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Bicyclization represents an effective method for the introduction of conformational constraints into small, biologically important peptides. Several strategies have been developed for the preparation of bicyclic lactam analogues of alpha-conotoxin SI, a 13-residue peptide neurotoxin found in cone snail venom. Four analogues of the natural regioisomer of alpha-conotoxin SI were designed and synthesized, each with one of the two paired cysteines of the parent peptide being replaced by a side-chain lactam bridged glutamic acid/lysine pair. Solid-phase lactamization was studied to determine rates of formation of the two possible loops and to document the extent of dimerization and higher oligomerization. Radioligand binding assays were carried out on all synthesized peptides, including the naturally occurring two-disulfide form, in order to determine their affinities for nicotinic acetylcholine receptors (nAChRs). Replacement of the Cys(2)-Cys(7) loop of alpha-conotoxin SI with a lactam bridge resulted in complete loss of activity, whereas replacement of the Cys(3)-Cys(13) disulfide loop resulted in a approximately 60-fold reduction in affinity for one orientation and a approximately 70-fold increase in affinity for the other. The two active lactam analogues retain the selectivity exhibited by the naturally occurring peptide for the alpha/delta subunit of nAChRs, as judged by competition experiments with the curariform antagonist metocurine.

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 cone snail venom that binds selectively to the muscle subtype of nicotinic acetylcholine receptors. The naturally occurring peptide has two 'interlocking' disulfide bridges connecting Cys2-Cys7 and Cys3-Cys13 (2/7&3/13), while in the two mispaired isomers the disulfide bridges connect Cys2-Cys13 and Cys3-Cys7 (2/13 & 3/7, 'nested') and Cys2-Cys3 and Cys7-Cys13 (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.