Introducing Regioselective Disulfide Linkages in Peptides under Pseudodilute Conditions by Harnessing Bro̷nsted Acid-Activated N-Chlorosuccinimide.

Methods that assemble multiple peptide disulfide bonds in the solid phase (pseudodilute conditions) are in high demand for synthesizing disulfide-rich peptides. Yet, the existing repertoire of disulfide-forming orthogonal chemistries in the solid phase is often hindered by additional steps for protecting group removals as well as the absolute necessity of rare, customized orthogonal cysteine (Cys) building blocks. We now describe a conceptually new while operationally simple on-resin method for disulfide assembly with widely accessible Cys protecting groups (Trt, Acm, and ^t Bu) using acid-activated N-chlorosuccinimide (NCS) in a single step. In the process, S- ^t Bu Cys emerged as a new orthogonal building block for the single-step assembly of the peptide disulfide. In our investigations, 2% TFA-activated NCS offered rapid (∼15 min) and a clean disulfide product in various peptides. Eventually, this novel strategy (2% TFA-NCS) was strategically merged in stepwise cross-linking with our previously described I₂/S₂O₈ ^2--mediated disulfide assembly protocol to leverage two regioselective disulfide bonds into conotoxin, gomesin, and a de novo sequence within ∼30 min. Invariably, this new method proved highly productive and operationally simple. DFT calculations also support the hypothesis of NCS activation that assists efficient disulfide assembly in crossing a low-lying energy barrier via sulfonium intermediates. This newly developed method for ^t Bu deprotection and concomitant disulfide assembly in the solid phase should find wide applications in de novo peptide disulfide synthesis.