Solid-phase synthesis of huwentoxin-I and its structure and bioactivity analysis.
Huwentoxin-I, a neurotoxic peptide from the spiderSelenocosmia huwena, was synthesized by solid-phase method with Fluorenylmethoxycarbonyl amino acid pentafluorophenyl esters (Fmoc-AA-OPfp). The carboxyl and the hydroxy groups were protected by tBu; the side chains of Lys and His were protected by Boc; the guanidine group of Arg was protected by Mtr and the mercaptan group of Cys was protected by Trt. The solid-phase carrier was ethylene diamine-polyethylene-polystyrene (DEA-PEG-PS) resin. 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 synthetic peptide was purified by HPLC and then characterized by amino acid composition and sequence analysis, peptide mapping and NMR. The biological activity of the synthetic product was tested by electrophysiological method using the isolated mouse phrenic nerve diaphragm preparation. The results indicated that the synthetic huwentoxin-I has the same chemical and conformational structure and biological activity as those of the native huwentoxin-I from the spider.
Department of Biology, Hunan Normal University, 410081, Changsha, China.
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