On-site reaction for PPARγ modification using a specific bifunctional ligand.

Site-specific labeling is an important methodology to elucidate the biological function of a target protein. Here, we report a strategy for site-specific chemical labeling, termed the "on-site reaction". We designed and readily synthesized a bifunctional ligand possessing two reaction sites, an enone and an azide moiety. This strategy involves an on-site conjugate addition reaction with protein followed by a Hüisgen cycloaddition reaction. We demonstrate this strategy by using fluorescein as a probe and peroxisome proliferator activated receptor γ (PPARγ) as a target protein. The reactions were evaluated by ESI-mass analysis and the binding site and modes of binding were revealed by X-ray crystallization analysis. The proposed methodology can easily convert a covalent ligand into chemical tool for protein functional analysis and the identification of drug targets.