Zinspy sensors with enhanced dynamic range for imaging neuronal cell zinc uptake and mobilization

Article abstract of DOI:10.1021/ja065759a

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

Full text of DOI:10.1021/ja065759a

Published on Web 11/08/2006
Zinspy Sensors with Enhanced Dynamic Range for Imaging
Neuronal Cell Zinc Uptake and Mobilization
Elizabeth M. Nolan,† Jubin W. Ryu,§ Jacek Jaworski,§,|,# Rodney P. Feazell,†
,|
Morgan Sheng,§, and Stephen J. Lippard*,†
Contribution from the Department of Chemistry, Picower Institute of Learning and Memory,
RIKENsMIT Neuroscience Research Center, and Howard Hughes Medical Institute,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Laboratory of
Molecular and Cellular Neurobiology, International Institute of Molecular and Cell Biology,
02-109 Warsaw, Poland
Received August 9, 2006; E-mail: lippard@mit.edu
Abstract: 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.
methyl-D-aspartate receptors,7-10 one major type of glutamater-
gic ion channel in the mammalian central nervous system, and
Introduction
The neurobiology of zinc has attracted significant attention.1,2
Zinc is the second most abundant d-block metal ion in the
human brain, with relatively high concentrations found in the
hippocampus and neocortex.3 Of particular interest to this work
is zinc found in the hippocampus, a region of the mammalian
forebrain involved in learning and memory formation. This zinc
can be divided into two types,4 protein bound and loosely bound,
the latter also termed histochemically observable, chelatable,
or mobile.5 Hippocampal loosely bound zinc is housed in the
synaptic vesicles of glutamatergic mossy fiber neurons. Co-
localization of zinc and glutamate in this subset of synaptic
vesicles was assumed for decades, but proof of such mutual
uptake was provided only recently.6
inhibition of Ca(II) channels.11 Zinc has also been implicated
in long-term potentiation (LTP), an electrophysiological phe-
nomenon thought to underlie certain forms of learning and
memory, in DG f CA3 synapses.12 Despite these observations,
the concept of vesicular zinc release into the synapse remains
controversial.13-19
Zinc pathology is also an area of current interest. High
concentrations of Zn(II) are neurotoxic. A number of zinc
transporter proteins20,21 and metallothionein22,23 maintain zinc
homeostasis under normal physiological conditions, but these
(7) Peters, S.; Koh, J.; Choi, D. W. Science 1987, 236, 589-593.
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Many studies point to the importance of Zn(II) in human
neurophysiology. Multiple neuromodulatory effects for Zn(II)
have been documented and include down-regulation of N-
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† Department of Chemistry, MIT.
§ Picower Institute of Learning and Memory, MIT.
RIKEN-MIT Neuroscience Research Center.
| Howard Hughes Medical Institute.
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# International Institute of Molecular and Cell Biology, Poland.
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J. AM. CHEM. SOC. 2006, 128, 15517-15528
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A R T I C L E S
Nolan et al.
mechanisms can fail during periods of stress. Uncontrolled zinc
release occurs from protein-bound and vesicular stores following
blunt head trauma and as a result of stroke or seizure, and it is
implicated in subsequent neurodegeneration.24-29 Evidence
exists for a role of Zn(II) in the onset and pathogenesis of
Alzheimer’s disease.30 There are several mechanisms to explain
Zn(II) neurotoxicity, including rapid influx into mitochondria,
which triggers apoptosis following uncontrolled release.28,31 Of
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growing link between Zn(II) toxicity and oxidative stress.27,31,32
Despite this knowledge, many details regarding the functional
significance of Zn(II) in neurobiology remain elusive. This
situation has motivated the design of many new Zn(II) imaging
tools suitable for studies in living cells. Initially, aryl sulfon-
amide-based sensors, including N-(6-methoxy-8-quinolyl)-p-
toluenesulfonamide (TSQ) and its derivatives, were employed
to visualize chelatable Zn(II) in the mammalian hippocampus
and in live cells.33-36 Over the past six years, in part guided by
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cal Zn(II) sensor design have been made.2,38-41 Many sensors,
including the ACF,42 ZnAF,43,44 Fluo- and Rhod-Zn,45,46
Zinpyr,47-49 and QZ50 probes, provide visible excitation and
bright fluorescence in the Zn(II)-bound forms. Some of these
Figure 1. Symmetrical and tertiary-amine-based Zinpyr (ZP) and Zinspy
(ZS) sensors.
sensors have been employed in studies of Zn(II) neurobiol-
ogy.17,44,49,50 Small-molecule ratiometric Zn(II) sensors have
been documented,46,51-55 several of which have been applied
in vivo.53,54 Protein-based56-59 approaches have also emerged
as useful Zn(II) sensing tools, and several peptide-based60-62
strategies have been reported. Nevertheless, the design and
implementation of new Zn(II) imaging reagents is required to
continue advances in this field. Several recent initiatives44,50,63
address the need for Zn(II) sensors with varying affinities for
use in vivo, since estimated endogenous Zn(II) concentrations
vary widely, depending on the tissue or cell type under study.
We previously reported the Zinpyr (ZP)47-49 and Zinspy
(ZS)64 families of Zn(II) sensors, several of which are depicted
in Figure 1. The thioether-for-pyridyl substitution that gives the
ZS sensors was made with the aims of lowering Zn(II) affinity
and potentially modulating metal-ion selectivity relative to the
parent ZP compounds, which employ the di(2-picolyl)amine
(DPA) chelate. Although the ZS probes exhibit improved Zn(II)
selectivity and lower affinity Zn(II) binding as compared to their
DPA-based counterparts, they generally suffer from relatively
poor photophysical properties, including high background
fluorescence and weak (e2-fold) fluorescence enhancement with
Zn(II) coordination, at least for the tertiary amine-based systems.
These features preclude facile application of these sensors for
biological imaging of Zn(II).
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In the present article, we describe Zinspy sensors with
enhanced dynamic range. On the basis of several observations
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Zinspy Sensors for Imaging Zn(II) in Neurobiology
A R T I C L E S
Scheme 1
300 or 500 MHz spectrophotometer operating at ambient probe
temperature, 283 K. The spectra were referenced to internal standards.
An Avatar FTIR instrument was used to obtain IR spectra, and the
data are included as Supporting Information. High-resolution mass
spectra were collected by staff at the MIT Department of Chemistry
Instrumentation Facility.
from our laboratory and a recent literature report that indicates
groups distant from a fluorophore contribute to PET quench-
ing,65 we reasoned that incorporating multiple aromatic hetero-
cycles in a tertiary amine-based ligand is important for achieving
relatively low background fluorescence and good Zn(II)-induced
fluorescence turn-on, at least for symmetrical probes such as
ZP1 and ZS1. Substitution of the thioether moiety in ZS1 or
ZS2 with a thiophene group is one way to accomplish this task.
Moreover, because thiophenes are less basic than pyridyl groups
and thioethers, we anticipated that the thiophene moiety would
be non-coordinating. The resulting N2O donor set for Zn(II)
binding would confer lower affinity Zn(II) coordination than
the N3O donor sets of the symmetrical ZP systems. In accord
with these expectations, the thiophene-for-thioether/pyridine
subsitution affords low-micromolar Zn(II) Kd values, improved
Zn(II) selectivity, and fluorescence enhancements suitable for
biological imaging. We describe extensive confocal microscopic
studies of ZS5 (Scheme 1) in cultured cells and in primary
cultures of neurons. ZS5 illuminates the mitochondria in HeLa
cells, affords a visual image of glutamate-dependent Zn(II)
uptake in dendrites of hippocampal neurons, and detects
endogenous Zn(II) release in dentate gyrus neurons resulting
from nitrosative stress. These findings indicate that ZS5 is a
versatile Zn(II) imaging tool with great potential for biological
use.
2-{6-Hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethylthiophen-2-yl-
methylamino)methyl]-3H-xanthen-9-yl}benzoic Acid (3, Zinspy-5,
ZS5). A portion of fluorescein 2 (50 mg, 0.13 mmol) was dissolved in
5 mL of DCE, and 30 µL of HOAc was added. Compound 1 (77 mg,
0.38 mmol) was dissolved in 5 mL of DCE and added to the fluorescein
solution in a dropwise manner. The cloudy and orange reaction mixture
was stirred for 1 h at room temperature. A portion of NaB(OAc)3H
(70 mg, 0.33 mmol) was added, and the solution clarified. The reaction
was stirred overnight at room temperature and became orange-brown.
The solution was diluted with 15 mL of CHCl3 and washed with water
(3 × 15 mL). The organic portion was dried over MgSO4, and the
solvent was removed in vacuo, which gave an orange solid. Preparative
TLC on silica gel (20:1 CHCl3/MeOH) gave the product as an orange-
red powder (37 mg, 38%); mp > 280 °C, dec. TLC: Rf ) 0.59 (silica,
1
9:1 CHCl3/MeOH). H NMR (CD3OD, 300 MHz): δ 3.90 (4H, q),
4.06 (4H, m), 4.15 (4H, m), 6.59 (2H, d), 6.90 (4H, m), 7.05 (2H, m),
7.12 (1H, d), 7.17 (2H, t), 7.25 (2H, d), 7.56 (4H, m), 7.65 (2H, t),
8.00 (1H, d), 8.35 (2H, d). HRMS (ESI): calcd [M + Na]+, 787.2019;
found, 787.2024.
2-{2,7-Dichloro-6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethyl-
thiophen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}benzoic Acid
(4, Zinspy-6, ZS6). Portions of 1 (200 mg, 0.979 mmol) and (CH2O)n
(50 mg, 1.7 mmol) were combined in 5 mL of MeCN and heated to
reflux for 45 min. A slurry of 2′,7′-dichlorofluorescein (131 mg, 0.327
mmol) in 7 mL of 1:1 MeCN/H2O was added to the reaction, and the
solution turned pink. The reaction was heated to reflux for 22 h, during
which time a pink precipitate formed, and cooled. The mixture was
filtered, and the precipitate was washed with 15 mL of Et2O. The pure
product was obtained as a light pink powder after recrystallization twice
from boiling EtOH (223 mg, 85%); mp ) 200-201 °C. 1H NMR
(DMSO-d6, 300 MHz): δ 3.96 (8H, m), 4.08 (4H, s), 6.56 (2H, s),
6.96 (4H, m), 7.33-7.43 (8H, m), 7.74-7.87 (5H, m), 7.99 (1H, d),
8.59 (2H, d). HRMS (ESI): calcd [M - H]-, 831.1264; found,
831.1265.
Experimental Section
Reagents. Acetonitrile was dried using columns of aluminum oxide,
and anhydrous 1,2-dichloroethane (DCE) was purchased from Aldrich.
Methanol was filtered through an aluminum oxide plug before use.
Ligand 166 and 4′,5′-fluorescein dicarboxaldehyde (2)47 were prepared
as previously described. 2′,7′-Difluorofluorescein was purchased from
Invitrogen and 2′,7′-dichlorofluorescein from Aldrich. The latter was
recrystallized from boiling EtOH before use. All other halogenated
fluorescein derivatives were prepared by acid-catalyzed condensation
of the appropriate resorcinol and substituted phthalic anhydride.
Additional reagents were obtained from Aldrich and used as received.
Materials and Methods. Merck F254 silica gel plates were used
for analytical TLC and were visualized with UV light. Preparative TLC
was performed on Whatman silica gel-60 plates of 1 mm thickness,
manufactured by EM Science. NMR spectra were acquired on a Varian
2-{2,7-Difluoro-6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethyl-
thiophen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}benzoic Acid
(5, Zinspy-7, ZS7). Portions of 1 (83 mg, 0.41 mmol) and (CH2O)n
(13 mg, 0.45 mmol) were combined in 5 mL of MeCN and heated to
reflux for 1 h. A slurry of 2′,7′-difluorofluorescein (50 mg, 0.14 mmol)
in 10 mL of 1:1 MeCN/H2O was added to the reaction, and the solution
turned orange. The reaction was heated to reflux for 22 h, and a light
(65) Sparano, B. A.; Shahi, S. P.; Koide, K. Org. Lett. 2004, 6, 1947-1949.
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Stephenson, K. A.; Babich, J. W.; Maresca, K. P.; Zubieta, J. Inorg. Chem.
2002, 41, 6417-6425.
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Nolan et al.
pink precipitate formed. The reaction was cooled to room temperature
and filtered, and the precipitate was recrystallized from boiling EtOH
twice (71 mg, 60%); mp ) 182-183 °C. H NMR (DMSO-d6 with
NaOD, 300 MHz): δ 3.74 (4H, s), 3.91 (8H, m), 6.26 (2H, d), 6.88-
7.00 (5H, m), 7.13 (2H, m), 7.31 (2H, d), 7.44 (2H, qd), 7.64 (4H, m),
7.98 (1H, dd), 8.36 (2H, d). HRMS (ESI): calcd [M - H]-, 799.1855;
found, 799.1866.
direct methods using the SAINTPLUS70 and SHELXTL71 software
packages. The structures were checked for higher symmetry by using
the PLATON software.72 All non-hydrogen atoms were located and
their positions refined with anisotropic thermal parameters by least-
squares cycles and Fourier syntheses. Unless otherwise noted, hydrogen
atoms were assigned to idealized positions and given thermal parameters
equivalent to either 1.5 (methyl hydrogen atoms) or 1.2 (all other
hydrogen atoms) times the thermal parameter of the carbon atom to
which they were attached.
1
2-{2,7-Dichloro-6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethylthio-
phen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}-3,4,5,6-tetrachloro-
benzoic Acid (6, Zinspy-Cl6, ZSCl6). Portions of 1 (200 mg, 0.979
mmol) and (CH2O)n (48 mg, 1.7 mmol) were combined in 5 mL of
MeCN and heated to reflux for 1 h. A slurry of 2′,3,4,5,6,7′-
hexachlorofluorescein in 10 mL of 1:1 MeCN/H2O was added to the
reaction, and the solution turned deep magenta. The reaction was heated
to reflux for 22 h (during which time a pink precipitate formed), cooled,
and filtered. The precipitate was washed with 10 mL of MeCN and
recrystallized twice from boiling EtOH, which afforded a light pink
powder (255 mg, 80%); mp ) 216-217 °C. 1H NMR (DMSO-d6, 300
MHz): δ 3.95 (8H, s), 4.05 (4H, s), 6.98 (4H, s), 7.26 (2H, s), 7.42
(6H, m), 7.86 (2H, m), 8.63 (2H, m). HRMS (ESI): calcd [M -H]-,
966.9705; found, 966.9705.
The structure of ZSF6 contains a dichloromethane molecule that was
modeled as disordered over four positions (10:50:15:25). The sulfur
and C3 carbon atoms of each thiophene group were modeled as
disordered over two positions (50:50). The angles and the 1-2 distances
around the sulfur and C3 carbon atoms were constrained to be the same
for both components of the disordered thiophene rings. The O1 and
O3 hydrogen atoms were located from the electron density map.
General Spectroscopic Methods. Aqueous solutions were prepared
with Millipore water. Puratonic grade KCl was purchased from
Calbiochem and molecular biology grade piperazine-N,N′-bis(2-ethane-
sulfonic acid) (PIPES) from Sigma. With the exception of the pKa
titrations, measurements were generally made at pH 7 in 50 mM PIPES,
100 mM KCl buffer. A buffer containing 50 mM CHES, 100 mM KCl
was employed for measurements at pH 9. Parallel experiments in buffer,
rigorously treated or not treated with Chelex resin (Bio-Rad, manu-
facturer protocol) to remove any potentially adventitious metal ions,
returned the same dynamic range and Kd values during trials that
employed ZS5 or ZS7. Untreated buffer was used for all subsequent
experiments. Excess EDTA was added to solutions of apo-ZS prior to
quantum yield and extinction coefficient determinations. Zinc stock
solutions (100 mM, 500 mM) were prepared from anhydrous 99.999%
ZnCl2 (Aldrich) and water. DMSO stock solutions (1 mM) of ZS sensors
were prepared, partitioned into ∼300-µL aliquots, stored at 4 or -25
°C, and thawed in the dark before use. A starting solution of 10 mM
KOH, 100 mM KCl, pH ∼12, was used for pKa titrations, and the pH
value of the solution was lowered by addition of 6, 2, 1, 0.5, or 0.1 N
HCl. Quantum yields were measured by using fluorescein in 0.1 N
NaOH (Φ ) 0.95) as the standard,73 as described previously.47
Excitation was provided at 490 (Zn(II)-bound ZS5 and ZS7), 492 (apo-
ZS5), 496 (apo-ZS7 and Zn(II)-bound ZS6), 499 (apo-ZS6), 500 (Zn(II)-
bound ZSF7), 503 (apo-ZSF7), 504 (Zn(II)-bound ZSF6), or 505 nm
(apo-ZSF6). Extinction coefficients were generally measured over a
concentration range of 1-10 µM for the metal-free and Zn(II)-bound
species. Apo-ZS5 was an exception, which gives an uncharacteristically
low (∼12 000 M-1 cm-1) extinction coefficient for symmetrical
fluorescein-based sensors such as ZP247 and QZ250 in the 3-10 µM
range under these experimental conditions. Its extinction coefficient
was therefore determined over a concentration range of 0-3 µM, which
returned an expected value (>30 000 M-1 cm-1). For Zn(II) selectivity
experiments, ZP147 was used as a control for the Fe(II)/Zn(II) studies.
Sensors ZS6 and ZSCl6 were not (ZSCl6) or only partially (ZS6)
characterized. These probes both suffer from low solubility in most
solvents, including DMSO, and poor dynamic range (∼2-fold or less),
and thus show little promise for biological imaging applications.
Experimental details for all spectroscopic measurements performed in
this study are available in the literature.47,74 All spectroscopic measure-
ments were repeated a minimum of three times using material from at
least two separate synthetic preparations, and the resulting averages
are reported.
2-{2,7-Dichloro-6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethylthio-
phen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}-3,4,5,6-tetrafluoro-
benzoic Acid (7, Zinspy-F6, ZSF6). Portions of 1 (200 mg, 0.971
mmol) and (CH2O)n (48 mg, 1.7 mmol) were combined in 5 mL of
MeCN and heated to reflux for 1 h. A slurry of 3,4,5,6-tetrafluoro-
2′,7′-dichlorofluorescein in 10 mL of 1:1 MeCN/H2O was added to
the reaction, and the solution turned dark pink. The reaction was
refluxed for 22 h, and a pink precipitate formed. The mixture was
filtered, and the pink precipitate was washed with 10 mL of H2O and
recrystallized twice from boiling EtOH (208 mg, 68%); mp > 250 °C,
1
dec. H NMR (DMSO-d6, 300 MHz): δ 3.94 (8H, s), 4.04 (4H, s),
6.95 (4H, m), 7.23 (2H, s), 7.41 (6H, m), 7.83 (2H, t), 8.59 (2H, d).
HRMS (ESI): calcd [M - H]-, 903.0887; found, 903.0893. Colorless
block crystals suitable for X-ray crystallography were grown at room
temperature by vapor diffusion of Et2O into a solution of ZSF6 in
CH2Cl2.
2-{2,7-Difluoro-6-hydroxy-3-oxo-4,5-bis[(pyridin-2-ylmethylthio-
phen-2-ylmethylamino)methyl]-3H-xanthen-9-yl}-3,4,5,6-tetrafluoro-
benzoic Acid (8, Zinspy-F7, ZSF7). Portions of 1 (207 mg, 1.01 mmol)
and (CH2O)n (50 mg, 1.7 mmol) were combined in 6 mL of MeCN
and heated to reflux for 30 min. A slurry of 2′,3,4,5,6,7′-hexafluoro-
fluorescein (148 mg, 0.336 mmol) in 10 mL of 1:1 MeCN/H2O was
added to the reaction, and the solution turned deep pink-red. The
reaction was heated to reflux for 24 h (during which time a pink
precipitate formed), cooled, and filtered. The precipitate was recrystal-
lized twice from boiling EtOH, which gave the product as a pink-orange
powder (185 mg, 63%); mp > 250 °C, dec. 1H NMR (DMSO-d6 with
NaOD, 300 MHz): δ 3.78 (4H, s), 3.93 (8H, s), 6.66 (2H, m), 6.89
(4H, m), 7.14 (2H, m), 7.33 (2H, d), 7.51 (2H, m), 7.61 (2H, m), 8.37
(2H, m). HRMS (ESI): calcd [M - H]-, 871.1478; found, 871.1492.
X-ray Crystallographic Studies. Single crystals were mounted on
the tips of glass fibers coated with paratone-N oil and cooled to -100
°C under a stream of N2, maintained by a KRYO-FLEX low-
temperature apparatus. Intensity data were collected on a Bruker APEX
CCD diffractometer with graphite-monochromated Mo KR radiation
(λ ) 0.71073 Å), controlled by a Pentium-based PC running the
SMART software package.67 Data collection and reduction protocols
are described elsewhere.68 Empirical absorption corrections were applied
by using the SADABS69 program, and the structures were solved by
(70) SAINTPLUS: Software for the CCD Detector System, version 5.01; Bruker
AXS: Madison, WI, 1998.
(71) SHELXTL: Program Library for Structure Solution and Molecular
Graphics, version 6.1; Bruker AXS: Madison, WI, 2001.
(72) Spek, A. L. PLATON, A Multipurpose Crystallographic Tool; Utrecht
University: Utrecht, The Netherlands, 2000.
(73) Brannon, J. H.; Madge, D. J. Phys. Chem. 1978, 82, 705-709.
(74) Nolan, E. M.; Burdette, S. C.; Harvey, J. H.; Hilderbrand, S. A.; Lippard,
S. J. Inorg. Chem. 2004, 43, 2624-2635.
(67) SMART: Software for the CCD Detector System, version 5.626; Bruker
AXS: Madison, WI, 2000.
(68) Kuzelka, J.; Mukhopadhyay, S.; Spingler, B.; Lippard, S. J. Inorg. Chem.
2004, 43, 1751-1761.
(69) Sheldrick, G. M. SADABS: Area-Detector Absorption Correction; Uni-
versity of Gottingen: Gottingen, Germany, 2001.
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Zinspy Sensors for Imaging Zn(II) in Neurobiology
A R T I C L E S
Fluorescence spectra were recorded on a Hitachi F-3010 spectro-
fluorimeter and UV-vis spectra on a Cary IE scanning spectropho-
tometer, both controlled by manufacturer-supplied software packages.
Circulating water baths were used during all acquisitions to maintain
the temperature at 25 ( 1 °C. Quartz cells (Starna) with a 1-cm path
length were used for all measurements.
Dissociation Constant Determination. The apparent zinc complex
dissociation constants for select ZS probes were determined by
fluorimetric analysis. In a typical experiment, a 1 µM solution of the
ZS compound was prepared (50 mM PIPES, 100 mM KCl, pH 7) and
its emission spectrum recorded. Various aliquots of 1 or 10 mM aqueous
ZnCl2 solutions were added, and the emission change was noted. The
data were integrated, normalized with respect to the full response, and
plotted against the total concentration of Zn(II) in solution. The data
were fit to a 1:1 metal-ligand binding model.75
ZS5 or ZP3 and were generally incubated with the reagents for 30 min
(37 °C, 5% CO2) before imaging.
A pGolgi-DsRed construct was employed to label the Golgi
apparatus. This plasmid expresses a fusion protein consisting of DsRed
and a sequence encoding the N-terminal 81 amino acids of human â-1,4-
galactosyltransferase (Clonetech). The latter sequence includes a
membrane-anchoring signal peptide, which directs the fusion protein
to the trans-Golgi region. Overexpression of pGolgi-DsRed results in
its presence in other compartments of the secretory pathway. HeLa
cells were passed and plated onto untreated 10-mm glass coverslips
36 h before transfection. The cells were transiently transfected with
pGolgi-DsRed using the Lipofectamine reagent (manufacturer protocol)
24-36 h prior to imaging.
Preparation of Embryonic Hippocampal Neuronal Cultures.
Hippocampal primary cultures were prepared from day 19 (E19)
Sprague-Dawley rat embryos according to a previously reported
protocol.77 Medium-density cultures (150 cells/mm2) were plated on
24-mm glass coverslips coated with poly-D-lysine (30 µg/mL) and
laminin (2.5 µg/mL). Hippocampal cultures were grown in Neurobasal
media (Invitrogen) supplemented with B27 (Invitrogen), glutamine (0.5
mM), and glutamate (12.5 µM). After 16-20 days in culture, the
medium was removed and replaced with fresh Neurobasal containing
no B27. The cells were stained by bath application of the appropriate
dye to the media and incubated at 37 °C under 5% CO2. The ZS5 stock
solution (1 mM in DMSO) was diluted 10-fold with either Neurobasal
or PBS before addition. The Zn(II)/pyrithione mix has poor solubility
in Neurobasal, so these DMSO stock solutions were diluted with either
PBS or serum-free DMEM before addition. In some instances, the
neurons were transfected with 3.6 µg of â-actin dsRed DNA using
Lipofectamine 2000 (manufacturer protocol). The neurons were incu-
bated with the DNA/lipofectamine mix for 1 h (37 °C, 5% CO2) on
DIV 16, washed with and bathed in the growth medium, and imaged
on DIV 19 or 20. Solutions of Zn(II)/glutamate (1:2 Zn(II)/glutamate
ratio) were prepared in PBS from aqueous solutions of ZnCl2 (10 mM)
and glutamate (10 mM) and used immediately. Experiments were
conducted in a minimum of four independently prepared cultures.
Preparation of Postnatal Dentate Gyrus Neuronal Cultures.
Primary cultures of dentate gyrus (DG) cells were prepared by
modification of a literature procedure.78 DG regions were dissected from
the hippocampi of 4-day-old Sprague Dawley rat pups. The cells were
plated on 24-mm glass coverslips (250 cells/mm3) coated with poly-
L-lysine (Sigma, 50 µg/mL). The cultures were maintained for the first
24 h in DMEM (Invitrogen) supplemented with 10% FCS (Invitrogen),
glutamine (2 mM), and penicillin-streptomycin mix (50 µg/mL).
Subsequently, the cells were kept in Neurobasal medium (Invitrogen)
supplemented with B27 (Invitrogen), glutamine (2 mM), potassium
chloride (20 mM), and penicillin-streptomycin mix (50 µg/mL). After
7 days in culture, the medium was removed and replaced with
Neurobasal containing no B27, and the cells were treated with ZS5
and Zn(II)/pyrithione as described above for the hippocampal neurons.
For experiments requiring SNOC, the reagent was prepared immediately
before use and added to the cells by application on the microscope
stage. Experiments were conducted with two independently prepared
cultures.
Stopped-Flow Kinetic Studies. Single-mixing stopped-flow kinetics
studies were performed with a Hi-Tech SF-61 DX2 double-mixing
stopped-flow apparatus equipped with a fluorescence detector. Excita-
tion was provided at 490 (ZS5, ZS7), 505 (ZS6), 510 (ZSF7), or 520
nm (ZSF6). A GG495 glass cutoff filter (<455 nm) was placed over
the exit of the photomultiplier tube, and the emission was monitored
from 455 to 700 nm. All solutions were prepared in 50 mM PIPES,
100 mM KCl, pH 7 buffer. Conditions for pseudo-first-order kinetics
were maintained by using at least a 10-fold excess of Zn(II) in all
experiments. Except for temperature-dependent studies, all experiments
were conducted at 4.3 ( 0.1 °C. The temperature inside the sample
chamber was monitored with an internal thermocouple. A series of
control experiments were conducted in which the initial ZS concentra-
tion was varied from 0.5 to 5 µM after mixing to ensure that the
observed rate constant was independent of initial dye concentration
(data not shown). Multiple shots were taken for each Zn(II) (or sensor)
concentration and averaged. Experiments were conducted three times
with fresh Zn(II), sensor, and buffer solutions, and the resulting averages
and standard deviations are reported. Observed rate constants were
obtained by fitting individual traces to monoexponentials. The Kinet-
Assyst software package (HiTech) was used to analyze the data. Further
experimental details are available elsewhere.50
Cell Culture. HeLa, COS-7, and HEK293-T cells were cultured in
Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) supplemented
with 10% fetal calf serum (FCS, Invitrogen), glutamine (2 mM),
penicillin (100 units/mL), and streptomycin (100 µg/mL). Two days
before imaging, the cells were passed and plated on untreated (HeLa,
COS-7) 10-mm glass coverslips or on glass coverslips coated with 0.2%
gelatin for at least 1 h at room temperature (HEK293-T). For labeling,
the growth medium was removed and replaced with DMEM containing
1% FCS. The cells were treated and incubated with 10 µM sensor at
37 °C under 5% CO2 for 30 min. The cells were washed once with
and bathed in DMEM containing no FCS prior to imaging and/or zinc
addition. Zinc was introduced to the cultured cells as the pyrithione
salt using a Zn(II)/pyrithione ratio of 1:1 or 1:2. Stock solutions of
ZnCl2 (10 mM) and sodium pyrithione (20 mM) in DMSO were
combined and diluted 10-fold with DMEM prior to addition. In a typical
experiment, a 100-µL aliquot of this solution was added to the cells
bathed in 2 mL of DMEM. A stock solution of TPEN (10 or 20 mM)
was also diluted 10-fold with DMEM containing no FCS prior to cell
treatment. S-Nitrosocysteine (SNOC, 10 mM) was prepared immediately
before use according to a literature procedure.76
Cytotoxicity Assays in HeLa Cells. To ascertain the cytotoxic effect
of sensor treatment over a 24-h period, HeLa cells were passed and
plated to ∼60% confluence in 12-well plates 12-16 h before treatment
and bathed in 1 mL of DMEM supplemented as described above. Prior
to ZP treatment, the DMEM was removed and replaced with fresh
DMEM, and aliquots of ZP1,47 ZP3,49 or ZS5 stock solutions (1 mM
DMSO) were added for final concentrations of 1, 5, and 10 µM. The
treated cells were incubated for 24 h at 37 °C and under 5% CO2.
Subsequently, the cells were treated with 5 mg/mL MTT (100 µL/
Lysotracker Red (Invitrogen) and Mitotracker Red (Invitrogen) were
employed for co-localization studies. In a typical experiment, a 1 mM
solution of either marker in DMSO was diluted 100- to 200-fold with
DMEM prior to cell treatment. The exact concentration of Lysotracker
or Mitotracker used varied with lot number and was in the range of
100-500 nM. The cells were co-treated with the organelle marker and
(75) Lim, M. H.; Xu, D.; Lippard, S. J. Nat. Chem. Biol. 2006, 2, 375-380.
(77) Brewer, G. J.; Toricelli, J. R.; Evege, E. K.; Price, P. J. J. Neurosci. Res.
1993, 35, 567-576.
(78) Figiel, I.; Kaczmarek, L. Neurochem. Int. 1997, 31, 299-240.
(76) Kro¨ncke, K.-D.; Kolb-Bachofen, V. Methods Enzymol. 1999, 301, 126-
135.
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A R T I C L E S
Nolan et al.
well) and incubated for an additional 2-4 h (37 °C, 5% CO2). The
DMEM was removed, the cells were dissolved in DMSO (500 µL/
well), and the absorbance at 550 nm was recorded. Three independent
trials were conducted, and the averages and standard deviations are
reported. The reported percent cell survival values are relative to
untreated control cells. Additional control studies indicated that the
DMSO added (1-10 µL) as a result of sensor treatment has no effect
on cell viability under these conditions. The HeLa cells used were at
P-10 to P-16, and the assays were repeated using a minimum of two
separate HeLa cell stocks.
Laser Scanning Confocal Microscopy. A Zeiss LSM510 micro-
scope equipped with a 63× or 100× oil-immersion objective was used
to obtain confocal images. For co-localization experiments, electronic
zoom (1.5×) was used in combination with the 100× objective. Samples
were excited at 488 nm with an Ar laser. The microscope stage was
outfitted with a CTI-3700 incubator, which maintained samples at 37
°C and under 5% CO2. With the exception of ZS5 treatment, which
was performed in a tissue culture hood, addition of all reagents was
carried out by bath application on the microscope stage.
Figure 2. ORTEP diagram for the structure of ZSF6, showing 40%
probability ellipsoids on all non-hydrogen atoms.
observed for the dinuclear ZP1:Zn(II) complex47 and ZPCl1.79
The hydrogen atoms of O1 and O3 were located from a
difference electron density map, and both form hydrogen bonds
with the nitrogen atoms of the pyridyl-amine-thiophene
moieties. The O1 proton is hydrogen-bonded to both the tertiary
amine N1 and pyridyl N2 atoms, with O1-N1 and O1-N2
distances of 3.190(3) and 2.599(3) Å, respectively. The O3
proton is hydrogen-bonded to the tertiary amine N3 atom, with
an O3-N3 distance of 2.634(3) Å. No hydrogen-bonding
interactions are observed with the thiophene moieties, as
expected given the poor basicity of this group.
Results and Discussion
Design and Syntheses of Probes. Two main considerations
influenced the design of the sensors described in this work. Our
primary goal was to improve the dynamic range and maintain
both the Zn(II) selectivity and relatively low Zn(II) affinity of
thioether-containing ZS sensors. We therefore substituted the
thioether moiety of our first-generation ZS sensors with a
thiophene group. We anticipated that replacement of an alkyl
chain with an aromatic heterocycle would result in lower
background emission and, in turn, greater fluorescence enhance-
ment following Zn(II) coordination. A second objective was to
prepare sensors in relatively few steps and high yield. For this
reason, we focus on symmetrical species with tertiary-amine-
based ligands, which can generally be obtained in six or fewer
steps from commercially available materials.
Photophysical Properties of Thiophene-Containing ZS
Sensors. Table 1 summarizes photophysical characterization and
thermodynamic parameters for ZS sensors, in addition to data
for the symmetrical ZP probes for comparison. In general,
halogenation of the fluorescein platform induces red-shifts in
both excitation and emission maxima. Chlorination or fluorina-
tion of the benzoate rings causes ca. 20 nm red-shifts, and
chlorination of the 2′ and 7′ positions of the xanthenone
framework also results in red-shifts relative to fluorescein and
its 2′,7′-difluoro derivative. These substitutions afford an
excitation and emission range of ∼35 nm, similar to that of the
halogenated ZP family.49 Although modest, this feature could
be useful for biological imaging experiments that require two
Zn(II)-specific probes or for dual Zn(II) and Ca(II) sensing.
Introduction of a thiophene group into the metal-binding unit
results in lower background emission and greater fluorescence
enhancement than observed for thioether-containing ZS1 and
ZS2. This trend is particularly evident for the ZS2/ZS7 pair,
where Φfree decreases from 0.39 to 0.25 (50 mM PIPES, 100
mM KCl, pH 7). As a result, the quantum yields and dynamic
ranges of thiophene-containing ZS more closely resemble those
of the ZP family, although Φfree is consistently higher and
indicates that DPA quenches the fluorescein excited state more
effectively than the pyridyl-amine-thiophene moiety. Depend-
ing on the substitution pattern on the fluorescein rings,
compounds 3-8 offer ∼2- (ZS6, ZSCl6) to >4.5-fold (ZSF7)
fluorescence turn-on following Zn(II) coordination. Representa-
tive emission spectra are given in Figures 3 and S1-S3
(Supporting Information). The optical absorption spectra of both
Zn(II)-free and -bound forms of the ZS sensors are characterized
by high molar extinction coefficients, and Zn(II) coordination
results in a slight (ca. 10 nm) blue-shift and increase in the molar
Scheme 1 illustrates the syntheses of symmetrical ZS sensors
3-8 from fluorescein precursors. Probes containing halogenated
fluorophores were prepared by Mannich reactions of the
fluorescein and the imminium ion condensation product of
paraformaldehyde and 1. In each case, the desired product
precipitated from the reaction solution, and multiple (two or
three) recrystallizations of the precipitates from boiling EtOH
yielded sensors 4-8 as light to bright pink powders in moderate
to high yields. Fluorescein-based ZS5 (3) was prepared from 1
and dialdehyde 2 (Scheme 1) using NaB(OAc)3H as the reducing
agent and obtained in moderate yield after workup and prepara-
tive TLC on silica gel
There are few reported crystal structures of fluorescein-based
compounds. This paucity most likely stems from the fact that
fluoresceins can adopt many isomers, including quinoid and
lactone forms, which makes crystallization difficult. Since we
could easily prepare relatively large (often >100 mg) quantities
of the ZS sensors described in this work, we were able, through
multiple attempts, to obtain X-ray-quality crystals of a ZS
sensor. Single crystals of ZSF6 were obtained by vapor diffusion
of Et2O into a solution of the sensor in CH2Cl2 at room
temperature. Figure 2 displays the ORTEP diagram of ZSF6,
and Tables S1 and S2 (Supporting Information) provide
crystallographic parameters and selected bond lengths and
angles, respectively. ZSF6 crystallized in the lactone form,
affording a colorless and non-fluorescent material, as previously
(79) Nolan, E. M.; Lippard, S. J. Unpublished results.
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15522 J. AM. CHEM. SOC. VOL. 128, NO. 48, 2006

Zinspy Sensors for Imaging Zn(II) in Neurobiology
A R T I C L E S
Table 1. Spectroscopic and Thermodynamic Data for Symmetrical ZP and ZS Sensorsa
absorption
max (nm), ꢀmax
104 (M-1 cm-
emission
1
λ
×
)
λ
max (nm), Φb
d
unbound
Zn(II)
unbound
Zn(II)
pKa1 (N)c
pKa2
ref
ZP1
ZP2
ZP3
ZPF1
ZPCl1
ZPF3
ZS1
ZS2
ZS5
515, 7.9
498, 4.4
502, 7.5
533, 9.9
534, 9.7
520, 8.7
510, 8.4
499, 6.7
497, 3.3
515, n.d.
532, 6.3
500, 6.2
521, 6.2
507, 8.4
490, 5.3
492, 8.5
525, 12.0
527, 12.0
510, 9.3
501, 7.5
489, 6.8
490, 4.2
505, n.d.
522, 7.0
490, 6.6
511, 7.0
531, 0.38
522, 0.25
521, 0.15
547, 0.11
550, 0.22
537, 0.14
531, 0.50
523, 0.39
522, 0.36
533, 0.44
549, 0.19
524, 0.25
535, 0.24
527, 0.87
517, 0.92
516, 0.92
544, 0.55
549, 0.50
533, 0.60
526, 0.70
516, 0.69
517, 0.80
527, 0.64
545, 0.63
518, 0.79
527, 0.68
8.4
9.4
6.8
6.9
7.0
6.7
7.7
7.7
8.0
7.8
7.1
7.0
6.9
2.8
3.9
2.0
1.8
1.9
1.9
2.0
2.7
4.6
3.3
2.0
2.7
2.5
47
47
49
49
49
49
64
64
this work
this work
this work
this work
this work
ZS6
ZSF6
ZS7
ZSF7
a All quantum yield and extinction coefficient measurements were conducted at pH 7 (50 mM PIPES, 100 mM KCl). b Quantum yields were measured
using fluorescein in 0.1 N NaOH as a standard (Φ ) 0.95, ref 73). c The pKa value associated with fluorescence enhancement as the pH is lowered. This
pKa is assigned to tertiary amine protonation. d The pKa value associated with fluorescence quenching as the pH is lowered.
Figure 4. Effect of pH on integrated emission (100 mM KCl, 25 °C). Top
plot: Representative pH titrations for ZS5 (circles), ZS6 (squares), and ZS7
(diamonds). Bottom plot: Representative pH titrations for ZSF6 (circles)
and ZSF7 (squares). For each titration, a 1 µM solution of ZS was prepared
Figure 3. Fluorescence response of 1 µM ZS5 to Zn(II). Top: Response
of ZS5 to Zn(II) at pH 7 (50 mM PIPES, 100 mM KCl) and 25 °C.
Bottom: Integrated emission vs Zn(II) concentration for ZS5. Aliquots of
1 and 10 mM ZnCl2 were added to afford 0, 0.33, 0.66, 1, 1.5, 2.0, 2.5,
3.0, 4.0, 6.0, 8.0, 10, 15, 20, 30, and 50 µM Zn(II). The circles depict the
(10 mM KOH, 100 mM KCl, pH ∼12), and the pH was lowered by
incremental addition of aqueous HCl. Data for pH >12 are excluded from
the plots, since no appreciable fluorescence change occurred in this range.
experimental data, and the line is the fit to a 1:1 metal-ligand binding
The pKa values, obtained from fitting the experimental data to a nonlinear
model. Excitation was provided at 490 nm.
model,47 are listed in Table 1.
absorptivity. This behavior parallels that of many previously
characterized ZS and ZP sensors and indicates that the phenol
moiety is involved in Zn(II) coordination.
pKa values are given in Table 1. We first consider ZS5-7, which
lack benzoate ring halogenation (Figure 4, top). Like ZP1-3,
the 2′,7′-dichloro (ZS6) and 2′,7′-difluoro platforms (ZS7) shift
the pH titration curve toward lower values relative to that of
the unsubstituted fluroescein (ZS5). This trend parallels the
known pH-dependent emission profiles and pKa values of the
parent fluorophores, with fluorescein having a phenolic pKa
value of 6.4,81 compared to 5.082 for 2′,7′-dichloro- and 4.883
The data in Table 1 reveal that substitution of the 2′ and 7′
positions of the xanthenone moiety causes systematic variations
in Φfree that are independent of ligand type. For both ZP and
ZS, the magnitude of Φfree decreases as X ) Cl > H > F.
Similar background fluorescence reduction has been observed
by others upon substitution of fluorescein with 2′,7′-difluoro-
fluorescein.43 Although the origin of this effect is unclear, it is
generally applicable to the design of fluorescein-based metal
ion sensors.
(80) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.;
McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515-
1566.
(81) Sjo¨back, R.; Nygren, J.; Kubista, M. Spectrochim. Acta Part A 1995, 51,
L7-L21.
The ZS sensors presented in this study operate via a PET
mechanism.80 Sensors of this type are often pH-sensitive. Figure
4 displays representative pH titrations for selected probes, and
(82) Leonhardt, H.; Gordon, L.; Livingston, R. J. Phys. Chem. 1971, 75, 245-
249.
(83) Sun, W.-C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org. Chem.
1997, 62, 6469-6475.
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J. AM. CHEM. SOC. VOL. 128, NO. 48, 2006 15523

A R T I C L E S
Nolan et al.
Table 2. Kinetic and Thermodynamic Parameters for Zn(II) Binding to Selected Sensorsa
1
b
1
c
d
1 e
k
on (M-1 s-
)
k
on (M-1 s-
)
Kd1
25
k
off (s-
)
∆
Hq
∆
Sq
(cal/mol‚
K)f
4.3
°
C
25
°
C
°
C
25
°
C
(kcal/mol)f
ZP1
ZP3
ZS5
ZS6
ZS7
ZSF6
ZSF7
(6.3 ( 0.1) × 105
(7.8 ( 0.1) × 105
(3.6 ( 0.2) × 105
(3.7 ( 0.1) × 105
(5.9 ( 0.1) × 105
(3.3 ( 0.1) × 105
(5.0 ( 0.1) × 105
(3.3 ( 0.4) × 106
(4.3 ( 0.3) × 106
(2.2 ( 0.1) × 106
(2.1 ( 0.1) × 106
(3.0 ( 0.1) × 106
(1.8 ( 0.1) × 106
(2.7 ( 0.1) × 106
0.7 ( 0.1 nM
0.7 ( 0.1 nM
1.5 ( 0.2 µM
n.d.
3.7 ( 0.2 µM
4.6 ( 0.2 µM
5.0 ( 0.3 µM
(2.3 ( 0.4) × 10-3
12.5 ( 0.1
11.6 ( 0.2
13.3 ( 0.1
12.9 ( 0.4
12.5 ( 0.3
12.6 ( 0.2
12.6 ( 0.2
13.2 ( 0.3
10.7 ( 0.7
15.2 ( 0.2
14.0 ( 0.7
12.9 ( 1.3
12.4 ( 0.9
13.0 ( 0.7
(2.9 ( 0.4) × 10-3
3.3
n.d.
11
8.3
13.5
a All measurements were made at pH 7 (50 mM PIPES, 100 mM KCl). Data for ZP1 and ZP3 are taken from ref 50. b Experimentally determined values
for kon at 4.3 ( 0.1 °C. c Experimentally determined values for kon at 25 ( 0.1 °C. d Dissociation constants obtained experimentally by fluorimetric titration
at 25 ( 1 °C. e Calculated koff for ZS at 25 °C. f Activation parameters were determined over a temperature range of ∼4 to ∼40 °C.
for 2′,7′-difluorofluorescein. As a result, the pKa transition
assigned to tertiary amine protonation decreases from 8.0 (ZS5,
X ) H) to 7.0 (ZS7, X ) F). Fluorescence quenching occurs at
pH <∼6 when the phenol group becomes protonated. These
pKa values range from 4.6 to 2.7 with X ) H > Cl ∼ F.
Titrations of bottom-ring-halogenated ZSF6 and ZSF7 are
given in the bottom panel of Figure 4. These profiles are both
characterized by two pKa transitions, with pKa1 ≈ 7 assigned
to tertiary amine protonation and pKa2 < 3 assigned to
protonation of the phenol. The pKa1 values are similar to those
observed for ZPF1 (pKa ) 6.9) and ZPF3 (pKa ) 6.7) and follow
the same trend of further diminution upon fluorination of the
2′ and 7′ positions. Scheme S1 depicts the protonation equibria
for the thiophene-containing ZS sensors proposed on the basis
of these experiments and on the low basicity of thiophene
moieties.
The titrations in Figure 4 illustrate that emission of the ZS
sensors is effectively quenched at high pH. The quantum yield
of apo-ZS5 drops from 0.36 at pH 7 to 0.04 at pH 9. As a result,
ZS5 shows >20-fold fluorescence enhancement following
Zn(II) coordination at pH 9, with ΦZn ) 0.73 (50 mM CHES,
100 mM KCl), as compared to ∼4-fold enhancement at pH 7
(50 mM PIPES, 100 mM KCl). This behavior is consistent with
the PET mechanism.
respectively.82,83 This behavior is somewhat evident in the Kd
values for the symmetrical ZP probes, which range from 0.5 to
1.1 nM.49
Thiophenes are weak bases with low affinity for many
divalent metal ions, including Zn(II). To establish that the
thiophene moiety is not involved in Zn(II) coordination, X-ray
crystallographic studies were conducted using a salicylaldehyde-
based model of ZS5. A report of this study is given as
Supporting Information. Single crystals were obtained from
small-scale crystallization trials, and X-ray structural determi-
nation of these complexes confirmed that the thiophene moiety
of the model ligand does not bind to Zn(II) or to other divalent
first-row transition metal ions, including Cu(II) (Figure S4).
Sensors 3-8 have two possible metal-binding sites. By
analogy to the symmetrical ZP systems and from chemical
intuition, we propose stepwise binding with formation of the
1:1 ZS:Zn(II) complexes at relatively low Zn(II) concentrations
and formation of 1:2 complexes at significantly higher concen-
trations of Zn(II). Fluorimetric titrations show that the fluores-
cence response of 1 µM ZS to Zn(II) maximizes at 10-50 µM
Zn(II), depending on the fluorophore platform, which indicates
that the second binding event has little influence on the
fluorescence response. For this reason, values for Kd2 were not
determined.
Metal-Binding Properties. I. Zinc Affinity and Coordina-
tion. Fluorescence spectroscopy was used to determine apparent
Zn(II) complex Kd values for ZS5, ZSF6, ZS7, and ZSF7, which
are listed in Table 2. Representative binding curves are given
in Figure 3 (ZS5) and as Supporting Information (ZSF6, ZS7,
ZSF7). Fluorescein-based ZS5 exhibits maximum fluorescence
in the presence of ∼10 equiv of Zn(II) and has a Kd value of
1.5 ( 0.2 µM. This value is ∼3 orders of magnitude greater
than the Kd1 value of ZP2 (Kd1 ) 0.5 ( 0.1 nM)47 and about
twice that of asymmetrical ZP9 (Kd ) 0.69 ( 0.04 µM).84
Halogenation of the xanthenone and benzoate moieties raises
the Kd significantly, with values for ZS7, ZSF6, and ZSF7 of
3.7 ( 0.4, 4.6 ( 0.2, and 5.0 ( 0.3 µM, respectively. We
attribute this behavior to variation in the donor ability of the
deprotonated phenol group. Incorporation of halogens reduces
the electron density on the oxygen atom, making it a poor donor
relative to X ) H. As a result, the Zn-O bond is weaker in
chlorinated and fluorinated ZS relative to ZS5. This trend is in
accord with the pH studies on fluorescein and its halogenated
derivatives, where the pKa of the phenol decreases with
chlorination and fluorination of the fluorescein platform,
II. Zinc Selectivity. One objective of this investigation was
to maintain the Zn(II) selectivity observed for thioether-
containing ZS. Figure 5 illustrates the selectivity of ZS5 for
Zn(II) in the presence of biologically relevant cations, divalent
first-row transition metal ions, Cd(II) and Hg(II). The data for
the halogenated sensors are generally analogous, although the
degree of quenching observed following Hg(II) addition varies
with fluorescein halogenation. As anticipated, the behavior of
ZS5 follows that of ZS1-4, maintaining the selectivity for
Zn(II) over Fe(II). The greater affinity of ZS5 for Zn(II) vs
Hg(II) is an improvement over the thioether-containing ZS
sensors because Zn(II) addition to solutions of ZS1-4 and
Hg(II) does not cause any fluorescence change.64 This difference
results from loss of a strong Hg-S bond between the metal ion
center and the thioether donor.
III. Kinetics and Thermodynamics of Zn(II) Association.
The fluorescence responses of the ZS probes are observable
immediately after Zn(II) addition. We therefore conducted
stopped-flow kinetic studies to determine the rate constants for
Zn(II) association, measured by fluorescence turn-on, for these
sensors. Table 2 lists the experimentally determined second-
order rate constants for Zn(II) association (kon) and, in most
instances, calculated rate constants for Zn(II) dissociation (koff).
(84) Nolan, E. M.; Jaworski, J.; Racine, M. E.; Sheng, M.; Lippard, S. J. Inorg.
Chem., published ASAP online October 31, 2006, http://dx.doi.org/10.1021/
ic061137c.
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Zinspy Sensors for Imaging Zn(II) in Neurobiology
A R T I C L E S
Figure 7. Comparison of subcellular compartmentalization of ZS5 (left)
and ZP3 (right) in HeLa cells. Both confocal images were obtained 5 min
after addition of 100 µM Zn(II)/pyrithione to cells treated with 10 µM ZS5
or ZP3 (37 °C, 5% CO2). Addition of Zn(II)/pyrithione has no effect on
sensor localization during this time frame. The scale bar is 25 µm.
and (ii) the rate of Zn(II) dissociation is significantly more
sensitive to loss of a DPA pyridyl group.44
Halogenation of the fluorescein platform has subtle effects
on the kon values. Both the ZP1/ZP3 and ZS6/ZS7 couples show
that fluorination of the 2′ and 7′ positions increases the
magnitude of kon relative to chlorination. Comparison of the
ZS couples ZS6/ZSF6 and ZS7/ZSF7 indicates that fluorination
of the benzoate moiety decreases kon to some degree.
Temperature variation experiments were conducted to deter-
mine the activation enthalpies and entropies for Zn(II) coordina-
tion to the ZS sensors, and these data are also included in Table
2. Figure S6 displays representative Eyring plots for each probe,
from which we compute ∆Hq to be 12.6-13.3 kcal/mol and
∆Sq to range from 12.4 to 15.2 cal/mol‚K. The former values
are indicative of a low activation barrier and fast association
rate; they are comparable to those determined for ZP family
members. The positive ∆Sq values are also typical of fluorescein-
based sensors designed in our laboratory, which we presume
reflect loss of water molecules or buffer components from the
Zn(II) center or loss of protons from ZS in the transition state
during binding.
Figure 5. Selectivity of ZS5 for Zn(II) at pH 7 (50 mM PIPES, 100 mM
KCl). Top plot: Fluorescence response of 1 µM ZS5 to 50 equiv of the
cation of interest: 1, Na(I); 2, Ca(II); 3, Mg(II); 4, Mn(II); 5, Fe(II); 6,
Co(II); 7, Ni(II); 8, Cu(II); 9, Zn(II); 10, Cd(II); 11, Hg(II). Bottom plot:
The light gray bars correspond to the bars in the top plot. The black bars
illustrate the fluorescence change that occurs immediately after 50 equiv
of Zn(II) is added to the solution containing 1 µM ZS5 and 50 equiv of the
cation of interest. All data (F) are normalized with respect to the emission
from the free sensor (Fo). Excitation was provided at 490 nm and 25 °C.
Biological Imaging Studies. I. Response of ZS5 to Zinc in
Cultured Cells and Subcellular Localization. Several features
of the ZS probes, including improved dynamic range relative
to the thioether-containing parent compounds and low-micro-
molar affinity for Zn(II), motivated us to consider their utility
for in vivo work. We began these investigations with ZS5, the
first compound we prepared, and quickly found that it is
amenable for such experiments. We therefore focused our
attention on this probe for biological imaging.
Figure 6. Plots of kobs vs Zn(II) concentration for ZS family members
obtained from stopped-flow fluorescence studies at pH 7 (50 mM PIPES,
100 mM KCl) and 4.3 °C. The concentration of ZS was 0.5 µM after mixing,
and the concentration of Zn(II) was varied from 0 to 400 µM after mixing.
Experimentally determined Zn(II) association (kon) and calculated Zn(II)
dissociation (koff) rate constants are given in Table 2.
Initial cell studies indicated that ZS5 permeates a number of
commercially available immortal cell lines and is Zn(II)-
responsive in vivo. Figure S7 exhibits representative images of
ZS5-treated HeLa, COS-7, and HEK293-T cells. In preliminary
work, the cells were treated with 10 µM ZS5 for varying
incubation times, which reveal 30 min (37 °C, 5% CO2) to be
optimal. Longer incubation periods (>1 h) are also acceptable,
but shorter (ca. 20 min or less) ones result in insufficient sensor
uptake. Addition of Zn(II) to the cells as the 1:1 or 1:2 Zn(II)/
pyrithione complex affords intracellular fluorescence enhance-
ment, which is reversed with TPEN treatment (Figure S7).
An important observation from these preliminary experiments
concerned the subcellular localization of ZS5 in HeLa cells.
Figure 7 shows a side-by-side comparison of HeLa cells treated
with 10 µM ZS5 or ZP3 and 100 µM Zn(II)/pyrithione. ZP3
Plots of kobs versus Zn(II) concentration are given in Figure 6,
which return kon values ranging from 3.3 × 105 to 5.9 × 105
M-1 s-1 at 4.3 °C. Data from temperature variation experiments
indicate kon values of 1.8 × 106 to 3.0 × 106 M-1 s-1 at 25 °C,
which are comparable to those of DPA-based probes such as
ZP50 and ZnAF44 family members. The dissociation rate
constants, koff, were calculated from the experimentally deter-
mined kon and Kd values and range from 3.3 to 13.5 s-1 at 25
°C, yielding t1/2 values of 50-210 ms. These kinetic parameters
demonstrate that the ZS sensors bind Zn(II) both rapidly and,
significantly, more reversibly than DPA-based ZP. Furthermore,
these data are in accord with rate constants obtained for ZnAF
probes, which indicated that (i) one pyridyl group in a DPA-
like chelate is sufficient for maintaining fast Zn(II) complexation
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A R T I C L E S
Nolan et al.
ZS5 and exogenous Zn(II). ZS5 enters and is Zn(II)-responsive
and reversible in both cell types.
A series of time-course experiments, in which cells were
treated with 10 µM ZS5 and 50 µM Zn(II)/pyrithione and
imaged over the course of 1-5 min, revealed that ZS5 responds
to exogenously added Zn(II) more quickly in neurons than in
other cell types, such as HeLa and COS-7. Whereas ca. 1 min
of incubation was sufficient to observe full fluorescence
enhancement in hippocampal neurons following treatment with
50 µM Zn(II)/pyrithione, ca. 5 min was required to observe
complete turn-on in HeLa cells. We believe that this variation
stems from the relative ability of the HeLa cells and neurons to
internalize the Zn(II)/pyrithione complex. From a molecular
standpoint, the difference in Zn(II)/pyrithione uptake might arise
from the greater diversity of ligand, voltage, and resting cation
membrane channels in neurons relative to HeLa cells.
We therefore questioned whether ZS5 would respond to
Zn(II) on a faster time scale in vivo, a feature that is necessary
for monitoring biological Zn(II) flux. Figure 9 shows time-series
images of a hippocampal neuronal cell body treated with 10
µM ZS5 before and after addition of 50 µM 1:1 Zn(II)/pyrithione
and 50 µM TPEN. The images were captured at ∼2.4-s intervals
and show that ZS5 responds to Zn(II) on this time scale in living
cells. We chose this time interval because it allowed for real-
time visualization of the signal on the computer screen without
any image manipulation. Control studies show that pretreatment
of the neurons with TPEN blocks any response of ZS5 to
Zn(II)/pyrithione (data not shown). The origin of the fluores-
cence rise that occurs in the ca. 4 s following TPEN addition is
unclear to us, but it is reproducible, and we have also observed
this phenomenon in HeLa cells. The value of these experiments
lies in their proof that ZS5 can respond to and release Zn(II)
rapidly in a biological system, which points toward its utility
for monitoring Zn(II) flux.
III. Effect of Glutamate on Exogenous Zinc Uptake.
Treating cells with Zn(II)/pyrithione is essentially a brute force
approach to generating a robust intracellular signal. Pyrithione
appears to deliver Zn(II) indiscriminately throughout the cell.
As a result, experiments that use Zn(II)/pyrithione have little
physiological or pathophysiological significance. We were
therefore motivated to investigate other routes of Zn(II) entry
that may be relevant to neurobiological signaling.
The Zn(II)-containing synaptic vesicles in the mammalian
hippocampus also house glutamate (E), an excitatory neu-
rotransmitter, and both species are released into the synaptic
cleft following exocytosis of vesicles at the pre-synaptic
membrane. Given this association, it is reasonable to think that
glutamate may modulate Zn(II) uptake into the post-synaptic
neuron or other nearby cells. Several reports indicate that
glutamate or kainate, a glutamate mimic, can trigger excessive
intracellular accumulation of Zn(II) in cortical neurons.86,88,89
We therefore co-treated ZS5-loaded hippocampal neurons with
ZnCl2 and glutamate (1:2 ratio, 50 µM zinc) and monitored the
intracellular response by fluorescence microscopy. Figure 10
displays representative images, which show significantly dif-
ferent staining than that observed for neurons treated with
Zn(II)/pyrithione. Fluorescence enhancements occur in several
Figure 8. Response of ZS5 to exogenously added Zn(II) in primary cultures
of hippocampal (row A) and dentate gyrus (row B) neurons. Left panels:
Neurons were treated with 10 µM ZS5 for 30 min (37 °C, 5% CO2), washed,
and imaged. Middle panels: Fluorescence change observed 1 min (hippo-
campal neurons) or 5 min (dentate gyrus neurons) after addition of 50 µM
Zn(II)/pyrithione (1:2 ratio). Right panels: Fluorescence decrease observed
1 min (hippocampal neurons) or 5 min (dentate gyrus neurons) after addition
of 50 µM TPEN. The scale bars represent 25 µm.
exhibits punctate staining, similar to that previously reported
for ZP1 in COS-7 cells,47 whereas the ZS5 pattern is much more
diffuse. Co-localization studies were therefore conducted in
HeLa cells to determine where ZS5 and ZP3 reside, and
representative images are provided in Figures S8 and S9.
Treatment of HeLa cells with ZS5 (free or zinc-bound) and
either Lysotracker or pGolgi-DsRed showed that this dye does
not localize to either the lysosomes or the Golgi apparatus.
Excellent co-localization of ZS5 and Mitotracker Red was
observed, which establishes that ZS5 is sequestered in the
mitochondria of healthy HeLa cells. In contrast, ZP3 shows
excellent co-localization with pGolgi-DsRed and no overlap with
the Lysotracker and Mitotracker markers, which establishes
Golgi compartmentalization of this probe in HeLa cells.
The observation that Zn(II) sensors can be directed to different
subcellular organelles as a result of structural modification is
intriguing and potentially advantageous. Although subcellular
compartmentalization of sensors has been described as prob-
lematic in the calcium sensor literature,37 we believe that a clear
understanding of sensor localization in the cell type under study
will be beneficial for studying Zn(II) entry into and trafficking
between certain organelles. Mitochondrial localization is relevant
from the standpoint of Zn(II) toxicity, because rapid influx of
Zn(II) into the mitochondria has been implicated in neuronal
death.28,31,85,86
II. Response of ZS5 to Zinc in Primary Neuronal Cultures.
We next focused our efforts on studying the behavior of ZS5
in primary neuronal cultures from pre- or postnatal rat. Two
different types of neuronal cultures were used in this work.87
Figure 8 exhibits representative images of both embryonic
hippocampal and postnatal dentate gyrus neurons treated with
(85) Sensi, S. L.; Yin, H. Z.; Carriedo, S. G.; Rao, S. S.; Weiss, J. H. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 2414-2419.
(86) Sensi, S. L.; Ton-That, D.; Weiss, J. H. Neurobiol. Dis. 2002, 10, 100-
108.
(87) The hippocampal cultures were prepared from prenatal rat and contain cells
from the entire hippocampus. The dentate gyrus cultures were prepared
from postnatal rats and contain cells only from the dentate gyrus
substructure. The dentate gyrus is the region of the hippocampus rich in
Zn(II)-filled vesicles, which makes these cultures particularly interesting
from the standpoint of Zn(II) neurobiology.
(88) Sensi, S. L.; Canzoniero, L. M. T.; Yu, S. P.; Ying, H. S.; Koh, J.-Y.;
Kerchner, G. A.; Choi, D. W. J. Neurosci. 1997, 17, 9554-9564.
(89) Colvin, R. A.; Davis, N.; Nipper, R. W.; Carter, P. A. J. Nutr. 2000, 130,
1484S-1487S.
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Zinspy Sensors for Imaging Zn(II) in Neurobiology
A R T I C L E S
Figure 9. Time-lapse imaging of Zn(II) addition to primary cultures of hippocampal neurons (DIV 18) pretreated with 10 µM ZS5. The ZS5-treated cell
was imaged (0-27.671 s); Zn(II)/pyrithione (50 µM, 1:1 ratio) was added during the image acquisition (29.937-34.530 s, top arrow), and a fluorescence
rise is observed. An aliquot of TPEN (50 µM) was subsequently added during the data collection (66.435-70.998 s, bottom arrow), and a fluorescence
decrease occurred at ca. 80 s.
Figure 10. Uptake of exogenous Zn(II) by hippocampal neurons as a result of glutamate (E) treatment. Hippocampal neurons were treated with 10 µM ZS5
for 30 min (37 °C, 5% CO2), washed, and imaged. The neurons were treated with 50 µM 1:2 Zn(II)/E and imaged 1 min following addition. A fluorescence
rise occurs in a number of neuronal projections, labeled by asterisks. The fluorescence increase is reversed 1 min after addition of 50 µM TPEN. Lysotracker
Red was used as the red marker in this experiment. The scale bar represents 25 µm.
neuronal projections 1 min after addition of the Zn(II)/E mix,
and this rise persists for at least 5 min (Figure S11). The staining
pattern consists of green dots that may be associated with
dendritic spines and/or GABA synapses, although detailed co-
localization studies must be conducted to address these pos-
sibilities. Control studies in which hippocampal neurons were
treated with only ZnCl2 (50 µM) showed no fluorescence change
over a 5-min time frame, indicating that Zn(II) alone does not
permeate the neuronal cell membrane over this time period
(Figure S11). Additional control studies, in which the ZS5-
loaded neurons were treated with glutamate (100 µM) for 5 min,
also showed no fluorescence change (Figure S11). This experi-
ment illustrates that extracellular glutamate modulates the uptake
of Zn(II) into hippocampal neurons rapidly and at specific
locales.
nitrosative stress. We chose neurons from the zinc-rich dentate
gyrus region of the rat hippocampus for this experiment. Figure
11 shows a time-course experiment in which dentate gyrus
neurons were loaded with 10 µM ZS5 and subsequently treated
with SNOC. Significant fluorescence enhancement occurs in
the neuron cell bodies and projections, and in surrounding glial
cells, at ca. 3 min following SNOC addition, and the response
maximizes at ca. 5 min.
To confirm that the observed changes in ZS5 fluorescence
intensity observed after SNOC treatment are due to the NO-
induced release of Zn(II) from native protein stores rather than
to general cell toxicity, we monitored neuronal cell viability
30 min after SNOC application by employing established
protocols for assaying cell death in dentate gyrus neurons.78 In
particular, we analyzed nuclear and dendritic morphology using
DAPI (nuclear stain) and MAP2 (dendritic marker), as described
in the Supporting Information. No statistically significant
difference was observed in a comparison of nuclear morphology
in untreated control and SNOC-treated cells (control, 96.7%
survival; SNOC, 95.1% survival), and dendritic fragmentation,
characteristic of apoptotic DG neurons, was not observed (Figure
IV. Imaging Endogenous Zinc Mobilization with ZS5. We
previously demonstrated that ZNP1, a ratiometric Zn(II) indica-
tor with a sub-nanomolar Kd value for this metal ion, can detect
endogenous Zn(II) mobilization in cultured COS-7 cells fol-
lowing SNOC treatment.54 We therefore wondered whether ZS5
would illuminate Zn(II) mobilization in live cells following
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A R T I C L E S
Nolan et al.
Figure 11. Imaging endogenous Zn(II) release in primary dentate gyrus neuronal cultures following nitrosative stress with ZS5. The cells were treated with
10 µM ZS5 (30 min, 37 °C, 5% CO2), washed, and imaged. An aliquot of SNOC was added (final concentration ) 1.5 mM), and the fluorescence change
was recorded at 1-min intervals. Time points for 0, 1, 3, 5, and 7 min are shown above. The right-most panel shows the fluorescence decrease that occurred
2 min after addition of 200 µM TPEN. The scale bar indicates 25 µm.
Table 3. Cytotoxicity Data (HeLa, 24 h)a
Zn(II) complexes. These sensors exhibit a number of advanta-
geous features. From a chemical perspective, they provide (i)
% cell survival
dissociation constants in the low micromolar range, (ii) fast
1
µ
M
5
µ
M
10 µM
Zn(II) association rates comparable to those observed for
Zn(II) sensors employing the DPA chelate but with more rapid
Zn(II) dissocation, and (iii) improved selectivity for Zn(II)
compared to the parent ZP and ZS compounds. Our studies
demonstrate that fluorescein halogenation is a general strategy
for modulating not only photophysical but also metal-binding
properties of fluorescent metal ion sensors. From the standpoint
of biological imaging, ZS5 is a robust and versatile tool for
illuminating Zn(II) uptake and mobilization in living cells,
including neurons. Mitochondrial probe localization occurs for
ZS5 in HeLa cells, in contrast to the Golgi localization of ZP3,
demonstrating that ligand modifications can direct fluorescein-
based sensors to specific locales. Glutamate-dependent Zn(II)
entry into embryonic hippocampal neurons was visualized, as
was Zn(II) release from native protein stores in dentate gyrus
neurons. We anticipate that ZS5, alone or in combination with
high-affinity Zn(II) probes such as ZP3, will be useful for studies
of Zn(II) physiology and pathology in a variety of contexts.
ZP1
ZP3
ZS5
96 ( 2
93 ( 4
68 ( 5
73 ( 8
87 ( 2
40 ( 6
57 ( 13
80 ( 4
101 ( 1
a Cell viability was quantified by the MTT assay (mean ( SD).
S10). Together, these experiments establish that dentate gyrus
neurons can release Zn(II) from native protein stores at
concentrations observable with a low-micromolar affinity probe
as a result of nitrosative stress.
Probe Cytotoxicity. Cytotoxicity is a potential side effect
of a probe that must be controlled when dealing with living
cells or tissues. Although we saw no evidence for cytotoxic
effects during the experiments described in this work, these
studies required relatively short time frames. We were therefore
interested in studying the possible consequences of a more
extended sensor treatment on cell viability. To date, we have
conduced cytotoxicity assays only in HeLa cells, but this
information should prove useful in the design of experiments
requiring other cell lines. Table 3 lists the cell viability data
for HeLa cells treated with ZP1, ZP3, and ZS5 as quantified
by the MTT assay, an established method of probing apoptotic
cell death. Corresponding bar graphs are given in Figure S12.
These data show that HeLa cells show ∼100% viability
following 24-h treatment with 1 µM ZP1, ZP3, or ZS5. Higher
sensor concentrations result in decreased cell survival, with 80%
( 4% viability observed following 24-h treatment with 10 µM
ZS5. These cytotoxicity tests indicate that sub- and low-
micromolar concentrations of ZP and ZS5 are essentially
nontoxic over at least a 24-h period and can safely be used for
biological studies that require prolonged sensor treatment.
Acknowledgment. This work was supported by Grant
GM65519 from the National Institute of General Medical
Sciences. Spectroscopic instrumentation at the MIT DCIF is
maintained with funding from NIH Grant 1S10RR-13886-01
and NSF Grants CH3-9808063, DB19729592, and CHE-
9808061. M.S. is an investigator of the Howard Hughes Medical
Institute. E.M.N. thanks the Whitaker Health Science Fund for
a graduate fellowship and Dr. D. Song for assistance with
collecting the X-ray data.
Supporting Information Available: Tables S1 and S2,
Scheme S1, Figures S1-S12, CIF files, experimental details
for X-ray crystallographic modeling studies and cytotoxicity
assays in dentate gyrus neurons, representative 1H NMR spectra,
and IR spectral data for all probes. This material is available
free of charge via the Internet at http://pubs.acs.org.
Summary
Turn-on fluorescent Zn(II) sensors of the Zinspy family give
improved dynamic range by substituting thioethers with thiophene
groups, which affords lower background fluorescence and bright
JA065759A
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