Absorption and emission study of 2',7'-difluorofluorescein and its excited-state buffer-mediated proton exchange reactions

Angel Orte, Luis Crovetto, Eva M Talavera, Noël Boens, Jose M Alvarez-Pez
Journal of Physical Chemistry. A 2005 February 10, 109 (5): 734-47
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 x 10(8) s(-1) for the monoanion and 2.47 x 10(8) s(-1) for the dianion, whereas the rate constant values of the buffer-mediated excited-state reaction were 9.70 x 10(8) and 1.79 x 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.

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