Zinc is an essential metal ion for human growth and development, the disruption of cellular Zn 2؉ homeostasis being implicated in several major disorders including Alzheimer's disease, diabetes, and cancer. The molecular mechanisms of Zn 2؉ physiology and pathology are insufficiently understood, however, owing in part to the lack of tools for measuring changes in intracellular Zn 2؉ concentrations with high spatial and temporal fidelity. To address this critical need, we have synthesized, characterized, and applied an intracellular fluorescent probe for the ratiometric imaging of Zn 2؉ based on a tautomeric seminaphthofluorescein platform.
Zinc is an indispensable element for sustaining life and is the second-most abundant transition metal in the human body (1, 2). Owing to its unique electronic and structural preferences, Zn 2ϩ plays a central role in regulating cellular metabolism (1, 3). Zn 2ϩ is an essential cofactor in all six classes of enzymes (1, 4) and several families of regulatory proteins, including those that control gene expression (3, 5), DNA repair (6, 7), and apoptosis (8).The physiological importance of Zn 2ϩ demands that cells exert strict control over the homeostasis of this ubiquitous metal ion (9), and most stores of intracellular Zn 2ϩ are tightly bound and serve as structural and͞or catalytic components of metalloprotein scaffolds (1, 2). Nevertheless, histochemical studies reveal that many mammalian organs accumulate pools of labile Zn 2ϩ under normal physiological conditions. Prominent examples include the brain (10), pancreas (11), and prostate (12). In addition, alterations of Zn 2ϩ homeostasis are implicated in a number of significant human disorders; disrupted patterns of intracellular Zn 2ϩ accumulation have been found in patients with Alzheimer's disease (13), diabetes (14), and cancer (15). Despite the far-ranging consequences of Zn 2ϩ homeostasis in human physiology and pathology, however, the mechanistic details surrounding intracellular Zn 2ϩ accumulation, trafficking, and function remain poorly defined even in the simplest singlecell organisms (16).Fluorescent chemosensors are well suited to meet the need for tools to map the spatial and temporal distribution of ionic Zn 2ϩ pools within living cells. Such reagents have revolutionized the study of calcium in cell biology (17) and hold much promise for enhancing our understanding of zinc in cell biology. Several families of fluorescent Zn 2ϩ probes have been described, including those with peptide-or protein-based scaffolds (18-22). There are also a number of cell-permeable, synthetic chemosensors that induce a change in fluorescence intensity upon Zn 2ϩ binding and do not require microinjection for intracellular use (23-30). Although these and related (31, 32) intensity-based fluorescent probes are of considerable practical value, they cannot provide quantitative information about changes in Zn 2ϩ concentrations, owing to variations in excitation intensity, emission collection efficiency, sample thickness, and artifacts assoc...