Efficient Chemical Synthesis and Oxidative Folding Studies of Scorpion Toxin Peptide WaTx

Coupling or condensation reagents that could be used to promote the condensation of carboxylic acids with amines to furnish amide bonds play crucial roles in solid phase peptide synthesis (SPPS) of peptides and peptide-based derivatives. Compared with traditional coupling reagents used in SPPS such as 1-hydroxy-7-azabenzotriazole (HOAt) and 1-hydroxybenzotriazole (HOBt), the novel N,N'-diisopropylcarbodiimide (DIC)/ethyl 2-cyano-2-(hydroxyimino) acetate (Oxyma) condensation system has advantages of inexpensiveness, safety, high coupling efficiency, low racemic rate, and compatibility with manual and automatic peptide synthesis represented by microwave-assisted SPPS. However, the effects of different reaction temperatures (e.g. 28, 50, and 75 degrees C) on the coupling efficiency of DIC/Oxyma and on the easily oxidized amino acids such as methionine (Met) remain to be further investigated. The transient receptor potential ankyrin 1 (TRPA1) channel plays an important role in temperature perception, auditory perception, and inflammatory pain. As a novel non-covalently TRPA1-specific agonist, Wasabi Receptor Toxin (WaTx) that consists of 33 amino acid residues and two pairs of disulfide bonds is considered as an important molecular tool to study the opening mechanism and function of TRPA1. In this study, 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexa-fluorophosphate (HCTU)/N,N'-diisopropylethylamine (DIEA) and DIC/Oxyma condensation systems were separately utilized to explore the synthetic efficiency of linear WaTx and the oxidative degree of Met residues at different temperatures. The robustness of DIC/Oxyma condensation system was validated by the rapid manual synthesis of linear WaTx. The one-step and two-step oxidative folding strategies were separately applied for the construction of two pairs of disulfide bridges, affording the active WaTx, which was further confirmed by circular dichroism and calcium fluorescence assay. In this study, a moderate, efficient synthesis and renaturation folding method of WaTx was established. Moreover, the effects of different reaction temperatures on the synthetic efficiency of DIC/Oxyma and on amino acid residues oxidization were compared for the first time. From the aspects of reaction efficiency (represented by the timescale of the overall Fmoc deprotection-washing-amino acid coupling SPPS cycle), amino acid residues oxidization and synthetic cost, n(Fmoc-amino acid):n(DIC):n(Oxyma)=3:6:3 under 50 degrees C should be the ideal reaction condition. This work provides both an imperative complement for SPPS and a particularly useful strategy for the manual efficient synthesis of disulfide-containing peptides with easily oxidized groups.

Automated summary

This article investigates the coupling reagents for promoting the condensation of carboxylic acids with amines and the impact of different reaction temperatures on the coupling efficiency and oxidation degree of amino acids. A stable and efficient condensation system is validated, and active peptides are successfully synthesized. The results provide important references for solid phase peptide synthesis and the synthesis of peptides with easily oxidizable groups.