A quantitative and site-specific chemoenzymatic glycosylation approach for PEGylated MUC1 peptides

Abstract

Full control over complex post-translational modifications (PTMs), such as O-glycosylation, is a prerequisite for testing and understanding the biological role of these modifications in protein function. Despite considerable progress over the last years, high throughput and easy-to-use methods for the synthesis of complex glycosylated peptides are still missing. We present here an efficient methodology to produce homogeneous site-specifically O-glycosylated peptides. Sequential chemoenzymatic glycosylation and separation from the reaction components are achieved via the temporary attachment of a monodisperse polyethylene glycol (PEG) polymer to the N-terminus of these peptides. Subsequent proteolytic removal of the PEG moiety allows quantitative recovery of homogeneous O-glycopeptides, suitable as building blocks for glycoprotein synthesis. Here, we demonstrate the preparation of glucuronylated variants of MUC1, a well-known member of the human mucin family. Homogeneously O-glycosylated variants were synthesized and will be used to study the role of O-linked glucuronic acid epitopes within the functional environment of the human MUC1 tandem repeat.

Glycosylation, an effective synthetic strategy to improve the bioavailability of therapeutic peptides

“…The glycosylated peptides were then recovered by precipitation and gel-permeation chromatography using a spin column. The presence of monodisperse PEG polymer allowed for quantifiable glycosylation reactions and easy recovery of the glycosylated products without intermediate purification steps 51. A more efficient method has been developed recently, in which a photocleavable auxiliary was attached to PEG polymer and cleanly removed by UV irradiation.…”

Chemical Methods for Encoding and Decoding of Posttranslational Modifications

“…Sometimes referred to as glycoprotein remodeling, recombinant enzymes with unique glycosyltransferase/glycosidase activity can be utilized in a stepwise fashion to produce more complex glycopeptide libraries that are out of reach of canonical chemical synthesis. For example, Bello and co-workers recently demonstrated the stepwise, chemoenzymatic synthesis of O-linked glycosylated MUC1 peptides utilizing Drosophila glycosyltransferases (Bello et al, 2014). In the area of N-linked glycoproteins, the Boons and Paulson labs collaborated to create a library of complex multi-antennary glycans by utilizing a chemically synthesized core oligosaccharide to which asymmetric, branched antenna were chemoenzymatically installed (Z.…”

A PEGylated Photocleavable Auxiliary Mediates the Sequential Enzymatic Glycosylation and Native Chemical Ligation of Peptides

“…[5] Glycosylation of Aux-MUC1(Tn) 10 with human C1GalT1 gave Aux-MUC1(T) 11 with a single T antigen (Galβ1-3GalNAcα disaccharide attached to Thr14) in excellent conversion (Figure 1 & S12A, B). The incubation with a mixture of ethanol and diethyl ether at −80° C induced the precipitation of 11 , which was then collected in 95% yield by centrifugation (Figure 1 & S12B) and used in the next glycosylation step without further purification.…”