Total Chemical Synthesis of an Intra‐A‐Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability

Abstract

Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non‐reducible cystine isostere, cystathionine, within the A‐chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra‐A‐chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next‐generation insulin analogues.

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin

“…Yields of insulin are thus low (typically <20 % in relation to the starting B‐chain concentration) because of the formation of non‐native disulfide isomers, covalent polymers, noncovalent aggregates and fibrils . In recent decades, insulin analogues have been sought with enhanced chain‐combination efficiency or stability; many of these excellent approaches are based on orthogonal Cys protection and regioselective disulfide formation or the use of tethers as “proinsulin” mimics. For example Wade, Hossain and co‐workers prepared a human insulin analogue in which the intrachain A6–A11 disulfide bond was replaced with cystathionine to obtain enhanced thermal stability .…”

“…In recent decades, insulin analogues have been sought with enhanced chain‐combination efficiency or stability; many of these excellent approaches are based on orthogonal Cys protection and regioselective disulfide formation or the use of tethers as “proinsulin” mimics. For example Wade, Hossain and co‐workers prepared a human insulin analogue in which the intrachain A6–A11 disulfide bond was replaced with cystathionine to obtain enhanced thermal stability . Kent and co‐workers engineered a labile ester bond between the side chains of Glu A4 and Thr B30 in the reduced and unfolded chains.…”

Insulin-like Growth Factor 1 Analogs Clicked in the C Domain: Chemical Synthesis and Biological Activities

“…Initially, an access of iodine in an acetic/hydrochloric acid mixture was used to cleave the Acm groups from Cys side chains in 2c (Figure S2). 30 However, this was ineffective, as the majority of the Acm groups remained uncleaved, even after 4 h. Therefore, silver triflate was employed for the removal of the Acm groups, without the purification of an intermediate product. Subsequently, the free −SH groups were "liberated" from the Ag + salt by the gaseous H 2 S, and the precipitated Ag 2 S was removed by centrifugation.…”

Recent Advances in the Chemical Synthesis of Insulin and Related Peptides

“…Directed disulfide bond formation has also recently enabled the synthesis of several novel insulin analogs containing disulfides isosteres. For example, Karas and coworkers substituted the A6-A11 disulfide with a thioether by replacing cystine with cystathionine [3,11]. The intrachain A6-A11 cystathionine containing A-chain was crosslinked through a B19S-pyridyl (S-Pyr) activated B-chain and a free cysteine at A20.…”