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Hironobu Hojo - One of the best experts on this subject based on the ideXlab platform.
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sialyl tn unit with tfa labile protection realizes efficient synthesis of sialyl glycoprotein
Chemistry: A European Journal, 2018Co-Authors: Naoki Takeda, Toshiki Takei, Yuya Asahina, Hironobu HojoAbstract:Amino acids bearing 4-methylbenzyl (MBn) and 4-methoxybenzyl (MPM)-protected sialic acid were synthesized and used for the 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis of a glycopeptide. The α-sialyl linkage of the MBn-protected unit was partially cleaved under the final deprotection by trifluoroacetic acid (TFA). In addition, the removal of several MBn groups were incomplete. On the other hand, the MPM-protected unit gave the desired glycopeptide without decomposition of the α-sialyl linkage. Using this unit, peptide Thioesters of the tandem repeat unit of MUC1 mucin were synthesized by the N-alkylcysteine (NAC) method and used for the one-pot ligation by the thioester method. As a result, the three tandem repeats of MUC1 carrying sialyl Tn antigens were successfully synthesized.
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the phenacyl group as an efficient thiol protecting group in a peptide condensation reaction by the thioester method
Organic and Biomolecular Chemistry, 2013Co-Authors: Hidekazu Katayama, Hironobu HojoAbstract:One of the condensation methods for the preparation of long-chain peptides, the so-called thioester method requires protecting groups for amino and thiol groups for regioselective ligation. In this study, we demonstrated that the phenacyl (Pac) group acts as an efficient protecting group of cysteine side chains. We synthesized a cysteine derivative carrying the Pac group at the side chain sulfur atom, and Pac-containing peptides and peptide Thioesters were synthesized using it by the ordinary 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis strategy. Pac-containing peptide segments could be condensed by the thioester method. After the condensation reaction, Pac groups could be removed by Zn/AcOH treatment. In addition, the azido group, which was used for the protection of lysine side chains, was simultaneously converted into an amino group, demonstrating that this protecting group scheme simplified the deprotecting reaction after the peptide condensation reaction to a single step.
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n methyl phenacyloxycarbamidomethyl pocam group a novel thiol protecting group for solid phase peptide synthesis and peptide condensation reactions
Organic and Biomolecular Chemistry, 2011Co-Authors: Hidekazu Katayama, Yoshiaki Nakahara, Hironobu HojoAbstract:In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of cysteine side chains, and synthesized Pocam-containing peptides and peptide Thioesters. These were condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine side chains, was also converted to an amino group, demonstrating that this protecting group strategy simplified the deprotecting reaction after the peptide condensation reaction to only one step.
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Application of a novel thioesterification reaction to the synthesis of chemokine CCL27 by the modified thioester method.
Organic & biomolecular chemistry, 2008Co-Authors: Hironobu Hojo, Yuichi Murasawa, Hidekazu Katayama, Tsuyoshi Ohira, Yuko Nakahara, Yoshiaki NakaharaAbstract:Aryl Thioesters of peptide segments were prepared by the conventional 9-fluorenylmethoxycarbonyl (Fmoc) strategy using a novel N-alkyl cysteine (NAC)-assisted thioesterification reaction. The peptide carrying NAC at its C-terminus was prepared by the Fmoc strategy and converted to the aryl thioester by 4-mercaptophenylacetic acid (MPAA) treatment without significant side reactions. The peptide thioester was used for the efficient preparation of 95-amino acid (AA) chemokine CCL27 by an Ag+-free thioester method.
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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, 1992Co-Authors: Hironobu Hojo, Saburo AimotoAbstract: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.
Akira Otaka - One of the best experts on this subject based on the ideXlab platform.
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sequence independent traceless method for preparation of peptide protein Thioesters using cpasey mediated hydrazinolysis
Chemical & Pharmaceutical Bulletin, 2020Co-Authors: Masahiro Ueda, Akira Shigenaga, Chiaki Komiya, Sayuki Arii, Kohshi Kusumoto, Masaya Denda, Keiichiro Okuhira, Akira OtakaAbstract:Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein Thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein Thioesters derived from expressed proteins represent Thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)3-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.
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traceless synthesis of protein Thioesters using enzyme mediated hydrazinolysis and subsequent self editing of the cysteinyl prolyl sequence
Chemical Communications, 2019Co-Authors: Chiaki Komiya, Akira Shigenaga, Jun Tsukimoto, Masahiro Ueda, Takuya Morisaki, Tsubasa Inokuma, Kohji Itoh, Akira OtakaAbstract:A traceless thioester-producing protocol featuring carboxypeptidase Y-mediated hydrazinolysis of cysteinyl prolyl leucine-tagged peptides has been developed. The hydrazinolysis followed by thioesterification affords cysteinyl prolyl Thioesters. Self-editing of the tag and subsequent trans-thioesterification yields peptide Thioesters. The developed protocol was successfully applied to the conversion of recombinant proteins to Thioesters.
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Resin-Bound Crypto-Thioester for Native Chemical Ligation
2018Co-Authors: Naoto Naruse, Tsubasa Inokuma, Akira Shigenaga, Kento Ohkawachi, Akira OtakaAbstract:The resin-bound N-sulfanylethylanilide (SEAlide) peptide was found to function as a crypto-thioester peptide. Exposure of the peptide resin to an aqueous solution under neutral conditions in the presence of thiols affords Thioesters without accompanying racemization of C-terminal amino acids. Furthermore, the resin-bound SEAlide peptides react with N-terminal cysteinyl peptides in the absence of phosphate salts to afford ligated products, whereas soluble SEAlide peptides do not. This unexpected difference in reactivity of the SEAlide peptides allows for a one-pot/three-fragment ligation using resin-bound and unbound peptides
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preparation of peptide Thioesters from naturally occurring sequences using reaction sequence consisting of regioselective s cyanylation and hydrazinolysis
Biopolymers, 2016Co-Authors: Rin Miyajima, Akira Shigenaga, Tsubasa Inokuma, Yusuke Tsuda, Miki Imanishi, Shiroh Futaki, Akira OtakaAbstract:The vital roles of peptide/protein Thioesters in protein chemistry, including chemical or semi-synthesis of proteins, have encouraged studies on the development of methods for the preparation of such chemical units. Biochemical protocols using intein or sortase have proved to be useful in protein chemistry as methods suitable for naturally occurring sequences, including recombinant proteins. Although chemical protocols are potential options for thioester preparation, only a few are applicable to naturally occurring sequences, because standard chemical protocols require an artificial chemical device for producing Thioesters. In this context, the chemical preparation of Thioesters based on a reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis was investigated. Regioselective S-cyanylation, which is required for cysteine-containing Thioesters, was achieved with the aid of a zinc-complex formation of a CCHH-type zinc-finger sequence. Free cysteine residues that are not involved in complex formation were selectively protected with a 6-nitroveratryl group followed by S-cyanylation of the zinc-binding cysteine. Hydrazinolysis of the resulting S-cyanopeptide and subsequent photo-removal of the 6-nitroveratryl group yielded the desired peptide hydrazide, which was then converted to the corresponding thioester. The generated thioester was successfully used in N-to-C-directed one-pot/sequential native chemical ligation using an N-sulfanylethylanilide peptide to give a 64-residue peptide toxin. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 531-546, 2016.
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one pot sequential native chemical ligation using n sulfanylethylanilide peptide
Chemical Record, 2012Co-Authors: Akira Otaka, Kohei Sato, Hao Ding, Akira ShigenagaAbstract:N-Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide Thioesters by 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide Thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N-terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three-fragment or unprecedented four-fragment ligation for efficient one-pot peptide/protein synthesis. Furthermore, dual-kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto-peptide Thioesters. DOI 10.1002/tcr.201200007
Akira Shigenaga - One of the best experts on this subject based on the ideXlab platform.
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sequence independent traceless method for preparation of peptide protein Thioesters using cpasey mediated hydrazinolysis
Chemical & Pharmaceutical Bulletin, 2020Co-Authors: Masahiro Ueda, Akira Shigenaga, Chiaki Komiya, Sayuki Arii, Kohshi Kusumoto, Masaya Denda, Keiichiro Okuhira, Akira OtakaAbstract:Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein Thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein Thioesters derived from expressed proteins represent Thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)3-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.
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traceless synthesis of protein Thioesters using enzyme mediated hydrazinolysis and subsequent self editing of the cysteinyl prolyl sequence
Chemical Communications, 2019Co-Authors: Chiaki Komiya, Akira Shigenaga, Jun Tsukimoto, Masahiro Ueda, Takuya Morisaki, Tsubasa Inokuma, Kohji Itoh, Akira OtakaAbstract:A traceless thioester-producing protocol featuring carboxypeptidase Y-mediated hydrazinolysis of cysteinyl prolyl leucine-tagged peptides has been developed. The hydrazinolysis followed by thioesterification affords cysteinyl prolyl Thioesters. Self-editing of the tag and subsequent trans-thioesterification yields peptide Thioesters. The developed protocol was successfully applied to the conversion of recombinant proteins to Thioesters.
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Resin-Bound Crypto-Thioester for Native Chemical Ligation
2018Co-Authors: Naoto Naruse, Tsubasa Inokuma, Akira Shigenaga, Kento Ohkawachi, Akira OtakaAbstract:The resin-bound N-sulfanylethylanilide (SEAlide) peptide was found to function as a crypto-thioester peptide. Exposure of the peptide resin to an aqueous solution under neutral conditions in the presence of thiols affords Thioesters without accompanying racemization of C-terminal amino acids. Furthermore, the resin-bound SEAlide peptides react with N-terminal cysteinyl peptides in the absence of phosphate salts to afford ligated products, whereas soluble SEAlide peptides do not. This unexpected difference in reactivity of the SEAlide peptides allows for a one-pot/three-fragment ligation using resin-bound and unbound peptides
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preparation of peptide Thioesters from naturally occurring sequences using reaction sequence consisting of regioselective s cyanylation and hydrazinolysis
Biopolymers, 2016Co-Authors: Rin Miyajima, Akira Shigenaga, Tsubasa Inokuma, Yusuke Tsuda, Miki Imanishi, Shiroh Futaki, Akira OtakaAbstract:The vital roles of peptide/protein Thioesters in protein chemistry, including chemical or semi-synthesis of proteins, have encouraged studies on the development of methods for the preparation of such chemical units. Biochemical protocols using intein or sortase have proved to be useful in protein chemistry as methods suitable for naturally occurring sequences, including recombinant proteins. Although chemical protocols are potential options for thioester preparation, only a few are applicable to naturally occurring sequences, because standard chemical protocols require an artificial chemical device for producing Thioesters. In this context, the chemical preparation of Thioesters based on a reaction sequence consisting of regioselective S-cyanylation and hydrazinolysis was investigated. Regioselective S-cyanylation, which is required for cysteine-containing Thioesters, was achieved with the aid of a zinc-complex formation of a CCHH-type zinc-finger sequence. Free cysteine residues that are not involved in complex formation were selectively protected with a 6-nitroveratryl group followed by S-cyanylation of the zinc-binding cysteine. Hydrazinolysis of the resulting S-cyanopeptide and subsequent photo-removal of the 6-nitroveratryl group yielded the desired peptide hydrazide, which was then converted to the corresponding thioester. The generated thioester was successfully used in N-to-C-directed one-pot/sequential native chemical ligation using an N-sulfanylethylanilide peptide to give a 64-residue peptide toxin. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 531-546, 2016.
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one pot sequential native chemical ligation using n sulfanylethylanilide peptide
Chemical Record, 2012Co-Authors: Akira Otaka, Kohei Sato, Hao Ding, Akira ShigenagaAbstract:N-Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide Thioesters by 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide Thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N-terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three-fragment or unprecedented four-fragment ligation for efficient one-pot peptide/protein synthesis. Furthermore, dual-kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto-peptide Thioesters. DOI 10.1002/tcr.201200007
Hidekazu Katayama - One of the best experts on this subject based on the ideXlab platform.
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the phenacyl group as an efficient thiol protecting group in a peptide condensation reaction by the thioester method
Organic and Biomolecular Chemistry, 2013Co-Authors: Hidekazu Katayama, Hironobu HojoAbstract:One of the condensation methods for the preparation of long-chain peptides, the so-called thioester method requires protecting groups for amino and thiol groups for regioselective ligation. In this study, we demonstrated that the phenacyl (Pac) group acts as an efficient protecting group of cysteine side chains. We synthesized a cysteine derivative carrying the Pac group at the side chain sulfur atom, and Pac-containing peptides and peptide Thioesters were synthesized using it by the ordinary 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis strategy. Pac-containing peptide segments could be condensed by the thioester method. After the condensation reaction, Pac groups could be removed by Zn/AcOH treatment. In addition, the azido group, which was used for the protection of lysine side chains, was simultaneously converted into an amino group, demonstrating that this protecting group scheme simplified the deprotecting reaction after the peptide condensation reaction to a single step.
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n methyl phenacyloxycarbamidomethyl pocam group a novel thiol protecting group for solid phase peptide synthesis and peptide condensation reactions
Organic and Biomolecular Chemistry, 2011Co-Authors: Hidekazu Katayama, Yoshiaki Nakahara, Hironobu HojoAbstract:In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of cysteine side chains, and synthesized Pocam-containing peptides and peptide Thioesters. These were condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine side chains, was also converted to an amino group, demonstrating that this protecting group strategy simplified the deprotecting reaction after the peptide condensation reaction to only one step.
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Application of a novel thioesterification reaction to the synthesis of chemokine CCL27 by the modified thioester method.
Organic & biomolecular chemistry, 2008Co-Authors: Hironobu Hojo, Yuichi Murasawa, Hidekazu Katayama, Tsuyoshi Ohira, Yuko Nakahara, Yoshiaki NakaharaAbstract:Aryl Thioesters of peptide segments were prepared by the conventional 9-fluorenylmethoxycarbonyl (Fmoc) strategy using a novel N-alkyl cysteine (NAC)-assisted thioesterification reaction. The peptide carrying NAC at its C-terminus was prepared by the Fmoc strategy and converted to the aryl thioester by 4-mercaptophenylacetic acid (MPAA) treatment without significant side reactions. The peptide thioester was used for the efficient preparation of 95-amino acid (AA) chemokine CCL27 by an Ag+-free thioester method.
Oleg Melnyk - One of the best experts on this subject based on the ideXlab platform.
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Insights into the Mechanism and Catalysis of Peptide Thioester Synthesis by Alkylselenols Provide a New Tool for Chemical Protein Synthesis
2021Co-Authors: Florent Kerdraon, Gemma Bogard, Benoît Snella, Hervé Drobecq, Muriel Pichavant, Vangelis Agouridas, Oleg MelnykAbstract:While thiol-based catalysts are widely employed for chemical protein synthesis relying on peptide thioester chemistry, this is less true for selenol-based catalysts whose development is in its infancy. In this study, we compared different selenols derived from the selenocysteamine scaffold for their capacity to promote thiol-thioester exchanges in water at mildly acidic pH and the production of peptide Thioesters from bis(2-sulfanylethyl)amido (SEA) peptides. The usefulness of a selected selenol compound is illustrated by the total synthesis of a biologically active human chemotactic protein, which plays an important role in innate and adaptive immunity
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native chemical ligation and extended methods mechanisms catalysis scope and limitations
Chemical Reviews, 2019Co-Authors: Vangelis Agouridas, Vincent Diemer, Ouafâa El Mahdi, Marine Cargoet, Jeanchristophe Monbaliu, Oleg MelnykAbstract:The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide Thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O, S-, N, S-, or N, Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N, S-acyl migrations and selective dechalcogenation reactions.
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Catalysis of Thiol–Thioester Exchange by Water-Soluble Alkyldiselenols Applied to the Synthesis of Peptide Thioesters and SEA-Mediated Ligation
2018Co-Authors: Marine Cargoët, Benoît Snella, Hervé Drobecq, Vangelis Agouridas, Vincent Diemer, Rémi Desmet, Annick Blanpain, Oleg MelnykAbstract:N-Alkyl bis(2-selanylethyl)amines catalyze the synthesis of peptide Thioesters or peptide ligation from bis(2-sulfanylethyl)amido (SEA) peptides. These catalysts are generated in situ by reduction of the corresponding cyclic diselenides by tris(2-carboxyethyl)phosphine. They are particularly efficient at pH 4.0 by accelerating the thiol–thioester exchange processes, which are otherwise rate-limiting at this pH. By promoting SEA-mediated reactions at mildly acidic pH, they facilitate the synthesis of complex peptides such as cyclic O-acyl isopeptides that are otherwise hardly accessible
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Synthesis of Thiazolidine Thioester Peptides and Acceleration of Native Chemical Ligation
Organic Letters, 2011Co-Authors: Julien Dheur, Nathalie Ollivier, Oleg MelnykAbstract:Thiazolidine thioester peptides were synthesized by reacting bis(2-sulfanylethyl)amido peptides with glyoxylic acid at pH 1. A significant increase in Native Chemical Ligation (NCL) rate was observed with thiazolidine Thioesters compared to 3-mercaptopropionic acid-thioester analogues. The method is of particular interest for accelerating valine-cysteine peptide bond formation.
