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

• Published in: Analytical chemistry (Washington) Vol. 87; no. 17; pp. 9061 - 9069
• Main Authors: Hirabayashi, Kazuhisa, Hanaoka, Kenjiro, Takayanagi, Toshio, Toki, Yuko, Egawa, Takahiro, Kamiya, Mako, Komatsu, Toru, Ueno, Tasuku, Terai, Takuya, Yoshida, Kengo, Uchiyama, Masanobu, Nagano, Tetsuo, Urano, Yasuteru
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.09.2015
• Subjects: Absorption; Analogue; Analytical chemistry; beta-galactosidase; chemical analysis; chemical equilibrium; Chemical synthesis; Derivatives; Fluorescein; Fluorescein - chemical synthesis; Fluorescein - chemistry; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Hydrogen-Ion Concentration; Lactones; Molecular Structure; Organic chemicals; Photochemical Processes; Probes; Scaffolds; Silicon; Silicon - chemistry; Spectra
• DOI: 10.1021/acs.analchem.5b02331
• ISSN: 0003-2700; 1520-6882

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 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 β-galactosidase.