Affinity determination of ricinus communis agglutinin ligands identified from combinatorial O- and S-,N-glycopeptide libraries.

Two combinatorial glycopeptide libraries were synthesized on solid support via the “split-and-mix” method combined with the ladder synthesis strategy. The O-glycopeptide library contained Gal(‚1-O)Thr, whereas the S-,N-glycopeptide library contained both Gal(‚1-S)Cys and Gal(‚1-N)Asn. In this model study, the two libraries were screened against the fluorescently labeled lectin Ricinus communisagglutinin (RCA120). The screening results showed that both O- and S -o rS-,N-glycopeptides were recognized by the lectin with similar amino acid recognition patterns. Surface plasmon resonance interaction studies demonstrated that both the selected S -o rS-,N-glycopeptide hits and the O-glycopeptides bound to the lectin with a similar affinity. Structure 19, containing two glycosylated cysteine residues, bound to the receptor with the highest affinity (KA ) 3.81 10 4 M -1 ), which is comparable to N-acetyllactosamine. Competition assays, in which some selected glycopeptides and methyl ‚-D-galactopyranoside competed for the binding site of immobilized RCA120, showed that the glycopeptide-lectin interaction was carbohydrate-specific. Incubation of the Oand S-,N-glycopeptides with ‚-galactosidase demonstrated the complete stability of S-,N-glycopeptides toward enzymatic degradation, whereas O-glycopeptides were not completely stable.