title: Analysis of chemical equilibrium of silicon-substituted fluorescein and its application to develop a scaffold for red fluorescent probes.
abstract: Fluorescein is a representative green fluorophore that has been widely used as a scaffold of practically useful green fluorescent probes. Here, we report synthesis and characterization of a silicon-substituted fluorescein, i.e., 2-COOH TokyoMagenta (2-COOH TM), which is a fluorescein analogue in which the O atom at the 10' position of the xanthene moiety of fluorescein is replaced with a Si atom. This fluorescein analogue forms a spirolactone ring via intramolecular nucleophilic attack of the carboxylic group in a pH-dependent manner. Consequently, 2-COOH TM exhibits characteristic large pH-dependent absorption and fluorescence spectral changes: (1) 2-COOH TM is colorless at acidic pH, whereas fluorescein retains observable absorption and fluorescence even at acidic pH, and the absorption maximum is also shifted; (2) the absorption spectral change occurs above pH 7.0 for 2-COOH TM and below pH 7.0 for fluorescein; (3) 2-COOH TM shows a much sharper pH response than fluorescein because of its pKa inversion, i.e., pKa1 > pKa2. These features are also different from those of a compound without the carboxylic group, 2-Me TokyoMagenta (2-Me TM). Analysis of the chemical equilibrium between pH 3.0 and 11.0 disclosed that 2-COOH TM favors the colorless and nonfluorescent lactone form, compared with fluorescein. Substitution of Cl atoms at the 4' and 5' positions of the xanthene moiety of 2-COOH TM to obtain 2-COOH DCTM shifted the equilibrium so that the new derivative exists predominantly in the strongly fluorescent open form at physiological pH (pH 7.4). To demonstrate the practical utility of 2-COOH DCTM as a novel scaffold for red fluorescent probes, we employed it to develop a probe for β-galactosidase.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Hirabayashi
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Hanaoka
authors.affiliation: Department of Life System, Institute of Technology and Science, The University of Tokushima , 2-1 Minami-josanjima, Tokushima 770-8506, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Takayanagi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Toki
authors.forename: Takahiro
authors.initials: T
authors.lastname: Egawa
authors.forename: Mako
authors.initials: M
authors.lastname: Kamiya
authors.forename: Toru
authors.initials: T
authors.lastname: Komatsu
authors.forename: Tasuku
authors.initials: T
authors.lastname: Ueno
authors.forename: Takuya
authors.initials: T
authors.lastname: Terai
authors.affiliation: Elements Chemistry Laboratory, and Advanced Elements Chemistry Research Team, Riken Center for Sustainable Resource Science, RIKEN , 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Yoshida
authors.affiliation: Elements Chemistry Laboratory, and Advanced Elements Chemistry Research Team, Riken Center for Sustainable Resource Science, RIKEN , 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Uchiyama
authors.affiliation: Drug Discovery Initiative, The University of Tokyo , Tokyo 113-0033, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
mesh_terms: D019793:Fluorescein / Q000138:chemical synthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D055668:Photochemical Processes; D012825:Silicon / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D019793:Fluorescein; D012825:Silicon