Synthesis and Optimization of Group B Streptococcus Capsular Polysaccharide Fragments Using the Glyconeer

Abstract

Group B Streptococcus (GBS) is a major neonatal pathogen and a leading cause of infant mortality worldwide. Management of GBS infection relies on the prophylactic and therapeutic use of antibiotics. This approach has limited efficiency and increases antibiotic resistance. Carbohydrate conjugate vaccines are consensually regarded as the best possible alternative and several formulations have been and are now in clinical trials. In general, carbohydrate vaccine development suffers from shortfalls associated with the availability oligosaccharide libraries. Chemically pure glycans enable the prosecution of structure-immunogenicity studies that elucidate minimal epitopes and the intermolecular interactions responsible for recognition and immunity. One of the most promising methodologys to produce libraries of glycans is Automated Glycan Assembly (AGA). In this work, glycans from GBS are produced by AGA using the commercial synthesizer Glyconeer. The entire process is optimized since the production of building blocks to the automated oligosaccharide synthesis process. Ready-to-use building blocks (BBs) are required in bulk to do automated experiments in a continuous way. The synthesis of BBs can be achieved with a variety of different methods and the choice is usually based on the chemist preference and experience. This work optimizes and streamlines the strategies for the synthesis of building blocks for AGA bearing a thiolate aglycon as a leaving group. The synthetic schemes are completed and upscaled with minimal column purifications. This thesis goes on to describing the optimization of elongation cycles that introduce each BB in the growing oligosaccharide chain during AGA. The first target is GBS type IV capsular polysaccharide. In the synthesis of GBS type IV, the major bottleneck was the assembly of the Glc-α-1,4-Gal cis linkage. A trisaccharide containing this linkage was successfully synthesized. However, the low yields obtained rendered the elongation of the chain unsuccessful. The other target structures were related with GBS type III. With only three different BBs, four different GBS type III fragments were assembled. A sialic acid moiety present in the native polysaccharide was introduced enzymatically, proving that the combination of enzymatic sialylation with AGA is an expeditious approach to the preparation of complex glycans for vaccine development. Overall, the work in this thesis is a contribution to the standardization of the chemical work involved in AGA and a steppingstone for the fast and cheap production of GBS related glycans.