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.

位点特异性标记是阐明靶蛋白生物学功能的重要方法。在这里，我们报告了一种针对特定地点进行化学标记的策略，称为“现场反应”。我们设计并易于合成具有两个反应位点，一个烯酮和一个叠氮部分的双功能配体。该策略涉及与蛋白质的现场共轭加成反应，然后进行Hüisgen环加成反应。我们通过使用荧光素作为探针和过氧化物酶体增殖物激活的受体γ证明了这一策略（PPARγ）作为靶蛋白。通过ESI质量分析评估反应，并通过X射线结晶分析揭示结合位点和结合方式。所提出的方法可以轻松地将共价配体转化为用于蛋白质功能分析和药物靶标鉴定的化学工具。