A two-photon benzocoumarin-NBD dyad for highly selective and sensitive ratiometric detection of H2S in biological samples

Article abstract of DOI:10.1016/j.dyepig.2020.108906

Hydrogen sulfide (H₂S), the third endogenous gas signaling transmitter, plays an important role in the basic signal conduction process of human physiology and pathology. It is critical to monitor the dynamic change of H₂S in living organisms. In this article, a two-photon ratiometric fluorescent probe (BCD) based on the benzocoumarin-NBD framework was first designed and synthesized. The probe showed large emission separation (about 115 nm), excellent selectivity, and high sensitivity. Moreover, it could be applied to ratiometric image endogenous H₂S biosynthesis in the living cells and zebrafish.

Full text of DOI:10.1016/j.dyepig.2020.108906

Journal Pre-proof
A two-photon benzocoumarin-NBD dyad for highly selective and sensitive ratiometric
detection of H S in biological samples
2
Jingshuo Gao, Xu Jia, Zhenbo Guo, Shuhan Yang, Lingyu Wei, Pingzhu Zhang, Chao
Wei, Guofeng Chen, Xiaoliu Li
PII:
S0143-7208(20)31603-X
DOI:
https://doi.org/10.1016/j.dyepig.2020.108906
DYPI 108906
Reference:
To appear in: Dyes and Pigments
Received Date: 9 May 2020
Revised Date: 1 September 2020
Accepted Date: 29 September 2020
Please cite this article as: Gao J, Jia X, Guo Z, Yang S, Wei L, Zhang P, Wei C, Chen G, Li X, A two-
photon benzocoumarin-NBD dyad for highly selective and sensitive ratiometric detection of H S in
2
biological samples, Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2020.108906.
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© 2020 Published by Elsevier Ltd.

Author Statement
Jingshuo Gao: Conceptualization, Synthesis, Spectra test, Data curation, Writing - original draft.
Xu Jia: Conceptualization, Bioimaging, Data curation. Zhenbo Guo: Cytotoxicity tests, Data
curation. Shuhan Yang: Resources, Investigation. Lingyu Wei: Investigation, Validation. Pingzhu
Zhang: Data curation, Formal analysis, Supervision. Chao Wei: Conceptualization, Writing -
review & editing, Funding acquisition. Guofeng Chen: Supervision, Project administration,
Funding acquisition. Xiaoliu li: Supervision, Language modification.

Highlights
•A two-photon ratiometric fluorescent probe was developed based on
the benzocoumarin-NBD framework.
•The probe exhibited two-photon excitation, large emission separation,
and high selectivity and sensitivity to H2S.
•The probe could be used to ratiometric image Cys and PPG induced
endogenous H2S biosynthesis in the living cells and zebrafish.

A two-photon benzocoumarin-NBD dyad for highly selective and sensitive
ratiometric detection of H2S in biological samples
Jingshuo Gao§, Xu Jia§, Zhenbo Guo, Shuhan Yang, Lingyu Wei, Pingzhu Zhang, Chao
Wei*, Guofeng Chen*, Xiaoliu Li
College of Chemistry and Environmental Science, Key Laboratory of Chemical Biology
of Hebei Province, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis
(Ministry of Education), Hebei University, Baoding 071002, P. R. China
Email address: weichao@hbu.cn (C. Wei), chenguofeng@hbu.cn (G. Chen).
§ These authors contributed equally to this work.
Abstract: Hydrogen sulfide (H2S), the third endogenous gas signaling transmitter,
plays an important role in the basic signal conduction process of human physiology
and pathology. It is critical to monitor the dynamic change of H2S in living organisms.
In this article, a two-photon ratiometric fluorescent probe (BCD) based on the
benzocoumarin-NBD framework was first designed and synthesized. The probe
showed large emission separation (about 115 nm), excellent selectivity, and high
sensitivity. Moreover, it could be applied to ratiometric image endogenous H2S
biosynthesis in the living cells and zebrafish.
Keywords: H2S; Dual-photon; Ratiometric; Benzocoumarin-NBD; Bioimaging.
1. Introduction
Hydrogen sulfide (H2S), the third endogenous gas transmitter following with nitric
oxide and carbon monoxide, takes part in the basic signal conduction process of
human physiology and pathology [1]. The imbalance of endogenous H2S is related to
many diseases, such as periodontitis [2], pouchitis [3], intestinal diseases [4],
Alzheimer’s disease [5] and cardiovascular disease [6]. Therefore, looking for an
effective method to monitor the dynamic change of endogenous H2S in the biological
system for the diagnosis of related diseases and the research of biological processes
is of great research significance.
Conventional detection methods for H2S, such as colorimetry [7], electrochemical
analysis [8], and gas chromatography [9], suffered from tedious preparation
sequence, somewhat time-consuming, and expensive equipment required.
Fluorescent probe stands out as one of the most attractive assay with its high
sensitivity, adjustable selectivity, non-destructive and simple operation, which is an
effective bioimaging technology in living cells, tissues, and in vivo [10-13]. At present,
based on the reducibility [14-18], nucleophilicity [19-23], and formation CuS
precipitation of H2S [24-26], numerous “off-on” and ratiometric fluorescent probes
have been reported. Especially, the ratiometric fluorescent probes with the
dual-wavelength calibration function could avoid interference of the external
environment, and further provide the accurately quantitive information of H2S in
cells and in vivo. As is well-known, Förster resonance energy transfer (FRET) was a
popular strategy to construct ratiometric probes with the FRET donor, FRET acceptor,
and nonconjugated linker. The FRET-based ratiometric probes are characterized by

adjustable excitation and emission wavelengths and flexible placement of the
recognition group in the donor or the receptor. Until now, azide and dinitrophenyl
ether modified fluorophore and anthocyanidin-analog dye have been used to
prepare FRET-based ratiometric H2S probes [27-29]. In 2013, The selective thiolysis of
NBD (7-nitro-1,2,3-benzoxadiazole) amine by H2S was first used to construct an
off-on H2S fluorescent probe based on the FRET mechanism by us [30]. And then,
two organelle-targeting ratiometric H2S fluorescent probes based on the
coumarin-NBD framework were separately reported by us and the Yi group [31,32].
Unfortunately, the two probes could not ratiometric image the endogenous H2S in
living cells. Meanwhile, two-photon fluorescent probes, excited using a long
wavelength (generally > 700 nm), could acquire deep-tissue imaging by improved
sensitivity[14,15]. For all we know, a two-photon FRET-based ratiometric H2S
fluorescent probe using NBD amine as recognition moiety has yet to be reported.
Herein, a new two-photon FRET-based ratiometric fluorescent probe (BCD) was
rationally designed and prepared using a benzocoumarin-NBD dyad. The effective
spectral overlap between the emission spectra of benzocoumarin and the absorption
spectra of NBD showed a high FRET efficiency of probe BCD. The probe showed a
large emission separation, obvious selectivity, and high sensitivity. Depends on the
two-photon absorption capacity of benzocoumarin [33], probe BCD was used to
ratiometric image endogenous H2S biosynthesis in the living cells and zebrafish.
2. Experimental section
2.1 Material and instrument
All the materials and solvents for the synthesis were of analytical grade, unless
otherwise specified, were used without further purification. Silica gel for column
chromatography was obtained from Qingdao Ocean Chemical Co., Ltd.
The melting points were obtained on a TECH X-4 micro melting point apparatus. 1H
13
NMR and C NMR spectra were determined on a Bruker Avance 400 or 600 MHz
spectrometers at 25 °C. The mass spectrum was recorded on an Agilent LC/MSD XCT
mass spectrometer. UV-vis spectra and fluorescence spectra were collected on
Purkinje TU-1900 and Hitachi-7000, respectively.
2.2 Synthesis of BCD
Compound 3 and 4 were synthesized according to the reported methods[31, 34]. A
mixture of
3
(120 mg, 0.50 mmol),
4
(125 mg, 0.50 mmol),
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (119 mg, 0.62
mmol) and N,N-Diisopropylethylamine (DIPEA) (110 μL, 0.63 mmol) was dissolved in
N,N-dimethylformamide (DMF) (5 mL). After stirred overnight, the reaction mixture
was poured into 100 mL ice water to obtain the orange precipitate. The crude was
chromatographed on silica gel to give an orange solid (83 mg, 35.21%), TLC (ethyl
o
1
acetate/petroleum = 1/1), Rf = 0.12. Mp: 159.2-161.5 C; H NMR (400 MHz,
DMSO-d6): δH 9.17 (s, 1H, CH=), 8.65 (d, J = 8.00 Hz, 1H, CH=), 8.56 (d, J = 8.00 Hz, 1H,

CH=), 8.32 (d, J = 8.00 Hz, 1H, CH=), 8.14 (d, J = 8.00 Hz, 1H, CH=), 7.82-7.79 (m, 1H,
CH=), 7.70 (t, J = 8.00 Hz, 3H, CH=), 6.71 (d, J = 8.00 Hz, 1H, CH=), 4.36 (s, 2H, NCH2),
4.22 (s, 2H, NCH2), 3.99 (s, 2H, NCH2), 3.86 (s, 2H, NCH2); 13C NMR (150 MHz,
DMSO-d6): 163.61, 157.72, 153.65, 145.41, 144.74, 139.15, 136.26, 134.19, 129.96,
128.97, 128.90, 128.57, 126.33, 123.28, 122.44, 121.55, 116.62, 112.68, 103.56,
49.42, 48.57, 48.30, 45.04, 41.21. m/z (ESI): 494.1 [M+Na]+.
2.3 UV-vis and fluorescence spectra measurement
Probe BCD (2 mM) was dissolved in absolute DMSO to prepare the stock solutions
and stored separately in the refrigerator. Anhydrous sodium sulfide was used as the
hydrogen sulfide resource. All UV-vis and fluorescence spectra were measured in PBS
solution (20 mM, 20% DMF, pH = 7.40) by excitation at 358 nm with the slit widths
were both 2.5 nm. The concentration of the probe for spectra measurement was
used at 10 μM.
2.4 Cell imaging
HeLa cells were cultured in high-glucose DMEM supplemented with 10% fetal
bovine serum at 37 °C and 5% CO2. For bioimaging of exogenous H2S, HeLa cells were
treated with BCD (10 μM) for 30 min and then treated with different concentrations
of Na2S (50 μM, 100 μM) for another 30 min. To image endogenous H2S biosynthesis,
the cells were incubated with DL-pmpargylglycine (PPG) (200 μM) and D-Cysteine
(500 μM) for 30 min, respectively, and then treated with BCD (10 μM) for 30 min. All
types of cells were rinsed with PBS buffer for confocal fluorescence microscopy. Cells
were imaged on a confocal microscope (Zeiss LSM 880) with a 20 × objective lens.
The fluorescence images were captured from the blue channel of 420-470 nm and
red channel of 525-625 nm with excitation at 405 nm.
2.6 Zebrafish imaging
Zebrafish were purchased from Eze-Rinka Company (Nanjing, China). The
three-day-old zebrafish were pre-treated with probe (10 μM) for 30 min and then
incubated with 50 μM Na2S or 200 μM Na2S respectively for another 30 min for
imaging exogenous H2S. The larvae were pre-incubated with Cys (500 μM) for 30 min
and then cultured with probe (10 μM) for an additional 30 min for imaging
endogenous H2S. Zebrafish were imaged on a confocal microscope (Zeiss LSM 880)
with two-photon excitation. The fluorescence images were captured from the blue
channel of 420-470 nm and red channel of 525-625 nm with excitation at 760 nm.
3. Results and discussion
3.1 Design and synthesis of BCD

Because of its deep penetration capacity and little interference from the
background fluorescence of biological tissues, a ratiometric fluorescent probe with
two-photo absorption ability has excellent application value in the field of bioimaging.
Benzocoumarin is a widely used fluorophore with a two-photon absorption ability.
The overlap between the emission spectra of compound 3 (FRET donor) and the
absorption spectra of compound 4 (FRET acceptor)showed the possibility of
occurrence of FRET between the benzocoumarin and NBD amine (Fig. S1). Thus, the
benzocoumarin-NBD dyad could be used to design the ratiometric H2S probe with
two-photon function. As shown in scheme 1, using benzosalicylaldehyde and diethyl
malonate as starting materials, compound 3 was prepared after Perkin and hydrolysis
reactions. After an amidation reaction with compound 4, probe BCD was obtained as
an orange solid. The structure of probe BCD was characterized by 1H NMR, 13C NMR,
and Electrospray ionization mass spectra (ESI-MS) (Supporting Information).
Scheme1. Synthesis of probe BCD for H2S detection
3.2 Spectral response of BCD towards H2S
We firstly investigated the UV-vis absorbance spectra change of probe BCD before
and after reaction with H2S in PBS buffer (20% DMF, pH=7.40). As shown in Fig. 1a,
the probe alone showed two absorption peaks at 360 nm and 490 nm, which belong
to benzocoumarin and NBD amine, respectively. After reaction with H2S, the
absorption peak at 490 nm was decreased and a new peak at 540 nm appeared. The
well-defined isosbestic point at 520 nm indicates a clean chemical reaction. The color
of the reaction solution changed from light yellow to red. The above phenomenon
implied the produce of nitrobenzofurazan thiol (NBD-SH), which is consistent with
our previous work [30-31]. The concentration-dependent fluorescence spectra
changes in the presence of H2S were also checked. Two emission bands centered at
440 nm and 555 nm belong to benzocoumarin and NBD amine, respectively. After
reaction with H2S, the emission peak at 555 nm gradually weakens, while the peak at
440 nm gradually strengthened (Fig. 1b). The distinct gap between the two bands
was 115 nm, which is favorable for the dual emission ratiometric detection of H2S.
Using quinine bisulfate as a reference, fluorescence quantum yield of probe BCD
before and after reaction with H2S was measured to be 0.14 and 0.40, respectively.

By fitting the ratio of emission intensities (I440 nm/I555 nm) and the concentration of H2S
( 0-150 μM), a good linear relationship appeared (r = 0.9940). The detection limit was
calculated to be 59 nM, which is low enough to effectively assay biological sulfides
(Fig. S2)[35].
Fig. 1. a) Time-dependent UV-vis absorption spectra of probe BCD (10 μM) upon
reaction with H2S (100 μM) at 25 °C. Inset: the photograph of probe solution before
and after reaction of H2S. b) Fluorescence spectra of probe BCD (10 μM) in the
presence of different amounts of H2S (0, 5, 10, 15, 20, 30, 40, 50, 75, 100, and 150
μM). Inset: The linear relationship between the ratio of I440 nm/I555 nm and the
concentrations of H2S in PBS (20% DMF, pH=7.40).
The selectivity of probe BCD for H2S was further investigated using some
2-
2-
biologically relevant species, including metal ions (Zn2+, Fe3+), anions (SO3 , SO4 ,
S2O3 , NO2 ) and the species with redox ability (H2O2, ClO-, Vc, Cys, Hcy, GSH). As
shown in Fig. 2, the ratiometric fluorescence exhibits a significant enhancement
(about 10 folds) when the probe is treated with H2S. While other species do not
induce obvious change. Furthermore, the fluorescent response in PBS at different pH
values was also investigated and found that probe BCD could detect H2S with a wide
pH range of 5.0-10.0 (Fig. S3). The above results implied that the probe is highly
selectivity and sensitivity to H2S, and could work efficiently at physiological
conditions.
2-
-
Fig. 2. a) Fluorescence responses of probe BCD (10 μM) to various biologicallu

relevant species in PBS (20% DMF, pH=7.40) for 1 h. Columns 1-14 correspond to: 1.
2-
2-
Probe only; 2. SO3 (100 μM); 3. SO4 (100 μM); 4. S2O32- (100 μM); 5. H2O2 (100 μM);
6. ClO- (100 μM); 7. NO2 (100 μM); 8. Zn2+ (100 μM); 9. Fe3+ (100 μM); 10. Vc (100
-
μM); 11. Cys (1 mM); 12. Hcy (1 mM); 13. GSH (5 mM); 14. H2S (100 μM). b)
2-
Fluorescence responses of probe BCD (10 μM) incubated with various species (SO3 ,
100 μM; Cys and Hcy, 1 mM; GSH, 5 mM) in the presence of H2S (100 μM).
3.3 One-photon fluorescence imaging of H2S in living cells
Encouraged by the positive properties of probe BCD, we decided to evaluate its
biological applications for imaging of H2S in living cells. BCD showed no obvious
cytotoxicity at concentrations below 25 μM using MTT assay (Fig. S4). HeLa cells were
first treated with BCD (10 μM) for 30 min and followed by the addition of different
concentrations of Na2S (50 μM, 100 μM) for another 30 min respectively. As shown in
Fig. 3a, the cells treated with the probe only exhibit no obvious fluorescence signal in
the blue channel and strong fluorescence in the red channel. After treated with Na2S,
the cells showed stronger fluorescence in the blue channel and weaker fluorescence
in the red channel (Fig. 3b and c). The significant fluorescence ratiometric changes
showed the probe BCD has good cell permeability.
Fig. 3. Fluorescence images of exogenous H2S in living HeLa cells. (a) The images of
HeLa cells were treated with probe BCD (10 μM) for 30 min. (b-c) The images of cells
first treated with BCD (10 μM) for 30 min, and then incubated with Na2S (50 μM and
100 μM) for another 30 min. (d) The ratiometric intensity of the blue channel and red
channel of a-c. (Excitation, 405 nm; Blue channel, 420-470 nm; Red channel, 525-625
nm; Scale bar, 20 μm)
The bioimaging of the formation and inhibition of endogenous H2S was further
performed and the results were shown in Fig. 4. Control HeLa cells showed moderate

fluorescence ratiometric change (Fig. 4a and 4d). It is reported
cystathionine-β-synthase (CBS) could catalyze the formation of endogenous H2S from
D-cysteine, and DL-pmpargylglycine (PPG) could inhibit the activity of CBS [36].
Therefore, the D-cysteine pre-treated cells showed the largest ratiometric
fluorescence changes (Fig. 4b and 4d), while the PPG pre-treated cells generate
minimal ratio change (Fig. 4c and 4d). The results implied that probe BCD could
image endogenous H2S in a ratiometric manner.
Fig. 4. Fluorescence images of endogenous H2S in living HeLa cells. (a) The images of
HeLa cells were treated with probe BCD (10 μM) for 30 min. (b, c) The images of cells
pretreated with a stimulant (Cys, 500 μM, b; PPG, 200 μM, c) for 30 min and then
incubated with BCD (10 μM) for another 30 min. (d) The ratiometric intensity of the
blue channel and red channel of a-c. (Excitation, 405 nm; Blue channel, 420-470 nm;
Red channel, 525-625 nm; Scale bar, 20 μm)
3.4 Two-photon fluorescence imaging of H2S in zebrafish.
The excitation of coumarin dye is generally in visible light region, which is difficult
to image H2S in zebrafish using one-photon excitation. Considering the two-photon
absorption property of probe BCD, the probe was eventually be used for imaging H2S
in vivo. Three-day old zebrafish larvae were used as the following experiments. The
zebrafish treated with the probe (10 μM) was used as the control group, and the
zebrafish first treated with the probe (10 μM) and then incubated with Na2S as the
experimental groups. When excited at 760 nm, the control zebrafish displayed strong
red fluorescent (Fig. 5a), while the experimental zebrafish showed the brighter blue
fluorescence and the weaker red fluorescence with increasing Na2S concentration
(Fig. 5b and 5c). The results demonstrated that probe BCD has good tissue
permeability and could be applied for the two-photo ratio imaging of exogenous H2S
in zebrafish.

Fig. 5. Two-photon fluorescence images of exogenous H2S in zebrafish. (a) The
images of zebrafish were treated with BCD (10 μM) for 30 min. (b, c) The images of
zebrafish pretreated with BCD (10 μM) for 30 min and then treated with Na2S ( 50
μM, b; 200 μM, c) and for another 30 min (Excitation, 760 nm; Blue channel, 420-470
nm; Red channel, 525-625 nm; Scale bar, 20 μm).
To further verify whether the probe could be used to monitor the dynamic change
of the endogenous of H2S in zebrafish. The larvae were firstly incubated with D-Cys
(500 μM) for 30 min and then treated with probe (10 μM) for an additional 30 min.
Surprisingly, using D-Cys to promote the production of H2S, obvious ratio
fluorescence can be observed with two-photon excitation at 760 nm (Fig. 6). The
results demonstrated that probe BCD is suitable for the imaging of the dynamic
change of endogenous H2S in vivo based on its deep tissue penetration.

Fig. 6. Two-photon fluorescence images of endogenous H2S in zebrafish. (a) The
images of zebrafish pretreated with BCD (10 μM) for 30 min; (b) The images of
zebrafish pretreated with D-Cys (500 μM) for 30 min and then incubated with BCD
(10 μM) for another 30 min (Excitation, 760 nm; Blue channel, 420-470 nm; Red
channel, 525-625 nm; Scale bar, 20 μm).
4. Conclusions
In summary, a two-photon ratiometric H2S fluorescent probe (BCD) was designed
and synthesized using the benzocoumarin-NBD framework, which has a large
emission separation (115 nm), good selectivity, and high sensitivity. The probe
showed excellent cell and tissue permeability and could be applied for monitoring
endogenous H2S biosynthesis in living cells and zebrafish. More small molecular
fluorescent probes with better imaging performance are being developed.
Acknowledgments
This work was supported by the Natural Science Foundation of Hebei Province
(B2018201234), the Colleges and Universities Science Technology Research Project of
Hebei Province (QN2017015), the second teaching reform project of College of
Chemistry and Environmental Science, Hebei University (H2-02010), and the
Post-graduate Innovation Fund Project of Hebei University (hbu2020ss018).
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Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: