Reactions And Immunochemistry Of Carbohydrates

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

Our research is currently aimed at developing a conjugate vaccine for cholera. Cholera is a serious enteric disease caused mainly by two strains of Vibrio cholerae O:1, Ogawa and Inaba. The work towards a potent vaccine for cholera involves, among other things, conjugation to proteins of synthetic oligosaccharides that mimic the structure of O-specific polysaccharides (O-PS) of the relevant Gram negative bacteria, and evaluation of the immunogenicity of the resulting neoglycoconjugates. The haptens required for conjugation result from multi step, sophisticated chemical synthesis, which is a formidable, very laborious process. For example, the synthesis of the spacer-equipped hexasaccharide fragment representing the terminus of the O-PS of Vibrio cholerae O:1, serotype Ogawa involves over thirty synthetic steps. It starts with the commercially available methyl a-D-mannopyranoside, which is converted to a disaccharide glycosyl acceptor and a disaccharide glycosyl donor. Condensation of these provides us, after further chemical manipulations, with the final hexasaccharide ready to be conjugated. The synthesis requires aglycon exchange in a methyl glycoside for the linker molecule via the intermediate trimethylsilylethyl (SE) glycoside. The preparation of SE-glycosides in the mannose series is notoriously problematic, giving only moderate yields of the desired products. According the recently improved protocol, using a glycosyl halide as the glycosyl donor and silver trifluoromethanesulfonate (AgOTf) as the promoter, the reported yield of the desired product was below 80%. Clearly, there is a need to improve preparation of SE-glycosides in the mannose series, especially when such compounds have to be prepared in a later stage of a multi step chemical synthesis. In order to improve the overall economy of our preparation of the required linker-equipped hexasaccharide, we have explored possibilities to generally improve the preparation of SE-glycosides in the mannose series. Unlike previous use of glycosyl halides as glycosyl donors in the reaction with trimethylsilyl ethanol (SE-OH), we used thioglycosides in conjunction with N-iodosuccinimide and AgOTf. During this work, we have found also that, in the mannose series, the nature of the protecting groups around the pyranose ring has a profound effect upon the outcome of the glycosylation of SE-OH. After carefully researching the proper combination of protecting groups, especially those at position 2 and 6 in thioglycosides, and optimizing some fundamental variables involved in the reaction of the aforementioned glycosyl donors with SE-OH, we were able to find the optimum reaction conditions. Using ethyl 1-thioglycosides as glycosyl donors, especially those bearing a pivaloyl or a nonparticipating group at O-2, the corresponding 2-(trimethylsilyl)ethyl a-D-mannopyranosides were obtained in excellent yields. In addition to improving the overall yields of substances we need for conjugation, our successful improvement of the preparation of SE-glycosides in the mannose series is generally useful in synthetic carbohydrate chemistry. During the past year, we have made also improvements in other aspects of the synthesis of oligosaccharides that mimic the structure of the O-PS of Vibrio cholerae O:1, serotype Inaba and Ogawa. This opens a more efficient way for exploring the structural requirements for a potential, medically acceptable vaccine for cholera.