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

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

Research output: Contribution to journalArticlepeer-review

48 Citations (Scopus)


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. (Chemical Equation Presented).

Original languageEnglish
Pages (from-to)9061-9069
Number of pages9
JournalAnalytical Chemistry
Issue number17
Publication statusPublished - 2015 Sept 1
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry


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