Vince Pozsgay

Synthesis of the monosaccharide units of the O-specific polysaccharide of Shigella sonnei☆

Abstract The monosaccharide components of the O-specific polysaccharide 1 of the lipopolysaccharide of the enteropathogenic bacterium Shigella sonnei were synthesized as their methyl glycosides 2 and 3 in their natural anomeric form. The key intermediate to the diaminotrideoxygalactose derivative 2 was ethyl 3-O-acetyl-2-deoxy-2-phthalimido-1-thio-β- d -glucopyranoside ( 9 ) that was converted to its ditosylate 10 . Regioselective deoxygenation at C-6 followed by nucleophilic displacement of the secondary tosyloxy group by azide afforded the 4-azido thioglycoside 13 . Methyl trifluoromethanesulfonate-assisted methanolysis of 13 gave the O-glycoside 14 . Replacement of the phthalimido by an acetamido group followed by catalytic reduction of the azido group led to the diamino-trideoxygalactose derivative 2 . The precursor to the l -altruronic acid derivative 3 was methyl α- l -glucopyranoside ( 19 ) that was routinely converted to the benzylidene-protected 2,3-anhydro-allopyranoside 22 . Regioselective opening of the epoxide ring by NaN 3 afforded the 2-azido derivative 23 that was benzylated at HO-3. Hydrolytic removal of the benzylidene group followed by TEMPO oxidation of C-6 and subsequent esterification with MeI gave the key l -azido-altruronic acid intermediate 29 that was transformed to the acetamidoaltruronic acid derivative 3 . High resolution NMR data of the altruronic acid derivatives indicate that the conformation of their pyranose ring is crucially dependent on the substitution pattern: the 2-azido altruronic acid derivatives prefer the 4 C 1 conformation whereas the 2-acetamido congeners exist preferentially in the 1 C 4 conformation.

Towards a Synthetic Glycoconjugate Vaccine Against Neisseria meningitidis A

Albumin conjugates of synthetic fragments of the capsular polysaccharide of the Gram-negative bacterium Neisseria meningitidis serogroup A were prepared. The fragments include monosaccharides 1 [alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)] and 2 [6-O-P(O)(O(-))(2)-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], disaccharide 3 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], and trisaccharide 4 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)]. Two monosaccharide blocks were employed as key intermediates. The reducing-end mannose unit featured the NHAc group at C-2, and contained the aminoethyl spacer as the aglycon for the final bioconjugation. The interresidual phosphodiester linkages were fashioned from an anomerically positioned H-phosphonate group in a 2-azido-mannose building block. The spacer-linked saccharides 1-4 were N-acylated with hepta-4,6-dienoic acid and the resulting conjugated diene-equipped saccharides were subjected to Diels-Alder-type addition with maleimidobutyryl-group functionalized human serum albumin to form covalent conjugates containing up to 26 saccharide haptens per albumin molecule. Complete (1)H, (13)C, and (31)P NMR assignments for 1-4 are given. Antigenicity of the neoglycoconjugates containing 1-4 was demonstrated by a double immunodiffusion assay which indicated that a fragment as small as a monosaccharide is recognized by a polyclonal meningococcus group A antiserum and that the O-acetyl group(s) present in the natural capsular material is not essential for antigenicity.

Synthetic Shigella Vaccines: A Carbohydrate–Protein Conjugate with Totally Synthetic Hexadecasaccharide Haptens

Advances in antibacterial glycoconjugate vaccines depend on the availability of defined fragments of the bacterial cell-surface polysaccharides. Oligosaccharide portions of the O-specific polysaccharide of the human pathogen Shigella dysenteriae type 1, have been assembled and attached to a human serum albumin (HSA; see below) to give semisynthetic vaccines that provide new directions against enteric diseases.

Synthesis and two-dimensional nuclear magnetic resonance analysis of a tetra- and a hexa-saccharide fragment of the O-specific polysaccharide of Shigella dysenteriae type 1☆

The synthesis of the tetra- and hexa-saccharide methyl glycosides alpha-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->3)- alpha-L-Rhap- OMe (1), and alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1--> 3)-alpha-D-GlcpNAc- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-OMe (3) is described, which represent various epitopes of the O-specific polysaccharide of Shigella dysenteriae type 1. The following monosaccharide intermediates were used: 1,3-di-O-acetyl-2-O-benzoyl-4-O-benzyl-alpha-L-rhamnopyranose (6 alpha), methyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (7), methyl 2,4-di-O-benzoyl-1-thio-alpha-L-rhamnopyranoside (8), 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl bromide (9), methyl 3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-1-thio-beta-D- galactopyranoside (13), methyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D- galactopyranoside (16), and 2-azido-4,6-O-benzylidene-3-O-bromoacetyl-2-deoxy-beta-D- glucopyranosyl chloride (19). A detailed analysis of the 1H and 13C NMR spectra of oligosaccharides 1 and 3 confirmed that the hexasaccharide 3 better approaches the conformation of the native polysaccharide, than either 1 or the homologous pentasaccharide 41.

A new strategy in oligosaccharide synthesis using lipophilic protecting groups: synthesis of a tetracosasaccharide

Abstract The use of lipophilic, acyl-type protecting groups in the synthesis of higher-membered oligosaccharides is described by the example of oligosaccharides corresponding to the O-specific polysaccharide (O-SP) of Shigella dysenteriae type 1. Thus, O -stearoylated and O -lauroylated l -rhamnose and d -galactose precursors, respectively, were synthesized and were combined together with a 2-azido-2-deoxy- d -glucopyranosyl donor to form a fully protected lipidated repeating unit of the O-SP. This module was condensed with another tetrasaccharide containing conventional blocking groups. The resulting lipidated octasaccharide was isolated in a pure form by adsorption to a reverse phase adsorbent from which it could be selectively desorbed by alcoholic solvents. Subsequent chain elongation using the conventionally protected tetrasaccharide module as glycosyl donor afforded oligosaccharides up to and including a tetracosasaccharide. The proposed approach can substantially alleviate the difficulties associated with the conventional silica gel chromatographic purification of protected oligosaccharide intermediates and utilizes environmentally friendly solvents that are less expensive than the solvents used for silica gel chromatography. A new, highly efficient method is also proposed for the synthesis of carbohydrate acetals and cyclic orthoesters employing scandium trifluoromethanesulfonate as the catalyst.

Synthesis of a tetrasaccharide building block of the O-specific polysaccharide of Shigella dysenteriae type 1

Abstract A glycosyl trichloroacetimidate derivative ( 1 ) of the tetrasaccharide α- d -Gal p -(1→3)-α- d -Glc p NAc-(1→3)-α-L-Rha p -(1→3)-α-L-Rha p was synthesized in a highly stereoselective, stepwise manner, using methyl 1-thioglycosides of L-rhamnose, 2-azido-2-deoxy-D-glucose and D-galactose, as major intermediates. The protecting group scenario in compound 1 permits regioselective deblocking at its “non-reducing end” unit. Therefore 1 is a suitable intermediate for the preparation of extended fragments of the title polysaccharide.

Saccharide/protein conjugate vaccines for Bordetella species: preparation of saccharide, development of new conjugation procedures, and physico-chemical and immunological characterization of the conjugates

Bordetellae are Gram-negative bacilli causing respiratory tract infections of mammals and birds. Clinically important are B. pertussis, B. parapertussis and B. bronchiseptica. B. pertussis vaccines have been successful in preventing pertussis in infants and children. Veterinary vaccines against B. bronchiseptica are available, but their efficacy and mode of action are not established. There is no vaccine against B. parapertussis. Based on the concept that immunity to non-capsulated Gram-negative bacteria may be conferred by serum IgG anti-LPS we studied chemical, serological and immunological properties of the O-specific polysaccharides (O-SP) of B. bronchiseptica and B. parapertussis obtained by different degradation procedures. One type of the B. parapertussis and two types of B. bronchiseptica O-SP were recognized based on the structure of their non-reducing end saccharide; no cross-reaction between the two B. bronchiseptica types was observed. Competitive inhibition assays showed the immunodominance of the non-reducing end of these O-SP. Conjugates of B. bronchiseptica and B. parapertussis O-SP were prepared by two methods: using the anhydro-Kdo residue exposed by mild acid hydrolysis of the LPS or the 2,5-anhydromannose residue exposed by deamination of the core glucosamine of the LPS, for binding to an aminooxylated protein. Both coupling methods were carried out at a neutral pH, room temperature, and in a short time. All conjugates, injected as saline solutions at a fraction of an estimated human dose, induced antibodies in mice to the homologous O-SP. These methodologies can be applied to prepare O-SP-based vaccines against other Gram-negative bacteria.

Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates.

There is no licensed vaccine for the prevention of shigellosis. Our approach to the development of a Shigella vaccines is based on inducing serum IgG antibodies to the O-specific polysaccharide (O-SP) domain of their lipopolysaccharides (LPS). We have shown that low molecular mass O-SP-core (O-SPC) fragments isolated from Shigella sonnei LPS conjugated to proteins induced significantly higher antibody levels in mice than the full length O-SP conjugates. This finding is now extended to the O-SPC of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1. The structures of O-SPC, containing core plus 1-4 O-SP repeat units (RUs), were analyzed by NMR and mass spectroscopy. The first RUs attached to the cores of S. flexneri 2a and 6 LPS were different from the following RUs in their O-acetylation and/or glucosylation. Conjugates of core plus more than 1 RU were necessary to induce LPS antibodies in mice. The resulting antibody levels were comparable to those induced by the full length O-SP conjugates. In S. dysenteriae type 1, the first RU was identical to the following RUs, with the exception that the GlcNAc was bound to the core in the beta-configuration, while in all other RUs the GlcNAc was present in the alpha-configuration. In spite of this difference, conjugates of S. dysenteriae type 1 core with 1, 2, or 3 RUs induced LPS antibodies in mice with levels statistically higher than those of the full size O-SP conjugates. O-SPC conjugates are easy to prepare, characterize, and standardize, and their clinical evaluation is planned.

Synthesis of di- to penta-saccharides related to the O-specific polysaccharide of Shigella dysenteriae type 1, and their nuclear magnetic resonance study

The syntheses of oligosaccharide fragments of the O-specific polysaccharide of the lipopolysaccharide of Shigella dysenteriae type 1 are described, including disaccharides methyl O-alpha-D-mannopyranosyl-(1-->2)-alpha-D-galactopyranoside (1), and methyl O-(2-deoxy-2-propionamido-alpha-D-glucopyranosyl)-(1-->3)-alpha-L- rhamnopyranoside (2), trisaccharide methyl O-alpha-D-galactopyranosyl-(1-->3)-O-(2-acetamido-2-deoxy-alpha-D- glucopyranosyl)-(1-->3)-alpha-L-rhamnopyranoside (3), tetrasaccharide methyl O-alpha-L-rhamnopyranosyl-(1-->2)-O-alpha-D-galactopyranosyl-(1-->3)- O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1-->3)-alpha-L-rhamno -pyranoside (4), and pentasaccharide methyl O-alpha-L-rhamnopyranosyl-(1-->3)-O-alpha-L- rhamnopyranosyl-(1-->2)-O-alpha-D-galactopyranosyl- (1-->3)-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1-->3)-alpha-L- rhamnopyranoside (5). The following monosaccharide building blocks were used as starting compounds: methyl 6-O-tert-butyldiphenylsilyl-3,4-O-isopropylidene-alpha-D-galact opy ranoside (8), methyl 3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-1-thio-beta-D- galactopyranoside (11), methyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-alpha- D-glucopyranoside (16), methyl 2-azido-4,6-O-benzylidene-2-deoxy-1-thio-alpha-D- glucopyranoside (18), methyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (21), methyl 2,3,4-tri-O-benzoyl-1-thio-alpha-L-rhamnopyranoside (22), 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl bromide (23), and methyl 4-O-benzyl-alpha-L-rhamnopyranoside (24). Nuclear magnetic resonance data indicate that oligosaccharides 4 and 5 partially mimic the conformation of the O-specific polysaccharide of S. dys. type 1.

CONFORMATIONAL STABILIZATION OF THE ALTRURONIC ACID RESIDUE IN THE O-SPECIFIC POLYSACCHARIDE OF SHIGELLA SONNEI/PLESIOMONAS SHIGELLOIDES

Complete assignments for the 1H- and the 13C-NMR spectra of the O-specific polysaccharide of S. sonnei/Plesiomonas shigelloides are reported. Evidence is presented that in this polysaccharide both pyranose residues exist preferentially in the 4C1 chair conformation and that the polysaccharide exists in the zwitterion form.