Dual Mechanism of an Intramolecular Charge Transfer (ICT)–FRET-Based Fluorescent Probe for the Selective Detection of Hydrogen Peroxide

Article abstract of DOI:10.1002/asia.201701382

A dual-mechanism intramolecular charge transfer (ICT)–FRET fluorescent probe for the selective detection of H₂O₂ in living cells has been designed and synthesized. This probe used a coumarin–naphthalimide hybrid as the FRET platform and a boronate moiety as the recognition group. Upon the addition of H₂O₂, the probe exhibited a redshifted (73 nm) fluorescence emission, and the ratio of fluorescence intensities at λ=558 and 485 nm (F₅₅₈/F₄₈₅) shifted notably (up to 100-fold). Moreover, there was a good linearity (R²=0.9911) between the ratio and concentration of H₂O₂ in the range of 0 to 60 μm, with a limit of detection of 0.28 μm (signal to noise ratio (S/N)=3). This probe could also detect enzymatically generated H₂O₂. Importantly, it could be used to visualize endogenous H₂O₂ produced by stimulation from epidermal growth factor.

Full text of DOI:10.1002/asia.201701382

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: The dual mechanism ICT-FRET-based fluorescent probe for the
selective detection of hydrogen peroxide
Authors: Xiao Liang, Xiaoyi Xu, Dan Qiao, Zheng Yin, and Luqing
Shang
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The dual mechanism ICT-FRET-based fluorescent probe for the
selective detection of hydrogen peroxide
Xiao Liang,[a] Xiaoyi Xu, [b] Dan Qiao,[a] Zheng Yin [a], and Luqing Shang *[a]
influence the measurements of fluorescence intensity and the
emission collection efficiency and excitation intensity, which may
further influence the accuracy and reliability of the fluorescence
intensity measurements.[10]
Abstract: We designed and synthesized the dual mechanism ICT-
FRET fluorescent probe for the selective detection of H2O2 in living
cells. This probe used a coumarin-naphthalimide hybrid as the FRET
platform and a boronate moiety as the recognition group. Upon
These above mentioned problems could be alleviated using
ratiometric fluorescent probes, which measure the ratio of
fluorescence intensities at two emissions, such that the signal
addition of H2O2, the probe exhibited
a redshifted (73 nm)
fluorescence emission, and the ratio of fluorescence intensities at
558 and 485 nm (F558 / F485) shifted notably (up to 100-fold). ratios would be independent of environmental effects.[10b, 11] One
Moreover, there was a good linearity (R2 = 0.9911) between the ratio
and the concentration of H2O2 in the range of 0 to 60 µM, with a limit
of detection of 0.28 µM (S/N = 3). This probe could also detect
enzymatically generated H2O2. Importantly, it could visualize
endogenous H2O2 produced by the stimulation of the epidermal
growth factor (EGF).
of the well established mechanisms for ratiometric fluorescent
probes is fluorescence Resonance Energy Transfer
(FRET).[10b,12] Although a few FRET-based fluorescent probes
for monitoring H2O2 have been designed in recent years,[13] there
is still much space for improvement. It is not easy to build these
FRET-based ratiometric fluorescent platforms, as they require a
considerable spectral overlap between the emission of the donor
and the absorption of the acceptor. However, Intramolecular
charge transfer (ICT) could trigger the absorption redshift and
quantum yield increase of the fluorophore.[14] It could be used for
changing the spectral overlap level of the FRET-based
fluorescent platforms for operating the FRET process on/off. To
our best knowledge, no ICT-FRET-based fluorescent probe has
yet been reported for the selective detection of H2O2 in living
cells. Therefore, there is still demand for a simple and efficient
ICT-FRET-based fluorescent probe for detecting H2O2.
Introduction
Hydrogen peroxide (H2O2), one of the most well-known reactive
oxygen species (ROS), has been associated with diverse
physiological processes.[1] H2O2 plays a critical role in cell
proliferation, differentiation, immune responses, and signalling
pathways under physiological conditions.[1b,2] It is believed that
H2O2 is generated by the activation of NADPH oxidase
complexes in living cells.[3] Additionally, H2O2 is engendered as a
metabolic by-product in many enzymatic reactions. For example,
the conversion of glucose to gluconolactone by the oxidization of
glucose oxidase can produce H2O2 in cells.[4] Moreover,
excessive H2O2 production is implicated in various diseases
including cancer, diabetes, DNA damage, cardiovascular
disease, neurodegenerative disorders and Alzheimer’s disease.[5]
Thus, it is of great importance to detect H2O2 in the biosystem.
Several chemical tools for the detection of intracellular H2O2
have been reported, such as mass probes, proteomics probes,
and fluorescent probes.[6] Fluorescent probes have been widely
used because of their high spatial and temporal resolution. In
recent years, many fluorescent probes have been reported for
detecting H2O2 in vitro and in vivo.[7] In particular, Chang group
have made great achievements in the development of new
fluorescent probes for detecting H2O2.[8] Most of these probes
are intensity-based turn-on fluorescent sensors. However,
explaining the results acquired with turn-on probes can be
complicated.[9] The probe location, variations in probe
concentration, probe environment, and excitation intensity may
In this work, we designed the novel ICT-FRET-based
fluorescent probe for the selective detection of H2O2. The FRET
system comprised
a coumarin donor and a naphthalimide
acceptor linked by a nonconjugated linker (CNP, Scheme 1).
The mechanism of the reaction of CNP with H2O2 is presented in
Scheme 1. Coumarin and naphthalimide are widely used as
fluorophores for their high photophysical properties.[15]
Furthermore, there is good spectral overlap between the
emission of coumarin and the absorption of naphthalimide.[14,16]
To obtain more efficient probes, we changed the overlap level by
adjusting the naphthalimide absorption wavelength to exert
on/off control over the FRET process. A boronate protecting
group was used because of its good sensitivity and selectivity to
H2O2. Additionally, the boronate group could be changed by
H2O2 to yield the hydroxyl products and lead to the absorption
redshift by the ICT process. As a result, the CNP probe can be
used to detect H2O2 by measuring the ratio of green and blue
fluorescence intensities based on the novel ICT-FRET
mechanism. The probe exhibited
a
redshifted (73 nm)
fluorescence emission, and the ratio of fluorescence intensities
at 558 and 485 nm (F558 / F485) shifted notably (up to 100-fold).
The response is more pronounced than lots of ratiometric
fluorescent probes that have been reported to detect H2O2.[13a, b]
Moreover, there was a good linearity with a limit of detection of
0.28 µM (S/N = 3). The probe featured good stability and high
selectivity and was successfully applied to detect H2O2 changes
in living cells.
[a]
[b]
X Liang, D Qiao, Z Yin, L Shang *
College of Pharmacy & State Key Laboratory of Elemento-Organic
Chemistry.Nankai University ,Tianjin 300071, China.
E-mail: shanglq@nankai.edu.cn
X Xu
Tianjin Medical University,Tianjin 300070, P. R. China.
Supporting information for this article is given via a link at the end of
the document.
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addition of H2O2 and the normalized fluorescence spectrum of CN-1 (EmCN-1, blue).
Data were acquired at 37°C in 20 mM PBS buffer containing 10% acetonitrile at pH
7.4.
To verify the feasibility of the CNP probe, we designed a
coumarin donor section (CN-1) and a naphthalimide acceptor
section (CN-2) (Figure 1A). The spectral overlap between the
coumarin emission (EmCN-1) and the naphthalimide absorption
(AbsCN-2) was negligible. The FRET process of the CNP probe
was suppressed. After the reaction of CN-2 with H2O2, the
absorption of naphthalimide (AbsCN-OH) showed a redshift and a
quantum yield increase via the ICT process (Figure S1). The
spectral overlap was dramatically increased because of the
redshift, and the FRET appeared theoretically (Figure 1B). When
excited at 485 nm (the maximum emission wavelength of the
donor CN-1, Figure S2B), CN-2 was in a colourless and
nonfluorescent state in the absence of H2O2. However, the
subsequent addition of H2O2 to the solution led to a gradual
increase in the fluorescence emission peak at 558 nm via the
ICT process (Figure 2). To elucidate the fluorescence response,
we decided to characterize the reduced product. Incubation of
CNP with H2O2 was confirmed to produce CNP-OH, as
determined by mass spectrometry (Figure S3). To further
understand the optical responses of CNP to H2O2, we performed
a density functional theory (DFT) calculation with the B3LYP/ 6-
311G++ (d, p) method by the Gaussian 09 program. The
coumarin−naphthalimide platform could possess remarkable
FRET efficiency with the suitable distance (14.13 Å) between the
two fluorophores (Figure S4). As shown in Figure S5, in the case
Scheme 1. The mechanism of the novel CNP ratiometric fluorescent probe with
H2O2.
Results and Discussion
Synthesis of the probe CNP
The synthesis route for target compound CNP is outlined in
Scheme 2 and the details are given in the Experimental Section.
The structure of CNP was confirmed using NMR spectroscopy
and HR-MS. The involved compounds CN-1 and CN-2 were also
prepared. Detailed structural characterization and NMR
spectroscopy are given in the ESI.†
of CNP and CNP-OH, the electrons on the HOMO was
located on the coumarin moiety, while the  electrons on the
LUMO located around the naphthalimide moiety. Further
comparison electrons distribution in the LUMO of CNP and
CNP-OH, the π electrons of CNP on the LUMO located around
the entire moiety, which indicated an ICT occurred through the
p-  conjugated from hydroxyl to naphthalimide unit. These
experimental results supported the proposed response
mechanism. Thus, the CNP detecting H2O2 was based on the
novel ICT- FRET mechanism.
Scheme 2. Synthetic route of the probe CNP.
Mechanism of the responses of CNP toward H2O2
Figure 2. A) Time-dependent emission intensity changes during the reaction
of CN-2 (5 µM) incubated with H2O2 (10 equiv.) from 0 to 120 min. Data were
acquired at 37°C in 20 mM PBS buffer, containing 10% acetonitrile, pH 7.4
with excitation at the maximum emission wavelength of the donor CN-1 (485
nm). (EX Slit:5.0 nm, EM Slit:5.0 nm). B) The changes fluorescence intensity
at 558 nm with time. The pseudo-first-order rate constant: kobs = 0.034 min-1
(R2=0.9954).
Figure 1. A) Structure of CN-1 and the reaction of CN-2 with H2O2. B) The
normalized absorption spectra of CN-2 before (AbsCN-2, pink) and after (AbsCN-OH, red)
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H2O2 from 0 to 60 μM (Figure 4B), suggesting that CNP was
potentially useful for quantitatively detecting H2O2.The detection
limit was calculated to be 0.28 μM (S/N = 3), indicating that CNP
was highly sensitive to H2O2.
Absorption and emission spectra of CNP in the presence
of H2O2
We hoped the CNP probe would be able to exhibit the designed
response in the presence of H2O2. First, CNP was evaluated
under in vitro conditions (in 20 mM PBS buffer containing 10%
acetonitrile at pH 7.4). The as-expected UV−vis spectra of CNP
are represented in Figure 3. The absorption at around 350 nm
was gradually decreased, and a maximum absorption peak in
the visible region centred at 450 nm was gradually increased
upon addition of H2O2 (10 equiv.) over 60 min. It is the similar to
the characteristic absorption changing of CN-2 with H2O2 (Figure
S1). This suggested that the structure of the probe had been
changed, with the enhanced absorption of the new chemical
entity caused by the ICT process (Scheme 1).
Figure 4. A) Concentration-dependent emission intensity changes during the
reaction of CNP (5 µM) incubated with H2O2 (0-100 μM) for 30 min. Data were
acquired at 37°C in 20 mM PBS buffer containing 10% acetonitrile at pH 7.4
with excitation at 420 nm. (EX Slit: 5.0 nm, EM Slit: 5.0 nm); Inset:
photographs of the cuvettes containing CNP before (left) and after (right)
addition of H2O2 under 365 nm excitation. B) Linear relationship between the
ratio of fluorescence intensities (F558 / F485) and the concentration of H2O2.
Selectivity of CNP
Considering the several species of ROS inside living cells, the
chemo-selectivity of the CNP probe to H2O2 over various ROS is
critical for the accurate measurement of H2O2 in living cells. The
selectivity experiment was under the above mentioned analytical
conditions, and the data were collected at several time points
over 60 min. As expected, only H2O2 notably increased the ratio
of fluorescence intensities (F558 / F485). The changes were barely
observable in the presence of other ROS, including hydroxyl
radical (.OH), tert-butylhydroperoxide (TBHP), tert-butoxy radical
(.OtBu), superoxide (O2-), hypochlorite (ClO-), nitric oxide (NO)
and peroxynitrite (ONOO-) (Figure 5). The results demonstrated
the excellent selectivity of the probe towards H2O2 over other
biologically relevant analytes.
Figure 3. Time-dependent absorption spectral changes during the reaction of
CNP (50 µM) incubated with H2O2 (10 equiv.) from 0 to 60 min. Data were
acquired at 37°C in 20 mM PBS buffer, containing 10% acetonitrile , pH 7.4.
We then explored the fluorescence emission of CNP (5 μM) in
20 mM PBS buffer (pH 7.4) with 10% acetonitrile. When excited
at 420 nm (the maximum absorption wavelength of the donor
CN-1, Figure S2A), CNP exhibited only the characteristic
fluorescence emission peak of the coumarin donor at
approximately 485 nm, with no fluorescence emission band of
the naphthalimide acceptor. However, the subsequent addition
of H2O2 to the CNP solution led to a gradual increase in the
fluorescence emission peak of the naphthalimide acceptor at
approximately 558 nm; meanwhile, the fluorescence emission
band of the coumarin donor gradually disappeared. CNP
exhibited a redshifted (73 nm) fluorescence emission upon
addition of H2O2 (Figure 4 and Figure S6). This result indicated
that CNP was transformed into CNP-OH in the presence of H2O2
and that the FRET platform was effectively caused by the ICT
process (Scheme 1). Moreover, the ratio of fluorescence
intensities at 558 and 485 nm (F558 / F485) varied from 0.143 in
the absence of H2O2 to 14.1 after H2O2 treatment for 120 min,
exhibiting an ~100-fold increase (Figure S6C). These results
demonstrated that CNP has excellent sensitivity for monitoring
H2O2. The concentration-dependent fluorescence response of
CNP was also investigated after the addition of H2O2 from 0–100
Figure 5. The ratios of fluorescence intensities (F558 / F485) of CNP (5 µM)
towards ROS represented the relative responses at 0, 10, 20, 30, 40, 50, and
60 min after the addition of each oxidant at 37°C in 20 mM PBS buffer
containing 10% acetonitrile at pH 7.4. Data were acquired at 200 μM H2O2,
excess NO, and at a concentration of 200 μM for all other oxidants, with
excitation at 420 nm. (EX Slit: 5.0 nm, EM Slit: 5.0 nm).
μM for 30 min (Figure 4A). There was a good linearity (R2
0.9911) between the F558 / F485 ratio and the concentration of
=
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fluorescence intensities (Fgreen / Fblue) was observed to be
consistent with the results when detecting H2O2 in vitro (Figure
7E). Thus, these results indicated that CNP is cell membrane
permeable and can effectively detect H2O2 in living cells.
Glucose detection by enzymatically generated H2O2
As H2O2 is an important metabolic by-product engendered in
many enzymatic reactions, the activity of the correlative
enzymes or the concentration of the substrates could be
evaluated by the generated H2O2. For example, conversion of
glucose to gluconolactone by the oxidization of glucose oxidase
can produce H2O2. Therefore, coupled with the reaction of
glucose oxidase with D-glucose to generate H2O2, CNP has
potential applicability to the accurate measurement of glucose.
Upon addition of D-glucose to a glucose oxidase solution
containing CNP, the fluorescence response quickly changed
because of the generation of H2O2. However, addition of D-
glucose to a glucose oxidase containing CNP in the presence of
catalase caused no obvious change in the fluorescence intensity,
because H2O2 was consumed by the catalase (Figure 6A).
These results proved that the CNP probe could be used to
evaluate glucose by detecting the enzymatically generated H2O2.
We also investigated the concentration-dependent fluorescence
response of CNP towards different amounts of D-glucose (0-50
μM) after reaction for 30 min. There was a good linearity
between the F558 / F485 ratios and the concentration of D-glucose
(Figure 6B and Figure S7).
Figure 6. A) Fluorescence detection of enzymatically generated H2O2 by CNP.
Red trace:
5 μM CNP, 50 μM D-glucose, 8
μg▪L-1 glucose oxidase with
excitation at 420 nm. Black trace: as in panel but in the presence of catalase
80 μg▪L-1. B) Linear relationship between the ratios of fluorescence intensities
(F558 / F485) and the concentration of D-glucose; conditions: 5 μM CNP and 8
μg▪L-1 glucose oxidase in 20 mM PBS buffer containing 0.1% DMSO, pH 7.4
with excitation at 420 nm.(EX Slit: 5.0 nm, EM Slit: 5.0 nm)
E
pH study and cytotoxicity
To study the practical applicability of CNP, the dependence of its
fluorescence response on pH was also investigated (Figure S8).
In the presence of H2O2, the F558 / F485 ratios significantly
increased in a pH range of 6–10 and reached a maximum near
pH 8. Moreover, CNP had excellent stability in various pH
buffers (Figure S9), which suggested that it could be used to
monitor H2O2 near physiological pH. Meanwhile, CNP had little
cytotoxicity for living cells as determined by the MTT assay
(Figure S10). These results indicated that CNP may be suitable
for the detection of H2O2 inside living systems.
Fluorescence imaging of H2O2 in living cells
Figure 7. Live-cell imaging with CNP (5 μM). CNP-loaded HeLa cells treated
with (A) vehicle control, (B) 2, (C) 10 and (D) 50 μM H2O2 for 60 min at
37 °C.blue channel (Ex = 405 nm, collected 450–510 nm); green channel ( Ex
We then examined whether CNP could be used as a responser
to image H2O2 in living cells. HeLa cells were initially incubated
with 5 µM CNP for 20 min at 37°C and then washed three times
with PBS buffer (pH 7.4). The fluorescence imaging exhibited
strong fluorescence in the blue channel and weak fluorescence
in the green channel. However, after treatment with various
concentrations of H2O2 for 60 min, the fluorescence in the blue
channel diminished and the fluorescence in the green channel
continuously increased (Figure 7 and Figure S11). The ratio of
=
405 nm, collected 540–600 nm); (Scalebar, 25 μm); (E) The ratio of
fluorescence intensities (Fgreen Fblue treated with the various H2O2
/
)
concentrations. Quantified relative fluorescence intensity was analyzed using
a Leica Application Suite X software and presented as mean ± SD, with n = 5
fields of cells.
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Experimental Section
Fluorescence imaging of endogenous H2O2 in living
cells
General Information
Finally, we examined the feasibility of using CNP to detect
endogenous H2O2 produced by the stimulation of the epidermal
growth factor (EGF) in living A431 cells as per the literature.[17]
Compared with the fluorescence in the cells before the
stimulation, the fluorescence imaging showed a fluorescence
Unless otherwise stated, all reagents used in this study were
commercial products of the highest available purity without
further purification. EGP was purchased from JinYan Biological
company. The HeLa and A431 cells were purchased from
Chinese academy of medical sciences. Catalase (Sigma) was
purchased from Aldrich, and glucose oxidase and D-glucose
were purchased from TCI Chemicals. Twice-distilled water was
used throughout all experiments.1H-NMR (400 MHz) and 13C-
NMR (100 MHz) spectra were recorded on a Bruker AVANCE-
400 (400MHz) (Bruker, Karlsruhe, Germany) NMR spectrometer
using CDCl3 as solvent and tetramethylsilane (TMS) as an
internal standard. Electrospray ionization mass spectroscopy
(ESI-MS) was performed on a Shimadzu LCMS-2020 (Shimadzu,
Kyoto, Japan). HRMS were recorded on a high-resolution ESI-
FTICR mass spectrometer (Varian 7.0 T, Varian, USA). UV-vis
decrease in the blue channel and
a remarkable emission
increase in the green channel (Figure 8). The result is consistent
with the previous literature.[17] These data indicated that CNP is
capable of imaging endogenous H2O2 in living A431 cells.
Bright
Blue
Green
Merge
spectra were measured by
a UH-5300 spectrophotometer
(Hitachi). Fluorescence measurements were recorded on a F-
7000 spectrophotometer (Hitachi). Fluorescence images of cells
were taken by alaser scanning confocal microscopy (Leica TCS
SP8).
Synthesis of 7-Diethylaminocoumarin-3-carboxylic acid
(1)
2-Hydroxybenzaldehyde (5.0 g, 26.0 mmol) and Meldrum’s acid
(3.8 g, 26.0 mmol) were suspended in the ethanol (50 mL). Then
the mixture was added piperidine (200 mg, 2.6 mmol) in a
portion and added dropwise acetic acid (20 drops). The mixture
was heated to 90 °C for overnight and monitored by TLC. The
precipitate formed was filtrated under vacuum (1) as a red solid
C
1
in 61.8%yield (4.2 g). H NMR (400 MHz, CDCl3) δ 12.35 (s, 1H),
8.62 (s, 1H), 7.44 (d, J = 9.0 Hz, 1H), 6.70 (dd, J = 9.0, 2.2 Hz,
1H), 6.51 (d, J = 2.0 Hz, 1H), 3.48 (q, J = 7.1 Hz, 4H), 1.26 (t, J
= 7.1 Hz, 6H).13C NMR (100 MHz, CDCl3) δ 165.56, 164.49,
158.06, 153.78, 150.28, 131.98, 110.94, 108.56, 105.52, 96.83,
45.37, 12.40.
Synthesis of
chromene-3-carbonyl)piperazine-1-carboxylate (CN-1)
tert-butyl-4-(7-(diethylamino)-2-oxo-2H-
Figure 8. A431 cells imaging with CNP (5 μM). A) treated with EGF
stimulation(500 ng • mL-1) then imaging with CNP (5 μM) after 60 min at 37 °C;
B) blue channel (Ex = 405 nm, collected 450–510 nm); green channel ( Ex =
405 nm, collected 540–600 nm); (Scale bar, 25 μm); C) The ratio of
Compound 1 (3.0 g, 11.5 mmol) and tert-butyl piperazine-1-
carboxylate (2.57 g, 13.8 mmol) were mixed in anhydrous DMF
(50 mL), HOBt (1.86 g, 13.8 mmol) was added and the mixture
was stirred at ambient temperature for 10 min, then EDC·HCl
(3.07 g, 16.1 mmol) and DIEA (2.85 mL, 17.25 mmol) was
added. The mixture formed was stirred at ambient temperature
in the dark overnight. The reaction mixture was diluted with
fluorescence intensities (Fgreen
experiment was repeated five times. *** : p < 0.001. Statistical analyses were
performed with the Student’s t-test.
/ Fblue) of the representative images. The
Conclusions
CH2Cl2, washed with 0.1
N HCl and brine, dried over
In conclusion, we have successfully designed and synthesized
the dual mechanism ICT-FRET-based fluorescent probe, CNP,
for the detection of H2O2 in living cells. The probe exhibited a
redshifted (73 nm) fluorescence emission upon addition of H2O2
accompanied by clear fluorescence emission ratio changes (up
to 100-fold) in the blue and green spectral regions. CNP
displayed high selectivity due to the adoption of a boronate
group, low cytotoxicity and good stability in the biological pH
range. This probe could also detect enzymatically generated
H2O2. Moreover, this probe was successfully applied to detect
both the exogenous and endogenous H2O2 in living cells. These
results indicate that this ICT-FRET-based fluorescent probe has
great potential for the detection of H2O2 in living systems.
Na2SO4 and concentrated. And CN-1 was isolated as a yellow
solid by silica-gel column chromatography in 91.1% yield (4.5 g).
1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.29 (d, J = 8.9 Hz,
1H), 6.59 (dd, J = 8.9, 2.4 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H), 3.68
(d, J = 20.5 Hz, 2H), 3.49 (d, J = 8.6 Hz, 4H), 3.43 (q, J = 7.1 Hz,
4H), 3.38 (d, 2H), 1.46 (s, 9H), 1.22 (t, J = 7.1 Hz, 6H).13C NMR
(100 MHz, CDCl3) δ 165.23, 159.15, 157.31, 154.60, 151.76,
145.34, 129.90, 115.96, 109.40, 107.72, 96.92, 80.18, 47.24,
44.96, 42.23, 28.37, 12.41.
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1
51.0% yield (180 mg). H NMR (400 MHz, CDCl3) δ 9.03 (t, J =
8.0 Hz, 1H), 8.45 (dd, J = 11.2, 8.7 Hz, 2H), 8.19 (t, J = 6.9 Hz,
1H), 7.83 (d, J = 9.3 Hz, 1H), 7.74 – 7.64 (m, 1H), 7.24 (d, J =
9.3 Hz, 1H), 6.53 (d, J = 8.6 Hz, 1H), 6.38 (s, 1H), 4.42 (t, J =
6.1 Hz, 2H), 3.69 (dd, 4H), 3.57 (t, J = 13.0 Hz, 2H), 3.36 (m, J =
6.6 Hz, 6H), 2.78 (dd, 2H), 1.40 (s, 12H), 1.20 – 1.12 (t, 6H).13C
NMR (100 MHz, CDCl3) δ 169.15, 165.37, 165.25, 164.17,
159.20, 157.34, 151.83, 145.82, 145.49, 135.71, 135.21, 135.18,
130.94, 129.97, 129.81, 127.77, 127.09, 124.36, 122.27, 115.57,
109.46, 107.72, 96.89, 84.63, 53.49, 44.96, 36.82, 31.63, 24.98,
24.82, 12.41.HRMS (ESI) m/z 707.3260 (M+H+), calc. For
C39H43N4O8B 707.3252.
Synthesis of 7-(diethylamino)-3-(piperazine-1-carbonyl)-2H-
chromen-2-one (2)
Compound CN-1 (3.4 g, 8.0 mmol) was dissolved by a solution
consisted of DCM (15 mL), TFA (6 mL), and then the mixture
was stirred at room temperature for 3 h. After that, the reaction
solution was removed under reduced pressure and (2) was
isolated as a yellow solid by silica-gel column chromatography in
84.6% yield (2.2 g). 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H),
7.27 (d, J = 9.9 Hz, 1H), 6.57 (d, J = 8.9 Hz, 1H), 6.45 (s, 1H),
3.71 (d, 2H), 3.41 (dd, J = 14.2, 7.1 Hz, 6H), 2.90 (d, J = 18.3 Hz,
4H), 1.85 (s, 1H), 1.20 (t, J = 6.9 Hz, 6H).13C NMR (100 MHz,
CDCl3) δ 164.98, 159.13, 157.21, 151.59, 144.72, 129.76,
116.62, 109.29, 107.77, 96.99, 48.72, 46.29, 45.75, 44.94, 43.42,
12.42.
MTT assay
Synthesis of 6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-
yl)benzo[de]isochromene-1,3-dione (3)
The Hala or A431 cells were cultured at 37 °C, 5% CO2 in
DMEM supplemented with 2.5 mM glutamine, 10% (v/v) fetal
bovine serum (FBS), penicillin (100 units/mL), and streptomycin
(100 μg/mL). One day before experiment, the Hala or A431 cells
(5×105 cells/mL) were passaged and plated in phenol red-free
medium in 96-well plate and allowed to grow to ~90%
confluence. Solutions of CNP in DMEM were prepared from a 10
mM stock solution in DMSO. The cells were covered in DMEM
buffer containing CNP at various concentrations (1-50 μM) or a
vehicle control. After incubated at 37 °C, 5% CO2 for 24 h, each
well of cells were treated with 20 μL MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) solution (5
mg/ml), and incubated for another 4 h. Then each well was
treated with 80 μL DMSO at 37 °C for 10 min, and then the
absorbance intensities at 570 nm were measured using a plate
reader. Each experiment was repeated five times.
A mixture of 4-bromo-1,8-naphthalic anhydride (277 mg,1.0
mmol),
bis(pinacolato)diboron
(380
mg,
1.5
mmol),
Pd(dppf)2Cl2 (73.2 mg, 0.1 mmol) and potassium acetate
(300 mg, 3.0 mmol) in dioxane (15 mL) was stirred at 90 °C
overnight under nitrogen atmosphere. After cooled to room
temperature, the reaction mixture was diluted with CH2Cl2,
washed with H2O and brine, and dried over Na2SO4. After
removal of solvent, (3) was isolated as a whitesolid by silica-gel
1
column chromatography in 60.2% yield (195 mg). H NMR (400
MHz, CDCl3) δ 9.21 (d, J = 8.4 Hz, 1H), 8.60 (d, J = 7.1 Hz, 1H),
8.56 (d, J = 7.3 Hz, 1H), 8.33 (d, J = 7.3 Hz, 1H), 7.89 – 7.78 (m,
1H), 1.46 (s, 12H).13C NMR (100 MHz, CDCl3) δ 160.80, 160.68,
136.54, 136.09, 135.39, 133.07, 131.90, 131.42, 129.87, 127.57,
120.70, 118.59, 84.91, 24.99.
Cell culture and fluorescence microscope experiments
Synthesis of 3-(1,3-dioxo-6-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)
propanoic acid (CN-2)
The cells cultured in Dulbecco’s modified eagle medium (DMEM)
supplemented with 2.5 mM glutamine, 10% (v/v) fetal bovine
serum (FBS), penicillin (100 units/mL), and streptomycin (100
μg/mL) in a humidified incubator under 5% CO2 in 95% air.
Under the protection of argon, a mixture of compound 3 (195 mg,
0.6 mmol), 3-aminopropanoic acid (72 mg, 0.78 mmol) and
DMAP (8 mg, 0 .06 mmol) in anhydrous ethanol (5 mL) was
stirred at 90 °C for 12 h. After cooled to room temperature, the
solvent was removed by rotary evaporation and the residue was
puried by silica gel column chromatography to afford a white
HeLa or A431 cells were seeded (5×105 cells/mL) onto a glass
base dish one day before imaging and allowed to grow to ~80%
confluence. A431 cells that were to be used for EGF stimulation
experiments were serum-starved in DMEM alone 18 h before
imaging. The medium replaced with PBS, and then 5 μM CNP
was loaded into the cells at 37°C for 60 min, washed three times
with PBS. HeLa cells were exposed containing H2O2 at various
concentrations. A431 cells were stimulated with 500 ng/mL EGF.
Fluorescence images were taken after 60 min incubation at
37 °C.
1
solid in 88.6% yield (210 mg). H NMR (400 MHz, CDCl3) δ 9.13
(d, J = 8.3 Hz, 1H), 8.61 (d, J = 7.1 Hz, 1H), 8.57 (d, J = 7.2 Hz,
1H), 8.30 (d, J = 7.2 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 4.51 (t, J =
7.5 Hz, 2H), 2.85 (t, J = 7.6 Hz, 2H), 1.45 (s, 12H).13C NMR (100
MHz, CDCl3) δ 175.56, 164.22, 135.80, 135.28, 131.12, 130.00,
127.90, 127.14, 124.40, 122.30, 84.64, 35.85, 32.28, 24.99.
Synthesis
of
2-(3-(4-(7-(diethylamino)-2-oxo-2H-
chromene-3-carbonyl)piperazin-1-yl)-3-oxopropyl)-6-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (CNP)
Acknowledgements
This work was supported by the National Basic Research
Program of China (973 program, Grant Nos. 2013CB911104,
2013CB911100), the National Natural Science Foundation of
China (Grant Nos. 21672115), and the "111" Project of the
Ministry of Education of China (Project No. B06005).
Compound 2 (165 mg, 0.5 mmol) and compound CN-2 (190 mg,
0.5 mmol) were mixed in anhydrous DMF (5 mL), HOBt (80 mg,
0.6 mmol) was added and the mixture was stirred at ambient
temperature for 10 min, then EDC·HCl (135 mg, 0.7 mmol) and
DIEA (125 μl, 0.75 mmol) was added. The mixture formed was
stirred at ambient temperature in the dark overnight. The
reaction mixture was diluted with CH2Cl2, washed with 0.1 N HCl
and brine, dried over Na2SO4 and concentrated. And CNP was
isolated as a yellow solid by silica-gel column chromatography in
Keywords: hydrogen peroxide • fluorescent probe • ratiometric
probe • ICT • FRET
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The dual mechanism
Xiao Liang, Xiaoyi Xu, Dan Qiao,
Zheng Yin , and Luqing Shang *
ICT-FRET
fluorescent
probe for the selective
detection of H2O2 in
living cells.
Page No. – Page No.
The dual mechanism ICT-FRET-
based fluorescent probe for the
selective detection of hydrogen
peroxide
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