Chemical Protein Synthesis via Direction-Switching One-Pot Peptide Ligation Enabled by Orthogonal Cysteinyl Protection

A one-pot multiple peptide ligation strategy accelerates the demanding processes of chemical protein synthesis. Ligation intermediates generated during peptide ligation differ depending on the order of peptide segment assembly, affecting the aggregation properties of the ligation mixture. Therefore, expanding the scope of the ligation order in one-pot methods contributes to optimizing a ligation scheme for each target protein in chemical protein synthesis. Herein, we present the first example of direction-switching one-pot peptide ligation, utilizing the two mutually orthogonal cysteinyl protections compatible with native chemical ligation (NCL): thiazolidine (Thz) and a new allylic thiol protecting group, <i>N</i>-(2,2,2-trifluoroethyl)-allyloxycarbonylaminomethyl (TfeAllocam). By optimizing the structure and metal-mediated deprotection conditions for the allylic protecting group, we established efficient methods for consecutive NCLs and deprotections in two direction-switching schemes with the same peptide segments: one scheme from C-terminus-to-N-terminus (C-to-N) to N-terminus-to-C-terminus (N-to-C) and the other from N-to-C to C-to-N. Using this ligation approach, we accomplished the semisynthesis of dimonoubiquitinated histone H3 bearing trimethyl-lysine by condensing five peptide segments in a one-pot manner.