title: Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates.
abstract: 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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Narae
authors.initials: N
authors.lastname: Shin
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Kenjiro
authors.identifier: 0000-0003-0797-4038
authors.initials: K
authors.lastname: Hanaoka
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Wen
authors.initials: W
authors.lastname: Piao
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Tokyo 113-8657, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.affiliation: Molecular Spectroscopy Laboratory, RIKEN , Wako 351-0198, Japan.
authors.forename: Tomotsumi
authors.initials: T
authors.lastname: Fujisawa
authors.affiliation: Molecular Spectroscopy Laboratory, RIKEN , Wako 351-0198, Japan.|Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), RIKEN , Wako 351-0198, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takeuchi
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Shodai
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.|PRESTO, Japan Science and Technology Agency , Saitama 332-0012, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Komatsu
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Takuya
authors.identifier: 0000-0002-3425-3589
authors.initials: T
authors.lastname: Terai
authors.affiliation: Molecular Spectroscopy Laboratory, RIKEN , Wako 351-0198, Japan.|Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), RIKEN , Wako 351-0198, Japan.
authors.forename: Tahei
authors.initials: T
authors.lastname: Tahara
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo , Tokyo 113-8657, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
authors.affiliation: Drug Discovery Initiative, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo , Tokyo 113-0033, Japan.|Graduate School of Medicine, The University of Tokyo , Tokyo 113-0033, Japan.|AMED CREST (Japan) Agency for Medical Research and Development , Tokyo 100-0004, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008856:Microscopy, Fluorescence; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D009601:Nitroreductases; D012235:Rhodamines / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; C021106:azoreductase