Scheme 1: Overview of templated peptide ligation strategies. (A) Peptides functionalized with bioorthogonal groups are templated together, increasing the effective concentration of two functional groups (FG 1 and FG 2 ). While this strategy increases reaction efficiency, the bioorthogonal groups remain bound to the ligated peptide, leaving "scars" that are unacceptable for most CPS applications. (B) With Click-Assisted NCL (CAN), peptides functionalized with traceless Lys linkers (Ddap) and either DBCO or azide are first templated together via SPAAC. These templated peptides then undergo a proximity-enhanced NCL, desulfurization (if desired), and gentle hydroxylamine treatment to cleave the Ddap linker and tracelessly afford the native peptide. Importantly, all steps can be performed in one pot.