Solution synthesis of mu-conotoxin GIIIB: optimization of the oxidative folding reaction.

mu-Conotoxin GIIIB, a skeletal muscle sodium channel specific blocker, was synthesized by the solution procedure. The whole molecule, which is composed of 22 amino acid residues including six cysteinyl and three trans-4-hydroxy-L-prolyl residues, was constructed from three segments. After removal of all protecting groups, the hexa-SH peptide was subjected to an oxidative folding reaction at a peptide concentration of 1 x 10(-5) M. Three major products 1, 2 and 3 were formed in a ratio of 1:4:3. Determination of the disulfide structures in each product revealed them to be disulfide isomers, with similar connectivities in the latter two. Study of the biological activities of the three products in mice indicated that only product 1, which is a minor component, has the same potency as the natural product. Analysis of the folding process at a peptide concentration of 1 x 10(-5) M showed that the disulfide bond between Cys10 and Cys15 was initially formed, thus leading to the predominant generation of products 2 and 3. The optimal conditions for the formation of product 1 (mu-conotoxin GIIIB) were obtained by increasing the peptide concentration in the oxidation reaction mixture or by using redox reagents, both of which functioned as promoters for the thiol-disulfide exchange reaction of the mismatched disulfide bond between Cys10 and Cys15.