A julolidine-fused coumarin-NBD dyad for highly selective and sensitive detection of H2S in biological samples

Article abstract of DOI:10.1016/j.dyepig.2018.12.064

A new julolidine-fused coumarin-NBD probe 2 for H₂S detection is rationally designed based on the DFT caculations. This improved probe exhibits faster and larger off-on response as well as higher sensitivity compared with the previous coumarin-NBD probe 1. Moreover, 2 possesses excellent selectivity and good biocompatibility, which can be employed to image H₂S in living cells and in zebrafish.

Full text of DOI:10.1016/j.dyepig.2018.12.064

Accepted Manuscript
A julolidine-fused coumarin-NBD dyad for highly selective and sensitive detection of
H S in biological samples
2
Ismail Ismail, Dawei Wang, Zhenghua Wang, Dan Wang, Changyu Zhang, Long Yi,
Zhen Xi
PII:
S0143-7208(18)32384-2
https://doi.org/10.1016/j.dyepig.2018.12.064
DYPI 7269
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 28 October 2018
Revised Date: 19 December 2018
Accepted Date: 28 December 2018
Please cite this article as: Ismail I, Wang D, Wang Z, Wang D, Zhang C, Yi L, Xi Z, A julolidine-fused
coumarin-NBD dyad for highly selective and sensitive detection of H S in biological samples, Dyes and
2
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2018.12.064.
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ACCEPTED MANUSCRIPT
A julolidine-fused coumarin-NBD dyad for highly selective and
sensitive detection of H S in biological samples
2
a
a
a
a
b
Ismail Ismail, Dawei Wang, Zhenghua Wang, Dan Wang, Changyu Zhang, Long
b,c*
a,c
Yi, and ZhenXi *
a
State Key Laboratory of Elemento-Organic Chemistry, and Department of Chemical Biology,
National Pesticide Engineering Research Center, College of Chemistry, Nankai University, Tianjin
3
00071, China. Fax: +86 022-23500952; Tel: +86 022-23504782. E-mail: zhenxi@nankai.edu.cn
b
Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, 15
Beisanhuan East Road, Chaoyang District, Beijing 100029, China. E-mail:
yilong@mail.buct.edu.cn
c
Collaborative Innovation Center of Chemical Science and Engineering, Nankai University,
Tianjin, China.
A B S T R A C T
A new julolidine-fused coumarin-NBD probe 2 for H S detection is rationally
2
designed based on the DFT caculations. This improved probe exhibits faster and
larger off-on response as well as higher sensitivity compared with the previous
coumarin-NBD probe 1. Moreover, 2 possesses excellent selectivity and good
biocompatibility, which can be employed to image H S in living cells and in
2
zebrafish.
Keywords: Julolidine-fused coumarin-NBD probe, H S detection, Cell imaging,
2
Zebrafish imaging
1

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1
. Introduction
H S has long been known as a toxic gas, but recent studies indicate that
2
endogenously produced H S has important physiological functions, which is named as
2
the third gasotransmitter after nitric oxide and carbon monoxide [1]. In mammals,
endogenous H S could be enzymatically produced by three distinctive pathways,
2
including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and
3
-mercaptopyruvate sulfur-transferase (3-MST) along with cysteine aminotransferase
(
CAT) [2]. Accumulative evidence suggests that H S influences a wide range of
2
physiological and pathological processes, including modulation of blood vessel tone
and cardioprotection [3], endogenous stimulator of angiogenesis [4], and
mitochondrial bioenergetics [5]. H S inhibits nuclear factor-kB activation in oxidized
2
low-density lipoprotein-stimulated macrophage [6]. H S also plays an important role
2
in tumor biology, and it is suggested that both inhibition of H S biosynthesis and
2
elevation of H S concentration beyond a certain threshold could exert anticancer
2
effects [4b,7]. In plants, H S participates in different processes including seed
2
germination, plant growth, development and acquisition of stress tolerance [8-10].
Nevertheless, the pharmacological characters of H S and the precise mechanisms by
2
which H S may be involved in vivo still remain largely unclear. Therefore, efficient
2
tools for visualization of biological H S should be useful in further exploring H S
2
2
biology and even for diagnosis of H S-related diseases.
2
Fluorescence-based method has recently emerged as an efficient approach for
in-situ and real-time detection of H S in living biological systems [11-19]. One major
2
challenge in the development of H S probes is the discovery of a chemical reaction to
2
effectively separate the reactivity of biothiols and H S in auqeous buffer. To address
2
this challenge, chemical reactions including nucleophilic addition [12], H S-triggered
2
reduction [13], copper precipitation [14], thiolysis of dinitrophenyl ether [15],
cleavage of C-N bonds (NBD amines) [16], thiolysis of NBD ethers/thioethers [17]
and cleavage of C=C bonds [18] have been successfully employed to develop these
molecular probes. For example, we reported the first H S-specific fluorescence probe
2
2

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1
based on cleavage of C-N bonds [16a]. Stoke shift, limit of detection (LOD) and
turn-on fluorescence fold (FI) of the probe 1 were 75 nm, 9 µM, and 45-fold,
respectively, which are potentially disadvantage for sensing the biological H S that
2
normally exists in the submicromolar level range.
Recently, Grimm et al. showed that replacing the N,N-dialkyl group with azetidine
group can greatly improve the brightness and quantum yield of the fluorophores [22].
Yang et al. reported that blocking the twisting N,N-diethylamino group of coumarin
can improve the fluorescence properties of the fluorophore, and in turn, enhance
sensing performances [23]. We envision that such twisting N,N-diethylamino group of
coumarin could also be employed for the development of improved NBD-based probe.
Herein, we rationally design a new julolidine-fused coumarin-NBD probe 2 for H2S
detection (Scheme. 1). To our delight, the probe 2 showed excellent sensing
performances with green-light emitting and was successfully used for bioimaging in
living cells and in zebrafish.
Scheme. 1 Rational design of an improved fluorescence probe for H S detection based on the
2
thiolysis of NBD amine. a) The probe 1 was used for H S-specific detection with production of
2
blue-emitting 1a [16a]. b) The julolidine-fused coumarin-NBD dyad 2 was used for H S-specific
2
detection with production of green-emitting 2a and more than 200-fold off-on response during H2S
activation.
2
. Experimental section
2
.1. Reagents and apparatus
3

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All chemicals and solvents used for synthesis were purchased from commercial
suppliers and used directly in the experiment without further purification (J&K
1
13
Scientific Ltd., Beijing, China). H NMR and C NMR spectra were recorded on a
Bruker 400 spectrometer with CDCl , CD Cl or DMSO-d as solvent. Chemical
3
2
2
6
shifts are reported in parts per million, relative to internal standard tetramethylsilane
δ = 0.00 ppm). High-resolution mass spectra (HRMS) were obtained on a 6520
(
Q-TOF LC/MS (Agilent, Santa Clara, CA). The UV-visible spectra were recorded on
a CARY 100 Bio (Varian, USA). Fluorescence study was carried out using Varian
Cary Eclipse spectrophotometer at 25 °C. The cells/zebrafish bioimaging were
performed on a confocal microscope (Olympus FV1000 UPLSAPO40X).
2
.2. Synthesis of probe2
Scheme 2. Synthesis route for probe 2. Reagents and conditions: a) diethyl malonate, piperidine,
CH CN, reflux; b) Conc. HCl; c) 1-boc-piperazine, DCC, DMAP, CH Cl ; d) trifluoroacetic acid,
3
2
2
CH Cl ; e) 4-chloro-7-nitro-1,2,3-benzoxadiazole, Et N, CH Cl .
2
2
3
2
2
A solution of 8-hydroxyjulolidine-9-carboxaldehyde (4.36 g, 20 mmol), diethyl
malonate (6.41 g, 40 mmol) and piperidine (2 mL) in ethanol (60 mL) was refluxed
for 24 h. After cooled to room temperature, the solvent was evaporated under vacuum
and the resulting residue was purified by column chromatography (petroleum ether:
1
ethyl acetate = 4: 1, v/v), yielding an orange solid 3 (4.95 g, 79%); mp 139-141 ºC; H
NMR (400 MHz, DMSO-d ) δ 8.38 (s, 1H), 7.17 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H),
6
3
.34-3.28 (m, 4H), 2.75-2.64 (m, 4H), 1.93-1.80 (m, 4H), 1.27 (t, J = 7.2 Hz, 3H).
A solution of 3 (2.0 g, 6.38 mmol) in concentrated HCl solution (20 mL) was
stirred at 25 ºC for 24 h. After the reaction was completed, water (100 mL) was added
to the solution. The mixture was extracted by ethyl acetate (50 mL) for three times,
the combined organic layer was washed by H O (50 mL) and brine, dried over
2
4

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anhydrous Na SO and concentered under reduced pressure to afford compound 4 as
2
4
1
orange solid (1.55 g, 85%), mp 223-225 ºC, H NMR (400 MHz, DMSO-d ) δ 12.20
6
(bs, 1H), 8.46 (s, 1H), 7.24 (s, 1H), 2.72 (dd, J = 13.2, 6.8 Hz, 4H), 2.51-2.50 (m, 4H),
1
5
.95-1.81 (m, 4H).
A solution of 4 (1.0 g, 3.5 mmol), 1-boc-piperazine (0.65 g, 3.5 mmol), DCC (1.1 g,
.26 mmol) and DMAP (4.9 mg, 0.04 mmol) in CH Cl (20 mL) was stirred at room
2
2
temperature for 12 h. After the reaction was completed, the precipitation was filtrated,
and the solvent was removed under reduced pressure. The resulting residue was
subjected to column chromatography on silica, yielding a yellow solid powder (1.46 g,
9
2%), which was further treated with 40 mL TFA/CH Cl (v/v= 1/1). After stirred at
2 2
room temperature for 3 h, the solvent was removed under the reduced pressure and
the residue was purified by the column to afford intermediate 2a (0.65 g, yield 83%),
1
mp 185-187 ºC, H NMR (400 MHz, CDCl ) δ 7.81 (s, 1H), 6.90 (s, 1H), 3.86 (bs,
3
2
H), 3.50 (bs, 2H), 3.31 (dd, J = 10.8, 5.2 Hz, 4H), 3.09 (bs, 4H), 2.87 (t, J = 6.4 Hz,
+
2
H), 2.76 (t, J = 6.4 Hz, 2H), 2.02-1.92 (m, 4H). HRMS (ESI): m/z 354.1815 [M+H]
+
(
calcd for C H N O , 354.1812).
2
0
24
3
3
A mixture of intermediate 2a (130 mg, 0.37 mmol), NBD-Cl (73.4 mg, 0.37 mmol)
and Et N (100 µL) in CH Cl (20 mL) was stirred at room temperature overnight.
3
2
2
After removed the solvent under reduced pressure, the resulting residue was subjected
to column chromatography on silica (1% MeOH in CH Cl ), yielding a red solid 2
2
2
1
(
173 mg, 91%), mp 249-251 ºC, H NMR (400 MHz, CD Cl ) δ 8.45 (d, J = 8.8 Hz,
2 2
1
3
2
1
1
2
H), 7.84 (s, 1H), 6.96 (s, 1H), 6.35 (d, J = 8.8 Hz, 1H), 4.21 (s, 4H), 3.97 (s, 2H),
.71 (s, 2H), 3.37-3.27 (m, 4H), 2.86 (t, J = 6.4 Hz, 2H), 2.77 (t, J = 6.0 Hz, 2H),
1
3
.06-1.94 (m, 4H). C NMR (101 MHz, CD Cl ) δ 165.78, 159.46, 152.56, 147.51,
2
2
46.11, 145.21, 145.13, 144.95, 135.23, 126.07, 124.01, 119.58, 113.99, 107.70,
06.12, 102.91, 50.24, 49.82, 29.76, 27.50, 21.28, 20.36, 20.17. HRMS (ESI): m/z
+
+
99.1006 [M+H] (calcd for C H F N O , 299.1002).
1
4
14
3
2
2
2
.3. General procedure for spectroscopic studies
Studies were carried out in degassed phosphate-buffer (PB, 20 mM, pH 7.4).
5

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Probes were dissolved into DMSO to prepare the stock solutions (5 mM). For the
selectivity study, biologically relevant molecules were prepared as stock solutions of
5
00 mM, then appropriate amount of species were added to separate portions of the
probe solution and mixed thoroughly. Each reaction mixture was shaken uniformly
before emission spectra were measured. All measurements were performed in a 3 mL
corvette with 2 mL solution. Excitation, 448 nm; emission, 496 nm. The fluorescent
quantum yields for probes 1, 1a, 2 and 2a were determined using
6
-amino-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (Φ = 0.13, PB, 20 mM, pH
7
.4) as the standard substance [20]. Time-resolved fluorescence decay measurements
were carried out at room temperature on a picosecond-diode-laser-based,
time-correlated single-photon-counting (TCSPC) fluorescence spectrometer (FLS
9
80). All tested samples were dissolved in MeOH solution. All the decays were fitted
to a double exponential function.
2
.4. DFT calculations
DFT calculations of selected compounds were performed using the Gaussian 09
software package [21]. Geometry optimizations and unscaled frequency calculations
were carried out at the B3LYP/6-31G* level of theory.
Compd. HOMO/ev LUMO/ev ∆E/ev λex/nm λ /nm LOD/µM
Φ
τ(ns)
em
1
-5.78
-5.44
-5.59
-5.19
-2.69
-1.61
-2.69
-1.50
3.08
3.83
2.90
3.69
405
405
449
449
480
480
496
496
9
/
0.000073 ND
1
a
0.017
0.00083
0.81
3.53
ND
2
0.9
/
2
a
6.27
Table 1. Summary of frontier molecular orbital energy and optical properties of 1, 1a, 2, 2a.
2
.5. Cell culture and fluorescence imaging
A549 cells were cultured at 37 ºC, 5% CO in F12 supplemented with 10% fetal
2
bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 4 mM
L-glutamine. HEK-293A cells were cultured at 37 ºC, 5% CO in DMEM/HIGH
2
GLUCOSE (GIBICO) supplemented with 10% fetal bovine serum (FBS), 100 U/ml
penicillin, 100 µg/ml streptomycin, and 4 mM L-glutamine. The cells were
6

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maintained in exponential growth, and then seeded in glass-bottom 35 mm plate at the
4
density about 2 × 10 /well. Cells were passaged every 2-3 days and used between
passages 3 and 10. For living cell imaging, cells were first incubated with Na S (100
2
µM) for 30 min at 37 °C. Then, removed the culture medium and washed the cells
with PB (10 mM, pH 7.4) for three times, and treated with probe 2 (10 µM) for 30
min at 37 ºC. While the control cells were only treated with probe 2 at 37 ºC for 30
min. Emission was collected at green channel (510-550 nm) under 405 nm excitation.
2
.6. Fluorescence imaging of zebrafish
Zebrafishes, 3-7 days postfertilization, were purchased from Eze-Rinka Company
(Nanjing, China). The zebrafishes were cultured in 5 mL of embryo medium
supplemented with 1-phenyl-2-thiourea (PTU) in 6-well plates for 24 h at 30 °C. Then
the zebrafish was incubated with Na S (200 µM) or L-Cys (200 µM) for 4 h. After
2
removed the embryo medium and washed the zebrafish with PB (10 mM, pH 7.4) for
three times, the zebrafish was further incubated with probe 2 (10 µM) for 1 h at 30 °C,
and then imaged.
3
. Results and discussion
Fig. 1.Optimized structures and frontier orbital energy of 1(a), 1a (b), 2(c), and 2a(d). Data were
calculated by Gaussian 09 with B3LYP/6-31G(d) level.
To better understand the superiority of probe 2 over probe 1 in this work, density
functional theory (DFT) calculations were performed. As shown in Table 1 and Fig. 1,
the energy difference (∆E) of the highest occupied molecular orbital (HOMO) and the
7

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lowest unoccupied molecular orbital (LUMO) of probes 1 and 2 are 3.08 eV and 2.90
eV, respectively, which are lower than that of 1a (∆E = 3.83 ev) and 2a (∆E = 3.69 ev),
suggesting that fluorescence resonance energy transfer (FRET) could occur between
coumarin and NBD within both probes. Fluorescence of coumarin and absorbance of
NBD have an obvious overlap (Fig. S1), which also supports the possible occurrence
of the FRET effect between the two fluorophores. The energy difference of
fluorophore 2a is lower than 1a, suggesting that under light excitation, the electrons
of 2a in HOMO orbital can be more easily transferred to LUMO orbital than 1a to
give longer emission wavelength. Moreover, the electron-density in the HOMO or
LUMO orbital is much different for 2a, implying that there is an electron flow within
coumarin with a large intramolecular charge transfer (ICT) effect. Inspired by the
calculation results, we determined to synthesize probe 2 for H S sensing.
2
Probe 2 can be smoothly prepared in a five-step synthesis by using julolidine-fused
phenyl 3-carbaldehydeas the starting material with a total yield of 47% (Scheme 2).
1
13
The probe 2 was well characterized by H and C{1H} NMR spectroscopy as well as
high-resolution mass spectrometry (HRMS). The sensing mechanism of the probe 2
should be the thiolysis of NBD amine [16], which was confirmed by HRMS (Fig. S2).
The time-dependent absorbance spectra of 2 with H S also implied the thiolysis
2
product NBS-SH (Fig. S3) [17b].
With probe 2 in hand, we first tested the fluorescence of probe 2 in the absence and
presence of H S. The result showed that probe 2 exhibited nearly non-fluorescence (Φ
2
=
0.00083) in the PB buffer (20 mM, pH 7.4) compared to that of its fluorophore 2a
(Φ = 0.81). This should be majorly due to the strong intramolecular FRET effect in 2,
though the photo-induced electron transfer (PET) between nitro group in NBD and
coumarin could also contribute to a certain quenching effect [16d]. Upon reaction
with H S, the FRET effect disappeared and significant fluorescence increase was
2
observed for the probe 2, implying the great potency for H S sensing. The emission
2
peak of fluorophore 2a was 496 nm, showing a notable red shift than that of 1a.
8

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Fig. 2. (A) Time-dependent spectra change of 2 (5µM) when treated with Na S (500 µM) in PB
2
buffer (20 mM, pH 7.4) at room temperature. Inset: photographs of 2 without (left) and with (right)
Na S under UV analyzer (λ = 365 nm). (B) Time-dependent fluorescent intensity at 496 nmof 2
2
ex
in the absence (red dot) or presence (back dot) of H S.The red line represents the best fitting to
2
-
1
give kobs of 0.23 min . (C) Spectra change of 2 (5 µM) when treated with Na S (0-500 µM) in PB
2
buffer (20 mM, pH 7.4) at room temperature. (D) The fluorescence life-time of 1a and 2a.
Encouraged by the primary results, we examined the time-dependent fluorescence
response of 2with H S (Na S was used as an equivalent). As shown in Fig. 2A, a
2
2
strong emission peak appeared at 496 nm. The fluorescent intensity of 2 increased
about 40-fold within 1 min; and the fluorescence reached the steady state at 15 min
with more than 200-fold intensity enhancement at 496 nm (Fig. 2B). The LOD of 2
for H S detection was determined as 0.9 µM using the 3σ/k method [16]. Compared to
2
its parent probe 1, probe 2 showed much improved response time (1: 45 min vs. 2: 15
min), LOD (1: 9 µM vs. 2: 0.9 µM) and fluorescent enhancement (1: 45 folds vs. 2:
2
00 folds). Specifically, compared with 1a (Φ = 0.017), the quantum yield of 2a (Φ =
0
.81) improved more than 47 folds, and the fluorescence life-time of 2a (τ = 6.27 ns)
9

ACCEPTED MANUSCRIPT
was also much longer than 1a (τ = 3.53 ns) (Fig. 2D and Table 1). These data are
consistent with the theoretical calculations, indicating that the twisting-blockage of
N,N-diethylamino group can significantly improve the performances of the
fluorescence probe 2.
Fig. 3. Fluorescence intensity at 496 nm of 2 (5µM) upon reacting with species (1 mM, black bar)
2
-
-
or H S and species (1 mM, red bar) in PB (pH 7.4) for 15 min: 1) 2 only; 2) SO ; 3) CH COO ; 4)
2
2
3
3
-
-
-
-
-
-
C O ; 5) F ; 6) NO ; 7) I ; 8) HCO ; 9) H O ; 10) ClO ; 11) Cys; 12) Hcy; 13) GSH; 14)
2
4
2
3
2
2
mercaptoethanol.
One of the major requirements for a fluorescent probe is that it must exhibit a
selective response towards the targeted analytes but not for other competing species.
In order to confirm that the turn-on response of 2 was selectively caused by H S, we
2
investigated the selectivity of 2 against other commonly encountered analytes (Fig. 3).
2
-
Different biologically relevant species, such as SO , mercaptoethanol, GSH, cysteine,
3
-
-
2-
-
-
-
-
Hcy, H O , ClO , CH COO , C O , NO , F , I and HCO were evaluated. The
2
2
3
2
4
2
3
results showed that probe 2 exhibited high selectivity toward H S among the selected
2
-
-
2-
analytes. Although four species (mercaptoethanol, HCO , I and SO ) induced a
3
3
slight fluorescence enhancement, which was much lower in comparison with that of
H S. We also tested the fluorescence of the probe with these analytes in the presence
2
of H S (Fig. 3, red bar). The results implied that all analytes did not interfere with the
2
1
0

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selective sensing of the probe toward H S. Furthermore, pH-dependent experiment
2
indicated that 2 could sense H S within a wide pH range 6-11 (Fig. S4). Obviously,
2
the largest fluorescence enhancement was appeared at around pH 7.4, suggesting that
2
could work efficiently at physiological conditions.
Inspired by the excellent properties of probe 2, we further explored its feasibility in
detection of H S in living biological systems. Firstly, methyl thiazolyl tetrazolium
2
(MTT) assays were carried out, which indicated that 2 did not possess obvious
cytotoxicity against HEK293A cells even at the concentration up to 20 µM (Fig. S5).
Subsequently, we examined whether 2 could be used to detect intracellular H S in
2
living cells. HEK293A cells were incubated with Na S (100 µM) for 30 min, washed
2
by PB, then stained with 2 (10 µM) and imaged using a confocal fluorescence
microscopy immediately. Cells incubated with 2 (10 µM) alone were used as control
experiment. The results indicated that no fluorescence was observed for 2-loaded cells
(
Fig. 4A), while cells treated with H S showed significantly bright green fluorescence
2
(Fig 4B). Similar tests were also performed in A549 cells (Fig. 4C, 4D). The results
suggest that probe 2 is cell-permeable and can be used for imaging of H S in both
2
A549 and HEK293A cells.
Fig. 4. Fluorescence images for exogenous H S detection in HEK293A and A549 cells using 2. A)
2
HEK293A Cells were incubated with 2 (10 µM) for 30 min and imaged in fluorescence (left) and
bright field (right). B) HEK293A Cells were incubated with Na S (100 µM) for 30 min, washed,
2
then treated with 2 (10 µM) for 30 min and imaged in fluorescence (left) and bright-field (right). C)
A549 Cells were incubated with 2 (10 µM) for 30 min and imaged in fluorescence (left) and bright
field (right). D) A549 Cells were incubated with Na S (100 µM) for 30 min, washed, then treated
2
1
1

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with 2 (10 µM) for 30 min and imaged in fluorescence (left) and bright-field (right).
Encouraging with these results, we further determined the applicability of probe 2
in visualizing H S in vivo, and zebrafish larvae were selected as the biological model
2
[
19c]. The zebrafish was first incubated with 2 for 1 h, washed, and then treated with
Na S (200 µM) for another 4 h. The zebrafish stained with 2 alone was used as
2
control group. As shown in Fig. 5A, no obvious fluorescence was observed in the
2
-loaded control group, whereas a strong green fluorescence was seen in venter and
eyes of the H S-treated zebrafish (Fig. 5B). These results imply that probe 2 can be
2
used to visualize H S in vivo. To further explore the potential of 2 in sensing
2
endogenous H S in situ, L-Cys (200µM) was introduced to the 2-stained zebrafish. To
2
our delight, a significant green fluorescence enhancement was observed in the eyes
and the venter of the larva (Fig. 5C). The locations of H S in zebrafish observed by us
2
are in consisted with the reported literature [24]. Taken together, the above results
indicated that probe 2 is an effective tool for H S detection in the living organisms.
2
Fig. 5. Confocal microscopy images for exogenous and L-Cys-induced endogenous H S detection
2
in zebrafish larvae. A) The zebrafish was incubated with probe 2 (10 µM) for 1 h and imaged in
green field (up) and bright field (down). B) The zebrafish was incubated with probe 2 (10 µM) for
1
h, then with Na S (200 µM) for 4 h and imaged in green field (up) and bright field (down). C)
2
The zebrafish was incubated with probe 2 (10 µM) for 1 h, then with L-Cys (200 µM) for another
h, and imaged in green field (up) and bright field (down).
4
1
2

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4
. Conclusion
In summary, we rationally designed a julolidine-fused coumarin-based probe 2
based on DFT calculations. This new probe is highly selective and sensitive towards
H S, and can be used for H S detection in living cells and in zebrafish. Compared to
2
2
its parent probe 1, probe 2 shows significantly improved properties, including lower
LOD, higher fluorescence response and higher sensitivity. This work further indicates
that the twisting-blockage of N,N-diethylamino group can significantly improve the
sensing performances of the fluorescence probes. Our work not only offers a useful
strategy to enhance the properties of H S fluorescence probes, but also provides an
2
excellent probe tool for imaging of H S in living biological systems.
2
Acknowledgements
This work was supported by the National Key R&D Program of China
(2017YFD0200500, 16JCYBJC20200), NSFC (21702111), 111 project (B14004).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.dyepig.2018.xx.xxx.
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Redox Signal 2014;20:770-82;
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.
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A new julolidine-fused coumarin-NBD probe for H S detection is rationally
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designed based on the DFT caculations.
This new probe exhibits faster and larger off-on response as well as higher
sensitivity compared with the previous coumarin-NBD probe.
This new probe possesses excellent selectivity and good biocompatibility, which
can be employed to image H S in living cells and in zebrafish.
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