Labelling of endogenous target protein via N-S acyl transfer-mediated activation of N-sulfanylethylanilide.
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.
Institute of Biomedical Sciences and Graduate School of Pharmaceutical Sciences, Tokushima University, Shomachi, Tokushima 770-8505, Japan. shigenaga.akira@tokushima-u.ac.jp aotaka@tokushima-u.ac.jp.
Masaya
M
Denda
Takuya
T
Morisaki
Taiki
T
Kohiki
Jun
J
Yamamoto
Kohei
K
Sato
Ikuko
I
Sagawa
Tsubasa
T
Inokuma
Youichi
Y
Sato
Aiko
A
Yamauchi
Akira
A
Shigenaga
Akira
A
Otaka
D000345:Affinity Labels / Q000737:chemistry*; D000813:Anilides / Q000737:chemistry*; D024401:Carbonic Anhydrase I / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D010047:Ovalbumin / Q000737:chemistry*; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000345:Affinity Labels; D000813:Anilides; D008024:Ligands; D013438:Sulfhydryl Compounds; D010047:Ovalbumin; D005982:Glutathione Transferase; D024401:Carbonic Anhydrase I; D010751:Phosphopyruvate Hydratase