Rational Design, Synthesis, and Spectroscopic and Photophysical Properties of a Visible-Light-Excitable, Ratiometric, Fluorescent Near-Neutral pH Indicator Based on BODIPY

• Published in: Chemistry : a European journal Vol. 17; no. 39; pp. 10924 - 10934
• Main Authors: Boens, Noël, Qin , Wenwu, Baruah, Mukulesh, De Borggraeve, Wim M., Filarowski , Aleksander, Smisdom, Nick, Ameloot, Marcel, Crovetto, Luis, Talavera, Eva M., Alvarez-Pez, Jose M.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 19-09-2011; WILEY‐VCH Verlag; Wiley Subscription Services, Inc
• Subjects: Adsorption; Aqueous solutions; Boron Compounds - chemical synthesis; Boron Compounds - chemistry; Chemistry; dyes/pigments; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; fluorescent probes; Hydrogen-Ion Concentration; Imidazoles - chemistry; Ligands; Light; pH indicators; photophysics; sensors; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201002280

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 Φf 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 kf of 2.4×108 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 pKa values of 6.0 and 12.6. The Φf 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–deprotonation 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 pKa 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 τ1=4.3 ns for the cationic and τ2=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 (H2PO4−/HPO42−) 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 (k01=2.4×108 s−1) and neutral (k02=3.0×108 s−1) forms of II.
Deeply BODIPY: A visible‐light‐excitable, ratiometric, brightly fluorescent indicator for near‐neutral pH measurements has been designed, synthesized by conjugatively linking a BODIPY fluorophore at the 3‐position to the pH‐sensitive ligand imidazole through an ethenyl bridge, and spectroscopically characterized. It emits intense green–yellow fluorescence at lower pH and intense orange fluorescence at higher pH (see figure).