Four new bright members of the ZinPyr zinc fluorescence sensor family for live cell imaging

As the second most abundant trace element, zinc plays numerous roles in the human body. Not only tightly bound as structural component and co-factor of more than 3000 proteins, but also as labile bound zinc, so-called mobile Zn (mZn). This mZn occurs especially in the central nervous system, where it plays a fundamental role in signal transduction. Accordingly, dysregulated zinc homeostasis is linked to the pathogenesis of neurodegenerative diseases. Fluorescence sensors have emerged as powerful tools to unravel its role on the molecular level. With 20 members, the most prominent sensor family is the ZinPyr family that exploits a fluorescein platform equipped with usually two bis(2-pyridylmethyl)amine (DPA) as zinc binding units. Within this article, we report four new bright members of the ZinPyr family, ZP1(5-en), ZP1(6-en), ZP1(5-Me₂en), and ZP1(6-Me₂en), which are derived from the known sensors ZP1(5-CO₂H) and ZP1(6-CO₂H). Modification of these parent sensors with ethane-1,2-diamine (en) or N¹,N²-dimethylethane-1,2-diamine (Me₂en) yielded cell-permeable sensors that combine the low quantum yield of the zinc-free state Φ_free (0.165(0) - 0.190(9)) of the parent sensors with a high turn-on (5) and dynamic range (4.2 - 5.4). These properties make the new sensors among the brightest sensors in the ZinPyr family. Live cell imaging demonstrated their ability to detect intracellular zinc with an approximate turn-on of 2-3. The sensors showed a vesicular localization, with ZP1(6-en) and ZP1(5-Me₂en) also localizing in the nuclei.