Synthesis and activity of a coumarin-based fluorescent probe for hydroxyl radical detection

Article abstract of DOI:10.1002/bio.3728

As a type of reactive oxygen species (ROS), hydroxyl radical (·OH) is closely associated with many kinds of diseases. The present study aimed to develo p a novel OH fluorescent probe based on coumarin, a new compound that has not been previously reported.

Full text of DOI:10.1002/bio.3728

Received: 13 August 2019
DOI: 10.1002/bio.3728
Revised: 30 September 2019
Accepted: 9 October 2019
R E S E A R C H A R T I C L E
Synthesis and activity of a coumarin-based fluorescent probe
for hydroxyl radical detection
Bin Zhang1 | Lijing Xu2 | Yindi Zhou1 | Weijian Zhang3 | Yuanhong Wang1
Yu Zhu4
|
1Department of Horticulture, Tianjin
Abstract
Agricultural University, Tianjin, China
2Department of Neurosurgery, Tianjin
Hospital, Tianjin, China
As a type of reactive oxygen species (ROS), hydroxyl radical (ꢀOH) is closely associ-
ated with many kinds of diseases. The present study aimed to develo p a novel OH
fluorescent probe based on coumarin, a new compound that has not been previously
reported. This probe exhibited good linear range and selectivity for ꢀOHl, and is able
to avoid interference from some metal ions and other kinds of ROS (H2O2, O2.-, 1O2,
and HClO). Meanwhile, this probe has been used to evaluate the ꢀOH-scavenging
efficiency of different compounds, such as isopropyl alcohol, cytosine, uracil, Tempo,
Glutathione (GSH), and dimethyl sulfoxide (DMSO). Therefore, the present study
shows that this probe not only can effectively measure the level of ꢀOH, but also can
assess the ꢀOH-scavenging efficiency of different compounds. Furthermore this cur-
rent study suggested that following further optimization, this probe may be poten-
tially applied in the diagnosis of oxidative stress in human body.
3Graduate School, Tianjin Medical University,
Tianjin, China
4Department of Clinical Laboratory, Huanhu
Hospital, Tianjin, China
Correspondence
Yuanhong Wang, Department of Horticulture,
Tianjin Agricultural University, 22 Jinjing
Road, Tianjin 300384, China.
Email: wangyh@tjau.edu.cn
Yu Zhu, Department of Clinical Laboratory,
Tianjin Huanhu Hospital, 6 Jizhao Road,
Tianjin 300350, China.
Email: zhuyutj@126.com
Funding information
K E Y W O R D S
Tianjin 131 Creative Talents Cultivation
Project; Tianjin Municipal Special Program of
Talents Development for Excellent Youth
Scholars, China, Grant/Award Number:
TJTZJH-QNBJRC-2-9; Tianjin Science and
Technology Key Project on Chronic Diseases
Prevention and Treatment, Grant/Award
Number: 16ZXMJSY-00020; Fund plan for
scientific research and development of Tianjin
Agricultural College, Grant/Award Number:
20190105; National Key Research and
Development Project of China, Grant/Award
Number: 2017YFD0201102
coumarin derivatives, fluorescent probe, hydroxyl radical, scavenging efficiency
1
|
INTRODUCTION
and even dissolution of lipid membranes.[9–12] ꢀOH can also change
the activity of phosphoric acid and induce the release of arachidonic
The hydroxyl radical (ꢀOH) is the most representative free radical in
the human body,[1,2] and it is closely related to the occurrence and
development of various diseases because of its strong oxidation abil-
ity, high reaction activity, and short half-time.[3–8] For example, ꢀOH
can induce a series of free radical reactions, causing lipid peroxidation
of membrane unsaturated fatty acids, resulting in the local destruction
acid (AA) from membrane phospholipids.[13] AA forms slow-reacting
substances and histamine under the action of lipid peroxidase,
resulting in increased thromboxane A2 (TXA2), microthrombosis, and
impaired microcirculation.[14] Therefore, to prevent the damage cau-
sed by ꢀOH, it is necessary to detect ꢀOH in vivo in a timely and effec-
tive manner.
As a new method to detect reactive oxygen species (ROS) in living
organisms, the development and application of fluorescent probes
Bin Zhang and Lijing Xu made an equal contribution.
Luminescence. 2019;1–7.
wileyonlinelibrary.com/journal/bio
© 2019 John Wiley & Sons, Ltd.
1

2
ZHANG ET AL.
have received extensive attention from experts in medicine, biology,
and chemistry.[15] As the existence of ꢀOH is very short, it is a chal-
lenge for scientists to develop fluorescent probes that can respond
quickly with ꢀOH. At present, the usual ꢀOH probes do not directly
detect ꢀOH, but need intermediates, such as DMSO.[16] Therefore, the
detection efficiencies of these probes are affected by the reaction
rates of ꢀOH and intermediates. This limitation requires researchers to
develop new reactive probes that can directly detect ꢀOH. We report
here the development of a novel fluorescence probe for ꢀOH, a
coumarin-based fluorescent probe named 5-isopropyl-3-(3-me-
thoxyphenyl)-8-methyl-2H– chromen-2-one (MOPC), which can
specifically detect ꢀOH in terms of an increase of fluorescence at
364 nm (λex, 245 nm), and the scavenging ability of various chemicals
to ꢀOH was further evaluated by MOPC.
1H-nuclear magnetic resonance (1H-NMR; 400-MR DD2;
400 MHz; Agilent Technologies, Inc.) and an electron ionization–
high resolution mass spectrometry (EI-HRMS; Micromass® GCT
Premier™; Waters Corporation). The target compound was
a
coumarin-based fluorescent probe and named 5-isopropyl-3-
(3-methoxyphenyl)-8-methyl-2H-chromen-2-one. All above reagent
was purchased from Sigma-Aldrich. The synthesis pathway is
shown Figure 1.
2.2
|
Fluorescence determination of MOPC for
different concentrations of ꢀOH
The appropriate excitation wavelength (EX, 245 nm) and emission
wavelength of MOPC(10 μM) was determined using RF-5301PC fluo-
rescence spectrophotometer (Olympus Corporation, Japan) and
Cytation 5 imaging reader. The ꢀOH was prepared using Fenton
reagent (FeSO4/H2O2 = 1/5, molar ratio, regarding [Fe2+] = [ꢀOH]).
The mechanism of the Fenton reaction is shown in eqn (1). To make
the reaction proceed smoothly, an addition system was applied. The
specific steps are as follows: firstly, H2O2 was added dropwise to the
MOPC solution and, after 10 sec, FeSO4 was added dropwise to the
above mixture, and the fluorescence spectrum was recorded after
5 min with an excitation at 245 nm.
2
|
EXPERIMENTAL
2.1
|
The synthesis of MOPC
Here, 30.0 g (200 mmol) 5-isopropyl-2-methylphenol was dissolved
in 200 ml of toluene under an argon atmosphere, and SnCl4 5.2 g
(20 mmol) and triethylamine (Et3N) 8.1
g (80 mmol) were
sequentially added using pipettes. The mixture was stirred for
20 min at room temperature. Paraformaldehyde (CH2O)n 13.2 g
(439 mmol) was then added, and the mixture was heated and
maintained at 100ꢁC for 8 h. After cooling to room temperature,
dilute hydrochloric acid was added to adjust the pH to 2. Ethyl
acetate was added, and the phases were separated. The aqueous
phase was extracted once again with Ethyl acetate (EA). The com-
bined organic phases were extracted once with a saturated sodium
chloride solution, dried with anhydrous magnesium sulfate, and fil-
tered with suction. The solvent was evaporated to give a purple-
black liquid that was subjected to column chromatography followed
by pentane/ether = 200/1) to provide the yellow liquid compound
2-hydroxy-6-isopropyl-3-methylbenzaldehyde (22.4 g, 125 mmol):
62.8%. Under an argon atmosphere, 10 mmol of the above
2-hydroxy-6-isopropyl-3-methylbenzaldehyde, 30 mmol acetic
2.3
|
Fluorescence determination of coumarin-
based probe for different ions and ROS
The influences of metal ions and ROS (ꢀOH: 80.65 μM Fe-EDTA
and 403.25 μM H2O2; HClO: 1 mM NaClO; H2O2: 1 mM H2O2;
−
O2ꢀ : 0.5 unit/ml xanthine oxidase and 1 mM xanthine; 1O2: 1 mM
NaClO and 1 mM H2O2; KCl: 1 mM KCl; MgSO4: 1 mM, MgSO4;
CaCl2: 1 mM CaCl2; Fe-EDTA: 1 mM Fe-EDTA) were confirmed by
adding them to the fluorescent probe solution (3.6 ml, 10 μM),
separately. The O2.-and 1O2 were prepared in the same procedure
as ꢀOH by the addition system. After the addition, fluorescence
intensity (λex, 245 nm) was recorded at the start (without adding
metal ions or ROS) and 5 min.
anhydride,
20
mmol
triethylamine,
and
11
mmol
2-(3-methoxyphenyl)acetic acid were added to a round-bottomed
flask. The mixture was heated and refluxed at 110ꢁC until thin
layer chromatography (TLC) showed completion of the reaction.
The reaction mixture was poured onto an ice-water mixture with
stirring and was stirred for an additional 20 min. The compound
structure was dissolved by deuterium oxide and analyzed using
2.4
|
Determination of ꢀOH-scavenging abilities of
some chemical entities using coumarin-based probe
Different amounts of isopropyl alcohol, cytosine, uracil, Tempo, GSH,
and DMSO were mixed separately with MOPC (10 μM) in 0.1 mM
potassium phosphate buffer at pH 7.4. Then, 40 μM Fenton reagent
FIGURE 1 Synthesis pathway of
coumarin-based fluorescent probe

ZHANG ET AL.
3
was sequentially added through addition system, and changes in the
fluorescence intensity under each condition were recorded at 5 min
with an excitation at 248 nm and emission at 364 nm.
J = 7.6 Hz, 1H), 7.33–7.32 (m, 2H), 7.21 (d, J = 7.6 Hz, 1H), 7.01 (d,
J = 7.6 Hz, 1H), 3.82 (s, 3H), 3.68–3.61 (s, 1H), 2.37 (s, 3H), 1.26–1.25
(m, 6H); 13C NMR (100 MHz, DMSO-d6) δ 159.3, 158.9, 151.6, 144.7,
137.1, 136.2, 132.8, 129.2, 125.8, 122.2, 121.0, 120.4, 116.4, 113.7,
55.1, 27.2, 23.6, 14.9. HRMS (ESI) m/z calcd for C20H20NaO3
(M + Na)+ 331.1310, found 331.1310. Therefore, the compound was
identified as the target compound.
2.5
|
Statistical analysis
Data are represented as mean standard deviation (SD) and were
analyzed using SPSS 11.0 software (SPSS, Inc.). One-way analysis of
variance (ANOVA) and the Tukey post hoc test was used for general
measurement data. P-value < 0.05 was considered to indicate a statis-
tically significant difference.
3.2
|
The MOPC belongs to a type of turn-on
probe and shows a linear relationship with OH
A good fluorescent probe should be able to respond efficiently to the
3
|
RESULTS AND DISCUSSION
| Structure identification of the probe
target. It can be seen from Figure 2 that with the addition of ꢀOH to
3.1
Yellow solid, melting point: 76–78ꢁC, productivity: 89%. 1H NMR
(400 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.39 (t,
FIGURE 3 Selectivity of the probe for ꢀOH over other biologically
relevant species (ꢀOH: 80.65 μM Fe-EDTA and 403.25 μM H2O2;
−
ClO−: 1 mM NaClO; H2O2: 1 mM H2O2; O2ꢀ : 0.5 unit/ml xanthine
oxidase and 1 mM xanthine; 1O2: 1 mM NaClO and 1 mM H2O2; KCl:
1 mM KCl; MgSO4: 1 mM, MgSO4; CaCl2: 1 mM CaCl2; Fe-EDTA:
1 mM Fe-EDTA). (a) Fluorescence spectrum of coumarin-based
fluorescent probe after adding different ions and ROS with excitation
at 245 nm. (b) Relative fluorescence intensity of coumarin-based
fluorescent probe after adding different ions and ROS, the
fluorescence spectra were recorded at 5 min with an excitation at
245 nm and emission at 364 nm. F0 refers to the fluorescence
intensity at of probe without adding any metal ions and ROS; F refers
to the fluorescence intensity of probe 5 mins after adding the metal
ions or ROS to the solution. The error bars represent the mean errors
from the results of five tests; *P < 0.05 when compared with the ꢀOH
group
FIGURE 2 Fluorescence intensity and linear of coumarin-based
fluorescent probe after adding different concentrations of ꢀOH. (OH
source: Fenton reagent). (a) Fluorescence spectra of MOPC (10 μM)
upon the addition of ꢀOH (0–94.34 μM), each spectrum was recorded
after 5 min in potassium phosphate buffer at pH 7.4, λex = 245 nm.
(b) Linear calibration graphs between fluorescence intensity and ꢀOH
concentration within 13.8–67.57 μM, the fluorescence spectra were
recorded at 5 min with an excitation at 245 nm and emission at
364 nm in potassium phosphate buffer. ꢀ

4
ZHANG ET AL.
the MOPC (10 μM) solution its fluorescence intensity at 433 nm
sharply decreased. At the same time when Fenton reagent added at
approximately 5 μM, a new peak at 364 nm appeared (the detection
limit of the probe was confirmed to be 5 μM). As the Fenton reagent
was continuously added to the MOPC (10 μM) solution, its fluores-
cence intensity at 364 nm increased synchronously. Subsequently,
when the concentration of Fenton reagent reached 94.34 μM in
MOPC solution (10 μM), its fluorescence intensity reached saturation,
which means the fluorescent probe was completely consumed
(Figure 2a), Further research showed that there is a linear relationship
between the ꢀOH and the probe. The regression equation is
y = 0.1869x − 2.123 (where y refers to the concentration of ꢀOH and
x refers to fluorescence intensity) (Figure 2b). Therefore, the results of
this experiment indicated that MOPC belongs to a type of turn-on
probe and the concentration of ꢀOH could be determined by a linear
relationship with fluorescence intensity over a specific concentration
range (75 μM > y > 10 μM).
of ROS. Therefore, to prevent the detection of ꢀOH from being
affected, the selectivity of the probe is crucial. Changes in the fluores-
cence intensity of coumarin-based fluorescent probe after adding dif-
ferent ions.
Note: None indicates the fluorescence intensity of coumarin-
based fluorescent probe coumarin-based fluorescent probe (17 μM).
KCl, MgSO4, CaCl, NaClO, Fe2+
, and H2O indicate the
fluorescence intensity after adding 20 μl 1 mM solution. OH indi-
cates the fluorescence intensity after adding 20 μl 10 mM H2O2
and Fe2+
.
As it shown in Figure 3(a, b), there were no apparent fluorescence
changes at 364 nm (λex, 245 nm) after the addition of species such as
KCl, MgSO4, CaCl2, NaClO, Fe2+, O2
,
1O2, and H2O2. In contrast, the
.-
fluorescence intensity at 364 nm increased greatly upon addition of
Fenton reagent, suggesting that MOPC had good selectivity to ꢀOH in
the presence of some metal ions and ROS.
3.4
|
MOPC can be used as a tool to evaluate the
3.3
|
The probe exhibits good selectivity for ꢀOH
ꢀOH-scavenging ability of compounds
compared with some metal ions and reactive oxygen
species
The simplest way to evaluate ꢀOH-scavenging abilities of substances
is to compare their reaction rate constants in documents. However,
due to differences in detection methods and conditions, even the
reaction rate constants of the same compound are quite different in
A good fluorescent probe should also have a good selectivity. Usually,
in biological environments, ꢀOH coexist with some ions or other kinds
TABLE 1 Second-order rate constants for the reactions of ꢀOH with some compounds
Compound
Isopropyl alcohol
Cytosine
k (L/mol/s)
2.1 × 109a
7.72 × 109b
2.9 × 109c
3.1 × 109c
4.5 × 109d
8.6 × 109c
6.4 × 109e
Uracil
Tempo
GSH
DMSO
aReference[36]
breference[17]
;
;
creference[18]
;
dreference;[19]
ereference [20]
;
f
.
Fe2þ þH2O2 ! Fe3þ þꢀOHþ −OHðIÞFenton reagent Equation 1
Equation 2

ZHANG ET AL.
5
different documents. For instance, in one document, the reaction con-
stant of uracil was 7.72 × 109 L/mol/s[17] and, in another study, its
reaction constant is 2.9 × 109 L/mol/s[18] (Table 1). This uncertainty is
especially pronounced when comparing compounds with similar rate
constants. For example, in the relevant documents, the
ꢀOH-scavenging abilities of GSH, Tempo, and DMSO are
8.6 × 109 L/mol/s,[18] 4.5 × 109 L/mol/s,[19] and 6.4 × 109[20] L/mol/s,
respectively. (Table 1) Therefore, if their ꢀOH-scavenging abilities
were judged according to the documents, the order of
ꢀOH-scavenging ability should be GSH > DMSO > Tempo. However,
both in Yang's study[21] and in our study, the order should be Tempo
> DMSO > GSH. The cause of this phenomenon may be the inconsis-
tency of the above-mentioned detection conditions and methods. In
conclusion, when the compounds and MOPC are mixed in the same
amount (10 μM), the scavenging efficiencies of those compounds
could be described as follows: Tempo > Cytosine > DMSO >
GSH > uracil.
ꢀOH can be detected by various methods. Among these, the fluo-
rescence method is an efficient method.[22] There are two strategies
for the design of fluorescent probes.[23] The first strategy is to detect
the ꢀOH through an intermediate. A commonly used intermediate is
DMSO, which can react with ꢀOH to form methyl radicals, which can
then be detected using fluorescent probes[23] (eqn 2). It is worth not-
ing that the detection limit of this strategy is influenced by the reac-
tion rate constants between ꢀOH to the intermediates and methyl
radical to the probes. The second strategy is to use the probe that can
FIGURE 4 Relative fluorescence intensity of coumarin-based fluorescent probe after adding scavenging capacity of compounds. The OH-
scavenging ability of isopropyl alcohol, cytosine, uracil, Tempo, GSH and DMSO with MOPC (10 μM) has been investigated in 0.1 mM potassium
phosphate buffer at pH 7.4. ꢀOH source: 40 μM Fenton reagent. Fluorescence spectra were recorded at 5 min with an excitation at 248 nm and
emission at 364 nm

6
ZHANG ET AL.
react directly with ꢀOH, and its fluorescence intensity should changes
with ꢀOH concentration.[23] The detection limit of the second strategy
is influenced by the reaction rate constants between probes and ꢀOH.
Because there are many molecules that can react with ꢀOH in biologi-
cal system, if the probe wants to detect ꢀOH effectively, the probe
should have a relatively high reaction constant. However, it is worth
noting that there are currently no probes that completely consume all
of the ꢀOH in a system. They can only react to certain degree with
ꢀOH contained in the biological system, which is why the probe has a
detection limit. An ideal probe should have a reaction rate much
higher than that of other molecules in the system, and its detection
limit should be very low. In this study, the detection limit of MOPC to
ꢀOH is about 10 μM, so the reaction rate constant of the probe should
reach a certain degree compared with other molecules. As it shown in
Figure 4, even if DMSO was added far more than the MOPC equiva-
lent, a MOPC response to ꢀOH still exists. This phenomenon indicated
that MOPC has a higher rate constant with ꢀOH than DMSO
(Figure 4). As the amount of DMSO in applications is in far excess,
DMSO can still function to capture ꢀOH via the first strategy.
The electron paramagnetic resonance (EPR) method is another
classic method for detection of ꢀOH. In this method, 5,5-dimethyl-1-
pyrroline Noxide (DMPO) or Tempo is usually used as a spin-trap (also
known as an intermediate).[24] For example, Aboul-eneim's group has
used DMPO as a spin-trap to evaluate the ꢀOH-scavenging ability of a
series of compounds using the EPR technique.[25–27] Similar to the
first strategy of the fluorescent probe method, the detection limit of
this reaction is also affected by the reaction constants of ꢀOH and
spin-traps. However, it is worth noting that the application of this
method is limited due to relatively expensive EPR equipment.
their reactivity is currently lacking, impeding the understanding of var-
ious fundamental processes in chemical and biological systems. Using
our probe , calibration and quantification of the scavenging efficiency
of reported scavengers would therefore be possible.
4
|
CONCLUSIONS
In conclusion, we developed a new coumarin-based fluorescent
probes for ꢀOH. The probe exhibited excellent selectivity for ꢀOH,
with good dynamic response ranges and micromolar applications
for determining the ꢀOH-scavenging abilities of several substances,
in which our probes demonstrated high sensitivity. Our studies will
facilitate further development of fluorescent probes that will aid in
investigating the pathophysiology of ꢀOH toxicity in living cells and
tissue.
ACKNOWLEDGEMENTS
The present study was supported by the Tianjin Science and Technol-
ogy Key Project on Chronic Diseases Prevention and Treatment (grant
no. 16ZXMJSY-00020), Tianjin Municipal Special Program of Talents
Development for Excellent Youth Scholars, China (grant no. TJTZJH-
QNBJRC-2-9), and Tianjin 131 Creative Talents Cultivation Project
(1st Class, 2016 Fund plan for scientific research and development of
Tianjin Agricultural College (grant no.20190105), National Key
Research and Development Project of China (grant no.
2017YFD0201102).
AVAILABILITY OF DATA AND MATERIALS
All data generated or analyzed during the present study are included
in this published article.
Chemiluminescence (CL) and electrochemiluminescence (ECL)
are also effective methods to evaluate the ꢀOH-scavenging ability of
compounds.[28,29] It is worth mentioning that, for these methods, the
luminophore is the basis of research.[30] For example, in Cai's study,
Eosin Y was used as a luminophore to measure the ꢀOH-scavenging
ability of daidzein.[28] In Liang's study, ꢀOH generated using an anode
was captured by palmatine, and palmatine concentration could be
determined using the displayed ECL.[29] They also used palmatine to
assess the OH-scavenging ability of salicylic acid and DMSO.
PATIENT CONSENT FOR PUBLICATION
Not applicable.
COMPETING INTERESTS
The authors declare that they have no competing interests.
In this study, a coumarin-based probe was used for detection of
ꢀOH. After adding ꢀOH at different concentrations, the notion that
fluorescence intensity is closely related to concentration of added
ꢀOH became apparent. This probe can accurately quantify ꢀOH on the
micromolar scale. To screen for fluorescent probes that only have rec-
ognition functions for ꢀOH, fluorescence detection experiments were
performed using different ions and ROS. These results indicated that
the MOPC probe can specifically detect ꢀOH and that detection is
barely affected using other ions. In addition, a good linear relationship
was also established between fluorescence intensity and the concen-
tration of ꢀOH.
ORCID
Yu Zhu
https://orcid.org/0000-0003-0499-0235
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