Pre-Assembled Coumarin–Rhodamine Scaffold for Ratiometric Sensing of Nitric Oxide and Hypochlorite

Article abstract of DOI:10.1002/chem.201703554

A new pre-assembled ratiometric sensing platform was constructed from a coumarin donor and a rhodamine acceptor designed for through-bond energy transfer (TBET). A phenylacetylene linker was installed to disrupt the planarity of the extended conjugated system but retaining the efficient energy transfer between the donor and acceptor motifs. To demonstrate its versatility as a sensing platform, we conjugated recognition motifs through amide coupling reactions to yield two TBET chemosensors capable of sensing either endogenously produced NO and ClO^?. Both probes possessed high selectivity for their analytes, exhibited good stability under physiological conditions, and performed well as bioimaging probes in living cells.

Full text of DOI:10.1002/chem.201703554

A Journal of
Accepted Article
Title: Preassembled Coumarin-Rhodamine Scaffold for Ratiometric
Sensing of Nitric Oxide and Hypochlorite
Authors: Jun Xiang Ong, Victoria Yu Ting Pang, Li Min Tng, and Wee
Han Ang
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To be cited as: Chem. Eur. J. 10.1002/chem.201703554
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10.1002/chem.201703554
Chemistry - A European Journal
Preassembled Coumarin-Rhodamine Scaffold for Ratiometric
Sensing of Nitric Oxide and Hypochlorite
Jun Xiang Ong,[a] Victoria Yu Ting Pang,[a] Li Min Tng,[a] and Wee Han Ang*[a, b]
[a] J. X. Ong, V. Y. T. Pang, L. M. Tng, Prof. W. H. Ang
Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore
117543 (Singapore)
E-mail: chmawh@nus.edu.sg
[b] Prof. W. H. Ang
NUS Graduate School for Integrative Sciences and Engineering, National University of
Singapore, 28 Medical Drive, Singapore 117456 (Singapore)
Abstract: A new preassembled ratiometric sensing platform was constructed from a coumarin
donor and a rhodamine acceptor designed for through-bond energy transfer (TBET). A
phenylacetylene linker was installed to disrupt the planarity of the extended conjugated system but
retaining the efficient energy transfer between the donor and acceptor motifs. To demonstrate its
versatility as a sensing platform, we conjugated recognition motifs through amide coupling
reactions to yield two TBET chemosensors capable of sensing either endogenously produced NO
and ClO-. Both probes possessed high selectivity for their analytes, exhibited good stability under
physiological conditions, and performed well as bioimaging probes in living cells.
1
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Introduction
In the past few decades, fluorescent probes based on small organic molecules have emerged
as powerful tools to study biomolecules and biologically-relevant metal ions in living systems.
These small-molecule fluorescent dyads have been widely employed in bioimaging owing to their
high sensitivity, selectivity and high spatial and temporal resolution.[1] However, most of these
probes function via changes to the intensity of a single emission signal. Hence, intensity-based
probes tend to be affected by factors such as variations in probe concentrations, excitation
intensities and environmental effects. To overcome challenges associated with intensity-based
probes, researchers have developed probes which can emit at two different emission wavelengths.
By taking the intensity ratios of the different emissions, these ratiometric probes provide a built-
in correction for the above-mentioned effects.[2] Fӧrster Resonance Energy Transfer (FRET) is a
mechanism commonly adopted for the design of ratiometric fluorescent probes. In a FRET system,
the donor and acceptor fluorophores are linked via non-conjugated spacers and energy transfer
from the donor to acceptor occurs through space. A substantial spectral overlap between the
emission spectrum of the donor and the absorption spectrum of the acceptor is required for efficient
energy transfer to take place but it limits the choice of donor and acceptor fluorophores available.[3]
There has been a growing interest in fluorescent probes based on Through Bond Energy Transfer
(TBET). As opposed to FRET, donor and acceptor fluorophores are linked via electronically
conjugated spacers in TBET systems and energy transfer occurs through the conjugated bond
without the need for spectral overlap.[4] This unique feature endows TBET fluorescent probes with
high energy transfer efficiencies and large pseudo-Stokes shifts.
TBET ratiometric chemosensors utilizing bright fluorogenic rhodamine acceptor scaffolds
have been reported for the detection of various analytes and their designs can be broadly divided
into two main strategies. In one strategy, the donor and acceptor motifs are not electronically-
conjugated when the rhodamine acceptor is in its inactivated closed-ring form. Upon activation
e.g. detection of an analyte, spiro-ring opening occurs on the rhodamine scaffold enabling TBET
via electronic conjugation.[5] In these designs, donor molecules are typically incorporated as part
of the sensing mechanism for the analytes which makes the probe susceptible to decomposition.
A more desirable strategy would be to construct TBET chemosensors from pre-assembled TBET
ratiometric scaffolds and to install the sensing mechanism at the last synthesis step. Few such
TBET ratiometric scaffolds have been reported. For example, Xiao and co-workers successfully
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fabricated two ratiometric fluorescent probes for the detection of mercury ions and nitric oxide
based on a phenyl-linked BODIPY-rhodamine TBET scaffold.[6] Peng and co-workers developed
a naphthalimide-rhodamine TBET platform and applied it for sensing of copper ions.[7] Tan and
co-workers developed two-photon naphthalene-rhodamine TBET chemosensors for imaging of
copper and palladium ions in living cells and tissues.[8] Apart from the aforementioned TBET
platforms, a handful of pre-assembled platforms based on FRET have also been developed for the
detection of an array of analytes.[9] There is a need to expand the repertoire of ratiometric TBET
scaffolds.
Hypochlorous acid (HClO)/hypochlorite (ClO-) is an important reactive oxygen species (ROS)
in living organisms produced by the reaction of hydrogen peroxide and chloride ions catalyzed by
myeloperoxidase (MPO).[10] It plays a key role in host immune defense system and is capable of
killing a wide range of pathogens.[11] Excessive HClO/ClO- generation can induce cellular damage
leading to cancer, neurodegeneration and cardiovascular diseases.[12] Nitric oxide (NO), an
important signal transduction molecule in pathological and physiological processes, plays a critical
role in cell signaling pathways in the central nervous system,[13] and acts as the endothelium-
derived relaxing factor in the cardiovascular system.[14] Misregulation of NO production is
implicated in various pathologies like stroke, heart disease, hypertension and neurodegenerative
diseases.[15] To the best of our knowledge, only one ratiometric probe fabricated from a pre-
assembled TBET platform has been reported for NO [6b] but none reported for ClO-.
Herein, we report the fabrication of a new coumarin-rhodamine platform designed for TBET
as shown in Fig. 1. The donor and acceptor motifs were connected by a phenylacetylene linker to
prevent the fragments from adopting planarity and behaving as a single conjugated fluorophore.
This was confirmed through computational modeling which showed that the coumarin and
rhodamine motifs adopt a twisted conformation favorable for TBET (Fig. S1). To demonstrate the
versatility of this new platform, we conjugated o-phenylenediamine and benzoylhydrazine as
sensing motifs for NO and ClO-, respectively, to access new ratiometric probes for detection of
NO and ClO- in living cells.
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Results and Discussion
Synthesis and Characterization
The coumarin-rhodamine ratiometric fluorescent dyads were prepared in accordance with
Scheme 1. The coumarin donor motif was synthesized in 2 steps from 4-bromophenyl-coumarin.
4-bromophenyl-coumarin was treated with trimethylsilylacetylene under Sonogashira coupling
conditions with Pd(PPh3)2Cl2 and CuI as co-catalysts to afford 4-(trimethylsilyl)-phenylacetylene-
coumarin in moderate yield. Deprotection of 4-(trimethylsilyl)-phenylacetylene-coumarin with
TBAF gave 4-phenylacetylene-coumarin in good yield. Conjugation of 4-phenylacetylene-
coumarin to 5’-Br-Rhodamine B via Sonogashira coupling afforded the TBET ratiometric platform
1 in moderate yield. 1 was treated sequentially with POCl3 and o-phenylenediamine or
benzoylhydrazine to prepare target compounds 1·NO and 1·ClO, respectively, and were fully
characterized by NMR and ESI-MS.
Photophysical properties and sensing responses of probes
We investigated for the photophysical properties and sensing responses. Firstly, the absorption
and emission properties of 1·NO was examined in CH3CN/PBS buffer (v/v=4:6, 2 mM, pH 7.4)
as shown in Fig. 2 (Fig. S2). Untreated 1·NO displayed an absorption band centered at 420 nm
which can be attributed to the characteristic absorption peak of the coumarin donor. Since the
rhodamine acceptor was in the closed-ring form, no absorption band of rhodamine was observed.
Upon addition of DEA NONOate (a commercially-available NO precursor), a new absorption band
centered at 555 nm appeared gradually indicating an NO-induced spiroring opening reaction
leading to the formation of the rhodamine chromophore. In addition, an obvious colour change
from yellow to pink was observed (Fig. 3). Similar to the absorption studies, upon excitation at
420 nm, untreated 1·NO displayed only a single emission band centered at 505 nm which can be
ascribed to the emission of coumarin donor. In the presence of increasing amounts of NO (0–50
eq.), the intensity of the emission band at 505 nm decreased in intensity and simultaneously a new
emission band at 580 nm that gradually increased in intensity was observed. This observation can
be attributed to the energy transfer from coumarin (donor) to rhodamine (acceptor) following ring
opening of the spirolactam induced by NO. Consistently, the emission colour of the solution turned
from bluish-green to orange-red (Fig. 3). The ratio of emission intensities of rhodamine to
coumarin (I580/I505) increased steadily with addition of increasing amounts of NO and good
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linearity was established between the emission ratios and NO concentrations from 0–500 μM,
suggesting that it is potentially useful for the quantitative determination of NO. The detection limit
(3σ/slope)[16] for NO was determined to be 0.73 μM. The energy transfer efficiency was
determined to be 81.5%, which was comparable to literature reports (Fig. S3).[7] Time-dependent
fluorescence response studies carried out revealed that the fluorescence intensity ratios increased
gradually in a linear fashion over time in the presence of NO and plateaued after 100 min (Fig.
S4).
The photophysical characteristics of 1·ClO was expected to be similar to 1·NO (Fig. 2, S5).
With increasing amounts of NaClO (0–10 eq.), 1·ClO displayed similar trends upon probe
activation with good linearity within the biologically-relevant range of 0–200 μM. The limit of
detection (3σ/slope) of 1·ClO was determined to be 0.41 μM. Notably, time-dependent
fluorescence response showed that the emission ratio reached a plateau within 2 min after the
addition of NaClO (Fig. S6). This indicated that 1·ClO could respond rapidly towards NaClO
making the probe an ideal tool for real-time imaging. Thus, we demonstrated that with our single
pre-assembled TBET ratiometric platform, we could generate at least two new chemosensors for
the ratiometric detection of NO and ClO-.
Selectivity and pH response of probes
Since biological systems contain a complex mixture of different reactive oxygen species
(ROS), reactive nitrogen species (RNS) and metal ions, it is imperative that chemosensors have
the abilities to detect target analytes without interference of other species commonly present in
such biological systems. We examined the selectivity of the probes in presence of various ROS
-
-
-
and RNS such as NO2 , NO3 , H2O2, ∙OH, tBuOOH and O2 , as well as metal ions such as Na+, K+,
Mg2+, Cu2+, Ni2+, Zn2+, Fe2+ and Fe3+. As shown in Fig. 4 (Fig. S7, S8), the presence of other
biological relevant species did not induce any changes to the fluorescence intensity ratios of 1·NO
and 1·ClO, revealing excellent selectivity towards NO and ClO-, respectively. The high selectivity
for NO and ClO- illustrated the potential of the probes for biological applications.
The effect of pH on the fluorescence responses of the probes was also studied. The fluorescence
intensity ratios (I580/I505) of untreated 1·NO and 1·ClO were not affected by variations in pH values
over a large pH range 3–11 as shown in Fig. 4. This showed that the probes existed predominantly
in the spirocyclic form in the pH range 3–11 making both probes compatible with physiological
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conditions. The activation of the probes in the presence of NO and ClO- is pH dependent, with
notable enhancement of fluorescence responses observed in the range of pH 3–9 for 1·NO and pH
range of 5–11 for 1·ClO compared to the free probes in buffer solutions. Since intracellular pH
typically ranges from 5–8, 1·NO and 1·ClO are expected to work in a cellular environment with
negligible background fluorescence.
Detection mechanisms of NO and ClO-
We postulated that the chemosensors were activated via spiroring-opening followed by
cleavage of the recognition motif to yield scaffold 1, triggering a fluorogenic response. The mode
of action is in keeping with previous reports. NO oxidises 1·NO to form an acylbenzotriazole
intermediate which decomposed in aqueous solution to yield 1 (Fig. S9).[17] Similarly, 1·ClO first
formed a diacyl diimide intermediate upon oxidation by ClO- followed by aqueous decomposition
to 1 (Fig. S10).[18] During the activation process, the probes were converted from the closed-ring
to the opened-ring form, allowing energy transfer to occur. To validate the proposed mechanisms,
we employed mass spectrometry to study plausible reaction products. Solutions of 1·NO and
1·ClO after treatment with DEA NONOate and NaClO, respectively, were analyzed using ESI-
MS. In both instances, a single peak of m/z 758.4 was observed which corresponded to the
formation of 1 (m/z 758.4 [M+H]+) (Fig. S11, S12). This is in good agreement with the absorption
and emission studies pointing to the formation of a common reaction product 1 upon treatment of
the respective probes with NO and ClO-.
Detection of exogenous NO and ClO- in cells via fluorescence imaging
To demonstrate the potential application of 1·NO and 1·ClO in biological systems, both
probes were applied for ratiometric fluorescence imaging in living cells. HeLa cells were incubated
with either 1·NO (10 μM) or 1·ClO (20 μM) in culture medium for 30 min at 37 °C. The cells
treated with the probes showed strong intracellular fluorescence arising from coumarin at λ505 and
negligible emission from rhodamine at λ580 indicating the cell membrane was permeable to the
probes and that they were stable under intracellular conditions. Upon treatment with DEA
NONOate (100 μM) for 2 h and NaClO (100 μM) for 1 h, a significant increase in λ580 was
observed accompanied by a decrease in λ505 (Fig. 5, S13). This phenomenon was in good
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agreement with the emission profiles of 1·NO and 1·ClO in aqueous solutions in the presence of
NO and ClO-, respectively.
Detection of endogenous NO and ClO- in RAW 264.7 cells via fluorescence imaging
The promising results of exogenous NO and ClO- ratiometric imaging in living cells prompted
us to investigate the feasibility of these probes in the detection of endogenous analytes. Murine
RAW 264.7 macrophages were chosen as the imaging model as they can be stimulated by the
treatment of lipopolysaccharides (LPS) and 12-myristate-13-acetate (PMA) to produce
endogenous ClO- in immune systems. In contrast, LPS and interferon-γ (IFN-γ) can stimulate
macrophages to produce NO. RAW 264.7 macrophage cells treated with probes only displayed
intense emission at λ505 and negligible emission at λ580. After treatment with LPS (1 μg mL-1) for
12 h and then further co-incubation with PMA (1 μg mL-1) and 1·ClO (20 μM) for 12 h at 37 °C,
decreased λ505 emission and a marked enhancement at λ580 was observed (Fig. 6). Similarly, co-
incubation of macrophage cells with LPS (1 μg mL-1), IFN-γ (1000 U mL-1) and 1·NO (10 μM)
for 12 h at 37 °C exhibited a similar profile (Fig. S14). Taken together, these data showed that the
probes are capable of detecting endogenously produced NO and ClO- in living cells.
Conclusion
In summary, we have successfully developed two ratiometric probes specific to NO and ClO-
based on a new TBET scaffold constructed from coumarin and rhodamine fluorophores. Both
probes demonstrated high selectivity over a panel of biologically-relevant ROS, RNS and metal
ions. The probes are stable over a wide pH range and possess good ratiometric fluorescence turn-
on ratios for detection of NO and ClO- at physiological pH. They are cell permeable and can be
applied for the ratiometric fluorescence imaging of exogenous and endogenous NO and ClO- in
living cells. The modular nature of this new TBET scaffold opens the door to a wider variety of
ratiometric fluorescent sensors by simply conjugating other recognition motifs of interest.
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Experimental Section
Materials and instrumentation
All reagents were purchased from commercial vendors and used without further purification.
5’-Br-Rhodamine B was synthesized according to literature methods.[19] 1H NMR spectra were
recorded on a Bruker Advance 300 MHz model. Chemical shifts are reported in parts per million
relative to residual solvent peaks. Electro-spray ionization mass spectrometry spectra were
obtained on a Thermo Finnigan MAT ESI-MS system. UV–vis spectra were recorded on a
Shimadzu UV-1800 UV–vis spectrophotometer. Emission fluorescence spectra were recorded on
a JOBIN YVON Fluorolog by HORIBA with iHR320 detector. Fluorescence images were
captured with Olympus FluoView FV1000 (Olympus, Japan) laser scanning confocal microscope,
with 405 nm laser as the excitation source. Computational modelling was performed using Maestro
docking program.
Synthesis of 4-bromophenyl-coumarin
To 4-bromophenylacetic acid (1.20 g, 5.61 mmol) and 4-diethylamino-salicylaldehyde (1.07
g, 5.58 mmol) was added pyridine (451 μL, 5.58 mmol). The reaction mixture was then dissolved
in acetic anhydride (1.05 mL) and refluxed for 12 h. After the reaction, solvent was removed and
the crude residue was purified by column chromatography (1:6 v/v ethyl acetate:hexane) to give
1
the product as yellow solid. Yield: 450 mg (21.7%). H NMR (300 Hz, CDCl3): δ 7.69 (s, 1 H,
Ar−H), 7.57 (dd, 4 H, Ar−H), 7.31 (d, 1 H, Ar−H), 6.60 (dd, 1 H, Ar−H), 6.52 (d, 1 H, Ar−H),
3.44 (q, 4 H, −CH2), 1.22 (t, 6 H, −CH3).
Synthesis of 4-(trimethylsilyl)-phenylacetylene-coumarin
To 4-bromophenyl-coumarin (330 mg, 0.89 mmol) was added Pd(PPh3)2Cl2 (63 mg, 0.089
mmol), CuI (17 mg, 0.089 mmol), PPh3 (48 mg, 0.180 mmol) and trimethylsilylacetylene (378 μL,
2.67 mmol). NEt3 (5 mL) and THF (25 mL) were then added and the reaction mixture was refluxed
for 12 h under an inert N2 environment. After the reaction, solvent was removed and the crude
residue was purified by column chromatography (1:6 v/v ethyl acetate:hexane) to give the product
as yellow solid. Yield: 307 mg (88.7%). 1H NMR (300 Hz, CDCl3): δ 7.72 (s, 1 H, Ar−H), 7.66
(d, 2 H, Ar−H), 7.51 (d, 2 H, Ar−H), 7.31 (d, 1 H, Ar−H), 6.63 (dd, 1 H, Ar−H), 6.55 (d, 1 H,
Ar−H), 3.45 (q, 4 H, −CH2), 1.23 (t, 6 H, −CH3), 0.26 (s, 9 H, −CH3).
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Synthesis of 4-phenylacetylene-coumarin
4-(trimethylsilyl)-phenylacetylene-coumarin (265 mg, 0.681 mmol) was dissolved in THF (20
mL) and 1 M TBAF solution in THF (0.681 μL, 0.681 mmol) was added. The reaction was stirred
at r.t. for 0.5 h. After the reaction, solvent was removed and the crude residue was purified by
column chromatography (1:3 v/v ethyl acetate:hexane) to give the product as yellow solid. Yield:
174 mg (80.6%). 1H NMR (300 Hz, CDCl3): δ 7.72 (s, 1 H, Ar−H), 7.66 (d, 2 H, Ar−H), 7.51 (d,
2 H, Ar−H), 7.31 (d, 1 H, Ar−H), 6.63 (dd, 1 H, Ar−H), 6.55 (d, 1 H, Ar−H), 3.44 (q, 4 H, −CH2),
3.12 (s, 1 H, −C≡C−H), 1.23 (t, 6 H, −CH3).
Synthesis of 1
5’-Br-Rhodamine B (233 mg, 0.448 mmol), 4-phenylacetylene-coumarin (142 mg, 0.448
mmol), PdCl2(PPh3)2 (32 mg, 0.045 mmol), PPh3 (23 mg, 0.264 mmol) and CuI (4.4 mg, 0.023
mmol) in THF/NEt3 (4:1) (25 mL) were refluxed for 18 h under N2. After the reaction, the solvent
was removed and the crude residue purified by column chromatography (100% dichloromethane
to 250:3:3 v/v dichloromethane:methanol:triethylamine to 95:5 v/v dichloromethane:methanol) to
1
give the product as purple-red solid. Yield: 172 mg (50.6%). H NMR (300 Hz, CDCl3): δ 7.69
(m, 5 H, Ar−H), 7.47 (d, 2 H, Ar−H), 7.35 (s, 1 H, Ar−H), 7.30 (d, 1 H, Ar−H), 6.70 (d, 2 H,
Ar−H), 6.58 (dd, 1 H, Ar−H), 6.49 (m, 3 H, Ar−H), 6.41 (m, 2 H, Ar−H), 3.40 (m, 12 H, −CH2),
1.19 (m, 18 H, −CH3). ESI-MS: m/z 758.5 [M+H]+.
Synthesis of 1·NO
1 (50 mg, 0.066 mmol) in 1, 2-dichloroethane (12 mL) was added POCl3 (0.3 mL) dropwise
over 5 min. The reaction mixture was left to reflux overnight. After the reaction, the solvent was
evaporated to give the crude acid chloride as violet-red oil. The crude acid chloride was dissolved
in DCM (8 mL) and added drop-wise to a solution of o-phenylenediamine (14.3 mg, 0.132 mmol)
and NEt3 (0.4 mL) in DCM (12 mL). The reaction mixture was stirred at r.t. overnight. After the
reaction, the solvent was removed and the crude residue purified by column chromatography (1:1
v/v ethyl acetate:hexane) to give the product as yellow solid. Yield: 20 mg (34.3%). 1H NMR (300
Hz, CDCl3): δ 8.05 (d, 1 H, Ar−H), 7.74 (m, 4 H, Ar−H), 7.50 (m, 2 H, Ar−H), 7.44 (s, 3 H, Ar−H),
7.05 (m, 2 H, Ar−H), 6.92 (m, 2 H, Ar−H), 6.60 (m, 2 H, Ar−H), 6.52 (m, 3 H, Ar−H), 6.39 (m, 1
9
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H, Ar−H), 3.43 (q, 12 H, −CH2), 1.24 (m, 18 H, −CH3). ESI-MS: m/z 874.4 [M+Na]+. Anal. Calcd.
for C55H53N5O4: C 77.90, H 6.30, N 8.26. Found: C 77.65, H 6.64, N 8.57.
Synthesis of 1·ClO
1 (50 mg, 0.066 mmol) in 1, 2-dichloroethane (12 mL) was added POCl3 (0.3 mL) dropwise
over 5 min. The reaction mixture was left to reflux overnight. After the reaction, the solvent was
evaporated to give the crude acid chloride as violet-red oil. The crude acid chloride was dissolved
in DCM (8 mL) and added drop-wise to a solution of benzoylhydrazine (18 mg, 0.132 mmol) and
NEt3 (0.4 mL) in DCM (12 mL). The reaction mixture was stirred at r.t. overnight. After the
reaction, the solvent was removed and the crude residue purified by column chromatography (1:1
v/v ethyl acetate:hexane) to give the product as yellow solid. Yield: 13 mg (22.7%). 1H NMR (300
Hz, CDCl3): δ 8.00 (d, 1 H, Ar−H), 7.69 (m, 5 H, Ar−H), 7.51 (m, 6 H, Ar−H), 7.36 (m, 6 H,
Ar−H), 6.57 (m, 2 H, Ar−H), 6.40 (bs, 2 H, Ar−H), 3.41 (m, 12 H, −CH2), 1.18 (m, 18 H, −CH3)
ppm. ESI-MS: m/z 876.3 [M+H]+. Anal. Calcd. for C56H53N5O5: C 76.78, H 6.10, N 7.99. Found:
C 77.11, H 6.45, N 8.38.
Fluorescence studies
1·NO and 1·ClO were dissolved in DMSO to obtain 1 mM stock solutions. For 1·NO,
fluorescence experiments were carried out by incubating the dyad (10 μM) dissolved in
CH3CN/PBS buffer (v/v=4:6, 2 mM, pH 7.4) with various amounts of DEA NONOate prepared
freshly in 0.01 M NaOH. For 1·ClO, fluorescence experiments were carried out by incubating the
dyad (20 μM) dissolved in CH3CN/Tris-HCl buffer (v/v=6:4, 2 mM, pH 7.4) with various amounts
of NaClO. The detection limits were calculated based on fluorescence titration experiments.
Fluorescence spectra of the probes were measured ten times to obtain standard deviations (σ) of
the blank measurements and the slopes were obtained from the plots of intensity ratios against the
concentrations of NO or ClO-. NaCl, KCl, MgCl2, CuSO4, ZnCl2, FeSO4 and FeCl3 were used as
the source of metal ions. NO was generated from DEA NONOate, ClO- was generated from
-
-
aqueous NaClO (4-4.99 % chlorine), NO2 was generated from NaNO2, NO3 was generated from
NaNO3, H2O2 was generated from aqueous H2O2 (30% w/w), •OH was generated from the Fenton
-
reaction, tBuOOH was generated from tBuOOH solution (70% wt. in H2O) and O2 was generated
from KO2. For 1·NO, selectivity tests were carried out by incubating the dyad (10 μM) with
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various ROS/RNS/metal ions (500 μM). For 1·ClO, selectivity tests were carried out by incubating
the dyad (20 μM) with various ROS/RNS/metal ions (200 μM). pH response studies of the probes
were measured from pH 3–11.6 by substituting buffers of different pH while keeping the final
buffer concentrations constant. pH 3–6 were measured using 200 mM CH3COOH/CH3COO-
buffer, pH 7.4 was measured using 10 mM PBS buffer or 50 mM Tris-HCl buffer and pH 8.4–11.6
-
were measured with 100 mM H+/HPO4 buffer.
Ratiometric fluorescence imaging of exogenous NO and ClO-
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% FBS and 1% antibiotics at 37 °C with 5 % CO2. Cells were seeded on cover slips in 6-
well plates at a density of 20 x 104 cells per mL and incubated overnight. The cells were washed
with PBS buffer twice and incubated with probes 1·NO (10 μM) or 1·ClO (20 μM) at 37 °C. After
30 min, the cells were washed with PBS buffer thrice and incubated with DEA NONOate (100
μM) or NaClO (100 μM) for 1 h and 2 h respectively at 37 °C. Subsequently, the cells were washed
with PBS buffer thrice, fixed with 4% paraformaldehyde for 15 min and then washed thrice with
PBS buffer. The control experiments were prepared by incubating the cells with probes only at 37
°C for 30 min, washed with PBS buffer thrice, fixed with 4% paraformaldehyde for 15 min and
then washed thrice with PBS buffer. The cover slips were mounted onto glass slides in a mounting
medium and fluorescence images were acquired with Olympus FluoView FV1000 (Olympus,
Japan) laser scanning confocal microscope, with 405 nm laser as the excitation source.
Ratiometric fluorescence imaging of endogenous NO and ClO-
RAW 264.7 macrophage cells were cultured in DMEM supplemented with 10% FBS and 1%
antibiotics at 37 °C with 5 % CO2. Cells were seeded on cover slips in 6-well plates at a density of
20 x 104 cells per mL and incubated overnight. The macrophage cells were washed twice with PBS
buffer and incubated with LPS (1 μg mL-1) at 37 °C. After 12 h, the cells were further co-incubated
with PMA (1 μg mL-1) and 1·ClO (20 μM) for 12 h at 37 °C. For detection of endogenous NO,
the macrophage cells were co-incubated with LPS (1 μg mL-1), IFN-γ (1000 U mL-1) and 1·NO
(10 μM) for 12 h at 37 °C. The cells were washed with PBS buffer thrice, fixed with 4%
paraformaldehyde for 15 min and then washed thrice with PBS buffer. The control experiment was
prepared by incubating the cells with probes only at 37 °C for 12 h, washed with PBS buffer thrice,
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10.1002/chem.201703554
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fixed with 4% paraformaldehyde for 15 min and then washed thrice with PBS buffer. The cover
slips were mounted onto glass slides in a mounting medium and fluorescence images were
acquired with Olympus FluoView FV1000 (Olympus, Japan) laser scanning confocal microscope,
with 405 nm laser as the excitation source.
Acknowledgements
The authors gratefully acknowledge financial support from Ministry of Education and
National University of Singapore (R143-000-680-114). The authors also thank Chemical,
Molecular and Materials Analysis Center (CMMAC) for performing elemental analysis
experiments.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Fluorescent Probes • Ratiometric • Through Bond Energy Transfer • Nitric Oxide •
Hypochlorite
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TMS
Br
Cl2
Pd(PPh3)2
PPh3
CuI,
,
Et2N
O
O
Et2N
O
O
NEt
TMSA,
3
Et2N
O
O
TBAF
Br
Cl2
Pd(PPh3)2
COO-
N+Et2
COO-
+
(1)
PPh3 NEt
CuI,
,
3
Et2N
O
Et2N
N+Et2
O
Et2N
O
O
POCl
POCl
1)
2)
3
1)
2)
3
O
NH2
NH2
NEt
,
3
NEt
,
3
NHNH2
O
O
O
O
Et2N
Et2N
NH2
O
O
O
N
N
N
H
(1·ClO)
(1·NO)
Et2N
NEt2
Et2N
NEt2
O
O
Scheme 1. Synthetic route of coumarin-rhodamine ratiometric dyads (1) for the detection of
nitric oxide and hypochlorite.
Figure 1. Design of a new coumarin-rhodamine TBET ratiometric platform and its applications
for ratiometric sensing of nitric oxide and hypochlorite.
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Figure 2. (a) Fluorescence spectra of 1·NO (10 μM) in response to the presence of NO (DEA
NONOate) (0–50 eq.) in CH3CN/PBS buffer (v/v=4:6, 2 mM, pH 7.4) (λex= 420 nm). Inset
showing the linear plot of fluorescence intensity ratios (I580/I505) against NO concentrations.
Figure 2. (b) Fluorescence spectra of 1·ClO (20 μM) in response to the presence of NaClO (0–10
eq.) in CH3CN/Tris-HCl buffer (v/v=6:4, 2 mM, pH 7.4) (λex= 420 nm). Inset showing the linear
plot of fluorescence intensity ratios (I580/I505) against NaClO concentrations.
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Figure 3. (a) Colour change before and after the addition of NO to 1·NO. (b) Fluorescence change
before and after the addition of NO to 1·NO.
Figure 4. (a) Fluorescence responses (I580/I505) of 1·NO (10 μM) upon addition of NO (DEA
NONOate) and other ROS/RNS in CH3CN/PBS buffer (v/v=4:6, 2 mM, pH 7.4). Inset:
Fluorescence responses (I580/I505) of 1·NO (10 μM) against pH in the absence (●) and presence (♦)
of NO (DEA NONOate).
Figure 4. (b) Fluorescence responses (I580/I505) of 1·ClO (20 μM) upon addition of NaClO and
other ROS/RNS in CH3CN/Tris-HCl buffer (v/v=6:4, 2 mM, pH 7.4). Inset: Fluorescence
responses (I580/I505) of 1·ClO (20 μM) against pH in the absence (●) and presence (♦) of NaClO.
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Figure 5. Confocal images of HeLa cells treated with 1·ClO in the absence and presence of
NaClO. (a) Bright field image of HeLa cells treated with 1·ClO (20 μM) only; (b) Fluorescence
image of (a) in the blue channel; (c) Fluorescence image of (a) in the red channel; (e) Bright field
image of HeLa cells treated with 1·ClO (20 μM) for 30 min and then further incubation with
NaClO (100 μM) for 1 h at 37 °C; (f) Fluorescence image of (e) in the blue channel; (g)
Fluorescence image of (e) in the red channel. (d) & (h) Ratio images of red and blue channels. λex=
405 nm.
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Figure 6. Confocal images of RAW 264.7 cells treated with 1·ClO in the absence and presence of
LPS and PMA. (a) Bright field image of RAW 264.7 cells treated with 1·ClO (20 μM) only; (b)
Fluorescence image of (a) in the blue channel; (c) Fluorescence image of (a) in the red channel;
(e) Bright field image of RAW 264.7 cells treated with LPS (1 μg mL-1) for 12 h and then further
co-incubation with PMA (1 μg mL-1) and 1·ClO (20 μM) for 12 h at 37 °C; (f) Fluorescence image
of (e) in the blue channel; (g) Fluorescence image of (e) in the red channel. (d) & (h) Ratio images
of red and blue channels. λex= 405 nm.
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TOC graphic
Fluorescent ratiometric platform: A pre-assembled ratiometric scaffold based on TBET mechanism was
developed to serve as a versatile chemosensor platform for the ready incorporation of various recognition
motifs through facile amide coupling chemistry.
20
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