Thioester

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Derek Macmillan - One of the best experts on this subject based on the ideXlab platform.

  • Investigation of Cysteine as an Activator of Side-Chain N→S Acyl Transfer and Tail-to-Side-Chain Cyclization
    Synlett, 2017
    Co-Authors: Durbis J. Castillo-pazos, Derek Macmillan
    Abstract:

    N→S Acyl transfer is a popular method for the postsynthesis production of peptide C α-Thioesters for use in native chemical ligation and for the synthesis of head-to-tail cyclic peptides. Meanwhile Thioester formation at the side chain of aspartic or glutamic acids, leading to tail-to-side-chain-cyclized species, is less common. Herein we explore the potential for cysteine to function as a latent Thioester when appended to the side chain of glutamic acid. Initial insights gained through study of C-terminal β-alanine as a model for the increased chain length were ultimately applied to peptide macrocyclization. Our results emphasize the increased barrier to acyl transfer at the glutamic acid side chain and indicate how a slow reaction, facilitated by cysteine itself, may be accelerated by fine-tuning of the stereoelectronic environment.

  • Investigation of peptide Thioester formation via N→Se acyl transfer
    Journal of peptide science : an official publication of the European Peptide Society, 2013
    Co-Authors: Anna L. Adams, Derek Macmillan
    Abstract:

    Native chemical ligation is widely used for the convergent synthesis of proteins. The peptide Thioesters required for this process can be challenging to produce, particularly when using Fmoc-based solid-phase peptide synthesis. We have previously reported a route to peptide Thioesters, following Fmoc solid-phase peptide synthesis, via an NS acyl shift that is initiated by the presence of a C-terminal cysteine residue, under mildly acidic conditions. Under typical reaction conditions, we occasionally observed significant Thioester hydrolysis as a consequence of long reaction times (~48 h) and sought to accelerate the reaction. Here, we present a faster route to peptide Thioesters, by replacing the C-terminal cysteine residue with selenocysteine and initiating Thioester formation via an NSe acyl shift. This modification allows Thioester formation to take place at lower temperatures and on shorter time scales. We also demonstrate how application of this strategy also accelerates peptide cyclization, when a linear precursor is furnished with an N-terminal cysteine and C-terminal selenocysteine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

  • shifting native chemical ligation into reverse through n s acyl transfer
    Israel Journal of Chemistry, 2011
    Co-Authors: Derek Macmillan, Anna L. Adams, Bhavesh Premdjee
    Abstract:

    Peptide Thioester synthesis by N→S acyl transfer is being intensively explored by many research groups the world over. Reasons for this likely include the often straightforward method of precursor assembly using Fmoc-based chemistry and the fundamentally interesting acyl migration process. In this review we introduce recent advances in this exciting area and discuss, in more detail, our own efforts towards the synthesis of peptide Thioesters through N→S acyl transfer in native peptide sequences. We have found that several peptide Thioesters can be readily prepared and, what’s more, there appears to be ample opportunity for further development and discovery.

  • Access to phosphoproteins and glycoproteins through semi-synthesis, Native Chemical Ligation and N→S acyl transfer
    Organic and Biomolecular Chemistry, 2010
    Co-Authors: Jinit Masania, Jiejin Li, Stephen J Smerdon, Derek Macmillan
    Abstract:

    Peptide Thioesters are important tools for protein synthesis and semi-synthesis through their use in Native Chemical Ligation (NCL). NCL can be employed to assemble site-specifically modified proteins that can help elucidate the mechanisms of biomolecular processes. In this article we explore the compatibility of phosphopeptide synthesis and glycopeptide synthesis with Thioester production through N→S acyl transfer.

John Offer - One of the best experts on this subject based on the ideXlab platform.

  • A Shortcut to the Synthesis of Peptide Thioesters.
    Methods in molecular biology (Clifton N.J.), 2020
    Co-Authors: Richard Raz, John Offer
    Abstract:

    Peptide Thioesters serve as fundamental building blocks for the synthesis of proteins and cyclic peptides. Classically, methods to synthesize Thioesters have been based on acid-labile amino-protecting groups for which final side-chain deprotection required the use of hazardous hydrogen fluoride (HF). Alternative protection schemes based on base-labile amino-protecting groups have become preferred methods but are not suitable due to the lability of Thioester bonds toward bases. In this method, we employ a trifluoracetic acid/trimethylsilyl bromide (TFA/TMSBr) protocol using a hydroxymethyl resin obviating the need for HF. TFA/TMSBr is volatile enough to be easily removed yet less hazardous than HF, making it more practical for general peptide chemists. We describe optimized cleavage procedures and appropriate protecting group schemes and discuss in situ neutralization protocols. The method is relatively simple, straightforward, and easily scalable, allowing the facile preparation of alkyl and aryl Thioesters.

  • in situ Thioester formation for protein ligation using α methylcysteine
    Chemical Science, 2014
    Co-Authors: Fabienne Burlina, George Papageorgiou, Caroline Morris, Peter D. White, John Offer
    Abstract:

    The progress of total chemical protein synthesis has been hampered by difficulties in preparing peptide Thioesters by standard Fmoc peptide synthesis. The amino acid, α-methylcysteine, sited at the C-terminus of a peptide can substitute for a Thioester in peptide ligation reactions. C-terminal α-methylcysteine is fully compatible with Fmoc peptide synthesis and its use in ligation is very simple and robust. Its potential is demonstrated with the synthesis of model proteins.

  • In situ Thioester formation for protein ligation using a-methylcysteine
    Chemical Science, 2013
    Co-Authors: Fabienne Burlina, George Papageorgiou, Caroline Morris, Peter D. White, John Offer
    Abstract:

    The progress of total chemical protein synthesis has been hampered by difficulties in preparing peptide Thioesters by standard Fmoc peptide synthesis. The amino acid, a-methylcysteine, sited at the C-terminus of a peptide can substitute for a Thioester in peptide ligation reactions. C-terminal a-methylcysteine is fully compatible with Fmoc peptide synthesis and its use in ligation is very simple and robust. Its potential is demonstrated with the synthesis of model proteins.

James C. Powers - One of the best experts on this subject based on the ideXlab platform.

  • [1] Peptide Thioester substrates for serine peptidases and metalloendopeptidases
    Methods in Enzymology, 2004
    Co-Authors: James C. Powers
    Abstract:

    Abstract Peptide Thioesters are sensitive substrates of various serine peptidases and metalloendopeptidases. Thioester substrates generally have high enzymatic hydrolysis rates and low background hydrolysis rates, and the hydrolysis rates can be easily monitored in the presence of thiol reagents such as 4,4′-dithiodipyridine or 5,5′-dithiobis (2-nitrobenzoic acid). Peptide Thioester substrates have been invaluable for the study of enzyme specificity and enzyme inhibitors, especially in cases where no other practical synthetic substrates are available. Tripeptide substrates of the type Boc-Ala-Ala-AA-SBzl, where AA is nearly all of the 20 common amino acids, have now been synthesized and should be useful for the subsite mapping of new serine peptidases and the study of crude cell preparations containing serine peptidases.

  • Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide Thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.
    Biochemistry, 1991
    Co-Authors: Shinjiro Odake, Lakshmi S. Narasimhan, Joseph T. Blake, Olivier Krahenbuhl, Juerg Tschopp, James C. Powers
    Abstract:

    : The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide Thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing Thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any Thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing Thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).

  • Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide Thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.
    Biochemistry, 1991
    Co-Authors: Shinjiro Odake, Lakshmi S. Narasimhan, Joseph T. Blake, Olivier Krahenbuhl, Juerg Tschopp, James C. Powers
    Abstract:

    : The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide Thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing Thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any Thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing Thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).

Saburo Aimoto - One of the best experts on this subject based on the ideXlab platform.

  • Epimerization in peptide Thioester condensation.
    Journal of peptide science : an official publication of the European Peptide Society, 2012
    Co-Authors: Kenta Teruya, Toru Kawakami, Takeyuki Tanaka, Kenichi Akaji, Saburo Aimoto
    Abstract:

    Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide Thioester. Peptide Thioester condensation, in which a C-terminal peptide Thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the Thioester is at risk of epimerization. During the preparation of peptide Thioesters by the Boc solid-phase method, no substantial epimerization of the C-terminal amino acid was detected. Epimerization was, however, observed during a Thioesterthiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, Thioesterthiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in Thioester peptide condensation. These results were applied to a longer peptide Thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the Thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

  • peptide Thioester preparation based on an n s acyl shift reaction mediated by a thiol ligation auxiliary
    Tetrahedron Letters, 2005
    Co-Authors: Toru Kawakami, Megumi Sumida, Kenichiroh Nakamura, Thomas Vorherr, Saburo Aimoto
    Abstract:

    Formation of peptide Thioesters, based on an N to S acyl shift mediated by an auxiliary, N-4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group, under acidic conditions, is described. The protected peptide was assembled on a hydroxymethylphenylacetamidomethyl resin via an N-Dmmb-amino acid residue according to standard Fmoc solid-phase peptide synthesis following treatment with trifluoroacetic acid. The peptide α-Thioester was released from the resin by reaction with 2-mercaptoethanesulfonic acid in the presence of N,N-diisopropylethylamine.

  • Sequential peptide chemical ligation by the Thioester method and extended chemical ligation
    Tetrahedron Letters, 2005
    Co-Authors: Toru Kawakami, Masahiro Tsuchiya, Ken’ichiroh Nakamura, Saburo Aimoto
    Abstract:

    Abstract The sequential chemical ligation of peptide Thioesters by a combination of the Thioester method and extended chemical ligation using a photoremovable auxiliary, 2-mercapto-1-(2-nitrophenyl)ethyl group, is described. The thiazolidine ring was used as a protecting group for the N-terminal 1,2-aminoethanethiol moiety of the auxiliary in the middle peptide Thioester. After the first Thioester coupling, the thiazolidine ring was opened by treatment with O -methylhydroxylamine. Second coupling by extended chemical ligation followed by UV irradiation gave the target polypeptide.

  • Polypeptide synthesis by the Thioester method.
    Biopolymers, 1999
    Co-Authors: Saburo Aimoto
    Abstract:

    A novel method for polypeptide synthesis, in which partially protected peptide Thioesters are used as building blocks, has been developed. Partially protected peptide Thioesters are easily prepared by solid-phase methodology. The Thioester moiety is converted to an active ester in the presence of a silver compound such as AgNO(3) or AgCl and an active ester component such as 1-hydroxybenzotriazole or 3,4-dihydro-3-hydro-4-oxo-1,2, 3-benzotriazine. Segment condensation can be accomplished using partially protected peptide segments. The consecutive condensation of the partially protected peptide segments is realized by the selective removal of the 9-flourenylmethoxycarbonyl group, for terminal amino protection, after segment condensation has been achieved. In this method, large peptide segments can easily be used. Thus, the products obtained by the Thioester method can be separated from by-products by reverse phase high performance liquid chromatography, even when no purification process was performed during the prior segment condensation procedures. This indicates that proteins that have no specific features such as enzymatic or biological activities can be obtained after isolation, solely based on their chromatographic profiles. Thus, the Thioester method will provide a new basis for protein studies including phosphorylated and glycosylated polypeptides.

  • protein synthesis using s alkyl Thioester of partially protected peptide segments synthesis of dna binding protein of bacillus stearothermophilus
    Bulletin of the Chemical Society of Japan, 1992
    Co-Authors: Hironobu Hojo, Saburo Aimoto
    Abstract:

    Using partially protected peptide Thioesters as building blocks, we synthesized HU-type DNA-binding protein of Bacillus stearothermophilus. Four peptide segments, Boc–[Lys(Boc)3]–HBs(1–15)–SCH2CH2CONH2, iNoc–[Lys(Boc)18,19,23,38]–HBs(16–39)–SCH2CH2CONH2, iNoc–[Lys(Boc)41,59]–HBs(40–60)–SCH2CH2CONH2, [Lys(Boc)75,80,83,86,90]–HBs(61–90) were prepared using peptides obtained by a solid-phase method. A partially protected peptide Thioester was condensed to a peptide with a free amino group by converting the Thioester to the corresponding active ester in the presence of silver ions and N-hydroxysuccinimide. Finally, highly pure synthetic HBs(1–90) was obtained.

Philip E. Dawson - One of the best experts on this subject based on the ideXlab platform.

  • chemical protein synthesis using a second generation n acylurea linker for the preparation of peptide Thioester precursors
    Journal of the American Chemical Society, 2015
    Co-Authors: Juan B Blancocanosa, Brunello Nardone, Fernando Albericio, Philip E. Dawson
    Abstract:

    The broad utility of native chemical ligation (NCL) in protein synthesis has fostered a search for methods that enable the efficient synthesis of C-terminal peptide-Thioesters, key intermediates in NCL. We have developed an N-acylurea (Nbz) approach for the synthesis of Thioester peptide precursors that efficiently undergo thiol exchange yielding Thioester peptides and subsequently NCL reaction. However, the synthesis of some glycine-rich sequences revealed limitations, such as diacylated products that can not be converted into N-acylurea peptides. Here, we introduce a new N-acylurea linker bearing an o-amino(methyl)aniline (MeDbz) moiety that enables in a more robust peptide chain assembly. The generality of the approach is illustrated by the synthesis of a pentaglycine sequence under different coupling conditions including microwave heating at coupling temperatures up to 90 C, affording the unique and desired N-acyl-N′-methylacylurea (MeNbz) product. Further extension of the method allowed the synthesis...

  • an efficient fmoc spps approach for the generation of Thioester peptide precursors for use in native chemical ligation
    Angewandte Chemie, 2008
    Co-Authors: Juan B Blancocanosa, Philip E. Dawson
    Abstract:

    The straightforward C-terminal modification of peptides assembled on a solid support remains a significant challenge in peptide and protein chemistry. In particular, C-terminal Thioester peptides are important intermediates for the generation of active esters, amides and hydrazides[1,2] and are an essential component of many synthetic strategies for protein synthesis.[3] Currently, the most effective approach for the synthesis of peptidyl Thioesters is the in situ neutralization protocol for Boc solid phase peptide synthesis (Boc-SPPS)[4] using Thioester linkers.[2,5] However, many laboratories use Fmoc-SPPS exclusively and such protocols are favored when synthesizing glyco- and phosphopeptides. The Thioester linkers used for Boc-SPPS have limited utility for Fmoc-SPPS due to the requirement for repeated Fmoc removal under basic conditions. Considerable effort has been applied to address this challenge[6] including optimized Fmoc deprotection cocktails,[7] thiol labile safety catch linkers,[8] activation of protected peptides in solution,[9] and recently Thioesters have been generated using O to S[10] or N to S[11] acyl transfer. Despite these notable advances, the synthesis of Thioester peptides by Fmoc-SPPS remains significantly more challenging than the synthesis of the corresponding acid or amide peptide.

  • Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives
    Journal of the American Chemical Society, 1997
    Co-Authors: Philip E. Dawson, Michael J. Churchill, M. Reza Ghadiri, Stephen B. H. Kent
    Abstract:

    In native chemical ligation, an unprotected peptide α-carboxy Thioester is reacted with a second peptide containing an N-terminal cysteine residue. It was anticipated that addition of thiophenol to a native chemical ligation reaction would keep cysteine side chains reduced, catalyze the reversal of unproductive Thioester formation, and generate a more reactive phenyl Thioester through thiol exchange. Several model peptide−α-Thioesters were treated with an excess of a competing thiol to investigate their susceptibility to thiol exchange:  a highly activated 3-nitro-4-carboxybenzyl α-Thioester was smoothly converted to the less activated benzyl α-Thioester through the addition of an excess of benzyl mercaptan; similarly, a peptide containing the benzyl α-Thioester group was converted to a more reactive phenyl α-Thioester by addition of thiophenol. Even a weakly activated peptide−α-Thioester was converted to a substantially more reactive species, as demonstrated by the conversion of peptide−αCOS-CH2COOH to p...