Polysaccharide antigens are T cell-independent antigens, and do not induce immune B cell memory. Consequently, vaccines based on polysaccharides have limited clinical usefulness and induce short-lasting antibody responses in adults. Their immunogenicity can be enhanced by conjugation to an immunogenic carrier protein, generating T cell-dependent glycoconjugate antigens able to induce immunological memory. However, these glycoconjugates suffer from some problems. Recent investigations have found a group of structurally distinct bacterial polysaccharides able to activate T cells in vivo and in vitro. They present a zwitterionic charge motif distributed along the chain and, for this reason, they are called zwitterionic polysaccharides (ZPSs). This zwitterionic charge motif is believed to be responsible for their particular immunological behavior. The integrity of the zwitterionic motif is essential for the biological activity of ZPS. However, it must be clarified if the introduction of the zwitterionic motif into a naturally non-zwitterionic polysaccharide confers to the resulting ZPS the ability to activate T cells without protein conjugation. To this end, zwitterionic oligomers must be obtained by chemical modification of fully synthetic, non-zwitterionic polysaccharide fragments. With these compounds in hand it would be possible to correlate structural and conformational properties of the ZPS with their biological activity, in terms of charge pattern and minimum molecular weight required for immunogenicity. For this purpose the capsular polysaccharide (CPS) of Salmonella typhi was chosen as a suitable model for our investigation. S. typhi is an encapsulated Gram-negative bacterium that causes typhoid fever. Its CPS, commonly named Vi antigen, is an anionic polymer composed of alpha-(1-4)-linked N-acetylgalactosaminuronic acid repeating units predominantly O-acetylated at position 3. Recent studies indicated the importance of the acetylation for the immunogenicity. The structure of the Vi antigen makes it an ideal candidate for our investigation, since its fragments can be easily converted into zwitterionic derivatives by formal N-deacetylation, without introducing huge structural modifications. We designed a flexible synthetic strategy in order to obtain, from common building blocks, two distinct series of oligomers: the ones corresponding to the natural structure and their zwitterionic derivatives. Moreover, the role of 3-O-acetylation will be investigated by the synthesis of both fully 3-O-acetylated and fully 3-non-O-acetylated oligosaccharides. We selected N-phenyltrifluoroacetimidate moieties as the best leaving group in the glycosyl donors. Moreover, all the oligomers were endowed with a suitable linker at the anomeric position of the reducing end in order to facilitate subsequent conjugation to multivalent scaffolds. In the first part of the work the synthesis of oligomers non acetylated at position 3 is described. The glycosyl donor and acceptor were obtained from commercially available D-galactosamine hydrochloride. Their glycosylation was performed by a slow addition of a diluted solution of the Lewis acid via a syringe pump, and complete alpha stereoselectivity was obtained. We also successfully applied a more efficient elongation strategy based on disaccharide donors to the synthesis of the Vi trisaccharide and its zwitterionic derivative. Evaluation of the biological behavior of the target compounds was also performed by ELISA competitive assay. The second part of the work was focused on the synthesis of oligomers acetylated at C-3. In particular, a different approach based on pre-oxidized galacturonate building blocks obtained via inversion of C-4 configuration of commercially available D-glucosamine hydrochloride is described.
SYNTHESIS OF FRAGMENTS OF SALMONELLA TYPHI CAPSULAR POLYSACCHARIDE AND THEIR ZWITTERIONIC ANALOGUES / M.m. Fusari ; tutor: L. Lay ; coordinator: E. Licandro. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2014 Nov 24. ((27. ciclo, Anno Accademico 2014.
|Titolo:||SYNTHESIS OF FRAGMENTS OF SALMONELLA TYPHI CAPSULAR POLYSACCHARIDE AND THEIR ZWITTERIONIC ANALOGUES|
|Supervisori e coordinatori interni:||LICANDRO, EMANUELA|
|Data di pubblicazione:||24-nov-2014|
|Parole Chiave:||oligosaccharide-based vaccines; glycoconjugate vaccines; zwitterionic polysaccharides; ZPS; Vi antigen; Salmonella typhi; typhoid fever; oligosaccharides; chemical synthesis; alpha-stereoselective glycosylations; disaccharide donors; ELISA competitive assay|
|Settore Scientifico Disciplinare:||Settore CHIM/06 - Chimica Organica|
|Citazione:||SYNTHESIS OF FRAGMENTS OF SALMONELLA TYPHI CAPSULAR POLYSACCHARIDE AND THEIR ZWITTERIONIC ANALOGUES / M.m. Fusari ; tutor: L. Lay ; coordinator: E. Licandro. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2014 Nov 24. ((27. ciclo, Anno Accademico 2014.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/fusari-matteo-maria_phd2014-11-24|
|Appare nelle tipologie:||Tesi di dottorato|