AVVISO DI SEMINARIO

Dott.ssa Claudia Bello

(Università degli Studi di Firenze)

Accessing Homogeneous Glycoproteins via Auxiliary-Assisted Sequential Glycosylation and Ligation of Peptides

The development of effective methodologies for the production of homogeneous proteins carrying complex PTMs is essential for studying the role of the PTMs in protein function and misfunction.

We established a strategy for the synthesis of peptides carrying multiple and complex PTMs, focusing at first on the preparation of homogenously glycosylated peptides. The tumor marker MUC1, a protein abundantly O-glycosylated in his extracellular domain, was chosen as a synthetic target.

Chemically synthesized mucin peptides are conjugated to a photocleavable ligation auxiliary, obtained via multistep synthesis, that supports NCL and carries a PEG polymer. This facilitates effective enzymatic glycosylation and recovery of the resulting glycopeptides without the need for chromatographic steps.It also allows the simple generation of selectively O-glycosylated MUC1 peptide hydrazides as masked thioester precursors, giving access to peptide α-thioesters that are otherwise inaccessible. The modified conjugates are combined to each other via auxiliary-mediated NCL and the ligated products are recovered as non-protected glycopeptides after UV irradiation.

I am building a library of mucin polypeptides with multiple PTMs that will be used in proteomic studies to provide new insights into the role of glycosylation in mucin function and cancer progression. We are also expanding our approach to sequential ligation, to ligation at sites with residues different from glycine and to other PTMs, in order to broaden the applicability of our methodology toward the synthesis of any kind of homogeneously posttranslationally modified protein.