Sequential Peptide Ligation by Combining the Cys–Pro Ester (CPE) and Thioester Methods and Its Application to the Synthesis of Histone H3 Containing a Trimethyl Lysine Residue

Abstract

A peptide thioester is a key building block for protein synthesis by a ligation method, such as the thioester method and native chemical ligation. Combining these ligation methods offers flexibility in the choice of the condensation sites in a sequential ligation strategy. We describe herein a novel strategy, in which native chemical ligation followed by the thioester method are utilized, based on the use of a peptide containing a CysPro ester (CPE) autoactivating unit at the C-terminus as a peptide thioester precursor. This sequential ligation strategy was applied to the synthesis of histone H3, which consists of 135 amino acid residues and contains a trimethyl Lys 9 residue.The ligation technique that utilizes peptide segments is a widely used procedure for protein synthesis.1 In the thioester method, a peptide thioester is used as a building block.2 The reaction can be performed at any ligation site, and it requires some protecting groups, activators such as silver salts, and organic solvents. In the native chemical ligation (NCL) method, a peptide thioester is also used as a building block, and can be carried out under neutral aqueous conditions without the need for protecting groups, although a cysteine residue is required at the condensation site.3 Several thiol auxiliary groups have been introduced that permit ligation to proceed without the need for a cysteine residue. 4 The combination of these ligation methods offers flexibility in the choice of condensation sites in multistep ligations for protein synthesis. We previously proposed the possibility of sequential chemical ligation, in which the thioester method followed by the thiol mediated extended chemical ligation is performed in a sequential manner. 5 Here we report on an alternative strategy, in which the thiol-mediated ligation, followed by the thioester method is performed using a cysteinyl proline ester (CPE) autoactivating unit, 6 and this strategy is applied to a synthesis of histone H3.Post-translational modifications play major roles in the regulation of numerous biological processes. Histone modifications, such as acetylation, methylation, phosphorylation, and ubiqutination, play a role in gene expression and silencing program. 7 A specifically modified histone library is extremely useful for functional studies of these molecules. So far, the synthesis of histone H2B 8 and H4, 9 which contain no Cys residues, has been reported based on the combination of NCL and a desulfurization strategy by several groups. On the other hand, methods for the synthesis of a Cys residue-containing histone H3 are limited, especially when the N-terminal tail region is modified, which is known to have complex modifications, and the syntheses were accompanied with Cys mutations for synthetic purposes by NCL. 9a,9c,9d,10 To the best of our knowledge, there are no reports of the synthesis of histone H3 without mutations, in which the tail region contains modifications. We started the synthesis of histone H3, which contains a total of 135 amino acid residues conta...

Chemistry‐Driven Epigenetic Investigation of Histone and DNA Modifications

“…Poirier and co‐workers also performed a similar approach to investigate the effect of phosphorylation at H3Y41 . In 2013, Kawakami and Aimoto synthesized K9‐trimethylated H3 by the combination of thioester ligation and NCL using CPE (Figure b) . Protection of the thiol groups and subsequent thioester ligation method enabled the synthesis of native H3.1, which contains two Cys residues.…”

Chemical synthesis of the ubiquitinated form of histone H3 and its effect on DNA methyltransferase 1

“…It is known that histones have many PTM sites and that they play an important role in epigenetic regulation . We previously reported on the synthesis of N ε ‐trimethyllysine‐containing histones H3 and H4 . Proteins with other small modifications, such as acetylation and phosphorylation, could be synthesized by the similar strategy.…”

One-pot native chemical ligation of peptide hydrazides enables total synthesis of modified histones

“…8 Meanwhile, Aimoto and coworkers accomplished the first Fmoc-based total synthesis of H3 by using the Cys-Pro ester autoactivating unit as a thioester precursor. 9 Notwithstanding these key advances, to meet the escalating needs in the studies of histone PTMs it is helpful to develop a more scalable and cost-effective method for the total synthesis of modified histones on a multi-milligram scale.…”