Fluorescein is a complex fluorophore that can exist in one or more of four different prototropic forms (cation, neutral, dianion, and monoanion) depending on pH. In the pH range 6-10, only the dianion and monanion forms are important. In a previous article, we showed by steady-state fluorescein measurements that an excited fluorescein molecule displays excited-state proton transfer reactions which interconvert the monoanion and dianion forms. However, we found that these reactions can occur only in the presence of a suitable proton donor-acceptor buffer such as phosphate buffer. Assuming that, at 1 M phosphate buffer concentration, the excited-state proton exchange reaction of fluorescein rapidly equilibrates during the lifetime of fluorescein, we were able to fit quantitatively steady-state fluorescence intensity vs pH titration graphs to a relatively simple reaction model. In this article, we use nanosecond emission (decay time) methods to study the excitedstate proton reactions of fluorescein in the pH range 6-10 and in the presence of a phosphate buffer concentration. Fluorescein is a challenging fluorophore for the study of excited-state proton reactions because of the strong overlap of the absorption and emission spectra of the monoanion and dianion forms of fluorescein. However by recording nanosecond emission graphs and using methods of analysis of high precision, we have been able to test kinetic mechanisms and evaluate the specific rate constants for the excited-state proton reactions as well as the lifetimes of the monoanion and dianion. Using these values for lifetimes and rate constants, we discuss the process of equilibration in the excited-state and derive expressions which allow us to predict how quickly the excited-state reactions can reach equilibrium. Moreover, we use the above kinetic and spectral parameters to calculate steady-state fluorescence intensity F S vs pH at 1 M phosphate buffer concentration and compare this theoretically calculated graph with the experimental graph.

2′,7′-Difluorofluorescein (Oregon Green 488) is a new fluorescein-based dye, which has found many applications, above all in biochemistry and neurosciences, and its use has become very popular in the last years. In recent years, we have been investigating the excited-state proton exchange reactions of fluorescein and the effect of suitable proton acceptors and donors which promote these reactions. The excited-state proton transfer reactions may appreciably influence the fluorescence results when using these dyes. We present steady-state emission evidence that acetate buffer species promote an excited-state proton transfer between neutral, monoanionic, and dianionic forms of 2′,7′-difluorofluorescein. The time course of the excited species in this reaction was characterized through time-resolved fluorescence measurements, and the kinetics of the reaction was solved by using the global compartmental analysis. A previous identifiability study on the compartmental system set the conditions to design the fluorescence decay surface. This is the first experimental system, studied within this kinetic model, solved under identifiability conditions through global compartmental analysis. The recovered rate constant values for deactivation were 2.94 × 10 8 s -1 for the monoanion and 2.47 × 10 8 s -1 for the dianion, whereas the rate constant values of the buffer-mediated excited-state reaction were 9.70 × 10 8 and 1.79 × 10 8 M -1 s -1 for the deprotonation and protonation, respectively. With these values, a pK a * ) 4.02 was obtained. In this work, we additionally provide an absorption study, including acid-base equilibria, determination of ground-state pK a values (1.02, 3.61, and 4.69), and recovery of molar absorption coefficients of every prototropic species, including absorption and NMR evidence for the existence of three tautomers in neutral species. Steady-state emission spectra of 2′,7′-difluorofluorescein in aqueous solution are also described, where the strong photoacid behavior of the cation is noteworthy.

The UV-vis electronic absorption and fluorescence emission properties of 8-halogenated (Cl, Br, I) difluoroboron dipyrrin (or 8-haloBODIPY) dyes and their 8-(C, N, O, S) substituted analogues are reported. The nature of the meso-substituent has a significant influence on the spectral band positions, the fluorescence quantum yields, and lifetimes. As a function of the solvent, the spectral maxima of all the investigated dyes are located within a limited wavelength range. The spectra of 8-haloBODIPYs display the narrow absorption and fluorescence emission bands and the generally quite small Stokes shifts characteristic of classic difluoroboron dipyrrins. Conversely, fluorophores with 8-phenylamino (7), 8-benzylamino (8), 8-methoxy (9), and 8-phenoxy (10) groups emit in the blue range of the visible spectrum and generally have larger Stokes shifts than common BODIPYs, whereas 8-(2-phenylethynyl)BODIPY (6) has red-shifted spectra compared to ordinary BODIPY dyes. Fluorescence lifetimes for 6, 8, and 10 have been measured for a large set of solvents and the solvent effect on their absorption and emission maxima has been analyzed using the generalized Catalán solvent scales. Restricted rotation about the C8-N bond in 7 and 8 has been observed via temperature dependent (1)H NMR spectroscopy, whereas for 10 the rotation about the C8-O bond is not hindered. The crystal structure of 8 demonstrates that the short C8-N bond has a significant double character and that this N atom exhibits a trigonal planar geometry. The crystal structure of 10 shows a short C8-O bond and an intramolecular C-H···π interaction. Quantum-chemical calculations have been performed to assess the effect of the meso-substituent on the spectroscopic properties.

Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1-9. In experiments with fluorescein-labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in practice. In this article we describe a general steady-state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH-sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor-acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion-dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor-acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 mM and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction, we have recorded titration graphs of fluorescence intensity versus pH for fluorescein solutions at low ( 5 mM) and high buffer (1 M) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state p k . The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pK, and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion-dianion reaction does not significantly occur at 5 mM phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The p&* of the excited monoanion-dia...