SOLID-PHASE SYNTHESIS OF HUWENTOXIN-I AND ITS STRUCTURE AND BIOACTIVITY ANALYSIS

Huwentoxin-I, a neurotoxic peptide from the spider Selenocosmia huwena , was synthesized by solid-phase method with Fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters (Fmoc-AA-OPfp). The carboxyl and t he hydroxy groups were protected by tBu; the side chains of Lys and Hi s were protected by Boc; the guanidine group of Arg was protected by M tr and the mercaptan group of Cys was protected by Trt. The solid-phas e carrier was ethylene diamine-polyethylene-polystyrene (DEA-PEG-PS) r esin. The synthetic peptide was cleaved from the resin and deprotected by a 90% TFA solution containing 5% thioanisole, 3% ethanedithiol and 2% anisole. The product was reduced with DTT and then incubated with GSSG and GSH to form the correct disulfide bond linkages. The syntheti c peptide was purified by HPLC and then characterized by amino acid co mposition and sequence analysis, peptide mapping and NMR. The biologic al activity of the synthetic product was rested by electrophysiologica l method using the isolated mouse phrenic nerve diaphragm preparation. The results indicated that the synthetic huwentoxin-I has the same ch emical and conformational structure and biological activity as those o f the native huwentoxin-I from the spider.