Efficient Synthesis and Oxidative Folding Studies of Centipede Toxin RhTx

The critical step for the synthesis of disulfide-containing peptides is the efficient construction of one or multiple disulfide bridges. Generally, the folding of disulfide bonds could be achieved by three chemical strategies, i.e. one-step oxidative folding strategy, multi-step oxidative folding strategy, and one-pot oxidative folding strategy. Because few comparative studies have been conducted on efficiencies of three strategies, the systematical research is desirable. Three folding strategies were separately applied for the preparation of centipede toxin RhTx. The results showed that the isolated yield of two-step oxidative folding strategy was higher than those of one-step and one-pot oxidative folding strategies. Besides, the one-pot oxidative folding strategy may induce severe misfoldings. The circular dichroism (CD) and activity tests indicated that disulfide bonds are critical for the structure and activity of RhTx. In addition, the efficient preparation of RhTx on tens of milligrams scale was achieved, affording molecular tools for the further biological and biophysical studies of RhTx targeting TRPV1. Overall, three mainstream oxidative folding strategies were systematically studied, which provided a valuable reference for the synthesis of disulfide-containing peptides.