Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates

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• 作者：Narae Shin ; Kenjiro HanaokaWen Piao ; Takuya Miyakawa ; Tomotsumi Fujisawa ; Satoshi Takeuchi ; Shodai Takahashi ; Toru Komatsu ; Tasuku Ueno ; Takuya Terai ; Tahei TaharaMasaru Tanokura ; Tetsuo Nagano ; Yasuteru Urano
• 刊名：ACS Chemical Biology
• 出版年：2017
• 出版时间：February 17, 2017
• 年：2017
• 卷：12
• 期：2
• 页码：558-563
• 全文大小：387K
• ISSN：1554-8937

文摘

Enzyme/substrate pairs, such as β-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the N═N bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron-donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at the single cell level.