Photoacid behaviour in a fluorinated green fluorescent protein chromophore:Ultrafast formation of anion and zwitterion states

The photophysics of the chromophore of the green fluorescent protein in Aequorea victoria (avGFP) are dominated by an excited state proton transfer reaction. In contrast the photophysics of the same chromophore in solution are dominated by radiationless decay, and photoacid behaviour is not observed. Here we show that modification of the pKa of the chromophore by fluorination leads to an excited state proton transfer on an extremely fast (50 fs) time scale. Such a fast rate suggests a barrierless proton transfer and the existence of a pre-formed acceptor site in the aqueous solution, which is supported by solvent and deuterium isotope effects. In addition, at lower pH, photochemical formation of the elusive zwitterion of the GFP chromophore is observed by means of an equally fast excited state proton transfer from the cation. The significance of these results for understanding and modifying the properties of fluorescent proteins are discusse

Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups

Ultrafast Excited-State Proton Transfer to the Solvent Occurs on a Hundred-Femtosecond Time-Scale

Steady-state and ultrafast time-resolved techniques were used to study a newly synthesized photoacid, phenol-carboxyether dipicolinium cyanine dye, QCy9. We found that the excited-state proton transfer (ESPT) to water occurs at the remarkably short time of about 100 fs, <i>k</i>_PT ≈ 1 × 10¹³ s^–1, the fastest rate reported up to now. On the basis of the Förster-cycle, the p<i>K</i>_a* value is estimated to be −8.5 ± 0.4. In previous studies, we reported the photoacidity of another superphotoacid, the QCy7 for which we found an ESPT rate constant of ∼1.25 × 10¹² s^–1, one-eighth that of the QCy9 compound. We found a kinetic isotope effect of the ESPT of about two