Design and synthesis of coumarin-based Zn²⁺ probes for ratiometric fluorescence imaging

The physiological roles of free Zn²⁺ have attracted great attention. To clarify those roles, there has been a need for ratiometric fluorescent Zn²⁺ probes for practical use. We report the rational design and synthesis of a series of ratiometric fluorescent Zn²⁺ probes. The structures of the probes are based on the 7-hydroxycoumarin structure. We focused on the relationship between the electron-donating ability of the 7-hydroxy group and the excitation spectra of 7-hydroxycoumarins, and exploited that relationship in the design of the ratiometric probes; as a result, most of the synthesized probes showed ratiometric Zn²⁺ -sensing properties. Then, we designed and synthesized ratiometric Zn ²⁺ probes that can be excited with visible light, by choosing adequate substituents on coumarin dyes. Since one of the probes could permeate living cell membranes, we introduced the probe to living RAW264 cells and observed the intracellular Zn²⁺ concentration via ratiometric fluorescence microscopy. As a result, the ratio value of the probe changed quickly in response to intracellular Zn²⁺ concentration.