Ligand-directed dibromophenyl benzoate chemistry for rapid and selective acylation of intracellular natural proteins.
Ligand-directed dibromophenyl benzoate chemistry for rapid and selective acylation of intracellular natural proteins.
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DOI：
10.1039/c5sc00190k
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发表时间：
2015-05-01
期刊：
影响因子：
8.4
通讯作者：
Hamachi I
中科院分区：
文献类型：
--
作者：
Takaoka Y;Nishikawa Y;Hashimoto Y;Sasaki K;Hamachi I
作为一种快速、选择性的蛋白质标记方法，LDBB化学是研究活细胞中天然蛋白质成像的有效工具。在配体定向化学的基础上，发展了一种快速、选择性的活细胞蛋白质酰化反应。通过对苯酯衍生物的反应性和稳定性进行微调，我们成功地确定了邻二溴苯甲酸酯为最佳反应基序。它在水缓冲液中足够稳定，13小时后水解率低于10%，但在活体哺乳动物细胞和体外（称为配体定向苯甲酸二溴苯酯（LDBB）化学）中具有足够的活性，可以高效和选择性地标记蛋白质。利用这种化学，各种荧光团可以直接连接到目标蛋白上，这就允许使用不同颜色的荧光团（包括香豆素、荧光素和罗丹明）在活细胞中对标记的蛋白质进行荧光可视化。此外，该标记不仅适用于过表达蛋白（大肠杆菌二氢叶酸还原酶），也适用于活细胞条件下的内源性人碳酸酐酶II和XII。LDBB化学是一种新的配体导向的蛋白质标记方法，对于活细胞中天然蛋白质的成像特别有用。
A rapid and selective protein labeling method, LDBB chemistry is a useful tool for natural protein imaging in living cells. A rapid and selective ligand-directed chemical reaction was developed for the acylation of proteins in living cells on the basis of ligand-directed chemistry. By fine tuning the reactivity and stability of the phenyl ester derivatives, we successfully identified ortho-dibromophenyl benzoate as the optimal reactive motif. It was sufficiently stable in an aqueous buffer, hydrolyzing less than 10% after 13 h of incubation, but reactive enough for efficient and selective protein labeling in living mammalian cells, as well as in vitro (referred to as ligand-directed dibromophenyl benzoate (LDBB) chemistry). Using this chemistry, various fluorophores can be tethered to the target protein directly, which allows fluorescence visualization of the labeled protein in live cells using different colored fluorophore groups (including coumarin, fluorescein and rhodamine). Furthermore, this labeling is applicable to not only an overexpressed protein (E. coli dihydrofolate reductase) but also endogenous human carbonic anhydrase II and XII under living cell conditions. LDBB chemistry is a new entry of ligand-directed protein labeling methods, and should be particularly useful for the imaging of natural proteins in living cells.