Trifluoroethanethiol: An Additive for Efficient One-Pot Peptide Ligation−Desulfurization Chemistry

Thompson, Robert E.; Liu, Xuyu; Alonso-García, Noelia; Pereira, Pedro José Barbosa; Jolliffe, Katrina A.; Payne, Richard J.

doi:10.1021/ja502806r

Cited by 133 publications

(132 citation statements)

References 48 publications

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“…Very recently, the application of the β-thiol Asp derivative 97 [122] has been reported in the one-pot, three-component total synthesis of madanin-1 123 (Scheme 30), a small, Cys-free thrombin inhibitory protein derived from the hard tick H. longicornis [124]. In this study, the target 60-amino acid protein 123 was disconnected at Asp and Ala ligation junctions using ligation-desulfurization chemistry mediated by β-thiol Asp and Cys, respectively.…”

Section: Aspartic Acidmentioning

confidence: 99%

“…In this study, the target 60-amino acid protein 123 was disconnected at Asp and Ala ligation junctions using ligation-desulfurization chemistry mediated by β-thiol Asp and Cys, respectively. Interestingly, for the construction of the target protein, the authors also reported the use of a novel thiol additive, trifluoroethanethiol (TFET) 124, which facilitates the facile application of a one-pot ligation-desulfurization protocol [124]. Previous attempts to streamline the ligation-desulfurization strategy into a one-pot process have been hampered by the use of aryl thiol ligation additives which, because of their radical quenching ability [125], complicate the radical desulfurization process [126][127][128].…”

Section: Aspartic Acidmentioning

confidence: 99%

“…The alkyl thiol TFET was designed as an alternative additive in ligation reactions to circumvent the issues posed by aryl thiol additives in radical desulfurization reactions while maintaining efficiency as a ligation catalyst [124]. Because the pK a of TFET (¼7.30) is comparable to the pK a of common aryl thiol ligation additives, it was postulated that this alkyl thiol would be sufficiently nucleophilic to promote initial transthioesterification with the unactivated C-terminal peptide thioester and would also maintain sufficient leaving group ability to promote the acylation of the Cys thiol moiety.…”

Section: Aspartic Acidmentioning

confidence: 99%

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Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

Malins

Payne

2014

Protein Ligation and Total Synthesis I

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Over the past 20 years, native chemical ligation has facilitated the synthesis of numerous complex peptide and protein targets, with and without post-translational modifications, as well as the design and construction of a variety of engineered protein variants. This powerful methodology has also served as a platform for the development of related chemoselective ligation technologies which have greatly expanded the scope and flexibility of ligation chemistry. This chapter details a number of important extensions of the original native chemical ligation manifold, with particular focus on the application of new methods in the total chemical synthesis of proteins. Topics covered include the development of auxiliary-based ligation methods, the post-ligation manipulation of Cys residues, and the synthesis and utility of unnatural amino acid building blocks (bearing reactive thiol or selenol functionalities) in chemoselective ligation chemistry. Contemporary applications of these techniques to the total chemical synthesis of peptides and proteins are described.

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Section: Aspartic Acidmentioning

confidence: 99%

Section: Aspartic Acidmentioning

confidence: 99%

Section: Aspartic Acidmentioning

confidence: 99%

See 1 more Smart Citation

Modern Extensions of Native Chemical Ligation for Chemical Protein Synthesis

Malins

Payne

2014

Protein Ligation and Total Synthesis I

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“…We thus aimed for employing ligation conditions which allow one-pot free-radical desulfurization. 16 Initial attempts with the native chemical ligation (NCL) catalyst trifluoroethanethiol (TFET) 17 resulted in large amounts of thioester hydrolysis of the branched peptide 1. We thus opted for methyl thioglycolate (MTG) as a thiol additive, 18 which resulted in slower ligation kinetics but which showed drastically reduced hydrolysis product formation, while still allowing one-pot desulfurization.…”

mentioning

confidence: 99%

Controlling the supramolecular assembly of nucleosomes asymmetrically modified on H4

2017

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In stem cells, H4 proteins carrying different modifications coexist within single nucleosomes. For functional studies, we report the synthesis of such asymmetric nucleosomes. Asymmetry is achieved by transiently crosslinking H4 by a traceless, protease-removable tag introduced via an isopeptide linkage. These nucleosomes are used to study Set8 activity, a key methyltransferase.Nucleosomes, the basic unit of chromatin, organize 147 bp of DNA wrapped around two of each core histone H3, H4, H2A and H2B.1 The histone proteins carry combinations of post-translational modifications (PTMs, or marks), which are implicated in regulating chromatin function. 2 In particular, methylation of lysine residues on H3 and H4 has clearly defined roles in gene activation and repression, with implication for cell differentiation, development and disease. 3Detailed MS studies found that key methyl-marks in embryonic stem cells (ESCs) exist in asymmetric nucleosomes, i.e. nucleosomes carrying two differently modified copies of H3 or H4. 4 These PTMs include H4 monomethylated at lysine 20 (H4K20me1), as well as H3K4me3, H3K36me3 and H3K27me3. 5 H4K20 methylation is a critical modification involved in heterochromatin silencing, and also in DNA replication and the DNA damage response. 6 The methyltransferase Set8 (also known as PR-Set7 or KMT5A) is responsible for H4K20 monomethylation, whereas two further methyltransferases, Suv4-20h1 and Suv4-20h2, catalyze di-and trimethylation of this residue. 6 Together, H4K20 methylation is involved in chromatin structure regulation, 7 and serves as a binding site for chromatin regulators, including 53BP1 8 and L3MBTL1. The establishment, maintenance and function of nucleosomes asymmetrically modified on H4 is not well understood. This is mainly due to the fact that such nucleosomes are not easily available for detailed in vitro mechanistic studies. Here, we thus developed a synthetic strategy enabling the traceless synthesis of nucleosomes containing differentially modified H4 proteins, with a focus on H4K20me1. While expressed protein ligation (EPL) methods readily enable the installation of combinations of marks on nucleosomes, 10 the reconstitution of asymmetric chromatin is not straightforward and is largely based on the attachment of affinity tags, followed by multi-step purification schemes. 4,11 We recently developed a chemical method to address this problem and control supramolecular nucleosome assembly. 12 Our synthetic approach was based on the inclusion of a link-and-cut (lnc)-tag that enabled transient crosslinking (by a disulfide bond) of H3 species during nucleosome reconstitution. This allowed us to synthesize asymmetrically modified nucleosomes, carrying H3K4me3 and H3K27me3 on different H3 copies. 12 After the formation of nucleosomes, the crosslink was reversed and the lnc-tag was removed using tobacco etch virus (TEV) protease (Scheme 1A-C).Based on these studies, we thus wondered if our synthetic method could be adapted to synthesize crosslinked versions of differentially mo...

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“…Molecular weight characterization of all polymers synthesized in this study were carried out using both size exclusion chromatography (Scheme 2B and 2C and Supporting Information) and MALDI-TOF mass spectrometry (see Supplementary Table 1 Polymers 11-13 bearing pseudo-cysteine end groups were next assessed as substrates for NCL reactions with model peptide thioester Ac-LYRANG-S(CH2)2CO2Et 14 (synthesized by Fmoc-SPPS, see Supplementary Information) to generate end-functionalized polymer-peptide conjugates. All NCL reactions were conducted at pH 7.0 using tris-carboxyethylphosphine (TCEP) as a reductant and trifluoroethane thiol (TFET) 30 as an additive to convert the unreactive S-propionate ethyl ester of 14 into the corresponding reactive S-trifluoroethyl ester to accelerate the rate of the reactions (Table 1). We initially assessed the efficiency of NCL reactions between pseudo-cysteine end-functionalized polymer 11 and peptide thioester 14 in a number of different buffer/solvent mixtures.…”

mentioning

confidence: 99%