Well-defined glycoconjugates via chemo-enzymatic synthesis of oligosaccharides

Abstract

Glycoconjugate vaccines are one of the most effective ways of preventing bacterial infections. They are generally composed of a poor immunogenic antigen that is covalently linked to a carrier protein to increase its immunogenicity and convert the response against saccharide antigens from T independent to T-dependent. Typically, such vaccines are obtained as heterogeneous and complex mixtures of poly or oligosaccharides by non-specific methods, rendering impossible quantitative correlations between structure and immunological properties. A breakthrough for carbohydrate-based vaccines occurred in 2004 when a Cuban research group introduced a synthetic oligosaccharide vaccine against Haemophilus influenzae type b in human. Besides lacking bacterial impurities, synthetic oligosaccharides present the advantage of bearing at their reducing end a chemical handle for chemoselective conjugation, minimal batch-to-batch variability and higher quality control standards during process manufacturing, in respect to carbohydrate from natural sources. New techniques such as solid phase automated synthesis and chemo-enzymatic synthesis reduced reaction time and the use of protective groups during synthetic steps. The thesis describe the use of chemical and chemo-enzymatic synthesis to obtain oligosaccharide fragments of Group B Streptococcus type III and Neisseria meningitidis serogroup X (MenX) respectively. In the first case, chemical synthesis was used to support structural studies on the minimal epitope, while chemo-enzymatic synthesis was used to produce a MenX fragment directly conjugable to the carrier protein, underlining the advantages of this procedure in comparison to polysaccharide purification from bacterial fermentation. Finally, with the aim of evaluating the role of oligosaccharide length and point of attachment to the carrier protein, a small MenX glycoconjugates library was synthesized with random or site-selective conjugation.