Fluorinated ligands to probe the binding of the O-polysaccharide of Vibrio cholerae 0:1 to antibodies

The internal parts of the O-antigen (O-specific polysaccharide, O-PS) of Vibrio cholerae O1, serotype Ogawa and Inaba are identical. They consist of (1→2)-linked moieties of 4-amino-4,6-dideoxy- a -D-mannopyranose (D-perosamine), the amino groups of which are acylated with 3-deoxy-L- glycero-tetronic acid. The Ogawa O-PS is terminated by a D-perosaminyl moiety whose O-2 is methylated. Cholera is lethal, infectious disease caused by bacterial pathogens of the Vibrio cholerae O:1 family. Systematic prevention of the disease has not been achieved because of lack of a protective vaccine. A part of our effort is directed towards development of a conjugate vaccine for cholera based on a synthetic carbohydrate antigen. Hydrogen bonding plays an important role in the recognition of carbohydrate antigens by their homologous antibodies. Understanding these phenomena is expected to provide structural criteria helpful in the design of a synthetic vaccine. The occurrence of hydrogen bonding interactions during binding can be probed by measuring ligand binding following hydroxyl group substitution by hydrogen. Cessation of binding of a ligand upon replacement of a hydroxyl group in it with hydrogen is indicative of involvement of that hydroxyl group in a critical hydrogen bonding interaction. Retention of binding following the OH → H substitution suggests noninvolvement of that hydroxyl group in binding. The source of hydrogen for such an interaction, the protein or the carbohydrate, can be deduced from binding/nonbinding following replacement of the same hydroxyl group with fluorine. The fluorine atom can not donate, but can accept hydrogen bonding. Thus, significant loss of binding following OH → F substitution in a ligand which had bound in its original form can be interpreted as hydrogen bond donation by the OH group which had been replaced. On the other hand, retention of binding, or only slightly diminished binding, indicates donation of hydrogen for hydrogen bonding interaction by the protein. Synthetic routes to analogs of the terminal tetronylated perosamine moiety of the O-PS of Vibrio cholerae O:1, specifically fluorinated at positions 2, 3, 2' and 4' have been explored. Various strategies have been applied to achieve introduction of the fluorine atom with the desired stereochemistry at a predetermined position in either a suitable derivative of 3-deoxy-L-glycero -tetronic acid or perosamine. The outlined chemistry produced seven out of the nine desired final compounds. All desired fluorinations were accomplished with the exception of the fluorination at the 3 position. Each final target compound involved a multi-step synthetic route. All new compounds were characterized by physicochemical constants and by NMR (1H and 13C) spectroscopy. NMR spectra was interpreted by first-order analysis, and where feasible, by two dimensional homo- and hetero-nuclear correlation experiments.