Akira Otaka

Elucidation of inhibitor-binding pockets of D-amino acid oxidase using docking simulation and N-sulfanylethylanilide-based labeling technology

Co-reporter:Taiki Kohiki;Yusuke Kato;Yusuke Nishikawa;Kazuko Yorita;Ikuko Sagawa;Masaya Denda;Tsubasa Inokuma;Akira Shigenaga;Kiyoshi Fukui

Organic & Biomolecular Chemistry 2017 vol. 15(Issue 25) pp:5289-5297

Publication Date(Web):2017/06/27

DOI:10.1039/C7OB00633K

Because of the relevance of D-serine (D-Ser) to schizophrenia, inhibitors of D-amino acid oxidase (DAO), which catalyzes degradation of D-Ser in the presence of flavin adenine dinucleotide (FAD), are expected to be anti-schizophrenia therapeutics. In this study, binding pockets of DAO to its inhibitor 4-bromo-3-nitrobenzoic acid were searched by combining in silico docking simulation and labeling experiments employing an N-sulfanylethylanilide-based labeling technology that we have developed. The results clearly demonstrated that there are two binding pockets: one is shared with D-Ser and FAD, and the other is an unexpected cleft between the subunits of a DAO dimer. These findings will provide insight to aid the development of new DAO inhibitors. In addition, it was also proved that our labeling technology could be applicable to elucidate the binding pockets of proteins.

One-Pot/Sequential Native Chemical Ligation Using Photocaged Crypto-thioester

Co-reporter:Keisuke Aihara, Kosuke Yamaoka, Naoto Naruse, Tsubasa Inokuma, Akira Shigenaga, and Akira Otaka

Organic Letters 2016 Volume 18(Issue 3) pp:596-599

Publication Date(Web):January 12, 2016

DOI:10.1021/acs.orglett.5b03661

A practical and efficient methodology for the chemical synthesis of peptides/proteins using a one-pot/sequential ligation is described. It features the use of photocleavable S-protection on an N-sulfanylethylaniline moiety. Removal of the S-protecting ligated materials under UV irradiation provides a readily usable mixture for subsequent native chemical ligation.

Development of an Anilide-Type Scaffold for the Thioester Precursor N-Sulfanylethylcoumarinyl Amide

Co-reporter:Mitsuhiro Eto, Naoto Naruse, Kyohei Morimoto, Kosuke Yamaoka, Kohei Sato, Kohei Tsuji, Tsubasa Inokuma, Akira Shigenaga, and Akira Otaka

Organic Letters 2016 Volume 18(Issue 17) pp:4416-4419

Publication Date(Web):August 16, 2016

DOI:10.1021/acs.orglett.6b02207

N-Sulfanylethylcoumarinyl amide (SECmide) peptide, which was initially developed for use in the fluorescence-guided detection of promoters of N–S acyl transfer, was successfully applied to a facile and side reaction-free protocol for N–S acyl-transfer-mediated synthesis of peptide thioesters. Additionally, 4-mercaptobenzylphosphonic acid (MBPA) was proven to be a useful catalyst for the SECmide or N-sulfanylethylanilide (SEAlide)-mediated NCL reaction.

An N-sulfanylethylanilide-based traceable linker for enrichment and selective labelling of target proteins

Co-reporter:Takuya Morisaki, Masaya Denda, Jun Yamamoto, Daisuke Tsuji, Tsubasa Inokuma, Kohji Itoh, Akira Shigenaga and Akira Otaka

Chemical Communications 2016 vol. 52(Issue 42) pp:6911-6913

Publication Date(Web):26 Apr 2016

DOI:10.1039/C6CC01229A

An N-sulfanylethylanilide-based traceable linker, developed to facilitate identification of target proteins of bioactive compounds, was introduced into an alkynylated target protein. Subsequent adsorption onto streptavidin beads allowed it to be treated with a cysteine–fluorophore conjugate in the presence of phosphate. This induced the N–S acyl transfer reaction of the N-sulfanylethylanilide unit. The subsequent native chemical ligation of the fluorophore resulted in cleavage of the linker for target elution and fluorescence labelling of the target, allowing it to be distinguished from non-target proteins.

Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1-Selective Inactivators

Co-reporter:Yukihiro Itoh; Keisuke Aihara; Paolo Mellini; Toshifumi Tojo; Yosuke Ota; Hiroki Tsumoto; Viswas Raja Solomon; Peng Zhan; Miki Suzuki; Daisuke Ogasawara; Akira Shigenaga; Tsubasa Inokuma; Hidehiko Nakagawa; Naoki Miyata; Tamio Mizukami; Akira Otaka;Takayoshi Suzuki

Journal of Medicinal Chemistry 2016 Volume 59(Issue 4) pp:1531-1544

Publication Date(Web):December 23, 2015

DOI:10.1021/acs.jmedchem.5b01323

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.

Labelling of endogenous target protein via N–S acyl transfer-mediated activation of N-sulfanylethylanilide

Co-reporter:Masaya Denda, Takuya Morisaki, Taiki Kohiki, Jun Yamamoto, Kohei Sato, Ikuko Sagawa, Tsubasa Inokuma, Youichi Sato, Aiko Yamauchi, Akira Shigenaga and Akira Otaka

Organic & Biomolecular Chemistry 2016 vol. 14(Issue 26) pp:6244-6251

Publication Date(Web):31 May 2016

DOI:10.1039/C6OB01014H

The ligand-dependent incorporation of a reporter molecule (e.g., fluorescence dye or biotin) onto a endogenous target protein has emerged as an important strategy for elucidating protein function using various affinity-based labelling reagents consisting of reporter, ligand and reactive units. Conventional labelling reagents generally use a weakly activated reactive unit, which can result in the non-specific labelling of proteins in a ligand-independent manner. In this context, the activation of a labelling reagent through a targeted protein–ligand interaction could potentially overcome the problems associated with conventional affinity-based labelling reagents. We hypothesized that this type of protein–ligand-interaction-mediated activation could be accomplished using N-sulfanylethylanilide (SEAlide) as the reactive unit in the labelling reagent. Electrophilically unreactive amide-type SEAlide can be activated by its conversion to the corresponding active thioester in the presence of a phosphate salt, which can act as an acid–base catalyst. It has been suggested that protein surfaces consisting of hydrophilic residues such as amino, carboxyl and imidazole groups could function as acid–base catalysts. We therefore envisioned that a SEAlide-based labelling reagent (SEAL) bearing SEAlide as a reactive unit could be activated through the binding of the SEAL with a target protein. Several SEALs were readily prepared in this study using standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase protocols. These SEAL systems were subsequently applied to the ligand-dependent labelling of human carbonic anhydrase (hCA) and cyclooxyganese 1. Although we have not yet obtained any direct evidence for the target protein-mediated activation of the SEAlide unit, our results for the reaction of these SEALs with hCA1 or butylamine indirectly support our hypothesis. The SEALs reported in this study represent valuable new entries to the field of affinity-based labelling reagents and are expected to show great utility in protein labelling.

Facile synthesis of C-terminal peptide thioacids under mild conditions from N-sulfanylethylanilide peptides

Co-reporter:Tatsuhiko Shimizu, Rin Miyajima, Kohei Sato, Ken Sakamoto, Naoto Naruse, Miku Kita, Akira Shigenaga, Akira Otaka

Tetrahedron 2016 Volume 72(Issue 7) pp:992-998

Publication Date(Web):18 February 2016

DOI:10.1016/j.tet.2015.12.070

A facile procedure has been developed for the synthesis of C-terminal peptide thioacids under mild conditions. A series of N-sulfanylethylanilide peptides prepared using Fmoc-based solid-phase peptide synthesis were successfully converted to the corresponding thioacids via a hydrothiolysis reaction in a phosphate buffer with only trace epimerization of the C-terminal amino acid.

Development of an Intein-Inspired Amide Cleavage Chemical Device

Co-reporter:Chiaki Komiya, Keisuke Aihara, Ko Morishita, Hao Ding, Tsubasa Inokuma, Akira Shigenaga, and Akira Otaka

The Journal of Organic Chemistry 2016 Volume 81(Issue 2) pp:699-707

Publication Date(Web):December 8, 2015

DOI:10.1021/acs.joc.5b02399

A photoresponsive amide cleavage device was developed based on the asparagine imidation-mediated cleavage of peptide bonds during intein-mediated protein splicing. The chemical environment of the protein splicing process was mimicked by the incorporation of geminal dimethyl groups and a secondary amine unit in asparagine scaffold. Furthermore, the resulting photoresponsive device could induce the phototriggered cleavage of an amide bond by the protection of the secondary amine unit with an o-nitrobenzyloxycarbonyl group.

The total chemical synthesis of the monoglycosylated GM2 ganglioside activator using a novel cysteine surrogate

Co-reporter:Kohei Sato, Keisuke Kitakaze, Takahiro Nakamura, Naoto Naruse, Keisuke Aihara, Akira Shigenaga, Tsubasa Inokuma, Daisuke Tsuji, Kohji Itoh and Akira Otaka

Chemical Communications 2015 vol. 51(Issue 49) pp:9946-9948

Publication Date(Web):13 May 2015

DOI:10.1039/C5CC02967H

We describe a novel peptide ligation/desulfurization strategy using a β-mercapto-N-glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 ganglioside activator protein bearing a monosaccharide on the native glycosylation site.

Photo-triggered fluorescent labelling of recombinant proteins in live cells

Co-reporter:Deokho Jung, Kohei Sato, Kyoungmi Min, Akira Shigenaga, Juyeon Jung, Akira Otaka and Youngeun Kwon

Chemical Communications 2015 vol. 51(Issue 47) pp:9670-9673

Publication Date(Web):15 May 2015

DOI:10.1039/C5CC01067E

A method to photo-chemically trigger fluorescent labelling of proteins in live cells is developed. The approach is based on photo-caged split-intein mediated conditional protein trans-splicing reaction and enabled background-free fluorescent labelling of target proteins with the necessary spatiotemporal control.

Liquid-Phase Synthesis of Bridged Peptides Using Olefin Metathesis of a Protected Peptide with a Long Aliphatic Chain Anchor

Co-reporter:Keisuke Aihara, Chiaki Komiya, Akira Shigenaga, Tsubasa Inokuma, Daisuke Takahashi, and Akira Otaka

Organic Letters 2015 Volume 17(Issue 3) pp:696-699

Publication Date(Web):January 28, 2015

DOI:10.1021/ol503718j

Bridged peptides including stapled peptides are attractive tools for regulating protein–protein interactions (PPIs). An effective synthetic methodology in a heterogeneous system for the preparation of these peptides using olefin metathesis and hydrogenation of protected peptides with a long aliphatic chain anchor is reported.

Design and synthesis of a hydrogen peroxide-responsive amino acid that induces peptide bond cleavage after exposure to hydrogen peroxide

Co-reporter:Miku Kita, Jun Yamamoto, Takuya Morisaki, Chiaki Komiya, Tsubasa Inokuma, Licht Miyamoto, Koichiro Tsuchiya, Akira Shigenaga, Akira Otaka

Tetrahedron Letters 2015 Volume 56(Issue 28) pp:4228-4231

Publication Date(Web):8 July 2015

DOI:10.1016/j.tetlet.2015.05.060

Oxidative stress-responsive compounds are attracting significant attention in the field of medicinal chemistry and chemical biology. Here, we disclose the development of a hydrogen peroxide (H2O2)-responsive amino acid that induces peptide bond cleavage in the presence of H2O2 that closely relates to oxidative stress. The H2O2-responsive amino acid possessing a boronate or boronic acid moiety was incorporated into a peptide using Fmoc-based solid-phase peptide synthesis or that with minor modifications, respectively, and the peptide bond cleavage of the obtained peptide was successfully triggered by the addition of H2O2.

Synthesis of lactam-bridged cyclic peptides using sequential olefin metathesis and diimide reduction reactions

Co-reporter:Keisuke Aihara, Tsubasa Inokuma, Chiaki Komiya, Akira Shigenaga, Akira Otaka

Tetrahedron 2015 Volume 71(Issue 24) pp:4183-4191

Publication Date(Web):17 June 2015

DOI:10.1016/j.tet.2015.04.093

A new approach has been developed for the synthesis of lactam-bridged cyclic peptides. Following the introduction of N-allyl glutamine and α-allylglycine into the peptide backbone, the side chains of these residues were subjected to a cyclization reaction by ring-closing metathesis (RCM). Reduction of the resulting peptide bearing olefin moiety was achieved using diimide, which was generated in situ from o-nitrobenzenesulfonyl hydrazine and piperidine, gave the corresponding saturated cyclic peptides.

Development of a Chemical Methodology for the Preparation of Peptide Thioesters Applicable to Naturally Occurring Peptides Using a Sequential Quadruple Acyl Transfer System

Co-reporter:Yusuke Tsuda;Dr. Akira Shigenaga;Kohei Tsuji;Masaya Denda;Kohei Sato;Keisuke Kitakaze;Takahiro Nakamura;Dr. Tsubasa Inokuma; Kohji Itoh ; Akira Otaka

ChemistryOpen 2015 Volume 4( Issue 4) pp:448-452

Publication Date(Web):

DOI:10.1002/open.201500086

Abstract

Peptide thioesters are very useful in protein chemistry, and chemistry- and biochemistry-based protocols are used for the preparation of thioesters. Among such protocols, only a few biochemistry-based approaches have been use for naturally occurring peptide sequences. The development of chemistry-based protocols applicable to natural sequences remains a challenge, and the development of such methods would be a major contribution to protein science. Here, we describe the preparation of peptide thioesters using innovative methodology that features nickel(II)-mediated alcoholysis of a naturally occurring peptide sequence, followed by O−N and N−S acyl transfers. This protocol involves sequential quadruple acyl transfer, termed SQAT. Notably, the SQAT system consists of sequential chemical reactions that allow naturally occurring peptide sequences to be converted to thioesters without requiring an artificial chemical unit.

Examination of native chemical ligation using peptidyl prolyl thioesters

Co-reporter:Takahiro Nakamura, Akira Shigenaga, Kohei Sato, Yusuke Tsuda, Ken Sakamoto and Akira Otaka

Chemical Communications 2014 vol. 50(Issue 1) pp:58-60

Publication Date(Web):22 Oct 2013

DOI:10.1039/C3CC47228K

Peptidyl prolyl thioesters, which have been regarded as too unreactive for practical use in native chemical ligations (NCLs), were proven to be versatile reagents in peptide coupling. Tuning the reactivity of prolyl thioesters enabled a one-pot/sequential NCL reaction based on kinetically controlled ligation.

Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins

Co-reporter:Jun Yamamoto, Masaya Denda, Nami Maeda, Miku Kita, Chiaki Komiya, Tomohiro Tanaka, Wataru Nomura, Hirokazu Tamamura, Youichi Sato, Aiko Yamauchi, Akira Shigenaga and Akira Otaka

Organic & Biomolecular Chemistry 2014 vol. 12(Issue 23) pp:3821-3826

Publication Date(Web):17 Apr 2014

DOI:10.1039/C4OB00622D

A traceable linker that is potentially applicable to identification of a target protein of bioactive compounds was developed. It enabled not only thiol-induced cleavage of the linker for enrichment of the target protein but also selective labelling to pick out the target from contaminated non-target proteins for facile identification.

Development of a fluoride-responsive amide bond cleavage device that is potentially applicable to a traceable linker

Co-reporter:Jun Yamamoto, Nami Maeda, Chiaki Komiya, Tomohiro Tanaka, Masaya Denda, Koji Ebisuno, Wataru Nomura, Hirokazu Tamamura, Youichi Sato, Aiko Yamauchi, Akira Shigenaga, Akira Otaka

Tetrahedron 2014 70(34) pp: 5122-5127

Publication Date(Web):

DOI:10.1016/j.tet.2014.05.110

Development of caged non-hydrolyzable phosphoamino acids and application to photo-control of binding affinity of phosphopeptide mimetic to phosphopeptide-recognizing protein

Co-reporter:Koji Ebisuno, Masaya Denda, Keiji Ogura, Tsubasa Inokuma, Akira Shigenaga, Akira Otaka

Bioorganic & Medicinal Chemistry 2014 22(11) pp: 2984-2991

Publication Date(Web):

DOI:10.1016/j.bmc.2014.04.002

Chemical Synthesis of Biologically Active Monoglycosylated GM2-Activator Protein Analogue Using N-Sulfanylethylanilide Peptide

Co-reporter:Kohei Sato;Dr. Akira Shigenaga;Keisuke Kitakaze;Ken Sakamoto;Dr. Daisuke Tsuji;Dr. Kohji Itoh ;Dr. Akira Otaka

Angewandte Chemie International Edition 2013 Volume 52( Issue 30) pp:7855-7859

Publication Date(Web):

DOI:10.1002/anie.201303390

Chemical Synthesis of Biologically Active Monoglycosylated GM2-Activator Protein Analogue Using N-Sulfanylethylanilide Peptide

Co-reporter:Kohei Sato;Dr. Akira Shigenaga;Keisuke Kitakaze;Ken Sakamoto;Dr. Daisuke Tsuji;Dr. Kohji Itoh ;Dr. Akira Otaka

Angewandte Chemie 2013 Volume 125( Issue 30) pp:8009-8013

Publication Date(Web):

DOI:10.1002/ange.201303390

One-Pot/Sequential Native Chemical Ligation Using N-Sulfanylethylanilide Peptide

Co-reporter:Akira Otaka;Kohei Sato;Hao Ding ;Akira Shigenaga

The Chemical Record 2012 Volume 12( Issue 5) pp:479-490

Publication Date(Web):

DOI:10.1002/tcr.201200007

Abstract

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

Labelling of endogenous target protein via N–S acyl transfer-mediated activation of N-sulfanylethylanilide

Co-reporter:Masaya Denda, Takuya Morisaki, Taiki Kohiki, Jun Yamamoto, Kohei Sato, Ikuko Sagawa, Tsubasa Inokuma, Youichi Sato, Aiko Yamauchi, Akira Shigenaga and Akira Otaka

Organic & Biomolecular Chemistry 2016 - vol. 14(Issue 26) pp:NaN6251-6251

Publication Date(Web):2016/05/31

DOI:10.1039/C6OB01014H

The ligand-dependent incorporation of a reporter molecule (e.g., fluorescence dye or biotin) onto a endogenous target protein has emerged as an important strategy for elucidating protein function using various affinity-based labelling reagents consisting of reporter, ligand and reactive units. Conventional labelling reagents generally use a weakly activated reactive unit, which can result in the non-specific labelling of proteins in a ligand-independent manner. In this context, the activation of a labelling reagent through a targeted protein–ligand interaction could potentially overcome the problems associated with conventional affinity-based labelling reagents. We hypothesized that this type of protein–ligand-interaction-mediated activation could be accomplished using N-sulfanylethylanilide (SEAlide) as the reactive unit in the labelling reagent. Electrophilically unreactive amide-type SEAlide can be activated by its conversion to the corresponding active thioester in the presence of a phosphate salt, which can act as an acid–base catalyst. It has been suggested that protein surfaces consisting of hydrophilic residues such as amino, carboxyl and imidazole groups could function as acid–base catalysts. We therefore envisioned that a SEAlide-based labelling reagent (SEAL) bearing SEAlide as a reactive unit could be activated through the binding of the SEAL with a target protein. Several SEALs were readily prepared in this study using standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase protocols. These SEAL systems were subsequently applied to the ligand-dependent labelling of human carbonic anhydrase (hCA) and cyclooxyganese 1. Although we have not yet obtained any direct evidence for the target protein-mediated activation of the SEAlide unit, our results for the reaction of these SEALs with hCA1 or butylamine indirectly support our hypothesis. The SEALs reported in this study represent valuable new entries to the field of affinity-based labelling reagents and are expected to show great utility in protein labelling.

The total chemical synthesis of the monoglycosylated GM2 ganglioside activator using a novel cysteine surrogate

Co-reporter:Kohei Sato, Keisuke Kitakaze, Takahiro Nakamura, Naoto Naruse, Keisuke Aihara, Akira Shigenaga, Tsubasa Inokuma, Daisuke Tsuji, Kohji Itoh and Akira Otaka

Chemical Communications 2015 - vol. 51(Issue 49) pp:NaN9948-9948

Publication Date(Web):2015/05/13

DOI:10.1039/C5CC02967H

We describe a novel peptide ligation/desulfurization strategy using a β-mercapto-N-glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 ganglioside activator protein bearing a monosaccharide on the native glycosylation site.

Examination of native chemical ligation using peptidyl prolyl thioesters

Co-reporter:Takahiro Nakamura, Akira Shigenaga, Kohei Sato, Yusuke Tsuda, Ken Sakamoto and Akira Otaka

Chemical Communications 2014 - vol. 50(Issue 1) pp:NaN60-60

Publication Date(Web):2013/10/22

DOI:10.1039/C3CC47228K

Peptidyl prolyl thioesters, which have been regarded as too unreactive for practical use in native chemical ligations (NCLs), were proven to be versatile reagents in peptide coupling. Tuning the reactivity of prolyl thioesters enabled a one-pot/sequential NCL reaction based on kinetically controlled ligation.

Photo-triggered fluorescent labelling of recombinant proteins in live cells

Co-reporter:Deokho Jung, Kohei Sato, Kyoungmi Min, Akira Shigenaga, Juyeon Jung, Akira Otaka and Youngeun Kwon

Chemical Communications 2015 - vol. 51(Issue 47) pp:NaN9673-9673

Publication Date(Web):2015/05/15

DOI:10.1039/C5CC01067E

A method to photo-chemically trigger fluorescent labelling of proteins in live cells is developed. The approach is based on photo-caged split-intein mediated conditional protein trans-splicing reaction and enabled background-free fluorescent labelling of target proteins with the necessary spatiotemporal control.

Development of a traceable linker containing a thiol-responsive amino acid for the enrichment and selective labelling of target proteins

Co-reporter:Jun Yamamoto, Masaya Denda, Nami Maeda, Miku Kita, Chiaki Komiya, Tomohiro Tanaka, Wataru Nomura, Hirokazu Tamamura, Youichi Sato, Aiko Yamauchi, Akira Shigenaga and Akira Otaka

Organic & Biomolecular Chemistry 2014 - vol. 12(Issue 23) pp:NaN3826-3826

Publication Date(Web):2014/04/17

DOI:10.1039/C4OB00622D

A traceable linker that is potentially applicable to identification of a target protein of bioactive compounds was developed. It enabled not only thiol-induced cleavage of the linker for enrichment of the target protein but also selective labelling to pick out the target from contaminated non-target proteins for facile identification.

Elucidation of inhibitor-binding pockets of D-amino acid oxidase using docking simulation and N-sulfanylethylanilide-based labeling technology

Co-reporter:Taiki Kohiki, Yusuke Kato, Yusuke Nishikawa, Kazuko Yorita, Ikuko Sagawa, Masaya Denda, Tsubasa Inokuma, Akira Shigenaga, Kiyoshi Fukui and Akira Otaka

Organic & Biomolecular Chemistry 2017 - vol. 15(Issue 25) pp:NaN5297-5297

Publication Date(Web):2017/05/05

DOI:10.1039/C7OB00633K

Because of the relevance of D-serine (D-Ser) to schizophrenia, inhibitors of D-amino acid oxidase (DAO), which catalyzes degradation of D-Ser in the presence of flavin adenine dinucleotide (FAD), are expected to be anti-schizophrenia therapeutics. In this study, binding pockets of DAO to its inhibitor 4-bromo-3-nitrobenzoic acid were searched by combining in silico docking simulation and labeling experiments employing an N-sulfanylethylanilide-based labeling technology that we have developed. The results clearly demonstrated that there are two binding pockets: one is shared with D-Ser and FAD, and the other is an unexpected cleft between the subunits of a DAO dimer. These findings will provide insight to aid the development of new DAO inhibitors. In addition, it was also proved that our labeling technology could be applicable to elucidate the binding pockets of proteins.

An N-sulfanylethylanilide-based traceable linker for enrichment and selective labelling of target proteins

Co-reporter:Takuya Morisaki, Masaya Denda, Jun Yamamoto, Daisuke Tsuji, Tsubasa Inokuma, Kohji Itoh, Akira Shigenaga and Akira Otaka

Chemical Communications 2016 - vol. 52(Issue 42) pp:NaN6913-6913

Publication Date(Web):2016/04/26

DOI:10.1039/C6CC01229A

An N-sulfanylethylanilide-based traceable linker, developed to facilitate identification of target proteins of bioactive compounds, was introduced into an alkynylated target protein. Subsequent adsorption onto streptavidin beads allowed it to be treated with a cysteine–fluorophore conjugate in the presence of phosphate. This induced the N–S acyl transfer reaction of the N-sulfanylethylanilide unit. The subsequent native chemical ligation of the fluorophore resulted in cleavage of the linker for target elution and fluorescence labelling of the target, allowing it to be distinguished from non-target proteins.

TOPICS

Abstract

Abstract