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Title: Rational Design, Synthesis, and Spectroscopic and Photophysical Properties of a Visible-Light-Excitable, Ratiometric, Fluorescent Near-Neutral pH Indicator Based on BODIPY
Authors: Boens, Noel
Qin, Wenwu
Baruah, Mukulesh
De Borggraeve, Wim M.
Filarowski, Aleksander
AMELOOT, Marcel 
Crovetto, Luis
Talavera, Eva M.
Alvarez-Pez, Jose M.
Issue Date: 2011
Source: CHEMISTRY-A EUROPEAN JOURNAL, 17(39). p. 10924-10934
Abstract: A visible-light-excitable, ratiometric, brightly fluorescent pH indicator for measurements in the pH range 5-7 has been designed and synthesized by conjugatively linking the BODIPY fluorophore at the 3-position to the pH-sensitive ligand imidazole through an ethenyl bridge. The probe is available as cell membrane permeable methyl ester 8-(4-carbomethoxyphenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1, 5,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (I) and corresponding water-soluble sodium carboxylate, sodium 8-(4-carboxylatophenyl)-4,4-difluoro-3-[2-(1H-imidazol-4-yl)ethenyl]-1,5 ,7-trimethyl-3a,4a-diaza-4-bora-s-indacene (II). The fluorescence quantum yield Phi(f) of ester I is very high (0.8-1.0) in the organic solvents tested. The fluorescence lifetime (ca. 4 ns) of I in organic solvents with varying polarity/polarizability (from cyclohexane to acetonitrile) is independent of the solvent with a fluorescence rate constant k(f) of 2.4 x 10(8) s(-1). Probe I is readily loaded in the cytosol of live cells, where its high fluorescence intensity remains nearly constant over an extended time period. Water-soluble indicator II exhibits two acid-base equilibria in aqueous solution, characterized by pK(a) values of 6.0 and 12.6. The Phi(f) value of II in aqueous solution is high: 0.6 for the cationic and anionic forms of the imidazole ligand, and 0.8 for neutral imidazole. On protonation-de-protonation in the near-neutral pH range, UV/Vis absorption and fluorescence spectral shifts along with isosbestic and pseudo-isoemissive points are observed. This dual-excitation and dual-emission pH indicator emits intense green-yellow fluorescence at lower pH and intense orange fluorescence at higher pH. The influence of ionic strength and buffer concentration on the absorbance and steady-state fluorescence of II has also been investigated. The apparent pK(a) of the near-neutral acid-base equilibrium determined by spectrophotometric and fluorometric titration is nearly independent of the added buffer and salt concentration. In aqueous solution in the absence of buffer and in the pH range 5.20-7.45, dual exponential fluorescence decays are obtained with decay time tau(1) = 4.3 ns for the cationic and tau(2) = 3.3 ns for the neutral form of II. The excited-state proton exchange of II at near-neutral pH becomes reversible on addition of phosphate (H(2)PO(4)(-)/HPO(4)(2-)) buffer, and a pH-dependent change of the fluorescence decay times is induced. Global compartmental analysis of fluorescence decay traces collected as a function of pH and phosphate buffer concentration was used to recover values of the deactivation rate constants of the excited cationic (k(01) = 2.4 x 10(8) s(-1)) and neutral (k(02) = 3.0 x 10(8) s(-1)) forms of II.
Notes: [Boens, N; Qin, WW; Baruah, M; De Borggraeve, WM; Filarowski, A] Katholieke Univ Leuven, Dept Chem, B-3001 Louvain, Belgium. [Qin, WW] Lanzhou Univ, Key Lab Nonferrous Met Chem & Resources Utilizat, Coll Chem & Chem Engn, Lanzhou 730000, Peoples R China. [Qin, WW] Lanzhou Univ, State Key Lab Appl Organ Chem, Coll Chem & Chem Engn, Lanzhou 730000, Peoples R China. [Filarowski, A] Univ Wroclaw, Fac Chem, PL-50383 Wroclaw, Poland. [Smisdom, N; Ameloot, M] Univ Hasselt, BIOMED, B-3590 Diepenbeek, Belgium. [Smisdom, N; Ameloot, M] Transnat Univ Limburg, B-3590 Diepenbeek, Belgium.
Keywords: dyes/pigments; fluorescent probes; pH indicators; photophysics; sensors;dyes/pigments; fluorescent probes; pH indicators; photophysics; sensors
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ISSN: 0947-6539
e-ISSN: 1521-3765
DOI: 10.1002/chem.201002280
ISI #: 000296262400019
Category: A1
Type: Journal Contribution
Validations: ecoom 2012
Appears in Collections:Research publications

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