Triple Function of 4‐Mercaptophenylacetic Acid Promotes One‐Pot Multiple Peptide Ligation

Abstract

One-pot multiple peptide ligation is akey technology to improve the efficiency of chemical protein synthesis.O nepot repetitive peptide ligation requires ac ycle of three steps: peptide ligation, removal of ap rotecting group,a nd inactivation of the deprotection reagent. However,p revious strategies are not sufficient because of harsh deprotection conditions, slow deprotection rates,a nd difficulty in quenching the deprotection reagent. To address these issues,w ed eveloped ar apid, efficient deprotection and subsequent quenching strategy using an allyloxycarbonyl group to protect the Nterminal cysteine residue.4 -Mercaptophenylacetic acid (MPAA), at hiol additive for native chemical ligation, functioned not only as as cavenger for p-allyl palladium complexes,but also as aquencher of palladium(0) complexes. By utilizing the multifunctionality of MPAA, we carried out ao ne-pot five-segment ligation to affordh istone H2AX (142 amino acids), whichw as isolated in 59 %yield.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Ru Catalyst Facilitates Total Chemical Protein Synthesis to Investigate the Effect of Epigenetic Modifications Decorated on Linker Histone H1.2 and Heterochromatin Protein 1α (HP1α)

“…13 C NMR (acetone-d6, 126 MHz) δ = 157.6, 156. 1,152.2,150.3,149.6,122.4,121.4,110.3,109.8,109.4,97.6,96.9,89.9,87.9,83.3,80.6,66.4,65.2,43.1. HRMS(ESI)…”

“…13 C NMR (acetone-d6, 126 MHz) δ = 168. 1,138.8,138.4,137.5,137.4,133.9,133.8,133.7,133.6,133.2,131.8,131.7,130.8,126.7,126.6,126.2,126.1,125.1,125.0,124.9,124.8,123.8,108.4,100.3,80.6,56.0,122 MHz) δ = 27.8,…”

“…8, 171.0, 156.3, 153.9, 144.3, 141.3, 137.9, 133.9, 130.5, 129.8, 128.2, 127.6, 125.6, 120.6, 118.7, 117.5, 66.0, 65.0, 54.4, 47.1, 42.0, 40.9, 32 following our previous report. 1 To peptide 1 solution in 15.0 μL denaturing buffer (6 M Gn•HCl aq containing 0.2 M NaH2PO4 at pH 7.0) (4.0 mM) were added denaturing buffer 5.0 μL, 2.4 μL TCEP aq (500 mM), 0.4 μL MgCl2 (500 mM), and 6.0 μL MPAA aq (500 mM). Then, to the mixture was added 1.2 μL Pd/phosphine solution (100 mM) and the reaction mixture were stirred for 10 min at room temperature under argon atmosphere.…”

Thiazolidine Deprotection by 2-Aminobenzamide-Based Aldehyde Scavenger for One-Pot Multiple Peptide Ligation

“…Among various one-pot peptide ligation strategies, [14][15][16][17][18][19][20][21][22][23] the use of protecting groups at N-terminal amine or thiol of Cys at internal segments was the simplest approach and reported in many research groups. For example, acetamidomethyl (Acm) group [22] and trifluoroacetamidomethyl (Tfacm) for thiol group, [24] 4-(dimethylamino)phenacyloxycarbonyl (Mapoc), [25] pborobenzyloxycarbonyl group (Dobz), [19] allyloxycarbonyl (Alloc) [26][27][28] and 9-fluorenylmethyloxycarboyl (Fmoc) for amine group [29] were successful N-terminal cysteinyl protecting groups in one-pot peptide ligation.…”

Convergent Hybrid Phase Ligation Strategy for Efficient Total Synthesis of Large Proteins Demonstrated for 212-residue Linker Histone H1.2

“…Typically, this has required division of a protein into accessible segments that can be stitched together using chemoligation strategies such as native chemical ligation (Agouridas et al, 2019; Dawson, Muir, & Kent, 1994; Kent, 2009; Müller & Muir, 2015) (NCL, resulting in a native peptide bond, most commonly at a cysteine site) followed by desulfurization (allowing expanded ligation sites, including alanine) (Jin, Li, Chow, Liu, & Li, 2017; Wan & Danishefsky, 2007; Yan & Dawson, 2001). Sequential (Raibaut, Ollivier, & Melnyk, 2012; Shimko, North, Bruns, Poirier, & Ottesen, 2011), one-pot (Bang & Kent, 2004; Kamo, Hayashi, & Okamoto, 2018; Li et al, 2014; Tang et al, 2015; Zuo, Zhang, Yan, & Zheng, 2018), and convergent (Bang, Pentelute, & Kent, 2006; Durek, Torbeev, & Kent, 2007; Fang, Wang, & Liu, 2012; Qi, He, Ai, Guo, & Li, 2017) ligation strategies have enabled the synthesis of small to medium-sized proteins (<150 residues) but have been limited in scope and practical application by the size and synthetic accessibility of the component peptide segments, the chemical or kinetic control of intermediate ligation specificity, and the number of purification steps required to achieve the final product. In theory, sequential ligation carried out on the solid phase should overcome these barriers, allowing facile recombination of peptide segments while eliminating the need for intermediate purification (Brik, Keinan, & Dawson, 2000; Canne et al, 1999; Jbara, Seenaiah, & Brik, 2014).…”