Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.
Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.
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DOI：
10.1021/jacs.2c08257
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发表时间：
2022-11
影响因子：
15
通讯作者：
Alexander V. West;Yuka Amako;Christina M. Woo
中科院分区：
文献类型：
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作者：
Alexander V. West;Yuka Amako;Christina M. Woo
在目标识别研究中，烷基二氮杂环化合物经常被用于光亲和标记来描绘小分子与蛋白质的相互作用。然而，烷基重氮类化合物可以优先标记酸性氨基酸和酸性蛋白质表面，这可能是通过一种反应性的烷基重氮中间体来实现的。在这里，我们探索使用环应变来改变这些反应性偏好，并报告了一种降低了pH依赖反应性的环丁烷二氮杂光亲和标记的开发，称为PALBOX。我们发现，PALBOX在体外与其他二氮杂环标签相比具有不同的反应性特征，并且很容易被结合到小分子中来描述它们在细胞中的结合作用。利用一组小分子片段和配体，我们证明了配备PALBOX的光亲和探针可以标记细胞中已知的蛋白质靶标，而减少了已知的烷基二氮杂环脱离靶标的标记。最后，我们证明了配备PALBOX的配体可以准确地定位小分子与蛋白质的结合位点。因此，PALBOX是一种基于二氮杂环的多功能光亲和标签，用于开发用于光亲和标记实验的化学探针，包括研究小分子与蛋白质的相互作用。
Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.