High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2′-Bipyridine Scaffold for Endogenous Zn(2+) and Its Application to Living Cells

Zinc is an essential trace element involved in many biological activities; however, its functions are not fully understood. To elucidate the role of endogenous labile Zn(2+), we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2′-bipyridin]-6-amine (6 (rBpyZ)) based on the 6-amino-2,2′-bipyridine scaffold, which acts as both the chelating agent for Zn(2+) and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of 6 (rBpyZ), and a ratiometric fluorescence response consisting of a decrease at the emission wavelength of 438 nm and a corresponding increase at the emission wavelength of 465 nm was observed. The ratiometric probe 6 (rBpyZ) exhibited a nanomolar-level dissociation constant (K(d) = 0.77 nM), a large Stokes shift (139 nm), and an excellent detection limit (0.10 nM) under physiological conditions. Moreover, fluorescence imaging using A549 human lung adenocarcinoma cells revealed that 6 (rBpyZ) had good cell membrane permeability and could clearly visualize endogenous labile Zn(2+). These results suggest that the ratiometric fluorescence probe 6 (rBpyZ) has considerable potential as a valuable tool for understanding the role of Zn(2+) in living systems.