Synthesis and Characterization of the R27S Genetic Variant of Insulin‐like Peptide 5

Abstract

We report the synthesis and in vitro bioactivity assessment for an insulin-like peptide 5 (INSL5) analogue that was recently discovered as a genetic mutation in an Amish population. The mutation was associated with improved metabolic status, and receptor-based antagonism was proposed as a potential mechanism for the altered phenotype. We determined the specific peptide analogue to be fully potent and of maximal efficacy at the human relaxin family peptide receptor 4 (RXFP4), suggesting an alternative basis for the observed effect. In preparation of this synthetically challenging hormone, we have introduced several improvements such as implementation of isoacyl chemistry for high-efficiency preparation of INSL5 B-chain and selective intramolecular A6-11 disulfide formation as a first step in sequential disulfide assembly.

Stepwise Construction of Disulfides in Peptides

“…Similar to the oxidation of two free thiols by DTNP and DTDP, these transformations proceed with rapid kinetics and high yields. The less‐reactive S t Bu group has also been reported to facilitate successful intrachain disulfide bond formation in neutral pH buffers …”

“…In the following step, the removal of Mmt initiates the on‐resin disulfide formation . The solution method uses Trt to protect this cysteine pair, resin cleavage followed by air oxidation or DTDP treatment forming the intra‐A chain disulfide . The initial interchain disulfide formation, between A7 and B7 or A20 and B19 is typically directed by SPy or SNPy activating groups.…”

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

“…The recent adoption of the isoacyl strategy to the synthesis of relaxin‐2 and INSLP‐5 has been highly fruitful. In both cases, the incorporation of the isoacyl motifs into their B‐chains not only enabled the purification of these peptides by using regular reversed‐phase columns, but also significantly improved the quality of peptide assembly (Scheme B and C) . Collectively, the introduction of the isoacyl strategy into the synthesis of insulin superfamily peptides has greatly enabled the generation of native sequences and their analogues.…”

High-Yield Synthesis of Human Insulin-Like Peptide 5 Employing a Nonconventional Strategy

“…24−27 These two challenges have been addressed with some success, but total yield still remains only marginally superior to the original report. 25 A fundamental challenge with the synthesis of INSL5 is presented by the poor physical properties of the synthetic intermediates that undermine high-yield disulfide bond formation, and their preparative purification. To eliminate the use of iodine, we employ tBucysteine as classical protection, and optimized reaction conditions for disulfide formation that circumvent the need for intermediate purification to produce INSL5 in 23% total yield (starting from individual chains), through a [1 + 1 + 1] synthetic scheme (see Figure 1A).…”

“…Consistent with our previous report, the reaction is regioselective for Cys12, relative to Cys21. 25 Once purified and lyophilized, A-chain 2 was treated with DTNP in DMSO to activate the remaining free cysteine. The reaction was quantitative and required no further purification.…”