configuration to the acetal carbon.Precise assignment of the signals was achieved a f t e r studying ^H-13 and -^C-n.m.r. spectra of oligosaccharides obtained by se-l e c t i v e degradative techniques (see Table IV.2.1) Methylation analysis.Methylation of K26 polysaccharide followed by reduction of the uronic e s t e r , h y d r o l y s i s , d e r i -v a t i z a t i o n as a l d i t o l acetates, and g.l.c.-m.s. analysis gave TABLE IV.2.1 N.M.R. DATA FOR KLEBSIELLA K26 CAPSULAR POLYSACCHARIDE AND DERIVED POLY- AND OLIGO-SACCHARIDES . H-n.m.r. Compound =-1,2 (Hz) Integral  proton Assignment Spectrum No. A-GlcA- -Man-OH a GlcA-—-Man-—-Man-OH a a GlcA-—-Wan-—^arr-—-<Gal-OH 5.32 5.20 4 .92 5-37 5.34 5.18 5.08 4.94 5.34 5 .29 5 . 1 7 5 . 0 7 4.64 2 . 5 2 2 2 .5 2 2 2 2 .5 2 2 2 7 } 1 1.0 0.6 0.4 2 .0 0.3 0.7 0 .3 2.0 0.3 1.0 0.7 G l c A - — a 3-Man OH a 3-Man-r-OH 3-Man-GlcA-unknown 3-Man OH a 3-Man—r-OH GlcA a 3-Man 3-Gal OH 2- Man-3- Gal-14 16 18 -OH TABLE IV.2.1 (cont.) G l c - — ^ l c - O H N-G a l 1 — ^ G l c ^ - ^ l c - O H N, G a l - — ^ G l c A - — \ a . n - — ^ G l y SH - ^ G a l - — ^ G l c A - — ^ M a n - — ^ a n 1 -6 Zj. a a a a 1 Glc 11 Glc H Gal 3 0.4 6 - G l c — O H 8 0.6 6-Glc-j-OH 20 8 1.0 Glc-3 0.4 6-Glc OH 7.5 0.6 6 - G l c — O H 7-5 0.4 4-Glc 1 7^Glc OH 22 H 7.5 0.6 4-Glc~%Jlc—£—OH ° 7-5 1.0 G a l — p 3 1.0 2-GlcA a 2 1.0 3-Man— 23 7 1.0 G a l — { 2.0 J 2-Glc 4 a 6-Glc- a 2 1.0 3-Man-et 2 1.0 2-Man-7.5 1.0 Gal-7.5 1.0 4-Glc-7.5 1.0 3-Gal-a TABLE IV.2.1 (cont.) - ^ G a l i — ^ G l c A — 2 3 ^ 1 2 1 Man-—%Ian— l l Glc 4 1 Gal 6 4 X H"3C C 0 2 H 5.50 5.28 5.08 4.7-4.5 1.65 13 C-n.m.r. Compound Chemical s h i f t  (P.P.m.) A 1 101.^2 94. 82 9^-33 102.86 2.0 1.0 1.0 3-0 3-0 J 4-GlcA I 6-Glc V a 3- Man a 2- Man a G a l — 4- G l c — 3- Gal 3 CH~—C 3 I C0 2H Assignment Spectrum No. GlcA a 3-Man OH 15 3 3-Man OH ci 3-Man TABLE IV. 2 . 1 (cont.) 1 0 1 . 3 6 9 3 . 5 6 9 3 . 3 5 A 3 1 0 3 . 0 6 1 0 1 . 3 3 9 7 . 2 3 9 5 . 3 8 9 5 - 1 1 9 3 . 1 0 N x 1 0 3 . 5 0 96.81 9 2 . 9 5 SH 105.41 1 0 0 . 9 2 1 0 0 . 2 2 K26 capsular poly- 105.41 saccharide -^Q3 7 0 103.24 GlcA 2-Man—^-0H 2-Man OH a 3-M? a GlcA 3-Gal—---OH 2-Marr-—-Gal-2-Man-=—^Gal-3-Gal OH a G l c — 6 - G l c — OH 6-Glc OH Gal--Man-2-GlcA-3- n a TABLE IV.2.1 ( c o n t . ) 101.00 6 - G l c — 100.76 2-GlcA— 4 99.87 2-Man— 25.77 CH^—C ( R c o n f i g n . ) C0 2H 114 the values shown i n Table IV.2.2 .column I.These r e s u l t s i n -dicate that the polysaccharide consists of a heptasaccharide repeating unit with a branch on the glucuronic acidithe t e r -minal g l y c o s y l residue i s a unit of galactose which has a 1-carboxyethylidene group present as an ac e t a l spanning 0-4 and 0 - 6 .Methylation of the carboxyl-reduced polysaccharide (see Table IV.2.2,columnII) showed the disappearance of the 3-0-methylglucose and the formation of 3.6-di-0-methylglucose confirming the glucuronic acid as the branch point.Removal of the modified acetal (reduction,methylation) and remethyl-a t i o n (Table IV.2.2,column III) confirmed the l o c a t i o n of t h i s residue by the formation of 2,3»4,6-tetra-0-methyl-galactose and the disappearance of 2,3-di-0-methylgalactose. P a r t i a l h y d r o l y s i s . P a r t i a l acid hydrolysis of the native polysaccharide was followed by separation of the a c i d i c and neutral frac t i o n s by ion-exchange chromatography.The neutral f r a c t i o n contained monosaccharides and a disaccharide (N^)f which were separated by gel-permeation chromatography.The a c i d i c f r a c t i o n contained three a c i d i c oligosaccharides (A^, Ag and A^) which were also separated by gel-permeation chro-matography (see Figure IV.2 ).On the basis of t h e i r n.m.r. spec t r a l data (see Table IV.2.1) and t h e i r a n a l y t i c a l and methylation data (see Table IV.2.3) the structures of these compounds were shown to be: A, GlcA -—2. Man a A 0 GlcA -— ^ M a n I —2 M a n -• a a 1 1 5 t e t r a -5 25 50 E l u t i o n volume (mL) Figure IV.2 Separation of a c i d i c oligosaccharides from p a r t i a l hydrolysis of K l e b s i e l l a K26 polysaccharide by gel-permeation chromatography (Bio-Gel P-2). TABLE IV.2.2 METHYLATION ANALYSIS OF K26 CAPSULAR POLYSACCHARIDE AND DERIVED PRODUCTS. Methylated sugars - Relative retention times — Mole % °-(as a l d i t o l acetates) SP-2340- ECNSS-M OV-225 I ^ II I l l 170° 170° 2,3,^,6 - Gal 1.14 1.25 1.19 — — ' 14.7 3.^.6 - Man 1.47 1.95 1.82 12.4 13.7 13.3 2,4,6 - Man 1.47 2.08 1.90 17. 8 16.0 14.0 2,4,6 - Gal 1.51 2.29 2.03 15.2 15.3 12.2 2,3.^ - Glc 1.62 2.50 2.26 15. k 17.5 16.8 2,3.6 - Glc 1.70 2.50 2.26 16.3 17.9 17.2 3.6 - Glc 2.04 4.40 3.70 — 11.2 11.7 2,3 - Gal 2.24 5.64 4.70 11.4 8.4 — 3 - Glc — 9.49 7.^0 11.5 — — - and - , as i n Table IV.1.2 .- Relative retention times referred to 2,3,4,6-Glc as 1.00. - Programmed at 160° for 4 min and then 2%>in to 230°. - I , o r i g i n a l capsular polysaccharide; II.methylation of the carbodiimide reduced capsular polysaccharide; I I I , methylation of the deacetalated methylated reduced capsular polysaccharide. 117 A 0 GlcA - — 2 M a n I—A M a n 1—1 j a a a N, Glc - — - Glc 1 6 Gal The aldotetraouronic acid (A^) obtained from p a r t i a l hy-d r o l y s i s has been previously i s o l a t e d from other K l e b s i e l l a capsular polysaccharides, ^ l ^ ^ a n d K 7 4 1 ' ^ ; gentiobiose (N^) has been also i s o l a t e d by p a r t i a l hydrolysis from K ^ l 1 ^ and Two possible structures are consistent with the r e s u l t s thus f a r obtained,either A or B : 2 l i ) G l c A I_Ji M a n 1_1 M a n G a l 1_1 G l c 1 _ £ de I-4(2) a a a B 1 Gal V or B 214) GlcA -—a M a n 1—2 M a n 4(2j 1 Glc 6 1_2 Gal 1| Glc 41 11 Gal v 118 In order to determine the possible structure,the carboxyl reduced polysaccharide was methylated,the modified acetal was s e l e c t i v e l y removed by mild acid treatment and the free hy-droxyl groups thus obtained were oxidized l 6^(DMSO/TFAA) to the dicarbonyl derivative.This product was subjected to alka-l i n e degradation and rernethylated.The formation of 2 , 3 . 4 , 6 -tetra-O-methylglucose and the disappearance of 2 , 3 . 6 - t r i - 0 -methylglucose indicated that the structure of K 2 6 polysaccha-ride i s B. Periodate oxidation,followed by Smith hydrolysis and r e -duction of the product gave an oligomer which on the basis of the n.m.r. and methylation analysis,was shown to be s Gal - — - GlcA -—2. Man - — - Glycer o l 3 a a This r e s u l t i s conclusive evidence i n favour of structure B, but s t i l l leaves two anomeric linkages unassigned ( one a and the other 3 ) Selective acid hydrolysis.Treatment of the polysaccharide with very d i l u t e acid (0.01M TFA ,95°»5h) and d i a l y s i s against d i s t i l l e d water afforded a non-dializable polymeric material and a dialyzate. The dialyzable material was shown by paper chromatography to be composed of galactose,a disaccharide ( i d e n t i c a l to N.^ ) and a trisaccharide (N 2).0n the basis of the n.m.r. spectral data ( see Table IV.2.1) and a n a l y t i c a l and methylation anal-y s i s (see Table IV.2.3) the structure of N 0 was shown to be: TABLE IV.2 . 3 ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF K26 POLYSACCHARIDE Oligosaccharide N-N, (water) + 64c + 79' +106c + 8 . 8 ° + 22 v Sugar analysis (as a l d i t o l acetates) (Molar proportions) Man (1) Glc(GlcA) (1) Man Gal Glc (2) Glc(GlcA) (1) Gal (1) Man (2) Glc(GlcA) (1) Glc (1) (2) Methylation analysis (as a l d i t o l acetates) (Molar proportions) 2,3,4 -Glc ( 1) 2,4,6 -Man ( 0.9) 2,3,4 -Glc ( 1) 2,4,6 -Man ( 1) 3,4,6 -Man ( 0.9) 2,3,4 -Glc ( 1) 2,4,6 -Man ( 1) 3,4,6 -Man ( 0.9) 2,4,6 -Gal ( 0.7) 2,3,4, 6 -Glc (1) 2,3,4 -Glc (0.9) 2,3,4, 6 -Gal (1) 2,3,6 -Glc (1) 2,3,4 -Glc (0.9) 120 N 0 Gal 1 — - Glc - Glc The structure of' N 2 demonstrates that the terminal galac-tose unit has the 8-configuration and thus,the side chain i s a -linked to the glucuronic acid.Methylation analysis of the polymeric material gave 3»^-di-0-methylglucose which i s d e r i -ved from the glucuronic a c i d , a f t e r removal of the side chain attached to po s i t i o n 4. The sum of these experiments permits the complete struc-ture of the polysaccharide to be written,but confirmation was made by carrying out a base-catalyzed uronic acid degradation. On methylation of the degraded product.hydrolysis.conversion into a l d i t o l acetates and g.l . c . a n a l y s i s . i t was possible to observe the formation of 2 ,3»4,6-tetra-Q-methylmannose d e r i -ved from methylation at po s i t i o n 3 of the mannose of the aldo-biouronic acid.A decrease was observed i n the amount of 2 , 3 . 4 -tri-O-methylglucose due to the degradation of th i s sugar on l i b e r a t i o n and exposure to the base. Conclusion. The structure of the capsular polysaccharide from K l e b s i e l l a serotype K26 i s thus based on the heptasac-charide repeating u n i t shown.This structure i s consistent 21 with the analysis reported by Nimmich and with the serolo-12 g i c a l cross-reaction observed with K l e b s i e l l a K21 .The l a t -t e r i s due to 4 , 6 -0-(l-carboxyethylidene)-D-galactopyranosyl units present i n both polymers. 121 IV.2.4 Experimental. General methods. The instrumentation used f o r n.m.r., g.1.c.-m.s.,infrared,c.d. and measurement of o p t i c a l r o t a t i o n has been described previously (Section III).Paper chromatogra-phy, ga s - l i q u i d chromatography,gel-permeation and ion-exchange chromatography were performed as described i n Section I I I . Preparation and properties of K26 capsular polysaccharide. A culture of K l e b s i e l l a K 26 (5884) was obtained from Dr. I. 0rskov,Copenhagen,and was grown by the procedure described i n Section III.7 . 1 (page 69 ).The polysaccharide was i s o l a t e d and p u r i f i e d as described i n Section III.7 . 1 . Y i e l d 12 g of poly-saccharide from 1 2 . 5 L of medium.The product had[al D+80 ( c O . 2 5 , water).The pu r i t y of the polysaccharide was determined by g e l -permeation chromatography and an average molecular weight of n 1 xiO' daltons was obtained. The "^H-n.m.r. spectrum on the o r i g i n a l polysaccharide i n DgO at 90° revealed signals corresponding to 7 anomeric pro-tons at 6 5 . 5 0 (2H,s), 6 5.28 (lH,s), $ 5 . 0 8 (lH,s) and signals between 6 4. 7 and 4 . 5 corresponding to 3H.At<5 I .65 (3H,s) there was a s i g n a l corresponding to the methyl group of an a-c e t a l linked pyruvic acid.On removal of the acetal by mild hydrolysis ( 0.1M TFA,30 min.,95°) a better 1H-spectrum was obtained (see Table IV.2.1 and spectrum N 0 J . 3 ) • ^ C-n.m.r.spec-trum showed 6 signals i n the anomeric region at 105.41,103.70, 103.24,101.00,100.76 and 99.87 P.P.m. with the s i g n a l at IO3.7 being twice the height of any of the other f i v e signals.A s i g -n a l at 25.77P.P-ni. corresponds to the pyruvate (TablelV.2.1) 122 Hydrolysis of the polysaccharide.Hydrolysis of a sample of K26 (20 mg) with 2M TFA overnight at 95°.removal of the acid by succesive evaporations with water .followed by paper chroma-, tography (solvent(A) and (B)) showed D-mannose,D-galactose,D-glucose,D-glucuronic acid and an aldobiouronic acid.The quan-t i t a t i v e sugar analysis of the carboxyl-reduced polysaccharide was performed as previously described (see page 7 1 ) . T h e a l d i -t o l acetates of mannose.galactose and glucose were i d e n t i f i e d by g.l.c. and found to be present i n a r a t i o of 1.0:1.0:1.5 . Preparative g . l . c . followed by measurement of the c d . spectra showed both mannitol and g l u c i t o l hexaacetates to be of the D-configuration. Methylation analysis.The capsular polysaccharide ( 2 9 0 mg) i n the free acid form (obtained by passing the sodium s a l t through a column of Amberlite IR-120(H +)resin) was dissolved i n 40 mL of dry DMS0 and was methylated by the Hakomori pro-cedure (see page 7 2 ).The product ( 3 0 0 mg).recovered a f t e r d i a l y s i s against tap water,showed complete methylation (no hydroxyl absorption i n the i . r . spectrum).Carboxyl-reduction of the f u l l y methylated polysaccharide ( 9 0 mg) with LiAlH^ i n anhydrous oxolane (see page 7k ).hydrolysis with 2M TFA and conversion into a l d i t o l acetates gave a mixture of p a r t i a l -l y methylated a l d i t o l acetates which was analyzed by g . l . c and g.l.c.-m.s. on columns (b) and (c) (see Table IV.2.2, column I).A good separation of 2,3»^-tri-0-methylglucose (R t1.62)and 2 , 3 , 6-tri-0-methylglucose ( R ^ l . 7 0 ) was obtained with column (a) programmed at 160° for 8 min. and then at 1 2 3 Z°/min up to 230° f o r 32 min.They were also characterized as the t r i m e t h y l s i l y l derivatives of the a l d i t o l s (column (e), 1?0° ).From preparative g . l . c . (column ( f ) f 2 1 5 ° ) 2 ,3-di-O-methylgalactose and 3-0-methylglucose were isolated.The galac-t i t o l derivative showed a p o s i t i v e c d . curve i n d i c a t i n g that the galactose has the D-configuration,and the g l u c i t o l d e r i -vative, showed a negative c d . curve,confirming the 3-0-methyl-D - g l u c i t o l pentaacetate 6 5. Carbodiimide reduction of capsular polysaccharide K 26. A sample of K26 polysaccharide ( Na*salt,640 mg) was dissolved i n 150 mL of HgO. l-Cyclohexyl - 3-(2-morpholinoethyl)-carbodi-imide metho-p_-toluenesulfonate ( CMC ,4.0 g) was added.As the reaction proceeded,the pH was maintained at 4 . 75 by t i t r a t -ing when necessary with hydrogen chloride (0.ION).When the con-sumption of HC1 ceased (7-50 mL) a f t e r 2 hours,an aqueous solution of NaBH^ (2M) was added slowly.Bubble formation was minimiffiized by a continuous flow of a i r blowing on the surface of the solution.A drop of 1-octanol was added p e r i o d i c a l l y to control the amount of foam.At the same time the NaBH^ was ad-ded, the pH of the s o l u t i o n was s t a b i l i z e d at 6 .5 with HC1 (4M). A t o t a l of 300 mL of sodium borohydride s o l u t i o n was added over a period of two hours.The f i n a l s o l u t i o n was concentrated and dialyzed against tap water during 48 h and freeze-dried.In a second treatment the consumption of HC1(0.10 N)was 2 .30 mL.A t o t a l of 520 mg of reduced polysaccharide was obtained. A sample of the reduced polysaccharide (15 mg) was hydro-lyzed with 2M TFA and a f t e r conversion of the sugars l i b e r a t e d 124 into a l d i t o l acetates,g.1 .c. showed mannitol,galactitol and g l u c i t o l i n a r a t i o of 0 . 9 5 : 1 . 0 0 : 1 . 4 5 i n d i c a t i n g almost com-plete reduction of the uronic acids. Estimation of the equivalent weight of the polysaccharide from the consumption of HC1 gives a value of 6 5 0 ( t h e o r e t i c a l , 640). A sample of the carboxyl-reduced polysaccharide ( 2 5 0 mg) was methylated by the Hakomori procedure to give 2 0 0 mg of f u l -l y methylated product.Part of the methylated material ( 2 5 mg) was hydrolyzed and g . l . c . analysis of the p a r t i a l l y methyl-ated a l d i t o l acetates i s shown i n Table IV.2 .2 ,column II .The r e s t of the product was heated on a steam-bath with 50?o acetic acid during 9 0 min. i n order to remove the 1-methoxyisopropyl-idene group.The excess acetic acid was evaporated with water. Part of the deacetalated product ( 2 0 mg) was remethylated by Hakomori procedure.Analysis of the hydrolyzate as a l d i t o l ace-tates gave on g. l . c . the r e s u l t s shown i n Table IV.2.2,column III. P a r t i a l hydrolysis.The K 2 6 polysaccharide ( 1 . 0 g) was dissolved i n 2M TFA ( 7 5 mL) and the s o l u t i o n was heated on a steam-bath f o r 2 h.After removal of the acid by successive e-vaporations with water,an a c i d i c and a neutral f r a c t i o n were separated on a column of ion-exchange r e s i n (Bio-Rad AG1-X2). The a c i d i c f r a c t i o n ( 4 3 0 mg) was separated on Bio-Gel P -2 to give 8 2 mg of a pure aldobiouronic acid (A 1),42 mg of a pure aldotriouronic acid (A 2) and 1 2 5 nig of a pure aldotetraouro-nic acid (A~).The neutral f r a c t i o n showed on paper chromato-1 2 5 graphy glucose.galactose and mannose and a neutral d i s a c c h a r i -de (N^ , 3 0 mg) which was also separated on Bio-Gel P-2.The a-nalyses performed on each oligosaccharide were,a) sugar analy-s i s (see Section III,page ?1 ),b) methylation analysis (see Section III,page 7 3 ).The r e s u l t s obtained for each oligosac-charide are given i n Table IV.2.3 and n.m.r. data are present-ed i n TablelV.2.1 . Oxidation of the methylated deacetalated polysaccharide. Part of the methylated and deacetalated carboxyl-reduced poly-saccharide ( 5 0 mg) was dissolved i n CHgClg ( 5 mL).Trifluoro-acetic anhydride ( 1 . 6 mL) was dissolved i n CHgClg (10 mL) and cooled to -60°. Dimethylsulfoxide (1.1 mL) i n CHgClg (10 mL ) was added to the TFAA solut i o n over a period of 10 min.The mixture was s t i r r e d during 10 minutes and the polysaccharide solu t i o n was added carefully,keeping the temperature below - 6 5 ° - A f t e r 10 minutes i t was allowed to warm up to room tem-perature and a f t e r 40 minutes triethylamine (4 mL) was added i n portions (1 mL) over a 10 minute period.After 10 min. three volumes of water were added and the dichloromethane s o l u t i o n was washed a further three times with water.The or-ganic layer was dried with anhydrous Na 2S0^ and the solvent was removed by evaporation.The carbon tetrachloride soluble f r a c t i o n ( 1 5 mg) showed on i . r . two carbonyl absorptions. Part of the product ( 5 mg) was hydrolyzed with 2M TFA and converted into a l d i t o l acetatesjg.1.c. indicated the disap-pearance of 2 , 3-di-0-methylgalactose which had been oxidized. The r e s t of the product (10 mg) was dissolved i n CH,C10 and 126 sodium ethoxide s o l u t i o n i n ethanol (1M,1.5 mL) was added . The solu t i o n was s t i r r e d during 1\ h. at room temperature a f t e r which i t was neutralized with hydrochloric acid,concentrated andcheated on a steam-bath during three hours.After extraction with chloroform,the product thus obtained was remethylated by the Hakomori procedure.Hydrolysis and conversion into a l d i t o l acetates showed on g.l.c. the appearance of 2 , 3 , ^ » 6-tetra-0-methylglucose and disappearance of 2 , 3 i6-tri-0-methylglucose. Periodate oxidation.A sol u t i o n of K26 polysaccharide(1.0 g) i n water (150 mL) was mixed with 0.1M NalO^ and 0.4M NaClO^ (150 mL).The so l u t i o n was kept i n the dark at 4 ° . A f t e r 240 h (consumption of periodate 5.2 moles per mole of K 26).ethylene-g l y c o l (10 mL) was added.The polyaldehyde was dialyzed over-night,reduced with NaBH^ (1 g) to the polyalcohol,neutralized with 20% acetic acid,dialyzed and freeze-dried to y i e l d the polyalcohol (600 mg).Eart of the polyalcohol (300 mg) was treated with 0.5M TFA f o r 20 h at room temperature ,the acid was removed by successive evaporations with water,and the pro-duct was reduced with NaBH^.The excess NaBH^ was destroyed with IR-120 (H +) r e s i n and borate was removed by evaporation with methanol.Paper chromatography of the products showed (solvent (C)) g l y c e r o l , e r y t h r i t o l and/or t h r e i t o l and an oligomer with a R^^O. 28. Hydrolysis with 2M TFA overnight and paper chromato-graphy of the hydrolyzate i n solvent (A) showed glycerol,ery-t h r i t o l and/or threitol,mannose.galactoseglucuronic acid and an aldobiouronic acid.Sugar analysis of the carboxyl-reduced product as a l d i t o l acetates showed on g.l.c. the presence of 12? g l y c e r o l , t h r e i t o l , e r y t h r i t o l t m a n n i t o l , g a l a c t i t o l and g l u c i t o l . The r a t i o of the hexitols was 1.0s1.0s1.1 respectively.The rest of the material was separated i n a column of Bio-Gel P-2.The el u t i o n p r o f i l e obtained indicated that the Smith hydrolysis did not go to completion,but 25 mg of a pure oligomer (SH) was isol a t e d . SH had [ a ] D + 6 7 ° (c 3,water) and R G a l 0.28 ( solvent ( C ) ) { n.m.r. data are given i n TablelV.2.1 .Sugar analysis of the carboxyl-reduced product as a l d i t o l acetate gave on g. l . c . , glycerol,mannose.galactose and glucose i n a r a t i o of l s l s l s l . Methylation analysis of the oligomer gave the following par-t i a l l y methylated a l d i t o l acetates $ 2 ,3 ,4,6-tetra-0-methyl-galactose,2 ,4,6-tri-0-methylmannose and 3»4-di-0-methylglucose. Selective p a r t i a l hydrolysis. K26 polysaccharide (500 mg) was dissolved i n 0.01M TFA (70 mL) and heated on a steam-bath for 7 hours.The acid was removed and the product was dialyzed against 1L of d i s t i l l e d water.A polymeric material (410 mg) and a dialyzate ( 80 mg) were obtained.Paper chromatography of the dialyzable f r a c t i o n showed pyruvic acid.galactose, a disaccharide ( i d e n t i c a l to N-^ ) and a trisaccharide (Ng) . This trisaccharide was i s o l a t e d by preparative paper chromato-graphy ,-yield 25 mg,[a] D+22° (c 2.5, water) .Sugar analysis i n -dicated glucose and galactose i n a r a t i o of 2si.Methylation hydrolysis,reduction and t r i m e t h y l s i l y l a t i o n of the a l d i t o l s gave (g.l.c. column (e)),2 , 3 , 4 , 6-tetramethylgalactose,2 , 3 , 6 -tri-0-methylglucose and 2 , 3 , 4-tri-0-methylglucose. Methylation analysis of the polymeric material,(g.1.c 128 column (c) and g.1.c.-m.s.)indicated the presence of 3>^-di-0-methylglucose i n l i e u of the 3-0-methylglucose found o r i g i n a l -l y . Uronic acid degradation.A sample of methylated K26 poly-saccharide (60 mg) was c a r e f u l l y dried and then subjected to a base-catalyzed uronic acid degradation (see page 75 ).Methyl iodide was the a l k y l a t i n g agent used.Hydrolysis and g.l.c of the a l d i t o l acetate derivatives gave 2 ,3»4,6-tetra-0-methyl-mannose ,3»^»6-tri-0-methylmannose ,2,4,6-tri-0-methylgalactose, 2 , 3 . 4-tri - 0-methylglucose , 2 , 3 , 6-tri - 0-methyglucose and 2 , 3-di-O-methylgalactose i n the r a t i o 1.0:1.0:0.9:0.4:1.0:1.0 . 1 2 9 IV.3 Bacteriophage degradation of K l e b s i e l l a polysaccharides  K 60 and K 46J"88 I V . 3 . 1 Introduction. Like many other organisms,bacteria are subject to i n f e c -t i o n by a range of viruses c a l l e d bacteriophages.Viral i n f e c -t i o n of bacteria has been known since 1 9 1 5 and since the early investigations,the high s p e c i f i c i t y f o r b a c t e r i a l host was ob-served. This i s rel a t e d to the recognition of s p e c i f i c receptors on the c e l l surface of the bacteria which can be: f l a g e l l a , p i l i , capsules,lipopolysaccharides,teichoic acid-peptidoglycan com -plexes,surface proteins,etc.Phages vary widely i n size and i n shape. They were c l a s s i f i e d by Bradley 1"^ 8 according to t h e i r mor-phological differences. The d i f f e r e n t steps i n a v i r a l i n f e c t i o n can be summarized i n the following cycle: i ) adsorption of the phage p a r t i c l e s onto the suscepti-ble 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 nucleic acid and phage pro-teins at the expense of the metabolic processes of the host, i v ) 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 . The i n j e c t i o n of the nucleic acid into the host c e l l needs the attachment of the phage to the cytoplasmic membrane.Al -. though i t was considered that capsulate bacteria were general-l y phage resistant.bacteriophages can be is o l a t e d for many cap-1 3 0 sulate species of bacteria.After the recognition and binding of the phage to the exopolysaccharide,the phage must fi n d i t s way through the exopolysaccharide layer.Attack of bacteriophage on exopolysaccharide producing bacteria i s often revealed by the fa c t that a halo i s formed surrounding the plaque.Within this h a l o 1 7 9 , b a c t e r i a are decapsulated,which can come from the r e -s u l t of the d i f f u s i o n of a phage-induced enzyme which hydro-lyses the capsule without k i l l i n g the bacteria.This enzyme ac-t i v i t y with hydrolysis of the capsule allows the movement of the phage v e r t i c a l l y and l a t e r a l l y , a l l o w i n g the phage to reach the cytoplasmic membrane (see Figure IV.3).Many other enzyma-t i c a c t i v i t i e s .catalyzing d i f f e r e n t degradation reactions of host surface polysaccharides may be associated with b a c t e r i a l virus p a r t i c l e s . The ease of isolation,manipulation and propagation of the bacteriophages has provided a p r o l i f i c source of enzymes which depolymerize b a c t e r i a l polysaccharides.These enzymes.acting on the exopolysaccharides y i e l d products corresponding to single repeating units of the polysaccharide as well as multiples thereof.The conditions of depolymerization are such that l a b i l e substituents.which by other degradative techniques ( p a r t i a l hy-1R1 T R? d r o l y s i s ) are lost,survive t h i s treatment * .This affords a valuable procedure for obtaining oligomers which can be used 39 i n numerous ways as, a) production of synthetic antigens^ 7, b) substrate for n.m.r. studies, c) source of new oligosaccha-r i d e s , etc. A large number of these viruses have been isol a t e d recent-1 3 1 Bacteriophage Li po polysaccharide j Cell membrane F i g u r e I V . 3 A t t a c k o f a b a c t e r i o p h a g e o n a n e n c a p s u l a t e d b a c t e r i a . 132 l y by Stirm and co-workers 1 8-^,which are s p e c i f i c for depoly-merizing the capsular polysaccharide produced by the host s t r a i n ( K l e b s i e l l a ) and i n some cases other capsular glycans of r e l a t e d structure. The r e s u l t s of the degradation of two K l e b s i e l l a capsular polysaccharides ( K 60 and K 46 )with th e i r respective bacte-riophages ( 0 60 and 0 46 .respectively) are presented here. IV.3.2 Results. K l e b s i e l l a bacteriophages were is o l a t e d from sewage and propagated on t h e i r host st r a i n s using nutrient broth as medi-um. Propagation was continued on an increasing scale u n t i l the 13 crude lysates contained a t o t a l of~ 1 0 J plaque-forming units, an amount s u f f i c i e n t to degrade one gram of polysaccharide.The crude lysates were concentrated to one fourth of the i r volume and dialyzed against running tap water for 2 days to remove low molecular weight materials from broth and c e l l lysates. The non-dialyzable f r a c t i o n was concentrated to a convenient volume and was added to a s o l u t i o n of the p u r i f i e d capsular polysaccharide i n water.The depolymerization was carried out at 37° during 2 d.After depolymerization,the sol u t i o n was con-centrated and dialyzed against d i s t i l l e d water ( 3 times).The combined dialyzates were concentrated and free z e - d r i e d , y i e l d -ing the crude mixture of oligomers which were further p u r i f i e d by treatment with Amberlite IR^120 (H +) resin.Table IV.3.2 shows the conditions used f o r depolymerization,and y i e l d s for the degradation of K60 and K46 K l e b s i e l l a capsular polysaccha-1 3 3 TABLE IV. 3^1 PROPAGATION OF BACTERIOPHAGES 060 AND 046. 060 046 Test tube l y s i s ; T i t r e (PFU/mL)-Volume ( mL ) Total 0 (PFU) Small f l a s k l y s i s ; T i t r e (PFU/mL) Volume ( mL ) Total 0 (PFU) Wash bottle l y s i s ; T i t r e (PFU/mL) Volume ( mL ) Total 0 (PFU) 1.3 * 10 14 10 ,11 6.3 * 10 100 10 ,12 2.8 x 10 900 10 2.0 x 10 6 10 1.8 x 10--1- 1 . 2x10 11 1.0 x 10 200 10 6.3 x l O A f c 2.0 x 10 12 l . O x 10 1000 10 2 . 5 x 1 0 1 3 1.0* 10 1 3 — PFU : plaque forming unit 134 TABLE-IV.3.2 DEPOLYMERIZATION OF KLEBSIELLA K60 AND K46 CAPSULAR POLYSACCHA- RIDES BY BACTERIOPHAGES 060 AND 046 RESPECTIVELY. 06OK6Q 046K46 Volume of phage (I) - ( mL ) 400 1000 T i t r e (PFU/mL) 2.8 * l O 1 0 l . O x i o 1 0 Volume of phage ( I I ) - ( mL ) 300 150 Weight of polysaccharide( g ) 1.0 0.83 Total volume ( mL ) 450 300 Y i e l d of oligomers ( % ) 76 78 -Volume of phage from the wash bottle l y s i s . - Volume of phage (I) a f t e r p u r i f i c a t i o n by d i a l y s i s against running tap water, and concentration. 1 3 5 rides with bacteriophage 060 and 046 respectively. Analysis of the products of depolymerization of K60.The purified products of K 6 0 depolymerization were added to a col-umn of Bio-Gel P-4 and eluted with water-pyridine-acetic acid ( 5 0 0 * 5 : 2 ) . T h e elution pattern is shown in Figure IV.4,where P 1 represents the repeating unit of the polysaccharide,P 2 the dimer of P^ and P^  represents several higher oligomeric pro-ducts. Components P^ and P 2 were analyzed by n.m.r. spectros-copy and methylation analysis. The ^C-n.m.r. of P.^  (see Table IV. 3 . 3 a ) demonstrates that i t is an heptasaccharide corresponding to one repeating unit.By comparison with the ^C spectrum of the original poly-saccharide (see Table I V . 3 . 3 a ) the signal at 103.07 p.p.m..as-signed to a —2. Glcp_ —g— .disappeared,indicating that the enzyme is a B -glucosidase.The signals corresponding to the anomeric carbons of the reducing end (96.74 and 9 3 . 0 3 p.p.m.) confirmed the glucose as the reducing end.The^H-n.m.r.( see Table IV . 3 . 3 b ) i s in agreement with this results,as the dou-blet at 6 5.04,corresponding to the B-glucose in the backbone of the original polysaccharide.disappeared. Confirmation of the reducing end and of the degree of polymerization (D.P.) of the repeating unit was obtained by 184 the method of Morrison .whereby the oligosaccharide is re-duced to the a l d i t o l and after hydrolysis,the free sugars are converted into the peracetylated aldononitriles;the reducing end is converted into the peracetylated alditol.Results are shown in Table IV.3.k. and indicate that glucitol.mannono-136 F i g u r e I V . 4 S e p a r a t i o n o f t h e d e p o l y m e r i z a t i o n p r o d u c t s o f K60 b y g e l - p e r m e a t i o n c h r o m a t o g r a p h y ( B i o - G e l P-4) 1 3 7 n i t r i l e . g l u c o n o n i t r i l e and ga l a c t o n o n i t r i l e are present i n a r a t i o of 1:1:4:1 ;before hydrolysis,the glucuronic acid r e s i -due was converted into a glucosyl residue by the normal pro-cedure (methanolysis-s.odium borohydride reduction) .Methyla-t i o n of P^,reduction with LiA1H^,hydrolysis and conversion into the a l d i t o l acetates showed on g.l . c . the r e s u l t s i n d i -cated i n Table I V . 3 . 5 . By comparison with the methylation a-naly s i s of the o r i g i n a l polysaccharide, 2 , 3-di - 0-methylglucose corresponding to the glucuronic acid residue appears,instead of the 2 - 0-methylglucose.indicating that the linkage cleaved by the bacteriophage born-enzyme i s : —2- D - G 1 C £ 2. D-GlcpA 1 f and that P 1 i s the following repeating unit of K60: D-Glc£ 4 1 — D-Glc£A — -2- D-Gal£ 2 ——2. n_ 1 D-G l C £ D-Man£ 2 ' l l D-GlC£ D-GlC£ Analysis of Pg by n.m.r. spectroscopy (see TablesIV. 3 . 3 a and I V . 3 . 3 b for re s u l t s and assignments ) confirmed the r e -su l t s obtained from the analysis of P^, showing the s i g n a l at 103.04 p.p.m. ( i n l 3c-n.m.r ) and at 5 4.99 ( i n ^ -n.m.r. ) TABLE IV.3.3a P.M.R. DATA FOR KLEBSIELLA K6Q CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS DERIVED  FROM BACTERIOPHAGE DEGRADATION. Compound A J i 2 I n t e g r a l Assignment (Hz) proton -2G1C- -2C] LcA1 —Avian— 4 I 5 2 a 2 a a 3 l 1 1 Gl -C Glc Glc G] LcA 1 •Avian 4 P 2 a 2 a 3 3 1 1 1 Glc Glc Glc Glc-OH 5M 2 2.0 5-37 2 1.0 5.04 7 1.0 4.85 7 1.0 4. 70 7 1.0 4.54 7 1.0 5.^9 s 1.0 5.46 s 1.0 5.^5 s 1.0 5.28 s 0.3 4.87 7 1.0 / G 1 ° — 3-Man-2 a 3-Gal-3-Glc-Glc-Glc-3-GlcA-4 { Glc-3-Man— 2 a 3-Gal 2 * 3-Glc OH a G l c - r -TABLE IV.3.3a (cont.) P2 4.75 7 1.0 4.64 7 0.7 4.45 7 1.0 5^9 s 2.0 5.46 s 2.0 5-45 s 2.0 5.28 s 0.3 4.99 8 1.0 4.74 8 2.0 4.70 8 2.0 4.66 8 0.7 4.45 8 2.0 G l c — 3- Glc-g—OH 4- GlcA--— / G 1 C — 3 - G a l — 2 a 3-Glc OH 3 - G l c — G l c — G l c — 3-Glc—OH j3TcA— 1.4-GlcA TABLE IV.3.3b N.M.R. ( 1 3 C ) DATA FOR KLEBSIELLA K60 CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS DERIVED FROM BACTERIOPHAGE DEGRADATION. Compound Chemical s h i f t (p.p.m.) Assignment — - G l c - — - G l c A - : — - k l a l - — 2 * 4 1 G B l l G l c Man-l l Glc 104.23 103.0? 102.44 100.60 99-56 3-GlcA-4 . Glc-3-Glc-Glc-Glc-3-Man-2 3-Gal-r l 104.29 102.31 100.54 99.67 { 4-GlcA-G l c — G l c — G l c -3-Man— 2 a TABLE IV.3.3b (cont.) P * - see Table IV.3.3a . - see text. 3-Glc OH B 3-Glc OH a 4-GlcA-4-GlcA-3 2x G l c — 3 - G l c -2x G l c — — 1 4 B G l c ^ A j l c A -a } 1 4 Glc^-^GlcA-a 2x3-Man 2 a 2 x 3 -Gal 2 a 3 - G l c — O H 3-Glc OH a 142 TABLE IV.3.4 DETERMINATION OF THE DEGREE OF POLYMERIZATION AND THE REDUCING  END OF K6Q OLIGOSACCHARIDES ( P^ AND Pg ). Peracetylated Relative retention Mole % derivative of time Pj P 2 OV-225 -Mannononitrile 1.00 15 15 Glucononitrile 1.2? 56 64 Galactononitrile I.38 14 14 G l u c i t o l 1.60 15 7 - isothermal at 210° 143 TABLE IV.3.5 METHYLATION ANALYSIS OF K60 OLIGOSACCHARIDES ( Pj^ AND Pg ) FROM BACTERIOPHAGE DEGRADATION. P a r t i a l l y methylated Relative retention Mole % -a l d i t o l acetates - time P-L Pg OV-225 ~ 2,3,4,6 - Glc 1.00 46.7 2,4,6 - Glc 1.83. 10.0 13-3 4,6 - Man 2.96 12.9 1^.0 4,6 - Gal 3-26 15.2 15-1 2,3 - Glc 4.63 15.3 7.4 2 - Glc 7.61 — 5.7 - 2,3,4,6 - Glc l , 5-di - 0-acetyl - 2 , 3 , 4 , 6-tetra - 0-methylglucitol, etc. — Values were corrected by use of carbon-response factors given by Albersheim et a l . 7 ^ — isothermal at 170°. 144 3 1 3 1 corresponding to the linkage — ^ Glc — r - * GlcA — 3 4 Methylation analysis as w e l l as the D.P. determination showed P 2 to be a dimer of P^^ (see Tables IV. 3 .4 and IV. 3 . 5 , r e s p e c t -i v e l y ) . Analysis of the products of depolymerization of K46.The p u r i f i e d products from the depolymerization of K l e b s i e l l a K46 capsular polysaccharide were eluted from a column of Bio-Gel P-4 with water-pyridine-acetic acid ( 5 0 0 : 5 * 2 ) . T h e e l u t i o n pat-tern i s shown i n Figure IV.5.where P^ represents the repeating unit of the K46 polysaccharide,P 2 a dimer of P-p and P^ higher oligomers.Components P^ and P 2 were analyzed by n.m.r. spec-troscopy and methylation analysis. The ^C-n.m.r. spectrum of P-^  ( see Table IV. 3 . 6 a ) , i n d i -cates that i t i s a hexasaccharide corresponding to one repeat-ing unit of the K46 polysaccharide.By comparison with the spec-trum of the o r i g i n a l polysaccharide (see Table I V . 3 . 6 a ) the s i g n a l at 1 0 3 , 1 6 , w h i c h was assigned to a 3 galactosyl residue, disappeared,indicating that the enzyme i s a 3 galactosidase. The anomeric carbons of the reducing end ( 9 7.^4 and 9 3 . 1 1 ) con-firmed galactose as the reducing sugar.The ^H-n.m.r. spectrum (see Table I V . 3 . 6 b ) i s i n agreement with these results.The reducing end being the galactose,as well as the D.P.of P^ were confirmed by the method of Morrison.Results are shown i n Table I V . 3 - 7 . G a l a c t i t o l and g l u c o n o n i t r i l e were found i n a r a t i o of 1 : 1 which i s the value expected for one repeating unit;glucuronic acid was not reduced i n t h i s case.Methylation 145 Figure IV.5 Separation of the depolymerization products of K46 by gel-permeation chromatography (Bio-Gel P-4) TABLE IV.3.6a P.M.R. DATA FOR KLEBSIELLA K46 CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS DERIVED FROM BACTERIOPHAGE DEGRADATION. Compound 2 Integral Assignment (Hz) proton —-GlcA-—-Man-—-kjal-" B ^Ian-ex a -Gal--Man !hP 3' 6 l l Glc G 4 cA-—-Man-=—-Gal-—-Gal-OH a a a Man 5>P 3' 6 1 Glc 5-29 5.20 5.05 4.88 4.62 1.47 5-31 5.21 5.20 5.06 4.71 2 2 s s 1.0 1.0 1.0 2.0 1.0 3.0 1.3 1.0 1.0 1.0 { 3-GlcA 4 3-Gal a 3-Man 3 - G a l - f 3-Man— r 4 6 6 V Glc-CH, C0 2H / 4-GlcA— 1 3 - G a l — { 3-Gal Gal OH a a 3-Gal Gal-r-OH a B 3-Man-3-Man-P a TABLE IV.3.6a (cont.) 4 . 6 6 4 . 6 5 1 . 5 2 P 2 5 - 3 0 5 . 1 9 5.06 4.85 4.69 4.65 1 . 5 3 1 . 5 2 - From ref.188 0 . 7 1.0 3 . 0 3-Gal OH B Glc—s-CH-—C 3 i C02H 2 . 3 < 4-GlcA-3-GlcA-4 k 3 - G a l -2 . 0 3 - G a l -2 . 0 3-Man-1. 0 3-Man1-1 . 0 3 . 7 3 - 0 3 - 0 -OH KJlcA 4 6 p ' V ' 3-Man-^GlcA 2 x C H 3 - ( [ C02H TABLE IV.3.6b N.M.R. ( 1 3 C ) DATA FOR KLEBSIELLA K46 CAPSULAR POLYSACCHARIDE AND THE OLIGOMERS DERIVED FROM BACTERIOPHAGE DEGRADATION. -2G: 4 l Compound Chemical s h i f t Assignment (p.p.m.) c A - — ^ M a n - — h a l - — A l a l — 103.16 3-Gal a a a 3 3 6 3 g 100.77 3 - g l c A -4^, a 100.16 / Glc-Man ;>P - — . ~ ^ — 6 3-Ms... 4 6 e 3 v W r Glc 97.16 3-Man 96.16 3-Gal ° 25.40 CH.,—C 3 i a C0 2H b P, 101.33 Glc 3 101.00 4-GlcA— a 100.37 3-Man—-4 6 B V TABLE IV.3.6b (cont.) P2 97.1^ 95.97 \ 95.81j 93.11 25.43 { 3-Man a 3-Gal-r-OH p 3-Gal 3-Gal—OH CH~—C 3 1 C0 2H 103.35 101.15 100.20 96.92 96. 80 93.10 25.34 3 - G a l — 3- G l c A — 4 4- G l c A — a 2x G l c - r — ( 2 x 3-Man—— 4 6 B V 2x3-Man a 3-Gal-^-OH 2x3-Gal 3-Gal OH 2 * 0H o—C 3 I C0 2H 1 5 0 TABLE IV.3.7 DETERMINATION OF THE DEGREE OF POLYMERIZATION AND THE REDUCING  END OF K46 OLIGOSACCHARIDES ( P 2 AND Pg ). Peracetylated derivative of Relative retention  time OV-225 -Mole % Mannononitrile Gluc ononi t r i l e G alactononitrile G a l a c t i t o l 1.00 1.27 1.38 1.51 34.0 21.8 22. 0 22.2 38.0 20.6 30.9 10.5 - as i n Table IV.3.4 1 5 1 TABLE IV.3.8 METHYLATION ANALYSIS OF K46 OLIGOSACCHARIDES ( P± AND Pg ) FROM BACTERIOPHAGE DEGRADATION. P a r t i a l l y methylated Relative retention Mole % — a l d i t o l acetates - time P n P. OV -225 -1 x2 2,3,4,6 - Glc 1 . 0 0 1 7 . 9 17.8 2,4,6 - Man 1 . 9 1 16 .0 16.1 2,4,6 - Gal 2 . 0 5 32.3 33 .0 2,3 - Glc 4.63 1 5 . 9 16.8 2 - Man 5 . 7 0 1 7 . 9 8.1 2 - Glc 7.61 — 8.2 -,- and - as i n Table I V . 3 . 5 152 analysis of P^^ showed on g.l.c. the r e s u l t s indicated i n Table IV.3.8. By comparison with the methylation analysis of the na-t i v e polysaccharide, 2,3-di-0-methylglucose corresponding to the glucuronic acic appeared instead of the 2-0-methylglucose from previously.This indicated that the linkage cleaved by the bacteriophage borne-enzyme of i s : — 2 D-Galp 1 . ^ D-GlcpA - — f and P^ i s the following hexasaccharide: k 6 D-Glc£ ~a D-Man£ D-Glc£A D-Man£ D-Galp. 1 -2 D-Galp_ Methylation analysis as well as the D.P. determination of P 2 confirmed i t to be the dimer of P^^ (see Tables IV. 3.7 and IV.3.8). Nuclear magnetic resonance spectroscopy (see Tables IV.3.6a and IV.3.6b ) confirmed the r e s u l t s from the analysis of P r IV.3.3 Discussion. The main purpose of the bacteriophage work ca r r i e d out by our research group i s to obtain large amounts of pure subunits of the polysaccharide degraded ( one repeating unit or/and the dimer),with the acid or base l a b i l e substituents present as i n the o r i g i n a l polysaccharide.In order to avoid the expensive 1 5 3 and time consuming p u r i f i c a t i o n of the phage,several p o s s i b i l -i t i e s have been investigated which would y i e l d i n a shorter pe-r i o d of time the amount of phage necessary f o r the degradation ( ~ 1 0 1 3 phages/g of polysaccharide) and i n such conditions as to simplify the p u r i f i c a t i o n of the degradation products.The best procedure,developed up to t h i s moment,is the one used i n the course of the present investigation.According to t h i s meth-od.the bacteriophage i s propagated i n broth as usual D t i l l the amount of phage i s s u f f i c i e n t to degrade the desired a-mount of polysaccharide.The crude phage so l u t i o n i s d i a l i z e d exhaustively to remove a l l low molecular weight materials i n -stead of p u r i f y i n g the phage by p r e c i p i t a t i o n with polyethyl-186 ene g l y c o l 6000 and isopycnic centrifugation.This solution, a f t e r concentration,is the source of bacteriophage for the degradation.The degradation i s car r i e d out i n d i s t i l l e d water, avoiding the use of PBS (buffered saline),as t h i s saline me-dium means more steps i n the p u r i f i c a t i o n . A f t e r degradation, d i a l y s i s affords the oligomers.The time has been reduced by a factor of four when we compare the two procedures,and there i s l i t t l e difference i n the q u a l i t y and quantity of products obtained. In order to demonstrate the s p e c i f i c i t y of the bacterio-phage-borne enzyme, 060 was used on the polysaccharide obtain-ed by removal of the three terminal glucosyl groups from K60 polysaccharide by Smith degradation. The v i r a l enzyme was to-t a l l y inactive on the Smith degraded polysaccharide (K60SH), due to changes on the environment of the g l y c o s i d i c linkage Figure IV. 6 Environment of the g l y c o s i d i c linkage which undergoes enzymic hydrolysis (K60) and the one that does not (K60SH) 1 5 5 which undergoes enzymic hydrolysis (see Figure IV.6) S p e c i f i c hydrolases were used on both repeating units of K60 and K 4 6 i n order to i s o l a t e smaller oligosaccharides,but no a c t i v i t y was observed.The enzymes used were a - and s -glu-cosidases which probably are active towards short chains of sugar residues such as d i - or tri-saccharides and have very l i t t l e or no action on longer chains 1 8'''. I V . 3 . 4 Experimental. Bacteriophage propagation and depolymerization. 0 6 0 and 0 4 6 were i s o l a t e d from sewage and propagated on the host str a i n s using nutrient broth as medium according to the stan-dard procedures of virology.Three successive propagations using increasing amounts of b a c t e r i a l cultures and bacterio-phage were necessary to obtain s u f f i c i e n t virus p a r t i c l e s with which to degrade the corresponding capsular polysaccharides. This involved: a) tube l y s i s ; 4 mL of nutrient broth, 0 . 5 mL of b a c t e r i a l culture, 0 . 5 mL of bacteriophage solution; b) small f l a s k l y s i s ; 4 8 mL of nutrient broth, 1 mL of bacte-r i a l culture and 1 mL of bacteriophage solu t i o n (from a ); c) wash bottle l y s i s ; 200 mL of nutrient broth, 10 mL of b a c t e r i a l culture and 2 5 mL of bacteriophage solu t i o n (from b). In the l a t t e r case,in order to control the propagation of phage,the b a c t e r i a l growth was monitored by o p t i c a l density ( see Figure IV.7).When the readings of o p t i c a l density were appropiate ( 0 . 5 - 0 . 6 ) the bacteriophage s o l u t i o n was added. One hour a f t e r l y s i s had occurred a few drops of chloroform 1 5 6 0.2 x 109 BC/mL 0.8 x 109 BC/mL 1.2 x 109 BC/mL 1.8x 109 BC/mL 2.4 x l O 9 BC/mL ( BC = b a c t e r i a l colonies ) Phage t i t r e : 3 x 10 (P.F.U /mL) 10 Time (h) Figure IV.7 Growth c u r v e — , and bacteriophage l y s i s .... of K l e b s i e l l a K60 bacteria . 1 5 7 were added i n order to prevent further b a c t e r i a l growth.Cen-t r i f u g a t i o n yielded a cl e a r phage solution which was then t i t r a t e d to determine the amount of phage.Table IV. 3 . 1 shows the concentrations obtained a f t e r each step. A volume of bacteriophage s o l u t i o n containing the amount T O of virus required to degrade the polysaccharide ( 1 0 7 g of polysaccharide) was concentrated by evaporation i n vacuo to one third,and dialyzed against running tap water f o r 2 days.After concentration to h a l f the volume,these phage solutions were added to the polysaccharide solutions i n d i s -t i l l e d water.The depolymerization was ca r r i e d out at 3 7 ° for 3 0 hours.Chloroform ( 3 mL) was added to avoid b a c t e r i a l growth. Is o l a t i o n and p u r i f i c a t i o n of degradation products.When the depolymerization was over,the solution was concentrated to a small volume ( 5 0 mL) and dialyzed against d i s t i l l e d wa-ter ( 1 L).This procedure was repeated three times.The three dialyzates were co l l e c t e d and concentrated to dryness. The s o l i d was redissolved i n HgO ( 5 0 mL) and I R - 1 2 0 (H +) r e s i n was added.The mixture was s t i r r e d , f i l t e r e d and freeze-dried and t h i s procedure was repeated u n t i l a c o l o r l e s s s o l u t i o n was obtained.The p u r i f i e d oligomers were obtained i n t h i s way. The amount of polysaccharide degraded.yield of oligosacchari-des, volumes used,etc.are shown i n Table I V . 3 . 2 . A portion of the depolymerization product ( 5 0 0 mg) was separated on Bio-Gel P-4 column using conditions as i n page 67 .The e l u t i o n p r o f i l e was obtained and f r a c t i o n s were c o l -lected. 1 5 8 Analysis of K60 degradation products. Two fractio n s were co l l e c t e d : ( 3 0 0 nig) and P 2 (14-5 mg),the r e s t corresponded to higher oligomers. P1 had an [ a] D + 6 l ° ( c 0.7 ,water)(calculated +6 2 ° ).For n.m.r. data see Table I V . 3 . 3 a and I V . 3 . 3 b (signals and assign-ment) and spectra N ° 2 5 and 2 6 . Determination of the reducing end and P.P. A sample of P 1 (lOmg) was dissolved i n HgO ( 5 mL) and NaBH^ ( 1 5 mg) was added.After s t i r r i n g f o r 2 h , the excess sodium borohydride was decomposed as usual. The dried reduced oligosaccharide was refluxed i n yfo methanolic HC1 overnight.After n e u t r a l i z a t i o n with Ag^CO^.and evaporation of the solvent a f t e r centrifugation,the uronic es-ters were reduced with NaBH^ ( 1 5 mg) i n anhydrous methanol (5mL). Hydrolysis was effected with 2M TFA on a steam-bath f o r 3 h and the excess TFA was removed by evaporations with water.A sol u t i o n ( 0 . 5 mL) of 5% hydroxylamine hydrochloride i n pyridine was added and heated on a steambath for 1 5 minutes.Acetic an-hydride ( 0 . 5 mL) was added to the cooled s o l u t i o n which was then heated f o r one hour on a steam-bath. G.l.c. of the mix-ture 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 was done on column (c) isothermally at 210°.Results are shown i n Table IV.3 .4. Methylation analysis^ P^ (10 mg) was methylated by the Hakomori procedure.Reduction with LiAlH^,hydrolysis,reduction and acet-y l a t i o n gave a mixture of p a r t i a l l y methylated a l d i t o l acetates which was separated and i d e n t i f i e d by g. l . c . and g.l.c.-m.s. Results are shown i n Table I V . 3 . 5 . 1 5 9 Enzvmic hydrolysis, a) B -glucosidase. P-j^  (40 mg) was dissolved i n acetate buffer (pH 5 . 3 ) and B -glucosidase (NBC)(8 mg) was added and the soluti o n incubated at 37° .Samples were taken at d i f f e r e n t times (up to a week) but no glucose was detected by-paper chromatography;the enzyme was however active on e e l l o -biose. b) a-glucosidase. P^ ( 1 5 mg)was dissolved i n water(5mL) and a -glucosidase (Type I,from Yeast,SIGMA Chem.Com.)(1 mg) was added.Incubated at 37° .Samples were taken at d i f f e r e n t i n -tervals (up to 5 days) but no glucose was detected by paper chromatography:the enzyme was active on maltose. P 2 had a n [ a l D + 4 5 ° (c 0 . 6 , w a t e r c a l c u l a t e d +56°).N.m.r. spectroscopy data are shown i n TablesIV. 3 - 3 a and I V . 3 . 3 b and spectrum NO.26. Determination of reducing end and D.P.was done as for P^ and the r e s u l t s are shown i n Table IV.3 - 4 . The re-su l t s of methylation analysis (as for P^) are indicated i n Table I V . 3 - 5 • Analysis of K46 degradation products. Two fractio n s were co l l e c t e d from g e l separation P^ (190 mg) and Pg (150 mg),the r e s t corresponded to higher oligomers. P 1 had a n [ a ] D + 6 9 ° (c 1 . 3 , water c a l c u l a t e d + 7 2 ° ) .N.m.r. spectroscopy data are shown i n TablesIV.3 •6a and IV.3 -6b and spectra N o 2 7 and 28.Pg had[<*] D + o > 0 ° ( c0.5,water c a l c u l a t e d +64° ).N.m.r. spectral data are shown i n TablesIV.3 .6a and IV.3.6b and spectrum N o 2 9 . The determination of the reducing end and D.P.of P^ and P^ was done by d i r e c t hydrolysis of the reduded oligosaccha-160 ride.conversion into the peracetylated a l d o n o n i t r i l e s and a l -d i t o l . R e s u l t s of the g . l . c . are shown i n Table I V . 3 - 7 . Methyl-a t i o n analysis of and Pg (as f o r K60 products) gave res u l t s shown i n Table I V . 3 . 7 . Enzvmic hydrolysis. P^ ( 1 3 mg) was dissolved i n acetate buffer (pH 5 . 3 ) and B-glucosidase (NBC, 3 m g ) was added.The solut i o n was l e f t at room temperature and samples were taken at d i f f e r -ent i n t e r v a l s (up to one week).No glucose was detected by paper chromatography. 161 V . STRUCTURAL STUDIES OF THE GUM EXUDATE OF CHORISIA SPECIOSA 162 V.- STRUCTURAL STUDIES OF THE GUM EXUDATE OF CHORISIA  SPECIOSA. V.1 Abstract. The p u r i f i e d gum exudate from Chorisia speciosa (Palo bor-racho ) was s t u d i e d . l t contains L-arabinose ( 1),L-rhamnose (2), D-mannose (1),D-galactose (9),D-glucuronic acid (3) and traces of D-xylose.The r e s u l t s from methylation analysis, 8-elimina -t i o n and p a r t i a l hydrolysis make possible a tentative assign-ment for the " average structure " of the gum polysaccharide. V.2 Introduction. The exudate gums from the bark and f r u i t of many trees and shrubs may be produced frequently at s i t e s of i n j u r y to the plant or even spontaneously.Many of these gums have found commercial applications such as, gum arabic (Acacia Senegal) and other Acacia gums, tragacanth (Astralgus sp.) .karaya gum (S t e r c u l i a urens),etc.Chemical examination of polysaccharide gum exudates has been carried out for many years.Much of the present knowledge of the chemical composition of plant gums 189 can be found i n monographs by, F.Smith and R.Montgomery , G . 0 . A s p i n a l l 1 9 0 , and G.O.Aspinall and A.M.Stephen 1 9 1.The struc-tures of the polysaccharide components of gums are a l l highly complex.In spite of the f a c t that newer a n a l y t i c a l techniques have been developed,it i s s t i l l very d i f f i c u l t to unravel the detailed structure of these polysaccharides. Certain s t r u c t u r a l patterns have been shown to p r e v a i l . 163 Stephen based on thi s a c l a s s i f i c a t i o n of the gum exudates. Three types were p r o p o s e d 1 9 2 and each can be described b r i e f l y i n the following way: Type A,consisting of a highly branched core of D-galactopyrano-s y l residues,mutually joined through B 1-3 and B 1-6 linkages, with residues of D-glucuronic acid.L-rhamnose and/or L-arabin -ose attached to i t ; Type B,consisting of an i n t e r i o r chain of D-galacturonic acid and L-rhamnose residues i n varying r e l a t i v e proportions and arrangments,with some other sugars present i n outer chains;and Type C,consisting of a chain of D-xylose r e -sidues with branches of L-arabinose,D-xylose and D-glucuronic acid residues. A s p i n a l l c l a s s i f i e d the gum exudates depending on the sug-ars forming the i n t e r i o r chains of the polysaccharides into, I) the galactan, II) the glucuronomannan, III) the galacturo-norhamnan, IV) the xylan and other minor groups. Group II) , as well as group I ) , i s included i n the Type A of Stephen's c l a s -s i f i c a t i o n , the reason being that although these gums have an i n t e r i o r chain of alt e r n a t i n g D-glucuronic acid and D-mannose residues,the outer chains are from the galactan type.Several examples are found among the gums from Prunus sp. The structure of the gum exudates has been studied for the i r p o t e n t i a l use i n chemical taxonomy of p l a n t s 1 9 2 , b u t , the conclusions up to the moment are that although i t i s not so clear at the l e v e l of orders,within a genus the d i f f e r e n t spe-cies have gums of the same type with many s t r u c t u r a l features i n common but not completely i d e n t i c a l (e.g. Acacia gums). 164 Chorisia speciosa S t . H i l . i s a large tree from the genus 193 Bombacaceae o r i g i n a l l y from Tropical South America 7-\ I t has 194 t y p i c a l bulging trunks allowing f o r water storage y .When the trunk s u f f e r s injury,a gum exudes.apparently to heal wounds. Although the le a f mucilage has been analyzed 1 9 5,no information i s available about the gum.Due to the r e l a t i o n s h i p of Bombaca-ceae to Sterculiaceae, as they both belong to the order of the Malvales.it i s therefore of i n t e r e s t to analyze the composi -t i o n of t h i s gum. V.3 Results and discussion. The gum polysaccharide from Chorisia speciosa S t . H i l . , aft e r p u r i f i c a t i o n by p r e c i p i t a t i o n with ethanol had[ a] D+l8°. By gel chromatography i t was shown to consist of two fractio n s a) 1 . 0 5 * IC-5 daltons (80#) and b) 4x10^ daltons ( 2 0 # ) . Hydro-l y s i s with 2M TFA yielded L-arabinose (0. 9)*L-rhamnose (1.8), D-mannose (1.0),D-galactose (?.8),and D-glucuronic acid (2.8) i n the proportions shown,together with traces of xylose. Methylation analysis of the gum gave the re s u l t s shown i n Table V.l,column I. P a r t i a l hydrolysis of the gum yielded monosaccharides, neutral and ac i d i c oligosaccharides ( N^.Ng.N^ and A-^ .Ag res -pectively) and a polymeric material (P^).The monosaccharides released were arabinose.rhamnose and galactose.The oligosac-charides were i s o l a t e d by preparative paper chromatography af t e r separation into a c i d i c and neutral components by ion-ex-change chromatography.The r e s u l t s of the analyses on each TABLE V . l METHYLATION ANALYSIS OF CHORISIA SPECIOSA GUM EXUDATE AND DERIVED PRODUCTS • d IV V Methylated sugars - Relative retention times II Mole % £ SP - 1 0 0 0 - III 2 , 3 . ^ -Rha 0 . 4 ? 0.46 1 0 . 9 4 . 3 2 , 3 . 4 -Ara 0 . 5 7 0 . 5 9 5 . 4 6 . 0 2 , 3 + 3 . 4 -Rha 0.84 3 - 6 2 , 3 -Ara 0 . 9 5 1 . 0 0 2 . 8 1 . 2 2 , 3 . 4 , 6 - G a l 1 . 0 0 1 . 0 0 4 . 8 3 4 . 0 4 . 1 42 . 6 3 5 . 0 3 . ^ . 6 -Man 1 . 3 2 1 . 4 4 — 40 . 9 — — 2 , 4 , 6 -Man 1 . 3 3 1 . 6 3 — 4 . 0 — 7 . 4 2 2 . 8 2 , 4 , 6 -Gal 1 . 3 9 1 . 6 9 7 . 8 1 2 . 5 1 . 2 3 1 . 5 3 3 - 3 2 , 3 . 4 -Glc 1 . 4 9 2 . 1 0 5 . 2 — 8 . 8 — —— 2 , 3 , 4 -Gal 1 . 9 3 2 . 1 0 2 1 . 8 2 8 . 8 5 - 2 — — 4 , 6 -Man 1 . 9 3 2 . 2 7 5 - 9 — 2 . 0 18 . 5 8 . 9 2 , 3 -Glc 2.08 2 . 9 8 1 3 . 6 — 3 7 . 8 — — 2 , 4 -Gal 2 . 2 0 3 . 5 6 1 0 . 0 9 - 2 2 -Gal 2 . 4 4 4 . 0 0 2 . 5 4 -Gal 2 . 5 7 4.24 2 . 3 as i n Table IV. 1 . 2 . - Programmed at 180° f o r 4 min,then at 2 % i i n to 2 3 0 ° , isothermal for 3 2 min . - I , o r i g i n a l gum exudate; II,product from s -elimination; III,methylation of P 1 ;IV,product A from Smith hydrolysis;V,product B from Smith hydrolysis. 166 oligosaccharide are given i n Table V.2,and indicated the f o l -lowing structures: N l Gal 1 I 3 s Gal N2 Gal 1 6 6 Gal N 3 Gal 1 3 B Gal and A1 GlcA 1-y 6- Gal A £ GlcA ^ y 6 - Gal Gal No mannose was detected either as the free sugar or as an i n t e g r a l part of the oligosaccharides,suggesting i t s presence i n the polymeric material. had[a ]^+20°,while the Hl-n.m.r. spectrum showed signals at 6 4 . 5 3 (J]_ 2 ®^z) and at 6 5.40 (s) i n a 1 : 1 ratio.Sugar analysis gave mannose.galactose and gluc-uronic acid i n a r a t i o of 3 - 9 s 1 . 0 : 4 . 1 .Methylation analysis (Table V.l,column III ) on the methylated and carboxyl-reduced P^ indicated mainly 3,4,6-tri-0-methylmannose and 2 , 3-di-0-methylglucose (derived from the glucuronic acid).These re-s u l t s suggested that the polymeric material was i n fact com-posed of alternating glucuronic acid and mannose residues.This was confirmed by the i s o l a t i o n and characterization (TableV . 3 ) of two a c i d i c oligosaccharides from p a r t i a l hydrolysis of P^. They were i d e n t i f i e d as A~ GlcA - — - Man 3 B A., GlcA ^ — - Man - — - GlcA 1 — - Man 4 B a B The r e s u l t s from methylation analysis and p a r t i a l hydro-l y s i s suggested the following s t r u c t u r a l features of the gum: TABLE V . 2 ANALYSIS OF THE OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF THE GUM EXUDATE. Oligosaccharide N-N, N, [ a ] D (water) + 1 3 ° - 3 . 0 ' - 3 6 ° - 8 ° + 40° + 14 + 2 2 Sugar analysis (as a l d i t o l acetates) Glc(GlcA) Gal Glc(GlcA) Gal Glc(GlcA) Man Glc(GlcA) Man Gal Gal Gal ( 1 . 0 ) ( 1 . 0 ) ( 1 . 0 ) ( 2 . 0 ) ( 1 . 0 ) ( 1 . 0 ) ( 1 . 0 ) ( 1 . 0 ) Methylation analysis (as a l d i t o l acetates) 2 , 3 . 4 - Glc ( 1 . 0 ) 2 , 3 , 4 - Gal ( 0 . 5 ) 2 , 3 . 5 - Gal ( 0 . 3 ) 2 , 3 , 4 - Glc ( 1 . 0 ) 2 , 3 . 4 - Gal ( 1 . 5 ) 2 , 3 , 5 - Gal ( 0 . 3 ) 2 , 3 . 4 - Glc ( 1 . 0 ) 3 , 4 , 6 - Man ( 0 . 9 ) 2 , 3 , 4 - Glc ( 1 . 0 ) 3 , 4 , 6 - Man ( 1 . 9 ) 2 , 3 - Glc ( 0 . 9 ) 2 , 3 , 4 , 6 - Gal(l.O) 2 , 4 , 6 - Gal ( 0 . 9 ) 2 , 3 . 4 , 6 - Gal(l.O) 2 , 3 , 4 - Gal ( 0 . 5 ) 2 , 3 . 5 - Gal ( 0 . 3 ) 2 , 3 , 4 , 6 - Gal(l.O) 2 , 4 , 6 - Gal ( 1 . 0 ) 2 , 3 , 4 - Gal ( 0 . 8 ) 168 a) a backbone of a l t e r n a t i n g glucuronic acid and mannose r e -sidues linked i n the following manner, 4 1 ? 1 4 1 ? 1 — - GlcA ±-r^- Man - — - GlcA ^ Man - — 6 a B a b) 6S% (approx.) of the mannosyl residues from branch points at p o s i t i o n 0-3 , c) the branches consist of a galactan framework of mainly 3 -(1-6) linked galactose residues which may also be subsituted at p o s i t i o n 0-3 , d) the main terminal non-reducing sugar i s L-rhamnose. The permethylated gum was subjected to a base-catalyzed uronic acid degradation gave the r e s u l t s shown i n Table V . l , column II,from which,the following conclusions may be drawn: a) the majority of the L-rhamnose residues are linked to 0-4 of glucuronic acid and are thus degraded during the reaction with base, b) the increase i n the amount of 2 , 3 ,4 ,6-tetra -0-methylgalac-tose i s due to the degradation of units of glucuronic acid at-tached to p o s i t i o n 0-6 of the galactose, c) the disappearance of 4,6-di-0-methylmannose and appearance of 2,4,6-tri-0-methylmannose i s i n accordance with the s t r u c -ture of the backbone.The small amount of trimethylmannose found i n the hydrolyzate i s due to incomplete degradation with base. The gum consumed 9.8 mmoles of NalO^ per gram ,upon ox-id a t i o n with 0.1M NalO,, solution,as expected from the methyl-169 ation results.Smith hydrolysis of the polyol and p r e c i p i t a t i o n with ethanol yielded a product (A) which upon methylation and methylation analysis gave the r e s u l t s shown i n Table V . l , c o l -umn IV. The presence of large amounts of 4,6-di-0-methylman-nose was explained by the resistance to hydrolysis of the r e -sidue of the oxidized glucuronic acid 1 9^.Treatment of A with 0.1M TFA on a steam-bath f o r an hour yielded a product (B)which upon methylation and methylation analysis (Table V.l,column V) showed an increase i n the proportion of 2,4,6-tri-O-methylman-nose compared to 4,6-di-0-methylmannose.The r e s u l t s of the Smith hydrolysis indicated that periodate r e s i s t a n t galactose residues are attached d i r e c t l y to the backbone at 0-3 of the mannose residues. The analysis of Chorisia speciosa gum indicates an "aver-age structure" that can be represented as i n Figure V.1 .This possible structure s a t i s f i e s the a n a l y t i c a l evidence,but r e -presents only one of the many that have the same characteris-t i c . A l l must contain the backbone of D-glucuronic acid and D-4 1 6 mannose,and the side chains ended i n Rha GlcA Gal — , some arabinose or galactose fragments.The side chains o f f e r many alternatives. There i s a clear difference with the related S t e r c u l i a 1 9 ^ gum.Those gums have D-galacturonic acid,rhamnose and galactose i n the central backbone,and i n them,D-glucuronic acid may be present as a minor component. I t would be i n t e r e s t i n g to extend the present study to other Chorisia species,as well as to other members of the Bom-TABLE V.3 N.M.R. ( 1H) DATA OF ACIDIC OLIGOSACCHARIDES FROM PARTIAL HYDROLYSIS OF THE GUM. Compound j . Integral Assignment Spectrum  proton No. GlcA 1—^Gal-' OH 5.27 2 0.4 6-• G a l — -OH B 4.61 8 0.6 6-• G a l — -OH 30 A l 4.52 8 1.0 GlcA-g GlcA^-^Gal 1-—^Gal-OH 5.25 2 0.4 6--Gal „ -OH B B 4.87 7 0.6 6--Gal g -OH A 2 4.52 7 1.0 GlcA—g— 4.42 8 1.0 6--Gal B G l c A - — ^ a n - OH 5.30 s 0.8 2--Man a -OH B 4.99 s 0.2 2--Man B -OH 31 A 3 4.55 8 1.0 GlcA— I GlcA-^-^Man- ^GlcA-^-^-Man-OH 5.40 s 1.0 2- Man— a B a B 5.29 s 0.8 2 -Man— a -OH A4 4.98 s 0.2 2 -Man—— B -OH 32 H 4.53 8 1.0 GlcA— 3~ 4.49 8 1.0 4 -GlcA-1 1 7 1 ^GA-—24M— GA: glucuronic acid M : mannose G : galactose R : rhamnose A i arabinose Figure V.1 One of the possible "average structures"for the gum of Chorisia speciosa, (Some of the configu-rations of the g l y c o s i d i c linkages are ten t a t i v e ) . 172 bacaceae,to further e s t a b l i s h the chemical r e l a t i o n s h i p of these plants. V.4 Experimental. General methods.The a n a l y t i c a l techniques and instrumen-t a t i o n used i n t h i s study have been already described i n Section III . P u r i f i c a t i o n of the gum. The gum ( 5 . 0 g) was treated as indicated i n Section I I I . 7 . 2 . (Yield 3.4 g). Molecular weight determination.A sample was analyzed by gel-permeation chromatography and was shown to consist of two f r a c t i o n s , one of F L 1 . 0 5 x l 0 5 daltons ( 8 0 % ) and the other of w 5f 4 X I C / * daltons ( 2 0 % ) . w Hydrolysis of the gum.A sample of the gum ( 3 0 mg) was heated with 2M TFA on a steam-bath.After 4 h of hydrolysis a sample was taken and a f t e r removal of the acid,the hydrolyzate was examined by paper chromatography i n solvent (A).Rhamnose, arabinose.galactose and several oligosaccharides were detected. After 4 8 h hydrolysis.rhamnose.arabinose,mannose,galactose and glucuronic acid were detected on p.c. (solvent (A)).Conversion of the neutral sugars into a l d i t o l acetates and g. l . c . gave rhamnose,arabinose,mannose and.galactose i n the r a t i o of 1 . 9 : 0 . 9 : 1 . 0 : 6 . 7 .Sugar analysis,as described before (see Section I I I . 8 ) , o n the gum showed rhamnose.arabinose,mannose.galactose and glucose i n the r a t i o of 1 . 8 : 0 . 9 : 1 . 0 : 7 . 8 : 2 . 8 . Methylation analysis.The gum ( 1 5 0 mg) was methylated by the Hakomori procedure followed by a single Purdie treatment 1 7 3 ( see Section II I . 9). The product (125 nig) showed no hydroxyl absorption i n the i . r . spectrum.Methylation analysis of this material gave the proportion of methylated sugars shown i n Table V.l,column I. Base-catalyzed uronic acid degradation.Part of the per-methylated gum ( 5 ° mg) was subjected to base-catalyzed uronic acid degradation as i n Section III.10 .Hydrolysis of the pro-duct and methylation analysis of the sugars released gave the r e s u l t s shown i n Table V.l,column II . P a r t i a l hydrolysis. The gum (450 mg) was hydrolyzed with 1M TFA on steam-bath for one hour.The acid was removed by e-vaporation with water and the product separated into a c i d i c (220 mg) and neutral (160 mg) components on a column of Bio-Rad AG1-X2. Two f r a c t i o n s , A^ (40 mg) and A 2 (25 mg) were is o l a t e d by paper chromatography from the a c i d i c fraction.Results of the analysis of these a c i d i c oligosaccharides are given i n Table V.2. The "Hi-n.m.r. spectrum of A^ showed signals at 65.27 (0.4H , J 1 2 2 H Z ) , 6 4.61 (O^H.J^ 28Hz) and 6 4.52 (l.OH.J^ 28Hz), while that of A 2 showed signals at 66 5-25 (0.4H,J 1 > 22Hz), 6 4.87 (0.6H,J l j 27Hz), 6 4.52 (1.0H,J l f 27Hz) and 6 4.42 (1.0H,J 1 28Hz). (see Table V . 3 and Spectrum N°30). A^ was i d e n t i f i e d as 6-0-( R -D-glucopyranosyl uronic acid)-D-galactose and the i d e n t i t y confirmed by co-chromatography with an authentic sample. A 2 was i d e n t i f i e d as 6-0-( g-D-glucopyran-osyl uronic a c i d ) - 6 - 0 - ( 8 -D-galactopyranosyl)-D-galactose. 1 7 4 Three neutral oligosaccharides, ( 1 0 mg),Ng ( 1 5 mg) and ( 5 mg) were is o l a t e d by preparative paper chromatography from the neutral fraction.Analysis from these oligosaccharides,given i n Table V.2,indicated the structures shown belows N-^  3 - 0 - ( 3 -D-galactopyranosyl)-D-galactose, N 2 6 - 0 - ( B-D-galactopyranosyl)-D-galactose, N^ 3 - 0 - ( B-D-galactopyranosyl) - 6 - 0 -( g -D-galactopyranosyl)-D-galactose. A sample of the gum ( 2 5 0 mg) was treated with IM TFA on a steam-bath f o r 1-f h.The acid was removed and the residue was dissolved i n water ( 1 0 mL) and dialyzed f o r 7 2 h against d i s t i l -led water (l.OL).The non-dialyzable material ( 5 0 mg) had[a] ^ + 2 0 ° (c 1 .7»water) and the "hi-n.m.r. spectrum i n DgO recorded signals a t 6 5 - 4 0 and 6 4 . 5 3 ( J ^ 2 ^Hz) i n a l s l ratio.Sugar analysis gave mannose,galactose and glucuronic acid i n a r a t i o of 3 - 9 s 1 . 0 : 4 . 1 .Methylation analysis gave the r e s u l t s shown i n Table V.l,column III.Hydrolysis of this material ( 2 0 mg) with 2M TFA on a steam-bath for 7 h showed by p.c. (solvent (A)), galactose,mannose.glucuronic acid,the aldobiouronic acid A^ , the aldobiouronic acid A^ and other higher oligomers.A^ (6 mg) was isol a t e d by paper chromatography and analyzed as shown i n Table V . 2 . The ^H-n.m.r. spectrum (see Table V . 3 and spectrum N ° 3 D showed signals at 6 5 . 3 0 ( 0 . 8 H , s ) , <S 4 . 99 ( 0 . 2 H,s) and 6 4 . 5 5 ( I . O H . J J L 2 8Hz).A^ was i d e n t i f i e d as 2 - 0 - ( 6 -D-gluco-pyranosyl uronic acid)-D-mannose.Another oligosaccharide,A^ ( 5 mg) was i s o l a t e d by paper chromatography from the hydro-1 7 5 lyzate and analyzed as shown i n Table V.2. The 1H-n.m.r. spec-trum (see Table V . 3 and spectrum N0.32) showed signals at 6 5.40 (l.OH.s), 6 5 . 2 9 ( 0 . 8 H . s ) , s 4 . 9 8 (0.2H,s), 5 4 . 5 3 (l.OH.J^ 2 8Hz) and <5 4 . 4 9 (1.0H,J 1 2 8Hz).A^ was hydrolyzed with 2M TFA for 4 h and p.c. of the hydrolyzate showed the aldo-biouronic acid Aj,mannose and glucuronic acid.This oligomer was i d e n t i f i e d as the dimer of A^. Periodate oxidation.A s o l u t i o n of the gum (200 mg) i n H20 (100 mL) was treated with 0.1M NalO^ (100 mL) for 9 6 h at 4 ° i n the dark.The periodate consumption was followed by the Fleury-Lange method.The f i n a l consumption of periodate was 9 . 8 mmoles per gram of the gum.Ethyleneglycol (10 mL) was added,the poly-aldehyde was dialyzed overnight,reduced with NaBH^ (1.0 g), neutralized with 5 ° % acetic acid,dialyzed and then freeze-dried to y i e l d the polyalcohol (120 mg).This product ( 5 mg) was hydrolyzed with 2M TFA on a steam-bath overnight and the sugars present i n the hydrolyzate (detected by p.c. i n solvent(A))were 1 found to be galactose and mannose.Quantitation (by g.l.c.) of the sugars as a l d i t o l acetates gave g a l a c t i t o l and mannitol i n the r a t i o of 2.8:1.0 . Smith hydrolysis of the polyalcohol (100 mg) was carried out with 0 . 7 5 M TFA during 20 h at room temperature.The acid was removed by evaporation and the residue a f t e r d i s s o l u t i o n i n water ( 3 mL) was preci p i t a t e d with ethanol (Yield of A, 2 5 mg).Methylation on part of t h i s material ( 5 mg) gave the re s u l t s shown i n Table V.l,column IV .The r e s t of the p r e c i -p i t a t e (20 mg) was heated with 0.1M TFA on a steam-bath for 1 7 6 one hour.After removal of the acid by evaporation the residue (B) was methylated by the Hakomori procedure and a f t e r hydro-l y s i s .reduction and acetylation gave the r e s u l t s shown i n Table V.l,column V. 1 7 7 VI. BIBLIOGRAPHY 1 7 8 BIBLIOGRAPHY 1 . " Surface Carbohydrates of the Prokaryotic C e l l " (ed.I. Sutherland).Academic Press,New Y o r k , ( 1 9 7 7 ) . 2 . J.A. Bordet, Annales de l ' I n s t i t u t Pasteur.1 1 .(1897). 1 7 7 - 2 1 3 -3 . J.R. Andrew, Amer. Chem. Soc. Symp. 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Lombardo, "Los Arboles Cultivados en los Paseos Publi-cos",Concejo Departamental de Montevideo, Montevideo ( 1 9 5 8 ) 1 9 4 . J . Hutchinson, " The Families of Flowering Plants" Vol I. Clarendon Press. O x f o r d . ( 1 9 6 9 ) . 1 9 5 . N.O. C a f f i n i and N.S. Pr i o l o de Lufano, Rev. Farm.(Buenos Aires) 1 2 1 ( 9 - 1 2 ) ( 1 9 7 8 ) 75-80. 1 9 6 . G.O. A s p i n a l l , V.P. Bhavanandan, and T.B. Christensen, J. Chem. Soc. ( 1 9 6 5 ) 2 6 7 7 - 2 6 8 4 . 1 9 0 A P P E N D I X I S T R U C T U R A L P A T T E R N S OF K L E B S I E L L A C A P S U L A R P O L Y S A C C H A R I D E S 191 X Uronic acid 0 Neutral sugar (X) 3-deoxy-L-glycero-pentulosonic acid ( x ) 4-0 (s)-l-carboxyethyl -D-glucuronic acid ( x ) 2R,3R-hex-4-enopyranosyluronic acid Pyruvate and acetate have been omitted Uronic acid absent. - 0 - 0 - 0 - 0 - - 0 - 0 - 0 - 0 - 0 I K32 , K72 0 K 5 6 (X) I - 0 - 0 - _ 0 - 0 - 0 - _ o - o I I I 0 0 0 1 I ® d > K3? K38 K22 Uronic acid i n chain, a) l i n e a r . _ X - 0 - 0 - - X - 0 - 0 - 0 - _ x - 0 - 0 -K l , K5, K63 K4, K6 K9*, K44 _ X - 0 - 0 - 0 - 0 - 0 -K70, K81 192 b) branch point on uronic acid i ) single unit side chain _ X - 0 - 0 - - X - 0 - 0 - 0 -I I 0 0 K l l , K5? K21, K24 i i ) two unit side chain - X - 0 - 0 - - X - 0 - 0 - 0 -1 I 0 0 1 I 0 0 K31 K46 i i i ) three unit side chain - X - 0 - 0 - 0 -I 0 I 0 K26 I 0 iv) plus branch points on neutral sugars X - 0 - 0 - 0 -I | | K60 0 0 0 c) branch not on uronic acid _ X - 0 - 0 - - x - O - O - O - - X - 0 I I I 0 0 0 K58 K7, K61, K62 K52, 193 • - x - 0 - 0 - • . - X - 0 - 0 - 0 -I I 0 0 K16, K54 K l ? d ) d o u b l e b r a n c h n o t o n u r o n i c a c i d 0 - X - 0 - 0 - 0 -I 0 U r o n i c a c i d i n s i d e c h a i n a ) s i n g l e u n i t s i d e c h a i n _ 0 - 0 - 0 - _ o - 0 - 0 - 0 -I I X X K2, K8 K9t K59 b ) t w o s i n g l e u n i t s i d e c h a i n - 0 - 0 - 0 - 0 - 0 -I I X 0 e x a c t l o c a t i o n o f s i d e K r , j c h a i n s n o t d e t e r m i n e d c ) t w o s i n g l e u n i t s s i d e c h a i n f o r m i n g a d o u b l e b r a n c h 0 0 1 I . 0 - 0 - 0 - - 0 - 0 - 0 - 0 -I I X X K30.K33 K27 194 d) two unit side chain i ) uronic acid terminal - 0 - 0 -I 0 K20, K23, K51. K55 X i i ) uronic acid non-terminal - 0 - 0 - - 0 - 0 - 0 - - 0 - 0 - 0 - 0 -I I I X X X I I I 0 0 0 K25, K47 K13. K74 K12, K28, K36 e) three unit side chain i ) uronic acid non-terminal - 0 - 0 - 0 - - 0 - 0 - 0 - 0 -I I 0 X 1 I X 0 I I 0 0 K18 K4l 195 APPENDIX II THE STRUCTURES OF KLEBSIELLA CAPSULAR POLYSACCHARIDES 196 K-type K l Structure K2 1 man— 3 a 1 G] .cA K4 -2G1C—^GlcA-—^Man^—2(ji cI_ K5 -AucA P A 1 4 1 V 1 2 B OAc K6 A - 2 - F u c - — ^ G l c - — ^ a n l — ^ 1 C A — a B B a Gal l l P 4 6-K7 — 2 < j l c A - — ^ M a r ^ — ^ a n ^ — ^ G l c - — ^ G l c — -a a GlcA 11 K8 - ^ G l c - — ^ G a l - — ^ G a l — B B 197 K9 GlcA -2ca 1 - — 2 - R h a - — — ^ R h a — K9* " - ^ G l c A 1 — ^ R h a 1 — ^ G a l 1 — ^ R h a 1 — ^ R h a 1 -K l l p < 4 G a l ^ I I -Ailc—-SlcA^—hal-K12 K13 Gal 1 4 P GlcA 1 a 3 •<Jalf— -Ailc-—2Rhal—2-Gal-^—^alf—a a a -^Glc-—^Man 1—^-Glc— 3 3 I I GlcA 4| PC 3 Gal K16 Gal 1 1 4 1 4 1 c ^ — ^ G l c A ^ — ^ F u c — 198 K17 K18 ha J ^ l c A l _ ^ R h a l ^ G l c 1 ^ 2 a p ^ a l 1 — - ^ G l c i — 2 R h a — l l Rha 2 l l GlcA l l Glc •Rha— a K20 GlcA 1 3 Gal 11 2 3 -=Man' + OAc K21 P-Cl Gal * 1 4 -4 IcA-—Aflanl—^an^—hal— 0 a a a B K22 <x> 1 6 Glc 11 4 -3d 199 K23 'Rha-—HJlc-2 l l Glc 6 e l GlcA Man 11 K24 -^GlcA- Man- -^Man-—2 Glc-K25 Glc 1 2 GlcA 11 P C £ Gal ^ 1 4 Glc 11 6 Glc 1 K26 4 -An cA^ 1 2l« a nl £war>i 2. Man^ kJal-200 K27 P A. kSal Glc 11 Gal-1 GlcA K28 - ^ a l - — ^ a n l — ^ a n -i l GlcA 31 1 — W Glc K30 -P C S Gal — % a n — OAc / i 6 l — G l c — l l GlcA P C S G L C 2 Man 1 K31 GlcA-1—1 Gal-K32 - 2 G a l -P A ^Rha 1 k :Rha 201 K33 P C S Gal ^ 11 OAc - ^ V l a n — ^ V l a n ^ — ^ G l c — l l GlcA K34 K36 Rha 11 -^Rha-—-Rha-—Ajicl—2^ a 1 A 1 — ^ R h a — -\al-—2Rhal—2-Rha1—^Rha— s 2 l l GlcA 4| P C S Glc K37 ® 1 6 6 Glc 1 a -4 all-W-202 (X) 2 K38 ^ l c l ^ a l l _ 4 a l l _ l l Glc Glc 1 K4l K44 6 G 1 41 GlcA - ^ G l c ^ — i R h a ^ — * G a l ^ - ^ G a l f ^ -a -^lcA^-^Rha^-2-Rha^—-2G1C^-^G1C^-8 a a B a + 0 A C K45 GlcA 11 Rha-—-Rha-—2. R h a. K46 Glc 1 3' Man ;"^P - ^ G a l - — 2 ( j a i l — ^ G l c A ^ — ^ M a n -203 K47 •Rha— a l l GlcA 1 Rha K48 3QJ^^1 3T»I 1 _ 1 2 T M — 1 'Rha-l l GalA =Glc- Rha-a a K49 GalA 11 % a n x ; G a l — a a + 0Ac K51 K52 GlcA 1 a 6 Glc 1 a G a l — a 1 2T -^Gal-—-Rha' a 1 I* GlcA- Ajal-—-Rha— l l Gal Rha 11 K53 - 3 Q IcA 1 2 Man- 1 2 1 Gal-—-Rha— 204 K54 -Glc 11 4 + 0F + 0Ac 1 4 lc^-r^GlcA-1 - ^ F u c i -K 5 5 Auc^-r^Rha— l l Gal 3 1 GlcA +0Ac K56 P ,4 6-Gal-^ G a l ^ Gal-11 Rha K57 K58 Man 11 • A u c 1 - * G a l A — — ^ a n 1 -a a GlcA P 1 4 1 F u c — 3 a a 1 Gal K 5 9 - -^Glc GlcA 11 1 _ 1 Gal^ ^ a n 1 - ^ +0Ac 1 Man-a a 2 0 5 Glc 1 - 2 G 1 C ^ — ^ G l c A ^ — ^ G a l - — 2 j v i a n l _ 2 8 a 2 a 6 1 1 Glc Glc Gal 11 1 2 ; - ^ l c A ^ - ^ M a n ^ — ' G l c ^ — * G l c -8 a 8 a - ^ G l c A ^ — h a r r— 2 G a l ^ - ^ G l c — l l Man —2<jalA——^Fuc-—2<j a l l_ +0Ac + 0F Rha 11 -A}lcA-—^anl—2-Glc^—^Man— a a 8 £ zr 1 PC£ G L C -\lcA^—-Rha^—2-Rha^—2-Glc-—^Gal^-& a a a —Rha Rha Rha Rha ^Glc— GlcA Glc 206 K72 A. - ^ G l c ^ - r ^ h a - — - R h a - — 2 R h a l _ K?4 K81 K83 P C £ Gal 1 4! GlcA -*Gal^- 1 2 1 ct a •^Rha 1—^Rha^-^lcA^^Rha 1--2Rha 1--^Gal 1--^Ga l - — - R h a — l l Gal 3 1 GlcA 20? The other possible structure i s : -^-GlcA 1—-Rha-—-Rha-—Ajail—lRha— P a a a a This structure i s l i k e K33 except OAc i s present every other repeating unit. This structure has been re-examined by E.H. M e r r i f i e l d ; Formate located on p o s i t i o n 4 of the terminal Glucose, and acetate on po s i t i o n 2 of Fucose. Acetate on p o s i t i o n 2 of Rhamnose. Acetate on p o s i t i o n 6 of Mannose,but not on a l l residues. Acetate located on p o s i t i o n 2 of Galacturonic acid,and Formate on p o s i t i o n 4 of the same sugar residue. Pyruvate on every other repeating unit. Tentative structure. 208 APPENDIX II BIBLIOGRAPHY Kl C. Erbing, L. Kenne, B. Lindberg, J. Lonngren and I. Sutherland, Carbohydr. Res. ,£0,(1976) 1 1 5 - 1 2 0 . K2 L.C. Gahan, P.A. Sandford and H.E. Conrad, Biochemistry, 6 , ( 1 9 6 7 ) 2 7 5 5 - 2 7 6 7 . K4 i ) E.H. 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Res., in press. K27 S.C. Churms, E.H. Merrifield, and A.M. Stephen,Carbohydr. Res.,81,(1980)49-58. K28 M. Curvall, B. Lindberg, J. Lonngren and W. Nimmich, Carbohydr. Res.,42, ( 1 9 7 5 ) 9 5 - 1 0 5 -K30 B. Lindberg, F. Lindh, J. Lonngren and I.W. Sutherland, Carbohydr. Res.,20, (1979)135-144. K31 CC. Cheng, S.L. Wong, and Y.M. Choy, Carbohydr. Res., 2 2 . ( 1 9 7 9 ) 1 6 9 - 1 7 4 . K32 . G.M. Bebault, G.G.S. Dutton, N. Funnel and K.L.Mackie, Carbohydr. Res. , 6 3 , ( 1 9 7 8 ) 1 8 3-192. K33 B. Lindberg, F. Lindh, J. Lonngren and W. Nimmich, Carbohydr. Res.,22,(1979)135-144. K34 J.P. Joseleau, personal communication. K38 B. Lindberg, B. Samuelson, and W. Nimmich,Carbohydr. Res., 2 0 . ( 1 9 7 3 ) 6 3 - 7 0 . 210 K4l J.P. Joseleau, M. Lapeyre, M. Vignon, and G.G.S. Dutton, Carbohydr. Res.,62,(1978)197-212. K44 G.G.S. Dutton and T.E. Folkman, Carbohydr. Res. ,28 , (1980) 3 0 5 - 3 1 5 . K46 G.G.S Dutton and K. Okutani, Carbohydr. R e s . , 8 6 , ( 1 9 8 0 ) 2 5 9 - 2 7 1 . K 4 7 H. Bjorndal, B. Lindberg, J. Lonngren, W. Nimmich and K. Rosell, Carbohydr. R e s . , 2 2 , ( 1 9 7 3 ) 2 7 2 - 2 7 8 . K48 J.P. Joseleau, personal communication. K49 J.P. Joseleau, personal communication. K51 A.K. Chakraborty and S. Stirm, Abst. Int. Symp. Carbohydr. Chem., 9 t h , London, (1978)4-39-440 K52 H. Bjorndal, B. Lindberg, J. Lonngren, M. Meszaros, J.L. Thompson and W. Nimmich, Carbohydr. Res.,31, ( 1 9 7 3 ) 93-100. K53 G.G.S. Dutton and M. Paulin, Carbohydr. Res.,82,(I98O) 1 0 7 - 1 1 7 . K54 i ) P.A. Sandford, J.R. Bamburg, J.D. Epley and T.J. Kindt, Biochemistry,£,(1966) 2808. ii)P.A. Sandford and H.E. Conrad, Biochemistry,£,(1966) 1 5 0 8 - 1 5 1 6 . K55 G.M. Bebault and G.G.S. Dutton, Carbohydr. Res.,64, ( 1 9 7 8 ) 1 9 9 - 2 1 3 . K 5 6 Y.M. Choy and G.G.S. Dutton,Can. J. Chem.,51.(1973) 3 0 2 1 - 3 0 2 6 . K 5 7 J.P- Kamerling, B. Lindberg, J. Lonngren and W. Nimmich, Acta Chem. Scand.,(B),2^,(1975) 5 9 3 -K58 G.G.S Dutton and A.V. Savage, Carbohydr. Res.,84.(1980) 2 9 7 - 3 0 5 . K59 B. Lindberg, J. Lonngren ,U. Ruden and W. Nimmich, Carbohydr. Res.,42, (1975) 8 3 - 9 3 . 211 K60 G.G.S. Dutton and J.L. Di Fabio,Carbohydr. R e s . . 8 7 . (1980) 1 2 9 - 1 3 9 . K61 i ) A.S.Rao, N. Roy and W. Nimmich, Carbohydr. R e s . , 6 7 , (1978) 4 4 9 - 4 5 6 . i i ) A . S . Rao, N. Roy and W. Nimmich, Carbohydr. R e s . , 7 6 , ( 1 9 7 9 ) 215-224 K62 G.G.S. Dutton and M.T. Yang, Carbohydr. R e s . , £ 2 , ( 1 9 7 7 ) 1 7 9 - 1 9 2 . K63 J.P. Joseleau and M.F. Marais, Carbohydr. Res , 2 2 » ( 1 9 7 9 ) 1 8 3 - 1 9 0 . K64 E.H. M e r r i f i e l d and A.M. Stephen,Carbohydr. Res. , 7 4 . ( 1 9 7 9 ) 241 - 2 5 7 . K?0 G.G.S. Dutton and K.L. Mackie, Carbohydr. R e s . , 6 2 , ( 1 9 7 8 ) 3 2 1 - 3 3 5 . K71 E.H. M e r r i f i e l d and A.M. Stephen, unpublished r e s u l t s . K72 Y.M. Choy and G.G.S. Dutton, Can. J . Chem. , £ 2 , ( 1 9 7 4 ) 684-687. K74 G.G.S. Dutton and M. Paulin, Carbohydr. Res. , 8 2 , ( 1 9 8 0 ) 1 1 9 - 1 2 7 . K81 M. Cu r v a l l , B. Lindberg, J . Lonngren, arid W. Nimmich, Carbohydr. Res.,42, ( 1 9 7 5 ) 73-82. K 8 3 B. Lindberg and w. Nimmich, Carbohydr. Res.,48, ( 1 9 7 6 ) 81-84. 212 APPENDIX III AND 13C-N.M.R. SPECTRA K'60 polysaccharide 1H n.m.r. 220 MHz, 90 C Spectrum No.1 Acetone i i 2.23 ! K 60 polysaccharide n.m.r. 20 MHz, amb.temp. 102.M+ Spectrum No.2 acetone 31 .07 ro H K 60 Compound A1 GlcA - 2 Gal~0H HOD H n.m.r. 100 MHz, 90°C . I . i i . i I I , i L I I I I I I I I I 1 I I I I It I I —U-l _L_L i I i i i i I i I I I I I I I i i i i Spectrum No.3 acetone K 60 Compound A1 GlcA-! ^ Gal^OH B C^ n.m.r. 20 MHz, amb.temp. Spectrum No> acetone 31 . 0 7 I 1 I ' I 1 I 1 I 1 I 1 1 1 I 1 I 1 I 1 i ' i ' i ' i K 60 Compound N1 Glc ^rr- Man Glcv/OH 13 C n.m.r* 20 MHz. amb.temp. 1 0 2 . 5 4 9 9 . 8 5 9 6 . 8 0 8 3 9 3 . 0 7 Spectrum No. 5 K 60, P 1 p o l y s a c c h a r i d e [ - J G I C A J hal- 2 M a n - ^ G l c — ] 8 a a B n 1 „ H n . m . r . 100 MHz, 90°C Spectrum No.6 5.33 5.27 K 60, P-| polysaccharide yZ n.m.r. 2 0 MHz, amb.temp. 101 . 4 2 Spectrum No.? acetone 31 .07 K 60 , Compound GlcA-} ^Qal- ^MarJ 2G1C~0H ^ B a a H n.m.r. 100 MHz, 90° C 5 . 5 . 3 1 Spectrum No. 11 K 60, Compound A^ ^C n.m.r. 2 0 MHz, amb.temp. 1 0 1 . 5 1 1 0 1 . 3 5 1 0 4 . 5 1 96.67 I I 9 3 Spectrum No.12 ro ro 1  K 26 , depyruvylated polysaccharide 1 H n.m.r 400 MHz , water n u l l 5 . ^ 9 4 . 5 1 5.rTs-01 4 . 6 3 [ 4 . 4 5 Spectrum No. 1 3 acetone 2 . 2 3 K 26,Compound GlcA -—^ Man~OH 1 a H n.m.r. 100 MHz, 90°C 5.32 5.20 4.92 »)0M^ -J 1 1 1 1 1 1 1 1 I I I I ' ' J 1 1 i r _i 1 1 1 1 1 J , L_l K 26,Compound GlcA - — ^ Man~OH 13 ° JC n.m.r. 20 MHz, amb.temp. Spectrum No.15 acetone 3 1 . 0 7 K 26,Compound A 2 GlcA - — ^ M a n 1—?. Man^ OH a a n.m.r. 2 0 MHz , amb.temp. 1 0 2 . 8 6 1 0 1 . 3 6 9 3 . 5 6 9 3 - 3 5 i ' i 1 i ' I 1 I 1 I r-r-' | i | Spectrum No.l? acetone 3 1 . 0 ? ro ro vo K 26, Compound Aj GlcA - — 2 . M a n 1—2 M a n 1—1 Gal~OH a a a 13 -^C n.m.r. 20 MHz , amb.temp. ' I ' I 1 I 1 l 1 l 1 I 1 I 1 1 1 I 1 I 1 I r Spectrum No.19 K 26, Compound N G l c — - Glc~OH 1 8 H n . m . r . 100 MHz ,90°C K 26 .Compound 1^ Glc - — - Glc~OH 1 3 r Spectrum No.21 B 'C n.m.r. 2 0 MHz , amb.temp. 1 0 3 . 5 0 96.81 6 1 . 6 0 acetone 3 1 * 0 ? ro 9 2 . 9 5 K 26,Compound N 2 Gal - — - Glc - — - Glc~OH *H n.m.r. 400 MHz , amb.temp. HOD 5.24 4.46 4.5 4.67* I Spectrum No.22 acetone 2 . 2 3 Spectrum No.23 K 26, Compound SH Gal GlcA 1 — ^ Man — - Gly 8 a a C^ n.m.r. 20 MHz ,amb.temp. Spectrum No. 24 acetone 31.07 0 6 0 K60, Compound X^C n.m.r. 20 MHz,amb.temp. Spectrum No.25 acetone 3 1 . 0 7 0 6 0 K60 , Compound P 2 n -^ C n.m.r. 20 MHz ,amb.temp. Spectrum NoZ6 046 K46,Compound ? 1  XH n.m.r. 400 MHz, water n u l l Spectrum No.2? acetone 2 . 2 3 ro V>) 1 . 5 2 046 K46,Compound P x -T n.m.r. 20 MHz ,amb.temp. 100.3? 97.14 101.00 101.33 95/ 97 95: 81 93.11 Spectrum No.28 ro -p-o acetone 31-0? 046 K46,Compound P 2  XH n.m.r. 400 MHz, water n u l l 5 . 1 9 4 . 8 5 4 . 6 5 5 . 3 0 5 . 0 6 4 . 6 9 Spectrum N 0 . 2 9 acetone 2 . 2 3 C. speciosa, Compound A GlcA - — - Man~OH 1 8 H n.m.r. 400 MHz, water null 5.30 Spectrum No.31 acetone 2 . 2 3 J C. speciosa, Compound GlcA - — - Man - — - GlcA - — - Man~OH , 3 a 3 H n.m.r. 400 MHz, water null HOD Spectrum No.32 acetone 2.23 245 APPENDIX IV USES OF PERACETYLATED ALDONONITRILES 246 USES OF PERACETYLATED ALDONONITRILES Peracetylated a l d o n o n i t r i l e s have been known since 1 8 9 3 . They were f i r s t used i n synthesis,a) the Wohl degradation"1' (pentoses are obtained from the peracetylated hexononitriles), b) formation of 1-amino-l-deoxyalditols ,etc.The synthesis of these derivatives has been studied and the best conditions ob-served were the treatment of the aldose with hydroxylamine hy-drochloride i n pyridine and then acetylation and dehydration done at the same time with acetic anhydride at high tempera -ture-^ ( i n the case of glucose.it has been observed that at low temperature a c y c l i c derivative i s p r e f e r e n t i a l l y formed). As derivatives of a n a l y t i c a l interest,the ,the trimethyl-4 s i l y l a t e d oximes were f i r s t used f o r g. l . c . separations.Lance and Jones-* used the peracetylated a l d o n o n i t r i l e s for g.l.c. se-paration of the methyl ethers of D-xylose.Several stationary phases have been employed since 1 9 7 1 to improve the separation 6—8 of the PAAN (peracetylated a l d o n o n i t r i l e s ) ~ . The g . l . c . retention times and g.l.c.-m.s. f o r the a c e t y l -9 6 1 ated a l d o n o n i t r i l e s of methyl ethers of mannose7 and glucose ' have been reported.The methylated sugars i n a methylation a-na l y s i s can now be completely characterized and i d e n t i f i e d by using g.l.c.-m.s. of the derived a l d o n o n i t r i l e s and a l d i t o l a-c e t a t e s . A l l methyl ethers of mannose can be separated by g.l.c. as a l d o n o n i t r i l e s ( see Table l ) . I n the course of t h i s inves-t i g a t i o n , the peracetylated a l d o n o n i t r i l e s of the methyl ethers were also used as means of analyzing the methylation products 247 TABLE 1 RELATIVE G.L.C. RETENTION-TIMES OF PERACETYLATED ALDONONITRILES  OF METHYL ETHERS OF D-GLUCOSE AND D-MANNOSE. Methyl ether — Retention times(on 5% of butanediol succinate) — D-Glucose D-Mannose 2 , 3 , ^ , 6-tetra- 0 .94 1 . 0 0 2,4,6 - t r i - 1.45 1.59 2 , 3 . 6 - t r i - 2 . 0 0 1.65 3 . 4 , 6 - t r i - 1.85 1.89 2 , 3 , 4 - t r i - 2 . 0 0 2 . 0 3 2,6 - d i - — 2.28 4,6 - d i - — 2.45 2 , 3 - d i - 3 - 5 0 2.55 3 , 6 - d i - — 2.85 2,4 - d i - 2.84 3 .16 3 , 4 - d i - 3 -58 3 .68 - 2 , 3 , 4 , 6 - t e t r a : 5 ^ 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l - D - g l u c o -n o n i t r i l e . e t c . - Relative to 5 - 0 - a c e t y l - 2 , 3 , 4 , 6 - t e t r a - 0 - m e t h y l -mannononitrile.Data from references 9) and 1 0 ) . TABLE 2 RELATIVE G.L.C. RETENTION TIMES OF PERACETYLATED ALDONONITRILES  OF METHYL ETHERS OF SUGARS. Methyl ether - Relative retention times OV-225 3% -2 , 3 ,4 ,6 - Glc 1.00 °-2 ,4 ,6 - Glc 1.39 2 ,4 ,6 - Man 1.59 2 ,4 ,6 - Gal 1.65 2 ,3 ,4 - Glc 1.85 2 ,3 ,6 - Glc 1. 98 3,4,6 - Man 1.98 4,6 - Man 2.20 4,6 - Gal 2.44 3,6 - Glc 2.59 2 ,3 - Glc 2.81 2,3 - Gal 2.81 2 — Glc 3.02 - 2 , 3 , 4 , 6 - Glc : 5 - 0-acetyl - 2 , 3 , 4 , 6-tetra - 0-methylglucononitrile. n i t r i l e . - Programmed at 165° for 4 min,then at 2°/min to 220°, isothermal for 32 min. - 6.6 min . 249 e s p e c i a l l y to separate the 2,3,4-and 2,3,6-tri-0-methylgluco-ses (see Table 2). Ketoses can also be characterized and separated by g.l . c . and g.l.c.-m.s. as the peracetylated oximes 1 1. In general,the PAAN have been used for examining neutral 12 13 sugars from polysaccharides .glycoproteins .mucopolysaccha-13 14 rid e s .and products r e s u l t i n g from Smith degradation .Another use for these d e r i v a t i v e s , i s the determination of the degree of polymerization of oligosaccharides and polysaccharides as well as the i d e n t i f i c a t i o n of the reducing end.The general proce -dure,known as the Morrison procedure 1-*, involves the reduction with NaBH^ of the reducing end,hydrolysis,and treatment of the hydrolyzate with hydroxylamine hydrochloride i n pyridine f o l -lowed by acetic anhydride.The free sugars are converted into the PAAN and the a l d i t o l into the a l d i t o l acetate.After g.l.c. separation and q u a n t i t a t i o n , i t i s possible to determine the r a t i o of free sugar/reducing end which gives the D.P.By iden-t i f i c a t i o n of the a l d i t o l acetate,the reducing end i s deter-mined. A l d i t o l acetates have been used for c d . measurements 1^, to determine the absolute configuration of t h e i r parent sug-ars ( D or L ).Sugars whose a l d i t o l s are meso compounds (galactose,xylose,etc.)cannot be studied,as they do not show c d . activity.The peracetylated aldononitriles,as they keep the c h i r a l i t y of the parent sugars and chromophores are pre-sent, show c d . a c t i v i t y and can be used to determine the ab-solute configuration of the parent sugars.They can be e a s i l y 2 5 0 TABLE 3 CIRCULAR DICHROISM OF THE PERACETYLATED ALDONONITRILES. Peracetylated Configuration Sign of the ald o n o n i t r i l e s c.d. curve Arabinose L -Arabinose D + Fucose L -Fucose D + Galactose D + Glucose D + Mannose D + 2 5 1 prepared and separated by preparative g . l . c . Results of this i n v e s t i g a t i o n are given i n Table 3« Experimental. Preparation of the peracetylated a l d o n o n i t r i l e s . The free sugars ( 5 - 1 0 mg) are dissolved i n hydroxylamine hydrochloride i n pyridine ( 5 ^ , 1 mL) and heated on a steam-bath f o r 1 5 min.The solution i s cooled to room temperature and acetic anhydride ( 1 mL) i s added and heated f o r 1 h on a steam-bath. Water ( 5 - 1 0 mL) i s added and the PAAN are extracted with CHCl^. After removal of the solvent,the samples are ready f o r g. l . c . Separation of the PAAN by g. l . c . The PAAN derived from the free sugars were separated on a column of 3% OV -225 on Gas Chrom Q and the temperature used was 2 1 0 ° isothermal.The PAAN derived from methylated sugars were separated on the same column,with the temperature pro-gramme 1 6 5 ° f o r 4 min,then 2 % i i n to 2 2 0 ° for 3 2 min. Ci r c u l a r dichroism measurements of the PAAN. Samples of the PAAN isola t e d by preparative g . l . c . on OV -225 3% (isothermal 2 1 0 ° ) were dissolved i n a c e t o n i t r i l e and the c d . curves were measured. 252 BIBLIOGRAPHY 1. A. Wohl, Ber.,26,(1893) 730. 2. C.H. Winestock and J.W.E. Plaut, J . Org. Chem.,26,(1961) 4456-4462. 3. V. Deulofeu, P. Cattaneo, and G. Mendivelza, J . Chem. S o c , (1934) I47rl48. 4. C C . Sweeley, R. Bentley, M. Makita, and W.W. Wells, J . Amer. Chem. S o c , 8 5 , ( I 9 6 3 ) 2497T2507. 5. D.G. Lance and J.K.N. Jones, Can. J . Chem. ,45_, (1967)1995^8. 6. B.A. Dmitriev, L.V. Backinowsky, O.S. Chizhov, B.M. Zolo-tarev, and N.K. Kochetkov, Carbohydr. Res. , 12 ,(1971)432-5. 7. J . Szafranek, CD. Pfaffenberger, and E.C. Horning, Anal. Lett. , 6 , ( 1 9 7 3 ) 4 7 9 - 4 9 3 . 8. R. Varma, R.S. Varma, and A.H. Wardi, J . Chromatog.,77. (1973) 222-227. 9. F.R. Seymour, R.D. Plattner, and M.E. Slodki, Carbohydr. Res. , 4 4 , ( 1 9 7 5 ) 181 -198. 10. F.R. Seymour, M.E. Slodki, R.D. Plattner,and A. Jeanes, Carbohydr. Res. , £ 2 , (1977) 153-166. 11. F.R. Seymour, J.E. Stouffer, and E.CM. Chen, Carbohydr. Res. ,83_, (1980) 201-242. 12. R. Varma, R.S. Varma, W.S. Allen,and A.H. Wardi,J. Chroma-tog. ,86.(1973)205r210. 1 3 a . T . P . Mawhinney, M.S. Feather, G.J. Barbero,and J.R. Marti-nez, Anal. Biochem..101.(1980) 112-117. 13b.R. Varma and R.S. Varma, J . Chromatog.,128,(1976) 4 5 r 5 2 . 14. J.K. Baird, M.J. Holroyde, and E.C. Ellwood, Carbohydr. Res.,22,(1973) 464^467. 15. I.M. Morrison, J. Chromatog., 108, (1975) 36lr-364. 2 5 3 G.M. Bebault, J.M. Berry, Y.M. Choy, G.G.S. Dutton, N. Funnel,L.D. Hayward, and A.M. Stephen, Can. J . Chem., £ 1 . ( 1 9 7 3 ) 3 2 4 . 3 2 6 . 

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