Total Chemical Synthesis of a Nonfibrillating Human Glycoinsulin

Abstract

Glycosylation is an accepted strategy to improve the therapeutic value of peptide and protein drugs. Insulin and its analogues are life-saving drugs for all type I and 30% of type II diabetic patients. However, they can readily form fibrils which is a significant problem especially for their use in insulin pumps. Because of the solubilizing and hydration effects of sugars, it was thought that glycosylation of insulin could inhibit fibril formation and lead to a more stable formulation. Since enzymatic glycosylation results in heterogeneous products, we developed a novel chemical strategy to produce a homogeneous glycoinsulin (disialo-glycoinsulin) in excellent yield (∼60%). It showed a near-native binding affinity for insulin receptors A and B in vitro and high glucose-lowering effects in vivo, irrespective of the route of administration (s.c. vs i.p.). The glycoinsulin retained insulin-like helical structure and exhibited improved stability in human serum. Importantly, our disialo-glycoinsulin analogue does not form fibrils at both high concentration and temperature. Therefore, it is an excellent candidate for clinical use in insulin pumps.

Recent Advances in the Chemical Synthesis of Insulin and Related Peptides

“…A century of advances in insulin science has established much improved therapy that elevates the level of efficacy and safety that must be exceeded by any further change in insulin structure. An example in how these new methods might be employed can be witnessed most recently by a report from Wade and associates to prepare an insulin analog with an additional cysteine at the N-terminal B-chain that following glycosylation displayed appreciable bioactivity devoid of fibril formation and with improved plasma stability [27]. Alternatively, the previously discussed report from Brunel et al illustrates one approach toward the chemical optimization of the hormone in pursuit of glucoseresponsive insulin analogs [18].…”

Chemical Glycosylation and Its Application to Glucose Homeostasis-Regulating Peptides

“…Using total chemical synthesis, insulin possessing an additional, orthogonally-protected Cys at the N-terminal of B-chain was synthesized to which a sialyloligosaccharide was selectively introduced. This sialo-glycoinsulin is an example of S-glycosylation, which, importantly, maintained native structure and in vivo blood glucose lowering activity (Hossain et al, 2020). Moreover, an S-glycan is reported to be more stable toward proteolysis than analogs of O-glycan (De Leon et al, 2017).…”

“…A long-acting insulin analog was obtained by modification of insulin with dendritic sialyl oligosaccharide by an enzymatic method (Sato et al, 2004). In addition, insulin modified with sialylundecasaccharide (Figure 2) demonstrated excellent stability against fibrillation at high concentration and high temperature (Hossain et al, 2020). Using total chemical synthesis, insulin possessing an additional, orthogonally-protected Cys at the N-terminal of B-chain was synthesized to which a sialyloligosaccharide was selectively introduced.…”

Recent Advances in the Chemical Biology of N-Glycans

“…As described above, the structure-activity relationship study of N-glycans on EPO demonstrates the importance of the N-glycan structure on the bioavailability and bioactivity of proteins [115]. Hossain and Wade et al reported that the physical properties of insulin can be improved by adding N-glycan to insulin, which originally has no glycans [148]. Introduction of sialic acid containing N-glycans to insulin successfully inhibited problematic fibril formation.…”