Turn-on Fluorescent Biosensors for Imaging Hypoxia-like Conditions in Living Cells.
Turn-on Fluorescent Biosensors for Imaging Hypoxia-like Conditions in Living Cells.
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DOI：
10.1021/jacs.2c01197
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发表时间：
2022-05-11
影响因子：
15
通讯作者：
Ribagorda, Maria
中科院分区：
文献类型：
--
作者：
Guisan-Ceinos, Santiago;Rivero, Alexandra R.;Romeo-Gella, Fernando;Simon-Fuente, Silvia;Gomez-Pastor, Silvia;Calvo, Natalia;Orrego, Alejandro H.;Manuel Guisan, Jose;Corral, Ines;Sanz-Rodriguez, Francisco;Ribagorda, Maria
我们介绍了无毒 3-偶氮共轭 BODIPY 染料作为类缺氧条件的掩蔽荧光生物传感器的合成、光物理性质和生物应用。该合成方法基于操作简单的 N=N 键形成方案，然后进行 Suzuki 偶联，从而可以直接获得简单且尚未充分开发的 3-偶氮取代 BODIPY。这些染料可以在化学和生物还原条件下开启其发射特性，包括细菌和人类偶氮还原酶，触发偶氮键断裂，产生荧光 3-氨基-BODIPY。我们还开发了一种实用的酶促方案，使用固定化细菌偶氮还原酶来评估这些偶氮基探针，并可用作不易获得且昂贵的人类还原酶 NQO1 的模型。量子力学计算揭示了偶氮部分还原后 S1 势能表面形貌的重构，并通过绘制前所未有的路径使荧光猝灭事件合理化。荧光显微镜实验表明，这些偶氮可用于可视化活细胞内的类似缺氧的条件。
We present the synthesis, photophysical properties, and biological application of nontoxic 3-azo-conjugated BODIPY dyes as masked fluorescent biosensors of hypoxia-like conditions. The synthetic methodology is based on an operationally simple N=N bond-forming protocol, followed by a Suzuki coupling, that allows for a direct access to simple and underexplored 3-azo-substituted BODIPY. These dyes can turn on their emission properties under both chemical and biological reductive conditions, including bacterial and human azoreductases, which trigger the azo bond cleavage, leading to fluorescent 3-amino-BODIPY. We have also developed a practical enzymatic protocol, using an immobilized bacterial azoreductase that allows for the evaluation of these azo-based probes and can be used as a model for the less accessible and expensive human reductase NQO1. Quantum mechanical calculations uncover the restructuration of the topography of the S1 potential energy surface following the reduction of the azo moiety and rationalize the fluorescent quenching event through the mapping of an unprecedented pathway. Fluorescent microscopy experiments show that these azos can be used to visualize hypoxia-like conditions within living cells.
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