The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene–tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene–tetrazole reaction product. This finding provides useful guidance to the future design of alkene–tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene–tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene–tetrazole chemistry in real-time imaging in live cells.

中文翻译：

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改进的光诱导氟代烯烃-四唑反应用于蛋白质标记

烯烃和四唑之间的1，3-偶极环加成反应代表了形成荧光团的生物正交化学的一个优雅而罕见的例子。对于活细胞的成像应用而言，这是一个有吸引力的反应，需要较少的密集洗涤步骤和/或需要时空分辨率。在目前的工作中，作为改善烯烃-四唑反应的荧光性质的努力，研究了芳香族烯烃（苯乙烯）作为双极性亲和剂。在水溶液中，反应产物的量子产率提高了30倍以上。根据我们的机理研究，观察到的改进可能是由于苯乙烯-四唑反应产物的质子化不足所致。这一发现为将来进行生物学研究的烯烃-四唑反应设计提供了有用的指导。在体外和体内都证明了使用苯乙烯-四唑反应进行荧光蛋白标记。这是通过遗传掺入含有苯乙烯部分的非天然氨基酸来实现的。可以预期，苯乙烯与不同的四唑衍生物的组合通常可以改善和拓宽烯烃-四唑化学在活细胞实时成像中的应用。