Analysis of Chemical Equilibrium of Silicon-Substituted Fluorescein and Its Application to Develop a Scaffold for Red Fluorescent Probes
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- 作者：Kazuhisa Hirabayashi ; Kenjiro Hanaoka ; Toshio Takayanagi ; Yuko Toki ; Takahiro Egawa ; Mako Kamiya ; Toru Komatsu ; Tasuku Ueno ; Takuya Terai ; Kengo Yoshida ; Masanobu Uchiyama ; Tetsuo Nagano ; Yasuteru Urano
- 刊名：Analytical Chemistry
- 出版年：2015
- 出版时间：September 1, 2015
- 年：2015
- 卷：87
- 期：17
- 页码：9061-9069
- 全文大小：588K
- ISSN：1520-6882
文摘
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鈥?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鈥?and 5鈥?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 尾-galactosidase.