Synthesis of relaxin‐2 and insulin‐like peptide 5 enabled by novel tethering and traceless chemical excision

Abstract

This report presents an entirely chemical, general strategy for the synthesis of relaxin-2 and insulin-like peptide 5. Historically, these two peptides have represented two of the more synthetically challenging members of the insulin superfamily. The key synthetic steps involve two sequential oxime ligations to covalently link the individual A-chain and B-chain, followed by disulfide bond formation under aqueous, redox conditions. This is followed by two chemical reactions that employ diketopiperazine cyclization-mediated cleavage and ester hydrolysis to liberate the connecting peptide and the heterodimeric product. This approach avoids the conventional iodine-mediated disulfide bond formation and enzyme-assisted proteolysis to generate biologically active two-chain peptides. This novel synthetic strategy is ideally suited for peptides such as relaxin and insulin-like peptide 5 as they possess methionine and tryptophan that are labile under strong oxidative conditions. Additionally, these peptides possess multiple arginine and lysine residues that preclude the use of trypsin-like enzymes to obtain biologically active hormones. This synthetic methodology is conceivably applicable to other two-chain peptides that contain multiple disulfide bonds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

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

“…The synthetic approach is not limited to oxime linkage and could conceivably utilize other linkage chemistries. A sagacious aspect of the reported syntheses is the use of DKP formation, an adverse reaction in peptide synthesis 25 as controlling element in the removal of the auxiliary crosslink 17,18 . Further refinements in the propensity to cyclize will broaden the ability to accelerate or delay reversal of the crosslink.…”

“…The Kent group described an insulinspecific linkage of GluA4-ThrB30, which was saponified following oxidative folding 16 . Subsequently, a sequence-agnostic approach to synthesis in the insulin-like peptide family was reported, which employed a reversible tethering of the A-chain N-terminus through a labile amide to the B-chain C-terminal ester 17,18 . In each instance, the strategies mimicked the linear order of the A-and B-chains found in proinsulin.…”

The Application of Isoacyl Structural Motifs in Prodrug Design and Peptide Chemistry

“…This method was then applied to the generation of insulin analogues with four disulfide bridges, through which the native hormone was engineered with an extra disulfide to improve its resistance to fibrillation . It was also adopted into newly established insulin synthetic methods; the most recent ones involved folding of a linear precursor joined together by oxime ligations of the isoacyl A‐ and B‐chains followed by chemical excision of the connecting region …”

“…[90] It was also adopted into newly established insulin syntheticm ethods; [91,92] the most recent ones involved folding of al inear precursor joined together by oxime ligations of the isoacyl A-and B-chains followed by chemical excisionoft he connectingr egion. [93][94][95] In the insulin superfamily,t here are seven additional members, relaxins1 -3 and insulin-like peptides (INSLPs)3 -6, which share the unique insulin-like two-chaint hree-disulfide-bond structure, and, to some extent, the hydrophobicity of the individualc hains as well. [96,97] In addition to the solubilityi ssue, assemblyo ft he B-chain of relaxin-2 and INSLP-5o nt he solid phase was known to be extremelyd ifficult.…”

Max Bergmann award lecture:Macromolecular medicinal chemistry as applied to metabolic diseases

“…We successfully demonstrated the utility of an oxime linked A:B heterodimer in which the linker was sequentially removed by a 2‐step diketopiperazine cyclization /alkaline hydrolysis sequence (Figure ). This strategy was subsequently validated for insulin and relaxin‐2 . These methodological advances along with refinement of existing directed disulfide chemistries to better accommodate oxidation sensitive amino acids were exemplified by the synthesis of insulin‐like peptide‐6 and relaxin‐2 .…”