Single fluorescent probe responds to H2O2, NO, and H2O2/NO with three different sets of fluorescence signals

Article abstract of DOI:10.1021/ja2100577

Hydrogen peroxide (H₂O₂) acts as a signaling molecule in a wide variety of signaling transduction processes and an oxidative stress marker in aging and disease. However, excessive H₂O₂ production is implicated w

Full text of DOI:10.1021/ja2100577

Article
pubs.acs.org/JACS
Single Fluorescent Probe Responds to H2O2, NO, and H2O2/NO with
Three Different Sets of Fluorescence Signals
Lin Yuan, Weiying Lin,* Yinan Xie, Bin Chen, and Sasa Zhu
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University,
Changsha 410082, P. R. China
S
* Supporting Information
ABSTRACT: Hydrogen peroxide (H2O2) acts as a signaling
molecule in a wide variety of signaling transduction processes and
an oxidative stress marker in aging and disease. However,
excessive H2O2 production is implicated with various diseases.
Nitric oxide (NO) serves as a secondary messenger inducing
vascular smooth muscle relaxation. However, mis-regulation of
NO production is associated with various disorders. To
disentangle the complicated inter-relationship between H2O2
and NO in the signal transduction and oxidative pathways,
fluorescent reporters that are able to display distinct signals to
H2O2, NO, and H2O2/NO are highly valuable. Herein, we present the rational design, synthesis, spectral properties, and living
cell imaging studies of FP-H2O2-NO, the first single-fluorescent molecule, that can respond to H2O2, NO, and H2O2/NO with
three different sets of fluorescence signals. FP-H2O2-NO senses H2O2, NO, and H2O2/NO with a fluorescence signal pattern of
blue−black−black, black−black−red, and black−red−red, respectively. Significantly, we have further demonstrated that FP-
H2O2-NO, a single fluorescent probe, is capable of simultaneously monitoring endogenously produced NO and H2O2 in living
macrophage cells in multicolor imaging. We envision that FP-H2O2-NO will be a unique molecular tool to investigate the
interplaying roles of H2O2 and NO in the complex interaction networks of the signal transduction and oxidative pathways. In
addition, this work establishes a robust strategy for monitoring the multiple ROS and RNS species (H2O2, NO, and H2O2/NO)
using a single fluorescent probe, and the modularity of the strategy may allow it to be extended for other types of biomolecules.
membrane bound isoenzymes that convert L-arginine to
citrulline and NO.5 NO serves as a secondary messenger by
activating soluble guanylyl cyclase (sGC), inducing a down-
stream pathway that elicits vascular smooth muscle relaxa-
tion.6,7 However, mis-regulation of NO production is associated
with various diseases ranging from stroke, heart disease,
hypertension, neurodegeneration, and erectile dysfunction,to
gastrointestinal distress.7
Notably, H2O2 and NO have interplaying roles in the
complex signal transduction and oxidative pathways.1a,c,d,4a,8−20
H2O2 and NO are concurrently present during various
physiological processes, and sometimes the generation of
H2O2 and NO are interdependent.8 Both H2O2 and NO play
interrelated roles as signaling molecules during the ABA-
dependent stomatal closure.9 H2O2 and NO are synthesized
within cardiac myocytes and play important roles in regulating
the cardiovascular signaling.10 A delicate balance between H2O2
and NO controls the process of defense against pathogens and
decides when the cell death is triggered. Cross-talk between the
two signaling molecules is required for the execution of
programmed cell death in some living systems. NO can
enhance the cytotoxic activity of H2O2 in human ovarian cancer
INTRODUCTION
■
Reactive oxygen species (ROS) are a class of radical or
nonradical oxygen-containing species that play critical roles in
many physiological and pathological processes.1 ROS are
endogenously generated from oxygen in the mitochondrial
respiration pathway.1a,b However, under stimulated by xeno-
biotics, infectious agents, and UV light, ROS can also be
exogenously produced.2 Hydrogen peroxide (H2O2), one of the
important ROS, acts as a signaling molecule in a wide variety of
signaling transduction processes, an oxidative stress marker in
aging and disease, and a defense agent in response to pathogen
invasion.3 It is believed that H2O2 is generated by activation of
NADPH oxidase complexes during cellular stimulation with
cytokines, peptide growth factors, and neurotransmitters.3a
H2O2 is involved in reversible oxidation of proteins that
ultimately regulate cellular processes ranging from protein
phosphorylation to gene expression.3a However, excessive
H2O2 production is implicated with various diseases including
cancer, diabetes, and cardiovascular and neurodegenerative
disorders.4
Reactive nitrogen species (RNS) are another class of
chemically reactive species that are essential to many biological
functions.1a,c,d,5 The prototypical RNS is nitric oxide (NO),
which is endogenously generated by nitric oxide synthases
(NOS), a group of evolutionarily conserved cytosolic or
Received: October 25, 2011
Published: December 8, 2011
© 2011 American Chemical Society
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Figure 1. Rational design strategy for probe FP-H2O2-NO, a unique type of a single fluorescent probe that can report H2O2, NO, and H2O2/NO
with three different sets of fluorescence signals: blue−black−black; black−black−red; and black−red−red.
cells,11 lymphoma cells,12 hepatoma cells,13 and several other
types of cells.14 In contrast, NO inhibits the H2O2-induced
apoptosis/stress in macrophages,4a,15 Chinese hamster fibro-
blasts cells,16 endothelial cells,17 and cardiomyoblasts.18 H2O2
can act as an upstream signal to induce production of NO in
some animal and plant cells.8b,c,e,9,10,19 On the other hand,
exposure to extracellular NO donor can increase the H2O2 level
in neuronal cells.20
To disentangle the complicated inter-relationship between
H2O2 and NO in the signal transduction and oxidative
pathways, reporters that are able to display distinct signals to
H2O2, NO, and H2O2/NO are highly valuable. An electron spin
resonance (ESR)-based method was employed to detect
production of ROS and NO in samples of plants and animals.21
However, this method requires that the biosamples be ground
and treated with spin trapping reagents before the ESR
measurement can be performed. Thus, the method is
destructive and not suitable for monitoring ROS and NO in
the native biological environment. By contrast, fluorescence
sensing, in combination with microscopy, offers an attractive
technique to study biomoleucles of interest in a noninvasive
manner with high spatial and temporal resolution.22 In recent
years, a number of well-designed synthetic fluorescent probes
23
specific for H2O2 or NO24 have been constructed, and good
progress has been made to study H2O2 or NO biology by
employing these synthetic fluorescent probes. As these
fluorescent probes are specific to H2O2 or NO, they are not
able to respond to H2O2, NO, and H2O2/NO with three
different sets of fluorescence signals. A partial solution to the
problem is to use several different types of fluorescent probes in
a cell.10,25 However, as pointed out by Suzuki et al.,26 the
combination of several fluorescent probes produces cross-talk, a
larger invasive effect, the different localization, and the different
metabolisms, making the scenario very complicated. Thus, a
single fluorescent probe which is capable of responding to
H2O2, NO, and H2O2/NO with distinct fluorescence signals is
highly desirable for dissecting the complicated roles of these
biomolecules in living systems. However, to our best
knowledge, the development of such a molecular probe is
still an unmet challenge.
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The aim of our work was to develop a single fluorescent
probe based on a 7-hydroxycoumarin−rhodamine platform
which can report H2O2, NO, and H2O2/NO with different
fluorescence signal patterns. Thus, the single fluorescent probe
can allow H2O2, NO, and H2O2/NO to be differentiated on the
basis of distinct fluorescence responses. Herein, we present the
rational design, synthesis, spectral properties, and living cell
imaging studies of FP-H2O2-NO (Figure 1), the first single-
fluorescent-molecule, that can respond to H2O2, NO, and
H2O2/NO with three different sets of fluorescence signals.
Significantly, FP-H2O2-NO is capable of simultaneously
detecting endogenously produced H2O2 and NO in living
cells by dual-color fluorescence imaging. FP-H2O2-NO is
promising as a unique molecular tool to report on the
production and dynamics of H2O2 and NO in the complex
interaction networks of the signal transduction and oxidative
pathways.
chemistry as the responsive site for NO. (4) Positioning of the
responsive sites of H2O2 and NO. To minimize the potential
interference (i.e., steric hindrance) between the two reaction
sites, they were rationally placed on the two ends of the FP-
H2O2-NO molecular scaffold. This setting should allow the two
reaction sites to be independently operated. (5) Synthetic
accessibility. The overall design allows FP-H2O2-NO to be
constructed in a few synthetic steps. (6) Modularity of the
design strategy based on the 7-hydroxycoumarin−rhodamine
platform. This flexible design allows a wide variety of reaction
sites to be incorporated on the 7-hydroxycoumarin−rhodamine
platform. We anticipate that the modular nature of the strategy
will afford a versatile approach for sensing multiple targets
based on a single fluorescent probe.
On the basis of the above design strategy, we reasoned that
FP-H2O2-NO could respond to H2O2, NO, and H2O2/NO
with distinct fluorescence signal patterns by exploiting the
unique optical properties of coumarin and rhodamine dyes.
Although 7-hydroxycoumarin has significant absorption at
around 400 nm, 7-alkoxycoumarin displays minimal absorption
at this wavelength. Rhodamine in the spirocyclic form has
essentially no absorption at around 550 nm and is almost
nonfluorescent, whereas rhodamine in the open form exhibits
intense absorption at around 550 nm and is highly fluorescent.
Thus, owing to the characteristic optical properties of coumarin
and rhodamine dyes, the free FP-H2O2-NO has no emission at
460 and 580 nm when excited at 400 nm and no emission at
580 nm when excited at 550 nm. The fluorescence signal
pattern for the free probe is black−black−black as shown in
Figure 1. However, when the probe is incubated with H2O2, the
boronate group of FP-H2O2-NO is removed to afford
compound FP-NO, which contains the 7-hydroxycoumarin
dye and the rhodamine in the spirocyclic form. FP-NO has
intense emission at 460 nm when excited at 400 nm and
exhibits no emission at 580 nm when excited at 400 or 550 nm.
Thus, the fluorescence signal pattern for the probe in the
presence of H2O2 is blue−black−black. Upon treatment of the
probe FP-H2O2-NO with NO, the phenylenediamine group of
FP-H2O2-NO is eliminated to give compound FP-H2O2, which
bears the 7-alkoxycoumarin dye and the rhodamine in the open
form. FP-H2O2 has no emission at 460 and 580 nm when
excited at 400 nm and exhibits strong emission at 580 nm when
excited at 550 nm. Thereby, the fluorescence signal pattern for
the probe FP-H2O2-NO in the presence of NO is black−
black−red. When the probe FP-H2O2-NO is incubated with
H2O2/NO, the boronate and phenylenediamine groups of FP-
H2O2-NO are removed to eventually provide compound 9,
which contains the 7-hydroxycoumarin dye and the rhodamine
dye in the open form. When compound 9 (the 7-
hydroxycoumarin−rhodamine platform) is excited at 400 nm,
the emission of 7-hydroxycoumarin should transfer to the
rhodamine dye by FRET (see Table S1 and the corresponding
Supporting Information). Thus, compound 9 has no emission
at 460 nm when excited at 400 nm and displays strong emission
at 580 nm when excited at 400 or 550 nm. Thus, the
fluorescence signal pattern for the probe in the presence of
H2O2/NO is black−red−red. Taken together, remarkably, a
single molecular probe, FP-H2O2-NO, may respond to H2O2,
NO, and H2O2/NO with three different sets of fluorescence
signals: an INHIBIT logic gate at 460 nm when excited at 400
nm and an AND or TRANSFER logic gate at 580 nm when
excited at 400 or 550 nm, respectively. This should serve as the
basis for independently or simultaneously detection of these
RESULTS AND DISCUSSIONS
■
Design and Synthesis of Fluorescent Probe FP-H2O2-
NO and Control Compounds FP-H2O2, FP-NO, and 9.
Since prominent reports by de Silva et al.,27 a number of
molecular logic gates28 and fluorescent sensors29 have been
developed by combining multibinding sites and one fluorescent
reporter in a single molecule for simultaneous sensing and
detection of multianalytes. Inspired by these elegant reports, in
this work, we present FP-H2O2-NO as a new type of single
fluorescent probe that can respond to H2O2, NO, and H2O2/
NO with three different sets of fluorescence signals. The design
strategy for FP-H2O2-NO (Figure 1) is formulated based on
the following considerations: (1) Selection of the fluorescent
dyes. 7-Hydroxycoumarin and rhodamine were chosen as the
fluorescent dyes. The absorption wavelengths of 7-hydrox-
ycoumarin and rhodamine dyes are at around 400 and 550 nm
(Supporting Information Figure S1), respectively. Thus, their
absorption wavelengths are well separated, and they can be
independently excited. Furthermore, the emission wavelengths
of 7-hydroxycoumarin and rhodamine dyes are at around 460
and 580 nm (Supporting Information Figure S2), respectively.
Thereby, their emission wavelengths are essentially resolved,
and essentially no cross-talk in the emission spectra is present.
This allows them to be employed simultaneously in optical
microscopy, key to the success of the two-color imaging of
H2O2 and NO. (2) Connection of the two fluorescent dyes. 7-
Hydroxycoumarin and rhodamine dyes were connected
through a rigid piperazine linker. The rigid linker and the
relatively short distance (R = 11.07 Å) between the two dyes
(Supporting Information Figure S3) ensure their efficient
Forster resonance energy transfer (FRET) in the 7-hydrox-
̈
ycoumarin−rhodamine platform (JDA = 3.62 × 1014 M−1 cm−1
nm4, R0 = 36.00 Å, E > 99%, see Table S1 and the
corresponding Supporting Information). This opens up
opportunities for multiple fluorescence signals. (3) Selection
of the responsive sites for H2O2 and NO. By exploiting the
boronate chemistry, Chang’s group has developed the H2O2-
specific fluorescent probes, and they have made significant
progress in studies of H2O2 biology using the boronate-based
fluorescent probes.30 Thus, the boronate moiety seems
biocompatible and is a good candidate for the H2O2 reaction
site. On the other hand, the phenylenediamine-based reaction
site has been utilized in the construction of fluorescent NO
probes for biological imaging in living cells.24a,d Thereby, we
decided to employ the chemospecific phenylenediamine-based
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a
Scheme 1. Synthesis of the Probe FP-H2O2-NO and the Control Compounds FP-H2O2, FP-NO, and 9
a
Reagents and conditions: (a) DCC, DMAP, CH2Cl2; (b) separation by column chromatography on silica (CH2Cl2:CH3OH = 9:1); (c) DCC,
NHS, then benzene-1,2-diamine, N(Et)3; (d) HCl/CH2Cl2, then N(Et)3, CH2Cl2; (e) N(Et)3, CH2Cl2; (f) CH2Cl2, Cs2CO3.
■
●
Figure 2. (a) Fluorescence intensity at 586 nm of the control compound FP-H2O2 in the presence of H2O2 ( ) or NO ( ) excited at 400 nm. (b)
■
●
The fluorescence intensity at 586 nm of the control compound FP-NO in the presence of NO ( ) or H2O2 ( ) excited at 550 nm.
key biomolecules in the signal transduction and oxidative
pathways.
diimide (DCC) in CH2Cl2 led to the formation of an activated
intermediate, which was further reacted with benzene-1,2-
diamine to afford the intermediate 4. Subsequently, the t-Boc
protecting group of compound 4 was removed under acidic
conditions to yield amine 5, which was then condensated with
the activated coumarin 6 under the standard coupling
conditions to provide the control compound FP-NO. Finally,
the hydroxyl group of FP-NO was alkylated with bromine 7 to
give the probe FP-H2O2-NO. Control compounds 9 and FP-
H2O2 were also prepared in a few steps. The t-Boc protecting
The synthesis of probe FP-H2O2-NO started with the
preparation of the intermediate 3a (Scheme 1). Condensation
of t-Boc-piperazyl 2 with the mixture of 4′- and 5′-carboxyrhod-
amines 1a/b by the standard coupling chemistry afforded the
mixture of 3a/b, which was separated by silica gel column
chromatography to give the individual compounds 3a and 3b in
the pure isomeric form. Treatment of 3a with coupling reagents
N-Hydroxysuccinimide (NHS) and N, N′-Dicyclohexyl carbo-
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◆
■
▲
●
Figure 3. Absorption (a) and emission (b, c) spectra of compounds FP-H2O2-NO ( ), FP-H2O2 ( ), FP-NO ( ), and 9 ( ) excited at 400 nm
(b) or 550 nm (c).
Figure 4. (a, b) Emission spectra of FP-H2O2-NO (1 μM) in the presence of different equivalents of H2O2 (0−50 equiv) excited at 400 nm (a) or
550 nm (b); (c, d) The emission spectra of FP-H2O2-NO (1 μM) in the presence of different equivalents of NO (0−200 equiv) excited at 400 nm
(c) or 550 nm (d).
group of compound 3a was removed under acidic conditions to
yield amine 8, which was then coupled with the activated
coumarin 6 to produce control compound 9 (the 7-
hydroxycoumarin−rhodamine platform). Alkylation of the key
intermediate 9 afforded another control compound, FP-H2O2.
The structures of FP-H2O2-NO and control compounds FP-
Figure 2a, FP-H2O2 displays a large turn-on fluorescence
response in the presence of H2O2, whereas it exhibits essentially
no fluorescence response to NO, indicating that the boronate-
based reaction site is highly selective for H2O2 over NO. As
displayed in Figure 2b, treatment of FP-NO with NO elicits
intense emission. By contrast, FP-NO has only minimal
fluorescence in the presence of H2O2, suggesting that the
phenylenediamine-based reaction site has high selectivity to
NO over H2O2. Taken together, these data imply that the
boronate-based and phenylenediamine-based reaction sites
could appropriately operate in the presence of H2O2 or NO,
respectively, critical to the success of the probe FP-H2O2-NO.
1
H2O2, FP-NO, and 9 were characterized by H NMR, 13C
NMR, MS (ESI), and HRMS (ESI).
Investigation of Spectral Properties of FP-H2O2-NO.
We first decided to examine the selectivity of the boronate-
based and phenylenediamine-based reaction sites to H2O2 and
NO by using control compounds FP-H2O2 and FP-NO (their
structures are shown in Figure 1 and Scheme 1). As shown in
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The spectral properties of the free probe FP-H2O2-NO were
then investigated. As shown in Figure 3a, FP-H2O2-NO has a
featured absorption peak at around 330 nm. However, it has no
absorption at around 400 nm as the 7-hydroxyl group of the
coumarin dye is alkylated. By sharp contrast, the control
compounds 9 and FP-NO have significant absorption at 400
nm. Furthermore, the free probe FP-H2O2-NO displays no
marked absorption at around 558 nm, as the rhodamine dye is
in the spirocyclic form. However, the control compounds 9 and
FP- H2O2 have strong absorption at 558 nm, as their
rhodamine dye is in the open form. Upon excitation at 400
nm, the free probe FP-H2O2-NO is almost nonfluorescent
(Figure 3b). By contrast, the control FP-NO has strong
emission at 460 nm, ascribed to the fluorescence of 7-
hydroxycoumarin dye. Upon excitation at 400 nm, the control 9
has an emission peak at 580 nm, attributed to the emission of
the rhodamine dye, as the excited energy is transferred from the
7-hydroxycoumarin to rhodamine dye by FRET. As shown in
Figure 3c, upon excitation at 550 nm, the free probe FP-H2O2-
NO is almost nonfluorescent, as its rhodamine dye is in the
spirocyclic form. However, the controls 9 and FP-H2O2 exhibit
a characteristic emission peak of rhodamine at 580 nm, as their
rhodamine dye is in the open form. Thus, the dramatic
distinctions in the optical properties of the free probe FP-
H2O2-NO and the controls 9, FP-H2O2, and FP-NO pave the
way for FP-H2O2-NO to respond to H2O2, NO, and H2O2/NO
with three different sets of fluorescence signals.
The titrations of the probe FP-H2O2-NO (1 μM) with NO
or H2O2 was conducted in pH 7.4 PBS buffer with 20%
CH3CN. As shown in Figure 4a, addition of gradually
increasing concentrations of H2O2 elicits intense emission at
around 460 nm when excited at 400 nm. By contrast, no
significant emission at 581 nm was observed with excitation at
400 (Figure 4a) or 550 nm (Figure 4b). Thus, in the presence
of H2O2, FP-H2O2-NO displays a fluorescence signal pattern of
blue−black−black, consistent with that shown in the design
scheme (Figure 1). On the other hand, when the probe was
incubated with NO, a large fluorescence turn-on at 581 nm was
noted upon excitation at 550 nm (Figure 4d). However, no
marked fluorescence variations at 460 and 581 nm were
detected with excitation at 400 nm (Figure 4c). Thereby, in the
presence of NO, FP-H2O2-NO exhibits a fluorescence signal
pattern of black−black−red, in accordance with that shown in
the design scheme (Figure 1). These data indicate that the
boronate-based and phenylenediamine-based reaction sites of
the probe FP-H2O2-NO selectively responded to H2O2 and
NO, respectively, as anticipated. It is worth noting that H2O2
and NO induce the formation of the emission peaks at 460 and
581 nm, respectively. Thus, the large difference in the emission
wavelengths, up to 121 nm, enables them to be suitable for
dual-color imaging of H2O2 and NO in tandem. The observed
rate constants of the probe to H2O2 and NO were determined
as 3.02 × 10−3 s−1 (Supporting Information Figure S4) and 1.18
× 10−3 s−1 (Supporting Information Figure S5), respectively,
under the pseudo-first-order conditions.
Figure 5. Time-dependent fluorescence intensity of FP-H2O2-NO at
■
●
460 nm excited at 400 nm ( ), at 581 nm excited at 400 nm ( ), and
▲
at 581 nm excited at 550 nm ( ). Notably, H2O2 (50 μM) was
introduced to FP-H2O2-NO (1 μM) at 0 min, and then NO (100 μM)
was further added at 20 min.
emission intensity at 581 nm with excitation at 400 or 550 nm
is almost no variation. These data are consistent with the
conversion of FP-H2O2-NO to FP-NO upon addition of H2O2
as shown in the design scheme (Figure 1). At the time point of
20 min, NO was further introduced to the solution. A
significant enhancement of fluorescence intensity at 581 nm
with excitation at 400 or 550 nm and a drastic drop of
fluorescence intensity at 460 nm with excitation at 400 nm was
observed, in good agreement with the transformation of FP-
NO to 9 upon further addition of NO (Figure 1). Thus, in the
presence of H2O2 and NO, FP-H2O2-NO could ultimately
exhibit a fluorescence signal pattern of black−red−red, in good
agreement with that shown in the design scheme (Figure 1).
To examine the selectivity, the probe FP-H2O2-NO was
incubated with various reactive oxygen species, reactive
nitrogen species, and other biologically relevant species
represented by hydrogen peroxide (H2O2), hypochlorous acid
(HOCl), superoxide (O2•‑), hydroxyl radical (OH•), tert-butyl
hydroperoxide (TBHP), tert-butoxy radical (•OtBu), NO,
NO3−, NO2−, ascorbic acid (AA), and dehydroascorbic acid
(DHA) at 100 μM. As shown in Figure 6a, upon excitation at
400 nm, H2O2 induces a large fluorescence enhancement at 460
nm, whereas HOCl, O2•‑, and other species trigger a small or
minimal fluorescence variation, indicating that the probe is
selective to H2O2 when monitored at 460 nm with excitation at
400 nm, as the boronate-based reaction site is selectively
cleaved by H2O2. On the other hand, upon excitation at 550
nm, only NO elicits a significant fluorescence increase at 581
nm (Figure 6b). By contrast, almost no change of fluorescence
intensity at 581 nm was noted when the probe was incubated
with other species, implying that the probe has a high selectivity
to NO when monitored at 581 nm with excitation at 550 nm, as
the phenylenediamine-based reaction site is selectively removed
by NO. Taken together, these results demonstrate that the
probe FP-H2O2-NO can be employed to separately and
simultaneously detect H2O2 and NO at the different emission
channels. Thus, the probe is promising for dual-color imaging
of H2O2 and NO in living cells.
We then investigated the fluorescence response of FP-H2O2-
NO in the presence of H2O2 and NO. As it is reported that
H2O2 can act as an upstream signal to induce production of NO
in some animal and plant cells,8b,c,e,9,10,19 we were interested to
know the fluorescence pattern of the probe when successively
treated with H2O2 and NO. As shown in Figure 5, addition of
H2O2 to FP-H2O2-NO triggers a large fluorescence enhance-
ment at 460 nm with excitation at 400 nm. However, the
Fluorescence Imaging of H2O2 and NO in HeLa Cells.
We proceeded to evaluate the ability of the probe FP-H2O2-
NO to operate in live cells. HeLa cells incubated with FP-
H2O2-NO (5 μM) for 30 min at 37 °C provide almost no
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Figure 7. Images of HeLa cells treated with probe FP-H2O2-NO. (a)
Bright field image of HeLa cells incubated with only probe FP-H2O2-
NO (5 μM) for 30 min. (b) Fluorescence image of (a) from blue
channel excited at around 405 nm. (c) Fluorescence image of (a) from
red channel excited at around 405 nm. (d) Fluorescence image of (a)
from red channel excited at around 540 nm. (e) Bright field image of
HeLa cells incubated with FP-H2O2-NO (5 μM) for 30 min and then
further incubated with H2O2 (50 μM) for another 30 min. (f)
Fluorescence image of (e) from blue channel excited at around 405
nm. (g) Fluorescence image of (e) from red channel excited at around
405 nm. (h) Fluorescence image of (e) from red channel excited at
around 540 nm. (i) Bright field image of HeLa cells incubated with
FP-H2O2-NO (5 μM) for 30 min and then further incubated with NO
donor diethylamine NONOate (50 μM) for another 30 min. (j)
Fluorescence image of (i) from blue channel excited at around 405
nm. (k) Fluorescence image of (i) from red channel excited at around
405 nm. (l) Fluorescence image of (i) from red channel excited at
around 540 nm. (m) Bright field image of HeLa cells preincubated
with FP-H2O2-NO (5 μM) for 30 min, then treated with H2O2 (50
μM) for another 30 min, and further loaded with NO donor
diethylamine NONOate (50 μM) for 30 min. (n) Fluorescence image
of (m) from blue channel excited at around 405 nm. (o) Fluorescence
image of (m) from red channel excited at around 405 nm. (p)
Fluorescence image of (m) from red channel excited at around 540
nm. Scale bar = 20 μm.
Figure 6. (a) Fluorescence intensity of the probe FP-H2O2-NO (1
μM) at 460 nm excited at 400 nm in the presence of various
biologically relevant species (100 μM). (b) The fluorescence intensity
of the probe FP-H2O2-NO (1 μM) at 581 nm excited at 550 nm in the
presence of various biologically relevant species (100 μM).
fluorescence in both the blue channel (Figure 7b) with
excitation at around 405 nm and the red channel with
excitation at either 405 (Figure 7c) or 540 nm (Figure 7d).
However, when the living HeLa cells were loaded with FP-
H2O2-NO and further treated with H2O2, they gave strong
fluorescence in the blue channel (Figure 7f) and essentially no
fluorescence in the red channel with excitation at around 405
nm (Figure 7g) or 540 nm (Figure 7h). These results imply
that FP-H2O2-NO is responsive to H2O2 in the living cells. On
the other hand, when the living HeLa cells were loaded with
FP-H2O2-NO and then treated with the NO donor diethyl-
amine NONOate, almost no fluorescence in the blue channel
(Figure 7j) and the red channel (Figure 7k) with excitation at
around 405 nm and a marked increase in the red emission
(Figure 7l) on excitation at around 540 nm were observed.
These data establish that FP-H2O2-NO can respond to
intracellular NO in living cells. We further examined the ability
of FP-H2O2-NO to respond to H2O2 and NO. The living cells
were pretreated with FP-H2O2-NO for 30 min, then incubated
with H2O2 for another 30 min, and further loaded with the NO
donor diethylamine NONOate for 30 min (Figure 7m). Almost
no fluorescence in the blue channel (Figure 7n) and a marked
increase in the red emission with excitation at around 405
(Figure 7o) or 540 nm (Figure 7p) were detected. Thus, the
overall results demonstrate that FP-H2O2-NO is cell membrane
permeable and can monitor intracellular H2O2 and NO with
three different sets of fluorescence signals as designed (Figure
1).
detect endogenously produced NO and H2O2 in a dual-color
manner. The RAW264.7 macrophage cells loaded with probe
FP-H2O2-NO display almost no fluorescence in the blue
channel (Figure 8b) and the red channel with excitation at
around 405 nm (Figure 8c) and 540 nm (Figure 8d). When the
RAW264.7 macrophage cells were coincubated with phorbol
12-myristate 13-acetate (PMA) and FP-H2O2-NO for 1 h, a
marked enhancement in the blue channel (Figure 8f) was
observed. However, the red channel is almost nonfluorescent
(Figures 8g,h). These results are in accordance with the
previous reports that PMA can effectively promote H2O2
production31 but induce only slight NO production32 in
RAW264.7 macrophage cells. Thus, the macrophage cells
loaded with PMA and FP-H2O2-NO display a fluorescence
pattern of blue−black−black (Figures 8f−h), consistent with
that shown in Figure 1 for the probe in the presence of H2O2.
We then investigated the fluorescence response of the probe
when treated with the RAW264.7 macrophage cells prestimu-
lated with lipopolysaccharide (LPS). When the RAW264.7
Fluorescence Imaging of Endogenously Produced
H2O2 and NO in RAW 264.7 Macrophages Cells.
Encouraged by the above promising results, we decided to
further examine the feasibility of the probe FP-H2O2-NO to
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Figure 9. Images of RAW 264.7 macrophages treated with probe FP-
H2O2-NO in the absence or presence of stimulants and scavengers. (a)
DIC image of RAW 264.7 macrophage cells incubated with LPS (1
μg/mL) for 12 h and then treated with FP-H2O2-NO (5 μM) for 1 h.
(b) Fluorescence image of (a) from blue channel excited at around
405 nm. (c) Fluorescence image of (a) from red channel excited at
around 405 nm. (d) Fluorescence image of (a) from red channel
excited at around 540 nm. (e) DIC image of RAW 264.7 macrophages
pretreated with LPS (1 μg/mL) for 6 h, then treated with PTIO (a
scavenger for NO) for 6 h, and further incubated with FP-H2O2-NO
(5 μM) for 1 h. (f) Fluorescence image of (e) from blue channel
excited at around 405 nm. (g) Fluorescence image of (e) from red
channel excited at around 405 nm. (h) Fluorescence image of (e) from
red channel excited at around 540 nm. (i) DIC image of RAW 264.7
macrophages pretreated with LPS (1 μg/mL) for 6 h, then loaded with
PEG-catalase (a scavenger for H2O2) for 6 h, and further incubated
with FP-H2O2-NO (5 μM) for 1 h. (j) Fluorescence image of (i) from
blue channel excited at around 405 nm. (k) Fluorescence image of (i)
from red channel excited at around 405 nm. (l) Fluorescence image of
(i) from red channel excited at around 540 nm. Scale bar = 20 μm.
Figure 8. Images of RAW 264.7 macrophages treated with probe FP-
H2O2-NO in the absence or presence of stimulants. (a) DIC image of
RAW 264.7 macrophages incubated with only probe FP-H2O2-NO (5
μM) for 1 h. (b) Fluorescence image of (a) from blue channel excited
at around 405 nm. (c) Fluorescence image of (a) from red channel
excited at around 405 nm. (d) Fluorescence image of (a) from red
channel excited at around 540 nm. (e) DIC image of RAW 264.7
macrophages coincubated with FP-H2O2-NO (5 μM), PMA (1 μg/
mL) for 1 h. (f) Fluorescence image of (e) from blue channel excited
at around 405 nm. (g) Fluorescence image of (e) from red channel
excited at around 405 nm. (h) Fluorescence image of (e) from red
channel excited at around 540 nm. (i) DIC image of RAW 264.7
macrophages incubated with LPS (1 μg/mL) for 12 h and then treated
with FP-H2O2-NO (5 μM) for 1 h. (j) Fluorescence image of (i) from
blue channel excited at around 405 nm. (k) Fluorescence image of (i)
from red channel excited at around 405 nm. (l) Fluorescence image of
(i) from red channel excited at around 540 nm. Notably, the
fluorescence responses of the probe in RAW264.7 macrophage cells
pretreated with PMA or LPS are in good agreement with those shown
in the design scheme (Figure 1). Scale bar = 20 μm.
with FP-H2O2-NO, a marked fluorescence enhancement in the
blue channel (Figure 9f) and essentially no fluorescence in the
red channel (Figures 9g, h) were noted. Comparison of the
results of the experiment LPS + PTIO with those of the
experiment LPS indicates that the fluorescence variations
observed are indeed triggered by NO. On the other hand, when
the RAW 264.7 macrophages cells were pretreated with LPS for
6 h, then treated with cell-permeable PEG-catalase, a scavenger
for H2O2,35 and further loaded with FP-H2O2-NO, almost no
fluorescence in the blue channel (Figure 9j) and in the red
channel with excitation at 405 nm (Figure 9k) and bright
fluorescence in the red channel with excitation at 540 nm
(Figure 9l) were detected. Comparison of the results of the
experiment LPS + catalase with those of the experiment LPS
demonstrates that the fluorescence variations observed are
indeed triggered by H2O2. Thus, taken together, these results
establish that FP-H2O2-NO is capable of simultaneously
monitoring endogenously produced NO and H2O2 in living
macrophage cells in multicolor imaging, suggesting the
potential utility of the probe FP-H2O2-NO in dissecting the
interplaying roles of NO and H2O2 in the complex interaction
networks of the signal transduction and oxidative pathways.
macrophage cells were treated with LPS for 12 h and then
incubated with FP-H2O2-NO for 1 h, essentially no
fluorescence in the blue channel (Figure 8j) excited at around
405 nm and a dramatic enhancement in the red emission
excited at 405 nm (Figure 8k) or 540 nm (Figure 8l) were
detected. These data are in line with the previous reports that
LPS can effectively induce the production of both H2O2 and
NO.33 Thus, the macrophage cells loaded with LPS and FP-
H2O2-NO exhibit a fluorescence pattern of black−red−red
(Figures 8j−l), consistent with that shown in Figure 1 for the
probe in the presence of both H2O2 and NO, validating the
design strategy. Significantly, to our best knowledge, this
represents the first report of simultaneous detection of
endogenously produced NO and H2O2 in dual-color imaging
using a single fluorescent probe.
To further confirm that the fluorescence changes of the cells
loaded with the probe observed above were indeed induced by
H2O2 and NO, the living RAW 264.7 macrophages were
incubated with FP-H2O2-NO in the absence or presence of the
stimulants and the corresponding scavengers. As anticipated,
the cells-loaded with the probe exhibit bright fluorescence in
the red channel excited at 405 (Figure 9c) or 540 nm (Figure
9d). However, when the RAW 264.7 macrophages cells were
pretreated with LPS (notably, LPS can effectively induce the
production of both H2O2 and NO33) for 6 h, then treated with
2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-
oxide (PTIO), a scavenger for NO,24e,34 and further loaded
CONCLUSION
■
In summary, we have described the rational design, synthesis,
optical properties, and living cell imaging applications of FP-
H2O2-NO, a unique type of a single fluorescent probe that can
report H2O2, NO, and H2O2/NO with three different sets of
1312
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Article
(ppm): 1.12 (12H), 3.31−3.92 (16H), 5.98 (d, J = 8.0 Hz, 1H), 6.27−
6.30 (3H), 6.38 (bs, 2H), 6.58−6.62 (3H), 6.75 (s, 1H), 6.84 (d, J =
8.0 Hz, 1H), 6.91 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 6.0 Hz, 1H), 7.54 (d,
J = 7.5 Hz, 1H), 7.72 (d, J = 6.0 Hz, 1H), 8.04 (s, 2H). 13C NMR
(CD3OD, 125 MHz) δ (ppm): 12.81, 45.33, 70.33, 99.19, 103.41,
109.44, 112.46, 115.19, 117.78, 118.29, 120.23, 122.08, 123.02, 126.24,
129.75, 129.88, 130.15, 131.74, 133.17, 133.54, 137.24, 145.83, 146.71,
150.61, 155.29, 155.48, 157.71, 160.45, 164.44, 166.69, 167.43, 171.60.
ESI-MS m/z 833.4 [M +H ]+. HRMS (ESI) m/z calcd for
C49H49N6O7 ([M + H]+): 833.3617. Found: 833.3657.
HeLa Cells Culture and Imaging. HeLa cells were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10% heat inactivated fetal calf serum (FCS), 2 mM L-glutamine, 50
μg/mL penicillin, and 50 μg/mL streptomycin in an atmosphere of 5%
CO2 and 95% air at 37 °C. One day before imaging, the cells were
plated on 6-well plates and allowed to adhere for 24 h. Subsequently,
the cells were incubated with FP-H2O2-NO (5 μM) for 0.5 h at 37 °C
and then washed with phosphate-buffered saline (PBS) three times.
After incubating with H2O2 (50 μM) or NO donor diethylamine
NONOate (50 μM) for another 0.5 h at 37 °C, the cells were rinsed
with PBS three times, and the fluorescence imaging was performed by
an Olympus inverted fluorescence microscope (IX71) equipped with a
cooled CCD camera (Figure 7).
fluorescence signals. FP-H2O2-NO responds to H2O2, NO, and
H2O2/NO with a fluorescence signal pattern of blue−black−
black, black−black−red, and black−red−red, respectively.
Significantly, we have further demonstrated for the first time
that FP-H2O2-NO, a single fluorescent probe, is capable of
simultaneously monitoring endogenously produced NO and
H2O2 in living macrophage cells in multicolor imaging. We
envision that FP-H2O2-NO will be a unique molecular tool to
investigate the interplaying roles of H2O2 and NO in many
important signaling and oxidative pathways. Further optimiza-
tion of this class of probes may render new opportunities in
studies of complicated ROS and RNS biology. In addition, this
work establishes a robust strategy for monitoring the multiple
ROS and RNS species (H2O2, NO, and H2O2/NO) using a
single fluorescent probe, and the modularity of the strategy may
allow it to be extended for other types of biomolecules by
judicious selection of the suitable reaction sites. Work along
these lines is under progress. We expect that this type of
fluorescent probe may have potential applications in flow
cytometry due to the multiple fluorescence signal patterns.
Raw 264.7 Murine Macrophages Culture and Imaging. Raw
264.7 murine macrophages were obtained from Xiangya hospital and
cultured in DMEM (Dulbecco’s Modified Eagle Medium) supple-
mented with 10% FBS (fetal bovine serum) in an atmosphere of 5%
CO2 and 95% air at 37 °C. For imaging studies, the cells were plated
on 6-well plates and allowed to adhere for 24 h. Subsequently, the
RAW 264.7 macrophage cells were coincubated with FP-H2O2-NO (5
μM) and PMA (1 μg/mL) for 1 h in an atmosphere of 5% CO2 and
95% air at 37 °C. Alternatively, the RAW 264.7 macrophage cells were
treated with LPS (1 μg/mL) for 12 h, then loaded with FP-H2O2-NO
(5 μM) for 1 h in an atmosphere of 5% CO2 and 95% air at 37 °C.
Immediately before the experiments, the cells were rinsed with PBS
three times, and the fluorescence images were acquired through an
Olympus inverted fluorescence microscope (IX71) equipped with a
cooled CCD camera (objective magnification: 20-fold, exposition-
acquisition times: 400 ms). The culture and imaging experiments in
the presence or absence of the stimulants and the corresponding
scavengers were conducted in an analogous manner.
EXPERIMENTAL SECTION
■
Synthesis of Compound FP-H2O2-NO. To a CH2Cl2 solution (5
mL) of compound FP-NO (40.0 mg, 0.048 mmol) were added
compound 7 (43.0 mg, 0.144 mmol) and Cs2CO3 (47.0 mg, 0.144
mmol) at room temperature. The mixture was heated and stirred
under reflux. After 0.5 h, the solvent was removed under reduced
pressure. The resulting residue was subjected to column chromatog-
raphy on silica (CH2Cl2:CH3OH = 200:1) to yield compound FP-
1
H2O2-NO as a white solid (34.1 mg, 67.5%). Mp 198−200 °C. H
NMR (CDCl3, 500 MHz) δ (ppm): 1.15 (t, J = 6.8 Hz, 12H), 1.35 (s,
12H), 3.32−3.91 (16H), 5.17 (s, 2H), 6.09 (d, J = 7.0 Hz, 1H), 6.27
(2H), 6.33 (2H), 6.41 (t, J = 7.5 Hz, 1H), 6.55 (d, J = 8.0 Hz, 1H),
6.64 (2H), 6.89 (s, 1H), 6.93−6.97 (2H), 7.33 (d, J = 7.5 Hz, 1H),
7.43 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 8.0 Hz,
1H), 7.84 (d, J = 7.5 Hz, 2H), 7.97 (s, 1H), 8.04 (s, 1H). 13C NMR
(CDCl3, 125 MHz) δ (ppm): 12.42, 24.81, 44.36, 70.56, 83.89, 98.02,
101.77, 107.99, 112.07, 114.07, 117.08, 118.25, 120.62, 121.80, 121.88,
125.04, 126.54, 128.54, 128.61, 128.81, 129.83, 131.80, 132.04, 135.17,
138.39, 144.33, 153.83, 156.17, 158.33, 162.98, 164.17, 165.32, 169.73.
ESI-MS m/z 1049.5 [M + H]+. HRMS (ESI) m/z calcd for
ASSOCIATED CONTENT
■
S
+
C62H6610BN6O9 ([M + H]+): 1048.5024. Found: 1048.5015.
* Supporting Information
Experimental procedures and some spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
Synthesis of Compound FP-H2O2. To a CH2Cl2 solution (5 mL)
of compound 9 (20.0 mg, 0.027 mmol), were added compound 7
(48.0 mg, 0.16 mmol) and Cs2CO3 (44.0 mg, 0.13 mmol) at room
temperature, and then the solution was stirred under reflux. After 0.5
h, the solvent was removed under reduced pressure. The resulting
residue was subjected to column chromatography on silica
(CH2Cl2:CH3OH = 9:1) to yield compound FP-H2O2 as a red
AUTHOR INFORMATION
■
Corresponding Author
weiyinglin@hnu.edu.cn
1
powder (12.0 mg, 46.3%). H NMR (CD3OD, 500 MHz) δ (ppm):
1.27 (t, J = 6.5 Hz, 12H), 1.34 (s, 9H), 3.51−3.88 (16H), 5.19 (s, 2H),
6.90−7.02 (5H), 7.22 (s, 2H), 7.36 (2H), 7.44 (d, J = 7.5 Hz, 1H),
7.59 (d, J = 8.5 Hz, 1H), 7.71−7.76 (3H), 8.06 (s, 1H), 8.21 (s, 1H).
13C NMR (CD3OD, 125 MHz) δ (ppm): 11.47, 23.85, 45.35, 74.46,
83.83, 95.71, 101.41, 112.17, 113.45, 113.63, 113.70, 120.10, 126.34,
126.49, 128.28, 128.67, 129.89, 130.11, 131.56, 134.31, 134.62, 136.49,
139.35, 144.01, 155.49, 156.01, 157.89, 158.74, 160.90, 163.12, 163.20,
165.01, 170.09, 170.69. ESI-MS m/z 959.4 [M]+. HRMS (ESI) m/z
ACKNOWLEDGMENTS
■
Funding was partially provided by NSFC (20872032,
20972044, 21172063), NCET (08-0175), the Doctoral Fund
of Chinese Ministry of Education (20100161110008), and the
Fundamental Research Funds for the Central Universities,
Hunan University.
+
calcd for C56H6010BN4O10 ([M]+): 958.4433. Found: 958.4432.
REFERENCES
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