A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides

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Abstract
We report a novel strategy for the preparation of neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-[α-Kdo-(2→4)-α-Kdo-(2→] and a Kdo trisaccharide [α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→] of Re-type and chlamydial LPS, respectively. Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined neoglycoconjugates. Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine. After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA. We have compared the immunological reactivity of such defined neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such neoglycoconjugates are immunogenic and well suited as antigens for the study of epitope specificities of monoclonal antibodies. In addition, we have compared these conjugates with those in which ligands were coupled by glutardialdehyde to BSA. Our approach proved to be superior since the latter led upon immunization of mice to a relatively high percentage of antibodies that reacted with glutardialdehyde derivatized BSA without the carbohydrate ligand. This was not the case for cysteamine-spacered ligands coupled via their isothiocyanate-derivatives.