An important step in elucidating the function of protein post-translational modifications (PTMs) is gaining access to site-specifically modified, homogeneous samples for biochemical characterization. Protein pyrophosphorylation is a poorly characterized PTM, and here a chemical approach to obtain pyrophosphoproteins is reported. Photo-labile phosphorimidazolide reagents were developed for selective pyrophosphorylation, affinity-capture, and release of pyrophosphoproteins. Kinetic analysis of the reaction revealed rate constants between 9.2 × 10–3 to 0.58 M–1 s–1, as well as a striking proclivity of the phosphorimidazolides to preferentially react with phosphate monoesters over other nucleophilic side chains. Besides enabling the characterization of pyrophosphorylation on protein function, this work highlights the utility of phosphoryl groups as handles for selective protein modification for a variety of applications, such as phosphoprotein bioconjugation and enrichment.
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机译：阐明蛋白质翻译后修饰（PTM）功能的重要步骤是获得针对位点修饰的均质样品的生物化学表征。蛋白质焦磷酸化是一种功能较弱的PTM，此处报道了一种化学方法来获得焦磷酸蛋白。开发了对光不稳定的磷酰亚胺咪唑试剂，用于选择性焦磷酸化，亲和捕获和释放焦磷酸蛋白。反应动力学分析表明，速率常数介于9.2×10 –3 至0.58 M –1 s -1 之间，并且具有惊人的倾向性磷酸咪唑啉化物与其他亲核侧链相比优先与磷酸单酯反应。除了能够表征蛋白质功能上的焦磷酸化作用外，这项工作还强调了磷酰基作为多种蛋白质（例如磷蛋白生物缀合和富集）的选择性蛋白质修饰处理的用途。
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