One-pot peptide ligation for accelerating chemical protein synthesis

Abstract

Chemical protein synthesis enables the preparation of homogeneous proteins bearing site-specific posttranslational modifications, unnatural amino acids, and other functional groups that are difficult to access by recombinant expression methods. In the synthetic schemes, peptide segments prepared by solid-phase synthesis are often assembled by native chemical ligation (NCL). However, synthesizing large proteins requires many segments, and the accompanying increase in purification steps lowers the overall yield and lengthens the synthesis. One-pot peptide ligation, in which three or more segments are ligated sequentially without intermediate purification, addresses this problem. This account summarizes the one-pot ligation methods our group has developed. We first established a one-pot ligation using N-allyloxycarbonyl (Alloc)-protected cysteine, exploiting the triple function of 4-mercaptophenylacetic acid (MPAA) as an NCL catalyst, a deprotection accelerator, and a quencher, achieving the first five-segment one-pot ligation for synthesizing histone H2AX (142 aa). Replacing the palladium complex with a highly active organoruthenium catalyst enabled quantitative Alloc removal with only 20 mol%, allowing the one-pot synthesis of proteins exceeding 200 residues. We also developed a thioester-compatible aldehyde scavenger, AMDBH, for repetitive thiazolidine (Thz) deprotection, enabling the one-pot synthesis of monoubiquitinated histone H2A.Z (209 aa). Finally, by combining an N–S acyl-shift thioester precursor protected with a newly designed allylic protecting group called TfeAllocam and the Thz/AMDBH system, we demonstrated the first direction-switching one-pot ligation and synthesized 287-residue dimonoubiquitinated histone H3. These strategies substantially improve the efficiency of chemical protein synthesis and provide a foundation for synthesizing increasingly complex proteins.