Preparation and application of hydrogen sulfide fluorescent probe based on thioxanthene

Abstract

The invention relates to preparation and application of a hydrogen sulfide (H2S) near-infrared fluorescent probe. The structural formula of the probe is # imgabs0. The invention provides a preparation method for synthesizing the fluorescent probe by taking benzoindole-thioxanthene dye, 4-chloro-7-nitrobenzofurazan and the like as raw materials. The fluorescent probe is a hydrogen sulfide fluorescent probe with large Stokes shift and long near infrared emission; firstly, the fluorescent probe shows good sensitivity to H2S, the linear range is 0.5-30 [mu] M, and the detection limit is 0.2 [mu] M; secondly, the fluorescent probe shows very high selectivity on H2S, and is not influenced by other metal ions, active nitrogen, active oxygen, amino acid, active sulfur and biological mercaptan; moreover, the fluorescent probe can quickly act with H2S, and the response time is within 10 minutes; in addition, the fluorescent probe can also be applied to detection of H2S content in living cells.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to the preparation and application of a hydrogen sulfide fluorescent probe based on thioxanthene. Background Art

[0002] Hydrogen sulfide (H2S) is an important endogenous gas signal molecule in the system, involved in a variety of physiological and pathological processes (Wang, R. Antioxid. Redox Signaling, 2003, 5, 493-501.). It has been identified as a physiological gasotransmitter with powerful cytoprotective effects in multiple organ systems, including regulatory effects in the cardiovascular system and regulation of the central nervous, respiratory and gastrointestinal systems (H. Kimura, Antioxid. Redox Signaling, 2010, 12, 1111; N. Skovgaard, A. Gouliaev, M. Aalling, U. Simonsen, Curr. Pharm, Biotechnol. 2011, 12, 1385; R. Wang, Physiol. Rev. 2012, 92, 791; G. Tang, G. Yang, B. Jiang, Y. Ju, L. Wu, R. Wang, Antioxid. Redox Signaling, 2013, 19, 1634.). At the same time, abnormal production of hydrogen sulfide can lead to a variety of diseases, including Down syndrome, liver cirrhosis, cardiovascular disease, diabetes, etc. (C. Szabo, Nat. Rev. Drug Discovery, 2007, 6, 917-935; M. Lavu, S. Bhushan, DJ Lefer, Clin. Sci. 2011, 120, 219-229; A. Martelli, L. Testai, M. C. Breschi, C. Blandizzi, A. Virdis, S. Taddei, V. Calderone, Med. Res. Rev. 2012, 32, 1093-1130; G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A. K. Mustafa, W. Mu, S. Zhang, S. H. Snyder and R. Wang, Science, 2008, 322, 587.). Therefore, it is very important to develop a convenient and reliable method to detect the changes of H2S levels in living cells in real time.

[0003] The detection of H2S by fluorescence method has attracted much attention due to its many advantages such as simple operation, high sensitivity, good selectivity, real-time and non-invasiveness (G. Deng, S. Li, Z. Sun, W. Li, L. Zhou, J. Zhang, P. Gong, L. Cai, Theranostics. 2018, 8, 4116-4128; G. Hong, S. Diao, J. Chang, A. L. Antaris, C. Chen, B. Zhang, S. Zhao, D. N. Tochin, P. L. Huang, K. I. Andreasson, C. J. Kuo, H. Dai, Nat. Photonics. 2014, 8, 723-730.). So far, some fluorescent probes for detecting H2S have been developed for real-time monitoring of H2S level changes in living cells (L. Yuan, QP Zuo, Sens. Actuators B, 2014, 196, 151-155; W. Chen, Armando Pacheco, Yoko Takano, Jacob J. Day, Kenjiro Hanaoka, M. Xian, Angew. Chem. Int. Ed. 2016, 55, 9993-9996; P. Ou, RL Zhang, ZJ Liu, XTTian, ​​GMHan, BH Liu, ZJ Hu, ZP Zhang, Angew. Chem. Int. Ed. 2019, 58, 2261-2265.). However, due to the short emission wavelength of these probes, the probes are easily interfered by their own background fluorescence, which hinders their application in biological systems. Therefore, it is crucial to design a fluorescent probe with near-infrared emission.

[0004] As a new type of fluorescent dye, thioxanthene has the advantages of large Stokes shift and good stability. And because the dye has near-infrared emission, it has a deeper tissue penetration depth, is not easily interfered by biological autofluorescence, and is more beneficial to biological imaging. Studies have found that fluorescent probes using thioxanthene have been successfully used to detect some targets, such as N2H4, Cys, etc. (ZMWang, Dyes Pigm., 2022, 203, 110382; STCai, J. Mater. Chem. B, 2022, 10, 1265). However, so far, there is no thioxanthene dye as a fluorescent probe to detect H2S. Therefore, it is very necessary to design and synthesize a fluorescent probe based on thioxanthene dye to detect H2S. Summary of the invention

[0005] According to the requirements put forward, the inventors have conducted in-depth research on this, and after paying a lot of creative work, provided a hydrogen sulfide near-infrared fluorescent probe based on benzindole-thioxanthene dye.

[0006] The technical solution of the present invention is a hydrogen sulfide fluorescent probe based on thioxanthene, the structural formula of which is as follows:

[0007]

[0008] A method for preparing a hydrogen sulfide fluorescent probe based on thioxanthene, the reaction steps are as follows:

[0009] 1.0 equivalent of SCy-OH, 1.0 to 3.0 equivalents of 4-chloro-7-nitrobenzofurazan, and 0.3 to 1.0 mL of triethylamine were added to a 100 mL round-bottom flask, and then 5 to 8 mL of anhydrous acetonitrile was added to dissolve it. The mixture was stirred at room temperature for 5 to 8 hours. After the reaction was completed, the crude product was freed from the solvent under reduced pressure and purified by column chromatography using a CH2Cl2 / CH3OH eluent with a volume ratio of 200:1 to 50:1 to obtain a blue-green solid compound SCy-H2S, which is the fluorescent probe.

[0010] The beneficial effect of the present invention is that a hydrogen sulfide fluorescent probe based on thioxanthene has good spectral response performance. First, the fluorescence spectrum properties of the probe are studied. The probe itself has no obvious near-infrared emission peak at 810nm; when H2S is added to the probe, an obvious near-infrared emission peak appears at 810nm. And as the concentration of H2S increases, the near-infrared fluorescence intensity of the probe continues to increase. When 30μM H2S is added, the fluorescence intensity is enhanced by about 7 times, so H2S can be detected well. The linear range of the probe is from 0.5μM to 30μM, and the detection limit is 0.2μM, which shows that the probe can detect H2S with high sensitivity. Next, the ultraviolet absorption spectrum of the probe is studied. When H2S is not added, the probe has an absorption peak at 650nm; after H2S is added, a new absorption peak appears near 760nm. Then, the selectivity of the probe is studied, and the probe and other interfering analytes such as metal ions (Na + Mg 2+ , Ca 2+ ), active nitrogen (NO, NO2 - 、NO3 - ), active oxygen (H2O2, ClO - 、ONOO - ), amino acids (Lys, Try, Leu, Phe, Met, Thr, Ile, Val), active sulfur (SO3 2- 、HSO3 - 、SO4 2-), biothiols (Cys, Hcy, GSH) and the fluorescence response of the test substance (H2S). The results showed that only H2S could cause changes in the fluorescence spectrum, and other test substances had no obvious effect on the fluorescence spectrum of the probe. Finally, the effect of pH value on the determination of H2S by the fluorescent probe was studied. When the pH value was between 7.0 and 9.0, it did not affect the determination of H2S by the fluorescent probe. In addition, the fluorescent probe responded relatively quickly, with a response time of less than 10 minutes.

[0011] Application of a hydrogen sulfide fluorescent probe based on thioxanthene. 4T1 cells were used for research. No obvious fluorescence was observed in the control group cells. When the fluorescent probe was added to the cells, strong fluorescence could be clearly observed, indicating that the cells contained H2S. After the thiols in the cells were removed by treatment with N-ethylmaleimide (NEM), the fluorescence in the cells was significantly weakened after the probe was added. However, after the thiols in the cells were removed by NEM, strong fluorescence signals were observed when exogenous H2S and fluorescent probes were added. These results show that the fluorescent probe can detect intracellular H2S, which provides a reliable means to monitor the level of hydrogen sulfide in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The synthetic route of fluorescent probe.

[0013] Figure 2 The fluorescence spectra of the fluorescent probe after reacting with different concentrations of H2S.

[0014] The horizontal axis is wavelength, and the vertical axis is fluorescence intensity. The concentration of the fluorescent probe is 10 μM, and the concentrations of H2S are: 0.5, 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 μM. The emission wavelength is 810 nm, and the corresponding excitation wavelength is 720 nm.

[0015] Figure 3 This is the fluorescence linear response diagram of the fluorescent probe to different H2S concentrations.

[0016] Figure 4 This is the UV-visible absorption spectrum of the fluorescent probe after reacting with H2S.

[0017] The horizontal axis is wavelength, and the vertical axis is absorbance. The concentration of the fluorescent probe is 10 μM, and the concentration of H2S is 30 μM.

[0018] Figure 5 Selectivity diagram of the fluorescent probe.

[0019] The concentration of the fluorescent probe was 10 μM, the concentration of H2S was 30 μM, and the concentration of other analytes was 200 μM.

[0020] Figure 6 This is a diagram showing the effect of pH on the fluorescent probe.

[0021] Figure 7 This is a curve diagram showing the relationship between the fluorescence intensity of the fluorescent probe and time after the action of H2S.

[0022] The horizontal axis is the wavelength, and the vertical axis is the fluorescence intensity. The concentration of the fluorescent probe is 10μM, and the concentration of H2S is 30μM.

[0023] Figure 8 This is a diagram of a cytotoxicity experiment. The horizontal axis is the concentration of the fluorescent probe, and the vertical axis is the cell survival rate.

[0024] Fig. 9 Cell imaging of the interaction between fluorescent probe and H2S.

[0025] Control group: cells were incubated with PBS for 30 min; SCy-H2S group: cells were stained with 10 μM SCy-H2S for 30 min; NEM+SCy-H2S group: cells were first treated with NEM for 60 min, and then treated with 10 μM SCy-H2S for 30 min; NEM+H2S+SCy-H2S group: cells were first treated with NEM for 60 min, and then treated with 40 μM NaSH and 10 μM SCy-H2S for 30 min.

[0026] Fig.10 The relative fluorescence intensity of cells. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto.

[0028] Embodiment 1:

[0029] Synthesis of fluorescent probes

[0030] Synthesis route such as Figure 1 . 1.0 equivalent of SCy-OH (92 mg, 0.20 mmol), 1.5 equivalent of 4-chloro-7-nitrobenzofurazan (40 mg, 0.20 mmol), and 0.4 mL of triethylamine were added to a 100 mL round-bottom flask, and then 5 mL of anhydrous acetonitrile was added to dissolve it. Stir at room temperature for 6 h. After the reaction was completed, extract with dichloromethane, remove the solvent under reduced pressure, and perform column chromatography using CH2Cl2 / CH3OH with a volume ratio of 100:1 as an eluent to obtain a blue-green solid SCy-H2S (82 mg, yield 65%), which is the fluorescent probe. 1H NMR (400MHz, CDCl3) δ8.68(d,J=8.3Hz,1H),8.34(d,J=14.6Hz,1H),8.23(dd,J=14.5,9.1Hz,1H),8.09(dd,J=17. 5,8.5Hz,1H),7.76(t,J=7.6Hz,1H),7.68–7.62(m,1H),7.55(d,J=8.4Hz,1H),7.43(d,J=10.0Hz,1H),7.33(s,2H) ,7.27(d,J=14.5Hz,1H),7.00(s,1H),6.67(d,J=7.5Hz,1H),5.90(d,J=6.8Hz,1H),4.65(d,J=10.6Hz,2H),2.80(d ,J=7.2Hz,2H),2.68(d,J=7.4Hz,2H),2.08(s,2H),1.70(d,J=6.7Hz,3H),1.43(s,3H),1.39(s,3H).HRMS(ESI)for C 37 H 31 N4O4S + :m / z found,[M] + 627.2061(calcd,627.2051).

[0031] Embodiment 2:

[0032] Fluorescent probe and H2S solution preparation

[0033] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in dimethyl sulfoxide to make 2×10 -4 M reserve solution. Add 500 μL of the probe reserve solution to a 10 mL volumetric flask and dilute to volume with PBS buffer solution to obtain a concentration of 1.0×10 -5 mol / L fluorescent probe solution. H2S was prepared into the following concentrations (0.5, 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 μM).

[0034] Embodiment 3:

[0035] Determination of fluorescence spectrum of fluorescent probe interacting with H2S

[0036] Figure 2This is the fluorescence spectrum of the fluorescent probe and H2S. The concentration of the fluorescent probe is 10μM, and the concentration of H2S is 0.5, 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0μM. The excitation wavelength used in the experiment is 720nm, and the emission wavelength range is 740-900nm. The slit width is 10.0nm / 10.0nm, and the fluorescence measurement instrument used is Hitachi F4600 fluorescence spectrophotometer. Figure 2 It can be seen that before the addition of H2S, the probe itself has almost no emission peak due to the quenching effect of the 4-chloro-7-nitrobenzofurazan group; with the addition of H2S, the 4-chloro-7-nitrobenzofurazan group is thiolyzed and restored to an electron-donating hydroxyl group, and the emission peak at 810nm is greatly enhanced, and with the increase of H2S concentration, the fluorescence intensity of the probe continues to increase. Figure 3 The linear response graph of the probe to different H2S concentrations. The fluorescence intensity is linearly related to the H2S concentration, with a linear range from 0.5μM to 30μM and a detection limit of 0.2μM. This shows that the probe can detect H2S with high sensitivity.

[0037] Embodiment 4:

[0038] Determination of UV-Visible Absorption Spectra of Fluorescent Probes Interacting with H2S

[0039] Figure 4 The figure is the UV-visible absorption spectrum after the fluorescent probe reacts with H2S. The concentration of the fluorescent probe is 10μM and the amount of H2S added is 30μM. The instrument used for UV-visible absorption spectrum measurement is Agilent Cary60 UV-visible spectrophotometer. Figure 4 It can be seen that the probe itself has an absorption band at 650nm; after adding H2S, the absorption peak at 650nm red-shifts, and a new strong absorption peak appears near 760nm.

[0040] Embodiment 5:

[0041] Selectivity of fluorescent probe for H2S determination

[0042] Figure 5 The selectivity of the fluorescent probe for H2S determination was investigated by adding metal ions (Na + Mg 2+ , Ca 2+ ), active nitrogen (NO, NO2 - 、NO3 - ), active oxygen (H2O2, ClO - 、ONOO - ), amino acids (Lys, Try, Leu, Phe, Met, Thr, Ile, Val), active sulfur (SO32- 、HSO3 - 、SO4 2- ), biothiols (Cys, Hcy, GSH), and the fluorescence response of the test substance (H2S). Figure 5 It can be seen that only H2S can cause a significant enhancement in the fluorescence spectrum, and other detection substances have no obvious effect on the fluorescence spectrum of the probe. These results show that the fluorescent probe has good selectivity for H2S.

[0043] Embodiment 6:

[0044] Effect of Solution pH on the Fluorescence Properties of Fluorescent Probes for Determination of H2S

[0045] The effect of pH on the fluorescence spectrum of H2S measured by fluorescent probe was investigated. Figure 6 . The pH range we studied was 4.0-11.0, the concentration of the fluorescent probe was 10μM, and the concentration of H2S was 30μM. As can be seen from the figure, the fluorescence intensity of the fluorescent probe remains basically unchanged as the pH changes, indicating that pH has no significant effect on the probe itself. However, after the addition of H2S, the fluorescence intensity ratio is significantly enhanced in the pH range of 7.0-9.0. In summary, when the pH value is 7.4, the probe can have a good determination effect on H2S, which is very beneficial for the use of the probe in the determination of H2S in actual samples.

[0046] Embodiment 7:

[0047] Determination of the response time of fluorescent probes to H2S

[0048] We studied the response time of the fluorescent probe to H2S, and the results are as follows Figure 7 As can be seen from the figure, the response time of the probe to H2S is 10 minutes, which can meet the requirements of real-time monitoring in actual samples. Figure 7 We can also see that after the fluorescence intensity reaches the maximum value, it no longer changes in the following period of time, which indicates that this fluorescent probe has good photostability.

[0049] Embodiment 8:

[0050] Application of fluorescent probes in living cells

[0051] First, we performed cytotoxicity assays, e.g. Figure 8 As shown. When 0-30 μM probe was added, the survival rate of mouse breast cancer 4T1 cells was above 90%. This shows that the fluorescent probe has low toxicity and can be used to detect H2S in living cells. Then, we studied the application of fluorescent probes in living cells and selected breast cancer 4T1 cells for confocal microscopy imaging. The results are shown in Fig. 9 , Fig.10 As shown. In the control group cells, almost no fluorescence was observed. Then the probe was added to the cells, and the fluorescence was observed to be significantly enhanced. When NEM was added to the cells to remove the intracellular thiol, the probe was added again, and it was found that the fluorescence in the cells almost disappeared. However, after removing the intracellular thiol with NEM in advance, adding exogenous H2S and fluorescent probe, a strong fluorescence signal can be observed. These results show that the probe can detect H2S in cells with high sensitivity.

Claims

1. A hydrogen sulfide fluorescent probe based on thioxanthene, namely SCy-H2S, characterized in that: The structure is as follows:

2. The method for preparing a hydrogen sulfide fluorescent probe based on thioxanthene according to claim 1, characterized in that: The reaction steps are as follows: 1.0 equivalent of SCy-OH, 1.0 to 3.0 equivalents of 4-chloro-7-nitrobenzofurazan, 0.3-1.0 mL of triethylamine was added to a 100 mL round-bottom flask, and then 5-8 mL of anhydrous acetonitrile was added to dissolve it. The mixture was stirred at room temperature for 5-8 hours. After the reaction was completed, the crude product was freed from the solvent under reduced pressure and purified by column chromatography using a CH2Cl2 / CH3OH eluent with a volume ratio of 200:1-50:1 to obtain a blue-green solid compound SCy-H2S, which is the fluorescent probe.

3. The use of a hydrogen sulfide fluorescent probe based on thioxanthene according to claim 1, characterized in that: The fluorescent probe is applied to the detection of hydrogen sulfide content in living cells.

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