Cyanine-based fluorescent probe for highly selective detection of glutathione in cell cultures and live mouse tissues

Article abstract of DOI:10.1021/ja412628z

Glutathione (GSH) plays a crucial role in human pathologies. Near-infrared fluorescence-based sensors capable of detecting intracellular GSH in vivo would be useful tools to understand the mechanisms of diseases. In this work, two cyanine-based fluorescent probes, 1 and 2, containing sulfonamide groups were prepared. Evaluation of the fluorescence changes displayed by probe 1, which contains a 2,4-dinitrobenzenesulfonamide group, shows that it is cell-membrane-permeable and can selectively detect thiols such as GSH, cysteine (Cys), and homocysteine (Hcy) in living cells. The response of 1 to thiols can be reversed by treatment with N-methylmaleimide (NMM). Probe 2, which possesses a 5-(dimethylamino)naphthalenesulfonamide group, displays high selectivity for GSH over Cys and Hcy, and its response can be reversed using NMM. The potential biological utility of 2 was shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can determine changes in the intracellular levels of GSH modualated by H₂O₂. The properties of 2 enable its use in monitoring GSH in vivo in a mouse model. The results showed that intravenous injection of 2 into a mouse generates a dramatic image in which strong fluorescence is emitted from various tissues, including the liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in mouse tissue cells promoted by excessive administration of the painkiller acetaminophen. The combined results coming from this effort suggest that the new probe will serve as an efficient tool for detecting cellular GSH in animals.

Full text of DOI:10.1021/ja412628z

Article
pubs.acs.org/JACS
Cyanine-Based Fluorescent Probe for Highly Selective Detection of
Glutathione in Cell Cultures and Live Mouse Tissues
Jun Yin,†,‡,⊥ Younghee Kwon,§,∥,⊥ Dabin Kim,† Dayoung Lee,† Gyoungmi Kim,† Ying Hu,†
Ji-Hwan Ryu,*,§,∥ and Juyoung Yoon*,†
†Department of Chemistry and Nano Science, Global Top 5 Research Program, Ewha Womans University, Seoul 120-750, Korea
‡Key Laboratory of Pesticide and Chemical Biology of the Ministry of Education, College of Chemistry, Central China Normal
University, Wuhan 430079, People’s Republic of China
∥
§Research Center for Human Natural Defense System and BK 21 Plus Project for Medical Science, Yonsei University College of
Medicine, Seoul 120-752, Korea
S
* Supporting Information
ABSTRACT: Glutathione (GSH) plays a crucial role in
human pathologies. Near-infrared fluorescence-based sensors
capable of detecting intracellular GSH in vivo would be useful
tools to understand the mechanisms of diseases. In this work,
two cyanine-based fluorescent probes, 1 and 2, containing
sulfonamide groups were prepared. Evaluation of the
fluorescence changes displayed by probe 1, which contains a
2,4-dinitrobenzenesulfonamide group, shows that it is cell-
membrane-permeable and can selectively detect thiols such as
GSH, cysteine (Cys), and homocysteine (Hcy) in living cells.
The response of 1 to thiols can be reversed by treatment with
N-methylmaleimide (NMM). Probe 2, which possesses a 5-(dimethylamino)naphthalenesulfonamide group, displays high
selectivity for GSH over Cys and Hcy, and its response can be reversed using NMM. The potential biological utility of 2 was
shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can determine changes in the intracellular
levels of GSH modualated by H2O2. The properties of 2 enable its use in monitoring GSH in vivo in a mouse model. The results
showed that intravenous injection of 2 into a mouse generates a dramatic image in which strong fluorescence is emitted from
various tissues, including the liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in
mouse tissue cells promoted by excessive administration of the painkiller acetaminophen. The combined results coming from this
effort suggest that the new probe will serve as an efficient tool for detecting cellular GSH in animals.
unique nucleophilic addition and substitution reactions.3
However, the design of a highly selective detection system
that discriminates between biothiols with similar structures and
reactivities (e.g., cysteine (Cys), homocysteine (HCy), and
glutathione (GSH)) is still a significant challenge.4
GSH, the most abundant intracellular nonprotein biothiol,
serves many cellular functions, including maintenance of
intracellular redox activity, xenobiotic metabolism, intracellular
signal transduction, and gene regulation. This important
biothiol protects cellular components from damage caused by
free radicals and reactive oxygen species (ROS) and maintains
exogenous antioxidants in their reduced forms.5 These
properties suggest that highly selective methods to monitor
and quantitate GSH under physiological conditions are of
significant interest in clinical medicine6 and for investigations
aimed at probing cellular functions.4
INTRODUCTION
■
Amino acids containing the thiol group are components of
many peptides that play crucial roles in maintaining biological
redox homeostasis in biological systems through an equilibrium
between reduced free thiol and oxidized disulfide forms.1
Importantly, numerous investigations have demonstrated that
abnormal levels of these amino acids are closely associated with
certain disease states, including liver damage, cancer, AIDS,
osteoporosis, Alzheimer’s disease, and heart, inflammatory
bowel, and cardiovascular diseases.2 Consequently, assessments
of the levels of thiol-containing substances in biological systems
may aid early diagnosis of some diseases. Over the past several
decades, a considerable effort has been devoted to development
of an effective strategy for the detection of thiols in living
systems (biothiols). Among the various analytical methods that
are available, molecular imaging based on fluorescence is
considered to be the most sensitive approach owing to its
sensitivity and simplicity. In recent years, numerous fluorescent
probes have been developed that can distinguish biothiols from
other amino acids by utilizing the fact that thiols undergo
Received: December 12, 2013
Published: March 20, 2014
© 2014 American Chemical Society
5351
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358

Journal of the American Chemical Society
Article
Thus far, several fluorescent probes based on organic dyes for
highly selective detection of GSH have been designed. For
example, in 2010 Yang and Chan described a promising GSH
probe that is based on a bis-spiropyran structure.7 Recently, a
resorufin-based probe was devised by Strongin to be used for
the detection of GSH in human blood plasma in a highly
selective manner.8 Yang has also developed a ratiometric,
monochlorinated BODIPY-based fluorescence sensor, which is
responsive to GSH and not Cys and Hcy and which can be
employed for detection of GSH in living cells.9
sulfonamide functional moiety. The results show that the
fluorescent turn-on probe 1 can be used to detect GSH, Cys,
and Hcy and that it is cell-membrane-permeable and applicable
for thiol detection in living cells. More important is the
observation that probe 2 displays a high selectivity for detection
of GSH over Cys and Hcy and that it can be used to monitor
GSH in cells. Furthermore, observations made in this effort
show that injection of probe 2 into a mouse gives rise to a
dramatic turn-on fluorescence image, with emission occurring
from various tissues such as the liver, kidney, lung, and spleen.
Considering the fact that GSH is a major endogenous
antioxidant present in the intracellular environment at
concentration levels in the range of 0.5−10 mM, its in vivo
detection using near-infrared (700−900 nm) fluorescent probes
would have important advantages owing to the less biological
damaging and deeper tissue penetration properties of light in
this wavelength region.10 Although several near-infrared,
cyanine-based fluorescent probes have been reported recently,
they suffer from low thiol selectivity.11 In the study described
below, we developed two near-infrared, cyanine-based fluo-
rescent probes (1 and 2, Scheme 1) each containing a
RESULTS AND DISCUSSION
■
Near-infrared fluorescent probes, and especially those that are
based on cyanine,12 have gained increasing interest recently. In
addition, the unique reactivity profile of thiols (e.g., promotion
of sulfonamide cleavage) is a significant feature of the design of
thiol-selective probes.3 In our design of the new thiol probes 1
and 2, we selected cyanine as the near-infrared fluorophore and
thiol-responsive sulfonamides as the reaction component. The
sulfonamides, linked to the cyanine moiety through a
piperazine bridge in 1 and 2, undergo cleavage in the presence
of thiols to form free piperazine 3 (Scheme 1) in concert with
turn-on fluorescence.13 The routes used to prepare 1 and 2,
outlined in Scheme 1, begin with reaction of IR-780 with
piperazine in DMF to produce intermediate 3. Treatment of
this substance with either 2,4-dinitrobenzene-1-sulfonyl chlor-
ide or dansyl chloride then efficiently leads to generation of the
respective cyanine-based probes 1 and 2.
Scheme 1. Structures and Synthesis of Cyanine-Based
a
Fluorescent Probes 1 and 2
Studies were carried out to explore the use of near-infrared
fluorescent probe 1 to detect amino acids containing a thiol
group. Analysis of UV/vis absorption spectroscopic changes
shows that addition of a thiol (such as GSH or Cys or Hcy, 100
μM) to a solution of 1 (10 μM) in HEPES buffer (10 mM, pH
7.4) containing 10% DMSO leads to an increase in absorbance
at 730 nm (Figure S1A, Supporting Information). Similarly,
mixing 1 with a thiol leads to production of a fluorescence
emission band with a maximum at 736 nm, while no obvious
changes take place when other amino acids are added (Figure
S1B). These observations suggest that 1 can be employed to
detect thiols.
a
Reagents and conditions: (a) piperazine, DMF, 85 °C, 85%; (b)
sulfonyl chloride, CH2Cl2/Et3N, 75−82%.
Figure 1. Confocal microscope images following addition of probe 1 to HeLa cells. Cell images were obtained using an excitation wavelength of 635
nm and a band-path (655−755 nm) emission filter. (A) Fluorescence image of HeLa cells. (B) Fluorescence image of HeLa cells incubated with
probe 1 (10 μM) for 20 min. (C) Fluorescence image of HeLa cells pretreated with N-methylmaleimide (NMM; 1 mM) for 20 min and incubated
with probe 1 (10 μM) for 20 min. (D) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min and then added to cysteine (100
μM) and incubated with probe 1 (10 μM) for 20 min. (E) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min and then
added to homocysteine (100 μM) and incubated with probe 1 (10 μM) for 20 min. (F) Fluorescence image of HeLa cells pretreated with NMM (1
mM) for 20 min and then added to glutathione (100 μM) and incubated with probe 1 (10 μM) for 20 min.
5352
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358

Journal of the American Chemical Society
Article
Figure 2. (A) Fluorescence response of probe 2 (10 μM) to various amino acids (100 μM). Each spectrum was recorded 20 min following addition
of the amino acid. (B) Time-dependent fluorescence changes of probe 2 (10 μM) upon addition of GSH, Cys, and Hcy (100 μM) in HEPES (10
mM, pH 7.4) containing 10% DMSO. λex = 730 nm, λem = 736 nm, and the slit width was 10 nm.
Figure 3. Confocal microscope images of probe 2 in HeLa cells. Cell images were obtained using an excitation wavelength of 635 nm and a band-
path (655−755 nm) emission filter. (A) Fluorescence image of HeLa cells. (B) Fluorescence image of HeLa cells incubated with probe 2 (20 μM)
for 20 min. (C) Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min and incubated with probe 2 (20 μM) for 20 min. (D)
Fluorescence image of HeLa cells pretreated with NMM (1 mM) for 20 min and then treated with GSH (100 μM) for 20 min and incubated with
probe 2 (20 μM) for 20 min.
To determine the biological relevance of the new near-
infrared fluorescent probe, studies were carried out to assess the
use of 1 in fluorescence imaging of cellular thiols. For this
purpose, HeLa cells were grown in RPMI 1640 (Roswell Park
Memorial Institute) supplemented with 10% heat-inactivated
fetal bovine serum, 100 U/mL penicillin, and 100 U/mL
streptomycin and were maintained in an incubator at 37 °C
with a 5% CO2/air environment. As illustrated by viewing the
confocal fluorescence microscope images displayed in Figure
1B and bright field images shown in Figure 1A, a significant red
fluorescence image is produced in the cytoplasm when HeLa
cells are incubated with probe 1 (10 μM) at 37 °C for 20 min
(Figure 1B). The results suggest that 1 is capable of permeating
into cells and reacting with resident thiols to produce
discernible fluorescence images.
A series of control experiments were performed to gain
support for this conclusion. The HeLa cells were pretreated
with the thiol-blocking reagent N-methylmaleimide (NMM)
for 20 min and then incubated with 1 (10 μM) for 20 min. The
resulting confocal microscope image (Figure 1C) does not
display red fluorescence. Moreover, addition of Cys, Hcy, or
GSH (100 μM) to the NMM-pretreated HeLa cells gives rise to
a significant increase in red emission (Figure 1D−F,
respectively). Thus, probe 1 can be employed to detect thiols
in living cells.
The results of studies, designed to assess the potential
selectivity of the response of the probe toward various thiol-
containing amino acids, show that 2 can be used to detect GSH
selectively. Specifically, treatment of a solution of probe 2 (10
μM) in HEPES buffer (10 mM, pH 7.4) containing 10%
DMSO with GSH (100 μM) results in significant turn-on
fluorescence at 736 nm (excitation at 730 nm) (Figure 2A) in a
GSH-concentration-dependent manner (Figure S3, Supporting
Information). The results of an experiment probing the time-
dependent fluorescence response of probe 2 (10 μM) to GSH
(100 μM) at 37 °C in HEPES buffer solution (10 mM, pH 7.4)
containing 10% DMSO show that the intensity of the 736 nm
emission band increases with time, reaching a maximum in less
than 15 min (Figure S4, Supporting Information). Compared
to GSH, the other thiol-based amino acids, Cys and Hcy, do
not promote as rapid a fluorescence response of probe 2
(Figure 2B). Subsequent investigation in human serum is
performed. According to the fluorescence spectra (Figure S5,
Supporting Information), we find that the fluorescence
intensity will increase along with the addition of human
serum owing to the existence of GSH in serum.
5353
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358

Journal of the American Chemical Society
Article
To gain support for the proposal that treatment of probes 1
and 2 with thiols promotes fluorescence through chemical
reactions that liberate free piperazine 3, the processes were
probed using mass spectrometry. For this purpose, a soft
ionization method, based on matrix-assisted laser ionization
(MALDI), was employed to follow reactions of the probes with
thiols. Formation of the thiolysis product 3 in reactions of
probe 1 with GSH, Cys, and Hcy was identified by using
MALDI-TOF mass spectrometry (in Figure S6, Supporting
Information). In addition, this method was used to show that
GSH cleaves the sulfonamide group of probe 2 to form 3 under
the same conditions (in Figure S7 and Scheme S1, Supporting
Information).
To demonstrate its cellular GSH fluorescence imaging
capability, probe 2 was incubated with living HeLa cells at 37
°C for 20 min. Red fluorescence emission was observed inside
the cells by using a confocal fluorescence microscope (Figure
3A,B), demonstrating that 2 is readily internalized into living
cells. NMM was employed as a thiol-blocking reagent in a
control experiment. A markedly lower fluorescence intensity
increase takes place when the HeLa cells are pretreated with
NMM for 20 min and then incubated with probe 2 (10 μM) for
20 min (Figure 3C). Moreover, addition of a high
concentration of GSH (100 μM) to this solution and
incubation for another 20 min leads to production of a strong
red fluorescence image (Figure 3D) in the same field of view.
These findings demonstrate that probe 2 has excellent cell
permeability and that it can be utilized to monitor GSH in
living cells. Even though the selectivity of probe 2 for GSH in
solution has been demonstrated, its ability to selectively detect
this thiol in cells requires investigation. Experiments in which
the thiol-blocking reagent NMM is employed were ideal for this
purpose. Accordingly, HeLa cells were pretreated with NMM
for 20 min and then incubated with probe 2 (10 μM) for 20
min. As expected, the confocal microscope image of the cells
treated in this manner does not display red fluorescence.
Moreover, addition of Cys or Hcy (100 μM) to the NMM-
pretreated HeLa cells give rise to only small changes in the
fluorescence intensity. In contrast, addition of GSH (100 μM)
does promote a significant increase in red emission (Figures S8,
Supporting Information).
could be determined. The results show that GSH does not
perturb the UV−vis absorption and fluorescence spectra of 4
(Figure S9, Supporting Information). In addition, we
demonstrated that GSH also does not promote changes in
the UV−vis and fluorescence spectra of 3 (Figure S10,
Supporting Information). These observations suggest that 3
and 4, which represent the independent components of probe
2, do not respond to GSH.
Results arising from earlier studies suggest that the
conformation of the piperazine ring is a very important factor
in governing sensing selectivities.14,15 The piperazine rings in
1−4, like that of cyclohexane, can exist in both chair and boat
conformations. It is possible that these conformational
preferences play a role in governing the selectivities in
responses to thiols.16 Consequently, a more detailed analysis
of the structure and conformation of probe 2 was carried out in
an attempt to gain insight into its selective response to GSH.
Accordingly, density functional theory (DFT) calculations were
used to optimize the structures of 1−4 at the B3LYP/6-31G*
level using a suite of Gaussian 09 programs.
The results of the calculations show that the piperazine ring
in 4 exists in a twist-boat conformation while the ring in 3
displays a classic chair conformation (Figure 4B,C). While the
The results of the experiments described above clearly
confirm that, in comparison to probe 1, probe 2 possesses a
high selectivity toward GSH in both solution and living cells.
Additional experiments were designed to gain a better
understanding of the source of this selectivity. As shown in
Scheme 2, probe 2 is comprised of two fragments represented
by model compounds 3 and 4, which contain a common
piperazine moiety. Compound 4 was prepared using the
previously described method14 so that its response to GSH
Figure 4. (A) Boat, twist-boat, and chair conformations of the
piperazine ring. Optimized, low-energy conformations of the
piperazine rings in 1−4 using DFT (B3LYP/6-31G*) calculations:
(B) compound 4, (C) compound 3, (D) probe 2, (E) probe 1.
piperazine rings in probe 2 and compound 3 (Figure 4D) also
possess chair conformations, the ring in probe 1 exists in a
twist-boat conformation (Figure 4E). Even though the
piperazine rings of 2 and 3 have similar chair conformations,
the N−H hydrogen in 3 is axially oriented while the dansyl
sulfonamide moiety in 2 is equatorially disposed. These
findings suggest that the preference for piperazine ring chair
conformations and the equatorial spatial disposition of the
sulfonamide groups in probe 2 might be a significant feature
determining the selective response to GSH.
Scheme 2. Fragments 3 and 4 of Probe 2
From another perspective, the sulfur−nitrogen bond (S−N)
lengths in these substances, which are likely related to bond
strenghts, differ in an interesting manner. For example, the
respective S−N bond lengths in 1 and 4 are 1.667 and 1.660 Å.
In contrast, probe 2 has a S−N bond length of 1.727 Å, which
suggests that the S−N bond in this substance is weaker than the
5354
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358

Journal of the American Chemical Society
Article
analogous bond in probe 1. Finally, the obvious structural
difference between GSH and the other thiols could be another
source of the differences seen in reactivities with the
sulfonamide moiety in the probes. GSH contains two amide
units, and it has a longer flexible backbone in comparison to
Cys and Hcy, which provides a possibility to form some
intermolecular interaction with the probe such as hydrogen
bonds. For that, we optimize the binding structure of probe 2
with GSH (Figure S12, Supporting Information), in which
amide units of GSH can form the relevant hydrogen bonds
(such as C−H···N and C−H···O) with the piperazine ring unit
of probe 2. Besides, there is a possible electrostatic interaction
between the indolium cation of probe 2 and the carbonate
anion of GSH from the binding structure. Therefore, these
intermolecular interactions will result in a closer distance of two
reactive sites (SH group and sulfonamide group), possibly a
reason for the higher reactivity of GSH. Moreover, it also
affords an idea to design probes with high selectivity from the
guest molecule’s viewpoint.
opolysaccharides (LPSs) are large molecules consisting of
lipid and polysaccharide units that act as endotoxins to elicit
strong immune responses in animals. Hence, the results of
investigations of the effects of LPS on cells provide a greater
understanding of the immune systems.17 As images displayed in
Figure 6 show, a dramatic fluorescence decrease occurs when a
Observations made in additional studies show that probe 2
can also be employed to detect the oxidant-governed redox
status of GSH in living cells. As seen by viewing the confocal
fluorescence microscope images displayed in Figure 5, addition
Figure 6. Confocal microscope images of probe 2 in RAW 264.7 cells.
Cell images were obtained using an excitation wavelength of 635 nm
and a band-path (655−755 nm) emission filter. (A) Fluorescence
image of RAW 264.7 cells. (B) Fluorescence image of RAW 264.7 cells
incubated with probe 2 (20 μM) for 20 min. (C) Fluorescence image
of RAW 264.7 cells pretreated with lipopolysaccharide (LPS; 1 μg/
mL) for 20 min, interferon γ (IFN-γ; 50 ng/mL) for 16 h, and phorbol
12-myristate 13-acetate (PMA; 2.5 μg/mL) for 30 min and incubated
with probe 2 (20 μM) for 20 min.
solution of HeLa cells is treated with LPS prior to the addition
of 2. This observation indicates that LPS as an inhibitor could
induce the fluorescence response in living cells.
To gain better insight into the molecular orbitals, the
structures and frontier molecular orbital profiles were
optimized by the DFT calculations at the B3LYP/6-31G*
level in a suite of Gaussian 09 programs. Figure S11
(Supporting Information) presents the frontier molecular
orbital profiles, which show that the different functional groups
on the piperazine ring had heavy effects, especially for probes 1
and 2 (Figure S13, Supporting Information), and the calculated
HOMO−LUMO energy gaps (2.14 and 2.13 eV for probes 1
and 2, respectively) were approximately the same as that of the
thiolysis product 3 (2.22 eV). The lower band gaps further
imply that two probes could be used as the near-infrared
fluorescent dyes for imaging in vivo.
The results obtained from cell studies suggest that probe 2
has the potential of being used to detect GSH in vivo. To
evaluate this proposal, mice were intravenously injected with
buffer solutions containing 2 (50 μM) and then after 20 min
observed using fluorescence imaging with an in vivo imaging
aystem (IVIS). Strong fluorescence was observed to emanate
from inside the mouse body (Figure 7). Moreover, when the
mice were pretreated with NMM (20 mM for 20 min) and then
injected with 2, a dramatically lower intensity fluorescence
image was observed (Figure 7). Mice not treated with probe 2
or treated with only NMM do not show specific fluorescence
under similar imaging conditions. To explore the location of
probe 2 based in vivo detection of GSH, four mice, which were
previously treated with 2, were dissected to isolate various
tissues. In accordance with the whole-body mouse images,
strong fluorescence was observed in various tissues, including
Figure 5. Confocal microscope images of probe 2 in HeLa cells. Cell
images were obtained using an excitation wavelength of 635 nm and a
band-path (655−755 nm) emission filter. (A) Fluorescence image of
HeLa cells. (B) Fluorescence image of HeLa cells incubated with
probe 2 (20 μM) for 20 min. (C) Fluorescence image of HeLa cells
pretreated with H2O2 (100 μM) for 20 min and incubated with probe
2 (20 μM) for 20 min.
of H2O2 (100 μM) to a solution containing HeLa cells and
probe 2 (Figure 5B) promotes a marked decrease in the
fluorescence intensity (Figure 5C). This finding is likely the
consequence of the alteration of the intracellular GSH
concentration through oxidation promoted by H2O2. An
experiment was performed to confirm this proposal. As
described above, compound 3 is the likely product of reaction
between probe 2 and GSH. When 3 is added to H2O2-treated
living HeLa cells, an evident red fluorescence can be observed
(Figure S11, Supporting Information). When the cells are
pretreated with NMM for 20 min and then incubated with
compound 3 (10 μM) for 20 min, no obvious fluorescence can
be observed. These results suggest that the fluorescence caused
by treating cells with probe 2 and GSH does not arise from the
reaction of 3 with intracellular H2O2. Therefore, the results of
this experiment show that probe 2 can be used as a tool to
monitor the level of intracellular GSH. Additionally, lip-
5355
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358

Journal of the American Chemical Society
Article
Figure 8. In vivo images of a mouse injected with probe 2 (50 μM) or
acetaminophen (APAP; 300 mg/kg in 200 μL of HEPES buffer
solution) via intravenous injection for 20 min. Fluorescence images of
(A) the mouse injected with probe 2 (2 only) and (B) the mouse
injected with probe 2 after being preinjected with APAP (APAP + 2).
Figure 7. In vivo images of a mouse injected with probe 2 (50 μM) or
NMM (20 mM) via intravenous injection for 20 min. Fluorescence
images of (A) the mouse not injected with probe 2 (no injection), (B)
the mouse injected with NMM (NMM only), (C) the mouse injected
with probe 2 (2 only), and (D) the mouse injected with probe 2 after
being preinjected with NMM (NMM + 2).
the liver, kidney, lung, and spleen. Importantly, fluorescence in
these tissues was attenuated when the mice were pretreated
with NMM (Figure S14, Supporting Information).
We have accumulated evidence that shows that probe 2 does
not initially react with SH-group-containing serum proteins and
biomolecules in the blood to form adducts that are then
delivered into different organs. For this purpose, liver, lung,
kidney, and spleen tissues were treated with probe 2 (20 μM
for 20 min). Strong fluorescence signals, having intensities that
are similar to those coming from probe 2 treated mice, were
observed to arise from the treated tissues (Figure S15,
Supporting Information). Moreover, when mice were pre-
treated with NMM (20 mM for 20 min) via intravenous
injection and then directly administered probe 2, lower
intensity fluorescence images were observed (Figure S15).
These results indicate that probe 2 can be employed to detect
different concentrations of thiols in specific tissues directly.
The use of excessive amounts of certain drugs often causes
damage to some tissues. For example, acetaminophen (APAP)
is a safe painkiller when utilized at therapeutic levels. However,
an overdose of this drug can cause severe liver damage18 and a
depletion of GSH in liver and kidney cells.19 Thus, we
examined the capability of probe 2 to detect acetaminophen-
promoted tissue cell damage associated with a decrease in the
level of GSH. Probe 2 based analysis of whole bodies and
various tissues (Figure 8) shows that pretreatment of mice by
intravenous injection with acetaminophen promotes a dramatic
decrease in the fluorescence intensities in liver, kidney, lung,
and spleen tissues (Figure 9). These observations clearly
demonstrate that 2 is an effective probe for cellular GSH at the
organism level.
Figure 9. In vivo images of tissues from a mouse intravenously
injected with probe 2 (50 μM) or acetaminophen (APAP; 300 mg/kg
in 200 μL of HEPES buffer solution). Fluorescence images of the (A)
liver, (B) kidney, (C) lung, and (D) spleen dissected from a mouse
injected with probe 2 (2 only) and a mouse injected with probe 2 after
being preinjected with APAP (APAP + 2).
which contains a 2,4-dinitrobenzenesulfonamide group, can be
used to detect the thiol-based amino acids GSH, Cys, and Hcy
in living cells. The other, possessing a 5-(dimethylamino)-
naphthalenesulfonamide unit, displays unique selectivity toward
GSH over Cys and Hcy. In addition, the latter probe is cell-
membrane-permeable, and as a result, it can be employed to
detect GSH in living cells. Finally, the latter near-infrared
fluorescent probe can be used to monitor GSH levels in mouse
tissues such as the liver, kidney, lung, and spleen. The results of
this effort strongly suggest that the GSH-selective probe
developed in this investigation can serve as an effective tool for
studies probing cellular functions that are related to GSH.
EXPERIMENTAL SECTION
■
General Methods. All reactions and assembly processes were
carried out under an argon atmosphere by using standard Schlenk
techniques, unless otherwise stated. All starting materials and reagents,
such as anhydrous N,N-dimethylformamide (DMF), dichloromethane
(DCM), triethylamine (Et3N), and human serum, were obtained
CONCLUSION
■
1
commercially. H and 13C NMR spectra used CDCl3 solutions and a
In summary, the study described above has resulted in the
development of two near-infrared fluorescent probes that
contain a cyanine fluorophore linked to sulfonamide functional
groups that undergo reactions with thiols. One of the probes,
Bruker AM-300 spectrometer with tetramethylsilane (TMS) as the
internal standard. Mass spectra were measured in the ESI or MALDI
mode. UV−vis spectra were obtained using a Scinco 3000
5356
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358

Journal of the American Chemical Society
Article
spectrophotometer (1 cm quartz cell) at 25 °C. Fluorescence spectra
were recorded on an RF-5301/PC (Shimada) fluorescence spec-
trophotometer (1 cm quartz cell) at 25 °C. Deionized water was used
to prepare all aqueous solutions.
for 20 min and then incubated with probes (20 μM) for 20 min. The
others were treated with additional GSH or Cys or Hcy (100 μM) at
the same field of view.
For the H2O2 experiment, the cells were treated with H2O2 (100
μM) for 30 min at 37 °C and then incubated with probe 2 (20 μM)
for 20 min at 37 °C.
RAW 264.7 cells were pretreated with LPS (1 μg/mL) for 20 min,
interferon γ (IFN-γ; 50 ng/mL) for 16 h, and PMA (phorbol 12-
myristate 13-acetate; 2.5 μg/mL) for 30 min.
HeLa cells were incubated with probes (10 μM) for 20 min at 37
°C, washed twice with DPBS, and imaged by using a confocal laser
scanning microscope. Cell images were acquired with an excitation 635
nm laser diode and emission filter with BA = 655−755 nm. Next the
HeLa cells were pretreated with NMM (1 mM) for 20 min and then
with GSH or Cys or Hcy (100 μM) for 20 min and incubated with
probes (20 μM) for 20 min.
Animals. We obtained 7−8 week old female C57BL/6 mice from
the Jackson Laboratory (Bar Harbor, ME). All animal experiments
were performed in a facility approved by the Association for
Assessment and Accreditation of Laboratory Animal Care using
protocols approved by the Department of Laboratory Animal
Resources, Yonsei Biomedical Research Institute, Yonsei University
College of Medicine.
In Vivo Imaging. C57BL/6 mice (female, 7−8 weeks old, Jackson
Laboratory) were intravenously injected with NMM (20 mM, in 200
μL of HEPES buffer solution (10 mM, pH 7.4)). After 20 min, the
mice were iv injected with probe 2 (50 μM, 200 μL). The back fur was
removed using a depilatory cream, and the mouse imaging was
performed with an IVIS Spectrum (Caliper Life Sciences, Hopkinton,
MA) in epifluorescence mode equipped with 675 and 720 nm filters
for excitation and emission, respectively, starting 20 min after probe 2
injection. Quantitative measurements of the fluorescence signal were
made with Living Image Software 4.3.1 (Caliper Life Sciences).
The mice were injected with APAP (300 mg/kg in 200 μL of
HEPES buffer solution). After 30 min, the mice were iv injected with
probe 2 (50 μM, 200 μL).
Synthesis of 3. To a solution of IR-780 iodine (134 mg, 0.2
mmol) in anhydrous DMF (10 mL) under an argon atmosphere was
added piperazine (69 mg, 0.8 mm). After being stirred for 4 h at 85 °C,
the mixture was cooled to room temperature. Removal of solvent
under reduced pressure and purification by silica gel column
chromatography with dichloromethane/methanol (40:1) as the eluent
1
generated 3 as a blue solid: yield 136 mg, 85%; H NMR (300 MHz,
CDCl3) δ 7.66 (d, J = 15.0 Hz, 2H), 7.26−7.32 (m, 4H), 7.09 (t, J =
6.0 Hz, 2H), 6.97 (d, J = 6.0 Hz, 2H), 5.80 (d, J = 15.0 Hz, 2H), 3.88
(m, 8H), 3.26 (t, J = 6.0 Hz, 4H), 2.44 (t, J = 6.0 Hz, 4H), 1.84 (m,
6H), 1.68 (s, 12H), 1.02 (t, J = 6.0 Hz, 6H); 13C NMR (75 MHz,
CDCl3) δ 173.3, 169.1, 142.8, 141.0, 140.3, 128.3, 123.7, 123.5, 122.2,
109.4, 85.9, 55.3, 48.2, 47.2, 45.2, 29.2, 25.0, 21.8, 20.3, 11.8; ESI MS
m/z 589.6 [M − I−]+, calcd exact mass 716.3.
Synthesis of 1. To a solution of 3 (143 mg, 0.2 mm) in anhydrous
dichloromethane (10 mL) under an argon atmosphere was added
Et3N (30 mg, 0.3 mm). After the resulting solution was stirred for 5
min, a solution of 2,4-dinitrobenzene-1-sulfonyl chloride (53 mg, 0.2
mm) in dichloromethane (5 mL) was added dropwise at 0 °C. Further
stirring for 4 h at room temperature was followed by solvent removal
under reduced pressure and purification by silica gel column
chromatography with dichloromethane/methanol (30:1) as the eluent
to produce 1 as a blue solid: yield 142 mg, 75%; 1H NMR (300 MHz,
CDCl3) δ 8.77 (m, 2H), 8.52 (s, 1H), 7.80 (d, J = 15.0 Hz, 2H), 7.35
(m, 4H), 7.21 (m, 2H), 7.06 (d, J = 9.0 Hz, 2H), 5.95 (d, J = 15.0 Hz,
2H), 3.96 (t, J = 6.0 Hz, 4H), 3.76 (d, J = 6.0 Hz, 4H), 3.69 (d, J = 6.0
Hz, 4H), 2.49 (t, J = 6.0 Hz, 4H), 1.86 (m, 6H), 1.66 (s, 12H), 1.05 (t,
J = 6.0 Hz, 6H); 13C NMR (75 MHz, CDCl3) δ 170.4, 169.9, 149.8,
147.9, 142.5, 141.9, 140.5, 136.8, 134.9, 128.6, 127.8, 126.3, 124.5,
122.2, 119.4, 110.0, 98.2, 54.0, 48.6, 47.8, 45.6, 28.9, 25.3, 21.5, 20.6,
11.7; MALDI-TOF MS m/z 820.5 [M − I−]+, calcd exact mass 946.3.
Synthesis of 2. To a solution of 3 (143 mg, 0.2 mm) in anhydrous
dichloromethane (10 mL) under an argon atmosphere was added
Et3N (30 mg, 0.3 mm). After the resulting solution was stirred for 5
min, a solution of dansyl chloride (54 mg, 0.2 mm) in dichloro-
methane (5 mL) was added dropwise at 0 °C. Further stirring for 4 h
at room temperature was followed by removal of solvent under
reduced pressure and purification by silica gel column chromatography
with dichloromethane/methanol (30:1) as the eluent to yield 2 as a
blue solid: yield 156 mg, 82%; 1H NMR (300 MHz, CDCl3) δ 8.76 (d,
J = 9.0 Hz, 1H), 8.60 (d, J = 9.0 Hz, 1H), 8.32 (d, J = 9.0 Hz, 1H),
7.59−7.70 (m, 5H), 7.36 (m, 2H), 7.18−7.28 (m, 4H), 7.08 (d, J = 9.0
Hz, 2H), 5.95 (d, J = 15.0 Hz, 2H), 3.99 (t, J = 6.0 Hz, 4H), 3.55 (d, J
= 6.0 Hz, 4H), 3.41 (d, J = 6.0 Hz, 4H), 2.96 (s, 6H), 2.47 (t, J = 6.0
Hz, 4H), 1.83 (m, 6H), 1.29 (s, 12H), 1.02 (t, J = 6.0 Hz, 6H); 13C
NMR (75 MHz, CDCl3) δ 170.4, 168.9, 152.0, 142.5, 141.7, 140.5,
132.0, 130.9, 130.1, 128.7, 128.6, 127.2, 124.4, 123.6, 122.0, 110.3,
98.8, 53.3, 48.3, 47.6, 45.8, 45.6, 28.2, 25.4, 21.5, 20.6, 11.7; MALDI-
TOF MS m/z 822.7 [M − I−]+, calcd exact mass 949.4.
For organ imaging, the mice were euthanized and the organs were
dissected after in vivo imaging experiments. For ex vivo organ imaging,
the liver, kidney, lung, and spleen were obtained from a mouse not
injected with probe 2, and the fluorescence image of those tissues
directly treated with probe 2 (20 μM for 20 min) was obtained. The
isolated liver, kidney, lung, and spleen were imaged with the IVIS
Spectrum.
ASSOCIATED CONTENT
* Supporting Information
■
S
UV/vis absorption and fluorescence spectra of probes 1 and 2,
mass spectra after treatment of probes 1 and 2 with thiols,
fluorescence images of mouse tissues, frontier molecular orbital
1
profiles, and H NMR, 13C NMR, and MS spectra of 1, 2, and
3. This material is available free of charge via the Internet at
http://pubs.acs.org.
Cell Culture. HeLa cells (human epithelial adenocarcinoma) and
RAW 264.7 cells (mouse leukemic monocyte macrophage) were
purchased from the Korean Cell Line Bank (Seoul, Korea). The cells
were grown in RPMI 1640 supplemented with 10% heat-inactivated
fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/
mL). All cells were maintained in an incubator at 37 °C with a 5%
CO2/air environment.
Confocal Microscope Imaging. Cells were seeded in 35 mm
glass-bottomed dishes at a density of 3 × 105 cells per dish in RPMI
1640 medium. After 24 h, probes (20 μM) were added to the cells, and
the cells were incubated for 20 min at 37 °C. After being washed with
Dulbecco’s phosphate-buffered saline (DPBS) twice to remove the
residual probe, the cells were imaged by using a confocal laser scanning
microscope (Fluoview 1200, Olympus, Japan). The cells were excited
by a 635 nm laser diode and detected at BA = 655−755 nm.
For control experiments, one group was treated with NMM (1
mM) for 20 min. Another group was pretreated with NMM (1 mM)
AUTHOR INFORMATION
Corresponding Authors
yjh@yuhs.ac
■
jyoon@ewha.ac.kr
Author Contributions
⊥J.Y. and Y.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by a grant from the National
Creative Research Initiative programs of the National Research
Foundation of Korea (NRF) funded by the Korean government
5357
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358

Journal of the American Chemical Society
Article
(13) Myochin, T.; Kiyose, K.; Hanaoka, K.; Kojima, H.; Terai, T.;
Nagano, T. J. Am. Chem. Soc. 2011, 133, 3401.
(14) Kim, J.; Lim, S.-H.; Yoon, Y.; Thangadurai, T. D.; Yoon, S.
(Ministry of Science, ICT and Future Planning, MSIP)
(2012R1A3A2048814) and by the Basic Science Research
Program through the NRF funded by the Ministry of Education
(Grant 2013R1A1A200), and by the Bio & Medical
Technology Development Program of the NRF funded by
MSIP (NRF-2013M3A9D5072551). We thank Dr. Zhiqian
Guo for his suggestion.
Tetrahedron Lett. 2011, 52, 2645.
(15) (a) Hilderbrand, S. A.; Lippard, S. J. Inorg. Chem. 2004, 43,
5294. (b) Guo, Z.; Zhu, W.; Shen, L.; Tian, H. Angew. Chem., Int. Ed.
2007, 46, 5549. (c) Srivastava, P.; Ali, R.; Razi, S. S.; Shahid, M.;
Patnaik, S.; Misra, A. Tetrahedron Lett. 2013, 54, 3688. (d) Biswal, B.;
Bag, B. Org. Biomol. Chem. 2013, 11, 4975.
(16) Carmona, P., Navarro, R., Hernanz, A., Eds. Spectroscopy of
Biological Molecules: Modern Trends; Springer: New York, 1997.
(17) Van de Bittner, G. C.; Bertozzi, C. R.; Chang, C. J. J. Am. Chem.
Soc. 2013, 135, 1783.
(18) Saito, C.; Zwinqmann, C.; Jaeschke, H. Hepatology 2010, 51,
246.
REFERENCES
■
(1) Wood, Z. A.; Schroder, E.; Robin Harris, J.; Poole, L. B. Trends
Biochem. Sci. 2003, 28, 32.
(2) For selected literature, see: (a) Herzenberg, L. A.; De Rosa, S. C.;
Dubs, J. G.; Roederer, M.; Anderson, M. T.; Ela, S. W.; Deresinski, S.
C.; Herzenberg, L. A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1967.
(b) Jacobsen, D. W. Clin. Chem. 1998, 44, 1833. (c) Shahrokhian, S.
Anal. Chem. 2001, 73, 5972. (d) Townsend, D. M.; Tew, K. D.;
Tapiero, H. Biomed. Pharmacother. 2003, 57, 145. (e) Tsien, R. Y. Nat.
Rev. Mol. Cell Biol. 2003, 4, SS16−SS21. (f) Meade, T. J.; Aime, S. Acc.
Chem. Res. 2009, 42, 821. (g) ilderbrand, S. A.; Weissleder, R. Curr.
Opin. Chem. Biol. 2010, 14, 71. (h) Ueno, T.; Nagano, T. Nat. Methods
2011, 8, 642. (i) Pittet, M. J.; Weissleder, R. Cell 2011, 147, 983.
(j) James, M. L.; Gambhir, S. S. Physiol. Rev. 2012, 92, 897. (k) Chang,
P. V.; Bertozzi, C. R. Chem. Commun. 2012, 48, 8864.
(19) Mitchell, D. B.; Acosta, D.; Bruckner, J. V. Toxicology 1985, 37,
127.
(3) For selected reviews, see: (a) Yin, L.-L.; Chen, Z.-Z.; Tong, L.-L.;
Xu, K.-H.; Tang, B. Chin. J. Anal. Chem. 2009, 37, 1073. (b) Chen, X.;
Zhou, Y.; Peng, X.; Yoon, J. Chem. Soc. Rev. 2010, 39, 2120. (c) Zhou,
Y.; Yoon, J. Chem. Soc. Rev. 2012, 41, 52. (d) Jung, H. S.; Chen, X.;
Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42, 6019. (e) Yin, C.; Huo,
F.; Zhang, J.; Martínez-Manez, R.; Yang, Y.; Lv, H.; Li, S. Chem. Soc.
́
̃
Rev. 2013, 42, 6032.
(4) For selected literature, see: (a) Zhang, M.; Yu, M.; Li, F.; Zhu,
M.; Li, M.; Gao, Y.; Li, L.; Liu, Z.; Zhang, J.; Zhang, D.; Yi, T.; Huang,
C. J. Am. Chem. Soc. 2007, 129, 10322. (b) Tang, B.; Xing, Y.; Li, P.;
Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666.
(c) Guy, J.; Caron, K.; Dufresne, S.; Michnick, S. W.; Skene, W. G.;
Keillor, J. W. J. Am. Chem. Soc. 2007, 129, 11969. (d) Sreejith, S.;
Divya, K. P.; Ajayaghosh, A. Angew. Chem., Int. Ed. 2008, 47, 7883.
(e) Lee, J. H.; Lim, C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am.
Chem. Soc. 2010, 132, 1216. (f) Yang, X.; Guo, Y.; Strongin, R. M.
Angew. Chem., Int. Ed. 2011, 50, 10690. (g) Lee, M. H.; Han, J. H.;
Kwon, P.-S.; Bhuniya, S.; Kim, J. Y.; Sessler, J. L.; Kang, C.; Kim, J. S. J.
Am. Chem. Soc. 2012, 134, 1316. (h) Shao, J.; Sun, H.; Guo, H.; Ji, S.;
Zhao, J.; Wu, W.; Yuan, X.; Zhang, C.; James, T. D. Chem. Sci. 2012, 3,
1049. (i) Guo, Z.; Nam, S.; Park, S.; Yoon, J. Chem. Sci. 2012, 3, 2760.
(j) Fu, N.; Su, D.; Cort, J. R.; Chen, B.; Xiong, Y.; Qian, W.-J.;
Konopka, A. E.; Bigelow, D. J.; Squier, T. C. J. Am. Chem. Soc. 2013,
135, 3567.
(5) For selected literature, see: (a) Meister, A.; Anderson, M. E.
Annu. Rev. Biochem. 1983, 52, 711. (b) Chen, X.; Tian, X.; Shin, I.;
Yoon, J. Chem. Soc. Rev. 2011, 40, 4783. (c) Feng, Y.; Cheng, J.; Zhou,
L.; Zhou, X.; Xiang, H. Analyst 2012, 137, 4885. (d) Hyman, L. M.;
Fanz, K. J. Coord. Chem. Rev. 2012, 256, 2333.
(6) Dalton, T. P.; Shertzer, H. G.; Puga, A. Annu. Rev. Pharmacol.
Toxicol. 1999, 39, 67.
(7) Shao, N.; Jin, J.; Wang, H.; Zheng, J.; Yang, R.; Chan, W.; Abliz,
C. J. Am. Chem. Soc. 2010, 132, 725.
(8) Guo, Y.; Yang, X.; Hakuna, L.; Barve, A.; Escobedo, J. O.; Lowry,
M.; Strongin, R. M. Sensors 2012, 12, 5940.
(9) Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.;
Yang, Q.-Z. J. Am. Chem. Soc. 2012, 134, 18928.
(10) Szaciłowski, K.; Macyk, W.; Drzewiecka-Matuszek, A.; Brindell,
M.; Stochel, G. Chem. Rev. 2005, 105, 2647.
(11) (a) Maity, D.; Govindaraju, T. Org. Biomol. Chem. 2013, 11,
2098. (b) Xu, K.; Qiang, M.; Gao, W.; Su, R.; Li, N.; Gao, Y.; Xie, Y.;
Kong, F.; Tang, B. Chem. Sci. 2013, 4, 1079.
(12) (a) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc.
Rev. 2013, 42, 622. (b) Guo, Z.; Park, S.; Yoon, J.; Shin, I. Chem. Soc.
Rev. 2014, 43, 16.
5358
dx.doi.org/10.1021/ja412628z | J. Am. Chem. Soc. 2014, 136, 5351−5358