Glycosylation is one of the most ubiquitous post-translational modifications observed in peptides and proteins. It affects the structural and functional features of these macromolecules, thereby profoundly influencing many biological processes. N-Glycans are expected to be beneficial modifiers for increasing the solubility and in vivo half-life of native bioactive peptides and proteins, as well as synthetic molecules such as peptide drug-conjugates, and for decreasing aggregation and immunogenicity. In a chemical approach, glycosylated Asn can serve as a building block for SPPS, and its aminoglycan can be used as a substrate for the Lansbury aspartylation reaction in glycopeptide/protein synthesis. Another approach, based on a chemoenzymatic strategy using ENGase, facilitates transglycosylation at the proximal GlcNAc position of target peptides and proteins. In addition to these, chemoselective glycosylation reactions that can be conducted post-synthesis and in aqueous conditions are promising for the high-throughput development of glyco-peptide/protein drugs.[1] This “glycoconjugation” approach is particularly advantageous in that manipulation of glycan protecting groups is not necessary, thereby allowing conjugation reactions to be carried out between target molecules and unprotected glycans. By providing a single glycosylation profile, i.e., glycan structure, number, and position, glycoconjugation not only allows the beneficial properties of N-glycans to be exploited, but also facilitates the investigation of N-glycan function.[2]
The development of therapeutic agents through the conjugation of glycans to peptides/proteins requires a reliable and consistent supply of considerable quantities of glycans. It is therefore practical to use soybean agglutinin (SBA) as a source of high Man-type N-glycans and sialylglycopeptide (SGP) as a source of complex-type N-glycans, obtained from abundant and inexpensive soybean flour and hen egg yolk, respectively. Asn-linked M9 and disialoglycans, obtained primarily by sequential digestion of SBA and SGP with amino/carboxypeptidases, can be converted to various glycan structures by chemical and enzymatic processes. These glycans can be used directly for chemical/chemoenzymatic glycosylation, or they can be converted into a conjugation-ready form and then used in glycoconjugation. However, the application of N-glycans to pharmaceuticals requires attention to their structural homogeneity from a quality control perspective, regardless of whether they are chemically synthesized or derived from natural sources. When hen egg yolk glycans are used for production, it should be noted that Asn(disialo) always contains a few percent of α2,3-linked sialic acid isomer, which is different from the α2,6-linked human form.[3]