Investigation of the Relationship between H2O2 and HClO in Living Cells by a Bifunctional, Dual-ratiometric Responsive Fluorescent Probe

Article abstract of DOI:10.1021/acs.analchem.9b05604

As two important reactive oxygen species, hydrogen peroxide (H2O2) and hypochlorous acid (HClO) play vital roles in many physiological and pathological processes. However, the relationship between these two species is seldom investigated, in part, because

Full text of DOI:10.1021/acs.analchem.9b05604

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Article
Investigation of the Relationship Between H2O2 and HClO in Living
Cells by a Bifunctional, Dual-ratiometric Responsive Fluorescent Probe
Jinliang Han, Xingjiang Liu, Haiqing Xiong, Jingpei Wang, Benhua Wang, Xiangzhi Song, and Wei Wang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b05604 • Publication Date (Web): 03 Mar 2020
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Analytical Chemistry
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Investigation of the Relationship Between H
2
O and HClO in Living
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Cells by a Bifunctional, Dual-ratiometric Responsive Fluorescent Probe
†
‡
†
†
†
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Jinliang Han, Xingjiang Liu, Haiqing Xiong, Jingpei Wang, Benhua Wang, Xiangzhi Song, * and
≠
,
Wei Wang *
†
College of Chemistry & Chemical Engineering, Central South University, Changsha 410083, Hunan Province, China.
Email: xzsong@csu.edu.cn.
‡
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, Henan Province, China.
Department of Pharmacology and Toxicology, College of Pharmacy, and BIO5 Institute, University of Arizona, Tucson,
≠
AZ, 85721, USA. Email: wwang@pharmacy.arizona.edu.
The corresponding author’s full contact information: Prof. Wei Wang, PhD, Department of Pharmacology and Toxicology,
College of Pharmacy, and BIO5 Institute, University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721-0207, USA.
Email: wwang@pharmacy.arizona.edu; phone: 1-520-626-1764.
ABSTRACT: As two important reactive oxygen species (ROS), hydrogen peroxide (H
vital roles in many physiological and pathological processes. However, the relationship between these two species is seldomly in-
vestigated, in part, because of the lack of robust molecular tools which can simultaneously visualize HClO and H in biosystems.
In this work, we present a design strategy to construct a single fluorescent probe that can detect H and HClO by simultaneously
monitoring two distinct detection channels. In the design, one phenothiazine-based coumarin serves as a chromophore and sensor
for HClO while a second coumarin precursor containing a boronate ester acts as a sensor for H . After a head-to-head screening
2
O
2
) and hypochlorous acid (HClO) play
2
O
2
2
O
2
2
O
2
of three candidates differing in their coumarin precursor moieties, probe CSU1 was found to have the optimal characteristics. As
shown experimentally, it is able to detect them selectively and sensitively to generate distinct fluorescence signals and patterns in
-
living cells. Furthermore, the endogenous generation of HClO from H
2
O
2
and Cl catalyzed by MPO (myeloperoxidase) enzyme in
living cells can be clearly monitored by the probe. These studies demonstrate the potential of the probe as a powerful tool to
investigate the interplay of HClO and H in oxidative stress.
2
O
2
4
As the important species within and between cells, reactive
ological and pathological conditions, the simultaneous inves-
-
•
•
1
oxygen species (ROS), such as O
2
, OH, O
2
, HClO and H
O
2 2
,
tigation of the activity of multiple ROS in cells is critically
important to help study their interactions and synergistic ef-
fects in complex environments. For example, HClO can be
play vital roles in cell signal transmission, differentiation,
migration, cellular immunity and body’s defense against path-
1-6
ogens.
H
2
O
2
, as the most described signaling molecule
endogenously produced from H
2
O
2
and chloride ion with the
among ROS families, is relatively stable and has various in-
dispensable functions in cell signal transduction and homeo-
catalysation of the myeloperoxidase enzyme (MPO) in leuko-
2
6, 27
cytes.
In organisms, the levels of HClO and H
2
2
O are
7-9
stasis.
H
2
O
2
is a precursor and a by-product in ROS chain
within a well specified range in order to maintain a relative
balance. Once the balance is disrupted, oxidative stress occurs
and thereby leading to aging and subsequent diseases such as
reactions, and its excessive production can lead to oxidative
damage and thereby cause aging and various diseases includ-
1
0
11
28-30
ing cardiovascular diseases, neurodegenerative diseases,
neurodegenerative Alzheimer’s and Parkinson’s diseases.
inflammation, cancer, etc.14-16 Strong oxidant HClO is a
1
2
13
As a result, the imaging tools capable of simultaneous detec-
microbicidal effector to cope with microbial invasion in im-
tion of HClO and H
2
O are highly valued and urgently needed.
2
1
7,18
mune system.
The abnormal accumulation of HClO can
Fluorescence imaging technique offers a high spatial and
temporal resolution and serves as an attractive tool to study
biological species in a noninvasive manner. Although nu-
merous fluorescent probes have been developed for the single
and specific detection of HClO or H
19
cause serious tissue damage to induce a series of diseases
including neurodegenerative diseases, atherosclerosis, cancer,
ischemia, reperfusion injury, rheumatoid rheumatism, and so
31
20-24
on.
In many cases, the delicate balance between oxidative
4, 32-40
2
O
2
,
as far as we are
stress and signal transduction in cells is strictly manipulated
by a group of reactive oxygen species, which are mutually
dependent and are often transformed into each other through
aware, there is no such probe that can simultaneously image
both HClO and H in vitro and in vivo, especially in a rati-
2
O
2
ometric manner. Ratiometric fluorescent probes are more de-
sirable because they can reduce the interference from the con-
centration of fluorescent probes, the instrument factor and the
2
5
intracellular reactions. Since ROS are very reactive and their
intracellular concentrations vary greatly, particularly in physi-
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Analytical Chemistry
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environments through self-calibration with two fluorescence
DMSO solutions at 490 nm was determined and the cell via-
bility was calculated using the following formula: cell viabil-
ity = (mean absorbance of test wells - mean absorbance of
medium control wells)/(mean absorbance of untreated wells -
mean absorbance of medium control wells) × 100%.
4
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signals. In light of the important roles that H
play in biosystems, it would be of high interest in a probe that
2
2
O and HClO
can simultaneously detect HClO and H
nals.
2
2
O with distinct sig-
Here, through the delicate design and after a head-to-head
screening of three candidates, we obtain a dual-ratiometric
Imaging of Exogenous H
2
2
O and HClO in MCF-7 Cells.
MCF-7 cells were treated with probe CSU1 (5.0 μM) for 20
min. Then CSU1-loaded MCF-7 cells were incubated with
fluorescent probe, CSU1, that can simultaneously detect H
and HClO from different channels (see its structure in Scheme
and working mechanism in Scheme 2). With the aid of con-
focal microscope, this probe enables to image HClO and H
in living cells, and particularly to observe the intracellular
2
O
2
H
2
2
O (500.0 μM) for 60 min and HClO (400.0 μM) for 20 min,
1
respectively. Cells were washed with PBS buffer three times
before fluorescence imaging. For the successive imaging of
2
O
2
0
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2
3
4
5
6
7
8
9
0
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2
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exogenous H
incubated with H
2
O
2
and HClO, CSU1-loaded cells were first
(500.0 μM) for 60 min, washed with PBS
-
generation of HClO from H
2
O
2
and Cl under the enzyme cata-
2
O
2
lysation by virtue of ratiometric fluorescence changes.
buffer three times, further treated with HClO (400.0 μM) for
another 20 min, and finally washed with PBS buffer three
times. A same procedure was use for the reverse addition or-
EXPERIMENTAL SECTION
der (from H
2
O
2
to HClO).
Materials and Instruments. The commercial chemicals
were used without further purification. The organic solvents
are analytically pure. NaClO was used as the source of HClO.
General Procedure for Monitoring HClO Generation
-
from H
2
O and Cl Induced by Myeloperoxidase (MPO) in
2
Solution and Living Cells. First, the measurement of MPO-
1
13
-
H and C NMR spectra were obtained using a Bruker 400
induced HClO from H
er (10.0 mM, pH 7.4, containing 50% CH
(5.0 μM), MPO (0.5 U/mL), NaCl (2.0 mM) and H
2
O
2
and Cl was carried out in PBS buff-
CN). Probe CSU1
(500.0
spectrometer with chemical shifts reported in ppm (TMS as an
internal standard). Mass spectra were obtained on a Bruker
Daltonics micr-OTOF-Q II mass spectrometer. Emission spec-
tra were recorded on a Hitachi F-7000 fluorometer, and UV-
vis absorption spectra were recorded on an Agilent UV-2450
spectrophotometer. A Leici PHS-3C meter was used for pH
measurements. The bioimaging experiments were carried out
on an Opera Phenix/Operetta CLS system from Perkin Elmer
and the analysis software was Harmony 4.5. Blue channel:
420-500 nm (excited at 355-385 nm); green channel: 515-580
nm (excited at 390-420 nm); red channel: 570-650 nm (excited
at 435-460 nm). MCF-7 cells were provided by the State Key
Laboratory of Chemo/Biosensing and Chemometrics, Hunan
University, China.
3
2
O
2
μM) were dissolved in the test solution. The mixture was in-
cubated at 37 °C for a total 27 min during which the fluores-
cence intensity at respective 409 nm and 520 nm was meas-
ured every 3 min. For monitoring intracellular MPO-induced
-
HClO from H
2
O
2
and Cl , cells were first treated with probe
CSU1 (5.0 μM), NaCl (2.0 mM) and MPO (0.5 U/mL) for 20
min and then incubated with H (500.0 μM) for another 30
min at 37 °C. For the real-time monitoring the process of the
2
O
2
-
endogenous HClO generation from H
2
O and Cl in the pres-
2
ence of MPO enzyme in living cells, cells was were treated
with probe CSU1 (5.0 μM), NaCl (2.0 mM) and MPO (0.5
U/mL) for 20 min, washed with PBS buffer three times and
Spectral Measurements. All the optical experiments ex-
cept pH measurements were performed in PBS buffer (10.0
mM, pH 7.4, containing 50% acetonitrile). The stock solutions
of fluorescent probes (CSU1, CSU2 and CSU3 in Scheme 1)
then observed under a microscope after the addition of H
(500.0 μM) at 37 °C.
2
O
2
Synthesis of Compounds 1, 2 and 5. Compounds 1, 2 and 5
were synthesized according to the reported methods (Scheme
(
0.1 mM) were prepared in acetonitrile (CH
3
CN). The solu-
42-44
1
).
tions of various testing species stock solutions (10.0 mM)
were prepared in twice-distilled water according to the litera-
ture methods (seen in ESI). For the titration experiments, a
Synthesis of Compound 3. To a solution of compound 2
(
(
790 mg, 2.0 mmol) in 20 mL of MeOH was added NaOH
240 mg, 6.0 mmol). The reaction mixture was refluxed for 1
certain amount of H
2
2
O or HClO was added into the solution
h. After removal of the solvent under a reduced pressure, the
residue was dissolved in CH Cl (50 mL) and acidified to pH
.0-4.0 using 10% aqueous HCl solution, and then washed
of a probe (5.0 μM) in a 2.0 mL total volume. The resulting
solutions were shaken well and then incubated for 20 min at
2
2
3
2
5 °C before the spectral measurements.
with water and brine. The organic layer was dried over anhy-
drous sodium sulfate and concentrated in vacuum to give a
pure oxblood solid (672 mg, 91%). HRMS (ESI) m/z calcd for
Cell Culture. DMEM was added with 10% fetal bovine se-
rum, 1% penicillin and 1% streptomycin as the culture medi-
um, and cells were placed in the medium at 37 °C under a
+
1
C
(
20
H
17NO
4
S [M+Na] : 390.0776; found 390.0784. H NMR
400 MHz, CDCl ) δ 8.67 (s, 1H), 7.29 (s, 1H), 7.21 (t, J = 7.7
Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H),
humidified atmosphere containing 5% CO and 95% air. Be-
2
3
fore imaging experiments, cells were cultured in an 18 mm
glass dish.
6
.94 (d, J = 8.2 Hz, 1H), 6.77 (s, 1H), 3.91 (t, J = 7.3 Hz, 2H),
1
3
Cytotoxicity Assay. MTT assay was performed using
MCF-7 cells, which were placed into 96-well plate and cul-
tured in a cell culture tank. After the cell attachment was com-
pleted, different concentrations (0.0, 5.0, 10.0, 15.0, 20.0 μM)
of probe CSU1 were added into cells in 96-well plate, which
1.88–1.76 (m, 2H), 1.51 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H). C
NMR (100 MHz, CDCl ) δ 164.6, 163.4, 156.3, 152.2, 149.7,
3
141.6, 128.0, 127.6, 126.7, 124.576, 122.9, 122.6, 116.5,
113.4, 109.9, 101.9, 48.6, 28.4, 20.1, 13.7.
Synthesis of Compounds SAB1-3. Compound SA1 (152 mg,
were then incubated in 5% CO humidified incubator for 24 h.
2
1
(
.0 mmol), 4-bromomethylphenylboronic acid pinacol ester
296 mg, 1.0 mmol), and K CO (414 mg, 3.0 mmol) were
CN (10.0 mL) under an argon atmos-
The MTT solution (1.0 mg/mL in PBS buffer) was then added
to each well and cells were incubated in a cell culture tank for
another 4 h. Finally, the MTT solutions were dumped and
DMSO (100.0 μL) was added to each well. The absorbance of
2
3
placed in anhydrous CH
3
phere. The reaction mixture was refluxed for 5 h. After cool-
ing to room temperature, 40 mL of water was added to the
2
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Page 3 of 10
Analytical Chemistry
mixture, which was extracted with 30.0 mL CH
times. The combined organic layers were dried over Na
and the solvent was evaporated under a reduced pressure. The
crude product was chromatographed on silica gel flash column
2
Cl
2
three
SO
7.9 Hz, 2H), 7.50-7.44 (m, 1H), 7.41 (d, J = 7.9 Hz, 2H), 7.07
(t, J = 7.6 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 5.20 (s, 2H), 4.46-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
4
,
1
3
4.41 (m, 2H), 3.96-3.91 (m, 2H), 1.35 (s, 12H). C NMR (100
MHz, CDCl ) δ 163.0, 158.4, 150.4, 139.0, 135.2, 135.2,
3
(
(
300-400 mesh; eluent, petroleum ether (PE) : ethyl acetate
129.5, 126.3, 121.4, 121.1, 112.9, 101.8, 84.0, 70.7, 67.9, 60.9,
24.9.
EA) = 5:1) to give SAB1 as a white solid (312 mg, 85%).
+
HRMS (ESI) m/z calcd for C21
found 391.1704. H NMR (400 MHz, CDCl
7
8
3
1
H25BO
5
[M+Na] : 391.1687;
Synthesis of Compounds CSU1-3. The mixture of com-
pounds 3 (37 mg, 0.1 mmol), SABC1 (48 mg, 0.1 mmol),
EDCI (14.1 mg, 0.15 mmol), and DMAP (2.0 mg, 0.015 mmol)
1
3
) δ 10.39 (s, 1H),
.84 (d, J = 9.9 Hz, 3H), 7.44 (d, J = 7.8 Hz, 2H), 6.55 (dd, J =
.7, 1.4 Hz, 1H), 6.48 (d, J = 1.8 Hz, 1H), 5.18 (s, 2H), 3.83 (s,
in dry CH
2
2
Cl (3.0 mL) was stirred at room temperature for 5
13
H), 1.35 (s, 12H). C NMR (100 MHz, CDCl
3
) δ 188.3,
h. Then solvent was removed under a reduced pressure and the
resultant solid was further purified by silica gel flash column
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
66.1, 162.7, 139.0, 135.2, 130.5, 126.4, 119.3, 106.2, 99.2,
83.9, 70.3, 55.7, 24.9.
(
300-400 mesh; eluent, PE:EA = 5:1) to afford the desired
SAB2 was prepared in a similar procedure as a white solid
product CSU1 as a red solid (57 mg, 69%). HRMS (ESI) m/z
+
(
322 mg, 83% yield). HRMS (ESI) m/z calcd for C24
H25BNO
4
calcd for C46
H45BN
2
O
10S [M+Na] : 852.2780; found 852.2786.
+
1
1
[M+Na] : 411.1738; found 411.1729. H NMR (400 MHz,
CDCl ) δ 10.99 (s, 1H), 9.29 (d, J = 8.5 Hz, 1H), 8.01 (d, J =
8.9 Hz, 1H), 7.85 (d, J = 7.6 Hz, 2H), 7.75 (s, 1H), 7.62 (t, J =
.1 Hz, 1H), 7.44 (dd, J = 17.0, 7.5 Hz, 3H), 7.30 (d, J = 9.0
H NMR (400 MHz, CDCl ), δ 8.82 (s, 1H), 8.45-8.40 (m, 2H),
3
3
7.81 (d, J = 7.9 Hz, 2H), 7.38 (d, J = 7.9 Hz, 2H), 7.20 (s, 1H),
7.19-7.14 (m, 1H), 7.11-7.07 (m, 1H), 6.98 (t, J = 7.4 Hz, 1H),
6.91 (d, J = 8.2 Hz, 1H), 6.68 (s, 1H), 6.61 (dd, J = 8.9, 2.1 Hz,
1H), 6.45 (d, J = 2.2 Hz, 1H), 5.18 (s, 2H), 4.61 (s, 4H), 3.89-
3.84 (m, 2H), 3.83 (s, 3H), 1.81 (dt, J = 14.9, 7.6 Hz, 2H),
1.47 (dd, J = 15.0, 7.4 Hz, 2H), 1.34 (s, 12H), 0.97 (t, J = 7.4
7
1
3
Hz, 1H), 5.36 (s, 2H), 1.35 (s, 12H). C NMR (100 MHz,
CDCl ) δ 192.2, 163.2, 139.0, 137.6, 135.3, 131.6, 130.0,
28.7, 128.3, 126.5, 126.4, 125.0, 125.0, 113.9, 109.5, 84.0,
3
1
7
1
3
1.4, 24.9.
Hz, 3H). C NMR (100 MHz, CDCl
1
1
3
) δ 165.8, 163.3, 160.6,
57.0, 156.7, 151.1, 149.3, 148.3, 142.4, 138.8, 135.2, 131.3,
27.8, 127.5, 126.5, 126.2, 123.9, 123.2, 121.2, 116.6, 116.2,
SAB3 was prepared in a similar procedure as a white solid
(
[
315 mg, 82% yield). HRMS (ESI) m/z calcd for C20
H
23BO
4
+
1
114.4, 112.8, 112.7, 106.7, 102.1, 99.7, 97.8, 83.9, 70.7, 63.4,
62.7, 55.7, 48.3, 28.5, 24.9, 20.1, 13.7.
M+Na] : 361.1582; found 351.1563. H NMR (400 MHz,
) δ 10.57 (s, 1H), 7.85 (dd, J = 7.6, 3.8 Hz, 3H), 7.51
dd, J = 11.3, 4.4 Hz, 1H), 7.44 (d, J = 7.8 Hz, 2H), 7.07-6.96
CDCl
(
(
3
CSU2 was prepared in a similar procedure as a red solid (65
1
3
m, 2H), 5.22 (s, 2H), 1.35 (s, 12H). C NMR (100 MHz,
) δ 189.8, 161.0, 139.1, 135.9, 135.2, 128.5, 126.4,
mg, 77%). HRMS (ESI) m/z calcd for C49
H
45BN
2
O S
9
+
1
CDCl
3
[M+Na] : 871.2752; found 871.2831. H NMR (400 MHz,
CDCl ) δ 8.88 (s, 1H), 8.44 (s, 1H), 7.90 (d, J = 9.0 Hz, 1H),
7.79 (t, J = 8.5 Hz, 4H), 7.59-7.53 (m, 1H), 7.44 (d, J = 7.4 Hz,
125.2, 121.1, 113.0, 83.9, 70.4, 24.9.
3
Synthesis of Compounds SABC1-3. To a solution of com-
pounds SAB1 (128 mg, 0.4 mmol) and compound 5 (65 mg,
0.5 mmol) in ethanol (4.0 mL) was added piperidine (11.0 μL,
3H), 7.28 (d, J = 5.2 Hz, 1H), 7.20-7.13 (m, 2H), 7.09 (d, J =
7.5 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H),
6.67 (s, 1H), 5.40 (d, J = 8.8 Hz, 2H), 4.68 (s, 4H), 3.90–3.83
0
.1 mmol). The reaction mixture was stirred at room tempera-
(
m, 2H), 1.80 (d, J = 5.8 Hz, 2H), 1.49-1.43 (m, 2H), 1.33 (s,
ture under an argon protection for 4 h. After removal of the
solvent under a reduced pressure, the obtained crude product
was chromatographed on silica gel flash column (300-400
mesh; eluent, PE:EA = 2:1) to give SABC1 as a pale yellow
1
3
1
1
1
1
2H), 0.97 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, CDCl ) δ
3
62.2, 156.7, 154.9, 153.3, 152.2, 151.2, 149.7, 142.8, 140.4,
27.8, 126.5, 126.2, 125.6, 123.8, 121.9, 121.8, 116.1, 115.0,
14.2, 112.8, 102.5, 83.9, 71.1, 56.0, 48.1, 32.7, 26.1, 24.9,
solid (132 mg, 79%). HRMS (ESI) m/z calcd for C26
H30BNO
7
+
1
20.1, 13.8.
[
M+Na] : 502.2008; found 502.1998. H NMR (400 MHz,
CDCl ) δ 8.81 (s, 1H), 8.45-8.36 (m, 1H), 7.84 (d, J = 6.8 Hz,
H), 7.41 (d, J = 7.0 Hz, 2H), 6.62-6.55 (m, 1H), 6.43 (d, J =
19.9 Hz, 1H), 5.18 (s, 2H), 4.52-4.39 (m, 2H), 3.90 (t, J = 12.2
3
CSU3 was prepared in a similar procedure as a red solid (50
2
mg, 61% yield). HRMS (ESI) m/z calcd for C45
H
43BN
2
O
9
SNa
[M+Na] : 821.2675; found 821.2587. H NMR (400 MHz,
CDCl ) δ 8.90 (s, 1H), 8.43 (s, 1H), 8.34 (d, J = 7.9 Hz, 1H),
+
1
1
3
Hz, 2H), 3.82 (t, J = 6.2 Hz, 3H), 1.35 (s, 12H). C NMR (100
MHz, CDCl ) δ 165.8, 163.7, 160.6, 149.3, 138. 8, 135.3,
31.2, 126.3, 116., 114.30, 106.7, 99.6, 97.5, 84.0, 70.7, 67.7,
3
3
7.82 (d, J = 7.7 Hz, 2H), 7.47 (t, J = 7.5 Hz, 1H), 7.39 (d, J =
7.6 Hz, 2H), 7.23-7.14 (m, 2H), 7.08 (t, J = 8.6 Hz, 2H), 7.01-
6.88 (m, 3H), 6.68 (s, 1H), 5.21 (s, 2H), 4.63 (s, 4H), 3.86 (t, J
1
6
1.0, 55.7, 24.9.
=
7.1 Hz, 2H), 1.80 (dd, J = 14.2, 7.3 Hz, 2H), 1.46 (dt, J =
SABC2 was prepared in a similar way as a yellow solid (149
mg, 86% yield). HRMS (ESI) m/z calcd for C29
1
3
1
4.0, 7.1 Hz, 2H), 1.34 (s, 12H), 0.97 (t, J = 7.3 Hz, 3H). C
NMR (100 MHz, CDCl ) δ 162.7, 162.5, 156.7, 150.4, 148.4,
42.2, 139.0, 135.2, 129.5, 127.8, 127.5, 126.5, 126.2, 124.0,
H30BNO
6
+
1
3
[
M+Na] : 522.2058; found 522.2039. H NMR (400 MHz,
CDCl ) δ 8.87 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 9.1 Hz, 1H),
.80 (dd, J = 15.7, 7.2 Hz, 3H), 7.75 (d, J = 8.4 Hz, 1H), 7.56
t, J = 7.6 Hz, 1H), 7.41 (dd, J = 17.1, 7.6 Hz, 3H), 7.27 (d, J =
.6 Hz, 1H), 5.39 (s, 2H), 4.61-4.41 (m, 2H), 4.12 (q, J = 7.1
1
1
8
3
23.0, 121.4, 121.0, 116.2, 113.0, 112.4, 102.0, 101.8, 99.9,
3.9, 70.6, 63.9, 62.6, 48.4, 28.4, 24.9, 20.1, 13.8.
7
(
8
Synthesis of Compound MC. Compound MC was synthe-
13
Hz, 1H), 3.97 (s, 2H), 1.34 (s, 12H). C NMR (100 MHz,
CDCl ) δ 162.5, 155.5, 152.2, 139.5, 135.2, 133.9, 131.7,
sized for the mechanism study according to the literature
4
5
3
method (Scheme 2).
128.8, 128.7, 128.1, 126.4, 124.8, 123.5, 115.2, 115.1, 114.3,
109.6, 84.0, 71.2, 68.1, 60.9, 24.9.
RESULTS AND DISCUSSION
SABC3 was prepared in a similar way as a yellow solid (149
mg, 85% yield). HRMS (ESI) m/z calcd for C25
H28BNO
6
Design and Synthesis of Probes CSU1-3. The rational de-
+
1
[M+Na] : 472.1907; found 472.1869. H NMR (400 MHz,
CDCl ) δ 8.87 (s, 1H), 8.31 (d, J = 7.8 Hz, 1H), 7.84 (d, J =
sign of the fluorescent probes is carried out by connecting a
fused phenothiazine-based coumarin and a precursor of an-
3
3
ACS Paragon Plus Environment

Analytical Chemistry
Page 4 of 10
other coumarin to form a single molecule CSU1-3, respective- induces the formation of coumarin MC to turn on a new blue
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ly (Scheme 1). The fused phenothiazine-based coumarin serv-
ers as a fluorophore as well as the chemoselective HClO oxi-
dizable sulfide moiety; and a coumarin precursor is designed
fluorescence while remaining the red fluorescence from com-
pound P2. Such a change can offer a method to determine
H
2
O
2
in a ratiometric manner using the red fluorescence as a
4
6
with boronate ester, a recognition group for H
2
O
2
.
It is ex-
built-in standard. With respect to the co-existence of H and
2
O
2
pected that phenothiazine-based coumarin possesses a long
wavelength emission, a relatively high quantum yield, a large
HClO, probe CSU1 can undergo these two independent reac-
tions to generate fluorophores P3 and MC regardless of the
47-49
stokes shift and good photo-stability.
HClO-mediated oxi-
addition order of H
green and blue fluorescence signals at the same time. When
adding H first, and then HClO to the solution of probe
2
2
O and HClO, which gives rise to mixed
dation of the ‘S’ atom can significantly alternate the ICT (in-
tramolecular charge transfer) effect and thereby resulting in a
ratiometric fluorescence change from red to green (Scheme 2).
2
O
2
CSU1, a fluorescence colour change from red to red-blue and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The precursor of the coumarin carrying with a H
2
O
2
-sensitive
to green-blue, would be observed. In a reverse addition order
boronate ester can be transferred into a blue-emitting in situ
(HClO first, and then H
green and to green-blue.
2
2
O ), the phenomena would be red to
5
0
formed coumarin in the presence of H
2
2
O through an oxida-
tion-cyclization cascade process, releasing the phenothiazine-
based coumarin at the same time. It’s noteworthy that these
three coumarins, phenothiazine-based coumarin and its oxi-
dized derivative, the newly formed coumarin, shall have well-
separated emissions to realize the ratiometric detection of
HClO and H
2
2
O by a single molecule. With these considera-
tions in mind, we designed three potential fluorescent probes
differing in coumarin precursors, CSU1-3, and wish to obtain
a bifunctional and dual-ratiometric fluorescent probe for HClO
and H
2
2
O .
Scheme 2. The proposed sensing mechanism of probe CSU1 for
HClO, H and HClO/H with distinct fluorescence readouts.
2
O
2
2 2
O
Encouraged by the rational design strategy, we synthesized
three probes, CSU1-3 (Scheme 1) and their optical properties
toward HClO and H
biological systems.
2
2
O were investigated in chemical and
The synthesis of probes CSU1-3 is described in Scheme 1.
Phenothiazine-based salicylaldehyde (compound 1) condenses
with malonate and then undergoes a cyclization reaction to
yield phenothiazine-based coumarin ester (compound 2). Phe-
nothiazine-based coumarin acid (compound 3) is obtained via
NaOH mediated saponification of compound 2 and subsequent
acidification with dilute aqueous HCl solution. Cyanoacetic
ester (compound 5) is synthesized by a Fisher esterification of
cyanoacetic acid (compound 4). The synthesis of SABC1-3
series can be achieved from corresponding salicylic aldehydes
SA1-3 via etherification and Knoevenagel condensation. Fi-
nally coupling of the two fragments 3 and SABC1-3 in the
presence of EDCI as coupling reagent delivers targets CSU1-3.
Scheme 1. Chemical structures and synthetic approaches to
probes CSU1-3. (a) piperidine, CH CH OH, rt for 2 h, 63% yield;
(b) NaOH, H O reflux for 1h, HCl, pH 3.0-4.0, 91% yield; (c)
TsOH, toluene, 110 °C for 4 h, 56% yield; (d) K CO , CH CN,
reflux for 5 h, 83-85% yield; (e) piperidine, CH CH OH, 25 °C
for 4 h, 79-86% yield; (f) EDCI, DMAP, CH Cl , 25 °C for 5 h,
1-77% yield.
1
13
These compounds are fully characterized by H and C NMR
and HRMS with >95% purify for the study of their properties
in chemical and biological systems.
3
2
2
2
3
3
3
2
Sensing Property of Probe CSU1. We took probe CSU1
as a representative to thoroughly investigate its optical proper-
2
2
6
ties toward HClO or/and H
First, we performed the absorption spectra of probe CSU1
in response to HClO and H in order to obtain appropriate
2
O
2
.
Taking probe CSU1 as an example, it is expected that it
emits a red signal (Scheme 2). HClO oxidizes the phenothia-
zine moiety to convert probe CSU1 into P1, which makes a
fluorescence change from red to green in a ratiometric man-
2
O
2
excitation wavelength for fluorescence study. Probe CSU1
exhibited two main absorption bands centred at 374 and 450
nm, respectively (Figure S1), which were characteristics of
4
4
4, 49
ner.
In contrast, the treatment of probe CSU1 with H
2
O
2
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Analytical Chemistry
phenothiazine-based coumarin and the precursor of another
coumarin, respectively. In the presence of H , the absorp-
Last, we explored probe CSU1 to sense both H
HClO (Figure 2). The mixture of probe CSU1 with H
hibited two kinds of fluorescence signals: red with λmax = 640
nm (λex = 440 nm) and blue with λmax = 409 nm (λex = 376 nm),
while the mixture of probe CSU1 with HClO only showed
green fluorescence with λmax = 520 (λex = 376 nm). The suc-
2
O
2
and
ex-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
O
2
2
O
2
tion band at 374 nm disappeared and a new 350 nm band oc-
curred, suggesting the formation of new coumarin MC. The
addition of HClO to the solution of probe CSU1 resulted in
the blue-shift in absorption from 450 nm to 384 nm, which
was in agreement with the oxidation phenomenon of pheno-
thiazine-based coumarin.
cessive addition of H
CSU1 resulted in a mixed blue and green fluorescence signals
376 nm excitation). The same result was obtained when probe
CSU1 was treated with these two species in a reverse order.
It’s noted that both of H and HClO could be detected quan-
titatively regardless of the addition order. For example, in the
addition order of H
2
O
2
and HClO to the solution of probe
(
Second, we studied the fluorescence responses of probe
CSU1 to H
2
2
O and HClO, respectively (Figure 1). Probe
2
O
2
CSU1 itself exhibited a red fluorescence with a peak at 640
nm under the excitation at 440 nm, and was weakly fluores-
cent when excited at 376 nm. Upon the incremental addition
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
O and HClO, the ratio of I520 nm/I409 nm
exhibited a good linear relationship with concentration of
HClO in a range of 100.0-275.0 μM. The ratio of I409 nm/I520 nm
of H
2
2
O to the solution of probe CSU1, the emission band at
6
40 nm remained stable under a 440 nm excitation, and a new
was linear with the concentration of H
when the addition order was HClO and H
clearly demonstrated that probe CSU1 could simultaneously
detect H and HClO through the dual-emission detection
2
O
2
(75.0-425.0 μM)
blue emission band centred at 409 nm appeared under a 376
nm excitation and increasingly enhanced. To our delight, the
intensity of red fluorescence from phenothiazine-based cou-
marin was nearly unchanged, which could serve as a built-in
2
O
2
. These results
2
O
2
without interference from the presence of each other.
reference signal for probe CSU1 to detect H
termining the ratio of fluorescence intensity at 409 nm and
40 nm (I409 nm and I640 nm). It was seen in Figure 1C that the
ratio of I409 nm/I640 nm increased paralleling with H concen-
tration. A linear relationship between the ratio of and the con-
centration of H (75.0-425.0 μM) was observed with a low
2
O
2
through de-
6
2
O
2
2
O
2
detection limit of 15 nM. When HClO was added to the solu-
tion of probe CSU1, we observed: i) a decrease in the 640 nm
emission peak and a new green emission band centred at 520
nm under a 440 nm excitation; and ii) only the green emission
at 520 nm notably increased under a 376 nm excitation (Figure
1D). The fluorescence intensity ratio of I520/I640 was linear to
the concentration of HClO (Figure 1F) in the concentration
range of 100.0-275.0 μM with a low detection limit (13 nM).
It’s noted that these three fluorescence signals (blue, green and
red) for probe CSU1 were well separated (over 110 nm differ-
ence), and thus avoiding the mutual interference in the detec-
tion H
2
O
2
and HClO. As a consequence, probe CSU1 could
and HClO with
sensitively and selectively respond to H
2
O
2
distinct fluorescence signal patterns. The features of probe
CSU1 made it suitable for the simultaneous, qualitative and
Figure 2. Fluorescence spectra (A) and fluorescence intensity
raitio (I520 nm/I409 nm) (B) of probe CSU1 (5.0 μM) treated with
100.0 equiv. H O and then incubated with various amounts of
2 2
HClO (0.0-100.0 equiv.). Fluorescence spectra (C) and fluores-
cence intensity ratio (I409 nm/I520 nm) (D) of probe CSU1 (5.0 μM)
treated with 80.0 equiv. HClO and then incubated with various
amounts of H O (0.0 -120.0 equiv.). Inset (B): the linear relation-
2 2
ship between I409 nm/I520 nm and the concentration of HClO. Excita-
tion wavelength: 376 nm.
quantitative detection of H
manner.
2
2
O and HClO in a ratiometric
Sensing Property of Probe CSU2. We then investigated
the spectral properties of probe CSU2 in response to H
2
O and
2
HClO (shown in Figure S7). In the presence of H , the blue
2
O
2
fluorescence at 450 nm (λex = 403 nm) increased while the red
fluorescence at 640 nm (λex = 440 nm) remained unchanged
Figures S7A and S7B), which was quite similar to the fluo-
(
rescence performance of probe CSU1. The new blue-emitting
coumarin HC, was isolated from the reaction mixture and was
validated by TLC, optical and HRMS analysis (Figures S8, S9,
S40 and S41). With the addition of HClO, the fluorescence of
probe CSU2 at 640 nm gradually disappeared, and a new
green fluorescence occurred when excited at 440 nm (Figure
S7E). Unexpectedly, under a 403 nm excitation, the blue fluo-
rescence at 450 nm was also observed in the solution of
Figure 1 Fluorescence spectra of probe CSU1 (5.0 μM) with the
addition of different concentrations of H (0.0–100.0 equiv.) (A,
2
O
2
B) and HClO (0.0–80.0 equiv.) (D, E) with the excitation at 440
nm and 376 nm, respectively. (C) The ratio of I409 nm/I640 nm vs the
concentration of H
ration of I409 nm/I640 nm and the concentration of H
2
O
2
, inset: the linear relationship between the
. (F) The ratio
2
O
2
of I520 nm/I640 nm vs the concentration of HClO, inset: the linear
relationship between I520 nm/I640 nm and the concentration of HClO.
5
probe CSU2 with HClO, indicating the formation of the new
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Analytical Chemistry
Page 6 of 10
coumarin, a cyclization product (Figure S7D). This result sug-
gested HClO could oxidize both phenoxazine moiety and
Selectivity and Kinetic Studies. To verify the selectivity of
probe CSU1 towards H and HClO over other related ROS,
probe CSU1 was incubated with various biologically relevant
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
O
2
5
1
cleave the borate ether bond as well. The successive addition
of H and then HClO to the solution of probe CSU2 still
‾
•
1
- •
•
•
2
O
2
species including NO, ONOO , OH, O
2
, O , ROO , t-BuO .
2
induced a fluorescence color change from red to blue-red, and
finally to blue-green (Figure S7C). In a reverse addition order
The fluorescence signals were collected through different col-
or channels. As seen in Figure 3A, under the excitation of 376
of HClO and then H
changed from red to blue-green toward HClO and remained
unchanged with the further addition of H because both of
the recognition sites were sensitive to HClO (Figure S7E). As
a result, probe CSU2 could still detect H and HClO indi-
vidually: red to blue-red fluorescence change for H , and
red to blue-green fluorescence change for HClO, but was una-
ble to sense H and HClO successively both in forward and
2
O
2
, however, fluorescence color first
nm, only H
2
2
O induced a remarkable fluorescence enhance-
ment at 409 nm (40-folds) while other analytes hardly caused
fluorescence intensity changes even within a 100 min incuba-
tion time. Similarly, under a 440 nm excitation, only HClO
resulted in a fluorescence enhancement at 520 nm and a de-
cline at 640 nm, while negligible fluorescence changes were
observed on the two emission bands for other test species
(Figure 3B). These experimental results demonstrated that
2
O
2
2
O
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
O
2
2
O
2
reverse addition orders.
probe CSU1 could be used to selectively detect H
2
2
O and
HClO in a ratiometric pattern.
Sensing Property of Probe CSU3. Finally, we investigated
the optical properties of probe CSU3 in a similar manner. As
shown in Figure S10, the addition of H
probe CSU3 didn’t affect the red fluorescence at 640 nm (λex
440 nm), and resulted in an 8-fold enhancement of the blue
2
2
O to the solution of
=
fluorescence at 420 nm (λex = 340 nm) (Figures S10A and
S10B). The response of probe CSU3 towards HClO was simi-
lar to that of probe CSU1 (Figures S10D and S10E), inducing
a red to green fluorescence change. Thus, probe CSU3 could
also selectively detect HClO and H
2
2
O individually. Consider-
ing the low fluorescence quantum yield of the new formed
blue-emitting coumarin, 2-oxo-2H-chromene-3-carbonitrile,
5
2
Figure 3. (A) Fluorescence spectra of probe CSU1 (5.0 μM) in
the presence of H (500.0 μM) and various interfering analytes
500.0 μM), excited at 376 nm; (B) Fluorescence spectra of probe
(Φ < 0.01) , probe CSU3 was not the ideal fluorescent probe.
f
2
O
2
The above screening studies of probes CSU1-3 indicated
that probe CSU1 exhibited the best performance. Hence, we
chose it for following studies.
(
CSU1 (5.0 μM) in the presence of HClO (400.0 μM) and the in-
terfering analytes (400.0 μM), excited at 440 nm. The interfering
-
•
1
- •
•
•
Mechanistic Studies. Probe CSU1 consists of two sensing
segments: SABC1 and compound 2 (Scheme 1). It is expected
2 2
analytes include NO, ONOO , OH, O , O , ROO and t-BuO .
that SABC1 derived segment would react with H
2
O
2
to form a
pH Effect. In order to investigate whether probe CSU1 is
capable of sensing H and HClO under physiological condi-
blue fluorescent coumarin, MC, while compound 2 would
response to HClO to form the green fluorescent phenothia-
zine-based coumarin. To confirm the sensing mechanism of
2
O
2
tions, we then studied pH effect on the fluorescence perfor-
mance of this probe. As shown in Figures S11 and S12, the
fluorescence behavior of probe CSU1 remained unchanged
within a wide pH range. In the presence of HClO, the probe
probe CSU1 to H
product MC and the mixture of SABC1 and H
tigated. It was shown in Figure S2 that MC and the mixture
SABC1 with H had nearly identical absorption and fluo-
2
O
2
, the optical properties of the expected
2
O
2
were inves-
showed a striking enhancement in fluorescence ratio (I520/I640
)
2
O
2
between pH 6.0 to 10.0. The mixture of H with probe
2
O
2
rescence spectra. Meanwhile, the fluorescence response of
compound 2 toward HClO was studied to prove the reaction
between probe CSU1 and HClO. Compound 2 displayed a
fluorescence change from red to green in response to HClO
(Figure S3), which was similar to the fluorescence behavior of
probe CSU1 before and after the treatment of HClO (Figure
CSU1 exhibited a large ratio of fluorescence intensities at 409
nm and 640 nm (I409/I640) between pH 6.0 to 10.0. These re-
sults indicated that probe CSU1 has an excellent stability and
exhibits a good performance to sense HClO and H
iological environments.
2
2
O in phys-
In Situ Monitoring of HClO Produced from H
2
O
2
and
Cl in Solution. It’s known that HClO can be endogenously
produced from the reaction of chloride ion with H cata-
1
D). To obtain more concrete evidence for the sensing mecha-
nism, mass spectral analysis was performed on probe CSU1
Figures S28-S30). As shown in Figure S28, the reaction mix-
ture of probe CSU1 with HClO had a mass peak at m/z =
67.2746, which well matched the exact molecular mass of the
-
2
O
2
(
lysed by MPO in biological systems. Thus, we used probe
CSU1 to investigate the above process in vitro. As illustrated
8
-
in Figure 4A, when H
2
2
O was added into the MPO/CSU1/Cl
+
anticipated compound P1 ([M+Na] = 867.2729) (Scheme 1).
For the case of probe CSU1 reacting with H , two peaks at
system, the fluorescence at 409 nm (λex = 390 nm) rose rapidly
in the beginning and reached a plateau within 15 min. At the
same time, the fluorescence at 520 nm (λex = 390 nm) occurred
and significantly increased, suggesting the generation of HClO
2
O
2
m/z = 434.1033 and 224.0320 were found, which correspond-
ed to the expected compounds P2 and MC, respectively (Fig-
ure S29). Moreover, the HR-MS spectrum of probe CSU1
in the MPO-catalysed process. However, the addition of H
2
O
2
with the addition of H
reverse reaction orders gave two mass peaks at 450.1000 and
24.0320, corresponding to the expected compounds P3 and
2
O
2
and HClO in both the forward and
-
to the CSU1/Cl system without MPO did not induce any fluo-
rescence at 520 nm, and only resulted in a fluorescence en-
hancement at 409 nm (Figure 4B). These results showed probe
2
MC, respectively (Figure S30). The above results clearly sup-
ported the working hypotheses, as illustrated in Scheme 1.
CSU1 was capable of monitoring the in-situ generation of
-
HClO from MPO/H
2
O
2
/Cl system.
6
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Analytical Chemistry
Figure 5. Bright and fluorescence images of living MCF-7 cells.
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5
6
7
8
9
(a-e) Cells only treated with probe CSU1 (5.0 μM) for 20 min. (f-j)
Cells incubated with probe CSU1 (5.0 μM) for 20 min and then
treated with HClO (400.0 μM) for another 20 min. (k-o) Cells
incubated with probe CSU1 (5.0 μM) for 20 min and then treated
with H
2
O
2
(500.0 μM) for another 1 h. (p-t) Cells treated with
(500.0
probe CSU1 (5.0 μM) for 20 min, incubated with H
2
O
2
μM) for 1 h, and further loaded with HClO (400.0 μM) for 20 min.
(u-y) Cells treated with probe CSU1 (5.0 μM) for 20 min, incu-
bated with HClO (400.0 μM) for 20 min, and further loaded with
H
3
2
O
2
(500.0 μM) for 1 h. Blue channel: 420-500 nm (excited at
55-385 nm). Green channel: 515-580 nm (excited at 390-420
nm). Red channel: 570-650 nm (excited at 435-460 nm). Scale bar:
Figure 4. (A) Time-dependent fluorescence of probe CSU1
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0
-
(
2 2 2 2
5.0μM) in MPO/H O /Cl system (500.0μM H O , 0.5 U/mL
2
0 μm.
MPO, 2.0 mM NaCl). (B) Time-dependent fluorescence of probe
-
CSU1 (5.0 μM) in H
2 2 2 2
O /Cl system (500.0 μM H O , 2.0 mM
NaCl). Excitation wavelength: 390 nm.
Fluorescence Imaging of Intracellular HClO Produced
by MPO System. Finally, we monitored the endogenous
-
Fluorescence Imaging of H
Encouraged by the favourable properties of probe CSU1 in
detecting of H and HClO in vitro, we determined to assess
its capability to image intracellular H and HClO. The cyto-
2
O
2
and HClO in Living Cells.
HClO generation from H
2
O and Cl in the presence of MPO
2
enzyme in living cells. The treatment of MCF-7 cells with
probe CSU1 and NaCl only triggered strong fluorescence sig-
nals in the red channel (Figures 6a-6e). When cells were treat-
2
O
2
2
O
2
toxicity of probe CSU1 was tested by the MTT assay using
MCF-7 cells and it was found that CSU1 had a low toxicity
ed with probe CSU1, H
2
2
O and NaCl, bright fluorescence
emerged in both the blue and red channels (Figures 6f-6j).
When cells were pretreated with probe CSU1, NaCl and MPO,
(
Figure S15). We collected the fluorescence signals in living
cells from three channels to detect H and HClO. Cells gave
2
O
2
and then incubated with H
2
2
O , strong fluorescence signals in
off strong fluorescence in the red channel (570-650 nm) when
only treated with probe CSU1 (Figure 5d). When CSU1-
treated cells were further incubated with HClO, a strong fluo-
rescence in the green channel (515-580 nm) was seen from
inside cells (Figure 5h). As expected, we observed strong fluo-
rescent signals in both blue (420-500 nm) and red (570-650
nm) channels upon the incubation of CSU1-treated cells with
the green channel, moderate fluorescence in the blue channel
and very weak signals in the red channel were observed (Fig-
ures 6k-6o).
H
2
O
2
(Figures 5l and 5n). We then further tested the fluores-
cence behaviour of probe CSU1 in imaging intracellular H
2
O
2
and HClO in living cells in different addition orders. Cells
were successively stained with probe CSU1 for 20 min, HClO
for 20 min and H
signals appeared from both blue and green channels (Figures
q and 5r). In the imaging experiment with a reverse addition
2
O
2
for another 1 h, and bright fluorescence
5
order of these two analytes, same results were observed (Fig-
ures 5v and 5w). These results demonstrated that probe CSU1
could selectively image intracellular H
through three different channels.
2
O
2
and/or HClO
Figure 6. Bright and fluorescence images of living MCF-7 cells.
(
a-e) Cells incubated with probe CSU1 (5.0 μM) and NaCl (2.0
mM) for 20 min. (f-j) Cells incubated with probe CSU1 (5.0 μM)
and NaCl (2.0 mM) for 20 min and then treated with H (500.0
2
O
2
μM) for another 30 min. (k-o) Cells incubated with CSU1 (5 μM),
NaCl (2.0 mM) and MPO (0.5 U/mL) for 20 min, and further
treated with H O (500.0 μM) for 30 min. Blue channel: 420-500
2 2
nm (excited at 355-385 nm). Green channel: 515-580 nm (excited
at 390-420 nm). Red channel: 570-650 nm (excited at 435-460
nm). Scale bar: 20 μm.
Lastly, probe CSU1 was used to monitor the real-time en-
-
dogenous generation of HClO from H
2
O
2
and Cl in the pres-
ence of MPO enzyme in living cells. As shown in Figure 7 and
the dynamic video (seen in SI), the original bright red fluores-
cence from cells treated with probe CSU1, NaCl and MPO
gradually decreased after the addition of H
2
O . At the same
2
time, green fluorescence occurred and was enhanced in a time-
dependent manner. These experiments clearly indicated the
-
fast generation of endogenous HClO from MPO/H
system in living cells.
2
2
O /Cl
7
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(4) Winterbourn, C. C. Reconciling the chemistry and biology of
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Figure 7. Real-time fluorescence images of MCF-7 cells (pre-
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channel: 570-650 nm (excited at 435-460 nm). Green channel:
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2 2
O . Red
(
0
1
2
3
4
5
6
7
8
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2
3
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CONCLUSION
50-62.
In summary, a new dual-ratiometric fluorescent probe has
(11) Wei, H.; Shang, T.; Wu, T.; Liu, G.; Ding, L.; Liu, X. Construc-
been built for simultaneously detecting HClO and H
solution and living cells for the first time. This probe displays
high selectivity and sensitivity for H and HClO. Further-
more, this probe has been successfully used to image H
2
O
2
in
tion of an ultrasensitive non-enzymatic sensor to investigate the dy-
namic process of superoxide anion release from living cells. Biosens.
Bioelectron. 2018, 100, 8-15.
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immunologist's guide to reactive oxygen species. Nat. Rev. Immunol.
2
O
2
2
O
2
and HClO in living cells using different fluorescence signal
2
013, 13(5), 349-361.
combinations. Importantly, it can be applied to monitor the
-
(13) Pussard, E.; Chaouch, A.; Said, T. Radioimmunoassay of free
plasma metanephrines for the diagnosis of catecholamine-producing
tumors. Clin. Chem. Lab. Med. 2014, 52(3), 437-444.
process of endogenous generation of HClO from H
2
2
O /Cl
/
MPO system. It is expected that the probe holds a geat
potential as a useful tool for biomedical scientists to
investigate and dissect the correlation of HClO and H en-
(
14) Barnham, K. J.; Masters, C. L.; Bush, A. I. Neurodegenerative
diseases and oxidative stress. Nat. Rev. Drug Discovery 2004, 3, 205-
14.
2
O
2
gaged biological processes in physiological and pathological
conditions.
2
(15) Lin, M. T.; Beal, M. F. Alzheimer's APP mangles mitochondria.
Nat. Med. 2006, 12(11), 1241-1243.
(16) Shah, A. M.; Channon, K. M. Free radicals and redox signalling
in cardiovascular disease. Heart 2004, 90(5), 486-487.
ASSOCIATED CONTENT
Supporting Information
(
17) Yue, Y.; Huo, F.; Yin, C.; Escobedo, J. O.; Strongin, R. M. Re-
Chemical structures of compounds, additional optical spectral
cent progress in chromogenic and fluorogenic chemosensors for hy-
pochlorous acid. Analyst 2016, 141(6), 1859-1873.
1
13
data, H NMR, C NMR and HRMS spectra of compounds. The
Supporting Information is available free of charge via the Internet
at http://pubs.acs.org.
(18) Whiteman, M.; Cheung, N. S.; Zhu, Y.-Z.; Chu, S. H.; Siau, J. L.;
Wong, B. S.; Armstrong, J. S.; Moore, P. K. Hydrogen sulphide: a
novel inhibitor of hypochlorous acid-mediated oxidative damage in
the brain? Biochem. Biophys. Res. Commun. 2005, 326(4), 794-798.
(19) Pattison, D. I.; Davies, M. J. Kinetic analysis of the reactions of
hypobromous acid with protein components:ꢀ implications for cellular
damage and use of 3-bromotyrosine as a marker of oxidative stress.
Biochem. 2004, 43(16), 4799-4809.
AUTHOR INFORMATION
Corresponding Authors
* Fax: +86-731-88836954; Tel: +86-731-88836954; E-mail:
xzsong@csu.edu.cn.
(20) Li, G.; Lin, Q.; Sun, L.; Feng, C.; Zhang, P.; Yu, B.; Chen, Y.;
*
Fax: +1-520-626-2466; Tel: +1-520-626-1764; E-mail：
Wen, Y.; Wang, H.; Ji, L.; Chao, H. A mitochondrial targeted two-
photon iridium (III) phosphorescent probe for selective detection of
hypochlorite in live cells and in vivo. Biomater. 2015, 53, 285-295.
wwang@pharmacy.arizona.edu.
Notes
(21) Pattison, D. I.; Hawkins, C. L.; Davies, M. J. Hypochlorous acid-
The authors declare no competing financial interest.
mediated oxidation of lipid components and antioxidants present in
low-density lipoproteins:ꢀ absolute rate constants, product analysis,
and computational modeling. Chem. Res. Toxicol. 2003, 16(4), 439-
449.
ACKNOWLEDGMENT
This work was supported by the National Natural Science Foundation
of China (No. U1608222), the State Key Laboratory of Fine Chemi-
cals (KF1606), the State Key Laboratory of Chemo/Biosensing and
Chemometrics (2016005) and the Fundamental Research Funds for
the Central Universities of Central South University (No. 202045010).
(22) Li, H.; Cao, Z.; Ray Moore, D.; Jackson, P.; Barnes, S.; Lam-
beth, J.; J Thannickal, V.; Cheng, G. Microbicidal activity of vascular
peroxidase 1 in human plasma via generation of hypochlorous acid.
Infect. Immun. 2012，80(7), 2528-2537.
(23) Hawkins, C. L.; Pattison, D. I.; Davies, M. J. Hypochlorite-
induced oxidation of amino acids, peptides and proteins. Amino Acids
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