Conformation‐Guided Disulfide Pairing Enables Efficient Folding of Disulfide‐Rich Peptides

Disulfide bond formation is critical for the structural integrity and biological functions of peptides and proteins. For disulfide-rich peptides (DRPs), particularly those containing ≥ 3 disulfide bonds, this process remains challenging due to misfolding and kinetic traps. Here, we report a conformation-guided "1 + 2" disulfide pairing strategy that enables rational control of oxidative folding in DRPs. Conformational sampling and statistical analysis identify a folding-promoting disulfide bond, whose early formation pre-organizes the peptide into a native-like conformation, thereby directing the efficient and selective closure of the remaining disulfide bonds. This principle is translated into a sequential, one-pot oxidative folding workflow that affords native disulfide connectivity across structurally and pharmacologically representative DRPs, with 57%-93% HPLC conversion. By reducing reliance on largely empirical solvent optimization and complex stepwise protocols, this approach addresses a key bottleneck in the synthesis of disulfide-rich peptide architectures.