Development of a coumarin-based fluorescent probe for hydrogen peroxide based on the Payne/Dakin tandem reaction

Article abstract of DOI:10.1016/j.dyepig.2021.109335

Hydrogen peroxide (H₂O₂) is associated with many physiological and pathological processes in biological systems. Here, we report 7-diethylamino-3-formyl-coumarin (Cou-CHO) as a turn-on fluorescent probe for H₂O₂. The proposed probe employs a tandem Payne/Dakin reaction to convert the electron-withdrawing aldehyde to the electron-donating hydroxyl group, thus hindering the formation of twisted intramolecular charge transfer (TICT) state of 7-diethylaminocoumarin, and thereby resulting in a fluorescence turn-on response. Cou-CHO features high specificity, excellent sensitivity and fast response toward H₂O₂. In particular, Cou-CHO enables the direct visualization of basal and endogenously produced H₂O₂ in living cells. The results demonstrate that the tandem Payne/Dakin reaction can provide a new choice to develop H₂O₂-selective probes.

Full text of DOI:10.1016/j.dyepig.2021.109335

Dyes and Pigments 190 (2021) 109335
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Development of a coumarin-based fluorescent probe for hydrogen peroxide
based on the Payne/Dakin tandem reaction
,
*
Fangfang Wua, Hanjie Yub, Qin Wangc, Jianjian Zhanga, Zheng Lib, Xiao-Feng Yanga
a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Materials Science, Northwest University,
Xi’an, 710127, PR China
b Laboratory for Functional Glycomics, College of Life Sciences, Northwest University, Xi’an, 710069, PR China
c Shaanxi Key Laboratory of Catalysis, School of Chemistry and Environment Science, Shaanxi University of Technology, Hanzhong, Shaanxi, 723000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hydrogen peroxide (H2O2) is associated with many physiological and pathological processes in biological sys-
tems. Here, we report 7-diethylamino-3-formyl-coumarin (Cou-CHO) as a turn-on fluorescent probe for H2O2.
The proposed probe employs a tandem Payne/Dakin reaction to convert the electron-withdrawing aldehyde to
the electron-donating hydroxyl group, thus hindering the formation of twisted intramolecular charge transfer
(TICT) state of 7-diethylaminocoumarin, and thereby resulting in a fluorescence turn-on response. Cou-CHO
features high specificity, excellent sensitivity and fast response toward H2O2. In particular, Cou-CHO enables the
direct visualization of basal and endogenously produced H2O2 in living cells. The results demonstrate that the
tandem Payne/Dakin reaction can provide a new choice to develop H2O2-selective probes.
Hydrogen peroxide
Fluorescent probe
Payne reaction
Dakin reaction
Coumarin
Cell imaging
1. Introduction
the utilization of boronate (or boronic acid) as a recognition unit for
ONOOꢀ [16,17] or benzoyl peroxide [18] have been reported. Similarly,
Hydrogen peroxide (H2O2), as an important reactive oxygen species
(ROS), is mainly generated in mitochondria by the activation of nico-
tinamide adenine dinucleotide phosphate (NADPH) oxidase complex
[1], and can diffuse across cellular membranes to regulate a large variety
of physiological processes from the immune response to cell signaling
[2]. However, aberrant production of H2O2 is involved in many diseases,
such as cardiovascular disorders, diabetes, Parkinson’s disease, Alz-
heimer’s disease, Huntington’s disease, metabolic diseases and cancers
[3,4]. Thus, it is of great importance to develop chemical tools to detect
H2O2 in living systems.
α
-ketoamide-based H2O2 probes were also reported for ONOOꢀ detec-
tion [19,20], because ONOOꢀ is reported to possess much stronger
nucleophilicity than H2O2 under physiological conditions [15]. There-
fore, the development of H2O2 fluorescent probes with new reaction
mechanisms is highly desirable.
Recently, Yang et al. reported an innovative probe for H2O2 based on
a tandem Payne/Dakin reaction coupled with the traditional rhodamine
decaging method [21]. The overall strategy employs a specific
H2O2-mediated transformation of benzaldehydes into the corresponding
phenols, which subsequently undergoes a spontaneous 1,6-elimination
and carbamate cleavage to release the fluorophore, resulting in a dra-
matic fluorescence enhancement. Inspired by the above strategy, we
envisioned that the above functionality transformation from the
electron-withdrawing aldehyde to the electron-donating hydroxyl group
might serve as a promising chemical handle for constructing H2O2
probes directly, because the pronounced change in electronic properties
of the function group may influence the photophysical states of a fluo-
rophore, which should in turn lead to observable changes in its optical
properties.
Fluorescent probes offer powerful chemical tools for monitoring
biological species in living biosystems, owing to their simplicity, high
sensitivity, high spatial resolution, and noninvasiveness. So far, several
fluorescent probes have been developed for H2O2 detection and imaging
[5–7], which employs boronic esters/acid [8,9], benzyl [10], aryl sul-
fonyl esters [11,12] and
α-ketoamide [13] as the recognition unit.
However, as H2O2 exhibits relatively mild reactivity among ROS, most
H2O2 probes give a sluggish response and have drawbacks in terms of
selectivity. For instance, some boronate-based probes respond to H2O2
more slowly when compare to other more reactive species, such as
peroxynitrite (ONOOꢀ ) and hypochlorous acid (HClO) [14,15]. In fact,
In this work, we report 7-diethylamino-3-formyl-coumarin (Cou-
CHO) as a new turn-on fluorescent probe for H2O2. In aqueous polar
* Corresponding author.
E-mail address: xfyang@nwu.edu.cn (X.-F. Yang).
https://doi.org/10.1016/j.dyepig.2021.109335
Received 18 January 2021; Received in revised form 15 March 2021; Accepted 16 March 2021
Available online 24 March 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

F. Wu et al.
Dyes and Pigments 190 (2021) 109335
solvents, Cou-CHO is weakly emissive due to the formation of the
twisted intramolecular charge transfer (TICT) state upon photoexcita-
tion. Upon mixing with H2O2 in the presence of trichloroacetonitrile
(CCl3CN), trichloroperoxyacetamidic acid (TCPAA) is formed from the
reaction of CCl3CN with H2O2 (Payne reaction), which then affords a
Dakin oxidation via nucleophilic attack of the aldehyde on the 3-posi-
tion of Cou-CHO by its anion form, and subsequent hydrolysis of the
intermediate gives the corresponding Cou-OH as the final product. This
tandem reaction converts the electron-withdrawing aldehyde moiety of
Cou-CHO to the electron-donating hydroxyl group, thus hindering for-
mation of the TICT state and thereby resulting in a significant fluores-
cence enhancement (Scheme 1). The fluorescent intensity at 502 nm
cells were pretreated with phorbol-12-myristate-13-acetate (PMA, 0.5
μ
g mL-1) or rotenone (RO, 0.5
μM) for 30 min, and then incubated with
the probe in the presence of CCl3CN (100
μM) for 30 min. For the
scavenging assay, the cells were co-incubated with the stimulator (PMA
or RO) and N-acetylcysteine (NAC, 1 mM) for 30 min, and then treated
with the probe and CCl3CN as described above. Cell images were
captured after the cells were washed with PBS three times.
3. Result and discussion
3.1. Development of fluorescent probes for H2O2
increases linearly with H2O2 concentration in the range 0.5–10 μM. The
Compared with other ROS, H2O2 is a relatively stable species and
exhibits mild reactivity. Therefore, fluorescent probes for H2O2 are
liable to be interfered upon by highly reactive species, such as ONOOꢀ .
Recently, Yang et al. reported a new approach to H2O2 recognition based
on a tandem Payne/Dakin reaction [21,23]. H2O2 can be activated by
the in situ formation of a sufficiently reactive trichloroperoxyacetamidic
acid (TCPAA) from its addition to trichloroacetonitrile (CCl3CN) in
neutral aqueous media [24]. Subsequently, TCPAA can afford a Dakin
reaction to give the corresponding phenol, which initiates the following
1,6-elimination to release the fluorescent conjugated xanthene ring and
emits strong fluorescence. The above synergistic Payne/Dakin reactions
afford the transformation of aldehydes (-CHO) into the corresponding
hydroxyl groups (-OH), which serves as the basis for sensitive and spe-
cific H2O2 detection. However, in the above design strategy, the func-
tionality transformation and the decaging of xanthene dyes have to be
integrated to achieve the sensing purpose, which makes the probe design
complicated and lengthy synthesis has to be carried out. This encourages
us to improve the above strategy to realize much advanced H2O2 probes.
In fact, the functional group transformation represents a new
approach for developing selective probes in biological systems. The
aldehyde is a typical electron-deficient group, while the hydroxyl group
is an electron-donating one. Thus, we envisioned that the above change
in electronic properties of the function group via tandem Payne/Dakin
reactions may influence the photophysical properties of a fluorophore,
thus producing a discernible change in its emission properties. This
might be an effective signaling mechanism and could be employed to
devise H2O2 fluorescent probes.
proposed probe exhibits excellent selectivity toward H2O2 over other
ROS, reactive nitrogen species (RNS) and related biological species. The
practical utility of Cou-CHO in biological contexts has been demon-
strated by imaging basal and endogenous H2O2 in living cells.
2. Experimental section
2.1. General procedure for spectral measurements
The stock solution of Cou-CHO (1 mM) was prepared in acetone.
H2O2 solution was dissolved in deionized water directly, and its con-
centration was identified from the absorption at 240 nm (ε = 43.6
Mꢀ 1cmꢀ 1). Other ROS/RNS were also prepared according to the previ-
ous reported method [22]. A typical test solution was prepared by
adding 25 μL of Cou-CHO (1 mM), 1.5 mL of ethanol, 0.5 mL of 0.2 M
phosphate buffer (0.2 M, pH 7.4), 50 μL of CCl3CN (100 μM), and a
required volume of test species sample solution. The above solution was
diluted to 5.0 mL with water and maintained at 37 ◦C for 20 min. And
then the absorption or fluorescence spectra were recorded.
2.2. Cell imaging experiments
HepG2 cells were plated on plastic Petri dishes and allowed to attach
for a day before the imaging experiment. The cells were then incubated
with Cou-CHO (5 μM) in FBS-free RPMI for 30 min then washed with
PBS (pH 7.4) three times. For imaging of exogenous H2O2, the cells were
then treated with different concentration of H2O2 (5, 10, 20 M) in the
presence of CCl3CN (100 M) in PBS buffer for 30 min. After washing the
μ
It is well-known that 7-dialkylaminiocoumarins are solvatochromic
dyes, which undergoes a nonradiative rotational (twisting) decay
mechanism that leads to full charge separation in the excited state [25].
In general, due to the formation of this twisted intramolecular charge
transfer (TICT) state, 7-dialkylaminiocoumarins are weakly emissive in
highly polar solvents [26]. It was further reported that the fluorescence
μ
cells with PBS buffer three times, cell imaging was then carried out
under a confocal fluorescence microscope with an objective lens ( × 40).
The fluorescence emission was collected at 470–520 nm upon excitation
at 405 nm. For imaging endogenous H2O2 generation in HepG2 cells, the
Scheme 1. Proposed reaction mechanism for sensing of H2O2 when using Cou-CHO.
2

F. Wu et al.
Dyes and Pigments 190 (2021) 109335
properties of these fluorophores can be varied by the strength of the
electron-withdrawing substituents at its 3-position [27,28]. A stronger
electron-withdrawing substituent would result in a greater charge
transfer from the electron-donating moiety of the aminocoumarin
molecule, thus affording weaker fluorescence emission in aqueous
media. We reasoned that the functionality transformation from the
electron-deficient aldehydes to the electron-donating hydroxyl groups
induced by the tandem Payne/Dakin reaction may reduce the
electron-withdrawing strength of the substituent, and the twisting
relaxational motion may not take place, which in turn leads to a large
wavelength shift in the emission spectrum of the probe as well as a
turn-on fluorescence response (Scheme 1). The detailed sensing mech-
anism is depicted in Scheme 1. Based on the above considerations,
Cou-CHO might be an ideal candidate for H2O2 detection via a tandem
Payne/Dakin reaction.
aldehyde group could be transformed to the hydroxyl group.
3.2. Sensing response to H2O2
To examine its usefulness for H2O2 detection, Cou-CHO was incu-
bated with H2O2 in phosphate buffer (20 mM, pH 7.4, with 30% ethanol,
100 μ
M CCl3CN) at 37 ◦C. As shown in Fig. 2a, the maxima absorption of
Cou-CHO is centered at 448 nm. However, with the addition of
increasing concentrations of H2O2 (0–15 M), its absorption maximum
μ
at 448 nm progressively decreased with the concomitant formation of a
new absorption band at around 378 nm. This spectral variation is
apparently due to the disruption of the electron donor-acceptor system
within the probe. Meanwhile, changes in the fluorescence spectra of
Cou-CHO upon treating with increasing concentrations of H2O2 were
investigated. As indicated in Fig. 2b, the solution of Cou-CHO is almost
non-fluorescent when excited at 391 nm. Upon addition of H2O2, the
emission intensity of Cou-CHO solution at 502 nm enhanced gradually
with increasing concentration of H2O2 until a plateau is reached at 10
To assess the validity of this assumption, compounds Cou-CHO and
Cou-OH were prepared (Scheme 2, the detailed synthetic procedures are
shown in Supplementary Data) and their photophysical data were
compared at the same conditions. Fig. 1a shows the UV–vis absorption
spectra of Cou-CHO and Cou-CHO in phosphate buffer (pH 7.4, 30%
ethanol). Cou-CHO exhibits intense absorption band at 448 nm, which is
apparently attributed to ICT. However, the absorption spectrum of Cou-
OH has a marked hypochromatic shift (~70 nm) when compared to
Cou-CHO. This blueshift is apparently attributed to interrupt of the ICT
process within the molecule. Furthermore, the fluorescence spectra of
compounds Cou-CHO and Cou-OH were recorded. It was observed that
they display similar emission maximum wavelengths (ca. 500 nm);
however, the emission intensity of Cou-OH is much higher than that of
Cou-CHO (Fig. 1b). The above results are compiled in Table 1. To our
delight, it was further observed that Cou-CHO exhibited almost no
fluorescence emission upon excitation at the absorption band of Cou-
OH, which is favorable for the determination of low concentration of
H2O2. The above results implied that Cou-CHO might be employed for
H2O2 detection with a turn-on fluorescence response, provided that its
μ
M. There is a ca. 34-fold increase in the fluorescence intensity in the
presence of 2 equiv. of H2O2. The fluorescence intensity at 502 nm
increased linearly with the concentration of H2O2 in the range 0.5–10
μ
M, and the detection limit was determined to be 31 nM (3δ). Clearly,
these results indicate that Cou-CHO can be used to detect H2O2 with
high sensitivity.
Further, the kinetic behavior of the reaction system was investigated.
With the addition of H2O2, the intensity of the reaction solution
increased significantly. A kinetic assay revealed that the fluorescence at
502 nm reached a maximum after approximately 20 min (Fig. 3). In
contrast, the Cou-CHO solution alone exhibited no observable fluores-
cence changes under identical conditions. In addition, time-dependent
absorption spectral changes of Cou-CHO in the presence of H2O2 were
recorded. As shown in Fig. S1, the increased of the absorption band at
378 nm and a gradual decline at 448 nm were observed simultaneously
as the reaction time prolonged, and they were saturated after ca. 20 min,
Scheme 2. Synthesis of Cou-CHO and control compounds J-Cou-CHO and Cou-OEt. Conditions: (a) POCl3, DMF; (b) CH2(COOEt)2, piperidine, reflux 6 h; (c)
concentrated HCl, CH3COOH, reflux 6 h; (d) POCl3, DMF; (e) n-butanol, CH3COOH, piperidine, reflux, 12 h; (f) SnCl2, HCl, room temperature, 2 h; (g) HCl, reflux, 4
h; (h) DMF, EtI, K2CO3, 60 ◦C, 7 h.
3

F. Wu et al.
Dyes and Pigments 190 (2021) 109335
Fig. 1. Absorption (a) and fluorescence spectra (b) of Cou-CHO and Cou-OH
(both 5 μM) in 20 mM phosphate buffer (pH 7.4, 30% ethanol).
Fig. 2. Absorption (a) and fluorescence spectra (b, λex = 391 nm) of Cou-CHO
Table 1
(5 μM) upon treating with increasing concentrations of H2O2 (0–15 μM) in 20
Photophysical characterization of Cou-CHO, Cou-OH, J-Cou-CHO and Cou-OEt
mM phosphate buffer (pH 7.4, 30% ethanol, 100 μM CCl3CN). The reaction was
in 20 mM phosphate buffer (pH 7.4, 30% ethanol).
Compounds
carried out at 37 ◦C. The spectra were recorded after incubation of Cou-CHO
with H2O2 for 20 min. Inset in (b): fluorescence intensity at 502 nm as a
function of H2O2 concentration.
c
λabs/nma
εmax/ × 104 Mꢀ 1cmꢀ 1
λem/nmb
ΦF
Cou-CHO
Cou-OH
448
378
464
380
4.55
1.05
4.70
1.01
499
502
513
489
0.017
0.26
0.58
0.24
J-Cou-CHO
Cou-OEt
fluorescent scaffolds. On the other hand, according to the reaction
mechanism, the initial step of Dakin oxidation involves the nucleophilic
attack of the aldehyde group by TCPAA in its anion form [30]. Thus, it
seems that an alkaline solution favors the Dakin oxidation. Therefore,
the effect of pH on the fluorescence signals of the present system was
investigated by carrying out the reaction at different pH phosphate
buffer solutions (pH: 5.0–9.18). As indicated in Fig. S3, the fluorescence
signal increased with pH in the range of 5.6–7.2, thereafter decreased
significantly. This is apparently due to the compromise of the above two
factors. A relatively strong fluorescence emission was observed in the pH
range of 6.5–7.8, which fulfills the requirement for monitoring of H2O2
at physiological pH conditions.
a
The maximal absorption of the dye.
The maximal emission of the dye.
b
c
ΦF is the relative fluorescence quantum yield, which was measured by using
quinine sulfate (ΦF = 0.55 in 0.05 M H2SO4) as a standard [29].
which was consistent with the results of the fluorescence measurement.
3.3. Effect of pH
The effect of pH on the fluorescence response of Cou-CHO to H2O2
was investigated. According to the reaction mechanism, the tandem
Payne/Dakin reaction should give Cou-OH as the final product. Since
Cou-OH contains a pH-sensitive hydroxyl group at its 3-position, we
initially investigate the effect of pH on its fluorescence emission
behavior in the pH range of 5.0–9.18. As shown in Fig. S2, the fluores-
cence signal of Cou-OH was strong and stable in the pH range of 5.0–7.0,
and decreased markedly when the solution pH is above 7.0. Based on the
above data, its pKa value was determined to be 7.65. The above results
indicate that an acidic environment favors the fluorescence measure-
ment of Cou-OH, which is unusual in the hydroxyl group-containing
3.4. Selectivity studies
The selectivity of Cou-CHO toward H2O2 was tested by treatment
with other potential interfering species in biological system. As shown in
Fig. 4 and S4, only H2O2 triggers a prominent fluorescence increment,
while other ROS and RNS (⋅OH, 1O2, O2⋅-, NO, ONOOꢀ , TBHP, TBO⋅ and
HClO), metal ions (Ca2+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Zn2+), and other
biological species (SO23ꢀ , GSH, Cys, Hcy, and His) afford no or minor
4

F. Wu et al.
Dyes and Pigments 190 (2021) 109335
Fig. 3. Time-dependent fluorescence intensity changes of Cou-CHO (5 μM) in
the presence of H2O2 (2 equiv.). The reaction was carried out in 20 mM
phosphate buffer (pH 7.4, 30% ethanol, 100
391/502 nm. Data are represented as mean ± SD (n = 3).
μ =
M CCl3CN) at 37 ◦C. λex/λem
changes under identical conditions. Notably, it was observed that HClO
at 100 M triggered a small fluorescence increment at 481 nm. The
μ
above phenomenon might be ascribed to the oxidation of aldehyde to
carboxylic group by HClO. However, this reaction has negligible impact
towards the selective H2O2 detection due to its different emission peak at
481 nm (cf.502 nm using H2O2). Although it was reported that bisulfite
can react with Cou-CHO to afford fluorescence enhancement, the
addition reaction has to be carried out in acidic medium (pH 5.0) [31].
Thus, the present sensing system can avoid the interference from
bisulfite by carrying out the measurement at physiological pH condi-
tions. In addition, Cou-CHO was also reported for Cys/Hcy detection
[32]. Nevertheless, it gives turn-off fluorescence response and thus
would not interfere with the H2O2 detection. Overall, these experiments
demonstrate that the present probe has high selectivity for H2O2
determination against ROS and RNS, metal ions, and other biological
species.
Fig. 4. Fluorescence spectra (a, λex = 391 nm) and emission intensity (b) of
Cou-CHO (5 μM) in the presence of various ROS/RNS (10 μM H2O2, 100 μM for
other species) in 20 mM phosphate buffer (pH 7.4, 30% ethanol, 100
μM
3.5. Mechanistic investigations
CCl3CN). Inset in (b): photographs of the solution of Cou-CHO after treating
with different reactive species under UV light at 365 nm. Data are represented
as mean ± SD (n = 3).
The sensing mechanism of the present system was investigated.
Initially, some control experiments were carried out to investigate the
reaction mechanism. It was observed that treatment of Cou-CHO with
CCl3CN or H2O2 alone gives no observable changes in fluorescence
emission; however, the mixture of CCl3CN and H2O2 exhibits pro-
nounced fluorescence enhancement at 502 nm (Fig. S5). The above re-
sults indicate the involvement of TCPAA in the sensing process. Next,
according the reaction mechanism, the treatment of Cou-CHO with
H2O2 in the presence of CCl3CN should give Cou-OH. To confirm this,
the reaction mixture of Cou-CHO and H2O2 was subjected to high res-
olution mass spectrometry. As shown in Fig. S6, a prominent peak at m/z
234.1100 corresponding to Cou-OH (calcd. 234.1124 for C13H16NO3) is
clearly observed in the HRMS data, indicating the generation of Cou-OH
in the reaction mixture. Additional proof of the above mechanism is
provided by comparing the absorption and emission spectra of the re-
action mixture and authentic Cou-OH. As shown in Fig. S7, the treat-
ment of Cou-CHO with H2O2 in the presence of CCl3CN gives a new
absorption band centered at 378 nm, which agrees well the absorption
peaks of Cou-OH. Further, the emission spectrum of the above reaction
solution is identical with that of Cou-OH. These results testified to the
proposed reaction mechanism shown in Scheme 1.
hydroxyl group formed in the sensing process could hydrogen bond to
the carbonyl in proximity, thus effectively modulating its fluorescence
[33–35]. To ascertain this possibility, we prepared Cou-OEt (Scheme 2,
synthesis and characterization details in Supplementary data), where
the hydroxyl group at the 3-position of Cou-OH was substituted by an
ethoxyl group, and thus precluded the formation of intramolecular
hydrogen bond. The fluorescence spectra of Cou-OEt and Cou-OH were
compared in 30% ethanol aqueous solution (pH 7.4). As indicated in
Fig. S8, the emission peak of Cou-OEt is blue-shifted about 7 nm when
compared to Cou-OH. However, their fluorescence intensity shows no
significant difference. These results indicate the present sensing system
is irrelevant to intramolecular hydrogen bond.
On the other hand, previous studies indicate the weak fluorescence
of 7-dialkylaminocoumarin is due to the formation of TICT state [26,
36]. To ascertain whether TICT is involved in the present sensing pro-
cess, we replaced the N,N-diethylamino group at the 7-position of
Cou-CHO with a bridged julolidinyl moiety (to form J-Cou-CHO as a
control compound, its synthesis and characterization details are shown
in Supplementary data), which would block the rotation of the dieth-
ylamino group with respect to the plane of the coumarin skeleton. Thus,
the involvement of the TICT state should be excluded [37,38]. The
Now, the key questions related to this reaction is that why the
transformation of aldehyde group to hydroxyl group affords a significant
turn-on fluorescence response. It was previously reported that the
5

F. Wu et al.
Dyes and Pigments 190 (2021) 109335
treatment of J-Cou-CHO with H2O2 was examined with the same con-
ditions described for Cou-CHO. As shown in Fig. S9, J-Cou-CHO gives
similar absorption variations as that of Cou-CHO; however, no fluo-
rescence increase was observed because J-Cou-CHO itself has prevented
the TICT state formation by incorporation of the nitrogen into a system
of fused rings. The above results indicate that the fluorescence
enhancement of Cou-CHO upon treatment with H2O2 is TICT-involved.
In addition to steric hindrance effect, TICT formation in a dye is also
governed by its intrinsically electronic effect. The aldehyde group is an
electron-withdrawing group, while the hydroxyl group is a typical
electron-donating one, thus we reasoned that the transformation of
aldehyde to hydroxyl group would increase the energy barrier to enter
the TICT state, thus suppressing TICT formation. Generally, the mole-
cules in the TICT excited state have a large dipole moment, and conse-
quently the TICT excited state is stabilized in a polar environment. With
the decrease of solution polarity, the energy barrier for torsional motion
leading to the TICT state increases; as a result, the TICT process is pro-
hibited, thus leading to an increased fluorescence emission [39,40].
Therefore, the fluorescence properties of Cou-CHO and Cou-OH were
investigated in solvents of different polarities. As shown in Fig. S10, an
increase of the 1,4-dioxane percentage in water from 0 to 99%, corre-
sponding to a decrease in solvent polarity, resulted in a hypochromatic
shift and fluorescence enhancement for Cou-CHO and Cou-OH. How-
ever, the fluorescence peak intensity of Cou-CHO increased 117-fold;
whereas for Cou-OH, only a 4.2-fold enhancement was observed
(Fig. S11), indicating that the former is more sensitive to polarity
changes than the latter. These data, therefore, indicate that the turn-on
fluorescence response of the present sensing system is apparently due to
the inhibition of TICT process within Cou-CHO via the functional groups
transformation.
evaluated in HepG2 cells by Cell Counting Kit-8 (CCK-8) assays
(Fig. S12), and the results suggests the low cytotoxicity of Cou-CHO in
the concentration range of 0–20 μM (cell viability > 89% for 24 h in-
cubation time). Next, to determine if Cou-CHO could react with H2O2 in
living cells, we stained HepG2 cells with Cou-CHO for 30 min, and then
exchanged cell media for new media containing varying concentrations
of H2O2 (5–20 μM). It was observed that cells treated with exogenous
H2O2 showed a dose-dependent fluorescence turn-on response after 30
min (Fig. 5a and b). There results indicate that Cou-CHO reacts with
H2O2 in living cells to generate a turn-on fluorescence response.
To further confirm its practical usefulness, we assessed the potential
ability of Cou-CHO for fluorescence imaging of endogenous H2O2 in
living cells. It was observed that the probe-loaded HepG2 cells displayed
observable fluorescence signal (Fig. 6a, first column). In contrast, the
cells pretreated with H2O2 scavenger NAC showed apparently decreased
fluorescence signals (Fig. 6a, second column). The outcome confirms
that Cou-CHO is capable of imaging intracellular basal H2O2. Next, Cou-
CHO was utilized to image the fluctuation of H2O2 in living HepG2 cells.
It was reported that PMA can trigger the up-regulation of intracellular
H2O2 through a cellular inflammation response [41]. Thus, PMA was
employed to stimulate the cells and then incubated with the probe. As
shown in the third column of Fig. 6a, PMA-stimulated cells showed high
levels of fluorescence intensity in comparison with the PMA free cells.
The fluorescence increment in the cells was efficiently suppressed by
pretreatment with NAC (Fig. 6a, fourth column and Fig. 6b). Further-
more, we stimulated the cells with RO, a high-affinity inhibitor of
complex I of the mitochondria electron-transfer chain known to induce
the generation of ROS, including H2O2 [42]. Similarly, the
RO-stimulated cells displayed strong fluorescence and the fluorescence
increment could be largely inhibited by NAC (Fig. 6a, fifth and sixth
column, Fig. 6b). Taken together, these results support the idea that
Cou-CHO can monitor endogenous H2O2 generation in living cells.
3.6. Fluorescence imaging of H2O2 in living cells
4. Conclusion
Having confirmed the efficiency of H2O2-triggered fluorescence turn-
on response, we next moved on to investigate the ability of Cou-CHO to
visualize H2O2 in living cells. Initially, cell cytotoxicity of Cou-CHO was
To summarize, we have developed a turn-on fluorescent probe for
Fig. 5. (a) Fluorescence images of HepG2 cells treated with Cou-CHO (5
CCl3CN (100 M) for 30 min λex = 405 nm, λem = 470–520 nm. Scale bar: 20
shown as mean ± SD (n = 3).
μ
M, 30 min) and exposed to a range of H2O2 concentrations (0–20
μM) in the presence of
μ
μ
m. (b) Relative fluorescence intensity shown in (a) was quantified. The results are
6

F. Wu et al.
Dyes and Pigments 190 (2021) 109335
Fig. 6. (a) Fluorescence imaging of endogenous H2O2 in HepG2 cells. Cells were pretreated with PBS only (as a control), NAC (1 mM), PMA (0.5
mM) + PMA (0.5 g/mL), RO (0.5 M, 0.5 h), NAC (1 mM) + RO (0.5 M) for 30 min, and further co-incubated with Cou-CHO (5 M, 30 min) and CCl3CN (100
for 30 min λex = 405 nm, λem = 470–520 nm. Scale bar: 20 m. (b) Relative fluorescence intensity shown in (a) was quantified. The results are shown as mean ± SD
(n = 3). Statistical significance was determined as **p < 0.001, ****p < 0.0001 by student’s test.
μ
g/mL), NAC (1
μ
μ
μ
μ
μ
M)
μ
detecting H2O2 in aqueous solutions and living cells. The proposed
probe employs a tandem Payne/Dakin reaction to selectively convert the
aldehyde to the corresponding hydroxyl group, thus hindering the for-
mation of TICT state of 7-diethylaminocoumarin, and thereby resulting
in selective fluorescence turn-on responses to biologically relevant
concentrations of H2O2 over potentially interfering species. Importantly,
the present probe is capable of imaging endogenous H2O2 in living cells.
We expect that the design strategy described herein can be extended to
construct future H2O2 fluorescent probes with improved properties.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109335.
References
[1] Kalyanaraman B, Cheng G, Hardy M, Ouari O, Bennett B, Zielonka J. Teaching the
basics of reactive oxygen species and their relevance to cancer biology:
mitochondrial reactive oxygen species detection, redox signaling, and targeted
therapies. Redox Biol 2018;15:347–62.
¨
[2] Rezende F, Brandes RP, Schroder K. Detection of hydrogen peroxide with
Credit author statement
fluorescent dyes. Antioxidants Redox Signal 2018;29:585–602.
[3] Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in
neurodegenerative diseases. Nature 2006;443:787–95.
Fangfang Wu: Conceptualization, Methodology; Investigation;
Writing - Original Draft; Hanjie Yu: Investigation, Data curation; Qin
Wang: Conceptualization, Investigation; Jianjian Zhang: Conceptuali-
zation, Methodology; Zheng Li: Resources; Xiao-Feng Yang: Writing -
Review & Editing; Project administration; Supervision.
[4] Brieger K, Schiavone S, Miller Jr FJ, Krause K-H. Reactive oxygen species: from
health to disease. Swiss Med Wkly 2012;142:w13659.
[5] Zheng D-J, Yang Y-S, Zhu H-L. Recent progress in the development of small-
molecule fluorescent probes for the detection of hydrogen peroxide. Trends Anal
Chem 2019;118:625–51.
[6] Wen Y, Huo F, Yin C. Organelle targetable fluorescent probes for hydrogen
peroxide. Chin Chem Lett 2019;30:1834–42.
Declaration of competing interest
[7] Bai X, Ng KK-H, Hu JJ, Ye S, Yang D. Small-molecule-based fluorescent sensors for
selective detection of reactive oxygen species in biological systems. Annu Rev
Biochem 2019;88:605–33.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[8] Chang MCY, Pralle A, Isacoff EY, Chang CJ. A selective, cell-permeable optical
probe for hydrogen peroxide in living cells. J Am Chem Soc 2004;126:15392–3.
[9] Lippert AR, Van de Bittner GC, Chang CJ. Boronate oxidation as a bioorthogonal
reaction approach for studying the chemistry of hydrogen peroxide in living
systems. Acc Chem Res 2011;44:793–804.
Acknowledgments
[10] Abo M, Urano Y, Hanaoka K, Terai T, Komatsu T, Nagano T. Development of a
highly sensitive fluorescence probe for hydrogen peroxide. J Am Chem Soc 2011;
133:10629–37.
This research was supported by the National Natural Science Foun-
dation of China (No. 22074120) and the Science and Innovation Capa-
bility Support Program of Shaanxi (No. 2020KJXX-030).
[11] Maeda H, Fukuyasu Y, Yoshida S, Fukuda M, Saeki K, Matsuno H, Yamauchi Y,
Yoshida K, Hirata K, Miyamoto K. Fluorescent probes for hydrogen peroxide based
on a non-oxidative mechanism. Angew Chem Int Ed 2004;43:2389–91.
[12] Xu K, Tang B, Huang H, Yang G, Chen Z, Li P, An L. Strong red fluorescent probes
suitable for detecting hydrogen peroxide generated by mice peritoneal
macrophages. Chem Commun 2005:5974–6.
7

F. Wu et al.
Dyes and Pigments 190 (2021) 109335
´ˇ
´
¨
ˇ
´
´
[13] Xie X, Yang X, Wu T, Li Y, Li M, Tan Q, Wang X, Tang B. Rational design of an
[28] Cigan M, Donovalova J, Szocs V, Gapsar J, Jakusova K, Gaplovský A. 7-
(Dimethylamino)coumarin-3-carbaldehyde and its phenylsemicarbazone: TICT
excited state modulation, fluorescent H-aggregates, and preferential solvation.
J Phys Chem A 2013;117:4870–83.
α
-ketoamide-based near-infrared fluorescent probe specific for hydrogen peroxide
in living systems. Anal Chem 2016;88:8019–25.
[14] Brewer TF, Garcia FJ, Onak CS, Carroll KS, Chang CJ. Chemical approaches to
discovery and study of sources and targets of hydrogen peroxide redox signaling
through NADPH oxidase proteins. Annu Rev Biochem 2015;84:765–90.
[15] Sikora A, Zielonka J, Lopez M, Joseph J, Kalyanaraman B. Direct oxidation of
boronates by peroxynitrite: mechanism and implications in fluorescence imaging of
peroxynitrite. Free Radical Biol Med 2009;47:1401–7.
[29] Parker CA, Rees WT. Correction of fluorescence spectra and measurement of
fluorescence quantum efficiency. Analyst 1960;85:587–600.
[30] Hocking MB, Ong JH. Kinetic studies of Dakin oxidation of o- and p-
hydroxyacetophenones. Can J Chem 1977;55:102–10.
[31] Cheng X, Jia H, Feng J, Qin J, Li Z. Reactive" probe for hydrogen sulfite: "turn-on"
fluorescent sensing and bioimaging application. J Mater Chem B 2013;1:4110–4.
[32] Kim T-K, Lee D-N, Kim H-J. Highly selective fluorescent sensor for homocysteine
and cysteine. Tetrahedron Lett 2008;49:4879–81.
[16] Chen X, Wang F, Hyun JY, Wei T, Qiang J, Ren X, Shin I, Yoon J. Recent progress in
the development of fluorescent, luminescent and colorimetric probes for detection
of reactive oxygen and nitrogen species. Chem Soc Rev 2016;45:2976–3016.
[17] Bezner BJ, Ryan LS, Lippert AR. Reaction-based luminescent probes for reactive
sulfur, oxygen, and nitrogen species: analytical techniques and recent progress.
Anal Chem 2020;92:309–26.
[33] Feuster EK, Glass TE. Detection of amines and unprotected amino acids in aqueous
conditions by formation of highly fluorescent iminium ions. J Am Chem Soc 2003;
125:16174–5.
[18] Chen W, Li Z, Shi W, Ma H. A new resorufin-based spectroscopic probe for simple
and sensitive detection of benzoyl peroxide via deboronation. Chem Commun
2012;48:2809–11.
[34] Xu C, Li H, Yin B. A colorimetric and ratiometric fluorescent probe for selective
detection and cellular imaging of glutathione. Biosens Bioelectron 2015;72:
275–81.
[19] Cheng D, Xu W, Yuan L, Zhang X. Investigation of drug-induced hepatotoxicity and
its remediation pathway with reaction-based fluorescent probes. Anal Chem 2017;
89:7693–700.
[35] Lee K-S, Kim H-J, Kim G-H, Shin I, Hong J-I. Fluorescent chemodosimeter for
selective detection of cyanide in water. Org Lett 2008;10:49–51.
[36] Barooah N, Mohanty J, Pal H, Bhasikuttan AC. Non-covalent interactions of
coumarin dyes with cucurbit uril macrocycle: modulation of ICT to TICT state
conversion. Org Biomol Chem 2012;10:5055–62.
[20] Li Y, Xie X, Yang X, Li M, Jiao X, Sun Y, Wang X, Tang B. Two-photon fluorescent
probe for revealing drug-induced hepatotoxicity via mapping fluctuation of
peroxynitrit. Chem Sci 2017;8:4006–11.
[37] Debnath T, Maity P, Lobo H, Singh B, Shankarling GS, Ghosh HN. Extensive
reduction in back electron transfer in twisted intramolecular charge-transfer
(TICT) coumarin-dye-sensitized TiO2 nanoparticles/film: a femtosecond transient
absorption study. Chem Eur J 2014;20:3510–9.
[21] Ye S, Hu J, Yang D. Tandem Payne/Dakin reaction: a new strategy for hydrogen
peroxide detection and molecular imaging. Angew Chem Int Ed 2018;57:10173–7.
[22] Wu B, Yang J, Zhang J, Li Z, Li H, Yang X-F. A ratiometric fluorescent probe for the
detection of endogenous hydroxyl radicals in living cells. Talanta 2019;196:
317–24.
[38] Van Gompel JA, Schuster GB. Photophysical behavior of ester-substituted
aminocoumarins: a new twist. J Phys Chem 1989;93:1292–5.
[39] Sun Q, Yang S-H, Wu L, Yang W-C, Yang G-F. A highly sensitive and selective
fluorescent probe for thiophenol designed via a twist-blockage strategy. Anal Chem
2016;88:2266–72.
[23] Ye S, Hananya N, Green O, Chen H, Zhao A, Shen J, Shabat D, Yang D. A highly
selective and sensitive chemiluminescent probe for real-time monitoring of
hydrogen peroxide in cells and animals. Angew Chem Int Ed 2020;59:14326–30.
[24] Arias LA, Adkins S, Nagel CJ, Bach RD. Epoxidation of alkenes with
trichloroacetonitrile/hydrogen peroxide in a neutral biphasic solvent system. J Org
Chem 1983;48:888–90.
[40] Jones II G, Jackson WR, Choi CY, Bergmark WR. Solvent effects on emission yield
and lifetime for coumarin laser dyes. Requirements for a rotatory decay
mechanism. J Phys Chem 1985;89:294–300.
[25] Nad S, Kumbhakar M, Pal H. Photophysical properties of coumarin-152 and
coumarin-481 dyes: unusual behavior in nonpolar and in higher polarity solvents.
J Phys Chem A 2003;107:4808–16.
[41] Larsen EC, DiGennaro JA, Saito N, Mehta S, Loegering DJ, Mazurkiewicz JE,
Lennartz MR. Differential requirement for classic and novel PKC isoforms in
respiratory burst and phagocytosis in RAW 264.7 cells. J Immunol 2000;165:
2809–17.
[26] Barik A, Kumbhaker M, Nath S, Pal H. Evidence for the TICT mediated
nonradiative deexcitation process for the excited coumarin-1 dye in high polarity
protic solvents. Chem Phys 2005;315:277–85.
[42] Zhou Q, Liu C, Liu W, Zhang H, Zhang R, Liu J, Zhang J, Xu C, Liu L, Huang S,
Chen L. Rotenone induction of hydrogen peroxide inhibits mTOR-mediated S6K1
and 4E-BP1/eIF4E pathways, leading to neuronal apoptosis. Toxicol Sci 2015;143:
81–96.
[27] Raju BB, Varadarajan TS. Substituent and solvent effects on the twisted
intramolecular charge transfer of three new 7-(diethylamino)coumarin-3-aldehyde
derivatives. J Phys Chem 1994;98:8903–5.
8