Synthesis of Shigella flexneri O-Antigenic Repeating Units - ACS

3 Synthesis of ShigellaflexneriO-Antigenic Repeating Units

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 19, 2017 | http://pubs.acs.org Publication Date: September 29, 1983 | doi: 10.1021/bk-1983-0231.ch003

Conformational Probes and Aids to Monoclonal Antibody Production D A V I D R. B U N D L E , M A R G A R E T A . J. G I D N E Y , S T A F F A N J O S E P H S O N , and H A N S - P E T E R W E S S E L National Research Council of Canada, Division of Biological Sciences, Ottawa, Ontario K1A 0R6, Canada

The primary structures of a large number of bacterial O -antigens are now well defined (1,2) and these data may be used to rationalize the serological classification of enterobacteriaceae (2). Since this classification is based upon antibody binding to c e l l wall polysaccharides, appreciation of the three dimensional structure of the antigen w i l l enhance the understanding of subtle serological interrelationships and cross-reactions. The inter pretation of the shapes of O-antigens of known primary structure is just beginning and in this chapter we describe how synthetic oligosaccharides are prepared and how models of O-antigen confor mation are inferred from NMR experiments on such compounds. Also outlined is the manner in which the same oligosaccharides may be incorporated into the protocol of the hybrid-myeloma (hybridoma) technique for production of monoclonal antibody toward O -antigens. In this way oligosaccharides for which the three dimensional structure is well established may be used to select specific antibodies. These are of both practical use in serodiagnosis, and are ideally suited to the study of the molecular and stereochemical basis of antibody binding. Structure and Conformation The structures of the O-antigens of Shigella flexneri were elucidated by Kenne and his co-workers (3-5). A tetrasaccharide [-2)Rha(α1-2)Rha(α1-3)Rha(α1-3)GlcNAc(βl-] is the repeating unit of the simplest of the S. flexneri O-antigens, the variant Y. The serogroups 1-5 possess this basic repeating unit struc ture but in addition carry a~^"glucopyranosyl and 0-acetyl substituents. Since the variant Y structure possessed glycosidic linkages involving only the ring hydroxyl groups and was devoid of either branching residues or charged groups, this 0antigen was considered a good model to investigate antigenantibody Interaction. This reasoning rested upon the assumption 0097-6156/83/0231-0049$06.00/0 Published 1983, American Chemical Society

Anderson and Unger; Bacterial Lipopolysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

50

BACTERIAL

LIPOPOLYSACCHARIDES

that the p r e f e r r e d s o l u t i o n conformation of an antigen was the most l i k e l y conformer to be bound by an antibody, and that the geometry of a simple, f a i r l y r i g i d polymer, composed of l i n e a r repeating u n i t s , would most e a s i l y provide t h i s conformational i n f o r m a t i o n . At the time t h i s p r o j e c t was s t a r t e d the work of Rees and h i s co-workers ( 6 7 ) * already e s t a b l i s h e d that homopolysaccharide and d i h e t e r o g l y c a n shape could be s a t i s f a c t o r i l y explained by c o n s i d e r a t i o n of the two g l y c o s i d i c t o r s i o n a l angles < ( > and ip. These define the conformation of a g l y c o s i d i c linkage i n v o l v i n g a secondary hydroxyl group. If the linkage involves the primary hydroxyl of a hexopyranoside a t h i r d t o r s i o n a l angle u> defines the extra degree of freedom that 1+6 linkages possess. As j>. f l e x n e r i 0-chains are devoid of 1+6 linkages the conformation of the Y 0-antigen could be modelled with some confidence, provided that due c o n s i d e r a t i o n was given to the exo-anomeric e f f e c t . Lemieux's work had e s t a b l i s h e d , that to a good approximation the g l y c o s i d i c conformation could be a n t i c i p a t e d by s e t t i n g the 0-1 to aglyconic carbon bond a n t i p e r i p l a n a r to the C - l to C-2 bond (8,9). A n a l y s i s of X-ray data (10) and t h e o r e t i c a l c a l c u l a t i o n s supported t h i s approach (11,12). In t h i s arrangement the non-bonded i n t e r a c t i o n s seemed to be best accommodated when the C - l to 0-1 bond e c l i p s e d the a g l y c o n i c carbon to hydrogen bond. Thus i t appeared that a reasonable model of the Y 0-antigen conformation could be p r e d i c t e d with some confidence, a view l a t e r substantiated by semi-empirical c a l c u l a t i o n s and NMR measurements ( 1 3 ) . n a c


S

Oligosaccharides corresponding to the a n t i g e n i c determi nants of the Y polysaccharide were required to e i t h e r map the antibody combining s i t e , or i f s u i t a b l y d e r i v a t i s e d , to e l i c i t a n t i b o d i e s . Such a r t i f i c i a l antigens (14) could then provide a n t i b o d i e s that would react with the 0-antigen and have a known binding s p e c i f i c i t y . We therefore synthesized a range of d i - , t r i - and t e t r a s a c c h a r i d e s corresponding to the Y 0-antigen. At the time these haptens were intended to provide p o l y c l o n a l but monospecific a n t i b o d i e s , but as a r e s u l t of subsequent develop ments the s t r u c t u r e s a l s o proved to be i n v a l u a b l e aids to the hybrid-myeloma (hydridoma) technique (15). Synthesis of Oligosaccharides The d e t a i l s of the s y n t h e t i c approach we have adopted f o r the preparation of a l l p o s s i b l e d i - and t r i s a c c h a r i d e sequences of the Y polysaccharide repeating unit have been published (1619), together with the syntheses of two of the four p o s s i b l e t e t r a s a c c h a r i d e s (19^20). The synthesis of a t h i r d t e t r a saccharide sequence not p r e v i o u s l y reported i s described below. Sequential Chain Extension. The general s y n t h e t i c approach adopted i n our published work was to employ s e q u e n t i a l chain extension r e a c t i o n s rather than block s y n t h e s i s . Since two of



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BUNDLE ET AL.

Shigella flexneri O-Antigenic Repeating Units

51

the four g l y c o s i d i c linkages i n the repeating unit were to the 0-2 p o s i t i o n of an crL-rhamnopyranose unit a general blocking procedure was required to provide t h i s unit i n s u i t a b l y protected form. This was e f f e c t i v e l y done by adopting the general concepts of Garegg and h i s co-workers (21) and Lemieux and h i s co-workers (22), whereby the base s t a b i l i t y of 1,2orthoesters was e x p l o i t e d to provide benzylated 1,2-orthoesters. These d e r i v a t i v e s possessing p e r s i s t e n t blocking groups at p o s i t i o n s 0-3, 0-4, and 0-6 and a l a t e n t acetate at 0-2 have considerable s y n t h e t i c v a l u e . Accordingly 3,4-di-0-benzyl-l,20-(l-methoxyethylidene)-3-L-rhamnopyranose could be q u a n t i t a t i v e l y converted to 2-(>-acetyl-3,4-di-CHbenzyl-a-Lrhamnopyranosyl c h l o r i d e (^) (18,23) and t h i s d e r i v a t i v e was p a r t i c u l a r l y e f f e c t i v e i n s i l v e r trlfluoromethanesulphonate ( t r i f l a t e ) promoted Koenigs-Kuorr r e a c t i o n s . Thus good y i e l d s of rhamnose o l i g o s a c c h a r i d e s c o n t a i n i n g both 1+2 and 1+3 linkages were obtained by c o u p l i n g t h i s g l y c o s y l h a l i d e with s e l e c t i v e l y blocked 3,4-di-0-benzyl-a-L-rhamnopyrano§ide d e r i v a t i v e s (4 and &), and with the 2,4-di-O-benzoyl-a-Lrhamnopyranoside (2) (18-20). The general scheme of t h i s chemistry i s i l l u s t r a t e d i n Figure 1, which describes the synthesis of the t e t r a s a c c h a r i d e sequence: GlcNAc(3l-2)Rha ( l-2)Rha( l-3)Rha(a)-0-R. Other features of the s y n t h e t i c scheme worthy of note are the s e l e c t i v e removal of acetate e s t e r s i n the presence of benzoate e s t e r s (18-20) and the s t a b i l i t y of the phthalimido f u n c t i o n a l i t y to t r a n s e s t e r i f i c a t i o n c o n d i t i o n s (16). Furthermore, the conversion of the phthalimido group to an acetainido group was achieved under s e l e c t i v e h y d r a z i n o l y s i s conditions (16), which preserved the i n t e g r i t y of the OJmethoxycarbonyloctyl ester grouping, an e s s e n t i a l element i n the e f f i c i e n t coupling of the carbohydrate hapten to p r o t e i n (14). The use of 3,4,6-tri-0-acetyl-2-deoxy-2-phthalimido-3-Dglucopyranosyl bromide (£) (24) had been d i c t a t e d by the low r e a c t i v i t y at the 0-2 p o s i t i o n of 3,4-di-O-benzyl-o-Lrhamnopyranosides (e.£. £) • For example, o>-methoxycarbonyloctyl 3,4-di-O-benzyl-a-L-rhamnopyranosides had f a i l e d to react with either 2-acetamido-3,4,6-tri-0-acetyl-2-deoxy-a-D-glucopyranosy1 c h l o r i d e or the corresponding 1,2-oxazoline d e r i v a t i v e (16); however, use of the bromosugar £ gave good y i e l d s of the d e s i r e d oligosaccharides• a

a

Block Synthesis. In contrast to t h i s published work we have r e c e n t l y i n v e s t i g a t e d the use of block synthesis to o b t a i n a r e l a t e d frame s h i f t e d t e t r a s a c c h a r i d e sequence, Rha(al-3)Rha ( l - 3 ) G l c N A c ( 3 l - 2 ) R h a ( ) - 0 - R , p r e v i o u s l y unavailable to us. This s t r u c t u r e was considered e s s e n t i a l since s e r o l o g i c a l screening had shown the t r i s a c c h a r i d e Kha( l-3)GlcNAc(3l-2)Rha( )-0-R to possess higher a c t i v i t y than e i t h e r of the p r e v i o u s l y synthesized t e t r a s a c c h a r i d e s , R h a ( l - 2 ) R h a ( l - 3 ) R h a ( l - 3 ) G l c N A c ( 3 ) - 0 - R or a

a

a

a

a

a

a



BACTERIAL LIPOPOLYSACCHARIDES

R=(CH ) C0 CH 2

8

2

3

Figure 1. Synthesis ofa chemical repeating unit of the Shigella flexneri variant Y O-antigen, employing a sequential chain extension strategy.


3.

BUNDLE ET AL.


GlcNAc(3l-2)Rha( l-2)Rha(al-3)Rha(a)-0-R. The synthesis of the new t e t r a s a c c h a r i d e i s summarized i n Figure 2. The synthesis of d i s a c c h a r i d e was accomplished by the r e a c t i o n of cu-methoxycarbonyloctyl 3,4-di-O-benzyl-a-Lrhamnopyranoside (19) with 3,4,6-tri-0-acetyl-2-deoxy-2phthalimido-fl-D-glucopyranosyl bromide (24) (7) as p r e v i o u s l y described (16,17). The s e l e c t i v e l y protected d i s a c c h a r i d e glycoside £ c o n s t i t u t e s one of the two b u i l d i n g u n i t s of the target t e t r a s a c c h a r i d e . In order to prepare the second d i s a c charide f o r the block s y n t h e s i s , methyl 2,4-di-O-benzoyl-or-Lrhamnopyranoside (^j^), made according to the general method of Garegg and Hultberg (25), was reacted with e i t h e r t r i - O - a c e t y l - a L-rhamnopyranosyl bromide (^) or 2-0-acetyl-3,4-di-0-benzyl-a-Lrhamnopyranosyl c h l o r i d e (1^), i n dichloromethane with respec t i v e l y mercury(II) cyanide or mercury(II) cyanide together with mercury(II) bromide. Disaccharides acid 13 were obtained i n 93% and 75% y i e l d s , r e s p e c t i v e l y , and t h e i r conversion to d i s a c charide g l y c o s y l h a l i d e s was I n v e s t i g a t e d under a v a r i e t y of c o n d i t i o n s . I n i t i a l l y the r e a c t i o n of 1£ with trimethylbromos i l a n e (23) was studied but even under the most f o r c i n g c o n d i tions examined, an eight molar excess of trimethylbromosilane i n toluene at 80° f o r 18 hr with or without c a t a l y t i c amounts of zinc bromide, the s t a r t i n g m a t e r i a l ^ was recovered unchanged. The most s a t i s f a c t o r y c o n d i t i o n s f o r conversion of 12 to U proved to be the r e a c t i o n with dibromomethyl methyl ether (27) i n absolute dichloromethane. In the presence of c a t a l y t i c amounts of zinc bromide ]£ gave ^4 i n 50% y i e l d , a f t e r 15 hr at -15° and 5 hr at 0 ° , followed by column chromatography on s i l i c a g e l . Monosaccharide bromides were produced as side products and these, together with the major product, were trapped and i s o l a t e d as the i s o p r o p y l g l y c o s i d e s ^ and i n order to confirm t h e i r s t r u c t u r e . Conversion of the d i s a c c h a r i d e g l y c o s i d e 13 to i t s corresponding g l y c o s y l h a l i d e was not p o s s i b l e using dibromo methyl methyl ether due to the l a b i l i t y of the benzyl ether groups. However, a c e t o l y s i s of gave the 1-acetate 1£ from which the corresponding g l y c o s y l h a l i d e could be r e a d i l y obtained. Use of a d e r i v a t i v e of t h i s type would provide a t e t r a s a c c h a r i d e intermediate from which a pentasaccharide could r e a d i l y be prepared i n a f a s h i o n analogous to the conversion of % v i a 6 to 8 (Figure 1). When bromide 14, i n molar excess, was reacted with the s e l e c t i v e l y blocked d i s a c c h a r i d e 9 i n dichloromethane i n the presence of mercury(II) cyanide as c a t a l y s t and 4A molecular sieve as the a c i d acceptor, a 90% y i e l d of the f u l l y blocked tetrasaccharide was obtained a f t e r column chromatography. Removal of b l o c k i n g groups was accomplished by the hydrogenolysis of the benzyl ether and benzylidene a c e t a l groups i n a c e t i c a c i d , followed by t r a n s e s t e r i f i c a t i o n . The deblocked t e t r a s a c c h a r i d e jJJ had NMR parameters i n agreement with i t s s t r u c t u r e . This could be confirmed by comparison with chemical s h i f t data f o r the a


53


BACTERIAL LIPOPOLYSACCHARIDES

0(CH ) 2

8

C0 CH 2

3

CH BzIO -

i

?

^

oi-i

H

Bzoryro-

r

HO

0

OBz

AcNH/ OH


C

10

F C H ^ T ^ O ^

OR

OAc

Z

I

OBz

CH R « B r ,R » A c 1

R*0-

2

R'»CI, R «Bzl

R 0

2

OAc

Z

R'.OCH(CH,) .R«.Ac

^

2

. ,

R

0

C

H

s

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2 ,

R

A

c

13

R «0CH ,

14

R • Br, R * A c

16

R

17

R • OAc, R « Bzl

1

3

1

R «Bzl 2

2

«0CH(CH3) ,R «Ac

1

2

2

1

2

OtCH^COzCHj

OtCH^ C 0 C H 2

CI

BzlO-J^p-^/

1

R,in

Q^O

AcNH/ 0

0 CHjT^O'/ A c O - J ^ — / AcO 18

OAc

AcNH/ 0

OBz

' 0

c

H

0

I OH

CHj ' Q

H

0

H

19

Figure 2. Block synthesis of a tetrasaccharide repeating unit of the Y O-antigen.


3

*

3.

BUNDLE ET AL.


55

p r e v i o u s l y synthesized o l i g o s a c c h a r i d e s . These have been exhaustively studied by *H and C NMR and the r e s u l t s c o r r e l a t e d with s t r u c t u r e and s o l u t i o n conformation (13). Horton and Samreth have reported the p r e p a r a t i o n of a d e r i v a t i v e s i m i l a r to 1^ but i t s conversion to a g l y c o s y l h a l i d e proceeded v i a a c e t o l y s i s and conventional treatment with hydrogen bromidea c e t i c a c i d . Their d i s a c c h a r i d e bromide gave only poor y i e l d s of a t r i s a c c h a r i d e on r e a c t i o n with a 2-acetainido-4,b-0-benzylidene2-deoxy-3-D-glucopyranoside (28). Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 19, 2017 | http://pubs.acs.org Publication Date: September 29, 1983 | doi: 10.1021/bk-1983-0231.ch003

1 3

Conformation

by NMR

and Semi-empirical C a l c u l a t i o n s

I n v e s t i g a t i o n of the p r e f e r r e d s o l u t i o n conformations of the o l i g o s a c c h a r i d e s synthesized by the above mentioned procedures was c a r r i e d out by semi-empirical c a l c u l a t i o n s and NMR methods (13)• The c a l c u l a t i o n s are a type of hard sphere treatment which take i n t o account an energy c o n t r i b u t i o n due to the exo-anomeric e f f e c t . The c a l c u l a t i o n s r e f e r r e d to as HSEA c a l c u l a t i o n s have been developed and used by Lemieux et a l . (29^,30) p a r t i c u l a r l y f o r the blood group determinants. The method and i t s use i n conjunction with *H and C NMR i s w e l l reviewed and explained by Bock (31). In o u t l i n e , experimental NMR measurements that provide conformational i n f o r m a t i o n , namely chemical s h i f t s , coupling constants, spin l a t t i c e r e l a x a t i o n rates (1/T.) and nuclear Overhauser enhancements (nOe), are used In combination with HSEA c a l c u l a t i o n s to i d e n t i f y c o n s i s t e n t o l i g o s a c c h a r i d e conformations. Heteronuclear three-bond, C-O-C-H coupling con stants ( Ji3£ i ) can provide information about the g l y c o s i d i c t o r s i o n a l angles (31,32). Proton chemical s h i f t d i f f e r e n c e s caused by short ( 2.7A) proton-oxygen distances can also be r e l a t e d to conformational features (13)• The magnitudes of both spin l a t t i c e r e l a x a t i o n rates and nOe parameters are i n t r i n s i c a l l y dependent upon proton-proton i n t e r n u c l e a r distances (33)* These d i s t a n c e s , e s p e c i a l l y those between protons on adjacent pyranose r e s i d u e s , can be r e l a t e d to the g l y c o s i d i c conformation. The anomeric proton and the a g l y c o n i c proton are always i n c l o s e proximity (8,9,29-31), and i f a second proton-proton distance between pyranose residues i s a v a i l a b l e these two distances can then define a unique pair of 4>,\|/ values for the g l y c o s i d i c linkage i n v o l v e d . An example of the q u a l i t a t i v e r e l a t i o n s h i p of T to conformation i s shown i n Figure 3. Since the c o n t r i b u t i o n by adjacent protons ( j ) to the r e l a x a t i o n of a p a r t i c u l a r proton ( i ) i s p r o p o r t i o n a l to r " , where r i s the i n t e r n u c l e a r d i s t a n c e between proton i and proton j , only those protons i n close proximity m a t e r i a l l y a f f e c t the r e l a x a t i o n rate (1/T.) of proton i (33). In the case of the t r i s a c c h a r i d e Rha(al-2)Rha( l-3) Rha(a)-0-R (a-b-c) H-l of rhamnose unit b r e c e i v e s a r e l a x a t i o n c o n t r i b u t i o n ( i n a d d i t i o n to that from H-2 of rhamnose b) from H-3 of the 'reducing rhamnose c. However the geometry of the terminal al+2 linkage places H-5 of rhamnose a close to H-l of b 1 3

3

H

1

6

a

1



Figure 3. Relaxation of the three anomeric protons ofa rhamnose trisaccharide sequence belonging to the Y O-antigen.



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BUNDLE ET AL.


57

(Figure 4). Therefore, i n comparison to the other anomeric protons H-la and H-lc, which receive only one s u b s t a n t i a l i n t e r r i n g r e l a x a t i o n c o n t r i b u t i o n , proton H-lb receives two such c o n t r i b u t i o n s ; H-5a/H-lb and H-lb/H-3c. Thus H-lb relaxes much f a s t e r than e i t h e r H-la or H-lc (13,18)* The dependence of nOe upon distance a l s o i n v o l v e s an r"^~"term and these measurements provide a more convenient method f o r estimating conformational preference, e s p e c i a l l y when performed i n the d i f f e r e n c e mode (34). This method was used e x t e n s i v e l y for the j>. f l e x n e r i o l i g o s a c c h a r i d e s and the Y-polysaccharide and the r e s u l t s were i n e x c e l l e n t agreement with HSEA c a l c u l a t i o n s of conformational preference (13). The shape of an octasaccharide derived from t h i s model and c o n s i s t i n g of two repeating u n i t s i s presented i n Figure 5. It can be n o t i c e d that the methyl groups C-6b, C-6a and C-6c bracket the polar acetamido group along one edge of the Y-polysaccharide s u r f a c e . We have proposed t h i s as a l i k e l y s i t e for antibody binding (13) and f o r t h i s reason we synthesized the t e t r a s a c c h a r i d e 1£. Monoclonal A n t i b o d i e s At the outset of t h i s work we had intended to r a i s e p o l y c l o n a l a n t i b o d i e s to our s y n t h e t i c haptens. However, developments i n immunology provided a dramatic advance when Koehler and M i l s t e i n (15) showed that myeloma antibody of predefined s p e c i f i c i t y could be s y s t e m a t i c a l l y generated v i a somatic c e l l f u s i o n techniques (15,35). The technique i s summarized i n Figure 6. A Balb/C mouse i s immunized with the antigen of i n t e r e s t e i t h e r as a pure antigen, or a component of a c e l l or a crude e x t r a c t . A f t e r a s u i t a b l e time i n t e r v a l to allow the development of a good immune response the spleen c e l l s (Blymphocytes) of the mouse are mixed with myeloma tumour c e l l s , adapted to t i s s u e c u l t u r e . The two c e l l populations are induced to fuse by polyethylene g l y c o l (PEG) and subsequently only h y b r i d c e l l s are permitted to grow because the s e l e c t i v e medium i n which the fused c e l l s are p l a t e d s e l e c t s against both parental c e l l types. The c u l t u r e supernatants from each micro-well are analyzed a f t e r two weeks f o r antibody a c t i v i t y against pure a n t i g e n . A c t i v e hybrid wells are cloned and re-screened f o r antibody a c t i v i t y . Since these cloned c e l l l i n e s possess the p r o p e r t i e s of tumour c e l l s they may be stored and propagated at will. As a s c i t e s these tumour c e l l l i n e s produce large q u a n t i t i e s (up to 100 mg/mouse) of monoclonal antibody. Such homo geneous molecules are i d e a l f o r s t r u c t u r a l studies and i n v e s t i gations of antibody-antigen b i n d i n g . The production of such hybridoma l i n e s to s p e c i f i c carbohydrate determinants i s f a c i l i t a t e d by the a v a i l a b i l i t y of chemically defined antigens and we have demonstrated t h i s f o r the human blood group B determinant (36)* However, for anti-LPS antibody we have a l s o incorporated the s y n t h e t i c antigens into a screening p r o t o c o l



Figure 4. Ball and stick (a) and CPK (b)plots of the disaccharide Rha(al-2)Rha showing the proximity of protons H5a to H lb and H la to H2b and H2a.


C/J

m

5

JO

>

η

Ο "Ό Ο

-v

> r r

2

m

00


Figure 5. CPKplot of an octasaccharide composed of two repeating units of the Y O-antigen. The φ,ψ pairs used to generate the model were derivedfrom published work ( 13) and correspond to the minimum energy conformers of each glycosidic linkage.


S'

I"

«"«..

!

i

i

η

X

ET

fD

Synthesis of Shigella flexneri O-Antigenic Repeating Units - ACS

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