Three-State 2‘,7‘-Difluorofluorescein Excited-State Proton Transfer Reactions in Moderately Acidic and Very Acidic Media

Abstract

2',7'-Difluorofluorescein (Oregon Green 488, OG488) is a novel fluorescein dye derivative which presents important advantages for improving the fluorimetric applications in the biomedical and biochemical sciences. In aqueous solution it displays four prototropic forms, namely cation (C), neutral (N), monoanion (M), and dianion (D). In previous works, we found (J. Phys. Chem. A 2005, 109, 734-747, 2840-2846) that OG488 undergoes excited-state proton transfer reactions, which may affect the results from applications using this dye. We established that the excited-state proton transfer (ESPT) reactions between neutral, monoanionic, and dianionic forms of OG488 are promoted by acetate buffer, and we characterized the ground and excited species involved. We also solved the kinetics of the prototropic reactions using global compartmental analysis. In the present paper, we extend our study on the ESPT reactions of OG488 to acidic media, in which only the three prototropic species cation, neutral, and monoanion coexist. We have solved the kinetics of the three-state ESPT reaction by means of global three-compartmental analysis of a fluorescence decay surface in moderately acidic media (pH between 1.1 and 3.0), recovering the kinetic and spectral parameters of this three-state system. This system is one of the most complex solved to date, due to the strong overlap of the absorption and emission spectra of the neutral and monoanionic forms of OG488. We also found that the cation behaves as "super" photoacid, showing a very high deprotonation rate constant (1.04 x 10(11) s(-1)) and an enhanced acidity. Therefore, we also carried out experiments at very high perchloric acid concentrations, dealing with some other effects which become noteworthy at these [H(+)]. The presence of xanthylium cation quenching due to "free" water molecules, and the reduction in the amount of water clusters acting as proton acceptors, are processes which alter notably the time course of the excited-species in this high [H(+)] range.

Coupled Excited-State Dynamics in N-Substituted 2-Methoxy-9-Acridones

“…where trueboldx˙(t) is the time derivative of vector x ( t ) and A is the 3 × 3 compartmental matrix, which contains all the kinetic processes of each species (Orte et al, 2005b; Boens and De Schryver, 2006). In this particular case, the matrix A has the form…”

“…The application of GCA to the study of the complex excited-state dynamics of fluorescent dyes yielded a remarkable improvement in terms of the capabilities and reliability of the obtained results. From simple two-state excited-state reactions (Boens and Ameloot, 2006) to buffer-mediated ESPT reactions (Boens et al, 2004; Crovetto et al, 2004; Orte et al, 2005a) and complex three-state systems (Orte et al, 2005b), the GCA provided invaluable tools to gain in-depth information on those photophysical systems. Such studies have also helped in the experimental design of experiments suitable enough to obtain unique values for all the rate constants involved in the dynamic systems (Boens et al, 2004; Boens and Ameloot, 2006).…”

“…Such studies have also helped in the experimental design of experiments suitable enough to obtain unique values for all the rate constants involved in the dynamic systems (Boens et al, 2004; Boens and Ameloot, 2006). To fully solve the system, at very high acidic concentrations, the reduction of available water molecules had to be considered, as previously done in the study of the superphotoacid 2′,7′-difluorofluorescein (Orte et al, 2005b). The complex excited-state dynamics found at acidic pH were successfully modeled, and all the rate constants were defined.…”

Synthesis and Photophysics of a New Family of Fluorescent 9‐Alkyl‐Substituted Xanthenones

“…At these high acidic concentrations, a simple emission band centered at 469 nm was detected, which rapidly changed to neutral emission with decreasing acid concentration. These features clearly indicate the excited‐state deprotonation of the cation form, since it turned to a super‐photoacid upon excitation, as occurs in fluoresceins,8b, 16 naphthol, and other phenols 17. 18…”

Effects of the anion salt nature on the rate constants of the aqueous proton exchange reactions

“…Xanthenic dyes have a peculiar property, the Excited-State Proton Transfer (ESPT) reaction of the phenolic group can only be achieved if some adequate proton donor/acceptor (buffer) is present in the medium at high enough concentration, and this peculiarity has been widely studied in previous papers. [1][2][3][4][5][6][7] Recently, we have also studied these buffer-mediated proton exchange reactions in the ground-state (Ground-State Proton Transfer, GSPT). 8 The obtained rate constant supported the uniformity of the process in the ground and the excited state, as well as the need of adequate character of the buffer species to be able to promote the excited state reaction, and to accelerate the ground-state reaction.…”