Development of a ratiometric fluorescent sensor for ratiometric imaging of endogenously produced nitric oxide in macrophage cells

Article abstract of DOI:10.1039/c1cc13047a

We described the development of Cou-Rho-NO as the first small-molecule suitable for ratiometric fluorescent imaging of endogenously produced nitric oxide in macrophage cells.

Full text of DOI:10.1039/c1cc13047a

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Development of a ratiometric fluorescent sensor for ratiometric imaging
of endogenously produced nitric oxide in macrophage cellsw
Lin Yuan, Weiying Lin,* Yinan Xie, Bin Chen and Jizeng Song
Received 24th May 2011, Accepted 27th June 2011
DOI: 10.1039/c1cc13047a
We described the development of Cou-Rho-NO as the first
small-molecule suitable for ratiometric fluorescent imaging of
endogenously produced nitric oxide in macrophage cells.
can be employed for ratiometric imaging of endogenously
produced NO in macrophage cells.
The novel ratiometric sensor Cou-Rho-NO was rationally
designed based on a coumarin-rhodamine scaffold as the
Forster resonance energy transfer (FRET) dyad. When the
¨
Nitric oxide (NO) is an endogenous signalling molecule produced
by inducible and constitutive nitric oxide synthases.1 Although
the half-life of nitric oxide is only several seconds at low
concentrations in biological systems,2 it is involved in many
critical biological processes including signal transduction,
smooth muscle relaxation, peristalsis, immune system control,
neurotransmission, blood pressure modulation, learning, and
memory.3,4 However, mis-regulation of NO production is
implicated with various diseases ranging from stroke, heart
disease, hypertension, neurodegeneration, erectile dysfunction, to
gastrointestinal distress.4
rhodamine dye is in the ring-closed form, it has no significant
absorption in the visible region and is non-fluorescent.
However, when the rhodamine dye is in the ring-opened form,
it has significant absorption in the visible region and is highly
fluorescent. The ring-opening process of the rhodamine dye
accompanied by optical property variations has been extensively
exploited to design turn-on type fluorescent sensors. Furthermore,
a rigid piperazine moiety was selected as the linker to avoid the
likely static fluorescence quenching due to the close contact of
the coumarin donor and rhodamine acceptor dyes in aqueous
environment. In addition, o-phenylenediamine was chosen
as the potential reaction site for NO. In the absence of NO,
the excitation energy of the coumarin donor could not be
transferred to the rhodamine acceptor which is in the closed
form. Thus, the FRET should be off in the free Cou-Rho-NO.
In other words, upon excitation at the coumarin donor, only
the emission of the coumarin dye is observed. However, we
envisioned that addition of NO may induce the rhodamine
acceptor to be in the ring-opened form via cascade reactions of
oxidation and hydrolysis as reported previously.7a Thus,
the FRET is switched on when the new ratiometric sensor
Cou-Rho-NO is incubated with NO. Obviously, we should
note the emission of the rhodamine acceptor upon excitation
at the coumarin donor.
Although a wide variety of techniques have been developed
to detect NO, fluorescence sensing has become the gold
standard due to its high sensitivity and simplicity.5 Thus, the
construction of small-molecule fluorescent sensors suitable for
specific NO detection in living systems has received great
attention.6,7 Metal-based and reaction-based sensors are the
two main classes of small-molecule sensing agents specific
for NO detection. The recently developed small-molecule
fluorescent NO sensors typically exhibit low fluorescence in
the absence of NO, but they display a considerable enhancement
in fluorescence intensity when incubated with NO. Thus, they
respond to NO with a fluorescence turn-on signal.
However, fluorescence intensity-based measurements are
influenced by sensor concentration, environment, and excitation
intensity, etc. By contrast, ratiometric measurements based on
the intensity ratios at two wavelengths may alleviate most of
these problems.8 Although ratiometric fluorescent NO sensors
suitable for cellular ratiometric imaging are highly sought,6
to our best knowledge, no such small-molecule ratiometric
fluorescent NO sensors have been reported to date.
The synthesis of compound Cou-Rho-NO started with the
preparation of the intermediate rhodamine acid 1a (Scheme S1w)
In this communication, we present compound Cou-Rho-NO
(Scheme 1) as the first small-molecule fluorescent sensor which
State Key Laboratory of Chemo/Biosensing and Chemometrics,
College of Chemistry and Chemical Engineering, Hunan University,
Changsha, Hunan 410082, P. R. China. E-mail: weiyinglin@hnu.cn;
Fax: (+)86 731 88821464; Tel: (+)86 731 88821464
w Electronic supplementary information (ESI) available: Experimental
procedures, characterization data, and some spectra. See DOI:
10.1039/c1cc13047a
Scheme 1 Synthesis of the ratiometric sensor Cou-Rho-NO. Reagents
and conditions: (a) DCC, DMAP, CH2Cl2; (b) phosphorus oxychloride,
1,2-dichloroethane; (c) o-diaminobenzene, triethylamine.
c
9372 Chem. Commun., 2011, 47, 9372–9374
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and piperazyl-coumarin 2 (Scheme S2w). Condensation of
3-(diethylamino)phenol 4 with commercially available 1,2,4-
benzenetricarboxylic anhydride 5 led to the formation of the
mixture of 40- and 50-carboxyrhodamines 1a/b (Scheme S1w).
However, the separation of the resulting mixture to afford
pure regioisomers 1a and 1b is very challenging due to their
quite similar structures and high polarity. Extensive optimization
was performed to identify suitable conditions for the separation,
and we eventually found that the mixture could be resolved by
preparative thin layer chromatography (CH2Cl2 :CH3OH, 6 : 1).
The 4- and 5-position regioisomers 1a and 1b were identified
by 1H NMR as they have distinct 1H NMR profiles, especially
for the three phenyl protons (Fig. S1w). The other key inter-
mediate, piperazyl-coumarin 2, was obtained in two steps using
coumarin acid 6 as the starting material (Scheme S2w). Coupling
of coumarin acid 6 with t-Boc-piperazine 7 followed by
deprotection under acidic conditions gave piperazyl-coumarin 2.
With the key intermediates 1a and 2 in hand, compound
Cou-Rho-NO was synthesized in two steps via the coumarin-
rhodamine intermediate 3 (Scheme 1). Condensation of
piperazyl-coumarin 2 with rhodamine acid 1a by the standard
coupling chemistry yielded compound 3. Interestingly, the
coupling almost exclusively occurred at the 4-position
carboxylic acid, in accordance with the results of our preliminary
studies (Scheme S3w). The high region-selectivity can be
ascribed to the less steric hindrance of the 4-position carboxylic
acid when compared to the 2-position one. Then, acid 3 was
converted into an acid chloride, which was further reacted with
o-diaminobenzene to afford the desired product, Cou-Rho-NO.
Then, we examined the spectral properties of the ratiometric
sensor Cou-Rho-NO (1 mM) in the absence or presence of NO.
As designed, in aqueous PBS buffer, the free sensor only
displayed the characteristic absorption peak of the coumarin
donor at around 416 nm but no featured absorption band of
the rhodamine dye at around 560 nm (Fig. S2w), as the
rhodamine acceptor is in the ring-closed form. However,
addition of DEA/NONOate (a commercially available NO
donor) elicited the formation of a significant absorption peak
at around 560 nm, suggesting that the rhodamine acceptor is
in the ring-opened form in the presence of NO.
Fig. 1 (a) Emission spectra of Cou-Rho-NO (1 mM) in the presence of
various amounts of NO (0–100 equiv.). The inset shows the visual
fluorescence color of compound Cou-Rho-NO (1 mM) before (left) and
after (right) addition of 100 equiv. of DEA/NONOate on excitation at
365 nm using a handheld UV lamp. (b) Emission intensity ratio
(I583/I473) of Cou-Rho-NO (1 mM) in the presence of various reactive
oxygen or nitrogen species (100 equiv. for NO anꢀd 1000 equiv. for
1
ꢁ
other analytes). 1, blank; 2, H2O2; 3, HClO; 4, O2 ; 5, OHꢁ; 6, O2;
7, NO3ꢀ; 8, NO2ꢀ; 9, NO; 10, AA; 11, DHA. The data were collected
from the emission spectra excited at 410 nm. The spectra were
recorded after incubation of Cou-Rho-NO with DEA/NONOate
and other analytes for 30 min at 37 1C in PBS (pH 7.4, containing
20% CH3CN as a cosolvent) with excitation at 410 nm.
supported by a drastic change of emission color from green to
orange (the inset in Fig. 1a). The detection limit (S/N = 3) of
the ratiometric sensor was determined to be 30 nM, indicating
that Cou-Rho-NO is potentially useful for monitoring NO
production in living cells.
Although the o-phenylenediamine-based reaction site has
been used in development of fluorescent turn-on type NO
sensors, it has not been employed in the context of ratiometric
sensor design. Based on the earlier literature report about the
turn-on sensing mechanism of o-phenylenediamine-based
fluorescent turn-on type NO sensors,7a,f a likely NO-induced
FRET sensing mechanism in Cou-Rho-NO is proposed
(Scheme S4w). To get insight into the proposed ratiometric
sensing mechanism, we decided to monitor the progress of the
reaction of Cou-Rho-NO with NO by both mass and fluorescence
spectroscopy. As shown in Fig. S3w, the free sensor Cou-Rho-NO
exhibited intense peaks at m/z 888.5 and 910.5 in the
ESI-MS spectrum, corresponding to (Cou-Rho-NO + H)+ and
(Cou-Rho-NO + Na)+, respectively. However, after incubation
of NO with the sensor for 0.5 min, a new peak at m/z 899.4
corresponding to the intermediate triazole was observed
(Fig. S4w). Longer incubation time (30 min) led to the complete
disappearance of the peak at m/z 899.4 and the formation of
two novel peaks at m/z 798.5 and 820.3, corresponding to (3)+
and (3 + Na ꢀ H)+ (Fig. S5w). In addition, there is a minor
time-dependent shift in the emission wavelength from around
600 to 583 nm upon treatment of NO with Cou-Rho-NO
(Fig. S6w), further supporting the presence of the intermediate
triazole in the titration process.7a Thus, the results of time-
dependent mass and fluorescence spectroscopic studies are
consistent with the proposed ratiometric sensing mechanism.
To examine the selectivity, ratiometric sensor Cou-Rho-NO
In good agreement with the results of the absorption
spectral studies, upon excitation at 410 nm, the free sensor
only exhibited the emission peak of the coumarin dye at 473 nm
but no typical emission of the rhodamine dye at around
583 nm, as the rhodamine acceptor is in the ring-closed form
and there is no FRET in the free Cou-Rho-NO. By sharp
contrast, as shown in Fig. 1a, addition of NO renders the
fluorescence intensity at around 473 nm significantly diminished
and simultaneous formation of a new red-shifted emission
band at around 583 nm, attributed to the emission of the
rhodamine acceptor. A well-defined isoemission point at 548 nm
in the emission spectra was observed. Remarkably, there is a
ca. 420-fold variation in the fluorescence ratio (I583/I473) from
0.061 in the absence of NO to 26.6 in the presence of
100 equiv. NO (Fig. 1b). It should be noted that such a big
change of signal ratios at two wavelengths is very desirable for
ratiometric fluorescent sensors, as the sensitivity as well as the
dynamic range of ratiometric sensors are controlled by the
ratios.8a The large ratiometric fluorescence response is further
was incubated with variousꢀreactive oxygen or nitrogen species
1
including H2O2, HClO, O2 , OHꢁ, O2, NO3ꢀ, NO2ꢀ, ascorbic
ꢁ
acid (AA), and dehydroascorbic acid (DHA) at up to 1 mM.
Importantly, these species do not elicit an observable change
in the fluorescence intensity ratio (Fig. 1b), indicating that the
ratiometric sensor is highly selective for NO. It is known that
c
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Chem. Commun., 2011, 47, 9372–9374 9373

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the likely NO-induced ratiometric sensing mechanism of
Cou-Rho-NO. Remarkably, the novel ratiometric fluorescent
sensor exhibited a very large variation (up to 420-fold) in the
fluorescence ratio (I583/I473). The other prominent features
of Cou-Rho-NO include high sensitivity, high specificity,
functioning-well at physiological pH, low cytotoxicity, and
good cell membrane permeability. Importantly, we have
demonstrated for the first time ratiometric imaging of
endogenously produced NO in macrophage cells by using
the novel ratiometric sensor Cou-Rho-NO. We expect that
the favorable features of Cou-Rho-NO will render it a very
useful ratiometric imaging agent for NO detection. Further-
more, the modular nature of the ratiometric sensor design and
synthesis holds great promise to access to a wide variety of
ratiometric fluorescent sensors by simply replacing the NO
reaction site with other reaction sites of interest.
Fig. 2 Images of RAW 264.7 macrophages treated with the ratio-
metric sensor Cou-Rho-NO. (a) DIC image of RAW 264.7 macro-
phages incubated with only Cou-Rho-NO (5 mM); (b) fluorescence
image of (a) from blue channel; (c) fluorescence image of (a) from red
channel; (d) DIC image of RAW 264.7 macrophages co-incubated
with 5 mM ratiometric sensor Cou-Rho-NO, 1.25 mg mLꢀ1 LPS, and
1000 U mLꢀ1 IFN-g; (e) fluorescence image of (d) from blue channel;
(f) fluorescence image of (d) from red channel.
This research was supported by NSFC (20872032, 20972044),
NCET (08–0175), and the Doctoral Fund of Chinese Ministry of
Education (No: 20100161110008).
DHA and AA often interfere with the NO detection in
o-diaminobenzene-based fluorescent turn-on NO sensors.7b,9
However, it is worthwhile to note that Cou-Rho-NO has
essentially no ratiometric response to both DHA and AA,
further supporting that Cou-Rho-NO is a superior sensing
agent for specific detection of NO. Furthermore, Cou-Rho-NO
can respond to NO over a wide pH range from 3 to 10 (Fig. S7w).
In addition, the MTT assays (see the ESIw, Fig. S8) suggest that
the sensor has low cytotoxicity to the cells.10
Notes and references
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We then assessed the ability of Cou-Rho-NO to operate in
live cells. For proof-of-concept, Cou-Rho-NO was initially
incubated with HeLa cells. Fig. S9w indicates that Cou-Rho-NO
can penetrate the cell membrane and respond to intracellular NO
in a ratiometric fashion. The promising results of the ratiometric
imaging of NO in HeLa cells encourage us to further evaluate the
feasibility of Cou-Rho-NO to detect endogenously produced NO.
Macrophage cells can generate NO in micromolar levels when
inducible nitric oxide synthase (iNOS) is activated in response to a
pathogenic attack.7g,11 Lipopolysaccharide (LPS) and interferon-g
(IFN-g) may stimulate expression of the iNOS gene leading to NO
production several hours later. The RAW264.7 macrophage cells
loaded with Cou-Rho-NO (5 mM) displayed a strong blue emission
(Fig. 2b) and almost no red emission (Fig. 2c). However, after the
RAW264.7 macrophage cells have been co-incubated with LPS
(1.25 mg mLꢀ1), IFN-g (1000 U mLꢀ1), and Cou-Rho-NO (5 mM)
for 12 h, a decrease in the blue emission (Fig. 2e) and a dramatic
enhancement in the red emission (Fig. 2f) was observed. The
ratiometric fluorescence images are shown in Fig. S10w. These
data indicate that Cou-Rho-NO is capable of ratiometric
fluorescent imaging of endogenously produced NO. To the
best of our knowledge, this represents the first report of
ratiometric fluorescent imaging of endogenously produced
NO in macrophage cells.
4 (a) S. P. L. Cary, J. A. Winger, E. R. Derbyshire and
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In summary, we have judiciously designed and synthesized
Cou-Rho-NO as the first small-molecule suitable for ratio-
metric fluorescent imaging of endogenously produced NO in
macrophage cells. In addition, we have preliminarily studied
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c
9374 Chem. Commun., 2011, 47, 9372–9374
This journal is The Royal Society of Chemistry 2011