Bright fluorescent chemosensor platforms for imaging endogenous pools of neuronal zinc

A series of new fluorescent Zinpyr (ZP) chemosensors based on the fluorescein platform have been prepared and evaluated for imaging neuronal Zn²⁺. A systematic synthetic survey of electronegative substitution patterns on a homologous ZP scaffold provides a basis for tuning the fluorescence responses of "off-on" photoinduced electron transfer (PET) probes by controlling fluorophore pK_a values and attendant proton-induced interfering fluorescence of the metal-free (apo) probes at physiological pH. We further establish the value of these improved optical tools for interrogating the metalloneurochemistry of Zn²⁺; the novel ZP3 fluorophore images endogenous stores of Zn²⁺ in live hippocampal neurons and slices, including the first fluorescence detection of Zn ²⁺ in isolated dentate gyrus cultures. Our findings reveal that careful control of fluorophore pK_a can minimize proton-induced fluorescence of the apo probes and that electronegative substitution offers a general strategy for tuning PET chemosensors for cellular studies. In addition to providing improved optical tools for Zn²⁺ in the neurosciences, these results afford a rational starting point for creating superior fluorescent probes for biological applications.