Zinspy Sensors with Enhanced Dynamic Range for Imaging Neuronal Cell Zinc Uptake and Mobilization

Thiophene moieties were incorporated into previously described Zinspy (ZS) fluorescent Zn(II)
sensor motifs (Nolan, E. M.; Lippard, S. J. Inorg. Chem. 2004, 43, 8310−8317) to provide enhanced
fluorescence properties, low-micromolar dissociation constants for Zn(II), and improved Zn(II) selectivity.
Halogenation of the xanthenone and benzoate moieties of the fluorescein platform systematically modulates
the excitation and emission profiles, pH-dependent fluorescence, Zn(II) affinity, and Zn(II) complexation
rates, offering a general strategy for tuning multiple properties of xanthenone-based metal ion sensors.
Extensive biological studies in cultured cells and primary neuronal cultures demonstrate 2-{6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethylthiophen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}benzoic acid (ZS5) to be
a versatile imaging tool for detecting Zn(II) in vivo. ZS5 localizes to the mitochondria of HeLa cells and
allows visualization of glutamate-mediated Zn(II) uptake in dendrites and Zn(II) release resulting from
nitrosative stress in neurons.