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

ABSTRACT 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.