Cell-trappable quinoline-derivatized fluoresceins for selective and reversible biological Zn(II) detection

Article abstract of DOI:10.1021/ic1012507

The synthesis and spectroscopic characterization of two new, cell-trappable fluorescent probes for Zn(II) are presented. These probes, 2-(4,5-bis(((6-(2- ethoxy-2-oxoethoxy)quinolin-8-yl)amino)methyl)-6-hydroxy-3-oxo-3H-8 xanthen-9-yl)benzoic acid (QZ2E)

Full text of DOI:10.1021/ic1012507

Inorg. Chem. 2010, 49, 9535–9545 9535
DOI: 10.1021/ic1012507
Cell-Trappable Quinoline-Derivatized Fluoresceins for Selective and
Reversible Biological Zn(II) Detection
Lindsey E. McQuade and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received June 23, 2010
The synthesis and spectroscopic characterization of two new, cell-trappable fluorescent probes for Zn(II) are
presented. These probes, 2-(4,5-bis(((6-(2-ethoxy-2-oxoethoxy)quinolin-8-yl)amino)methyl)-6-hydroxy-3-oxo-3H-8
0
0
xanthen-9-yl)benzoic acid (QZ2E) and 2,2 -((8,8 -(((9-(2-carboxyphenyl)-6-hydroxy-3-oxo-3H-xanthene-4,5-diyl)bis-
methylene))bis(azanediyl))bis(quinoline-8,6-diyl))bis(oxy))diacetic acid (QZ2A), are poorly emissive in the off-state
(
but exhibit dramatic increases in fluorescence upon Zn(II) binding (120 ( 10-fold for QZ2E, 30 ( 7-fold for QZ2A).
This binding is selective for Zn(II) over other biologically relevant metal cations, toxic heavy metals, and most first-row
transition metals and is of appropriate affinity (K (QZ2E) = 150 ( 100 μM, K (QZ2E) = 3.5 ( 0.1 mM, K (QZ2A) =
d1
d2
d1
2
20 ( 30 μM, K (QZ2A) = 160 ( 80 μM, K (QZ2A) = 9 ( 6 μM) to reversibly bind Zn(II) at physiological levels. In
d2
d3
live cells, QZ2E localizes to the Gogli apparatus where it can detect Zn(II). It is cell-membrane-permeable until
cleavage of its ester groups by intracellular esterases produces QZ2A, a negatively charged acid form that cannot
cross the cell membrane.
1
2
13,14
Introduction
including ischemia, Alzheimer’s disease,
and prostate
15,16
cancer.
To understand the biological roles of mobile Zn(II), it is
desirable to have tools for its detection invivo.
Zinc is a ubiquitous and essential metal in biology. It is both
tightly associated with proteins, where it plays structural and
1
7-21
1
Although
catalytic roles, and loosely bound in pools of so-called “mobile
2
many methods are available for sensing metal ions, fluores-
cence detection by microscopy has proved to be an invaluable
technique for noninvasive imaging of mobile Zn(II). Fluo-
rescent probes can be designed to be biologically compatible,
that is, water-soluble, cell-membrane-permeable, nontoxic,
and excitable at low-energy wavelengths that do not induce
interfering autofluorescence or harm the cell. Their photo-
physical properties can be tuned such that large differences
occur in luminescence for the metal-free and metal-bound
states, through changes in intensity and/or wavelength.
Sensor characteristics such as binding affinity can be adjusted
zinc”. The latter occur in abundance in certain regions of the
3
4
body, including the hippocampus and olfactory bulb in the
5
,6
7
brain, the mitochondria of the prostate, and the pancreas,
where release of Zn(II) plays an important role in physiological
signaling and other functions. As for any signaling molecule,
maintaining homeostasis is critical to proper function, and zinc
2
,8,9
levels are highly regulated by transporters
and zinc-binding
Disruption of Zn(II)
homeostasis has been associated with several pathologies,
10,11
proteins, such as metallothionein.
*
To whom correspondence should be addressed. E-mail: lippard@
mit.edu.
(
13) Bush, A. I.; Pettingell, W. H.; Multhaup, G.; Paradis, M. D.;
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2) Frederickson, C. J.; Koh, J.-Y.; Bush, A. I. Nat. Rev. Neurosci. 2005, 6,
Vonsattel, J.-P.; Gusella, J. F.; Beyreuther, K.; Masters, C. L.; Tanzi,
R. E. Science 1994, 265, 1464–1467.
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r 2010 American Chemical Society
Published on Web 09/17/2010
pubs.acs.org/IC

9
536 Inorganic Chemistry, Vol. 49, No. 20, 2010
McQuade and Lippard
Figure 1. Select symmetrical, fluorescein-based Zn(II) probes.
2
3,27,33,34
to match the physiological concentration of the analyte of
interest, and in the case of metal ion sensing, the chelating
group can be chosen to impart selectivity to the probe.
Many such Zn(II)-selective sensors have been designed and
utilized for both ex vivo and in vivo imaging. Figure 1 portrays
a selection of symmetrical, fluorescein-based probes of this
pH.
Symmetrical sensors tend to have lower extinction
coefficients than their asymmetrical counterparts, thereby
lowering fluorescence emission in the metal-free state and
increasing the effective dynamic range.
One of the symmetrical probes illustrated in Figure 1,
QZ2, displays particularly interesting sensing characteris-
tics by comparison to the first-generation Zinpyr (or ZP)
family of probes.
23,25
25
22-31
kind developed in our laboratory.
Fluorescein is an ideal
22,23,28-31,35-39
reporter because it is biologically compatible, very bright
For example, QZ2 has a
32
in the on-state (φ = 0.95 in 0.1 N NaOH), and compatible
with most fluorescent microscopes. The pH dependence of
fluorescein can be modified such that proton-induced fluores-
cence enhancement of a probe is minimized at physiological
much lower affinity for Zn(II) (Kd1 ∼ midmicromolar) than
ZP1 (Kd1 ∼ picomolar) and can therefore detect higher con-
centrations of mobile Zn(II) reversibly and without probe
saturation. It also has a larger dynamic range (∼150-fold
turn-on) than any of the ZP-type probes, which is beneficial
for in vivo imaging, where poor signal-to-noise ratios can
limit data interpretation. One limitation of QZ2 is that it is
(
22) Burdette, S.; Walkup, G. K.; Spingler, B.; Tsien, R. Y.; Lippard, S. J.
J. Am. Chem. Soc. 2001, 123, 7831–7841.
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(

Article
Inorganic Chemistry, Vol. 49, No. 20, 2010 9537
an atmosphere of H was applied and the reaction was stirred at
not cell-trappable. Other similar quinoline-derivatized fluo-
rescein probes, such as the NO sensor CuFL1,
2
40,41
readily
room temperature for 16 h. The resulting solution was then
purged with N and filtered, and the solvent was removed. The
diffuse out of cells after loading under continual perfusion of
media. Because media flow is a requirement for many biolog-
ical imaging experiments, a cell-trappable version of QZ2
would render it useful for more diverse sensing applications.
Herein we report the synthesis, photophysical characteri-
zation, and live-cell imaging of the desired new probe for
Zn(II), QZ2E, which employs the ester/acid strategy for cell
trappability, as well as its cell-impermeable acid counterpart,
QZ2A. QZ2E and QZ2A display dramatic fluorescence
enhancements in the presence of excess Zn(II), which are
selective for the metal over other biologically relevant alkali
and alkaline-earth metal ions, toxic heavy-metal ions, and
most first-row transition-metal ions. The binding of Zn(II) by
QZ2E and QZ2A is weak with Kd values ranging from
micromolar to millimolar, making the sensors suitable for
detecting mobile Zn(II) at high physiological concentrations.
2
dark-brown solid was purified by column chromatography on
silica using 3:1 hexanes/ethyl acetate as the eluant to afford a
pure, light-brown solid (227 mg, 75%). TLC: R = 0.20 (silica,
f
1
1
:1 Hex/EtOAc). Mp: 92-94 °C. H NMR (CD Cl , 300 MHz):
2
2
δ 1.29 (3H, t, J = 7.2 Hz), 4.26 (2H, q, J = 7.2 Hz), 4.69 (2H, s),
.08 (2H, broad s), 6.39 (1H, d, J = 2.7 Hz), 6.61 (1H, d, J = 2.4
Hz), 7.33 (1H, dd, J = 8.4 and 4.2 Hz), 7.95 (1H, dd, J = 8.4 and
5
1
3
1
1.5 Hz), 8.59 (1H, dd, J = 4.2 and 1.5 Hz). C{ H} NMR
(CD Cl , 125 MHz): δ 14.51, 61.84, 65.78, 95.62, 101.40, 122.51,
2
2
1
30.12, 135.34, 135.97, 145.97, 146.16, 157.58, 169.22. HRMS
þ
(
[M þ H] ). Calcd: m/z 247.1077. Found: m/z 247.1079.
2
methyl)-6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic Acid (QZ2E, 3).
-(4,5-Bis(((6-(2-ethoxy-2-oxoethoxy)quinolin-8-yl)amino)-
0
0
,5 -Fluoresceindicarboxaldehyde (43.9 mg, 113 μmol) and
4
ethyl[(8-aminoquinolin-6-yloxy)acetate] (2; 54.8 mg, 223 μmol)
were suspended in methanol (5 mL) and stirred at room tem-
perature for 1 h. The dark-red suspension was cooled to 0 °C,
and sodium borohydride (21.5 mg, 568 μmol) was added. The
reaction clarified to a dark-red solution, which was stirred for
1 h as it warmed to room temperature. The reaction solution was
quenched with water (5 mL), and the methanol was removed
under reduced pressure. The aqueous phase was extracted with
Experimental Section
0
0
Synthetic Materials and Methods. 4 ,5 -Fluoresceindicar-
boxaldehyde and ethylenediamine tetraacetate ethyl ester
2
2
4
2
(
EDTA-(Et)
dures. All other chemicals were used as received. Silica gel
SiliaFlash F60, Silicycle, 230-400 mesh) was used for column
4
)
were prepared by previously reported proce-
5
dried over MgSO and filtered, and the solvent was removed.
ꢀ 10 mL of dichloromethane, the combined extracts were
(
4
The resulting residue was purified by column chromatography
on silica (gradient from 99:1 CH Cl /CH OH to 19:1 CH Cl /
chromatography. Thin-layer chromatography (TLC) was per-
formed on EMD Chemicals F254 silica gel-60 plates (1 mm
thickness) and viewed by either UV light or ninhydrin staining.
2
2
3
2
2
CH
.67 (silica). Mp: 148-149 °C. H NMR (DMSO-d
δ 1.19 (6H, t, J = 4.2 Hz), 4.16 (4H, q, J = 8.4 and 4.2 Hz), 4.75
4H, s), 4.79 (4H, d, J = 3.6 Hz), 6.40 (2H, d, J = 1.5 Hz), 6.53
3
OH) to afford a dark-red solid (25.6 mg, 27%). TLC: R
f
=
1
1
13
1
0
6
, 500 MHz):
H and C{ H} NMR spectra were obtained on either a Varian
00 or 500 MHz spectrometer. Chemical shifts are reported in
3
(
parts per million (δ) and are referenced to residual protic solvent
resonances. The following abbreviations are used in describing
NMR couplings: (s) singlet, (d) doublet, (dd) doublet of dou-
blets, (t) triplet, (q) quartet, (m) multiplet. High-resolution mass
spectra were measured by staff at the MIT Department of
Chemistry Instrumentation Facility.
(2H, d, J = 5.4 Hz), 6.66 (2H, d, J = 1.5 Hz), 6.71 (2H, d, J =
5.4 Hz), 6.77 (2H, t, J = 3.3 Hz), 7.34 (1H, d, J = 4.5 Hz), 7.37
(2H, dd, J = 4.8 and 2.4 Hz), 7.74 (2H, dt, J = 12 and 4.2 Hz),
7.98 (1H, d, J = 4.5 Hz), 8.02 (2H, d, J = 4.8 Hz), 8.48 (2H, d,
1
3
1
J = 2.4 Hz). C{ H} NMR (DMSO-d
1
6
, 125 MHz): δ 13.98,
4.13, 60.62, 64.61, 77.13, 83.42, 93.18, 93.38, 96.26, 96.43,
109.90, 111.96, 122.14, 124.66, 126.31, 127.05, 127.73, 129.13,
130.14, 134.65, 134.87, 145.28, 150.49, 152.08, 157.19, 157.56,
6
-Hydroxy-8-nitroquinoline (1). 4-Amino-3-nitrophenol
5.00 g, 32.4 mmol) was suspended at 95 °C in 37% hydro-
chloric acid (35 mL) and 85% phosphoric acid (15 mL). Acrolein
2.4 mL, 36 mmol) was added dropwise, and the reaction mixture
(
þ
(
168.55, 168.61. HRMS ([M þ Na] ). Calcd: m/z 871.2586.
was stirred while heating for 4 h, during which time it changed
color from yellow to orange to dark red. The reaction mixture was
cooled, filtered, and washed with water. Ammonium hydroxide
Found: m/z 871.2570.
0
0
Synthesis of 2,2 -((8,8 -(((9-(2-Carboxyphenyl)-6-hydroxy-3-
oxo-3H-xanthene-4,5-diyl)bis(methylene))bis(azanediyl))bis-
(quinoline-8,6-diyl))bis(oxy))diacetic Acid (QZ2A, 4). QZ2E (3,
121.9 mg, 144 μmol) was dissolved in aqueous sodium hydrox-
ide (0.5 N, 15 mL) and methanol (5 mL) and heated to 90 °C for
12 h. The solution was cooled to room temperature, and the pH was
adjusted with concentrated hydrochloric acid until an orange precip-
itate formed. The solid was filtered, washed with water, and dried to
(
28%, 50 mL) was added to the filtrate, and the resulting red
precipitate was filtered and washed with water to yield pure
product (1.11 g, 18%). TLC: R = 0.68 (silica, 9:1 CH Cl
f
2
2
/
1
CH OH). Mp: 162-163 °C (dec). H NMR (DMSO-d , 300
MHz): δ 7.45 (1H, d, J = 2.7 Hz), 7.58 (1H, dd, J = 8.4 and 4.2
Hz), 7.80 (1H, d, J = 2.7 Hz), 8.35 (1H, dd, J = 8.4 and 1.2 Hz),
3
6
1
3
1
.78 (1H, dd, J = 4.2 and 1.8 Hz). C{ H} NMR (DMSO-d
8
6
, 125
yield the product (77.6 mg, 68%). TLC: R = 0.63 (silica, 100%
f
1
MHz): δ 112.34, 115.45, 123.19, 129.82, 133.50, 134.76, 148.63,
CH OH). Mp: 139-141 °C (dec). H NMR (DMSO-d , 300
3
6
þ
1
49.34, 154.42. HRMS ([M þ H] ). Calcd m/z 191.0451. Found:
MHz): δ 4.62 (4H, s), 4.71 (4H, s), 6.46 (2H, s), 6.50 (2H, s), 6.59
(2H, d, J = 8.7 Hz), 6.75 (2H, d, J = 8.4 Hz), 7.39-7.42 (3H, m),
7.70-7.82 (2H, m), 8.00 (2H, d, J = 7.2 Hz), 8.17 (2H, d, J =
m/z 191.0450.
Ethyl [(8-Aminoquinolin-6-yloxy)acetate] (2). To a solution of
1
3
1
6
-hydroxy-8-nitroquinoline (1; 234 mg, 1.23 mmol) in ethanol
7.8 Hz), 8.44 (2H, d, J = 3.9 Hz), 10.55 (2H, broad s). C{ H}
(
10 mL) was added ethyl bromoacetate (400 μL, 3.6 mmol) and
NMR (DMSO-d , 125 MHz): δ 36.40, 64.51, 93.39, 93.76, 98.31,
6
N,N-diisopropylethylamine (650 μL, 3.7 mmol), and the reac-
tion was heated to reflux for 24 h. The reaction was then cooled
and the solvent removed. The resultant crude product was taken
up in fresh ethanol (25 mL) containing 10% palladium on
109.80, 111.22, 112.27, 121.85, 122.20, 124.33, 124.70, 126.10,
127.59, 127.91, 129.70, 130.16, 130.37, 135.62, 142.83, 143.67,
;
150.61, 152.52, 157.75, 168.78, 169.96. HRMS ([M - H] ). Calcd:
m/z 791.1995. Found: m/z 791.1994.
0
carbon (203 mg) and purged with N
2
for 15 min, after which
Spectroscopic Materials and Methods. Piperazine-N,N -bis(2-
ethanesulfonic acid) (PIPES) was purchased from Calbiochem.
Potassium chloride (99.999%) was obtained from Aldrich.
Buffer solutions (50 mM PIPES, 100 mM KCl, pH 7) were
prepared in Millipore water, chelexed, filtered, and used for all
spectroscopic studies. Zinc chloride (Aldrich, 99.999%) stock
solutions of 1 M, 100 mM, 10 mM, and 1 mM were prepared in
(
40) Lim, M. H.; Wong, B. A.; Pitcock, W. H., Jr.; Mokshagundam, D.;
Baik, M.-H.; Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 14364–14373.
(
(
41) Lim, M. H.; Xu, D.; Lippard, S. J. Nat. Chem. Biol. 2006, 2, 375–380.
42) Csuk, R.; Barthel, A.; Brezesinski, T.; Raschke, C. Eur. J. Med.
Chem. 2004, 39, 975–988.

9
538 Inorganic Chemistry, Vol. 49, No. 20, 2010
McQuade and Lippard
Millipore water. Stock solutions of 1 mM ligands were prepared
in DMSO and stored in aliquots at -80 °C. Stock solutions
of 40 mM ethylenediamine tetraacetate (EDTA) and 40 mM
untreated control lane and a 5% DMSO in complete DMEM lane)
for another 24 h. The medium was removed and replaced with
200 μL of a 20:1 mixture of FBS-free DMEM and (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
5 mg/mL stock in PBS). The cells were incubated for 4 h, and then
the medium was removed and replaced with DMSO (100 μL/well).
The absorbance of each well was recorded at 555 nm on a micro-
plate reader (BioTek, Synergy HT), and the percent cell survival
values are reported relative to those of untreated control cells.
0
0
N,N,N ,N -tetrakis(2-pyridylmethyl)ethylenediamine(TPEN) were
prepared in DMSO, aliquotted, and stored at -20 °C. UV-
visible spectra were acquired on a Cary 50-Bio spectrometer
using spectrosil quartz cuvettes from Starna Cells Inc. (3.5 mL
volume, 1 cm path length). Acquisitions were made at 25.00 (
0
.05 °C. Fluorescence spectra were obtained on a Quanta
Master 4 L-format scanning spectrofluorimeter (Photon Tech-
nology International) at 25.0 ( 0.1 °C using 1 μM QZ2E or
QZ2A unless otherwise noted. All fluorescence experiments
were repeated in triplicate.
Results and Discussion
Design Considerations. The new trappable Zn(II)
probes were based on the QZ2 scaffold, because this
ligand offers many advantages as a Zn(II) detector.
Cell Culture and Imaging Materials and Methods. HeLa cells
were obtained from American Type Cell Collection (ATCC)
and cultured in Dulbecco’s Modified Eagle Medium (DMEM;
Cellgro, MediaTek, Inc.) supplemented with 10% fetal bovine
serum (FBS; PAA Laboratories), 2 mM glutamine (GIBCO;
Invitrogen), 100 U/mL penicillin (Cellgro, MediaTek, Inc.), and
2
5
First, it employs fluorescein as the reporter, which im-
parts good sensing characteristics (vide supra). Second,
QZ2 detects Zn(II) with an average ∼150-fold dynamic
range and is selective for Zn(II) over most biologically
relevant cations, toxic heavy metals, and first-row transition-
metal ions, with the exception of Cu(II) and Ni(II),
neither of which is available as a mobile M(II) species at
high concentrations in vivo. Lastly, QZ2 is capable of
detecting Zn(II) using one- and two-photon fluorescence
microscopy in live cells, where it reports variable fluores-
cence over a wide range of Zn(II) concentrations. Im-
portantly, the low binding affinity of QZ2 (dissociation
constant Kd1 = 41 ( 3 μM) relative to other Zn(II)-
specific probes such as ZP1 (K =0.04pM,K =1.2nM)
d1 d2
makes QZ-type probes suitable for in vivo imaging under
conditions of high zinc levels, such as in synaptic clefts
during neurotransmission in the hippocampus
1
00 μg/mL streptomycin (Cellgro, MediaTek, Inc.). For imag-
ing studies, cells were plated in poly(D-lysine)-coated plates
MatTek) containing 2 mL of complete DMEM and incubated
(
2
overnight at 37 °C under 5% CO .
For Zn(II) detection studies, the HeLa cells were incubated
with 5 μM QZ2E or QZ2A for ∼18 h. Thirty minutes prior to
imaging, the cells were coincubated with 4.5 μM Hoechst 33258
(
1
Sigma) and either 200 nM Mitotracker Red (Invitrogen) or
00 nM BOPIDY TR Ceramide (Invitrogen) for 30 min. Cells
were washed twice with 1 mL of phosphate-buffered saline
PBS; Cellgro, MediaTek, Inc.) before imaging and then bathed
in 2 mL of serum-free DMEM during imaging. Stock solutions
of 10 mM ZnCl , prepared in Millipore water, and 20 mM
2
5
3
9
(
2
sodium pyrithione (2-pyridinethiol-1-oxide), prepared in
DMSO, were combined in a 1:1 ratio and diluted in serum-free
DMEM to a 5 mM stock concentration. Zn(II) was added to
cells directly on the microscope stage at a final concentration of
43,44
45
or from the pancreatic β cells during insulin secretion,
where it is estimated that labile Zn(II) concentrations may
approach millimolar concentrations. Because QZ2 binds
Zn(II) with a micromolar dissociation constant and its
fluorescence does not saturate until millimolar concen-
trations of Zn(II) are present, QZ2 Zn(II) binding and
subsequent fluorescence enhancement should be revers-
ible under these physiological conditions, making the
probe particularly suitable for in vivo imaging.
To impart cell trappability to QZ2, esters were incor-
porated onto the quinoline rings. Esters maintain cell
permeability until the probe is internalized by cells, where
intracellular esterases hydrolyze the substituents to nega-
tively charged carboxylates. The two negative charges on
the probe scaffold make QZ2E incapable of recrossing the
cell membrane, rendering it cell-trappable.
Synthesis. The synthetic routes to QZ2E and QZ2A are
presented in Scheme 1. 6-Hydroxy-8-nitroquinoline, 1, was
prepared in low yield by the Skraup-Doebner-von Miller
reaction between acrolein and 4-amino-3-nitrophenol. Ester-
ification of the quinoline using ethyl bromoacetate and
H u€ nig’s base followed by reduction of the nitro group by
hydrogenation with palladium on carbon afforded ethyl [(8-
aminoquinolin-6-yloxy)acetate], 2, in moderate yield. Con-
1
imaging. Stock solutions of 40 mM ethylenediamine tetraace-
00 μM and allowed to incubate with cells for 5 min prior to
4
2
tate ethyl ester (EDTA-(Et)4) and 40 mM TPEN were pre-
pared in DMSO, aliquotted, and stored at -20 °C. Aliquots
were thawed immediately prior to use and diluted in serum-free
DMEM to a 4 mM stock concentration. The chelators were
added to the cells on the microscope stage at a final concentra-
tion of 150 μM for 5 min prior to imaging.
For diffusion studies, HeLa cells were incubated with 5 μM
QZ2E for 18 h and with 4.5 μM Hoechst 33258 added 30 min
prior to imaging for nuclear staining. Cells were washed twice
with 1 mL of PBS prior to imaging and then bathed in 2 mL of
serum-free DMEM during imaging. To mimic media flow, the
serum-free DMEM was removed, and the cells were washed
three times with 1 mL of fresh serum-free DMEM and then
bathed in 2 mL of fresh serum-free DMEM. This process was
repeated every 10 min during imaging.
Images were acquired on a Zeiss Axiovert 200 M inverted
epifluorescence microscope equipped with an EM-CCD digital
camera (Hamamatsu) and a MS200 XY Piezo Z stage (Applied
Scientific Instruments), illuminated by an X-Cite 120 metal-halide
lamp (EXFO). Plates were maintained on the microscope stage in
2
an INC-2000 incubator kept at 37 °C under 5% CO . Differential
interference contrast (DIC) and red, blue, and green fluor-
escent images were obtained using an oil immersion 63ꢀ
objective lens, with 2.00 s exposure times for green fluorescent
images. The microscope was operated with Volocity software
0
0
densation of the aminoquinoline with 4 ,5 -fluoresceindi-
2
2
carboxaldehyde in methanol followed by reduction of the
imine using sodium borohydride resulted in product for-
mation. Purification by column chromatography on silica
(Improvision). All fluorescent images were corrected for
background.
Cytotoxicity Assays. HeLa cells were seeded into 96-well plates
(43) Assaf, S. Y.; Chung, S.-H. Nature 1984, 308, 734–736.
(44) Howell, G. A.; Welch, M. G.; Frederickson, C. J. Nature 1984, 308,
(
100 μL total volume/well, 2500 cells/well) in complete DMEM
and incubated at 37 °C under 5% CO for 24 h. The medium was
replaced and the cells incubated with QZ2E (1-50 μM, with an
2
7
36–738.
(45) Chimienti, F.; Favier, A.; Seve, M. Biometals 2005, 18, 313–317.

Article
Inorganic Chemistry, Vol. 49, No. 20, 2010 9539
Scheme 1. Syntheses of QZ2E and QZ2A
Table 1. Photophysical Properties of QZ2, QZ2E, and QZ2A
4
-1
-1
b
absorbance λ (nm), ε ꢀ 10 (M cm
)
emission λ (nm), φ (%)
c
d
unbound
Zn(II)
unbound
Zn(II)
DR
ref
a
QZ2
QZ2E
QZ2A
499, 3.72
499, 2.72(9)
498, 6.41(3)
489, 3.36
496, 1.6(3)
492, 4.0(2)
∼520, 0.5
519, 0.4(1)
515, 1.2(1)
518, 70
514, 73(3)
515, 51(1)
∼150
25
this work
this work
120(10)
30(7)
a
b
c
2
Measurements were performed in 50 mM PIPES, 100 mM KCl at pH 7.0. Referenced to fluorescein (φ = 0.95 in 0.1 N NaOH). 20 mM ZnCl was
d
0
added. DR is the dynamic range, IZn(II)/I .
afforded the trappable ligand, QZ2E, 3, in fair yield. Sub-
sequent hydrolysis of QZ2E using aqueous sodium hydrox-
ide in methanol and precipitation with acid afforded the
cell-impermeable ligand, QZ2A, 4, in moderate yield without
the need for further purification.
this similarity, it is likely that QZ2A and FL2A have
comparable pK values (pK for FL2A = 5.9). For QZ2,
a
a
FL2, and FL2A, protonation causes only an ∼2-fold
increase in fluorescence, indicating that the ligands are
fairly insensitive to pH in the biologically relevant range,
especially by comparison to Zn(II)- or NO-promoted
turn-on.
Metal Binding of QZ2E and QZ2A. The emission pro-
files of QZ2E and QZ2A are almost identical to that of
QZ2. The emission maxima and quantum yields of the
free ligands are given in Table 1. Buffered solutions of QZ2E
exhibit significant fluorescence enhancements when treated
Spectroscopic Properties of QZ2E and QZ2A. QZ2E
and QZ2A exhibit spectroscopic features similar to those of
QZ2 (Table 1). The absorbance spectral maxima occur at
25
4
-1
-1
4
99 nm for QZ2E (ε=2.72 ( 0.09 ꢀ 10 M cm ) with a
4
-1 -1
shoulder at 467 nm (ε=1.2 ( 0.2 ꢀ 10 M cm ) and at
4
-1
-1
4
98 nm for QZ2A (ε=6.41 ( 0.03 ꢀ 10 M cm ) with a
4
-1 -1
shoulder at 464 nm (ε=2.49 ( 0.2 ꢀ 10 M cm ). Upon
addition of excess ZnCl , the maximum absorbance shifts
2
with excess ZnCl , leading to a 120 ( 10-fold increase in inte-
2
4
hypsochromically to 489, 496, and 492 nm (ε=3.36ꢀ10
grated emission upon saturation and a quantum yield in-
crease to 73% (Figure 2a and Table 1). By comparison, addi-
tion of excess Zn(II) to QZ2 causes an ∼150-fold increase in
integrated emission and a quantum yield increase to 70%.
The differences in fluorescent dynamic range can be accoun-
ted for by the difference in molar absorptivities of the
two ligands. Comparing the product of the molar absopr-
-
-
1
1
-1
4
-1
-1
4
M
M
cm , 1.6 ( 0.3 ꢀ 10 M cm , and 4.0 ( 0.2 ꢀ 10
-1
cm for QZ2, QZ2E, and QZ2A, respectively) (Figure
S1 in the Supporting Information, SI). This blue shift is
typical for metal coordination by fluorescein-based sensors
and is presumably caused by perturbation of the fluorescein
π system upon cation binding to the phenolic oxygen atoms
of the xanthenone ring.
-
1
-1
tivity and quantum yield (ε ꢀ φ) of QZ2E (∼114 M cm
-
1
-1
Esterified ligands become hydrolyzed during studies of
their pH-dependent properties. We therefore did not attempt
to measure the pK values of QZ2E. The pK of the secondary
in the off-state, ∼11 680 M cm in the on-state) with that
- -1 -1 -1
1
of QZ2 (∼186 M cm in the off-state, ∼23 520 M cm
in the on-state) yields dynamic ranges ((εꢀ φ) on/(εꢀ φ) off)
of ∼102 and ∼126, respectively. The dynamic ranges ob-
served by fluorescence (∼120 and ∼150, respectively) are
larger, but the ratio of the dynamic range of QZ2E to that of
QZ2 is ∼0.8 using either (ε ꢀ φ) or fluorescence emission.
a
a
25
amine nitrogen atom of QZ2 is 7.0, and an NO probe with a
similar structure, FL2, has the same pK value. Because of
46
a
(
46) McQuade, L. E.; Lippard, S. J. Inorg. Chem. 2010, 49, 7464–7471.

9
540 Inorganic Chemistry, Vol. 49, No. 20, 2010
McQuade and Lippard
Figure 2. Normalized fluorescence response in the presence of excess ZnCl
2
of (a) 1 μM QZ2E, 0-20 mM ZnCl
2
and (b) 1 μM QZ2A, 0-1 mM ZnCl
2
in
2
50 mM PIPES, 100 mM KCl, pH 7.0, T = 25 °C. Insets: Integrated fluorescence vs ZnCl concentration, normalized and adjusted for the basal fluorescence
of the sensor in the absence of Zn(II).
Therefore, the difference observed is accounted for by the
differences in molar absorptivities. Despite the slight
lowering of dynamic range upon esterification of the
quinoline, an ∼120-fold change in fluorescence intensity
is still much greater than that exhibited by Zn(II) sensors
and I , I , and I are the proportionality constants
1 2 3
Ka1
QZ2E þ ZnðIIÞ h QZ2EðZnðIIÞÞ,
½
QZ2EðZnðIIÞÞꢁ
1
9
Ka1
¼
¼ 1=Kd2
ð2Þ
ð3Þ
of the ZP family and more than adequate for cellular
imaging. Hydrolysis of QZ2E to QZ2A causes a signifi-
cant reduction in dynamic range upon exposure to excess
Zn(II) (30 ( 7-fold increase in integrated emission;
Figure 2b) because both the molar absorptivity and
quantum yield of apo-QZ2A are significantly higher
that those of either QZ2 or QZ2E. The quantum yield
of the Zn(II)-saturated QZ2A is also lowered to 51%
versus ∼70% for QZ2 and QZ2E; however, a 30-fold
turn-on is significantly greater than that of many useful
½
QZ2Eꢁ½ZnðIIÞꢁ
Ka2
QZ2EðZnðIIÞÞ þ ZnðIIÞ h QZ2EðZnðIIÞÞ2,
½
QZ2EðZnðIIÞÞ ꢁ
2
Ka2 ¼
¼ 1=Kd1
½
QZ2EðZnðIIÞÞꢁ½ZnðIIÞꢁ
F ¼ I1½QZ2Eꢁ þ I2½QZ2EðZnðIIÞÞꢁ
þ I ½QZ2EðZnðIIÞ ꢁ
2
2
Zn(II)-specific probes such as ZP1 and is more than
sufficient for live-cell imaging.
ð4Þ
ð5Þ
3
2
The strength of Zn(II) binding by a probe is an im-
portant feature that determines its use under specific
biological conditions. Because biological mobile Zn(II)
concentrations vary widely depending on location and
physiological events, it is essential to have fluorescent
sensors with a wide array of dissociation constants for
Zn(II) in order to ensure reversible detection without
probe saturation. As discussed in the design consider-
ations, the QZ-type probes are weak Zn(II) binders, with
½
QZ2Eꢁ ¼ ½QZ2Eꢁ þ ½QZ2EðZnðIIÞÞꢁ
t
þ ½QZ2EðZnðIIÞ2ꢁ
that account for the relative contributions of each QZ2E
species to the overall fluorescence emission, according to eq 4
(Figure 2, inset). Equation 1 was determined by substituting
into eq 4, using eqs 2, 3, and 5. Because the binding of Zn(II)
2
5
by QZ2E is weak, [Zn(II)] can be approximated by [Zn(II)]t,
K values in the micromolar range. This affinity holds
d
the total concentration of Zn(II) corrected for dilution.
Applying the fit to the normalized fluorescence data yields
true for QZ2E, which requires millimolar Zn(II) for
saturation of the fluorescence emission. The strength of
QZ2E Zn(II) binding was determined by fitting the
fluorescence data to a two-site model according to eq 1,
where [Zn(II)] is the concentration of free Zn(II) cor-
values of K = 7000 ( 5000 M and K = 287 ( 8 M.
a1
a2
Substituting into eqs 2 and 3 yields dissociation constants of
K =3.5 ( 0.1 mM (from K ) and K =150 ( 100 μM
d1
a2
d2
(
from K ). The large error associated with the K determi-
rected for dilution, [QZ2E] is the total concentration of
t
a1 a1
nation is probably due to the small changes in fluorescence
intensity observed upon coordination of the first Zn(II) equiv-
alent (Figure S2a in the SI, micromolar region), a postulate
2
½
QZ2Eꢁ ðI þ I K ½ZnðIIÞꢁ þ I K K ½ZnðIIÞꢁ Þ
t
1
2
a1
3
a1 a2
F ¼
2
ð1 þ K ½ZnðIIÞꢁ þ K K ½ZnðIIÞꢁ Þ
substantiated by the fact that the second proportionality
a1
a1 a2
4
constant, I , is similar to the first (I =1.1 ( 0.3 ꢀ 10 , I =
ð1Þ
the probe corrected for dilution, Ka1 and Ka2 are the asso-
ciation constants for the reactions in eqs 2 and 3, respectively,
2
4
1
2
3 ( 4 ꢀ 10 ) and that this value is subject to a large error.
On the other hand, the large fluorescence changes asso-
6
ciated with the second binding event (I
=1.44 ( 0.01ꢀ10 ;
3

Article
Inorganic Chemistry, Vol. 49, No. 20, 2010 9541
Figure S2a in the SI, millimolar region) allow for a more
precise estimation of K . It is useful to note that the order of
clash, it is possible that the Zn(II)-binding pocket of QZ2E
is different from that of QZ2. We hypothesize that, instead
of chelating Zn(II), the pyridine rings of TPEN form a com-
plex with Zn(II)-bound QZ2E, thereby stabilizing a fluores-
cent species. By comparison, the Zn(II)-induced fluores-
cence of QZ2A is fully reversed by both TPEN and EDTA
(Figure 3c,d), suggesting that removal of the ethyl groups is
sufficient to relieve the purported steric clash. Because an
effective chelator (EDTA) was found for QZ2E, TPEN
binding was not further pursued.
a2
magnitude of binding, ∼100 μM, is of the same order as that
estimated for QZ2. The K value for QZ2 was determined
d1
by taking kinetic data measured by stopped-flow spectros-
copy and fitting it to a one-site binding model, assuming that
the second Zn(II) binding event is of significantly lower
affinity. When the fluorescence data obtained for QZ2E
were fit to a one-site model (eq 6), the K value was 2.9 mM,
d
suggesting that the second binding event is responsible for
Selectivity of QZ2E and QZ2A for Zn(II). In order for a
probe to be useful for cellular detection of an analyte, it must
be specific for that analyte over other competing biologically
relevant species. One advantage of the QZ scaffold over the
ZP scaffold is that, although the dipicolylamine chelating
unit of the ZP family binds Zn(II) very tightly, it is not as
specific for Zn(II) compared to the aminoquinoline Zn(II)-
½
QZ2Eꢁ ðI2 - I1Þ½ZnðIIÞꢁ
t
F - F0 ¼
ð6Þ
Kd þ ½ZnðIIÞꢁ
most of the fluorescence enhancement. The poor data fit,
however, indicates that a one-site model is inadequate to
describe the system (Figure S2b in the SI).
When the same analysis was repeated for QZ2A using
the two-site model, the data could not be fit within a
reasonable degree of error (Kd1 = 2 mM ( 9 mM,
Kd2 = 400 μM ( 300 μM; Figure S3a in the SI). A three-
site model was therefore employed, as described by eq 7,
19,25
binding unit of QZ2.
Both families of sensors are specific
for Zn(II) over the biologically relevant Na(I), Ca(II), and
Mg(II), the first-row transition element Mn(II), and the
toxic heavy metals Hg(II) and Cd(II). The QZ family, how-
2
5
ever, is also selective for Zn(II) over Fe(II) and Co(II). We
therefore performed a fluorescence selectivity study to
ensure that modification of the quinoline ring did not
alter the metal binding properties of QZ2E or QZ2A
significantly enough to affect its selectivity (Figure 4). As
anticipated, Zn(II) can displace from the new probes all
the metal ions tested except for Ni(II) and Cu(II), in
agreement with the selectivity shown by QZ2, confirm-
ing that modification of the quinoline ring does not alter
metal-binding selectivity.
2
F ¼ f½QZ2Eꢁ ðI þ I K ½ZnðIIÞꢁ þ I K K ½ZnðIIÞꢁ
t
1
2
a1
3
a1 a2
3
þ I4Ka1Ka2Ka3½ZnðIIÞꢁ Þg=f1 þ Ka1½ZnðIIÞꢁ
þ Ka1Ka2½ZnðIIÞꢁ þ Ka1Ka2Ka3½ZnðIIÞꢁ g ð7Þ
because a similar ligand diacid such as one in the Cu(II)-
binding NO probe FL2A binds a third metal cation.
Fitting the fluorescence data for QZ2A to eq 7 gave three
2
3
47
Zn(II) Detection in Live Cells. The ability of QZ2E to
detect Zn(II) in live cells was investigated using pyrithione as
an ionophore to load the metal ion into cells. HeLa cells were
incubated with 5 μM QZ2E for 18 h (Figure 5); 4.5 μM
Hoechst 33258 was added 30 min prior to imaging to stain
the cell nuclei (Figure 5b). Very little background fluores-
cence was observed (Figure 5c) in the absence of Zn(II), but
upon addition of a physiologically relevant concentration of
values of K =220 ( 30 μM, K =160 ( 80 μM, and K =
d1
d2
d3
9
( 6 μM, all of which have a significant error, but the result
was much better than that afforded by a two-site model
Figure S3b in the SI). Most importantly, although Zn(II)
binding strength to the quinoline pockets cannot be distin-
(
guished from Zn(II) affinity for the diacids, all K values are
d
around the same order of magnitude as that of QZ2E (∼100
μM) and appropriate for reversible Zn(II) sensing at phy-
siological concentrations.
100 μM Zn/pyrithione (1:1), an immediate increase in fluores-
cence occurred that maximized within 5 min (Figure 5d). To
ensure that Zn(II) binding of QZ2E induced this fluores-
cence response, EDTA-(Et) , a cell-permeable variant of
Finally, because of the micromolar to millimolar affinity
of QZ2E and QZ2A for Zn(II), it was expected that, like
QZ2, their Zn(II)-induced fluorescence would be readily
4
-
15.6
48
EDTA, was incubated with the cells. For EDTA-(Et) -
4
reversed by the Zn(II) chelator TPEN (K =10
d
M).
When 100 μMZnCl wasaddedtoa1μM solution of QZ2E,
induced Zn(II) chelation, and therefore reduction in the
fluorescence signal, to occur, the ester moieties of the
chelator must be hydrolyzed intracellularly. Addition of
2
however, the resulting fluorescence was barely affected by
the addition of 100 μM TPEN (Figure 3a). When the same
-
16.4
48
M) as
150 μM EDTA-(Et) did not cause an immediate reduction
4
experiment was repeated using EDTA (K =10
d
of fluorescence, but after an ∼5 min incubation period, the
fluorescence in the green channel was reduced nearly to
background levels (Figure 5e). This result demonstrates that
the observed fluorescence is Zn(II)-dependent. A longer
the Zn(II) chelator, the fluorescence was diminished to near-
baseline levels (Figure 3b). Repeating the experiment with
tripicolylamine (TPA) versus diethylenetriaminepentaacetic
-
18.7
48
acid (DTPA; K =10
d
M) gave similar results (data not
incubation period of EDTA-(Et) would presumably lead
4
shown). It is unlikely that TPEN and TPA are incapable of
chelating Zn(II), given their superior affinity for the metal
ion over QZ2E. The basic, pyridyl-based chelators seemingly
interact with QZ2E to elicit a fluorescence response by a
different, unknown mechanism. Because esterification of the
quinoline rings of QZ2E presumably bends the quinolines
out of plane with respect to the fluorescein to avoid a steric
to complete hydrolysis of the chelator and therefore com-
plete reduction of the fluorescence signal in the green
channel; however, both the chelator and the ethanol bypro-
duct of hydrolysis are toxic to cells, and longer EDTA-(Et)4
incubation periods resulted in cell death.
Cell Trappability of QZ2E. To demonstrate trappability
of the probe, HeLa cells were incubated with 5 μM
QZ2E for ∼18 h to ensure sufficient time for hydrolysis
of the ester moieties to produce the cell-impermeable acids
(Figure 6). Addition of 4.5 μM Hoechst 33258 30 min
(
47) McQuade, L. E.; Ma, J.; Lowe, G.; Ghatpande, A.; Gelperin, A.;
Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 8525–8530.
48) Paoletti, P.; Ascher, P.; Neyton, J. J. Neurosci. 1997, 17, 5711–5725.
(

9
542 Inorganic Chemistry, Vol. 49, No. 20, 2010
McQuade and Lippard
Figure 3. Normalized fluorescence response of(a) 1 μM QZ2E to100 μM TPENafter fluorescence is elicited by additionof100 μM ZnCl
to 100 μM EDTA after fluorescence is elicited by addition of 100 μM ZnCl , (c) 1 μM QZ2A to 100 μM TPEN after fluorescence is elicited by addition of
00 μM ZnCl , and (d) 1 μM QZ2A to 100 μM EDTA after fluorescence is elicited by addition of 100 μM ZnCl
2
, (b) 1 μM QZ2E
2
1
2
2
.
prior to imaging was used to stain the cell nuclei
Figure 6b). Addition of 100 μM Zn(II)/pyrithione (1:1)
washing the cells and imaging, no fluorescent signal was
present before or after Zn(II)/pyrithione (1:1) addition
(Figure S4 in the SI). This result is consistent with the
hypothesis that the negative charges of the deprotonated
carboxylates would prevent QZ2A from entering cells.
QZ2E Localization in HeLa Cells. Like many other
fluorescein-based probes, QZ2E shows localization pat-
(
resulted in a change in fluorescence from background
levels (Figure 6c) to bright green (Figure 6d) within 5 min.
During this time and throughout the experiment, cells
were maintained in a microscope incubator at 37 °C under
5
% CO to preserve cell viability. Over the course of 45
2
2
7,47
min, the cells were periodically washed to mimic condi-
tions of media perfusion. The serum-free DMEM bath
was carefully removed by pipet while the plate remained
on the microscope, and then fresh serum-free DMEM
was added. The serum-free DMEM was removed and
replaced three times to ensure good washing of the cells
and then removed permanently and replaced with 2 mL of
fresh serum-free DMEM. Imaging every 10 min over the
course of the experiment (Figure 6e-h) revealed that,
under these conditions, QZ2E is retained in HeLa cells.
As a further demonstration of cell trappability, HeLa
cells were loaded with 5 μM QZ2A for ∼18 h with 4.5 μM
Hoechst 33258 applied 30 min prior to imaging. Upon
terns in live cells.
Punctate staining was observed with
clustering in the region surrounding the nuclei, suggesting
that QZ2E subcellularly localizes in HeLa cells. To test this
hypothesis and determine the organelle of localization,
HeLa cells were first coincubated with QZ2E (5 μM),
Hoechst 33258 (4.5 μM, nuclear stain), and Mitotracker
Red (200 nM, mitochondrial stain), because a probe with
a similar structure, Cu (FL2E), localizes to mitochondria
2
4
7
in SK-N-SH cells. Zn(II)/pyrithione (100 μM, 1:1) was
added because the green fluorescence could not be visual-
ized adequately without inducing fluorescence enhance-
ment. The results are illustrated in Figure 7a-e. QZ2E
does not enter the nucleus (Figure 7b,c), as expected,

Article
Inorganic Chemistry, Vol. 49, No. 20, 2010 9543
Figure 4. Selectivity of (a) QZ2E and (b) QZ2A for Zn(II) compared to Na(I), Ca(II), Mg(II), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Cd(II), and Hg(II).
Normalized fluorescence response relative to the emission of QZ2E or QZ2A in 50 mM PIPES, 100 mM KCl, pH 7.0, T = 25 °C.
Figure 5. HeLa cells coincubated with 5 μM QZ2E and 4.5 μM Hoechst 33258 for 18 h. (a) DIC image, (b) emission from the blue channel (nuclear stain),
(c) emission from the green channel (Zn(II) probe), (d) emission from the green channel after addition of 100 μM Zn/pyrithione (1:1), and (e) emission from
the green channel after addition of 150 μM EDTA-(Et) . Scale bars 25 μm.
4
Figure 6. Cell imaging experiments demonstrating retention of QZ2E in HeLa cells coincubated with 5 μM QZ2E and 4.5 μM Hoechst 33258 for 18 h. (a)
DIC image, (b) emission from the blue channel (nuclear stain), (c) emission from the green channel (Zn(II) probe), and (d) emission from the green channel
after addition of 100 μM Zn/pyrithione (1:1) at 5 min, (e) 15 min, (f) 25 min, (g) 35 min, and (h) 45 min. Scale bars 25 μm.
because the negatively charged acids produced upon
esterase cleavage of the esters should block passage
across the nuclear membrane. An overlay of the green
probe fluorescence (Figure 7c) and the red mitochondrial
stain fluorescence (Figure 7d) indicates very little overlap
(Figure 7e). Most of the red fluorescence persists in the

9
544 Inorganic Chemistry, Vol. 49, No. 20, 2010
McQuade and Lippard
Figure 7. HeLa cells coincubated with 5 μM QZ2E, 4.5 μM Hoechst 33258, and 200 nM Mitotracker red for 18 h. (a) DIC image, (b) emission from the
blue channel (nuclear stain), (c) emission from the green channel (Zn(II) probe) after addition of 100 μM Zn/pyrithione (1:1), (d) emission from the red
channel (mitochondrial stain), and (e) overlay of the green and red channels. Scale bars 25 μm.
Figure 8. HeLacells coincubatedwith5 μM QZ2E, 4.5 μM Hoechst 33258, and 100nMBODIPYTRCeramide for 18h. (a) DIC image, (b) emissionfrom
the blue channel (nuclear stain), (c) emission from the green channel (Zn(II) probe) after addition of 100 μM Zn/pyrithione (1:1), (d) emission from the red
channel (Golgi aparatus stain), and (e) overlay of the green and red channels. Scale bars 25 μm.
overlay except for a small region of yellow immediately
surrounding the nucleus. These data are inconsistent with
QZ2E localization to mitochondria.
QZ2E in DMSO. Incubation of HeLa cells with 50 μM
QZ2E requires a 5% loading of DMSO, which is itself
toxic, and therefore cell survival at this concentration
over 24 h was only ∼30%. A control incubation with 5%
DMSO and no QZ2E showed similar results (∼20% cell
survival over 24 h), confirming that the addition of
DMSO associated with QZ2E application is a limiting
factor in QZ2E cytotoxicity.
The Golgi apparatus is another subcellular compart-
2
7
ment to which fluorescein-based probes can localize and
is located in close proximity to the nucleus. Therefore, the
localization experiments were repeated by incubating HeLa
cells with QZ2E (5 μM), Hoechst 33258 (4.5 μM, nuclear
stain), and BODIPY TR Ceramide (100 nM, Golgi appa-
ratus stain) and then inducing adequate green fluores-
cence for visualization with Zn(II)/pyrithione (100 μM,
Summary
The synthesis and spectroscopic characterization of the cell-
trappable, fluorescein-based Zn(II) probe QZ2E and its cell-
impermeable counterpart, QZ2A, are described. Modifications
of the quinoline ring of QZ2E and QZ2A do not drastically
alter the Zn(II) binding capacities of the probes when com-
1
:1; Figure 8a-e). In contrast to the results of the mitochon-
drial staining experiment, an overlay of the green probe
fluorescence (Figure 8c) and the red Golgi stain fluorescence
(Figure 8d) revealed excellent colocalization (Figure 8e).
Essentially all of the red fluorescence observed in Figure 8d
appeared yellow when overlaid with the green QZ2E fluo-
rescence, indicating that QZ2E localizes subcellularly to the
Golgi apparatus in HeLa cells. The small overlap of green
and red fluorescence observed in Figure 7e is probably due to
mitochondrial and Golgi overlap along the z axis of the cell
and not true localization of QZ2E to mitochondria.
pared to the first-generation probe, QZ2 (K (QZ2) = 41 (
d1
3
μM, K (QZ2E) = 3.5 ( 0.1 mM, K (QZ2E) = 150 (
d1
d2
1
00 μM, K (QZ2A)=220 ( 30 μM, K (QZ2A)=160 ( 80
d1
d2
μM, K (QZ2A) = 9 (6 μM), nor are their emissive properties
d3
altered before and after ZnCl addition to buffered solutions
2
(
0
φ(QZ2) = 0.5%, φ(QZ2 þ Zn(II))=70%; φ(QZ2E)=0.4 (
.1%, φ(QZ2E þ Zn(II))=73 ( 3%; φ(QZ2A)=1.2 ( 0.1%,
Cytotoxicity of QZ2E. Over the course of the cell imag-
ing experiments, there was no visual cell death caused by
QZ2E at the concentration used (5 μM); however, the
experiments were performed at low probe concentration.
To investigate the potential cytotoxic effects of QZ2E at
higher concentrations, which may be needed for certain
φ(QZ2A þ Zn(II))=51 ( 1%). QZ2E has a slightly reduced
dynamic range with respect to QZ2, ∼120-fold enhancement
versus ∼150-fold enhancement, respectively, but maintains
selectivity for Zn(II) over other biologically relevant cations,
toxic heavy metals, and most first-row transition-metal ions.
The dynamic range of QZ2A is more significantly reduced, to
4
7
biological applications such as in tissue slices, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
∼
30-fold enhancement, but this value is more than adequate
4
MTT) assays were performed over a 24 h period. The
9
for in vivo visualization. QZ2E is a competent Zn(II) sensor in
live HeLa cells and is retained intracellularly despite numerous
washings. Unlike QZ2, QZ2E localizes to the Golgi apparatus
in HeLa cells, a difference that may reflect its greater negative
charge following intracellular hydrolysis of its ester moieties to
become QZ2A.
(
results indicate that QZ2E is minimally cytotoxic to HeLa
cells at concentrations under 25 μM, with ∼85% cell sur-
vival over 24 h for 1 μM probe loading and ∼70% cell
survival over 24 h for 10 μM QZ2E. At higher concentra-
tions, the probe becomes more toxic. For example, at
2
The cytotoxicity assays are limited by the insolubility of
5 μM QZ2E the cell survival over 24 h drops to ∼50%.
Acknowledgment. This work was supported by Grant
CHE-0907905 to S.J.L. from the National Science Foun-
dation (NSF). Spectroscopic instrumentation at the MIT
(
49) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.

Article
Inorganic Chemistry, Vol. 49, No. 20, 2010 9545
Department of Chemistry Instrument Facility is main-
tained with funding from NSF Grants DBI-9729592 and
CHE-9808061. We thank Drs. Elisa Tomat and Daniela
Bucella for insightful discussions.
normalized fluorescence response of QZ2E in the presence
of excess Zn(II) fit to eqs 1 and 6, fits with residuals of
normalized fluorescence response of QZ2A in the presence of
excess Zn(II) fit to eqs 1 and 7, QZ2A detection of Zn(II)/
pyrithione (1:1) in HeLa cells, and full list of authors for ref 16.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Supporting Information Available: Absorbance spectra for
Zn(II) binding of QZ2E and QZ2A, fits with residuals of