ID 111885
著者
Denda, Masaya Tokushima University
Morisaki, Takuya Tokushima University
Kohiki, Taiki Tokushima University
Yamamoto, Jun Tokushima University
Sato, Kohei Tokushima University|Shizuoka University
重永, 章 Tokushima University|Japan Science and Technology Agency (JST) 徳島大学 教育研究者総覧 KAKEN研究者をさがす
資料タイプ
学術雑誌論文
抄録
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.
掲載誌名
Organic & Biomolecular Chemistry
ISSN
14770520
14770539
cat書誌ID
AA1168650X
AA12084375
出版者
The Royal Society of Chemistry
14
26
開始ページ
6244
終了ページ
6251
発行日
2016-05-31
権利情報
© The Royal Society of Chemistry 2016
EDB ID
出版社版DOI
出版社版URL
フルテキストファイル
言語
eng
著者版フラグ
著者版
部局
技術支援部
薬学系