Deep-Red-Fluorescent Zinc Probe with a Membrane-Targeting Cholesterol Unit

Article abstract of DOI:10.1021/acs.inorgchem.0c01376

Organelle-targeting fluorescence probes are valuable because they can provide spatiotemporal information about the trafficking of analytes of interest. The spatiotemporal resolution can be improved by using low-energy emission signals because they are barely contaminated by autofluorescence noises. In this study, we designed and synthesized a deep-red-fluorescent zinc probe (JJ) with a membrane-targeting cholesterol unit. This zinc probe consists of a boron-azadipyrromethene (aza-BODIPY) fluorophore and a zinc receptor that is tethered to a tri(ethylene glycol)-cholesterol chain. In aqueous solutions buffered to pH 7.4, JJ exhibits weak fluorescence with a peak wavelength of 663 nm upon excitation at 622 nm. The addition of ZnCl2 elicits an approximately 5-fold enhancement of the fluorescence emission with a fluorescence dynamic range of 141000. Our electrochemical and picosecond transient photoluminescence investigations indicate that the fluorescence turn-on response is due to the zinc-induced abrogation of the formation of a nonemissive intramolecularly charge-separated species, which occurs with a driving force of 0.98 eV. The fluorescence zinc response was found to be fully reversible and to be unaffected by pH changes or the presence of biological metal ions. These properties are due to tight zinc binding with a dissociation constant of 4 pM. JJ was found to be nontoxic to HeLa cells up to submicromolar concentrations, which enables cellular imaging. Colocalization experiments were performed with organelle-specific stains and revealed that JJ is rapidly internalized into intracellular organelles, including lysosomes and endoplasmic reticula. Unexpectedly, probe internalization was found to permeabilize the cell membrane, which facilitates the influx of exogens such as zinc ions. Such permeabilization does not arise for a control probe without the tri(ethylene glycol)-cholesterol chain (JJC). Our results show that the membrane-targeting cholesterol unit can disrupt membrane integrity.

Full text of DOI:10.1021/acs.inorgchem.0c01376

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Deep-Red-Fluorescent Zinc Probe with a Membrane-Targeting
Cholesterol Unit
Jin Ju Kim,† Jayeon Hong,† Seungyeon Yu, and Youngmin You*
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ABSTRACT: Organelle-targeting fluorescence probes are valuable
because they can provide spatiotemporal information about the
trafficking of analytes of interest. The spatiotemporal resolution can
be improved by using low-energy emission signals because they are
barely contaminated by autofluorescence noises. In this study, we
designed and synthesized a deep-red-fluorescent zinc probe (JJ) with
a membrane-targeting cholesterol unit. This zinc probe consists of a
boron-azadipyrromethene (aza-BODIPY) fluorophore and a zinc
receptor that is tethered to a tri(ethylene glycol)−cholesterol chain.
In aqueous solutions buffered to pH 7.4, JJ exhibits weak
fluorescence with a peak wavelength of 663 nm upon excitation at
622 nm. The addition of ZnCl2 elicits an approximately 5-fold
enhancement of the fluorescence emission with a fluorescence
dynamic range of 141000. Our electrochemical and picosecond transient photoluminescence investigations indicate that the
fluorescence turn-on response is due to the zinc-induced abrogation of the formation of a nonemissive intramolecularly charge-
separated species, which occurs with a driving force of 0.98 eV. The fluorescence zinc response was found to be fully reversible and
to be unaffected by pH changes or the presence of biological metal ions. These properties are due to tight zinc binding with a
dissociation constant of 4 pM. JJ was found to be nontoxic to HeLa cells up to submicromolar concentrations, which enables cellular
imaging. Colocalization experiments were performed with organelle-specific stains and revealed that JJ is rapidly internalized into
intracellular organelles, including lysosomes and endoplasmic reticula. Unexpectedly, probe internalization was found to
permeabilize the cell membrane, which facilitates the influx of exogens such as zinc ions. Such permeabilization does not arise for a
control probe without the tri(ethylene glycol)−cholesterol chain (JJC). Our results show that the membrane-targeting cholesterol
unit can disrupt membrane integrity.
zinc-induced fluorescence responses with high spatiotemporal
resolution and large dynamic ranges.42−55
INTRODUCTION
■
The human body contains a pool of free, mobile zinc.1−4
Intracellular levels of mobile zinc are tightly regulated between
the picomolar and micromolar concentrations by the actions of
metallothionein and the systems involving zinc-transporter
proteins.1,5−7 Failures in zinc homeostasis are intimately linked
to the genesis and development of pathological conditions.8−23
Growing evidence also indicates that free zinc mediates
physiological actions, including immune and brain func-
tion,24−33 gene transcription,34,35 apoptosis regulation,36−38
mammalian reproduction,39,40 and redox modulation.41 These
findings are part of a general consensus that the knowledge of
zinc biochemistry can provide useful insights into the
development of theranostic tools.
One promising strategy for maximizing the sensing
capability of zinc probes is their targeted delivery at specific
locations. Probe localization prevents the alteration of zinc
homeostasis in probe-free regions. The use of localizable zinc
probes also minimizes fluorescence artifacts due to environ-
mental effects such as pH and viscosity that vary over the range
of intracellular organelles. A particular advantage of localizable
zinc probes is the improved spatial resolution of zinc-releasing
events within the region of interest.
Previous research has established a library of organelle-
specific probes capable of reporting zinc in mitochondria,56−60
The study of zinc biology requires fundamental information
about the location, concentration, and trafficking of free zinc.
However, the d10 electronic configuration of Zn2+ precludes the
use of the multifarious spectroscopic techniques available for
the study of biological transition metals. Fluorescent probe
techniques have been the most successful for the study of free
zinc, as they are compatible with live specimens and afford
Received: May 11, 2020
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lysosomes,60−64 endoplasmic reticula,60,65,66 and the Golgi
apparatus.67,68 It is anticipated that combinations of these
probes will aid the research into intracellular zinc signaling
between organelles. Cell-membrane-targeting zinc probes have
a particular advantage in this area.69 Probes anchored to the
outer surface of the plasma membrane can monitor
intercellular zinc signaling between cells. In a central example
of such techniques, the fluorescence detection of synaptic
transmission that is mediated by the efflux of vesicular zinc
from presynaptic neurons has been reported.70 In addition, the
secretion of insulin can be traced by attaching fluorescence
zinc probes to the plasma membranes of pancreatic cells.71
These examples demonstrate the particular utility of
membrane-targeting zinc probes.
The majority of membrane-targetable zinc probes contain
alkyl moieties. The hydrophobic interactions of these moieties
with lipids result in the adhesion of the probe to the
extracellular surfaces of the plasma membranes of cells. Li
and co-workers pioneered this design strategy by adding two
dodecyl chains to fluorescein that possessed a zinc receptor at
its bottom ring.71 This zinc probe can be used to monitor the
exocytotic release of pancreatic zinc upon depolarization with
KCl. Similar strategies have recently been pursued by the
groups of Lippard and Xu.72,73 Watkinson and co-workers
explored synthetic approaches to creating membrane-targeting
zinc probes.60,74 A membrane-targetable, two-photon-excitable
fluorescence probe has also been fabricated through the
addition a nonyl chain.75 As an alternative to simple alkyl
chains, cholesterol has been found to facilitate the localization
of fluorescence probes on the outer membranes of cells. Taki
and co-workers demonstrated the fluorescence visualization of
extracellular zinc by using a fluorescein-based zinc probe with
appended cholesterol.76 The Li group recently extended this
strategy by replacing the hydrophobic units with a HaloTag
substrate. This genetically encoded zinc probe was found to be
capable of the visualization of secretory granular zinc in
pancreatic islet β cells.77 In a later study, the Li group further
modified the probe with truncated exendin-4, which binds
strongly with the glucagon-like peptide 1 receptors present on
the surfaces of β cells.78 An earlier study by Palmer and co-
workers revealed the membrane localization of a dyad
containing a cyan fluorescent protein (CFP) and a yellow
fluorescent protein (YFP) linked through a His4 motif.79
As outlined above, the design to date of membrane-
localizable probes has mainly focused on the targeting
moieties. The choice of fluorogenic scaffolds remains narrow;
only blue- or green-fluorescent platforms, including fluores-
cein,71,72,76−78 naphthyldiimide,60,73,74 and quinaldine,72 have
been demonstrated. Obviously, these fluorophores are not
perfect for bioimaging as they rely on relatively high-energy
excitation beams. Although the use of two-photon excitable
fluorescence reduces the photocytotoxicity,75 its applications
are limited because of the requirement of sophisticated laser
optics. These limitations indicate that zinc probes must exhibit
low-energy fluorescence emission upon single-photon excita-
tion.
Figure 1. Chemical structures of the fluorescence zinc probe (JJ) and
its control molecules (JJC and aza-BODIPY).
fold fluorescence turn-on response to zinc ions with a peak
emission wavelength of 663 nm under photoexcitation at 622
nm in aqueous buffers. Our spectroscopic and electrochemical
results indicate that the fluorescence turn-on signaling is
mediated by the zinc-induced abrogation of the photoinduced
one-electron transfer, which occurs with a driving force as large
as 0.98 eV even for a narrow bandgap energy (1.87 eV). This
fluorescence zinc signaling is not affected by changes in pH or
the presence of biological metal ions. JJ binds zinc tightly with
a zinc dissociation constant of 4 pM, which ensures high zinc
selectivity. The bioimaging utility was demonstrated by
treating HeLa cells with our probe. Our fluorescence
microscopic experiments show that JJ is promptly internalized
into intracellular regions while the cells remain live.
Unexpectedly, we found that this probe internalization
permeabilizes the cell membrane, which facilitates the entry
of extracellular zinc without the need for a pyrithione
ionophore.
RESULTS AND DISCUSSION
■
Design and Syntheses. Figure 1 depicts the chemical
structures of JJ and its control compounds. Aza-BODIPY was
chosen as a fluorescent scaffold because of its outstanding
emission properties in the deep-red emission region.81 An N-
(4-imidazolyl)-N-(2-picolyl)amino (IPA, hereafter) unit teth-
ered to a tri(ethylene glycol) (TEG, hereafter) linker serves as
the zinc chelator. The incorporation of the TEG moiety
improves the solubility of JJ in aqueous solutions. Finally,
cholesterol was introduced at the other end of the TEG linker
in order to direct the probe to the outer surfaces of the plasma
membranes of mammalian cells.82 A control zinc probe
without a TEG−cholesterol unit (JJC in Figure 1) was also
synthesized to assess the localization ability of JJ.
JJ was synthesized with a ten-step synthesis; Scheme 1
illustrates the synthetic route. The preparation of JJ
commenced with the tosylation of cholesterol. Nucleophilic
substitution of the p-toluenesulfonate group with tri(ethylene
glycol) afforded the TEG−cholesterol adduct (2) in a yield of
59%. The free hydroxyl group in the adduct was tosylated
again, followed by substitution with imidazole-4-carbaldehyde
in the presence of K2CO3 in CH3CN. Reductive amination of
the substitution product (4) and 2-picolylamine afforded the
zinc receptor (5) in a moderate yield. The synthesis of the
Herein, we report the design, synthesis, and zinc-sensing
applications of a deep-red-fluorescent probe tethered to a
membrane-targeting cholesterol unit (JJ, Figure 1). Cholesterol
interacts strongly with the plasma membrane of mammalian
cells, due to its high rigidity and lipophilicity. The unique
membrane anchoring ability was exploited to create mem-
brane-localizable probes of copper and zinc.76,80 JJ exhibits a 5-
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a
Scheme 1. Synthesis of JJ
a
The synthetic route for JJC is shown in the Supporting Information, Scheme S1.
deep-red fluorophore (aza-BODIPY) started with the Michael
addition reaction of nitromethane into trans-chalcone. A three-
component condensation of the Michael product (6) and
NH4OAc in trifluoroethanol yielded the free-base aza-BODIPY
(7). Borylation of 7 with BF3·OEt2 furnished aza-BODIPY (8)
in a yield of 51%. In order to introduce 5 into aza-BODIPY, 8
was formylated through the Villsmeyer−Haack reaction.
Finally, JJ was obtained through reductive amination between
5 and the formylated aza-BODIPY (9). The details of the
synthesis and structural characterization data are summarized
in the Experimental Details section.
JJ exhibits high solubility in polar organic solvents, such as
ethers, acetonitrile, and chlorinated solvents, but has moderate
solubility in water. To prevent the aggregation of probes, an
aqueous buffer (pH 7.4; 25 mM piperazine-N,N′-bis(2-
ethanesulfonic acid) (PIPES), 100 mM KCl) containing 75
vol % DMSO was employed in the photophysical inves-
tigations. A solution of 100 mM KCl was employed to simulate
the ionic strength of the biological milieu. Lower volume
fractions of DMSO in buffers led to probe aggregation,
resulting in inferior fluorescence zinc responses (Supporting
Information, Figure S1). The partition coefficient (clogP),
which was determined with the relationship clogP = log(C0/
Cw), where C0 and Cw are the concentrations of JJ in n-octane
and the PIPES-buffered solution (pH 7.4, 100 mM KCl)
respectively, is −0.47. This clogP value is much lower than that
of aza-BODIPY (2.95), which indicates that JJ has better
solubility in aqueous solutions.
Zinc-Induced Fluorescence Turn-On. Figure 2a shows
the steady-state electronic absorption and emission spectra of 5
μM JJ in aqueous buffers (pH 7.4). JJ exhibits a strong
absorption band with a peak wavelength (λabs) of 643 nm
(molar absorbance (ε) = 3.68 × 104 M−1 cm−1). Upon
photoexcitation at 622 nm, the zinc-free JJ solution exhibits
deep-red fluorescence emission with a peak wavelength (λems
)
of 663 nm and a photoluminescence quantum yield (PLQY) of
0.038. The electronic spectra of JJ are reminiscent of those of
aza-BODIPY (λabs = 655 nm (ε = 3.02 × 104 M−1 cm−1); λems
= 682 nm (PLQY = 0.30)), except for the low fluorescence
intensity (Figure S2). The addition of 100 equiv of ZnCl2 to
the JJ solution results in a hypsochromic shift of the absorption
peak wavelength to 637 nm (ε = 3.62 × 104 M−1 cm−1). The
zinc addition also elicits an approximately 5-fold enhancement
in the fluorescence intensity. The zinc-rich JJ solution has a
fluorescence peak wavelength of 662 nm with a PLQY of 0.21
(photoexcitation wavelength = 622 nm). The fluorescence
dynamic range, which is defined as the fluorescence turn-on
ratio × ε622 nm, is as large as 141000. The fluorescence sensing
ability is not perturbed by the presence of the TEG−
cholesterol unit because virtually identical zinc-induced
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recovery of the fluorescence spectrum to the zinc-free state
upon the addition of N,N,N′,N′-tetrakis(2-picolyl)-
ethylenediamine (TPEN), a strong zinc chelator (Figure 3).83
In order to identify the photophysical origin of the
fluorescence response, we performed transient photolumines-
cence experiments in CH3CN (Figure S4). The fluorescence
decay traces for 5 μM JJ were acquired with time-correlated
single-photon-counting techniques after picosecond pulsed
laser photoexcitation at 375 nm. A zinc-free solution of JJ
produces a biphasic exponential decay with a weighted average
lifetime (τobs) of 0.23 ns. The τobs value increases to 1.3 ns
upon the addition of 100 equiv of zinc ions. The zinc-induced
increase in τobs corresponds to a one order of magnitude
decrease in the nonradiative decay rate (knr) from 42 × 108 s−1
(zinc-free state) to 6.1 × 108 s−1 (zinc-bound state), while the
radiative decay rate (kr) remains relatively unaltered (zinc-free
state, 1.7 × 108 s−1; zinc-bound state, 1.6 × 108 s−1).
Qualitatively identical changes were also found for JJC (zinc-
free state, kr = 2.9 × 108 s−1, knr = 53 × 108 s−1; zinc-bound
state, kr = 3.0 × 108 s−1, knr = 8.4 × 108 s−1). This nonradiative
control indicates the presence of a fluorescence-quenching
process that is abolished by zinc binding.
Figure 4 compares the cyclic and differential pulse
voltammograms of aza-BODIPY, JJC, and JJ. Aza-BODIPY
exhibits reversible one-electron reduction at a potential (Ered)
of −0.80 V vs Fc+/Fc. The oxidation process is irreversible,
and the differential pulse voltammogram shows that the
oxidation potential (Eox) is at 0.81 V vs Fc+/Fc. The
corresponding electrochemical bandgap energy (1.61 eV) is
comparable to the optical bandgap energy (ΔEg, 1.82 eV)
determined from the fluorescence peak wavelength (λems = 682
nm). For JJC, the first Eox is at 0.04 V vs Fc+/Fc, i.e., it is
cathodically shifted from that (0.81 V vs Fc+/Fc) of aza-
BODIPY. It is reasonable to assign this oxidation to originate
in the tertiary amino group in the zinc receptor. In contrast,
the first reduction potential of JJC is identical to that of aza-
BODIPY. This electrochemical alignment permits photo-
induced one-electron transfer from the zinc receptor to the
excited-state aza-BODIPY. The excited-state reduction poten-
Figure 2. UV−vis absorption (dotted lines) and fluorescence (solid
lines) spectra of (a) 5 μM JJ and (b) 5 μM JJC in the absence (black)
and presence (red) of 100 equiv of ZnCl2. Samples were dissolved in
aqueous solutions containing 25 mM PIPES, 100 mM KCl, and 75 vol
% DMSO buffered at pH 7.4. The peaks marked with an asterisk (*)
correspond to the excitation beam (λex = 622 nm).
fluorescence turn-on (4.8) and dynamic range (153000) values
are found for JJC (Figure 2b). The results indicate that the
TEG−cholesterol unit does not perturb zinc coordination.
Similarly, zinc responses with greater turn-on ratios were
observed in CH3CN (Figure S3). Finally, the fluorescence zinc
response of JJ is fully reversible, as is demonstrated by the
Figure 3. Reversible fluorescence zinc sensing of JJ. (a) Fluorescence spectra and (b) the corresponding integrated fluorescence intensities of 5 μM
JJ in the absence (black) and presence (red) of 1 equiv of ZnCl2 and after subsequent addition of 2 equiv of TPEN (gray). Samples were dissolved
in aqueous solutions containing 25 mM PIPES, 100 mM KCl, and 75 vol % DMSO buffered at pH 7.4. The peaks marked with an asterisk (*)
correspond to the excitation beam (λex = 622 nm).
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Figure 4. Driving force for intramolecular photoinduced one-electron transfer. (a) Cyclic (CV, solid curves) and differential pulse (DPV, dashed
curves) voltammograms of 2.0 mM aza-BODIPY (blue), 2.0 mM JJC (red), and 2.0 mM JJ in the absence (black) and presence (gray) of 10 equiv
of Zn(ClO4)2 obtained in Ar-saturated THF/DMSO (4:1, v/v) containing 0.10 M TBAPF6 supporting electrolyte. A Pt disk and a Pt wire were
used as the working and counter electrodes, respectively, and an Ag/AgNO3 (10 mM) pseudo-reference electrode was used (scan rate = 0.10 V s−1
(CV) and 4 mV s−1 (DPV)). The vertical blue and red dotted lines indicate the one-electron oxidation of aza-BODIPY and the tertiary amino units
in JJ and JJC, respectively. (b) Electrochemical potentials of the photoexcited JJC (left) and the charge-separated state of JJC (right). The black
arrow indicates photoinduced one-electron transfer with a driving force of 0.98 eV.
Table 1. Photophysical Parameters of JJ, JJC, and Aza-BODIPY
λabs (nm)
a
ab
,
/ε (104 M−1 cm−1
)
λems (nm) /PLQY
τobs (ns)
kr (108 s−1
)
knr (108 s−1
)
100 equiv
of ZnCl2
100 equiv
of ZnCl2
turn-on
ac
(100 equiv of Zn2+
(100 equiv of Zn2+
(100 equiv of Zn2+
dg
,
de
,
df
,
,
no ZnCl2
no ZnCl2
ratio/DR
/no Zn2+)
/no Zn2+)
/no Zn2+)
JJ
JJC
aza-BODIPY
643/3.68
636/3.93
655/3.02
637/3.62
629/3.69
−
663/0.038
657/0.053
682/0.30
662/0.21
655/0.26
−
5.4/141000
4.8/153000
−
1.3/0.23
1.6/1.7
3.0/2.9
−
6.1/42
8.4/53
−
0.88/0.18
h
h
h
h
h
h
−
a
5 μM in aqueous solutions containing 25 mM PIPES, 100 mM KCl, and 75 vol % DMSO buffered at pH 7.4. Data obtained with differed volume
b
fractions of DMSO are collected in Table S1. λex = 622 nm. Photoluminescence quantum yield (PLQY) determined relative to the 9,10-
diphenylanthracene standard (toluene, PLQY = 1.0).85 Dynamic range (DR) corresponds to DR = turn-on ratio × ε622 nm (zinc-free). 5 μM in
c
d
e
Ar-saturated CH3CN. Weighted average fluorescence lifetime, τobs = (Σai × τi2)/ (Σai × τi) (i = 1−2), where ai and τi are the pre-exponential
f
g
factor and the time constant, respectively. Radiative decay rate, kr = PLQYCH3CN/τobs. Nonradiative decay rate, knr = (1 − PLQYCH3CN)/τobs. The
PLQYCH3CN values in kr and knr were determined in Ar-saturated CH3CN: JJ, 0.0055 (no Zn2+) and 0.35 (100 equiv of Zn2+); JJC, 0.0049 (no
h
Zn2+) and 0.23 (100 equiv of Zn2+). Not determined.
tial (E*red) of aza-BODIPY (or JJC) was calculated to be 1.02
V vs Fc+/Fc by using the relationship E*red = Ered + ΔEg. This
E*red value is more positive than the Eox value (0.04 V vs Fc+/
Fc) of the zinc receptor. The driving force for one-electron
transfer (−ΔGeT) is as large as 0.98 eV according to the
Rehm−Weller equation −ΔGeT = e × [Eox (zinc receptor) −
E*red (aza-BODIPY)], where e is the elementary charge.84 JJ is
predicted to enable the same photoinduced intramolecular
electron transfer because it exhibits a bandgap energy and
electrochemical potentials identical to those of JJ (Figure 4a).
The addition of zinc ions into the JJ solution results in the
disappearance of the first oxidation (Eox = 0.04 V vs Fc+/Fc),
which suggests thermodynamic disallowance of the electron
transfer. Taking these results together, we conclude that the
zinc-induced fluorescence turn-on response is due to the
abrogation of intramolecular photoinduced electron transfer. It
has been challenging to modulate photoinduced electron
transfer involving deep-red emitters.86−88 Therefore, our
demonstration is a significant one. The photophysical data
for JJ, JJC, and aza-BODIPY are summarized in Table 1.
Fluorescence Zinc Detection. A fluorescence Job plot
indicates that the binding of JJ with zinc ions is 1:1 (Figure
S5). In order to assess the zinc detection ability, zinc titration
experiments were performed. Figure 5 shows the fluorescence
spectra of 5 μM JJ recorded in various zinc-buffered solutions
at pH 7.4 (free zinc concentration ([Zn]free) = 0.15 pM to 120
nM; 100 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesul-
fonic acid) (HEPES), 100 mM NaNO3, 10 mM N-(2-
hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid
(HEDTA), and ZnSO4). The corresponding fluorescence
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Figure 5. Determination of the zinc dissociation constant (Kd (Zn)) of JJ. (a) Fluorescence spectra of 5.0 μM JJ obtained in 15 zinc-buffered
aqueous solutions (pH 7.4; 100 mM HEPES, 100 mM NaNO3, 10 mM HEDTA, and ZnSO4). (b) Corresponding fluorescence zinc titration
isotherm (black squares) and a nonlinear least-squares fit to a 1:1 binding model (red curve). The peak marked with an asterisk (*) in (a)
corresponds to the excitation beam (λex = 622 nm). The error bars were obtained from three experiments.
titration isotherm has a typical sigmoidal form. A nonlinear
least-squares fit of the isotherm to a 1:1 binding model
indicated that the zinc dissociation constant (Kd (Zn)) is 4
pM. A similar Kd (Zn) was found for JJC (3 pM) (Figure S6).
These tight-binding behaviors are comparable to those of a
previous BODIPY-based probe bearing a structurally similar
di(2-picolyl)amine zinc receptor.89 Note that the zinc-binding
affinity depends sensitively on the volume fraction of DMSO.
A nonsigmoidal behavior is found for the fluorescence zinc
titration isotherm at an increased (80 vol %) DMSO fraction
(Figure S7).
The fluorescence zinc signaling of JJ is hardly perturbed by
pH changes. The fluorescence intensity is weak at basic pH
values and starts to increase for pH < 7. As shown in Figure 6,
pH titration experiments show that pKa is as low as 5.9. The
pKa of JJC is 6.1, which is close to that of JJ (Figure S8). This
similarity suggests that the fluorescence increase results from
the protonation of the zinc receptor. However, it is inferred
from the pKa value that the protonation-induced fluorescence
artifact is minimal at physiologically relevant pH values (∼7.4).
Note that the fluorescence turn-on response is preserved at low
pH values (Figure 6c). This pH tolerance is ascribed to tight
zinc binding; the zinc association is 300000-fold stronger than
the proton association according to the ratio Ka/Kd(Zn).
The fluorescence signaling of JJ is selective to zinc over
biological metal ions. The presence of 1.0 mM NaCl, 100 μM
MgCl2, and 1.0 mM CaCl2 does not interfere with the
fluorescence zinc response of 5 μM JJ (Figure 7). The presence
of 10 μM biologically important transition-metal ions,
including Cr, Mn, Fe, Co, and Ni, does not perturb zinc
binding. The Cu ion competes with zinc for coordination with
JJ, but zinc coordination appears stronger than copper binding
because a prompt turn-on response is elicited upon the
addition of zinc to a mixture of JJ and CuCl2. Finally, the
fluorescence zinc response is not affected by the presence of
Cd and Hg ions, which are strong competitors of zinc toward
coordination. Virtually identical zinc selectivity is observed for
JJC (Figure S9). From the results of the cuvette experiments,
we conclude that JJ exhibits turn-on responses exclusively to
zinc ions.
Fluorescence Visualization of Cellular Zinc. To assess
the usefulness of our probe in bioimaging, live HeLa cells were
treated with JJ. NucBlue Live/NucGreen Dead assays indicate
that the cytotoxicity of JJ is low; NucBlue Live and NucGreen
Dead selectively stain live and dead cells, respectively. Figure
8a demonstrates that the majority of HeLa cells are stained
blue (i.e., live) when they are incubated with 0.4 and 0.8 μM JJ
(10 min). Increases in the JJ concentration to 2 and 20 μM (10
min) lead to cell death, as can be seen in the emergence of cells
stained green. Similar results were obtained in MTT cell
viability assays (Figure 8c). The cell survival curve starts to
decay for JJ concentrations >1 μM (2 h incubation at 37 °C).
These viability results and the red fluorescence emission of JJ
(Figure 8a) ensures that there is perceptible zinc imaging after
incubation at submicromolar concentrations.
Figure 9 compares the fluorescence micrographs of HeLa
cells coincubated with organelle-specific fluorescence stains,
including nuclear stain, endoplasmic reticulum stain, lysosomal
stain, mitochondrial stain, and membrane stain. The overlay
images reveal that the fluorescent puncta of JJ overlap with
those of lysosomes and endoplasmic reticula. Pearson’s
colocalization coefficients are 0.82 and 0.61 for the former
and latter, respectively. In contrast, the Pearson’s colocalization
coefficient for the membrane stain is 0.12. JJC, the control
probe devoid of the TEG−cholesterol unit, is also found to
localize within the endoplasmic reticula and lysosomes (Figure
S10). The results suggest that the aza-BODIPY−zinc receptor
unit (i.e., JJC moiety) is responsible for the localization within
the lysosomes and endoplasmic reticula.
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Figure 6. pH tolerance of the fluorescence zinc responses of JJ. (a) Fluorescence spectra of 10 μM JJ in an aqueous solution (75 vol % DMSO)
containing 100 mM KCl for various pH values (10.30 to 4.59). (b) The corresponding fluorescence pH titration isotherm (black squares) and its
nonlinear least-squares fit to the equation FI = (AKa + B[H+])/([H+] + Ka) (red curve), where FI is the integrated fluorescence intensity and A and
B are fitting constants. (c) Integrated fluorescence intensities of 10 μM JJ in the absence (gray bars) and presence (black bars) of 10 equiv of ZnCl2
at various pH values.
This poor localization at the plasma membrane indicates
that the internalization of JJ is rapid after extracellular
membrane anchoring. Actually, live imaging experiments reveal
prompt anchoring of JJ at the plasma membrane of HeLa cells,
followed by subsequent uptake into cytoplasm (Figure S11 and
Movie S1). To determine the internalization mechanism, HeLa
cells were treated with 0.4 μM JJ under various conditions.
Figure 10 shows that the intracellular uptake of JJ is retarded
upon incubation at 4 °C or upon pretreatment with metabolic
inhibitors (50 mM 2-deoxy-D-glucose/5 μM oligomycin) or
endocytic inhibitors (5 μM chloroquine or 50 mM
NH4Cl).90,91 This retardation suggests that the cellular entry
of JJ occurs via various mechanisms, including passive
diffusion, active transport, and endocytosis. The rapid uptake
of JJ could be a corollary of the presence of a multitude of
intracellular uptake pathways.
because it is not anticipated that ZnCl2 will penetrate the cell
membrane without the help of a pyrithione (PT) ionophore.
Subsequent treatments with 200 μM ethylenediaminetetra-
acetic acid (EDTA), a cell-impermeable metal chelator,
restored the fluorescence intensity to the original state.
Identical behaviors were also found for JJ-treated DLD-1
cells (Figure S12). The observed fluorescence switching with
ZnCl2 and EDTA indicates that JJ permeabilized the cell
membrane. In sharp contrast, JJC devoid of the TEG−
cholesterol moiety does not evoke such permeabilization
(Figure 11b). Fluorescence turn-on responses are produced
only when ZnCl2 is present together with the PT ionophore.
These results suggest that the membrane-targeting TEG−
cholesterol unit has an antagonistic effect. This observation is
̈
consistent with the results of the groups of Stadler and
Wustner, where cholesterol has large effects on membrane
̈
Finally, the fluorescence zinc detection of JJ was evaluated
(Figure 11a). HeLa cells were incubated with 0.2 μM JJ for 20
min. JJ was internalized into the cells and found to exhibit
weak fluorescence intensities. An addition of 20 μM ZnCl2 into
the extracellular medium elicits an approximately 2-fold
fluorescence enhancement. This zinc response is unexpected
permeability.82,92 Anchored cholesterol can increase the free
volume of a lipid bilayer and, thereby, facilitates the diffusion
and transport of exogens.93 Our research shows that the
judicious use of cholesterol is essential for the immobilization
of probes.
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intercellular zinc trafficking. We have designed a deep-red
fluorescent zinc probe (JJ) with a focus on bioimaging utility.
Aza-BODIPY was modified with a N-(4-imidazolyl)-N-(2-
picolyl)amino zinc receptor, which was tethered to a
membrane-targeting cholesterol unit through a tri(ethylene
glycol) linker. JJ exhibits a reversible, approximately 5-fold
turn-on response to zinc ions in the deep-red emission region
(λems = 662 nm) in aqueous solutions buffered to pH 7.4. We
demonstrated that the fluorescence zinc signaling of our probe
is not affected by changes in pH and the presence of biological
metal ions. This high zinc selectivity is due to tight zinc
binding with a dissociation constant of 4 pM. Our
spectroscopic and electrochemical results show that electron
transfer occurs from the zinc receptor to photoexcited aza-
BODIPY to form a nonemissive, intramolecularly charge-
separated species with a driving force of 0.98 eV. The zinc
binding of JJ abrogated the nonradiative photoinduced
electron transfer to restore the inherent emission of aza-
BODIPY. JJ is nontoxic to HeLa cells, which implies that it can
be used for bioimaging in the deep-red-fluorescence region.
Unexpectedly, JJ was found to leak across the outer surface of
the plasma membrane and to internalize rapidly in the
lysosomes and endoplasmic reticula of HeLa cells. Our cellular
experiments have shown that the intracellular entry of JJ
involve a multitude of uptake mechanisms. This probe
internalization permeabilizes the plasma membrane, which
permits the influx of exogenous zinc ions into the intracellular
Figure 7. Fluorescence selectivity of JJ for zinc over biological metal
ions. Integrated fluorescence intensities of 5 μM JJ in the absence
(light gray bars) and presence (dark gray bars) of competing metal
ions and after the subsequent addition of 10 equiv of ZnCl2 (black
bars) (25 mM PIPES, 100 mM KCl, and 75 vol % DMSO buffered at
pH 7.4): 1.0 mM NaCl, 100 μM MgCl2, 1.0 mM CaCl2, and 10 μM
other metal chlorides.
SUMMARY AND CONCLUSIONS
■
Fluorescence zinc probes targeting specific organelles are
valuable because they enable the monitoring of intra- and
Figure 8. Cytotoxicity of JJ. (a) Micrographs showing the NucBlue Live/NucGreen Dead assay of HeLa cells treated with 0, 0.4, 0.8, 2, and 20 μM
JJ for 10 min. From the left, bright-field images, micrographs showing JJ fluorescence (λex = 633 nm, λem = 640−677 nm), NucBlue Live
fluorescence (λex = 488 nm, λem = 415−460 nm), and NucGreen Dead fluorescence (λex = 561 nm, λem = 499−552 nm), and overlay images are
shown. Scale bar = 100 μm. (b) Corresponding percent cell survival results. % cell survival = 100 × (number of live cells)/(number of total cells).
(c) MTT cell proliferation assay results. HeLa cells were treated with 0.1−40 μM JJ at 37 °C for 2 h.
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Figure 9. Intracellular localization of JJ in HeLa cells. First column panels, bright images; second column panels, fluorescence images of organelle-
specific stains; third column panels, fluorescence images of JJ (0.2 μM, 10 min, λex = 633 nm/λem = 646−684 nm); fourth column panels, overlay
images. From the top: nuclear stain, DAPI (300 nM, 5 min, λex = 488 nm/λem = 417−456 nm); endoplasmic reticulum (ER) stain, ER−Tracker
Green (1 μM, 20 min, λex = 488 nm/λem = 498−553 nm); lysosomal stain, LysoTracker Green (75 nM, 30 min, λex = 488 nm/λem = 498−553 nm);
mitochondrial stain, MitoTracker Green (200 nM, 15 min, λex = 488 nm/λem = 498−553 nm); membrane stain, CellMask Green Plasma
Membrane (×1000, 5 min, λex = 488 nm/λem = 498−553 nm). Pearson’s colocalization coefficients: nuclei, 0.17; endoplasmic reticula, 0.61;
lysosome, 0.82; mitochondria, 0.26; plasma membrane, 0.12. Scale bar = 50 μm.
a magnetic stir bar. After the slow addition of p-toluenesulfonyl
chloride (54.2 g, 284 mmol), the reaction mixture was stirred for 3
days at room temperature. Water (400 mL) was poured onto the
solution, and the crude product was extracted with diethyl ether (400
mL × three times). The collected organic layer was dried over
anhydrous MgSO4. Concentration under reduced pressure afforded a
compartments. A control probe that does not contain a
cholesterol unit was found not to elicit such permeabilization.
These results demonstrate that careful structural control is
required to obtain fluorescence signaling without altering
cellular function.
1
white powder in an 86% yield. Rf = 0.7 (CH2Cl2). H NMR (300
EXPERIMENTAL SECTION
Materials and Structural Identification. Commercially avail-
MHz, CDCl3) δ (ppm): 0.65 (s, 3H), 0.86 (dd, J = 6.6, 1.5 Hz, 6H),
0.90 (d, J = 6.6 Hz, 3H), 0.96 (s, 3H), 2.45 (s, 3H), 4.32 (m, 1H),
5.30 (d, J = 5.4 Hz, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 8.1 Hz,
2H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm): 11.8, 18.7, 19.2,
21.0, 21.7, 22.6, 22.8, 23.8, 24.3, 28.0, 28.2, 28.6, 31.7, 31.9, 35.8,
36.2, 36.4, 36.9, 38.9, 39.5, 39.7, 42.3, 49.9, 56.1, 56.7, 82.4, 123.5,
127.7, 129.7, 134.7, 138.9, 144.4.
Synthesis of 2. Compound 1 (40.0 g, 74.0 mmol) and
tri(ethylene glycol) (20.0 mL, 151 mmol) were combined in a 1 L
one-necked round-bottom flask. The reaction mixture was dissolved
■
1
able chemicals were used as received unless otherwise stated. H and
13C{1H} NMR spectra were collected with Bruker Ultrashield 500
and 300 plus NMR spectrometers. Chemical shifts were referenced to
(CH3)4Si. High-resolution mass spectra (positive mode, FAB, m−
NBA) were obtained by employing a JEOL, JMS-600W mass
spectrometer.
Synthesis of 1. Cholesterol (100 g, 259 mmol) was dissolved in
pyridine (350 mL) in a one-necked round-bottom flask equipped with
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Figure 10. Intracellular uptake of JJ. (a) Micrographs showing HeLa cells treated with 0.4 μM JJ (10 min) under various conditions. Top panels,
bright-field images; middle panels, fluorescence images (λex = 633 nm, λem = 640−677 nm); bottom panels, overlay images. Scale bar = 50 μm. (b)
Corresponding fluorescence intensities of the HeLa cells. Error bars were determined from five dishes treated independently.
Figure 11. Visualization of intracellular zinc ions. (a) Top panels, bright-field (1st panel) and fluorescence micrographs (2nd to 4th panels) of
HeLa cells treated with 0.2 μM JJ (20 min), after subsequent incubation with 20 μM ZnCl2 (5 min), and finally, after the addition of 200 μM
EDTA. Bottom panels, schematic representation of the JJ-induced permeabilization of the cell membranes. (b) Top panels, bright-field (1st panel)
and fluorescence micrographs (2nd and 3rd panels) of HeLa cells treated with 0.2 μM JJC (20 min) and after subsequent incubation with 20 μM
ZnCl2 (15 min); bottom panels, bright-field (1st panel) and fluorescence micrographs (2nd and 3rd panels) of HeLa cells treated with 0.2 μM JJC
(20 min) and after subsequent incubation with 20 μM ZnPT2 (15 min). (c) The corresponding fluorescence intensities of the fluorescence
micrographs. Error bars were determined from five dishes treated independently.
in dioxane (400 mL) and refluxed for 3 days. After it cooled to room
temperature, the reaction mixture was poured onto water (400 mL).
The crude product was recovered by extracting it with EtOAc (400
mL × three times). The organic layer was washed with a 5% aqueous
solution of KHCO3 and water, dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. Purification via silica gel
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column chromatography was performed with employing diethyl
ether/EtOAc = 8:2 (v/v) as an eluent to give a white solid in a 59%
yield. Rf = 0.3 (diethyl ether/EtOAc = 8:2, v/v). 1H NMR (300 MHz,
CDCl3) δ (ppm): 0.67 (s, 3H), 0.86 (dd, J = 6.6, 1.5 Hz, 6H), 0.91
(d, J = 6.6 Hz, 3H), 1.00 (s, 3H), 3.19 (m, 1H), 3.68 (m, 12H), 5.34
(d, J = 5.4 Hz, 1H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm):
11.9, 18.7, 19.4, 21.1, 22.6, 22.8, 23.8, 24.3, 28.0, 28.2, 28.3, 31.9,
32.0, 35.8, 36.2, 36.9, 37.2, 39.0, 39.5, 39.8, 42.3, 50.2, 56.2, 56.8,
61.8, 67.2, 70.4, 70.6, 70.9, 72.5, 80.0, 121.6, 140.9. HR MS (FAB+,
m−NBA): calcd for C33H58O4Na ([M + Na]+), 541.4233; found,
541.4229.
Synthesis of 3. Compound 2 (18.8 g, 36.3 mmol) was dissolved
in a mixture of pyridine (60 mL) and chloroform (120 mL) in a 500
mL one-necked round-bottom flask. p-Toluenesulfonyl chloride (8.99
g, 47.1 mmol) was added to the stirred solution at 0 °C. The reaction
mixture was stirred at 0 °C for 6 h and at room temperature for an
additional 11 h. The solution was concentrated under reduced
pressure, and the concentrate was poured onto water (400 mL). The
crude product was recovered with EtOAc (400 mL × three times).
The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. Purification via silica gel
column chromatography was performed by varying the eluent from
EtOAc/hexane = 1:4 (v/v) to 100% EtOAc to increase its polarity. A
pale yellow oil was obtained in an 83% yield. Rf = 0.6 (EtOAc/hexane
= 1:4, v/v). 1H NMR (300 MHz, CDCl3) δ (ppm): 0.68 (s, 3H), 0.86
(dd, J = 6.6, 1.2 Hz, 6H), 0.91 (d, J = 6.6 Hz, 3H), 0.99 (s, 3H), 2.45
(s, 3H), 3.17 (m, 1H), 3.60 (m, 8H), 3.69 (t, J = 4.8 Hz, 2H), 4.16 (t,
J = 4.8 Hz, 2H), 5.34 (d, J = 5.4 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H),
7.80 (d, J = 8.1 Hz, 2H). 13C{1H} NMR (126 MHz, CDCl3) δ
(ppm): 11.9, 18.7, 19.4, 21.1, 21.7, 22.6, 22.8, 23.8, 24.3, 28.0, 28.2,
28.4, 31.9, 32.0, 35.8, 36.2, 36.9, 37.2, 39.1, 39.5, 39.8, 42.3, 50.2,
56.2, 56.8, 67.3, 68.7, 69.2, 70.6, 70.8, 70.9, 79.5, 121.6, 128.0, 129.8,
133.0, 141.0, 144.8. MS (FAB+, m−NBA): calcd for C40H65O6S ([M
+ H]+), 673.45; found, 673.00.
CH3OH = 9:1 (v/v) to increase its polarity. A yellow oil was isolated
with a 10% yield. Rf = 0.1 (CH2Cl2/CH3OH = 5:1, v/v). H NMR
1
(300 MHz, CDCl3) δ (ppm): 0.67 (s, 3H), 0.86 (dd, J = 6.6, 1.2 Hz,
6H), 0.91 (d, J = 6.6 Hz, 3H), 0.99 (s, 3H), 3.17 (m, 1H), 3.61 (m,
8H), 3.73 (t, J = 5.1 Hz, 2H), 3.82 (s, 2H), 4.00 (s, 2H), 4.06 (t, J =
5.1 Hz, 2H), 5.33 (d, J = 5.1 Hz, 1H), 6.93 (s, 1H), 7.16 (m, 1H),
7.37 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 1.2 Hz, 1H), 7.64 (td, J = 7.8,
1.8 Hz, 1H), 8.55 (m, 1H). 13C{1H} NMR (126 MHz, CDCl3) δ
(ppm): 11.9, 18.7, 19.4, 21.1, 22.6, 22.8, 23.8, 24.3, 28.0, 28.2, 28.4,
29.7, 31.9, 31.94, 35.8, 36.2, 36.9, 37.2, 39.1, 39.5, 39.8, 42.3, 46.7,
47.1, 50.2, 54.2, 56.2, 56.8, 67.3, 70.5, 70.6, 70.7, 71.0, 79.5, 117.1,
121.6, 122.0, 122.4, 136.5, 137.3, 140.2, 140.9, 149.2, 159.1. HR MS
(FAB+, m−NBA): calcd for C43H69N4O3 ([M + H]+), 689.5370;
found, 689.5364.
Synthesis of 6. A mixture of trans-chalcone (1.00 g, 4.80 mmol),
nitromethane (1.30 mL, 24.0 mmol), and diethylamine (2.49 mL,
24.0 mmol) was added to a 10 mL one-necked round-bottom flask.
The reaction mixture was heated at 60 °C for 7 h. After it cooled to
room temperature, the reaction mixture was poured onto EtOAc (50
mL) and washed with brine (50 mL × twice). The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo. A
pale yellow solid was obtained in a 98% yield and used in the next step
without further purification. Rf = 0.2 (CH2Cl2/hexane = 1:1, v/v). 1H
NMR (300 MHz, CDCl3) δ (ppm): 3.46 (dd, J = 12.7, 7.5 Hz, 2H),
4.23 (tt, J = 7.5, 6.9 Hz, 1H), 4.77 (dd, J = 12.5, 6.9 Hz, 2H), 7.32 (m,
5H), 7.46 (m, 2H), 7.58 (m, 1H), 7.92 (m, 2H). 13C{1H} NMR (126
MHz, CDCl3) δ (ppm): 39.5, 41.7, 79.8, 124.8, 147.7, 128.1, 128.2,
128.9, 128.94, 129.3, 133.8, 136.6, 139.3, 197.0. HR MS (FAB+, m−
NBA): calcd for C16H16NO3 ([M + H]+), 270.1130; found, 270.1131.
Synthesis of 7. Compound 6 (0.500 g, 1.86 mmol) was dissolved
in 10 mL of CF3CH2OH in a 50 mL one-necked round-bottom flask.
NH4OAc (8.82 g, 114 mmol) was added to a stirred solution of the
reaction mixture, and the solution was refluxed for 2 h. The reaction
mixture was cooled to room temperature and poured onto CH2Cl2
(300 mL). The organic solution was washed with water (300 mL ×
three times), 1 M NaOH(aq) (300 mL), and water (300 mL). The
organic layer was dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. Silica gel column chromatog-
raphy was performed by varying the eluent from CH2Cl2/hexane =
1:1 (v/v) to 100% CH2Cl2 to increase its polarity. A black solid was
Synthesis of 4. Compound 3 (14.0 g, 20.7 mmol) and 4-
imidazolecarboxaldehyde (1.88 g, 19.5 mmol) were combined in a
500 mL one-necked round-bottom flask and dissolved in 180 mL of
acetonitrile. After the addition of K2CO3 (3.51 g, 25.4 mmol), the
reaction mixture was refluxed for 24 h. After it cooled to room
temperature, the reaction mixture was poured onto water (300 mL)
and neutralized by using 1.0 N HCl(aq). The crude product was
extracted with CH2Cl2 (300 mL × three times). The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo.
Silica gel column chromatography was performed by varying the
eluent from EtOAc/CH3OH = 99:1 (v/v) to EtOAc/CH3OH = 9:1
(v/v) to increase its polarity. A pale yellow oil was isolated with a 48%
1
obtained in a 42% yield. Rf = 0.6 (CH2Cl2/hexane = 1:1, v/v). H
NMR (300 MHz, CDCl3) δ (ppm): 7.21 (s, 2H), 7.46 (m, 12H), 7.97
(m, 4H), 8.07 (m, 4H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm):
115.1, 126.8, 128.2, 128.5, 129.3, 129.4, 130.3, 132.4, 133.9, 142.8,
149.8, 155.3. HR MS (FAB+, m−NBA): calcd for C32H24N3 ([M +
H]+), 450.1970; found, 450.1966.
1
yield. Rf = 0.3 (EtOAc/CH3OH = 9:1, v/v). H NMR (300 MHz,
Synthesis of 8. Compound 7 (0.150 g, 0.334 mmol) was diluted
in triethylamine (0.820 mL) in a 10 mL one-necked round-bottom
flask. BF3·OEt2 (0.810 mL, 6.75 mmol) was carefully added into the
stirred solution. After it was heated at 90 °C for 5 h, the solution was
cooled to room temperature. The reaction mixture was poured onto
CH2Cl2 (150 mL) and washed with 1 M HCl(aq) (150 mL) and
brine (150 mL × twice). The organic layer was dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. Purification via silica gel
column chromatography was performed with varying the eluent from
hexane to CH2Cl2/hexane = 1:1 (v/v) to increase its polarity. A black
solid was isolated in a 44% yield. Rf = 0.4 (CH2Cl2/hexane = 1:1, v/
CDCl3) δ (ppm): 0.67 (s, 3H), 0.86 (dd, J = 6.6, 1.2 Hz, 6H), 0.91
(d, J = 6.6 Hz, 3H), 0.99 (s, 3H), 3.15 (m, 1H), 3.60 (m, 8H), 3.79 (t,
J = 5.1 Hz, 2H), 4.17 (t, J = 5.1 Hz, 2H), 5.34 (d, J = 5.1 Hz, 1H),
7.64 (d, J = 1.2 Hz, 1H), 7.74 (d, J = 1.2 Hz, 1H), 9.88 (s, 1H).
13C{1H} NMR (126 MHz, CDCl3) δ (ppm): 11.9, 18.7, 19.4, 21.1,
22.6, 22.8, 23.8, 24.3, 28.0, 28.2, 28.4, 29.7, 31.9, 31.94, 35.8, 36.2,
36.9, 37.2, 39.1, 39.5, 39.8, 42.3, 47.8, 56.1, 56.8, 67.3, 70.0, 70.6,
70.8, 71.0, 79.5, 121.6, 125.2, 139.2, 140.9, 142.3, 186.3. HR MS
(FAB+, m−NBA): calcd for C37H61N2O4 ([M + H]+), 597.4631;
found, 597.4636.
Synthesis of 5. Compound 4 (1.36 g, 2.28 mmol) and 2-
picolylamine (0.246 g, 2.28 mmol) were dissolved in 16 mL of
acetonitrile in a 50 mL one-necked round-bottom flask. A drop of
acetic acid was added to the stirred solution, and the reaction mixture
was refluxed for 7.5 h. After the mixture cooled to 0 °C, NaBH4
(0.215 g, 5.70 mmol) was slowly added to the reaction mixture. The
reaction mixture was stirred for an additional 22 h at room
temperature. The reaction was quenched by adding 200 mL of
water onto the solution, and the crude product was extracted with
CH2Cl2 (200 mL × three times). The organic layer was dried over
anhydrous MgSO4, filtered, and concentrated under reduced pressure.
Purification via silica gel column chromatography was performed by
varying the eluent from CH2Cl2/CH3OH = 99:1 (v/v) to CH2Cl2/
1
v). H NMR (300 MHz, CDCl3) δ (ppm): 7.05 (s, 2H), 7.47 (m,
12H), 8.06 (m, 8H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm):
119.3, 128.8, 128.9, 129.6, 129.7, 129.8, 129.8, 131.1, 131.8, 132.5,
144.4, 145.8, 159.8. HR MS (FAB+, m−NBA): calcd for C32H22BF2N3
([M+]), 497.1875; found, 497.1880.
Synthesis of 9. POCl3 (7.30 mL, 78.3 mmol) was slowly added
into anhydrous DMF (7.30 mL, 94.9 mmol) in a 50 mL two-necked
round-bottom flask under an Ar atmosphere at 0 °C. The reaction
mixture was stirred at 0 °C for 5 min and then at room temperature
for 30 min. Compound 8 (110 mg, 0.220 mmol) dissolved in CH2Cl2
(8.0 mL) was added slowly into the stirred solution which was heated
at 70 °C for 6 h. The reaction was quenched by adding saturated
NaHCO3(aq) (100 mL) at 0 °C. The crude product was extracted
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1
with CH2Cl2 (200 mL × three times). The recovered organic layer
was dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. Purification via silica gel column chromatography
was performed with varying the eluent from EtOAc/hexane = 1:19
(v/v) to EtOAc/hexane = 1:2 (v/v) to increase its polarity. Dark blue
powders were obtained with a 51% yield. Rf = 0.3 (EtOAc/hexane =
31% yield. Rf = 0.2 (CH2Cl2/CH3OH = 5:1, v/v). H NMR (300
MHz, CD2Cl2) δ (ppm): 1.04 (t, J = 6.9 Hz, 3H), 3.20 (s, 2H), 3.33
(q, J = 6.9 Hz, 2H), 3.36 (s, 2H), 3.48 (t, J = 5.1 Hz, 2H), 3.64 (s,
2H), 3.85 (t, J = 5.1 Hz, 2H), 7.58 (d, J = 6.3 Hz, 2H), 7.71 (m, 2H),
7.85 (m, 2H), 7.95 (m, 2H), 8.27 (d, J = 3.6 Hz, 1H). 13C{1H} NMR
(126 MHz, CD2Cl2) δ (ppm): 15.4, 47.8, 48.6, 51.2, 59.5, 67.1, 70.0,
118.6, 119.5, 122.1, 123.4, 126.7, 128.3, 128.5, 128.7, 129.0, 129.1,
129.3, 129.6, 129.7, 130.1, 130.2, 131.4, 131.8, 132.0, 132.1, 132.6,
132.8, 136.6, 137.0, 138.4, 144.7, 145.1, 145.3, 148.9, 160.3, 161.4.
HR MS (FAB+, m−NBA): calcd for C47H43BF2N7O ([M + H]+),
770.3590; found, 770.3594.
1
1:2, v/v). H NMR (300 MHz, CDCl3) δ (ppm): 7.25 (s, 1H), 7.54
(m, 12H), 7.75 (m, 2H), 7.88 (m, 2H), 8.10 (m, 4H), 9.80 (s, 1H).
13C{1H} NMR (126 MHz, CDCl3) δ (ppm): 121.7, 125.8, 127.9,
128.0, 128.2, 128.4, 128.5, 129.1, 129.2, 129.4, 129.8, 128.9, 130.0,
130.4, 130.5, 130.9, 131.0, 131.1, 132.2, 133.0, 143.4, 147.7, 149.0,
159.6, 166.5, 186.4. HR MS (FAB+, m−NBA): calcd for
C33H23BF2N3O ([M + H]+), 526.1902; found, 526.1904.
Caution: Perchlorate salts are potentially explosive. Only small
amounts of materials should be cautiously handled at a time.
Spectroscopic Measurements. Milli-Q grade water (18.2 MΩ·
cm) was used to prepare the solutions for spectroscopic measure-
ments. PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid), ≥99%)
was purchased from Aldrich. A pH 7.4 buffer solution was prepared by
dissolving PIPES (25 mM) and KCl (100 mM) in Milli-Q water and
adjusting the pH with standard KOH solution (45 wt %, Aldrich) or
concentrated HCl (Aldrich). The buffer solution was further treated
with Chelex 100 resin (BIO-RAD) to remove trace metal ions and
filtered through a membrane (pore size = 0.45 μm); its pH was
confirmed prior to use. Fresh metal stock solutions (typically, 0.10 or
0.010 M except for CrCl3·6H2O) were prepared in Milli-Q water by
using the corresponding chloride salts: CuCl2 (99.999%, Aldrich),
NaCl (≥99.5%, Aldrich), KCl (Puratronic grade, Calbiochem),
MgCl2 (99.99%, Aldrich), CaCl2 (99.99%, Aldrich), CrCl3·6H2O
(98%, Aldrich), MnCl2 (99.99%, Aldrich), FeCl2 (99.99%, Aldrich),
CoCl2 (99.9%, Aldrich), NiCl2 (99.99%, Aldrich), CdCl2 (99.99%,
Aldrich), and ZnCl2 (99.999%, Aldrich). A TPEN solution was
prepared by dissolving N,N,N′,N′-tetrakis(2-picolyl)ethylenediamine
(≥99%, Sigma) in DMSO (99.9%, Aldrich). Zn(ClO4)2·6H2O
(Aldrich) was dissolved in CH3CN (spectrophotometric grade,
Aldrich) at concentrations of 1.0, 10, and 100 mM. Sensor solutions
were prepared by dissolution in DMSO at a concentration of 10 mM
and were partitioned into Eppendorf tubes. The sensor stock solution
was stored in a refrigerator at −10 °C and were thawed before the
measurements. Typically, 3.0 mL of the buffer and 3 μL of the sensor
stock solution (10 mM in DMSO) were mixed to give a 10 μM
solution. A 1 cm × 1 cm fluorimeter cell (Hellma) was used in the
steady-state photophysical measurements. UV−vis absorption spectra
were collected on a Varian Cary 300 spectrophotometer at room
temperature. Photoluminescence spectra were obtained by using a
Quanta Master 400 scanning spectrofluorimeter at room temperature.
The photoluminescence quantum yields (Φ) were relatively
Synthesis of JJ. Compounds 5 (0.110 g, 0.160 mmol) and 9
(0.060 g, 0.114 mmol) were dissolved in 12 mL of anhydrous CH2Cl2
in a 25 mL two-necked round-bottom flask. NaBH(OAc)3 (0.041 g,
0.193 mmol) was then added into the stirred solution. After the
addition of a drop of acetic acid, the reaction mixture was refluxed for
48 h. The solution was cooled to room temperature and poured onto
water (100 mL). The crude product was extracted with CH2Cl2 (100
mL × three times). The recovered organic layer was dried over
anhydrous MgSO4, filtered, and concentrated under reduced pressure.
Preparative TLC techniques were employed with CH2Cl2/CH3OH =
5:1 (v/v) as the eluent to purify JJ. A black solid was isolated in a 12%
1
yield. Rf = 0.3 (CH2Cl2/CH3OH = 5:1, v/v). H NMR (300 MHz,
CD3CN) δ (ppm): 0.58 (s, 3H), 0.86 (m, 12H), 2.99 (m, 1H), 3.28
(s, 2H), 3.45 (m, 10H), 3.64 (t, J = 4.8 Hz, 2H), 3.71 (s, 2H), 3.98 (t,
J = 4.8 Hz, 2H), 5.21 (d, J = 5.1 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H),
7.10 (m, 1H), 7.22 (s, 1H), 7.53 (m, 15H), 7.65 (d, J = 7.2 Hz, 2H),
7.80 (m, 2H), 7.95 (dd, J = 7.2, 1.8 Hz, 2H), 8.08 (m, 2H), 8.36 (d, J
= 3.6 Hz, 1H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm): 13.1,
14.3, 18.9, 19.6, 21.3, 22.8, 22.9, 23.0, 24.0, 24.5, 25.0, 27.4, 28.2,
28.4, 28.6, 29.3, 29.5, 29.6, 29.7, 29.8, 29.9, 32.1, 33.7, 36.0, 36.4,
37.1, 37.4, 39.3, 39.7, 40.0, 42.5, 50.4, 56.4, 57.0, 67.5, 70.8, 70.9,
71.2, 79.7, 121.8, 128.1, 128.4, 128.8, 129.4, 129.7, 130.1, 130.2,
131.2, 136.7, 141.1, 148.6. HR MS (FAB+, m−NBA): calcd for
C76H91BF2N7O3 ([M + H]+), 1198.7245; found, 1198.7244.
Synthesis of 10. 2-Bromoethyl ethyl ether (2.85 g, 17.7 mmol)
and 4-imidazolecarboxaldehyde (1.00 g, 10.4 mmol) were dissolved in
100 mL of acetonitrile in a 250 mL one-necked round-bottom flask.
K2CO3 (2.16 g, 15.6 mmol) was delivered into the stirred solution,
which was then refluxed for 26 h. The reaction mixture was cooled to
room temperature and was poured onto water (300 mL). The crude
product was extracted with CH2Cl2 (300 mL × three times). The
recovered organic layer was dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. Silica gel column chromatography was
performed with varying the eluent from CH2Cl2/CH3OH = 99:1 (v/
v) to CH2Cl2/CH3OH = 49:1 (v/v) to increase its polarity, which
afforded a pale yellow oil in a 57% yield. Rf = 0.3 (CH2Cl2/CH3OH =
9:1, v/v). 1H NMR (300 MHz, CDCl3) δ (ppm): 1.19 (t, J = 6.9 Hz,
3H), 3.49 (q, J = 6.9 Hz, 2H), 3.69 (t, J = 4.8 Hz, 2H), 4.15 (t, J = 4.8
Hz, 2H), 7.62 (d, J = 0.9 Hz, 1H), 7.71 (d, J = 0.9 Hz, 1H), 9.88 (s,
1H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm): 15.0, 47.9, 66.9,
69.1, 125.2, 139.2, 142.3, 186.3. HR MS (FAB+, m−NBA): calcd for
C8H13N2O2 ([M + H]+), 169.0977; found, 169.0977.
determined according to following standard equation: Φ = Φref
×
(I/Iref) × (Aref/A) × (n/nref)2, where A, I, and n are the absorbance at
the excitation wavelength, the integrated photoluminescence intensity,
and the refractive index of the solvent, respectively. 9,10-
Diphenylanthracene in a deaerated toluene solution was used as the
external reference (Φref = 1.00).85
Transient Photoluminescence Measurements. Photolumines-
cence decay traces were acquired based on time-correlated single-
photon counting (TCSPC) techniques by using a FluoTime 200
instrument (PicoQuant, Germany). A 377 nm diode laser
(PicoQuant, Germany; pulse energy = 35 pJ) with a repetition rate
of 125 kHz was used as the excitation source. The signals at 654 and
652 nm were obtained by using an automated motorized
monochromator and recorded with a NanoHarp unit. The decay
profiles were analyzed (OriginPro 8.0, OriginLab) by using single or
double exponential decay models.
Electrochemical Characterization. Cyclic and differential pulse
voltammetry measurements were carried out by using a CH
Instruments, CHI630 B potentiostat with a three-electrode-cell
assembly. A Pt wire and a Pt microdisc were used as the counter
electrode and the working electrode, respectively. An Ag/AgNO3 (10
mM) couple was used as the pseudo reference electrode. Measure-
ments were carried out in Ar-saturated THF/DMSO (4:1, v/v) with
using 0.10 M tetra-n-butylammonium hexafluorophosphate
(TBAPF6) as the supporting electrolyte at scan rates of 0.1 mV s−1
Synthesis of 11. A method identical to the synthetic procedure
for compound 5 was employed, except that compound 10 (0.600 g,
3.57 mmol) was used instead of compound 4. A pale brown oil was
1
obtained in a 16% yield. Rf = 0.1 (CH2Cl2/CH3OH = 5:1, v/v). H
NMR (300 MHz, CDCl3) δ (ppm): 1.18 (t, J = 6.9 Hz, 3H), 3.47 (q,
J = 6.9 Hz, 2H), 3.65 (t, J = 5.4 Hz, 2H), 3.82 (s, 2H), 4.00 (s, 2H),
4.04 (t, J = 5.1 Hz, 2H), 6.92 (s, 1H), 7.15 (m, 1H), 7.37 (d, J = 7.8
Hz, 1H), 7.47 (d, J = 1.2 Hz, 1H), 7.64 (td, J = 7.8, 1.8 Hz, 2H), 8.55
(m, 1H). 13C{1H} NMR (126 MHz, CDCl3) δ (ppm): 15.0, 46.7,
47.2, 54.4, 66.8, 69.7, 116.9, 121.9, 122.3, 136.5, 137.2, 140.5, 149.2,
159.5. HR MS (FAB+, m−NBA): calcd for C14H21N4O ([M + H]+),
261.1715; found, 261.1716.
Synthesis of JJC. A method identical to the synthetic procedure
for JJ was employed, except that compound 11 (0.092 g, 0.353 mmol)
was used instead of compound 5. A dark blue solid was obtained in a
L
https://dx.doi.org/10.1021/acs.inorgchem.0c01376
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
pubs.acs.org/IC
Article
(cyclic voltammetry) and 4.0 mV s−1 (differential pulse voltammetry).
A ferrocenium/ferrocene (Fc+/Fc) couple was employed as the
external reference.
AUTHOR INFORMATION
■
Corresponding Author
Youngmin You − Division of Chemical Engineering and
Materials Science, Ewha Womans University, Seoul 03760,
Republic of Korea; orcid.org/0000-0001-5633-6599;
Email: odds2@ewha.ac.kr
Determination of Kd. Zinc-buffered solutions (pH 7.4; 100 mM
HEPES, 100 mM NaNO3, and 10 mM HEDTA) were employed to
determine the dissociation constant (Kd). The procedure reported by
Nagano and co-workers was modified to prepare 15 solutions of free
zinc with concentrations in the range 0.15 pM to 120 nM.94 A 1.5 μL
sensor solution (10 mM, DMSO) was added to each zinc-buffered
solution (3 mL), which was then stored for at least 3 h. The
fluorescence spectrum of each solution was recorded and the intensity
was calculated by integrating the spectrum from 550 to 850 nm. A
zinc binding titration isotherm plotting the fluorescence intensity as a
function of the free zinc concentration was constructed and fitted to a
1:1 binding equation.94
Authors
Jin Ju Kim − Division of Chemical Engineering and Materials
Science, Ewha Womans University, Seoul 03760, Republic of
Korea
Jayeon Hong − Division of Chemical Engineering and Materials
Science, Ewha Womans University, Seoul 03760, Republic of
Korea
Seungyeon Yu − Division of Chemical Engineering and
Materials Science, Ewha Womans University, Seoul 03760,
Republic of Korea
Cell Cultures. HeLa and DLD-1 cells were obtained from Korean
Cell Line Bank and cultured in DMEM supplemented with 10% fetal
bovine serum and 1% penicillin/streptomycin at 37 °C in a
humidified incubator under 5% CO2. Two days before imaging,
cells were passed and plated onto poly(D-lysine)-coated glass-bottom
culture dishes.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01376
Confocal Laser Scanning Microscopy. Microscopic experi-
ments were performed at the Korea Basic Science Institute, Seoul,
Korea. Sensor solutions were prepared by dissolving 10 mM stock
solution (DMSO) in serum-free DMEM. A 100 mM ZnCl2(aq)
solution was prepared in Milli-Q grade water. A 100 mM sodium
pyrithione solution was prepared in DMSO, prior to the microscopic
experiments. Serum-free DMEM solutions containing 200 μM EDTA
were also prepared. Typically, after washing the cells with PBS,
medium containing 0.2 μM sensor was added to a culture dish. The
cells were incubated for 10 min at 37 °C. The incubated cells were
washed with PBS and fresh DMEM (serum-free), and fluorescence
micrographs were obtained by using a Carl Zeiss LSM 510 META
confocal laser scanning microscope with a Newport MaiTai eHP
DeepSee multiphoton excitation system. An excitation beam (633
nm) was focused onto the dish, and the signals were acquired through
32 emission channels covering the range 640−677 nm. After
removing the cell medium, the cells were washed with fresh
DMEM and incubated in DMEM containing 20 μM ZnCl2 for 5
min. The cells were imaged after washing with FBS and supplemented
with fresh DMEM. Finally, the cells were treated with 200 μM EDTA,
and microscopic visualization was performed. Photoluminescence
images and mean intensities were processed by using the software
ZEN2000 and ImageJ, respectively.
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Ministry of Science,
Information, and Communication Technology (ICT) and
Future Planning (MSIP) of Korea through the GFP grant
(CISS-2012M3A6A6054204) and by the Midcareer Research
Program (NRF2019R1A2C2003969) through the National
Research Foundation grant. The authors thank Dr. Seung-Hae
Kwon at the Korea Basic Science Institute for the assistance
with the microscopic experiments.
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ASSOCIATED CONTENT
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sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01376.
(Table S1) Photophysical parameters of JJ; (Scheme S1)
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DLD-1 cells, and H and 13C{1H} NMR spectra (PDF)
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