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.

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, 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.

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.

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.

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.

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.

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.

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.

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.

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.