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

Abstract

A Le x 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 BF 3·OEt 2. In turn, this trisaccharide was conjugated to BSA lysine side chains through a squarate–mediated coupling. This BSA-Le x glycoconjugate displayed 35 Le x haptens per BSA molecule. The relative affinity of the anti-Le x monoclonal antibody SH1 for the Le x antigen and analogues of Le x 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 Le x antigen, only the analogue of Le x 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 Le x analogues by the anti-Le x mAb SH1 did not result from different conformations adopted by the analogues when compared to the native Le x 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 Le x antigen. The identification of a trisaccharide analogue that does not cross-react with Le x but still retains the same conformation as Le x constitutes the first step to the design of a safe anti-cancer vaccine based on the dimeric Le x tumor associated carbohydrate antigen.