Synthesis of MUC4 glycopeptide antigen structures : Strategic synthesis of MUC4 glycopeptide using Fmoc-Thr-OAll for the TN-antigen incorporation

Glycosylation is the most abundant form of post-translational modification. In particular, changes in the protein O-glycosylation structures are associated with multiple diseases. This study is important as mucin glycoproteins are highly abundant on epithelial cells, are crucial for the protection of human organs, but also become key players in cancer progression. The mucins serve as diagnostic biomarkers for different diseases. Only a few studies have been done to evaluate MUC4 as a potential cancer biomarker, and often without considering the dense and aberrant mucin glycosylation observed on tumor cells. In contrast, the role of mucin MUC1 in tumor progression is rather well explored. This project aims to synthesize a ThomsenNouveau antigen (TN-antigen) glycosylated Fmoc-Threonine (Thr) amino acid building block and a few MUC4 glycopeptide antigen structures, which later will be conjugated to an immune carrier protein MUC4, a relevant target for pancreas and lung cancer. The long-term aim is that these vaccine constructs will be immunized in mice and used to generate polyclonal antibodies and later to develop monoclonal antibodies directed against these unique MUC4 glycopeptide epitopes presented on tumor cells. For this study, the TN-antigen was used, an Oglycan structure with a GalNAc on a serine or threonine structure, which is linked to a poor prognosis in cancer patients. The Fmoc-GalNAc-Thr was successfully prepared in 6 steps starting from Galactose (Gal) and Thr. Here, a new synthesis strategy using the O-allyl group was evaluated for the protection of the Thr C-terminal instead of the standard tert-butyl group previously applied. This would allow for orthogonal protecting group flexibility to use the building block in later synthesis of more complex glycosylated amino acids besides the FmocGalNAc-Thr, which here was used to successfully prepare 4 MUC4 glycopeptide antigens and their corresponding unglycosylated peptides by Fmoc-Solid-Phase Peptide Synthesis, FmocSPPS. These peptides will be ready for immune carrier protein conjugation and antibody epitope mapping.