Directed Oxidative Folding of Disulfide-Rich Peptides (DRPs) Using a Pseudoproline Unit: Ion Mobility-Mass Spectrometry (IM-MS) Reveals the Conformational Landscape of the DRP

Abstract Disulfide-rich peptides (DRPs) are attractive drug development scaffolds due to their exceptional stability and specificity. Regioselective disulfide bond formation is often achieved via orthogonal Cys protecting groups or strategic Pro placement. However, Cys protection strategies are labor-intensive and low-yielding, while strategic Pro placement imposes sequence constraints. To broaden the chemical diversity of DRPs, we developed an alternative approach using an acid-stable oxazolidine pseudoproline to guide disulfide formation in a multicyclic model peptide. This pseudoproline was incorporated as a monomer during automated Fmoc solid-phase peptide synthesis (SPPS), without pseudoproline dipeptide synthesis. The pseudoproline remained intact under standard peptide synthesis cleavage conditions (95% TFA), allowing it to direct peptide folding. Oxidation of the cleaved peptide strongly favored one regioisomer, Cys(I-IV)/Cys(II-III), with 86% selectivity. Ion mobility-mass spectrometry (IM-MS) revealed that this regioisomer exhibited two dominant conformational families. Experiments involving multiple rounds of IM separation and collision activation (CA) (IM-CA-IM) showed that these families interconvert in the gas phase, likely via cis/trans isomerization at the pseudoproline amide bond. This study provides an alternative to Pro for directing disulfide bond formation, relevant to medicinal chemistry projects. It also demonstrates the power of IM-MS to characterize DRP conformational landscapes and dynamics.