Synthesis of a BSA-Lex glycoconjugate and recognition of Lex analogues by the anti-Lex monoclonal antibody SH1: The identification of a non-cross reactive analogue

A Lex trisaccharide functionalized with a cysteamine arm was prepared and this synthesis provided additional information on the reactivity of N-acetylglucosamine O-4 acceptors when they are glycosylated with trichloroacetimidate donors activated with excess BF3·OEt2. In turn, this trisaccharide was conjugated to BSA lysine side chains through a squarate–mediated coupling. This BSA-Lex glycoconjugate displayed 35 Lex haptens per BSA molecule. The relative affinity of the anti-Lex monoclonal antibody SH1 for the Lex antigen and analogues of Lex in which the d-glucosamine, l-fucose or d-galactose residues were replaced with d-glucose, l-rhamnose and d-glucose, respectively, was measured by competitive ELISA experiments. While all analogues were weaker inhibitors than the Lex antigen, only the analogue of Lex in which the galactose residue was replaced by a glucose unit showed no binding to the SH1 mAb. To confirm that the reduced or loss of recognition of the Lex analogues by the anti-Lex mAb SH1 did not result from different conformations adopted by the analogues when compared to the native Lex antigen, we assessed the conformational behavior of all trisaccharides by a combination of stochastic searches and NMR experiments. Our results showed that, indeed, the analogues adopted the same stacked conformation as that identified for the Lex antigen. The identification of a trisaccharide analogue that does not cross-react with Lex but still retains the same conformation as Lex constitutes the first step to the design of a safe anti-cancer vaccine based on the dimeric Lex tumor associated carbohydrate antigen.

Figure optionsDownload as PowerPoint slide