Membrane-permeable and -impermeable sensors of the Zinpyr family and their application to imaging of hippocampal zinc in vivo

Esterification of fluorescent biosensors is a common strategy used to trap probes within the cell. Zinpyr-1 (ZP1) is a fluorescein-based bright fluorescent sensor for divalent zinc that is cell permeable without prior modification. We describe here the synthesis and characterization of ZP1 sensors containing a carboxylic acid or ethyl ester functionality at the 5 or 6 position of the fluorescein. The presence of an electronegative carboxylate decreases the proton-induced background fluorescence of the probe by lowering the pK _a of the benzylic amines responsible for fluorescence quenching. The charged species ZP1(6-CO ₂ ^-) is membrane-impermeant, whereas the permeability of the neutral ZP1(5/6-CO ₂Et) is similar to that of the parent sensor. Intracranial microinfusion of ZP1(6-CO ₂Et) into rat hippocampus produces reduced staining of vesicular zinc in neuropil and very clear delineation of zinc-positive injured neuronal somata and dendrites as compared with ZP1.