Chemical synthesis of kurtoxin, a T-type calcium channel blocker
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Kurtoxin isolated from the venom of scorpion,Parabuthus transvaalicus, is a 63-residue peptide with four intramolecular disulfide bonds which inhibits low-threshold T-type Ca2+ channels. Kurtoxin was synthesized by native chemical ligation involving the coupling of (1–26)-thioester peptide and Cys27-(28–63)-peptide. The former was synthesized by standard solid-phase peptide synthesis (SPPS) with Boc chemistry, while the latter was sequentially assembled from three protected segments onto a resin-bound C-terminal segment in a chloroform-phenol mixed solvent followed by deprotection reaction using HF. Each protected segment used for the coupling on a solid support was prepared on anN-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) resin and detached from the resin by treatment with 20% Et3N in DMF to produce it in the form of an α-carboxylic acid. Synthetic kurtoxin obtained after the oxidative folding reaction was found to be identical with the natural product by means of several analytical procedures, and its disulfide structure was determined for the first time to be Cys12-Cys61, Cys16-Cys37, Cys23-Cys44 and Cys27-Cys46 by peptide mapping, sequence analysis and mass measurements.
Key wordsa mixture of chloroform and phenol DIC/HOAt method epimerization kurtoxin segment condensation reaction T-type calcium channel
α-cyano-4-hydroxy cinaminic acids
convergent solid phase peptide synthesis
capillary zone electrophoresis
N-[(1H-benzotriazol-1-yl) (dimethylamino)methylmethanaminium hexafluorophosphateN-oxide
N-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid
solid phase peptide synthesis
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