Recent progress in chemical protein synthesis has been allowed to generate various artificial proteins. Especially, histone proteins containing posttranslational modifications (PTMs) and chemical labels have so far been synthesized through native chemical ligation (NCL) and applied to epigenetic research. For example, we prepared H2A-Y57ph using chemical protein synthesis. Although PTMs that affect the interaction between H2A and H2B have not been reported, our synthetic H2A-Y57ph showed strong regulation of the stability of the histone H2A–H2B dimer for the first time.<br/>In the chemical synthesis of large artificial proteins, one-pot multiple peptide ligation would be effective for improvement of synthesis efficiency. However, two serious difficulties still remain in one-pot multiple peptide ligation. In multiple ligation process of peptide segments, the N-terminal Cys of middle peptide segments should be protected to avoid polymerization and/or cyclization. In addition, rapid deprotection under mild conditions and complete quenching of deprotection reagents without affecting the subsequent NCL reaction are required for one-pot multiple peptide ligation. Therefore, we selected allyloxycarbonyl (Alloc) group for protection of N-terminal, which are removed with a palladium(0) complex. 4-Mercaptophenylacetic acid (MPAA), a thiophenol additive to accelerate NCL reaction, functioned as the nucleophile for the π-allyl palladium complex. MPAA was also utilized for quenching of the palladium complexes after deprotection. Using these functions of MPAA, efficient one-pot multiple peptide ligation was accomplished through the repetitive cycles of three steps: NCL, removal of the Alloc groups, and quenching of the palladium complexes. According to this procedure, we synthesized histone H2AX, a variant of histone H2A, in high yield through one-pot five-segment ligation.<br/>We also report a new concept of peptide ligation strategy, in which photocleavable peptide−oligodeoxynucleotide conjugates were employed. The peptide bond formation in this strategy accelerated greatly under highly diluted condition due to the hybridization of oligodeoxynucleotides. The oligodeoxynucleotides was removed by photocleavage to accomplish the precisely ordered and simultaneous ligation of multiple synthetic peptide segments through the novel DNA-programmed peptide ligation strategy.

Reaction design for highly efficient chemical protein synthesis

2019 Spring National Meeting

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