Ultrafast Biomimetic Oxidative Folding of Cysteine‐rich Peptides and Microproteins in Organic Solvents

Abstract Disulfides in peptides and proteins are essential for maintaining a properly folded structure. Their oxidative folding is invariably performed in an aqueous‐buffered solution. However, this process is often slow and can lead to misfolded products. Here, we report a novel concept and strategy that is bio‐inspired to mimic protein disulfide isomerase (PDI) by accelerating disulfide exchange rates many thousand‐fold. The proposed strategy termed organic oxidative folding is performed under organic solvents to yield correctly folded cysteine‐rich microproteins instantaneously without observable misfolded or dead‐end products. Compared to conventional aqueous oxidative folding strategies, enormously large rate accelerations up to 113,200‐fold were observed. The feasibility and generality of the organic oxidative folding strategy was successfully demonstrated on 15 cysteine‐rich microproteins of different hydrophobicity, lengths (14 to 58 residues), and numbers of disulfides (2 to 5 disulfides), producing the native products in a second and in high yield.