Zinc mobilized from endogenous stores serves as a signaling agent in the brain, pancreas, prostate, and other organs. In the central nervous system, high concentrations of mobile zinc of poorly defined function are heterogeneously distributed throughout the brain. Elucidating mobile zinc neurobiology requires sophisticated probes that selectively report its time-dependent location and concentration within live cells, tissues, and animals. The invention and application of luminescent probes allow one to track the spatiotemporal properties of mobile zinc, providing insight into its functions. This proposal delineates strategies for generating advanced fluorescent zinc sensors that overcome the limitations of current probes, mainly pH sensitivity, adventitious localization, and an inability t operate effectively in the near-infrared (NIR) region.
The first aim i s to install zinc- reactive esters onto the fluorophore scaffold to greatly improve the dynamic range, pH profile, and targetability of the sensors. Esterified probes can be made impervious to unwanted proton- or esterase-induced turn-on, rendering them essentially non-fluorescent, and can be delivered to discrete locales in live cells. Zinc-promoted de-esterification restores fluorescence, generating a bright, Zn-selective signal. In the second aim, a related but distinctive strategy will provide mobile zinc sensors that operate at long wavelengths required for deep tissue penetration. Zinc-induced fluorescence will be generated by promoting a major change in molecular structure of a probe from a dark form to an emissive, extensively conjugated isomer via ring-opening of benzooxazinoindoles and related molecules. The resulting chemistry will generate red and NIR fluorescent products in a reversible, pH-insensitive, and biocompatible manner. With these new reaction-based sensing mechanisms, the first two aims address the issues of dynamic range, pH-sensitivity, and imaging depth that have limited progress in mobile zinc sensing.
The third aim i ntroduces peptide- and protein-based targeting vectors that localize small-molecule zinc sensors to discrete subcellular locales. This strategy overcomes uncontrolled localization inherent to existing small-molecule fluorescent probes and provides spatial information not available via optical microscopy by limiting turn-on only to situations where Zn2+ arrives at a programmed target. These platforms also offer access to ratiometric sensors with variable zinc-binding fluorophores to tune their Kd and emission wavelength, as well as access to localization modules.
The final aim applies the foregoing novel constructs to address the action of mobile zinc in neural progenitor cells and sensory circuits. With sensors at programmed cellular locales, changes in zinc pools during neural progenitor cell differentiation will be determined. Through collaborations, targetable, reaction-based probes will be applied in live tissues and animals to help elucidate the functions of mobile zinc. Because the strategies outlined here can be applied to visualize analytes apart from zinc, they will impact other research areas in neurochemistry and offer fundamental insights into the physiology and pathology of metals in the brain.

Public Health Relevance

Mobile forms of zinc are thought to play critical roles in the physiology of memory formation, the differentiation of neural stem cells, and the processing of sensory information. When mobilized in an uncontrolled manner, for example during optic nerve damage, zinc contributes to neurodegeneration, a process that can be prevented and even reversed by rapid chelation with zinc-specific reagents. This research program will supply tools for revealing the spatiotemporal distribution of mobile zinc, apply them to map its cellular sources and targets during electrophysiological and chemical stimulation in neural progenitor cells, live brain slices, and animals, and provide knowledge essential for the development of novel protocols and compounds to treat disorders stemming from mobile zinc dysregulation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Program Officer
Anderson, Vernon
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
Zip Code
Zhang, Daniel Y; Azrad, Maria; Demark-Wahnefried, Wendy et al. (2015) Peptide-based, two-fluorophore, ratiometric probe for quantifying mobile zinc in biological solutions. ACS Chem Biol 10:385-9
Rivera-Fuentes, Pablo; Lippard, Stephen J (2014) SpiroZin1: a reversible and pH-Insensitive, reaction-based, red-fluorescent probe for imaging biological mobile zinc. ChemMedChem 9:1238-43
Khan, Mustafa; Goldsmith, Christian R; Huang, Zhen et al. (2014) Two-photon imaging of Zn2+ dynamics in mossy fiber boutons of adult hippocampal slices. Proc Natl Acad Sci U S A 111:6786-91
Chyan, Wen; Zhang, Daniel Y; Lippard, Stephen J et al. (2014) Reaction-based fluorescent sensor for investigating mobile Zn2+ in mitochondria of healthy versus cancerous prostate cells. Proc Natl Acad Sci U S A 111:143-8
Loas, Andrei; Radford, Robert J; Lippard, Stephen J (2014) Addition of a second binding site increases the dynamic range but alters the cellular localization of a red fluorescent probe for mobile zinc. Inorg Chem 53:6491-3
Radford, Robert J; Chyan, Wen; Lippard, Stephen J (2014) Peptide Targeting of Fluorescein-Based Sensors to Discrete Intracellular Locales. Chem Sci 5:4512-4516
Lin, Wei; Buccella, Daniela; Lippard, Stephen J (2013) Visualization of peroxynitrite-induced changes of labile Zn2+ in the endoplasmic reticulum with benzoresorufin-based fluorescent probes. J Am Chem Soc 135:13512-20
Radford, Robert J; Lippard, Stephen J (2013) Chelators for investigating zinc metalloneurochemistry. Curr Opin Chem Biol 17:129-36
Radford, Robert J; Chyan, Wen; Lippard, Stephen J (2013) Peptide-based Targeting of Fluorescent Zinc Sensors to the Plasma Membrane of Live Cells. Chem Sci 4:3080-3084
Huang, Zhen; Zhang, Xiao-an; Bosch, Miquel et al. (2013) Tris(2-pyridylmethyl)amine (TPA) as a membrane-permeable chelator for interception of biological mobile zinc. Metallomics 5:648-55

Showing the most recent 10 out of 55 publications