A tautomeric zinc sensor for ratiometric fluorescence imaging: Application to nitric oxide-induced release of intracellular zinc

^*Department of Chemistry, ^†Picower Center for Learning and Memory, and ^‡Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Stephen J. Lippard, December 5, 2003

Abstract

Zinc is an essential metal ion for human growth and development, the disruption of cellular Zn²⁺ homeostasis being implicated in several major disorders including Alzheimer's disease, diabetes, and cancer. The molecular mechanisms of Zn²⁺ physiology and pathology are insufficiently understood, however, owing in part to the lack of tools for measuring changes in intracellular Zn²⁺ 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 Zn²⁺ based on a tautomeric seminaphthofluorescein platform. Zin-naphthopyr 1 (ZNP1) affords single-excitation, dual-emission ratiometric detection of intracellular Zn²⁺ through Zn²⁺-controlled switching between fluorescein and naphthofluorescein tautomeric forms. The probe features visible excitation and emission profiles, excellent selectivity responses for Zn²⁺ over competing Ca²⁺ and Mg²⁺ ions at intracellular concentrations, a dissociation constant (K_d) for Zn²⁺ of <1 nM, and an 18-fold increase in fluorescence emission intensity ratio (λ₆₂₄/λ₅₂₈) upon zinc binding. We demonstrate the value of the ZNP1 platform for biological applications by imaging changes in intracellular [Zn²⁺] in living mammalian cells. Included is the ratiometric detection of endogenous pools of intracellular Zn²⁺ after NO-induced release of Zn²⁺ from cellular metalloproteins. We anticipate that ZNP1 and related probes should find utility for interrogating the biology of Zn²⁺.

Footnotes

↵ § To whom correspondence should be addressed. E-mail: lippard{at}lippard.mit.edu.
Abbreviations: ZNP1, Zin-naphthopyr 1; HRMS, high-resolution MS; ESI, electrospray ionization; TPEN, N,N,N′,N′-tetra(2-picolyl)ethylenediamine; SNOC, S-nitrosocysteine.