Chemoenzymatic synthesis of glycosylated and sulfated CCR5 N-terminal peptide library

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Co-localization of Tyrosine (Tyr) sulfation and O-glycosylation (CSOG) is an emerging global pattern of post-translational modifications (PTMs). CSOG is the major PTM pattern on the N- terminal peptide of CC chemokine receptor 5 (CCR5). CCR5 is involved in critical human diseases such as cancers and inflammatory diseases, and the N-terminal peptide of CCR5 is essential for the binding between CCR5 and its ligands. The access to sulfated and glycosylated N-terminal peptides of CCR5 (CCR5-SGNTPs) and any other peptides with CSOG is hindered by diversified patterns of glycosylation and sulfation, lability of sulfation, and complex sugar structures. This proposal aims to develop efficient chemoenzymatic methods for synthesizing glycopeptides containing O-glycans and Tyr sulfation. Therefore, we are aiming to efficiently synthesize CCR5-SGNTPs to build up the access to CCR5-SGNTPs and further provide new clues for related biomedical research. Lability of sulfate group during synthesis and complexity of sugar structures are the major obstacles to the achievement of the library of CCR5-SGNTPs. We provide a novel and efficient chemoenzymatic approach, merging two well- studied synthetic methodologies to overcome the obstacles. One of the two methodologies is solid-phase site-selective sulfation, and the other one is chemoenzymatic synthesis of glycopeptides. Site-selective sulfation with divergently protected Tyrs can efficiently build diversified sulfation patterns, and chemoenzymatic synthesis of glycopeptides can synthesize glycopeptides with complex sugar structures in high regio- and stereo-selectivity with high fidelity. Notably, the mild conditions (slightly basic) of glycosyltransferases catalyzed reactions are ideal for keeping the integrity of the labile sulfate group. Glycosylated-amino acids (GAAs) with core sugars and Fmoc-Tyrs with divergent protecting groups can be synthesized efficiently with current synthetic methodologies. Then the two classes of building blocks will be selectively incorporated into solid phase peptide synthesis (SPPS) to obtain protected sulfated and glycosylated peptides. After removal of protecting groups, glycosyltransferases will be used to extend the core sugars to generate larger and more complex glycopeptides following biosynthetic pathways. After binding study of the synthesized library, detailed information about how the two PTMs co-regulate binding processes will be obtained. Moreover, a peptide library with structure-defined CCR5-SGNTPs will provide standards for proteomics and glycomics research of CCR5-SGNTPs.