Synthesis of a Lewis X trisaccharide glycoconjugate and competitive binding studies with Lewis X analogues

The optimized synthesis of a Lewis X trisaccharide carrying a cysteamine adduct is described. Glycosylation at O-4 of the L-Fuc-alpha-(1→3)- D-GlcNAc-beta-(1→O)-All disaccharide with a trichloroacetimidate galactosyl donor was achieved with a 64% yield using BF3·OEt 2 under controlled conditions. The conjugations of Lewis X and analogues of Lewis X were accomplished and the hapten incorporations were determined. The antigens were coupled to BSA employing the squarate linker system or by reductive amination, with the highest incorporation number obtained for Lewis X with 35 haptens per BSA. Lewis X was additionally conjugated to Tetanus toxoid using the adipate linker system. The relative binding energies of methyl Lewis X analogues were determined using competitive ELISA procedures. The binding experiments revealed that substitutions of N-acetylglucosamine for glucose and fucose for rhamnose did not completely abolish cross reactivity with native Lewis X. In contrast, substitution of galactose for glucose eliminated recognition by anti-Lewis X mAb SH1.