An Auxiliary-Mediated Approach for the Chemoenzymatic Synthesis of Homogenous Glycopeptides

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 [1] 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 its extracellular domain, was chosen as a synthetic target. Attachment of a monodisperse PEG polymer to the N-terminus of our MUC1 peptides changes their solubility properties and allows sequential chemoenzymatic glycosylation of the peptides in solution and their separation from glycosyltransferases and low molecular weight components by simple techniques such as spin column gel permeation chromatography (GPC) and precipitation. This method proved to be high-yielding and versatile, as it could be coupled with native chemical ligation (NCL), thereby giving access to longer site-specifically O-glycosylated peptides. Herein we present the synthesis and application of a photocleavable auxiliary, based on the 1-nitrophenyl-2-sulfanylethyl scaffold previously described by the groups of Aimoto [2] and Dawson [3], that allows attachment of the PEG polymer for efficient enzymatic glycosylation, provides the functional groups for NCL, and is cleanly removed by UV irradiation [4]. Upon photolytic removal a native glycine residue remains at the ligation site (Figure 1B).