A novel two-fluorophore approach to ratiometric sensing of Zn(2+)

We present a small molecule ratiometric Zn2+-sensing system based on two fluorophores excited by visible light, a Zn2+-insensitive reporter fluorophore, coumarin 343, and a Zn2+-sensitive fluorescein-based compound, ZPA-1. The two fluorophores are linked by an ester to give Coumazin-1, a membrane-permeable, essentially nonfluorescent compound. Upon exposure to esterases, Coumazin-1 is hydrolyzed to its constituent fluorophores. Measurement of the ratio of coumarin emission at 488 nm (lambdaexc = 445 nm) and comparison with ZPA-1 emission at 534 nm (lambdaexc = 505 nm) affords information about the amount of sensor present as well as the amount of Zn2+ present. A generally applicable synthetic route to amide-functionalized ZP1 sensors is also described. The Zn2+-sensing properties of one member of this class are similar to those of the parent ZP1 sensor, with slightly tighter binding and lower background signal.