Protein-based biomacromolecules have been broadly studied for their essential roles in organisms. An important strategy for protein production is chemical peptide synthesis, in which native chemical ligation (NCL) is one of the most powerful and frequently used methods employed. However, as for the bulky amino acid sites (e.g. Pro, Val, Ile, etc.) at C-termini, their reaction rates are constrained, limiting the relevant peptide synthesis. 

To address the problem above, we have developed mild and efficient internal activation protocols of peptidyl prolyl thioesters and valinyl benzimidazolinones in NCL, which respectively improve the ligation efficiency at the Pro-Xaa sites and Val-Xaa sites. For the prolyl thioesters, the introduction of a 4-mercaptan substituent improves the reactivity via the formation of a bicyclic thiolactone intermediate. Benifiting from the ring strain, the ligtion could go smoothly with high efficiency. Based on the same concept, the efficiency of the liagation at the Val-Xaa sites was increased by the generation of thiolactone form penicillamine derived precusor.