A highly selective fluorescent probe for hydrogen polysulfides in living cells based on a naphthalene derivative

Article abstract of DOI:10.1016/j.saa.2019.117579

Hydrogen polysulfides (H₂S_n, n > 1) are members of reactive sulfur species (RSS) and signaling molecules derived from hydrogen sulfide (H₂S). Recently, the functions of H₂S_n in physiological and patho

Full text of DOI:10.1016/j.saa.2019.117579

Journal Pre-proof
A highly selective fluorescent probe for hydrogen polysulfides in living cells based on
a naphthalene derivative
Chunyan Wang, Junhong Xu, Qiujuan Ma, Yu Bai, Meiju Tian, Jingguo Sun, Zhijuan
Zhang
PII:
S1386-1425(19)30969-2
https://doi.org/10.1016/j.saa.2019.117579
SAA 117579
DOI:
Reference:
To appear in: Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Received Date: 7 August 2019
Revised Date: 15 September 2019
Accepted Date: 28 September 2019
Please cite this article as: C. Wang, J. Xu, Q. Ma, Y. Bai, M. Tian, J. Sun, Z. Zhang, A highly
selective fluorescent probe for hydrogen polysulfides in living cells based on a naphthalene
derivative, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2019), doi: https://
doi.org/10.1016/j.saa.2019.117579.
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2019 Published by Elsevier B.V.

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A highly selective fluorescent probe for hydrogen polysulfides in living cells based on a
naphthalene derivative
a
b
a,*
a
a
a
Chunyan Wang , Junhong Xu , Qiujuan Ma , Yu Bai , Meiju Tian , Jingguo Sun and Zhijuan
a,*
Zhang
a
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, PR China
b
Department of Dynamical Engineering, North China University of Water Resources and
Electric Power, Zhengzhou 450011, PR China
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*Corresponding author, E-mail: maqiujuan104@126.com; zhangyan0204@126.com; Tel:
+86-371-65676656; Fax: +86-371-65680028.
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Abstract
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Hydrogen polysulfides (H S , n>1) are members of reactive sulfur species (RSS) and
2 n
signaling molecules derived from hydrogen sulfide (H S). Recently, the functions of H S in
2
2 n
physiological and pathological processes have been increasingly recognized. However, their
biological effects and detailed mechanisms of action are still little known. Therefore, there is
an urgent need to develop highly selective and sensitive techniques for monitoring hydrogen
polysulfides (H S ) in living cells. In this study, we designed and synthesized a fluorescent
2
n
probe based on a naphthalene derivative for the detection of hydrogen polysulfides. A
naphthalene derivative was applied as the fluorescent main structure and the
2-fluoro-5-nitrobenzoate group was chosen as the recognition unit. In the absence of
hydrogen polysulfides, the fluorescent probe displayed almost no fluorescence. In the
presence of hydrogen polysulfides, the fluorescent probe exhibited strong fluorescence. The
sensing mechanism was based on H S -mediated aromatic substitution-cyclization reactions.
2
n
The linear range of the response concentration of the probe to hydrogen polysulfide was
-7
-5
-1
acquired in a concentration range of H S from 7.5×10 to 2.5×10 mol·L . The detection
2
n
-7
-1
limit was evaluated to be 5.0×10 mol·L for H S . The fluorescent probe can applied in a
2
n
wide pH range including physiological condition pH. The fluorescent probe showed high
specificity for H S over other reactive sulfur species (RSS). Moreover, the fluorescent probe
2
n
has been successfully applied to confocal imaging of hydrogen polysulfides in HepG2 cells
without cell cytotoxicity. All of such good qualities indicated that it could be used to detect
H S in living cells.
2
n
Keywords: fluorescent probe; hydrogen polysulfides; naphthalene; cell imaging
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1. Introduction
Reactive sulfur species (RSS) are sulfur-containing molecules [1,2], which play an
important regulatory role in biological system. Reactive sulfur species mainly include
glutathione (GSH), cysteine (Cys), hydrogen sulfide (H S), hydrogen polysulfides (H Sn, n >
2
2
1), persulfides, sulfur dioxide (SO ), sulfenic acids and so on. The performance of RSS
2
involves many aspects of cell biology, from protein function to redox signal transduction [3].
However, RSS disorders lead to a variety of diseases, including neurodegenerative damage,
inflammation and cancer [4-6]. Among them, hydrogen sulfide (H S) is considered to be an
2
important endogenous gas delivery signal molecule [7], and its various functions in
physiological and pathological processes have been widely studied and applied [8-12].
However, as members of RSS family and H S derivatives, hydrogen polysulfides (H S , n>1)
2
2 n
have attracted little attention. Recent evidences suggest that H S may play a role as a
2
n
practical medium in certain physiological and pathological processes associated with H S.
2
H S have a high S-thiol efficiency, which exert good roles in regulating enzyme activity and
2
n
play potentially important roles in H S signal transduction [13,14]. Moreover, H S are more
2
2 n
potent than H S in activating transient receptor potential ankyrin 1 (TRPA1) channels to
2
2
+
induce Ca flow in astrocytes, and in tumor suppressors and transcription factors [15-17].
The significance of H S molecules in biology has recently been recognized, but their
2
n
biological effects and detailed mechanisms of action are still little known. Therefore, it is
crucial to develop an accurate and sensitive method to detect H S in biological systems.
2
n
At present, traditional methods for detecting hydrogen polysulfides are ultraviolet-visible
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spectrophotometry (UV-Vis) [13], mass spectrometry (MS) [14, 18], and high performance
liquid chromatographic (HPLC) [19]. The absorption peaks at 290-300 nm and 370 nm by
UV-visible spectrophotometry are directly used for H S determination. The UV-Vis detection
2
n
method is simple and clear, but the sensitivity is not high. The MS-based method is not
suitable for the detection of H S at near-neutral pH [15]. The HPLC-based method required
2
n
the derivation of sample with monobromobimane to form the corresponding adducts. And
these above methods require sample pretreatment, which can cause cell lysis or destroy tissue,
so they are not suitable for rapid, accurate and real-time determination of biological samples.
It highlights the need for fluorescent probes, which have become the chemical tools needed to
detect H S in cells due to their high sensitivity, high selectivity and real-time detectability
2
n
[20-23]. Since Xian et al. reported the first fluorescent probe for H S in the year 2014 [24],
2
n
several fluorescent probes for H S have been extensively studied based on several
2
n
H S -mediated specific reactions. The H S fluorescent probes are designed mainly based on
2
n
2 n
H S -mediated aromatic substitution-cyclization reactions [25-29], H S -mediated ring
2
n
2 n
opening of aziridine [30] and H S -mediated reduction of nitro groups [31-34]. However,
2
n
some limitations of these fluorescent probes are still present, such as disturbance from other
biological species [35], and need of surfactant addition [25, 30, 35]. Thus, novel H2Sn
fluorescent probes with high selectivity and sensitivity are still in demand.
Naphthalene derivatives with a donor-π-acceptor (D-π-A) structure are widely used in
designing fluorescent probes because they display good spectroscopic properties with
excellent photo- and chemo-stability and high fluorescence quantum yield. Recently, the
naphthalene derivatives with a D-π-A structure have been developed to sense thiophenols [36],
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hydrogen peroxide [37], peroxynitrite [38], hypochlorous acid [39], cysteine [40], nitroxyl [41]
and fluoride ions [42] via fluorogenical signals. Herein, we developed a highly selective
fluorescent probe for H S based on a naphthalene derivative. 2-Fluoro-5-nitrobenzoate group
2
n
was used as the recognition unit for H S based on aromatic substitution-cyclization reaction.
2
n
In the absence of H S , the probe exhibited almost no fluorescence because
2
n
2-fluoro-5-nitrobenzoate group quenched the fluorescence. When H S was present, the
2
n
fluorescence emission intensity of centered 502 nm displayed an obvious increase. The probe
also showed a high selectivity for H S over other biological species. A detection limit of
2
n
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-1
5.0×10 mol·L was achieved for H S , which indicated that it could highly sensitively
2
n
determine H S . The more important, the probe also displayed low cell cytotoxicity and could
2
n
be successfully utilized to image H S in living cells.
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n
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2. Experimental
2.1. Materials and Instruments
2-Aminothiophenol and 6-hydroxy-2-naphthaldehyde were purchased from Energy
Chemical. p-Toluenesulfonic acid monohydrate was obtained from Sinopharm Chemical
Reagent Company. 2-Fluoro-5-nitrobenzoic acid, dimethyl sulfoxide (DMSO), thionyl
chloride and triethylamine were purchased from Heowns Biochemical Technology Company
and Tianjin Fuyu Fine Chemical Company, respectively. Hydrogen peroxide (H O ) was
2
2
obtained from 30% aqueous solutions. Hydroxyl radical (·OH) was formed by reaction of 1
2
+
1
mM Fe with 200 µM H O . Singlet oxygen ( O ) was prepared by the reaction of 1 mM
2
2
2
−
-
OCl with 200 µM H O . Peroxynitrite (ONOO ) solution was generated by the reaction of
2
2
H O and NaNO , and its concentration was obtained from absorption at λ=302 nm (ɛ=1670
2
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L·mol ·cm ) [35]. Before being used, dichloromethane was freshly subjected to distillation
from CaH . The reagents used in high performance liquid chromatography (HPLC)
2
experiments are all chromatographically pure and purchased from Tianjin Siyou Fine
Chemical Company. The other chemical reagents used are analytical pure reagents, obtained
from commercial suppliers and directly used in experiments without further purification. The
silica gel used in TLC is 60 F254 and the silica gel used in column chromatography is
200-300 meshes. Both of them were purchased from Qingdao Ocean Chemical Company.
Water is purified by SZ-93 automatic double pure water distiller (Shanghai Yarong
Biochemical Instrument Factory).
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The NMR spectra were conducted on Bruker's DRX-500 spectrometer using
tetramethylsilane (TMS) as an internal standard. Mass spectra were recorded on an Agilent
Technologies 6420 Triple Quad LC/MS high resolution mass spectrometer. Fluorescence tests
were performed on a Hitachi F-7000 fluorescence spectrophotometer equipped with a 1 cm
quartz absorption cell (Tokyo, Japan) with an excitation wavelength of 360 nm and entrance
and exit slits of 5 nm. The UV-visible absorption spectrum was measured by an
EVOLUTION 260 BIO UV-Vis spectrophotometer with a 1 cm quartz absorption cell. The
pH in the experiment was measured by a pH meter (METTLER TOLEDO). Fluorescence
imaging of living cells was recorded by an Olympus FV1200-MPE multiphoton laser
confocal microscope. High performance liquid chromatograms were obtained with a Waters
LC 2695-2998 HPLC/UV instrument equipped with an XBP-C18 column (5 µm, 4.6×250
mm). Data processing is mainly obtained in SigmaPlot software. All data obtained by
fluorescence spectrophotometry and UV-visible spectrophotometry were measured in 0.01 M
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PBS buffer (dimethyl sulfoxide /water = 1:9, V/V, pH = 7.40).
2.2. Syntheses
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The synthetic route for fluorescence probe 1 is shown in Scheme 1.
Insertion of Scheme 1
2.2.1. Synthesis of Compound
2
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Compound 2 was synthesized according to the reported literature [34]. 2-Aminothiophenol
(0.69 g, 5.5 mmol) was dissolved in 5 mL of ethanol. The above solution was dropwisely
added to a solution containing 6-hydroxy-2-naphthaldehyde (0.86 g, 5 mmol) and
p-toluenesulfonic acid monohydrate (1.90 g, 10 mmol) in 35 mL of ethanol at room
temperature. The reaction mixture was heated under reflux for 12 h, then the solvent was
evaporated under reduced pressure to give crude product. The obtained solid was purified by
silica gel column chromatography, with petroleum ether: ethyl acetate (4:1, V/V), thus
yielding a yellow intermediate 2 (1.05 g, 76%).
2.2.2. Synthesis of Compound
3
2-Fluoro-5-nitrobenzoic acid (1.02 g, 5.0 mmol) was dissolved in 10 mL of
dichlorosulfoxide under nitrogen flow condition and heated for reflux reaction for 12 h. The
solvent was evaporated to dryness under reduced pressure to give a white compound 3.
2.2.3. Synthesis of Compound
1
Compound 2 (0.55 g, 2 mmol) was dissolved in 25 mL of dry dichloromethane under a
nitrogen flow. 10 mL Anhydrous dichloromethane containing triethylamine (1.4 mL, 10 mmol)
and 2-fluoro-5-nitrobenzoyl chloride (0.49 g, 2.4 mmol) was added to the above solution. The
mixtures were reacted at room temperature for 24 h, followed by spinning off the solvent. The
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crude product was purified by silica column chromatography with elution with
dichloromethane/ethyl acetate (100:1, V/V), thus yielding the pure compound 1 as a yellow
1
solid after solvent removal (0.64 g, 72%). H NMR (500 MHz, DMSO-d ), δ(ppm)：8.89 (1H,
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s), 8.79 (1H, s), 8.65 (1H, d, J = 9.3 Hz), 8.33-8.29 (2H, m), 8.20 (1H, d, J = 8.5 Hz),
8.16-8.11 (2H, m), 8.02 (1H, s), 7.79 (1H, t, J = 9.3 Hz), 7.67 (1H, d, J = 9.6 Hz), 7.60-7.57
+
+
(1H, m), 7.51-7.48 (1H, m). MS (ESI) m/z: 445.0000 (M+H) , 467.2000 (M+Na) .
2.3. Cytotoxicity Assay
Cytotoxicity testing is a significant element in measuring the performance of fluorescent
probe bioimaging applications. To study the cytotoxicity of the probe, we used a tetrazolium-
based colorimetric assay (MTT assay) for cytotoxicity test [43]. HepG2 cells were cultured in
a Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum. When
4
the cells were in good condition, about 1×10 cells were seeded in each well of a 96-well
plate. The cells in the 96-well plates were cultured in an incubator containing 5% CO at 37 °
2
C for 24 h. After that, we removed the medium and washed it 3 times with Dulbecos
̕
phosphate buffer (DPBS). The medium containing different concentrations of probe 1 and
compound 2 was then incubated with HepG2 cells for 4 h, the drug-containing medium was
aspirated, and the fresh medium was added for 24 h. After that, the cells were incubated with
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100 µL of MTT (0.5 mg·mL ) for 3 h to ensure the formation of fromazan, and the
supernatant was aspirated. Then, 150 µL of DMSO was added and the 96-well plate was
shaken for 10 min to ensure sufficient dissolution of the formazan. Finally, the 96-well plate
was measured for absorbance at 490 nm using a microplate reader (Synergy 2, Bio Tek
Instruments Inc) and calculated survival rate according to A/A ×100% (A and A are the
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absorbance of experimental group and control group, respectively).
2.4. Confocal Imaging in Living Cells
HepG2 cells were cultured in a 20-mm laser scanning confocal microscope (LSCM) dish
for 24 h and ensured good growth. In one control experiment, 0.5 mM N-methylmaleimide
(thiol masking agent) was added to the cells and allowed for 40 min incubation, after which
DMEM was aspirated, the cells were washed with DPBS, incubated with the probe (5 µM) for
30 min and imaged. In one experiment group, fetal bovine serum medium containing probe 1
(5 µM) was added to the cells and allowed for 30 min incubation, after which the
probe-loaded medium was displaced by DPBS, the cells were cleaned with DPBS three times
and imaged. In another experiment group, fetal bovine serum medium containing probe 1 (5
µM) was added to the cells and allowed for 30 min incubation, after which the cells were
cleaned with DPBS for three times, incubated with 0.5 mM hydrogen polysulfides for another
30 min and imaged. The fluorescence confocal imaging pictures were observed by Olympus
FV1200-MPE multiphoton confocal microscopy with 60× objective lens in different channels.
3. Results and Discussion
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3.1. Spectroscopic Analytical Performance of Probe 1 towards Hydrogen Polysulfides
First, we researched fluorescence response of probe 1 to hydrogen polysulfides in 0.01 M
PBS buffer (pH=7.40, DMSO/water=1:9, V/V) (Figure 1). Considering the mutual
transformation of H
2
S
n
and H
2
S
2
under physiological conditions, Na
2
S
4
was chosen as H
S
2 n
donor in this study. As expected, probe 1 exhibited almost no fluorescence in the absence of
hydrogen polysulfides at 502 nm (Figure 1). The 2-fluoro-5-nirobenzoate group is a
quenching group and quenches the fluorescence, thus yielding the relatively weak background
fluorescence. However, the fluorescence intensity of the probe at 502 nm enhanced with the
increasing of the H
2
S
n
concentration until they reached a maximum plateau with the addition
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of 25 µM of hydrogen polysulfides. In the presence of 25 µM of hydrogen polysulfides, an
approximately 18-fold enhancement in the fluorescence emission intensity at 502 nm was
observed. When hydrogen polysulfides were present, the probe could be converted into
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-(2-benzothiazolyl)-2-naphthalenol (compound 2) with electron-donor hydroxyl moiety, thus
providing a strong fluorescence. It demonstrated that probe 1 could work as a detection tool
for hydrogen polysulfides in an efficient response.
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Insertion of Figure 1
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In addition, we also studied the UV-vis absorption spectra of fluorescent probe 1 (15.0 µM)
before and after addition of Na S (100 µM), and compound 2 (15 µM) Figure 2. As can be
2
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seen from Figure 2, fluorescent probe 1 had a maximum absorption at 325 nm and a shoulder
peak at 367 nm, the reaction mixture of probe 1 with Na S showed a maximum absorption at
2
4
338 nm, and compound 2 exhibited a maximum absorption at 338 nm. The new absorption
peak of probe 1 after addition of Na S should be attributed to the compound 2 generated via
2
4
the reaction of H S to compound 1.
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n
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Insertion of Figure 2
3.2. Principle of Operation and the Basis of Quantitative Assay
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Herein, different concentrations of Na S were fluorescently titrated to study the linear
2
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relationship of probe 1 to H S response (Figure 3). As shown in Figure 3, when the Na S
2 4
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n
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concentration is 7.5×10 -2.5×10 mol·L , the fluorescence intensity of the probe at 502 nm
has a good linear relationship with the concentration. The linear regression equation was I
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=13.5069 + 41.7445×10 ×C (R=0.9973). Here I is the fluorescence intensity of probe 1 at
502 nm, C represents the concentration of H S and R is the linear correlation coefficient. The
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n
detection limit was calculated according to the 3 S /m criterion [43], where S is the standard
B
B
deviation of the blank (n=10) and m is the slope for the range of the linearity. The detection
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limit of the probe for H S was estimated to be 5×10 mol·L which is lower than that of
2
n
,
formerly developed fluorescent probes for H S [27].
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Insertion of Figure 3
3.3. Effect of pH
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To investigate the effect of probe 1 on H S in different pH environments, the fluorescence
2 n
intensity of probe 1 at 502 nm before and after the addition of H S was performed at different
2
n
pH values (Figure 4).From Figure 4, the fluorescence intensity of probe 1 (5.0 µM) is quite
stable in the pH range from 2.00 to 9.00, while it shows significant enhancement with
treatment of H S (25 µM) from pH 2.00 to 9.00. The range covers the pH of the
2
n
physiological environment, indicating that the detection of hydrogen polysulfides in living
cells is also suitable. Therefore, the probe has great potential for efficient detection of H S in
2
n
organisms.
Insertion of Figure 4
3.4. Selectivity
The specificity of the fluorescent probe determines its effectiveness in the actual sample.
We studied the fluorescence signals of probe 1 on hydrogen polysulfides (25 µM) and other
interfering substances (potentially competitive reactive oxygen species (ROS), reactive
nitrogen species (RNS) and reactive sulfur species (RSS)) at pH 7.40 to evaluate the
selectivity of the probe for H S (Figure 5A). As illustrated in Figure 5 A, only the addition of
2
n
-
-
H S can trigger a strong fluorescence reaction, and other bio-related substances (NO , NO ,
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n
2
3
-
-
1
ONOO , H O , ClO , ·OH, GSH, Cys, Hcy, Na S, Na S O , Na SO , Na SO , O ) cannot
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2
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induce a significant increase in fluorescence. Moreover, when hydrogen polysulfides (25 µM)
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-
-
and other substances (NO , NO , ONOO , H O , ClO , ·OH, GSH, Cys, Hcy, Na S, Na S O ,
2
3
2
2
2
2
2
3
1
Na SO , Na SO , O ) coexisted in the solution containing the probe, we still observed the
2
3
2
4
2
equally strong fluorescence response (Fig. 5B). In other words, different interfering
substances have no effect on monitoring of hydrogen polysulfides. These results demonstrate
1
1

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that the probe is highly selective for H S and expected to detect endogenous H S in complex
2 n 2 n
biological circumstances.
Insertion of Figure 5
3.5. Proposed Mechanism
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According to the relevant literature [44-46], we suspect that the fluorescence enhancement
of compound 1 in the presence of hydrogen polysulfides is caused by the reaction of
compound 1 with H S to form compound 2 (scheme 2). In the absence of H S , the
2
n
2 n
2-fluoro-5-nitrobenzoate group of probe 1 quenched the fluorescence and the probe displayed
almost no fluorescence. After the addition of H S , the 2-fluoro-5-nitrobenzoate group of
2
n
probe 1 was struck by H S via aromatic nucleophilic substitution and a
̶
SSH-containing
2
2
intermediate product was produced, then the
̶ SSH underwent an intramolecular cyclization to
form compound 2 with hydroxyl groups. Compound 2 has the D-π-A structure and the
fluorescence intensity increases significantly. In order to further verify the suspected response
mechanism, we performed high performance liquid chromatography (HPLC) to analyze probe
1, the reaction mixture of the probe and hydrogen polysulfides, and compound 2 (Figure 6).
As can be seen in Figure 6A, the retention time of the free probe was 10.80 min. And the
reaction mixture of probe 1 and hydrogen polysulfides showed a peak at 5.32 min (Figure 6C),
which was the nearly same as that of compound 2 (Figure 6B). It is further explained that the
reaction between probe 1 and hydrogen polysulfides forms compound 2, which is consistent
with the conclusion obtained by UV-vis absorption spectroscopy in Figure 2. The above
results proved the correctness of the proposed mechanism.
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Insertion of Scheme 2
Insertion of Figure 6
3.6. Cytotoxicity Assays and Confocal Imaging in Living Cells
Cytotoxicity is the key factor for the probe to monitor endogenous hydrogen polysulfides.
Thus, we used MTT method to evaluate the cytotoxicity of probe 1 and compound 2 on
1
2

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
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2
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2
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HepG2 cells (Figure 7). As shown in Figure 7, the survival rate of HepG2 cells was higher
than 85%. When the concentration of probe 1 and compound 2 ranged from 0 to 16 µM, it
showed that probe 1 and compound 2 had almost no cytotoxicity and could be used for cell
imaging.
Insertion of Figure 7
Next, HepG2 cells were cultured in a medium containing fluorescent probe 1 (5 µM) and
N-methylmaleimide (0.5 mM) for 30 min, HepG2 cells exhibited nearly no fluorescent
emission (Figure 8b). In one experiment group, HepG2 cells were cultured for 30 min only in
culture medium containing fluorescent probe 1 (5 µM), showing weak green fluorescence
(Figure 8e), which was attributable to endogenous hydrogen polysulfides. In another
experiment group, cells were treated with 0.5 mM hydrogen polysulfides in probe 1 (5 µM),
which showed strong green fluorescence (Figure 8h).The experimental results show that
probe 1 emits green fluorescence of different intensities under different concentrations of
hydrogen polysulfides. Thereby fluorescence imaging of hydrogen polysulfides in living cells
is achieved.
Insertion of Figure 8
4. Conclusions
In summary, we designed and synthesized a novel fluorescent probe based on a naphthalene
derivative for the detection of hydrogen polysulfides. The probe uses a
2-fluoro-5-nitrobenzoate group as a recognition site. In the absence of hydrogen polysulfides,
the probe exhibits almost no fluorescence. When hydrogen polysulfides is present, the probe
undergo aromatic nucleophilic substitution and an intramolecular cyclization to produce
1
3

2
3
3
3
3
99
00
01
02
03
compound 2 with the D-π-A structure. Thus, in presence of hydrogen polysulfides the probe
displays strong fluorescence and the detection of hydrogen polysulfides can be realized. The
probe has the advantages of stability under physiological pH conditions, high selectivity and
excellent sensitivity for hydrogen polysulfides. Meanwhile, the probe has good
biocompatibility and is suitable for the detection of hydrogen polysulfides in living cells.
3
3
04
05
Acknowledgments
This work was supported by the Key Science and Technology Programme of Henan
3
3
3
06
07
08
Province (182102310101), National Natural Science Foundation of China (21807027), the
Fundamental Research Funds for the Provincial Universities (2014KYYWF-QN04) and
Graduate Innovation Fund in Henan College of Chinese Medicine (YJS2018A05).
3
09
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Captions of figures
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Scheme 1 The synthetic route of probe 1: (a) ethanol, p-toluenesulfonic acid
monohydrate, reflux, 12 h, 76%；(b) sulfoxide dichloride, reflux, 12 h；(c) anhydrous
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dichloromethane, triethylamine, room temperature, 24 h,72%.
Scheme 2 Proposed reaction mechanism of probe 1 with H S .
2
n
Figure 1 Fluorescence emission spectra of the probe 1 (5.0 µM) against the different
concentrations of Na S : 0, 0.75, 1, 2.5, 3.5, 5, 7, 10, 15, 20, 25 µM from 1 to 11
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(λ =360 nm).
ex
Figure 2 UV-vis absorption spectra of fluorescent probe 1 (15 µM), the reaction
mixture of probe 1 (15 µM) with Na S (100 µM), and compound 2 (15 µM). The solid
2
4
line (—) , medium dash (
̶ ̶ ̶ ) and dotted line (···) are probe 1, the reaction mixture of
probe 1 (15 µM) with Na S (100 µM), and compound 2 (15 µM), respectively.
2
4
Figure 3 A plot of the fluorescence intensity obtained from the reaction of the probe
(5µM) with the Na S as a function of the concentration of Na S from 0.75 µM to 25
2
4
2 4
µM (λ = 502 nm).
em
Figure 4 The effect of pH on the fluorescence intensity of 5.0 µM probe 1 without
addition (closed circles) and addition of 100 µM Na S (open circles).
2
4
Figure 5 (A) Fluorescence reaction of probe 1(5.0 µM) toward hydrogen polysulfides
-
and other substances at pH = 7.4. Each reaction substance is: (1) blank, (2) NO , (3)
2
-
-
-
NO , (4) ONOO , (5) H O , (6) ClO , (7) ·OH, (8) GSH, (9) Cys (25 µM), (10) Hcy,
3
2
2
1
(11) Na S, (12 ) Na S O (13) Na SO , (14) Na SO ,(15) O , (16) H S (25 µM).
2
2
2
3,
2
3
2
4
2
2 n
Interfering substances were at a concentration of 100 µM unless otherwise stated. (B)
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Fluorescence response of probe 1 (5.0 µM) to hydrogen polysulfides in the presence
-
-
-
-
of other substances: (1) NO , (2) NO , (3) ONOO , (4) H O , (5) ClO , (6) ·OH, (7)
2
3
2
2
GSH, (8) Cys (25 µM), (9) Hcy, (10) Na S, (11 ) Na S O (12) Na SO , (13) Na SO ,
2
2
2
3,
2
3
2
4
1
(14) O ,(15) H S (25 µM). Interfering substances were at a concentration of 100 µM
2
2 n
unless otherwise stated.
Figure 6 High performance liquid chromatogram of (A) compound 1 (20 µM), (B)
compound 2 (20 µM) and (C) the reaction product of compound 1 (20 µM) with
hydrogen polysulfides (100 µM). HPLC conditions: 1.0 mL/min flow rate, Agela
Technologies Venusil XBP-C18: 5µm, 4.6×250 mm column, acetonitrile/water= 90:
10 (pH=7.40, V/V), and detected at 335 nm.
Figure 7 MTT assay of probe 1 (A) and compound 2 (B). Different concentrations (0,
2, 4, 8, 16 µM) of probe 1 (A) and compound 2 (B) were incubated for 24 h at 37 ° C,
respectively.
Figure 8 Fluorescence confocal imaging of HepG2 cells treated with 5 µM probe 1. (a)
Transmission images of bright fields of HepG2 cells incubated with 0.5 mM
N-methylmaleimide for 40 min followed by incubation with probe 1 for 30 min; (b)
fluorescence image from green channel of image (a); (c) superimposed images of (a) and
(b); (d) brightfield transmission image of HepG2 cells incubated with 5 µM probe 1
for 30 min; (e) fluorescence image from green channel of image (d); (f) overlaid image of
(d) and (e); (g) brightfield transmission image of HepG2 cells incubated with 5 µM
probe 1 for 30 min and then incubated with 0.5 mM hydrogen polysulfides for 30 min;
(h) fluorescence image from green channel of image (g); (i) superimposed image of (g)
and (h).
4
36
1
8

N
CHO
H2N
HS
a
+
S
HO
HO
2
O
O
F
O N
2
O N
2
Cl
OH
b
F
3
N
O
S
c
2
+ 3
O
O N
2
F
1