Synthesis of disulfide-rich heterodimeric peptides through an auxiliary N, N-crosslink

Abstract

Insulins, relaxins, and other insulin-like peptides present a longstanding synthetic challenge due to their unique cysteine-rich heterodimeric structure. While their three disulfide signature is conserved within the insulin superfamily, sequences of the constituent chains exhibit considerable diversity. As a result, methods which rely on sequence-specific strategies fail to provide universal access to these important molecules. Biomimetic methods utilizing native and chemical linkers to tether the A-chain N-terminus to the B-chain Cterminus, entail complicated installation, and require a unique proteolytic site, or a two-step chemical release. Here we present a strategy employing a linkage of the A-and B-chains Ntermini offering unrestricted access to these targets. The approach utilizes a symmetrical linker which is released in a single chemical step. The simplicity, efficiency, and scope of the method are demonstrated in the synthesis of insulin, relaxin, a 4-disulfide insulin analog, two penicillamine-substituted insulins, and a prandial insulin lispro.

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

“…[14][15][16] Six out of eight Cys residues are at the conserved positions in insulin superfamily, whereas the positions of the other two Cys residues are not conserved; for example, both at the B chain in the isopod crustacean AGHs [11][12][13] and one at the A chain and the other one at the B chain in cone snail insulins. [8][9][10] Although a few four-disulfide ILPs have been synthesized, [17][18][19][20] the synthesis of such ILPs is still a challenging issue.…”

Chemical synthesis of the crustacean insulin‐like peptide with four disulfide bonds

“…In the former, A and B chains without protecting group are mixed in an aqueous solution containing some additives, generating thermodynamically stable native form . Four disulfide human insulin analogs were recently synthesized using an auxiliary crosslinking the N‐termini of A and B chains . A quite recent report demonstrated the improved oxidative folding method, by which human insulin could be obtained in 49% yield, whereas its applicability is still limited.…”

“…[11][12][13][14] Four disulfide human insulin analogs were recently synthesized using an auxiliary crosslinking the N-termini of A and B chains. 15 A quite recent report demonstrated the improved oxidative folding method, 16 by which human insulin could be obtained in 49% yield, whereas its applicability is still limited. Actually, Val A16 variant of human insulin that is known as a nonfoldable insulin analog 17 could not be obtained by this method.…”

Recent Advances in the Chemical Synthesis of Insulin and Related Peptides

“…This synthetic strategy is agnostic to the specific peptide sequence, a feature demonstrated by successful application to two well recognized insulin-like peptides of sizable synthetic difficulty, relaxin-2 and insulin-like peptide-5 [25]. The attraction of the dipeptide-based DKP cyclization as a synthetic step was significantly advanced through the introduction of a traceless, symmetrical linker between the N-termini of the A-and B-chains that was released in a single chemical step [26]. The A-and B-chains were prepared with site-specific isoacyl bonds, that had first been separately demonstrated by Liu and associates in 2014 to improve chain-synthesis.…”