Analysis of Chemical Equilibrium of Silicon-Substituted Fluorescein and Its Application to Develop a Scaffold for Red Fluorescent Probes

作者：Hirabayshi Kazuhisa; Hanaoka Kenjiro^*; Takayanagi Toshio; Toki Yuko; Egawa Takahiro; Kamiya Mako; Komatsu Toni; Ueno Tasuku; Terai Takuya; Yoshida Kengo; Uchiyama Masanobu; Nagano Tetsuo; Urano Yasuteru

来源：Analytical Chemistry, 2015, 87(17): 9061-9069.

DOI：10.1021/acs.analchem.5b02331

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 0 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 pK(a) inversion, i.e., pK(a1) > pK(a2). 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 beta-galactosidase.

• 出版日期2015-9-1
• 单位RIKEN