Chem Biol Drug Des 2014 Research Article
A High-Sensitivity Coumarin-Based Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells Qianqian Qiu1, Xin Deng1, Lei Jiao1, Tianxiao Zhao1, Fanfei Meng2, Wenlong Huang1,* and Hai Qian1,* 1
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China 2 Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China *Corresponding authors: Hai Qian, [email protected]; Wenlong Huang, [email protected] A novel coumarin-based fluorescence probe has been constructed for the selective and sensitive detection of hydrogen sulfide (H2S). This probe displays high sensitivity and linearity to H2S in degassed PBS buffers and fetal bovine serum. It reacts with H2S with high selectivity over Cys, GSH, and other anions. Meanwhile, successful detection of H2S in living cells was also demonstrated. Key words: bioimaging, coumarin, Fluorescence probe, hydrogen sulfide Received 22 October 2014, revised 9 November 2014 and accepted for publication 20 November 2014
Hydrogen sulfide (H2S), stepping with NO and CO, is the third endogenously generated gaseous signaling molecule (1). It plays a vital role in regulating the intracellular redox status and other fundamental signaling processes involved in human health and disease (2–7). The physiologically relevant H2S concentration is estimated to range from nano to millimolar levels (8). Once the level overruns the normal physiological range, H2S would lead to correlative diseases, such as Alzheimer’s disease (9), diabetes (10), Down syndrome (11), and other diseases of mental deficiency (2). Therefore, accurate and reliable detection of H2S concentration in vivo has become the significant subject of current chemical research. As the traditional gas chromatography (12), colorimetry (13), electrochemical analysis (14), and metal-induced sulfide precipitation (15) are with a variety of disadvantages and difficult to implement for in situ detection, fluorescence spectroscopy might become the most attractive technique for in vivo detection of biorelated species by ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12483
virtue of its high-sensitivity, real-time spatial imaging, and non-damaging detection in living cells or tissues (16–18). The design of fluorescent probes for H2S is mainly based on specific chemical reactions, such as the reduction of azide, nitro, or hydroxyl amine group (19–21), Cu2+ quencher removal (22), nucleophilic reaction (23), and thiolysis of dinitrophenyl ether (24). Among the methods mentioned above, reduction reactions exhibit more outstanding detection ability of H2S and Chang and Wang group firstly reported this kind of H2S-selective fluorescent probes (20,25). Shortly after their initial publication appeared, the concept of H2S-mediated reduction of azides to amines was extended to other fluorophore scaffolds by several laboratories (26,27), including the probe C-7Az of Tang’s group (28). C-7Az (Figure 1) was a coumarin-based fluorescence chemoprobe that displayed selective and sensitive detection of hydrogen sulfide in degassed PBS buffers and fetal bovine serum, even could visualize endogenous H2S in cardiac tissues of normal rats and atherosclerosis rats. However, the quantum yield of the fluorescent species C-7Am (kEx/Em = 352/440 nm) is comparatively low (Φ = 0.72 0.03) in aqueous solutions, which was perhaps correlated with little Stocks’ shift (88 nm). Herein, we introduce the electron-withdrawing group, trifluoromethyl to position 4 to obtain a new fluorescent probe CF3-N3 (Figure 1), which emits at a different wavelength (kEx/Em = 375/492 nm), possesses a higher fluorescence quantum yield (Φ = 0.89 0.02), and detects H2S more sensitivity in phosphate buffer.
Methods and Materials Materials and instruments All chemicals and solvents used for synthesis were purchased from Aladdin, J&K, and applied directly in the experiment without further purification. C-7Az and C-7Am were purchased from Sigma-Aldrich, St. Louis, MO, USA. All the buffers used are degassed with N2. 1H NMR and 13 C NMR spectra were recorded on a Bruker model DPX300 MHz NMR spectrometer (Bruker Instruments, Inc., Billerica, MA, USA), values are in ppm relative to tetramethylsilane. Mass spectra (MS) were measured with Waters liquid chromatography–mass spectrometer system (ESI). IR was tested on Nicolet Impact-410. Elemental analysis was recorded on CHN-O-Rapid instrument. Melting points were measured using a Mel-TEMP II melting 1
Qiu et al.
These above solutions were then immediately used for further fluorescence analysis and selectivity test by mixing with CF3-N3 and NaHS or other species working solutions. For each group of experiments, all the solutions were freshly prepared.
Figure 1: Fluorescent probe CF3–N3 for the detection of H2S.
point apparatus, which was uncorrected. Thin-layer chromatography (TLC) was performed on GF/UV 254 plates, and the chromatograms were visualized under UV light at 254 and 365 nm. Fluorescence data were collected fluorescence spectrophotometer (RF-5301 PC, Shimadzu, Tokyo, Japan). pH values of the buffers were obtained on Mettler Toledo Five Easy pH FE20. Confocal laser scanning microscopy was taken on Olympus FluoView-300 confocal laser scanning system (Olympus, Tokyo, Japan).
Cell lines Human leukemia cell lines K562 were kindly provided by Prof. Yunman Li (Department of Physiology, China Pharmaceutical University, Nanjing, China). The cell lines were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified incubator with 5% CO2 in air growth. All experiments were performed with cells in exponential growth.
Cytotoxicity assay K562 cells were grown in 96-well microtitre plates at 4– 5 9 103 cells per well and incubated for 24 h. In the assay of cytotoxic evaluation, a graded dose of CF3-NH2 and CF3-N3 diluted with medium was added into the wells. And the exponentially growing cancer cells were incubated for 48 h in an atmosphere of 95% air with 5% CO2 at 37 °C. Then, MTT was added directly to the cells. After additional incubation for 4 h at 37 °C, the absorbance at 492 nm was read on a microplate reader (Thermo Fisher Scientific) (29).
Visualization of H2S in K562 cells To prepare the experimental setup, approximately 105 cells were seeded in a confocal dish (35 mm) with the medium and allowed to adhere to the dish for 24 h. After the removal of cell medium, the cells were incubated with 10 lM CF3-N3 in phenol red-free media for 60 min at 37 °C, followed by washing the cells with degassed PBS buffer. The treated cells were further incubated for another 60 min with blank phenol red-free media or 50 and 200 lM NaHS in phenol-free media, respectively. Fluorescence imaging was then carried out with confocal laser scanning fluorescence microscopy (Olympus fluoview300).
Results and Discussion Fluorescence analysis and selectivity tests PBS buffer (10 mM, pH = 7.4) used in this study was first degassed with N2 for 30 min. 50 mM stock solution of CF3-N3 was first prepared in DMSO, which was further diluted to 100 lM in degassed PBS buffer for following measurements. 10 mM NaHS was prepared in degassed PBS buffer used as a hydrogen sulfide resource in all the following experiments and was further diluted to 100 lM solution for following experiments. For all other species, 10 mM stock solutions in degassed PBS buffer were prepared, which were further diluted into 1 mM solutions in degassed PBS buffer for use.
Chemistry By the Sandmeyer reaction, we got the target compounds generally prepared from m-aminophenol (30). The synthetic route target compounds CF3-NH2 and CF3-N3 are shown in Scheme 1 (31).
Synthesis of 7-amino-4-(trifluoromethyl)-2Hchromen-2-one(CF3-NH2) The m-aminophenol a (5.46 g, 50 mmol), ethyl trifluoroacetoacetate (11.05 g, 60 mmol), anhydrous zinc chloride (13.6 g, 100 mmol), and ethanol (30 mL) were refluxed for
Scheme 1: Synthesis of CF3–NH2 and CF3–N3. Reagents and conditions: (i) anhydrous zinc chloride, ethanol; (ii) Con.HCl, sodium nitrite and sodium azide, -5 C to 5 C.
2
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12 h. The cooled reaction mixture was poured into 100 mL of 0.1 N aqueous hydrochloric acid. The precipitated product was filtered, washed with water, and dried overnight at room temperature. The crude was reaystallized with EtOH to obtain the pure green solid (6.18 g, 53.9% yield). mp: 221–223 °C; 1H NMR (300 MHz, DMSO-d6): d: 7.37 (d, J = 8.4 Hz, 1H, ArH), 6.66 (d, J = 8.7 Hz, 1H, ArH), 6.53 (s, 2H, NH2), 6.52 (s, 1H, CH), 6.44 (s, 1H, ArH); 13C NMR (75 MHz, DMSO-d6): d: 159.35, 156.52, 154.05, 125.72, 112.15, 107.41, 107.34, 101.69, 98.90; IR (KBr, cm 1): 3453.1, 3360.6 (s, NH2), 3250.7 (w, C=CH), 1708.8 (m, C=O); ESI-MS m/z: 230.2 ([M + H]+);Anal. calcd. for C10H6F3NO2 (229.16): C, 52.41; H, 2.64; N, 6.11%. Found: C, 52.63; H, 2.59; N, 6.04%.
Synthesis of 7-azido-4-(trifluoromethyl)-2Hchromen-2-one(CF3-N3) CF3-NH2 (1 g, 4.36 mmol) was dissolved in 30 mL concentrated hydrochloric acid. In ice–salt baths, sodium nitrite (0.6 g, 8.72 mmol) was batch-added into this solution, and the temperature kept between 0 and 5 °C. The reaction mixture was vigorously stirred for 3 h. During 5 to 0 °C, sodium azide (680 mg, 10.46 mmol) was batch-added into it. The resulting solution was stirred for 4 h. The reaction was monitored by TLC. After the reaction was completed, it was poured into ice water and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, A
filtered, and concentrated. The crude product was purified by silica gel chromatography (ethyl acetate: petroleum ether = 3:97) to obtain the pure product as pale yellow solid (810 mg, 72.26% yield). mp: 95–97 °C; 1H NMR (300 MHz, DMSO-d6): d: 7.66 (d, J = 8.6 Hz, 1H, ArH), 7.27 (d, J=2.1 Hz, 1H, ArH), 7.17 (dd, J = 8.6, 2.1 Hz, 1H, ArH), 6.96 (s, 1H, CH); 13C NMR (75 MHz, DMSO-d6): d: 158.07, 154.89, 144.58, 126.14, 123.34, 119.69, 116.43, 115.65, 109.90, 107.65; IR (KBr, cm 1): 3091.5 (w, C=CH) 2112.1 (m, N3), 1737.8 (s, C=O); ESI-MS m/z: 256.0 ([M + H]+); Anal. calcd. for C10H4F3N3O2 (255.16): C, 47.07; H, 1.58; N, 16.47%. Found: C, 47.22; H, 1.71; N, 16.28%.
Fluorescence response between CF3-N3 and H2S First of all, the test of fluorescence response between CF3-N3 and H2S was conducted. Upon the addition of NaHS, CF3-N3 solution exhibited a robust increase in fluorescent intensity at 490 nm with excitation at 379 nm. As shown in Figure 2B, the fluorescence intensity of 100 lM CF3-N3 solution increased up to 43-fold within 10 min with the addition of 50 lM NaHS. The fluorescence enhancement reached more than 137-fold within 60 min under the same conditions, which appeared obvious fluorescent green under 365 nm-UV in contrast to the CF3-N3 solution before addition NaHS. And during 60 min to 2 h, there was no difference between the fluorescence intensity that indicated 60 min was the end of fluorescence reaction (Figure 2B). All the above B
Figure 2: (A) 100 lM CF3–N3 before (right) and after reaction 60min (left) with 50 lM NaHS in degassed PBS. (B) Fluorescence response of 100 lM CF3-N3 to 50 lM NaHS in 10 mM degassed PBS buffer (pH 7.4) at 37 C with excitation at 375 nm. Emission was collected from 400 to 600 nm at 0, 1, 5, 10, 20, 30, 45, and 60 min after the addition of 50 lM NaHS.
Figure 3: (A) Fluorescence responses of 100 lM CF3–N3 to relevant species in degassed PBS (10 mM, pH 7.4). Bars represent the mean fluorescence responses at 60 min after the addition of 100 lM H2S or 1 mM of all other. (B) H2S concentrationdependent fluorescence intensity determined using fluorescence spectrophotometer (RF5301 PC, SHIMADZU). CF3-N3: 100 lM, NaHS: 0.25, 0.5, 1, 5, 10, 25, 50, 100 lM in degassed PBS (•) and commercial fetal bovine serum (▲) (37 C, λEx/Em= 375/492 nm).
Chem Biol Drug Des 2014
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Figure 4: (A) The mean fluorescence intensity of 100 lM CF3-N3 in PBS with different pH values upon the addition of 50 lM H2S. (B) Photostability of 100 lM CF3N3 in degassed PBS solution with UV light irradiation at different time and the addition of 50 lM NaHS for 30 min (the last black column).
certificated this probe to sense H2S in real time. The reaction product has been confirmed to be CF3-NH2 by NMR, MS, and IR analysis.
Selectivity and linearity of CF3-N3 In complex biological systems, the selectivity of CF3-N3 is the major challenge for the distinct detection of H2S over other cellular molecules. The investigation indicated that only HS (100 lM) induces a dramatic increment at 492 nm with 100 lM CF3-N3 in PBS (10 mM, pH = 7.4), while other anions (HCO3 , F , Cl , Br , I , NO2 , NO3 , N3 , 1 mM), inorganic reactive sulfur species (SO24 , SO23 , S2 O23 , SCN , 1 mM), reactive oxygen species (H2O2, HOCl), and other biological thiols (GSH, Cys, 1 mM) induce only a neglectable enhancement of emission (Figure 3A). The excellent selectivity for H2S shows that CF3-N3 has potential applications for the detection of H2S in a biological environment. According to the further study, there was a good linearity between the fluorescence intensity and the concentrations of H2S in the range 0–10 lM in degassed PBS buffers and fetal bovine serum at 60 min (Figure 3B). The limit of detection (LOD) was as low as 1 lM with a signal-to-noise ratio (S/N) of 3:1, and the limit of quantification (LOQ) was 4.2 lM with S/N of 10:1. Various proteins present in serum are the primary cause for the relatively lower fluorescent intensity in fetal bovine serum, which may be scavengers for H2S (20).
pH and photo stability of CF3-N3 In addition to the good linearity, pH stability of CF3-N3 also has grand effect on biological H2S detection. We then evaluated its fluorescent stability in degassed PBS buffers with different pH values (pH 5.9, 6.4, 7.4, and 7.9) (Figure 4A). For a couple of hours before the addition of NaHS, no detectable change of 100 lM CF3-N3 solution was observed in all the degassed buffers, which reveals that different pH did not have any effect on the optical properties of CF3-N3 itself. With the addition of NaHS, the robust fluorescence enhancement of CF3-N3 was readily detectable within 10 min in comparison with those with no NaHS in all the buffers and the enhancement increased in the buffers with the elevated pH values (pH = 5.9, 6.4, 7.4, and 7.9). The photostability of 4
Figure 5: Cell viability with different concentration of CF3-N3 (0.5, 1, 5, 10, 20, 40, 60, 80 and 100 lM) for K562 cells through MTT assay in 48 h at 37 C.
CF3-N3 was also evaluated with a UV lamp at 365 nm, on account of photolysis of arylazide reagents (32). 100 lM CF3-N3 PBS solution was exposed to irradiation, and fluorescence intensity was recorded at different time. Within 30 min, there was a slight increment at 492 nm, which could be ignorable comparing to the H2S-triggered fluorescence for 30 min (Figure 4B). The assays concerned with stability of CF3-N3 demonstrated it a reliable probe for H2S even in extreme conditions.
Cytotoxicity assay To attest the application of CF3-N3, we next investigated the ability of the developed probe to visualize H2S in living cells. Firstly, the MTT assays for CF3-N3 and the reduced product CF3-NH2 were proceeded, and the results showed that both compounds have scarcely any cytotoxicity and a high IC50 value (>100 lM) after a long period (48 h) (Figure 5). Thus, the probe at 10 lM was selected for imaging in K562 cells.
Visualization of H2S in K562 cells K562 cells were pretreated with only 10 lM CF3-N3 for 60 min at 37 °C in phenol red-free media and exhibited weak fluorescence at 492 nm (Figure 6B), indicating that hardly any intracellular species could metabolize CF3-N3 Chem Biol Drug Des 2014
A High-Sensitivity Probe for Hydrogen Sulfide A
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Figure 6: Fluorescence imaging of H2S in K562 cells incubated with 10 lM CF3-N3. Cells were incubated with CF3-N3 for 60 min followed by PBS washing, after which phenol red free media, 50 lM or 200 lM NaHS was added. After further incubation for 60 min the cells were imaged. Brightfield image of K562 cells incubated with CF3-N3 with absence of (A), 50 lM (D) and 200 lM (G) NaHS; (B), (E), (H) refer fluorescent image of K562 cells in (A), (D), (G) respectively; (C) the overlaid image of (A) and (B); (F) the overlaid image of (D) and (E); (I) the overlaid image of (G) and (H).
under the assay conditions. However, the addition of NaHS (50 lM) to the above cells resulted in an evident change in the observed fluorescence after incubation for another 60 min (Figure 6E). With higher concentrations of NaHS, the fluorescence intensity shows a dose-dependent manner (Figure 6H). Above all, these apparently suggested that CF3-N3 can be used for imaging H2S in living cells. Chem Biol Drug Des 2014
Conclusion We have constructed a novel coumarin-based fluorescence chemoprobe for monitor H2S in PBS and fetal bovine serum, which exhibited remarkable sensitivity to H2S and excellent selectivity over other biological species at more than biologically relevant concentrations. Even though storage at different pH and exposure of UV light, 5
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the optical properties of CF3-N3 were relatively stable. Upon the addition of H2S, distinct fluorescence increments compared to that with no H2S were observed. The potential for biological applications of CF3-N3 was ascertain by employing it for fluorescence imaging of H2S in living cells.
Acknowledgment This study was supported by National Natural Science Foundation of China (Nos 81172932 and 81273376).
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27. Yu F., Li P., Song P., Wang B., Zhao J., Han K. (2012) An ICT-based strategy to a colorimetric and ratiometric fluorescence probe for hydrogen sulfide in living cells. Chem Commun;48:2852–2854. 28. Chen B., Li W., Lv C., Zhao M., Jin H., Jin H., Du J., Zhang L., Tang X. (2013) Fluorescent probe for highly selective and sensitive detection of hydrogen sulfide in living cells and cardiac tissues. Analyst;138:946–951. 29. Jabbar S., Twentyman P., Watson J. (1989) The MTT assay underestimates the growth inhibitory effects of interferons. Br J Cancer;60:523.
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A High-Sensitivity Coumarin-Based Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells Qianqian Qiu1, Xin Deng1, Lei Jiao1, Tianxiao Zhao1, Fanfei Meng2, Wenlong Huang1,* and Hai Qian1,* 1
Center of Drug Discovery, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China 2 Department of Pharmaceutics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China *Corresponding authors: Hai Qian, [email protected]; Wenlong Huang, [email protected] A novel coumarin-based fluorescence probe has been constructed for the selective and sensitive detection of hydrogen sulfide (H2S). This probe displays high sensitivity and linearity to H2S in degassed PBS buffers and fetal bovine serum. It reacts with H2S with high selectivity over Cys, GSH, and other anions. Meanwhile, successful detection of H2S in living cells was also demonstrated. Key words: bioimaging, coumarin, Fluorescence probe, hydrogen sulfide Received 22 October 2014, revised 9 November 2014 and accepted for publication 20 November 2014
Hydrogen sulfide (H2S), stepping with NO and CO, is the third endogenously generated gaseous signaling molecule (1). It plays a vital role in regulating the intracellular redox status and other fundamental signaling processes involved in human health and disease (2–7). The physiologically relevant H2S concentration is estimated to range from nano to millimolar levels (8). Once the level overruns the normal physiological range, H2S would lead to correlative diseases, such as Alzheimer’s disease (9), diabetes (10), Down syndrome (11), and other diseases of mental deficiency (2). Therefore, accurate and reliable detection of H2S concentration in vivo has become the significant subject of current chemical research. As the traditional gas chromatography (12), colorimetry (13), electrochemical analysis (14), and metal-induced sulfide precipitation (15) are with a variety of disadvantages and difficult to implement for in situ detection, fluorescence spectroscopy might become the most attractive technique for in vivo detection of biorelated species by ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12483
virtue of its high-sensitivity, real-time spatial imaging, and non-damaging detection in living cells or tissues (16–18). The design of fluorescent probes for H2S is mainly based on specific chemical reactions, such as the reduction of azide, nitro, or hydroxyl amine group (19–21), Cu2+ quencher removal (22), nucleophilic reaction (23), and thiolysis of dinitrophenyl ether (24). Among the methods mentioned above, reduction reactions exhibit more outstanding detection ability of H2S and Chang and Wang group firstly reported this kind of H2S-selective fluorescent probes (20,25). Shortly after their initial publication appeared, the concept of H2S-mediated reduction of azides to amines was extended to other fluorophore scaffolds by several laboratories (26,27), including the probe C-7Az of Tang’s group (28). C-7Az (Figure 1) was a coumarin-based fluorescence chemoprobe that displayed selective and sensitive detection of hydrogen sulfide in degassed PBS buffers and fetal bovine serum, even could visualize endogenous H2S in cardiac tissues of normal rats and atherosclerosis rats. However, the quantum yield of the fluorescent species C-7Am (kEx/Em = 352/440 nm) is comparatively low (Φ = 0.72 0.03) in aqueous solutions, which was perhaps correlated with little Stocks’ shift (88 nm). Herein, we introduce the electron-withdrawing group, trifluoromethyl to position 4 to obtain a new fluorescent probe CF3-N3 (Figure 1), which emits at a different wavelength (kEx/Em = 375/492 nm), possesses a higher fluorescence quantum yield (Φ = 0.89 0.02), and detects H2S more sensitivity in phosphate buffer.
Methods and Materials Materials and instruments All chemicals and solvents used for synthesis were purchased from Aladdin, J&K, and applied directly in the experiment without further purification. C-7Az and C-7Am were purchased from Sigma-Aldrich, St. Louis, MO, USA. All the buffers used are degassed with N2. 1H NMR and 13 C NMR spectra were recorded on a Bruker model DPX300 MHz NMR spectrometer (Bruker Instruments, Inc., Billerica, MA, USA), values are in ppm relative to tetramethylsilane. Mass spectra (MS) were measured with Waters liquid chromatography–mass spectrometer system (ESI). IR was tested on Nicolet Impact-410. Elemental analysis was recorded on CHN-O-Rapid instrument. Melting points were measured using a Mel-TEMP II melting 1
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These above solutions were then immediately used for further fluorescence analysis and selectivity test by mixing with CF3-N3 and NaHS or other species working solutions. For each group of experiments, all the solutions were freshly prepared.
Figure 1: Fluorescent probe CF3–N3 for the detection of H2S.
point apparatus, which was uncorrected. Thin-layer chromatography (TLC) was performed on GF/UV 254 plates, and the chromatograms were visualized under UV light at 254 and 365 nm. Fluorescence data were collected fluorescence spectrophotometer (RF-5301 PC, Shimadzu, Tokyo, Japan). pH values of the buffers were obtained on Mettler Toledo Five Easy pH FE20. Confocal laser scanning microscopy was taken on Olympus FluoView-300 confocal laser scanning system (Olympus, Tokyo, Japan).
Cell lines Human leukemia cell lines K562 were kindly provided by Prof. Yunman Li (Department of Physiology, China Pharmaceutical University, Nanjing, China). The cell lines were grown in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified incubator with 5% CO2 in air growth. All experiments were performed with cells in exponential growth.
Cytotoxicity assay K562 cells were grown in 96-well microtitre plates at 4– 5 9 103 cells per well and incubated for 24 h. In the assay of cytotoxic evaluation, a graded dose of CF3-NH2 and CF3-N3 diluted with medium was added into the wells. And the exponentially growing cancer cells were incubated for 48 h in an atmosphere of 95% air with 5% CO2 at 37 °C. Then, MTT was added directly to the cells. After additional incubation for 4 h at 37 °C, the absorbance at 492 nm was read on a microplate reader (Thermo Fisher Scientific) (29).
Visualization of H2S in K562 cells To prepare the experimental setup, approximately 105 cells were seeded in a confocal dish (35 mm) with the medium and allowed to adhere to the dish for 24 h. After the removal of cell medium, the cells were incubated with 10 lM CF3-N3 in phenol red-free media for 60 min at 37 °C, followed by washing the cells with degassed PBS buffer. The treated cells were further incubated for another 60 min with blank phenol red-free media or 50 and 200 lM NaHS in phenol-free media, respectively. Fluorescence imaging was then carried out with confocal laser scanning fluorescence microscopy (Olympus fluoview300).
Results and Discussion Fluorescence analysis and selectivity tests PBS buffer (10 mM, pH = 7.4) used in this study was first degassed with N2 for 30 min. 50 mM stock solution of CF3-N3 was first prepared in DMSO, which was further diluted to 100 lM in degassed PBS buffer for following measurements. 10 mM NaHS was prepared in degassed PBS buffer used as a hydrogen sulfide resource in all the following experiments and was further diluted to 100 lM solution for following experiments. For all other species, 10 mM stock solutions in degassed PBS buffer were prepared, which were further diluted into 1 mM solutions in degassed PBS buffer for use.
Chemistry By the Sandmeyer reaction, we got the target compounds generally prepared from m-aminophenol (30). The synthetic route target compounds CF3-NH2 and CF3-N3 are shown in Scheme 1 (31).
Synthesis of 7-amino-4-(trifluoromethyl)-2Hchromen-2-one(CF3-NH2) The m-aminophenol a (5.46 g, 50 mmol), ethyl trifluoroacetoacetate (11.05 g, 60 mmol), anhydrous zinc chloride (13.6 g, 100 mmol), and ethanol (30 mL) were refluxed for
Scheme 1: Synthesis of CF3–NH2 and CF3–N3. Reagents and conditions: (i) anhydrous zinc chloride, ethanol; (ii) Con.HCl, sodium nitrite and sodium azide, -5 C to 5 C.
2
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12 h. The cooled reaction mixture was poured into 100 mL of 0.1 N aqueous hydrochloric acid. The precipitated product was filtered, washed with water, and dried overnight at room temperature. The crude was reaystallized with EtOH to obtain the pure green solid (6.18 g, 53.9% yield). mp: 221–223 °C; 1H NMR (300 MHz, DMSO-d6): d: 7.37 (d, J = 8.4 Hz, 1H, ArH), 6.66 (d, J = 8.7 Hz, 1H, ArH), 6.53 (s, 2H, NH2), 6.52 (s, 1H, CH), 6.44 (s, 1H, ArH); 13C NMR (75 MHz, DMSO-d6): d: 159.35, 156.52, 154.05, 125.72, 112.15, 107.41, 107.34, 101.69, 98.90; IR (KBr, cm 1): 3453.1, 3360.6 (s, NH2), 3250.7 (w, C=CH), 1708.8 (m, C=O); ESI-MS m/z: 230.2 ([M + H]+);Anal. calcd. for C10H6F3NO2 (229.16): C, 52.41; H, 2.64; N, 6.11%. Found: C, 52.63; H, 2.59; N, 6.04%.
Synthesis of 7-azido-4-(trifluoromethyl)-2Hchromen-2-one(CF3-N3) CF3-NH2 (1 g, 4.36 mmol) was dissolved in 30 mL concentrated hydrochloric acid. In ice–salt baths, sodium nitrite (0.6 g, 8.72 mmol) was batch-added into this solution, and the temperature kept between 0 and 5 °C. The reaction mixture was vigorously stirred for 3 h. During 5 to 0 °C, sodium azide (680 mg, 10.46 mmol) was batch-added into it. The resulting solution was stirred for 4 h. The reaction was monitored by TLC. After the reaction was completed, it was poured into ice water and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, A
filtered, and concentrated. The crude product was purified by silica gel chromatography (ethyl acetate: petroleum ether = 3:97) to obtain the pure product as pale yellow solid (810 mg, 72.26% yield). mp: 95–97 °C; 1H NMR (300 MHz, DMSO-d6): d: 7.66 (d, J = 8.6 Hz, 1H, ArH), 7.27 (d, J=2.1 Hz, 1H, ArH), 7.17 (dd, J = 8.6, 2.1 Hz, 1H, ArH), 6.96 (s, 1H, CH); 13C NMR (75 MHz, DMSO-d6): d: 158.07, 154.89, 144.58, 126.14, 123.34, 119.69, 116.43, 115.65, 109.90, 107.65; IR (KBr, cm 1): 3091.5 (w, C=CH) 2112.1 (m, N3), 1737.8 (s, C=O); ESI-MS m/z: 256.0 ([M + H]+); Anal. calcd. for C10H4F3N3O2 (255.16): C, 47.07; H, 1.58; N, 16.47%. Found: C, 47.22; H, 1.71; N, 16.28%.
Fluorescence response between CF3-N3 and H2S First of all, the test of fluorescence response between CF3-N3 and H2S was conducted. Upon the addition of NaHS, CF3-N3 solution exhibited a robust increase in fluorescent intensity at 490 nm with excitation at 379 nm. As shown in Figure 2B, the fluorescence intensity of 100 lM CF3-N3 solution increased up to 43-fold within 10 min with the addition of 50 lM NaHS. The fluorescence enhancement reached more than 137-fold within 60 min under the same conditions, which appeared obvious fluorescent green under 365 nm-UV in contrast to the CF3-N3 solution before addition NaHS. And during 60 min to 2 h, there was no difference between the fluorescence intensity that indicated 60 min was the end of fluorescence reaction (Figure 2B). All the above B
Figure 2: (A) 100 lM CF3–N3 before (right) and after reaction 60min (left) with 50 lM NaHS in degassed PBS. (B) Fluorescence response of 100 lM CF3-N3 to 50 lM NaHS in 10 mM degassed PBS buffer (pH 7.4) at 37 C with excitation at 375 nm. Emission was collected from 400 to 600 nm at 0, 1, 5, 10, 20, 30, 45, and 60 min after the addition of 50 lM NaHS.
Figure 3: (A) Fluorescence responses of 100 lM CF3–N3 to relevant species in degassed PBS (10 mM, pH 7.4). Bars represent the mean fluorescence responses at 60 min after the addition of 100 lM H2S or 1 mM of all other. (B) H2S concentrationdependent fluorescence intensity determined using fluorescence spectrophotometer (RF5301 PC, SHIMADZU). CF3-N3: 100 lM, NaHS: 0.25, 0.5, 1, 5, 10, 25, 50, 100 lM in degassed PBS (•) and commercial fetal bovine serum (▲) (37 C, λEx/Em= 375/492 nm).
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Figure 4: (A) The mean fluorescence intensity of 100 lM CF3-N3 in PBS with different pH values upon the addition of 50 lM H2S. (B) Photostability of 100 lM CF3N3 in degassed PBS solution with UV light irradiation at different time and the addition of 50 lM NaHS for 30 min (the last black column).
certificated this probe to sense H2S in real time. The reaction product has been confirmed to be CF3-NH2 by NMR, MS, and IR analysis.
Selectivity and linearity of CF3-N3 In complex biological systems, the selectivity of CF3-N3 is the major challenge for the distinct detection of H2S over other cellular molecules. The investigation indicated that only HS (100 lM) induces a dramatic increment at 492 nm with 100 lM CF3-N3 in PBS (10 mM, pH = 7.4), while other anions (HCO3 , F , Cl , Br , I , NO2 , NO3 , N3 , 1 mM), inorganic reactive sulfur species (SO24 , SO23 , S2 O23 , SCN , 1 mM), reactive oxygen species (H2O2, HOCl), and other biological thiols (GSH, Cys, 1 mM) induce only a neglectable enhancement of emission (Figure 3A). The excellent selectivity for H2S shows that CF3-N3 has potential applications for the detection of H2S in a biological environment. According to the further study, there was a good linearity between the fluorescence intensity and the concentrations of H2S in the range 0–10 lM in degassed PBS buffers and fetal bovine serum at 60 min (Figure 3B). The limit of detection (LOD) was as low as 1 lM with a signal-to-noise ratio (S/N) of 3:1, and the limit of quantification (LOQ) was 4.2 lM with S/N of 10:1. Various proteins present in serum are the primary cause for the relatively lower fluorescent intensity in fetal bovine serum, which may be scavengers for H2S (20).
pH and photo stability of CF3-N3 In addition to the good linearity, pH stability of CF3-N3 also has grand effect on biological H2S detection. We then evaluated its fluorescent stability in degassed PBS buffers with different pH values (pH 5.9, 6.4, 7.4, and 7.9) (Figure 4A). For a couple of hours before the addition of NaHS, no detectable change of 100 lM CF3-N3 solution was observed in all the degassed buffers, which reveals that different pH did not have any effect on the optical properties of CF3-N3 itself. With the addition of NaHS, the robust fluorescence enhancement of CF3-N3 was readily detectable within 10 min in comparison with those with no NaHS in all the buffers and the enhancement increased in the buffers with the elevated pH values (pH = 5.9, 6.4, 7.4, and 7.9). The photostability of 4
Figure 5: Cell viability with different concentration of CF3-N3 (0.5, 1, 5, 10, 20, 40, 60, 80 and 100 lM) for K562 cells through MTT assay in 48 h at 37 C.
CF3-N3 was also evaluated with a UV lamp at 365 nm, on account of photolysis of arylazide reagents (32). 100 lM CF3-N3 PBS solution was exposed to irradiation, and fluorescence intensity was recorded at different time. Within 30 min, there was a slight increment at 492 nm, which could be ignorable comparing to the H2S-triggered fluorescence for 30 min (Figure 4B). The assays concerned with stability of CF3-N3 demonstrated it a reliable probe for H2S even in extreme conditions.
Cytotoxicity assay To attest the application of CF3-N3, we next investigated the ability of the developed probe to visualize H2S in living cells. Firstly, the MTT assays for CF3-N3 and the reduced product CF3-NH2 were proceeded, and the results showed that both compounds have scarcely any cytotoxicity and a high IC50 value (>100 lM) after a long period (48 h) (Figure 5). Thus, the probe at 10 lM was selected for imaging in K562 cells.
Visualization of H2S in K562 cells K562 cells were pretreated with only 10 lM CF3-N3 for 60 min at 37 °C in phenol red-free media and exhibited weak fluorescence at 492 nm (Figure 6B), indicating that hardly any intracellular species could metabolize CF3-N3 Chem Biol Drug Des 2014
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Figure 6: Fluorescence imaging of H2S in K562 cells incubated with 10 lM CF3-N3. Cells were incubated with CF3-N3 for 60 min followed by PBS washing, after which phenol red free media, 50 lM or 200 lM NaHS was added. After further incubation for 60 min the cells were imaged. Brightfield image of K562 cells incubated with CF3-N3 with absence of (A), 50 lM (D) and 200 lM (G) NaHS; (B), (E), (H) refer fluorescent image of K562 cells in (A), (D), (G) respectively; (C) the overlaid image of (A) and (B); (F) the overlaid image of (D) and (E); (I) the overlaid image of (G) and (H).
under the assay conditions. However, the addition of NaHS (50 lM) to the above cells resulted in an evident change in the observed fluorescence after incubation for another 60 min (Figure 6E). With higher concentrations of NaHS, the fluorescence intensity shows a dose-dependent manner (Figure 6H). Above all, these apparently suggested that CF3-N3 can be used for imaging H2S in living cells. Chem Biol Drug Des 2014
Conclusion We have constructed a novel coumarin-based fluorescence chemoprobe for monitor H2S in PBS and fetal bovine serum, which exhibited remarkable sensitivity to H2S and excellent selectivity over other biological species at more than biologically relevant concentrations. Even though storage at different pH and exposure of UV light, 5
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the optical properties of CF3-N3 were relatively stable. Upon the addition of H2S, distinct fluorescence increments compared to that with no H2S were observed. The potential for biological applications of CF3-N3 was ascertain by employing it for fluorescence imaging of H2S in living cells.
Acknowledgment This study was supported by National Natural Science Foundation of China (Nos 81172932 and 81273376).
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