A method for the one-pot regioselective folding of the heat-stable bacterial enterotoxin ST peptide residues 5–18

Abstract

A one-pot method for the regioselective formation of the three disulfide bridges of the heat-stable bacterial enterotoxin STa, residues 5–18, employing a temperature-controlled orthogonal protecting group scheme is reported. The protecting groups trityl, t-butyl and 4-methylbenzyl were chosen for the selective formation of the three cystine residues. Cleavage of the peptide from the solid support afforded a partially S-protected crude product with two free thiol groups. The first disulfide linking Cys 6 to Cys 14 was formed by oxidation of the dithiol peptide in a mixture of water/acetonitrile/DMSO. Following solvent removal, the second disulfide between Cys 5 and Cys 10 was formed directly by simultaneous cleavage and oxidation of the t-butyl groups in TFA/DMSO/anisole at room temperature. Subsequent heating of this solution initiated cleavage of the 4-methylbenzyl groups with concomitant oxidation of Cys 9 and Cys 17 yielding the desired three disulfide product.