Reversible near-infrared fluorescent probe introducing tellurium to mimetic glutathione peroxidase for monitoring the redox cycles between peroxynitrite and glutathione in vivo

Article abstract of DOI:10.1021/ja401360a

The redox homeostasis between peroxynitrite and glutathione is closely associated with the physiological and pathological processes, e.g. vascular tissue prolonged relaxation and smooth muscle preparations, attenuation hepatic necrosis, and activation matrix metalloproteinase-2. We report a near-infrared fluorescent probe based on heptamethine cyanine, which integrates with telluroenzyme mimics for monitoring the changes of ONOO^-/GSH levels in cells and in vivo. The probe can reversibly respond to ONOO^- and GSH and exhibits high selectivity, sensitivity, and mitochondrial target. It is successfully applied to visualize the changes of redox cycles during the outbreak of ONOO^- and the antioxidant GSH repair in cells and animal. The probe would provide a significant advance on the redox events involved in the cellular redox regulation.

Full text of DOI:10.1021/ja401360a

Article
pubs.acs.org/JACS
Reversible Near-Infrared Fluorescent Probe Introducing Tellurium to
Mimetic Glutathione Peroxidase for Monitoring the Redox Cycles
between Peroxynitrite and Glutathione in Vivo
Fabiao Yu, Peng Li, Bingshuai Wang, and Keli Han*
State Key Laboratory of Moleclar Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457
Zhongshan Road, Dalian, China 116023
S
* Supporting Information
ABSTRACT: The redox homeostasis between peroxynitrite
and glutathione is closely associated with the physiological and
pathological processes, e.g. vascular tissue prolonged relaxation
and smooth muscle preparations, attenuation hepatic necrosis,
and activation matrix metalloproteinase-2. We report a near-
infrared fluorescent probe based on heptamethine cyanine,
which integrates with telluroenzyme mimics for monitoring the changes of ONOO−/GSH levels in cells and in vivo. The probe
can reversibly respond to ONOO− and GSH and exhibits high selectivity, sensitivity, and mitochondrial target. It is successfully
applied to visualize the changes of redox cycles during the outbreak of ONOO− and the antioxidant GSH repair in cells and
animal. The probe would provide a significant advance on the redox events involved in the cellular redox regulation.
useful in the detection of disease states but also they will allow
for a screening-type analysis of potential signal transduction
pathways in cells. As known, intracellular ONOO− redox
homeostasis is a complex redox process in living systems. The
process includes signal transduction, homeostasis regulation,
and oxidative stress and antioxidant repair in time in living cells.
Therefore, we strive to develop a probe that can respond
reversibly to the changes in the redox homeostasis regulation
via a redox-based mechanism for visualizing states of this redox
cycles.
Intracellular thiols provide abundant reducing sources that
are central to cellular redox homeostasis in the antioxidant
defense systems. Among these biothiols, glutathione (GSH) is
the most abundant endogenous thiol, whose concentration
ranges from 1 to 15 mM depending on the cell types.7 GSH
manipulates the redox homeostasis through the equilibrium
between the reduced form (GSH) and disulfides form
(GSSG).8 Especially in mitochondria, the primary site of
formation and reactions of intracellular peroxynitrite,1,9 GSH
exists predominantly in the reduced form involved a GSH/
GSSG molar ratio of >100:1.8,10 ONOO− can significantly
perturb the mitochondrial GSH/GSSG ratio and further take
the responsibility for the irreversible damage to respiration.
Interpreting the regulation and interplay between peroxynitrite
and GSH also can reveal the physiological and pathological
roles of endogenous peroxynitrite. In order to distinct the roles
of ONOO−, it is very crucial to establish a real-time
visualization method for monitoring the redox homeostasis
changes between peroxynitrite and glutathione in vivo.
INTRODUCTION
■
Endogenous peroxynitrite (ONOO−) has been recognized as a
strong oxidant agent in vivo. This chemical property of
peroxynitrite makes it a central biological pathogenic factor in a
variety of diseases, such as cardiovascular, neurodegenerative,
and inflammatory disorders.1 Peroxynitrite has also shown as a
nitrating agent causing nitrative stress in cells, but more
investigations on endogenous ONOO− have been devoted to
modulate signal transduction pathways via its ability to nitrate
biomolecules including nitrated tyrosine residues, 8-nitro-
guanosine, and nitro fatty acids and thereby influencing cellular
processes.2 Peroxynitrite can also be as a potential candidate for
anticancer drugs.3 Intracellular peroxynitrite is produced by the
diffusion-controlled reaction of nitric oxide (NO) and super-
oxide (O2•−) radicals at rate of ∼1010 M−1 s−1.3 The
endogenous abnormally high concentration of peroxynitrite
has been estimated up to 100 μM per min in immune system
cells (notably in macrophages) when against invading micro-
organisms (e.g., Chagas’ disease and bacterial infection),4
whereas the steady-state concentration of peroxynitrite is
assessed to be in the nanomolar scale range for hours’ time.5
To provide interpretation for these paradoxes that mentioned
above, we consider the intracellular redox homeostasis between
peroxynitrite and endogenous antioxidants. Cells employ the
expression of ONOO−-specific scavengers glutathione perox-
idase (GPx, a selenoenzyme), to maintain the concentration of
ONOO− below a toxic threshold. However, many recent efforts
are focused on the GPx-like telluroenzyme mimics to explore
how peroxynitrite acts in physiological and pathological
processes. Small artificial organic molecule probes have
emerged as one of the most powerful biotechniques to detect
physiologically active species in living system with high
temporal and spatial resolution.6 Not only can they prove
Received: February 11, 2013
Published: April 27, 2013
© 2013 American Chemical Society
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After confirming the key point of our research, we design and
synthesize a single fluorescent probe Cy-NTe for the reversible
detection of ONOO− and GSH (Scheme 1). The organic small
the UV or visible region, which can cause interference from
biological autofluorescence. Therefore, it is necessary to
develop a reversible fluorescent probe that has near-infrared
(NIR) absorption and emission for peroxynitrite detection in
vivo. Recently, our group has focused on the development of a
selenium-containing reversible fluorescent probe for monitor-
ing cellular redox changes.14 Our overall strategy for imaging
the redox cycle in live biological systems is inspired by
exploiting a mimic of selenoenzyme, GPx, as redox reservoirs in
cells.15 It is reported that the GPx-like activity of telluroenzyme
analogue displays much higher activity than the organoselenium
analogue.16 Next, we selected 2-(phenyltellanyl)benzohydrazide
(NTe) as fluorescent modulator because the organotellurium
analogue undergone a reversible spirocyclization, which could
protect the telluroenzyme mimics moiety from overoxidation
by ONOO−.17 We chose heptamethine cyanine (Cy) as a signal
transducer because this fluorophore platform possessed near-
infrared (NIR) absorption and emission profiles to maximize
tissue penetration while minimizing the absorbance of heme in
hemoglobin and myoglobin, water, and lipids.18 Our design
strategy to the fluorescent probe for intracellular ONOO−/
GSH redox cycles was illustrated in Scheme 1. We reasoned
that the detection mechanism relied on a fast photoinduced
electron transfer (PET) process.14,18b,19 Scheme 2 summarized
the synthesis of Cy-NTe from the appropriately fluorophore
Cy.7.Cl and receptor NTe. The optimization of diphenyl
ditelluride (2) started from readily available bromobenzene and
tellurium;20 2-(methoxycarbonyl) benzenediazonium tetrafluor-
oborate (3) was steadily prepared as an isolable arenediazo-
nium salt.7a Our pivotal material was methyl 2-(phenyltellanyl)-
benzoate (4), which followed a nucleophilic aromatic addition−
elimination reaction with release of N2.21 Reaction of hydrazine
hydrate with 4 gave 2-(phenyltellanyl)benzohydrazide (5)
smoothly. Treatment of 5 with fluorophore Cy.7.Cl in
anhydrous acetonitrile resulted in the formation of our probe
Cy-NTe.22 The synthetic details of these compounds were
shown in the Supporting Information.
Scheme 1. Proposed Reaction Mechanism, Structures of Cy-
NTe and Its Oxidized Product Cy-NTeO
molecule fluorescent probe Cy-NTe features a telluroenzyme
(NTe) fluorescent modulator that is integrated into heptame-
thine cyanine platform (Cy). The probe is found to be selective
for the reversible monitoring of ONOO− and GSH redox cycles
with a detection limit of 9.17 × 10−7 M in potassium phosphate
solution. The fluorescent response of Cy-NTe to ONOO− and
GSH could complete within 10 and 5 min, respectively. These
features play a crucial role in the redox cycle detection on
account of the fast metabolism in homeostasis regulation.
RESULTS AND DISCUSSION
■
Design and Synthesis of Fluorescent Probe Cy-NTe.
Conceiving sensitive and selective fluorescent probes for
peroxynitrite is of critical importance for understanding its
roles in disease and signal transduction. To date, some
fluorescent probes for ONOO− detection have been reported
but can only respond irreversibly to a single initial detection
event.11 In contrast, small molecule probes that can respond
reversibly to changes in redox homeostasis regulation would be
much more valuable for visualizing cycles of redox signaling,
stress, repair, and their dynamic interconversion.12 Only a few
redox-sensitive reversible fluorescence probes for redox stress
have been developed.12,13 Additionally, the fluorescence
excitation and emission of the reported probes most locate in
a
Scheme 2. Strategy and Synthesis of Probe Cy-NTe
a
Notes: (a) Mg, anhydrous THF, reflux 2 h; (b) phenylmagnesium bromide (1) was added into suspension of Te, anhydrous THF, reflux 4 h, after
added saturated NH4Cl, bubbled air 12 h; (c) BF3·Et2O, isoamyl nitrite, anhydrous CH2Cl2, −15 °C, 1 h; (d) diphenyl ditelluride (2), Zn, dimethyl
carbonate, 85 °C, 12 h; (e) 85% hydrazine hydrate, ethanol, reflux 24 h; (f) 2-(phenyltellanyl)benzohydrazide (5), anhydrous acetonitrile, 50 °C, 12
h.
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Investigation of Spectral Properties of Probe Cy-NTe.
We examined the spectral properties of our probe Cy-NTe
under simulated physiological conditions (50 mM PBS pH 7.4,
10 μM). The probe exhibits λmax of absorption and emission at
793 nm (ε793 nm = 95 390 M−1 cm−1) and 820 nm respectively
in the NIR window. In response to ONOO−, Cy-NTe shows an
increase in fluorescence intensity (part b of Figure 1)
Figure 2. Time-dependent fluorescence response of 10 μM Cy-NTe to
ONOO− and other biologically relevant reactive oxygen and nitrogen
species. Bars represent fluorescence intensity at 820 nm during 0, 5,
10, 15, 20, 25, and 30 min after addition of various compounds. 1,
ONOO− (10 μM); 2, NO (300 μM); 3, H2O2 (200 μM); 4, tert-butyl
hydroperoxide (t-BuOOH, 250 μM); 5, cumene hydroperoxide
(CuOOH, 300 μM); 6, methyl linoleate hydroperoxide (MeLOOH,
•
.−
400 μM); 7, OH (200 μM); 8, O2 (200 μM); 9, ClO− (200 μM).
All data were acquired in 50 mM PBS (acetonitrile 10% v/v) at pH 7.4
(λex = 793 nm, λem = 820 nm).
chose the mouse macrophage cell line RAW264.7 as a bioassay
model to assess the specific nature of Cy-NTe toward ONOO−
in cells. The test results were exhibited in Figure 3. Macrophage
cells were treated with the following inducers, and then loaded
with 1 μM Cy-NTe for 15 min, then washed the cells three
times with RPMI-1640 before imaging. A high amount of
intracellular peroxynitrite can be produced by inducible nitric
Figure 1. Absorption (a) and fluorescence emission (b) spectral
changes of Cy-NTe (10 μM) after 10 min upon addition of 0, 2, 4, 6,
8, 10 μM peroxynitrite. Spectra were acquired in 50 mM PBS (pH 7.4,
acetonitrile 10% v/v) with excitation at 793 nm and emission colleting
window from 700 to 900 nm. Inset: Fluorescence intensities at 820 nm
response to peroxynitrite concentration.
corresponding to a quantum yield increase from Φ = 0.0032
for the apo dye Cy-NTe to Φ = 0.0431 for the peroxynitrite
oxidized probe Cy-NTeO. During the off−on fluorescence
processes, the absorbance spectra have no significant change
(part a of Figure 1). To evaluate the ability of Cy-NTe in the
determination of ONOO− concentration under simulated
physiological conditions, the probe is treated with ONOO−
under various concentrations (0 to 10 μM). The fluorescence
intensity at 820 nm was linearly proportional to the ONOO−
concentration in the given range (inset in part b of Figure 1, r =
0.963) indicating the suitability of Cy-NTe for quantitative and
qualitative detection of ONOO− potentially. The detection
limit was determined to be 9.17 × 10−7 M under the
experimental conditions.
Selectivity of Cy-NTe toward Peroxynitrite in Solution
and in Living Cells. The specificity of Cy-NTe toward
ONOO− was verified. The probe was tested against
physiological relevant reactive oxygen and nitrogen species in
solution and in living cells. The fluorescence response of Cy-
NTe was found to be good selective to ONOO− over other
biologically relevant reactive oxygen and nitrogen species
(Figure 2). After exposure of the probe to ONOO−, the
fluorescence signal changes could reach saturation within 10
min. As Figure 2 displayed, NO, H2O2, tert-butyl hydroperoxide
(t-BuOOH), cumene hydroperoxide (CuOOH), and hypo-
chlorite (ClO−) triggered limited fluorescence responses within
30 min of the reaction. However, the fluorescence intensity
increase was far weaker than that caused by ONOO−. Next, we
Figure 3. Fluorescence confocal microscopic images of RAW264.7
cells exposed to oxidative stress. Macrophage cells were treated with
various inducers and then loaded with 1 μM Cy-NTe for 15 min (a)
control. (b) LPS (1 μg/mL) and IFN-γ (50 ng/mL) for 4 h then PMA
(10 nM) for 0.5 h. (c) AG (1 mM), LPS (1 μg/mL), and IFN-γ (50
ng/mL) for 4 h then PMA (10 nM) for 0.5 h. (d) PMA (10 nM) for
0.5 h. (e) Cells treated with 200 μM paraquat for 8 h. (f) The image
after the administration of 50 μM NOC-5 for 3 min, (g) cells treated
with 100 μM NaClO for 0.5 h. (h) Graph showing quantification of
mean fluorescence intensity in a−g correspondingly. Scale bar = 20
μm.
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oxide synthase 2 (NOS2) by bacterial products such as
interferon-gamma (IFN-γ) and lipopolysaccharide (LPS).23
Macrophages are additionally stimulated with phorbol 12-
myristate 13-acetate (PMA) to provide an overproduction of
superoxide during the peroxynitrite formation. The inducer,
PMA, can also produce hydroxyl radical.24 Qaraquat can trigger
elevations in intracellular H2O2 through disruption of the
mitochondrial electron transport chain.25 NO is generated in
form of 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-tria-
zene (NOC-5).26 The hypochlorite (ClO−) is directly from
the sodium hypochlorite. There was almost no fluorescence in
the absence ofstimulant (part a of Figure 3). However, strong
fluorescence appeared after treatment with lipopolysaccharide
(LPS 1 μg/mL) and interferon-γ (IFN-γ 50 ng/mL) for 4 h
followed by additional stimulation with phorbol 12-myristate
13-acetate (PMA, 10 nM) for 0.5 h (part b of Figure 3).
Whereas the cells were pretreated with aminoguanidine (AG),
an inhibitor of nitric oxide synthase, much weaker fluorescence
was detected in stimulated cells (part c of Figure 3). The
hydroxyl radical (·OH) was produced by incubated cells with
(PMA, 10 nM) for 0.5 h, and there was no fluorescence
observed in part d of Figure 3. RAW264.7 cells were incubated
with 200 μM paraquat for 8 h (part e of Figure 3). Together
with RAW264.7 cells loaded with 50 μM NOC-5 for 3 min and
100 μM NaClO for 30 min also showed faint fluorescence
(parts f and g of Figure 3). Part h of Figure 3 showed
quantification of mean fluorescence intensity of each condition
shown in parts a−g of Figure 3. Taken together, these
selectivity assays demonstrate that the chemoselective oxidation
of Cy-NTe can be used for fluorescence selective detection of
ONOO− under simulated physiological conditions and in cells.
Detection of Mitochondrial Peroxynitrite in Macro-
phage Cells. Mitochondria are the cellular power plants. They
are also involved in signaling, cellular differentiation, cell death,
as well as the control of the cell cycle and cell growth.27
Endogenous peroxynitrite are produced via the enzymatic
activity of inducible nitric oxide synthase 2 (NOS2) and
NADPH oxidase. NOS2 is expressed primarily in macrophages
after induced by interferon-gamma (IFN-γ) and lipopolysac-
charide (LPS). Additionally, mitochondrial electron leak is a
main pathway to produce superoxide anion via NADPH
oxidase. Therefore, mitochondrial respiration is a major source
of peroxynitrite in macrophage cells.1,9 We next assessed
whether our probe Cy-NTe could respond to the mitochondrial
peroxynitrite in living cells. Our probe Cy-NTe can exhibit a
significant ability to target mitochondria because of the
ammonium cation in cyanine fluorophore.28 Co-localization
can describe the existence of two or more molecular dyes in
precisely the same space. Now we employed the colocalization
method to confirm Cy-NTe sublocated in mitochondria. Co-
localization experiments were performed by costaining
RAW264.7 cells with rhodamine 123, a mitochondria tracker.29
RAW264.7 cells were treated with LPS (1 μg/mL) and
interferon-γ (IFN-γ 50 ng/mL) for 4 h followed by additional
stimulation with phorbol 12-myristate 13-acetate (PMA, 10
nM) for 0.5 h. After washing with RPMI-1640, cells were
loaded with 1 μM Cy-NTe and 5 μg/mL rhodamine 123 for 15
min. The image in part a of Figure 4 merged well with the
image of staining with rhodamine 123 (part b of Figure 4),
indicating the peroxynitrite oxidized probe Cy-NTeO could
specifically localize in mitochondria. The intensity profiles of
the linear regions of interest across RAW264.7 cells costained
with Cy-NTeO and rhodamine 123 (red arrow in part c of
Figure 4. Cy-NTe and rhodamine 123 dye colocalization at
mitochondria in RAW264.7 cells. Cells treated with LPS (1 μg/mL)
and IFN-γ (50 ng/mL) for 4 h and then with PMA (10 nM) for 0.5 h,
then cells were washed with RPMI-1640. The cells were loaded with 1
μM Cy-NTe (a), and 5 μg/mL rhodamine 123 (b) for 15 min.
Fluorescence confocal microscopic images constructed from 790 to
860 nm for (a) and from 500 to 600 nm for (b) fluorescence
collection windows, λex = 780 and 488 nm, respectively. (c) Merged
red channel (a) and green channel (b). (d) Displayed the
colocalization areas of the red and green channels selected. (e) Co-
localization plot of (a) and (b), z axis represents the frequency that
color pair exists in (c), y and x axis represent the intensity of each pixel
in (d). (f) Intensity profile of regions of interest (red arrow in (c)
across RAW264.7 cells.
Figure 4) are varying in close synchrony (part f of Figure 4).
The Pearson’s coefficient Rr = 0.94 and the Manders’
coefficients m1 = 0.99, m2 = 0.98 were evaluated using Image-
Pro Plus software. We also employed this software to carry a
color intensity correlation analysis of parts a and b of Figure 4.
We plotted the intensity of stain Cy-NTeO against that of
rhodamine 123 for each pixel to assess the intensity distribution
of the two colocalization dyes. The dependent staining in parts
a and b of Figure 4 resulted in highly correlated plots (parts d
and e of Figure 4).30 All of these results made our probe a
strong candidate for real-time imaging mitochondrial redox
status changes caused by ONOO−. In addition, to prove
whether Cy-NTe had toxicity to cells, the MTT assay was
performed. The result showed IC50 was 160 μM. It was
demonstrated that the cytotoxicity of Cy-NTe was low under
experimental conditions.
Investigation of the Reversibility of the Probe in
Solution and in Cells. Next, we discussed the reversibility of
the reaction between Cy-NTeO and GSH in solution and in
cells. Considering the complex and diverse antioxidants
contained in cells, an additional important work of the probe
was carried out to determine which antioxidant could trigger
the fluorescent switch off efficaciously. As shown in part a of
Figure 5, Cy-NTeO exhibited a quick decrease in fluorescence
intensity upon reaction with GSH and cysteine. The
fluorescence recovery rate could be up to 97% within 5 min.
Other protein thoils including thioedoxinand and metal-
lothionein induced limited fluorescent response, whereas the
responses toward other antioxidants, such as vitamin C, vitamin
E, uric acid, and histamine were negligible during the 30 min
experiment (part a of Figure 5). Because the concentration of
mitochondrial GSH was far higher than that of mitochondrial
cysteine, protein thoils, and other endogenous antioxidants,9,31
we considered that the fluorescence signal could be regarded as
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Figure 6. Fluorescence confocal microscopic images of RAW264.7
cells loaded with 1 μM Cy-NTe and exposed to redox cycles between
peroxynitrite and GSH. (a) Cells were incubated under 37 °C for 5
min in RPMI-1640 containing 1 μM Cy-NTe, then washed with
RPMI-1640, and placed in fresh RPMI-1640. (b) Dye-loaded cells
treated with LPS (1 μg/mL) and IFN-γ (50 ng/mL) for 4 h then PMA
(10 nM) for 0.5 h. (c) Cells were washed with RPMI-1640, added AG
(1 mM) and GST (250 units/mL), then incubated in the fresh
complete medium (RPMI-1640 + 20% FBS) for 15 min (d) cells were
washed with RPMI-1640 and placed in fresh RPMI-1640, then treated
with 100 μM SIN-1 for 20 min (e) cells were washed with RPMI-1640,
added L-cysteine (1 mM) and GST (250 units/mL), then incubated in
the fresh complete medium (RPMI-1640 + 20% FBS) for 15 min (f)
quantification of mean fluorescence intensity in (a−e) correspond-
ingly. Scale bar = 20 μm.
Figure 5. (a) Fluorescence recovery ratio of Cy-NTeO (10 μM) with
the addition of different biological reductants during 30 min. Bars
represent fluorescence intensity at 0, 3, 5, 20, and 30 min. All
reductants at 500 μM. 1, L-cysteine; 2, glutathione; 3, thioedoxin; 4,
metallothionein; 5, vitamin C; 6, vitamin E; 7, uric acid; 8, histamine.
Fluorescence recovery ratio = (F − F0)/F; where F is the fluorescence
intensity of Cy-NTeO (10 μM), F0 is the fluorescence intensity of the
probe after adding different biological reductants. (b) Normalized
fluorescence responses of Cy-NTe (10 μM) to redox cycles. Cy-NTe
was oxidized by 1 equiv of added ONOO−. Ten minutes later, the
solution was treated with 2 equiv of GSH. When fluorescence returned
to the starting levels, another 1 equiv of ONOO− was added to the
mixture. The redox cycle was repeated 5 times. ONOO− is from a
peroxynitrite donor, 3-morpholinosydnonimine (SIN-1). All data were
acquired in 50 mM PBS (acetonitrile 10% v/v) at pH 7.4 (λex= 793
nm, λem = 820 nm).
units/mL) in the fresh complete medium (RPMI-1640 + 20%
FBS) for the further culture 15 min, the image exhibited a large
fluorescence decrement (part e of Figure 6). Part f of Figure 6
showed quantification of mean fluorescence intensity of each
condition shown in parts a−e of Figure 6. Taken together,
these results indicate that Cy-NTe allows the reversible
detection of the mitochondrial ONOO− reversible redox cycles
with high sensitivity and reversibility.
a response of Cy-NTeO to mitochondrial GSH when the probe
is used in living cells. Part b of Figure 5 showed that the
reversible oxidation−reduction cycle could be repeated at least
5 times with only a modest fluorescence decrement (25% of
Cy-NTeO was bleached during the process). Taken together,
Cy-NTe was suitable for mitochondrial peroxynitrite reversible
monitoring in living cells.
Visualization of ONOO− Redox Reversibility in Living
Mice. Cy-NTe will be favorable for bioimaging applications in
living animals because NIR light can penetrate tissue deeply
and minimize the interference form background autofluor-
escence. We next sought to apply Cy-NTe to fluorescent
imaging of ONOO− redox cycles in vivo. We utilized BALB/c
mice as biological models. Cy-NTe (1 μM, 50 μL in 1:9
DMSO/saline v/v) was injected into intraperitoneal (i.p.)
cavity of BALB/c mice, and the fluorescent signal produced by
our probe was imaged using a CCD camera after 30 min (part b
of Figure 7). Another mice in part a of Figure 7 was injected
with Cy-NTe (1 μM, 50 μL in 1:9 DMSO/saline v/v) and L-
cysteine (1 mM, 100 μL in saline). As shown in part f of Figure
7, the data suggest that Cy-NTe may be sensitive enough to
detect basal levels of ONOO− that are endogenously produced
without stimulating ONOO− production highlighting the
potential utility of Cy-NTe for ONOO− detection in vivo.
Mice in part c of Figure 7 were injected i.p. with LPS (10 μg/
mL) and IFN-γ (200 ng/mL) for 4 h and then with PMA (100
nM) for 0.5 h and next loaded with 1 μM Cy-NTe for 30 min.
We were pleased to observe that the drug-treated animals give a
significantly increased fluorescent signal compared to the
control animals in part a of Figure 7. Mice in part d of Figure
7 were treated as part c of Figure 7 described continued to
inject into i.p. with AG (1 mM, 100 μL in 1:9 DMSO/saline v/
v), GST (250 units/mL, 300 μL in saline), and L-cysteine (1
Because the probe Cy-NTe exhibited high redox sensitivity,
selectivity, and reversibility, we next verified whether it could
monitor ONOO− reversible redox cycles in the mitochondria
of living cells using confocal fluorescence microscopy. The
experimental results were shown in Figure 6. Macrophage cells
were loaded with 1 μM Cy-NTe for 15 min, and then washed
the cells three times with RPMI-1640. There was almost no
fluorescence (part a of Figure 6). Strong fluorescence appeared
after treatment with LPS (1 μg/mL) and interferon-γ (IFN-γ
50 ng/mL) for 4 h followed by additional stimulation with
phorbol 12-myristate 13-acetate (PMA, 10 nM) for 0.5 h (part
b of Figure 6), whereas the cells were pretreated with
aminoguanidine (AG), an inhibitor of nitric oxide synthase,
and the reactive oxygen species scavenger glutathione S-
transferase (GST, EC 2.5.1.18; 250 units/mL) much weaker
fluorescence was detected (part c of Figure 6). Addition of an
aliquot of 100 μM 3-morpholinosydnonimine (SIN-1, a
common peroxynitrite donor) oxidant resulted in another
burst of oxidative stress and an increase in intracellular
fluorescence (part d of Figure 6). The same cells were washed
with RPMI-1640, then added L-cysteine (1 mM) and GST (250
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental conditions, supplementary methods for chemical
synthesis and characterization of compounds, supplementary
experiments and data. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: klhan@dicp.ac.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NSFC Nos. 21273234, 21203192,
and 2013CB834604.
Figure 7. Imaging of redox cycles between peroxynitrite and GSH in
peritoneal cavity of the mice BALB/c. (a) Cy-NTe (1 μM, 50 μL in
1:9 DMSO/saline v/v) and L-cysteine (1 mM, 100 μL in saline) were
injected in the i.p. cavity. (b) Mice injected i.p. with Cy-NTe (1 μM,
50 μL in 1:9 DMSO/saline v/v). (c) Mice injected i.p. with LPS (10
μg/mL) and IFN-γ (200 ng/mL) for 4 h and then with PMA (100
nM) for 0.5 h, then loaded with 1 μM Cy-NTe for 30 min (d) Mice
treated as (c) described, then injected i.p. with AG (1 mM, 100 μL in
1:9 DMSO/saline v/v), GST (250 units/mL, 300 μL in saline), and L-
cysteine (1 mM, 200 μL in saline). (e) Mice treated as d) described,
then injected i.p. with SIN-1 (1 mM, 1 mL in saline) for 20 min (f)
Quantification of total photon flux from each mouse (a−e). The total
number of photons from the entire peritoneal cavity of the mice (a−e)
was integrated. Images constructed from 790 to 860 nm fluorescence
collection window, λex = 735 nm.
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mM, 200 μL in saline) for 30 min, the mice exhibited a large
fluorescence decrement compared to the animals in part c of
Figure 7. The final group mice were first treated as part d of
Figure 7 described then injected i.p. with SIN-1 (1 mM, 1 mL
in saline) for 20 min. The mice exhibited a significantly higher
fluorescence increase. Part f of Figure 7 showed quantification
of mean fluorescence intensity of each condition shown in parts
a−e of Figure 7. These results establish that Cy-NTe is sensitive
enough to visualize the redox cycles between peroxynitrite and
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■
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In summary, we have presented a new reversible near-infrared
fluorescent probe Cy-NTe to evaluate the ONOO−/GSH
redox status in cells and in vivo. We design the probe through
the synthesis of telluroenzyme, a GPx-like analogue that
interacts with ONOO−/GSH to trigger the fluorescent
response cycles. This mitochondria-targeted fluorescent probe
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that is selective over a variety of reactive nitrogen and oxygen
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