SpiroZin1: A reversible and pH-insensitive, reaction-based, red-fluorescent probe for imaging biological mobile zinc

Article abstract of DOI:10.1002/cmdc.201400014

A reversible, reaction-based sensor for biological mobile zinc was designed, prepared, and characterized. The sensing mechanism of this probe is based on the zinc-induced, ring-opening reaction of spirobenzopyran to give a cyanine fluorophore that emits i

Full text of DOI:10.1002/cmdc.201400014

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DOI: 10.1002/cmdc.201400014
SpiroZin1: A Reversible and pH-Insensitive, Reaction-
Based, Red-Fluorescent Probe for Imaging Biological
Mobile Zinc
Pablo Rivera-Fuentes and Stephen J. Lippard*[a]
A reversible, reaction-based sensor for biological mobile zinc
was designed, prepared, and characterized. The sensing mech-
anism of this probe is based on the zinc-induced, ring-opening
reaction of spirobenzopyran to give a cyanine fluorophore that
emits in the deep-red region of the electromagnetic spectrum.
This probe is not activated by protons and operates efficiently
in aqueous solution at pH 7 and high ionic strength. The
mechanism of this reaction was studied by using a combination
of kinetics experiments and DFT calculations. The biocompati-
bility of the probe was demonstrated in live HeLa cells.
Introduction
Zinc is an essential trace element in biology.[1,2] Although most
biological zinc occurs as a structural component of proteins,[3]
there are pools of readily chelatable, or mobile, zinc in specific
organs such as the brain,[4,5] pancreas,[6] and prostate.[7] In the
hippocampus, high concentrations of mobile zinc stored in
vesicles at glutamatergic synapses[8] have been conclusively as-
sociated with long-term potentiation.[9] Mobile zinc also plays
an important role in sensory perception.[10–13] In the pancreas,
insulin crystallization depends on the expression of the zinc
transporter ZnT8,[14] and zinc can be used as a surrogate for
imaging insulin secretion.[15] Mobile zinc accumulates in epithe-
lial cells in the prostate,[16] and a firm link has been established
between intracellular zinc levels and prostate cancer.[17]
long wavelengths. We recognized that switching reactions
offer an ideal platform for sensor development because of
their reversibility. The equilibrium between spirobenzopyrans
and their cyanine forms provides an excellent example of
a thermodynamically reversible switching reaction that has
been exploited extensively in materials science.[27] This reaction
is generally driven by light, but examples of chelation-induced
switching have also been reported.[28,29] Herein we report the
design, synthesis, and mechanistic study of SpiroZin1, a sensor
that comprises dipicolylamine as the zinc-binding unit, ap-
pended to a spirobenzopyran scaffold as well as its application
in live cell imaging.
The development of luminescent sensors for mobile zinc has
provided important tools for investigating its many biological Results and Discussion
roles.[18,19] Although these probes use various sensing mecha-
Synthesis and characterization
nisms, most rely on the modulation of photoinduced electron
transfer (PET) for their fluorescence turn-on response.[20] PET-
based probes are often sensitive to pH, however,[21,22] and do
not operate efficiently at long wavelengths (>700 nm). These
limitations restrict the information that can be obtained using
fluorescent sensors, especially because long-wavelength-emit-
ting probes are preferred for studies in vivo.[23,24]
SpiroZin1 was synthesized in a one-pot procedure starting
from commercially available dipicolylamine (DPA) and the read-
ily accessible building blocks 1 and 2 (Scheme 1A). The reac-
tion involves nucleophilic attack of DPA on the benzylic chlor-
ine of 2, followed by Knoevenagel condensation of the inter-
mediate aldehyde with 1. SpiroZin1 was isolated as a pale-
pink, waxy solid that forms nearly colorless solutions in
common organic solvents such as CH3OH, CH3CN, CH2Cl2, and
DMSO. 1H NMR spectroscopic measurements in CD3OD (Sup-
porting Information Figures SI-1 and SI-2) revealed two diaste-
reotopic singlets for the two methyl groups on the dihydroin-
dole ring and two diastereotopic doublets for the methylene
that connects the DPA arm (Scheme 1B) with the spirobenzo-
Probes that emit a luminescent signal following a chemical
reaction have been developed recently.[25,26] A limitation of
such “reaction-based” probes, however, is their irreversibility,
which precludes their application to real-time monitoring of
concentration changes.
In the present study, we sought to prepare a reaction-based
sensor for Zn2+ that is reversible, pH-insensitive, and emits at
pyran core (Figure SI-3). In addition, the two vinylic hydrogen
3
atoms on the benzopyran ring have a coupling constant JH-H
=
[a] Dr. P. Rivera-Fuentes, Prof. S. J. Lippard
Department of Chemistry, Massachusetts Institute of Technology
77 Massachusetts Ave, Cambridge, MA 02139 (USA)
E-mail: lippard@mit.edu
7.5 Hz, consistent with cis stereochemistry. These observations
demonstrate that SpiroZin1 exists predominantly as the spiro-
benzopyran isomer (Scheme 1B). Addition of one equivalent of
ZnCl2 turned the color of SpiroZin1 to a deep red. 1H NMR
spectra of this solution revealed only one singlet for the
Homepage: http://web.mit.edu/lippardlab/
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400014.
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508 nm produced fluorescence
in the deep-red region at lmax
=
650 nm (f=0.0042(7), Figure 1).
Despite the low quantum yield
of the probe in the zinc-bound
form, SpiroZin1 efficiently de-
tects mobile Zn2+ because of
the very low background in both
the absorption and emission
spectra of the apo form.
Titration of SpiroZin1 with
ZnCl2 revealed formation of a 1:1
complex (Figure SI-5). We pro-
pose a binding mode involving
the three nitrogen atoms of the
DPA arm and the oxygen atom
Scheme 1. A) One-pot synthesis of SpiroZin1. Reagents and conditions: a) K2CO3, EtOH, reflux, 2 h, 40%. B) Reversi-
on the cresol ring, which triggers
ble reaction of SpiroZin1 with Zn2+
.
the spirobenzopyran ring-open-
ing reaction. This binding mode
methyl groups on the indolenine ring and a singlet for the
methylene group that connects DPA with the rest of the mole-
cule (Scheme 1B). The coupling constant of the vinylic hydro-
gen atoms also changed to 3JH-H =16.5 Hz, consistent with
trans geometry of the alkene. These NMR signals prove that, in
the presence of Zn2+, the spirobenzopyran ring is opened to
produce the cyanine form.
maintains the [N3O] coordination sphere of previously reported
sensors, such as those in the ZP family,[30,31] ZPP1,[32] and the re-
cently reported ZBR probes.[33] SpiroZin1 chelates Zn2+ tightly
with an apparent dissociation constant (Kd) of 21(1) pm (Fig-
ure SI-6). A water molecule may be bound to zinc as well.
The selectivity of SpiroZin1 for biologically relevant metal
cations was investigated. Alkaline and alkali-earth metals pro-
duce no change in the fluorescence or absorbance spectra of
SpiroZin1 (Figure SI-7). Paramagnetic transition metals in-
creased the absorbance but not the fluorescence, suggesting
that they cause ring opening, but their partially filled d-orbitals
quench the fluorescence of the resulting cyanine. Cadmium in-
creases both fluorescence and the absorbance, but to a lesser
extent than zinc (Figure SI-7).
Sensing properties
The photophysical properties of SpiroZin1 were investigated in
aqueous buffer (50 mm PIPES, 100 mm KCl, pH 7). Under these
conditions, SpiroZin1 absorbs only weakly in the region be-
tween 400–700 nm (Figure 1). No fluorescence could be de-
tected for this solution upon excitation at 500 nm. Following
addition of 10 equivalents of ZnCl2, however, a strong band
appeared at lmax =508 nm (1.40(3)ꢁ104 mÀ1 cmÀ1). Excitation at
An advantage of SpiroZin1 is its ability to operate at low pH.
The fluorescence of SpiroZin1 turns on at pH 4, 5, and 6
(Figure 2), but this increase is very small compared with the
turn-on induced by Zn2+ at these pH values (Figure 2).
Mechanistic studies
SpiroZin1 is unique among reaction-based probes because of
its reversibility. We investigated the reaction kinetics of both
the turn-on event induced by Zn2+ and the turn-off process
caused by its removal following addition of the chelating
agent ethylenediaminetetraacetic acid (EDTA). The turn-on pro-
cess is very rapid and was monitored by stopped-flow spectro-
photometry. Similar results were obtained by stopped-flow
fluorescence spectroscopy (Figure SI-8). This study revealed
that turn-on occurs in two steps, each of which could be mod-
eled by a single exponential function (Figure 3). The first pro-
cess is fast with t1/2 =5.89(9) ms and the second step is slower,
with t1/2 =1.17(2) s. Overall, the turn-on event occurs within
seconds under physiological conditions. The turn-off process,
on the other hand, can be modeled by a single-exponential
decay as determined by fluorescence spectroscopy (Figure 3)
and is slow, with t1/2 =8.55(1) min.
Figure 1. Electronic absorption (solid lines) and fluorescence (dashed lines)
spectra of SpiroZin1 before (grey lines) and after (black lines) addition of
10 equiv of ZnCl2. All measurements were performed with 5 mm SpiroZin1 in
aqueous buffer (50 mm PIPES, 100 mm KCl, pH 7).
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qualitatively with the fast step observed by stopped-flow ki-
netics. The second step of the reaction is isomerization of the
alkene (D) to give the final trans cyanine (E). The higher calcu-
lated activation energy of this process is also consistent with
the slower kinetics measured. In the opposite direction, the
slow cis/trans isomerization occurs first and is the rate-deter-
mining step, which agrees with the single exponential decay
of fluorescence observed experimentally.
Live cell imaging
The ability of SpiroZin1 to report on the arrival of mobile zinc
in live cells was assessed by using human cervical cancer cells
(HeLa). HeLa cells were incubated at 378C with 5 mm SpiroZin1
for 30 min before imaging. Considerable intracellular red fluo-
rescence was detected under these conditions (Figure 5B). The
reason for this high background fluorescence might be the rel-
atively high concentration of endogenous mobile Zn2+ in HeLa
cells (~100 pm)[34] and the high Zn2+ affinity of SpiroZin1
(21(1) pm). The intracellular fluorescence, however, increased
further upon incubation of the cells with 20 mm ZnCl2 and
50 mm concentrations of the Zn2+ ionophore 2-mercaptopyri-
dine oxide (pyrithione) (Figure 5C). Quantification of the fluo-
rescence intensity revealed that addition of the exogenous
zinc elicited a 2.5(5)-fold turn-on. Addition of the cell-permea-
ble chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine
(TPEN) decreased the fluorescence, confirming that the fluores-
cence turn-on is induced by zinc (Figure 5D).
Figure 2. Integrated fluorescence intensity of 5 mm SpiroZin1 at various pH
values before (white bars) and after (grey bars) addition of 10 equiv of ZnCl2.
Intensities were normalized with respect to 5 mm SpiroZin1 at pH 7 before
addition of zinc.
To understand the mechanism of the sensing reaction of Spi-
roZin1, we carried out an intrinsic reaction coordinate analysis
using density functional theory (DFT) at the BP86/TZVP/
LANL2DZ level of theory. We decided to model only the elec-
tronic ground state of each reaction intermediate because the
experimental observations demonstrate chelation of Zn2+ to
be the main driving force of the reaction. We note that the
presence of light might influence the reaction mechanism and
that our calculations are meant to provide only a qualitative
representation.
Summary and Conclusions
We describe the design, synthesis, and characterization of
a red-emitting sensor for mobile zinc. This sensor, SpiroZin1, is
based on the ring-opening reaction of spirobenzopyran to
The reaction was modeled starting from a zinc complex of
SpiroZin1 in the spirobenzopyran form (Figure 4, structure A).
The first step in the reaction is ring opening (B) to afford a cis-
cyanine as an intermediate (C). The calculated activation
energy for this process is only 2.8 kcalmolÀ1, which agrees
form a fluorescent cyanine. This reaction is induced by Zn2+
,
can be reversed upon treatment with a chelator, and operates
even at low pH. Studies using stopped-flow kinetics, fluores-
Figure 3. A) Increase in absorbance at 508 nm of 5 mm SpiroZin1 in aqueous buffer (50 mm PIPES, 100 mm KCl, pH 7) after addition of 100 equiv of ZnCl2.
B) Decrease in integrated fluorescence (550–800 nm, lex =508 nm) of 5 mm zinc-bound SpiroZin1 in aqueous buffer (50 mm PIPES, 100 mm KCl, pH 7) after ad-
dition of 100 equiv of EDTA.
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(LRMS) were acquired on an Agi-
lent 1100 Series LC/MSD Trap spec-
trometer (LCMS), using electro-
spray ionization (ESI). High-resolu-
tion mass spectrometry (HR-ESI-
MS) was conducted by staff at the
MIT Department of Chemistry In-
strumentation Facility on a Bruker
Daltonics APEXIV 4.7 T FT-ICR-MS
instrument. The IUPAC name of
SpiroZin1 is provided and was de-
termined using CambridgeSoft
ChemBioDraw Ultra 12.0.
1-(Pyridin-2-yl)-N-(pyridin-2-yl-
methyl)-N-((1’,3’,3’,6-tetramethyl-
spiro[chromene-2,2’-indolin]-8-
yl)methyl)methanamine
Zin1): Compound
0.728 mmol) and K2CO3 (302 mg,
2.18 mmol) were added to
(Spiro-
(150 mg,
2
Figure 4. Intrinsic reaction coordinate analysis of the zinc-induced, ring-opening reaction of SpiroZin1: A) ring-
closed starting structure; B) ring-opening transition state; C) open, cis-cyanine intermediate; D) cis/trans isomeriza-
tion transition state; E) open, trans-cyanine product.
a Schlenk flask that was evacuated
and refilled with nitrogen three
cence spectroscopy, and DFT calculations provided mechanistic
details of the sensing mechanism. The biocompatibility of this
sensor was assessed in cultured HeLa cells.
times. A solution of dipicolylamine (290 mg, 1.46 mmol) in EtOH
(7 mL) was added via syringe. The mixture was stirred at 258C for
1 h. Compound 1 (219 mg, 0.728 mmol) was added as a solid in
one portion, and the mixture was heated at reflux for 1 h. The sus-
pension was filtered, and the filtrate was concentrated. The residue
was purified by flash column chromatography (CH2Cl2, 100%) to
give SpiroZin1 as a pale-pink solid (148 mg, 40%): mp: 50–518C;
The modular synthesis of SpiroZin1 allows modification of
the metal-binding site, which will be useful for tuning its affini-
ty and selectivity. We envision that this design principle will
inform the construction of probes that emit at even longer
wavelengths.
1
Rf =0.24 (CH2Cl2); H NMR (300 MHz, CD3OD): d=1.13 (s, 3H), 1.24
(s, 3H), 2.20 (s, 3H), 2.66 (s, 3H), 3.33 (d, J=18.1 Hz, 1H), 3.52 (d,
J=18.2 Hz, 1H), 3.59 (s, 4H), 5.71 (d, J=7.5 Hz, 1H), 6.47 (d, J=
7.5 Hz, 1H), 6.75–6.87 (m, 2H), 6.81 (s, 1H), 6.99 (s, 1H), 7.00–7.10
(m, 2H), 7.18–7.23 (m, 2H), 7.45 (d, J=6.1 Hz, 2H), 7.67–7.70 (m,
2H), 8.34 ppm (d, J=6.0 Hz, 2H); 13C NMR (75 MHz, CD3OD): d=
19.4, 19.6, 25.2, 28.1, 51.3, 52.0, 59.7, 104.4, 106.6, 118.99, 119.01,
121.2, 122.5, 123.3, 123.4, 126.5, 127.4, 128.9, 129.7, 131.2, 136.9,
137.5, 148.0, 148.2, 150.4, 159.3 ppm; HRMS-ESI: m/z calcd for
[C33H35N4O]+: 503.2811, found: 503.2818.
Experimental Section
Chemistry
General: All reagents were purchased from commercial sources
and used as received. Compounds 1[35] and 2[36] were prepared ac-
cording to published methods. Solvents were purified and de-
gassed by standard procedures. Thin-layer chromatography and
flash column chromatography were performed using Brockmann 1
basic Al2O3. NMR spectra were acquired on a Varian Inova-300 in-
Spectroscopic methods
1
strument. H and 13C NMR chemical shifts (d) are reported in parts
per million (ppm) relative to SiMe4 (d=0 ppm) and were refer-
enced internally with respect to residual solvent proton peaks
(1Hd=3.31 ppm and 13Cd=49.00 ppm for CH3OH). Coupling con-
stants (J) are reported in Hertz (Hz). Low-resolution mass spectra
All aqueous solutions were prepared using de-ionized water with
resistivity 18.2 mWcmÀ1, obtained using a Milli-Q water purification
system. Solvents were procured from Aldrich and used as received.
Piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) and 99.999% KCl
Figure 5. Microscopy images of live HeLa cells incubated with 5 mm SpiroZin1: A) differential interference contrast (DIC) image; B) red channel image before
addition of zinc pyrithione; C) red channel image after incubation with 20 mm zinc pyrithione; D) red channel image after incubating for 30 min with 50 mm
TPEN. Scale bar=25 mm.
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were purchased from Calbiochem. Stock solutions of SpiroZin1 in
DMSO were prepared at a concentration of 5 mm and stored at
À208C in 1 mL aliquots and thawed immediately before each ex-
periment. All spectroscopic measurements were conducted in
aqueous buffer containing 50 mm PIPES (pH 7) and 100 mm KCl.
UV/Vis spectra were acquired on a Cary 50 spectrometer using
quartz cuvettes from Starna (1 cm path length). Fluorescence spec-
tra were acquired on a Photon Technology International fluorime-
ter. All measurements were conducted at 258C maintained by cir-
culating water baths. Extinction coefficients were determined in
the 1–5 mm range in aqueous buffer. Fluorescence quantum yields
were determined by using 1–5 mm solutions of sensor in aqueous
buffer, exciting at 508 nm. Fluorescence emission spectra were in-
tegrated from 550 to 800 nm. The quantum yield calculation was
standardized to resorufin, which has a reported quantum yield of
0.74 at pH 9.5, lex =572 nm.[37]
10 s). A dead time determination indicated that the first usable
data points occur between 1 and 2 ms and the raw data was trun-
cated accordingly. The data used for analysis are averages of six in-
dependent measurements and were fit to a double exponential
function: A=A0 +B1[k t] +B2[k t], where A is the measured absorb-
ance, A0 the initial absorbance, k1 and k2 the rate constants of the
two steps, t is time, and B1 and B2 are constants. Minimization of
this equation gave the values k1 =178(3) sÀ1, k2 =0.59(1) sÀ1, R2 =
0.9997.
1
2
The ring-closing process was monitored by fluorescence spectros-
copy. A solution containing 5 mm SpiroZin1 and 5 mm ZnCl2 was
prepared and allowed to equilibrate in the dark for 1 h. The fluo-
rescence of this solution was measured and 100 equiv of EDTA
were added. The fluorescence intensity was measured every 30 s.
Fluorescence emission spectra at each time point were integrated
from 550 to 800 nm and plotted against time. The data were fit to
a single exponential function: F=F0 +B1[k t], where F is the integrat-
1
ed fluorescence, F0 the initial integrated fluorescence, k1 the rate
constant, t is time, and B1 a constant. Minimization of this equation
gave the values k1 =0.001351(1) sÀ1, R2 =0.99992.
pH Titration, metal selectivity, and dissociation constant
Buffered solutions at pH 3, 4, 5, 6, and 7 were prepared by mixing
different volumes of 0.1m citric acid and 0.2m Na2HPO4 in water
(Table SI-1) and the pH was verified using a Mettler Toledo FE20 pH
meter. The fluorescence of 5 mm SpiroZin1 in these buffered solu-
tions was measured before and after addition of 10 equiv of ZnCl2
and the intensities were normalized with respect to 5 mm SpiroZin1
at pH 7 before addition of ZnCl2.
DFT calculations
All calculations were carried out using ORCA 2.9.1[39] employing the
BP86 functional, the TZVP basis set for C, N, O, and H, and the
LANL2DZ basis set for Zn. The resolution of the identity (RI) ap-
proximation, with the appropriate auxiliary basis sets, was used in
all calculations. Geometry optimizations were performed in solu-
tion by applying the implicit conductor-like screening model
(COSMO) for water. The nature of the stationary points was charac-
terized by harmonic vibrational analysis. All minima showed zero
imaginary frequencies, whereas all transition states gave only one
imaginary frequency along the reaction coordinate. Cartesian coor-
dinates of all structures are provided in the Supporting Informa-
tion.
To determine the metal selectivity of SpiroZin1, PIPES-buffered sol-
utions (pH 7) containing 5 mm SpiroZin1 were prepared. To each
solution, 100 equiv of NaCl, MgCl2, CaCl2, CdCl2, NiCl2, CuCl2, MnCl2,
or CoCl2 were added and the fluorescence intensity measured. Cu+
was added as a solution of [Cu(CH3CN)4]PF6 in CH3CN to 5 mm Spi-
roZin1 in CH3CN under nitrogen. To the same solutions, 100 equiv
of ZnCl2 were added and the fluorescence intensity recorded. In
each case, the fluorescence intensity was normalized with respect
to 5 mm SpiroZin1 in aqueous buffer.
To determine the apparent dissociation constant (Kd), solutions
containing 1 mm 1,2-diaminohydroxypropanetetraacetic acid
(DHPTA) and ZnCl2 were prepared. The total concentration of Zn2+
was varied from 0.1 mm to 0.9 mm, to give buffered free Zn2+ con-
centrations ranging from 1.3ꢁ10À12 m to 1.1ꢁ10À10 m.[38] 5 mm
SpiroZin1 was added to each of these solutions, which were then
equilibrated in the dark for 24 h. The fluorescence of these solu-
tions was measured and normalized with respect to 5 mm Spiro-
Zin1 in the presence of 100 equiv of ZnCl2. The normalized fluores-
cence responses (R) were plotted against the concentration of free
Zn2+ [Zn] and the resulting data were fit to the equation
R=B[Zn]/(Kd+[Zn]), where B=1. Minimization of this equation
gave the values B=1.03(1), Kd =2.1(1)ꢁ10À11 m, and R2 =0.995.
Cell culture and staining procedures
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM; Cellgro, MediaTec Inc.), supplemented with 10% fetal
bovine serum (FBS; HyClone), 1% penicillin–streptomycin, 1%
sodium pyruvate, and 1% l-glutamine. The cells were grown to
90% confluence at 378C with 5% CO2 before being passed and
plated onto poly-d-lysine-coated plates 24 h prior to imaging. Cells
used were at passage number 7. A confluence level of 50% was
reached at imaging. The growth medium was replaced with dye-
free DMEM containing 5 mm SpiroZin1 and 2 mm Hoechst 33528
nuclear stain, and the cells were incubated for 30 min. Cells were
rinsed with PBS buffer (2ꢁ2 mL) before addition of fresh dye-free
DMEM (2 mL) and mounted on the microscope.
Kinetic experiments
Zinc-induced ring opening of SpiroZin1 was monitored by
stopped-flow UV/Vis spectroscopy configured with a single-wave-
length photomultiplier and a tungsten lamp. Stopped-flow data
were obtained using a Hi-Tech Scientific SF-61 DX2 stopped-flow
spectrophotometer. The optical change at 508 nm was recorded to
assess formation of the cyanine isomer.
Fluorescence microscopy
Imaging experiments were performed using a Zeiss Axiovert 200m
inverted epifluorescence microscope equipped with an EM-CCD
digital camera (Hamamatsu) and a MS200 XY Piezo Z stage (Ap-
plied Scientific Instruments). The light source was an X-Cite 120
metal-halide lamp (EXFO) and the fluorescence images were ob-
tained using an oil-immersion objective at 63ꢁ magnification. The
fluorescence filters sets used are defined as blue: excitation G
365 nm, beamsplitter FT 395 nm, emission BP 445/50 nm; red: BP
Solutions containing 5 mm SpiroZin1 and 500 mm ZnCl2 were pre-
pared in aqueous buffer (50 mm PIPES, 100 mm KCl, pH 7). The re-
actions were maintained at 258C with a circulating water bath.
Data points were collected on a logarithmic time scale (0.001–
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[12] S. M. Jo, M. H. Won, T. B. Cole, M. S. Jensen, R. D. Palmiter, G. Danscher,
Brain Res. 2000, 865, 227–236.
550/25 nm, beamsplitter FT 570 nm, emission BP 605/70 nm. The
microscope was operated using Volocity software (PerkinElmer).
[13] T. Perez-Rosello, C. T. Anderson, F. J. Schopfer, Y. Zhao, D. Gilad, S. R. Sal-
vatore, B. A. Freeman, M. Hershfinkel, E. Aizenman, T. Tzounopoulos, J.
Neurosci. 2013, 33, 9259–9272.
[14] K. Lemaire, M. A. Ravier, A. Schraenen, J. W. M. Creemers, R. Van de Plas,
M. Granvik, L. Van Lommel, E. Waelkens, F. Chimienti, G. A. Rutter, P.
Gilon, P. A. in’t Veld, F. C. Schuit, Proc. Natl. Acad. Sci. USA 2009, 106,
14872–14877.
[15] D. Li, S. Chen, E. A. Bellomo, A. I. Tarasov, C. Kaut, G. A. Rutter, W.-h. Li,
Proc. Natl. Acad. Sci. USA 2011, 108, 21063–21068.
[16] L. C. Costello, R. B. Franklin, P. Feng, Mitochondrion 2005, 5, 143–153.
[17] V. Kolenko, E. Teper, A. Kutikov, R. Uzzo, Nat. Rev. Urol. 2013, 10, 219–
226.
[18] E. L. Que, D. W. Domaille, C. J. Chang, Chem. Rev. 2008, 108, 1517–1549.
[19] Z. Huang, S. J. Lippard, Methods Enzymol. 2012, 505, 445–468.
[20] Z. Liu, W. He, Z. Guo, Chem. Soc. Rev. 2013, 42, 1568–1600.
[21] B. A. Wong, S. Friedle, S. J. Lippard, J. Am. Chem. Soc. 2009, 131, 7142–
7152.
The exposure times for acquisition of fluorescence images were
kept constant for each series of images at each channel. To mea-
sure analyte-induced fluorescence changes, the cells were treated
with dye-free DMEM containing 20 mm ZnCl2 and 50 mm pyrithione
for 10 min. To reverse the effect of zinc, the cells were exposed to
dye-free DMEM containing 50 mm TPEN for 15 min. All these experi-
ments were carried out on the stage of the microscope. Quantifica-
tion of fluorescence intensity was performed using ImageJ (version
1.45, NIH). The whole cell was selected as the region of interest.
The integrated fluorescence from the background region was sub-
tracted from the cell body region.
Acknowledgements
[22] Y. Koide, Y. Urano, K. Hanaoka, T. Terai, T. Nagano, ACS Chem. Biol. 2011,
6, 600–608.
This work was supported by US National Institutes of Health
(NIH) grant GM065519 from the US National Institute of General
Medical Sciences (NIGMS). Spectroscopic instrumentation at the
Massachusetts Institute of Technology Department of Chemistry
Instrumentation Facility (MIT–DCIF) is maintained with funding
from the US NIH (1S10RR13886-01). P.R.-F. thanks the Swiss Na-
tional Science Foundation for a postdoctoral fellowship, and Dr.
Patricia Marquꢀs Gallego, Dr. Wei Lin, and Ms. Alexandria D.
Liang are gratefully acknowledged for insightful discussions.
[23] J. V. Frangioni, Curr. Opin. Chem. Biol. 2003, 7, 626–634.
[24] S. A. Hilderbrand, R. Weissleder, Curr. Opin. Chem. Biol. 2010, 14, 71–79.
[25] J. Chan, S. C. Dodani, C. J. Chang, Nat. Chem. 2012, 4, 973–984.
[26] Y. Yang, Q. Zhao, W. Feng, F. Li, Chem. Rev. 2013, 113, 192–270.
[27] V. I. Minkin in Molecular Switches, 2nd edition, (Eds.: B. L. Feringa, W. R.
Browne), Wiley-VCH, Weinheim, 2011, p. 37–80.
[28] J.-F. Zhu, H. Yuan, W.-H. Chan, A. W. M. Lee, Org. Biomol. Chem. 2010, 8,
3957–3964.
[29] J.-F. Zhu, W.-H. Chan, A. W. M. Lee, Tetrahedron Lett. 2012, 53, 2001–
2004.
[30] S. C. Burdette, G. K. Walkup, B. Spingler, R. Y. Tsien, S. J. Lippard, J. Am.
Chem. Soc. 2001, 123, 7831–7841.
[31] E. M. Nolan, S. J. Lippard, Acc. Chem. Res. 2009, 42, 193–203.
[32] X.-a. Zhang, D. Hayes, S. J. Smith, S. Friedle, S. J. Lippard, J. Am. Chem.
Soc. 2008, 130, 15788–15789.
Keywords: bioinorganic chemistry · biosensors · coordination
chemistry · spirobenzopyran
[1] B. L. Vallee, K. H. Falchuk, Physiol. Rev. 1993, 73, 79–118.
[2] S. L. Kelleher, N. H. McCormick, V. Velasquez, V. Lopez, Adv. Nutr. 2011, 2,
101–111.
[3] W. Maret, Met. Ions Life Sci. 2013, 12, 479–501.
[4] C. J. Frederickson, J. Y. Koh, A. I. Bush, Nat. Rev. Neurosci. 2005, 6, 449–
462.
[33] W. Lin, D. Buccella, S. J. Lippard, J. Am. Chem. Soc. 2013, 135, 13512–
13520.
[34] Y. Qin, J. G. Miranda, C. I. Stoddard, K. M. Dean, D. F. Galati, A. E. Palmer,
ACS Chem. Biol. 2013, 8, 2366–2371.
[35] Y. Ueno, J. Jose, A. Loudet, C. Perez-Bolivar, P. Anzenbacher, Jr., K. Bur-
gess, J. Am. Chem. Soc. 2011, 133, 51–55.
[5] A. Takeda, M. Nakamura, H. Fujii, H. Tamano, Metallomics 2013, 5, 417–
423.
[36] P. Chirakul, P. D. Hampton, Z. Bencze, J. Org. Chem. 2000, 65, 8297–
8300.
[6] C. G. Taylor, BioMetals 2005, 18, 305–312.
[7] R. B. Franklin, B. Milon, P. Feng, L. C. Costello, Front. Biosci. 2005, 10,
2230–2239.
[8] P. Paoletti, A. M. Vergnano, B. Barbour, M. Casado, Neuroscience 2009,
158, 126–136.
[37] C. Bueno, M. L. Villegas, S. G. Bertolotti, C. M. Previtali, M. G. Neumann,
M. V. Encinas, Photochem. Photobiol. 2002, 76, 385–390.
[38] J. L. Vinkenborg, T. J. Nicolson, E. A. Bellomo, M. S. Koay, G. A. Rutter, M.
Merkx, Nat. Methods 2009, 6, 737–740.
[39] F. Neese, WIREs Comput. Mol. Sci. 2012, 2, 73–78.
[9] E. Pan, X.-a. Zhang, Z. Huang, A. Krezel, M. Zhao, C. E. Tinberg, S. J. Lip-
pard, J. O. McNamara, Neuron 2011, 71, 1116–1126.
[10] S. Redenti, R. L. Chappell, Vision Res. 2005, 45, 3520–3525.
[11] S. Redenti, H. Ripps, R. L. Chappell, Exp. Eye Res. 2007, 85, 580–584.
Received: January 3, 2014
Published online on && &&, 0000
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 7
&
6
&
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These are not the final page numbers!

FULL PAPERS
Seeing red! A red-emitting and pH-in-
sensitive probe for mobile zinc is report-
ed. The sensing mechanism is based on
a reversible ring-opening reaction that
is selectively induced by Zn2+. The bio-
compatibility of this probe was validat-
ed by fluorescence microscopy in live
cells.
P. Rivera-Fuentes, S. J. Lippard*
&& – &&
SpiroZin1: A Reversible and pH-
Insensitive, Reaction-Based, Red-
Fluorescent Probe for Imaging
Biological Mobile Zinc
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 7
&
7
&
ÞÞ
These are not the final page numbers!