Controlled syntheses of natural and disulfide-mispaired regioisomers of α-conotoxin SI

Methods are reported for the unambiguous syntheses of all three possible disulfide regioisomers with the sequence of alpha-conotoxin SI, a tridecapeptide amide from marine cane snail venom that binds selectively to the muscle sub type of nicotinic acetylcholine receptors. The naturally occurring peptide has two 'interlocking' disulfide bridges connecting Cys(2)-Cys(7) and Cys(3)-Cys(13) (2/7%3/13), while in the two mispaired isomers the disulfide bridges connect Cys(2)-Cys(13) and Cys(3)-Cys(7) (2/13 & 3/7, 'nested') and Cys(2)-Cys(3) and Cys(7)-Cys(13)(2/3&7/13 'discrete'), respectively. Alignment of disulfide bridges was controlled at the level of orthogonal protection schemes for the linear precursors, assembled by Fmoc solid-phase peptide synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-chain protection of cysteine was provided by suitable pairwise combination of the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups. The first disulfide bridge was formed from the corresponding bis(thiol) precursor obtained by selective deprotection of S-Xan, and the second disulfide bridge was formed by orthogonal co-oxidation of S-Acm groups on the remaining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers.