- Email: [email protected]
Design, synthesis and evaluation of a novel fluorescent probe to accurately detect H2S in hepatocytes and natural waters
Journal Pre-proof Design, synthesis and evaluation of a novel fluorescent probe to accurately detect H2S in hepatocytes and natural waters Dadong Shen, Jian Liu, Li Sheng, Yonghui Lv, Guofeng Wu, Pu Wang, Kui Du PII:
S1386-1425(19)31080-7
DOI:
https://doi.org/10.1016/j.saa.2019.117690
Reference:
SAA 117690
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: 14 September 2019 Revised Date:
21 October 2019
Accepted Date: 22 October 2019
Please cite this article as: D. Shen, J. Liu, L. Sheng, Y. Lv, G. Wu, P. Wang, K. Du, Design, synthesis and evaluation of a novel fluorescent probe to accurately detect H2S in hepatocytes and natural waters, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2019), doi: https:// doi.org/10.1016/j.saa.2019.117690. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Design, synthesis and evaluation of a novel fluorescent probe to accurately detect H2S in hepatocytes and natural waters Dadong Shen a, Jian Liu b, Li Sheng c, Yonghui Lv c, Guofeng Wu c, Pu Wang a* and Kui Du b* a
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou
310014, China. E-mail: [email protected] b
School of Chemistry and Chemical Engineering, Shaoxing University, Shaoxing
312000, China, Email: [email protected] c
Research & Development Center, Zhejiang Medicine Co. Ltd. Shaoxing 312500,
China, Email: [email protected] Abstract: Design and synthesis of fluorescent probe with fast response, excellent water solubility and good hepatocyte-targeting capacity to detect hydrogen sulfide (H2S) in hepatocytes and water samples is of great significance. Here, a novel fluorescent probe QL-Gal-N3 for detection of H2S was designed and synthesized based on H2Smediated azide reduction strategy. This sensor demonstrated low toxicity, fast response (within 1 min), high selectivity and low detection limit (as low as 126 nM in water) for the detection of H2S. HeLa, A549 and HepG-2 cells were chosen to investigate the hepatocyte-targeting ability of QL-Gal-N3 respectively. The results indicated that the specific recognition of ASGPR over-expressed in hepatocytes by galactose group was an important reason for the good targeting ability of probe QLGal-N3. Furthermore, due to the introduction of glycosyl moiety, the water solubility of fluorescent probe was enhanced obviously. It was successfully applied for the detection of H2S in environmental water samples including river water, tap water, lake water and waste water. 1
Keywords:
H2S;
Azide
reduction;
Fluorescent
probe;
Hepatocyte-targeting;
Environmental water samples 1. Introduction Hydrogen sulfide (H2S) is an important gas signal molecule, which is second only to nitric oxide and carbon monoxide in human body [1]. It can effectively remove hydrogen peroxide, superoxide anion and so on [2, 3]. However, the safety window of H2S between physiological level and toxicological level is narrow with less than two folds in the brain tissues [4]. And it is predominantly generated in the liver due to the tissue-specific distribution of Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), which responsible for the production of H2S with L(homo)cystein as the substrate [5]. In addition, H2S is soluble in water and can directly cause organism death after reaching a certain concentration, which is one of the important parameters for water quality detection [6]. Therefore, it is of great significance to detect the content of H2S in organisms especially liver and natural waters efficiently. Over the past few decades, electrochemical analysis [7, 8], chromatography [9], titrimetry [10], colorimetry [11] and fluorescence analysis [12] had been used for quantitative measurement of H2S. Among these detection methods, fluorescence analysis had attracted widespread attention due to its great advantages such as high sensitivity, simple operation, fast detection speed, simple fluorescence imaging and in situ detection [13-19]. Numerous small-molecule fluorescent probes based on fluorophore such as fluorescein, naphthalimide, quinoline, rhodamine, cyanine and coumarin were designed through reaction-based strategies for the detection of H2S [20-24]. And among the main reaction-based strategies for H2S, H2S-mediated azide reduction had been widely reported for its fast response, low detection limit and good 2
selectivity [25-27]. However, the targeting capacity and water solubility of these sensors still need to be improved to afford the applications of H2S detection in liver and water samples [28]. Recently, several small-molecule fluorescent probes with hepatocyte-selective imaging of H2S had been reported through introducing a glycosyl moiety (as a targeting agent) to the fluorescence probe [29-35]. This strategy provided a good direction for designing of fluorescent probes with good performance in detection of H2S in liver. However, the application of these sensors for the detection of H2S in water samples are rare and still in highly demand. Here we have reported a new smallmolecule fluorescent probes for H2S through the introduction of a glycosyl moiety to quinoline fluorophore. This new sensor displayed fast response (within 1 min), high sensitivty, low detection limit (as low as 126.3 nM in water) and excellent water solubility. In addition, it also demonstrated good application prospects for accurate detection of H2S in hepatocytes and natural water.
Scheme 1 Synthesis of QL-Gal-N3
3
2. Experimental 2.1. Materials and apparatus Unless otherwise specified, chemicals were purchased from J&K Scientific Co., Ltd. and used without further purification. During the experiment, water samples were taken directly from Shaoxing area. Bruker Avance-400 spectrometer was choosen to get the 1 H NMR and
13
C NMR spectra with CDCl3 or d6-DMSO used as a solvent
and tetramethylsilane (TMS) as an internal standard. Human AGSPR ELISA Kit was purchased from AMEKO.The absorbance of MTT was measured by Synergy HT Multi-Mode Microplate reader (Biotek). Fluorescent measurements and UV spectra were measured with SpectraMax M5. The confocal images of cells were captured by Zeiss Fluorescence Microscope (A1 Axiovert) with x40 objective lens. 2.2. Synthesis of QL-Gal-N3 The synthetic route of QL-Gal-N3 was shown in Scheme 1. The detailed synthesis process of compounds 2–5 were provided in the supporting information. Compound 5 (0.5g, 1.34 mmol), TMSN3 (0.185g, 1.6 mmol) and Tert-butyl nitrite(0.21g, 2.0 mmol) were dissolved in THF(50 mL). The reaction mixture was stirred at room temperature for 24h. After the reaction, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography with CH3OH and DCM (4:1) as eluent to give target QL-Gal-N3. White solid (0.1g, 19%) M p: 125-127 ℃; 1H NMR (400 MHz, DMSO) δ 9.12 (dd, J = 8.7, 1.6 Hz, 1H), 8.99 (dd, J = 4.1, 1.6 Hz, 1H), 8.84 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.74 (dd, J = 8.7, 4.1 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 5.61 (d, J = 9.2 Hz, 1H), 5.36 (d, J = 5.8 Hz, 1H), 5.10 (d, J = 5.5 Hz, 1H), 4.73 (t, J = 5.6 Hz, 1H), 4.67 (d, J = 5.3 Hz, 1H), 4.22-4.11 (m, 1H), 3.79 (t, J = 5.6 Hz, 2H), 3.63-3.53 (m, 3H). 13C NMR 4
(101 MHz, DMSO) δ 149.66, 145.12, 142.25, 136.11, 135.45, 127.58, 126.82, 125.32, 123.40, 123.14, 119.58, 88.35,78.48, 73.48, 69.24, 68.42, 60.39. HRMS (ESI+): found [M+H]+ 400.1380 C17H18N7O5 requires [M+H]+ 400.1379 2.3. Preparations of test solutions and detection procedure The stock solution of QL-Gal-N3 (1 mM) was prepared in deionized water and sodium sulfide (10 mM) solution was prepared through dissolving in PBS (10 mM, pH 7.4). Stock solutions of cysteine (Cys), glucose (Glu), glutathione (GSH), glycine (Gly), L-alanine (L-Ala), D-alanine (D-Ala), lysine (Lys), homocysteine(Hcy) as well as ions, including Cl- , Br-, F-, NO3-, SO42-, SO32-, K+ and Ca2+ were prepared in deionized water with concentration as 10 mM and kept at 2°C in a refrigerator. Before testing, 985 µL of PBS (10 mM, pH 7.4) and 5 µL of the stock solution of QL-Gal-N3 were mixed, followed by addition of different concentrations of analytes. Then the final volume was adjusted to 1 mL. After incubation at 37 °C for 30 min, 150 µL of the mixture was transferred to 96-well plates to measure fluorescent spectra. The excitation wavelength was 365 nm, and the emission wavelength was in the range from 400 nm to 650 nm with both excitation and emission slits of 5.0 nm. The quantum yield of probe QL-Gal-N3 and probe after reaction with Na2S was determined in PBS buffer using rhodamine B (ɸ, 0.69) as reference. 2.4. Water samples analysis Tap water, river water and lake water were collected from our laboratory, Keqiao District and Guazhu Laker in Shaoxing city, respectively. And the artificial waste water was prepared according to reference [28, 36, 37]. Furthermore, these water samples had not undergone any other special treatment before use. The standard addition method was chosen to determine the H2S content of the sample. The
5
fluorescence spectra were recorded by adding different concentrations of sodium sulfide into water samples. 2.5. Cell Culture and cytotoxicity test HeLa cells, A549 cells and HepG-2 cells were cultured in DMEM high glucose media supplemented with 10% fetal bovineserum, 1% Penstrep, 0.2% Amphotericin B at 37°C incubator with 5% CO2, respectively. And MTT assay was performed to assess the viability of above three cells. The cells were seeded at a density of 10,000 cells per well and grown overnight at 37°C incubator with 5% CO2 . Then different concentrations of probe QL-Gal-N3 were added into 96-well plate for 48h. After that, 10 µL of MTT solution (5mg/ml in PBS) was added into each well and solubilized using 100 µL of MTT solubilizing agent (DMSO) after 2h. Finally, the absorbance at 590 nm with a reference filter of 620 nm were taken using Synergy HT Multi-Mode Microplate reader (Biotek). 2.6 Confocal imaging of Hela cells, A549 cells and HepG-2 cells As for the cell image of HeLa cells, HeLa cells were incubated probe QL-GalN3 (5 µM) for 30 min and washed with PBS for three times, and images were acquired using Zeiss Fluorescence Microscope (A1 Axiovert) with x40 objective lens. After which HeLa cells were treated with different concentrations of H2S (0 µM, 10 µM and 50 µM) and incubated for 30 min, then the cell image were obtained under the excitation with 405nm for collection at 460–540nm. And we choosing 50 µM H2S for the investigation of time-dependent imaging in HeLa cells. In order to get the information of endogenous hydrogen sulfide in HeLa cells, A549 cells and HepG-2 cells, the above cell lines were incubated probe QL-Gal-N3 (5 µM) for 30 min respectively. And the cell image were obtained under the excitation with 405nm for collection at 460–540nm. 6
3. Result and disscussion 3.1. Selective detection of probe QL-Gal-N3 to H2S over other analytes In order to investigate the application of probe QL-Gal-N3 on H2S detection for biological and water samples, the selectivity was conducted firstly. Various species (100 µM) were selected to culture with probe QL-Gal-N3 (5 µM) and fluorescent emission spectra were measured after 30 min. As shown in Fig. 1a, a significant change of QL-Gal-N3 (ɸ, 0.003) was introduced by H2S in fluorescence at 521 nm, and almost 102-folds enhancement in intensity was achieved (compound 5 ɸ, 0.308). Although, probe QL-Gal-N3 exhibited a weak fluorescence intensity with Cys and GSH, it will introduce little influence on the detection process. At the same time, we increased the concentration of GSH to 5 mM, but no obvious fluorescence enhancement at 521 nm was observed (Fig. S6). It demonstrated that QL-Gal-N3 can specifically interact with H2S. The interference experiments of other species on QLGal-N3 was also conducted by us, Fig. 1b. It shown that the co-existent species have little effect on the fluorescent intensity of probe QL-Gal-N3 with H2S. These results indicated that probe QL-Gal-N3 has an excellent selectivity towards different analytes. (a)
(b)
Fig. 1. (a) Fluorescent emission spectra of QL-Gal-N3 (5 µM) in PBS buffer (pH=7.4) to H2S or other species (100 µM) for 30 min; (b) Fluorescent intensity of QL-Gal-N3
7
(5 µM) in the presence of 100µM interfering species including (1) Cl- , (2) Br-, (3) F-, (4) Hcy, (5) NO3-, (6) Cys, (7) SO42-, (8) SO32-, (9)K+, (10) Ca2+, (11) GSH, (12) Gly, (13)L-Ala, (14) D-Ala, (15)Lys, (16) Glu; λEx = 365 nm, λ Em = 521 nm. 3.2. Time-dependent and pH-fluorescence intensity of probe QL-Gal-N3 The pH dependence and response time of QL-Gal-N3 to H2S in biological atomosphere and water are important parameters for the applications. In order to investigate the pH effect on probe QL-Gal-N3, intensity of free QL-Gal-N3 and QLGal-N3 with H2S were obtained at the different range of pH. As shown in Fig. 2a, the fluorescence emission intensity was much lower in acidic environments, which indicating that the thiolysis of the azido bond can not be introduced in strongly acidic solutions [27]. Probe QL-Gal-N3 with H2S maintaining a good pH stability rang from pH=4-10, especially reach the highest intensity when pH=7. The value of saturated hydrogen sulfide aqueous solution is pH=4.14, which indicated that Probe QL-GalN3 is suitable for detecting H2S in water sample.
Fig. 2 (a) pH-dependent fluorescence intensity of QL-Gal-N3 (5 µM) with H2S (100 µM) in PBS buffer (pH= 7.4); (b) Time-dependent fluorescent intensity of QL-GalN3 (5 µM) with H2S (100 µM) in PBS buffer; λ Ex = 365 nm, λ Em = 521 nm. In addition, the time-dependent fluorescence signal of QL-Gal-N3 to H2S at 521 nm was checked (Fig. 2b). The results shown that the fluorescent intensity increased 8
quickly and achieved the plateau within about 1 min, and indicating that probe QLGal-N3 could response rapidly with H2S under physiological conditions. 3.3. The absorbance and concentration-dependent detection of H2S by QL-Gal-N3 In order to titrate the detection capacity of
probe QL-Gal-N3 to H2S, the
absorbance and emission spectra were examined (Fig. S1). The absorption spectrum of QL-Gal-N3 to H2S demonstrated a maximum band at 365 nm, and the intensity increased linearly with the addition of H2S from 0 to 100µM. Furthermore, as shown in Fig. 3b, the fluorescence emission intensity also demonstrated an excellent linearity with the concentration of H2S from 0 to 100µM. The detection limit of probe QL-Gal-N3 to H2S was calculated as 126 nM in PBS buffer through using the formula 3σ/k (σ is obtained from ten blank measurement and k is slope of linear). These results indicated that QL-Gal-N3 had an extremely low detection limit and was suitable for the detection of H2S in water samples. (a)
(b)
Fig. 3 (a) Fluorescent spectra of QL-Gal-N3 (5 µM) in PBS buffer (pH= 7.4) with the addition of H2S. (b) Linearity between the fluorescence intensity of QL-Gal-N3 and the concentration of H2S from 0 to 140µM; λEx = 365 nm.
3.4. Application of probe QL-Gal-N3 in water samples with different concentrations of H2S 9
Table 1 Detection of H2S in tap water, lake water, river water and waste water Samples
Na2S added
Na2S found
RSD
Recovery
(µM)
(µM)
(%)
(%)
1
−
−
−
−
2
1.5
1.52
1.13
102
3
3.0
3.06
1.02
100
4
4.5
4.52
0.96
97
Lake
1
−
−
−
−
water
2
1.5
1.52
1.83
100
3
3.0
3.04
1.25
101
4
4.5
4.48
0.72
94
River
1
−
−
−
−
water
2
1.5
1.54
3.17
118
3
3.0
3.11
2.15
108
4
4.5
4.74
1.23
99
Waste
1
−
−
−
−
water
2
1.5
1.95
6.95
122
3
3.0
3.34
5.21
111
4
4.5
4.86
3.87
108
Tap water
Number
In light of above fluorescent properties of probe QL-Gal-N3, we further investigated its application for H2S in different water samples. We collected river water from Keqiao District, Shaoxing City, which is the largest textile center in Asia. And the tap water in our laboratory, lake water in the Lake of Guazhu were collected by us respectively. Furthermore, the artificial waste water was prepared according to reference [28]. The standard addition method was chosen for the application of probe QL-Gal-N3 in water samples with different concentrations of H2S. As shown in Table 1, the recoveries were in the range of 94%–122% and RSD is less than 7%, demonstrating that the probe QL-Gal-N3 as a highly selective and sensitive fluorescent sensor for H2S detection in water samples. 3.5. Target imaging of H2S in cells by QL-Gal-N3
10
In order to investigate the applicability of the probe QL-Gal-N3 in cell imaging, the cytotoxicities of QL-Gal-N3 for HeLa cells, A549 cells and HepG-2 cells lines were determained. As shown in Fig. S2, 87%, 92% and 85% cells survived was achieved after incubation with QL-Gal-N3 (5 µM) for 24 h, respectively, indicated probe QL-Gal-N3 was low cytotoxicity for the above three cell lines. And then the HeLa was chosen to examine the imaging of probe QL-Gal-N3 (5 µM) with different concentration of H2S. As shown in Fig. 4, when HeLa cells incubated with free probe QL-Gal-N3 for 30 min, little fluorescence displayed. However, co-incubation of QL-Gal-N3 with different concentrations of H2S(10µM and 50µM) for 30min, a gradual increase of fluorescent intensity was detected. These results indicated QL-Gal-N3 can be applied to investigate the change of H2S in cells.
Fig. 4 Confocal imaging of fluorescence and bright field images of probe QL-Gal-N3 (5 µM) in HeLa cells treated with different concentration of H2S for 30 min; λ
Ex
=
405 nm, λ Em = 460-540 nm In order to investigate the time-dependent imaging of QL-Gal-N3 toward H2S in living cells, co-incubation of QL-Gal-N3(5 µM ) and H2S (50 µM ) in HeLa cells was conducted (Fig. 5). The results shown that the fluorescence intensity gradually increased and reaching a maximum after 3 min. Furthermore, the intensity displayed
11
no obvious increase after 3 min, which indicated that QL-Gal-N3 is a rapid responsive and highly sensitive fluorogenic probe to detect H2S in cancer cells.
Fig. 5 Confocal imaging of probe QL-Gal-N3 (5 µM) in Hela cells treated with H2S (50 µM) for 1.0 min, 2.0 min and 3.0 min; λ Ex = 405 nm, λ Em = 460-540 nm Subsequently, the target imaging of H2S in cells by QL-Gal-N3 was tested by us. Before imaging, an enzyme linked immunosorbent assay ( ELISA) was conducted to determine the ASGPR level in HepG-2, A549 and Hela in this study. As shown in Fig. S5 and Table S1, HepG-2 cells showed the highest ASGPR level with 168.15 pg/mL, while A549 and Hela cells displayed low level with 3.48 and 3.23 pg/mL, respectively. After co-incubation of QL-Gal-N3 (10 µM) with HepG-2 cells, A549 cells and HeLa cells for 10 min, a strong fluorescence intensity was observed in HepG-2 cells, as shown in Fig. 6. And demonstrated obviously selective for HepG-2 cells than A549 cells and HeLa cells. These results suggested that the presence of the galactosyl moiety facilitated the selective internalization of the probe QL-Gal-N3 through ASGPr-mediated endocytosis.
12
Fig. 6 Confocal imaging of probe QL-Gal-N3 (10 µM) in HepG-2 cells, A549 cells and HeLa cells for 10 min; λ Ex = 405 nm, λ Em = 460-540 nm 3.6. Comparison of probe QL-Gal-N3 with reported H2S sensors which based on the H2S-mediated azide reduction strategy According to the HRMS spectra of the reaction of QL-Gal-N3 with H2S (Fig. S7) and related literatures [29, 37-51], the specific recognition of QL-Gal-N3 for H2S is based on the reaction mechanism of H2S-mediated reduction of aryl azides. In recent years, the effective design of H2S fluorescent probes through H2S-mediated azide reduction strategy had attracted extensive attention from the researchers (Table 2). the Compared with the reported H2S sensors, probe QL-Gal-N3 demonstrated a good hepatocyte-targeting capacity, while many reported sensor were lack of targeting ability [39-41, 43, 44, 46-52]. QL-Gal-N3 also displayed relatively good detection performances for H2S than most of the reported sensors with a very short response time and an excellent detection limit. More importantly, QL-Gal-N3 showed good water solubility, and was successfully used for the determination of H2S in water samples, however, almost all of the reported fluorescent probes had not been used for the detection of H2S in water samples.
13
Table 2 Comparison of QL-Gal-N3 with other reported H2S sensors based on the H2S-mediated azide reduction strategy Ref.
Targeting
Detection
Response
limit
time
Media
H2S detection in water samples
29
hepatocyte
7.8×10-7 M
2 min
Aqueous solution
No
37
lysosomal
2.14×10-7 M
1 min
PBS (10% DMSO)
No
38
No
1.8×10-8 M
30 min
PBS (50% DMSO)
No
39
No
No discuss
10 min
PBS (1% CH3CN)
No
40
No
No discuss
1h
HEPES
No
41
lysosomal
No discuss
40 min
HEPES (50% CH3CN)
No
42
No
No discuss
10 min
Water(25% Ethanol)
No
43
No
8.6×10-8 M
60 min
PIPES buffer
No
44
lysosomal
5.0×10-7 M
> 60 min
PBS (10% DMF)
No
45
No
7.8×10-10 M
3 min
PBS buffer
No
46
No
1.12×10-7 M
30 min
PBS buffer
No
47
No
No discuss
No discuss
HEPES (33% CH3CN)
No
48
No
2.2×10-7 M
1 min
PBS (75% Acetone)
No
49
No
3.1×10-6 M
60 min
PBS buffer
No
50
No
No discuss
1 min
PBS (50% CH3CN)
No
51
No
8.0×10-8 M
10 min
HEPES
No
QL-
hepatocyte
1.26×10-7 M
1 min
PBS buffer
Yes
Gal-N3
4. Conclusion In summary, a novel hepatocyte-targeting fluorescent probe QL-Gal-N3 with good water solubility was designed and synthesized. QL-Gal-N3 displayed good sensitivity and selectivity toward H2S with the detection limit reached 126 nM. 14
Furthermore, probe QL-Gal-N3 demonstrated low toxicity and quick response (about 1 min) to detect H2S. Because of the introduction of glycosyl moiety, the water solubility and hepatocyte-targeting ability were enhanced obviously. The fluorescent probe was successfully applied for the detection of H2S in water samples and hepatocyte. Acknowledgements We are grateful to the National New Drug Innovation Program (No. 2008ZX09101048) for providing financial support. Conflicts of interest There are no conflicts to declare. References [1] G. Muyzer, A.J.M. Stams, The ecology and biotechnology of sulphate-reducing bacteria, Nat. Rev. Microbiol. 6 (2008) 441-454. [2] K. Shatalin, E. Shatalina, A. Mironov, E. Nudler, H2S: A Universal Defense Against Antibiotics in Bacteria, Science 334 (2011) 986-990. [3] K.R. Olson, Y. Gao, F. Arif, K. Arora, S. Patel, E.R. DeLeon, T.R. Sutton, M. Feelisch, M.M. Cortese-Krott, K.D. Straub, Metabolism of hydrogen sulfide (H2S) and Production of Reactive Sulfur Species (RSS) by superoxide dismutase, Redox Biol. 15 (2018) 74-85. [4] M.W. Warenycia, L.R. Goodwin, C.G. Benishin, R.J. Reiffenstein, D.M. Francom, J.D. Taylor, F.P. Dieken, Acute hydrogen sulfide poisoning. Demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels, Biochem. Pharmacol. 38 (1989) 973–981.
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a
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21
. . . .
H2S fluorescent probe with excellent hepatocyte-targeting capacity
Low toxicity, good selectivity and excellent water solubility
Fast response (within 1 min) and low detection limit (126 nM in water)
Successfully applied for the detection of H2S in water samples and hepatocyte.
There are no conflicts to declare.
S1386-1425(19)31080-7
DOI:
https://doi.org/10.1016/j.saa.2019.117690
Reference:
SAA 117690
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: 14 September 2019 Revised Date:
21 October 2019
Accepted Date: 22 October 2019
Please cite this article as: D. Shen, J. Liu, L. Sheng, Y. Lv, G. Wu, P. Wang, K. Du, Design, synthesis and evaluation of a novel fluorescent probe to accurately detect H2S in hepatocytes and natural waters, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2019), doi: https:// doi.org/10.1016/j.saa.2019.117690. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Design, synthesis and evaluation of a novel fluorescent probe to accurately detect H2S in hepatocytes and natural waters Dadong Shen a, Jian Liu b, Li Sheng c, Yonghui Lv c, Guofeng Wu c, Pu Wang a* and Kui Du b* a
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou
310014, China. E-mail: [email protected] b
School of Chemistry and Chemical Engineering, Shaoxing University, Shaoxing
312000, China, Email: [email protected] c
Research & Development Center, Zhejiang Medicine Co. Ltd. Shaoxing 312500,
China, Email: [email protected] Abstract: Design and synthesis of fluorescent probe with fast response, excellent water solubility and good hepatocyte-targeting capacity to detect hydrogen sulfide (H2S) in hepatocytes and water samples is of great significance. Here, a novel fluorescent probe QL-Gal-N3 for detection of H2S was designed and synthesized based on H2Smediated azide reduction strategy. This sensor demonstrated low toxicity, fast response (within 1 min), high selectivity and low detection limit (as low as 126 nM in water) for the detection of H2S. HeLa, A549 and HepG-2 cells were chosen to investigate the hepatocyte-targeting ability of QL-Gal-N3 respectively. The results indicated that the specific recognition of ASGPR over-expressed in hepatocytes by galactose group was an important reason for the good targeting ability of probe QLGal-N3. Furthermore, due to the introduction of glycosyl moiety, the water solubility of fluorescent probe was enhanced obviously. It was successfully applied for the detection of H2S in environmental water samples including river water, tap water, lake water and waste water. 1
Keywords:
H2S;
Azide
reduction;
Fluorescent
probe;
Hepatocyte-targeting;
Environmental water samples 1. Introduction Hydrogen sulfide (H2S) is an important gas signal molecule, which is second only to nitric oxide and carbon monoxide in human body [1]. It can effectively remove hydrogen peroxide, superoxide anion and so on [2, 3]. However, the safety window of H2S between physiological level and toxicological level is narrow with less than two folds in the brain tissues [4]. And it is predominantly generated in the liver due to the tissue-specific distribution of Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), which responsible for the production of H2S with L(homo)cystein as the substrate [5]. In addition, H2S is soluble in water and can directly cause organism death after reaching a certain concentration, which is one of the important parameters for water quality detection [6]. Therefore, it is of great significance to detect the content of H2S in organisms especially liver and natural waters efficiently. Over the past few decades, electrochemical analysis [7, 8], chromatography [9], titrimetry [10], colorimetry [11] and fluorescence analysis [12] had been used for quantitative measurement of H2S. Among these detection methods, fluorescence analysis had attracted widespread attention due to its great advantages such as high sensitivity, simple operation, fast detection speed, simple fluorescence imaging and in situ detection [13-19]. Numerous small-molecule fluorescent probes based on fluorophore such as fluorescein, naphthalimide, quinoline, rhodamine, cyanine and coumarin were designed through reaction-based strategies for the detection of H2S [20-24]. And among the main reaction-based strategies for H2S, H2S-mediated azide reduction had been widely reported for its fast response, low detection limit and good 2
selectivity [25-27]. However, the targeting capacity and water solubility of these sensors still need to be improved to afford the applications of H2S detection in liver and water samples [28]. Recently, several small-molecule fluorescent probes with hepatocyte-selective imaging of H2S had been reported through introducing a glycosyl moiety (as a targeting agent) to the fluorescence probe [29-35]. This strategy provided a good direction for designing of fluorescent probes with good performance in detection of H2S in liver. However, the application of these sensors for the detection of H2S in water samples are rare and still in highly demand. Here we have reported a new smallmolecule fluorescent probes for H2S through the introduction of a glycosyl moiety to quinoline fluorophore. This new sensor displayed fast response (within 1 min), high sensitivty, low detection limit (as low as 126.3 nM in water) and excellent water solubility. In addition, it also demonstrated good application prospects for accurate detection of H2S in hepatocytes and natural water.
Scheme 1 Synthesis of QL-Gal-N3
3
2. Experimental 2.1. Materials and apparatus Unless otherwise specified, chemicals were purchased from J&K Scientific Co., Ltd. and used without further purification. During the experiment, water samples were taken directly from Shaoxing area. Bruker Avance-400 spectrometer was choosen to get the 1 H NMR and
13
C NMR spectra with CDCl3 or d6-DMSO used as a solvent
and tetramethylsilane (TMS) as an internal standard. Human AGSPR ELISA Kit was purchased from AMEKO.The absorbance of MTT was measured by Synergy HT Multi-Mode Microplate reader (Biotek). Fluorescent measurements and UV spectra were measured with SpectraMax M5. The confocal images of cells were captured by Zeiss Fluorescence Microscope (A1 Axiovert) with x40 objective lens. 2.2. Synthesis of QL-Gal-N3 The synthetic route of QL-Gal-N3 was shown in Scheme 1. The detailed synthesis process of compounds 2–5 were provided in the supporting information. Compound 5 (0.5g, 1.34 mmol), TMSN3 (0.185g, 1.6 mmol) and Tert-butyl nitrite(0.21g, 2.0 mmol) were dissolved in THF(50 mL). The reaction mixture was stirred at room temperature for 24h. After the reaction, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography with CH3OH and DCM (4:1) as eluent to give target QL-Gal-N3. White solid (0.1g, 19%) M p: 125-127 ℃; 1H NMR (400 MHz, DMSO) δ 9.12 (dd, J = 8.7, 1.6 Hz, 1H), 8.99 (dd, J = 4.1, 1.6 Hz, 1H), 8.84 (s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.74 (dd, J = 8.7, 4.1 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 5.61 (d, J = 9.2 Hz, 1H), 5.36 (d, J = 5.8 Hz, 1H), 5.10 (d, J = 5.5 Hz, 1H), 4.73 (t, J = 5.6 Hz, 1H), 4.67 (d, J = 5.3 Hz, 1H), 4.22-4.11 (m, 1H), 3.79 (t, J = 5.6 Hz, 2H), 3.63-3.53 (m, 3H). 13C NMR 4
(101 MHz, DMSO) δ 149.66, 145.12, 142.25, 136.11, 135.45, 127.58, 126.82, 125.32, 123.40, 123.14, 119.58, 88.35,78.48, 73.48, 69.24, 68.42, 60.39. HRMS (ESI+): found [M+H]+ 400.1380 C17H18N7O5 requires [M+H]+ 400.1379 2.3. Preparations of test solutions and detection procedure The stock solution of QL-Gal-N3 (1 mM) was prepared in deionized water and sodium sulfide (10 mM) solution was prepared through dissolving in PBS (10 mM, pH 7.4). Stock solutions of cysteine (Cys), glucose (Glu), glutathione (GSH), glycine (Gly), L-alanine (L-Ala), D-alanine (D-Ala), lysine (Lys), homocysteine(Hcy) as well as ions, including Cl- , Br-, F-, NO3-, SO42-, SO32-, K+ and Ca2+ were prepared in deionized water with concentration as 10 mM and kept at 2°C in a refrigerator. Before testing, 985 µL of PBS (10 mM, pH 7.4) and 5 µL of the stock solution of QL-Gal-N3 were mixed, followed by addition of different concentrations of analytes. Then the final volume was adjusted to 1 mL. After incubation at 37 °C for 30 min, 150 µL of the mixture was transferred to 96-well plates to measure fluorescent spectra. The excitation wavelength was 365 nm, and the emission wavelength was in the range from 400 nm to 650 nm with both excitation and emission slits of 5.0 nm. The quantum yield of probe QL-Gal-N3 and probe after reaction with Na2S was determined in PBS buffer using rhodamine B (ɸ, 0.69) as reference. 2.4. Water samples analysis Tap water, river water and lake water were collected from our laboratory, Keqiao District and Guazhu Laker in Shaoxing city, respectively. And the artificial waste water was prepared according to reference [28, 36, 37]. Furthermore, these water samples had not undergone any other special treatment before use. The standard addition method was chosen to determine the H2S content of the sample. The
5
fluorescence spectra were recorded by adding different concentrations of sodium sulfide into water samples. 2.5. Cell Culture and cytotoxicity test HeLa cells, A549 cells and HepG-2 cells were cultured in DMEM high glucose media supplemented with 10% fetal bovineserum, 1% Penstrep, 0.2% Amphotericin B at 37°C incubator with 5% CO2, respectively. And MTT assay was performed to assess the viability of above three cells. The cells were seeded at a density of 10,000 cells per well and grown overnight at 37°C incubator with 5% CO2 . Then different concentrations of probe QL-Gal-N3 were added into 96-well plate for 48h. After that, 10 µL of MTT solution (5mg/ml in PBS) was added into each well and solubilized using 100 µL of MTT solubilizing agent (DMSO) after 2h. Finally, the absorbance at 590 nm with a reference filter of 620 nm were taken using Synergy HT Multi-Mode Microplate reader (Biotek). 2.6 Confocal imaging of Hela cells, A549 cells and HepG-2 cells As for the cell image of HeLa cells, HeLa cells were incubated probe QL-GalN3 (5 µM) for 30 min and washed with PBS for three times, and images were acquired using Zeiss Fluorescence Microscope (A1 Axiovert) with x40 objective lens. After which HeLa cells were treated with different concentrations of H2S (0 µM, 10 µM and 50 µM) and incubated for 30 min, then the cell image were obtained under the excitation with 405nm for collection at 460–540nm. And we choosing 50 µM H2S for the investigation of time-dependent imaging in HeLa cells. In order to get the information of endogenous hydrogen sulfide in HeLa cells, A549 cells and HepG-2 cells, the above cell lines were incubated probe QL-Gal-N3 (5 µM) for 30 min respectively. And the cell image were obtained under the excitation with 405nm for collection at 460–540nm. 6
3. Result and disscussion 3.1. Selective detection of probe QL-Gal-N3 to H2S over other analytes In order to investigate the application of probe QL-Gal-N3 on H2S detection for biological and water samples, the selectivity was conducted firstly. Various species (100 µM) were selected to culture with probe QL-Gal-N3 (5 µM) and fluorescent emission spectra were measured after 30 min. As shown in Fig. 1a, a significant change of QL-Gal-N3 (ɸ, 0.003) was introduced by H2S in fluorescence at 521 nm, and almost 102-folds enhancement in intensity was achieved (compound 5 ɸ, 0.308). Although, probe QL-Gal-N3 exhibited a weak fluorescence intensity with Cys and GSH, it will introduce little influence on the detection process. At the same time, we increased the concentration of GSH to 5 mM, but no obvious fluorescence enhancement at 521 nm was observed (Fig. S6). It demonstrated that QL-Gal-N3 can specifically interact with H2S. The interference experiments of other species on QLGal-N3 was also conducted by us, Fig. 1b. It shown that the co-existent species have little effect on the fluorescent intensity of probe QL-Gal-N3 with H2S. These results indicated that probe QL-Gal-N3 has an excellent selectivity towards different analytes. (a)
(b)
Fig. 1. (a) Fluorescent emission spectra of QL-Gal-N3 (5 µM) in PBS buffer (pH=7.4) to H2S or other species (100 µM) for 30 min; (b) Fluorescent intensity of QL-Gal-N3
7
(5 µM) in the presence of 100µM interfering species including (1) Cl- , (2) Br-, (3) F-, (4) Hcy, (5) NO3-, (6) Cys, (7) SO42-, (8) SO32-, (9)K+, (10) Ca2+, (11) GSH, (12) Gly, (13)L-Ala, (14) D-Ala, (15)Lys, (16) Glu; λEx = 365 nm, λ Em = 521 nm. 3.2. Time-dependent and pH-fluorescence intensity of probe QL-Gal-N3 The pH dependence and response time of QL-Gal-N3 to H2S in biological atomosphere and water are important parameters for the applications. In order to investigate the pH effect on probe QL-Gal-N3, intensity of free QL-Gal-N3 and QLGal-N3 with H2S were obtained at the different range of pH. As shown in Fig. 2a, the fluorescence emission intensity was much lower in acidic environments, which indicating that the thiolysis of the azido bond can not be introduced in strongly acidic solutions [27]. Probe QL-Gal-N3 with H2S maintaining a good pH stability rang from pH=4-10, especially reach the highest intensity when pH=7. The value of saturated hydrogen sulfide aqueous solution is pH=4.14, which indicated that Probe QL-GalN3 is suitable for detecting H2S in water sample.
Fig. 2 (a) pH-dependent fluorescence intensity of QL-Gal-N3 (5 µM) with H2S (100 µM) in PBS buffer (pH= 7.4); (b) Time-dependent fluorescent intensity of QL-GalN3 (5 µM) with H2S (100 µM) in PBS buffer; λ Ex = 365 nm, λ Em = 521 nm. In addition, the time-dependent fluorescence signal of QL-Gal-N3 to H2S at 521 nm was checked (Fig. 2b). The results shown that the fluorescent intensity increased 8
quickly and achieved the plateau within about 1 min, and indicating that probe QLGal-N3 could response rapidly with H2S under physiological conditions. 3.3. The absorbance and concentration-dependent detection of H2S by QL-Gal-N3 In order to titrate the detection capacity of
probe QL-Gal-N3 to H2S, the
absorbance and emission spectra were examined (Fig. S1). The absorption spectrum of QL-Gal-N3 to H2S demonstrated a maximum band at 365 nm, and the intensity increased linearly with the addition of H2S from 0 to 100µM. Furthermore, as shown in Fig. 3b, the fluorescence emission intensity also demonstrated an excellent linearity with the concentration of H2S from 0 to 100µM. The detection limit of probe QL-Gal-N3 to H2S was calculated as 126 nM in PBS buffer through using the formula 3σ/k (σ is obtained from ten blank measurement and k is slope of linear). These results indicated that QL-Gal-N3 had an extremely low detection limit and was suitable for the detection of H2S in water samples. (a)
(b)
Fig. 3 (a) Fluorescent spectra of QL-Gal-N3 (5 µM) in PBS buffer (pH= 7.4) with the addition of H2S. (b) Linearity between the fluorescence intensity of QL-Gal-N3 and the concentration of H2S from 0 to 140µM; λEx = 365 nm.
3.4. Application of probe QL-Gal-N3 in water samples with different concentrations of H2S 9
Table 1 Detection of H2S in tap water, lake water, river water and waste water Samples
Na2S added
Na2S found
RSD
Recovery
(µM)
(µM)
(%)
(%)
1
−
−
−
−
2
1.5
1.52
1.13
102
3
3.0
3.06
1.02
100
4
4.5
4.52
0.96
97
Lake
1
−
−
−
−
water
2
1.5
1.52
1.83
100
3
3.0
3.04
1.25
101
4
4.5
4.48
0.72
94
River
1
−
−
−
−
water
2
1.5
1.54
3.17
118
3
3.0
3.11
2.15
108
4
4.5
4.74
1.23
99
Waste
1
−
−
−
−
water
2
1.5
1.95
6.95
122
3
3.0
3.34
5.21
111
4
4.5
4.86
3.87
108
Tap water
Number
In light of above fluorescent properties of probe QL-Gal-N3, we further investigated its application for H2S in different water samples. We collected river water from Keqiao District, Shaoxing City, which is the largest textile center in Asia. And the tap water in our laboratory, lake water in the Lake of Guazhu were collected by us respectively. Furthermore, the artificial waste water was prepared according to reference [28]. The standard addition method was chosen for the application of probe QL-Gal-N3 in water samples with different concentrations of H2S. As shown in Table 1, the recoveries were in the range of 94%–122% and RSD is less than 7%, demonstrating that the probe QL-Gal-N3 as a highly selective and sensitive fluorescent sensor for H2S detection in water samples. 3.5. Target imaging of H2S in cells by QL-Gal-N3
10
In order to investigate the applicability of the probe QL-Gal-N3 in cell imaging, the cytotoxicities of QL-Gal-N3 for HeLa cells, A549 cells and HepG-2 cells lines were determained. As shown in Fig. S2, 87%, 92% and 85% cells survived was achieved after incubation with QL-Gal-N3 (5 µM) for 24 h, respectively, indicated probe QL-Gal-N3 was low cytotoxicity for the above three cell lines. And then the HeLa was chosen to examine the imaging of probe QL-Gal-N3 (5 µM) with different concentration of H2S. As shown in Fig. 4, when HeLa cells incubated with free probe QL-Gal-N3 for 30 min, little fluorescence displayed. However, co-incubation of QL-Gal-N3 with different concentrations of H2S(10µM and 50µM) for 30min, a gradual increase of fluorescent intensity was detected. These results indicated QL-Gal-N3 can be applied to investigate the change of H2S in cells.
Fig. 4 Confocal imaging of fluorescence and bright field images of probe QL-Gal-N3 (5 µM) in HeLa cells treated with different concentration of H2S for 30 min; λ
Ex
=
405 nm, λ Em = 460-540 nm In order to investigate the time-dependent imaging of QL-Gal-N3 toward H2S in living cells, co-incubation of QL-Gal-N3(5 µM ) and H2S (50 µM ) in HeLa cells was conducted (Fig. 5). The results shown that the fluorescence intensity gradually increased and reaching a maximum after 3 min. Furthermore, the intensity displayed
11
no obvious increase after 3 min, which indicated that QL-Gal-N3 is a rapid responsive and highly sensitive fluorogenic probe to detect H2S in cancer cells.
Fig. 5 Confocal imaging of probe QL-Gal-N3 (5 µM) in Hela cells treated with H2S (50 µM) for 1.0 min, 2.0 min and 3.0 min; λ Ex = 405 nm, λ Em = 460-540 nm Subsequently, the target imaging of H2S in cells by QL-Gal-N3 was tested by us. Before imaging, an enzyme linked immunosorbent assay ( ELISA) was conducted to determine the ASGPR level in HepG-2, A549 and Hela in this study. As shown in Fig. S5 and Table S1, HepG-2 cells showed the highest ASGPR level with 168.15 pg/mL, while A549 and Hela cells displayed low level with 3.48 and 3.23 pg/mL, respectively. After co-incubation of QL-Gal-N3 (10 µM) with HepG-2 cells, A549 cells and HeLa cells for 10 min, a strong fluorescence intensity was observed in HepG-2 cells, as shown in Fig. 6. And demonstrated obviously selective for HepG-2 cells than A549 cells and HeLa cells. These results suggested that the presence of the galactosyl moiety facilitated the selective internalization of the probe QL-Gal-N3 through ASGPr-mediated endocytosis.
12
Fig. 6 Confocal imaging of probe QL-Gal-N3 (10 µM) in HepG-2 cells, A549 cells and HeLa cells for 10 min; λ Ex = 405 nm, λ Em = 460-540 nm 3.6. Comparison of probe QL-Gal-N3 with reported H2S sensors which based on the H2S-mediated azide reduction strategy According to the HRMS spectra of the reaction of QL-Gal-N3 with H2S (Fig. S7) and related literatures [29, 37-51], the specific recognition of QL-Gal-N3 for H2S is based on the reaction mechanism of H2S-mediated reduction of aryl azides. In recent years, the effective design of H2S fluorescent probes through H2S-mediated azide reduction strategy had attracted extensive attention from the researchers (Table 2). the Compared with the reported H2S sensors, probe QL-Gal-N3 demonstrated a good hepatocyte-targeting capacity, while many reported sensor were lack of targeting ability [39-41, 43, 44, 46-52]. QL-Gal-N3 also displayed relatively good detection performances for H2S than most of the reported sensors with a very short response time and an excellent detection limit. More importantly, QL-Gal-N3 showed good water solubility, and was successfully used for the determination of H2S in water samples, however, almost all of the reported fluorescent probes had not been used for the detection of H2S in water samples.
13
Table 2 Comparison of QL-Gal-N3 with other reported H2S sensors based on the H2S-mediated azide reduction strategy Ref.
Targeting
Detection
Response
limit
time
Media
H2S detection in water samples
29
hepatocyte
7.8×10-7 M
2 min
Aqueous solution
No
37
lysosomal
2.14×10-7 M
1 min
PBS (10% DMSO)
No
38
No
1.8×10-8 M
30 min
PBS (50% DMSO)
No
39
No
No discuss
10 min
PBS (1% CH3CN)
No
40
No
No discuss
1h
HEPES
No
41
lysosomal
No discuss
40 min
HEPES (50% CH3CN)
No
42
No
No discuss
10 min
Water(25% Ethanol)
No
43
No
8.6×10-8 M
60 min
PIPES buffer
No
44
lysosomal
5.0×10-7 M
> 60 min
PBS (10% DMF)
No
45
No
7.8×10-10 M
3 min
PBS buffer
No
46
No
1.12×10-7 M
30 min
PBS buffer
No
47
No
No discuss
No discuss
HEPES (33% CH3CN)
No
48
No
2.2×10-7 M
1 min
PBS (75% Acetone)
No
49
No
3.1×10-6 M
60 min
PBS buffer
No
50
No
No discuss
1 min
PBS (50% CH3CN)
No
51
No
8.0×10-8 M
10 min
HEPES
No
QL-
hepatocyte
1.26×10-7 M
1 min
PBS buffer
Yes
Gal-N3
4. Conclusion In summary, a novel hepatocyte-targeting fluorescent probe QL-Gal-N3 with good water solubility was designed and synthesized. QL-Gal-N3 displayed good sensitivity and selectivity toward H2S with the detection limit reached 126 nM. 14
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. . . .
H2S fluorescent probe with excellent hepatocyte-targeting capacity
Low toxicity, good selectivity and excellent water solubility
Fast response (within 1 min) and low detection limit (126 nM in water)
Successfully applied for the detection of H2S in water samples and hepatocyte.
There are no conflicts to declare.