High-Affinity Ratiometric Fluorescence Probe Based on 6-Amino-2,2'-Bipyridine Scaffold for Endogenous Zn 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, we developed a novel ratiometric fluorescence probe, 5-(4-methoxyphenyl)-4-(methylsulfanyl)-[2,2'-bipyridin]-6-amine ( ()) based on the 6-amino-2,2'-bipyridine scaffold, which acts as both the chelating agent for Zn and the fluorescent moiety. The methoxy group acted as an electron donor, enabling the intramolecular charge transfer state of (), 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 () exhibited a nanomolar-level dissociation constant ( = 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 () had good cell membrane permeability and could clearly visualize endogenous labile Zn. These results suggest that the ratiometric fluorescence probe () has considerable potential as a valuable tool for understanding the role of Zn in living systems.