Parenterally-administered bioactive peptides often have limited pharmacological efficacy due to their rapid degradation by enzymes, leading to a short circulating plasma half-life. Several measures for half-life extension have been investigated, e.g., acylation, PEGylation, protein fusion and glycosylation as well as traditional rational design employing peptide synthetic technology. One of the most promising methods is to link a glycan to the peptide of interest, since glycosylation is generally considered to provide peptides and proteins with prolonged circulating plasma half-life, improved solubility and/or lowered immunogenicity. Asn-linked complex type disialoundecasaccharide [Asn(disialo)] can be readily obtained from hen egg yolk as a building block. Different glycan structures can be produced by remodeling Asn(disialo) via simple chemical/enzymatic processes to remove and re-add sugar units, even those not found on the initial structure such as α(2,3) linked sialic acid and core fucose. Through these processes it is possible to generate a library of over 50 different N-glycan structures that can be used to glycosylate target molecules.
Incorporation of Fmoc-Asn(glycan) onto the growing peptide chain can be done without the need to protect its hydroxyl groups, except for the carboxyl group on each sialic acid. Moreover, a bromoacetamidyl glycan prepared from Fmoc-Asn(glycan) without protecting groups on its hydroxyl or carboxyl groups can be coupled with the SH group of Cys on a free peptide to produce a glycosylated peptide analog having a native complex-type glycan structure. Applying this glycosylation strategy, bioactive peptide/proteins such as somatostatin, excendin 4 and interferon β were modified by glycan(s) optimized in terms of glycan structure, number and position to produce therapeutic candidates which are superior to their respective parent molecules in pharmacokinetic properties. We also applied our glycosylation approach to the development of a reversibly glycosylated peptide by coupling the parent peptide and glycan through a spontaneously cleavable linker. This enabled extended release of the parent peptide upon cleavage of the linker in aqueous physiological conditions, offering a useful method of enhancing the lifetime of peptides in vivo.