Glycosylation affects various aspects of peptides/proteins, including folding that defines their conformation, solubility, biodegradability, aggregability, immunogenicity and biological activity. Taking advantage of the beneficial characteristics of glycans, therefore, we have attached human N-glycans to the sulfhydryl group of Cys residue(s) newly incorporated or substituted into bioactive peptides/proteins to improve not only their original pharmacokinetic properties but also physicochemical ones. Glycosylated Asn bearing a disialylundecasaccharide [Asn(disialo)] obtained from hen egg yolks can be remodeled into more than 50 different glycan structures by simple chemical and/or enzymatic processes.1) Such glycans can be derivatized into bromoacetamidyl glycans that are used in a post-synthetic glycosylation procedure without protection of their functional groups,2) allowing high-throughput construction of glycopeptide libraries when used in combination with Cys-scanning across any peptide/protein sequences. Using this chemical glycosylation technology, we have been able to successfully develop a number of glycopeptide analogs superior to the original molecules, such as exendin-4, somatostatin, GLP-1 and interferon-β, with enhanced drug properties.3) Thus, N-glycans have proved to be useful modifier molecules for improving the in vivo half-lives of various bioactive peptides/proteins that have seen limited clinical application due to their short duration of action in the blood.
The N-glycans used in our research have structures that already exist in the human body and are highly biodegradable and biocompatible. Therefore, from a quality control perspective they must be structurally homogeneous when they are applied to pharmaceuticals. It should be noted, however, that Asn(disialo) isolated from hen egg yolks is always contaminated by a few percent of α2,3-linked sialic acid isomer,4,5) which is different from the α2,6-linked human form. By removing such α2,3-sialosides with an α2,3-sialidase, we can obtain Asn(disialo) in high purity. We are currently examining a more economical way to purify Asn(disialo) using a simple chemical process that does not rely on costly α2,3-sialidases.