Oxygen- and pH-Dependent Photophysics of Fluorinated Fluorescein Derivatives: Non-Symmetrical vs. Symmetrical Fluorination

Fluorescein, and derivatives of fluorescein, are often used as fluorescent probes and sensors. In systems where pH is a variable, protonation/deprotonation of the molecule can influence the pertinent photophysics. Fluorination of the xanthene moiety can alter the molecule's pK_a such as to render a probe whose photophysics remains invariant over a wide pH range. Di-fluorination is often sufficient to accomplish this goal, as has been demonstrated with compounds such as Oregon Green in which the xanthene moiety is symmetrically difluorinated. In this work, we synthesized a non-symmetrical difluorinated analog of Oregon Green which we call Athens Green. We ascertained that the photophysics and photochemistry of Athens Green, including the oxygen-dependent photophysics that results in the sensitized production of singlet oxygen, O₂(a¹Δ_g), can differ appreciably from the photophysics of Oregon Green. Our data indicate that Athens Green will be a more benign fluorescent probe in systems that involve the production and removal of O₂(a¹Δ_g). These results expand the available options in the toolbox of fluorescein-based fluorophores.