Disulfide bonds are an essential structural feature of many bioactive peptides however their chemical synthesis according to a defined connectivity remains a challenge. In cases of structurally complex molecules having multiple closely packed disulfide bonds the determination of the native connectivity already presents a problem due to ambiguities encountered using 2D NMR methods. In principle, the regioselective synthesis of disulfide-bonded peptides is an effective method of clarifying their connectivity. However, in practice current strategies are limited by the availability of robustly orthogonal cysteine protecting groups.
We have developed a safety-catch type protecting group (Msbh) for cysteine thiols to expand the scope of current strategies for regioselective synthesis of disulfide rich peptides. The Msbh group is stable to standard peptide synthesis conditions of both Fmoc- and Boc- chemistries and is orthogonal to other commonly used cysteine protecting groups. Here we apply the Msbh group for the regioselective synthesis of a four disulfide bond containing peptide.
We have recently discovered conotoxin Mi045 from the venom of Conus miles, a 33-residue peptide with a previously unknown cysteine framework. NMR analysis revealed a structurally unique conotoxin however the disulfide bond connectivity of its eight cysteines could not be confidently assigned. The regioselective synthesis of the four disulfide bonds of Mi045 was therefore undertaken according to one of the possible connectivities proposed by the preliminary NMR structure. Using our synthetic approach we were able to confirm that this connectivity was indeed that of the native molecule, which allowed disulfide restraints to be imposed and a high-resolution structure to be calculated.