Abstract
Carbohydrates play vital roles in all forms of life. Attached to proteins and lipids they serve as recognition elements in several biological interaction processes, such as intercellular communication, immune response, viral and parasitical infections, signal transduction, and development. Synthetic mimics of natural oligosaccharides have contributed to a better understanding of
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these complex recognition events. In addition, these mimics can be used to modulate, or intervene in undesired (pathological) interaction processes. Glycopeptides have been demonstrated to be efficient oligosaccharide mimics. Moreover, they are readily prepared in a library format, which provides rapid access to a variety of potential high-affinity ligands for carbohydrate-binding proteins. This thesis describes the preparation of combinatorial solid-phase glycopeptide libraries, and their use for the identification of glycopeptides that mimic oligosaccharides in binding to a receptor. Several glycosylated amino acid building blocks having a nonnatural S- or N-glycosidic linkage were synthesized. These building blocks were used to prepare S-,N-glycopeptides that may have an enhanced stability towards acidic conditions and enzymatic degradation. Two combinatorial solid-phase glycopeptide libraries containing Gal(b1-O)Thr, and Gal(b1-S)Cys and Gal(b1-N)Asn, respectively, were prepared according to the split-and-mix method, combined with the ladder synthesis strategy. Their screening with Ricinus communis agglutinin indicated that O-, S-, and S-,N-glycopeptides were recognized by the lectin with similar recognition patterns, and with a similar affinity. In addition, the S-,N-glycopeptides were completely stable towards enzymatic degradation by a b-galactosidase, whereas O-glycopeptides were not completely stable. The two galactosylated glycopeptide libraries, and a library containing Lac(b1-O)Thr were screened with galectin-1 and galectin-3 to identify ligands that may have the potential to inhibit malignant galectin-carbohydrate interactions. The screening of the galactosylated glycopeptide libraries primarily yielded peptide hits, whereas the lactosylated glycopeptide library predominantly yielded glycopeptide hits. Subsequent SPR-based inhibition assays indicated that the lactosylated glycopeptides were more potent galectin inhibitors than lactose. The (galactosylated glyco)peptides were able to inhibit the binding of h-Gal-3, but displayed a different effect on the binding of h-Gal-1, which may be attributed to aggregation of h-Gal-1. Subsequent solid-phase inhibition assays indicated that some (glyco)peptides were more efficient galectin inhibitors than lactose, and intrafamily cross-reactivity was observed. Additionally performed cell assays with cells of human tumour lines indicated that some lead compounds could effectively inhibit the cell surface binding of the galectins. A glycopeptide library containing Fuc(b1-S)Cys and Fuc(b1-N)Asn was prepared and screened with a-L-fucose-specific Ulex europaeus agglutinin-I to study the effect of the configuration of the S- or N-glycosidic linkage on the strength and specificity of carbohydrate-protein interactions. The results indicated that the lectin was able to recognize nonnatural b-linked S-,N-fucosylated glycopeptides. Some RCA120- and h-Gal-1-specific glycopeptides were used to develop SPR-based biosensors for the sensitive detection of carbohydrate-binding proteins in solution. The best sensitivity was obtained on glycopeptide-derivatized self-assembled monolayers consisting of a 9:1 ratio of 3-mercapto-1-propanol/11-mercaptoundecanoic acid. Detection limits of 0.13 and 0.9 nM were obtained for RCA120 and h-Gal-1, respectively. Additionally, the effect of interfering compounds on the sensitivity of the optimized biosensor was investigated, demonstrating remarkably different behavior of the respective analytes in the presence of a model protein mixture.
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