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

Methods are reported for the unambiguous syntheses of all three possible di sulfide regioisomers with the sequence of alpha-conotoxin SI, a tridecapept ide amide from marine cane snail venom that binds selectively to the muscle sub type of nicotinic acetylcholine receptors. The naturally occurring pep tide 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') an d Cys(2)-Cys(3) and Cys(7)-Cys(13)(2/3&7/13 'discrete'), respectively. Alig nment of disulfide bridges was controlled at the level of orthogonal protec tion schemes for the linear precursors, assembled by Fmoc solid-phase pepti de synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-c hain protection of cysteine was provided by suitable pairwise combination o f the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups . The first disulfide bridge was formed from the corresponding bis(thiol) p recursor obtained by selective deprotection of S-Xan, and the second disulf ide bridge was formed by orthogonal co-oxidation of S-Acm groups on the rem aining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice ve rsa). The highest overall yields were obtained when both disulfides were fo rmed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overa ll yields and poorer selectivities towards the desired isomers.