Addition of a second binding site increases the dynamic range but alters the cellular localization of a red...

We report the synthesis and photophysical properties of ZBR4 and ZR1, two resorufin-based ditopic probes for mobile zinc. Upon binding Zn(2+), the sensors display 14- and 41-fold enhancements of their red fluorescence emission, respectively. In contrast to ZR1 and other members of the ZBR family, which accumulate in the endoplasmic reticulum, ZBR4 spontaneously localizes to the mitochondria of HeLa cells. The modular approach in designing the constructs facilitates a homologation strategy aimed at tuning the zinc-binding and intracellular targeting properties of future probes.