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

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journal contribution
posted on 2016-12-30, 00:00 authored by Narae Shin, Kenjiro Hanaoka, Wen Piao, Takuya Miyakawa, Tomotsumi Fujisawa, Satoshi Takeuchi, Shodai Takahashi, Toru Komatsu, Tasuku Ueno, Takuya Terai, Tahei Tahara, Masaru Tanokura, Tetsuo Nagano, Yasuteru Urano
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.