For both the naturally occurring and de novo designed constrained peptides, the correct pairing of disulfide bonds is primarily driven by the amino acid sequences. Thus, existing constrained peptide scaffolds are, in principle, not compatible with the design of sequence-randomized libraries, where the sequences are extensively manipulated and the disulfide pairing becomes unpredictable. Our previous work on orthogonal disulfide pairing has shown the unique effect of the incorporation of CXC motif (cysteine-any-cysteine) and/or penicillamine (Pen) on the oxidative folding of peptides1-3. However, the essentials of directing the oxidative folding of peptide into specific isomers with up to three disulfide bonds have not yet been grasped. Here we introduce the effort of de novo designing C/Pen-mixed peptide frameworks with one or two CXC motifs that are able to precisely fold into a subset of specific disulfide connectivities fully covering all possible isomers oxidatively folding from typical cysteine-rich frameworks. All designs can generate as low as four specific isomers, and a subset of them are able to fold into one to two specific ones. Even certain triple-disulfide connectivities that have been considered to be formidable in topology can be generated with high yields (e.g., the knotted connectivity). These topologically-formidable scaffolds are of particular interest to further designing constrained peptides with new structures and functions, because they are hyper-constrained and new peptides created based on them should be exceptionally resistant to proteolysis and chemical denaturation. Our results demonstrate the power of combining two conceptually-different orthogonal disulfide pairing technologies for de novo designing constrained and sequence-independent peptide scaffolds without recourses to naturally occurring disulfide-rich peptides.
References
1. C. Wu, et al., Nat. Chem., 2012, 4, 1044.
2. Y. Zheng, et al., J. Am. Chem. Soc, 2015, 137, 15094.
3. Y. Zheng, et al., Chem. Sci, 2017, 8, 2547.