Phospha-Michael Addition as a New Click Reaction for Protein Functionalization
Phospha-Michael Addition as a New Click Reaction for Protein Functionalization
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DOI：
10.1002/cbic.201500697
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发表时间：
2016-03-15
期刊：
影响因子：
3.2
通讯作者：
Liu, Wenshe R.
中科院分区：
文献类型：
--
作者：
Lee, Yan-Jiun;Kurra, Yadagiri;Liu, Wenshe R.
建立了一种新型的烷基磷化氢与丙烯酰胺之间的点击反应，并将其应用于体外和活细胞中的位点特异性蛋白标记。丙烯酰胺是一种小的亲电烯烃，易于与烷基膦进行磷酸-迈克尔加成反应。动力学研究表明，在pH7.4条件下，丙烯酰胺与三（2-羧基乙基）膦反应的二阶速率常数为0.07m(-1)s（-1）。为了证明其在蛋白质功能化中的应用，我们使用了丹酰-膦偶联物成功地标记了位点特异性安装有n-丙烯酰-赖氨酸的蛋白质，并使用了生物素-膦偶联物选择性地探测了代谢标记有n-丙烯酰-半乳糖胺的人类蛋白质。
A new type of click reaction between an alkyl phosphine and acrylamide was developed and applied for site-specific protein labeling in vitro and in live cells. Acrylamide is a small electrophilic olefin that readily undergoes phospha-Michael addition with an alkyl phosphine. Our kinetic study indicated a second-order rate constant of 0.07m(-1)s(-1) for the reaction between tris(2-carboxyethyl)phosphine and acrylamide at pH7.4. To demonstrate its application in protein functionalization, we used a dansyl-phosphine conjugate to successfully label proteins that were site-specifically installed with N-acryloyl-l-lysine and employed a biotin-phosphine conjugate to selectively probe human proteins that were metabolically labeled with N-acryloyl-galactosamine.