ID 111885
Author
Denda, Masaya Tokushima University
Morisaki, Takuya Tokushima University
Kohiki, Taiki Tokushima University
Yamamoto, Jun Tokushima University
Sato, Kohei Tokushima University|Shizuoka University
Shigenaga, Akira Tokushima University|Japan Science and Technology Agency (JST) Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Content Type
Journal Article
Description
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.
Journal Title
Organic & Biomolecular Chemistry
ISSN
14770520
14770539
NCID
AA1168650X
AA12084375
Publisher
The Royal Society of Chemistry
Volume
14
Issue
26
Start Page
6244
End Page
6251
Published Date
2016-05-31
Rights
© The Royal Society of Chemistry 2016
EDB ID
DOI (Published Version)
URL ( Publisher's Version )
FullText File
language
eng
TextVersion
Author
departments
Technical Support Department
Pharmaceutical Sciences