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

Kishore Thalluri,Vasily Gelfanov,Richard D Dimarchi,Joseph R Chabenne,John P Mayer

doi:10.1038/s42004-018-0036-9

Kishore Thalluri, Vasily Gelfanov + Show 3 more

Open Access

https://doi.org/10.1038/s42004-018-0036-9

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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 C-terminus, 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 N-termini 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.

Highlights

Insulins, relaxins, and other insulin-like peptides present a longstanding synthetic challenge due to their unique cysteine-rich heterodimeric structure
We report the synthesis of insulin and a set of related peptides by a synthetic protocol that employs a reversible crosslink of the two N-termini through parallel extension of the respective A- and B-chains made by conventional Solid Phase Peptide Synthesis (SPPS)
We explored the chemical synthesis of insulin, relaxin-2, and four insulin analogs (Fig. 1) through reversible crosslink of the two N-termini by parallel extension of insulin A- and B-chains made by conventional SPPS

Summary

Results

This properly folded, single-chain insulin was obtained in a 45% combined yield for ligation, disulfide-formation, and RP-HPLC purification (Supplementary Figure 19). The N-terminal residue of the A-chain was introduced as Gln, which was subsequently cyclized to pGlu. The Boc-Lys(iBu)Gly-OH dipeptide, PEG8 and bis-Boc-aminooxyacetic acid were introduced as reported in the insulin synthesis (Supplementary Figures 22–25). This analog should be accessible by an enzyme-based approach as a synthesis that is DKP mediated To prove this point and assess the relative efficiency in the removal of the auxiliary N,N-crosslink, insulin 6 was synthesized (Supplementary Figure 8) as described for native sequence, but with replacement of the DKPsusceptible dipeptide with a Gly–Lys dipeptide. Oxime ligation and disulfide formation in insulin 6 were achieved as with native hormone in 46% yield (Supplementary Figures 49–52). The overall yield of 6 as produced by enzyme cleavage was 30% from purified A-chain, which is identical to the yield of 1 obtained by DKP-mediated chemical cleavage

Discussion

F V NQHL CGS HL VEALYL VCG ERGF FYTPKTOHO

Methods