Artificial disulfide-rich peptide scaffolds with precisely defined disulfide patterns and a minimized number of isomers.

Disulfide-rich peptides are emerging as potential templates for drug design applications. However, the synthesis and reengineering of disulfide-rich peptides are challenging, owing to the complexity of the oxidative folding process involving a number of diverse isomeric structures. Novel disulfide-rich peptide scaffolds that are not besieged by their disulfide isomers are still greatly desired. In this work, we report the design and synthesis of a novel class of artificial disulfide-rich peptide scaffolds with precisely defined disulfide patterns and a minimized number of isomers. In theory, natural peptides with three disulfide bonds have 15 possible isomers. By rationally engineering the thiol-framework of a peptide containing six cysteines with penicillamines and a dithiol amino acid, we demonstrated, for the first time, that the total number of isomers formed after oxidative folding can be decreased to a minimum of two (i.e., from 15 to 2). As fewer isomeric folds are involved in the oxidative folding, the pathway of the folding becomes more concise and the yield of the artificial scaffolds is substantially increased compared to that of its six-cysteine-containing analogue, which makes the artificial disulfide-rich scaffolds (with only 2 predefined isomeric folds) extremely promising for being exploited as structurally complex templates for the design of peptide therapeutics and ligands.