A FRET-based approach to ratiometric fluorescence detection of hydrogen peroxide

We report the synthesis, properties, and biological applications of Ratio-Peroxyfluor-1 (RPF1), a new ratiometric fluorescent reporter for hydrogen peroxide. RPF1 is comprised of a two-fluorophore cassette, where the spectral overlap between coumarin donor and fluoran/fluorescein acceptor partners can be controlled by the chemoselective peroxide-mediated deprotection of boronic ester pendants on the acceptor dye. RPF1 features good selectivity for hydrogen peroxide over a variety of reactive oxygen species, including superoxide and nitric oxide, a ca. 8-fold increase in fluorescence intensity ratio (λ₅₁₇/λ₄₆₄) upon H₂O₂ reaction, and excitation and emission profiles in the visible region. Experiments with viable yeast mitochondria show that RPF1 can monitor and quantify endogenous production of H₂O₂, establishing the potential utility of this approach for probing peroxide biology in living systems.