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Labelling of Endogenous Target Protein via N–S Acyl Transfer-Mediated Activation of N-Sulfanylethylanilide 00813891 傳田, 将也 デンダ, マサヤ Denda, Masaya 傳田 デンダ Denda 将也 マサヤ Masaya モリサキ, タクヤ モリサキ, タクヤ Morisaki, Takuya コヒキ, タイキ コヒキ, タイキ Kohiki, Taiki ヤマモト, ジュン ヤマモト, ジュン Yamamoto, Jun サトウ, コウヘイ サトウ, コウヘイ Sato, Kohei 佐川, 幾子 サガワ, イクコ Sagawa, Ikuko 佐川 サガワ Sagawa 幾子 イクコ Ikuko 40541272 猪熊, 翼 イノクマ, ツバサ Inokuma, Tsubasa 猪熊 イノクマ Inokuma 翼 ツバサ Tsubasa 10363160 佐藤, 陽一 サトウ, ヨウイチ Sato, Youichi 佐藤 サトウ Sato 陽一 ヨウイチ Youichi 30122253 山内, あい子 ヤマウチ, アイコ Yamauchi, Aiko 山内 ヤマウチ Yamauchi あい子 アイコ Aiko 10423394 重永, 章 シゲナガ, アキラ Shigenaga, Akira 重永 シゲナガ Shigenaga 章 アキラ Akira 20201973 大髙, 章 オオタカ, アキラ Otaka, Akira 大髙 オオタカ Otaka 章 アキラ Akira open access © The Royal Society of Chemistry 2016 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 Royal Society of Chemistry 2016-05-31 eng journal article AM 313229 https://tokushima-u.repo.nii.ac.jp/records/2005050 https://doi.org/10.1039/C6OB01014H 10.1039/C6OB01014H 14770520 14770539 AA1168650X AA12084375 Organic & Biomolecular Chemistry 14 26 6244 6251 https://tokushima-u.repo.nii.ac.jp/record/2005050/files/orbiochem_14_26_6244.pdf application/pdf 732 KB 2018-07-13 https://tokushima-u.repo.nii.ac.jp/record/2005050/files/orbiochem_14_26_6244_si.pdf application/pdf 628 KB 2018-07-13