A novel mitochondria-targeting fluorescent probe for hydrogen sulfide in living cells

Article abstract of DOI:10.1111/cbdd.12948

A novel mitochondria-targeting fluorescent probe compound S-N₃ for hydrogen sulfide (H₂S) in living cells has been designed and synthesized in this study. This article contained the chemosynthesis and some studies on bioactivity of the target compound in living cells. Compound S-N₃ is easy to synthesize and can remain stable under the effect of pH, system and photostability. In addition, it shows low cytotoxicity in cell imaging. And it can react with H₂S highly selective in PBS or FBS solution, which would cause the obvious increase in fluorescence intensity. Therefore, the low-cost detection method for H₂S is allowed for monitoring the quantity of H₂S existed in the mitochondria.

Full text of DOI:10.1111/cbdd.12948

PROF. HAI QIAN (Orcid ID : 0000-0002-3827-0992)
Received Date : 10-Nov-2016
Revised Date : 27-Dec-2016
Accepted Date : 09-Jan-2017
Article type
: Research Article
A Novel Mitochondria Targeting Fluorescent Probe as the Monitor of Hydrogen Sulfide in Living
Cells
Wei Shi1, Miaobo Pan1, Hao Qiang1, Qianqian Qiu1, Wenlong Huang1,2, Haiyan Lin3, Hai Qian1,2*, Liang
Ge1*
1 Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University,
24 Tongjiaxiang, Nanjing 210009, PR China
2 Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, PR China
3
Department of Biochemistry and Molecular Biology, Nanjing Medical University, 140 Hanzhong Road,
Nanjing 210029, PR China.
*Corresponding author:
Hai Qian, qianhai24@163.com; Liang Ge , geliang1981@hotmail.com
Abstract:
A novel mitochondria targeting fluorescent probe compound S-N3 as the monitor of Hydrogen Sulfide
(H2S) in living cells has been designed and synthesized in this paper. This article contained the
chemosynthesis and some studies on bioactivity of the target compound in living cells. Compound S-N3 is
easy to synthesize and can remain stable under the effect of pH, system and photo.
In additional, it shows low cytotoxicity in cell imaging. And it can react with H2S highly selective in PBS
or FBS solution, which would cause the obvious increase of fluorescence intensity. Therefore, the low-cost
detection method for H2S is allowed for the monitoring the quantity of H2S existed in the mitochondria.
Keywords: Fluorescent probe, Hydrogen sulfide, Mitochondria targeting, Selectively
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12948
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Introduction
Hydrogen sulfide (H2S) existing in agriculture, husbandry[1], organic manure fermentation[2], water
treatment[3] and industrial production was recognized as the third well-known gas signal molecule stepping
with NO and CO in vivo[4]. Some studies have suggested that excess H2S in vivo can greatly harm the
health of human and animals, leading to headaches, dizziness, fatigue, nausea and even death. However,
recently it has been found that a balanced amount of H2S plays an active role in the regulation of the
nervous system[5], inflammatory stress[6] and the cardiovascular system[7], etc[8]. In addition, H2S is also an
important class of disease markers, such as Alzheimer's disease, hypertension and diabetes[9].
At present, there are many detection methods of H2S, such as ultraviolet absorption method[10]
,
electrochemical method[11] and chromatography[12]. However, compared to the complex, expensive
equipment and reagents, the method of fluorescent probe which does not damage the biological samples is
more suitable for the detection[13]. Recently, a number of fluorescent probes for the detecting and imaging
of H2S in living cells have been reported[14-16]
.
Furthermore, mitochondria, as a kind of important organelles, possesses its physiological-function closely
related to the H2S[17]. Selective tracking of H2S in mitochondria is crucial to elucidate the complex
contributions of H2S for improving the activity of mitochondrial ATPase and the resistance to oxidation,
etc[18]. Therefore, quantitative and accurate detection of the H2S changes in mitochondrial with fluorescent
probe is in strong need for elucidating the complex contributions of H2S and studying some diseases and
the damage of mitochondria. Up to now, several innovative fluorescent probes targeting to mitochondria
for H2S based on different reaction mechanisms have been revealed in the literature. Wu et al. have
synthesized a fluorescent for a highly selective H2S probe for mitochondria and lysosome, respectively[19]. Chen
et al. and Wang et al. have reported mitochondrial targeting fluorescent probes based on coumarin merocyanine
and cyanine scaffold for H2S detection[20,21]. Xu et al. have reviewed the progress of some small molecule
fluorescent probes targeting different organelles[22]
.
To improve the flatness and solubility the of ring-fused 1,8-naphthalic anhydride and the medicinal
properties, we designed and synthesized a new fluorescence probe targeting the mitochondria to detect H2S
in it, which emited at a different wavelength (λEx/Em = 460/545 nm), and then determined its activity under
the condition of different effects [23, 24]. Results showed that the mitochondria targeting compound S-N3 had
higher selectivity and lower cytotoxicity to H2S. In addition, cells was valuable for further developing. The
structure of the target compound was shown below (Fig. 1).
Results and Discussion
Design and Synthesis of S-N3
By reacting, we got the target compound (S-N3) prepared from 5-Nitroacenaphthene (compound 1) [25,26]
The synthetic route is shown in Scheme 1 [27]
.
.
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Fluorescence response between S-N3 and H2S
Firstly, 10μM S-N3 solution was added into the cuvette to detect the excitation wavelength and emission
wavelength. Results showed that upon the addition of NaHS (50μM), S-N3 solution exhibited a robust
increase in fluorescent intensity at 545 nm with the excitation at 460 nm testing by the Fluorescence
Spectrophotometer and Perkin Elmer EnSpireTM, respectively. And the relationship between the response
time and Rel.Emission intensity of S-N3 (10μM) in NaHS (50 μM) solution at 37 , as well as the
detection of the most suitable excitation wavelength of S-N3 (10μM) with emission at 545 nm were shown
in Fig. 2, the fluorescence intensity of S-N3 10μM increased fast within 10 min after adding NaHS (50
μM), and obvious fluorescent yellow can be observed at 365 nm-UV compared to the S-N3 solution
without NaHS. The fluorescence intensity was increasing and approximately trended to be stable at 10 min.
Therefore, the equilibrium time between the probe and H2S would be chosen as 10 min.
Biological selectivity and linearity assessment
In complex organisms, the selectivity of S-N3 is the major issues for the further monitor of H2S over other
molecules. At 10 min, by comparing with different influence factors, the investigation indicated that only
-
-
HS- (100μM) caused a dramatic enhancement at 545 nm, while other anions (F-, Cl-, Br-, I-, NO2 , NO3 ,
2-
-
2-
2-
2-
CO3 , HCO3 , 1mM), inorganic reactive sulfur species (SO4 , S2O3 , SO3 , 1mM), and other biological
thiols (GSH, Cys, 1mM) had just a weak increment of fluorescence intensity at 25 (Fig. 3). The results
showed that S-N3 had potential research value in a biological environment for the detection of H2S. A
further study found that there was a great linearity between fluorescence intensity and concentrations of
H2S in the range of 5-40μM in PBS buffers at 10 min, 25 (Fig. 4). In this linear range, the obvious
spectral response can be got. And when the concentration of H2S up to 50μM, the response mainly did not
increase any more. Thus far, all the selectivity and linearity studies suggested this probe is very suitable for
the detection of sulfide in biological samples. For a further test, the limit of detection (LOD) was as low as
1μM with a signal-to-noise ratio (S/N) of 3:1 and the limit of quantification (LOQ) was 4.2μM with S/N of
10:1.
pH and photo stability
Furthermore pH and photo stability of S-N3 also had a tremendous influence on detection of H2S in
biological systems. Then we tested its stability with different pH values (pH= 1, 2, 3,……, 13) at 10 min,
25 , 545 nm, and the fluorescence intensity for different time was shown in Fig. 5(A)(B), respectively.
The fluorescence intensity of S-N3 (100μM) had no detectable change in all the buffers at pH 3-11
primarily, which indicated that different pH values between 3 and 11 did not have any effect on the
photometric characteristics of S-N3 without the adding of NaHS. In other word, this probe had a wide
application scope of pH. Moreover, the photo stability of S-N3 was also estimated with a UV lamp at
365nm. S-N3 (100μM) was exposed to the irradiation and the fluorescence response was obtained at
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different time within 10 min. Upon the addition of NaHS, there was a prominent enhancement to others
which were stable without NaSH (Fig. 5B), which demonstrated that within 10 min the probe was stable to
the photo (365 nm) itself, but it was sensitive to H2S and there was a great response between them.
Stability in different systems
In order to get further research on the stability of S-N3, we next determined the fluorescence intensity of
the target compound in different environments, such as the systems of degassed PBS buffers and
commercial fetal bovine serum (FBS). At different aging time within 30 min, the fluorescence intensity of
S-N3 with concentration of 30μM and100μM at 25 was recorded in the two systems shown in Fig. 6.
With the variation of the time, the same variation trend can be seen in both two systems. What's more, the
increase of fluorescence intensity was relatively stable by increasing the aging time but turned stronger by
increasing the concentration of S-N3. While in FBS solutions, the response was relatively lower than that in
the PBS buffers, which may be on account of various proteins in serum. The study focused on the stability
of S-N3 explained this probe for detection of H2S was a conclusive one in different testing environment.
Cytotoxicity assay
In order to get the further confirmation for the application of S-N3, the cytotoxicity of the targeted
compound in living cells was determined firstly. Above all, the MTT assays for S-N3 and the reduced
product S-NH2 were carried out, the inhibition rate of S-N3 and S-NH2 to MCF-7 cells at different
concentrations (3.125, 6.25, 12.5, 25, 50, 100 μM) , 37 was demonstrated in Table 1, and the results
showed that both the two compounds have scarcely any cytotoxicity and a high IC50 value (82.5 and
83.25μM) after 48 h by calculating. Thus, the probe at 20μM was selected for imaging in MCF-7 cells [24]
Mitochondria Visualization of H2S in MCF-7
In this paper, the capability of mitochondria targeting of S-N3 was verificated with the visualization of
MCF-7 cells [29]. As described in Fig. 7, cells incubated alone with Mito Tracker RED dye with red
fluorescence were collected from the red channel (580 nm), and cells cultivated by S-N3 and NaHS emited
green fluorescence were collected from the green channel (460nm). The overlaid image was in yellow
fluorescence. The significant fluorescence in green channel proved that probe S-N3 can be used for
imaging H2S in living cells. In addition, the result of colocalization of S-N3 and Mito Tracker RED dye at
37 was shown in Fig. 8 which showed the high repetition between S-N3 and the dye for dyeing to the
mitochondria. According to the results above, the target compound S-N3 had the same trend to
mitochondria for dyeing. That is to say, the S-N3 showed the similar activity of targeting to mitochondria,
which suggested probe S-N3 was mitochondria targeted as the famous Mito Tracker RED dye.
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CONCLUSION
In summary, a novel mitochondria targeting fluorescence probe was developed for detection of H2S. The
probe was found to be obvious sensitive to H2S among some anions tested with a detection limit of 1μM
with a signal-to-noise ratio of 3:1 and the limit of quantification (LOQ) was 4.2μM with S/N of 10:1. And
with the addition of H2S, distinct fluorescence increments compared to that with no H2S were obtained.
What's more, this probe was easy to synthesize and can remain stable under the effect of pH, system and
photo. In additional, it showed low cytotoxicity in cell imaging. Consequently, we feel that this
mitochondria targeting, selective, efficient, and low-cost detection method for H2S will be very useful in
the booming research field.
EXPERIMENTAL SECTION
Reagents and Instruments
All reagents used for synthesis of compounds were bought from Aladdin, J&K, and were directly applied
in the experiment without other purification. pH values of the buffers were recorded on the instrument
Metter Toledo Five Easy pH FE20. The purity of each tested compounds (>95%) was determined on a
Waters UPLC/MS instrument using a Waters C18 column (1.7 μm2.1×50 mm, with a flow rate of 0.2
mL/min and detection at 254 nm) employing a 10–90% acetonitrile/water/0.1% formic acid gradient. The
spectra of 1H NMR and 13C NMR were analyzed by the Bruker model DPX-300 MHz NMR spectrometer
(Bruker Instruments, Inc, Billerica, MA, USA). Elemental analysis was analyzed on CHN-O-Rapid
instrument. The result of Mass spectra (MS) was obtained by Waters liquid chomatography-mass
spectrometer system (ESI). Thin-layer chromatography (TLC) was visualized on GF/UV 254 plates, and
the fluorescent points were shown under UV light with the wavelength 254 and 365 nm. Fluorescent data
was presented by fluorescence spectrophotometer (RF-5301 PC, Shimadzu, Tokyo, Japan) and Perkin
Elmer EnSpireTM. Laser scanning confocal microscopy was taken on Olympus FluoView-300 confocal
laser scanning system (Olympus, Tokyo, Japan).
Cell Lines
Human breast cancer cell lines MCF-7 were kindly provided by our laboratory. The cell lines were
cultivated in hyclone DMEM medium with 10% fetal bovine serum (FBS) and incubated in the incubator
with 5% CO2 at 37
beforehand.
in air growth. All experiments were conducted with the cells we cultivated
Fluoresence analysis, biological selectivity and stability test
PBS buffer used in this study was 10mM and pH 7.4, and in the study of pH stability, the pH value was
adjusted with 1 N NaOH solution. 50mM stock solution of S-NH2 and S-N3 were dissolved in DMSO
firstly, and then diluted with PBS we prepared before to 100 and 30μM as the mother liquor. 10mM NaHS
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was prepared in the PBS buffer as the donor of H2S, and then diluted with PBS solution to different
-
-
2-
-
2-
concentrations as shown below. In addition, the ions of F-, Cl-, Br-, I-, NO2 , NO3 , CO3 , HCO3 , SO4 ,
2-
S2O3 , SO32- and the biological mercaptan ( such as GSH and Cys ) were prepared with PBS buffer to the
concentration of 1mM for the test of biological selectivity. These solutions above were then used for
further study in fluoresence analysis, biological selectivity and stability test.
Cytotoxicity assay
MCF-7 cells (cells per well) were seeded in 96-well plates. After 24 h incubation, cells were treated with
various concentrations of S-N3 for 48 h in an atmosphere of 95% air with 5% CO2 at 37 . Then, MTT
was added directly to the cells. After additional incubation for 4 h at 37 , the absorbance at 490 nm was
read on a microplate reader (Thermo Fisher Scientific). The IC50 values of the S-N3 for cytotoxicity were
calculated by GraphPadPrism 5.0 software from the dose-response curves. Experiments were conducted in
triplicates and repeated three times independently.
Mitochondria Visualization of H2S in MCF-7
Firstly, the cells were pretreated with the target compound (20μM, 1 ml) and the Mito Tracker RED dye for
mitochondria (250nM) for 1 h at 37 , respectively. And then washed by the PBS buffers (1 ml ×3). Next
the NaHS (20μM, 1 ml) was added to the cells in different systems mentioned above with reacting for 5
min. The cells then were set under the laser scanning confocal microscope with the two-channel
fluorescent signal of 460 nm and 580 nm [30]
.
Synthesis of (5-((2-(6-Amino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)amino)-5
-oxopentyl)triphenylphosphonium bromide (S-NH2)
The mixtures of compound 7 (9.2mmol), Zn (46.1mmol) and NH4Cl (92.2mmol) were dissolved with
solvent of EtOH and water (40ml, 3:1) and refluxed at 85 for 5 h. Then filtered with kieselguhr, and
solution was extracted with DCM (40 mL×3). The combined organic layers were washed with saturated
sodium bicarbonate solution and brine, dried over Na2SO4, and concentrated in vacuo. An orange powder
1
(S-NH2) was obtained. Yield: 64%; Orange powder; H NMR (300 MHz, DMSO-d6) δppm: 8.43 (d, J =
8.0 Hz, 2H), 8.05 (s, 1H), 7.92 (s, 5H), 7.76 (s, 12H), 5.78 (s, 1H), 4.10 (s, 2H), 3.74 (s, 2H), 3.35 (s, 2H),
2.42 (m, 4H), 1.64 (m, 4H); 13C NMR (75 MHz, DMSO-d6)δ: 174.88, 162.53, 158.17, 135.25, 133.77,
132.63, 130.69, 130.33, 130.16, 125.94, 124.87, 115.97, 113.87, 111.70, 40.75, 38.66, 37.48, 25.34, 23.06,
22.34; ESI-MS m/z: 600.24 [M+H]+. Anal. calcd. For C37H35N3O3PBr: C, 65.30; H, 5.18; N, 6.17; Found:
C, 65.39; H, 5.52; N, 6.14.
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Synthesis of (5-((2-(6-Azido-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)amino)-5
-oxopentyl)triphenylphosphonium bromide (S-N3).
S-NH2 (4.4mmol) was dissolved in 60% Con.H2SO4 (20 ml) at -10 , then adding NaNO2 (8.8mmol) in
batches while stirring for 30 min, and the temperature must be controlled within -5-0 . Finally, NaN3
(8.8mmol) was added into the system, and reacted for about 1 h. Then the reaction solution was diluted
with water (20 ml), and extracted with DCM (25 mL×5). The combined organic layers were washed with
saturated sodium bicarbonate solution and brine, dried over Na2SO4, and concentrated in vacuo. The MS
1
spectrum of S-N3 was shown in Fig. 9. The target S-N3 was obtained. Yield: 74%; orange powder; H
NMR (300 MHz, DMSO-d6) δppm: 1H NMR (300 MHz, DMSO) δ 8.45 (d, J = 7.9 Hz, 3H), 7.89 (s, 5H), 7.77
(s, 12H), 5.76 (s, 1H), 4.08 (s, 2H), 2.02 (s, 2H), 1.50 (s, 4H), 1.24 (s, 4H). 13C NMR (75 MHz, DMSO-d6)δ:
174.88, 162.53, 149.12, 135.25, 133.77, 132.78, 131.10, 129.81, 129.15, 125.39, 120.84, 115.97, 40.75,
38.66, 37.48, 25.34, 23.06, 22.34; ESI-MS m/z: 626.3 [M+H]+. Anal. calcd. For C37H33N5O3PBr: C, 62.90;
H, 4.71; N, 9.91; Found: C, 62.86; H, 4.78; N, 9.83.
Conflict of interest
Declare none.
Acknowledgements
This work was supported by the National Science and Technology Major Project of the Ministry of Science
and Technology of China (No. 2009ZX09102-033), National Natural Science Foundation of China (NO.
81273376 and 81673299) and Natural Science Foundation of Jiangsu Province (No. BK20131398).
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Figure Legends
Fig. 1 New fluorescent probe (S-N3) for the detection of H2S.
Scheme 1: Synthesis of target compound S-N3.
Reagents and conditions: (a) K2Cr2O7, CH3COOH, reflux, 110 , 4 h, 62%; (b) K2CO3, Di-tert-butyl
pyrocarbonate, CH3OH, r.t., 24 h, 59%; (c) Anhydrous EtOH, N2, reflux, 80 , 12 h, 65%; (d) Hydrogen
chloride saturated ethyl acetate, 3 h, 92%; (e) (4-Caboxybutyl)- triphenylphosphonium bromide, (COCl)2,
DCM, 24 h, 55%; (f) Zn, NH4Cl, ethanol, water, 85 , 5 h, 81%; (g) NaNO2, NaN3, 60% H2SO4, -5-0 , 1
h, 74%
Fig. 2 (A) The relationship of S-N3 10μM to NaHS (50μM) between Rel.Emission intensity and the time
at 37 with emission at 545 nm (B) The detection of the most suitable excitation wavelength of S-N3
(10μM) with emission at 545 nm, 37
Fig. 3 Biological selectivity of S-N3 at 10 min at 25
Fig. 4 Linearity between fluorescence intensity and concentrations of H2S at 25
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Fig. 5(A) pH stability of 100μM S-N3 in PBS buffers at 10 min, 25 without the adding of NaHS (B)
Photo stability of 100μM S-N3 in PBS buffers with UV light irradiation at different time and the addition
of 50μM NaHS for 10 min at 25 (the last column)
Fig. 6 Stability of 30μM and 100μM S-N3 in PBS and FBS systems at different time 25 within 30 min
Table 1 Inhibition rate of S-N3 and S-NH2 to MCF-7 cells at different concentrations, 37
Fig. 7 Visualization of MCF-7 cells by dyeing with S-N3 and Mito Tracker RED dye at 37 . (A) image of
MCF-7 cells dyeing by probe S-N3; (B) image of MCF-7 cells dyeing by Mito Tracker RED dye; (C) the
overlaid image of (A) and (B); (D) image of MCF-7 cells in dark field
Fig. 8 Colocalization of S-N3 and Mito Tracker RED dye
Fig. 9 The MS profile of S-N3
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