Emission Ratiometric Imaging of Intracellular Zinc: Design of a Benzoxazole Fluorescent Sensor and Its Application in Two-Photon Microscopy

Article abstract of DOI:10.1021/ja039073j

Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and pr

Full text of DOI:10.1021/ja039073j

Published on Web 12/24/2003
Emission Ratiometric Imaging of Intracellular Zinc: Design of a Benzoxazole
Fluorescent Sensor and Its Application in Two-Photon Microscopy
Masayasu Taki,† Janet L. Wolford,‡ and Thomas V. O’Halloran*,†,‡
Department of Chemistry and Department of Biochemistry, Molecular Biology, and Cell Biology,
Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208
Received October 15, 2003; E-mail: t-ohalloran@northwestern.edu
The inorganic physiology of intracellular zinc is poorly under-
stood but of emerging importance in understanding a variety of
disorders in humans. Histochemical studies of mammalian tissues
including prostate,1 insulin secreting pancreatic islets,2 and granule
neurons of the hippocampus3-5 reveal patterns of Zn(II) accumula-
tion that are disrupted in some types of prostate cancer, diabetes,
and neurodegenerative disorders, respectively. The function of zinc
in these tissues or even within single-cell organisms such as S.
A mixture of aqueous buffer (50 mM HEPES, pH 7.20, 0.1 M
KNO3) and DMSO (5%, v/v) was used for the UV titration of
cereVisiae remains controversial.5-8 Confocal fluorescence micros-
copy has proved to be a central tool in understanding calcium
biology and has the potential to resolve these issues in zinc biology.
Two-photon excitation (TPE) fluorescence microscopy provides
significant advantages over standard laser confocal approaches by
providing deeper sectioning, less phototoxicity, and selective
excitation of a smaller focal volume (i.e., femtoliter), thus decreasing
background fluorescence.9 Such advances can be applied to zinc
physiology; however, this requires the parallel development of new
zinc-specific chemical probes that operate within cells.
Protein-based zinc probes are useful in a variety of physiological
experiments,10,11 but several families of cell permeable organic
compounds that undergo a change in fluorescence intensity upon
metal binding can be used without microinjection into each
cell.2,12-23 Compounds that also exhibit a shift in the fluorescence
excitation or emission maxima upon formation of a 1:1 complex
can be used as probes in ratiometric imaging experiments, allowing
quantitative measurements of intracellular changes in metal ion
concentration. Photophysical studies of bidentate, zinc-responsive
benzazoles show promise in fluorescence ratio approaches.20
Benzofuran-based zinc probes such as ZnAF-R1 and ZnAF-R2 have
been used for excitation ratio imaging of a zinc loaded cell,19 and
the coumazin compound also has some excitation ratiometric
properties.23 Here, we describe an emission ratiometric probe for
intracellular zinc and demonstrate its utility in TPE microscopy of
mammalian cells. The fluorescent zinc reporter Zinbo-5 is cell
permeable, binds free Zn2+ with a Kd in the nanomolar range, and
shows significant zinc-induced changes in quantum yield and in
both the excitation and the emission maxima. We further show that
Zinbo-5 is well suited for two-photon experiments that readily reveal
changes in intracellular zinc within single cells.
Zinbo-5 with Zn2+. A significant decrease in the absorption band
of the apo at 337 nm (ꢀ ) 2.3 × 104 M-1 cm-1) and an increase
of a new band at 376 nm (ꢀ ) 1.9 × 104 M-1 cm-1) was observed
with a distinct isosbestic point at 356 nm (Figure S1). The
absorption bands at 337 and 376 nm linearly decreased and
increased, respectively, up to a 1:1 [Zn2+]/[Zinbo-5] ratio, indicating
formation of a 1:1 complex (Figure S1).
Figure 1A shows the emission spectra of Zinbo-5 when excited
at the isosbestic point of the absorption titration spectrum (356 nm),
at various free Zn2+ concentrations. The apo form exhibits a
characteristic band at 407 nm (Φ ) 0.02) that shifts to 443 nm (Φ
) 0.10) upon binding with Zn2+. Comparisons within the Zinbo
family indicate that the hydroxyl function gives rise to a larger
shift relative to the unsubstituted forms (data not shown) consistent
with the model studies of Farhni and co-workers.25 In contrast to
those model compounds which exhibit a blue shift in emission upon
zinc binding,20 Zn2+ binding to Zinbo-5 leads to a red shift in the
emission, suggesting a different photophysical mechanism for the
polydentate Zinbo complexes.
The apparent dissociation constant (Kd ) 2.2 ( 0.1 nM at pH
7.20) for Zn2+ was determined by plotting the fluorescence intensity
ratio between 395 and 443 nm (or UV absorbance) against log-
[Zn2+
]
and fitting these data as previously described14 (Figures
free
1B and S2). The corresponding pZn value (-log[Zn2+])14 is 9.3,
comparable to the affinity of Zinquin,14 Zinpyr,26 and ZnAF19 probes
(Table S1). The Zn2+ affinity of Zinbo-5 is much higher than that
of di(2-picolyl)amine (DPA), indicating that the phenolate oxygen
and benoxazole nitrogen are likely chelating the metal as the third
and fourth ligands.
A survey of physiologically relevant metal ions reveals that only
Zn2+ and Cd2+ induced an emission shift as shown in Figure 1A,
The Zinbo family compounds are built around the highly
fluorescent benzoxazole core substituted with a variety of Zn-
chelating groups.24 The synthesis of Zinbo-5, which employs the
aminomethyl pyridine moiety, involves reaction of a MOM-
protected phenol aldehyde derivative with amino-m-cresol and
oxidation with barium manganate to provide the benzoxazole
derivative. Bromination of the methyl group using NBS, coupling
with 2-aminomethyl pyridine, and deprotection by p-toluenesulfonic
acid yielded Zinbo-5.
whereas other heavy metal ions, Mn2+, Co2+, Ni2+, Fe2+, and Cu2+
quenched the fluorescence of Zinbo-5 (Figure S3). Na+, K+, Ca2+
,
,
and Mg2+ afforded no fluorescent response even at metal concen-
trations as high as 5 mM. Neither high nor low concentrations of
alkali metal and alkaline earth metal ions altered the fluorescent
response of Zinbo-5 to Zn2+, suggesting that this probe may be
useful in a wide range of biological and microscopic applications.
Two-photon images of mouse fibroblast cells treated with
Zinbo-5 show that intracellular zinc can be readily gauged with
this probe. Given that no differences are observed between one-
and two-photon-excited fluorescence emission or excitation spectra
† Department of Chemistry.
‡ Department of Biochemistry, Molecular Biology, and Cell Biology.
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10.1021/ja039073j CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
of cells with an excess of the cell-permeable, high affinity zinc
chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN),
decreases the intracellular ratio to equal that of the unbound probe
(Figure 2D). This and control experiments on untreated cells indicate
that autofluorescence does not contribute to the ratio images.
These emission ratio imaging experiments reveal that Zinbo-5
can readily reveal changes in intracellular zinc availability. The
photophysical data further suggest that Zinbo compounds can also
be used in epifluorescence excitation ratio imaging approaches to
studying zinc in tissues and cells. These methods are particularly
amenable to investigation of live tissue samples in real time and
are being applied to studies of hippocampus, where physiological
fluctuations in synaptic zinc concentration are estimated to be as
high as 100-300 µM or as low 2 nM.5
Figure 1. (A) Emission spectra of Zinbo-5 with the excitation at 356 nm
in Zn2+/EGTA buffered system (50 mM HEPES, pH 7.20, 0.1 M KNO3;
10 mM EGTA, 1-9 mM zinc sulfate) at 0, 0.14, 0.29, 0.66, 1.1, 1.8, 2.6,
4.0, 6.1, and 11 nM free Zn2+, respectively. (B) Plots of the fluorescence
intensity ratio between 395 and 443 nm (I443/I395) with a best curve for a
dissociation constant of 2.2(1) ( 0.1 × 10-9 M.
Acknowledgment. We thank NIH DK52627 and GM38784
(T.V.O.), JSPS (M.T.), the Robert H. Lurie Comprehensive Cancer
Center of Northwestern University, the Rice Foundation, and NSF
(CHE-9810378) for financial support, and N. Brown and E.
Kawamoto for advice.
Supporting Information Available: Synthesis and characterization
of Zinbo-5, UV-vis titration for Zn2+, and experimental details (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Figure 2. Emission ratio images of fibroblast [L(TK)-] cells in Zinbo-5.
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